U.S. patent application number 10/185991 was filed with the patent office on 2003-01-30 for compositions comprising alpha-1c specific compounds.
This patent application is currently assigned to Synaptic Pharmaceutical Corporation. Invention is credited to Branchek, Theresa A., Chiu, George, Forray, Carlos C., Gluchowski, Charles, Hartig, Paul R., Wetzel, John M..
Application Number | 20030022900 10/185991 |
Document ID | / |
Family ID | 26936480 |
Filed Date | 2003-01-30 |
United States Patent
Application |
20030022900 |
Kind Code |
A1 |
Gluchowski, Charles ; et
al. |
January 30, 2003 |
Compositions comprising alpha-1C specific compounds
Abstract
A method of treating benign prostatic hyperplasia in a subject
which comprises administering to the subject a therapeutically
effective amount of a compound which binds to a human
.alpha..sub.1C adrenergic receptor with a binding affinity greater
than ten-fold higher than the binding affinity with which the
compound binds to a human .alpha..sub.1A adrenergic receptor, a
human .alpha..sub.1B adrenergic receptor, and a human histamine
H.sub.1 receptor, and, binds to a human .alpha..sub.2 adrenergic
receptor with a binding affinity which is greater than ten-fold
lower than the binding affinity with which the compound binds to
such .alpha..sub.1C adrenergic receptor. Compounds meeting these
criteria are provided.
Inventors: |
Gluchowski, Charles;
(Danville, CA) ; Forray, Carlos C.; (Paramus,
NJ) ; Chiu, George; (Bridgewater, NJ) ;
Branchek, Theresa A.; (Teaneck, NJ) ; Wetzel, John
M.; (Elmwood Park, NJ) ; Hartig, Paul R.;
(Pennington, NJ) |
Correspondence
Address: |
John P. White
Cooper & Dunham LLP
1185 Avenue of the Americas
New York
NY
10036
US
|
Assignee: |
Synaptic Pharmaceutical
Corporation
|
Family ID: |
26936480 |
Appl. No.: |
10/185991 |
Filed: |
June 28, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10185991 |
Jun 28, 2002 |
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09444783 |
Nov 22, 1999 |
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09444783 |
Nov 22, 1999 |
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08244354 |
Apr 1, 1997 |
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6015819 |
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08244354 |
Apr 1, 1997 |
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PCT/US93/10950 |
Nov 12, 1993 |
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PCT/US93/10950 |
Nov 12, 1993 |
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07975867 |
Nov 13, 1992 |
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5403847 |
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Current U.S.
Class: |
514/252.17 ;
514/215; 514/252.14; 514/293; 514/317; 514/318; 514/649 |
Current CPC
Class: |
A61K 31/505 20130101;
A61K 31/445 20130101; A61P 13/02 20180101; C07D 495/06 20130101;
A61K 31/454 20130101; C07C 215/50 20130101; C07D 405/12 20130101;
A61K 31/4515 20130101; A61K 31/55 20130101; A61K 31/4409 20130101;
A61K 31/135 20130101; A61K 31/517 20130101; A61P 43/00 20180101;
C07C 2602/08 20170501; C07C 2602/12 20170501; A61K 31/00 20130101;
C07C 217/58 20130101; C07D 211/58 20130101; C07D 401/12 20130101;
C07C 215/52 20130101; C07C 211/30 20130101; A61K 31/137 20130101;
A61K 31/4545 20130101; C07C 2602/10 20170501; A61K 31/44 20130101;
A61P 15/00 20180101; C07C 217/60 20130101 |
Class at
Publication: |
514/252.17 ;
514/252.14; 514/317; 514/215; 514/293; 514/318; 514/649 |
International
Class: |
A61K 031/55; A61K
031/517; A61K 031/513; A61K 031/4745; A61K 031/137 |
Claims
What is claimed is:
1. A method of treating benign prostatic hyperplasia in a subject
which comprises administering to the subject a therapeutically
effective amount of a compound which: a. binds to a human
.alpha..sub.1C adrenergic receptor with a binding affinity greater
than ten-fold higher than the binding affinity with which the
compound binds to a human .alpha..sub.1A adrenergic receptor, a
human .alpha..sub.1B adrenergic receptor, and a human histamine
H.sub.1 receptor; and b. binds to a human .alpha..sub.2 adrenergic
receptor with a binding affinity which is greater than ten-fold
lower than the binding affinity with which the compound binds to
such .alpha..sub.1C adrenergic receptor.
2. A method of claim 1, wherein the compound additionally binds to
a calcium channel with a binding affinity which is greater than
ten-fold lower than the binding affinity with which the compound
binds to the .alpha..sub.1C adrenergic receptor.
3. A method of claim 1 or 2, wherein the compound additionally
binds to a dopamine D.sub.2 receptor with a binding affinity which
is greater than ten-fold lower than the binding affinity with which
the compound binds to the .alpha..sub.1C adrenergic receptor.
4. A method of claim 1, 2, or 3, wherein the compound additionally
binds to any serotonin receptor with a binding affinity which is
greater than ten-fold lower than the binding affinity with which
the compound binds to the .alpha..sub.1C adrenergic receptor.
5. A method of claim 1, 2, 3, or 4, wherein the compound
additionally binds to a dopamine D.sub.3, D.sub.4, or D.sub.5
receptor with a binding affinity which is greater than ten-fold
lower than the binding affinity with which the compound binds to
the .alpha..sub.1C adrenergic receptor.
6. A method of claim 1, 2, 3, 4, or 5 wherein the compound
additionally does not cause orthostatic fall in blood pressure at
dosages effective to alleviate benign prostatic hyperplasia.
7. The method of claim 6 wherein the compound additionally does not
cause orthostatic fall in blood pressure in rats at a dosage of 10
micrograms of compound per kilogram of rat.
8. A method of claim 1, wherein the compound has the structure:
5
9. A method of claim 1, wherein the compound has the structure:
6
10. A method of claim 1, wherein the compound has the structure:
7
11. A method of claim 1, wherein the compound has the structure:
8
12. A method of inhibiting contraction of prostate tissue which
comprises contacting the prostate tissue with an effective
contraction-inhibiting amount of a compound which: a. binds to a
human .alpha..sub.1C adrenergic receptor with a binding affinity
greater than ten-fold higher than the binding affinity with which
the compound binds to a human .alpha..sub.1A adrenergic receptor, a
human .alpha..sub.1B adrenergic receptor, and a human histamine
H.sub.1 receptor; and b. binds to a human .alpha..sub.2 adrenergic
receptor with a binding affinity which is greater than ten-fold
lower than the binding affinity with which the compound binds to
such .alpha..sub.1C adrenergic receptor.
Description
BACKGROUND OF THE INVENTION
[0001] Benign Prostatic Hyperplasia (BPH), also called Benign
Prostatic Hypertrophy, is a progressive condition which is
characterized by a nodular enlargement of prostatic tissue
resulting in obstruction of the urethra. This results in increased
frequency of urination, nocturia, a poor urine stream and hesitancy
or delay in starting the urine flow. Chronic consequences of BPH
can include hypertrophy of bladder smooth muscle, a decompensated
bladder and an increased incidence of urinary tract infection. The
specific biochemical, histological and pharmacological properties
of the prostate adenoma leading to the bladder outlet obstruction
are not yet known. However, the development of BPH is considered to
be an inescapable phenomenon for the aging male population. BPH is
observed in approximately 70% of males over the age of 70.
Currently, in the United States, the method of choice for treating
BPH is surgery (Lepor, H. Urol. Clinics North Amer., 17, 651
(1990)). Over 400,000 prostatectomies are performed annually (data
from 1986). A medicinal alternative to surgery is clearly very
desirable. The limitations of surgery for treating BPH include the
morbidity rate of an operative procedure in elderly men,
persistence or recurrence of obstructive and irritative symptoms,
as well as the significant cost of surgery.
[0002] .alpha.-Adrenergic receptors are specific neurcreceptor
proteins located in the peripheral and central nervous systems on
tissues throughout the body. These receptors are important switches
for controlling many physiological functions and, thus, represent
important targets for drug development. in fact, many
.alpha.-adrenergic drugs have been developed over the past 40
years. Examples include clonidine, phenoxybenzamine and prazosin
(treatment of hypertension), naphazoline (nasal decongestant), and
apraclonidine (treating glaucoma). .alpha.-Adrenergic drugs can be
broken down into two distinct classes: agonists (clonidine and
naphazoline are agonists), which mimic the receptor activation
properties of the endogenous neurotransmitter norepinephrine, and
antagonists (phenoxybenzamine and prazosin are antagonists), which
act to block the effects of norepinephrine. Many of these drugs are
effective but also produce unwanted side effects (for example,
clonidine produces dry mouth and sedation in addition to its
antihypertensive effects).
[0003] During the past 15 years a more precise understanding of
.alpha.-adrenergic receptors and their drugs has evolved through
increased scientific scrutiny. Prior to 1977, only one
.alpha.-adrenergic receptor was known to exist. Between 1977 and
1988, it was accepted by the scientific community that at least two
.alpha.-adrenergic receptors--.alpha..sub.1 and
.alpha..sub.2--existed in the central and peripheral nervous
systems. Since 1988, new techniques in molecular biology have led
to the identification of at least six .alpha.-adrenergic receptors
which exist throughout the central and peripheral nervous systems:
.alpha..sub.1A, .alpha..sub.1B, .alpha..sub.1C, .alpha..sub.2A,
.alpha..sub.2B and .alpha..sub.2C (Bylund, D. B., FASEB J., 6, 832
(1992)). It is not known precisely which physiological responses in
the body are controlled by each of these receptors. In addition,
many .alpha.-adrenergic drugs that were developed before 1992 are
not selective for any particular .alpha.-adrenergic receptor. Many
of these drugs produce untoward side effects which may be
attributed to their poor .alpha.-adrenergic receptor
selectivity.
[0004] Since the mid 1970's, nonselective .alpha.-antagonists have
been prescribed to treat BPH. In 1976, M. Caine, et al. (Brit. J.
