IL129935A - Chloropyrimidine intermediates for preparing 2-aminopurine nucleoside analogues and their preparation - Google Patents

Description

129935/2 Chloropyrimidine intermediates for preparing 2-aminopurine nucleoside analogoues and their preparation The Wellcome Foundation Limited C. 117193 129935/4 The present invention relates to certain novel pyrimidine intermediates, processes for their preparation and their conversion to substituted diamino-pyrimidines. A number of 2-aminopurine nucleoside analogues have been shown to be useful in the treatment or prophylaxis of viral infections, for example the compound of formula (A) is described as having potent activity against human immunodeficiency virus (HIV) and hepatitis B virus (HBV) (EP 0434450, corresponding to IL 90752).

Processes have been proposed for the preparation of 9-substituted-2-aminopurines, generally starting from a pyrimidine compound, coupling with a sugar analogue residue, and cyclisation to form the imidazole ring and introduction of any suitable 6-substituent.

Pyrimidine compounds which have been identified as being useful in the preparation of 9-substimted-2-aminopurines include 2,5-diammo-4,6-dicUoropyrirnidine, N,N'-(4,6-dichloro-2,5-pyrimidinediyl)bis formamide and also N-2-acylated pyrimidine derivatives such as the 2-acetamido and 2-isobutryamide derivatives (US Patent 5087697 corresponding to IL 90752 and IL 96748.

Processes for the synthesis of these intermediates generally involve a number of steps of which some are difficult to perform and produce poor yields, preventing any practical scale up of these processes above a few grams, and are thus difficult and uneconomical.

Processes for the synthesis of the intermediate 2,5-dlan-ino-4J6-dichJoropyi nidine include the direct chlorination of readily available 2,5-dammo-4,6-dihydroxypyrir£dciine using phosphorus oxychioride. The original examination of this reaction was carried out by Temple et al. (J. Org. Chem. 1975, 40: 3141-3142). These workers concluded that the reaction was un-mccessfuL apparently because of degradation of the pyrimidine ring system. Hanson (SmithKline Beecham, WO 91/01310, US Patent 5216161) subsequently described a process for the direct chlorination of 2,5-dianmo^,6-dmydroxypyrirmdine by refluxing with phosphorus oxychioride in the presence of large molar excesses of quaternary ammonium chlorides or amine hydrochlorides. We have examined this process and have obtained, repeatedly, much lower yields (<10%) of crude 2,5-diamino-4,6- dic oropyrimidine than those specified in the Smili line Beecham patent specification.. The extensive decomposition of the 2,5-diammo-4,6-dmydroxypyrirniciine to tars which coat the equipment, combined with the problems of dealing with the copious solids due to the insoluble amine salts, constitute significant drawbacks and make scale-up of such a process impractical The modifications of Legraverend et al. (Synthesis 1990: 587-589), namely using acetonitrile as a solvent and adding phosphorus pentac oride to the phosphorous oxychioride and quaternary ammonium chloride, result, in our experience in the isolation of approximately 30% (after chromatographic purification) of 2,5-diamino- 4,6-dicMoropyrimidine on a 2-5 gram scale. Again, scale-up beyond a few grams is impractical due to the formation of tarry precipitates.

A recent Lonza AG patent specification (EP 0 552 758 corresponding to IL 104478) suggests that higher yields (35- 65%) may be obtained with phosphorus oxychioride chlorination when the 5-amino group of 2,5-diamino-4,6-dmydroxypyrimidine is protected with an alkoxycarbonyi protecting group. This modification is claimed to simplify the chlorination step in that the amines and phosphorus pentachloride, employed in the prior processes discussed above are not required. This creates a new problem, namely the need to remove the alkoxycarbonyi protecting groups in order to be able to convert the pyrimidine intermediates to purines. Indeed, the Lonza AG specification does not show that such 5-protected 2,5-diarnino-4,6-cUcUoropyrimidines may be converted to purines in an advantageous manner, 129935/2 3 In one aspect of this invention we provide the following novel intermediates of the formula (X): wherein RX is N=CHNR'R2 or N¾ and RY is Ν=ΟΗΝ ¾2 or NHCHO, and R1 and R2 which may be the same or different, are selected from Cl-Salkyl, C3-8 . cycloalkyl, and optionally substituted aryl, with the exclusion of compounds of formula (X) wherein RX is N=CHNR!R2 and RY is NHCHO'.' 129935/1 3a These intermediates may be utilized in the synthesis of 2-aminopurines, namely compounds of formulae (I), (II) and (III); CI JHCHO (III) H2N N •ci 4 wherein R* and which be the same or.different, are selected from Ci-g straight-chain alkyl, Ci-8 branched alkyl, C3.8 cyclo alkyl, and aryl groups (such as phenyl or naphthyl), which may be optionally substituted, for example by C1. alkyl or halogen (e.g. CI). In a preferred embodiment of the invention * and ^ are both methyl.

These novel intermediates can be readily prepared in good yields and are useful for the preparation of a wide variety of different types of 2-ammopurines including the nucleoside analogue of formula (A), famciclovir (EP 0182024), penciclovir (EP 0141927), H2G (EP 0343133) corresponding to IL 90166), (l'S^'S,4'S)-2-amino-l,9-dihydro-9-[3,4- dihydroxy-3-hydroxymethyl-l-cyclopentyl]-6H-purin-6-one (EP 0420518), and other 9- substituted-2-aminopurines provided that the 9-substituent is not attached by a glycosidic bond.

In a further aspect of this invention we provide processes for the synthesis of the novel intermediates of formulae (Γ), (Π) and (HI), and the known intermediate 2,5-diamino-4,6-dicWoropyi-imdme(IV). These processes are illustrated in the simplified diagram below which is designed for illustration only of the possible ways of synt esising these intermediates: 2, - - The present invention also provides a process for the preparation of compounds of formula (I) which comprises chlorination of 2,5-dammo-4,6-dmydroxypyriimciine with a halomethylenimmim'um salt (Vilsmeier reagent) of formula (V). wherein and are as defined above Compounds of formula (V), may be prepared from a variety of formamides of secondary amines by reaction: with a variety of acid halides, such as phosphorus oxychloride, phosphorus pentachloride, thionyl chloride, phosgene, and oxalyl chloride, for example as detailed in a review by C. M. Marson, Tetrahedron 1992, 48: 3660-3720 and references therein.

