CA2203672A1 - L-ribofuranosyl nucleosides - Google Patents

Abstract

This invention relates to .alpha. and .beta. L-ribofuranosyl nucleosides, processes for their preparation, pharmaceutical compositions containing them, and methods of using them to treat various diseases in mammals.

Description

~ -Ribofur~no-yl Nucl~osid-s Field of the In~ention This invention relates to L-ribofuranosyl nucleosides and intermediate or derivatives thereof useful in the synthesis of such nucleosides, processes for their preparation, pharmaceutical compositions containing such, and methods of using such compounds to treat various diseases, particularly cancer,in mammals.

B~c~u.~r,d of the In~ention Perigaud, C., et al, Nucleosides and Nucleotides, 11(2-4), 903-945 (1992), provide a useful overview of the current state of the art relating to the use of nucleosides and/or nucleotides as chemotherapeutic agents (including use as anticancer, antiviral and antibacterial agents). As described in this review article, the term "nucleoside(s)" relates to naturally-occurring nucleosides which are distinguished depending on the base, for example, adenine and guanine (A and G, respectively~ have a purine base, whereas cytosine, uracil, thymine and hypoxanthine (C, U, T and H, respectively) have a pyrimidine base.

Nagasawa, N., et al., J. Or~. Chem., 32, 251-252 (1967), describe the production of certain D-ribopyranosyl nucleosides (particularly 9-(2'-Deoxy-~-D-ribopyranosyl) adenosine).

Fucik, V., et al., Nucleic Acids Research, Vol. 1, No. 4 (1974) 639-644, describe structural effects of chemical modification upon the affinity of purine nucleosides to cytidine-transport system in Bacillus subtilis using a series of modified derivatives including certain ribopyranosyl nucleosides.

Baud, M.V., et al., Tetrahedron Letters, Vol. 31, No. 31, pp. 4437-4440 (1990), describes the synthesis of certain 2'-deoxyribonucleoside compounds starting from available sugars (2-deoxyribofuranosyl or pyranosyl). The compounds described in this paper are all D-isomers.

Spadari, S., et al, J. Med. Chem., 35, pp. 4214-4220 (1992), describes certain L-~-nucleosides useful for treating viral infections including Herpes Simples Virus Type I.

Holy, A., Nucleic Acid Chemistr~, Vol. 1, 347-353 (1978), describes the synthesis of 2'-deoxy-L-uridine.

WO 92/08727 describes certain 2'-L-desoxyuridines and their use for treating viruses.

As is well known, sugars found in natural nucleic acids are D-ribose and D-deoxyribose in almost all cases. Much research has been done to investigate the chemical and biological activities of the D-isomers of ribonucleotides and ribonucleosides, however, far less work has been done with the L-isomers. This is primarily due to the fact that the synthesis of the L-isomers is much more difficult, often involving the optical resolution of the D,L-isomers of nucleosides with the aid of microorganisms and enzymes. (See generally, Asai, M., et al., Chem. Pharm. Bull., 15(12), 1863-1870 (1967).) The known activity of D-nucleoside compounds, and the successful commercialization of several of such D-sugar-nucleoside compounds, (see Perigaud, C., et al., supra, for a discussion of D-nucleoside analogs which have gained commercial acceptance) led in-part to the present work relating to the L-isomers of certain nucleoside analogs.

Perha~c the best known commercial nucleobase analog is 5-fluorc~~a- l (5-FU) the structure of which is shown below:

,~D,\N~H
o (s~

5-FU is commercially available from Roche and is one of the most commonly used drugs for treating certain types of cancer. The high acceptance of this drug is due in part to its extreme cytotoxic effects. However, it also has a narrow margin of safety and is, therefore, associated with many serious side effects including, for example, nausea, vomiting, diarrhea, alopecia, leukopenia, thrombocytopenia, etc. Additionally, 5-FU is primarily used in an intravenous formulation.

5-FU is currently dosed at short intervals due to the damage it does to normal cells. The patient is taken off chemotherapy for a time to allow recovery from the cytotoxic effects of the treatment. It is contemplated that if a drug is developed that is less cytotoxic to healthy cells it would no longer be necessary to treat the patient in periodic intervals, which may be associated with the development of multiple drug resistance often exhibited in treated cancer cells. Specifically, as a tumor is being killed the cells that are most resistant to the drug die slower and, therefore, when the treatment is stopped (often because of the toxicity to normal cells) the more resistant tumor cells are left to multiply.

A significant commercial nucleoside analog is azidothymidine (AZT), commercially available as Retrovir from Burroughs Wellcome. AZT, a ~-D-deoxy-ribofuranosyl derivative of the formula:

~ ~ CH3 (AZT

is useful as an antiviral agent, particularly against the virus responsible for the Acquired Immune Deficiency Syndrome (AIDS).

This compound, like 5-FU, is associated with a number of undesirable side effects including hematologic toxicity such as granulocytopenia and/or severe anemia.

Without intending to be limited, applicants believe that the L-nucleoside compounds as claimed in the present invention may be beneficial over compounds such as 5-FU and AZT since it is believed that L-nucleosides (as claimed) exhibit selective permeability to compromised cells. By compromised cells we mean cells such as cancer cells or other infected cells, whether the infection is bacterial, fungal, viral or parasitic. It is believed that the L-nucleosides of the present invention may be transported into or permeate these compromised cells, whereas in normal cells the L-nucleosides would not permeate. (See for example, Lin, T.S., et al., Abstract entitled "Synthesis and Biological Evaluation of 2l,3'-Dideoxy-L-Pyrimidine Nucleosides as Potential Antiviral Agents against HIV and HBV~ published J. Med. Chem., 37 (1994) 798-803; and Spadari, S., et al., J. Med. Chem., ~ (1992) 4214-4220.) Therefore, to the extent these L-nucleosides are selective for compromised cells, they are less harmful to normal cells than compounds like 5-FU.

In addition to this concept of selective permeability, in viral-infected cells where therapeutic compounds often have an inhibitory mechanism related to the RNA of the cell, it is contemplated that the enzymes of such viral-infected cells may be less specific than in a normal cell and, therefore, if you can permeate the cell with an L-nucleoside, a more primitive enzyme such as an organic phosphorylases, kinases or thymidilate synthase may recognize the compound in such a way as to cause inhibition.

Therefore, although certain nucleoside analogs and/or nucleobase analogs have been commercialized for indications such as cancer and/or AIDs treatment, there is a need for a nucleoside analog which is perhaps as cytotoxic as 5-FU or is less cytotoxic but more specific than 5-FU for cancer therapy and/or a compound which is more effective and/or better tolerated than AZT for treatment of viruses.

The present invention relates to a novel group of such L-ribofuranosyl nucleosides which have interesting activity as anticancer, antiviral, antiparasitic, antifungal, antibacterial and/or antimicrobial agents. These compounds are generally water soluble, which suggests that oral deliver may be achieved, and the activity of these compounds may be more selective for compromised cells as compared to normal cells.

W O96/13512 PCTrUS95/13716 Det~ilod De~criDtion of the Inv-nt$on There is provided by this invention ribofuranosyl nucleoside compounds having the formula (I):
B

O ~ R3 RO ~l ~

or a pharmaceutically acceptable salt thereof, wherein:
B is a naturally-occurring nucleobase (A, G, C, U, hypoxanthine or T) or a modified base comprising one or more substitutior.s selected from the group consisting of H, hzlogen, C1-C6 alky_, C2-C6 alkenyl, C1-C6 alkoxy, C3-C6 cycloalkv'-C1-C6 alko~.~, C3-C8 cycloalkyloxy, C3-C8 cycloalkylthio, C1-C6 alkylthio, a substituted amino group, an aryl, aralkyl, aryloxy, aralkoxy, arylthio, aralkylthio, a heterocyciic ring and an amino group, provided that when the base is a pyrimidine, the atom at position 4 in the base can be sulfur, and that when the base is a purine, the atom at posil c~ 6 in the base may be sulfur;

R is H, CGR-, P(5) R~R or 503H (wherein R5 is alkyl of 1-5 carbon a~sms cr an aromatic ring structure, R6 and R, are each H or alkyl of 1-5 carbon atoms and n is 2 or 3);

R. and R~ are independe..lly H, halogen, mono- or di-difluro, ORE
or B (wherein R~ is H, CORg, P(O)mRioR;l (wherein Rg is H2, substituted or unsubstituted alkyl of 1-5 carbon atoms or a substituted or unsubstituted aromatic ring structure, R1Q and R are each H or alkyl of 1-5 carbon atoms and m is 2 or 3)), provided that when R~ is OH, R2 and B can combine to form a 5-membered cyclic ring structure;

R3 and R~ are independently B, H or OR~2 (where R;2 is H, COR 3, P(O)FR;~R:~ (wherein R-. is substituted or unsubstituted alkyl of 1-5 carbor. atoms or a substituted or unsubstituted aromatic ring structure, R;~ and Rls are each H or alkyl of C1-C5 carbon atoms and p is 2 or 3)), provided that:

only one of R,-R4 can be B;

when R=H, R~ =OH, R2=H, R3=H and R4=B, then B cannot be ~, C, T, 5-FU, hypoxanthine, A, or G;

when R=H, Rl=OH, R2=OH, R3=B and R~=H, then B cannot be C;

when R=H, R~=OH, R2=OH, R3=H and R4=B, then B cannot be 5-FU, C, U, A or hypoxanthine;

when R=H, R;=OH, R,=H, R3=B and R4=H, then B cannot be 5-FU, A, C, G, T, U or hypoxanthine;

when R=H, R~=H, R2=H, R.=B and R~=H, then B cannot be A, C, G, ~, U, 5-FU, or hypoxanthine; and wher, R=H, R =H, R2=H, R3=H and R~=B, then B cannot be A, C, G, T, U, 5-FU or hypoxanthine.

Preferred compounds of the present invention include those compounds of formula (~) wherein:

R~or P.; is ~ an~i the other is H, such that when R3 is B the series is afid when R~ is B, the series is ~;

B is C, T, U, G, I, A, 5-fluorouracil, 6-thioguanine or 4-thiouracil;

R is H; and R-R~ are each H or OH, or when R2 is OH, R2 and B combine to form a five-membered cyclic ring.

Specifically preferred compounds of the present invention are the followina: ~-L-ribofuranosyluracil; 1-(2,3,5-tri-0-benzoyl-~-L-ribofuranosyl)-4-thiouracil; ~-L-ribofuranosyl-4-thiouracil; 1-(3,5-di-O-benzoyl-2-deoxy-~-L-~ibofuranosyl)-4-thiouracil; 2'-~-L-deox~rib^furanosyl-4-thiouracil; ~-L-ribofuranosyl-5-fluorouracil;

~-L-ribofuranosyl guanine; ~-L-ribofuranosyl-6-thioguanine and pharmaceutically acceptable salts thereof.

Also proYided by this invention are processes for the preparation of the compounds of formula (I), r~r~Aceutical compositions contAi~ing the compounds of formuls (I) _nd methods of using the compounds of formula (I) for the treatment of c ncer in a r~mmAl, ~s well as methods of using the compounds of formula (I) as antivirAls, antiparasitics, antibacterials, _ntifungals and ~ntimicrobial agents in a m~mmal.

The present invention describes a series of L-ribofuranosyl nucleosides usefut for treating various dise~ses (including cAncer and certain viruses). Compounds of this invention may be orally a~tive based on their water solubility.

The compounds of this invention wherein the nucleoside has a pyrimidine base (U, T, C or substituted pyrimidine base) which is linked to the ribofuranosyl sugar via ~ linkage (B is R, in a compound cf Formula (I)) can be made by the general Scheme I.

