U.S. patent application number 15/385512 was filed with the patent office on 2017-06-22 for metabolism resistant fenfluramine analogs and methods of using the same.
This patent application is currently assigned to ZOGENIX INTERNATIONAL LIMITED. The applicant listed for this patent is ZOGENIX INTERNATIONAL LIMITED. Invention is credited to Brooks M. BOYD, Stephen J. FARR.
Application Number | 20170174614 15/385512 |
Document ID | / |
Family ID | 59065966 |
Filed Date | 2017-06-22 |
United States Patent
Application |
20170174614 |
Kind Code |
A1 |
FARR; Stephen J. ; et
al. |
June 22, 2017 |
METABOLISM RESISTANT FENFLURAMINE ANALOGS AND METHODS OF USING THE
SAME
Abstract
Metabolism-resistant fenfluramine analogs are provided. The
subject fenfluramine analogs find use in the treatment of a variety
of diseases. For example, methods of treating epilepsy by
administering a fenfluramine analog to a subject in need thereof
are provided. Also provided are methods of suppressing appetite in
a subject in need thereof. Pharmaceutical compositions for use in
practicing the subject methods are also provided.
Inventors: |
FARR; Stephen J.; (Orinda,
CA) ; BOYD; Brooks M.; (Berkeley, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ZOGENIX INTERNATIONAL LIMITED |
Berkshire |
|
GB |
|
|
Assignee: |
ZOGENIX INTERNATIONAL
LIMITED
Berkshire
GB
|
Family ID: |
59065966 |
Appl. No.: |
15/385512 |
Filed: |
December 20, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62271168 |
Dec 22, 2015 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07C 215/64 20130101;
C07D 207/08 20130101; A61P 25/08 20180101; C07D 221/18 20130101;
C07C 2601/04 20170501; C07D 211/22 20130101; A61K 45/06 20130101;
A61K 31/137 20130101; C07C 211/29 20130101; C07C 2601/14
20170501 |
International
Class: |
C07C 211/29 20060101
C07C211/29; A61K 45/06 20060101 A61K045/06; A61K 31/137 20060101
A61K031/137 |
Claims
1. A metabolism-resistant fenfluramine analog compound of formula
(II): ##STR00053## wherein: R.sub.1 is CF.sub.3 or SF.sub.5;
R.sub.2, R.sub.3, R.sub.4, and R.sub.5 are independently selected
from hydrogen, halogen, alkoxy, acyl, substituted acyl, carboxy,
cyano, hydroxy, substituted alkoxy, alkyl, substituted alkyl, aryl,
substituted aryl, heterocycle, heteroaryl, substituted heteroaryl
and substituted heterocycle, wherein R.sub.2 and R.sub.5 or R.sub.2
and R.sub.4 are optionally cyclically linked together to form a
cycloalkyl ring, a heterocycle ring, an aryl ring or a heteroaryl
ring that is optionally substituted; X.sub.1-X.sub.5 are each
independently H, D, F, an alkyl or a substituted alkyl; and n is 1
or 2, wherein when n is 2 the nitrogen is positively charged; or a
salt thereof.
2. The compound of claim 1, having the formula (III): ##STR00054##
wherein X.sub.6-X.sub.7 are each independently H, D or F.
3. The compound of claim 1, having the formula (IV): ##STR00055##
wherein X.sub.8-X.sub.10 and each X is independently H, D or F,
provided at least one X.sub.8-X.sub.10 or X is F.
4. The compound of claim 1, having one of the following structures:
##STR00056##
5. The compound of claim 1, having one of formulae (IVa)-(IVc):
##STR00057## wherein each X are each independently H, D or F; and
R.sub.11-R.sub.16 are each independently an alkyl or a substituted
alkyl.
6. A pharmaceutical composition, comprising a therapeutically
effective amount of a compound having the formula (I): ##STR00058##
wherein: R.sub.1 is CF.sub.3 or SF.sub.5; R.sub.2, R.sub.3, R.sub.4
and R.sub.5 are independently selected from hydrogen, halogen,
alkoxy, acyl, substituted acyl, carboxy, cyano, hydroxy,
substituted alkoxy, alkyl, substituted alkyl, aryl, substituted
aryl, heterocycle, heteroaryl, substituted heteroaryl and
substituted heterocycle, where R.sub.2 and R.sub.5 or R.sub.2 and
R.sub.4 are optionally cyclically linked together to form a
cycloalkyl ring, a heterocycle ring, an aryl ring or a heteroaryl
ring that is optionally substituted; X.sub.1-X.sub.5 are each
independently H, D, F an alkyl or a substituted alkyl; and n is 1
or 2, wherein when n is 2 the nitrogen is positively charged; or a
pharmaceutically salt thereof
7. A method of treating epilepsy or a neurological related disease,
comprising administering to a patient in need thereof a
therapeutically effective amount of a metabolism-resistant
fenfluramine analog of formula (I): ##STR00059## wherein: R.sub.1,
R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6 and R.sub.7 are
independently selected from hydrogen, halogen, X.sub.1, X.sub.2,
alkoxy, acyl, substituted acyl, carboxy, cyano, hydroxy, alkoxy,
substituted alkoxy, alkyl, substituted alkyl, aryl, substituted
aryl, heterocycle, heteroaryl, substituted heteroaryl, substituted
heterocycle, or together with a second R.sup.1-R.sup.7 group form a
cycloalkyl ring, a heterocycle ring, an aryl ring or a heteroaryl
ring that is optionally substituted wherein R.sub.2 and R.sub.5,
R.sub.2 and R.sub.4, R.sub.1 and R.sub.5, R.sub.6 and R.sub.7,
and/or R.sub.3 and R.sub.6 are cyclically linked; X.sub.1-X.sub.5
are each independently H, D, F, alkyl or substituted alkyl; m is
0-4; and n is 1 or 2, wherein when n is 2 the nitrogen is
positively charged; or a pharmaceutically acceptable salt
thereof.
8. The method of claim 7, wherein the compound is a compound
according to one of claims 1-5.
9. The method of claim 7, further comprising co-administering to
the subject an antiepileptic agent.
10. A method of suppressing appetite in a subject, comprising
administering to the subject in need thereof an appetite
suppressing-amount of a compound of formula (I): ##STR00060##
wherein: R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6 and
R.sub.7 are independently selected from hydrogen, halogen, X.sub.1,
X.sub.2, alkoxy, acyl, substituted acyl, carboxy, cyano, hydroxy,
alkoxy, substituted alkoxy, alkyl, substituted alkyl, aryl,
substituted aryl, heterocycle, heteroaryl, substituted heteroaryl,
substituted heterocycle, or together with a second R.sup.1-R.sup.7
group form a cycloalkyl ring, a heterocycle ring, an aryl ring or a
heteroaryl ring that is optionally substituted wherein R.sub.2 and
R.sub.5, R.sub.2 and R.sub.4, R.sub.1 and R.sub.5, R.sub.6 and
R.sub.7, and/or R.sub.3 and R.sub.6 are cyclically linked;
X.sub.1-X.sub.5 are each independently H, D, F, alkyl or
substituted alkyl; m is 0-4; and n is 1 or 2, wherein when n is 2
the nitrogen is positively charged; or a pharmaceutically
acceptable salt thereof.
11. The method of claim 10, wherein the compound is a compound
according to one of claims 1-5.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] Pursuant to 35 U.S.C. .sctn.119 (e), this application claims
the benefit of priority to U.S. Provisional Patent Application Ser.
No. 62/271,168, filed Dec. 22, 2015, the disclosure of which
application is hereby incorporated by reference herein in its
entirety.
FIELD
[0002] This invention relates generally to the field of compounds
structurally related to the amphetamine drug fenfluramine and their
use in the treatment of neurological related diseases.
INTRODUCTION
[0003] Fenfluramine is an amphetamine drug that was once widely
prescribed as an appetite suppressant to treat obesity.
Fenfluramine is devoid of the psychomotor stimulant and abuse
potential of D-amphetamine and interacts with the
5-hydroxytryptamine (serotonin, 5-HT) transporters to release 5-HT
from neurons. Fenfluramine has been investigated for anticonvulsive
activity in the treatment of Dravet Syndrome, or severe myoclonic
epilepsy in infancy, a rare and malignant epileptic syndrome. This
type of epilepsy has an early onset in previously healthy
children.
[0004] Anorectic treatment with fenfluramine has been associated
with the development of cardiac valvulopathy and pulmonary
hypertension, including the condition cardiac fibrosis which led to
the withdrawal of fenfluramine from world-wide markets. Interaction
of fenfluramine's major metabolite norfenfluramine with the 5-HT2B
receptor is associated with heart valve hypertrophy. In the
treatment of epilepsy, the known cardiovascular risks of
fenfluramine are weighed against beneficial anticonvulsive
activity.
SUMMARY
[0005] Metabolism-resistant fenfluramine analogs are provided. The
subject fenfluramine analogs find use in the treatment of a variety
of diseases. For example, methods of treating epilepsy by
administering a fenfluramine analog to a subject in need thereof
are provided. Also provided are methods of suppressing appetite in
a subject in need thereof. Pharmaceutical compositions for use in
practicing the subject methods are also provided.
[0006] These and other objects, advantages, and features of the
invention will become apparent to those persons skilled in the art
upon reading the details of the metabolism-resistant fenfluramine
analogs and methods of using the same as more fully described
below.
DEFINITIONS
[0007] As used herein, the term "subject" refers to a mammal.
Exemplary mammals include, but are not limited to, humans, domestic
animals (e.g., a dog, cat, or the like), farm animals (e.g., a cow,
a sheep, a pig, a horse, or the like) or laboratory animals (e.g.,
a monkey, a rat, a mouse, a rabbit, a guinea pig, or the like). In
certain embodiments, the subject is human. "Patient" refers to
human and non-human subjects, especially mammalian subjects.
[0008] As used herein, the terms "treatment," "treating," and the
like, refer to obtaining a desired pharmacologic and/or physiologic
effect. The effect may be prophylactic in terms of completely or
partially preventing a disease or symptom thereof and/or may be
therapeutic in terms of a partial or complete cure for a disease
and/or adverse effect attributable to the disease. As used herein,
the terms "treating," "treatment," "therapeutic," or "therapy" do
not necessarily mean total cure or abolition of the disease or
condition. Any alleviation of any undesired signs or symptoms of a
disease or condition, to any extent can be considered treatment
and/or therapy. Furthermore, treatment may include acts that may
worsen the patient's overall feeling of well-being or appearance.
"Treatment," as used herein, covers any treatment of a disease in a
mammal, particularly in a human, and includes: (a) preventing the
disease from occurring in a subject which may be predisposed to the
disease but has not yet been diagnosed as having it; (b) inhibiting
the disease, i.e., arresting its development; and (c) relieving the
disease, i.e., causing regression of the disease.
[0009] As used herein, the term pKa refers to the negative
logarithm (p) of the acid dissociation constant (Ka) of an acid,
and is equal to the pH value at which equal concentrations of the
acid and its conjugate base form are present in solution.
[0010] "Alkyl" refers to monovalent saturated aliphatic hydrocarbyl
groups having from 1 to 10 carbon atoms and such as 1 to 6 carbon
atoms, or 1 to 5, or 1 to 4, or 1 to 3 carbon atoms. This term
includes, by way of example, linear and branched hydrocarbyl groups
such as methyl (CH.sub.3--), ethyl (CH.sub.3CH.sub.2--), n-propyl
(CH.sub.3CH.sub.2CH.sub.2--), isopropyl ((CH.sub.3).sub.2CH--),
n-butyl (CH.sub.3CH.sub.2CH.sub.2CH.sub.2--), isobutyl
((CH.sub.3).sub.2CHCH.sub.2--), sec-butyl
((CH.sub.3)(CH.sub.3CH.sub.2)CH--), t-butyl ((CH.sub.3).sub.3C--),
n-pentyl (CH.sub.3CH.sub.2CH.sub.2CH.sub.2CH.sub.2--), and
neopentyl ((CH.sub.3).sub.3CCH.sub.2--).
[0011] The term "substituted alkyl" refers to an alkyl group as
defined herein wherein one or more carbon atoms in the alkyl chain
have been optionally replaced with a heteroatom such as --O--,
--N--, --S--, --S(O).sub.n-- (where n is 0 to 2), --NR-- (where R
is hydrogen or alkyl) and having from 1 to 5 substituents selected
from the group consisting of alkoxy, substituted alkoxy,
cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted
cycloalkenyl, acyl, acylamino, acyloxy, amino, aminoacyl,
aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo,
thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy,
thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy,
aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl,
heterocyclooxy, hydroxyamino, alkoxyamino, nitro, --SO-alkyl,
--SO-aryl, --SO-heteroaryl, --SO.sub.2-alkyl, --SO.sub.2-aryl,
--SO.sub.2-heteroaryl, and --NR.sup.aR.sup.b, wherein R' and R''
may be the same or different and are chosen from hydrogen,
optionally substituted alkyl, cycloalkyl, alkenyl, cycloalkenyl,
alkynyl, aryl, heteroaryl and heterocyclic.
[0012] "Acyl" refers to the groups H--C(O)--, alkyl-C(O)--,
substituted alkyl-C(O)--, alkenyl-C(O)--, substituted
alkenyl-C(O)--, alkynyl-C(O)--, substituted alkynyl-C(O)--,
cycloalkyl-C(O)--, substituted cycloalkyl-C(O)--,
cycloalkenyl-C(O)--, substituted cycloalkenyl-C(O)--, aryl-C(O)--,
substituted aryl-C(O)--, heteroaryl-C(O)--, substituted
heteroaryl-C(O)--, heterocyclyl-C(O)--, and substituted
heterocyclyl-C(O)--, wherein alkyl, substituted alkyl, alkenyl,
substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl,
substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl,
aryl, substituted aryl, heteroaryl, substituted heteroaryl,
heterocyclic, and substituted heterocyclic are as defined herein.
For example, acyl includes the "acetyl" group CH.sub.3C(O)--
[0013] The term "acyloxy" refers to the groups alkyl-C(O)O--,
substituted alkyl-C(O)O--, cycloalkyl-C(O)O--, substituted
cycloalkyl-C(O)O--, aryl-C(O)O--, heteroaryl-C(O)O--, and
heterocyclyl-C(O)O-- wherein alkyl, substituted alkyl, cycloalkyl,
substituted cycloalkyl, aryl, heteroaryl, and heterocyclyl are as
defined herein.
[0014] "Aryl" or "Ar" refers to a monovalent aromatic carbocyclic
group of from 6 to 18 carbon atoms having a single ring (such as is
present in a phenyl group) or a ring system having multiple
condensed rings (examples of such aromatic ring systems include
naphthyl, anthryl and indanyl) which condensed rings may or may not
be aromatic, provided that the point of attachment is through an
atom of an aromatic ring. This term includes, by way of example,
phenyl and naphthyl. Unless otherwise constrained by the definition
for the aryl substituent, such aryl groups can optionally be
substituted with from 1 to 5 substituents, or from 1 to 3
substituents, selected from acyloxy, hydroxy, thiol, acyl, alkyl,
alkoxy, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, substituted
alkyl, substituted alkoxy, substituted alkenyl, substituted
alkynyl, substituted cycloalkyl, substituted cycloalkenyl, amino,
substituted amino, aminoacyl, acylamino, alkaryl, aryl, aryloxy,
azido, carboxyl, carboxylalkyl, cyano, halogen, nitro, heteroaryl,
heteroaryloxy, heterocyclyl, heterocyclooxy, aminoacyloxy,
oxyacylamino, thioalkoxy, substituted thioalkoxy, thioaryloxy,
thioheteroaryloxy, --SO-alkyl, --SO-substituted alkyl, --SO-aryl,
--SO-heteroaryl, --SO.sub.2-alkyl, --SO.sub.2-substituted alkyl,
--SO.sub.2-aryl, --SO.sub.2-heteroaryl and trihalomethyl.
