AU597483B2 - Desazapurine-nucleoside derivatives, processes for the preparation thereof, pharmaceutical compositions containing them and the use thereof for nucleic acid sequencing and as antiviral agents - Google Patents

Abstract

Novel deazapurine nucleosides of the formula I <IMAGE> in which X denotes nitrogen or a methine group, W denotes nitrogen or the group <IMAGE> R<1>, R<2>, R<3> and R<4> can be the same or different, and denote hydrogen, halogen, a lower alkyl, hydroxyl, mercapto, lower alkylthio, lower alkyloxy, aralkyl, aralkyloxy, aryloxy or an optionally mono- or disubstituted amino group, R<5> denotes hydrogen or a hydroxyl group, R<6> and R<7> each denote hydrogen or one of the two radicals R<6> and R<7> denotes halogen, a cyano, azido or an optionally mono- or disubstituted amino group, where one of the radicals R<6> and R<7> can also represent a hydroxyl group if X denotes a methine group, and R<5> and R<7> together can additionally represent a further bond between C-2' and C-3' and Y represents hydrogen, a monophosphate, diphosphate or triphosphate group, and possible tautomers and salts and nucleic acids which contain one or more compounds of the formula I as building block. The compounds according to the invention exhibit antiviral properties and can further be used in DNA sequencing, where they lead to chain termination and/or prevent band compression.

Description

-T _s 1

I

COMMO H WRALT OF AUS TRALIA PATENT ACT 1952 COMPLETE SPECIFICATION 597483

(ORIGINAL)

FOR OFFICE USE CLASS INT. CLASS Application Number: Lodged: 4* 4 4s 4 O 4 Complete Specification Lodged: Accepted: Published: Priority: Related Art-: I doAumnnort cootAlto tib aneradnrltwu made under Santloe 49.

aod Is oorrfct ar printing.

4i ri NAME OF APPLICANT: ADDRESS OF APPLICANT: BOEHRINGER MANNHEIM GMBH 6800 Mannheim 31, Federal Republic of Germany NAME(S) OF INVENTOR(S) Frank SEELA Heinz-Peter MUTH Klaus KAISER Werner BOURGEOIS Klaus MUHLEGGER Herbert VON DER ELTZ Hans-Georg BATZ ADDRESS FOR SERVICE: DAVIES COLLISON, Patent Attorneys 1 Little Collins Street, Melbourne, 3000.

COMPLETE SPECIFICATION FOR THE INVENTION ENTITLED:- "DESAZAPURINE-NUCLEOSIDE DERIVATIVES, PROCESSES FOR THE PREPARATION THEREOF, PHARMACEUTICAL COMPOSITIONS CONTAINING THEM AND THE USE THEREOF FOR NUCLEIC ACID SEQUENCING AND AS ANTIVIRAL AGENTS" The following statement is a full description of this invention, including the best method of performing.it known to us -1- -2- The present invention is concerned with new desazapurine-nucleoside derivatives, processes for the preparation thereof, as well as the use thereof in the sequencing of nucleic acids, and also as anti-viral agents.

The new desazapurine-nucleoside derivatives according to the present invention are compounds of the general formula:-

R

1

R

3

N

SI W (I) 2

X

R x

N

R

7

R

Y-0 0o 6 wherein X is a nitrogen atom or a methine group, W is a nitrogen atom or a C-R 4 radical, R 1

R

2

R

3 and R c" which can be the same or different, are hydrogen or halogen atoms, hydroxyl or mercapto groups, lower alkyl, lower alkylthio, lower alkoxy, aralkyl, aralkoxy or aryloxy radicals or amino groups which are unsubstituted S or substituted once or twice by aralkyl or lower alkyl which, in turn, can be substituted by lower alkoxy, halogen, amino or amino substituted once or twice by aralkyl or lower alkyl; or in case, when the amino group is substituted twice, both substituents together represent an alkylidene radical which, in turn, can be substituted by lower alkoxy, halogen, amino or amino substituted once or twice by aralkyl or lower alkyl, AA4 I Ir 2a

R

5 is a hydrogen atom or a hydroxyl group and R 6 and R 7 are each hydrogen atoms or one of them is a halogen atom or a cyano or azido group or an amino group which is unsubstituted or substituted once or twice by aralkyl or lower alkyl which, in turn, can be substituted by lower alkoxy, halogen, amino or amino substituted once or twice by aralkyl or lower alkyl; or in case, when the amino group is substituted twice, both substituents together represent an alkylidene radical which, in turn, can be substituted by lower alkoxy, halogen, amino or amino substituted once or twice by aralkyl or lower alkyl, whereby one of R 6 and R 7 can also be a hydroxyl group when d 0 X is a methine radical and, in addition, R 5 and R 7 can together 0 0 0 0 9 0000 0 00 00 0 0 0 6o a 0 6* 4 0 o 0 0 0* 4* -3represent a further valency bond between C-2' and C-3' and Y is a hydrogen atom or a mono-, di- or triphosphate group, as well as the tautomers and salts thereof and nucleic acids which contain compounds of general formula I as structural components.

The lower alkyl radicals in the definition of the substituents R 1

R

2

R

3 and can be saturated or unsaturated, straight-chained or branched and contain up to 7 and preferably up to 4 carbon atoms. This 1 definition of the alkyl radicals also applies to the alkyl moieties which occur in the definitions of the lower alkylthio and lower alkoxy radicals. The methyl and ethyl radicals are quite especially preferred.

By halogen in the definition of the substituents

R

I

R

2

R

3

R

4

R

6 and R 7 are to be understood fluorine, chlorine, bromine and iodine.

The aralkyl and aralkoxy radicals in the definitions of the substituents R R2 R and R contain an alkyl moiety with up to 5 and preferably with up to 3 carbon atoms which are substituted one or more times with an aromatic radical, for example a phenyl or naphthyl radical. The aromatic radicals can, in turn, be substituted one or more times by an alkyl or alkoxy radical. The benzyl radical is especially preferred.

As aryloxy radical in the definition of R

I

R

2

R

3 and R 4 the phenyloxy radical is especially preferred which can optionally be substituted one or more times by

L

-4further substituents, for example nitro groups and alkyl and alkoxy radicals.

The amino group occurring in the definition of R,

R

2

R

3

R

4

R

6 and R 7 which can optionally be substituted once or twice, contains, as possible substituents, preferably alkyl radicals with up to 5 and preferably up to 3 carbon atoms which, in turn, can be substituted by alkoxy radicals, halogen atoms or amino groups optionally substituted once or twice. These substituents can also 10 represent an aralkyl radical. The two nitrogen substituents can together also represent an alkylidene radical and preferably a methylidene radical which, in turn, can be substituted by alkoxy, substituted amino groups or I halogen atoms. A quite especially preferred substituent 15 of this kind is the dimethylaminomethylidene radical.

The monophosphate group is the -PO(OH) 2 group, the diphosphate group is the -P 2 0 3

(OH)

3 group and the triphosphate group is the P 3 0 5

(OH)

4 group.

As possible salts, there are especially preferred -ne alkali metal, alkaline earth metal and ammonium salts of the phosphate groups. The alkaline earth metal salts are especially the magnesium and calcium salts.

SBy ammonium salts, according to the present invention there are to be understood salts which contain the ammonium ion which can be substituted up to four times by alkyl radicals containing up to 4 carbon atoms and/or by aralkyl radicals, preferably the benzyl radical. The i! I NM-

I

substituents can hereby be the same or different. The salts of the phosphates can be converted in known manner into the free acids.

The compounds of general formula I can contain basic groups, especially amino groups, which can be converted into acid addition salts with appropriate acids. As acids for this purpose, there can be used, S' for example, hydrochloric acid, hydrobromic acid, C| sulphuric acid, phosphoric acid, fumaric acid, succinic 10 acid, tartaric acid, citric acid, lactic acid, maleic acid and methanesulphonic acid.

The compounds of general formula I are new. They can be prepared analogously to known, related compounds.

For the preparation of the compounds of general formula I, a process has proved to be especially preferred in which a compound of the general formula:- 1 3 R R

(II)

W

R

2 X N

H

1 2 3 in which X, W, R R and R have the same meanings as above, is reacted with a compound of the general formula:- 7' 5 R- R (RIII) R'-O

(II)

-6in which R 5 has the above-given meaning, R 6 and R 7 each represent hydrogen atoms or one of these two symbols represents an azido group or a hydroxyl group protected by an oxygen protection group, R' is an oxygen protection group and Z is a roAIeit group, to give a compound of the general formula:- R 1 3 R R pR 0 0 o hu- i wi ,r R o o 4 I in which X, W, R, R 2

R

3

R

5

R

6 R7' and R' have the ,o above-given meanings, and oxygen protective groups R X o i hl N (IV) o.

Po R 00 above-given meanings, and oxygen protective groups possibly present are split off and thereafter a compound thus obtained, in which R or R is a hydroxyl group, after selective protection of the 5'-hydroxyl group, is optionally converted with a halide, cyanide or azide in known manner into a compound of general formula I in which R 7 R or R is a halogen atom or a cyano or azido group or, in known manner, is deoxygenated to give a compound of general formula I, in which R6a\d R7 hydrogen atom or a compound thus obtained of general formula I, in which R 6 or R is an azido group, is reduced in known manner to a compound of general formula I in which R 6 or R is an amino group and, if desired, a compound of -7general formula I, in which Y is a hydrogen atom, is converted in known manner into a mono-, di- or triphosphate and, if desired, a free base or acid obtained is converted into an appropriate salt or a salt obtained is converted into the corresponding free base or acid.

The compounds of general formula II are reacted with the compounds of general formula III especially advantageously under phase transfer conditions. Under the conditions of phase transfer catalysis, the bases 1' 0 of general formula II are converted into a corresponding anion, for example by means of a 50% aqueous solution of t sodium hydroxide. The anion thus obtained is hydrophobed by a phase transfer catalyst, for example tris-(2-(2methoxyethoxy)-ethyl]-amine, and transported into the 15 organic phase in which it reacts with the reactive S. compound of general formula III.

As reactive groups Z in the compounds of general formula III, there are preferably used halogen atoms and alkoxy radicals. In the case of this reaction, the hydroxyl groups of the sugar residue are protected in i the usual way by conventional oxygen protective groups, Sfor example toluoyl, benzoyl or acetyl radicals. After ~completion of the reaction, the oxygen protective groups can again be split off in known manner under alkaline conditions, a 1M methanolic menthanolate solution preferably being used.

During the reaction, it can also be preferable to nx~FI iiu-a- t keep the radicals R R 2

R

3 and R 4 protected by appropriate protective groups.

Another advantageous method for the preparation of compounds of general formula IV is the solid-liquid phase transfer process with the use of solid, powdered i potassium hydroxide, the above-mentioned kryptand, as well as compounds of general formulae II and III in an i aprotic solvent.

i 6 7 Compounds of general formula I, in which R or R S* 10 is a halogen atom or an azido group, are preferably '0 prepared by starting from a compound of general formula I, in which R or R is a hydroxyl group. The hydroxyl group in the 5'-position is first to be selectively i protected. For this purpose, too, known processes are ii a a S" 15 available. For example, in nucleotide chemistry, the I 4,4'-dimethoxy-triphenylmethyl radical has proved to be useful. After the reaction has taken place, this can 4 ,44 again be easily split off by mild acid hydrolysis, whereas the also acid-labile glycosidic bond is not hydrolysed under these conditions. The reaction of the nucleoside to be protected with the oxygen protective group reagent for the 5'-hydroxyl group is carried out Sin an appropriate organic solvent, preferably in dry pyridine, with a small excess of the oxygen protective group reagent, as well as possibly of an appropriate adjuvant base, for example N-ethyldiisopropylamine.

The so protected compound of general formula I is r -r I -9reacted with a halide, preferably with an alkali metal halide or an organic halide, or with an azide, preferably with an alkali metal azide, in known manner. The hydroxyl group on the C-3' atom is thereby nucleophilically substituted by the halide or azide.

Compounds of general formula I, in which R or R is a hydroxyl group, can also, after previous protection of the 5'-hydroxyl group in the above-described manner, be desoxygenated by known methods to give compounds of general formula I, in which R and R are hydrogen atoms.

