Note: Descriptions are shown in the official language in which they were submitted.
O~;Z
This invention relates to 9-(2-hydroxyethoxy-
methyl)guanine monophosphate, in particular to
pharmaceutically acceptable salts thereof. The invention
also relates to methods of preparing this compound and
its salts, and also to pharmaceutical compositions
containing them.
9-(2-Hydroxyethoxymethyl) derivatives of purines
are known to have antiviral activity against various
classes of D~A and RNA viruses both in in vitro and in vivo
experiments, see U.K. Patent No. 1,523,865. In parti-
cular these compounds are active as antiviral agents
against Vaccinia, and herpes viruses, including simplex,
zoster and varicella in mammals, which viruses cause
such diseases as herpetic keratitis in rabbits and
herpetic encephalitis in mice.
It has now been found that the monophosphate
ester of 9-(2-hydroxyethoxymethyl)guanine is not only as
active as the unphosphorylated compound but also has the
selective advantage of much greater solubility at least
at a pH of from 1 to 7.5 compared with the correspond-
ing unphosphorylated compound.
According to the present invention there is
provided an 9-hydroxyethoxymethyl guanine derivative of
formula (I~:
OH
Hz ~
CH2 . O ~ CH2 C~2 ~ I
01
- 2 -
10~ 40fi Z YC/77
wherein W and Z are the same or different and each
represents an hydrogen atom or pharmaceutically acceptable
cation.
The pharmaceutically acceptable cation may be
selected from a group comprising sodium, potassium, lithium,
calcium/2, magnesium~2, aluminium/3 or ammonium.
Compounds of formula (I) wherein Z is sodium, potassium
or ammonium and W is hydrogen are preferred, and compounds
of formula (I) wherein Z is sodium or ammonium and W i5
hydrogen are particularly preferred.
In the above definition of W and Z, the polyvalent
cations are expressed as calcium/2, magnesium/2 and
aluminium/3, which is intended to mean the cation divided
. ~ ~/2 and Al~*/3. This is meant
by its valence le. Ca /2, Mg
to indicate that calcium or magnesium cations are in ionic
association with two phosphate oxygens, arld aluminium with
three.
In a second aspect of the present invention there
is provided a method of preparing a compound of formula (I),
as de~ined above characterised in that:-
(a) a compound of formula (II)
OH
H2N ~ ~
~2-0-CH2-cH2.OH
is reacted ~ h a phosphorylating agent to produce a
compound of formula (I) wherein l~oth ~ and Z are hydrogen
lO9~0fiZ
atoms,
(b) a compound of formula (III),
M
N ~ ~ (III)
G N N
CH20.CH2.CH2.O-P-OH
OH
wherein either M is a 6-hydroxy group and G is an atom or
group that can be replaced or converted to an amino group
by selective ammonolysis, or G is a 2-amino group and M is
an atom or group that can be replaced or converted to an
hydroxy group by selective hydrolysis, is converted to a
compound of formula (I) and optionally converting a compound
of formula (I) wherein W and Z are both hydrogen into a
compound wherein either or both of W and Z is a pharmaceuti-
cally acceptable cation, by reaction with a base or a salt
containing the desired cation.
In method (a) derivativés of phosphoric acid
having one to three hydroxy groups replaced by halogen atoms,
e.g. chlorine, such as phosphorus oxychloride, are
preferred for phosphorylation. Up to two of the hydroxy
groups can also be substituted to form alkoxy groups
optionally carrying further substitutions to form for
instance benzyloxy groups. SUch phosphohalic derivatives
or phosphates are applied under the usual neutral or alka-
line conditions, the latter preferably requiring activation
for instance by carbodiimide, e.g. dicyclohexylcarbodiimide
except when it is presented in
~ Z yC/77
the form of the anhydride.
Where at least two of the hydroxy groups in the
phosphoric acid derivative are replaced by halogen,
then after reaction with the compound of formula (II)
it is necessary to remove the free halogens by fairly
mild aqueous hydrolysis, using for example a molar
equivalent of water in a water miscible solvent such as
alcohol.
Substituted or unsubstituted alkoxy groups
introduced with a phosphate may be hydrolysed in a
suitable aqueous medium in the presence of bases in a
subsequent step. Aromatically substituted alkoxy groups
such as benzyloxy can also be subjected to hydrogenolysis,
preferably in the presence of a catalyst, according to
the usual techniques of reductive cleaving.
A preferred method of phosphorylating the
intermediate compounds of this invention involves reaction
of a compound of formula ~II), as defined above, with
phosphorus oxychloride in the presence of a trialkyl
phosphate and preferably at a temperature of about 0C
or less.
Other useful methods for preparing the mono-
phosphate include reaction of a compound of formula (II)
with phosphorus oxychloride in dry pyridine.
