Antiproliferative and neurotrophic molecules

Neurotrophic and antiproliferative compounds related to the antiepileptic drug valproate are provided. These compounds are useful for promoting neuronal function as in neurodegenerative disorders and for treating neoplastic disease.

FIELD OF THE INVENTION
 This invention provides methods and compositions useful for the prevention
 and/or treatment of neurodegenerative and proliferative diseases. The
 compositions of the invention promote neuronal cell maturation and retard
 their proliferation. In particular, this invention relates to nonprotein
 neurotrophic molecules capable of passing the blood brain barrier to
 provide therapeutic effects.
 BACKGROUND
 Proper function of the nervous system requires the maturation and
 maintenance of neuronal cells. In addition, the establishment of proper
 synaptic connections allows for the communication between different
 neurons. Deficits in the survival of neurons, or the ability to maintain
 synaptic connections is associated with neurodegenerative disorders
 including Alzheimer's disease, Huntington's disease, amyotrophic lateral
 sclerosis (ALS), Parkinson's disease, stroke and degeneration of neurons
 due to diabetic neuropathy and trauma.
 Many of the neurodegenerative disorders are associated with the loss or
 degeneration of a particular class of neuronal cells. For example, in
 Parkinson's disease dopaminergic neurons of the substantia nigra
 degenerate. Whereas ALS is associated with the loss of motor neurons.
 Wernicke-Korsakoff syndrome, commonly associated with chronic alcoholism,
 causes amnesia due to damage to the mammillary bodies and medial dorsal
 nucleus of the thalamus. Butters N., Seminar Neurol. (1984) 4:226-244.
 Alzheimer's disease appears to be associated with the degeneration of
 certain cholinergic neurons. The severance of axons as a result of trauma
 may cause retrograde degeneration and neuronal death.
 The association between neurodegeneration and the development of disease
 has prompted the search for neurotrophic agents capable of retarding,
 preventing, or reversing such neurodegeneration. To date, much emphasis in
 this area has focused on the identification and characterization of
 neurotrophic polypeptides. For example, attention has been given to
 studying the effects of nerve growth factor (NGF), ciliary neurotrophic
 factor (CNTF), brain drive neurotrophic factor (BDNF) and others. The
 general neurotrophic effect of CNTF and, in particular, its trophic action
 on motor neurons has led to its investigation as a useful agent in the
 treatment of ALS and other neurodegenerative disorders. See, for example,
 Collins et al. U.S. Pat. No. 5,141,856 and Masiakowski WO 91/04316 which
 are incorporated herein by reference. NGF which has been shown to promote
 neuronal outgrowth from central cholinergic neurons has been suggested as
 a useful agent in the treatment of Alzheimer's disease. Most of the
 neurotrophic polypeptides identified to date are active on relatively
 restricted populations of neuronal cells. Whereas others such as CNTF are
 active on a greater number of neuronal cell types.
 It has generally been observed that agents which induce maturation or
 differentiation of neuronal cells in culture, also inhibit their
 proliferation. Normal proliferating embryonic precursors to sympathetic
 and sensory neurons are induced to mature and stop dividing in the
 presence of certain growth factors such as NGF. The association between
 neuronal maturation or differentiation and anti-mitotic action has also
 been observed for certain neoplastic cells which are responsive to
 neurotrophic factors. For example, rat pheochromocytoma, PC12, cells in
 the presence of NGF develop long neurites and stop dividing. Green L A and
 Tischler A S, Proc. Natl. Acad. Sci. USA (1976) 72:2424-2428. Similar
 effects have been observed with other neuronal cells.
 Cells in the nervous system give rise to a variety of potentially fatal
 neoplastic diseases. For example, neuroblastoma and pheochromocytoma are
 believed to arise from cells having an origin in the neural crest.
 Non-neuronal cells of the nervous system including glial cells, astrocytes
 and Schwann cells also give rise to different types of turnors. Most
 present agents used for chemotherapy involving neuronal cells are
 cytotoxic and have relatively poor specificity and penetrability.
 Treatment of neoplastic disease through agents causing maturation has been
 a long sought for goal. Aaronson, S. A. Science (1991) 254:1146-1153.
 Although neurotrophic polypeptides may eventually prove useful for treating
 certain neurodegenerative, and proliferative disorders, they are
 characterized by poor bioavailability resulting from their relatively
 large size making them resistant to passing through the blood brain
 barrier. This poor penetration into the relevant target tissue raises
 substantial difficulties in their use for treating neurodegenerative
 disorders and neoplastic disease of the central nervous system.
 The anticonvulsant sodiutr valproate (VPA) is a branched chain carboxylic
 acid effective in the treatment of primary generalized seizures,
 especially those of the absence type. Pinder, R. M. et al., Drugs (1977)
 13:81-123. Recently, VPA has been reported to be a teratogen and has been
 suggested as potentially causing neural tube defects in 1% to 2% of
 exposed fetuses (Robert E. and Rosa F. W., "Maternal valproic acid and
 neural tube defects," Lancet (1982) 2:937). In addition, a number of other
 defects are also induced by valproic acid treatment during pregnancy (Nau
 et al. J. Pharmacol. Exp. Ther. (1981) 219:768-777. Spina bifida aperta, a
 most serious birth defect, can now also be induced by valproic acid in an
 animal model (Ehlers et al., 1992 a,b). Like the neurotrophic
 polypeptides, valproic acid also shows very limited transfer into the
 central nervous system of the human (Loscher et al., Epilepsia (1988)
 29:311-316). For reviews of clinical and experimental valproic acid
 teratogenesis. cf. Nau et al., Pharmacol. Toxicol. (1991) 69:310-321; Nau,
 CIBA Foundation Symposium 181, pp. 615-664; Marcel Dekker, 1993.
 Studies in vitro have demonstrated valproate to potently inhibit the rate
 of neural derived cell proliferation at concentrations within its
 therapeutic plasma level (Regan, C., Brain Res. (1985) 347:394-398). This
 antiproliferative action of valproate is restricted to a defined point in
 the G.sub.1 phase of the cell cycle. Martin M. and Regan C., Brain Res.
 (1991) 554:223-228. In the presence of valproate, cells assume a
 differentiated phenotype as judged by morphology, increased
 cell-substratum adhesivity and decreased affinity for concanavalin A
 lectin coated surfaces (Martin et al., Toxic in Vitro (1988) 2:43-48;
 Martin et al., Brain Res. (1988) 459:131-137; Maguire and Regan, Int. J.
 Devl. Neurosci. (1991) 9:581-586; Regan, C., Brain Res. (1985)
 347:394-398. These actions of valproate are likely to be restricted to
 cells of the developing neural tube as, in in vivo experimental models,
 valproate has been shown to increase the incidence of neural tube defects
 and sequester specifically into the neuroepithelium where it generates
 cellular disarray (Dencker et al., Teratology (1990) 41:699-706; Ehlers et
 al., Teratology (1992) 45:145-151; Ehlers et al., Teratology (1992)
 46:117-130; Kao et al., Teratogen. Mutagen. Carcinogen. (1981) 1:367-382;
 Turner et al., Teratology (1990) 41:421-442.
 Hyperthermia, which induces neural tube defects (Chernoff and Golden,
 Teratology (1988) 37:37-42; Edwards, Teratogen. Mutagen. Carcinogen.
 (1986) 6:563-582; Shiota, Am J. Med. Genet. (1982) 12:281-288; Finnell et
 al., Teratology (1986) 33:247-252), also arrests neural cells in the
 G.sub.1 phase of the cell cycle both in vivo and in vitro (Martin et al.
 Brain Res. (1991) 554:223-228; Walsh and Morris, Teratology (1989)
 40:583-592); and produces similar pro-differentiative effects to those
 observed with valproate (Martin and Regan, Brain Res. (1988) 459:131-137).
 Thus, a coincident anti-proliferative and pro-differentiative action may
 identify agents which are capable of inducing neural tube defects yet
 provide a basis for the development of compounds useful for treatment or
 prevention of neurodegenerative diseases.
 The studies of the structure activity relationship of teratogenic
 valproate-related compounds suggest a strict structural requirement for
 high teratogenic potency. Nau, H. et al., Pharmacol. & Toxicol. (1991)
 69:310-321. Studies of structure-activity relationships were possible as a
 result of previous work demonstrating that the parent drug molecule--at
 least in the case of valproic acid--and not metabolite(s) proved
 responsible for the teratogenic action (Nau, Fundam Appl Toxicol, (1986)
 6:662-668. Molecules which are highly teratogenic were reported to require
 an alpha-hydrogen atom, a free carboxyl function, and branching on carbon
 atom 2 with two chains containing three carbons each for maximum
 teratogenic activity. (Nau and Loscher, 1986; Nau and Scott, 1986).
 Substances which do not conform with these strict structural requirements
 are of very low or negligible teratogenic activity, but still often
 exhibit good anticonvulsant activity in several experimental models. These
 compounds may therefore be valuable antiepileptic agents (Nau et al.,
 Neurology (1984) 34:400-402; Loscher and Nau, Neuropharmacol (1985)
 24:427-435; Wegner and Nau, Neurology (1992) 42 (Supp. 5):17-24; Elmazar
 et al., J. Pharm. Sci. (1993) 82:1255-1258. Teratogenic activity also
 demonstrated stereoisomeric preferences suggesting a stereoselective
 interaction between the drugs and a specific structure within the embryo.
