WO2009053654A2

WO2009053654A2 - Gemcitabine phosphoester prodrugs as anticancer agents

Info

Publication number: WO2009053654A2
Application number: PCT/FR2008/051874
Authority: WO
Inventors: Christian Perigaud; Suzanne Peyrottes; Charles Dumontet
Original assignee: Hospices Civils De Lyon
Priority date: 2007-10-17
Filing date: 2008-10-17
Publication date: 2009-04-30
Also published as: FR2922551A1; WO2009053654A3; FR2922551B1

Abstract

The present invention relates to gemcitabine phosphoester prodrugs with antitumour activity, of formula (I) in which n, Y, Z and Ri are as defined in claim 1, and also the pharmaceutical compositions comprising such a compound, in combination with at least one pharmaceutically acceptable excipient, and the use of such a compound for preparing an antitumour or anticancer medicament.

Description

PRODRUGS PHOSPHOESTERS OF THE MCITABI G E E AS ANTI-CANCER AGENTS

The present invention relates to novel prodrugs of gemcitabiπe useful as anti-cancer agents.

Gemcitabine, an anti-cancer nucleoside, of formula:

is administered in an unphosphorylated form, but is phosphorylated in vivo in the form of active monophosphate, diphosphate or triphosphate metabolites.

Gemcitabine has recently been added to the anti-cancer pharmacopoeia and is currently widely used in the treatment of lung, pancreatic or bladder cancers.

The mode of action of this therapeutic agent requires, after its membrane penetration, an intracellular metabolism in its phosphorylated forms (nucleotides), which then interfere with various enzymatic systems involved in nucleic acid biosynthesis. Gemcitabine can be administered intravenously, combined with other anticancer agents with different mechanisms of action. The chemotherapy "cures" are administered at intervals varying according to the indications: one to three courses of high intensity in case of acute leukemia, 6 to 12 treatments administered every four weeks in the majority of other indications. Patients receiving gemcitabine for treatment of a solid tumor will benefit in a number of cases from these treatments, but this is never a cure and all patients will die of their disease.

In addition, the clinical use of gemcitabine comes up against the appearance of resistance phenomena. The mechanisms involved in resistance to gemcitabine can not be reduced to the expression of a single enzymatic system. These mechanisms, although poorly known today, are of multifactorial origin. The last decade has seen the cloning of several genes key factors involved in the cytotoxic action of this compound: the balanced (hENT) and concentrative (hCNT) transporters of nucleosides, the inactivating enzymes such as 5'-nucleotidases and other proteins. During the same period, studies carried out on lines made resistant in vitro to cytotoxic nucleoside analogues and on samples from patients with hematological malignancies or solid tumors made it possible to identify parameters of clinical importance. The "early" steps leading to the intracellular accumulation of active triphosphate derivatives can thus be modified in patients with neoplasias and be at the origin of chemoresistance phenomena to nucleoside analogues. A deficit of hENT1, the main transporter of gemcitabine, is thus associated with a lack of anticancer activity of these drugs in patients with pancreatic cancer. A deficiency of deoxycytidine kinase (dCK, an enzyme involved in the monophosphorylation of gemcitabine analogues) has been observed by many investigators in various resistance models, both in resistant lines in vitro, and in patients with leukemia. acute myeloid (AML) or chronic lymphatic leukemia (CLL) and not responding favorably to treatment.

Extra-or intracellular catabolism (deamination), lack of membrane penetration, or ineffective anabolism (phosphorylation) are all limitations to the activity of gemcitabine. In the absence of an adequate intracellular concentration in its phosphorylated forms, it is indeed not possible to obtain a satisfactory antimitotic activity.

The inventors have demonstrated, in the context of previous publications "Sensitization of ara-C-resistant lymphoma cells by a similar pronucleotide". CM. Galmarini, ML Clarke, CL. Santos, L. Jordheim, C. Périgaud, G. Gosselin, E. Cros, 3. R. Mackey and C. Dumontet. IntemationaIJournai of Cancer, 2003, 1OZ (1), 149-154; Characterization of a gemcitabine-resistant murine leukemic cell line: reversion of in vitro resistance by a mononucleotide prodrug. LP Jordheim, E. Cros, M.-H. Gouy, CM. Galmarini, S. Peyrottes, JR Mackey, C. Périgaud and C Dumontet. C / Inica / Cancer Research, 2004, 10 (16), 5614-5621), that the in vitro use of nucleotide prodrugs (pronucleotides) incorporating two enzymolabile groups protecting the phosphate function of the same nature and of the S-acyl-2-thioethyl type (SATE), such as the compound of formula (A) below:

Nu: analog nucleoside

mononucleoside bis (SATE) phosphotriester

led to the selective intracellular release of the first phosphorylated meta-bolide (mononucleotide) of a nucleoside analog.

By way of example of this type of compound, mention may be made of the pronucleotide of araC incorporating, as enzymolabile protections for the phosphate function, the S-pivaloyl-2-thioethyl (.BuSATE) group of structure (B).

The comparative evaluation of this pronucleotide (B) in two cell lines resistant to araC and gemcitabine has demonstrated its ability to bypass, at least in part, the cellular resistance processes. This result is associated with the selective release of 5'-mononucleotide (araCMP) within the tumor cells leading to a restoration of intracellular levels in phosphorylated forms.

Nevertheless, pharmacological studies carried out in animals with a certain number of pronucleotides bis (SATE), have shown the relative enzymatic instability of this type of structures when administered orally. This pre-systemic degradation may be associated with the presence of significant ester activity in the entire digestive tract and liver. These studies also demonstrated the transient formation of a 2-mercaptoethylphosphodiester, as shown in SCHEME 1 below, which shows the pronucleotide decomposition process bis (SATE) and the metabolites observed in pharmacological studies in different animal models. SCHEME 1 l ^ene step (fast): loss of the first array enzymolabile

SATE I I

"Chemical decomposition _Λ _ fl fl fi spontaneous esterase activity

OSATE O ^"

5'-mononucleotide 2-mercaptoethyl phosphodi ester

O protein conjugates (dalrance)

This 2-mercaptoethylphosphodiester is derived from the decomposition of the second SATE group, and can lead to protein conjugates via the likely creation of disulfide bridges. The identification of this phosphodiester as one of the main metabolites in the administration of bis pronucleotides (SATE) reflects a decrease in the rate of SN, leading to the 5'-mononucleotide, associated with the presence of the negative charge of the phosphate function.

The invention proposes to provide novel prodrugs of gemcitabine which make it possible to avoid a number of the limitations associated with the emergence of resistance during the use of this cytotoxic nucleoside analogue, by being:

- resistant to chemical and enzymatic degradation (catabolism);

- likely to cross the cell membranes by a process not dependent on carriers (simple diffusion);

capable of releasing intracellularly the first phosphorylated metabolite (mononucleotide) independently to the expression of cellular kinases (metabolism).

In the context of the invention, the inventors have developed new prodrugs of gemcitabine with anti-tumor activity, called "mixed", which characterized by the presence of two enzymolabile groups protecting the phosphate function of different nature.

In particular, the subject of the invention is the mixed phosphoester compounds, with anti-tumor activity, of formula (I):

in which :

n is 1, 2, 3 or 4,

Z represents O or S

Y represents a group chosen from alkyl or aryl groups, said alkyl or aryl groups possibly being substituted, for example by one or more substituents chosen from -OH, -SH or -NH ₂ ,

- Ri represents a group -NRaRb in which Ra and Rb, identical or different, represent a hydrogen atom, an alkyl or aryl group, said alkyl or aryl groups possibly being substituted, or R ₃ and R _b are linked between to form, with the nitrogen atom to which they are bonded, a cyclic amine selected, for example, from pyrrolidinyl, piperidinyl, morpholynyl, piperazinyl, pyridyl optionally substituted.