Urol., 48, 255 (1976)), reported that the nonselective
.alpha.-antagonist phenoxybenzamine was useful in relieving the
symptoms of BPH. This drug may produce its effects by interacting
with .alpha.-receptors located on the prostate. However, this drug
also produces significant side effects which severely limit its use
in treating patients on a chronic basis. More recently, the
.alpha.-adrenergic antagonists prazosin and terazosin have also
been found to be useful for treating BPH. However, these drugs also
produce untoward side effects. The most recently approved drug
Proscar.TM. (Merck) prescribed for BPH is not an .alpha.-adrenergic
antagonist, but rather acts by blocking 5-.alpha.-reductase. While
Proscar is able to relieve symptoms, it is effective in only 30% of
all patients, and requires a period of up to 6 months to show
results.
[0005] From binding studies using cloned rat .alpha..sub.1A,
hamster .alpha..sub.1B, and bovine .alpha..sub.1C receptors, and
functional studies of antagonism in vitro using human prostrate, I.
Marshall, et al., concluded that the receptor mediating contraction
of the human prostrate is of the .alpha..sub.1C subtype (Marshall,
I., et al., Brit. Pharmacol. Soc., (1992)).
[0006] Furthermore, using cloned human receptors the binding
characteristics of the known BPH drugs to various receptor subtypes
have been determined, as described more fully hereinafter. Based
upon such binding information and additional data, it has been
observed that the side effects which occur with the drugs prazosin
and terazosin may be due to their poor selectivity for specific
.alpha.-adrenergic receptors. In contrast, indoramin is a drug
which is slightly selective for the human .alpha..sub.1C receptor
relative to the other human .alpha.-adrenergic receptors, but it
also interacts at human histamine H1 receptors. This compound
produces untoward side effects which may be attributed to its
activity at such H.sub.1 receptors.
[0007] It would be desirable to provide methods and compounds which
allow the treatment of BPH but which avoid the production of side
effects observed for all currently used medications.
[0008] From the binding information described hereinafter, it has
unexpectedly been discovered that compounds which are specific for
an .alpha..sub.1C adrenergic receptor with a binding affinity
greater than ten-fold higher than the binding affinity with which
the compounds bind to an .alpha..sub.1A adrenergic receptor, a
human .alpha..sub.1B adrenergic receptor, and a human histamine
H.sub.1 receptor, and (b) bind to an .alpha..sub.2 adrenergic
receptor with a binding affinity which is greater than ten-fold
lower than the binding affinity with which the compounds bind to
such .alpha..sub.1C adrenergic receptor are effective for the
treatment of BPH.
[0009] Furthermore, we have characterized several antagonists
selective for the .alpha..sub.1C adrenergic receptor using a rat
orthostatic hypotension model to ascertain the vascular effects of
drugs which may be indicative of their ability to produce dizziness
in patients, and observed that while nonselective alpha 1
antagonists produce significant effects on orthostatic hypotension,
selective alpha 1c antagonists do not produce significant
effects.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] A more complete understanding of the invention and many of
its advantages will become apparent by reference to the detailed
description which follows when considered in conjunction with the
accompanying drawings, wherein:
[0011] FIG. 1 illustrates compounds which are potent antagonists of
the cloned human .alpha..sub.1C receptor.
[0012] FIG. 2 illustrates the correlation of inhibition constants
(pK.sub.i) for a series of .alpha..sub.1 antagonists at the cloned
human .alpha..sub.1A, .alpha..sub.1B, and .alpha..sub.1C receptors
with efficiency of blocking contraction of human prostate tissue
(pA.sub.2).
SUMMARY OF HE INVENTION
[0013] The present invention provides a method of treating benign
prostatic hyperplasia in a subject which comprises administering to
the subject a therapeutically effective amount of a compound which
(a) binds to a human .alpha..sub.1C adrenergic receptor with a
binding affinity greater than ten-fold higher than the binding
affinity with which the compound binds to a human .alpha..sub.1A
adrenergic receptor, a human .alpha..sub.1B adrenergic receptor,
and a human histamine H.sub.1 receptor, and (b) binds to a human
.alpha..sub.2 adrenergic receptor with a binding affinity which is
greater than ten-fold lower than the binding affinity with which
the compound binds to such .alpha..sub.1C adrenergic receptor.
[0014] The present invention also provides a method of inhibiting
contraction of prostate tissue which comprises contacting the
prostate tissue with an effective contraction-inhibiting amount of
a compound which (a) binds to a human .alpha..sub.1C adrenergic
receptor with a binding affinity greater than ten-fold higher than
the binding affinity with which the compound binds to a human
.alpha..sub.1A adrenergic receptor, a human .alpha..sub.1B
adrenergic receptor, and a human histamine H.sub.1 receptor, and
(b) binds to a human .alpha..sub.2 adrenergic receptor with a
binding affinity which is greater than ten-fold lower than the
binding affinity with which the compound binds to such
.alpha..sub.1C adrenergic receptor.
DETAILED DESCRIPTION OF THE INVENTION
[0015] The present invention provides a method of treating benign
prostatic hyperplasia in a subject which comprises administering to
the subject a therapeutically effective amount of a compound which
(a) binds to a human .alpha..sub.1C adrenergic receptor with a
binding affinity greater than ten-fold higher than the binding
affinity with which the compound binds to a human .alpha..sub.1A
adrenergic receptor, a human .alpha..sub.1B adrenergic receptor,
and a human histamine H.sub.1 receptor, and (b) binds to a human
.alpha..sub.2 adrenergic receptor with a binding affinity which is
greater than ten-fold lower than the binding affinity with which
the compound binds to such .alpha..sub.1C adrenergic receptor.
[0016] Desirably, the compound used to practice the method of the
invention additionally binds to a calcium channel with a binding
affinity which is greater than ten-fold lower than the binding
affinity with which the compound binds to the .alpha..sub.1C
adrenergic receptor.
[0017] Alternatively or incrementally, the compound used to
practice the method of the invention also binds to a dopamine
D.sub.2 receptor with a binding affinity which is greater than
ten-fold lower than the binding affinity with which the compound
binds to the .alpha..sub.1C adrenergic receptor.
[0018] Alternatively or incrementally, the compound used to
practice the method of the invention additionally binds to a
dopamine D.sub.2 receptor with a binding affinity which is greater
than ten-fold lower than the binding affinity with which the
compound binds to the .alpha..sub.1C adrenergic receptor.
[0019] Alternatively or incrementally, the compound used to
practice the method of the invention additionally binds to any
serotonin receptor with a binding affinity which is greater than
ten-fold lower than the binding affinity with which the compound
binds to the .alpha..sub.1C adrenergic receptor.
[0020] Alternatively or incrementally, the compound used to
practice the method of the invention also binds to a human dopamine
D.sub.3 receptor with a binding affinity which is greater than
ten-fold lower than the binding affinity with which the compound
binds to the .alpha..sub.1C adrenergic receptor.
[0021] Alternatively or incrementally, the compound used to
practice the method of the invention also binds to a human dopamine
D.sub.4 with a binding affinity which is greater than ten-fold
lower than the binding affinity with which the compound binds to
the .alpha..sub.1C adrenergic receptor.
[0022] Alternatively or incrementally, the compound used to
practice the method of the invention also binds to a human dopamine
D.sub.5 receptor with a binding affinity which is greater than
ten-fold lower than the binding affinity with which the compound
binds to the .alpha..sub.1C adrenergic receptor.
[0023] Alternatively or incrementally, the compound used to
practice the method of the invention also does not cause
orthostatic fall in blood pressure at a dosage effective to
alleviate benign prostatic hyperplasia.
[0024] Alternatively or incrementally, the compound used to
practice the method of the invention also does not cause
orthostatic fall in blood pressure in rats at a dosage 10
ug/kg.
[0025] A number of compounds have been identified or synthesized
which are useful in the practice of the invention. For example, the
compound has the structure: 1
[0026] In another example, the compound has the structure: 2
[0027] In still another example, the compound has the structure:
3
[0028] In an additional example, the compound has the structure:
4
[0029] Included within the scope of the method of treating BPH in
accord with the invention are the use of both R and S enantiomers
of the compounds described which possess stereogenic centers, as
well as the use of pharmaceutically acceptable salts and complexes
thereof.
[0030] The invention also provides a method of inhibiting
contraction of prostate tissue which comprises contacting the
prostate tissue with an effective contraction-inhibiting amount of
a compound which (a) binds to a human .alpha..sub.1C adrenergic
receptor with a binding affinity greater than ten-fold higher than
the binding affinity with which the compound binds to a human
.alpha..sub.1A adrenergic receptor, a human .alpha..sub.1B
adrenergic receptor, and a human histamine H.sub.1 receptor, and
(b) binds to a human .alpha..sub.2 adrenergic receptor with a
binding affinity which is greater than ten-fold lower than the
binding affinity with which the compound binds to such
.alpha..sub.1C adrenergic receptor.
[0031] The activity of compounds at the different human receptors
was determined in vitro using cultured cell lines that selectively
express the receptor of interest. These cell lines were prepared by
transfecting the cloned cDNA or cloned genomic DNA or constructs
containing both genomic DNA and cDNA encoding the human
.alpha.-adrenergic, serotonin, histamine, and dopamine receptors as
further described in detail in Example 9 hereinbelow.
[0032] In connection with this invention, a number of cloned human
receptors discussed herein, either as plasmids or as stably
transfected cell lines, have been made pursuant to, and in
satisfaction of, the Budapest Treaty on the International
Recognition of the Deposit of Microorganisms for the Purpose of
Patent Procedure, and are made with the American Type Culture
Collection, 12301 Parklawn Drive, Rockville, Md. 20852.
Specifically, these deposits have been accorded ATCC Accession
Numbers as follows:
1 Designation ATCC Accession No. Date L-.alpha..sub.1A CRL 11138
Sep. 25, 1992 L-.alpha..sub.1B CRL 11139 Sep. 25, 1992
L-.alpha..sub.1C CRL 11140 Sep. 25, 1992 L-.alpha..sub.2A CRL 11180
Nov. 6, 1992 L-NGC-.alpha..sub.2B CRL 10275 Oct. 25, 1989
L-.alpha..sub.2C CRL 11181 Nov. 6, 1992 pcEXV-H.sub.1 75346 Nov. 6,
1992 pcEXV-H.sub.2 75345 Nov. 6, 1992 pcEXV-D.sub.2 75344 Nov. 6,
1992
[0033] The data shown in the accompanying Tables 1 and 2 indicate
that the .alpha..sub.1C-specific receptor antagonists which satisfy
the criteria as defined herein have significant efficacy in the
inhibition of contraction of human prostate tissue. This in vitro
property is recognized in the art as correlating with efficacy in
treating benign prostatic hyperplasia in vivo.