 In the case of 4-en-VPA (2-n-propyl-4-pentenoic acid) (Hauck and Nau,
 Toxicol Lett (1989) 49:41-48) and 4-yn-VPA (2-n-propyl-4-pentynoic acid)
 (Hauck and Nau, Pharm. Res. (1992) 9:850-855) the S-enantiomers proved to
 be more potent teratogens than the corresponding R-enantiomers. This
 stereoselective teratogenicity was due to differing intrinsic teratogenic
 potencies of the enantiomers, and not due to differences in
 pharmacokinetics as both enantiomers of a given pair reached the target
 tissue to the same degree, but one was more potent than the other (Hauck
 et al., Toxicol. Lett (1992) 60:145-153). Other examples supported the
 pronounced stereoselectivity of the teratogenic, but not the
 anticonvulsant and sedative effect (Hauck et al., Life Sci. (1990)
 46:513-518; Nau et al., Pharmacol. & Toxicol. (1991) 69:310-321. Carbon
 chains connected to carbon atom 2 of valproate which were shorter or
 longer than 3 carbons reduced teratogenic activity. Nau et al. Id.
 Valproate's antimitotic activity has been suggested as being related to
 its teratogenic potential rather than as a potential therapeutic asset, as
 the non-teratogenic valpromide analogue is not antiproliferative (Regan et
 al., Toxic in Vitro (1991) 5:77-82). Teratogenic analogs of valproate have
 been synthesized to date for the purpose of producing more desirable
 antiepileptic agents having fewer or no side effects and have not been
 suggested as being useful in their own right for other therapeutic
 purposes.
 Despite continued efforts to identify compounds useful for treating
 neurodegenerative and proliferative disorders there is still a great need
 for useful compounds of increased efficacy and potency.
 SUMMARY OF THE INVENTION
 This invention provides compounds, pharmaceutical compositions and methods
 useful for promoting neuronal function and inhibiting cell mitosis.
 Accordingly, this invention also provides methods of preventing and
 treating neurodegenerative and proliferativie disorders.
 The compounds of this inmention have the general formula (I)
 ##STR1##
 wherein
 R.sup.1 is --C.ident.CH, --CH.dbd.CH.sub.2 or --CH.sub.2 --CH.sub.3,
 R.sup.2 is a saturated, unsaturated, branched or unbranched C.sub.1
 -C.sub.30 alkyl group which is optionally substituted with a C.sub.3
 -C.sub.9 aliphatic or aromatic cyclohydrocarbon or heterocyclic group.
 M is a hydrogen or a metal atom. Formula I is not 2-n-propyl-4-pentynoic
 acid (4-yn-VPA) or 2-n-propyl-4-pentenoic acid (4-en-VPA) and when R.sup.1
 is --CH.sub.2 --CH.sub.3, R.sup.2 is C.sub.5 to C.sub.30.
 This invention also provides a method of making the compounds of the
 invention.
 This invention also provides pharmaceutical compositions useful for
 inhibiting cell mitosis and/or promoting neuronal function comprising
 effective amounts of the compounds suitable for use in the treatments of
 the invention with a pharmaceutical carrier suitable for administration to
 an individual.
 In addition, this invention relates to methods of promoting neuronal
 function and/or survival, and in particular to methods of treating
 individuals with neurodegenerative disorders. The compounds useful for
 treating neurodegenerative disorders include those of formula I as
 described above including 2-n-propyl-4-pentenoic acid and
 2-n-propyl-4-pentynoic acid, as well as those of formula II
 ##STR2##
 wherein R.sup.3 and R.sup.4 are independently of one another C.sub.1
 -C.sub.30 saturated or unsaturated, branched and/or unbranched aliphatic
 hydrocarbon, optionally substituted by a C.sub.3-9 aliphatic or aromatic
 cyclohydrocarbon, or heterocyclic group. M is hydrogen or a metal atom.
 The compounds and compositions of this invention which are neurotrophic may
 be used to promote the survival and function of neurons which would
 otherwise have diminished function, degenerate or die. Accordingly, in
 addition to treating individuals diagnosed with a neurodegenerative
 disorder, the compounds and compositions of this invention may also be
 used prophylactically to prevent or retard the onset of neurodegenerative
 disorders in individuals identified as being at risk for developing such
 disorders.
 In another embodiment of this invention, the compounds and compositions
 useful for treating neurodegenerative disorders may also be used to treat
 proliferative disorders. The antiproliferative activity of the compounds
 and compositions may be used to prevent or retard the formation of a wide
 variety of tumors by administering the compounds and compositions to a
 person in need of treatment. This treatment is especially useful for
 treating tumors of neuronal or glial origin given that these compounds
 penetrate the CNS.
 It is an object to this invention to provide neurotrophic compounds useful
 for enhancing the survival of neurons and glial cells.
 It is another object of this invention to provide compounds and
 compositions useful for promoting the expression of characteristics
 associated with mature functioning neuronal or glial cells.
 By promoting the survival and function of neuronal or glial cells, it is an
 object of this invention to provide compounds and compositions useful for
 the prevention and/or treatment of a variety of neurodegenerative
 disorders.
 Another object of this invention is to provide compounds and compositions
 useful for inhibiting the pathologic proliferation of neuronal, glial or
 related cells.

DETAILED DESCRIPTION OF THE INVENTION
 This invention relates to derivatives of valproic acid, methods of their
 preparation and pharmaceutical compositions comprising these compounds.
 This invention also relates to a method of promoting neuronal function and
 differentiation which is useful for preventing and treating
 neurodegenerative disorders. The anti-mitotic activity of the compounds
 and compositions of the invention are useful for arresting cells in a
 specific stage of the cell cycle and for the prevention and treatment of
 neoplastic disease.
 The objects of this invention are accomplished by providing potent
 teratogenic analogs of valproic acid which penetrate the CNS as
 neurotrophic/neuroprotective agents capable of treating and retarding the
 onset of neurodegenerative diseases. The compounds and compositions of
 this invention are also useful for controlling the cell proliferative rate
 and the metastatic potential of neoplastic or potentially neoplastic
 cells.
 Accordingly, the compounds of this invention have the general formula (I)
 ##STR3##
 wherein
 R.sup.1 is --C.ident.CH, --CH.dbd.CH.sub.2 or --CH.sub.2 --CH.sub.3,
 R.sub.2 is independently a saturated, unsaturated with at least one double
 or triple bond, branched or unbranched C.sub.1-30 alkyl group, optionally
 substituted with an aliphatic or aromatic C.sub.3-9 cyclohydrocarbon or
 heterocyclic group; with the proviso that when R.sup.1 is CH.sub.2
 --CH.sub.3, R.sup.2 is C.sub.5-30, and that formula I is not
 2-n-propyl-4-pentynoic acid or 2-n-propyl-4-pentenoic acid (4-en-VPA).
 M is a hydrogen or a metal atom.
 This invention also includes the racemic mixtures and the separate
 enantiomeric R and S forms of the compounds and pharmaceutical acceptable
 salts thereof.
 Preferably, R.sup.1 is --C.ident.CH and R.sup.2 is an unbranched saturated
 C.sub.2 -C.sub.10 alkyl group. More preferred, R.sup.2 is an unbranched,
 saturated C.sub.4 -C.sub.6 alkyl group. Examples of preferred substituents
 for R.sup.2 include --(CH.sub.2).sub.1-9 --CH.sub.3, more preferred is
 --(CH.sub.2).sub.3-6 --CH.sub.3, and most preferred is
 --(CH.sub.2).sub.4-5 --CH.sub.3. Most preferred compounds
 2-n-butyl-4-pentynoic acid (R.sup.1 =--C.ident.CH; R.sup.2
 =--(CH.sub.2).sub.3 --CH.sub.3)), 2-n-pentyl-4-pentynoic acid
 (R.sup.1.dbd.--C.ident.CH; R.sup.2.dbd.--(CH.sub.2).sub.4 --CH.sub.3) and
 2-n-hexyl-4-pentynoic acid (R.sup.1.dbd.--C.ident.CH;
 R.sup.2.dbd.--(CH.sub.2).sub.5 --CH.sub.3). In addition, although both
 enantioners and their racemic mixtures are considered within the scope of
 this invention, the S-enantiomeric form is preferred. Preferred metal
 atoms are sodium or other alkali metals, as well as alkaline earth metals
 such as, for example, calcium or magnesium.
 The teratogenic, antiprolilerative and prodifferentiative potencies of the
 preferred compounds are much higher than of the antiepileptic drug
 valproic acid.
 Further branching of R.sup.1 or R.sup.2 reduces the potency of the
 corresponding compounds. This is demonstrated by the low teratogenic,
 antiproliferative and prodifferentiative potency of the following
 compound.
 ##STR4##
 Unsaturation between C.sub.2 and C.sub.3 (IV) as well as methylation of the
 C.sub.5 (V, VI) also lowers, but does not abolish, the above mentioned
 cellular neurotrophic and antiproliferative activity
 ##STR5##
 In agreement with our basic hypothesis, compound IV (Nau et al., Neurology
 (1984) 34:400-402; Nau and Loscher, Fundam Appl. Toxicol. (1986)
 6:669-676; Nau and Scott, Nature (1986) 323:276-278; Vorhees et al.,
 Teratology (1991) 43:583-590; Ehlers et al., Devel. Pharmacol. Ther.
