WO2007083173A2

WO2007083173A2 - Mononucleotides and mononucleosides for use in therapy

Info

Publication number: WO2007083173A2
Application number: PCT/HU2007/000004
Authority: WO
Inventors: János ARADI; László FÉSÜS; Zoltán BECK
Original assignee: University Of Debrecen
Priority date: 2006-01-19
Filing date: 2007-01-19
Publication date: 2007-07-26
Also published as: WO2007083173A3; HU0600042D0; HUP0600042A2; HUP0600042A3

Abstract

The present invention relates to thiolated pyrimidine-mononucleotides and pyrimidine- mononucleosides and their use as therapeutic agents. More closely, the present invention relates to therapeutic applications of 4-thio-uridine, its analogues, ribo- and deoxyribonucleotide derivatives and their various further derivatives, and also to these compounds for use in the therapeutic applications. Preferably, the subject of this invention is the use of these compounds and preparations containing these compounds as antiproliferative and/or antiviral agents.

Description

MONONUCLEOTIDES AND MONONUCLEOSIDES FOR USE IN THERAPY

The present invention relates to thiolated pyrimidine-mononucleotides and pyrimidine- mononucleosides and their use as therapeutic agents. More closely, the present invention relates to therapeutic applications of 4-thio-uridine, its analogues, ribo- and deoxyribonucleotide derivatives and their various further derivatives, and also to these compounds for use in the therapeutic applications. Preferably, the subject of this invention is the use of these compounds and preparations containing these conipounds as antiproliferative and/or antiviral agents.

While therapeutic use of various mononucleotide-analogues is well known, especially as antivirals, much less information is available in the prior art on any biological effect of 4-thio-uridine derivatives, and other 4-thio-uridine-containing mononucleotides.

A comprehensive work was published on the utilisation of nucleotide analogues as antivirals, edited by J. C. Martin [Martin, J. C. (Ed.) (1989): Nucleotide analogues as antiviral agents (ACS Symposium Series, 401.) American Chemical Society, Washington, DC. 190 pages (ISBN 0-8412-1659-1)]

The European patent application published as EP 1 431 304 A2 (Gilles G. et al.), describes several mononucleotides, which are active against hepatitis B virus, for example β-L-2'-deoxyuridine. The biological activity of the compound is attributed to the L sugar residue, since natural nucleotides contain D-pentoses.

When one of the compounds (2'-deoxy- β-L-cytidine) was synthesized by a stereo-selective method an intermediate, 3',5'-di-0-benzoyl-2'-deoxy β-L-4-thiouridine, was also prepared, however its potential therapeutic application had not been mentioned.

The European patent EP 0 806 956 (Fahrig, R. et al.) mentions only the combined application of (E)5-

(2-bromovinyl)-2'-deoxy-4'-thiouridine with cytostatic agents. The role of the supplementary treatment with this compound is to decrease the chance of or to prevent the development of resistance, possibly occurring after applying cytostatic therapy. The 4'-thiouridine utilized in EP 0 806 956 is a derivative of the earlier developed proposed anti-herpes agent, (E)5-(2-bromovinil)-2'-deoxyuridine, thiolated on the ribose moiety.

These experiments are not convincing concerning the therapeutic activity of the compound as a single agent. The US patent US 4 954 485 (Yoshioka H et al.), reports the antiviral activity of 2',3'-dideoxy-4-thio- uridine derivatives. The antiviral activity of these compounds was very low compared to AZT. The dideoxy nucleotide is capable of entering the cell, wherein it can be phosphorylated to the triphosphate derivative, and used in this form by the HIV reverse transcriptase. Thereby the nucleotide is built into the elongating nucleotide chain but causes chain termination therein, which might be a cause of the observed activity, even if very low.

Horvath, A. et al. [Virology 334, 214-23 (2005)] reported that the 35-mer oligo-4-thiouridylate has potent antiretroviral activity, which was substantially decreased with decreased number of nucleotides in the chain. Later, they prepared the 8-mer congener which did not have detectable antiviral activity.

However, the present inventors surprisingly found that the monomer unit, 4-thio-deoxyuridine- monophosphate (4-thio-dUMP) unexpectedly showed favourable activity, not only against retroviruses but also inhibiting the cell-proliferation without any detectable toxicity even in high concentration. Unnecessary to emphasize that there is an urgent need in the art for the development of new agents in this and other medical fields, especially for agents with low cost and simple production.

Further studies unexpectedly proved that beside the 4-thio-dUMP and 4-thio-UMP their diphosphate and triphosphate derivatives are also active. Furthermore, the 4-thio-uridine nucleoside itself was found to be also active. It was a realistic expectation that the analogues and derivatives of these compounds may also be active.

It should be noted that the 4-thiouridine itself has been known for a long time as a component of tRNA [Lipsett, M.N., The isolation of 4-thiouridylic acid from the soluble ribonucleic acid of Escherichia coli. J. Biol. Chem. 240, 3975-3978. (1965)]

BRIEF DESCRIPTION OF THE INVENTION

We briefly describe the invention below.

According to a first aspect of the invention a compound is provided for use in therapy wherein said compound is represented by general formula (I):

wherein in the formula above:

- X is O, S, NH, CH₂ or nothing,

Z and Y are, separately and independently from each other O, S, NH, CH₂ or nothing,

- W is O, S, NH or CH₂ - Q is O or S,

Ri, R₂, R₃ are, separately and independently from each other - H,

- a protecting group, preferably a protecting group applied in nucleotide chemistry

- H-[PO₃H]_n or any synthetic analogue thereof, wherein n = 1, 2 or 3, - a substituted or unsubstituted Ci_-J0 organic moiety, preferably Ci_-6 organic moiety, preferably alkyl, highly preferably substituted or not substituted methyl, ethyl, propyl, isopropyl, butyl, isobutyl or tert-butyl, or any unsaturated derivatives thereof,

- R₄ and R₅ are, separately and independently from each other H, substituted or not substituted C_I-i0 organic moiety, preferably Ci.₆ organic moiety, more preferably substituted or not substituted methyl, ethyl, propyl, isopropyl, butyl, isobutyl, or tert-butyl, or any unsaturated derivatives thereof,

- with the proviso that if Z and Y are nothing, and R₂ and R₃ are H, then R] is a polar group, preferably a negatively charged group, highly preferably H-[PO₃H]_n or any synthetic analogue thereof. The compound according to the invention is thus preferably a thiouridine derivative. Preferably, in general formula (I) above i) not more than two of R_h R₂ and R₃, preferably only one of R₁, R₂ and R₃ is H-[PO₃H]_n or any synthetic analogue thereof, ii) if Z is nothing, then R₂ is a moiety which is different from H-[PO₃H]_n or any synthetic analogue thereof, preferably H, and if

Y is nothing, then R₃ is a moiety which is different from H-[PO₃H]_n or any synthetic analogue thereof, preferably R₃ is H, iii) if both Z and Y are nothing, then Ri is H-[PO₃H]_n or any synthetic analogue thereof,

Preferably Q is O. Preferably both X and W represent O.

Preferably, the R_x group is different from a protecting group, preferably different from 2-bromovinyl 5' hydroxyl group.

According to a preferred version any one of R₄ or R₅ is H and the other one is substituted or not substituted methyl, ethyl, propyl or iso-propyl, or their any unsaturated derivatives: preferably methyl or ethyl, more preferably methyl. Highly preferably the thio-uridine derivative according to the invention, is thio-thymidine or a thio-thymidine derivative. According to a further preferred variant both R₄ and R₅ are H.

According to a preferred embodiment of the present invention the compound of the invention is represented by general formula (II),

wherein R₄ is H or substituted or not substituted methyl, ethyl, propyl, isopropyl, or any unsaturated variant thereof, the other groups being any other groups defined herein.

The substituted alkyl can be for example hydroxy-alkyl, thio-alkyl or aminoalkil, for example primer, secunder or tertier amino-alkyl; halogenized, carboxylized or sulfonated alkyl; ester, ether or acid-amide. The ester or ether group can be attached directly to the 5', 3' or 2' oxigens (when X, Z and/or Y is/are O). Furthermore, the alkyl may comprise a heterocycle as a substituent, preferably a naturally occurring heterocyclic compound as a substituent or a derivative of the naturally occurring heterocyclic compound as a substituent.

The substituted alkyl may contain more than one substituents, for example may contain more than one hidroxy-, amino-, thio-, ether or ester as substituents. As an advantageous variant, the substituted alkyl can be di-, tri-, terra-, penta- or hexahydroxyl alkyl. Furthermore, it can be ethylene-glycol or its oligomerized version.

