U.S. patent number 5,861,405 [Application Number 08/335,108] was granted by the patent office on 1999-01-19 for s-substituted 1,3,7-trialkyl-xanthine derivatives.
This patent grant is currently assigned to The United States of America as represented by the Department of Health and Human Services. Invention is credited to Bilha Fischer, Carola Gallo-Rodriguez, Kenneth A. Jacobson, Yishai Karton, Michel Maillard, Philip J. M. van Galen.
United States Patent |
5,861,405 |
Jacobson , et al. |
January 19, 1999 |
S-substituted 1,3,7-trialkyl-xanthine derivatives
Abstract
The present invention provides 8-substituted
1,3,7-trialkylxanthines useful as A.sub.2 -selective adenosine
receptor antagonists and compositions comprising such compounds.
Examples of the 8-substituted 1,3,7-trialkyl xanthines include:
##STR1## In compound (a), R.sub.1, R.sub.3, and R.sub.7 are methyl
and X is one to three substituents, which may be the same or
different and selected from the group consisting of amino, C.sub.1
-C.sub.4 alkylcarbonylamino, carboxy C.sub.2 -C.sub.4
alkylcarbonylamino, halo, C.sub.1 -C.sub.3 alkyloxy, amino C.sub.1
-C.sub.4 alkyloxy, C.sub.1 -C.sub.4 alkyloxy carbonylamino, amino
C.sub.1 -C.sub.4 alkenyloxy, isothiocyanato, and diazonium
tetrafluoroborate. In compound (b), R.sub.1, R.sub.3, and R.sub.7
are methyl, R.sub..beta. is hydrogen or methyl, and X is selected
from the group consisting of R, C(.dbd.O)OR, and C(.dbd.O)NH--R,
wherein R is a C.sub.1 -C.sub.6 alkyl.
Inventors: |
Jacobson; Kenneth A. (Silver
Spring, MD), Karton; Yishai (Nes Zionz, IL),
Gallo-Rodriguez; Carola (Rockville, MD), Fischer; Bilha
(Silver Spring, MD), van Galen; Philip J. M. (Rockville,
MD), Maillard; Michel (Cambridge, MA) |
Assignee: |
The United States of America as
represented by the Department of Health and Human Services
(Washington, DC)
|
Family
ID: |
22008413 |
Appl.
No.: |
08/335,108 |
Filed: |
November 7, 1994 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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57086 |
May 3, 1993 |
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Current U.S.
Class: |
514/263.34;
544/229; 544/273; 544/267; 544/272; 544/271; 514/263.35;
514/263.36; 514/151 |
Current CPC
Class: |
C07D
473/06 (20130101); C07D 473/10 (20130101); C07D
473/12 (20130101) |
Current International
Class: |
C07D
473/00 (20060101); C07D 473/06 (20060101); C07D
473/10 (20060101); C07D 473/12 (20060101); C07D
473/12 (); C07D 473/06 (); A61K 031/52 () |
Field of
Search: |
;544/267,271,272,273,224
;514/263 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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0 215 736 |
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Mar 1987 |
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EP |
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565377 |
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Oct 1993 |
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EP |
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590919 |
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Apr 1996 |
|
EP |
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WO 92/06976 |
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Apr 1992 |
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WO |
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94/01114 |
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Jan 1994 |
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WO |
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94/03456 |
|
Feb 1994 |
|
WO |
|
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|
Primary Examiner: Berch; Mark L.
Attorney, Agent or Firm: Leydig, Voit, & Mayer, Ltd.
Parent Case Text
This is a continuation of application Ser. No. 08/057,086 filed on
May 3, 1996 abandoned.
Claims
What is claimed is:
1. An 8-styryl xanthine having the formula: ##STR11## wherein
R.sub.1, R.sub.3, and R.sub.7 are methyl and X is one to three
substituents, which may be the same or different, selected from the
group consisting of amino, C.sub.1 -C.sub.4 alkylcarbonylamino,
succinylamino, halo, amino C.sub.1 -C.sub.4 alkyloxy, amino C.sub.1
-C.sub.4 alkenyloxy, isothiocyanato, and diazonium
tetrafluoroborate.
2. The 8-styryl xanthine of claim 1, wherein X is at a position
selected from the group consisting of 3, 4, 5, and combinations
thereof.
3. The 8-styryl xanthine of claim 2, wherein X is selected from the
group consisting of 3-amino, 3-C.sub.1 -C.sub.4 alkylcarbonylamino,
3-succinylamino, 3-halo, 3,5-dihalo, 3-isothiocyanato, and
3-diazonium tetrafluoroborate.
4. The 8-styryl xanthine of claim 1, wherein said C.sub.1 -C.sub.4
alkylcarbonylamino is acetylamino, said halo is bromo, chloro,
fluoro, or iodo, said amino C.sub.1 -C.sub.4 alkyloxy is
4-amino-butyloxy, and said amino C.sub.1 -C.sub.4 alkenyloxy is
4-amino-2-trans-buten-1-oxy.
5. The 8-styryl xanthine of claim 4, wherein X is at a position
selected from the group consisting of 3, 4, 5, and combinations
thereof.
6. The 8-styryl xanthine of claim 5, wherein X is selected from the
group consisting of 3-amino, 3-iodo, and 3-diazonium
tetrafluoroborate.
7. The 8-styryl xanthine of claim 5, wherein X is selected from the
group consisting of 3-acetylamino, 3-succinylamino, 3-fluoro,
3-chloro, 3,5-difluoro, and 3-isothiocyanato.
8. An 8-styryl xanthine having the formula: ##STR12## wherein
R.sub.1 and R.sub.3 are propyl, R.sub.7 is methyl, and X is one or
two amino substituents.
9. An 8-substituted xanthine having the formula: ##STR13## wherein
R.sub.1, R.sub.3, and R.sub.7 are methyl, R.sub..beta. is hydrogen
or methyl, and X is selected from the group consisting of R,
C(.dbd.O)OR, and C(.dbd.O)NH--R, wherein R is a C.sub.1 -C.sub.6
alkyl.
10. The 8-substituted xanthine of claim 9, wherein X is
n-propyl.
11. The compound of claim 9, wherein said compound is selected from
the group consisting of
8-(trans-2-tert-butoxycarbonylvinyl)-1,3,7-trimethylxanthine, and
8-(2-n-propylvinyl)-1,3,7-trimethylxanthine.
12. A compound selected from the group consisting of
1,3,7-trimethyl-8-(3-(di-(tert-butyloxycarbonyl)amino)styryl)xanthine,
1,3,7-trimethyl-8-(4-((tert-butyloxycarbonyl)
aminobutyloxy)styryl)xanthine,
1,3,7-trimethyl-8-(3-tert-butyloxycarbonyl aminostyryl)xanthine,
and
1,3,7-trimethyl-8-(3,5-dimethoxy-4-(4-amino-butyloxy)styryl)xanthine.
13. A pharmaceutical composition comprising a pharmaceutically
acceptable carrier and at least one of the compounds of claim
1.
14. A pharmaceutical composition comprising a pharmaceutically
acceptable carrier and at least one of the compounds of claim
6.
15. A pharmaceutical composition comprising a pharmaceutically
acceptable carrier and at least one of the compounds of claim
7.
16. A pharmaceutical composition comprising a pharmaceutically
acceptable carrier and at least one of the compounds of claim
9.
17. A pharmaceutical composition comprising a pharmaceutically
acceptable carrier and at least one of the compounds of claim
10.
18. The 8-substituted xanthine of claim 9, wherein X is selected
from the group consisting of C(.dbd.O)OC(CH.sub.3).sub.3 and
C(.dbd.O)NH-alkyl.
19. A pharmaceutical composition comprising a pharmaceutically
acceptable carrier and at least one of the compounds of claim
18.
20. The 8-styryl xanthine of claim 1, wherein X is a member
selected from the group consisting of 3-halo, 3-amino, and
3-acetylamino.
21. An 8-styryl xanthine having the formula: ##STR14## wherein
R.sub.1, R.sub.3, and R.sub.7 are methyl and X is two substituents,
one of which is methoxy and the other of which is selected from the
group consisting of amino, C.sub.1 -C.sub.4 alkylcarbonylamino,
succinylamino, halo, amino C.sub.1 -C.sub.4 alkyloxy, amino C.sub.1
-C.sub.4 alkenyloxy, isothiocyanato, and diazonium
tetrafluoroborate.
22. The compound of claim 1, wherein said compound is selected from
the group consisting of 1,3,7-trimethyl-8-(3-aminostyryl)xanthine,
1,3,7-trimethyl-8-(3-acetylaminostyryl)xanthine, and
1,3,7-trimethyl-8-(3-succinylaminostyryl)xanthine.
23. A compound selected from the group consisting of
1,3,7-trimethyl-8-(3,5-dialkyloxy-4-(amino-C.sub.1 -C.sub.4
-alkyloxy)styryl)xanthine and
1,3,7-trimethyl-8-(3,5-dialkyloxy-4-(amino-C.sub.1 -C.sub.4
-alkenyloxy)styryl)xanthine.
24. A pharmaceutical composition comprising a pharmaceutically
acceptable carrier and at least one of the compounds of claim
21.
25. A pharmaceutical composition comprising a pharmaceutically
acceptable carrier and at least one of the compounds of claim
22.
26. A pharmaceutical composition comprising a pharmaceutically
acceptable carrier and the compound of claim 12.
27. A pharmaceutical composition comprising a pharmaceutically
acceptable carrier and at least one of the compounds of claim
23.
28. The 8-styryl xanthine of claim 20 where X is 3-chloro.
Description
TECHNICAL FIELD OF THE INVENTION
The present invention relates to 8-substituted
1,3,7-trialkyl-xanthine derivatives and their use as A.sub.2
-selective adenosine receptor antagonists.
BACKGROUND OF THE INVENTION
Xanthine alkaloids, which include caffeine, theophylline, and
theobromine, are ubiquitously distributed in plants, such as the
seeds of Coffea arabica and related species, the leaves of Thea
sinensis, the seeds of Theobroma cacao, the nuts of the tree Cola
acuminata, and the like. Extracts of these naturally occurring
substances have been used throughout history as beverages and the
pharmacologically significant nervous system stimulant properties
of such concoctions have long been recognized.
Xanthine, itself, is 3,7-dihydro-1H-purine-2,6-dione. Chemically,
therefore, xanthine and its derivatives are structurally related to
uric acid and purine. Caffeine (1,3,7-trimethylxanthine),
theophylline (1,3-dimethylxanthine), and theobromine
(3,7-dimethylxanthine) represent the alkaloids most frequently
associated with the expression "xanthine." However, numerous other
xanthine derivatives have been isolated or synthesized. See, for
example, Bruns, Biochem. Pharmacol., 30, 325-333 (1981), which
describes more than one hundred purine bases and structurally
related heterocycles with regard to adenosine antagonism, and Daly,
J. Med. Chem., 25(3), 197-207 (1982).
Pharmacologically, the xanthines represent an important class of
therapeutic agents. Observed pharmacological actions include
stimulation of the central nervous system, relaxation of smooth
muscle constrictions of the smaller bronchi and other smooth
muscles, dilation of the small pulmonary arteries, stimulation of
cardiac muscle with increased cardiac output, and the promotion of
mild diuresis. Available evidence indicates that the therapeutic
actions of these drugs involve blockade or antagonism of adenosine
receptors.
It now has been recognized that there are not one but at least two
classes of extracellular receptors involved in the action of
adenosine. One of these has a high affinity for adenosine and has
been found to be coupled to a number of secondary messenger
systems, including inhibition of adenylate cyclase, inhibition of
calcium entry, stimulation of potassium flux, and phosphoinositide
metabolism (Van Galen et al., Medicinal Res. Rev., 12, 423-471
(1992)). This class has been termed by some as the A.sub.1
receptors. The other class of receptors has a low affinity for
adenosine and has been found to elicit a range of physiological
responses, including the inhibition of platelet aggregation (Lohse
et al., Naunyn Schmiedeberg's Arch. Pharmacol., 337, 64-68 (1988)),
dilation of blood vessels (Ueeda et al., J. Med. Chem., 34,
1340-1344 (1991)), erythropoietin production (Ueno et al., Life
Sciences, 43, 229-237 (1988)), and depression of locomotor activity
(Nikodijevic et al., J. Pharm. Exp. Therap., 259, 286-294 (1991)).
This class has been termed the A.sub.2 receptors.
Subtypes of A.sub.2 receptors also have been identified. For
example, A.sub.2a receptors, which are linked via G.sub.S guanine
nucleotide binding proteins to the stimulation of adenylate
cyclase, are present in high density in the striatum of the CNS.
They are also present on platelets, pheochromocytoma cells, and
smooth muscle cells. A.sub.2b receptors (Bruns et al., Mol.
Pharmacol., 29, 331-346 (1986)) are found in the brain,
fibroblasts, and intestines (Stehle et al., Mol. Endocrinol., 6,
384-393 (1992)).
Characterization of the adenosine receptors is now possible with a
variety of structural analogues. Adenosine analogues resistant to
metabolism or uptake mechanisms have become available. These are
particularly valuable, since their apparent potencies are less
affected by metabolic removal from the effector system than other
adenosine analogues. The adenosine analogues exhibit different rank
order of potencies at A.sub.1 and A.sub.2 adenosine receptors,
providing a simple method of categorizing a physiological response
with respect to the nature of the adenosine receptor. The blockade
of adenosine receptors, i.e., antagonism, provides another method
of categorizing a response with respect to the involvement of
adenosine receptors.
Adenosine, perhaps, represents a general regulatory substance,
since no particular cell type or tissue appears uniquely
responsible for its formation. In this regard, adenosine is unlike
various endocrine hormones. Furthermore, there is no evidence for
storage and release of adenosine from nerve or other cells. Thus,
adenosine is unlike various neurotransmitter substances.
Although adenosine can affect a variety of physiological functions,
particular attention has been directed over the years to those
functions that might lead to clinical applications. Preeminent has
been the cardiovascular effects of adenosine, which lead to
vasodilation and hypotension but which also lead to cardiac
depression. The antilipolytic, antithrombotic, and antispasmodic
actions of adenosine have also received some attention. Adenosine
stimulates steroidogenesis in adrenal cells, probably via
activation of adenylate cyclase, and inhibits neurotransmission and
spontaneous activity of central neurons. Finally, the
bronchoconstrictor action of adenosine and its antagonism by
xanthines represents an important area of research.
Although theophylline and other xanthines, such as caffeine, are
relatively weak adenosine antagonists, having affinity constants in
the range of 10-50 micromolar, they owe many of their
pharmacological effects to blockage of adenosine-mediated functions
at the A.sub.1 and A.sub.2 receptor sites. The A.sub.1 -adenosine
receptor is inhibitory to adenylate cyclase and appears involved in
antilipolytic, cardiac, and central depressant effects of
adenosine. The A.sub.2 -adenosine receptor is stimulatory to
adenylate cyclase and is involved in hypotensive, antithrombotic,
and endocrine effects of adenosine. Some xanthines, such as
3-isobutyl-1-methylxanthine, not only block adenosine receptors but
also have potent inhibitory effects on phosphodiesterases.
The brochodilator effects of the xanthines, particularly,
theophylline, have received considerable commercial attention and
various preparations of theophylline, such as the anhydrous base or
salts thereof, including sodium acetate, sodium benzoate, sodium
salicylate, calcium salicylate, etc., are available as tablets,
capsules, and elixirs including sustained released forms. Other
related xanthines, such as dyphyllin, have received widespread
usage. Caffeine has been used alone and in combination with other
drugs in the treatment of headaches.
Many of the xanthines, however, such as theophylline, have
undesirable side effects. Some of these side effects may be due to
actions at sites other than adenosine receptors. It is also likely
that some side effects are associated with blockade of the
adenosine receptors, themselves. It appears that at least some of
the side-effects caused by the adenosine receptor antagonists could
be avoided by the development of more potent blockers of such
receptors which, because of their increased blocking action, could
be employed in lower doses and, thus, would be less likely to
produce side-effects not associated with the adenosine receptor
blockade. Additionally, where the therapeutic effect is due to
blockade of one subtype of adenosine receptor, while side-effects
relate to blockade of a different subtype of adenosine receptor,
drugs, which are extremely potent at one receptor and substantially
less active at another adenosine receptor, also should have a
reduced likelihood of side-effects.
Potent and A.sub.2 -selective adenosine antagonists, suitable as
pharmacological tools, have long been lacking. A.sub.2 -selective
antagonists also may have application as therapeutic agents, e.g.,
in the treatment of Parkinson's disease (Schiffman et al., Drug
Dev. Res., 28, 381-385 (1993)). The slightly selective,
non-xanthine antagonist CGS 15943 was under development as an
antiasthmatic (Jacobson et al., J. Med. Chem., 35, 407-422 (1992)).
A low affinity antagonist, 3,7-dimethyl-1-propargylxanthine (DMPX),
was reported to be A.sub.2 -selective but by less than one order of
magnitude (Ukena et al., Life Sci., 39, 743-750 (1986)). It was
relatively weak in blocking the in vivo effects of N.sup.6
-cyclohexyladenosine (CHA) compared to those of
5'-N-ethylcarboxamidoadenosine (NECA), suggesting some A.sub.2
selectivity. Several non-xanthine antagonists of the
triazoloquinazoline class, including CGS 15943, are A.sub.2
-selective but also by only one order of magnitude (Francis et al.,
J. Med. Chem., 31, 1014-1020 (1988)). The locomotor activity of
several members of this class was described previously (Griebel et
al., NeuroReport, 2, 139-140 (1991)). A triazoloquinozaline
derivative, CP66,713, was found to be 12-fold selective in binding
assays at rat brain A.sub.2a - vs. A.sub.1 -receptors (Sarges et
al., J. Med. Chem., 33, 2240-2254 (1990)). Low selectivity,
interspecies differences in affinity, and low water solubility
precluded extensive use of this compound. In one study, partial
antagonism of A.sub.2 depression of locomotor activity was achieved
in vivo using CP66,713 (Nikodijevic et al., 1991, supra). At the
same dose CP66,713 had no effect on A.sub.1 depression of locomotor
activity.
It was only recently that 8-styrylxanthines were reported as the
first potentially useful compounds by Shimada et al. (J. Med.
Chem., 35, 2342-2345 (1992)). These authors found that 8-styryl
derivatives of 1,3-dimethylxanthines were the most selective for
A.sub.2 receptors (selectivities greater than 5000-fold were
reported), but the affinities of the corresponding 1,3-propyl
analogues at both subtypes were greater (the most potent compound
having a K.sub.i value of 7.8 nM at A.sub.2 receptors).
The literature is replete with examples of 8-substituted xanthine
derivatives, including 8-substituted 1,3,7-trialkyl-xanthines, such
as 8-styryl-1,3,7-trialkyl-xanthines. For example, U.S. Pat. No.
