FI68402C - REFERENCE TO A FRAMEWORK FOR THERAPEUTIC CONTAINING 9- (2-HYDROXIETOXIMETHYL) GUANIN MONONOPHOSPHATIVES - Google Patents

Description

... ADVERTISEMENT 68402

jWgflA 11 UTLÄGGNINGSSKRIFT

# 2¾¾ C (45) Patent granted 10 09 1985 J Patent aseddelat ^, (51) Kv.ik.4 / int.a.4 C 07 F 9/65 // C 07 D A73 / 18 FINLAND - FINLAND (21) Patent application - Patentansökning 780626 (22) Filing date - Ansökningsdag 2 A 02 78 (Fl) (23) Starting date - Giltighetsdag 2A.02.78 (41) Published public - Bllvlt often tl ig 2ζ 08 78

National Board of Patents and Registration ^ Date of Date of Publication and Publication—. pr or

Patent- och registerstyrelsen '1 Ansökan utlagd ooh utl.skriften publicerad -> -vj -vj (32) (33) (31) Privilege claimed - Begärd priority 2 A. 02.7 7 USA (US) 771778 Toteennaytetty-Styrkt 2A.12.77 English -Eng 1and (GB) 53905/77 Proven-Styrkt (71) The Wellcome Foundation Limited, 183 ~ 193 Euston Road, London NW1,

Engl ant i-Eng land (GB) (72) Howard John Schaeffer, Raleigh, North Carolina, USA (US) (7A) Oy Kolster Ab (5A) Method for preparing a therapeutically useful human monophosphate derivative of 3- (2-hydroxyethoxymethyl) guan - For the purposes of this Regulation, a monophosphate derivative of 9- (2-hydroxyethoxymethyl) -guan

The invention relates to a process for the preparation of a therapeutically useful 9- (2-hydroxyethoxymethyl) guanine monophosphate derivative of the formula

N-Yv

u y> f1 * H2N o

II

CH_.O.CH „.CH„ .O-P-OZ 2 2 2 1

OW

wherein W and Z are the same or different and represent hydrogen or a pharmaceutically acceptable cation.

68402

It is known that 9- (2-hydroxyethoxymethyl) derivatives of purines have antiviral activity against various classes of DNA and RNA viruses in both in vitro and in vivo experiments (GB patent application 38178/74). In particular, these compounds are active as antiviral agents against Vaccinia and Herpes viruses, such as mammalian Herpes simplex, zoster, and varicella viruses, which cause herpes zoster in rabbits and herpes encephalitis in mice.

It has now been found that the monophosphate ester of 9- (2-hydroxyethoxymethyl) guanine, in addition to having good antiviral activity, has the advantage of significantly better solubility at least in the pH range of 1-7.5 than the corresponding non-phosphorylated compound.

An alcohol corresponding to a phosphate ester derivative of the formula I is known from FI patent publication 60709. The antiviral activity of the phosphate compound now described (and also its monosodium salt) is about 50% of that of the non-phosphorylated compound (0.2 and 0.1 pM). However, the value of the phosphate compound is relatively high compared to other esters of the parent compound (acyclovir) as shown in the table below.

OH

li>

H2N ^ N ^ NX

CH20 (CH2) 2 OCOR

Antiviral activity R ED5Q (pM) a) H 0.03 b) -CH3 0.17 c) -CH2CH3 0.7 d) - (CH2) 2CH3 0.37 e) -C (CH3) 3 0.8 f) - (CH2) 4CH3 0.08 g) 30 3 68402

Antiviral activity is expressed as ED ^ Q using ja and units and was determined by the Plagne Reduction Assay. The results shown in the table show that differences in the size and structure of the ester group lead to antiviral activities that differ significantly from each other, and therefore it is not foreseeable whether a particular ester has high or low antiviral activity. This was also the case for the phosphate ester compounds now described, which have a relatively high antiviral activity compared to the average activity of the esters shown in the table. Some of the esters shown in the table are more active than the corresponding phosphate compounds, but it should be noted that the compounds now described have excellent water solubility in addition to good antiviral activity. As can be seen from the table, the acetate ester (compound b) has a somewhat better antiviral activity than phosphate, but it is poorly soluble in water.