[0015] "Heteroaryl" refers to an aromatic group of from 1 to 15
carbon atoms, such as from 1 to 10 carbon atoms and 1 to 10
heteroatoms selected from the group consisting of oxygen, nitrogen,
and sulfur within the ring. Such heteroaryl groups can have a
single ring (such as, pyridinyl, imidazolyl or furyl) or multiple
condensed rings in a ring system (for example as in groups such as,
indolizinyl, quinolinyl, benzofuran, benzimidazolyl or
benzothienyl), wherein at least one ring within the ring system is
aromatic and at least one ring within the ring system is aromatic,
provided that the point of attachment is through an atom of an
aromatic ring. In certain embodiments, the nitrogen and/or sulfur
ring atom(s) of the heteroaryl group are optionally oxidized to
provide for the N-oxide (N.fwdarw.O), sulfinyl, or sulfonyl
moieties. This term includes, by way of example, pyridinyl,
pyrrolyl, indolyl, thiophenyl, and furanyl. Unless otherwise
constrained by the definition for the heteroaryl substituent, such
heteroaryl groups can be optionally substituted with 1 to 5
substituents, or from 1 to 3 substituents, selected from acyloxy,
hydroxy, thiol, acyl, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl,
cycloalkenyl, substituted alkyl, substituted alkoxy, substituted
alkenyl, substituted alkynyl, substituted cycloalkyl, substituted
cycloalkenyl, amino, substituted amino, aminoacyl, acylamino,
alkaryl, aryl, aryloxy, azido, carboxyl, carboxylalkyl, cyano,
halogen, nitro, heteroaryl, heteroaryloxy, heterocyclyl,
heterocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy, substituted
thioalkoxy, thioaryloxy, thioheteroaryloxy, --SO-- alkyl,
--SO-substituted alkyl, --SO-aryl, --SO-heteroaryl,
--SO.sub.2-alkyl, --SO.sub.2-substituted alkyl, --SO.sub.2-aryl and
--SO.sub.2-heteroaryl, and trihalomethyl.
[0016] "Heterocycle," "heterocyclic," "heterocycloalkyl," and
"heterocyclyl" refer to a saturated or unsaturated group having a
single ring or multiple condensed rings, including fused bridged
and spiro ring systems, and having from 3 to 20 ring atoms,
including 1 to 10 hetero atoms. These ring atoms are selected from
the group consisting of nitrogen, sulfur, or oxygen, wherein, in
fused ring systems, one or more of the rings can be cycloalkyl,
aryl, or heteroaryl, provided that the point of attachment is
through the non-aromatic ring. In certain embodiments, the nitrogen
and/or sulfur atom(s) of the heterocyclic group are optionally
oxidized to provide for the N-oxide, --S(O)--, or
--SO.sub.2-moieties.
[0017] Examples of heterocycles and heteroaryls include, but are
not limited to, azetidine, pyrrole, imidazole, pyrazole, pyridine,
pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole,
dihydroindole, indazole, purine, quinolizine, isoquinoline,
quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline,
cinnoline, pteridine, carbazole, carboline, phenanthridine,
acridine, phenanthroline, isothiazole, phenazine, isoxazole,
phenoxazine, phenothiazine, imidazolidine, imidazoline, piperidine,
piperazine, indoline, phthalimide, 1,2,3,4-tetrahydroisoquinoline,
4,5,6,7-tetrahydrobenzo[b]thiophene, thiazole, thiazolidine,
thiophene, benzo[b]thiophene, morpholinyl, thiomorpholinyl (also
referred to as thiamorpholinyl), 1,1-dioxothiomorpholinyl,
piperidinyl, pyrrolidine, tetrahydrofuranyl, and the like.
[0018] In addition to the disclosure herein, the term
"substituted," when used to modify a specified group or radical,
can also mean that one or more hydrogen atoms of the specified
group or radical are each, independently of one another, replaced
with the same or different substituent groups as defined below.
[0019] In addition to the groups disclosed with respect to the
individual terms herein, substituent groups for substituting for
one or more hydrogens (any two hydrogens on a single carbon can be
replaced with .dbd.O, .dbd.NR.sup.70, .dbd.N--OR.sup.70,
.dbd.N.sub.2 or .dbd.S) on saturated carbon atoms in the specified
group or radical are, unless otherwise specified, --R.sup.60, halo,
.dbd.O, --OR.sup.70, --SR.sup.70, --NR.sup.80R.sup.80,
trihalomethyl, --CN, --OCN, --SCN, --NO, --NO.sub.2, .dbd.N.sub.2,
--N.sub.3, --SO.sub.2R.sup.70, --SO.sub.2O.sup.-M.sup.+,
--SO.sub.2OR.sup.70, --OSO.sub.2R.sup.70,
--OSO.sub.2O.sup.-M.sup.+, --OSO.sub.2OR.sup.70,
--P(O)(O.sup.-).sub.2(M.sup.+).sub.2,
--P(O)(OR.sup.70)O.sup.-M.sup.+, --P(O)(OR.sup.70).sub.2,
--C(O)R.sup.70, --C(S)R.sup.70, --C(NR.sup.70)R.sup.70,
--C(O)O.sup.-M.sup.+, --C(O)OR.sup.70, --C(S)OR.sup.70,
--C(O)NR.sup.80R.sup.80, --C(NR.sup.70)NR.sup.80R.sup.80,
--OC(O)R.sup.70, --OC(S)R.sup.70, --OC(O)O.sup.-M.sup.+,
--OC(O)OR.sup.70, --OC(S)OR.sup.70, --NR.sup.70C(O)R.sup.70,
--NR.sup.70C(S)R.sup.70, --NR.sup.70CO.sub.2.sup.-M.sup.+,
--NR.sup.70CO.sub.2R.sup.70, --NR.sup.70C(S)OR.sup.70,
--NR.sup.70C(O)NR.sup.80R.sup.80, --NR.sup.70C(NR.sup.70)R.sup.70
and --NR.sup.70C(NR.sup.70)NR.sup.80R.sup.80, where R.sup.60 is
selected from the group consisting of optionally substituted alkyl,
cycloalkyl, heteroalkyl, heterocycloalkylalkyl, cycloalkylalkyl,
aryl, arylalkyl, heteroaryl and heteroarylalkyl, each R.sup.70 is
independently hydrogen or R.sup.60; each R.sup.80 is independently
R.sup.70 or alternatively, two R.sup.80's, taken together with the
nitrogen atom to which they are bonded, form a 5-, 6- or 7-membered
heterocycloalkyl which may optionally include from 1 to 4 of the
same or different additional heteroatoms selected from the group
consisting of O, N and S, of which N may have --H or
C.sub.1-C.sub.3 alkyl substitution; and each M.sup.+ is a counter
ion with a net single positive charge. Each M.sup.+ may
independently be, for example, an alkali ion, such as K.sup.+,
Na.sup.+, Li.sup.+; an ammonium ion, such as
.sup.+N(R.sup.60).sub.4; or an alkaline earth ion, such as
[Ca.sup.2+].sub.0.5, [Mg.sup.2+].sub.0.5, or [Ba.sup.2+].sub.0.5
("subscript 0.5 means that one of the counter ions for such
divalent alkali earth ions can be an ionized form of a compound of
the invention and the other a typical counter ion such as chloride,
or two ionized compounds disclosed herein can serve as counter ions
for such divalent alkali earth ions, or a doubly ionized compound
of the invention can serve as the counter ion for such divalent
alkali earth ions). As specific examples, --NR.sup.80R.sup.80 is
meant to include --NH.sub.2, --NH-alkyl, N-pyrrolidinyl,
N-piperazinyl, 4N-methyl-piperazin-1-yl and N-morpholinyl.
[0020] In addition to the disclosure herein, substituent groups for
hydrogens on unsaturated carbon atoms in "substituted" alkene,
alkyne, aryl and heteroaryl groups are, unless otherwise specified,
--R.sup.60, halo, --O.sup.-M.sup.+, --OR.sup.70, --SR.sup.70,
--S.sup.-M.sup.+, --NR.sup.80R.sup.80, trihalomethyl, --CF.sub.3,
--CN, --OCN, --SCN, --NO, --NO.sub.2, --N.sub.3,
--SO.sub.2R.sup.70, --SO.sub.3.sup.-M.sup.+, --SO.sub.3R.sup.70,
--OSO.sub.2R.sup.70, --OSO.sub.3.sup.-M.sup.+, --OSO.sub.3R.sup.70,
--PO.sub.3.sup.-2(M.sup.+).sub.2, --P(O)(OR.sup.70)O.sup.-M.sup.+,
--P(O)(OR.sup.70).sub.2, --C(O)R.sup.70, --C(S)R.sup.70,
--C(NR.sup.70)R.sup.70, --CO.sub.2.sup.-M.sup.+,
--CO.sub.2R.sup.70, --C(S)OR.sup.70, --C(O)NR.sup.80R.sub.80,
--C(NR.sup.70)NR.sup.80R.sup.80, --OC(O)R.sup.70, --OC(S)R.sup.70,
--OCO.sub.2.sup.-M.sup.+, --OCO.sub.2R.sup.70, --OC(S)OR.sup.70,
--NR.sup.70C(O)R.sup.70, --NR.sup.70C(S)R.sup.70,
--NR.sup.70CO.sub.2.sup.-M.sup.+, --NR.sup.70CO.sub.2R.sup.70,
--NR.sup.70C(S)OR.sup.70, --NR.sup.70C(O)NR.sup.80R.sup.80,
--NR.sup.70C(NR.sup.70)R.sup.70 and
--NR.sup.70C(NR.sup.70)NR.sup.80R.sup.80, where R.sup.60, R.sup.70,
R.sup.80 and M.sup.+ are as previously defined, provided that in
case of substituted alkene or alkyne, the substituents are not
--O.sup.-M.sup.+, --OR.sup.70, --SR.sup.70, or
--S.sup.-M.sup.+.
[0021] In addition to the groups disclosed with respect to the
individual terms herein, substituent groups for hydrogens on
nitrogen atoms in "substituted" heteroalkyl and cycloheteroalkyl
groups are, unless otherwise specified, --R.sup.60,
--O.sup.-M.sup.+, --OR.sup.70, --SR.sup.70, --S.sup.-M.sup.+,
--NR.sup.80R.sup.80, trihalomethyl, --CF.sub.3, --CN, --NO,
--NO.sub.2, --S(O).sub.2R.sup.70, --S(O).sub.2O.sup.-M.sup.+,
--S(O).sub.2OR.sup.70, --OS(O).sub.2R.sup.70,
--OS(O).sub.2O.sup.-M.sup.+, --OS(O).sub.2OR.sup.70,
--P(O)(O.sup.-).sub.2(M.sup.+).sub.2,
--P(O)(OR.sup.70)O.sup.-M.sup.+, --P(O)(OR.sup.70)(OR.sup.70),
--C(O)R.sup.70, --C(S)R.sup.70, --C(NR.sup.70)R.sup.70,
--C(O)OR.sup.70, --C(S)OR.sup.70, --C(O)NR.sup.80R.sup.80,
--C(NR.sup.70)NR.sup.80R.sup.80, --OC(O)R.sup.70, --OC(S)R.sup.70,
--OC(O)OR.sup.70, --OC(S)OR.sup.70, --NR.sup.70C(O)R.sup.70,
--NR.sup.70C(S)R.sup.70, --NR.sup.70C(O)OR.sup.70,
--NR.sup.70C(S)OR.sup.70, --NR.sup.70C(O)NR.sup.80R.sup.80,
--NR.sup.70C(NR.sup.70)R.sup.70 and
--NR.sup.70C(NR.sup.70)NR.sup.80R.sup.80, where R.sup.60, R.sup.70,
R.sup.80 and M.sup.+ are as previously defined.
[0022] In addition to the disclosure herein, in a certain
embodiment, a group that is substituted has 1, 2, 3, or 4
substituents, 1, 2, or 3 substituents, 1 or 2 substituents, or 1
substituent.
[0023] It is understood that in all substituted groups defined
above, polymers arrived at by defining substituents with further
substituents to themselves (e.g., substituted aryl having a
substituted aryl group as a substituent which is itself substituted
with a substituted aryl group, which is further substituted by a
substituted aryl group, etc.) are not intended for inclusion
herein. In such cases, the maximum number of such substitutions is
three. For example, serial substitutions of substituted aryl groups
specifically contemplated herein are limited to substituted
aryl-(substituted aryl)-substituted aryl.
[0024] Unless indicated otherwise, the nomenclature of substituents
that are not explicitly defined herein are arrived at by naming the
terminal portion of the functionality followed by the adjacent
functionality toward the point of attachment. For example, the
substituent "arylalkyloxycarbonyl" refers to the group
(aryl)-(alkyl)-O--C(O)--.
[0025] As to any of the groups disclosed herein which contain one
or more substituents, it is understood, of course, that such groups
do not contain any substitution or substitution patterns which are
sterically impractical and/or synthetically non-feasible. In
addition, the subject compounds include all stereochemical isomers
arising from the substitution of these compounds.
[0026] The term "pharmaceutically acceptable salt" means a salt
which is acceptable for administration to a patient, such as a
mammal (salts with counterions having acceptable mammalian safety
for a given dosage regime). Such salts can be derived from
pharmaceutically acceptable inorganic or organic bases and from
pharmaceutically acceptable inorganic or organic acids.
"Pharmaceutically acceptable salt" refers to pharmaceutically
acceptable salts of a compound, which salts are derived from a
variety of organic and inorganic counter ions well known in the art
and include, by way of example only, sodium, potassium, calcium,
magnesium, ammonium, tetraalkylammonium, and the like; and when the
molecule contains a basic functionality, salts of organic or
inorganic acids, such as hydrochloride, hydrobromide, formate,
tartrate, besylate, mesylate, acetate, maleate, oxalate, and the
like.
[0027] The term "salt thereof" means a compound formed when a
proton of an acid is replaced by a cation, such as a metal cation
or an organic cation and the like. Where applicable, the salt is a
pharmaceutically acceptable salt, although this is not required for
salts of intermediate compounds that are not intended for
administration to a patient. By way of example, salts of the
present compounds include those wherein the compound is protonated
by an inorganic or organic acid to form a cation, with the
conjugate base of the inorganic or organic acid as the anionic
component of the salt.
[0028] "Solvate" refers to a complex formed by combination of
solvent molecules with molecules or ions of the solute. The solvent
can be an organic compound, an inorganic compound, or a mixture of
both. Some examples of solvents include, but are not limited to,
methanol, N,N-dimethylformamide, tetrahydrofuran,
dimethylsulfoxide, and water. When the solvent is water, the
solvate formed is a hydrate.
[0029] "Stereoisomer" and "stereoisomers" refer to compounds that
have same atomic connectivity but different atomic arrangement in
space. Stereoisomers include cis-trans isomers, E and Z isomers,
enantiomers, and diastereomers.
[0030] "Tautomer" refers to alternate forms of a molecule that
differ only in electronic bonding of atoms and/or in the position
of a proton, such as enol-keto and imine-enamine tautomers, or the
tautomeric forms of heteroaryl groups containing a
--N.dbd.C(H)--NH-- ring atom arrangement, such as pyrazoles,
imidazoles, benzimidazoles, triazoles, and tetrazoles. A person of
ordinary skill in the art would recognize that other tautomeric
ring atom arrangements are possible.