The advantage of protecting the diamiriopyrimidine from extensive decomposition during chlorination is achieved by the in situ protection of the amino groups with two molar equivalents of Vilsmeier reagent (V) to give a bis-formamidine intermediate (detected by thin-layer chromatography), which is subsequently chlorinated to a compound of formula (I) as the reaction with additional equivalents of Vilsmeier reagent proceeds. The improved solubility of such bis-formamidine derivatives is an added advantage of this process, facilitating the subsequent chlorination to compounds of formula (I) and their isolation and simple purification.

The disadvantage of the use of 5-alkoxycarbonyl protecting groups, as described in the Lonza specification (EP 0552758) is avoided since the formamidine groups in compounds of formula (I) are readily hydrolysed. under mild conditions in a step-wise manner to form the intermediates (Π) and (ΠΓ); or alternatively compounds of formula (T) can be directly hydrolysed to compounds of formula (ΠΓ).

The compound 2,5-diarrimo-4,6-dicMoropyrimidine (TV) can be prepared by:- ASB/JJ/13th January 1995 - - A) the hydrolysis of a compound of -formula (I); B) the hydrolysis of a compound of formula (Tf); or C) the hydrolysis of a compound of formula (ΠΙ).

The hydrolysis of (Γ), (Π), or (ΓΓΓ) to 2,5-diammo^,6-(_UcMoropyriniidine is conveniently carried out at pH 3 +/- 0.5 by adding a water-miscible cosolvent, such as ethanol. The hydrolysis is more efficient at pH 1-2, with shorter reaction times required than at a higher pH. It is advisable at pH 1-2, however, to protect 2,5-dammo-4,6-dc oropyrimi(iine from hydrolysis to hydroxypyrimidines by extraction, as it is formed, into an organic layer which is not miscible with the aqueous acid. When the pH of the aqueous layer is below 1, extraction of the product into the organic layer is inefficient (the pKa of (TV) was found to be ca 0.5 and the pyrimidine ring is thus significantly protonated below pH 1). Preferably, the acid used for this hydrolysis should be one which is not appreciably soluble in the organic layer, e.g. phosphoric or sulfuric acid. The organic solvent should be one which is stable to aqueous acid and in which (37V) is soluble. Satisfactory solvents for the organic layer include toluene and halocarbon solvents such as methylene chloride, chloroform, and 1,2-dichloroethane. At completion, the organic layer is simply washed, e.g. with saturated aqueous bicarbonate, dried and concentrated to provide (TV) with no purification required.

Compounds of formula (TJX) can be prepared by A) selective hydrolysis of a compound of formula (T); or B) selective hydrolysis of a compound of formula (Tf).

The hydrolysis of compounds of formula (Γ) or (Tf) to (ΤΠ) is most efficiently carried out in dilute aqueous acid, preferably in dilute aqueous mineral acid such, as sulfuric acid, hydrochloric acid, or phosphoric acid. Prolonged exposure to pH below 1 should be avoided as the cUoropyrimidine ring is protonated significantly below pH 1 and may ASB/JJ/13th January 1995 therefore undergo attack by water, generating undesired hydroxypyrimidine by-products. Preferably, the pH is maintained above 2 and optimally at 3 +/- 0.5 for the efficient formation of (HI). In this optimal pH range, the formamidine groups of (I) and (Π) are selectively hydrolysed to give (ΠΓ) in approximately 70% yield. As the hydrolysis of the formamidine groups of (I) and (Π) proceed, the secondary amine from which the Vilsmeier reagent (V) was formed (H R*R2) is liberated and causes the pH of the solution to rise, thus slowing, the hydrolyses. In addition, with certain reactive aliphatic amines HNR R2} such as Ν,Ν-dimethylamine, it is necessary to maintain a pH sufficiently low to prevent the chloro groups of the pyrimidine ring from displacement by the secondary amine. We have found that maintaining the pH of the reaction mixtures below 4 avoids significant displacement of the chloro groups by the secondary amine, even with amines as reactive as Ν,Ν-dimethylamine. It was thus found optimal to buffer the hydrolyses of (I) and (Π) to (ΠΓ) at pH 3 +/- 0.5 or to add increments of acid throughout the hydrolyses in order to maintain the pH in this range.

Optimally, the hydrolysis of compounds of formula (I) or (Π) to (ΠΙ) is carried out in a minimum of water with the pH controlled as described above. Under these conditions, (HI) precipitates as formed and is simply filtered off and washed with water. The hydrolysis is carried out at gentle reflux for 4 hours, or at lower temperatures for longer periods of time.

The"- compounds of formula (Π) can be prepared by the selective hydrolysis of the compounds of formula (Γ). Preferably the selective hydrolysis is carried out with slightly more than two molar equivalents of mineral acid in water or ethanol and warmed for 15-30 minutes.

The compounds of formula (!) can be prepared by reacting 2,5-diamino-4,6-d&yfroxypyrinudine with a Vilsmeier reagent of formula (V).

The compound 2,5-dammo ,6-dmydroxypyriniidine is commercially available (Sigma, Maybridge BTB, Pfaltz and Bauer, Polyorganix).

ASB/JJ/13th January 1995 The novel bis-formamidines of formula (I). are formed and isolated conveniently in high yield when the 2,5-diamino^,6-dmydroxypyrimidine (or a salt thereof, such as the hydrochloride or the hemisulfate) is treated with at least 4 molar equivalents of a Vllsmeier reagent (V). These chlorination reactions proceed under extremely mild conditions without " the formation of copious tarry 'precipitates which characterises direct cMorinations, as previously described with phosphorus oxychloride and phosphorus oxychloride / quaternary ammonium halides. The Vilsmeier chlorination of 2,5-diamino-4,6-d ydroxypyrirrridine may be carried out in an inert solvent, such as toluene, chloroalkenes, or chloroalkanes (such as methylene chloride, chloroform or 1,2-dichloroethane). Preferably the solvent is J,2-dichloroethane, chloroform, or methylene chloride. The chlorination may be carried out at 0 to 110°C, preferably at 40-100°C, conveniently at reflux for the solvent used. Reaction times are typically 12 to 48 hours. Isolation of compounds of formula (Ί) is simple and can be readily scaled-up, involving simply washing the reaction solution with an aqueous solution containing sufficient base, such as sodium bicarbonate, to neutralize any hydrogen chloride formed and then concentrating the dried organic layer to obtain the novel chlorinated pyrimidines of formula (I). The compounds of formula (Γ) are generally stable and may be precipitated from a variety of solvents, such as ethyl acetate, and stored or used without further purification.

