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. 60/050,960, 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
-1-
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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-
Adrenoreceptors: Molecular Bioloey. Biochemistry and Pharmacolo~v,
(Progress in Basic and Clinical Pharmacolo~v 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 1a/ld),
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. Pltarm~col. {1995) 352:1-10.
The differences in the alpha adrenergic receptor subtypes
have relevance in pathophysiologic conditions. Benign 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 sufl'lciently
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 92/161213,
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 1d, alpha 1b, or alpha la 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 lb receptors in the peripheral vasculature, e.g., hypotension
and syncope. -
The recent cloning of the human alpha la adrenergic
receptor (ATCC CRL 11140) and the use of a screening assay utilizing
the cloned human alpha la 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/I0989, published 26 May 1994] As
disclosed in the instant patent disclosure, a cloned human alpha 1a
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 1a adrenergic receptor antagonists useful for
treating BPH. The instant patent disclosure discloses novel compounds
which selectively bind to the human alpha 1a 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 receptor.
It is an object of the present invention to identify compounds
which bind to the alpha la 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 1a 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 1d 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 effects related to peripheral
adrenergic blockade. Such side effects include hypotension, syncope,
lethargy, etc. The compounds of the present invention have the
structure:
R15R~6 m24
n E 2s R
N C Q
R1-N
.J P R3 q
R17 R~s
wherein Q is selected from
-5-
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~ X)s ~/(X)s ~(X)s
I ~ ~I ~ O
O~ O
R8
N N Ra ~ N N RRs ~ N N
R~ ~ I R~ ~ 1o R~ S
O O R O I' Rg
O N R , R1o ,
H
(X)S
R22 R13 p
I
7
O \
,N
I \ (X)S ~~N NRg
O / , R~
R 5 N ~O
H
~ /X)s / (X)s
I I
O \ p O \ R11 12
II R
~~N~N ~~N~N
R~ ~ I W R~ ~ I W
O N 7~ R ~ 1
H R12 , O H 0 ,
R13
R1a R13 O
w 8 R 1 a (X)s
Y O R.N ~-N
~ O
R8 , 0i _N or O ~Sv
O
5 E, G, L and M are each independently selected from hydrogen, CZ-g
alkyl, C3-g cycloalkyl, (CH2)0-408,6, (CH2)0-4N(R19)2, (CH2)p_4CN,
(CH2)p_4CFg, (CH2)0-4C02R19, (CH2)p_4CON(R19)2, (CH2)p_4S02R19, or
(CH2)0-4S02N(R19)2'>
-s-
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J is selected from hydrogen, C1-g alkyl, C3-g cycloalkyl, (CH2)1-4OR6,
(CH2)1-4N(R19)2~ (CH2)1-4CN~ (CH2)0-4CF3~ (CH2)p_4C02R19,
(CH2)0_4CON(Rl9)2~ (CH2)0-4SO2R19, or (CH2)0-4S02N(Rl9)2;
R1 is selected from unsubstituted, mono- or poly-substituted phenyl
wherein the substituents on the phenyl are independently selected from
halogen, CF3, cyano, nitro, N(R19)2, NR19COR20, NR19CON(R20)2,
NR19S02R20, NR19S02N(R20)2, OR6, (CH2)0_4CO2R19, oxadiazolyl-, C1_
4 alkyl oxadiazolyl-, (CH2)0_4CON(Rl9)2, (CH2)0-4S02N(Rl9)2, (CH2)0-
4S02R6 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, (CH2)p-4CO2R19, (CH2)0_4CON(Rl9)2, (CH2)0-4S02N(R19)2~
(CH2)0-4s02R6~ phenyl, OR6, halogen, C1-4 alkyl or C3-g cycloalkyl;
R2, R3, R8, R10, R15~ R,16~ R17 and R1g are each independently selected
from hydrogen, C1-g alkyl, C3-g cycloalkyl, (CH2)2_4086 or (CH2)0-4CF3~
R4 is selected from hydrogen, (CH2)0-4COR6, (CH2)0-4CN, (CH2)0-4CF3~
(CH2)0_4CO2R19, (CH2)0-4CON(Rl9)2~ (CH2)0-4S02R6 or
(CH2)0-4S02N(Rl9)2;
R5 is selected from hydrogen, C1-g alkyl, C3-g cycloalkyl, (CH2)1-4086 or
(CH2)0_4CF3;
R6 is selected from hydrogen, C1-g alkyl, C3-g cycloalkyl or
(CH2)p_4CF3;
R7 is selected from hydrogen, C1-g alkyl, C4-g cycloalkyl,
(CH2)p_4CO2R19, (CH2)0-4CON(Rl9)2~ (CH2)0-4COR19, (CH2)2-40R6~
(CH2)1-4CF3~ (CH2)0-4S02R6, (CH2)p_4S02N(Rl9)2 or (CH2)1-4CN;
-7
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R9 is selected from hydrogen, C1-g alkyl, Cg-g cycloalkyl, C02R6,
CON(R6)2, (CH2)1-4086 or (CH2)p-4 CF3;
R11 and R12 are each independently selected from hydrogen,
C1-g alkyl or C3-g cycloalkyl;
R13 and R14 are each independently selected from hydrogen, C1-g alkyl,
C3-g cycloalkyl, (CH2)1-408,6, (CH2)0-4CF3~ unsubstituted, mono- or
poly-substituted phenyl wherein the substituents on the phenyl are
independently selected from halogen, CF3, cyano, nitro, amino, OR6,
(CH2)0-4CON(R19)2, (CH2)0-4C02R19 or C1-4 alkyl; or unsubstituted,
mono- or poly-substituted: pyridyl, thienyl, furanyl or naphthyl wherein
the substituents on the pyridyl, thienyl, furanyl or naphthyl are
independently selected from CF3, phenyl, OR6, halogen, C1-4 alkyl or
C3_g cycloalkyl;
R19 and R20 are each independently selected from hydrogen, C1-g alkyl,
C3-g cycloalkyl or (CH2)1-4CF3~
R22 is selected from hydrogen, C1-g alkyl, C3-g cycloalkyl, (CH2)0-4086
or (CH2)0-4CF3~
R24 and R26 are each independently selected from hydrogen or OR28;
R28 is selected from hydrogen, C1-g alkyl, C3-g cycloalkyl, or
(CH2)0_4CF3~
W is O or NR11;
each X is independently selected from halogen, cyano, nitro, C1-g alkyl,
C3-g cycloalkyl, (CH2)0-4086 or (CH2)0-4CF3;
Y is C-R19 or N;
_g-
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Z is hydrogen, oxygen or sulphur;
m, p and q are each independently an integer from zero to two, provided
that when q is zero, R26 is hydrogen;
n, o, and s are each independently an integer from zero to four; and
v is an integer from zero to one;
and the pharmaceutically acceptable salts thereof.
In a first embodiment of the invention is the compound
having the structure
16
R~5 R ' m R2
n /)E
N C C~
R N ~ ~ls
o G R»R~a p R
L
wherein R1 is selected from unsubstituted, mono- or poly-substituted
phenyl wherein the substituents on the phenyl are independently
selected from halogen, CF3, cyano, vitro, N(Rl9)2, NR19COR20,
NR19CON(R20)2, NR19S02R20, NR,19S02N(R20)2~ OR,6~ (CH2)0-
4C02R19, (CH2)0-4CON(Rl9)2~ (CH2)p_4S02N(R19)2~ (CH2)0-4S02R6 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, (CH2)0-4C02R19, (CH2)0-4CON(R19)2~ (CH2)0-4s02N(Rl9)2~
(CH2)0-4S02R6, phenyl, OR6, halogen, C1-4 alkyl or C3-g cycloalkyl;
R4 is selected from (CH2)0-4CORS, (CH2)0-4CN, (CH2)0-4CF3~
(CH2)0-4C02R19~ (CH2)0-4CON(Rl9)2, (CH2)0-4S02R6 or
(CH2)p_4S02N(Rl9)2~
R5 is selected from hydrogen, C1-g alkyl, Cg-g cycloalkyl, (CH2)2-4086 or
(CH2)p_4CF3~
_g_
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R9 is selected from hydrogen, C1-g alkyl, C3-g cycloalkyl, (CH2)2-4086 or
(CH2)0-4CF3~ and
all other variables are as defined above; and the pharmaceutically
acceptable salts thereof.
In a second embodiment of the invention is the compound of
the formula
M R~5Rls m24
n E 2s R
N C Q
R~_N J I
3
GJ R» Rie ~ R
L
wherein
E, G, L, M and J are each independently selected from hydrogen,
C1-g alkyl, C3-g cycloalkyl, or (CH2)0-4CF3;
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(R19)2, NR19COR20, NR19CON(R20)2,
~19S02R,20~ Ng,19S02N(R20}2, OR6, (CH2)0-4C02R19, oxadiazolyl-,
C1-4 alkyloxadiazolyl, (CH2)0-4CON(R19)2, (CH2)0-4S02N(Rl9)2, (CH2)0-
4S02R6 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, (CH2)0-4CO2R,19, (CH2)0-4CON(R19)2, (CH2)0-4S02N(Rl9)2~
(CH2)0-4S02R6~ phenyl, OR6, halogen, C1-4 alkyl or C3-g cycloalkyl;
R7 is selected from hydrogen, C1-g alkyl, C4-g cycloalkyl or
(CH2)1-4CF3~
-10-
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R13 and R14 are each independently selected from hydrogen, C1-g alkyl,
C3-g cycloalkyl, (CH2)1-4086, (CH2)0-4CF3~ substituted, mono-, di- or
tri-substituted phenyl wherein the substituents on the phenyl are
independently selected from halogen, CF3, cyano, nitro, amino, OR6,
(CH2)0-4 CON(Rl9)2, (CH2)0-4C02R1~ or C1-4 alkyl; or unsubstituted,
mono-, di- or tri--substituted: pyridyl, thienyl, furanyl or naphthyl
wherein the substituents on the pyridyl, thienyl, furanyl or naphthyl are
independently selected from CF3, phenyl, OR6, halogen, C1_4 alkyl or
C3_g cycloalkyl; and
n is an integer from zero to two;
and all other variables are as originally defined above;
and the pharmaceutically acceptable salts thereof.
In a third embodiment of the invention is the compound of
the formula
Ris
m R2
I
R1_N l N ~ W
~J ~P ~R3 q
G Ri7R~a
wherein E, G, L, M and J are each independently selected from
hydrogen, C1-g alkyl, C3-g cycloalkyl, or (CH2)0-4CF3~
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(R19)2, NR19COR20, NR19CON(R20)2,
NR19S02R20, NR19S02N(R20)2, OR6, (CH2)0-4C02R19~
(CH2)0-4CON(R19)2, (CH2)0-4S02N(R19)2~ (CH2)0-4S02R6 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,
-11-
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vitro, (CH2)0-4C02R19, (CH2)0-4CON(R19)2, (CH2)0-4S02N(Rl9)2~
(CH2)0-4S02R6~ phenyl, OR6, halogen, C1_4 alkyl or C3_g cycloalkyl;
R7 is selected from hydrogen, C 1-g alkyl, C4-g cycloalkyl or
(CH2)1-4CF3~
R13 and R14 are each independently selected from hydrogen, C1-g alkyl,
C3-g cycloalkyl, (CH2)1-4086, (CH2)0-4CF3~ substituted, mono-, di- or
tri-substituted phenyl wherein the substituents on the phenyl are
independently selected from halogen, CF3, cyano, vitro, amino, OR6,
(CH2)0-4CON(R19)2, (CH2)0-4C02R19 or C1_4 alkyl; or unsubstituted,
mono-, di- or tri--substituted: pyridyl, thienyl, furanyl or naphthyl
wherein the substituents on the pyridyl, thienyl, furanyl or naphthyl are
independently selected from CF3, phenyl, OR6, halogen, C1_4 alkyl or
C3_g cycloalkyl;
n is an integer from zero to two;
and all other variables are as defined in the first embodiment;
and the pharmaceutically acceptable salts thereof.
In a first class of the invention is the compound selected
from
R 24 R 24
R1-N~N R2s R2s
(CH2) Q R'-N N (CH2~Q
4
R R24
R'-N N ~ R2s R R2s
( H2)q Q ~~ R' N\~(CH2)q D
wherein Q is selected from
-12-
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~~ X)s \ (X)s ~~(X)s
I i ~ ~ ( i
O R4 O ~Ra ~ s
~ R
~~N~N y ~ 9 ~~N N
R I N N R R~ S
R~ ~ ~ Rlo O~ s
O H , O 0 R~oR
R 22 R 13 ''
R7
,N
or ~ I (X)S ;
o ~J
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, OR6, (CH2)0-2C02R,19, (CH2)0-2CON(R19)2,
(CH2)0-2SO2N(R19)2, (CH2)0-2SO2R6> C1-4 ~kYl oxadiazolyl- or C1-4
alkyl; or unsubstituted, mono-, or di-substituted pyridyl wherein the
substituents on the pyridyl are independently selected from halogen,
CF3, cyano, nitro, OR6, (CH2)0-2C02R,19, (CH2)0-2CON(R19)2,
(CH2)0-2SO2N(R19)2, (CH2)0-2SO2R6 or C1_4 alkyl;
15
R4 is selected from hydrogen, CORE, C02R19, S02R6 or CON(R19)2;
R~ is selected from hydrogen, C1-g alkyl, C3-6 cycloalkyl, (CH2)1-3086 or
(CH2)0-3CF3;
R6 is selected from hydrogen, C1-g alkyl, C3-g cycloalkyl or
(CH2)0_3CF3~
Rg and R10 are each independently selected from hydrogen,
C1-g alkyl, C3-6 cycloalkyl, (CH2)2-4086 or (CH2)0-3CF3;
R9 is selected from hydrogen, C1-6 alkyl, C3-g cycloalkyl, C02R6,
CON(R6)2, (CH2)1-4086 or (CH2)0-3CF3~
-13-
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R13 is selected from hydrogen, C1-6 alkyl, Cg-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, nitro, amino, OR6, C02R19 or C1-4
alkyl;
R19 is selected from hydrogen, C1-g alkyl, Cg-6 cycloalkyl or
(CH2)1-3CF3~
R22 is selected from hydrogen, C1-s alkyl, Cg-6 cycloalkyl, (CH2)0-4086
or (CH2)0-3CF3~
R28 is selected from hydrogen, C1-6 alkyl, C3-6 cycloalkyl, or
(CH2)0-3CF3~
q is an integer from zero to two; and
s is an integer from zero to three;
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 selected
from
(CH2~Q
R'- N~ N- , R'- N N (C H2}q (~
~/ ~/ ,
(CH2r-Q
R~-N~N \
~/ or R ~-N~ N~~""' (C H2~Q ,
wherein Q is selected from
-14-
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~~ X)s \ (X)s ~~~X)s
~ i ~ ~ ~ i
O O ~ O
~~N~N R4 y ~ R8 9 ~~N~N R
N N ~ R R~ S
O N R5 R7 ~O Rlo O~~C s
O R~oR
R~
Of i
~~N
_ ~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, nitro, ORS, (CH2)0-2C02R19, (CH2)0-2CON(R19)2,
(CH2)0-2S02N(R19)2, (CH2)0-2S02R6 or C1_4 alkyl; or unsubstituted,
mono-, or di-substituted pyridyl wherein the substituents on the pyridyl
are independently selected from halogen, CF3, cyano, nitro, OR6,
(CH2)0-2C02R19~ (CH2)0-2CON(R19)2~ (CH2)0_2S02N(R19)2~
(CH2)0-2S02R6 or C1_4 alkyl;
R4 is selected from CORE, C02R19, S02R6 or CON(Rl9)2;
R5 is selected from hydrogen, C1-6 alkyl, C3-g cycloalkyl, (CH2)1-3086 or
(CH2)0_3CF3~
R6 is selected from hydrogen, C1-g alkyl, Cg-6 cycloalkyl or
(CH2)0-3CF3~
Rg, R9 and R10 are each independently selected from hydrogen,
C1-g alkyl, C3-s cycloalkyl, (CH2)2-4086 or (CH2)0-3CF3;
R13 is selected from hydrogen, C1-g alkyl, C3-6 cycloalkyl,
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(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, nitro, amino, OR6, C02R19 or C1-4
alkyl;
R19 is selected from hydrogen, C 1-6 alkyl, C3-6 cycloalkyl or
(CH2)1_3CF3~
R22 is selected from hydrogen, C1-6 alkyl, C3-6 cycloalkyl, (CH2)0-4086
or (CH2)0-3CF3~
q is an integer from zero to two;
s is an integer from zero to three;
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 wherein
Q is selected from
'~ X)s ~~ (X)s
/ (X)S
R4 ~~ ~ O
N N H N Rs ~\ ~ I
Rs O~O °r H ~s
N R
H
R13 is selected from hydrogen, C1_4 alkyl or unsubstituted, mono- or di-
substituted phenyl wherein the substituents on the phenyl are
independently selected from halogen, CFg, cyano, nitro, amino, OR6,
C02R19 or
C 1_4 alkyl;
each X is a halogen;
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and all other variables are as defined previously in the first class; and
the pharmaceutically acceptable salts thereof.
