U.S. patent number RE38,551 [Application Number 10/058,634] was granted by the patent office on 2004-07-06 for anticonvulsant enantiomeric amino acid derivatives.
This patent grant is currently assigned to Research Corporation Technologies, Inc.. Invention is credited to Harold Kohn.
United States Patent |
RE38,551 |
Kohn |
July 6, 2004 |
**Please see images for:
( Reexamination Certificate ) ( PTAB Trial Certificate
) ** |
Anticonvulsant enantiomeric amino acid derivatives
Abstract
The present invention is directed to a compound in the R
configuration about the asymmetric carbon in the following formula:
##STR1## pharmaceutical compositions containing same and the use
thereof in treating CNS disorders in animals.
Inventors: |
Kohn; Harold (Chapel Hill,
NC) |
Assignee: |
Research Corporation Technologies,
Inc. (Tucson, AZ)
|
Family
ID: |
25226163 |
Appl.
No.: |
10/058,634 |
Filed: |
January 28, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
Reissue of: |
818688 |
Mar 17, 1997 |
05773475 |
Jun 30, 1998 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07C
237/06 (20130101); C07C 237/52 (20130101) |
Current International
Class: |
A61K
31/165 (20060101); C07C 233/05 (20060101); C07C
233/00 (20060101); A61K 031/165 (); C07C
233/05 () |
Field of
Search: |
;514/616
;564/155,158 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
Other References
Anderson, et al. J. Am. Chem. Soc. 89:19 pp. 5012-5017, (1967).
.
Kohn, Harold, et al. "Preparation and anticonvulsant activity of a
series of functionalized. alph.-heteroatom-substituted amino
acids", J. Med. Chem. 34, 2444-2452 (1991). .
Kohn, Harold, et al. "Marked stereospecificity in a new class of
anticonvulsants", Chemical Abstracts, 109 (1988) Abstract No.
183045. .
Choi, Daeock, et al. "Synthesis and Anticonvulsant Activities of
N-Benzyl-2-acetamidopropionamide Derivatives", J. Med. Chem., 39:
1907-1916 (1996)..
|
Primary Examiner: Kumar; Shailendra
Attorney, Agent or Firm: Scully, Scott, Murphy &
Presser
Government Interests
.Iadd.GOVERNMENT SUPPORT
This invention was made with Government support under
Grant/Contract No. NIH MS 15604 awarded by the National Institute
of Health. The Government has certain rights in the invention.
Parent Case Text
RELATED APPLICATION
This application claims priority from U.S. provisional application
No. 60/013,522 filed on Mar. 15, 1996..Iaddend.
Claims
What is claimed is:
1. A compound in the R configuration having the formula:
##STR11##
wherein Ar is phenyl which is unsubstituted or substituted with at
least one halo group; Q is lower alkoxy, and Q.sub.1 is methyl.
2. The compound according to claim 1 which is substantially
enantiopure.
3. The compound according to claim 1 wherein Q is lower alkoxy
containing 1-3 carbon atoms.
4. The compound according to claim 3 wherein Q is methoxy.
5. The compound according to claim 1 wherein Ar is unsubstituted
phenyl.
6. The compound according to claim 1 wherein halo is fluoro.
7. The compound according to claim 1 wherein Q is alkoxy containing
1-3 carbon atoms and Ar is unsubstituted phenyl.
8. The compound according to claim 1 which is (R)-N-Benzyl
2-Acetamido-3-methoxypropionamide.
9. The compound according to claim 8 which contains at least 90%
(w/w) R stereoisomer.
10. A therapeutic composition comprising an anticonvulsant
effective amount of a compound according to any one of claims 1-9
and a pharmaceutical carrier therefor.
11. A method of treating central nervous system disorders in an
animal comprising administering to said animal in need thereof an
anticonvulsant effective amount of a compound according to any one
of claims 1-9.
12. The method according to claim 11 wherein the animal is a
mammal.
13. The method according to claim 12 wherein the mammal is a human.
Description
FIELD OF THE INVENTION
The present invention relates to novel enantiomeric compounds and
pharmaceutical compositions useful in the treatment of epilepsy and
other CNS disorders.
BACKGROUND OF THE INVENTION
The predominant application of anticonvulsant drugs is the control
and prevention of seizures associated with epilepsy or related
central nervous system disorders. Epilepsy refers to many types of
recurrent seizures produced by paroxysmal excessive neuronal
discharges in the brain; the two main generalized seizures are
petit mal, which is associated with myoclonic jerks, akinetic
seizures, transient loss of consciousness, but without convulsion;
and grand mal which manifests in a continuous series of seizures
and convulsions with loss of consciousness.
The mainstay of treatment for such disorders has been the long-term
and consistent administration of anticonvulsant drugs. Most drugs
in use are weak acids that, presumably, exert their action on
neurons, glial cells or both of the central nervous system. The
majority of these compounds are characterized by the presence of at
least one amide unit and one or more benzene rings that are present
as a phenyl group or part of a cyclic system.
Much attention has been focused upon the development of
anticonvulsant drugs and today many such drugs are well known. For
example, the hydantions, such as phenytoin, are useful in the
control of generalized seizures and all forms of partial seizures.
The oxazolidinediones, such as trimethadione and paramethadione,
are used in the treatment of non-convulsive seizures. Phenacemide,
a phenylacetylurea, is one of the most well known anticonvulsants
employed today, while much attention has recently been dedicated to
the investigation of the diazepines and piperazines. For example,
U.S. Pat. Nos. 4,002,764 and 4,178,378 to Allgeier, et al. disclose
esterified diazepine derivatives useful in the treatment of
epilepsy and other nervous disorders. U.S. Pat. No. 3,887,543 to
Nakanishi, et al. describes a thieno [2,3-e][1,4]diazepine compound
also having anticonvulsant activity and other depressant activity.
U.S. Pat. No. 4,209,516 to Heckendorn, et al. relates to triazole
derivatives which exhibit anticonvulsant activity and are useful in
the treatment of epilepsy and conditions of tension and agitation.
U.S. Pat. No. 4,372,974 to Fish, et al. discloses a pharmaceutical
formulation containing an aliphatic amino acid compound in which
the carboxylic acid and primary amine are separated by three or
four units. Administration of these compounds in an acid pH range
are useful in the treatment of convulsion disorders and also
possess anxiolytic and sedative properties.
U.S. Pat. No. 5,378,729 to Kohn, et al. discloses compounds and
pharmaceutical compositions having central nervous system (CNS)
activity which are useful in the treatment of epilepsy and other
CNS disorders having the following general formula: ##STR2##
R is hydrogen, lower alkyl, lower alkenyl, lower alkynyl, aryl,
aryl lower alkyl, heterocyclic, heterocyclic lower alkyl, lower
alkyl heterocyclic, lower cycloalkyl, lower cycloalkyl lower alkyl,
and R is unsubstituted or is substituted with at least one electron
withdrawing group, or electron donating group.
R.sub.1 is hydrogen or lower alkyl, lower alkenyl, lower alkynyl,
aryl lower alkyl, aryl, heterocyclic lower alkyl, heterocyclic,
lower cycloalkyl, lower cycloalkyl lower alkyl, each unsubstituted
or substituted with an electron donating group or an electron
withdrawing group and
R.sub.2 and R.sub.3 are independently hydrogen, lower alkyl, lower
alkenyl, lower alkynyl, aryl lower alkyl, aryl, heterocyclic,
heterocyclic lower alkyl, lower alkyl heterocyclic, lower
cycloalkyl, lower cycloalkyl lower alkyl, or Z--Y wherein R.sub.2
and R.sub.3 may be unsubstituted or substituted with at least one
electron withdrawing group or electron donating group;
Z is O, S, S (O).sub.a, NR.sub.4, PR.sub.4 or a chemical bond;
Y is hydrogen, lower alkyl, aryl, aryl lower alkyl, lower alkenyl,
lower alkynyl, halo, heterocyclic, or heterocyclic lower alkyl, and
Y may be unsubstituted or substituted with an electron donating
group or an electron withdrawing group, provided that when Y is
halo, Z is a chemical bond, or
ZY taken together is NR.sub.4 NR.sub.5 R.sub.7, NR.sub.4 OR.sub.5,
ONR.sub.4 R.sub.7, OPR.sub.4 R.sub.5, PR.sub.4 OR.sub.5, SNR.sub.4
R.sub.7, NR.sub.4 SR.sub.7, SPR.sub.4 R.sub.5, PR.sub.4 SR.sub.7,
NR.sub.4 PR.sub.5 R.sub.6, PR.sub.4 NR.sub.5 R.sub.7, ##STR3##
R.sub.4, R.sub.5 and R.sub.6 are independently hydrogen, lower
alkyl, aryl, aryl lower alkyl, lower alkenyl, or lower alkynyl,
wherein R.sub.4, R.sub.5 and R.sub.6 may be unsubstituted or
substituted with an electron withdrawing group or an electron
donating group,
R.sub.7 is R.sub.6, COOR.sub.8 or COR.sub.8,
R.sub.8 is hydrogen, lower alkyl, or aryl lower alkyl, and the aryl
or alkyl group may be unsubstituted or substituted with an electron
withdrawing group or an electron donating group and
n is 1-4 and
a is 1-3.
Unfortunately, despite the many available pharmacotherapeutic
agents, a significant percentage of the population with epilepsy or
related disorders are poorly managed. Moreover, none of the drugs
presently available are capable of achieving total seizure control,
and most have disturbing side effects. Toxicities may appear upon
repeated dosing that are not apparent with acute administration.
Because many drugs which require chronic administration ultimately
place an extra burden on the liver, including for example, liver
enzyme induction or oxidative metabolism that may generate reactive
species, many anticonvulsants have associated therewith liver
toxicity.
Research is continuing in this area to find better and more
effective anticonvulsant agents, especially for long term treatment
(chronic administration). Obviously, the ideal drug is one that has
high pharmacological activity, minimal side effects and is
relatively non-toxic and safe to the animal that is being treated.
More specifically, the ideal anticonvulsant drug is one that
satisfies the following four criteria: (1) has a high
anticonvulsant activity, (expressed as a low ED.sub.50); (2) has
minimal neurological toxicity, (as expressed by the median toxic
dose (TD.sub.50)), relative to its potency; (3) has a maximum
protective index (sometimes known as selectivity or margin of
safety), which measures the relationship between the doses of a
drug required to produce undesired and desired effects, and is
measured as the ratio between the median toxic dose and the median
effective dose (TD.sub.50 /ED.sub.50); and (4) is relatively safe
as measured by the median lethal dose (LD.sub.50) relative to its
potency and is non-toxic to the animal that is being treated, e.g.,
it exhibits minimal adverse effects on the remainder of the treated
animal, its organs, blood, its bodily functions, etc. even at high
concentrations, especially during long term chronic administration
of the drug. Thus, for example, it exhibits minimal, i.e., little
or no liver toxicity. Although not as critical in short term or
acute administration of an anti-convulsant, since the animal may
tolerate some low levels of toxicity, the fourth criteria outlined
above is extremely important for an anti-convulsant which is to be
taken over a long period of time (chronic administration) or in
high dosage. It may be the most important factor in determining
which anti-convulsant to administer to a patient, especially if
chronic dosing is required. Thus, an anti-convulsant agent which
has a high anti-convulsant activity, has minimal neurological
toxicity and maximal P.I. (protective index) may unfortunately
exhibit such toxicities which appear upon repeated high levels of
administration. In such an event, acute dosing of the drug may be
considered, but it would not be used in a treatment regime which
requires chronic administration of the anti-convulsant. In fact, if
an anti-convulsant is required for repeated dosing in a long term
treatment regime, a physician may prescribe an anti-convulsant that
may have weaker activity relative to a second anti-convulsant, if
it exhibits relatively low toxicity to the animal. An
anti-convulsant agent which meets all four criteria is very
rare.
However, the present inventor has found such a group of compounds
that is generally potent, exhibit minimal neurological toxicity,
has a high protective index and is relatively non-toxic to the body
organs, including the liver upon multiple dosing.
SUMMARY OF THE INVENTION
Accordingly, the present invention is directed to
N-benzyl-2-acetamido propionamide derivatives in the R
configuration having the formula: ##STR4##
wherein
Ar is aryl which is unsubstituted or substituted with halo;
Q is lower alkoxy; and
Q.sub.1 is CH.sub.3.
The present invention contemplates employing the compound of
Formula I in a pharmaceutical composition. Moreover, the
administration of an effective amount of the present compounds in
their pharmaceutically acceptable forms provides an excellent
regime for the treatment of epilepsy, nervous anxiety, psychosis,
insomnia, and other related central nervous disorders.
These drugs exhibit high anti-convulsant activity, minimal
neurological toxicity, high P.I. and minimal toxicity. These
anti-convulsants are utilized in a treatment regime requiring acute
dosing, and especially chronic dosing thereof to the patient.
As shown hereinbelow, the compounds of the present invention
exhibit minimal effects on liver, which is in contrast to other
anti-convulsant compounds.
DETAILED DESCRIPTION OF THE INVENTION
As used herein the term "alkoxy" refers to an O-alkyl group
attached to the main chain through an oxygen bridge, wherein alkyl
is as defined hereinabove. The alkoxy groups are lower alkoxy
groups containing one to six carbon atoms, and more preferably, one
to three carbon atoms. The most preferred alkoxy groups are
propoxy, isopropoxy, ethoxy and especially methoxy.
The term "aryl", when used alone or in combination, refers to a
phenyl group which is unsubstituted or substituted with halo.
The term halo includes fluoro, chloro, bromo, iodo and the like.
The preferred halo is fluoro.
It is preferred that Q in the compound of formula I is alkoxy
having 1-3 carbon atoms. The most preferred alkoxy group is
propoxy, isopropoxy, ethoxy and especially methoxy.
The Ar group as defined herein, is phenyl, which may be
unsubstituted or substituted as defined herein. It is most
preferred that the aryl group, i.e., phenyl, is unsubstituted or
substituted with only one halo group. It is more preferred that if
substituted, the halo substituent is in the para or meta position.
It is even more preferred that the phenyl group is
unsubstituted.
Examples of the compounds of the present invention include:
(R)-N-Benzyl-2-acetamido-3-methoxy propionamide,
(R)-N-(3-Fluorobenzyl)-2-acetamido-3-methoxypropionamide,
(R)-N-(4-Fluorobenzyl)-2-acetamide-3-methoxypropionamide,
(R)-N-Benzyl-2-acetamido-3-ethoxy propionamide.
