U.S. patent application number 09/925947 was filed with the patent office on 2002-03-07 for reduced dipeptide analogues as calcium channel antagonitsts.
Invention is credited to Rafferty, Michael Francis, Song, Yuntao.
Application Number | 20020028801 09/925947 |
Document ID | / |
Family ID | 22246680 |
Filed Date | 2002-03-07 |
United States Patent
Application |
20020028801 |
Kind Code |
A1 |
Rafferty, Michael Francis ;
et al. |
March 7, 2002 |
Reduced dipeptide analogues as calcium channel antagonitsts
Abstract
The present invention provides novel N-type calcium channel
blockers, compositions comprising them, and methods of their use.
The compounds of the invention are useful in the treatment of
stroke, cerebral ischemia, pain, epilepsy, and head trauma. The
novel compounds provided by this invention have the Formula I: 1
wherein R.sup.1 is H or methyl, R.sup.2 is H, azepanylcarbonyl,
C.sub.1-C.sub.7 alkyl, --(CH.sub.2).sub.n-phenyl, wherein the
phenyl is unsubstituted or substituted with C.sub.1-C.sub.4 alkyl,
C.sub.1-C.sub.4 alkoxy, or halo, R.sup.3 is C.sub.1-C.sub.5 alkyl,
X is --NR.sup.4R.sup.5 or --OR.sup.7, R.sup.4 and R.sup.5 are
independently H, C.sub.1-C.sub.5 alkyl, or R.sup.4 and R.sup.5
together with the nitrogen to which they are both bound form: 2
R.sup.6 is --(CH.sub.2).sub.n-phenyl, wherein the phenyl is
unsubstituted or substituted with C.sub.1-C.sub.4 alkyl,
C.sub.1-C.sub.4 alkoxy, or halo, A and B are independently --CO--
or --CH.sub.2--, provided that A and B are not both --CO--, R.sup.7
is C.sub.1-C.sub.5 alkyl, Z is --CH.sub.2--, --O--, --S--, or
--N(R.sup.8)--, R.sup.8 is H or C.sub.1-C.sub.6 alkyl, and n is 1
or 2.
Inventors: |
Rafferty, Michael Francis;
(Ann Arbor, MI) ; Song, Yuntao; (Ann Arbor,
MI) |
Correspondence
Address: |
Warner-Lambert Company
2800 Plymouth Road
Ann Arbor
MI
48105
US
|
Family ID: |
22246680 |
Appl. No.: |
09/925947 |
Filed: |
August 9, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09925947 |
Aug 9, 2001 |
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09381983 |
Dec 27, 1999 |
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09381983 |
Dec 27, 1999 |
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PCT/US99/12275 |
Jun 2, 1999 |
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60094703 |
Jul 30, 1998 |
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Current U.S.
Class: |
514/211.01 ;
514/183; 514/217.11; 514/218; 514/227.5; 514/237.5; 514/255.01;
514/423; 540/467; 540/470; 540/544; 540/575; 540/607; 544/176;
544/386; 544/59; 548/530; 560/39; 564/158 |
Current CPC
Class: |
C07D 295/215 20130101;
C07C 237/20 20130101; C07D 295/13 20130101 |
Class at
Publication: |
514/211.01 ;
514/218; 514/255.01; 514/227.5; 514/237.5; 514/423; 514/183;
514/217.11; 540/467; 540/470; 540/544; 540/575; 540/607; 544/59;
544/176; 548/530; 544/386; 560/39; 564/158 |
International
Class: |
A61K 031/553; A61K
031/554; A61K 031/55; A61K 031/551; A61K 031/495; A61K 031/541;
A61K 031/5375; A61K 031/40 |
Claims
What is claimed is:
1. A compound having structural Formula I: 35wherein R.sup.1 is H
or methyl, R.sup.2 is H, azepanylcarbonyl, C.sub.1-C.sub.7 alkyl,
--(CH.sub.2).sub.n-phenyl, wherein the phenyl is unsubstituted or
substituted with C.sub.1-C.sub.4-alkyl, C.sub.1-C.sub.4 alkoxy, or
halo, R.sup.3 is C.sub.1-C.sub.5 alkyl, X is --NR.sup.4R.sup.5 or
--OR.sup.7, R.sup.4 and R.sup.5 are independently H,
C.sub.1-C.sub.5 alkyl, or R.sup.4 and R.sup.5 together with the
nitrogen to which they are both bound form: 36 R.sup.6 is
--(CH.sub.2).sub.n-phenyl, wherein the phenyl is unsubstituted or
substituted with C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 alkoxy, or
halo, A and B are independently --CO-- or --CH.sub.2--, provided
that A and B are not both --CO--, R.sup.7 is C.sub.1-C.sub.5 alkyl,
Z is --CH.sub.2--, --O--, --S--, or --N(R.sup.8)--, R.sup.8 is H or
C.sub.1-C.sub.6 alkyl, and n is 1 or 2.
2. The compound according to claim 1, wherein R.sup.4 and R.sup.5
are independently H, C.sub.1-C.sub.5-alkyl, or R.sup.4 and R.sup.5
taken together with the nitrogen to which they are both bound form
1-pyrrolidinyl, 1-piperidinyl, or 1-azepanyl, or R.sup.4 and
R.sup.5 taken together with the nitrogen to which they are both
bound are: 37and Z is --S-- or --O--.
3. A compound according to claim 1 selected from the group
consisting of
2-((2-(Azaperhydroepinylcarbonylamino)-4-methylpentyl)amino)-N-(tert-buty-
l)-3-(4-(phenylmethoxy)phenyl)-(2S)-propanamide;
[S-(R*,R*)]-Azepane-1-car- boxylic acid
{1-[2-(4-benzyloxy-phenyl)-1-tert-butoxy-methyl-ethylcarbamoy-
l]-3-methyl-butyl}-amide; [S-(R*,R*)]Azepane-1-carboxylic acid
(1-{[2-(4-benzyloxy-phenyl)-1-tert-butyl-carbamoyl-ethylamino]-methyl}-3--
methyl-butyl)-amide; [S-(R*,R*)]-2-Dimethylamino-4-methyl-pentanoic
acid [2-(4-benzyloxy-phenyl) 1-morpholin-4-ylmethyl-ethyl]-amide;
[S-(R*,R*)]-Azepane-1-carboxylic acid
{1-[2-(4-benzyloxy-phenyl)-1-morpho-
lin-4-ylmethyl-ethylcarbamoyl]-3-methyl-butyl}-amide;
[S-(R*,R*)]Azepane-1-carboxylic acid
{1-[2-(4-benzyloxy-phenyl)-1-(tert-b-
utyl-amino-methyl)-ethylcarbamoyl]-3-methyl-butyl}-amide;
(S,S)-Azepane-1-carboxylic acid
{1-[2-(4-benzyloxy-phenyl)-1-diethylamino-
methyl-ethylcarbamoyl]-3-methyl-butyl}-amide;
(S)-2-(2-Amino-4-methyl-pent-
ylamino)-3-(4-benzyloxy-phenyl)-N-tert-butyl-propionamide; and
2-(2-Amino-4-methyl-pentylamino)-3-(4-benzyloxy-phenyl)-N-tert-butyl-prop-
ionamide.
4. A therapeutic composition comprising an effective calcium
channel blocking amount of the compound of claim 1 with a
pharmaceutically acceptable carrier.
5. A therapeutic composition comprising an effective amount of the
compound of claim 0 with a pharmaceutically acceptable carrier.
6. A method of blocking calcium channels, the method comprising
administering to a mammal in need of calcium channel blocking, a
therapeutically effective amount of a compound of claim 1 to block
calcium channels.
7. A method of blocking calcium channels, the method comprising
administering to a mammal in need of calcium channel blocking, a
therapeutically effective amount of a compound of claim 3 to block
calcium channels.
8. A method of treating stroke, the method comprising administering
to a mammal having a stroke a therapeutically effective amount of a
compound of claim 1.
9. A method of preventing a stroke, the method comprising
administering to a mammal at risk of having a stroke a
therapeutically effective amount of a compound of claim 1.
10. A method of treating stroke, the method comprising
administering to a mammal having a stroke a therapeutically
effective amount of a compound of claim 3.
11. A method of preventing a stroke, the method comprising
administering to a mammal at risk of having a stroke a
therapeutically effective amount of a compound of claim 3.
12. A method of treating cerebral ischemia, the method comprising
administering to a mammal having cerebral ischemia a
therapeutically effective amount of a compound of claim 1.
13. A method of treating cerebral ischemia, the method comprising
administering to a mammal having cerebral ischemia a
therapeutically effective amount of a compound of claim 3.
14. A method of treating head trauma, the method comprising
administering to a mammal having head trauma a therapeutically
effective amount of a compound of claim 1.
15. A method of treating head trauma, the method comprising
administering to a mammal having head trauma a therapeutically
effective amount of a compound of claim 3.
