Note: Descriptions are shown in the official language in which they were submitted.
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TITLE OF THl~ rNVENTION
ALPHA lb ADRENERGIC RECEPTOR ANTAGONISTS
FIELD OF THE INVENTION:
S This invention relates to certain novel compounds and
derivatives thereof, their synthesis, and their use as selective alpha 1
adrenoceptor antagonists, especially alpha lb selective antagonists.
More particularly, the compounds of the present invention are useful
for treating hypertension.
BACKGROUN~ 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 catechol~min~s,
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
~nim~l and tissue sources. As a result, alpha and beta adrenergic
receptors were further subdivided into al, oc2, r~l, and r~2 subtypes.
Functional differences between ocl and oc2 receptors have been
recognized, and compounds which exhibit selective binding between
these two subtypes have been developed. Blockade of ocl receptors
inhibits vasoconstriction induced by endogenous catecholamines;
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vasodilation may occur in both arteriolar resistance vessels and veins.
The result is a fall in blood pressure because of decreased vascular
resistance. a2 adrenergic receptors play an important role in regulation
of the activity of the sympathetic nervous system, both peripherally and
centrally. Blockade of oc2 adrenergic receptors with selective
antagonists increase sympathetic outflow and potentiate the release of
norepinephrine from nerve endings, leading to activation of al and i31
receptors in the heart and peripheral vasculature with a consequent rise
in blood pressure [B. Hoffman and R.J. Lefkowitz, Adrenergic Receptor
Antagonists, in Goodman & Gilman's The Pharmocological Basis of
Therapeutics (8th ed., 1990)]. Thus, selective ~~1 adrenergic receptor
antagonists have found use in the treatment of hypertension.
Hypertension, or high blood pressure, is a major public
health concern in developed countries, it being common, asymptomatic,
readily detectible and often l~-ling to lethal complications if left
untreated. Patients with hypertension die prematurely, the most
common cause of death is heart disease, with stroke and renal failure
also frequent. [Harrison's Principles of Internal Medicine (12th ed.,
1991)]
Two selective ~~1 adrenergic receptor antagonists (i.e.,
selective for 0~1 versus oc2 receptors) useful as antihypertensives are
prazosin (i.e., 1-(4-amino-6,7-dimethoxy-2-quinazolinyl-4-(2-
furanylcarbonyl)piperazine) and terazosin (i.e., 1-(4-amino-6,7-
dimethoxy-2-quinazolinyl-4-(2-tetrahydrofuroyl)piperazine). In WO
92/0073, the selective ability of the R(+) enantiomer of terazosin to
selectively bind to adrenergic receptors of the alpha 1 subtype was
reported. The al/or~2 selectivity of this compound was disclosed as
being significant because agonist stimulation of the oc2 receptors was
said to inhibit secretion of epinephrine and norepinephrine, while
antagonism of the oc2 receptor was said to increase secretion of these
hormones. Thus, the use of non-selective alpha-adrenergic blockers,
such as phenoxyben7~mine and phentolamine, is limited by their 0~2
adrenergic receptor mediated induction of increased plasma
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catecholamine concentration and the attendant physiological sequelae
~increased heart rate and smooth muscle contraction).
For a general background on the a-adrenergic receptors,
the reader's attention is directed to Robert R. Ruffolo, Jr., a-
S Adrenoreceptors: Molecular Biolo~y. Biochemistry and Pharmacolo~y.(Progress in Basic and Clinical Pharmacolo~y series, Karger, 1991),
wherein the basis of al/a2 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
10 alpha adrenergic receptors), agonist structure-activity relationships,
receptor functions, and therapeutic applications for compounds
exhibiting a-adrenergic receptor affinity was explored.
The cloning, sequencing and expression of alpha receptor
subtypes from ~nim~l tissues has led to the subcl~ification of the al
receptors into ala~ (Lomasney, et al., J. Biol. (:~hem.~ 266:6365-6369
(1991), rat ala; Bruno et al., ~BRC. 179:1485-1490 (1991), hllm~n
ala), alb (Cotecchia, et al., PNAS. ~s5;7159-7163 (1988), hamster alb;
Libert, et al., Science. (1989), dog alb; Ramarao, et al., J. Biol.
Chem.. 267:21936-21945 (1992), hllm~n alb)~ and most recently, in a
20 study using bovine brain, a new alc subtype was proposed (Schwinn, et
al.. J. Biol. Chem.. 265:8183-8189 (1990); Hirasawa et al., BBRC
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
25 Pstl restriction fragment polymorphism in the alC adrenergic receptor
gene; another study suggests that there may even be an alpha 1 d
receptor subtype, see Perez et al., Mol. Pharm., 40:876-883, 1992).
Each al receptor subtype exhibits its own pharmacologic and tissue
specificities. Schwinn and coworkers noted that the cloned bovine oclc
30 receptor exhibited ph~rm~ological properties proposed for the ala
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.
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The differences in the a--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
S with increasing age. The symptoms of the condition include, 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. Recently, it has been determined that
the al adrenergic receptor that mediates hllm~n prostatic smooth
muscle contraction in hllm~n prostate has the pharmacological
properties of the cloned hllm~n alc subtype [Forray, C. et al., Mol.
Pharmacol., 45, 703-708 (1994)].
Effects on blood pressure, on the other hand, are mediated
by binding to subtypes other than the oc1c receptor (i.e., alb, ala) It
has now been found that compounds which selectively bind to the alb
adrenergic receptor sybtype are effective therapeutic agents for treating
cardiovascular disease conditions such as hypertension and congestive
heart failure without side effects associated with non-subtype selective
agents such as Prazosin or Terazosin caused by binding to the alC and
ala receptor sybtypes (e.g., relaxation of urethral smooth muscle).
Typically, identification of active compounds is
accomplished through use of ~nim~l tissues known to be enriched in
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~ns. This results in substantial
wastage of time and effort, particularly where high volume compound
screening programs are employed. There is also the danger that
compounds, which might be highly effective in humans, would be
missed because of their absence of appreciable affinity for the
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heterologous ~nim~l receptors. In this regard, it has been noted that
even single amino acid changes between the sequence 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
the homologous rat receptor. They further showed, in studies with
mllt~nt receptors, that substitution of only two amino acid residues was
both necessary and sufficient to reproduce the rat receptor's antagonist
binding afflnity in the hllm~n receptor. Oksenberg et al., (Nature~
360:161-163, 1992) showed that a single amino-acid difference confers
major pharmacological variation between the human and the rodent 5-
hydroxytryptamine 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
difflculty and unpredictability has resulted in a need for a compound
screen which will identify compounds that will be active in hllm~n~.
These problems were solved by cloning the hllm~n
adrenergic receptor subtypes (i.e., ala, alb and alC) and the use of a
screening assay which enables identification of compounds which
specifically interact with the desired human al adrenergic receptor
subtype [PCT 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 alb
adrenergic receptor and a method for identifying compounds which
bind the hllm~n alb receptor has now made possible the identification
of selective hllm~n alb adrenergic receptor antagonists useful for
treating hypertension. The instant patent disclosure describes novel
compounds which selectively bind to the human alb receptor. These
compounds are further tested for binding to other human alpha 1
receptor subtypes, as well as counterscreened against other types of
receptors, thus defining the speci~lcity of the compounds of the present
invention for the human alb adrenergic receptor.
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Because of their ability to selectively antagonize ~~lb
adrenergic receptors, the compounds of this invention are useful for
reducing blood pressure without causing ancillary effects due to binding
to the ala and alC receptor subtypes (e.g., inducing relaxation of
urethral smooth muscle).
