Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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TIT~E OF THE INVENTION
ALPHA la ADRENERGIC RECEPTOR ANTAGONISTS
~l~;LD OF THE INVENTION:
This invention relates to certain novel compounds and
derivatives thereof, their synthesis, and their use as selective 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
15 peripheral sympathetic nervous system upon binding of catecholamines,
norepinephrine and epinephrine.
Norepinephrine is produced by adrenergic nerve en~ling.s,
while epinephrine is produced by the adrenal medulla. The binding
affinity of adrenergic receptors for these compounds forms one basis of
2 0 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
2 5 opposed to responses in~ ced by beta receptor bin~lin~.
Subsequently, the functional distinction between alpha and
beta receptors was further highlighted and refined by the
pharmacological characterization of these receptors from various
~nim~l and tissue sources. As a result, alpha and beta adrenergic
3 0 receptors were further subdivided into al, a2, ~1, and ~2 subtypes.
Functional differences between al and a2 receptors have been
recognized, and compounds which exhibit selective binding between
these two subtypes have been developed. Thus, in WO 92/0073, the
selective ability of the R(+) enantiomer of terazosin to selectively bind
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to adrenergic receptors of the alpha 1 subtype was reported. The al/a2
selectivity of this compound was disclosed as being significant because
agonist stin~ tion of the a2 receptors was said to inhibit secretion of
epinep~rine and norepinephrine, while antagonism of the a2 receptor
was saicl to increase secretion of these hormones. Thus, the use of non-
selective alpha-adrenergic blockers, such as phenoxyben7~min~ and
phentol~lmine, is limited by their a2 adrenergic receptor me~ t~l
induction of increased plasma catechol~mine concentration and the
attendant physiological sequelae (increased heart rate and smooth muscle
10 contraction).
For a general background on the a-adrenergic receptors,
the reader's attention is directed to Robert R. Ruffolo, Jr., a-
Adrenoreceptors: Molecular Biolo~y~ Biochemistry and Pharmacolo~y.
(Pro~ress in Basic and Clinical Pharmacolo~y series, Karger, 1991),
15 wherein the basis of al/a2 subclassification, the molecular biology,
signal tr;msduction (G-protein interaction and location of the significant
site for this and ligand binding activity away from the 3'-termimls of
alpha adrenergic receptors), agonist structure-activity relationships,
receptor functions, and therapeutic applications for compounds
2 o exhibiting a-adrenergic receptor affinity was explored.
The cloning, sequencing and expression of alpha receptor
subtypes from ~nim~l tissues has led to the subclassification of the al
receptors into a1a, (Lomasney, et al., J. Biol. Chem.. 266:6365-6369
(1991), rat a1a; Bruno et al., BBRC, 179:1485-1490 (1991), hllm~n
25 ala)~ a1b (Cotecchia, et al., PNAS~ 85;7159-7163 (1988), hamster a1b;
Libert, et al., Science. (1989), dog alb; Ramarao, et al., J. Biol.
Chem.. 267:21936-21945 (19'32), hl-m~n alb)~ and most recently, in a
study using bovine brain, a new alc subtype was proposed (Schwinn, et
al.. J. Biol. Chem.. 265:8183-X189 (1990); Hirasawa et al., BBRC
3 0 195:902-909 (1993), described the cloning, functional expression and
tissue distribution of a human alC adrenergic receptor; Hoehe et al.,
Human Mol. Genetics 1(5):349 (8/92) noted the existence of a two-allele
Pstl restriction fragment polymorphism in the a1C adrenergic receptor
gene; another study suggests that there may even be an alpha ld
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receptor subtype, see Perez et al., Mol. Pharm., 40:876-883, 1992).
Each a1 receptor subtype exhibits its own pharmacologic and tissue
specificities. Schwinn and coworkers noted that the cloned bovine a1C
receptor exhibited pharmacological properties proposed for the a1a
subtype. Nonetheless, based on its non-expression in tissues where the
ala subtype is expressed, and its sensitivity to chloroethylclonidine, the
receptor was given a new designation.
The differences in the a-adrenergic receptor subtypes have
relevance in pathophysiologic conditions. Benign prostatic hyperylasia,
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, but are not
limited to, increased difficulty in urination and sexual dysfunction.
These synlytollls 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. Conco~ nil~ltly~ 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.
2 0 In benign prostatic hypelplasia, the male hormone 5a-
dihydrotestosterone has been identified as the principal culprit. The
continual production of Sa-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
2 5 enlarged gland begins to obstruct the urethra with the pathologic
symptoms noted above.
The elucidation of the mech~ni~m s-lmm~rized 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
3 0 these agents is Merck & Co., Inc.s' product PROSCAR~) (finasteride).
The effect of this compound is to inhibit the en~yme testosterone 5-
alpha reductase, which converts testosterone into Sa-dihydrotesterone,
resulting in a reduced rate of prostatic enlargement, and often reduction
in prostatic mass.
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The development of such agents as PROSCAR(~) bodes wel]
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
5 suffer, .md may in fact lose hope that the agents are working sufficiently
rapidly
In response to this problem, one solution is to identify
pharmaceutically active compounds which complement slower-acting
therapeutics by providing acute relief. Agents which induce relaxation
10 of the urethral smooth muscle, by binding to alpha 1 adrenergic
receptorLs, thus reducing the increased adrenergic tone due to the
disease, would be good candidates for this activity. Thus, one such
agent is alfuzosin, which is reported in EP 0 204597 to induce urination
in cases of prostatic hyperplasia. Likewise, in WO 92/0073, the
1 c, selective ability of the R(+) enantiomer of terazosin to bind to
adrener.gic receptors of the a1 subtype was reported. In addition, in
WO 921'161213, hereby incorporated by reference, combinations of 5-
alpha-reductase inhibitory compounds and alpha l-adrenergic receptor
blockers (terazosin, doxazosi~n, prazosin, bunazosin, indoramin,
2C alfuzosin) were disclosed. However, no information as to the a1a, a
or a1C subtype specificity of these compounds was provided as ~is 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
2 5 mesylate (Cardura, Pfizer). These non-selective antagonists suffer from
side effects related to antagonism of the alpha 1 a and alpha lb receptors
in the peripheral vasculature, e.g., orthostatic hypotension and syncope.
Typically, identification of active compounds is
accomplished through use of ~nim~l tissues known to be enriched in
3 0 adrenergic receptors. Thus, rat tissues have been used to screen for
potential adrenergic receptor antagonists. However, because of species
variability, compounds which appear active in ~nim~l tissue may not be
active or sufficiently selective in hllm~n~. This results in subst~n~
wastage of time and effort, particularly where high volume compound
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screening programs are employed. There is also the danger that
compounds, which might be highly effective in hllm~n.s, would be
missed because of their absence of appreciable affinity for the
heterologous ~nim~l receptors. In this regard, it has been noted that
5 even single amino acid changes between the se4uence of biologically
active proteins in one species may give rise to substantial
pharmacological differences. Thus, Fong et al., (J. Biol. Chem..
267:25668-25671, 1992) showed that there are 22 divergent amino acid
residues between the sequence of the human neurokinin-l receptor and
1 CI the homologous rat receptor. They further showed, in studies with
mutant receptors, that substitution of only two arnino acid residues was
both necessary and sufficient to reproduce the rat receptor's antagonist
binding affinity in the hl~m~n receptor. Oksenberg et al., (Nature~
360:161-163, 1992) showed that a single amino-acid difference confers
1 ~ major ph~rm~cological variation between the human and the rodent 5-
hydroxytrypt~min~ receptors. Likewise, Kuhse et al., (Neuron. 5:867-
873, 1990) showed that a single amino-acid exchange alters the
pharmacology of the neonatal rat glycine receptor subunit. This
difficulty and unpredictability has resulted in a need for a compound
2 o screen which will identify compounds that will be active in hllm~n~.
