CA1264738A - Treatment of tumors in mammals - Google Patents

Abstract

Abstract A series of difluoronucleosides have been found to be useful antineoplastic agents. In the compounds, the base portion may be, for example, a pyrimidine or purine base, and the sugar portion may be, for example, a difluoroxylose or difluororibose.
The compounds are used to treat both solid and non-solid tumors, including, for example, leukemia, myeloma and lymphosarcoma.

Description

IME~ROVEMENTS IN THE TREATMENT OF TUMORS IN ~MALS

While the treatment of cancer was once consid-ered impossible, great strides have been made during the past ten years in controlling the ravages of this often fatal disease. Several drugs which contribute to -the increasing rate of survival are now routinely used clinically. The most commonly employed antitumor agents include methotrexate, doxorubicin and the vinca alka-loids such as vincristine. However, research continuesto develop more effective compounds with greater safety for subjects under treatment. This invention provides valuable improvements in the treatment of tumors.
The present invention provides a method of treating susceptible neoplasms in mammals comprising administering to said mammal a pharmaceutically effec-tive amount of a compound of formula (I):

R 1 0~2C~ /~
~ T (I) t~
HO
in which:
ll R1 is hydrogen, C1-C~ alkyl or -C-Rs;

~ ~Çi~7~3 R2 is a base defined by one of the formulae ~ `~ RaHN/ ~ O ~

~ l~HR3 0~ ~;, P~_ X is N or C-R4;
O
R3 is hydrogen, Cl-C4 alkyl or ~C-R5;
R4 is hydrogen, C1-C4 alkyl, amino, bromo, fluoro, chloro or iodo;
each R5 independently is hydrogen or C1-C4 alkyl; or a pharmaceutically-acceptable salt thereof.
Thus, the present invention provides for the use of a compound of formula (I) for the manufacture of a medicament for the treatment of susceptible neoplasms.
The present invention also provides a novel compound of formula (II):

R OH2C\ ~ \ (II) H( ) Ç~J L~ 7;~ ~

in which:
Rt~ is hydrogen or Cl-C4 alkyl;
R7 is a pyrirnidine or purine base of one of the formulae ~HR8 c ~ 5~H~/ \'1'1'7 0~ ~ R3HN/ ~

~0, lS 0~ ~ R~

Q is N, C-(C2-C4 alkyl) or C-amino X is N or C-R4;
R8 is hydrogen or Cl-C4 alkyl;
R4 is hydrogen, C1-C4 alkyl, amino, bromo, fluoro, chloro and iodo; or a pharmaceutically-acceptable salt thereof;
with the proviso that Rfi and R8, both may be hydrogen only when X is N.

i47~8 The present invention further provides a com-pound of formula (III):

T t (III) HO~

in which:
R6 is hydrogen or C1-C4 alkyl;

/6~

o~ 4\~ .
H

a pharmaceutically-acceptable salt thereof.
Further, there is provided a process for preparing a compound of formula (II) or (III), as defined above, which comprises:
(a) coupling a pyrimidine base of the formula R7H, or a protected derivative thereof, with a carbohydrate of formula IV, or a protected derivative thereof:

R~OCI~ \ /\
T ICH (Leav) ~ (IV) H

7;~3 X-6615~ -5-in which the R7H pyrimidine base and R6 are as defined above and Leav is a leaving group, and if desired, removing any protecting group present to pro-duce the pyrimidine base product; and (b) if a formula (III) or formula (II) compound in which the base is a purine is desired, reacting with ammonia a corresponding purine nucleoside com-pound in which a C-2 and/or C-6 sub-stituent of the purine portion of the compound is halogen, and, if desired, alkylating the product; and where desired, forming a pharmaceutically acceptable salt of the compound of formula (II) or (III).
The present invention also provides pharma-ceutical formulations useful for treating susceptible neoplasms in mammals comprising a compound of formulae II
ox III in combination with a suitable pharmaceutically acceptable carrier, diluent or excipient therefor.
Further, there is provided a compound of formula (II) or (III) for use in the chemotherapy of a mammal.
The compounds employed in the present inven-tion are preferably prepared by reacting a D-glyceral-dehyde ketonide with a C1-C4 alkyl bromodifluoroacetate to afford an alkyl 3-dioxolanyl-2,2-difluoro-3-hydroxy-propionate. The hydroxypropionate is hydrolyzed to a lactone which is protected and reduced to afford a

2-desoxy-2,2-dif]uororibose or xylose derivative. The hydroxy group of this compound is provided with a leaving group, and the resulting carbohydrate is coupled with an appropriate base. The resulting protected .. .., .
,~

7;3~3 nucleoside is finally deprotected to provide the desired compound. The overall reaction scheme is illustrated as follows:
R ~ R \ j ~ I C, C. 31k~j :

(Prot)OH2f/O\ HOH2f~ 0 t T= Protect T t=
~- t ~~ ~ t-f (Prot)~ ~ HO

~ reduction (Prot)OH2fjO \ Leaving (Prot)OH2~/ 0 t T~H Group ~ t T-o(Leav) ,~ ., ,~ ,~ ,~
(Prot)~ (Prot)O I
l R2H

HOH2f 0 (Prot)OH2f 0 f \ o_p2 , Deprotect f \t - p2 t~ t~ ~ ~-HO ~ (Prot)~ :

wherein R1~ and R11 independently are C1-C3 alkyl, "Prot"

is a hydroxy protecting group and "Leav" is a leaving group.

7;~8 It generally is desirable to convert freehydroxy groups to protected hydroxy groups during coupllng o the 2-d~soxy-2,2-difluorocarbohy~rate to a base. The protecting groups are those commonly used in synthetic organic chemistry. Chemists are accustomed to choosing groups which can be placed efficiently on hydroxy groups, and which can be removed easily when the reaction is complete. Suitable groups may be those described in standard textbooks, such as Chapter 3 of Protective Groups in Orqanic Chemistry, McOmie, Ed., Plenum Press, New York (1973); and Chapter 2 of Protective Groups in Organic Synthesis, Greene, John Wiley & Sons, New York (1981).
Hydroxy-protecting groups commonly employed O
include formyl, -C-(Cl-C4 alkyl), 2-chloroacetyl, benzyl, diphenylmethyl, triphenylmethyl, 4-nitrobenzyl, phenoxycarbonyl, C1-C4 alkyl such as t-butyl, methoxy-methyl, tetrahydropyranyl, allyl, tetrahydrothienyl,2-methoxyethoxymethyl, methoxyacetyl, phenoxyacetyl, isobutyryl, ethoxycarbonyl, and benzyloxycarbonyl.
Silyl hydroxy protecting groups are particularly conve-nient because most are cleaved easily by contact with water or an alcohol. Such groups may include especially trimethylsilyl, as well as isopropyldimethylsilyl, methyldiisopropylsilyl, or triisopropylsilyl. The t-butyldimethylsilyl group is a special case and is preferred as the protecting gro~p in this synthesis; it is more difficult to cleave, requiring a reagent such as a hydrohalic acid to remove it from thé hydroxy groups.

Ribose or xylose has a hydroxy group at the l-position of its ring. In order to react the carbohy-drate with the base, to form the compounds employed in this invention, a leaving group must be placed at the 1-position. The leaving groups are those typically used in organic synthesis. The preferred leaving groups are sulfonates, of which the most preferred is methanesul-fonate. Other typical leaving groups such as toluene-sulfonate, ethanesulfonate, isopropanesulfonate, 4-methoxybenzenesulfonate, 4-nitrobenzenesulfonate, 2-chlorobenzenesulfonate, chloro and bromo also may be used.
The carbohydrates employed in the synthesis of the compounds employed in the present invention are prepared by reacting a D-glyceraldehyde ketonide of the formula ~o R ~ ~ -t R~ 0 ~0 wherein Rl and R1l are as defined ahove with a C1-C4 alkyl bromodifluoroacetate, preferably the ethyl ester.
The preferred glyceraldehyde ketonide is the ~5 acetonide in which Rl and Rll are both methyl (see Fischer and Baer, Helv. Chim. Acta. 17, 622 (1934)).
Ethyl bromodifluoroacetate was prepared first by Morel and Dawans, Tet. 33, 1445 (1977). The reaction of the ketonide and the haloacetate is carried out in the presènce of an activated metal such as magnesium or preferably zinc. Activation is obtained most easily by applying ultrasonic energy to the reaction mixture.

