DK168443B1 - Pyrimid-2-on-1-yl compounds, pharmaceutical preparations which comprise these compounds, and a process for preparing the compounds - Google Patents

Abstract

A compound of formula (I), wherein the radicals A, X, R<1>, R<2>, and R<3> are defined as follows: A: formula (a) or formula (b); X: (a) O; (b) S; (c) CH2; R<1>: H; alkyl containing 1-3 carbon atoms; -CH=CH2; -CH=CH-CH3; -CH2-CH=CH2; formula (II); -C=CH; R<2>: H; or R<2> constitutes together with R<3> a carbon-carbon bond; R<3>: H; F; Cl; Br; I; N3; CN; C=CH; OH; OCH3; CH2OH; and when R<3> is F; Cl; Br; I; N3; CN; C=CH; OH; OCH3 or CH2OH it may have either the cis-configuration or trans-configuration relative to the hydroxymethyl function at position 4', or R<3> constitutes together with R<2> a carbon-carbon bond, and therapeutically acceptable salts thereof, for use in therapy, in particular for the treatment of HIV virus infections.

Description

in DK 168443 B1

The present invention relates to chemical compounds of the formula I as claimed in claim 1 and their therapeutically acceptable salts for use in therapeutic and prophylactic control and treatment of acquired immunodeficiency syndrome (AIDS), human immunodeficiency virus infections, hepatitis B virus infections and retroviruses infections in animals and humans, therapeutic compositions containing such compounds, and methods for their preparation.

10 In the latter part of the 1970s, a new disease was reported, later referred to as acquired immunodeficiency syndrome (AIDS). It is now generally accepted that a retrovirus designated HIV (human immunodeficiency virus), previously designated human T-cell lymphotropic virus (HTLV-III) or lymphadenopathy-associated virus (LAV) plays a crucial role in the etiology of AIDS.

AIDS is characterized by a severe immune deficiency of 20 due to low numbers of a subset of lymphocyte T helper cells that are a target of HIV infection. The severe immune deficiency in AIDS patients makes these patients highly susceptible to a whole range of random infections of bacterial, fungal, protozoal or viral aetiology. The etiologic agents among viral random infections are often found within the herpes virus group, viz. Herpes simplex virus (HSV), varicella zoster virus (VZV), Epstein-Barr virus (EBV) and especially cytomegalovirus (CMV). Other retroviruses affecting humans are 30 HTLV-I and II and examples of retroviruses affecting animals are cat leukemia virus and equine infectious anemia virus.

Hepatitis B virus infections cause serious diseases such as acute hepatitis, chronic hepatitis and 35 fulminant hepatitis in a significant number of people. It has been estimated that there are 200 million patients with chronic hepatitis B infection in the world. A significant number of the 2 DK 168443 B1 chronic cases go into liver cirrhosis and liver tumors. In some cases, the hepatitis infections also take a severe and rapid turn to fulminant B hepatitis with approx. 90% mortality. Currently there is known in-gene effective treatment of hepatitis B infections.

A large number of nucleoside analogues exhibit up to several anti-metabolic activities. This is done by replacing or competing with naturally occurring nucleosides.

Recently, certain nucleoside analogs have been described that inhibit cell culture multiplication of human immunodeficiency virus (HIV, also referred to as HTLV-III, LAV), which is the causative agent of AIDS and the AIDS-related complex (ARC). These compounds are, for example, azidothymidine, dideoxycytidine and dideoxyadenosine. These and other described HIV antimetabolic nucleoside analogues have the same geometric relationship between the nucleoside base and the glycoside moiety as the naturally occurring nucleosides, ie. they are island anomers.

The following β-anomeric compounds are known: 1. Compounds of formula 25 Å ^ wr 1F ^ R3 30 / \ Η0_1 ^ 0/3 1 where R represents OH and R represents the following: 35 R ^ is H and CH ^: T Nishimura, B. Shinizu, I. Iwai, Chem.

Pharm. Bull. (Tokyo) 12 (1964, 1471) 3 DK 168443 B1 R is C2H5: m · Swierkowski, D. Shugar, J. Med. Chem.

12 (1969), 533 R "is n-CgH ^: A. Szaboles, J. Sågi, L. Otvos, J. Carbohydrates, Nucleosides, Nucleosides 2 (1975), 197-211 in R -CgH 2: M. Draminski, A. Zgit-Wroblewska, Polish J. Chemistry 54 (1980), 1085 R 1 is C = CH: P. J. Barr, A. S. Jones, P. Serafinowski, R. Walker, J. Chem. Perkin I (1978), 1263-1267 and wherein R is Ng and R is CH 2: M. Imezawa, F. Eckstein, J. Org. Chem. 43 (1978), 3044-3048.

2. Compounds of formula NH-20. 2 n ^ Y'R1

OH

"A

H0_I

R1 is C = CH is described by P.J. Barr, A.S. Jones, P. Serafinowski, R. Walker, J. Chem. Soc. Perkin I (1978), 1263-1267 R is H is described by J.J. Fox, N.C. Yung, I. Wempen, and 35 M. Hoffer, J. Am. Chem. Soc., Vol. 83 (1961), 4066-4070.

Both groups 1 and 2 relate only to compounds with the 3 'group and the 4'-hydroxymethyl group in trans configuration.

In addition, C.A. Vol. 92 (1980) 208903d the anti-viral action of the α-anomer of 5-isopropyl 2'-deoxyuridine.

FR Patent No. 2,040,177 and DK Patent Application No. 3722/83 disclose β-anomeric 5-propyl-2'-des-10-oxyribosyluracil derivatives and α-anomeric 5-haloethyl-2'-deoxyuridine derivatives exhibiting antiviral activity.

All of the aforementioned known α-anomeric compounds exhibit activity against Herpes virus, whereas these compounds do not have antiviral activity against HIV and hepatitis B virus. A study of the effects of the known compounds Acyclovir and Ganciclovir on hepatitis B virus is listed below in Table 2.

20 Surprisingly, it has now been found that certain novel α-anomeric pyrimid-2-one-1-yl compounds are potent inhibitors of HIV multiplication, but not of cell division.

These compounds of the invention with the formula I of claim 1, which are α-anomers, and their therapeutically acceptable salts can be used as therapeutic and / or prophylactic agents for the control and treatment of HIV viral infections in mammals and humans.

More generally, the compounds of formula I may be used as therapeutic and / or prophylactic agents for the control and treatment of infections caused by retroviruses and hepatitis B virus in mammals and humans.

35 All retroviruses, including HIV, require the enzyme reverse transcriptase in their natural reproductive cycle.

5 DK 168443 B1

Hepatitis B virus (HBV) is a DNA virus with a unique circular double-stranded DNA genome that is partially single-stranded. It contains a specific DNA polymerase needed for viral copying. This DNA polymerase 5 also acts as a reverse transcriptase during the copying of HBV DNA via an RNA intermediate.

The compounds of formula I inhibit the activity of reverse transcriptase from retroviruses, including HIV, as well as the activity of DNA polymerase from hepatitis B virus.

The invention thus relates to: 1. the compounds defined in claims 1 to 8; 2. pharmaceutical compositions comprising a compound according to claim 1 as the active ingredient, according to claim 11 20; 3. a compound according to claim 1 for use as a medicament according to claim 9 4. a compound according to claim 1 for use in the therapeutic treatment of infection in humans caused by HIV virus, according to claim 10, and in the manufacture of a medicament for the therapeutic and / or prophylactic treatment of infections. caused by a retrovirus, including HIV, or by hepatitis B virus, as claimed in claims 12 and 30. A method of preparing the compounds of the invention as claimed in claim 13.

The term "alkyl containing 1-3 carbon atoms" for the group R1 means CH3, CgHg, CH2CH2CH3 and CH (CH3) 2.

Formula I is F or Cl 2 OH, and both may have either cis configuration or trans configuration relative to the hydroxymethyl group at position 4 '.

Examples of preferred compounds are: 0 A / T j 10 ° An R3

H

15

R 1 is CH 3; R 3 is CH 2 OH R 1 is CH 3; R 3 is F R 1 is C 2 H 5; R 3 is CH 2 OH 20 R 1 is C 2 H 3; R3 is F

nh2 nAX.

lake

A

HO_I

R 3 is CH 2 OH or F.

