JP2002506036A - Novel nucleoside analogs and their use in treating diseases - Google Patents

Abstract

  (57) [Summary] The present invention relates to novel nucleosides and nucleoside dimers containing an L-sugar in at least one of the nucleosides, and pharmaceutical compositions thereof.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] This application is related to US patent application Ser. No. 08 / 531,875 filed Sep. 21, 1995.
Is a continuation-in-part application of the issue

BACKGROUND OF THE INVENTION Field of the Invention The present invention relates to novel nucleoside and dinucleoside dimers and derivatives of these compounds, wherein the sugar moiety of at least one nucleoside has an L-conformation. Deoxyribofuranosyl nucleoside phosphodiester dimer). These compounds are highly effective in treating various diseases. These can be used to treat parasitic infections, such as those caused by Plasmodium falciparum, which is the causative agent responsible for the most lethal form of malaria. They can also be used to treat bacterial, viral, and fungal infections, and can also be used to treat cancer.

[0003] Modified nucleoside analogs are an important class of anti-neoplastic and anti-viral drugs. This application is based on Disclosed are novel compounds of this type for use in treating falciparum infections and other parasitic infections. Pl
Asmodium falciparum is the most lethal form of malaria (1
It is a etiological factor responsible for the disease affecting 200 to 300 million people (all forms) annually, including the death of more than one million children.
In addition, more than 40% of the world's population lives in areas where malaria is prevalent. Due to the unusual morbidity and mortality associated with malaria and other parasitic infections, related research has been strongly promoted in the last decade in the desperate search for effective treatments. Safe and effective vaccines do not yet exist. Instead, many victims have to resort to chemotherapy.

[0004] These modified nucleoside analogs can also be used to treat a variety of other parasitic, bacterial, fungal, viral, and cancerous infections.

[0005] These chemotherapeutic agents can be divided into two groups: those that act post-translationally and those that act by interfering with nucleic acid synthesis.

[0006] Most drugs are in the first group, which indicates that they exert their therapeutic effect by interfering with cellular protein synthesis and thus its metabolism (but not its nucleic acid synthesis). means. Examples of this group of drugs include the following: antifolate compounds, which inhibit dihydrofolate reductase, and sulfonamide drugs, which inhibit dihydropteroate synthetase. However, these drugs have significant disadvantages. For example, malaria-causing protozoa develop resistance to these drugs very quickly. The reason is that resistance is an adaptive mutation (ad) in successive generations of the parasite.
One or two point mutations are often sufficient to confer resistance, as they occur through active mutation. Bacterial, viral, and fungal infections are also often susceptible to these types of resistant mutations.

[0007] The second group of compounds includes nucleic acid intercalators such as acridine, phenanthrene, and quinoline. These intercalators
In part, it mimics the biochemical activity of nucleic acids and, therefore, protozoal or cellular,
Once incorporated into nucleic acids (DNA and RNA), once incorporated, it does not permit further nucleic acid synthesis, and thus its effectiveness. At the same time, these intercalators also interfere with host nucleic acid synthesis, thus producing toxic side effects. Because of the potential for toxic side effects, these drugs can very often be given only in very small doses. Again, a resistive pattern can occur. For example,
Many protozoa are known to develop "cross-resistance", which means that parasites develop resistance to other classes of drugs (even if they are exposed to different classes of drugs). Also means).

[0008] In fact, all currently known drugs or drug candidates that utilize the delivery of cytotoxic pyrimidine or purine biosynthesis inhibitors to invading cells are highly toxic.
Thus, while drugs of this type (ie, those that interfere with nucleic acid synthesis) are effective, they lack selectivity. This latter parameter is what must be maximized in the development of a safe and effective drug. In other words, such drugs target host tissues that are infected or cancerous, while leaving the host tissues unchanged.

[0009] Recent advances in our understanding of the biochemistry of parasite cells serve as valuable examples for the design of effective therapies. A researcher (H. Ginsburg, Bioc)
hem. Pharmacol. 48, 1847-1856 (1994)) observe that normal and parasite-infected erythrocytes show significant differences in purine and pyrimidine metabolism in a single enzyme and in all branches of the relevant pathway. did. The parasite satisfies all of its purine requirements via the scavenger pathway; while host cells lack the enzymes necessary to utilize this pathway, and therefore, their pyrimidine requirements largely Must be satisfied by de novo synthesis. Stated another way, parasites are more effective than normal or host cells. Because it can synthesize a nucleic acid binding block.

[0010] Other researchers (G. Beaton, D. Pellinqer, WS Mars)
hall & M. H. Caruthers, In: Oligonucleotides
es and Analogues: A Practical Approac
h, F. Edited by Eckstein, IRL Press, Oxford, 109-
136 (1991) disclose that malaria-infected erythrocytes are not naturally occurring for use in nucleic acid synthesis.
-As opposed to "nucleosides"). In addition,
Normal mammalian cells are not permeable to this class of compounds. this is,
Suggests that the L-nucleoside is not toxic to normal mammals or host cells. Thus, derivatives of these compounds can be used as highly selective drugs for parasitic infections or as highly selective drugs for any other type of cell or organism utilizing L-nucleosides. Chemical modifications of L-nucleosides are generally such that the nucleosides are still recognized by the nucleic acid synthesis machinery of the invading cell or organism, and therefore are incorporated into the nucleic acid strand, but once this incorporation occurs, further synthesis occurs. Modifying the nucleoside so that it does not occur.

[0011] Currently, there are no therapeutic compounds for use based on dimers of these nucleoside analogs. While dimers of naturally occurring D-deoxyribofuranosyl nucleosides are well known, dimers in which one or both of the nucleosides are in the non-naturally occurring L-conformation are largely unknown. And their use in treating neoplastic and viral diseases is unknown.

In the synthesis of DNA-related oligomers, nucleoside dimer types are synthesized as part of the overall process. These dimers usually contain bases derived from the naturally occurring DNA or RNA sequence. Nucleoside monophosphate dimers are well known in the art. Many of these compounds have been synthesized and are commercially available. However, these dimers are made from nucleosides that contain a sugar moiety in the D-conformation.

Reese, C.A. B. , Tetrahedron 34 (1978) 3143
Describes the synthesis of fully protected dinucleoside monophosphates by means of the phosphotriester approach.

[0014] Littauer, U.S.A. Z. And Soreg, H .; (1982) The
Enzymes, Vol. XV, Academic Press, NY, 517
Page is a standard reference describing the enzymatic synthesis of dinucleotides.

Heikkilo, J. et al. Strid, S .; Obberg, B .; And Cha
ttpodhyaya, J. et al. , Acta Chem. Scand. B39 (1
985) 657-669 provides examples of the methodology used in the synthesis of various ApG nucleoside phosphate dimers. 3 ′ → 5 ′ phosphate and 2
References and methods for the synthesis of '→ 5' phosphate by solution phase chemistry are included.

Gait, M .; , "Oligoncleotide Synthesis"
, IRL Press, Ltd. , Oxford, England, 1984, is a general reference and is useful for reviewing oligonucleotide synthesis. The method is applicable to both dimer synthesis, both solution phase and solid phase methods. Both phosphite triester and phosphotolyester methods of attaching nucleosides are described. Solid phase methods are useful for dimer synthesis.

Gulyawa, V .; And Holly, A .; , Coll. Czec. Chem
. Commun. 44 613 (1979) describes the enzymatic synthesis of a series of dimers by the reaction of a 2'-3 'cyclic phosphate donor with a ribonucleoside acceptor. This reaction was catalyzed by a non-specific RNase. The donor is phosphorylated at the 5 'position, resulting in the following compound: donor nucleoside-
(3 ′ → 5 ′) Acceptor nucleoside. The dimer is converted to the acceptor β-
L-cytidine, β-L-adenosine, and 9 (α-L-lyxofuranosyl (l
yxofuranosyl)) made using adenine. Also, a number of dimers with the D-nucleoside in the 5 'position of the acceptor were made.

[0018] Holly, A. et al. Sorm, F .; , Collect. Czech. Chem.
Commun. , 34, 3383 (1969) describe β-D-guanylyl- (3 ′
→ 5 ′)-β-L-adenosine and β-D-guanylyl- (3 ′ → 5 ′)-β
The enzymatic synthesis of L-cytidine is described.

Schirmeister, H .; And Pfleiderer, W.C. , Hel
v. Chim. Acta 77, 10 (1994) describes all trimer synthesis and intermediate dimers, forms from β-D-nucleosides. They used the phosphoramidite method which gave good yields.

Therefore, a dimer having an L-deoxyribofuranosyl moiety at any position is represented by L
-The ribofuranosyl moiety is new, as is the dimer linked at the 3 'position of the phosphate intermediate.

[0021] Modified nucleoside analogs represent an important class of compounds in available weapons for anti-neoplastic and anti-viral drugs. Anticancer drug 5-fluorodeoxyuridine (floxyuridine), cytarabine, and deoxycoformicin;
And antiviral agents 3 'azidedeoxythymidine (AZT), dideoxycytidine (ddC), dideoxyinosine (ddI), acyclovir, 5-iododeoxyuridine (idoxuridine) fludarabine phosphate and vidarabine (adenine arabinoside / ara A) Is representative of this class of monomeric nucleoside-derived compounds used therapeutically.

More recently, “antisense” oligonucleotide analogs having modified base and / or phosphodiester backbones have been actively sought as antiviral and antitumor agents. Clinically approved drugs have not yet emerged from this class of compounds, but remain a very active area of research. Recently,
Antipodal L-sugar-based nucleosides also find application as potent antiviral agents. Because they can inhibit viral enzymes without affecting mammalian enzymes, resulting in agents with selective antiviral activity without concurrent mammalian cytotoxicity.

[0023] Most naturally occurring nucleosides have a D-conformation in the sugar moiety.
While the chemical properties of L-nucleosides are similar to those of their β-D-enantiomers, they show a very different biological profile in mammalian cells and exhibit normal D-nucleoside transport. Do not interfere. For example, β-
L-uridine is not phosphorylated at the 5 ′ position by human prostate phosphotransferase, which readily phosphorylates the enantiomer β-D-uridine. Clearly, L-nucleosides are not substrates for normal human cell kinases, but they cannot be phosphorylated by viral and cancer cell enzymes, enabling their use in the design of selective antiviral and anticancer agents. I do.

[0024] L-nucleoside based oligonucleotides have been previously studied. α
Octamers derived from-and β-L-thymidine were found to be resistant to fungal nucleases and bovine spleen phosphodiesterase, which readily degrades the corresponding β-D-oligonucleotide. . Fujimori et al. Fujimori, K .; Shudo, Y .; Hashimoto, J .; Am
. Chem. Soc. , 112, 7436 are enantiomer poly-α-DNA
Recognizes that it recognizes complementary RNA but not complementary DNA. This principle has been used in the design of nuclease resistant antisense oligonucleotides for potential therapeutic applications.

Thus, compounds based on L-nucleosides have potential as drugs against neoplastic, fungal and viral diseases and against parasitic infections. Although L-sugar-derived nucleosides and their oligonucleotides have been extensively evaluated for such activity, little is known about the biological activity of shorter oligomers such as dimers obtained by L-nucleoside substitution. Not been.

The present invention includes novel L-nucleoside derived antitumor, antiviral, antibacterial, antifungal, and antiparasitic agents. A novel L-nucleoside-derived dinucleoside monophosphate based on L-α-5-fluoro-2′-deoxyuridine showed a significantly higher potency activity profile in in vitro assays. This indicates a unique mechanism of action involving inhibition of telomerase. Thus, L-nucleosides can act as building blocks for new drugs with the particular advantage of low toxicity.

SUMMARY OF THE INVENTION [0027] Further embodiments of the invention include cancer, viral infection, parasite infection, fungal infection,
And the administration of a therapeutically effective amount of a compound of the present invention for the treatment of a bacterial infection.

Other and further objects, features, and advantages will be apparent from the following description of preferred embodiments of the invention, which are provided for purposes of disclosure when considered in conjunction with the accompanying drawings.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS It will be readily apparent to those skilled in the art that various substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. It is.

As used herein, the term “dimer” is defined by the structure shown in FIG. These compounds are dinucleoside monophosphates derived from L-nucleosides. The B ₁ and B ₂ units consist of either β-D, β-L or α-L nucleosides, and at least one of B ₁ or B ₂ is β-L or α-L. R ₁ and R ₂ are pyrimidine bases cytosine, thymine, uracil or 5-fluorouridine (5-FUdR), other 5-halo compounds, or purine bases, adenosine, guanosine or inosine. As can be seen in FIG. 1, this dimer can be linked by various bonds. Possible combinations include:
5'-3 ', 3'-5', 3'-3 ', 5'-5', 2'-3 ', 3'
-2 ', 2'-2', 2'-5 ', 5'-2', or any other stereochemically possible linkage. The sugar moiety of the nucleoside can be completely oxidized or can be in deoxy or dideoxy form.

Particular anti-disease compounds useful in the present invention include 3′-O- (α-L-5-fluoro-2′-deoxyuridinyl) -β-D-5-fluoro-2′- Deoxyuridine, (L-102), 3′-O- (β-D-5-fluoro-2′-deoxyuridinyl) -α-L-5
-Fluoro-2'-deoxyuridine, (L-103), 3'-O- (β-D-5-fluoro-2'-deoxyuridinyl) -α-L-2
'-Deoxyuridine, (L-107), 3'-O- (α-L-5-fluoro-2'-deoxyuridinyl) -α-L-5
-Fluoro-2'-deoxyuridine, (L-108), 3'-O- (β-L-5-fluoro-2'-deoxyuridinyl) -β-L-5
-Fluoro-2'-deoxyuridine, (L-109), 3'-O- (β-D-5-fluoro-2'-deoxyuridinyl) -β-L-5
-Fluoro-2'-deoxyuridine, (L-110), 3'-O-([beta] -D-5-fluoro-2'-deoxyuridinyl)-[alpha] -L-2.
'-Deoxyuridine, (L-111), 3'-O- (β-D-5-fluoro-2'-deoxyuridinyl) -2'-deoxy-β-L-cytidine (L-113), 3′-O- (2′-deoxy-β-L-cytidinyl) -β-D-5-fluoro-
2′-deoxyuridine (L-114), 3′-O- (2′-deoxy-α-L-cytidinyl) -β-D-5-fluoro-
2′-deoxyuridine (L-115), 3′-O- (β-D-5-fluoro-2′-deoxyuridinyl) -β-L-2
'-Deoxyuridine (L-117), 3'-O- (β-L-5-fluoro-2'-deoxyuridinyl) -α-L-5
-Fluoro-2'-deoxyuridine (L-119), 3'-O- (β-D-5-fluoro-2'-deoxyuridinyl) -α-L-5
-Fluoro-2'-deoxyuridine (3 ', 3') (L-122), 3'-O- (3'-deoxy-β-D-adenosinyl) -β-L-2'-deoxyuridine (L -150), 3'-O- (3'-deoxy-β-D-adenosinyl) -β-L-2'-deoxyadenosine (L-151), 3'-O- (3'-deoxy-β- D-adenosinyl) -α-L-2′-deoxyuridine (L-152), 3′-O- (3′-deoxy-β-D-adenosinyl) -β-L-2′-deoxycytidine (L- 153), 3′-O- (3′-deoxy-β-D-adenosinyl) -α-L-2′-deoxyuridine (L-154), 3′-O- (3′-deoxy-β-D) -Adenosinyl) -β-L-2′-deoxyadenosine (L-155), 3′-O- (2′-deoxy-β-D-adenosine) Le) -β-L-2'- deoxyadenosine (L-210), or therapeutically acceptable salts of these aforementioned compounds may be mentioned. In a currently preferred embodiment, 3'-O-([beta] -D-5-fluoro-2'-deoxyuridinyl)-[alpha] -L-5-fluoro-2'-deoxyuridine (L-103).
Is used.

The term “internucleotide binder” or “IBA” refers to a backbone linkage that joins nucleosides together. However, one of ordinary skill in the art will readily appreciate that a variety of other scaffolds are available and useful in the present invention. See, for example, FIG. Here, methoxy phosphotriester, methyl phosphonate, phosphorodithioate, phosphorothioate, silyl ether, sulfonate and ethylene dioxy ether are indicated. The 3′-5 ′ IBA schematically shown is
Sugar 5'-3 ', 3'-5', 3'-3 ', 5'-5', 2'-3 ', 3'-2'
, 2'-2 ', 2'-5', 5'-2 ', or any other stereochemically possible linkage. The sugar can be completely oxidized or, if acceptable, in deoxy or dideoxy form. In a preferred embodiment, the IBA of the compound is either a phosphodiester or a phosphorothioate. As used herein, the term “anti-disease” refers to any activity (anti-tumor, anti-neoplastic, anti-cancer, anti-parasitic activity) of a compound of the present invention to affect a disease state. And antiviral activity).

A compound or composition is said to be “pharmacologically acceptable” if administration of the compound or composition can be tolerated by the recipient mammal. Such an agent is "a therapeutically effective amount" if the amount administered is physiologically significant.
It is said to be administered at An agent is physiologically significant if the presence of the agent causes a technological change in the physiology of the recipient mammal. For example, in the treatment of cancer or neoplastic disease, compounds that inhibit tumor growth or reduce tumor size are therapeutically effective; while, in the treatment of viral diseases, they slow disease progression An agent that completely treats a disease is considered therapeutically effective.

(Dosage and Formulation) The anti-disease compound (active ingredient) of the present invention can be used in various disease states (tumor, tumor, Neoplasia, cancer, bacterial, fungal, parasitic and viral diseases) and can be administered. The anti-disease compound may be administered by any conventional means available for use with pharmaceuticals, either as individual therapeutic active ingredients or in a combination of therapeutic active ingredients.
The anti-disease compound can be administered alone, but will generally be administered with a pharmaceutical carrier selected based on the chosen route of administration and standard pharmaceutical practice.

The dosages given by way of example herein are the dosages usually used in treating tumors, neoplasias and cancers. Low doses can also be used. Dosages for antiparasitic and antiviral applications are generally between 10 and 5 for anticancer applications.
0% dosage.

The dosage administered is a therapeutically effective amount of the active ingredient, and is, of course, known in the art (eg, the pharmacokinetic characteristics of the particular active ingredient and its dosage form and route; Age, sex, health and weight; the nature and extent of the condition; the type of concurrent treatment, the frequency of treatment and the desired effect). Generally, a daily dosage (therapeutically effective amount) of the active ingredient may be from about 5-400 milligrams per kilogram of body weight. Usually, 10 to 200, and preferably 10 to 50 milligrams per kilogram per day, given in 2 to 4 divided doses per day, or in a sustained release form, may be necessary to achieve the desired result. It is valid.

Dosage forms (composition) suitable for internal administration contain from about 1.0 to about 500 milligrams of active ingredient per unit. In these pharmaceutical compositions, the active ingredient will ordinarily be present in an amount of about 0.05-95% by weight based on the total weight of the composition.

The active ingredient can be orally administered in solid dosage forms (eg, capsules, tablets and powders) or liquid dosage forms (eg, elixirs, syrups, emulsions and suspensions). The active ingredient may also be formulated for parenteral administration by injection, rapid infusion, nasopharyngeal absorption or dermal absorption. The agent can be administered intramuscularly, intravenously, or as a suppository.

Gelatin capsules contain the active ingredient and powdered carriers, such as lactose, sucrose, mannitol, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like. Compressed tablets may be made using similar diluents. 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 disintegration in the gastrointestinal tract obtain.

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

In general, water, a suitable oil, saline, water-soluble glucose (glucose), and related sugar solutions and glycols (eg, propylene glycol or polyethylene glycol) are suitable carriers for parenteral solutions. . Solutions for parenteral administration preferably contain a water-soluble salt of the active ingredient, suitable stabilizing agents, and if necessary, buffer substances. Antioxidants, such as sodium bisulfate, sodium sulfite or ascorbic acid, either alone or in combination, are suitable stabilizers. Also used are citric acid and its salts and sodium EDTA. In addition, parenteral solutions can contain preservatives, such as benzalkonium chloride, methyl or propylparaben, and chlorobutanol. Suitable pharmaceutical carriers are the standard reference texts in this field, Remington's P
described in Pharmaceutical Sciences.

