U.S. patent number 3,907,799 [Application Number 05/440,989] was granted by the patent office on 1975-09-23 for xanthine oxidase inhibitors.
This patent grant is currently assigned to ICN Pharmaceuticals, Inc.. Invention is credited to Darrell E. O'Brien, Roland K. Robins, Lionel N. Simon.
United States Patent |
3,907,799 |
O'Brien , et al. |
September 23, 1975 |
Xanthine oxidase inhibitors
Abstract
Compounds of the following structure are disclosed which are
effective inhibitors of the enzyme xanthine oxidase: ##SPC1## In
which Y.sub.1 and Y.sub.2 are C or N; R.sub.1 is H or an alkali
metal or ammonium; R.sub.2 is H, CH.sub.3, a halogen, phenylazo or
NO.sub.2 ; R.sub.3 is OR.sub.1, H, or a halogen; And R.sub.4 is H,
NO.sub.2 or a halogen. When Y.sub.1 is C, Y.sub.2 is N and when
Y.sub.1 is N, Y.sub.2 is C.
Inventors: |
O'Brien; Darrell E. (Mission
Viejo, CA), Robins; Roland K. (Santa Ana, CA), Simon;
Lionel N. (Santa Ana, CA) |
Assignee: |
ICN Pharmaceuticals, Inc.
(Irvine, CA)
|
Family
ID: |
26867835 |
Appl.
No.: |
05/440,989 |
Filed: |
February 11, 1974 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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172196 |
Aug 16, 1971 |
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Current U.S.
Class: |
544/281; 534/752;
534/887 |
Current CPC
Class: |
C07D
487/04 (20130101) |
Current International
Class: |
C07D
487/00 (20060101); C07D 487/04 (20060101); C07D
239/00 () |
Field of
Search: |
;260/256.4F |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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375,947 |
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Jun 1962 |
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JA |
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76,677 |
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Oct 1970 |
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DL |
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Primary Examiner: Daus; Donald G.
Assistant Examiner: Turnipseed; James H.
Attorney, Agent or Firm: Thomson; William E. Steffin;
William C. Boswell; Kay H.
Parent Case Text
This is a continuation, of application Ser. No. 172,196, filed Aug.
16, 1971 now abandoned.
Claims
We claim:
1. A compound of the structure ##SPC5##
wherein Y.sub.1 and Y.sub.2 are C or N;
R.sub.1 is H or an alkali metal or ammonium;
R.sub.2 is H, CH.sub.3, halogen, or NO.sub.2 ;
R.sub.3 is OR.sub.1, H, or halogen;
and R.sub.4 is H, NO.sub.2 or halogen; provided that when one of
Y.sub.1 or Y.sub.2 is C, the other is N;
that when Y.sub.1 is C, Y.sub.2 is N and R.sub.2 is H, R.sub.3 is
H, halogen or OR.sub.1 where R.sub.1 is an alkali metal or ammonium
and R.sub.2 is H, CH.sub.3, NO.sub.2 or halogen; and that when
Y.sub.1 is N and Y.sub.2 is C, R.sub.4 is H.
2. The compound of claim 1 in which Y.sub.1 is C and Y.sub.2 is N,
and R.sub.1, R.sub.2 and R.sub.4 are H and R.sub.3 is OH.
3. The compound of claim 1 in which Y.sub.1 is C and Y.sub.2 is N,
and R.sub.1, R.sub.2 and R.sub.4 are H and R.sub.3 is Cl.
4. The compound of claim 1 in which Y.sub.1 is C and Y.sub.2 is N,
and R.sub.1 and R.sub.2 are H, R.sub.3 is OH and X is NO.sub.2.
5. The compound of claim 1 in which Y.sub.1 is C and Y.sub.2 is N,
and R.sub.1 and R.sub.2 are H, R.sub.3 is OH and R.sub.4 is Cl.
6. The compound of claim 1 in which Y.sub.1 is C and Y.sub.2 is N,
and R.sub.1 and R.sub.2 are H, R.sub.3 is OH and X is Br.
7. The compound of claim 1 in which Y.sub.1 is N and Y.sub.2 is C,
and R.sub.1, R.sub.2, and R.sub.4 are H, and R.sub.3 is OH.
8. The compound of claim 1 in which Y.sub.1 is N and Y.sub.2 is C,
and R.sub.1 and R.sub.4 are H, and R.sub.2 is CH.sub.3 and R.sub.3
is OH.
9. The compound of claim 1 in which Y.sub.1 is N and Y.sub.2 is C,
and R.sub.1 and R.sub.4 are H, R.sub.2 is Br and R.sub.3 is OH.
10. The compound of claim 1 in which Y.sub.1 is N and Y.sub.2 is C,
and R.sub.1 and R.sub.4 are H, R.sub.2 is NO.sub.2 and R.sub.3 is
OH.
Description
BACKGROUND OF THE INVENTION
It is now well established that the enzyme xanthine oxidase is
implicated in the production of uric acid by the body, converting
hypoxanthine into xanthine and xanthine, in turn, into uric acid.
Under normal conditions, uric acid (2,6,8-trioxypurine) is found in
the body in only small amounts, a concentration in the blood on the
order of about one to about 3 micrograms per 100 milliliters. Under
certain pathological conditions, however, as for example gout, the
concentration of uric acid increases significantly.
Gout, of course, is metabolic disturbance in the body resulting
from an overproduction of uric acid, chronic hyperuricemia
(elevated blood uric acid), and progressive accumulation of uric
acid in the tissues. The body may also progressively lose its
capacity to excrete uric acid and is, therefore, in a constant
state of uric acid imbalance, accumulating a greater and greater
excess. Its concentration in the blood is high, and, because of its
low solubility, it tends to precipitate and form deposits at
various sites where the blood flow is least active, particularly
joints and cartilaginous tissues.