By way of example according to the invention, the compounds of formula (I) may have one or more of the following characteristics, when they do not exclude each other:

Y represents a methyl or tetf-butyl group,

n is equal to 2,

R 1 represents a group -NR ₃ R _b with R ₃ and R _b as defined for formula (I). In particular, R ₃ represents a hydrogen atom and R _b represents a benzyl or substituted benzyl, isopropyl or n-butyl group,

The present invention also relates to pharmaceutical compositions comprising a compound of formula (I) as defined previously, in combination with at least one pharmaceutically acceptable excipient.

According to another of its aspects, the invention relates to the use of a compound of formula (I) as defined above for the preparation of an anti-tumor or anti-cancer drug, in particular intended for treatment or prevention benign tumor lesions, or cancers, including haematological malignancies and solid tumors, including tumors of glandular, mesenchymal, genital, cutaneous and neurological origin.

In the context of the invention, certain definitions of the terms used are given below:

Alkyl means a linear or branched saturated hydrocarbon monovalent radical containing, unless otherwise specified, from 1 to 10 carbon atoms, preferably from 1 to 4 carbon atoms. By way of example, mention may be made of methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, sec-butyl, n-pentyl and n-hexyl groups.

By aryl group is meant a mono-, bi- or polycylic carbocycle comprising at least one aromatic group. Aryl groups comprising from 6 to 12 carbon atoms are preferred. As the aryl, there may be mentioned phenyl, 1-naphthyl, 2-naphthyl, indanyl, indenyl, biphenyl, bicyclo [4.2.0] octa-1,3,5-triene, phenyl groups being preferred.

When it is indicated that a group is optionally substituted, without more precision, it means that it carries one or more identical or different substituents, in particular chosen from halogens, nitrooxocarboxy, cyano, alkyl, trifluoroalkyl, alkenyl, alkynyl, cycloalkyl, aryl, heterocycloalkyl, amino, alkylamino, dialkylamino, hydroxy, O-alkyl, O-aryl. By halogen atom is meant a chlorine, bromine, iodine or fluorine atom. The term alkenyl corresponds to an alkyl group as defined above comprising at least one double bond. Examples of alkenyl groups are, for example, vinyl, allyl, isopropenyl, 1-, 2- or 3-butenyl, pentenyl, hexenyl. The term alkynyl corresponds to an alkyl group as defined above comprising at least one triple bond. The term cycloalkyl denotes a cyclic saturated hydrocarbon chain comprising from 3 to 10 carbon atoms, for example cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, bridged cycloalkyl groups such as adamantyl, bicyclo [3.2.1] octanyl groups. The term heterocycloalkyl means a cycloalkyl as defined above, incorporating one or more heteroatoms selected from nitrogen, oxygen and sulfur. Exemplary heterocycloalkyl groups include piperidine, pyrrolidine, azetidine, morpholine, tetrahydropyran, tetrahydrothiophene.

Also, benzyl and substituted benzyl are examples of substituted alkyl groups.

By way of example of a compound according to the invention, mention may be made of prodrugs of gemcitabine incorporating an S-pivaloyl-2-thioethyl (/ BuSATE) group and an amine residue derived from 7-butylamine of formula (1.1) , of Isopropylamine of formula (1.2), benzylamine of formula (1.3):

and also compounds incorporating a ^L (2,2-dimethyl-3-hydroxypropionyl) -2-thioethyl (Hydroxy-fBuSATE) moiety and an amino residue derived from benzylamine of formula (1.4):

In the case of the compounds according to the invention as defined for formula (I), a corresponding 5'-mononucleotide decomposition is observed, under the successive action of esterases, then probably of a "phosphoramidase activity" allowing avoid the formation of metabolites capable of covalently associating with serum proteins.

Moreover, the great structural variability that can be brought about by the nature of the - {CH2) nSC (= Z) Y groups as Ri substituents makes it possible to modulate the enzyme stability of the prodrug in relation to its lipophilicity and thus to optimize its pharmacokinetic properties. . In particular, the choice of the groups R 1 and, for example, of the group -NR ₃ R _b selected makes it possible to modulate the physicochemical properties of the compound of formula (I), in particular its solubility in water and its lipophilicity, and its stability in function of the affinities of phosphoramidases.

The compounds of formula (I) according to the invention may be used as anti-cancer agents. Indeed, the compounds according to the invention are insensitive to intracellular diffusion or activation problems, which makes it possible to broaden their spectrum of indications with respect to conventional cytotoxic nucleosides and significantly increase their efficacy.

In general, the compounds according to the invention may be used to prepare a medicament for the treatment, as a curative or preventive, of any type of cancer or precancerous state. In particular, the compounds of formula (I) may be used for the manufacture of anti-tumor or anti-cancer drugs, in particular for the treatment or prevention of benign tumor lesions, or cancers, including haematological malignancies and solid tumors. , including tumors of glandular, mesenchymal, genital, cutaneous and neurological origin. Mention may be made, as cancers that can be treated with the compounds of the present invention, of carcinomas, hematological malignancies of the myeloid and lymphoid lineages, tumors of mesenchymal origin, sarcomas, tumors of the central and peripheral nervous system, melanomas, seminomas, teratocarcinomas, osteosarcomas, xeroderma pigmentosum, chératoacantum, endocrine neoplasias, and Kaposi's sarcoma.

The subject of the present invention is therefore also the compounds of formula (I), as medicaments, as well as pharmaceutical compositions containing an effective dose of a compound of formula (I), and suitable excipients.

Said excipients are chosen according to the pharmaceutical form and the desired mode of administration. Acceptable pharmaceutical excipients being, for example, defined by the European Pharmacopoeia.

In the pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, topical, intratracheal, intranasal, transdermal, rectal or intraocular administration, the active ingredients of formula (I) above, may be administered in unit dosage forms, in admixture with conventional pharmaceutical carriers, to animals and humans for the prophylaxis or treatment of the above disorders or diseases. Suitable unit dosage forms include oral forms such as tablets, capsules, powders, granules and oral solutions or suspensions, sublingual, buccal, intratracheal, intranasal, dosage forms. subcutaneous, intramuscular or intravenous administration and forms of rectal administration. For topical application, the compounds according to the invention can be used in creams, ointments, lotions or eye drops.

In order to obtain the desired prophylactic or therapeutic effect, each unit dose may contain from 0.1 to 10,000 mg of compound according to the invention in combination with a pharmaceutical carrier. This unit dose can be administered 1 to 5 times per day so as to administer a daily dosage to obtain the desired effect.

When preparing a solid composition in tablet form, the main active ingredient is mixed with a pharmaceutical carrier, such as gelatin, starch, lactose, magnesium stearate, talc, gum arabic or the like. The tablets can be coated with sucrose, a cellulose derivative, or other suitable materials or they can be treated in such a way that they have prolonged or delayed activity and continuously release a quantity of predetermined active ingredient.

A preparation in capsules is obtained by mixing the active ingredient with a diluent and pouring the resulting mixture into soft or hard gelatin capsules.

The pharmaceutical compositions containing a compound of the invention may also be in liquid form, for example, solutions, emulsions, suspensions or syrups. Suitable liquid carriers may be, for example, water, organic solvents such as glycerol or glycols, as well as their mixtures, in varying proportions, in water.