Particularly preferred examples of the compounds of formulae (I), (Π) and (ΠΓ) are: a) 4,6-Pic oro-2,5-bis-[(dimethylammo)methyleneammo]py^ b) 2-Anmo^,6-dicUoro-5-[(dimethylan.mo)methyleneamm c) N-(2-Ammo-4,6-dic oro-5-pyrimic^yl)formamide According to a further aspect of this invention the novel intermediate of formula (ΤΠ) can be used in the synthesis of 2-amino-6-chloropurines. In addition compounds of formula (I) or (Π) may also be used in the synthesis of 2-amino-6-chloropurine nucleosides, provided that the amine HN !R2 (where R1 and R2 are defined earlier) liberated, during the conversion of the pyrimidine intermediate to the purine, is sufficiently unreactive towards the displacement of the chloro group of the 2-arnino-6-chloropurines generated.

ASB/JJ/23rd January 1995 The compounds of formula (ΠΓ) share with the previously described N-2-acylated derivatives the property of greater reactivity than 2,5-diarrjmo-4,6-dic oropyrirmdine toward displacement of a chloro group by an appropriate primary amine or protected hydroxylamine. However, such condensations with (HI) (described in more detail below) may be carried out under milder conditions at lower temperatures and with shorter reaction times than with compound (TV), thus resulting in less decomposition of the amines. The condensation products (VI) are isolated in greater yield and purity than the corresponding products (VET) formed in condensations with 2,5-diamino-4,6-(HcMoropyrirmdine (TV). Another advantage of the use of the intermediate (ΠΓ) over the previously described N-2-acylated derivatives, in addition to greater ease of synthesis, is that the purines generated from (TH) do not require deprotection, i.e. hydrolysis of the N-2-acyl group (these longer processes are described in US Patents 5,087,697 and 5,159,076).

Wherein is hereinafter defined.

ASB/JJ/13th January 1995 The compound of formula (III) can be used to prepare the novel intermediates of formula (VI) which are described and claimed in the parent Israel Application No. 112539: wherein R3 mav ¾e hydrogen or any group which is not attached by a glycosidic bond.

Preferably is a hydroxyl or a protected hydroxyl; or a carbocyclic group (e.g. 03. carbocyclic), an acyclic group (e.g. C2_g hydrocarbyl) wherein carbon atoms may be substituted by one or more heteroatoms such as N, O or S, or a heterocyclic group (e.g. C _7 heterocyclic) in which at least one carbon atom is replaced by a N, O, or S atom, or a substituted analogue of any thereof (e.g. such substituents are independently selected from Ci_4alk i, Ci_4 alkoxy, hydroxyl or protected hydroxyl, azido, phosphonyl, or halogen), provided that such groups are not attached by a glycosidic bond .

Preferred groups forR3 are hydroxyl or protected hydroxyl.

Further preferred groups for R3 are b. c.

ASB/JJ/13th January 1995 d. (AcOCH2)2CHCH2CH2- ; e. H0CH2CH2CHCH2- ; CEL- H A further preferred group for R3 is ; Suitable groups forR3 are selected from a; b; c; d; e; and f, as defined above.

By "hydrocarbyl" it is meant a group containing only hydrogen and carbon atoms, which may contain double and/or triple bonds and which may be straight, branched, cyclic or aromatic.

ASB/JJ/23rd January 1995 IL Application No. 112539 also provides a process for the preparation of compounds of formula (VI) which comprises reacting a compound of formula (III) with an amine of formula R3NE2, where R3 is defined above. Such condensations are preferably carried out at reflux in a solvent such as ethanol, butanol, water or acetonitrile in the presence of at least one equivalent of a base, such as triallsylamine or potassium or sodium carbonate.

Subsequent references to compounds of formula (Via, b, c, d, e, f, g, or h) denote a compound of formula (VI) in which R3 is a group- of a, b, c, d, e, f, g, or h as defined abover A particularly preferred compound of formula/VI) is (lS,4R)-4-[(2-amino-6-chloro-5-forn armdo-4-pyrimidmyl)amino]-2-cyclopentene- 1 -methanol (Via) The novel intermediates (VI) can be converted by ring closure to the corresponding compounds of formula (VTf):- wherein R3 is defined above.

Ring closure of (VI) to (VTf) is conveniently carried out in trialkylorthoformates (e.g. triethylorthoformate or trimethylorthoformate) with concentrated aqueous acid (e.g. 2-4 molar equivalents of hydrochloric, sulfuric acid or methane sulfonic). For example, the hydrochloride salt of (Vila) i.e. wherein R3 represents group a, begins to precipitate from such orthoformate solutions of (Via) within minutes and yields above 90% may be achieved by filtration of the precipitate, optimally after several hours at ambient temperature.