These problems were solved by cloning the hllm7~n
adrenergic receptor of the alC subtype (ATCC CRL 11140) and the use
of a screening assay which enables identification of compounds which
specifically interact with the hllm~n alc adrenergic receptor. [PCT
2 5 International Application Publication Nos. W094/08040, published 14
April 1994 and WO94/10989, published 26 May 1994] As disclosed in
the instant patent disclosure, a cloned human a1C adrenergic receptor
and a method for identifying compounds which bind the hllm~n a1C
receptor has now made possible the identification of selective hllm~n
3 0 a1 C adrenergic receptor antagonists useful for treating BPH. The
instant patent disclosure discloses novel compounds which selectively
bind to the hllm~n alC receptor. These compounds are further tested
for binding to other hllm~n alpha 1 receptor subtypes, as well as
counterscreened against other types of receptors, thus defining the
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specific:ity of the compounds of the present invention for the hllm~n
alC adrenergic receptor.
Meperidine and normeperidine are known opioid receptor
ligands useful as analgesics. LJanssen, P.A. et al, J. Med. Chem. 1(4),
c; 309 (1959)]. It has now been found that the compounds of the present
invention, which represent structurally modified normeperidine
derivatives, are selective alpha la receptor antagonists devoid of opioid
binding properties.
Compounds of this invention are thereful useful for
1 C reducin,g the acute symptoms of BPH without side effects caused by
ancillary opioid receptor binding. Thus, compounds of this invention
may be used alone or in conjunction with a more long-term anti-BPH
therapeutics, such as testosterone S-alpha reductase inhibitors, including
PROSCAR(~) (finasteride). Aside from their utility as anti-BPH agents,
15 these compounds may be used to induce highly tissue-specific, localized
a1C 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 lc receptor mediated
central nervous system events.
NOMENCLATURE
Recently, a new a1 adrenergic receptor (a1-AR)
classification scheme similar to that proposed by Ford, et al. [~1-
Adrenoc eptor Classification: Sharpening Occam's Razor. Trends in
2 5 Pharm ,Sci. 1994, 15, 167-170] was adopted at the August, 1994
meeting of the International lJnion of Pharmacology (IUPHAR) in
MontreaLI, C~n~ . The al-AR genes formerly known as ala/d~ alb
and alC were renamed a1d, alb and ala, respectively. This new
naming system reflects the correspondence between the proteins
3 0 encoded by the ala and alb genes (new IUPHAR nomenclature) and
the receptors characterized by traditional pharmacological means as
a1A and a1g, respectively, in the literature. Recombinant receptors
and receptors characterized ph~ cologically in tissues are
distingu.ished by lowercase and uppercase subscripts, respectively.
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The above discussion contained in the Background section
used the former classification scheme (i-e-, a1a/d, alb and a1c);
however, hereinafter, the new classification scheme will be utilized (i.e.,
ald, alb and ala)- Thus, what was formerly referred to as the alC
5 receptor (and alC receptor antagonists) will hereinafter be referred to
tili7.in~ the new nomenclature as the a1a receptor (and ala receptor
antagonists).
SUMMARY OF THE ~VENTION
The present invention provides a method of treating a
condition which is susceptible to treatment by antagonism of the alpha
la adrenergic receptor which comprises a(l-nini~tering to the subject a
therapeutically effective amount of a compound of the formula:
,X~
A~R3
1 5 wherein
A is selected from C-R2 or N;
X is C or N, provided that when X is N, then Rl is absent;
R l is selected from hydrogen, halogen, C1 8 alkyl, mono-, di- or tri-
halogenated Cl ~s alkyl, C1 6 alkoxy, cyano, CoNR4R5 or
C3-8 cycloalkyl;
2 5 R2 is selected from hydrogen, cyano, CoNR4R5 or C02R4;
R3 is selected from hydrogen, cyano, CoNR4R5, C02R4 or S02R4;
and
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R4 and RS are each independently selected from hydrogen, C1 8 alkyl
or C3-8 cycloalkyl;
and the pharmaceutically acceptable salts thereof. Preferably, the
5 compound used in the method is selected from
?~ or [~
H H
wherein X, Rl and R3 are as defined above.
In one embodiment of the invention is the method of
treating a condition which is susceptible to treatment by antagonism of
10 the alpha la adrenergic receptor wherein
R l is selected from hydrogen, halogen, C'l -6 alkyl, mono-, di- or tri-
halogenated Cl 6 alkyl, Cl 4 alkoxy, cyano, CONH2 or
C3-6 cycloaLkyl;
R3 is selected from hydrogen, cyano or C02R4; and
15 R4 and ]RS are each independently selected from hydrogen, C1 6 alkyl
or C3-6 cycloalkyl;
and the pharmaceutically acceptable salts thereof.
In a class of the invention is the method of treating a
condition which is susceptible to treatment by antagonism of the alpha
20 la adrenergic receptor wherein
xisC;
Rl is selected from hydrogen, chloro, Cl 4 alkyl, tri-halogenated
Cl 4 aLkyl, Cl 4 alkoxy, cyano or CONH2;
R2 is se]lected from hydrogen, cyano, CONH2 or C02R4;
2 5 R3 is selected from hydrogen, cyano or Co2cH2cH3; and
R4 is se;lected from hydrogen, Cl 4 alkyl or C3-6 cycloaLkyl;
and the pharmaceutically acceptable salts thereof.
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In a subclass of the invention is the method of treating a
condition which is susceptible to treatment by antagonism of the alpha
la adrenergic receptor wherein the compound has the formula
1R~
,J~ R3
5 wherein
R 1 is selected from hydrogen, chloro, methyl, trifluoromethyl,
methoxy, cyano or CONH2; and
R2 is selected from hydrogen, cyano, CONH2, C02H, C02CH3,
C02CH2CH3, C02(CH2)3CH3 or C02cyclohexyl;
10 and the pharmaceutically acceptable salts thereof.
Illustrative of the invention is the method of treating a
condition which is susceptible to treatment by antagonism of the alpha
la adrenergic receptor wherein the compound is selected from
1RJ~ 1RJ~R2
~,CN ~
15 wherein R2 is selected from hydrogen, cyano, C02H or C02NH2;
and the ph~ reutically acceptable salts thereof.
An illustration of the invention is the method of treating a
condition which is susceptible to treatment by antagonism of the alpha
la adrenergic receptor wherein the compound has the structure
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- 10 -
R~
H~R2
and the ph~rm~ceutically acceptable salts thereof.
Exemplifying the invention is the method wherein the
compound is selected from
5 4-cyano-4-(2-trifluoromethylphenyl)piperidine;
4-cyano-4-(2-methylphenyl)piperidine;
4-(2-chlorophenyl)-4-cyanopiperidine or
4-(2-chlorophenyl)-4-(methoxycarbonyl)piperidine
and the pharmaceutically acceptable salts thereof.
Examples of conditions which are susceptible to treatment
by antagronism of the alpha la adrenergic receptor include, but are not
limited lo, benign prostatic h;yperplasia, urinary obstruction, impotence
and high intraocular pressure The compounds useful in the methods of
the present invention selectively antagonize the hllm~n alpha la
15 adrenergic receptor at nanomolar concentrations while exhibiting at
least five fold lower affinity for the alpha l d and alpha lb hllm~n
adrenergic receptors and many other G-protein coupled receptors. Use
of selective alpha la adrener~;ic receptor antagonists in the methods of
the instant invention results in reduced side effects related to peripheral
2 0 adrenergic blockade. Such side effects include hypotension, syncope,
lethargy, etc.
Further illustrating the invention is a method of treating
benign prostatic hyperplasia in a subject in need thereof which
comprise s ~lmini~tering to the subject a therapeutically effective
2 5 amount of any of the compounds desribed above.
Further exemplifying the invention is a method of relaxing
urethral smooth muscle in a subject in need thereof which comprises
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~lmini~tering to the subject a therapeutically effective amount of any of
the compounds desribed above.
Additional illustrations of the invention are the methods of
treating BPH or of relaxing urethral smooth muscle wherein the
', compound additionally does not cause a fall in blood pressure at dosages
effective to alleviate benign prostatic hyperplasia or relax ul~lh
smooth muscle.
More specifically exemplifying the invention are the
methods of treating BPH or of relaxing urethral smooth muscle wherein
the compound is ~lmini~tered in combination with 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 I testosterone 5-alpha reductase
inhibitor and a type 2 testosterone 5-alpha reductase inhibitor) or a dual
type 1 and type 2 testosterone 5-alpha reductase inhibitor. More
preferably, the testosterone 5-alpha reductase inhibitor is a type 2
testosterone 5-alpha reductase inhibitor. Most preferably, the
testosterone 5-alpha reductase inhibitor is finasteride.
