U.S. patent application number 11/903147 was filed with the patent office on 2008-03-20 for compounds specific to adenosine a1 receptors and uses thereof.
This patent application is currently assigned to OSI Pharmaceuticals, Inc.. Invention is credited to Arlindo L. Castelhano, Bryan McKibben, David J. Witter.
Application Number | 20080070936 11/903147 |
Document ID | / |
Family ID | 26667435 |
Filed Date | 2008-03-20 |
United States Patent
Application |
20080070936 |
Kind Code |
A1 |
Castelhano; Arlindo L. ; et
al. |
March 20, 2008 |
Compounds specific to adenosine A1 receptors and uses thereof
Abstract
This invention pertains to compounds which specifically inhibit
the adenosine A.sub.1 receptor and the use of these compounds to
treat a disease associated with A.sub.1 adenosine receptors in a
subject.
Inventors: |
Castelhano; Arlindo L.; (New
City, NY) ; McKibben; Bryan; (White Plains, NY)
; Witter; David J.; (Putman Valley, NY) |
Correspondence
Address: |
John P. White, Esq.;Cooper & Dunham LLP
1185 Avenue of the Americas
New York
NY
10036
US
|
Assignee: |
OSI Pharmaceuticals, Inc.
|
Family ID: |
26667435 |
Appl. No.: |
11/903147 |
Filed: |
September 20, 2007 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10718280 |
Nov 20, 2003 |
|
|
|
11903147 |
Sep 20, 2007 |
|
|
|
10000280 |
Nov 30, 2001 |
6680324 |
|
|
10718280 |
Nov 20, 2003 |
|
|
|
60250895 |
Dec 1, 2000 |
|
|
|
Current U.S.
Class: |
514/265.1 |
Current CPC
Class: |
C07D 487/04 20130101;
A61P 11/00 20180101; A61K 31/519 20130101; A61P 9/00 20180101; A61P
25/00 20180101 |
Class at
Publication: |
514/265.1 |
International
Class: |
A61K 31/519 20060101
A61K031/519; A61P 11/00 20060101 A61P011/00; A61P 25/00 20060101
A61P025/00; A61P 9/00 20060101 A61P009/00 |
Claims
1-60. (canceled)
61. A method for treating a subject afflicted with a disease
associated with an A.sub.1 adenosine receptor in need of such
treatment, comprising administering to the subject a
therapeutically effective amount of a compound having the
structure: or ##STR289## or a ##STR290## pharmaceutically
acceptable salt thereof so as to thereby treat the subject, wherein
the disease is antidiuresis, bradycardia, bronchitis,
bronchoconstriction, Alzheimer's disease, cardiac arrythmias,
cardiac hypoxia, congestive heart failure, hypertension,
inflammation, negative cardiac inotropy and dromotropy, renal
failure, sedation or is associated with transmitter release,
respiratory epithelia, contraction of smooth muscle underlying
respiratory epithelia, vasoconstriction or mast cell
degranulation.
62. The method of claim 61, wherein the subject is a mammal.
63. The method of claim 62, wherein the mammal is a human.
64. The method of claim 61, wherein the disease is congestive heart
failure.
65. A method for inhibiting the activity of an A1 adenosine
receptor in a cell, which comprises contacting the cell with a
compound having the structure: ##STR291## or a pharmaceutically
acceptable salt thereof.
66. The method of claim 65, wherein the cell is a human cell.
67. A method for treating a subject having a respiratory disorder
associated with the A.sub.1 adenosine receptor in need of such
treatment, comprising administering to the subject a
therapeutically effective amount of a compound having the
structure: ##STR292## or a pharmaceutically acceptable salt
thereof, so as to thereby treat the subject.
68. The method of claim 67, wherein the respiratory disorder is
asthma, chronic obstructive pulmonary disease, allergic rhinitis,
or an upper respiratory disorder.
69. The method of claim 67, wherein the subject is a human.
70. A pharmaceutical composition comprising a compound having the
structure: ##STR293## or a pharmaceutically acceptable salt
thereof, and a pharmaceutically acceptable carrier.
71. The pharmaceutical composition of claim 70, further comprising
at least one of either a steroid, .beta.2 agonist, glucocorticoid,
leukotriene antagonist, or an anticolinergic agonist.
72. The pharmaceutical composition of claim 70, wherein the
pharmaceutical composition is formulated for administration as a
periocular, retrobulbar or intraocular injection.
73. The pharmaceutical composition of claim 70, wherein the
pharmaceutical composition is formulated for systemic
administration.
74. The pharmaceutical composition of claim 70, wherein the
pharmaceutical composition is formulated for administration as a
surgical irrigating solution.
75. A packaged pharmaceutical composition for treating a subject
suffering from a disease associated with an A1 adenosine receptor,
comprising the pharmaceutical composition of claim 70 and
instructions for using the composition for treating the subject.
Description
[0001] This application is a continuation of U.S. application Ser.
No. 10/000,280, filed Nov. 30, 2001, which claims the benefit of
U.S. Provisional Application No. 60/250,895, filed Dec. 1, 2000,
the entire contents of which are hereby incorporated by
reference.
[0002] Provisional Application No. 60/250,895, filed Dec. 1, 2000,
the entire contents of which are hereby incorporated by
reference.
BACKGROUND OF THE INVENTION
[0003] Adenosine is an ubiquitous modulator of numerous
physiological activities, particularly within the cardiovascular
and nervous systems. The effects of adenosine appear to be mediated
by specific cell surface receptor proteins. Adenosine modulates
diverse physiological functions including induction of sedation,
vasodilation, suppression of cardiac rate and contractility,
inhibition of platelet aggregability, stimulation of
gluconeogenesis and inhibition of lipolysis. In addition to its
effects on adenylate cyclase, adenosine has been shown to open
potassium channels, reduce flux through calcium channels, and
inhibit or stimulate phosphoinositide turnover through
receptor-mediated mechanisms (See for example, C. E. Muller and B.
Stein "Adenosine Receptor Antagonists: Structures and Potential
Therapeutic Applications," Current Pharmaceutical Design, 2:501
(1996) and C. E. Muller "A.sub.1-Adenosine Receptor Antagonists,"
Exp. Opin. Ther. Patents 7(5):419 (1997)).
[0004] Adenosine receptors belong to the superfamily of purine
receptors which are currently subdivided into P.sub.1 (adenosine)
and P.sub.2 (ATP, ADP, and other nucleotides) receptors. Four
receptor subtypes for the nucleoside adenosine have been cloned so
far from various species including humans. Two receptor subtypes
(A.sub.1 and A.sub.2a) exhibit affinity for adenosine in the
nanomolar range while two other known subtypes A.sub.2b and A.sub.3
are low-affinity receptors, with affinity for adenosine in the
low-micromolar range. A.sub.1 and A.sub.3 adenosine receptor
activation can lead to an inhibition of adenylate cyclase activity,
while A.sub.2a and A.sub.2b activation causes a stimulation of
adenylate cyclase.
[0005] A few A.sub.1 antagonists have been developed for the
treatment of cognitive disease, renal failure, and cardiac
arrhythmias. It has been suggested that A.sub.2a antagonists may be
beneficial for patients suffering from Morbus Parkinson
(Parkinson's disease). Particularly in view of the potential for
local delivery, adenosine receptor antagonists may be valuable for
treatment of allergic inflammation and asthma. Available
information (for example, Nyce & Metzger "DNA antisense Therapy
for Asthma in an Animal Model" Nature (1997) 385: 721-5) indicates
that in this pathophysiologic context, A.sub.1 antagonists may
block contraction of smooth muscle underlying respiratory
epithelia, while A.sub.2b or A.sub.3 receptor antagonists may block
mast cell degranulation, mitigating the release of histamine and
other inflammatory mediators. A.sub.2b receptors have been
discovered throughout the gastrointestinal tract, especially in the
colon and the intestinal epithelia. It has been suggested that
A.sub.2b receptors mediate cAMP response (Strohmeier et al., J.
Bio. Chem. (1995) 270:2387-94).
[0006] Adenosine receptors have also been shown to exist on the
retinas of various mammalian species including bovine, porcine,
monkey, rat, guinea pig, mouse, rabbit and human (See, Blazynski et
al., "Discrete Distributions of Adenosine Receptors in Mammalian
Retina," Journal of Neurochemistry, volume 54, pages 648-655
(1990); Woods et al., "Characterization of Adenosine
A.sub.1-Receptor Binding Sites in Bovine Retinal Membranes,"
Experimental Eye Research, volume 53, pages 325-331 (1991); and
Braas et al., "Endogenous adenosine and adenosine receptors
localized to ganglion cells of the retina," Proceedings of the
National Academy of Science, volume 84, pages 3906-3910 (1987)).
Recently, Williams reported the observation of adenosine transport
sites in a cultured human retinal cell line (Williams et al.,
"Nucleoside Transport Sites in a Cultured Human Retinal Cell Line
Established By SV-40 T Antigen Gene," Current Eye Research, volume
13, pages 109-118 (1994)).
[0007] Compounds which regulate the uptake of adenosine have
previously been suggested as potential therapeutic agents for the
treatment of retinal and optic nerve head damage. In U.S. Pat. No.
5,780,450 to Shade, Shade discusses the use of adenosine uptake
inhibitors for treating eye disorders. Shade does not disclose the
use of specific A.sub.3 receptor inhibitors. The entire contents of
U.S. Pat. No. 5,780,450 are hereby incorporated herein by
reference.
[0008] Additional adenosine receptor antagonists are needed as
pharmacological tools and are of considerable interest as drugs for
the above-referenced disease states and/or conditions.
SUMMARY OF THE INVENTION
[0009] The present invention is based on compounds which
selectively bind to adenosine A.sub.1 receptor, thereby treating a
disease associated with A.sub.1 adenosine receptor in a subject by
administering to the subject a therapeutically effective amount of
such compounds. The diseases to be treated are associated with
cognitive disease, renal failure, cardiac arrhythmias, respiratory
epithelia, transmitter release, sedation, vasoconstriction,
bradycardia, negative cardiac inotropy and dromotropy,
branchoconstriction, neutropil chemotaxis, reflux condition, or
ulcerative condition.
[0010] The present invention is based, at least in part, on the
discovery that certain N-6 substituted 7-deazapurines, described
infra, can be used to treat a N-6 substituted 7-deazapurine
responsive state. Examples of such states include those in which
the activity of the adenosine receptors is increased, e.g.,
bronchitis, gastrointestinal disorders, or asthma. These states can
be characterized in that adenosine receptor activation can lead to
the inhibition or stimulation of adenylate cyclase activity.
Compositions and methods of the invention include enantiomerically
or diastereomerically pure N-6 substituted 7-deazapurines.
Preferred N-6 substituted 7-deazapurines include those which have
an acetamide, carboxamide, substituted cyclohexyl, e.g.,
cyclohexanol, or a urea moiety attached to the N-6 nitrogen through
an alkylene chain.
[0011] The present invention pertains to methods for modulating an
adenosine receptor(s) in a mammal by administering to the mammal a
therapeutically effective amount of a N-6 substituted
7-deazapurine, such that modulation of the adenosine receptor's
activity occurs. Suitable adenosine receptors include the families
of A.sub.1, A.sub.2, or A.sub.3 receptors. In a preferred
embodiment, the N-6 substituted 7-deazapurine is an adenosine
receptor antagonist.
[0012] The invention further pertains to methods for treating N-6
substituted 7-deazapurine disorders, e.g., asthma, bronchitis,
allergic rhinitis, chronic obstructive pulmonary disease, renal
disorders, gastrointestinal disorders, and eye disorders, in a
mammal by administering to the mammal a therapeutically effective
amount of a N-6 substituted 7-deazapurine, such that treatment of
the disorder in the mammal occurs. Suitable N-6 substituted 7
deazapurines include those illustrated by the general formula I:
##STR1## and pharmaceutically acceptable salts thereof. R.sub.1 and
R.sub.2 are each independently a hydrogen atom or a substituted or
unsubstituted alkyl, aryl, or alkylaryl moiety or together form a
substituted or unsubstituted heterocyclic ring. R.sub.3 is a
substituted or unsubstituted alkyl, aryl, or alkylaryl moiety.
R.sub.4 is a hydrogen atom or a substituted or unsubstituted alkyl,
aryl, or alkylaryl moiety. R.sub.5 and R.sub.6 are each
independently a halogen atom, e.g., chlorine, fluorine, or bromine,
a hydrogen atom or a substituted or unsubstituted alkyl, aryl, or
alkylaryl moiety or R.sub.5 is carboxyl, esters of carboxyl, or
carboxamides, or R.sub.4 and R.sub.5 or R.sub.5 and R.sub.6
together form a substituted or unsubstituted heterocyclic or
carbocyclic ring.
[0013] In certain embodiments, R.sub.1 and R.sub.2 can each
independently be a substituted or unsubstituted cycloalkyl or
heteroarylalkyl moieties. In other embodiments, R.sub.3 is a
hydrogen atom or a substituted or unsubstituted heteroaryl moiety.
In still other embodiments, R.sub.4, R.sub.5 and R.sub.6 can each
independently be heteroaryl moieties. In a preferred embodiment,
R.sub.1 is a hydrogen atom, R.sub.2 is a cyclohexanol, e.g.,
trans-cyclohexanol, R.sub.3 is phenyl, R.sub.4 is a hydrogen atom,
R.sub.5 is a methyl group and R.sub.6 is a methyl group. In still
another embodiment, R.sub.1 is a hydrogen atom, R.sub.2 is ##STR2##
R.sub.3 is phenyl, R.sub.4 is a hydrogen atom and R.sub.5 and
R.sub.6 are methyl groups.
[0014] The invention further pertains to pharmaceutical
compositions for treating a N-6 substituted 7-deazapurine
responsive state in a mammal, e.g., asthma, bronchitis, allergic
rhinitis, chronic obstructive pulmonary disease, renal disorders,
gastrointestinal disorders, and eye disorders. The pharmaceutical
composition includes a therapeutically effective amount of a N-6
substituted 7-deazapurine and a pharmaceutically acceptable
carrier.
[0015] The present invention also pertains to packaged
pharmaceutical compositions for treating a N-6 substituted
7-deazapurine responsive state in a mammal. The packaged
pharmaceutical composition includes a container holding a
therapeutically effective amount of at least one N-6 substituted
7-deazapurine and instructions for using the N-6 substituted
7-deazapurine for treating a N-6 substituted 7-deazapurine
responsive state in a mammal.
[0016] The invention further pertains to compounds of formula I
wherein [0017] R.sub.1 is hydrogen; [0018] R.sub.2 is substituted
or unsubstituted cycloalkyl, substituted or unsubstituted alkyl, or
R.sub.1 and R.sub.2 together form a substituted or unsubstituted
heterocyclic ring; [0019] R.sub.3 is unsubstituted or substituted
aryl; [0020] R.sub.4 is hydrogen; and [0021] R.sub.5 and R.sub.6
are each independently hydrogen or alkyl, and pharmaceutically
acceptable salts thereof. The deazapurines of this embodiment may
advantageously be selective A.sub.3 receptor antagonists. These
compounds may be useful for numerous therapeutic uses such as, for
example, the treatment of asthma, kidney failure associated with
heart failure, and glaucoma. In a particularly preferred
embodiment, the deazapurine is a water soluble prodrug that is
capable of being metabolized in vivo to an active drug by, for
example, esterase catalyzed hydrolysis.
[0022] In yet another embodiment, the invention features a method
for inhibiting the activity of an adenosine receptor (e.g.,
A.sub.3) in a cell, by contacting the cell with N-6 substituted
7-deazapurine (e.g., preferably, an adenosine receptor
antagonist).
[0023] In another aspect, the invention features a method for
treating damage to the eye of an animal (e.g., a human) by
administering to the animal an effective amount of an N-6
substituted 7-deazapurine of formula I. Preferably, the N-6
substituted 7-deazapurine is an antagonist of A.sub.3 adenosine
receptors in cells of the animal. The damage is to the retina or
the optic nerve head and may be acute or chronic. The damage may be
the result of, for example, glaucoma, edema, ischemia, hypoxia or
trauma.
[0024] The invention also features a pharmaceutical composition
comprising a N-6 substituted compound of formula I. Preferably, the
pharmaceutical preparation is an ophthalmic formulation (e.g., an
periocular, retrobulbar or intraocular injection formulation, a
systemic formulation, or a surgical irrigating solution).
[0025] In yet another embodiment, the invention features a compound
having the formula II: ##STR3## wherein X is N or CR.sub.6; R.sub.1
and R.sub.2 are each independently hydrogen, or substituted or
unsubstituted alkoxy, aminoalkyl, alkyl, aryl, or alkylaryl, or
together form a substituted or unsubstituted heterocyclic ring,
provided that both R.sub.1 and R.sub.2 are both not hydrogen;
R.sub.3 is substituted or unsubstituted alkyl, arylalkyl, or aryl;
R.sub.4 is hydrogen or substituted or unsubstituted C.sub.1-C.sub.6
alkyl; L is hydrogen, substituted or unsubstituted alkyl, or
R.sub.4 and L together form a substituted or unsubstituted
heterocyclic or carbocyclic ring; R.sub.6 is hydrogen, substituted
or unsubstituted alkyl, or halogen; Q is CH.sub.2, O, S, or
NR.sub.7', wherein R.sub.7' is hydrogen or substituted or
unsubstituted C.sub.1-C.sub.6 alkyl; and W is unsubstituted or
substituted alkyl, cycloalkyl, aryl, arylalkyl, biaryl, heteroaryl,
substituted carbonyl, substituted thiocarbonyl, or substituted
sulfonyl; provided that if R.sub.3 is pyrrolidino, then R.sub.4 is
not methyl. The invention also pertains to pharmaceutically
acceptable salts and prodrugs of the compounds of the
invention.
[0026] In an advantageous embodiment, X is CR.sub.6 and Q is
CH.sub.2, O, S, or NH in formula II, wherein R.sub.6 is as defined
above.
[0027] In another embodiment of formula II, X is N.
[0028] The invention further pertains to a method for inhibiting
the activity of an adenosine receptor (e.g., an A.sub.2b adenosine
receptor) in a cell by contacting the cell with a compound of the
invention. Preferably, the compound is an antagonist of the
receptor.
[0029] The invention also pertains to a method for treating a
gastrointestinal disorder (e.g., diarrhea) or a respiratory
disorder (e.g., allergic rhinitis, chronic obstructive pulmonary
disease) in an animal by administering to an animal an effective
amount of a compound of formula II (e.g., an antagonist of
A.sub.2b). Preferably, the animal is a human.
[0030] This invention also features a compound having the
structure: ##STR4## [0031] wherein R.sub.1 is trans-4-hydroxy
cyclohexyl, 2-methylamino carbonylamino cyclohexyl, acetamido
ethyl, or methylamino carbonylamino ethyl; [0032] wherein R.sub.3
is a substituted or unsubstituted four to six membered ring.
[0033] In one embodiment of the compound, R.sub.3 is phenyl,
pyrrole, thiophene, furan, thiazole, imidazole, pyrazole,
1,2,4-triazole, pyridine, 2(1H)-pyridone, 4(1H)-pyridone, pyrazine,
pyrimidine, pyridazine, isothiazole, isoxazole, oxazole, tetrazole,
naphthalene, tetralin, naphthyridine, benzofuran, benzothiophene,
indole, 2,3-dihydroindole, 1H-indole, indoline, benzopyrazole,
1,3-benzodioxole, benzoxazole, purine, coumarin, chromone,
quinoline, tetrahydroquinoline, isoquinoline, benzimidazole,
quinazoline, pyrido[2,3-b]pyrazine, pyrido[3,4-b]pyrazine,
pyrido[3,2-c]pyridazine, pyrido[3,4-b]-pyridine,
1H-pyrazole[3,4-d]pyrimidine, pteridine, 2(1H)-quinolone,
1(2H)-isoquinolone, 1,4-benzisoxazine, benzothiazole, quinoxaline,
quinoline-N-oxide, isoquinoline-N-oxide, quinoxaline-N-oxide,
quinazoline-N-oxide, benzoxazine, phthalazine, cinnoline, or having
a structure: ##STR5## [0034] wherein Y is carbon or nitrogen;
[0035] wherein R.sub.20 and R.sub.21 are independently H,
substituted or unsubstituted alkyl, substituted or unsubstituted
aryl, halogen, methoxy, methyl amino, or methyl thio; [0036]
wherein R.sub.5 is H, alkyl, substituted alkyl, aryl, arylalkyl,
amino, substituted aryl, wherein said substituted alkyl is
--C(R.sub.7)(R.sub.8)XR.sub.9, wherein X is O, S, or NR.sub.10,
wherein R.sub.7 and R.sub.8 are each independently H or alkyl,
wherein R.sub.9 and R.sub.10 are each independently alkyl or
cycloalkyl, or R.sub.9, R.sub.10 and the nitrogen together form a
substituted or unsubstituted ring of between 4 and 7 members;
[0037] wherein R.sub.6 is H, alkyl, substituted alkyl, cycloalkyl;
or a pharmaceutically acceptable salt, or a prodrug derivative, or
a biologically active metabolite; with the proviso that when
R.sub.1 is acetylamino ethyl, R.sub.3 is not 4-pyridyl.
[0038] This invention also pertains to a compound having the
structure: ##STR6## [0039] wherein R.sub.3 is aryl, substituted
aryl, or heteroaryl; [0040] wherein R.sub.5 is H, alkyl,
substituted alkyl, aryl, arylalkyl, amino, substituted aryl,
wherein said substituted alkyl is
--C(R.sub.7)(R.sub.8)NR.sub.9R.sub.10, wherein R.sub.7 and R.sub.8
are each H or alkyl, wherein R.sub.9 and R.sub.10 are each alkyl or
cycloalkyl, or R.sub.9, R.sub.10 and the nitrogen together form a
ring system of between 4 and 7 members; and [0041] wherein R.sub.6
is H, alkyl, substituted alkyl, or cycloalkyl. This invention also
features a method for inhibiting the activity of an A.sub.1
adenosine receptor in a cell, which comprises contacting said cell
with the above-mentioned compounds.
DETAILED DESCRIPTION
[0042] The features and other details of the invention will now be
more particularly described and pointed out in the claims. It will
be understood that the particular embodiments of the invention are
shown by way of illustration and not as limitations of the
invention. The principle features of this invention can be employed
in various embodiments without departing from the scope of the
invention.
[0043] The present invention pertains to methods for treating a N-6
substituted 7-deazapurine responsive state in a mammal. The methods
include administration of a therapeutically effective amount of a
N-6 substituted 7-deazapurine, described infra, to the mammal, such
that treatment of the N-6 substituted 7-deazapurine responsive
state in the mammal occurs.
[0044] The language "N-6 substituted 7-deazapurine responsive
state" is intended to include a disease state or condition
characterized by its responsiveness to treatment with a N-6
substituted 7-deazapurine of the invention as described infra,
e.g., the treatment includes a significant diminishment of at least
one symptom or effect of the state achieved with a N-6 substituted
7-deazapurine of the invention. Typically such states are
associated with an increase of adenosine within a host such that
the host often experiences physiological symptoms which include,
but are not limited to, release of toxins, inflammation, coma,
water retention, weight gain or weight loss, pancreatitis,
emphysema, rheumatoid arthritis, osteoarthritis, multiple organ
failure, infant and adult respiratory distress syndrome, allergic
rhinitis, chronic obstructive pulmonary disease, eye disorders,
gastrointestinal disorders, skin tumor promotion, immunodeficiency
and asthma. (See for example, C. E. Muller and B. Stein "Adenosine
Receptor Antagonists: Structures and Potential Therapeutic
Applications," Current Pharmaceutical Design, 2:501 (1996) and C.
E. Muller "A.sub.1-Adenosine Receptor Antagonists," Exp. Opin.
Ther. Patents 7(5):419 (1997) and I. Feoktistove, R. Polosa, S. T.
Holgate and I. Biaggioni "Adenosine A.sub.2B receptors: a novel
therapeutic target in asthma?" TiPS 19; 148 (1998)). The effects
often associated with such symptoms include, but are not limited
to, fever, shortness of breath, nausea, diarrhea, weakness,
headache, and even death. In one embodiment, a N-6 substituted
7-deazapurine responsive state includes those disease states which
are mediated by stimulation of adenosine receptors, e.g., A.sub.1,
A.sub.2a, A.sub.2b, A.sub.3, etc., such that calcium concentrations
in cells and/or activation of PLC (phospholipase C) is modulated.
In a preferred embodiment, a N-6 substituted 7-deazapurine
responsive state is associated with adenosine receptor(s), e.g.,
the N-6 substituted 7-deazapurine acts as an antagonist. Examples
of suitable responsive states which can be treated by the compounds
of the invention, e.g., adenosine receptor subtypes which mediate
biological effects, include central nervous system (CNS) effects,
cardiovascular effects, renal effects, respiratory effects,
immunological effects, gastrointestinal effects and metabolic
effects. The relative amount of adenosine in a subject can be
associated with the effects listed below; that is, increased levels
of adenosine can trigger an effect, e.g., an undesired
physiological response, e.g., an asthmatic attack.
[0045] CNS effects include decreased transmitter release (A.sub.1),
sedation (A.sub.1), decreased locomotor activity (A.sub.2a),
anticonvulsant activity, chemoreceptor stimulation (A.sub.2) and
hyperalgesia. Therapeutic applications of the inventive compounds
include treatment of dementia, Alzheimer's disease and memory
enhancement.
[0046] Cardiovascular effects include vasodilation (A.sub.2a),
(A.sub.2b) and (A.sub.3), vasoconstriction (A.sub.1), bradycardia
(A.sub.1), platelet inhibition (A.sub.2a), negative cardiac
inotropy and dromotropy (A.sub.1), arrhythmia, tachycardia and
angiogenesis. Therapeutic applications of the inventive compounds
include, for example, prevention of ischaemia-induced impairment of
the heart and cardiotonics, myocardial tissue protection and
restoration of cardiac function.
[0047] Renal effects include decreased GFR (A.sub.1), mesangial
cell contraction (A.sub.1), antidiuresis (A.sub.1) and inhibition
of renin release (A.sub.1). Suitable therapeutic applications of
the inventive compounds include use of the inventive compounds as
diuretic, natriuretic, potassium-sparing,
kidney-protective/prevention of acute renal failure,
antihypertensive, anti-oedematous and anti-nephritic agents.
[0048] Respiratory effects include bronchodilation (A.sub.2),
bronchoconstriction (A.sub.1), chronic obstructive pulmonary
disease, allergic rhinitis, mucus secretion and respiratory
depression (A.sub.2). Suitable therapeutic applications for the
compounds of the invention include anti-asthmatic applications,
treatment of lung disease after transplantation and respiratory
disorders.
[0049] Immunological effects include immunosuppression (A.sub.2)
neutrophil chemotaxis (A.sub.1), neutrophil superoxide generation
(A.sub.2a) and mast cell degranulation (A.sub.2b and A.sub.3)
Therapeutic applications of antagonists include allergic and non
allergic inflammation, e.g., release of histamine and other
inflammatory mediators.
[0050] Gastrointestinal effects include inhibition of acid
secretion (A.sub.1) therapeutic application may include reflux and
ulcerative conditions. Gastrointestinal effects also include
colonic, intestinal and diarrheal disease, e.g., diarrheal disease
associated with intestinal inflammation (A.sub.2b).
[0051] Eye disorders include retinal and optic nerve head injury
and trauma related disorders (A.sub.3). In a preferred embodiment,
the eye disorder is glaucoma.
[0052] Other therapeutic applications of the compounds of the
invention include treatment of obesity (lipolytic properties),
hypertension, treatment of depression, sedative, anxiolytic, as
antileptics and as laxatives, e.g., effecting motility without
causing diarrhea.
[0053] The term "disease state" is intended to include those
conditions caused by or associated with unwanted levels of
adenosine, adenylyl cyclase activity, increased physiological
activity associated with aberrant stimulation of adenosine
receptors and/or an increase in cAMP. In one embodiment, the
disease state is, for example, asthma, chronic obstructive
pulmonary disease, allergic rhinitis, bronchitis, renal disorders,
gastrointestinal disorders, or eye disorders. Additional examples
include chronic bronchitis and cystic fibrosis. Suitable examples
of inflammatory diseases include non-lymphocytic leukemia,
myocardial ischaemia, angina, infarction, cerebrovascular
ischaemia, intermittent claudication, critical limb ischemia,
venous hypertension, varicose veins, venous ulceration and
arteriosclerosis. Impaired reperfusion states include, for example,
any post-surgical trauma, such as reconstructive surgery,
thrombolysis or angioplasty.
[0054] The language "treatment of a N-6 substituted 7-deazapurine
responsive state" or "treating a N-6 substituted 7-deazapurine
responsive state" is intended to include changes in a disease state
or condition, as described above, such that physiological symptoms
in a mammal can be significantly diminished or minimized. The
language also includes control, prevention or inhibition of
physiological symptoms or effects associated with an aberrant
amount of adenosine. In one preferred embodiment, the control of
the disease state or condition is such that the disease state or
condition is eradicated. In another preferred embodiment, the
control is selective such that aberrant levels of adenosine
receptor activity are controlled while other physiologic systems
and parameters are unaffected.
[0055] The term "N-6 substituted 7-deazapurine" is art recognized
and is intended to include those compounds having the formula I:
##STR7## "N-substituted 7-deazapurine" includes pharmaceutically
acceptable salts thereof, and, in one embodiment, also includes
certain N-6 substituted purines described herein.
[0056] In certain embodiments, the N-6 substituted 7-deazapurine is
not N-6 benzyl or N-6 phenylethyl substituted. In other
embodiments, R.sub.4 is not benzyl or phenylethyl substituted. In
preferred embodiments, R.sub.1 and R.sub.2 are both not hydrogen
atoms. In still other preferred embodiments, R.sub.3 is not a
hydrogen atom.
[0057] The language "therapeutically effective amount" of an N-6
substituted 7-deazapurine, described infra, is that amount of a
therapeutic compound necessary or sufficient to perform its
intended function within a mammal, e.g., treat a N-6 substituted
7-deazapurine responsive state, or a disease state in a mammal. An
effective amount of the therapeutic compound can vary according to
factors such as the amount of the causative agent already present
in the mammal, the age, sex, and weight of the mammal, and the
ability of the therapeutic compounds of the present invention to
affect a N-6 substituted 7-deazapurine responsive state in the
mammal. One of ordinary skill in the art would be able to study the
aforementioned factors and make a determination regarding the
effective amount of the therapeutic compound without undue
experimentation. An in vitro or in vivo assay also can be used to
determine an "effective amount" of the therapeutic compounds
described infra. The ordinarily skilled artisan would select an
appropriate amount of the therapeutic compound for use in the
aforementioned assay or as a therapeutic treatment.
[0058] A therapeutically effective amount preferably diminishes at
least one symptom or effect associated with the N-6 substituted
7-deazapurine responsive state or condition being treated by at
least about 20%, (more preferably by at least about 40%, even more
preferably by at least about 60%, and still more preferably by at
least about 80%) relative to untreated subjects. Assays can be
designed by one skilled in the art to measure the diminishment of
such symptoms and/or effects. Any art recognized assay capable of
measuring such parameters are intended to be included as part of
this invention. For example, if asthma is the state being treated,
then the volume of air expended from the lungs of a subject can be
measured before and after treatment for measurement of increase in
the volume using an art recognized technique. Likewise, if
inflammation is the state being treated, then the area which is
inflamed can be measured before and after treatment for measurement
of diminishment in the area inflamed using an art recognized
technique.
[0059] The term "cell" includes both prokaryotic and eukaryotic
cells.
[0060] The term "animal" includes any organism with adenosine
receptors or any organism susceptible to a N-6-substituted
7-deazapurine responsive state. Examples of animals include yeast,
mammals, reptiles, and birds. It also includes transgenic
animals.
[0061] The term "mammal" is art recognized and is intended to
include an animal, more preferably a warm-blooded animal, most
preferably cattle, sheep, pigs, horses, dogs, cats, rats, mice, and
humans. Mammals susceptible to a N-6 substituted 7-deazapurine
responsive state, inflammation, emphysema, asthma, central nervous
system conditions, or acute respiratory distress syndrome, for
example, are included as part of this invention.
[0062] In another aspect, the present invention pertains to methods
for modulating an adenosine receptor(s) in a mammal by
administering to the mammal a therapeutically effective amount of a
N-6 substituted 7-deazapurine, such that modulation of the
adenosine receptor in the mammal occurs. Suitable adenosine
receptors include the families of A.sub.1, A.sub.2, or A.sub.3. In
a preferred embodiment, the N-6 substituted 7-deazapurine is an
adenosine receptor antagonist.
[0063] The language "modulating an adenosine receptor" is intended
to include those instances where a compound interacts with an
adenosine receptor(s), causing increased, decreased or abnormal
physiological activity associated with an adenosine receptor or
subsequent cascade effects resulting from the modulation of the
adenosine receptor. Physiological activities associated with
adenosine receptors include induction of sedation, vasodilation,
suppression of cardiac rate and contractility, inhibition of
platelet aggregbility, stimulation of gluconeogenesis, inhibition
of lipolysis, opening of potassium channels, reducing flux of
calcium channels, etc.
[0064] The terms "modulate", "modulating" and "modulation" are
intended to include preventing, eradicating, or inhibiting the
resulting increase of undesired physiological activity associated
with abnormal stimulation of an adenosine receptor, e.g., in the
context of the therapeutic methods of the invention. In another
embodiment, the term modulate includes antagonistic effects, e.g.,
diminishment of the activity or production of mediators of allergy
and allergic inflammation which results from the overstimulation of
adenosine receptor(s). For example, the therapeutic deazapurines of
the invention can interact with an adenosine receptor to inhibit,
for example, adenylate cyclase activity.
[0065] The language "condition characterized by aberrant adenosine
receptor activity" is intended to include those diseases, disorders
or conditions which are associated with aberrant stimulation of an
adenosine receptor, in that the stimulation of the receptor causes
a biochemical and or physiological chain of events that is directly
or indirectly associated with the disease, disorder or condition.
This stimulation of an adenosine receptor does not have to be the
sole causative agent of the disease, disorder or condition but
merely be responsible for causing some of the symptoms typically
associated with the disease, disorder, or condition being treated.
The aberrant stimulation of the receptor can be the sole factor or
at least one other agent can be involved in the state being
treated. Examples of conditions include those disease states listed
supra, including inflammation, gastrointestinal disorders and those
symptoms manifested by the presence of increased adenosine receptor
activity.
[0066] Preferred examples include those symptoms associated with
asthma, allergic rhinitis, chronic obstructive pulmonary disease,
emphysema, bronchitis, gastrointestinal disorders and glaucoma.
[0067] The language "treating or treatment of a condition
characterized by aberrant adenosine receptor activity" is intended
to include the alleviation of or diminishment of at least one
symptom typically associated with the condition. The treatment also
includes alleviation or diminishment of more than one symptom.
Preferably, the treatment cures, e.g., substantially eliminates,
the symptoms associated with the condition.
[0068] The present invention pertains to compounds, N-6 substituted
7-deazapurines, having the formula I: ##STR8## [0069] wherein
R.sub.1 and R.sub.2 are each independently a hydrogen atom or a
substituted or unsubstituted alkyl, aryl, or alkylaryl moiety or
together form a substituted or unsubstituted heterocyclic ring;
[0070] R.sub.3 is a hydrogen atom or a substituted or unsubstituted
alkyl, aryl, or alkylaryl moiety; [0071] R.sub.4 is a hydrogen atom
or a substituted or unsubstituted alkyl, aryl, or alkylaryl moiety.
[0072] R.sub.5 and R.sub.6 are each independently a halogen atom,
e.g., chlorine, fluorine, or bromine, a hydrogen atom or a
substituted or unsubstituted alkyl, aryl, or alkylaryl moiety or
R.sub.4 and R.sub.5 or R.sub.5 and R.sub.6 together form a
substituted or unsubstituted heterocyclic or carbocyclic ring. Also
included, are pharmaceutically acceptable salts of the N-6
substituted 7-deazapurines.
[0073] In certain embodiments, R.sub.1 and R.sub.2 can each
independently be a substituted or unsubstituted cycloalkyl or
heteroarylalkyl moieties. In other embodiments, R.sub.3 is a
hydrogen atom or a substituted or unsubstituted heteroaryl moiety.
In still other embodiments, R.sub.4, R.sub.5 and R.sub.6 can each
be independently a heteroaryl moiety.
[0074] In one embodiment, R.sub.1 is a hydrogen atom, R.sub.2 is a
substituted or unsubstituted cyclohexane, cyclopentyl, cyclobutyl
or cyclopropane moiety, R.sub.3 is a substituted or unsubstituted
phenyl moiety, R.sub.4 is a hydrogen atom and R.sub.5 and R.sub.6
are both methyl groups.
[0075] In another embodiment, R.sub.2 is a cyclohexanol, a
cyclohexanediol, a cyclohexylsulfonamide, a cyclohexanamide, a
cyclohexylester, a cyclohexene, a cyclopentanol or a
cyclopentanediol and R.sub.3 is a phenyl moiety.
[0076] In still another embodiment, R.sub.1 is a hydrogen atom,
R.sub.2 is a cyclohexanol, R.sub.3 is a substituted or
unsubstituted phenyl, pyridine, furan, cyclopentane, or thiophene
moiety, R.sub.4 is a hydrogen atom, a substituted alkyl, aryl or
arylalkyl moiety, and R.sub.5 and R.sub.6 are each independently a
hydrogen atom, or a substituted or unsubstituted alkyl, aryl, or
alkylaryl moiety.
[0077] In yet another embodiment, R.sub.1 is a hydrogen atom,
R.sub.2 is substituted or unsubstituted alkylamine, arylamine, or
alkylarylamine, a substituted or unsubstituted alkylamide,
arylamide or alkylarylamide, a substituted or unsubstituted
alkylsulfonamide, arylsulfonamide or alkylarylsulfonamide, a
substituted or unsubstituted alkylurea, arylurea or alkylarylurea,
a substituted or unsubstituted alkylcarbamate, arylcarbamate or
alkylarylcarbamate, a substituted or unsubstituted alkylcarboxylic
acid, arylcarboxylic acid or alkylarylcarboxylic acid, R.sub.3 is a
substituted or unsubstituted phenyl moiety, R.sub.4 is a hydrogen
atom and R.sub.5 and R.sub.6 are methyl groups.
[0078] In still another embodiment, R.sub.2 is guanidine, a
modified guanidine, cyanoguanidine, a thiourea, a thioamide or an
amidine.
[0079] In one embodiment, R.sub.2 can be ##STR9## wherein
R.sub.2a-R.sub.2c are each independently a hydrogen atom or a
saturated or unsaturated alkyl, aryl or alkylaryl moiety and
R.sub.2d is a hydrogen atom or a saturated or unsaturated alkyl,
aryl, or alkylaryl moiety, NR.sub.2eR.sub.2f, or OR.sub.2g, wherein
R.sub.2e-R.sub.2g are each independently a hydrogen atom or a
saturated or unsaturated alkyl, aryl or alkylaryl moieties.