Actlvation by that means compensates for the presence of a small amount of water in the reaction mixture, avoid-ing the necessity to maintain anhydrous conditions, and also avoids the necessity to prepare and carefully store activated metals. However, if desired, the metal may be activated by the customary methods known in the art.
Approximately an equimolar amount of metal is the most advantageous amount.
The reaction has been performed in ethers such as tetrahydrofuran and diethyl ether, at moderate tem-peratures. However, other organic solvents which are inert to the reaction conditions may be used, including halogenated alkanes such as chloroform, dichloromethane, or trichloroethane, and aromatic solvents including benzene, toluene and the xylenes. Temperatures in the range of from about ambient temperature to about 150C
may be used; temperatures from about ambient temperature to about 80~C are preferred, however. Economically-acceptable yields have been obtained in reaction times ranging from a few minutes to a few hours. One should note that the reaction is exothermic, and the mixture may need to be cooled, depending on the scale of the reaction and the rate at which the reactants are added.
The product of the first reaction is an alkyl

3-dioxolanyl-2,2-difluoro-3-hydroxypropionate of the formula \ / ~
Ç02(C1-C~ alkyl) Rl 1/ \01 1 l l '~ 1--F
o H

in which R10 and R11 are as described above.

i47;38 The ratio of the 3-R-hydroxy intermediate to its 3-S-hydroxy enantiomer is usually about 3:1. The 3-R-hydroxy enantiomer has the proper stereochemistry to produce the ribose derivative in its natural configura-tion, and so it is the desired enantiomeric product ofthe first step. The 3-R-hydroxy enantiomer can general ly be separated cleanly from the 3-Senantiomer by chromatography on silica gel, eluting with chloroform containing 0.5% methanol.
The hydroxypropionate, in either form, is hydrolyzed using very mild conditions to form the lactone of the formula HOHzl~/O

Proper control of the hydrolysis step will cleave the ketonide function and the ester group, providing the lactone in a single step. The hydrolysis reagent preferably is a mildly acidic ion exchange resin, of which "Dowex 50W-X12" *(Dow Chemical Company) is most highly preferred. Other mild hydrolytic reagents may be employed although larger amounts of by-products may be obtained. For example, aqueous acetic acid, or other relatively strong acids swch as propionic acid, formic acid, chloroacetic acid, or oxalic acid may be used for the hydrolysis.
The hydroxy groups of the lactone should be protected before its keto oxygen is reduced. The usual * Trademark lX~7;~3 reaction conditions are used, depending on the protect-lng groups chosen. For example, -the t-butyldimeth~lsilyl group is most conveniently provided in the form of its trifluoromethanesulfonate, and the protection reaction is carried out in the presence of a base such as lutidine, pyridine and the like. Acyl protecting groups such as acetyl, benzoyl and the like are added by reacting the lactone with an acylating agent such as an acyl chlo-ride, bromide, cyanide or azide, or with an appropriate anhydride. The reactions are conveniently carried out in a basic solvent such as pyridin~, quinoline or isoquinoline, or in a tertiary amine solvent such as triethylamine, tributylamine, or methylpiperidine. The reaction also may be carried out in an inert solvent, to which an acid scavenger, such as a tertiary amine, has been added. Acylation catalysts such as 4-dimethylamino-pyridine or 4-pyrrolidinopyridine may be used in the reaction, if desired. The acylation reactions which provide protecting groups on the hydroxy groups are Garried out at moderate temperatures in the range of from -25C to 100C. Such acylations also may be performed by acid-catalyzed reactions of the appropriate carboxylic acids, in inert organic solvents or neat.
Acid catalysts such as sulfuric acid, polyphosphoric acid, or methanesulfonic acid may be used.
Acyl protecting groups may also be provided by forming an active ester of the appropriate acid, for example esters formed by reaction with reagents such as dicyclohexylcarbodiimide, acylimidazoles, nitrophenols, pentachlorophenol, N-hydroxysuccinimide and l-hydroxybenzotriazole.

7;~8 Protected groups of the ether type are pro-duced by reacting the lactone with, for example, an appropriate diazo compound, such as diazomethane, phenyldiazomethane or a silyldiazomethane. Such reac-tions commonly are carried out in solvents includingesters such as ethyl acetate, halogenated solvents including dichloromethane and chloroform, and ethers including diethyl ether and tetrahydrofuran. The process is usually carried out at low temperatures from about -50C to about 0C. Such ether-forming reactions may also be carried out with the assistance of reagents such as trimethyloxosulfonium hydroxide, trimethylsul-fonium hydroxide and trimethylselenonium hydroxide, in solvents such as dimethylsulfoxide, dimethylformamide, hexamethylphosphoramide, acetone, or acetonitrile.
The silyl protecting groups discussed above are placed on the hydroxy groups ~y the conventional methods, such as by reaction with the appropriate silylcarboxamide or bis(substituted-silyl)carboxamide, or an appropriately substituted silazane. Suitably substituted silyl methanesulfonates, toluenesulfonates and the like are useful also. An equivalent amount of a base is usually necessary in the reaction mixture, unless a basic solvent is used in the reaction.
When the hydroxy groups have been protected, the keto oxygen of the lactone is reduced to the alco-hol, forming the protected 2-desoxy-2,2-difluororibose or xylose. The most preferred reducing agent is diiso-butyl aluminum hydride, used at a low temperature in the range of about -100C to -20C. The reduction must be 7;18 ~-6615A -13-performed -~ery carefully -to avoid conditions so vigorous that the ring is opened a-t the oxygen atom. Other metal hydrides, such as the widely used lithium aluminum hydride, can also be used for the reduction, but it is necessary to keep the temperature quite low and to assure that the hydride is destroyed before the tempera-ture is allowed to rise above about -20C. Accordingly, a solvent with a very low freezing point, such as toluene, must be used in the reduction step. Other solvents, of course, can be used, including lower alkanols, especially ethanol, or ethers such as diethyl ether.
To obtain e~ficient reaction with the base, an appropriate leaving group must be placed at the l-position of the carbohydrate. The preferred leaving group is methanesulfonyl, and the compound with this leaving group is readily provided by reaction with methane- -sulfonyl chloride in the presence of an equivalent amount of a suitable acid scavenger such as triethyl-amine and the like. Other sulfonyl leaving groups are-provided in the same way by reaction with the appropri-ate sulfonyl halide.
When a chloro or bromo leaving group is to be used, it is frequently advantageous first to make the l-acetate derivative, as by reaction with acetic anhy-dride, or another source of acetyl groups, in the presence of an equivalent amount or more of an acid scavenger. The acetate grol1p then is displaced, at a low temperature such as about -50C to about 0C, with gaseous hydrogen bromide or hydrogen chloride. Because 7~3~3 the gaseous hydrogen halide may tend to remove the protecting groups, especially silyl protecting groups, operating this step at low temperatures and adding the hydrogen halide slowly in small increments is necessary.
The compounds employed in the present inven-tion having a base portion which is composed of a purine substrate are preferably synthesized by reacting the 1-hydroxy analog of the carbohydrate having protecting groups at the 3- and 5-position with the base in the presence of diethyl azodicarboxylate and triphenylphos-phine. Standard modifications are then made to the purine substrate if desired.
The bases used to form the compounds employed in the present invention are known to those skilled in the art, and no discussion of their synthesis is neces-sary. The primary amino groups present on some of the bases, however, should be protected before the base is coupled with the carbohydrate. The usual amino-protecting groups are employed, including silyl groups such as have been discussed, as well as such typical groups as t-butoxycarbonyl, benzyloxycarbonyl, 4-methoxy-benzyloxycarbonyl, 4-nitrobenzyloxycarbonyl, formyl, or acetyl.
Converting the keto oxygen atoms on the bases to the enol form, in order to make the base more highly aromatic and allowing a more ready attack of the base by the carbohydrate is advisable. Enolization is provided most conveniently by producing the silyl protecting groups. The usual silyl protecting groups, as discussed above, may be used for this purpose.