2 0 More particularly illustrating the invention is the use of any
of the compounds described above in the preparation of a medicament
for the treatment of benign prostatic hyperplasia, or for relaxing
urethral smooth muscle.
Another example of the invention is a drug which is useful
2 5 for treating benign prostatic hyperplasia or for relaxing urethral
smooth muscle, the effective ingredient of the said drug being any of the
compounds descibed above.
DETAILED DESCRIPTION OF THE ~VENTION
3 0 Representative compounds of the present invention exhibit
high selectivity for the human alpha la adrenergic receptor. One
implication of this selectivity is that these compounds display selectivity
for lowering intraurethral pressure without subst~nti~lly affecting
diastolic blood pressure.
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Representative compounds l-tili7ed in the methods of this
invention display submicrornolar affinity for the hum~n alpha la
adrenergic receptor subtype while displaying at least five-fold lower
affinity for the human alpha ld and alpha lb adrenergic receptor
subtypes, and many other G-protein coupled hnm~n receptors.
Preferred compounds of this invention exhibit nanomolar affinity for
the hl1rn~n alpha la adrenergic receptor subtype while displaying at least
10 fold lower affinity for the hnm~n alpha ld and alpha lb adrenergic
recepto,r subtypes, opioid receptors and many other G-protein coupled
hl~m~n receptors.
These compounds are a~lmini~tered in dosages effective to
antagonize the alpha la receptor where such tre~tment is n~e~le~l, as in
BPH. For use in medicine, 1he 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 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 ~,vith a
2 () solution of a pharmaceutically acceptable acid such as hydrochloric acid,sulphuric acid, fumaric acid, maleic acid, succinic acid, acetic acid,
benzoic acid, oxalic acid, citric acid, tartaric acid, carbonic acid or
phosphoric acid. Furthermore, where the compounds of the invention
carry an acidic moiety, suitable ph~rm~ceutically acceptable salts
2 ~, thereof may include alkali metal salts, e.g. sodium or potassium salts;
~Ik~line earth metal salts, e.g. calcium or m~nesium salts; and salts
formed with suitable organic ligands, e.g. quaternary ammonium salts.
Thus, r~presentative ph~rm~ceutically acceptable salts include the
followillg:
3 C Acetate, Benzenesulfonate, Benzoate, Bicarbonate,
Bisulfate, Bitartrate, Borate, Bromide, Calcium, Camsylate, Carbonate,
Chloride, Clav~ n~t~, Citrate, Dihydrochloride, Edetate, Edisylate,
Estolate, Esylate, Fumarate, Gluceptate, Gluconate, Glllt~m~te,
Glycoll~ylars~nil~te, Hexylresorcinate, Hydrabamine, Hydrobromide,
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Hydrochloride, Hydroxynaphthoate, Iodide, Isothionate, Lactate,
Lactobionate, LauMte, Malate, Maleate, Mandelate, Mesylate,
Methylbromide, Methylnitrate, Methylsulfate, Mucate, Napsylate,
Nitrate~ N-methylglllc~mine ammonium salt, Oleate, Oxalate, Pamoate
s j (Embonate), Palmitate, Pantothenate, Phosphate/diphosphate,
Polygalacturonate, Salicylate, Stearate, Sulfate, Subacetate, Succinate,
Tannate, Tartrate, Teoclate, Tosylate, Triethiodide and Valerate.
The present invention includes within its scope prodrugs of
the compounds of this invention. In general, such prodrugs will be
10 functional derivatives of the compounds of this invention which are
readily convertible in vivo into the required compound. 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
15 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
2 0 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 polymoIphs and as such are intended to be included in the
2 ~ 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
3 C ~lk~nes of one to ten total carbon atoms, or any number within this
range (i.e., methyl, ethyl, l-propyl, 2-propyl, n-butyl, s-butyl, t-butyl,
etc.).
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The term "alkenyl" shall mean straight or branched chain
alkenes of two to ten total carbon atoms, or any number within this
range.
The term "aryl" as used herein, except where otherwise
', specifically defined, refers to unsubstituted, mono- or poly-substituted
aromatic groups such as phenyl or naphthyl.
The term "cycloalkyl" shall mean cyclic rings of alkanes of
three to eight total carbon atoms (i.e., cyclopropyl, cyclobutyl,
cyclopentyl, cyclohexyl, cycloheptyl or cyclooctyl).
Whenever the term "alkyl" or "aryl" or either of their
prefix roots appear in a name of a substituent (e.g., araL~coxyaryloxy) it
shall be interpreted as including those limit~tions given above for
"alkyl" and "aryl." Design~ted numbers of carbon atoms (e.g., C1-10)
shall re~fer independently to Ihe number of carbon atoms in an alkyl or
1', 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
2 C degrees of substitution by a named subs~ilulellt. The term "poly-
substituted" as used herein shall include di-, tri-, tetra- and penta-
substitution by a named substituent.
Where multiple substituent moieties are disclosed or
claimed, the substituted compound can be independently sub~~iluled by
2 5 one or nnore of the disclosed or claimed substituent moieties, singly or
plurally.
The term heterocycle or heterocyclic ring, as used herein,
represents an unsubstituted or substituted stable 5- to 7-membered
monocyclic ring system which may be saturated or Im~tllrated, and
3 0 which consists of carbon atoms and from one to three heteroatoms
selected from N, O or S, and wherein the nitrogen and sulfur
heteroat~ms 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
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- 15 -
stable structure. Examples of such heterocyclic groups include, but is
not limited to, piperidinyl, piperazinyl, oxopiperazinyl, oxopiperidinyl,
oxopyrrolidinyl, oxoazepinyl, azepinyl, pyrrolyl, pyrrolidinyl, furanyl,
thienyl, pyrazolyl, pyrazolidinyl, imidazolyl, imidazolinyl,
imidazolidinyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, oxazolyl,
oxazolidinyl, isooxazolyl, isoxazolidinyl, morpholinyl, thiazolyl,
thiazolidinyl, isothiazolyl, thi~ 701yl, tetrahydropyranyl,
thiamorpholinyl, thiamorpholinyl sulfoxide, thiamorpholinyl sulfone,
and o~ 7.olyl. Morpholino is the same as morpholinyl.
The term "subject," as used herein refers to an ~nim~l,
preferably a m~mm~l, most preferably a hllm~n, who has been the
object of treatment, observation or experiment.
The term "therapeutically effective amount" as used
herein means that amount of active compound or ph~rm~ceutical
15 agent that elicits the biological or medicinal response in a tissue,
system, ~nim~l 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
2 0 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
25 suppositories; for oral, parenteral, intr~n~l, sublingual or rectal
?~lmini.ctration, or for ~-lmini~tration by inh~l~tion or insufflation.
Alternatively, the compositions may be presented in a form suitable for
once-weekly or once-monthly ~llrnini.~tration; for example, an insoluble
salt of the active compound, such as the decanoate salt, may be adapted
3 0 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
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- 16 -
other pharmaceutical diluents, e.g. water, to form a solid
preformulation composition cont~ining a homogeneous mixture of a
compolmd of the present invention, or a ph~rm~ceutically acceptable
salt thereof. When referling to these preformulation compositions as
', homogeneous, it is meant that the active ingredient is dispersed evenly
through,out the composition so that the composition may be readily
subdivilded into equally effective unit dosage forms such as tablets, pills
and caplsules. This solid preformulation composition is then subdivided
into unit dosage forms of the type described above cont~ining from 0.1
l(J to about 500 mg of the active ingredient of the present invention. The
tablets or pills of the novel composition can be coated or otherwise
compounded to provide a dosage form affording the advantage of
prolonged action. For example, the tablet or pill can comprise an inner
dosage and an outer dosage c omponent, the latter being in the for~n of
1~ an envelope over the former. The two components can be separated by
an entelic layer which serves to resist tli~integration in the stomach and
permits the inner component to pass intact into the duo~len-lm 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
2 0 acids and mixtures of polymeric acids with such materials as shellac,
cetyl alc ohol and cellulose acetate.
The liquid forms in which the novel compositions of the
present invention may be incorporated for ~lmini.stration orally or by
injection include aqueous solutions, suitably flavoured syrups, aqueous
2 5 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 sim-ilar pharmaceut;ical vehicles. Suitable dispersing or suspending
agents for aqueous suspensions include synthetic and natural gums such
as tr~g~C~ntll~ acacia, alginate, dextran, sodium carboxymethylcellulose,
3 o methylcellulose, polyvinyl-pyrrolidone or gelabin.