[0034] The present invention therefore provides a method of
treating benign prostatic hyperplasia, which comprises
administering a quantity of any of the .alpha..sub.1C receptor
antagonists defined as herein in a quantity effective against BPH.
The drug may be administered to a patient afflicted with benign
prostatic hyperplasia by any conventional route of administration,
including, but not limited to, intravenous, intramuscular, oral,
subcutaneous, intratumoral, intradermal, and parenteral. The
quantity effective against BPH is between 0.001 mg and 10.0 mg per
kg of subject body weight.
[0035] The method of treating BPH disclosed in the present
invention may also be carried out using a pharmaceutical
composition comprising any of the .alpha..sub.1C receptor
antagonists as defined herein and a pharmaceutically acceptable
carrier. The composition may contain between 0.05 mg and 500 mg of
an .alpha..sub.1C receptor antagonist, and may be constituted into
any form suitable for the mode of administration selected.
Compositions suitable for oral administration include solid forms,
such as pills, capsules, granules, tablets, and powders, and liquid
forms, such as solutions, syrups, elixers, and suspensions. Forms
useful for parenteral administration include sterile solutions,
emulsions, and suspensions.
[0036] The drug may otherwise be prepared as a sterile solid
composition which may be dissolved or suspended at the time of
administration using sterile water, saline, or other appropriate
sterile injectable medium. Carriers are intended to include
necessary and inert binders, suspending agents, lubricants,
flavorants, sweeteners, preservatives, dyes, and coatings.
[0037] Optimal dosages to be administered may be readily determined
by those skilled in the art, and will vary with the particular
.alpha..sub.1C receptor antagonist in use, the strength of the
preparation, the mode of administration, and the advancement of the
disease condition. Additional factors depending on the particular
patient being treated will result in a need to adjust dosages,
including patient age, weight, diet, and time of
administration.
[0038] The following Experimental Details are set forth to aid in
an understanding of the invention, and are not intended, and should
not be construed, to limit in any way the invention set forth in
the claims which follow thereafter.
[0039] Experimental Details.
[0040] Prazosin, 5-methylurapidil, and S-niguldipine were obtained
from Research Biochemicals, Inc. A30360
(4-fluoro-4-(8-fluoro-1,3,4,5-tetrahyd-
ro-2H-pyrido[4,3-b]indol-2-yl)butyrophenone hydrochloride) was
obtained from Aldrich Chemical Co. Other compounds were prepared
according to the examples which follow.
EXAMPLE 1
Synthesis of Terazosin Hydrochloride
N-(2-Furoyl)piperazine
[0041] This compound and its preparation has been described in
Great Britain Patents 1,390,014 and 1,390,015. Piperazine
hexahydrate (194 g, 1 mole) was dissolved in 250 ml H.sub.2O. The
solution was acidified to pH 4.5 with 6 N HCl. Furoyl chloride
(130.5 g, 1 mole, Aldrich) was added along with 10% NaOH solution
at such a rate that the pH was maintained at 4.5. After 1 hour, the
solution was made basic (pH=8.5) with NaOH solution. The reaction
mixture was continuously extracted with chloroform for 36 hours.
The CHCl.sub.3 extract was dried over MgSO.sub.4, and filtered.
Distillation gave 108.2 g product (60%), b.p.
132.degree.-138.degree. C./0.6 mm Hg, m.p. 69.degree.-70.degree.
C.
N-(Tetrahydro-2-furoyl)piperazine
[0042] The furoylpiperazine of Example 1 was converted to the
hydrobromide salt (m.p. 173.degree.-175.degree. C.). This salt
(39.0 g) in 250 ml methyl alcohol and 9.0 g Raney nickel was
hydrogenated at 3 atm. After uptake of H.sub.2 ceased, the catalyst
was filtered, the solvent concentrated, and the residue
crystallized from isopropyl alcohol to give 35.2 g.
tetrahydrofuroylpiperazine HBr, m.p. 152.degree.-156.degree. C.
This was suspended in 20 ml H.sub.2O. Then 10.5 g 50%, NaOH
solution was added slowly followed by 2.0 g solid Na.sub.2CO.sub.3.
This was extracted with 4.times.100 ml portions of warm CHCl.sub.3.
The CHCl.sub.3 extractions were distilled to give 22.5 g
tetrahydrofurolylpiperazine, b.p. 120.degree.-125.degree. C./0.2 mm
Hg.
2[4-(Tetrahydro-2-furoyl)piperazinyl]-4-amino-6,7-dimethoxyquinazoline
hydrochloride
[0043] To 7.00 g 2-chloro-4-amino-6,7-dimethoxyquinazoline
(Lancaster Synthesis) in 50 ml methoxyethanol was added 10.8 g,
tetrahydrofurolylpiperazine, and the mixture refluxed 3 hours. The
clear solution was concentrated and an aqueous solution of
potassium bicarbonate was added. The resultant solid that formed
was filtered and washed with water. It was then added to methanol
and the resulting suspension was acidified with a solution of
hydrogen chloride in isopropyl alcohol. The resulting solution was
concentrated and the residue crystallized from isopropyl alcohol
giving 8.12 g. of product, m.p. 278.degree.-279.degree. C.
EXAMPLE 2
Preparation of Indoramin
4-Benzamido-1-[2-(3-indolyl)ethylpyridinium Bromide
[0044] A solution of 4-benzamidopyridine (1.98 g) and
3-(2-bromoethyl)indole (2.24 g) in EtOH (15 ml) was refluxed for 2
hours, and the crystallized product (3.13 g, mp 264-266.degree. C.)
was collected by filtration from the hot reaction mixture.
Recrystallization gave the hydrate.
3-[2-4-Benzamidopiperid-1-yl)ethyl]indole (Indoramin)
[0045] 4-Benzamido-1-[2-(3-indolyl)ethyl]pyridinium bromide (3.0 g)
in 91% EtOH (300 ml) containing Et.sub.3N (0.8 g) was hydrogenated
in the presence of freshly prepared W-7 Raney Ni catalyst (ca. 3 g)
at 28.12 kg/cm.sup.2 and 50.degree. for 4 hours. After filtering
off the catalyst, the filtrate was evaporated and the residue was
shaken with CHCl.sub.3 and 2 N NaOH. The resulting insoluble
material (1.61 g, mp 203-206.degree. C.) was collected and dried.
Recrystallization from EtOH gave the product (1.34 g), as colorless
needles.
EXAMPLE 3
Preparation of 1-(3-benzoylpropyl)-4-benzamidopiperidine
[0046] A mixture of 4-chlorobutyrophenone (447 mg, 2.45 mmol),
4-benzamidopiperidine (500 mg, 2.45 mmol) and K.sub.2CO.sub.3 (338
mg, 2.45 mmol) was heated up in boiling water bath for 1 hour. The
reaction mixture was portioned between water and CHCl.sub.3. The
organic layer was separated and dried over Na.sub.2SO.sub.4. After
filtration and removal of solvent, the residue was purified by
chromatography (SiO.sub.2, MeOH:CHCl.sub.3, 5:95).
Recrystallization from AcOEt/hexane gave a white powder (78 mg,
8.2%). mp 143-144.degree. C.; .sup.1H NMR (CD.sub.3OD, 400 MHz)
.delta.1.65 (dq, J.sub.1=3.16 Hz, J.sub.2=11.9 Hz, 2H), 1.90-2.00
(m, 4H), 2.18 (t, J=11.9 Hz, 2H), 2.48 (m, 2H), 3.00-3.10 (m, 4H),
3.88 (m, 1H), 7.40-8.00 (m, 10H); Mass spectrum (M+1).sup.+ at m/z
351.
EXAMPLE 4
Preparation of
1-[3-(4-chlorobenzoyl)propyl]-4-benzamidopiperidine
[0047] A mixture of 3-(4-chlorobenzol)propyl bromide (640 mg, 2.45
mmol), 4-benzamidopiperidine (500 mg, 2.45 mmol) and
K.sub.2CO.sub.3 (1.01 g, 7.34 mmol) in 50 ml of acetone was heated
up to refluxing condition for 48 hours. The solid was removed by
filtration. Concentration of filtrate in vacuo gave a yellowish
solid, which was purified by chromatography (SiO.sub.2,
MeOH:CHCl.sub.3, 5:95). 320 mg (33.9%) of white powder was obtained
.sup.1H NMR (CDCl.sub.3, 300 mHz) .delta.1.46 (dq, J.sub.1=1.0 Hz,
J.sub.2=8.4 Hz, 2H), 1.90-2.10 (m, 4H), 2.16 (m, 2H), 2.43 (t,
J=6.9 Hz, 2H), 2.80-2.90 (m, 2H), 2.97 (t, J=6.9 Hz, 2H), 3.97 (m,
1H), 5.92 (d, J=7.8 Hz, 1H, N-H), 7.40-8.00 (m, 9H); Product was
converted to HCl salt and recrystallized with MeOH/Et.sub.2O, mp
243-244.degree. C.; Calcd for
C.sub.22H.sub.25ClN.sub.2O.sub.2.HCl.H.sub.2O: C, 60.15, H, 6.37,
N, 6.37; Found: C, 60.18, H, 6.34, N, 6.29.
EXAMPLE 5
Preparation of SKF-104856
1-[(4-Chlorophenyl)thio}-2-propanone
[0048] Chloroacetone (32.3 g, 0.347 mol) was added to a mixture of
4-chlorothiophenol (50 g, 0.347 mmol) and sodium hydroxide (14 g,
0.347 mol) in water (400 ml) and the mixture was stirred at
25.degree. C. for 1 hour. The mixture was extracted with ethyl
ether and the organic phase was washed with water, dried with
magnesium sulfate and concentrated to give 69 g (99%) of
1-[(4-chlorophenyl)thio]-2-propanone.