 (1992) 19:196-204 and VI (Nau et al., Phamacol. & Toxicol. (1991)
 69:310-321; Elmazar et al., J. Pharm. Sci. (1993) 82:1255-1258 has very
 low or undetectable teratogenic activities, but good anticonvulsant
 properties in experimental models.
 The compounds and compositions of this invention are more potent
 teratogenic analogues of valproate and exhibit greater antiproliferative
 and neurotrophic/neuroprotective activity than the parent. In contrast to
 saturated valproate analogues (where both chains must contain 3 carbon
 atoms each for maximal activity) a double or triple bond in the .omega.
 position of one chain exhibits higher activities when the other chain
 contains 4 to 10 carbon atoms. The 2-n-propyl-4-pentynoic acid,
 2-n-butyl-4-pentynoic acid, 2-n-pentyl-4-pentynoic acid,
 2-n-hexyl-4-pentynoic acid, 2-n-hepta-4-pentynoic acid and
 2-n-octa-4-pentynoic acid are the most potent valproate-related teratogens
 synthesized. 2-n-butyl-4-pentynoic acid, 2-n-pentyl-4-pentynoic,
 2-n-hexyl-4-pentynoic acid, 2-n-hepta-4-pentynoic acid and
 2-n-octa-4-pentynoic acid are more preferred. Most preferred are
 2-n-pentyl-4-pentynoic acid and 2-n-hexyl-4-pentynoic acid.
 The preferred compounds for use with this invention possess a chiral
 alpha-carbon. As a result of chirality, the efficacy and potency of
 different enantiomeric forms may differ. For example,
 S-2-n-propyl-4-pentynoic acid has significantly greater teratogenic
 potential than the R-enantiomeric form. Hauck and Nau, Pharm. Res. (1992)
 9:850-855; Hauck et al. Toxicol. Lett. (1992) 60:145-153. See Nau et al.
 Pharmacol. Toxicology (1991) 69:310-321 which is incorporated herein by
 reference. Although there is no general rule of the above-identified
 compounds, the S enantioneric form is preferred.
 The compounds of this invention are prepared by reacting an appropriately
 substituted malonic acid diethylesler with an appropriate unsaturated
 alkylating agent such as a straight-chain alkylhalide. The product is then
 hydrolyzed and decarboxylated.
 This reaction can also be carried out in the reciprocal manner in that a
 malonic acid diethylester, substituted with an unsaturated function is
 reacted with an appropriate alkylhalide. This reaction is again followed
 by hydrolysis and decarboxylation.
 The novel compounds of this invention may be produced according to the
 method of this invention. In one embodiment, the method of synthesizing
 the compounds comprises combining a malonic acid diester reactant with a
 first halide reactant having the general formula
EQU R.sup.2 --X (VII)
 wherein R.sup.2 is a saturated or unsaturated branched or unbranched
 C.sub.1 -C.sub.30 alkyl group and X is a halide. This first reaction
 produces a 2-alkyl-malonic acid diester. The 2-alkyl-malonic acid diester
 is then further combined with a second halide reactant having the general
 formula
EQU R.sup.1 --CH.sub.2 --X (VIII)
 wherein R.sup.1 is --C.ident.CH, --CH.dbd.CH.sub.2 or --CH.sub.2 --CH.sub.3
 to produce compounds with the general formula
 ##STR6##
 wherein R.sup.5 is an alkyl group.
 The resulting diesters are then hydrolyzed, decarboxylated and optionally
 converted into a salt.
 In an alternative embodiment, the order of carrying out the reactions is
 reversed, such that the R.sup.1 --CH.sub.2 --X is combined with the
 malonic acid diester followed by further reaction with the R.sup.2 --X.
 In a preferred method of preparing the compounds of this invention, malonic
 acid diethylester is treated with a base, for example, sodium ethylate, to
 deprotonate carbon 2. Subsequent treatment of the resulting deprotonated
 ester with an alkylating agent in the form of a straight-chain alkyl
 halide yields a 2-n-alkyl-malonic acid diethylester.
 ##STR7##
 This product is further alkylated with sodium ethylate and either
 2-propynehalide to yield XI
 ##STR8##
 The diesters (XI) and (XII) and (XIII) are hydrolyzed and decarboxylated
 with potassium hydroxide in ethanol/water with heat treatment.
 Another embodiment of this invention is the promotion of neural function by
 contacting neural cells with a neurotrophic amount of a compound of
 formula (II)
 ##STR9##
 wherein R.sup.3 and R.sup.4 are independently of each other saturated or
 unsaturated, branched, or unbranched, C.sub.1 -C.sub.30 aliphatic
 hydrocarbons, optionally possessing at least one double or triple bond.
 Preferably R.sup.3 and R.sup.4 are unbranched, and R.sup.3 is less than or
 equal to a three carbon chain. R.sup.4 preferably is a saturated alkyl
 group and is preferrably from C.sub.2 -C.sub.10, as in for example
 --(CH.sub.2).sub.1-9 --CH.sub.3, and more preferably from C.sub.4 to
 C.sub.6, as in for example --(CH.sub.2).sub.3-5 --CH.sub.3. In addition to
 the compounds stated above in connection with formula I, other compounds
 which are useful for the promotion of neuronal function and inhibition of
 cell mitosis are described in Nau et al. PCT application PCT/DE93/00861
 published as WO94/06743, and which is incorporated herein by reference.
 Preferred compounds useiful for promoting neuronal function include for
 example, 2-n-propyl-4-pentynoic acid (R.sup.3.dbd.--CH.sub.2 --C.ident.CH;
 R.sup.4.dbd.--(CH.sub.2).sub.2 --CH.sub.3); valproic acid
 (R.sup.3.dbd.R.sup.4.dbd.--(CH.sub.2).sub.2 --CH.sub.3);
 2-n-propylhexanoic acid (R.sup.3.dbd.--(CH.sub.2).sub.3 --CH.sub.3,
 R.sup.4.dbd.--(CH.sub.2).sub.2 --CH.sub.3); and 2-n-butylhexanoic acid
 (R.sup.3.dbd.R.sup.4.dbd.--(CH.sub.2).sub.3 --CH.sub.3).
 The promotion of neuronal function is particularly useful for preventing
 and treating neurodegenerative disorders. Neurodegenerative disorders
 include any disorder resulting in neuronal degeneration which is
 responsive to at least one of the valproate analogues or valproate itself.
 The neurotrophic activity associated with valproate and its analogues may
 be determined based on in vitro indices of differentiation, including
 inhibition of mitosis, increase in neurite outgrowth, and NCAM expression.
 For example, the ability to promote neurite outgrowth is correlated with
 enhanced survival of certain cultured neural cells including embryonic
 sensory and sympathetic neurons. Proliferating immature neuroblasts, in
 vitro, have a rounded shape and are loosely adherent to culture surfaces.
 In the presence of a neurotrophic factor, these cells become more adherent
 and sprout processes known in the art as neurites. Accordingly, in vitro
 neurite outgrowth may be used as an assay for determining concentrations
 of compound in contact with target cells which would be expected to
 achieve desirable neuroprotecting effects.
 Methods of assessing neurite outgrowth in vitro are well known in the art
 and, for example, may be assessed through direct microscopic visual
 inspection or through the use of computer aided image processing.
 Another characteristic of neurotrophic factors which may be used to assess
 the neuroprotective action of the compounds and compositions of this
 invention is their ability to promote survival of certain specific cell
 types. For example, NGF is required in vitro for the survival of certain
 specific cell types which die in the absence of NGF. Such NGF dependent
 cells include neurons of the chick dorsal rat ganglia at about embryonic
 day E5 to E8.
 Scanning electron microscopy illustrates the cells ability to increase
 cell-substratum adhesivity. They eliminate rounded and clustered growth,
 typical of tumor cells, and induce a flattening and greater interaction
 with the substratum (FIG. 2). In vivo, it is generally believed that these
 neurites further differentiate into axons and dendrites and form synapses
 with other neurons. During diseases involving neurodegeneration, there may
 be a loss of synapses and degeneration of axons and dendrites resulting in
 a deficit of neuronal function.
 Another index of differentiation resulting from the neurotrophic activity
 of valproate analogues is an increase in NCAM expression. Further,
 increases in NCAM prevalence enhances neurite outgrowth. Doherty et al.,
 Nature (1990) 343:464-466. NCAM has been reported as playing a fundamental
 role in memory formation as intraventricular infusion of anti-NCAM during
 consolidation of a recent learning event induces an amnesia. Doyle et al.,
 J. Neurochem. (1992) 59:1570-1573, which is incorporated herein by
 reference. Rapid endocytosis of the Aplysia NCAM homologue was reported
 following a serotonin-induced change in synapse structure in vitro. Bailey
 et al., Science (1992) 256:645-649.
 During development of individual brain regions, or in adults exhibiting
 ongoing neurogenesis, NCAM transiently increases its sialylation state.
 See review, Regan, Int. J. Biochem. (1991) 23:513-523, which is
 incorporated herein by reference, Rougon (1993) Eur. J. Cell Biol.