In a second subclass of the invention is the compound
wherein (a is selected from
X)s ~~ (X)s
I / p I / _(
O
O
R4 ~~ N N Rs
H N I H N
~ O ~~ H 13 '
O' _ N Rs O R
H
R13 is selected from hydrogen, C1_4 alkyl or unsubstituted, mono- or di-
substituted phenyl wherein the substituents on the phenyl are
independently selected from halogen, CFg, cyano, vitro, amino, OR6,
C02R19 or C1-4 alkyl;
each X is a halogen;
and all other variables are as defined previously in the second class;
and the pharmaceutically acceptable salts thereof.
In a first illustration of the invention is the compound
selected from
(R21)r
R 24
I~N N ~
~R26
~~(CH2)q-Q
(R21)r R24
R2s
N N CH Q
( 2~
-A
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(R21)r
R 24
--N N R2s
'(C H2)q-Q
(R21)r
R 26
N N (CH2}-Q
a
-A
wherein A is C-R21 or N;
each R21 is independently selected from hydrogen, halogen, cyano,
OC1_4 alkyl, OCF3, OCH2CF3, C02CH3, CONH2, S02NH2 or
S02C1_4 alkyl;
R24 and R26 are each independently selected from hydrogen or OR28,
wherein R28 is hydrogen or C1_4 alkyl;
each X is fluorine;
r is an integer from zero to two; and
q is an integer from zero to one;
and all other variables are as defined previously in the first subclass;
and the pharmaceutically acceptable salts thereof.
In a second illustration of the invention is the compound
selected from
(R21)r (CH2~Q
~- N N
-A
(R2~)r
-N N (CH2~Q
-A
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(R21)r
(C H2}q-Q
--- N N
-A or
(R21 )r
~~ --N N\~--(CH2~-Q
-A
wherein A is C-R21 or N;
each R21 is independently selected from hydrogen, halogen, cyano, OC1_
4 alkyl, OCF3, OCH2CF3, C02CH3, CONH2, S02NH2 or S02C1-4 alkyl;
each X is fluorine;
r is an integer from zero to two;
q is an integer from zero to one;
and all other variables are as defined previously in the second subclass;
and the pharmaceutically acceptable salts thereof.
In a first exemplification of the invention is the compound
wherein
Q is selected from
F F
F I \ F
\ F
O I / O / F
O /
R4 ~~ N~N I
H N I H ~ ~~ N \
~ O ~r H 13 '
O' _N R5 O
H
R4 is C02R19;
R5 is (CH2)1-3086;
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and all other variables are as defined previously in the first illustration;
and the pharmaceutically acceptable salts thereof.
In a second exemplification of the invention is the
compound wherein
(1 is selected from
F F
F ~ F
/ F
O / F
II O
~N~N R4 ~N N
H ~ H ~ ~ ~ N
O~ N Rs O O or H Ry3 ,
H
R4 is C02R19;
R5 is (CH2)1-3086;
and all other variables are as defined previously in the second
illustration;
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 carrier.
Another example of the invention is 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
component combination comprising any of the compounds described
above combined with both a type 1 testosterone 5-alpha reductase
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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.
More particularly exemplifying the invention is a method of
treating a disease which is susceptible to treatment by antagonism of the
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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.
DETAILED DESCRIPTION OF THE INVENTION
Representative compounds of the present invention exhibit
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 affinity for the human alpha la adrenergic receptor
subtype while displaying at least ten-fold lower afl'-lnity for the human
alpha ld and alpha 1b 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
fold lower affinity for the human alpha ld and alpha lb 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 la receptor where such treatment is needed, as in
BPH. For use in medicine, the salts of the compounds of this invention
refer to non-toxic "pharmaceutically acceptable salts." Other salts may,
however, be useful in the preparation of the compounds according to the
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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 acid, sulphuric
acid, fumaric 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.
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.
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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 in 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 are intended to be included in the
present invention. In addition, 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.
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.
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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., C1-10) 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 (e.g., phenyl) 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, R19, R20) at a particular location in a molecule be
independent of its definitions elsewhere in that molecule. Thus, -
N(R19)2 represents -NH2, -NHCHg, -NHC2H5, -N(CHg)C2H5, etc. and
R24
~_~~m represents for m=2
OH OH OH OH
a
i .sr'v , i .r~ , i s~~ i s~ , 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
can be readily synthesized by techniques known in the art as well as
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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 "Z is hydrogen," when refering to the "Q" group
R13
R14
~Y
O
Z N
R8
refers to the moiety
R13
R14
~Y
~N O
R8
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, pyl-idyl, 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.
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The term "thienyl," as used herein, refers to the group
S
The terms "(+)-DHP" and "DHP" as used herein, refer to a
dihydropyrimidinone group of the formula
(X)q
O
~N
O~ N R5
H
for example:
F
F
O ~ O
N Oi
~ O
O' _ N
H
The term "activated (+)-DHP," as used herein, refers to a N-
3-(activated)carbamate of the desired dihydropyrimidinone where the
activating group is, for example, a p-nitrophenyloxy group. A specific
example of an activated (+)-DHP is 4-(3,4-difluorophenyl)-5-
methoxycarbonyl-6-methoxymethyl-2-oxo-1,2,3,4-tetrahydropyrimidine-
3-carboxylic acid (4-nitrophenyl ester), also referred to as the compound
8.
The term "(S)-oxa" as used herein, refers to an
oxazolidinone group of the formula
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~x)q
O /Ra Rs
~,~ N 10
R
~'''O
for example,
F
F
O
~~ N
-O
O
The term "activated (S)-oxa" as used herein, refers to an N-
(activated)carbamate of the desired oxazolidinone where the activating
group is, for example, a p-nitrophenyloxy group. A specific example of
an activated (S)-oxa group is 4-(3,4-difluorophenyl)-2-oxo-oxazolidine-3-
carboxylic acid 4-nitrophenyl ester (i.e., compound 19).
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 la adrenergic
receptor as compared to the human alpha 1b, 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.
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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
suspensions, metered aerosol or liquid sprays, drops, ampoules, auto-
injector devices or suppositories; for oral, parenteral, intranasal,
sublingual or rectal administration, or for administration by inhalation
or insufflation. 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 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
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dosage and an outer dosage component, the latter being in the form of an
envelope over the former. 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 or by
injection include aqueous solutions, suitably flavoured syrups, aqueous
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
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,
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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 Groins 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 methods known from the art.
The specificity 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 la receptor and
membranes from cell lines or tissues known to express other types of
alpha (e.g., alpha ld, alpha lb) or beta adrenergic receptors. 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. 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 1a receptor is compared against the binding
af~lnities to other types of alpha or beta adrenergic receptors. The
human alpha adrenergic receptor of the la 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 la
receptor, when expressed in mammalian cell lines, is used to discover
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ligands that bind to the receptor and alter its function. Expression of the
cloned human alpha 1d, 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 Ia
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. See 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
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,
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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 those 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.
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
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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.
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-
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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 according to dosage regimens
established in the art whenever specific blockade of the human alpha
la adrenergic receptor is required.
The daily dosage of the products may be varied over a wide
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
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 20 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 7 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
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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 93/23050; W093/23038, ; W093/23048; W093/23041;
W093/23040; W093/23039; W093/23376; W093/23419, EP 0572165;
W093/23051.
The dosages of the alpha la adrenergic receptor and
testosterone 5-alpha reductase inhibitors are adjusted when
combined to achieve desired effects. As those skilled in the art will
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 la 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
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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 4,713-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 1 or 2 inhibitor are administered in separate oral,
systemic, or parenteral dosage formulations. See, e.g., U.S. Patent
No.'s 4,377,584 and 4,760,071 which describe dosages and
formulations for 5a-reductase inhibitors.
I5 Abbreviations used in the instant specification, particularly
the Schemes and Examples, are as follows:
AcOH or HOAc = acetic acid
BCE = bromochloroethane
Boc or BOC = t-butyloxycarbonyl
BOC20 = di-tert-butyl dicarbonate
BOPCl = bis(2-oxo-3-oxazolidinyl)phosphinic chloride
BuOH = butanol
Cbz-Cl = benzyloxycarbonyl chloride
DAST = diethylaminosulfurtrifluoride
DEAD = diethylazodicarboxylate
DMF = N,N-dimethylformamide
DMSO = dimethylsulfoxide
DPPA = diphenylphosphoryl azide
EDCI = 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide
hydrochloride
Et = ethyl
Et3N = triethylamine
EtOAc = ethyl acetate
EtOH = ethanol
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FABLRMS = fast atom bombardment low resolution mass
spectroscopy
FABMS = fast atom bombardment mass spectroscopy
HMPA = hexamethylphosporamide
HPLC = high performance liquid chromatography
HOBt = 1-hydroxy benzotriazole hydrate
i-Pr or iPr = isopropyl
i-PrOH = 2-propanol
i-Pr2NEt = diisopropylethylamine
LAH = lithium aluminum hydride
mCPBA = meta-chloroperbenzoic acid
Me = methyl
MeOH = methanol
n-BuLi = n-butyl lithium
NMR = nuclear magnetic resonance
PCTLC = preparative centrifugal thin layer
chromatography
PEI = polyethylenimine
Ph = phenyl
RT = retention time
TEBAC = benzyltriethylammonium chloride
TFA = trifluoroacetic acid
THF = tetrahydrofuran
TLC = thin layer chromatography
TMS = trimethylsilyl
Tos20 or TOS20 = p-toluenesulfonic anhydride
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.
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The preparation of some desired compounds are described
in Schemes 1-11. 3-(tent-Butyloxycarbonyl)amino piperidine was
prepared from nipecotic acid in three steps, Scheme 1. Subsequent
reductive amination with a piperidone derivative, followed by Boc
deprotection provided the desired acylation/alkylation precursor which
after treatment with activated (+)-DHP, (S)-oxa, or difluorophenyl acetic
acid derivatives produced the alpha la antagonists. When desired, the
ketone starting materials can be further elaborated, for instance, via
enolate alkylation to provide the alpha substituted ketones.
The 3-aminomethyl piperidinyl derivatives were assembled
as outlined in Scheme 2 starting with reductive amination of 3-hydroxy
methyl piperidine and a piperidone derivative. The hydroxy group was
converted to a tosylate, displaced with azide and reduced to the amino
compound. Acylation or alkylation under standard conditions produced
the targeted analogs.
The 3-aminopyrrolidinyl analogs were constructed by
reductive amination of 3-(tent-butoxycarbonyl)amino pyrrolidine and a
piperidone derivative, Scheme 3. Acidic deprotection of the carbamate
produced the 3-amino pyrrolidinyl intermediate which was acylated
under standard conditions.
In Scheme 4, the 3-aminomethyl pyrrolidinyl analogs were
assembled in an analogous manner to the 3-aminomethyl piperidinyl
analogs illustrated in Scheme 2.
Similarly, Scheme 5 describes the preparation of the 3-
aminoazetidinyl analogs.
Examples of the preparation of some desired compounds
are outlined in Scheme 6. Selective Boc protection of 4-aminomethyl
piperidine (1) on the piperidine nitrogen provided (2) and CBZ protection
of the primary amine resulted in (3). TFA treatment of (3) produced (4)
which after reductive amination with ketone (5) yielded (6).
Hydrogenolysis of (6) left the primary amine (7) which clearly acylated
with (8) which produced the DHP derivative (9). Amine (7) was also
acylated with the oxazolidinone (19) producing (28).
Other derivatives were prepared via the route outlined in
Schemes 7 to 8. N-protected 3-hydroxy azetidine (10) was tosylated
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providing (11). Azide displacement produced (12) followed by reduction
with PPh3/H20 resulted in N-protected 3-amino azetidine (13). Boc
protection provided (14) which after hydrogenation produced (15).
Reductive amination with (5) yielding (16), followed by TFA treatment set
up the requisite amine (17). Acylation with activated DHP (8), oxa (19)
and acids (29) and (31) produced (18), (20), (30) and (32), respectively.
The synthesis of some 3-substituted piperidinyl analogs was
accomplished starting with reductive amination of 3-hydroxymethyl
piperidine (21) and (5) providing (22) as shown in Scheme 8. Tosylation,
azide displacement and subsequent reduction provided the 3-
aminomethyl piperidinyl intermediate (25). Acylation with activated
DHP (8) and oxa (19) produced (26) and (27). These diastereomeric
mixtures were separated using a Chiracel AD column providing the
pairs (26a) & (26b) and (27a) & (27b).
Antagonists with cycloalkyl linking chains can be
assembled by reductive amination of the prerequisite amino alcohol and
a ketone, for example, N-(2-cyanophenyl)piperidin-4-one, Scheme 9.
Conversion of the hydroxy to a tosylate with tosyl anhydride, followed by
displacement by the sodium or lithium salt of the desired Q group
completes the synthesis of the targeted antagonists.
The 3-aminomethyl azetidine analogs were assembled
starting from the N-protected 3-cyanoazetidine (33) by LAH (lithium
aluminum hydride) reduction (34), amine protection with BOC20 (35)
and azetidine deprotection (36) under catalytic hydrogenation conditions,
Scheme 10. Reductive amination with (5) produced (37), which after Boc
deprotection with TFA liberated the required amine (38) for coupling
with activated species (8) and (19) providing (39) and (40), respectively.
Selective acylation of the primary amines was accomplished
by treatment of the amines with nearly equimolar quantities of the
activated termini species (i.e., the "Q" groups). The activated termini
species comprising the "Q" groups are readily prepared by one of
ordinary skill in the art. For example, unsubstituted, alkyl- and
cycloalkyl-substituted oxazolidinones are prepared and activated in
general by published and well developed chemistry, in particular, of
Evans. [Evans, D.A.; Nelson, J.V.; Taber, T.R. Top. Stereochem. 13, 1
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(1982)] The starting materials, in general, are natural and unnatural
amino acids. For instance, some of the preferred compounds are
prepared from substituted phenyl glycine 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).
Oxazolidinones substituted with carboxylate, carboxamide,
and hydroxymethyl are prepared by hydroxyamination of olefins to
provide protected aminoalcohols, using procedures as described in
Sharpless et al., Angew. Chem. Int. Ed. Engl., 35, 2813 (1996).