Compound of formula (II) can be considered as
intermediate in the synthesis of compounds of formula ~I)
~and can be prepared according to the methods described in
;Z
U.K. Patent No. 1,523,86S.
Conversion of a compound of formula (III), by
method (b) can be achieved in several ways, for example,
when G is a halogen atom, mercapto or an alkylthio group,
such as methylthio, it can be converted to an amine group
by ammonolysis. This method, together with other processes
well known in the art can be found in "Heterocyclic
compounds - Fused Pyrimidines Part II Purines ed. by D. J.
Brown (1971) published by Wiley - Interscience".
M can represent a halogen atom, mercapto or alkyl-
thio group, which may be converted to an hydroxy group by
hydrolytic methods described in the aforementioned book.
Compounds of formula (III) can be considered as
intermediates in the synthesis of compounds of formula (I)
and can be analogously prepared according to method (a),
which compounds can in turn be analogously prepared accord-
ing to the methods described in U.K. Patent No. 1,523,865.
Pharmaceutically acceptable salts of 9-(2-hydroxy-
ethoxymethyl)guanine monophosphate may be prepared by
neutralizing the monophosphate in its acidic form with an
equivalent (i.e. equinormal) amount of a base such as an
hydroxide, bicarbonate, carbonate which contains the desired
cation, that is sodium, potassium, ammonium, calcium, lithium,
magnesium or aluminium. Alternatively they may be prepared
by exchange reactions whereby one salt of the monophosphate
is treat~d with a solution, preferably aqueous, of a salt
containing the desired cation. For example the slightly
, ~ . . ,
,, - . .
10~40fi2
soluble barium salt of 9-(2-hydroxyethoxymethyl)guanine
monophosphate is treated in aqueou~ suspension with
sodium sulphate to remove the barium as the very insoluble
barium sulphate, leaving sodium 9-(2-hydroxyethoxymethyl)-
guanine monophosphate in solution.
In another aspect of the invention there is
provided a pharmaceutical composition comprising a compound
of formula (I) as hereinbefore defined together with a
pharmaceutically acceptable carrier therefor.
Pharmaceutically acceptable carriers are materials
useful for the purpose of administering the composition,
and may be solid, liquid or gaseous materials, which are
otherwise inert and medically acceptable and are compatible
with the active ingredient. These pharmaceutical composi-
tions may be administered orally, parenterally, used as a
suppository or pessary, applied topically as an ointment,
cream, aerosol, or powder, or given as eye or nose drops,
depending on whetHer the preparation is used to treat
internal or external viral infections.
For internal infections the compound of formula
(I) are administered at dose levels of 0.1 to 250 mg per
kg, calculated as the free phosphate form, preferably 1.0
to 50 mg per kg of mammal body weight, and are used in man
in a unit dosage form, administered for example a few
times daily, as one or more unit doses in an amount of 1
to 800 mg per unit dose, preferably 1 to 250 mg per unit
dose, most preferably 10 to 200 mg per unit dose.
lO ~ ~CH~2 yC/77
Por p~renteral administration, or administration
topically as drops, e.g. for eye infections, compounds of
formula (I) may be presented in aqueous solutions at a
concentration of from about 0.1 to 10~ w/v, preferably 0.1
to 7%, most preferably 0.2 to 5% w/v.
Alternatively, for infections of the eye or other
external tissue, e.g. mouth and skin, solution, ointment
or cream topical formulations are preferred. Concentrations
of from about 0.1 to 10~, preferably 0.3 to 6~, most
preferably 3~ may be used.
In yet a further aspect of the invention there is
provided a method of treating viral infections in mammals
which comprises the administration of an effective non-
toxic antiviral amount of a compound of formula (I) as
hereinbefore defined. As used herein the term "effective
non~toxic antiviral amount" is denoted to mean a predeter-
mined antiviral amount sufficient to be effective ag&inst
the virus in vivo.
The invention will now be illustrated with reference
to the following Examples.
10~ ~~Z YC/77
EXAMPLE 1
9-(2-l~ydroxyethox~ethyl)guanine monophosphate
Phosphorus oxychloride (0.03 ml) was added in one
portion to a stirred suspension of 2-chloro-9-(2-hydroxy-
ethoxymethyl)hypoxanthi~e (20 mg) in triethyl phosphâte
(0.3 ml) at -8C. The temperature was allowed to rise to
0C over 30 minutes. The reaction mixture was then stirTed
at 0C for 40 minutes and at ~5C for 50 minutes. It was
then poured onto ice, and the pH was adjusted to 7 with 2N
potassium hydroxide. The resulting solution was extracted
twice with chloroform (2 x 2 ml). The aqueous phase was
adjusted to pH 8-8.5 with 2N potassium hydroxide, and barium
acetate (105 mg) was added. The resulting barium phosphate
precipitate was removed by filtration. The supernatant was
treated with a large excess of ethanol, precipi~ating crude
barium 2-chloro-9-(2-hydroxyethoxymethyl) hypoxanthine
monophosphate. The solid was collected by filtration and
suspended in ethanol. The ethanolic suspension was then
heated on a steam bath for several minutes, cooled and
filtered. The collected precipitate was washed with
anhydrous ether and dried, giving barium 2-chloro-9-(2-
hydroxyethoxymethyl)hypoxallthine monophosphate (26 mg).