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
CA 0223~370 1998-04-20
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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
5 formation with an optically active acid, such as (-)-di-p-toluoyl-d-
tartaric acid and/or (+)-di-p-toluoyl-l-tartaric acid followed by
fractional cryst~lli7.~tion and regeneration of the free base. The
compounds may also be resolved by formation of diastereomeric esters
or amides, followed by chromatographic separation and removal of the
10 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
15 concerned. This may be achieved by means of conventional protecting
groups, such as those described in Protective Groups in Or~anic
Chemistry~ ed. J.F.W. McOmie, Plenum Press, 1973; and T.W. Greene
& P.G.M. Wuts, Protective Groups in Or anic Synthesis. John Wiley &
Sons, lg91. The protecting groups may be removed at a convenient
2 0 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
25 of alpha (e.g., alpha ld, alpha lb) or beta adrenergic receptors.
Expression of the cloned hllm~n alpha ld, alpha lb, and alpha la
receptors and comparison of their binding properties with kno~vn
selective antagonists provides a rational way for selection of compounds
and discovery of new compounds with predictable ph~rm~cological
3 0 activities. Antagonism by these compounds of the human alpha la
adrenergic receptor subtype may be functionally demonstrated in
anesthetized ~nim~l~. These compounds may be used to increase urine
flow without exhibiting orthostatic hypotensive effects.
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The ability of compounds of the present invention to
specifically bind to the alpha la receptor makes them useful for the
tre~tment of BPH. The specificity of binding of compounds showing
affinity for the alpha la receptor is compared against the binding
affinities to other types of alpha or beta adrenergic receptors. The
human alpha adrenergic receptor of the 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, each of which is
1 0 hereby mcorporated by reference. The cloned human alpha la
receptor, when expressed in m~mm~ n cell lines, is used to discover
ligands that bind to the receptor and alter its function. Expression of
the cloned hllm~n alpha ld, alpha lb, and alpha la receptors and
comparison of their binding properties with known selective antagonists
1 5 provides a rational way for selection of compounds and discovery of
new compounds with predictable ph~rm~cological activities.
Compounds of this invention exhibiting selective hl-m~n
alpha 1a adrenergic receptor antagonism may further be defined by
counterscreening. This is accomplished according to methods known in
2 o the art using other receptors responsible for me~ ting diverse
biological functions. rSee e.~., PCT Intemational Application
Publication No. WO94/10989, published 26 May 1994; U.S. Patent No.
5,403,847, issued April 4, 1995, the contents of which are hereby
incorporated by reference]. Compounds which are both selective
2 5 amongs1 the various hllm~n alpha 1 adrenergic receptor subtypes and
which have low affinity for other receptors, such as the alpha2
adrenergic receptors, the ~-adrenergic receptors, the muscarinic
receptors, the serotonin receptors, and others are particularly
~r~felled. The absence of these non-specific activities may be
3 o con~rmed by using cloned and expressed receptors in an analogous
fashion l:o the method disclosed herein for identifying compounds which
have hig;h affinity for the various hllm~n alpha 1 adrenergic receptors.
Furthermore, functional biological tests are used to co-lrilln the effects
of identified compounds as alpha la adrenergic receptor antagonists.
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The present invention also has the objective of providing
suitable topical, oral, systemic and parenteral pharmaceutical
form~ tions for use in the novel methods of treatment of the present
invention. The compositions cont~ining compounds of this invention
'j as the active ingredient for use in the specific antagonism of h~ n
alpha la adrenergic receptors can be ~-lmini.~tered in a wide variety
of therapeutic dosage forms in conventional vehicles for systemic
?~lmini~tration. For example, the compounds can be ~-lmini.ctered in
such oral dosage forms as tablets, capsules (each including timed
10 release and sustained release formulations), pills, powders, granules,
elixirs, tinctures, solutions, suspensions, syrups and emulsions, or by
injectiom. Likewise, they may also be ~ inistered in intravenous
(both bolus and infusion), intraperitoneal, subcutaneous, topical with
or without occlusion, or intramuscular form, all using forms well
15 known to those of ordinary skill in the ph~ ceutical arts. An
effective but non-toxic amount of the compound desired can be
employed as an alpha 1 a antagonistic agent.
Advantageously, compounds of the present invention
may be ~(lmini~tered in a single daily dose, or the total daily dosage
2 o may be ~lmini~tered in divided doses of two, three or four times
daily. Purthermore, compounds for the present invention can be
~-~mini~tered in intranasal fonn via topical use of suitable intranasal
vehicles, or via transderrnal routes, using those forms of transdermal
skin patches well known to those of ordinary skill in that art. To be
25 ~lmini~tered in the form of a transdermal delivery system, the
dosage ~ mini~tration will, of course, be continuous rather than
intermittent throughout the dosage regimen.
The dosage regimen lltili7:ing the compounds of the
present invention is selected in accordance with a variety of factors
3 0 including type, species, age, weight, sex and medical condition of the
patient; the severity of the condition to be treated; the route of
~mini~tration; 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
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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
5 availability to target sites. This involves a consideration of the
distribution, equilibrium, and elimin~tion of a drug.
In the methods of the present invention, the compounds
herein described in detail can form the active ingredient, and are
typically ~(lmini~tered in admixture with suitable pharmaceutical
10 diluents, excipients or carriers (collectively referred to herein as
"carrier" materials) suitably selected with respect to the inten~le-l
form of adrnini.ctration, that is, oral tablets, capsules, elixirs, syrups
and the like, and consistent with conventional pharmaceutical
practices.
For instance, for oral ~(lministration 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
2 0 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, tr~g~c~nth or sodium alginate,
carboxymethylcellulose, polyethylene glycol, waxes and the like.
Lubricants used in these dosage forms include, without limit~tion,
sodium oleate, sodium stearate, m~gnP.sium stearate, sodium
ben70~te, sodium acetate, sodium chloride and the like.
Disintegrators include, without limit~tion, starch, methyl cellulose,
agar, bentonite, x~nth~n gum and the like.
3 0 The li~uid 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 di~ye~sillg
agents which may be employed include glycerin and the like. For
parenteral ~tlmini~tration, sterile suspensions and solutions are desired.
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Isotonic preparations which generally contain suitable preservatives are
employed when intravenous ~-lmini~tration is desired.
The compounds of the present invention can also be
nini.~tered in the form of liposome delivery systems, such as small
~i lmil~mPllar 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
1 C 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-
~mitlephenol, polyhydroxy-ethylaspartamidephenol, or polyethyl-
15 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,
2 0 polyacetals, polydihydro-pyrans, polycyanoacrylates and cross-linked
or amphipathic block copolymers of hydrogels.
Compounds of this invention may be ~ ni~ctered in
any of the foregoing compositions and according to dosage regimens
established in ~e art whenever specific blockade of the hnm~n alpha
25 la adrenergic receptor is required.
The dai~y dosage of the products may be varied over a wide
range from 0.01 to 1,000 mg per adult hllm~n per day. For oral
~mini~tration, the compositions are preferably provided in the form of
tablets cont~ining 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0,
3 0 50.0, 100 and 500 milligrams of the active ingredient for the
s~lnptolllatic adjustment of the dosage to ~e patient to be treated. A
medicament typically contains from about 0.01 mg to about 500 mg of
~e active ingredient, preferably, from about 1 mg to about 100 mg of
active ingredient. An effective amount of ~e drug is ordinarily
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- 22 -
supplield at a dosage level of from about 0.0002 mg/kg to about 250
m~/kg of body weight per day. Preferably, the range is from about
0.001 to 100 mg/kg of body weight per day, and especially from about
0.001 mg/lcg to 7 m~/kg of body weight per day. The compounds may
S be ~llmini.ctered on a regimen of 1 to 4 times per day.