As indicated by the asterisk in formula I, the compounds of the
present invention contain at least one asymmetric carbon. The
stereochemistry of the asymmetric carbon at the asterisk is in the
R configuration. The inventor has found that the R stereoisomer at
the asymmetric carbon at the asterisk is significantly more
efficacious than the corresponding S enantiomer or a racemic
mixture thereof.
It is preferred that the compound of the present invention be
substantially pure, i.e., substantially free from impurities. It is
most preferred that the compounds of the present invention be at
least 75% pure (w/w) and more preferably greater than about 90%
pure (w/w) and most preferably greater than about 95% pure
(w/w).
It is also preferred that the compounds of the present invention be
substantially enantiomerically pure, i.e., substantially free from
the corresponding S isomer. It is more preferred that the compounds
of the present invention contain at least 90% (w/w) R stereoisomer,
and most preferably greater than about 95% (w/w) in the R
stereoisomer. Thus, the present invention contemplates compounds
having at most about 10% S isomer (w/w), and even more preferably
less than about 5% S isomer (w/w).
The compounds of the present invention in the R form are prepared
by art recognized techniques from commercially available starting
materials.
An exemplary procedure is outlined in Scheme 1 hereinbelow:
##STR5##
A D serine molecule (1) is esterified under acylation conditions
with an alcohol, such as acidic methanol, to provide the
corresponding ester (2). 2 is reacted with ArCH.sub.2 NH.sub.2,
such as benzylamine, under acylation conditions to form the
corresponding amide (3). Acylation of the free amino group, with an
acylating derivative of ##STR6##
such as acetic acid, or lower alkyl ester of acetic acid, or acetic
anhydride provides the hydroxymethyl derivative, i.e., ##STR7##
The enantiopurity of 4 was determined by techniques known in the
art, including melting point, optical rotation and .sup.1 H NMR
upon addition of an organic acid in the R-configuration, such as
R(-)- mandelic acid. Crystallization of 4 was repeated until the
desired enantiopurity thereof was achieved. The product of 4 is
converted to the ether under Williamson conditions by reacting it
with QX, wherein Q is as defined herein above and X is good leaving
groups, such as OTs, OMs, or halide (e.g., CH.sub.3 I) and the like
in the presence of base (e.g., Ag.sub.2 O) to form the product (5)
having Formula I.
Another variation is depicted in Scheme 2. ##STR8##
For example, beginning with D-serine (1), treatment with an
acylating derivative of acetic acid such as acetic anhydride in
acetic acid, gives the corresponding amide 6 which is then reacted
with ArCH.sub.2 NH.sub.2 under mixed anhydride coupling reaction
conditions, as described by Anderson, et al., in JACS, 1967, 89,
5012-5017, the contents of which are incorporated herein by
reference, to give the corresponding compound of the formula:
##STR9##
e.g., 7. Alkylation of this R-product in the presence of base under
Williamson conditions, such as methyl iodide in Ag.sub.2 O,
provides a product of Formula I (8).
An alternative route is depicted in Scheme 3. ##STR10##
D Serine (1) is protected with a N-protecting group known in the
art, by standard techniques. Thus, for example, it is reacted with
carbobenzoxy chloride (CBZ-cl, benzyl chloroformate) generating the
N-protected CBZ-D-serine adduct 9. The product serine adduct is
converted to the corresponding ether under Williamson conditions by
reacting it with QX wherein Q and X are defined hereinabove (e.g.,
CH.sub.3 I) in the presence of base (e.g., Ag.sub.2 0) to form an
ether 10. Under these conditions, the acid is also esterified.
Subsequent hydrolysis of the ester group in 10 permits amide
coupling with ArCH.sub.2 NH.sub.2 using amide coupling methodology
(e.g., mixed anhydride 1,1' Carbonyldiimidazole) to give the amide
12. Deprotection of the N-protecting group provide the free amine
13 which is then reacted with an acylating agent such as acetic
anhydride in base, (e.g., pyridine) to provide the product
(R)-8.
If necessary, in any of the procedures described hereinabove, the
optical purity of the product may be enhanced by further separation
of the S enantiomer from the R enantiomer, by standard techniques
known in the art, such as chiral chromatography using a standard
chiral support known in the art.
Alternatively, in any of the procedures provided hereinabove, a
racemic D serine may be utilized as the starting material.
Following the procedures in any of the schemes outlined hereinabove
would provide the racemic mixture, which can be resolved into the R
isomer by standard techniques known in the art such as chiral
chromatography.
The active ingredients of the therapeutic compositions and the
compounds of the present invention exhibit excellent anticonvulsant
activity when administered in amounts ranging from about 1 mg to
about 100 mg per kilogram of body weight per day. This dosage
regimen may be adjusted by the physician to provide the optimum
therapeutic response. For example, several divided doses may be
administered daily or the dose may be proportionally reduced as
indicated by the exigencies of the therapeutic situation. A decided
practical advantage is that the active compound may be administered
in an convenient manner such as by the oral, intravenous (where
water soluble), intramuscular or subcutaneous routes.
The active compound may be orally administered, for example, with
an inert diluent or with an assimilable edible carrier, or it may
be enclosed in hard or soft shell gelatin capsules, or it may be
compressed into tablets, or it may be incorporated directly into
the food of the diet. For oral therapeutic administration, the
active compound may be incorporated with excipients and used in the
form of ingestible tablets, buccal tablets, troches, capsules,
elixirs, suspensions, syrups, wafers, and the like. Such
compositions and preparations should contain at least 1% of active
compound. The percentage of the compositions and preparations may,
of course, be varied and may conveniently be between about 5 to
about 80% of the weight of the unit. The amount of active compound
in such therapeutically useful compositions is such that a suitable
dosage will be obtained. Preferred compositions or preparations
according to the present invention are prepared so that an oral
dosage unit form contains between about 5 and 100 mg of active
compound.
The tablets, troches, pills, capsules and the like may also contain
the following: A binder such as gum tragacanth, acacia, corn starch
or gelatin; excipients such as dicalcium phosphate; a
disintegrating agent such as corn starch, potato starch, alginic
acid and the like; a lubricant such as magnesium stearate; and a
sweetening agent such as sucrose, lactose or saccharin may be added
or a flavoring agent such as peppermint, oil of wintergreen, or
cherry flavoring. When the dosage unit form is a capsule, it may
contain, in addition of materials of the above type, a liquid
carrier. Various other materials may be present as coatings or to
otherwise modify the physical form of the dosage unit. For
instance, tablets, pills, or capsules may be coated with shellac,
sugar or both. A syrup or elixir may contain the active compound,
sucrose as a sweetening agent, methyl and propylparabens as
preservatives, a dye and flavoring such as cherry or orange flavor.
Of course, any material used in preparing any dosage unit form
should be pharmaceutically pure and substantially non-toxic in the
amounts employed. In addition, the active compound may be
incorporated into sustained-release preparations and formulations.
For example, sustained release dosage forms are contemplated
wherein the active ingredient is bound to an ion exchange resin
which, optionally, can be coated with a diffusion barrier coating
to modify the release properties of the resin.
The active compound may also be administered parenterally or
intraperitoneally. Dispersions can also be prepared in glycerol,
liquid polyethylene glycols, and mixtures thereof and in oils.
Under ordinary conditions of storage and use, these preparations
contain a preservative to prevent the growth of microorganisms.
The pharmaceutical forms suitable for injectable use include
sterile aqueous solutions (where water soluble) or dispersions and
sterile powders for the extemporaneous preparation of sterile
injectable solutions or dispersions. In all cases the form must be
sterile and must be fluid to the extent that easy syringability
exists. It must be stable under the conditions of manufacture and
storage and must be preserved against the contaminating action of
microorganisms such as bacteria and fungi. The carrier can be a
solvent or dispersion medium containing, for example, water,
ethanol, polyol (for example, glycerol, propylene glycol, and
liquid polyethylene glycol, and the like), suitable mixtures
thereof, and vegetable oils. The proper fluidity can be maintained,
for example, by the use of a coating such as lecithin, by the
maintenance of the required particle size in the case of
dispersions and by the use of surfactants. The prevention of the
action of microorganisms can be brought about by various
antibacterial and antifungal agents, for example, parabens,
chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In
many cases, it will be preferable to include isotonic agents, for
example, sugars or sodium chloride. Prolonged absorption of the
injectable compositions can be brought about by the use in the
compositions of agents delaying absorption, for example, aluminum
monostearate and gelatin.
Sterile injectable solutions are prepared by incorporating the
active compound in the required amount in the appropriate solvent
with various of the other ingredients enumerated above, as
required, followed by filtered sterilization. Generally,
dispersions are prepared by incorporating the various sterilized
active ingredient into a sterile vehicle which contains the basic
dispersion medium and the required other ingredients from those
enumerated above. In the case of sterile powders for the
preparation of sterile injectable solutions, the preferred methods
of preparation are vacuum drying and the freeze-drying technique
which yield a powder of the active ingredient plus any additional
desired ingredient from previously sterile-filtered solution
thereof.
As used herein, "pharmaceutically acceptable carrier" includes any
and all solvents, dispersion media, coatings, antibacterial and
antifungal agents, isotonic and absorption delaying agents, and the
like. The use of such media and agents for pharmaceutical active
substances is well known in the art. Except insofar as any
conventional media or agent is incompatible with the active
ingredient, its use in the therapeutic compositions is
contemplated. Supplementary active ingredients can also be
incorporated into the compositions.
It is especially advantageous to formulate parenteral compositions
in dosage unit form for ease of administration and uniformity of
dosage. Dosage unit form as used herein refers to physically
discrete units suited as unitary dosages for the mammalian subjects
to be treated; each unit containing a predetermined quantity of
active material calculated to produce the desired therapeutic
effect in association with the required pharmaceutical carrier. The
specifics for the novel dosage unit forms of the invention are
dictated by and directly, dependent on (a) the unique
characteristics of the active material and the particular
therapeutic effect to be achieved, and (b) the limitations inherent
in the art of compounding such an active material for the treatment
of disease in living subjects having a diseased condition in which
bodily health is impaired as herein disclosed in detail.
The principal active ingredient is compounded for convenient and
effective administration in effective amounts with a suitable
pharmaceutically acceptable carrier in dosage unit form as
hereinbefore described. A unit dosage form can, for example,
contain the principal active compound in amounts ranging from about
5 to about 1000 mg. Expressed in proportions, the active compound
is generally present in from about 1 to about 750 mg/ml of carrier.
In the case of compositions containing supplementary active
ingredients, the dosages are determined by reference to the usual
dose and manner of administration of the said ingredients.
Unless indicated to the contrary, percentages are by weight.
As used herein, the term lower alkyl refers to an alkyl group
containing 1-6 carbon atoms which may be straight chained or
branched.
For a better understanding of the present invention reference is
made to the following description and examples.
GENERAL METHODS
Melting points were determined with a Thomas Hoover melting point
apparatus and are uncorrected. Infrared spectra (IR) were run on
Perkin-Elmer 1330, 283 and a Mattson Genesis spectrometer and were
calibrated against the 1601 cm.sup.-1 bond of polystyrene.
Absorption values are expressed in wave-numbers (cm.sup.-1). Proton
(.sup.1 H NMR) and carbon (.sup.13 C NMR) nuclear magnetic
resonance spectra were taken on Nicolet NT-300 and General Electric
QE-300 NMR instruments. Chemical shifts (.delta.) are in parts per
million (ppm) relative to Me.sub.4 Si and coupling constants (J
values) are in hertz. All chemical ionization mass spectral
investigations were conducted on Finnegan MAT TSQ-70 instrument.
Microanalyses were provided by Atlantic Microlab Inc. (Norcross,
Ga). Thin layer chromatography was performed on precoated silica
gel GHLF microscope slides (2.5.times.10 cm; Analtech No.
21521).
EXAMPLE 1
(R)-N-Benzyl-2-Acetamide-3-methoxypropionamide
Hydrochloric acid (8.00 g, 219.4 mmol) was passed into MeOH (250
mL) and then D-Serine (20.00 g, 190.3 mmol) was added. The reaction
solution was heated at reflux (18 hours), benzylamine (81.6 mL, 761
mmol) was added and then the reaction was heated for an additional
eighteen hours. The solvent was removed under reduced pressure, the
insoluble salts filtered, and the excess benzylamine was removed
under high vacuum (Kugelrohr). The residue was dissolved in water
(100 mL), and the product was extracted with CHCl.sub.3
(8.times.200 mL). The organic layers were combined, dried (Na.sub.2
SO.sub.4), and the solvent was removed under reduced pressure. The
residue was triturated with Et.sub.2 O (150 mL) and filtered to
give 10.0 g (27%) of the product R-enriched N-benzyl
2-aminohydracrylamide, as a white solid: mp 74.degree.-78.degree.
C.; [.alpha.].sub.D.sup.23 (c=1, MeOH)=1.6.degree., R.sub.f 0.30
(10% MeOH--CHCl.sub.3); .sup.1 H NMR (DMSO-d.sub.6) .delta.1.87 (br
s, NH.sub.2), 3.23 (t, J=5.4 Hz, CH), 3.39-3.55 (m, CH.sub.2 OH),
4.28 (d, J=5.7 Hz, NHCH.sub.2) 4.76 (t, J=5.4 Hz, CH.sub.2 OH),
7.18-7.32 (m, 5PhH), 8.34 (t, J=5.7 Hz, NH), .sup.13 C NMR
(DMSO-d.sub.6) 41.8 (NHCH.sub.2), 56.9 (CH), 64.3 (CH.sub.2 OH),
126.6 (C.sub.4 '), 127.0 (2C.sub.2 ' or 2C.sub.3 '), 128.1
(2C.sub.2 ' or 2C3'), 139.5 C.sub.1 '), 173.3 (C(O)NH) ppm, MS
(+Cl) (rel intensity), 195 (M.sup.+ +1, 53), 117 (100), Mr(+Cl)
195.113 56 (M.sup.+ +1) (calcd. for C.sub.10 H.sub.15 N.sub.2
O.sub.2, 195.11335).