[0031] It will be appreciated that the term "or a salt or solvate
or stereoisomer thereof" is intended to include all permutations of
salts, solvates and stereoisomers, such as a solvate of a
pharmaceutically acceptable salt of a stereoisomer of subject
compound.
[0032] "Pharmaceutically effective amount" and "therapeutically
effective amount" refer to an amount of a compound sufficient to
treat a specified disorder or disease or one or more of its
symptoms and/or to prevent the occurrence of the disease or
disorder. In reference to tumorigenic proliferative disorders, a
pharmaceutically or therapeutically effective amount comprises an
amount sufficient to, among other things, cause the tumor to shrink
or decrease the growth rate of the tumor.
DETAILED DESCRIPTION
[0033] Before the present compounds and methods are described, it
is to be understood that this invention is not limited to
particular compounds and methods described, as such may, of course,
vary. It is also to be understood that the terminology used herein
is for the purpose of describing particular embodiments only, and
is not intended to be limiting, since the scope of the present
invention will be limited only by the appended claims.
[0034] Where a range of values is provided, it is understood that
each intervening value, to the tenth of the unit of the lower limit
unless the context clearly dictates otherwise, between the upper
and lower limits of that range is also specifically disclosed. Each
smaller range between any stated value or intervening value in a
stated range and any other stated or intervening value in that
stated range is encompassed within the invention. The upper and
lower limits of these smaller ranges may independently be included
or excluded in the range, and each range where either, neither or
both limits are included in the smaller ranges is also encompassed
within the invention, subject to any specifically excluded limit in
the stated range. Where the stated range includes one or both of
the limits, ranges excluding either or both of those included
limits are also included in the invention.
[0035] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, some potential and preferred methods and materials are
now described. All publications mentioned herein are incorporated
herein by reference to disclose and describe the methods and/or
materials in connection with which the publications are cited. It
is understood that the present disclosure supercedes any disclosure
of an incorporated publication to the extent there is a
contradiction.
[0036] It must be noted that as used herein and in the appended
claims, the singular forms "a", "an", and "the" include plural
referents unless the context clearly dictates otherwise. Thus, for
example, reference to "a compound" includes a plurality of such
compounds and reference to "the method" includes reference to one
or more methods and equivalents thereof known to those skilled in
the art, and so forth.
[0037] The publications discussed herein are provided solely for
their disclosure prior to the filing date of the present
application. Nothing herein is to be construed as an admission that
the present invention is not entitled to antedate such publication
by virtue of prior invention. Further, the dates of publication
provided may be different from the actual publication dates which
may need to be independently confirmed.
Metabolism-Resistant Fenfluramine Analogs
[0038] The present disclosure is related to structural and/or
functional analogs of fenfluramine that are resistant to systemic
metabolism. As used herein, the term "fenfluramine analog" refers
to a structural and/or functional analog of fenfluramine.
Functional analogs of fenfluramine are not necessarily structural
analogs. Unless explicitly stated, as used herein a fenfluramine
analog includes both functional and structural analogs. In some
cases, the subject fenfluramine analogs are resistant to metabolism
to de-ethylated norfenfluramine analogs in vivo.
[0039] Fenfluramine is an effective appetite suppressant drug that
was withdrawn from the drug market because of increased incidents
of heart disease. Fenfluramine is metabolized in vivo into
norfenfluramine. Such metabolism includes cleavage of an N-ethyl
group to produce norfenfluramine as shown below.
##STR00001##
[0040] De-ethylated norfenfluramine metabolite can have undesirable
biological activities that cause side effects such as increased
pulmonary hypertension and aortic valvular disease.
[0041] The present disclosure provides compounds that are
stabilized against such undesirable metabolism. As used herein, the
term "metabolism-resistant" refers to a stability of a fenfluramine
against any of the metabolic pathways of fenfluramine that reduces
the intended pharmacologic effect of the compound. One metabolic
pathway of interest against which the subject compounds can
resistant is the deethylation that can occur via P450 enzyme(s) in
the liver. In some cases, the fenfluramine analog is referred to as
being metabolically stable.
Metabolism into Norfenfluramine
[0042] Fenfluramine is metabolized in vivo into norfenfluramine by
metabolizing enzymes such as cytochrome P450 enzymes in the liver.
The enzymes in human liver that convert fenfluramine to
norfenfluramine include CYP1A2, CYP2B6, and CYP2D6; CYP2C9,
CYP2C19, and CYP3A4 also play a role.
Fenfluramine Analogs
[0043] Aspects of the present disclosure include analogs of
fenfluramine that are resistant to N-dealkylation, e.g., via action
of metabolizing enzymes (e.g., as described herein). In some
embodiments, the fenfluramine analogs are resistant to cytochrome
P450 enzymes. In certain cases, the fenfluramine analogs are
resistant to a P450 enzyme selected from CYP2D6, CYP2C19, CYP1A2,
CYP2B6, CYP3A4 and CYP2C9. In certain cases, the fenfluramine
analogs are resistant to a P450 enzyme selected from CYP1A2, CYP2B6
and CYP2D6. In some instances, the subject fenfluramine analogs
include a secondary, tertiary or quaternary amino group that is
stabilized against de-alkylation to a primary amine in vivo.
[0044] In some cases, the analogs are fluorinated compounds, e.g.,
analogs of fenfluramine that include one or more fluorine
substituents. In some instances, the analogs include fluorine
substituents at position(s) adjacent to the amine nitrogen of
fenfluramine. By "adjacent to" is meant substitution at a carbon
atom position that is located alpha, beta or gamma to the amine
nitrogen. In some embodiments, the fenfluramine analog further
includes one or more additional non-fluorine substituents which
impart upon the compound resistance to cytochrome P450 enzymes. In
certain cases, the fenfluramine analogs further includes one or
more additional non-fluorine substituents which impart upon the
compound resistance to a P450 enzyme selected from CYP2D6, CYP2C19,
CYP1A2, CYP2B6 CYP3A4 and CYP2C9. In certain cases, the
fenfluramine analogs further includes one or more additional
non-fluorine substituents which impart upon the compound resistance
to a P450 enzyme selected from CYP1A2, CYP2B6 and CYP2D6. In
certain instances, the analog further includes one or more
additional non-fluorine substituents which impart upon the compound
resistance to CYP2D6 metabolism.
[0045] In general terms, changes in pKa can have a predominant
effect on P450 enzyme (e.g., CYP2D6) substrate binding. P450 enzyme
substrates that include more basic amine groups are associated with
higher affinities and catalytic efficiencies. The present
disclosure provides fluorine substituted analogs where the pKa of
the amino group is lowered (i.e., lower basicity) relative to the
amine group of fenfluramine.
[0046] In addition, high lipophilicity of substituent groups in a
metabolizing enzyme, such as a P450 enzymes can be associated with
high affinity and catalytic efficiencies. In certain cases, the a
P450 enzyme is selected from CYP2D6, CYP2C19, CYP1A2, CYP2B6 CYP3A4
and CYP2C9. The present disclosure provides substituted analogs of
fenfluramine which include one or more hydrophilic substituents not
present in fenfluramine which impart on the compound a desirable
reduced liphophilicity.
[0047] Deuteration of biologically active compounds of interest can
produce analogs with improved pharmacokinetics (PK),
pharmacodynamics (PD), and/or toxicity profiles. In some
embodiments, fenfluramine analogs of interest include a deuterium
substituent at any convenient location (e.g., as described herein)
adjacent to the amine nitrogen atom. In certain instances, the
fenfluramine analog includes 2 or more deuterium substituents, such
as 3 or more, 4 or more, or 5 or more deuterium substituents. In
some cases, the deuterium substituents are located on carbon atoms
adjacent to the amino N atoms on the compound, e.g., the
alpha-carbon atom. In some cases, the two or more deuterium
substituents are located on the same carbon atom adjacent to the
amino nitrogen.
[0048] Other modifications of interest that can be incorporated
into the subject fenfluramine analogs include, but are not limited
to, replacement of the --CF.sub.3 aryl substituent of fenfluramine,
e.g., with an isosteric or isoelectronic group, and introduction of
quaternary amino group. In some instances, the fenfluramine
includes a --SF.sub.5 aryl substituent. In certain instances, the
fenfluramine analog is an N-alkylated analog where the amine
nitrogen is a quartery amine, i.e., a positively charged ammonium
group. In some embodiments, the analog includes an N-methylated
amine group.
[0049] Aspects of the present disclosure include fenfluramine
analogs including an additional substituent on the amino N atom
that sterically hinders the binding of the compound to a
metabolizing enzyme. Any convenient substituents may be included at
the N atom of the subject compounds to provide a sterically bulky
group. In some cases, the N-substituent of interest is a N-alkyl or
N-substituted alkyl group. In some cases, the N-substituent is a
N-aryl, N-heteroaryl, N-substituted aryl or N-substituted
heteroaryl group. Substituents of interest include, but are not
limited to, methyl, ethyl, propyl, isopropyl, butyl, tert-butyl,
trifluoromethyl, phenyl, benzyl and substituted benzyl. In some
embodiments, the analog includes an N-t-butyl group.
[0050] In some embodiments, the fenfluramine analog is a compound
having the formula (I):
##STR00002##
wherein:
[0051] R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6 and
R.sub.7 are independently selected from hydrogen, halogen, X.sub.1,
X.sub.2, alkoxy, acyl, substituted acyl, carboxy, cyano, hydroxy,
alkoxy, substituted alkoxy, alkyl, substituted alkyl, aryl,
substituted aryl, heterocycle, heteroaryl, substituted heteroaryl,
substituted heterocycle, or together with a second R.sup.1-R.sup.7
group form a cycloalkyl ring, a heterocycle ring, an aryl ring or a
heteroaryl ring that is optionally substituted wherein R.sub.2 and
R.sub.5, R.sub.2 and R.sub.4, R.sub.1 and R.sub.5, R.sub.6 and
R.sub.7, and/or R.sub.3 and R.sub.6 are cyclically linked;
[0052] X.sub.1-X.sub.5 are each independently H, D, F, alkyl or
substituted alkyl;
[0053] m is 0-4; and
n is 1 or 2, wherein when n is 2 the nitrogen is positively
charged; or a salt thereof.
[0054] In some embodiments of formula (I), R.sub.2 and R.sub.5 are
cyclically linked together to form a cycloalkyl ring, a heterocycle
ring, an aryl ring or a heteroaryl ring that is optionally
substituted. In some embodiments of formula (I), R.sub.2 and
R.sub.4 are cyclically linked together to form a cycloalkyl ring, a
heterocycle ring, an aryl ring or a heteroaryl ring that is
optionally substituted. In some embodiments of formula (I), R.sub.1
and R.sub.5 are cyclically linked together to form a cycloalkyl
ring, a heterocycle ring, an aryl ring or a heteroaryl ring that is
optionally substituted. In some embodiments of formula (I), R.sub.6
and R.sub.7 are cyclically linked together to form a cycloalkyl
ring, a heterocycle ring, an aryl ring or a heteroaryl ring that is
optionally substituted. In some embodiments of formula (I), R.sub.3
and R.sub.6 are cyclically linked together to form a cycloalkyl
ring, a heterocycle ring, an aryl ring or a heteroaryl ring that is
optionally substituted.
[0055] In some embodiments, the fenfluramine analog is a compound
having the formula (II):
##STR00003##
wherein:
[0056] R.sub.1 is an alkyl, a substituted alkyl (e.g., CF.sub.3) or
SF.sub.5;
[0057] R.sub.2, R.sub.3, R.sub.4 and R.sub.5 are independently
selected from hydrogen, halogen, alkoxy, acyl, substituted acyl,
carboxy, cyano, hydroxy, alkoxy, substituted alkoxy, alkyl,
substituted alkyl, aryl, substituted aryl, heterocycle, heteroaryl,
substituted heteroaryl and substituted heterocycle, where R.sub.2
and R.sub.5 or R.sub.2 and R.sub.4 are optionally cyclically
linked;
[0058] X.sub.1-X.sub.5 are each independently H, D, F, alkyl or
substituted alkyl; and
n is 1 or 2, wherein when n is 2 the nitrogen is positively
charged; or a salt thereof. In some embodiments of formula
(I)-(II), R.sub.2 and R.sub.4 are independently selected from
hydrogen, alkyl, substituted alkyl, aryl, substituted aryl,
heterocycle, heteroaryl, substituted heteroaryl and substituted
heterocycle. In some embodiments of formula (II), R.sub.2 and
R.sub.5 are cyclically linked together to form a cycloalkyl ring, a
heterocycle ring, an aryl ring or a heteroaryl ring that is
optionally substituted. In some embodiments of formula (II),
R.sub.2 and R.sub.4 are cyclically linked together to form a
cycloalkyl ring, a heterocycle ring, an aryl ring or a heteroaryl
ring that is optionally substituted. In certain cases, R.sub.2 is
cyclically linked to R.sub.5, e.g., to form a 5, 6 or 7-membered
carbocyclic or heterocyclic ring, which may be saturated or
unsaturated. In some embodiments of formula (I)-(II), R.sub.5 is
selected from hydrogen, halogen, alkoxy, acyl, substituted acyl,
carboxy, cyano, substituted alkoxy, alkyl and substituted alkyl. In
certain cases, R.sub.5 is cyclically linked to R.sub.2, e.g., to
form a 5, 6 or 7-membered carbocyclic or heterocyclic ring, which
may be saturated or unsaturated. In some embodiments of formula
(I)-(II), each R.sub.3 is selected from hydrogen, alkyl and
substituted alkyl. In some embodiments of formula (I)-(II), n is 1.
In some embodiments of formula (I)-(II), n is 2. In some
embodiments of formula (I), R.sub.3 is H. In some embodiments of
formula (I)-(II), R.sub.3 is H. In some cases, R.sub.3 is selected
from heteroaryl, substituted aryl, substituted heteroaryl. In some
cases, R.sub.3 is selected from methyl, ethyl, propyl, isopropyl,
butyl, tert-butyl, trifluoromethyl, phenyl, benzyl and substituted
benzyl. In some embodiments of formula (I)-(II), R.sub.3 is t-butyl
and n is 1.
[0059] In some embodiments of Formulae (I)-(II), the fenfluramine
analog has the formula (III):
##STR00004##
wherein X.sub.1-X.sub.7 are each independently H, D or F, and
R.sub.1, R.sub.3 and R.sub.4 are as defined in any of the
embodiments of formula (I). In some embodiments of formula (III),
R.sub.3 is selected from hydrogen, alkyl and substituted alkyl. In
some embodiments of formula (III), R.sub.3 is H. In some
embodiments of formula (III), R.sub.3 is selected from heteroaryl,
substituted aryl, substituted heteroaryl. In some cases, R.sub.3 is
selected from methyl, ethyl, propyl, isopropyl, butyl, tert-butyl,
trifluoromethyl, phenyl, benzyl and substituted benzyl. In some
embodiments of formula (III), R.sub.3 is t-butyl.
[0060] In some embodiments of formula (III), X.sub.1-X.sub.7 are
each independently H or F. In some embodiments of formula (III), at
least one of X.sub.1-X.sub.7 is F. In some embodiments of formula
(III), at least two of X.sub.1-X.sub.7 is F. In some embodiments of
formula (III), at least three of X.sub.1-X.sub.7 is F. In some
embodiments of formula (III), at least four of X.sub.1-X.sub.7 is
F. In some embodiments of formula (III), at least five of
X.sub.1-X.sub.7 is F. In some embodiments of formula (III), at
least six of X.sub.1-X.sub.7 is F.
