Note: Descriptions are shown in the official language in which they were submitted.
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TITLE OF THE INVENTION
ALPHA la ADRENERGIC RECEPTOR ANTAGONISTS
This application claims the benefit of U.S. Provisional
Application No. 60/050,126, filed June 18, 1997.
FIELD OF THE INVENTION:
This invention relates to certain novel compounds and
derivatives thereof, their synthesis, and their use as alpha la
adrenoceptor antagonists. More particularly, the compounds of the
present invention are useful for treating benign prostatic hyperplasia
(BPH?.
BACKGROUND OF THE INVENTION
Human adrenergic receptors are integral membrane
proteins which have been classified into two broad classes, the alpha
and the beta adrenergic receptors. Both types mediate the action of the
peripheral sympathetic nervous system upon binding of
catecholamines, norepinephrine and epinephrine.
Norepinephrine is produced by adrenergic nerve endings,
while epinephrine is produced by the adrenal medulla. The binding
affinity of adrenergic receptors for these compounds forms one basis of
the classification: alpha receptors bind norepinephrine more strongly
than epinephrine and much more strongly than the synthetic compound
isoproterenol. The binding affinity of these hormones is reversed for the
beta receptors. In many tissues, the functional responses, such as
smooth muscle contraction, induced by alpha receptor activation are
opposed to responses induced by beta receptor binding.
Subsequently, the functional distinction between alpha and
beta receptors was further highlighted and refined by the
pharmacological characterization of these receptors from various
animal and tissue sources. As a result, alpha and beta adrenergic
receptors were further subdivided into alpha 1~ alpha 2~ 131, and 132
subtypes. Functional differences between alpha 1 and alpha 2 receptors
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have been recognized, and compounds which exhibit selective binding
between these two subtypes have been developed.
For a general background on the alpha adrenergic
receptors, the reader's attention is directed to Robert R. Ruffolo, Jr., ~,
,A,drenoreceutors~ Molecular BioloQV Biochemistr~i and Pharmacoloev,
(Proeress in Basic and Clinical Pharmacology series, Karger, 1991),
wherein the basis of alpha 1/alpha 2 subclassification, the molecular
biology, signal transduction (G-protein interaction and location of the
significant site for this and ligand binding activity away from the 3'-
terminus of alpha adrenergic receptors), agonist structure-activity
relationships, receptor functions, and therapeutic applications for
compounds exhibiting alpha-adrenergic receptor affinity was explored.
The cloning, sequencing and expression of alpha receptor
subtypes from animal tissues has led to the subclassification of the
alpha 1 receptors into alpha ld (formerly known as alpha la or la/1d),
alpha lb and alpha la (formerly known as alpha lc) subtypes. Each
alpha 1 receptor subtype exhibits its own pharmacologic and tissue
specificities. The designation "alpha 1a" is the appellation recently
approved by the IUPHAR Nomenclature Committee for the previously
designated "alpha lc" cloned subtype as outlined in the 1995 Receptor
and Ion Channel Nomenclature Supplement (Watson and Girdlestone,
1995). The designation alpha la is used throughout this application to
refer to this subtype. At the same time, the receptor formerly designated
alpha la was renamed alpha ld. The new nomenclature is used
throughout this application. Stable cell lines expressing these alpha 1
receptor subtypes are referred to herein; however, these cell lines were
deposited with the American Type Culture Collection (ATCC) under the
old nomenclature. For a review of the classification of alpha 1
adrenoceptor subtypes, see, Martin C. Michel, et al., Naunyn-
Schmiedeberg's Arch. Pharmacol. ( 1995 ) 352:1-10.
The differences in tl- ~ alpha adren«rgic receptor subtypes
have relevance in pathophysiolog. : conditions. . ~~nign prostatic
hyperplasia, also known as benign prostatic hypertrophy or BPH, is an
illness typically affecting men over fifty years of age, increasing in
severity with increasing age. The symptoms of the condition include,
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but are not limited to, increased difficulty in urination and sexual
dysfunction. These symptoms are induced by enlargement, or
hyperplasia, of the prostate gland. As the prostate increases in size, it
impinges on free-flow of fluids through the male urethra.
Concommitantly, the increased noradrenergic innervation of the
enlarged prostate leads to an increased adrenergic tone of the bladder
neck and urethra, further restricting the flow of urine through the
urethra.
In benign prostatic hyperplasia, the male hormone 5alpha-
dihydrotestosterone has been identified as the principal culprit. The
continual production of 5a-dihydrotestosterone by the male testes
induces incremental growth of the prostate gland throughout the life of
the male. Beyond the age of about fifty years, in many men, this
enlarged gland begins to obstruct the urethra with the pathologic
symptoms noted above.
The elucidation of the mechanism summarized above has
resulted in the recent development of effective agents to control, and in
many cases reverse, the pernicious advance of BPH. In the forefront of
these agents is Merck & Co., Inc's product PROSCAR~ (finasteride).
The effect of this compound is to inhibit the enzyme testosterone 5-a
reductase, which converts testosterone into 5a-dihydrotesterone,
resulting in a reduced rate of prostatic enlargement, and often reduction
in prostatic mass.
The development of such agents as PROSCAR~ bodes well
for the long-term control of BPH. However, as may be appreciated from
the lengthy development of the syndrome, its reversal also is not
immediate. In the interim, those males suffering with BPH continue to
suffer, and may in fact lose hope that the agents are working sufficiently
rapidly.
In response to this problem, one solution is to identify
pharmaceutically active compounds which complement slower-acting
therapeutics by providing acute relief. Agents which induce relaxation
of the lower urinary tract tissue, by binding to alpha 1 adrenergic
receptors, thus reducing the increased adrenergic tone due to the
disease, would be good candidates for this activity. Thus, one such agent
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is alfuzosin, which is reported in EP 0 204597 to induce urination in
cases of prostatic hyperplasia. Likewise, in WO 92/0073, the selective
ability of the R(+) enantiomer of terazosin to bind to adrenergic receptors
of the alphal subtype was reported. In addition, in WO 92//61213,
combinations of 5a-reductase inhibitory compounds and alphal-
adrenergic receptor blockers (terazosin, doxazosin, prazosin, bunazosin,
indoramin, alfuzosin) were disclosed. However, no information as to
the alpha ld, alpha lb, or alpha 1a subtype specificity of these
compounds was provided as this data and its relevancy to the treatment
of BPH was not known. Current therapy for BPH uses existing non-
selective alpha 1 antagonists such as prazosin (Minipress, Pfizer),
Terazosin (Hytrin, Abbott) or doxazosin mesylate (Cardura, Pfizer).
These non-selective antagonists suffer from side effects related to
antagonism of the alpha ld and alpha 1b receptors in the peripheral
vasculature, e.g., hypotension and syncope.
The recent cloning of the human alpha 1a adrenergic
receptor (ATCC CRL 11140) and the use of a screening assay utilizing
the cloned human alpha 1a receptor enables identification of compounds
which specifically interact with the human alpha la adrenergic
receptor. [PCT International Application Publication Nos. W094/08040,
published 14 April 1994 and W094/10989, published 26 May 1994] As
disclosed in the instant patent disclosure, a cloned human alpha la
adrenergic receptor and a method for identifying compounds which bind
the human alpha la receptor has now made possible the identification of
selective human alpha la adrenergic receptor antagonists useful for
treating BPH. The instant patent disclosure discloses novel compounds
which selectively bind to the human alpha la receptor. These
compounds are further tested for binding to other human alpha 1
receptor subtypes, as well as counterscreened against other types of
receptors (e.g., alpha 2), thus defining the specificity of the compounds
of the present invention for the human alpha 1a adrenergic rec otor.
It is an object of the present invention to identify cc ..pounds
which bind to the alpha 1a adrenergic receptor. It is a further object of
the invention to identify compounds which act as antagonists of the
alpha 1a adrenergic receptor. It is another object of the invention to
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identify alpha la adrenergic receptor antagonist compounds which are
useful agents for treating BPH in animals, preferably mammals,
especially humans. Still another object of the invention is to identify
alpha la adrenergic receptor antagonists which are useful for relaxing
lower urinary tract tissue in animals, preferably mammals, especially
humans.
It has now been found that the compounds of the present
invention are alpha 1a adrenergic receptor antagonists. Thus, the
compounds of the present invention are useful for treating BPH in
mammals. Additionally, it has been found that the alpha la adrenergic
receptor antagonists of the present invention are also useful for relaxing
lower urinary tract tissue in mammals.
SUMMARY OF THE INVENTION
The present invention provides compounds for the
treatment of urinary obstruction caused by benign prostatic hyperplasia
(BPH). The compounds antagonize the human alpha la adrenergic
receptor at nanomolar and subnanomolar concentrations while
exhibiting at least ten fold lower affinity for the alpha ld and alpha lb
human adrenergic receptors and many other G-protein coupled
receptors. This invention has the advantage over non-selective alpha 1
adrenoceptor antagonists of reduced side eiI'ects related to peripheral
adrenergic blockade. Such side effects include hypotension, syncope,
lethargy, etc. The compounds of the present invention have the
structure:
R~s
M 2 R15 m O
n E R q R2 I~
w
N CRs ~Q
RLN J ~
13~p
R Rya
wherein Q is selected from
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(X)s ' (X)s ' (X)s
/ / /
Ra ~~ Rs ~~ Rs
~~N N Rs N
Rio S
O N R5 O O ~ O~~ s
H , Rio R
(X)s (X)s
/~
p RlyR~2
~~N W ~~N W
O N 7~R» O H
H R~2 , O
//(X)s
R2 Ris
t 1 ~~ (X)s ~ NRs
i
or R5 N O
H
R1 is selected from unsubstituted, mono- or poly-substituted phenyl
wherein the substituents on the phenyl are independently selected from
halogen, CFg, cyano, vitro, N(R7)2, NR7COR19, NR7CON(R19)2,
NR7S02R19, NR7S02N(R19)2, OR,6, (CH2)o~CO2R,7,
(CH2)o-4CON(R7)2, or C1_4 alkyl; or unsubstituted, mono- or poly-
substituted pyridyl, pyrazinyl, thienyl, thiazolyl, furanyl, quinazolinyl or
naphthyl wherein the substituents on the pyridyl, pyrazinyl, thienyl,
thiazolyl, furanyl, quinazolinyl or naphthyl are independently selected
from CF3, cyano, vitro, amino, (CH2)o-4C02R7, (CH2)~4CON(R7)2,
(CH2)o~S02N(R7)2, (CH2)o~S02R,s, phenyl, ORS, halogen, C1~ alkyl or
C3_g cycloalkyl;
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E, G, L and M are each independently selected from hydrogen,
C1-g alkyl, Cg-g cycloalkyl, (CH2)o-408,6, (CH2)o-4IV(R7)2,
(CH2)o-4CN~ (CH2)o-4CF3, (CH2)o~C02R,7~ (CH2)o-4CON(R7)2~
(CH2)o..4S02R7, or (CH2)o-4S02N(R7)2~
J is selected from hydrogen, C1-g alkyl, C3-g cycloalkyl, (CH2y-4086,
(CH2)1-4N(R7)2~ (CH2u-4CN~ (CH2)o-4CF3~ (CH2)o-4C02R7~
(CH2)o-4CON(R7)2, (CH2)O~S02R7, or (CH2)o..4S02N(R7)2;
R2 is independently selected from hydrogen, C1-g alkyl,
C4-g cycloalkyl, (CH2)o-4CO2R7> (CH2)o-4CON(R7)2, (CH2)o_4COR7,
(CH2)2-4OR6~ (CH2)1_4CF3, (CH2)o-4S02R7, (CH2)o-4sO2N(R7)2 or (CH2)~-
4CN;
R3 , R8, R9, 810, R14~ 815 ~d 816 ~.e each independently selected from
hydrogen, C1-g alkyl, Cg-g cycloalkyl, (CH2)2-4086 or (CH2)o-4CF3;
R4 is selected from hydrogen, (CH2)o-4COR6, (CH2)o-4CN, (CH2)o-4CF3,
(CH2)o-4C02R7~ (CH2)o-4CON(R7)2~ (CH2)o-48028,6, or
(CH2)o-4SO2N(R7)2~
R5 is selected from hydrogen, C1-g alkyl, Cg-g cycloalkyl,
(CH2y-4086 or (CH2)o-4CF3~
R6 is selected from hydrogen, C1-g alkyl, C3-g cycloalkyl or
(CH2)o_4CF3~
R7 and 819 are each independently selected from hydrogen, C1-g alkyl,
C4-g cycloalkyl or (CH2)1-4CF3~
811 and 812 are each independently selected from hydrogen,
C1-g alkyl or Cg-g cycloalkyl;
813 is selected from hydrogen, C 1-g alkyl, C3-g cycloalkyl,
(CH2)2-408.6, OR6 or (CH2)0-4CF3~
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R18 is selected from hydrogen, C1-g alkyl, C3-g cycloalkyl,
(CH2)1~OR6, (CH2)0-4CF3, unsubstituted, mono- or poly-substituted
phenyl wherein the substituents on the phenyl are independently
selected from halogen, CF3, cyano, vitro, C02R7, OR6,
(CH2)0-4CON(R7)2, (CH2)0-4C02R7 or C~_4 alkyl; or unsubstituted,
mono- or poly-substituted: pyridyl, pyrazinyl, thienyl, furanyl or
naphthyl wherein the substituents on the pyridyl, pyrazinyl, thienyl,
furanyl or naphthyl are independently selected from CF3, phenyl, OR6,
halogen, C1-4 alkyl or C3_g cycloalkyl;
R20 is selected from hydrogen, C 1-g alkyl, C3-g cycloalkyl,
(CH2)o-4086 or (CH2)o-4CF3;
WisOorNR.ll;
R26 is selected from hydrogen or OR2g;
R28 is selected from hydrogen, C 1-g alkyl, C3-g cycloalkyl, (CH2)2-4086
or (CH2)0-4CF3;
X is selected from halogen, cyano, vitro, C1-g alkyl, C3-g cycloalkyl,
(CH2)o~OR,s or (CH2)o~CF3
m, p and q are each independently an integer of from zero to three,
provided that when q is zero, R26 is hydrogen;
n, o, s and t are each independently an integer of from zero to four;
and the pharmaceutically acceptable salts thereof.