SC~EME I
General procedure to make ~-L-ribofuranosyl pyrimidines:

1. HMDS ClSiM~/ ~ \
BzO ~ ~ Ac + Base Snc ~ CH3CN ~ H

To a mixture of l-O-acetyl-2,3,5-tri-0-benzoyl-~-L-ribofur~nose ~1 mol) and pyrimidine base (1 mol) in ~nh~drous MeCN are successively added HMDS (1 mol), ClSiMe3 (O.8 mol) nd SnC14 (1.2 mol). The resulting clear solution is refluxed for 1 hour when TLC indie~tes completion of the reaction. The ~olvent is ev~porated ~nd the residue dissolved in EtOAc, washed with NaXC03 and H20. The EtOAc layer is dried, filtered and evaporated to give the crude product, which is either crystallized or purified on a silica gel column to obtain the pure 2,3,5-tri-0-benzoyl-~-L-ribofuranosyl pyrimidine compounds These oompounds are stirred with NH3/~eOH to give pure SUBSTITUTE SHEET (RULE 26~

PCT~S95/13716 ~-L-ribofuranosyl pyrimidines after purification and crystallization.

A more detailed schematic ior ~-linked pyrimidine compounds within the scope of the present invention is shown in SchPme I-A.

SCHEME I-A

L,R~x~e l.MeOH~2SO~ ~ ~ Mc 1.HE~HOA~n~O

2.BzCVPz ~ ~ 2.HOA~AQO ~ o~

OSiMc 2+ N ~ TMSOTf . ~ ~ NH~eOH ~ ~ ~
Mc3SiO 1 N ~ C1CH2CH2C1 ~ ~ \ HO ~ ~ \
0~_~ O~

o 4 H~ H~S/CISiM~ ~\ NH~lMeOH >
o~H SnC~CH3CN \ HO

5 ~P 6 NH~

2+ ~ H~DS/CIS~ f~ N]H~ OH r ~ SllC~CH3CN
0~ 0~

The compounds of this invention wherein the nucleoside has a purine base (A, I, G or su~stituted purine base such as thio-G) which is SU~STITUTE SHEET (RULE 26) linked to the ribofuranosyl sugar via ~ linkage (B is R, in a compound of Formula (I)) can be m~de by the general scheme II below.

SCHEME ~I
General procedure to make ~-L-ri~ofuranosyl purines:

Silylated l.TMSOTf/C~C~CH2Cl ~ \
B ~ ~ Ac + Base 2. ~eOH HB~

A mixture of purine base (2 mol) and (NH,)2SO, (cat~lytic amount) in ~MDS is refluxed until the solution becomes cle~r. The resulting clear solution is concentrated to yield silylated base to which anhydrous dichloroethane is added and the solution is cooled to 0C.
~nder nitrogen atmosphere a solution of 1-0-acetyl-2,3,5-tri-0-benzoyl-~-L-ribofuranose in dichloroethane (1 mol) nd TMSOTf (2.1 mol) are added to the above solution and stirred at room temperature for 16 hours. The rea~tion is quenched with saturated NaHCO3 solution and the solvent is evaporated. The residue is dissolved in EtOAc, washed with water and brine. After drying and evaporating the solvent, the residue obtained is separated on a silica gel column to give pure 2,3,5-tri-0-benzoyl-~-L-ribofur nosyl purines, which after stirring hith NH /Me~H and usual purification give pure ~-L-ribofuranosyl purines.

A more detailed schematic for the synthesis of ~-linked purine compounds within the scope of the present invention is provided in Scheme II-A.

SUBSTITUTE SHEET (RULE 26) W O96/13512 PCTrUS95/13716 SCHEME II-A

~ ~ l.DM~JN~/80C
Nl ~ - Nl TMSOTft ~ 2.MeOH/NH3 N / ~ ~ ClCH2CH2CL ~ \ HO ~ ~ \

N ~ N N ~ N

t NH~

NaOMeJ
MeOH

e~?\
~ N ~ N ~

11 ~ NH

C~
N~N TMSOTf/ ~ / \
~NJ~NJ\NH~C

3 ~ N~NHAc 12 --~N

NaOMe /\ l.l~ol~an~OH
MeOH CH OH / ~ .NH3~eOH

HO ~ \ HO J ~
~N~l~N~NH2 ~N~N~NH2 N ` NH N~NH

SUBSTITUTE SHEET (RULE 26) Wo96/13512 PCT~S95/13716 The compounds of this invention wherein the nucleoside has a pyrimidine base (U, T, C or substituted pyrimidine base ~uch as 5-F~
or thio-U) which is linked to the ribofuranosyl ~ugar ~ia ~ linkage (B is R3 in a compound of Formula (I)) can be made by the general Scheme III below.

SC~ME III
General procedure to make c-L-ribofuranosyl pyrimidines:
B~
~SPh Silylated 1. ~BS/M.S.4AIC~C12 ,~ \
~ ~ + B~ 2.H~/P~C~OH HO

A mixture of pyrimidine base (2 mol) in HMDS and ~mmonium sulfate (catalytic amount) is refluxed until the solution becomes clear.
The resulting clear solution is concentrated in vacuo to yield silylated base. To this silylated base in anhydrous CH2Cl2 under nitrogen atmosphere, l-thio-2,3,5-tri-0-benzyl-L-ribofuranoside (2 mol) 4A molecular sieves and NBS (l.l mol) are added. The reaction mixture is stirred at room temperature overnight and quenched with addition of Na,S;Oj solution. The organic layer is washed with water brine a~nd dried over Na250~. ~vaporation of the solvent gives the crude product which is pu-ified on a silica gel column to obtain pure 2,3,5-tri-0-benzyl-c-L-ribofuranosyl pyrimidines. These compounds are subjected to H2/Pd~C reduction, followed by purification and crystallization to give pure c-L-ribofuranosyl pyrimidines.

A more detailed schematic for the synthesis of ~-linked pyrimidines within the scope of the present invention is provided in c~h~me III-A.

SUBSTITUTE SHEET (RULE 26) W 096/13512 PCTtUS95tl3716 NHk ~NHC ~ --C- OCH3~ ~ H~ot CN 50% E~OH ~ ~ 0.2 ~ Ha o ~ C~H5CN .
CH~CN~3N ~ ~ P2S5ÇPy /
H~ E~\~ ~/

NH~eOH / \ NE~cOH
100C / \ r.~

NHl ~ 5 H~ H~J

~` PhSFVSnC~4 . ~ ~ s~ 2 N~H~h~31DMF~ ~ s~

~' ~ Ba3/CB2a2 ~/

NBSI~LS.4A~ ~ 2C12 H

The compounds of this invention wherein the nucleoside has a purine base whi~h is linked to the ribofurAnosyl sugar ~ia ~ linkage (B is R3 in a compound of formula (I)) and only one of Rl or R2 is OH, or where R2 is O~ and com~ines with B to form a cyclic ring structure, can be made by the general Scheme IV below.

SUBSTITUTE SHEET (RULE 26J

SCHE~E IV
General procedure to make ~-L-2'-Deoxyribofuranosyl purines:

Cl \ Silylated 1. NaH,/C~}CN ~/ \
~Tol ~ 2. ~eOH HO
B~

A mixture of purine ~ase (2 mol) ~nd NaH ~2.2 mol) is ~tirred in anhydrous C~3CN under nitrogen atmosphere at room temperature for 30 min. 1-Chloro-2-deoxy-3,~-di-0-p-toluoyl-c-L-pentofuranose is added to the reaction mixture and stirred for 2 hours. The reaction mixture is diluted with CHCl3 and filtered through Celite~. The filtrate is conce~trated, redissolved in EtOAc ~nd washed with water and brine. After drying and evaporatiny the solvent, the residue obtained is purified on a silica gel column to give pure 2'-deoxy-3,5-di-0-p-toluoyl-~-L-ribofuranosyl purines. These compounds Are treated with NH,/MeOH and then purified and crystallized to give pure ~-L-2'-deoxyribofuranosyl purines.

A detailed schematic for similar ~-linked deoxy-ribofuranosyl having pyrimidine bases is sho~ in Scheme IV-A.

SUBSTITLITE SHEET (RULE 26 W O 96/13512 PCTrUS95/13716 SCHEME IV-A

NH2CN ~----~N HC=C~--~CH~
,h;-~e N~

HO~ BzCN, ~ HCI/DM~'.
DMI' N
27 o 28 9 AIBN ~ ~ NaOMe/MeOH. H~
~ 3 Bu3 Sn~VBenz~e ~ 31 P~Ss~o~ane 2.H2,P~C

~ ~ NH3/MeOH ~ H~

34 ~ 32 - ~ 35 NH3/MeOH
lOoC

o\

H~/~/

~N~ 33 SUBSTITUTE SHEET (RULE 26) W O96/13512 PCTrUS9S/13716 The compounds of this invention comprising a-L-2'deoxy ribofur nosyl ~pyrimidines and purines (including 5-FU analogs of such) are shown in Scheme V, whereas Scheme V-A ~hows methods for making ~-linked L-2~-deoxy-ribofuranosyl compounds.

SC~ V
General procedure to make -L-2'-doexyribofuranosyl pyrimidines and purlnes:
B~
/(~)2 ~ ~ 1,3~ ~ O~}1~,3,3-t~la- o / ~
H ~ ~ ~opn~l di~ n~ PhCXC(S) ~H ~ ~ ~ D~LAP~
6~ )2 ~H
36a ~ 2 ~)2 0/ ~<o\~jCc B~Sn~VAIBN, ~ i~\~/ Toluene ' i~o W ~ H

'.-~)2 (~ OPn ~ )2 39a 38a 37a - ~ 3 l ~ 5 ~ -o- ( l, l, 3 ~ 3 -~-tr~ G~ 3-~llyl)-~-L-~r~blnofur~no~yll-~ur~n- or ~yr~ (36~) To a stirred suspension of (~ 1 mol) in pyridine is added 1,3-di~hloro-1,1,3,3-tetraisopropyldisiloxane (1.2 mol). This is stirred at room temperature until the completion of the reaction (five hours), the solvent is evaporated ~nd the residue is dissolved in ~tOAc and washed with water, 5~ HCl, water, ~aturated ~gueous NaHCO3 and brine. After drying over ~nhydrous Na2SO, it was filtered and evaporated to give the crude produot (~6~) which is used in the next step without further purifi~ation.

1-12'-O-P~ h~ocar~onyl-3',S'-0-(1,1,3,3-t-tr~
~1-llox n~ 3-~lyl)-t-L-~rab~nofur~nol~yl~-~urln- or ~y~
(~7~) To a solution of (~g) (1 mol) in anhydrous CH3CN is added 4-dimethly amino pyridine (DMAP) (1.9 mol) and phenyl lS
SUBSTITUTE SHEET (RULE 26) chlorothionoformate (1.1 mol). The solution is stirred at room temperature for 24 hours. Then the solvent is evaporated and the residue dissolved in EtOAc and washed with water, 5~ HCl, water, saturated aqueous NaHCO, and brine. The EtOAc layer is dried (Na2SO~), filtered and evaporated. The residue is purified on a silica gel column to give pure (37c).

3~,5~-0~ ,3,3-T--traioovro~yldi-iloxane-1,3-diyl)-~-~-~urine or ~yrimudine (~g) To a mixture of (~1~) (1 mol), AIBN (0.2 mol) in dry toluene is added Bu~SnH (5 mcl). The solution is deoxygenated with oxygen-free Ar then heated at 75C for four hours. The solvent is then evaporated and the residue is purified on a silica gel column to yield pure (38~!.

2~-Deoxy-~-L-~rine or pyrimudine (39~) A mixture of (~) (1 mo ) and TBAF (2 mol) in THF is stirred at room temperature. After completion of the reaction the solvent is evaporated anc the residue is dissolved in water and washed with ether. The water is evaporated and the residue purified on a silica gel colu~. to give pure 2'-Deoxy-c-L-purine or pyrimidine.