16. A method of treating epilepsy, the method comprising
administering to a mammal having epilepsy a therapeutically
effective amount of a compound of claim 1.
17. A method of treating epilepsy, the method comprising
administering to a mammal having epilepsy a therapeutically
effective amount of a compound of claim 3.
18. A method of treating pain, the method comprising administering
to a mammal having pain a therapeutically effective amount of a
compound of claim 1.
19. A method of treating pain, the method comprising administering
to a mammal having pain a therapeutically effective amount of a
compound of claim 3.
20. A method of inhibiting N-type calcium channels in a cell,
comprising contacting a cell having one or more N-type calcium
channels with a compound of claim 1.
21. A method of inhibiting N-type calcium channels in a cell,
comprising contacting a cell having one or more N-type calcium
channels with a compound of claim 3.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to novel compounds,
pharmaceutical compositions thereof and methods of using them in
the treatment of stroke, cerebral ischemia, pain, epilepsy, and
head trauma. In particular, the present invention relates to
reduced backbone dipeptide compounds that are potent antagonists of
N-type calcium channels.
SUMMARY OF THE RELATED ART
[0002] The entry of excessive amounts of calcium ions into neurons
following an ischemic episode or other neuronal trauma has been
well documented. Uncontrolled high concentrations of calcium in
neurons initiate a cascade of biochemical events that disrupts
normal cellular processes. Among these events are the activation of
proteases and lipases, breakdown of neuronal membranes, and the
formation of free radicals, which may ultimately lead to cell
death. Several types of calcium channels have been discovered: the
L, N, P, Q, R, and T types. Each type possesses distinct structural
features, functional properties, and cellular/subcellular
distributions. N-type calcium channels are tissue specific,
restricted to the central and peripheral neurons of the forebrain
and areas rich in synaptic connections. They have well defined
roles, e.g., regulation of calcium flux necessary for
depolarization-evoked release of transmitter from synaptic endings;
and they can be selectively blocked by high-affinity ligands, like
.omega.-conotoxins and synthetic analogs. Bowersox S. S., et al.,
Drug News and Perspective, 1994;7:261-268.
SUMMARY OF THE INVENTION
[0003] The present invention provides novel compounds and
compositions that are capable of blocking N-type calcium channels.
The compounds are useful in a method for treating afflictions
associated with increased calcium ion uptake through N-type calcium
channels, including such systemic effects as stroke, cerebral
ischemia resulting from cardiac arrest, head trauma, closed head
injury, pain, and epilepsy. Further advantages of this invention
will be clear to one skilled in the art from the reading of the
description that follows.
[0004] The present invention comprises new compounds and, more
particularly, novel reduced backbone dipeptide compounds that are
useful as N-type calcium channel blockers in mammals. The novel
compounds of the present invention are represented by the following
structural Formula I: 3
[0005] wherein A, B, X, R.sup.1, R.sup.2, R.sup.3, and R.sup.6 are
defined in more detail below and generally form a reduced dipeptide
backbone.
[0006] The invention also includes novel compositions of matter
containing the above-defined compound that are useful as
neuroprotective agents for the treatment of afflictions associated
with increased calcium ion uptake through N-type calcium channels
in mammals, as well as the methods of treatment using such
compositions.
[0007] The foregoing merely summarizes certain aspects of the
present invention and is not intended, nor should it be construed,
as limiting the invention in any manner. All patents and other
publications referenced herein are hereby incorporated by reference
in their entirety.
DETAILED DESCRIPTION OF THE INVENTION
[0008] The present invention comprises novel reduced backbone
dipeptide compounds and pharmaceutical compositions thereof that
are useful as N-type calcium channel antagonists.
[0009] The novel compounds of the present invention have the
following generic structural Formula I: 4
[0010] wherein
[0011] R.sup.1 is H or methyl,
[0012] R.sup.2 is H, azepanylcarbonyl, C.sub.1-C.sub.7 alkyl,
--(CH.sub.2).sub.n-phenyl, wherein the phenyl is unsubstituted or
substituted with C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 alkoxy, or
halo,
[0013] R.sup.3 is C.sub.1-C.sub.5 alkyl,
[0014] X is --NR.sup.4R.sup.5 or --OR.sup.7,
[0015] R.sup.4 and R.sup.5 are independently H, C.sub.1-C.sub.5
alkyl, or
[0016] R.sup.4 and R.sup.5 together with the nitrogen to which they
are both bound form: 5
[0017] R.sup.6 is --(CH.sub.2).sub.n-phenyl, wherein the phenyl is
unsubstituted or substituted with C.sub.1-C.sub.4 alkyl,
C.sub.1-C.sub.4 alkoxy, or halo,
[0018] A and B are independently --CO-- or --CH.sub.2--, provided
that A and B are not both --CO--,
[0019] R.sup.7 is C.sub.1-C.sub.5 alkyl,
[0020] Z is --CH.sub.2--, --O--, --S--, or --N(R.sup.8)--,
[0021] R.sup.8 is H or C.sub.1-C.sub.6 alkyl, and
[0022] n is 1 or 2.
[0023] In a preferred embodiment of the compound of Formula I,
[0024] R.sup.4 and R.sup.5 are independently H,
C.sub.1-C.sub.5-alkyl, or
[0025] R.sup.4 and R.sup.5 taken together with the nitrogen to
which they are both bound form 1-pyrrolidinyl, 1-piperidinyl, or
1-azepanyl, or
[0026] R.sup.4 and R.sup.5 taken together with the nitrogen to
which they are both bound are: 6
[0027] and
[0028] Z is --S--or --O--.
[0029] Also provided are pharmaceutically acceptable salts, esters,
amides, and pro-drugs of the compounds of the Formula I.
[0030] The term "alkyl" means a straight or branched chain
hydrocarbon. Representative examples of alkyl groups are methyl,
ethyl, propyl, isopropyl, isobutyl, butyl, tert-butyl, sec-butyl,
pentyl, hexyl, (CH.sub.3).sub.2CHCH.sub.2CH.sub.2--,
(CH.sub.3).sub.3CCH.sub.2CH.sub.2--- , and heptyl.
[0031] The term "alkoxy" means an alkyl group attached to an oxygen
atom. Representative examples of alkoxy groups include methoxy,
ethoxy, tert-butoxy, propoxy, and isobutoxy.
[0032] In a preferred embodiment, compounds of the invention are
selected from the group consisting of:
[0033]
2-((2-(Azaperhydroepinylcarbonylamino)-4-methylpentyl)amino)-N-(ter-
t-butyl)-3-(4-(phenylmethoxy)phenyl)-(2S)-propanamide;
[0034] [S-(R*,R*)]-Azepane-1-carboxylic acid
{1-[2-(4-benzyloxy-phenyl)-1--
tert-butoxymethyl-ethylcarbamoyl]-3-methyl-butyl}-amide;
[0035] [S-(R*,R*)]Azepane-1-carboxylic acid
(1-{[2-(4-benzyloxy-phenyl)-1--
tert-butylcarbamoyl-ethylamino]-methyl}-3-methyl-butyl)-amide;
[0036] [S-(R*,R*)]-2-Dimethylamino-4-methyl-pentanoic acid
[2-(4-benzyloxy-phenyl)-1-morpholin-4-ylmethyl-ethyl]-amide;
[0037] [S-(R*,R*)]-Azepane-1-carboxylic acid
{1-[2-(4-benzyloxy-phenyl)-1--
morpholin-4-ylmethyl-ethylcarbamoyl]-3-methyl-butyl}-amide;
[0038] [S-(R*,R*)]Azepane-1-carboxylic acid
{1-[2-(4-benzyloxy-phenyl)-1-(-
tert-butylamino-methyl)-ethylcarbamoyl]-3-methyl-butyl}-amide;
[0039] (S,S)-Azepane-1-carboxylic acid
{1-[2-(4-benzyloxy-phenyl)-1-diethy-
laminomethyl-ethylcarbamoyl]-3-methyl-butyl}-amide;
[0040]
(S)-2-(2-Amino-4-methyl-pentylamino)-3-(4-benzyloxy-phenyl)-N-tert--
butyl-propionamide dihydrochloride; and
[0041]
2-(2-Amino-4-methyl-pentylamino)-3-(4-benzyloxy-phenyl)-N-tert-buty-
l-propionamide dihydrochloride.
[0042] The compounds of the invention may be readily prepared as
set forth in the following reaction scheme(s) which employ general
synthetic methods well-known to those skilled in organic chemistry.
The following definitions apply:
1 H.sub.2SO.sub.4 Sulfuric acid HBTU
O-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium-
hexafluorophosphate DMAP 4-(Dimethylamino)pyridine DMF
N,N-Dimethylformamide Et.sub.2O Diethylether HCl Hydrochloric acid
NaBH.sub.3CN Sodium cyanoborohydride MeOH Methanol TFA
Trifluoroacetic acid DMC 4,4'-Dichloro-.alpha.-methylbenzhydrol
i-Pr.sub.2Net Diisopropyl ethylamine LAH Lithium aluminum hydride
HNMeOMe Methoxymethylamine Pd/C Palladium on carbon catalyst
[0043] 7
[0044] Wherein R.sup.a-R.sup.g are any chemical moiety consistent
with the definitions of R.sup.1-R.sup.7.