 61:197-207. The synapse specific NCAM isoform (NCAM 180) which is
 associated with differentiated neurons increases its sialylation state
 during later stages of development until the period of synaptogenesis is
 complete. Breen et al., J. Neurochem (1988) 50:712-716. A similar
 isoform-specific sialylatior of NCAM 180 occurs during consolidation of a
 passive-avoidance response. Doyle et al., J. Neurosci Res., (1992)
 31:513-523.
 We have observed the ability of these compounds and compositions to exert
 an in vivo neurotrophic action in acute and chronic studies employing
 adult male Wistar rats. The acute studies determined their ability to
 reverse the amnesic effect of a 6 hour post-training scopolamine lesion in
 a one trial passive avoidance paradigm as has been employed for other
 neuroprotective agents (Doyle et al., J. Neurochem. 1993 61: 266-272;
 Doyle and Regan, J. Neural Transm. 1993 92: 33-49).
 Accordingly, the methods of treatment and prevention of neurodegenerative
 diseases rely on the ability of valproate and its analogues to possess
 neurotrophic activity such as promoting neurite outgrowth and survival of
 neuronal cells and NCAM expression.
 It is contemplated that the methods of treatment may provide benefits to
 persons with neurodegeneration from disorders including, but not limited
 to ALS, Alzheimers disease, Parkinson's disease, Huntington's disease,
 diabetic neuropathy and stroke. In addition, the neurite promoting
 activity of the disclosed compounds and compositions would also provide
 benefits to individuals with traumatic nerve injury.
 In another embodiment of this invention, methods are provided for arresting
 cells in a specific stage of the cell cycle which leaves the cells in a
 differentiated state by contacting cells with a mitotic inhibitory amount
 of a compound of formula II as described above. Preferred substituents for
 R.sup.3, R.sup.4 and M for inhibiting mitosis are the same as those for
 promoting neuronal function, with the proviso that formula II is not
 valproate if simply used to inhibit cell mitosis. Preventing mitosis in
 this manner is useful for enhancing the expression of specific proteins
 associated with the differentiated phenotype. This enhanced expression
 facilitates purification of such proteins. In addition, arresting or
 retarding mitosis is useful for treating proliferative disorders by
 administering to individuals in need of treatment valproate and/or another
 of its anti-mitotic analogues.
 We have observed sensitivity to valproate or its anti-mitotic analogues in
 all cells tested. Such cell types include: primary astrocytes, human
 astrocytoma, and those from cardiac, renal, and immune systems.
 Accordingly, the antiproliferative action of valproate and its other
 analogues described herein should have broad applicability for a wide
 variety of tumors derived from a variety of cell types and particularly
 those mentioned above.
 The neurotrophic and/or anti-mitotic effective amounts of valproate and
 active analogues may be determined using standard dose-response curves.
 Accordingly, representative cells may be cultured in vitro in the presence
 of varying concentration of test compound. At an appropriate time, the
 cells under the different conditions are examined for the appropriate
 parameter (for example, cell number for anti-mitotic activiity; neurite
 outgrowth for neurotrophic activity) and the ED.sub.50 may be determined.
 The preferred compounds of this invention exert a most profound
 antiproliferative action with ED50 values well below (&lt;0.5 mM) those
 observed with valproate (FIG. 1). Thus, these compounds may be expected to
 act at concentrations which will be devoid of the sedative and hepatotoxic
 side effects of valproate. The preferred compounds also exert the
 prodifferentiative action observed with valproate. In the neuro-2a
 neuroblastoma cell line they induce a marked neuritogenic response which
 correlates with their antiproliferative potential (FIG. 2).
 In addition, the more potent of these compounds increase neural cell
 adhesion molecule (NCAM) prevalence (FIG. 3). This cell recognition system
 regulates neural plasticity during development and, later, during
 information storage in the adult animal by altering its prevalence and
 glycosylation state (Doyle et al., J. Neurosci Res., (1992) 31:513-523).
 Drugs which reverse scopolamine-induced amnesia, such as piracetim-related
 compounds, appear to act through a neuroprotective mechanism which
 involves a non-specific increase in NCAM glycosylation and/or prevalence
 (Doyle et al., J. Neurochem. (1993) 61:266-272). Consequently agents which
 would induce NCAM expression may be predicted to have neuroprotective
 potential.
 This invention also provides pharmaceutical compositions useful for
 treating neurodegenerative or proliferative disorders comprising a
 compound selected from formulas I or II as described above. In addition to
 the compounds of formula I or II, the pharmaceutical composition may also
 comprise adjuvant substances and carriers. The compositions may be in the
 form of tablets, capsules, powders, granules, lozenges, suppositories,
 reconstitutable powders, or liquid preparations such as oral or sterile
 parenteral solutions or suspensions.
 In order to obtain consistency or administration it is preferred that a
 composition of the invention is in the form of a unit dose.
 Unit dose presentation forms for oral administration may be tablets and
 capsules and may contain conventional excipients such as binding agents,
 for example syrup, acacia, gelatin, sorbitol, tragacanth, or
 polyvinylpyrrolidone, fillers, for example lactose, sugar, maize-starch,
 calcium phosphate, sorbitol or glycine; disintegrants, for example starch,
 polyvinylpyrrolidone, sodium starch glycolate or microcrystalline
 cellulose; or pharmaceutically acceptable wetting agents such as sodium
 lauryl sulphate.
 The solid oral compositions may be prepared by conventional methods of
 blending, filling, tabletting or the like. Repeated blending operations
 may be used to distribute the active agent throughout those compositions
 employing large quantities of fillers. Such operations are of course
 conventional in the art. The tablets may be coated according to methods
 well known in normal pharmaceutical practice, in particular with an
 enteric coating.
 Oral liquid preparations may be in the form of, for example, emulsions,
 syrups, or elixirs, or may be presented as a dry product for
 reconstitution with water or other suitable vehicle before use. Such
 liquid preparations may contain conventional additives such as suspending
 agents, for example sorbitol syrup, methyl cellulose, gelatin,
 hydroxyethylcellulose, carboxymethylcellulose, aluminum stearate gel,
 hydrogenated edible fats; emulsifying agents, for example lecithin,
 sorbitan monooleate, or acacia; non-aqueous vehicles (which may include
 edible oils), for example almond oil fractionated coconut oil, oily esters
 such as esters of glycerine, propylene glycol, or ethyl alcohol;
 preservatives, for example methyl or propyl p-hydroxybenzoate or sorbic
 acid; and if desired conventional flavoring or coloring agents.
 For parenteral administration, fluid unit dosage forms are prepared
 utilizing the compound and a sterile vehicle, and, depending on the
 concentration used, can be either suspended or dissolved in the vehicle.
 In preparing solutions the compound can be dissolved in water for
 injection and filter sterilized before filling into a suitable vial or
 ampoule and sealing. Advantageously, adjuvants such as a local
 anaesthetic, a preservative and buffering agents can be dissolved in the
 vehicle. To enhance the stability, the composition can be frozen after
 filling into the vial and the water removed under vacuum. Parenteral
 suspensions are prepared in substantially the same manner, except that the
 compound is suspended in the vehicle instead or being dissolved, and
 sterilization cannot be accomplished by filtration. The compound can be
 sterilized by exposure to ethylene oxide before suspending in the sterile
 vehicle. Advantageously, a surfactant or wetting agent is included in the
 composition to facilitate uniform distribution of the compound.
 The dose of the compound used in the treatment of such disease will vary in
 the usual way with the seriousness of the disorders, the weight of the
 sufferer, and the relative efficacy of the compound.
 Antiproliferative and neuroprotective actions should be sufficient to
 achieve the desired inhibition of mitosis or neuroprotection without
 serious hetaptotoxic side effects. The plasma concentrations to be
 achieved will be sufficient to provide therapeutically effective
 concentrations of compound in contact with the target cells. Standard
 clinical techniques may be used to determine the effective amount of
 compound to be administered to achieve the desired therapeutic effect.
 Dose response curves may be determined first in vitro in a relevant animal
 model to determine ranges of expected therapeutic concentrations in
 humans. For example, mitosis of mouse neuro-2a-neuroblastoma cells is
 inhibited by valproate with an ED.sub.50 of 1.0-1.3 mM. Other cell lines,
 including those of human origin may be used to assesses activity as well.
 EXAMPLE 1
 0.1 mol n-butyl malonic acid diethylester and 0.1 mol 3-bromo-1-propine
 were placed in a dry argon flushed flask and heated to 60.degree. C. To
 this mixture was added 0.1 mol sodium ethanolat (prepared from 0.1 mol
 sodium and 50 mol dry ethanol) dropwise such as to keep the mixture
 boiling. After completion of the addition, the mixture was heated until
 TLC (Silica alu sheets, hexane/ethylacetate 7.5/1) showed absence of
 starting material (usually 1-2 hours). The ethanol was evaporated under
 reduced pressure, the remaining salts were dissolved in water and the
 product was extracted three times with CH.sub.2 Cl.sub.2. The organic
 phase was dried over sodium sulfate and evaporated. The distillation under
 reduced pressure resulted in the unsymmetrically substituted malonic acid
 diethylester.
 bp.sub.0.3 mbar : 78-82.degree. C.