CA 02203672 l997-04-24 SC~EME V-A

O \ ~ h ~ 1,3~i~hln~}1~1,3,3-teba- ~ \
HOJ ~o~u~ si~n~ ~ , O ~ ~ PhOC(S)CU
O ~,~ N ' ~OJ ~ DMAp/py 6 l ~ 36 h /(~

~ Toluene ~ f ~ TBAF~HF

37 P~ , ~ ~p 38 P~

R =--C-OPh A general schematic for the synthesis of e and ~-L-2'-3'-dideoxyribofuranosyl pyrimidines and purines is pro~ided below in Scheme VI. Detailed schematics for ~-linXed 2'3' dideoxy pyrimidines and ~-linked 2'deoxyinosine are shown in Sch~mes VI-A
and VII.

SC~EME VI
General procedure to make e and ~-L-2',3'-dideoxyribofuranosyl pyrimidines and purines:

UTE S~ET`~R~2~) OAc Silyl~tcd EtAlC~ r 0~ c~
~i + Basc CH2C~2 1. Scparation 2.TBAFr~HF and H

A mixture of purine or pyrimidine base (1 mol) in HMDS and G onium sulfate (catalytic amount) is refluxed until the solution becomes clear. The resulting clear solution is concentrated in vacuo to yield silylated base. To a solution of this silylated base in nhydrous CHaCl2 under nitrogen atmosphere, a solution of l-~-acetyl-5-O-(tert-butyldiphenylsilyl)-2,3-dideoxy-L-ribofurnose is ~dded followed by the addition of EtAlCl2. The reaction mixture is stirred at room temperature for an hour and then poured into an ice cold mixture of CH2Cl, and saturated NaHCO3 solution. The mixture is stirred for lO m.n and filtered through Celite0. The organic l~yer is washed ~ith saturated NaHCO3 solution and brine. After evaporating the solvent, the c,0 crude product is separated on a silica gel column to give pure ~ and 0-5'-O-(tert-butyldiphenylsilyl)-2',3'-dideoxy purines and pyrimidines. These compounds are treated with TBAF to remove the ~ilyl protection, and then purified on a silica gel column to give pure ~ ~nd ~-dideoxy purines and pyrimidines.

The compounds of this invention wherein the nucleoside ~s ~
pyrimidine base (U, T, C or substituted pyrimidine base) which is linked to the ribofuranosyl sugar via ~ linka~e (B is R, in a compound of formula (I)) and are 2',3~-dideoxy compounds, can be made by the yeneral Scheme VI-A below.

SUBSTITUTE SHEET (RULE 26~

SCHEME VI-A

H~/ MM~ ~1 P~OC(S) DMUP~

31 ~ 40 ~ ~ 4 B~SnHyAlHN B0~ HOA~ , H~ lJ~O~ ~ H~
~1 ~ r.L 2~4CPhOPOC~h ',~
~ ~ ~ 4.N ~ sOH N~

42 O 43 O 44 NE~

H~ T~DMSC~ , TRn~ ~ NaOHyC~/B~CH~CN ~ r~
~0~ DMSO

6 ~ 45 ~ 46 B~SnH ~DM~ ~ l.~C H~
AIHN2.TBAF~rHF R=~--C~-so~o~cN

~ U~p o 48 Compounds of this in~ention wherein the nu~leoside is inosine (hypoxanthine) linked to the sugar via ~-linkage ~re shown in the general scheme VII below.

SUBSTITUTE SHEET (RULE 26) ~ - L - 2 ' -Deoxyino s ine C~O

L~ binn~:e BnOH~ICl ~ ~0~
~ C~Ph p-TsOH~DMF
HO

0~0 0~0 I~\o \~ NaHlCS2/MeI l~\o ~ BU3SnH
~1 ~ OC~h THF ~1 ~ OC~Ph Toluene OH O-C-SC~

O ~ O HO

oc~2Ph 2Ø5M HCl 2.p-TolCVPY

D~_OC~ HcvEther ~Tol ~Tol ~ N Na~CN
O~Tol / + ~ O~Tol /
~TolO~ ~ / ~ N ~ ~ ~TolO~ ~ /

56 ~ ~ N
HSCH2CH20H/H ~ 57 Cl NaOMe/MeOH

SUBSTITUTE SHEET (RULE 26 other compounds within the scope of the invention can be made based on the teachings of the schematics provided herein, as well as the specific examples incorporated herein and in view with what is known to the skilled artisan.

In addition to the teachings provided herein, the skilled artisan will readily understand how to make compounds within the scope of the present invention by applying well known techniques such as those described in Nucleic Acid Chemistrv. Imnroved ~nd New Svnthetic Procedures. Methods and Techniaues, Edited by Leroy B.
Townsend and R. Stuart Tipson, John Wiley & Sons, New York (1978);
and Chemistrv of Nucleosides and Nucleotides, Edited by Leroy B.
Townsend, Ne~ York, Plenum Press (1988-1991). Suitable methods for making various substitutions on purine nucleosides are provided in WO90/08147. Suitable methods for making substitutions on pyrimidine nucleosides are provided in W088/04662. The disclosure of both such applications being readily available to those skilled in the art and incorpGrate~ herein. Suitable methods for making substitutions within the sugar moiety of the presently claimed compounds are known to those skilied in the art and are described in various publica~ions including: US Patent 4,880,782; W088/00050; EP 199,451 A2; ~S Patent 3,817,982; Lange, P., et al, Progress in Antimicrobial and ~-~icancer Chemotherapy, Proceedinqs of the 6th International Cc~ ess -f C~ .hera~v, Univ. Park Press, England, 1970, Vol. II, p. 3~ ?; and To~send, et al., supra, all of which are inc_-p--ated herein by reference.

This in~JentiOn can be further understood by referring to the fo'lo~ins Examples and Tables below:

~xDeriment~l Fx~mnle 1 L-RibofuranosYll~racil Ste~ A
l-O-Acetyl-2, 3, 5-tri-0-benzoyl-~-L-ribose (~) To a solution of L-ribose (5.0 g, 33.31 mmol) in MeOH (150 ml), concentrated H25O~ (0.5 ml) was added and refluxed for 2 hours.
After cooling the reaction mixture, pyridine (30 ml) was added and the solven~s were evaporated. To the residue another 30 ml of pyridine was added and evaporated to dryness. The residue was dissolved in pyridine (5G ml) and CH2Cl2 (25 ml) then cooled to 0C
~i W O 96/13512 PCT~US9~/13716 and benzoyl chloride (19 ml, 166.55 mmol) was added dropwise and stirred at room temperature overnight. The solvents were evaporated and the residue dissolved in CHCl~ and washed with H~O and NaHCO3 and dried over anhydrous Na2SO4. After evaporating the CHCl3, the residue was coevaporated with toluene to give a brown residue. The brown residue was dissolved in 30% HBr/OHAc (67 ml) and the solution was stirred at room temperature for 30 minutes, after which time glacial acetic acid (47 ml) was added and the mixture was cooled to 8~C (internal temperature) in an ice-salt bath. Stirring and cooling were continued while H2O (34 ml) was added dropwise. The mixture was removed from the cooling bath and stirred another 30 minutes. Then the solvents were evaporated and the residue was dissolved in CHCl3 and washed with H2O and NaCHO3. The CHC13 was evaporated do~ to 50 ml, pyridine (50 ml) and acetic anhydride (9.5 ml) were added and stirred overnight at room temperature. Then the solvents we-e evaporated and the residue dissolved in CHCl;, washed with H O and NaHCO.. After evaporation of the solvent a ~rown residue was obtained and it was coevaporated with toluene. The brown residue was triturated with EtOH to give light brown crystals.
This was recrystallized from EtOH/EtOAc (5:2) to give 2 (8.15 g, g8.5~) as whi~e crystals: m.p. 126 - 127CC.

Ste~ B
1- (2, 3, ~-Tr ~ - ^-be~,zcy 7-~ -ribcfura..osyl ) uraci1 (~) A mixture of uracil (Q.4~ g, 3.96 mmol) and (NH~)2SO4 (catalytic amour.t) ir. HMDS (25 ml) was refluxed for five hours. The resulting solution was concer.trated under anhydrous conditions to yield silylated uracil. To a cooled (0C) and stirred solution of silylated uracil and 2 (1.0 g, 1.98 mmol) in dry dichloroethane (50 ml~, TMSCmf (0.77 ml, 3.96 mmol) was added. The reaction mixture was warmed to room temperature and stirred for 16 hours. The reaction was quenched with saturated NaCHO3 solution (5 ml) and evaporated to dryness. The residue was dissolved in EtOAc (100 ml), washed with water, brine, dried, filtered and evaporated to give a solid residue and it was purified on a silica gel column using EtOAc/CHCl~ (30 - 40~) to give a white solid which was crystallized from EtOH/petroleum ether to give pure 3 (0.914 g, 82.7~) as white crystals: m.p. 143 - 144C.

Ste~ C
~ L-Ribofuranosyluracil (4) Compound 3 (0.87 g, 1.56 mmol) in NH3/MeOH (100 ml) was stirred at room temperature overnight. The solvent was evaporated and the residue was dissolved in H2O (50 ml), washed with ether (3 X 25 ml) and evaporated to give a white solid and it was crystallized from 95% EtOH to give pure compound 4 (0.335 g, 87.2~) as white crystal:
m.p. 165 - 166C.

~xamDle 2 ~ L-Ribofuranosvl-5-fluorouracil Ste~ A
1-(2,3,5-Tri-O-benzoyl-~-L-ribofuranosyl)-5-fluorouracil (~) To a mixture of 5-fluorouracil (0.85 g, 6.54 mmol) and compound (3.0 g, 5.94 mmol) in anhydrous MeCN (100 ml) were successively adaed HMDS (1.25 ml, 5.95 mmsl), ClSiMe~ (0.6 ml, 4.76 mmol) and SnC1; (0.83 ml, 7.13 mmol). The resulting clear solution was refluxed for 1 hcur when TLC indicated completion of the reaction.
The solvent w2~ evaporated and the residue dissolved in EtOAc t250 ml), washed with NaHCO, and H~O. The EtOAc layer was dried, filtered and evaporated to give a white solid and it was crystallized from CHCl !MeOH (1 - 2~) to give compound 5 (2.11 g, 61.9~) as white crystals: m.p. 20& - 209'C.

Ste~ B
l-~-L-~ibofura.._syl-5-fluorouracil (6) Compound 5 (0.75 g, 1.30 mmol) in NH./MeOH (100 ml) was stirred at room temperature overnight and worked up as ln Example 1, Step C to give pure 6 (0.33 g, 96~) as white crystals: m.p. 147 - 148C.

F~mnle 3 1-~-L-Ribofuranosvlcvtosine Stec A
1-(2,3,~-~ri-0-benzoyl-~-L-ribofuranosyl)-~-acetylcytosine r7) To a mixture of N~-acetylcytosine (0.18 g, 1.19 mmol) and compound 2 (0.50 g, 0.99 mmol) in anhydrous MeCN (30 ml) were successively added HMDS (O.17 ml, 0.79 mmol), ClSiMe3 (0.10 ml, 0.79 mmol) and SnCl~ (0.14 ml, 1.19 mmol). The resulting clear solution was refluxed for one hour. Then the solvent was evaporated and the residue dissolved in EtOAc (100 ml), washed with NaHCO, and H2O.
After evaporaticn of the solvent the residue was purified on a W O96/13512 PCTrUS95/13716 silica gel colu~n using EtOAc/petroleum ether (70~) to give pure 7 (0.41 g, 70~) as a white foam.