Deprotection under standard conditions followed by a phosgene
equivalent to mediate cyclization provides the substituted oxazolidinone
ring system. Deprotonation with a strong base, for example, lithium
bis(trimethylsilyl)amide, and addition to a THF solution of p-
nitrophenylchloroformate produces the stable, isolatable "activated"
oxazolidinone.
Dihydropyrimidinones are prepared by condensation
reaction of the aldehyde, urea and a 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. I74-175. 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 the unsubstituted, alkyl-
and cycloalkyl-substituted oxazolidinones were synthesized
independently in racemic form, and then separated utilizing preparative
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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.
The oxazolidinones substituted with carboxylate,
carboxamide, and hydroxymethyl were prepared in enantiomer-
enriched form and the assignments of (4S,5R) were made in accordance
with Sharpless et al., Angew. Chem. Int. Ed. Engl., 35, 2813 (1996)
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SCHEME 1
AcOH or
\ Ti(OiPr)4 I /
NaCNBH3 N
/ N MeOH CN NHBOC
CN ~O HN NHBOC ~ HCI _
EtOAc
-NH ~) CBZCI
2) DPPA
C02H Et3NItBuOH
3) H2/Pd-C
/
NC
N
N HO \ F
I
/ F
NH2
EDC / \
HOBt
N
NC
N H
N
\ F
O / F
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SCHEME 1 (CONT"D)
/ r
\ I \ F
NC
N 02N / O /
\ I O' _N C02Me
~ I
N 8 O' _ N
H O
NH2 I \ F / I C02Me
F / N F H \ Oi
CN ~ N NH
F ~ ~ N
O O
O II N I1 0
O O
02N 19
F
/ F
CN ~N N N O
O O
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SCHEME 2
\ Ac0 H \
I NaCNBH3 I
/ N MeOH . / N
CN HN OH CN ~ ~
O v 'N OH
1 ) Tos20 I /
N
2) NaN3
3} Ph3P/H20 CN N NH2
F
\ I \ F
I
/ N O / 19
CN N N ~N C02Me
H ~ I O
O N
H
1F
\ I \ F
I / o /
CN N N- _N
H
O O
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SCHEME 2 (Cont'd)
I
N
CN
N -NH2
EDCI/HOBt
HO ~ F
I
O
F
N O ~ I F
CN N N ~ F
H
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SCHEME 3
\ AcOH I /
NaCNBH3 N
MeOH NC NHBOC
NC N NHBOC N~ HCI
HN~ EtOAc
~O
F
\ -
\
I / N .~ 9 I F
CN N NHZ CN N N N O
,~'~ N
O
O
\ ' F / I C02Me
I
/ N F H \ Oi
CN N N~N~NH
IOI I IO
EDC I/HOBt
HO / F
I
O v 'F
I\
/ N
H
CN N N \ F
I
O / F
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SCHEME 4
AcOH \
I NaCNBH3 I
N MeOH / N
CN ~O HN OH CN N OH
1 ) Tos20 I /
N
2) NaN3 CN
3) Ph3P/H20 N'~-~ NH2
F
I \ I \ 19
N O /
CN N N- _N C02Me
H
O~ N Ow
H
F
I \ I \ F
/ O /
CN ~
N N- 'N
'/_~ H
/~- O
O
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SCHEME 4 (Cont'd)
I\
/ N
CN
N~~_~ N H2
EDCI/HOBt
HO \ F
I
\ O v 'F
I
/ N O / I F
CN N N \ F
'/_~ H
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SCHEME 5
Ac0 H
NaCNBH3
MeOH I /
N
N CN
CN NHBOC
O HN~ NHBOC
1 ) Tos20 HCI
2) NaN3 EtOAc
Ph 3) Ph3P
>--N OH 4) BOC20 I /
Ph ~~ 5) H2~Pd-C N
CN
N
NH2
C02Me
/ I
(
19 / N F \ ~ O
H
CN N\~N~N~NH
IOI ~(O
F
~ F ~ /
CN ~ N N O
N;~- ~
O O
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SCHEME 5 (Cont'd)
N
CN N
NH2
EDCI/HOBt
R 22
R22 = H, i-Pr
HO ~ F
I
O
~N F
NC
N~ N R 22
F
O ~ F
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SCHEME 6
O
BOC20 ~-N~ Cbz-CI
H N~ -- O ~/ N H2
NH2
/
O
-N~ O TFA
p HN~ O
O ~J 'N--~
4 H 'p
/ CN
NaCNBH3 I
\
/ CN 5
I o
\ N /
/ cN ~ I
\ ( Hz N H
. N u0
N I lO
6
NH2
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SCHEME 6 (cont'd)
/ CN
I
N
H
N O~N Oi
~N N I O~
O j O
I
F
F
I/
~CN
N
CN
N
N
19 N O O
NH2 H
N N
7
O
28
/ F
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SCHEME 7
\ i\
/
/
TOS20 NaN
N \ _ O N I \
I n
~ ~ S-O
HO " 11
O
I\ I\
/ /
P h3P BOC20
N I \ H20 N i \ -'
N3 H2N
12 13
I \ CN
\i
H2 N
O N i \ --~ O 5 O
/ ~N
O N O H
H 14 NaCNBH3
/ CN / CN
\( \i
N
TFA
Na ~ -' N
NH
16 H O 17 2
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SCHEME 7 (cont'd)
/ CN
\ I
N
H
/ CN 8 NV- H O~ N Oi
\ I ~ N N I O\
N
18 O = O
N~ 19 / C N / I
i7 NH~ I \ F
N
F
N N O~-.-O
~-NJ
20 O
/ F 29 / F I / F
\I \I F
HO F HO v ~ F
31
/ CN
\I
N
O / F
CN N~ I
\ I H \ F
N
F
N~ O / I 30
32 H \ F
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SCHEME 8
CN
I / CN
OH ~ N ~
N OH TOS20
O
N
H N 22
NaCNBH3
21
CN / CN
~I o SIN
O S ~ ~ N3
N p NaN3 N
23 24
CN
I N O N Oi ~ 25
w
N ~ I
N ~ Ow
O = O
~I
26 F Chiracel AD
F
26a Diastereomer A
26b Diastereomer B
I CN ph3P
N NH2
N
8
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SCHEME 8 (cont'd)
CN
N H ~-O
N NH2 N N
19 N
N ~ p
25 31 ~
~F
Chiracel AD F
31a Diastereomer A
31b Diastereomer B
CN
I
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SCHEME 9
AcOH or
Ti(OiPr)4 I
I NaCNBH3 / N
/ N MeOH ~ _2
~-2 CN N (CH2)o-20H
CN CH OH
O HN- ( 2)0-2
02
0-2
Tos20
F
F \ I I / N
1-2
O nBuLi CN N (CH2)o-20Tos
H
)_
O F 02
/ N F \ I
1-2
CN O
N . ~0_2 N II
0-2 O
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SCHEME 9 (cont'd)
ig R14
R R14 / N 1-2 R13 O
H~Y CN ~ ~ s
O N - ~ ~0-2 Y~NR
Z N
R 8 ~ 0-2 Z
R13 \
s R14 I Ra
R~N NaH / N O N Ris
O 1-2
N CN ~ 14
O H N ~ ~0-2 N R
O (X)s 0-2 O
/\
HN I / I \
O S~ / N _ O (X)s
O CN 1 2
N ~ .0-2 N, I /
;S~
0-2 . ~ O
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SCHEME 10
\ /
LAH \ BOC20
N
N AIC13 I /
/ H2N
NC
33 34
CN
H~ N \ N
N ~ \ 10% Pd-C 5 O
/ BOCHN
BOCHN NaCNBH3
36
CN / CN
/
\ \ N
N
TFA
N N,~
NHBOC ~NH2
37 38
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PCT/US98/12672
SCHEME 10 (Cont'd)
CN
N
H
N H O N Oi
N ~ ~ O
~N
39 O = O
F
~$ 9 / CN F
N
N H O~-- O
~N N
J
o
_F
F
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SCHEME 11
0
0
O BnOCONHz NH O
O H MeOH Me tgcOCl, NaOH ~ Me
v - w - v
F ' HZSO.~ F (DHQ)zPHAL F dH
K,OsOz(OH)z
F
NHz O F
Hz, Pd/C LiHMDS
O H Me ~~ph~ ,~OzMe
F ' Pr~NEt H p-NOzPhOCOCI
o
F R 2~ F
F
~ Rz~ F
02N ~~NHz
gar OzMe O
O ~pr~~ ~,~OzMe
O
F F
F 1 ~ Rz~ , F
/~ CHC13/NH3 ~
N~N~ ~ ~~ OpMe ~N~ O ~~O N H2
N silica gel
H
O O
1
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Scheme 11 (cont'd)
F F
F
F ~ F ' 2,3 dihydropyran
LiB H4
N 02Me ----~' ,r-O H CSA, CHZCl2
THF R
O O
F
F
R~
LiHMDS -_ 02N
F ~~N~N H
p-N02PhOCOCt I C 'Pr~NEt
F F
2~ F R2~ F
\ ~~ ~N r"''-0 PTsOH ~N~ ~ ~,,-0 H
MeOH ~~~ ~,,~~I N
O
The following examples are provided to further define the
invention without, however, limiting the invention to the particulars of
these examples.
EXAMPLE 1
4 Aminomethvl pineridine 1 carboxylic acid tert-butyl 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
water, dried over MgS04, filtered and the solvents removed in vacuo to
give the title compound
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1H NMR (CDC13, 400 MHz) 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
4-(Benzyloxycarbonylamino-methyl)-piperidine-1-carboxylic acid tert-
butyl ester (3)
To a solution of 2 (2.4 g, 1I.8 mmol) in 60 ml of ethyl acetate
was added 60 ml of saturated K2C03. To this biphasic solution was
added benzyl chloroformate (2.03 ml, 14.23 mmol) dropwise. After
stirring at room temperature for 3 hours the layers were separated and
the organic layer dried over MgS04, filtered, and the solvents removed in
vacuo. The crude product was purified by chromatography on silica gel
(3:1 hexane:ethyl acetate) to give the title compound.
1H NMR (CDC13, 400 MHz) 7.39-7.30 (m, 5H), 5.09 (s, 1H),
4.90-4.80 (m, 1H), 4.15-4.05 (m, 2H), 3.09 (br t, 2H J=5.62 Hz), 2.66 {m, 2H),
1.70-1.62 (m, 3H), 1.45 (s, 9H), 1.20-1.00 (m, 2H).
EXAMPLE 3
Piperidin-4-vlmethyl-carbamic acid benzvl ester (4)
To a solution of 3_ (4.4 g, 13 mmol) in 90 ml of methylene
chloride was added 45 ml of TFA. After stirring at room temperature for
24 hours, the solvents were removed in vacuo and the residue 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, 400 MHz) 7.35 (s, 5H), 5.09 (s, 2H), 4.97 (br
s, 1H), 3.36 (br s, 1H), 3.20-3.00 (m, 3H), 2.60 (t, 2H, J=11.96 Hz), I.75-
1.50
(m, 3H), 1.25-1.05 (m, 3H).
EXAMPLE 4
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[1'-(2-Cyano-phenyl)-[1,4']bipiperidinyl-4-ylmethyl]-carbamic acid benzyl
ester (6)
To a solution of 2-(4-oxo-piperidin-1-yl)-benzonitrile (5_~ (653
mg, 3.26 mmol) and 4 (890 mg, 3.58 mmol) in 40 ml of methanol was
added 4 g of powdered 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 1M solution of
NaCNBH3 in THF (6.0 ml 5.6 mmol) was added slowly with a syringe
pump over 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, 400 MHz) 7.54 (dd 1H, J=7.5Hz, J=1.46Hz),
7.45 (m, 1H), 7.40-7.30 (m, 5H), 7.0-6.95 (m, 2H), 5.09 (s, 2H), 4.89 (m, 1H),
3.65 (d, 2H, J=11.9 Hz), 3.10 (t, 2H, J=6.4Hz) 2.99 (d, 2H, J=10.62Hz)2.80 (t,
2H, J=10.8Hz), 2.52 (br t, 1H, J=11.16Hz), 2.27 (t, 2H, J=10.81Hz), 1.98-1.70
(m, 6H), 1.60-1.45 (m, 1H), 1.40-1.25 (m, 2H).
EXAMPLE 5
2-(4-Aminomethvl-f 1,4'lbi~ineridinvl-1'-vl)-benzonitrile (7
A suspension of 6_ (100 mg, 0.232 mmol) and 10% Pd/C (30
mg) in 50 ml of ethyl acetate was hydrogenated at atmospheric pressure
for 6 days. The suspension was subsequently filtered through celite and
the solvent removed in vacuo to give the title compound.
1H NMR (CDC13, 400 MHz) 7.53 (dd, 1H, J=7.69Hz, J=l.4Hz)
7.45, (m, 1H), 7.05-6.90 (m, 2H), 3.66 (d, 2H, J=11.71Hz), 2.98 (d, 2H,
J=11.36 Hz), 2.80 (t, 2H, J=11.9Hz), 2.59 (d, 2H, J=5.86Hz), 2.50-2.40 (m,
1H), 2.30-2.15 (m, 2H), 2.00-1.75 (m, 6H), 1.35-1.10 (m, 4H).
EXAMPLE 6
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3-([1'-(2-Cyano-phenyl)-[1,4']bipiperidinyl-4-ylmethyl]-carbamoyl)-4-(3,4-
difluoro-phenyl)-6-methoxymethyl-2-oxo-1,2,3,4-tetrahydro-pyrimidine-5-
_carboxvlic acid methvl ester trifluoroacetic acid salt (9)
To a solution of 7 in 30 ml of chloroform was added dropwise
8 (100 mg, 0.232 mmol) 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 (CD30D, 400 MHz) 9.15 (br s, 1H), 7.70-7.55 (m,
2H), 7.30-7.10 (m, 5H), 6.63 (s, 1H), 4.66 (AB q, 2H, J=39Hz, J=15.02), 3.72
(s, 3H), 3.80-3.60 (m, 3H), 3.50-3.30 (m, 3H), 3.44 (s, 3H), 3.07 (t, 2H,
J=10.8Hz), 2.90 (t, 2H, J=11.54Hz), 2.23 (d, 2H, J=9.89 Hz), 2.10-2.80 (m,
6H), 1.60-1.42 (m, 2H). MS (FAB) 637 (M+1)
Analysis calculated for C33 H3g N6 05 F2 0.80 H20, 1.40
TFA: C, 53.03; H, 5.10; N, 10.37 Found: C 53.03 H 5.12 N 10.37.
EXAMPLE 7
Toluene 4 sulfonic acid 1 benzhvdrvl azetidin-3-vl ester (11)
To a cooled (0oC) solution of 10 (7 g, 29 mmol) in 100 ml of
chloroform was added paratoluene sulfonic anhydride (11.5 g,
35.2mmo1), and triethylamine (12 ml, 88mmo1). The resulting solution
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, 400 MHz) 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 8
3 Azido 1 benzhydrvl azetidine (~ 2)
A solution of 11 (11.5 g, 31.8 mmol) and sodium azide (4.12 g,
64 mmol) in 250 ml of DMF was heated to 70oC for 24 hours. After
cooling to room temperature the solvent was removed in vacuo and the
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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) to give the title compound.
1H NMR (CDC13, 400 MHz) 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 9
1 Benzhvdrvl azetidin 3 vlamine (13)
A solution of 12 (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 CHC13:
MeOH:NH40H).
1H NMR. (CDC13, 400 MHz) 7.41-7.15 (m, 10H), 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).
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EXAMPLE 10
1-Benzhvdrvl-azetidin-3-v~carbamic acid tert-butvl ester (14
A solution of 13 (3.5 g, 14.7 mmol) di-tert-butyl dicarbonate
(3.8 g, 17.6 mmol) and triethyl amine (4.0 ml, 29 mmol) in 100 ml of
chloroform 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 product was purified by
chromatography on silica gel (3:1 hexane:ethyl acetate) to give the title
compound.