[0061] In some embodiments of formula (III), X.sub.1-X.sub.7 are
each independently H or D. In some embodiments of formula (III), at
least one of X.sub.1-X.sub.7 is D. In some embodiments of formula
(III), at least two of X.sub.1-X.sub.7 is D. In some embodiments of
formula (II), at least three of X.sub.1-X.sub.7 is D. In some
embodiments of formula (III), at least four of X.sub.1-X.sub.7 is
D. In some embodiments of formula (III), at least five of
X.sub.1-X.sub.7 is D. In some embodiments of formula (III), at
least six of X.sub.1-X.sub.7 is D.
[0062] In some embodiments of Formulae (I)-(II), the fenfluramine
analog has the formula (IV):
##STR00005##
wherein X.sub.8-X.sub.10 and each X are independently H, D or F,
provided at least one X.sub.8-X.sub.10 or X is F. In some
embodiments of Formula (IV), each X is F. In some embodiments of
Formula (IV), each X is D. In some embodiments of Formula (IV),
each X is H. In some embodiments of Formula (IV), each X is F. In
some embodiments of Formula (IV), X.sub.8 is F. In some embodiments
of Formula (IV), X.sub.9 and X.sub.10 are each F.
[0063] In some embodiments of Formula (IV), the fenfluramine analog
has one of the following structures:
##STR00006##
[0064] In some embodiments of Formula (I), the fenfluramine analog
has one of the formulae (IVa)-(IVc):
##STR00007##
[0065] wherein X.sub.11, X.sub.12 and each X is independently H, D
or F; and
[0066] R.sub.11-R.sub.16 are each independently an alkyl or a
substituted alkyl.
In some embodiments of formulae (IVa)-(IVc), each X is F. In some
embodiments of formulae (IVa)-(IVc), X.sub.11 is F and X.sub.12 is
H. In some embodiments of formulae (IVa)-(IVc), X.sub.11 is F and
X.sub.12 is F. In some embodiments of formulae (IVa)-(IVc),
X.sub.11 is F and X.sub.12 is D. In some embodiments of formulae
(IVa)-(IVc), X.sub.11 is D and X.sub.12 is D. In some embodiments
of formulae (IVa)-(IVc), R.sub.11 is a substituted alkyl (e.g., as
described herein). In some embodiments of formulae (IVa)-(IVc),
R.sub.12 is a substituted alkyl (e.g., as described herein). In
some embodiments of formulae (IVa)-(IVc), R.sub.13 is selected from
methyl, ethyl, propyl, isopropyl, butyl, tert-butyl,
trifluoromethyl, phenyl, benzyl and substituted benzyl. In some
embodiments of formulae (IVa)-(IVc), R.sub.13 is a tert-butyl. In
some embodiments of formulae (IVa)-(IVc), R.sub.15 and R.sub.16 are
each an alkyl, such as methyl.
[0067] In some embodiments of Formula (I), the fenfluramine analog
has the formula (V):
##STR00008##
wherein R.sub.1, R.sub.3, R.sub.5, R.sub.6, R.sub.7 and m are as
defined above, p is 0, 1 or 2, and R.sub.8 and R.sub.9 are
independently selected from hydrogen, halogen, alkoxy, acyl,
substituted acyl, carboxy, cyano, hydroxy, alkoxy, substituted
alkoxy, alkyl, substituted alkyl, aryl, substituted aryl,
heterocycle, heteroaryl, substituted heteroaryl and substituted
heterocycle, or R.sub.8 and R.sub.9 are cyclically linked to form a
5 or 6-membered cycloalkyl, heterocycle, aryl or heteroaryl ring,
that is optionally further substituted, where the dashed bond
represents a single or double covalent bond.
[0068] In some embodiments of Formula (V), R.sup.8 and R.sup.9 are
not cyclically linked. In some embodiments of Formula (V), p is 0.
In some embodiments of Formula (V), p is 1. In some embodiments of
Formula (V), p is 2. In some embodiments of formula (V), each
R.sub.1 is independently selected from halogen, CF.sub.3, SF.sub.5,
acyl, substituted acyl, carboxy, alkyl ester, substituted alkyl
ester, cyano, hydroxy, alkoxy, substituted alkoxy, alkyl,
substituted alkyl; R.sub.7 is hydrogen, hydroxy, alkoxy,
substituted alkoxy, alkylcarbonyloxy, or substituted alkyl
carbonyloxy; R.sub.3 is hydrogen, alkyl or substituted alkyl; m is
0-4; and p is 0 or 1. In some embodiments of Formula (V), R.sub.6
is aryl or substituted aryl. In some embodiments of Formula (V),
R.sub.6 is phenyl or substituted phenyl. In some embodiments of
Formula (V), R.sub.6 is heteroaryl or substituted heteroaryl.
[0069] In some embodiments of Formula (V), R.sup.8 and R.sup.9 are
cyclically linked and together form an aryl or substituted aryl
ring, e.g., a fused benzene ring. In some embodiments of Formula
(V), R.sub.6 are R.sub.6 are hydrogen, fluoro or deuteron. In some
embodiments of Formula (V), R.sub.6 are R.sub.6 are hydrogen.
[0070] In some embodiments of Formula (V), R.sup.8 and R.sup.9 are
cyclically linked and together form an aryl or substituted aryl
ring, and R.sub.8 and R.sub.5 are also cyclically linked and
together form a 6-membered carbocycle ring, e.g., a partially
unsaturated fused ring. In some cases, the compound includes a
4-ring system of fused carbocyclic and heterocyclic rings. In some
embodiments of Formula (V), R.sub.6 are R.sub.6 are hydrogen,
fluoro or deuterium. In some embodiments of Formula (V), R.sub.6
are R.sub.6 are hydrogen.
[0071] In some embodiments of Formula (V), the fenfluramine analog
has the formula (VI):
##STR00009##
wherein R.sub.1, R.sub.3, R.sub.7, and m are as defined above, and
p is 0, 1 or 2.
[0072] In some embodiments of formula (VI), each R.sub.1 is
independently selected from halogen, CF.sub.3, SF.sub.5, acyl,
substituted acyl, carboxy, alkyl ester, substituted alkyl ester,
cyano, hydroxy, alkoxy, substituted alkoxy, alkyl, substituted
alkyl; R.sub.7 is hydrogen, hydroxy, alkoxy, substituted alkoxy,
alkylcarbonyloxy, or substituted alkyl carbonyloxy; R.sub.3 is
hydrogen, alkyl or substituted alkyl; each m is 0-4; and p is 0 or
1. In certain embodiments of formula (VI), each R.sub.1 is
independently selected from halogen, CF.sub.3, SF.sub.5, carboxy,
cyano, hydroxy, alkoxy, substituted alkoxy, alkyl, substituted
alkyl; R.sub.7 is hydrogen or hydroxy; R.sub.3 is hydrogen, alkyl
or substituted alkyl; each m is 0, 1 or 2; and p is 0 or 1. In
certain embodiments of formula (VI), R.sub.7 is hydrogen or
hydroxy; R.sub.3 is hydrogen, alkyl or substituted alkyl; each m is
0; and p is 0 or 1. In certain embodiments of formula (VI), R.sub.7
is hydrogen. In certain embodiments of formula (VI), R.sub.7 is
hydroxy. In certain embodiments of formula (VI), R.sub.3 is
hydrogen. In certain embodiments of formula (VI), R.sub.3 is alkyl
or substituted alkyl. In certain embodiments of formula (VI), each
m is 0. In certain embodiments of formula (VI), p is 0. In certain
embodiments of formula (VI), p is 1.
[0073] In certain embodiments of formula (VI), the fenfluramine
analog has one of the following structures:
##STR00010##
or a prodrug thereof, or a stereoisomer thereof, or a salt
thereof.
[0074] In some embodiments of formula (V), the fenfluramine analog
has formula (VII):
##STR00011##
or a prodrug thereof, or a stereoisomer thereof, or a salt
thereof.
[0075] In certain embodiments of formula (VII), each R.sub.1 is
independently selected from halogen, CF.sub.3, SF.sub.5, carboxy,
cyano, hydroxy, alkoxy, substituted alkoxy, alkyl, substituted
alkyl; R.sub.7 is hydrogen or hydroxy; R.sub.3 is hydrogen, alkyl
or substituted alkyl; and each m is 0, 1 or 2. In certain
embodiments of formula (VII), R.sub.3 is hydrogen, alkyl or
substituted alkyl; and each m is 0. In certain embodiments of
formula (VII), R.sub.3 is hydrogen. In certain embodiments of
formula (VII), R.sub.3 is alkyl or substituted alkyl. In certain
embodiments of formula (VII), each m is 0.
[0076] In certain embodiments of formula (VII), the fenfluramine
analog has the structure:
##STR00012##
or a prodrug thereof, or a stereoisomer thereof, or a salt
thereof.
[0077] In certain instances, the fenfluramine analog is apomorphine
or a structural analog or derivative thereof. Apomorphine
structural analogs and derivatives of interest, include but are not
limited to, those compounds described in EP1496915 by Holick et al.
In certain instances, the fenfluramine analog is
N-propylnorapomorphine.
[0078] In some embodiments of Formula (I), the fenfluramine analog
has the formula (VIII):
##STR00013##
wherein R.sub.1-R.sub.4, R.sub.6, R.sub.7 and m are as defined
above.
[0079] In certain embodiments of formula (VIII), each R.sub.1 is
independently selected from halogen, CF.sub.3, SF.sub.5, carboxy,
cyano, hydroxy, alkoxy, substituted alkoxy, alkyl, substituted
alkyl; and m is 0, 1 or 2. In certain embodiments of formula
(VIII), R.sub.3 is hydrogen, alkyl or substituted alkyl. In certain
embodiments of formula (VIII), R.sub.3 is hydrogen. In certain
embodiments of formula (VIII), R.sub.3 is alkyl or substituted
alkyl. In certain embodiments of formula (VIII), m is 0. In certain
embodiments of formula (VIII), m is 1 and R.sub.1 is a
4-substituent. In certain embodiments of formula (VIII), R.sub.7 is
hydrogen. In certain embodiments of formula (VIII), R.sub.2 is
alkyl or substituted alkyl. In certain embodiments of formula
(VIII), R.sub.2 is hydrogen. In certain embodiments of formula
(VIII), R.sub.4 is hydrogen. In certain embodiments of formula
(VIII), R.sub.4 is alkyl or substituted alkyl. In certain
embodiments of formula (VIII), R.sub.6 is a cycloalkyl, a
substituted cycloalkyl, an aryl, a substituted aryl, a heterocycle
or a substituted heterocycle. In certain embodiments of formula
(VIII), R.sub.6 and R.sub.7 are cyclically linked to form a 4, 5 or
6-membered cycloalkyl ring, optionally substituted with one or more
R.sub.1.
[0080] In some embodiments of Formula (I) and (VIII), the
fenfluramine analog has the formula (IX):
##STR00014##
wherein R.sub.1, R.sub.3, R.sub.4, and each m are as defined
above.
[0081] In certain embodiments of formula (IX), each R.sub.1 is
independently selected from halogen, CF.sub.3, SF.sub.5, carboxy,
cyano, hydroxy, alkoxy, substituted alkoxy, alkyl, substituted
alkyl; and each m is 0, 1 or 2. In certain embodiments of formula
(IX), R.sub.3 is hydrogen, alkyl or substituted alkyl. In certain
embodiments of formula (IX), R.sub.3 is hydrogen. In certain
embodiments of formula (IX), R.sub.3 is alkyl or substituted alkyl.
In certain embodiments of formula (IX), each m is 0 or 1. In
certain embodiments of formula (IX), each m is 0 or 1 and R.sub.1
is a 4-substituent. In certain embodiments of formula (IX), R.sub.4
is hydrogen. In certain embodiments of formula (IX), R.sub.4 is
alkyl or substituted alkyl.
[0082] In certain embodiments, the fenfluramine analog is
Desvenlafaxine or a structural analog or derivative thereof. In
certain embodiments, the fenfluramine analog is Desvenlafaxine or
O-desmethylvenlafaxine. In certain embodiments of formula (VII),
the fenfluramine analog has the structure:
##STR00015##
or a prodrug thereof, or a stereoisomer thereof, or a salt
thereof.
[0083] In some embodiments of Formula (I) and (VIII), the
fenfluramine analog has the formula (X):
##STR00016##
wherein R.sub.1-R.sub.4, and each m are as defined above, and q is
0, 1 or 2.
[0084] In certain embodiments of formula (X), each R.sub.1 is
independently selected from halogen, CF.sub.3, SF.sub.5, carboxy,
cyano, hydroxy, alkoxy, substituted alkoxy, alkyl, substituted
alkyl; and each m is 0, 1 or 2. In certain embodiments of formula
(X), R.sub.2 is alkyl or substituted alkyl. In certain embodiments
of formula (X), R.sub.2 is hydrogen. In certain embodiments of
formula (X), R.sub.3 is hydrogen, alkyl or substituted alkyl. In
certain embodiments of formula (X), R.sub.3 is hydrogen. In certain
embodiments of formula (X), R.sub.3 is alkyl or substituted alkyl.
In certain embodiments of formula (X), each m is 0 or 1. In certain
embodiments of formula (X), each m is 0 or 1 and R.sub.1 is a
4-substituent. In certain embodiments of formula (X), R.sub.4 is
hydrogen. In certain embodiments of formula (X), R.sub.4 is alkyl
or substituted alkyl.
[0085] In some embodiments of Formula (X), the fenfluramine analog
has the formula (XIa) or (XIb):
##STR00017##
[0086] In certain embodiments of formulae (XIa)-(XIb), each R.sub.1
is independently selected from halogen, CF.sub.3, SF.sub.5,
carboxy, cyano, hydroxy, alkoxy, substituted alkoxy, alkyl,
substituted alkyl; and m is 0, 1 or 2. In certain embodiments of
formula (XIa)-(XIb), each R.sub.1 is independently selected from
halogen, CF.sub.3, SF.sub.5 and hydroxy; and m is 0 or 1. In
certain embodiments of formula (XIa)-(XIb), R.sub.2 is alkyl or
substituted alkyl. In certain embodiments of formula (XIa)-(XIb),
R.sub.2 is hydrogen. In certain embodiments of formula (XIa)-(XIb),
R.sub.3 is hydrogen. In certain embodiments of formula (XIa)-(XIb),
R.sub.3 is alkyl or substituted alkyl. In certain embodiments of
formula (XIa)-(XIb), each m is 0 or 1. In certain embodiments of
formula (XIa)-(XIb), each m is 0 or 1 and R.sub.1 is a
4-substituent. In certain embodiments of formula (XIa)-(XIb),
R.sub.4 is hydrogen. In certain embodiments of formula (XIa)-(XIb),
R.sub.4 is alkyl or substituted alkyl.
[0087] In certain instances, the fenfluramine analog is sibutramine
or a structural analog or derivative thereof. Sibutramine
structural analogs and derivatives of interest, include but are not
limited to, didesmethyl sibutramine. In certain embodiments of
formula (XIb), the fenfluramine analog has the structure:
##STR00018##
or a prodrug thereof, or a stereoisomer thereof, or a salt
thereof.
Preparation of Fenfluramine Compounds
[0088] Any convenient methods of preparing fenfluramine can be
adapted in the preparation of the subject compounds. Exemplary
methods of interest which can be adapted for use in the preparation
of the subject fenfluramine analogs are described below.
[0089] Fenfluramine has been synthesized in many ways, some going
through the synthesis of the intermediate
1-(3-trifluoromethyl)phenyl-propan-2-one. This U.S. Pat. No.
