U.S. patent number 4,593,095 [Application Number 06/467,894] was granted by the patent office on 1986-06-03 for xanthine derivatives.
This patent grant is currently assigned to The Johns Hopkins University. Invention is credited to Robert F. Bruns, John W. Daly, Solomon H. Snyder.
United States Patent |
4,593,095 |
Snyder , et al. |
June 3, 1986 |
Xanthine derivatives
Abstract
Novel 8-phenylxanthines which are potent adenosine receptor
antagonists.
Inventors: |
Snyder; Solomon H. (Baltimore,
MD), Daly; John W. (Bethesda, MD), Bruns; Robert F.
(Ann Arbor, MI) |
Assignee: |
The Johns Hopkins University
(Baltimore, MD)
|
Family
ID: |
23857582 |
Appl.
No.: |
06/467,894 |
Filed: |
February 18, 1983 |
Current U.S.
Class: |
544/272;
544/267 |
Current CPC
Class: |
A61P
7/10 (20180101); A61P 11/08 (20180101); A61P
25/26 (20180101); A61P 9/04 (20180101); A61P
43/00 (20180101); C07D 473/08 (20130101); C07D
473/06 (20130101) |
Current International
Class: |
C07D
473/00 (20060101); C07D 473/06 (20060101); C07D
473/08 (20060101); C07D 239/36 (); A61K
031/52 () |
Field of
Search: |
;544/267 ;424/253
;514/263 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
Other References
Robert F. Bruns, Adenosine Antagonism by Purines, Pteridines and
Benzopteridines in Human Fibroblasts, Biochemical Pharmacology,
vol. 30, pp. 325-333, (1981)..
|
Primary Examiner: Rizzo; Nicholas S.
Attorney, Agent or Firm: Cushman, Darby & Cushman
Government Interests
The invention described herein was made in the course of work under
grant or award from the U.S. Department of Health and Human
Services.
Claims
We claim:
1. A compound, which in its free base form, has the formula
##STR4## wherein X is NH;
R.sub.1 is allyl, alkyl of 2-6 carbons, or cycloalkyl of 3 to 6
carbons, the lower alkyl or cycloalkyl being optionally substituted
with hydroxy, lower alkoxy or cyano;
R.sub.2 is hydrogen, allyl, lower alkyl or cycloalkyl, the lower
alkyl or cycloalkyl being optionally substituted as hereinafter
described,
R.sub.3 is NH.sub.2 or OH;
R.sub.4 is halogen, halo-lower alkyl, phenyl, amino, carboxy, lower
alkyl, cycloalkyl, lower alkoxy, cycloalkoxy, lower alkoxy amino,
lower alkyl amino or cycloalkylamino, the lower alkoxy, lower alkyl
or cycloalkyl in each instance being optionally substituted with
hydroxy, amino, methylamino, dimethylamino or carboxy; and
R.sub.5, which may be the same or different, are hydrogen, lower
alkyl, lower alkoxy, halogen, hydroxy, nitro or amino.
2. A compound according to claim 1 wherein X is NH, R.sub.1 and
R.sub.2 are alkyl of 3 or more carbons, R.sub.3 is amino, R.sub.4
is halogen and R.sub.5 is hydrogen.
3. A compound according to claim 1 wherein R.sub.1 and R.sub.2 are
alkyl of at least 3 carbons.
4. A compound according to claim 3 wherein R.sub.1 and R.sub.2 are
both propyl.
5. A compound according to claim 1 which is
1,3-dipropyl-8-(2-amino-4-chlorophenyl)xanthine.
Description
The present invention relates to certain novel 8-arylxanthines
which are potent adenosine receptor antagonists or blockers.
Xanthines are well known drugs which are used clinically as
bronchodilators, cardiotonics, diuretics and central nervous system
stimulants. Available evidence indicates that the therapeutic
actions of these drugs involves blockade or antagonism of adenosine
receptors. However, many of the xanthines, such as theophylline
(1,3-dimethylxanthine), have undesirable side-effects. Some of
these side-effects may be due to actions at sites other than
adenosine receptors. However, 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
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 should also have a reduced likelihood of side-effects.
The principal object of the present invention is to provide a novel
group of xanthines which are highly potent as inhibitors or
antagonists of adenosine receptors.
A more specific object of the invention is to provide a series of
8-arylxanthines,specifically 8-phenylxanthines, which are in
general much more potent as adenosine receptor blockers than
previously known xanthines.
Other objects will also be hereinafter apparent.
The novel 8-arylxanthines of the invention may be structurally
described as compounds of Formula I ##STR1## or the
pharmaceutically-acceptable salts, esters, amides, glycosides or
formaldehyde complexes thereof, wherein either
(a):
X is NH, O or S;
R.sub.1 is allyl, lower alkyl or cycloalkyl, the lower alkyl or
cycloalkyl being optionally substituted with hydroxy, lower alkoxy
or cyano;
R.sub.2 is hydrogen, allyl, lower alkyl or cycloalkyl, the lower
alkyl or cycloalkyl being optionally substituted as hereinafter
described,
R.sub.3 is NH.sub.2 or OH;
R.sub.4 is halogen, halo-lower alkyl (e.g. trifluoromethyl),
phenyl, amino, hydroxy, carboxy, lower alkyl, cycloalkyl, lower
alkoxy, cycloalkoxy, lower alkoxyamino, lower alkylamino or
cycloalkylamino, the lower alkoxy, lower alkyl or cycloalkyl in
each instance being optionally substituted with hydroxy, primary
amino, secondary amino, tertiary amino or carboxy provided that
R.sub.3 and R.sub.4 are not both amino when R.sub.1 and R.sub.2 are
both methyl; and
R.sub.5, which may be the same or different, are hydrogen, lower
alkyl, lower alkoxy, halogen, hydroxy, nitro or amino; or
(b)
X, R.sub.1, R.sub.2 and R.sub.5 have the meanings stated in
(a):
R.sub.3 is hydrogen; and
R.sub.4 is hydrogen or has the meaning stated in (a), except that
R.sub.1 is other than methyl or ethyl when R.sub.4 is hydrogen,
halogen, C.sub.1 -C.sub.3 alkoxy, amino or alkylamino and R.sub.5
is hydrogen or halogen.