Compounds of this patent disclosure may be used alone at
a~l~ro~iate dosages defined by routine testing in order to obtain
optimal antagonism of the hllm~n alpha la adrenergic receptor while
minimi7ing any potential toxicity. In addition, co-~-lmini~tration or
10 sequential ~ ini.~tration of other agents which alleviate the effects of
BPH is desirable. Thus, in one embodiment, this includes
~lmini~tration of compounds of this invention and a hllm~n testosterone
5-a reductase inhibitor. Included with this embodiment are inhibitors
of 5-alpha reductase isoenzyme 2. Many such compounds are now well
1 c; 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, hereby incorporated by reference). In addition
to PROSCAR@~), which is principally active in prostatic tissue due to its
selectivity for hllm~n 5-a reductase isozyme 2, combinations of
2 o 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 Sa-reductase inhibitors
have been described in W093/23420, EP 0572166; WO 93/23050;
25 W093/~!3038,; W093/23W8; WO93/23041; W093/23040;
W093/23039; W093/23376; WO93/23419, EP 0572165; WO93/23051,
each of which is hereby incorporated by reference.
The dosages of the alpha la adrenergic receptor and
testosterone 5-alpha reductase inhibitors are adjusted when combined
3 0 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 optimi7s~1 and
combined to achieve a synergistic result wherein the pathology is
reduced more than it would be if either agent were used alone. In
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accordance with the method of the present invention, the individual
components of the combination can be ~-lministered separately at
different times during the course of therapy or concurrently in
divided or single combination forms. The instant invention is
5 therefore to be understood as embracing all such regimes of
simultaneous or alternating treatment and the term "~flmini~tering" is
to be interpreted accordingly.
Thus, in one preferred embodiment of the present
invention, a method of treating BPH is provided which comprises
1 C ~lmini.~tering to a subject in need of tre~tment any of the compounds
of the present invention in combination with finasteride effective to
treat BPH. The dosage of finasteride ~dministered 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
15 dosage of finasteride in the combination is about 0.2 mg per subject
per day to about 10 mg per subject per day, more preferably, about
1 to about 7 mg per subject to day, most preferably, about 5 mg per
subject per day.
For the treatment of benign prostatic hyperplasia,
2 0 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 4,7~-dimethyl-4-aza-Sa-cholestan-3-
one, in a single oral, systemic, or parenteral pharmaceutical dosage
2 5 formulation. Alternatively, a combined therapy can be employed
wherein the alpha la adrenergic receptor antagonist and the 5a-
reductase 1 or 2 inhibitor are ~lmini~tered 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
3 0 formulal:ions for Sa-reductase inhibitors.
Abbreviations used in the instant specification, particularly
the Schemes and Examples, are as follows:
ACE-Cl = alpha-chloroethylchloroformate
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- 24 -
Boc or BOC = t-butyloxycarbonyl
BOPCI = bis(2-oxo-3-oxazolidinyl)phosphinic chloride
Cbz-CI = benzyloxycarbonyl chloride
DAST = diethylaminosulfurtrifluoride
DEAD = diethylazodicarboxylate
DMF = N,N-dimethylformamide
EDCI= 1-(3-dimethylaminopropyl)-3-ethylcarbodimide
hydrochloride
Et = ethyl
Et3N = triethylamine
EtOAc = ethyl acetate
EtOH = ethanol
FABLRMS = fast atom bombardment low resolution mass
spectroscopy
HPLC = high performance liquid chromatography
HOAc = acetic acid
HOBt = l-hydroxy benzotriazole hydrate
i-PrOH= 2-propanol
i-Pr2NEt = diisopropylethylamine
2 o Me = methyl
MeOH = methanol
NMP = 1-methyl-2-pyrrolidinone
NMR = nuclear m~ tic resonance
PCTLC = preparative centrifugal thin layer
2 5 chromatography
PEI = polyethyle~ le
Ph = phenyl
RT = retention time
TFA = trifluoroacetic acid
3 0 THF = tetrahydrofuran
TLC = thin layer chromatography
The compounds of the present invention can be prepared
readily according to the following reaction schemes and examples, or
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WO 97/17967' PCT/US96/18321
modifications thereof, using readily available starting materials,
reagents and conventional synthesis procedures. In these reactions, it is
also possib~e to make use of variants which are themselves known to
those of ordinary skill in this art, but are not mentioned in greater
5 detail. IJnless otherwise indicated, all variables are as defined above.
The preparation of 4,4-disubstituted piperidines 4 was
accomplished via spiro annulation of substituted acetonitrile derivatives
1 with N-Boc bischloroethyl amine under basic conditions, typically
NaH in DMF at 80 ~C in good yields, Scheme 1. The resulting 4-cyano
10 4-phenylpiperidines 3 were either: 1) N-Boc deprotected with HCl-
EtOAc or 2) the nitrile hydrolyzed to the corresponding carboxylic acid
with concomitant N-Boc deprotection with concentrated aqueous HCl
and converted to esters and ~mitles via simple coupling reactions to
targeted derivatives 4. In some instances the amino acids derived from
15 treatment of 3 with concentrated aqueous HCl required N-Boc
protection prior to the coupling reactions to limit oligomerization.
Scheme 1.
Cl CN
C~ + NBOC THF/DMF e~NBOC
R1 Cl 2 60~C R1 3
(R2=CN) ~2
C~NBOC Q{~NH-HCI
R1 2
(R =CO2R Or CONH2) 4
3 1 ) COnC aq HCI
2) ROH/tlCI(9~
Or ROH/BOPCI
Or NH3/BOPCI
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- 26 -
The preparation of 3,4-disubstituted piperidines 8 and 10
was accomplished in a simple two step fashion, Scheme 2. The vinyl
triflate 6, derived from N-Boc 3-carboxyethyl 4-piperidinone was
coupled under Pd catalysis to aryl boronic acids and aryl st~nn~n~s in
, excellent yields. The resulting a,~-unsaturated esters 7 were reduced
with hydrogen and Pd-C providing the (t)-cis enantiomeric pair 8. At
this stage several pathways were pursued: (1) the (+)-cis racemate 8 was
deprotected yielding 9, (2) the (i)-cis enantiomeric pair 8 was
converted to (+)-trans 10 via epimerization to the diequitorial isomer
10 wi~ the use of sodium ethoxide, then the resulting 10 (+)-trans was
deprotected providing 11, (3) the mixture (+)-cis enantiomers 8 was
separated using normal phase chiral HPLC providing 8a (-)-cis and 8b
(+)-cis and 8a was then deprotected providing 9a.
Scheme 2.
, ~ CO2CH2CH3 1~ 31 CO2CH2CH3
TfO~ K PO ~
B(O H)2 + ~ 4 , ~ NBOC
or NR THFlreflux -
SnMe3 or
- - Pd(OAc)2 7
5a Ar(BOH2) 6 R=BOC NMP/-15-0 C
5b .ArSnMe3
H2 l~jl CO2CH2CH3 ~ CO2CH2CH3
10 % Pd-C ~ NaOEt ~
7 EtOH . ~1 EtOH ~ ~ NR
Chiralcel OD R=BOC
HCI ~ 8 R=BOC 8a (-)-cis 10 (+)-trans R=BOC
EtOAc ¦~ g R=H-Hcl 8b (+)-cis HCI
R=H-HCI 11 R=H-HCI EtOAc
9a (-)-cis
9b (+)-cis
The commercially available amino alcohol 12 was
converted to the corresponding chloride 13 via treatment with thionyl
CA 02235370 1998-04-20
W O 97117967 PCT~US96118321.
chloride, Scheme 3. The chloride 13 was displaced by aniline in
toluene at 180 ~C providing anilinoethylbenzyl amine 14. Treatment of
14 with 2-chloroacrylonitrile in DMF provided the desired cyano
piperazine 15. N-benzyl deprotection was accomplished using ACE-CI
~, in dichloroethane followed by treatment wi~ methanol supplying the
cyano piperazine 16. Piperazines 15 and 16 were separated easily
tili7.ing chiral HPLC technology and each enantiomer was evaluated
individually, 15a and 15b, and 16a and 16b, respectively.