One approach to the control of gout commonly used in the past has
been the prescription of drugs which tended to prevent the
accumulation of uric acid in the body and thus diminsh the
liklihood of acute recurrences. Such drugs are identified as
"uricosuric agents" and promote the excretion of uric acid in the
urine. Examples of such drugs include p-dipropylsulfamyl benzoic
acid and sulfinpyrazone. These drugs cannot, however, be
administered in conjunction with aspirin or any other salicylate,
which might be given to relieve pain, because the agents and
salicylates are mutually antagonistic, i.e., each tends to offset
the action of the other.
A second approach to the treatment of gout which has become popular
is the use of the drug allopurinol, ##SPC2##
Which blocks the production of uric acid by the body by inhibiting
the enzyme xanthine oxidase, which, as noted previously, is
responsible for converting hypoxanthine into xanthine and xanthine
into uric acid. While allopurinol is effective to inhibit the
enzyme xanthine oxidase, nevertheless there are disadvantages which
limit its suitability.
First, the toxicity is higher than desirable, having a lethal
dosage level, LD.sub.50 (the dose required to kill 50% of a group
of mice in 2 weeks when injected into the intraperitoneal cavity)
on the order of 150 milligrams per kilogram of body weight.
Moreover, allopurinol is gradually metabolized in vivo to
4,6-dihydroxy pyrazolo ?3,4-d!pyrimidine, which is not as inhibitor
as is allopurinol. In addition to the foregoing disadvantages,
allopurinol, because of its chemical nature, must compete with
xanthine to occupy a place on the enzyme xanthine oxidase in order
to inhibit the enzyme and thus prevent the formation of uric acid
by the body, which likewise limits its efficiency. It is also known
that acute attacks of gouty arthritis occur in the early treatment
with allopurinol. It is accordingly necessary to give colchicine
during the initial period of therapy to prevent such acute attacks.
There have also been reports of the development of a pruritic rash
in some patients and of the occasional occurence of drowsiness when
allopurinol is administered. In view of the foregoing, it is
apparent that xanthine oxidase inhibitors which are of acceptable
toxicity and at the same time possess increased inhibition
efficiency as compared to allopurinol are highly desirable.
SUMMARY OF THE INVENTION
The present invention thus relates to xanthine oxidase inhibitors
comprising imidazo ?1,2,a! and pyrazolo ?1,5,a! pyrimidine
compounds of the following general structure: ##SPC3##
Y.sub.1 and Y.sub.2 are carbon or nitrogen; R.sub.1 is H or an
alkali metal or ammonium; R.sub.2 is H, CH.sub.3, a halogen,
phenylazo or NO.sub.2 ;
R.sub.3 is OR.sub.1, H, or a halogen; and R.sub.4 is H NO.sub.2 or
a halogen. When Y.sub.1 is carbon, Y.sub.2 is nitrogen, thereby
forming the pyrazolo compounds, and when Y.sub.1 is nitrogen,
Y.sub.2 is carbon, thus providing the imidazo compounds.
DETAILED DESCRIPTION OF THE INVENTION
The xanthine oxidase inhibitors of this invention are represented
by the foregoing structure. As will be seen from the illustrative
examples which follow, such compounds, especially compounds in
which R.sub.2 is H or phenylazo, demonstrate inhibitory activity
significantly greater than that of allopurinol. Moreover, such
compounds are stable to enzymatic oxidation and also are
noncompetitive inhibitors of the enzyme xanthine oxidase.
The method of preparing the compounds of the present invention will
be described in detail in certain of the illustrative examples. In
general, an efficient and comparatively straightforward process is
used for the preparation of such compounds with the precise process
varying depending upon whether an imidazo or pyrazolo compound is
produced.
The imidazo pyrimidines are preferably prepared from an imidazole
derivative such as amino imidazolinium sulfate, rather than by the
more common synthetic procedure for the preparation of imidazo
pyrimidines involving the condensation of aminopyrimidine
derivative with an .alpha.-halocarbonyl compound, which reaction is
limited by the nature of the substituents on the pyrimidine
starting material. For example, although reaction of
2-amino-4,6-dimethyl-pyrimidine with .alpha.-haloacetaldehyde
yields satisfactory quantities of 5,7-dimethyl imidazo?1,2,a!
pyrimidine, when 2-amino-4,6-dihydroxypyrimidine is reacted with
.alpha.-haloacetaldehyde, only polymeric materials are obtained.
This is believed to be due to the fact that pyrimidines, with three
strong electron donating substituents in the 2, 4, and 6 positions,
readily undergo electrophilic attack at the aromatic 5 position of
the pyrimidine ring.
Consequently, as indicated above, the imidazopyrimidines of the
present invention are produced from an imidazole derivative.
2-amino imdazolinium sulfate may be condensed with diethyl malonate
in the presence of sodium ethoxide in ethanol with acidification of
the alcoholic mixture with ethanolic hydrogen chloride to produce
the intermediate 2-amino-1-(ethoxymalonyl) imidazole. When this
compound is heated at 135.degree.C, it cylcizes to 5,7-dihydroxy
imidazo?1,2,a!pyrimidine, which may be readily purified by
recrystalization from water. Alternatively, the
imidazo?1,2,a!pyrimidine compound may be prepared directly from the
imidazole derivative 2-amino-imidazolinium sulfate without
isolation of the intermediate compound if the crude sodium salt
which separates from the initial reaction mixture does not come in
contact with hot water (in excess of 70.degree.C).
It will, of course, be understood that other of the described
5,7-imidazopyrimidines of the present invention can be prepared by
the above-described process if appropriate materials are used in
place of diethylmalonate. For the sake of brevity of disclosure,
however, the preparation of such compounds will not be specified in
greater detail at this point.
Treatment of the 5,7-dihydroxy imidazo?1,2,a!pyrimidine with an
aqueous solution of benzene diazonium chloride produces the
corresponding 6-phenylazo derivative. This compound may also be
produced by base catalyzed condensation of 2-amino imidazolinium
sulfate with diethyl phenylazomalonate. If the 5,7-dihydroxy
imidazo?1,2,a!pyrimidine compound is treated with bromine in
glacial acetic acid, 6-bromo-5,7-dihydroxyimidazo ?1,2,a!pyrimidine
is produced. Similarly, the 6-nitro derivative may be prepared by
base catalyzed condensation of the sulfate with diethyl
nitromalonate.
