EA040630B1 - NEW DERIVATIVES OF 2' AND/OR 5' AMINO ACID ESTERS OF 3'-DEOXYADENOSINE PHOSPHOROAMIDATES AS ANTI-CANCER COMPOUNDS - Google Patents

Description

The present invention relates to chemical compounds, to compounds for use in a method of treatment, in particular in a method for the prevention or treatment of cancer, to a method for preparing the compounds, and to pharmaceutical compositions containing said compounds.

In particular, although not exclusively, the present invention relates to chemical compounds for use in the treatment of leukemia, lymphoma and/or solid tumors in homo sapiens.

Cordycepin is a 3'-deoxyadenosine (3'dA). It is the nucleoside analog of adenosine, which lacks a 3'-hydroxyl group on the ribose moiety.

Cordycepin is one of the main bioactive substances produced by Cordyceps militaris, a parasitic fungus used in traditional Chinese medicine for its immune-activating, anti-aging and anti-cancer effects. See reference, Tuli, H. S. et al. 3 Biotech (2014) 4:1-12.

Cordycepin can be obtained synthetically from adenosine. For reference to such synthetic methods, see Robins, J. R. et al. J. Org. Chem. 1995, 60, 7902-7908 and Aman, S. et al. Organic Process Research & Development 2000, 4, 601-605.

Cordycepin has been most widely studied as an anti-cancer agent.

Due to their structure, 3'dA and its triphosphate form may possibly interfere with a process that requires the participation of adenosine or adenosine triphosphate (ATP), respectively.

However, after administration, 3'dA is rapidly deaminated by adenosine deaminase (ADA) and rapidly metabolized to the inactive metabolite 3'-deoxyinosine in vivo. See reference to Tsai, Y-J et al. J. Agri. food chem. 58 4638-43 (2010).

As described in Glazer, R. et al. Cancer Research 38, 2233-2238 (1978), cordycepin has been shown to have anticancer activity when used in combination with an adenosine deaminase inhibitor such as pentostatin (2-deoxycoformycin, dCF). Other ADA inhibitors have also been proposed as alternative co-medications for administration with cordycepin, but this combination of 3'dA-dCF has been used in clinical trials. However, as recognized by Wehbe-Janek, H. et al. Anticancer Research 27: 3143-3146 (2007), 2deoxycoformycin is known to be a relatively toxic drug.

2-fluorocordycepin (3'-deoxy-2-fluoroadenosine) is also known to be cytotoxic (see, for example, Montgomery et al., J. Med. Chem., 1969, 12(3), 498-504 and Dickinson et al, J. Med. Chem., 1967, 10(6), 1165-1166).

2-chlorocordycepin (3'-deoxy-2-fluoroadenosine) has been evaluated for its antiviral activity (Rosowsky et al. J. Med. Chem., 1989, 32, 1135-40).

The aim of the present invention is to solve the problem of increasing the efficacy of purine-based 3'deoxynucleosides, exemplified by cordycepin (3'-deoxyadenosine), when used in a prophylactic or treatment method, in particular, though not only, in anticancer chemotherapy, including chemotherapy in the treatment of leukemia, lymphoma and/or solid tumors.

Another object of the present invention is to provide a solution to the problem of purine-based 3'deoxynucleosides, as exemplified by cordycepin (3'-deoxyadenosine), which upon administration are deaminated by ADA and then rapidly metabolized to an inactive metabolite.

Another object of the present invention is to provide a solution to the problem of purine-based 3'deoxynucleosides, as exemplified by cordycepin (3'-deoxyadenosine), which upon administration are deaminated by ADA and then rapidly metabolized to an inactive metabolite, so as to completely eliminate or reduce to some extent, the need for co-administration of an ADA inhibitor when a purine-based 3'-deoxynucleoside is used in a prophylactic or treatment method, in particular, though not limited to, cancer chemotherapy, including chemotherapy for the treatment of leukemia, lymphoma, and/or solid tumors.

According to a first aspect of the present invention, there is provided a compound which is a compound of formula (Ia) x

O I ^w 2 (la) where W1 and W ₂ are each independently selected from the group consisting of -P(=O)(U)(V) and H, provided that at least one of W1 and W ₂ is -P(=O)(U)(V), where U and V, independently for each of W1 and W ₂ , are selected from the group consisting of:

(a) U is -OAr in combination with V is -NR ₄ -CR1R ₂ -C(=O)OR ₃ ,

- 1 040630 where Ar is selected from the group consisting of C _b-30 aryl and C _5-30 heteroaryl, each of which is optionally substituted;

each of R ₁ and R ₂ is independently selected from H and the group consisting of C _1-20 alkyl, C _b-30 arylC _1-b alkyl,

C _2-20 alkenyl, C _1-20 alkoxy, C _1-20 alkoxyC _1-20 alkyl, C _1-20 alkoxyC _6-30 aryl, C _2-20 alkynyl,

C _3-20 cycloalkyl C _b-30 aryl, C _b-30 aryloxy and C _5-20 heterocyclyl, any of which is optionally substituted;

R ₃ is selected from H and the group consisting of C _1-20 alkyl, C _b-30 arylC _1-20 alkyl, C _2-20 alkenyl, C _1-20 alkoxyC _1-20 alkyl, C _1-20 alkoxyC _b-30 aryl , C _2-20 alkynyl, C _3-20 cycloalkylC _6-30 aryl, and C _5-20 heterocyclyl, any of which is optionally substituted;

R ₄ is selected from H and the group consisting of C _1-20 alkyl, C _b-30 arylC _1-20 alkyl, C _2-20 alkenyl, C _1-20 alkoxy, C _1-20 alkoxyC _1-20 alkyl, C _{1- 20} alkoxyC _b-30 aryl, C _2-20 alkynyl, C _3-20 cycloalkylC _b-30 aryl, C _b-30 aryloxy, and C _5-20 heterocyclyl, any of which is optionally substituted; and (b) each of U and V is independently selected from -NR ₅ R ₆ where R ₅ is selected from the group consisting of H and C _1-6 alkyl and R6 is -CR ₇ R ₈ CO ₂ R ₉ where R ₇ and R8 are independently selected from the group consisting of side chains including H, naturally occurring alpha amino acids, and R ₉ is selected from H and the group consisting of C _1-20 alkyl, C _6-30 arylC _1-20 alkyl-, C _2-20 alkenyl , C _1-20 alkoxyC _1-20 alkyl, C _1-20 alkoxyC _6-30 aryl, C _2-20 alkynyl, C _3-20 cycloalkylC _6-30 aryl, and C _5-20 heterocyclyl, any of which is optionally substituted; or

R ₅ and R ₆ together with the N atom to which they are attached, form a ring group containing from 5 to 8 ring atoms;

Q is selected from the group consisting of O, S and CR ₁₀ Rn, where R ₁₀ and R11 are independently selected from H, F and C _1-6 alkyl;

each Xu Z is independently selected from the group consisting of H, OH, F, Cl, Br, I, C _1-6 alkyl, -NR ₁₂ R ₁₃ , where each of R ₁₂ and R ₁₃ is independently selected from H and C _{1- 6} alkyl, and -SR ₁₄ where R ₁₄ is selected from the group consisting of H and C _1-6 alkyl; And

Y is selected from the group consisting of H, OH, F, Cl, Br, I, -OC _1-b alkyl, C _1-6 alkyl, C _2-8 alkynyl, -NR ₁₅ R ₁₆ , where each of R ₁₅ and R ₁₆ is independently selected from H and C _1-6 alkyl, and -SRi ₇ , wherein R ₁₇ is selected from the group consisting of H and C _1-6 alkyl, or a pharmaceutically acceptable salt, ester, ester salt, solvate, or prodrug of a compound of formula (Ia).

The compounds of the present invention are purine-based 3'-deoxynucleosides in which each of the positions of the substituent 3' on the sugar moiety of the nucleoside is occupied by H.

In a further embodiment of the invention, the compound of the invention may be a compound of formula (Ib)

where W1 and W2 are each independently selected from the group consisting of -P(=O)(U)(V) and H, provided that at least one of W ₁ and W ₂ is -P(=O )(U)(V), where U and V, independently for each of W ₁ and W ₂ , are selected from the group consisting of:

(a) U is -OAr and V is -NR ₄ -CR1R ₂ -C(=O)OR ₃ where Ar is selected from the group consisting of C _6-30 aryl and C _5-30 heteroaryl, each of which is optionally substituted;

each of R1 and _R2 is independently selected from H and the group consisting of C _1-20 alkyl, C _6-30 arylC _1-6 alkyl, C _2-20 alkenyl, C _1-20 alkoxy, C _1-20 alkoxyC _1-20 alkyl, C _1-20 alkoxyC _6-30 aryl, C _2-20 alkynyl, C _3-20 cycloalkyl, C _6-30 aryl, C _6-30 aryloxy, and C _5-20 heterocyclyl, any of which is optionally substituted;

R ₃ is selected from H and the group consisting of C _1-20 alkyl, C _6-30 arylC _1-20 alkyl, C _2-20 alkenyl, C _1-20 alkoxyC _1-20 alkyl, C _1-20 alkoxyC _6-30 aryl , C _2-20 alkynyl, C _3-20 cycloalkyl, C _6-30 aryl and C _5-20 heterocyclyl, any of which is optionally substituted;

R ₄ is selected from H and the group consisting of C _1-20 alkyl, C _6-30 arylC _1-20 alkyl, C _2-20 alkenyl, C _1-20 alkoxy, C _1-20 alkoxyC _1-20 alkyl, C _{1- 20} alkoxyC _b-30 aryl, C _2-20 alkynyl, C _3-20 cycloalkyl, C _6-30 aryl, C _6-30 aryloxy, and C _5-20 heterocyclyl, any of which is optionally substituted; And

- 2 040630 (b) U and V are each independently selected from -NR ₅ R ₆ , where R ₅ is selected from the group consisting of H and

_C1 . ₆ alkyl, and R ₆ is -CR ₇ R ₈ CO ₂ R ₉ , wherein R ₇ and R ₈ are independently selected from the group consisting of side chains including H, naturally occurring alpha amino acids, and R ₉ is selected from H and the group consisting of C _1-20 alkyl, C ₆ . ₃₀ aryls ₁ . ₂₀ alkyl-, C _2-20 alkenyl, C ₁ . ₂₀ alkoxyC ₁ . ₂₀ alkyl, C ₁ . ₂₀ alkoxyC ₆ . ₃₀ aryl,

From ₂ . ₂ oalkynyl, C _3-20 cycloalkyl, C _6-30 aryl, and C _5-20 heterocyclyl, any of which is optionally substituted; or

R5 and R6 together with the N atom to which they are attached form a ring group containing 5 to 8 ring atoms;

X is selected from NR ¹² R ¹³ where R ₁₂ and R ₁₃ are each independently selected from H and C _1-6 alkyl; and -SR ₁₄ where R ₁₄ is selected from the group consisting of H and C _1-6 alkyl;

Z is independently selected from the group consisting of H, OH, F, Cl, Br, I, C _1-6 alkyl, -NR ₁₂ R ₁₃ , and -SR ₁₄ , where R ₁₄ is selected from the group consisting of H and C _1-6 alkyl; And

Y is selected from the group consisting of H, OH, F, Cl, Br, I, -OC _1-6 alkyl, C _1-6 alkyl, C _2-8 alkynyl, -NR ₁₅ R ₁₆ , where each of R ₁₅ and R ₁₆ is independently selected from H and C _1-6 alkyl, and -SR ₁₇ where R ₁₇ is selected from the group consisting of H and C 1-6 alkyl, or a pharmaceutically acceptable salt, ester, ester salt, solvate, or prodrug

where Ar, Y, Z, R ₂ and R ₃ have the meanings described above for formula (Ib), and where X represents -NR ¹² R ¹³ .

The compound of formula (Ib) may be a compound of formula (III)

where Ar, R ₂ and R ₃ have the meanings described above for formula (Ib), and where Y is selected from H, F, Cl and OMe.

The following definitions refer to compounds of any of the formulas (Ia), (Ib), (II) and (III). These definitions are independent and interchangeable. In other words, any of the features described in any of the following definitions may (where chemically acceptable) be combined with the features described in one or more of the other definitions below. In particular, when a compound is shown in the examples or illustrated in this specification, any two or more of the following definitions that describe a feature of that compound, expressed at any level of generality, may be combined to represent the subject matter of the invention, which is considered to form part of the description of this specification. inventions in this specification.

As used herein, the term natural alpha amino acid means an amino acid which may have L or D stereochemistry selected from the group consisting of glycine, alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, tryptophan, serine, threonine, lysine, arginine, histidine, aspartic acid, glutamic acid, asparagine, glutamine, cysteine and methionine. As used herein, the natural alpha amino acid side chain is thus a member selected from the group consisting of H, CH3, -CH(CH3)2, -CH2CH(CH3)2, -CH(CH3)(CH2CH3), CH2Ph, CH2Ph-OH, -CH2SH, -CH2CH2SCH3, -CH2OH, -(CH3)(OH), CH2CH2CH2CH2NH3+, CH2CH2CH2NHC(=NH2+)NH2, -CH2C(O)O-, CH2CH2COO-, CH2C(O)NH2, CH2CH2C(O) )NH2,

- 3 040630

It may be that W ₁ is -P(=O)(U)(V) and W ₂ is H and the compound of the invention is the 5'-phosphoramidate from the parent 3'-deoxynucleoside. In some preferred embodiments, W ₁ is -P(=O)(U)(V), where U is -OAr and V is -NR ₄ -CR ₁ R ₂ -C(=O)OR ₃ , and W ₂ is H.

It may be that W ₁ is H and W ₂ is -P(=O)(U)(V) and the compound of the invention is 2'-phosphoramidate from the parent 3'-deoxynucleoside. In some preferred embodiments, W ₁ is H and W ₂ is -P(=O)(U)(V), where U is -OAr and V is -NR ₄ -CR ₁ R ₂ -C( =O)OR ₃ .

It may be that each of W ₁ and W ₂ is -P(=O)(U)(V) and the compound of the invention is 2',5'-phosphoramidate from the parent 3'-deoxynucleoside. In some preferred embodiments of the invention, where each of W ₁ and W ₂ represents -P(=O)(U)(V), U represents -OAr and V represents -NR ₄ -CR ₁ R ₂ -C( =O)OR ₃ . In some preferred embodiments of the invention W _i is the same as W ₂ .

Ar may be unsubstituted. Ar may be substituted. When Ar is substituted, it may be substituted with one, two, three, four, or five substituents. The substituents may be selected from: halogen, C ₁ -C ₄ alkyl, C ₁ -C ₄ alkoxy, nitro and cyano.

Ar, either substituted or unsubstituted, may be selected from the group consisting of phenyl, pyridyl, naphthyl, and quinolyl. In some preferred embodiments, Ar is selected from the group consisting of phenyl and naphthyl. In further preferred embodiments of the invention, when Ar is naphthyl, the attachment to -O-P occurs at the 1-position on the naphthyl. In further preferred embodiments of the invention, Ar is unsubstituted phenyl or unsubstituted naphthyl, attached to -O-P at the 1-position on the naphthyl.

R ₁ and R ₂ can be chosen such that the CR ₁ R ₂ COO- group corresponds to the natural alpha amino acid group.

It may be that each of R ₁ and R ₂ is independently selected from Me and H. In some preferred embodiments of the invention, one of R ₁ and R ₂ is Me and one of R ₁ and R ₂ is H, so that the C atom bearing R ₁ and R ₂ has the same absolute configuration as L-alanine.

It may be that R ₁ is H. It may be that R ² is C ₁ C4 alkyl. It may be that R2 is methyl. It may be that the C atom bearing R1 and _R2 has the same absolute configuration as L-alanine.

It may be that each of R1 and R2 is Me. It may be that each of R1 and R2 is H.

It may be that R ₃ is selected from the group consisting of C ₆ _ ₃₀ arylC ₁ _ ₆ alkyl and unsubstituted C _1-20 alkyl. In some preferred embodiments of the invention, R ₃ is selected from the group consisting of benzyl (-CH ₂ -Ph), unsubstituted methyl (-CH ₃ ) and unsubstituted n-pentyl (-n-C ₅ H ₁₁ ). In further preferred embodiments, R ₃ is benzyl.

R ₄ may be H.

U and V may be independently selected from -NR ₅ R ₆ . Preferably, U and V are each the same. In further preferred embodiments, R8 is H and _R7 is selected from the group consisting of H, methyl, iso-propyl, -CH ₂ Ph, -CH ₂ CH(CH ₃ ) ₂ and -CH (CH ₃ ) (CH ₂ H ₅ ). In further preferred embodiments, R ₇ is methyl. In further preferred embodiments, the stereochemistry of the C atom bearing R ₇ and R 8 may have the same absolute configuration as L-alanine. Alternatively, the stereochemistry of the C atom bearing R ₇ and R 8 may have the same absolute configuration as D-alanine. In some preferred embodiments, R _g is selected from the group consisting of branched and straight chain C ₁ -C ₁₃ acyclic alkyl, C ₃ -C ₁₈ cyclic alkyl, and C _{6-30 arylC 1-6} alkyl, any of which is optionally substituted. In some preferred embodiments, R _g is benzyl.

In some embodiments, the compound of the present invention contains U and V, where U and V are each independently selected from -NR ₅ R ₆ , where R ₅ and R ₆ together with the N atom to which they are attached form a ring group containing from 5 to 8 ring atoms. U and V can be the same.

Q may represent O.

It may be that W ₁ is -P(=O)(U)(V), where U is -O-1-naphthyl and V is -NH-(L)CH(CH ₃ )-C(= O)-O-CH ₂ -Ph, W ₂ is H and Q is O.

It may be that each of X and Z is independently selected from the group consisting of H, OH, F, Cl, NH ₂ , SH and -SC ₁ . ₆ αkyl, and Y is selected from the group consisting of H, OH, F, Cl, -OC ₁ . ₆ alkyl, NH ₂ , C _2-8 alkynyl, SH and -SC ₁ . ₆ alkyl. It may be that X is NR ¹² R ¹³ , for example NH ₂ . In some preferred embodiments, Z is H. In further preferred embodiments, X is NH ₂ and Z is H. B

- 4 040630 preferred embodiments of the invention X is NH ₂ , Y is H and Z is H; X is NH ₂ , Y is F and Z is H; X is NH ₂ , Y is Cl and Z is H; or X is NH ₂ , Y is -OCH ₃ and Z is H. In some preferred embodiments of the invention, X is NH ₂ , Y is H and Z is H and thus representing compounds of the invention which are derivatives of cordycepin (3'dA).

In some particularly preferred embodiments, Ar is phenyl, R ₃ is benzyl, and R 2 is methyl.

Compounds of the present invention wherein, when P is asymmetric, the compound may exist as an R _p diastereoisomer, an S _p diastereoisomer, or a mixture of R _p and S _p diastereoisomers.

Preferred compounds of the invention include:

(2S)-benzyl 2-(((((2S,4R,5R)-5-(6-amino-9H-purin-9-yl)-4-hydroxytetrahydrofuran-2yl)methoxy)(naphthalene-1-yloxy) phosphoryl)amino)propanoate;

benzyl 2-(((((2S,4R,5R)-5-(6-amino-9H-purin-9-yl)-4-hydroxytetrahydrofuran-2yl)methoxy)(phenoxy)phosphoryl)amino)acetate;

(2S)-pentyl 2-(((((2S,4R,5R)-5-(6-αmino-9H-purin-9-yl)-4-hydroxytetrahydrofuran-2yl)methoxy)(naphthalene-1-yloxy) phosphoryl)amino)-4-methylpentanoate;

methyl 2-(((((2S,4R,5R)-5-(6-amino-9H-purin-9-yl)-4-hydroxytetrahydrofuran-2yl)methoxy)(naphthalene-1-yloxy)phosphoryl)amino) -2-methylpropanoate;

(2S)-benzyl 2-(((((2S,4R,5R)-5-(6-amino-9H-purin-9-yl)-4-hydroxytetrahydrofuran-2-yl)methoxy)(2(3- ethoxy-3-oxopropyl)phenoxy)phosphoryl)amino)propanoate;

(2S)-benzyl 2-(((((2R,3R,5S)-2-(6-amino-9H-purin-9-yl)-5-(hydroxymethyl)tetrahydrofuran-3yl)oxy)(phenoxy)phosphoryl )amino)propanoate;

benzyl 2-(((((2S,4R,5R)-5-(6-αmino-9H-purin-9-yl)-4-((((1-(benzyloxy)-1-oxopropan-2yl)amino )(phenoxy)phosphoryl)oxy)tetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate;

(2S)-benzyl 2-(((((2R,3R,5S)-2-(6-amino-9H-purin-9-yl)-5-(hydroxymethyl)tetrahydrofuran-3yl)oxy)(naphthalene-1 -yloxy)phosphoryl)amino)propanoate;

benzyl 2-[( {[5-(6-amino-9H-purin-9-yl)-4-hydroxyoxolan-2-yl]methoxy}( {[1-(benzyloxy)-1oxopropan-2-yl]amino } )phosphoryl)amino]propanoate;

(2S)-benzyl 2-((((2S,4R,5R)-5-(6-αmino-2-methoxy-9H-purine-9-ul)-4-hydroxytetrahydrofuran-2yl)methoxy)(naphthalene-1 -yloxy)phosphorylamino)propanoate;

(2S)-benzyl 2-((((2S,4R,5R)-5-(6-αmino-2-methoxy-9H-purine-9-ul)-4-hydroxytetrahydrofuran-2yl)methoxy)(phenoxy)phosphorylamino ) propanoate;

(2S)-benzyl 2-(((((2S,4R,5R)-5-(6-αmino-2-fluoro-9H-purin-9-yl)-4-hydroxytetrahydrofuran-2yl)methoxy)(phenoxy) phosphoryl)amino)propanoate;

(2S)-hexyl 2-(((((2S,4R,5R)-5-(6-αmino-2-fluoro-9H-purin-9-yl)-4-hydroxytetrahydrofuran-2yl)methoxy)(phenoxy) phosphoryl)amino)propanoate;

(2R)-benzyl 2-((((2S,4R,5R)-5-(6-αmino-2-chloro-9H-purin-9-yl)-4-hydroxytetrahydrofuran-2yl)methoxy)(naphthalene-1 -yloxy)phosphorylamino)propanoate;

3'-deoxyadenosine-5'-O-[phenyl(benzyloxy-L-αlaninyl)]phosphate;

2-O-methyl-3'-deoxyadenosine-5'-O-[1-naphthyl(1-pentyloxy-L-leucinyl)]phosphate;

2-O-methyl-3'-deoxyadenosine-5'-O-[phenyl (1-hexyloxy-L-αlaninyl)]phosphate;

2-fluoro-3'-deoxyadenosine-5'-O-[1-naphthyl(benzyloxy-L-αlaninyl)]phosphate;

2-fluoro-3'-deoxyadenosine-5'-O-[1-naphthyl(1-pentyloxy-L-leucinyl)]phosphate;

2-chloro-3'deoxyadenosine 5'-O-[1-phenyl(2,2-dimethylpropoxy-L-αlanine)]phosphate;

2-chloro-3'deoxyadenosine 5'-O-[1-naphthyl(2,2-dimethylpropoxy-L-αlanine)]phosphate;

2-chloro-3'deoxyadenosine 5'-O-[1-phenyl (ethoxy-L-αlanine)]phosphate; and (2S)-isopropyl-2-(((((2S,4R,5R)-5-(6-amino-9H-purin-9-yl)-4-hydroxytetrahydrofuran-2yl)methoxy)(phenoxy)phosphoryl) amino)propanoate and their pharmaceutically acceptable salts, esters, ester salts, solvates or prodrugs.

In some embodiments, the compound of the invention is not:

(2S)-isopropyl-2-(((((2S,4R,5R)-5-(6-amino-9H-purin-9-yl)-4-hydroxytetrahydrofuran-2yl)methoxy)(phenoxy)phosphoryl)amino ) propanoate.

According to a second aspect of the present invention, there is provided a compound of the present invention for use in a method of treatment. The compound may be used to prevent or treat cancer.

According to a third aspect of the present invention, there is provided the use of a compound of the present invention in the manufacture of a medicament for the prevention or treatment, in particular, though not exclusively, of cancer.

According to the fourth aspect of the present invention, a method for preventing

- 5 040630 or treatment, in particular, although not exclusively, of a malignant neoplasm, comprising administering to a patient, in need of such treatment, an effective dose of a compound of the present invention.

With regard to each of the second, third and fourth aspects of the present invention, embodiments of the invention include a malignancy selected from among hematological and solid tumors.

In particular, the cancer may be selected from the group consisting of leukemia, multiple myeloma, liver cancer, breast cancer, head and neck cancer, neuroblastoma, thyroid cancer, skin cancer (including melanoma), oral squamous cell carcinoma, urinary tract cancer. bladder, Leydig cell tumor, colon cancer, colorectal cancer, lung cancer (non-small cell and small cell), gallbladder cancer, pancreatic cancer, sarcoma, prostate cancer, central nervous system cancer, Ewing's sarcoma, cholangiocarcinoma and gynecological cancers, including ovarian cancer, uterine cancer, and cervical cancer, including cervical epithelial cancer. In preferred embodiments, the cancer is a leukemia or lymphoma, such as a cancer selected from the group consisting of acute lymphoblastic leukemia, acute myeloid leukemia, acute promyelocytic leukemia, acute lymphocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, monoblastic leukemia, hairy cell leukemia, Hodgkin's lymphoma and non-Hodgkin's lymphoma. In further preferred embodiments, the malignant neoplasm is acute lymphoblastic leukemia.

Each of the second, third and fourth aspects of the invention may include cancer treatment options used in combination with other cancer therapy. Examples of other cancer therapy include radiation therapy and/or other chemotherapy. Without being bound by theory or mechanism, it has been reported (e.g., Robertson, J. B. et al. Int. J. Radiat. Biol. Relat. Stud. Phys. Chem. Med. 1978 34(5): 417-29, Hiraoka, W. et al. Radiat. Res. (1988) 114(2):231-9, and Hiraoka, W. et al. J. Radiat. Res. (Tokyo) (1990) 31(2):156-61) that 3'-deoxyadenosine inhibits the repair of DNA damage caused by X-rays. In some embodiments of each of the second, third and fourth aspects of the present invention, the compounds of the invention are for use in, or are used in, a method of treating cancer comprising administering to a patient in need of such treatment a compound of the present invention in combination with radiation therapy.

With respect to each of the second, third, and fourth aspects of the present invention, further embodiments of the invention include compounds of the invention that are for use in, or are used in, a method for the prevention or treatment of myelodysplastic syndrome.

Without reference to theory or mechanism: Tuli et al. (supra) reported that cordycepin, in addition to having anticancer activity and apoptotic activity, also exhibits antioxidant, anti-inflammatory, antimalarial, antifungal, immunomodulatory, antidiabetic/hypoglycemic, steroidogenesis and anti-aging activities; Vodnala, S. K. et al. J. Med. Chem. 2013, 56, 9861-9873 reported that both cordycepin and 2-fluorocordycepin each exhibit antiparasitic activity; Ann, Y. J. et al. J. Agric. food chem. 2000 48 (7) 2744-8 reported that cordycepin exhibits antibacterial activity and de JulianOrtiz J. V. et al. J. Med. Chem. 1999 42(17) 3308-14 reported that cordycepin exhibits antiviral activity; Sugar et al, Antimicrob. agents. Chemother. 1998 42(6) 1424-7 showed that cordycepin has antifungal activity. With respect to each of the second, third, and fourth aspects of the present invention, embodiments of the invention include compounds of the invention that are for use in, or are used in, a method for the prevention or treatment of a patient with a disease or condition requiring at least one treatment selected from the group , including antioxidant, anti-inflammatory, antimalarial, antifungal, immunomodulatory, antidiabetic/hypoglycemic, steroidogenic, anti-aging, antiparasitic, antibacterial and antiviral activity.

With respect to each of the second, third and fourth aspects of the invention, embodiments of the invention include compounds of the present invention that are intended for use or are used in a method of prevention or treatment, where the method does not involve the administration of an additional drug that is an adenosine deaminase inhibitor. Unlike the parent cordycepin compound, which typically needs to be co-administered with an ADA inhibitor in order to be effective, it may be possible that the compounds of the invention will not require such co-administration.

An ADA inhibitor, however, may be used as an additional drug, if desired, in relation to each of the second, third and fourth aspects of the invention. A suitable ADA inhibitor for co-administration with a compound of the present invention is hydroxyurea or pentastatin.

According to a further aspect of the present invention, there is provided a pharmaceutical composition comprising a compound of the present invention in combination with a pharmaceutically acceptable carrier, diluent or excipient.

According to a further aspect of the present invention, there is provided a method for preparing a pharmaceutical composition, comprising the step of bringing into association a compound of the present invention with a pharmaceutically acceptable carrier, diluent or excipient.

According to a further aspect of the present invention, there is provided a process for preparing a compound of formula (Ia) with (a) a compound of formula V

by reacting a compound of formula IV or (b) POCl ₃ , then with an N ⁺ R ₅ R ₆ H ₂ salt, where Wi, W ₂ , Q, X, Y, Z, Ar, R _b R ₂ , R ₃ , R ₄ , R ₅ and R ₆ have the meanings given herein with respect to formula (Ia).

Surprisingly, the compounds of the present invention have been found to have enhanced pharmaceutical activity, in particular enhanced antitumor activity, compared to the parent purine-based 3'-deoxynucleoside (i.e. when W1 and _W2 are H), especially when used in the treatment of leukemia, lymphoma and/or solid tumors.

An increase in activity was found in the absence of administration of an additional drug to inhibit adenosine deaminase, compared to the parent purine-based nucleoside administered in the absence of administration of an additional drug to inhibit ADA.

Thus, the present invention surprisingly provides a means for using a 3'-deoxyadenosine derivative or a 3'-deoxyadenosine analogue derivative as a pharmaceutical agent, in particular as an anti-cancer agent, which alleviates the problem of deamination by adenosine deaminase while completely avoiding, if desired, the use of an additional drug that is an adenosine deaminase inhibitor, including the relatively toxic 2-deoxycoformycin.

Without being bound by any theory, the efficacy, especially antitumor efficacy, exhibited by the compounds of the present invention demonstrates that the 3'-deoxynucleoside compounds of the present invention are phosphorylated intracellularly to 3'-deoxyadenosine triphosphate or to 3'-deoxyadenosine triphosphate. When the compounds of the present invention have W1 as -P-(=O)(U)(V), enzymatic cleavage of U and V in the cell is believed to convert the compounds directly to 3'-deoxyadenosine monophosphate or 3'-deoxyadenosine monophosphate analog prior to phosphorylation to triphosphate.

None of the above intracellular activities of the compounds of the present invention could be predicted in advance.

The above benefits are in addition to increased cell permeability.

- 7 040630 membranes with phosphoramidate nucleosides of the present invention compared to 3'-deoxyadenosine parent compound or 3'-deoxyadenosine analog parent compound, where the increased permeability of the cell membrane is due to the phosphoramidate structure of the compound. The advantage of increased cell membrane permeability cannot, moreover, be assumed a priori for any nucleoside phosphoramidate. The compounds of the present invention are believed to be the first example of a 3'-deoxynucleoside phosphoramidate demonstrating enhanced antitumor activity over its parent 3'-deoxynucleoside. Thus, the cell membrane permeability effect of the compounds of the present invention is unexpected.

Preferred embodiments of the compounds of the present invention collectively have the features set forth above with respect to the embodiments of the compounds of the invention.

Each of Ar, R ₁ , R ₂ , R ₃ and R4 may be substituted with one, two, three, four or five substituents.