Scheme 3
~~ CHCI3 ~~ H ~HCI ~NH2
12 13 Toluene, 130~C
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- 28 -
=( N CN ~Cl 1 C813CoHC2CH2CI~
~NHHCl .~N~ + ~=O 2. MeOH, r.t. ~N~CN
N~ DMF, (i-Pr)2NEt ~13 Cl lN6 . HCI
14 ~ Chiralcel OD ~
~N~,CN ~N~CN
15a~l 15C
¢~ ~
N C N Chiralcel OD ~ ~ +
16 16a 16b
rj Preferred compounds of the present invention are shown
below im Table 1. The compounds of the present invention can be
prepared by one of ordinary skill in the art following the schemes and
examples set forth herein, substituting the a~lo~-iate readily available
starting materials.
lCI
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- 29 -
TABLE 1
R~r~R2
~A~,~,~ R3
R1 R2 R3 A R2/R3
4a H H H C NA
4b H CO2CH 3 H C NA
4c H CO2CH2CH3 H C NA
4d H C02(CH2)3CH3 H C NA
4e H CO2cyclohexyl H C NA
4f H CN H C NA
4g H CONH2 H C NA
4h H CO2H H C NA
4i CH3 CN H C NA
4; Cl C N H C NA
4k Cl CO2CH3 H C NA
41 CF3 C N H C NA
4m OCH3 C N H C NA
4n CN CN H C NA
CONH2 C N H C NA
9 H H CO2CH2CH3 C (i)-cis
11 H H CO2CH2CH3 C (+)-trans
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- 30 --
gb H H CO2CH2CI 13 C (+)-cis
9a H H CO2CH2CH3 C (-)-cis
1 6a H - C N N isomer A
16b H - C N N isomer B
In addition to the compounds disclosed above in Table 1,
4-cyano4-(2-pyridyl)piperidine was also prepared.
The following examples are provided to further define the
invention without, however, limiting the invention to the particulars of
these examples.
EXAMPLE 1
1~ Bis(2-c:hloroethyl)-N-(l~1-dimethylethoxy)carbonyl amine (2)
A solution of N-(2,2'-bischloro)diethyl amine (23.0 g,
0.130 mol) and di-tert-butyl dicarbonate (28.8 g, 0.130 mol) in CH2CI,~
(150 mL) was treated with N,N-diisopropylethylamine (22.52 ml, 0.720
mol) at room temperature (1.5 h). The solvent was removed in vacuo
and the residue was triturated with ether (300 ml). The ether solution
was collected and concentrated in vac~lo affording N42,2'-bischloro)-
diethyl-N-(1,1-dimethylethoxy)carbonyl amine as a clear oil.
lH NMR (CDCl3, 400 MHz) d 3.65 (m, 8H), 1.52 (s, 9H)
FABLRMS (3:1 mixture of dithiothreitol and
dithioerythritol in MeOH) m/e 242 g/mole (M++ H, C25H29N205SCl -
242.2 gJmole.)
EXAMPLE 2
2';
4-Cyano-N-( 1 ~1 -dimethylethoxy)carbonyl-4-(2-methylphenyl)piperidine
A solution of bis(2-chloroethyl)-N-(1,1-
dimethylethoxy)carbonyl amine (1.438 g, 5.94 mmol) and 2-
3 0 methylphenyl acetonitrile (600 mg, 3.96 mmol) in a 4:1 mixture of
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- 31 -
THF/DMF (15 mL) was treated with NaH (357.9 mg, 8.7 mmol) at 60
~C (3 d). The solvent was removed in vacuo, the residue dissolved in
EtOAc (200 ml) and washed with saturated NaHCO3 (50 ml), H20 (2 x
50ml), and saturated aqueous NaCI (50 ml), dried (Na2S04) and
5 concentrated in vacuo. PCTLC (SiO2, 6mm, 100% hexane) afforded 4-
cyano-. \7-(1,1 -dimethyletho~cy)carbonyl-4-(2-methylphenyl) piperidine
as a yellow/orange oil.
1H NMR (CDCl3, 400 MHz) d 7.25 (m,4H), 4.28 (br s,
2H), 3.28 (br t, 2H), 2.65 (s, 3H), 2.32 (d, 2H, J = 13.0 Hz), 2.32 (dt
1~~ 2H, J = 4.1, 13.0 Hz), 1.48 (s, 9H).
HPLC (Vydac; C18; diameter = 4.6 mm; length = 15 cm;
gradient = CH3CN [0.1% TFA] - H20 [0.1% TFA], S~o - 95%, 95 - 5%
over 20 min. 1.5 ml/min flow rate; RT = 11.730 min; focus = 215 nm;
75% pure.
11~
EXAMPLE 3
4-Cyano-4-(2-methylphenyl)piperidine hydrochloride (4i)
2 () A solution of EtOAc saturated with HCl (200 ml) was
added to 4-cyano-N-(I,l-dimethylethoxy)carbonyl-4-(2-
methylphenyl)piperidine (31 mg, 0.097 mmol). The resulting mixt~lre
was allowed to react for 1 hour at room temperature. The EtOAc was
removed in vacuo affording 4-cyano4-(2-methylphenyl)piperidine
2 '; hydrochloride (4i) as a white solid.
lH NMR (CD30D, 400 MHz) d 7.37 (m, lH), 7.32 (m,
3H), 3.64 (dd, 2H, J = 2.2, 11.4 Hz), 3.46 (t, 2H, J = 13.5 Hz), 2.64
(m, 2H'I, 2.65 (s, 3H), 2.28 (t d, 2H, J = 3.7 Hz, 13.5 Hz).
FABLRMS (3:1 mixture of dithiothreitol and
3 0 dithioerythritol in MeOH) m/e 201 g/mole (M++ H, C13H16N2 = 201.3
g/mole.)
HPLC (Vydac; C18; ~ meter = 4.6 mm; length =15 cm;
gradienlt = CH3CN [0.1% TFA] - H20 [0.1% TFAI, 5% - 95%, 95 5%
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-- 32 --
over 20 min. 1.5 ml/min flow rate; RT = 5.82 min; focus = 215 nm;
100% pure.
Anal. Calcd for Cl3Hl6N2. HCl and 0.30 H20 and 0.25
CH2C12: C = 60.42, H = 6.93, N = 10.64. Found: C = 60.37, H = 6.83,
5 N= 1l.09.
EXAMPLE 4
4-(2-Chlorophenyl)-4-cyano-N-(I .1 -dimethylethoxycarbonyl)piperidine
A solution of bis(2-chloroethyl)-N-(l,l-
dimethylethoxy)carbonyl amine (9.298 g, 38.4 mmol) and 2-
chlorophenylacetonitrile (5.0 g, 32.0 mmol) in a 4:1 mixture of THF/
DMF (15 mL) was treated with NaH (2.82 g, 70.4 mmol) at 60 ~C (7
1 5 d). The solvent was removed in vaclw, the residue dissolved in EtOAc
(200 ml) and washed with saturated NaHCO3 (50 ml), H20 (2 x 50ml),
and saturated aqueous NaCI (50 ml), dried (Na2SO4) and concentrated
in vac~o. Trituration of the residue with 100% MeOH afforded 4-(2-
chlorophenyl)-4-cyano-N-(1,1-dimethylethoxy) carbonyl piperidine as a
2 l~ white solid.
1H NMR (CD30D, 400 MHz) d 7.53 (m, 2H), d 7.41 (m,
2H), 4.28 (br dd, 2H, J = 13.4 Hz), 3.26 (m, 2H), 2.52 (dd, 2H, J = 2.2,
11.2 H;z), 2.03 (dt, 2H, J = 4.0, 9.2 Hz), 2.03 (s, 9H).
FABLRMS (3:1 mixture of dithiothreitol and
2 5 dithioel~thritol in MeOH) m/e 321 g/mole (M++ H, C17H21N202CI =
320.8 glmole.)
HPLC (Vydac; C18; diameter = 4.6 mm; leng~ = 15 cm;
gradient = CH3CN [0.1% TFA] - H20 [0.1% TFA], 5% - 95%, 95 - 5%
over 20 min. 1.5 ml/min flow rate; RT = 11.70 min; focus = 215 nm;
3 ~ 97.4% pure.
EXAMPLE 5
4-(2-Chlorophenyl)-4-cyanopiperidine hydrochloride (4j)
3c;
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A solution of EtOAc saturated with HCI (200 ml) was
added l:o 4-(2-chlorophenyl)-4-cyano-N-(l,l-
dimethylethoxycarbonyl)piperidine, (880 mg, 2.74 mmol). The
resultinLg mixture was allowed to react for 1 hour at room temperature.