3,641,010 (Schweiss et al.) discloses
1,3-dialkyl-7-methyl-8-styryl-xanthines and describes the compounds
as cerebral stimulants of the caffeine type. WO 92/06976 discloses
alkyl-substituted 8-styryl-xanthines as selective A.sub.2
-adenosine receptor antagonists useful in the treatment of asthma
and osteoporosis. 1-methyl-3,7-disubstituted-8-benzyl-xanthine
derivatives useful in the treatment of asthma and bronchitis are
disclosed in European Patent Application 0 215 736. The
administration of methylxanthines, which are described as adenosine
antagonists, to alleviate asystole and cardiac arrhythmia
associated with resuscitation is described in U.S. Pat. No.
4,904,472. Various substituted theophyllines/xanthines are
disclosed in U.S. Pat. Nos. 2,840,559, 3,309,271, 3,624,215,
3,624,216, 4,120,947, 4,297,494, 4,299,832, 4,546,182, 4,548,820,
4,558,051, 4,567,183, and 4,883,801, although only the U.S. Pat.
Nos. 4,593,095, 4,612,315, 4,696,932, 4,755,517, 4,769,377,
4,783,530, 4,879,296, 4,981,857, 5,015,647, and 5,047,534 describe
the disclosed compounds as potent adenosine receptor antagonists.
Although a number of these references disclose xanthine compounds
and describe them as "potent" and/or "selective" A.sub.2 -adenosine
receptor antagonists, the potency and/or selectivity actually
realized is not that significant. Accordingly, there remains a need
for highly selective and potent A.sub.2 -adenosine receptor
antagonists. Such compounds would reduce, if not completely
eliminate, the side effects associated with A.sub.2 -adenosine
receptor antagonists of reduced potency or selectivity by
increasing blocking activity at one receptor, significantly, if not
completely, eliminating blocking activity at non-A.sub.2 -adenosine
receptors and, consequently, enabling the employment of reduced
dosages.
An object of the present invention is to provide A.sub.2 -adenosine
receptor antagonists of high potency and/or selectivity. Another
object of the present invention is to provide a pharmaceutical
composition comprising one or more of the present inventive
adenosine receptor antagonists. Yet another object of the present
invention is to provide a method of selectively antagonizing
A.sub.2 adenosine receptors in a mammal in need of selective
antagonism of its A.sub.2 adenosine receptors. By means of these
objects, the present invention offers advantages over currently
available A.sub.2 -adenosine receptor antagonists by providing
A.sub.2 -selective adenosine receptor antagonists of increased
potency and/or specificity. Accordingly, the present invention also
provides an improved pharmaceutical composition comprising A.sub.2
-selective adenosine receptor antagonists and an improved method
for the selective antagonism of A.sub.2 adenosine receptors in a
mammal in need of such selective antagonism. The method, since it
involves the use of A.sub.2 -selective adenosine receptor
antagonists having increased potency and/or selectivity over
currently available antagonists, is expected to reduce, if not
completely eliminate, the side effects associated with the A.sub.2
-adenosine receptor antagonists by enabling the employment of
reduced dosages.
BRIEF SUMMARY OF THE INVENTION
The present invention provides novel 8-substituted
1,3,7-trialkyl-xanthines. Preferably, the 8-substituted
1,3,7-trialkyl-xanthine is a 1,3,7-trialkyl-8-styryl-xanthine
having the formula: ##STR2## wherein R.sub.1, R.sub.3, and R.sub.7
are methyl and X is one to three substituents, which may be the
same or different and are preferably positioned at positions 3, 4,
5, and combinations thereof, such as amino, C.sub.1 -C.sub.4
aliphatic saturated monoacyl amino, C.sub.1 -C.sub.4 aliphatic
saturated diacyl amino, halo, C.sub.1 -C.sub.3 alkyloxy, amino
C.sub.1 -C.sub.4 alkyloxy, amino C.sub.1 -C.sub.4 alkenyloxy,
isothiocyanato, and a diazonium salt. Even more preferred is a
1,3,7-trimethyl-8-styryl-xanthine, wherein X is selected from the
group consisting of 3-amino, 3-C.sub.1 -C.sub.4 aliphatic saturated
monoacyl amino, 3-C.sub.1 -C.sub.4 aliphatic saturated diacyl
amino, 3-halo, 3,5-dihalo, 4-alkoxy, 3,5-dialkoxy, 4-(amino-C.sub.1
-C.sub.4 -alkyloxy)-3,5-dialkoxy, 4-(amino-C.sub.1 -C.sub.4
-alkenyloxy)-3,5-dialkoxy, 3-isothiocyanato, and 3-diazonium salt.
The C.sub.1 -C.sub.4 aliphatic saturated monoacyl amino is
preferably acetylamino, the C.sub.1 -C.sub.4 aliphatic saturated
diacyl amino is preferably succinylamino, the halo is preferably
bromo, chloro, fluoro, or iodo, the C.sub.1 -C.sub.3 alkyloxy is
preferably methoxy, the amino C.sub.1 -C.sub.4 alkenyloxy is
preferably 4-amino-2-trans-buten-1-oxy, and the 3-diazonium salt is
preferably N.sub.2.sup.+ BF.sub.4.sup.-.
Also provided by the present invention is a
1,3,7-trialkyl-8-styryl-xanthine having the formula: ##STR3##
wherein R.sub.1 and R.sub.3 are propyl, R.sub.7 is methyl, and X is
one or two substituents, which may be the same or different and are
preferably positioned at positions 3, 4, 5, or combinations
thereof, such as amino, halo, and C.sub.1 -C.sub.3 alkoxy.
Preferably, X is 3-amino, 3-halo, 3,5-dihalo, 3,4-dialkoxy, and
3,5-dialkoxy. Even more preferably, X is 3-amino, 3-fluoro,
3,5-difluoro, 3,4-dimethoxy, and 3,5-dimethoxy.
The present invention also provides a
1,3,7-trialkyl-8-substituted-xanthine having the formula: ##STR4##
wherein R.sub.1, R.sub.3, and R.sub.7 are methyl, R.sub..beta. is
hydrogen or methyl, and X is R, C(.dbd.O)OH, C(.dbd.O)OR, or
C(.dbd.O)NH--R, wherein R is a C.sub.1 -C.sub.6 alkyl or phenyl,
with the proviso that R.sub..beta. is not hydrogen when X is
phenyl. Preferably, X is n-propyl, C(.dbd.O)OH, or C(.dbd.O)OC
(CH.sub.3).sub.3.
A pharmaceutical composition comprising a pharmaceutically
acceptable carrier and a therapeutically effective amount of one or
more of the above described compounds as well as a method of
selectively antagonizing A.sub.2 adenosine receptors in a mammal in
need of such antagonism are also provided.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a schematic diagram, which shows the synthesis route of
8-styryl-xanthine derivatives.
FIG. 2 is a schematic diagram, which shows the synthesis route of
8-(4-hydroxy-3,5-dimethoxystyryl)-xanthines and derivatives
thereof.
FIG. 3 is a schematic diagram, which shows the use of a
palladium-catalyzed Heck reaction to attach an 8-vinyl or 8-styryl
group to a xanthine.
FIG. 4 is a graph of the K.sub.i of 7-methyl analogues (nM) versus
the K.sub.i of 7-H analogues (nM), which shows the correlation of
affinity at adenosine receptors for 7-H versus 7-methyl analogues
of 1,3-dimethyl-8-styryl-xanthine derivatives.
FIG. 5 is a graph of the K.sub.i of 7-methyl analogues (nM) versus
the K.sub.i of 7-H analogues (nM), which shows the correlation of
affinity at adenosine receptors for 7-H versus 7-methyl analogues
of 1,3-dipropyl-8-styryl-xanthine derivatives.
FIG. 6A is a graph of IC.sub.50 versus % dimethylsulfoxide (DMSO),
which shows the dependence of observed IC.sub.50 on the
concentration of DMSO in competitive radioligand binding of
1,3-dipropyl-8-(3,5-dimethoxy-styryl)-xanthine.
FIG. 6B is a graph of absorption units at 345 nm versus theoretical
concentration, which shows the UV absorption of water solutions
following the addition of
1,3-dipropyl-8-(3,5-dimethoxy-styryl)-xanthine dissolved in
DMSO.
FIGS. 7A, B, C and D are graphs of bound radioligand (% control)
versus 8-(3-isothiocyanatostyryl)-caffeine (ISC) concentration
(.mu.M), which show the dose-dependent inhibition by ISC of
radioligand binding at A.sub.1 - and A.sub.2a -adenosine receptors
in rat, guinea pig, bovine, and rabbit striatal membranes,
respectively.
FIG. 8 is a graph of inhibition of binding (%) versus time (min),
which shows the time course for inhibition of rabbit striatal
A.sub.2a -adenosine receptors at 25.degree. C. by 2 .mu.M ISC.
FIG. 9A is a graph of CGS 21680 bound (f mol/mg protein) versus CGS
21680 concentration (nM), which shows the saturation curve for the
binding of [.sup.3 H]CGS 21680 to A.sub.2a -adenosine receptors in
rat striatal membranes.
FIG. 9B is a Scatchard transformation for the binding of [.sup.3
H]CGS 21680 to A.sub.2a -adenosine receptors in rat striatal
membranes.
FIG. 10 is a bar graph of inhibition of [.sup.3 H]CGS 21680 binding
(% of control) versus ISC concentration, which shows theophylline
protection of rat striatal A.sub.2a receptors from ISC
inhibition.
FIG. 11A is a graph of total distance traveled (cm/30 min) versus
concentration of 1,3,7-trimethyl-8-(3-chlorostyryl)-xanthine (CSC,
mg/kg), which shows the locomotor activity in male NIH Swiss mice
by CSC.
FIG. 11B is a graph of total distance traveled (cm/30 min) versus
concentration of
2-[(2-aminoethylamino)-carbonylethylphenylethylamino]-5'-N-ethylcarboxamid
oadenosine (APEC, .mu.g/kg), which shows the locomotor depression
in male NIH Swiss mice by APEC.
FIG. 12 is a bar graph of total distance traveled (cm/30 min)
versus the treatment methods of control, CSC,
8-cyclopentyl-1,3-dipropyl-xanthine (CPX), and CPX+CSC, which shows
the synergism of CPX and CSC in stimulating locomotor activity in
mice.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides novel 8-substituted 1,3,7-trialkyl
xanthines. Preferably, the 8-substituted 1,3,7-trialkyl xanthine is
a 1,3,7-trialkyl-8-styryl-xanthine having the formula: ##STR5##
wherein R.sub.1, R.sub.3, and R.sub.7 are methyl and X is one to
three substituents, which may be the same or different, selected
from the group consisting of amino, C.sub.1 -C.sub.4 aliphatic
saturated monoacyl amino, C.sub.1 -C.sub.4 aliphatic saturated
diacyl amino, halo, C.sub.1 -C.sub.3 alkyloxy, amino C.sub.1
-C.sub.4 alkyloxy, amino C.sub.1 -C.sub.4 alkenyloxy,
isothiocyanato, and a diazonium salt. Preferably, X is at a
position selected from the group consisting of 3, 4, 5, and
combinations thereof. Also, X is preferably 3-amino, 3-C.sub.1
-C.sub.4 aliphatic saturated monoacyl amino, 3-C.sub.1 -C.sub.4
aliphatic saturated diacyl amino, 3-halo, 3,5-dihalo, 4-alkoxy,
3,5-dialkoxy, 4-(amino-C.sub.1 -C.sub.4 -alkyloxy)-3,5-dialkoxy,
4-(amino-C.sub.1 -C.sub.4 -alkenyloxy)-3,5-dialkoxy,
3-isothiocyanato, or 3-diazonium salt. The C.sub.1 -C.sub.4
aliphatic saturated monoacyl amino is preferably acetylamino,
whereas the C.sub.1 -C.sub.4 aliphatic saturated diacyl amino is
preferably succinylamino, the halo is preferably bromo, chloro,
fluoro, or iodo, the C.sub.1 -C.sub.3 alkyloxy is preferably
methoxy, the amino C.sub.1 -C.sub.4 alkyloxy is preferably
4-amino-butyloxy, the amino C.sub.1 -C.sub.4 alkenyloxy is
preferably 4-amino-2-trans-buten-1-oxy, and the 3-diazonium salt is
preferably N.sub.2.sup.+ BF.sub.4.sup.-.
The 1,3,7-trimethyl-8-styryl-xanthines, wherein X is 3-amino,
3-iodo, 3-diazonium salt, 4-methoxy,
4-(4-amino-butyloxy)-3,5-dimethoxy, or
4-(4-amino-2-trans-buten-1-oxy)-3,5-dimethoxy, are preferred for
functionalized congeners for coupling to other molecules.
Also provided by the present invention is a
1,3,7-trialkyl-8-styryl-xanthine having the formula: ##STR6##
wherein R.sub.1 and R.sub.3 are propyl, R.sub.7 is methyl, and X is
one or two substituents, which may be the same or different,
selected from the group consisting of amino, halo, or C.sub.1
-C.sub.3 alkoxy.
Preferably, X is at a position selected from the group consisting
of 3, 4, 5, and combinations thereof. Also, X is preferably
3-amino, 3-halo, 3,5-dihalo, 3,4-dialkoxy, or 3,5-dialkoxy. Even
more preferably, X is 3-amino, 3-fluoro, 3,5-difluoro,
3,4-dimethoxy, or 3,5-dimethoxy.
The present invention also provides an 8-substituted
1,3,7-trialkylxanthine having the formula: ##STR7## wherein
R.sub.1, R.sub.3, and R.sub.7 are methyl, R.sub..beta. hydrogen or
methyl, and X is R, C(.dbd.O)OH, C(.dbd.O)OR, or C(.dbd.O)NH--R,
wherein R is a C.sub.1 -C.sub.6 alkyl or phenyl, with the proviso
that R.sub..beta. is not hydrogen when X is phenyl.
The compounds of the present invention may be synthesized by any
suitable means. However, the 8-styryl-xanthine derivatives of the
present invention are preferably synthesized by condensation of a
trans-cinnamic acid with a 1,3-dialkyl-5,6-diaminouracil to form an
amide, which is cyclized under strongly basic conditions to give
the 7-H xanthine derivative, which is subsequently methylated,
using methyl iodide, for example. Aryl amino substituents are
preferably obtained via Zn/HOAc reduction of the corresponding
nitro derivative or, in the case of tertiary aniline, by direct
incorporation of the corresponding cinnamic acid. The details of
the synthesis of these derivatives are set forth in FIGS. 1 and 2
and Example 1.
The other 8-substituted xanthine derivatives of the present
invention are preferably synthesized using a palladium-catalyzed
Heck reaction. The details of the synthesis of these derivatives
are set forth in FIG. 3 and Example 2.
The potency of the present compounds as adenosine receptor
antagonists may be determined by a standard screening procedure
(Bruns et al., PNAS USA, 77(9), 5547-5551 (September 1980)).
The compounds of the present invention may be used as is or in the
form of their pharmaceutically acceptable salts and derivatives,
and may be used alone or in appropriate combination with one or
more other 8-substituted xanthine derivatives or other
pharmaceutically active compounds.
The present invention also provides pharmaceutical compositions
comprising a pharmaceutically acceptable carrier and a
therapeutically effective amount of one or more of the
8-substituted 1,3,7-trialkyl-xanthine derivatives of the present
invention, i.e., one or more of the
1,3,7-trimethyl-8-styryl-xanthines of Formula I,
1,3-dipropyl-7-methyl-8-styryl-xanthines of Formula II, and
1,3,7-trimethyl-8-substituted xanthines of Formula III described
above, as well as their pharmaceutically acceptable salts and
derivatives.
Examples of pharmaceutically acceptable acid addition salts for use
in the present inventive pharmaceutical compositions include those
derived from mineral acids, such as hydrochloric, hydrobromic,
phosphoric, metaphosphoric, nitric and sulphuric acids, and organic
acids, such as tartaric, acetic, citric, malic, lactic, fumaric,
benzoic, glycolic, gluconic, succinic, and arylsulphonic, for
example p-toluenesulphonic acids. The xanthine derivative may be
present in the pharmaceutical composition in any suitable quantity.
The pharmaceutically acceptable excipients described herein, for
example, vehicles, adjuvants, carriers or diluents, are well-known
to those who are skilled in the art and are readily available to
the public. It is preferred that the pharmaceutically acceptable
carrier be one which is chemically inert to the active compounds
and one which has no detrimental side effects or toxicity under the
conditions of use.
The choice of excipient will be determined in part by the
particular compound, as well as by the particular method used to
administer the composition. Accordingly, there is a wide variety of
suitable formulations of the pharmaceutical composition of the
present invention. The following formulations for oral, aerosol,
parenteral, subcutaneous, intravenous, intramuscular,
interperitoneal, rectal, and vaginal administration are merely
exemplary and are in no way limiting.
Formulations suitable for oral administration can consist of (a)
liquid solutions, such as an effective amount of the compound
dissolved in diluents, such as water, saline, or orange juice; (b)
capsules, sachets, tablets, lozenges, and troches, each containing
a predetermined amount of the active ingredient, as solids or
granules; (c) powders; (d) suspensions in an appropriate liquid;
and (e) suitable emulsions. Liquid formulations may include
diluents, such as water and alcohols, for example, ethanol, benzyl
alcohol, and the polyethylene alcohols, either with or without the
addition of a pharmaceutically acceptable surfactant, suspending
agent, or emulsifying agent. Capsule forms can be of the ordinary
hard- or soft-shelled gelatin type containing, for example,
surfactants, lubricants, and inert fillers, such as lactose,
sucrose, calcium phosphate, and corn starch. Tablet forms can
include one or more of lactose, sucrose, mannitol, corn starch,
potato starch, alginic acid, microcrystalline cellulose, acacia,
gelatin, guar gum, colloidal silicon dioxide, croscarmellose
sodium, talc, magnesium stearate, calcium stearate, zinc stearate,
stearic acid, and other excipients, colorants, diluents, buffering
agents, disintegrating agents, moistening agents, preservatives,
flavoring agents, and pharmacologically compatible excipients.
Lozenge forms can comprise the active ingredient in a flavor,
usually sucrose and acacia or tragacanth, as well as pastilles
comprising the active ingredient in an inert base, such as gelatin
and glycerin, or sucrose and acacia, emulsions, gels, and the like
containing, in addition to the active ingredient, such excipients
as are known in the art.
The compounds of the present invention, alone or in combination
with other suitable components, can be made into aerosol
formulations to be administered via inhalation. These aerosol
formulations can be placed into pressurized acceptable propellants,
such as dichlorodifluoromethane, propane, nitrogen, and the like.
They also may be formulated as pharmaceuticals for non-pressured
preparations, such as in a nebulizer or an atomizer.