In any case, according to this invention the substituted alkyl is preferably hydrophilic. Preferably, in general formula (I) the synthetic analogue of H-[PO₃H]_n can be, for example, a phosphorothioate, phosphorodithioate, methylphosphonate, phosphoramidate. According to a highly preferred embodiment, in general formula (I) H-[PO₃H]_n or any of its synthetic analogues is represented by any of general formulae Pl, P2 or P3,

Qi Qi Q₂ Il Il Il

B - P - (P1) B - P - A₁ - P - (P2)

I I I

OH OH OH

Qi Q₂ Q3

Il Il Il

B - P - A₁ - P - A₂ - P - (P3),

I I I OH OH OH wherein in the formulae (with reference to the respective formula) Qi, Q₂ and Q₃ are, separately and independently from each other, O or S, B is -OH or methyl, - Ai, A₂, or A₃ is O or methyl.

Preferably, the moiety according to general formulae (Pl) (P2) or (P3) are a monophosphoryl moiety, a diphosphoryl moiety or a triphosphoryl moiety, respectively.

According to a further preferred variant R₁, and/or R₂ is a group capable of forming covalent bonds. According to a further preferred embodiment the present invention relates to a compound represented by general formula (I) for use

synthetic analogue thereof represented by general formulae CPl\ (P2) or (^"P3^") above, where n = 1, 2 or 3. According to a highly preferred embodiment R₁, R₂ or R₃, more preferably Ri or R₂ is monophosphoryl, diphosphoryl or triphosphoryl group.

According to a preferred embodiment X and Y; X and Z, or X, Y and Z are uniformly O. According to another preferred variant at least one of Z and Y is/are nothing, optionally both of them are nothing. In this case, preferably R₂ and/or R₃ is/are H.

Preferably, the compound represented by general formula I (thio-uridine derivative) is a mononucleotide.

Highly preferably the invented compound is deoxyribo-mononucleotide preferably 4-thio-dUMP, 4-thio-dUDP 4-thio-dUTP

4-thio-3'dUMP, 4-thio-3'dUDP, 4-thio-3'dUTP 4-thio-dTMP, 4-thio-dTDP, 4-thio-dTTP 4-thio-3'dTMP, 4-thio-3'dTDP, 4-thio-3'dTTP; 2',3'-dideoxyribomononucleotide, preferably 4-thio-ddUMP, 4-thio- ddUDP, 4-thio-ddUTP

4-thio- ddTMP, 4-thio- ddTDP, 4-thio-ddTTP. A further preferred embodiment of the present invention is a compound represented in general formula I for use in therapy, wherein in the formula at least one of Z and Y is/are O, S or NH, preferably at least one is O, more preferably at least one of Z-R₂ and Y-R₃ is OH.

In a preferred variant of this embodiment at least one of R^. R? and RT is H-[POiHl_n or any synthetic analogue thereof, where n = 1, 2 or 3. Highly preferably, R₁, R₂ or R₃ is, more preferably R₁ or R₂ is monophosphoryl, diphosphoryl or triphosphoryl.

Preferably, the compound according to the present invention is a ribo- or deoxyribo-mononucleotide, highly preferably, any of the compounds listed below:

4-thio-UMP, 4-thio-UDP, 4-thio-UTP, 4-thio-3'UMP, 4-thio-3'-UDP, 4-thio-3'UTP, 4-thio-2'-UMP, 4-thio-2'-UDP, 4-thio-2'-UTP,

4-thio-dUMP, 4-thio-dUDP, 4-thio-dUTP, 4-thio-3'dUMP, 4-thio-3'dUDP, 4-thio-3'dUTP, 4-thio-TMP, 4-thio-TDP, 4-thio-TTP,

4-thio-3'TMP, 4-thio-3'-TDP, 4-thio-3'TTP, 4-thio-2'-TMP, 4-thio-2'-TDP, 4-thio-2'-TTP, 4-thio-dTMP, 4-thio-dTDP, 4-thio-dTTP,

4-thio-3'dTMP, 4-thio-3'-dTDP, 4-thio-3'dTTP,

According to a preferred variant of these embodiment in general formula (I) RN R? and RsJs. separately and independently from each other. H or substituted or not substituted methyl, ethyl, propyl or isopropyl group, or any unsaturated variant thereof, with the proviso that if Y is nothing, then R₂ is H, and if Z is nothing then R₃ is H.

Preferably, the compound is a nucleoside represented in general formula (I), preferably any of the following nucleosides: 4-thio-uridine, 4-thio-deoxyuridine, 4-thio-thymidine, 4-thio-deoxythymidine.

A preferred embodiment of the first aspect of the invention is a compound represented by general structural formula (I) for use in the treatment of diseases with accompanied by cell proliferation. Preferably, the invention relates to the treatment of tumor diseases and malignancies with any of the compounds described herein.

Preferably, the invention relates to any of the compounds according to the invention for use in the treatment of autoimmune diseases accompanied by cell proliferation. Furthermore, preferably, according to a further preferred embodiment, the invention relates to the compounds represented in general structural formula I for use as antiviral agents, preferably against HIV infection.

Furthermore, the compounds represented in general structural formula I can be used for the preparation of antiviral medicines, preferably against HIV infection.

According to a further aspect the present invention relates to uses of the compounds of general formula I for the preparation of a medicine for inhibition of cell proliferation.

wherein in the formula

- X is O, S, NH, CH₂ or nothing,

Z and Y are, separately and independently from each other O, S, NH, CH₂ or nothing, - W is O, S, NH or CH₂

- Q is O or S,

Ri, R₂, R₃ are, separately and independently from each other - H,

- a protecting group, preferably a protecting group applied in nucleotide chemistry - H-[PO₃H]_n or any synthetic analogue thereof, wherein n = 1, 2 or 3,

- a substituted or unsubstituted C_1-10 organic moiety, preferably C^ organic moiety, preferably alkyl, highly preferably substituted or not substituted methyl, ethyl, propyl, isopropyl, butyl, isobutyl or tert-butyl, or any unsaturated derivatives thereof, - R₄ and R₅ are, separately and independently from each other H, substituted or not substituted C]_-10 organic moiety, preferably C_1-O organic moiety, more preferably substituted or not substituted methyl, ethyl, propyl, isopropyl, butyl, isobutyl, or tert-butyl, or any unsaturated derivative thereof. According to this aspect of the invention, the compound of general formula (I) can be, for example, a compound defined according to the first aspect of the invention. Preferably, in general formula (I) above i) not more than two of R_b R₂ and R₃, preferably only one of Rj, R₂ and R₃ is H-[PO₃H]_n or any synthetic analogue thereof, ii) if Z is nothing, then R₂ is a moiety which is different from H-[PO₃H]_n or any synthetic analogue thereof, preferably Z is H, and if

Y is nothing, then R₃ is a moiety which is different from H-[PO₃H]_n or any synthetic analogue thereof, preferably R₃ is H. Preferably Q is O.

Preferably both X and W represent O. Preferably, the R₁ group is different from a protecting group, preferably different from 2-bromovinyl ' hydroxyl group. According to a preferred variant any one of R₄ or R₅, is H and the other one is substituted or not substituted methyl, ethyl, propyl or isopropyl, or any unsaturated derivative thereof: preferably methyl or ethyl, highly preferably methyl. A further preferred version is wherein both R₄ and R₅ are H.

Preferably, in the general formula (I) the H-[PO₃H]_n or its any synthetic analogue is for example phosphorothioate, phosphorodithioate, methylphosphonate, phosphoramidate.

According to a highly preferred embodiment, in general formula (I) H-[PO₃H]_n or any of its synthetic analogues is represented by any of general formulae Pl, P2 or P3,

Qi Qi Q₂ H I

I l Ill H Il

B - P - (P1) B - P - Ai - - P - (P2)

I I _I I

OH OH OH

Q₁ Q₂ Q3 Il Il Il

B - P - Ai - P - A₂ - P - (P3),

I I I I I

OH OH OH wherein in the formulae (with reference to the respective formula) Qi. Q₂ and Q₃ are, separately and independently from each other, O or S, - B is -OH or methyl, - Ai, A₂, or A₃ is O or methyl.

According to a further preferred variant at least one of Z and Y is O, S, NH, preferably O; more preferably at least one of Z-R2 and Y-R3 is OH. According to a further preferred embodiment at least one of Z and Y is nothing, optionally both of them are nothing. In this case, preferably R₂ and/or R₃ is/are H.