In a first embodiment of the invention are compounds
having the str -'.~ure
_g_
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R~s
M 2 R's m 0
R '~
w
n E N CRs q N~Q
RL J ~
t3~P
b L G R Rla
wherein R4 is selected from (CH2)o-4COR6, (CH2)o-4CN, (CH2)o_4CFg,
(CH2)o-4C02R7, (CH2)o-4CON(R7)2, (CH2)o-4S02R6, or (CH2)o_
4S02N(R7)2;
R13 is selected from hydrogen, C1-g alkyl, Cg-g cycloalkyl,
(CH2)2-4086 or (CH2)0-4CF3~
and all other variables are as previously defined; and the
pharmaceutically acceptable salts thereof.
In a second embodiment of the instant invention is the
compound of the formula:
R~s
M 2 R'5 m0
E R qR2
~v
N CR3 ~Q
R -N
J 13~~P
R Rya
wherein Q is selected from
-9-
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'~x)S \~x)S ,~x)S
I~ I~ I~
Ra ~~ Rs ~~ Ra
~~N N R9 N
I ~ Rio S
O N R 5 O O , O'
H ~ Rio R
R2~~a
or ~ t \ ~~ (x)S
R1 is selected from unsubstituted, mono-, di- or tri-substituted phenyl
wherein the substituents on the phenyl are independently selected from
halogen, CF3, cyano, vitro, N(R~)2, NR?CORl9, NR7CON(R19)2,
NR7S02R19, NR7S02N(R19)2, ORS, (CH2)0_4C02R7,
(CH2)0-4CON(R7)2, or C1~ alkyl; or unsubstituted, mono-, di- or tri-
substituted pyridyl, pyrazinyl, thienyl, thiazolyl, furanyl, quinazolinyl or
naphthyl wherein the substituents on the pyridyl, pyrazinyl, thienyl,
thiazolyl, furanyl, quinazolinyl or naphthyl are independently selected
from CFg, cyano, vitro, amino, (CH2)0-4C02R7, (CH2)0-4CON(R?)2,
(CH2)0-4S02N(R7)2, (CH2)0-4SO~R6, phenyl, OR6, halogen,
C1_4 alkyl or C3_g cycloalkyl;
E, G, L, M and J are each independently selected from hydrogen,
C1-g alkyl, C3-g cycloalkyl, or (CH2)0_4CF3;
R2 is selected from hydrogen, C1-g alkyl, C4-g cycloalkyl or
(CH2)1-4CF3~
R3 , R8, R9, R10, Rl~ x,15 ~d R16 ~.e each independently selected from
hydrogen, C1-6 alkyl, l.,g-6 cycloalkyl, (CH2)2-4086 or
(CH2)p~CFg;
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R13 is selected from hydrogen, C1-6 alkyl, Cg-6 cycloalkyl,
(CH2)2-4086, OR6 or (CH2)0-4CF3~
R18 is selected from hydrogen, C1-g alkyl, Cg-g cycloalkyl,
(CH2)1-408,6, (CH2)0-4CF3~ substituted, mono-, di- or tri-substituted
phenyl wherein the substituents on the phenyl are independently
selected from halogen, CFg, cyano, vitro, C02R7, OR6, (CH2)0-
4CON(R7)2, (CH2)0-4C02R7 or C1-4 alkyl; or unsubstituted, mono-, di- or
tri-substituted: pyridyl, pyrazinyl, thienyl, furanyl or naphthyl wherein
the substituents on the pyridyl, pyrazinyl, thienyl, furanyl or naphthyl
are independently selected from CFg, phenyl, OR6, halogen, C1-4 alkyl
or C3_g cycloalkyl; and
R20 is selected from hydrogen, C 1-6 alkyl, C3-6 cycloalkyl,
(CH2)o-4086 or (CH2)o-4CF3;
R28 is selected from hydrogen, C1-6 alkyl, Cg-6 cycloalkyl, (CH2)2-4086
or (CH2)0-4CF3~
m, n and p are each independently an integer from zero to two, provided
that when q is zero, R28 is hydrogen;
and all other variables are as originally defined;
and the pharmaceutically acceptable salts thereof.
In a third embodiment of the instant invention is the
compound of the formula:
R~s
M 2 R~5 m0
w
E N CR3 q ~Q
L
R J
R R~4
wherein to is selected from
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,~x)S '~x)s ,~x)s
li li li
R8
~.N Ra ~~N R Rs ~~N
( ~ R1o S
O N R5 O O , O~ s
H ~ R1o R
R2~18
Of ~ t ~ \~ ~x)s
v i ~ ,
R1 is selected from unsubstituted, mono-, di- or tri-substituted phenyl
wherein the substituents on the phenyl are independently selected from
halogen, CF3, cyano, nitro, N(R7)2, NR7COR19, NR7CON(R19)2,
NR7S02R19, NR7S02N(R19)2, OR6, (CH2)0_4C02R7,
(CH2)0-4CON(R7)2, or C1_4 alkyl; or unsubstituted, mono-, di- or tri-
substituted pyridyl, pyrazinyl, thienyl, thiazolyl, furanyl, quinazolinyl or
naphthyl wherein the substituents on the pyridyl, pyrazinyl, thienyl,
thiazolyl, furanyl, quinazolinyl or naphthyl are independently selected
from CF3, cyano, nitro, amino, (CH2)0_4C02R7, (CH2)0-4CON(R7)2,
(CH2)0_4S02N(R7)2, (CH2)p~S02R6, Phenyl, OR6, halogen,
C1_4 alkyl or C3_g cycloalkyl;
E, G, L, M and J are each independently selected from hydrogen,
C1-g alkyl, C3-g cycloalkyl, or (CH2)0-4CF3;
R2 is selected from hydrogen, C1-g alkyl, C4-g cycloalkyl or
(CH2)1_4CF3;
R3 , R8, R9, R10, R ~ ~~, Rl'~ =;,15 ~d R16 ~.e each independently selected
from hydrogen, C1-6 alkyl, ~.3-6 cycloalkyl, (CH2)2~OR6 or (CH2)o-4CF3;
R18 is selected from hydrogen, C1-g alkyl, C3-g cycloalkyl,
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(CH2)1_4086, (CH2)p~CFg, unsubstituted, mono-, di- or tri-substituted
phenyl wherein the substituents on the phenyl are independently
selected from halogen, CF3, cyano, vitro, C02R7, OR6, (CH2)0_
4CON(R~)2, (CH2)0_4C02R7 or C1-4 alkyl; or unsubstituted, mono-, di- or
tri-substituted: pyridyl, pyrazinyl, thienyl, furanyl or naphthyl wherein
the substituents on the pyridyl, pyrazinyl, thienyl, furanyl or naphthyl
are independently selected from CF3, phenyl, OR6, halogen, C 1-4 alkyl
or Cg-g cycloalkyl; and
R20 is selected from hydrogen, C 1-6 alkyl, C3-6 cycloalkyl,
(CH2)o-4086 or (CH2)o-4CF3~
m, n and p are each independently an integer from zero to two;
and all other variables are as defined previously in the first embodiment;
1~ and the pharmaceutically acceptable salts thereof.
In a first class of the invention is the compound of the
formula
n /
R2 R26
R1_N N~CH2 q ~ N D
R~s ~ P
wherein Q is selected from
'~X)S \~x)s
H Rya
4
~~N R ~~N i ~X)s
or ~ t ~ ~
O N R5 O O
H '
R1 is selected from unsubstituted, mono-, di or tri-substituted phenyl
wherein the substituents on the phenyl are independently selected from
halogen, CF3, cyano, vitro, N(R7)2, OR6, (CH2)0_2C02R7,
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(CH2)0-2CON(R7)2, or C1_4 alkyl; or unsubstituted, mono- or di-
substituted pyridyl wherein the substituents are independently selected
from halogen, CFg, cyano, vitro, amino, OR6, C02R7, CON(R7)2 or C1-4
alkyl;
R2 is selected from hydrogen, C1-6 alkyl, C4-6 cycloalkyl or
(CH2)1-4CF3~
R4 is selected from hydrogen, CORE, (CH2)0-2C02R7, S02R6 or
(CH2)0-2CON(R7)2;
R5 is selected from hydrogen, C1-g alkyl, C3-6 cycloalkyl,
(CH2)1-3086 or (CH2)0-3CF3; and
1~ R6 is selected from hydrogen, C 1-6 alkyl, Cg-6 cycloalkyl or
(CH2)p_2CF3~
R7 is selected from hydrogen, C 1-6 alkyl, C4-6 cycloalkyl or
(CH2)1_2CF3~
R13 is selected from hydrogen or OR6;
R18 is selected from hydrogen, C1-6 alkyl, C3-6 cycloalkyl,
(CH2)2-4086, (CH2)0-2CF3 or unsubstituted, mono- or di-substituted
phenyl wherein the substituents on the phenyl are independently
selected from halogen, CFg, cyano, vitro, amino, OR6, C02R7,
CON(R7)2 or C1_4 alkyl;
R26 is selected from hydrogen or OR28, wherein R28 is selected from
hydrogen or C 1-6 alkyl;
m, n and p ~ : a each indepE dently an integer from zero to one; and
t is an integer from one to two;
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and all other variables are as defined previously in the second
embodiment;
and the pharmaceutically acceptable salts thereof.
In a second class of the invention is the compound of the
formula
2
n
w
N CH q N
R>_N
lp
wherein Q is selected from
~X)s
/ ~/
H R's
4
~N R ~~N t i ~x)S
5 ~ or ~ 1 /
O N R O O
H '
R1 is selected from unsubstituted, mono-, di or tri-substituted phenyl
wherein the substituents on the phenyl are independently selected from
halogen, CF3, cyano, vitro, N(R7)2, OR6, (CH2)0-2C02R7,
(CH2)0-2CON(R7)2, or C1_4 alkyl; or unsubstituted, mono- or di-
substituted pyridyl wherein the substituents are independently selected
from halogen, CFg, cyano, vitro, amino, OR6, C02R7, CON(R7}2 or C1-4
alkyl;
R2 is selected from hydrogen, C1-6 alkyl, C4-6 cycloalkyl or
(CH2)1-4CF3~
R4 is selected from CORE, (CH2)0-2C02R7, S02R6 or
(CH2)0-2CON(R7)2~
R5 is selected from hydrogen, C1-6 alkyl, C3-6 cycloalkyl,
(CH2)1-3086 or (CH2)0-3CF3; and
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R6 is selected from hydrogen, C1-6 alkyl, C3-6 cycloalkyl or
(CH2)0-2CF3~
R7 is selected from hydrogen, C1-6 alkyl, C4-6 cycloalkyl or
(CH2)1-2CF3~
R1g is selected from hydrogen, C1-g alkyl, C3-g cycloalkyl,
(CH2)2-4086, (CH2)0-2CF3 or unsubstituted, mono- or di-substituted
phenyl wherein the substituents on the phenyl are independently
selected from halogen, CF3, cyano, vitro, amino, OR6, C02R7,
CON(R?)2 or C1_4 alkyl;
m, n and p are each independently an integer from zero to one;
1~ t is an integer from one to two;
and all other variables are as defined previously in the third
embodiment;
and the pharmaceutically acceptable salts thereof.
In a first subclass of the invention is the compound of the
formula
~R 1 ~)r
m
~N N
-A R2 \ / N II Q
P
O
wherein A is C-R17 or N;
R2 is selected from hydrogen or CH2CF3;
R13 is selec~.ed from hydrogen a nydroxy;
each R17 is independently selected from hydrogen, halogen, CF3, cyano,
vitro, amino, OR6, C02R7, CON(R.7)2 or C~-4 alkyl;
- is -
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R26 is selected from hydrogen or hydroxy;
each X is halogen;
q and r are each independently an integer from zero to two, provided that
when q is zero, R26 is hydrogen; and
s is an integer from zero to three;
and all other variables are as previously defined in the first class;
and the pharmaceutically acceptable salts thereof.
In a second subclass of the invention is the compound of the
formula
~R »~
q R2s
-- N N
-A R2 ~~N~Q
R~3 ~ J IIP
O
20
wherein A is C-R17 or N;
R2 is selected from hydrogen or CH2CFg;
each R17 is independently selected from hydrogen, halogen, CFg, cyano,
vitro, amino, OR6, C02R7, CON(R7)2 or Cl-q. alkyl;
each X is halogen;
q and r are each independently an integer from zero to two; and
s is an integer from zero to three;
and all other variables are as previously defined in the second class;
and the pharmaceutically acceptable salts thereof.