The terms "alkyl", "lower alkyl", "alkoxy" or "lower alkoxy" as
used above are intended to represent any alkyl or alkoxy of 1-6
carbon atoms, straight or branched, e.g., methyl, ethyl, propyl,
isopropyl, butyl, isobutyl, pentyl or hexyl.
Any of the halogens are contemplated as R.sub.4 and R.sub.5 values.
Thus, as an example, R.sub.4 may be chloro, bromo or iodo and
R.sub.5 may be the same or different, e.g. fluoro or bromo although
R.sub.5 is preferably hydrogen.
Representative cycloalkyl substituents include cyclopropyl,
cyclobutyl, cyclopentyl or cyclohexyl.
The optional substitution on the R.sub.2 alkyl or cycloalkyl values
may include hydroxy, methoxy, amino, methylamino, dimethylamino,
carboxy, methylcarboxylate, ethylcarboxylate, carboxamide,
dimethylcarboxamide, ureido, cyano and glycosyl. The glycosyl group
may be attached to the alkyl chain by an ester, amide, ether, or
glycosidic bond.
As indicated, pharmaceutically-acceptable salts, esters, amides and
formaldehyde complexes of the indicated compounds, as well as the
glycosides thereof, are contemplated. Typical salts include the
alkali metal or alkaline earth metal salts although it is to be
appreciated that other nontoxic salts are also intended. The
xanthines where X is NH can form anions at alkaline pH (pK.about.9)
and thus can be advantageously administered as Na salts, choline
salts, ethylenediamine complexes, etc. The 7-thiaxanthines and
7-oxoanthines do not form anions although many of the R substituent
groups contemplated herein can form anions or cations. Hence a wide
variety of suitable salts may be formed.
As noted in connection with the optional substitution referred to
above for the R.sub.2 substituent, the glycosides may be linked to
the 3-position of the xanthine by glycosidic, amide or equivalent
bond. On the other hand, complexes with formaldehyde (or other
aldehyde) alone or with an amine may be formed through the
7-position nitrogen as shown by Formulas II and III: ##STR2##
It is to be noted that the provisos included in the foregoing
generic definition of the compounds of the invention (Formula I)
are intended to exclude previously known 8-phenylxanthines or even
some new compounds, which though new, demonstrate inferior potency
as antagonists for adenosine receptors.
With respect to the compounds represented by Formula I, overall
properties, such as water-solubility, blocking potency, etc., can
be varied by appropriate selection of the R.sub.1 -R.sub.5
substituents. For example, compounds where R.sub.1 is methyl and
R.sub.2 is isobutyl, appear to be potent phosphodiesterase
inhibitors.
The scope of permissible variation appears to be relatively narrow
for the R.sub.1 substituent. However, greater breadth of variation
seems to be possible in the case of the R.sub.2 substituent.
Accordingly, the R.sub.2 position may be used to carry substituents
which are strongly hydrophilic in order to improve water-solubility
without substantially affecting the potency of the resulting
compound as an adenosine receptor antagonist.
The nature of the substitution in the R.sub.3 and R.sub.4 positions
appears to be important for reasons of solubility and/or
potency.
Specific examples of xanthines according to the invention include
the following:
1,3-dipropyl-8-(2-amino-4-chlorophenyl)xanthine
1,3-dipropyl-8-(2,4-diaminophenyl)xanthine
1,3diethyl-8-(2-amino-4-chlorophenyl)xanthine
1,3-dipropyl-8-phenyl xanthine
1,3-dipropyl-8-(2-amino-4-chlorophenyl)-7-thiaxanthine
1,3-dipropyl-8-(2-amino-4-carboxyphenyl)xanthine
1,3-dipropyl-8-[2-amino-4-(carboxymethyl)phenyl]xanthine.
Of the above listed compounds, the first two (designated herein as
B256 and B262 for convenience) demonstrate particularly outstanding
activity as blockers of adenosine receptors.
A further particularly advantageous compound according to the
invention is 1,3-diallyl-8-(2-amino-4-chlorophenyl)xanthine. This
compound demonstrates useful activity as an adenosine receptor
antagonist and is also useful as an intermediate or as a precursor
to provide, for example, a tritium-labeled version of
1,3-dipropyl-8-(2-amino-4-chlorophenyl)xanthine.
There is a considerable amount of prior art relating to xanthines,
including 8-phenylxanthines. As representative, it is noted that
East German Pat. No. 31772 (Derwent 14969) of Oct. 31, 1961
describes various xanthines including, for example,
8-phenyltheophylline (i.e. 1,3-dimethyl-8-phenylxanthine) and
processes for making the same. Belgium Pat. No. 616174 (Derwent
13790) of Oct. 15, 1964 and British Pat. No. 982,079 appear to be
equivalent to the East German disclosure. These patents do not
appear to describe the use of the compounds disclosed therein as
adenosine receptor antagonists.
The following 8-phenylxanthines, among others, are believed to be
known from the prior art using Formula I (where X is NH and R.sub.5
is hydrogen) for ease of reference:
______________________________________ R.sub.1 R.sub.2 R.sub.3
R.sub.4 ______________________________________ (1) CH.sub.3
CH.sub.3 H H (2) CH.sub.3 CH.sub.3 H OCH.sub.3 or isopropoxy (3)
CH.sub.3 CH.sub.3 H NO.sub.2 (4) H CH.sub.3 H H (5) CH.sub.3 H H H
(6) H H H H (7) phenyl phenyl H H (8) CH.sub.3 CH.sub.3 H N(C.sub.2
H.sub.5).sub.2 (9) CH.sub.3 CH.sub.3 H N(CH.sub.3).sub.2 (10)
CH.sub.3 CH.sub.3 Cl Cl (11) H CH.sub.3 H Cl (12) H CH.sub.3 H
OCH.sub.3 (13) CH.sub.3 CH.sub.3 H CH.sub.3 (14) CH.sub.3 CH.sub.3
H F (15) CH.sub.3 CH.sub.3 H Cl (16) H H H Cl (17) H H H OCH.sub.3
(18) CH.sub.3 CH.sub.3 H Br (19) H H H NO.sub.2 (20) C.sub.2
H.sub.5 C.sub.2 H.sub.5 H H (21) CH.sub.3 CH.sub.3 COOH H (22)
CH.sub.3 CH.sub.3 NH.sub.2 H (23) CH.sub.3 CH.sub.3 NHCH.sub.3 H
(24) CH.sub.3 CH.sub.3 NO.sub.2 H
______________________________________
The above list is representative only and is not intended to
include all previously disclosed 8-phenylxanthines. In any case,
the compounds of the invention are distinguishable from the prior
art compounds in respect of at least one of the substituents
R.sub.1 -R.sub.5 or combinations thereof.