A preparation in the form of syrup or elixir or for administration in the form of drops may contain the active ingredient together with a sweetener, preferably acaloric, methylparaben and propylparaben as antiseptic, as well as a flavoring agent. and a suitable dye. Water-dispersible powders or granules may contain the active ingredient in admixture with dispersants or wetting agents, or suspending agents, such as polyvinylpyrrolidone, as well as with sweeteners or scavengers disgust.

For rectal administration, suppositories are used which are prepared with binders melting at the rectal temperature, for example cocoa butter or polyethylene glycols. For parenteral administration, aqueous suspensions, isotonic saline solutions or sterile and injectable solutions which contain pharmacologically compatible dispersing agents and / or wetting agents, for example propylene glycol or butylene glycol, are used. The active ingredient may also be formulated in the form of microcapsules, optionally with one or more supports or additives, or with matrices such as a polymer or a cyclodextrin (patch, sustained-release forms).

The compositions of the present invention may contain, in addition to the products of formula (I), for example, active ingredients which may be useful in the treatment of the disorders or diseases indicated above. Thus, the subject of the present invention is also pharmaceutical compositions containing several active ingredients in combination, one of which is a compound according to the invention.

The invention therefore relates to their use as medicaments that can be used alone or in combination with treatments such as chemotherapy or radiotherapy possibly implementing other active substances.

Furthermore, in general, the same preferences as those indicated above for the compounds of general formula (I) are applicable mutatis mutandis to the drugs, pharmaceutical compositions and use using the compounds according to the invention.

The examples below have no limiting character and allow to illustrate the invention. The compounds according to the invention are prepared according to techniques well known to those skilled in the art. One of his methods is illustrated in Scheme 2 below wherein n, Y and Z are as defined for compounds of formula (I), R 'is a hydroxy protecting group and R' is a protecting group of the amino function:

SCHEME 2

The hydroxyl functions carried by the osidic residue (sugar) and the exocyclic amine carried by the heterocycles of these nucleosides are transiently protected in the intermediates (II) and (Ia). The condensation of the compound (III) with these suitably protected nucleosides leads to the derivatives (II). The amidative oxidation of these precursors with derivatives of the HNR ₉ R _{b type} thus generates totally protected prodrugs, noted (Ia). A last step of hydrolysis of the protective groups is then necessary to lead to the desired prodrugs (I).

The starting compounds (IV) are prepared according to techniques well known to those skilled in the art.

This process is illustrated in the following examples.

EXAMPLE 1

Preparation of the orodroαue of αerncitabine of formula fl.ll:

Intermediate (IV.1): S-pivaloyl-2-thioethanol is obtained according to a protocol described in an earlier publication, I. Lefevbre, C. Périgaud et al., Journal of Medicinal Chemistry, 1995, 38, (20), 3941.

Intermediate (III.1): .9 Pivaloyl-2-thioethyl hydrogenophosphonate monoester, sodium salt

S-pivaloyl-2-thioethanol (IV, 1. 2.7 g, 16.76 mmol) is dissolved in a mixture of anhydrous pyridine (16 mL) and dioxane (50 mL). To the resulting solution is added dropwise a solution of 2-chloro-5,6-benzo-1,2,3-dioxaphosphorin-4-one (4.0 g, 20.00 mmol) in anhydrous dioxane. (16 mL). After stirring for 45 minutes at room temperature, a solution of triethylammonium bicarbonate (TEAB, 8 mL, 1M, pH 7) is added to the mixture. reaction. After stirring for 30 minutes, the reaction medium is concentrated under reduced pressure and two co-evaporations with toluene are carried out. The residue obtained is purified by column chromatography on silica gel (dichloromethane / triethylamine, 99/1, v / v) using as eluent a gradient of 0 to 10% methanol in dichloromethane containing 1% triethylamine. After passing through a column of Dowex resin (50 WX 2, Na ⁺ form) and lyophilization, the sodium salt of the compound (III.1) is obtained in the form of a white powder (2.7 g, 64%).

¹ H NMR (DMSOd ₆ , 400 MHz) δ 6.53 (d, 1H, Jp, _H = 573, pH), 3.62 (m, 2H,

CH ₂ O) ₁ 2.96 (t, 2H, J = 6.6, SCH _2), 1.16 (s, 9H, C (CH ₃ J ₃₎

¹³ C NMR (DMSO-dn, 100 MHz)? 205.6 (Is, C = O), 60.9 (Id, CH ₂ O) ₁ 45.9 (Is,

C (CH ₃ J ₃ ), 29.3 (Id, 5 CH ₂ ), 27.0 (Is, C (CH ₃ ) ₃ )

³¹ P NMR (DMSO-d, 81 MHz) δ 2.3

FAB MS> 0 (GT) / 77 / z497 (2M + H) ⁺ , 271 (M + Na) ⁺ , 249 (M + H) ⁺

FAB <0 (GT) m / z 473 (2M-Na) ^', 225 (M-Na) ^{"Microanalysis} Calcd for C ₇ Hi ANSP ₄ O _4: C: 33.87; H, 5.68; S: 12.92

Found: C, 33.79; H, 5.69; S: 12.90

When synthesizing the <> (5 ^: pivaloyl-2-thioethyl) hydrogenophosphonate monoester, it is the triethylammonium salt which is obtained after the chromatographic column on silica gel and which can be used directly in the coupling reactions. This triethylammonium salt is converted into its soda form by passage over a Dowex-type ion exchange resin in order to obtain the physicochemical characterizations.

Intermediate (IL1): 0 (5 ^L pivaloyl-2-thioethyl) -α5 '- (yv-4-O3'-bis (tert-butoxycarbonyl) gemcitabine hydrogenphosphonate diester

/ V-4-α-3'-bis (tert-butoxycarbonyl) gemcitabine (obtained according to ZW Guo, JM GaIIo, J. Org. Chem., 1999, 64, 8319) (0.5 g, 1, 08 mmol) and O- (5-pivaloyl-2-thioethyl) hydrogenophosphonate monoester, triethylammonium salt (III.1) (0.7 g, 2.16 mmol) are co-evaporated with pyridine and then dissolved in anhydrous pyridine (14 ml) and then pivaloyl chloride (0.33 ml, 2.69 mmol) are then added dropwise. After 12h, the reaction is stopped by addition of ammonium acetate (1 ml, 0.5 M), the reaction mixture is diluted in dichloromethane (10 ml) and then washed with ammonium acetate (10 ml, 0.01 M). The organic phase is dried over Na 2 SO 4, filtered and then evaporated under reduced pressure to give a yellow oil which is then coevaporated 3 times with toluene. Purification by column chromatography, using a dichloromethane / ethyl acetate mixture, 8/2, V / V + 0.2% acetic acid, as eluent, gives C3- (5-pivaloyl-2). -thioethyl) -05 '- (/ V-4-O-3'-bis (tert-butoxycarbonyl) gemcitabine hydrogenphosphonate diester (IL1) (0.2 g, 36%) as a colorless oil .