For this purpose, the compound of general formula I, in which R or R is a hydroxyl group and in which the hydroxyl group has been protected in the above-described S, way and other functional radicals also carry protective i 15 groups, is first converted into a o° *derivative which is subsequently reduced radically with a 44 tributyl tin hydride. Such methods for the desoxygen- Sation of 2'-desoxynucleosides to give 2',3'-didesoxynucleosidss are known, the Barton desoxygenation method having proved to be especially favourable Chem. Soc., Perkin Trans. I (1975), 1574).

Compounds of general formula I, in which R or R is an amino group, are preferably prepared by reducing a compound of general formula I, in which the substituent R 6 or R is an azido group. This reduction of the azido group to the amino group can be carried out by various generally known methods, tae reduction with ~i~sil3L i hydrogen in the presence of a palladium-charcoal catalyst having proved to be especially advantageous.

The phosphate groups are introduced into compounds of general formula I, in which Y is a hydrogen atom, in known manner. The monophosphates are obtained, for example, by phosphorylating compounds of general formula I, in which Y is a hydrogen atom, with phosphorus oxychloride in trimethyl phosphate. The triethylammonium Ssalts obtained in this way can be converted in known 10 manner into other salts by transsalification. The diand triphosphates are obtained according to known methods, preferably from the monophosphates, by reaction with orthophosphates or pyrophosphates. Their various salts g can also be prepared by known methods.

o 15 Compounds of general formula II are either known or can be prepared analogously to known compounds. Such methods of preparation are described, for example, in Chemische Berichte, 110, 1462/1977; J. Chem. Soc., 1960, 131; and Tetrahedron Letters, 21, 3135/1980.

Some of the compounds of general formula IIlare also known. Compounds which have not hitherto been described can be prepared completely analogously to the known compounds. The preparation of such compounds is described, for example, in Chem. Ber., 93, 2777/1960 and in Synthesis, 1984, 961.

The new compounds according to the present invention possess valuable pharmacological properties.

C I r -11- In particular, by inhibition of the enzyme reverse transcriptase, the multiplication of retroviruses is prevented, i.e. the compounds according to the present invention possess especially cytostatic, as well as antiviral properties.

The structural units of nucleic acids contain, as glycosidic components, either the P-D-ribofuranosyl So*O radical or the 2-desoxy derivative thereof. Besides o

J

these aglyconic radicals, modified D-ribofuranosyl 0 0o° 10 derivatives are also found in nucleoside antibiotics.

Thus, for example cordycepin, which can be isolated from 9oo, 0 1 culture filtrates of Cordyceps militaris, contains the monosaccharide cordycepose. Besides this or 3'- .o,0 desoxy derivative of the ribonucleosides, some considero' 15 able time ago, 2',3'-didesoxynucleosides have been prepared synthetically. They have an anti-viral action 0o*o and can, in particular, via the inhibition of the enzyme S, reverse trr.-scriptase, inhibit the multiplication of retroviruses (cf. Proc. Natl. Acad. Sci. USA, 83, 1911/ 1986 and Nature, 325, 773/1987). The inhibitory action on the HIV virus, the cause of AIDS, is of especial therapeutic interest. However, they have the disadvantage that they are also inhibitors of cellular DNA polymerase so that they act cytotoxically. Furthermore, they can be deactivated by cellular enzymes. The compounds of general formula I do not display these disadvantages.

They have an antiviral action without being cytotoxic.

A

'3 2' ia:Le^B~a ot i y ^aaa~&~a -12- 4o 0099, C, a 0 00 009 @0 4 00 0 000 0 0 o 00 00000 04 6 0 400 ant 0 00 0 0'0 00 ~J 00 The compounds of general formula I according to the present invention can also be advantageously used for DNA sequencing according to Sanger's method. The sequencing of d(G-C)-rich DNA fragments is, in particular, made difficult by the formation of secondary structures which lead to a band compression in the region of d(G-C) clusters. The reason for this is the Hoogsteen base pairing of guanosine molecules. By means of the replacement of 2'-desoxyguanosine triphosphate by the compounds 10 according to the present invention, in which R 6 is a hydroxyl group, the band compression is largely overcome.

The compounds of general formula I according to the present invention, in which R 6 and R 7 are hydrogen atoms, are used in DNA sequencing by Sanger's method as chain 15 terminators instead of the known 2',3'-didesoxy crnpounds.

Nucleic acids which, as structural components, contain one or more compounds of general formula I, can be prepared according to known processes (for example as described in Nucleic Acids Research, 14(5), 2319 et seq./1986). However, they also result, for example, in the case of the DNA sequencing. If compounds of general formula I, in which R 6 is a hydroxyl group, are used as structural components, then a nucleic acid can contain several such structural components; if, as structural component, a compound of general formula I is used, in which R 6 is a hydrogen atom, then such a structural component can only be incorporated once, namely, on the -13end of the chain. The nucleic acids according to the present invention are made up of 2 to 1000 and preferably of 8 to 50 nucleotide structural components, nucleic acids with 15 to 30 nucleotide structural components being especially preferred.

These nucleic acids can also be used as antiviral agents. As so-called anti-sense nucleic acids, these >nucleic acids hybridise with the ssDNA/RNA of the virus 4 o, ;and make diificult the transcription to the virus DNA.

S' 10 Such nucleic acids can be used especially as agents

O',

*i 1 against AIDS since they are not decomposed or only |i decomposed with difficulty by the cell's own restriction i i enzymes.

I For the preparation of pharmaceutical compositions, the compounds of general formula I, the pharmacologically .acceptable salts thereof or nucleic acids containing them are mixed in known manner with appropriate pharmaceutical Scarrier substances, aroma, flavouring and colouring materials and formed, for example, into tablets or dragees or, with the addition of appropriate adjuvants, are suspended or dissolved in water or an oil, for example olive oil.

The compounds according to the present invention can be administered enterally or parenterally in liquid or solid form. As injection medium it is preferred to use water which contains the additives usual in the case of injection solutions, such as stabilising agents, J -14solubilising agents and/or buffers.

Such additives inclu'e, for example, tartrate and citrate buffers, ethanol, complex formers (such as ethylenediamine-tetraacetic acid and the non-toxic salts thereof) and high molecular weight polymers (such as liquid polyethylene oxide) for viscosity regulation.

Solid carrier materials include, for example, starch, ,o lactose, mannitol, methyl cellulose, talc, highly dispersed silicic acids, high molecular weight fatty acids 10 (such as stearic acid), gelatine, agar-agar, calcium 4 0 00' "phosphate, magnesium stearate, animal and vegetable fats and solid high molecular weight polymers (such as polyethylene glycols). Compositions suitable for oral administration can, if desired, contain flavouring and 15 sweetening agents.

The compounds according to the present invention Goa" are usually administered in an amount of from 1 to 0 4, 100 mg. and preferably of from -2 to 80 mg. per day and 4 B 4 per kg. body weight. It is preferred to divide up the daily dose into 2 to 5 administrations, in which case each administration comprises 1 or 2 tablets with a Scontent of active material of from 5 to 1000mg. The tablets can also be retarded, in which case the number of administrations per day can be reduced to from 1 to 3. The active material content of the retarded tablets can be from 20 to 2000 mg. The active material can also be administered by injection one to eight times per day or by continuous infusion, in which case amounts of from 500 to 4000 mg./day normally suffice.

The following Examples are given for the purpose of illustrating the present invention:- Example 1.

2-Amino-7-desaza-2',3'-didesoxy-9-0-D-ribofuranosylpurin-6-one.

a) 4 4 '-Dimethoxytriphenylmethyl)-amino]-7-desaza- 2'-desoxy-5'-0-(4,4'-dimethoxytriphenylmethyl)-9-3-Dribofuranosylpurin-6-one.

g. (3.8 mMole) 7-desaza-2'-desoxyguanosine is evaporated twice with dry pyridine and then suspended in ml. pyridine. 4.0 g. (11.8 mMole) 4,4'-dimethoxytriphenylmethyl chloride and 2.5 (14.6 mMole) Hinig base (N-ethyldiisopropylamine) are added thereto and the reaction mixture is stirred for 3 hours at ambient 0 6 temperature.

The reaction mixture is subsequently introduced into 150 ml. of a 5% aqueous solution of sodium S 20 bicarbonate and extracted twice with 150 ml. amounts of dichloromethane. The combined organic extracts are dried Sover anhydrous sodium sulphate, filtered and chromatographed on silica gel 60 H (column 10 x 4 cm., dichloromethane/acetone 9:1 After evaporation of the main zone, the residue is dissolved in a little dichloromethane and added dropwise to a mixture of n-hexane/diethyl ether (1:1 After filtration, there are obtained 2.04 g.

r, -16- (61% of theory) of the desired colourless, amorphous compound. TLC (silica gel, dichloromethane/acetone (8:2 Rf 0.7; UV (methanol: max 272, 283 nm (shoulder) 18800, 16400).

1 H-NMR ([D 6 ]DMSO): 1.75 1.86 2 3.09 3.79 4.10 5.19 3'-OH, J 4.3 Hz), 5.61 (pt, J 6.5 Hz), 6.16 6-H, J 3.5 Hz), 6.62 5-H, J 3.5 Hz), S10.35 NH).

i 10 Analysis for C 53

H

50

N

4 0 8 871.0) calc.: C 73.07; H 5.79; N 6.43 found: 73.02; 5.98; 6.34 b) 2-[(4,4'-Dimethoxytriphenylmethyl)-amino]-7-desaza- 2'-desoxy-3'-0-phenoxythiocarbonyl-5'-0-(4,4'-dimethoxytriphenylmethyl)-9-p-D-ribofuranosylpurin-6-one.

A suspension of 1.0 g. (1.1 mMole) of the compound of la) in 15 ml. dry acetonitrile is mixed with 300 mg.

mMole) E-dimethylaminopyridine and 300 pi. (2.2 mMole) phenoxythiocarbonyl chloride and stirred for 16 20 hours at ambient temperature. The reaction mixture is evaporated and the residue chromatographed on a silica gel 60 H column (column 10 x 4 cm., dichloromethane/ acetone; 8:2 The residue obtained by evaporation of the main zone is dissolved in a little dichloromethane and precipitated out by the dropwise addition of a mixture of n-hexane/diethyl ether (1:1 v/v) to give 0.99 g. (89% of theory) of a colourless, amorphous 1 -17substance. TLC (silica gel, methylene dichloride/ acetone (8:2 Rf 0.8; UV (methanol): 7 mx 269, 282 nm (shoulder) (E 19300, 16000).

1 H-NMR ]DMSO): A=2.06 (in, 2 2.34 (in, 2 '-Ha) 3.26 (mn, 4.25 (in, 5.61 (in, 3'-H and 6.23 6-H, J 3.5 Hz), 6.67 5-H, J 3.5 Hz), 10.41 NH).

Analysis for C 60

H

54 N 4 0 9 S 1007.2) caic. C 71.77, Hi 5.40, N 5.56, S 3.18 found: 71.26, 5.43, 5.52, 3.11 c) 2

-I.

4 4 -Dimethoxytriphenylmethyl)-aininol-7-desaza- 2' 3 '-didesoxy-5'-0-(4,4'-dinethoxytriphenylmethyl)- 3 -D-ribofuranosylpurin-6-one.

500 mg. (0.5 inMole) of the compound of lb) in 20 ml.

15 freshly distilled toluene are mixed with 30 mng. (0.2 mMole) 2,2'-azo-bis-(2-inethylpropionic acid nitrile) and 300 pli. (1.1 mMole) tributyl tin hydride and stirred for 3 hours under an atmosphere of argon at 80'C. (TLC monitoring, chloroform/methanol 97:3 After 0 20 completion of the reaction, the reaction mixture is evaporated and the residue chromatographed on silica gel H (column 30 x 4 cm.; dichloroinethane/inethanol 99:1 After evaporation of the main zone and taking up in a little dichloromethane, 320 mng. (75% of theory) of the desired amorphous, colourless compound is precipitated out by dropping into n-hexane/diethyl ether. TLC (silica gel, mnethylene chloride/methanol, 95:5 v/v): Rf r '1 -18- H-NMR ([D6]DMSO): 1.63, 1.80 (2 m, 2'-H and 3.07 4.06 5.43 6.11 6-H, J 3.5 Hz), 6.65 5-H, J 3.5 Hz), 10.34

NH).

d) 2-Amino-7-desaza-2',3'-didesoxy-9--D-ribofuranosylpurine-6-one.