7~
X-6~15A -15-The reaction between the protected carbGhy-drate and -the base preferably is performed neat at a temperature in the range of from about 50C to about 200~C. ~se of relatively high-boiling solvents for the reaction, such as dimethylformamide, dimethylacetamide, or hexamethylphosphoramide, however, is po~sible. If the coupling reaction is carried out at elevated pres-sures to avoid distiilation of a low-boiling solvent, any convenient inert reaction solvent can be used.
The coupling reaction may be done at low temperatures if a reaction initiator, such as a tri-fluoromethanesulfonyloxysilane, is used~ The usual inert reaction solvents, as discussed above, may be used at temperatures in the range of from about ambient temperature to about 100C.
The final step of the reaction sequence is the removal of the protecting groups. Most silyl protecting groups are cleaved easily by contact with water or an alcohol. The t-butyldimethylsilyl protecting group requires acid conditions, such as contact with gaseous hydrogen halide, for its removal.
Acyl protecting groups are removed by simple hydrolysis with strong or moderately strong bases, such as alkali metal hydroxides, at temperatures from about ambient temperature to about 100C. At least one equivalent amount of base is needed ~or each protecting group. Such hydrolyses conveniently are carried out in hydroxylic solvents, especially aqueous alkanols. The reactions also may be carried out, however, in any convenient solvent, such as polyols including ethylene 7~8 glycol, ethers such as tetrahydrofuran, ketones such as acetone and methyl ethyl ketone aLld other polar solvents such as dimethylsulfoxide. The cleavage of acyl pro-tecting groups may also be performed with other bases, including, for example, sodium methoxide, potassium _-butoxide, hydrazine, hydroxylamine, ammonia, alkali metal amides and secondary amines such as diethylamine.
The acyl protecting groups also can be removed with acid catalysts, such as methanesulfonic acid, hydrochloric acid, hydrobromic acid, sulfuric acid, or with acidic ion exchange resins. Carrying out such hydrolyses at a relatively high temperature, such as the reflux tempera-ture of the mixture is preferred, but temperatures as low as ambient may be used when particularly strong acids are used.
The removal of protecting groups which are ethers is carried out by known methods, for example, with ethanethiol and aluminum chloride.
Compounds of the invention possessing hydroxy or amino acyl or alkyl groups can, of course, be either selectively deprotected, or such groups may be removed and selectively replaced by standard conditions.
None of the reaction steps require unusual excesses of the reactants. As usual in organic synthe-ses, use of a moderate excess, in the range of 1.05X to2X, is advisable.
The compounds employed in -this invention are capable of forming pharmaceutically-acceptable addition salts. Such salts are to be construed as included within the scope of this invention and may include y7;38 hydrobromide, hydrochloride, mono-, di- or triphosphate esters and sodium sal-ts of such phosphates, sulfate, the sodium, po~assium, lithium or ammonium salts, as well as others well-known to those skilled in the art. "Phar-maceutlcally-acceptable salts~' are those salts useful in the chemotherapy of warm-blooded animals.
The structural drawings defining the compounds employed in the present invention do not indicate their stereochemistry. Compounds of all configurations are believed to be useful, and the stereochemistry of the compound is not to be construed as a limitation. The preferred compounds possess the configuration of natu-rally occurring ribose, e.g., HOhz~ /

The configuration at the juncture between the ribose and the base is preferably as follows:
o\l ~ ~ ~

One skilled in the art would be aware of the bases which are used in the synthesis of the nucleosides employed in the present invention, but the following 47~38 X-6615A 1~-specific nucleosides are given to further elaborate the type of agents which may be used in this invention.

1-(2/4-dioxo-lH,3~-pyrimidin-1-yl)-2-desoxy-2,2-difluororibose 1-(4-amino-5-chloro-2-oxo-lTH-pyrimidin-l-yl)-2-desoxy-2,2-difluororibose 1-(4-amino-5-bromo-2-oxo-lH-pyrimidin-l-yl)-2-desoxy-2,2-difluororibose 1-(4-amino-2-oxo-lH-pyrimidin-l-yl)-2-desoxy-2,2-difluororibose 1-(4-amino-5-iodo-2-oxo-lH-pyrimidin-l-yl)-2-desoxy-2,2-difluororibose 1-(4-amino-5-methyl-2-oxo-lH-pyrimidin-l-yl)-2-desoxy-2,2-difluororibose 1-(2-amino-6-oxo-lH,9H-purin-9-yl)-2-desoxy-2,2-difluororibose 1-(6-amino-9H-purin-9-yl)-2-desoxy-2,2-difluororibose 1-~4-amino 5-fluoro-2-oxo-lH-pyrimidin-l-yl)-2-desoxy-2,2-difluororibose 1-(4-amino-5-chloro-2-oxo-lH-pyrimidin-l-yl)-2-desoxy-2,2-difluoroxylose 1-(4-amino-2-oxo-lH-pyrimidin-l-yl)-2-desoxy-2,2-difluoroxylose 1-(4-amino-5-fluoro-2-oxo-lH-pyrimidin-l-yl)-2-desoxy-2,2-diEluoroxylose 1-(4-amino-5-methyl-2-oxo-lH-pyrimidin-l-yl)-2-desoxy-2,2-difluoroxylose 1-(2-amino-6-oxo-lH,9H-purin-9-yl)-2-desoxy-2,2-difluoroxylose 7;~8 X-6615A -l9-1-(6-amino-9H-purin 9-yl)-2-desoxy-2,2-difluoroxylose or the pharmaceutically-acceptable salts thereof.
The following non-limiting Examples are provided to further illustrate -the invention.

Example 1 1-(4-Amino-2-oxo-lH-pyrimidin-1-yl)-2-desoxy-2,2-difluororibose To 47.3 g ~0.1 mol) of 3,5-bis(t-butyldimethyl-siloxy)-1-methanesulfonyloxy-2-desoxy-2,2-difluororibose in 940 ml of dry 1,2-dichloroethane was added 48.0 g (0.16 mol) of bis-trimethylsilyl N-acetylcytosine followed by 39.23 g (0.177 mol) of trifluoromethane-sulfonyloxytrimethylsilane. The reaction mixture was refluxed under a nitrogen atmosphere for about 15 hours, cooled to room temperature, and diluted by the addition of 16 ml of methanol. The resulting mixture was stirred for 30 minutes, concentrated under vacuum to about one-half the original volume and cooled in ice. The precipitated solid was collected by filtration and the filtrate was shaken one time with about 300 ml of 10%
sodium bicarbonate and one time with brine. The organic layer was separated and concentrated to dryness ln vacuo at 45C. The residue was dissolved into 1.3 1. of methanol saturated with ammonia and the resulting solution was stirred overnight. The volatiles were removed in vacuo at 45C to provide 32 g of residue.

1~47~38 The residue was dissolved into 275 ml of methanol and100 g of Biorad cation exchange resin (AG50WX8)*was added to the resulting solution. The suspension was stirred at ambient temperature overnight. The resin was removed by filtration and rinsed one time with 100 ml of methanol. The filtrate was discarded and the resin was suspended in 100 ml of methanol and 50 ml of concentrat-ed ammonium hydroxide. This mixture was stirred vigor-ously for 15 minutes and the resin was filtered. This procedure was repeated two times with additional fresh methanolic ammonia. The basic methanolic filtrates were.
combined and evaporated at 45C ln vacuo to yield a brown foam weighing 13.8 grams. This material was chromatographed with the use of a "Waters Prep 500~**cl8 15 reverse phase column with 100% water to yield 1.26 g of 1-(4-amino-2-oxo-lH-pyrimidin-l-yl)-2-desoxy-2,2-~ difluororibose.
- NMR (CD30D, 90 mHz, ~) 3.7-4.65 (ml 4~), 4.83 (s, 4H), 5.97 (d, J = 8Hz, lH), 6.24 (t, J = 7Hz, lH, 20 7.88 (d, J = 8Hz, lH).
~ Mass spec. m/e = 263 = P

Example 2 1-(4-Amino-5-iodo-2-oxo-lH-pyrimidin-l-yl)-2-desoxy-2,2-difluororibose To l.9g g (0.0042 mol) of 3,5-bis(t-butyl-dimethylsiloxy~-1-methanesulfonyloxy-2-desoxy-2,2-difluororibose in 35 ml of dry 1,2-dichloroethane was added 2.08 g (0.0046 mol) of tris-trimethylsilyl-5-* Trademark ** Trademark A

1~i4~38 iodocytosine followed by 1.11 g (0.005 mol) of trifluoro-methanesulfonyloxytrimethylsilane. The reaction mixture was refluxed for about 16 hours under a nitrogen atmo-sphere and cooled to room temperature. Five mllliliters of methanol were added to the reaction mixture and the mixture was stirred for an additional 30 minutes. The mixture was filtered and the precipitated solid was collected by filtration. The filtrate was evaporated to dryness under reduced pressure, and the resulting residue was dissolved in 20 ml of dichloromethane saturated with anhydrous hydrogen bromide. This mixture was stirred for about 3 hours. The volatiles were removed ln vacuo at 45C. The residue was dissolved in 15 ml of water, neutralized to pH 7-8 with 10% sodium bicarbonate, and the resulting solution was washed once with 10 ml of ethyl acetate. The aqueous layer was chromatographed on a "Whatman Prep ODS-3"* reverse phase column in 2.0 ml portions using water/methanol (9:1, v:v) to afford 30 mg of 1-(4-amino-5-iodo-2-oxo-lH-pyrimidin-1-yl)-2-desoxy-2,2-difluororibose.
NMR (CD30D, 90 mHz, ~) 3.47-4.66 (m, 4H), 4.78 (s, 4H), 6.14 (t, J = 7Hz, lH), 8.32 (s, lH).
Mass spec. m/e = 389 = P