Illustrative of the invention is the compound selected from
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CN
H
N O~ N i
O
H N N I O
O = O or
_F
F
CN
N
O
-O
N NJ
O
F
F
and the pharmaceutically acceptable salts thereof.
An illustration of the invention is a pharmaceutical
composition comprising a therapeutically effective amount of any of the
compounds described above and a pharmaceutically acceptable carrier.
An example of the invention is a pharmaceutical composition made by
combining any of the compounds described above and a
pharmaceutically acceptable carrier. Another illustration of the
invention is a process for making a pharmaceutical composition
comprising combining any of the compounds described above and a
pharmaceutically acceptable carrie
Another example of the -e~ntion _ y the composition further
comprising a therapeutically effective amount of a testosterone 5-alpha
reductase inhibitor. Preferably, the testosterone 5-alpha reductase
inhibitor is a type 1, a type 2, both a type 1 and a type 2 (i.e., a three
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component combination comprising any of the compounds described
above combined with both a type 1 testosterone 5-alpha reductase
inhibitor and a type 2 testosterone 5-alpha reductase inhibitor) or a dual
type 1 and type 2 testosterone 5-alpha reductase inhibitor. More
preferably, the testosterone 5-alpha reductase inhibitor is a type 2
testosterone 5-alpha reductase inhibitor. Most preferably, the
testosterone 5-alpha reductase inhibitor is finasteride.
More specifically illustrating the invention is a method of
treating benign prostatic hyperplasia in a subject in need thereof which
comprises administering to the subject a therapeutically effective
amount of any of the compounds (or any of the compositions) described
above.
Further exemplifying the invention is the method of
treating BPH wherein the compound (or composition) additionally does
not cause a fall in blood pressure at dosages effective to alleviate BPH.
Another illustration of the invention is the method of
treating benign prostatic hyperplasia wherein the compound is
administered in combination with a testosterone 5-alpha reductase
inhibitor. Preferably, the testosterone 5-alpha reductase inhibitor is
finasteride.
Further illustrating the invention is a method of inhibiting
contraction of prostate tissue or relaxing lower urinary tract tissue in a
subject in need thereof which comprises administering to the subject a
therapeutically effective amount of any of the compounds (or any of the
compositions) described above.
More specifically exemplifying the invention is the method
of inhibiting contraction of prostate tissue or relaxing lower urinary
tract tissue wherein the compound (or composition) additionally does not
cause a fall in blood pressures at dosages effective to inhibit contraction
of prostate tissue.
More particularly illustrating the invention is the method of
inhibiting contraction of prostate tissue or relaxing lower urinary tract
tissue wherein the compound (or composition) is administered in
combination with a testosterone 5-alpha reductase inhibitor; preferably,
the testosterone 5-alpha reductase inhibitor is finasteride.
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More particularly exemplifying the invention is a method of
treating a disease which is susceptible to treatment by antagonism of the
alpha la receptor which comprises administering to a subject in need
thereof an amount of any of the compounds described above effective to
treat the disease. Diseases which are susceptible to treatment by
antagonism of the alpha la receptor include, but are not limited to, BPH,
high intraocular pressure, high cholesterol, impotency, sympathetically
mediated pain, migraine (see, K.A. Vatz, Headache 1997:37: 107-108)
and cardiac arrhythmia.
An additional illustration of the invention is the use of any
of the compounds described above in the preparation of a medicament
for: a) the treatment of benign prostatic hyperplasia; b) relaxing lower
urinary tract tissue; or c) inhibiting contraction of prostate tissue; in a
subject in need thereof.
An additional example of the invention is the use of any of
the alpha 1a antagonist compounds described above and a 5-alpha
reductase inhibitor for the manufacture of a medicament for: a) treating
benign prostatic hyperplasia; b) relaxing lower urinary tract tissue; or c)
inhibiting contraction of prostate tissue which comprises an effective
amount of the alpha la antagonist compound and an effective amount of
5-alpha reductase inhibitor, together or separately.
DETAILED DESCRIPTION OF THE INVENTION
Representative compounds of the present invention exhibit
high selectivity for the human alpha la adrenergic receptor. One
implication of this selectivity is that these compounds display selectivity
for lowering intraurethral pressure without substantially affecting
diastolic blood pressure.
Representative compounds of this invention display
submicromolar ai~nity for the human alpha la adrenergic receptor
subtype while displaying at least ten-fold lower affinity for the human
alphald and alphalb adrenergic receptor subtypes, and many other G-
protein coupled human receptors. Particular representative compounds
of this invention exhibit nanomolar and subnanomolar affinity for the
human alpha la adrenergic receptor subtype while displaying at least 30
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fold lower affinity for the human alphald and alphalb adrenergic
receptor subtypes, and many other G-protein coupled human receptors
(e.g., serotonin, dopamine, alpha 2 adrenergic, beta adrenergic or
muscarinic receptors).
These compounds are administered in dosages effective to
antagonize the alpha 1a receptor where such treatment is needed, as in
BPH. For use in msdicine, the salts of the compounds of this invention
refer to non-tonic "pharmaceutically acceptable salts." Other salts may,
however, be useful in the preparation of the compounds according to the
invention or of their pharmaceutically acceptable salts. Suitable
pharmaceutically acceptable salts of the compounds of this invention
include acid addition salts which may, for example, be formed by
mixing a solution of the compound according to the invention with a
solution of a pharmaceutically acceptable acid such as hydrochloric
1~ acid, sulphuric acid, fuznaric acid, malefic acid, succinic acid, acetic
acid, benzoic acid, citric acid, tartaric acid, carbonic acid or phosphoric
acid. Furthermore, where the compounds of the invention carry an
acidic moiety, suitable pharmaceutically acceptable salts thereof may
include alkali metal salts, e.g. sodium or potassium salts; alkaline
earth metal salts, e.g. calcium or magnesium salts; and salts formed
with suitable organic ligands, e.g. quaternary ammonium salts. Thus,
representative pharmaceutically acceptable salts include the following:
Acetate, Benzenesulfonate, Benzoate, Bicarbonate,
Bisulfate, Bitartrate, Borate, Bromide, Calcium, Camsylate, Carbonate,
Chloride, Clavulanate, Citrate, Dihydrochloride, Edetate, Edisylate,
Estolate, Esylate, Fumarate, Gluceptate, Gluconate, Glutamate,
Glycollylarsanilate, Hexylresorcinate, Hydrabamine, Hydrobromide,
Hydrochloride, Hydroxynaphthoate, Iodide, Isothionate, Lactate,
Lactobionate, Laurate, Malate, Maleate, Mandelate, Mesylate,
Methylbromide, Methylnitrate, Methylsulfate, Mutate, Napsylate,
Nitrate, N-methylglucamine ammonium salt, Oleate, Pamoate
(Embonate), Palmitate, Pantothenate, Phosphate/diphosphate,
Polygalacturonate, Salicylate, Stearate, Sulfate, Subacetate, Succinate,
Tannate, Tartrate, Teoclate, Tosylate, Triethiodide and Valerate.
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Compounds of this invention are used to reduce the acute
symptoms of BPH. Thus, compounds of this invention may be used
alone or in conjunction with a more long-term anti-BPH therapeutics,
such as testosterone 5-a reductase inhibitors, including PROSCAR~
(finasteride). Aside from their utility as anti-BPH agents, these
compounds may be used to induce highly tissue-specific, localized alpha
la adrenergic receptor blockade whenever this is desired. Effects of this
blockade include reduction of intra-ocular pressure, control of cardiac
arrhythmias, and possibly a host of alpha la receptor mediated central
nervous system events.
The present invention includes within its scope prodrugs of
the compounds of this invention. In general, such prodrugs will be
functional derivatives of the compounds of this invention which are
readily convertible ~ vivo into the required compound. Thus, in the
methods of treatment of the present invention, the term "administering"
shall encompass the treatment of the various conditions described with
the compound specifically disclosed or with a compound which may not
be specifically disclosed, but which converts to the specified compound in
vivo after administration to the patient. Conventional procedures for the
selection and preparation of suitable prodrug derivatives are described,
for example, in "Design of Prodrugs," ed. H. Bundgaard, Elsevier, 1985.
Metabolites of these compounds include active species produced upon
introduction of compounds of this invention into the biological milieu.
Where the compounds according to the invention have at
least one chiral center, they may accordingly exist as enantiomers.
Where the compounds according to the invention possess two or more
chiral centers, they may additionally exist as diastereoisomers. It is to
be understood that all such isomers and mixtures thereof are
encompassed within the scope of the present invention. Furthermore,
some of the crystalline forms for compounds of the present invention
may exist as polymorphs and as such ar~_ intended to be included in the
present invention. In adc on, some of the compounds of the present
invention may form solvates with water (i.e., hydrates) or common
organic solvents. Such solvates are also encompassed within the scope
of this invention.
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The term "alkyl" shall mean straight or branched chain
alkanes of one to ten total carbon atoms, or any number within this
range (i.e., methyl, ethyl, 1-propyl, 2-propyl, n-butyl, s-butyl, t-butyl,
etc.).
The term "alkenyl" shall mean straight or branched chain
alkenes of two to ten total carbon atoms, or any number within this
range.
The term "aryl" as used herein, except where otherwise
specifically defined, refers to unsubstituted, mono- or poly-substituted
aromatic groups such as phenyl or naphthyl.
The term "cycloalkyl" shall mean cyclic rings of alkanes of
three to eight total carbon atoms (i.e., cyclopropyl, cyclobutyl,
cyclopentyl, cyclohexyl, cycloheptyl or cyclooctyl).
Whenever the term "alkyl" or "aryl" or either of their prefix
roots appear in a name of a substituent (e.g., aralkoxyaryloxy) it shall be
interpreted as including those limitations given above for "alkyl" and
"aryl." Designated numbers of carbon atoms (e.g., C1_1p) shall refer
independently to the number of carbon atoms in an alkyl or cyclic alkyl
moiety or to the alkyl portion of a larger substituent in which alkyl
appears as its prefix root.
The term "halogen" shall include iodine, bromine, chlorine
and fluorine.
The term "substituted" shall be deemed to include multiple
degrees of substitution by a named substituent. The term "poly-
substituted" as used herein shall include di-, tri-, tetra- and penta-
substitution by a named substituent. Preferably, a poly-substituted
moiety is di-, tri- or tetra-substituted by the named substituents, most
preferably, di- or tri-substituted.
It is intended that the definition of any substituent or
variable (e.g., X, R6, R7, R19) at a particular location in a molecule be
independent of its definitions elsewhere in that molecule. Thus, -
N(R19)2 represents -NH2, -NHCH3, -NHC2H5, -N(CH3)C2H5, etc. It is
understood that substituents and substitution patterns on the
compounds of the instant invention can be selected by one of ordinary
skill in the art to provide compounds that are chemically stable and that
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can be readily synthesized by techniques known in the art as well as
those methods set forth below.
Where multiple substituent moieties are disclosed or
claimed, the substituted compound can be independently substituted by
one or more of the disclosed or claimed substituent moieties, singly or
plurally.
The term heterocycle or heterocyclic ring, as used herein,
represents an unsubstituted or substituted stable 5- to 7-membered
monocyclic ring system which may be saturated or unsaturated, and
which consists of carbon atoms and from one to three heteroatoms
selected from N, O or S, and wherein the nitrogen and sulfur
heteroatoms may optionally be oxidized, and the nitrogen heteroatom
may optionally be quaternized. The heterocyclic ring may be attached at
any heteroatom or carbon atom which results in the creation of a stable
structure. Examples of such heterocyclic groups include, but is not
limited to, piperidinyl, piperazinyl, oxopiperazinyl, oxopiperidinyl,
oxopyrrolidinyl, oxoazepinyl, azepinyl, pyrrolyl, pyrrolidinyl, furanyl,
thienyl, pyrazolyl, pyrazolidinyl, imidazolyl, imidazolinyl,
imidazolidinyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, oxazolyl,
oxazolidinyl, isooxazolyl, isoxazolidinyl, morpholinyl, thiazolyl,
thiazolidinyl, isothiazolyl, thiadiazolyl, tetrahydropyranyl,
thiamorpholinyl, thiamorpholinyl sulfoxide, thiamorpholinyl sulfone,
and oxadiazolyl. Morpholino is the same as morpholinyl.
The terms "(+)-DHP" and "DHP" as used herein, refers to a
dihydropyrimidinone group of the formula
(X)q
O
Ra
O' N
H
for example:
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F
F
O ~ O
~~N
~ I O
O~ N
H
The term "activated (+)-DHP," as used herein, refers to a N-
3-(activated)carbamate of the desired dihydropyrimidinone where the
activated group is, for example, a p-nitrophenyl group. A specific
example of an activated (+)-DHP is the compound 6 (see, e.g., Scheme 3).
The term "(S)-oxa" as used herein, refers to an
oxazolidinone group of the formula
~x>q
/
R8 Rs
fit, N ~R~o
-O
O
for example,
F
F
I
O
~t,~N
--O
O
The term "activated (S)-oxa" as used herein, refers to an N-
(activated)carbamate of the desired oxazolidinone where the activated
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group is, for example, a p-nitrophenyl group. A specific example of an
activated (S)-oxa group is the compound 13 (see, e.g., Scheme 4).
The term "thienyl," as used herein, refers to the group
S
The term "selective alpha la adrenergic receptor
antagonist," as used herein, refers to an alpha la antagonist compound
which is at least ten fold selective for the human alpha 1a adrenergic
receptor as compared to the human alpha lb, alpha ld, alpha 2a, alpha
2b and alpha 2c adrenergic receptors.