300 mg. (0.35 mMole) of the compound from Ic) are dissolved in 10 ml. 80% acetic acid and stirred for minutes at ambient temperature. Subsequently, the solvent is stripped off at oil pump vacuum and the residue evaporated several times with water. The crude product is chromatographed on silica gel 60H (column x 4 cm., dichloromethane/methanol 9:1 The foamy substance obtained by evaporation of the main fraction is crystallised from a little methanol to give mg. (57% of theory) of colourless needles; m.p.

228 0 C. (decomp.). TLC (silica gel, dichloromethane/ methanol 9:1 Rf 0.3.

UV (methanol): Amax 261, 281 nm (shoulder) (E 13300, S 20 7800).

1 H-NMR ([D 6 ]DMSO): S 1.96 2.08, 2.27 1i (2 m, 2 '-Ha and 2 3.48 3.97 S4.86 5'-OH, J 5.4 Hz), 6.12 (pt, J 5.5 Hz), 6.24 NH 2 and 6.92 5-H, J 3.5 Hz), 10.34

NH).

Analysis for C 11

H

14

N

4 0 3 250.3) calc. C 52.79, H 5.64, N 22.39 found 52.98, 5.80, 22.55 4 7 19- I III t t dl r I, If 44 L w 4t4- 4 0# 9 0 O 00 0O 04 4 4~# In an analogous manner, via the corresponding 2'desoxynucleosides and subsequent deoxygenation as in c), .there are obtained the following compounds: A) 3,7-didesaza-2' ,3'-didesoxy-.9-f-D-ribofuranosylpurine UV (0.1 N HCl): )a 224, 274 nm Analysis for C 12

H

14 N 2 0 2 218.2) caic. C 66.0, H 6.4, N 12.8 found 66.1, 6.4, 12.6 10 B) 3,7-didesaza-2' ,3'-didesoxy-9-13-D-ribofuranosylpurin-6-one UV (methanol) max 264 nm E= 11600) 282 nm(= 8000), 295 nm (E 5200) Analysis for C 12

H

14 N 2 0 3 234.2) 15 calc. C 61.5, H 6.0, N 11.95 found 61.3, 6.1, 11.8 C) 2-chloro-6-methoxy-3,7-didesaza-2' ,3'-didesoxy-_9-0- D-ribofuranosylpurine UV (methanol): ma 271, 280 nm Analysis for C 13

H

15

N

2 0 3 C 282.6) caic. C 55.2, H 5.3, N 9.9 found 55.1, 5.3, 9.9 D) 6-amino-3,7-didesaza-2' ,3'-didesoxy-9-13-D-ribofur ano syl purime Analysis for C 12H 15N 302 233.2) calc.. C 63.65, H 6.16 N 17.13 found 63.62, 6.11, 17.01

I

4rA UV (methanol) S, max =271 nm S. 12800) E) 3,7-didesaza-2' ,3'-didesoxy-9-1 3 -D-ribofuranosylpurine- 2,6-dione Analysis for C 12

H

14 N 2 0 4 250.2) caic. C 57.55 H 5.6 N 11.2 found 57.50 5.7 11.2 Example 2.

2 -{j(Dimethylamino)-methylene3-amino>.-7-desaza-2' didesoxy-9-p-D-ribofuranosylpurin-6-one.

a) 2-[(Dimethylamino)-methylenel-amino-7-desaza-2'- 8 I I Ott desoxy-9- 3 -D-ribofuranosylpurin-6-one.

270 mg. (1.01 mMole) 7-desaza-2'-desoxyguanosine o in 5 ml. dry, amine-free dimethylformamide are mixed with 2 ml. (11.7 mMole) N,N-dimethylformnamide diethyl acetal and stirred for 1 hour at 50'C. under an atmosphere of 8 argon. Subsequently, the reaction mixture is evaporated 1 0 in a vacuum and the residue chromatographed on silica gel 60 H (column 10 x 4 cm., dichloromethane/methanol to 9:1 By evaporation of the solvent, from the main zone there are obtained 230 mg. of theory) of a a t pale yellow, amorphous substance. TLC (silica gel, dichloromethane/methanol 9:1 R f 0.3.

UV (methanol): S.ma 240, 311 nm 18300, 17400).

1 H-Nt4R (D 6 ]DMSO: ~=2.15 (in, 2 2.41 (in, 2'-H a), 3.02, 3.15 2 CH 3 3.52 (in, 3.79 (mn, 4.32 (in, 4.91 5'-OH, J 5.4 Hz), 5.27 3'-OH, J 3.5 Hz), 6.34 6-H, J 3.5 Hz), -21- 6.45 (pt, J 7.0 Hz), 7.07 5-H, J 3.5 Hz), 8.56 NH=C), 11.04 NH).

Analysis for C 14

H

19

N

5 0 4 321.3) calc. C 52.33, H 5.96, N 21.79 found 52.48, 6.14, 21.69 b) 2-{[(Dimethylamino)-methylene]-amino}-7-desaza-2'desoxy-5'-0-(4,4'-dimethoxytriphenylmethyl)-9-0-Dribofuranosylpurin-6-one.

100 mg. (0.31 mMole) of the compound from 2a) are dissolved in 2 ml. dry pyridine, mixed with 170 mg.

mMole) 4,4'-dimethoxytriphenylmethyl chloride and 0.2 ml. (1.2 mMole) Hunig base and stirred for 3 hours at ambient temperature. Subsequently, the reaction mixture is evaporated and the residue chromatographed on silica gel 60 H (column 10 x 2.5 cm., elution agent chloroform/methanol 99:1 The residue obtained by Sevaporation of the main fraction is dissolved in methylene chloride and, by dropping into a mixture of n-hexane/diethyl ether (1:1 160 mg. (84% of theory) of a colourless, amorphous substance are precipitated out. TLC (silica gel, methylene chloride/ S methanol 9:1 Rf 0.6.

UV (methanol): max 236, 311 nm (E 38200, 18100).

1 H-NMR ([D 6 ]DMSO): S= 2.23 2 2.42 2'-Ha), 3.03 CH 3 3.14 5'-H and CH 3 3.90 4.33 5.34 3'-OH, J 4.3 Hz), 6.34 (d, 6-H, J 3.5 Hz), 6.49 (pt, J 6.8 Hz), 6.90 (d,

I'.

-22- J 3.5 Hz), 8.58 NH=C), 11.07 NH).

Analysis for C 35

H

3 7

N

5 0 6 623.7) calc. C 67.40, H 5.98, N 11.23 found 67.31, 6.00, 11.17 c) 2-{[(Dimethylamino)-methylene]-amino)-7-desaza-2'desoxy-3'-0-phenoxythiocarbonyl-5'-0-(4,4'-dimethoxytriphenylmethyl)-9-0-D-ribofuranosylpurin-6-one.

900 mg. (1.4 mMole) of the compound from 2b), dissolved in 15 ml. dry dichloromethane, are mixed with 340 mg. (2.8 mMole) p-dimethylaminopyridine and 250 pl.

(1.8 mMole) phenoxythiocarbonyl chloride and stirred for o 16 hours at ambient temperature. The solution is evaporated in a vacuum and the residue chromatographed on Q040 silica gel 60 H (column 20 x 4 cm., chloroform/acetone 7:3 The residue obtained by evaporation of the main zone is taken up in a little dichloromethane and 64 ooo the desired colourless, amorphous compound precipitated out by dropping into n-hexane/diethyl ether (1:1 v/v).

TLC (silica gel, methylene chloride/methanol 95:5 v/v): R f UV (methanol): 235, 277 (shoulder), 283, 312 nm max (e 41300, 11400, 12600, 17000).

1 H-NMR ([D 6 ]DMSO): S= 2.73 2 2.97 2'-H 3.01, 3.10 2 CH 3 3.37 4.33 5.90 6.40 6-H, J 3.5 Hz), 6.55 (pt, 6.98 5-H, J 3.5 Hz), 8.58 CH=N), 11.30 NK).

i 5"1~ -r LI -23- Analysis for C 4 2

H

41

N

5 0 7 S 759.9) calc. C 66.39, H 5.44, N 9.22, S 4.22 found 66.49, 5.55, 9.25, 4.29 d) 2-{[(Dimethylamino)-methylene]-amino}-7-desaza- ?',3'-didesoxy-5'-0-(4,4'-dimethoxytriphenylmethyl)- 9-3-D-ribofuranosylpurin-6-one.

500 mg. (0.7 mMole) of the compound from 2c), dissolved in 20 ml. freshly distilled toluene, are mixed with 25 mg. (0.15 mMole) 2,2'-azo-bis-(2-methylpropionic acid nitrile) and 500 pl. (1.9 mMole) tributyl tin hydride and stirred for 16 hours at 80 0 C. under an atmosphere of argon. Subsequently, the reaction mixture is evaporated under oil pump vacuum and the residue chromatographed on silica gel 60 H (column 20 x 4 cm., dichloromethane/acetone 9:1 v/v, chloroform/acetone /:3 v/v, chloroform/acetone 6:4 The residue obtained 0 by evaporation of the main fraction is dissolved in a o*00* little dichloromethane and precipitated out by dropping into n-hexane/diethyl ether to give 320 mg.'(80% of 20 theory) of the desired colourless, amorphous compound.

TLC (silica gel, methylene chloride/methanol 95:5 v/v): Rf 0.3.

UV (methanol): max. 236, 277 (shoulder), 284, 312 nm 37200, 12000, 13500, 18000).

1 H-NMR 6 DMSO): S 2.02 2.20, 2.3j (m, 2'-H a and 2 3.02, 3.13 2 CH3), 3.08 4.17 6.21 6-H, J 3.5 Hz), 6.38 r ''t -r 2 -24- 6.92 5-H, J 3.5 Hz), 8.61 CH=N), 11.03 NH).

Analysis for C 35

H

37

N

5 0 7 607.7) calc. C 69.18, H 6.14, N 11.52 found 69.23, 6.24, 11.61 e) 2-{[(Dimethylamino)-methylene]-amino}-7-desaza- 2',3'-didesoxy-9-0-D-ribofuranosylpurin-6-one.

130 mg. (0.21 mMole) of the compound from 2d) are dissolved in 80% acetic acid and stirred for 15 minutes at ambient temperature. Subsequently, the acetic acid is evaporated off under oil pump vacuum and the residue is chromatographed on silica gel 60 H (column 20 x 2 cm., dichloromethane/methanol 95:5 The residue obtained by evaporation of the main fraction is foamed up by repeated evaporation with acetone to give 43 mg.

(67% of theory) of the desired colourless, amorphous compound. TLC (silica gel, dichloromethane/methanol 9:1 Rf UV (methanol): ma 239, 282 (shoulder), 311 nm max.

17400, 10500, 16900).

1 H-NMR ([D 6 ]DMSO): 2.06, 2.32 2'-H and 3.01, 3.14 2 CH 3 3.51 4.00 4.87 6.33 l'-H and 6-H, J 3.3 Hz), 7.05 5-H, J 3.3 Hz), 8.59 CH=N), 11.02 NH).

Analysis for C 14

H

1 9

N

5 0 3 (M.W.305.3) calc. C 55.07, H 6.27, N 22.94 found 55.23, 6.41, 22.75

I

S II *r 4 Iit 4 t I Example 3.

2-Amino-6-methoxy-7-desaza-2',3'-didesoxy-9-0-Dribofuranosylpurine.

a) 2-Amino-6-methoxy-7-desaza-2'-desoxy-9-P-Dribofuranosylpurine.