IH NMR (CDC13, 400 MHz) 7.38-7.15 (m, lOH), 4.81-4.90 (br
s, 1H), 4.28 (s, 1H), 3.52 (t, 2H, J=6.96Hz) 2.81 (m, 2H), 1.42 (s, 9H).
EXAMPLE 11
I5
Azetidin-3-vl-carbamic acid tent-butyl ester acetic acid salt (15)
A suspension of 14 (4.6 g, 13.6 mmol) and 10% Pd/C (2.0 g) in
200 ml of 10:2:1 ethanol:water:acetic acid was hydrogenated at 50 psi for
24 hours. The suspension was subsequently filtered through celite and
the solvents removed in vacuo to give the title compound.
1H NMR (CD30D, 400 MHz) 4.55-4.45 (m, 1H), 4.20-4.10 (m,
2H), 4.10-4.00 (m, 2H), 1.91 (s, 3H), 1.44 (s, 9H).
EXAMPLE 12
(1-[1-(2-Cyano-phenyl)-piperidin-4-yl]-azetidin-3-yl?-carbamic acid tert-
butvl ester (16)
The title compound was prepared from 15 (1.0 g, 4.9 mmol)
and 3 (1.0 g, 4.3 mmol) using the procedure described for the preparation
of 6_ to give the title compound.
1H NMR (CDC13, 400 MHz) 7.54-7.51 (m, 1H,), 7.46-7.40 (m,
1H), 7.00-6.92 (m, 2H), 5.00-4.85 (br s, 1H), 4.35-4.21 (br s, 1H), 3.64 (t,
2H,
J=6.83Hz), 3.55-3.42 (m, 2H), 2.95-2.80 (m, 4H), 2.25-2.10 (m, 1H), 1.90-1.75
(m, 2H), 1.62-1.50 (m, 2H), 1.45 (s, 9H).
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EXAMPLE 13
2-f4-(3-Amino-azetidin-1-vl)-pineridin-1-vll-benzonitrile (17)
The title compound was prepared from 16 ( 1.5 g) using the
procedure described for the preparation of 4_.
1H NMR (CDC13, 400 MHz) 7.53 (dd, 1H, J=7.57Hz, 1.46Hz),
7.50-7.37 (m, 1H), 7.05-6.90 (m, 2H), 3.70-3.55 (m, 3H), 3.55-3.45 (m, 2H),
2.95-2.85 (m, 2H), 2.70-2.60 (m, 2H), 2.20-2.10 (m, 1H), 1.90-1.77 (m, 2H),
1.70-1.30 (m, 4H).
EXAMPLE 14
3-t 1-[ 1-( 2-Cyano-phenyl)-piperidin-4-yl]-azetidin-3-ylcarbamoyl~-4-(3,4-
difluoro-phenyl)-6-methoxymethyl-2-oxo-1,2,3,4-tetrahydro-pyrimidine-5-
carboxylic acid methyl ester (18)
The title compound was prepared from 17 (1.0 g, 3.9 mmol)
and 8_ (520 mg, 1.1 mmol) using the procedure described for ~.
1H NMR (CDC13, 400 MHz} 9.04 (d, 1H, J=6.6Hz), 7.72 (s,
1H), 7.55-7.50 (m, 1H), 7.40-7.50 (m, 1H), 7.21-6.90 (m, 5H), 6.65 (s, 1H),
4.68 (s, 2H), 4.55-4.50 (m, 1H), 3.75-3.65 (m, 2H), 3.71 (s, 3H), 3.55-3.42
(m,
2H), 3.50 (s, 3H}, 2.95-2.82 (m, 4H), 2.30-2.15 (m, 1H), 2.87-2.75 (m, 2H),
1.63-1.45 (m, 2H).
MS (FAB) 595 (M+1).
Analysis calculated for C30 H22 N6 ~5 F2 1.00 H20:
C, 58.81; H, 5.59, N, 13.72. Found: C, 58.58; H, 5.20; N, 13.59.
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EXAMPLE 15
4-{3,4-Difluoro-phenyl)-2-oxo-oxazolidine-3-carboxylic acid {1-[1-(2-cyano-
phenyl)-piperidin-4-yl]-azetidin-3-yl}-amide triflouroacetic acid salt 20
The title compound was prepared from 17 (100 mg, 0.4
mmol) and 19 (171 mg, 0.48 mmol) using the procedure described for _9.
1H NMR (CD30D, 400 MHz) 7.65-7.55 (m, 2H), 7.35-7.25 (m,
2H) 7.15-7.20 (m, 3H), 5.45-5.50 (m, 1H), 4.85-4.78 (m, 1H), 4.75-4.62 (br s,
1H), 4.50-4.35 (m, 4H), 4.32-4.22 (m, 1H), 3.63 (d, 2H, J=12.27Hz), 3.50-3.40
(m, 1H), 2.88 (t, 2H, J=12.27), 2.16 (d, 2H, J=10.99Hz), 1.75-1.60 (m, 2H).
MS (FAB) 482 (M+1).
Analysis calculated for C25 H25 N5 03 F2 1.05 H20, 1.35
TFA: C, 50.84; H, 4.38; N, 10.70. Found: C, 50.83; H, 4.29; N, 11.09.
EXAMPLE 16
2-(3-Hvdroxvmethvl-f 1 4'lbipineridinvl-1'-vl)-benzonitrile (22)
The title compound was prepared from 21 (1.13 g, 5.6 mmol)
and 5_ (750 mg, 3.75 mmol) using the procedure described in 16 to give the
title compound.
1H NMR (CDC13, 400 MHz) 7.37-7.35 (m, 1H), 7.50-7.41 (m,
1H), 7.20-6.93 (m, 2H), 3.77-3.55 (m, 4H), 2.98-2.90 (m, 1H), 2.90-2.67 (m,
3H), 2.57-2.23 (m, 4H), 1.98-1.50 (m, 8H), 1.27-L08 (m, 1H).
EXAMPLE 17
p-Tolyl-methanesulfonic acid 1'-(2-cyano-phenyl)-[1,4']bipiperidinyl-3-
vlmethvl ester (23)
The title compound was prepared from 22 (1.1 g, S.Ommo1)
and para-toluene sulfonic anhydride (2.48 g, 7.58 mmol) using the
procedure described in 11.
1H NMR (CDCl3, 400 MHz) 7.80 (d, 2H, J=8.24Hz), 7.55-7.53
(m, 1H), 7.45 (m, 1H), 7.40-7.32 (m, 2H), ?.02-6.95 (m, 2H), 3.95-3.85 (m,
2H), 3.63 (d, 2H, J=11.71Hz), 2.82-2.70 (m, 3H), 2.45 (s, 3H), 2.45-2.35 (m,
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1H), 2.27-2.18 (m, 1H), 2.07-1.90 (m, 2H), 1.85-1.57 (m, 7H), 1.57-1.45 (m,
1H), 1.10-0.97 (m, 1H).
EXAMPLE 18
2-(3-Azidomethvl-f 1,4'lbipiperi dinvl-1'-vl)-benzonitrile 24
The title compound was prepared from 23 (1.37 g, 3.0 mmol)
and sodium azide (0.4 g, 6.0 mmol) according to the procedure described
in 12 to give the title compound.
1H NMR (CDC13, 400 MHz) 7.55-7.52 (m, 1H), 7.50-7.42 (m,
1H), 7.02-6.94 (m, 2H), 3.66 (d, 2H, J=11.97Hz), 3.30-3.15 (m, 2H), 2.95-2.75
(m, 5H), 2.55-2.40 (m, 1H), 2.30-2.20 (m, 1H), 2.06 (t, 1H, J=10.01Hz), 1.95-
1.65 (m, 6H), 1.65-1.50 (m, 1H), 1.10-0.95 (m, 1H).
EXAMPLE 19
2-(3-Aminomethvl-f 1.4'lbinineridinvl-1'-vl)-benzonitrile (25
The title compound was prepared from 2~ (560 mg, 1.73
mmol) and triphenylphosphine (900 mg, 3.4mmo1) using the procedure
described for _13.
1H NMR (CDC13, 400 MHz) 7.55-7.53 (m, 1H), 7.47-7.43 (m,
1H), ?.00-6.95 (m, 2H), 3.66 (d, 2H, J=11.72Hz), 3.02 (d, 1H, J=10.25Hz),
2.91 (d, 1H, J=10.98Hz), 2.81 (t, 2H, J=11.9Hz), 2.62 (br s, 2H), 2.60-2.45
(m, 1H), 2.30-2.20 (m, 1H), 2.05-1.52 (m, 11H), 1.00-0.87 (m, 1H).
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EXAMPLE 20
3-([1'-(2-Cyano-phenyl)-[1,4']bipiperidinyl-3-ylmethyl]-carbamoyl}-4-(3,4-
difluoro-phenyl)-6-methoxymethyl-2-oxo-1,2,3,4-tetrahydro-pvrimidine-5-
carboxylic acid methyl ester trifluoroacetic acid salt (26)
The title compound was prepared from 25 (I75 mg, 0.58
mmol) and 8_ (140 mg, 0.29 mmol) using the procedure described for 9_.
1H NMR (CD30D, 400 MHz) 9.20-9.10 (m, 1H), 7.67-7.55 (m,
2H), 7.25-7.10 (m, 5H), 6.61-6.59 (m, 1H), 4.66 (AB q, 2H, J=39.37Hz,
J=15.2Hz), 3.72 (s, 3H), 3.70-3.20 (m, 4H), 3.00-2.73 (m, 5H), 2.25-1.70 (m,
IOH), 1.35-1.20 (m, 1H).
MS (FAB) 637 (M+1).
Analysis calculated for C33 H3g N6 05 F2 0.35 H20, 1.65
TFA: C, 52.45; H, 4.89; N, 10.11. Found: C, 52.45; H, 4.88; N, 10.13.
EXAMPLE 21
4-(3,4-Difluoro-phenyl)-2-oxo-oxazolidine-3-carboxylic acid [1'-(2-cyano-
phenvl)-f1,4'lbipiperidinvl-3-vlmethvll-amide trifluoroacetic acid salt (27)
The title compound was prepared from 25 (175 g, 0.58 mmol)
and 19 (I05 mg, 0.28 mmol) using the procedure described for 9_.
1H NMR (DMSO-d6, 400 MHz) 8.10-8.00 (m, IH), 7.78-7.70
(m, 1H), 7.65-7.57 (m, 1H), 7.50-7.37 (m, 2H), 7.21-7.08 (m, 3H), 5.47-5.39
(m, 1H), 4.76 (t, 1H, J=8.79Hz), 4.23-4.13 (m, 1H), 3.70-2.60 (m, 12H), 2.25-
1.57 (m, 8H), 1.23-1.03 (m, 1H).
MS (FA$) 524 (M+1).
Analysis calculated for C2g H31 N5 03 F2 1.30 TFA:
C, 54.70; H, 4.85; N, 10.43. Found: C, 54.72; H, 4.77; N, 10.11.
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EXAMPLE 22
4-(3,4-Difluoro-phenyl}-2-oxo-oxazolidine-3-carboxylic acid [1'-(2-cyano-
phenvl)-f 14'lbipiveridinyl-4-vlmethvll-amide triflouroacetic acid salt (28)
The title compound was prepared from 7 (140 mg, 0.47
mmol) and 19 (170 mg, 0.47 mmol) using the procedure described for 9_.
1H NMR (CD30D, 400 MHz) 7.65-7.52 (m, 2H), 7.35-7.25 (m,
2H), 7.20-7.15 (m, 3H), 5.27-5.20 (m, 1H), 4.77 (t, 1H, J=8.97Hz), 4.27-4.20
(m, 1H), 3.74-3.58 (m, 4H), 3.40-3.30 (m, 1H), 3.25-3.15 (m, 2H), 3.10-3.00
(m, 2H), 2.95-2.82 (t, 2H, J=11.71Hz), 2.24 (d, 2H, J=11.54), 2.10-1.80 (m,
5H), 1.58-1.42 (m, 2H).
MS (FAB) 524 (M+1).
Analysis calculated for C2g H31 N5 03 F2 0.30 H20, 1.85
TFA: C, 51.45; H, 4.56; N, 9.47. Found: C, 51.47; H, 4.58; N, 9.36.
EXAMPLE 23
H2N~'~N
~/
3-Aminometh~ N-diphenvlmethvl azetidine (34)
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 33 (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 vacuo. The crude product was not purified.
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1H NMR (CDCl3, 300 MHz) 7.41-7.13 (m, 10 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).
EXAMPLE 24
BOCHN\'~N
3-N-( 1,1-dimethylethoxycarbonyl)aminomethyl N diphenylmethyl
a zetidine. (35
The title compound was prepared from 34 using the
procedure described for the preparation of 14.
1H NMR (CDC13, 300 MHz) 7.40-7.14 (m, 10 H), 4.84 (s, 1 H),
4.31 (s, 1 H), 3.50-3.21 (m, 4 H), 2.85 (t, 2 H), 2.59-2.48 (m, 1 H), 1.44 (s,
9
H).
EXAMPLE 25
BOCHN\~~NH
3-N-f(1,1-dimethvlethoxvcarbonvl)methvllaminomethvl azetidine (36)
The title compound was prepared from 35 using the
procedure described for the preparation of 1~.
1H NMR (CD30D, 300 MHz) 4.92-4.85 (m, 4 H), 4.05 (br m, 2
H), 3.88-3.86 (br m, 2 H), 1.94-1.90 (m, 1 H), 1.46 (s, 9 H)
EXAMPLE 26
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CN
N
N~~NHBOC
2-(4-(3-N-( 1,1-Dimethylethoxycarbonyl)aminomethyl-azetidin-1-yl)-
pineridin-1-yll-benzonitrile, (37)
The title compound was prepared from 36 using the
procedure described for the preparation of 16.
1H NMR (CDC13, 300 MHz) 7.57-7.40 (m, 2 H), 7.05-6.92 (m, 2
H), 4.89 (s, 1 H), 3.53-3.46 (m, 3 H), 3.39-3.27 (m, 4 H), 2.95-2.85 (m, 4 H),
2.64-2.58 (m, 1 H), 2.25-2.19 (m, 1 H), 1.85-1.79 (m, 2 H), 1.60-1.44 (m, 10
H).
EXAMPLE 27
CN
N
N~~ N H2
2-f4-(3-Aminomethvl-azetidin-1-yll-piperidin-1-yll-benzonitrile, (38)
The title compound was prepared from ~ using the
procedure desrcibed for the preparation of ~.
1H NMR (CDC13, 300 MHz) 7.54-7.40 (m, 2 H), 7.00-6.91 (m, 2
H), 3.55-3.46 (m, 2 H), 3.44-3.38 (m, 2 H), 2.93-2.83 (m, 6 H), 2.58-2.46 (m,
1
H), 2.23-2.14 (m, 1 H), 1.85-1.80 (m, 2 H), 1.60-1.48 (m, 2 H), 1.01-0.95 (br
s,
2 H).
EXAMPLE 28
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WO 98/57640 PCT/US98/12672
CN F
O O
I~ F
N
N 'N N NH
O O
Compound (39)
The title compound 39 was prepared from 38 using the
procedure described for the preparation of 18.
1H NMR (CDC13, 400 MHz) 8.98-8.95 (t, 1 H), 7.72 (s, 1 H),
7.54-7.52 (dd, 1 H), 7.46-7.42 (m, 1 H), 7.09-7.06 (m, 2 H), 6.99-6.93 (m, 2
H),
6.66 (s, 1 H), 4.68 (s, 2 H), 3.71 (s, 3 H), 3.55-3.39 (m, 10 H), 3.02-2.98
(t, 2
H), 2.88-2.73 (m, 3 H), 2.28-2.26 (m, 1 H), 1.83-1.80 (m, 2 H), 1.61-.1.56(m,
2
H).