[0045] The compounds of the invention are also useful research
tools for studying the biological, cellular effects of blocking
N-type calcium channels.
[0046] The invention further comprises a pharmaceutical composition
for the treatment of illnesses induced by uncontrolled high
concentrations of calcium in neurons, which composition comprises a
compound of Formula I as defined above or, a pharmaceutically
acceptable salt, solvent or pro-drug thereof, in an amount
effective in the treatment of said illnesses, and a
pharmaceutically acceptable carrier. The invention also comprises a
pharmaceutical composition for the treatment of afflictions
associated with increased calcium ion uptake through N-type calcium
channels, including such systemic effects as stroke, pain, cerebral
ischemia, head trauma, and epilepsy, which comprises a compound of
Formula I as defined above in an amount effective in the treatment
of said disorders, and a pharmaceutically acceptable carrier.
Preferred compositions of the invention are those containing
preferred compounds of Formula I as described above. The compounds
will be administered to mammals at the rate of about 0.01 to about
200 mg/kg. Typical daily doses will be about 5 to about 500 mg per
patient.
[0047] The term "pro-drug" refers to compounds that are rapidly
transformed in vivo to yield the parent compound of the above
formulae, for example, by hydrolysis in blood. A thorough
discussion is provided in T. Higuchi and V. Stella, "Pro-drugs as
Novel Delivery Systems," Vol 14 of the A.C.S. Symposium Series, and
in Bioreversible Carriers in Drug Design, ed. Edward B. Roche,
American Pharmaceutical Association and Pergamon Press, 1987, both
of which are incorporated herein by reference.
[0048] The invention further relates to a method for the treatment
of illnesses induced by uncontrolled high concentrations of calcium
in neurons by administering to a subject in need of such treatment
a compound of Formula I as defined above in an amount effective in
such treatment, and a method for the treatment of stroke, pain,
cerebral ischemia, head trauma, and epilepsy utilizing a compound
of Formula I as defined above in an amount effective in such
treatment. Preferred methods of the invention are those
administering a preferred compound of the Formula I as described
above.
[0049] The compounds of the present invention may be administered
to a patient either alone or as part of a pharmaceutical
composition. The compositions may be administered to patients
either orally, rectally, parenterally (intravenously,
intramuscularly, or subcutaneously), intracistemally,
intravaginally, intraperitoneally, intravesically, locally
(powders, ointments, or drops), or as a buccal or nasal spray.
[0050] Compositions suitable for parenteral injection may comprise
physiologically acceptable sterile aqueous or non-aqueous
solutions, dispersions, suspensions or emulsions, and sterile
powders for reconstitution into sterile injectable solutions or
dispersions. Examples of suitable aqueous and non-aqueous carriers,
diluents, solvents or vehicles include water, ethanol, polyols
(propyleneglycol, polyethyleneglycol, glycerol, and the like),
suitable mixtures thereof, vegetable oils (such as olive oil), and
injectable organic esters such as ethyl oleate. 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.
[0051] These compositions may also contain adjuvants such as
preserving, wetting, emulsifying, and dispensing agents. Prevention
of the action of microorganisms can be ensured by various
antibacterial and antifungal agents, for example, parabens,
chlorobutanol, phenol, sorbic acid, and the like. It may also be
desirable to include isotonic agents, for example sugars, sodium
chloride, and the like. Prolonged absorption of the injectable
pharmaceutical form can be brought about by the use of agents
delaying absorption, for example, aluminum monostearate and
gelatin.
[0052] Solid dosage forms for oral administration include capsules,
tablets, pills, powders, and granules. In such solid dosage forms,
the active compound is admixed with at least one inert customary
excipient (or carrier) such as sodium citrate or dicalcium
phosphate or (a) fillers or extenders, as for example, starches,
lactose, sucrose, glucose, mannitol, and silicic acid, (b) binders,
as for example, carboxymethylcellulose, alignates, gelatin,
polyvinylpyrrolidone, sucrose, and acacia, (c) humectants, as for
example, glycerol, (d) disintegrating agents, as for example,
agar-agar, calcium carbonate, potato or tapioca starch, alginic
acid, certain complex silicates, and sodium carbonate, (e) solution
retarders, as for example, paraffin, (f) absorption accelerators,
as for example, quaternary ammonium compounds, (g) wetting agents,
as for example, cetyl alcohol, and glycerol monostearate, (h)
adsorbents, as for example, kaolin bentonite, and (i) lubricants,
as for example, talc, calcium stearate, magnesium stearate, solid
polyethylene glycols, sodium lauryl sulfate, or mixtures thereof.
In case of capsules, tablets, and pills, the dosage forms may also
comprise buffering agents.
[0053] Solid compositions of a similar type may also be employed as
fillers in soft and hard-filled gelatin capsules using such
excipients as lactose or milk sugar as well as high molecular
weight polyethyleneglycols, and the like.
[0054] Solid dosage forms such as tablets, dragees, capsules,
pills, and granules can be prepared with coatings and shells, such
as enteric coatings and others well known in the art. They may
contain opacifying agents, and can also be of such composition that
they release the active compound or compounds in a certain part of
the intestinal tract in a delayed manner. Examples of embedding
compositions that can be used are polymeric substances and waxes.
The active compounds can also be in micro-encapsulated form, if
appropriate, with one or more of the above-mentioned
excipients.
[0055] Liquid dosage forms for oral administration include
pharmaceutically acceptable emulsions, solutions, suspensions,
syrups, and elixirs. In addition to the active compounds, the
liquid dosage forms may contain inert diluents commonly used in the
art, such as water or other solvents, solubilizing agents and
emulsifiers, as for example, ethyl alcohol, isopropyl alcohol,
ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate,
propyleneglycol, 1,3-butyleneglycol, dimethylformamide, oils (in
particular, cottonseed oil, groundnut oil, corn germ oil, olive
oil, castor oil, and sesame oil), glycerol, tetrahydrofurfuryl
alcohol, polyethyleneglycols and fatty acid esters of sorbitan or
mixtures of these substances, and the like.
[0056] Besides such inert diluents, the composition can also
include adjuvants such as wetting agents, emulsifying and
suspending agents, sweetening agents, flavoring agents, and
perfuming agents.
[0057] Suspensions, in addition to the active compounds, may
contain suspending agents, as for example, ethoxylated isostearyl
alcohols, polyoxyethylene sorbitol and sorbitan esters,
microcrystalline cellulose, aluminum metahydroxide, bentonite,
agar-agar, and tragacanth, or mixtures of these substances, and the
like.
[0058] Compositions for rectal or intravaginal administrations are
preferably suppositories which can be prepared by mixing the
compounds of the present invention with suitable non-irritating
excipients or carriers such as cocoa butter, polyethyleneglycol, or
a suppository wax, which are solid at ordinary temperatures but
liquid at body temperature and therefore, melt in the rectum or
vaginal cavity and release the active compound.
[0059] Dosage forms for topical administration of a compound of
this invention include ointments, powders, sprays, and inhalants.
The active component is admixed under sterile conditions with
physiologically acceptable carrier and any preservatives, buffers,
or propellants as may be required. Ophthalmic formulations, eye
ointments, powders, and solutions are also contemplated as being
within the scope of this invention.
[0060] The term "pharmaceutically acceptable salts, esters, amides,
and pro-drugs" as used herein refers to those carboxylate salts,
amino acid addition salts, esters, amides, and pro-drugs of the
compounds of the present invention which are, within the scope of
sound medical judgment, suitable for use in contact with the
tissues of patients without undue toxicity, irritation, allergic
response, and the like, commensurate with a reasonable benefit/risk
ratio, and effective for their intended use, as well as the
zwitterionic forms, where possible, of the compounds of the
invention. The term "salts" refers to the relatively non-toxic,
inorganic and organic acid addition salts of compounds of the
present invention. These salts can be prepared in situ during the
final isolation and purification of the compounds or by separately
reacting the purified compound in its free base form with a
suitable organic or inorganic acid and isolating the salt thus
formed. Representative salts include the hydrobromide,
hydrochloride, sulfate, bisulfate, nitrate, acetate, oxalate,
valerate, oleate, palmitate, stearate, laurate, borate, benzoate,
lactate, phosphate, tosylate, citrate, maleate, fumarate,
succinate, tartrate, naphthylate mesylate, glucoheptonate,
lactiobionate and laurylsulphonate salts, and the like. These may
include cations based on the alkali and alkaline earth metals, such
as sodium, lithium, potassium, calcium, magnesium, and the like, as
well as nontoxic ammonium, quaternary ammonium, and amine cations,
including, but not limited to, ammonium, tetramethylammonium,
tetraethylammonium, methylamine, dimethylamine, trimethylamine,
ethylamine, triethylamine, and the like. See, for example, Berge S.