3 In all examples of preferred compounds, R at position 3 'and hydroxymethyl at position 4' have trans configuration.

In clinical practice, the compounds of formula I will normally be administered orally, by injection or by infusion in the form of a pharmaceutical composition comprising the active ingredient in the form of the subject compound or optionally in the form of a pharmaceutically acceptable salt thereof together. with a pharmaceutically acceptable carrier which may be a solid, semi-solid or liquid diluent or an edible capsule. The compound can also be used without carriers. Examples of pharmaceutical compositions include tablets, dragees, capsules, granules, suspensions, elixirs, syrups and solutions. Usually, the active compound constitutes between 0.05 and 20% of preparations for injection and between 10 and 90% of preparations for oral administration.

In the treatment of patients suffering from retroviral infection, especially HIV or hepatitis B viral infections, it is preferred to administer the compounds in any appropriate manner, including by oral, parenteral, rectal, nasal, topical and vaginal route of administration. The parenteral route includes subcutaneous, intra-muscular, intravenous and sublingual administration. The topical pathway includes buccal and sublingual administration.

The dose with which the active ingredients are administered may vary within wide limits and depends on 25 different factors such as e.g. the severity of the infection, the patient's age, etc. and can be adjusted individually. One possible range of administration of the subject compounds or their physiologically acceptable salts is per. day from approx. 10 mg to approx. 10,000 mg, preferably 100-30 500 mg for intravenous administration and preferably 100-3000 mg for oral administration.

Examples of pharmaceutically acceptable salts of the compounds of formula I include base salts, i. derived from a suitable base such as e.g. alkali metal (as sodium), alkaline earth metal (as magnesium) salts, ammonium and NX + (where X is alkyl). Physiologically acceptable salts of an hydrogen atom or an amino group include salts of organic carboxylic acids such as acetic acid, lactic acid, gluconic acid, citric acid, tartaric acid, maleic acid, malic acid, pan-totenoic acid, glacial acid, succinic acid, oxalic acid and lactobionic acid; organic sulfonic acids such as methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-chlorobenzenesulfonic acid and p-toluenesulfonic acids and inorganic acids such as hydrochloric acid, hydrogen iodide acid, sulfuric acid, phosphoric acid and sulfamic acid. Physiologically acceptable salts of a compound having a hydroxy group include the anion of the compound together with a suitable cation such as e.g. Na +, NH4 + and NX4 + (where X is a C1-4 alkyl group).

The administered compounds can also be used in therapy with other drugs, e.g. 9 - [(2-Hydroxy-1- (hydroxymethyl) ethoxy) methyl] guanine, 9- (2-hydroxy-ethoxymethyl) guanine (acyclovir), 2-amino-9- (2-hydroxyethoxymethyl) purine , interferon, interferon, interleukin II and phosphonoformate or in combination with immunomodulatory therapy, including bone marrow or lymphocyte transplants or medication with e.g. levamisole or thymosin which will increase the number of lymphocytes and / or their function depending on what is appropriate.

The method according to the invention is characterized by the characterizing part of claim 13.

The compounds of the invention can thus be prepared by one of the following general methods.

A. Condensation of a glycoside of formula II, wherein the hydroxyl groups may be optionally protected, to the N1 position of a pyrimidine derivative corresponding to the group A of formula I by known methods described in the literature, followed by separation of the α-anomers and the removal of any protecting group (s). Such methods are described e.g. in "Basic Principles in Nucleic 9 DK 168443 B1

Acid Chemistry ", Vol. 1 (Academic Press, 1974, ed.

P.O.P.Ts'o), in "Nucleoside Analogues, Chemistry, Biology and Medical Applications" (Pharma Press, 1979, ed. R.T. Walker, E. De Clercq, and F. Eckstein) and in Nucleic Acids 5 Research Vol. 12, 1984, pages 6827-6837 (A. J. Hubbard, A. S. Jones and R. T. Walker). An example of this method is given in the case of a uracil base analog: 10 R 50/3 '3', 3 HnA

\ y — Hal] I

R «4 JL Γ -

(CH 3) 3 SiO 2 N / r 4 I

r5o_jO

II

Wherein R 1 is F or CHOOR 2, R 2 is a protecting group, e.g. p-toluoyl, acetyl, trityl and benzyl. R is as defined above.

25 30 35 10 DK 168443 B1 B. Anomerization of an island anomer of the formula

A

s RVX \ |

P

Wherein A and X are as defined in claim 1; R is F or 5 C wherein R is H or a hydroxy protecting group, to a mixture of α and β anomers, after which the α-anomer is separated and any protecting groups removed. The anomerization can be carried out by known methods, e.g. with an optionally protected β-nucleoside, e.g. a silylated nucleoside with a catalyst such as trimethylsilyltrifluoromethanesulfonate OSiMe, 0SiMe ~ XJ - ^ 0 Xj -. R, R and R are as defined above.

30 C. A transglycosylation reaction in which the sugar moiety forming a bond, or O-, to a nucleoside base is transferred to the desired pyrimidine base. The reaction is carried out with a catalyst, e.g. trimethylsilyl trifluoromethanesulfonate and is followed by separation of the e-anomer and removal of the protection.

11 DK 168443 B1 OSHCHJ, R * 0 o BIV fUCH3> 3 Ύ Ί rY n "Vr1 5 Γ ^ rj 10 'R50 — l \ o / + other products 15 14 5 wherein R, R and R are as defined above. The group B is a pyrimidine or purine base, the base used being not essential.

20 D · Insertion of F or CH90H into the 3 'position as a substitute for a suitable leaving group, R, followed by removal of any OH protecting groups.

25 30 35 12 DK 168443 B1

0 O

AA A r1

HN Y HN ^ VR

5 oAnJ X J · R7 _> ° "A r9 <, r5ojC / 'r, Λ.Τ_>", jcJ Jj

R O — 1 N (K

R 7 and R are defined above; R is an easily leaving group such as e.g. trifluoromethanesulfonyloxy, R is F βία or a synthesis precursor for the C R is an appropriate protecting group.

ISLAND

An alternative route for introducing the R function is the reaction of the 2.31-anhydrous α-anomer.

0 0 nAr1 Jl r1 .17 Η 4ΓΛ1 35 R50 — r50— · 0 ^ 13 DK 168443 B1 15 8 where k, Ra and R ° are as defined above.

The main principles of Methods A-D set forth above are applicable to the synthesis of both uridine and cytidine analogues of formulas I (a) and (b), although the formulas illustrate only the reactions with uridine analogs.

E. Conversion of the uracil group of the 5-substituted or unsubstituted α-uridine compounds to a cytosine group of the corresponding α-cytidine analogs. This is done in a known manner, the principles being described e.g. by W.L. Sung (J. Chem. Soc. Chem. Commun. 1981, p. 1089 and J. Organic Chemistry 1982, vol. 47, pp. 3623-3628) and by P. Herdewijn et al. (J. Medicinal Chemistry 1985, vol.

15 28, pages 550-555).

The following examples further illustrate the invention. Preparation of intermediates 20 A. Preparation of 3'F-S'-deoxy-S * -O-acetylthymidine (VSB 423) 45 mg of 31F-31-deoxythymidine (0.184 mmol) in 2.0 ml of acetic anhydride was heated with stirring. in an oil bath at 80 ° C for 7 hours. The solution was evaporated in vacuo and the residual acetic anhydride and acetic acid were removed by several additions and repeated evaporation of benzene-toluene (1: 1). The residue was used without further purification.

Preparation of the compounds of the invention Example 14

Preparation of 1- (3-F-2,3-dideoxy-tt-D-ribofuranosyl) thymine (VSA 419) (Method B) 23 mg of thymine (0.18 mmol) and S'F-S'- deoxy-S'-O-acetylthymidine was slurried in 1.2 ml of acetonitrile and 0.35 ml of N, O-Bis (trimethylsilyl) acetamide was added. The mixture was stirred at room temperature for one and a half hours. 0.05 ml of trimethylsilyl trifluoromethanesulfonate was then added. After stirring at room temperature for 192 hours, the mixture was poured with stirring into a 1: 1 (v / v) mixture of 20 ml of 10% aqueous KHCO 3 -ethyl acetate. The two phases were separated and the aqueous phase was extracted with 15 ethyl acetate (3 x 10 mL). The combined ethyl acetate phase was filtered and evaporated in vacuo. The residue was dissolved in dichloromethane-ethyl acetate 1: 1 and applied to a column of silica gel, and the column was eluted with dichloromethane-ethyl acetate 1: 1 to give 28 mg (53%) of starting material (VBS 20 423) (Rf 0.37 on TLC silica gel C ^ Clg-EtoAc 1: 1) and 18 mg (34%) 1- (3-F-2,3-dideoxy-5-O-acetyl-αD-ribofuranosyl) -thymine (VBS 424) (Rf 0.29 on TLC silica gel C ^-CE-EtoAc 1: 1).