According to a further preferred embodiment the compound of general formula (I) is a mononucleotide or nucleoside, highly preferably a compound selected from the following compounds:

4-thio-uridine, 4-thio-UMP, 4-thio-UDP, 4-thio-UTP, 4-thio-3'UMP, 4-thio-3'UDP, 4-thio-3'UTP, 4-thio-2'UMP, 4-thio-2'UDP, 4-thio-2'UTP,

4-thio-deoxy-uridine, 4-thio-dUMP, 4-thio-dUDP, 4-thio-dUTP, 4-thio-3'dUMP, 4-thio-3'dUDP, 4-thio-3'dUTP, 4-thio-dideoxy-uridine, 4-thio-ddUMP, 4-thio-ddUDP, 4-thio-ddUTP, 4-thio-thymidine, 4-thio-TMP, 4-thio-TDP, 4-thio-TTP, 4-thio-3'TMP, 4-thio-3'TDP, 4-thio-3'TTP, 4-thio-2'TMP, 4-thio-2'TDP, 4-thio-2'TTP,

4-thio-deoxy-thymidine, 4-thio-dTMP, 4-thio-dTDP, 4-thio-dTTP, 4-thio-3'dTMP, 4-thio-3'dTDP, 4-thio-3'dTTP, 4-thio-dideoxy-thymidine, 4-thio-ddTMP, 4-thio-ddTDP, 4-thio-ddTTP. According to a further embodiment of the present invention the compounds of the invention are used for preparation of therapeutic compositions for the treatment of malignant conditions.

Preferably, the therapeutic composition prepared according to this invention, may be suitable for the treatment of solid tumors and leukemias. According to a further embodiment the compounds of the present invention are used for the preparation of a therapeutic composition for sensitizing tumors. Preferably, in this case any therapeutic composition or kit of compounds comprises a compound described or defined herein in combination with a chemotherapeutic agent for therapeutic use.

Furthermore, the invention relates to a use in accordance with the invenion for the preparation of a therapeutic composition for the treatment of any disease or condition where killing the transformed or malignant cells are necessary, wherein the compounds described in this invention are capable of sensitizing the cells to cell killing agents.

The invention further relates to a use in accordance with the invention for the preparation therapeutic or therapeutic compositions for use in the treatment of conditions, where during the treatment apoptosis induction is required in patient cells.

According to a further aspect of this invention therapeutic compositions or kits are provided, said compositions or kits comprising any of the compounds according to the invention in combination with a chemotherapeutic agent, cytostatic, cell killing or apoptosis inducing agents together wih an other regularly used pharmaceutically acceptable excipient. Optionally the active compounds or agents are formulated as individual units (kit).

According to a further aspect of this invention a method to inhibit cell proliferation is provided, said method characterized in that the patients are treated with any compounds according to the invention in appropriate concentrations.

By the method of the invention preferably malignant diseases are treated with the compounds described herein, preferably solid tumors or leukemias.

According to a further embodiment of the invented method the thio-uridine derivatives disclosed by the invention are used for killing proliferating cells in combination with another therapeutic procedure. For example the thio-uridine derivatives of this invention can be applied in combination with other chemotherapeutic agents or radiation therapy. The treatment may be carried out in the same time with the administration of chemotherapeutic agents and/or radiation therapy, or before or after of said administration. Appropriately, in this case the treatment protocol can be fitted to the treatment regime with chemotherapeutic agents or radiotherapy.

In a further preferred embodiment of the inventive method thio-uridine derivatives are used in the treatment of solid tumors as supplementary treatment to surgical removal, shortly after the operation. The invention also relates to antiviral treatment of a patient, preferably treatment against retroviral infection in particular a treatment against HIV infection.

According to a further aspect of the invention a method for the induction of apoptosis is provided, characterized in that the thio-uridine compounds represented by general formula (I) are contacted to cells in which apoptosis induction is to be effected. The subject of this invention is the preparation of apoptosis inducing reagent kits containing thio-uridine derivatives according to the general structure (I). BRIEF DESCRIPTION OF THE FIGURES OF THE INVENTION

Figure 1 : in this experiment OCM-I cells were treated with various concentrations of S⁴UMP and the cell viability determined by MTT assay. Figure 2: the effect of S⁴UMP is shown on the colony forming activity of primary human acute myeloid leukemic (AML) cells treated with S⁴UMP. The dose response curve shows the % of inhibition on colony forming activity of the treated cells based on the control.

Figure 3: The effect of S⁴UMP on the colony forming activity of JY human B cells transplanted into SCID mice. Figure 3a: Colony forming activity of bone marrow blast cells from SCID mice injected with JY cells.

Figure 3b: Number of total tumor stem cells in the femur of JY-injected SCID mice. Figure 4: Effect of S⁴UMP on the morphology of OCM-I cells. The cells were dyed with May- Griinwald-Giemsa-solution and observed by Zeiss Axivert 135 microscope, at a magnification of 20Ox.

A. Control OCM-I cells after 48 hours of propagation; B. OCM-I cells after 48 hours of treatment with S⁴UMP (150 μM, i.e., 50 μg/ml); C. OCM-I cells after 72 hours of treatment with S⁴UMP (150 μM, i.e., 50 μg/ml).

Figure 5: DNA fragmentation observed in OCM-I cells after treatment with 30 μM (10 μg/ml) S⁴UMP. The lanes: (1) control; (2) 24 hours after treatment; (3) 48 hours after treatment.

Figure 6: Study of the fragmentation of DNA in HL-60 cells upon the treatment with 30 μM s⁴dU (7.3 μg/ml) and S⁴UMP (10 μg/ml). A characteristic DNA ladder was found. (1) after 24 hours of the treatment; (2) after 48 hours after treatment.

Figure 7: Activation of caspase-9 is shown, 24, 48 and 72 hours after the treatment and without treatment. A. HL-60 cells (10 μg/ml S⁴UMP), (B) He-La cells (30 μg/ml S⁴UMP), (C) OCM-I cells (30 μg/ml S⁴UMP).

DEFINITION OF CERTAIN TERMS USED HEREIN

According to this description those chemical groups are called "protecting group" herein which can be attached to a defined group of a molecule, and under certain condition it does not change whereas prevents the change of the defined group of the molecule, however, under other suitable conditions or upon the effect of certain reagents it can be cleaved off, whereby the defined group of the molecule, to which it was attached, getting free, in the form defined by predetermined conditions. In the present invention preferably protecting groups useful in peptide chemistry or nucleotide chemistry, highly preferably in nucleotide chemistry are used, for example protecting groups suitable for the protection of hydroxyl-, amino-, thio-, or phosphate groups. Such protecting groups are well known for the skilled person [see for example Townsend, L.B. (Dd.)

Chemistry of nucleosides and nucleotides. Plenum Press, New York.: Vol. 1 (1988), Vol. 2 (1991), Vol. 3 (1994), Vol. 4 (1995); VorbrQggen, H. And Ruh-Pohlenz, C. (2001): Handbook of nucleoside synthesis. John Wiley and Sons, New York.]. Under the expression "a thio-uridine derivative of the invention" any of the compounds are meant which can be utilized in any methods or uses described herein and exerts an effect shown in this invention, or defined in any of the claims, and which contains the moiety defined by the general formula (III):

wherein in the formula Q, R₄ and R₅ are any groups or substituents defined anywhere in the present description.

DETAILED DESCRIPTION OF THE INVENTION As will be seen below several examples for the compounds according to the invention are described as well as for diseases which can be treated according to the invention. It is obvious for a person skilled in the art that the protection is not limited only to these compounds; the examples are intended merely to illustrate the various possibilities according to the invention, helping the skilled experts to reduce the solution into practice. The solution according to this invention is based on the unexpected finding that some long- known thio-uridine derivatives (for example nucleosides, among others, 4-thio-uridine and 4-thio-deoxy-uridine, furthermore mononucleotides containing these necleosides) exert anti-proliferative and/or antiviral activity on transformed cell lines, while their toxicity is very low; actually, the present inventors were not able to detect any toxicity in respect of these compounds under the conditions used. Based on data obtained so far certain are contemplated as preferably applicable in the present solution.

These compounds are listed below, including their abbreviations and complete names:

4-thio-U S⁴U 4-thio-uridine

4-thio-UMP S⁴UMP 4-thio-uridine-5 ' -monophosphate

4-thio-UDP S⁴UDP 4-thio-uridine-5 ' -diphosphate

4-thio-UTP S⁴UTP 4-thio-uridine-5'-triphosphate

4-thio-3'UMP s⁴-3'UMP 4-thio-uridine-3 '-monophosphate

4-thio-3'UDP s⁴-3'UDP 4-thio-uridine-3 '-diphosphate

4-thio-3'UTP s⁴-3'UTP 4-thio-uridine-3 '-triphosphate

4-thio-2'UMP s⁴-2'UMP 4-thio-uridine-2 ' -monophosphate

4-thio-2'UDP s⁴-2'UDP 4-thio-uridine-2 ' -diphosphate

4-thio-2'UTP s⁴-2'UTP 4-thio-uridine-2 ' -triphosphate

4-thio-dU s⁴dU 4-thio-deoxy-uridine

4-thio-dUMP s⁴dUMP 4-thio-deoxy-uridine-5'-monophosphate

4-thio-dUDP s⁴dUDP 4-thio-deoxy-uridine-5 ' -diphosphate

4-thio-dUTP s⁴dUTP 4-thio-deoxy-uridine-5'-triphosphate 4-thio-3'dUMP s⁴-3'dUMP 4-thio-2 ' -deoxy-uridine-3 ' -monophosphate