, The EtOAc was removed in vacuo affording 4-(2-Chlorophenyl)4-
cyanopiperidine hydrochloride (4j) as a white solid.
lH NMR (CD30D, 400 MHz) d 7.53 (dd, lH, J = 2.0, 4.3
Hz), 7.:5 (dd, lH, J = 2.0, 5.3 Hz), 7.40 (ddd, 2H, J = 2.0, 6.0, 7.9 Hz),
3.14 (dldd, 4H, J = 2.2, 10.8, 12.4 Hz), 2.51 (dd, 2H, J = 2.2, 13.5 Hz),
10 2.00 (dt, 2H, J = 3.4, 13.5 Hz).
FABLRMS (3:1 mixture of dithiothreitol and
dithioelythritol in MeOH) rnJe 221 ~/mole (M++ H, C13H16N2 =
220.7 ~Jmole.)
HPLC (Vydac; C18; diameter - 4.6 mm; length =1 5 cm;
15 gradient = CH3CN [0.1% TFA] - H20 [0.1% TFA], 5% - 95%, 95 - 5
over 20 min. 1.5 ml/min flow rate; RT = 5.744 min; focus = 215 nm;
99.04% pure.
Anal. Calcd for C12H13N2Cl. HCI and 0.60 H2O: C =
62.26,H=6.18,N= 12.10. Found:C=62.29,H=5.69,N= 11.71.
2()
EXAMPLE 6
N-Benzyl-N-(2-chloroethyl)arnine hydrochloride (13)
2 ', A solution of N-benzyl-2-aminoethanol (12, 79.05 g, 0.523
mole) in chlorofolm (400 mL) was treated with thionyl chloride (80
mL, 1.09 mole) at room temperature. The resulting mixture was heated
to reflu ~ (6 h). The solvent was removed in vacuo, the residue
dissloved in hot ethanol (1.3 L) and cooled overnight. The solids were
3 0 collected via suction f1ltration and dried in vacuo at 65~C (24 h) to
afford t]he title compound 13 as white plates.
EXAMPLE 7
35 N-Benzyl-N'-phenyl-1.2-~ rninoethane hydrochloride (14)
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A solution of 13 (8.618 g, 42.0 mmol) and ~niline (11.75
g, 126 mmol) in toluene (40 mL) was heated to 130~C (1.5 h). The
mixture was triturated with dichloromethane (200 mL) and filtered.
Recryst~11i7-~tion from ethanol (220 mI,) afforded the title compound
14 as white crystals.
lH NMR (DMSO, 400 MHz) ~ 7.59 (dd, 2H, J = 1.93, 7.64
Hz), 7.42 (m, 3H), 7.10 (t, 2H, 7.98 Hz), 6.59 (m, 3H), 5.95 (s, lH),
4.17 (s. 2H), 3.41 (t, 2H, J = 6.38 Hz), 3.04 (t, 2H, J = 6.55 Hz).
FABLRMS m/e 241 g/mole (M++H, Cl6H2"N2=241
g/mole )
HPLC (Vydac; C18; diameter = 4.6 mm; length = 150 mm;
gradient = H2O [0.1% H3PO4] - CH3CN, 95% - 5%, 5% - 95%, over 16
minutes, 2 ml/min flow rate) RT = 7.371 min; focus = 215 nm; 99.3%
1 ', pure.
EXAMPLE 8
4-Benzyl-2-cyano- 1 -phenylpiperazine (15)
2()
A solution of 14 (5.07 g, 17.0 mmol) and
diisopropylethylamine (4.97 g, 38.4 mmol) in DMF (40 mL) was
treated with a solution of 2-cyanoacrylonitrile (2.30 g, 26.3 mmol) in
DMF (40 mL) at room temperature (14 d). The solvent was removed
2 'i in vacuo and the residue dissolved in dichloromethane alld washed with
water, brine, and dried (Na2SO4). Concentrated in vac~o, the residue
was reclystallized from e~anol to afford 15 as white crystals.
IH NMR (CDCl3, 400 MHz) ~ 7.34 (m, 7H), 7.00 (t, 3H, J
= 8.31 Hz), 4.53 (s, lH), 3.72 (d, lH, J = 13.26 Hz), 3.59 (d, lH, 13.43
3C Hz), 3.40 (d, lH, J = 11.91 Hz), 3.28 (td, lH, J = 3.13, 11.59 Hz), 3.13
(dt, lH, J = 2.26, 11.42 Hz), 3.01 (dq, lH, J = 2.43, 11.08 Hz), 2.53
(dd, lH, J = 3.19, 11.41 Hz), 2.39 (td, lH, J = 3.36, 11.25 Hz).
FABLRMS mte 278 g/mole (M++H, Cl8H20N3 = 278
g/mole. )
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HPLC (Vydac; C18; diameter = 4.6 mm; length = 150 mm;
gradient = H2O [0.1% H3PO4] - CH3CN, 95% - 5%, 5% - 95%, over 16
minllte~" 2 ml/min flow rate) RT = 7.936 min; focus = 215 nm; 100%
pure.
S Anal. Calcd for Cl8HIgN3 ~ 0.15 H2O: C = 77.19, H = 6.95
N = 15 00. Found: C = 77.14, H = 6.90, N = 14.88.
EXAMPLE 9
2-Cyano-1-phenylpiperazine (16)
A solution of 15 (3.40 g, 12.25 mmol) in 1,2-
dichloroethane (60 mL) was treated with alpha-chloroethyl-
chloroformate (3.78 g, 26.44 mmol) at 0~C. The reaction mixtllre was
1c, heated to 100~C (8 h). The solvent was removed in vacuo and the
residue dissolved in methanol (80 mL) at room temperature
(overnight). The solvent was removed in vacllo and the residue was
recrystallized from ethanol (160 mL) to afford crystalline 16.
IH NMR (DMSO, 400 MHz) ~ 9.74 (s, lH), 7.36 (m, 2H),
7.12(d,2H,J=7.89Hz),7.()3(t, lH,J=7.31 Hz),5.48(d, lH,J=
4.03 Hz), 3.75 (d, 2H, J = 13.09 Hz), 3.43 (dd, lH, J = 4.53, 13.43 Hz),
3.38 (d, 2H, J = 10.74 Hz), 3.07 (m, 2H).
FABLRMS m/e 188 g/mole (M++H, Cl,HI4N3= 188
g/mole.')
HPLC (Vydac; C18; diameter = 4.6 mm; length = 150 mm;
gradient = H2O [0.1% H3PO4] - CH3CN, 95% - 5%, 5% - 95%, over 16
minlltes, 2 ml/min flow rate) RT = 3.563 min; focus = 215 nm; 100%
pure.
Anal. Calcd for ClIHl3N3 ~ 1.00 HCl: C = 59.06, H = 6.31,
N = 18.'79. Found: C = 59.27, H = 6.37, N = 18.42.
EXAMPLE 10
Enantioxners of 15 (15~ and 1 5h).
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The enantiomeric mixture was separated lltili7ing norrnal
phase chiral HPLC (Chiralcel OD~ 2.0 cm x 25 cm, 65 hexanes/35 2-
propanol/0.2 diethyl amine, 2.5 mL/min). Each enantiomer possessed
identical spectroscopic properties but opposite signs of optical rotation.
~, Chiral purity was assessed lltili7in~ similar HPLC conditions (Chiralcel
OD, 4.6 mm x 25 cm, 65 hexanes/35 2-propanol/0.2 die~yl amine, 0.7
mL/min).
EXAMPLE 1 l
1()
Fn~ntiomers of 16 (16~ and 16h).
The enantiomeric mixture was separated ~ ing normal
phase chiral HPLC (Chiralcel OD, 2.0 cm x 25 cm, 65 hexanes/35 2-
15 propanol/0.2 diethyl amine, 2.5 mL/min). Each enantiomer possessedidentica,l spectroscopic properties but opposite signs of optical rotation.
Chiral purity was assessed lltili~ing similar HPLC conditions (Chiralcel
OD, 4.6 mm x 25 cm, 65 hexanes/35 2-propanol/0.2 diethyl amine, 0.7
mL/min).
2~3
EXAMPLE 12
As a specific embodiment of an oral composition, 100 mg
of the compound of Example 3 is formulated with sufficient finely
2 5 divided lactose to provide a total amount of 580 to 590 mg to fill a size
O hard gel capsule.