NMR (CDgOD) 61.95 (s, 3H, CH3 ~), 2.12 (s, 3H, CH3COC, 2.3-3.0 (m, 2H, H-2'a, b), 4 , 15 (d, 2H, J4 ', 5' = 4.4 Hz, H-5'a, b), 4.81 (dt, 1H, J3'F, 4 '= 30.0 Hz, J4', 5 '= 4.6 Hz, H-4'), 5.23 (dd, 1H, J3'F, 3 '= 53.7 Hz> J2', 3 '= 5.0, H-3'), 6.36 (d, 1H, J 1 ', 21 = 7.57 Hz, H-1'), 7.27 (d, 1H, J H-6, 30 CH 3 = 1.47, H-6) 13 C ( CD30D) 612.80 (CH3), 20.87 (CH3CO), 39.40 (d, J-20.8 Hz, C-2 '), 63.37 (d, J = 12.2 Hz, C 5 '), 84.65 (d, J = 24.4 Hz, C-4'), 86.50 (s, C-1 ') 93.82 (d, J = 178 Hz, C-3') , 111.12 (C-5), 135.07 (d, J = 6.1 Hz, C-6), 150.48 (C-2), 163.68 (C-4), 170.30 (CRjCO).

DK16443 B1 16 mg of the compound VSB 424 was dissolved in 5 ml of saturated methanolic ammonia and left overnight at room temperature. The solution was evaporated and the residue was treated with acetone benzene (1: 4) to give crystals of the desired compound, VSA 419 (9.4 mg, 69%) UV Mnax (H2O) 269 nm.

NMR (DMSO-d6) Ή 61.79 (d, 3H, J CH 3, H-6 = 1.2 Hz CH 2), 2.16-2.90 (m, 2H, H-2 '), 3.2 -3.6 (m, 2H, H-5 '), 4.61 (dt, 10H, J3'F, 4' = 23.4 Hz, J4 ', 5' = ~ 4 Hz, H-4 ' ), 5.06 (t, 1H, J5 ', OH = 5.6 Hz, OH), 5.32 (dd, 1H, J3'F, 3' = 54.2 Hz J2'3 '= 4.9 Hz, H-3 '), 6.18 (dd, 1H, Jl'2' = 7.7 Hz and 2.1 Hz, H-1 '), 7.39 (d, 1H, J CH3, H- 6 = 1.2 Hz, H-6) 13C (DMSO-d6) 612.46 (CH3), ~ 39 (C-2 '), 61.17 (d, J = 11.0

Hz, C-5 '), 85.85 (C-1'), 87.15 (d, J = 20.8 Hz, C-4 *), 94.75 (d, J = 173 Hz, C 3 '), 109.15 (C-5), 135.63 (d, J = 6.1 Hz, C-6), 150.53 (C-2), 163.95 (C-4) EXAMPLE 2

Preparation of 1- (3-F-2,3-dideoxy-eD-ribofuranosyl) -5-propyluracil (VSA 409) (Method C) 56 mg of 5-propyluracil and 47 mg of 3 '-F-3' deoxythymidine were suspended in 1.2 ml of acetonitrile and 0.35 N, O-Bis (trimethylsilyl) acetamide were added. The mixture was stirred at room temperature for one and a half hours. 0.05 ml of trimethylsilyl trifluoromethanesulfonate was added. After stirring at room temperature for 138 hours, the mixture was evaporated in vacuo and 0.5 ml of 0 0 filtered and washed with 0.5 ml of 0 0. The combined aqueous phase was applied to a C ^ g column (HPLC) and eluted with methanol-water (35:65) at a rate of 7.0 ml / min. The λ anomer eluted after 35 12.9 minutes and the desired α anomer, VSA 409, after 18.0 minutes. Yield 9.3 mg (18%) UV λ max (H30) 269 nm, MS M + 272 (10%), 154 (100%), 119 (76%).

EXAMPLE 3

Preparation of 1- (3-F-2,3-dideoxy-gD-ribofuranosyl) -5-ethyluracil (VSA 411) (Method C) 5 51 mg of 5-ethyluracil and 48 mg of 3 '-F-3' deoxythymidine were obtained. slurried in 1.2 ml of acetonitrile and 0.35 ml of N, O-Bis (trimethylsilyl) acetamide were added. The mixture was stirred at room temperature for one and a half hours. 0.05 ml of trimethylsilyl trifluoromethanesulfonate was added. After stirring at room temperature for 161 hours, the mixture was evaporated in vacuo, 0.5 ml of water was added and filtered and washed with 0.5 ml of water. The combined aqueous phase was applied to a Cig column (HPLC) and eluted with methanol-water (1: 3) at a rate of 8.0 ml / min. The island anomer eluted after 12.3 minutes and the desired α anomer, VSA 411, after 16.4 minutes. Yield 13.1 mg (26%). UV λ max (H 2 O) 267.5 nm. MS M + 258 (9%), 140 (100%), 119 (67%).

Compounds of formula I have been synthesized where X

3 is CH 2 and O and R is CH 2 OH; R has trans configuration relative to the hydroxymethyl function at position 4 '. These compounds are all according to the invention and have the general formula: B1 f-fO-i / V-A X is CH2 and O

The synthesis of the compounds where X is CH 2 is depicted in the following reaction scheme. The compounds in which X is 10 C12-2 are separated into the enantiomers by separation methods of enantiomers well known to those skilled in the art. IN

the examples are set forth in detail in the preparation of compounds wherein R is H or CHQ, a compound wherein 1 JR is ethyl, propyl, isopropyl, ethenyl, prop-1-enyl, prop-2-enyl, isopropenyl, ethylnyl and the like. can also be manufactured.

The carbocyclic nucleoside analogs (X = CH 2) of the reaction scheme were constructed by forming a pyrimidine ring of the 20 amino group of 1 - [(3,4-trans-C-dibenzyloxymethyl) cyclopentyl] amine (compound 8). Addition of an alkoxypropenoate isocyanate derivative which can be prepared in known manner followed by acid catalyzed cyclization gives the dibenzyloxy protected carbocyclic nucleoside analogues 10 and 16. 8).

The uracil base of compound 10 can be further functionalized by reacting compound 10 under well-known conditions to those skilled in the art and such as e.g. described by Bergstrom et al. (J. Am. Chem. Soc. 1976, vol. 98, pp. 1587-1589) and Bigge et al. (Ibid. 1980, vol. 102, pp. 2033-2038). In this way, compounds are prepared wherein R * is ethenyl, propenyl, isopropenyl or ethynyl.

18 DK 168443 B1

The cyclopentylamine, compound 8, is built up from sodium cyanide and diethyl maleate. The ketone formed (compound 2) is protected, the two carboxylic acid ester groups are isomerized to trans, reduced and the hydroxy groups formed are protected (compound 5). The protection is selectively removed from the ketone, an oxime is formed and reduced to the amine, compound 8. This amine can be separated into the enantiomers by methods well known to those skilled in the art.

The carbocyclic nucleoside analogs (X = CH 2) where R is CH 2 OH are stereochemically depicted in cis position relative to the 4'CH 2 OH group and have been prepared in a manner analogous to that described in the reaction scheme. but using starting materials with opposite stereochemistry. In this way, the two compounds of Examples 10 and 11 were prepared.

Example 10 A Gr 25 Λ / '30

Example 11A is tf-fJ

35 I

19 DK 168443 B1

The trans-3,4-dihydroxymethyl compounds of formula I wherein X is 0 are prepared by reacting a persilylated pyrimidine base with methyl (3-hydroxymethyl) -2,3-dideoxy-D-erythro-pentofuranoside containing two hydroxy-protected-5 the groups (benzoyl). The reaction is catalyzed with a silyl triflate ester to give the α and δ anomers in approximately equal amounts. After condensation, the benzoyl groups are removed. The preparation of the α and β anomers of cytosine analogs is given in Example 5 and the preparation of 10 α and β anomers of thymine analogs is given in Example 6.

γ. The μµ, 0 B ί RO-β / 20 3-hydroxymethylpentofuranoside ring is remodeled by reacting a mono-protected epoxide of 1,4-butanediol with allyl magnesium bromide (the reaction scheme is set forth in the experimental part describing the synthesis of the starting materials of Example 5 and 6). Then, the allyl group is oxidized with osmium tetraoxide and sodium periodate, cyclized to form the pentofuranoside ring, and the hydroxy groups protected.