Alternatively, R.sub.2a and R.sub.2b together can form a
carbocyclic or heterocyclic ring having a ring size between about 3
and 8 members, e.g., cyclopropyl, cyclopentyl, cyclohexyl
groups.
[0080] In one aspect of the invention, both R.sub.5 and R.sub.6 are
not methyl groups, preferably, one of R.sub.5 and R.sub.6 is an
alkyl group, e.g., a methyl group, and the other is a hydrogen
atom.
[0081] In another aspect of the invention, when R.sub.4 is
1-phenylethyl and R.sub.1 is a hydrogen atom, then R.sub.3 is not
phenyl, 2-chlorophenyl, 3-chlorophenyl, 4-chlorophenyl,
3,4-dichlorophenyl, 3-methoxyphenyl or 4-methoxyphenyl or when
R.sub.4 and R.sub.1 are 1-phenylethyl, then R.sub.3 is not a
hydrogen atom or when R.sub.4 is a hydrogen atom and R.sub.3 is a
phenyl, then R.sub.1 is not phenylethyl.
[0082] In another aspect of the invention, when R.sub.5 and R.sub.6
together form a carbocyclic ring, e.g., ##STR10## or
pyrimido[4,5-6]indole, then R.sub.3 is not phenyl when R.sub.4 is
1-(4-methylphenyl)ethyl, phenylisopropyl, phenyl or 1-phenylethyl
or when R.sub.3 is not a hydrogen atom when R.sub.4 is
1-phenylethyl. The carbocyclic ring formed by R.sub.5 and R.sub.6
can be either aromatic or aliphatic and can have between 4 and 12
carbon atoms, e.g., naphthyl, phenylcyclohexyl, etc., preferably
between 5 and 7 carbon atoms, e.g., cyclopentyl or cyclohexyl.
Alternatively, R.sub.5 and R.sub.6 together can form a heterocyclic
ring, such as those disclosed below. Typical heterocyclic rings
include between 4 and 12 carbon atoms, preferably between 5 and 7
carbon atoms, and can be either aromatic or aliphatic. The
heterocyclic ring can be further substituted, including
substitution of one or more carbon atoms of the ring structure with
one or more heteroatoms.
[0083] In still another aspect of the invention, R.sub.1 and
R.sub.2 form a heterocyclic ring. Representative examples include,
but are not limited to, those heterocyclic rings listed below, such
as morpholino, piperazine and the like, e.g., 4-hydroxypiperidines,
4-aminopiperidines. Where R.sub.1 and R.sub.2 together form a
piperazino group, ##STR11## wherein R.sub.7 can be a hydrogen atom
or a substituted or unsubstituted alkyl, aryl or alkylaryl
moiety.
[0084] In yet another aspect of the invention R.sub.4 and R.sub.5
together can form a heterocyclic ring, e.g., ##STR12## wherein the
heterocyclic ring can be either aromatic or aliphatic and can form
a ring having between 4 and 12 carbon atoms, e.g., naphthyl,
phenylcyclohexyl, etc. and can be either aromatic or aliphatic,
e.g., cyclohexyl, cyclopentyl. The heterocyclic ring can be further
substituted, including substitution of carbon atoms of the ring
structure with one or more heteroatoms. Alternatively, R.sub.4 and
R.sub.5 together can form a heterocyclic ring, such as those
disclosed below.
[0085] In certain embodiments, the N-6 substituted 7-deazapurine is
not N-6 benzyl or N-6 phenylethyl substituted. In other
embodiments, R.sub.4 is not benzyl or phenylethyl substituted. In
preferred embodiments, R.sub.1 and R.sub.2 are both not hydrogen
atoms. In still other preferred embodiments, R.sub.3 is not H.
[0086] The compounds of the invention may comprise water-soluble
prodrugs which are described in WO 99/33815, International
Application No. PCT/US98/04595, filed Mar. 9, 1998 and published
Jul. 8, 1999. The entire content of WO 99/33815 is expressly
incorporated herein by reference. The water-soluble prodrugs are
metabolized in vivo to an active drug, e.g., by esterase catalyzed
hydrolysis. Examples of potential prodrugs include deazapurines
with, for example, R.sub.2 as cycloalkyl substituted with
--OC(O)(Z)NH.sub.2, wherein Z is a side chain of a naturally or
unnaturally occurring amino acid, or analog thereof, an .alpha.,
.beta., .gamma., or .omega. amino acids, or a dipeptide. Preferred
amino acid side chains include those of glycine, alanine, valine,
leucine, isoleucine, lysine, .alpha.-methylalanine,
aminocyclopropane carboxylic acid, azetidine-2-carboxylic acid,
.beta.-alanine, .gamma.-aminobutyric acid, alanine-alanine, or
glycine-alanine.
[0087] In a further embodiment, the invention features deazapurines
of the formula (I), wherein R.sub.1 is hydrogen; R.sub.2 is
substituted or unsubstituted cycloalkyl, substituted or
unsubstituted alkyl, or R.sub.1 and R.sub.2 together form a
substituted or unsubstituted heterocyclic ring; R.sub.3 is
unsubstituted or substituted aryl; R.sub.4 is hydrogen; and R.sub.5
and R.sub.6 are each independently hydrogen or alkyl, and
pharmaceutically acceptable salts thereof. The deazapurines of this
embodiment may potentially be selective A.sub.3 receptor
antagonists.
[0088] In one embodiment, R.sub.2 is substituted (e.g., hydroxy
substituted) or unsubstituted cycloalkyl. In an advantageous
subembodiment, R.sub.1 and R.sub.4 are hydrogen, R.sub.3 is
unsubstituted or substituted phenyl, and R.sub.5 and R.sub.6 are
each alkyl. Preferably R.sub.2 is mono-hydroxycyclopentyl or
mono-hydroxycyclohexyl. R.sub.2 also may be substituted with
--NH--C(.dbd.O)E, wherein E is substituted or unsubstituted
C.sub.1-C.sub.4 alkyl (e.g., alkylamine, e.g., ethylamine.).
[0089] R.sub.1 and R.sub.2 may also together form a substituted or
unsubstituted heterocyclic ring, which may be substituted with an
amine or acetamido group.
[0090] In another aspect, R.sub.2 may be -A-NHC(.dbd.O)B, wherein A
is unsubstituted C.sub.1-C.sub.4 alkyl (e.g., ethyl, propyl,
butyl), and B is substituted or unsubstituted C.sub.1-C.sub.4 alkyl
(e.g., methyl, aminoalkyl, e.g., aminomethyl or aminoethyl,
alkylamino, e.g., methylamino, ethylamino), preferably when R.sub.1
and R.sub.4 are hydrogen, R.sub.3 is unsubstituted or substituted
phenyl, and R.sub.5 and R.sub.6 are each alkyl. B may be
substituted or unsubstituted cycloalkyl, e.g., cyclopropyl or
1-amino-cyclopropyl.
[0091] In another embodiment, R.sub.3 may be substituted or
unsubstituted phenyl, preferably when R.sub.5 and R.sub.6 are each
alkyl. Preferably, R.sub.3 may have one or more substituents (e.g.,
o-, m- or p-chlorophenyl, o-, m- or p-fluorophenyl).
[0092] Advantageously, R.sub.3 may be substituted or unsubstituted
heteroaryl, preferably when R.sub.5 and R.sub.6 are each alkyl.
Examples of heteroaryl groups include pyridyl, pyrimidyl,
pyridazinyl, pyrazinyl, pyrrolyl, triazolyl, thioazolyl, oxazolyl,
oxadiazolyl, furanyl, methylenedioxyphenyl and thiophenyl.
Preferably, R.sub.3 is 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl
or 3-pyrimidyl.
[0093] Preferably in one embodiment, R.sub.5 and R.sub.6 are each
hydrogen. In another, R.sub.5 and R.sub.6 are each methyl.
[0094] In a particularly preferred embodiment, the deazapurines of
the invention are water-soluble prodrugs that can be metabolized in
vivo to an active drug, e.g. by esterase catalyzed hydrolysis.
Preferably the prodrug comprises an R.sub.2 group which is
cycloalkyl substituted with --OC(O)(Z)NH.sub.2, wherein Z is a side
chain of a naturally or unnaturally occurring amino acid, an analog
thereof, an .alpha., .beta., .gamma., or .omega. amino acid, or a
dipeptide. Examples of preferred side chains include the side
chains of glycine, alanine, valine, leucine, isoleucine, lysine,
.alpha.-methylalanine, aminocyclopropane carboxylic acid,
azetidine-2-carboxylic acid, .beta.-alanine, .gamma.-aminobutyric
acid, alanine-alanine, or glycine-alanine.
[0095] In a particularly preferred embodiment, Z is a side chain of
glycine, R.sub.2 is cyclohexyl, R.sub.3 is phenyl, and R.sub.5 and
R.sub.6 are methyl.
[0096] In another embodiment, the deazapurine is
4-(cis-3-hydroxycyclopentyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]p-
yrimidine.
[0097] In another embodiment, the deazapurine is
4-(cis-3-(2-aminoacetoxy)cyclopentyl)amino-5,6-dimethyl-2-phenyl-7H-pyrro-
lo[2,3d]pyrimidine trifluoroacetic acid salt.
[0098] In another embodiment, the deazapurine is
4-(3-acetamido)piperidinyl-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidi-
ne.
[0099] In another embodiment, the deazapurine is
4-(2-N'-methylureapropyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyri-
midine.
[0100] In another embodiment, the deazapurine is
4-(2-acetamidobutyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidin-
e.
[0101] In another embodiment, the deazapurine is
4-(2-N'-methylureabutyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrim-
idine.
[0102] In another embodiment, the deazapurine is
4-(2-aminocyclopropylacetamidoethyl)amino-2-phenyl-7H-pyrrolo[2,3d]pyrimi-
dine.
[0103] In another embodiment, the deazapurine is
4-(trans-4-hydroxycyclohexyl)amino-2-(3-chlorophenyl)-7H-pyrrolo[2,3d]pyr-
imidine.
[0104] In another embodiment, the deazapurine is
4-(trans-4-hydroxycyclohexyl)amino-2-(3-fluorophenyl)-7H-pyrrolo[2,3d]pyr-
imidine.
[0105] In another embodiment, the deazapurine is
4-(trans-4-hydroxycyclohexyl)amino-2-(4-pyridyl)-7H-pyrrolo[2,3d]pyrimidi-
ne.
[0106] In yet another embodiment, the invention features a method
for inhibiting the activity of an adenosine receptor (e.g.,
A.sub.1, A.sub.2A, A.sub.2B, or, preferably, A.sub.3) in a cell, by
contacting the cell with N-6 substituted 7-deazapurine (e.g.,
preferably, an adenosine receptor antagonist).
[0107] In another aspect, the invention features a method for
treating damage to the eye of an animal (e.g., a human) by
administering to the animal an effective amount of an N-6
substituted 7-deazapurine. Preferably, the N-6 substituted
7-deazapurine is an antagonist of A.sub.3 adenosine receptors in
cells of the animal. The damage is to the retina or the optic nerve
head and may be acute or chronic. The damage may be the result of,
for example, glaucoma, edema, ischemia, hypoxia or trauma.
[0108] In a preferred embodiment, the invention features a
deazapurine having the formula II: ##STR13## [0109] wherein X is N
or CR.sub.6; [0110] R.sub.1 and R.sub.2 are each independently
hydrogen, or substituted or unsubstituted alkoxy, aminoalkyl,
alkyl, aryl, or alkylaryl, or together form a substituted or
unsubstituted heterocyclic ring, provided that both R.sub.1 and
R.sub.2 are both not hydrogen; [0111] R.sub.3 is substituted or
unsubstituted alkyl, arylalkyl, or aryl; [0112] R.sub.4 is hydrogen
or substituted or unsubstituted C.sub.1-C.sub.6 alkyl; [0113] L is
hydrogen, substituted or unsubstituted alkyl, or R.sub.4 and L
together form a substituted or unsubstituted heterocyclic or
carbocyclic ring; [0114] R.sub.6 is hydrogen, substituted or
unsubstituted alkyl, or halogen; [0115] Q is CH.sub.2, O, S, or
NR.sub.7', wherein R.sub.7' is hydrogen or substituted or
unsubstituted C.sub.1-C.sub.6 alkyl; and [0116] W is unsubstituted
or substituted alkyl, cycloalkyl, alkynyl, aryl, arylalkyl, biaryl,
heteroaryl, substituted carbonyl, substituted thiocarbonyl, or
substituted sulfonyl, provided that if R.sub.3 is pyrrolidino, then
R.sub.4 is not methyl.
[0117] In one embodiment, in compounds of formula II, X is CR.sub.6
and Q is CH.sub.2, O S, or NH. In another embodiment, X is N.
[0118] In a further embodiment of compounds of formula II, W is
substituted or unsubstituted aryl, 5- or 6-member heteroaryl, or
biaryl. W may be substituted with one or more substituents.
Examples of substituents include: halogen, hydroxy, alkoxy, amino,
aminoalkyl, aminocarboxyamide, CN, CF.sub.3, CO.sub.2R.sub.8,
CONHR.sub.8, CONR.sub.8R.sub.9, SOR.sub.8, SO.sub.2R.sub.8, and
SO.sub.2NR.sub.8R.sub.9, wherein R.sub.8 and R.sub.9 are each
independently hydrogen, or substituted or unsubstituted alkyl,
cycloalkyl, aryl, or arylalkyl. Preferably, W may be substituted or
unsubstituted phenyl, e.g., methylenedioxyphenyl. W also may be a
substituted or unsubstituted 5-membered heteroaryl ring, e.g.,
pyrrole, pyrazole, oxazole, imidazole, triazole, tetrazole, furan,
thiophene, thiazole, and oxadiazole. Preferably, W may be a
6-member heteroaryl ring, e.g., pyridyl, pyrimidyl, pyridazinyl,
pyrazinyl, and thiophenyl. In a preferred embodiment, W is
2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, or
5-pyrimidyl.
[0119] In one advantageous embodiment of compounds of formula II, Q
is NH and W is a 3-pyrazolo ring which is unsubstituted or
N-substituted by substituted or unsubstituted alkyl, cycloalkyl,
aryl, or arylalkyl.
[0120] In another embodiment of compounds of formula II, Q is
oxygen, and W is a 2-thiazolo ring which is unsubstituted or
substituted by substituted or unsubstituted alkyl, cycloalkyl,
aryl, or arylalkyl.
[0121] In another embodiment of compounds of formula II, W is
substituted or unsubstituted alkyl, cycloalkyl e.g., cyclopentyl,
or arylalkyl. Examples of substituents include halogen, hydroxy,
substituted or unsubstituted alkyl, cycloalkyl, aryl, arylalkyl, or
NHR.sub.10, wherein R.sub.10 is hydrogen, or substituted or
unsubstituted alkyl, cycloalkyl, aryl, or arylalkyl.
[0122] In yet another embodiment, the invention features a
deazapurine of formula II wherein W is
--(CH.sub.2).sub.a--C(.dbd.O)Y or --(CH.sub.2).sub.a--C(.dbd.S)Y,
and a is an integer from 0 to 3, Y is aryl, alkyl, arylalkyl,
cycloalkyl, heteroaryl, alkynyl, NHR.sub.11R.sub.12, or, provided
that Q is NH, OR.sub.13, wherein R.sub.11, R.sub.12 and R.sub.13
are each independently hydrogen, or unsubstituted or substituted
alkyl, aryl, arylalkyl, or cycloalkyl. Preferably, Y is a 5- or
6-member heteroaryl ring.
[0123] Furthermore, W may be --(CH.sub.2).sub.b--S(.dbd.O).sub.jY,
wherein j is 1 or 2, b is 0, 1, 2, or 3, Y is aryl, alkyl,
arylalkyl, cycloalkyl, alkynyl, heteroaryl, NHR.sub.14R.sub.15,
provided that when b is 1, Q is CH.sub.2, OR.sub.16, and wherein
R.sub.14, R.sub.15, and R.sub.16 are each independently hydrogen,
or unsubstituted or substituted alkyl, aryl, arylalkyl, or
cycloalkyl.
[0124] In another embodiment, R.sub.3 is selected from the group
consisting of substituted and unsubstituted phenyl, pyridyl,
pyrimidyl, pyridazinyl, pyrazinal, pyrrolyl, triazolyl, thioazolyl,
oxazolyl, oxadiazolyl, pyrazolyl, furanyl, methylenedioxyphenyl,
and thiophenyl. When R.sub.3 is phenyl, it may be substituted with,
for example, hydroxyl, alkoxy (e.g., methoxy), alkyl (e.g., tolyl),
and halogen, (e.g., o-, m-, or p-fluorophenyl or o-, m-, or
p-chlorophenyl). Advantageously, R.sub.3 may be 2-, 3-, or
4-pyridyl or 2- or 3-pyrimidyl.
[0125] The invention also pertains to a deazapurine wherein R.sub.6
is hydrogen or C.sub.1-C.sub.3 alkyl. Preferably, R.sub.6 is
hydrogen.
[0126] The invention also includes deazapurines wherein R.sub.1 is
hydrogen, and R.sub.2 is substituted or unsubstituted alkyl or
alkoxy, substituted or unsubstituted alkylamine, arylamine, or
alkylarylamine, substituted or unsubstituted aminoalkyl, amino
aryl, or aminoalkylaryl, substituted or unsubstituted alkylamide,
arylamide or alkylarylamide, substituted or unsubstituted
alkylsulfonamide, arylsulfonamide or alkylarylsulfonamide,
substituted or unsubstituted alkylurea, arylurea or alkylarylurea,
substituted or unsubstituted alkylcarbamate, arylcarbamate or
alkylarylcarbamate, or substituted or unsubstituted alkylcarboxylic
acid, arylcarboxylic acid or alkylarylcarboxylic acid.
[0127] Preferably, R.sub.2 is substituted or unsubstituted
cycloalkyl, e.g., mono- or dihydroxy-substituted cyclohexyl or
cyclopentyl (preferably, monohydroxy-substituted cyclohexyl or
monohydroxy-substituted cyclopentyl).
[0128] Advantageously, R.sub.2 may be of the following formula:
##STR14## wherein A is C.sub.1-C.sub.6 alkyl, C.sub.3-C.sub.7
cycloalkyl, a chain of one to seven atoms, or a ring of three to
seven atoms, optionally substituted with C.sub.1-C.sub.6 alkyl,
halogens, hydroxyl, carboxyl, thiol, or amino groups; wherein B is
methyl, N(Me).sub.2, N(Et).sub.2, NHMe, NHEt,
(CH.sub.2).sub.rNH.sub.3+, NH(CH.sub.2).sub.rCH.sub.3,
(CH.sub.2).sub.rNH.sub.2, (CH.sub.2).sub.rCH(CH.sub.3)NH.sub.2,
(CH.sub.2).sub.rNHMe, (CH.sub.2).sub.rOH, CH.sub.2CN,
(CH.sub.2).sub.mCO.sub.2H, CHR.sub.18R.sub.19, or CHMeOH, wherein r
is an integer from 0 to 2, m is 1 or 2, R.sub.18 is alkyl, R.sub.19
is NH.sub.3+ or CO.sub.2H or R.sub.18 and R.sub.19 together are:
##STR15## [0129] wherein p is 2 or 3; and R.sub.17 is
C.sub.1-C.sub.6 alkyl, C.sub.3-C.sub.7 cycloalkyl, a chain of one
to seven atoms, or a ring of three to seven atoms, optionally
substituted with C.sub.1-C.sub.6 alkyl, halogens, hydroxyl,
carboxyl, thiol, or amino groups.
[0130] Advantageously, A is unsubstituted or substituted
C.sub.1-C.sub.6 alkyl. B may be substituted or unsubstituted
C.sub.1-C.sub.6 alkyl.
[0131] In a preferred embodiment, R.sub.2 is of the formula
-A-NHC(.dbd.O)B. In a particularly advantageous embodiment, A is
--CH.sub.2CH.sub.2-- and B is methyl.
[0132] The compounds of the invention may comprise water-soluble
prodrugs which are metabolized in vivo to an active drug, e.g., by
esterase catalyzed hydrolysis. Examples of potential prodrugs
include deazapurines with, for example, R.sub.2 as cycloalkyl
substituted with --OC(O)(Z)NH.sub.2, wherein Z is a side chain of a
naturally or unnaturally occurring amino acid, or analog thereof,
an .alpha., .beta., .gamma., or .omega. amino acid, or a dipeptide.
Preferred amino acid side chains include those of glycine, alanine,
valine, leucine, isoleucine, lysine, .alpha.-methylalanine,
aminocyclopropane carboxylic acid, azetidine-2-carboxylic acid,
.beta.-alanine, .gamma.-aminobutyric acid, alanine-alanine, or
glycine-alanine.
[0133] In another embodiment, R.sub.1 and R.sub.2 together are:
##STR16## wherein n is 1 or 2, and wherein the ring may be
optionally substituted with one or more hydroxyl, amino, thiol,
carboxyl, halogen, CH.sub.2OH, CH.sub.2NHC(.dbd.O)alkyl, or
CH.sub.2NHC(.dbd.O)NHalkyl groups. Preferably, n is 1 or 2 and said
ring is substituted with --NHC(.dbd.O) alkyl.
[0134] In one advantageous embodiment, R.sub.1 is hydrogen, R.sub.2
is substituted or unsubstituted C.sub.1-C.sub.6 alkyl, R.sub.3 is
substituted or unsubstituted phenyl, R.sub.4 is hydrogen, L is
hydrogen or substituted or unsubstituted C.sub.1-C.sub.6 alkyl, Q
is O, S or NR.sub.7', wherein R.sub.7' is hydrogen or substituted
or unsubstituted C.sub.1-C.sub.6 alkyl, and W is substituted or
unsubstituted aryl. Preferably, R.sub.2 is -A-NHC(.dbd.O)B, wherein
A and B are each independently unsubstituted or substituted
C.sub.1-C.sub.4 alkyl. For example, A may be CH.sub.2CH.sub.2. B
may be, for example, alkyl (e.g., methyl), or aminoalkyl (e.g.,
aminomethyl). Preferably, R.sub.3 is unsubstituted phenyl and L is
hydrogen. R.sub.6 may be methyl or preferably, hydrogen.
Preferably, Q is O, S, or NR.sub.7' wherein R.sub.7' is hydrogen or
substituted or unsubstituted C.sub.1-C.sub.6 alkyl, e.g., methyl. W
is unsubstituted or substituted phenyl (e.g., alkoxy, halogen
substituted). Preferably, W is p-fluorophenyl, p-chlorophenyl, or
p-methoxyphenyl. W may also be heteroaryl, e.g., 2-pyridyl.
[0135] In a particularly preferred embodiment, the deazapurine is
4-(2-acetylaminoethyl)amino-6-phenoxymethyl-2-phenyl-7H-pyrrolo[2,3d]pyri-
midine.
[0136] In a particularly preferred embodiment, the deazapurine is
4-(2-acetylaminoethyl)amino-6-(4-fluorophenoxy)methyl-2-phenyl-7H-pyrrolo-
[2,3d]pyrimidine.
[0137] In a particularly preferred embodiment, the deazapurine is
4-(2-acetylaminoethyl)amino-6-(4-chlorophenoxy)methyl-2-phenyl-7H-pyrrolo-
[2,3d]pyrimidine.
[0138] In a particularly preferred embodiment, the deazapurine is
4-(2-acetylaminoethyl)amino-6-(4-methoxyphenoxy)methyl-2-phenyl-7H-pyrrol-
o[2,3d]pyrimidine.
[0139] In a particularly preferred embodiment, the deazapurine is
4-(2-acetylaminoethyl)amino-6-(2-pyridyloxy)methyl-2-phenyl-7H-pyrrolo[2,-
3d]pyrimidine.
[0140] In a particularly preferred embodiment, the deazapurine is
4-(2-acetylaminoethyl)amino-6-(N-phenylamino)methyl-2-phenyl-7H-pyrrolo[2-
,3d]pyrimidine.
[0141] In a particularly preferred embodiment, the deazapurine is
4-(2-acetylaminoethyl)amino-6-(N-methyl-N-phenylamino)methyl-2-phenyl-7H--
pyrrolo[2,3d]pyrimidine.
[0142] In a particularly preferred embodiment, the deazapurine is
4-(2-N'-methylureaethyl)amino-6-phenoxymethyl-2-phenyl-7H-pyrrolo[2,3d]py-
rimidine.
[0143] The invention further pertains to a method for inhibiting
the activity of an adenosine receptor (e.g., an A.sub.2b adenosine
receptor) in a cell by contacting the cell with a compound of the
invention. Preferably, the compound is an antagonist of the
receptor.
[0144] The invention also pertains to a method for treating a
gastrointestinal disorder (e.g., diarrhea) in an animal by
administering to an animal an effective amount of a compound of the
invention (e.g., an antagonist of A.sub.2b). Preferably, the animal
is a human.
[0145] In another embodiment, the invention relates to a
pharmaceutical composition containing an N-6 substituted
7-deazapurine of the invention and a pharmaceutically acceptable
carrier.
[0146] The invention also pertains to a method for treating a N-6
substituted 7-deazapurine responsive state in an animal, by
administering to a mammal a therapeutically effective amount of a
deazapurine of the invention, such that treatment of a N-6
substituted 7-deazapurine responsive state in the animal occurs.
Advantageously, the disease state may be a disorder mediated by
adenosine. Examples of preferred disease states include: central
nervous system disorders, cardiovascular disorders, renal
disorders, inflammatory disorders, allergic disorders,
gastrointestinal disorders, eye disorders, and respiratory
disorders.
[0147] The term "alkyl" refers to the radical of saturated
aliphatic groups, including straight-chain alkyl groups,
branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl
substituted cycloalkyl groups, and cycloalkyl substituted alkyl
groups. The term alkyl further includes alkyl groups, which can
further include oxygen, nitrogen, sulfur or phosphorous atoms
replacing one or more carbons of the hydrocarbon backbone, e.g.,
oxygen, nitrogen, sulfur or phosphorous atoms. In preferred
embodiments, a straight chain or branched chain alkyl has 30 or
fewer carbon atoms in its backbone (e.g., C.sub.1-C.sub.30 for
straight chain, C.sub.3-C.sub.30 for branched chain), and more
preferably 20 or fewer. Likewise, preferred cycloalkyls have from
4-10 carbon atoms in their ring structure, and more preferably have
5, 6 or 7 carbons in the ring structure.
[0148] Moreover, the term "alkyl" as used throughout the
specification and claims is intended to include both "unsubstituted
alkyls" and "substituted alkyls", the latter of which refers to
alkyl moieties having substituents replacing a hydrogen on one or
more carbons of the hydrocarbon backbone. Such substituents can
include, for example, halogen, hydroxyl, alkylcarbonyloxy,
arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy,
carboxylate, alkylcarbonyl, alkoxycarbonyl, aminocarbonyl,
alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato,
cyano, amino (including alkyl amino, dialkylamino, arylamino,
diarylamino, and alkylarylamino), acylamino (including
alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),
amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,
sulfates, sulfonato, sulfamoyl, sulfonamido, nitro,
trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an
aromatic or heteroaromatic moiety. It will be understood by those
skilled in the art that the moieties substituted on the hydrocarbon
chain can themselves be substituted, if appropriate. Cycloalkyls
can be further substituted, e.g., with the substituents described
above. An "alkylaryl" moiety is an alkyl substituted with an aryl
(e.g., phenylmethyl (benzyl)). The term "alkyl" also includes
unsaturated aliphatic groups analogous in length and possible
substitution to the alkyls described above, but that contain at
least one double or triple bond respectively.
[0149] The term "aryl" as used herein, refers to the radical of
aryl groups, including 5- and 6-membered single-ring aromatic
groups that may include from zero to four heteroatoms, for example,
benzene, pyrrole, furan, thiophene, imidazole, benzoxazole,
benzothiazole, triazole, tetrazole, pyrazole, pyridine, pyrazine,
pyridazine and pyrimidine, and the like. Aryl groups also include
polycyclic fused aromatic groups such as naphthyl, quinolyl,
indolyl, and the like. Those aryl groups having heteroatoms in the
ring structure may also be referred to as "aryl heterocycles",
"heteroaryls" or "heteroaromatics". The aromatic ring can be
substituted at one or more ring positions with such substituents as
described above, as for example, halogen, hydroxyl, alkoxy,
alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,
aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkoxycarbonyl,
aminocarbonyl, alkylthiocarbonyl, phosphate, phosphonato,
phosphinato, cyano, amino (including alkyl amino, dialkylamino,
arylamino, diarylamino, and alkylarylamino), acylamino (including
alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),
amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,
sulfates, sulfonato, sulfamoyl, sulfonamido, nitro,
trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an
aromatic or heteroaromatic moiety. Aryl groups can also be fused or
bridged with alicyclic or heterocyclic rings which are not aromatic
so as to form a polycycle (e.g., tetralin).
[0150] The terms "alkenyl" and "alkynyl" refer to unsaturated
aliphatic groups analogous in length and possible substitution to
the alkyls described above, but that contain at least one double or
triple bond respectively. For example, the invention contemplates
cyano and propargyl groups.
[0151] Unless the number of carbons is otherwise specified, "lower
alkyl" as used herein means an alkyl group, as defined above, but
having from one to ten carbons, more preferably from one to six
carbon atoms in its backbone structure, even more preferably one to
three carbon atoms in its backbone structure. Likewise, "lower
alkenyl" and "lower alkynyl" have similar chain lengths.
[0152] The terms "alkoxyalkyl", "polyaminoalkyl" and
"thioalkoxyalkyl" refer to alkyl groups, as described above, which
further include oxygen, nitrogen or sulfur atoms replacing one or
more carbons of the hydrocarbon backbone, e.g., oxygen, nitrogen or
sulfur atoms.
[0153] The terms "polycyclyl" or "polycyclic radical" refer to the
radical of two or more cyclic rings (e.g., cycloalkyls,
cycloalkenyls, cycloalkynyls, aryls and/or heterocyclyls) in which
two or more carbons are common to two adjoining rings, e.g., the
rings are "fused rings". Rings that are joined through non-adjacent
atoms are termed "bridged" rings. Each of the rings of the
polycycle can be substituted with such substituents as described
above, as for example, halogen, hydroxyl, alkylcarbonyloxy,
arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy,
carboxylate, alkylcarbonyl, alkoxycarbonyl, aminocarbonyl,
alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato,
cyano, amino (including alkyl amino, dialkylamino, arylamino,
diarylamino, and alkylarylamino), acylamino (including
alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),
amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,
sulfates, sulfonato, sulfamoyl, sulfonamido, nitro,
trifluoromethyl, cyano, azido, heterocyclyl, alkyl, alkylaryl, or
an aromatic or heteroaromatic moiety.
[0154] The term "heteroatom" as used herein means an atom of any
element other than carbon or hydrogen. Preferred heteroatoms are
nitrogen, oxygen, sulfur and phosphorus.
[0155] The term "amino acids" includes naturally and unnaturally
occurring amino acids found in proteins such as glycine, alanine,
valine, cysteine, leucine, isoleucine, serine, threonine,
methionine, glutamic acid, aspartic acid, glutamine, asparagine,
lysine, arginine, proline, histidine, phenylalanine, tyrosine, and
tryptophan. Amino acid analogs include amino acids with lengthened
or shortened side chains or variant side chains with appropriate
functional groups. Amino acids also include D and L stereoisomers
of an amino acid when the structure of the amino acid admits of
stereoisomeric forms. The term "dipeptide" includes two or more
amino acids linked together. Preferably, dipeptides are two amino
acids linked via a peptide linkage. Particularly preferred
dipeptides include, for example, alanine-alanine and
glycine-alanine.
[0156] It will be noted that the structure of some of the compounds
of this invention includes asymmetric carbon atoms and thus occur
as racemates and racemic mixtures, single enantiomers,
diastereomeric mixtures and individual diastereomers. All such
isomeric forms of these compounds are expressly included in this
invention. Each stereogenic carbon may be of the R or S
configuration. It is to be understood accordingly that the isomers
arising from such asymmetry (e.g., all enantiomers and
diastereomers) are included within the scope of this invention,
unless indicated otherwise. Such isomers can be obtained in
substantially pure form by classical separation techniques and by
stereochemically controlled synthesis.
[0157] The invention further pertains to pharmaceutical
compositions for treating a N-6 substituted 7-deazapurine
responsive state in a mammal, e.g., respiratory disorders (e.g.,
asthma, bronchitis, chronic obstructive pulmonary disorder, and
allergic rhinitis), renal disorders, gastrointestinal disorders,
and eye disorders. The pharmaceutical composition includes a
therapeutically effective amount of a N-6 substituted
7-deazapurine, described supra, and a pharmaceutically acceptable
carrier. It is to be understood, that all of the deazapurines
described above are included for therapeutic treatment. It is to be
further understood that the deazapurines of the invention can be
used alone or in combination with other deazapurines of the
invention or in combination with additional therapeutic compounds,
such as antibiotics, antiinflammatories, or anticancer agents, for
example.
[0158] The term "antibiotic" is art recognized and is intended to
include those substances produced by growing microorganisms and
synthetic derivatives thereof, which eliminate or inhibit growth of
pathogens and are selectively toxic to the pathogen while producing
minimal or no deleterious effects upon the infected host subject.
Suitable examples of antibiotics include, but are not limited to,
the principle classes of aminoglycosides, cephalosporins,
chloramphenicols, fuscidic acids, macrolides, penicillins,
polymixins, tetracyclines and streptomycins.
[0159] The term "antiinflammatory" is art recognized and is
intended to include those agents which act on body mechanisms,
without directly antagonizing the causative agent of the
inflammation such as glucocorticoids, aspirin, ibuprofen, NSAIDS,
etc.
[0160] The term "anticancer agent" is art recognized and is
intended to include those agents which diminish, eradicate, or
prevent growth of cancer cells without, preferably, adversely
affecting other physiological functions. Representative examples
include cisplatin and cyclophosphamide.
[0161] When the compounds of the present invention are administered
as pharmaceuticals, to humans and mammals, they can be given per se
or as a pharmaceutical composition containing, for example, 0.1 to
99.5% (more preferably, 0.5 to 90%) of active ingredient in
combination with a pharmaceutically acceptable carrier.
[0162] The phrase "pharmaceutically acceptable carrier" as used
herein means a pharmaceutically acceptable material, composition or
vehicle, such as a liquid or solid filler, diluent, excipient,
solvent or encapsulating material, involved in carrying or
transporting a compound(s) of the present invention within or to
the subject such that it can perform its intended function.
Typically, such compounds are carried or transported from one
organ, or portion of the body, to another organ, or portion of the
body. Each carrier must be "acceptable" in the sense of being
compatible with the other ingredients of the formulation and not
injurious to the patient. Some examples of materials which can
serve as pharmaceutically acceptable carriers include: sugars, such
as lactose, glucose and sucrose; starches, such as corn starch and
potato starch; cellulose, and its derivatives, such as sodium
carboxymethyl cellulose, ethyl cellulose and cellulose acetate;
powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa
butter and suppository waxes; oils, such as peanut oil, cottonseed
oil, safflower oil, sesame oil, olive oil, corn oil and soybean
oil; glycols, such as propylene glycol; polyols, such as glycerin,
sorbitol, mannitol and polyethylene glycol; esters, such as ethyl
oleate and ethyl laurate; agar; buffering agents, such as magnesium
hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water;
isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer
solutions; and other non-toxic compatible substances employed in
pharmaceutical formulations.
[0163] As set out above, certain embodiments of the present
compounds can contain a basic functional group, such as amino or
alkylamino, and are, thus, capable of forming pharmaceutically
acceptable salts with pharmaceutically acceptable acids. The term
"pharmaceutically acceptable salts" in this respect, refers to the
relatively non-toxic, inorganic and organic acid addition salts of
compounds of the present invention. These salts can be prepared in
situ during the final isolation and purification of the compounds
of the invention, or by separately reacting a purified compound of
the invention in its free base form with a suitable organic or
inorganic acid, and isolating the salt thus formed. Representative
salts include the hydrobromide, hydrochloride, sulfate, bisulfate,
phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate,
laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate,
fumarate, succinate, tartrate, napthylate, mesylate,
glucoheptonate, lactobionate, and laurylsulphonate salts and the
like. (See, e.g., Berge et al. (1977) "Pharmaceutical Salts", J.
Pharm. Sci. 66:1-19).
[0164] In other cases, the compounds of the present invention may
contain one or more acidic functional groups and, thus, are capable
of forming pharmaceutically acceptable salts with pharmaceutically
acceptable bases. The term "pharmaceutically acceptable salts" in
these instances refers to the relatively non-toxic, inorganic and
organic base addition salts of compounds of the present invention.
These salts can likewise be prepared in situ during the final
isolation and purification of the compounds, or by separately
reacting the purified compound in its free acid form with a
suitable base, such as the hydroxide, carbonate or bicarbonate of a
pharmaceutically acceptable metal cation, with ammonia, or with a
pharmaceutically acceptable organic primary, secondary or tertiary
amine. Representative alkali or alkaline earth salts include the
lithium, sodium, potassium, calcium, magnesium, and aluminum salts
and the like. Representative organic amines useful for the
formation of base addition salts include ethylamine, diethylamine,
ethylenediamine, ethanolamine, diethanolamine, piperazine and the
like.
[0165] The term "pharmaceutically acceptable esters" refers to the
relatively non-toxic, esterified products of the compounds of the
present invention. These esters can be prepared in situ during the
final isolation and purification of the compounds, or by separately
reacting the purified compound in its free acid form or hydroxyl
with a suitable esterifying agent. Carboxylic acids can be
converted into esters via treatment with an alcohol in the presence
of a catalyst. Hydroxyl containing derivatives can be converted
into esters via treatment with an esterifying agent such as
alkanoyl halides. The term is further intended to include lower
hydrocarbon groups capable of being solvated under physiological
conditions, e.g., alkyl esters, methyl, ethyl and propyl esters.
(See, for example, Berge et al., supra.)
[0166] The invention further contemplates the use of prodrugs which
are converted in vivo to the therapeutic compounds of the invention
(see, e.g., R. B. Silverman, 1992, "The Organic Chemistry of Drug
Design and Drug Action", Academic Press, Chapter 8). Such prodrugs
can be used to alter the biodistribution (e.g., to allow compounds
which would not typically enter the reactive site of the protease)
or the pharmacokinetics of the therapeutic compound. For example, a
carboxylic acid group, can be esterified, e.g., with a methyl group
or an ethyl group to yield an ester. When the ester is administered
to a subject, the ester is cleaved, enzymatically or
non-enzymatically, reductively or hydrolytically, to reveal the
anionic group. An anionic group can be esterified with moieties
(e.g., acyloxymethyl esters) which are cleaved to reveal an
intermediate compound which subsequently decomposes to yield the
active compound. In another embodiment, the prodrug is a reduced
form of a sulfate or sulfonate, e.g., a thiol, which is oxidized in
vivo to the therapeutic compound. Furthermore, an anionic moiety
can be esterified to a group which is actively transported in vivo,
or which is selectively taken up by target organs. The ester can be
selected to allow specific targeting of the therapeutic moieties to
particular reactive sites, as described below for carrier
moieties.