3,198,833 describes some syntheses of this ketone starting from
3-trifluoromethylphenylacetonitrile or from the corresponding
alcohol.
[0090] Fenfluramine can be obtained from
1-(3-trifluoromethyl)phenyl-propan-2-one through reductive
amination with an amine, for instance as described in Hungarian
Patent HU T055343.
[0091] U.S. Pat. No. 5,811,586 describes a process for
manufacturing 1-(3-trifluoromethyl)phenyl-propan-2-one intermediate
in the synthesis of fenfluramine that includes reacting the
diazonium salt of 3-trifluoromethylaniline with isopropenyl acetate
in a polar solvent in the presence of a catalytic amount of a
cuprous salt and, optionally, of a base and purifying the crude
product through the bisulfite complex or distillation under
vacuum.
Methods of Use
[0092] The above-described compounds may be employed in a variety
of methods. As summarized above, aspects of the method include
administering to a subject in need thereof a therapeutically
effective amount of a fenfluramine analog (e.g., a structural or
functional analog of fenfluramine as described herein) to treat or
prevent a disease or condition of interest. By "therapeutically
effective amount" is meant the concentration of a compound that is
sufficient to elicit the desired biological effect (e.g., treatment
or prevention of epilepsy). Diseases and conditions of interest
include, but are not limited to, epilepsy, particularly intractable
forms of epilepsy including Dravet syndrome, Lennox Gastaut
syndrome and Doose syndrome, neurological related diseases, obesity
and obesity related diseases.
[0093] In some embodiments, the subject method includes
administering to a subject a subject compound to treat a
neurological related disease. Neurological related diseases of
interest include, but are not limited to, epilepsy, and severe
myoclonic epilepsy in infancy (Dravet syndrome), Lennox-Gastaut
syndrome and Doose syndrome. In certain embodiments, the subject is
human. In certain instances, the subject suffers from Dravet
syndrome. In certain embodiments, the compound is administered as a
pharmaceutical preparation.
[0094] Thus, according to a still further aspect of the present
invention, there is provided a method of stimulating one or more
5-HT receptors in the brain of a patient by administering an
effective dose of a fenfluramine analog to said patient, said one
or more 5-HT receptors being selected from one or more of
5-HT.sub.1, 5-HT.sub.1A, 5-HT.sub.1B, 5-HT.sub.1C, 5-HT.sub.1D,
5-HT.sub.1E, 5-HT.sub.1F, 5-HT.sub.2, 5-HT.sub.2A, 5-HT.sub.2B,
5-HT.sub.2C, 5-HT.sub.3, 5-HT.sub.4, 5-HT.sub.5, 5-HT.sub.5A,
5-HT.sub.5B 5-HT.sub.6, and 5-HT.sub.7 amongst others. In certain
embodiments of this aspect of the invention, the patient has been
diagnosed with Dravet Syndrome.
[0095] In embodiments of the invention, any effective dose of the
fenfluramine analog can be employed. In certain embodiments, a
daily dose of less than about 10 mg/kg/day is employed, such as
about 9 mg/kg/day, about 8 mg/kg/day, about 7 mg/kg/day, about 6
mg/kg/day, about 5 mg/kg/day, about 4 mg/kg/day, about 3 mg/kg/day,
about 2 mg/kg/day, about 1 mg/kg/day, about 0.9 mg/kg/day, about
0.8 mg/kg/day, about 0.7 mg/kg/day, about 0.6 mg/kg/day, or about
0.5 mg/kg/day is employed. In some cases, a daily dose of between
about 1 mg/kg/day and about 10 mg/kg/day is employed, such as
between about 2 mg/kg/day and about 10 mg/kg/day, between about 3
mg/kg/day and about 10 mg/kg/day, between about 4 mg/kg/day and
about 10 mg/kg/day, or between about 5 mg/kg/day and about 10
mg/kg/day. In some cases, a daily dose of between about 0.5
mg/kg/day and about 1.0 mg/kg/day is employed. In certain
embodiments, a daily dose of less than about 1.0 mg/kg/day is
employed. In some cases, a preferred dose is less than about 0.5 to
about 0.01 mg/kg/day.
[0096] As indicated above the dosing is based on the weight of the
patient. However, for convenience the dosing amounts may be preset
such as in the amount of 1 mg, 2.5 mg, 5 mg, 10 mg, 15 mg, 20 mg,
30 mg, 40 mg, or 50 mg. In general the smallest dose which is
effective should be used for the particular patient.
[0097] The dose of fenfluramine analog administered in the methods
of the present invention can be formulated in any pharmaceutically
acceptable dosage form including, but not limited to oral dosage
forms such as tablets including orally disintegrating tablets,
capsules, lozenges, oral solutions or syrups, oral emulsions, oral
gels, oral films, buccal liquids, powder e.g. for suspension, and
the like; injectable dosage forms; transdermal dosage forms such as
transdermal patches, ointments, creams; inhaled dosage forms;
and/or nasally, rectally, vaginally administered dosage forms. Such
dosage forms can be formulated for once a day administration, or
for multiple daily administrations (e.g. 2, 3 or 4 times a day
administration).
[0098] Administration of the subject compounds may be systemic or
local. In certain embodiments, administration to a mammal will
result in systemic release of a subject compound (for example, into
the bloodstream). Methods of administration can include enteral
routes, such as oral, buccal, sublingual, and rectal; topical
administration, such as transdermal and intradermal; and parenteral
administration. Suitable parenteral routes include injection via a
hypodermic needle or catheter, for example, intravenous,
intramuscular, subcutaneous, intradermal, intraperitoneal,
intraarterial, intraventricular, intrathecal, and intracameral
injection and non-injection routes, such as intravaginal rectal, or
nasal administration. In certain embodiments, the subject compounds
and compositions are administered orally. In certain embodiments,
it may be desirable to administer a compound locally to the area in
need of treatment. In some embodiments, the method of
administration of the subject compound is parenteral
administration. This may be achieved, for example, by local
infusion during surgery, topical application, e.g., in conjunction
with a wound dressing after surgery, by injection, by means of a
catheter, by means of a suppository, or by means of an implant,
said implant being of a porous, non-porous, or gelatinous material,
including membranes, such as sialastic membranes, or fibers.
[0099] In some embodiments, the subject method includes
administering to a subject an appetite suppressing amount of the
subject compound to treat obesity. Any convenient methods for
treating obesity may be adapted for use with the subject
fenfluramine analogs. Any of the pharmaceutical compositions
described herein can find use in treating a subject for obesity.
Combination therapy includes administration of a single
pharmaceutical dosage formulation which contains the subject
compound and one or more additional agents; as well as
administration of the subject compound and one or more additional
agent(s) in its own separate pharmaceutical dosage formulation. For
example, a subject compound and an additional agent active with
appetite suppressing activity (e.g., phentermine or topiramate) can
be administered to the patient together in a single dosage
composition such as a combined formulation, or each agent can be
administered in a separate dosage formulation. Where separate
dosage formulations are used, the subject compound and one or more
additional agents can be administered concurrently, or at
separately staggered times, e.g., sequentially. In some
embodiments, the method further includes co-administering to the
subject with the subject fenfluramine analog, an antiepileptic
agent. Antiepileptic agents of interest that find use in methods of
co-administering include, but are not limited to, Acetazolamide,
Carbamazepine, Clobazam, Clonazepam, Eslicarbazepine acetate,
Ethosuximide, Gabapentin, Lacosamide, Lamotrigine, Levetiracetam,
Nitrazepam, Oxcarbazepine, Perampanel, Piracetam, Phenobarbital,
Phenytoin, Pregabalin, Primidone, Retigabine, Rufinamide, Sodium
valproate, Stiripentol, Tiagabine, Topiramate, Vigabatrin and
Zonisamide.
[0100] In some embodiments, the subject method is an in vitro
method that includes contacting a sample with a subject compound.
The protocols that may be employed in these methods are numerous,
and include but are not limited to, serotonin release assays from
neuronal cells, cell-free assays, binding assays (e.g., 5HT2B
receptor binding assays); cellular assays in which a cellular
phenotype is measured, e.g., gene expression assays; and assays
that involve a particular animal model for a condition of interest
(e.g., Dravet syndrome, Lennox-Gastaut syndrome or Doose
syndrome).
Pharmaceutical Preparations
[0101] Also provided are pharmaceutical preparations.
Pharmaceutical preparations are compositions that include a
compound (either alone or in the presence of one or more additional
active agents) present in a pharmaceutically acceptable vehicle.
The term "pharmaceutically acceptable" means approved by a
regulatory agency of the Federal or a state government or listed in
the U.S. Pharmacopeia or other generally recognized pharmacopeia
for use in mammals, such as humans. The term "vehicle" refers to a
diluent, adjuvant, excipient, or carrier with which a compound of
the invention is formulated for administration to a mammal.
[0102] The choice of excipient will be determined in part by the
particular compound, as well as by the particular method used to
administer the composition. Accordingly, there is a wide variety of
suitable formulations of the pharmaceutical composition of the
present invention.
[0103] The dosage form of a fenfluramine analog employed in the
methods of the present invention can be prepared by combining the
fenfluramine analog with one or more pharmaceutically acceptable
diluents, carriers, adjuvants, and the like in a manner known to
those skilled in the art of pharmaceutical formulation.
[0104] By way of illustration, the fenfluramine analog can be
admixed with conventional pharmaceutically acceptable carriers and
excipients (i.e., vehicles) and used in the form of aqueous
solutions, tablets, capsules, elixirs, suspensions, syrups, wafers,
and the like. Such pharmaceutical compositions contain, in certain
embodiments, from about 0.1% to about 90% by weight of the active
compound, and more generally from about 1% to about 30% by weight
of the active compound. The pharmaceutical compositions may contain
common carriers and excipients, such as solubilizers, isotonic
agents, suspending agents, emulsifying agents, stabilizers,
preservatives, colorants, diluents, buffering agents, surfactants,
moistening agents, flavoring agents and disintegrators, and
including, but not limited to, corn starch, gelatin, lactose,
dextrose, sucrose, microcrystalline cellulose, kaolin, mannitol,
dicalcium phosphate, sodium chloride, alginic acid, vegetable or
other similar oils, synthetic aliphatic acid glycerides, esters of
higher aliphatic acids or propylene glycol, corn starch, potato
starch, acacia, tragacanth, gelatin, glycerin, sorbitol, ethanol,
polyethylene glycol, colloidal silicon dioxide, croscarmellose
sodium, talc, magnesium stearate and stearic acid. Disintegrators
commonly used in the formulations of this invention include
croscarmellose, microcrystalline cellulose, corn starch, sodium
starch glycolate and alginic acid. The compounds can be formulated
into preparations for injection by dissolving, suspending or
emulsifying them in an aqueous or nonaqueous solvent, such as
vegetable or other similar oils, synthetic aliphatic acid
glycerides, esters of higher aliphatic acids or propylene glycol;
and if desired, with conventional additives such as solubilizers,
isotonic agents, suspending agents, emulsifying agents, stabilizers
and preservatives.
[0105] In some embodiments, formulations suitable for oral
administration can include (a) liquid solutions, such as an
effective amount of the compound dissolved in diluents, such as
water, or saline; (b) capsules, sachets or tablets, each containing
a predetermined amount of the active ingredient, as solids or
granules; (c) suspensions in an appropriate liquid; and (d)
suitable emulsions. Tablet forms can include one or more of
lactose, mannitol, corn starch, potato starch, microcrystalline
cellulose, acacia, gelatin, colloidal silicon dioxide,
croscarmellose sodium, talc, magnesium stearate, stearic acid, and
other excipients, colorants, diluents, buffering agents, moistening
agents, preservatives, flavoring agents, and pharmacologically
compatible excipients. Lozenge forms can include the active
ingredient in a flavor, usually sucrose and acacia or tragacanth,
as well as pastilles including the active ingredient in an inert
base, such as gelatin and glycerin, or sucrose and acacia,
emulsions, gels, and the like containing, in addition to the active
ingredient, such excipients as are described herein.
[0106] In some cases, the compound is formulated for oral
administration. In some cases, for an oral pharmaceutical
formulation, suitable excipients include pharmaceutical grades of
carriers such as mannitol, lactose, glucose, sucrose, starch,
cellulose, gelatin, magnesium stearate, sodium saccharine, and/or
magnesium carbonate. For use in oral liquid formulations, the
composition may be prepared as a solution, suspension, emulsion, or
syrup, being supplied either in solid or liquid form suitable for
hydration in an aqueous carrier, such as, for example, aqueous
saline, aqueous dextrose, glycerol, or ethanol, preferably water or
normal saline. If desired, the composition may also contain minor
amounts of non-toxic auxiliary substances such as wetting agents,
emulsifying agents, or buffers.
[0107] Particular formulations of the invention are in a liquid
form. The liquid may be a solution or suspension and may be an oral
solution or syrup which is included in a bottle with a pipette
which is graduated in terms of milligram amounts which will be
obtained in a given volume of solution. The liquid solution makes
it possible to adjust the solution for small children which can be
administered anywhere from 0.5 mg to 15 mg and any amount between
in half milligram increments and thus administered in 0.5, 1.0,
1.5, 2.0 mg, etc.
[0108] A liquid composition will generally consist of a suspension
or solution of the compound or pharmaceutically acceptable salt in
a suitable liquid carrier(s), for example, ethanol, glycerine,
sorbitol, non-aqueous solvent such as polyethylene glycol, oils or
water, with a suspending agent, preservative, surfactant, wetting
agent, flavoring or coloring agent. Alternatively, a liquid
formulation can be prepared from a reconstitutable powder.
EXAMPLES
[0109] The following examples are put forth so as to provide those
of ordinary skill in the art with a complete disclosure and
description of how to make and use the present invention, and are
not intended to limit the scope of what the inventors regard as
their invention nor are they intended to represent that the
experiments below are all or the only experiments performed.
Efforts have been made to ensure accuracy with respect to numbers
used (e.g. amounts, temperature, etc.) but some experimental errors
and deviations should be accounted for. Unless indicated otherwise,
parts are parts by weight, molecular weight is weight average
molecular weight, temperature is in degrees Centigrade, and
pressure is at or near atmospheric.
Example 1
Preparation of 1-(3-Trifluoromethyl)phenyl-propan-2-one
[0110] 35 mL of water and 45 g of 37% (w/w) aqueous hydrochloric
acid are put in a flask equipped with stirrer and dropping funnel.
24.25 Grams (0.151 moles) of m-trifluoromethylaniline are added
after having cooled to 10 degree C. with an ice bath and then, at 5
degree C., an aqueous solution containing 12.43 g (0.180 moles) of
sodium nitrite in 150 ml of water is slowly added. The reaction
mixture is stirred for 30 minutes and then is poured during 30
minutes into a mixture made by 90 ml of water, 1.35 g (0.014 moles)
of cuprous chloride, 2.30 g (0.013 moles) of cupric chloride
dihydrate, 50 ml of acetone, 40.8 g (0.300 moles) of sodium acetate
trihydrate and 23 g (0.230 moles) of isopropenyl acetate while
keeping the reaction temperature at 30 degree C. After further 30
minutes of stirring, the reaction mixture is brought to 20 degree
C., 50 ml of methylene chloride are added and the two layers are
separated.