M., et al., "Pharmaceutical Salts," J. Pharm. Sci., 1977;66:1-19,
which is incorporated herein by reference.
[0061] Those skilled in the art are easily able to identify
patients having a stroke or at risk of having a stroke, cerebral
ischemia, head trauma, or epilepsy. For example, patients who are
at risk of having a stroke include, but are not limited to,
patients having hypertension or undergoing major surgery.
[0062] The invention will be described in greater detail in
conjunction with the following specific examples. These examples
are provided for illustrative purposes only and are not intended,
nor should they be construed, as limiting the invention in any
manner. Those skilled in the art will appreciate that modifications
and variations of the following examples can be made without
exceeding the spirit or scope of the present invention and
claims.
EXAMPLE 1
[S-(R*,R*)]-Azepane-1-carboxylic acid
{1-[2-(4-benzyloxy-phenyl)-1-tert-bu-
toxymethyl-ethylcarbamoyl]-3-methyl-butyl}-amide
[0063] 8
[0064] Step A
(S)-2-[(Azepane-1-carbonyl)-amino]-4-methyl-pentanoic acid benzyl
ester
[0065] 9
[0066] A solution of triphosgene (15.7 g, 52.9 mmol) in
CH.sub.2Cl.sub.2 (600 mL) was cooled to -10.degree. C. under a
nitrogen atmosphere. The solution was treated dropwise with a
solution of (S)-2-amino-4-methyl-pen- tanoic acid benzyl ester
(28.1 g, 0.127 mol) and pyridine (26 mL, 0.32 mol) in 150 mL of
CH.sub.2Cl.sub.2. The resulting solution was stirred at -10.degree.
C. for 90 minutes and then treated with a solution of
hexamethyleneimine (22 mL, 0.38 mmol) in 75 mL of CH.sub.2Cl.sub.2.
The resulting solution was stirred for 48 hours at room
temperature. The reaction mixture was concentrated, and the residue
was dissolved in ether and washed with 1N HCl solution, water, and
saturated aqueous CuSO.sub.4 solution. The organic layer was dried
(Mg.sub.2SO.sub.4), treated with activated charcoal, and filtered.
The filtrate was concentrated to approximately 1/2 volume and
treated with hexane. The resulting suspension was stored overnight
at -10.degree. C. The solid was collected by filtration, washed
with hexane, and dried under vacuum to give
(S)-2-[(azepane-1-carbonyl)-amino]-4-methyl-pentanoic acid benzyl
ester as a white solid (38.6 g, 88%); mp 87-88.degree. C.
[0067] APCI-MS m/z 347 (MH.sup.+).
[0068] Step B
(S)-2-[(Azepane-1-carbonyl)-amino]-4-methyl-pentanoic acid
[0069] 10
[0070] A solution of
(S)-2-[(azepane-1-carbonyl)-amino]-4-methyl-pentanoic acid benzyl
ester (38.5 g, 111 mmol) in 600 mL of THF was hydrogenated at 50
psi over 20% Pd/C (2.00 g) for 17 minutes. The reaction mixture was
filtered through celite and concentrated to dryness. The residue
was heated in 50 mL of hexane. The resulting suspension was cooled,
and the solid was collected by filtration and washed with hexane.
The solid was dried at room temperature under vacuum to give
(S)-2-[(azepane-1-carbonyl- )-amino]-4-methyl-pentanoic acid as a
white solid (26.6 g, 93%); mp 88-89.degree. C. Microanalysis for
C.sub.13H.sub.24N.sub.2O.sub.3:
[0071] Calculated: C, 60.91; H, 9.44; N, 10.93.
[0072] Found: C, 60.99; H, 9.46; N, 10.85.
[0073] Step C
(S)-1-(4-Benzyloxy-benzyl)-2-tert-butoxy-ethylamine
[0074] 11
[0075] A mixture of 2.573 g (10 mmol)
(S)-.beta.-amino-4-(phenylmethoxy)-b- enzenepropanol (Advanced
Chemtech, Louisville, Ky. 40218), 50 mL 2-methylpropene, 2 mL
concentrated sulfuric acid, and 50 mL 1,4-dioxane were shaken in a
glass pressure vessel at 25.degree. C. for 62 hours. The
milky-white suspension was poured into a rapidly stirred mixture
containing 3.6 g 85% KOH, 75 mL ice-water, and 50 mL diethyl ether.
After layers separated, the aqueous phase was extracted with two 50
mL portions of ethyl acetate. The combined extracts were washed
with 100 mL brine, dried over anhydrous sodium sulfate, and
concentrated in vacuo giving 288 mg of a pale amber oil which was
purified by column chromatography (SiO.sub.2) with 12:1
chloroform:methanol to give 131 mg (4%) of
(S)-1-(4-benzyloxy-benzyl)-2-tert-butoxy-ethylamine as a clear,
colorless oil, that was used directly in the next reaction.
[0076] Step D
[0077] A 3.degree. C. solution of 106 mg (0.41 mmol)
(S)-2-[(azepane-1-carbonyl)-amino]-4-methyl-pentanoic acid (Example
1, Step B) and 0.137 mL (0.41 mmol) 4-methylmorpholine in 2 mL
N,N-dimethylformamide was treated with 165 mg (0.44 mmol)
O-benzotriazol-1-yl-N,N,N',N'-bis(tetramethylene)uronium
hexafluorophosphate (Novabiochem, La Jolla, Calif.), and stirred
for 30 minutes, at which time a solution of
(S)-1-(4-benzyloxy-benzyl)-2-tert-bu- toxy-ethylamine in 3 mL
dimethylformamide was added and the mixture stirred at 3.degree. C.
for 40 minutes. The clear solution was poured into 15 mL diethyl
ether and washed successively with 15 mL of each of the following:
5% aqueous HCl, brine, saturated aqueous NaHCO.sub.3, brine (twice)
then dried over anhydrous magnesium sulfate. The solvent was
removed in vacuo affording 180 mg of white solid, which was
recrystallized from hexane to give 1 16 mg (51%) of the title
compound as a white solid; mp 98-99.degree. C. APCI-MS: m/z 552.0
[MH.sup.+].
EXAMPLE 2
[S-(R*,R*)]Azepane-1-carboxylic acid
(1-{[2-(4-benzyloxy-phenyl)-1-tert-bu-
tylcarbamoyl-ethylamino]-methyl}-3-methyl-butyl)-amide
dihydrochloride salt
[0078] 12
[0079] Step A
N-tert-Butoxycarbonyl-L-leucinal
[0080] 13
[0081] N-tert-Butoxycarbonyl-L-leucinal was synthesized according
to the procedure published in Goel, et al., Org. Syn.,
1988;67:69.
[0082] Step B
(S)-[2-[(1,1-Dimethylethyl)amino]-2-oxo-1-(phenylmethyl)ethyl]-carbamic
acid 1,1-dimethylethyl ester
[0083] 14
[0084] A solution of N-(tert-butyloxycarbonyl)-O-benzyl-L-tyrosine
(Bachem, 2.00 g, 5.38 mmol) in 20 mL of DMF was cooled to 0.degree.
C. and treated with iPr NEt (1.5 mL) followed by
O-benzotriazol-1a-yl-N,N,N'- ,N'-bis(tetramethylene)-uronium
hexafluoro phosphate (2.04 g, 5.38 mmol, Novabiochem, La Jolla,
Calif.). The resulting suspension was stirred for 30 minutes at
0.degree. C. and then treated with tert-butylamine (0.48 g, 6.56
mmol). The reaction mixture was stirred for 1 hour at 0.degree. C.
and warmed to room temperature. The reaction mixture was poured
into Et O and washed sequentially with saturated aqueous
NaHCO.sub.3 solution, and saturated aqueous NaCl solution. The
organic phase was dried (MgSO.sub.4), filtered, and concentrated.
The crude residue was purified by chromatography (silica gel, 3:1
heptane/ethyl acetate) to give
(S)-[2-[(1,1-dimethylethyl)amino]-2-oxo-1-(phenylmethyl)ethyl]-carbamic
acid 1,1-dimethylethyl ester (2.65 g). MS (CI) 427 (MH).
[0085] Step C
(S)-2-Amino-3-(4-benzyloxy-phenyl)-N-tert-butyl-propionamide
[0086] 15
[0087] A solution of
(S)-[2-(4-benzyloxy-phenyl)-1-tert-butylcarbamoyl-eth- yl]-carbamic
acid tert-butyl ester (6.0 g, 14.1 mmol, Example 2, Step B) in
CH.sub.2Cl.sub.2 (28 mL) was treated with trifluoroacetic acid (28
mL). The resulting solution was stirred for 20 minutes and then
concentrated. The residue was diluted with EtOAc (300 mL), washed
with saturated bicarbonate solution (2.times.300 mL) and brine (300
mL), dried over Na.sub.2SO.sub.4, and concentrated to give 4.2 g
(91%) of
(S)-2-amino-3-(4-benzyloxy-phenyl)-N-tert-butyl-propionamide. MS:
328 (M.sup.+1 for C.sub.20H.sub.26N.sub.2O.sub.3). TLC: Silica gel,
R 0.43 (10% MeOH/CHCl).