[0167] Wetting agents, emulsifiers and lubricants, such as sodium
lauryl sulfate and magnesium stearate, as well as coloring agents,
release agents, coating agents, sweetening, flavoring and perfuming
agents, preservatives and antioxidants can also be present in the
compositions.
[0168] Examples of pharmaceutically acceptable antioxidants
include: water soluble antioxidants, such as ascorbic acid,
cysteine hydrochloride, sodium bisulfate, sodium metabisulfite,
sodium sulfite and the like; oil-soluble antioxidants, such as
ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated
hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol,
and the like; and metal chelating agents, such as citric acid,
ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid,
phosphoric acid, and the like.
[0169] Formulations of the present invention include those suitable
for oral, nasal, topical, transdermal, buccal, sublingual, rectal,
vaginal and/or parenteral administration. The formulations may
conveniently be presented in unit dosage form and may be prepared
by any methods well known in the art of pharmacy. The amount of
active ingredient which can be combined with a carrier material to
produce a single dosage form will generally be that amount of the
compound which produces a therapeutic effect. Generally, out of one
hundred percent, this amount will range from about 1 percent to
about ninety-nine percent of active ingredient, preferably from
about 5 percent to about 70 percent, most preferably from about 10
percent to about 30 percent.
[0170] Methods of preparing these formulations or compositions
include the step of bringing into association a compound of the
present invention with the carrier and, optionally, one or more
accessory ingredients. In general, the formulations are prepared by
uniformly and intimately bringing into association a compound of
the present invention with liquid carriers, or finely divided solid
carriers, or both, and then, if necessary, shaping the product.
[0171] Formulations of the invention suitable for oral
administration may be in the form of capsules, cachets, pills,
tablets, lozenges (using a flavored basis, usually sucrose and
acacia or tragacanth), powders, granules, or as a solution or a
suspension in an aqueous or non-aqueous liquid, or as an
oil-in-water or water-in-oil liquid emulsion, or as an elixir or
syrup, or as pastilles (using an inert base, such as gelatin and
glycerin, or sucrose and acacia) and/or as mouth washes and the
like, each containing a predetermined amount of a compound of the
present invention as an active ingredient. A compound of the
present invention may also be administered as a bolus, electuary or
paste.
[0172] In solid dosage forms of the invention for oral
administration (capsules, tablets, pills, dragees, powders,
granules and the like), the active ingredient is mixed with one or
more pharmaceutically acceptable carriers, such as sodium citrate
or dicalcium phosphate, and/or any of the following: fillers or
extenders, such as starches, lactose, sucrose, glucose, mannitol,
and/or silicic acid; binders, such as, for example,
carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone,
sucrose and/or acacia; humectants, such as glycerol; disintegrating
agents, such as agar-agar, calcium carbonate, potato or tapioca
starch, alginic acid, certain silicates, and sodium carbonate;
solution retarding agents, such as paraffin; absorption
accelerators, such as quaternary ammonium compounds; wetting
agents, such as, for example, cetyl alcohol and glycerol
monostearate; absorbents, such as kaolin and bentonite clay;
lubricants, such a talc, calcium stearate, magnesium stearate,
solid polyethylene glycols, sodium lauryl sulfate, and mixtures
thereof; and coloring agents. In the case of capsules, tablets and
pills, the pharmaceutical compositions may also comprise buffering
agents. Solid compositions of a similar type may also be employed
as fillers in soft and hard-filled gelatin capsules using such
excipients as lactose or milk sugars, as well as high molecular
weight polyethylene glycols and the like.
[0173] A tablet may be made by compression or molding, optionally
with one or more accessory ingredients. Compressed tablets may be
prepared using binder (for example, gelatin or hydroxypropylmethyl
cellulose), lubricant, inert diluent, preservative, disintegrant
(for example, sodium starch glycolate or cross-linked sodium
carboxymethyl cellulose), surface-active or dispersing agent.
Molded tablets may be made by molding in a suitable machine a
mixture of the powdered compound moistened with an inert liquid
diluent.
[0174] The tablets, and other solid dosage forms of the
pharmaceutical compositions of the present invention, such as
dragees, capsules, pills and granules, may optionally be scored or
prepared with coatings and shells, such as enteric coatings and
other coatings well known in the pharmaceutical-formulating art.
They may also be formulated so as to provide slow or controlled
release of the active ingredient therein using, for example,
hydroxypropylmethyl cellulose in varying proportions to provide the
desired release profile, other polymer matrices, liposomes and/or
microspheres. They may be sterilized by, for example, filtration
through a bacteria-retaining filter, or by incorporating
sterilizing agents in the form of sterile solid compositions which
can be dissolved in sterile water, or some other sterile injectable
medium immediately before use. These compositions may also
optionally contain opacifying agents and may be of a composition
that they release the active ingredient(s) only, or preferentially,
in a certain portion of the gastrointestinal tract, optionally, in
a delayed manner. Examples of embedding compositions which can be
used include polymeric substances and waxes. The active ingredient
can also be in micro-encapsulated form, if appropriate, with one or
more of the above-described excipients.
[0175] Liquid dosage forms for oral administration of the compounds
of the invention include pharmaceutically acceptable emulsions,
microemulsions, solutions, suspensions, syrups and elixirs. In
addition to the active ingredient, the liquid dosage forms may
contain inert dilutents commonly used in the art, such as, for
example, water or other solvents, solubilizing agents and
emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl
carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate,
propylene glycol, 1,3-butylene glycol, oils (in particular,
cottonseed, groundnut, corn, germ, olive, castor and sesame oils),
glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty
acid esters of sorbitan, and mixtures thereof.
[0176] Besides inert dilutents, the oral compositions can also
include adjuvants such as wetting agents, emulsifying and
suspending agents, sweetening, flavoring, coloring, perfuming and
preservative agents.
[0177] Suspensions, in addition to the active compounds, may
contain suspending agents as, for example, ethoxylated isostearyl
alcohols, polyoxyethylene sorbitol and sorbitan esters,
microcrystalline cellulose, aluminum metahydroxide, bentonite,
agar-agar and tragacanth, and mixtures thereof.
[0178] Formulations of the pharmaceutical compositions of the
invention for rectal or vaginal administration may be presented as
a suppository, which may be prepared by mixing one or more
compounds of the invention with one or more suitable nonirritating
excipients or carriers comprising, for example, cocoa butter,
polyethylene glycol, a suppository wax or a salicylate, and which
is solid at room temperature, but liquid at body temperature and,
therefore, will melt in the rectum or vaginal cavity and release
the active compound.
[0179] Formulations of the present invention which are suitable for
vaginal administration also include pessaries, tampons, creams,
gels, pastes, foams or spray formulations containing such carriers
as are known in the art to be appropriate.
[0180] Dosage forms for the topical or transdermal administration
of a compound of this invention include powders, sprays, ointments,
pastes, creams, lotions, gels, solutions, patches and inhalants.
The active compound may be mixed under sterile conditions with a
pharmaceutically acceptable carrier, and with any preservatives,
buffers, or propellants which may be required.
[0181] The ointments, pastes, creams and gels may contain, in
addition to an active compound of this invention, excipients, such
as animal and vegetable fats, oils, waxes, paraffins, starch,
tragacanth, cellulose derivatives, polyethylene glycols, silicones,
bentonites, silicic acid, talc and zinc oxide, or mixtures
thereof.
[0182] Powders and sprays can contain, in addition to a compound of
this invention, excipients such as lactose, talc, silicic acid,
aluminum hydroxide, calcium silicates and polyamide powder, or
mixtures of these substances. Sprays can additionally contain
customary propellants, such as chlorofluorohydrocarbons and
volatile unsubstituted hydrocarbons, such as butane and
propane.
[0183] Transdermal patches have the added advantage of providing
controlled delivery of a compound of the present invention to the
body. Such dosage forms can be made by dissolving or dispersing the
compound in the proper medium. Absorption enhancers can also be
used to increase the flux of the compound across the skin. The rate
of such flux can be controlled by either providing a rate
controlling membrane or dispersing the active compound in a polymer
matrix or gel.
[0184] Ophthalmic formulations, eye ointments, powders, solutions
and the like, are also contemplated as being within the scope of
this invention. Preferably, the pharmaceutical preparation is an
ophthalmic formulation (e.g., an periocular, retrobulbar or
intraocular injection formulation, a systemic formulation, or a
surgical irrigating solution).
[0185] The ophthalmic formulations of the present invention may
include one or more deazapurines and a pharmaceutically acceptable
vehicle. Various types of vehicles may be used. The vehicles will
generally be aqueous in nature. Aqueous solutions are generally
preferred, based on case of formulation, as well as a patient's
ability to easily administer such compositions by means of
instilling one to two drops of the solutions in the affected eyes.
However, the deazapurines of the present invention may also be
readily incorporated into other types of compositions, such as
suspensions, viscous or semi-viscous gels or other types of solid
or semi-solid compositions. The ophthalmic compositions of the
present invention may also include various other ingredients, such
as buffers, preservatives, co-solvents and viscosity building
agents.
[0186] An appropriate buffer system (e.g., sodium phosphate, sodium
acetate or sodium borate) may be added to prevent pH drift under
storage conditions.
[0187] Ophthalmic products are typically packaged in multidose
form. Preservatives are thus required to prevent microbial
contamination during use. Suitable preservatives include:
benzalkonium chloride, thimerosal, chlorobutanol, methyl paraben,
propyl paraben, phenylethyl alcohol, edetate disodium, sorbic acid,
polyquaternium-1, or other agents known to those skilled in the
art. Such preservatives are typically employed at a level of from
0.001 to 1.0% weight/volume ("% w/v").
[0188] When the deazapurines of the present invention are
administered during intraocular surgical procedures, such as
through retrobulbar or periocular injection and intraocular
perfusion or injection, the use of balanced salt irrigating
solutions as vehicles are most preferred. BSS.RTM. Sterile
Irrigating Solution and BSS Plus.RTM. Sterile Intraocular
Irrigating Solution (Alcon Laboratories, Inc., Fort Worth, Tex.,
USA) are examples of physiologically balanced intraocular
irrigating solutions. The latter type of solution is described in
U.S. Pat. No. 4,550,022 (Garabedian, et al.), the entire contents
of which are hereby incorporated in the present specification by
reference. Retrobulbar and periocular injections are known to those
skilled in the art and are described in numerous publications
including, for example, Ophthalmic Surgery: Principles of Practice,
Ed., G. L. Spaeth. W. B. Sanders Co., Philadelphia, Pa., U.S.A.,
pages 85-87 (1990).
[0189] As indicated above, use of deazapurines to prevent or reduce
damage to retinal and optic nerve head tissues at the cellular
level is a particularly important aspect of one embodiment of the
invention. Ophthalmic conditions which may be treated include, but
are not limited to, retinopathies, macular degeneration, ocular
ischemia, glaucoma, and damage associated with injuries to
ophthalmic tissues, such as ischemia reperfusion injuries,
photochemical injuries, and injuries associated with ocular
surgery, particularly injuries to the retina or optic nerve head by
exposure to light or surgical instruments. The compounds may also
be used as an adjunct to ophthalmic surgery, such as by vitreal or
subconjunctival injection following ophthalmic surgery. The
compounds may be used for acute treatment of temporary conditions,
or may be administered chronically, especially in the case of
degenerative disease. The compounds may also be used
prophylactically, especially prior to ocular surgery or noninvasive
ophthalmic procedures, or other types of surgery.
[0190] Pharmaceutical compositions of this invention suitable for
parenteral administration comprise one or more compounds of the
invention in combination with one or more pharmaceutically
acceptable sterile isotonic aqueous or nonaqueous solutions,
dispersions, suspensions or emulsions, or sterile powders which may
be reconstituted into sterile injectable solutions or dispersions
just prior to use, which may contain antioxidants, buffers,
bacteriostats, solutes which render the formulation isotonic with
the blood of the intended recipient or suspending or thickening
agents.
[0191] Examples of suitable aqueous and nonaqueous carriers which
may be employed in the pharmaceutical compositions of the invention
include water, ethanol, polyols (such as glycerol, propylene
glycol, polyethylene glycol, and the like), and suitable mixtures
thereof, vegetable oils, such as olive oil, and injectable organic
esters, such as ethyl oleate. Proper fluidity can be maintained,
for example, by the use of coating materials, such as lecithin, by
the maintenance of the required particle size in the case of
dispersions, and by the use of surfactants.
[0192] These compositions may also contain adjuvants such as
preservatives, wetting agents, emulsifying agents and dispersing
agents. Prevention of the action of microorganisms may be ensured
by the inclusion of various antibacterial and antifungal agents,
for example, paraben, chlorobutanol, phenol sorbic acid, and the
like. It may also be desirable to include isotonic agents, such as
sugars, sodium chloride, and the like into the compositions. In
addition, prolonged absorption of the injectable pharmaceutical
form may be brought about by the inclusion of agents which delay
absorption such as aluminum monostearate and gelatin.
[0193] In some cases, in order to prolong the effect of a drug, it
is desirable to slow the absorption of the drug from subcutaneous
or intramuscular injection. This may be accomplished by the use of
a liquid suspension of crystalline or amorphous material having
poor water solubility. The rate of absorption of the drug then
depends upon its rate of dissolution which, in turn, may depend
upon crystal size and crystalline form. Alternatively, delayed
absorption of a parenterally-administered drug form is accomplished
by dissolving or suspending the drug in an oil vehicle.
[0194] Injectable depot forms are made by forming microencapsule
matrices of the subject compounds in biodegradable polymers such as
polylactide-polyglycolide. Depending on the ratio of drug to
polymer, and the nature of the particular polymer employed, the
rate of drug release can be controlled. Examples of other
biodegradable polymers include poly(orthoesters) and
poly(anhydrides). Depot injectable formulations are also prepared
by entrapping the drug in liposomes or microemulsions which are
compatible with body tissue.
[0195] The preparations of the present invention may be given
orally, parenterally, topically, or rectally. They are of course
given by forms suitable for each administration route. For example,
they are administered in tablets or capsule form, by injection,
inhalation, eye lotion, ointment, suppository, etc. administration
by injection, infusion or inhalation; topical by lotion or
ointment; and rectal by suppositories. Oral administration is
preferred.
[0196] The phrases "parenteral administration" and "administered
parenterally" as used herein means modes of administration other
than enteral and topical administration, usually by injection, and
includes, without limitation, intravenous, intramuscular,
intraarterial, intrathecal, intracapsular, intraorbital,
intracardiac, intradermal, intraperitoneal, transtracheal,
subcutaneous, subcuticular, intraarticular, subcapsular,
subarachnoid, intraspinal and intrasternal injection and
infusion.
[0197] The phrases "systemic administration," "administered
systematically," "peripheral administration" and "administered
peripherally" as used herein mean the administration of a compound,
drug or other material other than directly into the central nervous
system, such that it enters the patient's system and, thus, is
subject to metabolism and other like processes, for example,
subcutaneous administration.
[0198] These compounds may be administered to humans and other
animals for therapy by any suitable route of administration,
including orally, nasally, as by, for example, a spray, rectally,
intravaginally, parenterally, intracisternally and topically, as by
powders, ointments or drops, including buccally and
sublingually.
[0199] Regardless of the route of administration selected, the
compounds of the present invention, which may be used in a suitable
hydrated form, and/or the pharmaceutical compositions of the
present invention, are formulated into pharmaceutically acceptable
dosage forms by conventional methods known to those of skill in the
art.
[0200] Actual dosage levels of the active ingredients in the
pharmaceutical compositions of this invention may be varied so as
to obtain an amount of the active ingredient which is effective to
achieve the desired therapeutic response for a particular patient,
composition, and mode of administration, without being toxic to the
patient.
[0201] The selected dosage level will depend upon a variety of
factors including the activity of the particular compound of the
present invention employed, or the ester, salt or amide thereof,
the route of administration, the time of administration, the rate
of excretion of the particular compound being employed, the
duration of the treatment, other drugs, compounds and/or materials
used in combination with the particular compound employed, the age,
sex, weight, condition, general health and prior medical history of
the patient being treated, and like factors well known in the
medical arts.
[0202] A physician or veterinarian having ordinary skill in the art
can readily determine and prescribe the effective amount of the
pharmaceutical composition required. For example, the physician or
veterinarian could start doses of the compounds of the invention
employed in the pharmaceutical composition at levels lower than
that required in order to achieve the desired therapeutic effect
and gradually increase the dosage until the desired effect is
achieved.
[0203] In general, a suitable daily dose of a compound of the
invention will be that amount of the compound which is the lowest
dose effective to produce a therapeutic effect. Such an effective
dose will generally depend upon the factors described above.
Generally, intravenous and subcutaneous doses of the compounds of
this invention for a patient, when used for the indicated analgesic
effects, will range from about 0.0001 to about 200 mg per kilogram
of body weight per day, more preferably from about 0.01 to about
150 mg per kg per day, and still more preferably from about 0.2 to
about 140 mg per kg per day.
[0204] If desired, the effective daily dose of the active compound
may be administered as two, three, four, five, six or more
sub-doses administered separately at appropriate intervals
throughout the day, optionally, in unit dosage forms.
[0205] While it is possible for a compound of the present invention
to be administered alone, it is preferable to administer the
compound as a pharmaceutical composition.
[0206] The present invention also pertains to packaged
pharmaceutical compositions for treating a N-6 substituted 7
deazapurine responsive state, e.g., undesirable increased adenosine
receptor activity in a mammal. The packaged pharmaceutical
compositions include a container holding a therapeutically
effective amount of at least one deazapurine as described supra and
instructions for using the deazapurine for treating the deazapurine
responsive state in the mammal.
[0207] The deazapurines of the invention can be prepared using
standard methods for organic synthesis. Deazapurines can be
purified by reverse phase HPLC, chromatography, recrystallization,
etc. and their structures confirmed by mass spectral analysis,
elemental analysis, IR and/or NMR spectroscopy.
[0208] Typically, synthesis of the intermediates as well as the
deazapurines of the invention is performed in solution. The
addition and removal of one or more protecting groups is also
typical practice and is known to those skilled in the art. Typical
synthetic schemes for the preparation of deazapurine intermediates
of the invention are outlined below in Scheme I.
[0209] This invention further provides a compound having the
structure (IV): ##STR17## [0210] wherein R.sub.1 is trans-4-hydroxy
cyclohexyl, 2-methylamino carbonylamino cyclohexyl, acetylamino
ethyl, or methylamino carbonylamino ethyl; [0211] wherein R.sub.3
is a substituted or unsubstituted four to six membered ring,
phenyl, pyrrole, thiophene, furan, thiazole, imidazole, pyrazole,
1,2,4-triazole, pyridine, 2(1H)-pyridone, 4(1H)-pyridone, pyrazine,
pyrimidine, pyridazine, isothiazole, isoxazole, oxazole, tetrazole,
naphthalene, tetralin, naphthyridine, benzofuran, benzothiophene,
indole, 2,3-dihydroindole, 1H-indole, indoline, benzopyrazole,
1,3-benzodioxole, benzoxazole, purine, coumarin, chromone,
quinoline, tetrahydroquinoline, isoquinoline, benzimidazole,
quinazoline, pyrido[2,3-b]pyrazine, pyrido[3,4-b]pyrazine,
pyrido[3,2-c]pyridazine, pyrido[3,4-b]-pyridine,
1H-pyrazole[3,4-d]pyrimidine, pteridine, 2(1H)-quinolone,
1(2H)-isoquinolone, 1,4-benzisoxazine, benzothiazole, quinoxaline,
quinoline-N-oxide, isoquinoline-N-oxide, quinoxaline-N-oxide,
quinazoline-N-oxide, benzoxazine, phthalazine, cinnoline, or having
a structure: ##STR18## [0212] wherein Y is carbon or nitrogen;
[0213] wherein R.sub.20 and R.sub.21 are independently H,
substituted or unsubstituted alkyl, substituted or unsubstituted
aryl, halogen, methoxy, methyl amino, or methyl thio; [0214]
wherein R.sub.5 is H, alkyl, substituted alkyl, aryl, arylalkyl,
amino, substituted aryl, wherein said substituted alkyl is
--C(R.sub.7)(R.sub.8)XR.sub.9', wherein X is O, S, or NR.sub.10',
wherein R.sub.7 and R.sub.8 are each independently H or alkyl,
wherein R.sub.9' and R.sub.10' are each independently alkyl or
cycloalkyl, or NR.sub.9'R.sub.10' is a substituted or unsubstituted
ring of between 4 and 7 members; [0215] wherein R.sub.6 is H,
alkyl, substituted alkyl, cycloalkyl; or a pharmaceutically
acceptable salt, a prodrug derivative, or a biologically active
metabolite, with proviso that when R.sub.1 acetylamino ethyl,
R.sub.3 is not 4-pyridyl.
[0216] In one embodiment of the compound having structure IV,
NR.sub.9'R.sub.10' is a substituted or unsubstituted ring of
between 4 and 7 members which is selected from the group consisting
of: ##STR19## wherein m is 0, 1, or 2, ##STR20## [0217] wherein n
is 0, 1, 2, or 3; wherein R.sub.24 is hydrogen, --OH, --CH.sub.2OH,
--C(.dbd.O)NR.sub.9R.sub.10, NHR.sub.22; wherein R.sub.22 is
--C(.dbd.O)CH.sub.3, or --SO.sub.2Me, or ##STR21## [0218] wherein R
is H, alkyl, or aryl.
[0219] In another embodiment of the compound having structure IV,
R.sub.3 has the structure: ##STR22## [0220] wherein Y is carbon or
nitrogen; wherein R.sub.23 is H, or halogen, --O-alkyl group, amine
group, or sulfide group; [0221] wherein R.sub.5 is H, alkyl,
substituted alkyl, aryl, arylalkyl, amino, substituted aryl,
wherein said substituted alkyl is
--C(R.sub.7)(R.sub.8)NR.sub.9'R.sub.10', wherein R.sub.7 and
R.sub.8 are each independently H or alkyl, wherein R.sub.9', and
R.sub.10' are each independently alkyl or cycloalkyl, or R.sub.9,
R.sub.10' and the nitrogen together form a substituted or
unsubstituted ring of between 4 and 7 members.
[0222] In another embodiment of the compound, Y is carbon.
[0223] In another embodiment of the compound, R.sub.23 is
hydrogen.
[0224] In another embodiment of the compound, R.sub.6 is
hydrogen.
[0225] In another embodiment of the compound, R.sub.5 is
hydrogen.
[0226] In another embodiment of the compound, R.sub.5, and R.sub.6
are each methyl.
[0227] In another embodiment of the compound, R.sub.5 is
--C(R.sub.7)(R.sub.8)NR.sub.9'R.sub.10', wherein R.sub.7 and
R.sub.8 are each independently H or alkyl, wherein R.sub.9' and
R.sub.10' are each independently alkyl or cycloalkyl, or R.sub.9',
R.sub.10' and the nitrogen together form a substituted or
unsubstituted ring of between 4 and 7 members.
[0228] In another embodiment of the compound, R.sub.23 is
halogen.
[0229] In another embodiment of the compound, Y is nitrogen.
[0230] In yet another embodiment of the compound, R.sub.23 is
hydrogen.
[0231] In a further embodiment of the compound, R.sub.5 and R.sub.6
are each hydrogen.
[0232] This invention also provides a compound having the structure
(V): ##STR23## [0233] wherein R.sub.3 is aryl, substituted aryl, or
heteroaryl; [0234] wherein R.sub.5 is H, alkyl, substituted alkyl,
aryl, arylalkyl, amino, substituted aryl, wherein said substituted
alkyl is --C(R.sub.7)(R.sub.8)NR.sub.9'R.sub.10', wherein R.sub.7
and R.sub.8 are each H or alkyl, wherein R.sub.9' and R.sub.10' are
each alkyl or cycloalkyl, or R.sub.9', R.sub.10' and the nitrogen
together form a ring system of between 4 and 7 members; and [0235]
wherein R.sub.6 is H, alkyl, substituted alkyl, or cycloalkyl.
[0236] In one embodiment of the compound having structure V,
R.sub.7 and R.sub.8 are each H; wherein R.sub.9' is H and R.sub.10'
is --R.sub.12C(.dbd.O)R.sub.13.
[0237] In another embodiment of the compound having structure V,
R.sub.7 and R.sub.8 are each H; wherein the ring system is
morpholino, thiomorpholino, N-4-substituted piperazino,
2-substituted piperazine, or R.sub.24 substituted pyrrolidino,
piperidine, wherein R.sub.24 is H, OH, CH.sub.2OH,
--C(.dbd.O)NR.sub.9R.sub.10, NR.sub.22, wherein R.sub.22 is
--C(.dbd.O)CH.sub.3, --SO.sub.2Me.
[0238] In another embodiment of the compound, the compound has the
following structure: ##STR24##
[0239] In another embodiment of the compound, the compound has the
structure: ##STR25##
[0240] In another embodiment of the compound, the compound has the
structure: ##STR26##
[0241] In another embodiment of the compound, the compound has the
structure: ##STR27##
[0242] In another embodiment of the compound, the compound has the
structure: ##STR28##
[0243] In another embodiment of the compound, the compound has the
structure: ##STR29##
[0244] In another embodiment of the compound, the compound has the
structure: ##STR30##
[0245] A compound having the structure: ##STR31## [0246] wherein
R.sub.3 is a 5-6 membered aromatic ring; wherein R.sub.5 and
R.sub.6 are independently H, or alkyl.
[0247] In one embodiment of the compound, the compound has the
structure: ##STR32##
[0248] In one embodiment of the compound, the compound has the
structure: ##STR33##
[0249] In another embodiment of the compound, the compound has the
structure: ##STR34##
[0250] In another embodiment of compound 1500, the compound has the
structure: ##STR35##
[0251] In a further embodiment of the compound, the compound has
the structure: ##STR36##
[0252] This invention also provides a compound having the
structure: ##STR37## [0253] wherein R.sub.3 is a 5-6 membered
aromatic ring; wherein R.sub.5 and R.sub.6 are independently H, or
alkyl; with the proviso that R.sub.3 is not 4-pyridyl.
[0254] In one embodiment of the compound, the compound has the
structure: ##STR38##
[0255] This invention further provides a compound having the
structure: ##STR39## [0256] wherein R.sub.3 is a substituted 5-6
membered aromatic ring; [0257] wherein R.sub.5 and R.sub.6 are
independently H, or alkyl.
[0258] In one embodiment of the compound, the compound has the
structure: ##STR40##
[0259] This invention also provides a compound having the
structure: ##STR41## [0260] wherein R.sub.3 is a 5-6 membered
aromatic ring; wherein Z is oxygen, or sulfur.
[0261] In one embodiment of the compound, the compound has the
structure: ##STR42##
[0262] This invention also provides a compound having the
structure: ##STR43## [0263] wherein R.sub.3 is a 5-6 membered
aromatic ring; wherein Z is oxygen, or sulfur.
[0264] In one embodiment of the compound, the compound has the
structure: ##STR44##
[0265] This invention further provides a method for treating a
disease associated with A.sub.1 adenosine receptor in a subject,
comprising administering to the subject a therapeutically effective
amount of a compound having the formula IV, V, VI, VII, VIII, IX,
or X.
[0266] In one embodiment of the method, the subject is a mammal. In
another embodiment of the method, the mammal is a human.
[0267] In another embodiment of the method, the A.sub.1 adenosine
receptor is associated with cognitive disease, renal failure,
cardiac arrhythmias, respiratory epithelia, transmitter release,
sedation, vasoconstriction, bradycardia, negative cardiac inotropy
and dromotropy, branchoconstriction, neutropil chemotaxis, reflux
condition, or ulcerative condition.
[0268] This invention also provides a combination therapy for
asthma, comprising compounds IV and V, and a steroid, b2 agonist,
glucocoticoid, lucotriene antagonist, or anticolinegic agonist.
Diseases associated with adenosine A.sub.1, A.sub.2a, A.sub.2b and
A.sub.3 receptors are disclosed in WO 99/06053 and WO-09822465,
WO-09705138, WO-09511681, WO-09733879, JP-09291089, PCT/US98/16053
and U.S. Pat. No. 5,516,894, the entire content of which are fully
incorporate herein by reference.
[0269] This invention also provides a water-soluble prodrug of a
compound having the structures IV, V, VI, VII, VIII, IX, or X,
wherein said water-soluble prodrug that is metabolized in vivo to
an active drug which selectively inhibit A.sub.1 adenosine
receptor.
[0270] In one embodiment of the prodrug, said prodrug is
metabolized in vivo by esterase catalyzed hydrolysis.
[0271] This invention also provides a pharmaceutical composition
comprising the prodrug and a pharmaceutically acceptable
carrier.
[0272] This invention further provides a method for inhibiting the
activity of an A.sub.1 adenosine receptor in a cell, which
comprises contacting said cell with a compound having the
structures IV, V, VI, VII, VIII, IX, or X.
[0273] In one embodiment of the method, the compound is an
antagonist of said A.sub.1 adenosine receptor.
[0274] This invention also provides for a method for treating a
gastrointestinal disorder in an subject, comprising administering
to the an effective amount of a compound having the structures IV,
V, VI, VII, VIII, IX, or X.
[0275] In one embodiment of the method, said disorder is
diarrhea.
[0276] In another embodiment of the method, the subject is a
human.
[0277] In another embodiment of the method, the compound is an
antagonist of A.sub.1 adenosine receptors.
[0278] This invention also provides a method for treating
respiratory disorder in a subject, comprising administering to the
subject an effective amount of a compound having the structures IV,
V, VI, VII, VIII, IX, or X.
[0279] In one embodiment of the method, said disorder is asthma,
chronic obstructive pulmonary disease, allergic rhinitis, or an
upper respiratory disorder.
[0280] In another embodiment of the method, the subject is a
human.
[0281] In another embodiment of the method, said compound is an
antagonist of A.sub.1 adenosine receptors.
[0282] This invention further provides a method for treating damage
to the eye of a subject which comprises administering to said
subject an effective amount of a compound having the structures IV,
V, VI, VII, VIII, IX, or X.
[0283] In one embodiment of the method, said damage comprises
retinal or optic nerve head damage.
[0284] In another embodiment of the method, said damage is acute or
chronic.
[0285] In another embodiment of the method, wherein said damage is
the result of glaucoma, edema, ischemia, hypoxia or trauma.
[0286] In another embodiment of the method, the subject is a
human.
[0287] In another embodiment of the method, the compound is an
antagonist of A.sub.1 adenosine receptors.
[0288] This invention also provides a pharmaceutical composition
comprising a therapeutically effective amount of a compound having
the structures IV, V, VI, VII, VIII, IX, or X, and a
pharmaceutically acceptable carrier.
[0289] In another embodiment of the pharmaceutical composition,
said therapeutically effective amount is effective to treat a
respiratory disorder or a gastrointestinal disorder.
[0290] In another embodiment of the pharmaceutical composition,
said gastrointestinal disorder is diarrhea.
[0291] In another embodiment of the pharmaceutical composition,
said respiratory disorder is asthma, allergic rhinitis, or chronic
obstructive pulmonary disease.
[0292] In another embodiment of the pharmaceutical composition,
said pharmaceutical composition is an ophthalmic formulation.
[0293] In another embodiment of the pharmaceutical composition,
said pharmaceutical composition is an periocular, retrobulbar or
intraocular injection formulation.
[0294] In yet another embodiment of the pharmaceutical composition,
said pharmaceutical composition is a systemic formulation.
[0295] In a further embodiment of the pharmaceutical preparation,
said pharmaceutical composition is a surgical irrigating
solution.
[0296] This invention also provides a packaged pharmaceutical
composition for treating a disease associated with A.sub.1
adenosine receptor in a subject, comprising: (a) a container
holding a therapeutically effective amount of an adenosine A.sub.1
specific compound; and (b) instructions for using said compound for
treating said disease in a subject.
[0297] As used herein, "A compound is A.sub.1 selective" means that
a compound has a binding constant to adenosine A.sub.1 receptor of
at least ten times higher than that to adenosine A.sub.2a, A.sub.2b
or A.sub.3.
[0298] This invention also provides a method of preparing the
compound having structure IV, comprising the steps of [0299] a)
reacting ##STR45## [0300] to provide ##STR46## [0301] wherein P is
a removable protecting group; [0302] b) treating the product of
step a) under cyclization conditions to provide ##STR47## [0303] c)
treating the product of step b) under suitable conditions to
provide ##STR48## [0304] d) treating the chlorinated product of
step c) with NH.sub.2R.sub.1 to provide ##STR49## [0305] wherein
R.sub.1 is trans-4-hydroxy cyclohexyl, 2-methylamino carbonylamino
cyclohexyl, acetylamino ethyl, or methylamino carbonylamino ethyl;
[0306] wherein R.sub.3 is a substituted or unsubstituted four to
six membered ring; [0307] wherein R.sub.6 is H, alkyl, substituted
alkyl, cycloalkyl; or [0308] a pharmaceutically acceptable salt, or
a prodrug derivative, or a biologically active metabolite; with the
proviso that when R.sub.1 is acetylamino ethyl, R.sub.3 is not
4-pyridyl.
[0309] This invention also provides a method of preparing the
compound having structure V, comprising the steps of [0310] a)
reacting ##STR50## [0311] to provide ##STR51## [0312] wherein P is
a removable protecting group; [0313] b) treating the product of
step a) under cyclization conditions to provide ##STR52## [0314] c)
treating the product of step b) under suitable conditions to
provide ##STR53## [0315] d) treating the chlorinated product of
step c) with ##STR54## [0316] to provide ##STR55## [0317] wherein
R.sub.3 is aryl, substituted aryl, heteroaryl; [0318] wherein
R.sub.5 is H, alkyl, substituted alkyl, aryl, arylalkyl, amino,
substituted aryl, wherein said substituted alkyl is
--C(R.sub.7)(R.sub.8)NR.sub.9R.sub.10, wherein R.sub.7 and R.sub.8
are each H or alkyl, wherein R.sub.9 and R.sub.10 are each alkyl or
cycloalkyl, or NR.sub.9R.sub.10 is a ring system of between 4 and 7
members; and [0319] wherein R.sub.6 is H, alkyl, substituted alkyl,
or cycloalkyl.
[0320] Compounds represented by formulas VI, VII, and VIII can be
synthesized by any of the Schemes I-VIII. Compounds represented by
formulas IX and X can be prepared by Scheme IX.
[0321] This invention further provides compounds having the
formula: ##STR56## [0322] wherein [0323] R.sub.1NR.sub.2 together
form a ring having the structure: ##STR57## [0324] or R.sub.1 is H
and R.sub.2 is: ##STR58## [0325] R.sub.5 is H, or substituted or
unsubstituted alkyl or alkylaryl.
[0326] In one embodiment the compound has the structure:
##STR59##
[0327] In a further embodiment the compound has the structure:
##STR60##
[0328] In a further embodiment the compound has the structure:
##STR61##
[0329] In a further embodiment the compound has the structure:
##STR62##
[0330] In a further embodiment the compound has the structure:
##STR63##
[0331] In a further embodiment the compound has the structure:
##STR64##
[0332] In a further embodiment the compound has the structure:
##STR65##
[0333] In a further embodiment the compound has the structure:
##STR66##
[0334] In a further embodiment the compound has the structure:
##STR67##
[0335] In a further embodiment the compound has the structure:
##STR68##
[0336] In a further embodiment the compound has the structure:
##STR69##
[0337] In a further embodiment the compound has the structure:
##STR70##
[0338] In a further embodiment the compound has the structure:
##STR71##
[0339] In a further embodiment the compound has the structure:
##STR72##
[0340] In a further embodiment the compound has the structure:
##STR73##
[0341] In a further embodiment the compound has the structure:
##STR74##
[0342] In a further embodiment the invention provides a method for
treating a disease associated with A.sub.1 adenosine receptor in a
subject, comprising administering to the subject a therapeutically
effective amount of a compound of formula XI, or compound 1601,
1602, 1605, 1606, 1611, 1614, 1619, 1621, 1623, 1624, 1625, 1626,
1627, 1628, 1629, or 1630.
[0343] In a further embodiment the invention provides the above
method, wherein the subject is a mammal.
[0344] In a further embodiment the invention provides the above
method, wherein the mammal is a human.
[0345] In a further embodiment the invention provides the above
method, wherein said A.sub.1 adenosine receptor is associated with
cognitive disease, renal failure, cardiac arrhythmias, respiratory
epithelia, transmitter release, sedation, vasoconstriction,
bradycardia, negative cardiac inotropy and dromotropy,
branchoconstriction, neutropil chemotaxis, reflux condition, or
ulcerative condition.
[0346] In a further embodiment the invention provides a
water-soluble prodrug of the compounds of formula XI, or compound
1601, 1602, 1605, 1606, 1611, 1614, 1619, 1621, 1623, 1624, 1625,
1626, 1627, 1628, 1629, or 1630, wherein the water-soluble prodrug
is metabolized in vivo to produce an active drug which selectively
inhibits A.sub.1 adenosine receptor.
[0347] In a further embodiment the invention provides, wherein said
prodrug is metabolized in vivo by esterase catalyzed
hydrolysis.
[0348] In a further embodiment the invention provides a
pharmaceutical composition comprising the above prodrug and a
pharmaceutically acceptable carrier.
[0349] In a further embodiment the invention provides a method for
inhibiting the activity of an A.sub.1 adenosine receptor in a cell,
which comprises contacting the cell with a compound of formula XI
or compound 1601, 1602, 1605, 1606, 1611, 1614, 1619, 1621, 1623,
1624, 1625, 1626, 1627, 1628, 1629, or 1630.
[0350] In a further embodiment the invention provides the above
method for inhibiting the activity of an A.sub.1 adenosine receptor
in a cell, wherein the compound is an antagonist of the A.sub.1
adenosine receptor.
[0351] In a further embodiment the invention provides the above
method for inhibiting the activity of an A.sub.1 adenosine receptor
in a cell, wherein the cell is human cell.
[0352] In a further embodiment the invention provides the above
method for inhibiting the activity of an A.sub.1 adenosine receptor
in a human cell, wherein the compound is an antagonist of A.sub.1
adenosine receptors.
[0353] In a further embodiment the invention provides a method for
treating a disease associated with A.sub.1 adenosine receptor in a
subject, wherein said disease is asthma, chronic obstructive
pulmonary disease, allergic rhinitis, or an upper respiratory
disorder.
[0354] In a further embodiment the invention provides a method for
treating a disease associated with A.sub.1 adenosine receptor in a
subject, wherein said disease is asthma, chronic obstructive
pulmonary disease, allergic rhinitis, or an upper respiratory
disorder and wherein the subject is a human.
[0355] In a further embodiment the invention provides a method for
treating the above disease, wherein said compound is an antagonist
of A.sub.1 adenosine receptors.
[0356] In a further embodiment the invention provides a combination
therapy for asthma, comprising a compound of formula XI, or
compound 1601, 1602, 1605, 1606, 1611, 1614, 1619, 1621, 1623,
1624, 1625, 1626, 1627, 1628, 1629, or 1630, and a steroid, b2
agonist, glucocorticoid, lucotriene antagonist, or anticolinergic
agonist.