The term "lower urinary tract tissue," as used herein,
refers to and includes, but is not limited to, prostatic smooth muscle, the
prostatic capsule, the urethra and the bladder neck.
The term "subject," as used herein refers to an animal,
preferably a mammal, most preferably a human, who has been the
object of treatment, observation or experiment.
The term "therapeutically effective amount" as used
herein means that amount of active compound or pharmaceutical
agent that elicits the biological or medicinal response in a tissue,
system, animal or human that is being sought by a researcher,
veterinarian, medical doctor or other clinician, which includes
alleviation of the symptoms of the disease being treated.
The present invention also provides pharmaceutical
compositions comprising one or more compounds of this invention in
association with a pharmaceutically acceptable carrier. Preferably
these compositions are in unit dosage forms such as tablets, pills,
capsules, powders, granules, sterile parenteral solutions or
sup~~nsions, metered aerosol or liquid sprays, drops, ampoules, auto-
in, r devices or suppositories; for oral, parenteral, intranasal,
su :goal or rectal administration, or for administration by inhalation
or insui~lation. Alternatively, the compositions may be presented in a
form suitable for once-weekly or once-monthly administration; for
example, an insoluble salt of the active compound, such as the
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decanoate salt, may be adapted to provide a depot preparation for
intramuscular injection. For preparing solid compositions such as
tablets, the principal active ingredient is mixed with a pharmaceutical
carrier, e.g. conventional tableting ingredients such as corn starch,
lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate,
dicalcium phosphate or gums, and other pharmaceutical diluents, e.g.
water, to form a solid preformulation composition containing a
homogeneous mixture of a compound of the present invention, or a
pharmaceutically acceptable salt thereof. When referring to these
preformulation compositions as homogeneous, it is meant that the
active ingredient is dispersed evenly throughout the composition so that
the composition may be readily subdivided into equally effective unit
dosage forms such as tablets, pills and capsules. This solid
preformulation composition is then subdivided into unit dosage forms of
the type described above containing from 0.1 to about 500 mg of the active
ingredient of the present invention. The tablets or pills of the novel
composition can be coated or otherwise compounded to provide a dosage
form affording the advantage of prolonged action. For example, the
tablet or pill can comprise an inner dosage and an outer dosage
component, the latter being in the form of an envelope over the farmer.
The two components can be separated by an enteric layer which serves to
resist disintegration in the stomach and permits the inner component to
pass intact into the duodenum or to be delayed in release. A variety of
materials can be used for such enteric layers or coatings, such
materials including a number of polymeric acids and mixtures of
polymeric acids with such materials as shellac, cetyl alcohol and
cellulose acetate.
As used herein, the term "composition" is intended to
encompass a product comprising the specified ingredients in the
specified amounts, as well as any product which results, directly or
indirectly, from combination of the specified ingredients in the specified
amounts.
The liquid forms in which the novel compositions of the
present invention may be incorporated for administration orally ar by
injection include aqueous solutions, suitably flavoured syrups, aqueous
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or oil suspensions, and flavoured emulsions with edible oils such as
cottonseed oil, sesame oil, coconut oil or peanut oil, as well as elixirs and
similar pharmaceutical vehicles. Suitable dispersing or suspending
agents for aqueous suspensions include synthetic and natural gums
such as tragacanth, acacia, alginate, dextran, sodium
carboxymethylcellulose, methylcellulose, polyvinyl-pyrrolidone or
gelatin.
Where the processes for the preparation of the compounds
according to the invention give rise to mixtures of stereoisomers, these
isomers may be separated by conventional techniques such as
preparative chromatography. The compounds may be prepared in
racemic form, or individual enantiomers may be prepared either by
enantiospecific synthesis or by resolution. The compounds may, for
example, be resolved into their component enantiomers by standard
I5 techniques, such as the formation of diastereomeric pairs by salt
formation with an optically active acid, such as (-)-di-p-toluoyl-d-tartaric
acid and/or (+)-di-p-toluoyl-1-tartaric acid followed by fractional
crystallization and regeneration of the free base. The compounds may
also be resolved by formation of diastereomeric esters or amides,
followed by chromatographic separation and removal of the chiral
auxiliary. Alternatively, the compounds may be resolved using a chiral
HPLC column.
During any of the processes for preparation of the
compounds of the present invention, it may be necessary and/or
desirable to protect sensitive or reactive groups on any of the molecules
concerned. This may be achieved by means of conventional protecting
groups, such as those described in Protective Groups in Organic
Chemistry. ed. J.F.W. McOmie, Plenum Press, 1973; and T.W. Greene
& P.G.M. Wuts, Protective Groups in Organic Synthesis. John Wiley &
Sons, 1991. The protecting groups may be removed at a convenient
subsequent stage using ~thod5 known from the art.
Tlne specific . .. y of binding of compounds showing affinity for
the alpha la receptor is shown by comparing affinity to membranes
obtained from tranfected cell Lines that express the alpha 1a receptor
and membranes from cell lines or tissues known to express other types
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of alpha (e.g., alpha 1d, alpha 1b) or beta adrenergic receptors.
Expression of the cloned human alpha ld, alpha 1b, and alpha la
receptors and comparison of their binding properties with known
selective antagonists provides a rational way for selection of compounds
and discovery of new compounds with predictable pharmacological
activities. Antagonism by these compounds of the human alpha la
adrenergic receptor subtype may be functionally demonstrated in
anesthetized animals. These compounds may be used to increase urine
flow without exhibiting hypotensive effects.
The ability of compounds of the present invention to
specifically bind to the alpha la receptor makes them useful for the
treatment of BPH. The specificity of binding of compounds showing
affinity for the alpha la receptor is compared against the binding
affinities to other types of alpha or beta adrenergic receptors. The
human alpha adrenergic receptor of the is subtype was recently
identified, cloned and expressed as described in PCT International
Application Publication Nos. W094/08040, published 14 April 1994 and
WO 94/21660, published 29 September 1994. The cloned human alpha 1a
receptor, when expressed in mammalian cell lines, is used to discover
ligands that bind to the receptor and alter its function. Expression of the
cloned human alpha ld, alpha lb, and alpha la receptors and
comparison of their binding properties with known selective antagonists
provides a rational way for selection of compounds and discovery of new
compounds with predictable pharmacological activities.
Compounds of this invention exhibiting human alpha la
adrenergic receptor antagonism may further be defined by
counterscreening. This is accomplished according to methods known in
the art using other receptors responsible for mediating diverse biological
functions. fSee e.e..e.e.. PCT International Application Publication No.
W094/10989, published 26 May 1994; U.S. Patent No. 5,403,847, issued
April 4, 1995]. Compounds which are both selective amongst the various
human alphal adrenergic receptor subtypes and which have low affinity
for other receptors, such as the alpha2 adrenergic receptors, the 13-
adrenergic receptors, the muscarinic receptors, the serotonin receptors,
and others are particularly preferred. The absence of these non-specific
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activities may be confirmed by using cloned and expressed receptors in
an analogous fashion to the method disclosed herein for identifying
compounds which have high affinity for the various human alphal
adrenergic receptors. Furthermore, functional biological tests are used
to confirm the effects of identified compounds as alpha la adrenergic
receptor antagonists.
The present invention also has the objective of providing
suitable topical, oral, systemic and parenteral pharmaceutical
formulations for use in the novel methods of treatment of the present
invention. The compositions containing compounds of this invention
as the active ingredient for use in the specific antagonism of human
alpha la adrenergic receptors can be administered in a wide variety
of therapeutic dosage forms in conventional vehicles for systemic
administration. For example, the compounds can be administered
in such oral dosage forms as tablets, capsules (each including timed
release and sustained release formulations), pills, powders,
granules, elixirs, tinctures, solutions, suspensions, syrups and
emulsions, or by injection. Likewise, they may also be administered
in intravenous (both bolus and infusion), intraperitoneal,
subcutaneous, topical with or without occlusion, or intramuscular
form, all using forms well known to those of ordinary skill in the
pharmaceutical arts. An effective but non-toxic amount of the
compound desired can be employed as an alpha la antagonistic
agent.
Advantageously, compounds of the present invention
may be administered in a single daily dose, or the total daily dosage
may be administered in divided doses of two, three or four times
daily. Furthermore, compounds for the present invention can be
administered in intranasal form via topical use of suitable intranasal
vehicles, or via transdermal routes, using those forms of
transdermal skin patches well known to =hose of ordinary skill in
that art. To be administered in the form of a transdermal delivery
system, the dosage administration will, of course, be continuous
rather than intermittent throughout the dosage regimen.
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The dosage regimen utilizing the compounds of the
present invention is selected in accordance with a variety of factors
including type, species, age, weight, sex and medical condition of the
patient; the severity of the condition to be treated; the route of
administration; the renal and hepatic function of the patient; and the
particular compound thereof employed. A physician or veterinarian
of ordinary skill can readily determine and prescribe the effective
amount of the drug required to prevent, counter or arrest the
progress of the condition. Optimal precision in achieving
concentration of drug within the range that yields efficacy without
toxicity requires a regimen based on the kinetics of the drug's
availability to target sites. This involves a consideration of the
distribution, equilibrium, and elimination of a drug.
In the methods of the present invention, the compounds
herein described in detail can form the active ingredient, and are
typically administered in admixture with suitable pharmaceutical
diluents, excipients or carriers (collectively referred to herein as
"carrier" materials) suitably selected with respect to the intended
form of administration, that is, oral tablets, capsules, elixirs, syrups
and the like, and consistent with conventional pharmaceutical
practices.
For instance, for oral administration in the form of a
tablet or capsule, the active drug component can be combined with an
oral, non-toxic pharmaceutically acceptable inert carrier such as
ethanol, glycerol, water and the like. Moreover, when desired or
necessary, suitable binders, lubricants, disintegrating agents and
coloring agents can also be incorporated into the mixture. Suitable
binders include, without limitation, starch, gelatin, natural sugars
such as glucose or beta-lactose, corn sweeteners, natural and
synthetic gums such as acacia, tragacanth or sodium alginate,
carboxymethylcellulose, polyethylene glycol, waxes and the like.
Lubricants used in these dosage forms include, without limitation,
sodium oleate, sodium stearate, magnesium stearate, sodium
benzoate, sodium acetate, sodium chloride and the like.
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Disintegrators include, without limitation, starch, methyl cellulose,
agar, bentonite, xanthan gum and the like.
The liquid forms in suitably flavored suspending or
dispersing agents such as the synthetic and natural gums, for example,
tragacanth, acacia, methyl-cellulose and the like. Other dispersing
agents which may be employed include glycerin and the like. For
parenteral administration, sterile suspensions and solutions are
desired. Isotonic preparations which generally contain suitable
preservatives are employed when intravenous administration is desired.
The compounds of the present invention can also be
administered in the form of liposome delivery systems, such as small
unilamellar vesicles, large unilamellar vesicles and multilamellar
vesicles. Liposomes can be formed from a variety of phospholipids,
such as cholesterol, stearylamine or phosphatidylcholines.
Compounds of the present invention may also be
delivered by the use of monoclonal antibodies as individual carriers to
which the compound molecules are coupled. The compounds of the
present invention may also be coupled with soluble polymers as
targetable drug carriers. Such polymers can include polyvinyl-
pyrrolidone, pyran copolymer, polyhydroxypropylmethacryl-
amidephenol, polyhydroxy-ethylaspartamidephenol, or polyethyl-
eneoxidepolylysine substituted with palmitoyl residues.
Furthermore, the compounds of the present invention may be
coupled to a class of biodegradable polymers useful in achieving
controlled release of a drug, for example, polylactic acid, polyepsilon
caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals,
polydihydro-pyrans, polycyanoacrylates and cross-linked or
amphipathic block copolymers of hydrogels.
Compounds of this invention may be administered in
any of the foregoing compositions and ac~~rding to dosage regimens
established in the art whenever specific kade of th~~ human alpl~
la adrenergic receptor is required.
The daily dosage of the products may be varied over a wine
range from 0.01 to 1,000 mg per adult human per day. For oral
administration, the compositions are preferably provided in the form of
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tablets containing 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0
and
100 milligrams of the active ingredient for the symptomatic adjustment
of the dosage to the patient to be treated. A medicament typically
contains from about 0.01 mg to about 500 mg of the active ingredient,
preferably, from about 1 mg to about 100 mg of active ingredient. An
effective amount of the drug is ordinarily supplied at a dosage level of
from about 0.0002 mg/kg to about 25 mg/kg of body weight per day.
Preferably, the range is from about 0.001 to 10 mg/kg of body weight per
day, and especially from about 0.001 mg/kg to ? mg/kg of body weight per
day. The compounds may be administered on a regimen of 1 to 4 times
per day.
Compounds of this patent disclosure may be used alone at
appropriate dosages defined by routine testing in order to obtain optimal
antagonism of the human alpha la adrenergic receptor while
minimizing any potential toxicity. In addition, co-administration or
sequential administration of other agents which alleviate the effects of
BPH is desirable. Thus, in one embodiment, this includes
administration of compounds of this invention and a human
testosterone 5-a reductase inhibitor. Included with this embodiment are
inhibitors of 5-alpha reductase isoenzyme 2. Many such compounds are
now well known in the art and include such compounds as
PROSCAR~, (also known as finasteride, a 4-Aza-steroid; see US Patents
4,377,584 and 4,760,071, for example). In addition to PROSCAR~, which
is principally active in prostatic tissue due to its selectivity for human 5-
a reductase isozyme 2, combinations of compounds which are
specifically active in inhibiting testosterone 5-alpha reductase isozyme 1
and compounds which act as dual inhibitors of both isozymes 1 and 2,
are useful in combination with compounds of this invention.