It is to be noted that compound (1) listed above is
8-phenyltheophylline and compound (6) is 8-phenylxanthine.
Elsewhere herein the symbols T and X are used to represent
theophylline and xanthine, respectively.
The inhibiting effect of xanthines on adenosine receptors is
referred to in a paper describing the binding of N.sup.6
-cyclohexyl[.sup.3 H]adenosine, and 1,3-diethyl-8-[.sup.3
H]phenylxanthine, also referred to as [.sup.3 H]CHA and [.sup.3
H]DPX, respectively, for convenience, to adenosine receptors in
brain membrane (Bruns et al, Proc. Nat'l. Acad. Sci. USA, Vol.
77,No. 9, pp. 5547-5551, September 1980). This paper discloses,
inter alia, the labeling of A.sub.1 subtype of adenosine receptors
in bovine brain membranes with [.sup.3 H]CHA and [.sup.3 H]DPX. The
potencies of various xanthines in displacing [.sup.3 H]CHA from
A.sub.1 adenosine receptors in brain membranes, representing the
inhibiting effect of these compounds on adenosine receptors
measured as IC.sub.50 nM values on a standard screen, are also
shown. Theophylline, 8-phenyltheophylline and
8-(p-sulfophenyl)theophylline are included among the xanthines so
evaluated.
A related paper by Bruns entitled "Adenosine Antagonism by Purines,
Pteridines and Benzopteridines In Human Fibroblasts", Biochemical
Pharmacology, Vol. 30, pp. 325-333 (1981) provides additional
information regarding the potencies as adenosine antagonists of
various xanthines (X) and theophyllines (T), including a number of
8-substituted theophyllines such as the 8-(p-chlorophenyl),
8-(p-bromophenyl)-, 8-(p-methoxyphenyl)-, 8-(p-nitrophenyl)-,
8-(p-dimethylaminophenyl)-, 8-(p-methylphenyl)-,
8-(3,4-dichlorophenyl)-, 8-(o-carboxyphenyl)- and
8-(2,6-dimethyl-4-hydroxyphenyl)-derivatives.
Another generally related paper by Snyder et al is entitled
"Adenosine Receptors and Behavorial Actions of Methylxanthines",
Proc. Nat'l. Acad. Sci. USA, Vol. 78, No. 5, pp. 3260-3264, May
1981.
The 8-phenylxanthines of the invention may be synthesized in any
convenient fashion, e.g. according to the abovementioned East
German No. 31772 or its equivalent Belgian Pat. No. 616,174 or
British Pat. No. 982,079. In a preferred method, the appropriate
5,6-diaminouracil, itself prepared by reduction of the
5-nitroso-6-amino-uracil, is acylated to form the corresponding
5-acylamino-6-amino-uracil which is then ring-closed. Conventional
acylating and ring-closing conditions may be used. For example, an
appropriately substituted benzoic acid may be employed to form the
5-acylamino- compound. Ring closure may be effected by, for
example, heating at the boil in 2.5N. NaOH for a sufficient period
of time, e.g. 5 minutes, or by heating in POCl.sub.3 for an
appropriate time, e.g. 20 minutes or so.
The potency of the present compounds as adenosine receptor
antagonists may be determined on the standard screen which involves
blocking N.sup.6 -cyclohexyl [.sup.3 H]adenosine binding to
adenosine receptors as described in the 1980 Bruns et al paper
referred to above. Briefly, the screen, as used herein, involved
the following:
10 mg. original tissue wet weight of bovine brain membranes were
incubated for 2 hours at 25.degree. C. with the test compound and
0.5 nM [.sup.3 H]CHA in 2 ml of 50 mM Tris.HCl pH 7.7. The test
compound and [.sup.3 H]CHA were added to the tube first, and the
incubation was initiated by addition of the tissue. Incubation was
terminated and samples were collected on GF/B filters under vacuum,
washed three times, and counted in a liquid scintillation counter.
Dose-inhibition curves were generated with four to eight
concentrations of the test compound in triplicate incubations.
IC.sub.50 values were computed from total binding (no compound),
nonspecific binding (10 .mu.M L-PIA), and the dose-inhibition data
using a non-linear least-squares fit to a competitive inhibition
model. K.sub.i values were calculated from the Cheng-Prusoff
equation (Biochem. Pharmacol. 22, 3099-3108 (1973)). Compounds with
K.sub.i values below 0.5 nM were tested in binding assays with only
2.5 mg wet weight of tissue in order to avoid conditions where the
receptor concentration exceeded the K.sub.i.
Tests of the present compounds in the foregoing screen indicate
that the most active compound of the invention (B256) has an
extraordinary adenosine receptor activity, with a K.sub.i for
adenosine A.sub.1 receptors of 2.2.times.10.sup.-11 M when using
bovine brain for test purposes. The compound accordingly appears to
be approximately 4,000,000 times more potent than xanthine itself
and 60,000 to 70,000 times more potent than theophylline.
In connection with the foregoing, it is noted that A.sub.1
receptors from bovine brain have an unusually high affinity for
8-phenylxanthines, and bovine brain was chosen for test purposes
for that reason, in order to ensure that even less potent analogs
would have IC.sub.50 values below their solubility limits. The more
"normal" A.sub.1 receptor in rat brain has a K.sub.i of 5 nM for
compound B256, 150 nM for 8-phenyltheophylline, and 10 .mu.M for
theophylline. Thus, although both 8-phenylxanthines are much less
potent in rat than in bovine brain,
1,3-dipropyl-8-(2-amino-4-chlorophenyl)xanthine (B256) is still
about 30-fold more potent than 8-phenyltheophylline and 2000-fold
more potent than theophylline using rat brain.