5-Chloro-3-methylbenzo(b)thiophene
[0049] 1-[(4-Chlorophenyl)thio}-2-propanone (50 g, 0.25 mol) was
added to polyphosphoric acid (300 g) and the mixture was stirred as
the temperature was gradually raised to 120.degree. C. as an
exotherm started. The mixture was stirred at 130.degree. C. for 1
hour, diluted with water, extracted with ethyl ether and the
organic phase was dried and concentrated. The residue was stirred
in methanol (200 ml), filtered and the filtrate concentrated to
give 17.5 g (40%) of 5-chloro-3-methylbenzo(b)thiophene: bp
120.degree. C. (0.6 mm Hg).
Ethyl5-chloro-3-methylbenzo(b)thiophene-2-carboxylate
[0050] n-Butyllithium in hexane (2.6 M, 2.3 ml) was added to a
solution of 5-chloro-3-methylbenzo(b)thiophene (1,0 g, 6 mmol) in
ethyl ether (20 ml) stirred at 0.degree. C. under argon. The
mixture was stirred for 30 minutes and transferred slowly under
argon pressure to a stirred solution of ethyl chloroformate (0.63
g, 6 mmol) in ethyl ether (20 ml). The mixture was stirred at
0.degree. C. for 30 minutes and at 25.degree. C. for 1.5 hours. The
mixture was treated with water and the organic phase was dried,
concentrated and triturated with hexane to give 1.0 g (67%) of
ethyl 5-chloro-3-methylbenzo(b)thiophene-2-carboxylate: mp 92.5-94
.degree. C.
Ethyl 3-bromomethyl-5-chlorobenzo(b)thiophene-2-carboxylate
[0051] A mixture of ethyl
5-chloro-3-methylbenzo(b)thiophene-2-carboxylate (9.0 g, 0.035
mol), N-bromosuccinimide (6.53 g, 0.037 mol) and benzoyl peroxide
(130 mg) in carbon tetrachloride (150 ml) was refluxed and
illuminated with sunlamp for 2 hours. The resulting suspension was
cooled, filtered and the filter cake was triturated with methanol
to give 9.9 g, (85%) of the methanol-insoluble ethyl
3-bromomethyl-5-chlorobenzo(- b)thiophene-2-carboxylate: mp
148-150.degree. C.
Ethyl
5-Chloro-3-[N-(2,2-dimethoxyethyl)-N-methyl(aminomethyl)]benzol(b)th-
iophene-2-carboxylate
[0052] A mixture of ethyl
3-bromomethyl-5-chlorobenzo(b)thiophene-2-carbox- ylate (11 g,
0.033 mol), methylaminoacetaldehyde dimethyl acetal (4.76 g, 0.04
mol) and potassium carbonate (11.4 g, 0.8 mol) in dry acetone (200
ml) was stirred for 48 hours, filtered and the filtrate
concentrated to give 11.8 g, (96%) of ethyl
5-chloro-3-(N-2,2-dimethoxyethyl)-N-methyl(am-
inomethyl)benzol(b)thiophene-2-carboxylate.
Ethyl
7-chloro-3,4-dihydro-4-methylthieno[4,3,2-ef]-[3]benzazepine-2-carbo-
xylate
[0053] Ethyl
5-chloro-3-[N-(2,2-dimethoxyethyl)-N-methyl(aminomethyl)]benz-
o[b]thiophene-2-carboxylate (3.0 g, 8.1 mmol) was added in portions
to trifluoromethanesulfonic acid (10 ml) stirred at 0.degree. C.
under argon. The mixture was stirred at 25.degree. C. for 45
minutes and diluted with water. The mixture was basified with
aqueous sodium hydroxide and extracted with ethyl ether to give
ethyl
7-chloro-3,4-dihydro-4-methylthieno-[4,3,2-ef][3]benzazepine-2-carboxylat-
e.
Ethyl
7-chloro-3,4,5,6-tetrahydro-4-methylthieno[4,3,2-ef][3]benzazepine-2-
-carboxylate
[0054] Diborane in tetrahydrofuran (1 M, 40 ml) was added to a
solution of ethyl
7-chloro-3,4-dihydro-4-methylthieno[4,3,2-ef][3]benzazepine-2-carbo-
xylate (2.8 g) in tetrahydrofuran (30 ml) stirred at 0.degree. C.
The mixture was refluxed for 3 hours and stirred at 25.degree. C.
for 18 hours, cooled, treated with methanol (50 ml), refluxed for
18 hours and concentrated. The residue was triturated with ethyl
ether-hexane (3:1) to give 1.6 g (84%) of ethyl
7-chloro-3,4,5,6-tetrahydro-4-methylthieno[4,3,-
2-ef][3]benzazepine-2-carboxylate:mp 138-140.degree. C. The free
base was treated with hydrogen chloride to give ethyl
7-chloro-3,4,5,6-tetrahydro--
4-methylthieno[4,3,2-ef][3]benzazepine-2-carboxylate hydrochloride:
mp 240.degree. C.
7-Chloro-3,4,5,6-tetrahydro-4-methylthieno[4,3,2-ef][3]benzazepine-2-metha-
nol
[0055] A solution of ethyl
7-chloro-3,4,5,6-tetrahydro-4-methylthieno[4.3.-
2-ef][3]benzazepine-2-carboxylate (4.0 g, 12.9 mmol), in ethyl
ether (48 ml) was treated with lithium aluminum hydride (0.53 g, 14
mmol). The mixture was stirred for 1.5 hours, cooled and treated
carefully with water (2.0 ml), 10% sodium hydroxide (1.0 ml) and
water (2.0 ml). The resulting mixture was filtered and the solvent
evaporated to give 1.9 g (57%) of
7-chloro-3,4,5,6-tetrahydro-4-methylthieno[4,3,2-ef][3]benzazepi-
ne-2-methanol: mp 184-185.degree. C.
7-Chloro-3, 4,5,6-tetrahydro-4-methylthieno-4,3,2-ef
][3]benzazepine-2-carboxaldehyde
[0056] A solution of
7-chloro-3,4,5,6-tetrahydro-4-methylthieno[4,3,2-ef][-
3]benzazepine-2-methanol (1.6 g, 6 mmol) in dichloromethane (150
ml) was stirred under argon with activated manganese dioxide (8.3
g) for 2 hours. The mixture was filtered through Celite.TM. and the
filtrate was dried with magnesium sulfate and concentrated to give
a 63% yield of
7-chloro-3,4,5,6-tetrahydro-4-methylthieno[4,3,2-ef[[3]benzazepine-2-carb-
oxaldehyde.
7-Chloro-2-ethenyl-3,4,5,6-tetrahdyro-4-methylthieno[4,3,2-ef][3]benzazepi-
ne (SKF-104856)
[0057] Sodium hydride (60% dispersion in mineral oil. 3.8 mmol) was
added to a stirred solution of methyltriphenylphosphonium bromide
(1.35 g, 3.8 mmol) in dry tetrahydrofuran (30 ml) and stirred for
15 minutes. The mixture was treated with a solution of
7-chloro-3,4,5,6-tetrahydro-4-meth-
ylthieno[4,3,2-ef][3]-benzazepine-2-carboxaldehyde, prepared as in
Example 3, (0.5 g, 1.9 mmol) in dimethylformamide (4 ml), stirred
at 25.degree. C. for 16 hours, quenched with ice and extracted with
ethyl acetate. The organic phase was washed, dried and concentrated
and the residue was chromatographed on silica gel eluted with a
gradient of methylene chloride to methanol-methylene chloride
(3.5:96.5). The product was treated with hydrogen chloride to give
0.2 g (35%) of
7-chloro-2-ethenyl-3,4,5,6-tetrahydro-4-methylthieno[4,3,2-ef][3]benzazep-
ine hydrochloride: mp 234-236.degree. C.
EXAMPLE 6
2-Hydroxymethyl-1,2,3,4-tetrahydronaphthalene
[0058] A solution of 1,2,34-tetrahydro-2-naphthoic acid (2.50 g,
14.2 mmol) in 100 ml THF was treated with LiAlH.sub.4 (681 mg,
17.04 mmol) and the reaction mixture was heated at reflux for 5
hours. The suspension was cooled to 0.degree. C. and quenched by
addition of solid Na.sub.2SO.sub.4.box-solid.10H.sub.2O. The
mixture was stirred at room temperature for 4 hours. The solid was
removed by filtration. Concentration of filtrate in vacuo gave a
yellowish oil (2.28 g, 98.8%) ; .sup.1H NMR (CDCl.sub.3, 300 MHz)
.delta.1.43 (m, 1H), 2.00 (m, 2H) 2.51 (dd, J.sub.1=16.5 Hz,
J.sub.2=10.8 Hz, 1H) , 2.85 (m, 3H), 3.65 (dd, J.sub.1=6.3 Hz,
J.sub.2=1.2 Hz, 2H), 7.09 (s, 4H).
2-Bromomethyl-1,2,3,4-tetrahydronaphthalene
[0059] A solution of 2-hydroxymethyl-1,2,3,4-tetrahydronaphthalene
(2.28 g, 14.0 mmol) in 100 ml of CH.sub.2Cl.sub.2 was treated with
PBr.sub.3 (1.28 g, 4.73 mmol) at 0.degree. C. The mixture was
stirred at room temperature for 72 hours then poured onto 100 g of
ice. The organic layer was isolated, washed with 10%
K.sub.2CO.sub.4 aqueous solution, H.sub.2O, sat'd brine, and then
dried over Na.sub.2SO.sub.4. After filtration and removal of
solvent, the residue was purified by chromatography (SiO.sub.2,
EtOAc:hexane, 1:10) to give a colorless oil (1.33 g, 41.6%);
.sup.1H NMR (CDCl.sub.3, 300 MHz) .delta.1.55 (m, 1H), 2.11 (m,
1H), 2.11 (m, 2H), 2.58 (dd, J.sub.1=16.2 Hz, J.sub.2=10.2 Hz, 1H),
2.80-3.10 (m, 3H), 3.45 (d, J=6.3 Hz, 2H), 7.10 (m, 4H).