Formulations suitable for parenteral administration include aqueous
and non-aqueous, isotonic sterile injection solutions, which can
contain anti-oxidants, buffers, bacteriostats, and solutes that
render the formulation isotonic with the blood of the intended
recipient, and aqueous and non-aqueous sterile suspensions that can
include suspending agents, solubilizers, thickening agents,
stabilizers, and preservatives. The compound may be administered in
a physiologically acceptable diluent in a pharmaceutical carrier,
such as a sterile liquid or mixture of liquids, including water,
saline, aqueous dextrose and related sugar solutions, an alcohol,
such as ethanol, isopropanol, or hexadecyl alcohol, glycols, such
as propylene glycol or polyethylene glycol, glycerol ketals, such
as 2,2-dimethyl-1,3-dioxolane-4-methanol, ethers, such as
poly(ethyleneglycol) 400, an oil, a fatty acid, a fatty acid ester
or glyceride, or an acetylated fatty acid glyceride with or without
the addition of a pharmaceutically acceptable surfactant, such as a
soap or a detergent, suspending agent, such as pectin, carbomers,
methylcellulose, hydroxypropylmethylcellulose, or
carboxymethylcellulose, or emulsifying agents and other
pharmaceutical adjuvants.
Oils, which can be used in parenteral formulations include
petroleum, animal, vegetable, or synthetic oils. Specific examples
of oils include peanut, soybean, sesame, cottonseed, corn, olive,
petrolatum, and mineral.
Suitable fatty acids for use in parenteral formulations include
oleic acid, stearic acid, and isostearic acid. Ethyl oleate and
isopropyl myristate are examples of suitable fatty acid esters.
Suitable soaps for use in parenteral formulations include fatty
alkali metal, ammonium, and triethanolamine salts and suitable
detergents include cationic detergents, for example, dimethyl
dialkyl ammonium halides, and alkyl pyridinium halides; anionic
detergents, for example, alkyl, aryl, and olefin sulfonates, alkyl,
olefin, ether, and monoglyceride sulfates, and sulfosuccinates;
nonionic detergents, for example, fatty amine oxides, fatty acid
alkanolamides, and polyoxyethylenepolypropylene copolymers; and
amphoteric detergents, for example, alkyl-.beta.-aminopropionates,
and 2-alkyl-imidazoline quaternary ammonium salts, as well as
mixtures.
The parenteral formulations will typically contain from about 0.5
to about 25% by weight of the active ingredient in solution.
Preservatives and buffers may be used. In order to minimize or
eliminate irritation at the site of injection, such compositions
may contain one or more nonionic surfactants having a
hydrophile-lipophile balance (HLB) of from about 12 to about 17.
The quantity of surfactant in such formulations ranges from about 5
to about 15% by weight. Suitable surfactants include polyethylene
sorbitan fatty acid esters, such as sorbitan monooleate and the
high molecular weight adducts of ethylene oxide with a hydrophobic
base, formed by the condensation of propylene oxide with propylene
glycol. The parenteral formulations can be presented in unit-dose
or multi-dose sealed containers, such as ampules and vials, and can
be stored in a freeze-dried (lyophilized) condition requiring only
the addition of the sterile liquid excipient, for example, water,
for injections, immediately prior to use. Extemporaneous injection
solutions and suspensions can be prepared from sterile powders,
granules, and tablets of the kind previously described.
Additionally, the compounds of the present invention may be made
into suppositories by mixing with a variety of bases, such as
emulsifying bases or water-soluble bases. Formulations suitable for
vaginal administration may be presented as pessaries, tampons,
creams, gels, pastes, foams, or spray formulas containing, in
addition to the active ingredient, such carriers as are known in
the art to be appropriate.
The present invention also provides for antagonizing A.sub.2
adenosine receptors by contacting such receptors with the
8-substituted 1,3,7-trialkyl-xanthine derivatives of the present
invention.
The method of the present invention can be practiced in vitro for
scientific and research purposes. For example, the present
inventive xanthine derivatives may be used to probe adenosine
receptors in order to isolate or characterize the receptors. In
this regard, the amine and carboxylic acid derivatized analogues
are most useful. For example, an amine congener (e.g. 22b or 40 of
Table I) of suitable high affinity may be converted to the
condensation product with the p-aminophenylacetyl (PAPA) group for
radioiodination and photoaffinity cross-linking to the receptor
protein. The cross-linking to the receptor may be carried out with
the photoaffinity cross-linking reagent SANPAH, or by conversion of
the aryl amino group to an azide, followed by photolysis in the
presence of the receptor. Alternately, a chemically reactive
bifunctional reagent, such as p-phenylene diisothiocyanate, may be
coupled to the amine congener, in a manner that leaves one
electrophilic group unreacted. Another type of reporter group, a
fluorescent dye, such as fluorescein isothiocyanate, may be coupled
to an amine congener to provide an affinity probe. These probes
obviate the need for radioactive ligands for receptor
characterization in studies utilizing membrane homogenates and
tissue slices. A carboxylic acid congener (e.g. 24 of Table I) may
be linked to an amine functionalized agarose matrix for the
affinity chromatography of A.sub.2a -receptors.
The method of the present invention has particular usefulness in in
vivo applications, such as the therapeutic treatment of Parkinson's
disease, Huntington's chorea, and other diseases of the central
nervous system (CNS), particularly those involving the dopaminergic
or GABA transmitter systems, both of which are modulated by
A.sub.2a adenosine receptors. A relationship between the striatal
dopaminergic and the adenosine A.sub.2 systems has been proposed
(reviewed in Ferre et al., Neuroscience, 51, 501-512 (1992)).
Activation of A.sub.2a receptors inhibits a dopaminergic pathway in
the striatum. D.sub.2 -dopamine receptors and A.sub.2a receptors
are colocalized on the subset of GABAergic neurons in the striatum,
which innervates the globus pallidus and expresses enkephalin.
Thus, an A.sub.2 antagonist would be expected to enhance
dopaminergic striatopallidal transmission. The other class of
striatal GABAergic neurons, those expressing substance P, are
located in the striatonigral pathway. An A.sub.1 antagonist would
not have a direct postsynaptic action on striatopallidal neurons,
but may still affect both striatopallidal and striatonigral
dopaminergic pathways by enhancing the release of dopamine in the
striatum. Activation of presynaptic A.sub.1 receptors is associated
with the inhibition of release of stimulatory neuro-transmitters in
the CNS (Ferre et al., supra). Accordingly, the present inventive
method is expected to have utility in the enhancement of
dopaminergic activity in the brain and, therefore, is potentially
useful in the treatment of diseases accompanied by a deficiency in
dopaminergic function, such as Parkinson's disease. The present
inventive method includes the administration to an animal, such as
a mammal, particularly a human, in need of selective antagonism of
its A.sub.2 adenosine receptors of a therapeutically effective
amount of one or more of the aforementioned present inventive
8-substituted 1,3,7-trialkyl-xanthines or pharmaceutically
acceptable salts or derivatives thereof, alone or in combination
with one or more other pharmaceutically active compounds.
Some of the compounds of the present invention, such as the
1,3,7-trimethyl-8-styryl xanthines, wherein X is 3-amino, 3-iodo,
3-diazonium salt, 4-methoxy, 4-(4-amino-butyloxy)-3,5-dimethoxy, or
4-(4-amino-2-trans-buten-1-oxy)-3,5-dimethoxy, may be utilized as
functionalized congeners for coupling to other molecules, such as
amines and peptides. The use of such congeners enables increased
potency, prolonged duration of action, specificity of action, and
prodrugs. Water solubility is also enhanced, which allows for
reduction, if not complete elimination, of undesirable binding to
plasma proteins and partition into lipids. Accordingly, improved
pharmacokinetics may be realized.
One skilled in the art will appreciate that suitable methods of
administering a compound of the present invention to an animal are
available, and, although more than one route can be used to
administer a particular compound, a particular route can provide a
more immediate and more effective reaction than another route.
Accordingly, the above-described methods are merely exemplary and
are in no way limiting.
The dose administered to an animal, particularly a human, in the
context of the present invention should be sufficient to effect a
prophylactic or therapeutic response in the animal over a
reasonable time frame. One skilled in the art will recognize that
dosage will depend upon a variety of factors including the strength
of the particular compound employed, the age, species, condition,
and body weight of the animal, as well as the severity of the
infection and stage of the disease. The size of the dose will also
be determined by the route, timing and frequency of administration
as well as the existence, nature, and extent of any adverse
side-effects that might accompany the administration of a
particular compound and the desired physiological effect.
Suitable doses and dosage regimens can be determined by
conventional range-finding techniques. Generally, treatment is
initiated with smaller dosages, which are less than the optimum
dose of the compound. Thereafter, the dosage is increased by small
increments until the optimum effect under the circumstances is
reached. For convenience, the total daily dosage may be divided and
administered in portions during the day if desired. In proper doses
and with suitable administration of certain compounds, the present
invention provides for a wide range of selective inhibition of
A.sub.2 -adenosine receptors, e.g., from little inhibition to
essentially full inhibition.
The following examples further illustrate the present invention
and, of course, should not be construed as in any way limiting its
scope.
EXAMPLE 1
This example describes the synthesis of 8-styryl-xanthine
derivatives substituted at the 1, 3, and 7 xanthine positions and
at various phenyl positions of the styryl moiety.
8-styryl-xanthine derivatives substituted at the 1, 3, and 7
xanthine positions and at various phenyl positions of the styryl
moiety were synthesized as shown in FIGS. 1 and 2. FIG. 1 is a
schematic diagram of the synthesis of 8-styryl xanthine
derivatives. In step (a) the reagents included
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDAC),
4-(N,N-dimethylamino)-pyridine (DMAP), and imidazole. Sodium
hydroxide (NaOH) was used in step (b), which was carried out at
80.degree. C. FIG. 2 is a schematic diagram of the synthesis of
8-(4-hydroxy-3,5-dimethoxystyryl)-xanthines and their derivatives.
In step (a), which was carried out at 160.degree. C., C.sub.6
H.sub.5 SNa was used. R'--Br was used in step (b). The structures
of those compounds that were synthesized in accordance with the
following methods are set forth in Table I. The numbers used to
refer to such compounds are those which appear in Table I.
TABLE I
__________________________________________________________________________
Affinities of 8-styryl xanthine derivatives in radioligand binding
assays at rat brain A.sub.1 and A.sub.2 receptors..sup.a ##STR8##
Compd. R.sub.1, R.sub.3 = R.sub.7 = X = K.sub.i (A.sub.1).sup.a
K.sub.i (A.sub.2a).sup.a A.sub.1 /A.sub.2a
__________________________________________________________________________
ratio 15a. Me H H 654 .+-. 170 291 .+-. 40 2.3 15b. Me Me H 3,890
.+-. 1,150 94 .+-. 36 41 16. Me H H(R.sub.a = F) 2,190 .+-. 400
2,110 .+-. 810 1.4 17a. Me H 2-methoxy 1,730 .+-. 420 645 .+-. 144
2.7 17b. Me Me 2-methoxy 4,760 .+-. 720 267 .+-. 84 18 18. Me H
3-hydroxy 702 .+-. 40 303 .+-. 55 2.4 19a. Me H 3-methoxy 1,830
.+-. 821 378 .+-. 155 4.8 19b. Me Me 3-methoxy 5,430 .+-. 1,470
84.8 .+-. 24.0 64 20a. Me H 3-trifluoromethyl 881 .+-. 251 343 .+-.
58 2.6 20b. Me Me 3-trifluoromethyl 3,330 .+-. 410 134 .+-. 44 25
21a. Me H 3-nitro 1,060 .+-. 150 438 .+-. 106 2.4 21b. Me Me
3-nitro 2,140 .+-. 480 195 .+-. 44 11 22a. Me H 3-amino 288 .+-. 60
202 .+-. 79 1.4 22b. Me Me 3-amino 1,690 .+-. 360 57 .+-. 3 30 23.
Me Me 3-(acetylamino) 9,470 .+-. 2,540 39 .+-. 21 240 24. Me Me
3-(succinylamino) 35,100 .+-. 11,700 143 .+-. 45 250 25. Me Me
3-t-butyloxycarbonylamino 23,600 .+-. 2,500 784 .+-. 100 30 26. Me
Me 3-di-(t-butyloxycarbonyl)amino 10,800 .+-. 1,300 740 .+-. 77 15
27a. Me H 3-fluoro 2,720 .+-. 360 516 .+-. 99 5.3 27b. Me Me
3-fluoro 15,780 .+-. 2,860 83 .+-. 18 190 28a. Me Me 3-chloro
28,200 .+-. 7,000 54 .+-. 19 520 28b. Me Me 3-bromo 3,520 .+-. 80
29.2 .+-. 3.1 120 28c. Me Me 3-iodo 2,370 .+-. 1,420 38.6 .+-. 12.5
61 28d. Me Me 3-diazonium (N.sub.2 .sup.+ BF.sub.4 .sup.-) 2,990
.+-. 560 64.8 .+-. 19.6 46 28e. Me Me 3-isothiocyanato 20,300 .+-.
1,700 111 .+-. 1 180 29a. Me H 4-methoxy 858 .+-. 320 472 .+-. 132
1.8 29b. Me Me 4-methoxy 14,200 .+-. 3,500 327 .+-. 75 44 30a. Me H
4-dimethylamino 3,030 .+-. 300 12,800 0.24 30b. Me Me
4-dimethylamino 5.6%.sup.b (3 .times. 10.sup.-5) 9,270 .+-. 150
>1 31a. Me H 2,3-dimethoxy 1,600 .+-. 250 600 .+-. 204 2.7 31b.
Me Me 2,3-dimethoxy 5,390 .+-. 1,020 716 .+-. 144 75 32a. Me H
3,4-dimethoxy 5,340 .+-. 1,440 1,100 .+-. 250 48 32b. Me Me
3,4-dimethoxy 13,790 .+-. 2,420 197 .+-. 33 70 33a. Me H
3,5-dimethoxy 3,044 .+-. 520 120 .+-. 36 25 33b. Me Me
3,5-dimethoxy 12.5 .+-. 6.3%.sup.b (10.sup.-5) 75.3 .+-. 29.1
>200 34a. Me H 3,5-difluoro 2,330 .+-. 830 366 .+-. 77 6.4 34b.
Me Me 3,5-difluoro 14,750 .+-. 3,890 65 .+-. 9 230 35. Me Me
3,5-dimethoxy-4-hydroxy 8,700 .+-. 4,100 450 .+-. 66 19 36. Me Me
4-acetoxy-3,5-dimethoxy 6,330 .+-. 1,680 68 .+-. 22 93 37. Me Me
4-(4-benzyloxy)-3,5-dimethoxy 4,120 .+-. 460 139 .+-. 7 30 38. Me
Me 4-(4-amino-butyloxy)-3,5-dimethoxy 6,170 .+-. 1,010 173 .+-. 43
36 39. Me Me 4-[4-t-(butyloxycarbonyl)amino-butyloxy- 11,031 265
.+-. 105 42 3,5-dimethoxy 40. Me Me
4-(4-amino-2-t(ans-buten-1-oxy)-3,5- 6,280 .+-. 1,580 228 .+-. 20
28 dimethoxy 41. Me Me 4-(4-acetylamino-2-trans-buten-1-oxyl)-3,5
17 .+-. 7%.sup.b (10.sup.-5) 216 .+-. 40 >50 dimethoxy 42. Me Me
4-[4-t-butyloxycarbonyl)amino-2-trans 11 .+-. 5%.sup.b (10.sup.-5)
353 .+-. 62 >40 buten-1-oxy]-3,5-dimethoxy 43a. Me H
2,3,4-trimethoxy 26 .+-. 10%.sup.b (10.sup.-5) 1,610 .+-. 260 >5
43b. Me Me 2,3,4-trimethoxy 6,920 .+-. 330 206 .+-. 81 34 44a. Me H
3,4,5-trimethoxy 2,280 .+-. 530 360 .+-. 170 6.3
[>100,000].sup.c [71].sup.c [>1100] 44b. Me Me
3,4,5-trimethoxy 9,200 .+-. 3,560 131 .+-. 54 70
[>100,000].sup.c [18].sup.c [>5600] 44c. Me Et
3,4,5-trimethoxy 6,290 .+-. 680 882 .+-. 239 7.1 44d. Me
hydroxyethyl 3,4,5-trimethoxy 26 .+-. 9%.sup.b (10.sup.-5)
22%.sup.b (10.sup.-5) -- 44e. Me propargyl 3,4,5-trimethoxy 4,040
.+-. 370 525 .+-. 220 7.7 44f. Me phenylethyl 3,4,5-trimethoxy 32
.+-. 9%.sup.b (10.sup.-5) 14%.sup.b (10.sup.-5) -- 45a. Et H
3,4,5-trimethoxy 852 .+-. 277 269 .+-. 7 3.2 45b. Et Me
3,4,5-trimethoxy 2,790 .+-. 960 81 .+-. 17 34 46. allyl Me
3,4,5-trimethoxy 1,930 .+-. 100 131 .+-. 69 13 [>100,000].sup.c
[15].sup.c [>6700] 47. Pr H H 55 .+-. 28 44 .+-. 19
1.3 [1800 or 22.sup.d ].sup.c [26 or 85.sup.d ].sup.c [69 or
0.26.sup.b ] 48. Pr Me 3-nitro 272 .+-. 68 56.2 .+-. 6.8 4.8 49. Pr
Me 3-amino 113 .+-. 21 18.9 .+-. 5.3 6.0 50a. Pr H 3-fluoro 78 .+-.
17 153 .+-. 31 0.51 50b. Pr Me 3-fluoro 301 .+-. 64 33 .+-. 15 9.1
51a. Pr H 3-chloro 167 .+-. 39 216 .+-. 66 0.77 51b. Pr Me 3-chloro
874 .+-. 222 61.3 .+-. 17.6 14 52a. Pr H 3,4-dimethoxy 71 .+-. 11
48.5 .+-. 8.6 1.3 [1700].sup.c [6700].sup.c [0.25] 52b. Pr Me
3,4-dimethoxy [KF17837] 577 .+-. 42 31.1 .+-. 11.8 19 [1500].sup.c
[7.8].sup.c [190] 53a. Pr H 3,5-dimethoxy 632 .+-. 152 210 .+-. 140
3.0 53b. Pr Me 3,5-dimethoxy 2,630 .+-. 20 24.0 .+-. 6.0 110 54a.
Pr H 3,5-difluoro 146 .+-. 25 346 .+-. 97 0.42 54b. Pr Me
3,5-difluoro 382 .+-. 40 53 .+-. 15 7.2 55a. Pr H 2,3,4-trimethoxy
97 .+-. 19 64.0 .+-. 15.6 1.5 55b. Pr Me 2,3,4-trimethoxy 379 .+-.
128 68.5 .+-. 12.6 5.5 56a. Pr H 2,4,5-trimethoxy 143 .+-. 19 323
.+-. 74 0.44 56b. Pr Me 2,4,5-trimethoxy 689 .+-. 239 327 .+-. 52
2.1
__________________________________________________________________________
a. Expressed in nM (single determination or mean .+-. S.E.M. for 3
or mor determinations) vs [.sup.3 H]PIA (1 nm) at rat A.sub.1
-receptors and vs [.sup.3 H]CGS21680 (5 nM) at rat striatal A.sub.2
-receptors b. Percent displacement of specific binding at the
concentration indicate in parentheses c. Values in brackets are
from Shimada et. al., J. Med. Chem., 35, 2342-2345 (1992) and
represent K.sub.i values vs. [.sup.3 H]NECA in rat striatum and vs.