The pyrazolo?1,5,a! pyrimidines described in this invention are
preferably prepared by the following procedure. The treatment of
3-aminopyrazole with diethylmalonate in the presence of sodium
ethoxide affords the disodium salt of
5,7-dihydroxypyrazolo?1,5,a!pyrimidine. This salt, when dissolved
in water and treated with hydrochloric acid, yields
5,7-dihydroxypyrazolo?1,5,a!pyrimidine. In an analogous manner, the
condensation of 3-amino-4-chloropyrazole, 3-amino-4-bromopyrazole,
and 3-amino-4-nitropyrazole with diethylmalonate in the presence of
sodium ethoxide affords 3-chloro, 3-bromo, and 3-nitro derivatives
of 5,7-dihydroxy pyrazolo?1,5,a!pyrimidine.
The condensation of 3-aminopyrazole with methyl diethylmalonate in
the presence of sodium ethoxide affords
5,7-dihydroxy-6-methylpyrazolo?1,5,a!pyrimidine. Similarly, the
condensation of 2-phenylazo diethylmalonate with 3-amino pyrazole
in the presence of sodium ethoxide yields
5,7-dihydroxy-6-phenylazopyrazolo?1,5,a!pyrimidine.
Refluxing a solution of 5,7-dihydroxypyrazolo?1,5,a!pyrimidine in
phosphorus oxychloride affords
5,7-dichloropyrazolo?1,5,a!pyrimidine (I). The ##SPC4##
treatment of (I) with 2.5 N sodium hydroxide solution yields
5-chloro-7-hydroxypyrazolo?1,5a!pyrimidine (II). The reductive
dehalogenation of (II) affords 7-hydroxypyrazolo?1,5,a! pyrimidine
(III). This product is identical in all respects to the product
obtained by the condensation of 3-aminopyrazole with the sodium
salt of ethyl malonaldehydic ester in the presence of sodium
ethoxide. (reaction IV).
The invention will be better understood by reference to the
following specific but illustrative examples. The ultraviolet
spectra was recorded on a Cary-15 spectrophotometer. Proton
magnetic resonance studies were carried out with a Hitachi
Perkin-Elmer R-20A spectrometer with DSS as internal reference.
EXAMPLE I
Preparation of Imidazo ?1,2a! pyrimidine -5,7-diol. (Method A)
A solution of sodium ethoxide was prepared by dissolving sodium
?8.1g (0.352 formula weights)! in 200 ml of absolute ethanol.
Diethylmalonate ?27.4g (0.171 mole)! and 2-amino imidazole
hemisulfate ?22.2g (0.168 mole)! were added to the sodium ethoxide
solution. The resulting solution was stirred and warmed slowly to
reflux. After reflux was obtained a sodium salt began to separate
from the solution. The mixture was stirred and heated at reflux for
8 hours. After cooling to room temperature, the sodium salt was
separated by filtration, washed with ethanol, and air dried. The
sodium salt was dissolved in 100 ml of water (30.degree.), and the
product precipitated from the solution by the addition of 6.sup.N
hydrochloric acid until a pH of 1-2 was obtained. The mixture was
cooled to 5.degree. and the product was separated by filtration,
washed with ice water, and dried at 100.degree.. Recrystallization
from water afforded 11.9g (47%) of analytically pure product that
had a Mp>360.degree.C; .lambda. max (pH 1) 216nm (.epsilon.
22,200) and 263nm (.lambda., 7,900): .lambda.max (pH 11) 223nm
(.epsilon.19,500) and 265nm (.epsilon., 7,600).
Anal. calcd. for C.sub.6 H.sub.5 N.sub.3 O.sub.2 : C, 47.7; H 3.31;
N, 27.8. Found: C, 47.8; H, 3.31; N, 27.7.
EXAMPLE II
Preparation of Imidazo ?1,2a!-pyrimidine-5,7-diol (Method B)
A. 2-Amino-1-(ethoxymalonyl) imidazole -- A solution of sodium
ethoxide was prepared by dissolving sodium ?2.3g co. 1 formula
weights)! in 150 ml of absolute ethanol. Diethylmalonate ?8.8g, 55
m moles! and 2-aminoimidazole, hemisulfate ?6.7g, 50 m moles! were
added to the sodium ethoxide solution. The solution was stirred and
slowly warmed to reflux, at which time a sodium salt began to
precipitate. The mixture was refluxed for 6 hours and then 50 ml of
anhydrous ethanolic hydrogen chloride was added dropwise. After the
addition was complete the mixture was refluxed for 20 minutes,
filtered, and the filtrate evaporated to dryness. The gummy residue
was dissolved in 30 ml of water and the pH of this solution brought
to 6 by the addition of saturated sodium carbonate solution to
afford a light yellow product. The product was separated by
filtration, washed with water, and dried at 80.degree..
Recrystallization of this product from water afforded 3.35g (34%)
of analytically pure product that had a melting point of
175.degree. (slowly melts and resolidifys).
Anal. calcd. for C.sub.8 H.sub.11 N.sub.3 O.sub.3 : C, 48.7; H,
5.58; N, 21.3. Found: C, 48.7; H, 5.69; N, 21.4.
B. Imidazo ?1,2a! pyrimidine-5,7-diol. -- 2-amino-1-(ethoxymalonyl)
imidazole ?1.97g, 10 m moles! was heated at 200.degree. for 45
minutes. The residue after cooling was recrystallized from water to
afford 1.13g (75%) of analytically pure imidazo ?1,2a!
pyrimidine-5.7-diol; Mp 360.degree.. The ultraviolet absorption
spectra of this product is identical to the ultraviolet absorption
spectra of the product obtained by Method A.
Anal. calcd. for C.sub.6 H.sub.5 N.sub.3 O.sub.2 ; C, 47.4; H,
3.31; N, 27.8. Found: C, 47.4; H, 3.43; N, 27.5.
EXAMPLE III
Preparation of 6-Bromoimidazo ?1,2a! pyrimidine-5,7-diol.