ExamPle 3 1-(2,4-Dioxo-lH,3H-pyrimidin-1-yl)-2-desoxy-2,2-difluororibose A solution of 190 mg (0.0007 mol) of 1-(4-amino-2-oxo-lH-pyrimidin-l-yl)-2-desoxy-2,2-difluoro-* Trademark i47;~8 ribose in 16 ml of glacial acetic acid and 4 ml of water was refluxed for approximately 24 hours. The reaction mixture was cooled to amblent temperature and the volatiles were evaporated under vacuum at about 60~-70C.
The residue was stirred with 5.0 ml of toluene and the resulting solution was evaporated several times. The residue was dlssolved in 12 ml of methanol, and the resulting mixture was cooled to -15C and saturated with anhydrous ammonia. The solution was stirred overnight at ambient temperature. The volatiles were removed ln vacuo at 45DC. The residue was suspended in about 5.0 ml of hot water and the insoluble material was removed by filtration. The filtrate was chromatoqraPhed on a Whatman 5Q cm "Partisil ODS-3"* reverse phase column using water/methanol (9:1, v:v) as the eluent to afford 0.05 g of product containing a small trace of unreacted starting material. The unreacted starting material was removed by passing a solution of 0.05 g of the mixture in about 5.0 ml of a solvent solution of methylene ~hloride/methanol (9:1, v:v) through a "Waters Silica "Sep-Pak."** The eluate was evaporated 1n vacuo at 45C to yield 0.036 g of 1-(2,4-dioxo-lH,3H-pyrimidin-1-yl~-2-desoxy-2,2-difluororibose.
NMR ~CD30D, 90 mHz, ~) 3.54-4.48 (m, 4H), 4.83 (s, 3H), 5.69 (d, J = BHz, lH), 6.10 (dd, J = 7Hz, 9Hz, lH), 7.8 (d, J = 8Hz, lH).
Mass spec. m/e = 264 = P
* Trademark ** Trademark 1~647;38 Example 4 1-(4-Amino-5-methyl-2-oxo-lH-pyrimidin-1-yl)-2-desoxy-2,2-difluororibose A solution of 1.86 g (0.0039 mol) of 3,5-bis~t-butyldimethylsilyloxy)-1-methanesulfonyloxy-2-desoxy-2,2-difluoroA bose, 1.87 g (0.0055 mol) of bis-trimethylsilyl-5 methylcytosine and 1.34 g (0.006 mol) of trifluoromethanesulfonyloxytrimethyl-silane in 37 ml of dry methylene chloride was refluxed overnight. The reaction mixture was cooled to room temperature and 1.0 ml of methanol was added thereto.
The precipitated solid was collected by filtration and the filtrate was evaporated ln vacuo at 45C. The residue was dissolved in 20 ml of water and the result-ing solution was concentrated to about 10 ml ln vacuo at 50C at which point a precipitate formed. The precipi- -i tated solid was collected by filtration and the filtrate 3 20 was concentrated ln vacuo at 50C to afford 2.2 g of residue. The residue was triturated several times with 10 ml portions of warm acetone. The decanted organic layers were combined and evaporated in vacuo at 45C to provide 1.67 g of a yellow oil. This material was dissolved into 15 ml of methanol/water (v:v, 1:1) and the resulting solution was stirred overnight with 5.0 g of "Biorad AG50WX8".* The suspension was saturated with anhydrous ammonia and stirred for 10 minutes. The resin was collected by filtration and suspended in 30 ml of methanol/ammonia (v:v, 2:1). The solution was stirred for 10 minutes. The resin was collected by vacuum * Trademark ,~

.

, -. .

' 7;~8 filtration, and the basic filtrates were combined andconcentrated in vacuo at 50C to provide 1.5 g of an orange oil. The oil was dissolved in 10 ml of water and chromatographed 2.0 ml per run on a"Whatman Partisil ODS-3"* 50 cm reverse phase prep column using water as the eluent to provide 0.07 g of 1-(4-amino-5-methyl-2-oxo-lH-pyrimidin-l-yl)-2-desoxy-2,2-difluororibose.
NMR (CD30D, 90 mHz, ~) 1.94 (s, 3H), 3.53-4.62 (m, 4H), 4.75 (s, 4H), 6.17 ~t, J = 8Hz, lH, 7.67 (s, lH).
Mass spec. m/e = 277 = P

Example 5 1-(4-Amino-2-oxo-lH-pyrimidin-1-yl)-2-desoxy 2,2-difluoroxylose Under a nitrogen atmosphere, to 17.89 g (0.0375 mol) of 3,5-bis(t-butyldimethylsiloxy)-1-methane-sulfonyloxy-2-desoxy-2,2-difluoroxylose in 300 ml of dry methylene chloride was added 23.0 g (0.063 mol) of tris-trimethylcytosine followed by 10.84 g (0.0488 mol) of trifluoromethanesulfonyloxytrimethylsilane. The solution was refluxed overnight and cooled to room temperature. Twenty milliliters of methanol were added to the reaction mixture and the resulting solution was stirred vigorously for about one hour. The precipitated solid was collected by filtration. The filtrate was charged with 100 ml of water and the suspension was stirred vigorously for 30 minutes. The organic layer was separated and concentrated ln vacuo at 45C to give * Trademark L~ 8 11.2 g of a brown oll. The oil was dissolved in 9S mlof methanol to which 33 g of Biorad AG50WX8 cation exchange resin had been added and the suspension was stirred overnight at ambient temperature. The resin was collected by filtration and washed with 50 ml of metha-nol. The resin was stirred vigorously with 100 ml of a solution of methanol/ammonia (v:v, 1:1). The resin was collected by filtration and again stirred in this solution. The resin was collected and the basic fil-trates were combined and concentrated 1n vacuo at 50Cto give 2.09 g of a yellow residue. This material was suspended in 25 ml of water and stirred vigorously for 15 minutes. The insoluble precipitate was filtered to yield 0.250 g of a compound labeled A. The filtrate was concentrated in vacuo at 50C to yield 0.86 g of a compound labeled B. Compound A was dissolved in 20 ml of methanol and stirred for 3 days with Biorad AG50WX8 at ambient temperature. The resin was collected by filtration and slurried in 30 ml of a solution of methanol/concentrated ammonium hydroxide ~v:v, 1:1).
The resin was collected by filtration and the filtrate concentrated in vacuo at 50C to give 0.14 g of 1-(2-desoxy-2,2-difluoro-~-D-xylofuranosyl)cytosine.
NMR (CD30D, 90 mHz, ~) 3.72-4.34 (m, 4H), 4.78 (s, 4H), 5.86 (d, J = 8Hz, lH), 6.17 (d, J = 15Hz, lH), 7.78 (d, J = 8Hz, lH).
Mass spec. m/e = 263 = P
The compound labeled B was chromatographed on a Whatman 50 cm ODS-3 reverse phase prep column using water/methanol (v:v, 1:1) as the eluent to afford 0.06 g of 1-(2-desoxy-2,2-difluoro-~-D-xylofuranosyl)cytosine.

. . .

:.".,.,,...,.,,;. ,.,. ~.

. .