[.sup.3 H]CHA in guinea pig brain, unless noted d. Affinities at
both A.sub.1 and A.sub.2 receptors measured in rat brain from
Erickson et. al., J. Med. Chem., 34, 1431-1435 (1991)
A trans-cinnamic acid (8, FIG. 1) was condensed with a
1,3-dialkyl-5,6-diamino-uracil, 7, such as
5,6-diamino-1,3-dimethyl-uracil to obtain an amide, 9. The
substituted cinnamic acid (1 equiv) was dissolved in a minimum
volume of DMF containing 1,3-dialkyl-5,6-diamino-uracil (1.5
equiv). 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide HCl (1
equiv) was added, followed by a catalytic amount (0.05 equiv) of
4-(N,N-dimethylamino)-pyridine and 0.05 equiv of imidazole. The
mixture was stirred at room temperature for 3 h, and saturated
sodium chloride solution was added (for 1,3-dipropyl derivatives,
water was used here), to form a precipitate or amorphous insoluble
fraction. The insoluble residue was filtered and dissolved in 4N
aqueous sodium hydroxide containing sufficient methanol to obtain a
clear solution. The mixture was heated at 60.degree. C. for 2 hours
or until the starting material completely disappeared, as judged
using thin layer chromatography (TLC) (silica plate, CHCl.sub.3 ;
CH.sub.3 OH; HOAc; 85:10:5 v/v). The mixture was cooled and
acidified to pH=1 with 6N aqueous hydrochloric acid solution. The
precipitate was washed with water, dried and further purified using
a preparative silica plate (85-95% CHCl.sub.3, 5-15% methanol; 1-5%
HOAc).
The resulting amide, 9, was cyclized under strongly basic
conditions to give the 7-H xanthine derivative, 10, which was
methylated using methyl iodide at 50.degree.-60.degree. C. An
8-styrylxanthine derivative (1 equiv) was dissolved in a minimum of
N,N-dimethylformamide (DMF). Excess finely powdered anhydrous
potassium carbonate was added and the solution was left for 10 min
in an ultrasonic bath. Methyl iodide (5 equiv) was added. The
mixture was stirred at 60.degree. C. for 30 minutes or until the
starting material completely disappeared as judged using TLC
(silica, chloroform:methanol:acetic acid; 95:4:1 v/v). The reaction
mixture was cooled, and excess concentrated aqueous ammonia
solution was added. The precipitate was washed with water, dried in
vacuo, and further purified, either by crystallization or by
chromatography on a preparative thin layer plate (85-95%
chloroform, 5-15% methanol; 1-5% acetic acid). The trans
orientation of the 8-styryl group was verified for each of the
derivatives based on the proton-proton coupling constants of the
olefinic protons (typically .gtoreq.15 Hz).
[3,5-dimethoxy-4-hydroxy]-8-styrylxanthines were demethylated and
then O-alkylated.
1,3,7-trialkyl-8-(3,4,5-trimethoxystyryl)-xanthine (1 equiv) was
dissolved in minimum DMF, and 1.5 equiv of sodium thiophenoxide
were added. The solution was heated to 150.degree.-160.degree. C.
for 20 min or when judged complete using TLC. An appropriate halide
(2 equiv for monohalide and 8 equiv for dihalide) was added,
followed by finely powdered, anhydrous K.sub.2 CO.sub.3. The
solution was left in an ultrasonic bath for 15 min and further
heated at 50.degree.-80.degree. C. for 2 h or until judged complete
using TLC. The reaction mixture was cooled and extracted with
petroleum ether. The crude product was precipitated by water (for
product of reaction with monohalides) or reacted further (for
dihalides) with concentrated aqueous ammonia and chromatographed on
preparative TLC using 90-95% chloroform:5-10% methanol and 1%
aqueous ammonia.
The 4-hydroxy intermediate, compound 35, was also isolated and
recrystallized. .sup.1 H NMR DMSO,d.sub.6 d 3.23 and 3.46 (each S,
3H, N.sub.1 and N.sub.3 CH.sub.3); 3.83 (S, 6H 3,5-di-OCH.sub.3);
4.03 (S, 3H, N.sub.7 CH.sub.3); 7.08 (S, 2H, Ar), 7.18 and 7.61
(each d, 1H, C.dbd.C, J=15.7 Hz), 8.82 (br s, 1H, ArOH).
Synthesis of hydroxyl ring-substituted 8-styrylxanthines was
attempted by the usual route (FIG. 1), starting with the 3- or
4-hydroxycinnamic acid. The intermediate amide was formed in low
yield, and the cyclization provided the desired xanthine in only
very low yield (e.g. 18). Carrying out the sequence with hydroxyl
protection in order to obtain a free hydroxyl group in the
p-position of the final product was attempted, but proved
unsatisfactory. Acetyl ester and p-methoxybenzyl ether derivatives
formed the amide intermediate, 9, but the cyclization step in 4N
NaOH failed. The attempted deprotection of mono-methoxy derivatives
in the series using sodium thiophenolate, trimethylsilyl iodide, or
nitrogen bases at high temperature was unsuccessful. It was,
however, possible to selectively demethylate
8-(3,4,5-trisubstituted)-styrylxanthines, 12 (FIG. 2), using sodium
thiophenolate in DMF at 160.degree. C. The position of the free
hydroxyl group (4-) in 13 was determined by proton NMR. This
hydroxyl group could be readily acylated or alkylated (in some
cases carried out in situ following the deprotection reaction) to
provide 14.
Aryl amino substituents were obtained via Zn/HOAc reduction of the
corresponding nitro derivative (e.g. 21) or, in the case of
tertiary aniline (e.g. 30a), by direct incorporation of the
corresponding cinnamic acid. The N-7 position of 30a was
selectively alkylated using methyl iodide at 50.degree.-60.degree.
C. to provide 30b. Catalytic hydrogenation of the nitrostyryl
derivative 21 afforded the saturated aniline analogue 57.
7-methoxy-2-benzofuranecarboxylic acid, trans-cinnamic acid and the
following derivatives thereof were obtained from Aldrich (St.
Louis, Mo.): .alpha.-fluoro, 2-methoxy, 3,4-dimethoxy,
3,5-difluoro, and 3,5-dimethoxy. .beta.-Methyl-3-nitrocinnamic acid
was obtained from the Sigma-Aldrich Library of Rare Chemicals
collection. 3- and 4-Methoxy derivatives of trans-cinnamic acid
were obtained from Fluka (Ronkonoma, N.Y.). The following
derivatives of trans-cinnamic acid were purchased from Lancaster
(Windham, N.H.): 2,3-dimethoxy, 3,4,5-trimethoxy, 2,3,4-trimethoxy,
2,4,5-trimethoxy, and 3-fluoro. The following derivatives of
trans-cinnamic acid were obtained from Janssen Chimica (Geel,
Belgium): 3-trifluoromethyl, 3-chloro, and 3-nitro.
2-Chloroadenosine was obtained from Research Biochemicals, Inc.
(Natick, Mass.). Compound 46 was the gift of Dr. Ray Olsson (Univ.
So. Florida, Tampa, Fla.). 8-Cyclohexylcaffeine, 2, was the gift of
Dr. John W. Daly (National Institutes of Health). Analytical TLC
plates and silica gel (230-400 mesh) were obtained from VWR
(Bridgeport, N.J.).
All xanthine derivatives were judged to be homogeneous using thin
layer chromatography following final purification. New compounds
were characterized (and resonances assigned) by 300 MHz proton
nuclear magnetic resonance mass spectroscopy using a Varian
GEMINI-300 FT-NMR spectrometer. Unless noted, chemical shifts are
expressed as ppm downfield from tetramethylsilane. Synthetic
intermediates were characterized by chemical ionization mass
spectrometry (NH.sub.3) and xanthine derivatives by fast atom
bombardment mass spectrometry (positive ions in a glycerol matrix)
on a JEOL SX102 mass spectrometer. In the EI mode accurate mass was
determined using a VG7070F mass spectrometer. C, H, and N analyses
were carried out by Atlantic Microlabs (Norcross, Ga.), and
.+-.0.4% was acceptable.
1,3-Dimethyl-8-(2-methoxystyryl)xanthine (17a)
Compound 17a was made from 2-methoxycinnamic acid and triturated
with hot methanol. mp above 300.degree. C. .sup.1 H NMR
DMSO-d.sub.6 d 3.27 (s, 3H N.sub.3 --CH.sub.3); 3.35 (s, 3H N.sub.7
--CH.sub.3); 3.5 (s, 3H OCH.sub.3); 3.9 (s, 3H, N.sub.7
--CH.sub.3); 7.1 (d, 1H, J=18 Hz); 7.0-7.2 (m, 2H); 7.4 (m, 1H);
7.7 (d, 1H, J=8 Hz); 7.8 (d, 1H, J=18 Hz). MS (CI/NH.sub.3) m/e 313
(MH.sup.+, base) 281, 117.
1,3,7-Trimethyl-8-(2-methoxystyryl)xanthine (17b)
Compound 17b was made from 17a. mp 238.degree.-240.degree. C.
.sup.1 H NMR DMSO-d.sub.6 d 3.24 (s, 3H N.sub.3 CH.sub.3); 3.48 (s,
3H N.sub.7 CH.sub.3); 3.90 (s, 3H OCH.sub.3); 4.06 (s, 3H, N.sub.7
CH.sub.3); 7.0-7.14 (m, 2H); 7.34 (d, 1H, J=16 Hz); 7.4 (m, 1H);
7.9 (d, 1H, J=8 Hz); 8.0 (d, 1H, J=16 Hz). MS (CI/NH.sub.3) m/e 327
(MH.sup.+) base peak.
1,3-Dimethyl-8-(3-trifluoromethylstyryl)xanthine (20a)
Compound 20a was made from 3-trifluoromethylcinnamic acid.
mp>300.degree. C. .sup.1 H NMR DMSO-d.sub.6 d 3.26 (s, 3H
N--CH.sub.3); 3.48 (s, 3H N--CH.sub.3); 7.19 (d, 1H J=16 Hz); 7.64
(t, 1H J=8 Hz); 7.70 (d, 1H J=7 Hz); 7.72 (d, 1H J=16 Hz); 7.94 (d,
1H J=8 Hz); 7.96 (s, 1H). MS (CI) m/e 350 (base), 329, 292.
1,3,7-Trimethyl-8-(3-trifluoromethylstyryl)xanthine (20b)
Compound 20b was made from 20a. mp 232.degree.-236.degree. C.
.sup.1 H NMR DMSO-d.sub.6 d 3.25 (s, 3H N--CH.sub.3); 3.49 (s, 3H
N--CH.sub.3); 4.09 (s, 3H N.sub.7 --CH.sub.3); 7.58 (d, 1H J=16
Hz); 7.67 (t, 1H J=8 Hz); 7.72 (d, 1H J=8 Hz); 7.78 (d, 1H J=16
Hz); 8.09 (d, 1H J=7 Hz); 8.26 (s,1H). MS (EI) m/e 364.
1,3-Dimethyl-8-(3-nitrostyryl)xanthine (21a)
Compound 21a was made from 3-nitrocinnamic acid (temperature raised
to 80.degree. C. for 3 h, recrystallized from methanol).
mp>300.degree. C. .sup.1 H NMR DMSO-d.sub.6 d 3.25 (s, 3H
N--CH.sub.3); 3,48 (s, 3H N--CH.sub.3); 7.22 (d, 1H J=16 Hz); 7.70
(t, 1H J=8 Hz); 7.76 (d,1H J=16 Hz); 8.10 (d, 1H J=8 Hz); 8.18 (d.
1H J=8 Hz); 8.41 (s, 1H). MS (EI) m/e327 (base), 310, 280.
1,3,7-Trimethyl-8-(3-nitrostyryl)xanthine (21b)
Compound 21b was made from 21a. mp 306.degree.-308.degree. C.
.sup.1 H NMR DMSO-d.sub.6 d 3.23 (s, 3H N--CH.sub.3); 3.47 (s, 3H
N--CH.sub.3); 4.08 (s, 3H N.sub.7 --CH.sub.3); 7.63 (d, .sup.1 H
J=16 Hz); 7.71 (t, 1H J=8 Hz); 7.80 (d, 1H J=16 Hz); 8.18 (d, 1H
J=8 Hz); 8.23 (d, 1H J=8 Hz); 8.70 (s, 1H). MS (EI) m/e 341 (base);
294.
1,3-Dimethyl-8-(3-aminostyryl)xanthine (22a)
Compound 22a was made from 21a reducing with Zn/acetic acid for 3
h. mp>300.degree. C. .sup.1 H NMR DMSO-d.sub.6 d 3.24 (s, 3H
N--CH.sub.3); 3.46 (s, 3H N--CH.sub.3); 5.19 (s, 2H --NH.sub.2);
6.56 (d, 1H J=8 Hz); 6.74 (d, 1H J=8 Hz); 6.76 (s, 1H); 6.84 (d, 1H
J=16 Hz); 7.05 (t, 1H J=8 Hz); 7.49 (d, 1H J=16 Hz). MS
(CI/NH.sub.3) m/e 315 (M+NH.sub.4.sup.+), 298 (MH.sup.+, base).
1,3,7-Trimethyl-8-(3-aminostyryl)xanthine (22b)
Compound 22b was made from 21b using Zn/acetic acid as reducing
agent for 3 h. mp 222.degree.-224.degree. C. .sup.1 H NMR
DMSO-d.sub.6 d 3.22 (s, 3H N--CH.sub.3); 3.46 (s, 3H N--CH.sub.3);
4.00 (s, 3H N.sub.7 --CH.sub.3); 5.14 (s, 2H --NH.sub.2); 6.58 (d,
1H J=8 Hz, H-4); 6.87 (s, 1H, H-2); 6.92 (d, 1H J=8 Hz, H-6); 7.07
(t, 1H J=8 Hz, H-5); 7.14 (d, 1H J=16 Hz); 7.51 (d, 1H J=16 Hz). MS
(CI/NH.sub.3) m/e 312 (MH.sup.+).
1,3,7-Trimethyl-8-(3-acetylaminostyryl)xanthine (23)
Compound 23 was made from 22b with acetic anhydride in DMF and DMAP
for 1 h. mp>300.degree. C. .sup.1 H NMR DMSO-d.sub.6 d 2.06 (s,
3H --COCH.sub.3), 3.23 (s, 3H N--CH.sub.3); 3.47 (s, 3H
N--CH.sub.3); 4.03 (s, 3H N.sub.7 --CH.sub.3); 7.24 (d, 1H J=16
Hz); 7.34 (t, 1H J=8 Hz); 7.50 (t, 1H J=8 Hz); 7.54 (d, 1H J=8 Hz);
7.61 (d, 1H J=16 Hz); 7.86 (s, 1H). MS (CI/NH.sub.3) m/e 354
(MH.sup.+).
1,3,7-Trimethyl-8-(3-succinylaminostyryl)xanthine (24)
Compound 24 was made from 22b with succinic anhydride in DMF and
DMAP. mp>300.degree. C. .sup.1 H NMR DMSO-d.sub.6 d 2.28 (t, 2H
J=7 Hz); 2.43 (t, 2H J=7 Hz), 3.23 (s, 3H N--CH.sub.3); 3.47 (s, 3H
N--CH.sub.3); 4.03 (s, 3H N.sub.7 --CH.sub.3); 7.24 (d, 1H J=16
Hz); 7.32 (t, 1H J=8 Hz); 7.45 (d, 1H J=8 Hz); 7.54 (d, 1H J=8 Hz);
7.61 (d, 1H J=16 Hz); 7.82 (s, 1H). MS (CI/NH.sub.3) m/e 394
(M--OH), 312, 209 (base). UV characteristics: .lambda..sub.max in
methanol 349 nm, log e=4.48. The maximal aqueous solubility
following dissolution in K.sub.2 HPO.sub.4 (0.1M) was determined to
be 19 mM.
1,3,7-Trimethyl-8-(3-tert-butyloxycarbonyl aminostyryl)xanthine
(25)
Compound 25 was made from 22b with di-tert-butyl dicarbonate and
DMAP in DMF. mp>300.degree. C. .sup.1 H NMR DMSO-d.sub.6 d 1.40
(s, 9H CH.sub.3 COO); 3.17 (s, 3H N--CH.sub.3), 3.41 (s, 3H
N--CH.sub.3); 3.89 (s, 3H N7-CH.sub.3); 7.23 (d, 1H J=16 Hz); 7.33
(d, 1H J=8 Hz); 7.51 (t, 1H J=8 Hz); 7.57 (s, 1 H); 7.67 (d, 1H
J=16 Hz); 7.75 (d, 1H J=8 Hz). MS (CI/NH.sub.3) 414 (M--CH.sub.3
+NH.sub.4.sup.+, base), 338, 314, 312.
1,3,7-Trimethyl-8-[3-[di-(tert-butyloxycarbonyl)
amino]styryl]xanthine (26)
Compound 26 was made from 22b with Di-tert-butyl dicarbonate and
DMAP in DMF. mp 175.degree.-177.degree. C. .sup.1 H NMR
DMSO-d.sub.6 d 1.39 (s, 18H CH.sub.3 COO); 3.23 (s, 3H
N--CH.sub.3), 3.46 (s, 3H N--CH.sub.3); 4.03 (s, 3H N.sub.7
--CH.sub.3); 7.17 (d, 1H J=8 Hz); 7.42 (t, 1H J=8 Hz); 7.43 (d, 1H
J=16 Hz); 7.67 (d, 1H J=16 Hz); 7.69 (d, 1H J=8 Hz); 7.74 (s, 1H).
MS (CI/NH.sub.3) 514 (M--CH.sub.3 +NH.sub.4.sup.+); 414 (base).
1,3-Dimethyl-8-(4-methoxystyryl)xanthine (29a)
Compound 29a was made from 4-methoxycinnamic acid,
m.p.>320.degree. C. .sup.1 H NMR DMSO-d.sub.6 3.24 (s, 3H
N.sub.3 CH.sub.3); 3.46 (s, 3H N.sub.7 --CH.sub.3); 3.78 (s, 3H
OCH.sub.3); 6.85 (d, 1H, J=16 Hz); 7.0 (d, 2H, J=8 Hz); 7.55 (d,
2H, J=8 Hz); 7.6 (d, 1H, J=16 Hz). MS (CI/NH.sub.3) m/e 313
(MH.sup.+, base) 172.
1,3,7-Trimethyl-8-(4-methoxystyryl)xanthine (29b)
Compound 29b was made from 29a, m.p.>320.degree. C. .sup.1 H NMR
DMSO-d.sub.6 d 3.22 (s, 3H N.sub.3 CH.sub.3); 3.45 (s, 3H N.sub.7
CH.sub.3); 3.8 (s, 3H OCH.sub.3); 4.0 (s, 3H, N.sub.7 CH.sub.3);
7.0 (d, 1H, J=8 Hz); 7.2 (d, 1H, J=16 Hz); 7.66 (d, 1H, J=16 Hz),
7.72 (d, 1H, J=8 Hz). MS (CI/NH.sub.3) m/e 327 (MH.sup.+, base)
205.