ASB/JT/23rd January 1995 The synthesis of 9-substitoted-2-animo-6-cMoropurm.es, such as compounds of 'formula (VII), in this manner represents a significant improvement over previously published syntheses utilizing triammopyrimidine intermediates such as (VET): as described US Patent 4,916,224. The previously-described routes to intermediates such as (VIE) are longer and, more importantly, the number of steps to the purine targets after incorporation of the group is greater. Also, triammopyrimidine intermediates such as (Vin) are air- and hght-sensitive and extremely difficult to purify due to their polarity and metal-chelating abilities (the isolation from the zinc reduction of diazo intermediates is especially problematic). The novel 5-formamido intermediates of formula (VI) are easily and directly attainable from compounds of formula (Tfl) in one step and are generally solids which are stable and easily-purified by precipitation from a suitable solvent. (rS,3'S,4'S)-2-Animo-l,9-Q¾ydro-9-[3,4-dmydroxy-3-hydroxymemyl-l-^ purin-6-one (TXh) (EP0420,518) may be prepared by condensation of the compound of formula(TJT) with 4-amino-3-cyclopentene-l -methanol (US patent 5,049,671) to form the compound of formula (VIg) followed by ring closure of the compound of formula (VIg) to prepare the compound of formula (VTIg), which may be hydroxylated, with osmium tetroxide/N-methyl-morpholine N-oxide to provide the compound of formula (VTTn). The compound of formula (Vllh) is hydrolysed to form the compound of formula (TXh).

ASB/JJ/13th January 1995 2-Ammo-6-cMoropurine (Vllb) may be prepared by ring closure of novel 2,4- Carbocyclic nucleosides may also be synthesized from the compound of formula (VTIb), for example by (Pd-catalyzed coupling with an appropriate carbocyclic intermediate as described in Mac Keith et al., J. Chem.So jerkin Trans 1. 1993: 313-314 and references therein.

The compounds of formula (Vila), (VTIc), (Vile), (VTIf), (VHg) and (V Ih) are conveniently hydrolyzed to the corresponding guanine compound by refluxing with aqueous base or acid.

We have found an alternative process for the synthesis of 2,6-diaminopurmes (wherein the 6-amino group is substituted by R4 and R^, which may be the same or different, and are selected from H, Ci.galkyl, C3_6cycloalk l, aryl (such as phenyl), in particular R4 is H and R5 is cyclopropyl) directly from (VI) which advantageously eliminates a step in the process. Such 2-aminopurine compounds can be synthesised directly from the intermediates (VI) by refluxing the compound of formula (VI) with an excess of the amine (H R4R5) in a refluxing solvent, such as ethanol, isopropanol, n-propanol, t-butanol or n-butanol.

In particular cases, it may be more useful to utilize 2,5-&amino-4,6-dichloropyrimidine(TV) to prepare compounds of formula (VIIT), useful in the synthesis of 8-modified 2-aminopurine nucleoside analogues, such as 8 -aza-2-aminopurines (which have broad-spectrum anti-herpes activities described in Storer et al., Spec. Publ. Roy. Soc. Chem (Rec. Adv. Chem. Anti-Infect. Agents) 1993, 119: 251-265); in such cases the intermediates (I), (Π) and (Tff) can be used to provide (TV).

ASB/JJ/23rd January 1995 Pharmaceutically acceptable esters of certain compounds of the invention may be prepared by esterification using conventional methods known in the art. Such methods include , for example, the use of an appropriate acid halide or anhydride.

The compounds of the invention, including -esters thereof, may be converted into pharmaceutically acceptable salts in a conventional manner by treatment with an appropriate acid or base. An ester or salt of a, compound of the invention may be converted into the parent compound, for example, by hydrolysis.

The following examples are intended for Hlustration only and are not intended to limit the scope of the invention in any way.

Example 1 4.6-DicMoro-2.5-bis-{rfdimethy 2,5-Diarnmo^,6-dmydroxypyrimidine hemisulfate (Sigma, 25.0 g, 0.131 mole) was stirred in chloroform (ARMallinckrodt, 400 mL) in a 2 L- 3 -necked round bottom flask equipped with a reflux condenser (with source of nitrogen connected to the top of the condenser) and an exit for HC1 gas connecting another neck of the flask to a NaOH trap. (Chloromethylene)dimethyl ammonium chloride (Vilsmeier reagent, Aldrich, 88.0 g, 0.651 mole as 95%) was washed into the flask with additional chloroform (400 mL). The reaction'-mixture was brought cautiously to reflux with nitrogen sweeping the HC1 evolved into the trap. hen the evolution of HC1 slowed after about 1 hour of reflux, the sweep.was stopped and the reaction kept under a gentle positive pressure of nitrogen from that point. Additional Vilsmeier reagent (50.0 g, 0.370 mole) was added after 24 hours and reflux continued for an additional 20 hours. The stirred reaction mixture (yellow solution with dark yellow solid) was cooled (ice bath) and diluted with water (sufficient to dissolve the solid, ca. 300 mL). The aqueous layer was adjusted to pH 7 with sodium hydroxide or solid sodium carbonate. The chloroform layer was separated, washed with water (3 x 400 mL), dried (sodium sulfate), and concentrated in vacuo to a dark yellow solid (36 g). This solid was dissolved in ethyl acetate (300 mL), stirred with charcoal (1 g), and filtered with a silica gel pad (3x3 in., packed in ethyl acetate). The pad was ASB/JJ/23rd January 1995 washed with additional ethyl acetate and eluents concentrated in vacuo to leave the title compound as a light tan solid (30.75 g, 81% ); nip. 116-119°C; 1H-NMR identical to that of recrystallized samples.

Anal: Calcd. for CioHi4N6Cl2.0.10 EtOAc: C, 41.92; H, 5.01; N, 28.20; O, 23.80. Found: C, ,42.23; H, 4.95; N, 28.46; CI, 24.11.

Recrystallization of such a sample from ethyl acetate gave the title compound as white granules; m,p. 123-125 °C; mass spectrum (CI/CH4): 291, 289 (M+l); 1H-N R (DMSO-d6) δ: 8.49 and 8.69 (both s, 1 each, 2CH), 3.16 (s, 3, CH3), 3.03 (s, 6, 2CH3), 2.97 (s, .3, CH3); UV (pH 7 phosphate buffer) max 296 nM (ε33,300), λπώι 248 (5200).

Anal. Calcd. for C10H14N6C12: C, 41.54; H, 4.88; N, 29.06; CI, 24.52. Found: C, 41.59; H, 4.91; N, 29.01; CI, 24.47.