[0088] Step D
[S-(R*,S*)]-(1-{[2-(4-Benzyloxy-phenyl)-1-tert-butylcarbamoyl-ethylamino]--
methyl}-3-methyl-butyl)-carbamic acid tert-butyl ester
[0089] 16
[0090] A solution consisting of 1.00 g (4.64 mmol)
N-tert-butoxycarbonyl-L- -leucinal and 1.52 g (4.64 mmol)
2-amino-3-(4-benzyloxy-phenyl)-N-tert-but- yl-propionamide (Example
2, Step C) in 20 mL methanol containing 1% acetic acid was stirred
at 25.degree. C. for 20 minutes, at which time a solution of 0.614
g (9.29 mmol) sodium cyanotrihydridoborate (Aldrich, Milwaukee,
Wis.) in 12 mL methanol was added and the resulting mixture stirred
for 24 hours. The reaction was quenched by the addition of 3 M HCl
in 3 mL increments until the vigorous bubbling subsided. The
mixture was neutralized with saturated NaHCO.sub.3 solution then
extracted with three 50 mL portions of ethyl acetate. The combined
extracts were washed with 100 mL brine, dried over anhydrous sodium
sulphate, and evaporated at reduced pressure to give a clear,
colorless oil which was chromatographed on a silica gel column
using 30% to 40% ethyl acetate:hexane to give 735 mg (30%) of
[S-(R*,S*)]-(1-{[2-(4-benzyloxy-ph-
enyl)-1-tert-butylcarbamoyl-ethylamino]-methyl}-3-methyl-butyl)-carbamic
acid tert-butyl ester as a clear foam. APCI-MS: m/z 526.3
[MH.sup.+]. Microanalysis for C.sub.31H.sub.47N.sub.3O.sub.4.0.25
H.sub.2O:
[0091] Calculated: C, 70.22; H, 9.03; N, 7.92.
[0092] Found: C, 70.27; H, 9.09; N, 7.78.
[0093] Step E
(S,S)-2-(2-Amino-4-methyl-pentylamino)-3-(4-benzyloxy-phenyl)-N-tert-butyl-
-propionamide
[0094] 17
[0095] A solution of 850 mg (1.6 mmol)
(1-{[2-(4-benzyloxy-phenyl)-1-tert--
butylcarbamoyl-ethylamino]-methyl}-3-methyl-butyl)-carbamic acid
tert-butyl ester in 6 mL dichloromethane was treated with 2 mL
2,2,2-triflouroacctic acid and the resulting solution stirred 1
hour at 25.degree. C. The mixture was concentrated at reduced
pressure to a viscous amber oil to which was dissolved in 8 mL
diethyl ether, and treated with 3 mL of diethyl ether which had
been saturated with hydrogen chloride gas. The solvent was removed
under reduced pressure, and the pale yellow oil thus obtained
((S,S)-2-(2-amino-4-methyl-pentylamino)-3-(-
4-benzyloxy-phenyl)-N-tert-butyl-propionamide) was used directly in
the next reaction.
[0096] Step F
Azepane-1-carbonyl chloride
[0097] 18
[0098] Azepane-1-carbonyl chloride was made according to the
procedure reported in Tetrahedron Lett., 1994;35:839.
[0099] Step G
[0100] A solution of 746 mg (1.6 mmol)
2-(2-amino-4-methyl-pentylamino)-3--
(4-benzyloxy-phenyl)-N-tert-butyl-propionamide, 0.408 mL (3.1 mmol)
hexahydro-azepinyl carbamoyl chloride, 20 mg (0.16 mmol)
4-dimethylaminopyridine (Aldrich, Milwaukee, Wis.), 0.818 mL (4.7
mmol) N,N-diisopropylethylamine (Aldrich, Milwaukee, Wis.), in 10
mL of benzene was heated under reflux for 16 hours, cooled to
25.degree. C. and filtered, and the solids washed with benzene. The
filtrate was concentrated under reduced pressure, and the residue
thus obtained was dissolved in 100 mL ethyl acetate, and washed
with 100 mL each saturated NaHCO.sub.3 solution and brine, dried
over anhydrous magnesium sulfate, then concentrated under reduced
pressure to give a pale amber oil that was purified by column
chromatography on silica gel with 1:1 ethyl acetate:chloroform as
eluant. The clear, colorless oil obtained was dissolved in 10 mL
diethyl ether, and treated with diethyl ether that had been
saturated with hydrogen chloride gas. The precipitate that formed
was collected by filtration, and the solids washed with ethyl
acetate and dried. The title compound was obtained as 440 mg
off-white solid (77%); mp 51-57.degree. C. APCI-MS: m/z 551.4
[MH.sup.+].
EXAMPLE 3
[S-(R*,R*)]-2-Dimethylamino-4-methyl-pentanoic acid
[2-(4-benzyloxy-phenyl)-1-morpholin-4-ylmethyl-ethyl]-amide
[0101] 19
[0102] Step A
(S)-2-Dimethylamino-4-methyl-pentanoic acid
[0103] 20
[0104] (S)-2-Dimethylamino-4-methyl-pentanoic acid was synthesized
according to the procedure described in J. Chem. Soc.,
1950:1342-1345.
[0105] Step B
N-tert-Butoxycarbonyl-O-benzyl-L-tyrosinal
[0106] 21
[0107] N-tert-Butoxycarbonyl-O-benzyl-L-tyrosinal was synthesized
according to the procedure published in Thompson, et al.,
Tetrahedron Lett., 1990;31:6819-6822.
[0108] Step C
[2-(4-Benzyloxy-phenyl)-1-morpholin-4-ylmethyl-ethyl]-carbamic acid
tert-butyl ester
[0109] 22
[0110] A solution consisting of 2.86 g (8 mmol)
N-tert-butoxycarbonyl-O-be- nzyl-L-tyrosinal and 0.701 mL (8 mmol)
morpholine (Aldrich, Milwaukee, Wis.) in 36 mL methanol containing
1% acetic acid was stirred at 25.degree. C. for 20 minutes, at
which time a solution of 1.06 g (16 mmol) sodium
cyanotrihydridoborate (Aldrich, Milwaukee, Wis.) in 12 mL methanol
was added and the resulting mixture stirred for 24 hours. The
reaction was quenched by the addition of 3 M HCl in 3 mL increments
until the vigorous bubbling subsided. The mixture was neutralized
with saturated NaHCO.sub.3 solution then extracted with three 100
mL portions of ethyl acetate. The combined extracts were washed
with 100 mL brine, dried over anhydrous sodium sulphate, and
evaporated at reduced pressure to give a clear, colorless oil which
was chromatographed on a silica gel column using ethyl
acetate:hexane, 1:1 as eluant. The A white, waxy solid was
recrystallized from 2,2,4-trimethylpentane to give 1.53 g (45%) of
[2-(4-benzyloxy-phenyl)-1-morpholin-4-ylmethyl-ethyl]-carbamic acid
tert-butyl ester as soft white crystals; mp 98-99.degree. C.
APCI-MS m/z 427.2 [MH.sup.+]. Microanalysis for
C.sub.34H.sub.47N.sub.3O.0.25H.sub.2O- :
[0111] Calculated: C, 78.80; H, 9.24; N, 8.11.
[0112] Found: C, 78.64; H, 9.56; N, 7.95.
[0113] Step D
(S)-2-(4-Benzyloxy-phenyl)-1-morpholin-4-ylmethyl-ethylamine
[0114] 23
[0115] A solution of 1.41 g (3.3 mmol)
[2-(4-benzyloxy-phenyl)-1-morpholin- -4-ylmethyl-ethyl]-carbamic
acid tert-butyl ester in 5 mL dichloromethane was treated with 1.5
mL 2,2,2-triflouroacetic acid and the resulting solution stirred 16
hours at 25.degree. C. The mixture was concentrated at reduced
pressure to a viscous amber oil to which was added 50 mL saturated
aqueous sodium bicarbonate solution and the resulting mixture
extracted with three 50 mL portions of ethyl acetate. The combined
extracts were washed with 100 mL brine, dried over anhydrous sodium
sulfate and concentrated at reduced pressure affording a soft
solid, that was recrystallized from 2,2,4-trimethylpentane
containing about 5% ethyl acetate to afford 804 mg (77%) of
(S)-2-(4-benzyloxy-phenyl)-1-morpholin-- 4-ylmethyl-ethylamine as
light tan crystals; mp 180-182.degree. C. MS: m/z 327.2
[MH.sup.+].