MS (FAB) 609 (M+1)
EXAMPLE 29
F F
CN _
N
N'/~ N N O
O O
Compound (40)
The title compound 40 was prepared from 38 using the
procedure described for the preparation of 20.
1H NMR (CDC13, 400 MHz) 8.01-7.98 (s, I H), 7.53-7.51 (d, 1
H), 7.45-7.41 (t, 1 H), 7.22-7.12 (m, 1 H), 7.07-7.00 (m, 1 H), 6.98-6.92 (m,
2
H), 5.41-5.40 {m, 1 H), 4.74-4.70 (t, 1 H), 4.27-4.23 (m, 1 H), 3.52-3.44 (m,
3
H), 3.40-3.31 (m, 3 H), 2.91-2.86 (m, 4 H), 2.67-2.64 (m, 1 H), 2.21 (br s, 1
H), 1.81-1.78 (m, 2 H), 1.58-1.53(m, 2 H).
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Anal. Calcd for C2gH27N5~3F2 ' 0.25 CHC13 + 0.20 H20 : C =
61.94, H = 5.48, N = 13.87. Found: C = 61.97, H = 5.55, N = 13.50.
MS (FAB) 496 (M+1)
Utilizing the methodology described in detail herein, the
following additional compounds shown in Tables 1 and 2 were made. In
Tables 1 and 2, (+)DHP is
F
F
O ~ O
.~~ N Oi
~ O
O' _ N
H
-77-
I
CA 02294346 1999-12-13
WO 98/57640 PCT/US98/12672
~6~~ ~b~~ ~b~
O d: ~ O O ~n
~ ~
~
M
_ _
U ~.. O ~~- a ~~
.- w ~
11
o It
~z u~ ~z ~~;z
"
~;~~
I~
_ C ~ ~ ~
O V'7 ~ Ov
M V~'1
o ~IW V ~I- 0 Ifyn
I ~ ~
~n
i Z 3~~ U 3x
Q '~
3xz
Zw ~_'~b~ O _'~b~
_
O oo V O O vp
~ ~1
~
~ ~ ~ O _
C w. w.
a~ ~ ,o ~ ~O ~ ,~
II ~' II ~
V ~ Il
U
Z ~ U e~
U
y Q U_7U~ U _~_U'~U7_U
of p ca p n7
Cd .fl a1 p
.a of
Z Uv~UO U v~UO UcnU
H
z c c a c
U _ ~E ~~ _
V E E
LL N vD Ov N
x 00 h 00 00
_N
O
O O ~ O
OD Op ~p CO
M .-. O~ ~f
N N M
h tn M M
a
o0 00 N N
L=r M N ~O 10
a m
v ~r
V Vr
A A
a ~ x
_7s_
CA 02294346 1999-12-13
WO 98157640 PCTNS98/12672
a
0
0 0
0
,_; N E .",
...
~z~
0
0
w o ~ r,
o
h ~; E
y v~
~n
O ~ a U U
tip
ea .O
p U rUO
W
~--Z a
d
~E
N
~D
00
d
O
bD
~T
N
M
O
- 79 -
i
CA 02294346 1999-12-13
WO 98/57640 PCTNS98/12672
O
T ~
~ ~
~ N ~ _ Q
~ N
~ ~
' O~
~ ti ~ p~ O w
O ...
.-.
o ~zz o_ ~z ' ~ gz
N ~ ~ v1 "
M W O ~O
O ~p ~ O
O ~ ~
p N ,n
~ ~'~ ~'~j'r'
~c~
Q U _ o 3== U
z 3zz ._ Z
__ _ ,V . ~ ~ . ~ ,U
Z bs b~
N _ O _ O O
O '-! O et N
N ~ ~
'~ O ~ cn rn ~ M O ~
r"~ M ~ ~
rt
C ,
w
Z ~
Z ~ ee U cc V ca
V U V
U U
y U ~ U U ~ U U 7
uj p vj U
N
Gz7 U cnUO U v~UO U rnUO
o
c
Z
_ ~
Z V ~ E
V
GL N O~O '~T
N ~ O~
U
_
U O O
O
~r ~ W GO
r.~ W
r. O~
Gi M N ~O
a
.r
o~
0
.,
a ~ z
-80-
CA 02294346 1999-12-13
WO 98!57640 PCT/US98/12672
v
a
0
u~~
'o °'
o Eoo_
voz"
x ~.~z
U ~~~
0 o vo 00
00 ~n ,~.; a;
o ~l ~'ii'
3~~
0 0~0.
~-' ~ 00 00
'" ~ V U
_7 _U
at O ~
U ~nUO
W
a
Q, .E
E~ ~''
et
0
E
00
0
0
h
N
d'
-
CA 02294346 1999-12-13
WO 98/57640 PCT/US98/12672
EXAMPLE 30
(4S, 5R)-4-(3,4-Difluorophenyl-3-(1-(1-(4-fluoro-2-(3-methyl-
[1,2,4]oxadiazol-5-yl)phenyl]piperidin-4-yl}-(3R)-pyrrolidin-3-
ylcarbamoyl)-2-oxo-oxazolidine-5-carboxylic acid methyl ester
F
F
F ~ N~N~ ~ ~~,C02M a
-N H ~O
O ~ /,O
Ma
Step A: trans-3,4-Difluorocinnamic acid methyl ester
To a solution of trans-3,4-difluorocinnamic acid (10 g, 54 mmol) in
300 mL methanol was added concentrated sulfuric acid (2 mL). The
solution was stirred 48 h at ambient temperature and then concentrated
in vacuo. The residue was taken up in ethyl acetate (500 mL) and
washed with saturated sodium bicarbonate (2 x 100 mL), brine (1 x I00
mL), dried with magnesium sulfate, and concentrated in vacuo to
provide trans-3,4-difluorocinnamic acid methyl ester (10.7 g, 54 mmol,
100%) as a white solid.
'H NMR dH (400 MHz, CDC13) 7.59 (d, 1H, J = 15.9), 7.34 (m, 1H),
7.24 (m, 1H) 7.18 (dd, 1H, J = 9.9, 2.0), 6.35 (d, 1H, J = 16.1), 3.81 (s,
3H).
Step B: (2R, 3S)-N-Benzyloxycarbonyl-3-amino-3-(3,4-
difluorophenyl)-2-hydroxypropionic acid methyl ester
A solution of NaOH (4.1 g, 103 mmol) was prepared in 175 mL
water. Potassium osmate dehydrate (491 mg, 1.3 mmol) was dissolved in
mL of this NaOH solution, resulting in a dark pink homogeneous
mixture. To a 1000 mL round bottom flask is added the remaining
NaOH solution prepared above, 135 mL n-propanol and benzyl cabamate
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(9.8 g, 110 mmol). The suspension was stirred at ambient temperature
for 30 min wherein the mixture was nearly homogeneous. The reaction
flask was placed in a room temperature water bath and the surrounding
lights were turned off. Freshly prepared t-butylhypochlorite (11.2 mL,
103 mmol) was added dropwise with vigorous stirring, and the reaction
stirred an additional 15 min. In a separate 250 mL round bottom flask
was suspended traps-3,4-difluorocinnamic acid methyl ester (6.6 g, 33.3
mmol) and (DHfI)2PHAL (1.3 g, 1.7 mmol) in 100 mL n-propanol. The
suspension was added to the above reaction mixture and the residue
rinsed into the reaction flask (2 x 10 mL). To the reaction was added the
above prepared solution of potassium osmate dihydrate. The resulting
green solution became amber/brown over 1 h. Sodium metabisulfite (66
g, 347 mmol) was added and the resulting suspension stirred 3 h when it
was poured into a separatory funnel containing ethyl acetate (200 mL)
and the layers separated. The aqueous layer was extracted witFl ethyl
acetate ( 150 mL) and the combined organics washed with brine ( 100 mL),
dried with magnesium sulfate, and concentrated in vacuo to provide a
pale yellow solid. The crude material was passed through silica (25%
ethyl acetate/hexane) to give (2R, 3S)-N-benzyloxycarbonyl-3-amino-3-
(3,4-difluorophenyl)-2-hydroxypropionic acid methyl ester contaminated
with benzyl carbamate.
Step C: (2R, 3S)-3-Amino-3-(3,4-difluorophenyl)-2-hydroxypropionic
acid methyl ester
(2R, 3S)-N-Benzyloxycarbonyl-3-amino-3-(3,4-difluorophenyl)-2-
hydroxypropionic acid methyl ester (>12.2 g, 33.3 mmol maximum) was
dissolved in 750 mL ethanol. The flask was purged and filled with argon
three times. Palladium on carbon (2 g, 10% wt) was added under argon
and the suspension was again purged and filled with argon three times.
The suspension was then purged, filled with hydrogen, and stirred 16 h.
The suspension was purged, filled with argon three times, filtered
through celite and concentrated in vacuo to give (2R, 3S)-3-amino-3-(3,4-
difluorophenyl)-2-hydroxypropionic acid methyl ester (5.8 g, 25 mmol,
75% from traps-3,4-difluorocinnamic acid methyl ester).
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'H NMR dH (400 MHz, CDC13) 7.26 (m, 1H), 7.15-7.08 (m, 2H), 4.28
(s, 2H), 3.82 (s, 3H), 2.48 (bs, 2H).
Step D: (4S, 5R)-4-(3,4-Difluorophenyl)-2-oxo-oxazolidine-5-
carboxylic acid methyl ester
To a solution of (2R, 3S)-3-amino-3-(3,4-difluorophenyl)-2-
hydroxypropionic acid methyl ester (5.8 g, 25 mmol) in 250 mL
tetrahydrofuran at 0°C was added N,N-diisopropylethylamine (8.75 mL,
50 mmol) and triphosgene (2.48 g, 8.4 mmol). The reaction was stirred
at 0°C for 30 min when it was poured over ethyl acetate (200 mL) and
saturated sodium carbonate solution ( 100 mL). The layers were
separated, the organic layer washed with saturated sodium carbonate
solution (1 x 100 mL), dried with magnesium sulfate, and concentrated
in vacuo to provide a pale yellow oil. The material was triturated with
25% ethyl acetate/hexane from dichloromethane to provide (4S, 5R)-4-
(3,4-difluorophenyl)-2-oxo-oxazolidine-5-carboxylic acid methyl ester.
The recovered mother liqour was passed through silica (50% ethyl
acetate/hexane) to give an additional 1.1 g (4.8 g total, 18 mmol, 75%)
'H NMR dH (400 MHz, CDCl3) 7.25-7.20 (m, 2H), 7.15 (m, 1H), 6.33
(bs, 1H), 4.98 (d, 1H, J = 5.1), 4.72 (d, 1H, J = 5.3), 3.89 (s, 3H).
FABMS M+H = 258
Step E: (4S, 5R)-4-(3,4-Difluorophenyl)-2-oxo-oxazolidine-3,5-
dicarboxylic acid methyl ester 3-(4-nitrophenyl) ester
To a solution of (4S, 5R)-4-(3,4-difluorophenyl)-2-oxo-oxazolidine-5-
carboxylic acid methyl ester (910 mg, 3.5 mmol) in anhydrous
tetrahydrofuran (50 mL), cooled to -78°C under argon, was added a 1 M
solution of lithium bis(trimethylsilyl)amide in tetrahydrofuran (3.5 mL,
3.5 mmol) dropwise. The reaction mixture was warmed to 0°C in an ice
bath, stirred 30 minutes, then returned to -78°C. In a separate flask,
p-
nitrophenylchloroformate (714 mg, 3.54 mmol) was dissolved in
anhydrous tetrahydrofuran (40 mL) under argon and cooled to -78°C.
The above prepared anion solution was added via cannula to the
_ g,4 _
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chloroformate solution and the reaction mixture was stirred 1 h at -
78°C. The reaction mixture was treated with ethyl acetate ( 150 mL) and
the resulting solution was washed with water (1 x 150 ml), brine (1 x 150
ml) and dried over magnesium sulfate and filtered. The volitiles were
removed under reduced pressure and the resulting oil was triturated
with diethyl ether. Ether was twice decanted from the resulting pale
yellow solid to give (4S, 5R)-4-(3,4-difluorophenyl)-2-oxo-oxazolidine-3,5-
dicarboxylic acid methyl ester 3-(4-nitrophenyl) ester (1.3 g, 3.1 mmol,
87%).
FAB MS: m/z=423 (M+H)
Step F: (4S, 5R)-4-(3,4-Difluorophenyl-3-(1-{1-[4-fluoro-2-(3-
methyl[1,2,4]oxadiazol-5-yl)phenyl]-piperidin-4-yl}-(3R)-
pyrrolidin-3-ylcarbamoyl)-2-oxo-oxazolidine-5-carboxylic
acid methyl ester
To a solution of 1-{1-[4-fluoro-2-(3-methyl[1,2,4]oxadiazol-5-
yl)phenyl]piperidin-4-yl}-(3R)-pyrrolidin-3-ylamine (105 mg, 0.23 mmol)
in dry, degassed N,N-dimethylformamide (2 mL) was added N,N-
diisopropylethylamine (120 ~.L, 0.69 mmol) followed by (4S, 5R)-4-(3,4-
difluorophenyl)-2-oxo-oxazolidine-3,5-dicarboxylic acid methyl ester 3-(4-
nitrophenyl) ester (98 mg, 0.23 mmol). The reaction mixture was stirred
at ambient temperature for 1 h when the volatiles were removed under
reduced pressure. The residue was dissolved in ethyl acetate (100 mL)
and washed with 10% aqueous sodium carbonate solution (8 x 100 mL),
brine ( 1 x 100 mL), dried over magnesium sulfate and filtered. The
volatiles were removed under reduced pressure and the resulting oil
was purified by pressurized silica gel chromatography (2% methanol in
ethyl acetate) to give a foam. The hydrochloride salt was prepared
according to standard procedures to afford (4S, 5R)trans-4-(3,4-
difluorophenyl-3-( 1-{1-[4-fluoro-2-(3-methyl-[1,2,4]oxadiazol-5-yl)phenyl]-
piperidin-4-yl}-(3R)-pyrrolidin-3-ylcarbamoyl)-2-oxo-oxazolidine-5-
carboxylic acid methyl ester as a solid.
HPLC retention time = 8.59 min, purity= 94%
FAB MS: m/z=629 (M+H)
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Analysis: Calcd for C30H31N6O6F3 ~ 2HC1 ~ 0.2EtOAc
C51.44,H4.85,N11.69.
Found: C 51.83, H 5.11, N 11.68.
The compounds in Examples 31 and 32 were prepared by procedures
substantially as described above for Example 30, Step F.
EXAMPLE 31
IO (4S, 5R)-4-(3,4-Difluorophenyl-3-{1-[1-(4-fluoro-2-
methoxyphenyl)piperidin-4-yl]-(3R)-pyrrolidin-3-ylcarbamoyl)-2-oxo-
oxazolidine-5-carboxylic acid methyl ester
F
F '
F ~ N~N~ ~ ~,C02M a
OMe H ~O
llO
FAB MS: m/z=577 (M+H)
Analysis: Calcd for C28H31N4O6F3~2F3CC02H
C 45.81, H 4.42, N 6.68.
Found: C 45.85, H 4.54, N 6.62.
EXAMPLE 32
(4S, 5R)3-{1-[1-(2-Cyanophenyl)piperidin-4-yl]-(3R)-pyrrolidin-3-
ylcarbamoyl}-4-(3,4-difluorophenyl)-2-oxo-oxazolidine-5-carboxylic acid
methyl ester
F
F '
/ N~N~ O ~~,C02M a
CN ~N -_ O
H
O
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FAB MS: m/z=554 (M+H)
Analysis: Calcd for C28H29N505F2 ~ HCl ~ 0.25H20 ~ 0.75dioxane
C 56.36, H 5.57, N 10.60.