Ammonium sulfate (3.96 mg) was added to a stirred
suspension of barium 2-chloro-~-(2-hydroxyethoxymethyl)
hypoxanthine monophosphate ~7 mg) in water (0.5 ml). The
mixture was stirred at ambient temperature for 15 m;nutes
and then cooled in an ice bath. The precipitated barium
sulfate was remo~ed by filtration and wasTIed Wit}l water
g
109 40fi 2 YC/77
(1 ml) and ethanol (10 ml). The combined filtrate and
washings were evaporated under reduced pressure, and
the resulting residue dissolved in methanol (3 ml).
The methanolic solution was transferred to a Teflo ~
lined stainless steel bomb~ and methanol ~ ml) saturated
with gaseous ammonia at ice bath temperature was also
added to the bomb. The sealed bomb was placed in a 122C
oven for 4 hours, chilled and opened. Solvent was
evaporated to minimal volume. The residual reaction
mixture was spotted on Eastman Chromatogram~ cellulose TLC
sheets which were then developed in n-propanol:water
~70:30 v/v). The bands at Rf 0.16 and 0.34 were excised,
suspended in Tris buffer (0.6 ml) at pH 8, and the
cellulose was removed by filtration.
These bands were shown to contain 9-(2-hydroxy-
ethoxymethyl)guanine monophosphate and 2-chloro-9-(2-hydroxy-
ethoxymethyl)hypoxanthine monophosphate by enzymatic
dephosphorylation with alkaline phosphatase to 9-(2-hydroxy-
ethoxymethyl)guanine and 2-chloro-9-(2-hydroxyethoxymethyl)
hypoxanthine, respectively. Alkaline phosphatase (2 ~1)
from E. coli was added to the filtrate and the mixture was
heated at 32C for 2 hours. It was then examined by thin
layer chromatography on Eastman Chromatogram~ cellulose
sheets in three solvent systems:-
(a) _-propanol:water (70:30 v/v)
(b) water
(c) n-propanol conc. ammonium hydroxide:water
(60:30:10 v/v)
-- 10 -
yC/77
` ` 10940fi2
two spots were present in each system, corresponding to
~-(2-hydroxyethoxymethyl)guanine (A) and 2-chloro-9-
(2-hydroxyethoxymethyl)hypoxanthine ~B).
Solvent System Rf (A) Rf (B) Rf of Reaction Product
~ (a) 0.51 0.64 0.51 and 0.65
(b) 0.68 0.97 0.67 and 0.97
(c) 0.51 0.71 0.51 and 0.71
EXAMPLE 2
9-(2-Hydroxyethoxymethyl)guanine monophosphate
Phosphorus oxychloride (0.76 ml) was added to a
stirred, cooled (-10C) mixture of 9-(2-hydroxyethoxymethyl)
guanine (0.225 g) and triethyl phosphate (5 ml). The
temperature of the reaction mixture was allowed to rise to
0C over 30 minutes and was held at this temperature for
2 hours. It was then poured onto a mixture of ice and water,
- and the pH was adjusted to 7 with 2N potassium hydroxide.
The resulting solution was extracted twice with chloroform
and once with ether. The pH of the remaining aqueous
solution was adjusted to 7.1 with 2N potassium hydroxide and
was then lyophylized. The resulting white solid was dissolved
in water (7 ml), and methanol (7 ml) was added to precipitate
the inorganic sal~s which were then removed by filtration.
Acetone ~70 ml) was added to the filtrate, precipitating a
white gum. The gum was dissolved in water (7 ml), ethanol
(7 ml) added and the mixture filtered. A large excess of
acetone (70 ml) was added, again precipitating the gum. The
YC/77
`` ` lO~Ofi2
gum was dissolved in ethanol (ca 20 ml) and the solvent
was removed by flash evaporation, giving a white powder
(2.6 g) which was a mixture of inorganic salts and the
desired phosphate. The solid was dissolved in water
B ~lo ml), applied to a Bio-Gel P-2 column ~200-400 mesh,
2.7 x 90 cm) and eluted with water. The majority of
the monophosphate was eluted in a 50 ml volume after 166
ml of eluate had been collected, as shown by thin layer
chromatography on Eastman Chromagram~ cellulose in _-
propanol:water ~70:30 v/v); Rf = 0.26 for 9-~2-hydroxy-
ethoxymethyl)guanine phosphate and Rf = 0.11 for potassium
9~2-hydroxyethoxymethyl)guanine phosphate. The eluate
was lyophilized to give 0.28 g of a solid which was shown
by ultraviolet spectroscopy to contain 0.2 g of mono-
phosphate product.