2-[(4-Methoxyphenethyl)aminomethyl]-1,2,3,4-tetrahydronaphthalene
(Compound 11)
[0060] A solution of 2-bromomethyl-1,2,3,4-tetrahydronaphthalene
(1.33 g, 5.91 mmol) and 4-methoxyphenethylamine (1.79 g, 11.8 mmol)
in 50 ml of EtOH was refluxed for 48 hours. After removal of EtOH
in vacuo, the residue was dissolved in 100 ml of CHCl.sub.3, washed
with 10% K.sub.2CO.sub.3, H.sub.2O, sat'd brine, and then dried
over Na.sub.2SO.sub.4. Filtration followed by evaporation of
solvent gave a yellow oil, which was purified by chromatography
(SiO.sub.2, MeOH:CHCl.sub.3, 5:95) to a give a yellowish oil (1.03
g, 58.9%). The product was converted to HCl salt, crystallization
with MeOH/Et.sub.2O gave a white powder. mp 274-275.degree. C.;
Calcd for C.sub.20H.sub.25NO.HCl: C, 72.37, H, 7.91, N, 4.22; Found
C, 72.40, H, 7.76, N, 4.13.
EXAMPLE 7
4,4-Diphenylpiperidine hydrochloride
[0061] A mixture of 4-piperidone monohydrate hydrochloride (15.0 g,
97.6 mmol, 1.00 equiv, Aldrich) and AlCl.sub.3 (130 g, 976 mmol,
10.0 equiv) in anhydrous benzene (600 mL) was stirred at reflux for
4 hours. Ice (300 g) and water (50 mL) were added, the mixture was
filtered, and the solid was washed with toluene and dried to afford
19.2 g (72%) of off-white solid, which was pure by .sup.1H NMR.
Recrystallization from ethanol gave the analytically pure sample:
m.p. 300-301.degree. C.; .sup.1H NMR (300 MHz, CD.sub.3OD)
.delta.2.65 (m, 4 H), 3.18 (m, 4 H), 7.18 (m, 2 H), 7.30 (m, 8 H);
Anal. Calcd. for C.sub.17H.sub.19N. HCl: C, 74.57; H. 7.36; N,
5.12. Found: C, 74.32; H, 7.34; N, 5.02. The free base was
generated by addition of the above salt to dilute aqueous sodium
hydroxide and extraction with CH.sub.2Cl.sub.2. The organic phase
was dried over MgSO.sub.4 and concentrated to give a light brown
solid: IR (neat) 2942.8, 1494.5, 1445.9 cm.sup.-1; CIMS (NH.sub.3)
m/e 238 (M+1).sup.+.
3-(4,4-Diphenylpiperidin-1-yl)propionitrile
[0062] To a suspension of 4,4-diphenylpiperidine hydrochloride (195
mg, 0.712 mmol, 1.0 equiv) in ETOh (1.5 mL) was added triethylamine
(0.25 mL, 1.83 mmol, 2.6 equiv) followed by acrylonitrile (0.13 mL,
2.01 mmol, 2.8 equiv). The resulting solution was stirred at room
temperature under argon for 15 minutes and then concentrated. Water
was added, and the mixture was extracted three times with EtOAc.
The combined organic extracts were dried over MgSO.sub.4 and
concentrated to give 170 mg (87%) of tan solid, which was used for
the next reaction without purification. m.p. 95-96.degree. C.;
.sup.1H NMR (300 MHz, CDCl.sub.3) .delta.2.37 (m, 2H), 2,46 (m,
4H), 2.52 (m, 6H), 7.12 (m, 2H), 7.23 (m, 8H); .sup.13C NMR (75
MHz, CDCl.sub.3) .delta.16.65, 36.71. 45.08, 50.78, 54.13, 119.70,
126.48, 127.78, 129.11, 147.87; IR (neat) 2944.4, 2821.0, 1495.5,
1445.9 cm.sup.-1.
1-(3-Aminopropyl)-4,4-diphenylpiperidine
[0063] To a stirred solution of
3-(4,4-diphenylpiperidine-1-yl)propionitri- le (2.00 g, 6.89 mmol,
1.0 equiv) in anhydrous THF (20 mL) under argon was added a
solution of BH.sub.3 in THF (1.0 M, 24.1 mL, 24 mmol, 3.5 equiv) at
room temperature. The mixture was refluxed for 4.5 hours and then
cooled to room temperature. Aqueous HCl (6 N, 50 mL) was added and
stirring was continued for 1 hour. The mixture was basified to pH 9
by addition of 6 N aq. NaOH, extracted 3 times with
CH.sub.2Cl.sub.2, dried over MgSO.sub.4 and concentrated. The
residue was purified by flash chromatography (SiO.sub.2,
EtOAc-MeOH, 9:1, followed by EtOAc-MeOH-isopropylamine (60:10:1),
followed by EtOAc-MeOH-isopropylamin- e (40:10:2) to give 1.35 g
(66%) of tan solid: m.p. 98-99.degree. C.; .sup.1H NMR (300 MHz,
CDCl.sub.3) .delta.1.64 (tt, J=7.7 Hz, 2H), 2.33 (br t, J=7.2 Hz,
2H), 2.50 (m, 8H), 2.76 (br t, J=6.5 Hz, 2H), 3.06 (br s, 2H), 7.13
(m, 2H), 7.26 (m, 8H); .sup.13C NMR (75 MHz, CDCl.sub.3)
.delta.29.79, 36.80, 41.41, 45.24, 51.25, 57.41, 126.30, 127.77,
128.97, 148.11; IR (neat) 3361.5 cm.sup.-1; CIMS (NH.sub.3) m/e 295
(M+1).sup.+.
Acetoacetic acid N-[3-(4,4-diphenylpiperidin-1-yl)propyl]amide
[0064] Diketene (0.44 mL, 5.68 mmol, 1.3 equiv, Aldrich) was added
at room temperature to a stirred solution of
1-(3-aminopropyl)-4-,4-diphenylpiper- idine (1.288 g, 4.37 mmol,
1.0 equiv) in anhydrous toluene (15 mL) under argon, and stirring
was continued for 48 hours. The mixture was concentrated to give
1.294 g (78%) of white solid, which was used for the next reaction
without purification: .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.1.70
(tt, J=6.4, 6.4 Hz, 2H), 2.23 (s, 3H), 2.44 (br t, J=6.5 Hz),
2.49-2.67 (m, 8H) , 3.32 (br t, J=5.8 Hz), 3.36 (s, 2H), 7.16 (m,
2H), 7.27 (m, 8H).
2,6-Dimethyl-4-(4-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylic
acid N-(3-(4,4-diphenylpiperidine-1-yl)propyl]amide methyl
ester
[0065] A solution of acetoacetic acid
N-[3-(4,4-diphenylpiperidin-1-yl)pro- pyl]amide (365 mg, 0.964
mmol, 1.0 equiv), methyl 3-aminocrotonate (138 mg, 1.20 mmol, 1.2
equiv, Aldrich), and 4-nitrobenzaldehyde (181 mg, 1.20 mmol, 1.2
equiv, Aldrich) in isopropanol was refluxed under argon for 60
hours. The mixture was cooled to room temperature and concentrated,
and the residue was diluted with CH.sub.2Cl.sub.2, washed with
water, dried over MgSO.sub.4, and concentrated. The residue was
purified by flash chromatography (SiO.sub.2, EtOAc, followed by
EtOAc-MeOH, 19:1 and 9:1) to give 147.8 mg (25%) of yellow solid:
.sup.1H NMR (300 MHz, CDCl.sub.3) .delta.1.55 (m, 2H), 2.14 (s, 3H)
, 2.15-2.50 (m, 10H) , 2.32 (s, 3H) , 3.20 (m, 1H), 3.37 (m, 1H),
3.54 (s, 3H), 5.00 (s, 3H), 5.48 (br s), 6.98 (br t, J=4.9 Hz, 1H),
7.14-7.30 (m, 10H), 7.39 (dm, J=8.7 Hz, 2H), 8.05 (dm, J=8.7 Hz,
2H); .sup.13C NMR (75 MHz, CDCl.sub.3) .delta.18.74, 20.64, 25.61,
36.77, 40.20, 42.26, 45.03, 51.16, 51.61, 58.08, 100.65, 109.71,
124.35, 126.46, 127.61, 128.84, 129.06, 135.52, 146.96, 147.10,
154.55, 168.22, 168.70; IR (neat) 1680, 1610, 1515, 1340 cm.sup.-1;
MS (FAB) m/e 609 (M+H).sup.+.
2,6-Dimethyl-4-(4-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylic
acid N-[3-(4,4-diphenylpiperidin-1-yl)-propyl]amide methyl ester
hydrochloride hydrate (Compound 2)
[0066] To a solution of
2,6-dimethyl-4-(4-nitrophenyl)-1,4-dihydro-pyridin-
e-3,5-dicarboxylic acid
N-[3-(4,4-diphenylpiperidin-1-yl)propyl]amide methyl ester (147.8
mg, 0.243 mmol, 1.0 equiv) in EtOH (2 mL) was added a solution of
HCl in ether (1.0 M, 0.24 mL, 0.24 mmol, 1.0 equiv). Addition of
ethyl acetate (3 mL) followed by heating gave a clear solution.
Slow cooling of this solution, followed by filtration gave 91 mg of
yellow crystalline solid: m.p. 182-183.degree. C.; Anal. Calcd. for
C.sub.36H.sub.40N.sub.4O.sub.5.HCI.H.sub.2O: C, 65.20, H, 6.54; N,
8.45. Found: C, 65.30; H, 6.28; N, 8.15.
EXAMPLE 8
3-(4,4-Diphenylpiperid-1-yl)-propanol
[0067] 4,4-Diphenylpiperidine (40 g), 3-bromopropanol (24.7 g,
Aldrich), powdered potassium carbonate (116.4 g) and approximately
1 g of potassium iodide (in 500 ml of a 1:1 mixture of dioxane and
1-butanol) were heated for about 48 hours under reflux and with
vigorous stirring. After cooling, the mixture was filtered, and the
filtrate was concentrated. The oily residue was taken up in ethyl
acetate, and the solution was filtered again. Concentrating the
filtrate to dryness yielded the product in the form of a yellowish,
oily residue which slowly solidifies to a wax-like product (yield:
44.8 g). Hydrochloric acid in ether produced the hydrochloride
(m.p.: 226.degree. to 227.degree. C.), which was recrystallized
from 2-propanol.