Theophylline is itself an adenosine antagonist which is used
clinically as a bronchodilator in the treatment of asthma. The
present compounds should also be useful in the same way as
theophylline or other known xanthines, based on the indicated
inhibition or blocking of adenosine receptors. This would include
not only use as bronchodilators in the treatment of asthma but also
use for cardiotonic effects in the treatment of heart failure, for
diuretic effects in the treatment of high blood pressure or renal
failure and for central nervous stimulant effects in treating
depression. However, because of their surprisingly greater potency
as adenosine receptor antagonists, the present compounds should be
effective to block adenosine receptors in substantially lower
amounts with consequent reduction in possible side effects.
It is contemplated that the present compounds would be used in the
form of conventional pharmaceutical compositions with the usual
types of carriers as in the case of the known xanthines or other
adenosine receptor antagonists or blockers. It is also contemplated
that these compositions, e.g. tablets or capsules for oral
administration or sterile solutions for injection, would contain
the usual amount of active component, e.g. from 0.01 to 0.5% by
weight, based on the weight of the composition although, as noted,
the dosages should be reduced to account for the generally greater
activity of the present compounds.
The invention is illustrated, but not limited, by the following
examples:
EXAMPLE 1
Synthesis of 1,3-Dipropyl-8-(2-Amino-4-Chlorophenyl)Xanthine
(B256)
1,3-dipropyl-8-(2-amino-4-chlorophenyl)xanthine was synthesized by
a modification of the method of Pfleiderer and Kempter (Ang Int.
Ed. 6:259-260, 1967). 2-nitro-4-chlorobenzoic acid 0.02 mol) was
dissolved in 30 ml of methanol.
1,3-dipropyl-5-nitroso-6-aminouracil (0.01 mol) was added with
stirring, followed by 0.2 mol diisopropylcarbodiimide (DICD).
After ten minutes, the white precipitate,
1,3-dipropyl-5-[(2-nitro-4-chlorobenzoyl)oxy]imino-6-(2-nitro-4-chlorobenz
oyl) iminouracil, was collected by filtration. To the dried
intermediate was added 15 ml of 22% ammonium sulfide. After ten
minutes, concentrated HCl was added to pH 8 in a hood and the
precipitate was collected by filtration. The product was roughly a
50:50 mixture of 1,3-dipropyl-8-(2-amino-4-chlorophenyl)xanthine
and
1,3-dipropyl-5-[(2-amino-4-chlorobenzoyl)amino]-6-aminouracil.
In order to complete the cyclization, the crude product was boiled
in 2.5N KOH for 20 minutes, neutralized, and filtered. The product
was purified once by dissolving in KOH and precipitating with HCl
and again by recrystallizing from dimethylformamide. The product
was identified by chemical ionization mass spectrometry and
elemental analyses. Yield was 2.1%.
EXAMPLE 2
Synthesis of 1,3-Dipropyl-8-(2,4-Diaminophenyl) Xanthine (B262)
1,3-dipropyl-5,6-diaminouracil (0.01 mol) was suspended in 30 ml
THF. N-trifluoroacetyl-4-nitroanthranilic acid-trifluroacetic acid
mixed anhydride (0.01 mol) was added and the suspension was stirred
at room temperature for 30 minutes, then evaporated in a rotary
evaporator at 37.degree. and then at 60.degree. . The solid was
boiled in 40 ml 2.5N KOH for five minutes, filtered hot, adjusted
to pH 8.0 with concentrated HCl, filtered, and washed with H.sub.2
O. The precipitate was dissolved in 20 ml 2.5N KOH, heated, 5 ml
22% ammonium sulfide added, boiled for one minute, brought to pH 8
with concentrated HCl, filtered, and washed with H.sub.2 O. Yield
7.2%. Chemical ionization mass spectroscopy with NH.sub.3 gave the
M+1 peak at M/e 343. Microanalysis was consistent with 75% product
and 25% thiol impurities. The product was not purified further
because the thiols appeared to protect the desired product from
oxidation.
EXAMPLE 3
Synthesis of 8-(2-Amino-4-Chlorophenyl) Theophylline (B246)
1,3-dimethyl-5,6-diaminouracil (0.01 mol) was suspended in 50 ml
methanol. 2-amino-4-chlorobenzoic acid (0.01 mol) was added,
followed by 0.01 mol of DICD. The reactants were stirred at room
temperature for 15 min, then filtered and washed with methanol. The
solid was boiled in 40 ml 2.5N NaOH for five min, filtered hot, and
the eluate was left to cool for three hours. The material which
precipitated on cooling was filtered without washing, redissolved
in 40 ml water, and precipitated by neutralization with
concentrated HCl. The solid was collected by filtration, washed
with H.sub.2 O, and dried. The product was purified by suspending
in 100 ml water, adding NaOH until the compound was dissolved,
filtering, precipitating the solid with HCl, filtering, washing
with H.sub.2 O, and drying. The product was identified by chemical
ionization mass spectrometry and elemental analysis. Yield
12.5%.
EXAMPLE 4
Synthesis of 1,3-Dipropyl-8-Phenylxanthine (B255)
1,3-dipropyl-5,6-diaminouracil (0.01 mol) was dissolved in 30 ml
methanol, followed by 0.01 mol of benzoic acid and then 0.01 mol of
DICD. The solution was stirred for 30 min at room temperature,
filtered, and washed with a small amount of methanol. The solid was
boiled for ten minutes in 2.5N KOH, filtered hot, and the liquid
neutralized with concentrated HCl. The solid was collected by
filtration, washed with water, redissolved in 100 ml with a minimum
amount of KOH, precipitated by neutralization with HCL, filtered,
washed with water, and dried. The product was identified by
chemical ionization mass spectrometry and elemental analysis. Yield
77%.