30 35 20 DK 168443 B1

Reaction Scheme for Preparation of Compounds in which X = CH out ,

\ / ~ noc «tCHtOH \ / \ QJ

fto / c '° ^ co ° ^ * L · L · 10

COOCHj H

is 100 - “^ /> 0 COOCU '* —ΟΗ A Jl 20 CWn _ w-> 25' -“ “r © * -« «c © S 6 / - '30 Q-OjO — r -Mjt OH. , * kÉy ±., \ Y ^ 35 Loc ^^ - ^) * —οα ^ ίτΟ)

i _L

DK 168443 B1 21

Scheme (continued) K

- ^? ' ^ ° -] - LftJc ^ o «· EtOCHr C-t-W = C = 0-v yf * 5 Lo6m L0 <b ^ ,? I S / <T / 2f * CH- ,. 'ix' ti- - ^ VJ —γ

—0¾ ^ —OH

15 ii lo R. * U Ji_ M 21 z '~ CH3 tICk 0-4Π) Λ / ^ α, ”i'-AT *' Jw ¢, 0 ^ Οη 25 · & ---> ^ / 16 \ _ / \ - / '^ / Z ft' - / * ^, / δ - & - ^ '= Η 30 ^ Y ^ S / ~ il *' - c / <3% / A) _ HO-i / ^ Λ

_> \ LJ

35]

-OH

Ilj R * ^ 20/2. ' EXAMPLE 4

Preparation of 1- [2 ', 3'-dideoxy-31-C- (hydroxymethyl) -α- and O-D-erythro-pentofuranosyl] -cytosine (Compounds 21a 5 and 21b)

A suspension of cytosine (120 mg, 1.08 mmol) and a small crystal (NH₂SO ^ in hexamethyldisilazane (2 ml) and trimethylchlorosilane (0.2 ml) was heated at reflux until a reflux was obtained). The solution was concentrated in vacuo and evaporated with dry xylene. The solid residue was dissolved in dry CH 2 Cl 2 (2 mL) under nitrogen and methyl 5-O-benzoyl-3 - [(benzoyloxy) methyl] -2 , 3-Dideoxy-D-erythro-pentofuranoside (170 mg, 0.46 mmol) was added followed by addition of t-butyldimethylsilyl triflate (0.22 ml, 0.96 mmol). at room temperature, the reaction was quenched by the addition of aqueous saturated NaHCO 3 and the resulting mixture was stirred for 30 minutes. The solution was diluted with CH 2 Cl 2 and washed with saturated NaHCO 3, dried, filtered and concentrated to give an anomeric mixture of the protected nucleoside. This mixture was treated with methanolic ammonia (20 ml, saturated) for 24 hours at room temperature. After concentration, the residue was dissolved in water and extracted with CH 2 Cl 2. The aqueous phase was concentrated to a small volume and applied to a semi-preparative C-18 reverse phase chromatographic column and eluted with water containing 2% methanol. First, the α-isomer was collected, followed by the isomer.

Thirty appropriate fractions were pooled and evaporated to give 33 mg of the α-anomer (30%) and 27 mg of the 0-anomer (24%).

et anomer: [α] 26 D - 54 ° (c 0.3 HoO); UV (HoO) K: 272 nm - D z z max 35 (e 10894); 1 H NMR (270 MHz, D 2 O): 1.92 (m, J 2, a 2 alpha = 13.5 Hz, J 2, a / 3, = 9 Hz, J 2'a, 1 '= 6.5 Hz' 1H ' 2'a), 2.5 (m, 1H, H-31); 2.7 (m, J 2, a 2, b = 13.5 Hz, = 23 H 2, J 2, a, = 6 Hz, 1H, H-2'b); 3.67, 8.69 (d and q, overlap, jJ, 3, = 6.2 Hz, J5, a 5tb = 12.5 Hz, J4.5, a = 5.3 Hz, 3H, H-6 'and H-5'a); 3.85 (q, Jg, a g, b = 12.5 Hz, = ^ Hz, 1H, H-5'b); 4.28 (ro, * 4T = ® Hz, ^ 41513 δ = 5.3 Hz, J4.5.b = 3 Hz, 1H, H-4 '); 6.1 (d and q, overlap, J5 6 = 7.3 Hz, J-p 2, = 6.5 Hz, 2H H-5 and H-1 '); 7.8 (d, J 5'6 - 7.3 Hz, 1H, 'H-6); 13 C NMR (25.05 MHz, D 2 O); 36.4 (C-2 '); 42.3 (C-3 *); 62.7, 63.6 (C-5 ', C-6'); 84.4, 88.2 (C-1 ', C-41); 96.6 (C-5); 141.9 (C-6); 158.1 (C-2); 66.8 (c-4).

O-Anomer: [α] 26π: + 64 ° (c 0.27 Ho0); UV (H + O) hi 272 nm (e 9208); 1 H NMR (270 MHz, D 2 O): δ 2.2-2.46 (ro, 3H, H-2 'and H-3'), 3.68 (d, J3, g, = 5.5 Hz, 2H, H-6 '), 3.76 (dd, 15 J4' 5'a = 5'5 Ez 'J5'a, 5'b = 12'5 Ez' 1H 'H_5'a)' 3'92 ( dd 'J4', 5 * b = 2'9 Hz ', J5'a, 5'b = 12.5 Ez' 1E 'H_5'b)' 4.01 (ro, Jo, 4, = 8.1 Hz, Jpja , 4 »= 5.5 Hz, Jøib 4» = 2.9

Hz, 1H, H-4 ''), 6.05 (d, J5 6 '= 7.3 Hz, 1H, H-5), 6.11 (dd, Jr 2, a = 7.0 Hz, J1 (2, b = 4.0 Hz, 1H, H-1 '), 7.91 (d, J5 6' = 7.3 Hz, 1H, H-6); 13 C-NMR (25.05 MHz, D20) δ 36.1 (C-2 '), 40.8 (C-3'), 62.7, 63.1 (C-5 'and C-6'), 84.7, 87.1 (C- 1 'and C-4'), 96.5 (C-5), 142.2 (C-6), 158.2 (C-2), 166.8 (C-4), Anal. ^ qH ^^ O ^ NOXO, 7

H 2 O: C, 47.3; H, 6.1; N, 16.6. Found C 47.2, H 5.8, N

16.4.

EXAMPLE 5

Preparation of 1- [2 *, 3'-dideoxy-3'-C- (hydroxymethyl) -α-30 and β-D-erythro-pentofuranosyl] -thymine (compound no.

22a and 22b)

Thymine (150 mg, 1.19 mmol) was condensed with methyl 3-C - [(benzoyloxy) methyl] -5-0-p-bromobenzyl-2,3-dideoxy-D-35 erythropentofuranoside (205 mg, 0 47 mmol) followed by the same method as described in Examples 1 and 2 to give an anomeric mixture of the protected nucleoside. The mixture was dissolved in ethanol containing NaHCO 3 (excess) and hydrogenated over Pd (10% on carbon, 1 atmosphere) for 3 hours. After working up the residue, it was further reacted with methanolic ammonia for 24 hours. After concentration, the residue was dissolved in water and extracted with CH 2 Cl 2 · The aqueous layer was concentrated to a small volume and subjected to semi-preparative C-18 reverse phase column chromatography and eluted with water containing 10% methanol. First, the α-isomer followed by the & isomer. Appropriate fractions were pooled and evaporated to obtain 40 mg of the α-anomer (33%) and 41 mg of the α-anomer (33%).