Compounds that are active as 5a-reductase inhibitors have been
described in W093/23420, EP 0572166; WO 93J23050; W093/23038, ;
W093123048; W093/2304 i; W093J23040; W093/23039; W093123376;
W093J23419, EP 0572165; W093J23051.
The dosages of the alpha 1a adrenergic receptor and
testosterone 5-alpha reductase inhibitors are adjusted when
combined to achieve desired effects. As those skilled in the art will
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appreciate, dosages of the 5-alpha reductase inhibitor and the alpha
la adrenergic receptor antagonist may be independently optimized
and combined to achieve a synergistic result wherein the pathology is
reduced more than it would be if either agent were used alone. In
accordance with the method of the present invention, the individual
components of the combination can be administered separately at
different times during the course of therapy or concurrently in
divided or single combination forms. The instant invention is
therefore to be understood as embracing all such regimes of
simultaneous or alternating treatment and the term
"administering" is to be interpreted accordingly.
Thus, in one preferred embodiment of the present
invention, a method of treating BPH is provided which comprises
administering to a subject in need of treatment any of the compounds
of the present invention in combination with finasteride effective to
treat BPH. The dosage of finasteride administered to the subject is
about 0.01 mg per subject per day to about 50 mg per subject per day
in combination with an alpha 1a antagonist. Preferably, the dosage
of finasteride in the combination is about _0.2 mg per subject per day to
about 10 mg per subject per day, more preferably, about 1 to about 7
mg per subject to day, most preferably, about 5 mg per subject per
day.
For the treatment of benign prostatic hyperplasia,
compounds of this invention exhibiting alpha la adrenergic receptor
blockade can be combined with a therapeutically effective amount of a
5a-reductase 2 inhibitor, such as finasteride, in addition to a 5a-
reductase 1 inhibitor, such as 4,7f3-dimethyl-4-aza-5a-cholestan-3-
one, in a single oral, systemic, or parenteral pharmaceutical dosage
formulation. Alternatively, a combined therapy can be employed
wherein the alpha la adrenergic receptor antagonist and the 5a-
reductase ' or 2 inhibitor are administ-:red in sE tarate oral,
systemic, pare--iteral dosage formulations. Spa, e.g., U.S. Patent
No.'s 4,37 r ,a~34 and 4,760,071 which describe dosages and
formulations for 5a-reductase inhibitors.
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Abbreviations used in the instant specification, particularly
the Schemes and Examples, are as follows:
AcOH or HOAc = acetic acid
BCE = bromochloroethane
BINAP = 2,2'-bis(diphenylphosphino)-1,1'-binaphthyl
Bn = benzyl
Boc or BOC = t-butyloxycarbonyl
BOPCl = bis(2-oxo-3-oxazolidinyl)phosphinic chloride
Cbz-Cl = benzyloxycarbonyl chloride
dba = dibenzylideneacetone
DEAD = diethylazodicarboxylate
DIBAL = diisobutylaluminum hydride
DMF = N,N-dimethylformamide
DMSO = dimethylsulfoxide
EDCI = 1-(3-dimethylaminopropyl)-3-ethylcarbodimide
hydrochloride
Et = ethyl
EtgN = triethylamine
EtOAc = ethyl acetate
EtOH = ethanol
FABLRMS = fast atom bombardment low resolution mass
spectroscopy
HPLC = high performance liquid chromatography
HOAc = acetic acid
HOBt = 1-hydroxy benzotriazole hydrate
i-PrOH = 2-propanol
i-Pr2NEt = diisopropylethylamine
LAH = lithium aluminum hydride
mCPBA = meta-chloroperbenzoic acid
Me = methyl
MeOH = methanol
NMR = nuclear magnetic resonance
PCTLC = preparative centrifugal thin layer
chromatography
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PEI = polyethylenimine
Ph = phenyl
RT = retention time
tBu = tertiary butyl
TEBAC = benzyltriethylammonium chloride
TFA = trifluoroacetic acid
THF = tetrahydrofuran
TLC = thin layer chromatography
TMS = trimethylsilyl
The compounds of the present invention can be prepared
readily according to the following reaction schemes and examples, or
modifications thereof, using readily available starting materials,
reagents and conventional synthesis procedures. In these reactions, it
is also possible to make use of variants which are themselves known to
those of ordinary skill in this art, but are not mentioned in greater
detail. Unless otherwise indicated, all variables are as defined above.
The preparation of key intermediates for the compounds of
the present invention was accomplished via either Pd mediated coupling
reactions or direct nucleophilic displacement as outlined in Schemes 1
and 2. The products, for instance, 4-(ketalized) oxo or 4-hydroxy
piperidines were converted to their corresponding piperidone derivatives
either by acid catalyzed deketalization or Swern oxidation, respectively.
The resulting ketones are further elaborated, for instance, via enolate
alkylation. Once the desired ketones were in hand, reductive amination
with the desired mono-blocked (protected) diamine was carried out by
reaction with acetic acid in methanol followed by slow addition of a THF
solution of sodium cyanoborohydride. On occasion, the newly generated
secondary amine was protected or alkylated, the terminal amine
deprotected and then coupled to the desired Q group vii alkylation,
acylation, reductive aminat a, etc.
The activated Termini species comprising .::.e "Cl" groups
are readily prepared by one of ordinary skill in the art. For example,
oxazolidinones are prepared and activated in general by published and
well developed chemistry, in particular, of Evans. [Evans, D.A.; Nelson,
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J.V.; Taber, R.R. Top. Stereochem. ,~, 1 (1982)] The starting materials,
in general, are natural and unnatural amino acids. For instance, some
of the preferred compounds are prepared from substituted phenylglycine
derivatives, which after reduction of the carboxylate and a phosgene
equivalent mediated cyclization provides the substituted oxazolidinone
ring system. Deprotonation with n-butyl lithium and addition to a THF
solution of p-nitrophenylchloroformate produces the stable, isolable
"activated"oxazolidinone (oxa).
Dihydropyrimidinones are prepared by condensation
reaction of the aldehyde, urea and 1,3-acetoacetate type derivative
catalyzed by a Lewis Acid, a copper (I) species and acetic acid.
Activation was accomplished by treatment with a strong base, for
instance, LiN(TMS)2, followed by addition to a THF solution of p-
nitrophenylchloroformate.
Hydantoins and cycloimide were prepared in two chemical
steps from ketones as outlined in the literature. More specifically,
hydantoins were prepared according to known methodology, e.g., J.J.
Edmunds et al., J. Med. Chem. 1995, 38, pp. 3759-3771; J.H. Poupart et
al., J. Chem. Res. 1979, pp. 174-175. Saccharins were prepared
according to known methods, e.g., page 40 and Examples 21 and 22 of
PCT International Application Publication No. W096/25934, published
August 29, 1996.
The dihydropyrimidinones and oxazolidinones were
synthesized independently in racemic form, and then separated
utilizing preparative chiral HPLC. Their optical rotations were
recorded. Then they were activated and reacted with prerequisite
amines. From the receptor binding studies, a preferred isomer was
identified, the (+) rotational isomer in each case. The absolute
configurations were determined to be (S) for both the
dihydropyrimidinones and oxazolidinones by correlating their optical
rotations with x-ray crystal structures obtained of fragments involved in
the production of the antagonists.
Examples of the preparation of some desired compounds
are outlined in Schemes 3-5. Selective Boc protection of 4-amino
methylpiperidine (1) followed by reductive amination with ketone (3)
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provided the terminally protected amino derivative (4). Acid mediated
deprotection of (4) followed by acylation with (6) and (22) provided (7) and
(23), Scheme 3 and 4.
Other derivatives were prepared via the routes outlined in
Schemes 5-7. For example, N-protected 3-hydroxy azetidine (8) was
tosylated providing (9). Displacement with sodium azide produced (10)
which after reduction with PPH3/H20 resulted in N-protected-3-
aminoazetidine (11). Reductive amination with ketone (3) produced (12)
which after Boc protection of the secondary amine, hydrogenolysis of the
N-protecting group, acylation and HCl/EtOAc mediated concomitant
deprotection and salt formation yields the desired products.
Still other derivatives were prepared via the routes outlined
in Schemes 8 and 9. The synthesis of the 3-aminomethylpyrrolidine
intermediate was accomplished from the commercially available lactam
as outlined Scheme 8. The ester was converted to the amide by a simple
two step process. Subsequent DIBAL reduction yielded the
monoprotected diamino intermediate. Reductive aminations with
cyclohexanones, followed by deprotection of the N-benzyl group, and
acyclation with preferred activated "Q"-groups furnished the final
targets.
The synthesis of the 4-amino-3-hydroxypyrrolidine
intermediate began with 3,4-pyrroline, Scheme 9. BOC protection of the
amine followed by mCPBA oxidation provided the epoxidation.
Subsequent sodium azide opening of the epoxide and
triphenylphosphine/water mediated reduction produced 4-amino-N-1-
(1,1-dimethylethoxycarbonyl)-3-hydroxypyrrolidine. The key amino
intermediate was alkylated by reductive amination reactions with
cyclohexanones. Following the cleavage of the BOC protecting group
acyclation with preferred activated "Q"-groups furnished the final
targets.
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SCHEME 1
n
HN
O
1
R'Br ~ OJ
Pd2(dba}3
KOtBu/P(o-tolyi)3
or BINAP
n
HN ~ ~ O R~ N n
R1 F ( ~ O C ~O Aq HCi A
o
OJ AcOH
neat, THF,
or DMF
R2 R16 R16
H-N-(CR32)q
1 n R13 ~ m n R2 R15 R16
R~N R14 P P R1-N ~ N-(CR32)q
m
( o O AcOH/MeOH o R13 N-P
A NaCNBH3 R14
P = BOC
HCI
EtOAc
P=H~HCI
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SCHEME 1 (Cont'd)
n R2 R1s R1s
R1-N~N-(CR32)q
~/ ~~ m
o R1s N-H
R14 P
O
Q~(CI, p-N02Ph0, OH]
Acyiating Agent
n R2 R15 R16
R1-N ~ N-(CR32)q )
m
o R13
R14 P II Q
O
SCHEME 2
n
HN '
R' ~ o O H
Br
Pd2(dba)3
KOtBu/P(o-tolyl)3
or BINAP
n
HN ~ R1 n
R1 N
F ~ o OH
o OH
neat, THF,
or DMF Swern
Oxidation
R 1 ~.1 n
N
o O
A
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SCHEME 3
HN BOC20 N
NH2 O NH2
/ CN
/ CN _ \ I
\ I N
N N
O H N O
O
NaCNBH3 TFA
/ CN
\I
N
H N
02N / O N Oi 5 H N H
O N I O
O = O
\ (
~F
CN F
/I
\ N
H
O N i
H ~ ~ I ~O
N~N O~
'OI = O
7 \ I
_F
F
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SCHEME 4
/ CN
N
N
H NH
O~--O
\ O NJ
02N / O
13 \ F
F
/ CN
N
O
O
i
N NJ
O
14
F
F
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SCHEME 5
~\ ~/
/
\ T~ N \ NaN3
N O I/
/ ~ ~ S_O
HO p
8 9
\ \
/ ~ /
Ph3P
N ~ \ H20 N I /
/ HN
N3 10 2 11
/ CN I \
11 / CN /
\ N
NaCNBH3 \ N \
O AcOH N ~ I /
3 N
H
_ q,3 _
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SCHEME 6
~ HPh2 LAH N CHPh2 3
N
AIC13 NaCNBH3
NC H2N
15 16
CN / CN
N BOC20 N
NH NBOC
~--~~NCHPh2 ~~NCHPh2
17 18 H2
10% Pd-C
/ CN
CN N
Activated Carbamates NBOC
~---~ N
Activated Acids 19 H
NBOC
~---~NCOQ
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SCHEME 7
O OH
OMe DIBAL 1.) Tos20
O -: 2.) NaN3
N CH2C12
3.) Ph3P/H20
24 25
R
/ N
CN N
3 H NBn
29
Ac0 H
26 R = OTos NaBH3CN
27 R = N3 MeOH
28R=NHZ
H2 / N
BOC20 N 10% Pd-C CN N NH
C~ CN N BOC
BOC~NBn 3~
/ N
activated
--~ CN
carbamates and BOC~NCOQ
aci d ~../s
32
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SCHEME 8
O
1 ) NaOH O
DIBAL
Me20C Ngn ~ ' 2HNOC ~ ~'
2 EDCI NBn
NH3
2HN + ~ / NaCNBH3
~'~'~~N B n N
CN AcOH, MeOH
O
\ ~ 1 ) H2/Pd-C Final
N
cN 2) Activated Products
H ~NBn (+)-DHP
or
(S}-OXA
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SCHEME 9
BOC20 mCPBA
CNH ~ / NBOC
NaHC03
HO
O NaN3 ~ PPh3
\~NBOC ~ 3N NBOC
DMF H20
HO
NaCNBH3
2HN NBOC + CN N AcOH, MeOH
'O
HO, 1 ) HCI Final
N
' Products
CN N NBOC 2) Activated
H (+)-DHP
or
(S)-OXA
The following examples are provided to further define the
invention without, however, limiting the invention to the particulars of
these examples.
