WO1996000585A1

WO1996000585A1 - Diphosphate or triphosphate analogue conjugates

Info

Publication number: WO1996000585A1
Application number: PCT/FR1995/000872
Authority: WO
Inventors: Christophe Creminon; Jacques Grassi; Philippe Pradelles; Charles Mioskowski; Luc Lebeau; Mourad Saady
Original assignee: Commissariat A L'energie Atomique; Centre National De La Recherche Scientifique
Priority date: 1994-06-30
Filing date: 1995-06-29
Publication date: 1996-01-11
Also published as: FR2721931A1; FR2721931B1

Abstract

Diphosphate or triphosphate analogue conjugates with antigenic enzymes or carriers and immunological assay using such conjugates. In these conjugates, the disphosphate or triphosphate analogue is based on formula (I) in which n is equal to 0 or 1, X?1 and X2¿ which can be CH¿2?, CHF, CF2, CC12, CHC1 or NR?6, R1, R2, R3 and R4¿ are a hydrogen atom, NH¿4?+, a quaternary ammonium ion or M+1/v, and R5 is a nucleoside-derived group, linked to the antigenic enzyme or carrier, either by the nucleoside-derived R5 group, or by one of R?1, R2, R3 or R4¿. Such conjugated compounds are useful for assaying anti-viral phosphorylated metabolites of antiviral agents such as AZT.

Description

Conjugates of dlphosphates or trlphosphates analogs

The invention relates to conjugated compounds formed from a diphosphate or triphosphate analog and from a molecule chosen from antigenic carriers and enzymes.

More specifically, it relates to conjugated compounds in which the analogs are analogs of diphosphates or triphosphates of nucleosides which are of great interest in biology, such as adenosine diphosphate (ADP) and adenosine triphosphate (ATP) . In fact, living cells capture, store, and transport energy in chemical form, essentially in the form of adenosine triphosphate (ATP). ATP can transmit its energy to certain other molecules and thus release its terminal phosphate group; the energy-rich ATP molecule then becomes adenosine diphosphate

(ADP) whose energy has decreased, but which can regain a phosphate group to become 1 ATP again thanks to a supply of solar energy in photosynthetic cells, or chemical energy in animal cells.

ATP is the main connecting link between two major sets of enzyme-catalyzed reactions in cells. One of these sets conserves chemical energy from the environment by causing phosphorylation of energy-poor ADP, energy-rich ATP. The other set uses the energy of ATP to biosynthesize cellular components from simple precursors, to do mechanical work. necessary for contraction and locomotion, or to perform osmotic work of membrane transport. These sets of enzyme catalyzed reactions are almost identical in all living species.

Also, ATP or ADP can be used in many techniques, for example to immobilize, orient or transport molecules of biological interest. However, these techniques are limited due to the low chemical stability of ATP and ADP (hydrolysis of phosphoric anhydride bonds). It would therefore be of great interest to have analogs of ATP or ADP, which are not hydrolysable to fulfill these functions.

Other analogs of diphosphates and triphosphates of interest are the diphosphates and triphosphates of anti-viral nucleoside agents such as 3'-azido-3'-deoxythymidine (AZT), dideoxyinosine (ddl), dideoxycytidine (ddC) and 2 ', 3' -dehydro-3'-deoxy-thymidine (d_] T), which are anti-HIV drugs.

Indeed, conjugate compounds obtained by coupling diphosphates or triphosphates analogs of anti-viral nucleoside agents with biological molecules would be of great interest for carrying out immunological assays of the phosphorylated metabolites of these anti-viral agents in subject patients. to treatment with such agents. One of the major problems encountered during therapeutic treatment of AIDS is the extreme variability of response from one patient to another, this being further complicated by the appearance of phenomena of resistance to drugs. Thus, one of the primary objectives identified during the last Berlin meeting on AIDS (9 ^th International AIDS Conference, Berlin, 7-11 June 1993) is good to quantify in patients the active metabolites of the various antiviral drugs administered. For almost all of these nucleoside molecules, the mechanism of action involves phosphorylation of nucleotide 5'-triphosphate under the action of cellular kinases. AZT, for example, penetrates the cell membrane and under the effect of kinases undergoes phosphorylations which makes it active to selectively inhibit HIV reverse transcriptase while also being incorporated at the 3 'end of the strands of viral DNA in the process of growth, thus playing a role of chain terminator. Aside from side effect issues, viral strains that have become resistant to AZT have also recently been isolated. This has led to the marketing of other drugs such as ddl or ddC whose mode of action is very similar to that of AZT. Many other modified nucleosides have also been shown to be potent inhibitors of HIV replication. All these compounds act according to the same mechanism as AZT, that is to say inhibition of reverse transcriptase in the form of nucleotide triphosphates produced by the action of cellular kinases. Their activity is therefore related to their phosphorylation speed and their affinity for reverse transcriptase, but also to their possible catabolism (under the action of phosphorylases, phosphatases, deaidases ...). Given this, it is clear that from one patient to another, there will be great variability in the response to antivirals. Thus the possibility of rapidly and effectively measuring the concentrations of phosphorylated metabolites in each treated individual should allow a better dosage adjustment and more effective therapeutic monitoring.

To date, a number of quantitative assays for AZT or other nucleositic antivirals have been published. The most numerous are based on high performance liquid chromatography (HPLC) (G. Molema et al. - J. Chromatogra. (1992), 579, 107-114). Radioimmunoassays (RIA) have been described (S. Tedepalli and R. Quinn in Clin. Chem. (1989), 35, 1187) and RIA assay kits have been marketed ("The Immunoassay Kit Directory" Vol. 1; part 4 (1992), E. Nays ith, ed. Kluwer Académie Publishers, Lankaster, UK.) As well as other immunoassay techniques based on the use of non-radioactive markers such as FPIA (Polarized immunological assay fluorescence) (GG Granich et al. - Anti icrob. Agents Chemother. (1989) 33, 1275-1279 or ELISA (Enzyme-linked immunosorbent assay) (S. Tedepally, R. Quinn - J. Acq. Imm. Def. Syndr. (1990) 3, 19-27) Finally, a microbiological technique has been described (H. Etesse-Carsenti et al. - AIDS (1990) 4, 265).

HPLC techniques are the most used. They have the advantage of allowing a quantitative assay of phosphorylated metabolites of AZT. However, they are limited in terms of sensitivity (~ lng / ml) (large blood test to have a sufficient number of cells) and they require the use of extraction methods (problems linked to plasma interference). The immuno-analytical techniques are more sensitive (~ 0, lng / ml), simpler to implement, but those described so far do not make it possible to discriminate AZT from phosphorylated metabolites for reasons related to the strategy for obtaining the antibodies necessary for the assay.