¹ H NMR (CDCl ₃ , 300 MHz) δ 7.77 (m, 1H, H-6), 7.23 (m, 1H, H-5), 6.40 (m, 1H, H-10, 6-89 (d, 1H). ,% _w = 720, pH), 5.08 (m, 1H, H-30, 4.39 (m, 2H, H-5 'and H-5 "), 4.27 (m, 1H, H-40, 4.11 (pq, 2H, J = 6.6, CH ₂ O), 3.08 (t, 2H, J = 6.6, CH ₂ S), 1.44 (s, 18H, CH ₃ Boc), 1.17 (s, 9H, CH ₃ SATE)

¹³ C NMR (CDCl ₃ , 75 MHz) δ 204.5 (CO SATE), 162.3 (C-4), 153.4 (CO Boc), 150.4 (C-2), 150.0 (CO Boc), 143.4 (C-6), 122.7, 119.2, 115.7 (C-20, 94.8 (C-5), 84.3 (C [CH ₃ J ₃ ), 83.4 (C-10, 81.9 (C [CH ₃ ) ₃ ), 77.1 (C-40, 71.2 ( C-30, 63.6 (CH ₂ O), 62.2 (C-50, 45.5 (C (CH ₃ J _3), 28.3 (CH ₂ S), 27.2 (CH _3), 26.4 (CH ₃₎ ³¹ P NMR (CDCl ₃ , 121 MHz) δ 8.0, 8.9 FAB SM> 0 (GT) m / z 672 (M + H) ⁺ , 408 (M-GemC) ⁺

FAB <0 (GT) m / z 670 (MH) ^', 526 (M- tBuSATE) ^"

Intermediate (Ia.l): N- (Λ-butylamino) -O (5 ^L -pivaloyl-2-thioethyl) -> 5 '- (/ V-4-O 3'-bis (tert-butoxycarbonyl) gemcitabine phosphoramidate diester 0- (Spivaloyl-2-thioethyl) -0-5 '- (/ V-4-O-3'-bis (f) -butoxycarbonyl) gemcitabine hydrogenphosphonate diester (IL1) (0.6 g) 0.894 mmol) is co-evaporated twice with anhydrous pyridine and then dissolved in carbon tetrachloride (22 ml) .N-butylamine (0.27 ml, 2.68 mmol) is added to this solution. The reaction mixture is stirred at room temperature After 15 minutes, the reaction mixture is diluted with dichloromethane (86 ml) and washed with a hydrochloric acid solution (86 ml, 0.1 N, pH = 1) until the reaction mixture is complete. that the pH of the aqueous solution is acidic (pH≈l) The organic phase is neutralized with an aqueous solution of NaHCO ₃ and then washed with water (86 ml) and dried over Na ₂ S0 ₄ , then filtered and concentrated under reduced pressure. Purification by column chromatography, eluting with ethyl acetate / petroleum ether (7/3, v / v) gave 5'-isopropylamino-L (5-valivaloyl-2-thioethyl) -0-5 ' (1H-α) -3'-bis (tert-butoxycarbonyl) gemcitabine phosphoramidate (Ia.1) (0.550 g, 83%) as a colorless oil.

¹ H NMR (CDCl ₃ , 400 MHz) δ 7.81 (dd, 2H, J = 7.1, H-6 and NH), 7.24 (d, 1H, J = 7.1, H-5), 6.38 (m, 1H, H -IO, 5.12 (m, 1H, H-30, 4.25 (m, 3H, H-4 ', H-5' and H-5 "), 4.02 (m, 2H, CH ₂ O), 3.08 (m, 2H, CH ₂ S), 2.87 (m, 3H, CH ₂ N and NH), 1.41 (m, 2OH, OZ ₂ CH ₂ N and CH ₃ Boc), 1.27 (m, 2H, CH ₂ CH 3), 1.21 ( s, 9H, CH ₃ SATE), 0.86 (m, 3H, CH ₃ / Bu)

¹³ C NMR (CDCl ₃ , 100 MHz) δ 205.7 (CO), 163.2 (C-4), 154.5 (C-2), 151.4 (CO Boc), 150.9 (CO Boc), 144.5 (C-6), 123.0 , 120.4, 117.8 (C-20, 95.6 (C-5), 84.7 (αCH ₃₎ _3), 84.6 (C-IO, 83.0 (C (CH ₃ J _3), 78.4 (C-40, 72.6 (C- 30, 65.0 (CH ₂ O), 63.7 (C-50, 46.5 (3CH ₃ J ₃ ), 41.2 (CH ₂ N), 31.7 (OH ₂ CH ₂ N), 28.7 (CH ₂ S), 27.9, 27.5, 27.3 (CH ₃ Boc and tBu), 19.7 (CH ₂ CH ₃ ), 13.7 (OH ₃ CH ₂ ) ³¹ P NMR (CDCl ₃ , 121 MHz) δ 9.6, 9.4 FAB MS> 0 (GT) m / z 743 ( M + H) ⁺ , 643 (M-BoC) ⁺

FAB <0 (GT) m / z 741 (MH) ^', 597 (M-tBuSATE) -, 523 (M-tBuSATE- nBuNH) ^", 497 ^{(M-tBuSATE-Boc)"}

Prodrug (1.1): / V - (/> butylamino) -O (5-pivaloyl-2-thioethyl) -05'-gemcitabine phosphoramidate diester

To a stirred solution of Λ- (2-butylamino) -C - (S-pivaloyl-2-thioethyl) -C> -5 '- (Λ) -4 -3'-bis (tetf-butoxycarbonyl) gemcitabine phosphoramidate (Ia.l) (0.55 g, 0.74 mmol) in dry dichloromethane (7 ml), a solution of TFA (7 ml, 50% in dichloromethane v / v) is added dropwise at ^0.degree. C. The reaction is complete after 1 hr at room temperature The reaction mixture is co-evaporated twice with CH ₂ Cl ₂ and 4 times with ethanol The purification of the residue on a RP18 column using gradient as eluent from 20% to 50% by volume of CH ₃ CN / H ₂ O makes it possible to obtain the desired derivative (1.1) (0.264 g, 66%) in the form of a white solid. ¹ H NMR (DMSOcI ₆ , 400 MHz) δ 7.55 (d, 1H, J = 7.5, H-6), 7.50, 7.45 (2s, 2H, NH 2), 6.44 (t, 1H, J = 6.1, OH), 6.18 (t, 1H, J = 8.0, HI ⁷ ), 5.80 (dd, 1H, J = 7.5, H-5), 5.11 (m, 1H, NH), 4.13 (m, 3H, H-3 ', H And 5.01 (m, 2H, CH ₂ O), 3.09 (t, 2H, J = 6.4, CH ₂ S), 2.75 (m, 2H, CH ₂ N), 1.37 (m, 2H, OZiCH ₂ N), 1.27 (m, 2H, CH ₂ CH ₃ ), 1.18 (s, 9H, CH ₃ tBu), 0.84 (t, 3H, J = 7.3 , CH ₃ nBu)

¹³ C NMR (DMSO-dd, 100 MHz) δ 205.2 (CO), 165.4 (C-4), 154.3 (C-2), 141.0 (C-6), 126.0, 122.6, 119.2 (C-20, 94.7 ( C-5), 83.8 (C-10, 78.3 (C-40, 69.4 (C-3 '), 63.7 (C-5' and CH ₂ O), 45.9 (αCH ₃ ) ₃ ), 40.3 (CH ₂ N) ), 33.3 (CH ₂ CH ₂ N), 28.4 (CH ₂ S), 26.8 (CH ₃ tBu), 19.2 (CH ₂ CH ₃ ), 13.6 (CH ₃ nBu)

³¹ P NMR (DMSO-de, 121 MHz) δ 10.7, 10.0

FAB MS> 0 (GT) m / z 534 (M + H) ⁺ , 399 (M-tBuSATE) ⁺

FAB <0 (GT) m / z 541 (MH) ^"

EXAMPLE 2

Preparation of the prodroul of qemcitabine of formula fl.2 ^:

Intermediate (IA.2): / V "(isopropylamino) - (> (S ^L pivaloyl-2-thioethyl) -05 '- (/ V-4-0-3'-bis (te / i-butoxycarbonyl) gemcitabine phosphoramidate diester O (S-pivaloyl-2-thioethyl) - (-> 5 '- (/ V-4-C>-3'-bis (tert-butoxycarbonyl) gemcitabine hydrogenphosphonate diester (IL1) (0.6 g, 0.894 mmol) is co-evaporated twice with anhydrous pyridine and then dissolved in carbon tetrachloride (22 ml) to this solution, isopropylamine (0.23 ml, 2.68 mmol) is added The reaction mixture is stirred at room temperature After 1H15, the reaction mixture is diluted with dichloromethane (86 mL) and washed with hydrochloric acid solution (86 mL, 0.1 N, pH = 1). until the pH of the aqueous solution is acidic (pH≈l) The organic phase is neutralized with an aqueous solution of NaHCO 3 and then washed with water (86 ml) and dried over Na ₂ SO ₄ , then filtered and concentrated under reduced pressure. Purification by column chromatography, eluting with ethyl acetate / petroleum ether (7/3, v / v), gives Λ-isopropylamino - (> (5-pivaloyl-2-thioethyl) -> 5 '(4H-3'-bis (tert-butoxycarbonyl) gemcitabine phosphoramidate (Ia.2) (0.323 g, 50%) as a colorless oil.

¹ H NMR (CDCl ₃ , 400 MHz) δ 7.81 (dd, 2H, J = 7.6, H-6 and NH), 7.21 (m, 1H, H-5), 6.34 (m, 1H, HI ¹ ), 5.17. (m, 1H, H-30, 4.24 (m, 3H, H-4 ', H-5' and H-5 "), 4.01 (m, 2H, CH ₂ O), 3.32 (m, 1H, NCH) 3.08 (m, 2H, CH ₂ S), 2.75 (m, 1H, NH), 1.44 (s, 18H, CH ₃ Boc), 1.16 (s, 9H, CH ₃ SATE), 1.10 (m, 6H, CH). ₃ iPr)

¹³ C NMR (CDCl ₃ , 100 MHz) δ 205.7 (CO), 163.1 (C-4), 154.5 (C-2), 151.4 (CO Boc), 150.9 (CO Boc), 144.5 (C-6), 122.9 , 120.4, 117.7 (C- ²⁷ ), 95.6 (C-5), 84.7 (αCH ₃ ) ₃ ), 84.0 (C-10, 83.0 (4CHs) ₃ ), 78.3 (G40, 72.6 (C-30, 65.0 (CH ₂ O), 63.7 (C-50, 46.5 (C [CH ₃ ) ₃ ), 44.1 (CHN), 29.7 (CH ₂ S), 28.7, 27.9, 27.5 (CH ₃ Boc and tBu), 25.3 (CH 3) ₃ iPr) ³¹ P NMR (CDCl ₃ , 121 MHz) δ 8.6, 8.3 FAB MS> 0 (GT) m / z 1457 (2M + H) ⁺ , 729 (M + H) ⁺ , 629 (M-BoC) ⁺

FAB <0 (GT) m / z 1455 (2M-H) ^- , 727 (MH) ^- , 583 (M-tBuSATE) ^- , 483 (M-tBuSATE-Boc) -

Prodrug (1.2): / V- (isopropylamino) -O (5-pivaloyl-2-thioethyl) -05'-gemcitabine phosphoramidate diester

To a stirred solution of ^ yv- isopropylamino) -0 (5 ^L pivaloyl-2-thioethyl) -0-5 '- (/ V-4-0-3'-bis (te / t-butoxycarbonyl) gemcitabine phosphoramidate ( Ia.2) (0.270 g, 0.37 mmol) in anhydrous dichloromethane (4 ml), a solution of TFA (4 ml, 50% in dichloromethane v / v) is added dropwise at 0 ^° C. The reaction is complete after Ihl5 at room temperature The reaction mixture is co-evaporated twice with CH ₂ Cl ₂ and 4 times with ethanol Purification of the residue on a RP18 column using as eluent a gradient of 20% to 60% by volume of CH ₃ CN / H ₂ O makes it possible to obtain the desired derivative (1.2) (0.170 g, 87%) in the form of a white solid. ¹ H NMR (DMSO-C- ₆ , 400 MHz) δ 7.55 (d, 1H, J = 7.6, H-6), 7.41, 7.37 (2s, 2H, NH 2), 6.40 (t, 1H, J = 6.1, OH), 6.18 (t, 1H, J = 8.0, H-10, 5.78 (dd, 1H, J = 7.6, H-5), 5.03 (m, 1H, NH), 4.21-4.12 (m, 3H, H); -3 ', H-5' and H-5 "), 4.05 (m, 1H, H-40, 3.94 (m, 2H, CH ₂ O), 3.24 (m, 1H, CHN) 3.11 (t, 2U ₁ J = 6.4, CH ₂ S), 1.20 (s, 9H, CH ₃ tBu), 1.10, 1.08 (2s, 6H, CH ₃ iPr)

¹³ C NMR (DMSO-d6, 100 MHz) δ 205.2 (CO), 165.6 (C-4), 154.5 (C-2), 141.0 (C-6), 122.6, 119.2 (C-20, 94.7 (C 5), 83.2 (C-IO, 78.3 (C-40, 69.5 (C-3 ^7), 63.7 (C-5 'and CH ₂ O), 46.0 (3chs) _3), 43.1 (CHN), 28.4 (CH ₂ S), 26.9 (CH ₃ tBu), 24.9, 24.8 (CH ₃ iPr)

³¹ P NMR (DMSO-de, 121 MHz) δ 10.1, 9.9

FAB MS> 0 (GT) m / z 1057 (2M + H) ⁺ , 529 (M + H) ⁺

EXAMPLE 3

Preparation of the prodroma of qemcitabine of formula (1.31:

Intermediate (Ia.3): N- (benzylamino) - (- (S-pivaloyl-2-thioethyl) -t-5 '- (/ V-4-O-3'-bis (tert-butoxycarbonyl) The gemcitabine phosphoramidate diester (> (5-pivaloyl-2-thioethyl) -O-5 '- (/ V-4 - (>3'-bis (fe / t ^L butoxycarbonyl) hydrogen phosphonate diester gemcitabine (II.1) (0 1 g, 0.156 mmol) is co-evaporated twice with anhydrous pyridine, then dissolved in carbon tetrachloride (3.7 ml), to which benzylamine (0.05 ml, 0.47 mmol) is added. The reaction mixture is stirred at room temperature After 15 minutes, the reaction mixture is diluted with dichloromethane (15 ml) and washed with a hydrochloric acid solution (15 ml, 0.1 N, pH = 0). 1) until the pH of the aqueous solution is acidic (pH≈l) The organic phase is neutralized with an aqueous solution of NaHCO ₃ and then washed with water (15 ml) and dried over Na ₂ SO 4, then filtered and concentrated under reduced pressure. Purification by column chromatography, eluting with a dichloromethane / ethyl acetate mixture (7/3, v / v) gives the 2-benzylamino-CK (pivaloyl-2-thioethyl) -0-5 '- (ΛM). -0-3'-bis (te / t-butoxycarbonyl) gemcitabine phosphor- amidate (IA.3) (0.084 g, 70%) as a colorless oil. ¹ H NMR (CDCl _3, 400 MHz) Δ 7.77 (m, 1H, H-6), 7.26 (m, 5H, Ph), 7.20 (m, 1H, H-5), 6.38 (m, 1H, H-10, 5.08 (m, 1H, H); 30, 4.22 (d, 2H, J = 6.1, CH ₂ O), 4.05 (m, 4H, H-5 ', H-5 "and CH ₂ N), 3.50 (m, 1H, H-4 ¹ ), 3.04 (m, 2H, CH ₂ S), 1.43 (s, 18H, CH ₃ BoC), 1.15 (s, 9H, CH ₃ SATE) ³¹ P NMR (CDCl ₃ , 121 MHz) δ 9.1, 8.8 FAB MS> 0 (GT) m / z 777 (M + H) ⁺ , 677 (M-BoC) ⁺