EXAMPLE 1
4-Aminometh ~~1-piperidine-1-carboxylic acid tent-bu~~l ester (2)
A solution of C-piperidin-4-yl-methylamine (1) (5.0 g, 44
mmol) and triethylamine (12 ml 88 mmol) in 150 ml of chloroform was
cooled to 0° C. To this solution was added dropwise
ditertbutyldicarbonate (8.6 g, 40 mmol) in 100 ml of chloroform. After
stirring at room temperature for 24 hours the solution was washed with
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water, dried over MgS04, filtered and the solvents removed in vacuo to
give the title compound
1H NMR (CDC13) 4.20-4.00 (br m, 2H), 2.75-2.62 (br t, 2H),
2.60 (d, 2H), 1.75-1.65 (br m, 2H), 1.50-1.30 (m, 3H), 1.63 (s, 9H), 1.20-1.00
(m, 2H).
EXAMPLE 2
N
CN
O
N(2-cvanoDhenvl)-4-nin .~r~nnP «)
A solution of 2-fluorobenzonitrile (2.75 g, 22.7 mmol) and 4-
piperidone ethylene ketal (4.25 g, 29.7 mmol} in DMF (40 mL) was heated
to 120°C for 4h. The resulting mixture was cooled to room temperature
overnight. The solvent was removed in vacuo and the residue dissolved
in ether and sodium bicarbonate solution. The aqueous layer was
extracted with two additional portions of ether and the combined organic
extracts were washed with brine, dried over Na2S04, and concentrated
under reduced pressure to afford the 4-piperidone ethylene ketal. The
crude product was used directly.
A solution of 4-piperidone ethylene ketal (533 mg, 2.18
mmol) in ether (10 mL) was treated with 5% aqueous HCl (20 mL). The
mixture was stirred at room temperature ( l ld). The reaction was
diluted with ether and neutralized with sodium bicarbonate solution.
The aqueous layer was extracted with two additional portions of ether
and the combined organic extracts were washed with brine, dried over
Na2S04, and concentrated under reduced pressure. PCTLC (Si02,
4mm, 20% EtOAc; 80S °~exane) afl'orr= i the title comF ud (3).
1H NMR ICDCl3, 400 M~:z) consistant wits: assigned
structure.
_ 4g _
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FABLRMS m/e 201 g/mole (M++H, C12H12N2~ = 201
g/mol a . )
HPLC (Vydac; C18; diameter = 4.6 mm; length = 150 mm;
gradient = H20 [0.1% HgP04] - CH3CN, 95% - 5%, 5% - 95%, over 16
minutes, 2 ml/min flow rate) RT = 7.32 min; focus = 215 nm; 97.5% pure.
EXAMPLE 3
4-{[ 1-(2-Cyano-phenyl)-piperidin-4-ylamino]-methyl}-
cvclohexanecarboxylic acid tent-butyl ester (4)
To a solution of 2-(4-oxo-piperidin-1-yl)-benzonitrile (3) (750
mg, 3.75 mmol) and 2 in 40 ml of methanol was added 4 g of powdered
0
4A molecular sieves. The resulting suspension was stirred at room
temperature for 24 hours. The suspension was subsequently acidified to
pH 5 with acetic acid and a IM solution of NaCNBH3 in THF (6.0 ml 5.6
mmol) was added slowly with a syringe pump aver 24 hours. When the
addition was complete, the solvent was removed in vacuo and the
residue taken up in chloroform and filtered. The solution was then
washed with 10% Na2C03, dried over MgS04, and the solvent removed in
vacuo to give the crude amine. The crude product was purified by
chromatography on silica gel (5% MeOH/CHC13) to give the title
compound.
1H NMR (CDC13) 7.54 (dd, 1H, J=7.56 Hz,1.46 Hz), 7.45, (t,
1H, J=7.94 Hz), 7.01-6.94 (m, 2H), 4.18-4.05, (m, 2H), 3.61-3.52 (m, 2H),
2.81-2.95 (m, 2H), 2.78-2.57 (m, 3H), 2.55 (d, 2H, J=6.59 Hz), 2.09-1.95 (m,
2H), 1.72 (br d, 2H, J=12.7 Hz), 1.70-1.50 (m, 3H), 1.45 (s, 9H), 1.45-1.30,
(m, 1H), 1.20-1.00 (m, 2H).
EXAMPLE 4
2-(4-f(Piueridin-4-vlmethvl)-aminol-giperidin-1-vll-benzonitrile (5)
To a solution of 4 (1.5 g, 3.76 mmol) in 30 ml of methylene
chloride was added 15 ml of TFA. After stirring at room temperature
for 24 hours, the solvents were removed in vacuo and the residue
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partitioned between chloroform and 10% Na2C03. The organics were
dried over MgS04, filtered, and the solvent removed in vacuo to give the
title compound.
1H NMR (CDC13) 7.54 (dd, 1H, J=7.57 Hz, J=1.47 Hz), 7.50-
7.40 (m, 1H), 7.03-6.95, (m, 2H), 3.61-3.55 (m, 2H), 3.30-3.20 (m, 2H), 2.87
(d of t, 2H, J=12.45 Hz, J=2.44 Hz), 2.74 (d of t, 2H, J=12.45Hz, J=2.68 Hz},
2.63-2.55 (m, 1H), 2.57 (d, 2H, J=6.83), 2.08-1.97 (m, 2H), 1.95-1.83 (m, 2H),
1.6?-1.50 (m, 3H), 1.42-1.25 (m, 2H}.
EXAMPLE 5
3-(4-{[1-(2-Cyano-phenyl)-piperidin-4-ylamino]-methyl)-piperidine-1-
carbonyl)-4-(3,4-difluoro-phenyl)-6-methoxymethyl-2-oxo-1,2,3,4-
tetrahvdro-nvrimidine-5-carboxylic acid methyl ester (7)
To a solution of 5 (280 mg, 0.94 mmol) in 30 ml of chloroform
was added dropwise 6 in 10 ml of chloroform. The resulting solution
was stirred for 20 minutes and the crude material purified by
chromatography on silica gel (5% MeOH/CHC13)
1H NMR (DMSO-dg 75°C) 7.63 (d, 1H, J=7.57 Hz), 7.55 (t, 1H,
J=7.57 Hz), ?.42-7.25 (m, 2H), 7.13 (d, 2H, J=8.31 Hz), 7.03 (t, 1H, J=7.57
Hz), 5.67 (s, 1H), 4.51 (AB q, 2H Ja=48.1 Hz, Jb=13.43Hz, 3.90-3.50 (m,
2H), 3.60 (s, 3H), 3.45 (d, 2H J=11.96Hz), 3.31 (s, 3H), 2.90-2.65 (m, 4H),
2.60-2.45 (m, 1H), 2.39 (d, 2H J=5.86Hz), 1.90 (d, 2H, J=10.74Hz)1.75-1.35
(m, 8H), 1.05-0.90 (m, 1H).
MS (FAB) 637 (M+1)
Analysis calculated for C33 H38 N6 Ob F2 0.50 CHC13, 0.25
H20: C, 57.40; H, 5.61; N, 11.99. Found: C, 57.40; H, 5.56; N, 12.18.
EXAMPLE 6
oluene-4-s~fonic ar~r~ 1-hpn~hv~,.yl-azeti.-'~ -~--3-vl ester (9)
To a cooled (0°C) solution of 8 ~ ~, 29 mmol) in 100 ml of
chloroform was added paratoluene sulfonic anhydride (1I.5 g,
35.2mmol), and triethylamine (12 ml, 88mmol). The resulting solution
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was stirred at room temperature for 24 hours. The solution was
subsequently washed with water, dried over MgS04, filtered, and the
solvent removed in vacuo. The crude material was purified by
chromatography on silica gel to give the desired product as an oil.
1H NMR (CDC13) 7.75 (d, 2H, J=8.3Hz), 7.35-7.15 (m, 12H),
4.95-4.82 (m, 1H), 4.32 (s, 1H), 3.50-3.40 (m, 2H), 3.10-3.00 (m, 2H), 2.43
(s,
3H).
EXAMPLE 7
3-Azido-1-benzhydrvl-azetidine (10)
A solution of 9 (11.5 g, 31.8 mmol) and sodium azide (4.12 g,
64 mmol) in 250 ml of DMF was heated to 70°C for 24 hours. After
cooling to room temperature the solvent was removed in vacuo and the
residue partitioned between chloroform and water. The organics were
dried over MgS04, filtered and the solvent removed in vacuo. The crude
product was purified by chromatography on silica gel (8:1 hexane: ethyl
acetate) afforded the title compound.
1H NMR (CDC13) 7.41-7.15 (m, lOH), 4.33 (s, 1H), 4.02-3.95
(m, 1H) 3.50-3.41 (m, 2H), 3.07-3.00 (m, 2H).
EXAMPLE 8
1-Benzhvdrvl-azetidin-3-vlamine (11)
A solution of 10 (5.7 g, 21.6 mmol), triphenylphosphine (11.3
g, 43 mmol) and water (5 ml) was heated to reflux for 24 hours. After
cooling to room temperature the solvent was removed in vacuo and the
residue purified by chromatography on silica gel (90:9:1
CHCI3:MeOH:NH40H).
1H NMR (CDC13) 7.41-7.15 (m, lOH), 4.27 (s, 1H), 3.65-3.55
(m, 1H), 3.55-3.50 (m, 2H), 2.65-2.60 (m, 2H) 1.44 (br s, 2H).
EXAMPLE 9
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2-[4-( 1-Benzhydryl-azetidin-3-ylamino)-piperidin-1-yl]-benzonitrile
trifluoroacetic acid salt (12)
The title compound was prepared from 3 (1.0 g, 3.9 mmol)
and 11 using the procedure described for 4.
1H NMR (CDC13) 7.56-6.90 (m, 14H), 4.32 (s, 1H), 3.62-3.45
(m, 5H), 2.90-2.87 (m, 2H), 2.75-7.65 (m, 2H), 2.64-2.58 (m, 1H), 1.91 (d,
2H, J=11.36Hz), 1.62-1.53 (m, 2H), 1.50-1.20 (br s, 1H).
MS (FAB) 423 (M+1).
Analysis calculated for C28 H3o N4 0.05 H20, 2.30 TFA:
C, 57.10; H, 4.76; N, 8.17.
Found: C, 57.10; H, 4.75; N, 8.25.
EXAMPLE 10
2-[4-({1-[4-(3,4-Difluoro-phenyl)-2-oxo-oxazolidine-3-carbonyl]-piperidin-4-
ylmethyl}-amino)-piperidin-1-yl]-benzonitrile trifluoroacetic acid salt (14)
The title compound was prepared from 5 (200 mg, 0.67
mmol) and 13 (121 mg, 0.67 mmol) using~the procedure described for 7.
~H NMR (CD30D) 7.70-7.55 (m, 2H), 7.50-7.40 (m, 1H), 7.35-
7.25 (m, 2H) 7.20-7.03 (m, 2H), 5.50 (t, 1H, J=9.03Hz), 4.75 (t, 1H,
J=8.54Hz), 4.30-4.13 (m, 3H), 3.65 (d, 2H, J=12.21Hz), 3.15-2.85 (m, 6H),
2.25 (d, 2H, J=10.26Hz), 2.10-1.75 (m, 6H), 1.50-1.10 (m, 2H).
MS (FAB) 524 (M+1).
Analysis calculated for CZ8 H31 N5 03 F2 1.30 TFA, 0.20 H20:
C, 54.41; H, 4.88; N, 10.37.
Found: C, 54.37; H, 4.88; N, 10.46.
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EXAMPLE 11
H2N~y~N
3-Aminomethvl N diphenylmethy~ azetidine. (16)
To a cooled solution of aluminum chloride (0.33 g, 2.41
mmol) in ether (50 mL) at -78° C was added lithium aluminum hydride
(2.41 ml, 2.41 mmol). After stirring 15 minutes at -78° C the slurry
was
added a solution of 15 (0.50 g, 2.01 mmol) in ether (10 mL) dropwise. The
resulting mixture was stirred at room temperature for 2 hours. The
solution was cooled to 0°C and quenched with water (10 mL) dropwise
followed by 25% NaOH solution (10 mL). The aqueous layer was
extracted with EtOAc. The organics were dried over Na2S04, filtered,
and removed in uaccuo. The crude product was not purified.
1H NMR (CDC13, 300 MHz} 7.41-7.13 (m, IO H), 4.32 (s, 1 H),
3.28 (t, 2 H), 2.88-2.79 (m, 4 H), 2.52-2.42(m, 1 H), 1.28 (s, 1 H).
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EXAMPLE 12
CN
/ N
N H~N
Compound ( 17 )
The title compound was prepared from 16 and 3 using the
procedure described for the preparation of 4.
1H IVMR (CDC13, 400 MHz) 7.54-7.51 (dd, 1 H), 7.46-7.38 (m, 5
H), 7.28-7.24 (m, 4 H), 7.18-7.15 (m, 2 H), 6.99-6.93 (m, 2 H), 4.33 (s, 1 H),
3.57-3.45 (m, 2 H), 3.35-3.31 (t, 2 H), 2.89-2.79 (m, 7 H), 2.64-2.56 (m, 2
H),
2.01-1.98 (m, 2 H), 1.62-1.53 (m, 2 H).