[0111] The aqueous layer is discarded while the organic layer is
concentrated under vacuum until an oil is obtained which is treated
with 35 g of sodium metabisulfite, 70 ml of water and 150 ml of
heptane under stirring at room temperature for 12 hours. The
suspension is filtered, the bisulfite complex is washed on the
filter with 50 ml of heptane and then suspended in a two-phase
mixture made by 100 ml of methylene chloride and 150 ml of a 10%
(w/v) aqueous solution of sodium hydroxide. The layers are
separated after one hour of stirring at room temperature, the
aqueous phase is discarded while the organic layer is washed with
water and evaporated under vacuum to give pure ketone.
Example 2
[0112] Method for Assaying Activity of Analogs Using Zebrafish
Model of Epilepsy
[0113] Zebrafish embryos (Danio rerio) heterozygous for the scn1Lab
mutation (scn1Lab+/-) are backcrossed with Tupfel longfin wildtype
(WT scn1Lab+/+). Adult zebrafish are housed at 28.0.degree. C., on
a 14/10 hour light/dark cycle under standard aquaculture
conditions. Fertilized eggs are collected via natural spawning.
Anaesthetized fish (tricaine 0.02%) are fin-clipped and genotyped
by PCR. After genotyping, samples are purified (MinElute PCR
Purification Kit) and sequenced by LGC Genomics. Age-matched Tupfel
longfin wildtype larvae are used as control group (WT scn1Lab+/+).
These embryos and larvae are kept on a 14/10 hour light/dark cycle
in embryo medium (Danieaus): 1.5 mM HEPES, pH 7.6, 17.4 mM NaCl,
0.21 mM KCl, 0.12 mM MgSO.sub.4, and 0.18 mM Ca(NO.sub.3).sub.2 in
an incubator at 28.0.degree. C.
[0114] To evaluate the locomotor activity of homozygous scn1Lab-/-
mutants and control WT scn1Lab+/+, zebrafish larvae are placed in a
96-well plate in 100 .mu.L of embryo medium from 4 to 8 dpf. Each
day the larvae are tracked in an automated tracking device
(ZebraBox.TM. apparatus; Viewpoint, Lyon, France) for 10 min after
30 min habituation (100-second integration interval). All
recordings are performed at the same time during daytime period.
The total distance in large movements is recorded and quantified
using ZebraLab.TM. software (Viewpoint, Lyon, France). Data are
pooled together from at least three independent experiments with at
least 24 larvae per condition.
[0115] Epileptiform activity is measured by open-field recordings
in the zebrafish larval forebrain at 7 dpf. Homozygous scn1Lab-/-
mutants and control WT scn1Lab+/+ are embedded in 2%
low-melting-point agarose (Invitrogen) to position a glass
electrode into the forebrain. This glass electrode is filled with
artificial cerebrospinal fluid (aCSF) made from: 124 mM NaCl, 2 mM
KCl, 2 mM MgSO.sub.4, 2 mM CaCl.sub.2, 1.25 mM KH.sub.2PO.sub.4, 26
mM NaHCO.sub.3 and 10 mM glucose (resistance 1-5 M.OMEGA.) and
connected to a high-impedance amplifier. Subsequently, recordings
are performed in current clamp mode, low-pass filtered at 1 kHz,
high-pass filtered 0.1 Hz, digital gain 10, at sampling intervals
of 10 s (MultiClamp 700B amplifier, Digidata 1440A digitizer, both
Axon instruments, USA). Single recordings are performed for 10 min.
Epileptiform activity is quantified according to the duration of
spiking paroxysms as described previously (Orellana-Paucar et al,
2012). Electrograms are analyzed with the aid of Clampfit 10.2
software (Molecular Devices Corporation, USA). Spontaneous
epileptiform events are taken into account when the amplitude
exceeded three times the background noise and lasted longer than 50
milliseconds (ms). This threshold is chosen due to the less
frequent observation of epileptiform events in wildtype ZF larvae
with a shorter duration than 50 ms.
[0116] Analogs (agonists) and antagonists can be chosen based on
their high and selective affinity for the different
5-HT.sub.subtype receptors (Ki in nanomolar range), and on their
logP value (i.e. >1, expected to exhibit a good bioavailability
in zebrafish larvae (Milan, 2003)). Compounds are dissolved in
dimethylsulfoxide (DMSO, 99.9% spectroscopy grade, Acros Organics)
and diluted in embryo medium to achieve a final DMSO concentration
of 0.1% w/v, which also served as a vehicle control (VHC).
[0117] To evaluate the maximal tolerated concentration (MTC) of
each compound, 6 dpf-old WT scn1Lab+/+ zebrafish larvae are
incubated in a 96-well plate (tissue culture plate, flat bottom,
FALCON.RTM., USA) with different concentrations of compound or VHC
at 28.degree. C. on a 14/10 hour light/dark cycle under standard
aquaculture conditions (medium is replenished daily). Each larva is
individually checked under the microscope during a period of 48
hours for the following signs of toxicity: decreased or no touch
response upon a light touch of the tail, loss of posture, body
deformation, edema, changes in heart rate or circulation and death.
The maximum tolerated concentration (MTC) is defined as the highest
concentration at which no signs of toxicity are observed in 12 out
of 12 zebrafish larvae within 48 hours of exposure to sample. The
MTC (Tables 1 and 2) is used throughout the work.
[0118] Scn1Lab-/- mutants and WT scn1Lab+/+ larvae are arrayed in
the same plate and treated at 6 days post fertilization (dpf) with
fenfluramine analogs (at their MTC) or VHC in individual wells of a
96-well plate. After incubation at 28.degree. C. on a 14/10 hour
light/dark cycle and 30-min chamber habituation 6 and 7 dpf larvae
are tracked for locomotor activity for 10 min (100-second
integration interval) under dark conditions. An incubation time of
1.5 hours is further referred as short treatment (6 dpf).
Furthermore these larvae are analyzed after more than 22 hours
incubation (7 dpf), i.e. long treatment. The total locomotor
activity is quantified using the parameter lardist and plotted in
cm. In some cases, data is pooled together from two (5-HT.sub.1B-,
5-HT.sub.1F-, 5-H.sub.T3--, 5-HT.sub.4-, 5-HT.sub.5A-,
5-HT.sub.6-agonist and all antagonists except 5-HT.sub.1B- and
5-HT.sub.7-antagonists) or three (fenfluramine compounds,
5-HT.sub.1A-, 5-HT.sub.1D-, 5-HT.sub.1E-, 5-HT.sub.2A-,
5-HT.sub.2B-, and 5-HT.sub.2C-agonist) independent experiments with
at least 9 larvae per treatment condition.
[0119] Epileptiform activity is measured by open-field recordings
in the zebrafish larval forebrain at 7 dpf, as described above.
Scn1Lab-/- mutants and WT scn1Lab+/+ larvae are incubated with
fenfluramine (25 .mu.M), the functional analogs (except for the
5-HT.sub.5A-agonist) that exhibited locomotor-reducing activity in
the previous assay (see below) (MTC), a negative control (3.125
.mu.M 5-HT2B-agonist) or VHC on 6 dpf for a minimum of 22 hours
(long treatment). Recordings of 7 dpf larvae, from at least 8
scn1Lab-/- mutant larvae are taken per experimental condition. For
treated WT scn1Lab+/+ larvae at least 5 per condition are analyzed,
due to the scarce observation of epileptiform activity in wildtype
larvae. Electrographic recordings are quantified for the different
treatment conditions.
[0120] The heads of 7 dpf-old zebrafish larvae are used to
determine the amount of the neurotransmitters dopamine,
noradrenaline and serotonin present. Six heads per tube are
homogenized on ice for one min in 100 .mu.l 0.1 M antioxidant
buffer (containing vitamin C). Homogenates are centrifuged at 15
000 g for 15 min at 4.degree. C. Supernatants (70 .mu.l) are
transferred to a sterile tube and stored at -80.degree. C. until
analysis.
[0121] The neurotransmitter determination is based on the microbore
LC-ECD method (Sophie Sarre, Katrien Thorre, Ilse Smolders, 1997).
The chromatographic system consists of a FAMOS microautosampler of
LC Packings/Dionex (Amsterdam, The Netherlands), a 307 piston pump
of Gilson (Villiers-le-Bel, France), a DEGASYS DG-1210 degasser of
Dionex and a DECADE II electrochemical detector equipped with a
.mu.-VT03 flow cell (0.7 mm glassy carbon working electrode,
Ag/AgCl reference electrode, 25 .mu.m spacer) of Antec
(Zoeterwoude, The Netherlands). The mobile phase is a mixture of
87% V/V aqueous buffer solution at pH 5.5 (100 mM sodium acetate
trihydrate, 20 mM citric acid monohydrate, 2 mM sodium
decanesulfonate, 0.5 mM disodium edetate) and 13% V/V acetonitrile.
This mobile phase is injected at a flow rate of 60 .mu.L/min. The
temperature of the autosampler tray is set on 15.degree. C. and the
injection volume is 10 .mu.L. A microbore UniJet C8 column
(100.times.1.0 mm, 5 .mu.m) of Bioanalytical Systems (West
Lafayette, Ind., United States) is used as stationary phase. The
separation and detection temperature is performed at 35.degree. C.,
with a detection potential of +450 mV vs Ag/AgCl. Data acquisition
is carried out by Clarity chromatography software version 3.0.2 of
Data Apex (Prague, The Czech Republic). The amount of
neurotransmitter (in nmol) is calculated based on the total mass of
six heads.
[0122] Statistical analyses are performed using GraphPad Prism 5
software (GraphPad Software, Inc.). The larval locomotor activity
is evaluated by using One-way ANOVA, followed by Dunnett's multiple
comparison tests. Values are presented as means.+-.standard
deviation (SD). LFP measurements (electrographic brain activity)
are analyzed by a Mann-Whitney test. Statistically significant
differences (p<0.05) between a treatment group and the
equivalent control groups (scn1Lab-/- mutant or WT scn1Lab+/+) are
considered indicative of a decrease or increase in locomotor or
electrographic brain activity of zebrafish larvae. The
neurotransmitter amount of scn1Lab-/- mutants is compared with WT
scn1Lab+/+ larvae by a Student's t-test because all data passed the
normality test (D'Agostino & Pearson omnibus normality
test).
Example 3
[0123] Phenotype-Based Antieplileptic Drug Screening in a Zebrafish
Model of Dravet Syndrome
[0124] Compounds provided by the present disclosure are assessed
for their anticonvulsant activity in vitro using a high-throughput
mutant zebrafish screening assay. The following methods can be
adapted for use in assessing the subject fenefluramine analogs.
[0125] Animals: Scn1A
[0126] Zebrafish are maintained in a light- and
temperature-controlled aquaculture facility under a standard 14:10
h light/dark photoperiod. Adult Heterozygous scn1Lab.+-.mutant
zebrafish are housed in 1.5 L tanks at a density of 5-12 fish per
tank and fed twice per day (dry flake and/or flake supplemented
with live brine shrimp). Water quality is continuously monitored to
maintain the following conditions: temperature, 28-30.degree. C.;
pH 7.4-8.0; conductivity, 690-710 mS/cm. Zebrafish embryos are
maintained in round Petri dishes (catalog #FB0875712, Fisher
Scientific) in "embryo medium" consisting of 0.03% Instant Ocean
(Aquarium Systems, Inc.) and 000002% methylene blue in reverse
osmosis-distilled water.
[0127] Larval zebrafish clutches are bred from wild-type (WT; TL
strain) or scn1Lab (didys552) heterozygous animals that have been
back-crossed to TL wild-type for at least 10 generations.
Homozygous mutants (n 6544), which have widely dispersed
melanosomes and appear visibly darker as early as 3 d
post-fertilization (dpf; FIG. 1b), or WT larvae (n=71) are used in
all experiments at 5 or 6 dpf. Embryos and larvae are raised in
plastic petri dishes (90 mm diameter, 20 mm depth) and density is
limited to 60 per dish. Larvae between 3 and 7 dpf lack discernible
sex chromosomes. The care and maintenance protocols comply with
requirements [outlined in the Guide for the Care and Use of Animals
(ebrary Inc., 2011) and are subject to approval by the
Institutional Animal Care and Use Committee (protocol
#AN108659-01D)].
[0128] Test Agents:
[0129] Compounds for screening are provided as 10 mM DMSO
solutions. Test agents for locomotion or electrophysiology studies
are dissolved in embryo media and are tested at an initial
concentration of 100 M, with a final DMSO concentration of 2%. In
all drug screen studies, compounds are coded and experiments are
performed by investigators who are blind to the nature of the
compound. Drug concentrations between 0.5 and 1 mM are used for
electrophysiology assays to account for more limited diffusion in
agar-embedded larvae.
[0130] Seizure Monitoring
[0131] Zebrafish larvae are placed individually into 1 well of a
clear flat-bottomed 96-well microplate (catalog #260836, Fisher
Scientific) containing embryo media. To study changes in
locomotion, microplates are placed inside an enclosed
motion-tracking device and acclimated to the dark condition for
10-15 min at room temperature. Locomotion plots are obtained for
one fish per well at a recording epoch of 10 min using a
DanioVision system running EthoVision XT software (DanioVision,
Noldus Information Technology); threshold detection settings to
identify objects darker than the background are optimized for each
experiment. Seizure scoring is performed using the following
three-stage scale (Baraban et al., 2005): Stage 0, no or very
little swim activity; Stage I, increased, brief bouts of swim
activity; Stage II, rapid "whirlpool-like" circling swim behavior;
and Stage III, paroxysmal whole-body clonus-like convulsions, and a
brief loss of posture. WT fish are normally scored at Stage 0 or I.
Plots are analyzed for distance traveled (in millimeters) and mean
velocity (in millimeters per second). As reported previously
(Winter et al., 2008; Baraban et al., 2013), velocity changes are a
more sensitive assay of seizure behavior.
[0132] Baseline recordings of seizure behavior are obtained from
mutants bathed in embryo media, as described above; a second
locomotion plot is then obtained following a solution change to a
test compound and an equilibration period of 15-30 min. Criteria
for a positive hit designation are as follows: (1) a decrease in
mean velocity of 44% (e.g., a value based on the trial-to-trial
variability measured in control tracking studies; FIG. 1c); and (2)
a reduction to Stage 0 or Stage I seizure behavior in the
locomotion plot for at least 50% of the test fish. Each test
compound classified as a "positive hit" in the locomotion assay is
confirmed, under direct visualization on a stereomicroscope, as the
fish being alive based on movement in response to external
stimulation and a visible heartbeat following a 60 min drug
exposure.
[0133] Toxicity (or mortality) is defined as no visible heartbeat
or movement in response to external stimulation in at least 50% of
the test fish. Hyperexcitability is defined as a compound causing a
44% increase in swim velocity and/or Stage III seizure activity in
at least 50% of the test fish. Hits identified in the primary
locomotion screen are selected and rescreened, again using the
method described above. Select compound stocks that are successful
in two primary locomotion assays, and are not classified as toxic
in two independent clutches of zebrafish are then subjected to
further testing in an electrophysiology assay.
[0134] Electrophysiology Assay:
[0135] Zebrafish larvae are briefly paralyzed with bungarotoxin (1
mg/ml) and immobilized in 1.2% agarose; field recordings are
obtained from forebrain structures. Epileptiform events are
identified post hoc in Clampfit (Molecular Devices) and are defined
as multi-spike or polyspike upward or downward membrane deflections
greater than three times the baseline noise level and 500 ms in
duration. During electrophysiology experiments zebrafish larvae are
continuously monitored for the presence (or absence) of blood flow
and heart beat by direct visualization on an Olympus BX51WI upright
microscope equipped with a CCD camera and monitor.
[0136] Data Analysis
[0137] Data are presented as the mean and SEM, unless stated
otherwise. Pairwise statistical significance is determined with a
Student's two-tailed unpaired t test, ANOVA, or Mann-Whitney rank
sum test, as appropriate, unless stated otherwise. Results are
considered significant at p 0.05, unless otherwise indicated.