[0357] In a further embodiment the invention provides a
pharmaceutical composition comprising a therapeutically effective
amount of a compound of formula XI, or compound 1601, 1602, 1605,
1606, 1611, 1614, 1619, 1621, 1623, 1624, 1625, 1626, 1627, 1628,
1629, or 1630, and a pharmaceutically acceptable carrier.
[0358] In a further embodiment the invention provides a method for
treating a respiratory disorder a compound of formula XI, or
compound 1601, 1602, 1605, 1606, 1611, 1614, 1619, 1621, 1623,
1624, 1625, 1626, 1627, 1628, 1629, or 1630, wherein said
respiratory disorder is asthma, allergic rhinitis, or chronic
obstructive pulmonary disease.
[0359] In a further embodiment the invention provides the above
pharmaceutical composition(s), wherein said pharmaceutical
composition is an periocular, retrobulbar or intraocular injection
formulation.
[0360] In a further embodiment the invention provides the above
pharmaceutical composition(s), wherein said pharmaceutical
composition is a systemic formulation.
[0361] In a further embodiment the invention provides the above
pharmaceutical composition(s), wherein said pharmaceutical
composition is a surgical irrigating solution.
[0362] In a further embodiment the invention provides a packaged
pharmaceutical composition for treating a disease associated with
A.sub.1 adenosine receptor in a subject, comprising: [0363] (a) a
container holding a therapeutically effective amount of a compound
of formula XI, or compound 1601, 1602, 1605, 1606, 1611, 1614,
1619, 1621, 1623, 1624, 1625, 1626, 1627, 1628, 1629, or 1630; and
[0364] (b) instructions for using said compound for treating said
disease in a subject.
[0365] In a further embodiment the invention provides a
pharmaceutically acceptable salt a compound of formula XI, or
compound 1601, 1602, 1605, 1606, 1611, 1614, 1619, 1621, 1623,
1624, 1625, 1626, 1627, 1628, 1629, or 1630.
[0366] In a further embodiment the invention provides the above
pharmaceutically acceptable salt, wherein the pharmaceutically
acceptable salt of compound 1611, 1619, 1625, 1628, or 1629
contains a cation selected from the group consisting of sodium,
calcium and ammonium.
[0367] In yet a further embodiment the invention provides a method
for treating a disease associated with A.sub.1 adenosine receptor
in a subject, wherein the A.sub.1 adenosine receptor is associated
with congestive heart failure.
[0368] The invention is further illustrated by the following
examples which in no way should be construed as being further
limiting. The contents of all references, pending patent
applications and published patent applications, cited throughout
this application, including those referenced in the background
section, are hereby incorporated by reference. It should be
understood that the models used throughout the examples are
accepted models and that the demonstration of efficacy in these
models is predictive of efficacy in humans.
[0369] This invention will be better understood from the
Experimental Details which follow. However, one skilled in the art
will readily appreciate that the specific methods and results
discussed are merely illustrative of the invention as described
more fully in the claims which follow thereafter.
EXPERIMENTAL DETAILS
[0370] The deazapurines of the invention can be prepared using
standard methods for organic synthesis. Deazapurines can be
purified by reverse phase HPLC, chromatography, recrystallization,
etc. and their structures confirmed by mass spectral analysis,
elemental analysis, IR and/or NMR spectroscopy.
[0371] Typically, synthesis of the intermediates as well as the
deazapurines of the invention is performed in solution. The
addition and removal of one or more protecting group is also
typical practice and is known to those skilled in the art. Typical
synthetic schemes for the preparation of deazapurine intermediates
of the invention are outlined below in Scheme I. ##STR75## [0372]
wherein R.sub.3, R.sub.5 and R.sub.6 are as defined above.
[0373] In general, a protected 2-amino-3-cyano-pyrrole can be
treated with an acyl halide to form a carboxyamido-3-cyano-pyrrole
which can be treated with acidic methanol to effect ring closure to
a pyrrolo[2,3d]pyrimidine-4(3H)-one (Muller, C. E. et al. J. Med.
Chem. 40:4396 (1997)). Removal of the pyrrolo protecting group
followed by treatment with a chlorinating reagent, e.g.,
phosphorous oxychloride, produced substituted or unsubstituted
4-chloro-7H-pyrrolo[2,3d]pyrimidines. Treatment of the
chloropyrimidine with amines afforded 7-deazapurines.
[0374] For example, as shown in Scheme I, a
N-(1-dl-phenylethyl)-2-amino-3-cyano-pyrrole was treated with an
acyl halide in pyridine and dichloromethane. The resultant
N-(1-dl-phenylethyl)-2-phenylcarboxyamido-3-cyano-pyrrole was
treated with a 10:1 mixture of methanol/sulfuric acid to effect
ring closure, resulting in a
dl-7H-7-(1-phenylethyl)pyrrolo[2,3d]pyrimidine-4(3H)-one. Removal
of the phenylethyl group by treatment of the pyrimidine with
polyphosphoric acid (PPA) followed by POCl.sub.3 afforded a key
intermediate, the 4-chloro-7H-pyrrolo[2,3d]pyrimidine. Further
treatment of the 4-chloro-7H-pyrrolo[2,3d]pyrimidine with various
amines listed in Table 1-A gives compounds of formula (I) and (II).
TABLE-US-00001 TABLE 1-A R M.sup.+ + H ##STR76## 343.2 ##STR77##
343.18 ##STR78## 337.21 ##STR79## 364.19 ##STR80## 330.18 ##STR81##
347.22 ##STR82## 350.28 ##STR83## 344.19 ##STR84## 394.16 ##STR85##
371.12 ##STR86## 359.39 ##STR87## 403.33 ##STR88## 351.49 ##STR89##
330.37 ##STR90## 407.23 ##STR91## 355.45 ##STR92## 441.33 ##STR93##
413.24 ##STR94## 372.48 ##STR95## 351.27 ##STR96## 430.35 ##STR97##
359.44 ##STR98## 404.32 ##STR99## 330.45 ##STR100## 339.47
##STR101## 353.41 ##STR102## 324.45 ##STR103## 359.38 ##STR104##
379.40 ##STR105## 387.41 ##STR106## 344.48 ##STR107## 337.53
##STR108## 295.2 ##STR109## 321.2 ##STR110## 337.53 ##STR111##
350.2 ##STR112## 343.2 ##STR113## 373.2 ##STR114## 307.2
[0375] A general approach to prepare 6-substituted pyrroles is
depicted in the following scheme (Scheme II). ##STR115## [0376]
wherein R.sub.1, through R.sub.5 are as defined above.
[0377] Transesterification and alkylation of ethyl cyanoacetate
with an .alpha.-haloketone affords a ketomethylester. Protection of
the ketone followed by treatment with an amidine (e.g., alkyl, aryl
or alkylaryl)hydrochloride produced the resultant ketal protected
pyrimidine. Removal of the protecting group, followed by
cyclization and treatment with phosphorous oxychloride afforded the
chloride intermediate which could be further treated with an amine
to afford an amine 6-substituted pyrrole. Additionally, alkylation
of the pyrrole nitrogen can be achieved under art recognized
conditions.
[0378] A general approach to prepare 5-substituted pyrroles is
depicted in the following scheme (Scheme III). ##STR116## [0379]
wherein R.sub.1 through R.sub.6 are defined as above and R is a
removable protecting group.
[0380] Condensation of malononitrile and an excess of a ketone
followed by bromination of the product afforded a mixture of
starting material, monobrominated and dibrominated products which
were treated with an alkylamine, arylamine or alkylarylamine. The
resultant amine product was acylated with an acid chloride and the
monacylated pyrrole was cyclized in the presence of acid to afford
the corresponding pyrimidine. The pyrrole protecting group was
removed with polyphosphoric acid and treated with phosphorous
oxychloride to produce a chlorinated product. The chlorinated
pyrrole could subsequently be treated with an amine to produce an
amino 5-substituted pyrrole. Alkylation of the pyrrole nitrogen can
be achieved under art recognized conditions.
[0381] Schemes IV and V depict methods for preparing the
deazapurines 1 and 2 of the invention. ##STR117## [0382] wherein
R.sub.5 and R.sub.6 are as described above, e.g., CH.sub.3.
Specific Preparation of 6-methyl pyrrolopyrimidines:
[0383] The key reaction toward 6-methylpyrrolopyrimidines (1)
[R.sub.5.dbd.CH.sub.3] was cyclization of a cyanoacetate with
benzamidine to a pyrimidine. It was believed methyl cyanoacetate
would cyclize more efficiently with benzamidine to a pyrimidine
than the corresponding ethyl ester. Therefore, transesterification
and alkylation of ethyl cyanoacetate in the presence of NaOMe and
an excess of an .alpha.-haloacetyl moiety, e.g., chloroacetone,
gave the desired methyl ester (3) in 79% yield (Scheme IV). The
ketoester (3) was protected as the acetal (4) in 81% yield. A new
cyclization method to the pyrimidine (5) was achieved with an
amidine hydrochloride, e.g., benzamidine hydrochloride, with 2
equivalents of DBU to afford the 5 in 54% isolated yield. This
method improves the yield from 20% using the published conditions,
which utilizes NaOMe during the cyclization with guanidine.
Cyclization to the pyrrole-pyrimidine (6) was achieved via
deprotection of the acetal in aqueous HCl in 78% yield. Reaction of
(6) with phosphorous oxychloride at reflux gave the corresponding
4-chloro derivative (7). Coupling with trans-4-aminocyclohexanol in
dimethyl sulfoxide at 135.degree. C. gave (1) in 57% from (7). One
skilled in the art will appreciate that choice of reagents allows
for great flexibility in choosing the desired substituent R.sub.5.
##STR118## Specific Preparation of 5-methylpyrrolopyrimidines
[0384] Knoevengel condensation of malononitrile and an excess
ketone, e.g., acetone in refluxing benzene gave 8 in 50% yield
after distillation. Bromination of 8 with N-bromosuccinimide in the
presence of benzoyl peroxide in chloroform yielded a mixture of
starting material, mono- (9), and di-brominated products (5/90/5)
after distillation (70%). The mixture was reacted with an
.alpha.-methylalkylamine or .alpha.-methylarylamine, e.g.,
.alpha.-methylbenzylamine, to deliver the aminopyrrole (10). After
passing through a short silica gel column, the partially purified
amine (31% yield) was acylated with an acid chloride, e.g., benzoyl
chloride to deliver mono- (11), and diacylated (12) pyrroles, which
were separated by flash chromatography. Acid hydrolysis of the
disubstituted pyrrole (12) generated a combined yield of 29% for
the acylpyrrole (11). Cyclization in the presence of concentrated
sulphuric acid and DMF yielded (13) (23%), which was deprotected
with polyphosphoric acid to (14). Reaction of (14) with phosphorous
oxychloride at reflux gave the corresponding 4-chloro derivative
(15). Coupling with trans-4-aminocyclohexanol in dimethyl sulfoxide
at 135.degree. C. gave (2) [R.sub.6.dbd.CH.sub.3] in 30% from (14)
(See Scheme V). One skilled in the art will appreciate that choice
of reagents allows for great flexibility in choosing the desired
substituent R.sub.6. ##STR119## ##STR120## Alternative Synthetic
Route to R.sub.6-Substituted Pyrroles, e.g., 5-methyl
pyrrolopyrimidines:
[0385] This alternative route to R.sub.6-substituted pyrroles,
e.g., 5-methylpyrrolopyrimidines, involves transesterification and
alkylation of ethyl cyanoacetate to (16) (Scheme VI). The
condensation of (16) with benzamidine hydrochloride with 2
equivalents of DBU affords the pyrimidine (17). Cyclization to the
pyrrole-pyrimidine (14) will be achieved via deprotection of the
acetal in aqueous HCl. Reaction of (14) with phosphorous
oxychloride at reflux gave the corresponding 4-chloro derivative
(15). Coupling with trans-4-aminocyclohexanol in dimethyl sulfoxide
at 135.degree. C. gives 2. This procedure reduces the number of
synthetic reactions to the target compound (2) from 9 to 4 steps.
Moreover, the yield is dramatically improved. Again, one skilled in
the art will appreciate that choice of reagents allows for great
flexibility in choosing the desired substituent R.sub.6.
##STR121##
[0386] A general approach to prepare des-methylpyrrole is depicted
in the following scheme (Scheme VII) ##STR122## ##STR123## [0387]
wherein R.sub.1 through R.sub.3 are defined as above.
[0388] Alkylation of an alkyl cyanoacetate with a diethyl acetal in
the presence of a base afforded a cyano diethyl acetal which was
treated with an amidine salt to produce a methyl pyrrolopyrimidine
precursor. The precursor was chlorinated and treated with an amine
to form the des-methyl pyrrolopyrimidine target as shown above.
[0389] For example, Scheme VIII depicts the synthesis of compound
(18). ##STR124##
[0390] Commercially available methyl cyanoacetate was alkylated
with bromoacetaldehyde diethyl acetal in the presence of potassium
carbonate and NaI to yield (19). Cyclization to the pyrimidine (20)
was achieved in two steps. Initially, the pyrimidine-acetal was
formed via reaction of (19) with benzamidine hydrochloride with 2
equivalents of DBU. The resultant pyrimidine-acetal was deprotected
without purification with aqueous 1 N HCl and the resultant
aldehyde cyclized to the pyrrolo-pyrimidine (20), which was
isolated by filtration. Reaction of (20) with phosphorous
oxychloride at reflux afforded the corresponding 4-chloro
derivative (21). Coupling of the chloro derivative with
trans-4-aminocyclohexanol in DMSO at 135.degree. C. gave compound
(18) from compound (21).
[0391] Schemes II-VIII demonstrate that it is possible to
functionalize the 5- and 6-position of the pyrrolopyrimidine ring.
Through the use of different starting reagents and slight
modifications of the above reaction schemes, various functional
groups can be introduced at the 5- and 6-positions in formula (I)
and (II). Table 2-A illustrates some examples. TABLE-US-00002 TABLE
2-A Selected list of 5- and 6-substituted pyrrolopyrimidines.
Starting Reagent R.sub.5 R.sub.6 ##STR125## H ##STR126## H
Substituted Ar ##STR127## H CH.sub.2C(O)OCH.sub.3 ##STR128##
C(O)OCH.sub.3 CH.sub.3 ##STR129## C(O)NHCH.sub.3 CH.sub.3
[0392] A skilled artisan will know that metabolism of the compounds
disclosed herein in a subject produces certain biologically active
metabolites which can serve as drugs.
[0393] The invention is further illustrated by the following
examples which in no way should be construed as being further
limiting. The contents of all references, pending patent
applications and published patent applications, cited throughout
this application, including those referenced in the background
section, are hereby incorporated by reference. It should be
understood that the models used throughout the examples are
accepted models and that the demonstration of efficacy in these
models is predictive of efficacy in humans.
EXEMPLIFICATION
Preparation 1
[0394] A modification of the alkylation method of Seela and Lupke
was used..sup.1 To an ice-cooled (0.degree. C.) solution of ethyl
cyanoacetate (6.58 g, 58.1 mmol) in MeOH (20 mL) was slowly added a
solution of NaOMe (25% w/v; 58.1 mmol). After 10 min, chloroacetone
(5 mL; 62.8 mmol) was slowly added. After 4 h, the solvent was
removed. The brown oil was diluted the EtOAc (100 mL) and washed
with H.sub.2O (100 mL). The organic fraction was dried, filtered,
and concentrated to a brown oil (7.79 g; 79%). The oil (3) (Scheme
IV) was a mixture of methyl/ethyl ester products (9/1), and was
used without further purification. .sup.1H NMR (200 MHz,
CDCl.sub.3) .delta..sub.--4.24 (q, J=7.2 Hz, OCH.sub.2), 3.91 (dd,
1H, J=7.2, 7.0 Hz, CH), 3.62 (s, 3H, OCH.sub.3), 3.42 (dd, 1H,
J=15.0, 7.1 Hz, 1.times.CH.sub.2); 3.02 (dd, 1H, J=15.0, 7.0 Hz,
1.times.CH.sub.2); 2.44 (s, 3H, CH.sub.3), 1.26 (t, J=7.1 Hz,
ester-CH.sub.3). .sup.1Seela, F.; Lupke, U. Chem. Ber. 1977, 110,
1462-1469.
Preparation 2
[0395] The procedure of Seela and Lupke was used..sup.1 Thus,
protection of the ketone (3) (Scheme IV; 5.0 g, 32.2 mmol) with
ethylene glycol (4 mL, 64.4 mmol) in the presence of TsOH (100 mg)
afforded (4) as an oil (Scheme IV; 5.2 g, 81.0) after flash
chromatography (SiO.sub.2; 3/7 EtOAc/Hex, R.sub.f 0.35). Still
contains .about.5% ethyl ester: .sup.1H NMR (200 MHz, CDCl.sub.3)
.delta..sub.--4.24 (q, J=7.2 Hz, OCH.sub.2), 3.98 (s, 4H, 2.times.
acetal-CH.sub.2), 3.79 (s, 3H, OCH.sub.3), 3.62 (dd, 1H, J=7.2, 7.0
Hz, CH), 2.48 (dd, 1H, J=15.0, 7.1 Hz, 1.times.CH.sub.2), 2.32 (dd,
1H, J=15.0, 7.0 Hz, 1.times.CH.sub.2); 1.35 (s, 3H, CH.sub.3), 1.26
(t, J=7.1 Hz, ester-CH.sub.3); MS (ES): 200.1 (M.sup.++1).
.sup.1Seela, F.; Lupke, U. Chem. Ber. 1977, 110, 1462-1469.
Preparation 3
[0396] A solution of acetal (4) (Scheme IV, 1 g, 5.02 mmol),
benzamidine (786 mg, 5.02 mmol), and DBU (1.5 mL, 10.04 mmol) in
dry DMF (15 mL) was heated to 85.degree. C. for 15 h. The mixture
was diluted with CHCl.sub.3 (30 mL) and washed with 0.5 N NaOH (10
mL) and H.sub.2O (20 mL). The organic fraction was dried, filtered
and concentrated to a brown oil. Flash chromatography (SiO.sub.2;
1/9 EtOAc/CH.sub.2Cl.sub.2, R.sub.f 0.35) was attempted, but
material crystallized on the column. The silica gel was washed with
MeOH. Fractions containing the product (5) (Scheme IV) were
concentrated and used without further purification (783 mg, 54.3%):
.sup.1H NMR (200 MHz, CDCl.sub.3) .delta. 8.24 (m, 2H, Ar--H), 7.45
(m, 3H, Ar--H), 5.24 (br s, 2H, NH.sub.2), 3.98 (s, 4H, 2.times.
acetal-CH.sub.2), 3.60-3.15 (m, 2H, CH.sub.2), 1.38 (s, 3H,
CH.sub.3); MS (ES): 288.1 (M.sup.++1).
[0397] Preparation of compound (20) (Scheme VIII): A solution of
acetal (19) (4.43 g, 20.6 mmol).sup.1, benzamine hydrochloride
(3.22 g, 20.6 mmol), and DBU (6.15 mL, 41.2 mmol) in dry DMF (20
mL) was heated to 85.degree. C. for fifteen hours. The mixture was
diluted with 100 mL of CHCl.sub.3, and washed with H.sub.2O
(2.times.50 mL). The organic fraction was dried, filtered, and
concentrated to a dark brown oil. The dark brown oil was stirred in
1N HCl (100 mL) for 2 hours at room temperature. The resulting
slurry was filtered yielding the HCl salt of (20) as a tan solid
(3.60 g, 70.6%); .sup.1H NMR (200 MHz, DMSO-d6) 11.92 (s 1H), 8.05
(m, 2H, Ar--H), 7.45 (m, 3H, Ar--H), 7.05 (s, 1H, pyrrole-H); MS
(ES): 212.1 (M.sup.++1).
Preparation 4
[0398] A solution of acetal (5) (700 mg, 2.44 mmol) in 1 N HCl (40
mL) was stirred for 2 h at RT. The resultant slurry was filtered
yielding the HCl salt of
2-phenyl-6-methyl-7H-pyrrolo[2,3d]pyrimidin-4(3H)-one as a tan
solid (498 mg, 78.0%): .sup.1H NMR (200 MHz, DMSO-d.sub.6) .delta.
11.78 (s, 1H), 8.05 (m, 2H, Ar--H), 7.45 (m, 3H, Ar--H), 6.17 (s,
1H, pyrrole-H), 2.25 (s, 3H, CH.sub.3); MS (ES): 226.1
(M.sup.++1).
Preparation 5
[0399] A modification of the Chen et al. cyclization method was
used..sup.1 To an ice-cooled (0.degree. C.) solution of bromide
(9), (Scheme V; 20.0 g, 108 mmol; 90% pure) in isopropyl alcohol
(60 mL) was slowly added a solution of .alpha.-methylbenzylamine
(12.5 mL, 97.3 mmol). The black solution was allowed to warm to RT
and stir for 15 h. The mixture was diluted with EtOAc (200 mL) and
washed with 0.5 N NaOH (50 mL). The organic fraction was dried,
filtered, and concentrated to a black tar (19.2 g; 94%). The
residue was partially purified by flash chromatography (SiO.sub.2;
4/96 MeOH/CH.sub.2Cl.sub.2, R.sub.f 0.35) to a black solid (6.38 g,
31%) as the compound
dl-1-(1-phenylethyl)-2-amino-3-cyano-4-methylpyrrole: MS (ES):
226.1 (M.sup.++1). .sup.1Chen, Y. L.; Mansbach, R. S.; Winter, S.
M.; Brooks, E.; Collins, J.; Corman, M. L.; Dunaiskis, A. R.;
Faraci, W. S.; Gallaschun, R. J.; Schmidt, A.; Schulz, D. W. J.
Med. Chem. 1997, 40, 1749-1754.
Preparation 6
[0400] To a solution of
dl-1-(1-phenylethyl)-2-amino-3-cyano-4,5-dimethylpyrrole.sup.1
(14.9 g, 62.5 mmol) and pyridine (10.0 mL) in dichloromethane (50.0
mL) was added benzoyl chloride (9.37 g, 66.7 mmol) at 0.degree. C.
After stirring at 0.degree. C. for 1 hr, hexane (10.0 mL) was added
to help precipitation of product. Solvent was removed in vacuo and
the solid was recrystallized from EtOH/H.sub.2O to give 13.9 g
(65%) of
dl-1-(1-phenylethyl)-2-phenylcarbonylamino-3-cyano-4,5-dimethylpyrrole.
mp 218-221.degree. C.; .sup.1H NMR (200 MHz, CDCl.sub.3)
.delta..sub.--1.72 (s, 3H), 1.76 (d, J=7.3 Hz, 3H), 1.98 (s, 3H),
5.52 (q, J=7.3 Hz, 1H), 7.14-7.54 (m, 9H), 7.68-7.72 (dd, J=1.4 Hz,
6.9 Hz, 2H), 10.73 (s, 1H); MS (ES): 344.4 (M.sup.++1).
[0401] .sup.1Liebigs Ann. Chem. 1986, 1485-1505.
[0402] The following compounds were obtained in a similar
manner.
Preparation 6A:
[0403]
dl-1-(1-phenylethyl)-2-(3-pyridyl)carbonylamino-3-cyano-4,5-dimeth-
ylpyrrole. .sup.1H NMR (200 MHz, CDCl.sub.3) .delta..sub.--1.83 (d,
J=6.8 Hz, 3H), 2.02 (s, 3H), 2.12 (s, 3H), 5.50 (q, J=6.8 Hz, 1H),
7.14-7.42 (m, 5H), 8.08 (m, 2H), 8.75 (m, 3H); MS (ES): 345.2
(M.sup.++1).
[0404]
dl-1-(1-phenylethyl)-2-(2-furyl)carbonylamino-3-cyano-4,5-dimethyl-
pyrrole. .sup.1H NMR (200 MHz, CDCl.sub.3) .delta. 1.84 (d, J=7.4
Hz, 3H), 1.92 (s, 3H), 2.09 (s, 3H), 5.49 (q, J=7.4 Hz, 1H), 6.54
(dd, J=1.8 Hz, 3.6 Hz, 1H), 7.12-7.47 (m, 7H); MS (ES): 334.2
(M.sup.++1), 230.1.
[0405]
dl-1-(1-phenylethyl)-2-(3-furyl)carbonylamino-3-cyano-4,5-dimethyl-
pyrrole. .sup.1H NMR (200 MHz, CDCl.sub.3) .delta. 1.80 (d, J=7 Hz
3H), 1.89 (s, 3H), 2.05 (s, 3H), 5.48 (q, J=7 Hz, 1H), 6.59 (s,
1H), 7.12-7.40 (m, 6H), 7.93 (s, 1H); MS (ES): 334.1 (M.sup.++1),
230.0.
[0406]
dl-1-(1-phenylethyl)-2-cyclopentylcarbonylamino-3-cyano-4,5-dimeth-
ylpyrrole. .sup.1H NMR (200 MHz, CDCl.sub.3) .delta. 1.82 (d, J=7.4
Hz, 3H), 1.88 (s, 3H), 2.05 (s, 3H), 1.63-1.85 (m, 8H), 2.63 (m,
1H), 5.43 (q, J=7.4 Hz, 1H), 6.52 (s, 1H), 7.05-7.20 (m, 5H); MS
(ES): 336.3 (M.sup.++1).
[0407]
dl-1-(1-phenylethyl)-2-(2-thienyl)carbonylamino-3-cyano-4,5-dimeth-
ylpyrrole, .sup.1H NMR (200 MHz, CDCl.sub.3) .delta. 1.82 (d, j=6.8
Hz, 3H), 1.96 (s, 3H), 2.09 (s, 3H), 5.49 (q, J=6.8 Hz, 1H),
7.05-7.55 (m, 8H); MS (ES): 350.1 (M.sup.++1), 246.0.
[0408]
dl-1-(1-phenylethyl)-2-(3-thienyl)carbonylamino-3-cyano-4,5-dimeth-
ylpyrrole.
[0409] .sup.1H NMR (200 MHz, CDCl.sub.3) .delta. 1.83 (d, J=7.0 Hz,
3H), 1.99 (s, 3H), 2.12 (s, 3H), 5.49 (q, J=7.0 Hz, 1H), 6.90 (m,
1H), 7.18-7.36 (m, 6H), 7.79 (m, 1H); MS (ES): 350.2 (M.sup.++1),
246.1.
[0410]
dl-1-(1-phenylethyl)-2-(4-fluorophenyl)carbonylamino-3-cyano-4,5-d-
imethylpyrrole.
[0411] .sup.1H NMR (200 MHz, CDCl.sub.3) .delta. 1.83 (d, J=7.4 Hz,
3H), 1.96 (s, 3H), 2.08 (s, 3H), 5.51 (q, J=7.4 Hz, 1H), 7.16-7.55
(m, 9H); MS (ES): 362.2 (M.sup.++1), 258.1.
[0412]
dl-1-(1-phenylethyl)-2-(3-fluorophenyl)carbonylamino-3-cyano-4,5-d-
imethylpyrrole.
[0413] .sup.1H NMR (200 MHz, CDCl.sub.3) .delta. 1.83 (d, J=7.4 Hz
3H), 1.97 (s, 3H), 2.10 (s, 3H), 5.50 (q, J=7.4 Hz, 1H), 7.05-7.38
(m, 7H), 7.67-7.74 (m, 2H); MS (ES): 362.2 (M.sup.++1), 258.1.
[0414]
dl-1-(1-phenylethyl)-2-(2-fluorophenyl)carbonylamino-3-cyano-4,5-d-
imethylpyrrole. .sup.1H NMR (200 MHz, CDCl.sub.3) .delta. 1.85 (d,
J=7.2 Hz, 3H), 1.94 (s, 3H), 2.11 (s, 3H), 5.50 (q, J=7.2 hz, 1H),
7.12-7.35 (m, 6H), 7.53 (m, 1H), 7.77 (m, 1H), 8.13 (m, 1H); MS
(ES): 362.2 (M.sup.++1), 258.0.
[0415]
dl-1-(1-phenylethyl)-2-isopropylcarbonylamino-3-cyano-4,5-dimethyl-
pyrrole. .sup.1H NMR (200 MHz, CDCl.sub.3) .delta. 1.19 (d, J=7.0
Hz, 6H), 1.82 (d, J=7.2 Hz, 3H), 1.88 (s, 3H), 2.06 (s, 3H), 2.46
(m, 1H), 5.39 (m, J=7.2 Hz, 1H), 6.64 (s, 1H), 7.11-7.36 (m, 5H);
MS (ES): 310.2 (M.sup.++1), 206.1.
[0416] In the case of acylation of
dl-1-(1-phenylethyl)-2-amino-3-cyano-4-methylpyrrole, monoacylated
dl-1-(1-phenylethyl)-2-benzoylamino-3-cyano-4-dimethylpyrrole and
diacylated pyrrole
dl-1-(1-phenylethyl)-2-dibenzoylamino-3-cyano-4-methylpyrrole were
obtained. Monoacylated pyrrole: .sup.1H NMR (200 MHz, CDCl.sub.3)
.delta..sub.--7.69 (d, 2H, J=7.8 Hz, Ar--H), 7.58-7.12 (m, 8H,
Ar--H), 6.18 (s, 1H, pyrrole-H), 5.52 (q, 1H, J=7.2 Hz,
CH--CH.sub.3), 2.05 (s, 3H, pyrrole-CH.sub.3), 1.85 (d, 3H, J=7.2
Hz, CH--CH.sub.3); MS (ES): 330.2 (M.sup.++1); Diacylated pyrrole:
.sup.1H NMR (200 MHz, CDCl.sub.3) .delta..sub.--7.85 (d, 2H, J=7.7
Hz, Ar--H), 7.74 (d, 2H, J=7.8 Hz, Ar--H), 7.52-7.20 (m, 9H,
Ar--H), 7.04 (m, 2H, Ar--H), 6.21 (s, 1H, pyrrole-H), 5.52 (q, 1H,
J=7.2 Hz, CH--CH.sub.3), 1.77 (d, 3H, J=7.2 Hz, CH--CH.sub.3), 1.74
(s, 3H, pyrrole-CH.sub.3); MS (ES): 434.1 (M.sup.++1).
Preparation 7
[0417] To a solution of
dl-1-(1-phenylethyl)-2-phenylcarboxyamido-3-cyano-4,5-dimethylpyrrole
(1.0 g, 2.92 mmol) in methanol (10.0 mL) was added concentrated
sulfuric acid (1.0 mL) at 0.degree. C. The resulted mixture was
refluxed for 15 hr and cooled down to room temperature. The
precipitate was filtered to give 0.48 g (48%) of
dl-5,6-dimethyl-2-phenyl-7H-7-(1-phenylethyl)pyrrolo[2,3d]pyrimidin-4(3H)-
-one. .sup.1H NMR (200 MHz, CDCl.sub.3) .delta..sub.--2.02 (d,
J=7.4 Hz, 3H), 2.04 (s, 3H), 2.41 (s, 3H), 6.25 (q, J=7.4 Hz, 1H),
7.22-7.50 (m, 9H), 8.07-8.12 (dd, J=3.4 Hz, 6.8 Hz, 2H), 10.51 (s,
1H); MS (ES): 344.2 (M.sup.++1). The following compounds were
obtained in a similar manner as that of Preparation 7:
[0418]
dl-5,6-dimethyl-2-(3-pyridyl)-7H-7-(1-phenylethyl)pyrrolo[2,3d]pyr-
imidin-4(3H)-one. .sup.1H NMR (200 MHz, CDCl.sub.3)
.delta..sub.--2.03 (d, J=7.2 Hz, 3H), 2.08 (s, 3H), 2.42 (s, 3H),
6.24 (q, J=7.2 Hz, 1H), 7.09-7.42 (m, 5H), 8.48 (m, 2H), 8.70 (m,
3H); MS (ES): 345.1 (M.sup.++1).
[0419]
dl-5,6-dimethyl-2-(2-furyl)-7H-7-(1-phenylethyl)pyrrolo[2,3d]pyrim-
idin-4(3H)-one. .sup.1H NMR (200 MHz, CDCl.sub.3) .delta. 1.98 (d,
J=7.8 Hz, 3H), 1.99 (s, 3H), 2.37 (s, 3H), 6.12 (q, J=7.8 Hz, 1H),
6.48 (dd, J=1.8 Hz, 3.6 Hz, 1H), 7.17-7.55 (m, 7H), 9.6 (s, 1H); MS
(ES): 334.2 (M.sup.++1).
[0420]
dl-5,6-dimethyl-2-(3-furyl)-7H-7-(1-phenylethyl)pyrrolo[2,3d]pyrim-
idin-4(3H)-one. .sup.1H NMR (200 MHz, CDCl.sub.3) .delta. 1.99 (d,
J=7 Hz, 3H), 2.02 (s, 3H), 2.42 (s, 3H), 6.24 (q, J=7 Hz, 1H), 7.09
(s, 1H), 7.18-7.32 (m, 5H), 7.48 (s, 1H), 8.51 (s, 1H); MS (ES):
334.2 (M.sup.++1).
[0421]
dl-5,6-dimethyl-2-cyclopentyl-7H-7-(1-phenylethyl)pyrrolo[2,3d]pyr-
imidin-4(3H)-one. .sup.1H NMR (200 MHz, CDCl.sub.3) .delta. 1.95
(d, J=7.4 Hz, 3H), 2.00 (s, 3H), 2.33 (s, 3H), 1.68-1.88 (m, 8H),
2.97 (m, 1H), 6.10 (q, J=7.4 Hz, 1H), 7.16-7.30 (m, 5H), 9.29 (s,
1H); MS (ES): 336.3 (M.sup.++1).
[0422]
dl-5,6-dimethyl-2-(2-thienyl)-7H-7-(1-phenylethyl)pyrrolo[2,3d]pyr-
imidin-4(3H)-one. .sup.1H NMR (200 MHz, CDCl.sub.3) .delta. 2.02
(d, J=7.2 Hz, 3H), 2.06 (s, 3H), 2.41 (s, 3H), 6.13 (q, J=7.2 Hz,
1H), 7.12 (dd, J=4.8, 2.8 Hz, 1H), 7.26-7.32 (m, 5H), 7.44 (d,
J=4.8 Hz, 1H), 8.01 (d, J=2.8 Hz, 1H) 11.25 (s, 1H); MS (ES): 350.2
(M.sup.++1).
[0423]
dl-5,6-dimethyl-2-(3-thienyl)-7H-7-(1-phenylethyl)pyrrolo[2,3d]pyr-
imidin-4(3H)-one. .sup.1H NMR (200 MHz, CDCl.sub.3) .delta. 2.00
(d, J=7.4 Hz, 3H), 2.05 (s, 3H), 2.43 (s, 3H), 6.24 (q, J=7.4 Hz,
1H), 7.24-7.33 (m, 5H), 7.33-7.39 (m, 1H), 7.85 (m, 1H), 8.47 (m,
1H), 12.01 (s, 1H); MS (ES): 350.2 (M.sup.++1).
[0424]
dl-5,6-dimethyl-2-(4-fluorophenyl)-7H-7-(1-phenylethyl)pyrrolo[2,3-
d]pyrimidin-4(3H)-one. .sup.1H NMR (200 MHz, CDCl.sub.3) .delta.
2.01 (d, J=6.8 Hz, 3H), 2.05 (s, 3H), 2.42 (s, 3H), 6.26 (q, J=6.8
Hz, 1H), 7.12-7.36 (m, 7H), 8.23-8.30 (m, 2H), 11.82 (s, 1H); MS
(ES): 362.3 (M.sup.++1).
[0425]
dl-5,6-dimethyl-2-(3-fluorophenyl)-7H-7-(1-phenylethyl)pyrrolo[2,3-
d]pyrimidin-4(3H)-one. .sup.1H NMR (200 MHz, CDCl.sub.3) .delta.
2.02 (d, J=7.4 Hz, 3H), 2.06 (s, 3H), 2.44 (s, 3H), 6.29 (q, J=7.4
Hz, 1H), 7.13-7.51 (m, 7H), 8.00-8.04 (m, 2H), 11.72 (s, 1H); MS
(ES): 362.2 (M.sup.++1).
[0426]
dl-5,6-dimethyl-2-(2-fluorophenyl)-7H-7-(1-phenylethyl)pyrrolo[2,3-
d]pyrimidin-4(3H)-one. .sup.1H NMR (200 MHz, CDCl.sub.3) .delta.
2.00 (d, J=7.2 Hz, 3H), 2.05 (s, 3H), 2.38 (s, 3H), 6.24 (q, J=7.2
Hz, 1H), 7.18-7.45 (m, 8H), 8.21 (m, 1H), 9.54 (s, 1H); MS (ES):
362.2 (M.sup.++1).
[0427]
dl-5,6-dimethyl-2-isopropyl-7H-7-(1-phenylethyl)pyrrolo[2,3d]pyrim-
idin-4(3H)-one.
[0428] .sup.1H NMR (200 MHz, CDCl.sub.3) .delta. 1.30 (d, J=6.8 Hz,
3H), 1.32 (d, J=7.0 Hz, 3H), 2.01 (s, 3H), 2.34 (s, 3H), 2.90 (m,
1H), 6.13 (m, 1H), 7.17-7.34 (m, 5H), 10.16 (s, 1H); MS (ES): 310.2
(M.sup.++1).
Preparation 8
[0429] A solution of
dl-1-(1-phenylethyl)-2-benzoylamino-3-cyano-4-methylpyrrole (785
mg, 2.38 mmol) with concentrated H.sub.2SO.sub.4 (1 mL) in DMF (13
mL) was stirred at 130.degree. C. for 48 h. The black solution was
diluted with CHCl.sub.3 (100 mL) and washed with 1 N NaOH (30 mL),
and brine (30 mL). The organic fraction was dried, filtered,
concentrated, and purified by flash chromatography (SiO.sub.2; 8/2
EtOAc/Hex, R.sub.f 0.35) to a brown solid (184 mg, 24%) as
dl-5-methyl-2-phenyl-7H-7-(1-phenylethyl)pyrrolo[2,3d]pyrimidin-4(3H)-one-
. .sup.1H NMR (200 MHz, CDCl.sub.3) .delta..sub.--8.18 (m, 2H,
Ar--H), 7.62-7.44 (m, 3H, Ar--H), 7.40-7.18 (m, 5H, Ar--H), 6.48
(s, 1H, pyrrole-H), 6.28 (q, 1H, J=7.2 Hz, CH--CH.sub.3), 2.18 (s,
3H, pyrrole-CH.sub.3), 2.07 (d, 3H, J=7.2 Hz, CH--CH.sub.3); MS
(ES): 330.2 (M.sup.++1).
Preparation 9
[0430] A mixture of
dl-1-(1-phenylethyl)-2-amino-3-cyano-4,5-dimethylpyrrole (9.60 g,
40.0 mmol) and of formic acid (50.0 mL, 98%) was refluxed for 5 hr.
After cooling down to room temperature and scratching the sides of
flask, copious precipitate was formed and filtered. The material
was washed with water until washings showed neutral pH to give
dl-5,6-dimethyl-7H-7-(1-phenylethyl)pyrrolo[2,3d]pyrimidin-4(3H)-one.