Bromine ?0.bg (0.00375 formula weights)! in 10 ml of acetic acid
was added dropwise to a solution of imidazo ?1,2a!
pyrimidine-5,7-diol ?0.5g, 3.3 m moles! in 20 ml of acetic acid
with good stirring at room temperature. After the addition was
complete, the mixture was stirred at room temperature for 3 hours.
The solid was separated by filtration, washed with ethanol 2(25
ml), and dried to afford 0.68g (90%) of product. Recrystallization
from water afforded in analytically pure sample that decomposed
above 225.degree.C. .lambda.max (pH 1) 220 nm (.epsilon., 27,100)
and 276 nm (.epsilon., 9,900) :.mu.max (pH 11) 226 nm
(.epsilon.,33,600) and 274 nm (.epsilon., 7,800)
Anal. calcd. for C.sub.6 H.sub.4 N.sub.3 O.sub.2 Br : c, 31.3; H,
1.69; N, 18.3. Found: C, 31.2; H, 1.68; N, 18.0.
EXAMPLE IV
Preparation of 6-Methylimidazo ?1,2a! pyrimidine-5,7-diol.
A solution of sodium ethoxide was prepared by dissolving sodium
?1.84g (0.08 formula weights)! in 100 ml of absolute ethanol.
Diethyl methyl malonate ?6.96g, 40 m moles! and 2-aminoimidazole,
hemisulfate ?5.28g, 40 m moles! were added to the sodium ethoxide
solution. The mixture was refluxed with stirring for 8 hours, and
then evaporated to dryness at reduced pressure. The residue was
dissolved in 100 ml of water (30.degree.) and the ph adjusted to 2
by adding 6.sup.N hydrochloric acid. The product was separated by
filtration, washed with cold water, and recrystallized from water
to yield 2.74g (40%) of analytically pure product that had a
melting point of 310-2.degree. (dec.); .mu.max (pH 1) 218 nm
(.epsilon., 28,800) and 275 nm (.epsilon., 11,900); .mu.max (pH 11)
223 nm (.epsilon., 28,000) and 276 nm (.epsilon., 11,700).
Anal. calcd. for C.sub.7 H.sub.7 N.sub.3 O.sub.2 : C, 50.9; H,
4.24; N, 25.4. Found: C, 50.9; H, 4.38; N, 24.9.
EXAMPLE V
Preparation of 6-Nitroimidazo ?1,2a! pyrimidine-5,7-diol.
A solution of sodium ethoxide was prepared by dissolving sodium
?4.6g (0.2 formula weights)! in 250 ml of absolute ethanol. Diethyl
nitromalonate ?20.5g (0.1 mole)! and 2-aminoimidazole, hemisulfate
?13.2g (0.1 mole)! were added to the sodium ethoxide solution. The
mixture was refluxed with stirring for 9 hours, and then evaporated
to dryness at reduced pressure. The residue was dissolved in 250 ml
of water, and the pH adjusted to 1-2 by the addition of 6N
hydrochloric acid. The product was separated by filtration, washed
thoroughly with water and dried. Purification was afforded by
reprecipitating the product from dilute sodium hydroxide solution
by the addition of 6N hydrochloric acid to afford 5.9g (30%) of
analytically pure product. Mp, 305.degree.-7.degree.(dec); .lambda.
max (pH 1) 207 nm (.epsilon., 23,300) and 320 nm (.epsilon.,
6,300); .lambda. max (pH 11) 226 nm (.epsilon., 13,300) and 342 nm
(.epsilon., 4,900).
Anal. calcd. for C.sub.6 H.sub.4 N.sub.4 O.sub.4 : C 36.7; H, 2.04;
N, 28.6. Found: C, 36.8; H, 2.16; N, 28.4.
EXAMPLE VI
Preparations of 6-phenylazoimidazo ?1,2a! pyrimidine-5,7-diol.
A. A solution of sodium ethoxide was prepared by dissolving sodium
?1.98g (0.086 formula weights)! in 150 ml of absolute ethanol.
Diethylphenylazomalonate ?11.35g, 43 m moles! and 2-aminoimidazole,
hemisulfate ?5.68g. 43 m moles! were added to the sodium ethoxide
solution. The mixture was refluxed with stirring for 8 hours, and
then evaporated to dryness at reduced pressure. The residue was
dissolved in 300 ml of water and the pH adjusted to 5-6 by the
addition of a glacial acetic acid. The product was separated by
filtration, washed with water, and purified by recrystallizing from
ethanol to afford 3.40g (31%) of analytically pure product that had
a melting point of 277.degree.-9.degree. (dec); .lambda. max (pH 1)
205 nm (.epsilon., 23,000) and 408 nm (.epsilon., 27,800); .lambda.
max (pH 11) 217 nm (.epsilon., 19,400) and 384 nm (.epsilon.,
20,900).
Anal. calcd. for C.sub.12 H.sub.9 N.sub.5 O.sub.2 : C, 56,5; H,
3,53; N, 27.4. Found: C, 56.7; H, 3.62; N, 26.6.
B. A solution of benzenediazonium chloride was prepared by treating
a solution of aniline ?0.31g, 3,3 m moles! in 7 ml of 1.7N
hydrochloric acid with a solution of sodium nitrite ?0.25g, 0.0036
formula weights! in 5 ml of water at 5.degree.. The benzene
diazonium chloride solution was added dropwise to a stirred
solution of imidazo ?1.2a! pyrimidine-5,7-diol ?0.5g, 3.3 m moles!
in 10 ml of 1.25 N sodium hydroxide solution at 10.degree.. After
the addition was complete, the mixture was stirred at 10.degree.
for 30 minutes and then at room temperature for 30 minutes. The
solid was separated by filtration, and purified by reprecipitating
from a dilute sodium hydroxide solution by the addition of acetic
acid. A final purification by recrystallizing from absolute ethanol
afforded 0.41g (40%) of analytically pure product that had a
melting point of 277.degree.-8.degree. (dec). The ultraviolet
absorption spectra of this product is identical to the spectra of
the compound prepared by Method A.