Found: C 56.33, H 5.39, N 10.61.
EXAMPLE 33
(4S, 5R)-4-(3,4-Difluorophenyl)-5-hydroxymethyl-2-oxo-oxazolidine-3-
carboxylic acid {1-[1-(2-cyanophenyl)piperidin-4-yl]-(3R)-pyrrolidin-3-
yl}amide
F
F
~ N~N~ H
CN N
H
Step A: (4S, 5R) 4-(3,4-Difluorophenyl)-5-hydroxymethyl-oxazolidin-
2-one
To a solution of (4S, 5R)-4-(3,4-difluorophenyl)-2-oxo-
oxazolidine-5-carboxylic acid methyl ester [(200 mg, 0.8 mmol) product of
Example 30 Step D] in tetrahydrofuran (10 mL) at 0°C was added a 2
M
solution of lithium borohydride in tetrahydrofuran (0.4 mL, 0.8 mmol).
After stirring for 20 min at 0°C, saturated sodium bicarbonate (20
mL)
was added and the mixture stirred at ambient temperature for 20 min.
Ethyl acetate (50 mL) was added and the layers separated. The organic
layer was washed with brine (1 x 10 mL), dried with magnesium sulfate,
filtered and concentrated in vacuo to provide (4S, 5R) 4-(3,4-
difluorophenyl)-5-hydroxymethyl-oxazolidin-2-one as a white solid (180
mg, 0.8 mmol, 100%)
'H NMR dH (400 MHz, CDC13) 7.23-7.15 (m, 2H), 7.10-7.07 (m, 1H),
6.48 (bs, 1H), 4.89 (d, 1H, J = 6.8), 4.31 (dt, 1H, J = 6.6, 2.9), 3.96 (dd,
1H, J
= 12.82, 2.75), 3.70 (bdd, 1H, J = 12.1, 2.2), 3.53 (bs, 1H).
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Step B: (4S, 5R) 4-(3,4-Difluorophenyl)-5-(tetrahydropyran-2-
yloxymethyl)-oxazolidin-2-one
To a solution of (4S, 5R) 4-(3,4-difluorophenyl)-5-hydroxymethyl-
oxazolidin-2-one (695 mg, 3.0 mmol) in dry dichloromethane (30 mL)
was added 2,3-dihydropyran (0.3 mL, 3.6 mmol) and camphorsulfonic
acid (70 mg, 0.3 mmol). The reaction mixture was stirred at ambient
temperature for 3 h. The reaction mixture was diluted with
dichloromethane (100 mL), washed with saturated sodium bicarbonate
solution (2 x 100 ml), brine (1 x 100 ml), dried over magnesium sulfate
and filtered. The volatiles were removed under reduced pressure and
the resulting solid was purified by pressurized silica gel
chromatography ( 1:1 then 2:1 ethyl acetate:hexane) to afford (4S, 5R)-4-
(3,4-difluorophenyl)-5-(tetrahydropyran-2-yloxymethyl)-oxazolidin-2-one
as a colorless oil (750 mg, 2.4 mmol, 80°l0).
FAB MS: m/z=314 (M+H)
Step C: (4S, 5R)-4-(3,4-Difluorophenyl)-2-oxo-5-(tetrahydropyran-2-
yloxymethyl)-oxazolidine-3-carboxylic acid 4-nitrophenyl
ester
To a solution of (4S, 5R)-4-(3,4-difluorophenyl)-5-(tetrahydropyran-
2-yloxymethyl)-oxazolidin-2-one (910 mg, 2.9 mmol) in anhydrous
tetrahydrofuran (60 mL) cooled to -78°C under argon, was added a 1 M
solution of lithium bis(trimethylsilyl)amide in tetrahydrofuran (2.9 mL,
2.9 mmol) dropwise. The reaction mixture was warmed to 0°C in an ice
bath, stirred 45 min, and then returned to -78°C. Meanwhile, in a
separate dried flask, the p-nitrophenylchloroformate (586 mg, 2.9 mmol)
was dissolved in anhydrous tetrahydrofuran (50 mL) under argon and
cooled to -78°C. The above prepared anion solution was added via
cannula to the chloroformate solution and reaction mixture was stirred
1 h at -78°C. The reaction mixture was treated with ethyl acetate (150
mL). The resulting solution was washed with water (1 x 150 ml), brine
(1 x 150 mL), dried over magnesium sulfate and filtered. The volatiles
were removed under reduced pressure to give (4S, 5R)-4-(3,4-
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difluorophenyl)-2-oxo-5-(tetrahydropyran-2-yloxymethyl)-oxazolidine-3-
carboxylic acid 4-nitrophenyl ester as a yellow foam (1.3g, 2.8 mmol,
96%).
FAB MS: m/z=479 (M+H)
Step D: (4S, 5R)-4-(3,4-Difluorophenyl)-2-oxo-5-(tetrahydropyran-2-
yloxymethyl)oxazolidine-3-carboxylic acid {1-[1-(2-
cyanophenyl)piperidin-4-yl]-(3R)-pyrrolidin-3-yl}amide
To a solution of 2-[4-(3-amino-(3R)-pyrrolidin-1-yl)piperidin-1-
yl]benzonitrile (166 mg, 0.44 mmol) in dry, degassed N,N-
dimethylformamide (4 mL) was added N,N-diisopropylethylamine (230
~L, 1.3 mmol) followed by (4S, 5R)-4-(3,4-difluorophenyl)-2-oxo-5-
(tetrahydropyran-2-yloxymethyl)-oxazolidine-3-carboxylic acid 4-
nitrophenyl ester (210 mg, 0.44 mmol). The reaction mixture was stirred
at room temperature for 4 h when the volatiles were removed under
reduced pressure, the residue dissolved in ethyl acetate (100 mL),
washed with 10% aqueous sodium carbonate solution (8 x 100 mL), brine
(1 x 100 mL), dried over magnesium sulfate and filtered. The volatiles
were removed under reduced pressure and the resulting oil was purified
by pressurized silica gel chromatography (1:1 then 2:1 ethyl
acetate:hexane) to afford (4S, 5R)-4-(3,4-difluorophenyl)-2-oxo-5-
(tetrahydropyran-2-yloxymethyl)oxazolidine-3-carboxylic acid {1-[1-(2-
cyanophenyl)piperidin-4-yl]-(3R)-pyrrolidin-3-yl}amide as a white foam
(156 mg, 0.26 mmol, 59%).
FAB MS: m/z=610 (M+H)
Step E: (4S, 5R)-4-(3,4-Difluorophenyl)-5-hydroxymethyl-2-oxo-
oxazolidine-3-carboxylic acid {1-[1-(2-cyanophenyl)piperidin-
4-yl]-(3R)-pyrrolidin-3-yl}amide
To a solution of (4S, 5R)-4-(3,4-difluorophenyl)-2-oxo-5-
(tetrahydropyran-2-yloxymethyl)oxazolidine-3-carboxylic acid {1-[1-(2-
cyanophenyl)piperidin-4-yl]-(3R)-pyrrolidin-3-yl}amide (156 mg, 0.26
mmol) in methanol (5 mL) was added p-toluenesulfonic acid (50 mg, 0.26
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mmol). The reaction mixture was stirred at room temperature for 17 h.
The volatiles were removed under reduced pressure, the residue taken
up in ethyl acetate (100 mL), washed with saturated sodium carbonate
solution (3 x 100 mL), brine (1 x 100 ml), dried over magnesium sulfate
and filtered. The volatiles were removed under reduced pressure to
afford (4S, 5R)-4-(3,4-difluorophenyl)-5-hydroxymethyl-2-oxo-oxazolidine-
3-carboxylic acid (1-[1-(2-cyanophenyl)piperidin-4-yl]-(3R)-pyrrolidin-3-
yl}amide. The hydrochloride salt was prepared according to standard
procedures to provide a white solid (123 mg, 0.22 mmol, 84%)
HPLC: retention time= 7.20 min, purity=95%
FAB MS: m/z=526 (M+H)
'H NMR HCl salt dH (400 MHz, CD30D) 7.97 (d, J= 7.33 Hz, 1H),
7.53 (d, J= 7.69 Hz, 1 H), 7.45 (t, J= 8.61, 1H), 7.16 (m, 2H), 7.04 (m, 1H),
6.96 (m, 2H), 5.26 (d, J=4.58 Hz, 1H), 4.39 (d, J=4.21, 1H), 4.25 (m, 1H),
3.95 (dd, J=8.61, 3.11, 1H), 3.82 (dd, J=9.16, 3.48, 1H), 3.57 (m, 2H), 2.88
(m. 3H), 2.77 (m, 1H), 2.68 (m, 1H), 2.47 (m, 1H), 2.22 (m, 3H), 1.95 (m,
2H), 1.78 (bm, 2H), L68 (bm, 1H)
Analysis: Calcd for C27 H29 N5 04 F2~0.35 HZO
C 60.97, H 5.63, N 13.17.
Found: C 61.02, H 5.59, N 13.12.
The compounds of Examples 34 and 35 were prepared by
procedures described in Example 30 Steps A-F, followed by pressurized
silica gel chromatography using an elution system containing
chloroform saturated with ammonia gas and methanol.
F F
F F
~N~N~N~ ~~OZMe ----~ N N~ ~,GONH2
~CN ~/ ~H CN H
O O
EXAMPLE 34
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(4S, 5R)3-{1-[1-(2-Cyanophenyl)piperidin-4-yl]-(3R)-pyrrolidin-3-
ylcarbamoyl}-4-(3,4-difluorophenyl)-2-oxo-oxazolidine-5-carboxamide
F
F '
N~~ O ~~,CONH2
N
C N H ~O
'/O
FAB MS: m/z=539 (M+H)
Analysis: Calcd for C27H28N604F2~HCl~0.9H20
C 54.85, H 5.25, N 14.22.
Found: C 54.83, H 5.02, N 14.05.
EXAMPLE 35
(4S, 5R)-4-(3,4-Difluorophenyl-3-{ 1-[1-(4-fluoro-2-
methoxyphenyl)piperidin-4-yl]-(3R)-pyrrolidin-3-ylcarbamoyl}-2-oxo-
oxazolidine-5-carboxamide
F
F '
F \ N~N~ O ~~,CONH2
N~ O
OMe H
O
FAB MS: m/z=562 (M+H)
Analysis: Calcd for C27H30N505F3~0.85H20
C56.21,H5.54,N12.14.
Found: C 56.20, H 5.20, N 12.05.
EXAMPLE 36
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
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F
F
~O
O N
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 Hz0 then neutralized with 10%
aqueous HCl solution. The organic layer was dried over Na2S04,
concentrated, and purified by PCTLC (7% MeOH in CHCl3 with 2%
NH40H) to afford a 2.65 g mixture of the title compounds (71% yield).
The 1H NMR was consistent with the assigned structure.
MS (FAB) 255 (M+1)
EXAMPLE 37
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
Ht
N~V~ O
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
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resulting mixture was refluxed at 90 °C for 16 hours. After cooling to
room temperature the solvent was removed in Uacuo. The solid was
dissolved in CH2C12 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 38
4S-4-(3,4-Difluorophenyl)-6-methoxymethyl-3-(4-nitrophenoxycarbonyl)-2-
oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid methyl ester
O
H
The title compound was prepared by treating the mixture
obtained from Example 36 or Example 37 (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 1H NMR was consistent with the
assigned structure.
EXAMPLE 39
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
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F F
i \ F I \ F
/ /
~O ~ ~O
O N ~ 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 36. A mixture of 2.0 g of the title compounds was
obtained in 50% yield. The 'H NMR was consistent with the assigned
structure.
MS (FAB) 255 (M+1)
Compounds of the invention can be prepared by reacting the
products obtained in Example 38 in accordance with procedures and
schemes described above. The compound of Example 38 can, for
example, be reacted with an aminopiperidine or aminoalkylpiperidine
as set forth in Schemes 1 and 2 to obtain the desired compounds.
Compounds of the invention can also be prepared by preparing a
nitrophenoxy derivative of the compound of Example 39 in accordance
with the procedure set forth in Example 38 and then reacting the
derivative with an aminopiperidine or aminoalkylpiperidine as set forth
in Schemes 1 and 2 to obtain compounds of the invention.
The following compounds were prepared in accordance with
procedures set forth in the foregoing Schemes and Examples.
EXAMPLE 40
N-( 1-{1-(2-Cyanophenyl)piperidin-4-yl)azetidin-3-yl)-2-(3,4-
difluorophenyl)acetamide
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N
CN N ~ / F
N F
H
1H NMR (CDC13, 400 MHz) consistent with assigned
structure.
FABLRMS m/e 411 g/mole (M++H, C23H24F2N401 = 410.47
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% pure.
Anal. Calcd for C23H24F5N503 ' 1.20 TFA: C = 55.74, H =
4.64, N = 10.24. Found: C = 55.75, H = 4.65, N = 10.34.
EXAMPLE 41
(2~-N-( 1-( 1-(2-Cyanophenyl)piperidin-4-yl)azetidin-3-yl)-2-(3,4-
difluorophenyl)-2-(2-propyl)acetamide
/)
N
CN N 0 / F
N ~ ~F
H
1H NMR (CDC13, 400 MHz) consistent with assigned
structure.
FABLRMS m/e 453 g/mole (M++H, C26H30F2N401 = 452.23
g/mole. )
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HPLC (Vydac; C 18; 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% pure.
Anal. Calcd for C26H3pF2N4O1 ~ 1.15 TFA ~ 0.15 H20: C =
57.96,H=5.41,N=9.56. Found:C=57.98,H=5.19,N=9.73.
EXAMPLE 42
(4S)-4-(3,4-Difluorophenyl)-2-oxo-oxazolidine-3-carboxylic acid {1-[1-(2-
cyanophenyl)-piperidin-4-yl]azetidin-3-yl}-amide
N
CN N O
H N
,,
F
F
1H NMR (CDC13, 400 MHz) consistent with assigned
structure.
FABLRMS m/e 482 g/mole (M++H, C25H25F2N503 = 481.51
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; >99% pure.
Anal. Calcd for C25H25F2N503 1.35 TFA and 1.05 H20: C =
50.84, H = 4.38, N = 10.70. Found: C = 50.83, H = 4.29, N = 11.09.
EXAMPLE 43
(4S)-3-{ 1-[ 1-(2-Cyanophenyl)piperidin-4-yl]azetidin-3-ylcarbamoyl}-4-(3,4-
difluorophenyl)-6-methoxymethyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5-
carboxylic acid methyl ester
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N
CN N O
N N NH
H
/
F
C02Me
F
structure.
g/mole. )
1H NMR (CDCl3, 400 MHz) consistent with assigned
FABLRMS m/e 595 g/mole (M++H, C3pH32F2N6O5 = 594.62
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; >97% pure.
Anal. Calcd for C3pH32F2N6O5 ' 1.55 TFA and 1.30 H20: C
= 50.02, H = 4.58, N = 10.58. Found: C = 49.99, H = 4.55, N = 10.59.
EXAMPLE 44
N-( 1-( 1-(2-Cyanophenyl)piperidin-4-yl)azetidin-3-ylmethyl)-2-(3,4-
difluorophenyl)acetamide
N
CN N H
~N ~ F
O v 'F
structure .
1H NMR (CDC13, 400 MHz) consistent with assigned
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FABLRMS m/e 425 g/mole (M++H, C24H26F2N401 = 424.49
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; 99% pure.
Anal. Calcd for C24H26F2N401 ~ 0.05 CH2C12: C = 67.37, H
= 6.14, N = 13.07. Found: C = 67.53, H = 6.15, N = 13.40.