NOMENCLATURE
Recently, a new a1 adrenergic receptor ((x1-AR)
classification scheme similar to that proposed by Ford, et al. [~1_
Adrenoceptor ClassificatioTl: Sharpenin Occam's Razor. Trends in
Pharm. Sci. 1994, 15, 167-170] was adopted at the August, 1994
meeting of the International Union of Ph~rm~cology (IUPHAR) in
Montreal, Canada. The ocl-AR genes formerly known as ala/d~ alb
and alC were renamed CCld, CClb and ala, respectively. This new
n~ming system reflects the correspondence between the proteins
encoded by the ala and alb genes (new IUPHAR nomenclature) and
the receptors characterized by traditional pha~nacological means as
oclA and oc1g, respectively, in the literature. Recombinant receptors
and receptors characterized ph~ rologically in tissues are
distinguished by lowercase and uppercase subscripts, respectively.
The above discussion contained in the Background section
used the former classification scheme (i.e., ~~la/d, alb and ~Clc);
however, hereinafter, the new classification scheme will be utilized (i.e.,
~Cld, OClb and CCla)~ Thus, what was formerly referred to as the oclc
receptor (and alc receptor antagonists) will hereinafter be referred to
tili7in~ the new nomenclature as the ala receptor (and ala receptor
antagonists).
SUMMARY OF THE INVENTION
The present invention provides compounds for the
treatment of hypertension. The compounds selectively antagonize the
human alpha 1 adrenergic receptors. Specifically, the compounds of the
present invention selectively bind to the alpha lb adrenergic receptor at
submicromolar concentrations while exhibiting at least five fold lower
af~mity for the alpha ld and alpha la human adrenergic receptors and
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many other G-protein coupled receptors (e.g., serotonin). The
compounds of the present invention have the structure:
1R R2
CH~ N ~A
CH~O/~ N
NH2
wherein A is selected from CR3R8, N-R3, O, S or SO2;
Rl and R2 are each independently selected from hydrogen, CN,
C(o)R4, CH20R4, CH2NR4RS, CoNR4R5, C02R4 or S02R4,
provided that Rl and R2 are not both hydrogen;
R3 is selected from hydrogen, CN, oR6, NR6R7, C(o)R4, C02R4,
CoNR4R5, Het or (CH2)mAr where Ar is unsubstituted, mono-, di- or
tri-substituted Ar and where the substituents on Ar are independently
selected from oR4, NR4RS, halogen, C1 8 alkyl, CF3, nitro or CN;
15 R4 and RS are each independently selected from hydrogen, CH2CF3,
C1 8 alkyl, C3-8 cycloaLkyl, Het or (CH2)mAr, where Ar is
unsubstituted, mono-, di- or tri-substituted Ar and where the
substituents on Ar are independently selected from oR6, halogen,
NR6R7, C1 8 alkyl, CF3 or C3-8 cycloaLkyl;
R6 and R7 are each independently selected from hydrogen, CH2CF3,
C1 8 alkyl or C3-8 cycloalkyl;
R8 is selected from hydrogen, C1 8 alkyl, CF3, C3-8 cycloalkyl, Het or
25 (CH2)mAr where Ar is unsubstituted, mono-, di- or tri-substituted Ar
and where the substituents on Ar are independently selected from oR4,
NR4R5, halogen, C1 8 alkyl, CF3, nitro or CN;
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Ar is selected from phenyl, naphthyl, furanyl, thiazolyl, pyrrolyl,
thienyl, 2-, 3- or 4-pyridyl, or chromanyl;
Het is an unsubstituted, mono- or di-substituted heterocyclic ring
5 selected from tetrahydrofuranyl, pyrrolidinyl, piperidinyl, piperazinyl
or morpholinyl, where the substituents on Het are independently
selected from hydroxyl, C1 8 aLkyl, CF3, halogen, CN, nitro,
Cl-4 alkoxy, arnino or C02-C1-4 alkyl;
10 m is an integer of from zero to three; and
n is an integer of from one to three;
and the pharmaceutically acceptable salts thereof.
In one embodiment of the invention is the compound
selected from
CH30~f ~A '
NH2 NH2
wherein Rl and R2 are each independently selected from CN, C(o)R4,
CH20R4, CH2NR4RS, CoNR4RS, Co2R4 or So2R4;
R4 is selected from hydrogen, CH2CF3, C1 6 alkyl, C3-6 cycloaIkyl,
20 Het or (CH2)mAr where Ar is unsubstituted, mono-, di- or tri-
substituted Ar and the substituents on Ar are independently selected
from oR6, halogen, NR6R7, Cl 5 alkyl, CF3 or C3-8 cycloalkyl; and
RS is selected from hydrogen, CH2CF3, C1 8 alkyl or C3-8 cycloalkyl;
25 where all other variables are as defined above;
and the pharmaceutically acceptable salts thereof.
In a class of the invention are the compounds wherein
A is selected from CR3R8 or N-R3;
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Rl and R2 are each independently selected from CN, CoNR4R5 or
C02R4;
R3 is selected from hydrogen, C(o)R4 or Co2R4;
R4 is selected from Cl-6 alkyl, C3-6 cycloalkyl, tetrahydrofuranyl or
(CH2)mAr where Ar is unsubstituted, mono-, di- or tri-substituted Ar
and the substituents on Ar are independently selected from oR6,
halogen, Cl 4 alkyl or C3-8 cycloalkyl;
R5 is selected from hydrogen, C1 6 alkyl or C3-6 cycloalkyl;
Ar is selected from phenyl, furanyl or chromanyl; and
m is an integer of from zero to two;
where all other variables are as defined above;
and the pharmaceutically acceptable salts thereof.
In a subclass of the invention are the compound of the
formula
CON H R5
~N~ R3
CH30~ ~r 'J
/~N
CH30 T
NH2
wherein R4 is selected from Cl 4 alkyl, benzyl, furanyl,
tetrahydrofuranyl or 4-oxo-chromene; and
R5 is selected from hydrogen, Cl 4 alkyl or C3-6 cycloalkyl; and where
25 all other variables are as defined above;
and the pharmaceutically acceptable salts thereof.
Illustrative of the invention is the compound of the formula
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- 10 -
CoNHR5
~N,R3
CH30~ ~r N~J
C H30~ N
NH2
where all other variables are as defined above;
and the ph~ ceutically acceptable salts thereof.
An illustration of the invention is the compound of the
5 formula
CONHtBu
~ ,R3
CH30~ N~ ~iJ
C H30~ N
NH2
where all other variables are as defined above;
and the ph~ ceutically acceptable salts thereof.
Exemplifying the invention is the compound selected from
(S)-1-(4-Amino-6,7-dimethoxy-2-quinazolinyl)-4-
[(benzyloxy)carbonyl]-3-(1,1-dimethylethylamino)carbonyl piperazine;
(S)- 1 -(4-Amino-6,7-dimethoxy-2-quinazolinyl)-3-( 1, I -
dimethylethylamino)carbonyl piperazine;
1 -(4-amino-6,7-dimethoxy-2-quinazolinyl)-3-( 1 ,1 -dimethyl-
1~ ethylamino)carbonyl-[(tetrahydro-2-furanyl)carbonyl]-piperazine;
(R)- 1 -(4-Amino-6,7-dimethoxy-2-quinazolinyl)-4-[( 1,1 -dimethyl-
ethoxy)carbonyl]-3-(1,1-dimethylethylamino)carbonyl piperazine;
(R)- 1 -(4-Amino-6,7-dimethoxy-2-quinazolinyl)-3 -(1,1 -
dimethylethylamino)carbonyl piperazine; or
4-(4-amino-6,7-dimethoxyquinazolin-2-yl)- 1 -(4-oxo-4H-chromene-2-
carbonyl)-piperazine-2-carboxylic acid tert-butylamide,
and the pharmaceutically acceptable salts thereof.