., 1~ ~L~7;~

~MR (CD30D, 90 mHz, ~) 3.53 3.9 (m, 2H),

4.1-~.57 (m, 2H) 4.83 (s, 4H), 5.9 (d, J = 8Hz, lH~, 6.3 (dd, J = 7Hz, 12Hz, lH) 7.55 (d, J = 8Hz, lH).
Mass spec. m/e = 263 = P

Example 6 1-(6-Amino-9H-purin-9-yl)-2-desoxy-2,2-difluororibose A. 1-(6-Chloro-9H-purin-9-yl)-3,5-bis(t-butyldimethylsiloxy)-2-desoxy-2,2-difluororibose To a solution of 0.77 g (5.0 mmol) of 6-chloro-purine in 50 ml of tetrahydrofuran was added 1.31 g (5.0 mmol) of triphenylphosphine and 0.87 g (5.0 mmol) of diethyl azodicarboxylate. To this solution was added a solution of l.99 g (5.0 mmol) of 3,5-bis(t-butyldi-methylsiloxy)-l-hydroxy-2-desoxy-2,2-difluororibose in tetrahydrofuran. The reaction mixture was stirred at room temperature for approximately 60 hours and an additional 0.66 g (1.7 mmol) of 3,5-bis(t-butyldimethyl-siloxy)-l-hydroxy-2-desoxy-2,2-difluororibose was added to the reaction mixture. The mixture was stirred for an additional 6 hours at room temperature. The solvent was evaporated under vacuum and the residue was stirred in a small amount of diethyl ether overnight. The precipi-tated solid was removed by vacuum filtration and the filtrate was concentrated under vacuum to dryness. The residue was chromatographed over 70 g of silica and eluted with chloroform. Fractions containing the major ~ 7 component were combined and the solvent was evaporated therefrom to provide 1.0 g of 1-(6-chloro-9H-purin-9-yl)-3,5-bis(t-butyldimethylsiloxy)-2-desoxy-2,2-difluororibose. The structure of the product was verified by NMR. Mass spec. = 477 [534-(t-butyl)]

B. 1-(6-Amino-9H-purin-9-yl)-3,5-bis(t-butyldimethylsiloxy)-2-desoxy-2,2-difluororibose A solution of 0.5 g (0.936 mmol) of 1~6-chloro-9H-purin-9-yl)-3,5-bis(t-butyldimethylsiloxy)-2-desoxy-2,2-difluororibose dissolved in 75 ml of absolute ethanol was saturated with anhydrous ammonia at about 0C. The reaction flask was sealed, and the mixture was allcwed to warm to room temperature. The mixture was stirred for about 72 hours at room temperature and the volatiles were evaporated under reduced pressure to provide 420 mg of 1-(6-amino-9H-purin-9-yl)~3,5-bis(t-butyldimethylsiloxy)-2-desoxy-2,2-difluororibose.
Mass spec = 458 [515-(t-butyl)]

C. A solution of 100 mg (0.194 mmol) of 1-(6-amino-9H-purin-9-yl)-3,5-bis(t-butyldimethyl-siloxy)-2-desoxy-2,2-difluororibose dissolved in 25 ml of methylene chloride cooled to about 0C with an external ice bath was saturated with anhydrous hydrogen bromide gas. The mixture was stirred at about 0C for about 4 hours, and nitrogen was bubbled through the reaction mixture. The mixture was filtered and the col-lected solid was washed with methanol to provide 110 mgof solid. The solid was purified by HPLC to provide ,; .

- ~ .

~2647~38 12.1 mg of ~-1-(6-amino-9H-purin-9-yl)-2-desoxy-2,2-difluororibose.
NM~ ~CD30D, 30 mHz, ~): 3.8-4.65 (m, 4H); 4.83 (bs, 4H); 6.33 (dd, lH); 8.22 (s, lH); 8.4 (s, lH). mass spec. m/e = 287 Example 7 A. 1-(2,6-Dichloro-9H-purin-9-yl)-3,5-bis(t-butyldimethylsiloxy)-2-desoxy-2,2-difluororibose To a solution of 1.89 g (10.0 mmol) of 2,6-dichloropurine in lO0 ml of tetrahydrofuran was added 2.62 g (10.0 mmol) of triphenylphosphine and 1.74 g (10.0 mmol) of diethyl azodicarboxylate. To this mixture was added a solution of 3.98 g (10.0 mmol) of 3,5-bis(t-butyldimethylsiloxy)-1-hydroxy-2-desoxy-2,2-difluororibose i~ 25 ml of tetrahydrofuran and the mixture was stirred at room temperature overnight. The precipitated solid was removed by vacuum filtration and the filtrate was concentrated under vacuum. The residue was dissolved in 100 ml of diethyl èther and the solu-tion was stirred at room temperature overnight. The mixture was filtered and the filtrate was evaporated to dryness under vacuum. The residue was dissolved in 25 ml of ethyl acetate, and the mixture was set in the refrigerator. The mixture was filtered and the fil-trate was chromatographed by HPLC while eluting with hexane/ethyl acetate (4/1, v/v). The first chromaphore containing fractions were combined and the solvent was evaporated therefrom to provide 2.5 g of 1-(2,6-~.2~i~7~38 dicl~loro-9H-purln-9-yl)-3,5-bis(t-butyldimethylsiloxy)-2-desoxy-2,2-difluororibose. m/e = [568-(t-butyl)] = 511 B. l-(2-Chloro-6-oxo-lH,9H-purin-9-yl)-2-desoxy-2,2-difluororibose and l-(2-chloro-6-bromo-9~-purin-9-yl)-2-desoxy-2,2-difluororibose.

A solution of 0.5 g (0.88 mmol~ of 1-(2,6-dichloro-9H-purin-9-yl)-3,5-bis~t-butyldimethylsiloxy)-2-desoxy-2,2-difluororibose dissolved in 100 ml of methylene chloride cooled to about 0C was saturated with anhydrous hydrogen bromide gas. The mixture was stirred at CC for about 7 hours and then at room temperature for about 16 hours. The mixture was fil-tered, and the precipitated solid was dissolved in methanol. The methanolic solution was concentration under vacuu~ to provide 160 mg of a mixture of 1-(2-chloro-6-oxo-lH,9H-purin-9-yl)-2-desoxy-2,2-difluoro-ribose and l-(2-chloro-6-bromo-9H-purin-9-yl)-2-desoxy-2,2~difluororibose as a light yellow solid. m/e = 322 and 386 respectively.

C. 1-(2-Chloro-6-oxo-lH,9H-purin-9-yl)-2-desoxy-2,2-difluororibose A mixture of 1.18 g (3 mmol) of 1-(2-chloro-6-oxo-lH,9H~purin-9-yl)-2-dèsoxy-2,2-difluororibose and 1-(2-chloro-6-bromo-9H-purin-9-yl)~2-desoxy-2,2-difluoro-ribose dissolved in 11 ml of 1.0 N sodium hydroxide was stirred at room temperature for three hours. The pH of the mixture was lowered to about 7 with 2N hydrochloric ''` ' -7;3~3 acid. The mixture was concentrated under vacuum at about 45C. The residue was slurried in warm methanol, filtered and this procedure was repeated. The filtrates were combined and the solution was concentrated under vacuum at 15C to provide 1.36 g of l-(2-chloro-6-oxo-lH,9H-purin-9-yl)-2-desoxy-2,2-difluororibose. m/e = 322.

D. This is the preferred synthesis of 1-(2-amino-6-oxo-lH,9H-purin-9-yl)-2-desoxy-2,2-difluoro-ribose. The material prepared by the following reaction was used as a reference standard for the subsequent synthesis of the compound which was biologically evaluated.
To a suspension of 1.3 g of 1-(2-chloro-6-oxo-lH,9H-purin-9-yl)-2-desoxy-2,2-difluororibose in 30 ml of absolute ethanol at a temperature of about 0C was added anhydrous ammonia. The mixture was placed in a closed reaction vessel and heated at about 150C over-night. The mixture was cooled and the solid was col-lected. The filtrate was suspended in 15 ml of hot methanol and the mixture was again filtered. The fil-trate was concentrated under vacuum and the residue was chromatographed by HPLC using water/methanol (9/1, v/v) as the eluent at a flow rate of 4 ml/minute to provide 10 mg of ~ (2-amino-6-oxo-lH,9H-purin-9-yl)-2-desoxy-2,2-difluororibose and 5 mg of ~-1-(2-amino-6-oxo-lH,9H-purin-9-yl)-2-desoxy-2,2-difluororibose. m/e = 303 The compounds which were biologically tested were prepared as follows:

~6~7~38 To 0.26 g of a mixture of 1-(2-chloro-6-oxo-lH,9H-purin-9-yl~-2-desoxy-2,2-difluorori~ose and 1-(2-chloro-6-bromo-9H-purin-9-yl)-2-desoxy-2,2-difluoro-ribose in 10 ml of absolute ethanol at about 0C was added anhydrous ammonia for 20 minutes. The flask was sealed and placed in an oil bath at about 150C for about 16 hours. The volatiles were evaporated under reduced pressure and the residue was purified by standard procedures to provide 9.6 mg of ~-1-(2-chloro-6-amino-9H-purin-9-yl)-2-desoxy-2,2-difluororibose having m/e = 322; 8.2 mg of ~-1-(2-chloro-6-amino-9H-purin-9-yl)-2-desoxy-2,2-difluororibose having ~/e = 322 and an NMR (CD30D, 300 mHz, ~) 3.8-4.65 tm, 4H); 4.93 (bs, 4H); 6.25 (dd, lH); 8.35 (s, lH); 6.5 mg of a mixture of ~- and ~-l-(2,6-diamino-9H-purin-9-yl)-2-desoxy-2,2-difluororibose having (m + l)/e = 304 and m/e calc. 303.1017; obs. 303.1009; 9.0 mg of 1-(2-amino-6-oxo-lH,9H-purin-9-yl)-2-desoxy-2,2-difluororibose having (m+H)/e and calc. 304.0857; obs. 304.0857; and NMR
~CD3OD, 300 mF~z, ~) 3.85-4.65 (m, 4~); 4.9 (bs, 5H);
6.15 (dd, lH); 7.98 (s, lH); and 9.0 mg of ~- and ~ (2,6-dioxo-lH,3H,9H-purin-9-yl)-2-desoxy-2,2-difluororibose having m/e = 304.
The present invention provides a method of treating susceptible neoplasms in mammals comprising administering to a mammal in need of such treatment a pharmaceutically effective amount of a compound of formula I. The method comprises administering the compound to the mammal by various routes including the oral, rectal, transdermal, subcutaneous, intravenous, intramuscular or intranasal routes.