EXAMPLE 5
The following compounds are also representative of the invention
and may be prepared in generally the same way as shown in the
preceding examples: ##STR3##
As indicated earlier, the potency of xanthines or other compounds
as inhibitors of adenosine receptors can be determined by testing
the compounds in the known screen referred to above and involving
the use of [.sup.3 H]CHA in bovine brain membrane. The activities
of the compounds of Examples 1 to 4 are compared below in Table I
with other structurally related compounds, at least some of which
(xanthine, B7, B80, B52, B87 and B70) are known compounds, in terms
of IC.sub.50 (nM) values determined by screening the compounds
against .sup.3 H]-cyclohexyladenosine in bovine brain:
TABLE I ______________________________________ IC.sub.50 (nM)
______________________________________ xanthine 200,000 B7
theophylline (1,3-dimethylxanthine) 3,000 B80 1,3-dipropylxanthine
200 B52 8-phenyltheophylline 3 B87 8-(o-aminophenyl)theophylline 5
B70 8-(p-chlorophenyl)theophylline 0.8 B211
8-(p-aminophenyl)theophylline 1.7 B232
8-(2,4-diaminophenyl)theophylline 8 B246
8-(2-amino-4-chlorophenyl)theophylline 0.15 B255
1,3-dipropyl-8-phenylxanthine 0.3 B256
1,3-dipropyl-8-(2-amino-4-chlorophenyl) 0.05 xanthine B262
1,3-dipropyl-8-(2,4-diaminophenyl) 0.2 xanthine
______________________________________
It will be noted that the compounds of Examples 1 to 4 (Compounds
B256, 262, 246 and 255, respectively) demonstrate IC50 (nM) values
which are significantly lower (indicative of greater potency as
adenosine receptor blockers or inhibitors) than those obtained with
the other listed compounds. Particularly active is B256 (Example 1)
whose activity is some 16 times greater than that of the most
active prior art compound (B70). It is also noted that the
inhibiting activity of the compound of Example 2 (B262) is
four-fold greater than that of B70.
While B256 is extremely potent, it is hydrophobic and very
water-insoluble. In certain cases, therefore, there may be an
advantage in incorporating water solubilizing groups in the
compound, e.g. in the 3-position (R.sub.2). The compound B262,
while substantially less active than B256, has much greater
water-solubility and offers this as an advantage over B256 in cases
where such solubility is important.
The results set forth in Table I indicate that the best results are
obtained when R.sub.3 and R.sub.4 in Formula I (i.e., the ortho and
para positions of the 8-phenyl substituent) are both substituted,
particularly with amino as R.sub.3 and chloro or other halogen as
R.sub.4, R.sub.5 being hydrogen in both instances and R.sub.1 and
R.sub.2 being lower alkyl. Increasing the length of the alkyl for
R.sub.1 and R.sub.2 appears to improve the potency of the xanthines
as inhibitors for adenosine receptors. Compare in this respect the
results obtained with xanthine itself; theophylline (B7); and
1,3-dipropylxanthine. It is a particularly surprising aspect of the
invention that while 8-phenyltheophylline is about 1000 times more
potent than theophylline and 1,3-dipropyl substituents enhance the
potency of theophylline by almost twenty times, the combination of
1,3-propyl substituents and the 8-phenyl substituent gives a
synergistic effect such that 1,3-dipropyl-8-phenylxanthine is about
10,000 times more potent than theophylline. Accordingly, it is
preferred for present purposes that R.sub.1 and R.sub.2 be the same
or different alkyl with at least 3 carbon atoms.
The substantial potency of the compounds of the present invention
is also evident from a comparison of the activities of such
compounds with other compounds as determined versus [.sup.3 H-CHA]
in bovine brain:
TABLE II ______________________________________ IC.sub.50 (nM)
______________________________________ xanthine (X) 200,000
3-methylX 150,000 1-methylX 6,000 1,7-dimethylX 30,000 8-nitroT
3,500 caffeine 20,000 7-(2-chloroethyl)T 5,000 7-(2-hydroxyethyl)T
100,000 7-(2,3-dihydroxypropyl)T 800,000 1,3-diethylX 3,000
8-(n-propyl)T 100 8-cyclopentylT 2 8-(p-methoxyphenyl)T 1.5
8-(o-nitrophenyl)T 80 8-(p-nitrophenyl)T 8
8-(2,6-dimethyl-4-hydroxyphenyl)T 30 8-(1-naphthyl)T 80
8-(3-indolyl)T 18 8-(p-bromophenyl)T 0.8 8-(p-dimethylaminophenyl)T
1.8 8-(p-methylphenyl)T 0.8 8-benzylT 1,500 8-cyclohexylT 3
1,3-diallylX 4,000 1-methyl-8-phenylX 2.5 8-(3,4-dichlorophenyl)T 5
8-(m-methoxyphenyl)T 20 8-(m-nitrophenyl)T 50
8-(m-dimethylaminophenyl)T 80 8-(m-methylphenyl)T 13
8-(p-hydroxyphenyl)T 2 8-(p-ethoxyphenyl)T 2 8-(2-pyridyl)T 100
8-(3-pyridyl)T 50 8-(4-pyridyl)T 35 8-(2-furyl)T 18
8-(o-carboxyphenyl)T 2,500 adenine 800,000
1-ethyl-3-propyl-7-thiaxanthine 8,000 8-methyladenine 35,000
alloxazine 1,500 1,3-dimethylalloxazine 25,000 8-(p-fluorophenyl)T
3.5 8-(p-iodophenyl)T 1.3 8-(3,4-dimethoxyphenyl)T 20
8-(p-isopropylphenyl)T 2.5 8-(2-thienyl)T 5 8-(m-bromophenyl)T 10
8-(m-hydroxyphenyl)T 6 8-(m-aminophenyl)T 10 8-(p-sulfophenyl)T 500
8-(p-ethylphenyl)T 0.8 8-(p-phenylphenyl)T 3.5
8-(3,5-dimethoxyphenyl)T 500 8-(2-naphthyl)T 5 8-(m-fluorophenyl)T
4 1,3-diethyl-8-phenylX 2.5 1,3-diethyl-8-(p-bromophenyl)X 1.0
8-(o-fluorophenyl)T 12 8-(o-hydroxyphenyl)T 10 8-(o-methoxyphenyl)T
350 8-(o-methylphenyl)T 6 8-(m-carboxyphenyl)T 1,000
8-(p-carboxyphenyl)T 50 8-(2,4-dimethoxyphenyl)T 200
8-(2-amino-4-nitrophenyl)T 2.5 8-(3-furyl)T 4 8-ferrocenylT 20
8-(5-bromo-2-furyl)T 50 8-(N--methyl-2-pyrrolyl)T 20
8-cyclopentylmethylT 30 1-allyl-3-methyl-8-phenylX 4
1-allyl-3-methyl-8-(2-amino-4-chlorophenyl)X 2 8-(p-butoxyphenyl)T
4 1,3-diethyl-8-(2-amino-4-chlorophenyl)X 0.8
1,3-diallyl-8-(2-amino-4-chlorophenyl)X 0.8
1-allyl-3-methyl-8-(2-amino-4-methylphenyl)X 7
8-(2-amino-4-methylphenyl)T 10 8-(5-methyl-2-thienyl)T 5
8-(p-methylthiophenyl)T 2
______________________________________
As noted, the most potent compounds of the invention appear to be
those of Formula I which include propyl substituents for R.sub.1
and R.sub.2 in combination with the 8-phenyl, whether the latter is
substituted or not. However, potent compounds are also obtained
when R.sub.1 and/or R.sub.2 are other than propyl (or higher
alkyl), provided the 8-phenyl group is substituted, preferably but
not necessarily with at least two substituents in the 8-phenyl
group, and most preferably with at least one such substituent in
the para position.