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In a second subclass of the invention is the compound of the
formula
CoNR4R5
~A
~O~Nq N~J
--o~f
NH2
where all other variables are as defined above;
S and the pharmaceutically acceptable salts thereof.
Illustrating this second subclass of the invention is the
compound wherein
A is selected from CR3R8 or N-R3; where all other variables are as
defined above;
10 and the pharmaceutically acceptable salts thereof.
Exemplifying this second subclass of the invention is the
compound of the formula
CON R4R5
--N
,O~N~N~J
--0~
NH2
wherein R3 is selected from hydrogen, C(o)R4 or C02R4;
Ar is selected from phenyl, furanyl or chromanyl; and
Het is tetrahydrofuranyl; where all other variables are as defined above;
and the pharmaceutically acceptable salts thereof.
An example of the invention is a ph~ ceutical
composition comprising a therapeutically effective amount of any of the
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- 12 -
compounds described above and a ph~rm~ceutically acceptable carrier.
Another 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
S invention is a process for making a ph~rm~ceutical composition
comprising combining any of the compounds described above and a
pharmaceutically acceptable carrier.
Further illustrating the invention is a method of treating
hypertension in a subject in need thereof which comprises ~lministering
10 to the subject a therepeutically effective amount of any of the
compounds or pharmaceutical compositions described above.
Further exemplifying the invention is a method of lowering
blood pressure in a subject in need thereof which comprises
~lministering to a subject in need thereof a therapeutically effective
lS amount of any of the compounds or pharmaceutical composition
described above.
More specifically illustrating the invention is a method of
treating a disease which is susceptible to treatment by selective
antagonism of the alpha lb receptor which comprises ~lministering to a
20 subject in need thereof an amount of any of the compounds or
ph~rm~cuetical compositions described above effective to treat the
disease. Diseases which are susceptible to treatment by selective
antagonism of the alpha lb receptor include, but are not limited to,
hypertension, high intraocular pressure, congestive heart failure and
25 cardiac arrhythmia.
More particularly exemplifying the invention is a method
of treating hypertension in a subject in need thereof which comprises
~1mini.stering to the subject a therapeutically effective amount of a
compound which binds to a human alpha lb adrenergic receptor with a
30 binding affinity greater than five-fold higher than the binding affinity
with which the compound binds to a hllm~n alpha la adrenergic
receptor, a hllm~n alpha ld adrenergic receptor, a human alpha 2a
adrenergic receptor, a human alpha 2b adrenergic receptor and a human
alpha 2c adrenergic receptor. Preferably, the compound utilized in the
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method of treating hypertension binds to the human alpha lb adrenergic
receptor with a binding affinity at least twenty-fold higher than the
binding affinity with which the compound binds to the hllm~n alpha la
adrenergic receptor, the human alpha ld adrenergic receptor, and the
5 human alpha 2a, alpha 2b and alpha 2c adrenergic receptors. More
preferably, the compound utilized in the method of treating
hypertension binds to the human alpha lb adrenergic receptor with a
binding affinity: at least 100-fold higher than the binding affinity with
which the compound binds to the hllm~n alpha la adrenergic receptor,
10 at least 25-fold higher than the binding affinity with which the
compound binds to the hllm~n alpha ld adrenergic receptor, and at least
100-fold higher than the binding affinity with which the compound
binds to the hllm~n alpha 2a, alpha 2b and alpha 2c adrenergic
receptors. Most preferably, the compound utilized in the method of
15 treating hypertension binds to the human alpha lb adrenergic receptor
with a binding affinity: at least 500-fold higher than the binding affinity
with which the compound binds to the hllm~n alpha la adrenergic
receptor, at least 25-fold higher than the binding affinity with which the
compound binds to the human alpha ld adrenergic receptor and at least
20 500-fold higher than the binding affinity with which the compound
binds to the human alpha 2a, alpha 2b and alpha 2c adrenergic
receptors.
More particularly illustrating the invention is the use of any
of the compounds described above in the preparation of a medicament
25 for the treatment and/or prevention of hypertension in a m~mm~l in
need thereof. Still further exemplifying the invention is the use of any
of the compounds described above in the preparation of a medicament
for lowering blood pressure in a m~mm~l in need thereof.
- Another exarnple of the invention is a drug which is useful
30 for treating and/or preventing hypertension in a m~mm~l in need
thereof, the effective ingredient of the said drug being any of the
compounds descibed above. More specifically exemplifying the
invention is a drug which is useful for lowering blood pressure in a
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- 14 -
m~mm~l in need thereof, the effective ingredient of the said drug being
any of the compounds described above.
DETAILED DE~CRIPTION OF THE INVENTION
Representative compounds of the present invention
exhibited high selectivity for the human alpha lb adrenergic receptor.
One implication of this selectivity is that these compounds displayed
selectivity for lowering blood pressure without, for example,
subst~n~i~lly affecting urethral pressure.
Representative compounds of this invention displayed
submicromolar affinity for the hllm~n alpha lb adrenergic receptor
subtype while displaying at least five-fold lower affinity for the human
allpha ld and alpha la adrenergic receptor subtypes, hllm~n alpha 2a,
alpha 2b and alpha 2c adrenergic receptor subtypes and many other G-
lS protein coupled hllm~n receptors (e.g., serotonin). Particular
representative compounds of this invention exhibited nanomolar affinity
for the hllm~n alpha lb adrenergic receptor subtype while displaying at
least 20-fold lower affinity for the hl1m~n alpha ld and alpha la
adrenergic receptor subtypes and the hllm~n alpha 2a, alpha 2b and
alpha 2c adrenergic receptor subtypes and many other G-protein
coupled human receptors (e.g., serotonin). Preferred compounds of
this invention exhibited Ki's for the hllm~n alpha lb adrenergic receptor
which were more than 25-fold lower than for the hllm~n alpha ld
receptor, and more than 100-fold lower than for the human alpha la,
alpha 2a, alpha 2b and alpha 2c adrenergic receptors, while exhibiting
selectivity for the human alpha lb adrenergic receptor over other
human G-protein coupled receptors tested (e.g., serotonin). The most
preferred compounds of the instant invention exhibited Ki's for the
human alpha lb adrenergic receptor which were more than 25-fold
lower than for the human alpha 1 d receptor and more than 500-fold
lower than for the hllm~n alpha la, alpha 2a, alpha 2b and alpha 2c
adrenergic receptors.
The compounds of the present invention are ~lmini~tered
in dosages effective to antagonize the alpha lb receptor where such
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- 15 -
treatment is needed, as in hypertension. For use in medicine, the salts
of the compounds of this invention refer to non-toxic "ph~rm~ceutically
acceptable salts." Other salts may, however, be useful in the
preparation of the compounds according to the invention or of their
pharmaceutically acceptable salts. Suitable pharmaceutically acceptable
salts of the compounds of this invention include acid addition salts which
may, for example, be formed by mixing a solution of the compound
according to the invention with a solution of a pharmaceutically
acceptable acid such as hydrochloric 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
phalmaceutically acceptable salts thereof may include alkali metal salts,
e.g. sodium or potassium salts; ~lk~line earth metal salts, e.g. calcium or
magnesium salts; and salts formed with suitable organic ligands, e.g.
quaternary ammonium salts. Thus, representative ph~rm~ceutically
acceptable salts include the following:
Acetate, Benzenesulfonate, Benzoate, Bicarbonate,
Bisulfate, Bitartrate, Borate, Bromide, Calcium, Camsylate, Carbonate,
Chloride, Clav~ n~te, Citrate, Dihydrochloride, Edetate, Edisylate,
Estolate, Esylate, Fumarate, Gluceptate, Gluconate, Gl-lt~m~te,
Glycollylars~nil~qte, Hexylresorcinate, Hydrabamine, Hydrobromide,
Hydrochloride, Hydroxynaphthoate, Iodide, Isothionate, Lactate,
Lactobionate, Laurate, Malate, Maleate, Mandelate, Mesylate,
Methylbromide, Methylnitrate, Methylsulfate, Mucate, Napsylate,
Nitrate, N-methylglucamine ammonium salt, Oleate, Oxalate, Pamoate
(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
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
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- 16 -
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
10 - 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
15 organic solvents. ~uch solvates are also encompassed within the scope
of this invention.