Substituents on Ar may be in ortho, meta, para, or other positions on aromatic groups. Substituents on Ar are independently selected from the group consisting of hydroxy, C _1-6 acyl, C _1-6 acyloxy, nitro, amino, carboxyl, C _2-6 ester, C _1-6 aldehyde, cyano, C _1-6 alkylamino, diC _1-6 alkylamino, thiol, chlorine, bromine, fluorine, iodine, C _1-6 alkyl, C _2-6 alkenyl, C _1-6 alkoxyC _1-6 alkyl, C _1-6 alkoxyC _6-10 aryl, C _{5 -7} cycloalkyl, C _5-11 cycloalkylC _1-6 alkyl, C _5-7 cycloalkenyl, C _8-12 cycloalkynyl, C _6-11 arylC _1-6 alkyl, C _1-6 alkylC _6-11 aryl, C _6-11 aryl, C _1-6 fluoroalkyl, C _2-6 fluoroalkenyl, SO ₃ H, SH and SR', wherein R' is independently selected from the same group as R ₁ above with respect to formula Ia. Each substituent may be substituted with any other substituent.

The substituents on R ₁ , R ₂ , R ₃ and R4 are independently selected from the group consisting of hydroxy, C _1-6 acyl, C _1-6 acyloxy, nitro, amino, amido, carboxy, C _2-6 ester, C _{1- 6} aldehyde, cyano, C _1-6 alkylamino, diC _1-6 alkylamino, thiol, chlorine, bromine, fluorine, iodine, C _5-7 cycloalkyl, C _5-7 cycloalkenyl, C _8-12 cycloalkynyl, C _6-11 aryl , C _6-11 arylC _1-6 alkyl, C _5-20 heterocyclyl, SO ₃ H, SH and SR', wherein R' is independently selected from the same group as R ₁ above with respect to formula Ib.

In some preferred embodiments, R ₁ and R ₂ are independently selected from the group consisting of H, C _1-10 alkyl, C _6-30 arylC _1-6 alkyl, C _2-10 alkenyl, C _2-10 alkoxyC _1-10 alkyl , C _1-10 alkoxyC _6-10 aryl, C _2-10 alkynyl, C _3-20 cycloalkyl, C _3-20 cycloalkenyl, C _8-20 cycloalkynyl, and C _5-10 heterocyclyl.

In some embodiments, R ₁ and/or R ₂ correspond to the side chain, including H, of a naturally occurring alpha amino acid that may have L or D stereochemistry. Thus, it may be that R ₁ and/or R ₂ (for example, separately or R ₁ and R ₂ ) are selected from the group consisting of H, CH3, -CH(CH3)2, CH2CH(CH3)2, -CH( CH3) (CH2CH3), C^PI, ^Ph-OH, -CH2SH, -^^S^, -CH2OH, CH(CH3)(OH), CH2CH2CH2CH2NH3 ⁺ , ^2^2^2^1^(=^ 2+)^2, CH2C(O)O-, CH2CH2C(O)O-, CH2C(O)NH2, CH2CH2C(O)NH2,

In some preferred embodiments of the invention, R1 and R2 are independently selected from the group consisting of H, -CH3 and -CH2CH(CH3)2. In further preferred embodiments, R1 and R2 together correspond to the L alanine side chains.

R ₃ may be selected from the group consisting of H, C _1-20 alkyl, C _6- zoarylC _1-6 alkyl, C _2-10 alkenyl, C _1-10 alkoxyC _1-10 alkyl, C _1-10 alkoxyC _6-10 aryl, C _2-10 alkynyl, C _3-20 cycloalkyl, C _3-20 cycloalkenyl, C 8-20 cycloalkynyl and C 5-20 heterocyclyl.

R ₃ may be selected from the group consisting of H, C _1-20 alkyl, C _6-30 arylC _1-6 alkyl, and C _3-20 cycloalkyl. R ₃ may be selected from the group consisting of C _6-30 arylC _1-6 alkyl and unsubstituted C _1-20 alkyl. In some preferred embodiments, R ₃ is selected from the group consisting of benzyl (-CH ₂ Ph), unsubstituted methyl (-CH ₃ ) and unsubstituted n-pentyl (-n-C ₅ H ₁₁ ). R ₃ may be benzyl.

R4 may be selected from the group consisting of H, C _1-20 alkyl, C _6-30 arylC _1-6 alkyl, C _2-10 alkenyl, C _1-10 alkoxy, C _1-10 alkoxyC _1-10 alkyl, C _{1 -10} alkoxyC _6-10 aryl, C _2-10 alkynyl, C _3-20 cycloalkyl,

C3-20 cycloalkenyl, C8-20 cycloalkynyl and C5-20 heterocyclyl.

R4 may be selected from the group consisting of H, C _1-18 alkyl, C _6-30 arylC _1-6 alkyl, C _3-20 cycloalkyl, and C 5-20 heterocyclyl. R4 may be selected from the group consisting of H, methyl, ethyl, propyl, butyl, pentyl, hexyl and cyclohexyl. R4 may be H.

This invention relates to a compound of the invention for use in targeting cancer stem cells.

This invention relates to the use of a compound of the invention in the manufacture of a medicament for targeting cancer stem cells.

The present invention relates to a method for targeting cancer stem cells, wherein the method

- 8 040630 includes providing a population of cancer stem cells with a sufficient amount of a compound of the invention to target such cancer stem cells.

Cancer stem cell targeting of the present invention can be used to prevent or treat cancer. In such embodiments, the cancer stem cell population may be cancerous or precancerous in a patient in need of such targeting, and the method may include administering a therapeutically effective amount of a compound of the invention to the patient.

This invention relates to a compound of the invention for use as a cancer stem cell drug. This use of the compounds of the invention may also be used in the prevention or treatment of cancer.

This invention relates to a method for determining whether a patient suffering from a malignant neoplasm or having a precancerous condition will be effective in preventing or treating a malignant neoplasm using a compound of the invention, wherein the method comprises:

analysis of a biological sample characteristic of a malignant neoplasm or precancerous condition in a patient for the presence of cancer stem cells; wherein the presence of cancer stem cells in a biological sample indicates that the patient would benefit from treatment with a compound of the invention.

This invention relates to a method for determining an appropriate treatment regimen for a patient suffering from a malignant neoplasm or having a precancerous condition, where the method includes:

analysis of a biological sample characteristic of a malignant neoplasm or precancerous condition in a patient for the presence of cancer stem cells; where the presence of cancer stem cells in a biological sample indicates that a suitable treatment regimen would include treating the patient with a compound of the invention.

This invention relates to a compound of the invention for use in the prevention or treatment of cancer in a patient selected for such treatment by a method comprising:

analysis of a biological sample characteristic of a malignant neoplasm or precancerous condition in a patient for the presence of cancer stem cells; wherein the presence of cancer stem cells in a biological sample indicates that the patient would benefit from treatment with a compound of the invention.

The methods described above may further include the step of preventing or treating a malignant neoplasm or precancerous condition using a compound of the invention.

In suitable embodiments of the methods of the invention, the cancer is recurrent or refractory cancer. The compound of the invention can be used in the treatment of such relapsed or refractory cancer.

This invention relates to a compound of the invention for use in the treatment of refractory cancer in a subject. The subject may be a human patient. The subject may be a pet, such as a mammal.

This invention relates to the use of a compound of the invention in the manufacture of a medicament for the treatment of relapsed or refractory cancer in a subject. The subject may be a pet, such as a mammal.

The present invention relates to a method of treating relapsed or refractory cancer in a subject, wherein the method comprises providing the subject with a therapeutically effective amount of a compound of the invention in need of such treatment. The subject may be a pet, such as a mammal.

This invention relates to a compound of the invention for use in the treatment of cancer, wherein the compound of the invention is for use at a dose in the range of about 25 and 4000 mg/m ² per week for at least one initial treatment cycle and then for use at a lower weekly dose in at least one additional cycle of treatment. The malignancy may be recurrent or refractory malignancy.

Various aspects of the invention are based on the fact that the compound of the invention is able to reduce the number of cancer stem cells and can reduce them preferentially compared to other cell types. This finding is surprising in that cancer stem cells are known to be resistant to many chemotherapeutic agents and it has not been previously suggested that either the compound of the invention or cordycepin or 2-fluorocordycepin, the parent prodrug from which the compound invention was obtained were able to target cancer stem cells. Thus, the discovery that a compound of the invention is able to target cancer stem cells and therefore reduce their number, a finding that has been validated by the inventors, applicable to a wide range of cancers, represents an amazing breakthrough that allows the use of a whole a number of novel therapeutic uses for the compounds of the invention.

The biological activity exhibited by the compounds of the invention, which have not been previously reported, indicates that these compounds are capable of providing a treatment that is likely to be effective in patients with relapsed or refractory cancer. Treatment of this kind with the compounds of the invention may result in a reduction in tumor size and/or a reduction in clinically relevant biomarkers, which may be associated with a better prognosis. In addition, treatment with a compound of the invention may help maintain a reduction in tumor size in patients with relapsed or refractory cancer. Accordingly, treatment with a compound of the present invention can provide a high reliable disease control rate (DCR) in patients with relapsed or resistant cancers.

Without wishing to be bound by any hypothesis, the inventors believe that the ability of the compounds of the invention to target cancer stem cells contributes to the therapeutic utility of these compounds in the treatment of relapsed or refractory cancer.

Except where the context requires otherwise, references within this specification to the use of the compounds of the invention in accordance with the invention may be taken to refer to any of the medical uses of the compounds of the invention described herein. Likewise, references to methods of the invention using a compound of the invention should be construed as referring to any of the methods described herein.

The ability of a compound of the invention to target cancer stem cells provides new therapies directed against those cancer cells that are considered the most difficult to treat and that are believed to play a major role in resistance, which limits the effectiveness of many existing cancer treatments. This ability also provides a way to target cells believed to be associated with the development, progression, recurrence, and spread of cancer. Accordingly, it will be recognized that this activity of the compounds of the invention against cancer stem cells provides advantages in those cases for which new effective treatments have long been sought.

Brief description of the figures

Embodiments of the invention are described below with reference to the accompanying drawings, in which:

fig. 1 Comparison of LD ₅₀ values for cordycepin, Compound A, 2-P-cordycepin, Compounds O, P, Q, and R. All analyzes were performed using KG1a cells and data are presented as mean (±SD) of five independent experiments;

fig. 2 - analysis of the targeting ability of leukemic stem cells (LSC) cordycepin and compound A. Previously obtained data (ii) are shown for comparison. All data represent the mean (±SD) of three independent experiments;

fig. 3 - LSC targeting ability analysis of 2-P-cordycepin and compounds O, P, Q and R. All data represent the mean (±SD) of three independent experiments;

fig. 4 - Comparison of LSC targeting ability of 2-P-cordycepin and each proTide. All data represent the mean (±SD) of three independent experiments.

Detailed description

As used herein, the term alkyl refers to a straight or branched saturated monovalent (unless the context otherwise requires) cyclic or acyclic hydrocarbon radical having the number of carbon atoms indicated (or, when not indicated, an acyclic alkyl group may contain 1-20, 1-18, 1-10, 1-6 or 1-4 carbon atoms, and the cyclic alkyl group may contain 3-20, 3-10 or 3-7 carbon atoms), optionally substituted with one, two or three substituents independently selected from the group described above with respect to substituents which may be present on R1, R2, R3 and R4. As non-limiting examples, alkyl groups may include methyl, ethyl, propyl, butyl, pentyl, hexyl, octyl, nonyl, and dodecyl.

As used herein, the term alkenyl refers to a straight or branched unsaturated monovalent (unless the context otherwise requires) acyclic or cyclic hydrocarbon radical having one or more C=C double bonds and having the specified number of carbon atoms (or, when not specified, an acyclic alkenyl group may contain 2-20, 2-10, 2-6, or 2-4 carbon atoms, and a cyclic alkenyl group may contain 3-20 or 5-7 carbon atoms), optionally substituted with one, two or three substituents independently selected from the group described above with respect to substituents which may be on R1, _R2 , R3 and R4. As non-limiting examples, alkenyl groups may include vinyl, propenyl, butenyl, pentenyl, and hexenyl.

- 10 040630

As used herein, the term alkynyl refers to a straight or branched unsaturated monovalent (unless the context otherwise requires) acyclic or cyclic hydrocarbon radical having one or more C=C triple bonds and having the specified number of carbon atoms (or, when not specified, an acyclic alkynyl group may contain 2-20, 2-10, 2-6 or 2-4 carbon atoms, and a cyclic alkynyl group may contain 8-20 carbon atoms), optionally substituted with one, two or three substituents, independently selected from the group described above with respect to the substituents that may be on R1, R ₂ , R ₃ and R4.

As used herein, the term alkoxy refers to an alkyl-O- group, where alkyl is as defined above, and where the alkyl moiety may optionally be substituted with one, two, or three substituents as described above for alkyl. Linking is via -O-. As non-limiting examples, alkoxy groups may include methoxy, ethoxy, n-propoxy, iso-propoxy, n-butoxy, t-butoxy, sec-butoxy, n-pentoxy, n-hexoxy, and 1,2dimethylbutoxy.

As used herein, the term aryloxy refers to an aryl-O- group, where aryl is as defined below, and where the aryl moiety may optionally be substituted with one, two, or three substituents as described above with respect to the Ar group. Linking is via -O-.

As used herein, the term alkoxyalkyl refers to an alkyl group having an alkoxy substituent. The bonding is via an alkyl group. The alkyl moiety and the alkoxy moiety have the meanings defined herein with respect to the definitions of alkyl and alkoxy, respectively. The alkoxy and alkyl moieties may each be substituted with one, two or three substituents as described above in terms of the definition of alkyl.

As used herein, the term arylalkyl refers to an alkyl group having an aryl substituent. The bonding is via an alkyl group. The aryl moiety and the alkyl group have the meanings defined herein with respect to the definitions of aryl and alkyl, respectively. The aryl and alkyl moieties may each be substituted with one, two or three substituents, where the substituents have the meanings defined herein when describing those substituents that may be present with respect to aryl and alkyl, respectively. In a preferred embodiment, the arylalkyl is benzyl, which is Ph-CH ₂ -.

As used herein, the term alkoxyaryl refers to an aryl group having an alkoxy substituent. The linkage is via an aryl group. The alkoxy moiety and the aryl moiety have the meanings defined herein with respect to the definitions of alkoxy and aryl, respectively. The alkoxy and aryl moieties may each be substituted with one, two, or three substituents, where the substituents have the meanings defined herein when describing those substituents that may be present with respect to alkoxy and aryl, respectively.

As used herein, the term cycloalkylaryl refers to an aryl group having a cyclic alkyl substituent. The linkage is via an aryl group. The cycloalkyl moiety and the aryl moiety have the meanings defined herein in relation to the definitions of cycloalkyl and aryl, respectively. The cycloalkyl moiety and the aryl moiety may each optionally be substituted with one, two, or three substituents as set forth herein in terms of the definitions of alkyl and aryl, respectively.

As used herein, the term aryl refers to a monovalent (unless the context otherwise requires) aromatic carbocyclic radical having one, two, three, four, five, or six rings and having the indicated number of carbon atoms (or, when not specified, 6 to 30, 6 to 12, or 6 to 11 carbon atoms). Preferably there are one, two or three rings. The aryl group may optionally be substituted with one, two, three, four or five substituents as described above with respect to the optional substituents that may be present on the Ar group. In preferred embodiments of the invention, the aryl group includes:

aromatic monocyclic ring containing 6 carbon atoms; aromatic fused bicyclic ring system containing 7, 8, 9 or 10 carbon atoms; or an aromatic fused tricyclic ring system containing 10, 11, 12, 13 or 14 carbon atoms. Non-limiting examples of aryl include phenyl and naphthyl. In a preferred embodiment, the optional group substituent on the aryl group can be independently selected from hydroxy, C _1-6 acyl, C _1-6 acyloxy, nitro, amino, carboxyl, cyano, C _1-6 alkylamino, diC _1-6 alkylamino, thiol , chlorine, bromine, fluorine, iodine, SO ₃ H, SH and SR', where R' is independently selected from the same group as R1 with respect to formula Ia.

As used herein, the term aryl refers to a monovalent (except where the context otherwise requires) aromatic carbocyclic radical having

- 11 040630 mu one, two, three, four, five or six rings and having the specified number of carbon atoms (or, when not specified, from 6 to 30, from 6 to 12 or from 6 to 11 carbon atoms). Preferably there are one, two or three rings. The aryl group may optionally be substituted with one, two, three, four or five substituents as described above with respect to the optional substituents that may be present on the Ar group. In preferred embodiments of the invention, the aryl group includes:

aromatic monocyclic ring containing 6 carbon atoms; aromatic fused bicyclic ring system containing 7, 8, 9 or 10 carbon atoms; or an aromatic fused tricyclic ring system containing 10, 11, 12, 13 or 14 carbon atoms. Non-limiting examples of aryl include phenyl and naphthyl. In a preferred embodiment, the optional group substituent on the aryl group can be independently selected from hydroxy, C _1-6 acyl, C _1-6 acyloxy, nitro, amino, carboxyl, cyano, C _1-6 alkylamino, diC _1-6 alkylamino, thiol , chlorine, bromine, fluorine, iodine, SO ₃ H, SH and SR', where R' is independently selected from the same group as R1 with respect to formula Ia.

As used herein, the term _5-30 heteroaryl refers to a monovalent (unless the context otherwise requires) unsaturated aromatic heterocyclic radical having 5 to 30 ring members as one, two, three, four, five, or six fused rings and at least one ring containing at least one heteroatom selected from the group consisting of N, O and S. In a preferred embodiment, there are one, two or three fused rings. Available carbon atoms and/or heteroatoms in the ring system may be substituted on the ring with one, two, three, four or five substituents as described above with respect to substituents that may be present on the Ar group. Heteroaryl groups may include an aromatic monocyclic ring system containing six ring members, of which at least one ring member is a N, O, or S atom, and which optionally contains one, two, or three additional N ring atoms; an aromatic monocyclic ring containing six members, of which one, two or three ring members represent an N atom; an aromatic bicyclic fused ring system containing nine members, of which at least one ring member is a N, O or S atom and which optionally contains one, two or three additional N ring atoms; or an aromatic bicyclic fused ring system containing ten ring members, of which one, two, or three ring members are an N atom. Examples include, but are not limited to, pyridyl and quinolyl.

As used herein, the term _5-20 heterocyclyl refers to a monovalent (unless the context otherwise requires) saturated or partially unsaturated heterocyclic radical having 5 to 20 ring members, wherein at least one ring member is selected from the group , including N, O and S, and presented in the form of one, two, three, four, five or six fused rings. In a preferred embodiment, the radical contains one, two or three rings. In a preferred embodiment, the radical contains from 5 to 10 ring members. Heterocyclyl radicals may include: a monocyclic ring system containing five ring members, of which at least one ring member is an N, O or S atom, and which optionally contains one additional O ring atom or one, two or three additional N ring atoms ; a monocyclic ring system containing six ring members, of which one, two or three ring members are an N atom and which optionally includes an O atom; a bicyclic fused ring system containing nine ring members, of which at least one ring member is a N, O, or S atom and which optionally contains one, two, or three additional N ring atoms; or a bicyclic fused ring system containing ten ring members, of which one, two, or three ring members are N. Examples include, but are not limited to, pyrrolinyl, pyrrolidinyl, 1,3-dioxolanyl, imidazolinyl, imidazolidinyl, pyrazolinyl, pyrazolidinyl , piperidinyl, morpholinyl or piperazinil.

The available ring carbons and/or ring heteroatoms of the heterocyclyl ring systems described above may be substituted with one, two, three, four or five substituents. When the ring(s) is(are) substituted with one or more heteroatoms, the heteroatomic substituents are selected from halogen (F, Cl, Br and I) and from oxygen, nitrogen and sulfur, where oxygen, nitrogen or sulfur form part of the substituent moiety. When the ring(s) is(are) substituted with one or more heteroatoms, it is preferred that 1, 2, 3 or 4 heteroatomic substituents are selected from the group consisting of oxygen, nitrogen, sulfur and halogen. Examples of substituent groups that may be present in the heterocyclic ring system may be independently selected from hydroxy, C ₁ . ₆ acyla, C ₁ . ₆ acyloxy, nitro, amino, carboxyl, cyano, C _1-6 alkylamino, diC _1-6 alkylamino, thiol, chlorine, bromine, fluorine, iodine, SO ₃ H, SH and SR', where R' is independently selected from the same groups as R ₁ with respect to formula Ia.

As used herein, the term acyl refers to straight or branched, saturated or unsaturated, substituted or unsubstituted, monovalent (excluding

- 12 040630 we eat cases when the context implies otherwise) to a radical that includes a moiety -C(=O)-, where the bonding is through the atom -C- of the moiety -C(=O)-, and contains the indicated number of atoms (or, when not specified, the acyl group has 1-6, or 1-4, or 1-2 carbon atoms, including the C atom of the -C(=O)- moiety, optionally substituted with one, two, or three substituents independently selected from the group, described above with respect to substituents which may be present on R1, _R2 , _R3 and R4. As non-limiting examples, acyl groups include HC(=O)-, CH ₃ C(=O)-, C2H ₅ C(=O)-, C ₃ H ₂ C(=O)-, C4H ₉ C(=O ) - and C ₅ H _n C(=O)-.

As used herein, the term acyloxy refers to a straight or branched, saturated or unsaturated, substituted or unsubstituted monovalent (unless the context indicates otherwise) radical that includes a moiety -C(=O)-O-, where the linkage is carried through the -O- atom, and contains the indicated number of atoms, including the C atom of the fragment -C (= O) -O - (or, when not indicated, the acyloxy group contains 1-6, 1-4 or 1-2 carbon atoms , including the carbon atom of the fragment -C(=O)-O)-), optionally substituted with one, two or three substituents, which may be present on R1, R ₂ , R ₃ and R ₄ . As non-limiting examples, acyloxy groups include HC(=O)-O-, CH ₃ C(=O)-O-, C ₂ H ₅ C(=O)-O-, C ₃ H ₇ C(=O) -O-, C ₄ H ₉ C (=O) -O- and C ₅ H _n C (=O) \u003d O-.

As used herein, the term C ₂ _ ₆ ester refers to a substituted or unsubstituted monovalent (unless the context otherwise requires) radical that contains R ₁₈ C(=O)-OR ₁₉ where R ₁₈ is selected from group containing H and C _1-4 alkyl, and R ₁₉ is selected from the group including C ₁ .5 alkyl, taking into account the maximum total number of C atoms, including the C atom of the moiety -C(=O)-O- from R ₁₈ C( =O)-OR ₁₉ equal to six. The bonding is through R ₁₈ or R19, with no H on the corresponding group, so that the alkyl group through which the bonding occurs is divalent, or, when R ₁₈ is H, through the C atom of the moiety -C(=O)-O -. In a preferred embodiment, the C2-6 ester, including the C atom of the -C(=O)-O moiety, has 2-5 carbon atoms. The C _2-6 ester may optionally be substituted with one, two or three substituents independently selected from the group described above with respect to the substituents that may be present on R1, R ₂ , R ₃ and R ₄ . By way of non-limiting example, the C ₂ _ ₆ ester can be -C ₂ H ₄ -C(=O)-O-C ₂ H ₅ , where the -C ₂ H ₄ moiety is -CH2-CH2-, and the binding carried out through the -C2H4- fragment.

As used herein, the term aldehyde refers to a straight or branched, saturated or unsaturated, substituted or unsubstituted monovalent (unless the context otherwise requires) radical that contains HC(=0)-R _2O -, where the linkage is through -R ₂₀ -, contains the indicated number of atoms, including the C atom of the fragment e -C(=O)- (or, when not indicated, the aldehyde group has 1-6, 1-4 or 1-2 carbon atoms, including the atom C fragment -C(=O)-), optionally substituted with one, two or three substituents, which may be present on R1, R ₂ , R ₃ or R ₄ . As non-limiting examples, aldehyde groups include HC(=O)-CH ₂ -, HC (=O)-C ₂ H ₄ -, HC (=O)-C ₃ H ₆ -, HC (=O)-C ₄ H ₈ - and HC (= 0) -C ₅ H _1O -.

As used herein, the term fluoroalkyl refers to an alkyl group, where the alkyl group is a straight or branched saturated monovalent (unless the context otherwise requires) cyclic or acyclic hydrocarbon radical having the indicated number of carbon atoms (or, when not specified, an acyclic alkyl group has 1-6 or 1-4 carbon atoms, and a cyclic alkyl group has 3-6 carbon atoms) substituted with 1 to 6 F atoms.

As used herein, the term fluoroalkenyl refers to an alkenyl group, where the alkenyl group is a straight or branched unsaturated monovalent (except where the context otherwise requires) acyclic or cyclic hydrocarbon radical having one or more C=C double bonds, and containing the specified number of carbon atoms (or, when not specified, an acyclic alkenyl group has 2-6 or 2-4 carbon atoms and a cyclic alkenyl group has 4-6 carbon atoms) substituted with 1 to 6 F atoms.

The process for preparing a compound of formula Ia or Ib is preferably carried out in the presence of a suitable solvent.

Suitable solvents include hydrocarbon solvents such as benzene and toluene; ether type solvents such as diethyl ether, tetrahydrofuran, diphenyl ether, anisole and dimethoxybenzene; halogenated hydrocarbon solvents such as methylene chloride, chloroform and chlorobenzene; ketone type solvents such as acetone, methyl ethyl ketone and methyl isobutyl ketone; alcohol type solvents such as methanol, ethanol, propanol, isopropanol, n-butyl alcohol and t-butyl alcohol; nitrile type solvents such as acetonitrile, propionitrile and benzonitrile; ester type solvents such as ethyl acetate and butyl acetate; carbonate type solvents such as ethylene carbonate and propylene carbonate; etc. They may be used alone, or two or more of them may be used in a mixture.

Preferably, an inert solvent is used in the process of the present invention.

- 13 040630

The term "inert solvent" means a solvent that is inert under the reaction conditions with which it is described, including, for example, benzene, toluene, acetonitrile, tetrahydrofuran, dimethylformamide, chloroform, methylene chloride (or dichloromethane), diethyl ether, ethyl acetate, acetone, methyl ethyl ketone, methanol , ethanol, propanol, isopropanol, t-butanol, dioxane, pyridine and the like. Tetrahydrofuran is particularly preferred.

Preferably, the process of the present invention is carried out under substantially dry conditions.

Phosphorochloridate can be prepared starting from aryloxy phosphorodichloidate and an appropriately protected amino acid derivative. Alternatively, phosphate synthesis can be applied using suitable condensing agents.

Preferably, the process for preparing a compound of formula Ib may include the step of protecting a free OH group on the nucleoside other than that to which the phosphoramidate is attached. For example, reacting a 3'-deoxynucleoside with the desired phosphorochloridate in the presence of t-BuMgCl produces 2'-phosphoramidate.

Reaction of the 3'-deoxynucleoside with POCl ₃ , then with the N ⁺ R ₅ R ₆ H ₂ salt affords compounds where each of U and V is -NR ₅ R ₆ . Suitable salts include chloride, tosylate, sulfonate, and ester salts such as 4-methylbenzenesulfonate. The subsequent addition of a base such as diisopropylethylamine may aid the process.

As used herein, the term stereoisomer defines all possible compounds consisting of the same atoms linked by the same bond sequence, but having different three-dimensional structures that the compounds of the present invention may have.

When the compounds according to the invention have at least one chiral center, they may accordingly exist as enantiomers. When the compounds have two or more chiral centers, they may furthermore be in the form of diastereoisomers. When the process for preparing compounds according to the invention results in a mixture of stereoisomers, these isomers can be separated by conventional methods such as preparative chromatography. The compounds may be prepared stereochemically mixed, or the individual enantiomers may be prepared by standard methods known to those skilled in the art, such as enantiospecific synthesis or resolution, formation of diastereomeric pairs by salting with an optically active acid followed by fractional crystallization and isolation of the free base. Compounds can also be resolved by formation of diastereomeric esters or amides followed by chromatographic separation and removal of the chiral auxiliary. Alternatively, compounds can be separated using a chiral HPLC column. It is to be understood that all such isomers and mixtures thereof are within the scope of the present invention.

In addition, it should be understood that the phosphate center is chiral in the compounds of the present invention, and the compounds may exist as diastereoisomers R _P and S _P . The compound may be a mixture of R _p and S _p or be a single pure diastereoisomer. In a preferred embodiment, the compound is a substantially pure diastereoisomer of either R _P or S _P . By substantially pure single diastereoisomer is meant that the compound consists of 98% or more of either the R _P diastereoisomer or the S _P diastereoisomer. In another embodiment, it may be a 1:1 mixture of diastereoisomers R _P and S _P . Alternatively, the compound may contain a mixture of R _P and S _P diastereoisomers in a ratio of R _P and S _P diastereoisomers from 1:90 to 90:1, from 1:50 to 50:1, from 1:20 to 20:1, from 1: 15 to 15:1, 1:10 to 10:1, 1:9 to 9:1, 1:8 to 8:1, 1:7 to 7:1, 1:6 to 6:1 , 1:5 to 5:1, 1:4 to 4:1, 1:3 to 3:1, or 1:2 to 2:1. In preferred embodiments, the compound of the invention may contain a mixture of R _p and Sp diastereoisomers greater than 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15, 1:20, 1:50, 1:90, 1:95 or 1:99 or vice versa.

The term solvate means a compound of formula Ia or formula Ib as described above, wherein suitable solvent molecules are embedded in the crystal lattice. A suitable solvent is physiologically tolerable at the dosage administered. Examples of suitable solvents are ethanol, water and the like. When the solvent is water, the molecule is referred to as a hydrate.

The compounds of the present invention may also be presented as pharmaceutically acceptable salts. For use in medicine, the salts of the compounds of the present invention are indicated as pharmaceutically acceptable salts. FDA-approved pharmaceutically acceptable salt forms (Ref. International J. Pharm. 1986, 33, 201-217; J. Pharm. Sci., 1977, Jan, 66 (1)) include pharmaceutically acceptable acid/anionic or basic/cationic salts.

Pharmaceutically acceptable acid/anionic salts include, but are not limited to, acetate, benzenesulfonate, benzoate, bicarbonate, bitartrate, bromide, calcium edetate, camsylate, carbonate, chloride, citrate, dihydrochloride, edetate, edisylate, estral, esylate, fumarate, glyceptate, gluconate, glutamate, glycolylarsanilate, hexylresorcinate, hydrabamine, hydrobromide, hydroxynaphthoate, iodide, isethionate, lactate, maleate, mandelate, mesylate, methyl bromide, methyl nitrate, methyl sulfate, mucat, napsilate, nitrate, pamoate, pantothenate, phosphate, diphosphate, polygalacturonate, salicylate, stearate, subacetate, succinate, sulfate, tannate, tartrate, theoglat, tosylate and triethiodide.

Pharmaceutically acceptable base/cationic salts include, but are not limited to, aluminum, benzathine, calcium, chlorprocaine, choline, diethanolamine, ethylenediamine, lithium, magnesium, potassium, procaine, sodium, and zinc salts.

The present invention includes within its scope prodrugs of the compounds of the present invention. Typically, such prodrugs are functional derivatives of compounds that are readily converted in vivo to the desired compound. Thus, in the methods of treatment of the present invention, the term administration will encompass the treatment of the various disorders described using a compound as specifically described, or using a compound that may not be specifically described, but which is converted in vivo to said compound upon administration to a subject. Conventional methods for selecting and preparing suitable prodrug derivatives are described, for example, in Design of Prodrugs, ed. H. Bundgaard, Elsevier, 1985.

Pharmaceutically acceptable ester derivatives in which one or more free hydroxy groups are esterified as a pharmaceutically acceptable ester are specific examples of prodrug esters that can be converted by solvolysis under physiological conditions to compounds of the present invention having free hydroxy groups.

Pharmaceutical compositions for use in accordance with the present invention may be formulated in the usual manner using one or more physiologically acceptable carriers containing excipients and adjuvants which facilitate the processing of the active compounds into formulations which can be used pharmaceutically. These pharmaceutical compositions may be prepared in a manner which is known per se, for example by known conventional methods of mixing, dissolving, granulating, panning, milling, emulsifying, encapsulating, entrapping or lyophilizing. Appropriate preparations depend on the chosen route of administration.

A compound or pharmaceutical composition according to the present invention may be administered to a patient, which may be homo sapiens or an animal, by any suitable means.