4-thio-3'dUDP s⁴-3'dUDP 4-thio-2'-deoxy-uridine-3'-diphosphate

4-thio-3'dUTP s⁴-3'dUTP 4-thio-2'-deoxy-uridine-3'-triphosphate

4-thio-ddU s⁴ddU 4-thio-dideoxy-uridine

4-thio-ddUMP s⁴ddUMP 4-thio-2',3'-dideoxy-uridine-5'-monophosphate

4-thio-ddUDP s⁴ddUDP 4-thio-2 ' ,3 ' -dideoxy-uridine -5 ' -diphosphate

4-thio-ddUTP s⁴ddUTP 4-thio-2 ' ,3 ' -dideoxy-uridine -5 ' -triphosphate

4-thio-T S⁴T 4-thio-thymidine

4-thio-TMP S⁴TMP 4-thio-thymidine-5'-monophosphate

4-thio-TDP S⁴TDP 4-thio-thymidine-5 '-diphosphate

4-thio-TTP S⁴TTP 4-thio-thymidine-5 ' -triphosphate

4-thio-3'TMP s⁴-3'TMP 4-thio-thymidine-3'-monophosphate

4-thio-3'TDP s⁴-3'TDP 4-thio-thymidine-3 '-diphosphate

4-thio-3'TTP s⁴-3'TTP 4-thio-thymidine-3 ' -triphosphate

4-thio-2'TMP s⁴-2'TMP 4-thio-thymidine-2'-monophosphate

4-thio-2'TDP s⁴-2'TDP 4-thio-thymidine-2'-diphosphate

4-thio-2'TTP s⁴-2'TTP 4-tio-thymidine-2'-triphosphate

4-thio-dT s⁴dT 4-thio-deoxy-thymidine

4-thio-dTMP s⁴dTMP 4-thio-2'-deoxy-thymidine-5'-monophosphate

4-thio-dTDP s⁴dTDP 4-thio-2'-deoxy-thymidine-5'-diphosphate

4-thio-dTTP s⁴dTTP 4-thio-2'-deoxy-thymidine-5'-triphosphate

4-thio-3'dTMP s⁴-3'dTMP 4-thio-2'-deoxy-thymidine-3'-monophosphate

4-thio-3'dTDP s⁴-3'dTDP 4-thio-2'-deoxy-thymidine-3'-diphosphate

4-thio-3'dTTP s⁴-3'dTTP 4-thio-2'-deoxy-thymidine-3'-triphosphate

4-thio-ddT s⁴ddT 4-thio-dideoxy-thymidine

4-thio-ddTMP s⁴ddTMP 4-thio-2',3'-dideoxy-thymidine-5'-monophosphate

4-thio-ddTDP s⁴ddTDP 4-thio-2 ' ,3 ' -dideoxy-thymidine -5 ' -diphosphate

4-thio-ddTTP s⁴ddTTP 4-thio-2 ' ,3 ' -dideoxy-thymidine -5 '-triphosphate

It has to be emphasized that the solution described in the patent is not limited to the molecules above, since it is obvious for a person skilled in the art that a wide variety of similar molecules, the modified compounds by substitution with analogous functional groups, protected and activated molecules in addition to the carrier-bound forms also can be used in all applications of the invention. Variations and modifications of these compounds are summarized in the brief description of the invention, above.

The molecules and their derivatives can be synthesized by organic chemistry as known for a skilled person. Modern techniques and the methods for production of a variety of nucleotides and derivates are well known in the art. The following scientific publications and reviews reveal some examples: W. W. Zorbach, R. S. Tipson (Eds.) (1968): Preparation of purines, pyrimidines, nucleosides, and nucleotides. Vol. 1. (Synthetic procedures in nucleic acid chemistry.) Interscience Publishers. A John Wiley Division, New York; . K. Kochetkov, E. I. Budovskii (1971): Organic chemistry of nucleic acids. Vol. A. Plenum Press, London. 268 pages; R. E. Harmon, R. K. Robins and L. B. Townsend (Eds.) (1978): Chemistry and biology of nucleosides and nucleotides. Academic Press, New York; R. T. Walker, E. De Clercq and F. Eckstein (Eds.) (1979): Nucleoside analogues. Chemistry, biology and medical applications. Vol. 26. (NATO Advanced Study Institute, Series A.) Plenum Press, New York ; E. Lukevics, A. Zablocka (1991): Nucleoside synthesis. Organosilicon methods. Ellis Horwood, London. 496 pages. H. Vorbrϋggen, C. Ruh-Pohlenz (2001): Handbook of nucleoside synthesis. John Wiley and Sons, New York. 631 pages; Townsend, L. B, (Ed.) Chemistry of nucleosides and nucleotides. Plenum Press, New York.; Vol. 1. (1988), VoI 2. (1991), Vol. 3. (1994), Vol. 4. (1995)

The produced nucleotides can be examined by well known analytical tools, e.g.: C. W. Gehrke, K. C. T. Kuo (Eds.) (1990): Analytical methods for major and modified nucleosides - HPLC, GC, MS, NMR, UV and FT-IR. Vol. A. (Chromatography and modification of nucleosides. Journal of chromatography library, 45A.) Elsevier, Amsterdam. 400 pages.

Since the compounds of this invention presumably exert their effects on the cell surface, according to our present knowledge of the inventors it is advantageous if sufficient polar groups are present in the molecule to prevent or at least to minimize the cellular uptake. Therefore, if some parts of the molecule contain apolar groups (e.g. an 5-methyl-group of thymidines or dideoxy derivates) it is useful to insert polar groups into an other part of the molecule.

The thio-uridin derivates of the invention, like all nucleotides, tend to undergo a ketho-enol tautomerization. The tautomers are in a steady state, but the balance is shifted to the direction of ketho form. The enol form has a -SH (thiol) group instead of =S (thiono) group in position 4. This -SH is a reactive group, which can form a covalent disulphide bridge with protein -SH groups as it was proven in case of (s⁴dU)₃₅ [Virology 334, 214-23 (2005)], in which study the compound "Suligovir" was covalently bound to thioredoxin.

The role of protein disulphide isomerase (PDI) on the cell surface is to catalyze the thiol+→disulfide transformation of the cell surface proteins, which ensures the activity of the receptors and transport proteins.

These catalytic reactions are more active on the surface of tumor cells than normal cells. The reduction of the two disulphide bridges of HIV-I gpl20 protein, which is also catalyzed by the cell surface PDI, is necessary for the entry of the virus into the target cell. Moreover, the second domain of the CD4 receptor, the main receptor for HIV infection, has two -SH groups that also undergo an oxidative-reductive change during the entry of the virus into the cell. Chemically feasible and it is very likely that the antiproliferative and antiviral

(e.g. anti-HIV) activity of the S⁴UMP is based on the inhibition of these oxidative-reductive changes.

Therefore, in the opinion of the present inventors, the two types of effects are probably related.

The experiments below provide evidence for the effect of the thiouridin derivates of the invention on a variety of different tumor cell lines. These activities also suggest the inhibition of an important basic mechanism in the proliferating cells or during the entry process of the virus into the target cell.

As described in the examples and explained among possible applications of the present invention, these molecules can be used in diseases where cell proliferation has to be inhibited, because these molecules inhibit the tumor cell growth, facilitate the apoptosis and sensitize the cell to other cell toxic effects (e.g. other compound or radiation). According to these data, preferably the thiouridin derivates can be given to the patient somewhat prior to or along with an antiproliferative, antitumor treatment (e.g. chemotherapy or radiation therapy).

The administration of the compound depends on the type of disease, tumor or virus. It can be administered locally, systemically, orally or intravenously as an injection. The methods of administration and targeting are well known to the persons skilled in the present field of the art. It is concluded that the compound of the invention or its derivates, described in the invention, can be adapted to standard antiproliferative or antitumor protocols. These therapies and protocols can be found in details e.g. in the following publications: Cvitkovic E, Droz JP, Armand JP, Khoury S, ed. Handbook of chemotherapy in clinical oncology. Jersey: Scientific Communication International Ltd., 109-1 19 (1993); Skeel RT. Handbook of Cancer Chemotherapy. Baltimore: Lippincott Williams & Wilkins, 41 1-459 (2003); Skeel RT Handbook of Chemotherapy 5^th edn Philadelphia: Lippincot and Williams; (1999).

Recently, several antiviral, e.g. anti-HIV treatments are available; the main tracks are summarized in the following book: Retroviruses. Coffin, John M.; Hughes, Stephen H.; Varmus, Harold E. Plainview (NY): Cold Spring Harbor Laboratory Press; cl997.

EXAMPLES

1. Materials and Methods

1.1. Chemicals

Chemicals were purchased from Sigma Aldrich and Amersham Pharmacia and were analytical grade or better. 1.2. Synthesis of 4-thio-UMP Cs⁴UMP")

The thiolated mononucleotide, S⁴UMP, was produced by H₂S treatment of cytidine-5-monophosphate as it was described by the members of the present research group [Horvath A., T6kes S., es Hartman T. et al.,

Virology 334, 214-223 (2005)], and purified by ion-exchange chromatography [Horvath A. and Aradi J.