 The dialkylated malonic esters were heated in a solution of 20.3 g (0.35
 mol) potassium hydroxide, 50 ml water and 100 ml ethanol. After completion
 of the saponification, ethanol was evaporated under reduced pressure. The
 remaining residue was diluted with water and washed with ether. The water
 layer was acidified with concentrated HCl (pH&lt;2) and extracted with
 ether. Drying over anhydrous sodium sulfate and concentration under
 reduced pressure yielded crude dialkyl malonic acid. Decarboxylation was
 achieved by heating of the crude product (120-180.degree. C.). The dark
 residue was distilled twice in vacuo resulted in the desired products.
 Overall yield: 18% bp.sub.0.1 mbar : 75-78.degree. C. 1.sub.H-NMR
 (CDCl.sub.3): 0.94 (3H, t, CH,), 1.34 (4H, m, 2.times.CH.sub.2), 1.72 (2H,
 m, CH.sub.2 --CHRCOOH), 2.04 (1H, t, C.ident.C--H), 2.36-2.68 (3H, m,
 CHRCOOH--CH.sub.2 --C.ident.C), 11.88 (1H, s, broad, COOH).
 EXAMPLE 2
 0.1 mol n-pentyl malonic acid dielthylester is reacted with 0.1 mol
 3-bromo-1-propine as described in example 1.
 Overall yield: 14% bp.sub.15 mbar : 135.degree. C. 1-.sub.H-NMR
 (CDCL.sub.3): 0.92 (3H, t, CH.sub.3), 1.32 (6H, m, 3.times.CH.sub.2), 1.72
 (2H, m, CH.sub.2 ---CHRCOOH), 2.04 (1H, t, C.ident.C--H), 2.40-2.72 (3H,
 m, CHRCOOH--CH.sub.2 --C.ident.C), 11.32 (1H, s, broad, COOH).
 EXAMPLE 3
 (+)-2-(2-propinyl)-Octanoic acid (Hexyl-4-yn)
 ##STR10##
 Synthesis is by the dianion method (Petragnani, Synthesis 521, 1982).
 All glassware was oven dried and the reaction apparatus was flushed with
 argon throughout the entire operation.
 Lithium-dianion (0.2 Mol) was prepared by adding 0.2 Mol n-butyl-lithium to
 a solution of 0.2 Mol freshly distilled diisopropylamine and 130 ml dry
 tetrahydrofurane at 0.degree. C. Octanoic acid (0.1 Mol) was added
 followed by 19 hexamethylphosphoric acid triamide to effect solution of
 the dianion. The resulting mixture was stirred at room temperature for 30
 min followed by cooling to -60.degree. C. and addition of 3-bromo-1-propin
 (0.1 Mol) quickly via a syringe. The temperature rose instantly. After
 cooling back to -60.degree. C., the reaction was stirred and monitored by
 TLC (Hexane:Ethylacetate=7.5:1 plus 5% acetic acid) until completion (ca
 1.5 h). Cooling was removed and 200 ml 10% HCl was added. The phases were
 separated and the water phase was extracted twice with ether. The combined
 organic phases were washed with half saturated NaCl solution and dried
 with Na.sub.2 SO.sub.4. Evaporation of the solvent yielded a yellow oil.
 Destillation yielded a colorless liquid (bp. 82-84.degree. C., 0.1 mbar).
 .sup.1 H NMR (CDCl.sub.3)=0.88 (3H, t, CH.sub.3), 1.40 (8H, mc, CH.sub.2),
 1.90 (2H, mc, CH.sub.2), 2.04 (1H, t, .ident.--H), 2.32-2.68 (3H, m,
 CH.sub.2, H.sub..alpha.), 12.04 (1H, s broad, COOH).
 EXAMPLE 4
 The following non-limiting preferred examples are compounds within the
 scope of this invention:
 2-n-propyl-4-pentynoic acid
 2-n-prop-1.sup.1 -enyl-4-pentynoic acid
 2-n-prop-2.sup.1 -enyl-4-pentynoic acid
 2-i-propyl-4-pentynoic acid
 2-i-propenyl-4-pentynoic acid
 2-n-butyl-4-pentynoic acid
 2-n-but-1.sup.1 -enyl-4-pentynoic acid
 2-n-but-2.sup.1 -enyl-4-pentynoic acid
 2-n-but-3.sup.1 -enyl-4-pentynoic acid
 2-(1.sup.1 -methylbutyl)-4-pentynoic acid
 2-(1.sup.1 -methylprop-1.sup.1 -enyl)-4-pentynoic acid
 2-(1.sup.1 -methylprop-2.sup.1 -enyl)-4-pentynoic acid
 2-(2.sup.1 -methylpropyl)-4-pentynoic acid
 2-(2.sup.1 -methylprop-1.sup.1 -enyl)-4-pentynoic acid
 2-(2.sup.1 -methylprop-2.sup.1 -enyl)-4-pentynoic acid
 2-tert.-butyl-4-pentynoic acid
 2-n-pentyl-4-pentynoic acid
 2-(1.sup.1 -methylbutyl)-4-pentynoic acid
 2-(2.sup.1 -methylbutyl)-4-pentynoic acid
 2-(3.sup.1 -methylbutyl)-4-pentynoic acid
 2-(1.sup.1,1.sup.1 -dimethylpropyl)-4-pentynoic acid
 2-(1.sup.1,2.sup.1 -dimethylpropyl)-4-pentynoic acid
 2-(2.sup.1,2.sup.1 -dimethylpropyl)-4-pentynoic acid
 2-n-hexyl-4-pentynoic acid
 2-n-hex-1.sup.1 -enyl-4-pentynoic acid
 2-n-hex-2.sup.1 -enyl-4-pentynoic acid
 2-n-hex-3.sup.1 -enyl-4-pentynoic acid
 2-n-hex-4.sup.1 -enyl-4-pentynoic acid
 2-n-hex-5.sup.1 -enyl-4-pentynoic acid
 2-(1.sup.1 -methylpentyl)-4-pentynoic acid
 2-(1.sup.1 -methylpent-1.sup.1 -enyl)-4-pentyroic acid
 2-(1.sup.1 -methylpent-2.sup.1 -enyl)-4-pentynoic acid
 2-(1.sup.1 -methylpent-3.sup.1 -enyl)-4-pentynoic acid
 2-(1.sup.1 -methylpent-4.sup.1 -enyl)-4-pentynoic acid
 2-(2.sup.1 -methylpentyl)-4-pentynoic acid
 2-(2.sup.1 -methylpent-1.sup.1 -enyl)-4-pentynoic acid
 2-(2.sup.1 -methylpent-2.sup.1 -enyl)-4-pentynoic acid
 2-(2.sup.1 -methylpent-3.sup.1 -enyl)-4-pentynoic acid
 2-(2.sup.1 -methylpent-4.sup.1 -enyl)-4-pentynoic acid
 2-(3.sup.1 -methylpentyl)-4-pentynoic acid
 2-(3.sup.1 -methylpent-1.sup.1 -enyl)-4-pentynoic acid
 2-(3.sup.1 -methylpent-2.sup.1 -enyl)-4-pentynoic acid
 2-(3.sup.1 -methylpent-3.sup.1 -enyl)-4-pentynoic acid
 2-(3.sup.1 -methylpent-4.sup.1 -enyl)-4-pentynoic acid
 2-(4.sup.1 -methylpentyl)-4-pentynoic acid
 2-(4.sup.1 -methylpent-1.sup.1 -enyl)-4-pentynoic acid
 2-(4.sup.1 -methylpent-2.sup.1 -enyl)-4-pentynoic acid
 2-(4.sup.1 -methylpent-3.sup.1 -enyl)-4-pentynoic acid
 2-(4.sup.1 -methylpent-4.sup.1 -enyl)-4-pentynoic acid
 2-(1.sup.1,1.sup.1 -dimethylbutyl)-4-pentynoic acid
 2-(1.sup.1,1.sup.1 -dimethylbut-2.sup.1 -enyl)-4-pentynoic acid
 2-(1.sup.1,1.sup.1 -dimethylbut-3.sup.1 -enyl)-4-pentynoic acid
 2-(1.sup.1,2.sup.1 -dimethylbutyl)-4-pentynoic acid
 2-(1.sup.1,2.sup.1 -dimethylbut-1.sup.1 -enyl)-4-pentynoic acid
 2-(1.sup.1,2.sup.1,dimethylbut-2.sup.1 -enyl)-4-pentynoic acid
 2-(1.sup.1,2.sup.1,dimethylbut-3.sup.1 -enyl)-4-pentynoic acid
 2-(1.sup.1,3.sup.1 -dimethylbutyl)-4-pentynoic acid
 2-(1.sup.1,3.sup.1 -dimethylbut-1.sup.1 -enyl)-4-pentynoic acid
 2-(1.sup.1,3.sup.1 -dimethylbut-2.sup.1 -enyl)-4-pentynoic acid
 2-(1.sup.1,3.sup.1 -dimethylbut-3.sup.1 -enyl)-4-pentynoic acid
 2-(2.sup.1,2.sup.1 -dimethylbutyl)-4-pentynoic acid
 2-(2.sup.1,2.sup.1 -dimethylbut-3.sup.1 -enyl)-4-pentynoic acid
 2-(2.sup.1,3.sup.1 -dimethylbutyl)-4-pentynoic acid
 2-(2.sup.1,3.sup.1 -dimethylbut-1.sup.1 -enyl)-4-pentynoic acid
 2-(2.sup.1,3.sup.1 -dimethylbut-2.sup.1 -enyl)-4-pentynoic acid
 2-(2.sup.1,3.sup.1 -dimethylbut-3.sup.1 -enyl)-4-pentynoic acid
 2-(3.sup.1,3.sup.1 -dimethylbutyl)-4-pentynoic acid
 2-(3.sup.1,3.sup.1 -dimethylbut-1.sup.1 -enyl)-4-pentynoic acid
 2-(1.sup.1,1.sup.1,2.sup.1 -trimethylpropyl)-4-pentynoic acid
 2-(1.sup.1,1.sup.1,2.sup.1 -trimethylprop-2.sup.1 -enyl)-4-pentynoic acid
 2-(1.sup.1,2.sup.1,2.sup.1 -trimethylpropyl)-4-pentynoic acid
 2-n-heptyl-4-pentynoic acid
 2-(1.sup.1 -methylhexyl)-4-pentynoic acid
 2-(2.sup.1 -methylhexyl)-4-pentynoic acid
 2-(3.sup.1 -methylhexyl)-4-pentynoic acid
 2-(4.sup.1 -methylhexyl)-4-pentynoic acid
 2-(5.sup.1 -methylhexyl)-4-pentynoic acid
 2-(1.sup.1,1.sup.1 -dimethylpentyl)-4-pentynoic acid
 2-(1.sup.1,2.sup.1 -dimethylpentyl)-4-pentynoic acid
 2-(1.sup.1,3.sup.1 -dimethylpentyl)-4-pentynoic acid
 2-(1.sup.1,4.sup.1 -dimethylpentyl)-4-pentynoic acid
 2-(2.sup.1,2.sup.1 -dimethylpentyl)-4-pentynoic acid