[0116] Step E
[0117] (S)-2-Dimethylamino-4-methyl-pentanoic acid (55.3 mg, 0.347
mmol) was dissolved in dry DMF (3 mL) under nitrogen atmosphere and
cooled to 0.degree. C. in an ice-water bath. To this solution were
added in succession N,N-diisopropylethylamine (0.180 mL, 1.04 mmol)
and solid O-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium
hexafluorophosphate (132 mg, 0.347 mmol, Novabiochem, La Jolla,
Calif.). The resulting reaction mixture was stirred at that
temperature for 30 minutes,
(S)-2-(4-benzyloxy-phenyl)-1-morpholin-4-ylmethyl-ethylamine (153
g, 0.347 mmol, Example 3, Step D) was then added. After an
additional 60 minutes stirring at 0.degree. C., the reaction
mixture was mixed with 60 mL of diethyl ether, and the resulting
mixture was successively washed with saturated aqueous NaHCO.sub.3
solution and brine and then dried over Na.sub.2SO.sub.4. The
solution was concentrated in vacuo, an oil was obtained. The crude
product was purified by chromatography (silica gel, 10% methanol in
chloroform) to give the title compound as a white solid (90 mg,
55%); mp 113-114.degree. C. APCI-MS: m/z 468.4 (MH.sup.+).
EXAMPLE 4
[S-(R*,R*)]-Azepane-1-carboxylic acid
{1-[2-(4-benzyloxy-phenyl)-1-morphol-
in-4-ylmethyl-ethylcarbamoyl]-3-methyl-butyl}-amide
[0118] 24
[0119] A solution of 87 mg (0.34 mmol) of
(S)-2-[(azepane-1-carbonyl)-amin- o]-4-methyl-pentanoic acid
(Example 1, Step B) and 0.131 mL (1.2 mmol) 4-methylmorpholine was
stirred at 25.degree. C. for 30 minutes, after which 136 mg (0.36
mmol) O-benzotriazol-1-yl-N,N,N',N'-bis(tetramethylene- )uronium
hexafluorophosphate in 4 mL dry DMF was added. The resulting
solution was cooled in an ice-bath and 150 mg (0.34 mmol)
2-(4-benzyloxy-phenyl)-1-morpholin-4-ylmethyl-ethylamine (Example
3, Step D) was added. The resulting solution was warmed to
25.degree. C. and stirred for 30 minutes. The mixture was then
poured into 35 mL diethyl ether, and washed sequentially with 25 mL
each of 2.5% aqueous HCl solution, brine, saturated aqueous sodium
bicarbonate solution, and finally twice with brine. The mixture was
then dried over anhydrous sodium sulfate and concentrated at
reduced pressure to give an amber oil that was purified by
preparative thin-layer chromatography on a 1000 .mu.m silica gel
plate with 50:50:1 ethyl acetate:hexane:methanol as eluant to give
46 mg (24%) of the title compound as a pale amber glass. APCI-MS:
m/z 565.5 [MH.sup.+]. Microanalysis for C.sub.33H.sub.48N.sub.4O-
.sub.4.0.25EtOAc:
[0120] Calculated: C, 69.59; H, 8.59; N, 9.55.
[0121] Found: C, 69.70; H, 8.67; N, 9.39.
EXAMPLE 5
[S-(R*,R*)]Azepane-1-carboxylic acid
{1-[2-(4-benzyloxy-phenyl)-1-(tert-bu-
tylamino-methyl)-ethylcarbamoly]-3-methyl-butyl}-amide
trifloroacetate salt (1:1)
[0122] 25
[0123] Step A
(S)-[2-(4-Benzyloxy-phenyl)-1-(tert-butylamino-methyl)-ethyl]-carbamic
acid tert-butyl ester
[0124] 26
[0125] A solution consisting of 2.86 g (8 mmol)
N-tert-butoxycarbonyl-O-be- nzyl-L-tyrosinal (Example 4, Step A)
and 0.846 mL (8 mmol) tert-butyl amine (Aldrich, Milwaukee, Wis.)
in 36 mL methanol containing 1% acetic acid was stirred at
25.degree. C. for 20 minutes, at which time a solution of 1.06 g
(16 mmol) sodium cyanotrihydridoborate (Aldrich, Milwaukee, Wis.)
in 12 mL methanol was added and the resulting mixture stirred for
48 hours. The reaction was quenched by the addition of 3 M HCl in 3
mL increments until the vigorous bubbling subsided. The mixture was
neutralized with saturated NaHCO.sub.3 solution then extracted with
three 100 mL portions of ethyl acetate. The combined extracts were
washed with 100 mL brine, dried over anhydrous sodium sulphate and
evaporated at reduced pressure to give a pale amber oil which was
chromatographed on a silica gel column using 1% to 2% methanol in
ethyl acetate as eluant to give 604 mg (18%) of the title compound
as waxy white crystals; mp 77-79.degree. C. APCI-MS m/z 413.2
[MH.sup.+].
[0126] Step B
(S)-3-(4-Benzyloxy-phenyl)-N.sup.1-tert-butyl-propane-1,2-diamine
[0127] 27
[0128] A solution of 1.41 g (3.3 mmol)
[2-(4-benzyloxy-phenyl)-1-morpholin- -4-ylmethyl-ethyl]-carbamic
acid tert-butyl ester in 5 mL dichloromethane was treated with 1.5
mL 2,2,2-triflouroacetic acid and the resulting solution stirred 16
hours at 25.degree. C. The mixture was concentrated at reduced
pressure to a viscous amber oil to which was added 50 mL saturated
aqueous sodium bicarbonate solution and the resulting mixture
extracted with three 50 mL portions of ethyl acetate. The combined
extracts were washed with 100 mL brine, dried over anhydrous sodium
sulfate and concentrated at reduced pressure affording a soft
solid, that was recrystallized from 2,2,4-trimethylpentane
containing about 5% ethyl acetate to afford 804 mg (77%) of the
title compound as light tan crystals; mp 180-182.degree. C. APCI-MS
m/z 382.2 [MH.sup.+].
[0129] Step C
[0130] A solution of 200 mg (0.79 mmol) of
(S)-2-[(azepane-1-carbonyl)-ami- no]-4-methyl-pentanoic acid
(Example 1, Step B) and 0.260 mL (2.4 mmol) 4-methylmorpholine in 4
mL DMF was stirred at 25.degree. C. for 0.5 hour, and 298 mg (0.79
mmol) O-benzotriazol-1-yl-N,N,N',N'-bis(tetramethylene)u- ronium
hexafluorophosphate was added, and the resulting solution cooled in
an ice-bath and stirred 30 minutes, and 236 mg (0.79 mmol) of
[2-(4-benzyloxy-phenyl)-1-(tert-butylamino-methyl)-ethylamine was
added and the resulting solution warmed to 25.degree. C. and
stirred 30 minutes. The mixture was poured into 50 mL diethyl
ether, and washed with 55 mL saturated aqueous sodium bicarbonate
solution, and finally twice with 75 mL brine, and dried over
anhydrous sodium sulfate and concentrated at reduced pressure to
give a pale amber oil that was purified by column chromatography on
silica gel with 3% methanol in chloroform as eluant to give 117 mg
(27%) of the title compound as a white foam. APCI-MS m/z 565.5
[MH.sup.+]. Microanalysis for
C.sub.33H.sub.50N.sub.4O.sub.3.TFA.0.5 H.sub.2O:
[0131] Calculated: C, 62.39; H, 7.78; N, 8.31; H.sub.2O, 1.34.
[0132] Found: C, 62.45; H, 8.17; N, 8.58; H.sub.2O, 1.57.
EXAMPLE 6
(S,S)-Azepane-1-carboxylic acid
{1-[2-(4-benzyloxy-phenyl)-1-diethylaminom-
ethyl-ethylcarbamoyl]-3-methyl-butyl}-amide
[0133] 28
[0134] Step A
(S)-[1(4-Benzyloxy-benzyl)-2-diethylamino-ethyl]-carbamic acid
tert-butyl ester
[0135] 29
[0136] A solution consisting of 2.86 g (8 mmol)
N-tert-butoxycarbonyl-O-be- nzyl-L-tyrosinal (Example 3, Step B)
and 0.832 mL (8 mmol) diethyl amine in 36 mL methanol containing 1%
acetic acid was stirred at 25.degree. C. for 20 minutes, at which
time a solution of 1.06 g (16 mmol) sodium cyanotrihydridoborate
(Aldrich, Milwaukee, Wis.) in 12 mL methanol was added and the
resulting mixture stirred for 48 hours. The reaction was quenched
by the addition of 3 M HCI in 3 mL increments until the vigorous
bubbling subsided. The mixture was neutralized with saturated
NaHCO.sub.3 solution then extracted with three 100 mL portions of
ethyl acetate. The combined extracts were washed with 100 mL brine,
dried over anhydrous sodium sulphate and evaporated at reduced
pressure to give an amber oil which was chromatographed on a silica
gel column using 50:50:1 ethyl acetate:hexane:methanol as eluant to
give 236 mg (7%) of
(S)-[1-(4-benzyloxy-benzyl)-2-diethylamino-ethyl]-carbamic acid
tert-butyl ester as a clear, colorless oil. APCI-MS: m/z 413.2
[MH.sup.+].