Research has therefore been continued to develop a method for assaying metabolites of anti-viral agents, meeting the following requirements:

- sensitive (to avoid too large a test portion and to minimize non-specific interference by dilution), - specific (to be able to specifically dose AZT and its phosphorylated metabolites, particularly 5'-triphosphate,

- simple (being able to avoid purification steps, being suitable for mass production assays, being able to transfer technology without difficulty), and

- inexpensive (with the exception of the investment to develop the tools of the method, it must subsequently require simple and inexpensive equipment).

In this perspective, immuno-analytical techniques are and remain unambiguously the most appropriate. They have so far been unsatisfactory for the determination of phosphorylated metabolites because the immunoanalysts have found themselves faced with the difficulty of obtaining antibodies specific for these metabolites. Indeed, AZT-5'-triphosphate of formula:

which will be taken as an example, belongs to the category of haptens.

It is necessary to covalently couple the molecule on an antigenic carrier (bovine serum albumin for example) and to administer the conjugate to an animal (rabbit or mouse, for example) to trigger an immune response capable of inducing synthesis specific hapten antibodies. If we want to direct the specificity towards the triphosphate chain, it will be necessary to couple to albumin another part of the molecule in order to present to the immune system the desired epitope

(whole triphosphate + sugar chain). The anhydride bonds of POP acid implied in the triphosphate chain being particularly unstable, it is completely random (taking into account the too short half-life of this compound), to hope to induce an immune response against a molecule of this unprocessed type.

The present invention specifically relates to conjugated compounds of analogs of diphosphates and triphosphates of nucleosides coupled to antigenic carriers, which allow precisely obtaining antibodies specific for the metabolites of these anti-viral agents. These compounds must have the double characteristic of: - have stable phosphate bonds (di or tri),

- to mimic as much as possible the natural phosphate bonds so that the antibodies induced recognize the natural nucleotides.

It also relates to conjugated compounds of analogs of diphosphates or triphosphates of nucleosides coupled to enzymes, which can be used as markers in enzymo-immunological assays of these metabolites.

According to the invention, the conjugate compound of a diphosphate or triphosphate analog and of a molecule chosen from antigenic carriers and enzymes, in which the analog corresponds to the formula:

where n is 0 or 1,

- X ¹ and X ² which may be identical or different, represent CH2, CHF, CF2, CCl2, CHC1 or NR ⁶ with R ⁶ being a hydrogen atom, an alkyl, aryl or aralkyl group, the two R ^ may be identical or different when n = 1 or capable of forming together a hydrocarbon chain comprising a phenyl ring,

- Ri, R ² , R3 and R ^ which may be the same or different, represent a hydrogen atom, NH4, a quaternary ammonium ion or an ion of formula M ⁺ ^ _v with M representing a metal and v being the state of valence of this metal, and

- R ^ is a group derived from a nucleoside, and in which the analog is coupled to said molecule, either via the group R ^ derived from nucleoside, either through one of the groups R ¹ , R ² , R-3 and R ⁴ , provided that X ¹ does not represent CH2 when n is equal to 0.

The use in this conjugate compound of a diphosphates or triphosphates analog corresponding to formula (I) given above, is very advantageous, because the replacement of the POP polyphosphate chain of the natural diphosphate or triphosphate by a PX ¹ chain -? or P-xi-PX ² - ?, leads to a stable, non-hydrolyzable compound. Preferably according to the invention, the analog is a triphosphate analog, n being equal to 1 in the above formula.

Indeed, in the case of nucleoside derivatives such as antiviral agents, it has been clearly demonstrated by various authors that their ultimate mode of action is the blocking of the transcription and replication mechanisms by insertion of the therapeutic agent , metabolized to triphosphate by different cellular kinases, in the DNA or RNA chain (Aids, the unanswered questions, Science 1993, 260, 1253-1293). Indeed, the 3 ′ position of the sugar, deprived of its hydroxyl group, does not allow the anchoring of another nucleotide and the therapeutic agent thus plays a role of "chain terminator". Consequently, it is absolutely obvious that the most interesting metabolite to be assayed is the nucleotide triphosphate which constitutes the biologically active species. The analog can also be a diphosphate analogue,

1 6 in which according to the invention n = 0 and X represents NR or a halomethylene group.

Indeed, although the CH2 group can mimic the oxygen atom of a pyrophosphate bridge by conferring on the molecular structure which contains it (a nucleotide for example) a very good resistance to enzymatic hydrolysis, such a modification of the structure can drastically modify the biological properties of the molecule in question (J. Biol. Chem. 1983, 258, 9536-9543. Proc. Natl. Acad. Sci. USA 1992, j}, 2370-2373. J Am. Chem. Soc. 1987, 109, 5544-5545. Adv. Enzymol. 1975, 43 1-56. Chem. Rev. 1977, 77, 349-367. Chem. Ind. (London) 1981, 134-138). This deterioration in biological properties is most often associated with a difference in acidity between phosphoric acids (contained in natural substances) and phosphoric and phosphinic acids (contained in analogs) or with differences in the geometry of the motif, the length of the phosphorus-carbon bond being slightly different from that of the phosphorus-oxygen bond. It is therefore essential to be able to modulate the pKa of the nalogues, on the one hand, and their intimate geometry, on the other. This can be achieved by exchanging the CH2 group (s) with halomethylene or amino groups, in accordance with the invention.

Thus, with the analogs of formula (I) it is possible to induce the synthesis of anti-triphosphate antibodies of anti-viral agent, for example of anti-AZT-5 '-triphosphate antibodies using this chemical analog where the polyphosphate chain is stabilized and whose chemical and conformational structure mimics that of the natural triphosphate chain so that the antibodies produced using this stable analog recognize with high affinity AZT-5 '-triphosphate . According to a first embodiment of the invention, the conjugated compound is an immunogen intended for the production of antibodies. In this case, the molecule coupled to the nucleoside diphosphate or triphosphate analog is an antigenic carrier. Generally, the coupling is carried out by means of a spacer arm grafted on the pyrimidine or purine base of the nucleoside, which comprises at its other end a group capable of reacting with the antigenic carrier, for example an H2 group. spacer arm on the nucleoside can be carried out either on an exocyclic H2 group in the case where the base of the nucleoside comprises such a group, or on a nitrogen atom present in the base cycle. It can also be carried out on sugar or on the phosphorus chain. read

The spacer arm is generally a saturated hydrocarbon chain. However, other spacers can be used, for example a polyoxyethyene (CH2-CH2-O) _n or a polyol - (CHOH) _n .

In some cases, the coupling can also be carried out directly on the nucleoside by means of a spacer fixed on the sugar part of the nucleoside.

In all cases, the group capable of reacting with the antigenic carrier is an NH2 group, but it is also possible to use other groups capable of reacting with the antigenic carrier, for example a COOH, maleimide, thil, aniline group.