Journal of Medicinal Chemistry 18 (1975), No. 7, pp. 721-726 suggests that phosphorylation of the primary hydroxy groups of nucleoside analogs enhances this. solubility of the compounds. However, the publication does not present an actual quantitative comparison between the aqueous solubilities of the nucleoside and the corresponding phosphate. On page 721, it has been found that the antiviral agent Ara-A is relatively insoluble in water and the solubility values of the compound are reported. With respect to phosphate derivatives, compounds 3 and 6 have been found to be "water soluble" on page 723. Compound 3 is a methyl phosphate analog of Ara-A and Compound 6 is a phosphate of a hypoxanthine compound. These compounds have been reported to be "water soluble". It should be noted, however, that most compounds are at least somewhat soluble in water. However, the publication does not indicate the extent to which water solubility has improved. No water solubility values have been reported for Ara-A monophosphate (Compound b) either. Therefore, the findings described in the publication cannot be directly applied to the acyclic derivatives now described. Thus, it was not foreseeable that the phosphate compounds now described would be more soluble in 4 68402 water than a compound containing a free hydroxy group with a water solubility of 0.125%. For example, the acetate analog (phosphate group replaced by acetoxy group, compound b in table) has a water solubility of 0.62% at room temperature, while the propionyloxy compound (compound c in table) has a water solubility of only 0.086%.

The free phosphate compound of formula I (Z = W = H) has the surprising ability to form highly water-soluble base salts. The water solubility of the free phosphate compound is 0.196%, while the water solubility of the monosodium salt (Z = Na, W = H) is 23.1%, i.e., the water solubility is increased 100-fold. By comparison, the corresponding hemisuccinate compound (the phosphate group replaced by -OCO (Cl 2)) has a water solubility of 0.19%. It should be emphasized that the hemisuccinate ester and the phosphate ester are similar in that they both contain an ionic ester group capable of forming base salts. However, the water solubility of the monosodium salt of the hemisuccinate compound is only 2.7%, which corresponds to an increase of about 10-fold compared to the free ester, while the phosphate compounds have an increase of at least 100-fold. The ability of the phosphate group to form highly water-soluble salts should be considered surprising. , water-solubility values of a chemically similar hemisuccinate analogue This very good solubility in water is also surprising given that the esters mentioned in the table above are poorly soluble in water.

The free phosphate compound of formula I (Z = W = H) is not as soluble in water as, for example, the monosodium salt, but since it is capable of forming highly water-soluble salts in suitable media, e.g. solutions of suitable bases, it must be considered very useful.

The 9-hydroxyethoxymethylguanidine monophosphate of formula I can be prepared by reacting (a) a compound of formula 5 qh 684 0 2 rVv H2N2 ^ N (II) CH2O.CH2.CH2OH with a phosphorylating agent to give a compound of formula I wherein W and Z are hydrogen, or (b) a compound of formula

M

IX>

G O

I 11

CH .O.CH .CH .O-P-OH 2 2 2 OH

wherein either M is a 6-hydroxy group and G is an atom or group that can be replaced by an amino group or converted to an amino group by selective ammonolysis, or G is a 2-amino group, and M is an atom or a group that can be replaced by a hydroxy group or converted to a hydroxy group by selective hydrolysis, converted to a compound of formula I; and, if desired, a compound of formula I wherein W and Z are hydrogen is converted to a compound of formula I wherein W and / or Z is a pharmaceutically acceptable cation by reaction with a base and / or a salt containing the desired cation.