EXAMPLE VII
Preparation of Pyrazolo ?1,5a! pyrimidine-5,7-diol.
This compound was prepared by the procedure of Y. Makisumi as
described in the Chemical and Pharmaceutical Bulletin (Tokyo), 10,
612 (1962). This procedure is as follows:
A solution of sodium ethoxide was prepared by dissolving sodium
?4.6g (0.2 formula weights)! in 300 ml of absolute ethanol.
Diethylmalonate ?16.0g, 0.1 mole! and 3-aminopyrazole ?8.3g, 0.1
mole! were added to the sodium ethoxide solution. The solution was
stirred and slowly warmed to reflux at which time a sodium salt
began to precipitate. The mixture was refluxed for 6 hours and then
allowed to cool to room temperature. The sodium salt was separated
by filtration, washed with absolute ethanol, and dried. The sale
was dissolved in 250 ml of water and the product precipitated by
the addition of 6 N hydrochloric acid until a pH of 1-2 was
obtained. The product was separated by filtration, washed with the
water, and recrystallized from water to afford 7.4g (49%) of
analytically pure product; mp 247.degree.-8.degree. (dec); .lambda.
max (pH 1) 218 nm (.epsilon., 14,700) and 276 nm (.epsilon.,
7,300); 229 nm (.epsilon., 22,200) and 270 nm (.epsilon.,
10,900).
Anal. calcd. for C.sub.6 H.sub.5 N.sub.3 O.sub.2 : C, 47.7; H,
3.31; N, 27.8. Found: C, 47.6; H, 3.42; N, 27.7.
EXAMPLE VIII
Preparation of 6-Methylpyrazolo ?1,5a! pyrimidine-5,7-diol.
A solution of sodium ethoxide was prepared by dissolving sodium
?2,3g (0.1 formula weights)! in 150 ml of absolute ethanol. Diethyl
methylmalonate ?8.7g, 50 m moles! and 3-aminopyrazole ?4.15g, 50 m
moles! were added to the sodium ethoxide solution. The mixture was
refluxed and stirred for 3 hours, and then evaporated to dryness at
reduced pressure. The residue was dissolved in 125 ml of water and
the pH adjusted to 1-2 by the addition of 6 N hydrochloric acid.
The product was separated by filtration, washed with water, and
dried at 100.degree.. Purification was afforded by reprecipitating
the product from dilute sodium hydroxide solution by the addition
of 6 N hydrochloric acid to afford 4.37g (54%) on analytically pure
product; mp, 358.degree.-60.degree. dec; .lambda. max (pH 1) 209 nm
(.epsilon., 21,300) and 275 nm (.epsilon., 10,700); .lambda. max
(pH 11 ) 226 nm (.epsilon., 22,700) and 280 nm (.epsilon.,
13400).
Anal. calcd. for C.sub.7 H.sub.7 N.sub.3 O.sub.2 : C, 50.9; H,
4.24; N, 25.4. Found: C, 50.9; H, 4.36; N, 25.1.
EXAMPLE IX
Preparation of 6-Phenylazopyrazolo ?1,5a! pyrimidine-5,7-diol.
A solution of sodium ethoxide was prepared by dissolving sodium
?1,52g (0.066 formula weights)! in 150 ml of absolute ethanol.
Diethyl phenylazomalonate ?8.71g, 33 m moles! and 3-aminopyrazole
?2.74g, 33 m moles! were added to the sodium ethoxide solution. The
mixture was stirred and heated at reflux for 6 hours, and then
evaporated to dryness at reduced pressure. The residue was
dissolved in 300 ml of water and the pH adjusted to 2 by the
addition of 6 N hydrochloric acid. The solid was separated by
filutration, washed with water and ethanol, and air dried.
Purification was afforded by reprecipitating the product from a
dilute sodium hydroxide solution by the addition of 6 N
hydrochloric acid to afford 2.34g (28%) of analytically pure
product; mp. 300.degree.-2.degree. (dec); .lambda. max (pH 1) 202
nm (.epsilon., 25,200), 246 nm (.epsilon., 9,700), and 406 nm
(.epsilon., 30,100 ); .lambda. max (pH 11) 210 nm (.epsilon.,
19,900) and 386 nm (.epsilon., 20,000).
Anal. calcd. for C.sub.12 H.sub.9 N.sub.5 O.sub.2 : C, 56.5; H,
3.53; N, 27.5. Found: C, 57.0; H, 3.84; N, 27.9.
EXAMPLE X
Preparation of 3-Chloropyrazolo ?1,5a! pyrimidine-5,7-diol.
A solution of sodium ethoxide was prepared by dissolving sodium
?0.98g (0.0428 formula weights)! in 50 ml of absolute ethanol.
Diethylmalonate ?3.50g, 21.9 m moles! and 3-amino-4-chloropyrazolo
?2.30g, 2.14 m moles! were added to the sodium ethoxide solution.
The mixture was stirred and slowly warmed to reflux. After
refluxing for 8 hours the mixture was allowed to cool to room
temperature and the sodium salt separated by filtration. The sodium
salt was dissolved in 100 ml of water and the pH adjusted to 1-2 by
the addition of 6 N hydrochloric acid. The product was separated by
filtration, washed with ice water, and dried. Recrystallization
from water afforded 1.48g (37%) of analytically pure product that
had a melting point of 270.degree.-2.degree. (dec); .lambda. max
(pH 1) 219 nm (.epsilon., 32,140) and 286 nm (.epsilon., 16,680);
.lambda. max (pH 11) 231 nm (.epsilon., 47,000) and 275 nm
(.epsilon., 18,520).
Anal. calcd. for C.sub.6 H.sub.4 Cl N.sub.3 O.sub.2 : C, 38.8; H,
2.16; N, 22.6. Found: C, 38.6; H, 2.46; N, 22.9.
EXAMPLE XI
Preparation of 3-Nitropyrazolo ?1,5a! pyrimidine-5,7-diol,
hemihydrate.
A solution of sodium ethoxide was prepared by dissolving sodium
?0.92g (0.04 formula weights)! in 50 ml of absolute ethanol.