FAB <0 (GT) / 77 / Z775 (MH) ^"

Prodrug (1.3): N-benzylamino-pivaloyl-thioethyl-OS'-gemcitabine phosphoramidate diester

To a stirred solution of M-benzylamino) - (pivaloyl-2-thioethyl) -? - 5 '- (? - 4-C? 3'-bis (tert-butoxycarbonyl) gemcitabine phosphoramidate (Ia.3 ) (0.041 g, 0.052 mmol) in anhydrous dichloromethane (0.5 ml), a solution of TFA (0.5 ml, 50% in dichloromethane v / v) is added dropwise at ^0.degree . The reaction is complete after 1 h at room temperature The reaction mixture is co-evaporated twice with CH ₂ Cl ₂ and 4 times with ethanol The purification of the residue on a chromatography column using as eluent with a dichloromethane mixture / ethanol (9/1, v / v) makes it possible to obtain the desired derivative (1.3) (0.026 g, 72%) in the form of a white solid.

¹ H NMR (DMSOd ₆ , 400 MHz) δ 7.71 (d, 1H, J = 6.9, H-6), 7.41, 7.04 (2s, 2H, NH 2), 7.3 (m, 5H, Ph), 6.50 ( s, 1H, OH), 6.16 (m, 1H, H-10, 5.91 (d, 1H, J = 6.9, H-5), 5.78 (m, 1H, NH), 4.15 (m, 3H, H-3). , H-5 'and H-5'0.396 (m, 5H, H-4', CH ₂ O and CH ₂ N) 3.05 (t, 2H, J = 6.3, CH ₂ S), 1.17 (s) , 9H, CH ₃ )

³¹ P NMR (DMSO-d ₆ , 121 MHz) δ 10.1, 9.9 FAB MS> 0 (GT) m / z 1053 (2M + H) ⁺ , 577 (M + H) ⁺ EXAMPLE 4

Preparation of the prodroαue of αemcitabine of formula (IA):

Intermediate (IV.2): 2,2-Dimethyl-3-triphenylmethoxy-5-2-ethylthio propanoate is obtained according to a protocol described in an earlier publication, A.-L. Villard, et al., Bioorganic and Medicinal Chemistry, 2008, 16, 7321.

Intermediate (III.2): O- [S- (2,2-dimethyl-3- [triphenylmethoxy-propionyl) -2-thioethyl] hydrogenphosphonate monoester, triethylammonium salt

To a solution of 2,2-dimethyl-3-triphenylmethoxy-S ^L 2 -ethylthiopropanoate (IV.2, 9.5 g, 22.6 mmol) in anhydrous pyridine (30 ml) is added an acid solution. phosphorous (18.5 g, 225.9 mmol) in pyridine (113 ml). To the resulting suspension is added dropwise pivaloyl chloride (15.3 mL, 124.2 mmol). After stirring for 3 hours at room temperature, a solution of triethylammonium bicarbonate (TEAB, 40 mL, 1M, pH 7) is added to the reaction mixture. The solution is diluted with ethyl acetate (250 mL) and triethylammonium bicarbonate solution (TEAB, 250 mL, 0.5M, pH 7). The mixture is decanted and the aqueous phase is extracted with ethyl acetate. The organic phases are combined, dried over Na ₂ SU ₄ , filtered and then evaporated under reduced pressure to give a yellow oil which is then coevaporated with toluene. The residue obtained is purified by column chromatography on silica gel (dichloromethane / triethylamine, 99/1, v / v) using as eluent a gradient of 0 to 10% methanol in dichloromethane containing 1% triethylamine. <> [S- (2,2-Dimethyl-3-α-triphenylmethoxy-propionyl) -2-thioethyl] hydrogenophosphonate monoester (salt triethylammonium, III.2) is obtained as a yellow oil (9.77 g, 74%).

¹ H NMR (CDCl ₃ , 300 MHz) δ 7.31 (m, 6H, Ph), 7.16 (m, 9H, Ph), 6.70 (d, 1H,

Jp. _H = 634, PH), 3.85 (m, 2H, CH ₂ O), 3.08 (m, 4H, C ₁₂ H ₂ OTr, ₅ CH ₂ ), 2.94 (q, 6H, J = 7.3, CH ₂ H) ₁ 1.18 (t, 9H, J = 7.3, CH ₂ N), 1.07 (s, 6H, CH ₃ )

¹³ C NMR (CDCl ₃ , 75 MHz) δ 203.6 (Is, C = O), 142.6, 127.7, 126.7, 126.1 (Ph),

85.3 (C (Ph) ₃ ), 66.1 (CH ₂ OTr), 61.5 (CH ₂ O), 49.3 (C (CH ₃ ) ₂ , 44.5 (CH ₂ N), 28.4 (SCH ₂ ), 21.8 (C (CH ₃ ) ₂ ), 7.5 (CH ₃ CH ₂ N)

³¹ P NMR (CDCl ₃ , 121 MHz) δ 3.4

FAB MS> 0 (GT) m / z 586 (M + H) ⁺ , 243 (Tr) ⁺

FAB <0 (GT) m / z 483 (M-hNET ₃₎ ^", 259 ^(Troh)", 181 (M-TrOSATE) ^"

Intermediate (II.2): 0 [5- _{(2/2-dimethyl-3-Otriphénylméthoxypropionyl)} -2-thioethyl] -0-5 '- (/ V-4 - (>3'-bis (te / t- butoxycarbonyl) gemcitabine hydrogenophosphonate diester

1,4-C, 3'-bis (tert-butoxycarbonyl) gemcitabine (obtained according to ZW Guo, JM GaIIo, J. Org Chem., 1999, 64, 8319) (4.2 g, 9.1 mmol). and O [5 ^L (2,2-dimethyl-3-mercenylmethoxypropionyl) -2-thioethyl] hydrogenophosphonate monoester (triethylammonium salt, III.2), (8.0 g, 13.6 mmol) are co-evaporated with pyridine, then dissolved in anhydrous pyridine (140 ml) and then pivaloyl chloride (2.8 ml, 22.7 mmol) is then added dropwise. After 3h, the reaction is stopped by addition of ammonium acetate (25 ml, 0.5M), the reaction mixture is diluted in dichloromethane (250 ml) and then washed with ammonium acetate (250 ml 0.01 M). The organic phase is dried over Na ₂ SO _-I, filtered and then evaporated under reduced pressure to give a yellow oil which was then coevaporated three times with toluene. Purification by column chromatography, using a dichloromethane / ethyl acetate mixture, 7/3, v / v + 0.2% acetic acid as eluent, gives the O- [5 ^L (2.2 -Dimethyl-3-C? -Triphenylmethoxypropionyl) -2-thioethyl] -C? -5? - (? - 4-c? 3'-bis (tert-butoxycarbonyl) gemcitabine hydrogenphosphonate diester (II.2) ( 6.9 g, 72%) as a white foam.