 2-(2.sup.1,3.sup.1 -dimethylpentyl)-4-pentynoic acid
 2-(2.sup.1,4.sup.1 -dimethylpentyl)-4-pentynoic acid
 2-(3.sup.1,3.sup.1 -dimethylpentyl)-4-pentynoic acid
 2-(3.sup.1,4.sup.1 -dimethylpentyl)-4-pentynoic acid
 2-(4.sup.1,4.sup.1 -dimethylpentyl)-4-pentynoic acid
 2-(1.sup.1,1.sup.1,2.sup.1 -trimethylbutyl)-4-pentynoic acid
 2-(1.sup.1,1.sup.1,3.sup.1 -trimethylbutyl)-4-pentynoic acid
 2-(1.sup.1,2.sup.1,3.sup.1 -trimethylbutyl)-4-pentynoic acid
 2-(2.sup.1,2.sup.1,3.sup.1 -trimethylbutyl)-4-pentynoic acid
 2-(2.sup.1,3.sup.1,3.sup.1 -trimethylbutyl)-4-pentynoic acid
 EXAMPLE 5
 Maintenance of Cell Lines
 The mouse neuro-2a neuroblastoma cell line (Klebe and Ruddle, 1969 J. Cell
 Biol., 43:69A) was cultured in Dulbecco's modified Eagle's medium (DMEM;
 Flow Laboratories) supplemented with 10% fetal bovine serum (Tissue
 Culture Services), 200 mM glutamine and 100 .mu.g/ml of gentamicin or 100
 units/ml and 100 .mu.g/ml of penicillin/streptomycin antibiotics (Sigma
 Chemicals). The cells were maintained in a water-humidified atmosphere of
 9% CO.sub.2 at 37.degree. C. Cells were passaged using 0.025% trypsin
 (Gibco) in DMEM, and were seeded at a density of 1.times.10.sup.4
 cells/cm.sup.2.
 Antiproliferative Assay
 Neuro-2a cells were seedled in 25 cm.sup.2 flasks (Costar) at a density of
 1.times.10.sup.4 cells/cm.sup.2. Following a recovery period of 24 h, the
 agent to be examined was added to the cells in a vehicle of dimethyl
 sulphoxide (DMSO), the volume of which was 0.2% of the total volume of
 medium bathing the cells. A flask containing the DMSO vehicle alone was
 employed as control. Following incubation for 48 h, cells were exaimined
 using an inverted phase contrast microscope (Leitz Diavert) and
 photographed (Ilford 50ASA film). Cells were then harvested by
 trypsinization anid were counted using a haemocytometer (improved Neubauer
 model). FIG. 1 shows the resultant decrease in cell proliferation.
 Scanning Electron Microscopy
 Cells which were to be examined by scanning electron microscopy were grown
 as previously described in 25 cm.sup.2 flasks. Following 48 h exposure to
 the agent, cells were fixed in a solution of 2.5% glutaraldehyde in 0.1M
 sodium phosphate buffer, pH 7.4, overnight at 4.degree. C. The cells were
 post-fixed subsequently in phosphate-buffered 1% osmium tetroxide for 1 h
 at room temperature, washed and were dehydrated gradually for 1 hour using
 a series of ethanol concentrations stepwise from 20, 40, 60, 80 to a final
 concentration 100%.
 Sections of the base of the tissue culture flask were removed and were
 critical point dried to minimize shrinkage and cracking. This was achieved
 by placing the samples in a Polaron critical point dryer and purging the
 chamber several times with CO.sub.2 to remove all traces of ethanol. After
 1 h the temperature and pressure were increased to 40.degree. C. and 1200
 lbs/in.sup.2, respectively, at which stage the critical point for carbon
 dioxide had been reached and drying was completed.
 Specimens were subsequently removed from the chamber, mounted on stubs
 suitable for scanning electron microscopy using conductive carbon cement
 (Neubauer) and were sputter coated with gold under vacuum
 (5.times.10.sup.-2 torr) in the presence of argon gas at a current of 20
 mA for 3 minutes (Polaron E5100). Following gold-coating, samples wvere
 examined in the scanning electron microscope (JEOL 35C) at an accelerating
 voltage of 15 kV. Images were recorded on film (Kodak Plus-X Pan 120 film)
 as shown in FIG. 2.
 Fluorescence Microscopy
 Cells were seeded in 24-well plates at a density of 1.times.10.sup.4
 cells/cm.sup.2. Following a recovery period of 24 h they were exposed for
 an additional 48 h to the drug under investigation. Cells were
 progressively fixed by six ten-minute incubations with DMEM containing
 increasing concentrations of neutral buffered formalin stepwise from 10,
 30, 50, 70, 90 to a final concentration of 100%. When fixation was
 complete, cells were washed three times with phosphate buffered saline pH
 7.4 over a 30 minute period. The cells were then incubated with a 1 in 50
 dilution of rabbit anti-NCAM antibody, (Pliophys et al. J. Neuropsychiatr.
 2:413-417, 1990) in phosphate buffered saline containing 1% (W/V) bovine
 serum albumin for 1 h at RT and washed three times with phosphate buffered
 saline, pH 7.4 for 30 minutes. Washed cells were then incubated for 1 h at
 RT with the secondary anti-rabbit antibody diluted 1 in 50 in phosphate
 buffered saline containing 1% (W/V) bovine serum albumin (Sigma) which was
 conjugated to rhodamine. The cells were again washed three times with
 phosphate buffered saline pH 7.4 and were then mounted using Citifluor
 (Agar Scientific) containing a fluorescence enhancer. Fluorescence of
 rhodamine was visualized using an excitatory wavelength of 535 nm (Leica
 filter block N2.1) on a Leitz DMRB fluorescence microscope. Fluorescence
 intensity was examined at points of cell-cell contact using a Quantimet
 500 Image Analysis System. Fluorescence intensity is expressed as grey
 level at points of cell contact relative to that observed in the control.
 FIG. 3 shows the increase in NCAM immunofluorescence.
 EXAMPLE 6
 Acute and Chronic in vivo Studies
 The ability of the compounds and compositions of this invention to exert an
 in vivo neurotrophic action was investigated in acute and chronic studies
 employing adult male Wistar rats. The acute studies determined their
 ability to reverse the amnesic effect of a 6 hour post-training
 scopolamine lesion in a one trial passive avoidance paradigm as has been
 employed for other neuroprotective agents (Doyle et al., J. Neurochem.
 1993 61:266-272; Doyle and Regan, J. Neural Transm. 1993 92:33-39.
 The 2-n-pentyl-4-pentynoic acid was given in the immediate 3 hour
 post-training period and followed by scopolamine at the 6 hour
 post-training time. All dosing was via the intraperitoneal route at the
 indicated post-training times. Additionally, 3 further groups of at least
 3 animals were administered 2-n-pentyl-4-pentynoic acid at 3 hours and
 vehicle at 6 hours to assess and control for any unwanted effects the
 compound may have in this paradigm. The data is presented as box plots
 which indicate the median and interquartile ranges and statistical
 significance was established by the Mann-Whitney U-test for non-parametric
 data.
 All animals, receiving vehicle only, exhibited good recall with a median
 latency value of 300 seconds to enter the dark, shock compartment,
 indicating good acquisition of the avoidance task (FIG. 4). Scopolamine
 administered at 6 hours post-training attenuated recall at 24 hours giving
 a median latency value of 65 seconds. This demonstrates the efficacy of
 this drug as a potent amnesic agent in this learning model. To assess the
 ability of these neurotrophic compounds to block or reduce these learning
 deficits, 2-n-pentyl-4-pentynoic acid was administered intraperitoneally 3
 hours post-training and scopolamine at 6 hours post-training as before.