Diethylmalonate ?3.2g, 20 m moles! and 3-amino-4-nitropyrazole
?2.56g, 20 m moles! were added to the sodium ethoxide solution. The
mixture was stirred and refluxed for 16 hours and then allowed to
cool to room temperature. The sodium salt was separated by
filtration, washed with absolute ethanol and air dried. The sodium
salt was dissolved in 100 ml of water and the pH of this solution
adjusted to 1-2 by the addition of 6 N hydrochloric acid. The
product was separated by filtration, washed with cold water, and
recrystallized with water to afford 3.0g (77%) of analytically pure
product; Mp, 226.degree.-8.degree.; .lambda. max (pH 1) 218 nm
(.epsilon., 17,600) and 319 nm (.epsilon., 8,700); .lambda.max (pH
11) 227 nm (.epsilon., 12,700) and 332 nm (.epsilon., 13,700).
Anal. calcd. for C.sub.6 H.sub.4 N.sub.4 O.sub.4 1 1/2H.sub.2 O: C,
35.1; H, 2.45; N, 27.3. Found: C, 35.1; H, 2.55; N, 27.6.
EXAMPLE XII
Preparation of 3-Bromopyrazolo ?1,5a!-pyrimidine-5,7-diol.
A solution of sodium ethoxide was prepared by dissolving sodium
?2.76g (0.120 formula weights)! in 50 ml of absolute ethanol.
Diethylmalonate ?9.6g, 60.2 m moles! and 3-amino-4-bromopyrazole
?9.75g (60.2 m moles)! were added to the sodium ethoxide solution.
The mixture was stirred at room temperature for 3 days, and the
sodium salt separated by filtration. The sodium salt was dissolved
in 100 ml of water and the pH of this solution adjusted to 1-2 by
the addition of 6 N hydrochloric acid. The product was separated by
filtration, washed with cold water, and dried. Purification was
accomplished by reprecipitating the product from a dilute sodium
hydroxide solution by the addition of 6 N hydrochloric acid. The
analytically pure material weighed 4.3g (31%) and decomposed
without melting at 265.degree.-7.degree.; .lambda. max (pH 1) 221
nm (.epsilon., 20,300) and 283 nm (.epsilon., 9,870); .lambda. max
(pH 1) 233 nm (.epsilon., 32,800) and 273 nm (.epsilon.,
12,850).
Anal. calcd. for C.sub.6 H.sub.4 Br N.sub.3 O.sub.2 : C, 31.3; H,
1.72; N, 18.2. Found: C, 31.2; H, 1.84; N, 18.3.
EXAMPLE XIII
Preparation of 5-Chloropyrazolo ?1,5a! pyrimidine-7-ol
A. 5,7-Dichloropyrazolo ?1,5a! pyrimidine
A mixture of pyrazolo ?1,5a!-pyrimidine-5,7-diol (32g) and
phosphorus oxychloride (160 ml) was stirred and heated at
110.degree.. At the end of 1 hour a complete solution was obtained,
and this solution was heated for an additional 11/2 hours. The
excess phosphorous oxychloride was removed at reduced pressure
using the steam bath as the source of heat. The red syrup was added
slowly to 400g of crushed ice with good stirring. This mixture was
stirred at 5.degree. for 30 minutes, and then extracted with
anhydrous ether 6(400ml). The etheral extract was washed with
water, washed with dilute sodium bicarbonate solution, and dried
over anhydrous sodium sulfate. Evaporation of the etheral extract
afforded 23g of slightly yellow product, which was sublimed at
reduced pressure (1 mm) and elevated temperature
(70.degree.-100.degree.) to afford 11.9g (29%) of analytically pure
product that had a melting point of 72.degree.-4.degree.; .lambda.
max (CH.sub.3 OH) 235 nm (.epsilon., 32,000) and 290 mm (.epsilon.,
2,200).
Anal. calcd. for C.sub.6 H.sub.3 Cl.sub.2 N.sub.3 : C, 38.3; H,
1.59; N, 22.3. Found: C, 38.2; H, 1.78; N, 22.4.
B. 5-Chloropyrazolo ?1,5a! pyrimidine-7-ol
A solution of 5,7-dichloropyrazolo ?1,5a! pyrimidine ?6.0g 31.9 m
moles! in 1.25N sodium hydroxide solution (120 ml) was heated at
95.degree. for 1 hour. The hot solution was treated with
decolorizing carbon and filtered. Acification of the filtrate with
6 N hydrochloric acid until a pH of 2 was obtained, afforded the
desired product. Recrystallization from a mixture of water and
ethylalcohol (1:1) gave 4.83g (89%) of analytically pure product
that had a melting point of 295.degree.-8.degree. (dec); .lambda.
max (pH 1) 223 nm (.epsilon., 34,000) 263 nm (.epsilon., 4,900) and
305 nm (.epsilon., 5,100); .lambda. max (pH 11) 227 nm (.epsilon.,
18,200), 281 nm (.epsilon., 8,000) and 303 nm (.epsilon.,
7,300).
Anal. calcd. for C.sub.6 H.sub.4 N.sub.3 O Cl: C, 42.5; H, 2.35; N,
24.8. Found: C, 42.3; H, 2.76; N, 24.7.
XIV
Preparations of Pyrazolo ?1,5a!-pyrimidine-7-ol.
A. A mixture of 5-chloropyrazolo ?1,5a!-2-pyrimidine-7-ol ?8.50g 50
m moles!; 150 ml of methanol, 10 ml of 1.25 N sodium hydroxide
solution, and 10% palladium or charcoal catalyst was placed in a
Paro hydrogenation apparatus and hydrogenated at a pressure of 42
lbs/in.sup.2. Within 5 hours, the calculated quantity of hydrogen
(Ca. 4.5 lbs./in.sup.2) had been absorbed. The mixture was filtered
and the filtrate acidified with 6 N hydrochloric acid until a pH of
2 was obtained. The acidified solution was evaporated to dryness
and the residue washed with cold water. The white crystalline
product was recrystallized from a mixture of methanol and water
(2.5:1) to afford 3,13g (46%) of analytically pure product that had
a melting point of 327.degree.-9.degree. (dec); .lambda. max (pH 1)
216 nm (.epsilon., 10,600), 260 nm (.epsilon., 6,900) and 295 nm
(.epsilon., 4,900); .lambda. max (pH 11) 226 nm (.epsilon., 4,900),
280 nm (.epsilon., 8,600) and 308 nm (.epsilon., 7,700).