In addition, standard pharmaceutical methods can be performed to control the duration of action. These are well known in the art and include controlled release preparations and suitable polymers, such as polymers, polyesters, polyamino acids, polyvinyl, pyrrolidone, ethylene vinyl acetate, methyl cellulose, carboxymethyl cellulose, or protamine sulfate. including. Polymer concentration and method of incorporation can be adjusted to control release. Further, the agent can be incorporated into particles of a polymeric substance, such as a polyester, polyamino acid, hydrogel, poly (lactic acid), or ethylene vinyl acetate copolymer. In addition to being incorporated, these agents can also be used to trap compounds in microcapsules.

Useful pharmaceutical dosage forms for administration of the compounds of the present invention may be illustrated below.

Capsules: 10 capsules each in a standard two-piece hard gelatin capsule
Prepared by filling 0 mg of powdered active ingredient, 175 mg lactose, 24 mg talc and 6 mg magnesium stearate.

Soft gelatin capsule: A mixture of the active ingredients in soybean oil is prepared and injected into the gelatin by a positive displacement pump to form a soft gelatin capsule containing 100 milligrams of active ingredient. The capsule is then washed and dried.

Tablets: Tablets are prepared by conventional procedures so that the dosage unit is 100 milligrams of active ingredient. 0.2 milligrams of colloidal silicon dioxide, 5 milligrams of magnesium stearate, 275 milligrams of microcrystalline cellulose, 11 milligrams of corn starch and 98.8 milligrams of lactose. Appropriate coatings may be applied to increase palatability or eliminate absorption.

Injectable: Parenteral compositions suitable for administration by injection are prepared by stirring 1.5% by weight of active ingredient in 10% by volume in propylene glycol and water. The solution is made isotonic with sodium chloride and sterilized.

Suspension: 5 milligrams each contain 100 milligrams of finely divided active ingredient, 200 milligrams of sodium carboxymethylcellulose, 5 milligrams of sodium benzoate, 1.0 gram of sorbitol solution U.S.P. S. P and 0.0
An aqueous suspension is prepared for oral administration to contain 25 millimeters of vanillin.

Summary of Compounds Synthesized The following examples are provided by way of illustration, and are not intended to limit the invention in any manner. Nucleosides and dimers incorporate any stereochemically possible linkage and may include various oxidized, deoxy, and dideoxy sugar ring forms. The synthetic nucleosides and dimers described in the examples may include any of the substitutions discussed above. Backbone and salt modifying groups can be added. Various substitutions enhance the affinity, chemical stability, and cellular uptake properties of specific dimer treatment.

Example 1 (Synthesis of 2′-deoxy-α-L-5-fluorouridine) β-D-5-fluoro-deoxyuridine is commercially available. The α-L-isomer, 2′-deoxy-α-L-5-fluorouridine, is not commercially available, and this dimer composition was synthesized from L-arabinose.

(1- (2 ′, 3 ′, 5′-Tri-O-benzoyl-α-L-arabinofuranosyl) -5-fluorouracil (3)) 5-Fluorouracil (4) mixed in anhydrous MeCN .01g, 30.87m
mol) and compound 2 (15.57 g, 30.87 mmol) were added to HMDS (
5.20 ml, 24.69 mmol), ClSiMe ₃ (3.10 ml, 24.69 mmol) and SnCl ₄ (4.30 ml, 37.04 mmol) were added successively. The resulting clear solution was refluxed for 1 hour. Then, the solvent was evaporated and the residue was dissolved in EtOAc (750 ml), washed with H ₂ O and saturated NaHCO ₃ solution. The EtOAc layer was dried over sodium sulfate, filtered, and evaporated to give a crude product. The crude product was purified on a silica gel column, eluting with 40-50% EtOAc / petroleum ether, to give purified product 3 (11.7 g, 66.0% yield) as a white foam.

[0052]

(Equation 1) (1-α-L-arabinofuranosyl-5-fluorouracil (4)) To a solution of compound 3 (11.7 g, 20.37 mmol) in MeOH (300 ml) was added NaOMe (4.2 ml of methanol 25% w). / V solution) and the solution was stirred until the reaction was complete. Then, the solvent was evaporated and the residue was dissolved in H ₂ O (200ml), washed with ether, and neutralized with Dowex 50 ion exchange resin. After filtration of the resin, the aqueous solution was evaporated to give compound 4 (4.92 g, 92% yield) as a white foam.

[0053]

(Equation 2) (1- [3 ', 5'-O- (1,1,3,3-tetraisopropyldisiloxy
San-1,3-diyl) -α-L-arabinofuranosyl] -5-fluorouracil
(5)) 4 (6.43 g, 24.52 mmol) in pyridine (200 ml) was stirred.
1,3-dichloro-1,1,3,3-tetraisopropyldisiloxy
Sun (10.3 ml, 29.43 mmol) was added. This is when the reaction is complete
And stirred at room temperature until complete (5 hours). The solvent is evaporated to a residue.
And the residue is dissolved in EtOAc and_TwoO, 5% HCl, H_TwoO, get tired
NaHCO_ThreeAnd washed successively with brine. Replace the EtOAc portion with Na_TwoSO _Four After drying in, the solution was filtered and evaporated to give crude product 5. This crude product was used in the next step without further purification.

(1 [2′-O-phenoxythiocarbonyl-3 ′, 5′-O- (1,1,3
, 3-tetraisopropyldisiloxane-1,3-diyl) -α-L-arabinofuraosyl] -5-fluorouracil (6)) To a solution of 5 (24.52 mmol) in anhydrous MeCN (300 ml) was added -Dimethylaminopyridine (DMAP) (5.80 g, 47.58 mmol) and phenylchlorothionoformate (3.85 ml, 26.98 mmol) were added. The solution was stirred at room temperature for 24 hours. Then, the solvent was evaporated to give a residue which was dissolved in EtOAc, and washed _{H 2 O, 5% HCl,} H 2 O, saturated NaHCO _3, and brine successively with. After drying the EtOAc portion over Na ₂ SO ₄ , the solution was filtered and evaporated,
An oil was obtained. The oil was purified on a silica gel column using 30% EtOAc / petroleum ether to give pure 6 (8.9 g, 56.7% yield) as a yellow foam.

[0055]

(Equation 3) (3 ′, 5′-O- (1,1,3,3-tetraisopropyldisiloxane-1
, 3-Diyl) -α-L-2′-deoxy-5-fluorouridine (7)) To a solution of 6 (8.92 g, 13.91 mmol) in anhydrous toluene (300 ml) was added AIBN (0.46 g, 2.78 mmol) followed by Bu ₃ SnH (20.0 ml, 69.35 mmol). Deoxygenate this solution with argon,
Then, it was heated at 75 ° C. for 4 hours. The solvent was then evaporated and the residue was purified on a silica gel column using 30% EtOAc / petroleum ether to give pure 7 (5.44 g, 80% yield) as a white foam. .

[0056]

(Equation 4) (2′-Deoxy-α-L-5-fluorouridine (8)) A solution of compound 7 (5.44 g, 11.13 mmol) and NH ₄ F (4.12 g, 111.3 mmol) in MeOH was added to an oil. Stirred in the bath at 60 ° C. for 3 hours. Silica gel (3 g) was added and the mixture was evaporated to give a dry powder. This powder is added to a silica column and 10-15% MeOH / CH
Elution with Cl ₃ yielded pure 8 (2.4 g, 87.6% yield) as a white foam.

[0057]

(Equation 5) (Example 2) (Synthesis of 2′-deoxy-α-L-uridine) (1- (2 ′, 3 ′, 5′-tri-O-benzoyl-α-L-arabinofuranosyl) uracil (9 )) Uracil (1.17 g, 10.49 mmol) in anhydrous MeCN (100 ml)
) And compound 2 (5 g) were added to HMDS (1.77 ml, 8.39 mm).
ol), ClSiMe ₃ (1.06 ml, 8.39 mmol) and SnCl ₄ (
1.47 ml, 12.58 mmol) were added continuously. The resulting clarified solution was refluxed for 1 hour. The solvent is then evaporated and the residue is washed with EtO
Dissolved in Ac (200 ml) and washed with H ₂ O, and saturated NaHCO ₃ solution. The EtOAc layer was dried over sodium sulfate, filtered, and evaporated to give a crude product. This crude product is subjected to 40-50% Et on a silica gel column.
Purification using OAc / petroleum ether gave pure 9 (3.66 g, 62.7 yield).
%) As a white foam.

[0058]

(Equation 6) (1-α-L-arabinofuranosyl-uracil (10)) Compound 8 (17.83 g, 32.03 mmol) in MeOH (400 ml).
NaOMe (5.0 ml of a 25% w / v solution of methanol) was added to the solution of
The solution was stirred until the reaction was completed. The solvent was then evaporated and the residue dissolved in H ₂ O (250 ml), washed with ether and neutralized with Dowex 50 ion exchange resin. After filtration of the resin, the aqueous solution was evaporated to give compound 10 (7.4 g, 94.6% yield) as a white foam. This was used in the next step without further purification.

(1- [3 ′, 5′-O- (1,1,3,3-tetraisopropyldisiloxane-1,3-diyl) -α-L-arabinofuranosyl] -uracil (11) ) To a stirred suspension of 10 (7.4 g, 30.3 mmol) in pyridine was added 1
, 3-Dichloro-1,1,3,3-tetraisopropyldisiloxane (12.7
4 ml, 36.36 mmol) was added. This was stirred at room temperature until the reaction was completed (5 hours). The solvent was evaporated and the residue was washed with EtOAC (500 m
It was dissolved in l), and _{H 2 O, 5% HCl,} H 2 O, saturated NaHCO _3, washed successively with and brine. After drying the EtOAc portion over Na ₂ SO ₄ , the solution was filtered and evaporated to give crude product 11. This crude product was used in the next step without further purification.

1- [2′-o-phenoxythiocarbonyl-3 ′, 5′-o- (1,1,3
, 3-Tetraisopropyldisiloxane-1,3-diyl) -α-L-arabinofuranosyl] -uracil (12) 4-dimethylaminopyridine (DMAP) in a solution of 11 (30.3 mmol) in anhydrous MeCN (7.2 g, 58.78 mmol) and phenylchlorothionoformate (4.7 ml, 33.33 mmol) were added. The solution was stirred at room temperature for 24 hours. Then the solvent was evaporated to give a residue, which was
Dissolved in Ac (750 ml) and washed sequentially with H ₂ O, 5% HCl, H ₂ O, saturated NaHCO ₃ , and brine. After drying the EtOAc portion over Na ₂ SO ₄ , the solution was filtered and evaporated to an oil. 30 oils
Purify on silica gel column using% EtOAc / petroleum ether to give pure 12%
(13.14 g, 74.5% yield) as a white foam.

[0061]

(Equation 7) 3 ′, 5′-o- (1,1,3,3-tetraisopropyldisiloxane-1,
3-Diyl) -α-L-2′-deoxyuridine (13) 12 (13.14 g, 21.09 mmol) in dry toluene (300 ml)
Was added to AIBN (0.69 g, 4.2 mmol), followed by Bu ₃ SnH (28.4 ml, 105.4 mmol). The solution was deoxygenated with argon and heated at 75 ° C. for 4 hours. Then the solvent was evaporated and the residue was reduced to 30%
Purify on silica gel column using EtOAc / petroleum ether to give pure 13 (
9.29 g, 88.4% yield) as a white foam.

[0062]

(Equation 8) 2′-Deoxy-α-L-uridine (14) A mixture of compound 13 (9.2 g, 18.63 mmol) and NH ₄ F (6.9 g, 186.3 mmol) in MeOH (200 ml) was treated with a 60 ° C. oil. Stirred in bath for 3 hours. Silica gel (5 g) was added and the mixture was evaporated to give a dry powder. Add this powder to a silica gel column and add 10-15% MeOH
Elution with / CHCl ₃ afforded pure 14 (3.70 g, 83% yield) as a white foam.

[0063]

(Equation 9) Example 3 (Synthesis of 2′-deoxy-α-L-cytidine) 3 ′, 5′-di-O-benzoyl-2′-deoxy-α-L-uridine (15
) BzCN in MeCN (10ml) (0.61g, a solution of 4.67 mmol), was added dropwise to a suspension of compound 14 in MeCN (10ml) (0.43g, 1.87mmol ), followed by Et ₃ N (0.1 ml) was added. The reaction was stirred at room temperature for 3 hours, after which the solvent was evaporated to dryness. 50% EtO
Purify on silica gel column using Ac / petroleum ether to give pure 15 (0.5
7 g, 70% yield) as a yellow foam.

[0064]

(Equation 10) 3 ', 5'-Di-O-benzoyl-2'-deoxy-4-thio-α-L-uridine (16) A boiling solution of compound 15 (0.54 g, 1.25 mmol) in anhydrous dioxane was treated with P ₂ Treated with S ₅ (0.61 g, 2.75 mmol), and the mixture was refluxed for 1 hour under a nitrogen atmosphere. The remaining solid was filtered from the warm solution and washed by filtration with additional dioxane. The filtrate was evaporated to dryness and the crude product was purified on a silica gel column using 30% EtOAc / petroleum ether to give pure 16 (0.42 g, 74% yield) as a yellow oil.

[0065]

[Equation 11] 2′-Deoxy-α-L-cytidine (17) Compound 16 (0.42 g, 9.28 mmol) was placed in a steel cylinder (steel).
bomb) in NH ₃ / MeOH (50 ml) at 100 ° C. for 10 hours. After cooling, the solvent was evaporated to dryness and the residue was washed with water (50 mL)
And washed with ether (3 × 50 ml). The aqueous layer was treated with charcoal, filtered through celite, and evaporated to dryness azeotropically with EtOH. The resulting semi-solid was crystallized from EtOH / ether to give compound 17 (0.18
g, 85.7% yield).

[0066]

(Equation 12) N^Four-Benzoyl-2'-deoxy-α-L-cytidine (18) Compound 17 (0.82 g, 3.6) in pyridine (50 ml) cooled in an ice bath.
1 mmol) to the stirred suspension over 30 minutes._Three(2.3 ml, 18.05 mmol) was added dropwise. Then, BzCl (2.1 ml, 18.
05 mmol) was added dropwise and the reaction mixture was cooled at room temperature for 2 hours. This reaction
The mixture was cooled again in an ice bath and cold water (10 ml) was added dropwise. 15 minutes later, concentrated NH _Four OH (10 ml) was added to obtain an approximately 2M ammonia solution. 30 minutes after the addition of the ammonia solution, the solvent is evaporated down, dissolved in water and added with ether.
Washed. Evaporation of this aqueous solution gives the crude product (18),
Was used in the next step without further purification.

Example 4 Synthesis of a Dimer A dimer was prepared from a monomeric material according to the general formula shown in Scheme 2.

(A. α-L, β-D5FUdR dimer)
Lysine (20a) α-L-5-fluoro-2'-deoxyuridine (8) (500 mg, 2.0
mmol) was dissolved in 10 ml of dry distilled pyridine. 4,4'-
Dimethoxytrityl chloride (813 mg, 2.4 mmol) and 4-dimethyl
Aminopyridine (DMAP) (50 mg, 0.4 mmol) was added. This mix
The mixture was stirred under an argon atmosphere for 16 hours. After this time, the pyridine was removed under vacuum
Removed. The residue was dissolved in EtOAc (50ml). Organic layer is saturated with NaHC
O_Three, Water, and brine. Organic layer_TwoSO_Four, Filtered and evaporated under vacuum to give a residue, which is 10% MeOH / CHCl _Three And purified on a silica gel column. Pure fractions were pooled and evaporated to give pure product as an off-white foam (679 mg, 86%
Yield). Rf = 0.48 (10% MeOH / CHCl_Three).

[0069]

(Equation 13) (5′-O-dimethoxytrityl-α-L-5-fluoro-2′-deoxyuridine-3′-N, N-diisopropylmethoxyphosphoramidite (21a) 5′-O-dimethoxytrityl-α-L- 5-Fluoro-2'-deoxyuridine (20a, 548 mg, 1 mmol) was dissolved in anhydrous dichloromethane (20 ml) Under an argon atmosphere, N, N-diisopropylethylamine (700
μl, 4 mmol) was added through a septum, followed by chloro-N, N-diisopropylmethoxyphosphine (290 μl, 1.5 mmol). The reaction was stirred for 30 minutes. The solvent was evaporated and the residue was washed with 80% EtOA.
Partitioned between the c / triethylamine mixture and brine. The organic layer was washed with saturated NaH
Washed with CO ₃ solution and brine. The organic residue was evaporated to dryness and the residue was washed with a mixture of dichloromethane, EtOAc and triethylamine (45%).
: 45:10; Rf = 0.69). The product (390 mg) was isolated as a yellow foam and used in the next step without further purification.

3′-acetoxy-β-D-5-fluoro-2′-deoxyuridine (24a
) Β-D-5-Fluoro-2′-deoxyuridine (500 mg, 2.2 mmol)
l) was dissolved in 10 ml of dry distilled pyridine. To this solution was added 4,4'-dimethoxytrityl chloride (813 mg, 2.4 mmol) and 4-dimethylaminopyridine (DMAP) (50 mg, 0.4 mmol). The mixture was stirred at room temperature for 16 hours. Pyridine was removed under vacuum. This residue was dissolved in dichloromethane (50 ml). The organic layer was washed with 0.3N HCl, brine, saturated N
Washed with aHCO ₃ , and again with brine. The organic layer was dried over Na ₂ SO ₄ , filtered and evaporated in vacuo to give a residue, which was purified on a silica gel column eluted with 10% MeOH / CHCl ₃ . Pure fractions were pooled and evaporated to give pure product as an off-white foam (685 mg, 86%
Yield). This material was dissolved in pyridine (12 ml) and treated with acetic anhydride (2.5 ml) at room temperature for 3 hours. The solvent was evaporated and the residue was dissolved in ethyl acetate. The ethyl acetate was washed as above, dried over sodium sulfate, and evaporated. The residue is then treated with 80% acetic acid (2.5 h at room temperature).
10 ml). The solvent was evaporated under vacuum and the residue was
Chromatography on silica gel eluting with MeOH / CHCl ₃ afforded pure 24a (422 mg) as a white foam.

[0071]

[Equation 14] 5′-O-dimethoxytrityl-3 ′-[O- (3′-O-acetyl) -β-
D-5-Fluoro-2′-deoxyuridinyl] -α-L-5-fluoro-2 ′
-Deoxyuridine (25a) 3'-O-acetyl-β-D-5-fluoro-2'-deoxyuridine (18
8 mg, 0.65 mmol) was dissolved in dry acetonitrile (5 ml). Sublimed 1H-tetrazole (80 mg) was added and the mixture was stirred for 15 minutes under an argon atmosphere. 21a (38) dissolved in 5 ml of dry acetonitrile
0 mg, 0.54 mmol) was added to the reaction solution via syringe over 5 minutes. The mixture was stirred at room temperature for 3 hours. The acetonitrile was evaporated under vacuum to give a residue. This residue was triturated with a 70% EtOAc / ether mixture. The insoluble tetrazole was removed by filtration, and the filtrate was evaporated to give a dry yellow foam (468 mg). This foam was used in the next step without further purification.

(3′-acetoxy-β-D-5-fluoro-2′-deoxyuridinyl) —
α-L-5-fluoro-2′-deoxyuridine methylphosphonate ester (
26a) The dimer, 25a (504 mg) was dissolved in 8 ml of THF containing 0.2 ml of water and 2 ml of pyridine. Crystals of iodine (26 mg) were added and the contents of the loosely stoppered flask were stirred for 2.1 hours. Excess iodine was removed by adding a few drops of saturated sodium thiosulfate. The reaction mixture was then evaporated to dryness. The crude product was dissolved in EtOAc, washed with saturated NaHCO ₃ solution and brine. The organic layer was dried over Na ₂ SO _4, filtered, and evaporated in vacuo. The residue (530 mg) was dissolved in 10 ml of 80% acetic acid / water solution and stirred until the reaction was completed. The solvent was evaporated and the residue using 20% MeOH / CHCl ₃ and purified on a silica gel column. Fractions containing a single spot by TLC (10% MeOH / CHCl ₃ Rf = 0.35) were pooled and evaporated to give pure product (316 mg).