When the group capable of reacting with the antigenic carrier is an NH2 group, the conjugated compound comprising a diphosphate or triphosphate analog coupled to an antigenic carrier can correspond to the following formula:

in which R ⁷ is a bivalent group derived from a nucleoside, n is equal to 0 or 1, X ¹ and X ² have the meanings given above, p is equal to 0 or is an integer ranging from 1 to 6 and Z is an antigenic carrier.

For example, R ⁷ - (CH2) _p - H- can answer the following formulas:

Preferably, in the analog of formula (I), R5 is derived from a nucleoside antiviral agent such as those of formulas:

The antigenic carriers capable of being coupled to the nucleoside diphosphate or triphosphate analog to form the conjugated compound serving as an munogen can be of different types. Proteins such as bovine albumin, ovalbumin, polylysine and β-lactoglobulin can be used in particular, for example 12

1 limpet hemocyanin (keyhole lympet hae ocyanin, KLH).

According to a second embodiment of the invention, the conjugate compound is an enzymatic label. In this case, the molecule coupled to the nucleoside diphosphate or triphosphate analog is an enzyme.

The enzymes that can be used are those that have interesting properties (sensitivity, detection threshold) for immunoassays.

As examples of such enzymes, o can cite peroxidases,, phosphatases and β

-galactosidase, in particular acetylcholinesterases such as acetylcholinesterase from Electrophorus electricus.

This coupling can also be carried out by means of suitable reagents on an NH2 group of the nucleoside of the diphosphate or triphosphate analog. As an example of such a conjugate, mention may be made of the conjugate of the triphosphate analog of formula:

coupled with an acetylcholinesterase. The analogs of nucleoside diphosphates and triphosphates used in the invention can be prepared by reaction of a diphosphate or triphosphate of formula:

in which X ^, X ² and n have the meanings given above, and R ^ is a hydrocarbon group, with a nucleoside derivative whose reactive functions are protected by appropriate groups except for the primary alcohol function of the sugar part of the nucleoside.

The diphosphates or triphosphates of formula III:

with n = 1 and X ¹ = X ² = CH ₂ can be prepared by reaction of a bis- (halomethylene) phophinate of formula: O

in which X is a halogen atom, with a mixed phosphite of formula:

in which R ^ is a hydrocarbon group and R ^ is a benzyl or allyl group to obtain a compound of formula (III) with X ¹ = X ² = CH ² and n = 1

In this process, the analog is prepared by a double Arbuzov reaction between a mixed phosphite and an aryl or alkyl bis- (halomethylene) phosphinate. The starting materials used in this reaction can be prepared by conventional methods.

Thus, bis- (halomethylene) phosphinate can be prepared according to the methods described by Ivanov et al in Zh. Obshch. Khim., 1967 37 (8), p.

1856-1862, and by Frank et al in Can. J. Chem., 1966,

44, p. 2593-2596. The mixed phosphite can be prepared by successive reactions of different alcohols R ⁸ OH, R ⁹ OH on PCI3 in the presence of a base.

Generally, the Arbuzov reaction is carried out at a temperature of 140 ° C., under low pressure, for example under 4 mm of mercury. Preferably, the reaction medium is further subjected to violent stirring to continuously remove the halide formed R ^ X.

The triphosphates of formula (III) in which X ¹ and X ² represent the group NR ^ with the two R ^ together forming a hydrocarbon chain as defined above, can be prepared by a process which comprises the following steps: a) reacting a chlorophosphate of formula:

with a diamine of formula: H ₂ NR ¹⁰ -NH ₂ in which R ⁸ is a hydrocarbon group, and R ¹⁰ is the hydrocarbon chain formed by 2R ⁶ , to form a bis-phosphoramide of formula:

O o

10 8

R ⁸ O- P NH —R —NH P — OR

¹ Ό OR ^s 0 * P b) cyclizing the bis phosphoramide thus obtained on a dichlorophosphite of formula:

Cl ₂ P-OR ⁸ in which R ⁸ is a hydrocarbon group, and c) oxidizing the cyclized product thus obtained.

The diamine used as the starting material in this reaction can be synthesized by conventional methods such as those described by Carpino in J. Org. Chem., 1986, 51, p. 4768-4779.

In this process, the first reaction corresponds to a diphosphorylation of the diamine and this reaction can be carried out in the presence of two equivalents of triethylamine. This reaction is very rapid and allows the diphosphoramide to be obtained with a high yield. The diphosphoramide is then cyclized with a dichlorophosphite in the presence of triethylamine and 4-dimethylaminopyridine, in an appropriate organic solvent such as tetrahydrofuran.

The oxidation of the cyclized product can then be carried out with 4-chloroperoxybenzoic acid.

After this reaction, a triphosphate analog of formula (I) is obtained in which X] _ and X2 are groups NR ^ and R ¹ , R ² , R ^ R ⁴ and R ⁵ are benzyl groups.

To prepare a triphosphate analog of formula (III) in which X ¹ and X ² are respectively the groups CH2 and NR ^, a process can be used which consists in reacting a methyl phosphonate of formula:

O

OF? P CH,

1 o

W 17

with a diphosphate analog of the formula:

Cl N -OR ^c

OR 8 OR ^£ to form the compound of formula:

The diphosphate analog used in this process can be prepared from the corresponding imidodiphosphate in which Cl is replaced by OH, by reaction with hydrochloric acid.

The diphosphates of formula (III) in which X ^ is a methylene group can be prepared by a process which consists in reacting a chlorophosphate of formula:

with a formula

The diphosphates of formula (III) in which X ¹ is the group NH can be prepared by a process which consists in reacting the compound of formula:

0

CI ₃ P: N Cl

Cl with a compound of formula R ⁸ OH to form the compound of formula:

The precursor pentachloride can be prepared by the method described by Emsley et al in J. Chem. Soc. A., 1971, p. 2873.

In the case of the diphosphates of formula (III) in which X represents the group NR ^, these can be prepared by reaction of a compound of formula:

OR ⁶ O

Cl — P —N -Cl

Cl Cl with an alcohol of formula R ⁸ OH.

By way of examples, the reaction scheme for the preparation of a non-hydrolyzable analogue of ATP provided with a spacer arm for coupling with an antigenic carrier or an enzyme is described below.

The coupling between the triphosphate analog and the modified nucleoside is carried out by activation of the alcohol in position 5 'on the sugar to form the

20 nucleoside triphosphate. The chlorine on the base of the nucleoside is then displaced by nucleophilic substitution with a diamine precursor. The final molecule is obtained by catalytic hydrogenation.