Studies with various substituents on the 8-phenyl ring of
8-phenyltheophylline further indicate that the nature and
positioning of such substituents can have a marked effect on the
receptor affinity or blocking activity of the resulting compound.
In general, these studies indicate that ortho substituents on the
8-phenyl ring generally reduce the receptor affinity of
8-phenyltheophylline, probably because the ortho substituent
creates steric hindrance with the N-7 and N-9 of the xanthine. In
agreement with this is the finding that ortho substitution with the
more bulky methoxy and nitro groups causes the largest decrements
in affinity. This suggests that the receptor prefers the 8-phenyl
ring to be in the same plane as the xanthine ring. Of the various
ortho substituents, the ortho amino causes the least reduction in
potency of 8-phenyltheophylline, perhaps because it hydrogen bonds
to the N-7 of the xanthine, stabilizing a conformation with the
8-phenyl and xanthine rings in the same plane.
Meta substituents generally reduce potency of 8-phenyltheophylline
3-100 fold. The 8-phenyl ring has two possible meta positions
(R.sup.5) and the ring rotates freely. If only one of the meta
positions was important for receptor interactions, then even
unfavorable substituents would reduce potency only about two-fold,
since the rotamer with the unsubstituted meta position in contact
with the receptor and the substituted meta in the "unimportant"
position would still have full affinity. The far greater reductions
in potency observed with meta substituents suggest that both meta
positions are important.
Para substituents can either increase or decrease the potency of
8-phenyltheophylline. Except for the p-carboxyl group, the changes
in potency are not large, being less than four-fold in all cases.
Hydrogen bonding to the receptor does not appear crucial since an
amino group, which can be both a donor and acceptor of hydrogen
bonds, and a chloro, which is neither a donor nor an acceptor of
such bonds, have similar effects. Development of a resonance
structure with a substituent does not appear to be crucial since a
methyl group, which does not provide a resonance form, considerably
enhances potency. Donation or withdrawal of electrons from the
8-phenyl ring does not appear of importance, since both electron
donating and withdrawing groups have similar effects. Accordingly,
it is most likely that optimal activity in this position is
associated largely with steric factors.
Though para substituents on the 8-phenyl ring produces very potent
agents, disubstitution of the 8-phenyl ring in the ortho and para
positions (R.sub.3 and R.sub.4) clearly give the compounds of
maximum potency, particularly when R.sub.1 and R.sub.2 are longer
chain alkyl than methyl or ethyl. An ortho amino group adds
hydrophilicity and although this group added alone to
8-phenyltheophylline lowers affinity in [.sup.3 H]CHA binding
slightly, it increases affinity three-fold or so when added to
8-(p-chlorophenyl)-theophylline. This apparently synergistic
interaction suggests that one group (probably the o-amino)
stabilizes a conformation which is favorable to the binding of the
other group.
The following additional data shows adenosine receptor affinities
of various xanthines in terms of inhibition of [.sup.3 H]CHA
binding to A.sub.1 adenosine receptors in bovine brain membranes
using the method described earlier herein.
TABLE III ______________________________________ Inhibition of
[.sup.3 H]CHA Binding Substituents K.sub.i, nM
______________________________________ None (xanthine) 99,000
1-Methyl 2,600 1,7-Dimethyl 7,400 1,3-Dimethyl (theophylline) 1,600
3,7-Dimethyl (theobromine) 68.000 1,3,7-Trimethyl (caffeine) 11,000
1,3-Diethyl 1,400 1,3-Dipropyl 100 1,3-Dimethyl-8-phenyl 1.2
1,3-Diethyl-8-phenyl (DPX) 2.0 1,3-Dipropyl-8-phenyl 0.12
______________________________________
The last compound in Table III is the only one listed in the table
which is representative of the invention. This compound, which
corresponds to Formula I when R.sub.1 and R.sub.2 are propyl, X is
NH and R.sub.3, R.sub.4 and R.sub.5 are all hydrogen, is clearly
much more potent as an inhibitor than other compounds.
The data set out below in Table IV shows the adenosine receptor
affinity of various 8-phenyltheophyllines with the indicated
substituents on the 8-phenyl ring:
TABLE IV ______________________________________ Inhibition of
[.sup.3 H]CHA Binding (K.sub.i, nM) Substituent on 8-Phenyl Ring at
Substituent ortho meta para ______________________________________
H 1.2 1.2 1.2 Bromo -- 4.0 0.34 Methyl 3.6 5.4 0.51 Methoxy 190 8.7
0.63 Chloro -- -- 0.64 Amino 2.3 5.8 0.69 Fluoro 6.8 2.4 1.8
Hydroxy 4.8 3.1 2.0 Nitro 49 22 4.0 Carboxyl 21,000 540 18
______________________________________
Table V shows the effects of disubstitution on the 8-phenyl ring of
8-phenyltheophylline with respect to adenosine receptor affinity as
determined by inhibition of [.sup.3 H]CHA binding to A.sub.1
adenosine receptors in bovine brain membrances.