The term "alkyl" shall mean straight or branched chain
alkanes of one to eight total carbon atoms, or any number within this
range (i.e., methyl, ethyl, l-propyl, 2-propyl, n-butyl, s-butyl, t-butyl,
20 etc.).
The term "alkenyl" shall mean straight or branched chain
alkenes of two to eight total carbon atoms, or any number within this
range.
The term "aryl" as used herein, except where otherwise
2~ specifically defined, refers to unsubstituted, mono- or poly-substituted
aromatic groups such as phenyl or naphthyl.
The term "cycloaLkyl" shall mean cyclic rings of alkanes of
three to eight total carbon atoms (i.e., cyclopropyl, cyclobutyl,
cyclopentyl, cyclohexyl, cycloheptyl or cyclooctyl).
Whenever the term "alkyl" or "aryl" or either of their
prefix roots appear in a name of a substituent (e.g., araLkoxyaryloxy) it
shall be interpreted as including those limitations given above for
"alkyl" and "aryl." Designated numbers of carbon atoms (e.g., C1-8)
shall refer independently to the number of carbon atoms in an aLkyl or
CA 02232138 1998-03-16
W O 97111698 PCT~US96/15223
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.
S The term "substituted" shall be deemed to include multiple
degrees of substitution by a named substitutent. The term "poly-
substituted" as used herein shall include di-, tri-, tetra- and penta-
sub~liLuLion by a named substituent.
Where multiple substituent moieties are disclosed or
claimed, the substituted compound can be independently substituted by
one or more of the disclosed or claimed substituent moieties, singly or
plurally.
The term heterocycle or heterocyclic ring, as used herein,
represents an unsubstituted or substituted stable S- to 7-membered
monocyclic ring system which may be saturated or unsaturated, and
which consists of carbon atoms and from one to three heteroatoms
selected from N, O or S, and wherein the nitrogen and sulfur
heteroatoms may optionally be oxidized, and the nitrogen heteroatom
may optionally be quaternized. The heterocyclic ring may be attached
at any heteroatom or carbon atom which results in the creation of a
stable structure. Examples of such heterocyclic groups include, but is
not limited to, piperidinyl, piperazinyl, oxopiperazinyl, oxopiperidinyl,
oxopyrrolidinyl, oxoazepinyl, azepinyl, pyrrolyl, pyrrolidinyl, furanyl,
thienyl, pyrazolyl, pyrazolidinyl, imidazolyl, imidazolinyl,
imidazolidinyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, oxazolyl,
oxazolidinyl, isooxazolyl, isoxazolidinyl, morpholinyl, thiazolyl,
thiazolidinyl, isothiazolyl, thi~ 7olyl, tetrahydropyranyl,
tetrahydrofuranyl, thiamorpholinyl, thiamorpholinyl sulfoxide,
thiamorpholinyl sulfone, and oxadiazolyl. Morpholino is the same as
morpholinyl.
The term "chromanyl," as used herein, refers to the group
CA 02232l38 l998-03-l6
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- 18 -
wherein the dotted line represents either a single or a double bond.
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
S object of treatment, observation or experiment.
The term "therapeutically effective amount" as used
herein means that amount of active compound or ph~rm~ceutical
agent that elicits the biological or medicinal response in a tissue,
system, ~nim~l or human that is being sought by a researcher,
10 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
15 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
~lminictration, or for ~lministration by inh~l~tion or insufflation.
20 Alternatively, the compositions may be presented in a form suitable for
once-weekly or once-monthly a-lministration; 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
25 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
preform~ tion composition cont~ining a homogeneous mixture of a
30 compound of the present invention, or a pharmaceutically acceptable
salt thereof. When referring to these preformulation compositions as
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- 19 -
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 preform~ tion composition is then subdivided
5 into unit dosage forms of the type described above cont~ining 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
10 dosage and an outer dosage component, the latter being in the form of
an envelope over the former. The t~,vo 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
15 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.
The liquid forms in which the novel compositions of the
present invention may be incorporated for ~llmini~tration orally or by
20 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
25 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
30 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
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- 20 -
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
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
10 desirable to protect sensitive or reactive groups on any of the molecules
concerned. This may be achieved by means of conventional protecting
groups, such as those described in Protective Groups in Organic
Chemistry. ed. J.F.W. McOmie, Plenum Press, 1973, and T.W. Greene
& P.G.M. Wuts, Protective Groups in Or~anic Synthesis. John Wiley &
15 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 lb receptor is shown by comparing affinity to membranes
obtained from tranfected cell lines that express the alpha lb receptor
20 and membranes from cell lines or tissues known to express other types
of alpha (e.g., alpha ld, alpha la) or beta adrenergic receptors.
Expression of the cloned hurnan alpha 1 d, alpha lb, and alpha 1 a
receptors and comparison of their binding properties with known
selective antagonists provides a rational way for selection of compounds
25 and discovery of new compounds with predictable pharmacological
activities. Antagonism by these compounds of the hllm~n alpha lb
adrenergic receptor subtype may be functionally demonstrated in
anesthetized ~nim~l~. These compounds may be used to decrease blood
pressure without exhibiting effects on urethral pressure.
The ability of compounds of the present invention to
specifically bind to the alpha lb receptor makes them useful for the
treatment of hypertension. l~e specificity of binding of compounds
showing affinity for the alpha lb receptor is compared against the
binding affinities to other types of alpha or beta adrenergic receptors.
CA 02232138 1998-03-16
W O 97/11698 PCT~US96/15223
The human alpha adrenergic receptor of the lb 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
hereby incorporated by reference. The cloned hllm~n alpha lb
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
provides a rational way for selection of compounds and discovery of
new compounds with predictable pharmacological activities.
Compounds of this invention exhibiting selective hllm~n
alpha lb adrenergic receptor antagonism may further be defined by
counterscreening. This is accomplished according to methods known in
the art using other receptors responsible for me~ tin~; diverse
biological functions. rSee e.g., PCT International 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
amongst the various hllm~n alpha 1 adrenergic receptor subtypes and
which have low afflnity for other receptors, such as the alpha 2
adrenergic receptors, the ~-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 alpha 1 adrenergic receptors.
Furthermore, functional biological tests are used to confirm the effects
of identif1ed compounds as alpha lb 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 con~ining compounds of this invention
as the active ingredient for use in the specif1c antagonism of hllm~n
CA 02232l38 l998-03-l6
W O 97/11698 PCTrUS96/15223
alpha lb 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~tered in
such oral dosage forms as tablets, capsules (each including timed
S release and sustained release formulations), pills, powders, granules,
elixirs, tinctures, solutions, suspensions, syrups and emulsions, or by
injection. Likewise, they may also be ~lmini~tered in intravenous
(both bolus and infusion), intraperitoneal, subcutaneous, topical with
or without occlusion, or intramuscular form, all using forms well
10 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 lb antagonistic agent.