543 mg. (10 mMole) finely powdered potassium hydroxide and 68 mg. (0.2 mMole) tetrabutylammonium hydrogen sulphate in 30 ml. anhydrous dichloromethane are stirred for 15 minutes at ambient temperature under an atmosphere of nitrogen. Subsequently, the reaction mixture is mixed with 330 mg. (2 mMole) 2-amino-6-methoxy- 7-desazapurine (2-amino-4-methoxy-7H-pyrrolo[2,3-d]pyrimidine) and stirred for a further 30 minutes. After the addition of 884 mg. (2.2 mMole) 2-desoxy-3,5-di-0p-toluoyl--D-erythro-pentofuranosyl chloride, stirring i is continued for a further 3 minutes. Insoluble components are filtered off with suction, washed with a little I dichloromethane and the filtrate concentrated to about ml. After mixing with 3 ml. 1M sodium methoxide in methanol, stirring is continued for 3 hours at ambient temperature. After neutralisation with acetic acid, the solvent is stripped off, the residue is taken up in hot water, filtered and the filtrate chromatographed on an exchanger column of Dowex (1 x 2 OH form, 30 x 3 cm.) (water/methanol 9:1 After stripping off the solvent and recrystallising from water, from the main zone there are obtained 260 mg. (63% of theory) of -26colourless crystals; m.p. 152 15400. TLC (silica gel, dichioromethane/methanol 9:1 R f 0.7.

UV (methanol): >max. 225, 259, 285 (~=24900, 3600, 7600 1 H-NMR ([D 6 6.27 (lH, d, J 3.7 Hz), 6.42 (11-, dd, Jl', 2 'a 8.4 Hz, Jl', 2 1b 5.9 Hz), 7.10 (lH, d, J 3.7 Hz) ppm.

13CNMRQL6 52.49 (OCH 82.37 98.85 119.45 ppm.

b) The compound 2-amino-6-methoxy-7-desaza-2'-desoxy- 9-p-D-ribofuranosylpurine obtained according to a) is 0 desoxygenated in the manner described in Example lc) to give 2-amino-6-methoxy-desaza-2' ,3'-didesoxy-9-p-D- 0 0 ribofuranosylpurine.

Example 4.

0*0 2-Amino-6-chloro-7-desaza-2' ,3'-didesoxy-9-3-Dribofuranosylpurine.

a) The compound is prepared, after acetylation of 2-amino-7-desaza-2' ,3'-didesoxy-9-f3-D-ribofuranosylpurin- 6-one (prepared according to Example ld) by halogenation according to the method described in Liebigs Ann. Chem., 1987, 15 19.

b) The resulting crude mixture is, for the removal of the acetyl protective group, left to stand for 3 hours in methanolic ammonia solution at ambient temperature, then evaporated to dryness and finally c).romatographed on silica gel with the elution agent chloroform/methanol.

r 7

-U

-27- After combining the main fractions and evaporating, the residue obtained is crystallised from water.

UV (methanol): max. 235, 258, 316 (E 27800, 4300, 5800).

Analysis for C 11 H1 3

N

4 0 2 C 268.7) caic. C 49.1, H 4.8, N 20.8, Cl 13.1 found 49.3, 4.85, 20.7, 13.: 0 1 Example 2-Amino-7-desaza-2' ,3'-didesoxy-9-3-D-ribofuranosvl-

I

0* S46 A 4 S4, purine.

268 mg. (1 mMole) 2 -amino-6.zhloro-7-desaza-2',3'didesoxy-9-f3-D-ribofuranosylpurine are dissolved in 25 ml. 70% aqueous methanol, added to a suspension of mg. prehydrogenated Pd/C in 25 ml. 70% aqueous 15 methanol and hydrogenated until the take up of hydrogen is complete. The solvent is stripped off and the residue is crystallised from methanol. Yield 180 mg. (77% of theory).

Analysis for C 11

H

14 N 4 0 2 234.3) calc. C 56.4, H 6.0, N 23.9 found 56.3, 6.0, 23.7 UV (methanol): max. 234, 256, 314 nm (E6 30600, 4100, 5200).

Example 6.

2-Amino-6-mercapto-7-desaza-2' ,3'-didesoxy-9-p-Dribofuranosylpurine.

536 mg. (2 mMole) 2-amino-6-chloro-7-desaza-2' t

I

7 l -28didesoxy-9-P-D-ribofuranosylpurine and 1.5 g. (20 mMole) thiourea are suspended in 30 ml. ethanol and heated under reflux for about 15 hours. Thereafter, the solvent is distilled off, the residue is taken up in about 25 ml. methanol and then chromatographed on silica gel 60 H (column 20 x 3 cm., dichloromethane/methanol 9:1 By evaporation of the main fraction and crystallisation from methanol/water, there are obtained 230 mg. (43% of theory) of the thio compound.

S.o 10 Analysis for C 11

H

14

N

4 0 2 S 266.3) calc. C 49.6, H 5.3, N 21.0 o S. found 49.4, 5.4, 21.1 UV (methanol): 235, 271, 345 nm( E 176000, max.

11700, 18700).

1 H-NMR ([D 6 ]DMSO): 1.9 2.1 2'-Hb), 2.34 2'-H 3.50 3.97 4.86 OH), 6.12 6.24 NH 2 and 8-H), 6.92 11.1 NH).

Example 7.

S 20 2,6-Diamino-7-desaza-2',3'-didesoxy-9-3-D-ribofuranosylpurine.

268 mg. (1 mMole) 2-amino-6-chloro-7-desaza-2',3'didesoxy-9-p-D-ribofuranosylpurine are mixed with 40 ml.

aqueous concentrated ammonia solution and heated for hours at 65 0 C. on a waterbath in a tightly closed vessel.

After evaporation of the solvent, the residue is chromatographed on a silica gel column, first with -29dichioromethane/methanol (9:1 v/v) (starting material) and then with chloroform/methanol (4:1 After crystallisation from water, there are obtained 120 mg.

(48% of theory) of the 'desired diamino compound.

Analysis for 0 11

H

15

N

5 0 2 249.3) caic. C 53.0, H 6.0, N 28.1 found 53.15, 5.9, 28.2 UV (methanol): max. 264, 284 nm (E =9800, 8000).

1 H-NMR 6 1DMSO: (in, 2.1, 2.4 (2 m, 21 -Hab), 3.4 (in, 3.8 (in, 4.8 5.6 NH 2 6.2 (dd, 6.3 6.7 NH 2 6.9 8-H).

feet tExample 8.

2-Methylthio-6-methoxy-7-desaza-2' ,3'-didesoxy-9-03- D-ribofuranosylpurine.

a) 2-Methylthio-6-methoxy-7-desaza-2'-desoxy-9-p-Dribofuranosylpurine.

500 mg. (2.56 inMole) 4-Methoxy-2-methylthio-7Hpyrrolo[2,3-dlpyrimidine and 400 mg. (1.75 inMole) 20 benzyltriethylanronium chloride are dissolved in 20 ml.

dichloromethane with 20 ml. of a 5% aqueous solution of sodium hydroxide as counterphase and briefly mixed up in a vibratory mixer. 1.2 g. (3.1 inMole) 2-desoi. di-0-( 2 toluoyl)-f-D-erythro-pentofuranosyl e in a little dichlorornethane, is added theret t' he vibratory mixing continued for 30 minutes. The organic phase is separated off and the aqueous phase shaken up with dichloromethane. The combined organic extracts are washed with water and dried with anhydrous sodium sulphate. After filtration, the filtrate is evaporated and the residue dissolved in 100 ml. IM sodium methanolate in methanol. The solution is stirred for about 12 hours at ambient temperature, then evaporated and the residue is taken up in water and adsorbed on a Dowex 1-X2 ion exchanger column (30 x 3 cm., OH form).

Elution with water-methanol (1:1 v/v) gives a main zone.

10 After evaporation of the solvent, the residue is recrystallised from water; yield 321 mg. (40% of theory) of colourless needles; m.p. 118 0 C. TLC (silica gel; t *dichloromethane/acetone 8:2 R 0.26.

UV (methanol): max. 283, 236 nm (E 13000, 15500).

1 H-NMR ([D 6 ]DMSO): S 2.20 2.40 o{ *2.56 CH3S), 3.50 5'-H 2 3.81 4.01 S(s, CH 3 4.35 4.90 5'-OH, J 5 Hz), 5.29 3'-OH, J 4 Hz), 6.48 5-H, J 4 Hz), 6.55 J 5 Hz), 7.47 6-H, J 4 Hz).

S, 20 Analysis for C 1 3

H

17

N

3 0 4 S 311.4) calc. C 50.15, H 5.50, N 13.50, S 10.30 found 50.28, 5.47, 13.56, 10.31 b) 2-Methylthio-6-methoxy-7-desaza-2',3'-didesoxy-9- P-D-ribofuranosylpurine.

This is prepared by deoxygenation of the 2'-desoxy compound obtained according to a) in the manner described in Example Ic).

-31- UV (methanol): >ma.=283, 236 1300, 15500) Analysis for C0 13

H

17

N

3 0 3 S 295.4) caic. C 52.8, 11 5.75, N 14.2 found 52.6, 5.70, 14.2 Example 9.

6-Methoxy-7-desaza-2' ,3'-didesoxy-9-0-D-ribofuranosyrlpurine.

a) 6 -Methoxy-7-desaza-2'-desoxy-9-p-D-ribofuranosy..

purine.

ftr 10 The synthesis of this compound takes place in the manner described in Liebigs Ann. Chem., 1985, 1360-1366.

b) The didesoxy derivative can be obtained by deoxygenation of the compound obtained in a) in the manner described in Example 1c).

An alternative way is the desulphurisation of 2rethylthio-6-methoxy-7-desaza-2' ,3'-didesoxy-9-p-Dribofuranosylpurine from Example 8, also in the manner described in Liebigs Ann. Chem., 1985, 1360 -1366.

TLC (dichloromethane/methanol 9:1 Rf 0.8.

UV (methanol): max 261 nm (log (Z 3.86).

1 H.NHR (DMSO-d 6 2.04 (in, 2.24 (in, 2 '-Hb 2.40 (in, 2'-H 3.55 (in, 4.04 OCH 4.07 a3 (mn, 41-H), 4.93 t, J 5.5 Hz, 6.47 (dd, J 4.4 and 6.8 Hz, 6.55 J 3.7 Hz, 7,66 J 3.7 Hz, 8.42 2-H).

Analysis for C 12

H

15

N

3 0 3 249.3) calc. C 57.8, H N 16.8 found 57.8, 6.05, 16.65 -32- Another possibility for the preparation of this compound is described in Example 24i).

Example 7-Desaza-2',3'-didesoxy-9-g-D-ribofuranosylpurin-6-one.

The preparation of this compound takes place via the 2'-desoxy compound as described in Liebigs Ann.

Chem., 1985, 312-320 and subsequent deoxygenation as described in Example Ic).

TTV (methanol): X max. 258, 280 (shoulder) (E 9200, 6400).

TLC (dichloromethane/methanol 9:1 R I 1 SH-NMR (DMSO-d 6 S 2.00 2.16 2 .Ot 2.37 2 3.49 (dd, J 4.9 and 11.6 Hz, 3.58 (dd, J 4.2 and 11.6 Hz, 4.05 6.33 (dd, J 4.2 and 6.9 Hz, 6.50 J 3.5 Hz, '441 7.36 J 3.5 Hz, 7.90 2H).

Analysis for C 11

H

1 3

N

3 0 3 235.2) °40 calc. C 56.1, H 5.5, N 17.8 found 56.0, 5.3, 18.0 20 A further possibility for the preparation of this compound is described in Example 24j).

Example 11.

7-Desaza-2',3'-didesoxy-9-0-D-ribofuranosylpurine- 2,6-dione.

The synthesis of this compound takes place via the 2'-desoxy compound as described in Liebigs Ann. Chem., 1985, 312-320 and subsequent deoxygenation as described in Example Ic).

f i -33-, 1£ we' 44t 461(55 4 1 p *4 4 4 8 4* o 9.

@4 0 0 @0 444.

o *4 0 4e UV (phosphate buffer; pH S ax 251, 280 rim 10500, 7400) Analysis~ for C 11

H

13 N 3 0 4 251.4) caic. C 52.5, H- 5.2, N 16.7 found 52.3, 5.1, 16.5 Example 12.

2,6-Dimethoxy-7-desaza-2 1 ,3'-didesoxy-9-0-Dribofuranosylpurine.