To a stirred methylene chloride suspension (100 ml) of R enriched
N-benzyl-2-aminohydracrylamide (10.00 g, 51.5 mmol) was added
acetic anhydride (5.8 mL, 61.8 mmol), and the reaction suspension
was stirred at room temperature (1 hour). The solvent was removed
under reduced pressure to give a white solid. The product was
triturated with Et.sub.2 O (250 mL) to give 7.60 g (62%) of
enriched R-N-benzyl-2-acetamidohydracrylamide as a white solid. The
reaction product was recrystallized (2.times.) using EtOH to give
3.50 g (29%) of the R-N-benzyl-2-acetamidohydracylamide mp
148.degree.-149.degree. C.; [.alpha.].sub.D.sup.23 (c=1,
MeOH)=+22.4.degree.; Rf 0.40 (10% MeOH--CHCl.sub.3); IR (KBr) 3295,
3090, 2964, 1642, 1533, 1376, 1281, 1051, 705 cm; .sup.1 H NMR
(DMSO-d.sub.6) .delta.1.86 (s, C(O)CH.sub.3), 3.57 (dd, J=5.7, 5.7
Hz, CH.sub.2 OH), 4.25-4.31(m, CH), 4.27 (d, J=5.7 Hz, NHCH.sub.2),
4.92 (t, J=5.7 Hz, CH.sub.2 OH), 7.18-7.32 (m, 5 PhH) 7.94 (d,
J=7.8Hz, NH), 8.38 (t, J=5.7 H, NH), addition of excess R-(-)
mandelic acid to a CDCl.sub.3 solution of R-N-benzyl
2-acetamidohydracrylamide prepared hereinabove gave only one signal
for the acetyl methyl protons; .sup.13 C NMR (DMSO-d.sub.6) 22.7
(C(O)CH.sub.3), 42.0 (CH.sub.2 NH), 55.6 (CH), 61.8(CH.sub.2 OH),
126.7 (C.sub.4 '), 127.0 (2C.sub.2 ' or 2C.sub.3 '), 128.2
(2C.sub.2 ' or 2C.sub.3 '), 139.4 (C.sub.1 '), 169.5 (C(O)CH.sub.3
or C(O)NH), 170.3 (C(O)CH.sub.3 or C(O)NH) ppm; MS (+Cl) rel
intensity) 237 (M.sup.+ +1, 100), 219(8); Mr(+Cl) 237.12388
[M.sup.+ +1] (calcd for C.sub.12 H.sub.17 N.sub.2 O.sub.3
237.12392); Anal (C.sub.12 H.sub.16 N.sub.2 O.sub.3), C,H,N.
To a stirred acetonitrile solution (300 mL) of
(R)-N-benzyl-2-acetamidohydroacrylamide (2.36 g, 10 mmol) was
successively added Ag.sub.2 O (11.59 g, 50 mmol) and methyl iodide
(6.2 mL, 100 mmol) at room temperature. The reaction mixture was
stirred at room temperature for 4 days. The insoluble salts were
filtered, and the solvents were removed in vacuo to give a white
solid. The residue was filtered with Et.sub.2 O (100 mL) to give
2.20 g (88%) of the above-identified product.
mp 143.degree.-144.degree. C.; [.alpha.].sub.D.sup.23 (c=1,
MeOH)=+16.4.degree.; Rf0.47 (10% MeOH--CHCl.sub.3); IR (KBr) 3289,
3086, 2923, 2876, 2819, 1636, 1547, 1138, 695 cm.sup.-1 ; .sup.1 H
NMR (CDCl.sub.3) .delta.2.04 (s, C(O)CH.sub.3), 3.38 (s,
OCH.sub.3), 3.43 (dd, J=7.8, 9.0 Hz, CHH'OCH.sub.3), 3.82 (dd,
J=4.2, 9.0 Hz, CHH'OCH.sub.3), 4.48(d, J=6.0 Hz, NHCH.sub.2),
4.51-4.57 (m,CH), 6.44 (br d, J=5.4 Hz, NH), 6.75 (br s, NH),
7.25-7.37 (m, 5 PhH), addition of excess (R)-(-)-mandelic acid to a
CDCl.sub.3 solution of (R)-18 gave only one signal for the acetyl
methyl and ether methyl protons; .sup.13 C NMR (CDCl.sub.3) 23.2
(C(O)CH.sub.3), 43.5 (CH.sub.2 NH), 52.4 (CH), 59.1 (OCH.sub.3),
71.7 (CH.sub.2 OCH.sub.3), 127.4 (C.sub.4 '), 127.5 (2C.sub.2 ' or
2C.sub.3 '), 128.7 (2C2' or 2C.sub.3 '), 137.9 (C.sub.1 '), 169.9
(C(O)CH.sub.3 or C(O)NH), 170.3 (C(O)CH.sub.3 or C(O)NH) ppm; MS
(+Cl) (rel intensity) 251 (M.sup.+ +1, 100), 219(6); Mr
(+Cl)251.139 76 [M.sup.+ +1] (calcd for C.sub.13 H.sub.19 N.sub.2
O.sub.3 251.139 57); Anal. (C.sub.13 H.sub.18 N.sub.2 O.sub.3) C,
H, N.
EXAMPLE 2
Another Synthesis of (R)-N-Benzyl 2-Acetamide-3-methoxy
propionamide
(a) Improved Synthesis of (R)-N-Benzyl
2-Acetamidohydracrylamide
To a stirred AcOH (20 mL) suspension of D-serine (5.26 g, 50 mmol)
was added Ac.sub.2 O (4.7 mL, 50 mmol), and then the reaction
suspension was stirred at room temperature (24 hours). The ACOH was
removed in vacuo to give an oily residue, and then THF (150 mL) was
added to the residue. The THF suspension was cooled to -78.degree.
C. under N.sub.2 and 4-methylmorpholine (11.0 mL, 100 mmol) was
added. After stirring for two minutes, isobutyl chloroformate (13.0
mL, 100 mmol) was added leading to the precipitation of a white
solid. The reaction was allowed to proceed for two additional
minutes and then benzylamine (10.4 mL, 100 mmol) was added at
-78.degree. C. The reaction mixture was allowed to stir at room
temperature (30 minutes) and the 4-methylmorpholine hydrochloride
salt was filtered. The organic layer was concentrated in vacuo. The
product was purified by flash column chromatography on SiO.sub.2
gel (10% MeOH--CHCl.sub.3) to give 3.89 g (33%) as a white solid mp
147.degree.-148.degree. C.; [.alpha.].sub.D.sup.23 (C=1,
MeOH)=+21.70; .sup.1 H NMR (DMSO-d.sub.6) .delta.1.86 (s, C(O)
CH.sub.3), 3.57 (dd, J=5.1, 5.1 Hz, CH.sub.2 O), 4.27-4.31 (m,
CH.sub.2 NH, CH), 4.90 (t, J=5.1 Hz, OH), 7.20-7.31 (m, 5 PhH),
7.93, (d, J=8.1 Hz, NH), 8.37 (t, J=6.0 Hz, NH), addition of excess
(R)-(-)-mandelic acid to a CDCl.sub.3 solution of the product of
(a) gave only one signal for the acetyl methyl protons.
(b) (R)-N-Benzyl-2-Acetamide-3-methoxypropionamide
To the compound prepared in (a) (1.42 g, 6 mmol) in a stirred
solution (300 ml) of CH.sub.3 CN was successively added Ag.sub.2 O
(6.95 g, 30 mmol) and methyl iodide (3.7 mL, 60 mmol) and stirred
at room temperature for 4 days. The insoluble salts were filtered
and the solvent was removed in vacuo to give a white solid. The
white solid was triturated with Et.sub.2 O (100 mL) to given 1.30 g
(87%) of the above-identified compound: mp 143.degree.-144.degree.
C., [.alpha.].sub.D.sup.23 (c=1, MeOH)=+16.0.degree.; .sup.1 H NMR
(CDCl.sub.3) .delta.2.04 (s, C(O)CH.sub.3), 3.38(s, OCH.sub.3),
3.44 (dd, J=7.5, 9.0 Hz, CH H.sup.1 OCH.sub.3), 3.81 (dd, J=4.2,
9.0 Hz, CHH'OCH.sub.3), 4.48 (d, J=5.7 Hz, NHCH.sub.2), 4.52-4.58
(m, CH), 6.46 (br d, J=5.7 Hz, NH), 6.78 (br, s, NH), 7.25-7.37 (m,
5 Ph H), addition of excess (R)-(-)-mandelic acid to a CDCl.sub.3
solution of the above-identified compound gave only one signal for
the acetyl and ether methyl protons.
EXAMPLE 3
R-N-(3-Fluorobenzyl)2-Acetamide-3-Methoxypropionamide
(a) R-N-(3-Fluorobenzyl)-2-Acetamide-hydracrylamide
Utilizing the procedure of Example 2(a) with the following amounts
of D-serine (5.26 g, 50 mmol), Ac.sub.2 O (5.7 mL, 60 mmol),
4-methylmorpholine (11.0 mL, 100 mmol), isobutyl chloroformate
(13.0 mL, 100 mmol) and substituting 3-fluorobenzylamine (11.8 mL,
100 mmol) for benzylamine, gave 4.20 g (33%) of the above compound
as a white solid after purification: mp 137.degree.-138.degree. C.;
[.alpha.].sub.D.sup.23 (c=1, MeOH)=+20.8.degree.; Rf0.32 (10%
MeOH--CHCl.sub.3); IR (KBr) 3282, 3101, 2944, 1636, 1542, 1252,
1050, 779, 690 cm.sup.-1 ; .sup.1 H NMR (DMSO-d.sub.6) .delta.1.87
(s,C(O)CH.sub.3), 3.56-3.63 (m, CH.sub.2 OH), 4.29 (d, J=6.0 Hz,
CH.sub.2 NH), 4.25-4.30 (m, CH), 4.95 (t, J=5.4 Hz, CH.sub.2 OH),
7.00-7.09 (m, 3 ArH), 7.29-7.30 (m, 1 ArH), 7.97 (d, J=8.1 Hz, NH),
8.44 (t, J=6.0 Hz, NH), addition of excess (R)-(-)-mandelic acid to
a CDCl.sub.3 solution of this product gave only one signal for the
acetyl methyl portions; .sup.13 C NMR (DMSO-d.sub.6) 22.7
(C(O)CH.sub.3), 41.6 (CH.sub.2 N), 53.4 (CH), 61.7 (CH.sub.2 OH),
113.3 (d, J.sub.CF =20.0 Hz, (C.sub.2 ' or C.sub.4 '), 113.6 (d,
J.sub.CF =20.7 Hz, C.sub.2 ' or C.sub.4 '), 122.9 (C.sub.6 '),
130.1 (d, J.sub.CF= 8.2 Hz, C.sub.5'), 142.6 (d, J.sub.CF =7.0 Hz,
C.sub.1'), 162.3 (d, J.sub.CF =241.4 Hz, C.sub.3'), 169.6
(C(O)CH.sub.3 or C(O)NH), 170.5 (C(O)CH.sub.3 or C(O)NH) ppm; MS
(+Cl) (rel. intensity) 255 (M.sup.+ +1, 100); M.sub.r (+Cl) 255.113
54 [M.sup.+ +1] (calcd. for C.sub.12 H.sub.16 FN.sub.2 O.sub.3
255.114 50); Anal. (C.sub.12 H.sub.15 FN.sub.2 O.sub.3) C, H,
N.
(b) (R)-(N-3-Fluorobenzyl)-2-Acetamide-3-methoxypropionamide
To the product of (a) (2.54 g, 10 mmol) in a stirred CH.sub.3 CN
solution was successively added Ag.sub.2 O (11.59 g, 50 mmol) and
MeI (6.2 mL, 100 mmol) at room temperature. The reaction mixture
was stirred at room temperature for 2 days. The insoluble salts
were filtered and the solvent was removed in vacuo to give a white
solid which was triturated with Et.sub.2 O (100 mL) to give a crude
product of the above identified compound. The product was further
purified by flash chromatography on SiO.sub.2 gel (10%
MeOH--CHCl.sub.3) to give 2.00 g (75%) of the above-identified
compound: mp 150.degree.-151.degree. C.; [.alpha.].sub.D.sup.23
(c=1, MeOH)=+16.5.degree. C.; R.sub.f 0.50 (10% MeOH--CHCl.sub.3);
IR (KBr) 3287, 3072, 2928, 2883, 1634, 1548, 1256, 1142, 785
cm.sup.-1 ; .sup.1 H NMR (CDCl.sub.3) .delta. 2.05 (s,
C(O)CH.sub.3), 3.40 (s, OCH.sub.3), 3.44-3.47 (m, CHH'OCH.sub.3),
3.81-3.85 (m, CHH'OCH.sub.3), 4.41-4.50 (m, NHCH.sub.2), 4.53-4.59
(m, CH), 6.42 (br s, NH), 6.81 (br s, NH), 6.93-7.05 (m, 3 PhH),
7.26-7.31 (m, 1 PhH); addition of excess (R)-(-)-mandelic acid to a
CDCl.sub.3 solution of the above identified compound gave only one
signal for the acetyl methyl protons and ether methyl protons;
.sup.13 C NMR (DMSO-d.sub.6) 22.8 (C(O)CH.sub.3), 42.7 (CH.sub.2
N), 52.6 (CH), 58.9 (OCH.sub.3), 72.0 (CH.sub.2 OCH.sub.3), 114.0
(d, J.sub.CF= 21.5 Hz, C.sub.2' and C.sub.4'), 122.7 (C.sub.6'),
129.9 (d, J.sub.CF =7.7 Hz, C.sub.5'), 140.6 (d, J.sub.CF =6.8 Hz,
C.sub.1'), 162.9 (d, J.sub.CF =244.4 Hz, C.sub.3'), 170.2
(C(O)CH.sub.3 or C(O)NH), 170.5 (C(O)CH.sub.3 or C(O)NH) ppm; MS
(+Cl) (rel. intensity) 269 (M.sup.+ +1, 100); M.sub.r (+Cl) 269.129
31 [M.sup.+ +1] (calcd for C.sub.13 H.sub.18 FN.sub.2 O.sub.3
269.130 15); Anal. (C.sub.13 H.sub.17 FN.sub.2 O.sub.3) C, H,
N.