The drugs used in the present invention may be administered by the oral or parenteral route, intravenous, intramuscular, intraperitoneal, subcutaneous, transdermal, air (aerosol), rectal, vaginal, and topical (including buccal and sublingual) administration.

For oral administration, the compounds of the invention are generally provided as tablets or capsules, as a powder or granules, or as an aqueous solution or suspension.

Tablets for oral use may contain the active ingredient mixed with pharmaceutically acceptable excipients such as inert diluents, disintegrating agents, binding agents, lubricating agents, sweetening agents, flavoring agents, coloring agents and preservatives. Suitable inert diluents include sodium and calcium carbonate, sodium and calcium phosphate, and lactose, with cornstarch and alginic acid being suitable disintegrating agents. Binding agents may include starch and gelatin, while the lubricant, if present, is usually magnesium stearate, stearic acid or talc. If desired, tablets may be coated with a material such as glyceryl monostearate or glyceryl distearate to delay absorption in the gastrointestinal tract.

Tablets for oral use may contain the active ingredient mixed with pharmaceutically acceptable excipients such as inert diluents, disintegrating agents, binding agents, lubricating agents, sweetening agents, flavoring agents, coloring agents and preservatives. Suitable inert diluents include sodium and calcium carbonate, sodium and calcium phosphate, and lactose, with cornstarch and alginic acid being suitable disintegrating agents. Binding agents may include starch and gelatin, while the lubricant, if present, is usually magnesium stearate, stearic acid or talc. If desired, tablets may be coated with a material such as glyceryl monostearate or glyceryl distearate to delay absorption in the gastrointestinal tract.

Capsules for oral administration include hard gelatin capsules in which the active ingredient is mixed with a solid diluent and soft gelatin capsules in which the active ingredient is mixed with water or an oil such as peanut oil, liquid paraffin or olive oil.

Formulations for rectal administration may be presented as suppositories with a suitable base containing, for example, cocoa butter or salicylate.

Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or aerosol preparations containing, in addition to the active ingredient, such carriers as are known in the art to be suitable.

For intramuscular, intraperitoneal, subcutaneous and intravenous administration, the compounds of the invention are generally provided as sterile aqueous solutions or suspensions buffered to suitable pH and isotonicity. Suitable aqueous drug vehicles include Ringer's solution and isotonic sodium chloride solution. Aqueous suspensions according to the invention may contain suspending agents such as cellulose derivatives, sodium alginate, polyvinylpyrrolidone and gum tragacanth, and a wetting agent such as lecithin. Suitable preservatives for aqueous suspensions include ethyl and n-propyl p-hydroxybenzoate.

The compounds of the invention may also be presented as liposome preparations.

Typically, a suitable dose will be in the range of 0.1 to 300 mg per kilogram of body weight of the recipient per day. A more suitable dose may be in the range of 0.5 to 150 mg per kilogram of recipient body weight per day, in the range of 0 to 5 to 100 mg per kilogram of recipient body weight per day, in the range of 1-50 mg per kilogram of body weight of the recipient. recipient per day, or in the range of 1 to 10 mg per kilogram of recipient body weight per day. A suitable lower dose may be 0.5 mg per kilogram of recipient body weight per day or 1 mg per kilogram of recipient body weight per day. Alternatively, a suitable dose may be in the range of 1 to 100 mg per m ² of the recipient's body surface area per day, or 5 to 50 mg per m ² of the recipient's body surface area per day. Suitable doses may be 6, 12, 24 or 48 mg per m ² of the recipient's body surface area per day. The desired dose may be presented and administered as a single daily dose or as two, three, four, five or six or more sub-doses administered at appropriate intervals throughout the day. Doses may be administered in unit dosage forms, for example containing 10 to 1500 mg, preferably 20 to 1000 mg, and most preferably 50 to 700 mg of the active ingredient per unit dosage form. The total daily dose is, respectively, 1000-3000 mg, regardless of whether it is taken as a single dose or as subdoses at intervals throughout the day.

Cancer stem cells.

Cancer stem cells, sometimes referred to as tumor-initiating cells, are well known to those skilled in the art. As used herein, the term cancer stem cell should be understood in accordance with its broad meaning, i.e., as a cell that has the ability to self-renew by asymmetric division, thereby initiating the formation of tumors, and also, when differentiated, gives rise to more mature non-stem progeny cells.

Cancer stem cells play an important role in the development, progression, recurrence and spread of malignant neoplasms. Accordingly, the finding that the compounds of the present invention can target cancer stem cells, and thus reduce their number, provides therapeutic options for the prevention or treatment of such activity.

As discussed in more detail elsewhere herein, cancer stem cells have been found in precancerous conditions, the presence of which is believed to contribute to the development of these conditions into malignant neoplasms.

Accordingly, the methods of treatment and medical use of the invention, which use the compound of the present invention to target cancer stem cells, can be used to reduce the number of cancer stem cells in precancerous conditions (such as myelodysplastic syndrome or other conditions discussed elsewhere in the description) and, thus, to prevent the progression of such precancerous conditions into malignancy.

As stated above, asymmetric cell division of cancer stem cells results in differentiated cancer non-stem cells. Thus, cancer stem cells are responsible for the formation and maintenance of the bulk of the tumor.

The accumulation of such non-stem cancer cells plays an important role in the progression of malignant neoplasms. Targeting a compound of the present invention to cancer stem cells can reduce the number of cancer stem cells, which in turn reduces the number of non-stem cancer cells in the offspring. Thus, the methods of treatment and medical use of the compound of the present invention in accordance with the present invention are effective in the treatment of cancer by preventing the progression of cancer. Such embodiments are described in more detail elsewhere in this specification.

Cancer stem cells are also able to act as a reservoir of cancer cells, which can cause cancer to recur after remission. Even when most of a patient's cancer cells have been destroyed (for example, by surgery, radiation therapy, or chemotherapy, either alone or in combination) and there is no evidence of cancer, the continued presence of cancer stem cells can lead to recurrence of malignancy. innovations over time.

Targeting cancer stem cells with a compound of the present invention provides a novel method by which the number of cancer stem cells can be reduced and the cancer stem cells destroyed. Accordingly, and as detailed elsewhere herein, in appropriate embodiments, the present invention relates to methods and medical uses in which a compound of the present invention prevents or delays the recurrence of cancer.

- 16 040630

In addition, the movement of cancer stem cells from a site of a malignant neoplasm to another site in the body may contribute to the spread of the malignant neoplasm, for example by giving rise to metastases. Therefore, the ability of a compound of the invention to target cancer stem cells thus provides new treatments and medical applications for preventing or treating the spread of cancer.

In addition to their biological activity, cancer stem cells can be identified by the expression of certain characteristic cell surface markers. Cancer stem cells identified in hematological malignancies are typically CD34+, while CD44+, CD133+ and CD90+ have been identified as cancer stem cell markers in solid tumors. The following table lists examples of known surface phenotypes of cancer stem cells. It is to be expected that the compound of the present invention can target each of these forms of cancer stem cells in accordance with the present invention, and therefore methods or uses using the compounds of the present invention can be used to prevent or treat malignancies associated with cancer. stem cells expressing any of these sets of markers.

Table I

Type of tumor Known cell surface markers of cancer stem cells solid tumors Breast SG4 4 ⁺ /SG2 4-/ ^low /line' /EZA ⁺ CNS CD133+ Colon CD133+ Colon _ЕЗ Аvysokaya / _С04 4+ / LINE- / (CD166 ⁺ ) Ewing's sarcoma CD133 ⁺ Head and neck CO44 ⁺ /line- Melanoma ABCB5 ⁺ Liver CD90+/CD45-/ (CD44 ⁺ ) Cholangiocarcinoma CD44 ⁺ /GLI1 ⁺ (glioma-associated oncogene homologue-1) ovaries CD44+/CD117+ Pancreas CD44 ⁺ /CD24 ⁺ /ESA ⁺ Pancreas CD133+ Non-small cell cancer lungs CD44 ⁺ /Ver-EP4 ⁺ bladder cancer CD4 ⁴⁺ /ALDHlAl ⁺ Hematological tumors Acute myeloid leukemia Lin-/CD34 ⁺ /CD38-/CD123 ⁺ B-cell acute lymphoblastic leukemia CD34+/CD10- or CD34+/CD19- B-cell acute lymphoblastic leukemia CD34 ⁺ /CD38-/CD19 ⁺ multiple myeloma CD34-/CD138- T-cell acute lymphoblastic leukemia CD34+/CD4- or CD34+/CD7-

The data presented in the examples show that the compound of the present invention is capable of targeting cancer stem cells of a leukemic cell line, specifically cancer stem cells present in the acute myeloid leukemia KG1a cell line. A small stem cell compartment with a distinct immunophenotype (Lin ^- /CD34 ⁺ /CD38 ^- /CD123 ⁺ ) was found in this cell line, which is targeted by the compound of the present invention. Accordingly, the methods of treatment or medical use of the compound of the present invention in accordance with the present invention can be used to prevent or treat leukemias and other types of malignancies associated with cancer stem cells expressing these characteristic markers.

The present invention also relates to methods and medical uses in which patients are selected for the prevention or treatment of cancer using a compound of the invention based on the identification of the presence of cancer stem cells in a biological sample characteristic of a patient suffering from malignancy or having a precancerous condition. The markers above provide suitable examples that

- 17 040630 can be used to identify the presence of cancer stem cells in accordance with such embodiments of the invention. Suitable methods by which the expression of these markers in a biological sample can be examined are discussed further in the present description, a biological sample characteristic of a patient suffering from a malignant neoplasm or having a precancerous condition.

Targeting cancer stem cells.

The first feature of the present invention is the fact that the compounds of the present invention can be used to target cancer stem cells. The ability of the compounds of the invention to target cancer stem cells is illustrated in the examples described herein.

As can be seen, if the compound of the present invention enters a population of malignant cells containing cancer stem cells, it will target the cancer stem cells present, resulting in a decrease in the total number of malignant cells. As already discussed elsewhere in this specification, some of the compounds of the present invention target cancer stem cells rather than tumor cell masses, and the activity of such compounds can not only reduce the total number of cancer cells present, but also reduce the proportion of the total number of cancer cells that exhibit phenotypic markers of cancer stem cells.

It is assumed that the compounds of the present invention penetrate into malignant cells and are integrated into the nucleic acids (RNA and/or DNA) of these cells. Without being bound by any theory, it is believed that the efficacy, especially anti-cancer efficacy, exhibited by the compounds of the present invention indicates that the compounds of the present invention are phosphorylated to cordycepin triphosphate or a cordyceptin derivative (e.g., 2-fluorocordyceptin or 2-C1- cordyceptin) and suggest that enzymatic cleavage within the cell converts the compound of the invention directly to 8-chloroadenosine monophosphate before being phosphorylated to triphosphate.

It is also believed that the compounds of the present invention have increased cell membrane permeability (compared to cordycepin) and this contributes to the increased anticancer activity of the compounds of the invention compared to the parent compound from which they are derived.

Without wishing to be bound by any hypothesis, the inventors believe that the reduction in the number of cancer stem cells results from the targeted destruction of cancer stem cells in a population of malignant cells. Thus, the compounds of the present invention are capable of inducing the death of cancer stem cells. In addition, the results reported herein indicate that certain compounds of the present invention appear to preferentially kill cancer stem cells over non-cancer stem cells, thereby causing not only cancer stem cell death, but also a reduction in the proportion of cancer cells. stem cells in the general population of malignant cells.

Although the present inventors believe that the compounds of the present invention, which preferentially target cancer stem cells, preferentially kill cancer stem cells over non-stem cancer cells, other mechanisms mediated by the compound of the present invention may also contribute to the reduction in the proportion of cancer stem cells. targeting these cells.

Just by way of example, treatment with a compound of the invention can result in increased differentiation of cancer stem cells, thereby reducing the numbers of cancer stem cells as well as their proportion of total cancer cells represented by cancer stem cells. Alternatively, the compound of the present invention can cause cancer stem cells to lose the stem cell phenotype, such as losing the ability to self-renew, thereby reducing the number of cancer stem cells.

The indications for targeting cancer stem cells in the present description should be interpreted accordingly. For purposes of this disclosure, targeting cancer stem cells can be understood to include any mechanism by which a compound of the present invention reduces the amount of cancer stem cells present in a cell population, both in vitro and in vivo. In particular, targeting cancer stem cells can be understood to include preferentially reducing the number of cancer stem cells compared to other cell types, in particular compared to non-stem cancer cells. Targeting indications herein may be understood to include killing, and possibly predominantly killing, cancer stem cells as compared to non-stem cancer cells.

Prevention and treatment of malignant neoplasm.

The invention relates to medical uses and methods of treatment in which the compound of the present invention is used to prevent or treat cancer. In the context of the present invention, the term cancer prevention

- 18 040630 should be considered as referring to the prophylactic use of the compound of the present invention, used before the development of a malignant neoplasm, and with the aim of stopping the development of a malignant neoplasm. On the other hand, the treatment of cancer should be understood to be the use of the compound of the present invention after the cancer has occurred to reduce the cancer by slowing down or stopping the proliferation of malignant cells and tumor growth. Preferably, treatment of a malignant neoplasm may cause a partial or complete reduction in the number of malignant cells and tumor size. Effective treatment of malignancy can result in the disease either stabilizing or responding according to the RECIST guidelines (criteria for evaluating response in solid tumors).

As described in more detail below, the prevention of malignancy in accordance with the present invention can be particularly effective in patients with a precancerous condition in which there is an increased likelihood of developing malignancy.

Prevention of malignant neoplasm.

Prevention of cancer in accordance with the present invention can be carried out by treating a precancerous condition using the compound of the present invention in accordance with various aspects or embodiments of the present invention, as described herein.

In particular, the prevention of cancer in the context of the present invention can be achieved by the methods or medical applications according to the present invention, in which a patient with a precancerous compound receives a compound of the present invention. The methods of treatment or medical use according to this embodiment can prevent the treated precancerous conditions from progressing to malignancy, thereby providing effective prevention of malignancy.

Indications for the prevention of cancer in the context of the present invention may also include other prophylactic uses of the compound of the present invention. For example, the ability of a compound of the invention to target cancer stem cells and thereby prevent the development of a cancer and/or prevent the progression of a cancer and/or prevent the recurrence of a cancer and/or prevent the spread of a cancer.

precancerous conditions.

A malignant neoplasm is often preceded by the development of a precancerous condition, which is not itself malignant, but is associated with an increased risk of developing a malignant neoplasm. The accumulation of genetic or epigenetic changes can lead to the development of previously normal cells into a cancer stem cell phenotype. Accordingly, cancer stem cells may also be present in such precancerous conditions as well as in malignancy.

It is believed that the presence of cancer stem cells in precancerous conditions contributes to the development of these conditions into malignancy. The methods and medical uses of the present invention can be used to target cancer stem cells present in precancerous conditions and thus to treat such conditions. It should be appreciated that the new and unexpected finding that the compounds of the invention target cancer stem cells means that treatment of precancerous conditions with such compounds can be used to prevent the treated conditions from developing into malignancy. This provides a way in which the compound of the present invention can be used from a medical point of view for the prevention of malignancy, as discussed in the present description.

Examples of precancerous conditions that may be treated according to the present invention include, but are not limited to, conditions selected from the group consisting of: senile keratosis, Barrett's esophagus, atrophic gastritis, dyskeratosis congenita, sideropenic dysphagia, lichen planus, oral submucosal fibrosis, solar elastosis, cervical dysplasia, leukoplakia, erythroplakia, monoclonal gammopathy of unknown origin (MGUS), monoclonal B-cell lymphocytosis (MBL), myelodysplastic syndromes, and precancerous gastric conditions such as atrophic gastritis, gastric ulcer, pernicious anemia , gastric stump, gastric polyps and Menetrier's disease. Of the listed precancerous conditions of the stomach, atrophic gastritis, pernicious anemia, gastric stump, and some types of gastric polyps may have a particularly increased risk of developing a malignant neoplasm.

Precancerous conditions often take the form of lesions containing dysplastic or hyperplastic cells. Accordingly, the presence of dysplasia or hyperplasia, alternatively or in addition to the presence of cells with expressed markers or a phenotype characteristic of cancer stem cells, can be used to identify precancerous conditions.

The severity of dysplasia can change in a number of different precancerous conditions or with the development

- 19 040630 a single precancerous condition over time. In general, the later the stage of dysplasia associated with a precancerous condition, the more likely it is that the precancerous condition will develop into a malignant neoplasm. Dysplasia is usually classified as mild, moderate, or severe. Severe dysplasia usually develops into malignancy if left untreated. Accordingly, methods of treatment or medical use using the compound of the present invention can thus be used to treat a patient with a precancerous condition associated with severe dysplasia.

In a corresponding embodiment of the present invention, the compound of the invention is used to treat a patient with severe cervical dysplasia. Severe cervical dysplasia can be diagnosed with a cervical smear. In another embodiment of the present invention, the compound of the present invention is used to treat severe esophageal dysplasia (Barrett's esophagus). Severe esophageal dysplasia can be diagnosed after tissue biopsy.

According to recent information, pre-malignant tumors can also be identified by detecting somatic mutations in the cells of individuals who are not known to have a malignant neoplasm. Specifically, age-related clonal hematopoiesis has been reported to be a common precancerous condition that is associated with increased overall mortality and an increased risk of cardiometabolic disease. Most of the mutations found in blood cells occur in three genes: DNMT3A, TET2 and ASXL1. Accordingly, patients who would benefit from the use of a compound of the invention for targeting cancer stem cells, and thus for treating precancerous conditions, can be identified by analyzing a sample containing blood cells for the presence of genetic mutations indicative of a precancerous condition, at least at least one of the following: DNMT3A and/or TET2 and/or ASXL1.

Precancerous conditions for which treatment with a compound of the invention by targeting cancer stem cells according to the invention can be effective can also be identified by detecting the presence of cancer stem cells with reference to any of the techniques based on the expression of markers characteristic of cancer stem cells or phenotype of cancer stem cells, as described elsewhere in the description.

Treatment of a malignant neoplasm.

One of skill in the art will appreciate that there are many parameters by which cancer treatment can be judged. Just by way of example, any reduction or prevention of cancer development, cancer progression, cancer recurrence, or cancer spread should be considered an indication of effective cancer treatment.

In some embodiments, a compound of the invention may be used: to reduce the proportion of cancer stem cells in a cancer cell population; and/or to inhibit tumor growth; and/or to reduce oncogenicity; and/or for the prevention or treatment of primary malignancy; and/or to prevent or treat recurrent cancer; and/or to prevent or treat metastatic or secondary malignancy; and/or for the treatment, prevention or inhibition of metastases and relapses; and/or to treat or prevent untreatable cancer.

The ability to reduce tumor size, as well as maintain a reduced tumor size during/after the treatment period, in the treatment of malignancy using the compound of the invention, is an extremely important indication of the effectiveness of the treatment of malignancy. As indicated in the examples, the treatment or medical application according to the present invention was surprisingly effective in this regard, even in models using cells representing relapsed or refractory cancers that were previously resistant to treatment with other types of therapeutic agents.

The data presented in the examples show that treatment with a compound of the present invention reduces the proportion of cancer stem cells in a population of cancer cells. Characteristic biological activities or cell surface markers by which cancer stem cells can be identified are described elsewhere in this specification. In a suitable embodiment, the treatment of cancer according to the present invention may result in a reduction in the proportion of cancer stem cells present in a patient suffering from cancer by at least 10%, at least 20%, at least 30%, or at least by 40%. In suitable embodiments, the cancer treatment of the present invention may result in a reduction in the proportion of cancer stem cells present in a patient suffering from cancer by at least 50%, at least 60%, at least 70%, or at least 80%. The treatment of malignancy according to the present invention may result in a reduction

- 20 040630 the proportion of cancer stem cells present in a patient suffering from a malignant neoplasm, at least 85%, at least 90%, or at least 95%. Indeed, the treatment of cancer according to the invention can result in a reduction in the proportion of cancer stem cells present in a patient's cancer by at least 96%, at least 97%, at least 98%, at least 99%. %, or even 100% (thus essentially no cancer stem cells remain).

The asymmetric division of cancer stem cells promotes the growth of tumors. Treatment of a malignant neoplasm with a compound of the invention in accordance with the present invention can result in inhibition of tumor growth by at least 10%, at least 20%, at least 30%, or at least 40%. Thus, treatment of a malignant neoplasm in accordance with the invention can lead to inhibition of tumor growth by at least 50%, at least 60%, at least 70%, or at least 80%. Treatment of a malignant neoplasm in accordance with the invention can lead to inhibition of tumor growth by at least 85%, at least 90%, or at least 95% in a patient receiving treatment. Indeed, the treatment of a malignant neoplasm in accordance with the invention can result in tumor growth inhibition of at least 96%, at least 97%, at least 98%, at least 99%, or even 100% of the treated tumor. .

Tumor growth can be assessed by any suitable method in which the change in tumor size is assessed over time. Applicable option, when the size of the tumor before the treatment of malignant neoplasms can be compared with the size of the same tumor during or after the treatment of malignant neoplasms. There are a number of ways in which tumor size can be estimated. For example, tumor size can be estimated by in situ imaging of the tumor in a patient. Appropriate methods, such as imaging techniques, may allow tumor volume to be determined and changes in tumor volume to be assessed.

As the results in the examples of the present disclosure show, the methods of treatment and medical use of the compound of the present invention are capable of not only delaying tumor growth, but are actually capable of reducing tumor volume in patients suffering from cancer, including patients with relapsed or refractory malignancy. neoplasm. Appropriate treatment of a malignant neoplasm in accordance with the invention can lead to a reduction in tumor volume of at least 10%, at least 20%, at least 30%, or at least 40%. In appropriate embodiments, the treatment of cancer according to the invention may result in a reduction in tumor volume of at least 50%, at least 60%, at least 70%, or at least 80%. Treatment of a malignant neoplasm of cancer in accordance with the invention can lead to a reduction in tumor volume of at least 85%, at least 90%, or at least 95%. Indeed, the treatment of a malignant neoplasm in accordance with the invention can lead to a reduction in tumor volume of at least 96%, at least 97%, at least 98%, at least 99%, or even 100%.

The reduction in tumor volume of the type described above can be calculated with reference to a suitable control. For example, in studies conducted in vitro or in vivo in suitable animal models, tumor volume reduction can be determined by directly comparing the volume of a tumor treated with a compound of the invention and that of a control tumor (which may be untreated, or may be treated with a compound other than the compound of the present invention). It should be borne in mind that such models requiring no tumor treatment may be ethically unacceptable in the context of clinical trials or therapeutic control of patients, in which case the reduction in tumor volume can be assessed by comparing the volume of the treated tumor and the volume of the tumor before treatment. or the predicted volume that would be achieved by the tumor in the absence of treatment.

Methods of treatment and medical use of the compounds of the present invention can lead to a decrease in the number of biomarkers indicative of malignancy. The reduction of such biomarkers provides an additional assessment by which one can demonstrate effective treatment of malignancy. Suitable examples of such biomarkers can be selected based on the type of cancer being treated: in the case of gynecological malignancies, CA125 is a suitable example of a biomarker, while in the case of pancreatic or bile duct cancer, CA19.9 is a suitable example of a biomarker, and in the case of colorectal cancer, CE may be a suitable biomarker.

Appropriate cancer treatment according to the invention can result in a reduction in cancer biomarkers of at least 10%, at least 20%, at least 30%, or at least 40%. In appropriate embodiments, the treatment of cancer in accordance with the invention may

- 21 040630 lead to a decrease in cancer biomarkers by at least 50%, at least 60%, at least 70%, or at least 80%. Treatment of cancer according to the invention can result in a reduction in cancer biomarkers of at least 85%, at least 90%, or at least 95%. Indeed, cancer treatment according to the invention can result in a reduction in cancer biomarkers of at least 96%, at least 97%, at least 98%, at least 99%, or even 100%.

Beneficial effects, such as a reduction in the proportion of cancer stem cells present, a reduction in tumor growth, or a reduction in tumor volume or malignancy biomarkers, observed with cancer treatment in accordance with the present invention, can be maintained for at least one month. Accordingly, such beneficial effects may persist for at least two months, at least three months, at least four months, at least five months, or at least six months. Indeed, such positive effects may persist for at least 12 months, at least 18 months, or at least 24 months. Accordingly, the beneficial effects may persist for at least three years, at least four years, at least five years, at least six years, at least seven years, at least eight years, at least nine years, or for ten years or more.

In a suitable embodiment of the present invention, the compound of the invention is used in a method for the prevention or treatment of a malignant neoplasm or precancerous condition by targeting cancer stem cells. In a suitable embodiment, the present invention relates to the use of a compound of the invention in a method for the prevention or treatment of a malignant neoplasm or precancerous condition, wherein the method reduces the tumorigenicity of one or more cancer stem cells. Accordingly, such methods can prevent the progression of cancer or inhibit tumor growth.

When a compound of the present invention is used in the methods or medical uses of the present invention to prevent or treat the progression of a cancer, such prevention or treatment may result in slowing, delaying or completely stopping the progression of the cancer.

The progression of a malignancy is usually determined by assessing the stage of the malignancy. Staging is usually done by assigning a number from I to IV to the cancer, where I is the isolated cancer and IV is the cancer that has spread to that limit, which is a scoring measure. Features of staging vary depending on the malignant neoplasm, but staging usually takes into account the size of the tumor, whether it has affected neighboring organs, how many regional (nearest) lymph nodes it has spread (if spread), and whether it has appeared in more distant places (metastasis).

Typically, stage I is localized to one part of the body and can be treated with surgical resection (for solid tumors that are quite small). Stage II is locally advanced and is treatable with chemotherapy, radiation therapy, surgery, or a combination. Stage III is also locally advanced, and designation stage II or stage III depends on the specific type of malignancy, although stage III is generally considered to be late locally advanced. Stage IV malignancy often metastasizes to a second organ. Treatment of cancer using a compound of the present invention in the methods or medical applications of the present invention can be used in the treatment of stage I, II, III or IV cancer by targeting cancer stem cells. Treatment with a compound of the invention can be used to prevent the progression of cancer from one stage to the next. In one embodiment, treatment with a compound of the present invention is used to prevent progression from stage I to stage II. In another embodiment, treatment with a compound of the present invention is used to prevent progression from stage II to stage III. In yet another embodiment, treatment with a compound of the present invention is used to prevent progression from stage III to stage IV.

Prevention or inhibition of cancer progression is particularly important to prevent the spread of cancer, such as progression from stage I to stage II when the cancer spreads locally, or progression from stage III to stage IV when the cancer metastasizes to other organs. Cancer stem cells are ongogenic and therefore are believed to play a critical role in the spread of malignancy, both locally and through metastases. The methods of treatment or medical use of the invention using a compound of the invention can therefore be used to prevent the spread of cancer by targeting and thus reducing the number of oncogenic cancer stem cells.

Malignant neoplasms.

The compounds of the present invention exhibit enhanced anticancer activity compared to the parent nucleoside from which they are derived. This increase in anti-cancer activity appears to be the result of increased activity against both cancer stem cells and non-stem cancer cells.

Cancer stem cells play a role in the biological activity of a wide range of malignant diseases. Accordingly, there is a wide range of cancers that can be prevented or treated in accordance with the present invention.

As described elsewhere in the specification, cancer stem cells are known to be present in many types of tumors, including fluid tumors (including hematologic tumors such as leukemias and lymphomas) and solid tumors (eg, breast, lung, colon tumors). , prostate, ovaries, skin, bladder, tumor of the biliary system and pancreas). Methods for the treatment and medical use of a compound of the invention by targeting cancer stem cells are thus expected to be effective in preventing or treating such cancers.

Thus, the compound of the invention can be used to prevent or treat cancer selected from the group consisting of: leukemia, lymphoma, multiple myeloma, lung cancer, liver cancer, breast cancer, head and neck cancer, neuroblastoma, thyroid cancer, skin cancer (including melanoma), oral squamous cell carcinoma, bladder cancer, Leydig cell tumor, biliary tract cancer such as cholangiocarcinoma or bile duct cancer, pancreatic cancer, colon cancer, colorectal cancer, and gynecological cancers, including ovarian cancer, endometrial cancer, fallopian tube cancer, uterine cancer and cervical cancer, including cervical epithelial cancer. In suitable embodiments, the malignancy is a leukemia and may be selected from the group consisting of: acute lymphoblastic leukemia, acute myeloid leukemia (also known as acute myeloid leukemia or acute non-lymphocytic leukemia), acute promyelocytic leukemia, acute lymphocytic leukemia, chronic myelogenous leukemia (also known as chronic myeloid leukemia, chronic myelocytic leukemia, or chronic granulocytic leukemia), chronic lymphocytic leukemia, monoblastic leukemia, and hairy cell leukemia. In other preferred embodiments, the malignancy is acute lymphoblastic leukemia. In a specific embodiment, the leukemia is a refractory TdT positive leukemia. In a particular embodiment, the cancer is a lymphoma, which may be selected from the group consisting of: Hodgkin's lymphoma, non-Hodgkin's lymphoma, Burkitt's lymphoma, and small cell lymphocytic lymphoma.

By appropriately targeting the cancer stem cells in such cancers, effective treatment of the cancer can be achieved by preventing or treating the development of the cancer, by preventing or treating the progression of the cancer, by preventing or treating the recurrence of the cancer, or by preventing or treating the spread of the cancer.

In a suitable embodiment, the present invention provides a compound of the invention for use in targeting cancer stem cells for the prevention or treatment of metastatic cancer.

In a suitable embodiment, the present invention relates to a compound of the invention for use in targeting cancer stem cells for the treatment of relapsed or refractory malignancy.

In a suitable embodiment, the present invention provides a compound of the invention for use in targeting cancer stem cells for the treatment of primary cancer. Accordingly, the primary cancer being treated may be an additional focus of the primary malignant neoplasm.

The present invention relates to a compound of the invention for use in targeting cancer stem cells for the treatment of secondary malignancy. In a suitable embodiment, the secondary malignancy is a metastatic malignancy.

In a suitable embodiment, the present invention relates to a compound of the invention for use in targeting cancer stem cells, wherein targeting the cancer stem cells prevents or inhibits: (i) cancer recurrence; (ii) the occurrence of an additional focus of the primary malignant neoplasm; or (iii) metastasis of a malignant neoplasm.

Methods of treatment or medical applications that use the compound of the present invention based on its ability to target cancer stem cells can be used to treat relapsed or refractory malignancy. The factors to be considered regarding relapsed or refractory cancer in such embodiments of the invention are, except where the context otherwise requires, the same factors as for the treatment of relapsed or refractory cancer according to aspects of the present invention.

Recurrent or refractory malignancy.

As mentioned above, certain aspects and embodiments of the present invention specifically relate to the use of a compound of the invention for the treatment of relapsed or refractory cancer.

For the purposes of the present invention, refractory malignancies can be considered as malignant neoplasms that demonstrate resistance to anticancer therapy other than therapy in which the compound of the present invention is used.

For example, a compound of the present invention may be used to treat refractory cancers that are resistant to radiation therapy. Alternatively, or additionally, a compound of the present invention may be used to treat refractory cancers that are resistant to biological agents used to treat cancer. In a suitable embodiment, the compound of the present invention may be used to treat refractory cancers that are resistant to treatment with chemotherapeutic agents other than the compound of the invention.

In particular, refractory cancers for which the methods of treatment or medical use of the invention using a compound of the present invention may be effective include those cancers that are resistant to cordycepin or 2-fluorocordycepin.

Recurrent malignancies (or recurrent malignancies) are those that return again after a period of remission during which no malignancy can be detected. Cancer recurrence can occur at the site of the original malignancy (local recurrence), at a site close to the original tumor (regional recurrence), or at a site distant from the original malignancy (distant recurrence). Cancer stem cells are believed to play a role in cancer recurrence by providing a source from which cancer relapse cells are formed.