α-Isomer: [et] 28: -3.60 (c 0.36 HoO); UV (HoO) h: 268 m u z z mcLx 15 nm (e 13756); 1 H-NMR (270 MHz, D 2 O): 1.89 (d, J - 1.1 Hz, 3H, 5-CH 3); 1.96 (m, J2'a, 2b '= 13/2 Hz' J2'a, 3 * = 9'9

Hz, J2, and lt »7.7 Hz, 1H, H-2'a); 2.47 (m, 1H, H-3 '); 2.6 (m 'J2'a, 2'b' = 13.2 Hz 'J2'b, 3' = 8.1 Hz 'J2'b, l' = 6/2

Hz, 1H, H-2'b); 3.64 and 3.68 (d and q overlap, Jg »a 4i = 20.5 Hz, J5, a 5, b = 12.1 Hz, Jg, 3, = 6.1 Hz, 8H, H- 5'a, H-6 '); 3.81 (q, = 2.9 Hz, J5, b5, a = 12.1 Hz, H-5'b); 4.24 (m, = 8.4 Hz, J 4 + 5ta = 5.5 Hz, J 4, 4-b - 2.9 Hz, 1H, H-4); 6.11 (q, 2, 2, a = 7.7 Hz, J ±, =

6.2 Hz, 1H, H-1 '); 7.59 (d, J = 1.1 Hz, 1H, H-6); 1SC NMR

25 (25.05 MHz, D 2 O); 12.5 (5-CH 3); 35.7 (C-2 '); 42.7 (C-3 '); 62.6, 63.5 (C-51, C-6 '); 84.2, 87.3 (C-1 \ C-4 '); 111.6 (C-5); 138.1 (C-6); 152.4 (C-2); 167.3 (C-4).

O-isomer: [α] D 26 + 17.8 ° (c 0.41H0O); UV (HoO) 268 nm (e 8516); 1 H-NMR (270 MHz, D 2 O): δ 1.91 (s, 3H, 5- CH 3), 2.2 (m, 2H, H-2 '), 2.5 (m, 1H, H-3') ), 3.69 (d, J3, 6, = 5.9 Hz, 2H, H-6 '), 3.76 (dd, J5 «b / 5.a B 12.4 Hz, J5'b, 4 '= 5fl Hz' lH 'H ~ 5'b)' 3'9 J5'a, 5'b = 12'4

Hz, J5.a 4, = 2.9 Hz, 1H, H-5'a), 3.99 m, J4, / 5, a = 2.9 Hz, J4.5, 'b »5.1 Hz, J4, 3, - 8.1 Hz, 1H, H-4) / 6.14 (dd,

J1 ', 2'a = 4'8 Hz' Jl '2'b = 6.6 HZ / 1H' H “1} '7'73 (d' J

= 1 ', 1 Hz, 1H, H-6). 13 C-NMR (25.05 MHz D 2 O) δ 12.5 (5- DK 168443 B1 CH3), 35.4 (c-2 '), 40.9 (C-3'), 62.8, 62, 9 (C-5 'and C-6'), 84.5, 86.1 (C-1 'and C-4'), 111.7 (C-5), 138.3 (C-6), 152 , 7 (C-2), 167.5 (C-4).

Analysis calculated for C1; LH1605N2x0.8 H2 O: C 48.8, H 6.6, N 10.4.

Found C 48.9, H 6.1, N 10.2.

10 15 20 25 30 35 26 DK 168443 B1

Manufacture of intermediates

The starting materials for compounds 21 and 22 of Examples 4 and 5 were prepared according to the following Scheme 5 ad: OR 6 OR 6 OR 6 <f '- f * [10 Ηθ · - \ OR 5 OR 5 OR 5 15 R 2 = p-bromobenzyl = p-bromobenzyl 5 5

R = H R ° = H

6 T

R = p-bromobenzyl R = benzoyl

r6o I CJ

- "

25 J

R 50 p = R-p-bromobenzyl 5 R = benzoyl in 6 R = R = benzoyl (1S) (2S, 3R) -10β-bromobenzyl-3-C- (2'-propenyl) - 1,2,4-butanetriol

To a cold solution of allyl magnesium bromide -50 ° C (20 5 ml, 1M) in 100 ml dry diethyl ether under a nitrogen atmosphere was added a solution of (2S, 3R) -3 - [[(4-bromobenzyl) oxy] methyl ] oxirane-2-methanol-1 (1.36 g, 5 mmol) in 140 ml dry diethyl ether over 30 minutes. The mixture was stirred vigorously for 30 minutes at -50 ° C and then quenched with saturated aqueous ammonium chloride. The organic phase was collected and the aqueous phase was extracted with diethyl ether. The organic phases were combined and washed with hydrochloric acid (1M), sodium hydrogen carbonate (saturated), dried, filtered, concentrated and separated by column chromatography (toluene: ethyl acetate, 1: 5) to give the title compound (1g, 64g). %), [α] β: + 1.56 ° (c 1.03 CHCl 3); 1 H-NMR (100 MHz, CDCl 3): 1.8 (m, 1H, H-3); 2.13 (t, Jlt 2, = 3 = 6/8 Hz, 2H, H-1 ') in 3.3 and 3.0 (wide, 2H, OH-2); 3.59 # (m, 4H, H-1, H-4); 4.0 (m, 1H, 20H-2); 4.48 (s, 2H, CH 2 Ph); 4.94 and 5.09 (m, 2H, H-3'a, H-3'b); 5.78 (m, 1H, H-2 '); 7.14-7.5 (m, 4H, aroma); 13 C NMR (25.05 (MHz, CDCl 3); 30.6 (Cl +); 42.3 (C-3); 63.3 (C-4); 71.9, 72.3, 72.5 (CH2Ph, Cl, C-2 '); U6.4 (C-3'); 121.5, 129.1, 131.3 (aromatic C); 136.4 (C-2 and aromatic C).

B) (2S, 3R) -4-O-Benzoyl-1- O -p-bromobenzyl-3-C- (21-propenyl) -1,2,4-butanetriol

Benzoyl chloride (3.21 ml, 27.6 mmol) was added dropwise to a solution of compound 2 (8.54 g, 27 mmol) in dry pyridine (50 ml) at 0 ° C. The reaction mixture was stirred for 15 minutes at 0 ° C. Water (5 ml) was added and the solvent was evaporated. The residue was dissolved in dichloromethane and washed with hydrochloric acid (1M), aqueous sodium hydrogen carbonate (saturated), dried, concentrated and purified by flash chromatography (toluene: ethyl acetate, 2: 1) to give the 97 title compound (9.77 g , 86%). [Α] D: + 8.5 ° 28 C 168443 B1 (c 0.71, CHCl 3); 1 H NMR (100 MHz, CDCl 3); 1.95-2.43 (m, 3H, H-3, H-1 '); 2.59 (d, JH (OH) 2 = 3'9 Hz '1H' OH '2)' * 3.54 (m, sec order, 2H, H-1a, H-1b); 3.95 ( m, 1H, H-2); 4.34 (d, J4 g = 5.1 Hz, 2H-4); 4.48 (s, 2H, CH2Ph); 5.14 and 5.0 (m, * 2H, H-3'a, H-3'b); 5.78 (m, 1H, H-2 '); 8.1-7.14 (m, 9H, arom); 13 C-NMR (25 , 05 MHz, CDCl3); 31.3 (C-1 '; 40.4 (C-3); 70.1 (C-2; 64.0 (C-4); 72.3 (CH2Ph); 72 , 5 (Cl); 116.3 (C-31); 121.4-134.7 (arom.); 136.4 (C-2 '); 166.1 (COPh).

C) Methyl 3-C - [(benzoyloxy) methyl] -5-O-p-bromobenzyl-2,3-dideoxy-D-erythro-pentofuranoside

To an ice-cold mixture of compound b (7.5 g, 17.9 mmol) and N-methylmorpholine N-oxide (4.8 g, 35.5 mmol) in tetrahydrofuran water (3: 1, 70 ml), put OsO 2 in t-butanol (18 mL, 0.02 M, treated with 1% TBHP, 0.36 mmol). After a few minutes, the ice bath was removed and the reaction mixture was stirred overnight at room temperature under nitrogen. NaHCSO4 (2 g) was added and the mixture was stirred for 15 minutes. The solvent was evaporated and the residue was diluted with ethyl acetate, washed with HCl (1M), NaHCO3 (saturated), dried, filtered and concentrated. The crude compound was dissolved in 25 tetrahydrofuran (3: 1, 200 ml) and treated with NalO 2 (7.65 g, 35.8 mmol). The cis diol was completely cleaved after 30 minutes at room temperature. The tetrahydrofuran was evaporated and the aqueous residue was saturated with NaCl and extracted with diethyl ether. The organic phase 30 was dried and concentrated. The residue was treated with methanolic HCl (0.05%, 50 ml) for 10 minutes, neutralized with Dowex 2x8 (HCO3), filtered, evaporated and the residue was purified by flash chromatography (toluene; ethyl acetate, 3: 1) to give a anomeric mixture of the title compound (6.63 g, 85%) as a colorless syrup.