.sup.1H NMR (200 MHz, CDCl.sub.3) .delta. 1.96 (d, J=7.4 hz, 3H),
2.00 (s, 3H), 2.38 (s, 3H), 6.21 (q, J=7.4 Hz, 1H), 7.11-7.35 (m,
5H), 7.81 (s, 1H), 11.71 (s, 1H); MS (ES): 268.2 (M.sup.++1).
Preparation 10
[0431]
dl-5,6-dimethyl-2-phenyl-7H-7-(1-phenylethyl)pyrrolo[2,3d]pyrimidi-
n-4(3H)-one (1.0 g, 2.91 mmol) was suspended in polyphosphoric acid
(30.0 mL). The mixture was heated at 100.degree. C. for 4 hr. The
hot suspension was poured onto ice water, stirred vigorously to
disperse suspension, and basified to pH 6 with solid KOH. The
resulting solid was filtered and collected to give 0.49 g (69%) of
5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidin-4(3H)-one. .sup.1H
NMR (200 MHz, DMSO-d.sub.6) .delta..sub.--2.17 (s, 3H), 2.22 (s,
3H), 7.45 (br, 3H), 8.07 (br, 2H,), 11.49 (s, 1H), 11.82 (s, 1H);
MS (ES): 344.2 (M.sup.++1).
[0432] The following compounds were obtained in a similar manner as
that of Preparation 10:
[0433] 5-methyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidin-4(3H)-one. MS
(ES): 226.0 (M+1).
[0434]
5,6-dimethyl-2-(3-pyridyl)-7H-pyrrolo[2,3d]pyrimidin-4(3H)-one. MS
(ES): 241.1 (M.sup.++1).
[0435]
5,6-dimethyl-2-(2-furyl)-7H-pyrrolo[2,3d]pyrimidin-4(3H)-one.
.sup.1H NMR (200 MHz, DMSO-d.sub.6) .delta. 2.13 (s, 3H), 2.18 (s,
3H), 6.39 (dd, J=1.8, 3.6 Hz, 1H), 6.65 (dd, J=1.8 Hz, 3.6 Hz, 1H),
7.85 (dd, J=1.8, 3.6 Hz, 1H), 11.45 (s, 1H), 11.60 (s, 1H); MS
(ES): 230.1 (M.sup.++1).
[0436] 5,6-dimethyl-2-(3-furyl)-7H-pyrrolo[2,3d]pyrimidin-4
(3H)-one. .sup.1H NMR (200 MHz, DMSO-d.sub.6) .delta. 2.14 (s, 3H),
2.19 (s, 3H), 6.66 (s, 1H), 7.78 (s, 1H), 8.35 (s, 1H), 11.3 (s,
1H), 11.4 (s, 1H); MS (ES): 230.1 (M.sup.++1).
[0437]
5,6-dimethyl-2-cyclopentyl-7H-pyrrolo[2,3d]pyrimidin-4(3H)-one.
.sup.1H NMR (200 MHz, DMSO-d.sub.6) .delta. 1.57-1.91 (m, 8H), 2.12
(s, 3H), 2.16 (s, 3H), 2.99 (m, 1H), 11.24 (s, 1H), 11.38 (s, 1H);
MS (ES): 232.2 (M.sup.++1).
[0438]
5,6-dimethyl-2-(2-thienyl)-7H-pyrrolo[2,3d]pyrimidin-4(3H)-one.
.sup.1H NMR (200 MHz, DMSO-d.sub.6) .delta. 2.14 (s, 3H), 2.19 (s,
3H), 7.14 (dd, J=3.0, 5.2 Hz, 1H), 7.70 (d, J=5.2 Hz 1H), 8.10 (d,
J=3.0 Hz, 1H), 11.50 (s, 1H); MS (ES): 246.1 (M.sup.++1).
[0439]
5,6-dimethyl-2-(3-thienyl)-7H-pyrrolo[2,3d]pyrimidin-4(3H)-one.
.sup.1H NMR (200 MHz, DMSO-d.sub.6) .delta. 2.17 (s, 3H), 2.21 (s,
3H), 7.66 (m, 1H), 7.75 (m, 1H), 8.43 (m, 1H), 11.47 (s, 1H), 11.69
(s, 1H); MS (ES): 246.1 (M.sup.++1).
[0440]
5,6-dimethyl-2-(4-fluorophenyl)-7H-pyrrolo[2,3d]pyrimidin-4(3H)-on-
e. .sup.1H NMR (200 MHz, DMSO-d.sub.6) .delta. 2.17 (s, 3H), 2.21
(s, 3H), 7.31 (m, 2H), 8.12 (m, 2H), 11.47 (s, 1H); MS (ES): 258.2
(M.sup.++1).
[0441]
5,6-dimethyl-2-(3-fluorophenyl)-7H-pyrrolo[2,3d]pyrimidin-4(3H)-on-
e. .sup.1H NMR (200 MHz, DMSO-d.sub.6) .delta. 2.18 (s, 3H), 2.21
(s, 3H), 7.33 (m, 1H), 7.52 (m, 1H), 7.85-7.95 (m, 2H), 11.56 (s,
1H), 11.80 (s, 1H); MS (ES): 258.1 (M.sup.++1).
[0442]
5,6-dimethyl-2-(2-fluorophenyl)-7H-pyrrolo[2,3d]pyrimidin-4(3H)-on-
e. .sup.1H NMR (200 MHz, DMSO-d.sub.6) .delta. 2.18 (s, 3H), 2.22
(s, 3H), 7.27-7.37 (m, 2H), 7.53 (m 1H), 7.68 (m, 1H), 11.54 (s,
1H), 11.78 (s, 1H); MS (ES): 258.1 (M.sup.++1).
[0443]
5,6-dimethyl-2-isopropyl-7H-pyrrolo[2,3d]pyrimidin-4(3H)-one.
.sup.1H NMR (200 MHz, DMSO-d.sub.6) .delta. 1.17 (d, J=6.6 Hz, 6H),
2.11 (s, 3H), 2.15 (s, 3H), 2.81 (m, 1H), 11.20 (s, 1H), 11.39 (s,
1H); MS (ES): 206.1 (M.sup.++1).
[0444] 5,6-dimethyl-7H-pyrrolo[2,3d]pyrimidin-4 (3H)-one. .sup.1H
NMR (200 MHz, DMSO-d.sub.6) .delta. 2.13 (s, 3H), 2.17 (s, 3H),
7.65 (s, 1H); MS (ES): 164.0 (M.sup.++1).
Preparation 11
[0445] A solution of
5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidin-4(3H)-one (1.0 g,
4.2 mmol) in phosphorus oxychloride (25.0 mL) was refluxed for 6 hr
and then concentrated in vacuo to dryness. Water was added to the
residue to induce crystallization and the resulting solid was
filtered and collected to give 0.90 g (83%) of
4-chloro-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine. .sup.1H
NMR (200 MHz, DMSO-d.sub.6) .delta..sub.--2.33 (s, 3H), 2.33 (s,
3H), 7.46-7.49 (m, 3H), 8.30-8.35 (m, 2H), 12.20 (s, 1H); MS (ES):
258.1 (M.sup.++1).
[0446] The following compounds were obtained in a similar manner as
that of Preparation 11:
[0447] 4-chloro-5-methyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine. MS
(ES): 244.0 (M.sup.++1).
[0448] 4-chloro-6-methyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine. MS
(ES): 244.0 (M.sup.++1).
[0449] 4-chloro-2-phenyl-7H-pyrrolo[2,3d]pyrimidine. .sup.1H NMR
(200 MHz, DMSO-d6) 8.35 (2, 2H), 7.63 (br s, 1H), 7.45 (m, 3H),
6.47 (br s, 1H); MS (ES): 230.0 (M.sup.++1).
[0450]
4-chloro-5,6-dimethyl-2-(3-pyridyl)-7H-pyrrolo[2,3d]pyrimidine. MS
(ES): 259.0 (M.sup.++1).
[0451]
4-chloro-5,6-dimethyl-2-(2-furyl)-7H-pyrrolo[2,3d]pyrimidine.
.sup.1H NMR (200 MHz, DMSO-d.sub.6) .delta. 2.35 (s, 3H), 2.35 (s,
3H), 6.68 (dd, J=1.8, 3.6 Hz, 1H), 7.34 (dd, J=1.8 Hz, 3.6 Hz, 1H),
7.89 (dd, J=1.8, 3.6 Hz, 1H); MS (ES) 248.0 (M.sup.++1).
[0452]
4-chloro-5,6-dimethyl-2-(3-furyl)-7H-pyrrolo[2,3d]pyrimidine.
.sup.1H NMR (200 MHz, DMSO-d.sub.6) .delta. 2.31 (s, 3H), 2.31 (s,
3H), 6.62 (s, 1H), 7.78 (s, 1H), 8.18 (s, 1H), 12.02 (s, 1H); MS
(ES): 248.1 (M.sup.++1).
[0453]
4-chloro-5,6-dimethyl-2-cyclopentyl-7H-pyrrolo[2,3d]pyrimidine.
.sup.1H NMR (200 MHz, DMSO-d.sub.6) .delta. 1.61-1.96 (m, 8H), 2.27
(s, 3H), 2.27 (s, 3H), 3.22 (m, 1H), 11.97 (s, 1H); MS (ES): 250.1
(M.sup.++1).
[0454]
4-chloro-5,6-dimethyl-2-(2-thienyl)-7H-pyrrolo[2,3d]pyrimidine.
.sup.1H NMR (200 MHz, DMSO-d.sub.6) .delta. 2.29 (s, 3H), 2.31 (s,
3H), 7.14 (dd, J=3.1 Hz, 4.0 Hz, 1H), 7.33 (d, J=4.9 Hz, 1H), 7.82
(d, J=3.1 Hz, 1H), 12.19 (s, 1H); MS (ES): 264.1 (M.sup.++1).
[0455]
4-chloro-5,6-dimethyl-2-(3-thienyl)-7H-pyrrolo[2,3d]pyrimidine.
.sup.1H NMR (200 MHz, DMSO-d.sub.6) .delta. 2.32 (s, 3H), 2.32 (s,
3H), 7.62 (dd, J=3.0, 5.2 Hz, 1H), 7.75 (d, J=5.2 Hz, 1H), 8.20 (d,
J=3.0 Hz, 1H); MS (ES): 264.0 (M.sup.++1).
[0456]
4-chloro-5,6-dimethyl-2-(4-fluorophenyl)-7H-pyrrolo[2,3d]pyrimidin-
e. .sup.1H NMR (200 MHz, DMSO-d.sub.6) .delta. 2.33 (s, 3H), 2.33
(s, 3H), 7.30 (m, 2H), 8.34 (m, 2H), 12.11 (s, 1H); MS (ES): 276.1.
(M.sup.++1).
[0457]
4-chloro-5,6-dimethyl-2-(3-fluorophenyl)-7H-pyrrolo[2,3d]pyrimidin-
e. .sup.1H NMR (200 MHz, DMSO-d.sub.6) .delta. 2.31 (s, 3H), 2.33
(s, 3H), 7.29 (m, 1H), 7.52 (m, 1H), 7.96 (m, 1H), 8.14 (m, 1H),
11.57 (s, 1H); MS (ES): 276.1 (M.sup.++1).
[0458]
4-chloro-5,6-dimethyl-2-(2-fluorophenyl)-7H-pyrrolo[2,3d]pyrimidin-
e. .sup.1H NMR (200 MHz, DMSO-d.sub.6) .delta. 2.34 (s, 3H), 2.34
(s, 3H), 7.33 (m, 2H), 7.44 (m, 1H), 7.99 (m, 1H), 12.23 (s, 1H);
MS (ES): 276.1 (M.sup.++1).
[0459]
4-chloro-5,6-dimethyl-2-isopropyl-7H-pyrrolo[2,3d]pyrimidine.
.sup.1H NMR (200 MHz, DMSO-d.sub.6) .delta. 1.24 (d, J=6.6 Hz, 6H),
2.28 (s, 3H), 2.28 (s, 3H), 3.08 (q, J=6.6 Hz, 1H), 11.95 (s, 1H);
MS (ES): 224.0 (M.sup.++1).
[0460] 4-chloro-5,6-dimethyl-7H-pyrrolo[2,3d]pyrimidine. .sup.1H
NMR (200 MHz, DMSO-d.sub.6) .delta. 2.31 (s, 3H), 2.32 (s, 3H),
8.40 (s, 1H); MS (ES): 182.0 (M.sup.++1).
[0461]
dl-4-chloro-5,6-dimethyl-2-phenyl-7H-7-(1-phenylethyl)pyrrolo[2,3d-
]pyrimidine.
Preparation 12
[0462] To a solution of dl-1,2-diaminopropane (1.48 g, 20.0 mmol)
and sodium carbonate (2.73 g, 22.0 mmol) in dioxane (100.0 mL) and
water (100.0 mL) was added di-tert-butyldicarbonate (4.80 g, 22.0
mmol) at room temperature. The resulted mixture was stirred for 14
hr. Dioxane was removed in vacuo. The precipitate was filtered off
and the filtrate was concentrated in vacuo to dryness. The residue
was triturated with EtOAc and then filtered. The filtrate was
concentrated in vacuo to dryness to give a mixture of
dl-1-amino-2-(1,1-dimethylethoxy)carbonylamino-propane and
dl-2-amino-1-(1,1-dimethylethoxy)carbonylamino-propane which were
not separable by normal chromatography method. The mixture was used
for the reaction in Example 8.
Preparation 13
[0463] To solution of Fmoc-.beta.-Ala-OH (1.0 g, 3.212 mmol) and
oxalyl chloride (0.428 g, 0.29 mL, 3.373 mmol) in dichloromethane
(20.0 mL) was added a few drops of N,N-dimethylformamide at
0.degree. C. The mixture was stirred at room temperature for 1 hr
followed by addition of cyclopropylmethylamine (0.229 g, 0.28 mL,
3.212 mmol) and triethylamine (0.65 g, 0.90 mL, 6.424 mmol). After
10 min, the mixture was treated with 1 M hydrochloride (10.0 mL)
and the aqueous mixture was extracted with dichloromethane
(3.times.30.0 mL). The organic solution was concentrated in vacuo
to dryness. The residue was treated with a solution of 20%
piperidine in N,N-dimethylforamide (20.0 mL) for 0.5 hr. After
removal of the solvent in vacuo, the residue was treated with 1 M
hydrochloride (20.0 mL) and ethyl acetate (20.0 mL). The mixture
was separated and the aqueous layer was basified with solid sodium
hydroxide to pH=8. The precipitate was removed by filtration and
the aqueous solution was subjected to ion exchange column eluted
with 20% pyridine to give 0.262 g (57%) of N-cyclopropylmethyl
.beta.-alanine amide. .sup.1H NMR (200 MHz, CD.sub.3OD)
.delta..sub.--0.22 (m, 2H), 0.49 (m, 2H), 0.96 (m, 2H), 2.40 (t,
2H), 2.92 (t, 2H), 3.05 (d, 2H); MS (ES): 143.1 (M.sup.++1).
Preparation 14
[0464] N-tert-butoxycarbonyl-trans-1,4-cyclohexyldiamine.
trans-1,4-cyclohexyldiamine (6.08 g, 53.2 mmol) was dissolved in
dichloromethane (100 mL). A solution of di-t-butyldicarbonate (2.32
g, 10.65 mmol in 40 mL dichloromethane) was added via cannula.
After 20 hours, the reaction was partitioned between CHCl.sub.3 and
water. The layers were separated and the aqueous layer was
extracted with CHCl.sub.3 (3.times.). The combined organic layers
were dried over MgSO.sub.4, filtered and concentrated to yield 1.20
g of a white solid (53%). .sup.1H-NMR (200 MHz, CDCl.sub.3):
.delta. 1.0-1.3 (m, 4H), 1.44 (s, 9H), 1.8-2.1 (m, 4H), 2.62 (brm,
1H), 3.40 (brs, 1H), 4.37 (brs, 1H0; MS (ES): 215.2
(M.sup.++1).
4-(N-acetyl)-N-tert-butoxycarbonyl-trans-1,4-cyclohexyl diamine
[0465] N-tert-butoxycarbonyl-trans-1,4-cyclohexyldiamine (530 mg,
2.47 mmol) was dissolved in dichloromethane (20 mL). Acetic
anhydride (250 mg, 2.60 mmol) was added dropwise. After 16 hours,
the reaction was diluted with water and CHCl.sub.3. The layers were
separated and the aqueous layer was extracted with CHCl.sub.3
(3.times.). The combined organic layers were dried over MgSO.sub.4,
filtered and concentrated. Recrystallization (EtOH/H.sub.2O)
yielded 190 mg of white crystals (30%). .sup.1H NMR (200 MHz,
CDCl.sub.3): .delta. 0.9-1.30 (m, 4H), 1.43 (s, 9H), 1.96-2.10 (m,
7H), 3.40 (brs, 1H), 3.70 (brs, 1H), 4.40 (brs, 1H), 4.40 (brs,
1H); MS (ES): 257.2 (M.sup.++1), 242.1 (M.sup.+-15), 201.1
(M.sup.+-56).
4-(4-trans-acetamidocyclohexyl)amino-5,6-dimethyl-2-phenyl-7H-(1-phenyleth-
yl)pyrrolo[2,3d]pyrimidine
[0466]
4-(N-acetyl)-N-tert-butoxycarbonyl-trans-1,4-cyclohexyldiamine (190
mg, 0.74 mmol), was dissolved in dichloromethane (5 mL) and diluted
with TFA (6 ml). After 16 hours, the reaction was concentrated. The
crude solid, DMSO (2 mL), NaHCO.sub.3 (200 mg, 2.2 mmol) and
4-chloro-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine (35 mg,
0.14 mmol) were combined in a flask and heated to 130.degree. C.
After 4.5 hours, the reaction was cooled to room temperature and
diluted with EtOAc and water. The layers were separated and the
aqueous layer was extracted with EtOAc (3.times.). The combined
organic layers were dried over MgSO.sub.4, filtered and
concentrated. Chromatography (silica preparatory plate; 20:1
CHCl.sub.3:EtOH) yielded 0.3 mg of a tan solid (1% yield). MS (ES):
378.2 (M.sup.++1).
4-(N-methanesulfonyl)-N-tert-butoxycarbonyl-trans-1,4-cyclohexyldiamine
[0467] trans-1,4-cyclohexyldiamine (530 mg, 2.47 mmol) was
dissolved in dichloromethane (20 ml) and diluted with pyridine (233
mg, 3.0 mmol). Methanesulfonyl chloride (300 mg, 2.60 mmol) was
added dropwise. After 16 hours, the reaction was diluted with water
and CHCl.sub.3. The layers were separated and the aqueous layer was
extracted with CHCl.sub.3 (3.times.). The combined organic layers
were dried over MgSO.sub.4, filtered and concentrated.
recrystallization (EtOH/H.sub.2O) yielded 206 mg of white crystals
(29%). .sup.1H-NMR (200 MHz, CDCl.sub.3): .delta. 1.10-1.40 (m,
4H), 1.45 (s, 9H), 2.00-2.20 (m, 4H), 2.98 (s, 3H), 3.20-3.50 (brs,
2H), 4.37 (brs, 1H); MS (ES) 293.1 (M.sup.++1), 278.1 (M.sup.+-15),
237.1 (M.sup.+-56).
4-(4-trans-methanesulfamidocyclohexyl)amino-5,6-dimethyl-2-phenyl-7H-(1-ph-
enylethyl)pyrrolo[2,3d]pyrimidine
[0468]
4-(N-sulfonyl)-N-tert-butoxycarbonyl-trans-1,4-cyclohexyldiamine
(206 mg, 0.71 mmol), was dissolved in dichloromethane (5 ml) and
diluted with TFA (6 ml). After 16 hours, the reaction was
concentrated. The crude reaction mixture, DMSO (2 ml), NaHCO.sub.3
(100 mg, 1.1 mmol) and
1-chloro-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine were
combined in a flask and heated to 130.degree. C. After 15 hours,
the reaction was cooled to room temperature, and diluted with EtOAc
(3.times.). The combined organic layers were dried over MgSO.sub.4,
filtered and concentrated. Chromatography (silica preparatory
plate, 20:1 CHCl.sub.3/EtOH) yielded 2.6 mg of a tan solid (5%
yield). MS (ES): 414.2 (M.sup.++1).
Example 1
[0469] A solution of
4-chloro-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine (0.50 g,
1.94 mmol) and 4-trans-hydroxy cyclohexylamine (2.23 g, 19.4 mmol)
in methyl sulfoxide (10.0 mL) was heated at 130.degree. C. for 5
hr. After cooling down to room temperature, water (10.0 mL) was
added and the resulted aqueous solution was extracted with EtOAc
(3.times.10.0 mL). The combined EtOAc solution was dried
(MgSO.sub.4) and filtered, the filtrate was concentrated in vacuo
to dryness, the residue was chromatographed on silica gel to give
0.49 g (75%) of
4-(4-trans-hydroxycyclohexyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]-
pyrimidine. mp 197-199.degree. C.; .sup.1H NMR (200 MHz,
CDCl.sub.3) .delta..sub.--1.25-1.59 (m, 8H), 2.08 (s, 3H), 2.29 (s,
3H), 3.68-3.79 (m, 1H), 4.32-4.38 (m, 1H), 4.88 (d, J=8 Hz, 1H),
7.26-7.49 (m, 3H), 8.40-8.44 (dd, J=2.2, 8 Hz, 2H), 10.60 (s, 1H);
MS (ES): 337.2 (M.sup.++1).
[0470] The following compounds were obtained in a similar manner to
that of Example 1:
[0471]
4-(4-trans-hydroxycyclohexyl)amino-6-methyl-2-phenyl-7H-pyrrolo[2,-
3d]pyrimidine. .sup.1H NMR (200 MHz, CDCl.sub.3)
.delta..sub.--11.37 (s, 1H, pyrrole-NH), 8.45 (m, 2H, Ar--H), 7.55
(m, 3H, Ar--H), 6.17 (s, 1H, pyrrole-H), 4.90 (br d, 1H, NH), 4.18
(m, 1H, CH--O), 3.69 (m, 1H, CH--N), 2.40-2.20 (m, 2H), 2.19-1.98
(m, 2H), 2.25 (s, 3H, CH3) 1.68-1.20 (m, 4H); MS (ES): 323.2
(M.sup.++1).
[0472]
4-(4-trans-hydroxycyclohexyl)amino-5-methyl-2-phenyl-7H-pyrrolo[2,-
3d]pyrimidine. .sup.1H NMR (200 MHz, CDCl.sub.3)
.delta..sub.--11.37 (s, 1H, pyrrole-NH), 8.40 (m, 2H, Ar--H), 7.45
(m, 3H, Ar--H), 5.96 (s, 1H, pyrrole-H), 4.90 (br d, 1H, NH), 4.18
(m, 1H, CH--O), 3.69 (m, 1H, CH--N), 2.38-2.20 (m, 2H), 2.18-1.98
(m, 2H), 2.00 (s, 3H, CH3) 1.68-1.20 (m, 4H); MS (ES): 323.2
(M.sup.++1).
[0473]
4-(4-trans-hydroxycyclohexyl)amino-2-phenyl-7H-pyrrolo[2,3d]pyrimi-
dine. mp 245.5-246.5.degree. C.; .sup.1H NMR (200 MHz, CD.sub.3OD)
.delta. 8.33 (m, 2H, Ar--H), 7.42 (m, 3H, Ar--H), 7.02 (d, 1H,
J=3.6 Hz, pyrolle-H), 6.53 (d, 1H, J=3.6 Hz, pyrolle-H), 4.26 (m,
1H, CH--O), 3.62 (m, 1H, CH--N), 2.30-2.12 (m, 2H), 2.12-1.96 (m,
2H), 1.64-1.34 (m, 4H); MS, M+1=309.3; Anal
(C.sub.18H.sub.20N.sub.4O) C, H, N.
[0474]
4-(4-trans-hydroxycyclohexyl)amino-5,6-dimethyl-2-(3-pyridyl)
-7H-pyrrolo[2,3d]pyrimidine. .sup.1H NMR (200 MHz, CDCl.sub.3)
.delta..sub.--1.21-1.54 (m, 8H); 2.28 (s, 3H); 2.33 (s, 3H); 3.70
(m, 1H), 4.31 (m, 1H), 4.89 (d, 1H), 7.40 (m, 1H), 8.61 (m, 2H),
9.64 (m, 1H); MS (ES): 338.2 (M.sup.++1).
[0475] 4-(4-trans-hydroxycyclohexyl)amino-5,6-dimethyl-2-(2-furyl)
-7H-pyrrolo[2,3d]pyrimidine. .sup.1H NMR (200 MHz, CDCl.sub.3)
.delta. 1.26-1.64 (m, 8H), 2.22 (s, 3H), 2.30 (s, 3H), 3.72 (m,
1H), 4.23 (m, 1H), 4.85 (d, 1H), 6.52 (m, 1H), 7.12 (m, 1H), 7.53
(m, 1H), 9.28 (s, 1H); MS (ES): 327.2 (M.sup.++1).
[0476] 4-(4-trans-hydroxycyclohexyl)amino-5,6-dimethyl-2-(3-furyl)
-7H-pyrrolo[2,3d]pyrimidine. .sup.1H NMR (200 MHz, CDCl.sub.3)
.delta.1.25-1.63 (m, 8H), 2.11 (s, 3H), 2.27 (s, 3H), 3.71 (m, 1H),
4.20 (m, 1H), 4.84 (d, 1H), 7.03 (m, 1H), 7.45 (m, 1H), 8.13 (m,
1H), 10.38 (m, 1H); MS (ES): 327.2 (M.sup.++1).
[0477]
4-(4-trans-hydroxycyclohexyl)amino-5,6-dimethyl-2-cyclopentyl-7H-p-
yrrolo[2,3d]pyrimidine. .sup.1H NMR (200 MHz, CDCl.sub.3) .delta.
1.26-2.04 (m, 16H), 2.26 (s, 3H), 2.27 (s, 3H), 3.15 (m, 1H), 3.70
(m, 1H), 4.12 (m, 1H), 4.75 (d, 1H); MS (ES): 329.2
(M.sup.++1).
[0478]
4-(4-trans-hydroxycyclohexyl)amino-5,6-dimethyl-2-(2-thienyl)-7H-p-
yrrolo[2,3d]pyrimidin-4-amine. .sup.1H NMR (200 MHz, CDCl.sub.3)
.delta. 1.28-1.59 (m, 8H), 2.19 (s, 3H), 2.29 (s, 3H), 3.74 (m,
1H), 4.19 (m, 1H), 4.84 (d, 1H), 7.09 (m, 1H), 7.34 (m, 1H), 7.85
(m, 1H), 9.02 (s, 1H); MS (ES): 343.2 (M.sup.++1).
[0479]
4-(4-trans-hydroxycyclohexyl)amino-5,6-dimethyl-2-(3-thienyl)
-7H-pyrrolo[2,3d]pyrimidine. .sup.1H NMR (200 MHz, CDCl.sub.3)
.delta. 1.21-1.60 (m, 8H), 1.98 (s, 3H), 2.23 (s, 3H), 3.66 (m,
1H), 4.22 (m, 1H), 7.27 (m, 1H), 7.86 (m, 1H), 8.09 (m, 1H), 11.23
(s, 1H); MS (ES): 343.2 (M.sup.++1).
[0480]
4-(4-trans-hydroxycyclohexyl)amino-5,6-dimethyl-2-(4-fluorophenyl)-
-7H-pyrrolo[2,3d]pyrimidine. .sup.1H NMR (200 MHz, CDCl.sub.3)
.delta. 1.26-1.66 (m, 8H), 1.94 (s, 3H), 2.28 (s, 3H), 3.73 (m,
1H), 4.33 (m, 1H), 4.92 (d, 1H), 7.13 (m, 2H), 8.41 (m, 2H), 11.14
(s, 1H); MS (ES): 355.2 (M.sup.++1).
[0481]
4-(4-trans-hydroxycyclohexyl)amino-5,6-dimethyl-2-(3-fluorophenyl)-
-7H-pyrrolo[2,3d]pyrimidine. .sup.1H NMR (200 MHz, CDCl.sub.3)
.delta. 1.26-1.71 (m, 8H), 2.06 (s, 3H), 2.30 (s, 3H), 3.72 (m,
1H), 4.30 (m, 1H), 4.90 (d, 1H), 7.09 (m, 1H), 7.39 (m, 1H), 8.05
(m, 1H), 8.20 (m, 1H), 10.04 (s. 1H); MS (ES): 355.2
(M.sup.++1).
[0482]
4-(4-trans-hydroxycyclohexyl)amino-5,6-dimethyl-2-(2-fluorophenyl)-
-7H-pyrrolo[2,3d]pyrimidine. .sup.1H NMR (200 MHz, CDCl.sub.3)
.delta. 1.30-1.64 (m, 8H), 2.17 (s, 3H), 2.31 (s, 3H), 3.73 (m,
1H), 4.24 (m, 1H), 4.82 (d, 1H), 7.28 (m, 2H), 8.18 (m, 1H), 9.02
(m, 1H), 12.20 (s, 1H); MS (ES): 355.3 (M.sup.++1).
[0483] 4-(4-trans-hydroxycyclohexyl)amino-5,6-dimethyl-2-isopropyl
-7H-pyrrolo[2,3d]pyrimidine .sup.1H NMR (200 MHz, CDCl.sub.3)
.delta. 1.31 (d, J=7.0 Hz, 6H), 1.30-1.65 (m, 8H), 2.27 (s, 3H),
2.28 (s, 3H), 3.01 (m, J=7.0 Hz, 1H), 3.71 (m, 1H), 4.14 (m, 1H),
4.78 (d, 1H); MS (ES): 303.2.
[0484]
dl-4-(2-trans-hydroxycyclohexyl)amino-5,6-dimethyl-2-isopropyl-7H--
pyrrolo[2,3d]pyrimidine H NMR (200 MHz, CDCl.sub.3) .delta.
1.31-1.42 (br, 4H), 1.75-1.82 (br, 4H), 2.02 (s, 3H), 2.29 (s, 3H),
3.53 (m, 1H), 4.02 (m, 1H), 5.08 (d, 1H), 7.41-7.48 (m, 3H), 8.30
(m, 2H), 10.08 (s, 1H); MS (ES): 337.2 (M.sup.++1).
[0485]
4-(3,4-trans-dihydroxycyclohexyl)amino-5,6-dimethyl-2-phenyl-7H-py-
rrolo[2,3d]pyrimidine. MS (ES): 353.2 (M.sup.++1).
[0486]
4-(3,4-cis-dihydroxylcyclohexyl)amino-5,6-dimethyl-2-phenyl-7H-pyr-
rolo[2,3d]pyrimidine. MS (ES): 353.2 (M.sup.++1).
[0487]
4-(2-acetylaminoethyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]-
pyrimidine.
[0488] mp 196-199.degree. C.; .sup.1H NMR (200 MHz, CDCl.sub.3)
.delta. 1.72 (s, 3H), 1.97 (s, 3H), 2.31 (s, 3H), 3.59 (m, 2H),
3.96 (m, 2H), 5.63 (br, 1H), 7.44-7.47 (m, 3H), 8.36-8.43 (dd, J=1
Hz, 7 Hz, 2H), 10.76 (s, 1H); MS (ES): 324.5 (M.sup.++1).
[0489]
dl-4-(2-trans-hydroxycyclopentyl)amino-5,6-dimethyl-2-phenyl-7H-py-
rrolo[2,3d]pyrimidine..sup.1 .sup.1 For preparation of
2-trans-hydroxycyclopentylamine, see PCT 9417090.
[0490] .sup.1H NMR (200 MHz, CDCl.sub.3) .delta..sub.--1.62 (m,
2H), 1.79 (br, 4H), 1.92 (s, 3H), 2.29 (s, 3H), 4.11 (m, 1H), 4.23
(m, 1H), 5.28 (d, 1H), 7.41-7.49 (m, 3H), 8.22 (m, 2H), 10.51 (s,
1H); MS (ES): 323.2 (M.sup.++1).
[0491]
dl-4-(3-trans-hydroxycyclopentyl)amino-5,6-dimethyl-2-phenyl-7H-py-
rrolo[2,3d]pyrimidine..sup.1 .sup.1 For preparation of
3-trans-hydroxycyclopentylamine, see EP-A-322242.
[0492] .sup.1H NMR (200 MHz, CDCl.sub.3) .delta. 1.58-1.90 (br,
6H,), 2.05 (s, 3H), 2.29 (s, 3H), 4.48-4.57 (m, 1H), 4.91-5.01 (m,
2H), 7.35-7.46 (m, 3H), 8.42-8.47 (m, 2H), 10.11 (s, 1H); MS (ES):
323.2 (M.sup.++1).
[0493]
dl-4-(3-cis-hydroxycyclopentyl)amino-5,6-dimethyl-2-phenyl-7H-pyrr-
olo[2,3d]pyrimidine..sup.1 .sup.1 For preparation of
3-cis-hydroxycyclopentylamine, see EP-A-322242.
[0494] .sup.1H NMR (200 MHz, CDCl.sub.3) .delta..sub.--1.82-2.28
(br, 6H), 2.02 (s, 3H), 2.30 (s, 3H), 4.53-4.60 (m, 1H), 4.95-5.08
(m, 1H), 5.85-5.93 (d, 1H), 7.35-7.47 (m, 3H), 8.42-8.46 (m, 2H),
10.05 (s, 1H); MS (ES): 323.2 (M.sup.++1).
[0495]
4-(3,4-trans-dihydroxycyclopentyl)amino-5,6-dimethyl-2-phenyl-7H-p-
yrrolo[2,3d]pyrimidine..sup.1 1H NMR (200 MHz, CDCl.sub.3)
.delta..sub.--1.92-1.99 (br, 2H), 2.14 (s, 3H), 2.20 (br, 2H), 2.30
(s, 3H), 2.41-2.52 (br, 2H), 4.35 (m, 2H), 4.98 (m, 2H), 7.38-7.47
(m, 3H), 8.38-8.42 (m, 2H), 9.53 (s, 1H); MS (ES): 339.2
(M.sup.++1). .sup.1 For preparation of
3,4-trans-dihydroxycyclopentylamine, see PCT 9417090.
[0496]
4-(3-amino-3-oxopropyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d-
]pyrimidine.
[0497] .sup.1H NMR (200 MHz, CDCl.sub.3) .delta..sub.--2.02 (s,
3H), 2.29 (s, 3H), 2.71 (t, 2H), 4.18 (m, 2H), 5.75-5.95 (m, 3H),
7.38-7.48 (m, 3H), 8.37-8.41 (m, 2H), 10.42 (s, 1H); MS (ES): 310.1
(M.sup.++1).
[0498]
4-(3-N-cyclopropylmethylamino-3-oxopropyl)amino-5,6-dimethyl-2-phe-
nyl-7H-pyrrolo[2,3d]pyrimidine. .sup.1H NMR (200 MHz, CD.sub.3OD)
.delta..sub.--0.51 (q, 2H), 0.40 (q, 2H), 1.79-1.95 (br, 1H), 2.36
(s, 3H), 2.40 (s, 3H), 2.72 (t, 2H), 2.99 (d, 2H), 4.04 (t, 2H),
7.58-7.62 (m, 3H), 8.22-8.29 (m, 2H); MS (ES): 364.2
(M.sup.++1).
[0499]
4-(2-amino-2-oxoethyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]-
pyrimidine .sup.1H NMR (200 MHz, CD.sub.3OD) .delta. 2.31 (s, 3H),
2.38 (s, 3H), 4.26 (s, 2H), 7.36 (m, 3H), 8.33 (m, 2H); MS (ES):
396.1 (M.sup.++1).
[0500]
4-(2-N-methylamino-2-oxoethyl)amino-5,6-dimethyl-2-phenyl-7H-pyrro-
lo[2,3d]pyrimidine. .sup.1H NMR (200 MHz, CDCl.sub.3)
.delta..sub.--1.99 (s, 3H), 2.17 (s, 3H), 2.82 (d, 3H), 4.39 (d,
2H), 5.76 (t, 1H), 6.71 (br, 1H), 7.41-7.48 (m, 3H), 8.40 (m, 2H),
10.66 (s, 1H); MS (ES): 310.1 (M.sup.++1).
[0501]
4-(3-tert-butyloxyl-3-oxopropyl)amino-5,6-dimethyl-2-phenyl-7H-pyr-
rolo[2,3d]pyrimidine. .sup.1H NMR (200 MHz, CDCl.sub.3)
.delta..sub.--1.45 (s, 9H), 1.96 (s, 3H), 2.29 (s, 3H), 2.71 (t,
2H), 4.01 (q, 2H), 5.78 (t, 1H), 7.41-7.48 (m, 3H), 8.22-8.29 (m,
2H); MS (ES): 367.2 (M.sup.++1).
[0502]
4-(2-hydroxyethyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyri-
midine. .sup.1H NMR (200 MHz, CDCl.sub.3) .delta. 1.92 (s, 3H),
2.29 (s, 3H), 3.81-3.98 (br, 4H), 5.59 (t, 1H), 7.39-7.48 (m, 3H),
8.37 (m, 2H), 10.72 (s, 1H); MS (ES): 283.1 (M.sup.++1).
[0503]
4-(3-hydroxypropyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyr-
imidine. .sup.1H NMR (200 MHz, CDCl.sub.3) .delta. 1.84 (m, 2H),
1.99 (s, 3H), 2.32 (s, 3H), 3.62 (t, 2H), 3.96 (m, 2H), 3.35 (t,
1H), 7.39-7.48 (m, 3H), 8.36 (m, 2H), 10.27 (s, 1H); MS (ES): 297.2
(M.sup.++1).
[0504]
4-(4-hydroxybutyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyri-
midine. .sup.1H NMR (200 MHz, CDCl.sub.3) .delta. 1.71-1.82 (m,
4H), 1.99 (s, 3H), 2.31 (s, 3H), 3.68-3.80 (m, 4H), 5.20 (t, 1H),
7.41-7.49 (m, 3H), 8.41 (m, 2H), 10.37 (s, 1H); MS (ES): 311.2
(M.sup.++1).
[0505]
4-(4-trans-acetylaminocyclohexyl)amino-5,6-dimethyl-2-phenyl-7H-py-
rrolo[2,3d]pyrimidine.
[0506]
4-(4-trans-methylsulfonylaminocyclohexyl)amino-5,6-dimethyl-2-phen-
yl-7H-pyrrolo[2,3d]pyrimidine.
[0507]
4-(2-acetylaminoethyl)amino-5,6-dimethyl-2-phenyl-7H-7-(1-phenylet-
hyl)pyrrolo[2,3d]pyrimidine.
[0508]
4-(4-trans-hydroxycyclohexyl)amino-5,6-dimethyl-2-phenyl-7H-7-(1-p-
henylethyl)pyrrolo[2,3d]pyrimidine.
[0509]
4-(3-pyridylmethyl)amino-5,6-dimethyl-2-phenyl-7H-7-(1-phenylethyl-
)pyrrolo[2,3d]pyrimidine.
[0510]
4-(2-methylpropyl)amino-5,6-dimethyl-2-phenyl-7H-7-(1-phenylethyl)-
pyrrolo[2,3d]pyrimidine.