TABLE V ______________________________________ 8-Phenyl Xanthine
Inhibition of [.sup.3 H]CHA binding Substituents Substituents
K.sub.i, nM ______________________________________ H 1,3-Dimethyl
1.2 2-Amino-4-nitro 1,3-Dimethyl 1.2 2,4-Diamino 1,3-Dimethyl 5.9
2-Amino-4-chloro 1,3-Dimethyl 0.20 H 1,3-Diethyl 2.0
2-Amino-4-chloro 1,3-Diethyl 0.32 H 1,3-Dipropyl 0.12 2,4-Diamino
1,3-Dipropyl 0.14 2-Amino-4-chloro 1,3-Dipropyl 0.022
______________________________________
The following methods were employed in the preparation of various
intermediates or final xanthine products referred to in the
foregoing examples or in Tables I-V:
1,3-Dialkyl-5-nitroso-6-aminouracils.
The 1,3-disubstituted 6-aminouracil was suspended (0.5M) with
vigorous stirring in water with one equivalent of sodium nitrite.
Concentrated HCl was added in small amounts to maintain the pH at
4.0. When the pH stopped rising, HCl was added to pH 2.5 and the
thick precipitate was filtered. The product was dried and used
without further characterization.
1,3-Dialkyl-5,6-diaminouracils:
Sodium Hydrosulfite Method
1,3-Diethyl-5,6-diaminouracil and 3-allyl-1-ethyl-5,6-diaminouracil
were prepared by reduction of the corresponding 5-nitroso compounds
with sodium hydrosulfite. The nitroso compound was suspended in
water (1M) and sodium hydrosulfite was added until the nitroso
color disappeared. An additional quantity of sodium hydrosulfite
was added and the solution was left at 4.degree. C. overnight. The
precipitated bisulfite salt of the product was collected by
filtration.
1,3-Dialkyl-5,6-diaminouracils
Ammonium Sulfide Method
To 0.01 mol of 1,3-dipropyl-5-nitroso-6-aminouracil or
1,3-diallyl-5-nitroso-6-aminouracil was added 10 ml of 22% light
ammonium sulfide in a fume hood. After about 2 minutes, the
suspension became hot and in some cases boiled violently. After 30
minutes, the ammonium sulfide was removed in a rotary evaporator.
The solid remaining had a strong sulfide stench but gave a
satisfactory coupling reaction with benzoic acid.
1,3-Dialkyl-5-acylamino-6-aminouracils
Method A
Fusion with the Carboxylic Acid
1,3-dimethyl-5,6-diaminouracil and the appropriate carboxylic acid
were heated above their mixed melting point (120-180.degree. C.)
until solid or until three hours had elapsed, whichever came
first.
1,3-Dialkyl-5-acylamino-6-aminouracils
Method B
Fusion with the Acyl Chloride
1,3-dimethyl-5,6-diaminouracil was suspended in an excess of the
appropriate acid chloride and heated to 120-160.degree. C. for 30
minutes to 2 hours.
1,3-Dialkyl-5-acylamino-6-aminouracils
Method C
EDAC in Water
1,3-dimethyl-5,6-diaminouracil was dissolved at 0.3M boiling water
and allowed to cool below 40.degree. C. One equivalent of the
appropriate carboxylic acid was added and the pH was raised slowly
with NaOH until the carboxylic acid was dissolved (pH 4 to 7). One
equivalent of EDAC was added with stirring and the pH was kept
constant by addition of HCl. When the pH stopped rising, the
precipitated amide was collected by filtration. In the case of
1,3-dimethyl-5-(p-sulfobenzoylamino)-6-aminouracil, the product was
precipitated by addition of MeOH.
1,3-Dialkyl-5-acylamino-6-aminouracils
Method D
DICD in Methanol
The 1,3-dialkyl-5,6-diaminouracil (free base or bisulfite salt) and
the appropriate carboxylic acid were dissolved or suspended at 0.3
M each in MeOH. One equivalent of DICD was added and the copious
amide precipitate was collected by filtration after 5 to 30
minutes. In a few cases (e.g., 1,3-diethyl-5,6-diaminouracil with
2-amino-4-chlorobenzoic acid) the amide was soluble in MeOH and had
to be collected by precipitation with water or evaporation of the
MeOH.
1,3-Dialkyl-5-acylamino-6-aminouracils
Method E
EDAC in Methanol
This method is the same as Method D, except that EDAC was used in
place of DICD.
1,3-Dialkyl-5-acylamino-6-aminouracils
Method F
Mixed Anhydride
To 2-amino-4-nitrobenzoic acid in a small volume of THF was added
two equivalents of trifluoroacetic anhydride. After 10 minutes,
trifluoroacetic acid and its anhydride were removed in a rotary
evaporator. The product 2-trifluoroacetamido-4-nitrobenzoic
acid-trifluoroacetic acid mixed anhydride (0.01 mol) was reacted
with 1,3-dialkyl-5,6-diaminouracil (0.01 mol) in THF for 60
minutes. In the case of the 1,3-dimethyl derivative, the product
1,3-dimethyl-5-(2-trifluoroacetamido-4-nitrobenzamido)-6-aminouracil
precipitated in 140 ml THF and was collected by filtration. The
1,3-dipropyl homolog was soluble in 30 ml THF and was collected in
a rotary evaporator. When the corresponding xanthines were produced
by ring closure in 2.5N KOH (see below), the trifluoroacetyl group
was lost.
8-Substituted Xanthines
Ring Closure in NaOH
The 1,3-dialkyl-5-acylamino-6-aminouracil (0.3 M) was boiled for 5
to 20 minutes in 2.5N NaOH (or KOH). Uracils which were insoluble
or had electron-donating groups on the acyl moiety required the
longest times.
Isolation of 8-Substituted Xanthines
When possible, the xanthine in boiling NaOH was filtered to remove
impurities which were insoluble in boiling NaOH. Xanthines which
were synthesized by Method A usually contained an alkali-insoluble
material of molecular weight 252. This step was omitted when the
xanthine was insoluble in boiling NaOH or when there was
precipitation during filtration. The solution of xanthine in NaOH
was cooled to 0.degree.. If the xanthine precipitated as the sodium
salt, it was collected by filtration without washing, redissolved
in distilled water, precipitated by neutralization (pH 7 to 9) with
concentrated HCl, filtered, and washed with water. If the xanthine
remained dissolved at 0.degree. in 2.5N NaOH, it was neutralized,
filtered and washed. The final wash was omitted for
8-(p-sulfophenyl)theophylline, which precipitated as the sodium
salt. For the 8-(carboxyphenyl) theophyllines, HCl was added to pH
6 in the precipitation step.