Accordingly, methods of treatment and medical use of the compound of the present invention in accordance with the present invention, which target cancer stem cells, can be highly effective in the context of recurrent malignancy. The ability of a compound of the invention to target cancer stem cells can be used to remove populations of such cells that are capable of relapse, thereby preventing recurrence of the cancer. The activity of the compound of the present invention against cancer stem cells can also be used to target cancer stem cells in malignancies that have recurred, and also likely to have a cytotoxic effect on non-cancerous stem cells, thereby providing treatment for recurrent malignancies.

In view of the foregoing, it will be appreciated that a compound of the present invention may be used in the methods or for use of the present invention for the prevention or treatment of recurrent cancer. The compound of the present invention may be used in the methods or for use of the invention for the prevention or treatment of local, regional or distant recurrent malignancies.

The compound of the present invention can be used in the methods or for the use of the invention to prevent recurrence of cancer, providing at least 2 months, at least 6 months, at least 12 months, at least 18 months, at least 24 months or at least 30 months of remission. Indeed, the compound of the invention can be used to prevent the recurrence of a malignant neoplasm, providing at least 4 years, at least 5 years, at least 6 years, at least 7 years, at least 8 years, at least 9 years or at least 10 years of remission.

The compound of the present invention may be used in the methods or for use of the invention for the treatment of recurrent cancer that recurs after at least 2 months, at least 6 months, at least 12 months, at least 18 months, at least 24 months or at least 30 months in remission. Indeed, the compound of the invention can be used to treat recurrent cancer that recurs after at least 4 years, at least 5 years, at least 6 years, at least 7 years, at least 8 years. , at least 9 years or at least 10 years of remission.

The ability of the compounds of the invention to target cancer stem cells enables these compounds to be used in the prevention or treatment of cancers in accordance with the medical uses or methods of treatment of the present invention. However, it should be noted that the compounds of the present invention also have a direct cytotoxic effect on cancerous non-stem cells, which constitute the bulk of tumors. While cancer stem cell activity may underlie much of the resistance that makes relapsed or refractory cancers so difficult to treat, cancerous non-stem cells are also a major component of such relapsed or refractory cancers.

The compounds of the present invention have a stronger cytotoxic effect on cancer non-stem cells than cordycepin or 2-fluorocordycepin, from whose chemotherapeutic molecule the compounds of the present invention are derived. Accordingly, the mechanism by which the compound of the invention acts in the treatment of relapsed or refractory cancer cannot be limited only to the activity of the compound against cancer stem cells, but may also utilize the effect of the compound of the invention on cancer non-stem cells. In this application, treatment with a compound of the invention will reduce the total number of both types of cancer stem cells and cancer non-stem cells. When using some of the compounds of the present invention, such treatments will preferably reduce the proportion of cancer stem cells that remain after treatment.

Therapeutically effective doses of a compound of the present invention.

A therapeutically effective amount of a compound of the present invention may be an amount sufficient to cause cancer cell death.

A therapeutically effective amount of a compound of the present invention may be an amount sufficient to induce the death of cancer stem cells. In some embodiments of the invention, particularly those relating to the treatment of relapsed or refractory cancer, a therapeutically effective amount of a compound of the present invention may be an amount sufficient to cause death of cancer stem cells as well as to cause death of cancer non-stem cells.

There are various methods by which the amount of a therapeutically effective compound, such as a compound of the invention, administered to a patient can be calculated and displayed. One such method, which is considered particularly suitable for doses of agents in the case of cancer prevention or treatment, is the amount of agent administered per unit surface area of the patient's body. Such doses are generally expressed as the amount of agent (which may be determined by weight) per square meter (m ² ) of surface area.

When using the compound of the present invention for the prevention or treatment of cancer, a weekly dose of 10 to 1000 mg/m ² can be used. In such treatment, for example, a weekly dose of 375 to 900 mg/m ² may be used. For example, effective treatment of relapsed or refractory cancer can be achieved when patients receive weekly doses of a compound of the invention that range from about 500 to 825 mg/m ² .

Without wishing to be bound by any hypothesis, the inventors believe that the ability of a compound of the invention to target cancer stem cells allows therapeutic efficacy to be achieved at lower doses of the compound than might be expected. Just by way of example, weekly doses of a compound of the invention as low as 825, 750, 600, or 500 mg/m ² may be therapeutically effective when used and in the methods of the invention.

The selected weekly dose of a compound of the invention may be provided by a single administration or multiple administrations over the course of a week. Thus, for example, a weekly dose of a compound of the invention may be administered twice, three times, or more. Thus, for a weekly dose of 750 mg/m ² , this can be achieved by administering 250 mg/m ² three times a week or 375 mg/m ² twice a week. Similarly, for a weekly dose of 600 mg/m ² , this can be achieved with three doses of 200 mg/m ² during the week or two doses of 300 mg/m ² during the week.

A suitable amount of the compound of the present invention, administered once per treatment to obtain the required dose of this compound over a weekly course, may be in the range from about 100 to 300 mg/m ² .

The weekly dose of the compound of the present invention may be reduced during the course of treatment. For example, treatment may begin with a weekly dose of approximately 1000, 900, 825, 750, or 725 mg/m ² and during the course of treatment the required dose may be reduced to approximately 750 mg/m ² (in those cases when the initial dose is above this value), about 650 mg/m ² , about 625 mg/m ² , or even about 500 mg/m ² or about 375 mg/m ² .

Doses of the compound of the invention may, of course, be in other forms. The most common of these is the amount of active agent given per unit body weight. For the average human patient, a dose of 1 mg/m ² was calculated to be equivalent to about 0.025 mg/kg of body weight. Accordingly, the data indicate that a compound of the present invention is effective in the treatment of relapsed or refractory cancer at doses ranging from about 6.25 to about 25 mg/kg.

A suitable dose may, for example, be in the range of about 9.5 to 22.5 mg/kg. In an appropriate embodiment, the compound of the present invention effectively treats relapsed or refractory cancer when patients take weekly doses in the range of about 12.5 to 20.5 mg/kg.

Considerations regarding compositions of a compound of the invention suitable for use in the prophylactic or therapeutic methods and medical uses of the present invention are described elsewhere in this specification. In the case of injectable formulations of the compound of the invention, they may be administered intravenously. Intravenous administration can be carried out for any suitable time, within, for example, a ten minute injection or the like.

Types of treatment.

In a suitable embodiment of the invention, the compound of the present invention can be used to target cancer stem cells as a first line therapy for malignancy.

However, the discovery that the compounds of the invention are able to target cancer stem cells and thus treat relapsed or refractory cancer indicates that the compound of the invention is capable of providing effective treatment for cancer in a context where other treatments have failed. . Thus, in a related embodiment, the present invention relates to a compound of the present invention for targeting cancer stem cells as a second line treatment for cancer. Indeed, in a suitable embodiment, the present invention relates to a compound of the present invention for targeting cancer stem cells as a third or subsequent line of cancer treatment.

In a suitable embodiment, the invention provides the use of a compound of the present invention as a neoadjuvant for the treatment of cancer. A neoadjuvant is an agent administered to a patient to reduce the size of a tumor prior to primary cancer therapy, such as surgical removal of the cancer. The compound of the present invention can be used as a patient who will subsequently undergo cancer surgery and/or cancer radiation therapy.

Alternatively or additionally, the invention provides a compound of the invention for use as an adjuvant in the treatment of cancer. An adjuvant agent is an agent administered to a patient after primary cancer therapy, such as surgical removal of the cancer, to prevent the return of the cancer after primary therapy. The compound of the present invention can be used as an auxiliary agent for a patient after cancer surgery and/or cancer radiation therapy.

The compound of the present invention may be used in the methods or may be used as a monotherapy of the present invention, i.e. for prophylaxis or treatment, in which the compound of the invention provides substantially the full therapeutic activity used for prophylaxis or treatment.

Alternatively, in the methods or uses of the present invention, a compound of the invention may be used in combination therapy. In such embodiments, a compound of the invention is used in combination with at least one adjunctive cancer therapy. Additional cancer therapy may include surgery and/or radiation therapy. In addition, or alternatively, further cancer therapy may include the use of at least one therapeutic agent that contributes to the prevention or treatment of cancer. Accordingly, such an agent may be a chemotherapeutic agent or a biological agent used in the prevention or treatment of cancer.

In a suitable embodiment of combination therapy, the compound of the invention and the additional therapeutic agent may be administered to the patient simultaneously. In a suitable example, the compound of the present invention and the additional therapeutic agent may be presented as part of the same pharmaceutical composition. Alternatively, the connection

- 26 040630 of the present invention and additional therapeutic agent can be included in independent compositions for administration to the patient, essentially at the same time.

In yet another embodiment of combination therapy, the compound of the invention and the additional therapeutic agent may be administered to the patient at different times. The compound of the present invention and the additional therapeutic agent may be administered sequentially to the patient. For example, a compound of the present invention may be administered to a patient prior to administration of an additional therapeutic agent. Alternatively, a compound of the present invention may be administered to a patient after administration of an additional therapeutic agent.

Additional Therapeutic Agents

The compound of the present invention can be used in combination with a wide range of additional therapeutic agents for the prevention or treatment of cancer. These include biological agents, immunotherapeutic agents, and chemotherapeutic agents that can be used to prevent or treat cancer.

Although the following paragraphs discuss specific examples of suitable additional agents, they should not be construed as limiting the range of additional therapeutic agents suitable for use with a compound of the invention. Indeed, the ability of a compound of the invention to target cancer stem cells indicates that it can be effectively used in combination with any additional therapeutic agent used to prevent or treat cancer, whether or not such additional agent targets cancer stem cells. , cancer non-stem cells, or other cells or components involved in the development, maintenance, recurrence or spread of a malignant neoplasm.

Examples of additional therapeutic agents that may be used in combination with a compound of the invention include:

(a) an anti-angiogenic agent, where, optionally, the anti-angiogenic agent is: (i) an inhibitor of the VEGF pathway, optionally bevacizumab; (ii) a tyrosine kinase inhibitor, optionally sorafenib, sunitinib, or pazopanib; or (iii) an MTOR inhibitor, optionally everolimus;

(b) an alkylating agent;

(c) an antimetabolite;

(d) an antitumor antibiotic;

(e) topoisomerase;

(f) a mitosis inhibitor;

(g) a monoclonal antibody;

(h) a metallic agent; or (i) active or passive immunotherapy.

Unless otherwise indicated by the context, the additional therapeutic agents mentioned in the previous list should all be considered for use in any of the embodiments of combination therapy with a compound of the invention as discussed above.

Patient selection.

The inventors' finding that a compound of the invention is capable of targeting cancer stem cells allows a number of methods by which to determine whether administering a compound of the invention is likely to be effective in treating or preventing a malignancy, such as relapsed or refractory cancer, in a particular patient.

Accordingly, the present invention relates to a method for determining whether a patient suffering from a malignancy or precancerous condition would benefit from preventing or treating cancer with a compound of the invention, wherein the method comprises: evaluating a biological sample indicative of a malignant neoplasm or precancerous condition in a patient for the presence of cancer stem cells; where the presence of cancer stem cells in a biological sample indicates that the patient will benefit from treatment with a compound of the invention.

The present invention also relates to a method for determining an appropriate treatment regimen for a patient suffering from a malignant neoplasm or precancerous condition, where the method includes: evaluating a biological sample characteristic of a malignant neoplasm or precancerous condition in a patient for the presence of cancer stem cells; where the presence of cancer stem cells in a biological sample indicates that a suitable treatment regimen would include treating the patient with a compound of the present invention.

The invention also relates to the use of a compound of the invention for the prevention or treatment of cancer in a patient, selected for such treatment by a method comprising: evaluating a biological sample indicative of a malignancy or precancerous condition in a patient for the presence of cancer stem cells; where the presence of cancer stem cells in the biological sample indicates that the patient is suitable for treatment with a compound of the invention.

In suitable embodiments, the implementation of cancer stem cells in a biological sample can be identified by the expression of characteristic sets of markers, as discussed earlier in the application.

One skilled in the art will appreciate that there are many suitable examples of biological samples that can be used in embodiments of the present invention, such as the markers mentioned above. Accordingly, such a sample may include cells of a malignant neoplasm or precancerous condition. A suitable biological sample may be a tissue sample, such as a sample for use in histology. Cells in such samples can be directly assessed for the expression of cancer stem cell markers, such as those listed above.

Alternatively or additionally, a suitable biological sample may contain target molecules typical of the gene expression of malignant neoplasm or precancerous cells. Examples of such target molecules include proteins encoded by expressed genes or nucleic acids such as mRNAs typical of gene expression.

Suitable examples of methods by which expression of cancer stem cell markers can be assessed can be selected with reference to the type of sample. Techniques for examining expressed markers are often used in the context of clinical assessments (eg, for diagnostic or prognostic purposes) and their use is well known to those skilled in the art who need to use them in the context of the present invention. Just as an example, in samples containing proteins, the presence of cancer stem cell markers can be assessed by suitable methods using antibodies that interact with the cancer stem cell markers of interest. Examples of such samples containing protein markers of cancer stem cells include histological samples (where the presence of markers can be visualized using appropriate immunocytochemistry methods) or samples obtained from circulating blood. However, the presence of circulating cancer stem cells (which are believed to contribute to the spread of malignancy through metastases) can be assessed using methods such as flow cytometry.

In samples containing nucleic acids typical for the expression of cancer stem cell markers, such expression can be assessed by appropriate molecular biological methods such as amplification-based polymerase chain reaction (PCR) using suitable primers.

Example 1. Methods of synthesis.

Compounds of the invention may be prepared according to or analogously to the following general synthetic methods and examples.

General method 1 (for connections A-F and L-U).

N-methylimidazole (1.0 mmol) and a solution of the corresponding phosphorochloridate (0.6 mmol) in anhydrous THF (2 ml) were added dropwise to a suspension of 3'-deoxyadenosine (0.20 mmol) or substituted 3'-deoxyadenosine in anhydrous THF (10 ml) and the reaction mixture was stirred at room temperature for 16 h. Purification by column chromatography and preparative TLC gave the desired compound as a white solid. The number of components used may vary, and the actual amounts are given in the examples below.

General method 2 (for connection J).

3'-Deoxyadenosine (0.80 mmol) was suspended in (CH ₃ O) ₃ PO (5 ml) and POCl ₃ (0.80 mmol) was added dropwise at -5°C. The reaction mixture was allowed to warm to room temperature and allowed to stir for 4 h. A solution of the appropriate amino acid ester salt (4.0 mmol) dissolved in anhydrous CH2Cl2 (5 mL) was added, followed by diisopropylethylamine (8.0 mmol) at -78 °C. After stirring at room temperature for 20 h, water was added and the layers were separated. The aqueous phase was extracted with dichloromethane and the organic phase was washed with brine. The combined organic layers were dried over Na ₂ SO ₄ and concentrated. The residue was purified by column chromatography (gradient elution with CH ₂ Cl ₂ /MeOH= 100/0 to 93/7) to give the desired product as a white foam. The number of components used may vary, and the actual amounts are given in the examples below.

General method 3 (for connection G-I).

3'-Deoxyadenosine (0.20 mmol) was suspended in anhydrous THF (5 ml) and tert-BuMgCl (1.0 M solution in THF, 0.22 mmol) was added dropwise at room temperature. A solution of the appropriate phosphorochloridate (0.6 mmol) in anhydrous THF (2 ml) was added dropwise and the reaction mixture was stirred at room temperature for 16 hours. Purification by column chromatography and preparative TLC gave the desired compound as a white solid. The number of components used may vary, and the actual amounts are given in the examples below.

- 28 040630

General method 4 (for connection V).

To a solution of the corresponding 3'-deoxyadenosine derivative (1 mol/eq) in anhydrous DMF was added tert-butyldimethylsilyl chloride (3.3 mol/eq) and imidazole 6.6 (mol/eq), and the reaction mixture was stirred at room temperature for nights (16-20 h). Then NH4C1 was added to the mixture and washed twice with ethyl acetate. The organic layers were combined, dried over Na ₂ SO ₄ and the solvent was removed in vacuo. Purification of the mixture by column chromatography gave intermediate C1. Intermediate C1 was then dissolved in an aqueous solution of THF/H2O/TFA 4/1/1 (6 ml/eq) and stirred at 0° C. for 4 h. The solution was then carefully neutralized with saturated aqueous NaHCO ₃ and the mixture was washed two times with ethyl acetate. The organic layers were combined, dried over Na ₂ SO ₄ and the solvent was removed in vacuo. Purification of the mixture by column chromatography gave intermediate C2. The general method B was then carried out and intermediate C3 was obtained. Intermediate C3 was dissolved in an aqueous solution of THF/H ₂ O/TFA 1/1/1 (6 ml/eq) at 0°C and stirred at room temperature for 24 h. Purification by chromatography gave the desired compounds as white solids.

General Method 5 (to obtain 3'-deoxyadenosine and 3'-deoxy-2-chloradenosine used in the examples).

A solution of H2O/CH3CN 1:9 and then a-AIBBr (4.0 mol/eq) were successively added to a suspension of dry adenosine or 2-chloroadenosine in anhydrous CH3CN and stirring was continued at room temperature (20°C). After 1 h, a saturated solution of NaHCO ₃ was carefully added and the solution was extracted with EtOAc. The combined organic phase was washed with brine. The aqueous phase was extracted with EtOAc and the combined organic phase was dried over Na ₂ SO ₄ , filtered and evaporated to give a white gum. The crude mixture was dissolved in anhydrous MeOH and stirred for 1 h with Amberlite resin (2xOH ^- ), pre-washed well with anhydrous MeOH. The solution was then filtered and the resin was carefully washed with anhydrous methanol. Evaporation of the combined filtrate gave 2',3'-dehydroadenosine or 2',3'-dehydro-2-chloroadenosine as a white solid.

A solution of LiEt ₃ BH (1 M in THF 4-4.3 mol/eq). Stirring was continued at 4° C. for 1 hour and at room temperature overnight (16 hours). The reaction mixture was carefully acidified (5% AcOH/H ₂ O), purged with N ₂ for 1 h (under a fume hood) to remove the self-igniting triethylborane and evaporated. The residue was subjected to chromatography to obtain 3'-deoxyadenosine or 3'-deoxy-2chloradenosine as a white powder.

Using General Method 5: 2',3'-dehydroadenosine was prepared starting from 10.0 g (37.4 mmol) adenosine, 7.5 ml H ₂ O/CH ₃ CN (1/9), 22 ml (149, 7 mmol) a-AIBBr in 500 ml anhydrous CH3CN and 300 ml Amberlite resin (2xOH ^- ) in 400 ml dry methanol. 2',3'-Dehydroadenosine was obtained as a white solid (9.12 g, 98%). 3'-Deoxyadenosine was prepared starting from 9.12 g (36.6 mmol) 2',3'dehydroadenosine and 159 ml (159 mmol) LiEt ₃ BH/THF 1M, in anhydrous DMSO/THF (1/10, 50 ml) . Purification by silica gel column chromatography (eluent system 3-18% MeOH in DCM) gave 3'-deoxyadenosine as a white powder (7.12 g, 77%).

¹ H NMR (500 MHz, DMSO-d6): δ 8.37 (s, 1H, H8), 8.17 (s, 1H, H2), 7.29 (br s, 2H, NH2), 5 .89 (d, J=2.5 Hz, 1H, H1'), 5.68 (d, J=4.5 Hz, 1H, OH-2'), 5.19 (t, J=6, 0 Hz, 1H, OH-5'), 4.63-4.58 (m, 1H, H2'), 4.404.34 (m, 1H, H4'), 3.71 (ddd, J=12.0, 6.0, 3.0 Hz, 1H, H5'), 3.53-3.49 (ddd, J=12.0, 6.0, 4.0 Hz, 1H, H5'), 2.302.23 (m, 1H, H3'), 1.98-1.90 (m, 1H, H3'). ¹³ C NMR (125 MHz, DMSO^) δ 156.00 (C6), 152.41 (C2), 148.82 (C4), 139.09 (C8), 119.06 (C5), 90.79 ( SG), 80.66 (C4'), 74.56 (C2'), 62.61 (C5'), 34.02 (C3)

Using general method 5: 2',3'-dehydro-2-chloradenosine was prepared starting from 5.0 g (16.6 mmol) 2-chloradenosine, 3.0 ml H ₂ O/CH ₃ CN (1/9), 9 .7 ml (66.2 mmol), a-AIBBr in 38 ml anhydrous CH3CN and 150 ml Amberlite resin (2xOH ^- ) in 200 ml anhydrous methanol. 2',3'-Dehydro-2-chloroadenosine was obtained as a white solid (3.03 g, 60%). 3'-Deoxy-2-chloradenosine was prepared starting from 2.18 g (7.68 mmol) 2',3'-dehydro-2-chloradenosine and 30.7 ml (30.7 mmol) LiEt ₃ BH/THF 1M in anhydrous DMSO/THF (1/10 ml, 30 ml). Purification by silica gel column chromatography (eluent system 2-20% MeOH in DCM) gave 3'-deoxy-2-chloroadenosine as a white powder (1.20 g, 55%).

¹ H NMR (500 MHz, CD3OD): δH 8.41 (s, 1H, H8), 5.93 (d, J=2.5 Hz, 1H, H1'), 4.68-4.66 (m , 1H, H2'), 4.56-4.52 (m, 1H, H4'), 3.95 (dd, J=3, 12.5 Hz, 1H, H5'), 3.70 (dd, J=3, 12.5 Hz, 1H, H5'), 2.39-2.33 (m, 1H, H3'), 2.08-2.03 (m, 1H, H3') ¹³ C NMR ( 125 MHz, CD3OD): δC 158.14 (C6), 155.19 (C2), 151.15 (C4), 141.30 (C8), 119.56 (C5), 93.58 (C1'), 82.80 (C4'), 76.81 (C2'), 64.01 (C5'), 34.33 (C3').

Preparation of 3'-deoxy-2-fluoroadenosine.

A solution of H ₂ O/CH ₃ CN (1:9; 1.4 ml) and then a-AIBBr (4.10 ml, 28.05 mmol) were added sequentially to a suspension of dry 2-fluoroadenosine (2.0 g, 7. 01 mmol) in anhydrous CH3CN (50 ml), and stirring

- 29 040630 continued at room temperature (20°C). After 1 h, a saturated solution of NaHCO ₃ was carefully added and the solution was extracted with EtOAc (2x100 ml). The combined organic phase was washed with brine (1x50 ml). The aqueous phase was extracted with EtOAc (2x50 ml) and the combined organic phase was dried over Na ₂ SO ₄ , filtered and evaporated to give a white gum. The crude mixture was dissolved in THF/H2O (4/1, 50 ml) and stirred for 1 h with 60 ml of Amberlite resin (2xOH ^- ) (previously well washed with THF). The solution was then filtered and the resin washed gently with THF. Evaporation of the combined filtrate and crystallization of the residue from EtOH gave 2',3'-dehydro-2-fluoroadenosine as a white solid (1.13 g, 60%).

To a cold (4°C, ice bath) solution of 2',3'-dehydro-2-fluoroadenosine (1.13 g, 4.18 mmol) in anhydrous DMSO/THF (1/10, 15 mL) under argon a solution of LiEt ₃ BH/THF (1M; 18.01 ml, 18.01 mmol) was added dropwise. Stirring was continued at 4°C for 1 h and at room temperature overnight (16 h). The reaction mixture was carefully acidified (5% AcOH/H ₂ O), purged with N2 for 1 h (under a fume hood) to remove the self-igniting triethylborane, and evaporated. The residue was chromatographed on silica gel (3-18% MeOH in DCM) to give 3'-deoxy-2-fluoroadenosine as a white powder (7.12 g, 77%).

¹⁹ F NMR (470 MHz, DMSO-d6): 5F -52.19-1H NMR (500 MHz, DMSO-d6) 5H 8.34 (s, 1H, H8), 7.80 (br s, 2H, NH2), 5.78 (d, J=2.25 Hz, 1H, H1'), 5.68 (br. s, 1H, OH-2'), 5.01 (br. s, 1H, OH- 5'), 4.55-4.51 (m, 1H, H2'), 4.39-4.32 (m, 1H, H4'), 3.73-3.76 (m, 1H, H5' ), 3.56-3.50 (m, 1H, H5'), 2.26-2.18 (m, 1H, H3'), 1.94-1.85 (m, 1H, H3'). ¹³ C NMR (125 MHz, DMSO-d6) 5C 158.51 (d, 1JC-F=202.7 Hz, C2), 157.55 (d, ³ Jc-f=21.2 Hz, C6), 150 .11 (d, ³ Jc-f=20.3 Hz, C4), 139.22 (d, ⁵ Jc-f=2.2 Hz, C8), 117.37 (d, ⁴ Jc-f=4, 1 Hz, C5), 90.67 (C1'), 80.90 (C4'), 74.73 (C2'), 62.35 (C5'), 33.89 (C3').

Preparation of 3'-deoxy-2-methoxyadenosine.

A solution of H2O/CH3CN (1:9; 1.4 ml) and then a-AIBBr (4.10 ml, 28.05 mmol) were added sequentially to a suspension of dry 2-fluoroadenosine (2.0 g, 7.01 mmol) in anhydrous CH ₃ CN (50 ml) and stirring was continued at room temperature (20°C). After 1 h, a saturated NaHCO3 solution was added carefully and the solution was extracted with EtOAc (2x100 ml). The combined organic phase was washed with brine (1x50 ml). The aqueous phase was extracted with EtOAc (2x50 ml) and the combined organic phase was dried over Na2SO4, filtered and evaporated to give a white gum. The crude mixture was dissolved in anhydrous MeOH (50 ml) and stirred for 1 h with 60 ml of Amberlite resin (2xOH ^- ) (previously well washed with anhydrous MeOH). The solution was then filtered and the resin washed gently with THF. Evaporation of the combined filtrate and crystallization of the residue from EtOH gave 2',3'-dehydro-2-methoxyadenosine as a white solid (1.57 g, 84%).

To a cold (4°C) solution of 2',3'-dehydro-2-methoxyadenosine (762 mg, 2.84 mmol) in anhydrous DMSO/THF (1/10, 15 ml) in an argon atmosphere was added dropwise a solution of LiEt ₃ BH (1M solution in THF; 8.53 ml, 8.53 mmol). Stirring was continued at 4° C. for 1 hour and at room temperature overnight (16 hours). The reaction mixture was carefully acidified (5% AcOH/H2O), purged with N2 for 1 h (under a fume hood) to remove the self-igniting triethylborane, and evaporated. The residue was chromatographed on silica gel (3-17% MeOH in DCM) to give 3'deoxy-2-methoxyadenosine as a white powder (650 mg, 81%).

1H NMR (500 MHz, CD3OD): 5H 8.20 (s, 1H, H8), 5.90 (d, J=2.4 Hz, 1H, H1'), 4.75-4.71 (m, 1H, H2'), 4.54-4.48 (m, 1H, H4'), 3.91 (dd, J=12.3, 2.5 Hz, 1H, H5'), 3.69 (dd , J=12.30, 4.0 Hz, 1H, H5'), 3.37 (s, 3H, OCH3), 2.43-2.35 (m, 1H, H3'), 2.08-2 .02 (m, 1H, H3'). ¹³ C NMR (125 MHz, CD3OD) 5C 163.68 (C2), 158.12 (C6), 151.94 (C4), 139.71 (C8), 116.64 (C5), 93.36 (C1 '), 82.53 (C4'), 76.59 (C2'), 64.24 (C5'), 55.29 (OCH ₃ ), 34.81 (C3').

Phosphorochloridates were prepared by the methods described starting from aryl phosphorodichloridates and amino acid ester hydrochlorides.

3'-Deoxyadenosine-5'-O-[phenyl(benzyloxy-L-alaninyl)]phosphate A

Compound A was prepared according to General Method 1 using 3'-deoxyadenosine (50 mg, 0.20 mmol), N-methylimidazole (80 μl, 1.0 mmol) and phenyl(benzyloxy-L-alaninyl) phosphorochloridate (212 mg , 0.6 mmol). Purification by column chromatography (eluent system CH3OH/CH2Cl2 0/100 to 7/93) with CH ₂ Cl ₂ /MeOH gradient (100 to 95:5%) and preparative TLC (1000 µm, eluent system CH3OH/CH2C12 5/95) gave the title compound as a solid

- 30 040630 white color (31 mg, 28%).

1H NMR (500 MHz, CD3OD): δH 8.26 (s, 0.5H, H8), 8.24 (s, 0.5H, H8), 8.22 (s, 0.5H, H2), 8 .21 (s, 0.5H, H2), 7.34-7.25 (m, 7H, Ar), 7.21-7.13 (m, 3H, Ar), 6.01 (d, J= 2.9 Hz, 1H, H1'), 6.00 (d, J=2.9 Hz, 1H, H1'), 5.155.04 (m, 2H, OCH ₂ Ph), 4.73-4.63 (m, 2H, H2', H4'), 4.43-4.35 (m, 1H, H5'), 4.27-4.20 (m, 1H, H5'), 4.03-3, 91 (m, 1H, CHCH3), 2.35-2.28 (m, 1H, H3'), 2.09-2.02 (m, 1H, H3'), 1.32 (d, J=7 .4 Hz, 1.5 H, CHCH3), 1.28 (d, J=7.4 Hz, 1.5 H, CHCH3).

¹³ C NMR (125 MHz, CD3OD): δΓ 174.84 (d, ³ Jc-p=4.5 Hz, C=O), 174.63 (d, ³ Jc-p=4.5 Hz, C= O), 157.32 (C6), 157.31 (C6), 153.86 (C2), 153.84 (C2), 152.13 (C4), 152.07 (C4), 150.20 (C -Ar), 150.18 (C-Ar), 140.47 (C8), 137.26 (C-Ar), 137.19 (C-Ar), 130.76 (CH-Ar), 130.74 (CH-Ar), 129.57 (CH-Ar), 129.32 (CH-Ar), 129.31 (CH-Ar), 129.29 (CH-Ar), 129.26 (CH-Ar) , 126.16 (CH-Ar), 126.14 (CH-Ar), 121.46 (d, ³ JC-P=4.7 Hz, CH-Ar), 121.38 (d, ³ Jc-p =4.7 Hz, CH-Ar) 120.54 (C5), 120.53 (C5), 93.24 (C1'), 93.18 (C1'), 80.43 (d, ³ Jc-p =3.6 Hz, C4'), 80.36 (d, ³ Jc-p=3.6 Hz, C4'), 76.62 (C2'), 68.62 (d, ² Jc-p=5 .3 Hz, C5'), 68.30 (d, ² Jc-p=5.3 Hz, C5'), 67.95 (OCH2Ph), 67.92 (OCH ₂ Ph), 51.74 (CHCH ₃ ), 51.60 (CHCH ₃ ), 34.91 (C3'), 34.70 (C3'), 20.45 (d, ³ J _CP = 7.0 Hz, CHCH3), 20.28 (d, ³ Jc-p=7, 0 Hz, CHCH3).

³¹ P NMR (202 MHz, CD3OD): δP 3.9, 3.7.

MS (ES+) m/z: Found: 569.2 (M+H+), 591.2 (M+Na+), 1159.4 (2M+Na+). C26H29N6O7P required: (M) 568.2.

HPLC. Reverse-phase HPLC eluting with 100/10 to 0/100 H2O/CH3CN over 30 min, 1 ml/min, l=254 nm, showed two diastereoisomer peaks with a tR of 14.02 min. and tR 14.26 min.

(2S)-Benzyl 2-(((((2S,4R,5R)-5-(6-amino-9Η-purin-9-yl)-4-hydroxytetrahydrofuran-2-yl)methoxy) (naphthalene-1 - yloxy)phosphoryl)amino)propanoate B

O

Using General Method 1 above, N-methylimidazole (240 μl, 3.0 mmol) and a solution of (2S)-benzyl 2-((chloro(naphthalene-1-yloxy)phosphoryl)amino)propanoate (727 mg, 1 8 mmol) in anhydrous THF (10 ml) was added dropwise to a suspension of 3'-deoxyadenosine (150 mg, 0.6 mmol) in anhydrous THF, and the reaction mixture was stirred at room temperature for 16 h. Purification by column chromatography ( eluent system CH ₃ OH/CH ₂ Cl ₂ 0/100 to 6/94) and preparative TLC (2000 μm, eluent system CH ₃ OH/CH ₂ Cl ₂ 5/95) gave the desired compound as a white solid ( 45 mg, 12%).