¹ H NMR (CDCl ₃ , 300 MHz) δ 7.85 (d, 1H, J = 6.3, H-6), 7.31 (m, 16H, Ph and H-5), 6.91 (d. , ¹ H, _JP-H = 719 PH) 6.50 (m, IH, H-IO, 5.15 (m, IH, H- 30, 4.34 (m, 3H, H-4 ', H-5' and H-5 "), 4.21 (m, 2H, CH ₂ O), 3.22 (m, 4H, CH ₂ OTr) and CH ₂ S), 1.53 (s, 18H, CH ₃ Boc), 1.25 (s, 6H, CH ₃ SATE)

¹³ C NMR (CDCl ₃ , 75 MHz) δ 203.4 (CO SATE), 163.7 (C-4), 153.8 (C-2), 151.8 (CO BOC) ₇ 151.1 (CO Boc), 145.1 (C-6), 143.4, 128.2, 127.8, 127.0 (Ph), 121.1 (C-20, 95.3 (C-5), 85, 8 (4Ph) ₃ ), 83.9 (C (CH ₃₎ ₃ , C-10, 81.3 (αCH ₃ ) ₃ ), 76.6 (C-40, 72.5 (C-30, 69, (CH ₂ OTr), 63.6 (C-50, 63.5 (CH ₂ O), 50.3 (C (CH ₃ ) ₂ ), 28.4 (CH ₂ S), 27.7, 27 , 1 (CH ₃ ), 22.3 (CH ₃ )

³¹ P NMR (CDCl ₃ , 121 MHz) δ 8.4, 8.8

FAB MS> 0 (GT) / 77 / Z930 (M + H) ⁺ , 243 (Tr) ⁺

FAB <0 (GT) Λ7 / z928 (MH) ^', 526 (M- TrOSATE) ^"

Intermediate (Ia.4): N- (benzylamino) -O [S ^L (2,2-dimethyl-3-C "-triphenylmethoxypropyl) -2-thioethyl] -05 '- (/ V-4-C>-3'-bis- (ήs / * - butoxycarbonyl) gemcitabine phosphoramidate diester

Ae CV [S- (2,2-dimethyl-3-alphatriphenylmethoxypropionyl) -2-thioethyl] -C -5 '- (/ V-4-α-3'-bis (tert-butoxycarbonyl) gemcitabine hydrogenphosphonate diester (II.2) (1.0 g, 1.07 mmol) was co-evaporated twice with anhydrous pyridine and then dissolved in carbon tetrachloride (25.5 ml). 0.35 ml, 3.22 mmol) is added dropwise The reaction mixture is stirred at room temperature After 15 minutes, the reaction mixture is diluted with dichloromethane (100 ml) and washed with a hydrochloric acid solution (100 ml, 0.1 N, pH = 1) until the pH of the aqueous solution is acidic (pH≈l) The organic phase is neutralized with an aqueous solution of NaHCO ₃ and then washed with water. water (100 ml) and dried over Na ₂ SO _I, then filtered and concentrated under reduced pressure. purification by column chromatography, eluting with a dichloromethane / ethyl acetate (7/3, v / v) gave the / V- b enzylamino-O [ ^5L (2,2-dimethyl-3-O-triphenylmethoxypropionyl) -2-thioethyl] -5 '- (N-4 -3'-bis (tert-butoxycarbonyl) gemcitabine phosphoramidate (Ia. 4) (0.907 g, 82%) as a white foam.

¹ H NMR (DMSO-d ₆ , 300 MHz) δ 10.6 (s, 1H, NH), 8.01 (d, 1H, J = 6.5, H-6), 7.28 (m, 2OH). , Tr and Ph), 7.08 (d, 1H, J = 6.5, H-5), 6.30 (m, 1H, H-10, 5.78 (m, 1H, NHBn), 5, (M, 1H, H-30, 4.42 (m, 1H, H-40, 4.23) (m, 2H, H-5 'and H-5 "), 3.95 (m, 4H, CH ₂ O and CH ₂ N), 3.05 (m, 4H, CH ₂ Otr and CH ₂ S), 1.45 (s, 18H, CH ₃ ), 1.11 (s, 6H, CH ₃ )

¹³ C NMR (DMSO-Cl ₆ , 100 MHz) δ 201.2 (CO), 161.4 (C-4), 151.5 (CO, Boc), 149.4 (C-2), 148.8 (CO, Boc), 142.5 (C-6), 141.1, 138.1, 125.9, 124.8, 124.4 (C-Ph), 118.8 (C-20, 93, 0 (C-5), 83.5 (C (Ph) ₃ ), 82.5 (C-10, 81.6, 79.1 (αCH ₃ ) ₃ ), 74.5 (C-40, 70, (C-30, 67.2 (CH ₂ O), 61.7 (C-5 '), 48.1 (αCH ₃ ) ₂ ), 41.9 (CH ₂ O), 39.8 (CH ₂ N), 26.1 (CH ₂ S), 24.8, 24.5 (CH ₃ Boc), 20.1 (CH ₃ tBu)

³¹ P NMR (DMSO-C ₁₆ , 121 MHz) δ 10.2, 10.0

FAB MS> 0 (GT) m / z 1035 (M + H) ⁺ , 793 (M-Tr) ⁺ , 464 (diBocGemC) ⁺ ,

243 (Tr) ⁺

FAB <0 (GT) m / z 1033 (MH) ^', 791 (M Tr) ^", 631 (M- ^TrOSATE)", 531 (M- TrOSATE-Boc) ^"

Prodrug (1.4): / V- (benzylamino) -O (5 ^L (2,2-dimethyl-3-hydroxypropionyl) -2-thioethyl) -O-5'-gemcitabine phosphoramidate diester

To a stirred solution of β- (benzylamino) -O- (5- (2,2-dimethyl-3-O-triphenylmethoxypropionyl) -2-thioethyl) -0-5 '- (ν-4 -3'-bis) (tert-butoxycarbonyl) gemcitabine phosphoramidate (Ia.4) (4.2 g, 4.06 mmol) in dry dichloromethane (45 ml), TFA solution (45 ml, 50% in dichloromethane v / v ) is added dropwise at 0 ^° C. The reaction is complete after 1 h at room temperature The reaction mixture is co-evaporated twice with CH ₂ Cl ₂ and 4 times with ethanol. residue on a chromatographic column of silica gel using as eluent with a dichloromethane / ethanol mixture (9/1, v / v) followed by RP18 reverse phase chromatography using as eluent a gradient of acetonitrile 20% to 50 % in water, allows to obtain the desired derivative (1.4) (1.59 g, 66%) in the form of a white solid.