Anal. calcd. for C.sub.6 H.sub.5 N.sub.3 O: C, 53.3; H, 3.70; N,
31.1. Found: C, 53.3; H, 3.97; N, 30.9.
B. A mixture of ethylacetate ?17.8g, 0.2 mole! and ethylformate
?14.8g (0.2 mole! was added dropwise to a suspension of finely cut
sodium ?4.6g (0.2 formula weights)! in 500 ml of absolute ether
with vigorous stirring. After the addition was complete the mixture
was stirred vigorously for 20 hours, during which time the sodium
salt of malonaldehydic ester precipitated. A solution of
3-aminopyrazole ?16.6g, 0.2 mole! in 250 m1 of absolute ethanol was
added to the suspension, and the resulting mixture heated at
70.degree. until the ether had evaporated. The resulting mixture
was refluxed for 8 hours and then evaporated to dryness at reduced
pressure. The residue was dissolved in 200 ml of water and this
solution treated with decolorizing carbon and filtered. The
filtrate was acidified to pH2 by the addition of 6 N hydrochloric
acid, and the precipitated product separated by filtration.
Recrystallization from a methanol and water mixture afforded 3.1g
(11%) of analytically pure product that had a melting point of
325.degree.-8.degree. (dec). The ultra-violet absorption spectra of
this compound is identical to the spectra of the compound prepared
by Method A.
EXAMPLE XV
In this example, the inhibitory activity of the compounds of this
invention was determined by recording spectral changes at a
constant wavelength of 290 nm, using a Cary Model 15 recording
spectrophotometer equipped with an 0-0.1 O.D. slidewire. To
facilitate dissolution in the incubation mixture, the compounds of
this invention were dissolved in DMSO (Pure spectral grade), which
is a good solvent and also one which does not inhibit the enzyme at
the indicated concentrations. After dissolving in DMSO, the
compounds were introduced into the incubation mixture containing
150 micromoles of tris HCl buffer (pH 7.83), 0.6 micromoles EDTA,
0.013 micromoles xanthine, and 20 to 40 micrograms of xanthine
oxidase in a final volume of 3.0 milliliters. The xanthine oxidase
was purified milk xanthine oxidase from Worthington Biochemical
Corporation and had a specific activity of 0.29 u/mg of protein.
The assay was carried out by mixing all the components in a cuvette
at 25.degree.C. The reaction was started by the addition of the
enzyme with the aid of a stirrer-adder. Changes in O.D. were
measured for several minutes, and the rate (.DELTA. O.D. per
minute) was calculated from the spectral change over the first 15
seconds.
In order to fully evaluate the inhibitory activity and to compare
the relative inhibitory abilities of the compounds of this
invention with allopurinol, experiments were carried out in which
the concentration of inhibitor was varied over a range of
approximately 10.sup.-.sup.4 to about 10.sup.-.sup.7 molar and the
initial velocity of xanthine oxidation was measured. From a plot of
the log of inhibitor concentration against the percent inhibition,
the concentration of inhibitor giving 50% inhibition, (I.sub.50),
was calculated. This value was obtained from a linear regression
analysis of the straight lines obtained in this graph. The data is
presented in Tables I and II which follows as (I/S).sub.0.5 values.
The lower the (I/S).sub.0.5 value, the more potent the inhibitory
capability of the compound.
Table I ______________________________________ Imidazo
?1,2,a!pyrimidines Compound No. R.sub.1 R.sub.2 R.sub.3 R.sub.4
(I/S).sub.0.5 ______________________________________ (1) H H OH H
3.57 * (2) NH.sub.2 H OH H 100.1 (3) H CH.sub.3 OH H 1.54 (4) H Br
OH H 0.92 (5) H phenyl- OH H 0.17 azo * (6) NH.sub.2 phenyl- OH H
55 azo * (7) NH.sub.2 Br OH H 7.65 (8) H NO.sub.2 OH H 2.80 * (9)
Allopurinol 0.58 ______________________________________ * Included
for comparison only; outside scope of invention.
TABLE II ______________________________________ Pyrazolo
?1,5,a!pyrimidines Compound No. R.sub.1 R.sub.2 R.sub.3 R.sub.4
(I/S).sub.0.5 ______________________________________ (1) H H OH H
0.447 (2) H CH.sub.3 OH H 5.37 (3) H phenyl- OH H 3.22 azo (4) H H
H H 1.08 (5) H H Cl H 2.42 *(6) NH.sub.2 H Cl H 32 *(7) NH.sub.2 H
OH H 33.4 *(8) Allopurinol 0.58
______________________________________ *Included for comparison
only; outside scope of invention.
Referring now more particularly to Table I, it will be observed
that Compounds (1), (3), (4), (5), and (8) demonstrated significant
inhibitory activity. Compound (5), the 5,7-dihydroxyimidazo
compound in which R.sup.2 is phenylazo, demonstrated exceptional
activity, being approximately 31/2 times superior to allopurinol,
which is also included in Table I for comparison. It will also be
observed that Table I includes compounds in which an amino group is
present at the 7 position. A comparison of the activity results
between Compounds (1) and (2), (5) and (6), and (4) and (7), shows
that the inhibitory activity is decreased significantly. A
comparison of Compounds (1) and (7) and (5) and (6) in Table II
shows similar results.
It should also be noted, with respect to Table II, that Compounds
(1), (2), (3), (4), and (5) demonstrated significant ability to
inhibit the enzyme xanthine oxidase. Compound (1) was especially
good, being superior to allopurinol, which has an (I/S).sub.0.5 of
0.58.
EXAMPLE XVI
Oxidation tests were also conducted on the compounds of the present
invention to determine their stability against enzyme oxidation.