1,3-Dimethyl-8-(4-dimethylaminostyryl)xanthine (30a)
A solution of 4-dimethylaminocinnamic acid (0.1 g, 0.52 mmol),
1-hydroxy benzotriazole (0.14 g, 1.04 mmol) and EDAC (0.19 g, 1.04
mmol) in DMF (1 ml) was sonicated for 1 h.
1,3-Dimethyl-5,6-diaminouracil (0.088 g, 0.52 mmol) was added and
the mixture was heated for 3 h at 80.degree. C. The dark red
solution was cooled to room temperature and the product was
obtained as a deep yellow precipitate (0.045 g). An additional crop
was obtained by cooling the mother liquor in an ice bath and adding
10 volumes of brine (combined yield 38%). .sup.1 H NMR CD.sub.3 OD
d 7.54 (d, 1H, J=15.5 Hz), 7.45 (d, 2H, 8.8 Hz), 6.74 (d, 2H, J=8.8
Hz), 6.56 (d, 1H, J=15.5 Hz), 3.42, 3.27 (s, 3H, CH.sub.3), 3.00
(s, 6H, N(CH.sub.3).sub.2). MS (CI) m/e 344 (MH.sup.+).
The above amide (0.045 g, 0.13 mmol) was suspended in methanol (1
ml) and 4N NaOH (1 ml) was added. The resulting solution was
stirred at 80.degree.-90.degree. C. for 1.5 h. 18% HCl was added
carefully to the ice cooled reaction solution to pH 7-8. A yellow
precipitate was obtained (0.018 g, 43%). 1H NMR DMSO-d.sub.6 d 7.54
(d, 1H, J=16 Hz), 7.44 (d, 2H, J=8.5 Hz), 6.74 (d, 2H, J=16 Hz),
6.738 (d, 2H, J=16 Hz), 3.47, 3.25 (s, 3H, CH.sub.3), 2.97 (s, 6H,
N(CH.sub.3).sub.2). MS (CI) m/e 326 (MH.sup.+).
1,3-Dimethyl-8-(2,3-dimethoxystyryl)xanthine (31a)
Compound 31a was made from 2,3-dimethoxycinnamic acid
(recrystallized from methanol). mp 299.degree.-301.degree. C.
.sup.1 H NMR DMSO-d.sub.6 d 3.25 (s, 3H N.sub.3 --CH.sub.3); 3.47
(s,3H N --CH.sub.3); 3.78 (s, 3H OCH.sub.3); 3.82 (s, 3H
OCH.sub.3); 7.05 (d, 1H J=17 Hz); 7.05 (dd,1H J=2 Hz J=8 Hz); 7.11
(t, 1H J=8 Hz) 7.26 (dd, 1H J=2 Hz J=8 Hz), 7.84 (d, 1H J=17 Hz).
MS (CI/NH.sub.3) m/e 360 (M+NH.sub.4.sup.+), 343 (base peak).
1,3,7-Trimethyl-8-(2,3-dimethoxystyryl)xanthine (31b)
Compound 31b was made from 31a, mp 233.degree.-235.degree. C.
.sup.1 H NMR DMSO-d.sub.6 d 323 (s,3H N--CH.sub.3); 3.47 (s,3H
N--CH.sub.3); 3.78 (s, 3H O--CH.sub.3); 3.83 (s, 3H O--CH.sub.3);
4.02 (s, 3H N.sub.7 --CH.sub.3); 7.06 (d,1H J=8 Hz); 7.10 (t, 1H
J=8 Hz); 7.32 (d, 1H J=16 Hz); 7.51 (d, 1H J=8 Hz); 7.90 (d, 1H
J=16 Hz). MS (EI) m/e 356 (base); 325.
1,3-Dimethyl-8-(3,4-dimethoxystyryl)xanthine (32a)
Compound 32a was made from 3,4-dimethoxycinnamic acid,
mp>320.degree. C. .sup.1 H NMR DMSO-d.sub.6 d 3.25 (s, 3H
N.sub.3 CH.sub.3); 3.46 (s, 3H N.sub.7 --CH.sub.3); 3.78 (s, 3H
OCH.sub.3); 3.82 (s, 3H, OCH.sub.3), 6.96 (d, 1H, J=16 Hz); 6.98
(d, 1H, J=8 Hz); 7.14 (d, 1H, J=8 Hz); 7.25 (s, 1H). MS
(CI/NH.sub.3) m/e 343 (MH.sup.+, 172 (base peak).
1,3,7-Trimethyl-8-(3,4-dimethoxystyryl)xanthine (32b)
Compound 32b was made from 32a, mp 230.degree.-232.degree. C.
.sup.1 H NMR DMSO-d.sub.6 d 3.29 (s, 3H N.sub.3 --CH.sub.3); 3.52
(s, 3H N.sub.7 CH.sub.3); 3.85 (s, 3H OCH.sub.3); 3.9 (s, 3H,
OCH.sub.3), 4.09 (s, 3H, N.sub.7 CH.sub.3); 7.05 (d, 1H, J=8 Hz);
7.25 (d, 1H, J=16 Hz); 7.30 (d, 1H, J=8 Hz), 7.48 (s, 1H), 7.66 (d,
1H, J=16 Hz). MS (CI) m/e 357 (MH.sup.+ base), 209.
1,3-Dimethyl-8-(3,5-dimethoxystyryl)xanthine (33a)
Compound 33a was made from 3,5-dimethoxycinnamic acid,
mp>320.degree. C. .sup.1 H NMR DMSO-d.sub.6 d 3.24 (s, 3H
N.sub.3 CH.sub.3); 3.46 (s, 3H N.sub.7 CH.sub.3); 3.78 (s, 6H
OCH.sub.3); 6.5 (s, 1H), 6.78 (s, 2H), 7.02 (d, 1H, J=16 Hz); 7.54
(d, 1H, J=16 Hz). MS (CI) m/e 343 (MH.sup.+ base), 166, 136.
1,3,7-Trimethyl-8-(3,5-dimethoxystyryl)xanthine (33b)
Compound 33b was made from 33a, mp 228.degree.-230.degree. C.
.sup.1 H NMR DMSO-d.sub.6 d 3.22 (s, 3H N.sub.3 CH.sub.3); 3.45 (s,
3H N.sub.3 CH.sub.3); 3.79 (s, 6H OCH.sub.3); 4.04 (s, 3H, N.sub.7
CH.sub.3), 6.5 (s, 1H), 6.97 (s, 2H), 7.32 (d, 1H, J=16 Hz), 7.58
(d, 1H, J=16 Hz).
1,3,7-Trimethyl-8-(3,5-dimethoxy-4-benzyloxystyryl) xanthine
(37)
Compound 37 was made from benzyl bromide, mp
190.degree.-195.degree. C. .sup.1 H NMR CDCl.sub.3 d 3.42 (s, 3H
N.sub.3 CH.sub.3), 3.63 (s, 3H N.sub.5 CH.sub.3); 3.89 (s, 6H
OCH.sub.3); 5.06 (s, 2H, OCH.sub.2), 6.8 (s, 2H); 6.78 (d, 1H, J=16
Hz); 7.3-7.5 (m, 5H); 7.7 (d, 1H, J=16 Hz). MS (CI) m/e 463
(MH.sup.+ base), 375, 357.
1,3,7-Trimethyl-8-[3,5-dimethoxy-4-[4-aminobutyloxy]styryl]xanthine
(38)
Compound 38 was made from 1,4-dibromobutane. MS (CI) m/e 444
(MH.sup.+ base), 373, 359.
1,3,7-Trimethyl-8-[3,5-dimethoxy-4-[4-(tert-butyloxycarbonylamino)butyloxy]
styryl]xanthine (39)
Compound 39 was made from 38 using di-tert-butyl dicarbonate in
CHCl.sub.3 (30 min). The chloroform was removed under a stream of
N.sub.2, and the crude product was purified using a preparative
plate (silica, ethyl acetate/petroleum ether 70:30). .sup.1 H NMR
CDCl.sub.3 d 1.41 (s, 9H CH.sub.3), 1.6-1.8 (m, 4H, CH.sub.2), 3.2
(m, 2H CH.sub.2 NH), 4.0 (m, 2H, OCH.sub.2), 3.39 (s, 3H, N.sub.3
CH.sub.3), 3.6 (s, 2H, N.sub.7 CH.sub.3), 3.88 (s, 6H, OCH.sub.3,
4.05 (s, 3H, N.sub.7 CH.sub.3), 6.74 (s, 2H), 6.75 (d, 1H, J=16
Hz), 7.7 (d, 1H, J=16 Hz). MS (CI) m/e 544 (MH.sup.+ base) 44,
359.
1,3,7-Trimethyl-8-[3,5-dimethoxy-4-[4-(amino-butyloxy)
styryl]xanthine (40)
Compound 40 was made from 1,4-dibromo-trans-2-butene. .sup.1 H NMR
CDCl.sub.3 d 3.41 (s, 3H N.sub.3 CH.sub.3); 3.63 (s, 3H N.sub.7
CH.sub.3); 3.91 (s, 6H OCH.sub.3); 4.06 (s, 3H, N.sub.7 CH.sub.3);
4.43 (s, 2H, CH.sub.2 NH.sub.2); 5.94 (s, 2H, OCH.sub.6); 6.78 (s,
2H), 6.79 (d, 1H, J=16 Hz). MS (CI) m/e 442 (MH.sup.+ base)
373,357,124.
7-Ethyl-1,3-trimethyl-8-[3,4,5-trimethoxystyryl]xanthine (44c)
Compound 44c was made from compound 44a, except that ethyl iodide
was used during methylation, instead of methyl iodide. .sup.1 H NMR
DMSO,d.sub.6 d 1.34 (t, 3H, CH.sub.3 Et, J=7 Hz); 3.25 and 3.47
(each s, 3H NCH.sub.3); 3.70 (s, 4H 4-OCH.sub.3); 3.86 (s, 6H
3,5-di-OCH.sub.3); 4.54 (q, 2H, N7-CH.sub.2); 7.13 (s, 2H, Ar),
7.30 and 7.68 (each d, 1H, C.dbd.C, J=16 Hz).
1,3-Dipropyl-7-methyl-8-styrylxanthine (47)
5-Amino-6-nitroso-1,3-dipropyluracil was suspended in DMF (10
mmol/100 ml) and hydrogenated over 5% Pd/C at 40 psi overnight. The
clear solution was filtered through Celite and could be stored at
-20.degree. C.
Trans-cinnamic acid (0.47 g) and EDAC (0.65 g) were added to 2.1
mmol of the above solution and stirred for 4 h. An additional 0.3 g
of EDAC was added. After 2 additional h, half-saturated NaCl
solution was added and the mixture was extracted with ethyl acetate
(6.times.). The organic layer was dried over Na.sub.2 SO.sub.4 and
evaporated to an oil, which was used without further
purification.
The above oil was dissolved in methanol (30 ml) and treated with 4N
NaOH (20 ml). After refluxing for 15 min, the mixture was cooled,
ice was added, and it was acidified using 6N HCl. A precipitate
formed and was recovered by filtration. The NMR and MS were
consistent with the assigned structure of 47. Recrystallized from
DMF/water.
e.sub.342 for 47 in methanol (.lambda..sub.max) was 35,100. A
smaller absorption peak was at 265 nm.
1,3,7-Trimethyl-8-[2-(3-aminophenyl)ethyl]xanthine (57)
Compound 54 was made from 21b with H.sub.2 /Pd 50 psi in DMF for 3
h. mp 158.degree.-160.degree. C. .sup.1 H NMR DMSO-d.sub.6 d 2.82
(t, 2H J=8 Hz); 2.96 (t, 2H J=8 Hz); 3.20 (s, 3H N--CH.sub.3); 3.42
(s, 3H N--CH.sub.3); 3.69 (s, 3H N.sub.7 --CH.sub.3); 4.95 (s, 2H
--NH.sub.2); 6.34-6.39 (3H, H-2 H-4 H-6); 6.90 (t, 1H J=8 Hz, H-5).
MS (CI/NH.sub.3) m/e 314 (MH+).
The physical characteristics and elemental analyses of the xanthine
derivatives are summarized in Table II.
TABLE II
__________________________________________________________________________
Characterization of xanthine derivatives and elemental analysis.
Yield mp Calculated: Found: Compd. % (.degree.C.) Formula C H N C H
N
__________________________________________________________________________
15a. 51 >280 C.sub.15 H.sub.14 N.sub.14 N.sub.4
O.sub.2.1/2H.sub.2 61.85 5.19 19.23 62.42 5.12 18.78.sup.b 15b. 81
220-222 C.sub.16 H.sub.16 N.sub.4 O.sub.2.1/4H.sub.2 O 63.88 5.53
18.62 63.93 5.68 17.60.sup.b 16. 57 >280 C.sub.15 H.sub.13
N.sub.4 O.sub.2 F 60.00 4.36 18.66 60.02 4.37 18.66 17a. 31 >300
C.sub.16 H.sub.16 N.sub. O.sub.3.2/5H.sub.2 O 60.14 5.30 17.53
60.44 5.13 17.11 17b. 74 238-240 C.sub.17 H.sub.18 N.sub..sub.4
O.sub.3 62.57 5.56 17.17 62.41 5.58 17.09 18. 3 >300 C.sub.15
H.sub.14 N.sub..sub.4 O.sub.3 60.40 4.73 18.78 19a. 65 >280
C.sub.16 H.sub.16 N.sub..sub.4 O.sub.3 19b. 61 212-215 C.sub.17
H.sub.18 N.sub..sub.4 O.sub.3.1/2H.sub.2 O 60.89 5.71 16.71 60.93
5.83 15.86.sup.b 20a. 55 >300 C.sub.16 H.sub.13 N.sub.4 O.sub.2
F.sub.3 54.86 3.74 15.99 54.74 3.76 15.84 20b. 84 232-236 C.sub.17
H.sub.15 N.sub.4 O.sub.2 F.sub.3.1/2H.sub.2 54.69 4.32 15.01 54.93
4.15 14.81 21a. 56 >300 C.sub.15 H.sub.13 N.sub.4 O.sub.4 55.05
4.00 21.40 55.06 4.08 21.22 21b. 84 306-308 C.sub.16 H.sub.15
N.sub.4 O.sub.4 56.30 4.43 20.52 56.31 4.50 20.46 22a. 85 >300
C.sub.15 H.sub.15 N.sub.5 O.sub.2.1/2H.sub.2 O 58.82 5.27 22.86
59.03 5.25 22.65 22b. 92 222-224 C.sub.16 H.sub.17 N.sub.5
O.sub.2.0.85H.sub.2 O 58.83 5.77 21.44 58.93 5.87 21.37.sup.c 23.
77 >300 C.sub.18 H.sub.19 N.sub.5 O.sub.3.3/5H.sub.2 O 59.36
5.59 19.23 59.21 5.48 18.99.sup.c 24. 78 >300 C.sub.20 H.sub.21
N.sub.5 O.sub.5.0.7H.sub.2 O 56.65 5.33 16.52 56.96 5.23
16.18.sup.c 25. 59 >300 C.sub.21 H.sub.25 N.sub.4
O.sub.4.1/2H.sub.2 O 59.99 6.23 16.66 59.94 5.87 16.00.sup.b,c 26.
27 175-177 C.sub.26 H.sub.33 N.sub.5 O.sub.6.2/5H.sub.2 O 60.20
6.57 13.50 61.47 6.57 13.05c 27a. 87 >310 C.sub.15 H.sub.13
N.sub.4 O.sub.2 F.1/2H.sub.2 O 58.25 4.56 18.11 58.68 4.39
17.58.sup.b,c 27b. 75 208-209 C.sub.16 H.sub.15 N.sub.4 O.sub.2 F
61.14 4.81 17.82 61.07 4.80 17.73 28. 10 205 C.sub.16 H.sub.15
N.sub.4 O.sub.2 CI 58.10 4.57 16.94 58.18 4.55 16.89 29a. 4 >320
C.sub.16 H.sub.16 N.sub.4 O.sub.3 61.53 5.16 17.94 61.35 5.11 17.89
29b. 55 220-222 C.sub.17 H.sub.18 N.sub.4 O.sub.3 62.57 5.56 17.17
62.43 5.58 17.08.sup.c 30a. 43 >230 C.sub.17 H.sub.19 N.sub.5
O.sub.2 c 30b. 29 >230 C.sub.18 H.sub.21 N.sub.5 O.sub.2 63.70
6.24 20.63 64.10 6.55 18.15.sup.b,c 31a. 32.sup.a 299-301 C.sub.17
H.sub.18 N.sub.4 O.sub.4 59.64 5.30 16.37 59.60 5.34 16.29 31b. 49
233.5-235 C.sub.18 H.sub.2 ON.sub.4 O.sub.4.1/2H.sub.2 O 59.17 5.79
15.33 59.45 5.64 15.30 32a. 4 >295 C.sub.17 H.sub.18 N.sub.4
O.sub.4 59.64 5.30 16.37 59.55 5.28 16.31.sup.c 32b. 63
230-232 C.sub.18 H.sub.2 ON.sub.4 O.sub.4.1/2H.sub.2 O 59.17 5.79
15.33 59.15 5.73 15.23 33a. 18 >320 C.sub.17 H.sub.18 N.sub.4
O.sub.4 59.64 5.30 16.37 59.56 5.34 16.35 33b. 63 228-230 C.sub.18
H.sub.2 ON.sub.4 O.sub.4 60.67 5.66 15.72 60.60 5.67 15.65 34a. 76
>310 C.sub.15 H.sub.12 N.sub.4 O.sub.2 F.sub.2 56.61 3.80 17.60
56.66 3.85 17.51 34b. 87 238-239 C.sub.16 H.sub.14 N.sub.4 O.sub.2
F.sub.2.3/4H.sub.2 55.57 4.52 16.20 55.94 4.64 16.15 35. 54
269.sup.d C.sub.20 H.sub.22 N.sub.4 O.sub.6 58.06 5.41 15.05 57.79
5.48 14.95.sup.c 36. 57 274-279 C.sub.18 H.sub.2 ON.sub.4
O.sub.5.1/4H.sub.2 O 57.34 5.41 13.37 57.18 5.33 13.46.sup.c 37. 75
190-194 C.sub.25 H.sub.26 N.sub.4 O.sub.5.1/2H.sub.2 O 63.68 5.77
11.88 63.51 5.71 11.47 38. 90.sup.e 207-212 C.sub.22 H.sub.29
N.sub.5 O.sub.5 c 39. 15.sup.d 184.5- C.sub.27 H.sub.37 N.sub.5
O.sub.7.0.5H.sub.2 O 58.68 6.93 12.67 58.54 6.84 12.30.sup.c 186.5
40. 18 200-206 C.sub.22 H.sub.27 N.sub.5 O.sub.5 58.66 6.27 14.55
59.28 6.33 14.83.sup.b,c 41. 71 .sup. 229-232.sup.d C.sub.24
H.sub.29 N.sub.5 O.sub.6 57.48 6.23 13.96 57.53 6.24 13.77.sup.c
42. 75 192-195 C.sub.27 H.sub.35 N.sub.5 O.sub.7 59.88 6.51 12.93
59.58 6.39 12.59.sup.c 43a. 72 165 C.sub.18 H.sub.20 N.sub.4
O.sub.5 58.06 5.41 15.05 58.34 5.58 14.06.sup.b 43b. 43 189-193
C.sub.19 H.sub.22 N.sub.4 O.sub.5.1/2H.sub.2 O 57.71 5.86 14.17
57.59 5.88 13.37.sup.b 44a. 63 >280 C.sub.18 H.sub.20 N.sub.4
O.sub.5 58.06 5.41 15.05 57.99 5.46 14.99 44b. 82 245-247 C.sub.19
H.sub.22 N.sub.4 O.sub.5 59.06 5.74 14.50 58.99 5.75 14.49 44c. 84
225-229 C.sub.20 H.sub.24 N.sub.4 O.sub.5 59.99 6.04 13.99 60.00
6.08 13.90 44d. 70 251-254 C.sub.20 H.sub.24 N.sub.4 O.sub.2 57.69
5.81 13.45 57.59 5.77 13.40 44e. 79 235-237 C.sub.21 H.sub.22
N.sub.4 O.sub.5 61.46 5.40 13.65 61.43 5.67 13.53 44f. 71 215-218
C.sub.26 H.sub.28 N.sub.4 O.sub.5 65.53 5.92 11.76 65.32 5.91 11.64
45a. 20.sup.a 286-289 C.sub.20 H.sub.24 N.sub.4 O.sub.5.1/4H.sub.2
O 59.32 6.10 13.84 59.69 5.98 13.56.sup.c 45b. 64 207-210 C.sub.21
H.sub.26 N.sub.4 O.sub.5 60.86 6.32 13.52 60.68 6.34 13.45.sup.c
47. 52.sup.a 257-260 C.sub.19 H.sub.22 N.sub.4 O.sub.2 67.44 6.55
16.56 67.52 6.58 16.49 48. 91 215-217 C.sub.20 H.sub.23 N.sub.4
O.sub.4 60.44 5.83 17.62 60.66 5.97 17.38 49. 92 145-148 C.sub.20
H.sub.25 N.sub.5 O.sub.2.3/4H.sub.2 O 63.06 7.01 18.38 63.08 6.62
18.37 50a. 61 264-265 C.sub.19 H.sub.21 N.sub.4 O.sub.2 F 64.03
5.94 15.72 63.89 5.97 15.65 50b. 83
155-157 C.sub.20 H.sub.23 N.sub.4 O.sub.2 F.1/4H.sub.2 O 64.07 6.32
14.94 63.97 6.26 14.89.sup.c 51a. 18.sup.a 257-259 C.sub.19
H.sub.21 N.sub.4 O.sub.2 Cl 61.21 5.68 15.03 61.31 5.74 15.09 51b.