EXAMPLE 4
(R)-N-(4-Fluorobenzyl)2-Acetamido-3-Methoxypropanamide
(a) (R)-N-(4-Fluorobenzyl)2-Acetamidohydracrylamide
Utilizing the procedure of Example 2(a) with the following amounts
of D-serine (5.26 g, 50 mmol), Ac.sub.2 O (5.7 mL, 60 mmol),
4-methylmorpholine (11.0 mL, 100 mmol), and isobutyl chloroformate
(13.0 mL, 100 mmol) and substituting 4-fluorobenzylamine (11.8 mL,
100 mmol) for benzylamine, the above-identified compound was
prepared as a white solid after purification (4.08 g, 32%); mp:
169.degree.-170.degree. C.; [.alpha.].sub.D.sup.23 (c=1,
MeOH)=+17.6.degree.; R.sub.f 0.31 (10% MeOH--CHCl.sub.3); IR (KBr)
3289, 3101, 3071, 2936, 1632, 1565, 1543 1508, 1214, 1053, 814
cm.sup.-1 ; .sup.1 H NMR (DMSO-d.sub.6) .delta.1.86 (s,
C(O)CH.sub.3), 3.56 (6, J=5.4 Hz, CH.sub.2 OH), 4.25 (d, J=6.0 Hz,
CH.sub.2 NH), 4.25-4.29 (m, CH), 4.91 (t, J=5.4 Hz, CH.sub.2 OH),
7.08-7.14 (m, 2C.sub.2'H), 7.25-7.29 (m, 2C.sub.3'H), 7.93 (d,
J=7.8 Hz, NH), 8.39 (d, J=6.0 Hz, NH), addition of excess
(R)-(-)-mandelic acid to a CDCl.sub.3 solution of the
above-identified compound gave only one signal for the acetyl
methyl protons; .sup.13 C NMR (DMSO-d.sub.6) 22.7 (C(O) CH.sub.3),
41.3 (CH.sub.2 N), 55.3 (CH), 61.7 (CH.sub.2 OH), 114.8 (d,
J.sub.CF =21.8 Hz, 2C.sub.3,), 128.9 (d, J.sub.CF =8.0 Hz,
2C.sub.2'), 135.6 (C.sub.1'), 161.1 (d, J.sub.CF =240.1 Hz,
C.sub.4'), 169.4 (C(O)CH.sub.3 or C(O)NH), 170.3 (C(O)CH.sub.3 or
C(O)NH) ppm; MS (+Cl) (rel. intensity) 255 (M.sup.+ +1, 100);
Mr(+Cl) 255.113 60 [M.sup.+ +1] (calcd for C.sub.12 H.sub.16
FN.sub.2 O.sub.3 255.114 50); Anal. (C.sub.12 H.sub.15 FN.sub.2
O.sub.3.0.2H.sub.2 O) C, H, N.
(b) R-N-(4-Fluorobenzyl)2-Acetamido-3-methoxypropanamide
Following the procedure of Example 3(b) to the product of Example
4(a) (2.54 g, 10 mmol) in a stirred CH.sub.3 CN solution (300 mL)
was successively added) Ag.sub.2 O (11.59 g, 50 mmol) and MeI (6.2
mL, 100 mmol) at room temperature and then stirred for 7 days. The
insoluble salts were filtered, and the solvent was removed in vacuo
to given a white solid. The white solid was triturated with
Et.sub.2 O (100 mL) to give a crude product. The crude product was
further purified by flash column chromatography (10%
MeOH--CHCl.sub.3) to give 2.00 g (75%) of the above product; mp:
144.degree.-145.degree. C.; [.alpha.].sub.D.sup.23 (c=1,
MeOH)=+12.0; R.sub.f 0.52 (10% MeOH--CHCl.sub.3); IR (KBr) 3281,
3102, 3072, 2959, 1632, 1547, 1513, 1223, 1100 cm.sup.-1 ; .sup.1 H
NMR (CDCl.sub.3) .delta.2.04 (s, C(O) CH.sub.3), 3.38 (s,
OCH.sub.3), 3.39-3.46 (m, CHH'OCH.sub.3), 3.80-3.84 (m,
CHH'OCH.sub.3), 4.44 (br d, J=5.4 Hz, CH.sub.2 NH), 4.48-4.56 (m,
CH), 6.42 (br s, NH) 6.76 (br s, NH), 6.99-7.05 (m, 2 PhH),
7.21-7.31 (m, 2 PhH), addition of excess (R)-(-)-mandelic acid to a
CDCl.sub.3 solution of the above-identified product gave only one
signal for the acetyl methyl portions and ether methyl portions,
.sup.13 C NMR (CDCl.sub.3) 22.9 (C(O)CH.sub.3), 42.6 (CH.sub.2 N),
52.5 (CH), 58.9 (OCH.sub.3), 72.0 (CH.sub.2 OCH.sub.3), 115.3 (d,
J.sub.CF =22.0 Hz, 2C.sub.3'), 129.0 (d, J.sub.CF =6.9 Hz,
2C.sub.2'), 133.7 (C.sub.1') 161.9 (d, J.sub.CF =245.3 Hz,
C.sub.4'), 170.1 (C(O)CH.sub.3 or C(O)NH), 170.4 (C(O)CH.sub.3 or
C(O)NH) ppm; MS (+Cl) (rel. intensity) 269 (M.sup.+ +1, 100);
M.sub.r (+Cl) 269.129 66 [M.sup.+ +1] (calcd for C.sub.13 H.sub.18
FN.sub.2 O.sub.3 269.130 15); Anal. (C.sub.13 H.sub.17 FN.sub.2
O.sub.3) C, H, N.
EXAMPLE 5
N-Benzyl 2-Acetamide-3-Methoxypropionamide
(a) Cbz-(D) Serine (9)
D-Serine (5 g) was dissolved in water (85 mL). To this was added
MgO (6 g), and ethyl ether (40 mL). The mixture was cooled in an
ice bath to 0.degree. C. To this ice-cold mixture was added slowly,
dropwise benzylchloroformate (95%, 11 mL). Upon complete addition,
the mixture was stirred at 0.degree. C. (2 h) and then allowed to
spontaneously warm to room temperature. Stirring was continued for
an additional 30 minutes. The mixture was filtered and the filtrate
washed with ethyl ether (2.times.25 mL). The aqueous layer was
separated and cooled in an ice bath to 0.degree. C. The pH of this
ice-cold aqueous layer was carefully adjusted to 3.0 using 5N HCl.
The acidified solution was stored in a refrigerator overnight. The
white crystalline solid product was isolated by filtration, and
dried in vacuo. The filtrate was extracted with ethylacetate
(2.times.50 mL). The combined ethyl acetate extracts were dried
(Na.sub.2 SO.sub.4), filtered and evaporated in vacuo to obtain
additional amounts of the white crystalline product. Total product
obtained was 7.51 g (68%): mp 118.degree.-120.degree. C.
(b) Methyl-2-(Carbobenzyloxyamino)-3-Methoxypropionate (10)
To a solution of 9 (1.72 g, 7.21 mmol) in acetonitrile (150 mL) was
added methyl iodide (10.23 g, 72.1 mmol, 4.5 mL) and silver(I)oxide
(8.4 g, 36 mmol) and the mixture was stirred in the dark at room
temperature for 24 hours. The insoluble salts and excess silver
oxide were removed by filtration and the filtrate was evaporated in
vacuo to obtain an oily residue which was subjected to flash column
chromatography (silica gel and 5% MeOH--CHCl.sub.3) to obtain pure
10 as a pale yellow oil (1.81 g, 94%): R.sub.f (10%
MeOH/CHCl.sub.3) 0.75.
(c) 2-(Carbobenzyloxyamino)-3-Methoxypropionic Acid (11)
Compound 10 (0.58 g) was dissolved in 80% aqueous methanol (3.0
mL). To this solution was added anhydrous K.sub.2 CO.sub.3 (0.5 g)
and the reaction mixture was stirred at room temperature (8 hours).
The methanol was evaporated in vacuo and the residue suspended in
water (50 mL). The aqueous suspension was washed with ethyl ether
(2.times.25 mL) and then acidified to pH 3.0 using 5N HCl. The
acidified aqueous phase was extracted with ethyl acetate
(3.times.25 mL). The ethyl acetate extracts were combined, dried
(Na.sub.2 SO.sub.4), filtered, and evaporated in vacuo to obtain
pure 11 as a clear viscous oil (0.52 g, 95%): R.sub.f 0.30 (10%
MeOH/CHCl.sub.3).
(d) N-Benzyl 2-(Carbobenzyloxyamino)-3-Methoxypropionamide (12)
A solution of 11 (0.52 g, 2.04 mmol) in dry tetrahydrofuran (10 mL)
was cooled to -78.degree. C. in a dry ice-acetone bath under a
N.sub.2 atmosphere. To this was added via a dry syringe
4-methylmorpholine (0.34 mL, 3.06 mmol). After stirring for 5
minutes, isobutyl chloroformate (0.4 mL, 3.06 mmol) was added via
dry syringe and then the mixture stirred for 5 minutes. This was
followed by the addition of benzylamine (0.32 mL, 3.06 mmol). After
stirring at -78.degree. C. for 5 minutes, the reaction was allowed
to warm to room temperature, and stirring was continued at room
temperature (30 min). The hydrochloride salt of 4-methyl morpholine
was removed from the reaction by filtration. The clear filtrate was
evaporated in vacuo and the residue was triturated with ethyl ether
(5.0 mL). The white crystalline product obtained was isolated by
filtration after washing with small amounts of ether and air-dried
(0.55 g, 78%): mp 112.degree.-114.degree. C., R.sub.f 0.6 (10%
MeOH/CHCl.sub.3)
(e) N-Benzyl-2-Amino-3-Methoxypropionamide (13)
To a solution of 12 (122.8 mg, 0.36 mmol) in methanol (2.0 mL) was
added 10% Pd--C (11 mg) and the mixture stirred at room temperature
in the presence of H.sub.2 gas for 75 min. Celite was added to the
reaction mixture and the catalyst was removed by filtration. The
clear filtrate was evaporated in vacuo to give pure 13 as a clear
viscous oil (72 mg, 97%): R.sub.f 0.30 (5% MeOH/CHCl.sub.3).
(f) N-Benzyl-2-Acetamido-3-Methoxypropionamide
To a solution of 13 (0.20 g, 0.98 mmol) in dry THF (2.0 mL) is
added pyridine (0.086 g, 1.08 mmol), and then acetic anhydride (0.2
g, 1.96 mmol) is added dropwise. The reaction is stirred at room
temperature for 18 hours. The solvent is evaporated in vacuo and
the residue purified by flash column chromatography to obtain the
above compound as the R isomer.
COMPARATIVE EXAMPLE 1
Preparation of N-Acetyl-D,L-alanine-N'-benzylamide
Acetic anhydride (2.20 g, 0.022 mol) was slowly added to a
methylene chloride solution (30 mL) of D,L-alanine-N-benzylamide
(3.80 g, 0.021 mol) and allowed to stir at room temperature (3 h).
The mixture was then successively washed with H.sub.2 O (15 mL),
dried (Na.sub.2 SO.sub.4) and concentrated in vacuo. The residue
was recrystallized from CH.sub.2 Cl.sub.2.
Yield: 2.50 g (54%). mp 139.degree.-141.degree. C.
.sup.1 H NMR (DMSO-d.sub.6): .delta.1.22 (d, J=7.1 Hz, 3H), 1.84
(s,3H), 4.04-4.50 (m,3H), 7.26 (s,5H), 8.11 (br d, J=7.3 Hz, 1H),
8.42 (br t, J=6 Hz, 1H).
.sup.13 C NMR (DMSO-d.sub.6): 18.2, 22.4, 41.9, 48.2, 126.5, 126.9,
128.1 139.4, 168.9, 172.4 ppm.
IR (CHCl.sub.3) 3440, 3300, 3005, 1660, 1515 cm.sup.-1.
Mass spectrum (CI mode), m/e: 221 (P+I); mol wt. 220.1208
(calculated for C.sub.12 H.sub.16 N.sub.2 O.sub.2, 220.1212).
COMPARATIVE EXAMPLES 2 AND 3
Preparation of N-Acetyl D and L-amino acid N-benzylamides
General procedure: The D or L amino acid amide (11 mmol) was
dissolved in dichloromethane (15 mL) and then acetic anhydride
(1.23 g, 1.40 mL, 12 mmol) was added dropwise. The solution was
stirred at room temperature (18 h) and then concentrated to
dryness. The residue was crystallized from chloroform/hexane.
COMPARATIVE EXAMPLE 2
N-Acetyl-D-alanine-N'-benzylamide
Yield: 1.36 g (56%). mp 139.degree.-141.degree. C.
[.alpha.].sub.D.sup.23 =+36.2 (c 2.5, MeOH).
.sup.1 H NMR (80 MHz, DMSO-d.sub.6): .delta.1.25 (d, J=7.1 Hz, 3H),
1.86 (s, 3H), 4.04-4.50 (m, 1H), 4.30 (d, J=6.0 Hz, 2H), 7.26 (s,
5H), 8.09 (d, J=7.3 Hz, 1H), 8.40 (t, J=6.0 Hz, 1H).
.sup.13 C NMR (80 MHz, DMSO-d.sub.6): 18.3, 22.5, 42.0, 48.4,
126.6, 127.0 (2C), 128.2 (2C), 139.4, 169.2, 172.5 ppm.
IR (KBr): 3290, 1635 (br), 1540, 1455, 700, 695 cm.sup.-1.
Mass spectrum, m/e (relative intensity): 221 (30), 114 (20), 106
(40), 91 (80), 87 (100), 77 (5), 72 (20), 65 (5).
Elemental analysis calculated for C.sub.12 H.sub.16 N.sub.2 O.sub.2
65.42% C; 7.34% H; 12.72% N. Found 65.31% C; 7.28% H; 12.63% N.
COMPARATIVE EXAMPLE 3
N-Acetyl-L-alanine-N'-benzylamide
Yield: 1.11 g (46%). mp 139.degree.-142.degree. C.
[.alpha.].sub.D.sup.23 =35.3 (c 2.5, MeOH). .sup.1 H NMR (80 MHz,
DMSO-d.sub.6): .delta.1.23 (d, J=7.2 Hz, 3H), 1.86 (s, 3H),
4.26-4.35 (m, 1H), 4.29 (d, J=5.8 Hz, 2H), 7.22-7.33 (s,5H), 8.10
(d, J=7.4 Hz, 1H), 8.42 (t, J=5.8 Hz, 1H).
.sup.13 C NMR (80 MHz, DMSO-d.sub.6): 18.3, 22.6, 42.0, 48.4,
126.7, 127.0 (2C), 128.3 (2C) 139.5, 169.2, 172.6 ppm.