MS (FAB) 437 (M+1).
EXAMPLE 13
_N
O
H2N O
O
~I-(2-benzamido)-4-niueridone ethylene ketal l21 )
A mixture of 2-fluorobenzamide (7.0 g, 50.0 mmol) and 4-
piperidone ethylene ketal (7.16 g, 50.0 mmol) was heated at 100° C (7
d).
The solvent was removed in uacuo and triturated with ether affording
the title cor and (21).
3 NMR (DMSO-dg, 300 MHz) consistant with assigned
structure.
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HPLC (Vydac; C18; diameter = 4.6 mm; length = 150 mm;
gradient = H20 [0.1% HgP04.] - CHgCN, 95% - 5%, 5% - 95%, over 16
minutes, 2 ml/min flow rate) RT = 4.49 min; focus = 215 nm; 100% pure.
EXAMPLE 14
N
H2N O O
N-(2-benzamido)-4-~ineridone (22
A solution of the ketal 21 (13.2 mg, 46.753 mmol) in acetic
acid (50 mL) and 6N aqueous HCl (50 mL) was heated at 60° C (12 h), 80%
conversion. The solvent was removed in vacuo, neutralized with 25%
aqueous NaOH, extracted with CHCIg (3 x 250 mL), the combined
organic extracts were washed with brine, dried over Na2S04, and
concentrated under reduced pressure to give (22) which was used
without further purification.
1H NMR (CDC13, 400 MHz) cansistant with assigned
structure .
EXAMPLE 15
O
NH2
/ N
N Hr~N
Compound (23)
The title compound was prepared from 16 and 22 using the procedure
described for the preparation of 4.
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1H NMR (CDC13, 400 MHz) 9.53 (s, 1 H), 8.16-8.13 (m, 1 H),
7.45-7.38 (m, 6 H), 7.29-7.21 (m, 2 H), 7.19-7.15 (m, 4 H), 5.76 (s, 1 H),
5.29
(s, 1 H), 4.33 (s, 1 H), 3.36-3.32 (t, 2 H), 3.20-3.17 (d, 2 H), 2.87-2.75 (m,
7
H), 2.64-2.55 (m, 2 H), 2.03-2.00 (d, 2 H), 1.55-1.45 (m, 2 H).
Anal. Calcd for C29H34N4O1 ~ 0.05 CHC13 + 0.60 H20 : C =
74.01, H = 7.54, N = 11.89. Found: C = 74.06, H = 7.51, N = 11.57.
MS (FAB) 455 (M+1)
EXAMPLE 16
~~ OH
N
.~Y-1-Benzvl-3- hvdroxpmeth~py~rrolidine (25)
A solution of Methyl 1-benzyl-5-oxo-3-pyrrolidinecarboxylate
(24) (10.0 g, 42.8 mmol) in 175 ml of dichloromethane was added
dropwise to a cooled solution of 1.5 M DIBAL-H (143 mL, 214 mmol) at -
78° C. After stirring at room temperature for 48 hours the solution was
cooled to 0° C and added 25 mL methanol dropwise. The solution was
added 250 mL saturated aqueous R,ochelle salts and extracted with
chloroform. The combined organics were dried over MgS04, filtered and
the solvents removed in vacuo to give the crude alcohol. The crude
product was purified by chromatography on silica gel (7%o MeOH/CHC13)
to afford the title compound.
1H NMR (CDC13, 400 MHz) 7.33-7.24 (m, 5 H), 3.70-3.66 (m, 1
H), 3.59-3.48 (m, 4 H), 2.86-2.80 (m, 1 H), 2.66-2.63 (d, 1 H), 2.51-2.47 (t,
1
H), 2.34-2.28 (m, 2H), 2.05-1.96 (gin, 1 H), 1.75-1.~7 (m, 1 H).
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EXAMPLE 17
0
~~ o- s
N O
Toluene-4-sulfonic acid 1-benzvl-Rvrrolidin-3-methyl ester (26)
To a cooled (0°C) solution of 25 (1.0 g, 5.2 mmol) in 100 ml of
chloroform was added para-toluenesulfonic anhydride (2.5 g, 7.8 mmol),
and triethylamine (2.1 ml, 15 mmol). The resulting solution was stirred
at room temperature for 24 hours. The solution was subsequently
washed with water, dried over MgSO~, filtered, and the solvent removed
in vacuo. The crude material was purified by chromatography on silica
gel to afford the desired product.
1H NMR (CDCl3, 400 MHz) 7.78-7.76 (d, 2 H), 7.33-7.23 (m, 7
H), 3.94-3.92 (m, 2 H), 3.53 (s, 2 H), 2.58-2.43 (m, 6 H), 2.27-2.24 (m, 1 H),
1.97-1.88 (m, 1 H), 1.44-1.36 (m, 1 H).
EXAMPLE 18
~~ Ns
N
N-1-Benzvl-3-azidomethvl nvrrolidine (27)
A solution of 26 (1.26 g, 3.64 mmol) and sodium azide (0.474
g, ?.29 mmol) in 100 ml of DMF was heated to 70°C for 24 hours. After
cooling to room temperature the solvent was removed in vacuo and the
residue partitioned between chloroform and water. The organics were
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dried over MgS04, filtered and the solvent removed in vacuo to afford the
azide.
1H NMR (CDC13, 400 MHz) 7.34-?.22 (m, 5 H), 3.60-3.59 (d, 2
H), 3.28-3.26 (m, 2 H), 2.70-2.66 (m, 1 H), 2.57-2.54 (t, 1 H), 2.44-2.38 (m,
2
H), 2.31-2.27 (m, 1 H), 2.04-L98 (m, 1 H), 1.51-1.45 (m, 1 H).
EXAMPLE 19
~~ NH2
N
N-1-Benzvl-3-aminomethvl ,pyrrolidine (28)
A solution of 27 (0.71 g, 3.2 mmol), triphenylphosphine (1.7
g, 6.5 mmol), THF (50 mL}, and water (5 mL) was heated to reflux for 24
hours. After cooling to room temperature the solvent was removed in
vacuo and the residue purified by chromatography on silica gel (90:9:1
CHCI3:MeOH:NH40H) to afford the amine.
1H NMR (CDCl3, 400 MHz) 7.33-7.22 (m, 5 H), 3.60-3.58 (d, 2
H), 2.76-2.72 (m, 1 H), 2.60-2.58 (m, 3 H}, 2.52-2.47 (m, 1 H), 2.24-2.17 (m,
2
H), 2.00-1.95 (m, 1 H), 1.48-1.42 (m, 1 H), 1.12 (br s, 2 H).
EXAMPLE 20
CN
N
N
N
Zp
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The title compound was prepared from 28 and 3 using the
procedure described for the preparation of 4.
1H NMR (CDC13, 400 MHz) 7.54-7.52 (d, 1 H), 7.46-7.42 (t, 1
H), 7.33-7.22 (m, 5 H), 6.99-6.93 (m, 2 H), 3.65-3.48 (m, 5 H), 3.89-2.83 (t,
2
H), 2.79-2.74 (t, 1 H), 2.66-2.59 (m, 4 H), 2.55-2.49 (q, 1 H), 2.34-2.28 (m,
1
H), 2.23-2.19 (t, 1 H), 2.01-1.98 (m, 3 H), 1.63-1.52 (m, 2 H), 1.50-1.45(m, 1
H).
Anal. Calcd for C24H32N4C12 ' 0.25 Hcl + 0.35 ETOAc : C =
62.59, H = 7.25, N = 11.50. Found: C = 62.60, H = 7.10, N = 11.46.
MS (FAB) 375 (M+1)
EXAMPLE 21
Mixture of 4S-4-(3,4-difluorophenyl)-6-methoxymethyl-2-oxo-1,2,3,4-
tetrahydropyrimidine and 4S-4-(3,4-difluorophenyl)-6-methoxymethyl-2-
oxo-2,3,4,5-tetrahydropyrimidine
H
To a solution of (+)-4-(3,4-difluorophenyl)-6-methoxymethyl-
2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid methyl ester (4.63 g,
14.7 mmol) in a methanol (100 ml) was added sodium hydroxide (2.94 g,
73.6 mmol). The resulting mixture was refluxed at 90 oC for 16 hours.
After cooling to room temperature the solvent was removed in vacuo.
The solid was dissolved in CHZC12 and H20 then neutralized with 10%
aqueous HCl solution. The organic layer was dried over Na2S0,~,
concentrated, and purified by PCTLC (7% MeOH in CHC13 with 2%
NH40H) to afford a 2.65 g mixture of the title compounds (71% yield).
The 'H NMR was consistent with the assigned structure.
MS (FAB) 255 (M+1)
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EXAMPLE 22
Mixture of 4S-4-(3,4-difluorophenyl)-6-methoxymethyl-2-oxo-1,2,3,4-
tetrahydropyrimidine and 4S-4-(3,4-difluorophenyl)-6-methoxymethyl-2-
oxo-2,3;4,5-tetrahydropyrimidine
H
To a solution of (+)-4-(3,4-difluorophenyl)-6-methoxymethyl-
2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid methyl ester (5.36 g,
17.0 mmol) in a methanol (150 ml) was added 1N NaOH (10 ml). The
resulting mixture was refluxed at 90 oC for 16 hours. After cooling to
room temperature the solvent was removed in uczcuo. The solid was
dissolved in CHZCl2 and H20 then neutralized with 10% aqueous HCl
solution. The organic layer was dried over Na2S04, concentrated, and
purified by PCTLC (7% MeOH in CHC13 with 2% NH40H) to afford a 2.35
g mixture of the title compounds (54% yield). The 'H NMR was
consistent with the assigned structure.
MS (FAB) 255 (M+1)
EXAMPLE 23
4S-4-(3,4-Difluorophenyl)-6-methoxymethyl-3-(4-nitrophenoxycarbonyl)-
2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid methyl ester
_g0_
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H
The title compound was prepared by treating the mixture
obtained from Example 21 or Example 22 (1.93 g, 7.59 mmol) with
lithium diisopropylamide (2.OM THF solution, 1.1 equivalents) in THF at
-78 °C for 20 minutes followed by the rapid addition of 4-nitrophenyl
chloroformate (1.5 equivalents) in THF. 0.488 g of the title compound
was obtained in a 15% yield. The 'H NMR was consistent with the
assigned structure.
EXAMPLE 24
Mixture of 4R-4-{3,4-difluorophenyl)-6-methoxymethyl-2-oxo-1,2,3,4-
tetrahydropyrimidine and 4R-4-(3,4-difluorophenyl)-6-methoxymethyl-2-
oxo-2,3,4,5-tetrahydropyrimidine
F F
F ~ F
HN
O_' 'N O~ O N
H
The title compounds were prepared from 4R-4-(3,4-
difluorophenyl)-6-methoxymethyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5-
carboxylic acid methyl ester (5.0 g, 17.7 mmol) using the procedure
described in Example 7. A mixture of 2.0 g of the title compounds was
obtained in 50% yield. The 'H NMR was consistent with the assigned
structure.
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MS (FAB) 255 (M+1)
Compounds of the invention can be prepared by reacting the
products obtained in Example 23 in accordance with procedures and
schemes described above. The compound of Example 23 can, for
example, be acylated with an arylpiperidinyl aminoalkylpiperidine in
accordance with Scheme 3 to obtain the desired compounds.
Compounds of the invention can also be prepared by preparing a
nitrophenoxy derivative of the compound of Example 24 in accordance
with the procedure set forth in Example 23 and then reacting the
derivative with an arylpiperidinyl aminoalkylpiperidine as set forth in
Scheme 3 to obtain compounds of the invention.
Examples 25 and 26 were prepared as outlined in Scheme 8.
EXAMPLE 25
(4S)-2-[4-( { 1-[4-(3,4-Difluorophenyl )-2-oxo-oxazolidine-3-carbonyl]-
pyrrolidin-3-ylmethyl}-amino)-piperidin-1-yl]-benzonitriie
N
CN
N
H
N
N
,U
,,
F
F
1H ~'MR, "'7C13, 400 MHz) consistent witi. assigned
structure.
FABLRMS m/e 510 g/mole (M++H, C27H29F2N5~3 = 509.55
g/mole. )
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HPLC (Vydac; C18; diameter = 4.6 mm; length = 150 mm;
gradient = H20 [0.1% H3P04] - CH3CN, 95% - 5%, 5% - 95%, over 16
minutes, 2 ml/min flow rate) focus = 215 nm; 100% pure.
Anal. Calcd for C27H2gF2N503: C = 57.16, H = 5.84, N =
11.11. Found: C = 57.24, H = 5.78, N = 11.12.
EXAMPLE 26
(4S)-3-(3-{[1-(2-Cyanophenyl)-piperidin-4-ylamino]-methyl}-pyrrolidine-1-
carbonyl~4-(3,4-difluorophenyl)-6-methoxymethyl-2-oxo-1,2,3,4-
tetrahydro-pyrimidine-5-carboxylic acid methyl ester
N
CN
H N
N NH
F ( ~ , i- O~
F / C02Me
1H NMR (CDCl3, 400 MHz) consistent with assigned
structure .
FABLRMS m/e 623 g/mole (M++H, C32H36F2N605 = 622.67
g/mole.)
HPLC (Vydac; C18; diameter = 4.6 mm; length = 150 mm;
gradient = H20 [0.1% H3P04] - CH3CN, 95% - 5%, 5% - 95%, over 16
minutes, 2 ml/min flow rate) focus = 215 nm; 95.3% pure.