The pharmaceutically acceptable cation may be one of the following: sodium, potassium, lithium, calcium / 2, magnesium / 2, aluminum / 3 or ammonium. Preferred compounds of formula I are those in which Z is sodium, potassium or ammonium and W is hydrogen and particularly preferred are compounds of formula I in which Z is sodium or ammonium and W is hydrogen.

In the above definition of W and Z, the terms calcium / 2, magnesium / 2 and aluminum / 3 were used for the multivalent cations, meaning that the cation is partitioned into valence, i.e. Ca ++ / 2, Mg ++ / 2 and Al +++ / 3. This is to show that calcium or magnesium cations are ionically associated with two phosphate acids or Aluminum with three phosphate acids.

6 68402

Process variant (a) preferably uses phosphoric acid derivatives for phosphorylation in which 1 to 3 hydroxy groups have been replaced by halogen atoms, e.g. chlorine, such as phosphorus oxychloride. 1-2 of the hydroxy groups may also form alkoxy groups which may be further substituted and form, for example, benzyloxy groups. Such phosphohalogen derivatives or phosphates are used under ordinary neutral or temporal conditions, and the latter preferably use, for example, a carbodiimide such as dicyclohexylcarbodiimide for activation, except when they are anhydrides.

If at least two of the hydroxy groups of the phosphoric acid derivative have been replaced by halogen, then after reaction with a compound of formula II it is necessary to remove free halogens by relatively mild aqueous hydrolysis using, for example, one molar equivalent of water in a water-miscible solvent such as alcohol. In the next step, the substituted or unsubstituted alkoxy groups attached to the phosphate can be hydrolyzed in a suitable aqueous medium in the presence of a base. Alkoxy groups substituted with aromatic groups, such as a benzyloxy group, can also be subjected to hydrogenolysis, preferably using a catalyst in the usual manner, whereby the cleavage takes place via reduction.

In a preferred embodiment, the compound of formula II is reacted with phosphorus oxychloride in the presence of a trialkyl phosphate, preferably at a temperature of at most 0 ° C.

In another suitable embodiment, the compound of formula II is reacted with phosphorus oxychloride in dry pyridine.

The compound of formula II can be prepared by the methods described in GB patent application 38278/74.

In process variant (b), the reaction of the compound of formula III can be carried out in several different ways, for example, when G is a halogen, mercapto or alkylthio group, such as methylthio, it can be converted into an amine group by ammonolysis. This method, like many others known in the art, is described in "Heterocyclic compounds Fused Pyrimidines Part II Purines, edited by D.J. Brown (1971), published by Wiley - Interscience".

7 68402 M may be a halogen atom, a mercapto or alkylthio group which can be converted into a hydroxy group by the hydrolysis methods described in the above-mentioned book.

Compounds of formula III may be prepared by methods analogous to method (a) described in GB Patent Application 38278/74.

Pharmaceutically acceptable salts of 9- (2-hydroxyethoxymethyl) guanine monophosphate can be prepared by neutralizing the acid monophosphate with an equivalent amount of a base such as hydroxide, bicarbonate, carbonate containing the desired cation, namely sodium, potassium, magnesium, ammonium, ammonium. Alternatively, they can be prepared by exchange reactions in which one salt of the monophosphate is treated with a solution of a salt containing the desired cation, preferably an aqueous solution. For example, the sparingly soluble barium salt of 9- (2-hydroxyethoxymethyl) guanine monophosphate is treated as an aqueous suspension with sodium sulfate to remove barium as highly insoluble barium sulfate, leaving sodium 9- (2-hydroxyethoxymethyl) guanine monophosphate in solution.

The compound of formula I may be used in pharmaceutical compositions in association with a pharmaceutically acceptable carrier.

Pharmaceutically acceptable carriers are substances that are suitable for pharmaceutical compositions for their dosing purposes. They may be solid, liquid or gaseous substances which are otherwise inert and medically acceptable and compatible with the active ingredient. These pharmaceutical compositions may be administered orally, parenterally, as suppositories or pessaries, or applied topically as ointments, creams, aerosols or powders, eye or nasal drops, depending on whether the internal or external viral infection is being treated.