Advantageously, compounds of the present invention
may be a-lmini~tered in a single daily dose, or the total daily dosage
15 may be ~rlmini~tered in divided doses of two, three or four times
daily. Furthermore, compounds for the present invention can be
~lminictered in intranasal forrn via topical use of suitable intranasal
vehicles, or via transdermal routes7 using those forms of transdermal
skin patches well known to those of ordinary skill in that art. To be
20 ~lminictered in the form of a transdermal delivery system, the
dosage ~lmini~tration will, of course, be continuous rather than
intermittent throughout the dosage regimen.
The dosage regimen utili7in~ the compounds of the
present invention is selected in accordance with a variety of factors
25 including type, species, age, weight, sex and medical condition of the
patient; the severity of the condition to be treated; the route of
~lmini~tration; the renal and hepatic function of the patient; and the
particular compound thereof employed. A physician or veterinarian
of ordinary skill can readily deterrnine and prescribe the effective
30 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
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- 23 -
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
S typically ~lmini~tered 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 ~lmini~tration, that is, oral tablets, capsules, elixirs, syrups
and the like, and consistent with conventional pharmaceutical
1 0 practices.
For instance, for oral ~lmini~tration 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
15 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,
20 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 limit~tion, starch, methyl cellulose,
25 agar, bentonite, ~nth~n 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
30 parenteral ~1mini~tration, sterile suspensions and solutions are desired.
~ Isotonic preparations which generally contain suitable preservatives are
employed when intravenous ~lmini~tration is desired.
The compounds of the present invention can also be
?~lmini~tered in the form of liposome delivery systems, such as small
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- 24 -
llnil~mellar vesicles, large unilamellar vesicles and multilamellar
vesicles. Liposomes can be formed from a variety of phospholipids,
such as cholesterol, stearylarnine 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-
10 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,
15 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 ~lmini~tered in
any of the foregoing compositions and according to dosage regimens
20 established in the art whenever specific blockade of the human alpha
lb 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
a-lmini~tration, the compositions are preferably provided in the form of
25 tablets cont~ining 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. An effective
amount of the drug is ordinarily supplied at a dosage level of from
about 0.0002 mg/kg to about 250 mg/kg of body weight per day.
30 Preferably, the range is from about 0.001 to 100 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 ~lmini~tered on a regirnen of
1 to 4 times per day.
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Compounds of this patent disclosure may be used alone
at appropriate dosages defined by routine testing in order to obtain
optimal antagonism of the hllm~n alpha lb adrenergic receptor while
minimi7ing any potential toxicity. In addition, co-~(1mini~tration or
S sequential ~lmini~tration of other agents which alleviate the effects
of hypetension (e.g., ~3-adrenergic blocking agent, diuretic, ACE
inhibitor) is desirable. Thus, in one embodiment, this includes
~flmini~tration of compounds of this invention and a thiazide
diuretic.
The dosages of the alpha lb adrenergic receptor and
diuretic (or ,13 blocker) are adjusted when combined to achieve
desired effects. As those skilled in the art will appreciate, dosages of
the diuretic (or ,13 blocker) and the alpha lb 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 ~lmini~tered 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 "~lmini~tering" is to be
interpreted accordingly.
Abbreviations used in the instant specification, particularly
the Schemes and Examples, are as follows:
Bn = benzyl
Boc or BOC = t-butyloxycarbonyl
BOPCI = bis(2-oxo-3-oxazolidinyl)phosphinic chloride
Cbz or CBZ = benzyloxycarbonyl
Cbz-Cl = benzyloxycarbonyl chloride
DAST= diethylaminosulfurtrifluoride
DEAD = diethylazodicarboxylate
DMF = N,N-dimethylformamide
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- 26 -
EDCI = 1-(3-dimethylaminopropyl)-3-ethylcarbodirnide
hydrochloride
Et = ethyl
Et3N = triethylarnine
EtOAc = ethyl acetate
EtOH = ethanol
FABHRMS = fast atom bombardment high resolution mass
spectroscopy
FABLRMS = fast atom bombardment low resolution mass
spectroscopy
HPLC = high performance liquid chromatography
HOAc = acetic acid
HOBt or HBT = l-hydroxy benzotriazole hydrate
iPr = isopropyl
i-PrOH or IPA = 2-propanol
i-Pr2NEt = diisopropylethylamine
Me = methyl
MeOH = methanol
NMR = nuclear magnetic resonance
PCTLC = preparative centrifugal thin layer
chromatography
Ph = phenyl
RT = retention time
SGC = silica gel chromatography
tBu = tert-butyl
TFA = trifluoroacetic acid
THF = tetrahydrofuran
TLC = thin layer chromatography
The compounds of the present invention can be prepared
readily according to the following reaction schemes and exarnples, 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
CA 02232138 1998-03-16
W O 97/11698 PCT~US96/1~223
those of ordinary skill in this art, but are not mentioned in greater
detail. Unless otherwise indicated, all variables are as defined above.
The commercially available 4-amino-2-chloro-6,7-
dimethoxyquinazoline, 1, allowed ready access to a variety of
S substituted cyclic amino derivatives 2 - 4. Typically thermolysis in a
sealed tube at 90 ~C for 12 - 24 hours provided analogs 2 in good to
excellent yield. In the case where the amino nucleophile was a
piperazine (C = N) bearing a protecting group such as CBZ, 2a,
hydrogenation was facile under standard conditions providing 3a. The
10 synthesis of the corresponding enantiomer was achieved from the
unprotected piperazine where the regiochemistry of addition was easily
dictated by electronic and steric factors of the 2-(~)-CONHtBu group.
The resulting deprotected piperazine 3a was further elaborated via
standard acylation protocols, either EDCI mediated coupling with
15 carboxylic acids 4b or treatment with the appropriate acid chlorides 4a.
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- 28 -
,Cl tR~A,R
2 2 I PA/90 ~,C~, N
R~A~R3 2 NH2
HNJ 2a R1 = H; R2 = (S)-CONHtBu; A = N; R3 =CO2Bn
2b R1 = (S)-CONHtBu; R2 = H; A = N; R3 = CO2tBu
2c R1 = (S)-CO2Me; R2 = H; A, R3 = absent
2d R1 = (S)-C02iPr; R2= H; A, R3 = absent
\for 3b A = N, R3 = H, R2 = (R)-CONHtBu
~,Pr2EtN/lPA/90 C
R2
1 R~ NH 1 ) R3 = ArCO2H or HetCO2H
R3 = CBZo ~N N~J EDCI/HOBt
2a H2/1 0 % Pd-C Tl I q' or
O ~f 2) R3 = ArCOCI or HetCOCI
NH2 iPr2NEtrrHF
3a R1 = H; R2= (S)-CONHtBu
3b R1 = H; R2 = (R)-CONHtBu
--N'
~0~ N~,N~J
~N
4 NH2
4a R1 = H; R2= (S)-CONHtBu; R3= C=0(2-furanyl)
4b R1 = H; R2= (S)-CONHtBu; R3= 2-Carboxy-chromone
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- 29 -
~NH HO~ EDCI, HET
MeO~ N~N~J +
MeO~N H ~
3a NH2 >~N O O
MeO Nq~N
MeO J~ N O
NH2
Receptor binding data for representative compounds of the
present invention in cloned human receptors is shown below in Table 1.
Table 1
CON HtBu
,R1
~O~ Nq~N~J 2a R1 isCO2CH2Ph
'o~N 3a R1is H
4a R1 is C=0(2-furanyl)
NH2
alpha adrenergic receptor subtype binding ~nM)
Compound 1 a 1 b 1 d 2a 2b 2c
2a 463 1.0 29 683 >10,000 715
3a 1193 2.7 1 11 1172 >10,000 736
4a >3675 3.4 97 65% @ 10 uM 23% @ 10 uM 71% @ 10 uM
23% @ 1 uM 16% @ 1 uM 42 % @ 1 uM
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- 30 -
The following examples are provided to further define the
invention without, however, limiting the invention to the particulars of
these examples.