[0137] .sup.1H NMR (CDCl.sub.3): 7.40-7.28 (5H, m), 7.08 (2H, d,
J=8.5 Hz), 6.86 (2H, d, J=8.5 Hz), 5.00 (2H, s), 3.77 (1H, bs),
2.77-2.37 (7H, m), 1.38 (9H, s), 0.96 (6H, bs).
[0138] Step B
(S)-3-(4-Benzyloxy-phenyl)-N.sup.1,N.sup.1-diethyl-propane-1,2-diamine
[0139] 30
[0140] A solution of 171 mg (0.4 mmol)
[2-(4-benzyloxy-phenyl)-1-(diethyla- mino-methyl)-ethyl]-carbamic
acid tert-butyl ester in 2 mL dichloromethane was treated with 0.5
mL 2,2,2-triflouroacetic acid and the resulting solution stirred 3
hours at 25.degree. C. The mixture was concentrated at reduced
pressure to a viscous amber oil to which was added 30 mL saturated
aqueous sodium bicarbonate solution and the resulting mixture
extracted with three 20 mL portions of ethyl acetate. The combined
extracts were washed with two 50 mL portions of brine, dried over
anhydrous sodium sulfate, and concentrated at reduced pressure and
the residue purified by column chromatography using ethyl acetate
containing 1% methanol as eluent. There was obtained 112 mg (90%)
of
(S)-3-(4-benzyloxy-phenyl)-N.sup.1,N.sup.1-diethyl-propane-1,2-diamine
as a clear, colorless oil. APCI-MS: m/z 313.2 [MH.sup.+].
[0141] 1H NMR (CDCl.sub.3): 7.37-7.25 (5H, m), 7.07 (2H, d, J=8.06
Hz), 6.85 (2H, d, J=7.8 Hz), 4.98 (2H, s), 3.05 (1H, bs), 2.67-2.24
(6H, m), 0.96 (6H, t).
[0142] Step C
[0143] A solution of 53 mg (0.20 mmol) of
(S)-2-[(azepane-1-carbonyl)-amin- o]-4-methyl-pentanoic acid
(Example 1, Step B) and 0.079 mL (7.2 mmol) 4-methylmorpholine in 4
mL DMF was stirred at 25.degree. C. for 0.5 hour, and 82 mg (0.21
mmol) 0-benzotriazol-1-yl-N,N,N',N'-bis(tetramethylene)ur- onium
hexafluorophosphate was added. The resulting solution was cooled in
an ice-bath and stirred 30 minutes, after which 64 mg (0.20 mmol)
of
(S)-3-(4-benzyloxy-phenyl)-N.sup.1,N.sup.1-diethyl-propane-1,2-diamine
in 2 mL DMF was added. The resulting solution was warmed to
25.degree. C. and stirred for 30 minutes. The mixture was then
poured into 30 mL diethyl ether, and washed with 25 mL each of 5%
HCl solution, brine, saturated aqueous sodium bicarbonate solution,
and finally twice with brine. The mixture was dried over anhydrous
sodium sulfate and concentrated at reduced pressure to give a
clear, colorless oil that was purified by preparative thin-layer
chromatography on a 500 gm silica gel plate using 50:50:1 ethyl
acetate:hexane:methanol as eluant to give 21.2 mg (19%) of the
title compound as a clear, colorless oil. APCI-MS: m/z 551.5
[MH.sup.+]. Microanalysis for C.sub.33H.sub.48N.sub.4O.sub.3.0.5
H.sub.2O:
[0144] Calculated: C, 70.81; H, 9.18; N, 10.01; H.sub.2O, 1.61.
[0145] Found: C, 70.64; H, 8.97; N, 9.73; H.sub.2O, 1.29.
EXAMPLE 7
(S)-2-(2-Amino-4-methyl-pentylamino)-3-(4-benzyloxy-phenyl)-N-tert-butyl-p-
ropionamide dihydrochloride
[0146] 31
[0147] Step A
(S)-2-Amino-3-(4-benzyloxy-phenyl)-N-tert-butyl-propionamide
monohydrochloride
[0148] 32
[0149] To a solution of
(S)-2-amino-3-(4-benzyloxy-phenyl)-N-tert-butyl-pr- opionamide
(Example 2, Step C) in ether was added excess amount of ethereal
HCl, solid formed was isolated via filtration, subsequent drying
under vacuum gave title compound as a yellow solid.
[0150] Step B
1-{[2-(4-Benzyloxy-phenyl)-1-tert-butylcarbamoyl-ethylamino]-methyl
}-3-methyl-butyl)-carbamic acid tert-butyl ester
[0151] (S)-2-Amino-3
-(4-benzyloxy-phenyl)-N-tert-butyl-propionamide monohydrochloride
(1.68 g, 4.65 mmol, Example 7, Step A) and BOC-Leu-CHO
((tert-butoxy)-N-[1-(2-methylpropyl)-2-oxoethyl]carboxamide) (1.00
g, 4.65 mmol, Peninsula Laboratories, Belmont, Calif.) were mixed
in CH.sub.2Cl.sub.2 (25 mL). After stirring at ambient temperature
under nitrogen atmosphere for 30 minutes, the solution was cooled
to 0.degree. C. in an ice-water bath. To this solution was added
sodium triacetoxyborohydride (1.50 g, 6.98 mmol). The resulting
reaction mixture was stirred for, in succession, 30 minutes at
0.degree. C. and 12 hours at ambient temperature. Twenty-five
milliliters of saturated aqueous NaHCO.sub.3 solution was added to
the reaction mixture, and the resulting mixture was stirred for 5
minutes. The two layers were separated, and the aqueous layer was
extracted with CH.sub.2Cl.sub.2 (2.times.30 mL). The combined
organic solution was dried over Na.sub.2SO.sub.4. The solution was
concentrated in vacuo affording a viscous oil. The reaction gave
two diastereomers which were purified twice by column
chromatography (eluant used in first column chromatography: 30%
EtOAc-4% MeOH-66% hexanes; eluant used in second column
chromatography: 100% acetonitrile). When the solvent system, 30%
EtOAc-66% benzene-4% MeOH, was used as eluant. The two
diastereomers gave different R.sub.f values on TLC (0.68 and 0.53).
The compound with a R.sub.f value of 0.68 was isolated by
preparative plate as a yellow oil (0.3 g) and used in used
step.
[0152] Step C
[0153] To a solution of
(1-{[2-(4-benzyloxy-phenyl)-1-tert-butylcarbamoyl--
ethylamino]-methyl}-3-methyl-butyl)-carbamic acid tert-butyl ester
(0.4 g, 0.76 mmol, Example 7, Step B) in CH.sub.2Cl.sub.2 (7.5 mL)
was added trifluoroacetic acid (2.5 mL) at ambient temperature
under nitrogen atmosphere. The resulting reaction mixture was
stirred for 25 minutes, then concentrated in vacuo. The viscous oil
obtained was dissolved in 40 mL of CH.sub.2Cl.sub.2 and
successively washed with saturated aqueous NaHCO.sub.3 solution
(2.times.40 mL), brine (2.times.40 mL), and was dried over
Na.sub.2SO.sub.4. The CH.sub.2Cl.sub.2 solution of free amine was
concentrated in vacuo to afford the crude product as a viscous oil
which was then dissolved in 10 mL of ethyl ether. To this solution
was added saturated HCl solution in ethyl ether until no more white
precipitate formed. Filtration and drying overnight under vacuum
afforded 0.23 g (61 %) of titled compound as a yellow solid; mp
228-230.degree. C. APCI-MS m/z 426.1 (MH.sup.+).