This derivative is synthesised by phase transfer glycosylation and subsequent deoxygenation as described in Example 1c).

UV (methanol): X~ max. 257, 271 nm (E 7300, 7400) Analysis for C 13

H

17

N

3 0 4 279.3) calc. C 55.85, H 6.1, N 15.0 15 found 55.7, 6.1 15.1 Example 13.

6-Amino-7-desaza-2' ,3'-didesoxy-9-03-D-ribofuranosylpurin-2-one.

This compound is obtained according to J. Chem.

20 Soc., Perkin Trans. 11, 1986, 525 530 by phase transfer glycosylation of 2-methoxy-6-amino-7-desazapurine, subsequent demethylation and finally deoxygenation analogously to Example 1c).

UV (methanol): max. 255, 305 nm (e 7600, 7200) Analysis for C 11H 14N 40 3(M.W. 250.2) calc. C 52.7, H 5.6, N 22.4 found 52.75, 5.5, 22.3 '-34- Example 14.

2-Amino-7-desaza-7-nethyl-2' ,3'-didesoxy-9-3-Dribofuranosylpurin-6-one.

This compound is synthesised via the 2'-desoxynucleoside described in Liebigs Ann. Chem., 1984, 708- 721 with subsequent deoxygenation as described in Example Ic).

UV (methanol): max. 224, 264, 285 nm (shoulder) (~=22500, 10500, 6500) Analysis for C 2

H

16

N

4 0 3 264.3) caic. C 54.5, H 6.05, N 21.2 found 54.3, 6.1, 21.1 Example '1 2-Amino-7-desaza-2' ,3'-didesoxy-3'-azido-9-p3-Dribofuranosylpurin-6-one.

0 This compound is prepared by glycosylation of 2amino-7-desazapurin-6-one with the azido sugar prepared 4 according to Byatkina/Az'hayev (Synthesis, 1984, 961-963).

UV (methanol): ax 261, 281 nm (shoulder) 20 (E =13300, 7800).

Analysis for C H 1 1 N 0 3 291.3) caic. C 45.3, H 4.45, N 33.65 found 45.4, 4.3, 33.4 Example 16.

3,7-Didesaza-2' ,3'-didesoxy-3'-azido-9-P-D-ribofuranosylpurine.

This compound is prepared by ribosidation of 3,7didesazapurine with the azido sugar prepared according to Byatkina/Azhayev (Synthesis, 1984, 961-963).

UV (methanol): max. 224, 274 nm.

Analysis for C 12

H

13 N 5 0 2 259.2) caic. C 55.55, H 5.0, N 27.0 found 55.4, 5.1, 26.8 Example 17.

6-Amino-8-aza-7-desaza-2' ,3'-didesoxy-9-p3-D-ribofuranosylpurine (4-amino-l-(2-desoxy-p3-D-erythro- 0 9 10 pentofuranosyl)-lH-pyrazolo[3,4-dlpyrimidine) 4-Benzoylamino-l-(2'-desoxy-9-p3-D-erythro-pentoo ~furanosyl-5 -dimethoxytriphenylinethyl )-lHpyrazolo[ 3,4-d ]pyrimidine.

6-Amino-8-aza-7-desaza-2 '-desoxy- p-D-ribofuranosyrlpurine is prepared in the manner described in Helv. Chim. Acta, 68, 563 570/1985. The benzoylation of the 4-amino group and the subsequent introduction of the dimethoxyzrityl protective group is carried out analogously to known methods.

20 b) 4-Benzoylamino-l-(2'-desoxy-j-D-erythro-pnto furanosyl -dimethoxytriphenylmethyl) 3' -0-phenoxythiocarbonyl-ll-pyrazolo[13 ,4-d 1pyrimidine.

200 mg. (0.3 mMole) of the product of Example 17a) are reacted in 4 ml. acetonitrile with 82 pl. (0.6 mMole) phenyl chlorothiocarbonate at ambient temperature for 16 hours in the presence of 90 mg. (0.75 mMole) 4- -36- (dimethylamino) -pyridine. After chromnatographic purification (silica gel, dichloroinethane/ethyl acetate 95:5 there are isolated 150 mng. of theory) of the desired product.

TLC (silica gel, dichiororiiethane/ethyl acetate, 95:5 v/v: R f 0.4.

1 H-NMTR ([D 6 ]DMSO): g=3.26 (mn, 3.69 2 x 00H 3 4.45 (mn, 5.98 (mn, 8.45 8.78 11.72 NH).

o~ It o 10 c) 4-Benzoylamiino-l-(2',3'-didesoxy-9-I 3 -D-glyceropentofuranosyl -diinethoxytriphenylinethyl) lH-pyrazolo [3 ,4-d Ipyriinidine.

&fee 200 ing. (0.25 in.Mole) of the product of Example 17b) are deoxygenated according to Barton's method in 7 ml.

toluene with 150 pl. (0.55 inMole) tri-N-butyl stannane I. at 80'C. under an atmosphere of argon. After chroinatography (silica gel, dichloromethane/ethyl acetate 95:5 there are obtained 120 mg. (757% of theory) of the desired colourless and amorphous product.

TLC (silica gel, dichloromethane/ethyl acetate 95:5 v/v): R =O0.

3 1 H-NMR [D 6 ]DMSO): g=2.16 (in, 2.49 (mn, 2.99 (mn, 3.65, 3.68 (2s, 2 x OCH 3 4.32 (in, 6.69 (in, 8.41 8.80 6-H), 11.66 NH).

d) 6-Aiino-8-aza-7-desaza-2' ,3'-didesoxy-9-03-D-ribofuranosylpurine (4-amino-l-(2' ,3'-didesoxy-P-Dglyceropentofuranosyl)-lH-pyrazolol3,4-dlpyrimidine)

LA

-T -r -37a) 300 mg. (0.47 mMole) of the product of Example' 17c) are treated in 40 ml. ammonia-saturated methanol at 600C. for 4 hours and then evaporated to dryness. There are obtained 200 mg. (81% of theory) 4-amino-l-(2',3'didesoxy-3-D-glyceropentofuranosyl)-5'-0-(4,4'dimethoxy-triphenylmethyl)-lH-pyrazolo[3,4-d]pyrimidine in the form of a colourless foam after chromatography on silica gel (dichloromethane/acetone 7:3 v/v).

TLC (silica gel, dichloromethane/acetone 8:2 v/v): S 10 R 0.25.

SH-NMR ]DMSO): S= 2.16 2.45 d6 S 2.99 3.69, 3.70 (2s, 2 x OCH 3 4.25 6.52 7.74 NH 2 8.06 8.24 6-H).

b) 110 mg. (0.2 mMole) of the above product are ao stirred for 20 minutes at ambient temperature in 10 ml.

80% acetic acid. After chromatography (silica gel, gdichloromethane/methanol 9:1 there is obtained the desired product in crystalline form. Subsequent re- 20 crystallisation from isopropanol/cyclohexane gives 40 mg.

of theory) of the desired product as a colourless solid.

UV (methanol): max. 260, 275 nm 9000, 10200).

Analysis for C10H13H502 235.25) calc. C 51.06, H 5.57, N 29.77 found 50.96, 5.65, 29.80

I

-38- 13 C-NMR 6 1DMSO): ~=133 100.3 158.1 156.1 153.6 84.4 30.4 27.4 81.7 64.3 TLC (silica gel, dichiorotnethane/inethanol 9:1 v/v): R f 0.4.

UV (methanol): max. 260, 275 un(E 9000, 10200 1 H--NMR ([D 6 ]DMSO): g=2.11 (in, 2.40 (in, 3.36 (in, 4.08 (in, 4.75 (in, 6.45 (in, 7.75 NH 2 8.14 8.13 6-H).

Example 18.

a) 4,6-Dichloro-l-(2'-desoxy-3' ,5'-di-0-p-toluoyl-p3- D-erythro-pento'urano'3yl)-lH-pyrrolo[3,2-clpyridine.

A solution of 300 mg. (1.6 mMole) 4,6-dichloro-lHpyrrolo[3,2-clpyridine in 75 ml. dry acetonitrile, which contains 450 mng. (8.0 mMole) potassium hydroxide and mng. (0.1 mMole) tris-112-(2-methoxyethoxy)-ethyl]amine, is stirred at ambient temperature for 30 minutes under an atmosphere of nitrogen. While stirring, 625 mg.

(1.6 inMole) c-chloro-2-desoxy-3,5-di-0-p-toluoyl-Derythro-pentofuranose are added thereto and stirring continued for 15 minutes. Insoluble material is then filtered off and the filtrate is evaporated in a vacuum.

j4 The oily residue is chromatographed on silica gel (column 17 x 4 cm., elution agent dichloromethane/ethyl acetate 97:3 There are obtained 762 mg. (90% of theory) of the colourless, amorphous product.

IH-NMR (Me 2 SO-d 6 =2.37 and 2.41 (2s, 2 CH 3 2.77 -39- 2.94 (in, 4.57 (mn, H1-4', 5.68 (in, 6.66 11-1), 6.71 J 3.5 Hz, H1-3), 8.00 H-7).

13 CMR(eS-') 36.8 64.2 74.9 81.7 85.6 102.0 106.1 123.1 129.7 140.0 140.6 142.4 (C-7a).

b) 4, 6-Dichloro-l-(2'-desoxy-Q-D-erythro-pentofuranosyl )-lH-pyrrolo[ 3,2-clpyridine.

500 mg. (0.93 inMole) of the compound of Example 18a) are dissolved in 30 ml. methanolic ammonia and stirred for 12 hours at' 5000. The solution is evaporated to dryness, the solid residue is adsorbed in silica gel H (2 and applied to a silica gel column (14 x 4 cm., 0 fai 15 elution agent chloroform/methanol 9:1 vr/v). From the U main fraction there is isolated the desired product in the form of a colourless oil which crystallises from~ Ii aqueous ethanol in the form of colourless needles.

Yield 101 mg. of theory); m.p. 18000.

1 11-NMR (Me 2 SO-d 6 2.28 (in, 11-2's), 2.43 (mn, H-2'a), 3.56 (in, 3.85 (mn, 4.38 (mn, 5.02 J 5.2 Hz, 5.34 J =4.1 Hz, 6.42 (pt, 6.67 J 3.4 Hz, '39 (d, J 3.4 Hz, 11-2), 7.96 11-7).

13C-NM (Me 2 SO-d 6 g= 40.6 61.5 70.5 85.5 87.6 101.3 106.1 123.1 129.7 139.7 140.4 142.0 (C-7a).

c) 4-Amino-6-chloro-l-(2'-desoxy--D-erythro-pentofuranosyl)-1H-pyrrolo[3,2-c]pyridine.

460 mg. (1.52 mMole) of the compound of Example 18b) are dissolved in 6 ml. dry hydrazine and heated to 800C. for 60 minutes. The hydrazine is removed under a vacuum and the oily residue evaporated twice with, in each case, 10 ml. ethanol. The residue is dissolved in ml. aqueous ethanol and then 2 g. Raney nickel are added thereto and the mixture heated to the boil for 2 hours, while stirring. The catalyst is filtered off and thoroughly washed with hot aqueous ethanol. The filtrate is evaporated to dryness, the residue is dissolved in methanol, adsorbed on 2 g. silica gel and the solvent removed under a vacuum. This silica gel is suspended in chloroform/methanol (9:1 v/v) and applied to a silica gel column (6 x 3 Elution with chloroform/ methanol (9:1 v/v) gives a colourless syrup from which, by crystallisation from methanol, the product can be obtained in the form of small, colourless crystals; j 20 m.p. 232 0 C. Yield: 207 g. (48% of theory).

TLC (chloroform/methanol 9:1 Rf 0.2.

UV (methanol): a 277 nm (E 14800 285 nm max.

13800).

1 H-NMR (Me 2 SO-d 6 2.20 2.40 H-2'a), 3.51 3.78 4.32 4.89 J 5 Hz, 5.26 J 4 Hz, 6.19 (pt, 6.55 NH 2 6.64 J 3 Hz, H-3), 6.83 7.36 J 3 Hz, H-2).