Anal. Biochem. 338, 341-343 (2005)]. In detail, in one of the cases the compound was prepared as follows, in accordance with scheme (I):

Scheme (I)

100 mg of CMP was dissolved in 0.5 ml of sterile water, then 0.5 ml of pyridine was added. This mixture was put in a stainless steel bomb with Teflon lining and placed to -70 for one hour. Ten ml liquid H₂S was added then the bomb closed and kept at 55⁰C for 10 days. Opening the bomb, to the yellow solid 4 ml of sterile water was added. A yellow solution was incubated at 4°C for 30 minutes, then the fine sulfur precipitate was removed by filtration through sterile Millipore filter. The solution was lyophilized and dissolved again in 1 ml of water and filtered through sterile Millipore filter. The clear solution obtained after filtration was purified on RESOURCE Q anion-exchange column. Parameters of purification: A buffer: 0.005 M TrisC10₄ (pH 7.8) B buffer: 0.005 M TrisC10₄ (pH 7.8) + 1 M NaClO₄ Flow rate: 1 ml/min Gradient increase: 1 mM/min

Nucleotide detected at: 300 nm

The obtained solution was lyophilized then mixed with cold (-20⁰C) acetone, this treatment resulted in S⁴UMP, as a yellow precipitate. The precipitate was collected by centrifugation and washed four times with acetone; each washing was followed by centrifugation. Finally, the precipitate was dried in lyophilizing equipment then dissolved in sterile distillated water.

Determination of concentration by UV measurement: 18 OD₃₃₀=I μg. MW: 340.25 g/mol Formula: C₉H₁₃N₂O₈PS.

1.3. Preparation of the other compounds The other mononucleotides and nucleosides were prepared as we have shown for 4-thio-UMP. i.e., the appropriate unthiolated mononucleotide or nucleoside was treated with H₂S in pyridine-water mixture.

1.4. Propagation of cells

OCM-I human uveal melanoma cells were obtained from Dr. Monique H. Hurks (Department of Ophthalmology, Leiden University Medical Center, Leiden, The Netherlands). Other human uveal melanoma cell lines were introduced by Monique H. Hurks at al. [Invest Ophthalmol. Vis. Sci. 42(13), 3081-4 (2001)].

The cells were cultured in RPMI containing 10% heat-inactivated FBS and antibiotics (100 units/ml penicillin and 100 μg/ml streptomycin), and were grown at 37°C in a humidified atmosphere containing 5% CO₂. Cells were harvested after incubation with PBS/trypsin (2.5 μg/ml).

The conditions for culturing OCM-I cells were applied for the other studied cell lines, with minor modifications according to the supplier: HL-60, NB-4, WEHI-3B, THP- 1.

For the propagation of He-La cells it was taken into consideration that it is a cell line growing in monolayer.

The He-La, HL-60 and THP-I cell lines are available from ATCC (American Type Culture Collection, http://www.atcc.org or in Europe from LGC Promochem, Queens Road, Teddington, Middlesex TWl 1 OLY UK or LGC Promochem, Mercator Str. 51, 46485 Wesel Germany; http://www.lgcpromochem-atcc.com , for example under the following identification numbers:

CRL-13011 Homo sapiens (human) HeLa NRl [ HeLaNRl ] CCL- 13 Homo sapiens (human) HeLa [ Chang Liver ] CCL-2.2 Homo sapiens (human) HeLa S3 CCL-2.1 Homo sapiens (human) HeLa 229

CRL- 1958 Homo sapiens (human) HlHeLa [ H-HeLa ; Hl HeLa ; Hl-HeLa ] CCL-2 Homo sapiens (human) HeLa CRL- 1964 Homo sapiens (human) Clone 15 HL-60 CRL-2258 Homo sapiens (human) HL-60/MX1 CRL-2257 Homo sapiens (human) HL-60/MX2 CCL-240 Homo sapiens (human) HL-60 TIB-202 Homo sapiens (human) THP-I.

The NB-4 (acute promyelocyte leukemia) cells were provided generously by Dr, Michel Lanotte, Ph.D. [Research Director (DRl CNRS). Head of Department. INSERM U685, Hospital Saint-Louis. 1, rue Claude Vellefaux-75495 Paris CEDEXlO].

The WEHI-3B cell [a monomyelocytic leukemia cell line, see J Nat Cancer Inst 1969;43:963; J Immunol 1982; 128:2393; Proc Nat Acad Sci USA; 1984; 81:1208; isolated from BALB/c mouse,] is available from DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1 b 38124 Braunschweig, Germany), catalogue number: DSMZ ACC 26.

1.5. MTT assay

To measure the cell viability 5xlO⁵ cells were seeded into 24 well plates. Cells were cultured for 6 hours in 1 ml of medium, as described above, and then treated with various concentrations of S⁴UMP. Aliquots of 100-100 μl each were removed in triplicate at the indicated time for MTT [3-(4,5- dimethylthiazolyl-2)-2, 5-diphenyl-tetrazolium bromide] assay. The assay was performed as described by Gerlier, D. and Thomass P102592et, N. [J.Immunol. Methods., 94; 57-63 (1986)] as also specified by the manual of ATCC (American Type Culture Collection).

For control sample, untreated cells were used, or in certain cases unmodified compounds (UMP).

1.6. DNA fragmentation analysis The cells (2xlO⁶) were treated with S⁴UMP for 24 and 48 hours as indicated, collected by centrifugation (1000 rpm, Jouan C/CR4-12, Horizontal Rotor; 10 min, 10°C), washed twice with PBS, then DNA was isolated and subjected to agarose gel-electrophoresis [Zhu, N. and Wang, Z. Anal. Biochem. 246. 155-8 (1997)]. The agarose gels were evaluated and archived by AlphaImager™2200.

1.7. Measurement of caspase-9 activity Cells treated with S⁴UMP were centrifuged at 1000 rpm for 10 minutes and washed twice with PBS.

Detection of the caspase-9 activity was performed using a Caspase-9/Mch6 Colorimetric Assay Kit, according to the instruction of manufacturer (Medical and Biological Laboratories Co., Ltd., Nagoya, Japan). The results represent the average of three independent experiments.

1.8. Flow cytometry Cells were cultured for up to 72 hours in the presence or absence of s4UMP. Cells (5x10⁵ cells/tube) were centrifuged (100 rpm, Jouan C/CR4-12, Horizontal Rotor) for 10 minutes at 10⁰C then resuspended in 0.5 ml binding buffer (25 Mm HEPES, 125 mM NaCl, 2.5 mM CaCl₂) and were labelled by 5 μl annexin- FITC and 5 μl propidium iodide according to the instruction of manufacturer (Medical and Biological Laboratories Co., Ltd.) Flow cytometric measurements were carried out on unfixed cells by a FacsCalibur flow cytometer (Becton Dickinson, Biosciences, San Jose, CA) using 488 nm argon laser excitation. Data were stored in "list mode" files and were analyzed by CellQuest software. In all cases fluorescence data of 20 000 events were collected. 1.9. Morphological studies

The treatment of the cells were completed separately with S⁴UMP or other compounds. The dyeing was performed according to the standard procedure, the soluble cells were centrifuged to a microscopic plate, known as cytospin protocol. The following steps were completed: 1. 50 μl cell culture + 200 μl PBS (Phosphate Buffered Saline),

2. Cytospin 5 minutes, 800 rpm (centrifugation),

3. Fixing with a few drops of methanol,

4. After drying, dyeing with May-Grϋnwald-Giemsa solution (100 μl May-GrUnwald + 100 μl Giemsa + 800 μl water; the May-GrUnwald and Giemsa solutions are available as ready-made solutions) 5. Rinsing with tap water and photographed under microscope.

1.10. Toxicity studies

Six treated and six untreated mice were used in this experiment. The first, control group was treated with 200 μl physiological salt solution, the second group was treated with S⁴UMP, in a quantity of lg/kg body weight living mice. The treatment was performed once by intravenous injection. Every day the weight of the mice was measured in the same time and after 30 days the animals were killed and an expert pathologist completed detailed morphological studies. No changes were detected after 30 days.

1.11. Determination of anti-HIV activity

The anti-HIV activity of the compounds were determined by measuring the reverse transcriptase (RT) activity in the supernatant of infected and treated MT-4 cells [Virology, 334, 214-223 (2005)]. . Beside MT-4 cells the measurement was also completed on PBMCs. PBMCs were isolated from blood from healthy donors; the cells were stimulated and treated with interleukin 2; in the fourth day the cells were washed and infected by virus and the RT activity determined on day 11. The control in both cases was infected but untreated cultures (100%).