1 H-NMR (100 MHz, CDCl 3); 1.7-2.9 (three m, 3H, H-3, H-2a, H-2b); 3.31, 3.35 (2s, 3H, OCH 3); 3.6 (m, 2H, H-5), 4.1 (m, 1H, H-4); 4.4 m, 2H, H-6); 4.6 (m, 2H, CH 2 Ph); 5.1 (m, 1H, H-1); 7.1-8.0 (m, 9H, aromatic); 13 C NMR (25.05 δ (MHz, CDCl 3); 35.6, 36.4 (C-2); 38.7, 39.3 (C-3); 54.3, 54.5 (OCH 3) ; 65.7, 66.6 (C-6); 71.5, 72.35, 72.37, 73.8 (C-5, (CH 2 Ph)); 79.9, 80.1 (C-4) ); 104.8 (Cl); 121.0-136.4 (aromatic); 165.8 (COPh).

D) Methyl 5-O-benzoyl-3-C - [(benzoyloxy) methyl] -2,3-dide-oxy-D-erythro-pentafuranoside

A solution of compound c (1 g, 2.3 mmol) in dry diethyl ether (3 ml) was dissolved in liquid ammonia (50 ml) in a Dewar flask. Sodium (300 mg, 13 mmol) was added portionwise over 5 minutes. The solution was stirred for 30 minutes and then quenched with solid NH ^ Cl. The ammonia was evaporated in a stream of nitrogen and the solid residue was diluted with ethyl acetate. The solid was filtered off and washed several times with ethyl acetate. The filtrate was concentrated and then evaporated along with dry toluene. The crude residue was dissolved in dry pyridine (30 ml). Benzoyl chloride (0.8 ml, 6.9 mmol) was added and the solution was stirred for 40 minutes at room temperature, then water (5 ml) was added and the mixture was concentrated to dryness. The residue was dissolved in CH 2 Cl 2 and washed with aqueous HCl (1M), aqueous NaHCO 3 (saturated), dried, filtered and evaporated to dryness. Flash chromatography (toluene: ethyl acetate, 3: 1) afforded the title compound (580 mg, 68%) as an anomeric mixture. A small sample of the compound was separated using si-licagel. 1 H-NMR and m.p. of the Æ anomers were in accordance with the above.

EXAMPLE 6 DK 168443 B1

Preparation of 1 - [(3,4-trans-C-dihydroxymethyl) cyclopentyl] uracil (Compound 11) 3,4-Dibenzylcyclamine (1.0 g, 3.1 mmol) (Compound 8) was dissolved in 10 ml of CgHg under nitrogen. C-Ethoxyacryloylisocyanate (Y.F. Shealy, J. Hetrocycl. Chem. 1976, Vol.

13, p. 1015) (0.38 M in CgHg, 8.2 mL, 3.1 mmol) was added at 20 ° C with vigorous stirring. After a reaction time of 20 minutes, the solution was evaporated and the residue was dissolved in trifluoroacetic acid (20 ml, 70% aqueous). The solution was heated at reflux for 30 minutes. The solution was cooled and diluted with 50 ml of water and extracted with Et 2 O / toluene 1: 1. The organic phase was washed with water (3 x 50 ml) and dried with Na 2 SO 4 and NaHCO 3.

Filtration and evaporation gave a light brown gum of 1-20 [(3,4-trans-C-dibenzylomethyl)) cyclopentyl] uracil (for compound 10). This gum was dissolved in CH 2 Cl 2 (5 mL) nitrogen and treated with trimethylsilyl iodide (0.72 mL, 5.11 mmol). The resulting cloudy yellow solution was stirred for 1 hour at 20 ° C. The solution was evaporated, dissolved in CH 2 Cl 2 and extracted with water (3 x 2 ml). The aqueous phase was washed with Et 2 O (3 x 5 mL) and evaporated.

Purification by flash chromatography (Merck Si60) and elution with ethyl acetate (7) -methanol (1) -H 2 O (0.5). Evaporation of the pure fractions gave the title compound as a colorless gum.

1 H NMR Uses 250 MHz, D 2 O; 1.5-2.4 ppm-CH2 CHN, 4H, CHCH2O, 2H. 3.4-3.8 ppm (CH2O4H (m) 4.7-4.9 ppm CHN,

1H, 5.8-5.9 ppm (CH = CH, 1H (d) 7.7-7.8 ppm CH = CH 1H

(D).

Example 7 31 DK 168443 B1

Preparation of 1- [(3,4-trans-C-dihydroxyinethyl-) -cyclopentyl] thymine (Compound 17)

The compound was prepared as described for compound 11 in Example 7 using methoxymethacryloyl isocyanate instead of ethoxyacryloyl isocyanate (prepared as described by 6. Shaw and RN War purer, 10 J. Chem. Soc. 1958, p. 157) ethoxyacryloylisocyanat.

1 H NMR Uses 250 MHz, D 2 O; 1.87 ppm-CH 3 (d) 3H, 1.9-2.3 CH 2 CHN (m) 4H, 3.5-3.7 CH 2 OH (m) 4H, 4.7-4.9 (CHN (m)), 7.57 CH = C (s), 1H.

EXAMPLE 8

Preparation of 1- [3,4-trans-C-dihydroxymethyl (cyclopentylcytosine (Compound 14)) 1- [(3,4-trans-C-dibenzyloxymethyl) cyclopentyl] uracil (Compound 10, prepared as described in Example 7), 0.5 g, 1.9 mmol was dissolved in CH 2 Cl 2 (20 mL) and triethylamine (0.31 mL, 3 mmol) mesitylene sulfonyl chloride (0.33 g, 1.5 mmol) and dimethylaminopyridine ( 0.037 g, 0.3 mmol) was added under a nitrogen atmosphere and stirred vigorously The solution was stirred for 2 hours at 40 ° C and 2-nitrophenol (0.67 g, 5.0 mmol) was added together with triethylamine ( The solution was stirred overnight, diethyl ether (50 ml) was added and the solution was washed with 0.5 N NaOH (3 x 20 ml). The organic phase was dried over Na 2 SO 4 and The residue was purified by flash chromatography on silica gel (Merck Si60) (elution with EtoAc / cyclohexane 1: 1). The pure fractions were collected and evaporated to a colorless gum. The gum was dissolved in THF (5 ml) and NH 2 OH aqueous (2 5%, 2 ml) was added and the mixture was stirred at 0.5 bar and overnight at 65 ° C. The solution was evaporated and the residue was purified on silica gel eluting with 8: 1 ethyl acetate / methanol. Evaporation of the pure fractions gave a gum.

5

The gum was dissolved in CH 2 Cl 2 (1 mL) and trimethylsilyl iodide (0.018 mL, 0.125 mmol) was added under nitrogen. The solution was stirred for 60 minutes at 20 ° C and evaporated.

10

The residue was dissolved in water (1 ml) and washed with Et 2 O (2 x 2 ml). The aqueous phase was evaporated to give the title compound as a solid white material.

15-h NMR Uses 250 MHz, D 2 O; 20 ° C: 1.4-2.4 ppm (CH 2 CHN, CHCH 2 O (m) 6H 3.4-3.8 ppm CH 2 OH (m) 4H, 4.6-4.8 (CHN) (m) 1H, 5 , 8-5.9 ppm CH = CH (d) 1H 7.7-7.8 CH = CH (d) 1H.

EXAMPLE 9 20

Preparation of 1 - [(3,4-trans-C-dihydroxymethyl) cyclopentyl] -5-methylcytosine (Compound 20)

The title compound was prepared from 1 - [(3,4-trans-25C-dibenzyloxymethyl) -cyclopentyl] -thymine (compound 16) in the same manner as described for compound 14 in Example 8.

EXAMPLE 10 33 DK 168443 B1 1 - [(3,4-dis-C-dihydroxymethyl (cyclopentyl)] cytosine 5 (mixture of α- and β-anomers) • JO-α) · T iol o o TLC: EtOAc / MeOH / H2 O 7: 2: 1, Silica Rf: 0.25 1 H-NMR (D 2 O): 87.65 (d, 1H, CH = CH), 5.95 (d, 1H, CH = 15 CH), 4.75-4.65 (m, 1H, H-1 '), 3.70-3.60 (dd, 2H, CH 2 OH), 3.55-3.45 (dd, 2H, CH 2 OH), 2, 35-1.35 (m, 6H, H-2 ', H-3', H-4 ', H-5').