1 -41- 13 0C-NMR (Me 2 SO-d 6 40 61.8 70.6 84.7 87.2 95.1 101.6 109.6 123.5 141.0 141.4 152.9 Analysis frC12 14 N3 03 Acalc. C 50.80, H 4.97, N 14.81, C1 12.50 found 50.91, 5.05, 34.75, 12.53 d) 4-Amino-l-(2'-desoxy-o3-D-erythro-pentofuranosyl)lH-pyrrololl3,2-clpyridine.

A solution of 200 mg. (0.7 mMole) of the compound from Example 18c) in 30 ml. methanol, which contains 0.4 ml. of ammonia-saturated methanol, is hydrogenated in the presence of palladium/charcoal (50 mg., 10%~ Pd) at ambient temperature for 30 hours. The catalyst is filtered off and the solvent removed in a vacuum.

Purification by flash chromatography (column 4 x 4 cm., I elution agent chloroform/methanol/triethylamine 7:3:2 v/v/v) and crystallisation from methanol gives 70 mg.

of theory) of the desired product in the form of colourless crystals; m.p. 205'C.

TLC (elution agent chloroform/mtethanol/triethylamine 7:3:2 R f 0.4.

UV (methanol): max. 271 nm E 12800).

1 HNM (Me 2 SO-d 6 8 2.20 (in, 2.42 (in, H-2'a), 3.51 (mn, 3.80 (mn, 4.32 (in, 4.91 (in, 5.32 (mn, 6.08 NH 2 6.23 (pt, 6.65 J 3 Hz, 6.75 J =6 Hz, H-7), C_ .iax- al II- i~L s- -i" r *t^'-sr 2 -42- 7.35 J 3 Hz, 7.55 J 6 Hz, H-6).

1 3 C-NMR (Me 2 SO-d 6 S 39.8 62.0 70.8 84.5 87.1 96.9 101.5 110.7 122.5 139.7 140.0 153.7 Analysis for C 1 2

H

1 5

N

3 0 3 calc. C 57.82, H 6.07, N 16.86 found 57.97, 6.12, 16.74 Example 19.

a) 6-Chloro-l-(2'-desoxy-o-D-erythro-pentofuranosyl)- 1H-pyrrolo[3,2-c]pyridin-4-one.

A solution of 400 mg. (1.32 mMole) of the compound of Example 18b) is heated to the boil for 30 hours in 2N aqueous sodium hydroxide solution with a small amount of 15 1,4-dioxan. The reaction mixture is neutralised with 2N hydrochloric acid, filtered and then applied to an Amberlite XAD 4 column (17 x 2 Inorganic salts are removed by washing with water and then the product is eluted with methanol. Crystallisation from water gives 158 mg. (42% of theory) of colourless crystals; m.p. 242 243 0

C.

TLC (chloroform/methanol 8:2 R UV (methanol): max 270 nm (E 11100), 292 nm 9300).

1 H-NMR (Me 2 SO-d 6 2.2z 2.38 H-2'a), 3.53 3.80 4.33 4.96 5.29 6.22 (pt, H- 6.54 (d, t r t ir I It I I t I II II I 111£ I~ 1: -43- J 3.3 Hz, 6.96 7.38 J 3.3 Hz, 11.81 (br. NH).

13 -NMR (Me SO-d 6 40.5 61.7 70.6 85.0 87.4 94.9 (C-7)1, 104.1 114.0 123.2 129.1 139.2 158.7 Analysis for C 12 H, 3 C1N 2 0 4 caic. :C 50.63, H 4.60, N 9.84, Cl 12.45 found 50.79, 4.74, 9.80, 12.69 b) 1-(2'-Desoxy-f3-D-erythro-pentofuranosyl)-IHpyrrolo[3,2-cjpyridin-4-one.

A solution of 100 mg. (0.35 mMole) of the compound of Example 19a) in 15 ml. methanol is mixed with 0.5 ml.

aqueous ammonia solition and hydrogenated in the presence of palladium/animal charcoal (10% Pd, 15 mrg.) for 3 hours at ambient temperature. The catalyst is filtered off and the filtrate evaporated to dryness.

The solid residue is crystallised from water. There are obtained 51 mg. (58% of theory) of the desired product; 420 m.p. 147 -14800.

TLC (elution agent chloroform/methanol 8:2 v/v): R f 0.3.

UV (methanol): max. 264 nm (E 11700), 282 nrn (sh, E 8000), 295 nm (sh, E 5100).

1 H-NMR (Me 2 SO-d 6 9 2.22 (in, 2.40 (mn, H-2's), 3.52 (mn, 3.81 (mn, 4.32 (in, 4.93 J 5.4 Hz, 5.32 H 4.3 Hz, 3'-OH), 'SiJfi 1 -44- 6.21 (pt, 6.54 J 3 Hz, 6.62 (d, J 7 Hz, 7.03 J 7 Hz, 7.34 J 3 Hz, 10.87 (br NH).

13C-NMR (Me 2 SO-d 6 g 40 superimposed by solvent signals), 61.8 70.7 84.8 87.4 93.8 104.6 115.9 122.0 127.8 139.0 159.6 Analysis for C 1 3

H

1 4

N

2 0 4 calc. C 59.08, H 6.10, N 10.60 found 59.09, 6.07, 10.65 Example a) 1-(2'-Desoxy-P-D-erythro-pentofuranosyl)-4,6dichloro-5'-0-(4,4'-dimethoxytrityl)-1H-pyrrolo- [3,2-c]pyridine.

500 mg. (1.65 mMole) of the compound of Example 18b) are evaporated to dryness with 10 ml. pyridine.

The material is dissolved in 10 ml. dry pyridine and 0.7 ml. (4.1 mMole) of Hunig's bases, as well as 690 mg. (2.0 mMole) 4,4'-dimethoxytrityl chloride, added thereto. The solution is stirred for 1 hour at ambient temperature. After the addition of 75 ml. of 5% aqueous sodium bicarbonate solution, it is extracted twice with, in each case, 75 ml. dichloromethane. The combined organic phases are dried over anhydrous sodium sulphate.

The sodium sulphate is filtered off and the filtrate evaporated. The residue is applied to a silica gel column (30 x 3 cm.; elution agent dichloromethane/

I

I

acetone 99:1 The product is obtained from the main fraction in the form of a yellowish amorphous mass. The product is dissolved in diethyl ether and precipitated out with n-hexane. Yield 740 mg. (74% of theory).

H-NMR (Me 2 SO-d 6 S 2.39 2.64 H-2'a), 3.09 3.72 2 OCH 3 3.96 4.42 5.41 J 4.8 Hz, 6.47 (pt, 6.65 J 3.5 Hz, 6.76 7.27 (aromat. 7.76 J 3.5 Hz, 7.89 H-7).

1 3 C-NMR (Me 2 SO-d 6 S 40 superimposed by solvent signals), 55.1 (2 OCH 3 63.6 70.05 85.0 85.5. 85.5 OCDMT), 101.3 106.2 i 123.2 129.1 139.8 140.5 142.3 (C-7a).

Analysis for C 33

H

3 0 C12N205 calc. C 65.46, H 4.99, Cl 11.71, N 4.63 found 65.47, 5.09, 11.78, 4.56 b) l-(2'-Desoxy-3-D-erythro-pentofuranosyl)-4,6dichloro-5'-0-(4,4'-dimethoxytrityl)-3'-0-phenoxythiocarbonyl-lH-pyrrolo[3,2-c]pyridine.

300 mg. (0.5 mMole) of the compound of Example are dissolved in 11 ml. dry acetonitrile and 350 mg.

(2.8 mMole) 4-dimethylaminopyridine and 150 pl. (1.1 mMole) phenyl chlorothiocarbonate added thereto and the solution is stirred for 16 hours at ambient temperature.

The reaction mixture is subsequently evaporated to dryness in a vacuum. The residue is chromatographed on

L

p 7-^ -46silica gel (elution agent dichloromethane). The colourless product is isolated from the main fraction.

Yield 310 mg. (84% of theory).

1H-NMR (Me 2 SO-d 6 S 2.92 3.35 3.72 2 OCH 3 4.43 5.89 6.61 (pt, 6.71 J 3.5 Hz, 6.81 7.52 (aromat. 7.76 J 3.5 Hz, 8.01 H-7).

13 C-NMR (Me 2 SO-d 6 S 37.0 5j.. (2 OCH 3 63.8 83.0, 84.2, 85.6, 86.0 OCDMT), 101.8 106.3 123.1 128.9 140.1 140.6 142.4 193.8 Analysis for C 40

H

34 C1 2

N

2 0 6

S

calc. C 64.78, H 4.62, Cl 9.55, N 3.77, S 4.32 I found 64.66, 4.59, 9.65, 3.70, 4.40 1 15 c) 4,6-Dichloro-l-(2',3'-didesoxy--D-glyceropentofuranosyl)-5'-0-(4,4'-dimethoxytrityl)-1H-pyrrolo- [3,2-c]pyridine.

170 mg. (0.23 mMole) of the compound of Example b) and 15 mg. (0.1 mMole) 2,2'-azo-bis-(2-methyl)propionitrile are dissolved in 10 ml. dry toluene under an atmosphere of argon. 140 il1. (0.51 mMole) tri-n-butyl stannane are added thereto, while stirring, and the reaction mixture is then further stirred for 3 hours at 8000C. The solvent is removed under a vacuum and the residue chromatographed on silica gel (elution agent dichloromethane). From the main fraction are isolated 115 mg. (85% of theory) of the desired product.

I i iii ii ill Ililllll. ilil. n l.m 1 I 1^ i 1 K M iill i ilii r i III! '-47- IH-NWIR (Me 2 SO-d 6 S'2.05 2.50 (H-21, superimposed by signals of the solvent), 2.90 3.15 (in, 4.25 (mn, 6.38 (mn, H-i 1 6.63 J 3.4 Hz, 6.69 7.30 (aroinat. 7.79 J 3.4 Hz, 7.89 H-7).

d) 2,6-Dichloro-3,7-didesaza-2' ,3'-didesoxy-9-03-Dribofuranosylpurine.

The dimethoxytrityl protective group is removed from the compound of Example 20c) analogously to Example 24f).

e) 6-Aiino-3,7-didesaza-2' ,3'-didesoxy-9-P-Dribofuranosylpurine.

The compound of Example 20d) is treated with hydrazine and subsequently reduced with Raney nickel in the manner described in Example 18c). There is thus obtained the compound described in Example 1lD).

f) 3,7-Didesaza-2' ,3'-didesoxy-9-0-D-ribofuranosylpurine.

The compound of Example 20d) is hydrogenated in the presence of palladium/animal charcoal/hydrogen analogously to Example 24g). There is obtained the compound already described in Example IA).

g) 3,7-Didesaza-2' ,3'-didesoxy-9-f3-D-ribofuranosvlpurin-6-one.

The compound of Example 20d) is treated with an aqueous solution of sodium hydroxide in the manner described in Example 19a) and subsequently hydrogenated -48in -the manner described in Example 19b). There is thus obtained the compound already described in Example lE) V Example 21.

2-Amino-(2' ,3'-didesoxy-p3-D-glyceropentofuranosyl)lH-pyrazolo[3,4-dlpyrimidin-4-one.

This compound is prepared analogously to the method described in Example 17 via 2-amino-(2'-desoxy-9- 3

-D-

ribofuranosyl)-1H-pyrazolo[3,4-dlpyrimidin-4-one and Barton deoxygenation of 2-amino-(2'-desoxy-3'-0-methoxy- [3,4-dlpyrimidin-4-one; m.p. 221'C.

Analysis for C H H0 10 13 503(MW25.) calc. C 47.81, H 5.22, N 27.88 found 48.01, 5.30, 27.83 ~1 E 15 1 C-NMR (DMSO-d 6 135.1 99.7 157.9 155.3 154.5 83.8 30.3 27.3 81.6 64.3 1 H-NM'R: 9' 6.19 (dd, J 6.9, 3.5 Hz), 2.06 (in, Example 22.