In internal infections, the compounds of the formula I are administered in doses of 0.1 to 250 mg / kg, calculated as free phosphate, preferably 1.0 to 50 mg / kg (mammalian body weight). The unit dose administered to a human one or more times a day is 1 to 800 mg, preferably 1 to 250 mg, and most preferably 10 to 200 mg.

8 68402

For parenteral or topical administration in the form of drops, e.g. ophthalmitis, the compounds of formula I may be water soluble at a concentration of about 1.0-10% w / v, preferably 0.1-7% and most preferably 0.2-5 %.

Infections of the eye or other external tissues, e.g. the mouth and skin, can preferably be treated with solutions, creams or Savings suitable for topical use. Their concentrations are about 1.0-10%, preferably 0.3-6% and most preferably 3%.

In another aspect, the invention provides a method of treating viral infections in a mammal by administering an antivirally effective, non-toxic amount of a compound of formula I as defined above. As used herein, the term "antivirally effective non-toxic amount" means a predetermined antiviral amount that is effective against viruses in vivo.

The invention is illustrated by the following examples.

Example 1 9- (2-Hydroxyethoxymethyl) guanine monophosphate To a stirred suspension of 2-chloro-9- (2-hydroxyethoxyacetyl) hypoxanthine (20 mg) in triethyl phosphate (0.3 ml) at -8 ° C was added phosphorus oxychloride (0.03 ml) in one portion. ). The temperature was allowed to rise to 0 ° C in 30 minutes. The mixture was then stirred at 0 ° C for 40 minutes and at + 5 ° C for 50 minutes. The mixture was then poured onto ice, the pH was adjusted to 7 with 2N potassium hydroxide. The resulting solution was extracted twice with chloroform (2 x 2 mL). The pH of the aqueous phase was adjusted to 8-8.5 with 2N potassium hydroxide, and then barium acetate or (105 mg) was added. The resulting barium phosphate precipitate was filtered. The filtrate was treated with a large excess of ethanol to precipitate crude barium 2-chloro-9- (2-hydroxyethoxymethyl) hypoxanthine monophosphate. The precipitate was collected by filtration and suspended in ethanol. The ethanol suspension was then heated on a steam bath for several minutes, cooled and filtered. The resulting precipitate was washed with anhydrous ether and dried to give barium 2-chloro-9- (2-hydroxyethoxymethyl) hypoxanthine monophosphate (26 mg).

To a stirred suspension of barium-2-chloro-9- (2-hydroxyethoxymethyl) hypoxanthine monophosphate (7 mg) in water (0.5 ml) was added ammonium sulfate (3.96 mg). The mixture was stirred at room temperature for 15 minutes, then cooled in an ice bath. The precipitated 9 68402 barium sulfate was filtered, washed with water (1 mL) and ethanol (10 mL). The filtrate and washings were evaporated under reduced pressure, and the residue was dissolved in methanol (3 ml). The methanol solution was transferred to a Teflon-lined stainless steel pressure vessel, to which methanol (8 mL) saturated with ammonia gas was also added at ice bath temperature. The sealed pressure vessel was heated at 122 ° C for 4 hours, cooled and opened.

The solvent was evaporated to a minimum. The residual reaction mixture was spotted onto cellulose thin layer plates (Eastman (S)

Chromatogram45) developed in n-propanol-water (70:30 v / v). The strips at Rf 0.16 and 0.34 were suspended in Tris buffer (0.6 ml) at pH 8, and the cellulose was removed by filtration.