EXAMPLE 1
(S)- 1 -(4-Amino-6,7-dimethoxy-2-quinazolinyl)-4-
r(benzyloxy)carbonyll-3-(1.1-dime~hylethylamino)carbonyl piperazine
A solution of 4-amino-2-chloro-6,7-dimethoxyquinazoline
(0.78 g, 3.2535 mmol) and (S)-4-[(benzyloxy)carbonyl]-3-(1,1-
dimethylethylamino) carbonyl piperazine (see Askin, D. et al.,
Tetrahedron Letters 1994, 35, 673-676) (1.04 g, 3.2535 mmol) in 2-
propanol (6 mL) was heated at 90 ~C (24 h). The solvent was removed
in vacuo and the residue subjected to SGC (SiO2, 40 mm x 240 mm, 0 -
10 % MeOH/CH2C12) which afforded (S)-1-(4-Amino-6,7-dimethoxy-
2-quinazolinyl)-4-[(benzyloxy)carbonyl]-3-(1,1-dimethylethylamino)-
carbonyl piperazine.
lH NMR (CDCl3, 400 MHz) for the major conformer (9:1)
o 7.30 - 7.40 (br m, 5 H), 6.87 (br s, 2 H), 6.39 (br s, I H), 5.48 (br s,
2 H), 5.20 (m, 2 H), 5.18 (br d, 1 H, J = 13 Hz), 4.71 (br s, 1 H), 4.54
(d, I H, J = 11.7 Hz), 4.10 (br s, 1 H), 3.96 (s, 3 H), 3.92 (s, 3 H), 3.31
(br d, 1 H, J = 13 Hz), 3.10 - 3.25 (br m, 2 H), 1.21 (s, 9 H).
FABHRMS 523.2860 (M+~ H, C27H34N605 requires
523.2669)
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 = 8.77 min; focus = 214
nm; 100 % pure.
Anal. Calcd for C27H34N6O5 and 0.1 H20 and 0.25
CH2Cl2: C = 59.98, H = 6.41, N = 15.40. Found: C = 59.95, H = 6.43,
N = 15.01.
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EXAMPLE 2
(S)- 1 -(4-Amino-6,7-dimethoxy-2-quinazolinyl)-
3-(1.1 -dimethylethvlamino)carbonyl piperazine
A solution of (S)-1-(4-Amino-6,7-dimethoxy-2-
quinazolinyl)-4-[(ben~;yloxy)carbonyl] -3-(1,1 -dimethylethylamino)-
carbonyl piperazine (1.11 g, 2.124 mmol) and 10 % Pd-C (111 mg, 10
weight %) in dry EtOH (8 mL) was evacuated under high vacuum and
10 purged to a H2 balloon (14 h). The mix'ture was filtered through Celite
(30 mm x 30 mm), the filter cake washed with EtOH and concentrated
in vacuo. PCTLC (sio2~ 4 mm, 0 - 10 % CH3OH/CH2C12) provided
the title compound.
lH NMR (CDC13, 300 MHz) for the major conformer (9:1)
~ 7.03 (br s, 1 H), 6.87 - 6.96 (br m, 2 H), 5.84 (br s, 2 H), 5.30 (s, 1
H), 4.68 (br dd, 1 H), 4.43 (br d, 1 H), 3.97 (s, 3 H), 3.95 (s, 3 H), 3.35
(dd, 1 H, J = 3.4, 9.1 Hz), 3.10 - 3.30 (br m, 2 H), 3.02 (m, 1 H), 2.95
(m, 1 H), 1.36 (s, 9 H).
FABHRMS 389.2366 (M++ H, ClgH2gN6O3 requires
389.2300949)
HPLC (Vydac; C18; diameter = 4.6 mm; length =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 = 4.73 min; focus = 214
nm; 100 % pure.
Anal. Calcd for ClgH2gN6O3 and 0.3 CH2C12: C = 55.99,
H = 6.96, N = 20.30. Found: C = 56.07, H = 6.96, N = 19.99.
EXAMPLE 3
1 -(4-amino-6,7-dimethoxy-2-quinazolinyl)-3-(1,1 -dimethyl-
ethylamino)carbonvl - r(tetrahvdro-2-furanyl)carbonyll -piperazine
A solution of (S)-1-(4-Amino-6,7-dimethoxy-2-
quinazolinyl)-3-(1,1 -dimethylethylamino)carbonyl piperazine (133.2
mg, 0.3429 mmol) was treated with 2-furoyl chloride in dry 1.0 mL
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THF (14 h). The reaction mixture was concentrated in vacuo and
submitted to PCTLC (SiO2, 2 mm, 0 - 10 % CH30H/CH2C12)
providing the desired amide.
lH NMR (CDC13, 300 MHz) ~ 7.53 (br s, 1 H), 7.15 (br s,
S lH), 6.89 (br s, lH), 6.83 (br s, 1 H), 6.68 (br s, 1 H), 6.52 (d, 1 H, J =1.7 Hz), 5.30 (br s, 2 H), 5.10 - 5.23 (br m, 2 H), 4.62 (br d, 1 H),
4.50 (br d, 1 H), 3.97 (s, 3 H), 3.94 (s, 3 H), 3.18 - 3.50 (br m, 3 H),
1.25 (s, 9 H).
FABLRMS 483 (M++ H, C24H30N6os requires 482.5439)
HPLC (Vydac; C18; diameter = 4.6 mm; length =15 cm;
gradient = CH3CN [0.1 % TFA] - H20 [O.I % TFA], 5 % - 95 %, 95 -
5 % over 20 min. 1.5 ml/min flow rate; RT = 6.57 min; focus = 214
nm; 99.4 % pure.
Anal. Calcd for C24H30N6os and 0.7 CH30H and 0.55
CH2C12: C = 54.97, H = 6.19, N = 15.23. Found: C = 54.96, H = 5.92,
N= 15.07.
EXAMPLE 4
(R)- 1 -(4-Amino-6,7-dimethoxy-2-quinazolinyl)-4-[(1,1 -dimethyl-
ethoxy)carbonyll-3-(1.1-dimethylethvlamino)carbonyl piperazine
A solution of 4-amino-2-chloro-6,7-dimethoxyquinazoline
(94.4 mg, 0.3939 mmol) and (R)-4-[(1,1-dimethylethoxy)carbonyl]-3-
(l,l-dimethylethylamino) carbonyl piperazine (see Askin, D. et al.,
Tetrahedron Letters 1994, 35, 673-676) (112.4 mg, 0.3939 mol) in 2-
propanol (1 mL) was heated at 90 ~C (24 h). The solvent was removed
in vacuo and the residue subjected to PCTLC (SiO2, 2 mm, 0 - 10 %
MeOH/CH2Cl2) which afforded ((R)-1-(4-Amino-6,7-dimethoxy-2-
quinazolinyl)-4-[(1,1 -dimethylethoxy)carbonyll -3-(1,1 -
dimethyle~ylamino)carbonyl piperazine.
lH NMR (CD30D, 300 MHz) ~ 7.37 (br s, 1 H), 6.95 (br
s, 1 H), 5.10 (br s, 1 H), 4.63 (br s, 1 H), 4.20 - 4.50 (brm, 2 H), 3.92
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(s, 3 H), 3.89 (s, 3 H), 3.60 (br m, 1 H), 3.10 - 3.25 (obscured by
CD30D, 2 H), 1.47 (s, 9 H), 1.30 (s, 9 H).