7;~8 The term "pharmaceutically effective amount"
refers to an appropriate amount of a compound of formula I which is capable of providing chemotherapy to mammals. The active compounds are effectlve over a wide dosage range. For example, dosages per day will normally fall within the range of about 0.1 to about 1200 mg/kg of body weight. In the treatment of adult humans, the range of about 0.1 to about 50 mg/kg, in single or divided doses, is preferred. However, it will be understood that the amount of compound actually administered will be determined by a physician, in the light of the relevant circumstances including the condition to be treated, the particular compound to be administered, the chosen route of administration, the age, weight, and response of the individual patient, and the severity of the patient's symptoms, and therefore the above dosage ranges are not intended to limlt the scope of the invention in any way.
The term "susceptible neoplasm", as defined herein, represents an abnormal growth of tissue in mammals capable of being treated by a compound of formula I. While the compounds of formula I are effec-tive against tumors, both solid and non-solid type, the compounds are effective in controlling the growth of rapidly dividing cells because of the compounds' cyto-toxic nature. It is a special feature of these com-pounds that they have a broad spectrum of activity, and are accordingly useful against a variety of tumors.
The compounds of the present method are preferably administered as a pharmaceutical formulation.
Therefore, as yet another embodiment of the present 73~

invention, a pharmaceutical formulation useful for treating susceptible neoplasms in mammals is provided comprising a compound of formulae II or III in combina-tion with a pharmaceutical carrier, diluent or excipient therefor.
The active ingredient will be present in the formulation in the range of about 1% to about 90% by weight. The active ingredient will usually be mixed with a carrier, or diluted by a carrier, or enclosed within a carrier which may be in the form of a capsule, sachet, paper or other container. When the carrier serves as a diluent, it may be a solid, semi-solid or liquid material which acts as a vehicle, excipient or medium for the active ingredient. Thus, the composi-tions can be in the form of tablets, pills, powders,lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), ointments containing for example up to 10% by weight of the active compound, soft and hard gelatin capsules, suppositories, sterile injectable solutions and sterile packaged powders.
Some examples of suitable carriers, excipi-ents, and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrroli-done, cellulose, water, syrup, methyl cellulose, methyl-and propylhydroxybenzoates, talc, magnesium stearate and mineral oil. The formulations can additionally include lubricating agents, wetting agents, emulsifying and suspending agents, preserving agents, sweetening agents .

.

1~ti47;~8 or flavoring agents. The compositions of the invention may be formulated so as to provide quick, sustained release of the active ingredient after administration to the patient by employing procedures well known in the art.
The compositions are preferably formulated in a unit dosage form, each dosage containing from about 5 to about 500 mg, more usually about 25 to about 300 mg, of the active ingredient. The term "unit dosage form"
refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association wi-th a suitable pharmaceutical carrier.
The following formulation examples represent specific pharmaceutical formulations employing compounds comprehended by the present method. The formulations may employ as active compounds any of the compounds of Formula I. The examples are illustrative only and are not intended to limit the scope of the invention in any way.

Formulation l Hard gelatin capsules are prepared using the following ingredients:

Quantity (mg/capsule) 1-(4-amino-5-methyl-2-oxo-lH-pyrimidin-1-yl)-2-desoxy-2,2-difluororibose 250 Starch dried 200 Magnesium stearate 10 The above ingredients are mixed and filled into hard gelatin capsules in 460 mg quantities.
Formulation 2 A tablet formula is prepared using the ingre-dients below:
Quantity (mg/tablet) 1-(2-oxo-4-amino-lH-pyrimidin-1-yl)-2-desoxy-2,2-difluoro-ribose 250 Cellulose, microcrystalline 400 Silicon dioxide, fumed 10 Stearic acid 5 The components are blended and compressed to form tablets each weighing 665 mg.

12~ 7~38 Formulation 3 An aerosol solution is prepared containing the following components:
Wei~ht %
1-(2,4-dioxo-lH,3H-pyrimidin-1-yl)-2-desoxy-2,2-difluoro-ribose 0.25 Ethanol 29.75 Propellant 22 70.00 (Chlorodifluoromethane) The active compound is mixed with ethanol and the mixture added to a portion of the propellant 22, cooled to -30C and transferred to a filling device.
The required amount is then placed in a stainless steel container and diluted with the remainder of the propel-lant. The valve units are then fitted to the container.
.

Formulation 4 Tablets each containing 60 rng of active ingredient are made up as follows:
1-~4-amino-2-oxo-lH-pyrimidin-1-yl)-2-desoxy-2,2-difluoro-ribose 60 mg Starch 45 mg Microcrystalline cellulose 35 mg Polyvinylpyrrolidone (as 10% solution in water) 4 mg Sodium carboxymethyl starch 4.5 mg Magnesium stearate 0.5 mg Talc 1 mg ,: . .. . .
'"' ~':

:, ~

7;~8 The difluoronucleoside starch and cellulose are passed through a No. 45 mesh U.S. sieve and mixed thoroughly. The solution of polyvinylpyrrolidone is mixed with the resultant powders which are then passed through a No. 14 mesh U.S. sieve. The granules so produced are dried at 50-60C and passed through a No.
18 mesh U.S. sieve. The sodium carboxymethyl starch, magnesium stearate and talc, previously passed through a No. 60 mesh U.S. sieve, are then added to the granules which, after mixing, are compressed on a tablet machine to yield tablets each weighing 150 mg.

Formulation 5 Capsules each containing 80 mg of medicament are made as follows:
1-~4-amino-2-oxo-lH-pyrimidin-1-yl)-2-desoxy-2,2-difluor-oxylose 80 mg Starch 59 mg Microcrystalline cellulose 59 mg Magnesium stearate 2 mg The active ingredient, cellulose, starch and magnesium stearate are blended, passed through a No. 45 mesh U.S. sieve, and filled into hard gelatin capsules in 200 mg quantities.

~47~8 Formulation 6 Suppositories each containing 225 mg of nucleoside are made as follows:
1-(2,4-dioxo-lH,3H-pyrimidin-1-yl)-2-desoxy-2,2-difluoro-ribose 225 mg Saturated fatty acid glycerides to 2 g The nucleoside is passed through a No. 60 mesh U.S. sieve and suspended in the saturated fat~y acid glycerides previously melted using the minimum heat necessary. The mixture is then poured into a supposi-tory mold of nominal 2 g capacity and allowed to cool.

Formulation 7 Suspensions each containing 50 mg of medica-0 ment per 5 ml dose are made as follows:1-(4-amino-5-methyl-2-oxo-lH-pyrimidin-l-yl~-2-desoxy-2,2-difluororibose 50 mg Sodium carboxymethyl cellulose 50 mg Syrup 1.25 ml Benzoic acid solution 0.10 ml Flavor q.v.
Color q.v.
Purified water to 5 ml The medicament is passed through a No. 45 meshU.S. sieve and mixed with the sodium carboxymethyl cellulose and syrup to form a smooth paste. The benzoic ~ , ~ , , , 7;~8 acid solution, flavor and color are diluted with some o~
the water and added, with stirring. Sufficient water,is then added to produce the required volume.