In case we want to prepare a

25 non-hydrolyzable analogue of AMT triphosphate, the following reaction scheme can be followed:

30

35 2 U

The coupling between the nucleoside and

The triphosphate analog is produced by activation of the primary sugar alcohol which then reacts with the triphosphate analog.

The final deprotection is carried out as previously by catalytic hydrogenation.

^• 30

In the case where it is desired to prepare a non-hydrolyzable analogue of AZT triphosphate, the following reaction scheme can be used:

• 35 20-1

30 21

In this case, the nucleoside is prepared in two stages from a thymidine derivative described in the literature. The coupling with the triphosphate analog is carried out as above. The final deprotection is carried out with triethyl silyl bromide (TMSBr) followed by passage through basic medium.

To couple these diphosphates or triphosphates analogs of nucleosides with an enzyme or an antigenic carrier, conventional techniques of coupling by covalent bond are used between a reactive group carried by the nucleoside and a reactive group carried by the enzyme or antigenic carrier such as lysyl, tyrosyl, histidyl, tryptophanyl or glutamyl groups. The reactive groups of the nucleoside can be constituted by acid, amino, phenol, hydroxyl or ketone functions. Coupling reactions can be carried out using reagents such as carbodiimides, gluraraldehyde, benzidine, mixed anhydrides, activated hapten esters and para-aminophenyl acetic acid.

The conjugate compounds of the invention can be used either as immunogens or as enzyme markers.

Also, the subject of the invention is also a process for the preparation of antibodies specific for a nucleoside diphosphate or triphosphate of formula:

22

in which n = 0 or 1 and R ^ represents a nucleoside derivative, which consists

- injecting a mammal with a conjugate compound of a nucleoside diphosphate or triphosphate analog of formula:

in which R ¹ , R ² , R ³ , R ⁴ , X ¹ and X ² have the meanings given above for the formula (I), and n and R ⁵ have the meanings given above for the formula (V ), coupled to an antigenic carrier, and

- then to draw blood from the immunized mammal to isolate the antibodies produced.

It also relates to a process for the preparation of monoclonal antibodies to nucleoside diphosphates or triphosphates analogs starting from the conjugate compound described above, by the technique of Kohler and Milstein. The invention also relates to a method for the immunological determination of a nucleoside diphosphate or triphosphate of formula:

23

in which n = 0 or 1 and R ' ⁵ represents a nucleoside derivative, which consists in carrying out a competition reaction between the diphosphate or triphosphate of formula (V) to be assayed and a compound composed of an enzyme and an analog of diphosphate or triphosphate of formula:

in which R ~> is an identical or different nucleoside derivative R ', has the meaning given for the formula (V), and R ¹ , R ² , R ⁴ , -'- and X ² have the meanings given for the formula (I), p limited sites of an antibody specific for said analog, then determining the amount of conjugated compound attached to the antibody. This assay method can be applied to the adenosine triphosphate (ATP) assay or to the assay of a metabolite of an anti-viral nucleoside ag such as anti-HIV drugs such as A

In the latter case, one can use for the conjugate dosage of a diphosphate or triphosphate analog of AMT or AZ. Other characteristics and advantages of the invention will appear better on reading the _. following examples given b understood by way of nonlimiting illustration, with reference to the single appended FIG.

The attached figure is a diagram illustrating the result of immunoassays carried out in accordance with the invention, that is to say the variation of the B / BO ratio in which B represents the enzymatic activity linked to the antibody in the presence of a substance to be assayed, and B0 represents the enzymatic activity linked to the antibody in the absence of 24

substance to be dosed, depending on the concentration (in pmol / ml) of substance to be dosed.

Example 1: Preparation of (dibenzyl phosphonomethyl)

(benzyl hydrogen phosphono-methyl) benzyl phosphinate (compound No. 1).

C30H33O8P3 PM: 614.50

a) Preparation of tribenzyl phosphite

(compound n ° 2) of formula:

To a solution of 25ml (286.5 mmol, 1 eq.) Of phosphorus trichloride in 1.5 liters of anhydrous ether, cooled to -78 ° C, 123.8 ml (888) is added dropwise over ten minutes. , 3 mmol, 3.1 eq.) Of 25

anhydrous triethylamine. The reaction medium is then treated dropwise with 92.95 g (859.6 mmol, 3 eq.) Of benzyl alcohol dissolved in 300 ml of ether. After 2 hours, the solution is warmed to room temperature and stirred under argon for 8 hours. The precipitate formed is filtered, the solvent is evaporated under vacuum, and the residue is chromatographed on a silica column (ethyl ether / hexane / trietylamine: 40/60/1); 90 g of a colorless oil are thus obtained (yield: 89%).

The characteristics of the product obtained are as follows: CCM

Rf = 0.5 (Et ₂ O / hexane / Et ₃ N: 50/50/1). NMR ¹ ?! : (CDC13) 200 MHz δ (ppm): 7.37 (m, 15H, aromatic protons); 4.94 (d,

^3J HP = 8 Hz, 6H, Ha).

¹³ C NMR: (CDCI3) 50 MHz δ (ppm): 138.21 (3C _b ); 128.37 (6C _d ); 127, 67 (3C _e ); 127.49 (6C _C ); 64.47 (d, ² JCP = 11.1 Hz, 3C _a ).

³¹ P NMR: (CDCl ₃ / H ₃ Pθ4) 81.015 MHz δ (ppm): 20.17 (s). IR (pure film) cm ^-1 3088; 3063; 3031; 2940; 2874; 1606; 1497; 1454;

1375; 1211; 994; 787. b) Preparation of bis (chloromethylene) chlorophospinate (compound No. 3) of formula: formula B p 108

C2H4CI3OP PM: 181.39 26

25 g (284.1 mmol, leq.) Of NaH2PO2 and 17.05 g (568.3 mmol, about 2 eq. Depolymerized) of paraformaldehyde are dissolved in 140 ml of distilled water. 50 ml of concentrated hydrochloric acid are added and the mixture is brought to reflux for 44 hours. After distillation of the water and then filtration of the salts, the filtrate is taken up in 50 ml of acetone and the salts formed are filtered again. The filtrate is concentrated, then it is taken up with 3 times 100 ml of benzene, which gives, after evaporation under vacuum, a yellow syrup. The residue is dissolved in 100 ml of benzene, then the mixture is brought to reflux, while 72.53 ml are added dropwise for 3 hours.

(994.4 mmol, 3.5 eq) of thionyl chloride (large gassing). After that, the reaction medium is left at reflux for another 12 hours. After evaporation of the solvent in vacuo, an oil is obtained which is purified by vacuum distillation to give 44.4 g of bis (chloromethylene) chlorophosphinate in the form of a colorless oil (86% yield).