EXAMPLE 45
N-(1-(1-(2-Cyanophenyl)piperidin-4-yl)azetidin-3-ylmethyl)-2,2-
spirocyclopropyl-2-(3,4-difluorophenyl)acetamide
N
CN
N
~N ~ F
O . v 'F
1H NMR (CDC13, 400 MHz) consistent with assigned
structure .
FABLRMS m/e 451 g/mole (M++H, C26H28F2N401 = 450.53
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; 98% pure.
Anal. Calcd for C26H28F2N401 ~ 1.9 HCl and 0.15 Hexane:
C = 60.64, H = 6.05, N = 10.52. Found: C = 60.63, H = 6.45, N = 10.78.
EXAMPLE 46
(4S)-3-{1-[1-(2-Cyanophenyl)piperidin-4-yl]azetidin-3-ylmethylcarbamoyl}-
4-(3,4-difluorophenyl)-6-methoxymethyl-2-oxo-1,2,3,4-
tetrahydropyrimidine-5-carboxylic acid methyl ester
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/ F
( F / O OMeOMe
N I
CN N
N NH
N
O O
1H NMR (CDC13, 400 MHz) consistent with assigned
structure.
FABLRMS m/e 609.26 g/mole (M++H, C31H34F2N605 =
608.64 g/mole.)
HPLC (Vydac; C18; diameter = 4.6 mm; length ' 150 mm;
gradient = H20 [0.1% H3P04] - CH3CN, 95°l0 - 5%, 5% - 95%, over 16
minutes, 2 ml/min flow rate) focus = 215 nm; 93.4% pure.
EXAMPLE 47
(4S)-4-(3,4-Difluorophenyl)-2-oxo-oxazolidine-3-carboxylic acid (1-[1-(2-
cyanophenyl)-piperidin-4-yl]azetidin-3-ylmethyl)-amide
/ F
~ I F ~
N I
CN
N~ H
~.N_ /N
~O
1H NMR (CDC13, 400 MHz) consistent with assigned
structure.
FABLRMS m/e 496.26 g/mole (M++H, C26H27F2N503 =
495.53 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 mllmin flow rate) focus = 215 nm; 94.8% pure.
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Anal. Calcd for C2gH32F5N505 ~ 0.05 CHC13 and 0.2 H20:
C = 61.94, H = 5.48, N = 13.87. Found: C = 61.97, H = 5.55, N = 13.50.
EXAMPLE 48
(4S)-4-(3,4-Difluorophenyl)-2-oxo-oxazolidine-3-carboxylic acid {1-[1-(2
cyanophenyl)-piperidin-4-yl]-3-hydroxy-azetidin-3-ylmethyl}-amide
/ F
I F /
N I
CN \
N~ H
N_ /N
O ~H
O
IH NMR (CDC13, 400 MHz) consistent with assigned
structure .
FABLRMS m/e 512 g/mole (M++H, C26H27F2N504 = 511.52
g/mole. )
HPLC (Vydac; C18; diameter = 4.6 mm; length = 150 mm;
gradient = H20 [0.1% H3P04] - CH3CN, 95% - 5%, 5% - 95%, over I6
minutes, 2 ml/min flow rate) focus = 215 nm; 95% pure.
EXAMPLE 49
(4S)-3-( 1-[1-( 2-Cyanophenyl)piperidin-4-yl]-3-hydroxy-azetidin-3-
ylmethylcarbamoyl}-4-(3,4-difluorophenyl)-6-methoxymethyl-2-oxo-
1,2,3,4-tetrahydropyrimidine-5-carboxylic acid methyl ester
F
\ ~ F / O OMeOMe
N
CN ~ I \
N~H N NH
IOHv N ' I
O O
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1H NMR (CDC13, 400 MHz) consistent with assigned
structure.
g/mole. )
FABLRMS m/e 625 g/mole (M++H, C3IH34F2N606 = 624.64
HPLC (Vydac; C18; diameter = 4.6 mm; length = 150 mm;
gradient = H20 [0.1% H3P04] - CH3CN, 95% - 5%, 5% - 95%, over 16
minutes, 2 mllmin flow rate) focus = 215 nm; 98°~o pure.
Anal. Calcd for C31H34F2N606 ' 1.65 HCl and 0.2 Et20: C
= 54.59, H = 5.42, N = 12.01. Found: C = 54.65, H = 5.25, N = 11.99.
EXAMPLE 50
(3R)-N-( 1-( I-(2-Nitrophenyl)piperidin-4-yl)pyrrolidin-3-yl)-2-(3,4-
difluorophenyl)acetamide
N 02
N N O F
N F
H
1H NMR (CDCl3, 400 MHz) consistent with assigned
structure.
g/mole. )
FABLRMS m/e 445 g/mole (M++H, C23H26F2N403 = 444.485
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; 98% pure.
Anal. Calcd for C23H26F2N403 ' 1.75 HCl: C = 54.35, H =
5.50, N = 11.02. Found: C = 54.29, H = 5.51, N = 10.63.
EXAMPLE 51
(Dias A) (3R)-N-(1-(1-(2-Nitrophenyl)piperidin-4-yl)pyrrolidin-3-yl)-2-(3,4-
difluorophenyl)acetamide
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N02
N~N cR~ O F
F
1H NMR (CDC13, 400 MHz) consistent with assigned
structure.
FABLRMS m/e 487 g/mole (M++H, C26H32F2N403 = 486.567
g/mole.)
HPLC (Vydac; C18; diameter = 4.6 mm; length = 150 mm;
gradient = H20 [0.1% H3P04] - CH3CN, 95°l0 - 5%, 5% - 95°~0,
over 16
minutes, 2 ml/min flow rate) focus = 215 nm; 100% pure.
Anal. Calcd for C26H32F2N4O3 ' 1.4 HCl: C = 58.08, H =
6.26, N = 10.42. Found: C = 58.34, H = 6.22, N = 10.55.
EXAMPLE 52
(Dias A) (3RD-N-(1-(1-(2-Cyanophenyl)piperidin-4-yl)pyrrolidin-3-yl)-2-
(3,4-difluorophenyl)acetamide
O / F
CN
N'
I \ N~ H ~ _F
1H NMR (CDC13, 400 MHz) consistent with assigned
structure.
FABLR.MS m/e 467 g/mole (M++H, C27H32F2N401 = 466.577
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; 95.4% pure.
EXAMPLE 53
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(Diast B)(3R)-N-(1-(1-(2-Cyanophenyl)piperidin-4-yl)pyrrolidin-3-yl)-2-
(3,4-difluorophenyl)acetamide
F
CN
N~
I \ N~ H ~ _ F
.,- w
structure.
g/mole. )
1H NMR (CDCl3, 400 MHz) consistent with assigned
FABLRMS m/e 467 g/mole (M++H, C27H32F2N401 = 466.577
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; 95.8% pure.
EXAMPLE 54
(3R,4S)-4-(3,4-Difluorophenyl)-2-oxo-oxazolidine-3-carboxylic acid {1-[1-(2-
nitrophenyl)-piperidin-4-yl]pyrrolidin-3-yl}-amide
F
F
I/
N02
N~
I \ N\J H N
-O
structure.
g/mole. )
1H NMR (CDC13, 400 MHz) consistent with assigned
FABLRMS m/e 516 g/mole (M++H, C25H27F2N505 = 515.522
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.
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Anal. Calcd for C25H27F2N505 ~ 1.35 HCl: C = 53.17, H =
5.06, N = 12.40. Found: C = 53.26, H = 5.09, N = 12.08.
EXAMPLE 55
(3R,4S)-4-(3,4-Difluorophenyl)-2-oxo-oxazolidine-3-carboxylic acid {1-[1-(2
[N-1-(N-3-methylureyl )]phenyl )-piperidin-4-yl]pyrrolidin-3-yl )-amide
F
HN~CH3 I ~ F
O
~NH
H N O
O
1H NMR (CDC13, 400 MHz) consistent with assigned
structure.
FABLRMS m/e 543 g/mole (M++H, C27H32F2N604 = 542.591
g/mole.)
HPLC (Vydac; C 18; 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% pure.
Anal. Calcd for C27H32F2N604 ~ 2.15 HCl: C = 52.22, H =
5.54, N = 13.53. Found: C = 52.20, H = 5.50, N = 13.22.
EXAMPLE 56
(3R,4S)-4-(3,4-Difluorophenyl)-2-oxo-oxazolidine-3-carboxylic acid {1-[1-(2-
(N-3-dimethylsulfonamido)aminophenyl)-piperidin-4-yl]pyrrolidin-3-yl}-
amide
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F
C H3 ~ F
H3C~N~S~ ~ /
O NH
N~
I \ NJ H N
O
O
structure.
1H NMR (CDC13, 400 MHz) consistent with assigned
FABLRMS m/e 593 g/mole (M++H, C27H34F2N605S =
592.666 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; 99.1% pure.
Anal. Calcd for C27H34F2N6O5S ~ 1.90 HCl: C = 48.99, H =
5.47, N = 12.70. Found: C = 49.06, H = 5.76, N = 12.69.
EXAMPLE 57
(3R,4S}-4-(3,4-Difluorophenyl)-2-oxo-oxazolidine-3-carboxylic acid {1-[1-(2-
(N-1-methanesulfonyl)aminophenyl)-piperidin-4-yl]pyrrolidin-3-yll-
amide
F
F
H3CvS,~
O~ ~NH O
N~~ ~
I \ N\J H N
-O
O
structure.
1H NMR (CDC13, 400 MHz) consistent with assigned
FABLRMS m/e 564 g/mole (M++H, C26H31F2N5O5S =
563.624 g/mole.)
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HPLC (Vydac; C 18; diameter = 4.6 mm; length = 150 mm;
gradient = H20 [0.1% H3P04] - CH3CN, 95% - 5°~0, 5% - 95%, over 16
minutes, 2 ml/min flow rate) focus = 215 nm; 100°~o pure.
Anal. Calcd for C26H31F2N5~5S ~ 2.0 HCl: C = 49.05, H =
5.23, N = 11.00. Found: C = 49.03, H = 5.18, N = 10.93.
EXAMPLE 58
(3R,4S)-4-(3,4-Difluorophenyl)-2-oxo-oxazolidine-3-carboxylic acid {1-[1-(2-
(N-1-acetyl )aminophenyl )-piperidin-4-yl]pyrrolidin-3-yl}-amide
F
F
O
NH
I ~ ~ H N
O
1H NMR (CDC13, 400 MHz) consistent with assigned
structure.
FABLR.MS m/e 528 g/mole (M++H, C27H31F2N504 = 527.576
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; 95.7% pure.
EXAMPLE 59
(4S)-3-{ 1-[1-(2-Nitrophenyl)piperidin-4-yl]-pyrrolidin-3-ylcarbamoyl}-4-
(3,4-difluorophenyl )-6-methoxymethyl-2-oxo-1,2,3,4-
tetrahydropyrimidine-5-carboxylic acid methyl ester
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N 02
H~ Hs
Hs
H
structure.
1H NMR (CDC13, 400 MHz) consistent with assigned
FABLRMS m/e 629 g/mole (M++H, C3pH34F2N6O7 = 628.64
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; 100% pure.
EXAMPLE 60
(4S)-3-(1-[1-(2-[N-1-(N-3-methylureyl)]phenyl)piperidin-4-yl]-pyrrolidin-3-
ylcarbamoyl)-4-(3,4-difluorophenyl)-6-methoxymethyl-2-oxo-1,2,3,4-
tetrahydropyrimidine-5-carboxylic acid methyl ester
HN~CH3
O~N H O
N
N i ~OCH3
~ OCH3
O' _N
1H NMR (CDC13, 400 MHz) consistent with assigned
structure.
g/mole. )
FABLRMS m/e 656 g/mole (M++H, C32H3gF2N7O6 = 655.709
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HPLC (Vydac; C 18; 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; 96.8% pure.
EXAMPLE 61
(4S)-3-( 1-[ 1-(2-(N-3-dimethylsulfonamido)aminophenyl)piperidin-4-yl]-
pyrrolidin-3-ylcarbamoyl}-4-(3,4-difluorophenyl)-6-methoxymethyl-2-oxo-
1,2,3,4-tetrahydropyrimidine-5-carboxylic acid methyl ester
CH3 F
H3C- ~S o ~ I
O~ ~NH
H
\ ~ H N ~ _OC 3
~ OCH3
O' _ N
H
1H NMR (CDC13, 400 MHz) consistent with assigned
structure.
FABLRMS m/e 706 g/mole (M++H, C32H41F2N707S =
705.783 g/mole.)
HPLC (Vydac; C 18; 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; 99.1% pure.
Anal. Calcd for C32H41F2N707 ' 2.10 HCI: C = 49.12, H =
5.55, N = 12.53. Found: C = 49.12, H = 5.60, N = 12.44.
EXAMPLE 62
(4S)-3-{1-[1-(2-(N-1-acetyl)aminophenyl)piperidin-4-yl]-pyrrolidin-3-
ylcarbamoyl}-4-(3,4-difluorophenyl)-6-methoxymethyl-2-oxo-1,2,3,4-
tetrahydropyrimidine-5-carboxylic acid methyl ester
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F
~H3
O
NH
N OCH
\ NJ H ~ ~ s
OCH3
O N
1H NMR (CDC13, 400 MHz) consistent with assigned
structure.
FABLRMS m/e 641 g/mole (M++H, C32H38F2N606 = 640.693
g/mole . )
HPLC (Vydac; C18; diameter = 4.6 mm; length = 150 mm;
gradient = H20 (0.1% H3P04] - CH3CN, 95% - 5°~0, 5% - 95%, over 16
minutes, 2 ml/min flow rate) focus = 215 nm; 100% pure.
EXAMPLE 63
(4S)-3-(1-[1-(2-(N-1-methanesulfonyl)aminophenyl)piperidin-4-yl]-
pyrrolidin-3-ylcarbamoyl}-4-(3,4-difluorophenyl)-6-methoxymethyl-2-oxo-
1,2,3,4-tetrahydropyrimidine-5-carboxylic acid methyl ester
H3C~ ,,O
O SAN H O
OCH
NJ H N ~ s
~ OCH3
-'' O~ N
H
1H NMR (CDC13, 400 MHz) consistent with assigned
structure.
FABLRMS m/e 677 g/mole (M++H, C31H38F2N6O7S =
676.747 g/mole.)
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HPLC (Vydac; C18; diameter = 4.6 mm; length = 150 mm;
gradient = H20 [0.1% H3P04] - CHgCN, 95% - 5%, 5°~0 - 95%, over 16
minutes, 2 ml/min flow rate) focus = 215 nm; 99.9% pure.
EXAMPLE 64
(Diast A) (4S)-4-(3,4-Difluorophenyl)-2-oxo-oxazolidine-3-carboxylic
acid{ 1-[ 1-( 2-cyanophenyl)piperidin-4-yl]-3-hydroxy-pyrrolidin-4-yl} amide
F
F
.O
CN O
N' ~ ~
N v _NI _N
I \ ~ H ~O
O
structure.
1H NMR (CDC13, 400 MHz) consistent with assigned
FABLRMS m/e 512 g/mole (M++H, C26H27F2N5O4 = 511.52
g/mole. )
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) focus = 215 nm; 96% pure.
EXAMPLE 65
(Diast B) (4S)-4-(3,4-Difluorophenyl)-2-oxo-oxazolidine-3-carboxylic
acid{1-[1-(2-cyanophenyl)piperidin-4-yl]-3-hydroxy-pyrrolidin-4-yl}amide
F
F
~H
CN
N~ 1
\ N ~H N
I ~-O
O
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1H NMR (CDC13, 400 MHz) consistent with assigned
structure .