These bands were shown by enzymatic dephosphorylation with alkaline phosphatase to contain 9- (2-hydroxyethoxymethyl) guanine monophosphate and 2-chloro-9- (2-hydroxyethoxymethyl) hypoxanthine monophosphate, which were converted to phosphatase by hydroxy- 2-chloro-9- (2-hydroxyethoxymethyl) guanine. Alkaline phosphatase (2 μol) from E. coli was added to the filtrate, and the mixture was heated at 32 ° C for 2 hours. It was then chromatographed on cellulose plates (Eastman Chromatogran®) with three solvent systems: (a) n-propanol-water (70:30 v / v) (b) water (c) n-propanol-concentrated NH 4 OH-water (60:30:10 v / v).

Two spots corresponding to 9- (2-hydroxyethoxymethyl) hypoxanthine (B) were obtained in each system.

Solvent system Rf (A) Rf (B) - Reaction product Rf (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 To a cooled (-10 ° C) mixture of 9- (2-hydroxyethoxymethyl) guanine (0.225 g) and triethyl phosphate (5 ml) was added phosphorus oxychloride (0.76 ml) with stirring. The temperature of the reaction mixture 68402 10 was allowed to rise from 30 minutes to 0 ° C and maintained there for 2 hours. The reaction mixture was then poured into 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 aqueous phase was adjusted to 7.1 with 2N potassium hydroxide and lyophilized. The obtained white solid was dissolved in water (7 ml), and methanol (7 ml) was added to the solution to precipitate inorganic salts, which were then filtered. Acetone (70 ml) was added to the filtrate, whereupon a white gum precipitated. This was dissolved in water (7 ml), ethanol (7 ml) was added and the mixture was filtered. A large excess of acetone (70 ml) was added and the gum precipitated again. The gum was dissolved in ethanol (ca. 20 ml) and the solvent removed by evaporation to give a white powder (2.6 g) which was a mixture of inorganic salts of the desired phosphate. It was dissolved in water (10 ml), the solution was applied to a Bio-Gel P-2 column (200-400 mesh, 2.7 x 90 cm) and eluted with water. Most of the monophosphate eluted in 50 ml of eluate. After collecting 166 ml of eluate, which could be detected by thin layer chromatography on a cellulose layer (Eastman Chromatography) with an n-propanol-water system (70:30 v / v); Rf = 9.26 for 9- (2-hydroxy-ethoxymethyl) guanine phosphate and Rf = 0.11 for potassium 9- (2-hydroxy-ethoxymethyl) guanine phosphate. The eluate was lyophilized to give 0.28 g of a solid which was shown by UV spectroscopy to contain 0.2 g of monophosphate.

Example 3 9- (2-Hydroxyethoxymethyl) guanine phosphate 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 carbon (Fischer 5-690B; 50-200 mesh, acid washed and deactivated with toluene). The carbon 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 monophosphate (0.048 g); Rf = 0.30 on Eastman cellulose, n-propanol-water (70:30 v / v).

11 68402

Example 4

Disodium 9- (2-hydroxyethoxymethyl) guanine phosphate To a cooled (-30 ° to -20 ° C) mixture of 9- (2-hydroxyethoxymethyl) guanine (25 g) and triethyl phosphate (250 ml) was added phosphorus oxychloride (54 mL). The temperature of the reaction mixture was then allowed to rise to 0 ° C over 45 minutes and held for a further 45 minutes. The mixture was then poured into a mixture of ice and water and the pH was adjusted to about 7 with 2N sodium hydroxide. The resulting solution was extracted once with chloroform and once with ether. The pH of the aqueous phase was adjusted to 6.8 with 2N sodium hydroxide and then to 7.3 with 10N sodium hydroxide to give a final volume of 2.5 L.