EXAMPLE 11 1 - [(3,4-cis-C-dihydroxymethyl (cyclopentyl)] thymine (mixture of α and β anomers) 25

A: LJ

0 V

TLC: EtOAc / MeOH / H2 O 7: 2: 1, Silica Rf: 0.35 1 H-NMR (D 2 O): 87.55 (s, 1H, C = .CH), 4.85-4.65 (m , 1H, H-1 '), 3.80-3.70 (dd, 2H, CH 2 OH), 3.65-3.55 (dd, 2H, CH 2 OH), 2.55-1.50 (m, 6H , H-2 ', H-3', H-4 ', H-5'), 1.85 (s, 3H, CH 3).

34 DK 168443 B1

Manufacture of intermediates

The starting materials for compounds 11, 17, 14 and 20 of Examples 6, 7, 8 and 9, respectively, were prepared as follows:

The carbocyclic ring was built using sodium cyanamide dimer in the diethyl maleate section as described by Dolly et al. [1].

i I io

Cyclopentanone-3,4-dicarboxylic acid dimethyl ester (Compound 1) was dissolved in ethylene glycol [2]. The solution was saturated with HCl. Ketalization was allowed to proceed overnight at room temperature. Then, the hydrochloric acid was removed at 12 mmHg pressure at room temperature. Distillation under reduced pressure (<1 mmHg) enabled 1-dioxolane-3,4-dicarboxylic acid dimethyl ester to be isolated.

The above compound is a 1: 1 mixture of cis and trans 20 dicarboxylic acid dimethyl esters (compound 2) which can be isomerized mainly to (30%) of the transform under the following conditions. The ketal is dissolved in toluene-methanol (5: 1, 10 ml / g) and sodium methylate (0.3 equivalents) is added and the whole mixture is heated at 50 ° C for 5 hours.

Then the reaction mixture is poured into water and extracted with ethyl acetate to give compound 3.

C NMR (CDCl 3): 173.5 (2) ester carbonyl; 114.9 (s) C-1; 64.0 (t, JCH = 147.74 (H3) dioxolano; 51.6 (q, JCH = 147.74 H3) OCHg, 43.5 (d, JCH = 173.3 H3) C-3 and C-4; 38.6 (t, JCH = 133.0 H3) C-2 and C-5.

1. L.J. Dolly. S. Esfandiari, C.A. Elliger, K.S. Marshall. J. Eng. Chem., 36, 1277 (1971).

2. B.G. Howard, R.V. Lindsey, J.Am. Chem. Soc., 82, 158 (1960).

B1 1-Dioxolano-3,4-trans-dicarboxylic acid dimethyl ester (for compound 3) is slurried in tetrahydrofuran (10 ml / g) and lithium aluminum hydride (0.32 g / g starting material) is added portionwise sufficiently with rapid stirring. to maintain a gentle reflux. Stirring is continued for 1 hour after the addition is complete. The reaction mixture is then poured into ethyl acetate and filtered over a Celite® bed. After evaporation of the solvent, the crude material is used in the next step.

10 1-Dioxolano-3,4-trans-C-dihydroxymethyl (compound 4) (7.8 g, 44.6 mmol) is dissolved in THF-DMF mixture (9: 1) and added to a suspension of 80% sodium hydride ( 2.81 g, 94 mmol) in the same solvent under reflux. Ca.

15 minutes later pure benzyl bromide is dropped under reflux. The reflux is maintained for 2 hours after the addition is complete. After cooling, the reaction mixture is poured into ethyl acetate and washed thoroughly with water.

20

The crude dibenzylated material (compound 5) is then dissolved in dichloromethane containing 15% isopropanol (5 ml / mmol) and toluene sulfonic acid monohydrate (1.05 equivalent) added. Stirring is continued for 4 hours, after which the reaction mixture is poured into 0.01 N NaOH and extracted with dichloromethane. Purification by column chromatography using hexane: ethyl acetate 1: 1 as eluent gives 1-oxo-3,4-trans-di-C-hydroxymethyl-3,4-di-0-benzyl-cyclopentane (Compound 6) (27.6 mmol, 9 g) in total yield of 62%.

1 H-NMR (CDCl 3); S, 7.30 (m, 10H) benzyl aromatic fractions; 4.5 (2.4H) benzyl CH 2; 3.5 (m, 4H) Cf 2.5 (m, 4H) H-2 and H-5; 2.2 (m, 2H) H-3 and H-4.

13 C-NMR (CDCl 3): 138.3, C-1; 127.9 and 126.8 benzyl aromatic carbon atoms; 73.0, benzyl CH 2; 72.2, -CH2O-; 41.7, 35 36 168163 B1 C-3 and C-4; 38.9, C-2 and C-5.

A solution of compound 6 (0.50 g, 1.54 iranol) in EtOH (30 mL) was refluxed and K 2 CO 3 (0/300 5 g, 2.2 mmol) was added followed by NK 26 g, 3.71 mmol) in small portions. The solution was cooled and diluted with 100 ml of water and extracted with CH 2 Cl 2 (3 x 50 ml). The combined extracts were dried over Na 2 SO 4 and evaporated. The residue was dissolved in THF (50 ml) and LAH 10 (0.23 g, 6.16 mmol) was carefully added. The solution was heated at reflux for 5 hours, cooled and quenched with water (1 ml). The solution was diluted with Et 2 O (200 mL) and filtered through Celite®.

The solution was washed with water (3 x 20 mL), extracted with aqueous hydrochloric acid (3 x 20 mL), extracted with aqueous hydrochloric acid (3 x 20 mL, 2M), and the aqueous phase adjusted to alkaline reaction with NaOH (aqueous) to pH 14.

The aqueous phase was extracted with Et 2 O (3 x 20 mL), the combined extracts were dried over Na 2 SO 4 and evaporated to give a yellow oil. The oil was purified by flash chromatography on silica gel (Merck Si60), eluting with OE CC / / MeOH / NHg 95: 3: 2. The pure fractions were evaporated to give 1 - [(3,4-trans-C-dibenzyloxymethyl) cyclopentyl] amine (compound 8) as an oil.

1 H-NMR Uses 250 MHz, (CDCl 3, 1.3-2.5 ppm CHCH20.2H, CH2CHNH2 4H, 3.3-3.7 CH2O, 4H, 4.4-4.7 (pH-CH 4H, CHNH2, 1H, NH2, 2H, 7.3-7.4 ppm CgHg-O ^ 10H.

Biological experiments

Study I. Effect of Compounds of Formula I on 35 HIV in H9 Cells 37 DK 168443 B1

Materials and Methods: HIV infection of H9 cells 5 H9 cells, 10 cells per well on a 24-well plate, suspended in 2 ml of RPMI medium containing 10% fetal calf serum, 100 µg / ml penicillin, 10 µg / ml streptomycin sulfate and 2 μ9 / πι1 polybrene were exposed to HIV (HTLV-IIIg) and different concentrations of the test compounds. The plates were incubated at 37 ° C in 5% CO 2 for 6-7 days. The contents of each well were then homogenized with a 10 pipette and transferred to a centrifuge tube. After centrifugation for 10 minutes at 1500 rpm. the supernatant portion was removed and the cell pellet analyzed by fixation in methanol on glass plates. Human HIV positive serum diluted 1:80 or 1: 160 was added and 15 were incubated at 37 0 for 30 minutes. Next, the plate was washed with phosphate buffer in saline (PBS) containing 2 + 2+ containing Ca and Mg. Sheep antihuman conjugate (FITC) was added and after a new incubation the plate was washed again with PBS. Contrast staining was done with Evans 20 blue, and after drying, the frequency of HIV antigen-containing cells was determined by microscope. The experimental results are given in Table 1.

25 30 35

Table 1 38 DK 168443 B1

Concentration (µM) for 50% inhibition (IC ^ q) of human immunodeficiency virus proliferation in cell culture 5 ----

Compounds IC5Q (µM)% Inhibition 1- (3-fluoro-2,3-dideoxy-10 α-D-ribofuranosyl-5-ethyluracil (USA 411) 0.1 50 1- (3-fluoro-2,3-dideoxy-αD) -ribofuranosyl-5- propyluracil (USA 409) 2,5 50 1- [2 ', 3'-dideoxy-3T C- (hydroxymethyl) - is -D-erythro-pentafuranosyl] - cytosine (compound 21a) 5 50 1- [21,3'-dideoxy-3'C- (hydroxymethyl) - "- D-erythro-pentafuranosyl] -thymine (compound 22a) 50

Table 1 shows that the compounds tested in question are active inhibitors of HIV virus propagation.

To show the effects of known antiherpes compounds Acyclovir and Ganciclovir on HSV-1 and HIV multiplication, the following experiments were performed, the results being given in Table 2 below.