FABLRMS m/e 512 g/mole (M++H, C26H27F2N504 = 511.52
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; 98% pure.
EXAMPLE 66
(Racemic G~ pyrrolidine) (4S)-3-(1-[1-(2-Cyanophenyl)piperidin-4-yl]-3-
hydroxy-pyrrolidin-4-ylcarbamoyl}-4-(3,4-difluorophenyl)-6-
methoxymethyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid
methyl ester
F
,OH ~ /
CN O
N
N j a ~H N I -OME
~J O' _ N
H
1H NMR (CDC13, 400 MHz) consistent with assigned
structure.
FABLRMS m/e 625.4 g/mole (M++H, C31H34F2N606 =
624.64 g/mole. )
HPLC (Vydac; C18; diameter = 4.6 mm; length = 150 mm;
gradient = H20 [0.1% H3P04] - CH3CN, 95% - 5°~0, 5% - 95%, over 16
minutes, 2 ml/min flow rate) focus = 215 nm; 100% pure.
EXAMPLE 67
(bias A) (4S)-3-{1-[1-(2-Cyanophenyl)piperidin-4-yI]-3-hydroxy-pyrrolidin-
4-ylcarbamoyl}-4-(3,4-difluorophenyl)-6-methoxymethyl-2-oxo-1,2,3,4-
tetrahydropyrimidine-5-carboxylic acid methyl ester
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F
CN
\ N N~H N ( OMe
I ~
O' _ N
H
1H NMR (CDC13, 400 MHz) consistent with assigned
structure .
FABLRMS m/e 625.4 g/mole (M++H, C31H34F2N606 =
624.64 g/mole.
HPLC (Vydac; C18; diameter = 4.6 mm; length = 150 mm;
gradient = H20 [0.1°lo H3P04] - CH3CN, 95% - 5%, 5°~0 - 95%,
aver 16
minutes, 2 ml/min flow rate) focus = 215 nm; 100% pure.
EXAMPLE 68
(Dias B) (4S)-3-(1-[1-(2-Cyanophenyl)piperidin-4-yl]-3-hydroxy-pyrrolidin-
4-ylcarbamoyl}-4-(3,4-difluorophenyl)-6-methoxymethyl-2-oxo-1,2,3,4-
tetrahydropyrimidine-5-carboxylic acid methyl ester
,O H
CN
N
I \ N N
H
H
1H NMR (CDC13, 400 MHz) consistent with assigned
structure.
FABLRMS m/e 625.4 g/mole (M++H, C31H34F2N6O6 =
624.64 g/mole. )
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HPLC (Vydac; C18; diameter = 4.6 mm; length = 150 mm;
gradient = H20 [0.1°~o H3P04] - CH3CN, 95% - 5%, 5% - 95%, over 16
minutes, 2 ml/min flow rate) focus = 215 nm; 100% pure.
EXAMPLE 69
(racemic) (4S)-4-(3,4-Difluorophenyl)-2-oxo-oxazolidine-3-carboxylic acid
( 1-[1-(2-cyanophenyl)-piperidin-4-yl]pyrrolidin-3-ylmethyl}-amide
F
CN
I /
N ~ O
H N
N
O
O
1H NMR (CDC13, 400 MHz) consistent with assigned
structure.
FABLRMS m/e 510.34 g/mole (M++H, C27H2gF2N5O3 =
509.55 g/mole.)
HPLC (Vydac; C 18; diameter = 4.6 mm; length = 150 mm;
gradient = H20 [0.1% H3P04] - CH3CN, 95% - 5%, 5°l0 - 95%, over 16
minutes, 2 ml/min flow rate) focus = 215 nm; 96% pure.
EXAMPLE 70
(racemic) (4S)-3-{1-[1-(2-Cyanophenyl)piperidin-4-yl]pyrrolidin-3-
ylmethylcarbamoyl}-4-(3,4-difluorophenyl)-6-methoxymethyl-2-oxo-
1,2,3,4-tetrahydropyrimidine-5-carboxylic acid methyl ester
F
N O
\ / ~ O O/
I/
N
N NH
N N \\
O
O
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1H NMR (CDC13, 400 MHz) consistent with assigned
structure.
FABLRMS m/e 623.5 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; 92% pure.
EXAMPLE 71
Diast A (4S)-3-(1-[1-(2-Cyanophenyl)piperidin-4-yl]pyrrolidin-3-
ylmethylcarbamoyl}-4-(3,4-difluorophenyl)-6-methoxymethyl-2-oxo-
1,2,3,4-tetrahydropyrimidine-5-carboxylic acid methyl ester
N F O O
Oi
N ~ \
N~NH
N'\~~~,""",~
O O
1H NMR (CDC13, 400 MHz) consistent with assigned
structure.
FABLRMS m/e 623.5 g/mole (M++H, C32H36F2N6O5 =
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; 92% pure.
EXAMPLE 72
(Diast B) (4S)-3-(1-[1-(2-Cyanophenyl)piperidin-4-yl]pyrrolidin-3-
ylmethylcarbamoyl}-4-(3,4-difluorophenyl)-6-methoxymethyl-2-oxo-
1,2,3,4-tetrahydropyrimidine-5-carboxylic acid methyl ester
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CN
N
-, N . .. .
N \
~,~i~ O
O
1H NMR (CDC13, 400 MHz) consistent with assigned
structure.
FABLRMS m/e 623.5 g/mole (M++H, C32H36F2N6O5 =
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; 92% pure.
EXAMPLE 73
(Racemic) (4S)-4-(3,4-Difluorophenyl)-2-oxo-oxazolidine-3-carboxylic acid
{ 1-[ 1-(2-cyanophenyl)-piperidin-4-yl]piperidin-3-ylmethyl}-amide
F F
CN
N
N O
N N \\
O O
IH NMR (CDC13, 400 MHz) consistent with assigned
structure.
FABLRMS m/e 524 glmole (M++H, C28H3IF2N503 = 523.58
g/mole. )
HPLC (Vydac; C18; diameter = 4.6 mm; length = I50 mm;
gradient = H20 [0.1% H3P04] - CH3CN, 95% - 5%, 5% - 95%, over 16
minutes, 2 ml/min flow rate) focus = 215 nm; >95% pure.
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EXAMPLE 74
(Dias A) (4S)-4-(3,4-Difluorophenyl)-2-oxo-oxazolidine-3-carboxylic acid
{ 1-[1-(2-cyanophenyl)-piperidin-4-yl]piperidin-3-ylmethyl}-amide
F F
CN
N
N O
N N
O O
1H NMR (CDC13, 400 MHz) consistent with assigned
structure.
FABLRMS m/e 524 g/mole (M++H, C28H31F2N503 = 523.58
g/mol a . )
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% pure.
EXAMPLE 75
(Dias B) (4S)-4-(3,4-Difluorophenyl)-2-oxo-oxazolidine-3-carboxylic acid {1-
[ 1-(2-cyanophenyl)-piperidin-4-yl]piperidin-3-ylmethyl}-amide
F F
CN
N
N O
N N 1,
O O
1H NMR (CDC13, 400 MHz) consistent with assigned
structure.
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FABLRMS m/e 524 g/mole (M++H, C28H31F2N503 = 523.58
g/mole.)
HPLC (Vydac; C18; diameter = 4.6 mm; length = 150 mm;
gradient = H20 [0.1°lo H3P04] - CH3CN, 95% - 5%, 5% - 95%, over 16
minutes, 2 mllmin flow rate) focus = 215 nm; >95% pure.
EXAMPLE 76
(racemic) (4S)-3-{1-[1-(2-Cyanophenyl)piperidin-4-yl]piperidin-3-
ylmethylcarbamoyl}-4-(3,4-difluorophenyl)-6-methoxymethyl-2-oxo-
1,2,3,4-tetrahydropyrimidine-5-carboxylic acid methyl ester
N
/ N O~
N~N~N H
~N
O O
structure.
g/mole. )
1H NMR (CDC13, 400 MHz) consistent with assigned
FABLRMS m/e 637 g/mole (M++H, C33H38F2N605 = 622.67
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% pure.
EXAMPLE 77
(Diast A) (4S)-3-{1-[1-(2-Cyanophenyl)piperidin-4-yl]piperidin-3-
ylmethylcarbamoyl}-4-(3,4-difluorophenyl)-6-methoxymethyl-2-oxo-
1,2,3,4-tetrahydropyrimidine-5-carboxylic acid methyl ester
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CN
\ ~ ~ 02Me
N
N NH
N N \\
O O
1H NMR (CDC13, 400 MHz) consistent with assigned
structure.
FABLRMS m/e 637 g/mole (M++H, C33H38F2N605 = 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% pure.
EXAMPLE 78
(Diast B) (4S)-3-{1-[1-(2-Cyanophenyl)piperidin-4-yl]piperidin-3-
ylmethylcarbamoyl}-4-(3,4-difluorophenyl)-6-methoxymethyl-2-oxo-
1,2,3,4-tetrahydropyrimidine-5-carboxylic acid methyl ester
CN
\ ~ ~ C02Me
N ~ Oi
N NH
N N \1
O O
1H NMR (CDC13, 400 MHz) consistent with assigned
structure.
FABLRMS m/e 637 g/mole (M++H, C33H38F2N605 = 622.67
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; >95% pure.
EXAMPLE 79
(4S)-4-(3,4-Difluorophenyl)-2-oxo-oxazolidine-3-carboxylic acid {1-[1-(2-
cyanophenyl)-piperidin-4-yl]piperidin-4-ylmethyl)-amide
N
F
N
N
H O
N
O I IO
1H NMR (CDCl3, 400 MHz) consistent with assigned
structure.
FABLRMS m/e 524 g/mole (M++H, C28H31F2N503 = 523.58
g/mole. )
HPLC (Vydac; C18; diameter = 4.6 mm; length = 150 mm;
gradient = H20 [0.1% H3P04] - CH3CN, 95% - 5%, 5% - 95°l0, over 16
minutes, 2 ml/min flow rate) focus = 215 nm; >95% pure.
EXAMPLE 80
(4S)-3-{ 1-[ 1-(2-Cyanophenyl)piperidin-4-yl]piperidin-4-
ylmethylcarbamoyl?-4-(3,4-difluorophenyl)-6-methoxymethyl-2-oxo-
1,2,3,4-tetrahydropyrimidine-5-carboxylic acid methyl ester
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N
F
/ N '
02Me
N
H NH
N
O I IO
1H NMR (CDC13, 400 MHz) consistent with assigned
structure.
FABLRMS m/e 637 g/mole (M++H, C33H38F2N605 = 622.67
g/mole. )
HPLC (Vydac; C 18; 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; >99% pure.
EXAMPLE 81
(3R,4S,5S)-4-(3,4-Difluorophenyl)-5-methyl-2-oxo-oxazolidine-3-carboxylic
acid {1-[1-(2-carboxymethylphenyl)-piperidin-4-yl)pyrrolid-3-yl~-amide
F
F
C02Me O
N
N H N
-O
O
1H NMR (CDC13, 400 MHz) consistent with assigned
structure.
HPLC (Vydac; C18; diameter = 4.6 mm; length = 150 mm;
gradient = H20 [0.1% H3P04] - CH3CN, 95% - 5%, 5% - 95%, over 16
minutes, 2 mUmin flow rate) focus = 215 nm; 100% pure.
Anal. Calcd for C28H32F2N405 ~ 1.25 EtOAc and 1.85 H20:
C=57.77,H=6.71,N=8.17. Found:C=57.75,H=6.95,N=8.16.
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EXAMPLE 82
(3R,4S,5R)-5-Carboxymethyl-4-(3,4-difluorophenyl)-2-oxo-oxazolidine-3
carboxylic acid {I-[1-(2-carboxymethylphenyl)-piperidin-4-yl]pyrrolid-3
yl}-amide
F
F
C02Me
N~
N N N -C02Me
H // O
O
1H NMR (CDC13, 400 MHz) consistent with assigned
structure.
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; 96% pure.
Anal. Calcd for C2gH32F2N4O7 ~ 2.1 HCl and 0.7 Et20: C =
53.41, H = 5.79, N = 7.84. Found: C = 53.39, H = 5.56, N = 7.82.
EXAMPLE 83
As a specific embodiment of an oral composition, 100 mg of
the compound of Example 6 (i.e., Compound ~ 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 84
Screening assay: Aloha la Adrenergic Receptor Binding
Membranes prepared from the stably transfected human
alpha la cell line (ATCC CRL 11140) were used to identify compounds
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that bind to the human alpha la adrenergic receptor. These competition
binding reactions (total volume = 200 ~l) contained 50 mM Tris-HCl pH.
7.4, 5 mM EDTA, 150 mM NaCl, 100 pM [125 I)_HEAT, membranes
prepared from the alpha la cell line and increasing amounts of
unlabeled Iigand. 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 < 50 nM.
EXAMPLE 85
Selective Binding assays
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 1a adrenergic receptor. These competition binding reactions (total
volume = 200 ~1) 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 86
EXEMPLARY COUNTERSCREENS
1. Assay Title: Dopamine D2, D3, D4 in vitro screen
Objective of the As av_~
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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 (IOmM Tris-HCl/5mM Mg, pH 7.4). Pellets obtained after
centrifuging these membranes (15' at 24,450 rpm) are resuspended in
50mM Tris-HCl pH 7.4 containing EDTA, MgCl[2], KCI, NaCl, CaCI[2]
and ascorbate to give a 1 Mg/mL suspension. The assay is initiated by
adding 50-75 ~g membranes in a total volume of 500 ~,1 containing 0.2 nM
[3H]-spiperone. Non-specific binding is defined using 10 ~.M
apomorphine. The assay is terminated after a 2 hour incubation at room
temperature by rapid filtration over GFlB filters presoaked in 0.3% PEI,
using 50mM Tris-HCl pH 7.4.
2. Assay Title: Serotonin 5HTla
Objective of the Assay
The objective of this assay is to eliminate agents which
specifically affect binding to cloned human 5HTla receptor
Metho
Modified from Schelegel and Peroutka Biochemical
Ph~rma~cology 35: 1943-1949 (1986).
Mammalian cells expressing cloned human SHTla
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-dipropylamino-1,2,3,4-tetrahydronaphthalene) in 50
mM Tris-HCl, 4 mM CaCl2 and lmg/ml ascorbate. Non-specific binding
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is defined using 10 ~.M propranolol. The assay is terminated after a 1
hour incubation at room temperature by rapid filtration over GF/Cfilters
EXAMPLE 87
EXEMPLARY FUNCTIONAL ASSAY
In order to confirm the specificity of compounds for the
human alpha 1a 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
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 solution if needed.
The tissue is placed in a Petri dish containing oxygenated
Krebs solution [NaCl, 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 ml jacketed tissue bath containing Krebs
buffer at 37°C, bubbled with 5% C02/95°l0 02. The tissues are
connected
to a Statham-Gould force transducer; 1 gram (rat, human) or I.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.
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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
minutes for one hour. Vehicle or antagonist is added to the bath and
5 allowed to incubate for one hour, then another cumulative concentration
response curve to the agonist is generated.
ECSp 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
10 concentrations of antagonist are tested, Kb values are calculated
according to the following formula Kb =~B ,
x-1
where x is the ratio of EC50 of agonist in the presence and absence of
antagonist and [B] is the antagonist concentration.
2. Measurement of Intra-Urethral Pressure in Anesthetized Dogs
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 efl'lcacy 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
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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 vena 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 --1/2 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
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 effects 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
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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. Effects of the alpha 1 antagonists on baseline arterial pressure are
also monitored. Comparison of the relative antagonist potency on
changes in arterial pressure and intra-urethral pressure provide
insight as to whether the alpha receptor subtype responsible for
increasing intra-urethral pressure 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
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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