[0073]

(Equation 15) 3′-O- (β-D-5-fluoro-2′-deoxyuridinyl) -α-L-
5-Fluoro-2'-deoxyuridine (27a) O-protected dimer, 26a (280 mg) was treated with 20 ml of saturated methanolic ammonia at room temperature, and the reaction was performed until the reaction was completed at room temperature. The solvent was removed in vacuo and the residue was purified by DEAE cellulose ion exchange column using a gradient of NH ₄ CO ₃ buffer 0.02～0.2M. Pure fractions were evaporated to dryness under high vacuum at 40 ° C. to give pure product (162 mg).

[0074]

(Equation 16) (B. β-D, α-L5FUdR dimer) 5′-O-dimethoxytrityl-3 ′-[O- (3′-O-acetyl) -α-
L-5-Fluoro-2'-deoxyuridinyl]-[beta] -D-5-fluoro-2 '
-Deoxyuridine (25b) 3'-O-acetyl-α-L-5-fluoro-2'-deoxyuridine, 24
b (188 mg, 0.65 mmol) was dissolved in dry acetonitrile (5 ml). Sublimed 1H-tetrazole (80 mg) was added and the mixture was stirred for 15 minutes under an argon atmosphere. 21b dissolved in 5 ml of dry acetonitrile
(380 mg, 0.54 mmol) was added to the reaction solution via syringe over 5 minutes. The mixture was stirred at room temperature for 3 hours. The acetonitrile was evaporated under vacuum to give a residue. This residue was triturated with a 70% EtOAc / ether mixture. The insoluble tetrazole was removed by filtration, and the filtrate was evaporated to give a dry yellow foam (484 mg). This foam was used in the next step without further purification.

(3′-acetoxy-α-L-5-fluoro-2′-deoxyuridinyl)-
β-D-5-fluoro-2′-deoxyuridine methyl phosphate (26b
) The dimer, 25b (526 mg), was added to 8 ml of THF containing 0.2 ml of water.
And 2 ml of pyridine. Crystals of iodine (26 mg) were added and the contents of the loosely stoppered flask were stirred for 1 hour. Excess iodine was removed by adding a few drops of saturated sodium thiosulfate. The reaction mixture was then evaporated to dryness. The crude was dissolved in EtOAc and washed with saturated NaHCO ₃ solution and brine. The organic layer was dried over Na ₂ SO ₄ , filtered and evaporated under vacuum. The residue (578 mg) was dissolved in 10 ml of 80% acetic acid / water solution and stirred for 3 hours. The solvent was evaporated and the residue was
% Was purified by a silica gel column using MeOH / CHCl _3. Fractions containing a single spot by TLC (10% MeOH / CHCl ₃ Rf = 0.35) were pooled and evaporated to give pure product (342 mg).

[0076]

[Equation 17] 3′-O- (α-L-5-fluoro-2′-deoxyuridinyl) -β-D-
5-Fluoro-2'-deoxyuridine (27b) O-protected dimer, 26b (170 mg) was treated with 20 ml of saturated methanolic ammonia at room temperature and this operation was performed until the reaction was completed. The solvent was removed in vacuo, and the residue was purified by DEAE cellulose ion exchange column using a gradient of NH ₄ CO ₃ buffer 0.02～0.2M. The pure fractions were evaporated to dryness under high vacuum at 40 ° C. to give the pure product (89 mg).

[0077]

(Equation 18) (C. α-L uridine, β-D5FUdR dimer) 5′-O- (di-p-methoxytrityl) -2′-deoxy-α-L-uridine (20c) α-L-2′-deoxy 25 ml of uridine (1.5 g, 6.57 mmol)
Was dissolved in dry distilled pyridine. To this solution was added 4,4′-dimethoxytrityl chloride (2.9 g, 7.89 mmol) and 4-dimethylaminopyridine (D
(MAP) (160 mg, 1.31 mmol) was added. The mixture was stirred for 16 hours under an argon atmosphere. After this time, the pyridine was removed under vacuum. This residue was dissolved in EtOAc (150ml). The organic layer was washed with saturated NaHCO ₃ , water, and again with brine. The organic layer was dried over Na ₂ SO ₄ , filtered and evaporated under vacuum to give a residue, which was purified on a silica gel column using 5% MeOH / CHCl ₃ . Pure fractions were pooled and evaporated to give pure product as an off-white foam (2.84 g, 81% yield).

[0078]

[Equation 19] 5′-O- (dimethoxytrityl) -α-L-2′-deoxyuridine-3 ′
—N, N-diisopropylmethoxyphosphoramidite (21c) 5′-O-dimethoxytrityl-α-L-2′-deoxyuridine (2.35
g, 4.43 mmol) were dissolved in anhydrous dichloromethane (50 ml). Under an argon atmosphere, N, N-diisopropylethylamine (3.1 ml, 17.72
mmol) was added through a septum, followed by chloro-N, N-diisopropylmethoxyphosphine (1.3 ml, 6.64 mmol). The reaction was stirred for 30 minutes. The solvent was evaporated and the residue was partitioned between 80% EtOAc / triethylamine mixture and brine. The organic layer was washed with saturated NaHCO ₃ solution and brine. The organic residue was evaporated to dryness and the residue was washed with dichloromethane, EtOAc and triethylamine (40:50:10; R
f = 0.69) and purified on a silica gel column. The product was quantitatively isolated as a yellow foam and was used in the next step without further purification.

5′-O-dimethoxytrityl-3 ′-[O- (3′-O-acetyl) -β-
D-5-Fluoro-2'-deoxyuridinyl] -2'-deoxy-α-L-uridine (25c) 3′-O-acetyl-β-D-5-fluoro-2′-deoxyuridine (0 .
95 g, 3.29 mmol) were dissolved in dry acetonitrile (125 ml).
Sublimated 1H-tetrazole (350 mg, 4.91) was added and the mixture was stirred for 15 minutes under an argon atmosphere. A solution of 21c (4.91 mmol) dissolved in 5 ml of dry acetonitrile was added to the reaction solution via syringe over 5 minutes. The mixture was stirred at room temperature for 3 hours. Acetonitrile was evaporated under vacuum to give a residue. This residue was triturated with a 70% EtOAc / ether mixture. The insoluble tetrazole was removed by filtration and the filtrate was evaporated to give a dry yellow foam. This compound was further purified on a silica gel column using 5% MeOH / CHCl ₃ to give pure product (2.81 g, 97% yield).

3′-acetoxy-β-D-5′-fluoro-2′-deoxyuridinyl)-
α-L-2′-deoxyuridine methyl phosphate (26c) dimer, 25c (2.81 g, 3.2 mmol) was added to THF: pyridine: water (
25: 6: 0.6). Crystals of iodine (150 mg) were added,
The contents of the loosely stoppered flask were stirred for 1 hour. Excess iodine was removed by adding a few drops of saturated sodium thiosulfate. The reaction mixture was then evaporated to dryness. The crude product was dissolved in EtOAc and washed with saturated NaHCO ₃ solution and brine. The organic layer was dried over Na ₂ SO ₄ , filtered and evaporated under vacuum. The residue (1.48 g) was dissolved in 25 ml of 80% acetic acid / water solution and stirred until the reaction was completed. The solvent was evaporated and the residue using 10% MeOH / CHCl ₃ and purified on a silica gel column. Fractions containing a single spot by TLC (10% MeOH / CHCl ₃ Rf = 0.4) were pooled and evaporated to pure product (0.465 g, 25
% Yield).

[0081]

(Equation 20) 3′-O- (β-D-5-fluoro-2′-deoxyuridinyl) -α-L-
50 ml of 2'-deoxyuridine (27c) O-protected dimer, 26c (465 mg, 0.78 mmol) at room temperature
Of saturated methanolic ammonia, and this operation was performed until the reaction was completed. The solvent was removed in vacuo, and the residue was purified by DEAE cellulose ion exchange column using a gradient of NH ₄ CO ₃ buffer 0.02～0.2M. The pure fractions were evaporated to dryness under high vacuum at 40 ° C. to give the pure product (370 mg,
87.7% yield).

[0082]

(Equation 21) (D. β-L, β-L5FUdR dimer) (5′-O-dimethoxytrityl-β-L-5-fluoro-2′-deoxyuridine (20d)) β-L-5-fluoro-2 ′ -Deoxyuridine (19d, 1.42 g, 5.
77 mmol) was dissolved in 25 ml of dry, distilled pyridine. To this solution, 4,4′-dimethoxytrityl chloride (2.34 g, 6.92 mmol)
) And 4-dimethylaminopyridine (DMAP) (140 mg, 1.15 mm
ol) was added. The mixture was stirred for 16 hours under an argon atmosphere.
After this point, the pyridine was removed in vacuo. The residue was treated with EtOAc (100 m
l). The organic layer was washed with saturated NaHCO ₃ and brine. The organic layer was dried over Na ₂ SO _4, filtered, and evaporated in vacuo to a residue and purify the residue on a silica gel column using 5% MeOH / CHCl _3. Pure fractions were pooled and evaporated to give pure product as an off-white foam (2.88 g, 88.7% yield).

[0083]

(Equation 22) (5′-O-dimethoxytrityl-β-L-5-fluoro-2′-deoxyuridine-3′-N, N-diisopropylmethoxyphosphoramidite (21d)
5) -O-Dimethoxytrityl-β-L-5-fluoro-2'-deoxyuridine (20d, 840mg, 1.53mmol) was added to anhydrous dichloromethane (50m
l). N, N-diisopropylethylamine (1.1 ml, 6.13
mmol) was added through a septum, followed by chloro-N, N-diisopropylmethoxyphosphine (0.42 ml, 2.3 mmol) under an argon atmosphere. The reaction was stirred for 30 minutes. The solvent was evaporated and the residue was partitioned between 80% EtOAc / triethylamine mixture and brine. The organic layer was washed with saturated NaHCO ₃ solution and brine. The organic residue was evaporated to dryness and the residue was purified on a silica gel column using a mixture of dichloromethane, EtOAc and triethylamine (45:45:10; Rf = 0.69). The product (700 mg, 65%) was isolated as a yellow foam and used in the next step without further purification.

(5′-O-dimethoxytrityl-3 ′-[O- (3′-O-acetyl) -β
-L-5-fluoro-2'-deoxyuridinyl] -β-L-5-fluoro-2
'-Deoxyuridine (25d)) 3'-acetyl-β-L-5-deoxyuridine, 24d (330 mg, 1.
15 mmol) was dissolved in dry acetonitrile (50 ml). Sublimated 1H-tetrazole (120 mg, 1.77 mmol) was added and the mixture was stirred under an argon atmosphere for 15 minutes. A solution of 21d (950 mg, 1.36 mmol) in 5 ml of dry acetonitrile was added via syringe to the reaction solution over 5 minutes. The mixture was allowed to stir at room temperature for 3 hours. Acetonitrile was evaporated in vacuo to a residue. This residue is converted to 70% E
Triturated with the tOAc / ether mixture. The undissolved tetrazole was removed by filtration and the filtrate was evaporated to give a dry yellow foam. The foam was purified on a silica gel column using 5% MeOH / CHCl ₃ to give the pure product (960 mg, 93% yield).

[0085]

(Equation 23) ((3′-acetoxy-β-L-5-fluoro-2′-deoxyuridinyl)
-Β-L-5-fluoro-2′-deoxyuridine methyl phosphate (26
d)) 25d of the dimer (960 mg, 1.07 mmol) was added to THF: pyridine: water (
12: 3: 0.3). Crystal of iodine (50mg)
Was added and the contents of the loosely stoppered flask were stirred for 1 hour. Excess iodine was removed by adding a few drops of saturated sodium thiosulfate. The reaction mixture was then evaporated to dryness. The crude product dissolved in EtOAc was washed with saturated NaHC
O ₃ solution and brine. The organic layer was dried over Na ₂ SO ₄ , filtered, and evaporated in vacuo. The residue (530 mg) was dissolved in 20 ml of 80% acetic acid /
Dissolved in the aqueous solution and stirred until the reaction was completed. The solvent was evaporated and was made fine by a silica gel column using 10% MeOH / CHCl ₃ to the residue. Fraction containing one spot by TLC (10% MeOH / CH
(Cl ₃ , Rf = 0.35) was pooled and evaporated to give pure product (310 mg, 46% yield).

[0086]

(Equation 24) (3′-O- (β-L-5-fluoro-2′-deoxyuridinyl) -β-L
-5-Fluoro-2'-deoxyuridine (27d)) O-protected dimer 26d (300 mg, 0.49 mmol) is completed at room temperature with 50 ml of saturated methanolic ammonia. Processed. The solvent was removed in vacuo and the residue using a gradient of 0.02~0.2M of NH ₄ CO ₃ buffer and purified on a DEAE cellulose ion exchange column. The pure fractions were evaporated to dryness at 40 ° C. under high vacuum to give pure product (240 mg, 85% yield).

[0087]

(Equation 25) (E. β-L, β-D5FUdR dimer) (5′-O-dimethoxytrityl-3 ′-[O- (3′-O-acetyl) -β
-D-5-Fluoro-2'-deoxyuridinyl] -β-L-5-fluoro-2
'-Deoxyuridine (25e)) 3'-O-acetyl-β-D-5-fluoro-2'-deoxyuridine (25
(0 mg, 0.97 mmol) was dissolved in dry acetonitrile (50 ml). Sublimated 1H-tetrazole (100 mg, 1.46 mmol) was added and the mixture was stirred under an argon atmosphere for 15 minutes. A solution of 21e (1.02 g, 1.46 mmol) in 5 ml of dry acetonitrile was added to the reaction solution via syringe over 5 minutes. The mixture was allowed to stir at room temperature for 3 hours. Acetonitrile was evaporated in vacuo to a residue. This residue was triturated with a 70% EtOAc / ether mixture. The undissolved tetrazole was filtered off and the filtrate was evaporated to give a dry yellow foam. The foam was purified on a silica gel column using 5% MeOH / CHCl ₃ to give a quantitatively pure product.

((3′-acetoxy-β-D-5-fluoro-2′-deoxyuridinyl)
-Β-L-5-fluoro-2′-deoxyuridine methylphosphonate ester (26e)) Reduced form of dimer 25 (700 mg, 0.78 mmol) in THF: pyridine: water (25: 6: 0.6) Was dissolved in the mixture containing Iodine crystal (10
0 mg) was added and the contents of the loosely stoppered flask were allowed to stir for 2.5 hours. Excess iodine was removed by adding a few drops of saturated sodium thiosulfate. The reaction mixture was then evaporated to dryness. The crude product dissolved in EtOAc was washed with saturated NaHCO ₃ solution and brine. The organic layer was dried over Na ₂ SO _4, filtered, and evaporated in vacuo. The residue was treated with 25 ml of 80% acetic acid /
Dissolved in the aqueous solution and stirred until the reaction was completed. The solvent was evaporated and was made fine by a silica gel column using 10% MeOH / CHCl ₃ to the residue. Fraction containing one spot by TLC (10% MeOH / CH
(Cl ₃ , Rf = 0.35) was pooled and evaporated to give pure product (340 mg, 71.4% yield).

[0089]

(Equation 26) (3′-O- (β-D-5-fluoro-2′-deoxyuridinyl) -β-L
-5-Fluoro-2'-deoxyuridine (27e)) O-protected dimer 26e (340 mg, 0.57 mmol) in 100 ml of saturation
Using methanolic ammonia,
Worked up at room temperature until the reaction was complete. The solvent was removed in vacuo and the residue was treated with NH _Four CO_ThreeUsing a 0.02-0.2M gradient of buffer, DEAE cellulose ion
The mixture was purified by an exchange column. Evaporate pure fractions to dryness at 40 ° C in high vacuum
To give the pure product (200 mg, 66.9% yield).

[0090]

[Equation 27] (5′-O- (dimethoxytrityl) -α-L-5-fluoro-2′-deoxyuridine-3′-N, N-diisopropylcyanoethyl phosphoramidite (
21f)) 5′-O-Dimethoxytrityl-α-L-5-fluoro-2′-deoxyuridine (1.48 g, 2.71 mmol) was dissolved in anhydrous dichloromethane (50 ml). N, N-diisopropylethylamine (1.9 ml, 10.84 mmol
l) is added through a septum, then 2'-cyanoethyl-N, N-diisopropylchlorophosphoramidite (0.78 ml, 3.52 mmol)
It was added under an argon atmosphere. The reaction was stirred for 30 minutes. The solvent was evaporated and the residue was partitioned between 80% EtOAc / triethylamine mixture and brine. The organic layer was washed with saturated NaHCO ₃ solution and brine. The organic residue was evaporated to dryness and the residue was purified using a mixture of dichloromethane, EtOAc and triethylamine (45:45:10; Rf = 0.7).
Purified on a silica gel column. The product was quantitatively isolated as a yellow foam and used in the next step without further purification.

(5′-O-dimethoxytrityl-3 ′-[O- (5′-O-dimethoxytrityl) -β-D-5-fluoro-2′-deoxyuridinyl] -α-L-5 -Fluoro-2'-deoxyuridine (25f)) 5'-O-dimethoxytrityl-β-D-5-fluoro-2'-deoxyuridine (0.44 g, 0.81 mmol) is dissolved in dry acetonitrile (20 ml). did. Sublimed 1H-tetrazole (90 mg) was added and the mixture was stirred for 15 minutes under an argon atmosphere. A solution of 21f (0.51 mg, 0.67 mmol) dissolved in 10 ml of dry acetonitrile was via syringe
Added to reaction solution over 5 minutes. The mixture was allowed to stir at room temperature for 3 hours. Acetonitrile was evaporated in vacuo to a residue. This residue is
Triturated with a% EtOAc / ether mixture. The undissolved tetrazole was filtered off and the filtrate was evaporated to a dry yellow foam (9
70 mg). This foam was used in the next step without further purification.

((Β-D-5-Fluoro-2′-deoxyuridinyl) -α-L-5-fluoro-2′-deoxyuridine cyanoethylphosphonate ester (26f)) Dimer 25f (970 mg) was obtained. , 16 ml of THF containing 0.4 ml of water
And dissolved in 4 ml of pyridine. Crystals of iodine (50 mg) were added and the contents of the loosely stoppered flask were allowed to stir for 1 hour. Excess iodine was removed by adding a few drops of saturated sodium thiosulfate. The reaction mixture was then evaporated to dryness. The crude product dissolved in EtOAc was washed with saturated NaHCO ₃ solution and brine. The organic layer was dried over Na ₂ SO _4, filtered, and evaporated in vacuo. The residue was dissolved in 20 ml of 80% acetic acid / water solution and stirred until the reaction was completed. The solvent was evaporated and the residue was 10-15%
Purified on a silica gel column using MeOH / CHCl ₃ . Fractions containing one spot by TLC (10% MeOH / CHCl ₃ , Rf = 0.35) were pooled and evaporated to give pure product (330 mg)
.

(3′-O- (β-D-5-fluoro-2′-deoxyuridinyl) -α-L
-5-Fluoro-2'-deoxyuridine (27f)) The O-protected dimer 26f (200 mg) was treated with 20 ml of concentrated ammonia solution until the reaction was complete. The solvent was removed in vacuo and the residue using a gradient of 0.02~0.2M of NH ₄ CO ₃ buffer and purified on a DEAE cellulose ion exchange column. The pure fractions were evaporated to dryness at 40 ° C. under high vacuum to give the pure product (79 mg).