EXAMPLE 8
2-(2-Amino-4-methyl-pentylamino)-3-(4-benzyloxy-phenyl)-N-tert-butyl-propi-
onamide dihydrochloride
[0154] 33
[0155] Step A
(1-{[2-(4-Benzyloxy-phenyl)-1-tert-butylcarbamoyl-ethylamino]-methyl}-3-me-
thyl-butyl)-carbamic acid tert-butyl ester
[0156] 34
[0157] (S)-2-Amino-3-(4-benzyloxy-phenyl)-N-tert-butyl-propionamide
monohydrochloride (1.68 g, 4.65 mmol, Example 7, Step A) and
BOC-Leu-CHO
((tert-butoxy)-N-[1-(2-methylpropyl)-2-oxoethyl]carboxamide) (1.00
g, 4.65 mmol, Peninsula Laboratories, Belmont, Calif.) were mixed
in CH.sub.2Cl.sub.2 (25 mL). After stirring at ambient temperature
under nitrogen atmosphere for 30 minutes, the solution was cooled
to 0.degree. C. in an ice-water bath. To this solution was added
sodium triacetoxyborohydride (1.50 g, 6.98 mmol). The resulting
reaction mixture was stirred for, in succession, 30 minutes at
0.degree. C. and 12 hours at ambient temperature. Twenty-five
milliliters of saturated aqueous NaHCO.sub.3 solution was added to
the reaction mixture, and the resulting mixture was stirred for 5
minutes. The 2 layers were separated, and the aqueous layer was
extracted with CH.sub.2Cl.sub.2 (2.times.30 mL). The combined
organic solution was dried over Na.sub.2SO.sub.4. The solution was
concentrated in vacuo affording a viscous oil. The reaction gave
two diastereomers which were purified twice by column
chromatography (eluant used in first column chromatography: 30%
EtOAc-4% MeOH-66% hexanes; eluant used in second column
chromatography: 100% acetonitrile). When the solvent system, 30%
EtOAC-66% benzene-4% MeOH, was used as eluant, The two
diastereomers gave different R.sub.f values on TLC (0.68 and 0.53).
The compound with a R.sub.f value of 0.53 was isolated by
preparative plate as a yellow oil (0.27 g) and used in used
step.
[0158] Step B
[0159] To a solution of
(1-{[2-(4-benzyloxy-phenyl)-1-tert-butylcarbamoyl--
ethylamino]-methyl}-3-methyl-butyl)-carbamic acid tert-butyl ester
(0.4 g, 0.76 mmol, Example 8, Step A) in CH.sub.2Cl.sub.2 (7.5 mL)
was added trifluoroacetic acid (2.5 mL) at ambient temperature
under nitrogen atmosphere. The resulting reaction mixture was
stirred for 25 minutes, then concentrated in vacuo. The viscous oil
obtained was dissolved in 40 mL of CH.sub.2Cl.sub.2 and
successively washed with saturated aqueous NaHCO.sub.3 solution
(2.times.40 mL), brine (2.times.40 mL), and was dried over
Na.sub.2SO.sub.4. The CH.sub.2Cl.sub.2 solution of free amine was
concentrated in vacuo to afford the crude product as a viscous oil
which was then dissolved in 10 mL of ethyl ether. To this solution
was added saturated HCl solution in ethyl ether until no more white
precipitate formed. Filtration and drying overnight under vacuum
afforded 0.15 g (59%) of titled compound as a yellow solid; mp
228-230.degree. C. APCI-MS m/z 426.1 (MH.sup.+).
EXAMPLE 9
Biological Assays
[0160] Measurement of N-type Ca.sup.2+Channel Blocking Potencies of
Compounds in IMR32 Cells Using the Fluorescent Ca.sup.2+Indicator
Indo-1
[0161] IMR-32 cells are a human tumoral cell line of neural origin.
The IMR-32 cell line has been shown to contain both N- and L-type
voltage sensitive calcium channels. Calcium flux into these cells
may be induced by stimulation with elevated potassium
concentrations. The L-channel component of calcium flux may be
blocked by adding 5 .mu.M nitrendipine. The remaining component of
calcium entry into the IMR-32 cells is due to calcium flux through
N-type calcium channels. Intracellular calcium concentrations are
measured using the fluorescent calcium indicator Indo-1. The effect
of drug concentration on calcium uptake is studied.
[0162] The IMR-32 cell line was obtained from the American Type
Culture Collection (Rockville, Md.). Cells were grown in Eagle's
Minimum Essential Medium with Earle's salts supplemented with 10%
fetal bovine serum, 2 mM L-Gln and antibiotic/antimicotic mixture
(Gibco). At approximately 80% confluency, differentiation was
induced by the addition of 1 mM dibutyryl cAMP and 2.5 .mu.M
bromodeoxyuridine to the medium. After 7 to 13 days of
differentiation, cells were detached using 0.5 mM EDTA and loaded
with 5 .mu.M Indo-1 acetoxymethyl ester (Molecular Probes, Eugene,
Oreg.) at 300.degree. C. for 45 minutes. Loaded cells were washed
twice, resuspended (-10.sup.7 cells/mL) in assay buffer (10 mM
HEPES/Tris pH 7.4 in Hank's Balanced Salt Solution without
bicarbonate or phenol red containing 0.5% bovine serum albumin) and
kept on ice until use. Fluorescence measurements were carried out
in a Photon Technology International (PTI, South Brunswick, N.J.)
Model RF-F3004 spectrofluorometer with dual emission monochromators
using excitation at 350 nm and emission at 400 and 490 nm. The
instrument was equipped with a thermostated cuvette holder with
stirring capabilities as well as with a computer-controlled pump
that allowed for reagent addition during measurement. Instrument
control and data collection was done by PTI's OSCAR software
running on an IBM compatible computer. Different concentrations of
the test compounds (60 .mu.L in dimethyl sulfoxide) were added to
5.94 mL of assay buffer containing approximately 3.times.10.sup.6
loaded cells, and 5 .mu.M Nitrendipine (in 30 .mu.L EtOH) to block
L-type Ca.sup.2+channels. Samples were incubated for 10 minutes at
30.degree. C. and then aliquoted into three 10.times.10 mm
disposable acrylic cuvettes. Emission signals at 400 and 409 nm
were acquired from each cuvette at 30.degree. C. for 50 seconds. At
20 seconds after the start of reading, cells were depolarized by
the addition of 160 .mu.L of stimulation solution (1M KCl, 68 mM
CaCl.sub.2) to the cuvette via the computer-controlled pump. Ratio
of dual emission signals (400 nm/409 nm), which is proportional to
intracellular Ca.sup.2+concentration, was plotted against time, and
the difference between maximal response after stimulation and basal
value (before stimulation) was determined. Values obtained in this
way were plotted as a function of drug concentration. IC.sub.50
values of test compounds were calculated by fitting a 4-parameter
logistic function to the data using the least squares method.
[0163] The following as the results for the IMR32 and FLP150
assays:
2 TABLE 1 Compound of IMR32 Example No. IC.sub.50 (.mu.M) 1 0.21 2
0.19 3 48% @ 10 .mu.M 6% @ 1 .mu.M 4 1.2 5 1.1 6 1.1 7 1.2 8 1
[0164] Audiogenic Seizure Model in DBA/2 Mice:
[0165] Compounds of the present invention were dissolved in water
using 10% (weight/volume)Emulphor (GAF Corp., Wayne, N.J.)
surfactant. Substances were admimistered by intravenous injection
into the retro-orbital venous sinus. All testing was performed 15
minutes or 45 minutes after drug injection. All the male mice, 3 to
4 weeks old, were obtained from Jackson Laboratories, Bar Harbour,
Me. Immediately before anticonvulsant testing, mice were placed
upon a wire mesh, 4 inches square suspended from a steel rod. The
square was slowly inverted through 180 degrees, and the mice were
observed for 30 seconds. Any mouse falling from the wire mesh was
scored as ataxic.
[0166] Mice were placed into an enclosed acrylic plastic chamber
(21 cm height, approximately 30 cm diameter) with a high-frequency
speaker (4 cm diameter) in the center of the top lid. An audio
signal generator (Protek model B-810) was used to produce a
continuous sinusoidal tone that was swept linearly in frequency
between 8 kHz and 16 kHz once each 10 msec. The average sound
pressure level (SPL) during stimulation was approximately 100 dB at
the floor of the chamber. Mice were placed within the chamber and
allowed to acclimatize for 1 minute. DBA/2 mice in the
vehicle-treated group responded to the sound stimulus (applied
until tonic extension occurred, or for a maximum of 60 seconds)
with a characteristic seizure sequence consisting of wild running
followed by clonic seizures, and later by tonic extension, and
finally by respiratory arrest and death in 80% or more of the mice.
In vehicle-treated mice, the entire sequence of seizures to
respiratory arrest lasts approximately 15 to 20 seconds.
[0167] The incidence of all the seizure phases in the drug-treated
and vehicle-treated mice was recorded, and the occurrence of tonic
seizures were used for calculating anticonvulsant ED values by
probit analysis. Mice were used only once for testing at each time
and dose point.
[0168] Compound 2 was found to give 100% protection at 30 mg/kg at
15 minutes posttreatment time, and Compound 1 gave 80% protection
at 30 mg/kg at 15 minutes posttreatment time.
3TABLE 2 Compound of Time Post Number of Mice Example No. Dose
Treatment Protected from Tonic Number (mg/kg, IV) (minutes)
Convulsions* 1 30 15 4/5 1 30 45 2/5 1 10 15 1/5 2 30 15 5/5 2 30
45 1/5 2 10 15 1/5 7 10 15 2/5 7 10 45 1/5 8 10 15 0/5 8 10 45 0/5
*Number of mice protected from tonic convulsions/Number of mice
tested
* * * * *