3, 7-Didesaza-2 '-desoxy-9- -D-ribofuranosylpurine (21 -desoxy-3 ,7-didesazanebular in) The compound of Example 18b) is hydrogenated in the presence of palladium/animal charcoal (10% Pd) in ammoniacal methanol. After filtering off the catalyst and evaporating the filtrate in a vacuum, the product is purified from inorganic salts by chromatography on '2 2 -49- Amberlite XAD (methanol/water), as well as by crystallisation from water; m.p. 175 176'C.

UV (0.1m aqueous hydrochloric acid): X~ max. 224, 274 nm 13 C-NMIR 6 ]DMSO): =126.9 101.7 125.5 143.3 140.6 105.9 139.2 84.6 70.8 87.8 61.9 1 H-NR (DMSO-d 6 2.23 (in, 2.29 (mn, 2'-Ha), 3.55 (mn, 5- H 2 3.8 (m 4.38 (mn, 4.99 5.37 6.42 (pt, 6.66 J 3 Hz, 3H), 7.62 J 6 Hz, 7.71 J 3 Hz, 8.21 J =6 Hz, 8.23 4-H).

Analysis for C 12H 14N 203 15 caic. C 61.53, H 6.02, N 11.96 found 61.55, 6.12, 12.02 t I t 9 1 4 Example 23.

a) 2-Chloro-6-methoxy-3,7-disaza-2'-desox-9- 3

-D-

ribofuranosylpurine.

The compound of Example 18b) is heated for hours in 1N inethanolic sodium 1 methanolate solution.

The reaction product is purified on Amberlite XAD by hydrophobic chromatography (methanol/water).

UV (methanol): A max. 271, 280 nm.

Analysis for C 13

H

15 C1 NO0 4 calc. C 52.27, H 5.06, Cl 11.87, N 9.3 found 52.24, 5.14, 12.05, 9.4 8 6 b) 2-Ghloro-3,7-didesaza-2'-desoxy-9-P-D-ribofuranosylpurin-6-one.

Heating the compound of Example 18b) for 30 hours in 2N aqueous sodium hydroxide solution/l,4-dioxan gives the desired compound.

UV (methanol): a.=262 nm Analysis for C 13

H

16 N 2 0 4 caic. C 59.08, H 6.10, N 10.60 found 59.09, 6.07, 10.65 1 H-NMR 6 DMSO): S 2.22 (in, 2 2.38 (mn, 2 a), 3.53 (in, 5'-H 2 3.80 (mn, 4.33 (mn, 4.96 5.29 6.22 (pt, 6.54 J 3 Hz, 6.96 7.38 J 3 Hz, 11.81

(NH).

Example 24.

a) 4-Ghloro-7-(2'-desoxy-3,5-di-0-(p-toluoyl)- 3

-D-

erythro-pentofuranosyl -7H-pyrrolo 12, 3-d IIpyrirnidine.

1 g. (17.8 mMole) powdered potassium hydroxide is introduced at ambient temperature into 60 ml. dry acetonitrile. 100 pl. (0.31 mMole) tris-[2-(2-inethoxyethoxy)-ethyl]-amine-are added thereto, while stirring.

After .5 minutes, 1.23 g. (8.01 mMole) 4-chloro-7Hpyrrolo[2,3-d]pyrimidine are dissolved in the reaction mixture which is stirred for a further 5 minutes.

c-hloro-2-desoxy-3,5-di-0-p-toluoyl-p-D-erythro-pentofuranose is then added thereto. After stirring for minutes, insoluble material is removed by filtration.

-51- The filtrate is evaporated to dryness in a vacuum and the residue chromatographed on a silica gel column x 7 cm., chloroform). After evaporation of the eluate in a vacuum there are obtained 3.26 g. (81% of theory) of product which crystallises from ethanol in the form of colourless needles; m.p. 120'C.

Further variants of the process of preparation: Solid-liquid glycosilation in the absence of a catalyst. The reaction is carried out as described above but witf.out the use of a catalyst. After working t500 mg. (3.26 mMole) 4-chloro-7H-pyrrolo[2,3-d]- I 44 pyrimidine are dissolved in 20 ml. dichloromethane.

9 ml. of 50% aqueous sodium hydroxide solution are 444e 4 added thereto. After the addition of 10 mg. (0.03 mMole) tetrabutylammonium hydrogen sulphate, the 4 .solution is stirred for 1 minute with a vibratory mixer.

Subsequently, 1.4 g. (3.61 mMole) of the above-described halogenose is added thereto and mixing continued for a further 3 minutes, whereafter the phases are separated.

The aqueous phase is extracted twice with, in each case, ml. dichloromethane. The combined organic phases are dried over anhydrous sodium sulphate. The filtrate is evaporated to dryness and the residue is chromatographed on silica gel (column 5 x 5 cm., chloroform).

1Ti C- U(nrr -52- Isolation of the product from the main fraction and crystallisation from ethanol gives 1.04 g. (63% of theory) of the desired product; m.p. 1180C.

TLC (cyclohexane/ethyl acetate 3:2 Rf 0.7.

UV (methanol): a 240 nm (log E 4.48).

max.

1H-NMR (DMSO-d 6 S 2.37, 2.40 2 CH 3 2.77 (m, 3.18 4.60 4'-H and 5.77 6.75 J 3.7 Hz, 6.78 7.34, 7.91 8 aromat. H and 8.65 2-H).

b) 4-Chloro-7-(2'-desoxy-p-D-erythro-pentofuranosyl)- 7H-pyrrolo[2,3-d]pyrimidine.

i, 2.4 g. (4.7 mMole) of the compound of Example 24a) are stirred for 24 hours at ambient temperature in 100 ml. methanol saturated with ammonia. The solution 't 15 is evaporated to dryness, the residue is adsorbed on 10 g. silica gel 60 H and applied to a silica gel column (4 x 10 cm., chloroform/methanol 95:5 The product is isolated from the main fraction as a colourless, o solid substance which crystallises from ethyl acetate as colourless needles. Yield 1.07 g. (84% of theory); m.p. 1620C.

TLC (chloroform/methanol, 9:1 R 0.6.

UV (methanol): >max. 273 nm (log E 3.69).

max.

1 H-NMR (DMSO-d 6 S 2.28 2.58 2'-Ha), 3.57 3.87 4.40 5.00 J 5.4 Hz, 5.35 J 4.2 Hz, 3'-OH), 6.66 6.72 J 3.8 Hz, 7.99 J 3.8 Hz, 8.66 2-H).

-53c) 4 -Chloro- 7 -(2'-desoxy--D-erythro-pentofuranosyl)- 5'-0-(4,4'-dimethoxytrityl)-7H-pyrrolo[2.3-d]pyrimidine.

1 g. (3.7 mMole) of the compound of Example is dried by evaporating with 10 ml. dry pyridine. The material is dissolved in 20 ml. dry pyridine. 2 ml.

(11.7 mMole) Hunig's base and 2 g. (5.9 mMole) 4,4'dimethoxytrityl chloride are added thereto. The solution is stirred for 3 hours at ambient temperature. After the addition of 80 ml. 5% aqueous sodium bicarbonate solution, the solution is extracted three times with 100 ml.

amounts of dichloromethane. The combined organic phases are dried over anhydrous sodium sulphate. After filtering off, the filtrate is evaporated in a vacuum.

The residue is purified by column chromatography (silica gel, elution agent dichloromethane and dichloromethane/ ethyl acetate 9:1 Isolation of the product from the main fraction, dissolving in diethyl ether and precipitation with petroleum ether gives 1.66 g. (78% of tl of the desired product in the form of a yellowish am ious substance.

Analysis for C32 H 30N 3 0 5 C1 572.1) calc. C 67.19, H 5.29, Cl 6.20, N 7.35 found 67.03, 5.47, 6.19 7.29 TLC (dichloromethane/acetone 9:1 Rf 0.3.

UV (methanol): Amax. 274 nm (log E 3.85).

'V -54lH-NMIR (DMSO-d 6 =2.36 (mn, 21 2.70 (mn, 2 t -Ha)ll 3.72 00H 3 3.18 J 4.5 Hiz, 51-H), 3.99 (mn, 4.45 (mn, 5.42 J 4.6 Hz, 3'-OH), 6.65 (mn, 11-11), 6.69 J =3.7 Hz, 5-H1), 7.81 (d, J =3.7 Hz, 8.64 2-H).

d) 4-Chloro-7-(2'-desoxy-O3-D-erythro-pentofuranosyl)- 5'-0-(4,4'-dimethoxytrityl)-3'-0-phenoxythiocarbonyl- 7H-pyrrolo[ 2,3-dlpyrimidine.

1 go (1.7 inMole) of the compound of Example 24c) is dissolved in 30 ml. dry acetonitrile, 500 mng. (4.1 inMole) 4-dimethylaminopyridine and 400 p1. (2.9 inMole) phenyl chiorothiocarbonate are added thereto and the solution is stirred for 16 hours at ambient temperature.

Subsequently, the reaction mixture is evaporated to dry- 15 ness in a vacuum and the residue applied to a silica gel 4 4 1 too column (3 x 15 cm., dichloromethane). From the main fraction, there are isolated 950 mng. (76% of theory) of colourless, amorphous product.

A7 alySiS for C 39 H 'C1N 0 6 S 708.2) calc. :C 66.14, H 4.84, Cl 5.01, N 5.93, S 4.53 found 66.22, 4.94, 5.12, 5.93, 4.46 TLC (dichloroinethane/acetone 95:5 Rf 0.8.

4UV (methanol): max. 274 nm (log E 3.87).

1 H-NMR (DMSO-d 6 9 2.84 (in, 2 3.21 (in, 2 3.37 (mn, 4.46 (in, 5.92 (mn, 6.70 (in, 6.76 J 3.8 Hz, 7.85 J= 3.8 Hz, 8.61 2-H).

e) 4-Chloro-7-(2' ,3'-didesoxy-f-D-glyrceropentofuranosyl)-5'-0-(4,4'-dimethoxytrity)-7Hpyrrolo.

[2,3-dlpyrimidine.

800 mg. (1.1 mMole) of the compound of Example 24d) and 40 mg. (0.2 mMole) 2 ,2'-azo-bis-(2-methyl)-propionitrile are dissolved in 40 ml. dry toluene under an I atmosphere of argon. 600 pl. (2.2 mMole) tri-n-butyl stannane are added thereto, while stirring, and the reaction is continued for 2 hours at 75'C. The solvent is removed in a vacuum and the residue chromatographed on silica gel (column 15 x 3 cm., dichloromethane/ethyl acetate 95:5 From the main fraction, there are obtained 470 mg. (75% of theory) of the desired product.

Analysis for C 32

H

0 C1N 0 4 556.1) 15 calc. :C 69.12, H 5.44, Cl 6.38, N 7.56 found 69.07, 5.53, 6.33, 7.58 TLC (dichloromethane/acetone 95:5 Rf UV (methanol): max 273 nm (log E 3.78).

eQ 1 H-NMR (DMSO-d 6 2.08 (in, 2.10 (in, 2 '-Hb) 2.43 (in, 2 3.11 J 4.4 Hz, 3.71

OCH

3 42 6 .55 (dd, J 6 and 6.9 Hz, 6.64 J 3.7 Hz, 7.83 J =3.7 Hz, 8.67 2-H).

f) 4-Chloro-7-(2 t ,3'-didesoxy-O-D-glyceropentofuranosyl)-7H-pyrrolo[2,3-dlpyrimidine.

400 mg. (0.7 mMole) of the compound of Example 24 .6)are dissolved in 15 ml. 807% aqueous acetic acid and -56stirred for 30 minutes at ambient temperature. The solvent is removed in a vacuum and traces of acetic acid are removed by evaporation with water. The residue is purified by column chromatography (dichloromethane and dichloromethane/methanol, 98:2 From the main fraction there are obtained 120 mg. (67% of theory) of product which, after crystallisation from ethyl acetate, is obtained in the form of colourless needles; m.p.

90 0

C.

Analysis for C 1 1

H

1 2 C1N 3 0 2 253.7) calc. C 52.08, H 4.77, C1 13.98, N 16.56 found 52.20, 4.81, 14.04, 16.54 TLC (dichloromethane/methanol 95:5 Rf SUV (methanol): 274 nm (log E 3.65).

r r' max.