Example 2 4,6-DicHoro-2,5-bis-{[( l-me1+iyl^^ (Example 1, 5.87g, .3 mmol) was dissolved in 95% ethanol (200 mL) and 6 N aqueous hydrochloric acid (13.5 mL) added. The solution was heated in an oil bath at 55 °C under nitrogen for 30 minutes, at which point TLC (silica gel, 5% methanol-chloroform) showed that starting material had been cleanly converted to a lower-Rf product. The cooled (ice bath) solution was adjusted to pH ~8 with concentrated ammonium hydroxide and the resulting mixture (white precipitate formed) concentrated on a rotary evaporator to ~5 mL to remove ethanol. Additional water (20 mL) was added and the cooled mixture was filtered. The white precipitate was washed with additional water (2 x 20 mL) and dried to give the title compound as a white powder (4.50 g, 95%), m.p. >dec 250 °C ; mass spectrum (CI/CH4): 236, 234 (M+l); !H-NMR (DMSO-d6)5: 7.59 (s, 1, CH), 6.90 (s, 2, NH2), 3.00 and 2.94 (both s, 3 each, 2CH3); UV (pH 7 phosphate buffer) Xmax: 328 nM (s 4500), 255 (15,800).

ASB/n/13th January 1995 - Anal. Calcd. for C7H9N5C12: C, 35.92; -H, 3.88: N, 29.92; CI, 30.29. Found: C, 35.66; H, 3.86; N, 29.74; CI, 30.54.

In another experiment, 2,5-diammo^,6-dmydroxypyrimidirie hemisulfate (Sigma, 48.0 g, 0.250 mole) was reacted as in Example -1 with less Vilsmeier reagent (7.2 molar equivalents) and the resulting 4,6-dic oro-2J5-bis-{[(dimethylammo)methylene]arnino} pyrimidine (92%), without recrystallization, was hydrolyzed in 95% ethanol (1 L) and 6 N aqueous hydrochloric acid (110 mL) to provide the title compound of the same purity (elemental analysis and 1H-NMR) as the characterized sample described above (44.2 g. 76% overall from 2,5-dammo-4,6-dmydroxypyrirnidine hemisulfate).

Example 3 N-(2-Ammo-4.6-dicMoro-5-pyrirmdmvi fo (UP A slurry of 2-ammo-4,6-&cMoro-5-{[(dimethylam (Example 2, 1.50 g, 6.41 mmol) and 1.5 M aqueous potassium phosphate buffer (35 mL, prepared by adjusting the pH of a 1.5 M solution of KH2P04 to 3.2 by addition of 85% phosphoric acid) was gently refluxed (in an oil bath at 125 °C). After 4 hours of reflux, the pH of the rnixture was adjusted from 4 to 3 by addition of 4 drops of 85% phosphoric acid. After a total of 6 hours of reflux, TLC(silica gel plates developed in 5% methanol-chloroform) showed that the starting material had been largely converted to a lower-Rf product. The solid was filtered and washed with water (5 mL), methanol (5 mL), and dried to give the title compound as a white solid (0.900 g, 68%), m.p. >250°C dec; mass spectrum (CI CH4): 209, 207 ( +l); 1H- MR (DMSO-d6)5: 9.81 and 9.46 (s and d, J = 11 Hz, total 1, NH), 8.25 and 8.00 (s and d, J = 11 Hz, total 1, CHO), 7.69 and 7.63 (both s, total 2, NH2).

Anal. Calcd for C5H4N40C12: C, 29.01; H, 1.95; N, 27.07; CL 34.25. Found: C, 29.12; H, 1.96; N, 27.13; CL 34.34.

ASB/JJ/13th January 1995 In another experiment, a slurry of 2-ammo-4,6-dicWoro-5-{[(dimethyl-arj3mo)me1 lene]amino}-pyriniidirie (Example 2, 25.0 g, 0.107 mol) in 1.5 M aqueous potassium phosphate buffer (300 mL, prepared as above) was gently refluxed for 4 hours. pH.was maintained at 3.2 by addition of 85% phosphoric acid, as required, throughout this period. The precipitate was filtered, washed with water (3 10 mL), methanol (2 x 10 mL), and dried (50°C, 25 mm Hg) to give the title compound as an off-white powder (16.0 g, 72%) with purity identical to that of the characterized sample described above (elemental analysis and 1H-NMR).

Example 4 2.5- Diammo-4.6-dcMoropyriniidine (TV) 4.6- DicMoro-2,5-bis-{[(dimethylernino)methylene]a.mino}pyrim (Example 1, 1.00 g, 3.36 mmol) in ethanol (25 mL) and pH 3.2 aqueous potassium phosphate buffer (1.5 M, 10 mL, prepared as described in Example 3) was refluxed for 24 hours. During reflux, the pH was maintained at ca. 3 by addition of 85% phosphoric acid, as required. The ethanol was evaporated in vacuo and water added (10 mL). This solution was extracted with chloroform (3 x 25 mL). The combined chloroform layers were dried (sodium sulfate) and chloroform evaporated to leave a solid (0.40 g). Crystallization of this solid from ethanol-water/ 4:1 gave the title compound (TV) as off-white needles (0.324 g, 52%); darkens and shrinks to black solid above 185°C, does not become fluid below 300°C ; .[Lit. 198°C (Legraverend et al., Synthesis 1990:587-589) and 188-191°C (Temple et al., J. Org. Chem. 1975, 40:3141-3142)]; mass spectrum (CI CH4): 181, 179 (M+l); 1H-NMR (DMSO-d6)5: 6.50 (br s, 2, NH2), 4.73 (br s, 2, NH2).

Anal. Calcd. for C4H4N4C12.0.12 EtOH: C, 27.60; H, 2.58; N, 30.36; CI, 38.42. Found: C, 27.99; H, 2.39; N, 30.42; d, 38.74.