Formulation 8 An intravenous formulation is prepared as follows:
1-(4-amino-2-oxo-lH-pyrimidin-1-yl)-2-desoxy-2,2-difluoro ribose 100 mg isotonic saline 1000 ml The solution of the above ingredients is administered intravenously at a rate of 1 ml/minute to a mammal in need of treatment from susceptible neoplasms.
The activity of representative compounds employed in the present invention has been demonstrated in standard screens commonly used by those in the art for testing compounds with potential antitumor activity.
For example, these screens have been used to demonstrate the antitumor activity of commercially available cancer drugs such as the vinca alkaloids. See, e.q., Miller et al., in J. Med. Chem. Vol. 20, No. 3 409 ~1977) and Sweeney, _ al., in Cancer Research 38, 2886 (1978).
The compounds represented by formula I are cytostatic in that they inhibit the growth of human leukemic cells (CCRF-CEM cell line). Table 1 below gives the results of the testing of several compounds representative of those of Formula I. In the Table, column 1 gives the name of the compound and column 2 the IC50 (concentration giving 50% growth inhibition) in mcg/ml.

l~gj~ 7~8 Table 1 Cytotoxicitv Screen Com~ound Name IC50 mcg/ml 1-(4-amino 2-oxo-lH-pyrimidin-1- 0.0039 yl)-2-desoxy-2,2-difluororibose 0.0057 0.0068 0.0260 1-(4-amino-2-oxo-lH-pyrimidin-1- 0.3 yl)-2-desoxy-2,2-difluoroxylose 1-(2,4-dioxo-lH,3H-pyrimidin-l-yl)- 5.4 2-desoxy-2,2-difluororibose 1-(4-amino-5-methyl-2-oxo-lH-pyrimidin- 0.3 l-yl)-2-desoxy-2,2-difluororibose ~-1-(6-amino-9H-purin-9-yl)-2-desoxy- 0.5 2,2-difluororibose ~ (6-amino-9H-purin-9-yl)-2-desoxy- 6.9 2,2-difluororibose ~-1-(2-chloro-6-amino-9H-purin-9- >20.0 yl)-2-desoxy-2,2-difluororibose ~-1-(2-chloro--6-amino-9H-purin-9~ 0.4 yl)-2-desoxy-2,2-difluororibose 1-(2,6-diamino-9H-purin-9-yl)-2- 0.075 desoxy-2,2-difluororibose 1-(2-amino-6-oxo-lH,9H-purin~9- 0.10 yl)-2-desoxy~2,2-difluororibose 1-(2,6-dioxo-lH,3H,9H-purin-9-yl)- 0.30 2-desoxy-2,2-difluororibose , ., .

~ 7 To further demonstrate the ability of the compounds o~ formula I to treat susceptible neoplasms in mammals, the compounds of Example 1, 1-(4-amino-2-oxo-lH-pyrlmidin-1-yl)-2-desoxy-2,2-difluororibose, Example 5, 1-(4-amino-2-oxo-lH-pyrimidin-l-yl)-2-desoxy-2, 2-difluoroxylose, and Example 6, 1-(6-amino-9H-purin-9-yl)-2-desoxy-2,2-difluororibose, were tested in animals bearing a tumor system representative of L1210V
lymphocytic leukemia.
The study testing the efficacy of these com-pounds against L1210V leukemia was initiated by an IP
inoculation of 1 X 106 cells. Trea-tment was begun 24 hours after inoculation. The response to therapy was determlned by comparing the mean life span of the ten treated animals to that of the ten control animals;
prolongation of life in the treated animals beyond that of controls is expressed as a percentage. Table 2 gives the results of several experiments in mice bearing this tumor. In the Table, column 1 gives the example number of the compound tested; column 2, the experiment number;
column 3, the dose level of the compound in mg/kg;
column 4, the route of administration; column 5, the dosage schedule, that is, the days on which the compound was administered to the mice; column 6, the average increase in life span of the treated mice as compared to the control mice; column 7, the toxic deaths over the number of mice in each group; and column 8, the indefi-nite survivors, that is, the number of 45 day survivors in each group.

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x z E ~ u~ ~o r7;38 The compounds of Example 1, 1-(4-amino-2-oxo-lH-pyrimidirl-1-yl)-2-deso~y-2,2-difluororibose, and Example 5, 1-(4-amino-2-oxo-lH-pyrimidin-l-yl)-2-desoxy-2,2-difluoroxylose, also demonstrated activity in additional tumor test systems. These systems include the 6C3H~D lymphosarcoma, also known as the Gardner lymphosarcoma (6C3HED); the CA~755 adenocarcinoma (CA755); the P1534J lymphatic leukemia (P1534J); and the X5563 plasma cell myeloma (X5563). Each of these systems is described in detail below.
6C3HED. - The 6C3HED lymphosarcoma was ob-tained in 1979 from the Division of Cancer Treatment, N.C.I., tumor bank maintained at E. G. and G. Mason Research (Worchester, MA). First passage tumor was stored in liquid nitrogen using standard techniques.
The transplanted tumor was re-established from the tumor bank every six months or as needed. The tumor is maintained by serial passage twice weekly in C3H mice (Charles River; Wilmington, MA).
CA755 - The adenocarcinoma 755 is an undiffer entiated mammary carcinoma which was obtained in 1980 from the Division of Cancer Treatment, N.C.I., tumor bank maintained at E. G. and G. Mason Research (Worchester, MA). First passage tumor was stored in liquid nitrogen using standard techniques. The trans-planted tumor was re established from the tumor bank every six months or as needed. The tumor is maintained by serial passage once a week in C57BL/6 emale mice (Jackson Laboratory; Bar Harbor, ME).

a~7~8 Pl53~J - The P1534J lymphocytic leukemia ~solid form) was obtalned in 1973 from the Jackson Laboratory ~Bar Harbor, ME). First passage tumor was stored in liquid ni~rogen using standard techniques.
Subsequent replenishment of the tumor bank with this tumor was accomplished from first passage tumor. The transplanted tumor was re-established from the tumor bank every six months or as needed. The tumor is maintained by serial passage once a week in DBA/2 mice (Charles River; Wilmington, MA).
X5563 Myeloma - the tumor is maintained in C3H
mlce .
The following procedure was employed in demon-strating the activity of these compounds against the tumor systems. The tumor was removed from passage animals and minced into l to 3 mm square fragments using sterile techniques. Tumor pieces were checked for sterility using both Antibiotic Medium 1 and Brain Heart Infusion (Difco; Detroit, MI). Recipient mice were shaved and tumor pieces were implanted subcutaneously in the axillary region by trocar. Drug therapy on the appropriate schedule was initiated on the day after tumor implant. The compound was dissolved in saline for all experiments. All animals were weighed at the beginning and end of drug treatment. Food and water were provided ad libitum. On days 10 to 12, two dimen-sional measurements (width and length) of all tumors were taken using vernier calipers. Tumor weights were calculated from these measurements using the following formula:

7~8 Tumor I.~eight ~mg) = Tumor Length (mm) X Tumor Width (mm) /2 For all data, the tumor weigh-t was rounded -to the neaxest tenth of a gram for analysis. No group is included in the analysis for therapeutic activity in which deaths attributable to drug toxicity exceeded 30 percent of the treated group.
In Table 3 which follows, column 1 gives the example number of the compound tested; column 2 provides the tumor system; column 3, the dose level; column 4, the route administered; column 5, the dosage schedule;
column 6, the percent inhibition of the tumor; and column 7, the toxic deaths observed prior to completion of the study~

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The compounds employed in the present method are also effective for the treatment of viral infec-tions, and more particularly in the treatment of infec-tions caused by viruses o the herpes genus. The compounds are effectively administered orally, topically or parenterally. In general, dosage rates in the range of from about 5 mg/kg to about 500 mg/kg are useful. It is more preferred to administer at rates in the range of from about lO mg/kg to about 100 mg/kg.

Claims (25)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE PROPERTY
OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A compound of formula (II):

(II) in which R6 is hydrogen or C1-C4 alkyl;
R7 is a pyximidine base of one of the formulae Q is N, C-(C2-C4 alkyl) or C-amino;
X is N or C-R4; and R8 is hydrogen or C1-C4 alkyl; or a pharmaceutically-acceptable salt thereof; with the proviso that R6 and R8, both may be hydrogen only when X is N.

2. A compound of formula (II):
(II) in which:
R6 is hydrogen or C1-C4 alkyl; or R7 is a purine base of the formula:

wherein R8 is hydrogen or C1-C4 alkyl; or a pharmaceutically-acceptable salt thereof; with the proviso that R6 and R8, both may not be hydrogen.

3. A compound of formula (II):

(II) in which:
R6 is hydrogen or C1-C4 alkyl;
R7 is a purine base of the formula:

wherein R8 is hydrogen or C1-C4 alkyl; and R4 is C1-C4 alkyl, amino, bromo, fluoro, chloro and iodo;
or a pharmaceutically-acceptable salt thereof.

4. A compound of formula (III):

(III) in which:

R6 is hydrogen or C1-C4 alkyl; and ; or a pharmaceutically-acceptable salt thereof.