15 1 15 -NMR (DMSO-d 6 S 2.04 2.28 2 I 2.46 2 3.57 4.11 4.95 J 5.5 Hz, 6.52 (dd, J 3.8 and 6.9 Hz, 6.69 J 3.8 Hz, 8.01 J 3.8 Hz, i 8.64 2-H).

g) ,3'-Didesoxy- 3 -D-glyceropentofuranosyl)- S7H-pyrrolo[2,3-d]pyrimidine.

A solution of 200 mg. (0.8 mMole) of the compound of Example 24f) in 20 ml. methanol, to which had been added 0.5 ml. (6.6 mMole) of concentrated aqueous ammonia solution, is stirred with palladium on animal charcoal mg., 10% Pd) in an atmosphere of hydrogen at ambient temperature for 3 hours. The catalyst is filtered off 4 t -57and the solvent removed in a vacuum. The residue is dissolved in water and chromatographed on an Amberlite XAD-4 column (1st elution agent water, 2nd elution agent water/methanol 8:2 Isolation of the product from the main zone gives 130 mg. (75% of theory) of the colourless product in the form of needles; m.p. 131OC.

Analysis for C 11

H

1 3 0 2

N

3 219.2) calc. C 60.26, H 5.98, N 19.17 *o0 found 60.19, 5.97, 19.18 1 o 10 TLC (dichloromethane/methanol 9:1 Rf 0.6.

Soo UV (methanol): Ax 270 nm (log E 3.56).

max.

.oo

I

1 H-NMR (DMSO-d 6 2.06 2.27 2 2.42 2'-H 3.55 4.09 4.93 0 oo J 5.5 Hz, 6.54 (dd, J 4.3 and 6.9 Hz, 0O 15 6.67 J 3.7 Hz, 7.86 J 3.7 Hz, o O0 8.79 9.01 2-H).

00oo00 h) 4-Amino-7-(2',3'-didesoxy-3-D-glyceropentofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine (2',3'-dideoxytubercidin) 200 mg. (0.8 mMole) of the compound of Example 24f) are stirred in 60 ml. 25% aqueous ammonia solution for 15 hours at 100 0 C. under pressure in a steel bomb.

The solvent is subsequently removed in a vacuum and the residue dissolved in 200 ml. water. This solution is purified on Dowex 1 x 2 (OH- form). The column is washed with water and the product eluted with water/ methanol (9:1 From the main zone are obtained s I ii;: r i Ir -~usu c- -58- 120 mg. (65% of theory) of product.

TLC (dichloromethane/methanol 9:1 Rf 0.3.

1 H-NMR (DMSO-d 6 2.03 2.22 2 2.33 2 3.53 4.04 4.99 6.35 6.51 J 3.6 Hz, 7.00 NH 2 7.34 J 3.6 Hz, 8.04 2-H).

i) 7-(2',3'-Didesoxy- 3 -D-glyceropentofuranosyl)-4methoxy-7H-pyrrolo[2,3-d]pyrimidine.

«o 170 mg. (0.7 mMole) of the compound of Example 24f) 10 are dissolved in 5 ml. LM methanolic methanolate solution 00 o O and stirred at ambient temperature for 4 hours. The solution is neutralised with 80% acetic acid, evaporated 4 0 in a vacuum and the residue applied to a silica gel column (elution agent dichloromethane/methanol 98:2 v/v).

S 15 Isolation of the main zone gives a colourless oil which, upon storing, crystallises in the form of needles.

Yield 130 mg. (78% of theory).

j) 7-(2',3'-Didesoxy- -D-glyceropentofuranosyl)-4Hpyrrolo[2,3-d]pyrimidin-4-one.

200 mg. (0.8 mMole) of the compound of Example 24f) are suspended in 10 ml. 2N aqueous sodium hydroxide solution and boiled under reflux for 5 hours. The solution is neutralised with 80% acetic acid and the insoluble material is removed by filtration. The filtrate is applied to an Amberlite XAD-4 column. The column is washed with 500 ml. of water and the product eluted with water/isopropanol (9:1 There are

I

a -59-7 obtained 180 mg. (80% of theory) of product.

Example 1-(2',-Ddsx--glcrp afuranosyrl)-lHpyr~zolo[3.4-dllpyrimidin-4-one.

The product of Example 17d) is deaminated with j adenosine deaminase from intestinal calf mucosa cells.

The progress of the reaction is monitored UV spectroscopically at 275 nm. The reaction gives the product Ott: quantitatively in the form of colourless crystals; m.p. 171'C.

VstUV (methanol): =251 nm (E=7700).

9 max.

*Ott TLC (silica gel, dichloromethane/methanol 9:1 v/v): R f 13 GCNMR [D 6 ]DMS0): 135.2 106.1 157.3 148.4 152.3 84.6 30.7 27.3 82.2 64.2 1 HNM 6 DMSO): 2.13 (mn, 2.40 (in, 3.40 (in, 4.09 (in, 4.73 (in, 6.43 (mn, 8.11 8.13 6-H).

Example 26.

2-Amino-7-desaza-2' -didesoxy-9-p3-D--ribofuranosylpurin-6-one Analysis for C 1 H 14 4

%V

3 Na 3 556.2) calc. P 16.7 found 16.4 UV (buffer, pHl max. 259 nm (E 13400) I, 31 P-NMR (D 2 -10.0 P-Li)o -21.5 P-13).

Example 27.

2-Amino-3,7-didesaza-2'-desoxy-9-P-D-ribofuranosyl-_ purin-6-one Analysis for C 12

H

15 N 3 O0 13

P

3 Na 3 555.2) caic. P 16.75 found 16.5 0UV (buffer, PH >ma.=272 nm (~=12400).

9max.

Example 28.

3,7-Didesaza-2' ,3'-didesoxy-9-5-D-ribofuranosylpurine '-triphosphate.

Anaysi fo C12 H14 N 2 0 11

P

3 Na 3 524.1) *calc. P 17.7 found 17.3 UV (buffer, pH max 224, 274 nm.

All of the triphosphates described in Examples 26 to 28 are prepared by phosphorylation of the corresponding nucleosides by the method described by Yoshikawa (Tetrahedron Letters, 50, 5065/1967) to give the 5'-monophosphates and subsequent conversion into the hates by the method of Hoard and Ott 87, 1785/1965).

Example 29.

Antiviral activit.

The stability of the N-glycosidic bond of didesoxynucleosides is bound up with the antiviral -61activity.

The hydrolysis of the bond was investigated at in three different concentrations of hydrochloric acid. For this purpose, the UV absorption (E t was measured at 258 nm. Via the absorption/time curve, there were determined the velocity constants of the hydrolysis and the half life times on the basis of the following equation: aaoo" k 1/t x In (E E Eo) S00 o a oo 9 0 0 0 10 E being the absorption at time t 0 and Eo being the 0 0 0 0 O0 o 0 ooo absorption after complete termination of the reaction.

0000 o There were compared 2',3'-didesoxyadenosine (a) 0 and 6-amino-8-aza-7-desaza-2',3'-didesoxy-9-p-D- *o ribofuranosylpurine at 25 0 C. The results obtained 15 are set out in the following Table: Table *oo 000 0s e a 0 0 00 o 20 IN HC1 0.1N HC1 0.01N HCI T/2 1.9 min. 31.5 min.

k 0.363 min 1 0.022 min 1 T/2 0.83 min. 20.4 min. 280 min.

k 0.85 min 1 0.033 min 1 0.0025 min 1 The above Table shows that the compound according to the present invention is more than 10 times more stable and thus more antivirally effective than

Claims (10)

1. Desazapurine-nucleoside derivatives of the general formula:- R 1 R 3 2X N R 7 R y-O 0 R 6 f wherein X is a nitrogen atom or a methine radical, W is 4 1 2 3 4 a nitrogen atom or a ,C-R radical, R, R 2 R and R, which can be the same or different, are hydrogen or halogen atoms, hydroxyl or mercapto groups, lower alkyl, lower alkylthio, lower alkoxy, aralkyl, aralkoxy or aryloxy radicals or amino groups which are unsubstituted or substituted once or twice by aralkyl or lower alkyl which, in turn, can be substituted by lower alkoxy, halogen, amino or amino substituted once or twice by aralkyl or lower alkyl; or in case, when the amino group J is substituted twice, both substituents together represent an alkylidene radical which, in turn, can be substituted by lower alkoxy, halogen, amino or amino substituted once or twice by aralkyl or lower alkyl, R 5 is a hydrogen atom or a hydroxyl group, R 6 and R 7 are each hydrogen atoms or one of them is a halogen atom or a A 0 C~4 hst<; lower alkyl which, in turn, can be substituted by lower alkoxy, halogen, amino or amino substituted once or twice by aralkyl or lower alkyl; or in case, when the amino group is substituted twice; both substituents together represent an alkylidene radical which, in turn, can be substituted by lower alkoxy, halogen, amino or amino substituted once or twice by aralkyl or lower alkyl, whereby one of R 6 and R 7 can also be a hydroxyl group when X is a methine radical and, in addition RI and R 7 can together also represent a further valency bond between C- ti 2' and C-3' and Y is a hydrogen atom or a mono-, di- or 'tri-phosphate group, as well as the tautomers and salts thereof and nucleic acids which contain one or more 0 I Ai 17 -63- compounds of general formula as structural components.

2. Desazapurine-nucleoside derivatives according to claim 1 which are hereinbefore specifically exemplified.

3. Process for the preparation of compounds according to claim 1, wherein a compound of the general formula:- 1 R 3 (II) 0 000 00 0 0 00 0004i 0t 0 r4 9P 00 0 0 0 0 0941l in which X, W, R 1 R 2 and R 3 have the meanings given in claim l,is reacted with a compound of the general formula:- 0 rr I 0 0 1t 4 00 R 7 R R'-O (III) S f t 4 j in which R 5 has the meaning given in claim 1, R 6 and R are each hydrogen atoms or one of them is an azido group or a hydroxyl group protected by an oxygen protective group, R' is an oxygen protective group and Z is a -s^aS group, to give a compound of the general formula:- I -64- R 1 R 3 R R (IV) 0 00 0 0, 0 0000i 010 000004 00 0 VI 0* tr4 4 0f i *t 4. 00 0~ 4 00tl '6' in which X, W, R 1 R 2 R 3 R 5 R 6 R 7 and R' have the above-given meanings, and oxygen protective groups possibly present are split off and thereafter, if desired, a compound thus obtained, in which R 6 or R is a hydroxyl group, is, after previous protection of the 5'-hydroxyl group, converted with a halide, cyanide or azide in known manner into a compound of general formula I, in which R 6 or R 7 is a halogen atom or a cyano or azido group or is desoxygenated in known manner to give a 6 7 .aves compound of general formula I, in which R ba-c R 7 3== hydrogen atoms or a compound of general formula I so obtained, in which R 6 or R 7 is an azido group, is converted in known manner into a compound of general formula I, in which R 6 or R 7 is an amino group, and, if desired, a compound of general formula I so obtained is converted in known manner into a mono-, di- or tri- phosphate and, if desired, a free base or acid obtained is converted into an appropriate salt or a salt obtained is converted into the corresponding free base or acid. L~

4. Process for the preparation of compounds accord- ing to claim 1, substantially as hereinbefore described and exemplified.

Compounds according to claim 1, whenever prepared by the process according to claim 3 or 4.

6. The use of compounds according to claim 1 for DNA sequencing.

7. The use of compounds according to claim 1 as o* antiviral agents. 10

8. Pharmaceutical compositions containing at least 0 one compound according to claim 1, as well as convent- oa ional carriers and adjuvants.

9. .he use of compounds according to claim 1 for the preparation of pharmaceutical compositions. 6 0 9*

-10. The ctops,-fgAUUroS,-compos i4-ions and..cmpounIc2 o referred to or indicated in the specifica c n/or Coc claims of this application -ntd-i'ually or collectively, Sand any and m-lS'b-iniations of any two or more of said a or foaturoe. SDATED this 8th day of April, 1988 BOEHRINGER MANNHEIM GMBH By Its Patent Attorneys DAVIES COLLISON