MS (ES+) m/z: Found: 619.2 (M+H+), 641.2 (M+Na+), 1259.4 (2M+Na+). C ₃₀ H ₃₁ N ₆ O ₇ P required: (M) 618.58.

³¹ P NMR (202 MHz, CH3OD): δP 4.3 (s), 4.1 (s).

1H NMR (500 MHz, CH3OD): δH 8.24 (s, 0.5H, H8), 8.22 (s, 0.5H, H8), 8.20 (s, 0.5H, H2), 8 .19 (s, 0.5H, H2), 8.14-8.09 (m, 1H, Ar), 7.89-7.85 (m, 1H, Ar), 7.70-7.67 ( m, 1H, Ar), 7.53-7.42 (m, 3H, Ar), 7.39-7.34 (m, 1H, Ar), 7.31-7.25 (m, 5H, Ar ), 5.99 (d, J=2.0 Hz, 0.5H, H1'), 5.98 (d, J=2.0 Hz, 0.5H, H1'), 5.10-5, 01 (m, 2H, CH2Ph), 4.72-4.61 (m, 2H, H2', H4'), 4.47-4.40 (m, 1H, H5'), 4.33-4, 24 (m, 1H, H5'), 4.09-3.98 (m, 1H, CH ala) 2.35-2.26 (m, 1H, H3'), 2.07-1.98 (m , 1H, H3'), 1.30-1.24 (m, 3H, CH ₃ ).

¹³ C NMR (125 MHz, CH3OD): δC 174.85 (d, ³ Jc-p=3.7 Hz, C=O), 174.56 (d, ³ Jc-p=3.7 Hz, C= O), 157.33 (C6), 157.31 (C6), 153.87 (C2), 153.85 (C2), 150.24 (C4), 150.23 (C4), 147.91 (d , ³ JC-P=7.5 Hz, 'ipso' Nap), 147.95, (d, ³ Jc-p=7.5 Hz, 'ipso' Nap), 140.56 (C8), 140.50 (C8), 137.22 (C-Ar), 137.17 (C-Ar), 136.28 (C-Ar), 129.55 (CH-Ar), 129.53 (CH-Ar), 129 .30 (CH-Ar), 129.25 (CH-Ar), 128.88 (CH-Ar), 128.82 (CH-Ar), 127.91 (d, ² Jc-p=6.25 Hz , C-Ar), 127.83 (d, ² Jc-p=6.25 Hz, C-Ar), 127.77 (CH-Ar), 127.75 (CH-Ar), 127.49 (CH -Ar), 127.45 (CH-Ar), 126.48 (CH-Ar), 126.47 (CH-Ar), 126.02 (CH-Ar), 125.97 (CH-Ar), 122 .77 (CH-Ar), 122.63 (CH-Ar), 120.58 (C5), 120.53 (C5), 116.35 (d, ³ Jc-p=3.75 Hz, CH-Ar ), 116.15 (d, ³ Jc-p=3.75 Hz, CH-Ar), 93.22 (C1'), 93.20 (C1'), 80.30 (d, ³ Jc-p = 2.75 Hz, C4'), 80.24 (d, ³ Jc-p=2.75 Hz, C4'), 76.51 (C2'), 76.44 (C2'), 68.87 (d , ² Jc-p=5.2 Hz, C5'), 68.64 (d, ² Jc-p=5.2 Hz, C5'), 67.93 (OCH2Ph), 51.82 (CH ala), 51.73 (CH ala), 35.01 (C-3'), 34.76 (C3'), 20.4 1 (d, ³ JC-P=6.7 Hz, CH ₃ ala), 20.22 (d, ³ J _CP =6.7, CH ₃ ala).

HPLC. Reverse phase HPLC eluting with H2O/CH3CN 100/10 to 0/100 over 30 min, 1 ml/min, 1=200 nm, showed two diastereoisomer peaks with a tR of 16.36 min. and tR 16.60 min.

Benzyl 2-(((((2S,4R,5R)-5-(6-amino-9Η-purin-9-yl)-4-hydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)acetate C

- 31 040630

Using General Method 1 above, N-methylimidazole (80 µl, 1.0 mmol) and a solution of benzyl 2-((chloro(phenoxy)phosphoryl)amino)acetate (204 mg, 0.6 mmol) in anhydrous THF (2 ml) was added dropwise to a suspension of 3'-deoxyadenosine (50 mg, 0.20 mmol) in anhydrous THF and the reaction mixture was stirred at room temperature for 16 h. Purification by column chromatography (eluent system CH ₃ OH/CH ₂ Cl ₂ 0/100 to 6/94) and preparative TLC (500 μM, eluent system CH ₃ OH/CH ₂ Cl ₂ 5/95) gave the desired compound as a white solid (21 mg, 19%).

(ES+) m/z, Found: 555.2 (M+H+), 577.2 (M+Na+), 1131.4 (2M+Na+). C ₂₅ H ₂₇ N ₆ O ₇ P required: (M) 554.2. ³¹ R NMR (202 MHz, CH3OD) δ 5.1, 4.9.

1H NMR (500 MHz, CH3OD) δ 8.27 (s, 0.5H, H8), 8.24 (s, 0.5H, H8), 8.22 (s, 0.5H, H2), 8, 21 (s, 0.5H, H2), 7.37-7.26 (m, 7H, Ph), 7.22-7.13 (m, 3H, Ph), 6.02 (d, J=1 .8 Hz, 0.5H, H1'), 6.00 (d, J=1.8 Hz, 0.5H, H1'), 5.14-5.11 (m, 2H, OCH2₽h), 4.73-4.64 (m, 2H, H2', H4'), 4.50-4.39 (m, 1H, H5'), 4.36-4.24 (m, 1H, H5') , 3.533.71 (m, 2H, CH2 gly), 2.39-2.25 (m, 1H, H3'), 2.13-2.02 (m, 1H, H3').

¹³ C NMR (125 MHz, CH3OD) δ 172.30 (d, ³ Jc-p=5.0 Hz, C=O), 172.27 (d, ³ Jc-p=5.0 Hz, C=O ), 157.34 (C6), 157.32 (C6), 153.88 (C2), 153.87 (C2), 152.08 (d, ³ Jc-p=7.5 Hz, C-Ar) , 152.05 (d, ³ Jc-p = 7.5 Hz, C-Ar), 150.20 (C4), 150.19 (C4), 140.52 (C8), 140.42 (C8), 137.15 (C-Ar), 130.79 (CH-Ar), 129.57 (CH-Ar), 129.55 (CH-Ar), 129.35 (CH-Ar), 129.34 (CH -Ar), 129.33 (CH-Ar), 126.22 (CH-Ar), 121.44 (d, Jc-p=3.7 Hz, CH-Ar), 121.40 (d, Jc- p=3.7 Hz, CH-Ar), 120.51 (C5), 120.49 (C5), 93.19, 93.14 (C1'), 80.46 (d, ³ Jc-p=4 .60 Hz, C4'), 80.39 (d, ³ JC-P=4.60, C4'), 76.66 (C2'), 68.68 (d, ² JC-P=5.42 Hz , С5'), 68.24 (d, ² JC-P=5.42 Hz, С5'), 67.95 (OCH2₽h), 67.93 (OCH2Ph), 43.90 (CH2 gly), 43 .83 (CH2 gly), 34.83 (C3'), 34.54 (C3').

HPLC. Reverse phase HPLC eluting with H2O/CH3CN 100/10 to 0/100 over 30 min, 1 ml/min, 1=200 nm showed two diastereoisomer peaks with a tR of 13.63 min. and tR 13.41 min.

(2S)-Pentyl 2-(((((2S,4R,5R)-5-(6-amino-9H-purin-9-yl)-4-hydroxytetrahydrofuran-2-yl)methoxy) (naphthalene-1 - yloxy)phosphoryl)amino)-4-methylpentanoate D

Using General Method 1 above, N-methylimidazole (76 μl, 0.95 mmol) and a solution of (2S)-pentyl 2-((chloro(naphthalene-1-yloxy)phosphoryl)amino)-4-methylpentanoate (250 mg , 0.6 mmol) in anhydrous THF (1 ml) was added dropwise to a suspension of 3'-deoxyadenosine (48 mg, 19 mmol) in anhydrous THF (5 ml) and the reaction mixture was stirred at room temperature for 16 h. Purification column chromatography (eluent system CH ₃ OH/CH ₂ Cl ₂ 0/100 to 5/95) and preparative TLC (1000 μM, eluent system CH3OH/CH2Cl2 4/96) gave the desired compound as a white solid ( 27 mg, 22%).

MS (ES+) m/z: Found: 641.3 (M+H+), 663.3 (M+Na+), 1303.6 (2M+Na+). C31H41N6O ₇ P required: (M) 640.3. ³¹ R NMR (202 MHz, CH3OD) δ 4.64, 4.37.

1H NMR (500 MHz, CH3OD) δ 8.28 (s, 0.5H, H-8), 8.25 (s, 0.5H, H-8), 8.21 (s, 0.5H, H -2), 8.20 (s, 0.5H, H-2), 8.17-8.12 (m, 1H, Nap), 7.88-7.83 (m, 1H, Nap), 7 .69-7.66 (m, 1H, Nap), 7.54-7.42 (m, 3H, Nap), 7.407.35 (m, 1H, Nap), 7.31-7.26 (m, 5H, Ar), 6.01 (d, J=2.1 Hz, 0.5H, H1'), 6.00 (d, J=2.1 Hz, 0.5H, H1'), 4.47 -4.67 (m, 2H, H2', H4'), 4.55-4.44 (m, 1H, H5'), 4.43-4.31 (m, 1H, H5'), 4, 00-3.87 (m, 3H, CH leu, CH2 Pen), 2.44-2.30 (m, 1H, H3'), 2.14-2.04 (m, 1H, H3'), 1 .66-1.39 (m, 5H, CH ₂ CH leu, CH ₂ Pen), 1.1.28-1.21 (m, 4H, CH ₂ CH ₂ Pen), 0.86-0.81 ( m, 3H, CH3 Pen), 0.81-0.68 (m, 6H, (CH3)2 leu).

¹³ C NMR (125 MHz, CH3OD) δ 175.42 (d, ³ Jc-p=2.5 Hz, C=O), 175.04 (d, ³ Jc-p=2.5 Hz, C=O ), 157.32 (C6), 153.87 (C2), 153.86 (C2), 150.23 (C4), 147.97 (d, ³ Jc-p=6.2 Hz, 'ipso' Nap ), 140.55 (C8), 136.30 (C-Ar), 136.29 (C-Ar), 128.89 (CH-Ar), 128.84 (CH-Ar), 127.95 (C -Ar), 127.91 (C-Ar), 127.84 (C-Ar), 127.78 (CHAr), 127.76 (CH-Ar), 127.46 (CH-Ar), 126.50 (C-Ar), 126.48 (C-Ar), 126.46 (C-Ar), 126.01 (CH-Ar), 125.91 (CH-Ar), 122.80 (CH-Ar) , 122.70 (CH-Ar), 120.58 (C5), 120.56 (C5), 116.40 (d, ³ Jc-p=3.7 Hz, CH-Ar), 116.01 (d , ³ Jc-p=3.7 Hz, CH-Ar), 93.31 (С1'), 93.27 (С1'), 80.35 (d, ³ Jc-p=3.5 Hz, С4' ), 80.29 (d, ³ Jc-p=3.5 Hz, C4'), 76.54 (C2'), 76.50 (C2'), 69.07 (d, ² JC-P=5 .5 Hz, C5'), 68.85 (d, ² JC-P=5.5 Hz, C5'), 66.33 (CH2 pent), 66.32 (CH2 pent), 54.81 (CH leu ), 54.71 (CH leu), 44.22 (d, ³ Jc-p=7.6 Hz, CH leu), 43.93 (d, ³ Jc-p=7.6 Hz, CH2 leu), 35.15

- 32 040630 (C3'), 34.86 (C3'), 29.32 (CH2 pent), 29.30 (CH2 pent), 29.11 (CH2 pent), 25.67 (CH leu), 25, 45 (CH leu), 23.30 (CH2 pent), 23.12 (CH3 leu), 23.02 (CH3 leu), 22.04 (CH3 leu), 21.78 (CH3 leu), 14.28 ( CH3 pent).

HPLC. Reverse-phase HPLC eluting with H ₂ O/CH ₃ CN 100/10 to 0/100 over 30 min, 1 ml/min, l=200 nm showed one peak of two overlapping diastereoisomers with tR

20.84 min.

Methyl 2-(((((2S,4R,5R)-5-(6-amino-9H-purin-9-yl)-4-hydroxy-tetrahydrofuran-2-yl)-methoxy) (naphthalene-1-yloxy)phosphoryl )amino)-2-methylpropanoate Е

Using General Method 1 above, N-methylimidazole (24 μl, 3.0 mmol) and a solution of methyl 2-((chloro(naphthalene-1-yloxy)phosphoryl)amino)-2-methylpropanoate (612 mg, 1.8 mmol) in anhydrous THF (1 ml) was added dropwise to a suspension of 3'-deoxyadenosine (150 mg, 0.6 mmol) in anhydrous THF (15 ml) and the reaction mixture was stirred at room temperature for 16 h. column chromatography (eluent system CH3OH/CH2Cl2 0/100 to 7/93) and preparative TLC (1000 μM, eluent system CH3OH/CH2Cl2 4/96) gave the desired compound as a white solid (20 mg, 6%).

MS (ES+) m/z: Found: 557.2 (M+H+), 579.2 (M+Na ⁺ ), 1135.4 (2M+Na+). C25H29N6O7P required: (M) 556.51. ³¹ R NMR (202 MHz, CH ₃ OD) δ 2.73.

1H NMR (500 MHz, CH3OD) δ 8.28 (s, 0.5H, H8), 8.25 (s, 0.5H, H8), 8.21 (s, 0.5H, H2), 8, 19 (s, 0.5H, H2), 8.18-8.14 (m, 1H, Nap), 7.90-7.84 (m, 1H, Nap), 7.71-7.66 (m , 1H, Nap), 7.53-7.47 (m, 3H, Nap), 7.41-7.35 (m, 1H, Nap), 6.03 (d, J=2.l Hz, 0 .5H, H1'), 5.99 (d, J=2.1 Hz, 0.5H, H1'), 4.76-4.67 (m, 2H, H2', H4'), 4.52 -4.44 (m, 1H, H5'), 4.42-4.33 (m, 1H, H5'), 3.65 (s, 1.5H, OCH3), 3.64 (s, 1, 5H, OCH3), 2.48-2.41 (m, 0.5H, H3'), 2.372.30 (m, 0.5H, H3'), 2.15-2.09 (m, 0.5H , H3'), 2.08-2.02 (m, 0.5H, H3'), 1.47-1.44 (m, 6H, CH3).

¹³ C NMR (125 MHz, CH3OD) δ 177.25 (d, ³ Jc-p=3.7 Hz, C=O), 157.53 (C6), 157.51 (C6), 153.86 (C2 ), 150.28 (C4), 150.25 (C4), 148.06 (d, ³ Jc-p=7.5 Hz, 'ipso' Nap), 148.04 (d, ³ Jc-p=7 .5, 'ipso' Nap), 140.67 (C8), 140.60 (C8), 136.28 (C-Ar), 136.27 (C-Ar), 128.82 (CH-Ar), 128.80 (CH-Ar), 127.93 (d, ² Jc-p=6.25 Hz, C-Ar), 127.92 (d, ² Jc-p=6.25 Hz, C-Ar) , 127.71 (CH-Ar), 127.69 (CH-Ar), 127.32 (CH-Ar), 126.44 (CH-Ar), 125.84 (CH-Ar), 122.93 ( CH-Ar), 120.56 (C5), 120.50 (C5), 116.38 (d, ³ JC-P=3.75 Hz, CH-Ar), 116.36 (d, ³ JC-P =3.75 Hz, CH-Ar), 93.25 (C1'), 80.40 (d, ³ Jc-p=8.0 Hz, C4'), 80.33 (d, ³ Jc-p= 8.0 Hz, C4'), 76.57 (C2'), 76.43 (C2'), 68.99 (d, ² JC-P=5.5 Hz, C5'), 68.84 (d , ² JC-P=5.5 Hz, C5'), 53.01 (OCH3), 35.22 (C-3'), 34.90 (C3'), 27.85 (d, ³ JC-P =6.0 Hz, CH3), 27.80 (d, ³ JC-P=6.0, CH3), 27.60 (d, ³ JC-P=6.0, CH3), 27.56 (d , ³ JC-P=6.0, CH3).

HPLC. Reverse-phase HPLC eluting with H ₂ O/CH ₃ CN from 100/10 to 0/100 over 30 min, 1 ml/min, l=254 nm, showed two peaks with tR 16.51 min, tR 16, 75 min.

(2S)-Benzyl 2-(((((2S,4R,5R)-5-(6-amino-9H-purin-9-yl)-4-hydroxytetrahydrofuran-2-yl)methoxy) (2-(3 -ethoxy-3-oxopropyl)phenoxy)phosphoryl)amino)propanoate F

Using General Method 1 above, N-methylimidazole (32 μl, 4.2 mmol) and a solution of (2S)benzyl 2-((chloro(2-(3-ethoxy-3-oxopropyl)phenoxy)phosphoryl)amino)propanoate (1.14 g, 2.5 mmol) in anhydrous THF (2 ml) was added dropwise to a suspension of 3'-deoxyadenosine (210 mg, 0.84 mmol) in anhydrous THF (10 ml) and the reaction mixture was stirred at room temperature. temperature for 16 h. Purification by column chromatography (eluent system CH ₃ OH/CHC1 ₃ 0/100 to 8/92) and preparative TLC (1000 μM, eluent system CH ₃ OH/CH ₂ Cl ₂ 5/95) gave the desired compound as a white solid (123 mg, yield=22%).

MS (ES+) m/z: Found: 669.3 (M+H+), 691.3 (M+Na ⁺ ), C31H37N6O9P, required: (M) 668.63.

³¹ P NMR (202 MHz, CH3OD): δP 3.95, 3.65.

1H NMR (500 MHz, CH3OD): δH 8.25 (s, 0.5H, H8), 8.21 (s, 1H, H8, H2), 8.20 (s, 0.5H, H2), 7 .35-7.29 (m, 6H, Ph), 7.25-7.21 (m, 1H, Ph), 7.16-7.07 (m, 2H, Ar), 6.00 (d, J=1.9 Hz, 0.5H, H1'), 5.98 (d, J=1.9 Hz, 0.5H, H1'), 5.17-5.05 (m, 2H, OC^ Ph), 4.76-4.73 (m, 0.5H, H2'), 4.70-4.59 (m, 1.5H, H2', H4'), 4.45-4.34 ( m, 1H, H5'), 4.30-4.22 (m, 1H, H5'), 4.08-3.96 (m, 3H, CH2CH3, CH ala), 2.98-2.92 ( m, 2H, CH2CH2), 2.62-2.56 (m, 2H,

- 33 040630

CH2CH2), 2.40-2.29 (m, 1H, H3'), 2.11-2.03 (m, 1H, H3'), 1.36 (d, J=6.9 Hz, 1, 5 H, CH3 ala), 1.33 (d, J=6.9 Hz,

1.5 N, CH ₃ ala), 1.17 (t, J=7.0 Hz, 1.5 N, CH ₂ CH ₃ ), 1.16 (t, J=7.0 Hz, 1.5 H, CH ₂ CH ₃ ).

¹³ C NMR (125 MHz, CH3OD): 8C 174.82 (d, ³ Jc-p=3.7 Hz, C=O), 174.62 (C=O), 174.58 (C=O), 174.55 (d, ³ Jc-p=3.7 Hz, C=O), 157.34 (C6), 157.32 (C6), 153.86 (C2), 153.84 (C2), 150 .48 (d, Jc-p=2.5 Hz, С-Ar), 150.44 (С4), 150.22 (d, Jc-p=2.5 Hz, С-Ar), 140.49 ( C8), 137.29 (C-Ar), 137.21 (C-Ar), 133.09 (d, J=7.5 Hz, C-Ar), 132.94 (d, J=7.5 Hz, C-Ar), 131.62 (CH-Ar), 131.59 (CH-Ar), 129.58 (CH-Ar), 129.34 (CH-Ar), 129.31 (CHAr), 129.28 (CH-Ar), 128.70 (d, J=5.0 Hz, CH-Ar), 128.69 (d, J=5.0 Hz, CH-Ar), 126.18 (CH -Ar), 121.02 (d, J=2.5 Hz, CH-Ar), 120.49 (d, J=2.5 Hz, CH-Ar), 120.58 (C5), 93.28 (C1'), 93.24 (C1'), 80.32 (d, ³ Jc-p=8.7 Hz, C4'), 76.57 (C2'), 68.86 (d, ² Jc- p=5.0 Hz, C5'), 68.53 (d, ² Jc-p=5.0 Hz, C5'), 67.98 (OCH2Ph), 67.95 (OCH2Ph), 61.57 (CH2CH3 ), 51.76 (CH ala), 51.65 (CH ala), 35.37 (CH2CH2), 35.30 (CH2CH2), 35.08 (C3'), 34.85 (C3'), 26, 77 (CH2CH2), 26.72 (CH2CH2), 20.55 (d, ³ Jc-p=6.2 Hz, CH3 ala), 20.33 (d, ³ Jc-p=6.2 Hz, CH3 ala ), 14.53 (CH2CH3).

HPLC. Reverse phase HPLC eluting with H2O/CH3CN 100/10 to 0/100 over 30 min, 1 ml/min, 1=245 nm showed one peak with a tR of 15.99 min.

(2S)-Benzyl 2-(((((2R,3R,5S)-2-(6-amino-9H-purin-9-yl)-5-(hydroxymethyl)tetrahydrofuran-3-yl)oxy)(phenoxy )phosphoryl)amino)propanoate G

ABOUT

Using General Method 3 above, 3'-deoxyadenosine (50 mg, 0.20 mmol) was suspended in anhydrous THF (5 ml) and tert-BuMgCl (1.0 M solution in THF, 0. 22 ml, 0.22 mmol). A solution of (28)-benzyl 2-((chloro(phenoxy)phosphoryl)amino)propanoate (212 mg, 0.6 mmol) in anhydrous THF (2 ml) was added dropwise and the reaction mixture was stirred at room temperature for 16 h Purification by column chromatography (eluent system cH ₃ OH/cH ₂ cl ₂ 0/100 to 8/92) and preparative TLC (500 μM, eluent system CH ₃ OH/CH ₂ Cl ₂ =5/95) gave the desired compound as a white solid (6 mg, 5%).

MS (ES+) m/z: Found: 569.2 (M+H+), 591.2 (M+Na+), 1159.4 (2M+Na+). C26H29N6O7P required: (M) 568.2.

³¹ P NMR (202 MHz, CH3OD): 8P 2.44 (s), 2.92 (s). 1H NMR (500 MHz, CH3OD): 8H 8.41 (s, 0.5H, H8), 8.28 (s, 0.5H, H8), 8.19 (s, 0.5H, H2), 8.18 (s, 0.5H, H2), 7.39-7.30 (m, 4H, Ar), 7.28-7.18 (m, 4H, Ar), 7.177.11 (m, 1H , Ar), 7.08-7.03 (m, 1H, Ar), 6.23 (d, J=2.0 Hz, 0.5H, H1'), 6.08 (d, J=3, 4 Hz, 0.5H, H1'), 5.52-5.43 (m, 1H, C2'), 5.19-5.12 (m, 1H, CH ₂ Ph), 5.07-4, 95 (m, 1H, CH2Ph), 4.48-4.42 (m, 1H, H4'), 4.05-3.97 (m, 1H, CH ala), 3.95-3.87 (m , 1H, H5'), 3.69-3.61 (m, 1H, H5'), 2.59-2.45 (m, 1H, H3'), 2.31-2.23 (m, 1H , H3'), 1.36-1.27 (m, 3H, CH3 ala).

¹³ C NMR (125 MHz, CH3OH): 8C 174.76 (d, ³ Jc-p=5.0 Hz, C=O), 174.52 (d, ³ Jc-p=5.0 Hz, C= O), 157.44 (C6), 153.76 (C2), 151.93 (C4), 150.06 (C-Ar), 149.93 (C-Ar), 141.38 (C8), 141 .18 (C8), 137.33 (C-Ar), 137.10 (C-Ar), 130.69 (CH-Ar), 130.79 (CH-Ar), 129.61 (CH-Ar) , 129.51 (CH-Ar), 129.40 (CH-Ar), 129.30 (CH-Ar), 129.23 (CH-Ar), 126.33 (CH-Ar), 126.16 ( CH-Ar), 121.53 (d, ³ Jc-p=4.5 Hz, CH-Ar), 121.20 (d, ³ Jc-p=4.5 H, CHAr), 120.76 (C5 ), 91.56 (d, ³ Jc-p=7.7 Hz, C1'), 91.45 (d, ³ Jc-p=7.7 Hz, C1'), 82.78 (C4'), 82.28 (C4'), 81.83 (d, ² Jc-p=4.7 Hz, C2'), 80.96 (2xd, ² Jc-p=4.7 Hz, C2'), 67, 95 (OCH2Ph), 67.92 (OCH2Ph), 64.13 (C5'), 63.59 (C5'), 51.88 (CH ala), 51.75 (CH ala), 33.75 (d, ³ Jc-p=3.0 Hz, С3'), 33.59 (d, ³ Jc-p=3.0 Hz, С3'), 20.33 (d, ³ Jc-p=7.1 CH3 ala ), 20.18 (d, ³ Jc-p=7.1 CH3 ala).

HPLC. Reverse phase HPLC eluting with H ₂ O/CH ₃ OH 90/10 to 0/100 over 30 min, 1 ml/min, 1=254 nm, showed two diastereoisomer peaks with a tR of 22.16 min. and tR 22.43 min.

Benzyl 2-(((((2S,4R,5R)-5-(6-amino-9H-purin-9-yl)-4-((((1-(benzyloxy)-1-oxopropan-2-yl )amino) (phenoxy)phosphoryl)oxy)tetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-amino)propanoate H

- 34 040630

Using general method 3 above, 3'-deoxyadenosine (50 mg, 0.20 mmol) was suspended in anhydrous THF (5 ml) and tert-BuMgCl (1.0 M solution in THF, 0. 22 ml, 0.22 mmol). A solution of (2S)-benzyl 2-((chloro(phenoxy)phosphoryl)amino)propanoate (212 mg, 0.6 mmol) in anhydrous THF (2 ml) was added dropwise and the reaction mixture was stirred at room temperature for 16 h Purification by column chromatography (eluent system CH ₃ OH/CH ₂ Cl ₂ 0/100 to 8/92) and preparative TLC (500 μM, eluent system CH ₃ OH/CH ₂ Cl ₂ 5/95) gave the desired compound as a white solid (19 mg, yield=11%).

MS (ES+) m/z, Found: 886.3 (M+H+), 1771.6 (2M+H+), 751.2 (molecule without M nucleobase). C42H45N7O11P2 required: (M+) 885.3.

³¹ P NMR (202 MHz, CH3OD): 5P 3.98, 3.88, 3.59, 3.12, 3.05, 2.45, 2.32.

1H NMR (500 MHz, CH3OD): 5H 8.24-8.13 (m, 2H, H8, H2), 7.39-7.08 (m, 20H, Ph), 6.27-6.23 ( m, 0.5H, H1'), 6.16-6.13 (m, 0.5H, H1'), 5.61-5.48 (m, 1H, H2'), 5.17-4, 91 (m, 4H, CH2Ph), 4.57-4.49 (m, 1H, H4'), 4.414.29 (m, 1H, H5'), 4.25-4.15 (m, 1H, H5 '), 4.10-4.01 (m, 1H, CH ala), 3.99-3.89 (m, 1H, CH ala), 2.57-2.41 (m, 1H, H3') , 2.28-2.17 (m, 1H, H3'), 1.38-1.23 (m, 6H, CH3 ala).

¹³ C NMR (125 MHz, CH3OD): 5C 174.88 (C=O), 174.83 (C=O), 174.79 (C=O), 174.73 (C=O), 174.61 (C=O), 174.57 (C=O), 174.53 (C=O), 157.36 (C6), 157.34 (C6), 157.32 (C6), 157.29 (C6 ), 154.04 (C2), 154.01 (C2), 153.97 (C2), 153.94 (C2), 152.09 (C4), 152.04 (C4), 152.02 (C4) , 151.97 (C4), 150.31 (C-Ar), 150.29 (CAr), 150.16 (C-Ar), 140.98 (C8), 140.91 (C8), 140.81 (C8), 137.31 (C-Ar), 137.28 (C-Ar), 137.22 (C-Ar), 137.09 (C-Ar), 130.86 (CH-Ar), 130 .78 (CH-Ar), 130.77 (CH-Ar), 129.65 (CH-Ar), 129.61 (CH-Ar), 129.58 (CH-Ar), 129.55 (CH- Ar), 129.44 (CH-Ar), 129.42 (CH-Ar), 129.38 (CH-Ar), 129.34 (CH-Ar), 129.32 (CHAr), 129.30 ( CH-Ar), 129.28 (CH-Ar), 129.23 (CH-Ar), 129.21 (CH-Ar), 12.42 (CH-Ar), 126.23 (CH-Ar), 126.20 (CH-Ar), 126.17 (CH-Ar), 121.65 (CH-Ar), 121.63 (CH-Ar), 121.61 (CH-Ar), 121.59 (CH -Ar), 121.52 (CH-Ar), 121.50 (CH-Ar), 121.47 (CH-Ar), 121.46 (CH-Ar), 121.40 (CH-Ar), 121 .39 (CH-Ar), 121.36 (CHAr), 121.35 (CH-Ar), 121.30 (CH-Ar), 121.28 (CH-Ar), 121.26 (CH-Ar) , 121.24 (CH-Ar), 1 20.61 (C5), 120.57 (C5), 120.56 (C5), 120.54 (C5), 91.56 (Cl ), 91.51 (C1'), 91.45 (C1' ), 91.25 (C1'), 91.20 (C1'), 81.84 (C2'), 81.82 (C2'), 81.79 (C2'), 81.27 (C2'), 81.22 (C2'), 81.18 (C2'), 80.49 (C4'), 80.43 (C4'), 80.06 (C4'), 79.99 (C4'), 68, 29 (C5', OCH2Ph), 68.25 (C5', OCH2Ph), 68.00 (C5', OCH2Ph), 67.96 (C5', OCH2Ph), 67.94 (C5', OCH2Ph), 67, 90 (C5', OCH ₂ Ph), 67.71 (C5', OCH ₂ Ph), 67.67 (C5', OCH ₂ Ph), 51.91 (CH ala), 51.74 (CH ala), 51.70 (CH ala), 51.59 (CH ala), 34.22 (C3'), 34.20 (C3'), 34.16 (C3'), 33.97 (C3'), 33, 94 (C3'), 33.91 (C3'), 20.44 (CH3 ala), 20.43 (CH ₃ ala), 20.39 (CH ₃ ala), 20.29 (CH ₃ ala), 20 .27 (CH ₃ ala), 20.24 (CH ₃ ala), 20.21 (CH ₃ ala), 20.19 (CH ₃ ala).

HPLC. Reverse phase HPLC eluting with H2O/CH3CN 100/10 to 0/100 over 30 min, 1 ml/min, l=254 nm showed one broad peak with a tR of 15.97 min.