1.12. Colony forming activity of blast cells This study was performed according to the publication. Benkd et al. FJ. Antimicrob. Chemother. 43.

672-81. (1999Yl.

2. Study of the viability of cells

In the experiment reported below the controls were untreated cells. The unmodified compound (UMP) proved to be ineffective.

2.1. Anti-proliferative effect of S⁴UMP on OCM-I cells

In preliminary experiments the S⁴UMP and s⁴dUMP proved to be equally active; therefore the experiments were completed with the cheaper s⁴dUMP.

First, we studied the dose response and the effect of the length of the treatment on proliferation, measuring the decreased viability by MTT assay. In these experiments the drug was added only once, at

"zero time" to the culture of OCM-I cells. The viability of the cells after 24 hours of treatment decreased in all of the four applied drug concentrations (6, 30, 150, and 300 μM which concentrations are equivalent to 2,

10, 50 and 100 μg/ml, respectively).

The effect was dose dependent; however, at high drug concentration it showed saturation kinetic (Figure 1.a). When 6 μM (i.e. 2 μg/ml S⁴UMP) inhibitor was used the cell viability was decreased in 24 hours by 20%. The nucleotide was not more active in 300 μM concentration then in 150 μM concentration (Figure l.a, 24 hours treatment). The observation that the inhibitory effect shows a saturation kinetic supports the assumed mode of action (see below): the inhibitory nucleotide interacts with cell surface proteins.

Another important conclusion which may be drawn from the viability studies is that the cells could metabolize the S⁴UMP. Applying 30 μM S⁴UMP (10 μg/ml) it was found that the decrease of the viability after 24, 48 and 72 hours were 32%, 40% and 9%. The 9% inhibition after72 hours indicate that the cells almost completely recovered from the treatment (Figure l.a). This result is in good agreement with the observed nontoxic nature of the S⁴UMP and related compounds.

2.2. Antiproliferative effect of S⁴UMP in HL-60. NB-4 and THP-I cells The experiments were completed similarly as described for OCM-I cells. The results, shown here, are the averages of five parallel determinations. The results are as follows:

HL-60 (myeloid leukemia cell line)

10 μg/ml S⁴UMP decreased the cell viability 39 ± 8% in 48 hours of treatment.

NB-4 (promyelocytic leukemia cell line) 10 μg/ml S⁴UMP decreased the cell viability 31 ± 7% in 48 hours of treatment.

THP-I (monocytic leukemia cell line)

10 μg/ml S⁴UMP decreased the cell viability 43 ± 5% in 48 hours of treatment.

2.3. Effect of 4-thio-uridine-di- and tri-phosphates on proliferation of HL-60 promyelocvtic leukemia cells The experiments were performed as described in the Materials and Methods section, and as described above.

Table 1

Effects of S⁴UDP, S⁴UTP, s⁴dUDP and s⁴dUTP in various concentration on proliferation of HL-60 promyelocytic leukemia cells.

From the above shown table it is obvious that the cell proliferation was inhibited even at a low concentration of the nucleotides. Furthermore it seems that the triphosphates are preferred among the nucleotides. It can also be seen that the use of deoxy-derivatives does not result in significantly different inhibitory effect. 2.4. A comparison of the effects of S⁴UMP and s⁴dϋ on HeLa. OCM-I. HL-60. NB-4 and WEHI-3B cells

In the experiments described below effects exerted by S⁴UMP and s⁴dU on various cell lines were compared. The cells were propagated as described in the Materials and Methods section and the protocols are also were the same or as described herein.

We found that the cell culture experiments are not very easily reproducible, therefore sometimes deviation of the results was relatively high. The inhibitory activity of the compounds depends on the cell culture medium and on the quality of fetal calf serum (FCS) added to the culture; although, they are theoretically the same (Medium: RPMI 1640; FCS: GIBCO product) but they may vary from batch to batch. This is the reason why significant differences were observed in various experiments. However, it was immediately evident in each case that we observed unambiguous inhibition, and the inhibitory tendencies were the same as the results obtained in other experiments.

In Table 2 we summarized the decreased viability of tumor cells as a percentage of control (untreated cells).

Table 2a Cell viability decrease of OCM-I cells (% of control)

Table 2e Cell viability decrease of WEHI-3B cells (% of control)

As shown above the S⁴UMP and s⁴dU in most of the cases have similar inhibitory activity. In several cases the treatment with 150 μM drug after 48 or 72 hours resulting in 80 or 90 % inhibition (!).

3. Effect of s4UMP on tumor stem cells

Aim and result of the study:

Population of tumor cells in the examined AML sample is heterogenic in respect of proliferative capacity of the tumor cells. This population consists stem cells with unlimited growth that can reproduce and maintain the population and other reproductive cells with limited number of cell division. Elimination of tumor stem cells, which constitute less than 1% (mainly 0.05-0.1%) of the tumor cells, is necessary for recovery of the patient. Differentiating these cells using only an antiproliferative assay based on measurement of decrement in cell number is not possible, because the death of only the more sensitive cells with limited capacity of division may occur. Demonstration of tumor stem cells can be done only with colony assays, in which the progeny cells of the stem ancestor stay together and form colonies in a special semi-soft agarose culture. Decrease of the copy number of the colonies obviously indicates the death of tumor stem cells.

3.1. Study of s4UMP effect on colony forming capacity of human primary acute myeloid leukemia cells.

The cells were cultured from the bone marrow of an acute myeloid leukemia (AML) patient's first sample taken at the diagnosis of the disease. The majority of the white cells of the blood consists leukemia cells, and also the bone morrow was full with tumor cells. Special blast colony assay with serial dilutions of the studied compound was performed to culture the bone morrow cells of the patient. There were two parallel cultures with each concentration of the S⁴UMP. The culturing period was 2 weeks. The colony numbers were correlated to the negative control, which was not treated with S⁴UMP and the number of colonies was considered as 100%. On the basis of dose-efficacy curve, the S⁴UMP inhibited the colony forming capacity of the myeloid leukemia stem cells. While there was a 50% inhibition on concentration of 30 μM, there was a complete inhibition above 300 μM of the compound (Figure 2).

3.2. Study of s4UMP effect on colony forming capacity of JY human B-cell leukemia cell line in SCID mice in vivo. Femoral bone morrow samples of SCID mice were studied. Mice were treated and studies as to anatomy in the animal house of Department of Dermatology, University of Debrecen. Treatment and process of examination.

The human cell-injected SCID mice were treated with the studied material in vivo. 2x10⁷ JY tumor cells (human B lymphoma/leukemia cell line) were injected into SCID mice. The human leukemia cells can grow in the severely immunodeficient mice, and these cells can be found e.g. in the bone morrow. The mice were treated with the selected dose of S⁴UMP, while the control animals were treated only with PBS on each day from the third day of experiment, ten times. The mice were dissected after the tenth treatment. The femur was cut out under sterile condition; the cells were extracted from the bone and cultured. The entire bone morrow material of the femur of the animals was washed out, and then the amount of tumor cells and colony forming capacity of leukemia cells were compared to the controls using a blast cell colony assay. Results

It can be seen in Figure 3a and 3b that the ratio of the colony forming JY blast cells decreased in the femoral bone morrow compared to the control group (p<0,05) after the treatment. Also the gross number of the JY stem cells in the femoral bone morrow decreased significantly. The data are shown in table 3.

Table 3.

4. Study of anti-HIV activity The anti-HIV activity was examined first in MT-4 cells as described in the materials and methods. The

IC₅₀ value was 10±3 μg/ml; this effect the includes the growth inhibitory activity of the compound on the MT-4 cells.

PBMCs isolated from healthy donors were also studied with reverse transcriptase assay in supernatants of cell cultures. The IC₅₀ value was 33±6 μg/ml in these experiments. Anti-HIV-1 activity of the monomeric compound (S⁴UMP) can be characterized as a 65% inhibition of

HIV activity in a concentration of 2 μg/ml and 83% inhibition in a concentration of 10 μg/ml. This activity was demonstrated in each experiment, the results were similar (IC₅₀=I 0-40 μg/ml depending the methods and virus strains). This relatively modest inhibitory effect is still impressive if we consider the non-toxic characteristic of this molecule (S⁴UMP). S⁴UMP is non-toxic in concentration of 1000 μg/ml (1 g/kg) intravenously injected to mice.

5. Study of toxicity

Toxicity of S⁴UMP was studied in a preliminary mouse experiment in vivo. The mice were treated intravenously one time. Weight of the mice was measured in the same time each day. The mice were anesthetized and examined in 30 days post injection.

The mice did not loose weight under treatment, but apparently felt poorly on the day of injection. On day 30 post injection pathological signs and structural changes were searched by morphological studies of liver, kidney, heart, brain, bone morrow, esophagus, gastric, muscles, and skin.