ASB/JJ/13th January 1995 - - Example 5 ■ ' .. 2 -Diammo-4.6-dicMoropyrimidme (ΊΥ) A _ mixture of 2-ammo-4,6-dicMoro-5-[(dimet^ (Example 2, 500 mg, 2.14 mmol), pH 3.2 aqueous potassium phosphate buffer (1.5 M, 6 mL, prepared as described in Example 3), water (1 mL), and ethanol (5 mL) was refluxed gently for 28 hours. During the reflux period, pH was maintained at ca. 3 by addition of 85% phosphoric acid. Volatiles were evaporated in vacuo and the residual solids partitioned between water (30 mL, adjusted to ph 8 with dilute ammonium hydroxide) and chloroform (75 mL). The chloroform layer was dried (sodium sulfate) and the chloroform evaporated to leave off-white solid (0.30 g). Crystallization of this solid from ethanohwater/ 4:1 gave the title compound (TV) as light pink needles (332 mg, 61%); darkens and shrinks to black solid above 185°C, does not become fluid below 300°C; 1H- MR (DMSO-d6) and mass spectra identical to those described in Example 4.

Anal. Calcd. for C4H4N4C12: C, 26.83; H, 2.25; N, 31.30; CI, 39.61. Found: C, 26.93; H, 2.25; N, 31.24; CL 39.52.

Example 6 2.5-DianTmo-4.6-dic oropyrin idine (TV) N-(2-Ammo-4,6-dicMoro-5-pyrin3idhyl)formamide (Example 3, 500 mg, 2.42 mmol) was dissolved in 0.1 N hydrochloric acid (5 mL, 2.5 mequiv) and ethanol (7 mL) at reflux. The solution was refluxed for 5 hours. Volatiles were removed in vacuo. The residue was partitioned between water (30 mL) adjusted to pH 8 with dilute ammonium hydroxide and ethyl acetate (75 mL). The ethyl acetate layer was dried (sodium sulfate). Evaporation of the ethyl acetate left pink solid (0.40 g). Recrystallization of the solid from 95% ethanol gave the title compound (TV) as light pink needles (280 mg, 65%); darkens and shrinks to black solid above 185°C, does not become fluid below 300°C ; lH-NMR (DMSO-d6) and mass spectra identical to those described in Example 4.

ASB/JJ/13th January 1995 Anal. Calcd. for C4H4N4CI2: C.26.83;. H.2.25; N.31.30; Cl.39.61. Found C.26.95; H.2.24; N. 31.19; CI. 39.53.

Example 7 (1 S.4R)-4-[f2-Ammo-6-cMoro-5-formamido^^^ n ethanol (Via.) N-(2-Am o^,6-dicMoro-5-pyrinrid^ (Example 3, 2.07 g, 10.0 mmol) was stirred in refluxing absolute ethanol (40 mL) under itrogen to achieve partial dissolution. To this stirred mixture was added a solution of freshly prepared (lS,4R)-4-amino-2-cyclopentene-1 -methanol (PCT Application 9204015.3, 1.57 g, 12.5 mmol as 90%) in ethanol (15 mL) followed by triethylamine (3.5 mL, 25 mmol, freshly distilled from calcium hydride). After 14 hours of reflux, the resulting dark solution was cooled and 1 N sodium hydroxide (10 mL) was added. The volatiles were evaporated in vacuo. The residual tan solid foam was dissolved in 5% methanol-ethyl acectate, and the solution was washed through a silica gel pad to give the title compound as an off-white solid (2.50 g, 88%), after evaporation of solvents. Recrystallization of the solid from ethyl acetate-methanol (20:1) gave the title compound (Via) as fine white crystals (2.29 g, 81%), m.p. 177-178°C; mass spectrum (CI CH4): 286, 284 (M+l), 190, 188 (B+H); 1H-NMR (DMSO-d6)5: 8.99 and 8.58 (s and d, J = 11.1 Hz, total 1, amide NH), 8.11 and 7.80 (s and d, J = 11.1 Hz, total 1, amide CH), 6.77 and 6.61 (two d, J = 8.0 Hz) overlapping ¾.60 and 6.48 (two br s, total 3, NH and NH2), 5.85 and 5,70 (two m, 1 each, CH=CH), 5.15-5.00 (m, 1, NCH), 4.71 (t, J = 5.1, 1, OH), 3.45-3.30 (m overlapping H20, OCH2), 2.80-2.65 (m, 1, CH), 2.45-2.25 and 1.45-1.30 (both m, 1 each, CH2); [a]20 589 +21.2°, [a]20 578 + 22.2°, [α]2° 546 + 25.2°, [a]20 436 + 41.4°, [a]20 365 + 48.3° (c 0.50, methanol).

Anal. Calcd. for C11H14N502C1: C, 46.57; H, 4.97; N, 24.69; CI, 12.50. Found: C, 46.63; H, 4.99; N, 24.58; CL 12.59.

ASB/JJ/23rd January 1 95 - - Example 8 (lS.4R -4-(2-Ammo-6-cMoro-9-H-purin-9-ylV2-cyclo Hydrochloride (Vila) A mixture of (lS,4R)-4-[(2-ammo-6¾hloro-5-fonnanri^ cyclopentene-1 -methanol (Example 7, 1.00 g, 3.50 mmol) and triethylorthoformate (Aldrich, Sure Seal, 18 mL) was stirred while concentrated hydrochloric acid (37%, 1.25 mL) was added in one portion. The resulting clear, colorless solution was stirred under nitrogen. A white precipitate began to form after 15 minutes. After 4 hours, TLC of a drop of the reaction mixture dissolved in methanol and neutralized with sodium hydroxide (silica gel plates developed in 10% methanol-chloroform, visualized in UV light) showed almost complete conversion of Via to a higher-Rf material. The precipitate was filtered, washed with t-butyl methyl ether (15 mL) and dried at 0.2 mm Hg 25°C for 18 hours to give the title compound as a white powder (975 mg, 92%), m.p. >300°C dec; mass spectrum (CI CH4): 266(M+1); 1H->MR (DMSO-d6)6: 8.18 (s, 1, purine CH), 7.2-6.7 (br s, NH2, OH overlapped by water), 6.20 and 5.90 (both m, 1 each, CH=CH), 5.48 (m, 1, NCH), 3.47 (d, J = 5.7 Hz, 2, CH20), 2.90 (m, 1, CH), 2.75-2.55 and 1.75-1.60 (both m, 1 each, CH2).

Anal. Calcd. for C11H12N50C1.HC1: C, 43.73; H, 4.34; N, 23.18; CI, 23.48. Found: C, 43.62; H, 4.34; N, 23.07; CL 23.53.