IR (KBr): 3290, 1635 (br), 1545, 1450, 700, 695 cm.sup.-1.
Mass spectrum, m/e (relative intensity): 221 (40), 114 (40), 106
(80), 106 (80), 91 (75), 87 (100), 77 (5), 72 (15), 65 (5).
Elemental analysis calculated for C.sub.12 H.sub.16 N.sub.2 O.sub.2
65.42% C; 7.34% H; 12.72% N. Found 65.58% C; 7.32% H; 12.43% N.
COMPARATIVE EXAMPLE 4
Preparation of D,L-2-Acetamido-N-benzyl-2-methoxyacetamide
To a methanolic solution (180 mL) of methyl
2-acetamide-2-methoxyacetate (8.73 g, 54 mmol) was rapidly added
benzylamine (8.68 g, 8.80 mL, 81 mmol) and then the mixture was
stirred at 50.degree. C. (3 days) during which time a beige
precipitate appeared. The solvent was removed in vacuo and the
resulting precipitate was recrystallized from tetrahydrofuran
(2.times.) to given 7.67 g (32%) of the desired product as beige
crystals: R.sub.f 0.35 (95:5 chloroform/methanol). mp
145.degree.-146.degree. C.
.sup.1 H NMR (300 MHz, CDCl.sub.3): .delta.2.06 (s, CH.sub.3 CO),
3.37 (2,CH.sub.3O), 4.40-4.35 (m, CH.sub.2), 5.52 (d, J=8.7 Hz,
CH), 7.12 (d, J=8.7 Hz, NH), 7.20-7.40 (m, Ph, NH).
.sup.13 C NMR (300 MHz, CDCl.sub.3): 23.03 (CH.sub.3 CO), 43.51
(CH.sub.2), 55.84 (CH.sub.3o), 78.94 (CH), 127.62 (C.sub.4 "),
127.70 (2C.sub.2 " or 2C.sub.3 '"), 128.70 (2C.sub.2 or 2C.sub.3
'), 137.45 (C.sub.1 "), 166.91 (COCH.sub.3), 171.57 (CONH) ppm.
IR (KBr): 1260, 1825 (br), 1550, 1505, 1435, 1390, 1370, 1230,
1120, 1050 935, 890, 690 cm.sup.-1.
Mass spectrum, m/e (relative intensity): 237 (1), 205 (2), 177 (2),
163 (4), 146 (1), 134 (1), 121 (2), 106 (26), 102 (98), 91 (95), 77
(13), 61 (100). Elemental analysis calculated for C.sub.12 H.sub.16
N.sub.2 O.sub.3 61.00% C; 6.83% H; 11.86% N. Found 60.91% C; 6.85%
H; 11.66% N.
COMPARATIVE EXAMPLES 5-7
Synthesis of Unsubstituted and
Substituted-.alpha.-Acetamido-N-benzyl-2-furanacetamides
General Procedure
4-Methylmorpholine (1 equiv) was added to a solution of
.alpha.-acetamido-2-furanacetic acid (1 equiv) in dry
tetrahydrofuran (75 mL/10 mmol) at -10.degree. to -15.degree. C.
under N.sub.2. After stirring (2 min.), isobutyl chloroformate (1
equiv) was added leading to the precipitation of a white solid. The
reaction was allowed to proceed for 2 additional minutes and then a
solution of the substituted benzylamine (1 equiv) in
tetrahydrofuran (10 mL/10 mmol) was added over 5 min. at
-10.degree. to -15.degree. C. The reaction mixture was allowed to
stir at room temperature for 5 min. and then the 4-methylmorpholine
hydrochloride salt filtered. The organic layer was concentrated in
vacuo, and the residue was triturated with ethyl acetate, and the
remaining white solid filtered. Concentration of the ethyl acetate
layer led to additional amounts of the white solid. The desired
product was purified by either recrystallization or flash
chromatography of the combined solid material.
COMPARATIVE EXAMPLE 5
(D,L)-.alpha.-Acetamido-N-benzyl-2-furanacetamide
Benzyl amine (0.27 g, 2.56 mmol) and racemic
.alpha.-acetamido-2-furanacetic acid (0.47 g, 2.56 mmol) gave the
desired compound. The product was recrystallized from ethyl acetate
to give a white solid.
Yield: 0.46 g (65%) R.sub.f 0.30 (98:2 chloroform/methanol). mp
177.degree.-178.degree. C.
.sup.1 H NMR (DMSO-d.sub.6) .delta.1.90 (s, CH.sub.3), 4.31 (d,
J=6.0 Hz, CH.sub.2), 5.58 (d, J=8.1 Hz, CH), 6.27-6.33 (m, C.sub.3
H), 6.40-6.44 (m, C.sub.4 H), 7.20-7.36 (m, 5 PhH), 7.60-7.64 (m,
C.sub.5 H), 8.57 (d, J=8.1 Hz, NH), 8.73 (t, J=6.0 Hz, NH).
COMPARATIVE EXAMPLE 6
(D)-(-).alpha.-Acetamido-N-benzyl-2-furanacetamide
Starting with D-.alpha.-acetamido-2-furanacetic acid (2.45 g, 13.38
mmol) and benzylamine (1.43 g, 13.38 mmol), the desired product was
obtained. Yield: 2.54 g (70%). The product was further
recrystallized from ethyl acetate to give the title compound.
Yield: 2.30 g mp 196.degree.-197.degree. C. [.alpha.].sup.26 D[c=1,
MeOH]=78.30. Addition of R(-)-mandelic acid to a CDCl.sub.3
solution the product gave only one signal for the acetamide methyl
protons. Mass spectrum, m/e (relative intensity) 272 (M+, 2), 184
(2), 165 (2), 140 (8), 139 (88), 138 (34), 97 (46), 96 (100), 91
(63).
Elemental analysis: calculated: 66.16% C; 5.92% H; 10.29% N. Found:
66.09% C; 6.01% H; 10.38% N.
COMPARATIVE EXAMPLE 7
(L)-(+)-.alpha.-Acetamido-N-benzyl-2-furanacetamide
L-.alpha.-acetamido-2-furanacetic acid (2.83 g, 15.46 mmol) and
benzylamine (1.65 g, 15.4 mmol) gave 3.80 g of the enriched desired
product. .sup.1 H NMR analysis with R(-)-mandelic acid showed that
it was greater than 80% enriched in the title compound. The pure
L-enantiomer was obtained by recrystallization from absolute
ethanol.
Yield: 1.60 g. mp 196.degree.-197.degree. C. [.alpha.].sup.26
D[c=1, MeOH]=+79.0.degree..
Mass spectrum, m/e (relative intensity) 273 (M.sup.+ +1,3) 229 (2),
214 (2), 184 (1), 165 (7), 157 (4), 140 (33), 139 (100), 138 (95),
97 (98), 96 (100), 91 (98).
Elemental analysis: calculated: 66.16% C; 5.92% H; 10.29% N. Found:
65.89% C; 5.86% H; 10.42% N.
COMPARATIVE EXAMPLE 8
Synthesis of N-Benzyl 2-Acetamidohydracrylamide
To an anhydrous THF solution (400 mL) of
methyl-.alpha.-acetamido-N-benzylmalonamate (14.4 g, 54.5 mmol) was
successively added dry LiCl (4.62 g, 109 mmol), NaBH.sub.4 (4.13 g,
109 mmol) and EtOH (200 mL). The reaction mixture was stirred at
room temperature (5h). The suspension was concentration in vacuo.
After continuous extraction (12h) of the product using CHCl.sub.3
(1000 mL) and H.sub.2 O (250 mL), the organic layer was collected,
dried (Na.sub.2 SO.sub.4), and removed in vacuo to give a crude
white solid. The crude product was triturated with Et.sub.2 O (500
mL) to give 11.45 g (89%) of the above compound: mp
201.degree.-203.degree. C.; R.sub.f 0.40 (10% MeOH--CHCl.sub.3); IR
(KBr) 3287, 3085, 2969, 2859, 1648, 1552, 1456, 1055, 697 cm.sup.-
; .sup.1 H NMR (DMSO-d.sub.6) 51.88 (s, C(O)CH.sub.3), 3.59 (dd,
J=5.7 Hz, 5.7 Hz, CH.sub.2 O), 4.19-4.35 (m, CH.sub.2 NH, CH), 4.92
(t, J=5.7 Hz, OH), 7.10-7.40 (m, 5 PhH), 7.94 (d, J=5.7 Hz, NH),
8.38 (t, J=5.7 Hz, NH); .sup.13 C NMR (DMSO-d.sub.6) 22.2
(C(O)CH.sub.3), 41.6 (CH.sub.2 N), 54.9 (CH), 61.3 (CH.sub.2 OH),
126.2 (C.sub.4'), 126.5 (2C.sub.2' or 2C.sub.3'), 127.7 (2C.sub.2'
or 2C.sub.3'), 138.9 (C.sub.1'), 169.1 (C(O)CH.sub.3 or C(O)NH),
169.9 (C(O)CH.sub.3 or C(O)NH) ppm; MS (+Cl) (relative intensity)
237 (M.sup.+ +1, 100), 219 (9); M.sub.r (+Cl) 237.123 88 [M.sup.+
+1] (calcd for C.sub.12 H.sub.17 N.sub.2 O.sub.3 237.123 92); Anal.
(C.sub.12 H.sub.16 N.sub.21 O3) C, H, N.
COMPARATIVE EXAMPLE 9
Synthesis of N-Benzyl 2-Acetamido-3-methoxypropionamide(racemic
mixture)
To an CH.sub.3 CN solution (500 mL) of the product of Comparative
Example 8 (2.36 g, 10 mmol) was successively added Ag.sub.2 O
(11.59 g, 50.0 mmol) and CH.sub.3 l (6.23 mL, 100 mmol) at room
temperature and then the reaction mixture was stirred at room
temperature (4 d). The insoluble salts were filtered, and the
solvent was removed in vacuo to give a white solid. The residue was
triturated with Et.sub.2 O (50 mL) to give 2.10 g (84%) of the
above-identified compound: mp 121.degree.-122.degree. C.; R.sub.f
0.47 (10% MeOH--CHCl.sub.3); IR (KBr) 3290, 3087, 2924, 2878, 2820,
1637, 1548, 1139, 695 cm.sup.-1 ; .sup.1 H NMR (CDCl.sub.3)
.delta.2.04 (s,C(O)CH.sub.3), 3.38 (s, OCH.sub.3), 3.43 (dd, J=7.8,
9.0 Hz, CHH'OCH.sub.3), 3.82 (dd, J=4.2, 9.0 Hz, CHH'OCH.sub.3),
4.48 (d, J=6.0 Hz, NHCH.sub.2), 4.51-4.57 (m,CH), 6.43 (br d, J=5.4
Hz, NH), 6.74 (br s, NH), 7.25-7.37 (m, 5 PhH); .sup.13 C NMR
(CDCl.sub.3) 23.2 (C(O)CH.sub.3), 43.5 (CH.sub.2 N), 52.4 (CH),
59.1 (OCH.sub.3), 71.7 (CH.sub.2 OCH.sub.3), 127.4 (C.sub.4' and
2C.sub.2' or 2C.sub.3'), 128.7 (2C.sub.2' or 2C.sub.3'), 137.8
(C.sub.1'), 170.0 (C(O)CH.sub.3 or C(O)NH), 170.3 (C(O)CH.sub.3 or
C(O)NH) ppm; MS (+Cl) (relative intensity) 251 (M.sup.+ +1, 100),
219 (100); M.sub.r (+Cl) 251.139 39 [M.sup.+ +1] (calcd for
C.sub.13 H.sub.19 N.sub.2 O.sub.3 251.139 57); Anal. (C.sub.13
H.sub.18 N.sub.2 O.sub.3) C, H, N.
COMPARATIVE EXAMPLE 10
(S)-N-Benzyl 2-Acetamidohydracrylamide
To a stirred AcOH (20 mL) suspension of L-serine (2.63 g, 25 mmol)
was added Ac.sub.2 O (2.5 mL, 26.3 mmol) and then the reaction
suspension was stirred at room temperature (24h). The AcOH was
removed in vacuo to given an oily residue, and then THF (150 mL)
was added to the residue. The THF suspension was cooled to
-78.degree. C. under N.sub.2 and 4-methylmorpholine (5.5 mL, 50
mmol) was added. After stirring (2 min.), isobutyl chloroformate
(6.5 mL, 50 mmol) was added leading to the precipitation of white
solid. The reaction was allowed to proceed for two additional
minutes and then benzylamine (5.5 mL, 50 mmol) was added at
-78.degree. C. The reaction mixture was allowed to stir at room
temperature (30 min.) and then the 4-methylmorpholine hydrochloride
salt was filtered. The organic layer was concentrated in vacuo. The
product was purified by flash column chromatography on SiO.sub.2
gel (10% MeOH--CHCl.sub.3) to given 2.20 g (37%) of the above
product as a white solid: mp 146.degree.-147.degree. C.;
[.alpha.].sub.D.sup.23 (c=1, MeOH)=-21.50; .sup.1 H NMR
(DMSO-d.sub.6) .delta.1.86 (s, C(O)CH.sub.3), 3.57 (dd, J=5.1 Hz,
5.1 Hz, CH.sub.2 O), 4.25-4.32 (m, CH.sub.2 NH, CH), 4.91 (t, J=5.1
Hz, OH), 7.20-7.33 (m, 5 PhH), 7.93 (d, J=8.1 Hz, NH), 8.37 (t,
J=5.7 Hz, NH), addition of excess (R)-(-) mandelic acid to a
CDCl.sub.3 solution of the above-identified compound gave only one
signal for the acetyl methyl protons.
COMPARATIVE EXAMPLE 11
(S)-N-Benzyl 2-Acetamido-3-methoxypropionamide
To a stirred CH.sub.3 CN solution (300 mL) of the compound produced
in Comparative Example 10 (1.18 g, 5 mmol) was successively added
Ag.sub.2 O (5.80 g, 25 mmol) and MeI (3.1 mL, 10 mmol) at room
temperature. The reaction mixture was stirred at room temperature
(4 d). The insoluble salts were filtered, and the solvent was
removed in vacuo to give a white solid. The white solid was
triturated with Et.sub.2 O (100 mL) to give 1.00 g (80%) of the
above-identified compound: mp 143.degree.-144.degree. C.