 2-n-pentyl-4-pentynoic acid was seen to dose-dependently reduce the
 scopolamine-induced imemory deficits observed at 24 hours post-training
 (FIG. 4). Reversal of scopolamnine-induced amnesia was afforded by doses
 of 2-n-pentyl-4-pentynoic acid in the dose range of 50.4-134 mgs/kg with a
 highly significant reversal being observed with a dose of 84 mg/kg. No
 adverse effects were observed at this dose. When 134.0 mg/kg was
 administered, the attenuation of scopolamine-induced amnesia was greater
 but more variable. In the three remaining groups dosed with
 2-n-pentyl-4-pentynoic acid and vehicle only, the anti-amnesic effect
 plateaued only at the highest concentration (134 mg/kg) tested. This was
 due to variation in the animals' recall ability suggesting a possible
 bell-shaped dose-response effect as no variation in locomotor activity was
 observed.
 The chronic studies evaluated the ability of these compounds and
 compositions to spare an age-dependent decline in a population of neural
 cell adhesion molecule polysialylated neurons located to the granule cell
 layer/hilar border of the hippocampal dentate gyrus. The frequency of
 activated polysialylated neurons in this region increases dramatically
 during memory formation, and, conversely, declines with ageing when memory
 deficits become pronounced (Fox and Regan, Neurochem. Res. 1995 20:
 521-526). As a consequence, they may be considered to be an index of
 memory-associated neuroplastic potential. Chronic intraperitoneal
 administration of 2-n-pentyl-4-pentynoic acid at 16.8 and 50.4 mgs/kg over
 the postnatal day 40-80 period, when an approximate 70% natural decline in
 the number of polysialylated neurons is observed, produced a significant
 sparing when compared to the control animals which received 16.8 mg/kg of
 2-(2-methylpropyl)-4-pentynoic acid, which is without the
 antiproliferative and prodifferentiative effect seen with
 2-n-pentyl-4-pentynoic acid (FIG. 5). This sparing amounted to
 approximately 25% at 50.4 mgs/kg, the highest dose evaluated, which
 represents approximately 2.5 years in human terms. In addition,
 polysialylated neurons were observed in the entorhinal cortex and
 extended, as a single band, through perirhinal cortex up to the level of
 the piriform cortex. This cortical cell population exhibited an
 approximate 2-fold sparing and/or activation following exposure to 50.4
 mgs/kg of 2-n-pentyl-4-pentynoic acid over the postnatal day 40-80 period
 when compared to the control animals which received 16.8 mgs/kg of
 2-(2-methylpropyl)-4-pentynoic acid (FIG. 5).
 The dramatic sparing and/or activation of the polysialylated neurons in the
 rhinal cortex may be related to the differential distribution of
 2-n-pentyl-4-pentynoic acid into the cortex as opposed to the other brain
 regions. In a single animal experiment, an intravenous bolus of
 2-n-pentyl-4-pentynoic acid (84 mgs/kg) exhibited a 2-4 fold greater
 distribution to the cortex as compared to the other brain regions examined
 (FIG. 6). This cortical penetration was equivalent to that observed in the
 kidney. In addition, analysis of the plasma indicated the free
 concentration of 2-n-pentyl-4-pentynoic acid to be approximately 60
 .mu.g/gm corresponding to an unbound fraction of 20%.
 No adverse effects were seen at either dose as indicated by an invariant
 weight gain between the animal groups (FIG. 5) and lack of any abnormal
 behaviour in open-field observations.
 Passive Avoidance Training
 A one-trial, step-through, light-dark model was developed and validated for
 learning/memory studies. The apparatus consisted of a box measuring 300 mm
 wide.times.260 mm deep.times.270 mm high. The front and top were
 transparent, allowing the experimenter to observe the behaviour of the
 animal inside the apparatus. The box was divided into 2 compartments,
 separated by a central shutter which contained a small opening 50 mm wide
 and 75 mm high. The smaller of the compartments measured 90 mm in width
 and contained a low power (6v) illumination source--the light compartment.
 The larger compartment measured 210 mm in width and was not illuminated.
 The floor in this dark compartment consisted of a grid of 16 horizontal
 stainless steel bars which were 5 mm in diameter and 12.5 mm apart. A
 current generator was used to supply 0.75 mA to the grid floor, scrambled
 once every 0.5 seconds across the 16 bars. A resistance range of 40 to 60
 kOhms was calculated for a group of rats (250-350 g) and the apparatus
 calibrated accordingly. An electronic circuit detecting the resistance of
 the animal ensured an accurate current delivery by automatic variation of
 the voltage with change in resistance.
 Animals were introduced to the test holding room 3 days prior to the
 commencement of all studies to allow time for adjustment to the new
 environment. After this period, animals were handled for 2 minutes for 3
 days under low level red light illumination. After each handling session,
 the animal was weighed and placed in the open field arena where ambulation
 and general behaviour was assessed. On the fourth day--training day--each
 rat was handled, weighed and assessed in the open field arena as before.
 However, following behavioral assessment, the animal was placed in to the
 small, light compartment of the passive avoidance training apparatus so
 that it faced the rear wall. The door was quickly and carefully closed.
 Once the rat turned around to face the front panel of the compartment
 (after an adaption time of usually less than 30 seconds) a timer was
 started and the latency to enter the larger dark compartment recorded.
 This time was usually less than 20 seconds. Once the animal entered the
 dark compartment, with all four paws on the steel grid floor, a current of
 0.75 mA was delivered remotely to the floor bars. A maximum shock time of
 5 seconds was set and the animal almost always returned to the safe, light
 compartment within this time, known as the escape latency.
 The escape latency was recorded for each rat and the animal returned to its
 home cage immediately. A control group of animals received no footshock
 but were allowed to explore the apparatus for a time which was paired with
 one of the shocked animals. Between each training session, both
 compartments were wiped down thoroughly to reduce the possibility of
 confounding olfactory cues.
 The animals were then tesited for recall--or the ability to remember not to
 go through to the dark compartment--at 24 hours following raining. Animals
 were handled, weighed and assessed as before and once again placed into
 the safe compartment. Once the animal turned to face the front panel, the
 timer was started and the latency to enter the dark compartment by head
 only, front two paws and finally all four paws was recorded. No current
 was applied to the bars and a cut-of or criterion time of 5 minutes was;
 allowed before the animal was removed and returned to its home cage. The
 time taken for all four of the animals' paws to enter the dark compartment
 was used as the basic assessment of the ability of the animal to remember
 and at least 6, animals per group were used to produce median and
 percentile ranges. A Mann-Whitney U-test for non-paramatric data estimated
 significance between groups.
 Open Field Behaviors
 Open field studies formed an essential part of the passive avoidance
 training. The open field apparatus was constructed out of black-painted
 wood 620 mm length.times.620 mm breadth.times.150 mm high. The
 white-painted floor of the apparatus was ruled from side to side every 77
 mm dividing it into a series of boxes 77.times.77 mm sq. Each animal was
 placed into the centre of the arena and allowed to move to a side of the
 apparatus at which point a timer was started. As a measure of locomotor
 activity, the number of lines crossed over the next 5 minutes was counted.
 Other behaviour assessed included rearing, grooming, piloerection,
 defecation and posture--all being a good indication of the state of health
 of the animal. body weight was noted as a matter of course. All
 observations were carried out in the quiet room under low-level
 illumination between 08.00 and 14.00 to minimise circadian influence. This
 behavioral assessment was invaluable for detecting animals not responding
 to the training schedule or unwanted interventive effects which may
 confound test results.
 Immunohistochemistry
 The frequency of neural cell adhesion molecule polysialylated neurons at
 the granule cell layer/hilar region of the hippocampal dentate gyrus was
 established by immunohistochemical techniques. Freshly dissected brains
 were coated immediately in an optimal cutting temperature compound (Gurr,
 U.K.), snap-frozen in liquid nitrogen-cooled n-hexane and stored at
 -80.degree. C. until required for further processing. Horizontal cryostat
 sections of 12 .mu.m were cut from frozen tissue using a MICROM (Series
 500) cryostat. Serial sections were obtained for analysis from the same
 point which was mid-way down the septo-temporal axis (equivalent to -5.6
 mm from Bregma for analysis of dentate cells and -8.10 mm from Bregma for
 analysis of cortical cells (Paxinos and Watson, The Rat Brain in
 Stereotaxic Coordinates, Academic Press, 1986)) and thaw-mounted onto 0.1%
 (w/v) poly-1-lysine coated glass slides. The sections were fixed in 70%
 (v/v) ethanol for 30 min, washed twice ior 10 min in a washing buffer of
 0.1M phosphate buffered 0.9% saline, pH7.4, (PBS) and incubated overnight
 (20 hours) in a humidified chamber at room temperature with anti-NCAM-PSA
 (Rougon et al., J. Cell Biol. 1986 103:2429-2437) diluted 1:500 in an
 incubation buffer composed of PBS containing 1% (w/v) bovine serum albumen
 (Sigma Chemical Co., U.K.) and 1% (v/v/) normal goat serum (DAKO, Denmark)
 in order to eliminate non-specific staining. The sections were washed
 again and exposed for 3 hours to fluorescein-conjugated goat anti-mouse
 IgM (Calbiochem. U.K.) diluted 1:100 with incubation buffer. The sections
 received a final wash before being mounted in Citifluor.RTM. (Agar, UK), a
 fluorescence-enhancing medium. The staining pattern was observed with a
 Letiz DM RB fluorescence microscope using an exciting wavelength of 495 nm
 and an emitting wavelength of 525 nm. Immunofluorescence staining was
 specific as it was eliminated completely by omission of either the primary
 of secondary antibody and by pre-absorbing anti-NCAM-PSA with colominic
 acid (1 mg/ml; Sigma Chemical Co., U.K.). which contains .alpha.2,8
 homopolymers of sialic acid. Where relevant, sections were counter-stained
 by a brief exposure (60 seconds) to propidium iodide (40 ng/ml PBS) which
 was detected using an excitation wavelength of 552 nm and an emission
 wavelength of 570 nm.