Example 4
[0138] Multi-Electrode Array Screening for Anticonvulsant
Activity
[0139] Test agents are assessed as therapeutic targets by measuring
their effects on electrophysiological parameters identified as
reflecting disease using multi-electrode arrays recording (MEAs)
across principal regions of hippocampal brain slices (CA1, CA3,
dentate gyrus) taken from untreated wild type and DS KO mice.
[0140] Tissue Preparation:
[0141] Male and female 129S wild-type and DS KO mice, are humanely
killed by cervical dislocation. No more than one slice per animal
is used to investigate any one experimental condition. Brains are
swiftly (<2 min) removed and placed in chilled, carboxygenated
(95% 02:5% CO2) artificial cerebrospinal fluid (aCSF), comprising
(mM) NaCl 124, KCl 3, KH2PO4 1.25, NaHCO3 36, MgSO4.6H2O 1,
d-glucose 10 and CaCl2 2. Conventional transverse hippocampal
slices (Egert et al., 2002a) are cut at a thickness of 450 m using
a Campden Vibroslice/M tissue slicer (Campden Instruments,
Loughborough, UK) and left to rest for at least 1 h in aCSF at room
temperature before recording commenced.
[0142] For Mg2+-free induction of epileptiform activity, MgSO4.6H2O
is omitted without substitution from the aCSF. Standard or
Mg2+-free aCSF is used throughout dissection and recording as
appropriate with positive controls, test agents and vehicle added
during recording from previously prepared aliquots of concentrated
stocks. Aliquots are frozen immediately after preparation and
individually thawed before use. All reagents and drugs are obtained
from Sigma-Aldrich (Poole, UK). Positive controls and test agents
are dissolved in DMSO at 1000.times. working concentration and
stored at -20.degree. C. until use before incorporation into aCSF;
maximum DMSO bath concentration is 0.1% 2.2.
[0143] MEA Recordings:
[0144] Electrical activity across each hippocampal slice is
monitored and recorded using MEAs (59 electrodes each 30.mu.
diameter with 200 .mu.m spacing and 100 .mu.m recording radius,
FIG. 1A; Multi Channel Systems, GmbH, Reutlingen, Germany).
[0145] Prior to recording, MEAs are cleaned with 5% (w/v)
Terg-A-Zyme (Cole-Palmer, London, UK), methanol and finally
distilled water.
[0146] Slices are adhered to MEAs using an applied (.about.41) and
evaporated cellulose nitrate solution in methanol (0.24%, w/v,
Protran Nitrocellulose Transfer Membranes; Schleicher & Schuell
Bioscience Inc., NH, USA; Ma et al., 2008). Slice position on the
MEA is ascertained by observation on a Nikon TS-51 microscope
(Nikon, Japan) at magnification .times.4, with images of the slice
and electrode positions being acquired via a Mikro-Okular camera
(Meade Instruments Corp., CA, USA; FIG. 1A) to a PC. Once attached,
slices are continually perfused with carboxygenated aCSF (.about.2
ml/min). Slices are maintained at 21.degree. C. in order to provide
good separation of synaptically mediated LFP components and
maximize slice viability time. Slice viability and contact with MEA
electrodes are assessed by applying voltage pulses (STG2004
stimulator, Multi Channel Systems GmbH, Reutlingen, Germany)
through MEA electrodes on the slice (200 s biphasic pulses,
.+-.0.5-3.0 V) to evoke local field potentials.
[0147] For each agent, 12-15 recordings are made per condition and
in each tissue type (mutant and wildtype). Signals are amplified
(1200.times. gain), by a 120-channel dual headstage amplifier
(MEA60 System, Multi Channel Systems GmbH, Reutlingen, Germany) and
simultaneously sampled at a minimum of 10 kHz per channel on all 60
channels. Data acquisition is to a PC using MC Rack software (Multi
Channel Systems GmbH, Reutlingen, Germany) to monitor and record
data for offline analysis.
[0148] Data Analysis:
[0149] Data analyses are performed using in-house scripts for
Matlab 6.5 and 7.0.4 (Mathworks, Natick, Mass., USA; Matlab scripts
used throughout this study available on request from authors).
Microsoft Excel (Microsoft, Redmond, USA) and MC DataTool (Multi
Channel Systems GmbH, Reutlingen, Germany) are also used to process
and present data.
[0150] Raw data are filtered using a 2 Hz high pass 2nd order
Butterworth filter to remove very low frequency artefacts
associated with bath perfusion. Changes in burst amplitude and
frequency during control experiments are assessed using in-house
Matlab 7.0.4 scripts. Data are downsampled from 10 kHz to 500 Hz,
whereupon a single Matlab script returned the peak amplitude (V) of
each burst within an experiment and the time at which the peaks
occurred (s). These data are used for representative plots of
amplitude and frequency.
[0151] Additionally, mean amplitudes are calculated at 10 min
intervals using the peak amplitudes of the ten bursts directly
preceding the time of interest. The same ten bursts are also used
to calculate frequency where: frequency=10/(time of last burst-time
of first burst). When data of this type from different electrodes
and slices are pooled, the first burst from each electrode
recording is considered to have occurred at 0 s; subsequent bursts
are offset by the same amount. This improved comparability between
recordings from different sources. In control experiments assessing
amplitude and frequency changes, mean amplitude and frequency
values are normalized to the value calculated at 30 min after
bursting commenced.
[0152] Burst propagation paths are determined by constructing
contour plots from raw data files at one frame per sampling point
(10 kHz) using an in-house adaptation of MEATools (Egert et al.,
2002b) in Matlab 6.5 and interpolated using a 5 point
Savitzky-Golay filter before export to JPEG image format.
Individual JPEGs are concatenated and converted into AVI animations
using Photolapse
(http://home.hccnet.nl/s.vd.palen/photolapsedlc.html) for later low
speed replay. After determining burst initiation site, termination
site and resulting propagation path, the time that burst peaks
occurred at electrode positions closest to burst initiation (CA3)
and termination (CA1) sites are ascertained in MC Rack. Initiation
to termination distances is calculated using ImageJ (Abramoff et
al., 2004) and propagation speed thusly derived from time and
distance values. Mean propagation speeds are derived from pooled
data.
[0153] Spectrograms are produced with Neuroexplorer 4.045
(NexTechnologies, Littleton, Mass., USA) using 20 ms window shifts,
8192fast Fourier transform (FFT) frequency divisions and a
frequency cut-off at 250 Hz. Spectrograms are normalized for
comparison by expression of spectral power as the log of the power
spectral density (dB). Power spectral density values are produced
using Neuroexplorer using 2048 FFT frequency divisions with a
frequency cut-off of 250 Hz. Representative power spectral density
plots are shown for the frequency range 0-50 Hz and are smoothed
using a five point Gaussian filter. However, the full unsmoothed
frequency range (0-250 Hz) is used for quantification of total
power changes in the absence and presence of anticonvulsant drugs.
Data sets of equal duration (.gtoreq.150 s) in epileptiform and
anticonvulsant treated states are used in the construction of power
spectra and subsequent quantification.
[0154] Changes in total power in the presence of anticonvulsant
drugs are expressed as a percentage of the total power in the
presence of 100M4-AP or absence of Mg2+ for each electrode.
Percentage values from individual electrodes are then pooled as
hippocampal regions (CA1, CA3 and dentate gyrus (DG)) before
averaging. Data from .gtoreq.4 electrodes from >1 slice
preparations are analyzed for each region. In experiments where
anticonvulsants are added, bursting is first induced by application
of 100M 4-AP or Mg2+-free aCSF; 30 min after bursting commenced,
phenobarbital or felbamate are applied. Differences in burst
frequency, amplitude and duration are thus assessed between the 10
bursts prior to drug application (at 30 min after bursting
commenced), and the 10 bursts 30 min after drugs had been applied.
If no bursts are recorded in the last 5 min of the 30 min after AED
application, bursting is considered to have been abolished. In this
instance, continuing slice viability and fidelity of contact with
the MEA are confirmed by evocation of field potentials via
electrode stimulation as described above.
[0155] Statistical significance is determined by non-parametric
Mann-Whitney U-test in the case of all normalised data and
comparisons of frequency and latency between models. The
significance of anticonvulsant drug effects on propagation speeds
is tested using a two-tailed paired Students t-test. p.ltoreq.0.05
is considered significant in all cases. All data are presented as
means.+-.S.E.M and data is given for each hippocampal region (DG,
CA3, CA1).
Example 5
[0156] Survival Studies
[0157] Dose-response curves are generated for candidate therapeutic
agents using wild-type and Scn1a.sup.-/- mutant knock-out mice.
[0158] Treatment of Animals:
[0159] 129S wild-type mice (controls) and 129S Scn1a knock-out mice
("DS KO mice") are obtained from The Jackson Laboratory (MMRRC
Stock No: 37107-JAX). All animal procedures comply with all
applicable animal welfare regulations.
[0160] Groups of 10-12 animals having roughly equal numbers of male
and females are treated with either vehicle, a positive control, or
a test agent. Animals are injected subcutaneously with 0.08 ml
injection volume twice daily at 0900 and 1600 hours from P8 until
sacrifice. Injection sites are rotated in the following order: left
shoulder, right shoulder, left hip, right hip. Dose concentrations
are adjusted to retain consistent volumes of administration.
[0161] Following treatment, animals are sacrificed at 2 days
post-weaning (P23/24) or sooner as required by mathematical model.
Animals are then assessed using Kolmogorov-Smirnov welfare scoring
(compared between groups (Massey, F. J. "The Kolmogorov-Smirnov
Test for Goodness of Fit." Journal of the American Statistical
Association. Vol. 46, No. 253, 1951, pp. 68-78) and Mantel-Cox or
Gehan-Breslow-Wilcoxon mortality tests (for the former, see Mantel
N., "Evaluation of survival data and two new rank order statistics
arising in its consideration" Cancer Chemother Rep. 1966 PMID:
5910392). Data obtained is subjected to statistical analysis (see
below) and is presented as median, IQR and max/min (except %
mortality).
Example 6
[0162] Assessment of Compounds as Potential Therapeutic Agents for
Dravet Syndrome Patients Using Induced Pluripotent Stem Cell
Cortical Neurons
[0163] The therapeutic efficacy of compounds of interest, alone and
in combination with other commonly used anti-convulsants, is
assessed using induced pluripotent stem cell (iPSC) cortical
neurons derived from Dravet syndrome patients who are known
responders to fenfluramine. Results obtained for test agents are
compared to those obtained for fenfluramine. The following methods
can be adapted for use in assessing the subject fenefluramine
analogs.
[0164] A. Materials and Methods
[0165] Differentiation of Human iPSCs into Cortical Neurons
[0166] iPSC cells taken from Dravet syndrome patients known to
respond to fenfluramine are differentiated into cortical
pyramid-like neurons and cortical interneurons.
[0167] Generation of Cortical Pyramidal-Like Neurons
[0168] iPSCs are first dissociated into single cells with Accutase
or 0.5 mM EDTA (Lonza), and plated onto gelatin-coated dishes for 1
h in hESC medium with 10 .mu.M ROCK inhibitor. Suspended iPSCs are
then re-plated on Matrigel-coated 12-well plates in MEF conditioned
hESC medium with 10 ng/ml FGF2. At 95% confluence, the medium is
changed to 3N medium supplemented with 1 .mu.M Dorsomorphin and 10
.mu.M SB431542. Cells are cultured for 8-11 d, and neural induction
is monitored by the appearance of "neural rosettes".
Neuroepithelial cells are dissociated with Dispase or 5 mM EDTA and
replated in 3N medium with 20 ng/ml FGF2 on Matrigelcoated plates.
After 2-4 d, FGF2 is withdrawn to promote differentiation. Cultures
are passaged with Accutase, replated at 5.times.10.sup.5 on 60 mm
Matrigel-coated plates seeded with rodent forebrain glia or human
iPS derived glia cells in 3N medium and maintained for up to 100 d
with medium changes every other day.
[0169] Generation of Cortical Interneurons
[0170] iPSC embryoid bodies (EBs) are plated in a neural induction
medium similar to that described above with TGF-.beta. inhibitors
until a neuroepithelial sheet forms. The neuroepithelial sheet are
then patterned to medial ganglion eminence (MGE)-like progenitors
using high concentrations of Pur and differentiate the MGE-like
progenitors to GABAergic interneurons. A nearly pure population
(90%) of GABAergic interneurons is generated, and confirmed after 7
weeks in culture by performing immunocytochemistry for GABA.
[0171] Measurement of Voltage-Gated Sodium Current and Action
Potential Firing in Whole Cells
[0172] The effects of compounds of interest, alone and in the
presence of known AEDs, on voltage-gated sodium current and action
potential firing in whole cell is measured using whole cell voltage
and current clamp recordings. As a first step, the effect of
increasing concentrations of test compounds on sodium current is
measured under voltage-clamp. The effect of test compounds on
evoked and spontaneous action potential firing is then measured
under current clamp.
[0173] Perfusion of Drugs During Voltage- or Current-Clamp
Experiments
[0174] Test compounds (at final concentrations of 10 .mu.M to 1 mM)
or vehicle (either sodium current recording solution or ACSF) are
perfused onto neurons following recordings of basal sodium current
levels or action potential firing. Effects of acute and long-term
drug applications are then measured.
[0175] Changes in voltage-sensitive sodium current and action
potential firing can be assessed in response to increasing
concentrations of a compound of interest, in the presence and
absence of the following AEDs frequently prescribed for Dravet
Syndrome, at the final concentration indicated: topiramate (200
.mu.M), stiripentol (100 .mu.M), valproic acid (250 .mu.M), and
clobazam (3 .mu.M).
[0176] Voltage-Clamp Recordings
[0177] Voltage-clamp recordings are performed as previously
described. See Brackenbury et. al, Abnormal neuronal patterning
occurs during early postnatal brain development of Scn1b-null mice
and precedes hyperexcitability. Proc Natl Acad Sci USA. 2013;
110(3):1089-94. PMCID: 3549092.
[0178] Isolated sodium currents are recorded from single neurons
(bipolar or pyramidal) at RT (21-22.degree. C.) in the presence of
a bath solution that contains (in mM): 120 NaCl, 1 BaCl2, 2 MgCl2,
0.2 CdCl2, 1 CaCl, 10 HEPES, 20 TEA-Cl and 10 glucose (pH 7.35 with
CsOH, Osmolarity: 300-305 mOsM). Fire-polished patch pipettes are
generated from borosilicate glass capillaries (Warner Instrument
Corp.) using a Sutter P-97 puller (Sutter Instrument Co.) and are
filled with an internal solution containing (in mM): 1 NaCl, 177
N-methyl-D-glucamine, 10 EGTA, 2 MgCl2, 40 HEPES, and 25
phosphocreatine-tris (pH 7.2 with H2SO4). Recordings are performed
within 10-120 min after the culture medium is replaced by bath
recording solution and the dish with cells is placed on the
recording setup. Experimental data collected includes
current-voltage relationships, current density, voltage-dependence
of activation, voltage-dependence of inactivation, and recovery
from inactivation.