The characteristics of this compound are as follows: T _eb = 85-88 ° C (lmmHg). NMR ¹ !!: (CDC1 ₃ ) 200 MHz δ (ppm): 4.09 (AB part, ABX system,

J _BX = 7.5 Hz, vA = 4.13, vB = 4.03, 4H). ¹³ C NMR: (CDC1 ₃ ) 50MHz δ (ppm): 36, 99 (d, ¹ cP = 88, 5Hz, 2C). ^31p NMR ^: (CDCI3 / H3PO4) 81.015MHz δ (ppm): 49.3 (s). IR (pure, film) cm ^-1 :

3020; 2998; 2942; 2870; 1490; 1384; 1258; 1200; 1120; 990. 27

c) Preparation of benzyl bis (chloromethylene) phosphinate (compound No. 4) of formula:

C9H11CI2O2P PM: 253.06

To 20g (110.3mmol, leq.) Of bis (chloromethylene) chlorophosphinate (compound n ° 2) dissolved in 400ml of anhydrous ether at 0 ° C, 16.9ml (121.3mmol, l, lq.) Of anhydrous triethylamine. Then added in 15 min, 13.11 g (121.3 mmol, 1.1 eq.) Of benzyl alcohol in 50 ml of anhydrous ether. After one hour at 0 ° C, the solution is warmed to room temperature and stirred for one hour. The precipitate formed is filtered, the filtrate is evaporated in vacuo and chromatographed (ethyl acetate / hexane: from 60/40 to 100/0); 24.6 g of a colorless oil are thus obtained (yield 88%). The characteristics of compound n ^c 4 are as follows: TLC:

Rf = 0.5 (Et ₂ 0). 1 H NMR: (CDCI3) 200 MHz δ (ppm): 7.45-7.37 (m, 5H, aromatic protons); 5.19 (d, ³ J _H _ _P = 9.3 Hz, 2H, H _a ); 3.66 (part AB, system ABX,

Hz, v _A = 3.72, v _B = 3.60, 4H, Hf). ¹³ C NMR: (CDCI3) 50MHz 28

δ (ppm): 134.93 (d, ³ J _c _ _P = 5, lHz, lCb);

128.63 (1Ce); 128.40 (2Cd); 127.97 (2Cc);

67.73 (d, ² J _c _ _P = 6.5 Hz, ICa); 32.63 (d, ¹ J _C _ _P = 104, 7Hz, 2Cf). ³¹ P NMR: (CDCI3 / H3PO4) 81., 015MHz

Ô (ppm): 41, 25 (s). IR (CH2CL2) c ^-1 :

3091; 3050; 3000; 2950; 2895; 1498; 1456; 1393; 1262; 1200; 1110; 1007; 847. d) Preparation of benzyl bis (dibenzylphosphonomethyl) phosphinate (compound No. 5) of formula:

C37H39O8P3 PM: 704.63

To 8 g (31.61 mmol, eq. Of benzyl bis (chloromethylene) phosphinate obtained in a), 44.5 g (126.4 mmol, 4 eq.) Of tribenzyl phosphite obtained in b) are added. The mixture is heated under vacuum to 140 ° C under 4 mm of mercury. And it is subjected to violent stirring in order to continuously remove the benzyl chloride. After 10 hours of stirring, the reaction mixture is chromatographed on a silica column (Et20 / ethyl acetate / Ethanol. From 100/0/0 to 0/80/20); 15.8 g of compound 4 are thus obtained in the form of a colorless oil (yield of 71%). 29

The characteristics of compound No. 4 are as follows:

CCM:

Rf = 0.4 (AcOEt). 1 H NMR: (CDC1 ₃ ) 200 MHz δ (ppm): 7.32-7.28 (m, 25H, aromatic protons); 5.13-4.95 (m, ^3J Hp = 8.5 Hz, 10 H, H _a and H _a ι); 2.84 (dd, ^2J H-pβ = 20.3 Hz, ² JH-Pα = 18.2 Hz, 4H _f ).

RM ¹³ C: (CDC13) 50 MHz δ (ppm): 135.77-135.57 (m, ^3J CP = 7.5 Hz,

4Cb 'and ÎC ^; 128.75-127.53 (80 ^, 2Cd>, 8C _C , 2C _c ι,

4C _e , lC _e .); 67.95 and 67.72 (2d, ^2J CP = 6.2 Hz, 4C _a) ; 66.79 (d, ^2J CP = 6.5 Hz, lC _a ; 28.65 (dd, ^1J CP = 130 Hz,

¹ CP = 87.5 Hz, 2C _f ).

³¹ P NMR: (CDC13 / H3P04) 121.4 MHz δ (ppm): 38.47 (t, ^2J p _α -Pβ ^{= 4} ' ^{5 Hz} ' Pα) and 20.90 (d, ^2J _Pα _ _P β = 4.5 Hz, _2P β)

MS: (IC / NH3) m / z: 721.8 (MNH4 +).

IR (pure, film) cm ^-1

3088; 3063; 3033; 2954; 2894; 1497; .1468; 1380; 1250; 1182; 998.

e) Preparation of (dibenzyl phosphonomethyl) (benzyl hydrogenphosphono-methyl) benzyl phosphinate (compound n "1).

A 0.796 g of 1,4-diazabicyclo [2,2,2] octane

(DABCO) (7.09 mmol, leq), 5 g of compound 4 are added

(7.09 mmol, leq) dissolved in 70 ml of anhydrous toluene. The reaction mixture is brought to reflux 30

during 2 hours. The solution is reduced in vacuo and the salt obtained is dissolved in 100 ml of a water / methanol mixture (30/70). To the above solution, an ion exchange resin loaded with H ₃ O ⁺ ions (Dowex 50X8) is added and then stirred for 12 hours. The resin is recovered by filtration, the filtrate is concentrated and it is taken up 3 times with 100 ml of toluene. 4.18 g of compound No. 12 are thus obtained in the form of a yellow oil which can be used without any purification (yield of 96%).

The characteristics of compound n ° 1 are the following 1 H-NMR: (DMSO d6) 200 MHz: δ (ppm): 7.37-7.31 (m, 20H, aromatic protons); 5.07 (d, ³ J _H _p = 7.2 Hz, 2H, H benzyls); 4.97 (d, ³ J _H _ _P = 7.4 Hz, 6H, benzyl H); 2.77 (t, ² J _H _ _P = 20, 1Hz, 4H, Hf). ¹³ C NMR: (CD ₃ OD) 50 MHz δ (ppm): 137, 82-137.38 (2Cb, lCb ', lCb "); 129, 60-128, 85 (4Cd, 2Cd', 2Cd", 2Ce, ICe ', ICe ", 4Cc, 2Cc', 2Cc"); 69.45 (d,

² J _C _ _P = 5.7 Hz) -68.75 (d, ² J _C _ _P = 5.7 Hz) 68.33 (d, ² J _C _ _P = 6.3 Hz) (2Ca, ICa * and lCa "); 29.62 (dd, ^x J _C _ _P = 87, 1Hz and ¹ J _c _ _P = 130.6Hz, 2Cf). ³¹ P NMR: (CDC1 ₃ / H ₃ P0 ₄ ) 81, 015MHz δ ( ppm): 41.21 (d, ² Jp_ _P = 4.7 Hz, Pα) and

18.59 (d, ² J _P _p = 5.08 Hz, Pβ and Pβ ').