Ste~ E
~ L-Ribofuranosylcytosine (8) Compound 7 (0.85 g, 1.42 mmol) in NH3/MeOH (100 ml) was stirred at room temperature overnight and worked up as in Example 1, Step C to give pure 8 (0.18 ~, 52~) as white crystals: m.p. 210C.

~mnl e 4 9-~-L-Ribofuranosvladenine SteD A
9-(2,3,5-Tri-O-benzoyl-~-L-ribofuranosyl)-6-chloropurine (9) A mixture of 6-chloropurine (1.22 g, 7.93 mmol) and (NH4)2SO4 (catalytic amount) in HMDS (25 ml) was refluxed for eight hours.
The resulting solution was concentrated under anhydrous conditions to yield silylated 6-chloropurine. To a cooled (0C) and stirred solution of si'ylated 6-chloropurine and 2 (2.0 g, 3.97 mmol) in dry dichloroethane (25 ml), TMSOTf (1.87 g, 1.6 ml, 7.93 mmol) was added. The reaction mixture was warmed to room temperature and stirred for 16 hours. The reaction was quenched with saturated NaHCO. solution (10 ml) and the solvent was evaporated. The residue was dissc ved in EtOAc (150 ml), washed with water, brine, dried, fiitered and evap^rated to give a solid residue and it was purified on a silica gel columLn using CHCl.:MeOH (5~) to give pure ~ (2.35 g, 99~) as foa-..

Ste~ B
g-~-T,-Ribofuranosyl~denine (10) A solution of 9 (1.00 g) in DME/NH; (50 ml) was heated at 80C in a steel bomb for 24 hours. After cooling, the solvent was evaporated and the solid obtained was stirred in NH,/MeOH (100 ml) overnight.
After the evaporation of the solvent, the residue was dissolved in water (50 ml), washed with CHC13 (2 x 25 ml) and ether (2 x 25 ml).
The water layer was evaporated and the residue crystallized from water to give pure 10 (0.30 g, 67~) as white crystals: m.p. 225C
(dec).

W O96/13~12 PCTAUS95/13716 Fx~mnle 5 9-~-L-Ribofuranosvlh~Doxanthine (~1) A mixture of 2 (1.05 g, 1.70 mmol), mercaptoethanol (0.48 ml, 6.9 mmol), and NaOMe in MeOH (100 ml) was refluxed for four hours. The reaction mixture was cooled, neutralized with glacial acetic acid and evaporated to dryness. The solid obtained was washed with CHCl3 and the residue was crystallized from EtOH to give pure 11 (6.198 g, 47~) as white crystals: m.p. 212 - 213C (dec).

F~xamnle 6 9-~-T,-RibofuranosvlcuAnine Ste~ A
9- (2, 3, 5-Tri-O-benzoyl-~-L-ribofuranosyl) -2-acetarnido-6-chloropurine (12 ) A mixture of 2-acetamido-6-chloropurine (1.68 g, 7.93 mmol) and (NH;) 253~ (catalytic amour,t) in HMDS (75 ml) was refluxed for 16 hours. The res~llting clear solution was concentrated under anhydrous cor,d_tions to yield silylated 2-acetamido-6-chloropurine.
To a cooled (0 C) and stirred solution of silylated 6-chloropurine and 2 (2.0 g, 3.96 mmol) in dry dichloroethane (100 ml), TMSOTf tl.6 ml, 7.93 mmol) was added. The reaction mixture was quenched with saturated NaHCO. solution (10 ml) and the solvent was evaporated.
The residue was dissolved in EtOAc (150 ml), washed with water, brine, dried, filtered and evaporated to give a solid residue and it was pu-ified on a silica gel colu~ using EtOAc/petroleum ether (40 - 50C) to give pure 12 (1.38 g, 53%) as white foam.

Ste~ E
9-~-L-Ri~of..ranosylgua.~ine (~) A mixture of 12 (0.58 g, 0.88 mmol), mercaptoethanol (0.25 ml, 3.53 mmol) and NaOMe (0.76 ml, 3.53 mmol, 25~ weight solution in MeOH (10 ml)) was refluxed for six hours. The reaction mixture was cooled, neutralized with glacial acetic acid and evaporated to dryness. The solid obtained was washed with CHC13 and the residue was crystallized from water to yield pure 13 (0.215 g, 86~) as white crystal: m.p. 248C (dec).

F~ATr~1e 7 9-~-L-Ribofuranosvl-6-thio~uanine (14) To a solution of 12 (0.68 g, 1.03 mmol) in anhydrous EtOH was added thio~rea (3.15 g, 2.06 mmol). The reaction mixture was refluxed for an hour and then the solvent was evaporated. The residue was dissolved in EtOAc and washed with water and dried. After evaporation of the solvent the crude product was purified on a silica gel column (5~ MeOH/CHCl3) to yield benzoylated thioguanine.
The product was debenzoylated by stirring with NH3/~eOH (100 ml) at room temperature overnight. After evaporating the solvent the solid obtained was dissolved in water and washed with CHCl3 (3 x 50 ml).
Then the water was concentrated and crystallized from water to give pure 14 (0.15 g, 58~) as yellow crystals: m.p. 227C (dec).

~Amnle 8 2-Amino-~-L-ribofurano~1'.2':4.5loxazoline (1~) A mixture of L-ribose (7.0 g, 46.63 mmol), cyanamide (3.92 g, 93.27 m~mcl) and lN NH~OH (20 ml) was heated in a 30 - 35~C water bath until the solids were dissolved. The reaction mixture was kept at room temperature for 30 minutes and heated again at 60C for an hour, during which time a white solid started to precipitate. After adding MeO~. (35 ml), this was kept in the refrigerator overnight, filtered and washed with MeOH and ether to give compound 15 (7.46 g, 92%) as white crystals: m.p. 195 - 196C (dec).

ExamDle 9 O ~--Anhvdrc-1-c-L-ribofuranosvluracil (~6) A mixture of compound 15 (7.86 g, 45.13 mmol) and methyl propiolate (14.04 mi, 15,.95 mmol) in 50% EtOH (100 ml) was refluxed for six hours. After cooling, the solvent was evaporated to dryness and coevaporated Iwice with EtOH. Then the solid obtained was boiled ir.
EtOH, cooled a~G filtered to give compound 16 (5.88 g, 57.6%) as white crystals: m.p. 215C (dec).

F:xAmnle 10 1-~-L-Ribofuranosvluracil tl7) A solution of compound 1~ (4.50 g, 19.89 mmol) in 30 ml of 0.2 N HCl was refluxed for two hours. After cooling, it was neutralized with Dowex (2 x 8-100) ion exchange resin. The resin was filtered and washed with water and the combined filtrates were evaporated and coevaporated with ethanol to give a hygroscopic foam, to which 1:1 mixture of acetone-ether (100 ml) was added and kept at room temperature fo~ two days. This was filtered to yield compound 17 (4.80 g, 86.6%) as white solid: m.p. 137~C.

W O96/13512 PCT~US95/13716 ~am~le 11 1-(2.3,5-Tri-O-benzovl-~-L-ribofuranosyl)-4-thiouracil (19) Ste~ A
1-(2,3,5-Tri-O-Benzoyl-~-L-ri~ofuranosyl)uracil rls) A solution of benzoyl cyanide (4.29 g, 32.76 mmol) in CH3CN (25 ml) was added dropwise to a suspension of compound 17 (2.0 g, 8.19 mmol) in CH3CN (50 ml) followed by Et3N. The mixture was stirred at room temperature for three hours and the solvent was evaporated to dryness. The crude material obtained was purified on a silica column using 50~ EtOAc/hexane as solvent to yield compound ~ (4.55 g, quantitative) as pale yellow foam.

Ste~ B
1-(2,3~5-Tri-O-benzoyl-~-L-ri~ofuranosyl)-4-thiouracil (12) Phosphorus pentasulfide (5.99 g, 26.95 mmol) was added to a solution of compound 18 (3.75 g, 6.73 mmol) in pyridine (70 ml) and was refluxed for four hours. After cooling, the solvent was evaporated anc the residue dissolved in CHCl~, washed with water and brine.
After drying over anhydrous Na2SO~, evaporation of the solvent gave the crude product which was purified on a silica gel column using 30% EtOAc/pelroleum ether as solvent to give compound ~2 (3.68 g, 95~) 2S golde~ yello~ foam.

Exæm~'e 12 ~ L-RibofuranosYlcYtosine (20~
CompGund 19 (3.6& g, 6.42 mmol) was treated with 200 ml of methanolic ammonia in a bomb at 100~C for 18 hours. After cooling to room tempera'ure, the solvent was evaporated to dryness and the residue dissolved in water (200 ml). The aqueous solution was extracted successively with CHCl3 and CCl4 (3 x 100 ml) to remove benzamide and methyl benzoate. The aqueous layer was treated with active charcoal, filtered through Celite~, evaporated to dryness and coevaporated with EtOH. The solid obtained was recrystallized from MeOH to give pu~e compound 20 (1.36 g, 81~) as white solid: m.p. 206 - 207^C (dec).

n le 13 ~ L-Ribofuranosvl-4-thiouracil (~1~
Compo~nd 19 (0.61 g, 1.06 mmol) in NH3/MeOH (40 ml) was stirred at room temperature overnight. The solvent was evaporated and the W O96/13512 PCT~US9~/13716 residue was purified on preparative plates using MeOH/CHCl3 (20~) to give pure compound ~ (0.185 g, 66.5~) as yellow foam.

~ m~le 14 l-~-L-ribofuranosyl-5-fluorouracil Ste~ A
1-Thio-2, 3, 5-tri-0-benzoyl-L-ribofuranoside (~) To a solution of 2 (0.50 g, 0.99 mmol) in CH2Cl2 (50 ml), thiophenol (0.11 ml, 1.09 mmol) was added and stirred at room temperature for 15 minutes. Then the reaction mixture was cooled in an ice bath and SnC1~ (O.07 ml, 0.59 mmol) was added dropwise and stirred at room temperature overnight. The reaction mixture was washed with 2N HCl (2 x 20 ml), water (25 ml), NaHCO3 solution (25 ml) and then with brine. After drying over Na2SO~, the solvent was evaporated and the residue was purified on a silica gel column using 15 - 20~
EtOA~/pet-Gle~. ether as solvent to give pure compound 22 (0.45 g, 82~) as an oil.

Ste~ B
1-Thio-2,3,~-tri-O-benzyl-L-ribofuranoside (~) To a sclution of 22 (0.45 g, 0.81 mmol) in MeOH (20 ml), NaOMe (0.03 ml, 0.16 mmcl) was added and stirred for 18 hours. The reaction mixture was ne-_.ralized by Dowex 50 ion exchange resin, filtered and evapcrated. ,_ this residue, DMF (20 ml) was added and cooled in an ice bath. To this cooled solution NaH (0.32 g, B.11 mmol) was added in portion and s.irred for 15 minutes. Benzyl bromide (0.96 ml, 8.11 mmol) was adde dropwise and stirred at 0~C for 2 - 3 hours.
The reaction was quenched with water after diluting with EtOAc. The EtOAc layer was washed with water (2 X 25 ml) and brine. After .
dryina and evaporation of the sol~ent, the crude product obtained was purified on a silica gel column using 5 - 10% EtOAc/petroleum ether as solver.t to yield pure Z3 (0.30 g, 73.5%) as an oil.