[.alpha.].sup.23 D (c=1, MeOH)=-16.4.degree.; .sup.1 H NMR
(CDCl.sub.3) .delta.2.03 (s, C(O)CH.sub.3), 3.38 (s, OCH.sub.3),
3.43 (dd, J=7.5, 9.0 Hz, CHH'OCH.sub.3), 3.81 (dd, J=4.2, 9.0 Hz,
CHH'OCH.sub.3), 4.47 (d, J=5.7 Hz, NHCH.sub.2), 4.52-4.59 (m,CH),
6.48 (br d, J=6.0 Hz, NH), 6.81 (br s, NH), 7.25-7.37 (m, 5 Ph),
addition of excess (R)-(-)-mandelic acid to a CDCl.sub.3 solution
of the above-identified compound gave only one signal for the
acetyl methyl and ether methyl protons.
COMPARATIVE EXAMPLE 12
(R)-N-Benzyl 2-Acetamidohydracylamide
This compound was prepared in accordance with the procedures
described in Examples 1 and 2.
COMPARATIVE EXAMPLE 13
N-Acetyl-D,L-phenylglycine-N-benzylamide
This compound was prepared in accordance with the procedure
described in U.S. Pat. No. 5,378,729, the contents of which are
incorporated by reference. The D,L-phenylglycine amide (11 mmol)
was dissolved in dichloromethane (15 mL) and then acetic anhydride
(1.23 g, 1.40 mL, 12 mmol) was added dropwise. The solution was
stirred at room temperature (4-6h) and then concentrated to
dryness. The residue was recrystallized from chloroform/hexane.
Yield: 2.05 g (66%) mp 202.degree.-203.degree. C.
.sup.1 H NMR (DMSO-d.sub.6) : .delta.1.91 (s, 3H) , 4.27 (d, J=5.6
Hz, 2H), 5.50 (d, J=7.9 Hz, 1H), 7.21 (s, 5H), 7.36 (s, 5H),
8.38-8.86 (m, 2H).
.sup.13 C NMR (DMSO-d.sub.6): 22.3, 42.0, 56.3, 126.6 (2C), 127.0,
127.1 (2C), 127.4 (2C), 128.1 (2C), 138.9, 139.0, 168.9, 169.9
ppm.
IR (KBr): 3020, 1635, 1580, 1540, 1450, 1265, 745, 690
cm.sup.-1.
Mass spectrum, m/e (relative intensity): 283(20), 264(21),
149(100), 131(20), 118(34), 106(92), 91(70), 79(56), 77(54),
65(45), 51(37).
Elemental analysis Calculated for C.sub.17 H.sub.18 N.sub.2 O.sub.2
72.31% C; 6.44% H; 9.92% N. Found 72.49% C; 6.47% H; 9.89% N.
The compounds of the present invention are useful for the treatment
of central nervous disorders, such as epilepsy, nervous anxiety ,
psychosis, insomnia and the like in animals, e.g., mammals, such as
man, in need thereof. They exhibit excellent anti-convulsant
activity, and of course and can thus be administered for short term
treatment. Moreover, the compounds of the present invention have
the added advantage of being useful in drug regimes for long-term
treatment. The compounds of the present invention are substantially
non-toxic, exhibiting minimal toxicity, if any, to the treated
animal, as shown below in the pharmacology section.
PHARMACOLOGY
Compounds were screened for anticonvulsant activity in both male
albino Carthworth Farms No. 1 mice (ip route) and male albino
Sprague Dawley rats [oral (po) route]. Activity was established
using the electrical (maximal electroshock or MES) test. In the MES
test, a drop of electrolyte solution with anesthetic (0.5%
butacaine hemisulfate in 0.9% sodium chloride) was used in the eyes
of the animals prior to positioning the corneal electrodes and
delivery of current. A 60 cycle alternating current was
administered for 0.2 sec. in both species, 50 mA in mice and 150 mA
in rats. Protection endpoints were defined as the abolition of the
hind limb tonic extensor component of the induced seizure. In mice,
effects of compounds on forced spontaneous motor activity were
determined using the rotorod test. The inability of animals to
maintain their balance for 1 min. on a 1 inch diameter knurled rod
at 6 rpms in 3 successive trials demonstrated motor impairment.
Normally under these conditions, mice maintain their balance almost
indefinitely. In rats, motor impairment is assessed by observing
for overt evidence of ataxia, abnormal gait and stance, and/or loss
of placing response and muscle tone. In the mouse identification
screening study all compounds were given at three dose levels (30,
100, 300 mg/kg) and two time periods (0.5 hours, 4 hours).
Typically, in the MES seizures test one animal was tested at 30
mg/kg and 300 mg/kg, and three animals at 100 mg/kg. In the rotored
toxicity test four animals were tested at 30 mg/kg, and 300 mg/kg,
and eight animals at 100 mg/kg. If activity was found at 30 mg/Kg,
then lower dosages were used to find the ED.sub.50 values.
The quantitative determination of the median effective (ED.sub.50)
and toxic doses (TD.sub.50) was conducted at previously calculated
times of peak effect. Groups of at least eight animals were tested
using different doses of test compound until at least two points
wee determined between 100 and 0% protection and minimal motor
impairment. The dose of candidate substance required to produce the
defined end-point in 50% of the animals in each test and the 95%
confidence interval was calculated.
TABLE 1 Physical and Pharmacological Data for Functionalized
N-Benzyl 2-Acetamidopropionamide Stereoisomers of the formula
ArCH.sub.3 NHC(O)CH(R.sup.2)NHC(O)CH.sub.3 mice (ip).sup.b rat
(po).sup.f Stere MES,.sup.c tox,.sup.d MES,.sup.c tox,.sup.d No.
obem. R.sup.2 Ar m p.sup.a ED.sub.50 TD.sub.50 Pl.sup.e ED.sub.50
TD.sub.50 Pl.sup.e Comp. (R, S) CH.sub.3 Ph 138-139 76.5 [1] 454
[0.5] 5.9 24.2 [1] .sub.--.sup.b >20.8 Ex. 1 (66.6-89.0)
(417-501) (32.0-71.8) Comp. (R) CH.sub.3 Ph 139-141 54.8 [0.5] 2.14
[0.5] 3.9 28.4 [4] .sub.--.sup.b >35.2 Ex. 2 (50.3-59.7)
(148-262) (22.4-35.0) Comp. (S) CH.sub.3 Ph 139-142 548 [0.5] 841
[0.5] 1.5 .sub.--.sup.i .sub.--.sup.i .sup.i Ex. 3 (50.3-59.7)
(691-954) Comp. (R, S) CH.sub.2 OCH.sub.3 Ph 121-122 8.3 [0.5] 42.9
[0.25] 5.2 3.8 [2] 386.8 [1] 101.8 Ex. 9 (7.9-9.8) (38.1-46.8)
(2.9-5.5) (316.0-514.6) Ex. (R) CH.sub.2 OCH.sub.3 Ph 143-144 4.5
[0.5] 26.8 [0.25] 6.0 3.9 [0.5] >500 [0.5] >128.2 1, 2
(3.7-5.5) (25.5-28.0) (2.6-6.2) Comp. (S) CH.sub.2 OCH.sub.3 Ph
143-144 >100, <300 >300 >30 >30 .sup.i Ex. 11 Comp.
(R, S) CH.sub.2 OH Ph 201-203 >100, 300 >300 .sub.--.sup.i
.sub.--.sup.i .sup.i Ex. 8 Comp. (R) CH.sub.2 OH Ph 148-149 53.4
[2] >500 [2] >9.4 .sub.--.sup.i .sub.--.sup.i .sup.i Ex. 12
(37.5-67.3) Ex. 3 (R) CH.sub.2 OCH.sub.3 Ph (m-F) 150-151 6.9
[0.25] 46.3 [0.25] 6.7 6.9 [0.5] >396 [0.5] >57.7 (6.1-8.0)
(40.4-54.5) (4.3-9.9) Ex. 4 (R) CH.sub.2 OCH.sub.3 Ph (p-F) 144-145
4.2 [0.5] 27.8 [0.25] 6.6 2.6 [2] >125, <250 .sup.i (3.5-5.1)
(22.4-33.5) (1.9-3.6) Comp. (R, S) OCH.sub.3 Ph 145-146 98.30
>100 <300 >1, <3 .sub.--.sup.i .sup.i .sup.i Ex. 4
Comp. (R) furyl Ph 190-197 3.3 23.8 >.2 .sub.--.sup.i
.sub.--.sup.i .sup.i Ex. 6 Comp. (S) furyl Ph 196-197 >25.
>200 .sub.--.sup.i .sub.--.sup.i .sup.i .sup.i Ex. 7 Comp. (R,
S) furyl Ph 178-179 10.3 .about.40 >3.9 .sub.--.sup.i
.sub.--.sup.i .sup.i Ex. 5 Comp. (D, L) Ph Ph 112-115 20.3 96.92
4.77 48.3 >1000 >20.7 Ex. 13 .sup.a Melting points (.degree.
C.) are uncorrected. .sup.b The compounds were administered
interperitoneally. ED.sub.50 and TD.sub.50 values are in mg/kg.
Numbers in parentheses are 95% confidence intervals. The dose
effect data was obtained at the "time of peak effect" [indicated in
hours in the brackets]. .sup.c MES = maximal electroshock seizure
test. .sup.d Tbx = neurologic toxicity determined from rotorod
test. .sup.e Pl = protective index (TD.sub.50 /MES Ed.sub.50)
.sup.f The compounds were administered orally. .sup.g No alaxia
observed up to 1000 mg/kg. .sup.i Data not available
The compounds of the present invention were compared with respect
to their efficacy and toxicity and PI values to compounds having a
structural similarity with the difference being the substituent at
R.sup.2. The protocols for these compounds is as described
hereinabove.
The results thereof are provided in Table I.
As clearly shown by the above data, the R enantiomers of the
present invention have quite potent anticonvulsant activity. The
inventor has also found that the R stereoisomer is unexpectedly
more potent than the corresponding S stereiosomer and the racemic
mixture.
The data in the table clearly demonstrate that the efficacy of the
comparative examples are significantly less than those of the
present invention. Only the 2-furyl derivative in the Table shows
comparable potency.
In addition, the compounds of the present invention have relatively
low neurological toxicity, considering the efficacy thereof. In
fact, as clearly shown by the data, the neurological toxicity is
significantly lower in rats in which the compounds were
administered orally than in the mice in which the compounds were
administered intraperitoneally. In fact, in rats, the neurological
toxicity of the compounds of the present invention is very low.
The PI values of the compounds of the present invention are quite
high in the mice model in which the compounds were administered
intraperitoneally and especially in the rat model in which the
compounds were administered orally. Of the compounds tested, the PI
values of the compounds of the present invention are generally
higher than that of the comparative examples, except for the
compound in which R.sup.2 is CH.sub.2 OH. However, the efficacy of
this latter compound is significantly less than that of compounds
of the present invention.
It is important to place the data in the table in proper
perspective. Looking at the data, it is quite apparent that the
compounds of the present invention exhibit an excellent drug
profile. On the other hand, based upon the data, except for the
furyl derivatives, the other comparative compounds are
significantly inferior drugs relative to the compounds of the
present invention. Although in some cases, the neurological
toxicity of the compounds of the comparative examples is low and
the PI value is satisfactory, the data cannot be viewed in a
vacuum. It is preferred that the drug not have a low potency, even
if it has a low neurological toxicity. After all, the objective is
to administer as little drug as possible to obtain an efficacious
result; the more drug administered to achieve a particular
efficacious result, the greater will be the risk that the drug
would have other effects, some of which are adverse, on other
bodily systems of the patient. Thus, except for the furyl
derivatives, based upon the data in the table the other comparative
examples have a significantly poorer drug profile relative to the
compounds of the present invention.
There is thus still another factor which must be taken into
consideration relating to the toxicity of the drug when
administered for extended periods to the animal. Obviously, even if
the drug has excellent anti-convulsant activity and an excellent PI
ratio, the drug will not be useful if the drug is toxic upon
chronic dosing to the patient. In the pharmaceutical industry
relating to anti-convulsants, one of the standards utilized to
measure a drug's toxicity to the animal is liver toxicity. The
objective is to find a drug having a relatively low or
substantially minimal liver toxicity.
Based upon the above data, both the furyl derivative and the
compounds of the present invention have an excellent drug profile;
and both could be used in acute administration. However even though
the furyl compound is quite active, as will be shown hereinbelow,
the furyl compound is more toxic to the animal, making it
considerably less undesirable for chronic administration than the
compounds of the present invention. On the other hand, the
compounds of the present invention as shown hereinbelow are
significantly less toxic than the furyl compound, and in fact
exhibit little, if any, toxicity to the animal. Thus, the compounds
of the present invention are useful for administration to the
treated animal for an extended period.
The following experiments measure the effect of a representative
compound of the present invention on the liver. The drug utilized
is the compound of Example 1, i.e.,
R-N-Benzyl-2-Acetamide-3-methoxypropionamide, hereinafter referred
to as BAMP.
I. Short term liver study
The protocol is as follows:
Four groups of 8 rats each were treated via p.O. administration
daily for 4 days with vehicle (groups 1 and 2), or 3.9 mg/kg of the
compound of Example 1 (group 3) or 100 mg/kg of the compound of
Example 1 (group 4). On day 5, animals in groups 2, 3 and 4
received 3.9 mg/kg of compound 1 (hereinafter "BAMP") and those in
group 1 received another dose of the vehicle.
To verify that the drug was effective, all groups were tested at
the time of peak effect (TPE) for drug efficacy against MES-induced
tonic extension, as described hereinabove.
Following the MES test, animals in group 4 received 96.1 mg/kg dose
of the compound of Example 1, a dose equal to the difference
between the ED.sub.50 and 100 mg/kg. On day 6, all groups were
tested for sleep time response (time from loss to or regaining, of
righting reflex) to a standard dose 100 mg/kg, i.p. of
hexobarbital. The hexobarbital sleep time provides an assessment of
hepatic drug metabolism. Following the performance of this test,
all animal groups received the same treatment as they received on
day 1. Day 7 had a similar dosing allocation except that group 2
received 100 mg/kg of BAMP. On days 8 and 9, four rats from each of
the four groups were euthanized. Blood was collected in cooled
tubes, allowed to clot, and then centrifuged to separate RBCs (red
blood cells). The serum was frozen at -70.degree. until serum
alanine aminotransferase (sALT) activity, indicative of potential
liver damage, was determined. The livers were perfused in situ with
ice cold saline, blotted dry, weighed, homogenized in 0.25M sucrose
and centrifuged to separate endoplasmic reticulum (i.e.,
macrosomes) and cytosol.