39 The inhibition of multiplication of HSV-1 (strain C-42) was determined in monkey kidney cells (GMK) according to the method described by Ejercito et al. (J. Gen. Virol. 2, p. 234-241, 1968) and the inhibition of HIV was determined by the method described by Koshida et al. (Antimicrob. Agents and Chemother. 33, p. 778-780, 1989). The inhibition value is given as the concentration (µM) that gives 50% inhibition of virus multiplication.

TABLE 2

Investigation of antiherpes compounds against HSV-1 and HIV virus 15 -

Nucleoside IC, -n

analog HSV-1 HIV

Acyclovir 0.3> 50

Ganciclovir 0.05> 50 9Π w The above results show that Acyclovir, a widely used and powerful agent for the control of herpes simplex virus infections, is completely inactive against HIV and retroviruses. The same is true of the other tested compound Gan-

OK

ciclovir.

30 35 40 DK 168443 B1

Study II. Cellular toxicity 7

Hg cells, 2x10 cells per plate, was incubated in RPMI-1640 medium containing 10% fetal calf serum, 70 mg / l penicillin, 100 mg / l streptomycin and 10 mM hepes, with or without test compounds. The number of cells per cell plate was determined after 48 hours. Cells incubated without test compound then underwent two cell division cycles.

10 F5000 cells that are human embryonic cells, 1x10B cells per plate, was incubated in Eagle's minimal essential medium, added to Earle's salty, non-essential amino acids, 10% fetal calf serum, 10 mM Hepes, 70 ml / 1 peicillin and 100 mg / 1 streptomycin, with or without test compounds. The number of cells per cell plate was determined after 48 hours. Cells incubated without test compounds then underwent a cell division cycle. The results are indicated as TCgg, which is the concentration of the ion in µM of a compound which gives 50% inhibition of cell proliferation.

25 30 35

Table 3 41 DK 168443 B1

Cellular toxicity to H9 and F5000 cells 5 TC5Q (> iM)

Compound H9 F5000 1- (3-Fluoro-2,3-dideoxy-αD-ribo-furanosyl-5-ethyluracil (USA 411) 400,500 1- (3-fluoro-2,3-dideoxy-αD-ribo-furanosyl) -5-Raethyluracil (USA 419) 250 1- (2, 31-Dideoxy-3 'C- (hydroxy methyl) -αD-erythro-pentafuranosyl] thymine (Compound 22a)> 100

Table 3 shows that the test compounds exhibit TC50 values that greatly exceed the concentration of ICgg of Table 1 for 50% inhibition of HIV virus proliferation.

25

In addition, the compound became 1 - [(1'a, 3'0,4'α) -3 ', 4'-dihydroxymethylcyclopent-1'-yl] thymine (compound of formula 1 wherein X = CH 2, R 3 = CH 2 OH ) tested against HIV-1 type of virus.

30

The virus multiplication in H9 cells was inhibited 42% if the above compound was added at a concentration of 1 µg / ml.

The study was conducted under the following conditions: 42 DK 168443 B1

Medium: RPMI 1640.5% FCS, penicillin / streptomycin 5 VDB (virus degradation buffer): 50 mM Tris-HCl pH = 7.6, 35 mM KCl, 4 mM DTT, 1 mM EDTA, 1.3% Triton X-100 10 RT test:

10 µl carbon cycle supernatant, 40 µl VDB and 50 µl reaction mixture to achieve a final concentration of 100 mM

Tris-HCl pH = 7.6, 100 mM KCl, 4 mM MgOU, 4 mM DTT, 275 Z 3

15 ng / ml BSA / ml, 5 ng (rA) n (άΤ) ^. · ^ / ®! about H * dTTP

(specific activity 18,000 cpm / pmol).

5 H9 cells are adjusted to 2 x 10 celler cells / ml medium and seeded on microplates (96 wells / plate) at 100 µl cell suspension / well, yielding 2 x 10 celler cells / well.

The compound to be tested is dissolved at 10 mg / ml in DMSO = stock solution (stored at 20 ° C).

The compound dissolved in DMSO is diluted 25 times in medium to give 400 ng / ml. Additional dilutions to 40 ng / ml and 4 ng / ml are made in microplates.

50 µl of the dilutions 40 ng / ml and 4 ng / ml are transferred to 30 the "cell-containing" microplate with a multi-channel pipette (Final concentration: 10 and 1 ng / ml).

Finally, 50 nine virus suspensions are added to each well (with a repetitive "Eppendorf multiple"). Each plate has 35 at least 4 wells with virus, but no drug (virus control) and two wells without virus (medium control).

43 DK 168443 B1

The plate is placed in a plastic bag to avoid evaporation and incubated for 6 days in a CC 2 atmosphere.

Determination of RT activity 5 µl supernatant from each well is transferred with a multi-channel pipette to a new microplate, where 40 µl VDB is added to each well. Addition of 50 µl RT reaction mixture gives a final volume of 100 µl / well.

10

After 60 seconds of incubation, the entire test volume is transferred with a cell harvester to a 5% TCA pre-moistened filter mat. The filter is washed in 5% TCA and rinsed once in ethanol. After drying the filter mat at 60 ° C for 30 minutes, each filter (96 µm) is punched and placed in counters, then 2 ml scintillation liquid is added and the samples are counted (1 min) or the whole filter mat is placed in a plastic bag, 10 ml scintillation liquid is added , and the filter mat is counted on a Beckman Betaplate counter.

% reduction of RT activity is determined by comparing RT activity of virus control with the measured RT activity for each dilution of the compound.

25 30 35

Claims (20)

44 DK 168443 B1 Patent claims: 1. Pyrimid-2-one-1-yl compounds of formula 5 3 A r where A means: J is a: (a) Ί "γ 0 ^ \ Ν / 20 or Jk r1 1,1 £ y X is O or CH2 is H, alkyl containing 1-3 carbon atoms, -CH = CH2, -CH = CH-CH3, -CH2 -CH = CH2, -C-CH3 or -C = CH, and CH2 35 3 168 DK 168443 B1 R means F or CH 2 OH, both of which may have either cis configuration or trans configuration in relation to the hydroxymethyl group at position 4 ', and therapeutically acceptable salts thereof A compound according to claim 1, characterized in that A is A * 1 The FN I of 15 wherein R is as defined in claim 1. A compound according to claim 2, characterized in that R · · · · is H, CH ^ or ¢ 2¾. A compound according to claim 3, characterized in that R at position 3 'and the hydroxymethyl group at position 4' have trans configuration. A compound according to claim 1, characterized in that A is NH 9 fY 30 O -Y where R 1 is as defined in claim 1. 35 A compound according to claim 5, characterized in that R 1 is H; CH3 or C2H5. 46 DK 168443 B1 A compound according to claim 6, characterized in that R 1 is H. A compound according to claim 7, characterized in that R at position 3 'and the hydroxymethyl group at position 4' have trans configuration. A compound according to claim 1 for use as a medicament. 10 The compound of claim 1 for use in the therapeutic treatment of infection in humans caused by HIV virus. A pharmaceutical composition, characterized by containing as an active ingredient a compound according to claim 1. A compound according to claim 1 for use in the manufacture of a medicament for the therapeutic and / or prophylactic treatment of infections caused by a retrovirus or hepatitis B virus. Process for the preparation of a compound 25 according to claim 1, characterized in that A) condenses an optionally protected glycoside of the formula 30 P-7 JL & 4,5 where R represents F or CH 2 OR and R represents H or a protecting group, to the N1 position of a pyrimidine derivative corresponding to the group A of formula I, after which the α-anomer of the compound I formed is separated and any protective groups removed; 5 B) anomerizes an ε anomer of the formula .AR 0 -i 10 X X Π1 15 4 5 wherein A, X, R and R are as defined above to a mixture of α and δ anomers, whereupon the a -anomers are separated and any protective groups removed? B 25 C) transglycosylates a nucleoside of the formula B r preparing a compound containing group A as defined above, then separating the .α anomer and removing any protecting groups; 7 D) substitutes the group R in a compound of formula A R7 20 5 / V R50-1 x 5 7 wherein A, X and R are as defined above and R is a leaving group, with F or C1- OH, then 25 any protective groups are removed; or E) converts the uracil group ly 30 HH Y 35. a compound of formula I to a cytosine group 49 wherein R * is as defined above, and then the compound of formula I optionally converted to a therapeutically acceptable salt. 15 20 25 30 35