5. A pharmaceutical formulation for use in treating susceptible neoplasms in mammals comprising as the active ingredient, a compound according to claim 1, 2 or 3 or a pharmaceutically-acceptable salt thereof, in combination with a suitable pharmaceutically-acceptable carrier, diluent or excipient therefor.

6. A pharmaceutical formulation for use in treating susceptible neoplasms in mammals comprising, as the active ingredient, a compound of claim 4, or a pharmaceutically-acceptable salt thereof, in combination with a suitable pharmaceutically-acceptable carrier, diluent or excipient therefor.

7. 1-(2-chloro-6-amino-9H-purin-9-yl)-2-desoxy-2,2-difluororibose, or a pharmaceutically-acceptable salt thereof.

8. 1-(2,6-dioxo-9H-purin-9-yl)-2-desoxy-2,2-difluororibose, or a pharmaceutically-acceptable salt thereof.

9. 1-(2,6-dioxo-1H, 3H, 9H-purin-9-yl)-2-desoxy-2,2-difluororibose, or a pharmaceutically-acceptable salt thereof.

10. A pharmaceutical formulation for use in treating susceptible neoplasms in mammals comprising as the active ingredient, the compound recited in claim 7, or a pharmaceutically-acceptable salt thereof, in association with a suitable pharmaceutically-acceptable carrier, diluent or excipient therefor.

11. A pharmaceutical formulation for use in treating susceptible neoplasms in mammal , comprising as the active ingredient the compound recited in claim 8, or a pharmaceutically-acceptable salt thereof, in association with a suitable pharmaceutically-acceptable carrier, diluent or excipient therefor.

12. A pharmaceutical formulation for use in treating susceptible neoplasms in mammals, comprising as the active ingredient the compound recited in claim 9, or a pharmaceutically-acceptable salt thereof, in association with a suitable pharmaceutically-acceptable carrier, diluent or excipient therefor.

13. A pharmaceutical formulation for use in treating susceptible neoplasms in mammals which comprises, as the active ingredient, a compound of formula (I) (I) in which:

R1 is hydrogen, C1-C4 alkyl or -C-R5;

R2 is a base defined by one of the formulae X is N or C-R4;
O

R3 is hydrogen, C1-C4 alkyl or -C-R5;
R4 is hydrogen, C1-C4 alkyl, amino, bromo, fluoro, chloro or iodo;
each R5 independently i5 hydrogen or Cl-C4 alkyl; or a pharmaceutically-acceptable salt thereof, in association with a suitable pharmaceutically-acceptable carrier, diluent or excipient therefor.

14. A pharmaceutical formulation according to claim 13 wherein the active ingredient is the compound 1-(4-amino-2-oxo-1H-pyrimidin-1-yl)-2-desoxy-2,2-difluororibose, or a pharmaceutically-acceptable salt thereof.

15. A pharmaceutical formulation according to claim 13 whereln the active ingredient is the compound 1-(4-amino-2-oxo-lH-pyrimidin-1-yl)-2-desoxy-2,2-difluoroxylose, or a pharmaceutically-acceptable salt thereof.

16. A pharmaceutical formulation according to claim 13 wherein the active ingredient is the compound 1-(2,4-dioxo-1H, 3H-pyrimidin-1-yl)-2-desoxy-2,2-difluororibose, or a pharmaceutically-acceptable salt thereof.

17. A pharmaceutical formulation according to claim 13 wherein the active ingredient is the compound 1-(4-amino-5-mathyl-2-oxo-1H-pyrimidin-1-yl)-2-desoxy-2,2-difluororibose, or a pharmaceutically-acceptable salt thereof.

18. A process for preparing a compound of formula (II) (II) in which:
R6 is hydrogen or C1-C4 alkyl;
R7 is a pyrimidine base of one of the formulae Q is N, C-(C2-C4 alkyl) or C-amino;
X is N or C-R4; and R8 is hydrogen or C1-C4 alkyl; or a pharmaceutically-acceptable salt thereof; with the proviso that R6 and R8, both may be hydrogen only when X is N, which comprises removing the protecting groups from a correspondingly protected nucleoside of formula (II); and where desired, forming a pharmaceutically-acceptable salt of said compound of formula (II).

19. A process for preparing a compound of formula (II) (II) in which:
R6 is hydrogen or C1-C4 alkyl;
R7 is a purine base of the formula wherein R8 is hydrogen or C1-C4 alkyl; or a pharmaceutically-acceptable salt thereofi with the proviso that R6 and R8, both may not be hydrogen, which comprises removing the protecting groups from a correspondingly protected nucleoside of formula (II); and where desired, forming a pharmaceutically-acceptable salt of said compound of formula (II).

20. A process for preparing a compound of formula (II) (II) in which:
R6 is hydrogen or C1-C4 alkyl;
R7 is a purine base of the formula wherein R8 is hydrogen or C1-C4 alkyl; and R4 is C1-C4 alkyl, amino, bromo, fluoro, chloro and iodo;
or a pharmaceutically-acceptable salt thereof, which comprises removing the protecting groups from a correspondingly protected nucleoside of formula (II); and where desired, forming a pharmaceutically-acceptable salt of said compound of formula (II).

21. A process for preparing a compound of formula (III) (III) in which:
R6 is hydrogen or C1-C4 alkyl; and ; or a pharmaceutically-acceptable salt thereof, which comprises removing the protecting groups from a correspondingly protected nucleoside of formula (III); and where desired, forming a pharmaceutically-acceptable salt of said compound of formula (III).

22. A process for preparing a compound of formula (II) (II) in which:
R6 is hydrogen or C1-C4 alkyl;
R7 is a pyrimidine base of one of the formulae Q is N, C-(C2-C4 alkyl) or C-amino;
X is N or C-R4; and R8 is hydrogen or C1-C4 alkyl; or a pharmaceutically-acceptable salt thereof; with the proviso that R6 and R8, both may be hydrogen only when X is N, which comprises:
coupling a pyrimidine base of the formula R7H, or a protected derivative thereof, with a carbohydrate of formula (IV), or a protected derivative thereof:

(IV) in which the R7H base and R6 are as defined in claim 1 and Leav is a leaving group, and if desired, removing any protecting group present to produce the pyrimidine base product; and where desired, forming a pharmaceutically-acceptable salt of the product so prepared.

23. A process for preparing a compound of formula (II) (II) in which:
R6 is hydrogen or C1-C4 alkyl;
R7 is a purine base of the formula wherein R8 is hydrogen or C1-C4 alkyl; or a pharmaceutically-acceptable salt thereof;
with the proviso that R6 and R8, both may not be hydrogen, which comprises:

(a) coupling a base of the formula R7H, or a protected derivative thereof, with a carbohydrate of formula (IV), or a protected derivative thereof:
(IV) in which the R7H base and R6 are as defined in claim 1 and Leav is a leaving group, and if desired, removing any protecting group present to produce the base product; and (b) reacting with ammonia a corresponding purine nucleoside compound in which a C-2 and/or C-6 substituent of the purine portion of the compound is halogen, and, if desired, alkylating the product; and where desired, forming a pharmaceutically-acceptable salt of the product so prepared.

24. A process for preparing a compound of formula (II) (II) in which:
R6 is hydrogen or C1-C4 alkyl;
R7 is a purine base of the formula wherein R8 is hydrogen or C1-C4 alkyl; and R4 is C1-C4 alkyl, amino, bromo, fluoro, chloro and iodo;
or a pharmaceutically-acceptable salt thereof, which comprises:
(a) coupling a base of the formula R7H, or a protected derivative thereof, with a carbohydrate of formula (IV), or a protected derivative thereof:
(IV) in which the R7H base and R6 are as defined in claim 1 and Leav is a leaving group, and if desired, removing any protecting group present to produce the base product; and (b) reacting with ammonia a corresponding purine nucleos-ide compound in which a C-2 and/or C-6 substituent of the purine portion of the compound is halogen, and, if desired, alkylating the product; and where desired, forming a pharmaceutically-acceptable salt of the product so prepared.

25. A process for preparing a compound of formula (III):
(III) in which:

R6 is hydrogen or C1-C4 alkyl; and R9 is ; or a pharmaceutically-acceptable salt thereof, which comprises:
(a) coupling a base of the formula R7H, or a protected derivative thereof, with a carbohydrate of formula (IV), or a protected derivative thereof:
( IV) in which the R7H base and R6 are as defined in claim 1 and Leav is a leaving group, and if desired, removing any protecting group present to produce the base product; and (b) reacting with ammonia a corresponding purine nuclaoside compound in which a C-2 and/or C-6 substituent of the purine portion of the compound is halogen, and, if desired, alkylating the product; and where desired, forming a pharmaceutically-acceptable salt of the product so prepared.