Acetoacetic acid 3-(4,4-diphenylpiperidin-1-yl)propyl ester
[0068] 23.6 g of 3-(4,4-diphenylpiperid-1-yl)-propanol were
dissolved in 100 ml of absolute toluene, and 16 ml of a 50%
strength solution of diketene in acetone were added with stirring.
After standing for several days at room temperature (monitored by
thin layer chromatography), the mixture was concentrated, and the
residue was dried under high vacuum. The pale yellow, viscous oil
which remains was employed without further purification for the
next stage.
2,6-Dimethyl-4-(4-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxy-ylic
acid [3-(4,4-diphenylpiperidin-1-yl)propyl] ester methyl ester
[0069] A solution of methyl 3-aminocrotonate (265 mg, 2.3 mmol, 1.0
equiv), 4-nitrobenzaldehyde (348 mg, 2.3 mmol, 1.0 equiv), and
acetoacetic acid 3-[4,4-diphenylpiperidin-1-yl)propyl] ester (872
mg, 2.3 mmol, 1.0 equiv) in isopropanol was refluxed under argon
with stirring for 68 hours. Cooling and removal of solvent gave a
residue, which was purified by flash chromatography (SiO.sub.2,
EtOAc-hexane, 1:1 and 1:2, followed by EtOAc) to afford 717 mg
(51%) of yellow solid: .sup.1H NMR (300 MHz, CDCl.sub.3)
.delta.1.73 (m, 2H), 2.22 (m, 2H), 2.30-2.51 (m, 8H) , 2.34 (s, 3H)
, 2.35 (s, 3H), 3.63 (s, 3H), 4.05 (dt, J=2.1, 7.9 Hz, 2H), 5.06
(s, 1H), 5.73 (br s, 1H), 7.14 (m, 2H), 7.27 (m, 8H), 7.42 (dm,
J=8.8 Hz, 2H) , 8.06 (dm, J=8.8 Hz, 2H) ; .sup.13C NMR (75 MHz,
CDCl.sub.3) .delta.15.30, 19.65, 26.32, 36.11, 39.88, 44.60, 50.60,
51.12, 55.34, 62.66, 102.99, 107.55, 123.39, 125.67, 127.12,
128.33, 128.65, 144.80, 144.93, 146.36, 147.50, 154.78, 166.91,
167.43; IR (neat) 1698.0, 1684.7, 1517.5, 1345.7 cm.sup.-1; CIMS
(NH.sub.3) 610 (M+1).sup.+, 553, 338.
2,6-Dimethyl-4-(4-nitrophenyl)-1 4-dihydropyridine-3,5-dicarboxylic
acid [3-(4,4-diphenylpiperidin-1-yl)propyl] ester methyl ester
hydrochloride (Compound 8)
[0070] To a solution of
2,6-dimethyl-4-(4-nitrophenyl)-1,4-dihydro-pyridin-
e-3,5-dicarboxylic acid [3-(4,4-diphenylpiperidine-1-yl)-propyl]
ester methyl ester (710 mg, 1.16 mmol, 1.0 equiv) in EtOH (5 mL)
was added a solution of HCl in ether (1.0 M, 1.5 mL, 1.5 mmol, 1.3
equiv). The solvents were removed and the residue was dissolved in
CH.sub.2Cl.sub.2. This solution was added dropwise to 25 mL of
ether to afford, after filtration, 500 mg of yellow crystalline
solid: m.p. 152-153.degree. C. Anal. Calcd. for
C.sub.36H.sub.39N.sub.3O.sub.6.HCl: C, 66.92; H, 6.24; N, 6.50.
Found: C, 66.70; H, 5.99; N, 6.27
EXAMPLE 9
Protocol for the Determination of the Potency of .alpha..sub.1
Antagonists
[0071] The activity of compounds at the different human receptors
was determined in vitro using cultured cell lines that selectively
express the receptor of interest. These cell lines were prepared by
transfecting the cloned cDNA or cloned genomic DNA or constructs
containing both genomic DNA and cDNA encoding the human
.alpha.-adrenergic, serotonin, histamine, and dopamine receptors as
follows:
[0072] .alpha..sub.1A Human Adrenergic Receptor: The entire coding
region of .alpha.1A (1719 bp) (Sequence I.D. No. 1), including 150
basepairs of 5' untranslated sequence (5' UT) and 300 bp of 3'
untranslated sequence (3' UT), was cloned into the BamHI and ClaI
sites of the polylinker-modified eukaryotic expression vector
pCEXV-3, called EXJ.HR. The construct involved the ligation of
partial overlapping human lymphocyte genomic and hippocampal cDNA
clones: 5' sequence were contained on a 1.2 kb SmaI-XhoI genomic
fragment (the vector-derived BamHI site was used for subcloning
instead of the internal insert-derived SmaI site) and 3' sequences
were contained on an 1.3 kb XhoI-ClaI cDNA fragment (the ClaI site
was from the vector polylinker). Stable cell lines were obtained by
cotransfection with the plasmid .alpha.1A/EXJ (expression vector
containing the .alpha.1A receptor gene) and the plasmid pGCcos3neo
(plasmid containing the aminoglycoside transferase gene) into
LM(tk.sup.-), CHO, and NIH3T3 cells, using calcium phosphate
technique. The cells were grown, in a controlled environment
(37.degree. C., 5% CO.sub.2), as monolayers in Dulbecco's modified
Eagle's Medium (GIBCO, Grand Island, N.Y.) containing 25 mM glucose
and supplemented with 10% bovine calf serum, 100 units/ml
penicillin g, and 100 .mu.g/ml streptomycin sulfate. Stable clones
were then selected for resistance to the antibiotic G-418 (1
mg/ml), and membranes were harvested and assayed for their ability
to bind [.sup.3H]prazosin as described below (see "Radioligand
Binding assays").
[0073] .alpha..sub.1B Human Adrenergic Receptor: The entire coding
region of .alpha.1B (1563 bp) (Sequence I.D. No. 3), including 200
basepairs and 5' untranslated sequence (5' UT) and 600 bp of 3'
untranslated sequence (3' UT), was cloned into the EcoRI site of
pCEXV-3 eukaryotic expression vector. The construct involved
ligating the full-length containing EcoRI brainstem cDNA fragment
from .lambda. ZapII into the expression vector. Stable cell lines
were selected as described above.
[0074] Human .alpha..sub.1C Adrenergic Receptor: The entire coding
region of .alpha.1C (1401 bp) (Sequence I.D. No. 5), including 400
basepairs of 5' untranslated sequence (5' UT) and 200 bp of 3'
untranslated sequence (3' UT), was cloned into the KpnI site of the
polylinker-modified pCEXV-3-derived eukaryotic expression vector,
EXJ.RH. The construct involved ligating three partial overlapping
fragments: a 5' 0.6 kb HincII genomic clone, a central 1.8 EcoRI
hippocampal cDNA clone, and a 3' 0.6 Kb PstI genomic clone. The
hippocampal cDNA fragment overlaps with the 5' and 3' genomic
clones so that the HincII and PstI sites at the 5' and 3' ends of
the cDNA clone, respectively, were utilized for ligation. This
full-length clone was cloned into the KpnI site of the expression
vector, using the 5' and 3' KpnI sites of the fragment, derived
from vector (i.e., pBluescript) and 3'-untranslated sequences,
respectively. Stable cell lines were selected as described
above.
[0075] Radioligand Binding Assays: Transfected cells from culture
flasks were scraped into 5 ml of 5 mM Tris-HCl, 5 mM EDTA, pH 7.5,
and lysed by sonication. The cell lysates were centrifuged at 1000
rpm for 5 min at 4.degree. C., and the supernatant was centrifuged
at 30,000.times.g for 20 min at 4.degree. C. The pellet was
suspended in 50 mM Tris-HCl, 1 mM MgCl.sub.2, and 0.1% ascorbic
acid at pH 7.5. Binding of the .alpha.1 antagonist
[.sup.3H]prazosin (0.5 nM, specific activity 76.2 Ci/mmol) to
membrane preparations of LM(tk-) cells was done in a final volume
of 0.25 ml and incubated at 37.degree. C. for 20 min. Nonspecific
binding was determined in the presence of 10 .mu.M phentolamine.
The reaction was stopped by filtration through GF/B filters using a
cell harvester. Inhibition experiments, routinely consisting of 7
concentrations of the tested compounds, were analyzed using a
non-linear regression curve-fitting computer program to obtain Ki
values.
[0076] .alpha..sub.2 Human Adrenergic Receptors: To determine the
potency of .alpha..sub.1 antagonists at the .alpha..sub.2
receptors, LM(tk-) cell lines stably transfected with the genes
encoding the .alpha..sub.2A, .alpha..sub.2B, and .alpha..sub.2C
receptors were used. The cell line expressing the .alpha..sub.2A
receptor is designated L-.alpha..sub.2A, and was deposited on Nov.
6, 1992 under ATCC Accession No. CRL 11180. The cell line
expressing the .alpha..sub.2B receptor is designated
L-NGC-.alpha..sub.2B, and was deposited on Oct. 25, 1989 under ATCC
Accession No. CRL10275. The cell line expressing the .alpha..sub.2C
receptor is designated L-.alpha..sub.2C, and was deposited on Nov.
6, 1992 under ATCC Accession No. CRL-11181. Cell lysates were
prepared as described above (see Radioligand Binding Assays), and
suspended in 25 mM glycylglycine buffer (pH 7.6 at room
temperature). Equilibrium competition binding assay were performed
using [3H]rauwolscine (0.5 nM), and nonspecific binding was
determined by incubation with 10 .mu.M phentolamine. The bound
radioligand was separated by filtration through GF/B filters using
a cell harvester.