(2S)-Benzyl 2-(((((2R,3R,5S)-2-(6-amino-9H-purin-9-yl)-5-(hydroxymethyl)tetrahydrofuran-3-yl)oxy)(naphthalene -1-yloxy)phosphoryl)amino)propanoate I

Using General Method 3 above, 3'-deoxyadenosine (50 mg, 0.20 mmol) was suspended in anhydrous THF (5 ml) and tert-BuMgCl (1.0 M solution in THF, 0. 3 ml, 0.3 mmol). A solution of (2S)-benzyl 2-((chloro(naphthalen-1yloxy)phosphoryl)amino)propanoate (323 mg, 0.8 mmol) in anhydrous THF (2 ml) was added dropwise and the reaction mixture was stirred at room temperature for 16 hours Purification by column chromatography

- 35 040630 (eluent system CH ₃ OH/CH ₂ Cl ₂ 0/100 to 6/94) and preparative TLC (500 μM, eluent system

CH ₃ OH/CH ₂ C1 ₂ 5/95) gave the desired compound as a white solid (14 mg, 11%).

(ES+) m/z, Found: 619.2 (M+H+), 641.2 (M+Na ⁺ ), 1259.4 (2M+Na ⁺ ). C ₃₀ H ₃₁ N ₆ O ₇ P required: (M)

618.20.

³¹ P NMR (202 MHz, CH3OD): δP 3.27 (s), 2.75 (s). ¹ H NMR (500 MHz, CH3OD): δH 8.37 (s, 1H, H8), 8.18 (s, 1H, H8), 8.14 (s, 1H, H2), 8.13-8, 11 (m, 0.5 N, Nap) 8.11 (s, 1H, H2), 7.94-7.90 (m, 0.5 N, Ar), 7.907.87 (m, 0.5 N , Ar), 7.86-7.82 (m, 0.5 H, Ar), 7.74-7.70 (m, 0.5 H, Ar), 7.66-7.61 (m, 0.5 H, Ar), 7.57-7.47 (m, 1.5 H, Ar), 7.46-7.37 (m, 2.5 H, Ar), 7.34-7, 27 (m, 4H, Ar), 7.25-7.17 (m, 1H, Ar), 6.19 (d, J=2.4 Hz, 0.5H, H1'), 6.04 (d , J=2.4 Hz, 0.5H, H1'), 5.60-5.54 (m, 0.5H, H2'), 5.50-5.42 (m, 0.5H, H2' ), 5.16-4.99 (m, 2H, OC^Ph), 4.46-4.40 (m, 0.5H, H4'), 4.36-4.30 (m, 0.5H , H4'), 4.13-4.04 (m, 1H, CH ala), 3.90-3.83 (m, 1H, H5'), 3.64-3.56 (m, 1H, H5 '), 2.61-2.54 (m, 0.5H, H3'), 2.49-2.41 (m, 0.5H, H3'), 2.35-2.27 (m, 0 .5H, H3'), 2.22-2.16 (m, 0.5H, H3'), 1.35-1.24 (m, 3H, CH3 ala).

¹³ C NMR (125 MHz, CH3OH): δC 174.52 (C=O), 174.49 (C=O), 157.27 (C6), 153.58 (C2), 149.97 (C4), 149.93 (C-4), 147.70 (d, ³ Jc-p=7.5, 'ipso' Nap), 147.48 (d, ³ Jc-p=7.5, 'ipso' Nap) , 141.36 (C8), 141.19 (C8), 137.25 (C-Ar), 137.05 (C-Ar), 136.31 (C-Ar), 136.20 (C-Ar) , 129.58 (CH-Ar), 129.48 (CH-Ar), 129.37 (CHAr), 129.26 (CH-Ar), 129.22 (CH-Ar), 128.88 (CH- Ar), 127.84 (CH-Ar), 127.75 (CH-Ar), 127.49 (CH-Ar), 127.44 (CH-Ar), 126.48 (CH-Ar), 126, 39 (CH-Ar), 126.26 (CH-Ar), 126.05 (CH-Ar), 122.76 (CH-Ar), 122.38 (CH-Ar), 120.68 (C5), 120.61 (C5), 116.64 (d, ³ Jc-p=3.75 Hz, CH-Ar), 116.13 (d, ³ Jc-p=3.75, CH-Ar), 91, 60 (d, ³ Jc-p=7.5 Hz, C1'), 91.43 (d, ³ Jc-p=7.5 Hz, C1'), 82.74 (C4'), 82.27 ( C4'), 81.99 (d, ² Jc-p=5.5 Hz, C2'), 81.12 (d, ² J _c . _p =5.5 Hz, C2'), 67.97 (OCH ₂ Ph), 67.94 (OCH ₂ Ph), 64.16 (C5'), 63.51 (C5'), 51.96 (CH ala), 51.89 (CH ala), 33.89 (d , ³ Jc-p=7.5 Hz, CH3 ala), 33.63 (d, ³ Jc-p=7.5 Hz, CH3 ala).

HPLC Reverse phase HPLC eluting with H ₂ O/CH ₃ OH 100/10 to 0/100 over 30 min, 1 ml/min, 1=200 nm, showed two diastereoisomer peaks with a tR of 24.84 min. and tR 25.43 min.

Benzyl 2- [( {[5 -(6-amino-9H-purin-9-yl)-4-hydroxyoxolan-2-yl]methoxy }({[1-(benzyloxy)-1oxopropan-2-yl]amino} )phosphoryl)amino]propanoate J

ABOUT

Using general method 2 above, 3'-deoxyadenosine (200 mg, 0.80 mmol) was suspended in (CH ₃ ) ₃ PO ₃ (5 ml) and POCl ₃ (75 µl, 0.80 mmol). The reaction mixture was allowed to warm to room temperature and allowed to stir for 4 h. in anhydrous CH ₂ Cl ₂ (5 ml), then diisopropylethylamine (1.4 ml, 8.0 mmol) at -78°C. After stirring at room temperature for 20 hours, water was added and the layers were separated. The aqueous phase was extracted with dichloromethane and the organic phase was washed with brine. The combined organic layers were dried over Na ₂ SO ₄ and concentrated. The residue was purified by column chromatography (gradient elution with CH ₂ Cl ₂ /MeOH= 100/0 to 93/7) to give a white foam (256 mg, 49%).

MS (ES+) m/z: Found: 654.2 (M+H ⁺ ), 676.2 (M+Na ⁺ ), 1329.5 (2M+Na+). C ₃₀ H ₃₆ N ₇ O ₈ P required: (M) 653.62. ³¹ P NMR (202 MHz, CH3OD) δ 13.9.

1H NMR (500 MHz, CH ₃ OD) δ 8.28 (s, 1H, H8), 8.22 (s, 1H, H2), 7.37-7.26 (m, 10H, Ph), 6, 00 (d, J=1.9 Hz, 1H, H1'), 5.15-5.05 (m, 4H, OCH2Ph), 4.74-4.70 (m, 1H, H2'), 4, 63-4.56 (m, 1H, H4'), 4.24-4.18 (m, 1H, H5'), 4.11-4.05 (m, 1H, H5'), 3.97- 3.87 (m, 1H, CH ala), 2.35-2.27 (m, 1H, H3'), 2.07-2.01 (m, 1H, H3'), 1.341.27 (m, ZN, CH ₃ ala).

¹³ C NMR (125 MHz, CH3OD) δ 175.40 (d, ³ _Jc.p = 5.0 Hz, C=O), 175.36 (d, J _C - _P 5.0 Hz, C=O) , 157.36 (C6), 153.91 (C2), 150.25 (C4), 140.64 (C8), 137.33 (C-Ar), 137.29 (C-Ar), 129.58 (CH-Ar), 129.57 (CH-Ar), 129.33 (CH-Ar), 129.31 (CH-Ar), 129.29 (CH-Ar), 120.55 (C5), 93 .18 (C1'), 80.67 (d, ³ Jc-p=8.4 Hz, C4'), 76.59 (C2'), 67.90 (OCH2Ph), 67.47 (d, ² Jc -p=5.2 Hz, С5'), 51.14 (d, ² Jc-p=1.7 Hz, CH ala), 51.11 (d, ² Jc-p=1.7 Hz, CH ala ), 35.08 (S3'), 20.77 (d, ³ Jc-p=6.5 Hz, CH3 ala), 20.59 (d, ³ Jc-p=6.5 Hz, CH3 ala).

HPLC Reverse phase HPLC eluting with H ₂ O/CH ₃ CN 90/10 to 0/100 over 30 min, 1 ml/min, 1=254 nm showed one peak with tR 13.87 min.

(2S)-Benzyl 2-((((2S,4R,5R)-5-(6-amino-2-methoxy-9H-purin-9-yl)-4-hydroxytetrahydrofuran-2-yl)methoxy)(naphthalene -1-yloxy)phosphorylamino)propanoate K

- 36 040630

Using General Method 1 above, N-methylimidazole (99 μl, 1.24 mmol) and a solution of (2S)-benzyl 2-((chloro(naphthalene-1-yloxy)phosphoryl)amino)propanoate (303 mg, 0. 75 mmol) in anhydrous THF (5 ml) was added dropwise to a suspension of 2-O-methyl-3'-deoxyadenosine (70 mg, 0.25 mmol) in anhydrous THF (10 ml) and the reaction mixture was stirred at room temperature in for 16 hours. Purification by column chromatography (eluent system CH ₃ OH/CH ₂ Cl ₂ 0/100 to 6/94) and preparative TLC (eluent system CH ₃ OH/CH ₂ Cl ₂ 5/95) gave the desired compound as a white solid (96 mg, 60%).

MS (ES+) m/z: Found: 649.2 (M+H+) C31H33N6O8P, required: 648.21(M).

³¹ P NMR (202 MHz, CD3OD): pP 4.38 (s), 4.08 (s).

1H NMR (500 MHz, CD3OD): bH 8.14-8.11 (d, J=8.0 Hz, 0.5H, Ar), 8.07 (d, J=8.0 Hz, 0.5H , Ar), 8.05 (s, 0.5H, H8), 8.02 (s, 0.5H, H8), 7.82-7.80 (m, 1H, Ar), 7.61 (d , J=7.0 Hz, Ar), 7.47-7.44 (m, 4H, Ar), 7.35-7.29 (m, 2H, Ar), 7.24-7.22 (m , 3H, Ar), 5.88 (s, 1H, H1'), 4.71-4.68 (m, 1H, H4'), 4.65-6.60 (m, 1H, H2'), 4.42-4.40 (m, 1H, H5'), 4.30-4.27 (m, 1H, H5'), 4.08-3.98 (m, 1H, CH ala) 3.88 (s, 1.5H, OCH3), 3.86 (s, 1.5H, OCH3), 2.372.33 (m, 1H, H3'), 2.04-2.01 (m, 1H, H3') , 1.27 (d, J=7.0 Hz, 1.5H, CH3), 1.24 (d, J=7.0 Hz, 1.5H, CH3).

¹³ C NMR (125 MHz, CH3OD): bC 174.83 (d, ³ JC-P=3.7 Hz, C=O), 174.60 (d, ³ JC-P=3.7 Hz, C= O), 163.70 (C-2), 158.10 (C6), 151.95 (C4), 147.95 (d, ³ Jc-p=7.5 Hz, 'ipso' Nap), 147, 91, (d, ³ Jc-p=7.5 Hz, 'ipso' Nap), 139.39 (C8), 139.37 (C8), 137.12, 137.17 (C-ipso CHPh), 136 .22 (C-Ar), 129.57, 129.54, 129.48, 129.32, 129.27, 129.12, 129.24 128.89, 128.83, (CH-Ar), 127 .85 (d, ² Jc-p=6.25 Hz, C-Ar), 127.86, 127.76, 127.51, 127.48, 126.49, 126.00, 125.97, 122, 73, 122.63 (CH-Ar), 116.86 (C5), 116.72 (C5), 116.29 (d, ³ Jc-p=3.75 Hz, CH-Ar), 116.22 ( e, ³ Jc-p=3.75 Hz, CH-Ar), 93.33 (C1'), 93.31(C1'), 80.24 (e, ³ Jc-p=2.75 Hz, C4 '), 76.29 (C2'), 76.26 (C2'), 69.09 (d, ² Jc-p=5.0 Hz, C5'), 68.16 (d, ² JC-P= 8.2 Hz, C5'), 67.95 (OCH ₂ Ph), 55.28, 55.32 (OCH ₃ ), 51.79 (CH ala), 51.71 (CH ala), 35.40 ( C-3'), 35.12 (C3'), 20.49 (d, ³ JC-P=6.7 Hz, CH3 ala), 20.35 (d, ³ JC-P=6.7, CH3 ala).

HPLC. Reverse phase HPLC eluting with 100/10 to 0/100 H2O/CH3CN over 30 min, F=1 ml/min, λ=280 nm showed two diastereoisomer peaks with a tR of 16.22 min. and tR 16.48 min.

(2S)-Benzyl 2-((((2S,4R,5R)-5-(6-amino-2-methoxy-9H-purin-9-yl)-4-hydroxytetrahydrofuran-2-yl)methoxy)(phenoxy )phosphorylamino)propanoate L

Using General Method 1 above, N-methylimidazole (99 μl, 1.24 mmol) and a solution of (2S)-benzyl 2-((chloro(phenoxy)phosphoryl)amino)propanoate (264 mg, 0.75 mmol) in anhydrous THF (2 ml) was added dropwise to a suspension of 2-O-methyl-3'-deoxyadenosine (70 mg, 0.25 mmol) in anhydrous THF, and the reaction mixture was stirred at room temperature for 16 h. Chromatography (eluent system CH ₃ OH/CH ₂ Cl ₂ 0/100 to 6/94) and preparative TLC (eluent system CH3OH/CH2Cl2 5/95) gave the desired compound as a white solid (13 mg, 10%) .

(ES+) m/z, Found: 599.2 (M+H+), C27H31N6O8P, required: 598.19 (M).

³¹ P NMR (202 MHz, CD3OD) b 3.97, 3.64.

1H NMR (500 MHz, CD3OD) b 8.06 (s, 0.5H, H8), 8.04 (s, 0.5H, H8), 7.33-7.28 (m, 7H, Ph), 7.20-7.14 (m, 3H, Ph), 5.92 (d, J=1.5 Hz, 0.5H, H1'), 5.90 (d, J=1.5 Hz, 0 .5H, H1'), 5.14-5.04 (m, 2H, OCH ₂ Ph), 4.78-4.76 (m, 0.5H, H4'), 4.74-4.72 ( m, 0.5H, H4'), 4.63-4.59 (m, 1H, H2'), 4.10-4.34 (m, 1H, H5'a), 4.25-4.20 (m, 1H, H5'b), 3.94, 3.95 (OCH3), 3.99-3.90 (m, 1H, CH ala), 2.40-2.37 (m, 1H, H3 '), 2.07-2.04 (m, 1H, H3'), 1.31 (d, J=7.0 Hz, CH3), 1.26 (d, J=7.0 Hz, CH3) .

¹³ C NMR (125 MHz, CD3OD) b 174.82 (d, ³ JC-P=3.7 Hz, C=O), 174.62 (d, ³ JC-P=3.7 Hz, C=O ), 163.80 (C2), 158.16, 158.13 (C6), 152.15 (C4), 152.05 (d, ³ JC-P=4.8 Hz, C-ipso Ph), 152 .00 (d, ³ JC-P=4.8 Hz, C-ipso Ph), 139.39 (C8), 137.30, 137.21 (C-ipso CH2Ph), 130.72, 129.57, 129.31.129.27, 126.122 (CH-Ar), 121.42 (d, Jc-p=4.5 Hz, CH-Ar), 121.37 (d, Jc-p=4.5 Hz, CH- Ar), 116.72 (C5), 116.69 (C5), 93.33, 93.24 (C1'), 80.26 (d, ³ Jc-p=8.87, C4'), 80, 19 (d, ³ Jc-p=8.87, C4'), 76.35 (C2'), 68.78 (d, ² Jc-p=5.0 Hz, C5'), 68.35 (d , ² Jc-p=5.0 Hz, С5'),

- 37 040630

67.94 (OCH ₂ Ph), 67.92 (OCH ₂ Ph), 55.25, 55.28 (OCH3), 51.69, 51.57 (CH ala), 35.23 (C3'), 34 .96 (C3'), 20.38 (d, ³ Jc-p=6.7, CH3 ala), 20.26 (d, ³ Jc-p=6.7, CH3 ala).

HPLC. Reverse phase HPLC eluting with 100/10 to 0/100 H2O/CH3CN over 30 min, F=1 ml/min, λ=280 nm showed two diastereoisomer peaks with a tR of 14.22 min. and tR 14.51 min.

2-O-Methyl-3'-deoxyadenosine-5'-O-[1-naphthyl(1-pentyloxy-L-leucinyl)]phosphate М

Compound M was prepared according to General Method 1 using 2-O-methyl-3'deoxyadenosine (70 mg, 0.25 mmol), N-methylimidazole (99 μl, 1.24 mmol) and naphthyl(pentyloxy-L-leucinyl)phosphorochloridate (330 mg, 0.75 mmol). Purification by column chromatography (0/100 to 6/94 CH ₃ OH/CH ₂ Cl ₂ gradient eluent system) and preparative TLC (2000 μM, CH3OH/CH2Cl2 7/93 eluent system) gave the title compound in as a white solid (50 mg, 30%).

³¹ P NMR (202 MHz, CD ₃ OD) 5P 4.53, 4.28.

1H NMR (500 MHz, CD3OD) 6H 8.04-7.96 (m, 1H, H8), 7.77-7.71 (m, 1H, Nap), 7.58-7.53 (m, 1H , Nap), 7.45-7.17 (m, 5H, Nap), 5.83-5.75 (m, 1H, H1'), 4.64-4.51 (m, 2H, H2', H4'), 4.40-4.16 (m, 2H, H5'), 3.88-3.75 (m, 6H, OCH3, O(CH2) ₄ CH3, CHCH2CH(CH3)2), 2, 38-2.24 (m, 1H, H3'), 2.00-1.91 (m, 1H, H3'), 1.53-1.05 (m, 11H, O(CH2) ₄ CH3, CHCH2CH (CH3)2), 0.77-0.55 (m, 9H, O(CH)4CH3, SNODSCCHCH3b).

¹³ C NMR (125 MHz, CD3OD) 5C 175.02 (d, ³ Jc-p=2.5 Hz, C=O), 174.78 (d, ³ Jc-p=2.5 Hz, C=O ), 163.76 (C2), 158.14 (C6), 151.03 (C4), 147.96 (d, ³ Jc-p=7.2, 'ipso' Nap), 138.96 (C8) , 136.30 (C-Ar), 136.28 (C-Ar), 136.22 (C-Ar), 128.93 (CH-Ar), 128.88 (CH-Ar), 128.81 ( CH-Ar), 128.48 (CH-Ar), 127.77 (CH-Ar), 127.73 (CH-Ar), 127.44 (CH-Ar), 127.42 (CH-Ar), 127.06 (CH-Ar), 126.86 (CH-Ar), 126.45 (CH-Ar), 126.44 (CHAr), 126.31 (CH-Ar), 125.98 (CH-Ar ), 125.88 (CH-Ar), 123.83 (CH-Ar), 123.43 (CH-Ar), 123.24 (CH-Ar), 122.81 (CH-Ar), 122.77 (CH-Ar), 122.69 (CH-Ar), 116.34 (d, ³ Jc-p=3.7 Hz, CH-Ar), 116.02 (d, ³ Jc-p=3.7 Hz, CH-Ar), 115.71 (C5), 93.42 (C1'), 93.32 (C1'), 80.22 (d, ³ Jc-p=5.3 Hz, C4'), 80.15 (d, ³ Jc-p=5.3 Hz, C4'), 76.29 (C2'), 76.27 (C2'), 69.22 (d, ² Jc-p=5.2 Hz, C5'), 69.028 (d, ² Jc-p=5.2 Hz, C5'), 66.31 (O(CH2)4CH3), 66.30 (O(CH2)4CH3), 55.29 ( OCH3), 55.24 (OCH3), 54.79 (CHCH2CH(CH3)2), 54.68 (CHCH2CH(CH3)2), 44.20 (d, ³ Jc-p=7.25 Hz, CHCH2CH( CH3)2), 43.93 (d, ³ Jc-p=7.25 Hz , CHCH2CH(CH ₃ ) ₂ ), 35.49 (C3'), 35.17 (C3'), 29.31 (O(CH ₂ ) ₄ CH ₃ ), 29.11 (O(CH ₂ ) ₄ CH ₃ ), 25.67 (CHCH ₂ CH(CH ₃ ) ₂ ), 25.44 (CHCH ₂ CH(CH ₃ ) ₂ ), 23.30 (O(CH2)4CH3), 23.10 (CHCH2CH(CH3) 2), 23.00 (CHCH2CH(CH3)2), 22.94 (CHCH2CH(CH3)2), 22.81 (CHCH2CH(CH3)2), 14.27 (O(CH2) ₄ CH3).

(ES+) m/z, Found: 671.3 (M+H+), C ₃₂ H ₄₃ N ₆ O ₈ P, required: 670.69 (M).

HPLC. Reverse-phase HPLC eluting with H2O/CH3CN 100/10 to 0/100 over 30 min, 1 ml/min, l=254 nm showed two diastereoisomer peaks with a tR of 20.83 min. and tR 20.93 min.

2-O-Methyl-3'-deoxyadenosine-5'-O-[phenyl (1-hexyloxy-L-alaninyl)]phosphate N

ABOUT

Compound N was prepared according to General Method 1 using 2-O-methyl-3'deoxyadenosine (70 mg, 0.25 mmol), N-methylimidazole (99 μl, 1.24 mmol) and phenyl(hexyloxy-Lalaninyl)phosphorochloridate (261 mg, 0.75 mmol). Purification by column chromatography (CH _{3 OH} /CH ₂ C1 ₂ gradient 0/100 to 6/94 eluent system) and preparative TLC (1000 μM, CH ₃ OH/CH ₂ C1 ₂ 7/93 eluent system) gave the title compound as a white solid (26 mg, 18%).

³¹ P NMR (202 MHz, CD3OD) 5P 3.87, 3.65.

1H NMR (500 MHz, CD3OD) δΠ 8.08 (s, 0.5H, H8), 8.07 (s, 0.5H, H8), 7.36-7.29 (m, 2H, Ph), 7.24-7.14 (m, 3H, Ph), 5.94 (d, J=2.0 Hz, 0.5H, H1'), 5.92 (d, J=2.0 Hz, 0 .5H, H1'), 4.81-4.76 (m, 1H, H2'), 4.71-4.62 (m, 1H, H4'), 4.48-4.43 (m, 0 .5H, H5'), 4.42-4.36 (m, 0.5H, H5'), 4.33-4.25 (m, 1H, H5'), 4.10-3.83 (m , 6H, _OCH3 , O(CH2)5CH3, CHCH3), 2.48-2.40 (m, 1H, H3'), 2.13-2.07 (m, 1H, H3'), 1.61 -1.51 (m, 2H, O(CH2)5CH3), 1.331.24 (m, 9H, O(CH2)5CH3, CHCH3), 0.89 (m, 3H, O(CH2)5CH3).

¹³ C NMR (125 MHz, CD3OD) 6C 175.13 (d, ³ Jc-p=4.3 Hz, C=O), 174.94 (d, ³ Jc-p=4.3 Hz, C=O ), 163.80

- 38 040630 (C2), 163.78 (C2), 158.17 (C6), 158.15 (C6), 152.17 (d, ² Jc-p=6.3 Hz, C-Ar), 152 .15 (d, ² Jc-p=6.3 Hz, C-Ar), 152.03 (C4), 151.99 (C4), 139.42 (C8), 139.39 (C8), 130, 75 (CH-Ar), 130.74 (CH-Ar), 126.13 (CH-Ar), 121.43 (CH-Ar), 121.41 (CH-Ar), 121.39 (CH-Ar ), 121.37 (CH-Ar), 116.74 (C5), 116.69 (C5), 93.40 (C1'), 93.27 (C1'), 80.30 (C4'), 80 .23 (C4'), 76.40 (C2'), 68.85 (d, ² Jc-p=5.2 Hz, C5'), 68.42 (d, ² Jc-p=5.2 Hz , C5'), 66.43 (OCHCH-CHS), 55.30 (OCH3), 55.26 (OCH3), 51.64 (CHCH3), 51.54 (CHCH3), 35.30 (C3'), 35.04 (C3'), 32.58 (O(CH2)5CH3), 29.67 (O(CH2)5CH3), 29.64 (O(CH2)5CH3), 26.61 (O(CH2)5CH3 ), 23.59 (O(CH2)5CH3), 20.56 (d, ³ Jc-p=6.4 Hz, CHCH3), 20.41 (d, ³ Jc-p=6.4 Hz, CHCH3) , 14.36 (O(CH2) _5CH3 ).

(ES+) m/z, Found: 593.3 (M+H ⁺ ), C ₃₂ H ₄₃ N ₆ O ₈ P, required: 592.58 (M).

HPLC Reverse phase HPLC eluting with 100/10 to 0/100 H2O/CH3CN over 30 min, 1 ml/min, 1=254 nm, showed two diastereoisomer peaks with a tR of 17.02 min. and tR 17.23 min.

2-Fluoro-3'-deoxyadenosine-5'-O-[1-naphthyl(benzyloxy-L-alaninyl)]phosphate О

Compound O was prepared according to General Method 1 using 2-fluoro-3'-deoxyadenosine (50 mg, 0.18 mmol), N-methylimidazole (74 μl, 0.93 mmol) and phenyl(benzyloxy-L-alaninyl) phosphorochloridate (196 mg, 0.56 mmol). Purification by column chromatography (eluent system with CH ₃ OH/CH ₂ Cl ₂ gradient from 0/100 to 6/94) and preparative TLC (500 μm, eluent system CH ₃ OH/CH ₂ cl ₂ 5/95) gave the title compound as a white solid (5 mg, 4%).

³¹ P NMR (202 MHz, CD3OD) 5P 4.33, 4.08.

1H NMR (500 MHz, CD3OD) 5H 8.17 (s, 0.5H, H8), 8.14 (s, 0.5H, H8), 8.14-8.09 (m, 1H, Ar), 7.89-7.85 (m, 1H, Ar), 7.70-7.66 (m, 1H, Ar), 7.54-7.42 (m, 4H, Ar), 7.40-7 .24 (m, 5H, Ar), 5.89 (d, J=2.3 Hz, 0.5H, H1'), 5.88 (d, J=2.3 Hz, 0.5H, H1' ), 5.08-5.01 (m, 2H, OCH2Ph), 4.70-4.60 (m, 2H, H2', C4'), 4.46-4.39 (m, 1H, C5' ), 4.32-4.24 (m, 1H, C5'), 4.09-3.97 (m, 1H, CHCH3), 2.36-2.25 (m, 1H, H3'), 2 .06-1.98 (m, 1H, H3'), 1.32-1.25 (m, 3H, cHcH3).

¹³ C NMR (125 MHz, CD3OD) 0C 175.54 (CO), 175.22 (CO), 161.02 (d, 4--, 207.3 Hz, C2), 160.89 (d, ¹ Jc -f=207.3 Hz, C2), 158.45 (d, ³ Jc-f=18.2 Hz, C6), 158.23 (d, ³ Jc-f=18.2 Hz, C6), 150 .63 (d, ³ Jc-f=18.4 Hz, C4), 140.67 (C8), 136.26 (C-Ar), 131.62, 131.54, 129.56 (CH-Ar) , 129.52 (CH-Ar), 129.37 (CH-Ar), 129.31 (CH-Ar), 129.26 (CH-Ar), 128.87 (CH-Ar), 128.81 (CH- Ar), 128.29 (CH-Ar), 128.02 (CH-Ar), 127.79 (CH-Ar), 127.76 (CH-Ar), 127.51 (CH-Ar), 127, 49 (CH-Ar), 127.47 (CH-Ar), 126.47 (CH-Ar), 126.33 (C-Ar), 126.27 (СAr), 125.97 (CH-Ar), 122.78 (CH-Ar), 122.74 (CH-Ar), 122.64 (CH-Ar), 122.62 (CH-Ar), 116.35 (d, ⁴ Jc-f=3.0 Hz, C5), 116.15 (d, ⁴ Jc-f=3.0 Hz, C5), 93.25 (C1'), 93.20 (C1'), 80.41 (d, ³ Jc-p =7.5 Hz, C4'), 80.33 (d, ³ Jc-p=7.5 Hz, C4'), 76.43 (C2'), 76.35 (C2'), 68.84 ( e, ² Jc-p=5.5 Hz, C5'), 68.45 (e, ² Jc-p=5.5 Hz, C5'), 67.92 (OCH ₂ Ph), 67.92 (OCH2Ph ), 51.75 (CHCH3), 51.52 (CHCH3), 34.97 (C3'), 34.74 (C3'), 20.42 (d, ³ Jc-p=6.7 Hz, CHCH3) , 20.20 (d, ³ Jc-p=6.7 Hz, CHCH3).

¹⁹ F NMR (470 MHz, CD3OD) 5F -53.14, -53.22.

(ES+) m/z, Found: 637.2 (M+H ⁺ ), C ₃₀ H ₃₀ FN ₆ O ₇ P, required: 636.57 (M).

HPLC. Reverse-phase HPLC eluting with H ₂ O/CH ₃ CN 100/10 to 0/100 over 30 min, 1 ml/min, 1=254 nm showed two diastereoisomer peaks with a tR of 17.09 min. and tR 17.34 min.

(2S)-Benzyl 2-(((((2S,4R,5R)-5-(6-amino-2-fluoro-9H-purin-9-yl)-4-hydroxytetrahydrofuran-2-yl)methoxy)( phenoxy)phosphoryl)amino)propanoate P

ABOUT

Using General Method 1 above, N-methylimidazole (74 µl, 0.93 mmol) and a solution of (2S)-benzyl 2-((chloro(phenoxy)phosphoryl)amino)propanoate (196 mg, 0.56 mmol) in anhydrous THF (2 ml) was added dropwise to a suspension of 2-fluoro-3'-deoxyadenosine (50 mg, 0.18 mmol) in anhydrous THF (5 ml) and the reaction mixture was stirred at room temperature for 16 h. column chromatography (eluent system CH ₃ OH/CH ₂ Cl ₂ 0/100 to 6/94) and preparative TLC (eluent system CH ₃ OH/CH ₂ C1 ₂ 5/95) gave the desired compound as a white solid (5 mg, 7%).

(ES+) m/z, Found: 587.1 (M+H ⁺ ), C ₂₆ H ₂₈ FN ₆ O ₇ P, required: 586.17 (M).

- 39 040630 ¹⁹ F NMR (470 MHz, CD3OD): δF -53.17, -53.23.

³¹ P NMR (202 MHz, CD3OD): δP 3.95 (s), 3.67 (s).

1H NMR (500 MHz, CDCI3): δH 8.19 (s, 0.5H, H8), 8.16 (s, 0.5H, H8), 7.36-7.27 (m, 7H, Ar) , 7.22-7.13 (m, 3H, Ar), 5.91 (d, J=1.5 Hz, 0.5H, H1'), 5.89 (d, J=1.7 Hz, 0.5H, H1'), 5.15-5.06 (m, 2H, OCH ₂ Ph), 4.73-4.58 (m, 2H, H2', H4'), 4.42-4, 34 (m, 1H, H5'), 4.02-3.90 (m, 1H, H5'), 3.27-3.24 (m, 1H, H3'), 2.08-2.00 ( m, 1H, H3'), 1.33 (d, J=7.1 Hz, 1.5H, CH3 ala), 1.29 (d, J=7.1 Hz, 1.5H, CH3 ala).