67 164-166 C.sub.20 H.sub.23 N.sub.4 O.sub.2 Cl 60.00 6.17 13.99
59.67 5.79 13.84.sup.c 52a. 48 250-253 C.sub.21 H.sub.26 N.sub.4
O.sub.4.1/4H.sub.2 O 62.59 6.63 13.90 62.82 6.63 13.44 52b. 78
164-164 C.sub.22 H.sub.28 N.sub.4 O.sub.4.3/4H.sub.2 O 62.03 6.98
13.15 62.26 6.75 12.79.sup.c 53a. 100 150-152 C.sub.21 H.sub.26
N.sub.4 O.sub.4.2/5H.sub.2 O 62.18 6.66 13.81 62.54 6.41
13.44.sup.c 53b. 59 166-167 C.sub.22 H.sub.28 N.sub.4 O.sub.4 64.06
6.84 13.58 64.20 6.90 13.42.sup.c 54a. 78 275-278 C.sub.19 H.sub.20
N.sub.4 O.sub.2 F.sub.2.3/4H.sub.2 58.83 5.59 14.44 59.09 5.26
14.30 54b. 85 161-163 C.sub.20 H.sub.22 N.sub.4 O.sub.2
F.sub.2.0.9H.sub.2 59.37 5.93 13.9 59.12 5.92 14.26 55a. 32 241-244
C.sub.22 H.sub.28 N.sub.4 O.sub.5 61.67 6.59 13.08 61.59 6.61 13.04
55b. 88 107.5-109 C.sub.23 H.sub.30 N.sub.4 O.sub.5 61.43 6.83
12.66 62.16 6.85 12.60 56a. 11 252-254 C.sub.22 H.sub.28 N.sub.4
O.sub.5 61.67 6.59 13.08
61.56 6.61 13.06 56b. 82 193-194 C.sub.23 H.sub.30 N.sub.4
O.sub.5.1/2H.sub.2 O 61.18 6.92 12.41 61.44 6.80 12.44 57. 67
158-160 C.sub.16 H.sub.19 N.sub.5 O.sub.2 61.33 6.11 22.35 61.40
6.14 22.32.sup.c 58a. 78 >280 C.sub.16 H.sub.14 N.sub.4
O.sub.4.1/2H.sub.2 O 57.31 4.51 16.71 57.51 4.42 16.46 58b. 99
273-275 C.sub.17 H.sub.16 N.sub.4 O.sub.4 60.00 4.74 16.46 59.88
4.86 16.26
__________________________________________________________________________
a. Yield calculated from 1,3dialkyl-6-amino-5-nitrosouracil. b.
Analyses: % N found (calcd.) 15b, 17.60 (18.62); 19b. 15.86
(16.71); 25, 16.00 (16.66); 27a, 17.58 (18.11); 30b, 18.15 (20.63);
43a, 14.06 (15.06); 43b, 13.37 (14.17); % C found (calcd.) 15a,
62.42 (61.85); 40, 58.66 (59.28). c. Accurate mass, measured (ppm
from calculated), in EI mode, unless noted: 18, 298.1055 (-3.7);
22b, 311.1373 (5.6); 23, 353.1483 (-1.4); 24, 411.1556 (3.2); 25
411.1894 (-3.1); 26, 511.2450 (3.7); 27a, 300.1018 (2.3); 29b,
326.1371 (-2.4); 30a, 325.1537 (-0.5); 30b, 339.1688 (-4.1); 32a,
342.1326 (-0.6); 35, 372.1436 (0.7); 36, 414.1543 (0.9); 38 (FAB),
444.2255 (0.8); 39, 543.2684 (-1.7); 40, 441.2001 (-2.5); 41,
483.2131 (2.7); 42, 541.2544 (1.4); 45a (FAB), 401.1812 (-1.3);
45b, 414.1898 (-1.3); 50b, 370.1795 (-2.7); 51b, 386.1492 (-4.5);
52b, 412.2110 (-0.1); 53a, 398.1937 (-4.3); 53b, 412.2093 (-4.3);
57, 313.1521 (-5.7). d. From compound 44b. e. From compound 39.
EXAMPLE 2
This example describes the use of a palladium-catalyzed Heck
reaction to attach an 8-vinyl or 8-styryl group to a xanthine.
8-styryl- and 8-vinyl-xanthine derivatives were synthesized as
shown in FIG. 3. FIG. 3 is a schematic diagram of the synthesis,
wherein methyl iodide and heat were used in the first step,
CH.sub.2 .dbd.CHCO.sub.2 C(CH.sub.3).sub.3, Pt(OAc).sub.2, and
(o-Tol).sub.3 P were used in the second step, and trifluoroacetic
acid (TFA) was used in the last step.
For example, a mixture of 8-bromo-caffeine (450 mg, 1.65 mmol),
tert-butylacrylate (0.390 ml, 2.69 mmol), Pd(AcO).sub.2 (3.7 mg,
16.5 .mu.mol), tri-o-tolylphosphine (20 mg, 66 .mu.mol),
triethylamine (2 ml) and acetonitrile (2 ml) was warmed at
100.degree. C. for 16 h with stirring in a capped tube. After
cooling to room temperature, CHCl.sub.3 was added and the mixture
was filtered. The organic layer was extracted twice with 1N HCl,
washed with brine several times, dried (MgSO.sub.4), and then
evaporated to dryness. The residue was created with MeOH (1 ml),
and hexane was added, to afford 152 mg of the crystalline product
8-(trans-2-tert-Butyloxycarbonylvinyl)-1,3,7-trimethylxanthine. The
mother liquors were evaporated, and the remaining product was
purified by preparative TLC (hexane:ethyl acetate 1:1) to give 49
mg (38% overall). mp: 214.degree.-215.degree. C. .sup.1 H NMR
DMSO-d.sub.6 : d 1.48 (s, 9H, CH.sub.3), 3.22 (s, 3H, NCH.sub.3),
3.42 (s, 3H, NCH.sub.3), 4.03 (s, 3H, N.sub.7 CH.sub.3), 6.73 (d,
1H, J=15 Hz), 7.51 (d, 1H, J=15 Hz). MS (CI NH.sub.3) m/e 321
(MH.sup.+).
8-(trans-2-tert-Butyloxycarbonylvinyl)-1,3,7-trimethylxanthine (76
mg, 238 .mu.mol) was dissolved in 3 ml TFA and stirred for 1 h.
After evaporation, the residue was triturated with ether to provide
the pure product 8-(trans-2-Carboxyvinyl)-1,3,7-trimethylxanthine
(55 mg, 88% yield). mp: 278d .degree.C. .sup.1 H NMR DMSO-d.sub.6 :
d 3.27 (s, 3H, NCH.sub.3), 3.44 (s, 3H, NCH.sub.3), 4.02 (s, 3H
N.sub.7 CH.sub.3), 6.78 (d, 1H, J=15.4 Hz), 7.55 (d, 1H, J=15.4
Hz), 8.4 (br s, 1H, COOH). MS (CI NH.sub.3) m/e 265 (MH.sup.+).
Alternatively, compound
8-(trans-2-Carboxyvinyl)-1,3,7-trimethylxanthine was prepared from
8-(trans-2-tert-Butyloxycarbonylvinyl)-1,3,7-trimethylxanthine in
DMF/water (1:1) solution by saponification with sodium hydroxide in
49% yield.
TABLE III ______________________________________ Affinities of
8-styryl xanthine derivatives radioligand binding assays at rat
brain A.sub.1 and A.sub.2 receptors,.sup.a wherein R.sub.1,
R.sub.3, and R.sub.7 are methyl, and R.sub..beta. is hydrogen or
methyl ##STR9## X = K.sub.i (A.sub.1).sup.a K.sub.i (A.sub.2).sup.a
A.sub.1 /A.sub.2 ratio ______________________________________
n-propyl(R.sub..beta. = H) 6,000 1,600 3.8 C(O)OC(CH.sub.3).sub.3
(R.sub..beta. = H) 18,000 590 31 C(O)OH(R.sub..beta. = H)
>100,000 30,000 >3 phenyl (R.sub..beta. = Me) 8,680 .+-.
1,420 .+-. 160 6 2300 C(O)NH-phenyl(R.sub..beta. = H) 50,000 2,530
.+-. 520 19.8 ______________________________________ a. Expressed
in nM (single determination or mean .+-. S.E.M. for 3 or mor
determinations) vs. [.sup.3 H]PIA (1 nM) at rat A.sub.1 -receptors
and vs [.sup.3 H]CGS21680 (5 nM) at rat striatal A.sub.2
-receptors.
EXAMPLE 3
This example describes a radioligand binding assay, which was used
to assess the affinity of the 1,3,7-trialkyl-8-substituted xanthine
compounds for adenosine receptors.
The 1,3,7-trialkyl-8-substituted xanthine compounds of the present
invention were tested in a radioligand binding assay for affinity
at adenosine receptors in rat brain membranes. The compounds were
assayed for affinity at rat A.sub.1 cortical receptors using
[.sup.3 H]N.sup.6 -phenylisopropyladenosine (Schwabe et al.,
Naunyn-Schmiedenberg's Arch. Pharmacol., 313, 179-187 (1980)) and
at rat striatal A.sub.2a receptors using [.sup.3 H]CGS 21680
(Tables I, III, and IV) (Jarvis et al., J. Pharmacol. Exp. Therap.,
251, 888-893 (1989)).
Rat cerebral cortical membranes and striatal membranes were
prepared (Francis et al., 1980, supra; and Sarges et al., 1990,
supra) and treated with adenosine deaminase (2 U/ml) for 30 min at
37.degree. C. prior to storage at -70.degree. C. Solid samples of
the adenosine derivatives were dissolved in DMSO and stored in the
dark at -20.degree. C. The stock solutions were diluted with DMSO
to a concentration of .gtoreq.0.1 mM prior to adding to the aqueous
medium. The final concentration of DMSO in the assay medium was
generally 2%.
Inhibition of binding of 1 nM [.sup.3 H]N.sup.6
-phenylisopropyladenosine (Dupont NEN, Boston, Mass.) to A.sub.1
receptors in rat cerebral cortex membranes was measured as
described (Schwabe et al., 1980, supra). Membranes (.about.100
.mu.g protein per tube) were incubated for 1.5 h at 37.degree. C.
in a total volume of 0.5 ml of 50 mM Tris hydrochloride, at pH 7.4.
Test drugs were dissolved in DMSO and added in 10 .mu.l aliquots,
resulting in a final DMSO concentration of 2%. Bound and free
radioligand were separated by addition of 3 ml of a buffer
containing 50 mM Tris hydrochloride, pH 7.4, at 5.degree. C.,
followed by vacuum filtration using a Brandel Cell Harvester
(Brandel, Gaithersburg, Md.) and a Whatman GF/B glass fiber filter
with additional washes totaling 9 ml of buffer. Non-specific
binding was determined with 10 .mu.M 2-chloroadenosine.
Inhibition of binding of 5 nM [.sup.3 H]CGS 21680
(2-[4-[(2-carboxyethyl)-phenyl]ethylamino]-5'-N-ethylcarboxamido-adenosine
) was carried out as follows. Membranes (.about.80 .mu.g protein
per tube, prepared according to Jarvis et al., 1989, supra) were
incubated for one hour at 25.degree. C. in a total volume of 0.5 ml
of 50 mM Tris hydrochloride 50 mM, containing 10 mM MgCl.sub.2 at
pH 7.4. Test drugs were dissolved in DMSO and added in 10 .mu.l
aliquots, resulting in a final DMSO concentration of 2%.
Non-specific binding was defined using 20 .mu.M 2-chloroadenosine.
Filtration was carried out using a Brandel Cell Harvester, as
above, using Tris HCl/MgCl.sub.2 as the washing buffer.
At least six different concentrations spanning three orders of
magnitude, adjusted appropriately for the IC.sub.50 of each
compound, were used. IC.sub.50 values, computer-generated using a
non-linear regression formula on the GraphPAD program (Institute
for Scientific Information), were converted to apparent K.sub.i
values using K.sub.D values (Francis et al., 1988, supra; and
Sarges et al., 1990, supra) of 1.0 and 14 nM for [.sup.3 H]PIA and
[.sup.3 H]CGS 21680 binding, respectively, and the Cheng-Prusoff
equation (Cheng et al., Biochem. Pharmacol., 22, 3099-3108
(1973)).
Small alkyl substituents at the 1 and 3 position were identical and
varied from methyl to propyl. Substituents at the 7-position varied
from H to 2-phenylethyl. A number of related xanthines (not
8-styryl) were prepared for comparison (Table IV). K.sub.i values
of nearly 10.sup.-8 M at A.sub.2 receptors and selectivities of
hundreds of fold were achieved.
TABLE IV ______________________________________ Affinities of
related xanthine derivatives in radioligand binding assays at rat
brain A.sub.1 and A.sub.2 receptors..sup.a ##STR10## Com- K.sub.i
A.sub.1 /A.sub.2 pound R.sub.7 = R.sub.8 = K.sub.i (A.sub.1).sup.a
(A.sub.2).sup.a ratio ______________________________________ 2. Me
cyclohexyl [28,000].sup.b 17,100 1.6 57. Me 2-(3-amino- 15%.sup.c
(10.sup.-5) 18,000 phenyl)ethyl 58a. H 7-methoxybenzofuran- 1,700
.+-. 70 3,900 .+-. 0.5 2-yl 940 58b. Me 7-methoxybenzofuran- 4,740
2-yl ______________________________________ a. Expressed in nM
(single determination or mean .+-. S.E.M. for 3 or mor
determinations) vs. [.sup.3 H]PIA (1 nM) at rat A.sub.1 -receptors
and vs [.sup.3 H]CGS21680 (5 nM) at rat striatal A.sub.2
-receptors. b. Shamim et al., J. Med. Chem., 32, 1231-1237 (1989)
c. Percent displacement of specific binding at the concentration
indicate in parentheses.
The greatest effect of elongating N--Me to N--Pr groups at the N-1
and N-3 positions was a substantial increase in A.sub.1 -affinity,
thus diminishing A.sub.2 -selectivity. A
1,3-diethyl-7-methylxanthine, 45b, was nearly as A.sub.2 -selective
(34-fold) as the 1,3-dimethyl analogue, 44b, which was 70-fold
selective. The corresponding diallyl analogue, 46 (reported
previously by Shimada et al., 1992, supra) to be >6700 A.sub.2
-selective), was only 13-fold selective in rat brain in this
study.
The N-7 position was either H-- or substituted with groups as large
as 2-phenylethyl (compound 44f). Only small, hydrophobic groups
(including ethyl and propargyl) at this position were tolerated in
binding to either receptor. The 7-methyl analogues were found to
exhibit the greatest degree of A.sub.2 -selectivity.
FIGS. 4 and 5 are graphs of K.sub.i (nm) for 7-methyl analogues
versus K.sub.i (nM) for 7-H analogues, which show correlations of
affinity at adenosine receptors for the 7-H to 7-Me modification,
which generally results in decreased A.sub.1 affinity and increased
A.sub.2 affinity. The correlations of affinity for the 7-H to
7-methyl modification in 1,3-dimethyl-8-styryl-xanthine derivatives
is shown in FIG. 4, whereas the correlations of affinity for the
7-H to 7-methyl modification in 1,3-dipropyl-8-styryl-xanthine
derivatives is shown in FIG. 5. In both figures, inhibition
constants in nM are given for A.sub.1 (.quadrature., FIG. 4;
.smallcircle., FIG. 5) and A.sub.2 (.box-solid., FIG. 4;
.circle-solid., FIG. 5) receptors. In general, among
8-styrylxanthine derivatives, the 1,3,7-trimethylxanthines were
A.sub.2 -selective by factors between 10 and 500-fold, whereas the
corresponding 1,3-dimethylxanthines were generally A.sub.2
-selective by factors of only 2 and 5-fold. The 7-hydroxyethyl and
phenylethyl substituents were nearly inactive, in addition to
having less favorable aqueous solubility. In the 1,3-dipropyl
series (FIG. 5), each 7-H analogue was relatively non-selective.
The selectivity of the 1,3-dipropyl-7-methyl-8-styryl xanthines
(resulting from decreased Al affinity upon methylation) was highly
dependent on the styryl substitution.