[0077] Human Histamine H.sub.1 Receptor: The coding sequence of the
human histamine H.sub.1 receptor, homologous to the bovine H.sub.1
receptor, was obtained from a human hippocampal cDNA library, and
was cloned into the eukaryotic expression vector pCEXV-3. The
plasmid DNA for the H.sub.1 receptor is designated pcEXV-H1, and
was deposited on Nov. 6, 1992 under ATCC Accession No. 75346. This
construct was transfected into COS-7 cells by the DEAE-dextran
method. Cells were harvested after 72 hours and lysed by sonication
in 5 mM Tris-HCl, 5 mM EDTA, pH 7.5. The cell lysates were
centrifuged at 1000 rpm for 5 min at 4.degree. C., and the
supernatant was centrifuged at 30,000.times.g for 20 min. at
4.degree. C. The pellet was suspended in 37.8 mM NaHPO.sub.4, 12.2
mM KH.sub.2PO.sub.4, pH 7.5. The binding of the histamine H.sub.1
antagonist [.sup.3H]mepyramine (1 nM, specific activity: 24.8
Ci/mM) was done in a final volume of 0.25 ml and incubated at room
temperature for 60 min. Nonspecific binding was determined in the
presence of 10 .mu.M mepyramine. The bound radioligand was
separated by filtration through GF/B filters using a cell
harvester.
[0078] Human Histamine H.sub.2 Receptor: The coding sequence of the
human H.sub.2 receptor was obtained from a human placenta genomic
library, and cloned into the cloning site of PCEXV-3 eukaryotic
expression vector. The plasmid DNA for the H.sub.2 receptor is
designated pcEXV-H2, and was deposited on Nov. 6, 1992 under ATCC
Accession No. 75346. This construct was transfected into COS-7
cells by the DEAE-dextran method. Cells were harvested after 72
hours and lysed by sonication in 5 mM Tris-HCl, 5 mM EDTA, pH 7.5.
The cell lysates were centrifuged at 1000 rpm for 5 min at
4.degree. C., and the supernatant was centrifuged at 30,000.times.g
for 20 min at 4.degree. C. The pellet was suspended in 37.8 mM
NaHPO.sub.4, 12.2 mM K2PO.sub.4, pH 7.5. The binding of the
histamine H.sub.2 antagonist [.sup.3H]tiotidine (5 nM, specific
activity: 70 Ci/mM) was done in a final volume of 0.25 ml and
incubated at room temperature for 60 min. Nonspecific binding was
determined in the presence of 10 .mu.M histamine. The bound
radioligand was separated by filtration through GF/B filters using
a cell harvester.
[0079] Human Serotonin Receptors:
[0080] 5HT.sub.1D.alpha., 5HT.sub.1D.beta., 5HT.sub.1E, 5HT.sub.1F
Receptors: The cell lysates of LM(tk-) clonal cell line stably
transfected with the genes encoding each of these 5HT
receptor-subtypes were prepared as described above. The cell line
for the 5HT.sub.1D.alpha. receptor, designated as Ltk-8-30-84, was
deposited on Apr. 17, 1990, and accorded ATCC Accession No. CRL
10421. The cell for the 5HT.sub.1D.beta. receptor, designated as
Ltk-11, was deposited on Apr. 17, 1990, and accorded ATCC Accession
No. CRL 10422. The cell line for the 5HT.sub.1E receptor,
designated 5 HT.sub.1E-7, was deposited on Nov. 6, 1991, and
accorded ATCC Accession No. CRL 10913. The cell line for the
5HT.sub.1F receptor, designated L-5-HT.sub.1F, was deposited on
Dec. 27, 1991, and accorded ATCC Accession No. ATCC 10957. These
preparations were suspended in 50 mM Tris-HCl buffer (pH 7.4 at
37.degree. C.) containing 10 mM MgCl.sub.2, 0.2 mM EDTA, 10 .mu.M
pargyline, and 0.1% ascorbate. The potency of .alpha..sub.1
antagonists was determined in competition binding assay by
incubation for 30 minutes at 37.degree. C. in the presence of 5 nM
[3H]serotonin. Nonspecific binding was determined in the presence
of 10 .mu.M serotonin. The bound radioligand was separated by
filtration through GF/B filters using a cell harvester.
[0081] Human 5HT.sub.2 Receptors: The coding sequence of the human
5HT.sub.2 receptor was obtained from a human brain cortex cDNA
library, and cloned into the cloning site of pCEXV-3 eukaryotic
expression vector. This construct was transfected into COS-7 cells
by the DEAE-dextran method. Cells were harvested after 72 hours and
lysed by sonication in 5 mM Tris-HCl, 5 mM EDTA, pH 7.5. This cell
line was deposited with the ATCC on Oct. 31, 1989, designated as
L-NGC-5HT.sub.2, and was accorded ATCC Accession No. CRL 10287. The
cell lysates were centrifuged at 1000 rpm for 5 minutes at
4.degree. C., and the supernatant was centrifuged at 30,000.times.g
for 20 minutes at 4.degree. C. The pellet was suspended in 50 mM
Tris-HCl buffer (pH 7.7 at room temperature) containing 10 mM
MgSO.sub.4, 0.5 mM EDTA, and 0.1% ascorbate. The potency of alpha-1
antagonists at 5HT2 receptors was determined in equilibrium
competition binding assays using [3H]ketanserin (1 nM). Nonspecific
binding was defined by the addition of 10 .mu.M mianserin. The
bound radioligand was separated by filtration through GF/B filters
using a cell harvester.
[0082] Human Dopamine D2 Receptors: The potency of .alpha..sub.1
antagonists at the D2 receptor was determined using membrane
preparations from COS-7 cells transfected with the gene encoding
the human D2 receptor. The coding region for the human D2 receptor
was obtained from a human striatum cDNA library, and cloned into
the cloning site of PCDNA 1 eukariotic expression vector. The
plasmid DNA for the D.sub.2 receptor is designated pcEXV-D2, and
was deposited on Nov. 6, 1992 under ATCC Accession No. ATC 75344.
This construct was transfected into COS-7 cells by the DEAE-dextran
method. Cells were harvested after 72 hours and lysed by sonication
in 5 mM Tris-HCl, 5 mM EDTA, pH 7.5. The cell lysates were
centrifuged at 1000 rpm for 5 minutes at 4.degree. C., and the
supernatant was centrifuged at 30,000.times.g for 20 minutes at
4.degree. C. The pellet was suspended in 50 mM Tris-HCl (pH 7.4)
containing 1 mM EDTA, 5 mM KCl, 1.5 mM CaCl.sub.2, 4 mM MgCl.sub.2,
and 0.1% ascorbic acid. The cell lysates were incubated with
[3H]spiperone (2 nM), using 10 .mu.M (+)Butaclamol to determine
nonspecific binding.
[0083] Other Dopamine receptors are prepared by known methods
(D.sub.3: Sokoloff, P. et al., Nature, 347, 146 (1990), and
deposited with the European Molecular Biological Laboratory (EMBL)
Genbank as X53944; D.sub.4: Van Tol, H. H. M., et al., Nature, 350,
610 (1991), and deposited with EMBL Genbank as X58497; D.sub.5:
Sunahara, R. K., et al., Nature, 350, 614 (1991), and deposited
with EMBL Genbank as X58454-HU HD 5DR).
[0084] Determination of the Activity of .alpha..sub.1 Antagonists
at Calcium Channels
[0085] The potency of .alpha..sub.1 antagonists at calcium channels
was determined in competition binding assays of [3H]nitrendipine to
membrane fragments of rat cardiac muscle, essentially as described
by Glossman and Ferry (Methods in Enzymology 109:513-550, 1985).
Briefly, the tissue was minced and homogenized in 50 mM Tris-HCl
(pH 7.4) containing 0.1 mM phenylmethylsulfonyl fluoride. The
homogenates were centrifuged at 1000 g for 15 minutes, the
resulting supernatant was centrifuged at 45,000 g for 15 minutes.
The 45,000 g pellet was suspended in buffer and centrifuged a
second time. Aliquots of membrane protein were incubated for 30
minutes at 37.degree. C. in the presence of [3H]nitrendipine (1
nM), and nonspecific binding was determined in the presence of 10
.mu.M nifedipine. The bound radioligand was separated by filtration
through GF/B filters using a cell harvester.
EXAMPLE 10
Functional Properties of .alpha..sub.1 Antagonists in the Human
Prostate
[0086] The efficacy of .alpha..sub.1 adrenergic antagonists for the
treatment of benign prostatic hyperplasia (BPH) is related to their
ability to elicit relaxation of prostate smooth muscle. An index of
this efficacy can be obtained by determining the potency of
.alpha..sub.1 antagonists to antagonize the contraction of human
prostatic tissue induced by an .alpha..sub.1 agonist "in vitro".
Furthermore, by comparing the potency of subtype selective
.alpha..sub.1 antagonists in binding assays using human
.alpha..sub.1 receptors with their potency to inhibit
agonist-induced smooth muscle contraction, it is possible to
determine which of the .alpha..sub.1 adrenergic receptor subtypes
is involved in the contraction of prostate smooth muscle.
[0087] Methods: Prostatic adenomas were obtained at the time of
surgery from patients with symptomatic BPH. These were cut into
longitudinal strips of 15 mm long and 2-4 mm wide, and suspended in
5 ml organ baths containing Krebs buffer (pH 7.4). The baths were
maintained at 37.degree. C. and continuously oxygenated with 5%
CO.sub.2 and 95% O.sub.2. Isometric tension was measured with a
Grass Instrument FT03 force transducer interfaced with a computer.
Tissue strips were contracted with varying concentrations of
phenylephrine after incubating for 20 minutes in the absence and
presence of at least three different concentrations of antagonist.
Dose-response curves for phenylephrine were constructed, and the
antagonist potency (pA.sub.2) was estimated by the dose-ratio
method. The concentration of some antagonists in the tissue bath
was assessed by measuring the displacement of [3H]prazosin by
aliquots of the bath medium, using membrane preparations of the
cloned human .alpha..sub.1C receptor. This control was necessary to
account for losses of antagonist due to adsorption to the tissue
bath and/or metabolism during the time the antagonists were
equilibrated with the prostate tissue.