Example 2
[0511] To a stirred suspension of triphenylphosphine (0.047 g,
0.179 mmol) and benzoic acid (0.022 g, 0.179 mmol) in THF (1.0 mL)
cooled to 0.degree. C. was added 4-(4-trans-hydroxycyclohexyl)
amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine (0.05 g,
0.149 mmol) at 0.degree. C. Diethyl azodicarboxylate (0.028 ml,
0.179 mmol) was then added dropwise over 10 minutes. The reaction
was then allowed to warm to room temperature. After reaction was
complete by TLC the reaction mixture was quenched with aqueous
sodium bicarbonate (3.0 mL). The aqueous phase was separated and
extracted with ether (2.times.5.0 mL). The organic extracts were
combined, dried, and concentrated in vacuo to dryness. To the
residue was added ether (2.0 mL) and hexane (5.0 mL) whereupon the
bulk of the triphenylphosphine oxide was filtered off.
Concentration of the filtrate gave a viscous oil which was purified
by column chromatography (hexane:ethyl acetate=4:1) to give 5.0 mg
(7.6%) of
4-(4-cis-benzoyloxycyclohexyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d-
]pyrimidine. MS (ES): 441.3 (M.sup.++1). The reaction also produced
50.0 mg (84%) of
4-(3-cyclohexenyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine.
MS (ES): 319.2 (M.sup.++1).
Example 3
[0512] To a solution of
4-(4-cis-benzoyloxycyclohexyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d-
]pyrimidine (5.0 mg, 0.0114 mmol) in ethanol (1.0 mL) was added 10
drops of 2M sodium hydroxide. After 1 hr, the reaction mixture was
extracted with ethyl acetate (3.times.5.0 mL) and the organic layer
was dried, filtered and concentrated in vacuo to dryness. The
residue was subjected to column chromatography (hexane:ethyl
acetate=4:1) to give 3.6 mg (94%) of 4-(4-cis-hydroxycyclohexyl)
amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine. MS (ES):
337.2 (M.sup.++1).
[0513] The following compounds were obtained in a similar manner as
that of Example 3:
[0514]
4-(3-N,N-dimethyl-3-oxopropyl)amino-5,6-dimethyl-2-phenyl-7H-pyrro-
lo[2,3d]pyrimidine. .sup.1H NMR (200 MHz, CDCl.sub.3) .delta. 2.01
(s, 3H), 2.31 (s, 3H), 2.73 (t, 2H), 2.97 (s, 6H), 4.08 (m, 2H),
6.09 (t, 1H), 7.41-7.48 (m, 3H), 8.43 (m, 2H), 10.46 (s, 1H); MS
(ES): 338.2 (M.sup.++1).
[0515]
4-(2-formylaminoethyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]-
pyrimidine. .sup.1H NMR (200 MHz, CDCl.sub.3) .delta. 2.26 (s, 3H),
2.37 (s, 3H), 3.59-3.78 (m, 2H), 3.88-4.01 (m, 2H), 5.48-5.60 (m,
1H), 7.38-7.57 (m, 3H), 8.09 (s, 1H), 8.30-8.45 (m, 2H), 8.82 (s,
1H); MS (ES): 310.1 (M.sup.++1).
[0516]
4-(3-acetylaminopropyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d-
]pyrimidine. MS (ES): 338.2 (M.sup.++1).
Example 4
[0517]
4-(3-tert-butyloxy-3-oxopropyl)amino-5,6-dimethyl-2-phenyl-7H-pyrr-
olo[2,3d]pyrimidine (70.0 mg, 0.191 mmol)) was dissolved in
trifluoroacetic acid:dichloromethane (1:1, 5.0 mL). The resulting
solution was stirred at room temperature for 1 hr. and then
refluxed for 2 hr. After cooling down to room temperature, the
mixture was concentrated in vacuo to dryness. The residue was
subjected to preparative thin layer chromatography (EtOAc:hexane:
AcOH=7:2.5:0.5) to give 40.0 mg (68%) of.
4-(3-hydroxy-3-oxopropyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyri-
midine. .sup.1H NMR (200 MHz, CD.sub.3OD) .delta. 2.32 (s, 3H),
2.38 (s, 3H), 2.81 (t, 2H), 4.01 (t, 2H), 7.55 (m, 3H), 8.24 (m,
2H); MS (ES): 311.1 (M.sup.++1).
[0518] The following compound was obtained in a similar manner as
that of Example 4:
[0519]
4-(3-aminopropyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrim-
idine. MS (ES): 296.1 (M.sup.++1), 279.1 (M+--NH.sub.3).
Example 5
[0520]
4-(3-hydroxy-3-oxopropyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,-
3d]pyrimidine (50.0 mg, 0.161 mmol) was dissolved in a mixture of
N,N-dimethylformamide (0.50 mL), dioxane (0.50 mL) and water (0.25
mL). To this solution was added methylamine (0.02 mL, 40% wt in
water, 0.242 mmol), triethylamine (0.085 mL) and
N,N,N'N'-tetramethyl uronium tetrafluoroborate (61.2 mg, 0.203
mmol). After stirring at room temperature for 10 min, the solution
was concentrated and the residue was subjected to preparative thin
layer chromatography (EtOAc) to give 35.0 mg (67%) of
4-(3-N-methyl-3-oxopropyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyr-
imidine. .sup.1H NMR (200 MHz, CDCl.sub.3) .delta. 1.92 (s, 3H),
2.30 (s, 3H), 2.65 (t, 2H), 4.08 (t, 2H), 5.90 (t, 1H), 6.12 (m,
1H), 7.45 (m, 3H), 8.41 (m, 2H), 10.68 (s, 1H); MS (ES): 311.1
(M.sup.++1).
[0521] The following compounds were obtained in a similar manner as
that of Example 5:
[0522]
4-(2-cyclopropanecarbonylaminoethyl)amino-5,6-dimethyl-2-phenyl-7H-
-pyrrolo[2,3d]pyrimidine. MS (ES): 350.2 (M.sup.++1).
[0523]
4-(2-isobutyrylaminoethyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2-
,3d]pyrimidine. MS (ES): 352.2 (M.sup.++1).
[0524]
4-(3-propionylaminopropyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2-
,3d]pyrimidine. .sup.1H NMR (200 MHz, CDCl.sub.3) .delta. 1.00-1.08
(t, 3H), 1.71-2.03 (m, 4H), 2.08 (s, 3H), 2.37 (s, 3H), 3.26-3.40
(m, 2H), 3.79-3.96 (m, 2H), 5.53-5.62 (m, 1H), .sub.--6.17-6.33 (m,
1H), 7.33-7.57 (m, 3H), 8.31-8.39 (m, 2H), 9.69 (s, 1H); MS (ES):
352.2 (M.sup.++1).
[0525]
4-(2-methylsulfonylaminoethyl)amino-5,6-dimethyl-2-phenyl-7H-pyrro-
lo[2,3d]pyrimidine. .sup.1H NMR (200 MHz, CDCl.sub.3) .delta. 2.18
(s, 3H), 2.27 (s, 3H), 2.92 (s, 3H), 3.39-3.53 (m, 2H), 3.71-3.88
(m, 2H), 5.31-5.39 (m, 1H), 6.17-6.33 (m, 1H), 7.36-7.43 (m, 3H),
8.20-8.25 (m, 2H), 9.52 (s, 1H); MS (ES): 360.2 (M.sup.++1).
Example 6
[0526] A mixture of
4-chloro-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine (0.70 g,
2.72 mmol) and 1,2-diaminoethane (10.0 mL, 150 mmol) was refluxed
under inert atmosphere for 6 hr. The excess amine was removed in
vacuo, the residue was washed sequentially with ether and hexane to
give 0.75 g (98%) of
4-(2-aminoethyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine.
MS (ES); 282.2 (M.sup.++1), 265.1 (M.sup.+-NH.sub.3).
Example 7
[0527] To a solution of
4-(2-aminoethyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine
(70.0 mg, 0.249 mmol) and triethylamine (50.4 mg, 0.498 mmol) in
dichloromethane (2.0 mL) was added propionyl chloride (25.6 mg,
0.024 mL, 0.274 mmol) at 0.degree. C. After 1 hr, the mixture was
concentrated in vacuo and the residue was subjected to preparative
thin layer chromatography (EtOAc) to give 22.0 mg (26%) of
4-(2-propionylaminoethyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyri-
midine. MS (ES): 338.2 (M.sup.++1).
[0528] The following compounds were obtained in a similar manner as
that of Example 7:
[0529]
4-(2-N'-methylureaethyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3-
d]pyrimidine. .sup.1H NMR (200 MHz, CDCl.sub.3) .delta. 2.13 (s,
3H), 2.32 (s, 3H), 3.53 (d, 3H), 3.55 (m, 2H), 3.88 (m, 2H), 4.29
(m, 1H), 5.68 (t, 1H), 5.84 (m, 1H), 7.42 (m, 3H), 8.36 (dd, 2H),
9.52 (s, 1H); MS (ES): 339.3 (M.sup.++1).
[0530]
4-(2-N'-ethylureaethyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d-
]pyrimidine. MS (ES): 353.2 (M.sup.++1).
Example 8
[0531] To a solution of
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (41.1
mg, 0.215 mmol), dimethylaminopyridine (2.4 mg, 0.020 mmol) and
pyruvic acid (18.9 mg, 0.015 mL, 0.215 mmol) in dichloromethane
(2.0 mL) was added
4-(2-aminoethyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine
(55.0 mg, 0.196 mmol). The mixture was stirred at room temperature
for 4 hr. Usual workup and column chromatography (EtOAc) then gave
10.0 mg (15%) of
4-(2'-pyruvylamidoethyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2-
,3d]pyrimidine. MS (ES): 352.2 (M.sup.++1).
Example 9
[0532] To a solution of
4-(2-aminoethyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine
(60.0 mg, 0.213 mmol) in dichloromethane (2.0 mL) was added
N-trimethylsilyl isocyanate (43.3 mg, 0.051 mL, 0.320 mmol). The
mixture was stirred at room temperature for 3 hr followed by
addition of aqueous sodium bicarbonate. After filtration through
small amount of silica gel, the filtrate was concentrated in vacuo
to dryness to give 9.8 mg (14%) of
4-(2-ureaethyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine.
MS (ES): 325.2 (M.sup.++1).
[0533] The following compounds were obtained in a similar manner as
that of Example 9:
[0534]
dl-4-(2-acetylaminopropyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2-
,3d]pyrimidine. .sup.1H NMR (200 MHz, CDCl.sub.3) .delta. 1.28-1.32
(d, J=8 Hz, 3H), 1.66 (s, 3H), 1.96 (s, 3H), 2.30 (s, 3H) 3.76-3.83
(m, 2H), 4.10-4.30 (m, 1H), 5.60-5.66 (t, J=6 Hz, 1H), 7.40-7.51
(m, 3H), 8.36-8.43 (m, 2H), 10.83 (s, 1H); MS (ES): 338.2
(M.sup.++1).
[0535]
(R)-4-(2-acetylaminopropyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[-
2,3d]pyrimidine. .sup.1H NMR (200 MHz, CDCl.sub.3) .delta. 1.31 (d,
3H), 1.66 (s, 3H) 1.99 (s, 3H), 2.31 (s, 3H), 3.78-3.83 (m, 2H),
4.17-4.22 (m, 1H), 5.67 (t, 1H), 7.38-7.5 (m, 3H), 8.39 (m, 2H),
10.81 (s, 1H); MS (ES): 338.2 (M.sup.++1).
[0536]
(R)-4-(1-methyl-2-acetylaminoethyl)amino-5,6-dimethyl-2-phenyl-7H--
pyrrolo[2,3d]pyrimidine. .sup.1H NMR (200 MHz, CDCl.sub.3) .delta.
1.41 (d, 3H), 1.68 (s, 3H), 2.21 (s, 3H), 2.34 (s, 3H), 3.46-3.52
(br, m, 2H), 4.73 (m, 1H), 5.22 (d, 1H), 7.41-7.46 (m, 3H),
8.36-8.40 (m, 2H), 8.93 (s, 1H); MS (ES): 338.2 (M.sup.++1).
[0537]
(S)-4-(2-acetylaminopropyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[-
2,3d]pyrimidine. .sup.1H NMR (200 MHz, CDCl.sub.3) .delta. 1.31 (d,
3H), 1.66 (s, 3H) 2.26 (s, 3H), 2.35 (s, 3H), 3.78-3.83 (m, 2H),
4.17-4.22 (m, 1H), 5.67 (t, 1H), 7.38-7.5 (m, 3H), 8.39 (m, 2H),
8.67 (s, 1H); MS (ES): 338.2 (M.sup.++1).
[0538]
(S)-4-(1-methyl-2-acetylaminoethyl)amino-5,6-dimethyl-2-phenyl-7H--
pyrrolo[2,3d]pyrimidine. .sup.1H NMR (200 MHz, CDCl.sub.3) .delta.
1.41 (d, 3H), 1.68 (s, 3H), 2.05 (s, 3H), 2.32 (s, 3H), 3.46-3.52
(m, 2H), 4.73 (m, 1H), 5.22 (d, 1H), 7.41-7.46 (m, 3H), 8.36-8.40
(m, 2H), 10.13 (s, 1H); MS (ES): 338.2 (M.sup.++1).
Example 10
[0539] Reaction of
4-chloro-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine with the
mixture of dl-1-amino-2-(1,1-dimethyl ethoxy)carbonylamino-propane
and dl-2-amino-1-(1,1-dimethyl ethoxy)carbonylamino-propane was run
in a similar manner as that of Example 1. The reaction gave a
mixture of
dl-4-(1-methyl-2-(1,1-dimethylethoxy)carbonylamino)ethylamino-5,6-dimethy-
l-2-phenyl-7H-pyrrolo[2,3d]pyrimidine and
dl-4-(2-methyl-2-(1,1-dimethylethoxy)carbonylamino)ethylamino-5,6-dimethy-
l-2-phenyl-7H-pyrrolo[2,3d]pyrimidine which were separated by
column chromatography (EtOAc:hexanes=1:3). The first fraction was
dl-4-(1-methyl-2-(1,1-dimethylethoxy)carbonylaminoethyl)amino-5,6-dimethy-
l-2-phenyl-7H-pyrrolo[2,3d]pyrimidine: .sup.1H NMR (200 MHz,
CDCl.sub.3) .delta. 1.29-1.38 (m, 12H), 1.95 (s, 3H), 2.31 (s, 3H)
3.34-3.43 (m, 2H), 4.62-4.70 (m, 1H), 5.36-5.40 (d, J=8 Hz, 1H),
5.53 (br, 1H), 7.37-7.49 (m, 3H), 8.37-8.44 (m, 2H), 10.75 (s, 1H).
MS 396.3 (M.sup.++1); The second fraction was
dl-4-(2-(1,1-dimethylethoxy)carbonylaminopropyl)amino-5,6-dimethyl-2-phen-
yl-7H-pyrrolo[2,3d]pyrimidine: .sup.1H NMR (200 MHz, CDCl.sub.3)
.delta. 1.26-1.40 (m, 12H), 2.00 (s, 3H), 2.31 (s, 3H) 3.60-3.90
(m, 2H), 3.95-4.10 (m, 1H), 5.41-5.44 (d, J=6.0 Hz, 1H), 5.65 (br,
1H), 7.40-7.46 (m, 3H), 8.37-8.44 (m, 2H), 10.89 (s, 1H); MS (ES):
396.2 (M.sup.++1).
[0540] The following compounds were obtained in a similar manner as
that of Example 10:
[0541]
(S,S)-4-(2-acetylaminocyclohexyl)amino-5,6-dimethyl-2-phenyl-7H-py-
rrolo[2,3d]pyrimidine. .sup.1H NMR (200 MHz, CDCl.sub.3)
.delta..sub.--1.43 (m, 4H), 1.60 (s, 3H), 1.83 (m, 2H), 2.18 (s,
3H), 2.30 (m, 2H), 2.32 (s, 3H), 3.73 (br, 1H), 4.25 (br, 1H), 5.29
(d, 1H), 7.43-7.48 (m, 3H), 8.35-8.40 (m, 2H), 9.05 (s, 1H).
[0542]
4-(2-methyl-2-acetylaminopropyl)amino-5,6-dimethyl-2-phenyl-7H-pyr-
rolo[2,3d]pyrimidine. .sup.1H NMR (200 MHz, CDCl.sub.3)
.delta..sub.--1.51 (s, 6H), 1.56 (s, 3H), 2.07 (s, 3H), 2.36 (s,
3H), 3.76 (d, 2H), 5.78 (t, 1H), 7.41-7.48 (m, 3H), 7.93 (s, 1H),
8.39 (m, 2H), 10.07 (s, 1H); MS (ES): 352.3 (M.sup.++1).
Example 11
[0543] dl-4-(1-methyl-2-(1,1-dimethylethoxy)carbonyl aminoethyl)
amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine (60.6 mg,
0.153 mmol) was treated with trifluoroacetic acid (0.5 mL) in
dichloromethane (2.0 mL) for 14 hr. The organic solvent was removed
in vacuo to dryness. The residue was dissolved in
N,N-dimethylformamide (2.0 mL) and triethylamine (2.0 mL). To the
solution at 0.degree. C. was added acetic anhydride (17.2 mg, 0.169
mmol). The resulted mixture was stirred at room temperature for 48
hr and then concentrated in vacuo to dryness. The residue was
subjected to preparative thin layer chromatography (EtOAc) to give
27.0 mg (52%) of
dl-4-(1-methyl-2-acetylaminoethyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[-
2,3d]pyrimidine. .sup.1H NMR (200 MHz, CDCl.sub.3) .delta.
1.38-1.42 (d, J=8 Hz, 3H), 1.69 (s, 3H), 2.01 (s, 3H), 2.32 (s, 3H)
3.38-3.60 (m, 2H), 4.65-4.80 (m, 1H), 5.23-5.26 (d, J=6 Hz, 1H),
7.40-7.51 (m, 3H), 8.37-8.43 (m, 2H), 10.44 (s, 1H); MS (ES): 338.2
(M.sup.++1).
Example 12
[0544]
(R,R)-4-(2-aminocyclohexyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[-
2,3d]pyrimidine, prepared in a similar manner as that of Example 1
from 4-chloro-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine
(0.15 g, 0.583 mmol) and (1R,2R)-(-)-1,2-diaminocyclohexane (0.63
g, 5.517 mmol), was treated with triethylamine (0.726 g, 7.175
mmol) and acetic anhydride (0.325 g, 3.18 mmol) in
N,N-dimethylformamide (10.0 mL) at room temperature for 2 hr. After
removal of solvent in vacuo, ethyl acetate (10.0 mL) and water
(10.0 mL) were added to the residue. The mixture was separated and
the aqueous layer was extracted with ethyl acetate (2.times.10.0
mL). The combined ethyl acetate solution was dried (MgSO.sub.4) and
filtered. The filtrate was concentrated in vacuo to dryness and the
residue was subjected to column chromatography (EtOAc:Hexane=1:1)
to give 57.0 mg (26%) of
(R,R)-4-(2-acetylaminocyclohexyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2-
,3d]pyrimidine. .sup.1H NMR (200 MHz, CDCl.sub.3)
.delta..sub.--1.43 (m, 4H), 1.60 (s, 3H), 1.84 (m, 2H), 2.22 (s,
3H), 2.30 (m, 2H), 2.33 (s, 3H), 3.72 (br, 1H), 4.24 (br, 1H), 5.29
(d, 1H), 7.43-7.48 (m, 3H), 8.35-8.39 (m, 2H), 8.83 (s, 1H); MS
(ES): 378.3 (M.sup.++1).
Example 13
[0545] To a solution of
4-(2-hydroxyethyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine
(40.0 mg, 0.141 mmol) in pyridine (1.0 mL) was added acetic
anhydride (0.108 g, 1.06 mmol) at 0.degree. C. The mixture was
stirred at room temperature for 4 hr and the solvent was removed in
vacuo. The residue was subjected to preparative thin layer
chromatography (EtOAc:hexane=1:1) to give 32.3 mg (71%) of
4-(2-acetyloxyethyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidin-
e. .sup.1H NMR (200 MHz, CDCl.sub.3) .delta..sub.--1.90 (s, 3H),
2.08 (s, 3H), 2.31 (s, 3H), 4.05 (m, 2H), 4.45 (t, 2H), 5.42 (m,
1H), 7.41-7.49 (m, 3H), 8.42 (m, 2H), 11.23 (s, 1H).
Example 14
[0546] A solution of Fmoc-.beta.-Ala-OH (97.4 mg, 0.313 mmol) and
oxalyl chloride (39.7 mg, 27.3 .mu.L, 0.313 mmol) in
dichloromethane (4.0 mL) with 1 drop of N,N-dimethylformamide was
stirred at 0.degree. C. for 1 hr followed by addition of
4-(2-aminoethyl)
amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine (80.0 mg,
0.285 mmol) and triethylamine (57.6 mg, 79.4 .mu.L, 0.570 mmol) at
0.degree. C. After 3 hr, the mixture was concentrated in vacuo and
the residue was treated with the solution of 20% piperidine in
N,N-dimethylforamide (2.0 mL) for 0.5 hr. After removal of the
solvent in vacuo, the residue was washed with diethyl ether:hexane
(1:5) to give 3.0 mg (3%) of
4-(6-amino-3-aza-4-oxohexyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]p-
yrimidine. MS (ES): 353.2 (M.sup.++1).
Example 15
[0547] A solution of
4-(2-aminoethyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine
(70.0 mg, 0.249 mmol) and succinic anhydride (27.0 mg, 0.274 mmol)
in dichloromethane (4.0 mL) with 1 drop of N,N-dimethylformamide
was stirred at room temperature for 4 hr. The reaction mixture was
extracted with 20% sodium hydroxide (3.times.5.0 mL). The aqueous
solution was acidified with 3 M hydrochloride to pH=7.0. The whole
mixture was extracted with ethyl acetate (3.times.10 mL). The
combined organic solution was dried (MgSO.sub.4) and filtered. The
filtrate was concentrated in vacuo to dryness to give 15.0 mg (16%)
of
4-(7-hydroxy-3-aza-4,7-dioxoheptyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo-
[2,3d]pyrimidine. MS (ES): 382.2 (M.sup.++1).
Example 16
[0548] To 10 mL of dimethylformamide (DMF) at room temperature were
added 700 mg of
4-(cis-3-hydroxycyclopentyl)amino-2-phenyl-5,6-dimethyl-7H-pyrr-
olo[2,3d]pyrimidine followed by 455 mg of N-Boc glycine, 20 mg of
N,N-dimethylaminopyridine (DMAP), 293 mg of hydroxybenzotriazole
(HOBT) and 622 mg of 1-(3-dimethylaminopropyl)-3-ethylcarboiimide
hydrochloride (EDCl). The reaction mixture was left stirring
overnight. DMF was then removed under reduced pressure and the
reaction mixture was partitioned between 20 mL of ethyl acetate and
50 mL of water. The aqueous portion was extracted further with
2.times.20 mL of ethyl acetate and the combined organic portions
were washed with brine, dried over anhydrous sodium sulfate,
filtered and concentrated. Purification on silica gel, eluting with
ethyl acetate/hexane gave 410 mg of the desired product:
4-(cis-3-(N-t-butoxycarbonyl-2-aminoacetoxy)cyclopentyl)amino-2-phenyl-5,-
6,-dimethyl-7H-pyrrolo[2,3d]pyrimidine, MS (ES) (M.sup.++1)=480.2.
The ester was then treated with 5 mL of 20% trifluoroacetic acid in
dichloromethane at room temperature, left over night and then
concentrated. Trituration with ethyl acetate gave 300 mg of an off
white solid; 4-(cis-3-(2-aminoacetoxy)cyclopentyl)amino-5,6-dimethy
1-2-phenyl-7H-pyrrolo[2,3d]pyrimidine trifluoroacetic acid salt, MS
(ES) (M.sup.++1)=380.1.
[0549] One skilled in the art will appreciate that the following
compounds can be synthesized by the methods disclosed above:
[0550]
4-(cis-3-hydroxycyclopentyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo-
[2,3d]pyrimidine MS (ES) (M.sup.++1)=323.1.
[0551]
4-(cis-3-(2-aminoacetoxy)cyclopentyl)amino-5,6-dimethyl-2-phenyl-7-
H-pyrrolo[2,3d]pyrimidinetrifluoroacetic acid salt MS (ES)
(M.sup.++1)=380.1.
[0552]
4-(3-acetamido)piperidinyl-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]p-
yrimidine MS (ES) (M.sup.++1)=364.2.
[0553]
4-(2-N'-methylureapropyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,-
3d]pyrimidine, MS (ES) (M.sup.++1)=353.4.
[0554]
4-(2-acetamidobutyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]py-
rimidine, MS (ES) (M.sup.++1)=352.4.
[0555]
4-(2-N'-methylureabutyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3-
d]pyrimidine MS (ES) (M.sup.++1)=367.5
[0556]
4-(2-aminocyclopropylacetamidoethyl)amino-2-phenyl-7H-pyrrolo[2,3d-
]pyrimidine MS (ES) (M.sup.++1)=309.1.
[0557]
4-(trans-4-hydroxycyclohexyl)amino-2-(3-chlorophenyl)-7H-pyrrolo[2-
,3d]pyrimidine MS (ES) (M.sup.++1)=342.8.
[0558]
4-(trans-4-hydroxycyclohexyl)amino-2-(3-fluorophenyl)-7H-pyrrolo[2-
,3d]pyrimidine MS (ES) (M.sup.++1)=327.2.
[0559]
4-(trans-4-hydroxycyclohexyl)amino-2-(4-pyridyl)-7H-pyrrolo[2,3d]p-
yrimidine MS (ES) (M.sup.++1)=310.2.
Example 17
[0560] ##STR130##
[0561] The pyrrole nitrogen of (7) (Scheme IX) was protected with
di-t-butyldicarbonate under basic conditions to yield the
corresponding carbamate (22). Radical bromination of (22) proceeded
regioselectively to yield bromide (23). In general, compound (23)
served as a key electrophilic intermediate for various nucleophilic
coupling partners. Displacement of the alkyl bromide with sodium
phenolate trihydrate yielded compound (24). Subsequent displacement
of the aryl chloride and removal of the t-butyl carbamate
protecting group occurred in one step yielding desired compound
(25). Detailed Synthesis of Compounds (22)-(25) in Accordance with
Scheme IX ##STR131##
[0562] Di-t-butyl dicarbonate (5.37 g, 24.6 mmol) and dimethyl
aminopyridine (1.13 g, 9.2 mmol) were added to a solution
containing (7) (1.50 g, 6.15 mmol) and pyridine (30 mL). After 20 h
the reaction was concentrated and the residue was partitioned
between CH.sub.2Cl.sub.2 and water. The CH.sub.2Cl.sub.2 layer was
separated, dried over MgSO.sub.4, filtered and concentrated to
yield a black solid. Flash chromatography (SiO.sub.2; 1/9
EtOAc/Hexanes, R.sub.f 0.40) yielded 1.70 g (80%) of a white solid
(22). .sup.1H NMR (200 MHz, CDCl.sub.3) .delta..sub.--8.50 (m, 2H,
Ar--H), 7.45 (m, 3H, Ar--H), 6.39 (s, 1H, pyrrole-H), 2.66 (s, 3H,
pyrrole-CH.sub.3), 1.76 (s, 9H, carbamate-CH.sub.3); MS, M+1=344.1;
Mpt=175-177.degree. C. ##STR132##
[0563] N-Bromosuccinimide (508 mg, 2.86 mmol) and AIBN (112 mg,
0.68 mmol) were added to a solution containing (22) (935 mg, 2.71
mmol) and CCl.sub.4 (50 mL). The solution was heated to reflux.
After 2 h the reaction was cooled to room temperature and
concentrated in vacuo to yield a white solid. Flash chromatography
(SiO.sub.2; 1/1 CH.sub.2Cl.sub.2/Hexanes, R.sub.f 0.30) yielded 960
mg (84%) of a white solid (23). .sup.1H NMR (200 MHz, CDCl.sub.3)
.delta. 8.52 (m, 2H, Ar--H), 7.48 (m, 3H, Ar--H), 6.76 (s, 1H,
pyrrole-H), 4.93 (s, 2H, pyrrole-CH.sub.2Br), 1.79 (s, 9H,
carbamate-CH.sub.3); MS, M+1=423.9; Mpt=155-157.degree. C.
##STR133##
[0564] Sodium phenoxide trihydrate (173 mg, 1.02 mmol) was added in
one portion to a solution of bromide (23) (410 mg, 0.97 mmol)
dissolved in CH.sub.2Cl.sub.2 (5 mL) and DMF (10 mL). After 2 h the
reaction solution was partitioned between CH.sub.2Cl.sub.2 and
water. The water layer was extracted with CH.sub.2Cl.sub.2. The
combined CH.sub.2Cl.sub.2 layers were washed with water, dried over
MgSO.sub.4, filtered and concentrated to yield a yellow solid.
Flash chromatography (SiO.sub.2; 1/6 EtOAc/Hexanes, R.sub.f 0.30)
yielded 210 mg (50%) of a white solid (24). .sup.1H NMR (200 MHz,
CDCl.sub.3) .delta. 8.53 (m, 2H, Ar--H), 7.48 (m, 3H, Ar--H), 7.34
(m, 2H, Ar--H), 7.03 (m, 3H, Ar--H), 6.83 (s, 1H, pyrrole-H), 5.45
(s, 2H, ArCH.sub.2O), 1.76 (s, 9H, carbamate-CH.sub.3); MS,
M.sup.+=436.2. ##STR134##
[0565] A solution containing (24) (85 mg, 0.20 mmol),
N-acetylethylenediamine (201 mg, 1.95 mmol) and DMSO (3 mL) was
heated to 100.degree. C. After 1 h the temperature was raised to
130.degree. C. After 3 h the reaction was cooled to room
temperature and partitioned between EtOAc and water. The water
layer was extracted with EtOAc (2.times.). The combined EtOAc
layers are washed with water, dried over MgSO.sub.4, filtered and
concentrated. Flash chromatography (SiO.sub.2; 1/10
EtOH/CHCl.sub.3, R.sub.f 0.25) yielded 73 mg (93%) of a white foamy
solid (25). .sup.1H NMR (200 MHz, DMSO-d.sub.6) .delta. 11.81 (br
s, 1H, N--H), 8.39 (m, 2H, Ar--H), 8.03 (br t, 1H, N--H), 7.57 (br
t, 1H, N--H), 7.20-7.50 (m, 5H, Ar--H), 6.89-7.09 (m, 3H, Ar--H),
6.59 (s, 1H, pyrrole-H), 5.12 (s, 2H, ArCH.sub.2O), 3.61 (m, 2H,
NCH.sub.2), 3.36 (m, 2H, NCH.sub.2), 1.79 (s, 3H, COCH.sub.3); MS,
M+1=402.6
[0566] The following compounds were obtained in a manner similar to
that of Example 17:
[0567]
4-(2-acetylaminoethyl)amino-6-phenoxymethyl-2-phenyl-7H-pyrrolo[2,-
3d]pyrimidine. mp 196-197.degree. C.; MS (ES): 401.6
(M.sup.++1).
[0568]
4-(2-acetylaminoethyl)amino-6-(4-fluorophenoxy)methyl-2-phenyl-7H--
pyrrolo[2,3d]pyrimidine. MS (ES): 420.1 (M.sup.++1).
[0569]
4-(2-acetylaminoethyl)amino-6-(4-chlorophenoxy)methyl-2-phenyl-7H--
pyrrolo[2,3d]pyrimidine. MS (ES): 436.1 (M.sup.++1).
[0570]
4-(2-acetylaminoethyl)amino-6-(4-methoxyphenoxy)methyl-2-phenyl-7H-
-pyrrolo[2,3d]pyrimidine. MS (ES): 432.1 (M.sup.++1).
[0571]
4-(2-acetylaminoethyl)amino-6-(N-pyridin-2-one)methyl-2-phenyl-7H--
pyrrolo[2,3d]pyrimidine. MS (ES): 403.1 (M.sup.++1).
[0572]
4-(2-acetylaminoethyl)amino-6-(N-phenylamino)methyl-2-phenyl-7H-py-
rrolo[2,3]pyrimidine. MS (ES): 400.9 (M.sup.++1).
[0573] 4-(2-acetylaminoethyl)amino-6-(N-methyl-N-phenylamino)
methyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine. MS (ES): 414.8
(M.sup.++1).
[0574]
4-(2-N'-methylureaethyl)amino-6-phenoxymethyl-2-phenyl-7H-pyrrolo[-
2,3d]pyrimidine. MS (ES): 416.9 (M.sup.++1).
Example 18
Synthesis of Adenosine A.sub.1 Antagonists
[0575] Compound 1319 and Compound 1320 (Table 13 below) can be
synthesized by the general procedures given below. ##STR135##
[0576] Compound 1319 (81%) .sup.1H-NMR (d.sub.6-DMSO) d 1.37 (m,
4H), 1.93 (m, 2H), 2.01 (m, 2H), 4.11 (brs, 1H), 4.61 (d, 1H, J=4.4
Hz), 6.59 (m, 1H), 7.09 (m, 1H), 7.21 (m, 2H), 7.49 (dd, 1H, J=8
Hz, 14 Hz), 8.03 (m, 1H), 8.18 (d, 1H, J=8 Hz), 11.55 (brs, 1H). MS
(ES): 327.0 (M.sup.++1).
[0577] Compound 1320 (31%) MS (ES): 343.1 (M.sup.++1).
Example 19
Synthesis of Adenosine A.sub.1 Antagonist
[0578] Compound 1321 (Table 13 below) can be synthesized by the
general procedures given below. ##STR136##
[0579] Compound 28 (10.93 g, 50.76 mmol) was dissolved in DMF (67
mL). 4-Amidinopyridine hydrochloride (8.0 g, 50.76 mmol) and DBU
(15.4 g, 101.5 mmol) were added sequentially and the reaction was
heated to 85.degree. C. After 22 hours, the reaction was cooled to
room temperature and the DMF was removed in vacuo. The dark oil was
diluted with 2M HCl (80 mL). The reaction was allowed to stand.
After 2 hours, the solution was cooled to 10.degree. C. and
filtered. The solid was washed with cold water and dried to yield
7.40 g of a yellow solid, Compound 29 (69%). .sup.1H-NMR (200 MHz,
d.sub.6-DMSO) d 6.58 (s, 1H), 7.27 (s, 1H), 8.53 (d, 2H, J=5.6),
9.00 (d, 2H, J=5.2 Hz), 12.35 (brs, 1H). MS (ES): 212.8
(M.sup.++1).
[0580] Compound 29 (7.4 g, 29.8 mmol) was diluted with POCl.sub.3
and heated to 105.degree. C. After 18 hours, the reaction is cooled
to room temperature and the POCl.sub.3 is removed in vacuo. The
thick dark oil is diluted with MeOH (75 mL) followed by ether (120
mL). The amorphous red solid is filtered and washed with ether to
yield 3.82 g of a red solid. The crude solid is approximately 80%
pure and used without further purification in the next reaction. MS
(ES): 230.7 (M.sup.++1).
[0581] Compound 1321 .sup.1H-NMR (15%) (200 MH, d.sub.6-DMSO) d
1.38 (m, 4H), 1.92 (brs, 2H), 2.02 (brs, 2H), 3.44 (brs, 1H), 4.14
(brs, 1H), 4.56 (d, 1H, J=4 Hz), 6.63 (m, 1H), 7.15 (m, 1H), 7.32
(d, 1H, J=6.2 Hz), 8.20 (d, 2H, J=4.4 Hz), 8.65 (d, 2H, J=4.4 Hz),
11.67 (brs, 1H). MS (ES): 310.2 (M.sup.++1).
[0582] Compound 1501 (Table 15 below) .sup.1H-NMR (70%) (200 MHz,
CD.sub.3OD) d 1.84 (s, 3H), 3.52 (t, 2H, J=6.0 Hz), 3.83, t, 2H,
J=6.0 Hz), 6.51 (d, 1H, J=3.4 Hz), 7.06 (d, 1H, J=3.8 Hz), 7.42 (m,
3H), 8.36 (m, 2H). MS (ES): 296.0 (M.sup.++1).
[0583] Compound 1502 (Table 15 below) MS (ES): 345.0
(M.sup.++1).
[0584] Compound 1500 (Table 15 below) .sup.1H-NMR (200 MHz,
CDCl.sub.3) .delta. 1.40-1.80 (m, 6H), 1.85-2.10 (m, 2H), 2.18 (s,
3H), 2.33 (s, 3H), 2.50 (d, 3H), 3.90-4.10 (m, 2H), 4.76 (m, 1H),
5.50 (d, 1H), 6.03 (m, 1H), 7.40 (m, 3H), 8.37 (m, 2H), 9.15 (brs,
1H). MS (ES): 393.3 (M.sup.++1).
Example 20A
Synthesis of Adenosine A.sub.1 Antagonist
[0585] Compound 1504 (Table 15 below) can be synthesized by the
general procedures given below. ##STR137##
[0586] Compound 31 (200 mg, 0.47 mmol) was dissolved in DCM (4 mL).
Triethylamine (51 mg, 0.5 mmol) and thiomorpholine (52 mg, 0.5
mmol) were added sequentially. The solution was mixed for several
minutes and allowed to stand for 72 hours. The reaction was diluted
with DCM and H.sub.2O and the layers were separated. The aqueous
layer was extracted with DCM. The combined DCM layers were dried
over MgSO.sub.4, filtered and concentrated. Ethyl ether was added
to the crude sample and the resulting solid was filtered to yield
100 mg of a white solid, 32 (62%). .sup.1HNMR (200 MHz, CDCl.sub.3)
.delta. 1.76 (s, 9H), 2.66 (brs, 2H), 2.79 (brs, 2H), 3.86 (s, 2H),
7.46 (m, 3H), 8.50 (m, 2H).
[0587] Compound 32 was combined with DMSO (3 mL) and
trans-4-aminocyclohexanol (144 mg, 1.25 mmol) and heated to
130.degree. C. for 4 hours. The reaction was cooled to room
temperature, and diluted with EtOAc and H.sub.2O. The layers were
separated and the aqueous layer was extracted with EtOAc
(2.times.). The combined organic layers were washed with H.sub.2O
and brine, dried over MgSO.sub.4, filtered and concentrated.
Chromatography (silica, 8:1 CHCl3/EtOH) yields 32 mg of a tan oil.
Ethyl ether was added and the resulting solid was filtered to yield
5 mg of a white solid (9%). OSIC-148265: .sup.1H-NMR (200 MHz,
CD.sub.3OD): d 1.44 (brm, 4H), 2.03 (brm, 2H), 2.21 (brm, 2H), 2.70
(brm, 8H), 3.63 (m, 4H), 3.92 (m, 1H), 4.26 (brs, 1H), 6.42 (s,
1H), 7.42 (m, 3H), 8.33 (m, 2H).