The neutralized solution was applied to a column containing 2000 g of Dowex 1x8 equilibrated with 50 mmol KHCO3. Eluted with 30 liters of a linear gradient of 50-500 mmol KHCO 3, then washed with 500 mmol KHCO 3. The product-containing fractions were combined and most of the KHCO 3 was removed by adding Dowex 50-H 2 O resin and removing CO 2 in vacuo. The solution was evaporated in vacuo to 2 liters and the product was precipitated at 4 ° C by the addition of 10 l of acetone. The dried precipitate (55 g) was applied to a 10 x 100 cm Bio-Gel P-2 column and eluted with water. 22 g of solid were obtained. It was crystallized as a hydrogen salt at 4 ° C at pH 3 from water, and more material was obtained from the mother liquor by crystallization from 20% ethanol at pH 3. The hydrogen salt was dissolved in the smallest possible volume of water, the pH was adjusted to 8.5 with NaOH and the product was precipitated at 4 ° C with 2 volumes of ethanol. This precipitate was dissolved in 100 ml of water and precipitated at 4 ° C with 9 volumes of ethanol to give 15.1 g of 9- (2-hydroxyethoxymethyl) guanidine sodium salt as the dihydrate.

The purity of the salt was determined by elemental analysis, high performance liquid chromatography and UV spectra.

Gross formula: C0H .. _NeO, .P 2Na 2H „0 o Iz Db z

Calculated: 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% UV spectra: Solvent Λ max t. A min sh 0.1-m HCl 254 12970 225 272 pH 7 299 14060 219 266 0.1-m NaOH 255-264 11760 228

HPLC purity = 99%, base / phosphate ratio = 1.00 / 1.01.

Claims (11)

68402 12 A process for preparing a therapeutically useful 9-hydroxy-ethoxymethylguanine monophosphate derivative having the formula OH jfV \ (i) Λ ,, λ / H_N NN O Process according to Claim 1, characterized in that the phosphorylating agent is a phosphoric acid derivative. 2. II CH_.O.CH_.CH ~ .O-P-OZ 2 2 2 | OW wherein W and Z are the same or different and represent hydrogen or a pharmaceutically acceptable cation, characterized in that (a) a compound of formula OH J * Y * \ ί I} un H Λη / CH2.O.CH2.CH2. OH is reacted with a phosphorylating agent to give a compound of formula I wherein W and Z are hydrogen, or (b) a compound of formula [1 '> N NX OGI II CH- .O.CH_.CH_.OP-OH Δ Δ λ j ÖH wherein either M is a 6-hydroxy group and G is an atom or group that can be replaced by an amino group 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 by a hydroxy group or converted to a hydroxy group by selective hydrolysis, converted to a compound of formula I; and, if desired, converting a compound of formula I wherein W and Z are hydrogen to a compound of formula I wherein W and / or Z is a pharmaceutically acceptable cation by reaction with a base or salt containing the desired cation. Process according to Claim 2, characterized in that 1 to 3 hydroxy groups in the phosphoric acid derivative are replaced by halogen atoms. Process according to Claim 3, characterized in that the phosphoric acid derivative is phosphorus oxychloride. Process according to Claim 2, characterized in that 1 to 2 hydroxy groups of phosphoric acid are substituted to form alkoxy groups which may have substituents. Process according to Claim 4, characterized in that the compound of the formula II is reacted with phosphorus oxychloride in the presence of a trialkyl phosphate at a temperature of at most 0 ° C. Process according to Claim 4, characterized in that the compound of the formula II is reacted with phosphorus oxychloride in dry pyridine. Process according to Claim 1, characterized in that the compound of the formula III in which M is a 6-hydroxy group and G is a halogen atom, a mercapto or alkylthio group is converted into a compound of the formula I by ammonolysis. Process according to Claim 1, characterized in that the compound of the formula III in which M is a halogen atom, a mercapto or alkylthio group and G is a 2-amino group is converted into a compound of the formula I by hydrolysis. Process according to one of Claims 1 to 9, characterized in that the base with which the compound of the formula I in which W and Z represent hydrogen is reacted is 68402 is a hydroxide, bicarbonate or carbonate containing the desired pharmaceutically acceptable cation. Process according to Claim 10, characterized in that the desired cation is sodium, potassium, ammonium, calcium, lithium, magnesium or aluminum. 15 Patentkrav 6 84 0 2