[0088] Results:
[0089] Table 1 shows that the pA.sub.2 values measured for a series
of .alpha..sub.1 antagonists in human prostate tissue correlate
closely (r=0.76) with the corresponding pK.sub.i values measured in
the .alpha..sub.1C receptor assays. In contrast, the human prostate
pA.sub.2 values correlate poorly with the pK.sub.i values measured
at the .alpha..sub.1A (r=-0.06) and .alpha..sub.1B (r=-0.24)
adrenergic receptors. (See FIG. 2.) Thus, antagonists which are
more potent at blocking the .alpha..sub.1C adrenergic receptor are
more effective at blocking the contraction of the human prostate
than antagonists which are more potent at the .alpha..sub.1A or
.alpha..sub.1 adrenergic receptors. In addition, antagonists which
are selective for the .alpha..sub.1C receptor will have a better
therapeutic ratio than nonselective .alpha. antagonists.
[0090] With SNAP 5036 (11), the low pA.sub.2 observed in the
prostate may be attributed to tissue absorption or metabolism.
[0091] Table 2 illustrates the cross reactivity of .alpha..sub.1
antagonists at other receptors such as .alpha..sub.2A,
.alpha..sub.2B, .alpha..sub.2C, histamine H.sub.1, H.sub.2,
serotonin 5-HT.sub.1D.alpha., 5-HT.sub.1D.beta., 5-HT.sub.1E,
5-HT.sub.1F, 5-HT.sub.2, and dopamine D.sub.2. Only compounds SNAP
5036, 5041, and 5089 have binding affinities which are greater than
ten-fold higher at .alpha..sub.1C receptors than the binding
affinities at other receptors.
2TABLE 1 COMPARISON OF THE BINDING POTENCY (pK.sub.1) of ALPHA-1
ANTAGONISTS IN CLONED HUMAN RECEPTORS AND THEIR PROTENCY (pA.sub.2)
TO INHIBIT PROSTATE SMOOTH MUSCLE CONTRACTION Human Alpha-1
Adrenergic (pK.sub.1) Human Compound a1A a1B a1C Prostate (pA) 1
Prazosin 9.48 9.26 9.23 9.08 2 Compound 2 5.98 6.57 8.87 8.94 3
A-30360 7.49 7.86 8.52 8.72 4 5-Methyl-Urapidil 7.79 6.77 8.35 8.38
5 Indoramin 6.74 7.39 8.35 7.86 6 SKF-104856 8.48 7.50 7.60 7.66 7
Compound 7 6.82 7.18 8.42 7.63 8 Compound 8 6.52 7.07 8.48 7.46 9
Compound 9 6.12 6.76 7.83 7.41 10 Terazosin 8.46 8.71 8.16 7.30 11
Compound 11 6.81 7.14 8.36 6.64
[0092]
3TABLE 2 CROSS REACTIVITY OF ALPHA-1 ANTAGONISTS AT CLONED HUMAN
RECETORS (pK,) Alpha-1 Adrenergic Alpha-2 Adrenergic Histamine
Serotonin Dopamine Calcium Compound a1A a1B a1C a2a a2b a2c H1 H2
5HT1Da 5HT1Db 5HT1E 5HT1F 5HT2 D2 Channel Terazosin 8.46 8.71 8.16
6.26 7.51 6.64 4.00 5.04 <6.0 <6.0 <5.0 <5.0 <5.0
<5.0 5.19 Prazosin 9.48 9.26 9.23 6.76 7.64 7.65 4.00 5.19
<5.0 <5.0 ND ND <6.0 <5.0 4.57 5-Methylurapidil 7.79
6.77 8.35 6.63 7.38 6.88 5.16 4.47 7.30 6.82 ND ND <6.0 <5.0
ND Indoramin 6.74 7.39 8.35 4.94 5.72 5.22 7.37 5.63 <6.0
<6.0 <5.0 <5.0 <7.0 <8.0 4.53 Compound 11 6.81 7.14
8.36 6.86 6.90 6.92 5.74 7.45 <6.0 <6.0 <5.0 <5.0
<7.0 <6.0 5.18 A-30360 7.49 7.86 8.52 6.69 6.37 6.23 6.03
5.77 <6.0 <6.0 <5.0 <5.0 <8.0 <9.0 5.26 Compound
7 6.82 7.18 8.42 6.19 6.07 6.09 7.59 6.02 <6.0 <5.0 <5.0
<5.0 <6.0 <7.0 4.79 Compound 9 6.12 6.76 7.83 5.80 5.69
5.90 7.29 5.44 <6.0 <6.0 <5.0 <5.0 <7.0 <7.0 4.44
SKF-104856 8.48 7.50 7.60 7.30 8.49 7.60 5.59 5.84 <7.0 <7.0
<6.0 <7.0 <6.0 <7.0 4.68 S-Niguldipine 6.72 7.07 8.75
6.19 5.24 6.43 6.78 6.24 ND ND ND ND <7.0 <7.0 8.04 Compound
8 6.52 7.07 8.48 5.99 6.12 5.77 6.67 6.11 <6.0 <5.0 <5.0
<5.0 <7.0 <6.0 6.87 Compound 2 5.98 6.57 8.87 5.48 5.93
5.88 7.16 7.48 <7.0 <6.0 <5.0 <5.0 <6.0 <7.0 6.13
ND = Not Determined
EXAMPLE 11
Functional Properties of .alpha..sub.1 Antagonists on Rat
Orthostatic Hypertension
[0093] We have identified a large series of compounds (well over
150 compounds, data not shown) which exemplify the hereinabove
described properties of antagonists highly selective for the
.alpha..sub.1C adrenergic receptor. That is, these compounds are
highly selective Alpha 1c antagonists which have less than 10 fold
the affinity at cloned human Alpha 1a, Alpha 1b, Alpha 2a, Alpha
2b, Alpha 2c, Histamine H1, Dopamine D2 and Serotonin receptors. In
addition, these compounds have 10 fold lower affinity at calcium
channels (data not shown). We designated five of these highly
selective antagonists for the .alpha..sub.1C adrenergic receptor as
drugs 21-25 and used them to further characterize highly selective
antagonists for the .alpha..sub.1C adrenergic receptor.
[0094] In addition, a number of these selective alpha 1c
antagonists are potent at inhibiting the phenylephrine stimulated
contraction of human prostate as described in Example 10. This is a
well established protocol for evaluation the efficacy of drugs
which may be useful for the treatment of BPH.
[0095] In addition, we have examined a number of selective alpha 1c
antagonists in an in vivo canine prostate model (Felson, D., et
al., J. Urol., 141, 1230-1233 (1989))which is a well characterized
model for evaluating the efficacy of BPH drugs (data not shown). In
this model, selective alpha 1c antagonists increase urethral
pressure at doses which do not produce significant decreases in
canine blood pressure. In contrast, nonselective alpha 1
antagonists do not have as large a separation between the effects
on urethral pressure and the effects on blood pressure. These
observations support our premise that a selective alpha 1c
antagonist will have a better safety profile than a nonselective
alpha 1 antagonist. We have further characterized selective alpha
1c antagonists in a rat orthostatic hypotension model. This model
gives information on the vascular effects of drugs which may be
indicative of their ability to produce dizziness in patients
(Hieble, J. P., et al., Cardiovascular Pharmacology, 15, 845
(1990)). Our objective was to characterize the effects of selective
alpha 1c antagonists on rat orthostatic hypotension and contrast
the results with those obtained using nonselective alpha 1
antagonists.
[0096] Methods
[0097] Rat Orthostatic Hypotension Model
[0098] Adult male Sprague-Dawley normotensive rats were
anesthetized with sodium pentobarbital (45 mg/kg, i.v.). The
femoral vein and artery of the right hindlimb were cannulated for
drug administration and blood pressure monitoring, respectively.
Heart rate was determined by a cardiotachometer triggered by the
blood pressure pulse. The rats were secured in the supine position
to a board that could be tilted 90 degrees. When blood pressure and
heart rate had stabilized, the rats were subjected to a 90 degree
vertical (head up) tilt for 60 seconds. Changes in blood pressure
and heart rate from pre-tilt levels were monitored continuously.
The rats were returned to the supine position and blood pressure
and heart rate were allowed to stabilize. Either an antagonist
selective for the .alpha..sub.1C adrenergic receptor (designated
drug 21, 22, 23, 24 or 25), an antagonist nonselective for the
.alpha..sub.1C adrenergic receptor (Prazosin or Terazosin) or
saline was then administered through venous cannula, either as an
i.v. bolus or as an infusion. When blood pressure had stabilized,
the rats were subjected to a second tilt and blood pressure and
heart rate were recorded as described above. Most saline treated
rats typically exhibit a greater ability to return their blood
pressure toward pre-tilt levels during the second tilt. Data from
the second tilt are used in statistical analysis.
[0099] Results
[0100] Table 3 shows that while nonselective alpha 1 antagonists
produce significant effects on orthostatic hypotension, selective
alpha 1c antagonists do not produce significant effects. More
specifically, Prazosin and Terazosin consistently cause orthostasis
at the lowest dose (10 ug/kg) and, in some rats, in a
dose-dependent manner. Drug 21 causes orthostasis only at the
highest dose (1000 ug/kg) in 2 out of 4 rats, while the other
antagonists selective for the .alpha..sub.1C adrenergic receptor
caused no orthostasis at the highest dose. Placebo and 22, 23, 24,
25 did not induce orthostasis at any dose. Taken all together, this
is a positive result since it is believed that orthostatic
hypotension contributes to the dizziness observed clinically with
nonselective alpha 1 antagonists. This further supports our premise
that a selective alpha 1c antagonist will have a better safety
profile than a nonselective alpha 1 antagonist.
4TABLE 3 Summary of Studies on Drug Effects on Orthostasis Dose 1
Dose 2 Dose 3 10 ug/kg 100 ug/kg 1000 ug/kg orthostatic BP
orthostatic BP orthostatic BP Drug n fall in BP fall fall in BP
fall fall in BP fall Notes Placebo (DMSO) 3 - - - - - - Prezosin 4
+ + ++ or +++ ++ ++ or +++ +++ Terazosin 2 + + ++ or +++ ++ ++ or
+++ +++ 21 4 - + - ++ +/- +++ (+ in 2/4) 22 3 - + - ++ - +++ 23 6 -
- - - - + 24 6 - - - +/- - + 25 4 - - +/- - - - (+ in 1/4) + and -
mean positive or negative findings, respectively +, ++ and +++ are
relative to doses of the same drug but not compared to other drugs
+/- positive findings found in some rats
[0101]
Sequence CWU 1
1
* * * * *