[0179] Current Clamp Recordings
[0180] Current clamp recordings are performed as described in Liu
et. al, Dravet syndrome patient-derived neurons suggest a novel
epilepsy mechanism. Ann Neurol. 2013; 74(1):128-39. PMCID:
3775921
[0181] For current-clamp recordings of action potentials in
iPSC-derived neurons, the patch pipette is filled with internal
solution consisting of (in mM): 135, K-gluconate; 4, NaCl; 0.5,
CaCl4; 10, HEPES; 5, EGTA, 2, Mg-ATP and 0.4, GTP (pH 7.3, adjusted
with KOH). iPSC neurons are bathed in a solution consisting of (in
mM): 115, NaCl; 2.5, KCl; 1, MgCl2; 1.25, KH2PO4; 26, NaHCO3; 2,
CaCl; 10 HEPES and 10, D-glucose (pH 7.4, adjusted with NaOH).
Individual action potentials are evoked from their resting membrane
potential by injection of a series of 1 ms depolarizing currents
beginning from the subthreshold level until consistent generation
of action potentials at 0.02 nA-increment. The minimal current
required for initiation of the first action potential is defined as
the threshold current. Repetitive spike firing is evoked by
injection of a 1500 ms depolarizing current (0.02 nA) from a
holding potential at their resting levels. Spontaneous firing is
recorded from neurons held at their resting membrane potential.
[0182] Quantitative data are presented as mean and SEM. Pairwise
statistical significance are determined with Student's two-tailed
paired/unpaired t-tests, or Mann-Whitney Rank Sum tests, as
appropriate. Multiple comparisons are made using ANOVA followed by
Tukey post-hoc analysis. Results are considered significant at
P<0.05.
[0183] Measurement of Spontaneous Action Potential Firing of iPSC
Cortical Neuron Clusters
[0184] Administration of Drugs During MEA Recordings:
[0185] Increasing concentrations of test compounds and vehicle are
added to the media of each well of a 96-well plate (6 wells per
subject per condition, with one control and one Dravet subject per
plate) containing human iPSC neurons following recordings of basal
activity levels. Each well contains 8 electrodes for extracellular
recordings of spontaneous action potentials. Recordings are made
for 5 minutes every 15 minutes over a 1-hour period, and are
repeated every week over a 3 week period during weeks 5-7 of
neuronal differentiation. All experiments are performed in
duplicate.
[0186] MEA Recordings
[0187] Control and Dravet Syndrome human iPSC-derived neurons are
cultured on the Axion 96-well MEA chips, which contain 8 electrodes
per well. Plates are coated with fibronectin and seeded with neural
progenitors at the density of 1.3.times.106 cells/ml. The
extracellular electrical signals detected by the MEA system are
amplified using the built-in Axion amplifier and sampling software,
and the closed system maintains the cells at 37 degrees C. and
allows for maintaining a 5% CO2 environment for prolonged
recordings. Action potential analyses are performed using
NeuroExplorer software. Spike rates per well and per electrode,
bursting rates, degree of synchronous discharges (i.e., occurring
simultaneously at multiple electrodes) and local field potential
morphology are determined.
[0188] Notwithstanding the appended claims, the disclosure set
forth herein is also defined by the following clauses:
[0189] Clause 1. A method of treating epilepsy or a neurological
related disease, comprising administering to a patient in need
thereof a therapeutically effective amount of a compound of formula
(I):
##STR00019##
[0190] wherein:
[0191] R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6 and
R.sub.7 are independently selected from hydrogen, halogen, X.sub.1,
X.sub.2, alkoxy, acyl, substituted acyl, carboxy, cyano, hydroxy,
alkoxy, substituted alkoxy, alkyl, substituted alkyl, aryl,
substituted aryl, heterocycle, heteroaryl, substituted heteroaryl,
substituted heterocycle, or together with a second R.sup.1-R.sup.7
group form a cycloalkyl ring, a heterocycle ring, an aryl ring or a
heteroaryl ring that is optionally substituted wherein R.sub.2 and
R.sub.5, R.sub.2 and R.sub.4, R.sub.1 and R.sub.5, R.sub.6 and
R.sub.7, and/or R.sub.3 and R.sub.6 are cyclically linked;
[0192] X.sub.1-X.sub.5 are each independently H, D, F, alkyl or
substituted alkyl;
[0193] m is 0-4; and
[0194] n is 1 or 2, wherein when n is 2 the nitrogen is positively
charged;
[0195] or a pharmaceutically acceptable salt thereof.
[0196] Clause 2. The method of clause 1, wherein the compound is a
compound having the formula (II):
##STR00020##
wherein:
[0197] R.sub.1 is an alkyl, a substituted alkyl (e.g., CF.sub.3) or
SF.sub.5;
[0198] R.sub.2, R.sub.3, R.sub.4 and R.sub.5 are independently
selected from hydrogen, halogen, alkoxy, acyl, substituted acyl,
carboxy, cyano, hydroxy, alkoxy, substituted alkoxy, alkyl,
substituted alkyl, aryl, substituted aryl, heterocycle, heteroaryl,
substituted heteroaryl and substituted heterocycle, where R.sub.2
and R.sub.5 or R.sub.2 and R.sub.4 are optionally cyclically
linked;
[0199] X.sub.1-X.sub.5 are each independently H, D, F, alkyl or
substituted alkyl; and
n is 1 or 2, wherein when n is 2 the nitrogen is positively
charged;
[0200] or a salt thereof.
[0201] Clause 3. The method of clause 1 or 2, wherein the compound
is a compound having the formula (III):
##STR00021##
wherein X.sub.1-X.sub.7 are each independently H, D or F, and
R.sub.1, R.sub.3 and R.sub.4 are as defined in any of the
embodiments of formula (I).
[0202] Clause 4. The method of clause 1 or 2, wherein the compound
is a compound having the formula (IV):
##STR00022##
wherein X.sub.8-X.sub.10 and each X are independently H, D or F,
provided at least one X.sub.8-X.sub.10 or X is F.
[0203] Clause 5. The method of clause 4, wherein the compound is a
compound having one of the following structures:
##STR00023##
[0204] Clause 6. The method of any one of clauses 1, 2 and 4,
wherein the compound is a compound having one of the formulae
(IVa)-(IVc):
##STR00024##
[0205] wherein X.sub.11, X.sub.12 and each X is independently H, D
or F; and
[0206] R.sub.11-R.sub.16 are each independently an alkyl or a
substituted alkyl.
[0207] Clause 7. The method of clause 1, wherein the compound is a
compound having the formula (V):
##STR00025##
wherein R.sub.1, R.sub.3, R.sub.5, R.sub.6, R.sub.7 and m are as
defined above, p is 0, 1 or 2, and R.sub.8 and R.sub.9 are
independently selected from hydrogen, halogen, alkoxy, acyl,
substituted acyl, carboxy, cyano, hydroxy, alkoxy, substituted
alkoxy, alkyl, substituted alkyl, aryl, substituted aryl,
heterocycle, heteroaryl, substituted heteroaryl and substituted
heterocycle, or R.sub.8 and R.sub.9 are cyclically linked to form a
5 or 6-membered cycloalkyl, heterocycle, aryl or heteroaryl ring,
that is optionally further substituted, where the dashed bond
represents a single or double covalent bond.
[0208] Clause 8. The method of clause 7, wherein the compound is a
compound having the formula (VI):
##STR00026##
wherein R.sub.1, R.sub.3, R.sub.7, and m are as defined above, and
p is 0, 1 or 2.
[0209] Clause 9. The method of clause 8, wherein the compound is a
compound having one of the following structures:
##STR00027##
or a prodrug thereof, or a stereoisomer thereof, or a salt
thereof.
[0210] Clause 10. The method of clause 7, wherein the compound is a
compound having formula (VII):
##STR00028##
or a prodrug thereof, or a stereoisomer thereof, or a salt
thereof.
[0211] Clause 11. The method of clause 10, wherein the compound has
the structure:
##STR00029##
or a prodrug thereof, or a stereoisomer thereof, or a salt
thereof.
[0212] Clause 12. The method of clause 1, wherein the compound is a
compound having the formula (VIII):
##STR00030##
wherein R.sub.1-R.sub.4, R.sub.6, R.sub.7 and m are as defined
above.
[0213] Clause 13. The method of clause 12, wherein the compound is
a compound having the formula (IX):
##STR00031##
wherein R.sub.1, R.sub.3, R.sub.4, and each m are as defined
above.
[0214] Clause 14. The method of clause 13, wherein the compound has
the structure:
##STR00032##
or a prodrug thereof, or a stereoisomer thereof, or a salt
thereof.
[0215] Clause 15. The method of clause 12, wherein the compound is
a compound having the formula (X):
##STR00033##
wherein R.sub.1-R.sub.4, and each m are as defined above, and q is
0, 1 or 2.
[0216] Clause 16. The method of clause 15, wherein the compound is
a compound having has the formula (XIa) or (XIb):
##STR00034##
[0217] Clause 17. The method of clause 16, wherein the compound has
the structure:
##STR00035##
or a prodrug thereof, or a stereoisomer thereof, or a salt
thereof.
[0218] Clause 18. The method of clause 7, further comprising
co-administering to the subject an antiepileptic agent.
[0219] Clause 19. A method of suppressing appetite in a subject,
comprising administering to the subject in need thereof an appetite
suppressing-amount of a compound of formula (I):
##STR00036##
[0220] wherein:
[0221] R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6 and
R.sub.7 are independently selected from hydrogen, halogen, X.sub.1,
X.sub.2, alkoxy, acyl, substituted acyl, carboxy, cyano, hydroxy,
alkoxy, substituted alkoxy, alkyl, substituted alkyl, aryl,
substituted aryl, heterocycle, heteroaryl, substituted heteroaryl,
substituted heterocycle, or together with a second R.sup.1-R.sup.7
group form a cycloalkyl ring, a heterocycle ring, an aryl ring or a
heteroaryl ring that is optionally substituted wherein R.sub.2 and
R.sub.5, R.sub.2 and R.sub.4, R.sub.1 and R.sub.5, R.sub.6 and
R.sub.7, and/or R.sub.3 and R.sub.6 are cyclically linked;
[0222] X.sub.1-X.sub.5 are each independently H, D, F, alkyl or
substituted alkyl;
[0223] m is 0-4; and
[0224] n is 1 or 2, wherein when n is 2 the nitrogen is positively
charged;
[0225] or a pharmaceutically acceptable salt thereof.
[0226] Clause 20. The method of clause 19, wherein the compound is
a compound having the formula (II):
##STR00037##
wherein:
[0227] R.sub.1 is an alkyl, a substituted alkyl (e.g., CF.sub.3) or
SF.sub.5;
[0228] R.sub.2, R.sub.3, R.sub.4 and R.sub.5 are independently
selected from hydrogen, halogen, alkoxy, acyl, substituted acyl,
carboxy, cyano, hydroxy, alkoxy, substituted alkoxy, alkyl,
substituted alkyl, aryl, substituted aryl, heterocycle, heteroaryl,
substituted heteroaryl and substituted heterocycle, where R.sub.2
and R.sub.5 or R.sub.2 and R.sub.4 are optionally cyclically
linked;
[0229] X.sub.1-X.sub.5 are each independently H, D, F, alkyl or
substituted alkyl; and
n is 1 or 2, wherein when n is 2 the nitrogen is positively
charged;
[0230] or a salt thereof.
[0231] Clause 21. The method of clause 19 or 20, wherein the
compound is a compound having the formula (III):
##STR00038##
wherein X.sub.1-X.sub.7 are each independently H, D or F, and
R.sub.1, R.sub.3 and R.sub.4 are as defined in any of the
embodiments of formula (I).
[0232] Clause 22. The method of clause 19 or 20, wherein the
compound is a compound having the formula (IV):
##STR00039##
wherein X.sub.8-X.sub.10 and each X are independently H, D or F,
provided at least one X.sub.8-X.sub.10 or X is F.
[0233] Clause 23. The method of clause 22, wherein the compound is
a compound having one of the following structures:
##STR00040##
[0234] Clause 24. The method of any one of clauses 19, 20 and 22,
wherein the compound is a compound having one of the formulae
(IVa)-(IVc):
##STR00041##
[0235] wherein X.sub.11, X.sub.12 and each X is independently H, D
or F; and
[0236] R.sub.11-R.sub.16 are each independently an alkyl or a
substituted alkyl.
[0237] Clause 25. The method of clause 19, wherein the compound is
a compound having the formula (V):
##STR00042##
wherein R.sub.1, R.sub.3, R.sub.5, R.sub.6, R.sub.7 and m are as
defined above, p is 0, 1 or 2, and R.sub.8 and R.sub.9 are
independently selected from hydrogen, halogen, alkoxy, acyl,
substituted acyl, carboxy, cyano, hydroxy, alkoxy, substituted
alkoxy, alkyl, substituted alkyl, aryl, substituted aryl,
heterocycle, heteroaryl, substituted heteroaryl and substituted
heterocycle, or R.sub.8 and R.sub.9 are cyclically linked to form a
5 or 6-membered cycloalkyl, heterocycle, aryl or heteroaryl ring,
that is optionally further substituted, where the dashed bond
represents a single or double covalent bond.
[0238] Clause 26. The method of clause 25, wherein the compound is
a compound having the formula (VI):
##STR00043##
wherein R.sub.1, R.sub.3, R.sub.7, and m are as defined above, and
p is 0, 1 or 2.
[0239] Clause 27. The method of clause 25, wherein the compound is
a compound having one of the following structures:
##STR00044##
or a prodrug thereof, or a stereoisomer thereof, or a salt
thereof.
[0240] Clause 28. The method of clause 27, wherein the compound is
a compound having formula (VII):
##STR00045##
or a prodrug thereof, or a stereoisomer thereof, or a salt
thereof.
[0241] Clause 29. The method of clause 28, wherein the compound has
the structure:
##STR00046##
or a prodrug thereof, or a stereoisomer thereof, or a salt
thereof.
[0242] Clause 30. The method of clause 19, wherein the compound is
a compound having the formula (VIII):
##STR00047##
wherein R.sub.1-R.sub.4, R.sub.6, R.sub.7 and m are as defined
above.
[0243] Clause 31. The method of clause 30, wherein the compound is
a compound having the formula (IX):
##STR00048##
wherein R.sub.1, R.sub.3, R.sub.4, and each m are as defined
above.
[0244] Clause 32. The method of clause 31, wherein the compound has
the structure:
##STR00049##
or a prodrug thereof, or a stereoisomer thereof, or a salt
thereof.
[0245] Clause 33. The method of clause 30, wherein the compound is
a compound having the formula (X):
##STR00050##
wherein R.sub.1-R.sub.4, and each m are as defined above, and q is
0, 1 or 2.
[0246] Clause 34. The method of clause 33, wherein the compound is
a compound having has the formula (XIa) or (XIb):
##STR00051##
[0247] Clause 35. The method of clause 34, wherein the compound has
the structure:
##STR00052##
or a prodrug thereof, or a stereoisomer thereof, or a salt
thereof.
[0248] The preceding merely illustrates the principles of the
invention. It will be appreciated that those skilled in the art
will be able to devise various arrangements which, although not
explicitly described or shown herein, embody the principles of the
invention and are included within its spirit and scope.
Furthermore, all examples and conditional language recited herein
are principally intended to aid the reader in understanding the
principles of the invention and the concepts contributed by the
inventors to furthering the art, and are to be construed as being
without limitation to such specifically recited examples and
conditions. Moreover, all statements herein reciting principles,
aspects, and embodiments of the invention as well as specific
examples thereof, are intended to encompass both structural and
functional equivalents thereof. Additionally, it is intended that
such equivalents include both currently known equivalents and
equivalents developed in the future, i.e., any elements developed
that perform the same function, regardless of structure. The scope
of the present invention, therefore, is not intended to be limited
to the exemplary embodiments shown and described herein. Rather,
the scope and spirit of present invention is embodied by the
appended claims.
* * * * *
References