[0094]

[Equation 28] (5′-O-dimethoxytrityl-3 ′-[O- (3′-O-acetyl) -β
-L-5-fluoro-2'-deoxyuridinyl] -α-L-5-fluoro-2
'-Deoxyuridine (25 g)) 3'-O-acetyl-β-D-5-fluoro-2'-deoxyuridine (0.
19g, 0.67mmol) was dissolved in dry acetonitrile (20ml). Sublimated 1H-tetrazole (70 mg) was added and the mixture was stirred for 15 minutes under an argon atmosphere. 21f dissolved in 10 ml of dry acetonitrile
(0.51 mg, 0.67 mmol) was added to the reaction solution via syringe over 5 minutes. The mixture was allowed to stir at room temperature for 3 hours. Acetonitrile was evaporated in vacuo to a residue. This residue is treated with 70% EtO
Powdered with Ac / ether mixture. The undissolved tetrazole was filtered off and the filtrate was evaporated to dry yellow foam (611 mg)
I got This foam was used for the next step without further purification.

((3′-acetoxy-β-L-5-fluoro-2′-deoxyuridinyl)
-Α-L-5-Fluoro-2′-deoxyuridine cyanoethylphosphonate ester (26 g) 25 g (611 mg) of the dimer were dissolved in 8 ml of THF containing 0.2 ml of water and 2 ml of pyridine. Crystals of iodine (30 mg) were added and the contents of the loosely stoppered flask were stirred for 1 hour. Excess iodine was removed by adding a few drops of saturated sodium thiosulfate. The reaction mixture was then evaporated to dryness. The crude product dissolved in EtOAc was washed with saturated NaHCO ₃ solution and brine. The organic layer was dried over Na ₂ SO _4, filtered, and evaporated in vacuo. The residue was dissolved in 20 ml of 80% acetic acid / water solution and stirred until the reaction was completed. The solvent was evaporated and the residue was 10-15% M
Purified on a silica gel column using OH / CHCl ₃ . Fractions containing one spot by TLC (10% MeOH / CHCl ₃ , Rf = 0.35) were pooled and evaporated to give pure product (200 mg).

(3′-O- (β-L-5-fluoro-2′-deoxyuridinyl) -α-L
-5-fluoro-2'-deoxyuridine (27 g) [α-L β-L5FUd
R dimer]) 26 g (200 mg) of the O-protected dimer were treated with 20 ml of concentrated ammonia solution until the reaction was complete. The solvent was removed in vacuo and the residue using a gradient of 0.02~0.2M of NH ₄ CO ₃ buffer and purified on a DEAE cellulose ion exchange column. The pure fractions were evaporated to dryness at 40 ° C. under high vacuum to give the pure product (134 mg).

[0097]

(Equation 29) (5′-O- (dimethoxytrityl) -β-L-2′-deoxyuridine-3
'-N, N-diisopropylmethoxy phosphoramidite (21h)) 5'-O-dimethoxytrityl-α-L-2'-deoxyuridine (1.0 g)
, 1.88 mmol) was dissolved in anhydrous dichloromethane (50 ml). N, N-
Diisopropylethylamine (1.31 ml, 7.55 mmol) was added through the septum, then chloro-N, N-diisopropylmethoxyphosphine (0.55 ml, 2.83 mmol) was added under an argon atmosphere. The reaction was stirred for 30 minutes. Evaporate the solvent and remove the residue with 80% EtOAc
/ Triethylamine mixture and brine. The organic layer was washed with saturated NaHC
O ₃ solution and brine. The organic residue was evaporated to dryness and the residue was washed with dichloromethane, EtOAc and triethylamine (50: 40: 1).
0; Rf = 0.8) using a mixture on a silica gel column. The product was quantitatively isolated as a yellow foam and used in the next step without further purification.

(5′-O-dimethoxytrityl-3 ′-[O- (3′-O-acetyl) -β
-D-5-fluoro-2'-deoxyuridinyl] -β-L-2'-deoxyuridine (25h)) 3'-O-acetyl-β-D-5-fluoro-2'-deoxyuridine ( 0.
54 g, 1.88 mmol) were dissolved in dry acetonitrile (50 ml). Sublimated 1H-tetrazole (200 mg) was added and the mixture was stirred for 15 minutes under an argon atmosphere. 21 dissolved in 15 ml of dry acetonitrile
h (1.88 mmol) was added via syringe to the reaction solution over 5 minutes. The mixture was allowed to stir at room temperature for 3 hours. Acetonitrile was evaporated in vacuo to a residue. This residue was triturated with a 70% EtOAc / ether mixture. The undissolved tetrazole was filtered off and the filtrate was evaporated to give a dry yellow foam (1.08 g). This foam was used in the next step without further purification.

((3′-acetoxy-β-D-5-fluoro-2′-deoxyuridinyl)
-Β-L-2′-deoxyuridine methylphosphonate ester (26h)) 25h (1.08g) of the dimer was converted to 15ml of TH containing 0.3ml of water.
F and dissolved in 3 ml of pyridine. Crystals of iodine (100 mg) were added and the contents of the loosely stoppered flask were stirred for 1 hour. Excess iodine was removed by adding a few drops of saturated sodium thiosulfate. The reaction mixture was then evaporated to dryness. The crude product dissolved in EtOAc was washed with saturated NaHCO ₃ solution and brine. The organic layer was dried over Na ₂ SO _4, filtered, and evaporated in vacuo. The residue was dissolved in 25 ml of 80% acetic acid / water solution and stirred until the reaction was completed. The solvent was evaporated and the residue was
Purified on silica gel column using% MeOH / CHCl ₃ . Fractions containing one spot by TLC (10% MeOH / CHCl ₃ , Rf = 0.4) were pooled and evaporated to give pure product (400 mg)
.

(3′-O- (β-D-5-fluoro-2′-deoxyuridinyl) -β-L
-2'-Deoxyuridine (27h)) The O-protected dimer 26h (400mg) was treated with 100ml of methanolic ammonia solution until the reaction was completed. The solvent was removed in vacuo and the residue, 0.02 to 0 of NH ₄ CO ₃ buffer
. Purified on a DEAE cellulose ion exchange column using a 2M gradient. The pure fractions were evaporated to dryness at 40 ° C. under high vacuum to give pure product (175 mg).

[0101]

[Equation 30] (5′-O-dimethoxytrityl-N ⁴ -benzoyl-2′-deoxy-β-L-cytidine (20i)) N ⁴ -benzoyl-2′-deoxy-β-L-cytidine (0.8 g, 2. 42) was dissolved in 50 ml of dry, distilled pyridine. For this solution, 4,4 '
-Dimethoxytrityl chloride (3.0 g, 8.85 mmol) and 4-dimethylaminopyridine (DMAP) (60 mg, 0.48 mmol) were added. The mixture was stirred for 16 hours under an argon atmosphere. After this point, the pyridine was removed in vacuo. The residue was dissolved in EtOAc (100ml). The organic layer was washed with water, saturated NaHCO ₃ and brine. Na ₂ S
Dried over O ₄ , filtered and evaporated in vacuo to a residue, which was purified on a silica gel column with 10% MeOH / CHCl ₃ . Pure fractions were pooled and evaporated to give pure product as an off-white foam (1.49 g, (97% yield), Rf = 0.48, 10% M
in OH / CHCl ₃ )

[0102]

(Equation 31) (5′-O- (dimethoxytrityl) -N ⁴ -benzoyl-2′-deoxy-β-L-cytidine-3′-N, N-diisopropylmethyl phosphoramidite (
21i)) 5′-O-dimethoxytrityl-N ⁴ -benzoyl-2′-deoxy-β-L-cytidine (0.6 g, 0.95 mmol) in anhydrous dichloromethane (50 ml)
Was dissolved. N, N-diisopropylethylamine (0.66 ml, 3.79 m
mol) was added through a septum, and then chloro-N, N-diisopropylmethoxyphosphine (0.28 ml, 1.42 mmol) was added under an argon atmosphere. The reaction was stirred for 30 minutes. The solvent was evaporated and the residue was partitioned between 80% EtOAc / triethylamine mixture and brine. The organic layer was washed with saturated NaHCO ₃ solution and brine. The organic residue was evaporated to dryness and the residue was purified on a silica gel column using a mixture of dichloromethane, EtOAc and triethylamine (60:30:10; Rf = 0.8). The product was quantitatively isolated as a yellow foam and used in the next step without further purification.

(5′-O-dimethoxytrityl-3 ′-[O- (3′-O-acetyl) -β
-D-5-fluoro-2'-deoxyuridylate isoxazolidinyl] -N ⁴ - benzoyl-2'-deoxy-beta-L-cytidine (25i)) 3'-O- acetyl-beta-D-5-fluoro - 2'-deoxyuridine (0.
23g, 0.78mmol) was dissolved in dry acetonitrile (30ml). Sublimated 1H-tetrazole (110 mg) was added and the mixture was stirred for 15 minutes under an argon atmosphere. 21 dissolved in 15 ml of dry acetonitrile
The solution of i (0.94 mmol) was added to the reaction solution via syringe over 5 minutes. The mixture was allowed to stir at room temperature for 3 hours. Acetonitrile was evaporated in vacuo to a residue. This residue was triturated with a 70% EtOAc / ether mixture. The undissolved tetrazole was filtered off and the filtrate was evaporated to give a dry yellow foam (0.73 g). This foam was used in the next step without further purification.

((3′-acetoxy-β-D-5-fluoro-2′-deoxyuridinyl)
-N ⁴ - benzoyl-2'-deoxy-beta-L-cytidine methylphosphonate an ester (26i)) dimer 25i a (0.73 g), of 20ml containing water of 0.4 ml TH
F and dissolved in 4 ml of pyridine. Crystals of iodine (100 mg) were added and the contents of the loosely stoppered flask were stirred for 1 hour. Excess iodine was removed by adding a few drops of saturated sodium thiosulfate. The reaction mixture was then evaporated to dryness. The crude product dissolved in EtOAc was washed with saturated NaHCO ₃ solution and brine. The organic layer was dried over Na ₂ SO _4, filtered, and evaporated in vacuo. The residue was dissolved in 25 ml of 80% acetic acid / water solution and stirred until the reaction was completed. The solvent was evaporated and the residue was
Purified on silica gel column using% MeOH / CHCl ₃ . Fractions containing one spot by TLC (10% MeOH / CHCl ₃ , Rf = 0.4) were pooled and evaporated to give pure product (108 mg).
.

(3′-O- (β-D-5-fluoro-2′-deoxyuridinyl) -2′-
Deoxy-β-L-cytidine (27i)) The O-protected dimer 26i (108 mg) was treated with 100 ml methanolic ammonia solution until the reaction was completed. The solvent was removed in vacuo and the residue, 0.02 to 0 of NH ₄ CO ₃ buffer
. Purified on a DEAE cellulose ion exchange column using a 2M gradient. The pure fractions were evaporated to dryness at 40 ° C. under high vacuum to give the pure product (56 mg).

[0106]

(Equation 32) (5'-O-dimethoxytrityl-3 '-[O- (3'-O-acetyl) -N ^Four -Benzoyl-2'-deoxy-β-L-cytidinyl) -β-D-5-fluoro-2'deoxyuridine (25j)) 3'-O-acetyl-β-D-5-fluoro-2'- Deoxyuridine (0.
25g, 0.67mmol) was dissolved in dry acetonitrile (30ml).
Sublimated 1H-tetrazole (94 mg) is added and the mixture is placed under an argon atmosphere
Stirred under for 15 minutes. 21j (0. 1) dissolved in 15 ml of dry acetonitrile.
51 g, 0.67 mmol) into the reaction solution via syringe over 5 minutes.
Just added. The mixture was allowed to stir at room temperature for 3 hours. Acetonitrile
Evaporation under reduced pressure gave a residue. This residue is treated with 70% EtOAc / E
Milled with tel mixture. The insoluble tetrazole is filtered off and the filtrate is
Evaporate to give a dry yellow foam (0.49 g). This foam is
Used in the next step without further purification.

(3′-acetoxy-N ⁴ -benzoyl-2′-deoxy-β-L-cytidinyl) -β-D-5-fluoro-2′-deoxyuridinyl cyanoethyl phosphonate ester (26j)) 2 The monomer, 25j (0.49 g) was dissolved in 8 ml of THF containing 0.2 ml of water and 2 ml of pyridine. Iodine crystals (30 mg) were added and the contents of the loosely stoppered flask were allowed to stir for 1 hour. Excess iodine was removed by the addition of a few drops of saturated sodium thiosulfate. Then the reaction mixture was evaporated to dryness. The crude product was washed with saturated NaHCO ₃ solution and brine with Et OAc. The organic phase was dried over Na ₂ SO ₄ , filtered and evaporated under reduced pressure. The residue was dissolved in 20 ml of 80% acetic acid / water solution and stirred until the reaction was completed. The solvent was evaporated and the residue was 10-15% M
Purified on a silica gel column using MeOH / CHCl ₃ . Fractions containing one spot by TLC (10% MeOH / CHCl ₃ Rf = 0.4) were pooled and evaporated to give pure product (188 mg).

(3′-O- (2′-deoxy-β-L-cytidinyl) -β-D-5-fluoro-2′-deoxyuridine (27j)) O-protected dimer, 26j (188 mg) Was treated with 100 ml of concentrated ammonia solution until the reaction was completed. The solvent was removed under reduced pressure and the residue was
. It was purified by DEAE cellulose ion exchange column using a gradient of NH ₄ CO ₃ in 02～0.2M. The pure fractions were evaporated to dryness at 40 ° C. under high vacuum to give the pure product (105 mg).

[0109]

[Equation 33] (5'-O-dimethoxytrityl-3 '-[O- (3'-O-acetyl) -N ^Four -Benzoyl-2'-deoxy-α-L-cytidinyl) -β-D-5-fluoro-2'-deoxyuridine (25k)) 3'-O-acetyl-β-D-5-fluoro-2 ' -Deoxyuridine (0.
19g, 0.51mmol) was dissolved in dry acetonitrile (30ml).
Sublimation 1H-tetrazole (80 mg) is added and the mixture is placed under an argon atmosphere
Stirred under for 15 minutes. 21k (0. 0) dissolved in 15 ml of dry acetonitrile.
45 g, 0.61 mmol) was added to the reaction solution via syringe over 5 minutes.
Just added. The mixture was allowed to stir at room temperature for 3 hours. Acetonitrile
Evaporation under reduced pressure gave a residue. This residue is treated with 70% EtOAc / E
Milled with tel mixture. The insoluble tetrazole is filtered off and the filtrate is
Evaporate to give a dry yellow foam (0.42 g). This foam is
Used in the next step without further purification.

(3′-acetoxy-N ⁴ -benzoyl-2′-deoxy-α-L-cytidinyl) -β-D-5-fluoro-2′-deoxyuridinyl cyanoethyl phosphonate ester (26k)) 2 The monomer, 25k (0.42 g), was dissolved in 10 ml of THF containing 0.2 ml of water and 2 ml of pyridine. Iodine crystals (45 mg) were added and the contents of the loosely stoppered flask were allowed to stir for 1 hour. Excess iodine was removed by the addition of a few drops of saturated sodium thiosulfate. Then the reaction mixture was evaporated to dryness. The crude product was washed with sat. NaHCO ₃ solution and brine with EtOAc. The organic phase was dried over Na ₂ SO ₄ , filtered and evaporated under reduced pressure. The residue was dissolved in 25 ml of 80% acetic acid / water solution and stirred until the reaction was completed. The solvent was evaporated and the residue was purified on a silica gel column using 10-15% MeOH / CHCl _3. Fractions containing one spot by TLC (10% MeOH / CHCl ₃ Rf = 0.4) were pooled and evaporated to give the pure product (125 mg).

(3′-O- (2′-deoxy-α-L-cytidinyl) -β-D-5-fluoro-2′-deoxyuridine (27k)) O-protected dimer, 26k (125 mg) Was treated with 100 ml of concentrated ammonia solution until the reaction was completed. The solvent was removed under reduced pressure and the residue was
. It was purified by DEAE cellulose ion exchange column using a gradient of NH ₄ CO ₃ in 02～0.2M. The pure fractions were evaporated to dryness at 40 ° C. under high vacuum to give the pure product (40 mg).

[0112]

(Equation 34) Example 5 In vitro dimer test in B16 melanoma and P388 leukemia, and in vitro dimer test in an inhibition assay for 293 progressive telomerase. The effect of monomeric 5-FUdR on cell lines and the B16 melanoma cell line was compared to the effect of monomeric 5-FUdR in an inhibition assay on 293 progressive telomerase. Telomerase is
It is a DNA-promoting enzyme that is not expressed in normal somatic cells, but is commonly expressed only in germline and fetal cells. In many types of cancer cells, the enzyme activity is reactivated (and so on). Thus, telomerase inhibitors are a valuable new class of anti-neoplastic agents. Table 2 shows the results.

TABLE 1 Inhibition of tumor cell growth and telomerase activity by 5-FUdR dinucleoside monophosphate

[0114]

[Table 1] The results show that the dimer of prototypes, to inhibit the growth of cultured murine leukemic L1210 cells and melanoma B16 cells greater potency (some IC ₅₀ values of less than 1 nM). This IC ₅₀ value is several times stronger than FUdR. These results are unexpected and therefore these compounds are truly unusual.

This preliminary result of telomerase inhibition is also intriguing. α-L, β-D
The dimer inhibited the enzyme by 84% compared to the control.

These data indicated that dimers containing L-sugars have very interesting biological profiles and represent a new class of potent anti-neoplastic agents. The profile of the activity of this L-dimer is different from that of the parent monomeric drug β-D-5FUdR.

The biological effect of this dimer was compared to the effect of monomeric 5-FUdR on P388 leukemia cell line and B16 melanoma cell line. Table 2 shows the results.

Table 2 In vitro test data

[0119]

[Table 2] These data indicate that β-D-5FU along with α-L or β-L-nucleoside
dinucleoside monophosphate compounds containing dR, as demonstrated by a lower an IC ₅₀ value indicates that exhibit excellent in vitro activity against murine P388 leukemia cell line and B16 melanoma cell line. This indicates that such nucleoside dimers may actually act or be metabolized and / or transported differently than 5-FUdR by a mechanism different from that of 5-FUdR.

Example 6 Measurement of Thymidylate Synthase Activity and Inhibition in Intact L1210 Leukemia Cells In Vitro Thymidylate synthase is one suspicious site of action of this compound. Therefore, the activity of selected compounds of the present invention, measured against thymidylate synthase activity measured in vitro, is a reliable indicator of the behavior of these compounds in an in vitro system.

[0121] Murine leukemia L1210 cells were harvested from cell culture flasks and cell concentrations were measured. The cells were then resuspended in the desired volume of medium to give a stock concentration of 5 × 10 ⁷ cells / mL. Serial dilutions of stock solutions of the compound to be tested were prepared (concentrations range from 10 ⁸ M to 10 ^-3 M). A solution of the compound to be tested at the desired concentration is pipetted into a microcentrifuge tube and incubated at 37 ° C. using a shaking water bath. The reaction was initiated by the addition of [5- ³ H] -2′-deoxycytidine (10 μL, stock solution concentration −10 ⁻⁵ M) after 30 or 60 minutes of pre-incubation with 80 μL of cell suspension. And continue for 30 minutes at 37 ° C. in a shaking water bath. The reaction is stopped by adding 100 μL of 10% charcoal in 4% HClO ₄ . Stir vigorously by vortexing the tube, then use the Beckman Microfug
e. Centrifuge for 10 minutes. The radioactivity of 100 μL of the supernatant fraction from each tube was measured using a toluene-based scintillation mixture in Packa
Count on an rd Tri-Carb (model 2450 or 3255) liquid scintillation spectrometer. Tritium release is expressed as a percentage of the total amount of radioactivity added. The IC ₅₀ value determined from the dose response curve indicates the concentration of inhibitor required for 50% inhibition of tritium release. Table 3 below shows the results of the analysis of tritium release and determination of the IC ₅₀ .