The compounds were tested for their ability to serve as alternate
substrate in two ways: (1) by substituting the test compound for
xanthine in the basic incubation mixture described with respect to
Example XV above and measuring the change in O.D. from 220 to 380
nm over a 60-minute period, using the repetitive scan accessory of
the Cary spectrophotometer, and (2) using the method of Johns,
Spector, and Robins, Bioch. P. Col., 18, p. 2371-83 (1969) in which
phenozine methosulfate and cytochrome C were substituted for oxygen
as the electron carrier. Changes in 0.D. of the test compound
(Method I) or of the phenozine methosulfate (Method 2) were
observed for 30 minutes at the appropriate wavelength values (550
nm for phenozine methosulfate).
As noted previously, it is known that allopurinol is oxidized to
4,6-dihydroxypyrazolo?3,4,d!pyrimidine by xanthine oxidase.
Because, of this, the ability of the compounds of this invention to
be oxidized by xanthine oxidase were determined. The reactions were
carried out in vitro using the methods described above. Under the
same conditions, allopurinol was slowly oxidized to
4,6-dihydroxypyrazolo ?3,4,d!pyrimidine when oxygen was used to
accept electrons and was completely oxidized to such compound in
the presence of phenozine methosulfate in less than 5 minutes
incubation at 25.degree.C. In contrast, the compounds of this
invention shown in Tables I and II above were completely stable to
enzymatic oxidation under either condition.
EXAMPLE XVII
The compounds of this invention shown in Tables I and II were next
subjected to kinetic analysis using the methods of Lineweaver and
Burke, or Hanes Enzymes, M. Dixon and E. C. Webb, 1964, Acad.
Press, New York, N.Y., to determine the type of inhibition they
were exhibiting. Allopurional has been shown to be a competitive
inhibitor of xanthine oxidase. As can be seen from Tables III and
IV which follow, the 5,7-dihydroxy compounds having the indicated
R.sub.2 substituents were non-competitive inhibitors. The K.sub.i
values shown in Tables III and IV were calculated from a Dixon-plot
(I) against 1/v at the two indicated substrate concentrations.
TABLE III
__________________________________________________________________________
Imidazo?1,2,a!pyrimidines Non-inhibited Reaction Type of R.sub.1
R.sub.2 R.sub.3 R.sub.4 Inhibition Km .times. 10.sup.6 Vm.sup.x
10.sup.6 K.sub.i .times. 10.sup.6
__________________________________________________________________________
H Br OH H Non-Comp. 2.83 13.2 11.9, 17.1 H phenyl- OH H " 3.98 13.2
2.75, 3.12 azo H NO.sub.2 OH H " 5.51 16.7 29.8, 38.3 H CH.sub.3 OH
H " 2.68 14.8 23.6, 26.2 H H OH H " 2.68 14.8 34.0, 51.8
__________________________________________________________________________
TABLE IV
__________________________________________________________________________
Pyrazolo?1,5,a!pyrimidine Non-inhibited Reaction Type of R.sub.1
R.sub.2 R.sub.3 R.sub.4 Inhibition Km .times. 10.sup.6 Vm.sup.x
10.sup.6 K.sub.i .times. 10.sup.6
__________________________________________________________________________
H H OH H Non-Comp. 1.99 11.3 6.08, 6.95 H phenyl- OH H " 1.10 10.8
101, 116 azo H CH.sub.3 OH H " 3.23 13.3 75.9, 90.8
__________________________________________________________________________
EXAMPLE XVIII
Using the general procedure and conditions described with respect
to Example XV, the inhibitory activity of the compounds set forth
in Table V was determined. The incubation mixture again had a total
reaction volume of 3.0 milliliters and the xanthine concentrations
was 1.32 .times. 10.sup.-.sup.5 M. It will be seen from Table V
that the compounds in which R.sub.4 is NO.sub.2, Cl or Br,
demonstrated highly significant inhibitory activity. Also shown in
Table V for comparison is Compound (1) of Table II. It will be
appreciated that the three other compounds shown in Table V
exhibited very good inhibition capability with the halogenated
compounds showing significantly better
TABLE V ______________________________________
Pyrazolo?1,5,a!pyrimidine R.sub.1 R.sub.2 R.sub.3 R.sub.4
(I/S).sub.0.5 ______________________________________ H H OH
NO.sub.2 1.14 H H OH Cl 0.106 H H OH Br 0.091 *H H OH H 0.447
______________________________________ *Compound (1) of Table II.
(I/S).sub.0.5 values than Compounds(1).
It should be further noted that the toxicity level of the compounds
of the present invention is less than allopurinol. As noted
previously, the LD.sub.50 for allopurinol is 150.+-. 80 milligrams
per kilogram.
At this point, it should be noted that the chemical structure of
Compound (1) of Table II has been previously reported, Chemical
& Pharmaceutical Bulletin (Tokyo), Vol. 10, No. 7, p. 612 et
seq. (1962) and Japanese Pat. No. 7983, dated July 11, 1962. To our
knowledge, however, there is no indication reported that this
compound has utility as an inhibitor of the enzyme xanthine
oxidase.
The xanthine oxidase inhibitors are administered as oral
preparation, in capsule or tablet form. The tables or capsules will
contain from about 5 to about 100 milligrams of the inhibitor per
tablet or capsule. The required dose of the inhibitor, of course,
will vary depending upon the condition of the patient, but will
normally range from approximately 20 to 600 milligrams per day. To
effectively inhibit the enzyme xanthine oxidase, a concentration in
the blood from about 1 to about 500 micrograms of the inhibitor is
required, preferably about 1 to 50 micrograms per milliliter.
The capsules will be the usual gelatin capsules and will contain in
addition to the inhibitor, a small quantity, for example less than
5% by weight preferably less than about 1%, magnesium stearate or
other flowing agent, such as "Avicel" (carboxymethylcellulose).
Tablets will include the inhibitor and, e.g., a binder, which may
be a gelatin solution, a starch paste in water, polyvinyl
pyrrilidone, polyvinyl alcohol in water, etc., with a typical sugar
coating.
* * * * *