FABLRMS 489 (M++ H, C24H36N605 requires
488.59172)
HPLC (Vydac; C18; diameter = 4.6 rnm; length =15 cm;
gradient = CH3CN [0.1 % TFA] - H2O [0.1 % TFA], 5 % - 95 %, 95 -
5 % over 20 min. 1.5 ml/min flow rate; RT = 8.42 min; focus = 214
nm; 97.3 % pure.
Anal. Calcd for C24H36N605 and 0.8 IPA and 1.7 CHC13:
C = 45.63, H = 6.01, N = 11.36. Found: C = 45.81, H = 5.60, N =
11.12.
EXAMPLE 5
(S)- 1 -(4-Amino-6,7-dimethoxy-2-quinazolinyl)-2-carboxymethyl
pyrrolidine and (S)-1-(4-Amino-6,7-dimethoxy-2-quinazolinyl)-2-
carboxyisopropyl pyrrolidine
A solution of 4-amino-2-chloro-6,7-dimethoxyquinazoline
(92.8 mg, 0.387 mmol), diisopropylethyl amine (50 mg, 0.387), and
(S)-proline methyl ester (50.0 mg, 0.387 mol) in 2-propanol (1 mL)
was heated at 90 ~C (24 h). The solvent was removed in vacuo and the
residue subjected to preparative HPLC Water Delta Prep 4000 (~18,
isocratic 50 % CH3CN [0.1 % TFA] - 50 % H2O [0.1 % TFA]) to
afford (S)-1-(4-Amino-6,7-dimethoxy-2-quinazolinyl)-2-carboxymethyl
pyrrolidine and (S)-1-(4-Amino-6,7-dimethoxy-2-quinazolinyl)-2-
carboxyisopropyl pyrrolidine.
For (S)-1-(4-Amino-6,7-dimethoxy-2-quinazolinyl)-2-carboxymethyl
pyrrolidine:
lH NMR (CD30D, 300 MHz) o 7.55 (br s, 1 H), 7.12 (br
s, 1 H), 4.85 (m, 1 H), 3.98 (s, 3 H), 3.92 (s, 3 H), 3.77 (s, 3 H), 3.72
(obscured by CH3, 2 H), 2.39 (br m, 1 H), 2.18 (br s, 3 H).
FABLRMS 333 (M++ H, C16H20N4O4 requires 332.36)
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HPLC (Vydac; C18; diameter = 4.6 mm; length =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 = 5.69 min; focus = 214
nm; 96 % pure.
Anal. Calcd for C16H20N4o4~ 0.25 ~2HO2F3, and 0.85
H20: C = 45.33, H = 4.72, N = 11.43. Found: C = 45.32, H = 4.49, N
= 11.73.
For (S)-1-(4-Amino-6,7-dimethoxy-2-quinazolinyl)-2-carboxyisopropyl
pyrrolidine:
lH NMR (CD30D, 300 MHz) o 7.56 (br s, 1 H), 7.13 (br
s, 1 H), 5.04 (m, 1 H), 4.80 (m, 1 H), 3.98 (s, 3 H), 3.92 (s, 3 H), 3.73
(br m, 2 H), 2.39 (br m, 1 H), 2.18 (br s, 3 H), 1.29 (d, 1 H, J = 6.1
Hz), 1.20 (d, 1 H, J = 6.1 Hz).
FABLRMS 361 (M++ H, C 1 gH24N4O4 requires 360.42)
HPLC (Vydac; C18; diameter = 4.6 mm; length =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 = 7.09 min; focus = 214
nm; 100 % pure.
Anal. Calcd for C1gH24N4O4, 1.9 C2H02F3, and 0.3
H2O: C = 44.95, H = 4.59, N = 9.62. Found: C = 44.95, H = 4.51, N =
9.84.
EXAMPLE 6
(R)- 1 -(4-Amino-6,7-dimethoxy-2-quinazolinyl)-
3-(1 ~ 1 -dimethvlethylamino)carbonyl piperazine
A solution of 4-amino-2-chloro-6,7-dimethoxyquinazoline
(3.40 g, 14.19 mmol), diisopropylethyl amine (1.84 g, 14.19) and (S)-3-
(1,1-dimethylethylamino) carbonyl piperazine (see Askin, D. et al.,
Tetrahedron Letters 1994, 35, 673-676) (2.63 g, 14.19 mol) in 2-
propanol (15 mL) was heated at 90 ~C (24 h). The white precipita~e
was filtered off and washed with cold 2-propanol affording (R)-1-(4-
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Amino-6,7-dimethoxy-2-quinazolinyl)-4-[(benzyloxy)carbonyl] -3-(1,1 -
dimethylethylamino)carbonyl piperazine.
lH NMR (DMSO-d6, 300 MHz) o 7.41 (s, 1 H), 7.25 (s, 1
H), 7.11 (br s, 2 H), 6.73 (s, 1 H), 4.65 (dd, 1 H, J = 2.4, 12.2 Hz), 4.41
S (brd, 1 H,J= 12.2Hz),3.83 (s,3 H),3.78(s,3H),3.08(dd, 1 H,J=
3.1, 10.4Hz),2.92(brd, 1 H,J= 11.9),2.50-2.85 (brm,4H), 1.28
(s, 9 H)-
FABLRMS 389 (M++ H, ClgH2gN6O3 requires 388.47)
HPLC (Vydac, C18; diameter = 4.6 mm; length =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 = 4.58 min; focus = 214
nm; 99.4 % pure.
Anal. Calcd for ClgH2gN6O3 and 0.25 CH2C12: C =
56.43, H = 7.01, N = 20.51. Found: C = 56.46, H = 6.91, N = 20.35.
EXAMPLE 7
4-(4-amino-6,7-dimethoxyquinazolin-2-yl)- 1 -(4-oxo-4H-chromene-2-
carbonyl)-piperazine-2-carboxylic acid tert-butylamide (6)
A solution of 3a (105 mg, 0.27 mmol), ~ (56 mg, 0.29
mmol), EDCI (59 mg, 0.31 mmol), and HBT (42 mg, 0.31 mmol) in
DMF (1 mL) was treated with diisopropylethyl~mine (87 mg, 0.67
mmol) at room temperature (24 h). The solvent was removed in vacuo
and the residue dissolved in EtOAc, washed with saturated NaHCO3,
H20, and brine, dried (Na2SO4) and concentrated in vacuo. PCTLC
(SiO2, 4mm, 10% EtOH; 90% CH2Cl2) afforded the title compound 6 as
a yellow powder.
lH NMR (DMSO, 400 MHz) o 8.22 (ddd, lH, J = 6.5 Hz),
7.73 (m, lH), 7.54 (d, lH, J = 8.4 Hz), 7.46 (dd, lH, J = 9.7, 7.4 Hz),
7.10 and 6.73 (two singlets, lH), 6.87 (d, lH, J = 6.1 Hz), 6.83 (s, lH),
6.81 and 6.67 (two singlets, lH), 5.28 (m, 3H), 4.91 and 4.72 (two
doublets, lH, J = 13.8 Hz), 4.50 (m, lH), 3.97 (s, 3H), 3.95 (s, 3H),
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3.80 (d, lH, J = 11.6 Hz), 3.40 (m, 2H), 3.13 (m, lH), 2.93 (m, lH),
2.09 (s, lH), 1.26 and 1.17 (two singlets, 9H).
FABLRMS m/e 561 g/mole (M++H, C29H32N6O6 = 561
g/mole.)
S HPLC (Vydac; C18; diameter = 4.6 m~n; length = 150 mm;
gradient = H2O [0.1% H3PO4] - CH3CN, 95% - 5%, 5% - 95%, over 16
minlltt~s, 2 ml/min flow rate) RT = 7.349 min; focus = 215 nm; 99.5%
pure.