[0122]

[Table 3] (Example 7) (In vivo test of dimer in P388 leukemia and B16 melanoma) (A. Experiment) (1. P388 leukemia) Ascites containing P388 cells was extracted from tumorized B6D2F1 mice into B6D2F1 mice. An inoculum of P388 mouse leukemia cells, prepared by centrifugation and then resuspending the leukemia cells in saline, was injected intraperitoneally (ip.
) Administered. Mice received 1 × 10 ⁸ P388 cells intraperitoneally on day 0. On day 1, the tumorigenic mice were treated with dimer or vehicle control.
The route of drug administration was intraperitoneal and the schedule selected was 5 times daily. The maximum tolerated dose (MTD) was 200 mg / kg for each dimer and was determined in a first dose experiment in non-tumorized mice. In actual experiments, L
-103 was given at 100 mg / kg and 50 mg / kg.

2. B16 Melanoma B6D2F1 mice were intraperitoneally (ip) administered an inoculum of B16 mouse melanoma blye prepared from B16 tumors growing subcutaneously (sc) of the mice. (0
Day). On day 1, the tumorigenic mice were treated with dimer or vehicle control. The route of drug administration was intraperitoneal and the schedule selected was 5 times daily. The maximum tolerated dose (MTD) is 200 mg / kg for each dimer,
And it was determined in a first dose experiment in non-tumorigenic mice. In actual experiments, L-103 was given at 100 mg / kg and 50 mg / kg.

3. Criteria for Survival The mean survival times of all groups were calculated and the results are expressed as mean survival of treated mice / mean survival of control mice (T / C) × 100%. A T / C value of 150 means that mice in the treatment group survived 50% longer than mice in the control group; this is sometimes referred to as increased lifespan, or ILS value.

In the P388 study, mice that survive 30 days are considered long-term survivors or cured; whereas in B16, mice that survive 60 days are considered long-term survivors or cured. The widely accepted activity cutoff in both models (used by the National Cancer Institute (NCI) for many years) is T / C = 125
It is. Traditional use of B16 and P388 over the years has defined the following levels of activity: T / C <125, no activity; T / C = 125-150, weak activity;
T / C = 150-200, moderate activity; T / C = 200-300, high activity;
T / C> 300, with long-term survivors; excellent, curative activity.

B. Results 1. P388 Leukemia L-103 showed moderate activity at all tested doses in P388 leukemia in mice (Table 5). L-103 given intraperitoneally 5 times daily at doses of 100 mg / kg and 50 mg / kg, 149 and 14 respectively.
A T / C value of 4 was obtained. Fluorodeoxyuridine (FUdR) was used as a positive drug control in this study; FUdR had a T / T in the P388 test.
C = 164 (Table 4). All drugs were well tolerated in this experiment; there was little or no weight loss and no toxic deaths were recorded.

(Table 4 L-103 vs. mouse P388 leukemia)

[0128]

[Table 4] (2. B16 melanoma) L-103 showed mild activity against B16 melanoma transplanted into mice (Table 6). L-103 (ip; 5 times daily) was 139 and 134, respectively.
T / C values were given. The positive control drug FUdR produced mild efficacy in the B16 test; a T / C value of 135 was obtained (Table 5). All drugs were well tolerated with little or no weight loss; no drug-related deaths occurred.

Table 5 L-103 vs mouse B16 melanoma

[0130]

[Table 5] L-103 showed mild activity against both P388 and B16 experimental mouse tumors at the two doses tested. L-103 is almost as active as the positive control drug FUdR in the B16 test, and P3
In 88 tests, the activity was somewhat lower than FUdR.

From the above, the importance of α- and β-enantiomer nucleosides, nucleotides and analogs thereof based on L-sugars as versatile and highly effective chemotherapeutic agents is clear. Our results for derivatives of 5-FUdR show that L
2 shows that isomers containing -5-FUdR are less toxic than isomers containing β-D-5FUdR. Because they are probably not phosphorylated and are not transported as the latter. We have found that dimeric derivatives designed and prepared from α-L-5FUdR have very potent activity against P388 leukemia cells and the B16 melanoma cell line (the activity of β-D-5FUdR). To surpass). These appear to have unusual mechanisms of action, including inhibition of telomerase.

Example 8 In Vivo Activity of Nucleoside Analogs A. In Vitro Culture of Malaria Parasites falciparum, FCQ27, by Tagger and Jensen
(W. Trager and JB Jensen, Science, 1936)
73-675 (1976)). Cultures containing a 2% hematocrit suspension of parasitized human O + erythrocytes in RPMI 1640 medium supplemented with 25 mM HEPES-KOH (pH 7.2), 25 mM NaHCO ₃ and 10% human O + serum (v / v) Maintain at 37 ° C. in a mixture of 5% O ₂ , 5% CO ₂ and 90% N _{2 in} a modular incubator chamber. P. used in these experiments. falcipar
The isolate for um was FCQ27, which was routinely maintained in synchronized in vitro or asynchronous in vitro culture at low hematocrit.

(In Vitro Toxicity to BP falciparum) The potential toxicity of the nucleoside analogs to F. falciparum was determined in microtiter plates over a range of drug concentrations.
Time tested. Procedures for monitoring parasite viability are well established (AM Gero, HV Scott, WJO'Sullivan and RI Christopherson, Mol. Biochem. Par.).
asitol. 34, 87-89 (1989)), which is based on the incorporation of radiolabeled hypoxanthine or radiolabeled isoleucine. [G-
3H] hypoxanthine. Incorporation of falciparum into nucleic acids was used to assess in vitro parasite viability. The microculture plate was filled with 225 μl of a 2% hematocrit culture of asynchronously parasitized erythrocytes (1
% Parasitized cells). Each plate containing various concentrations of the drug to be studied (for the initial screening, up to a final concentration of 200 μM) was incubated for 24 hours at 37 ° C. in a mixture of 5% O ₂ , 5% CO ₂ and 90% N ₂ . . At this point, [G-3H] hypoxanthine was added to each well and incubation was continued under the same conditions for an additional 18-20 hours. Control infected cells (i.e., no drug) typically reached 6-8% parasitemia prior to collection. The drug sensitivity assay was simplified by a 96-well plate counter using lactate dehydrogenase (LK Basco, F.C.
Marguet, M .; T. Makler and J.M. Lebbras, Exp. P
arasitol. 80, 260-271 (1995); T. Makler
And D. J. Hinrichs; Am. J. Trop. Med. Hyg. 48
, 205-210 (1993)). This assay gave the same results as the hypoxanthine technique. In addition, in each experiment, microscopic counting of thin slides stained with Giemsa was used as a control.

(Transport and Metabolism in CP Falciparum-Infected Erythrocytes) The metabolism of L-nucleoside conjugate was examined by HPLC analysis. The main purpose is
It was to determine their ability to be catalyzed by the parasite purine salvage enzyme. Some effects on the purine metabolic pool were also observed.

For each HPLC measurement, 200 μL of packed cells of 80-90% vegetatively infected cells were used. These include the synchronization of parasites in in vitro cultures using sterile D-sorbitol (L. Lambros and JP Vande).
rberg, Parasitol. 65, 418-420 (1979)), followed by the previous (AM Gero, HV Scott, WJO'Sulliv.
an and R.A. I. Christopherson, Mol. Biochem.
Parasitol. 34, 87-89 (1989)).
Isolated from in vitro cultures by separation of trophozoites from uninfected erythrocytes by an 11 gradient. The trophozoites are given at 37 ° C for 2 hours with each compound to be tested.
Incubated for hours. These compounds were incubated with both infected whole cells (as whole cells) and lysed uninfected normal red blood cells, and the following a) -c
A) Invasion into cells (whether they were transported) b) Metabolic effects in cells (whether this compound was metabolized) c) Disrupted or lysed cells Ability to catalyze a compound that may not be able to enter a non-cell (ie, is this compound metabolized to an active form when transported into the cell)? Upston and Gero (JM Upston and AM Gero,
Biochem. Biophys. Acta. 1236, 249-258 (19
95)), the drug incubation was terminated by centrifugation through silicon oil. This procedure separated the intact trophozoite from extracellular, non-transported drug solution.

The metabolism of nucleosides with potential chemotherapeutic activity was determined by reversed-phase ion pair high performance liquid chromatography (RS Togusov, YV Tikhonov).
, A. M. Pimenov, V .; Prokudin, Journal of Ch
romatography, I. et al. Biomedical Appl. 434,4
47-453 (1988)). Nucleotides, nucleosides, and bases were separated by this HPLC method.

Purine nucleoside transport and metabolism is determined by normal human erythrocytes and Plasmo.
It differs significantly from human red blood cells infected with D. falciparum.
Malaria parasites are unable to synthesize purine de novo and therefore have to rely on the salvage pathway to obtain the purines necessary for their growth and division (LW.
Scheibel and T.W. W. Sherman, Malaria: Princ
iples and practices of Malliology (W.
H. Wernsdorfer and I.S. McGregor ed.) 1,234-
242 (1988)). Furthermore, normal human erythrocytes do not contain significant levels of pyrimidine nucleotides (E. Szabodos and RI Christop).
Herson, Biochem. Edu. 19, 90-94 (1991)), and this parasite is unable to obtain pyrimidine bases by the salvage pathway and must also rely on de novo synthesis (LW Scheibel and TW Sherman, Malaria: Principles and
practices of Malliology (WH Warnsdor)
fer and I.S. McGregor ed.) 1,234-242 (1988))
. These alterations to the metabolic pathways of infected erythrocytes, along with alterations in their transport system, exhibit significant variability from normal erythrocytes and may provide the parasite with an opportunity to selectively use toxic compounds.

Nucleosides have attracted investigators as potential therapeutics. Naturally occurring nucleosides are usually in β-D form. Thus, most of the nucleoside analogs designed for the treatment of cancer, viral and parasitic diseases,
It has been synthesized in this stereochemical configuration. Due to recent findings in our labs at the University of Georgia, Iowa and Yale and universities in France and Italy, most L-nucleosides show that normal cells
Since it was not used for the production of NA or DNA, and was not metabolized, it was confirmed to exhibit low toxicity.

Recently, Dr. Gero and coworkers have reported that non-physiological β-L-adenosine has been falciparum has been found to be able to be selectively transported into erythrocytes infected with it (AM Gero and JM Upston, Pyrine &
Pyrimidine Metabolism in Man VIII (A
. Sahota and M.S. Taylor) Plenum Press, NY,
495-498 (1995)). Normal red blood cells and other cell types are completely impermeable to this compound.

During the Phase I study, we developed a non-natural nucleoside analog for selective transport into malaria-infected cells to create a class of non-toxic antimalarial drugs based on newly synthesized L-nucleosides. You used this unique ability. Our working hypothesis is the design and biological evaluation of novel chemical entities consisting of both the known inhibitors of thymidylate synthase, 5-fluorodeoxyuridine (FUdR) and L-nucleosides or derivatives thereof. Based. “Dimers” consisting of α- or β-L isomer modifications of physiological nucleosides or their derivatives were linked to FUdR via phosphate or pro-phosphate linkages. Along with anticancer activity, FudR has potential as an antimalarial agent (S.
A. Queen, D.C. L. Vander Jagt and P.M. Reyer, An
timicrobial Agents & Chemotherapy. 34
, 1393-1398 (1990)). Unfortunately, the toxicity of FUdR limits its use. Theoretically, binding of the FUdR to the L-nucleoside unit has no effect on normal cells, but gives rise to entities that can selectively transport the active ingredient to infected cells.

During the Phase I study, a total of 42 L-nucleoside analogs were falcip
arum in an in vitro assay. These compounds are shown in FIG. 31 of these 42 compounds are Lipitek Inte
Available for screening from rnation's library, 11 was specially synthesized for the purposes of this project. The detailed synthesis of the 11 L-nucleoside conjugates is described in Methods and Procedures. These are 1
Prepared on a scale of 00 mg and analyzed by analytical methods (NMR, HPLC, mass spectrometry, TLC
). The 42 compounds tested represented L-nucleoside monomers or four different types of L-nucleoside conjugates. The conjugates tested were a) dinucleoside phosphate, b) dinucleoside phosphorothioate, c) SATE derivative of L-nucleoside, and d) L-nucleoside conjugate of nitrobenzylthionosin (NBMPR). Still more diversification resulted from exploiting the 3 ′ → 5 ′ vs. 5 ′ → 3 ′ phosphodiester linkages characteristic of nucleosides and purine and pyrimidine variations in both parts of the dimer. Should be emphasized.

For biological screening, the protozoan P. fal
Evaluation of the compound for C. parium was included. A range of drug concentrations was used independently in the two assays. On the one hand, Falciparum radiolabeled hypoxanthine incorporation into nucleic acids, the other being a more convenient assay, a 96-well plate sensitivity assay using lactate dehydrogenase. Both assays gave identical results. In addition, microscopic counting of Giemsa-stained thin slides was used as a control. The results of the biological assays are shown in Table 1.
An example of the experimental curve is attached in Appendix 2. Biological tests were performed at several concentrations. The highest concentration was 200 μM. These compounds were considered active at concentrations below 40 μM.

(Discussion) (In vitro activity) Screened 42 by careful analysis of the data shown in Table 6 (below).
Show that 9 analogs had an IC50 of less than 40 μM (see FIG. 14 for the structures of the compounds tested). The most active representative dinucleoside phosphates are L-101, L-103, L-110, L-111, L-113.
, L-133 and L-138.

(Table 6. In vitro test results)

[0145]

[Table 6] The 14 L-nucleoside monomers in α and β form, and even α-L-FUdR, have falciparum. Because of this, further work on the monomer was discontinued (see Table 6).

A dimer containing only the nucleoside in the “unnatural” isomeric form (L-109) showed no activity.

Careful analysis of the data in Table 6 indicates that the β-D-isomer of FUdR is the active component of the dimeric molecule. The position of the active ingredient in the dimer is important. β-D-FUdR has a phosphodiester bond to its 5′-OH,
It is necessary to be linked to the 3'-OH end of L-nucleoside. FUdR
Compounds linked via the 3′-OH of are much less active (Table 7).
See). This indicates that the substitution pattern of β-D-FUdR is important for the activity of the dimer, and most likely that the mechanism involves thymidylate synthase inhibition. It is well known that FUdR TS inhibitors have very rigid structural requirements and do not tolerate any substitutions at the 3 'end.

Table 7. Activity of L-nucleoside containing dimers versus position of FUdR binding.

[0149]

[Table 7] Table 8. Dimer activity versus L-nucleoside chemical configuration.

[0150]

[Table 8] In the case of purine nucleosides, attachment of the α-L nucleoside to the β-D-FUdR monomer reduces the activity of the dimer as compared to a dimer containing β-L units (
See Table 8, L-125 and L138). In the case of pyrimidine nucleosides, there is no apparent difference in activity (L-103 and L-110, L-111 and L-113).

Table 9. Activity of dimers versus nature of binding between two nucleoside analogs

[0152]

[Table 9] The different activities of the dimer depend on the structure of the second nucleoside.

The plausible pathways and / or modes of action of metabolic activation of the tested dimeric molecules are as follows: (1) The dimer is a phosphate bond or prophosphate between two monomer units Without hydrolysis of the bond, it can act as a new chemical entity; (2) Hydrolysis to L-nucleosides and FUdR nucleotides can occur, in which case the dimer is a prodrug. L-nucleoside is β-D-FU
Used to protect dR monophosphate or increase its bioavailability. It is also important to note that hydrolysis can occur inside and outside the cell.

In the last decade, enduring efforts have been directed to the synthesis of oligonucleotide analogs with modified phosphodiester linkages. The goal is to improve the stability of duplex and triplex forms, improve its cellular uptake, and reduce the rate of degradation of oligonucleotides by endonucleases and exonucleases that cleave phosphodiester bonds Was that. We have selected one such chemical modification for the study. As a result, 2
Several dimers with phosphorothioate linkages between two nucleosides were synthesized and tested. This phosphorothioate is a phosphodiester linked phosphorus, containing an oxygen to sulfur substitution (for the corresponding dimer structure, see Appendix 1
See). The S homolog has been shown to be more resistant to cellular nucleases and to be more easily taken up by cells (M. Matsukuura, K.
. Shinozuka, G .; Zon, H .; Mitsuya, M .; Reitz, J
. S. Cohen, L .; M. Neckers, Proc. Natl. Acad.
Sci. USA. 84.7706 (1987)). Several oligonucleotides of this type are currently in clinical trials (ISIS Pharmaceuticals).
als).

Table 10. Activity of β-D-FUdR and some possible products of its metabolism

[0156]

[Table 10] Displacement of the phosphate bond by a phosphorothioate bond in a dimer containing two β-D-FUdR units increases the activity of the compound by a factor of 10 (Table 9,
L-101 and L-128 data). The only active phosphorothioate analog appears to be compound L-128. The activity of L-128 is high compared to all possible hydrolysates (see Table 10). Further, L-128
Was the most active compound tested.

(Table 11. Activity of SATE derivative of FUdR)

[0158]

[Table 11] Incorporation of phosphorothioate into the molecule of a dimer containing "unnatural" nucleoside isomers was unsuccessful: the activity of this compound was dramatically reduced (Table 9, L-103)
, L-117 and L-138). As discussed previously, one of the possible mechanisms of dimer action is its involvement in the metabolic pathway of the entire non-hydrolyzed molecule. In this case, the increase in dimer stability due to the introduction of a phosphorothioate linkage
This results in increased activity of L-101. For dimers, including "unnatural" isomeric variants of nucleosides, metabolism of the entire non-hydrolyzed molecule is probably impossible.

It is well established that most nucleoside analogs are dependent on kinase-mediated activation to make bioactive nucleotides, and ultimately nucleoside triphosphates (C. Periqaud, G. Goss).
elin, J .; L. Imbach, Nucleoside Nucleotid
es. 11, 903 (1992)). Activation occurs in the cytoplasm after nucleoside incorporation, and involves three consecutive viral and / or cellular kinases. The first is highly specific (MC Sta.
mes, Y .; C. Cheng, J .; Biol. Chem. 262,988 (19
87)). One possibility for improving the efficiency of a nucleoside analog may be that the therapeutic agent bypasses the phosphorylation step. Unfortunately, nucleoside monophosphate itself cannot efficiently cross cell membranes because of its polarity (K.
C. Leibman, C .; J. Heidelberg, J .; Biol. Chem
. 216, 823 (1995)). Thus, the more lipophilic derivative, which temporarily masks or reduces the negative charge of the phosphate by neutral substituents, which is expected to return to the nucleoside monophosphate once inside the cell The idea of producing.

One possible structural modification for the kinase bypass is the use of bis-S-acetylthioethyl (SATE) derivatives, pioneered by: -L
. Imbach (I. Lefebvre, C. Periaud, A. Pomp)
on, A. M. Aubert, J .; L. Gradet, A .; Kirn, G .;
Gosselin and J.M. L. Imbach, J .; Med. Chem. 38,
3941-3950 (1995); Perlgaud, G .; Gosseli
n, I. Lefebvre, J .; L. Girardet, S.M. Benzaria
, I .; Barber and J.W. L. Imbach, Bio. Org. Med. C
hem. Lett. 3, 2521-2526 (1933)). Several SATE derivatives of the FUdR isomer have been synthesized and falciparum was tested for in vitro activity. The results obtained are listed in Table 11.

It should be emphasized that all SATE derivatizations were performed on FUdR monomers with different conformations. Therefore, derivatives of α and βD and L-FUdR were prepared. Three types of SATE analogs were made:
a) 5 'modified analogs of nucleosides, b) 3' modified analogs of nucleosides, and c) analogs modified at both 3 'and 5' of nucleosides to effect two substitutions. 15 L-nucleoside dimers from Lipitek's library (L-101, L-103, L-103A, L
-107, L-110, L-111, L-112, L-114, L-117, L
-120, L-122, L-124, L-125, L-133, and L-13
8) was submitted to the US Army Antimalarial Testing Program for in vitro screening (see FIG. 7 for compound structure). These compounds have two P.O. falciparum strains: tested for activity against D6 (chloroquine resistant) and W2 (chloroquine resistant). Seven of the compounds tested were
At less than 40 μM, Falciparum showed activity against both strains.
The most active dimers are L-101, L-110, L-112, L-117, L-
133, and L-138.