MS: (IC / NH ₃ ) m / z: 631.9 (MNH ⁺ ); 524.2 (M-Bn + H ⁺⁾ • IR (pure, film) cm-l _:

3064-2300; 1693; 1610; 1498; 1455; 1382; 1215; 997; 854; 734.

Example 2: Preparation of 6-chloro-2 ', 3'-O-benzylidene (5'-hydroxy) purine-9H (compound n "6) of formula: 31

C17H15C1N404 PM: 374.78

a) Preparation of 2 ', 3'-O-benzylidene inosine (compound 7)

Ci7Hι ₆ N ₄ 0 ₅ PM: 356.34

20 g (74.5 mmol, 1 eq.) Of inosine are added to 56.7 g (372.5 mmol, 5 eq.) Of benzaldehyde dimethylacetal dissolved in 400 ml of anhydrous acetonitrile and the mixture is cooled to 0 °. vs. The mixture is treated dropwise with 13.9 ml (149 mmol, 2 eq.) Of freshly distilled phosphorus oxychloride. After 1 hour at 0 ° C, the medium is stirred for a further 2 hours at room temperature; the solution becomes yellow and clear then becomes cloudy. Then pour the 32

reaction mixture very slowly over a mixture of water saturated with NaHC ₃ / ice and the mixture is stirred for approximately 3/4 of an hour. A white precipitate appears, it is filtered, then washed with water and ether. After drying under vacuum, 23.9 g of 2 ′, 3′-O-benzylidene inosine are obtained (yield of 90%).

The characteristics of this compound are as follows: TLC: Rf = 0.3 (CH ₂ Cl ₂ / EtOH: 90/10)

F ° = 254-255 ° C RM ^ -H (CD3OD) 200MHz δ (ppm): 8,33 (s, 1H, H ₈ ), 8,08 (s, 1H, H2); 7,54-7, 6 (m, 5H, aromatic protons) 6.26 (d, ³ J _H1 ._ _H2 '= 3Hz, 1H, H _λ 1); 6, 01 (s, 1H, H _a ) 5.40 (dd, ³ J _H2 '-H'l = ³ Hz, ³ J _H2 - - _H 3 <= 5Hz, 1H, H ₂ •)

4.60 (m, ³ J _H4 ._ _H3 ι = 2.5Hz, 1H, H4.); 3.75 (m, 2H, H _5. ). ¹³ C NMR: (DMSO-d6) 50 MHz δ (ppm): 156.42 (1C ₆ ); 147.75 (104);

146.02 (1C ₂ ); 138.81 (1C ₈ ) 136.12 (1Cb); 129.71 (ICe); 128.37 (2Cd); 126.82 (2Cc) 124.42 (1C ₅ ); 106.58 (1Ca); 89.55 (10! '); 86.53 (1C _4. ) 84.44 (1C ₂ >), 82, 61 (1C _3. ); 61.46 (lC ₅ ι). IR (KBr) cm ^_1 :

3392; 3048; 2876; 1700; 1589; 1550; 1508; 1419; 1214; 1098; 974. b) Preparation of 2, 3 '-O-benzy-lidene (5'-acetyl) inosine (compound No. 8) of formula: 33

C19H18N O6 PM: 398.37

Is added to 24 g (67.35 mol, leq.) Of the 2,3'-0-benzylidene inosine obtained above, dissolved in 500 ml of anhydrous acetonitrile, 11.26 ml (80.82 mmol, 1.2 eq.) of anhydrous triethylamine, 7 ml (74.09 mmol, 1.1 eq.) of acetic anhydride and 822 mg (6.74 mmol, 0.1 eq.) of 4-dimethylaminopyridine (DMAP), and stirred for 2 hours at room temperature. The solvent is evaporated to obtain an oil. The product is then precipitated using the diethyl ether / CH2Cl2: 90/10 mixture. The precipitate is filtered, washed with ether and dried in vacuo. 23.34 g of

2 ', 3'-0-benzylidene (5'-acetyl) inosine in the form of a white powdery solid (87% yield).

The characteristics of this compound are as follows: TLC: Rf = 0.5 (CH2Cl2 / EtOH: 90/10). F ° = 170-171 ° C 1 H NMR: (CDCI3) 200 MHz δ (ppm): 8.27 (s, 1H, H ₈ ); 7.95 (s, 1H, H ₂ ); 7.60-7.43 (m, 5H, aromatic protons); 6.27 (d, ³ J _H1 ._H2 ' ^{= 1} _/ 9Hz, 1H, H _x .); 6.21 (s, 1H, Ha); 34

5.54

³ J _H2 <-H3 '= 6.5Hz, 1H,

H ₂ >); 5.13 (dd, ³ J _H 3'-H4 '=, Hz, ³ J _H3 ι _ _H2 ' = 6, 5Hz; IH,

H31); 4.67 (part X of an ABX, 1H, H.); 4.33 (AB system part ABX, J _a j -, = ll, 9Hz, J _A χ = 4.3Hz, J _B χ = 6, 1Hz, vA = 4.39, vB = 4.28.2H, H51); 2.02 (s, 3H; Hg). ¹³ C NMR: (DMSO-d6) 50 MHz δ (ppm): 169.87 (lCf); 156.46 (1C ₆ );

147.65 (1C) 146.02 (1C ₂ ) 139.02 (1C ₈ ) 135.88 (1Cb); 129.78 (1Ce) 128.37 (2Cd) 126.85 (2Cc) 124.42 (1C ₅ ); 106, 85 (1Ca) 89.09 (10 !!) 84.34 (IC4.) 83, 64 (1C ₂ -), 81.99 (1C _3. ) 63.56 (1C _5. ); 20.32 (lCg).