¹³ C NMR (125 MHz, CD3OD): δC 175.85 (d, ³ Jc-p=3.7 Hz, C=O), 174.63 (d, ³ Jc-p=5.0 Hz, C= O), 160.58 (d, 1Jc-F=207.5 Hz, C2), 160.53 (d, 1Jc-F=207.5 Hz, C2), 159.06 (d, ³ Jc-f= 18.7 Hz, C6), 159.05 (d, ³ Jc-f=17.5 Hz, C6), 152.11 (d, ² Jc-p=8.75 Hz, C-Ar), 152, 08 (d, ² Jc-p=8.7 Hz, C-Ar), 151.58 (d, ³ Jc-f=19.7 Hz, C4), 151.56 (d, ³ Jc-f=19 .5 Hz, C4), 140.63 (C8), 137.28 (C-Ar), 137.21 (C-Ar), 130.78 (CH-Ar), 130.75 (CH-Ar), 129.58 (CH-Ar), 129.38 (CH-Ar), 129.34 (CH-Ar), 129.32 (CH-Ar), 129.28 (CH-Ar), 128.3 (CH -Ar), 128.02 (CH-Ar), 121.16 (CH-Ar), 121.18 (CH-Ar), 121.47 (CH-Ar), 121.51 (CH-Ar), 121 .42 (CH-Ar), 121.39 (CH-Ar), 121.36 (CH-Ar), 118.75 (d, ⁴ Jc-f=3.7 Hz, C5), 118.72 (d , ⁴ Jc-f=3.7 Hz, C5), 93.25 (C1'), 93.18 (C1'), 80.48 (d, ³ Jc-p=8.3 Hz, C4'), 80.46 (d, ³ Jc-p=8.1 Hz, C4'), 76.51 (C2'), 76.49 (C2'), 68.54 (d, ² Jc-p=5.2 Hz, C5'), 68.18 (d, ² Jc-p=5.6 Hz, C5'), 67.94 (CH2 Bn), 67.91 (CH2 Bn), 51.71 (CH ala), 51.56 (CH ala), 34.85 (C3'), 34.64 (C3'), 20.42 (d, ³ Jc-p=7.1 Hz, CH3 ala), 20.25 (d, ³ Jc-p=7.5 Hz, CH3 ala).

HPLC. Reverse-phase HPLC eluting with H2O/CH3CN 100/10 to 0/100 over 30 min, 1 ml/min, 1=280 nm showed two diastereoisomer peaks with a tR of 14.98 min. and tR 15.12 min.

2-Fluoro-3'-deoxyadenosine-5'-O-[1-naphthyl(1-pentyloxy-L-leucinyl)]phosphate Q

Compound Q was prepared according to General Method 1 using 2-Fluoro-3'-deoxyadenosine (50 mg, 0.18 mmol), N-methylimidazole (74 μl, 0.93 mmol) and naphthyl(pentyloxy-L-leucinyl) phosphorochloridate (246 mg, 0.56 mmol). Purification by column chromatography (eluent system CH ₃ OH/CHC1 ₃ 0/100 to 6/94) and preparative TLC (1000 μm, eluent system CH ₃ OH/CH ₂ C1 ₂ 5/95) gave the title compound in as a white solid (65 mg, 53%). ³¹ P NMR (202 MHz, CD3OD): 4.60, 4.35.

1H NMR (500 MHz, CD3OD): δH 8.23 (s, 0.5H, H8), 8.20 (s, 0.5H, H8), 8.18-8.12 (m, 1H, Ar) , 7.92-7.86 (m, 1H, Ar), 7.73-7.68 (m, 1H, Ar), 7.57-7.46 (m, 3H, Ar), 7.42- 7.36 (m, 1H, Ar), 5.93-5.91 (m, 1H, H1'), 4.744.62 (m, 2H, H2', H4'), 4.55-4.50 ( m, 0.5H, H5'), 4.49-4.44 (m, 0.5H, H5'), 4.43-4.37 (m, 0.5H, H5'), 4.36- 4.31 (m, 0.5H, H5'), 4.02-3.86 (m, 3H, CHCH2CH(CH3)2, O(SH)4CH3), 2.43-2.29 (m, 1H , H3'), 2.12-2.04 (m, 1H, H3'), 1.67-1.20 (m, 11H,O(CH2)4CH3, CHCH ₂ CH(CH3) ₂ ), 0, 89-0.67 (m, 9H, O(SH)4CH3, CHCH ₂ CH(CH3) ₂ ).

¹³ C NMR (125 MHz, CD3OD): δC 175.03 (d, ³ Jc-p=2.5 Hz, C=O), 174.93 (d, ³ Jc-p=2.5 Hz, C= O), 161.45 (d, 1JC-F=205.5 Hz, C2), 160.39 (d, 1JC-F=205.5 Hz, C2), 158.33 (C6), 151.60 ( C4), 147.92 (C-Ar), 140.69 (C8), 136.30 (C-Ar), 128.88 (CH-Ar), 128.83 (CH-Ar), 127.80 ( CH-Ar), 127.76 (CH-Ar), 127.49 (CH-Ar), 127.46 (CH-Ar), 126.48 (CH-Ar), 126.45 (CH-Ar), 126.02 (CH-Ar), 125.91 (CH-Ar), 123.03 (C-Ar), 122.81 (CH-Ar), 122.69 (CH-Ar), 116.39 (d , ³ Jc-p=2.9 Hz, CH-Ar), 116.28 (С5), 116.26 (С5), 115.97 (d, ³ Jc-p=2.9 Hz, CH-Ar) , 93.29 (C1'), 93.23 (C1'), 80.45 (d, ³ Jc-p=6.0 Hz, C4'), 80.38 (d, ³ Jc-p=6, 0 Hz, C4'), 76.45 (C2'), 76.41 (C2'), 68.99 (d, ² Jc-p=5.4 Hz, C5'), 68.78 (d, ² Jc-p = 5.4 Hz, C5'), 66.31 (OCHCH^CH3), 66.29 (O(CH2)4CH3), 54.78 (CHCH2CH(CH3)2), 54.66 (CHCH2CH( CH3)2), 44.16 (d, ³ Jc-p=7.25 Hz, CHCH2CH(CH3)2), 43.84 (d, ³ Jc-p=7.3 Hz, CHCH2CH(CH3)2) , 35.09 (C3'), 34.79 (C3'), 29.31 (O(CH ₂ )4CH3), 29.12 (O(CH ₂ )4CH3), 25.65 (CHCH2CH(CH3)2 ), 25.41 (CHCH ₂ CH(CH ₃ ) ₂ ), 23.33 (O (CH2)4CH3), 23.11 (CHCH2CH(CH3) ₂ ), 23.00 (CHCH2CH(CH3)2), 21.95(CHCH2CH( _CH3 )2), 21.68(CHCH2CH ₍ CH3) ) ₂ ), 14.29 (O(CH ₂ )4CH3).

¹⁹ F NMR (470 MHz, CD3OD): δF -53.15, -53.20.

(ES+) m/z, Found: 659.3 (M+H+), C ₃₁ H ₄₀ FN ₆ O ₇ P, required: 658, 66 (M).

HPLC. Reverse phase HPLC eluting with H2O/CH3CN 100/10 to 0/100 over 30 min, 1 ml/min, 1=254 nm showed one peak of overlapping diastereoisomers with a tR of 21.95 min.

(2B)-Hexyl 2-(((((2S,4R,5R)-5-(6-amino-2-fluoro-9H-purin-9-yl)-4-hydroxyteΊrahydrofuran-2-yl)methoxy)( phenoxy)phosphoryl)amino)propanoate R

- 40 040630

Using General Method 1 above, N-methylimidazole (74 µm, 0.93 mmol) and a solution of (2S)-hexyl 2-((chloro(phenoxy)phosphoryl)amino)propanoate (196 mg, 0.56 mmol) in anhydrous THF (2 ml) was added dropwise to a suspension of 2-fluoro-3'-deoxyadenosine (50 mg, 0.18 mmol) in anhydrous THF (5 ml) and the reaction mixture was stirred at room temperature for 16 h. column chromatography (eluent system CH ₃ OH/CH ₂ Cl ₂ 0/100 to 6/94) and preparative TLC (eluent system CH ₃ OH/CH ₂ Cl ₂ 5/95) gave the desired compound as a white solid (5 mg, 7%).

(ES+) m/z, Found: 587.1 (M+H+), C26H28FN6O7P, required: 586.17 (M).

¹⁹ F NMR (470 MHz, CD3OD): 5 F -53.15, -53.20.

³¹ P NMR (202 MHz, CD3OD): 3.91 (s), 3.73 (s).

Ή NMR (500 MHz, CDCl ₃ ): 5H 8.21 (s, 0.5H, H8), 8.20 (s, 0.5H, H8), 7.37-7.29 (m, 7H, Ar ), 7.26-7.13 (m, 3H, Ar), 5.94-5.91 (m, 1H, H1'), 4.76-4.64 (m, 2H, H2', H4' ), 4.49-4.44 (m, 0.5H, H5'), 4.43-4.37 (m, 0.5H, H5'), 4.33-4.26 (m, 1H, H5'), 4.11-3.99 (m, 2H, CH ₂ Hex), 3.97-3.83 (m, 1H, CH ala), 2.41-2.32 (m, 1H, H3 '), 2.13-2.06 (m, 1H, H3'), 1.62-1.52 (m, 2H, CH2 Hex), 1.37-1.23 (m, 9H, CH ₃ ala , CH2 Hex), 0.92-0.85 (m, 3H, CH3 Hex).

¹³ C NMR (125 MHz, CD3OD): 5C 175.15 (d, ³ Jc-p=3.7 Hz, C=O), 174.96 (d, ³ Jc-p=5.0 Hz, C= O), 160.59 (d, Jc-f=207.5 Hz, C2), 160.56 (d, Jc-f=207.5 Hz, C2), 159.09 (d, ³ Jc-f= 21.2 Hz, C6), 159.08 (d, ³ Jc-f=20.0 Hz, C6), 152.16 (d, ² Jc-p=7.5 Hz, C-Ar), 152, 14 (d, ² Jc-p=6.3 Hz, C-Ar), 151.71 (d, ³ Jc-f=20.0 Hz, C4), 151.67 (d, ³ Jc-f=20 .0 Hz, C4), 140.70 (d, ⁵ Jc-f=2.5 Hz, C8), 140.68 (d, ⁵ Jc-f=2.5 Hz, C8), 130.77 (CH -Ar), 130.74 (CH-Ar), 126.16 (CH-Ar), 126.24 (CH-Ar), 121.48 (CH-Ar), 121.44 (CH-Ar), 121 .41 (CH-Ar), 121.37 (CH-Ar), 118.80 (d, ⁴ Jc-f = 3.1 Hz, C5), 118.77 (d, ⁴ Jc-f = 3.7 Hz, C5), 93.37 (C1'), 93.25 (C1'), 80.52 (d, ³ Jc-p=3.7 Hz, C4'), 80.45 (d, ³ Jc- p=4.1 Hz, C4'), 76.52 (C2'), 76.49 (C2'), 68.69 (d, ² Jc-p=5.4 Hz, C5'), 68.30 (d, ² Jc-p=4.9 Hz, C5'), 66.46 (CH2 Hex), 51.68 (CH ala), 51.57 (CH ala), 35.02 (C3'), 34 .80 (C3'), 32.58 (CH2 Hex), 29.65 (CH2 Hex), 26.61 (CH2 Hex), 23.59 ( _CH2 Hex), 20.60 (d, ³ Jc-p =7.1 Hz, CH3 ala), 20.43 (d, ³ Jc-p=7.5 Hz, CH ₃ ala), 14.35 (CH ₃ Hex).

HPLC. Reverse phase HPLC eluting with H ₂ O/CH ₃ CN 100/10 to 0/100 over 30 min, 1 ml/min, 1=280 nm showed two diastereoisomer peaks with a tR of 17.83 min. and tR 18.02 min.

(2R)-Benzyl 2-((((2S,4R,5R)-5-(6-amino-2-chloro-9H-purin-9-yl)-4-hydroxytetrahydrofuran-2-yl)methoxy)(naphthalene -1-yloxy)phosphorylamino)propanoate S

To a stirred solution of 2-chloro-3'-deoxyadenosine (100 mg, 1.0 mol/eq.) in 10 ml of anhydrous THF was added dropwise 424 mg of (2S)-benzyl 2-(chloro(naphthalen-1-yloxy)phosphorylamino )propanoate (3.0 eq/mol) dissolved in 10 ml of anhydrous THF. To this reaction mixture at room temperature under argon was added dropwise 0.14 ml of NMI (5 mol/eq.). The reaction mixture was stirred for 88 hours. The solvent was removed under reduced pressure and the residue was purified by column chromatography with eluent gradient (CH ₃ OH/CH ₂ Cl ₂ 0/100 to 5/95) to give the desired product as a yellow solid. colors. (7 mg, yield=3%).

MS (ES+) m/z: Found: 653 (M+H ⁺ ), 675 (M+Na ⁺ ) C30H30ClN6O7P, required: 652.16 (M);

³¹ P NMR (202 MHz, CD3OD): 5P 4.39 (s), 4.12 (s); 1H NMR (500 MHz, CD3OD): 5H 8.10 (s, 0.5H, H8), 8.07 (s, 0.5H, H8), 8.02-7.97 (m, 3H, CH2Ph and Naph), 7.43-7.14 (m, 9H, CH2Ph and Naph), 5.80-5.81 (m, 1H, H1'), 4.89-4.97 (m, 2H, CH2Ph) 4.49-4.53 (m, 2H, H4' and H2'), 4.30-4.35 (m, 1H, H5'), 4.15-4.21 (m, 1H, H5') , 3.873.95 (m, 1H, CHCH ₃ ), 2.12-2.23 (m, 1H, H3'), 1.86-1.93 (m, 1H, H3'), 1.14-1 .17 (m, 3H, CHCH3);

¹³ C NMR (125 MHz, CD ₃ OD): 0C 174.85 (d, Jcp=4.0 Hz, C=O), 174.55 (d, Jcp=4.3 Hz, C=O), 158 .07, 158.04 (C6), 155.31, 155.28 (C2), 151.34, 151.31 (C4), 149.69 (C-Ar), 147.96 (d, ³ Jcp= 7.25 Hz, C-ipso Naph), 147.90 (d, ³ Jcp=7.0 Hz, C-ipso Naph), 140.70 (C8), 137.21, 137.16 (C-ipso CH2Ph ), 136.26 (C-Ar), 130.92, 130.80, 129.56, 129.53, 129.31, 129.27, 129.25, 128.88, 128.81 (CH-Ar ), 127.78 (d, Jcp=4.7 Hz, CH-Ar), 127.50 (d, Jcp=6.2 Hz, CH-Ar), 126.48, 126.02, 125.97 ( CH-Ar), 119.46, 119.42 (C5), 116.33 (d, Jcp=3.0, CH-Ar), 116.16 (d, Jcp=3.4, CH-Ar), 93.30, 93.27 (C1'), 80.56 (d, J=8.3 Hz, C4'), 80.51 (d, J=8.4 Hz, C4'), 76.61, 76.54

- 41 040630 (C2'), 68.74 (d, Jcp=5.3 Hz, C5'), 68.54 (d, Jcp=5.1 Hz, C5'), 67.93, 67.90 ( CH2₽h), 51.81, 51.70 (CHCH3),

34.79, 34.53 (C3'), 20.42 (d, Jcp=6.5 Hz, CHCH3), 20.23 (d, Jcp=7.7 Hz, CHCH3).

HPLC. Reverse phase HPLC eluting with H ₂ O/CH ₃ CN 90/10 to 0/100 over 30 min, F=1 ml/min, l=254 nm, tR 18.03 min.

2-Chloro-3' deoxyadenosine 5'-O-[1-phenyl(2,2-dimethylpropoxy-L-alanine)]phosphate T

Compound T was prepared according to General Method 1 using 2-chloro-3'-deoxyadenosine (350 mg, 1.25 mmol), N-methylimidazole (490 μl, 6.15 mmol) and phenyl(2,2-dimethylpropoxy- L-alaninyl) phosphorochloridate (1231 mg, 3.69 mmol). Purification by column chromatography (0/100 to 5/95 CH ₃ OH/CH ₂ Cl ₂ eluent system) and preparative TLC (1000 µm, 4/96 CH3OH/CH2Cl2 eluent system) gave the title compound as a solid. white (181 mg, 25%).

³¹ P NMR (202 MHz, CD3OD): 5P 3.93, 3.72.

1H NMR (500 MHz, CD3OD): 5H 8.12 (s, 0.5H, H8), 8.10 (s, 0.5H, H8), 7.19-7.23 (m, 2H, Ph) , 7.03-7.12 (m, 3H, Ph), 5.84 (d, J=2, 0.5H, Hl'), 5.83 (d, J=2, 0.5H, Hl' ), 4.54-4.60 (m, 2H, H4'and H2'), 4.34-4.38 (m, 0.5H, H5'), 4.27-4.31 (m, 0 .5H, H5'), 4.16-4.23 (m, 1H, H5'), 3.80-3.90 (m, 1H, CHCH3), 3.57-3.73 (m, 2H OCH2C (CH3)3), 2.18-2.28 (m, 1H, H3'), 1.94-1.99 (m, 1H, H3'), 1.20-1.24 (m, 3H, CHCH3), 0.81 (s, 4.5H OCH2(CH3)3), 0.79 (s, 4.5H OCH2(CH3)3).

¹³ C NMR (125 MHz, CD3OD): 5C 175.09 (d, ³ Jcp=4.75 Hz, C=O), 174.90 (d, ³ J _cp =5.37 Hz, C=O), 158.10, (C6), 155.31, 155.28 (C2), 152.14 (d, ² Jcp=6.37 Hz, C-ipso Ph), 152.13 (d, ² Jcp=6, 25 Hz, C-ipso Ph), 151.33, 151.30 (C4), 140.87, 140.76 (C8), 130.78, 130.77 (CH-Ar), 126.17, 126, 42 (CH-Ar), 121.45 (d, ³ Jcp=11.75 Hz, CH-Ar), 121.41 (d, ³ Jcp=11.75 Hz, CH-Ar), 119.52, 119 .48 (C5), 93.49, 93.35 (Cl'), 80.67 (d, ³ J=8.62 Hz, C4'), 80.65 (d, ³ J=8.25 Hz, C4'), 76.70, 76.67 (C2'), 75.43, (OCH2C(CH3)3), 68.68 (d, ² Jcp=5.12 Hz, C5'), 68.42 ( e, ² Jcp=5.12 Hz, C5'), 51.77, 51.60 (CHCH3), 34.94, 34.67 (C3'), 32.36, 32.32 (OCH2C(CH3)3 ), 26.78, 26.76 (OCH2C(CH3)3), 20.83 (d, Jcp=6.25 Hz, CHCH3), 20.61 (d, Jcp=7.12 Hz, CHCH3).

MS (ES+) m/z: Found: 583 (M+H+), 605 (M+Na ⁺ ) C24H32ClN ₆ O ₇ P, required: 582.18 (M).

HPLC. Reverse phase HPLC eluting with H2O/CH3CN 90/10 to 0/100 over 30 min, F=1 ml/min, l=254 nm, tR 16.37, 16.55 min.

2-Chloro-3'deoxyadenosine 5'-O-[1-naphthyl(2,2-dimethylpropoxy-G-alanine)] phosphate U

Compound U was prepared according to General Method 1 using 2-chloro-3'-deoxyadenosine (350 mg, 1.25 mmol), N-methylimidazole (4×90 μm, 6.15 mmol) and naphthyl(2,2-dimethylpropoxy -Galaninyl)phosphorochloridate (1416 mg, 3.69 mmol). Purification by column chromatography (0/100 to 5/95 CH ₃ OH/CH ₂ Cl ₂ eluent system) and preparative TLC (1000 µm, 4/96 CH3OH/CH2Cl2 eluent system) gave the title compound as a solid. white (264 mg, 34%).

³¹ P NMR (202 MHz, CD3OD): 5P 4.35, 4.20.

1H NMR (500 MHz, CD3OD): 0H 8.23 (s, 0.5H, H8), 8.21 (s, 0.5H, H8), 8.11-8.16 (m, 1H, Naph) , 7.867.89 (m, 1H, Naph), 7.69-7.70 (m, 1H, Naph), 7.54-7.46 (m, 3H, Naph), 7.37-7.41 ( m, 1H, Naph), 5.95 (d, J=2, 0.5H, Hl'), 5.94 (d, J=1.5, 0.5H, Hl'), 4.67-4 .73 (m, 2H, H4'and H2'), 4.34-4.55 (m, 2H, H5'), 4.00-4.08 (m, 1H, CHCH3), 3.66- 3.81 (m, 2H OCH2C (CH3)3), 2.28-2.41 (m, 1H, H3'), 2.03-2.10 (m, 1H, H3'), 1.31- 1.34 (m, 3H, CHCH3), 0.90 (s, 4.5H OCH2C(CH3)3), 0.89 (s, 4.5H CHCH3)3).

¹³ C NMR (125 MHz, CD3OD): 5C 175.11 (d, Jcp=4.1 Hz, C=O), 174.85 (d, Jcp=5.0 Hz, C=O), 158.10 , 158.04 (C6), 155.32, 155.30 (C2), 151.33 (C4), 147.96 (d, ² Jcp=7.25 Hz, C-ipso Naph), 147.93 ( e, ² Jcp=7.25 Hz, C-ipso Naph), 140.84, 140.76 (C8), 136.29 (C-Ar), 128.87, 128.82 (CH-Ar), 127 .85 (C-Ar), 127.77, 127.74, 127.48, 127.45, 126.47, 125.99, 125.96, 122.74, 122.66 (CH-Ar), 119 .47 (C5), 116.29 (d, ³ Jcp=3.4 Hz, CH-Ar), 116.17 (d, ³ Jcp=2.9 Hz, CH-Ar), 93.42, 93, 34 (Cl'), 80.57 (d, ³ Jcp=8.1 Hz, C4'), 80.53 (d, ³ Jcp=5.1 Hz, C4'), 76.61, 76.53 ( C2'), 75.41, 75.38 (OCH2C(CH3)3), 68.95 (d, ² Jcp=5.3 Hz, C5'), 68.82 (d, ² Jcp=5.2 Hz , C5'), 51.84,

- 42 040630

51.73 (CHCH3), 35.04, 34.75 (C3'), 32.29 (OCH ₂ C(CH3)3), 26.70 (OCH ₂ C(CH3)3), 20.76 (d , ³ Jcp=6.4 Hz,

CHCH3), 20.55 (d, ³ Jcp=7.2 Hz, CHCH3).

MS (ES+) m/z: Found: 633 (M+H ⁺ ), 655 (M+Na ⁺ ) C28H34CIN6O7P, required: 652.16 (M).

HPLC. Reverse phase HPLC eluting with H ₂ O/CH ₃ CN 90/10 to 0/100 over 30 min, F=1 ml/min, l=254 nm, tR 19.16 min.

2-Chloro-3'deoxyadenosine 5'-O-[1-phenyl (ethoxy-L-alanine)] phosphate V

Compound V was prepared according to General Method 4 using 2-chloro-3'-deoxyadenosine (343 mg, 0.66 mmol), tert-butyldimethylsilyl chloride (328 mg, 2.18 mmol), imidazole (297 mg, 4. 36 mmol). Purification by column chromatography (eluent system CH3OH/CH2Cl2 0/100 to 12/88) gave intermediate 1 in quantitative yield. Next, Intermediate 1 (970 mg, 1.89 mmol) was reacted with 12 ml of a THF/H2O/TFA 4/1/1 solution. Purification by column chromatography (eluent system CH3OH/CH2Cl2 0/100 to 12/88) gave intermediate 2 (544 mg, 72%). Intermediate 2 (204 mg, 0.51 mmol) was then reacted with tert-butyl magnesium chloride and a solution of phenyl(ethyloxy-L-alaninyl)phosphorochloridate (348.56 mg, 1.02 mmol) in anhydrous THF (5 ml). Purification by column chromatography (eluent system CH3OH/CH2Cl2 0/100 to 8/92) gave intermediate 3 (93 mg, 28%). Finally, intermediate 3 (93 mg, 0.14 mmol) was reacted with a THF/TFA/H2O 1/1/1 solution (3 mL). Purification by preparative TLC (2000 µm, CH3OH/CH2Cl2 4/96 eluent system) gave the title compound as a white solid (50 mg, 66%). (Total yield 13%).

³¹ P NMR (202 MHz, CD3OD): 6P 3.93, 3.72.

1H NMR (500 MHz, CD ₃ OD): 8H 8.12 (s, 0.5H, H8), 8.11 (s, 0.5H, H8), 7.18-7.23 (m, 2H, Ph), 7.03-7.12 (m, 3H, Ph), 5.85 (d, J=1.5, 0.5H, H1'), 5.84 (d, J=2, 0, 5H, H1'), 4.55-4.62 (m, 2H, H4'and H2'), 4.34-4.38 (m, 0.5H, H5'), 4.28-4.32 (m, 0.5H, H5'), 4.16-4.22 (m, 1H, H5'), 3.93-4.03 (m, 2H, OCH ₂ CH ₃ ), 3.70-3 .84 (m, 1H, CHCH3), 2.20-2.28 (m, 1H, H3'), 1.95-1.99 (m, 1H, H3'), 1.15-1.21 ( m, 3H, CHCH3), 1.06-1.11 (m, 3H, OCH2CH3).

¹³ C NMR (125 MHz, CD3OD): bC 173.66 (d, ³ Jcp=4.5 Hz, C=O), 173.65 (d, ³ Jcp=5.3 Hz, C=O), 156 .68, 156.70 (C6), 153.93, 153.88 (C2), 150.72 (d, ² Jcp=6.7 Hz, C-ipso Ph), 150.71 (d, ² Jcp= 6.5 Hz, C-ipso Ph), 149.89, 149.94 (C4), 139.41, 139.35 (C8), 129.33 (CH-Ar), 124.74, 124.73 ( CH-Ar), 120.03 (d, ³ Jcp=4.75 Hz, CH-Ar), 119.97 (d, ³ Jcp=4.87 Hz, CH-Ar), 118.07, 118.03 (C5), 92.02, 91.88 (C1'), 79.26, 79.19 (C4'), 75.26, 75.24 (C2'), 67.18 (d, ² Jcp=5 .25 Hz, C5'), 66.81 (d, ² Jcp=5, 12 Hz, C5'), 60.96 (OCH ₂ CH ₃ ), 50.23, 50.12 (CHCH ₃ ), 33, 46, 33.21 (C3'), 19.16 (d, ³ Jcp=6.3 Hz, CHCH3), 18.97 (d, ³ Jcp=7.2 Hz, CHCH ₃ ), 13.10, 13 .07 (OCH2CH3).

MS (ES+) m/z: Found: 541 (M+H ⁺ ), 563 (M+Na ⁺ ), C21H26ClN6O7p, required: 540 (M).

HPLC. Reverse phase HPLC eluting with H2O/CH3CN 90/10 to 0/100 over 30 min, F=1 ml/min, l=254 nm, tR 12.41, 12.83 min.

(2S)-Isopropyl-2-(((((2S,4R,5R)-5-(6-amino-9H-purin-9-yl)-4-hydroxytetrahydrofuran-2yl)methoxy)(phenoxy)phosphoryl)amino ) propanoate W

N-methylimidazole (240 μm, 5 mmol) and a solution of (2S)-isopropyl 2 ((chloro(phenoxy)phosphoryl)amino)propanoate (546 mg, 3 mmol) in anhydrous THF (5 ml) were added dropwise to the suspension (2R ,3R,5S)-2-(6-Amino-9H-purin-9-yl)-5-(hydroxymethyl)tetrahydrofuran-3-ol (150 mg, 0.6 mmol) in anhydrous THF (3 ml), and the reaction mixture was stirred at room temperature for 16 hours. Purification by column chromatography (eluent system CH3OH/CH2Cl2 0/100 to 6/94) and preparative TLC (2000 µm, eluent system CH3OH/CH2Cl2 5/95) gave the desired compound as a white solid (40 mg, 13%).

MS (ES+) m/z: Found: 521.2 (M+H ⁺ ), 543.3 (M+Na ⁺ ), 1063.4 (2M+Na+). C ₃₁ H ₃₃ N ₆ O ₈ P required: 520.18(M).

³¹ P NMR (202 MHz, CD3OD): bP 3.99 (s), 3.82 (s). 1H NMR (500 MHz, CD3OD): bH 8.16 (s, 0.5H, H8), 8.15 (s, 0.5H, H8), 8.11 (s, 1H, H-2) 7, 23-7.20 (m, 2H, Ph), 7.11-7.03 (m, 3H, Ph), 5.91 (d, J=2.0 Hz, 0.5H, H1'),

- 43 040630

5.90 (d, J=2.0 Hz, 0.5H, H1'), 4.85-4.79 (m, 1H, CH(CH3) ₂ , 4.64-4.63 (m, 1H , H4'), 4.60-6.57 (m, 1H, H2'), 4.374.33 (m, 1H, H5'), 4.31-4.28 (m, 1H, H5'), 3 .74-4.22-4.17 (m, 1H, H5'), 3.70 (m, 1H, CH ala), 2.02-1.97 (m, 1H,

H3'), 2.04-2.01 (m, 1H, H3'), 1.18-1.14 (m, 3H, CH ₃ ), 1.24 (m, 6H, CH(CH ₃ ) ₂ ) HPLC Reversed phase

HPLC eluting with 100/10 to 0/100 H2O/CH3CN over 30 min, F=1 ml/min, λ=200 nm, showed two diastereoisomer peaks with a tR of 11.58 min. and tR 11.92 min.

Solvents and reagents. The following anhydrous solvents were purchased from Sigma-Aldrich: dichloromethane (CH ₂ Cl ₂ ), trimethyl phosphate ((CH ₃ O) ₃ PO). Commercially available amino acid esters were purchased from Sigma-Aldrich. All commercially available reagents were used without further purification.

Thin layer chromatography (TLC). Aluminum-backed pre-coated plates (60 F254, 0.2 mm thick, Merck) were visualized in both short and long wavelength ultraviolet light (254 and 366 nm) or by incineration using the following TLC indicators: i) ammonium molybdate cerium sulfate ; (Ii) potassium permanganate solution. Preparative TLC plates (20x20 cm, 500-2000 µm) were purchased from Merck.

Flash column chromatography. Flash column chromatography was performed using silica gel supplied by Fisher (60A, 35-70 µm). Glass columns were packed with a suspension using the appropriate eluent, loading the sample in the form of a concentrated solution in the same eluent or previously adsorbed on silica gel. Fractions containing product were identified by TLC and pooled and the solvent was removed in vacuo.

High performance liquid chromatography (HPLC). The purity of the target compounds was determined by HPLC analysis to be >95% using either I) ThermoSCIENTIFIC, SPECTRA SYSTEM P4000, SPECTRA SYSTEM UV2000 detector, Varian Pursuit XRs 5 C18, 150x4.6 mm (as analytical column), or II) Varian Prostar (LC Workstation-Varian Prostar 335 LC detector), Thermo SCIENTIFIC Hypersil Gold C18, 5µ, 150x4.6mm (as analytical column). See the experimental part for the elution method.

Nuclear magnetic resonance (NMR). 1H NMR (500 MHz), ¹³ C NMR (125 MHz), ³¹ P NMR (202 MHz) and ¹⁹ F NMR (470 MHz) were recorded on a Bruker Avance 500 MHz spectrometer at 25°C. Chemical shifts (δ) are given in parts per million (ppm) relative to internal standard MeOH-d4 (δ 3.34 ¹ H-NMR, δ 49.86 ¹³ C-NMR) and CHCl3-d4 (δ 7.26 Ή NMR, δ 77.36 ¹³ C NMR) or external standard 85% H ₃ PO ₄ (δ 0.00 ³¹ R NMR). Coupling constants (J) are measured in hertz. When assigning NMR signals, the following abbreviations are used: s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), shir. s (wide singlet), dd (doublet doublet) dt (triplet doublet), each. (apparent). Assignment of signals in ¹ H NMR and ¹³ C NMR was performed based on the analysis of binding constants and additional two-dimensional experiments (COSY, HSQC, HMBC, PENDANT).

Mass spectrometry (MS). Low resolution mass spectra were recorded on a Bruker Daltonics microTof-LC (atmospheric pressure ionization, electron sputtering mass spectrometry) in positive or negative modes.

The purity of the final compounds. >95% purity of all final compounds was confirmed by HPLC analysis.

Example 2 Cytotoxicity.

Exemplary compounds of the present invention were evaluated for their anticancer activity by the following methods.