Transport and Metabolic Studies It has been established that transport and uptake in parasite-invaded cells is different from normal red blood cell transport and uptake (AM Gero and AM).
. Wood, Pyrine & Pyrimidine Metabolis
Min Man VII, Part A. (RA Harkness et al., Eds.) Plenum Press, NY, 169-172 (1991)). Invasion by malaria parasites involves the cell membrane and allows the penetration of non-natural substances of various sizes and shapes. In contrast, normal cells are very selective in uptake. L-nucleosides and their derivatives have been shown to readily penetrate invading cells. On the other hand, despite their invasion, they have a very slow rate of uptake into normal cells. To obtain preliminary data on transport, uptake, and metabolism, the following 10 Lipitek compounds were analyzed using HPLC methods: L-101, L-103, L-109, L-
111, L-117, L-133, L-138, GCI 1007, GCI 1
027, GCI 1069. The HPLC retention times for standard compounds purchased from Sigma are shown in Table 12.

(Table 12. HPLC retention time for standard compounds)

[0164]

[Table 12] (Table 13. HPLC retention time for Lipitek compounds) (Vegetative incubation)

[0165]

[Table 13] These compounds were selected for identification of possible metabolites. In this experiment, compounds were incubated with both whole infected cells and non-infected and lysed uninfected cells, followed by separation of unreacted compounds and H
PLC analysis was performed. The results are presented in Table 13: Column 1 shows the retention time of the original compound (no incubation with any cells). Column 2 shows the retention time of the original compound remaining after incubation with whole cells infected with the parasite. Column 3 shows metabolites (ie, new peaks due to conversion of the original compound or alteration of the natural purine or pyrimidine profile of infected cells).

Any L-nucleoside (L-101, L-103, L-111, L-117
, L-133, and L-138) and the tested L-nucleoside monomer analogs (GCI 1007, GCI 1027 and GCI 1069)
All nucleoside monophosphate dimers, including the β-D-FUdR unit in combination with, entered the infected cells. All of these compounds are described in f
Toxicity to alciparum. L-, which is a combination of two L dimers
109 was unable to invade infected cells and was also not toxic.

Compounds L-101, L-133, and L-138 appear to be metabolized by infected cells, each producing at least one new peak (see Table 13). It is possible that L-138 and L-133 can be cleaved into nucleotides and nucleosides.

None of the above 10 compounds were found to enter normal erythrocytes. Metabolism of any of the above compounds did not occur in human erythrocyte or lymphocyte lysates. Therefore, even if these compounds could invade normal cells, normal cells would not be able to metabolize these compounds into active ingredients. This further emphasizes the low toxicity and selectivity of the L-nucleoside conjugates disclosed and claimed in this application.

Example 9 Synthesis of N ⁶ -Benzoyl-3′-deoxy-β-D-adenosine 3′-deoxyadenosine (2.10 g) in pyridine (80 ml) cooled in an ice bath.
(0 g, 7.96 mmol), ClSiMe ₃ (5.0 ml, 39.8 mmol) was added dropwise and stirred for 30 minutes. Then benzoyl chloride (3.7 ml, 31.84 mmol) was added dropwise and the reaction mixture was stirred at room temperature for 2 hours. This was cooled in an ice bath and water (16 ml) was added dropwise. After 15 minutes, concentrated NH ₄ OH (16 ml) was added to give an approximately 2M solution in ammonia. After 30 minutes, the solvent was evaporated and the residue was dissolved in water and washed with ether. The aqueous layer was concentrated and the compound crystallized from water as a white solid (2.32 g)
, 82%).

Example 10 Synthesis of N ⁶ -Benzoyl-5′-O- (di-p-methoxytrityl) -3′-deoxy-β-D-adenosine Compound N in pyridine (100 ml) ^To a solution of ⁶ -benzoyl-3′-deoxy-β-D-adenosine 1 (2.32 g, 6.53 mmol) was added 4,4′-
Dimethoxytrityl chloride (3.32 g, 9.79 mmol) and DMAP
(0.24 g, 1.96 mmol) was added and stirred at room temperature under argon for 2 hours. To complete the reaction, additional DMTCl (0.5 g) was added and stirred for another 2 hours. The reaction was quenched by the addition of MeOH (5 ml) and the solvent was evaporated. The residue is taken from EtOA
and washed with water, NaHCO ₃ , and brine. After drying over Na ₂ SO ₄ , the EtOAc layer was evaporated and the crude compound was purified on a silica gel column using 80% EtOAc / CHCl ₃ as solvent to give pure compound 2 (4.33 g). , 83%) as a white foam.

Example 11 Synthesis of N ⁶ -Benzoyl-2′-O-acetoxy-β-D-3′-deoxyadenosine N ⁶ -Benzoyl-5′-O- in pyridine (100 ml) (Di-p-methoxytrityl) -3′-deoxy-β-D-adenosine (4.3 g, 6.58 mmol)
l) in a solution of acetic anhydride (1 ml, 9.87 mmol), and DMAP (0.
08 g, 0.65 mmol) and stirred at room temperature for 15 minutes. The solvent was then evaporated and the residue was dissolved in EtOAc, water, N
Washed with aHCO ₃ , brine and dried over NA ₂ SO ₄ . After evaporation of EtOAc, the crude was then dissolved in 80% AcOH (50 ml) and stirred at room temperature for 1 hour. The solvent was then evaporated, then co-evaporated with toluene and 3-5
Purified on a silica gel column using 5% MeOH / CHCl ₃ to give pure N ⁶ -benzoyl-2′-O-acetoxy-β-D-3-deoxyadenosine (2.
11 g, 81%) as a foam.

(Example 12) (Synthesis of β-L-dU, cordycepin dimer (L-150)) β-L-dU (1.0 g, 4.38 mmol) was added to dry pyridine (50 ml).
And 4,4′-dimethoxytrityl chloride (1.78 g, 5
. 25 mmol) and DMAP (0.1 g, 0.87 mmol).
This was stirred at room temperature under argon for 2 hours and quenched with MeOH (5 ml). The solvent was evaporated and the residue was dissolved in EtOAc, water, N
Washed with aHCO ₃ and brine. After drying and evaporation of the solvent, the crude material was purified on a silica gel column using 60-80% EtOAc / CHCl ₃ as the solvent to give pure 5′-Odimethoxytrityl-β-L-
2′-Deoxyuridine (2.2 g, 94.8%) was obtained as a white foam.

Dimethoxytrityl-β-L-2′-deoxyuridine (1.5 g, 2.83
mmol) was dissolved in anhydrous dichloromethane (50 ml). N, N-diisopropylethylamine (2.0 ml, 11.3 mmol) is added under argon, followed by 2′-cyanoethyl-N, N-diisopropylchlorophosphoramidite (
0.82 ml, 3.68 mmol). The reaction was stirred for 30 minutes,
And this solvent was evaporated. This residue was dissolved in 80% EtOAc / Et ₃ N (75 ml) and washed with water, NaHCO ₃ , and brine. The organic layer was evaporated and purified on a short silica gel column using a mixture of EtOAc, CH ₂ Cl ₂ , and Et ₃ N (40:50:10)
5′-O-dimethoxytrityl-β-L-2′-deoxyuridine-3′-N, N-diisopropylcyanoethyl phosphoramidite was obtained in a quantitative yield.

5′-O-dimethoxytrityl-3 ′-[O- (2′-O-acetyl) -N ⁶ -benzoyl-β-D-3′-deoxyadenosinyl] -2′-deoxy-β-
L-uridine cyanoethyl phosphite (7).

To a solution of the compound 5′-O-dimethoxytrityl-β-L-2′-deoxyuridine-3′-N, N-diisopropylcyanoethyl phosphoramidite (2.83 mmol) in anhydrous acetonitrile (60 ml). , Acetonitrile (40m
N ⁶ -Benzoyl-2′-O-acetoxy-β-D-3-deoxyadenosine (1.12 g, 2.83 mmol) in l) was added and stirred for 10 minutes under argon. To this solution, sublimated 1H-tetrazole (0.6 g, 8.5 mm
ol) and stirred overnight. The solvent was evaporated and the residue was triturated with 70% EtOAc / ether and filtered. The filtrate was evaporated, 5'-O-dimethoxytrityl -3 '- [O- (2'- O- acetyl) -N ⁶ - benzoyl-beta-D-3'-deoxy adenovirus shea sulfonyl] -2' Deoxy-β-L-uridine cyanoethyl phosphite was obtained as a foam and used in the next step without further purification.

5′-O-dimethoxytrityl-3 ′-[O- (2′-O-acetyl) -N ⁶ -benzoyl-β-D-3′-deoxyadenosinyl] -2′-deoxy-β-
L-uridine cyanoethyl phosphite was dissolved in THF (24 ml), pyridine (6 ml) and water (0.6 ml). Iodine crystals (1.0 g)
It was added in small portions until the iodine color persisted. The reaction mixture was stirred for a further 15 minutes and excess iodine was removed by the addition of saturated sodium thiosulfate. The solvent was evaporated and the residue was dissolved in EtOAc, and washed with water, NaHCO _3, and brine. The EtOAc layer was evaporated and the residue was dissolved in 80% acetic acid / water solution (40 ml) and 1%
Stirred for hours. The solvent was then evaporated and the crude product was purified on a silica gel column using 8-15% MeOH / CHCl ₃ as solvent to give pure (2′-acetoxy-N ^6- Benzoyl-3′-deoxy-β-D-adenosinyl) -β-L-2′-deoxyuridinyl cyanoethyl phosphate (0.75 g) was obtained as a foam.

The dimer (2′-acetoxy-N ⁶ -benzoyl-3′-deoxy-β-D-adenosinyl) -β-L-2′-deoxyuridinyl cyanoethyl phosphate (0.75 g) was obtained. , And treated with ammonium hydroxide solution (100 ml) overnight.
The solvent was evaporated and the residue was eluted with a NH ₄ HCO ₃ buffer gradient (0.
05-0.2M) using a DEAE cellulose ion exchange column. The pure fractions were collected and lyophilized to give pure 3′-O- (3′-deoxy-β-D-adenosinyl) -β-L-2′-deoxyuridine (L-15).
0) (0.486 g) was obtained as a white solid.

Example 13 Synthesis of β-L-dA, Cordycepin Dimer (L-151) 2′-Deoxy-β-L-adenosine in pyridine (75 ml) cooled in an ice bath To a stirred solution of 2.05 g (8.16 mmol) was added ClSiMe ₃ (5.17 ml, 40.8 mmol) dropwise and stirred for 30 minutes. Then benzoyl chloride (4.7 ml, 40.8 mmol) was added dropwise and the reaction mixture was stirred at room temperature for 2 hours. This was cooled in an ice bath and water (15 ml) was added dropwise. After 15 minutes, concentrated NH ₄ OH (15 ml) was added to give an approximately 2M solution in ammonia. After 30 minutes, the solvent was evaporated and the residue was dissolved in water and washed with ether. The aqueous layer was concentrated and N ⁶ -benzoyl-2′-deoxy-β-L-adenosine was crystallized from water as a white solid (2.48 g, 85.8%).

To a solution of N ⁶ -benzoyl-2′-deoxy-β-L-adenocin (2.48 g, 6.98 mmol) in pyridine (100 ml) was added 4,4′-dimethoxytrityl chloride (3.55 g). , 10.47 mmol) and DMAP (0.25
g, 2.09 mmol) were added and stirred at room temperature for 2 hours under argon. To complete the reaction, additional DMTCl (1.3 g) was added and stirred for another 2 hours. The reaction was quenched by the addition of MeOH (5 ml) and the solvent was evaporated. This residue was dissolved in EtOAc,
Washed with water, NaHCO ₃ , and brine. After drying over Na ₂ SO ₄ , the EtOAc layer was evaporated and the crude compound was dissolved in 3-5% M as solvent.
Purify on silica gel column using OH / CHCl to give pure N ⁶ -benzoyl-5′-O- (di-p-methoxytrityl) -2′-deoxy-β-L-
Adenosine (3.42 g, 74.5%) was obtained as a pale yellow foam.

Dissolve N ⁶ -benzoyl-5′-O- (di-p-methoxytrityl) -2′-deoxy-β-L-adenosine (1.64 g, 2.5 mmol) in anhydrous dichloromethane (50 ml). did. N, N-diisopropylethylamine (1.75 ml
, 10.0 mmol) was added under argon, followed by 2'-cyanoethyl-N,
N-diisopropylchlorophosphoramidite (0.8 ml, 3.25 mmol
) Was added. The reaction was stirred for 30 minutes and the solvent was evaporated. This residue was dissolved in 80% EtOAc / Et ₃ N (75 ml) and washed with water, NaHCO ₃ , and brine. The organic layer was evaporated and purified on a short silica gel column using a mixture of EtOAc, CH ₂ Cl ₂ , and Et ₃ N (40:50:10) to give a quantitative yield of N ⁶ -benzoyl-5′-O- (dimethoxytrityl) -β-L-2′-deoxyadenosine-3
'-N, N-diisopropylcyanoethyl phosphoramidite was obtained.

N ⁶ -benzoyl-5′-O- (dimethoxytrityl) -β-L-2′-deoxyadenosine-3′-N, N-diisopropylcyanoethyl phosphoramidite (2 ml) in anhydrous acetonitrile (60 ml). 0.5 mmol), N ⁶ -benzoyl-2′-O-acetoxy-β-D-3′-deoxyadenosine (0.94 g, 2.36 mmol) in acetonitrile (40 ml) is added and under argon. For 10 minutes. The sublimated 1H-tetrazole (0
. (5 g, 7.2 mmol) was added and stirred overnight. The solvent was evaporated and the residue was triturated with 70% EtOAc / ether and filtered. The filtrate was evaporated to give N ⁶ -benzoyl-5′-O- (dimethoxytrityl) -3 ′-[O- (2′-O-acetyl) -N ⁶ -benzoyl-β-D-3-deoxyadenoate. [Synyl] -2'-deoxy-β-L-adenosine cyanoethyl phosphite was obtained as a foam, which was used in the next step without further purification.

The dimer N ⁶ -benzoyl-5′-O- (dimethoxytrityl) -3 ′-[O- (2′-O-acetyl) -N ⁶ -benzoyl-β-D-3-deoxyadenosine 2] -deoxy-β-L-adenosine cyanoethyl phosphite
Dissolved in THF (24 ml), pyridine (6 ml), and water (0.6 ml). Crystals of iodine (0.63 g) were added in small portions until the iodine color persisted. The reaction mixture was stirred for a further 15 minutes and excess iodine was removed by the addition of saturated sodium thiosulfate. The solvent was evaporated and the residue was dissolved in EtOAc, and washed with water, NaHCO _3, and brine. The EtOAc layer was evaporated and the residue was dissolved in an 80% acetic acid / water solution (50 ml) and stirred for 1 hour. The solvent was then evaporated and the crude product was purified on a silica gel column using 5-10% MeOH / CHCl ₃ as solvent to give pure (2′-acetoxy-N ⁶ -benzoyl). -3'-deoxy-β-D-adenosinyl) -N ⁶ -benzoyl-β-L-2'-deoxyadenosinyl cyanoethyl phosphate (0.97 g)
Was obtained as a foam.

The dimer (2′-acetoxy-N ⁶ -benzoyl-3′-deoxy-β-D-adenosinyl) -N ⁶ -benzoyl-β-L-2′-deoxyadenosinyl cyanoethyl phosphate ( 0.97 g) in an ammonium hydroxide solution (100 ml).
) Overnight. The solvent was evaporated and purified on DEAE cellulose ion exchange column The residue using a gradient of NH ₄ HCO ₃ buffer (0.05~0.2M). The pure fractions were collected and lyophilized to give pure compound 3'-O- (3'-deoxy-β-D-adenosinyl) -β-L-2'-deoxyadenosine (L-151) (0.55 g) was obtained as a white solid.

Example 14 Synthesis of α-L-dU, Cordycepin Dimer (L-152) α-L-dU (1.04 g, 4.5 mmol) was dissolved in dry pyridine (50 ml). And 4,4′-dimethoxytrityl chloride (2.4 g, 6.8).
6 mmol) and DMAP (0.11 g, 0.91 mmol). This was stirred at room temperature under argon for 2 hours and quenched with MeOH (5 ml). The solvent was evaporated and the residue was dissolved in EtOAc, water, Na
Washed with HCO ₃ and brine. After drying and evaporating the solvent, the crude material was purified on a silica gel column using 3-5% MeOH / CHCl ₃ as solvent and pure 5′-O-dimethoxytrityl-α-L- 2'-
Deoxyuridine (2.4 g, 99%) was obtained as a white foam.

5′-O-dimethoxytrityl-α-L-2′-deoxyuridine (1.73
g, 3.26 mmol) in anhydrous dichloromethane (30 ml).
Diisopropylethylamine (2.3 ml, 13.04 mmol) was added under argon, followed by 2′-cyanoethyl-N, N-diisopropylchlorophosphoramidite (0.95 ml, 4.23 mmol). 30
Stir for a minute and evaporate the solvent. This residue is treated with 80% EtOA
Dissolved in c / Et ₃ N (75 ml) and washed with water, NaHCO ₃ , and brine. It was evaporated organic layer, and EtOAc, and purified by CH ₂ Cl _2, and Et ₃ N (40:50:10) on a short silica gel column using a mixture of, 5'-O-dimethoxytrityl -α -L-2'-deoxyuridine-
3′-N, N-diisopropylcyanoethyl phosphoramidite (2.26 g,
95%) as a foam.

5′-O-Dimethoxytrityl-α-L in anhydrous acetonitrile (60 ml)
To a solution of -2'-deoxyuridine-3'-N, N-diisopropylcyanoethyl phosphoramidite (2.26 g, 3.09 mmol) was added acetonitrile (4.
N ⁶ in 0 ml) - benzoyl -2'-O-acetoxy -β over D-3'deoxy adenosine (1.35 g, 3.4 mmol) was added and 1 under argon
Stirred for 0 minutes. To this solution was added sublimated 1H-tetrazole (0.65 g, 8.
5 mmol) was added and stirred overnight. The solvent was evaporated and the residue was triturated with 70% EtOAc / ether and filtered. The filtrate was evaporated and 5'-O-dimethoxytrityl-3 '-[O- (2'-
O- acetyl) -N ⁶ - benzoyl-beta-D-3'-deoxy adenovirus sheet sulfonyl] - 2'-deoxy-.alpha.-L-uridine cyanoethyl phosphite esters, obtained as a foam, and further purification Used in the next step without doing.

The dimer 5′-O-dimethoxytrityl-3 ′-[O- (2′-O-acetyl)
-N ⁶ - benzoyl-beta-D-3'-deoxy adenovirus shea sulfonyl] -2'-deoxy-.alpha.-L-uridine cyanoethyl phosphite, THF (24ml), pyridine (6 ml) and water (0.6 ml ). Crystal of iodine (0.7 g
) Was added in small portions until the iodine color persisted. The reaction mixture was stirred for a further 15 minutes and excess iodine was removed by the addition of saturated sodium thiosulfate. The solvent was evaporated and the residue was dissolved in EtOAc, and washed with water, NaHCO _3, and brine. The EtOAc layer was evaporated and the residue was dissolved in an 80% acetic acid / water solution (50 ml) and stirred for 1 hour. The solvent was then evaporated and the crude product was purified on a silica gel column using 8-15% MeOH / CHCl ₃ as solvent to give pure (2′-acetoxy-N ⁶ -benzoyl). -3′-Deoxy-β-D-adenosinyl) -α-L-2′-deoxyuridinyl cyanoethyl phosphate (1.29 g) was obtained as a foam.