The effects of substitution of the 8-styryl group could be compared
within the 1,3-dimethyl series and within the 1,3,7-trimethyl
series. The unsubstituted styryl analogue 15a (7-H) was
non-selective, but was moderately selective (41-fold) following
methylation (15b). Fluorine substitution in the .alpha.-position
resulted in diminished potency at both A.sub.1 - (3-fold) and
A.sub.2 -receptors (7-fold). Monomethoxy substitution of the phenyl
ring (compounds 17, 19, and 29) resulted in selectivity of 18- to
63-fold in the 7-Me series, but did not result in significant
A.sub.2 -selectivity in the 7-H series. Compound 19, the meta
derivative, was the most potent and selective monomethoxy
derivative, with a K.sub.i value of 85 nM at A.sub.2 -receptors.
The analogue bearing a 3-hydroxystyryl group in the 7-H series, 18,
was equipotent with the methoxy compound, 19b, at A.sub.2
-receptors and more potent at A.sub.1 -receptors.
The A.sub.2 -potency of 1,3,7-trimethyl-xanthines having a variety
of styryl 3-position substituents varied in the order:
acetylamino>chloro, amino>fluoro,
methoxy>H>trifluoromethyl>nitro. Although the 3-chloro
derivative (28, K.sub.i value of 54 nM) was slightly less potent
than the 3-acetylamino derivative (23, K.sub.i value of 39 nM,
240-fold selective), it was more selective (520-fold). It was
equipotent to the amino derivative, 22b, but considerably more
selective. Very bulky substituents at the 3-position (urethanes 25
and 26) reduced potency at A.sub.2 -receptors roughly 20-fold, but
moderate A.sub.2 -selectivity remained. A water-solubilizing
3-succinylamino group (24) resulted in decreased potency (134 nM)
but high selectivity (250-fold).
For comparison to the methoxy group at the styryl 4-position, a
highly electron donating group, e.g. dimethylamino, was
incorporated and resulted in greatly diminished potency at both
receptors. Only the 7-Me form, 30b, displayed A.sub.2
-selectivity.
Dimethoxy substitutions at various positions of the phenyl ring
were compared, and substantial differences were observed. The order
of both potency and selectivity was 3,5>3,4>2,3. In the
1,3,7-trimethyl series, 3,5-dimethoxy or 3,5-difluoro substituents
(33b and 34b, respectively) resulted in >200-fold
selectivity.
In the 1,3-dipropyl-7-methyl- series, A2-selectivity was generally
merely 5- to 19-fold, with only one exception (53b). The
3-chlorostyryl analogue, 51b, analogous to the most selective agent
in the 1,3,7-trimethyl series, was only 14-fold selective.
1,3-Dipropyl-7-methyl-8-(3,5-dimethoxystyryl)xanthine, 53b, proved
to be a potent (K.sub.i vs. [.sup.3 H]CGS 21680 was 24 nM) and
A.sub.2 -selective (110-fold) adenosine antagonist, i.e., 5-fold
more selective than the corresponding 3,4-dimethoxy analogue, 52b.
Compound 52b was prepared by Shimada et al. (1992, supra,
[KF17837]) and was reported to be 190-fold selective, versus
19-fold in this study.
High selectivities were also observed among 1,3,7-dimethylxanthines
that were trisubstituted on the phenyl ring.
1,3,7-trimethyl-8-(3,4,5-trimethoxy)-styryl-xanthine, 44b, was
70-fold A.sub.2 -selective in binding in the rat brain (versus
>5600-fold reported by Shimada et al., 1992, supra). The
corresponding 1,3-dimethyl analogue was only 10-fold A.sub.2
-selective. In general, the order of both potency and selectivity
for trisubstituted phenyl substituents was 3,4,5>2,3,4>2,4,5.
Among 3,4,5-substituted analogues there was considerable
substitution of the 4-methoxy group tolerated at A.sub.2
-receptors. The moderately selective 3,5-dimethoxy-4-hydroxy
analogue, 35, was acylated (36) and alkylated (37, 38), resulting
in enhanced A.sub.2 -selectivity and potency. The
4-acetoxy-3,5-dimethoxy analogue, 36, was 93-fold A.sub.2
-selective. Functional groups that also tended to increase water
solubility, such as alkyl amines (38 and 40) were included. These
amino derivatives may serve as functionalized congeners (Jacobson,
J. Med. Chem., 32, 1043-1051 (1989a)) since it appears that long
chain extension is possible without disrupting receptor binding.
Moderately potent and selective acylated derivatives were prepared
from the amine functionalized. Butyl versus trans-butenyl amine
were compared to examine the effect of altering conformational
flexibility at this distal site. No major differences in potency or
selectivity between butyl and butenyl analogues were found.
In an attempt to account for the discrepancy in K.sub.i values
between the present study and Shimada et al. (1992, supra), the
effects of varying concentrations of DMSO in the assay medium were
examined. DMSO was needed because of the limited aqueous solubility
(in the range of 10.sup.-5 M) of most of the 8-styrylxanthines
tested. To avoid precipitation associated with serial aqueous
dilutions, the only point at which DMSO was added to aqueous medium
was immediately prior to the incubation.
The effects of varying concentrations of DMSO (ranging from 0.5-6%)
on the apparent affinity of compound 53b (FIG. 6A) was measured.
FIG. 6A is a graph of IC.sub.50 (nM, mean.+-.S.E.M. for 3 or more
determinations) versus % DMSO for [.sup.3 H]PIA (1 nM) at rat A
receptors (squares) and [.sup.3 H]CGS21680 (5 nM) at rat striatal
A.sub.2 receptors (circles), which shows the dependence of observed
IC.sub.50 values on DMSO concentration in competitive radioligand
binding assays. The apparent affinity of compound 53b at A.sub.2
receptors was constant within the range of 0.5% to 6% DMSO. In
addition, the total specific binding of [.sup.3 H]CGS 21680 to
striatal membranes was maintained, even at 6% DMSO. However,
A.sub.1 affinity appeared to be somewhat dependent on DMSO
concentration (at 0.5 and 1% DMSO), and at 6% DMSO the total
specific binding of [.sup.3 H]PIA (data not shown) diminished to
roughly 30% of its value at 1%. At the lowest concentration (0.5%
DMSO), higher concentrations of the drug were required to displace
[.sup.3 H]PIA. This effect of increase in the apparent K.sub.i
value at .ltoreq.1% DMSO most likely relates to the xanthine
precipitating from the solution, since the UV absorption does not
increase in a linear fashion with the amount of xanthine added to a
fixed aqueous volume as shown in FIG. 5B, which is a graph of
absorption units at 345 nm versus theoretical concentration, which
shows the UV absorption of water solutions following addition of
1,3-dipropyl-8-(3,5-dimethoxystyryl)-xanthine dissolved in 0.5%
DMSO (theoretical final concentration assuming complete dissolution
given on abscissa), with a peak absorption occurring at 345 nm with
a molar extinction coefficient (.epsilon.) of 13,200. The UV
absorption decreases beyond 20 .mu.M, suggesting
supersaturation.
Related, non-styryl xanthines (Table IV) were tested in adenosine
receptor binding for comparison to the 8-styryl derivatives.
Cyclohexylcaffeine, 2, which was found to be A.sub.2 selective in
effects on adenylate cyclase (Shamim et al., J. Med. Chem., 32,
1231-1237 (1989)), was non-selective in binding. The saturated
aniline derivative 57 was .about.300 fold-less potent at A.sub.2
receptors than the corresponding styryl derivative, 22b.
Ring-constrained styryl analogues, 58, containing a
8-(2-benzofuran) group were synthesized. Both the 7-H and 7-Me
analogues were only weak antagonists of binding at adenosine
receptors (Table IV).
The selectivity factors in the present study were generally much
less than in Shimada et al. (1992, supra). The principal reason may
be that A.sub.1 -affinity in this study was measured in the same
species as A.sub.2 -affinity (rat), whereas Shimada et al. measured
A.sub.1 affinity in guinea pig brain and A.sub.2 affinity in rat
brain. The species dependence of affinity of alkylxanthines at both
A.sub.1 and A.sub.2a receptors is well documented (Ukena et al.,
FEBS Letters, 209, 122-128 (1986a); Stone et al., Drug Dev. Res.,
15, 31-46 (1988)). Invariably, A.sub.1 affinity is higher in the
rat than in the guinea-pig, but the affinity ratios have been found
to vary from only 2-fold for theophylline to as much as 20-fold for
8-phenyltheophylline (Ukena et al., 1986a, supra). Indeed, the
A.sub.1 affinities in rat reported here differ even more: up to
33-fold (e.g., compound 47: A.sub.1 affinity in rat is 55 nM versus
1800 nM in guinea-pig (Shimada et al., 1992, supra); Erickson et
al., J. Med. Chem., 34, 1431-1435 (1991) have determined a K.sub.i
value at rat A.sub.1 receptors of 22 nM). Thus, comparing
guinea-pig A.sub.1 values to rat A.sub.2 affinities results in
artificially high selectivity ratios. Therefore, the affinities
reported by Shimada et al. are inaccurate, given that same-species
comparisons were not performed. In addition, some unexplained and
substantial differences (e.g. compound 50a) were observed between
K.sub.i -values versus [.sup.3 H]CGS 21680 in this study and versus
[.sup.3 H]NECA in Shimada et al. (1992, supra) (both having been
measured in rat striatal membranes).
Another potential reason for discrepancies with previous results in
binding assays was the amount of DMSO present. Shimada et al.
(1992, supra) utilized approximately 1% DMSO in the assay medium,
whereas 2% was used in this study. At 0.5% DMSO a
1,3-dipropyl-7-methylxanthine derivative, 53b, did not remain
dissolved in aqueous solution at concentrations greater than 10
.mu.M (FIG. 5A). This would affect, in particular, A.sub.1
displacement curves for many compounds in this study, for which
data points beyond xanthine concentrations of 10 .mu.M are
required. Thus, the addition of insufficient DMSO to the medium (or
serial aqueous dilutions) might tend to overestimate the
selectivity of the A.sub.2 -selective xanthines, but would not be
expected to alter the apparent affinity at A.sub.2 receptors (FIG.
5A).
In summary, the position of styryl ring substitution (meta favored)
is a determinant of potency and selectivity (compare 17b, 19b, and
29b). Increasing the size of small alkyl groups at the 1- and
3-xanthine position (e.g. 45b versus 44b) increases potency at both
receptors and decreases A.sub.2 selectivity. A.sub.2 -selectivity
and moderate affinity are maintained with long chain extension from
the para-position of the styryl ring (e.g. 41). It would seem that
this position of the 8-styryl group, when bound to the receptor, is
located in a relatively insensitive region. A.sub.2 -selectivities
of thousands of fold reported previously (Shimada et al., 1992,
supra) were not observed in this study, although the selectivities
of up to 520-fold (compound 28a), promise to be useful in
physiological studies. A.sub.2 -antagonists of particular interest
are: compounds 23, 24, 27b, 28a, 33b, and 34b (A.sub.2 -selectivity
of 200-fold or greater); compounds 23, 28a, 49, 50b, 52b, 53b, and
54b (A.sub.2 -affinity 50 nM or less); compounds 22b, 38, and 40
(amine functionalized). Compound 24 also has enhanced water
solubility; the maximal solubility in a 0.1M potassium phosphate
solution at pH 7.4 was 19 mM.
EXAMPLE 4
This example describes the synthesis of
8-(3-isothiocyanatostyryl)-caffeine, which is a selective
irreversible inhibitor of binding to A.sub.2a -adenosine
receptors.
2-Chloroadenosine was obtained from Research Biochemicals, Inc.
(Natick, Mass.). [.sup.3 H]N.sup.6 -phenylisopropyladenosine, and
[.sup.3 H]CGS 21680 were obtained from Dupont NEN (Boston,
Mass.).
1,3,7-trimethyl-8-(3-aminostyryl)-xanthine (50 mg, 0.16 mmol) was
dissolved in 2 ml chloroform, and saturated sodium bicarbonate
solution (1 ml) was added. After cooling the mixture in an ice
bath, thiophosgene (0.1 ml, 1.3 mmol) was added at once with
vigorous stirring. After 5 min, the reaction was complete, and
additional solvent was added to break the emulsion. The phases were
separated, and the organic phase was washed several times with
water and dried (MgSO.sub.4). The solvent was evaporated, and the
solid yellow residue was recrystallized from
chloroform/acetonitrile to provide 32 mg (57% yield) of the
homogeneous product, 8-(3-isothiocyanatostyryl)caffeine (ISC) or
1,3,7-trimethyl-8-(3-isothiocyanatostyryl)xanthine hemi-hydrate
(TLC system chloroform: methanol: acetic acid, 95:4:1, R.sub.f
=0.41). Mp 268.degree.-271.degree. C. .sup.1 H NMR CDCl.sub.3 d
3.43 (s, 3H N--CH.sub.3); 3.63 (s, 3H N--CH.sub.3); 4.07 (s, 3H
N7-CH.sub.3); 6.93 (d, 1H J=16 Hz, olefin); 7.21 (d, 1H J=8 Hz);
7.39 (t, 1H J=8 Hz, C5 arom); 7.44 (s, 1H, C2 arom); 7.47 (d, 1H
J=8 Hz); 7.75 (d, 1H J=16 Hz, olefin). MS (EI) M.sup.+ 353. IR
(NaBr) 2124 cm.sup.-1. Elemental analysis (C.sub.17 H.sub.15
N.sub.5 O.sub.2 S.0.5 H.sub.2 O): calculated, 56.34% C, 4.45% H,
19.33% N; found 56.43% C, 4.16% H, 19.07% N.
EXAMPLE 5
This example describes the radioligand binding assay that was used
to assess the irreversible, inhibitory activity of ISC at A.sub.2
-adenosine receptors.
Striatal tissue was isolated by dissection of rabbit, bovine, and
rat brain, obtained frozen from Pel-Freeze Biologicals Co. (Rogers,
Ark.), and guinea pig brain, obtained frozen from Keystone
Biologicals (Cleveland, Okla.). Membranes were homogenized in 20
volumes of ice cold 50 mM Tris HCl (pH 7.4) using a Polytron
(Kinematica, GmbH, Lucerne, Switzerland) at a setting of 6 for 10
sec. For each species except rat, the homogenization was carried
out in the presence of protease inhibitors (5 mM EDTA, 0.1 mM
phenylmethanesulfonyl fluoride, 0.01 mg/ml soybean trypsin
inhibitor, 5 .mu.g/ml leupeptin, 1 .mu.g/ml pepstatin A). The
membrane suspension was then centrifuged at 37,000.times.g for 10
min at 4.degree. C. The pellet was resuspended (20 mg tissue/ml) in
the above buffer solution, preincubated at 30.degree. C. for 30 min
with 3 IU/ml of adenosine deaminase, and the membranes were again
homogenized and centrifuged. Finally the pellet was suspended in
buffer (100 mg wet weight per ml) and stored frozen for no longer
than two weeks at -70.degree. C. Protein was determined using the
BCA protein assay reagents (Pierce Chemical Co., Rockford,
Ill.).
Striatal membranes were treated with inhibitor as follows.
Membranes were incubated with ISC in pH 7.4 Tris buffer containing
adenosine deaminase for 1 h at 25.degree. C., and subjected to
three washing cycles, which consisted of centrifugation at
37,000.times.g and resuspension of the pellet in Tris buffer, prior
to radioligand binding. For kinetic experiments with the affinity
label, aliquots were removed periodically and quenched with a large
volume of buffer solution (30.times.) prior to radioligand binding.
For protection experiments, membranes were preincubated with
theophylline at 25.degree. C. for 20 min, and then ISC was added
immediately for an additional incubation at 25.degree. C. for 30
min. At the end of this sequence, the membranes were washed by
repeated centrifugation and resuspension and subjected to [.sup.3
H]CGS 21680 binding.
Washing cycles for inhibition experiments required resuspending the
membrane pellet by gentle vortex mixing. At the final step, prior
to radioligand binding, the membranes were homogenized manually
using a glass tissue grinder.
In competition studies, to avoid precipitation of the xanthine in
the 100 .mu.M concentration range, the tubes in that range
containing all components were warmed to .about.50.degree. C.,
prior to the incubation carried out for 90 min at 37.degree. C.
For saturation and competition studies, B.sub.max, K.sub.d, and
IC.sub.50 values were determined using the Ligand and Inplot
(Graphpad, San Diego, Calif.) computer programs. IC.sub.50 values
were converted to apparent K.sub.i values using K.sub.D values in
rat striatum of 1.0 and 15 nM for [.sup.3 H]PIA and [.sup.3 H]CGS
21680 binding, respectively, and the Cheng-Prusoff equation (Cheng
and Prusoff, 1973, supra).
Competition by ISC of binding of [.sup.3 H]CGS 21680 (an A.sub.2a
-selective agonist) and [.sup.3 H]R-PIA (an A.sub.1 -selective
agonist) in striatal membranes from four species was measured
(Table V) under "reversible" conditions. Major species differences
have been noted previously for xanthines binding at A.sub.2a
-adenosine receptors (Stone et al., 1988, supra). In rat striatum,
the IC.sub.50 at A.sub.2a -receptors was found to be 146 nM
(corresponding to an apparent K.sub.i value of 111 nM, assuming
reversibility). At A.sub.1 -receptors the IC.sub.50 was found to be
43 .mu.M (corresponding to a K.sub.i value of 20 .mu.M). Thus, the
selectivity ratio of ISC for A.sub.2a - versus A.sub.1 -receptors
in the rat based on IC.sub.50 values was 290-fold (180-fold, based
on K.sub.i values). The selectivity ratio in guinea pig striatum
was nearly identical. In other species, A.sub.2a -selectivity was
maintained (bovine, 120-fold, and rabbit, 180-fold), although the
affinity was diminished. At rabbit A.sub.2a receptors, the apparent
K.sub.i value of ISC was 290 nM based on the reported K.sub.d value
of 28.6 nM for binding of [.sup.3 H]CGS 21680 (Jacobson et al.,
Mol. Pharmacol., 42, 123-133 (1992)). The Hill coefficients for
displacement of binding of [.sup.3 H]CGS 21680 in the four species
were approximately equal to 1. The A.sub.2 -selectivity of ISC was
consistent with the previously determined A.sub.2 -selectivity of
the amino precursor and the 3-chloro derivative (30-fold and
520-fold selectivity, respectively, based on K.sub.i values).
TABLE V ______________________________________ Potencies of ISC in
inhibiting radioligand binding at central A.sub.1 and A.sub.2a ,
receptors in four mammalian species..sup.a Species IC.sub.50
(A.sub.1).sup.a IC.sub.50 (A.sub.2).sup.a A.sub.1 /A.sub.2 ratio
______________________________________ Rat 42,600 .+-. 3600.sup.b
146 .+-. 2.6.sup.c 291 Guinea pig 51,400 .+-. 17,700 160 .+-. 1.6
320 Bovine 63,400 .+-. 5,900 516 .+-. 64 122 Rabbit 75,600 .+-. 12
413 .+-. 135.sup.d 183 ______________________________________ a.