In vitro viability assays were performed to evaluate the effects of compounds on cell viability in 7 selected cell lines over 72 hours using the CellTiterGlo assay (CTG, Promega-G7573). The assays were run in repeat, compound treatment at 9 points, 3.16 fold titration in 96-well plates for ~72 hours. Initial compound concentrations were 198 mM. Cell viability assay was performed using CellTiterGlo in a 96-well plate. Treatment with the compound was carried out for 72 hours under standard growth conditions and in duplication. Compounds were dissolved to 40 mM with 100% thawing. Compounds were serially diluted 3.16 times with thawed DMSO and heated to 37° C. before being dissolved in medium (2+200 µl). The compounds were then dissolved in the medium (the medium was also heated to 37°C). The medium containing the compounds was heated to 37° C. in an incubator and then the compounds in the medium were added to the cell plates (50 µl + 50 µl) in duplication. Final compound concentrations ranged from 198 M to 19.9 nM. The solubility of all compounds was checked and recorded again, then the plates were immediately transferred to a CO2 tissue culture incubator and incubated for 3 days. The final concentration of DMSO was 0.5%.

The results of the primary screening are presented in table. II. A is a relative IC ₅₀ of 0.1 to 5 µM, B is a relative IC _{50 of} over 5 µM and up to 15 µM, C is a relative IC50 of over 15 µM and up to 100 µM; and D is a relative IC50 over 100 μM.

- 44 040630

Table II

Comm. MOLT-4 ³ KG-l ^b HL- ^60s CCRF-CEM ^d K562 ^e IC ₅ o ^f M.1.%9 1C ₅ about M.1.% 1C ₅ about | M.1.% 1C ₅ about | M.1.% 1C ₅ about M.1.% cordycepin With 52 WITH 78 WITH 88 WITH 12 WITH 88 & A 98 V 92 IN 97 & 100 A 92 V A 100 V 100 V 100 & 100 A 97 A 93 D 65 82 IN 94 IN 92 D A 100 IN 100 V 100 V 100 V 99 G With 75 63 57 With 65 With 78 H With 100 69 80 With 100 With 89 I With 100 93 99 With 99 With 87 E IN 97 WITH 69 With 74 With 90 D 81 A 100 WITH 29 With 98 With 100 WITH 97 F A 100 WITH 98 With 100 S 99 WITH 91

^a MOLT-4: acute lymphoblastic leukemia; ^b KG-l: acute myelogenous leukemia; ^c HL-60: acute promyelocytic leukemia; ^d CCRF-CEM: acute lymphoblastic leukemia; ^e K562: [chronic myeloid leukemia. ^f IC ₅₀ μM: relative 1C ₅₀ ; em.1.%: maximum percentage inhibition of cell viability.

Table II (continued)

Compound MCF- ^{7h HepG2T} 1С ₅ 0 M.1.% 1С ₅₀ M.1.% cordycepin WITH 78 WITH 66 A A 94 IN 76 V A 99 IN 95 With IN 87 With 59 D A 100 V 99 G V 97 With 67 H A 94 V 55 I V 99 With 90 E With 78 With 59 J With 97 With 55 F V 99 With 84

^h MCF-7: breast adenocarcinoma;

ⁱ HepG2: hepatocellular carcinoma.

A portion of the compounds of the invention were then assayed for their cytotoxic activity in a wider variety of different solid tumors and hematologic malignancies using the following assay.

Analysis of solid tumors and hematological malignancies.

An in vitro viability assay was performed to evaluate the effects of compounds on cell viability in selected cell lines for 72 hours using the CellTiterGlo assay (CTG, Promega-G7573). Tests were performed in replicate, treating compounds at 9 points, 3.16 fold titration in 96-well plates for ~72 h. Initial compound concentrations were 198 mM. Cell viability assay was performed using CellTiterGlo in a 96-well plate. Treatment with the compound was carried out for 72 h under standard growth conditions in duplicate. Compounds were dissolved to 40 mM with thawed 100%. Compounds were serially diluted 3.16 times with thawed DMSO and heated to 37° C. before being dissolved in medium (2+200 µl). After the compounds were dissolved in the medium, the medium containing the compounds was heated to 37° C. in an incubator and then the compounds in the medium were added to the cell plates (50+50 µl) in duplication. Final compound concentrations ranged from 198 M to 19.9 nM. The solubility of all compounds was checked and recorded again, then the plates were immediately transferred to a CO ₂ tissue culture incubator and incubated for 3 days. The final concentration of DMSO was 0.5%.

The following cell lines were investigated and the data are shown in table. IV further.

- 45 040630

Table III

Cell culture malignant tumor Cell culture malignant tumor MOLT-4 Acute lymphoblastic leukemia HEL92.1.7 erythroleukemia CCRFCEM Acute lymphoblastic leukemia HL-60 Promyelocytic leukemia RL Non-Hodgkin's lymphoma MV4-11 Biphenotypic B- cellular myelomonocytic leukemia HS445 Lymphoma Hodgkin HepG2 Hepatocellular carcinoma RPMI8226 multiple myeloma human HT29 Colon adenocarcinoma K562 Chronic myelogenous leukemia VkhRS-3 pancreatic cancer glands KG-1 Spicy MCF-7 Breast adenocarcinoma myelogenous leukemia glands TNR-1 Acute monocytic leukemia MiaPaCa2 breast adenocarcinoma Z-138 lymphoma from cells mantle zone SW620 Colon adenocarcinoma NCI-H929 plasmacytoma Yurkata acute T-cell leukemia

The results of additional screening are presented in table. IV-VII. For table. IV-VI: A is an absolute IC ₅₀ from 0.1 to 5 µM, B is an absolute IC ₅₀ over 5 µM and up to 15 µM, C is an absolute IC ₅₀ over 15 µM and up to 100 µM; and D is an absolute IC ₅₀ over 100 μM. For table. VII: A represents an absolute EC ₅₀ from 0.1 to 5 μM, B represents an absolute EC ₅₀ over 5 and up to 15 μM, C represents an absolute EC ₅₀ over 15 and up to 100 μM; and D is an absolute EC50 over 100 μM.

Table IV

CCRFCEM MOLT-4 KG-1 Jurkat Comm. 1С ₅₀ М1% 1С ₅₀ М1% 1С ₅₀ М1% 1C ₅₀ М1% Cordycepin D 41 D 46 D 69 D 20 A A 100 A 9Θ WITH 100 A 100 IN A 100 A 98 IN 97 A 100 With IN 100 A 92 WITH 102 IN 100 F A 101 A 98 With 95 A 100 E A 100 A 98 WITH 100 IN 95 TNR-1 RL HS445 NCI-H929 Comm. yu ₅₀ М1% yu ₅₀ М1% 1С ₅₀ М1% ts ₅₀ М1% Cordycepin D -3 D 17 D 2 D 24 A WITH 74 A 93 WITH 98 IN 100 IN With 99 A 96 V 96 A 99 With WITH 99 IN 100 With 102 IN 104 F With 100 IN 90 With 92 IN 100 E D 43 IN 88 WITH 85 With 98 RPMI-8226 MV4-11 HEL92,1,7 K562 Comm. ic ₅₀ М1% 1C ₅₀ М1% ic ₅₀ М1% 1C _5O М1% Cordycepin D 1 D 1 With 88 With 78

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A IN 96 A 99 IN 100 A 96 IN IN 102 A 99 A 98 A 99 With With 103 IN 106 A 99 IN 100 F With 106 A 100 IN 99 A 93 E With 89 IN 101 With 101 IN 90

HL-60 Z138 VkhRS-3 HepG2 Comm. 1С ₅₀ М1% IC50 М1% 1C ₅₀ М1% 1С ₅₀ М1% Cordycepin D 61 IN 95 D 22 D 13 A IN 99 With 95 WITH 81 WITH 75 IN A 99 IN 100 IN 90 IN 98 With IN 100 V 100 With 99 With 99 F IN 96 With 76 WITH 78 WITH 79 E IN 95 With 93 With 71 With 67

HT2 9 MCF7 MiaPaCa-2 SW620 Comm. 1C ₅₀ М1% TS ₅₀ М1% IC50 М1% 1C ₅₀ М1% Cordycepin D 44 D 77 D 35 D 10 A IN 93 A 99 IN 96 WITH Θ5 IN IN 98 A 96 A 98 IN 93 With With 99 A 102 IN 106 With 100 F With 89 IN 103 IN 101 WITH 90 E With 76 IN 89 WITH 91 WITH 74

Table V

CCRFCEM KG-1 K562 MOLT-4 Comm. 1C ₅₀ M.I. % IC50 M.I. % 1C ₅₀ M.I. % 1C ₅₀ M.I. % 2-OMe- Cordycepin D -4 D 1 D 40 D 1 L With 100 D 92 With 104 With 101 TO IN 102 With 99 With 100 With 104 M IN 100 With 102 IN 100 IN 101 N With 93 With 96 With 78 With 97 HT2 9 MCF7 NCI-H929 RL Comm. 1C ₅ about M.I. % IC50 M.I. % 1С ₅₀ M.I. % 1С ₅₀ M.I.% 2-OMe-Cordycepin D 19 D 8 D 51 D 0 L WITH 81 WITH 90 With 104 WITH 97 TO With 98 IN 99 IN 102 With 104 M With 100 With 100 IN 99 IN 100 N With 69 With 73 With 99 With 88

Table VI

HepG2 HL-60 HT2 9 Comm. TS ₅₀ M.I. % yu ₅₀ M.I.% ts ₅₀ M.I. % 2-F-Cordycepin With 82 IN 96 WITH 73 P Q R Comm. 2-FCordycepin R Q R CCI ic ₅₀ A V V V 1FCEM M.I. 101 99 99 101 100 83 94 82 % A A A HEL92,1,7 1C ₅₀ I B A A A A 101 100 99 100 4.1% 99 100 99 99 99 c c c c KG-1 1C ₅₀ V c c c A 84 87 88 92 M.I. % 99 99 97 103 96 MiaPaCa-2 MCF7 K562 Comm. IC50 M.I.% 1C ₅₀ M.I. % 1C ₅ about M.I. % 2-F-Cordycepin With 100 With 97 With 97 0 A 100 A 95 A 99 R A 98 A 95 A 97 Q A 100 A 99 A 100 R IN 98 A 87 IN 96

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Table VII

VkhRS-3- Luc CCRF- CEM HEL,92, 1.7 HepG2 HL-60 HS445 HT2 9 K562 KG-1 2-C1cordice pin eu ₅₀ D WITH IN D WITH D D D D М1% -1.3 98.1 94.3 31.1 84.4 -1.9 4.4 37.6 4.3 Comm. S eu ₅₀ WITH IN IN WITH IN WITH WITH With WITH М1% 82.6 100 100.4 84.6 101.9 105.9 88.1 101.4 99 MCF-7 Mia-pa- Sa-2 MOLT-4 MV4-11 NCI- 11929 RL RPMI- 8226 SW620 TNR-1 Z-138 2-C1 cordycepin eu ₅₀ D D D WITH IN WITH D D D WITH М1% -0.9 17.7 80.4 100.2 93.6 84.1 29 36, 3 24.1 99.5 Comm. S eu ₅₀ WITH WITH IN IN IN IN WITH With WITH WITH М1% 100.9 98.2 100.1 100.9 102.3 100.2 99.6 92.1 97 90.2

All tested compounds exhibited cytotoxic activity against the tested cell lines. In most cases, the compounds of the invention were more potent than the parent nucleoside against all cell lines.

Example 3 Evaluation of cytotoxicity and activity of cancer stem cells.

An additional comparative toxicity analysis of the compounds in the KG1a acute myeloid leukemia (AML) cell line was performed over an extended dose range and the relative effect of the compounds on the leukemia stem cell compartment (LSC) in the KG1a cell line over the entire dose range was evaluated.

Materials and methods.

Conditions for cell culture KG1a.

The KG1a cell line was maintained in RPMI medium (Invitrogen, Paisley, UK) supplemented with 100 units/ml penicillin, 100 μg/ml streptomycin and 20% fetal bovine serum. Cells were then aliquoted (105 cells/100 µl) in 96-well plates and incubated at 37°C in a humidified atmosphere with 5% carbon dioxide for 72 h in the presence of nucleoside analogs and their respective proTides at concentrations that were determined experimentally for each series of connections. In addition, control cultures were carried out to which no drug was added. Cells were then harvested by centrifugation and analyzed by flow cytometry using annexin V assay.

Measurement of apoptosis in vitro.

The cultured cells were collected by centrifugation and then resuspended in 195 μl of calcium-rich buffer. Then, 5 μl of Annexin V (Caltag Medsystems, Botolph Claydon, UK) was added to the cell suspension and the cells were incubated in the dark for 10 min before washing. The cells were finally resuspended in 190 µl of calcium rich buffer along with 10 µl of propidium iodide. Apoptosis was assessed by two-color immunofluorescent flow cytometry as described above. Then, for each nucleoside analogue and ProTide, LD50 values (dose needed to kill 50% of cells in culture) were calculated.

Leukemic stem cell compartment identification by immunophenotyping.

KG1a cells were cultured for 72 hours in the presence of a wide range of concentrations of each analyte. Cells were then harvested and labeled with a cocktail of anti-antibodies (PE-cy7), anti-CD34 (FITC), anti-CD38 (PE) and anti-CD123 (PerCP Cy5) antibodies. The subpopulation expressing the LSC phenotype was then identified and expressed as a percentage of the total number of viable cells remaining in culture. The percentage of stem cells remaining was then plotted on a dose-response plot and the effects of the compounds were compared with each other as well as with the parent nucleoside.

Statistical analysis.

The data obtained in these experiments were evaluated using one-way analysis of variance. All data were validated as Gaussian or Gaussian fit using the K2 omnibus test. LD ₅₀ values were calculated from non-linear regression and analysis with optimization of sigmoidal dose-response curves. All statistical analyzes were performed using GraphPad Prism 6.0 software (GraphPad Software Inc., San Diego, CA).

Results.

In vitro drug sensitivity was measured using the Annexin V/propidium iodide assay. Compound A showed increased in vitro activity compared to cordycepin (P<0.0001). 2-E-cordycepin was significantly more potent than cordycepin (P<0.0001) and all ProTides tested showed increased activity compared to the parent nucleoside (FIG. 1).

These experiments confirmed that Compound A has increased activity against the stem cell compartment at concentrations above 1 mM. As can be seen in FIG. 2, compound A has demonstrated the ability not only to reduce the number of cancer stem cells in the total

- 48 040630 complexity, but also to reduce the number of such cells in relation to the total number of malignant cells present in the culture. This indicates the ability of Compound A to primarily target cancer stem cells. At higher concentrations tested (1 mM and above), Compound A's ability to preferentially target LSCS was significantly greater than that of the parent compound.

The 2-P-cordycepin compounds proTidesP, Q, and R also showed preferential targeting by LSCS, which was significantly higher than the parent nucleoside. In contrast, while compound O was able to reduce the proportion of LSCS present in the treated cell populations (indicative of the ability to target LSCS), its activity was not significantly different from that of 2-F-cordycepin in any concentration study. In FIG. 3 shows a comparison of 2-F-cordycepin and all proTides tested, while individual comparisons are shown in the images of FIG. 4.

Example 4 Additional Cytotoxicity Evaluation and Inhibition Assay.

Some of the compounds of the invention were subjected to further studies to test the cytotoxic activity of some of the compounds of the invention, as well as to evaluate their activity against 4 lines of hematological malignant cells.

TdT positive CEM (ALL human),

TdT negative K562 (human CML),

TdT negative HL-60 (human ANLL),

RL (CRL-2261) non-HD lymphoma.

The concentrations of the active metabolite dATP (cordycepin triphosphate) were also measured in these cell lines.

Cytotoxic activity and intracellular concentrations of 3'-dATP were also studied in the presence of hENT1, adenosine kinase (AK), and adenosine deaminase, which are pharmacological inhibitors of the CEM and RL cancer cell lines. These inhibitors mimic the known resistance mechanisms of malignant neoplasms.

Ways.

Cell culture.

Leukemia cell lines HL-60 (ATCC® CCL-240™), K562 (ATCC® CCL-243™), CCRF-CEM (ATCC® CRM-CCL-119™) and RL (ATCC® CRL-2261™) obtained from the American Type Culture Collection (ATCC), Middlesex. The HL-60 and K562 cell lines are deoxynucleotide transferase negative (TdT-ve), while the KVOP-CEM cell line is TdT+ve.

The HL-60 cell line is an acute promyelocytic leukemia cell line; K562 is a CML cell line, CCRF-CEM cell line is an acute lymphoblastic leukemia (ALL) cell line; a RL is a non-Hodgkin's lymphoma cell line.

Maintenance of cell lines.

Cell lines HL-60, K562, CCRF-CEM and RL were cultured in RPMI-1640 medium (Sigma Aldrich, UK) supplemented with 10% fetal bovine serum (FBS) (PAA Laboratories), 1% amphotericin B (5.5 ml) and 1% penicillin/streptomycin (5.5 ml) (PAA Laboratories), and grown in flasks at 37° C. in a 5% CO ₂ incubator.

Analysis of adenosine 5'-triphosphate (ATP).

The amount of ATP was used as a measure of cell number and cell viability. ATP ViaLight™ plus assay kit (Lonza, USA: serial number LT07-121) for the detection of ATP in cells treated in luminescence-compatible 96-well plates (starting cell concentration was 1x104 cells/well) together with cordycepin and ProTides at concentrations: 0, 0.1, 0.5, 1.5 and 10 μM followed by incubation for 72 h at 37°C in a 5% CO2 incubator. For inhibitor studies, 10 μM NbTi or 1 μM EHNA or A-134974 was added and left for 5 min before drug addition (see Section 5 for more information on inhibitors).

After incubation, 50 µl cell lysis reagent to release intracellular ATP was added to 96-well plates followed by 100 µl ATP control reagent (AMR). Luminescence values in each well were determined using a FLUOstar OPTIMA microplate reader (BMG Labtech), which converts ATP to light using the luciferase enzyme. Thus, the amount of luminescence produced was directly proportional to the amount of ATP.

Processing of cells and extraction of samples for analysis of intracellular triphosphate.

Cell lines 5x106 cell/ml were used. Cells were treated with 1 μl of 50 μM each of cordycepin and compounds A, B, D, E and F, and incubated for 2 h at 37°C with 5% CO ₂ . After incubation, cells were centrifuged (ambient temperature, 1200 rpm, 5 min), culture medium supernatants were removed, and cell pellets were washed with 1 ml PBS and centrifuged (ambient temperature, 1200 rpm, 5 min). The supernatants were removed; the beads were reconstituted in 100 µl PBS and 100 µl 0.8 M perchloric acid and mixed on a Vortex and stored on

- 49 040630 ice for 30 min. It was then centrifuged (ambient temperature, 1200 rpm, 5 min) and 180 μl of the supernatant was transferred to new tubes and stored at -80° C. prior to analysis.

During the analysis, 90 μl of the extract was transferred to fresh tubes. 25 µl was added to the extract.

1M ammonium acetate and then neutralized by adding 10 µl of 10% ammonium and 5 µl of deionized water, then transferred to LC-MS vials and 10 µl injected into the SELC-MS/MS system.

Inhibitor research.

Cell lines were treated in the same manner as described above, but before starting drug treatment, several inhibitors were added:

1) nitrobenzylthioinosine (NbTi) (Sigma-Aldrich, St. Louis, MO, serial number N2255) blocks nucleotide transporters,

2) EHNA hydrochloride (Sigma-Aldrich, St. Louis, MO, serial number E114) blocks adenosine deaminase,

3) adenosine kinase inhibitor A-134974 dihydrochloride hydrate (Sigma-Aldrich, St. Louis, MO, serial number A2846): blocks adenosine kinase.

Cells were treated with 10 μM NbTi or 1 μM EHNA or A-134974 and left for 5 min before drug addition. The cells were then incubated for 2 h at 37°C with 5% CO2.

LC-MS/MS analysis.

Analytes were dissolved using an ultra high performance liquid chromatography system (Accela SELC, Thermo Scientific, UK) equipped with Biobasic Ax5 µm, a 50x2.1 mm column (Thermo Electron Corporation, Murrieta, CA, USA) and a mobile phase consisting of a mixture of 10 mM NH ₄ Ac in ACN/H2O (30:70 v/v), pH 6.0 (A) and 1 mM NH ₄ Ac in ACN/H2O (30:70 v/v), pH 10.5 (B ). A gradient mobile phase was used containing: Buffer A=95% at 0-0.5 min, 95 to 0% for 1.25 min, maintained at 0% for 1.75 min, 0 to 95% in within 0.1 min, finishing 95% within 2.9 min, all at a flow rate of 500 µl/min.

Eluted compounds of interest were detected using a Vantage triple quadrupole mass spectrometer system (Thermo Scientific, UK) equipped with an electrospray ion source. Samples were analyzed by monitoring multiple reactions, negative ion modes at a spray voltage of 3000V. Nitrogen was used as a shell and auxiliary gas at a flow rate of 50 and 20 conventional units, respectively. Argon was used as the collision gas at a pressure of 1.5 mTorr. The optimal masses of transient daughter ions and collision energy for each analyte were: 3'ATP 490.1^392.1 (collision energy 19V) and internal standard ChloroATP 539.9^442.2 (collision energy 24V).

Statistical analysis.

Dose-response curves of cytotoxicity of drugs were determined using a non-linear regression analysis of percent cell viability as a function of concentration, and EC50 values were obtained. Intracellular analysis was performed in five repetitions for each condition. The intracellular assay was evaluated using a double t-test (two-tailed) 3'ATP/ATP concentration assay and p-values were obtained. All analyzes used Prism (GraphPad Software) and Microsoft Powerpoint® 2013 to plot results.

Results.

Summary table IC ₅₀ (µM)

Cordycepin A (FD) B (FD) D (FD) E (FD) F(FD) CEM (TdT ^+ve ) 19.5 0.87 (22) 0.13 (150) 6, 1 (3) 100 (2) 4.3 (5) K562 10, 9 2.4 (5) 0.21 (52) 4.2 (3) 13, 9 (1) 6, 3 (2) HL-60 11.4 4, 6 (3) 2, 6 (4) 5, 4 (2) 10.2 (1) 8.3(1) CRL 24.5 2.1 (12) 0.4 (61) 4.8 (5) 11.0 (2) 3.4 (7)

(FD)=fold difference compared to cordycepin=cordycepin IC50/ProTide IC50.

- 50 040630

Summary mean levels of intracellular 3'-dATP (µg/mL)

Compounds A and B were the best options with IC _50s ranging from 3 to 150 times better than cordycepin. Compounds A and B gave intracellular concentrations of 3'-dATP 3 to 56 times better than cordycepin.

________ IC ₅₀ summary table (all in µM) _______

CEM (FD) CRL (FD) Cordycepin Control 11.5 7.3 NBTI 57.7 (5) 17.9 (2) EHNA 0.7 (-16) 13.2(2) AK 29.2(3) 28.6 (4) A Control 1.4 3, 6 NBTI 2.0 (1) 2.6(1) EHNA 3.1 (2.2) 2.9(1) AK 3.6(3) 10.2(3) IN Control 0.9 3, 1 NBTI 1.4 (1) 2.6(1) EHNA 1.3 (1) 5.2(2) AK 1.3 (1) 2.8(1) E Control 9, 9 8.2 NBTI 17.1(2) 8.2(1) EHNA 13.4 (1) 5.9(1) AK 10.3 (1) 7.1(1)

(EP)=fold difference compared to control.

Summary mean levels of intracellular 3'-dATP (µg/mL)

CEM (FD) CRL (FD) Cordycepin Control 0.24 0.10 NBTI 0.14(1) 0.06 (1) EHNA 9.01 (38) 1.85 (19) AK 0.31 (1) 0.16 (1) A IN Control 1.30 0.31 NBTI 0.99 (1) 0.32(1) EHNA AK Control NBTI EHNA AK 1.35 (1) 1.20 (1) 4.07 3.14(1) 3.62 (1) 2.99 (1) 0.27(1) 0.30 (1) 0.59 0.68 (1) 0.67(1) 0.77 (1) E Control 0.32 0.08 NBTI 0.17(1) 0.12 (1) EHNA 0.21 (1) 0.07 (1) AK 0.19 (1) 0.06 (1)

(BP)=fold difference compared to control.

NbTi, AK and EHNA do not affect the intracellular 3'-dATP generated by the three tested compounds of the invention, indicating that these inhibitors do not affect the metabolism by which the compound of the invention generates the 3'-dATP active agent within cells. -

Claims (23)

of hematological malignancies used in this study. Since these inhibitors mimic known cancer resistance mechanisms, these results indicate that the compounds of the present invention will be less sensitive to cancer resistance mechanisms than cordycepin. CLAIM 1. Compound of formula (Ib) where W ₁ is -P(=O)(U)(V), where U is -OAr and V is -NR4-CR1R2-C(=O)OR3; W ₂ is H; X is NH2; Z is H; Y is selected from the group consisting of H, F, Cl and -OCH ₃ ; R1 and R ₂ are independently selected from the group consisting of H, -CH ₃ and -CH ₂ CH(CH ₃ ) ₂ ; R ₃ is selected from the group consisting of C ₆ . ₃₀ aryls ₁ . ₆ alkyl and unsubstituted C _1-20 alkyl; R ₄ is H; And Ar is selected from the group consisting of phenyl and naphthyl, each of which is optionally substituted, where when Ar is substituted, the substituents are independently selected from R ₁₈ C(=O)-OR ₁₉ where R ₁₈ is C ₁ . ₄ alkyl and R ₁₉ is Sc5 alkyl, or a pharmaceutically acceptable salt of a compound of formula (Ib). 2. The compound according to claim 1, where the compound of formula (Ib) is a compound of formula (II) 3. The compound according to claim 1, where the compound of formula (Ib) is a compound of formula (III) 4. A compound according to any one of claims 1 to 3, wherein Ar is selected from phenyl and naphthyl. 5. A compound according to any one of claims 1 to 4, wherein R ₂ is methyl. 6. A compound according to claim 5, wherein the C atom bearing R1 and _R2 has the same absolute configuration as L-alanine. 7. A compound according to any one of claims 1 to 6 wherein R ₃ is selected from the group consisting of benzyl and unsubstituted C1. ₂₀ alkyl. 8. A compound according to claim 7 wherein R ₃ is selected from the group consisting of benzyl, unsubstituted methyl and unsubstituted n-pentyl. 9. A compound according to claim 8, wherein R ₃ is benzyl. 10. A compound according to any one of claims 1 to 9, wherein Y is H. - 52 040630 11. A compound according to any one of claims 1 to 9, wherein Y is F. 12. A compound according to any one of claims 1 to 9, wherein Y is Cl. 13. A compound according to any one of claims 1 to 9, wherein Y is OMe. 14. The compound according to claim 1, where the compound of formula (Ib) is selected from: (2B)-benzyl 2-(((((28.4L,5L)-5-(6-amino-9H-purin-9-yl)-4-hydroxytetrahydrofuran-2yl)methoxy)(naphthalene-1-yloxy) phosphoryl)amino)propanoate; benzyl 2-(((((28.4L,5L)-5-(6-amino-9H-purin-9-yl)-4-hydroxytetrahydrofuran-2yl)methoxy)(phenoxy)phosphoryl)amino)acetate; (2B)-pentyl 2-(((((28.4L,5L)-5-(6-amino-9H-purin-9-yl)-4-hydroxytetrahydrofuran-2yl)methoxy)(naphthalene-1-yloxy) phosphoryl)amino)-4-methylpentanoate; methyl 2-(((((28.4L,5L)-5-(6-amino-9H-purin-9-yl)-4-hydroxytetrahydrofuran-2yl)methoxy)(naphthalene-1-yloxy)phosphoryl)amino) -2-methylpropanoate; (2B)-benzyl 2-(((((28.4L,5L)-5-(6-amino-9H-purin-9-yl)-4-hydroxytetrahydrofuran-2-yl)methoxy)(2(3- ethoxy-3-oxopropyl)phenoxy)phosphoryl)amino)propanoate; (2B)-benzyl 2-((((28.4L,5L)-5-(6-amino-2-methoxy-9H-purin-9-yl)-4-hydroxytetrahydrofuran-2yl)methoxy)(naphthalene-1 -yloxy)phosphorylamino)propanoate; (2B)-benzyl 2-((((28.4L,5L)-5-(6-amino-2-methoxy-9H-purin-9-yl)-4-hydroxytetrahydrofuran-2yl)methoxy)(phenoxy)phosphorylamino ) propanoate; (2B)-benzyl 2-(((((28.4L,5L)-5-(6-amino-2-fluoro-9H-purin-9-yl)-4-hydroxytetrahydrofuran-2yl)methoxy)(phenoxy) phosphoryl)amino)propanoate; (2B)-hexyl 2-(((((28.4L,5L)-5-(6-amino-2-fluoro-9H-purin-9-yl)-4-hydroxytetrahydrofuran-2yl)methoxy)(phenoxy) phosphoryl)amino)propanoate; (2H)-benzyl 2-((((28.4L,5L)-5-(6-amino-2-chloro-9H-purin-9-yl)-4-hydroxytetrahydrofuran-2yl)methoxy)(naphthalene-1 -yloxy)phosphorylamino)propanoate; 2-O-methyl-3'-deoxyadenosine-5'-O-[1-naphthyl(1-pentyloxy-L-leucinyl)]phosphate; 2-O-methyl-3'-deoxyadenosine-5'-O-[phenyl (1-hexyloxy-L-alaninyl)]phosphate; 2-fluoro-3'-deoxyadenosine-5'-O-[1-naphthyl(benzyloxy-L-alaninyl)]phosphate; 2-fluoro-3'-deoxyadenosine-5'-O-[1-naphthyl(1-pentyloxy-L-leucinyl)]phosphate; 2-chloro-3'-deoxyadenosine 5'-O-[1-phenyl(2,2-dimethylpropoxy-L-alanine)]phosphate; 2-chloro-3'-deoxyadenosine 5'-O-[1-naphthyl(2,2-dimethylpropoxy-L-alanine)]phosphate; 2-chloro-3'-deoxyadenosine 5'-O-[1-phenyl (ethoxy-L-alanine)]phosphate; and (28)-isopropyl-2-(((((28.4L,5R)-5-(6-amino-9H-purin-9-yl)-4-hydroxytetrahydrofuran-2yl)methoxy)(phenoxy)phosphoryl) amino) propanoate. 15. A compound according to claim 1, wherein the compound of formula (Ib) is (S _p )-3'-deoxyadenosine-5'O-[phenyl(benzyloxy-L-alaninyl)]phosphate. 16. A compound according to claim 1, wherein the compound of formula (Ib) is (R _p )-3'-deoxyadenosine-5'O-[phenyl(benzyloxy-L-alaninyl)]phosphate. 17. The use of a compound according to any one of claims 1 to 16 for the prevention or treatment of cancer. 18. Use according to claim 17, wherein the cancer is selected from the group consisting of: leukemia, lymphoma, multiple myeloma, lung cancer (including non-small cell lung cancer and small cell lung cancer), liver cancer, breast cancer, bladder cancer, prostate cancer gland, head and neck cancer, neuroblastoma, sarcoma (including Ewing's sarcoma), thyroid cancer, skin cancer (including melanoma), oral squamous cell carcinoma, bladder cancer, Leydig cell tumor, gallbladder cancer such as cholangiocarcinoma or cancer bile duct, pancreatic cancer, colon cancer, colorectal cancer and gynecological cancers including ovarian cancer, endometrial cancer. 19. Use according to claim 18, wherein the cancer is leukemia or lymphoma. 20. Use according to claim 19, wherein the leukemia is selected from the group consisting of acute lymphoblastic leukemia, acute myeloid leukemia, acute promyelocytic leukemia, acute lymphocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, monoblastic leukemia, hairy cell leukemia, Hodgkin's lymphoma and non-Hodgkin's lymphoma . 21. Use according to claim 20, wherein the leukemia is acute lymphoblastic leukemia. 22. A method for preventing or treating cancer, comprising administering to a patient in need thereof an effective dose of a compound according to one of claims 1-16. 23. A pharmaceutical composition containing a compound according to any one of claims 1 to 16 in combination with a pharmaceutically acceptable carrier, diluent or excipient. -