8-(o-Hydroxyphenyl)theophylline
Ring Closure in POCl.sub.2
8-(o-hydroxyphenyltheophylline could not be prepared by the usual
NaOH ring closure, even when
1,3-dimethyl-5-(acetylsalicyloyl)amino-6-aminouracil was used as
intermediate. Instead,
1,3-dimethyl-5-(acetylsalicyloyl)amino-6-aminouracil was refluxed
for 10 minutes in POCl.sub.2. The cooled POCL.sub.2 solution was
added slowly to a large volume of ice cold water with vigorous
stirring. After the POCl.sub.2 was completely hydrolyzed, the
solution was neutralized with KOH pellets and filtered. The
filtrate was a mixture of the xanthine and the uncycled amide. The
latter was eliminated by boiling for 5 minutes in 2.5N KOH and the
xanthine was collected by neutralization and filtration.
1,3-Dipropyl-8-(2,4-diaminophenyl)xanthine
Reduction of Nitro Derivative
Method G
1,3-dipropyl-8-(2-amino-4-nitrophenyl)xanthine (0.007 mol) was
dissolved in 20 ml boiling 2.5N KOH. Five ml of 22% ammonium
sulfide was added, and the solution was removed from heat after 1
minute. HCl was added to pH 8 in a hood, and the product was
collected by filtration and washed with water. About 25% of the
product was a sulfur-containing impurity. Since this impurity
appeared to protect the xanthine from oxidation, no attempt was
made to further purify the xanthine.
8-(2,4-diaminophenyl)theophylline was synthesized in the same
way.
1,3-Dialkyl-8-(2-amino-4-chlorophenyl)xanthine
From Acylated Nitrosouracil
Method H
1,3-dipropyl-8-(2-amino-4-chlorophenyl)xanthine and
1,3-diallyl-8-(2-amino-4-chloropheny)xanthine were synthesized by
the method of Pfleiderer and Kempter. 2-nitro-4-chlorobenzoic acid
(0.02 mol) was dissolved in 30 ml of MeOH.
1,3-dialkyl-5-nitroso-6-aminouracil (0.1 mol) was added with
stirring, followed by 0.02 mol DICD. After 10 minutes, the white
precipitate, 1,3-dialkyl-5-[2-nitro-4-chlorobenzoyl)
oxy]imino-6-(2-nitro-4-chlorobenzoyl)iminouracil, was collected by
filtration. To the dried intermediate was added 15 ml of 22%
ammonium sulfide. After 10 minutes, concentrated HCl was added to
pH 8 in a hood and the precipitate was collected by filtration. The
product was roughly a 50:50 mixture of
1,3-dialkyl-8-(2-amino-2-chlorophenyl)xanthine and
1,3-dialkyl-5-[2-amino-4-chlorobenzoyl)amino]-6-aminouracil. In
order to complete the cyclization, the crude product was boiled in
2.5N KOH for 20 minutes, neutralized, and filtered.
Product Purification
When microanalysis for a xanthine did not agree with theoretical
values, the xanthine was suspended at 0.1 M in water and dissolved
with a minimal amount of KOH. After filtration, the xanthine was
neutralized, collected by filtration, and washed with water. If
microanalysis was still incorrect, the compound was recrystallized
from dimethylformamide.
Characterization of Products
All products gave correct parent ions (M+1) on NH.sub.3 chemical
ionization spectrometry. Except for the carboxyphenyltheophyllines,
M+18 peaks were not seen. This allowed easy detection of the M+19
uncyclized amide. The structure of 8-p-sulfophenyltheophylline was
confirmed by proton magnetic resonance in deuterated DMSO.
Compounds were dried before elemental analysis. Most compounds were
purified until satisfactory microanalyses were achieved, but a few
had to be used without purification because of the small amount of
material available.
Solubility of 8-Phenylxanthines
ALL of the uncharged 8-phenylxanthines were quite insoluble in
water. 8-phenyltheophylline was soluble at 10 .mu.M in water, and
1,3-diethyl-8-phenylxanthine was soluble at 3 .mu.M. The more
hydrophobic analogs appeared to be considerably less soluble in
water. 8-phenyltheophylline was soluble at 1 mM in DMF and in 0.01N
NaOH, but was almost insoluble in ethanol. More hydrophobic analogs
were less soluble in NaOH but more soluble in DMF. Unlike most
8-phenylxanthines, 1,3-dipropyl-8-(2-amino-4-chlorophenyl)xanthine
was soluble at 1 mM in ethanol. It was soluble at 30 mM in DMF and
at 1 mM in hot 0.1N KOH.
Stock solutions of the 8-phenylxanthines were made up in 0.1N KOH
or DMF and stored at 4.degree. C. pending their testing. KOH
solutions were stable for about 3 weeks and DMF solutions were
stable longer. KOH solutions sometimes precipitated irreversibly if
frozen. Dilutions were made up fresh from stock. Solutions were
diluted directly to 1 .mu.M or 10 .mu.M in distilled water and (if
possible) immediately diluted further.
In summary, the compounds of Formula I, particularly those where X
is NH, and R.sub.1 and R.sub.2 are lower alkyl of at least 3
carbons, R.sub.3 is NH.sub.2, R.sub.4 is halogen, particularly
chlorine, and R.sub.5 is hydrogen, are extremely potent adenosine
receptor antagonists and they should be useful as, for example,
bronchodilators, cardionics, diuretics, and central nervous system
stimulants. In addition, when labelled with tritium, iodine-125, or
some other radiolabel, the present compounds may be used as
radioligands for binding to adenosine receptors. Such a radioligand
may be used for measurement of adenosine receptor levels or for
measurement of levels of adenosine or adenosine analogs. Such
measurements are useful as research tools and as diagnostic
tests.
It is also contemplated that at least some of the present compounds
will be potent inhibitors of cyclic GMP phosphodiesterase.
The invention described herein was made in the course of work under
a grant or award from the Department of Health and Human
Services.
The scope of the invention is defined in the following claims
wherein.
* * * * *