[3′-O- (3′-deoxy-β-D-adenosinyl) -α-L-2′-deoxyuridine (6) (L-152)] Dimer (2′-acetoxy-N) ⁶ -Benzoyl-3′-deoxy-β-D-adenosinyl) -α-L-2′-deoxyuridinyl cyanoethyl phosphate (1.29 g) was treated with an ammonium hydroxide solution (100 ml) overnight. .
The solvent was evaporated and the residue was eluted with a NH ₄ HCO ₃ buffer gradient (0.
05-0.2M) using a DEAE cellulose ion exchange column. The pure fractions were collected and lyophilized to give pure [3'-O]-(3 '
-Deoxy-β-D-adenosinyl) -α-L-2′-deoxyuridine (L-
152) (0.856 g) was obtained as a white solid.

Example 15 Synthesis of β-L-dC, Cordycepin Dimer (L-153) β-L-dCBz (1.7 g, 5.22 mmol) in pyridine (100 ml)
4) -Dimethoxytrityl chloride (2.65 g, 7.83 m)
mol) and DMAP (0.13 g, 1.04 mmol) were added and stirred under argon for 2 hours at room temperature. Additional DMT to complete the reaction
Cl (0.9 g) was added and stirred for another 2 hours. This reaction was carried out with MeOH (5 m
Quenched by the addition of l) and the solvent was evaporated. This residue is treated with EtO
Dissolved in Ac and washed with water, NaHCO ₃ and brine. After drying over Na ₂ SO ₄ , the EtOAc layer was evaporated and the crude compound was removed as solvent by 3-5
% MeOH / CHCl ₃ and purified on a silica gel column to give pure N ⁴ -benzoyl-5′-O- (di-p-methoxytrityl) -2′-deoxy-β-L-.
Cytidine (2.98 g, 90%) was obtained as a pale yellow foam.

[N ⁴ -benzoyl-5′-O- (dimethoxytrityl) -β-L-2′-deoxycytidine-3′-N, N-diisopropylcyanoethyl phosphoramidite (3)] N ⁴ -benzoyl -5'-O- (di-p-methoxytrityl) -2'-deoxy-β-L-cytidine (1.7 g, 2.68 mmol) was added to anhydrous dichloromethane (
50 ml) and dissolved in N, N-diisopropylethylamine (1.9 ml, 10 ml).
. 72 mmol) under argon and then 2'-cyanoethyl-N, N
-Diisopropylchlorophosphoramidite (0.85 ml, 3.5 mmol)
Was added. The reaction was stirred for 30 minutes and the solvent was evaporated.
The residue was dissolved in _{80% EtOAc / Et 3 N (} 75ml), and washed with water, NaH CO ₃ and brine. It was evaporated organic layer, and purified by a short silica gel column using EtOAc, mixtures of CH ₂ Cl ₂ and Et ₃ N and (30:60:10), N ⁴ - Benzoyl-5'-O-(dimethoxytrityl) -Β-L-2′-deoxycytidine-3′-N, N-diisopropylcyanoethyl phosphoramidite (2.06 g, 92%) was obtained as a foam.

N ⁴ -Benzoyl-5′-O- (dimethoxytrityl) -β-L-2′-deoxycytidine-3′-N, N-diisopropylcyanoethyl phosphoramidite (100 g) in anhydrous acetonitrile (100 ml). 06G, a solution of 2.47 mmol), N ⁶ in acetonitrile (40 ml) - benzoyl -2'-O-acetoxy Shi-beta-D-3'-deoxyadenosine (1.08 g, a 2.72 mmol) was added And stirred under argon for 10 minutes. To this solution was added sublimed 1H-tetrazole (0.52 g, 7.4 mmol) and stirred overnight. The solvent was evaporated and the residue was triturated with 70% EtOAc / ether and filtered. The filtrate was evaporated, N ⁴ - Benzoyl-5'-O - dimethoxytrityl -3 '- [O- (2'- O- acetyl) -N ⁶ - benzoyl le-beta-D-3'-deoxy adenovirus sheet sulfonyl] -2'-Deoxy-β-L-cytidine-cyanoethyl phosphite was obtained as a foam and used in the next step without further purification.

Dimer N ⁴ -benzoyl-5′-O-dimethoxytrityl-3 ′-[O- (2′-O-acetyl) -N ⁶ -benzoyl-β-D-3 ′-'deoxyade Nosinyl-2′-deoxy-β-L-cytidine-cyanoethyl phosphite was dissolved in THF (24 ml), pyridine (6 ml) and water (0.6 ml). Crystals of iodine (0.55 g) were added partially until the iodine color fixed. The reaction mixture was stirred for a further 15 minutes and excess iodine was removed by the addition of saturated sodium thiosulfate. The solvent was evaporated and the residue was dissolved in EtOAc, and washed with water, NaHCO ₃ and brine. The EtOAc layer was evaporated and the residue was treated with 80% acetic acid / water solution (50 ml)
And stirred for 1 hour. The solvent is then evaporated and the crude product is purified on a silica gel column using 5-10% MeOH / CHCl ₃ as solvent to give the pure compound (2′-acetoxy-N ⁶ -benzoyl-3 ′). -Deoxy-β-D-adenosinyl) -N ⁴ -benzoyl-β-L-2′-deoxycytidinyl cyanoethyl phosphate (1.46 g) was obtained as a foam.

3′-O- (3′deoxy-β-D-adenosinyl) -β-L-2′-deoxycytidine (6) (L-153) Dimer (2′-acetoxy-N ⁶⁾ -Benzoyl-3'-deoxy-β-D-adenosinyl) -N ⁴ -benzoyl-β-L-2'-deoxycytidinyl cyanoethyl phosphate (1.46 g) in ammonium hydroxide solution (100
ml) overnight. The solvent was evaporated, and the residue using a gradient of NH ₄ H CO ₃ buffer (0.05~0.2M) was purified by DEAE cellulose ion exchange column. The pure fractions were collected and lyophilized, pure 3′-O- (3′-deoxy-β-D-adenosinyl) -β-L-2′-deoxycytidine (L-153) (0 .81 g) as a white solid.

Example 16 Synthesis of α-L-dC, Cordycepin Dimer (L-154) A solution of α-L-dC (1.6 g, 4.88 mmol) in pyridine (100 ml) was prepared. 4,4'-dimethoxytrityl chloride (2.43 g, 7.2 mmol
) And DMAP (0.13 g, 1.04 mmol) were added and stirred under argon for 2 hours at room temperature. To complete the reaction, additional DMTCl (
1.0 g) was added and stirred for another 2 hours. The reaction was quenched by the addition of MeOH (5 ml) and the solvent was evaporated. This residue was dissolved in EtOAc and washed with water, NaHCO ₃ and brine. After drying over Na ₂ SO ₄ , the EtOAc layer was evaporated and the crude compound was purified with 3-5% M as solvent.
Purify on a silica gel column using OH / CHCl ₃ and purify pure N ⁴ -benzoyl-5′-O- (di-p-methoxytrityl) -2′-deoxy-α-L-cytidine (2.34 g, 76 %) As a pale yellow foam.

N ⁴ -benzoyl-5′-O- (di-p-methoxytrityl) -2′-deoxy-α-L-cytidine (1.84 g, 2.9 mmol) was treated with anhydrous dichloromethane (
50 ml). N, N-diisopropylethylamine (2.0 ml, 1
1.6 mmol) under argon and then 2'-cyanoethyl-N, N
-Diisopropylchlorophosphoramidite (0.85 ml, 3.5 mmol)
Was added. The reaction was stirred for 30 minutes and the solvent was evaporated.
The residue was dissolved in _{80% EtOAc / Et 3 N (} 75ml), and washed with water, NaH CO ₃ and brine. It was evaporated organic layer, and EtO Ac, a mixture of hexane and Et ₃ N (50:40:10) using purified by short silica Kagerukaramu, N ⁴ - Benzoyl-5'-O-(dimethoxytrityl) - α-L-2′-Deoxycytidine-3′-N, N-diisopropylcyanoethyl phosphoramidite (2.01 g, 83%) was obtained as a foam.

The compound N ⁴ -benzoyl-5′-O- (dimethoxytrityl) -α-L-2′-deoxycytidine-3′-N, N-diisopropylcyanoethyl phosphoramidite (2 ml) in anhydrous acetonitrile (100 ml) .01 g, 2.4 mmol)
To a solution of N ⁶ -benzoyl-2′-O-acetoxy-β-D-3′-deoxyadenosine (1.05 g, 2.65 mmol) in acetonitrile (40 ml).
Was added and stirred for 10 minutes under argon. The sublimated 1H-
Tetrazole (0.5 g, 7.2 mmol) was added and stirred overnight. The solvent was evaporated and the residue was triturated with 70% EtOAc / ether and filtered. The filtrate was evaporated, N ⁴ - Benzoyl-5'-O-(dimethoxytrityl) -3 '- [O- (2'- acetyl) -N ⁶ - benzoyl-beta-D-3'-deoxy adenovirus sheet sulfonyl] -2'-Deoxy-α-L-cytidine-cyanoethyl phosphite ester was obtained as a foam, which was used in the next step without purification.

The dimer N ⁴ -benzoyl-5′-O- (dimethoxytrityl-3 ′-[O- (2′-acetyl) -N ⁶ -benzoyl-β-D-3′-deoxyadenosine) Nyl] -2'-deoxy-α-L-cytidine-cyanoethyl phosphite ester was dissolved in THF (24 ml), pyridine (6 ml) and water (0.6 ml) .Iodine crystals (0.5 g) were dissolved in iodine. The reaction mixture was stirred for a further 15 minutes and the excess iodine was removed by addition of saturated sodium thiosulfate. The sulfate was evaporated and the residue was evaporated with EtOAc. And washed with water, NaHCO ₃ and brine The EtOAc layer was evaporated and the residue was treated with 80% acetic acid / water solution (50 ml).
) And stirred for 1 hour. The solvent was then evaporated and the crude product was purified on a silica gel column using 5-10% MeOH / CHCl ₃ as solvent to give pure (2′-acetoxy-N ⁶ -benzoyl-3′-). Deoxy-β-D-adenosinyl) -N ⁴ -benzoyl-α-L-2-deoxycytidinyl cyanoethyl phosphate (1.8 g) was obtained as a foam.

The dimer (2′-acetoxy-N ⁶ -benzoyl-3′-deoxy-β-D-adenosinyl) -N ⁴ -benzoyl-α-L-2-deoxycytidinyl cyanoethyl phosphate ( 1.8 g) in an ammonium hydroxide solution (100 ml).
) Overnight. The solvent was evaporated, and the residue using a gradient of NH ₄ HCO ₃ buffer (0.05~0.2M), DEAE Cellulose
Purified on an ion exchange column. The pure fractions are collected and lyophilized,
Pure 3′-O- (3′deoxy-β-D-adenosinyl) -α-L-2′-deoxycytidine (L-154) (1.08 g) was obtained as a white solid.

Example 17 Synthesis of α-L-dA, Cordycepin Dimer (L-155) 2′-Deoxy-α-L-adenosine (75 ml) in pyridine cooled in an ice bath To a stirred solution of 2.05 g, 8.16 mmol) was added ClSiMe ₃ (5.17 ml, 40.8 mmol) dropwise and stirred for 30 minutes. Then benzoyl chloride (4.7 ml, 40.8 mmol) was added dropwise and the reaction mixture was stirred for 2 hours at room temperature. This was cooled in an ice bath and water (15 ml
) Was added dropwise, and 15 minutes later, concentrated NH ₄ OH (15 ml) was added to obtain an about 2 M ammonia solution. After 30 minutes, the solvent was evaporated and the residue was dissolved in water and washed with ether. The aqueous layer was concentrated and N ⁶ -benzoyl-2′-deoxy-α-L-adenosine was crystallized from water as a white solid (2.
48 g, 85.8%).

Compound N in pyridine (100 ml)⁶Benzoyl-2'-deoxy-α-L-adenosine (2.48 g, 6.98 mmol) in 4,4'-dimethoxy
Citrityl chloride (3.55 g, 10.47 mmol) and DMAP (0
. 25g, 2.09mmol) and stirred at room temperature under argon for 2 hours.
Stirred. To complete the reaction, additional DMTCl (1.3 g) was added and
And stirred for an additional 2 hours. The reaction was quenched by the addition of MeOH (5 ml).
And the solvent was evaporated. This residue was dissolved in EtOAc, water, Na
HCO_ThreeAnd washed with brine. Na_TwoSO_FourAfter drying with EtOAc, the EtOAc layer was evaporated and the crude compound was removed as solvent with 3-5% MeOH / CHCl _Three And purified on a silica gel column using pure N⁶-Benzoyl-5'-O- (di
-P-methoxytrityl) -2'-deoxy-α-L-adenosine (3.42 g)
, 74.5%) as a pale yellow foam.

Dissolve N ⁶ -benzoyl-5′-O- (di-p-methoxytrityl) -2′-deoxy-α-L-adenosine (1.64 g, 2.5 mmol) in anhydrous dichloromethane (50 ml). did. N, N-diisopropylethylamine (1.75 ml
, 10.0 mmol) is added under argon and then 2'-cyanoethyl-N
, N-diisopropylchlorophosphoramidite (0.8 ml, 3.25 mmol
l) was added. The reaction was stirred for 30 minutes and the solvent was evaporated. The residue was dissolved in 80% EtOAc / Et ₃ N (75 ml) and washed with water, NaHCO ₃ and brine. The organic layer was evaporated and purified on a short silica gel column using a mixture of EtOAc, CH ₂ Cl ₂ and Et ₃ N (40:50:10) to give N ⁶ -benzoyl-5 in quantitative yield. '-O- (dimethoxytrityl) -α-L-2'-deoxyadenosine-3'-N, N-diisopropylcyanoethyl phosphoramidite was obtained.

N ⁶ -Benzoyl-5′-O- (dimethoxytrityl) -α-L-2′-deoxyadenosine-3′-N, N-diisopropylcyanoethyl phosphoramidite in anhydrous acetonitrile (60 ml). 5 mmol), N ⁶ -benzoyl-3′-O-acetoxy-β-D-2′-deoxyadenosine (0.94 g, 2.36 mmol) in acetonitrile (40 ml) is added and under argon. Stir for 10 minutes. To this solution, sublimated 1H-tetrazole (0.
(5 g, 7.2 mmol) was added and stirred overnight. The solvent was evaporated and the residue was triturated with 70% EtOAc / ether and filtered. The filtrate is evaporated to give N ⁶ -benzoyl-5′-O-dimethoxytrityl-3 ′-[O- (2 ′)-acetyl) -N ⁶ -benzoyl-β-D-3′-deoxyadenosinyl]- The 2′-deoxy-α-L-adenosine cyanoethyl phosphite ester was obtained as a foam and was used in the next step without further purification.

The dimer N ⁶ -benzoyl-5′-O-dimethoxytrityl-3 ′-[O- (2 ′-)-acetyl) -N ⁶ -benzoyl-β-D-3′-deoxyadenoadeno [Cinyl] -2'-deoxy-α-L-adenosine cyanoethyl phosphite ester was dissolved in THF (24 ml), pyridine (6 ml) and water (0.6 ml). Crystals of iodine (0.63 g) were added partially until the iodine color fixed. The reaction mixture was stirred for a further 15 minutes and excess iodine was removed by the addition of saturated sodium thiosulfate. The solvent was evaporated and the residue was dissolved in EtOAc, and washed with water, NaHCO ₃ and brine. The EtOAc layer was evaporated and the residue was treated with 80% acetic acid / water solution (50 m
1) and stirred for 1 hour. The solvent was then evaporated and the crude product was purified on a silica gel column using 5-10% MeOH / CHCl ₃ as solvent to give the pure compound (2′-acetoxy-N ⁶ -benzoyl-3). '-Deoxy-β-D-adenosinyl) -N ⁶ -benzoyl-α-L-2'-deoxyadenosinyl cyanoethyl phosphate (0.97 g) was obtained as a foam.

The dimer (2′-acetoxy-N ⁶ -benzoyl-3′-deoxy-β-D-adenosinyl) -N ⁶ -benzoyl-α-L-2′-deoxyadenosynyl cyanoethyl phosphate The ester (0.97 g) was added to an ammonium hydroxide solution (10
0 ml) overnight. The solvent was evaporated, and the residue using a gradient of NH ₄ HCO ₃ buffer (0.05~0.2M) was purified by DEAE Cellu lose ion exchange column. The pure fractions were collected and lyophilized as pure white as pure {3} [3 ']-O- (3'-deoxy-β-D
-Adenosinyl) -β-L-2′-deoxyadenosine (0.55 g) (L-1
55) was obtained.

Example 18 Synthesis of β-L-dA, β-D-dA (L-210) 2′-Deoxy-β-L-adenosine in pyridine (75 ml) cooled in an ice bath (2.05 g, 8.16 mmol) to a stirred solution of ClSiMe ₃ (5.17 ml, 40.8 mmol) was added dropwise and stirred for 30 minutes. Then benzoyl chloride (4.7 ml, 40.8 mmol) was added dropwise and the reaction mixture was stirred for 2 hours at room temperature. This was cooled in an ice bath and water (15 ml
) Was added dropwise, and after 15 minutes, concentrated NH ₄ OH (15 ml) was added to obtain an about 2 M ammonia solution. After 30 minutes, the solvent was evaporated and the residue was dissolved in water and washed with ether. The aqueous layer was concentrated and N ⁶ -benzoyl-2′-deoxy-β-L-adenosine was crystallized from water as a white solid (2.48 g, 85.8%).

N in pyridine (100 ml)⁶-Benzoyl-2'-deoxy-β-L-adenosine (2.48 g, 6.98 mmol) was added to a solution of 4,4′-dimethoxytri
Tyl chloride (3.55 g, 10.47 mmol) and DMPA (0.25 g)
g, 2.09 mmol) and stirred at room temperature under argon for 2 hours
. To complete the reaction, additional DMTCl (1.3 g) was added and
The mixture was further stirred for 2 hours. The reaction was quenched by the addition of MeOH (5 ml) and
The solvent was evaporated. This residue was dissolved in EtOAc, water, NaHCO _Three And washed with brine. Na_TwoSO_FourAfter drying with EtOAc, the EtOAc layer was evaporated and the crude compound was removed as solvent with 3/5% MeOH / CHCl_ThreePurified on silica gel column using pure N⁶-Benzoyl-5'-O- (di-p-methoxytrityl) -2'-deoxy-β-L-adenosine (3.42 g, 7
4.5%) as a pale yellow foam.

Dissolve N ⁶ -benzoyl-5′-O- (di-p-methoxytrityl) -2′-deoxy-β-L-adenosine (1.71 g, 2.61 mmol) in anhydrous dichloromethane (50 ml). did. N, N-diisopropylethylamine (1.8 ml
, 10.34 mmol) are added under argon and then 2'-cyanoethyl-
N, N-diisopropylchlorophosphoramidite (1.0 ml, 4.47 mm
ol) was added. The reaction was stirred for 30 minutes and the solvent was evaporated. The residue was dissolved in 80% EtOAc / Et ₃ N (75 ml) and washed with water, NaHCO ₃ and brine. Was evaporated organic layer, and EtOAc, the purified short have a silica gel column using a mixture of hexane and _{Et 3 N (50:40:10), N} 6 as a foam - benzoyl-5'-O-( Dimethoxytrityl) -β-L-2′-deoxyadenosine-3′-N, N-diisopropylcyanoethyl phosphoramidite (1.87 g, 84%) was obtained.

N ⁶ -Benzoyl-5′-O- (dimethoxytrityl) -β-L-2′-deoxyadenosine-3′-N, N-diisopropylcyanoethyl phosphoramidite (1. 87 g (2.18 mmol) of a solution of N ⁶ -benzoyl-3′-O-acetoxy-β-D-2′-deoxyadenosine (0.95 g, 2.4 mmol) in acetonitrile (40 ml). And stirred under argon for 10 minutes. To this solution was added sublimed 1H-tetrazole (0.46 g, 6.6 mmol) and stirred overnight. The solvent was evaporated and the residue was triturated with 70% EtOAc / ether and filtered. The filtrate was evaporated and N ⁶ -benzoyl-5′-O-dimethoxytrityl-3 ′-[O- (3′-O′acetyl) -N ⁶ -benzoyl-β-D-2′-deoxy as a foam. Adenosinyl] -2'-deoxy-β-
The L-adenosine cyanoethyl phosphite ester was obtained and used for the next step without further purification.