EXAMPLE 3
Ammonium 9-~2-hydroxyethoxymethyl)guanine monophosphate
9-~2-Hydroxyethoxymethyl)guanine phosphate ~0.28 g)
was dissolved in water ~30 ml) and the pH of the solution
was adjusted to 6 with 6N hydrochloric acid. The product
was adsorbed onto 14 ml of packed charcoal (Pischer 5-690B,
50-200 mesh, acid washed and deactivated with toluene). The
charcoal was washed well with water and eluted with 70 ml
of 50~ aqueous ethanol containing 2~ concentrated ammonium
hydroxide. The solvent was evaporated under reduced pressure
to give ammonium 9-~2-hydroxyethoxymethyl)guanine mono-
phosphatc ~0.048 g); Rf = 0.30 on Eastman cellulose in
~ ~aJ~ n~
- 12 -
YC/77
10~0~
n-propanol:water (70:~0 v/v).
EXAMPLE 4 - Tablet
Sodium 9-(2-hydroxyethoxymethyl)guanine phosphate 100 mg
Lactose 200 mg
Starch . 50 mg
Polyvinylpyrrolidone 5 mg
Magnesium stearate 4 mg
Total weight 359 mg
EXAMPLE 5 - Ophthalmic Solution
10Sodium 9-(2-hydroxyethoxymethyl)guanine phosphate 1.0 g
Sodium chloride, analytical reagent 0.9 g
Thimerosal 0.001 g
Purified water, ~.s. to lOO. ml
pH adjusted to 5.5-7.5
EXAMPLE 6 - Injectable Solution
Sodium 9-(2-hydroxyethoxymethyl)guanine phosphate 0.775 g
Sterile, pyrogen-free, pH7 phosphate buffer ~s to 25 ml
EXA~IPLE 7
Disodium 9~2-hydroxyethox~ymethyl)guanine phosphate
20Phosphorus oxychloride ~54 ml) was addcd over a 3
hour period to a stirred~ cooled (-30 to -20C) mixture
of 9-(2-hydroxyethox~neth}rl)guanine (25 g) and triethyl
- 13 -
10~ 4~fi 2 YC/77
phosphate (250 ml). The temperature of the reaction
mixture was allowed to rise to 0C over 45 minut~s and
was held at that temperature for an additional 45 minutes.
It was then poured into a mixture of ice and water, and
the pH was adjusted to about 1 with 2N sodium hydroxide.
The resulting solution was extracted once with chloroform
and once with ether. The pH of the remaining aqueous
solution was adjusted to 6.8 with 2N sodiuln hydroxide and
subsequently to 7.3 with lON sodium hydroxide, giving a
final volume of 2.5 liters.
The neutralized solution was applied to a column
B containing 2,000g of Dowex 1 x 8 which had been equilibrated
with 50 mM KHC03. Elution was by a 30 Q linear gradient
of 50-500 mM KHC03 followed by a 30 ~ wash of 500 mM KH~03.
The fractions containing product were combined and most of
the KHC03 was removed by adding ~owex~ 50-H and removing C02
under vacuum. The volume was reduced to 2 Q in vacuo and
the product was precipitated at 4C by the addition of 10
of acetone. The dr ed precipitate 55 g was applied to a
10 x 110 cm Bio-Gel P-2 column and eluted with E~20. 22 g of
solid was obtained. The material was recrystallized at 4~C
as the hydrogen salt from a pH 3 solution of water and more
material was obtained from the mother liquor by crystalli~ing
from 20~ ethanol at pH 3. The hydrogen salt was dissolved in
a minimal ~ol~ne of H20 brought to pH 8.5 with NaOE~ and
precipitated with 2 volume of ethanol at 4C. This preci-
pitate was dissolved in 100 ml H20 and precipitated ~ith 9
volumec of ethanol at 4C to give 15.1 g of 9-~2-hy~roxy-
~ rr~d~
10~?40fiZ
ethoxymethyl)guanine monophosphate disodium salt
dihydrate.
Purity was confirmed by elemental analysis, high
pressure liquid chromatography and W spectra.
Empirical formula : C8H12N5O6P 2Na 2H2O
Theory: C 24.94%; H 3.66%, N 18.19%: P 8.04%
Found : C 25.20%, H 3.63%, N 18.10%; P 7.89%
W Spectra: Solvent ~ max ~ ~ min sh
O.LM HCl 254 12970 225 272
ph 7 299 14060 219 266
0.lm NaOH 255-264 11760 228
High pressure liquid chromatography purity = 99%
Base/phosphate ratio = 1.00/1.01