Surprisingly, the S⁴UMP was not toxic even in 1 g/kg(!) dose intravenously injected into mice. There was no morphological change of the examined organs on day 30. These apparently contradictory data (S⁴UMP is non-toxic, but apoptotic) can be explained by the observations that the molecule is metabolized, and the cells can regenerate after a longer incubation time (see experiments of the 2.4 section). When only small dose ofs⁴UMP or s⁴dU were added, the decrease of cell viability was moderated or disappeared after a short period of time (table 2.a - 2.e). The reason for this phenomenon may be the metabolism of the tested active agents.

6. Study on the mode of mechanism In order to get some information about the mode of action of S⁴UMP we studied the morphology of

OCM-I cells upon the treatment (Figure 4). The S⁴UMP decreased the cell number and the change cell morphology, suggesting that the treatment induced apoptosis.

All of the other cells were studied similarly and apoptotic changes were observed in each case. The most characteristic apoptotic morphological changes were observed in HL-60 cells. A characteristic sign of apoptosis is the degradation of DNA and the formation of the DNA-ladder with about 180 nucleotide increments. We treated OCM-I cells with S⁴UMP and after 24 and 48 hours DNA was isolated and analyzed with agarose-gel-electrophoresis. The DNA fragmentation was dependent on the length of treatment, and showed characteristic apoptotic degradation pattern (Figure 5).

In another experiment HL-60 cells were studied by treatment of the cells with s⁴dU and S⁴UMP and the DNA analyzed as described above. The characteristic DNA ladder was also observed here (Figure 6).

To further analyze the mode of action of S⁴UMP we studied the change of caspase-9 activity after 24, 48 and 72 hours of treatment, in HL-60, HeLa and OCM-I cells (Figure 7). The caspase-9 activity was doubled after 48 hours of treatment clearly indicating that S⁴UMP induced apoptosis.

After these experiments we determined the ratio of annexin-positive and negative cells before and after treatment, as well the ratio of propidium-iodide positive and negative cells. The results indicate that the main mode of action of S⁴UMP is the induction of apoptosis in the treated cells and necrosis may be induced only as a secondary event. Table 4

Change of the ratio of Annexin⁺ and Annexin-PI⁺* cells upon the treatment of S⁴UMP

*Annexin-Propidium-iodide

At the surface of apoptotic cells the phosphatidyl-serine appears (turning out from the inner surface) and binds annexin. The PI (propidium-iodide) dyes the DNA in necrotic cells, and the necrotic cells are also labeled with annexin in a certain stage of necrosis. It must also be noted that apoptosis may be followed by necrosis. Nevertheless, the data summarized in Figure 4 rather indicate an apoptotic processes.

7. Inhibition of glvceraldehyde-phosphate dehydrogenase (GAPDH) by S⁴UMP

An essential cysteine is located in the active centre of GAPDH. We measured the GAPDH activity in the presence of various concentrations of S⁴UMP and UMP. While the enzyme was inhibited by S⁴UMP, its closed structural analogue, UMP, was completely inactive on GAPDH. This experiment is a convincing one indicating that S⁴UMP is able to inhibit the function of -SH groups and suggests that its antiproliferative and anti-HIV activity is based on this mechanism. This assumption is in good agreement with the well known fact that nucleotides do not penetrate into the cells due to the polar phosphate group. Thus, S⁴UMP could exert its effect on the cell surface.

Table 5

8. Effect of combination of s UMP and adriamvcin

The above described experiments indicate that the S⁴UMP is not a highly aggressive agent but it has a rather wide spectrum of inhibitory activity on various tumor cells. We concluded from these results that it can advisably be utilized in combination with other cytotoxic agents. Using the above described methods we studied the inhibition of proliferation of THP-I cells with S⁴UMP in the presence of various concentrations of adriamycin. We found that 10 μg/ml S⁴UMP doubled the effect of low concentration (30 ng/mθ of adriamvcin. However, when high concentration of S⁴UMP was applied the effect of adriamycin decreased. The adriamycin has an effect on the cell membrane, too, thus, it could be assumed that in high concentration the two agents decreased the effects of each other by competing for the same binding sites on the membrane. This experiment support our hypothesis that S⁴UMP could be applied as a sensitizing agent in tumor therapy and also provides a new data verifying the assumed mechanisms.

It can also be concluded from this experiment that in a combination therapy it is advisable to use S⁴UMP together with agents exerting their inhibitory effects not on the cell membrane but acting rather inside the cells. As a matter of course, such treatments include radiotherapy.

9. Planning a chemotherapeutic protocol

When a treatment protocol for a tumor patient is devised a chemotherapeutic agent with well known mode of action exerting its inhibitory activity inside the cells should advisably be chosen. Such agents, for example, are alkylating agents, anti-metabolites, drugs interacting with DNA, inhibitors of topo-isomerases, drugs interacting with microtubules and drugs cause amino-acid depletion. Such agents are well known in the art [for example: Peter Nygren, Acta Oncologica, Volume 40, Number 2-3/ March 1, Pages: 166-174 (2001)].

During the therapeutic protocol the active agent should advisably be applied at a lower concentration than usual (for example 1/4 1/3, 1/2, 3/4 part of the usual amount and such quantities are considered during planning the protocol).

The thiouridine-derivatives described in this invention, for example S⁴UMP is added in an effective dose. The effective drug concentration can be determined in an appropriate cell-line model or in animal model. From these experiments we can conclude for the right dose mg/kg body weight. It is advisable to use initially low dose then it can be increased if necessary. Since in this application protocol the thiouridine derivative is utilized as a sensitizing agent, and it is metabolized after certain time, it is advisable to administer the thiouridine-derivative simultaneously with the chemotherapeutic agent or shortly before the application thereof to the patient.

Since the S⁴UMP is a nucleotide and relatively polar molecule, it is likely that it does not penetrate into the cells, rather it acts on the cell surface; therefore, its apoptosis inducing activity may be due to the interaction between the cell surface proteins and the modified nucleotide. This possible mechanism is chemically feasible since the 4-thiono group has a propensity toward tautomeric conversion [Simuth J et al. Biochim Biophys Acta, 204. 371-80 (1970)] to form reactive -SH group at position 4, which may be able to interact cell surface -SH containing proteins forming disulfide bridge.

Large number of -SH groups are present on the cell surface with a significance in the receptor functions. The reductive function of the cell surface is well known mediated by certain cell surface proteins including protein disulfide isomerase [Mandel R et al. Proc Natl Acad Sci USA 90, 4112-16 (1993); Terada

K et al. J Biol Chem 270, 20410-16 (1995)]. These -SH containing proteins are the most likely targets of

S⁴UMP.

We studied the inhibition of PDI by S⁴UMP. The experiment could not be performed properly because the S⁴UMP reacted with the substrate, also carrying -SH groups. However, we could show that S⁴UMP inhibits the glyceraldehyde-phosphate-dehydrogenase (GAPDH) enzyme, which contains an essential -SH group in its active centre. Furthermore, it was observed that insulin, also carrying -SH groups, interacts with the invented nucleotide. These observations make quite likely that the thio-uridine derivatives of this invention exert their antiproliferative activity by inhibiting the functions of cell surface thiols. It should be noted that the appearance of thioredoxin on the cell surface assumed as a marker of the propensity toward active cell proliferation [Hogg, PJ. Disulfide bonds as switches for protein function. Trends Biochem Sci. 28, 210-14 (2003)]. This is a further direction explaining the low toxicity of the compounds described herein beside their significant antiproliferative activity. This mechanism could also explain the anti-HIV activity of the compounds, since before the entry process the cell surface PDI reduces two disulfide bridges, out of nine, of the viral gpl20 protein. Furthermore, the second domain of CD4 receptor is temporally reduced during viral entry [Matthias LJ, Hogg PJ. Redox control on the cell surface: implication for HIV-I entry. Antioxid. Redox Signal. 5, 133-138 (2003)]. This reduction is performed by thioredoxin; the PDI itself is composed of thioredoxin domains. As we noticed earlier the (s⁴dU)₃₅, the deoxy-oligomer of 4-thio-uridylate, is able to form covalent interaction with cell surface thioredoxin [Hotvath, A. et al. Virology 334, 214-23 (2005)].

The DNA degradation, the activation of caspase-9, the FACS analysis is in good agreement with the assumed apoptotic mechanism, although, the exact mechanism of action is not known. Therefore, it should be emphasized that the above described mechanism must be considered as a well supported theory, but we are not wishing to be bond and limited by this theory; however, the knowledge of this theory may help the professional experts, to apply the invention in a wide scope of applications.

It is likely that the 4-thio-UMP does not produce biologically active degradation product because its degradation is initiated by loosing the sulfur spontaneously. The product formed is UMP (uridine- monophosphate). Before loosing the sulfur the dephosphorylation of the molecule is not likely, because, due to the chemical modification, it is not a good substrate of phosphatases. The UMP is a natural compound and is not toxic et all.