Anal. Calcd for C29H32N6O6 ~ 0.45 CH2Cl2: C = 59.06, H =
5.54, N = 14.04. Found: C = 59.37, H = 5.42, N = 13.65.
EXAMPLE 8
As a specific embodiment of an oral composition, 100 mg
of the compound of Example 7 is formulated with sufflcient finely
divided lactose to provide a total amount of 580 to 590 mg to fill a size
O hard gel capsule.
EXAMPLE 9
Screenin assay: Alpha lb Adrenergic Receptor Binding
Membranes prepared from the stably transfected hllm~n
alpha lb cell line (ATCC CRL 11139) were used to identify compounds
that bind to the hllm~n alpha lb adrenergic receptor. These competition
binding reactions (total volume = 200 ,ul) contained 50 mM Tris-HCl pH
7.4, 5 mM EDTA, 150 mM NaCl, 100 pM [125 I]-HEAT, membranes
prepared from the alpha lb cell line and increasing amounts of
unlabeled ligand. Reactions were incubated at room temperature for
one hour with shaking. Reactions were filtered onto Wh~ 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). Representative compounds of the present invention
were found to have Ki values < 7 nM.
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~XAMPLE 10
Selective Bindin~ assays
Membranes prepared from stably transfected human
alpha ld and alpha la cell lines (ATCC CRL 11138 and CRL 11140,
respectively) were used to identify compounds that selectively bind to
the hllm~n alpha lb adrenergic receptor. These competition binding
reactions (total volume = 200 ~l) contained 50 mM Tris-HCl pH 7.4, 5
mM EDTA, 150 rnM NaCl, 100 pM [125 I]-HEAT, membranes
10 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
~h~king. Reactions were filtered onto Wh~tm~n GF/C glass fiber filters
with a Inotec 96 well cell harvester. Filters were washed three tirnes
15 with ice cold buffer and bound radioactivity was determined (Ki).
Representative compounds of the present invention were folmd to bind
to the human alpha lb adrenergic receptor with binding affinities
greater than twenty-fold higher than the binding affinities with which
they bind to the hl-m~n alpha ld and alpha la adrenergic receptors.
EXAMPLE 1 1
EXEMPLARY COUNTERS(~REENS
25 1. Assay Title: Dopamine D2, D3, D4 in vitro screen
Objective of the Assav:
The objective of this assay is to elimin~e agents which
specifically affect binding of [3H] spiperone to cells expressing hllm~n
30 dopamine receptors D2, D3 or D4.
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- 3g -
Method:
Modified from VanTol et al (1991); Nature (Vol 350) Pg
610-613.
Frozen pellets cont~inin~ specific dopamine receptor
S subtypes stably expressed in clonal cell lines are lysed in 2 ml lysing
buffer (lOmM Tris-HCl/5mM Mg, pH 7.4). Pellets obtained after
centrifuging these membranes (15' at 24,450 rpm) are resuspended in
50mM Tris-HCl pH 7.4 cont~ining EDTA, MgCl[2], KCl, NaCl, CaCl[2]
and ascorbate to give a 1 Mg/mL suspension. The assay is initiated by
~flcling 50-75 ~g membranes in a total volùme of 500 ,ul cont~inin~; 0.2
nM [3H]-spiperone. Non-specific binding is defined using 10 ,uM
apomorphine. The assay is te~nin~d 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.
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 hl~m~n 5HTla receptor.
Method:
Modified from Schelegel and Peroutka Biochemical
Pharmacology 35: 1943-1949 (1986).
~mm~ n cells expressing cloned hllm~n 5HT1a 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 38,000Xg 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 CaC12 and lmg/ml ascorbate. Non-specific
binding is defined using 10 ,uM propranolol. The assay is terrnin~ted
after a 1 hour incubation at room temperature by rapid filtration over
GF/C filters
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E~AMPLE 12
EXEMPLARY FIJNCTIONAL ASSAYS
s
In order to confirm the specificity of compounds for the
hllm~n alpha lb adrenergic receptor and to define the biological activity
of the compounds, the following functional tests may be performed:
10 1. In vitro Rat. Do~ and Human Prostate and Do~ 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
15 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
20 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 cont~ining oxygenated
Krebs solution [NaCl, 118 mM; KCl, 4.7 mM, CaC12, 2.5 mM;
KH2PO4, 1.2 mM; MgSO4, 1.2 mM, NaHCO3, 2.0 mM; dextrose, 11
25 mM] warmed to 37~C. Excess lipid material and connective tissue are
carefully removed. Tissue se~ment~ 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% CO2/95~o ~2- The
tissues are connected to a St~th~m-Gould force transducer; 1 gram (rat,
30 human) or 1.5 gram (dog) of tension is applied and the tissues are
allowed to equilibrate for one hour. Contractions are recorded on a
Hewlett-Packard 7700 series strip chart recorder.
After a single priming dose of 3 ~M (for rat), 10 ,uM (for
dog) and 20 ~lM (for hllm~n) of phenylephrine, a cumulative
35 concentration response curve to an agonist is generated; the tissues are
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washed every 10 minutes for one hour. Vehicle or antagonist is added
to the bath and allowed to incubate for one hour, then another
cumulative concentration response curve to the agonist is generated.
E~so values are calculated for each group using GraphPad
5 Inplot software. pA2 (-log Kb) values were obtained from ~child plot
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
10 where x is the ratio of EC~50 of agonist in the presence and absence of
antagonist and [B] is the antagonist concentration.
2. Measurement of Intra-Urethral Pressure in Anesthetized Do~s
1~ PU~POSE: 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
20 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 the
alpha la receptor subtype has been identified as the predominent
subtype in the hllm~n prostate, it is now possible to specifically target
2~ this receptor to inhibit prostatic contraction without concomitant
changes in the vasculature. Alternatively, since it is now believed that
the alpha lb receptor subtype is predominantly responsible for
mediating changes in the vasculature, it is now possible to specifically
target the alpha lb receptor subtype to lower arterial pressure without
30 concomitant changes in urethral pressure. The following model is used
to measure adrenergically mediated changes in intra-urethral pressure
and arterial pressure in anesthetized dogs in order to evaluate the
efficacy and potency of selective alpha adrenergic receptor antagonists.
The goals are to: I) identify the alpha 1 receptor subtypes responsible
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for prostatic/urethral contraction and vascular responses, and 2) use this
model to evaluate novel selective alpha adrenergic antagonists. Novel
and standard alpha adrenergic antagonists may be evaluated in this
manner.
s
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
~nim.~l ventilated with room air using a Harvard instruments positive
displacement large ~nim~l 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 ~clministration of drugs, respectively. A supra-pubic
incision ~1/2 inch lateral to the penis is made to expose the urethers,
15 bladder and urethra. The urethers are ligated and c~nmll~tt-d 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
20 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
25 the prostate and noting the large change in urethral pressure.
Phenylephrine, an alpha 1 adrenergic agonist, is
lmini~tered (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 a(lministration of increasing doses of an alpha
30 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 ~nim~l
(one control, three or four doses of antagonist or vehicle). The relative
antagonist potency on phenylephrine induced changes in arterial and
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intra-urethral pressure are determined by Schild analysis. The family
of averaged curves are fit simultaneously (using ALLFIT software
package) with a four paramenter logistic equation constraining the
slope, minimllm response, and maximum response to be constant among
5 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/l~g, iv) determined.
The Kb (dose of antagonist causing a 2-fold rightward shift of the
10 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
15 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
arterial pressure is also present in urethral smooth muscIe. According
to this method, one is able to confirm the selectivity of alpha lb
20 adrenergic receptor antagonists that prevent the increase in arterial
pressure to phenylephrine without any activity in inter-urethral
pressure.
While the foregoing specification teaches the principles of
25 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.