EXAMPLE 13
3 C Screening assay: Alpha la Adrenergic Receptor Bin¢lin~
Membranes prepared from the stably transfected hllm~n
alpha la cell line (ATCC CRL 11140) were used to identify compounds
that bind to the hllm~n alpha la adrenergic receptor. These competition
binding reactions (total volume = 200 ~l) contained 50 mM Tris-HCl
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pH. 7.4, 5 mM EDTA, 150 mM NaCI, 1()0 pM [125 I]-HEAT,
membr;mes prepared from the alpha la cell line and increasing amounts
of unlalbeled ligand. Reactions were incubated at room temperature for
one hour with ~h~king Reactions were filtered onto Wh~tm~n GF/C
t, glass fiber filters with a Inotec 96 well cell harvester. Pilters 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 ~ 500 nM.
EXAMPLE 14
Selective Bindin,~ assays
Membranes prepared from stably transfected hllm~n alpha
ld and alpha lb cell lines (ATCC CRL 11 138 and CRL 11139,
15 respectively) were used to identify compounds that selectively bind to
the hllm~n alpha la adrenergic receptor. These competition binding
reactions (total volume = 20() ,ul) 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
2 C expressiion plasmid and increasing amounts of unlabeled ligand.
Reactions were incubated at room temperature for one hour with
sh~kin~. Reactions were filtered onto Whatnn~n 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 15
EXEMPLARY COIJNTERSCREENS
3 o 1. Assay Title: Dopamine D2, D3, D4 in vitro screen
Objective of the Assay:
The objective of this assay is to elimin~te agents which
specifically affect binding of [3H] spiperone to cells expressing hl-m~n
dopamine receptors D2, D3 or D4.
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Method;
Modified from VanTol et al (1991); Nature (Vol 350) Pg
610-613.
Frozen pellets cont~ining specific dopamine receptor
5 subtypes stably expressed in clonal cell lines are Iysed in 2 ml Iysing
buffer (lOmM Tris-HCV5mM Mg, pH 7.4). Pellets obtained after
centrifilging these membranes (lS' at 24,450 rpm) are resuspended in
50mM Tris-HCI pH 7.4 cont~ining EDTA, MgCI[2~, KCI, NaCI, CaCI[2]
and ascorbate to give a 1 Mg/mL suspension. The assay is initiated by
1 ~ ~dding 50-75 ,ug membranes in a total volume of 500 ,ul con~ining 0.2
nM [3H]-spiperone. Non-specific binding is defined using 10 ,uM
apomorphine. The assay is termin~ted after a 2 hour incubation at
room temperature by rapid filtration over GF/B filters presoaked in
0.3% PEI, using 50mM Tris-HCl pH 7.4.
l''j
2. Assay Title: Serotonin SHTla
Objective of the Assay
The objective of this assay is to elimin~te agents which
specifically affect binding to cloned human SHTla receptor
2()
Method:
Modified from Schelegel and Peroutka Biochemical
Pharmacology 35: 1943- 1949 (1986).
mm~ n cells expressing cloned hllm~n SHTla receptors
2 'j 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 38,000Xg for 30'. The binding assay contains 0.25 nM [3H]8-
OH-DPAT (8-hydroxy-2-dipropylamino- 1,2,3,4-tetrahydronaphthalene )
3 CI in 50 mM Tris-HCl, 4 mM CaCl2 and lmg/ml ascorbate. Non-specific
binding is defined using 10 ,uM propranolol. The assay is tern~in~ted
after a 11 hour incubation at room temperature by rapid filtration over
GF/Cfilters
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EXAMPLE 16
EXEMPLARY FUNCTIONAL ASSAYS
In order to confirm the specificity of compounds for the
hllm~n alpha la adrenergic receptor and to define the biological activity
of the compounds, the following functional tests may be performed:
1. In vitro Rat, Dog and Human Prostate and Dog Urethra
Taconic Farms Sprague-Dawley male rats, weighing 250-
1~ 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.
2 0 The tissue is placed in a Petri dish cont~ining oxygenated
Krebs solution [NaCl, 118 mM; KCI, 4.7 mM; CaCl2, 2.5 mM;
KH2PC)4, 1.2 mM; MgSO4, 1.2 mM; NaHCO3, 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
2 ', with 4-0 surgical silk and placed in a 5 ml jacketed tissue bath
cont~in-in~ Krebs buffer at 37~C, bubbled with 5% C02/95% O2. The
tissues are connected to a St~th~m-Gould force transducer; 1 gram (rat,
hllm~n) or 1.5 gram (dog) of tension is applied and the tissues are
allowedl to equilibrate for one hour. Contractions are recorded on a
3 () Hewletli-Packard 7700 series strip chart recorder.
After a single priming dose of 3 ~uM (for rat), 10 ~M (for
dog) and 20 ~M (for hllm~n) of phenylephrine, a cumulative
concentration response curve to an agonist is generated; the tissues are
washed every 10 minutes for one hour. Vehicle or antagonist is added
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to the bath and allowed to incubate for one hour, then another
cumulative concentration response curve to the agonist is generated.
EC50 values are calculated for each group using GraphPad
Inplot software. pA2 (-log Kb) values were obtained from Schild plot
5 when three or more concentrations were tested. When less than three
concentrations of antagonist are tested, Kb values are calculated
according to the following formula Kb = rBl,
x-l
where x is the ratio of EC50 of agonist in the presence and absence of
10 antagonist and [B3 is the antagonist concentration.
2. Measurement of ~tra-Urethral Pressure in Anesthetized Dogs
PURPOSE: Benign prostatic hyperplasia causes a decreased
urine flow rate that may be produced by both passive physical
15 obstruction of the prostatic urethra from increased prostate mass as well
as active obstruction due to prostatic contraction. Alpha adrenergic
receptorantagonists 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 l
2 0 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
concolllila,lt changes in the vasculature. The following model is used to
2 5 measure adrenergically mediated changes in intra-urethral pressure and
arterial pressure in anesthetized dogs in order to evaluate the efficacy
and potency of selective alpha adrenergic receptor antagonists. The
goals are to: 1) identify the alpha 1 receptor subtypes responsible for
prostati~/urethral contraction and vascular responses, and 2) use this
3 o model to evaluate novel selective alpha adrenergic antagonists. Novel
and standard alpha adrenergic antagonists may be ev~ ted in this
m~nner.
METHODS: Male mongrel dogs (7-12 kg) are used in
this study. The dogs are anesthetized with pentobarbital sodium (35
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mglkg, i.v. plus 4 mg/kglhr iv infusion). An endotracheal tube is
inserted and the ~nim~l ventilated with room air using a Harvard
instruments positive displacement large ~nim~l ventilator. C~theters
(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 ~lmini.~tration 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 c~nn~ ted so that urine flows freely into beakers. The
1 () dome of the bladder is retracted to facilitate dissection of the proximaland 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 s-uture) 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.
2 o Phenylephrine, .m alpha 1 adrenergic agonist, is
lmini~tered (0.1-100 ug/kg, iv; 0.05 mllkg volume) in order to
construct dose response curves for changes in intra-urethral and arterial
pressure. Following ~11mini.~tration of increasing doses of an alpha
adrenergic antagonist (or vehicle), the effects of phenylephrine on
2 ~, arterial pressure and intra-urethral pressure are re-evaluated. Four or
five phenylephrine dose-response curves are generated in each ~nim~l
(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 farnily
3 C of averaged curves are fit simultaneously (using ALLFIT software
package) with a four paramenter logistic equation constraining ~e
slope, mi~ response, and maximllm 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
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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 c~l-sing 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.
EXAMPLE 17
2 0 Opioid receptor binding assay
3H-Naloxone binds with high affinity to opiate receptors in
brain tissue (Creese and Snyder, J. Pharm Expt. Ther., 194: 205-219,
1975). The potency of a compound to inhibit the specific binding of
this radioligand gives a measure of the affinity for these receptors.
2 5 3H-Naloxone binding in rat brain membranes was performed as
described by Creese and Snyder (J. Pharm. Expt. Ther., 194, 205-219,
1975) using Tris buffer alone or in the presence of 150 mM NaCl.
Naloxone binding for representative examples of the present invention
are 2 6 ~M.
3 0 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 wi~in the scope of the following claims and their equivalents.