Ste~ C
1- (2, 3, ~-Tri-O-benzyl-~-L-ribOfUranOsyl) -5-fluorouracil (~) A mixture of 5-fluorouracil (0.15 g, 1.17 mmol) in hexamethyldisilazane (30 ml) and ammonium sulfate (catalytic amount) was refluxed for four hours. The resulting clear solution was concentrated in vacuo to yield silylated 5-fluorouracil as colorless oil. To a solution of silylated 5-fluorouracil in CH2C12 (20 ml) unde- nitrogen atmosphere were added NBS (0.11 g, 0.64 mmol), 4A

W O 96/13512 PCT~US95/13716 molecular sieves (0.21 g) and compound ~ (0.30 g, 0.58 mmol) in CH2Cl, (20 ml). The reaction mixture was stirred at room temperature overnight and quenched with the addition of Na2S203 solution. The organic layer was washed with water, brine and dried over Na2SO4.
Evaporation of the solvent gave the crude product and it was purified on a silica gel column using 5% MeOH/CH2Cl2 as solvent to give the pure c isomer ~ (0.23 g, 74.5%) as yellow oil.

Stez D
l-c-L-ribofuranosyl-5-fluorouracil (2~) To a solution of ~ (1.0 g, 1.87 mmolJ in CH2C12 (50 ml), at -78C
under nitrogen atmosphere, 1 M solution of BCl3 (20 ml, 20.57 mmol) was added dropwise. The reaction mixture was stirred at -78C for four hours, a 1:1 mixture of CH2Cl2/MeOH (50 ml) was added and the reaction mixture was brought to room temperature and the solvents were evaporated to dryness. The residue was coevaporated with MeOH
(25 ml) 5 times. The residue obtained was dissolved in water and washed with CH~_- (2 x 50 ml). The water layer was evaporated to give a white sclid which was crystallized from EtOH/ether to give the pure 2S (0.41 g, 83%) as white crystals: m.p. 150C.

Exam~le 15 2-~mino-~-L-arabinofurano~1' 2':4 5loxazoline (~6) A mixture of L-ara~inose (10.0 g, 66.60 mmol), cyanamide (5.60 g, 133.20 mmol), methanol (30 ml) and NH~OH (3.3 ml) was stirred at room temperature for three days and then kept at -10C overnight The product was ccllec~ed with suction, washed with methanol and ether to give compound 26 (9.60 g, 82.7%) as white crystals: m.p.
175C.

~mnle 16 C~,O--Anh~dro-~-L-arabinofuranos~luracil (27) A solution of compound 26 (15.0 g, 85.15 mmol) and methyl propiolate (23.0 ml, 261.75 mmol), in 50% aqueous ethanol (250 ml) was refluxed for five hours. After cooling, the solvent was evaporated to dryness and the solid obtained was coevaporated with EtOH twice.
Then the residue was dissolved in hot EtOH, cooled and filtered tG
give 27 (12.52 g, 65~) as white solid: m.p. 236C.

F.x~mnle 17 2'-Deoxv-~-T,-uridine (31) Ste~ A
3 ~, 5 ~, -Di-O-benzoyl -02, O' -anhydro-~-L-uridine (~8) A suspension of compound 27 (12.52 g, 55.31 mmol) and benzyl cyanide (15.96 g, 121.77 mmol), in DMF (100 ml) was treated dropwise with triethylamine (1.9 ml). The mixture dissolved rapidly and spontaneously deposited the product. The mixture was diluted with DMF (50 ml), stirred for three hours and finally diluted with ethanol (15 ml). This was poured into ether (250 ml), the precipitate collected with suction, washed with ether and dried to give 28 (21.92 g, 88%) as white solid: m.p. 260C.

Ste~ B
3 ~, 5 ~, -Di - O-benzoyl -2 ' -chl oro -2 ' -deoxy-~-L-uridine (~2) A mixture of compound 28 (21.72 g, 50.46 mmol), DMF (200 ml) and 6 M
HCl in DMF (4C.5 ml) was stirred at 100C for 90 minutes under exclusion cf a~mospheric moisture, cooled down and poured under stirring intc 1.5 L of water. The precipitate was collected with su~tio,., washed with 1 L of water and recrystallized from ethanol (60G m') ~c give pure 29 (19.9 g, 83.7%) as white crystals: m.p. 166 Ste~ ' 3 ,~ De.._-yl-2~-deoxy-~1-T-uridine (30) A m x~_-e -~ co.mpour.d 29 (18.89 g, 39.93 mmol), tri-a-butyltin hyd~_d~ .l. 159.7 mmol), benzene (400 ml), and azob_s~sob~tyronitrile (0.160 g) was refluxed under stirring for one hour. ~fter cooling, the solid was filtered and washed with benzen~. This solid was portion-wise recrystallized from ethanol (3.2 L~ t- yield pure 30 (15.9 g, 91.3~) as white crystals: m.p. 223 - 224-C.

Ste~ D
2'-Deoxy-~-L-uridine (31) To a solution of compound 30 (5.75 g, 13.17 mmol) in MeOH (75 ml) was added 4.62 M NaOMe (3.17 ml) and the reaction mixture was stirred at room temperature overnight. Then the solvent was evaporated and the residue was dissolved in water (250 ml) and washed with ether (3 x 100 ml). The aqueous layer was neutralized with Dowex 50(H ) ion exchange resin, filtered and evaporated. The WO96/13512 PCT~S9S/13716 .

crude product obtained was coevaporated with ethanol and crystallized from ethanol to give ~ (2.53 g, 84.3%) as white crystals: m.p. 162 - 163C.

~mnle 18 3~.5~.-Di-O-benzoYl-2'~eoxv-4-thio B-T,-uri~ine (32) The boiling solution of compound ~ (5.0 g, 11.45 mmol) in anhydrous dioxane was treated with phosphorus pentasulfide (2.58 g, 12.83 mmol) and the mixture refluxed under nitrogen atmosphere for 30 minutes. The mixture was then treated with additional phosphorus pentasulfide (2.85 g), refluxed for another 30 minutes, filtered while hot, and the solid washed with dioxane. The filtrate was evaporated to dryness and the crude product obtained was purified on a silica gel column using 20 - 30% EtOAc/petroleum ether as solvent to give an oil, which was coevaporated with ethanol and then crysta'iizea from ethanol-petroleum ether to give pure 32 (2.62 g, 50.5~) as yellow crystals: m.p. 136 - 137C.

~xam~le 19 2~-Deoxv-B-L-cvtidine (33) Comp~un 32 (3.0 g, 6.63 mmol) was treated with 200 ml of methanolic ammonia in a bomb at 10CC for 10 hours. After cooling to room tempC-2~ure, the solven~ was evaporated to dryness and the residue dis~-:.e_ ir. water (2GC ml). The aqueous layer was washed with ethe- ~ x 100 r, ) and treated with active charcoal, filtered thrc_~ e'ite~, and evaporated to dryness and coevaporated with ethan-:. T;r.e solid obtained was recrystallized from ethanol-aceto~ ile mixture (1:10) to give 33 (1.13 g, 75%) as white crystalc: m.~. 210~C.

~xamrle 20 2~-Deoxv-6-L-4-thiouridine (34) Compound 32 (0.25 g, 0.55 mmol) in NH3/MeOH (30 ml) was stirred at room temperature overnight. The solvent was evaporated and the residue was purified on preparative plates using MeOH/CHCl3 (20%) to give pure 34 (0.12 g, 88%) as yellow oil.

~x~mnle 21 2'-Deoxv-~-L-thvmidine (35~
Compound 31 (0.5 g, 2.19 mmol) was heated at 60 - 70C for six days in a mixture of 1.2 mi 37~ aqueous formaldehyde ana 1.2 ml lM KO~.

WO96/13512 PCT~S95/13716 ~very 24 hours the reaction mixture was treated with an additional 0.55 ml of lM KOH and 0.55 ml of aqueous formaldehyde. The solution was then diluted with water, adjusted to pH4 with Dowex 50(H~), filtered, the filtrate evaporated in vacuo and the residue coevaporated several times with ethanol. The final residue was dissolved in ethanol and made alkaline (pH10) by the addition of triethylamine, evaporated and coevaporated several times with toluene. The residue was dissolved in 22 ml ethanol and 50 ,ul concentrated hydrochloric acid was added. The solution was refluxed for five hours. Then the reaction mixture was made alkaline with triethylamine, evaporated and the residue was purified by flash-chromatography (20~ methanol-chloroform). 240 mg pure product and 280 mg mixture (containing starting material) was obtained. The product was dissolved in 20 ml ethanol, acidified with 27 ~ul concentrated hydrochloric acid and hydrogenated over 55 mg of 10%
palladium on cha~coal catalyst overnight under atmospheric pressure.
The mixture wac filtered, the filtrate made alkaline with triethylamine a~d evaporated to dryness. Flash-chromatography of the residue (20~ ethanol-chloroform) gave 35 (0.18 g, 34% overall yield) as a white powder.

Exam~le 22 2'-Deoxv-~-L-5-fluorouridine (39) Ste~ ~
1-[3',5'-0-(l,i,',3-Tetraisopropyldlsiloxane-1,3-diyl)-~-L-ribGfuranos~l]-~-fluorouracil (36) To a stirred suspension of (1.0 g, 3.90 mmol) in pyridine (40 ml) was addea 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane tl.65 ml, 4.68 mmol). This was stirred at room temperature until the completion o, the reaction tfive hours), the solvent was evaporated and the residue was dissolved in EtOAc and washed with water, 5%
HCl, water, saturated aqueous NaHCO3 and brine. After drying over anhydrous Na2SO4 it was filtered and evaporated to give the crude product 36 and it was used in the next step without further purification.

WO96/13512 PCT~S95/13716 Ste~c B
1-~2~-0-Phenoxythiocarbonyl-3',5'-0-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-~-L-ribofuranolsyl]-5-fluorouracil r37) To a solution of 36 (3.90 mmol) in anhydrous CH3CN (50 ml) was added 4-(dimethyl amino) pyridine (DMAP) (0.92 g, 7.56 mmol) and phenyl chlorothionoformate (0.6 ml, 4.29 mmol). The solution was stirred at room temperature for 24 hours. Then the solvent was evaporated and the residue was dissolved in EtOAc and washed with water, 5%
HCl, water, saturated agueous NaHCO3 and brine. The EtOAc layer was dried (Na2SO~), filtered and evaporated. The resultant oil was purified on a silica gel column using MeOH/CHCl3 (5~) to give pure 37 (0.47 g) as white foam.

~te~ C
3~,51-G-(1,1,3,3-~e t'-2i sopropyldisiloxane-1,3-diyl)-~-L-5-flucrouridlne (38' To a mixture c r 37 (0.46 g, C.72 mmol), AIBN (0.02 g, 0.14 mmol) in dry toluene (3C ml) was added Bu3SnH (2.0 ml, 5.05 mmol). The solution was deoxygenated with oxygen-free air and then heated at 75CC for four hours. Then the solvent was evaporated and the residue was purified on a silica gel column using EtOAc/petroleum ethe~ (30~) to yield pure 38 (0.34 g, 97~) as white foam.

Ste~ D
2~-Deoxy~ - fluorou~idine (39) A mixture of 38 (0.32 g, 0.66 mmol) and TBAF (1.4 ml, l M solution) in THF (15 ml) was stirred at room temperature. After completion of the reaction the solvent was evaporated and the residue was dissolved in water and washed with ether. The water was evaporated and the residue was purified on a silica gel column using MeOH/CHCl.
(l0~) to yield pure 30 (1.30 g, 80~) as an oil.