Express in nM (single determination or mean .+-. S.D. for 3 or more
determinations vs. [.sup.3 H]PIA (1 nM) at striatal A.sub.1
-receptors an vs [.sup.3 H]CGS 21680 (5 nM) at striatal A.sub.2a
-receptors. Nonspecifi binding was determined in the presence of 10
.mu.M 2chloroadenosine. b. Corresponds to K.sub.i value of 20,300
.+-. 1700 nM. c. Corresponds to K.sub.i value of 111 .+-. 0.5 nM
and a selectivity rati of 182. d. Corresponds to K.sub.i value of
347 .+-. 112 nM.
ISC was examined for the ability to irreversibly inhibit A.sub.2a
-receptors. Preincubation of rat striatal membranes with ISC caused
a dose-dependent, irreversible antagonism of the binding of 5 nM
[.sup.3 H]CGS 21680 (an A.sub.2a -selective agonist), with an
IC.sub.50 value of 2.7 .mu.M (FIG. 7A). This IC.sub.50 value was
18-times greater than the IC.sub.50 value in competitive
displacement of [.sup.3 H]CGS 21680 in the same tissue (Table V).
Preincubation with 20 .mu.M ISC resulted in the loss of
approximately 80% of the specific binding of [.sup.3 H]CGS 21680.
The irreversible nature of inhibition by the isothiocyanate
derivative was demonstrated by the failure of repeated washing to
regenerate the A.sub.2a -receptor binding site. Nearly all of the
binding of [.sup.3 H]N.sup.6 -phenylisopropyladenosine (PIA) to
striatal A.sub.1 receptors was recovered following washout by
repeated cycles (4.times.) of centrifugation and resuspension of
the membranes in fresh buffer. Thus, at A.sub.1 -adenosine
receptors in rat striatal membranes, ISC at a high concentration of
20 .mu.M was barely effective as an irreversible inhibitor. At this
concentration only 12.+-.2.9% of [.sup.3 H]PIA binding was lost
compared to 81.+-.1.6% of [.sup.3 H]CGS 21680 binding.
Exposure of the ISC-treated striatal membranes to the weak
adenosine antagonist 3-isobutyl-1-methyl-xanthine (IBMX, 100 .mu.M)
overnight also did not regenerate any A.sub.2a -receptor binding
(data not shown). Treatment with IBMX was used to remove
non-chemically bound ligand from the membranes in a previous study
of chemically reactive xanthines as irreversible inhibitors of
A.sub.1 -receptors (Jacobson et al., 1989, supra). Such treatment
was found to be unnecessary, since no difference in binding was
observed. Increasing the temperature of pre-incubation with ISC to
37.degree. C. also did not affect significantly the fraction of
binding irreversibly inhibited (data not shown).
The irreversibility was examined in three other species (FIGS. 7B,
C, and D). FIGS. 7A, B, C, and D show the dose-dependent inhibition
by 8-(3-isothiocyanatostyryl)-caffeine (ISC) of radioligand binding
at A.sub.1 - and A.sub.2a -adenosine receptors in rat, guinea pig,
bovine, and rabbit striatal membranes (n=4 or more), respectively.
The preincubation with ISC or control was carried out for 1 h at
25.degree. C. and the subsequent binding assay involved a 90 min
incubation followed by rapid filtration. The radioligand binding
step consisted of incubation (n=3) with 5 nM [.sup.3 H]CGS 21680
for A.sub.2a -receptors or 1 nM [.sup.3 H]PIA for A.sub.1
-receptors. In the guinea pig striatum, the inhibition occurred at
concentrations similar to those used with rat striatum (EC.sub.50
value 2.8 .mu.M). In rabbit and bovine striatum, ISC caused an
irreversible inhibition of A.sub.2a receptors, but at considerably
higher concentrations than in rat striatum. The EC.sub.50 values
for ISC irreversibly inhibiting bovine and rabbit A.sub.2a
receptors were 8 and 10 .mu.M, respectively.
The irreversibility is likely due to the presence of the chemically
reactive isothiocyanate group, since the binding of the
corresponding analogue in which the isothiocyanate was replaced by
a chloro group was completely reversible (data not shown).
The time course for inactivation of rat A.sub.2a receptors by 2
.mu.M ISC is shown in FIG. 8. FIG. 8 is a graph of % inhibition of
binding versus time (min), which shows the time course for
inhibition of rabbit striatal A.sub.2a -adenosine receptors at
25.degree. C. by 2 .mu.M ISC. The membranes were washed by
centrifugation (3.times.) prior to radioligand binding. [.sup.3
H]CGS 21680 was used at a concentration of 5 nM. The curve
represents the data from three separate experiments. The time
course for inactivation was rapid, although the degree of
irreversible inhibition was not complete even after 2 h.
Approximately 3 min was required for inhibition of 50% of its final
value at 2 h (at 2 h approximately 55% of the specific [.sup.3
H]CGS 21680 binding relative to control membranes was lost). This
concentration was only 14-fold greater than the IC.sub.50 value for
ISC in the "competitive" binding assay vs. [.sup.3 H]CGS 21680
(Table V). The fraction of receptors inactivated by this
isothiocyanate derivative increased as the concentration of ISC was
raised (FIG. 7).
Saturation of binding of [.sup.3 H]CGS 21680 to rat striatal
receptors following treatment with ISC and washing was measured and
is shown in FIGS. 9A and B. FIG. 9A is a graph of CGS 21680 bound
(f mol/mg protein) versus CGS 21680 concentration (nM), which
represents the saturation curve for the binding of [.sup.3 H]CGS
21680 to A.sub.2a adenosine receptors in control (.smallcircle.)
and experimental (.circle-solid., i.e., following preincubation at
25.degree. C. for 1 h with 2 .mu.M ISC) rat striatal membranes.
FIG. 9B is a Scatchard transformation for the binding of [.sup.3
H]CGS 21680 to A.sub.2a adenosine receptors in rat striatal
membranes. The volume of incubation for radioligand binding
(approximately 150 .mu.g protein/tube) was 1 ml. Membranes were
incubated with radioligand at 25.degree. C. for 90 min. Specific
binding in control and treated membranes is shown. Non-specific
binding in control and treated membranes was nearly identical and
amounted to 8-10% of total binding at 5 nM [.sup.3 H]CGS 21680.
Following a preincubation resulting in partial inhibition, the
B.sub.max value relative to control membranes at the remaining
A.sub.2a sites was reduced without a significant effect on the
K.sub.d value. Following treatment with 5 .mu.M ISC, the K.sub.d
value for [.sup.3 H]CGS 21680 binding was 15.7 nM, and the
B.sub.max value was 450 fmol/mg protein, compared to 14.3 nM and
900 fmol/mg protein for control. When the ISC-treated membranes
were stored for one day at -20.degree. C. prior to the saturation
experiment, a reduction in B.sub.max (to 27 nM) was noted, while
the affinity of CGS 21680 in the control membranes was
unchanged.
Inhibition of binding of [.sup.3 H]CGS 21680 at A.sub.2a -receptors
by ISC could be prevented by the adenosine receptor antagonist
theophylline. The receptor was protected in the presence of 1 mM
theophylline, with degrees of protection of 45% and 37% at 0.5
.mu.M and 2 .mu.M ISC, respectively (FIG. 10). FIG. 10 is a bar
graph of inhibition of [.sup.3 H]CGS 21680 binding as a percentage
of control versus 0.5 .mu.M and 2 .mu.M concentration of ISC, which
shows theophylline protection of rat striatal A.sub.2a receptors
from ISC inhibition. The percent irreversible inhibition relative
to the level of specific binding of 5 nM [.sup.3 H]CGS 21680 in
control membranes is shown (n=3). Shaded bars are for ISC alone, at
the indicated concentration. Solid bars are for the combination of
ISC and theophylline (1 mM).
ISC appears to be moderately selective for A.sub.2a -versus A.sub.1
-receptors in four species. The chemical mechanism for the
irreversibility is presumably acylation by the reactive
isothiocyanate group of a nucleophilic group located on or in the
vicinity of the antagonist binding site of the receptor protein.
Following partial inactivation, the remaining rat A.sub.2a -binding
sites retained the same K.sub.d value for saturation by [.sup.3
H]CGS 21680. Thus, the inhibition is all-or-none, consistent with
covalent anchoring of the ligand in its usual binding site.
EXAMPLE 6
This example shows that 1,3,7-trimethyl-8-(3-chlorostyryl) xanthine
(CSC) is a highly selective A.sub.2 -adenosine receptor antagonist
in vivo.
CSC and
2-[(2-aminoethylamino)-carbonylethylphenylethylamino]-5'-N-ethylcarboxamid
oadenosine (APEC) were synthesized as described (Jacobson et al.,
J. Med. Chem., in press; Jacobson et al., J. Mol Recognit., 2,
170-178 (1989b)). All other xanthines and adenosine analogs are
commercially available.
Biochemical activity of CSC was determined as follows. Antagonism
of NECA-elicited stimulation of adenylate cyclase via an A.sub.2a
receptor in rat pheochromocytoma (PC12) cell membranes or in human
platelets was assayed as described (Ukena et al., Life Sc., 38,
797-807 (1986b)). Antagonism of N.sup.6
-phenylisopropyladenosine-elicited inhibition of adenylate cyclase
via an A.sub.1 receptor in rat adipocyte membranes was assayed as
described (Ukena et al., supra). K.sub.B values were calculated
using the Schild equation from the ratio of EC.sub.50 values for
agonist in the presence and absence of antagonist.
Locomotor activity of CSC was determined as follows. Adult male
mice of the NIH (Swiss) strain weighing 25-30 g were housed in
groups of 10 animals per cage with a light-dark cycle of 12:12 h.
The animals were given free access to standard pellet food and
water and were acclimatized to laboratory conditions for 24 h prior
to testing. Each animal was used only once in the activity
monitor.
Locomotor activity of individual animals was studied in an open
field using a Digiscan activity monitor (Omnitech Electronics Inc.,
Columbus, Ohio) equipped with an IBM-compatible computer. The
computer-tabulated measurements represent multivariate locomotor
analysis with specific measures, such as simultaneous measurements
of ambulatory, rearing, stereotypical, and rotational behaviors.
Data were collected in the morning, for three consecutive intervals
of 10 minutes each, and analyzed separately and as a group.
Statistical analysis was performed using the Student t test. The
results are reported as mean .+-. standard error for each point.
All drugs were dissolved in a 20:80 v/v mixture of Alkamuls EL-620
(Rhone-Poulenc, Cranbury, N.J.) and phosphate-buffered saline,
except for CSC, which was dissolved initially in DMSO and diluted
in at least 20 volumes of vehicle. Drugs were administered i.p. in
a volume corresponding to 5 ml/kg body weight. Where applicable,
the antagonist was injected 10 minutes before the agonist.
ED.sub.50 values were determined using regression analysis on the
InPlot software (GraphPAD, San Diego, Calif.). The results are
shown in Table VI.
TABLE VI
__________________________________________________________________________
Receptor affinities and effects of various xanthines on adenosine
agonist-elicited inhibition (A.sub.1) or stimulation (A.sub.2) of
adenylate cyclase. Values are means or means .+-. S.E.M. (n = 3-4).
Inhibition of Binding (K.sub.i, .mu.M) Adenylate Cyclase (K.sub.B,
.mu.M) Rat cortex.sup.a Rat striatum.sup.b Rat adipocytes Human
platelets Rat PC12 cells Behavorial Compound A.sub.1 A.sub.2a
A.sub.1 A.sub.2a A.sub.2a stimulation.sup.d
__________________________________________________________________________
caffeine 44 41 59.sup.a 30 37 +++(20) DMPX 45 16 94.sup.a 4.0 9.6
+++(10) CPT 0.024 1.4 n.d. 0.14 n.d. ++.sup.c (10) CPX 0.0009 0.47
0.0006.sup.b 0.14 0.25 -(1) CSC 28 0.054 1.32 .+-. 0.26 0.26 .+-.
0.07 0.060 .+-. 0.014 +(5)
__________________________________________________________________________
.sup.a vs. agonist ligand [.sup.3 H]N.sup.6
-phenylisopropyladenosine .sup.b vs. agonist ligand [.sup.3
H]Nethylcarboxamidoadenosine, except vs agonist ligand [.sup.3
H]CGS 21680 for CSC .sup.c stimulatory effect disappears within 20
min postinjection (Baumgol et al., Biochem. Pharmacol., 43, 889-894
(1992)) .sup.d degree of stimulation indicated by + through +++,
with a typical dose (mg/g, i.p.) shown in parentheses n.d. not
determined
In reversing adenosine agonist effects on adenylate cyclase (Table
VI), CSC was 22-fold selective for A.sub.2a receptors in rat
pheochromocytoma (PC12) cells versus A.sub.1 receptors in rat
adipocytes. CSC displayed a lower potency in adenylate cyclase
effects at A.sub.2a receptors in human platelets (K.sub.B 260 nM)
than at rat A.sub.2a receptors in PC12 cells (K.sub.B 60 nM). This
probably reflects the species difference: large differences in
potency of xanthines at adenosine receptors of different species
have been noted previously (Stone et al., 1988, supra).
The locomotor effects in mice of CSC alone or in combination with
the potent and A.sub.2a -selective agonist APEC (Nikodijevic et
al., 1991, supra) were examined. CSC administered i.p. at a maximum
soluble dose of 1 mg/kg was found to nearly completely reverse the
locomotor depression elicited by APEC at its previously determined
(Nikodijevic et al., supra) ED.sub.50 of 16 .mu.g/kg i.p. as shown
in FIG. 11A, which is graph of total distance traveled (cm/30 min)
versus CSC (mg/kg), which shows the locomotor activity in male NIH
Swiss mice (6 weeks) by the A.sub.2 -selective adenosine antagonist
CSC alone (.smallcircle.) or in the presence of the A.sub.2
-selective agonist APEC at 16 .mu.g/kg (.circle-solid.). A dose of
CSC of 5 mg/kg (injected as a suspension, since the solubility was
exceeded at 1 mg/ml of injection vehicle) was found to cause
significant locomotor stimulation by 22% over vehicle control
value. The total distance traveled in CSC animals was 4223.+-.496
cm/30 min (n=13) versus 3449.+-.198 cm/30 min (n=8) in controls.
This stimulation was most pronounced (56% increase versus control)
in the last 10 minutes of the 30 min monitoring period. Since CSC
was not very efficacious in stimulating locomotor activity at the
highest tested dose, the ED.sub.50 for CSC alone was not
determined. The concurrent administration of a 16 .mu.g/kg of dose
of APEC with 5 mg/kg CSC had no effect on the locomotor activity.
The drug combination resulted in a total distance traveled of
3949.+-.284 cm/30 min (n=14). This level of locomotor activity
represents a 73% increase versus APEC alone with 2277.+-.229 cm/30
min (n=13).
CSC (5 mg/kg) had no effect on locomotor depression elicited by the
potent Alagonist CHA at its determined ED.sub.50 value of 100
.mu.g/kg i.p. Coadministration of both drugs resulted in a total
distance traveled of 2029.+-.250 cm/30 min (n=8) versus 2090.+-.438
cm/30 min (n=9) for the CHA control.
Dose response curves for locomotor depression by APEC in the
absence and presence of CSC are presented in FIG. 11B, which is a
graph of total distance traveled (cm/30 min) versus APEC
(.mu.g/kg), which shows the locomotor depression in mice by APEC
alone (.DELTA.) or in the presence of CSC at 1.0 mg/kg
(.tangle-solidup.), where n=6-19. The following p values are as
indicated: * less than 0.005, ** less than 0.01, and *** less than
0.025. The ED.sub.50 for locomotor depression elicited by APEC was
right shifted from20 .mu.g/kg i.p. to 190 .mu.g/kg following
administration of 1 mg/kg CSC.
The A.sub.1 -selective antagonist CPX was administered alone and in
combination with CSC is shown in FIG. 12, which is a bar graph of
total distance traveled (cm/30 min) versus treatment method of
control, CSC, CPX, and CPX together with CSC, which shows the
synergism of an A.sub.1 selective antagonist, namely CPX (0.25
mg/kg, i.p.), and an A.sub.2 selective antagonist, namely CSC (1.0
mg/kg, i.p.) in stimulating locomotor activity in mice (n=9-19; *
represents a p value of less than 0.001 versus CSC alone). CPX
alone resulted in a total distance traveled of 3035.+-.330 cm/30
min (n=14) (i.e., a minimal depressant effect on locomotor activity
compared to control). CSC alone (1 mg/kg) had no significant effect
on locomotor activity, with a total distance traveled of
3550.+-.230 cm/30 min (n=19). However, the combination of the two
antagonists, each at a subthreshold dose, stimulated locomotor
activity by 37% (p<0.001) over CSC alone (total distance
traveled of 4861.+-.243 cm/30 min, n=9), suggesting a synergism of
A.sub.1 - and A.sub.2 -antagonist effects in the CNS. Following
coadministration, the average distance per move was increased by
approximately 30%, and clockwise and anti-clockwise rotations were
increased in the range of 30-60% (data not shown).
Since at the highest dose administered there was essentially no
effect on the locomotor depression elicited by CHA, CSC is a
functionally specific antagonist at A.sub.2a versus A.sub.1
receptors in mice in vivo.
Selective A.sub.1 and A.sub.2a antagonists alone are either
non-stimulatory or weakly stimulatory in locomotor activity (Table
VI), but the combination (as shown for subthreshold doses of CSC
and CPX) causes substantial stimulation (FIG. 12). An increase in
rotational movement, seen with the combination of A.sub.1 and
A.sub.2a antagonists, is also observed with maximal stimulant doses
of caffeine (unpublished results). This suggests the possibility
that enhancement of dopaminergic action by blocking both
presynaptic (A.sub.1) and postsynaptic (A.sub.2a) mechanisms might
be required for substantial locomotor stimulation by xanthines. The
pronounced enhancement of locomotor activity by non-selective
xanthines (Table VI), such as caffeine and theophylline (Snyder et
al., PNAS USA, 78, 3260-3264 (1981); Nikodijevic et al., 1991,
supra), is consistent with this view. The moderate, but transient,
locomotor stimulation by CPT (8-cyclopentyltheophylline) may result
from its non-selectivity in vivo at high doses (Table VI). The
synergistic behavioral depressant effects of A.sub.1 agonists in
combination with A.sub.2 agonists (Nikodijevic et al., 1991, suPra)
is also consonant with this view.
All publications, patents, and patent applications cited herein are
hereby incorporated by reference to the same extent as if each
individual document were individually and specifically indicated to
be incorporated by reference and were set forth in its entirety
herein.
While this invention has been described with emphasis upon
preferred embodiments, it will be obvious to those of ordinary
skill in the art that the preferred embodiments may be varied. It
is intended that the invention may be practiced otherwise than as
specifically described herein. Accordingly, this invention includes
all modifications encompassed within the spirit and scope of the
appended claims.
* * * * *