The protein concentration of both of these subcellular fractions
was determined by the Lowry method described in Lowry, et al., in
J. Biol Chem. 193, 265-275, (1951) and the yield of microsomal
protein calculated. The protein concentration of these two
subcellular fractions provide the basis for calculation of all
enzyme concentrations and activities.
Changes in a wide range of drug metabolizing enzymes known to be
variously altered by drug treatments were sought. Both microsomal
and cytosolic Phase I (cytochrome P450 catalyzed oxidations and
quinone oxidoreductase activity, respectively) and microsomal
(glucuronidation) and cytosolic (glutathione and sulfate
conjugation) Phase II conjugation reactions were evaluated, in
accordance with the procedure described in Arch Biochem. Biophys,
143, 318-329 (1971), the contents of which are incorporated by
reference. BAMP showed no evidence of causing liver necrosis.
Collectively, the results obtained from a battery of liver enzyme
studies suggest that the liability for serious drug-drug
interactions and liver toxicity is relatively low for this
compound.
Since the compound showed minimal liver toxicity in a 48 hours
study, a much longer study was performed over 30 days.
The methodology is as follows:
II. Five groups of Crl:CD.RTM. BR Charles River rats were each
exposed to BAMP or a control substance (0.5% methylcellulose [400
Cps] aqueous solution in distilled water) according to the
following dosage schedule:
Group 1--vehicle control (10 males, 10 females), 0 mg/kg/day
Group 2--low (10 males, 10 females), 10 mg/kg/day
Group 3--mid low (10 males, 10 females), 30 mg/kg/day
Group 4--mid high (10 males, 10 females), 100 mg/kg/day
Group 5--high (10 males, 10 females), 300 mg/kg/day
Exposure was by oral gavage, once daily, for a period of at least
30 consecutive days, after which all animals were sacrificed for
pathologic evaluation.
All animals were weighed once prior to initiation of dosing and
weekly thereafter. Food-fasted (overnight) blood samples for
clinical chemistry and hematology were collected at termination.
Blood samples were collected from the orbital venous plexus using
carbon dioxide (mixed with oxygen) as an anesthetic.
All animals were sacrificed, at the appropriate time, by
exsanguination, under barbiturate anesthesia, and all were
subjected to a necropsy examination.
Clinical observations were reviewed at necropsy, and all grossly
observed abnormalities were entered directly into the computerized
data collection system. Adrenals, brain with brainstem, heart,
kidneys, liver, ovaries, pituitary, testes with epididymides, and
thyroid with parathyroids were weighted from each animal. The
pituitary and thyroid with parathyroids were weighed after fixation
and all of the other organs were weighed at the time of necropsy.
The changes in the weight of the liver is depicted in Table 3.
As required by the protocol, histologic evaluations were conducted
on liver only from all animals of Groups 1 (control) and 5 (high).
All histologic findings were entered directly into the computerized
data capture system. Lesions were graded as to relative severity or
degree of involvement (1=minimal, 2=slight, 3=moderate,
4=moderately severe, 5=severe). In general, minimal represents the
least consistently recognizable degree of any given
histoniorphologic alteration, while severe would represent the most
extreme degree reasonably possible, with the other three grades
occupying a continuum between the two extremes. The grades are
subjective, comparative evaluations, based on morphology alone and
are not intended by themselves to imply any degree of functional
impairment.
Results
Gross Findings--There were few gross abnormalities reported. All
were frequently encountered in normal populations of rats of this
strain and age; none were suggestive of any effect of treatment.
The data is found in Table 2.
TABLE 2 30-DAY RANGE-FINDING ORAL TOXICITY STUDY OF BAMP IN RATS
GROSS PATHOLOGY INCIDENCE SUMMARY TABLE INCLUDES: SEX = ALL; GROUP
= ALL; WEEKS = ALL DEATH = ALL; SUBSET = ALL NUMBER OF ANIMALS
AFFECTED SEX: MALE FEMALE GROUP: 1 2 3 4 5 1 2 3 4 5 ORGAN AND
KEYWORD(S) OR PHRASE NUMBER: 10 10 10 10 10 10 10 10 10 10
PARATHYROID (PT) NUMBER EXAMINED: 10 10 10 10 10 10 10 10 10 10 NOT
REMARKABLE: 10 10 10 10 10 10 10 10 10 10 ESOPHAGUS (ES) NUMBER
EXAMINED: 10 10 10 10 10 10 10 10 10 10 NOT REMARKABLE: 10 10 10 10
10 10 10 10 10 10 TRACHEA (TR) NUMBER EXAMINED: 10 10 10 10 10 10
10 10 10 10 NOT REMARKABLE: 10 10 10 10 10 10 10 10 10 10 LUNG (LU)
NUMBER EXAMINED: 10 10 10 10 10 10 10 10 10 10 NOT REMARKABLE: 10
10 10 10 10 10 10 10 10 10 HEART (HT) NUMBER EXAMINED: 10 10 10 10
10 10 10 10 10 10 NOT REMARKABLE: 9 10 10 10 10 10 10 10 10 10
ENLARGED 1 0 0 0 0 0 0 0 0 0 SPLEEN (SP) NUMBER EXAMINED: 10 10 10
10 10 10 10 10 10 10 NOT REMARKABLE: 10 10 10 10 10 10 10 10 10 10
LIVER (LI) NUMBER EXAMINED: 10 10 10 10 10 10 10 10 10 10 NOT
REMARKABLE: 10 10 10 10 9 10 10 10 10 10 ENLARGED 0 0 0 0 1 0 0 0 0
0
Histopathology--All were of the kinds frequently encountered in
normal populations of rats in this strain and age. None presented
in a dropwise pattern suggestive of a treatment effect.
TABLE 3 30 DAY RANGE-FINDING ORAL TOXICITY STUDY OF BAMP IN RATS
ORGAN WEIGHT DATA TABLE INCLUDES: SEX = ALL; GROUP = ALL; WEEKS =
ALL DEATH = ALL; SUBSET = ALL LIVER ORGAN-TO- TERMINAL ORGAN
ORGAN-TO- BRAIN WT DOSE BODY WT WEIGHT BODY WT RATIO SEX GROUP (g)
(g) (%) RT M 1 NUMBER IN GROUP: 10 10 10 10 MEAN: 356.3 11.76 3.323
5.760 STANDARD DEV: 42.8 1.34 0.350 0.577 M 2 NUMBER IN GROUP: 30
10 10 10 MEAN: 342.0 11.10 3.248 5.388 STANDARD DEV: 26.6 0.96
0.161 0.391 M 3 NUMBER IN GROUP: 10 10 10 10 MEAN: 349.9 11.48
3.278 5.619 STANDARD DEV: 21.4 1.26 0.265 0.666 M 4 100 mg/kg/day
.times. 30 days NUMBER IN GROUP: 10 10 10 10 MEAN: 351.5 11.79
3.351 5.702 STANDARD DEV: 29.3 1.38 0.223 0.725 M 5 300 mg/kg/day
.times. 30 days NUMBER IN GROUP: 10 10 10 10 MEAN: 358.8 14.45*
4.016* 7.028* STANDARD DEV: 28.4 2.24 0.430 1.141 TERMINAL ORGAN
ORGAN-TO- ORGAN-TO- DOSE BDDY WT WEIGHT BODY WT BRAIN WT SEX GROUP
(g) (g) (%) RATIO F 1 NUMBER IN GROUP: 10 10 10 10 MEAN: 202.5 6.56
3.247 3.374 STANDARD DEV: 13.1 0.54 0.266 0.296 F 2 NUMBER IN
GROUP: 10 10 10 10 MEAN: 214.1 6.91 3.237 3.665 STANDARD DEV: 12.2
0.76 0.379 0.440 F 3 NUMBER IN GROUP: 10 10 10 10 MEAN: 207.0 6.71
3.244 3.453 STANDARD DEV: 17.7 0.66 0.185 0.350 F 4 100 mg/kg/day
.times. 30 days NUMBER IN GROUP: 10 10 10 10 MEAN: 218.3 7.88*
3.608 4.179* STANDARD DEV: 16.7 1.00 0.310 0.312 F 5 300 mg/kg/day
.times. 30 days NUMBER IN GROUP: 10 10 10 10 MEAN: 205.9 7.88*
3.832* 4.313* STANDARD DEV: 14.3 0.88 0.419 0.457 *significantly
different from control value p .ltoreq. 0.05 RT - Date analyzed
following rank transformation.
Conclusions
The liver of rats exposed to BAMP by oral gavage for at least 30
days showed no histologic evidence of an adverse effect at the
highest dose level employed (300 mg/kg/day).
The results were compared with the comparative examples in Table I
which showed the greatest efficacy and greatest PI values, viz.,
the compound of Comparative Example 1 (hereinafter referred to as
Compound A), Comparative Example 6 (hereinafter referred to as
Compound B) and Comparative Example 13 (hereinafter referred to as
Compound C).
COMPARATIVE EXAMPLE 14
Compound C, i.e., N-acetyl-D, L-phenylglycine N-benzylamide was
subjected to 5-day chronic treatment on anticonvulsant activity
(maximal electroshock). 3 groups of 8 animals each were treated as
follows. One group was given the MES ED.sub.50 of the test drug for
5 days; the second group was given the requisite volume of the
vehicle (0.04 ml/10 g body wt.) for 4 days and a single dose (MES
ED.sub.50) of the test drug on day 5; and the third group was given
the requisite volume of the vehicle daily for 5 days. At the time
of peak effect of the candidate substance on day 5, all groups were
subjected to the MES test and the number of animals protected
recorded. Seizure components of the unprotected animals were timed
to the nearest 10th of a second and the extensor/flexor (E/F)
ratio, S.E., and p value determined. Since extensor duration
decreases and flexor duration increases as a maximal seizure is
attenuated, the E/F ratio provides a measure of seizure
severity.
All rats subjected to the 5-day tolerance studies were maintained
in their home cage for 24 hours and then subjected to the
hexobarbital sleep time test (day 6). Each rat in each of the 3
groups was given 100 mg/kg of hexobarbital (i.p.) and the sleep
time measured to the nearest minute. The mean sleep time and S.E.
for each group were calculated. If the mean sleep time of the
treated group was significantly less than that of the
treated-control group, it was considered suggestive of metabolic
tolerance.
Two of the three groups of animals subjected to the hexobarbital
sleep time test (chronically treated and vehicle control groups)
were continued on their respective original treatment regimen for
two days (days 6 and 7) and 24 hours later (day 8) subjected to
liver microsomal studies. The rats were decapitated and the livers
perfused with 0.9% sodium chloride solution. The livers were
removed, weighed, and homogenized in 0.25M sucrose. Microsomes were
prepared and their drug metabolizing capabilities (microsomal
protein yield; cytochrome P-450 concentration; p-nitroanisole
O-demethylase and NADPH cytochrome c reductase activities;
norbenzphetamine MI complex formation; and glucuronyl transferase,
erythromycin demethylase, and ethylmorphine demethylase activities)
measured (Arch. Biochem. Biophys. 143: 318-329, 1971).
The chronic studies in rats demonstrate that 5 daily doses of 48
mg/kg of Compound C does not affect either the anticonvulsant
activity or hexobarbital sleep time. In contrast, chronic
administration of Compound C induces some liver microsomal enzyme
systems as indicated by the significant increases in p-nitroanisole
O-demethylase, ethylmorphine demethylase, and NADPH cytochrome c
reductase activities. See Table 4.
TABLE 4 ANTICONVULSANT SCREENING PROJECT TEST RESULTS PHASE VII
EVALUATION, Rats, p.o. CMPD C Solvent: 30% PEG (M&P, SB) Ave.
Animal Wt: 123.3 g. TPE 4 hrs, ED50 48 mg/kg B. EFFECT OF CHRONIC
ADMINISTRATION ON THE LIVER MICROSOMAL SYSTEM Parameter Control
Treated 7 Days Body Weight (g) 148.8 .+-. 4.3 146.3 .+-. 3.8 Liver
Weight (g) 8.36 .+-. 0.26 8.54 .+-. 0.26 Total Protein (mg) 30.0
.+-. 1.6 31.7 .+-. 1.4 Cytochrome P-450 0.60 .+-. 0.02 0.71 .+-.
0.06 (nmoles/mg) p-nitroanisole O-demethylase 0.56 .+-. 0.04 0.82
.+-. 0.12* (nmoles/mg/min) NADPH Cytochrome c reductase 137.8 .+-.
9.2 160.3 .+-. 5.2* (nmoles/mg/min) Norbenzphetamine MI Complex
0.013 .+-. 0.002 0.023 .+-. 0.003 (nmoles/mg/min) Glucoronyl
Transferase 9.10 .+-. 0.20 9.77 .+-. 0.23 (nmoles/mg/min)
Erythromycin demethylase 0.55 .+-. 0.04 0.64 .+-. 0.07
(nmoles/mg/min) Ethylmorphine demethylase 5.59 .+-. 0.37 6.75 .+-.
0.37* (nmoles/mg/min) *Significantly different from control, p <
0.05
These findings suggest that the compound C has an adverse effect on
the liver.
As shown by the data, Compound C has a relatively less than
desirable longterm, i.e., 7-day dose, profile in inducing liver
enzyme. At 48 mg/kg/day (which is its effective one-time dose in
preventing MES convulsion) p.o..times.7 days, the data clearly show
that hepatic involvement was observed in liver enzyme induction. It
should be noted that if the MES-ED.sub.50 dose were continued for
30 days rather than 7 days, there is a high probability that more
profound changes would likely have occurred, suggesting that a
safety ratio of only 1 could be anticipated in a 30-day dosing
schedule.
COMPARATIVE EXAMPLE 15
Compound A, i.e., N-Acetyl-D,L-alanine-N'-benzylamide was tested
for its liver toxicity in accordance with the procedure described
in Comparative Example 14.