Anal. Calcd for C32H36F'2N605 ' 1.3 HCl and 0.55 H20: C =
56.62, H = 5.69, N = 12.36. Found: C = 56.53, H = 5.69, N = 12.32.
Examples 27 and 28 were prepared as outlined in Scheme 9.
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EXAMPLE 27
(racemic) (4S)-2-[4-({1-[4-(3,4-Difluorophenyl)-2-oxo-oxazolidine-3-
carbonyl]-3-hydroxy-pyrrolidin-4-ylmethyl}-amino)-piperidin-1-yl]-
benzonitrile
\ ( HQ
CN ~ N
NH N O
F
F
1H NMR, (CDC13, 400 MHz) consistent with assigned
structure.
FABLRMS m/e 501.2 g/mole (M++H, C26H3pF2N4O4 =
500.54 g/mole. )
HPLC (Vydac; C18; diameter = 4.6 mm; length = 150 mm;
gradient = H20 [0.1% H3P04] - CH3CN, 95%~- 5%, 5% - 95%, over 16
minutes, 2 ml/min flow rate) focus = 215 nm; 87% pure.
EXAMPLE 28
(racemic) (4S)-3-(4-{[1-(2-Cyanophenyl)-piperidin-4-ylamino]-methyl}-3-
hydroxy-pyrrolidine-1-carbonyl)-4-(3,4-difluorophenyl)-6-methoxymethyl-
2-oxo-1,2,3,4-tetrahydro-pyrimidine-5-carboxylic acid methyl ester
\ i Hc~
CN ~ N--il
NH v NH
F \ , . / O~
F I / C02Me
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1H NMR (CDC13, 400 MHz) consistent with assigned
structure.
FABLRMS m/e 625.88 g/mole (M++H, C31H34F2N606 =
624.65 g/mole. )
HPLC (Vydac; C18; diameter = 4.6 mm; length = 150 mm;
gradient = H20 [0.1% H3P041- CH3CN, 95% - 5%, 5% - 95%, over 16
minutes, 2 ml/min flow rate) focus = 215 nm; 98% pure.
EXAMPLE 29
As a specific embodiment of an oral composition, 100 mg of
the compound of Example 9 (i.e., Compound 14) is formulated with
sufficient finely divided lactose to provide a total amount of 580 to 590 mg
to fill a size O hard gel capsule.
EXAMPLE 30
Screening assay: Alpha 1a Adrenergic Receptor Binding
Membranes prepared from the stably transfected human
alpha la cell line (ATCC CRL 11140) were used to identify compounds
that bind to the human alpha la adrenergic receptor. These competition
binding reactions (total volume = 200 ~1) contained 50 mM Tris-HCl pH.
7.4, 5 mM EDTA, 150 mM NaCI, 100 pM [125 I]-HEAT, membranes
prepared from the alpha la cell line and increasing amounts of
unlabeled ligand. Reactions were incubated at room temperature for
one hour with shaking. Reactions were filtered onto Whatman GF/C
glass fiber filters with a Inotec 96 well cell harvester. Filters were
washed three times with ice cold buffer and bound radioactivity was
determined (Ki). Representative compounds of the present invention
were found to have Ki values 5 50 nM.
EXAMPLE 31
Selective Binding assays
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Membranes prepared from stably transfected human alpha
ld and alpha lb cell lines (ATCC CRL 11138 and CRL 11139, respectively)
were used to identify compounds that selectively bind to the human
alpha la adrenergic receptor. These competition binding reactions (total
volume = 200 ~tl) contained 50 mM Tris-HCl pH. 7.4, 5 mM EDTA, 150
mM NaCl, 100 pM [125 I]-HEAT, membranes prepared from cell lines
transfected with the respective alpha 1 subtype expression plasmid and
increasing amounts of unlabeled ligand. Reactions were incubated at
room temperature for one hour with shaking. Reactions were filtered
onto Whatman GF/C glass fiber filters with a Inotec 96 well cell
harvester. Filters were washed three times with ice cold buffer and
bound radioactivity was determined (Ki).
EXAMPLE 32
EXEMPLARY COUNTERSCREENS
~. Assav Title: Dopamine D2, D3, D4 in vitro screen
Objective of the Assay
The objective of this assay is to eliminate agents which
specifically affect binding of [3H] spiperone to cells expressing human
dopamine receptors D2, D3 or D4.
Method:
Modified from VanTol et al (1991); Nature (Vol 350) Pg 610-
613.
Frozen pellets containing specific dopamine receptor
subtypes stably expressed in clonal cell lines are lysed in 2 ml lysing
buffer (lOmM Tris-HCl/5mM Mg, pH 7.4). Pellets obtained after
~-ifuging these membranE. (15' at 24,4'7 rpm) are resuspended in
:VI Tris-HCl pH 7.4 contav=ring EDTA agCl[2], KCI, NaCl, CaCI[2]
a~.a ascorbate to give a 1 Mg/mL suspension. The assay is initiated by
adding 50-75 ~tg membranes in a total volume of 500 ~1 containing 0.2
nM [3H]-spiperone. Non-specific binding is defined using 10 ~.M
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apomorphine. The assay is terminated after a 2 hour incubation at
room temperature by rapid filtration over GFB filters presoaked in 0.3%
PEI, using 50mM Tris-HCl pH 7.4.
2. Assay Title: Serotonin SHTla
S?biective of the Assav
The objective of this assay is to eliminate agents which
specifically affect binding to cloned human 5HTla receptor
Modified from Schelegel and Peroutka Biochemical
Pharmacology 35: 1943-1949 (1986).
Mammalian cells expressing cloned human 5HTla
receptors are lysed in ice-cold 5 mM Tris-HCl , 2 mM EDTA (pH 7.4) and
homogenized with a polytron homogenizer. The homogenate is
centrifuged at 1000Xg for 30', and then the supernatant is centrifuged
again at 38,OOOXg for 30'. The binding assay contains 0.25 nM [3H]8-OH-
DPAT (8-hydroxy-2-dipropylawino-1,2,3,4-tetrahydronaphthalene) in 50
mM Tris-HCl, 4 mM CaCl2 and lmg/ml ascorbate. Non-specific binding
is defined using 10 ~.M propranolol. The assay is terminated after a 1
hour incubation at room temperature by rapid filtration over GF/Cfilters
30
EXAMPLE 33
EXEMPLARY FUNCTIONAL ASSAYS
In order to confirm the specificity of compounds for the
human alpha la adrenergic receptor and to define the biological activity
of the compounds, the following functional tests may be performed:
1. In vitro Rat, Dog and Human Prostate and Dog Urethra
Taconic Farms Sprague-Dawley male rats, weighing 250-
400 grams are sacrificed by cervical dislocation under anesthesia
(methohexital; 50 mg/kg, i.p.). An incision is made into the lower
abdomen to remove the ventral lobes of the prostate. Each prostate
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removed from a mongrel dog is cut into 6-8 pieces longitudinally along
the urethra opening and stored in ice-cold oxygenated Krebs solution
overnight before use if necessary. Dog urethra proximal to prostate is
cut into approximately 5 mm rings, the rings are then cut open for
contractile measurement of circular muscles. Human prostate chips
from transurethral surgery of benign prostate hyperplasia are also
stored overnight in ice-cold Krebs solutaon if needed.
The tissue is placed in a Petri dish containing oxygenated
Krebs solution [NaCI, 118 mM; KCl, 4.7 mM; CaCl2, 2.5 mM; KH2P04,
1.2 mM; MgS04, 1.2 mM; NaHC03, 2.0 mM; dextrose, 11 mM] warmed
to 37°C. Excess lipid material and connective tissue are carefully
removed. Tissue segments are attached to glass tissue holders with 4-0
surgical silk and placed in a 5 mI jacketed tissue bath containing Krebs
buffer at 37°C, bubbled with 5% C02/95% 02. The tissues are connected
to a Statham-Gould force transducer; 1 gram (rat, human) or 1.5 gram
(dog) of tension is applied and the tissues are allowed to equilibrate for
one hour. Contractions are recorded on a Hewlett-Packard 7700 series
strip chart recorder.
After a single priming dose of 3 ~M (for rat), 10 ~.M (for dog)
and 20 ~M (for human) of phenylephrine, a cumulative concentration
response curve to an agonist is generated; the tissues are washed every
10 minutes for one hour. Vehicle or antagonist is added to the bath and
allowed to incubate for one hour, then another cumulative concentration
response curve to the agonist is generated.
EC50 values are calculated for each group using GraphPad
Inplot software. pA2 (-log Kb) values were obtained from Schild plot
when three or more concentrations were tested. When less than three
concentrations of antagonist are tested, Kb values are calculated
according to the followinff formula Kb =~B ,
x_t
where x is the ratio of ' p of a q.: onist in tl presence and absen ~ =~ of
antagonist. and (B] is th: .ntagonist concentration.
2. Measurement of Intra-Urethral Pres ure in Ane thetized Do~~
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PURPOSE: Benign prostatic hyperplasia causes a decreased urine flow
rate that may be produced by both passive physical obstruction of the
prostatic urethra from increased prostate mass as well as active
obstruction due to prostatic contraction. Alpha adrenergic receptor
antagonists such as prazosin and terazosin prevent active prostatic
contraction, thus improve urine flow rate and provide symptomatic
relief in man. However, these are non-selective alpha 1 receptor
antagonists which also have pronounced vascular effects. Because we
have identified the alpha la receptor subtype as the predominent subtype
in the human prostate, it is now possible to specifically target this
receptor to inhibit prostatic contraction without concomitant changes in
the vasculature. The following model is used to measure adrenergically
mediated changes in intra-urethral pressure and arterial pressure in
anesthetized dogs in order to evaluate the efficacy and potency of
selective alpha adrenergic receptor antagonists. The goals are to: 1)
identify the alpha 1 receptor subtypes responsible for prostatic/urethral
contraction and vascular responses, and 2) use this model to evaluate
novel selective alpha adrenergic antagonists. Novel and standard alpha
adrenergic antagonists may be evaluated in this manner.
METHODS: Male mongrel dogs (7-12 kg) are used in this study. The
dogs are anesthetized with pentobarbital sodium (35 mg/kg, i.v. plus 4
mg/kg/hr iv infusion). An endotracheal tube is inserted and the animal
ventilated with room air using a Harvard instruments positive
displacement large animal ventilator. Catheters (PE 240 or 260) are
placed in the aorta via the femoral artery and versa cava via the femoral
veins (2 catheters, one in each vein) for the measurement of arterial
pressure and the administration of drugs, respectively. A supra-pubic
incision -lJ2 inch lateral to the penis is made to expose the urethers,
bladder and urethra. The urethers are ligated and cannulated so that
urine flows freely into beakers. The dome of the bladder is retracted to
facilitate dissection of the proximal and distal urethra. Umbilical tape
is passed beneath the urethra at the bladder neck and another piece of
umbilical tape is placed under the distal urethra approximately 1-2 cm
distal to the prostate. The bladder is incised and a Millar micro-tip
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pressure transducer is advanced into the urethra. The bladder incision
is sutured with 2-0 or 3-0 silk (purse-string suture) to hold the
transducer. The tip of the transducer is placed in the prostatic urethra
and the position of the Millar catheter is verified by gently squeezing the
prostate and noting the large change in urethral pressure.
Phenylephrine, an alpha 1 adrenergic agonist, is
administered (0.1-100 ug/kg, iv; 0.05 ml/kg volume) in order to construct
dose response curves for changes in intra-urethral and arterial
pressure. Following administration of increasing doses of an alpha
adrenergic antagonist (or vehicle), the et~'ects of phenylephrine on
arterial pressure and intra-urethral pressure are re-evaluated. Four or
five phenylephrine dose-response curves are generated in each animal
(one control, three or four doses of antagonist or vehicle). The relative
antagonist potency on phenylephrine induced changes in arterial and
intra-urethral pressure are determined by Schild analysis. The family
of averaged curves are fit simultaneously (using ALLFIT software
package) with a four paramenter logistic equation constraining the
slope, minimum response, and maximum response to be constant
among curves. The dose ratios for the antagonist doses (rightward shift
in the dose-response curves from control) are calculated as the ratio of
the ED50's for the respective curves. These dose-ratios are then used to
construct a Schild plot and the Kb (expressed as ug/kg, iv) determined.
The Kb (dose of antagonist causing a 2-fold rightward shift of the
phenylephrine dose-response curve) is used to compare the relative
potency of the antagonists on inhibiting phenylephrine responses for
intra-urethral and arterial pressure. The relative selectivity is
calculated as the ratio of arterial pressure and intra-urethral pressure
Kb's. Efi'ects of the alpha 1 antagonists on baseline arterial pressure are
also monitored. Comparison of the relative antagonist potency on
3~' ~hanges in arteris assure and infra-urethral pressure provide
,nsight as to whet: ie alpha rceptor subtype responsible for
increasing infra-ur:. _ ral pressz is also present in the systemic
vasculature. According to this method, one is able to confirm the
selectivity of alpha la adrenergic receptor antagonists that prevent the
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increase in intra-urethral pressure to phenylephrine without any
activity at the vasculature.
While the foregoing specification teaches the principles of
the present invention, with examples provided for the purpose of
illustration, it will be understood that the practice of the invention
encompasses all of the usual variations, adaptations and/or
modifications as come within the scope of the following claims and their
equivalents.
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