N, which is a dimer⁶-Benzoyl-5'-O-dimethoxytrityl-3 '-[O- (3'-O'-acetyl) -N⁶-Benzoyl-β-D-2'-deoxyadenosinyl] -2'-deoxy-β-L-adenosine cyanoethyl phosphite
The stell was added to THF (24 ml), pyridine (6 ml) and water (0.6 ml).
Dissolved. Add iodine crystals (0.5 g) partially until the iodine color is fixed
did. The reaction mixture is stirred for a further 15 minutes and the excess iodine is
Removed by addition of sodium osulphate. The solvent was evaporated and the residue
Dissolve the distillate in EtOAc and add water, NaHCO_ThreeAnd washed with brine. The EtOAc layer was evaporated and the residue was treated with 80% acetic acid / water solution (50 m
1) and stirred for 1 hour. The solvent is then evaporated, and
The crude product was treated with 5-10% MeOH / CHCl as solvent_ThreeAnd purified as a foam using the pure compound (3'-acetoxy-N ⁶ -Benzoyl-2'-deoxy-β-D-adenosinyl) -N⁶-Benzoyl-
β-L-2′-deoxyadenosinyl cyanoethyl phosphate (0.13 g
) Got.

Dimer (3′-acetoxy-N ⁶ -benzoyl-2′-deoxy-β-D-adenosinyl) -N ⁶ -benzoyl-β-L-2′-deoxyadenosine cyanoethyl phosphate The ester (1.3 g) was added to an ammonium hydroxide solution (100 g).
ml) overnight. The solvent was evaporated and the residue using a gradient of NH ₄ HC O ₃ buffer (0.05~0.2M), and purified by DEAE cellulo se ion exchange column. The pure fractions are collected and lyophilized to give pure 3'-O- (2'deoxy-β-D-adenosinyl) as a white solid.
-Β-L-2'-deoxyadenosine (L-210) (0.640 g) was obtained.

[0211] All patents and publications mentioned herein are indicators of the level of ordinary skill in the art to which this invention belongs. All patents and publications are herein specifically and incorporated by reference to the same extent as if each individual publication was individually and individually indicated.

Those skilled in the art will readily appreciate that the present invention is well suited to carrying out the objects and obtains the objects and advantages mentioned as well as the particulars herein. The dimers, pharmaceutical compositions, treatments, methods, procedures and techniques described herein are presently representative of preferred embodiments and are intended to be exemplary and not limiting the scope. Not intended. Variations and other uses herein will occur to those skilled in the art, which are encompassed within the spirit of the invention or defined by the appended claims. Certain features of the present invention may be exaggerated on scale or depicted schematically according to conventional practice in the field of biochemistry.

[Brief description of the drawings]

FIG. 1 is a schematic diagram of an example of a dinucleotide dimer of the present invention.

FIG. 2 is a schematic diagram of an example of a synthesis scheme according to the present invention.

FIG. 3 is a schematic diagram of an example of a synthesis scheme according to the present invention.

FIG. 4 is a schematic diagram of an example of a synthesis scheme according to the present invention.

FIG. 5 is a schematic diagram of an example of a synthesis scheme according to the present invention.

FIG. 6 is a schematic diagram of an example of a synthesis scheme according to the present invention.

FIG. 7 is a schematic diagram of an example of a synthesis scheme according to the present invention.

FIG. 8 is a schematic diagram of an example of a synthesis scheme according to the present invention.

FIG. 9 is a schematic diagram of an example of a synthesis scheme according to the present invention.

FIG. 10 is a schematic diagram of an example of a synthesis scheme according to the present invention.

FIG. 11 is a schematic diagram of an example of a synthesis scheme according to the present invention.

FIG. 12 is a schematic diagram of an example of a synthesis scheme according to the present invention.

FIG. 13 is a schematic diagram of an example of a synthesis scheme according to the present invention.

FIG. 14 is a schematic diagram of an example of a synthesis scheme according to the present invention.

FIG. 15A is a schematic diagram of an example of a dinucleoside phosphate dimer containing an alternative backbone.

FIG. 15B is a schematic diagram of an example of a dinucleoside phosphate dimer containing an alternative backbone.

FIG. 16A is a schematic diagram of the dinucleoside phosphate dimer used in the examples.

FIG. 16B is a schematic diagram of the dinucleoside phosphate dimer used in the examples.

FIG. 16C is a schematic diagram of the dinucleoside phosphate dimer used in the examples.

FIG. 16D is a schematic diagram of the dinucleoside phosphate dimer used in the examples.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 31/12 A61P 31/12 33/00 33/00 33/06 33/06 35/00 35/00 C07H 19/067 C07H 19/067 19/073 19/073 19/167 19/167 19/173 19/173 19/207 19/207 (31) Priority claim number 09/220, 307 (32) Priority date Heisei 10 December 23, 1998 (December 23, 1998) (33) Priority Claimed States United States (US) (81) Designated States EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG) , AP (GH, GM, KE, LS, MW, SD, SL, SZ, UG, Z W), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN , CU, CZ, DE, DK, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL , TJ, TM, TR, TT, UA, UG, UZ, VN, YU, ZW (72) Inventor Weiss, Alexander El. United States Texas 78230, San Antonio, Old Wick Road 12718 (72) Inventor Purentilan, Kilpasabi United States Texas 78251, San Antonio, Village Parkway 2514 (72) Inventor Jero, Annette M. Australia New South Wales 2090, Cremon, Rangers Road 4F Term (reference) 4C057 LL08 LL18 LL19 LL27 LL41 LL42 LL44 LL46 4C086 AA01 AA02 AA03 EA16 MA01 MA04 NA14 ZB26 ZB32 ZB33 ZB35 ZB

Claims (83)

[Claims] 1. A method for treating a viral, bacterial, fungal, cancer or parasite infection in a mammal, the method comprising the following formula: Or a pharmaceutically acceptable salt thereof, wherein B ₁ and B ₂ are each a group consisting of β-D, β-L and α-L nucleosides And at least one of B ₁ or B ₂ must be an L-nucleoside; R ₁ and R ₂ are purine or pyrimidine bases; and wherein R ₁ and R ₂ are The same base or a different base, wherein B ₁ or B ₂ binds to an internucleotide binding agent (IBA) and the B ₁ or B ₂ is an L-nucleoside, the R ₁ or R ₂ bonded to can not be a cytosine; and IBA is phosphodiester, phosphorothioate, methoxy phosphotriester, methylphosphonate, phosphoro Thioate, phosphorothioate, silyl ethers are selected from the linking group consisting of sulfonates and ethylenedioxy ether method. 2. The compound according to claim 1, wherein the compound is as follows: 3′-O- (α-L-5-fluoro-2′-deoxyuridinyl) -β-D-5
-Fluoro-2'-deoxyuridine (L-102); 3'-O-([beta] -D-5-fluoro-2'-deoxyuridinyl)-[alpha] -L-5.
-Fluoro-2'-deoxyuridine (L-103); 3'-O-([beta] -D-5-fluoro-2'-deoxyuridinyl)-[alpha] -L-2.
'-Deoxyuridine (L-107); 3'-O- (α-L-5-fluoro-2'-deoxyuridinyl) -α-L-5
-Fluoro-2'-deoxyuridine (L-108); 3'-O-([beta] -L-5-fluoro-2'-deoxyuridinyl)-[beta] -L-5.
-Fluoro-2'-deoxyuridine (L-109); 3'-O-([beta] -D-5-fluoro-2'-deoxyuridinyl)-[beta] -L-5.
-Fluoro-2'-deoxyuridine (L-110); 3'-O-([beta] -D-5-fluoro-2'-deoxyuridinyl)-[alpha] -L-2.
3′-O- (β-D-5-fluoro-2′-deoxyuridinyl) -2′-deoxy-β-L-cytidine (L-113); 3′-deoxycytidine (L-111); '-O- (2'-deoxy-β-L-cytidinyl) -β-D-5-fluoro-
2′-deoxyuridine (L-114); 3′-O- (2′-deoxy-α-L-cytidinyl) -β-D-5-fluoro-
2′-deoxyuridine (L-115); 3′-O- (β-D-5-fluoro-2′-deoxyuridinyl) -β-L-2
'-Deoxyuridine (L-117); 3'-O- (β-L-5-fluoro-2'-deoxyuridinyl) -α-L-5
-Fluoro-2'-deoxyuridine (L-119); 3'-O-([beta] -D-5-fluoro-2'-deoxyuridinyl)-[alpha] -L-5.
-Fluoro-2'-deoxyuridine (3 ', 3') (L-122); 3'-O- (3'-deoxy-β-D-adenosinyl) -β-L-2'-deoxyuridine (L 3′-O- (3′-deoxy-β-D-adenosinyl) -β-L-2′-deoxyadenosine (L-151); 3′-O- (3′-deoxy-β- D-adenosinyl) -α-L-2′-deoxyuridine (L-152); 3′-O- (3′-deoxy-β-D-adenosinyl) -β-L-2′-deoxycytidine (L- 153); 3′-O- (3′-deoxy-β-D-adenosinyl) -α-L-2′-deoxycytidine (L-154); 3′-O- (3′-deoxy-β-D) -Adenosinyl) -β-L-2′-deoxyadenosine (L-155); 3′-O- (2′-deoxy-β-D-adenosine) Le) is-beta-L-2'-deoxyadenosine (L-210), or is selected from the group consisting of or a pharmaceutically acceptable salt thereof, The method according to claim 1 3. The formula is I, B ₁ is β-D, B ₂ is α-L, R ₁ and R ₂ are both 5FUdR, and IBA is a phosphodiester. The method of claim 1. 4. The formula is II, B ₁ is α-L, B ₂ is β-D, R ₁ and R ₂ are both 5FUdR, and IBA is a phosphodiester. The method of claim 1. 5. The method according to claim 1, wherein the compound is 3′-O- (β-D-5-fluoro-2′-
Deoxyuridinyl) -α-L-5-fluoro-2′-deoxyuridine (L-
103) The method according to claim 1, wherein the method is (103). 6. The method of claim 1, wherein said IBA is a phosphodiester linking group. 7. The following formula: Or a pharmaceutically acceptable salt thereof, wherein R ₁ is —CH ₂ OH in the L configuration; R ₂ and R ₃ are from the group consisting of —H or —OH And X is a nitrogenous base selected from the group consisting of purines and pyrimidines;
A compound or a pharmaceutically acceptable salt thereof. 8. The compound of claim 7, wherein R ₂ and R ₃ are each —OH, and X is adenine. 9. The method according to claim 1, wherein R ₃ is equatorial, wherein R ₂ is axially oriented and the adenine is axially oriented,
A compound according to claim 8. 10. The compound according to claim 7, wherein R ₂ is —OH, R ₃ is —H, and X is 5-fluorouracil. 11. The compound according to claim 10, wherein R ₂ is axially oriented and 5′-fluorouracil is axially oriented. 12. The compound of claim 7, wherein R ₂ and R ₃ are each —OH, and X is guanine. 13. The compound of claim 12, wherein said R ₂ and R ₃ are each axially oriented and said guanine is equatorially oriented. 14. The compound of claim 7, wherein R ₂ and R ₃ are each —OH, and X is adenine. 15. The compound of claim 14, wherein said R ₂ and R ₃ are each axially oriented and said adenine is equatorially oriented. 16. The compound of claim 7, wherein R ₂ and R ₃ are each —OH, and X is inine. 17. The compound of claim 16, wherein said R ₂ and R ₃ are each axially oriented and said inine is equatorially oriented. 18. The compound according to claim 7, wherein R ₂ and R ₃ are each —OH, and X is mercaptoguanine. 19. The compound of claim 18, wherein said R ₂ and R ₃ are each axially oriented and said mercaptoguanine is equatorially oriented. 20. The compound of claim 7, wherein said R ₂ is -OH, said R ₁ is -H, and X is adenine. 21. The compound of claim 20, wherein said R ₂ is axially oriented and said adenine is equatorially oriented. 22. The compound of claim 7, wherein said R ₂ is -OH, said R ₁ is -H, and X is deoxyinine. 23. The compound of claim 22, wherein said R ₂ is axially oriented and said deoxyinosine is attached to a β hydrogen on a ribose ring. 24. wherein R ₂ is —OH, and R ₃ is —OH;
The compound according to claim 7, wherein X is adenine. 25. The compound of claim 24, wherein said R ₂ and R ₃ are each axially oriented and said adenine is axially oriented. 26. The method of claim 26, wherein R ₂ is —OH, R ₃ is —H, and X is 3-aminopyrine, and the point of attachment of the aminopurine to the ribose ring is hydrogen. The compound according to claim 7, which is 3. 27. The compound of claim 26, wherein said R ₂ is axially oriented and said aminopurine is axially oriented. 28. The compound of claim 7, wherein said R ₂ is -OH, and said R ₃ is -H, and X is guanine. 29. The compound of claim 28, wherein said R ₂ is axially oriented and said guanine is equatorially oriented. 30. The compound of claim 7, wherein R ₂ and R ₃ are each —H, and X is adenine. 31. The compound of claim 30, wherein said adenine is equatorially oriented. 32. The compound of claim 7, wherein R ₂ and R ₃ are each —H, and X is adenine. 33. The adenine is axially oriented.
The compound according to the above. 34. The compound of claim 7, wherein R ₂ and R ₃ are each —OH, and X is 6-thiopurine. 35. The method of claim 34, wherein said R ₂ and R ₃ are each axially oriented and said 6-thiopurine is equatorially oriented.
The compound according to the above. 36. The compound of claim 7, wherein R ₂ is —OH, R ₃ is —H, and X is 5-fluorouracil. 37. The compound of claim 36, wherein said R ₂ is equatorially oriented and said 5-fluorouracil is attached to the α-hydrogen of the ribose ring. 38. The compound of claim 7, wherein R ₂ and R ₃ are each —OH, and X is 5-fluorouracil. 39. The compound of claim 38, wherein said R ₂ is axially oriented and said 5′-fluorouracil is axially oriented. 40. A nucleoside dimer having the formula: R ₁ -XR ₂ ,
Here, X is located in a suitable part for chemically linking the R ₁ and R _2; R ₁ and R ₂ are nucleosides and R ₁ and R _2, via the -OH group A nucleoside dimer linked to X. 41. The compound of claim 40, wherein said X is selected from the group consisting of PO ₄ and S = PO ₃ . 42. The compound of claim 40, wherein R ₁ is β-D-deoxyfluorouridine. 43. The compound of claim 40, wherein said R ₁ is α-L-deoxyfluorouridine. 44. The compound of claim 40, wherein said R ₁ is β-L-deoxyfluorouridine. 45. The compound of claim 40, wherein R ₁ is α-L-deoxycytosine. 46. The compound according to claim 40, wherein said R ₁ is β-L-deoxycytosine. 47. The compound of claim 40, wherein R ₁ is β-L-deoxyuridine. 48. The compound of claim 40, wherein said R ₁ is β-L-deoxyguanosine. 49. The compound of claim 40, wherein said R ₁ is β-L-deoxyadenosine. 50. The compound according to claim 40, wherein said R ₁ is α-L-deoxyadenosine. 51. The compound according to claim 40, wherein said R ₁ is nitrobenzylthionosin. 52. A nucleoside dimer comprising β-D-deoxyfluorouridine, β-L-adenosine, and a suitable moiety for linking the two nucleosides. 53. Nitrobenzylthionosin. 54. The following non-stereospecific formula: Wherein R ₁ and R ₂ are each (CH ₃ COSCH ₂ CH ₂ O) ₂ P ＝ O or-
H; and X is a purine or a pyrimidine. 55. R ₁ is _{(CH 3 COSCH 2 CH 2 O} ) 2 P = O, R ₂ is -H, wherein -OR ₁ is oriented in Equatorial, -OR ₂ is ,
55. The compound of claim 54, wherein the compound is axially oriented and -X is axially oriented. 56. R ₁ is —H and R ₂ is (CH ₃ COSCH ₂ CH ₂ O) ₂ P
= O, where -OR ₁ is equatorially oriented and -OR ₂ is
55. The compound of claim 54, wherein the compound is axially oriented and -X is axially oriented. 57. Each of R ₁ and R ₂ is (CH ₃ COSCH ₂ CH ₂ O) ₂ P = O, wherein -OR ₁ is equatorially oriented and -OR ₂ is axially 55. The compound of claim 54, wherein the compound is oriented and -X is axially oriented. 58. R ₁ is —H and R ₂ is (CH ₃ COSCH ₂ CH ₂ O) ₂ P
= 0, where -OR ₁ is axially oriented, -OR ₂ is equatorially oriented, and -X is equatorially oriented,
A compound according to claim 54. 59. R ₁ and R ₂ are each (CH ₃ COSCH ₂ CH ₂ O) ₂ P ＰO, wherein -OR ₁ is axially oriented and -OR ₂ is equatorially 55. The compound of claim 54, wherein the compound is oriented and -X is equatorially oriented. 60. R ₁ is —H and R ₂ is (CH ₃ COSCH ₂ CH ₂ O) ₂ P
55. The compound of claim 54, wherein ＝ O, wherein -OR ₁ is axially oriented, -OR ₂ is equatorially oriented, and -X is axially oriented. 61. R ₁ and R ₂ are each (CH ₃ COSCH ₂ CH ₂ O) ₂ P ＝ O, wherein -OR ₁ is axially oriented and -OR ₂ is equatorially 55. The compound of claim 54, wherein the compound is oriented and -X is axially oriented. 62. Formula: A compound having a R ₁ -X-R _2, wherein, R ₁ is selected from the group consisting of purine and pyrimidine; R _{2 is, (CH 3 COSCH 2 CH 2} O X) is a suitable linking group; ₂ ) P = O; 63. A compound having the formula: R ₁ -XR ₂ , wherein R ₁ is selected from the group consisting of purines and pyrimidines; R ₂ is selected from the group consisting of purines and pyrimidines And X is a suitable linking group; 64. A method for treating Plasmodium falciparum infection, comprising administering to a diseased mammal a therapeutic dose of a compound of claims 7-63. 65. A method for treating Plasmodium falciparum infection, comprising administering to a diseased mammal a therapeutically effective amount of a compound of the formula R1-X-R2.
A compound having, X is located in a suitable part for chemically linking the R ₁ and R _2; R ₁ and R ₂ are nucleosides and R ₁ and R _2, -OH Administering a compound linked to X via a group. 66. The method of claim 64, wherein said X is selected from the group consisting of PO ₄ and S = PO ₃ . 67. The method of claim 64 or claim 65, wherein said R ₁ is β-D-deoxyfluorouridine. 68. The method of claim 64 or claim 65, wherein said R ₁ is α-L-deoxyfluorouridine. 69. The method of claim 64 or claim 65, wherein said R ₁ is β-L-deoxyfluorouridine. 70. The method of claim 64 or claim 65, wherein said R ₁ is α-L-deoxycytosine. 71. The method according to claim 64, wherein said R ₁ is β-L-deoxycytosine. 72. The method of claim 64 or 65, wherein R ₁ is β-L-deoxyuridine. 73. The method according to claim 64 or 65, wherein R ₁ is β-L-deoxyguanosine. 74. The method of claim 64 or 65, wherein said R ₁ is β-L-deoxyadenosine. 75. The method of claim 64 or 65, wherein R ₁ is α-L-deoxyadenosine. 76. The method of claim 64 or claim 65, wherein said R ₁ is nitrobenzylthionosin. 77. A method for treating a disease in a mammal, comprising administering to the affected mammal a therapeutically effective amount of deoxyadenosine or dideoxyadenosine. 78. The method of claim 77, wherein said disease is cancer. 79. The method of claim 78, wherein said disease is malaria. 80. The method of claim 79, wherein said disease is caused by a viral infection. 81. The method of claim 79, wherein said disease is caused by a bacterial infection. 82. The method of claim 79, wherein said disease is caused by a parasitic infection. 83. The method of claim 79, wherein said disease is caused by a fungal infection.