Example 9 (lS.4R)-4-[2-Ammo-6- (^clopropy1aminoV9H-purm-9-yl]-2-cvclopen^ ( i A solution of (lS,4R)-4-c oro-5-formamido-6-{[(4-hydroxymethyl)-2-cyclopenten-l-yl]ammo}pyTirridine (Example 8, 250 mg, 0.883 mmole) was refluxed gently (in an oil bath maintained at 130°C) in n-butanol (dried over 4 A molecular sieves, 5 mL) under nitrogen with cyclopropylamine (Aldrich, 0.30 mL, 4.4 mmol) for 16 hours. A second portion of cyclopropylamine (0.15 mL) was added and reflux continued for an additional 5 hours. The volatiles were removed and the residual oil redissolved in ethanol-water (1:1) ASB/JJ/13th January 1995 - - with 1 N sodium hydroxide (0.5 mL).-- Volatiles were again removed and the residue chromatographed on a silica gel flash column (1x10"). (IS, 4R)-[(2,5-Diamino-6-chloro-4-pyrimidinyl)amino]-2-cyclopentene-l-methanol (Villa, 35 mg, 16%) eluted with 5% methanol-ethyl acetate. Continued elution with 10% methanol-ethyl acetate gave (IS, 4R)-4-[2-ammo-6-(cyclopropylammo)-9H-pu^ as a light tan solid foam (160 mg, 60%); H-NMR (DMSO-d6)5: 7.58 (s, 1, purine CH), 7.25 (d, J = 4.5 Hz, 1, NH), 6.10 (m, 1, =CH), 5.80-5.75 (m, 3, =CH and H2), 5.40 (m, 1, NCH), 4.72 (m, 1, OH), 3.45 (m, 2, OCH2), 3 A ( br m, 1, CH of cyclopropyl), 2.80 (br m, 1, CH), 2.70-2.50 (m overlapping solvent, CH), 1.50-1.05 (m,l, CH), 0.70-0.50 (m, 4, 2 CH2 of cyclopropyl).

Anal. Calcd. for C14H18N6O.0.20 H2O.0.40 CHS OH: C, 57.32; H, 6.35; N, 27.85. Found: C, 57.59; H, 6.48; N, 27.70.

Example 10 (1 S.4RV4-[2-A mo-6-(cyclopropyIammoV9H-pui½-9-yl]-2-c^^ 1 -methanol (TXa) (1 S,4R)-4-(2-Ammo-6-cHoro-9-H-pum-9-yl)-2-cyclopentene-l-methanol (US Patent 5,206,435) or the hydrochloride salt thereof (Example 8) was refluxed in ethanol with 10 molar equivalents of cyclopropylamine for 2 hours. The resulting solution was cooled to ambient temperature and 1 N sodium hydroxide (1 or 2 molar equivalents, depending on whether the starting material was Vila or the hydrochloride salt of VHa) was added. The volatiles were evaporated in vacuo. (lS,4R)-4-[2-Arnmo-6-(cyclopropylarnmo)-9H-purk-9-yl]-2-cyclopentene -1 -methanol (TXa) was washed from a silica gel pad eluted with 5% methanol-chloroform or 10% methanol-ethyl acetate and isolated as a white solid foam (80%); spectra identical to those of the product of Example 9.

ASB/JJ/13th January 1995 Example 11 niS-3'S.4'SV2-Ammo-L9-dmvdro-9^^^ puriii-6-one a) ( 4E0-4- Γ 2- A minn-6-cMoro-5-formamidc^-pyrimidmv -cvclopentene- 1 - methanol By the method of Example 7, N-(2-Ammo^,6-dicMoro-5-pyimiidmyl)foimamide (Example 3, 2.56g, 52.4mmol) was reacted with (4R)-4-amino-l -cvclopentene- 1- -methanol (1.4g, 52.4mmol), available from (-)-2-azabicyclo[2.2.1]hept-5-en-3-one (Chiroscience) by methods described in Examples 1-4 and 42 of U.S. Patent 5,049,671. Crystallization from ethyl acetate - methanol gave title compound as white crystals, mp. 148-150°C; mass spectrum (CI CH4): 286, 284 (M+l), 190, 188 (B+H); iH-NMR (DMSO-d6)5: 8.97 and 8.55 (s and d with J = 11.3 Hz, total 1, MJCHO), 8.12 and 7.80 (s and d with J = 11.5 Hz, total 1, CHO), 7.00 and 7.78 (both d, J = 7.4 Hz, total 1, NH), 6.60 and 6.40 (both 8, total 2, NE¾), 5.48 (s, 1, = CH), 4.74 (t, J = 5.5 Hz, 1, OH), 4.74-4.60 (m, 1, NCH), 4.0-3.90 (m, 2, CH20), 2.75-2.55 and 2.40-2.15 (both m, 2 each, 2CH2); [ ]58 20^.4θ} [α]57820-5.2θ, [α]54620-4.8θ, [α]43620-20.0°, [ ]36520-60.4θ (c 0.25, methanol).

Anal. Calcd. for Ci 1H14N502C1: C, 46.57; H, 4.97; N, 24.69; CI, 12.50. Found: C, 46.64; H, 5.01; N, 24.60; CI, 12.45. b) (4R -4-(2-Ammo-6-cMoro-9H-purin-9-ylV 1 -cvclopentene- 1 -methanol A mixture of (4R)-4-[(2-ammo-6-cUoro-5-forman-ddo-4-pyrimidm cyclopentene-l-methanol (Part a, 1.60'g, 5.65mmol) and triethylorthoformate (29mL) was stirred while concentrated hydrochloric acid (37%, 2.0mL) was added in one portion. The resulting clear, colourless solution was stirred under nitrogen. After 5 hours the resulting precipitate was filtered and washed with t-butyl methyl ether (3 x lOmLO and dried to provide white powder (1.25g). This powder was ASB/JJ/23rd January 1995 dissolved in water and the pH adjusted to 3 by addition of IN hydrochloric acid. The solution was heated at 60°C for 4 hours, cooled, neutralized, and evaporated to a solid which was chromatographed on silica gel. Title compound was eluted . with 5% methanol chloroform and crystallized from ethanol-ethyl acetate to white crystals, m.p. 145-147°C; mass spectrum (CI/CH4): 268, 266 ( +l)» 172, 170 (B+H); !H-NMR (DMSO-d^S: 8.09 (s, 1, purine CH), 6.9 (br s, 2, N-¾), 5.64 (m, 1, = CH), 5.2-5.0 (m, 1, NCH), 4.87 (t, J = 5.5 Hz, 1, OH), 4.05 (m, 2, CH20), 3.0-2.5 (m, 4, 2 CH2).