 The total number of NCAM-PSA-immunoreactive neurons in the dentate granule
 cell layer and at the hilar border were counted in ten alternate 12 .mu.m
 sections, to preclude double counting of the 5-10 .mu.m perikarya, divided
 by the total area of the granule cell layer, which included all propidium
 iodide labelled cells, and multiplied by the average granule cell area
 which was 0.15.+-.0.01 mm.sub.2 at this level, and the mean.+-.sem for
 each animal group calculated. These means were used to establish the
 mean.+-.sem for each animal group. Area measurements were performed using
 a Quantimet 500 Image Analysis System. Statistical analysis employed the
 Students' t-test.
 GC-MS Analysis of 2-n-pentyl-4pentynoic Acid
 An indwelling cannula (0.5 mm internal diameter) was placed into the left
 jugular vein under sodium pentobarbitone (60 mgs/kg) anaesthesia, 4 days
 prior to drug administration. The cannula was maintained patent with 20%
 polyvinylpyrrolidone containing 50 units/ml heparin and flushed twice
 daily. A bolus of 2-n-pentyl-4-pentynoic acid 84 (mg/kg) was administered
 once daily for 10 days. The animal was sacrificed 30 mins following final
 administration of the drug, the brain was removed, dissected into
 individual regions, placed in pre-weighed vials and stored frozen at
 -80.degree. C. until required. The liver and kidney were treated in a
 similar manner. The blood was obtained by cardiac puncture, collected into
 heparinised tubes and the plasma prepared by centrifugation at 1500 rpm
 for 10 min. The supernatant was aliquoted and stored frozen in pre-weighed
 vials.
 To extract the 2-n-pentyl-4-pentynoic acid, the tissue was homogenised by
 ultrasonication into 2-10 vols H.sub.2 O. Aliquots of 100-200 .mu.l were
 extracted by the addition of 50 .mu.l of 1N NaH.sub.2 PO.sub.4 buffer (pH
 5.0) and 1 ml of ethylacetate containing 1 .mu.g/ml
 2-methyl-2-ethylhexanoic acid as an internal standard. The mixture was
 shaken for 15 min, centrifuged for 1 min and the supernatant concentrated
 to approximately 200 .mu.l by a stream of nitrogen at 20.degree. C. and a
 100 .mu.l aliquot of acetonitrile added. The extraction was repeated with
 ethylacetate alone and the combined extracts evaporated to a final volume
 of 10-20 .mu.l. Trimethylsilylation was accomplished by addition of 30
 .mu.l of pyridine and 30 .mu.l of
 N-methyl-N-trimethylsilyl-trifluoroacetamide at room temperature for at
 least 30 min and 1 .mu.l aliquots were injected into the gas
 chromatographic-mass spectrometer (GC-MS). Unbound 2-n-pentyl-4-pentynoic
 acid was measured after ultrafiltration of serum aliquots with the Amicon
 MPS-1 Ultrafiltration advice (YMT-membrane, cut-off 30 kDa). GC-MS
 analysis was carried out using a Perkin-Elmer F22-9-C, coupled via a Jet
 separator to a Finnigan MAT CH-7-A mass spectrometer operated by a 2100D
 Superincos. A fused silica Megabore column was used (30 m.times.0.53 mm
 internal diameter, 1 .mu.m film thickness), coated with DB1701. The
 temperature of the injector was held at 220.degree. C. The initial over
 temperature was 80.degree. C. After injection, the temperature was held at
 80.degree. C. for 1 min, rapidly raised to 120.degree. C., and then at a
 rate of 4.degree. C./min to 190.degree. C. Detection took place in the
 selective ion monitoring mode with the following ions: m/z 225 for
 2-n-pentyl-4-pentynoic acid and ion m/z 215 for the
 2-methyl-2-ethylhexanoic acid internal standard. Linear calibration graphs
 were used to determine the individual tissue concentrations.
 EXAMPLE 7
 Teratogenicity of Valproic Acid and Valproic Acid-Related Carboxylic Acids
 The teratogenicity of valproic acid and valproic acid-related carboxylic
 acids with variations in the length of one side chain in NMRI mice was
 determined. Tables 1 and 2 set forth the results for the following
 compounds: VPA (valproic acid), ethyl-4-yn-VPA (2-n-ethyl-4-pentynoic
 acid), 4-yn-VPA (2-n-propyl-4-pentynoic acid), butyl-4-yn-VPA
 (2-n-butyl-4-pentynoic acid), pentyl-4-yn-VPA (2-n-pentyl-4-pentynoic
 acid) and hexyl-4-yn-VPA (2-n-hexyl-4-pentynoic acid). Each carboxylic
 acid compound was administered in doses of mmol sodium salt/kg. The
 embryolethality values reflect a percentage of total implants, and the
 exencephaly values reflect a percentage of live fetuses. The number of
 affected fetuses are shown in parentheses. The teratogeinic effect of the
 carboxylic acids according to the present invention was determined by the
 mouse exencephaly model described by Nau in Toxicol. Appl. Pharmacol. 80,
 243-250 (1985) and U.S. Ser. No. 08/344,810, filed Nov. 23, 1994 which is
 herein incorporated by reference. Female NMRI mice are mated with male
 NMRI mice between 6.00 and 9.00 hours. The first 24 hours after conception
 are regarded as day zero of gestation. The solution of the sodium salts of
 the carboxylic acids were injected intraperitoneally into the mice on the
 morning of day eight of gestation. On day 18 of gestation, between 9.00
 and 12.00 hours, the animals were anesthetized with diethyl ether and
 subsequently the uterus was removed. The number of implantation sites and
 the resorptions and dead fetuses (embryolethality) was determined. Each
 live fetus was weighed and examined for exencephaly.
 TABLE 1
 Fetal weight
 Dose.sup.1 No. of No. of live (mean .+-. SD)
 Embryolethality.sup.2 Exencephaly.sup.3
 Carboxylic acid (mmol/kg) litters fetuses (g)
 (%) (%)
 VPA 3.0 8 60 1.07 .+-. 0.10
 49 42
 Ethyl-4-yn-VPA 1.85 6 73 1.12 .+-. 0.09
 5 0
 4-yn-VPA 1.85 7 21 0.92 .+-. 0.12
 76 81(17)
 1.23 13 89 1.03 .+-. 0.13
 16 12
 Butyl-4-yn-VPA 1.85 8 27 0.88 .+-. 0.06
 69 96(26)
 1.25 9 63 0.99 .+-. 0.09
 45 71(45)
 Pentyl-4-yn-VPA 1.85 9 26 0.84 .+-. 0.11
 78 81(21)
 1.25 6 44 1.02 .+-. 0.09
 40 60(26)
 Hexyl-4-yn-VPA 1.85 3 0 -- 100
 --
 1.5 6 12 0.86 .+-. 0.07
 84 100(12)
 1.25 7 29 0.96 .+-. 0.06
 67 79(23)
 1.0 7 37 1.03 .+-. 0.05
 54 70(26)
 0.5 13 165 1.25 .+-. 0.10
 18 7(9)
 Controls 14 152 1.19 .+-. 0.12
 11 0
 .sup.1 The doses are in mmol sodium salt of each carboxylic acid/kg. All
 substances were injected intraperitoneally on the morning of day 8 of
 gestation.
 .sup.2 % of total implants.
 .sup.3 % of live fetuses. Number of affected fetuses are shown in
 parentheses.
 TABLE 2
 Fetal weight
 No. of No. of live (mean .+-. SD)
 Exencephaly
 Substance.sup.1 litters Embryolethality fetuses (g)
 (%)
 4-yn-VPA 13 16 89 1.03 .+-. 0.13
 12
 Butyl-4-yn-VPA 9 45 63 0.99 .+-. 0.09
 71 (45)
 Pentyl-4-yn-VPA 6 40 44 1.02 .+-. 0.09
 60 (26)
 Hexyl-4-yn-VPA 7 67 29 0.96 .+-. 0.06
 79 (23)
 .sup.1 1.25 nmol sodium salt of each carboxylic acid per kg body weight was
 administered on the morning of day 8 of gestation.
 While we have hereinbefore described a number of embodiments of this
 invention, it is apparent that the basic constructions can be altered to
 provide other embodiments which utilize the methods of this invention.
 Therefore, it will be appreciated that the scope of this invention is
 defined by the claims appended hereto rather than by the specific
 embodiments which have been presented hereinbefore by way of example.