A FEW EXAMPLES FOR INDUSTRIAL APPLICATION OF THE INVENTION In the above described studies we used several types of tumor models, among them resistant, very aggressive tumor cell lines (For example, uveal melanoma) were applied, furthermore we examined tumor cells obtained from patients in order to get information about the effects of the invented compounds on tumor blast cells. The compounds described in this invention, and probably their many analogues and derivatives are anti-tumor agents, with minimal toxicity, actually we were not able to detect any toxic effects of them.

Considering the observed antiproliferative effects of the invented compounds on immortal cell lines, and their minimal toxicity proved by in vivo studies, the compounds can be applied for complex treatment of malignant proliferative processes.

The compounds of this invention could be utilized especially preferably as a supporting therapeutic agent to sensitize tumor cells. In this case they can suitably used together with other chemotherapeutic agents or radiation. The apoptosis inducing activity of these agents makes them excellent candidates for this propose. Here, it should be noted that according to best knowledge of the inventors this is the first report on the apoptosis inducing activity of S⁴UMP and related compounds.

The low toxicity is highly advantageous for those patients who could not tolerate the aggressive therapy, who are old, or are in co morbid state.

For the same reasons, the described treatment can excellently applied in low, malignancy chronic proliferative processes, thus, can be included preferably in the therapeutic protocols of chronic-leukemias. In the treatment of solid tumors, the compounds, described herein, can be utilized supporting the surgery, because their low side-effect-profϊles shortly after operation can be introduced.

According to their proved activity (see for example their activity against tumor stem cells), it is assumed that they could be utilized preferably to prevent the formation of metastasis and after surgery to eliminate the tumor cells from the circulating blood.

The compounds described in this invention, including S⁴UMP, inhibit the entry of those viruses, which require thiol <→ disulfide exchange during the entry process. Thus, compounds described in this invention exert anti-HIV activity.

Claims

1. A compound of general formula (I):

wherein in the formula above

- X is O, S, NH, CH₂ or nothing,

- W is O, S, NH or CH₂

- Q is O or S,

Ri, R₂, R₃ are, separately and independently from each other - H,

- H-[PO₃H]_n or any synthetic analogue thereof, wherein n = 1, 2 or 3,

- a substituted or unsubstituted C_1-10 organic moiety, preferably C ₁.₆ organic moiety, preferably alkyl, highly preferably substituted or not substituted methyl, ethyl, propyl, isopropyl, butyl, isobutyl or tert-butyl, or any unsaturated derivatives thereof,

- R₄ and R₅ are, separately and independently from each other H, substituted or not substituted C_1-10 organic moiety, preferably C₁^ organic moiety, more preferably substituted or not substituted methyl, ethyl, propyl, isopropyl, butyl, isobutyl, or tert-butyl, or any unsaturated derivative thereof,

- with the proviso that if Z and Y are nothing and R₂ and R₃ are H, then R₁ is a polar group, preferably a negatively charged group, highly preferably H-[PO₃H]_n or any synthetic analogue thereof, for use in therapy.

2. The compound according to claim 1 for use in therapy wherein in general formula (I) above

- Q, X and W are O,

- any one OfR₄ or R₅ is H and the other one is substituted or not substituted methyl, ethyl, propyl or iso-propyl, or any unsaturated derivative thereof; preferably methyl or ethyl, more preferably methyl, or

- both R₄ and R₅ are H.

3. The compound according to claim 1 for use in therapy wherein in general formula (I) any OfR₁, R₂ or R₃ is

H-[PO₃H]_n or a synthetic analogue thereof represented by any of general formulae Pl, P2 or P3,

Qi Q₁ Q₂ Il

B - P - (P1) B - P - A₁ - P - (P2) I I I OH OH OH Qi Q₂ Q₃ Il Il Il

B - P - A₁ - P - A₂- P - (P3),

S I I I

OH OH OH

wherein in the formulae (with reference to the respective formula) Qii Q₂ and Q₃ are, separately and independently from each other, O or S, 0 - B is -OH or methyl,

A₁, A₂, or A₃ is O or methyl; preferably, ,

Preferably, Ri, R₂ or R₃ is a monophosphoryl moiety, a diphosphoryl moiety or a triphosphoryl moiety.

4. The compound according to claim 3 for use in therapy, wherein said compound is any of the following: 5 - a ribo-mononucleotide, preferably

4-thio-UMP, 4-thio-UDP, 4-thio-UTP,

4-thio-3'UMP, 4-thio-3'UDP, 4-thio-3'UTP, 4-thio-2'UMP, 4-thio-2'UDP, 4-thio-2'UTP,

4-thio-TMP, 4-thio-TDP, 4-thio-TTP,

4-thio-3'TMP, 4-thio-3'TDP, 4-thio-3'TTP, 4-thio-2'TMP, 4-thio-2'TDP, 4-thio-2'TTP, - a deoxyribo-mononucleotide, preferably

4-thio-dUMP, 4-thio-dUDP 4-thio-dUTP

4-thio-3'dUMP, 4-thio-3'dUDP, 4-thio-3'dUTP

4-thio-dTMP, 4-thio-dTDP, 4-thio-dTTP

4-thio-3'dTMP, 4-thio-3'dTDP, 4-thio-3'dTTP; - a 2 ' ,3 ' -dideoxyribo-mononucleotide, preferably

4-thio-ddUMP, 4-thio-ddUDP, 4-thio-ddUTP

4-thio-ddTMP, 4-thio-ddTDP, 4-thio-ddTTP.

5. The compound according to any of claims 1 or 2 for use in therapy, wherein in general formula (I) Ri, R₂ and R₃ is, separately and independently from each other H, or substituted or not substituted methyl, ethyl, propyl or isopropyl, or any unsaturated variant thereof, with the proviso that

- at least one of X-Ri, Z-R₂ and Z-R₃ is OH,

- if Y is nothing, then R₂ is H, and if Z is nothing then R₃ is H.

6. The compound of claim 5 for use in therapy, wherein said compound is a nucleoside, preferably any of the following: 4-thio-uridine, 4-thio-deoxy-uridine, 4-thio-thymidine, 4-thio-deoxy-thymidine.

7. The compound according to any of claims 1 to 6

- for use in the treatment of diseases accompanied by cell proliferation, preferably in the treatment of tumour diseases and malignancies; or

- for use as antiviral agents, preferably against HIV infection.

8. Use of a compound of general formula (I) for the preparation of a medicament or a therapeutic composition for inhibiting cell proliferation,

wherein in the formula above - X is O, S, NH, CH₂ or nothing,

Q is O or S,

R₁, R₂, R₃ are, separately and independently from each other - H,

- a substituted or unsubstituted Ci.₎₀ organic moiety, preferably C_1-6 organic moiety, preferably alkyl, highly preferably substituted or not substituted methyl, ethyl, propyl, isopropyl, butyl, isobutyl or tert-butyl, or any unsaturated derivatives thereof, - R₄ and R₅ are, separately and independently from each other H, substituted or not substituted C_1-I0 organic moiety, preferably Ci_-6 organic moiety, more preferably substituted or not substituted methyl, ethyl, propyl, isopropyl, butyl, isobutyl, or tert-butyl, or any unsaturated derivative thereof.

9. The use according to any of claim 8, wherein the compound is any of the following:

4-thio-deoxy-uridine, 4-thio-dUMP, 4-thio-dUDP 4-thio-dUTP

4-thio-3'dUMP, 4-thio-3'dUDP, 4-thio-3'dUTP

4-thio-dideoxy-uridine, 4-thio-ddUMP, 4-thio-ddUDP, 4-thio-ddUTP

4-thio-thymidine, 4-thio-TMP, 4-thio-TDP, 4-thio-TTP, 4-thio-3'TMP, 4-thio-3'TDP, 4-thio-3TTP, 4-thio-2'TMP, 4-thio-2'TDP, 4-thio-2'TTP,

4-thio-deoxy-thymidine, 4-thio-dTMP, 4-thio-dTDP, 4-thio-dTTP

4-thio-3'dTMP, 4-thio-3'dTDP, 4-thio-3'dTTP;

4-thio-dideoxy-thymidine, 4-thio-ddTMP, 4-thio-ddTDP, 4-thio-ddTTP.

10. The use according to any of claims 8 or 9 for the preparation of a medicament or therapeutic composition in the treatment of malignant conditions, e.g of solid tumour or leukemia.

11. The use according to any of claims 8 to 10, wherein the compound of general formula (I) is used in combination with a further agent useful in the treatment of the malignant condition, preferably in combination with a chemotherapeutic agent.

12. A therapeutic composition or a therapeutic kit comprising the compound of general formula (I) as defined in claim 8 or 9 in combination with a cell killing agent, e.g. an agent for treating a malignant condition, preferably a chemotherapeutic agent together with an other pharmaceutically acceptable excipient, wherein optionally the active compounds or agents are formulated as individual units.

13. A kit for inducing apoptosis, wherein said kit comprises the compound of general formula (I) as defined in claim 8 or 9.