~xample 23 2~-~eoxv-~-L-5-fluorollri~ine (49~
Following the method of Example 22, this compound was made starting from l-~-L-ribcfuranosyl-5-flUorouraCil (25): m.p. 150C.
NMR: (DMSOd~) ~ l.90 (m,lH, H-2'a) 2.55 (m, lH, H-2'b), 3.33 (m, 2H, H-5'), 4.l9 (m, 2H, H-3' ~ 4'), 4.85 (br s, lH, OH), 5.43 (br s, lH, OH), 6.10 (dd, lH, H-l~), 8.15 (d, l:-, H-6), ll.78 (br s, lH, N-H) W O96/13512 PCTrUS95/13716 ~x~mn le 24 2l.3l-DideoxY-~-L-uridine (43) Ste~ A
2'-Deoxy-5'-0-(4-monomet~oxytrityl)-~-L-uridine (~) To a solution of compound ~ (2.0 g, 9.16 mmol) in pyridine (100 ml), 4-monomethoxytrityl chloride (3.11 g, 10.08 mmol) was added and stirred at room temperature for 24 hours. Then the solvent was evaporated and the residue was dissolved in EtOAc, washed with water, NaHCO3 and brine. After drying over anhydrous Na2SO~, the solvent was evaporated to give the crude product and it was purified on a silica gel column (5~ MeOH/CHCl3) to yield 40 (4.13 g, 94~) as white foam.

Ste~ B
2'-Deoxy-~'-0-(4-monomethoxytrityl)-3'-0-phenoxythiocarbonyl-~-L-uridine (41~
To a solution cf 40 (4.13 g, 8.25 mmol) in anhydrous CH3CN (100 ml) wzs added D1~ (0.46 g, 3.76 mmol) and phenyl chlorothiono formate (1.26 ml, 9.07 mmol). The solution was stirred at room temperature for 24 hours. Then the solvent was evaporated and the residue was dissolved in EtOAc and washed with water, 5% HCl, water, saturated aqueous Na~C5 and brine. Evaporation of the EtOAc layer gave the crude product and it was purified on a silica gel column (3.5%
MeOH/CHCl.) to yield pure 41 (3.95 g, 75~) as foam.

Ste C
2',3'-Dideoxy-~1-0-(4-monomethoxytrityl)-~-L-uridine (42) To a mixture Gf 41 (3.95 g, 6.20 mmol), AIBN (0.20 g, 1.24 mmol) in dry toluene (150 ml) was added Bu3SnH (16.7 ml, 62.0 mmol). The solution was deo~genated with oxygen-free air and then heated at 75C for five hours. The solvent was evaporated and the residue was chromatographed on a silica gel column (50 - 60~ EtOAc/petroleum ether) to yield pure 42 (2.36 g, 78.8~) as white foam.

Ste~ D
2',3'-Dideoxy-~-L-uridine (~3) Compound 42 (0.37 g, 0.76 mmol) in 80% acetic acid (5.0 ml) was stirred at room temperature for two hours, the solvent was evapcrated and the residue was coevaporated with toluene. The residue was pur~fied on a silica gel column (15% MeOH!CHCl,) to give W O 96/13512 PCTrUS95/13716 pure 43 and it was crystallized from MeOH/ether to give ~ (0.118 g, 73~) as white crystals: m.p. 122C.

F~mnle 25 2'-Deoxv-~-T,-inosine (58 Ste~ A
9-(2-Deoxy-3,5-di-0-~-toluoyl-~-L-ribofuranosyl)-6-chloropurine (57) A mixture of 6-chloropurine (0.40 g, 2.62 mmol) and sodium hydride (60% in oil, 0.11 g, 2.88 mmol) in anhydrous CH3CN (50 ml) was stirred under a nitrogen atmosphere for 30 minutes at room temperature. Crystalline compound 56, made according to the procedure described in Tetrahedron 43, 2355-2368, 1987, for the D-isomer (0.85 g, 2.18 mmol), was added and stirring was continued for 2 hours. After addition of CHCl3 (50 ml), the mixture was filtered through Celite~. The filtrate was evaporated and the crude was purified on a silica gel column using 50~ EtOAc/petroleum ether to give pure compound 57 (0.55 g, 50~) as white solid.

Ste~ B
2'-Deoxy-~-~-inosine (45) A mixture of 57 (0.20 g, 0.39 mmol), mercaptoethanol (0.11 ml, 1.55 mmoll, and NaOMe (0.34 ml, 1.55 mmol) in MeOH was refluxed for four hours. The reaction mixture was cooled, neutralized with glacial acet_c acid an_ evaporated to dryness. The solid obtained was washed with CH_'- and the residue was crystallized from H2OtMeOH to give pure 58 (C.070 g, 71~) as white crystals: m.p. 219 - 220C.

~tilitY
In ~itro activity against certain human tumor cell lines.

CELL LINES: Eight different established human cell lines CALU
(lung), COLO320 (colon), H578St ~breast), HT-29 (colon), MCF-7 (breast), OM-l (colon), SKLU (lung) and SKMES(lung), and two control cell lines (bone marrow and/or fibroblast cells) were utilized. All cell lines were obtained from the Tumor Cloning Laboratory, Institute for Drug Development, Cancer Therapy and Research Center, San Antonio, Texas. All cell lines grew as monolayers in the appropriate culture medium supplemented with heat-inactivated calf serum. All reagents were obtained from Grand Island Riological Co., Grand Island, Ne~ York.

WO96/13512 PCT~S95/13716 IN V~T~O EXPOSURE OF TUMOR CELLS TO COMPOUNDS: Stock solutions of intravenous (iv) formulations of certain of the compounds of the present invention (as shown in Table I below), as well as intravenous formulations of 5-FU (control) were used. The iv formulations of the compounds of the present invention were prepared with sterile buffered saline and stored at -70C until required for testing. The 5-F~ control formulation was prepared as suggested in the product literature.

Following trypsinization, tumor cells were suspended in tissue culture medium and exposed to the antitumor agents continuously at three different concentrations: l0, l and 0.l ~g/ml.

RADIOMETRIC MEAS'JREMENT OF GROWTH INHIBITION: Growth inhibition was assessed with the BACTEC System 460 (Johnston Laboratories, Towson, MD) after addi.ion of the antitumor agent to the cell in the respective growth medium containing '6C-glucose at a final concentrat.on of 2 uCi/ml. (See generally, C. Arteaga, et al-, k Radiometric Method for Evaluation of Chemothera~v Sensitivitv:
Results of Screenina a Panel of Hl~m~n Breast C~ncer Cell Tlines, Cancer Research, 47, 6248-6253 (l987).

Two mis of the tumor cell suspension containing radioactive glucose were seeded int~ sterile, disposable 15 ml vials by injection through self-sealing rubber-aluminum caps. For each cell line, the optimal number c r tumor cells needed per vial in order to show significantly measurable growth in this radiometric system varied.
The seeded vials were then incubated at 37C. Measurement of the release of 1~CO2 resulting from the metabolism of 1~C-glucose were performed on days 6, 9, 12 and 15 in the BACTEC instrument. This instrument flushes the ~'CO2 containing air out of the vials into an ionization chamber that converts dpm to growth index values.
Chemotherapy sensitivity was calculated by comparing the growth index values of drug-treated vials to that observed in control vials. Each data point represents triplicate values.

Results are shown in Table I below.

TART.~ I
COMPOUND~ SURVIVAL BONE~ SURVIVAL IC 50 MARRO~ TUMOR

5-FU 1.9 CALU 2.2 <0.6 COLO3201.0 <0.6 HS578T43.5 <0.6 HT29 1.2 0.613 MCF-7 0.8 <0.6 OM-1 1.7 1.47 SKLU 5.0 1.05 SKMES11.2 <0.6 ~-l- 96.6 CALU 89.1 >10 ribofuranosyl-uracil 2-amino--L- 114.6 OM-l 2.6 0.026 ribofurano [1',2':4,5]
oxazoline 2-amino-c-L- 109.5 CALU 89 44.6 arabinofurans MCF-7 88.8 187 [1',2':4,5' OM-1 41.3 5 oxazoline Oi,Oi-anhy-rc_~_ 70.9 OM-1 46.4 2.97 c-L-ribofuranosyl uracll ~-L- 120.1 COLO32082.3 34.9 ribcfuranosyl SKLU 88.8 240 cytosine 1-(2,3,5-tri-G- 85.9 HT29 75 >10 be.,zcyl-~
ribofuranosyl~-4-thiourac~_ 1-(3,5-di-C- 102.1 MCF-7 84.5 >10 benzoyl-2- OM-1 35.7 0.9&
deoxy-~-L- SKLU 77.7 88.7 ribofuranosyl)-4-thiouracii 2'-~-L-deo~ 98.2 OM-1 82.3 34.3 ribofuranosyl- SKLU 79.4 >10 4-thiouracil ~-L- 94.6 OM-1 0.4 0.87 ribofuranosy,- SKLU 71 43.4 5-fluorouracil ~-L- 129.9 COLO32081.9 188 ribofuranosyl- HT29 64.7 51 5-fluorouracil OM-1 71.9 37.8 SKLU 69.7 32.2 ~-L- 72.6 OM-1 39.2 5.9 ribofuranosyl cytosine WO 96/13512 PCTrUS95/13716 COMPOUND~ SURVIVAL BONE% SURVIVAL IC 50 MARROW TUMOR
~-L- 169.8 HT29 80.9 >10 ribofuranosyl OM-1 44.7 0.097 guanine 2~-~-L-deoxy 104 HT29 81.7 85.2 ribofuranosyl- MCF-7 66.9 16.6 5-fluorouracil OM-1 66.7 62.9 SKLU 83.9 32.5 2'-~-L-deoxy 158.9 COLO32067.0 57.4 ribofuranosyl HT29 72.2 30.4 thymine OM-1 37.6 6.5 SKLU 72.4 38.2 ~-L- 112.5 HT29 78.7 >10 ribofuranosyl OM-1 30.8 0.094 uracil 2',3'-~-L- 79.4 CALU 0.4 0.623 dideo~y ribofuranos~' ura-il ~-L- 91.2 OM-1 50.9 4.23 ribofuranos~_ adenine ~-L- 118.4 COLO32068.2 17.9 ribofuranosv' ~S578T93.4 >10 hypoxanthine MCF-7 96.8 38.1 OM-1 87.3 82.7 SKLU 82.5 48.1 ~-L- 97.4 HS578T66.6 17.1 ribcFuranosv_- OM-1 83.1 42.7 6-~hioguanine SKLU 45.1 8.7 2'-~-L-deoxy 108.8 COLO32019.6 6.56 ribofuranosyl- HT29 42.5 8.46 5-fiuorourac l MCF-7 86.9 60.7 OM-1 85.2 56.1 SKLU 86.2 59.9 2'-~-L-deoxy 64.9 CALU 28.9 0.696 ribofuranosyl adenine 2'-~-L-deoxy 109.6 OM-1 83.1 >10 ribofuranosyl hypoxanthine The data presented in Table I are compared to results achieved with 5-FU as the control. All compounds were dosed on an equimilimolar basis. Inhibitory concentration (IC 50) is defined as the concentration recl~ired to kill 50~ of the untreated cancer cells.

WO96/13512 PCT~S95/13716 Although the IC 50 of certain of the compounds listed in Table I may be higher than that for 5-FU (the control), the compounds of the present invention are generally less toxic to normal cells such as bone marrow or fibroblasts. This implies that the compounds of the present invention may have advantages over known cancer therapies as the claimed compounds may be less toxic and/or more selective for the tumor cells, thereby causing less serious side effects.
Additionally, because of their lower toxicity to normal cells, it is anticipated that the present compounds may be dosed at a higher rate to selectively increase toxicity to the cancer cells. In this regard, a therapeutic ratio for a given compound is typically determined by the following calculation.
survival bone marrow ~ survival tumor A therapeutic ratio of <80~ is considered ac'ive.

~n vi~o ~v~uation Representative compounds of the present invention are being tested in a variety of preclinical tests of anti-cancer activity which are indicative of clinical utility. For example, certain compounds will be tested i.~ vivo agai-.st human tumors xenografted into nude mice, specifically B15, MX-l and P388 Leukemia tumor lines were used.