Example 20B
Synthesis of Adenosine A.sub.1 Antagonist
[0588] Compound 1503 (Table 15 below) can be synthesized by the
general procedures given below. ##STR138##
[0589] The bromide, compound 31 (220 mg, 0.47 mmol) was dissolved
in 1:1 DMF:Dichloromethane (5 mL). To this was added
K.sub.2CO.sub.3 (71 mg, 0.52 mmol) and morpholine (0.047 mL, 0.47
mmol). The mixture was allowed to stir at room temperature
overnight. Solvents were removed in vacuo and the residue was
partitioned between H.sub.2O and dichloromethane. The organic layer
was dried with MgSO.sub.4, filtered, and concentrated to give an
off white solid which upon trituration with ether/hexanes gave 175
mg of a white solid, 33 (84%). .sup.1H-NMR (200 MHz, CDCl.sub.3):
(1.9 (9H, s), 2.54 (4H, s), 3.65 (4H, s), 3.85 (1H, s), 6.59 (1H,
s), 7.45 (3H, m), 8.5 (2H, m).
[0590] Compound 33 (50 mg, 0.11 mmol) and trans-4-aminocyclohexanol
(105 mg, 0.91 mmol) were taken up in DMSO (2 mL). The resultant
solution was sparged with N.sub.2 and then heated to 100.degree. C.
in an oil bath and stirred overnight. The crude reaction mixture
was poured into water and extracted twice with ethyl acetate (50
mL). The combined organic layers were washed with H.sub.2O. After
drying with MgSO and filtering, the organic layer was concentrated
in vacuo to give an orange solid. Chromatography (silica, 10%
CH.sub.3OH in CH.sub.2Cl.sub.2) yielded 15 mg (33%). .sup.1H-NMR
(200 MHz, CDCl.sub.3): (1.24-1.62 (4H, m), 1.85 (2H, m), 2.10 (2H,
m), 2.26 (4H, m), 3.53 (4H, m), 4.22 (1H, m), 4.73 (1H, m), 5.85
(1H, d), 6.15 (1H, s), 7.25 (3H, m), 8.42 (2H, M), 10.0 (1H, s). MS
(ES): 408 (M.sup.++1).
[0591] Compounds 1500, 1501, and 1502 can be synthesized using
similar preparation steps of Example 20 by treating compound 32
with an appropriately substituted amine.
Example 21
Synthesis of
1-[6-(4-Hydroxy-4-phenyl-piperidin-1-yl-methyl)-2-phenyl-7H-pyrrolo[2,3-d-
]pyrimidin-4-yl]-pyrrolidine-2-carboxylic Acid Amide (1601)
[0592] Compound 1601 was synthesized in a manner similar to that of
Example 17 using synthesis scheme IX with L-prolineamide and
4-phenyl-piperidin-4-ol to obtain: ##STR139##
[0593] .sup.1H-NMR (d.sub.6-DMSO) d 1.53 (s, 1H), 1.60 (s, 1H),
1.84-2.30 (m, 6H), 2.66 (m, 2H), 3.60 (s, 2H), 3.88 (m, 1H), 4.02
(m, 1H), 4.66 (d, 1H, J=6.8 Hz), 4.73 (s, 1H), 6.44 (s, 1H), 6.94
(s, 1H), 7.12-7.50 (m, 10H), 8.35 (m, 2H), 11.6 (brs, 1H); MS (ES):
305.1 (M.sup.++1); mp=234-235.degree. C.
Example 22
Synthesis of
[N-(2-Phenyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)(L)-prolinamide
(1602)
[0594] Compound 1602 was synthesized using synthesis scheme VII
with L-prolineamide to obtain: ##STR140##
[0595] .sup.1H-NMR (DMSO-d.sub.6) .delta. 2.05 (m, 4H), 3.85 (m,
1H), 4.05 (m, 1H), 4.70 (d, 1H, J=8.0 Hz), 6.58 (brs, 1H), 6.95
(brs, 1H), 7.15 (d, 1H, J=3.4 Hz), 7.40 (m, 3H), 7.50 (brs, 1H),
8.40 (m, 2H), 11.6 (brs, 1H); MS (ES): 308.3 (M.sup.++1).
mp=236-238.degree. C.
Example 23
Synthesis of
[N-(2-phenyl-6-methoxymethyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-(L)-prolina-
mide (1605)
[0596] Compound 1605 was synthesized using precursor compound 23 of
synthesis scheme IX to obtain: ##STR141##
[0597] Bromide 23 (4.23 g, 10 mmol) is dissolved in anhydrous
methanol (60 mL) and DCM (120 mL) and treated with
AgO.sub.2CCF.sub.3 under N.sub.2 at rt for 1 h. The solid is
removed by filtration and washed with DCM (2.times.20 mL). The
filtrate is concentrated in vacuo. The residue is redissolved in
DCM (80 mL). The resulted solution is then washed with saturated
NaHCO.sub.3 solution and brine, dried over MgSO.sub.4, filtered and
concentrated to give 3.71 g (4, 99%) off white solid. .sup.1H-NMR
(CDCl.sub.3) d 1.75 (s, 9H), 3.51 (s, 3H), 4.83 (s, 2H), 6.70 (s,
1H), 7.47 (m, 3H), 8.52 (m, 2H). ##STR142##
[0598] Aryl chloride 4 (2.448 g, 6.55 mmol), DMSO (15 mL),
L-prolineamide (4.0 g, 35.0 mmol) and NaHCO.sub.3 (2.9 g) are
combined and heated to 120.degree. C. under nitrogen. After 4 h,
the reaction is cooled to room temperature and diluted with water
(60 ml). The resulted slurry is extracted with DCM (10.times.). The
combined organic layers are washed with saturated NaHCO.sub.3
solution and brine, dried over MgSO.sub.4, filtered and
concentrated to give 2.48 g brown solid. Pure product (1.86 g, 81%)
is obtained after flash column as white solid. White crystals are
gotten from THF/hexane. M.p.=213-215.degree. C. .sup.1H-NMR
(CDCl.sub.3) .delta. 2.15 (m, 3H), 2.52 (m, 1H), 3.26 (s, 3H), 3.92
(m, 1H), 4.10 (m, 1H), 4.42 (s, 2H), 5.08 (d, 1H, J=8.2 Hz), 5.49
(brs, 1H), 6.48 (s, 1H), 7.08 (brs, 1H), 7.42 (m, 3H), 8.38 (m,
2H), 9.78 (brs, 1H); MS (ES): 352.2 (M.sup.++1).
Example 24
Synthesis of
4-Hydroxy-1-(2-phenyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl]-pyrrolidine-2-carb-
oxylic Acid Amide (1606)
[0599] Compound 1606 was obtained with synthesis scheme VII using
cis-hydroxy prolineamide to obtain: ##STR143##
[0600] .sup.1H-NMR (d.sub.6-DMSO) d 1.90 (m, 1H), 3.85 (d, 1H,
J=9.2 Hz), 4.08 (m, 1H), 4.37 (s, 1H), 4.67 (dd, 1H, J=8.8, 4.0
Hz), 5.30 (s, 1H), 6.55 (s, 1H), 7.15 (s, 2H), 7.37 (m, 3H), 7.64
(s, 1H), 8.37 (m, 2H), 11.65 (brs, 1H); MS (ES): 324.2 (M.sup.++1);
mp=268-271.degree. C.
Example 25
Synthesis of
3-[4-((S)-2-Carbamoyl-pyrrolodin-1-yl)-2-phenyl-7H-pyrrolo[2,3-d]pyrimidi-
n-6-yl]-propionic Acid (1611)
[0601] Compound 1611 was obtained using precursor compound 23 of
synthesis scheme IX to obtain: ##STR144##
[0602] The tert-butoxycarbonyl protected aryl bromide 23 (4.0 g,
9.5 mmol), dry DMSO (25 ml), NaH.sub.2PO.sub.4 (454 mg, 3.79 mmol)
and Na.sub.2HPO.sub.4 (1.62 g, 11.4 mmol) were combined and heated
to 50.degree. C. under argon for approximately 3.5 h. The mixture
was then poured into water (200 ml) and extracted with three 100 ml
portions of EtOAc. The combined organic layers were thoroughly
washed with water, brine, dried over MgSO.sub.4, filtered and
concentrated to give a yellow solid which was purified by
triturating with ethanol. to give 1.55 g of a pale yellow solid
(7). The mother liquor was purified by flash chromatography (10%
EtOAc in hexane) to give an additional 454 mg (60%). .sup.1H-NMR
(CDCl.sub.3) .delta. 1.77 (s, 9H), 7.25 (s, 1H), 7.48 (m, 3H), 8.52
(m, 2H) 10.39 (s, 1H); m.p.=156.degree. C. (dec). ##STR145##
[0603] Aldehyde 7 (600 mg, 1.7 mmol) was dissolved in dry THF (20
ml) and cooled to 0.degree. C. under argon. To this was added a
0.degree. C. solution of
(tert-butoxycarbonylmethylene)-triphenylphosphorane (694 mg, 1.8
mmol) in 10 ml of dry THF dropwise through a cannula. After 3 h the
mixture was concentrated and purified by triturating with ethanol
to give 565 mg (73%) of a white solid (8). .sup.1HNMR (CDCl.sub.3)
.delta. 1.58 (s, 9H), 1.79 (s, 9H), 6.46 (d, 1H), 6.95 (s, 1H),
7.48 (m, 3H), 8.09 (d, 1H), 8.56 (m, 2H). ##STR146##
[0604] A solution of compound 8 (565 mg 1.2 mmol) in 5 ml THF was
diluted to 100 ml with EtOAc. After adding 600 mg of catalyst (5%
wt Pd, 50% H.sub.2O) and purging with argon, the mixture was
hydrogenated under atmospheric pressure. After 8 h the mixture was
filtered, concentrated and purified with flash chromatography (10%
EtOAc in hexane) to isolate 200 mg (35%) of 9 as a clear oil that
crystallized upon standing. .sup.1HNMR (CDCl.sub.3) .delta. 1.42
(s, 9H), 1.75 (s, 9H), 2.65 (t, 2H), 3.32 (t, 2H), 6.41 (s, 1H)
7.45 (m, 3H), 8.51 (m, 2H). ##STR147##
[0605] Aryl chloride 9 (200 mg, 0.44 mmol), DMSO (10 ml) and
L-prolinamide (440 mg, 4.4 mmol) were combined and heated to
85.degree. C. under argon. After 14 hours the mixture is cooled to
room temperature and partitioned between water and ethyl acetate.
The layers were separated and the aqueous layer washed with EtOAc
(3.times.). The combined organic layers were thoroughly washed with
water (3.times.), brine, dried over MgSO.sub.4, filtered and
concentrated to give 10 as a yellow film which was purified by
flash chromatography (2.5% MeOH in CH.sub.2Cl.sub.2). 185 mg (97%).
MS (ES): 435.8 (M.sup.++1). ##STR148##
[0606] Ester 10 (30 mg, mmol) in 5 ml dioxane was hydrolyzed by
adding 0.5 ml concentrated HCl. After 3 hours the mixture was
concentrated in vacuo and recrystallized in EtOH/EtOAc to obtain
1611 as a white solid (20 mg, 61%). MS (ES): 380 (M.sup.++1).
Example 26
Synthesis of [N-(2-phenyl-6-aminocarbonyl
methoxymethyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-(L)-prolinamide
(1614)
[0607] Compound 1614 was obtained using precursor compound 23 of
synthesis scheme IX to obtain: ##STR149##
[0608] Bromide 23 (1.27 g, 3 mmol) and molecular sieve (5 g) are
stirred in anhydrous methyl glycolate (5.8 g, 60 mmol) and DCM (40
mL). The solution is treated with AgOTf under N.sub.2 and allowed
to stir for 3 h. The solid is removed by filtration and washed with
DCM (2.times.20 mL). The filtrate is concentrated in vacuo. The
residue is redissolved in DCM (80 mL). The resulted solution is
then washed with water, saturated NaHCO.sub.3 solution and brine,
dried over MgSO.sub.4, filtered and concentrated to give 1.35 g
(99%) off white solid (12). .sup.1H-NMR (CDCl.sub.3) .delta. 1.75
(s, 9H), 3.80 (s, 3H), 5.0 (s, 2H), 6.78 (s, 1H), 7.47 (m, 3H),
8.52 (m, 2H). ##STR150##
[0609] Aryl chloride 12 (177 mg, 0.41 mmol), DMSO (10 mL),
L-prolinamide (466 mg, 4 mmol) and NaHCO.sub.3 (500 mg) are
combined and heated to 120.degree. C. under nitrogen. After 4 h,
the reaction is cooled to room temperature and diluted with water
(60 ml). The resulted slurry is extracted with DCM (5.times.30 mL).
The combined organic layers are washed with saturated NaHCO.sub.3
solution and brine, dried over MgSO.sub.4, filtered and
concentrated to give brown solid. Pure product (154 mg, 92%) is
obtained after flash column as white solid (13). .sup.1H-NMR
(CDCl.sub.3) .delta. 2.15 (m, 3H), 2.52 (m, 1H), 3.55 (s, 3H), 4.58
(s, 2H), 5.08 (s, 1H,), 5.85 (brs, 1H), 6.48 (s, 1H), 7.08 (brs,
1H), 7.42 (m, 3H), 8.40 (m, 2H), 10.58 (brs, 1H); MS (ES): 410.1
(M.sup.++1). ##STR151##
[0610] Methyl ester 13 (124 mg, 0.3 mmol) is dissolved in
HOCH.sub.3 (15 mL) Ammonia is bubbled through the solution for 0.5
h. The reaction mixture is then stirred for another 3 h at rt.
After removal of solvent 111 mg of a white solid (1614, 93%) is
obtained. .sup.1H-NMR (CDCl.sub.3) .delta. 1.82 (m, 3H), 2.20 (m,
1H), 2.80 (m, 1H), 3.10 (m, 1H), 3.63 (dd, 2H, J.sub.1=13.8 Hz,
J.sub.2=19.4 Hz), 3.87 (m, 1H), 4.07 (m, 1H), 4.97 (m, 1H), 5.96
(m, 2H), 6.35 (s, 1H), 6.86 (brs, 1H), 7.11 (brs, 1H), 7.37 (m,
3H), 8.28 (m, 2H), 11.46 (brs, 1H); MS (ES): 394.8 (M.sup.++1).
Example 27
Synthesis of
[4-(2-Carbamoylpyrrolidin-1-yl)-2-phenyl-7H-pyrrolo[2,3-d]pyrimidine-6-ca-
rboxylic Acid] (1619)
[0611] Compound 1619 was synthesized using precursor compound 15 of
synthesis scheme VII to obtain: ##STR152##
[0612] To a suspension of sodium hydride (780 mg of a 60% oil
suspension, 19.5 mmol) in dry DMF (20 mL), cooled by an ice/water
bath, under nitrogen, is added a solution of the pyrrolopyrimidine
15 (2.00 g, 7.52 mmol) in DMF (10 mL) over 5 min. After 15 min,
benzenesulfonyl chloride (1.2 mL, 9.40 mmol) is added, then the
cooling bath is removed. After 4 h, the reaction mixture is poured
into a mixture of ice and sat. NaHCO.sub.3 sol., the precipitated
solid is filtered off and triturated with acetone (3) and methanol
(2), yielding 2.37 g of a beige solid. This solid (16) contains
approx. 10 mol-% DMF (based on that 83% yield) and can be used in
the next step; a pure sample can be obtained by chromatography on
silica gel using acetone as eluent. .sup.1H-NMR (CDCl.sub.3):
.delta. 6.70 (d, J=4.2 Hz, 1H), 7.47-7.68 (m, 6H), 7.76 (d, J=4.2
Hz, 1H), 8.24-8.32 (m, 2H), 8.48-8.56 (m, 2H); IR (solid): n=3146
cm.sup.-1, 1585, 1539, 1506, 1450, 1417, 1386, 1370, 1186, 1176,
1154, 1111, 1015, 919, 726, 683, 616, 607; MS (ES): 372/370
(MH.sup.+); mp=226-227.degree. C. ##STR153##
[0613] To a solution of the N-sulfonyl compound 16 (337 mg, 0.911
mmol) in dry THF (34 mL), cooled by dry ice/acetone, is added
LDA.THF (1.0 mL, 1.5M solution in cyclohexane, 1.5 mmol). After 45
min, carbon dioxide is bubbled into the solution for 5 min, then
the cooling bath is removed. When the solution has reached ambient
temp., the solvents are evaporated, yielding 398 mg of the salt 17,
containing 0.5 equiv. of (iPr).sub.2NCO.sub.2Li, as yellow solid.
The salt is used without purification in the next step. .sup.1H-NMR
(D.sub.6-DMSO): d=6.44 (s, 1H), 7.50-7.75 (m, 6H), 8.33-8.40 (m,
2H), 8.53 (dd, J=8.0, 1.6 Hz, 2H). ##STR154##
[0614] A solution of the lithium salt 17 (50 mg) and L-prolinamide
(122 mg, 1.07 mmol) in DMSO (1.5 mL) is heated under nitrogen to
80.degree. C. for 15.5 h. 4% aq. acetic acid (10 mL) is added to
the cooled solution, and the mixture is extracted with EtOAc (5'10
mL). The combined organic layers are washed with 4% aq. acetic acid
(10 mL), water (10 mL) and brine (10 mL) and are dried over MgSO4.
Filtration and concentration gives 40 mg of 18 as a yellowish
solid, which is used without purification in the next step.
.sup.1H-NMR (CD.sub.3OD): d=1.95-2.36 (m, 4H), 3.85-3.95 (m, 1H),
3.95-4.17 (m, 1H), 4.72 (brs, 1H), 7.14 (s, 1H), 7.35-7.45 (m, 3H),
7.45-7.70 (m, 3H), 8.33-8.50 (m, 4H). ##STR155##
[0615] A solution of sodium hydroxide in methanol (1.5 mL, 5M, 7.5
mmol) is added to a solution of the pyrrolopyrimidine 18 (40 mg,
0.081 mmol) in methanol (2 mL). After 2 h, the pH is adjusted to 5,
most of the methanol is evaporated, the mixture is extracted with
EtOAc (5 10 mL), the combined organic layers are washed with brine
and dried over MgSO.sub.4. Filtration and concentration yields 24
mg of a pale yellow solid, which is triturated with
toluene/EtOAc/MeOH to yield 15.6 mg (55%) of the acid 1619 as
slightly yellowish solid. .sup.1H-NMR (CD.sub.3OD): d=2.05-2.20 (m,
4H), 3.95-4.10 (m, 1H), 4.15-4.25 (m, 1H), 4.85 (brs, 1H), 7.14 (s,
1H), 7.35-7.42 (m, 3H), 8.38-8.45 (m, 2H); IR (solid): n=3192
cm.sup.-1, 2964, 2923, 2877, 1682, 1614, 1567, 1531, 1454, 1374,
1352, 1295, 1262, 1190, 974, 754, 700; MS (ES): 352 (M.sup.++1);
m.p.=220.degree. C. (decomp.).
Example 28
Synthesis of
1-(6-methyl-2-phenyl-7H-pyrrolo[2,3-d]pyrimidine-4-yl)-(S)-pyrrolidine-2--
carboxylic Acid Amide (1621)
[0616] Compound 1621 was synthesized by the following steps:
##STR156##
[0617] Aryl chloride 20 (3 g, 10.7 mmol), DMSO (50 ml) and
(S)-prolinamide were combined and heated to 85.degree. C. under
argon. After stirring overnight (14 hrs), the mixture was cooled to
room temperature and poured into 800 ml of water. This was
extracted with three 200 ml portions of EtOAc. The combined organic
layers were thoroughly washed with water (3.times.300 ml), brine,
dried over MgSO.sub.4, filtered and concentrated to give a dark
brown solid. The solid was recrystallized twice from EtOAc to yield
1.95 g (57%) of a tan solid (1621). .sup.1HNMR (DMSO-d.sub.6) d
1.8-2.2 (m, 4H), 2.3 (s, 3H), 3.8 (m, 1H), 4.0 (m, 1H), 4.6 (d, 1H)
6.2 (s, 1H), 6.9 (s, 1H), 7.2 (m, 3H), 7.3 (s, 1H), 8.4 (m, 2H),
11.5 (s, 1H); MS (ES): 322 (M.sup.++1)
Example 29
Synthesis of 1-[6-(2-Hydroxy-ethoxymethyl)
-2-phenyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl]-pyrrolidine-2-carboxylic
Acid Amide (1623)
[0618] Compound 1623 was synthesized in a manner similar to that of
Example 17 using synthesis scheme IX with L-prolineamide and
ethane-1,2-diol to obtain: ##STR157##
[0619] MS (ES): 382 (M.sup.++1).
Example 30
Synthesis of
4-(6-Imidazol-1-ylmethyl-2-phenyl-7H-pyrrolo[2,3-d]pyrimidin-4-ylamino)-c-
yclohexanol (1624)
[0620] Compound 1624 was synthesized in a manner similar to that of
Example 17 using synthesis scheme IX with N-6 amino cyclohexanol
and imidazole to obtain: ##STR158##
[0621] MS (ES): 389 (M.sup.++1)
Example 31
Synthesis of
4-(4-Hydroxy-cyclohexylamino)-2-phenyl-7H-pyrrolo[2,3-d]pyrimidine-6-carb-
oxylic Acid (1625)
[0622] Compound 1625 was synthesized in a manner similar to that of
Example 27 using synthesis scheme IX with N-6 amino cyclohexanol to
obtain: ##STR159##
[0623] MS (ES): 353 (M.sup.++1)
Example 32
Synthesis of
4-[6-(2-Hydroxy-ethoxymethyl)-2-phenyl-7H-pyrrolo[2,3-d]pyrimidin-4-ylami-
no]-cyclohexano 1 (1626)
[0624] Compound 1626 was synthesized in a manner similar to that of
Compound 1623 using synthesis scheme IX with N-6 amino cyclohexanol
to obtain: ##STR160##
[0625] MS (ES): 383 (M.sup.++1)
Example 33
Synthesis of
4-(4-Hydroxy-cyclohexylamino)-2-phenyl-7H-pyrrolo[2,3-d]pyrimidine-6-carb-
oxylic Acid Methyl Ester (1627)
[0626] ##STR161##
[0627] A solution of the lithium salt 17 (0.13 mmol) in dry DMF (4
mL) is stirred with methyl iodide (0.1 mL, 1.6 mmol) at 20.degree.
C. under argon for 3 h. DMF is evaporated, and aqueous ammonium
chloride solution is added (15 mL). The mixture is extracted with
EtOAc (3'15 mL), the combined organic layers are washed with water
(2'10 mL) and brine (10 mL) and are dried over MgSO.sub.4.
Filtration and concentration gives 21 mg (38%) of the methyl ester
22. ##STR162##
[0628] A solution of the methyl ester 22 (24.5 mg, 0.057 mmol) and
4-trans-aminocyclohexanol (66 mg, 0.57 mmol) in DMSO (1.5 mL) is
heated under nitrogen to 80.degree. C. for 5 h, then the heating is
stopped, and stirring at 20.degree. C. is continued for 13.5 h. 4%
aq. acetic acid (10 mL) is added to the cooled solution, and the
mixture is extracted with EtOAc (3'10 mL). The combined organic
layers are washed with 4% aq. acetic acid (10 mL), water (10 mL) 2N
NaOH (10 mL), water (10 mL), and brine (10 mL) and are dried over
MgSO.sub.4. To a solution of the crude material obtained after
filtration and concentration (1H NMR indicates about 50% removal of
the benzenesulfonyl group) in THF (2 mL) is added a solution of
NaOH in MeOH (0.5 mL of 5M solution, 2.5 mmol) at ambient
temperature. After 20 min, water and sat. NaHCO.sub.3 solution (5
mL each) are added, and the mixture is extracted with EtOAc (4'15
mL). The combined organic layers are washed with 2N NaOH (10 mL),
water (10 mL), and brine (10 mL) and are dried over MgSO.sub.4.
Chromatography of the crude material obtained after filtration and
concentration on silica gel, eluting with hexanes/EtOAc 1:1.RTM.1:2
yields 8.6 mg (41%) of 1627 as a white solid, mp. 225-227.degree.
C. .sup.1H-NMR (CD.sub.3OD): d=1.38-1.62 (m, 4H), 1.95-2.10 (m,
2H), 2.10-2.25 (m, 2H), 3.55-3.70 (m, 1H), 3.91 (s, 3H), 4.20-4.35
(m, 1H), 7.32 (s, 1H), 7.35-7.47 (m, 3H), 8.35-8.42 (m, 2H); IR
(solid): n=3352 cm.sup.-1, 3064, 2935, 2860, 1701, 1605, 1588,
1574, 1534, 1447, 1386, 1333, 1263, 1206, 1164, 1074, 938, 756,
705; MS (ES): 367 (MH.sup.+).
Example 34
Synthesis of
[4-(2-Carbamoyl-pyrrolidin-1-yl)-2-phenyl-7H-pyrrolo[2,3-d]pyrimidin-6-yl-
methoxy]-acetic Acid Methyl Ester (1628)
[0629] Compound 1628 was synthesized in a manner similar to example
26 using precursor compound 12 to obtain: ##STR163##
[0630] MS (ES): 410 (M.sup.++1)
Example 35
Synthesis of
[4-(2-Carbamoyl-pyrrolidin-1-yl)-2-phenyl-7H-pyrrolo[2,3-d]pyrimidin-6-yl-
methoxy]-acetic Acid (1629)
[0631] Compound 1629 was synthesized in a manner similar to
compound 1628 wherein the methyl ester group was hydrolized with a
base to obtain: ##STR164##
[0632] MS (ES): 396 (M.sup.++1)
Example 36
Synthesis of
4-(4-Hydroxy-cyclohexylamino)-2-phenyl-7H-pyrrolo[2,3-d]pyrimidine-6-carb-
oxylic Acid Amide (1630)
[0633] ##STR165##
[0634] Gaseous ammonia is condensed into a solution of the
pyrrolopyrimidine 23 (7.8 mg, 0.021 mmol) in methanol (6 mL),
cooled by dry ice/acetone, until a total volume of 12 mL is
reached. After stirring for 10d at 20.degree. C., the solvents are
evaporated, and the residue is purified by preparative TLC on
silica gel, eluting with 5% MeOH in CH.sub.2Cl.sub.2. The material
thus obtained is triturated with ether to yield 6.5 mg (88%) of the
amide 1630 as white solid, mp. 210-220.degree. C. (decomp.).
.sup.1H-NMR (CD.sub.3OD): .delta.=1.40-1.60 (m, 4H), 2.00-2.15 (m,
2H), 2.15-2.25 (m, 2H), 3.55-3.70 (m, 1H), 4.20-4.35 (m, 1H), 7.16
(s, 1H), 7.35-7.47 (m, 3H), 8.34-8.40 (m, 2H); IR (solid): n=3358
cm.sup.-1, 3064, 3025, 2964, 2924, 2853, 1652, 1593, 1539, 1493,
1452, 1374, 1326, 1251, 1197, 1113, 1074, 1028, 751, 699; MS (ES):
352 (MH.sup.+).
Activity of Compounds
[0635] Adenosine 1 (A.sub.1) receptor subtype saturation and
competition radio ligand binding were carried out for compounds
1601, 1602, 1605, 1606, 1611, 1614, 1619, 1621, 1623, 1624, 1625,
1626, 1627, 1628, 1629, 1630 and 1631 as described herein and inter
alia, on pages 192-193 of this specification. All of the
above-referenced compounds equaled or surpassed the A.sub.1
receptor binding affinity of reference compounds 1318 or 1319 as
described herein and, inter alia, in Table 13, on pages 209-212 of
the specification.
[0636] The water solubilities of the above compounds listed in
Table 1 are expected to be better than reference compounds 1318 or
1319 due to their cLogP values, which were calculated using the
computer program CS ChemDraw, ChemDraw Ultra ver. 6.0 .COPYRGT.1999
as provided by CambridgeSoft Corporation, 100 Cambridge Park Drive,
Cambridge, Mass. 02140.
[0637] The compounds specific to the A.sub.1 receptor listed in
Table 1 had lower cLogP values, between about 1.5 to about 3.4, as
compared to reference compounds 1318 or 1319 with a cLogP value
about 3.8. It was not predicted that the more polar A.sub.1
receptor compounds listed in Table 1 having lower cLogP values than
the reference compounds 1318 or 1319 would still retain the potency
and A.sub.1 receptor binding selectivity as compared to those
reference compounds. TABLE-US-00003 TABLE 1 Compound cLogP 1601 4.1
1602 3.0 1605 2.88 1606 2.1 1611 2.9 1614 1.5 1619 2.7 1621 3.37
1623 2.4 1624 2.8 1625 3.1 1626 2.8 1627 3.4 1628 2.4 1629 2.2 1630
2.4 1631 2.05
[0638] Yeast .beta.-Galactosidase reporter gene assays for human
adenosine A.sub.1 and A.sub.2a receptor: Yeast strains (S.
cerevisiae) were transformed with human adenosine A.sub.1
(A.sub.1R; CADUS strain CY12660) or human A.sub.2a (A.sub.2a; CADUS
strain CY8362) and the addition of a lacZ(.beta.-Galactosidase)
reporter gene to utilize as a functional readout. A complete
description of the transformations is listed below (see Yeast
Strains). NECA (5'-N-ethylcarboxamidoadenosine), a potent adenosine
receptor agonist with similar affinity for A1 and A.sub.2a
receptors, was used as a ligand for all assays. Test compounds were
examined at 8 concentrations (0.1-10,000 nM) for ability to inhibit
NECA-induced .beta.-Galactosidase activity by CY12660 or
CY8362.
[0639] Preparation of Yeast Stock Cultures: Each of the Respective
yeast strains, CY12660 and CY8362, were streaked onto an LT agar
plate and incubated at 30.degree. C. until colonies were observed.
Yeast from these colonies were added to LT liquid (pH 6.8) and
grown overnight at 30.degree. C. Each yeast strain was then diluted
to an OD.sub.600=1.0-2.0 (approximately 1-2.times.10.sup.7
cells/ml), as determined spectrophotometrically (Molecular Devices
VMAX). For each 6 ml of yeast liquid culture, 4 ml of 40% glycerol
(1.5:1 vol:vol) was added ("yeast/glycerol stock"). From this
yeast/glycerol stock, ten 1 ml aliquots were prepared and stored at
-80.degree. C. until required for assay.
[0640] Yeast A.sub.1R and A.sub.2aR Assay: One vial each of CY8362
and CY12660 yeast/glycerol stock was thawed and used to inoculate
Supplemented LT liquid media, pH 6.8 (92 ml LT liquid, to which is
added: 5 ml of 40% glucose, 0.45 ml of 1M KOH and 2.5 ml of Pipes,
pH 6.8). Liquid cultures were grown 16-18 hr (overnight) at
30.degree. C. Aliquots from overnight cultures were then diluted in
LT media, containing 4 U/ml adenosine deaminase (Type VI or VII
from calf intestinal mucosa, Sigma), to obtain OD.sub.600=0.15
(1.5.times.10.sup.6 cells/ml) for CY8362 (A2aR) and OD.sub.600=0.50
(5.times.10.sup.6 cells/ml) for CY12660 (A.sub.1R).
[0641] Assays were conducted with a final volume of 100 ul in
96-well microtiter plates, such that a final concentration of 2%
DMSO was achieved in all wells. For primary screening, 1-2
concentrations of test compounds were utilized (10 uM, 1 .mu.M).
For compound profiling, 8 concentrations were tested (10000, 1000,
500, 100, 50, 10, 1 and 0.1 nM). To each microtiter plate, 10 ul of
20% DMSO was added to "Control" and "Total" wells while 10 ul of
Test Compound (in 20% DMSO) was added to "Unknown" wells.
Subsequently, 10 ul of NECA (5 uM for A.sub.1R, 1 uM for A.sub.2aR)
were added to "Total" and "Unknown" wells; 10 ul of PBS was added
to the "Control" wells. In the final addition, 80 ul of yeast
strain, CY8362 or CY12660, were added to all wells. All plates were
then agitated briefly (LabLine orbital shaker 2-3 min) and allowed
to incubate for 4 hrs. at 30.degree. C. in a dry oven.
[0642] .beta.-Galactosidase activity can be quantitated using
either calorimetric (e.g., ONPG, CPRG), luminescent (e.g.,
Galacton-Star) or fluorometric substrates (e.g., FDG, Resorufin)
substrates. Currently, fluorescence detection is preferred on the
basis of superior signal:noise ratio, relative freedom from
interference and low cost. Fluorescein digalactopyranoside (FDG,
Molecular Probes or Marker Gene Technologies), a fluorescent
.beta.-Galactosidase substrate, was added to all wells at 20
ul/well (final concentration=80 uM). Plates were shaken for 5-6 sec
(LabLine orbital shaker) and then incubated at 37.degree. C. for 90
min (95% O.sub.2/5% CO.sub.2 incubator). At the end of the 90 min
incubation period, .beta.-Galactosidase activity was stopped using
20 ul/well of 1M Na.sub.2CO.sub.3 and all plates shaken for 5-6
sec. Plates were then agitated for 6 sec and relative fluorescence
intensity determined using a fluorometer (Tecan Spectrafluor;
excitation=485 nm, emission=535 nm).
[0643] Calculations: Relative fluorescence values for "Control"
wells were interpreted as background and subtracted from "Total"
and "Unknown" values. Compound profiles were analyzed via
logarithmic transformation (x-axis: compound concentration)
followed by one site competition curve fitting to calculate
IC.sub.50 values (GraphPad Prism).
[0644] Yeast strains: Saccharomyces cerevisiae strains CY12660
[far1*1442 tbt1-1 fus1-HIS3 can1 ste14::trp1::LYS2 ste3*1156 gpa1
(41)-G.alpha.i3 lys2 ura3 leu2 trp1: his3; LEU2
PGKp-Mf.alpha.1Leader-hA1R-PHO5term 2mu-orig REP3 Ampr] and CY8362
[gpa1p-rG.alpha.sE10K far1*1442 tbt1-1 fus1-HIS3 can1
ste14::trp1:LYS2 ste3*1156 lys2 ura3 leu2 trp1 his3; LEU2
PGKp-hA2aR 2mu-ori REP3 Ampr] were developed.
[0645] LT Media: LT (Leu-Trp supplemented) media is composed of 100
g DIFCO yeast nitrogen base, supplemented with the following: 1.0 g
valine, 1.0 g aspartic acid, 0.75 g phenylalanine, 0.9 g lysine,
0.45 g tyrosine, 0.45 g isoleucine, 0.3 g methionine, 0.6 g
adenine, 0.4 g uracil, 0.3 g serine, 0.3 g proline, 0.3 g cysteine,
0.3 g arginine, 0.9 g histidine and 1.0 g threonine.
Construction of Yeast Strains Expressing Human A.sub.1 Adenosine
Receptor
[0646] In this example, the construction of yeast strains
expressing a human A.sub.1 adenosine receptor functionally
integrated into the yeast pheromone system pathway is
described.
I. Expression Vector Construction
[0647] To construct a yeast expression vector for the human A.sub.1
adenosine receptor, the A.sub.1 adenosine receptor cDNA was
obtained by reverse transcriptase PCR of human hippocampus mRNA
using primers designed based on the published sequence of the human
A.sub.1 adenosine receptor and standard techniques. The PCR product
was subcloned into the NcoI and XbaI sites of the yeast expression
plasmid pMP15.
[0648] The pMP15 plasmid was created from pLPXt as follows: The
XbaI site of YEP51 (Broach, J. R. et al. (1983) "Vectors for
high-level, inducible expression of cloned genes in yeast" p.
83-117 in M. Inouye (ed.), Experimental Manipulation of Gene
Expression. Academic Press, New York) was eliminated by digestion,
end-fill and religation to create Yep51NcoDXba. Another XbaI site
was created at the BamHI site by digestion with BamHI, end-fill,
linker (New England Biolabs, # 1081) ligation, XbaI digestion and
re-ligation to generate YEP51NcoXt. This plasmid was digested with
Esp31 and NcoI and ligated to Leu2 and PGKp fragments generated by
PCR. The 2 kb Leu2 PCR product was generated by amplification from
YEP51Nco using primers containing Esp31 and BglII sites. The 660
base pair PGKp PCR product was generated by amplification from
pPGK.alpha.s (Kang, Y.-S. et al. (1990) Mol. Cell. Biol.
10:2582-2590) with PCR primers containing BglII and NcoI sites. The
resulting plasmid is called pLPXt. pLPXt was modified by inserting
the coding region of the a-factor pre-pro leader into the NcoI
site. The prepro leader was inserted so that the NcoI cloning site
was maintained at the 3' end of the leader, but not regenerated at
the 5' end. In this way receptors can be cloned by digestion of the
plasmid with NcoI and XbaI. The resulting plasmid is called
pMP15.
[0649] The pMP15 plasmid into which was inserted the human A.sub.1
adenosine receptor cDNA was designated p5095. In this vector, the
receptor cDNA is fused to the 3' end of the yeast a-factor prepro
leader. During protein maturation the prepro peptide sequences are
cleaved to generate mature full-length receptor. This occurs during
processing of the receptor through the yeast secretory pathway.
This plasmid is maintained by Leu selection (i.e., growth on medium
lacking leucine). The sequence of the cloned coding region was
determined and found to be equivalent to that in the published
literature (GenBank accession numbers S45235 and S56143).
II. Yeast Strain Construction
[0650] To create a yeast strain expressing the human A.sub.1
adenosine receptor, yeast strain CY7967 was used as the starting
parental strain. The genotype of CY7967 is as follows:
[0651] MAT.alpha. gpaD1163 gpa1(41)Gai3 far1D1442 tbt-1 FUS1-HIS3
can1 ste14::trp1::LYS2 ste3D1156 lys2 ura3 leu2 trp1 his3
[0652] The genetic markers are reviewed below: TABLE-US-00004 TABLE
2 MATa Mating type a. gpa1D1163 The endogenous yeast G-protein GPA1
has been deleted. gpa1(41)G.alpha.i3 gpa1(41)-Gai3 was integrated
into the yeast genome. This chimeric Ga protein is composed of the
first 41 amino acids of the endogenous yeast Ga subunit GPA1 fused
to the mammalian G-protein Gai3 in which the cognate N-terminal
amino acids have been deleted. far1D1442 FAR1 gene (responsible for
cell cycle arrest) has been deleted (thereby preventing cell cycle
arrest upon activation of the pheromone response pathway). tbt-1
strain with high transformation efficiency by electroporation.
FUS1-HIS3 a fusion between the FUS1 promoter and the HIS3 coding
region (thereby creating a pheromone inducible HIS3 gene). can 1
arginine/canavinine permease. ste14::trp1::LYS2 gene disruption of
STE14, a C-farnesyl methyltransferase (thereby lowering basal
signaling through the pheromone pathway). ste3D1156 endogenous
yeast STR, the a factor pheromone receptor (STE3) was disrupted.
lys2 defect in 2-aminoapidate reductase, yeast need lysine to grow.
ura3 defect in orotidine-5'-phosphate decarboxylase, yeast need
uracil to grow leu2 defect in b-isopropylmalate dehydrogenase,
yeast need leucine to grow. trp1 defect in
phosphoribosylanthranilate, yeast need tryptophan to grow. his3
defect in imidazoleglycerolphosphate dehydrogenase, yeast need
histidine to grow.