Anal. Calcd. for CHH12N5OCI: C, 49.06; H, 4.64; N, 26.01; CI, 13.16. Found: C, 49.18; H, 4.63; N, 26.11; CI, 13.19. riS.2S.4R -4-f2-Aminn-6-cMoro-9H-purm-9-ylV2-rhvdroxymethvD-1.2- cyclopentanediol (4R)-4-(2-Ammo-6-c oro-9H-puim-9-yl)-l-cyclopentene-l-methanol (Part b, 501mg, 1.89mmol), N-methylmorpholine N-oxide (60% aqueous solution, Aldrich, 0.33mL, 1.89mmol), osmium tetroxide (2.5% in t-butyl alcohol, Aldrich, 0.47mL), and t-butyl alcohol (12mL) were heated at 60°C for 1.5 hours. Volatiles were evaporated and the residual solids were chromatographed on silica gel. Title compound was eluted with 10% methanol-chloroform as tan solid (210mg) and resolidified from absolute ethanol to give white powder, m.p. 217-219°C; mass spectrum (CI/CH4): 302, 300 (M+l), 172, 170 (B+H); !H-NMR (DMSO-d6)5: 8.29 (s, 1, purine CH), 6.9 (br s, 2, NH2)» 5.15-4.90 (m, 1, NCH), 4.80 (d, J = 3.9 Hz) overlapping 4.78 (t, J - 3.5 Hz, total 2, 2 OH), 4.30 (s) overlapping 4.3-4.2 (m, total 2, OH and OCH), 3.45-3.35 (m, overlapping water, CH2OH), 2.25-2.05 (m, 4, 2 CH2).

Anal. Calcd. for CnH^ sOsCl: C, 44.08; H, 4.71; N, 23.37; CI, 11.83. Found: C, 43.89; H, 4.80; N, 23.16; CI, 11.73.

ASB/JJ/23rd January 1995 d) rrS.3^.4'SV2-Aiiiino-1.9-d&vdro-9-r3.4-dihvdroxy-3-hvdroxym cyclopentylV6H-purm-6-one (iS>2S)4 )-4-(2-Amino-6-cMoro-9H-pOrm-9-yl)-2-(hydroxyme^ tanediol (Part c, 90mg, 0.27mmole) was refluxed in IN hydrochloric acid (2.7mL) for 45 minutes. Volatiles were evaporated in vacuo. Portions of water were evaporated and the residue was redissolved in water. The pH was adjusted to 5 with hydrochloric acid and the resulting mixture cooled, filtered, and the —precipitate dried to provide the title compound as an off-white powder (5 lmg, 68%), m.p. >300°dec; mass spectrum (CI/CH4): 283 (M+l); !H-NMR(DMSO- c¼) identical with that described in U.S. Patent 5,233,041.

Example 12 N-(2.4-Di3TriiTin- -cMoro-5-pyrirnidmyl)fom N-(2-ATnino-4,6-dicMoro-5-pyrimidinyl)fonTiarnide (Example 3, 500mg, 2.14mmol) and ammonia (150mL) was stirred in a Parr bomb at 50°C for 18 hours. The ammonia was evaporated and the residual solid triturated with water (lOmL). The solid was dried to give the title compound as red powder (400mg, 89%), m.p 300°C; mass spectrum (CI CH4): 190, 188 (M+l); iH-NMRpMSO-d^S: 9.05 and 8.60 (both br s, total 1, MICHO), 8.1 and 7.8 (bothbr s, total 1, NHCHO), 6.80-6.20 (4 br s, total 4, 2 NE¾).

Anal. Calcd. for C5H6N5OCI: C, 32.01; H, 3.22; N, 37.34; CI, 18.90. Found: C, 3.1.97; H, 3.23; N, 37.26; CI, 19.00.

Passages of the description which are not within the scope of fhe claims do not constitute part of the invention.

ASB/JJ/23rd January 1995

Claims (9)

129935/3 26 C L A I M S:

1. A compound of formula (X) wherein RX is N=CHNR'R2 or NH2 and RY is N=CHNR1R2 or NHCHO, and R1 and R2 which may be the same or different, are selected from C 1-8alkyl, C3.8 cycloalkyl, and optionally substituted aryl, with the exclusion of compounds of formula (X) wherein RX is N=CHNR'R2 and RY is NHCHO.

2. A compound of formula (1) wnsrsin R1 and R2 are as defined in claim 1

3. A compound of formula (l) as claimed in claim 2 wherein R1 and R2 sre both Ci-a alky I.

4. A compound of formula (II) wherein R1 and R2 are as dsrlnsd in claim 1 or 3. PB1517 IL2 129935/2 27

5. A compound of formula (III) ■

6. A process for the preparation of a compound of formula (I) as defined in claim 2 comprising of reacting 2,5-diamino-4,6-dihydroxypyrimidine with a compound of formula (V) wherein R and R2 are as defined in claim 1 or 3.

7. A process for the preparation of a compound of formula (II) wherein R1 and R2 are defined in claim 1 or 3; comprising hydrolysing a compound of formula (I).

8. A process for the preparation of a compound of formula (III) by hydrolysing a compound of formula (I) or (i!). P81517 IL2 129935/2 28 '

9. A process for the preparation of 2,5-diamino-4,6-dichloropyrimidine by the hydrolysis of a compound of formula (I), (II), or (III). For the Appl i cants , REINHOLD COHN AND PARTNERS ** TOTAL PAGE.07 **