IR (CH ₂ Cl ₂ ) cm-l:

3370; 3066; 2990; 2900; 1745; 1689; 1588;

1547; 1513; 1460; 1374; 1229; 1093; 977. c) Preparation of the

6-chloro-2 ', 3' -O-benzylidene (5'-acetyl) purine-9H (compound n °) of formula:

C ₁ 9H17CLN4O5 PM: 416.82

20 g (50.20 mmol, leq.) Of 2 ', 3' -O-benzylidene (5 '-acetyl) inosine are added to a mixture of 150 ml of phosphorus oxychloride and 6.37 ml (50 , 20 mmol, leq.) Of N, N '-dimethylaniline. An oil bath is heated beforehand to 120 ° C. We shake it 35

vigorously mixed, it is brought for 1 minute and 30 seconds to reflux, then it is cooled to 0 ° C. and immediately distilled under vacuum with a water pump 100 ml of phosphorus oxychloride. The remaining solution is poured very slowly over a mixture of water saturated with NaHCO ₃ / ice, and stirred for approximately 30 minutes. The aqueous solution is extracted with methylene chloride. The organic phase is dried over sodium sulphate, reduced under vacuum and purified by chromatography on a silica column (ethyl ether / hexane: from 80/20 to 100/0); 16.11 g of 6-chloro-2 ', 3'-O-benzylidene (5'-acetyl) purine-9H are thus obtained (yield of 77%).

The characteristics of this compound are as follows:

CCM: Rf = 0.5 (Et2θ). 1 H-NMR: (CDC1 ₃ ) 200 MHz δ (ppm): 8.78 (s, 1H, H ₈ ); 8.26 (s, 1H, H ₂ ) 7.56-7.39 (m, 5H, aromatic protons) 6.32

IH, H ₂ . ); 6.06 (s, IH, Ha) 5.64 (dd, ³ J _H2 '_Hl' = ² , lHz, ³ J _H2 ._ _H3 . = 6, 5Hz, IH, H ₂ -)

4.70 (part X of an ABX, 1H, H ₄ 1); 4.30 (part AB syst.ABX, J _AB = 20Hz, J _7Λ χ = 4.4Hz, J _BX = 6Hz, vA = 4.37, vB = 4.25, 2H, H51); 1.97 (s, 3H; Hg). ¹³ C NMR: {CDCI3) 50 MHz δ (ppm): 1.69.94 (lCf); 152.00 (1C ₆ ); 151.54 (1C ₂ or IC4); 150.82 (1C ₂ or IC4); 144.23 (1C ₈ ); 135.36 (1Cb) 131.41 (1C ₅ ). 129.95 (1Ce); 128.46 (2Cd); 126.54 (2Cc) 108.01 (1Ca); 90.85 (lCι.); 8, 60 (104. And 1C ₂ •) 82.30 (1C ₃ ); 63.52 (IC51); 20.37 (lCg). d) Preparation of the

6-chloro-2 ', 3'-O-benzylidene (5'-hydroxy) purine-9H

(compound n ° 6). 36

8 g (19.19 mmol) of 6-chloro-2 ', 3'-O-benzylidene (5'-acetyl) purine-9H obtained previously are dissolved in 140 ml of a methanol solution saturated with ammonia, and the mixture is stirred for 2 hours at room temperature. The solution is reduced in vacuo and purified directly by chromatography (ethyl ether / hexane: 60/40 to 100/0); 5.89 g of 6-chloro-2 ', 3'-O-benzylidene (5'-hydroxy) purine-9H are thus obtained in the form of a white solid (yield of 82%).

The characteristics of this compound are as follows:

CCM:

Rf = 0.4 (Et ₂ O).

F ° = 190-191 ° C.

1 H-NMR: (CDC1 ₃ ) 200 MHz δ (ppm): 8.79 (s, 1H, H ₈ ); 8.22 (s, 1H, H ₂ );

7.60-7.46 (m, 5H, aromatic protons); 6.12 (d, Jhl'-H2 ^{I =} 4.6Hz, 1H, Hi.); 6.09 (s, 1H, Ha); 5.36 (dd, ³ J _H 2 '-HL' ^{= 4 6Hz>} ^3j H2 '-H3 ^{= 6'} ^2Hz ^'1H H ₂ <); 5.23 (dd, ³ J _H3 .-. _H 2 '= 6.2Hz, ³ J _H3 . _ _H. = 1, 3 Hz, 1H, H _3. ); 5.09 (dd, ³ J _Hf _ _H5 . = 2.7Hz, ³ J _Hf _ _H5 . = 9.8Hz, 1H, Hf); 4.73 (m, 1H, H ι); 3.93 (m, 2H, H _5. ). ¹³ C NMR: (DMSO-d6) 50 MHz δ (ppm): 151.58 (1 C ₆ ); 151.29 (1C ₂ or IC4)

149.29 (1C ₂ or IC4); 145.74 (1C ₈ ); 136.03 (lCb)

131.38 (1C ₅ ); 129, 66 (ICe); 128.31 (2Cd); 126.77 (2Cc) 106.38 (ICa); 90.32 (10 !.); 87.13 (104-); 84.29 (1C ₂ ') 82.59 (1C ₃ '); 61.33 (1C _5. ). MS: (IC / HN ₃ ) m / z: 375 (MH +). IR (KBr) cm ^-1 :

3400; 3069; 2926; 2876; 1593; 1565; 1493; 1434; 1406; 1339; 1201; 1111; 1072. 37

Example Preparation of

6-chloro-2 ', 3' -O-benzylidene 5 '[tetrabenzyl-α, β: ι

-dimethylene triphosphate] purine-9H (compound No. 10).

1

C47H46CLN4OHP3 PM: 971.27

To a solution of 1.19 g (1.93 mmol, leq.) Of compound No. 1, 726 mg (1.93 mmol, 1 eq.) Of 6-chloro-2 ', 3' -O-benzylidene ( 5 '-hydroxy) purine-9H (compound n ° 6) and 1.27 g (4.82 mmol, 2.5 eq.) Of triphenylphosphine dissolved in 30 ml of anhydrous THF, 759 μl is added dropwise (4 , 82 mmol, 2.5 eq.) Of ethyl azodicarboxylate at room temperature. After 1 hour, the solvent is evaporated off under reduced pressure and the residue is chromatographed on a silica column (ethyl ether / ethyl acetate / ethanol: from 100/0/0 to 0/90/10); 1.32 g of compound 10 are thus obtained in the form of a white powder (yield of 70%).