Bl6 Melanoma B6D2Fl mice receive i.p. inocula of Bl6 murine melanoma brei prepared from B'6 tumors growing s.c. in mice (day 0). On day l, tumored mice are treated with drugs or vehicle controli the drugs, route of drug administration and schedule are selected as appropriate for the study in question. If dosing information for agents is not available, the maximum tolerated dose (MTD) is determined in initial dose-finding experiments in non-tumored mice.
In a typical experiment, drugs are given at their MTD and l/2 MTD
doses i.p. on a daily x 5 schedule.

W O96113~12 PCTAUS95/13716 The mean survival times of all groups are calculated and results are expressed as mean survival of treated mice/mean survival of control mice (T~C) x 100. A T/C value of 150 means that the treated group lived 50~s longer than the control group; this is sometimes referred to as the increase in life span, or ILS value.

Mice that survive for 60 days are considered long term survivors, or cures, in the B16 model. The universally accepted cut-off for activity in this model, which has been used for years by the NCI, is T/C=125. Conventional use of B16 over the years has set the following levels of activity: T/C<125, no activity; T/C=125-150, weak activity; T/C=150-20C, modest activity; T/C=200-300, high activity; T/C>300, with long term survivors~ excellent, curative activity.

Statistics are performed o-. the data using primarily the log rank p-value test.

P388 Leu~.emia This tes~ is conducted in exactly the same way as the B16 test. The tumor inoculu~, is prepared by removing ascites fluid containing P388 cells fr_.. tumored DB~'2 mice, centrifuging the cells, and then resuspe~ ng the leukemi2 cells in saline. Mice receive 1 x 105 P3~ c~::c i.~. or day C.

M~ a.. Ereact Tumor Xeno~raft Nude m~ re a-e implanted s.c. by trocar with fragments of MX-1 mammary carcinomas harvested from s.c. growing MX-1 tumors in nude mice hosts. When tumors are approximately 5 mm x 5 mm in size (usually about ten days after inoculation), the animals are pair-matched into treatment and control groups. Each group contains 10 tumored mice, each of which is ear-tagged and followed individually throughout the experiment. The administration of drugs or vehicle begins the day the animals are pair-matched (day 1). The doses, route of dru~ administration and schedule are selected as appropriate for the study in question. If the MTD dose of an agent is not kno~., it is determined in an initial dosing experiment in non-tumored mice. In a typical experiment, drugs are given at their MTD and i'2 MTD doses i.p. on a daily x 5 schedule.

The experiment is usually terminated when control tumors reach a size of 2-3 g. Mice are weighed twice weekly, and tumor measurements are taken by calipers twice weekly, starting on day 1.
These tumor measurements are converted to mg tumor weight by a well-known formula, and from these calculated tumor weights the termination date can be determined. Upon termination, all mice are weighed, sacrificed, and their tumors excised. Tumors are weighed and the mean tumor weight per group is calculated. In this model, the mean control tumor weight/mean treated tumor weight x 100~ (C/T) is subtracted from 100% to give the tumor growth inhibition (TGI) for each group.

Some drugs cause tumor shrinkage in the MX-1 model. With these agents, the final weight of a given tumor is subtracted from its own weight at the start of treatment on day 1. This difference divided by the initial tumor weight is the ~ shrinkage. A mean % tumor shrinkage can be calculated from data from the mice in a group that experienced M~-1 regressions. If the tumor completely disappears in a mouse, this is considered a complete regression or complete tumor shrinkage. If desired, mice with partial or total tumor regressions can be kept alive past the termination date to see whether they live to become iong term, tumor-free survivors.

S~ali~tics are pe-formed on the data using primarily the log rank p-value tes'.

Protocols for ~Iv-l In~cti~tion 8tudi~c General protocols for the testing of compounds in in vitro antiviral screens are disclosed in the following references:

1) Perez, V.L., Rowe, T., Justement, J.S., Butera, S.T., June, C.H. and Folks, T.M., An HIV-1-infected T cell clone defective in IL-2 production and Ca'~ mobilization after CD3 stimulation, J. Immuncl . 147:3145-3148, 1991.

2) Folks, T.M., Justement, J., Kinter, A., Dinarello, C. and Fauci, A.S., Cytokine-induced expression of HIV-1 in a chronically infected promonocyte cell line, Science 238:800-802, 1987.

W O96/13S12 PCT~US95/13716 3) Folks, T.M., Clouse, K.A., Justemer.t, J., Rabson, A., Duh, E., Kehrl, J.H. and Fauci, A.S., Tumor necrosis factor a induces expression of human immunodeficiency virus in a chronically infected T-cell clone, Proc. Natl. Acad. Sci. USA 86:2365-2368, 1989.

4) Clouse, K.A., Powell, D., Washington, I., Poli, G., Strebel, K., Farrar, W., Barstad, P., Kovacs, J., Fauci, A.S. and Folks, T.M., Monokine regulation of human immunodeficiency virus-1 expression in a chronically infected human T cell clone, J. I~unol . 142:431-438, 1989.

1. Inactivation of c~ fr~- ~IV-l.
Cell-free HIV-1 stocks are derived from culture supernatants of H-9 human T cells chronically infected with the HTLV-IIIB strain of HIV-1. Other HI~- strains including the MN and some African strains ma~ be used iate- for confirmatory purposes.
a) Ce'l-f-ee HTLV-III~:
Cell-free HIV-1 (5 x 10 to 1 x 10~ TCIDs0/ml, or median tissue culture infectious dose~ is either left untreated, or treated with RPr'_ 1640 culture medium, or with different concen~ra.ions of antivirals for various time intervals at _7'C, or at a temperature to be determined. After incubation, the trea~ed and untreated stocks are added to 5 x 105 washed ar,d pelle'ed target MT-4 cells. After 1 h incubation at 37C, the M.-4 ce~is are washed three times with RPMI 1604, resuspenae- in RPMI 1640 supplemented with 15~ fetal bovine serum (FB'`, and cultured in a 5~ CO2 humidified incubator at 37C. Cell viability is determined on day 7 of culture by the addition of the 3-(4,5-dimethyl-thiazol-2-yl)-2,5-d.phenyitetrazolium bromide ~MTT) dye, which changes in color in the presence of live mitochondria. All determinations are aone in triplicates.

b) Cell-free J~-CSF:
In addition to assessing the effects of antivirals on a lab strain of HIV-1 (HTLV-IIIB), it is also important to determine antiviral effects on a primary isolate of HIV-1 (JR-CSF), wr.ich only infects primary human peripheral mononuclear cells W O96/13512 PCTrUS95/13716 (PBMCs). Human PBMCs activated with phytohemagglutinin A
(PHA, Sigma Chemical Co.) are prepared by culturing PBMCs in RPMI 1640 culture medium supplemented with 10~ FBS (complete medium) and 2.0~g of PHA/ml for 1 day before used in infectivity studies. HIV-l (JR-CSF) untreated or treated as above are added to PHA-activated human PBMCs and incubated for 1 h at 37~C. After incubation, 1.0 ml of complete RPMI 1640 culture medium is added to the cells. Culture supernatants are collected on days 3, 6 and 9 of culture, and the amounts of HIV-1 p24 core protein are determined in triplicate by the HIV-1 p24 antigen capture assay (Coulter Immunology, FL, or NEN-DuPont, Wilmington, DE).

2. Inact~vat~on of cell~ oc~at-d xrv-l.
HIV-1-infected human cells to be used include the chronically infected H-9 cells (HTLV-IIIB or MN strains), and human PBMCs infected with HTLV-IIIB or with JR-CSF. HTLV-IIIB and MN infected H-9 cell lines are available from various laboratories including those listed in the references cited above. For infected human PBMCs, fresh human PBMCs are obtained from normal volunteers and stimulated with PHA, then infected with HTLV-IIIB or JR-CSF, as described above. On day 7 after in ~itro infection, infectivity is checked by testing for the presence of HIV-1 p24 in the culture supernatants. Infected cultures are divided in equal aliquots. One set is then treated with antivirals at different concentrations for va-ious time intervals, whereas one set is left untreated. Culture supernatants collected on days 3, 6 and 9 of culture will be assessed for HIV-1 p24 levels by the p24 antigen capture assay kit.
Cells from these cultures can also be used in immunofluorescence (IF) studies to determine the percentage of cells expressing HIV-1 antigen(s).

3. Inact~vation of ~IV-l lat--ntly ~nf--ct--~ c~ll~.
These assays are designed to study the effects of antivirals on HIV-1 latently infected cells. One or more of the following HIV-1 latently infected human cell lines can be used (J1-1, U1/HIV and ACH-2 obtained from the NIH AIDS Research and Reagent Reference Program, Rockville, MD). These cells are characterized by HIV-1 infe_ticr. without significant HIV-1 viral replication unless they are stimulated with different cytokines which results in a 10 - 100 fold increase in HIV-1 replication. J1-1, or U1/HIV, or ACH-2 cells are seeded in 96-well round-bottom tissue culture plates to give 5 x 105/well in RPMI 1640 supplemented with 15% fetal bovine serum (FBS). The cells are either left untreated or treated with different concentrations of antivirals for various time intervals.
Subsequent to treatment, treated and untreated cells are washed three times in RPMI 1640 and are stimulated as follows.

The J1-1 cells are stimulated with 1000 U of ~ tumor necrosis factor (-TNF, Genzyme) for 48 h at 37C as previously described (Reference 1) .

The U1/HIV-1 cells are stimulated with 20~5 - 40~5 PHA-culture supernatant (Electronucleonics) for 48 h at 37C (Reference 2). The PHA-superna_z..t will either be purchased from Electronucleonics or will be prepare~ in our laboratory. To prepare PHA-supernatant, normal human PBM_ will be cultured at a cell density of 10~ cells/ml in RPMI 1640 supplemented with 15~ FBS and 10 ,ug/ml of phytohemagglu inin A (PHA, Sigma Chemical Co.). The culture supernatant will be harvested, filtered through a 2 ~m filter and used tc stimulate the U1/HIV cells as described above.

The ACH-2 cells will be stimulated by addition of 1.0 ~M of phorbal 12-myristate _^~ acetate (PMA, Sigma Chemical Co.) for 48 h at 37~C
as described (References 3 and 4 above). At the end of the stimulation period, culture supernatants are collected and HIV-1 expression is assessed by the HIV-1 p24 antigen capture ELISA
(DuPont) and by the reverse transcriptase (RT).

In ina~'ivation of cell-associated HIV-1 experiments, the treated and un.reated cells could also be submitted to PCR analysis.

4. Inhibition of ~IV~ c-~ syncytiu~ form~t~on.
HIV-1-infected H-9 cells are left untreated or treated with antiviral as described above. Treated and untreated cells (5 x 10 cells/well) are added to 96-well flat-bottom microtiter tissue culture plates containing 1 x 105 indicator SupT1 human T cells/well in comple.e RPMI 1640 culture medium. Following overnight W O96/13512 PCTrUS9S/13716 incubation at 37C, syncytium formation is scored by two independent people using an inverted microscope scope.

5. Cytotoxicity studl--s.
The cytotoxicity of the antivirals can be tested on a variety of cell types. All of the cell lines used above and normal human PBMCs are incubated with different antiviral concentrations for various time intervals as described above. Cytotoxicity is determined by the MTT dye method (see above) and by [3H]thymidine uptake and scintillation counting.

Dosaae ~nd Formulation The antitumor compounds (active ingredients) of this invention can be administered to inhibit tumors by any means that produces contact of the active ingredient with the agent~s site of action in the body of a mammal. They can be administered by any conventional means available fGr use in conjunction with pharmaceuticals, either as individual therapeutic active ingredients or in a combination of therapeutic a- ive ingredients. They can be administered alone, but are generally administered with a pharmaceutical carrier selected on the basis of the chosen route of administration and standard pharmaceutical practice.