¹ H NMR (DMSO-d6 400 MHz) δ 7.54 (m, 3H, H-6 and NH ₂ ), 7.28 (m, 5H, Ph), 6.44 (s, 1H, OH), 6.17 (t, IH, J = s ₁ H-IO, 5.76 (m, 2H, H-5 and NH), 4.96 (s, IH, OH), 4.16 (m, 3H, H-3 ', H-5' and H-5'0, 3.97 (m, 3H, H-4 'and CH ₂ N), 3.89 (m, 2H, CH ₂ O), 3.70 (s, 2H, QH ₁ QH), 3.04 (m, 2H, CH ₂ S), 1.11 (s, 6H, CH ₃ ) ¹³ C NMR (DMSOd ₆ , 100 MHz) δ 204.5 (CO), 165.7 (C-4), 154.5 (C-2), 141.6 (C-6), 140.9, 128 7, 127.6, 127.3 (C-Ph), 120.5 (C-20, 95.3 (C-5), 84.2 (C-IO, 78.8 (C-4 ⁷⁾ , 69.9 (C-3 ^7), 68.8 (CH _2), 66.8 (CH ₂ OH), 64.4 (C-50 _ι 52.2 (αCH ₃₎ _2), 44.7 ( CH ₂ N), 28.7 (CH ₂ S), 22.3 (CH ₃ )

³¹ P NMR (DMSO-dl, 121 MHz) δ 10.1, 9.9

FAB MS> 0 (GT) m / z 1035 (M + H) ⁺ , 793 (M-Tr) ⁺ , 464 (diBocGemC) ⁺ ,

243 (Tr) ⁺

FAB <0 (GT) m / z 1033 (MH) -, 791 (M Tr) ^", 631 (M- ^TrOSATE)", 531 (M- TrOSATE-Boc) ^"

Microanalysis Calcd for C ₂₃ H ₂ F _3I N ₄ O ₈ PS: C: 46.62; H, 5.27; N, 9.46; S, 5.41

Found: C, 46.94; H, 5.53; N, 8.77; S: 4.83

Evaluation of the antitumor activity of the compounds (1.1) to (1.4) in different gemcitabine resistant cell lines (Table 1), shows their ability to bypass, at least in part, the cellular resistance processes.

Evaluation of the cytotoxic activity of the compounds is carried out by an in vitro cytotoxicity test on tumor lines. These lines are cultured under sterile conditions for 72 hours in culture medium with 10% fetal calf serum in the presence of different concentrations of the compound of interest. After 72 hours, cell proliferation is evaluated by measuring the metabolic activity by reducing MTT to formazan crystals. These crystals are solubilized in an isopropanol / 1N HCl (90:10, v: v) mixture and optical density values are obtained spectrophotometrically. These values allow the determination of the 50% inhibitory concentrations, corresponding to the concentration of compound causing a growth inhibition of 50% compared to the control (identical line cultivated in the absence of compound).

Messa and Messa / IOK

Messa is a line of a patient with soft tissue sarcoma. Messa 1OK was produced by prolonged exposure to gemcitabine and is devoid of deoxycytidine kinase (Jordheim LP, CM Galmariπi, Dumontet C. Gemcitabine resistance to deoxycytidine kinase deficiency can be reverted by fruitfly deoxynucleoside kinase, DmdNK, in human uterine sarcoma cells. Cancer Chemother Pharmacol. 2006; 58: 547-54.)

L1210 and L1210 IQK

L1210 is a line of murine leukemia. L1210 1OK has been produced by prolonged exposure to gemcitabine and is devoid of deoxycytidine kinase (Jordheim LP, Cros E, Gouy MH, CM Galmarini, Peyrottes S, Mackey J, et al., Characterization of a gemcitabine-resistant murine leukemic cell line: Revision of In Vitro Resistance by Mononucleotide Prodrug Clin Cancer Res., 2004; 10: 5614-21)

EMC and EMC R

CEM is a line of human leukemia. CEM R is a carrier-free resistant line for nucleoside analogues. These two lines were provided by the authors who described this line (Gourdeau H, Clarke ML, Ouellet F, Mowles D, Selner M, Richard A, Lee N, JR Mackey, Young JD, Jolivet J, Lafrenière RG, Cass CE. Mechanisms of uptake and resistance to troxacitabine, a novel deoxycytidine nucleoside analogue, in human leukemic and solid tumor cell lines.Cancer Res 2001 Oct 1; 61 (19): 7217-24.)

MCF7 and MCF7 IK

MCF7 is a line of human breast cancer. MCF7 was developed by the inventors and shows an increased level of expression of ribonucleotide reductase, an important target of gemcitabine (Jordheim LP, Guittet O, Lepoivre M, Galmarini CM, Dumontet C. Increased expression of the large subunit of ribonucleotide Reductase is involved in resistance to gemcitabine in human mammary adenocarcinoma cells, Mol Cancer Ther., 2005; 4: 1268-76)

A2780 and AG6000

A2780 is a line of ovarian cancer. AG6000 is a line lacking deoxycytin kinase. These lines were provided by the authors having described this line (Al-Madhoun AS, van der WiIt CL, Loves WJ, Padron JM, Eriksson S, Talianidis I, Peters GJ Detection of an alternative spliced form of deoxycytidine mRNA kinase in the 2'-2'-difluorodeoxycytidine (gemcitabine) -resistant human ovarian cancer cell line AG6000. Biochem Pharmacol. 2004 Aug 15; 68 (4): 601-9.)

Table 1. Chemo susceptibility of a non-resistant (Messa wt) and resistant (10K) cell line exposed to different concentrations of gemcitabine or a prodrug (I)

The asymmetric compound (1.3) is slightly more active than gemcitabine in resistance models with dCK deficiency (Table 2a and 2B). In the MCF7 IK model, the compounds (1.3) and its hydroxylated derivative (1.4) are comparable to gemcitabine (Tables 2a, 2b and 3a, 3b). Table 2a

Table 2b

Table 3a

Compounds (1.1) and (1.2) are more active than gemcitabine in models of dCK deficiency (Table 4a, 4b). Table 4a

Table 4b

The IC ₅ O are obtained by averaging the results obtained on three experiments and are expressed in μM ± standard deviation. IC ₅₀ and the values of the relative resistance ratio are obtained from the computer generated concentration effect curves. RR: relative resistance ratio (IC ₅₀ Messa 10K / Messa wt). nm: not measurable.

Claims

1 - Phosphoester prodrugs of gemcitabine, with anti-tumor activity, of formula (I):

in which :

n is 1, 2, 3 or 4,

Z represents O or S

Y represents a group chosen from alkyl or aryl groups, said alkyl or aryl groups possibly being substituted, for example by one or more substituents chosen from -OH, -SH or -NH _2;

- Ri represents a group -NR ₃ R _b in which Ra and R _{b /} identical or different, represent a hydrogen atom, an alkyl or aryl group _/ said alkyl or aryl groups which may be optionally substituted, or R ₃ and Rb are bonded together to form, with the nitrogen atom to which they are bonded, a cyclic amine selected from optionally substituted pyrrolidinyl, piperidinyl, morpholynyl, piperazinyl, pyridyl groups.

2 - Compounds according to claim 1 characterized in that Y = methyl or te / t-butyl.

3 - Compounds according to claim 1 characterized in that Y = 2,2-dimethyl-3-hydroxypropionyl.

4 - Compounds according to claim 1, 2 or 3 characterized in that n = 2.

5 - Compounds according to one of claims 1 to 4 characterized in that Ri = -NR ₉ Rb with R ₃ and Rb as defined in claim 1.

6 - Compounds according to claim 5 characterized in that R ₃ = H and R _b = benzyl, isopropyl or n-butyl.

7 - Compounds according to claim 1 of formula (1.1), (1.2), (1.3) or (1.4) below:

8 - Pharmaceutical compositions comprising a compound according to one of claims 1 to 7, in combination with at least one pharmaceutically acceptable excipient.

9 - Use of a compound according to one of claims 1 to 8 for the preparation of an anti-tumor or anti-cancer drug.

10 - Use according to claim 9 for the preparation of an anti-tumor or anti-cancer drug for the treatment or prevention of benign tumor lesions, or cancers, including haematological malignancies and solid tumors, including tumors of glandular, mesenchymal, genital, cutaneous and neurological origin.