The results are as follows:
The 5-day chronic studies in rats demonstrate that 5 daily doses of
48 mg/kg does not induce tolerance to the anticonvulsant effects
(MES Test) of Compound A within this period of time. This
interpretation is supported by the similar effectiveness of
Compound A by the MES test, the increased hexobarbital sleep time,
and the unaltered liver microsomal enzyme activity. In view of the
increased hexobarbital sleep time in the 5-day treated animals, it
was thought important to determine the in vitro effect of Compound
A on p-nitroanisole O-demethylase activity. The low inhibitory
potency of Compound A (I.sub.50 =5000 .mu.M) suggests that there is
little interference by the compound itself on hexobarbital
metabolism in the sleep test. This may indicate that the
potentiation of hexobarbital sleep time is central and not
peripheral.
5-day tolerance studies (MES and hexobarbital sleep time tests) and
7-day liver microsomal enzyme studies in rats, indicate that
tolerance was not induced by 5 daily doses of the MES ED.sub.50 (48
mg/kg) of Compound A (4/8 protected in the single dose acute
control group; 3/8 protected in the chronically treated group);
5-day chronic treatment increased hexobarbital sleep time from that
induced by a single acute dose (31.7.+-.1.7, 34.3.+-.1.1, and
44.4.+-.1.9 minutes in solvent control, acute control, and 5-day
treated, respectively). There was no significant change in body
weight (148.8.+-.5.9 vs 140.0.+-.4.6 g), liver weight (7.71.+-.0.22
vs 7.22.+-.0.45 g), total microsomal protein (32.3.+-.0.56.+-.0.04
nmoles/mg), p-nitroanisole O-demethylase activity (0.50.+-.0.04 vs
0.62.+-.0.07 nmoles/mg/min, NADPH cytochrome c reductase activity
(95.3.+-.11.0 vs 105.0.+-.4.1 nmoles/mg/min) in solvent control and
7-day treated, respectively. The candidate substance (Compound A)
had very little inhibitory potency (I.sub.50 : c.5000 .mu.M) for in
vitro p-nitroanisole demethylation.
However, there was found little, if any liver enzyme induction in
the 7-day study, and the compound was advanced to a 30 day dose
ranging toxicology study, as that described hereinabove.
More specifically, 50 male and 50 female Crl:CoBs.RTM. CD(SD)
selected from 68 male and 68 female rats (4 weeks old) were used as
test animals in the study 1. The rats were housed individually in
elevated wire mesh cages with food (Purina Certified Rodent
Chow.RTM. 5002) and tap water (via an automated watering system)
available ad libitum. Each batch of feed utilized was analyzed by
the manufacturer for concentrations of specified heavy metals,
aflatoxin, chlorinated hydrocarbons, organophosphates, and
specified nutrients. The tap water was routinely analyzed on a
retrospective basis for specified microorganisms, pesticides, heavy
metals, alkalinity, and halogens for contamination. None were
present in the animal feed or water at levels sufficient to
interfere with this study.
During the quarantine and study periods, the room temperature and
relative humidity were recorded twice daily and ranged from
64.degree. to 77.degree. F. and 12 to 51%, respectively. An
artificial light cycle of 12 hours light and 12 hours dark was
maintained.
The rats were selected for use on the study using a
computer-generated weight randomization procedure and assigned to
the following groups:
Compound A No. of Rats Dosage Levels Group Male Female mg/kg/day 1
(Control) 10 10 0 2 (low) 10 10 30 3 (Mid-1) 10 10 100 4 (Mid-2) 10
10 300 5 (High) 10 10 1000
Following randomization, the rats were identified with an ear tag
bearing a unique permanent identification number. The rats were
randomly assigned to treatment groups by first eliminating the ones
with extreme body weights (.+-.2 standard deviations from the mean
body weight). Body weights at initiation ranged from 188.9 to 215.4
grams for the males and 141.8 to 158.8 grams for the females.
Compound Preparation and Administration
The desired amount of carboxymethyl cellulose was weighed on an
appropriate (milligram) balance, transferred to a precalibrated
beaker containing two-thirds of the total value of distilled water,
and stirred on a magnetic stirrer until a solution formed.
Distilled water was then added to final volume and stirred to
achieve a 0.5% w/v solution.
Compound A was first ground into a powder. The desired amount for
each dose level as weighed on an appropriate (milligram) balance
and transferred into a precalibrated beaker. A small amount (0.5 to
4ml) of 0.5% carboxymethyl cellulose was added to Compound A and
mixed to form a paste. Carboxymethyl cellulose (0.5%) was added to
the final volume and mixed with a Tekmar.RTM. Tissumizer.RTM. for 2
to 3 minutes then mixed with a magnetic stirrer for 2 to 3 minutes.
Fresh suspensions of Compound A were prepared daily and fresh
solutions of 0.5% carboxymethyl cellulose were prepared weekly and
stored refrigerated.
Each rat received Compound A at a dosage factor of 10 milliliters
per kilogram of body weight via gavage between 9 a.m. and noon each
day. The dosing volume for each rat was calculated and adjusted
weekly by the computer from the most recently recorded individual
body weight.
Compound A was administered orally.
Reserve samples of carboxymethyl cellulose (1 gram), distilled
water (10 milliliters), and Compound A (1 Gram) were taken at
initiation and stored at room temperature.
All rats were observed twice daily for mortality and moribundity.
Clinical observations were made prior to dosing and at 1 and 4
hours after dosing. All signs were recorded as they were observed.
Individual body weights were recorded at initiation of treatment,
at weekly intervals, and at termination while food consumption was
recorded weekly.
Sacrifice and Gross Pathology
Following 30 or 31 days of treatment, surviving rats were weighed,
anesthetized, and exsanguinated under sodium pentobarbital
anesthesia. Complete necropsies were performed on each rat by
appropriately trained personnel using procedures approved by
board-certified pathologists. Necropsy included examination of the
following:
External surface
All orifices
Cranial cavity
Carcass
External surface of the brain and spinal cord (postfixation)
Nasal cavity and paranasal sinuses
Thoracic, abdominal, and pelvic cavities and their viscera
Cervical tissues and organs
All findings were recorded.
Gross Pathology
Individual gross pathology findings are as follows:
A possible compound-related effect on the kidneys was observed.
Dilated pelves were noted in three males and two females in Group
5, one male each in Groups 3 and 4, and one female in Group 1.
Other observations noted which appear to be incidental and not
compound-related included dark areas on the lungs, liver, thymus,
stomach, and cecal mucose, granular spleen, raised area on the
liver, fluid-distended uterus, fluid in the cranial cavity, and a
small, soft testis.
Organ Weights and Organ/Body Weight Ratios
Various organs were weighted and compared to the control, e.g.,
brain with stem, heart, spleen, kidney, liver, sex organs. Only the
liver weights were significantly different from the control value
as shown in Table 5.
TABLE 5 PATH/TOX SYSTEM OUTPUT THIRTY-DAY DOSE RANGE FINDING STUDY
OF COMPOUND A IN RATS ORGAN TO TERMINAL BODY WEIGHT RATIO MEANS (%)
TABLE INCLUDES: SEX = ALL; GROUP = ALL; WEEKS = ALL DEATH = ALL;
SUBSET = ALL SEX: MALE FEMALE GROUP: 1 2 3 4 5 1 2 3 4 5 NUMBER: 10
10 10 10 10 10 10 10 10 10 BR - BRAIN W/STEM # IN GRP: 10 10 10 10
10 10 10 10 10 10 MEAN: .588 .593 .599 .581 .571 .918 .887 .899
.934 .891 STAND DEV: .034 .065 .037 .019 .035 .048 .043 .044 .073
.079 HT - HEART # IN GRP: 10 10 10 10 10 10 10 10 10 10 MEAN: .321
.313 .314 .328 .343 .358 .372 .345 .373 .347 STAND DEV: .027 .021
.037 .055 .057 .029 .039 .035 .028 .033 SP - SPLEEN #IN GRP: 10 10
10 10 10 10 10 10 10 10 MEAN: .176 .172 .166 .163 .176 .200 .204
.200 .198 .189 STAND DEV: .029 .027 .020 .013 .021 .034 .022 .021
.036 .016 KD - KIDNEY # IN GRP: 10 10 10 10 10 10 10 10 10 10 MEAN:
.732 .719 .694 .695 .770 .753 .773 .768 .770 .763 STAND DEV: .038
.043 .055 .047 .063 .063 .063 .043 .043 .054 LI - LIVER # IN GRP:
10 10 10 10 10 10 10 10 10 10 MEAN: 2.846 2.849 3.059* 3.101*
3.683* 3.004 3.122 3.166 3.172 3.739* STAND DEV: .183 .167 .238
.147 .173 .249 .211 .147 .167 .242 TP - TESTIS/EPIDID # IN GRP: 10
10 10 10 10 0 0 0 0 0 MEAN: 1.251 1.265 1.219 1.212 1.202 STAND
DEV: .171 .118 .136 .098 .099 *Significantly different from the
control value, (p .ltoreq. .05).
Thus, the mean liver weight was increased in the males of Groups 4,
(300 mg/kg/day.times.30 days) and 5 (1000 mg/kg/day.times.30 days)
in the Group 5 females. This was reflected by increases in the
liver/body weight ratios. The liver/body weight ratio was also
noted for the Group 3 (100 mg/kg/day.times.30 days) males. The only
other change of note was a small increase in the mean kidney weight
of the Group 5 males.
Thus, using a daily dose of 100 mg/kg as a threshold dose to a
potentially hepatotoxic dose, this would give a liver safety ratio
against the anti-convulsant dose of 2.1.
COMPARATIVE EXAMPLE 16
Compound C, i.e.,
D-(-)-.alpha.-acetamido-N-benzyl-2-furanacetylamide was evaluated
for liver toxicity using the procedures described hereinabove. More
specifically, various dosages such as 25 mg/kg, 100 mg/kg, 500
mg/kg of the drug was administered by oral gavage to rats for a set
period of time. The rats were housed separately. The rats were
periodically viewed for mortality and moribundity. At the
termination of the study, the surviving rats were anesthetized, and
exsanguinated under anesthesia. Complete necropsies were performed
by appropriately trained personnel using procedures approved by
board certified pathologists and the results were recorded.
When the D-furyl derivative of Comparative Example 6 was
administered to the rat, hepatocellular necrosis was evident at 100
and 25 mg/kg in rats treated for 13 weeks.
The data respecting these compounds tested BAMP, compounds A, B and
C are summarized in Table 6.
TABLE 6 RAT Liver Pathology MOUSE oral, multiple doses
anticonvulsant dose anticonvulsant Safety Route i.p. Ratio Oral
Ratio daily ED.sub.50 (95% C.L.) to ED.sub.50 (95% C.L.) Ratio
dose; mg/kg MES mg/kg to MES MES Neurotox (p.2) MES Neurotox MES
Dose Pathology Report (p.2) Compound C 20.3 96.9 4.8 48.3 >1000
20.7 48 mg/kg/day .times. liver enzyme induction -1 D.L-phenyl
(16.8- (79.8- (3.31- 7 days 24.5) 118) 68) Compound A 76.5 454
(417- 5.9 48.2 >1000 20.8 100 mg/kg/ liver to body wt. 2.1 D,L
methyl (66.6- 501) (32.0- day .times. ratio increased 89.0) 71.8)
30 days male 300 mg/kg/ absolute weight liver -6.3 day .times. inc.
- male 30 days Compound B 3.3 23.8 7.2 1.97 333 (259- 175 25 mg/kg/
hepatocellular 12.7 D-furan (2.8- (17.2- (1.07- 411) day .times. 30
necrosis 3.9) 30.7) 3.3) days BAMP 4.46 26.8(25.5- 6.0 3.90 >500
>128 100 mg/kg/ liver to brain wt. 25.6 D-methoxy- (3.72- 28.0)
(2.58- day .times. 30 ratio increased- methyl 5.46) 6.20) days
female 300 mg/kg no adverse histologic >76.9 day .times. 30
effects days MES - Maximal Eletrochock Seizure test Neurotox -
rotorod test determined at peak effect
As clearly, shown by the data in Table 6, the ED.sub.50 value in
the MES test for BAMP is significantly less (significantly more
effective) than that of Compounds A and C and is of the same order
of magnitude with respect to Compound B. Moreover, the PI ratio of
BAMP is significantly greater than that of Compounds A and C.
However, and most importantly, BAMP had no histopathologic
indications at the higher dose (300 mg/kg/day for 30 days) and
exhibited a minor deviation at the lower dosage. This is in
complete contrast to the liver pathology of Compounds A, C and
especially B. All of the comparative examples showed significantly
greater liver toxicity than BAMP. This is seen in the safety ratio
daily dose of MES, shown in the last column in the table. In this
table, the daily dose given for multiple consecutive days at which
the first indications of liver toxicity is noted. That ratio,
expressed against the oral anticonvulsant MES single dose, is a
safety index for onset of liver problems upon chronic
administration of drug. As shown in the table, the safety ratio for
the dose to signs of liver enlargement following 30 days medication
relative to an oral anti-convulsant dose was 2.1 for Compound A,
but was 25.6 for BAMP. For histologic signs of liver toxicity,
including, for example, hepatocellular necrosis, the safety ratio
was 12.7 for Compound B. In contrast, 30 days chronic dosing with
BAMP caused no adverse histologic effects at 76.9 times its
anti-convulsant dose.
Thus, the toxicity of the compounds of the present invention when
administered for extended periods to the animal is an important
parameter. Even when an anti-convulsant has active efficacy, if it
shows toxicity to the animal, it is unlikely that it will be a
candidate for use in chronic dosing. Thus, in selecting an
anti-convulsant it is not only important that is satisfies the
three criteria outlined hereinabove (high efficiency, low
neurological toxicity, high P.I.) but also the fourth criteria, low
toxicity. The compounds of the present invention meet these
criteria.
Thus, as clearly shown by the data the compounds of the present
invention have low liver toxicity required of drugs to be used in
chronic administration and are thus quite safe. The compounds of
the present invention exhibit none or minimal effects on the
liver.
Thus, the compounds of the present invention exhibit an excellent
drug profile. They meet all of the four characteristics outlined
heretofore, high potency, low neurological toxicity relative to its
potency, high protective index and minimal liver toxicity. The
compounds of the present invention are substantially non-toxic to
the liver. These compounds of the present invention exhibit
advantages that have not heretofore been realized. They therefore
can be used in a treatment regimen requiring administration thereof
over extended periods of time (chronic administration).
The above preferred embodiments and examples are given to
illustrate the scope and spirit of the present invention. The
embodiments and examples described herein will make apparent to
those skilled in the art other embodiments and examples. These
other embodiments and examples are within the contemplation of the
present invention. Therefore, the present invention should be
limited only by the appended claims.
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