The dosage ad.-,-nistered will be a tumor-inhibiting amount of active ingredient and will, of course, vary depending upon known factors, such as the pharmacodymanic characteristics of the particular active ingredient and its mode and route of administration; age, health and weight of the recipient; nature and extent of symptoms; kind of concurrent treatment, frequency of treatment and the effect desired.
Usually a dai'y dosage (therapeutic effective amount or cancer-inhibiting amount) of active ingredient can be about 5 to 400 milligrams per kilogram of body weight. Ordinarily, 10 to 200, and preferably 10 to 50, milligrams per kilogram per day given in divided doses 2 to 4 times a day or in sustained release form is effective to obtain desired results.

Dosage forms (compositions) suitable for internal administration contain from about 1 milligram to about 500 milligrams of active ingredient per unit. In these pharmaceutical compositions the W O96/13S12 PCTrUS9~tl3716 active ingredient will ordinarily ~e present in an amount of about 0.05 - 95~ by weight, based on the total weight of the composition.

The active ingredient can be administered orally in solid dosage forms such as capsules, tablets and powders, or in liquid dosage forms such as elixirs, syrups and suspensions. It can also be administered parenterally, in sterile liquid dosage forms.

Gelatin capsules contain the active ingredient and powdered carriers such as lactose, sucrose, mannitol, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like. Similar diluents can be used to make compressed tablets. Both tablets and capsules can be manufactured as sustained release products to provide for continuous release of medication over a period of hours. Compressed tablets can be sugar coated or film coated to mask any unpleasant taste and protect the tablet from the atmosphere, or enteric coated for selective dis~ntegration in the gastrointestinal tract.

Liquid dosage forms for oral administration can contain coloring and flavoring to increase patient acceptance.

In general, water, a su1table oil, salinej aqueous dextrose (glucose), and related sugar solutions and glycols such as propylene glycol or polyethylene glycols are suitable carriers for parenteral solu.ions. So'utions for parenteral administration contain, preferably, a water soluble salt of the active ingredient, suitable stabilizing agents and, if necessary, buffer substances.
Antioxidizing agents such as sodium bisulfate, sodium sulfite or ascorbic acid, either alone or combined are suitable stabilizing agents. Also used are citric acid and its salts, and sodium EDTA.
In addition, parenteral solutions can contain preservatives such as benzalkonium chloride, methyl- or propyl-para~en and chlorobutanol.

Suitable pharmaceutical carriers are described in Pemington's Pharmaceutical Sciences, Mack Publishing Company, a standard reference text in this field.

Useful pharmaceutical dosage forms for administration of the compounds of this invention can be illustrated as follows.

WO96/13512 PCT~S95/13716 Capsules: Capsules are prepared by filling standard two-piece hard gelatin capsulates each with lO0 milligrams of powdered active ingredient, 175 milligrams of lactose, 24 milligrams of talc and 6 milligrams magnesium stearate.

Soft Gelatin Capsules: A mixture of active ingredient in soybean oil is prepared and injected by means of a positive displacement pump into gelatin to form soft gelatin capsules containing lO0 milligrams of the active ingredient. The capsules are then washed and dried.

Tablets: Tablets are prepared by conventional procedures so that the dosage unit is lO0 milligrams of active ingredient, 0.2 milligrams of colloidal silicon dioxide, 5 milligrams of magnesium stearate, 275 milligrams of microcrystalline cellulose, ll milligrams of cornstarch and 98.8 milligrams of lactose.
Appropriate coatings may be applied to increase palatability or to delay absorp.icr..

Injectable: A parenteral composition suitable for administration by injection is prepared by stirring l.5% by weight of active ingredients in lO~ by volume propylene glycol and water. The solution is made isotonic with sodium chloride and sterilized.

Suspensicn: An ac,uecus suspension is prepared for oral administraticn so that each 5 millimeters contain lO0 milligrams of finely divided active ingredient, 200 milligrams of sodium carboxymethyl cellulose, 5 milligrams of sodium benzoate, l.0 grams of sorbitol sclution U.S.P. and 0.025 millimeters of vanillin.

In the present disclosure it should be understood that the specified materials and conditions are important in practicing the invention but that unspecified materials and cor.ditions are not excluded so long as they do not prevent the benefits of the invention from being realized.

Claims (16)

1. A compound of the formula:

(I) or a pharmaceutically acceptable salt thereof, wherein:
R is H, COR5, P(O)nR6R7, or SO3H wherein R5 is alkyl of 1-5 carbon atoms or an aromatic ring structure, R6 and R7 are each H or alkyl of 1-5 carbon atoms and n is 2 or 3;

R1 and R2 are independently H, halogen, mono- or di-difluoro or OR8 wherein R8 is H, COR9, P(O)mR10R11 wherein R9 is H2, substituted or unsubstituted alkyl of 1-5 carbon atoms or a substituted or unsubstituted aromatic ring structure, R10 and R11 are each H or alkyl of 1-5 carbon atoms and m is 2 or 3, provided that when R2 is OH, R2 and B can combine to form a 5-membered cyclic ring structure;

R3 and R4 are independently B, H or OR12 where R12 is H, COR13, P(O)pR14R15 wherein R13 is substituted or unsubstituted alkyl of 1-5 carbon atoms or a substituted or unsubstituted aromatic ring sturcture, R14 and R15 are each H or alkyl of C1-C5 carbon atoms and p is 2 or 3;

B is a naturally-occurring nucleobase or a modified base consisting of one of more substituents selected from the group consisting of H, halogen, C1-C6 alkyl, C2-C6 alkenyl, C1-C6 alkoxy, C3-C6 cycloalkyl-C1-C6 alkoxy, C3-C8 cycloalkyloxy, C3-C8 cycloalkylthio, C1-C6 alkylthio, a substituted amino group, an aryl, aralkyl, aryloxy, aralkoxy, arylthio, aralkylthio, a heterocyclic ring and an amino group, provided that when the base is a pyrimidine, the atom at position 4 of the base can be sulfur and further provided that when the base is a purine, the atom at position 6 of the base may be sulfur;
provided that:
only one of R3 or R4 can be B and there is only one B;

when R=H, R1=OH, R2=H, R3=H and R4=B, then B cannot be U, C, T, 5-FU, hypoxanthine, A or G;

when R=H, R1=OH, R2=OH, R3=B and R4=H, then B cannot be C, when R=H, R1=OH, R2=OH, R3=H and R4=B, then B cannot be 5-FU, C, U, A, or hypoxanthine;

when R=H, R1=OH, R2=H, R3=B and R4=H, then B cannot be 5-FU, A, C, G, T, U or hypoxanthine;

when R=H, R1=H, R2=H, R3=B and R4=H, then B can not be A, C, G, T, U, 5-FU or hypoxanthine;

when R=H, R1=H, R2=H, R3=H and R4=B, then B can not be A, C, G, T, U, 5-FU or hypoxanthine;

when R=H or P(O)mR10R11, where R10=H and R11=H and m=3, and R1 and R2 are independently H or F; and R3=H and R4=B, then B
cannot be U, C, T, A, G or hypoxanthine; and when R=H or P(O)mR10R11, where R10=H, R11=H, and m=3; and R1 and R2 are independently H or F; and R3=B and R4=H, then B
cannot be U, C, T, A, G or hypoxanthine.

2. A compound of Claim 1 wherein R3 is defined as B and R4 is H.

3. A compound of Claim 1 wherein R4 is defined as B and R3 is H.

4. A compound of Claim 1 wherein B is a nucleobase selected from the group consisting of C, T, U, G, A, hypoxanthine, 6-thioguanine, 4-thiouracil and 5-fluorouracil.

5. A compound of Claim 1 wherein R is H.

6. A compound of Claim 1 wherein R1 and R2 are each independently H or OH.

7. A compound of Claim 6 wherein R2 is OH and combines with B to form a 5-membered cyclic ring.

8. A compound of Claim 1 wherein R3 is B; B is a nucleobase selected from the group consisting of C, T, U, G, A, hypoxanthine, 6-thioguanine, 4-thiouracil and 5-fluorouracil; R
is H; R1 and R2 are each independently H or OH; and R4=H.

9. A compound of Claim 1 wherein R4 is B; B is a nucleobase selected from the group consisting of C, T, U, G, A, hypoxanthine, 6-thioguanine, 4-thiouracil and 5-fluorouracil; R
is H; R1 and R2 are each independently H or OH; and R3=H

10. The compound of Claim 1 which is selected from the group consisting of a-L-ribofuranosyluracil; 1-(2,3,5-tri-O-benzoyl-a-L-ribofuranosyl)-4-thiouracil; a-L-ribofuranosyl-4-thiouracil; 1-(3,5-di-O-benzoyl-2-deoxy-b-L-ribofuranosyl)-4-thiouracil; 2'-b-L-deoxyribofuranosyl-4-thiouracil; a-L-ribofuranosyl-5-fluorouracil;b-L-ribofuranosylguanine; b-L-ribofuranosyl-6-thioguanine; or a pharmaceutically acceptable salt thereof.

11. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of one or more of the compounds of Claim 1.

12. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of one or more of the compounds of Claim 10.

13. A method of treating cancer in a mammal, the method comprising administering to a mammal bearing a cancer, a cancer-inhibiting amount of a compound of the formula:

(I) or a pharmaceutically acceptable salt thereof, wherein:
R is H, COR5, P(O)nR6R7 or SO3H (wherein R5 is alkyl of 1-5 carbon atoms or an aromatic ring structure, R6 and R7 are each H or alkyl of 1-5 carbon atoms and n is 2 or 3);

R1 and R2 are independently H, halogen, mono- or di-difluoro or OR8 (wherein R8 is H, COR9, P(O)mR10R11 (wherein R9 is H2, substituted or unsubstituted alkyl of 1-5 carbon atoms or a substituted or unsubstituted aromatic ring structure, R10 and R11 are each H or alkyl of 1-5 carbon atoms and m is 2 or 3)), provided that when R2 is OH, R2 and B can combine to form a 5-membered cyclic ring structure;

R3 and R4 are independently B, H or OR12 (where R12 is H, COR13, P(O)pR14R15 (wherein R13 is substituted or unsubstituted alkyl of 1-5 carbon atoms or a substituted or unsubstituted aromatic ring structure, R14 and R15 are each H or alkyl of C1-C5 carbon atoms and p is 2 or 3));

B is a naturally-occurring nucleobase or a modified base consisting of one or more substituents selected from the goup consisting of H, halogen, C1-C6 alkyl, C2-C6 alkenyl, C1-C6 alkoxy, C3-C6 cycloalkyl-C1-C6 alkoxy, C3-C8 cycloalkyloxy, C3-C8 cycloalkylthio, C1-C6 alkylthio, a subsituted amino group, an aryl, aralkyl, aryloxy, aralkoxy, arylthio, aralkylthio, a heterocyclic ring and an amino group, provided that when the base is a pyrimidine, the atom at position 4 of the base can be sulfur and further provided that when the base is a purine, the atom at a position 6 of the base may be sulfur;

14. A method of treating cancer in a mammal, the method comprising administering to a mammal bearing a cancer, a cancer-inhibiting amount of a compound of Claim 10.

15. An anhydride derivative of a compound of Claim 1, selected from the group consisting of 2-amino-a-L-ribofurano [1',2':4,5]oxazoline, and O2,O2-anhydro-1-a-L-ribofurano- syluracil.

16. A method of treating cancer in a mammal, the method comprising administering to a mammal bearing a cancer, a cancer-inhibiting amount of a compound selected from the group consisting of 2-amino-a-L-ribofurano[1',2':4,5] oxazoline, O2,O2-anhydro-1-a-L-ribofuranosyluracil and O2,O2-anhydro-1-a-L-ribofuranosyl uracil.