[0653] Two plasmids were transformed into strain CY7967 by
electroporation: plasmid p5095 (encoding human A.sub.1 adenosine
receptor; described above) and plasmid p1584, which is a
FUS1-.beta.-galactosidase reporter gene plasmid. Plasmid p1584 was
derived from plasmid pRS426 (Christianson, T. W. et al. (1992) Gene
110:119-1122). Plasmid pRS426 contains a polylinker site at
nucleotides 2004-2016. A fusion between the FUS1 promoter and the
.beta.-galactosidase gene was inserted at the restriction sites
EagI and XhoI to create plasmid p1584. The p1584 plasmid is
maintained by Trp selection (i.e., growth on medium lacking
leucine).
[0654] The resultant strain carrying p5095 and p1584, referred to
as CY12660, expresses the human A.sub.1 adenosine receptor. To grow
this strain in liquid or on agar plates, minimal media lacking
leucine and tryptophan was used. To perform a growth assay on
plates (assaying FUS1-HIS3), the plates were at pH 6.8 and
contained 0.5-2.5 mM 3-amino-1,2,4-triazole and lacked leucine,
tryptophan and histidine. As a control for specificity, a
comparison with one or more other yeast-based seven transmembrane
receptor screens was included in all experiments.
Construction of Yeast Strains Expressing Human A2a Adenosine
Receptor
[0655] In this example, the construction of yeast strains
expressing a human A.sub.2a adenosine receptor functionally
integrated into the yeast pheromone system pathway is
described.
I. Expression Vector Construction
[0656] To construct a yeast expression vector for the human A2a
adenosine receptor, the human A2a receptor cDNA was obtained from
Dr. Phil Murphy (NIH). Upon receipt of this clone, the A2a receptor
insert was sequenced and found to be identical to the published
sequence (GenBank accession # S46950). The receptor cDNA was
excised from the plasmid by PCR with VENT polymerase and cloned
into the plasmid pLPBX, which drives receptor expression by a
constitutive Phosphoglycerate Kinase (PGK) promoter in yeast. The
sequence of the entire insert was once again sequenced and found to
be identical with the published sequence. However, by virtue of the
cloning strategy employed there were three amino acids appended to
the carboxy-terminus of the receptor, GlySerVal.
II. Yeast Strain Construction
[0657] To create a yeast strain expressing the human A2a adenosine
receptor, yeast strain CY8342 was used as the starting parental
strain. The genotype of CY8342 is as follows: MATa far1D1442 tbt1-1
lys2 ura3 leu2 trp1 his3 fus1-HIS3 can1 ste3D1156 gpaD1163
ste14::trp1::LYS2 gpa1p-rG.sub..alpha.sE10K (or
gpa1p-rG.sub..alpha.sD229S or gpa1p-rG.sub..alpha.sE10K+D229S)
[0658] The genetic markers are as described above, except for the
G-protein variation. For human A2a receptor-expression, yeast
strains were utilized in which the endogenous yeast. G protein GPA1
had been deleted and replaced by a mammalian G.sub..alpha.s. Three
rat G.sub..alpha.s mutants were utilized. These variants contain
one or two point mutations which convert them into proteins which
couple efficiently to yeast .beta..gamma.. They are identified as
G.sub..alpha.sE10K (in which the glutamic acid at position ten is
replaced with lysine), G.sub..alpha.sD229S (in which the aspartic
acid at position 229 is replaced with serine) and
G.sub..alpha.sE10K+D229S (which contains both point mutations).
[0659] Strain CY8342 (carrying one of the three mutant rat
G.sub..alpha.s proteins) was transformed with either the parental
vector pLPBX (Receptor.sup.-) or with pLPBX-A2a (Receptor.sup.+). A
plasmid with the FUS1 promoter fused to .beta.-galactosidase coding
sequences (described in above) was added to assess the magnitude of
activation of the pheromone response pathway.
Functional Assay Using Yeast Strains Expressing Human A.sub.1
Adenosine Receptor
[0660] In this example, the development of a functional screening
assay in yeast for modulators of the human A.sub.1 adenosine
receptor is described.
I. Ligands Used in Assay
[0661] Adenosine, a natural agonist for this receptor, as well as
two other synthetic agonists were utilized for development of this
assay. Adenosine, reported to have an EC.sub.50 of approximately 75
nM, and (-)--N6-(2-phenylisopropyl)-adenosine (PIA) with a reported
affinity of approximately 50 nM were used in a subset of
experiments. 5'-N-ethylcarboxamido-adenosine (NECA) was used in all
growth assays. To prevent signaling due to the presence of
adenosine in the growth media, adenosine deaminase (4 U/ml) was
added to all assays.
II. Biological Response in Yeast
[0662] The ability of the A.sub.1 adenosine receptor to
functionally couple in a heterologous yeast system was assessed by
introducing the A.sub.1 receptor expression vector (p5095,
described above) into a series of yeast strains that expressed
different G protein subunits. The majority of these transformants
expressed G.sub..alpha. subunits of the G.sub..alpha.i or
G.sub..alpha.o subtype. Additional G.sub..alpha. proteins were also
tested for the possible identification of promiscuous
receptor-G.alpha. protein coupling. In various strains, a STE18 or
a chimeric STE18-G.gamma.2 construct was integrated into the genome
of the yeast. The yeast strains harbored a defective HIS3 gene and
an integrated copy of FUS1-HIS3, thereby allowing for selection in
selective media containing 3-amino-1,2,4-triazole (tested at 0.2,
0.5 and 1.0 mM) and lacking histidine. Transformants were isolated
and monolayers were prepared on media containing
3-amino-1,2,4-triazole, 4 U/ml adenosine deaminase and lacking
histidine. Five microliters of various concentrations of ligand
(e.g., NECA at 0, 0.1, 1.0 and 10 mM) was applied. Growth was
monitored for 2 days. Ligand-dependent growth responses were tested
in this manner in the various yeast strains. The results are
summarized in Table 3 below. The symbol (-) indicates that
ligand-dependent receptor activation was not detected while (+)
denotes ligand-dependent response. The term "LIRMA" indicates
ligand independent receptor mediated activation. TABLE-US-00005
TABLE 3 Yeast G.gamma. Strain strain G.alpha. subunit subunit
Variants Result CY1316 GPA.sub.1 STE18 - GPA41-G.sub..alpha.i1 +
GPA41-G.sub..alpha.i2 + GPA41-G.sub..alpha.i3 +
GPA41-G.sub..alpha.i2-G.sub..alpha.OB LIRMA
GPA41-G.sub..alpha.SE10K - GPA41-G.sub..alpha.SD229S - CY7967
GPA41-G.sub..alpha.i3- STE18 +++ integrated CY2120 GPA.sub.1 STE18
sst2.DELTA. + GPA41-G.sub..alpha.i1 + GPA41-G.sub..alpha.i2 +
GPA41-G.sub..alpha.i3 + GPA41-G.sub..alpha.i2-G.sub..alpha.OB LIRMA
GPA41-G.sub..alpha.SE10K - GPA41-G.sub..alpha.SD229S - CY9438
GPA.sub.1 STE18-G.gamma.2 - GPA41-G.sub..alpha.i1 +
GPA41-G.sub..alpha.i2 + GPA41-G.sub..alpha.i3 +
GPA41-G.sub..alpha.i2-G.sub..alpha.OB LIRMA
GPA41-G.sub..alpha.SE10K - GPA41-G.sub..alpha.SD229S - CY10560
GPA.sub.1-integrated STE18-G.gamma.2 sst2.DELTA. ++
[0663] As indicated in Table 3, the most robust signaling was found
to occur in a yeast strain expressing the
GPA.sub.1(41)-G.sub..alpha.i3 chimera.
III. fus1-LacZ Assay
[0664] To characterize activation of the pheromone response pathway
more fully, synthesis of .beta.-galactosidase through fus1LacZ in
response to agonist stimulation was measured. To perform the
.beta.-galactosidase assay, increasing concentrations of ligand
were added to mid-log culture of human A.sub.1 adenosine receptor
expressed in a yeast strain co-expressing a Ste18-G.gamma.2 chimera
and GPA.sub.41-G.sub..alpha.i3. Transformants were isolated and
grown overnight in the presence of histidine and 4 U/ml adenosine
deaminase. After five hours of incubation with 4 U/ml adenosine
deaminase and ligand, induction of .beta.-galactosidase was
measured using CPRG as the substrate for .beta.-galactosidase.
5.times.10.sup.5 cells were used per assay.
[0665] The results obtained with NECA stimulation indicated that at
a NECA concentration of 10.sup.-8 M approximately 2-fold
stimulation of .beta.-galactosidase activity was achieved.
Moreover, a stimulation index of approximately 10-fold was observed
at a NECA concentration of 10.sup.-5 M.
[0666] The utility of this assay was extended by validation of the
activity of antagonists on this strain. Two known adenosine
antagonist, XAC and DPCPX, were tested for their ability to compete
against NECA (at 5 mM) for activity in the .beta.-galactosidase
assay. In these assays, .beta.-galactosidase induction was measured
using FDG as the substrate and 1.6.times.10.sup.5 cells per assay.
The results indicated that both XAC and DPCPX served as potent
antagonists of yeast-expressed A.sub.1 adenosine receptor, with
IC.sub.50 values of 44 nM and 49 nM, respectively.
[0667] In order to determine if this inhibitory effect was specific
to the A.sub.1 subtype, a series of complementary experiments were
performed with the yeast-based A.sub.2a receptor assay. Results
obtained with the A.sub.2a yeast-based assay indicated that XAC was
a relatively effective A.sub.2a receptor antagonist, consistent
with published reports. In contrast, DPCPX was relatively inert at
this receptor, as expected from published reports.
IV. Radioligand Binding
[0668] The A.sub.1 adenosine receptor assay was further
characterized by measurement of the receptor's radioligand binding
parameters. Displacement binding of [.sup.3H]CPX by several
adenosine receptor reference compounds, XAC, DPCPX, and CGS, was
analyzed using membranes prepared from yeast expressing the human
A.sub.1 adenosine receptor. The results with yeast membranes
expressing the human A.sub.1 adenosine receptor were compared to
those from yeast membranes expressing the human A2a adenosine
receptor or the human A3 receptor to examine the specificity of
binding. To perform the assay, fifty mg of membranes were incubated
with 0.4 nM [.sup.3H]CPX and increasing concentrations of adenosine
receptor ligands. Incubation was in 50 mM Tris-HCl, pH 7.4, 1 mM
EDTA, 10 mM MgCl.sub.2, 0.25% BSA and 2 U/ml adenosine deaminase in
the presence of protease inhibitors for 60 minutes at room
temperature. Binding was terminated by addition of ice-cold 50 mM
Tris-HCl, pH 7.4 plus 10 mM MgCl.sub.2, followed by rapid
filtration over GF/B filters previously soaked with 0.5%
polyethyenimine, using a Packard 96-well harvester. Data were
analyzed by nonlinear least square curve fitting procedure using
Prism 2.01 software. The IC.sub.50 values obtained in this
experiment are summarized in Table 4, below: TABLE-US-00006 TABLE 4
IC.sub.50 [nM] Compound hA1R hA2aR hA3R XAC 6.6 11.7 53.1 DPCPX 8.5
326.4 1307.0 CGS-15943 13.1 15.8 55.5 NECA 215.5 294.9 34.9 R-PIA
67.6 678.1 23.6 IB-MECA 727.7 859.4 3.1 Alloxozine 1072.0 1934.0
8216.0
[0669] These data indicate that the reference compounds have
affinities consistent with those reported in the literature. The
data further indicate that the yeast-based assays are of sufficient
sensitivity to discriminate receptor subtype specificity.
Functional Assay Using Yeast Strains Expressing Human A2a Adenosine
Receptor
[0670] In this example, the development of a functional screening
assay in yeast for modulators of the human A.sub.1 adenosine
receptor is described.
I. Ligands Used in Assay
[0671] The natural ligand adenosine, as well as other thoroughly
characterized and commercially available ligands were used for
study of the human A2a receptor functionally expressed in yeast.
Three ligands have been used in the establishment of this assay.
They include: TABLE-US-00007 Ligand Reported K.sub.i Function
Adenosine .sup. 500 nM agonist 5'-N-ethylcarboxamidoadenosine 10-15
nM agonist (NECA) (-)-N6-(2-phenylisopropyl)- 100-125 nM agonist
adenosine (PIA)
[0672] To prevent signaling due to the presence of adenosine in the
growth media, adenosine deaminase (4 U/ml) was added to all
assays.
II. Biological Response in Yeast
[0673] A2a receptor agonists were tested for the capacity to
stimulate the pheromone response pathway in yeast transformed with
the A2a receptor expression plasmid and expressing either
G.sub..alpha.sE10K, G.sub..alpha.sD229S or
G.sub..alpha.sE10K+D229S. The ability of ligand to stimulate the
pheromone response pathway in a receptor dependent manner was
indicated by an alteration in the yeast phenotype. Receptor
activation modified the phenotype from histidine auxotrophy to
histidine prototrophy (activation of fus1-HIS3). Three independent
transformants were isolated and grown overnight in the presence of
histidine. Cells were washed to remove histidine and diluted to
2.times.10.sup.6 cells/ml. 5 .mu.l of each transformant was spotted
onto nonselective media (including histidine) or selective media (1
mM AT) in the absence or presence of 4 U/ml adenosine deaminase.
Plates were grown at 30.degree. C. for 24 hours. In the presence of
histidine both Receptor.sup.+ (R.sup.+) and Receptor.sup.-
(R.sup.-) strains were capable of growth. However, in the absence
of histidine only R.sup.+ cells grew. Since no ligand had been
added to these plates two explanations were possible for this
result. One possible interpretation was that the receptor bearing
yeast were at a growth advantage due to Ligand Independent Receptor
Mediated Activation (LIRMA). Alternatively the yeast could have
been synthesizing the ligand adenosine. To distinguish between
these two possibilities, an enzyme which degrades the ligand,
adenosine deaminase (ADA), was added to the growing yeast and
plates. In the presence of adenosine deaminase R.sup.+ cells no
longer grew in the absence of histidine, indicating that the yeast
were indeed synthesizing ligand.
[0674] This interpretation was confirmed by an A.sub.2a growth
assay in liquid. In this experiment R.sup.+ yeast (a
G.sub..alpha.sE10K strain expressing the A2a receptor) were
inoculated at three densities (1.times.10.sup.6 cell/ml;
3.times.10.sup.5 cells/ml; or 1.times.10.sup.5 cells/ml) in the
presence or absence of adenosine deaminase (4 U/ml). The stringency
of the assay was enhanced with increasing concentrations (0, 0.1,
0.2 or 0.4 mM) of 3-amino-1,2,4-triazole (AT), a competitive
antagonist of imidazoleglycerol-P dehydratase, the protein product
of the HIS3 gene. In the presence of adenosine deaminase and
3-amino-1,2,4-triazole yeast grew less vigorously. However in the
absence of 3-amino-1,2,4-triazole, adenosine deaminase had little
effect. Thus adenosine deaminase itself had no direct effect upon
the pheromone response pathway.
[0675] An alternative approach to measuring growth and one that can
be miniaturized for high throughput screening is an A2a receptor
ligand spot assay. A G.sub..alpha.sE10K strain expressing the A2a
receptor (A2aR+) or lacking the receptor (R-) was grown overnight
in the presence of histidine and 4 U/ml adenosine deaminase. Cells
were washed to remove histidine and diluted to 5.times.10.sup.6
cells/ml. 1.times.10.sup.6 cells were spread onto selective plates
containing 4 U/ml adenosine deaminase and 0.5 or 1.0 mM
3-amino-1,2,4-triazole (AT) and allowed to dry for 1 hour. 5 .mu.l
of the following reagents were applied to the monolayer: 10 mM
adenosine, 38.7 mM histidine, dimethylsulfoxide (DMSO), 10 mM PIA
or 10 mM NECA. Cells were grown 24 hours at 30.degree. C. The
results showed that cells without receptor could only grow when
histidine was added to the media. In contrast, R.sup.+ cells only
grew in areas where the A2a receptor ligands PIA and NECA had been
spotted. Since the plates contained adenosine deaminase, the lack
of growth where adenosine had been spotted confirmed that adenosine
deaminase was active.
III. fus1 LacZ Assay
[0676] To quantitate activation of the yeast mating pathway,
synthesis of .beta.-galactosidase through fus1LacZ was measured.
Yeast strains expressing G.sub..alpha.sE10K, G.sub..alpha.sD229S or
G.sub..alpha.sE10K+D229S were transformed with a plasmid encoding
the human A2a receptor (R+) or with a plasmid lacking the receptor
(R-). Transformants were isolated and grown overnight in the
presence of histidine and 4 U/ml adenosine deaminase.
1.times.10.sup.7 cells were diluted to 1.times.10.sup.6 cells/ml
and exposed to increasing concentrations of NECA for 4 hours,
followed by determination of the .beta.-galactosidase activity in
the cells. The results demonstrated that essentially no
.beta.-galactosidase activity was detected in R- strains, whereas
increasing amounts of .beta.-galactosidase activity were detected
in R+ strains expressing either G.sub..alpha.sE10K,
G.sub..alpha.sD229S or G.sub..alpha.sE10K+D229S as the
concentration of NECA increased, indicating a dose dependent
increase in units of .beta.-galactosidase detected in response to
exposure to increased ligand concentration. This dose dependency
was only observed in cells expressing the A2a receptor. Furthermore
the most potent G.sub..alpha.s construct for the A2a receptor was
G.sub..alpha.sE10K. The G.sub..alpha.sD229S construct was the
second-most potent G.sub..alpha.s construct for the A2a receptor,
while the G.sub..alpha.sE10K+D229S construct was the least potent
of the three G.sub..alpha.s constructs tested, although even the
G.sub..alpha.sE10K+D229S construct stimulated readily detectable
amounts of .beta.-galactosidase activity.
[0677] For a further description of the assays identified, see
International Application No. WO 99/63099, entitled "Functional
Expression of Adenosine Receptors in Yeast", published Dec. 9,
1999, the entire contents of which are hereby incorporated herein
by reference.
Pharmacological Characterization of the Human Adenosine Receptor
Subtypes
Material and Methods
[0678] Materials. [.sup.3H]-DPCPX [Cyclopentyl-1,3-dipropylxantine,
8-[dipropyl-2,3-.sup.3H(N)] (120.0 Ci/mmol); [.sup.3H]-CGS 21680,
[carboxyethyl-.sup.3H(N)] (30 Ci/mmol) and [.sup.125I]-AB-MECA
([.sup.125I]-4-Aminobenzyl-5'-N-Methylcarboxamideoadenosine) (2,200
Ci/mmol) were purchased from New England Nuclear (Boston, Mass.).
XAC (Xantine amine congener); NECA
(5'-N-Ethylcarboxamidoadenosine); and IB-MECA from Research
Biochemicals International (RBI, Natick, Mass.). The Adenosine
Deaminase and Complete protease inhibitor cocktail tablets were
purchased from Boehringer Mannheim Corp. (Indianapolis, Ind.).
Membranes from HEK-293 cells stably expressing the human Adenosine
2a [RB-HA2a]; Adenosine 2b [RB-HA2b] or Adenosine 3 [RB-HA3]
receptor subtypes, respectively were purchased from Receptor
Biology (Beltsville, Md.). Cell culture reagents were from Life
Technologies (Grand Island, N.Y.) except for serum that was from
Hyclone (Logan, Utah).
[0679] Yeast strains: Saccharomyces cerevisiae strains CY12660
[far1*1442 tbt1-1 fus1-HIS3 can1 ste14::trp1::LYS2 ste3*1156
gpa1(41)-G.alpha.i3 lys2 ura3 leu2 trp1:his3; LEU2
PGKp-Mf.alpha.Leader-hA1R-PHO5term 2mu-orig REP3 Ampr] and CY8362
[gpa1p-rG.alpha.sE10K far1*1442 tbt1-1 fus1-HIS3 can1
ste14::trp1:LYS2 step3*1156 lys2 ura3 leu2 trp1 his3; LEU2
PGKp-hA2aR 2mu-ori REP3 Ampr] were developed as described
above.
[0680] Yeast culture: Transformed yeast were grown in Leu-Trp [LT]
media (pH 5.4) supplemented with 2% glucose. For the preparation of
membranes 250 ml of LT medium were inoculated with start titer of
1-2.times.10.sup.6 cells/ml from a 30 ml overnight culture and
incubated at 30.degree. C. under permanent oxygenation by rotation.
After 16 h growth the cells were harvested by centrifugation and
membranes were prepared as described below.
[0681] Mammalian Tissue Culture: The HEK-293 cells stably expressed
human Adenosine 2a receptor subtype (Cadus clone # 5) were grown in
Dulbeco's minimal essential media (DMEM) supplemented with 10%
fetal bovine serum and 1.times. penicillin/streptomycin under
selective pressure using 500 mg/ml G418 antibiotic, at 37.degree.
C. in a humidified 5% CO.sub.2 atmosphere.
[0682] Yeast Cell Membrane Preparations: 250 ml cultures were
harvested after overnight incubation by centrifugation at
2,000.times.g in a Sorvall RT6000 centrifuge. Cells were washed in
ice-cold water, centrifuged at 4.degree. C. and the pellet was
resuspended in 10 ml ice-cold lysis buffer [5 mM Tris-HCl, pH 7.5;
5 mM EDTA; and 5 mM EGTA] supplemented with Protease inhibitor
cocktail tablets (1 tablet per 25 ml buffer). Glass beads (17 g;
Mesh 400-600; Sigma) were added to the suspension and the cells
were broken by vigorous vortexing at 4.degree. C. for 5 min. The
homogenate was diluted with additional 30 ml lysis buffer plus
protease inhibitors and centrifuged at 3,000.times.g for 5 min.
Subsequently the membranes were pelleted at 36,000.times.g (Sorvall
RC5B, type SS34 rotor) for 45 min. The resulting membrane pellet
was resuspended in 5 ml membrane buffer [50 mM Tris-HCl, pH 7.5;
0.6 mM EDTA; and 5 mM MgCl.sub.2] supplemented with Protease
inhibitor cocktail tablets (1 tablet per 50 ml buffer) and stored
at -80.degree. C. for further experiments.
[0683] Mammalian Cell Membrane Preparations: HEK-293 cell membranes
were prepared as described previously (Duzic E et al.: J. Biol.
Chem., 267, 9844-9851, 1992) Briefly, cells were washed with PBS
and harvested with a rubber policeman. Cells were pelted at
4.degree. C. 200.times.g in a Sorvall RT6000 centrifuge. The pellet
was resuspended in 5 ml/dish of lysis buffer at 4.degree. C. (5 mM
Tris-HCl, pH 7.5; 5 mM EDTA; 5 mM EGTA; 0.1 mM Phenylmethylsulfonyl
fluoride, 10 mg/ml pepstatin A; and 10 mg/ml aprotinin) and
homogenized in a Dounce homogenizer. The cell lysate was then
centrifuged at 36,000.times.g (Sorvall RC5B, type SS34 rotor) for
45 min and the pellet resuspended in 5 ml membrane buffer [50 mM
Tris-HCl, pH 7.5; 0.6 mM EDTA; 5 mM MgCl.sub.2; 0.1 mM
Phenylmethylsulfonyl fluoride, 10 mg/ml pepstatin A; and 10 mg/ml
aprotinin) and stored at -80.degree. C. for further
experiments.
[0684] The Bio-Rad protein assay kits, based on the Bradford
dye-binding procedure, (Bradford, M.: Anal. Biochem. 72:248 (1976))
were used to determine total protein concentration in yeast and
mammalian membranes.
[0685] Adenosine 1 receptor subtype saturation and competition
radioligand binding: Saturation and competition binding on
membranes from yeast cell transformed with human A.sub.1 receptor
subtype were carried out using antagonist [.sup.3H] DPCPX as a
radioactive ligand. Membranes was diluted in binding buffer [50 mM
Tris-HCl, pH 7.4; containing 10 mM MgCl.sub.2; 1.0 mM EDTA; 0.25%
BSA; 2 U/ml adenosine deaminase and 1 protease inhibitor cocktail
tablet/50 ml] at concentrations of 1.0 mg/ml.
[0686] In saturation binding membranes (50 .mu.g/well) were
incubate with increasing concentrations of [.sup.3H] DPCPX (0.05-25
nM) in a final volume of 100 .mu.l of binding buffer at 25.degree.
C. for 1 hr in the absence and presence of 10 .mu.M unlabeled XAC
in a 96-well microtiter plate.
[0687] In competition binding membranes (50 .mu.g/well) were
incubate with [.sup.3H] DPCPX (1.0 nM) in a final volume of 100
.mu.l of binding buffer at 25.degree. C. for 1 hr in the absence
and presence of 10 .mu.M unlabeled XAC or increasing concentrations
of competing compounds in a 96-well microtiter plate.
[0688] Adenosine 2a receptor subtype competition radioligand
binding: Competition binding on membranes from HEK293 cell stably
expressing the human A2a receptor subtype were carried out using
agonist [.sup.3H] CGS-21680 as a radioactive ligand. Membranes was
diluted in binding buffer [50 mM Tris-HCl, pH 7.4; containing 10 mM
MgCl.sub.2; 1.0 mM EDTA; 0.25% BSA; 2 U/ml adenosine deaminase and
1 protease inhibitor cocktail tablet/50 ml] at concentrations of
0.2 mg/ml. Membranes (10 .mu.g/well) were incubate with [.sup.3H]
CGS-21680 (100 nM) in a final volume of 100 .mu.l of binding buffer
at 25.degree. C. for 1 hr in the absence and presence of 50 .mu.M
unlabeled NECA or increasing concentrations of competing compounds
in a 96-well microtiter plate.
[0689] Adenosine 3 receptor competition radioligand binding:
Competition binding on membranes from HEK293 cell stably expressing
the human A3 receptor subtype were carried out using agonist
[.sup.125I] AB-MECA as a radioactive ligand. Membranes was diluted
in binding buffer [50 mM Tris-HCl, pH 7.4; containing 10 mM
MgCl.sub.2; 1.0 mM EDTA; 0.25% BSA; 2 U/ml adenosine deaminase and
1 protease inhibitor cocktail tablet/50 ml] at concentrations of
0.2 mg/ml. Membranes (10 .mu.g/well) were incubate with [.sup.125I]
AB-MECA (0.75 nM) in a final volume of 100 .mu.l of binding buffer
at 25.degree. C. for 1 hr in the absence and presence of 10 .mu.M
unlabeled IB-MECA or increasing concentrations of competing
compounds in a 96-well microtiter plate.
[0690] At the end of the incubation, the A.sub.1, A.sub.2a and
A.sub.3 receptor subtypes radioligand binding assays was terminated
by the addition of ice-cold 50 mM Tris-HCl (pH 7.4) buffer
supplemented with 10 mM MgCl.sub.2, followed by rapid filtration
over glass fiber filters (96-well GF/B UniFilters, Packard)
previously presoaked in 0.5% polyethylenimine in a Filtermate 196
cell harvester (Packard). The filter plates were dried coated with
50 .mu.l/well scintillation fluid (MicroScint-20, Packard) and
counted in a TopCount (Packard). Assays were performed in
triplicate. Non-specific binding was 5.6.+-.0.5%, 10.8.+-.1.4% and
15.1.+-.2.6% of the total binding in a A1R, A2aR and A3R binding
assay, respectively.
[0691] Adenosine 2b receptor subtype competition radioligand
binding: Competition binding on membranes from HEK293 cell stably
expressing the human A2b receptor subtype were carried out using
A.sub.1 receptor antagonist [.sup.3H] DPCPX as a radioactive
ligand. Membranes was diluted in binding buffer [10 mM Hepes-KOH,
pH 7.4; containing 1.0 mM EDTA; 0.1 mM Benzamidine and 2 U/ml
adenosine deaminase] at concentrations of 0.3 mg/ml. Membranes (15
.mu.g/well) were incubate with [.sup.3H] DPCPX (15 nM) in a final
volume of 100 .mu.l of binding buffer at 25.degree. C. for 1 hr in
the absence and presence of 10 .mu.M unlabeled XAC or increasing
concentrations of competing compounds in a 96-well microtiter
plate. At the end of the incubation, the assay was terminated by
the addition of ice-cold 10 mM Hepes-KOH (pH 7.4) buffer followed
by rapid filtration over glass fiber filters (96-well GF/C
UniFilters, Packard) previously presoaked in 0.5% polyethylenimine
in a Filtermate 196 cell harvester (Packard). The filter plates
were dried coated with 50 .mu.l/well scintillation fluid
(MicroScint-20, Packard) and counted in a TopCount (Packard).
Assays were performed in triplicate. Non-specific binding was
14.3.+-.2.3% of the total binding.
[0692] Specific binding of [.sup.3H] DPCPX; [.sup.3H] CGS-21680 and
[.sup.125I] AB-MECA was defined as the difference between the total
binding and non-specific binding. Percent inhibition of the
compounds was calculated against total binding. Competition data
were analyzed by iterative curve fitting to a one site model, and
K.sub.I values were calculated from IC.sub.50 values (Cheng and
Prusof, Biochem. Pharmacol. 22, 3099-3109, 1973) using the GraphPad
Prizm 2.01 software.
Results
[0693] A primary function of certain cell surface receptors is to
recognize appropriate ligands. Accordingly, we determined ligand
binding affinities to establish the functional integrity of the
Adenosine 1 receptor subtype expressed in yeast. Crude membranes
prepared from Saccharomyces cerevisiae transformed with human
Adenosine 1 receptor subtype construct exhibited specific saturable
binding of [.sup.3H] DPCPX with a K.sub.D of 4.0.+-.0.19 nM. The
K.sub.D and B.sub.max value were calculated from the saturation
isotherm and Scatchard transformation of the data indicated a
single class of binding sites. The densities of adenosine binding
sites in the yeast membrane preparations were estimated to
716.8.+-.43.4 fmol/mg membrane protein.
[0694] The pharmacological subtype characteristics of the
recombinant yeast cells transformed with human A.sub.1 receptor
subtype were investigated with subtype selective adenosine ligands
(XAC, DPCPX; CGS-15943; Compound 600; Compound 1002; NECA, (R)-PIA;
IB-MECA and Alloxazine) that competed with [.sup.3H] DPCPX in the
expected rank order. Displacement curves recorded with these
compounds show the typical steepness with all the ligands, and the
data for each of the ligands could be modeled by a one-site fit.
The apparent dissociation constants estimated for the individual
compound from the curves (Table 5) are consistent with value
published for the receptor obtained from other sources.
TABLE-US-00008 TABLE 5 Ki values for membranes from yeast cells
transformed with human A.sub.1 receptor subtype Ligands K.sub.I
(nM) XAC 5.5 DPCPX 7.1 CGS-1594 10.8 NECA 179.6 (R)-PIA 56.3
IB-MECA 606.5 Alloxazine 894.1 Compound 600 13.9 Compound 1002
9.8
[0695] Tables 6 through 12 demonstrate the efficacy and structure
activity profiles of deazapurines of the invention. Tables 13 and
14 demonstrate selectivity can be achieved for human adenosine
receptor sites by modulation of the functionality about the
deazapurine structure. Table 14 also demonstrates the surprising
discovery that the compounds set forth therein have subnanomolar
activity and higher selectivity for the A.sub.2b receptor as
compared to the compounds in Table 13. TABLE-US-00009 TABLE 6
Effect of N.sub.6-Substituent ##STR166## A1 Com- Binding Yeast
pound R Ki (nM) IC50 (nM) 600 ##STR167## 13.9 97.2 601 ##STR168##
1423 >10,000 602 ##STR169## 483.5 >10,000 603 ##STR170##
196.6 4442.0 604 ##STR171## >10,000 >10000 605 ##STR172##
>10000 >10000 606 ##STR173## 297.9 >10000 607 ##STR174##
309.7 >10000 608 ##STR175## 29.1 609 ##STR176## 193.9 610
##STR177## 411.5 611 ##STR178## 785.6 >10000 612 ##STR179## 64.8
613 ##STR180## 6726.0 614 ##STR181## 32.1 615 ##STR182## 816.9
2577.0 616 ##STR183## 34.3
[0696] TABLE-US-00010 TABLE 7 Effect of C.sub.2-Substituent
##STR184## A1 Binding Yeast Compound R Ki (nM) IC50 (nM) 700
##STR185## 604.5 >10000 701 ##STR186## 157.7 763.1 702
##STR187## 198.5 2782.5 703 ##STR188## 443.6 >10000 704
##STR189## 61.1 297.0 705 ##STR190## 30.1 194.7 706 ##STR191## 19.9
707 ##STR192## 62.8 708 ##STR193## 2145 709 ##STR194## 48.7
[0697] TABLE-US-00011 TABLE 8 Effect of Pyrrole Ring Substituent
##STR195## A1 Yeast Binding IC50 Compound R R' R'' R''' Ki (nM)
(nM) 800 ##STR196## Me Me Me 3311 >10000 801 ##STR197## H Me H
22.3 148.3 802 ##STR198## H H Me 8.9 803 ##STR199## ##STR200## Me
Me 2210 >10000 804 ##STR201## ##STR202## Me Me 863.1 805
##STR203## ##STR204## Me Me 4512 806 ##STR205## ##STR206## Me Me
8451 807 ##STR207## ##STR208## Me Me 35.3
[0698] TABLE-US-00012 TABLE 9 ##STR209## A1 Binding Yeast IC50
Compound R Ki (nM) (nM) 900 ##STR210## 863.1 901 ##STR211## 4512
902 ##STR212## 8451 903 ##STR213## 35.3
[0699] TABLE-US-00013 TABLE 10 Effect of N.sub.6-Substituent
##STR214## A1 Com- Binding Yeast pound R Ki (nM) IC50 (nM) 1000
##STR215## 1789 >10000 1001 ##STR216## 54.4 1865 1002 ##STR217##
9.8 82.8 1003 ##STR218## 26.7 195.7 1004 ##STR219## 32.8 545.8 1005
##STR220## 147.5 3972 1006 ##STR221## 151.7 2918 1007 ##STR222##
692.5 >10000 1008 ##STR223## 93.1 3217 1009 ##STR224## 475.3
>10000 1010 ##STR225## 674.9 9376.0 1011 ##STR226## 121.9 2067.5
1012 ##STR227## 233.9 3462 1013 ##STR228## 270.1 3009.5 1014
##STR229## 384.9 2005 1015 ##STR230## 179.3 3712 1016 ##STR231##
176.1 5054
[0700] TABLE-US-00014 TABLE 11 Effect of N.sub.6-Substituent
##STR232## A1 Com- Binding Yeast pound R Ki (nM) IC50 (nM) 1100
##STR233## 9.8 115.4 1101 ##STR234## 53.9 551.0 1102 ##STR235##
10.3 101.3 1103 ##STR236## 71.1 3217 1104 ##STR237## 6.5 58.7 1105
##STR238## 105.4 472.1 1106 ##STR239## 27.8 162.4 1107 ##STR240##
126.5 1297.0 1108 ##STR241## 2.3 1109 ##STR242## 9.0 1110
##STR243## 17.3 1111 ##STR244## 2.5 1112 ##STR245## 213
[0701] TABLE-US-00015 TABLE 12 "Retro-Amide" Analogues ##STR246##
A1 Com- Binding Yeast pound R Ki (nM) IC50 (nM) 1200 ##STR247##
16.5 189.4 1201 ##STR248## 7.4 45.7 1202 ##STR249## 95.8 3345.0
1203 ##STR250## 529.1 4040.0 1204 ##STR251## 1060.0 >10000 1205
##STR252## 1272 >10000 1206 ##STR253## 50.8 4028 1207 ##STR254##
48.5 701.5
[0702] TABLE-US-00016 TABLE 13 Profile of Selective Adenosine
Antagonists ##STR255## Binding Ki (nM) Compound R A1 A2a A2b A3
1300 ##STR256## 9.8-25.1 18.0-48.6 80.3 513.0 1301 ##STR257## 27.8
50.7 84.6 429.8 1302 ##STR258## 20.2 75.6 20.1 4.3 1303 ##STR259##
17.4 111.3 120.6 44.6 1304 ##STR260## 13.9-30.9 933.7 138.0 21.5
1305.sup.1 ##STR261## 46.6 730.9 30% 9.9 1306.sup.2 ##STR262## 16.4
766.3 168.3 71.7 1307 ##STR263## 29.1 190.6 1143.0 3.1 1308
##STR264## 180 230 670 1.0 1309 ##STR265## 40 109 109 0.3 1310
##STR266## 255 76% 275 .ltoreq.2.6 1311 ##STR267## 531 981 736 5.3
1312 ##STR268## 443 2965 375 .ltoreq.6.2 1313.sup.3 ##STR269## 30%
65% 515 24 1314 ##STR270## 87 204 30 0.02 1315 ##STR271## 75,000
720,000 3,400 507 1316 ##STR272## 333 710,000 710,000 97 1317
##STR273## 710,000 710,000 720,000 369 1318.sup.4 ##STR274## 3.7
.+-. 0.5 630 .+-. 56.4 2307 .+-. 926 630 .+-. 76 1319.sup.4,5
##STR275## 1.8 206 802 270 1320.sup.4,6 ##STR276## 8.0 531 530 419
1321.sup.4,7 ##STR277## 8.0 131 1031 54%.sup.8
.sup.12-thienyl-2-yl; .sup.2C.sub.5-H; .sup.3water soluble;
.sup.4R.sub.5 and R.sub.6 are hydrogen; .sup.5R.sub.3 is
3-fluorophenyl; .sup.6R.sub.3 is 3-chlorophenyl; .sup.7R.sub.3 is
4-pyridyl; .sup.8% activity @ 10 .mu.M
[0703] TABLE-US-00017 TABLE 14 Profile of Selective A.sub.2b
Antagonists ##STR278## Binding Data K.sub.i (nM) Compound XR.sub.1
R.sub.2 A.sub.1 A.sub.2a A.sub.2B A.sub.3 1400 --O--Ph Me 41.7 21
10.3 14.6 1401 --O--Ph(p)F Me 33 58 8.8 18 1402 --O--Ph(p)Cl Me 825
591 22 60 1403 -N-pyridin- Me 60 41 18 48 2-one 1404 --NH--Ph Me 49
31 4.6 57
[0704] TABLE-US-00018 TABLE 15 Adenosine A.sub.1 Receptor Selective
Compounds Relative Relative Relative Compound Structure Ki-A.sub.1
Ki-A.sub.2a Ki-A.sub.2b Ki-A.sub.3 706 ##STR279## * 1318 ##STR280##
* 1319 ##STR281## * 1320 ##STR282## * 1500 ##STR283## * 1321
##STR284## * 1501 ##STR285## * 1502 ##STR286## * 1503 ##STR287## *
1504 ##STR288## * *at least 10 times more selective than other
three subtypes.
INCORPORATION BY REFERENCE
[0705] All patents, published patent applications and other
references disclosed herein are hereby expressly incorporated
herein by reference.
EQUIVALENTS
[0706] Those skilled in the art will recognize, or be able to
ascertain, using no more than routine experimentation, many
equivalents to specific embodiments of the invention described
specifically herein. Such equivalents are intended to be
encompassed in the scope of the following claims.
* * * * *