The characteristics of this compound are as follows: TLC: 38

Rf = 0, 5 (AcOEt / EtOH: 5/5). F ° = 49-50 ° C 1 H NMR: (CDCl3) 200 MHz, mixture of diastereoisomers: δ (ppm): 8, 75-8, 46 (m, 2H, H ₈ and H ₂ ); 7, 61-7, 2 (m, 25H, aromatic protons); 6.41-6.27 (4d, ³ J _H1 ._ _H 2 '= 2.6Hz, 1H, Hl'); 6.01-5.94 (6s, 1H, Hf); 5.58-5.52 (m, 1H, H2 '); 5.40-5.33 (m, 1H, H3.); 5.30-4.85 (m, 8H, Ha, Ha 'and Ha "); 4.60 (m, 1H, H4'); 4.38-4.04 (m, 2H, H5 '); 2 , 95-2, 55 (m, 4H, Hk and Hk '). ¹³ C NMR: (CDCI3) 50 MHz, mixture of diastereoisomers: δ (ppm): 151.58 (1C ₆ ); 151.15-150.96 (1C ₂ and 1C4); 144, 44-144, 35 (1C8); 135, 66-135, 18 (2Cb, lCb ', lCb "and lCg); 132.08-131.39 (105);

129.88-129.87 (2Ce, ICe ', ICe "and lCj), 128, 52-127, 92 (4Cd, 2Cd', 2Cd", 4Cc, 2Cc ', 2Cc ", 2Ch and 2Cj); 107, 92 (1Cf); 90, 70-90, 58 (ICI '); 90.23-90.22 (1C4 *);

85, 05-84, 44 (1C2 '); 82, 05-81, 78 (1C3'); 68, 41-61, 83 (2Ca, ICa ', ICa "and 1C5'); 30, 76-25, 70 (lCk and lCk ').

IR (CCl4) cm ^_1 :

3088; 3063; 3033; 2954; 2894; 1593; 1505; 1468; 1380; 1250; 1108; 1074. 39

Example 4: Preparation of N ⁶ - (4 -azidobutyl) -2 ', 3' -0- benzylidene-5 '- (tetrabenzyl-α, β: β, γ-dimethylene triphosphate) adenosine (compound No. ll) of formula :

C51H5

To a solution of 0.59 g (0.61 mmol, leq.) Of compound No. 10 in 20 ml of anhydrous methanol is added 0.20 g (1.74 mmol, 2.8 eq.) Of 4-azido-l-butylamine, to ambient temperature. The reaction mixture is stirred for 36 h, then it is reduced under vacuum and the residue is chromatographed on silica (ethyl acetate / ethanol: 100/0 to 90/10). 0.51 g of compound No. II is thus obtained in the form of a glassy solid (yield of 80%).

The characteristics of this compound are as follows:

TLC: Rf = 0.7 (ethyl acetate / ethanol: 90/10). 1 H NMR (CDCl 3) 200 MHz mixture of diastereoisomers: d (ppm) 8.36-7.95 (m, 2H, H8, H2); 7.41-7.26 (m, 25H, aromatic protons ¹ ); 6.31-5.92 (m, 3H, NH, Hl ', Hf); 5.53 (m, 1H, H2 '); 5.12-4.86 (m, 8H, Ha, Ha ', Ha "); 4.58 (m, 1H, H3'); 4.37-3.92 (m, 3H, H4 ', H5'); 3.62 (m, 2H, Ho); 3.25 (m, 2H, Hl); 2.95-2.65 (m, 4H, Hk, Hk '); 1.66 (m, 4H, Hm, Hn).

IR (CHC13) cm-1: 3291, 2942, 2096, 1619, 1458, 1251, 998, 828. 40

Example 5: Preparation of N ⁶ - (4-aminobutyl) -5'- (α.β .γ-dimethylene triphosphate) adenosine (compound No. 12) of formula:

C16H29N6O11P3 PM: 574

To a solution of 76 mg (72.5 μmol, leq.) Of compound No. ll in 3 ml of an ethanol / water mixture: 9/1, 76 mg of Pd / C at 10% and 460 mg (7.2 mmol, 100 eq) are added. .) of ammonium formate. The mixture is brought to reflux for 2 hours with the addition, in portions, of 3 ml of additional water in the medium. The mixture is filtered on paper and then the product is precipitated by the addition of ethanol. Centrifuge, the supernatant is discarded and the pellet is redissolved in 2 ml of water, then lyophilized. 34 mg of compound No. 12 are thus obtained in the form of a white hygroscopic powder (yield of 82%).

The characteristics of this compound are as follows:

1 H NMR (D20) 200 MHz: δ (ppm) 8.07 (s, 1H, H2 or H8);

7.78 (s, 1H, H2 or H8); 5.92 (d, ³ J _H ι '-H2' = 4.6Hz, Hl '); 4.50 (m, 1H, H2 '); 4.34 (m, 1H, H3 '); 3.98 (m, 2H, 41

H5 ^f ); 3.42 (m, 2H, Ha); 2.77 (m, 2H, Hd); 2.26-1.96 (m, 4H, He, Hf); 1.58 (m, 4H, Hb, Hc).

Example 6: Preparation of 3'-deoxy-3'-azido-5'- (tetrabenzyl-α, β: β, γ-dimethylene triphosphate) thymidine (compound No. 13) of formula:.

C40H43N5OHP3 PM: 862

To a solution of 165 mg (0.62 mmol, eq.) Of AZT in 5 ml of anhydrous toluene is added 380 mg

(0.62 mmol, eq.) Of compound No. l. The solution is evaporated to dryness, taken up with 5 ml of anhydrous toluene, then re-evaporated to dryness. The residue is dissolved in 2.5 ml of anhydrous THF and 0.5 ml (2.53 mmol, 4, leq.) Of diisopropyl azodicarboxylate is added. To this solution, 42

648 mg (2.47 mmol, 4 eq.) of triphenylphosphine dissolved in 2 ml of anhydrous THF are slowly added dropwise. The addition is carried out in 1 hour and the solution is further stirred for 3 hours at room temperature. The solvent is evaporated under vacuum and the residue is chromatographed on a silica column (ethyl acetate / ethanol: 100/0 to 85/15). 172 mg of a glassy solid are thus obtained (yield of 32%).

The characteristics of this compound are as follows:

TLC: Rf = 0.2 (ethyl acetate).

¹ H NMR (CDC1 ₃ ) 200 MHz, mixture of diastereoisomers: δ (ppm) 7.30 (m, 21H, aromatic protons, Ha); 6.22-6.01 (m, 1H, Hl ¹ ); 5.11-4.94 (m, 9H, Ha ', Ha ", Ha"', H3 '); 4.44-3.85 (m, 3H, H4 *, H5 '); 3.20-2.65 (m, 4H, Hc, Hd); 2.40-2.18 (m, 2H, H2 *); 2.07 (s, 3H, Hb).

43

Example 7: Preparation of 3'-deoxy-3'-amino-5'- (α, β: β, γ-dimethylene triphosphate) thymidine (compound No. 14) of formula:

Cl2H22 30ϋP ₃ PM: 477

To a solution of 82 mg (95, l μmol, leq.) Of compound No. 100 in 10 ml of ethanol is added 3 ml of THF and 69 mg of Pd / C at 10%. The mixture is carefully purged with hydrogen and stirred at room temperature for 1.5 hours under a pressure of 1 atmosphere of hydrogen. 5 ml of water are then added and the mixture is reduced by half under vacuum. The medium is purged with hydrogen and the mixture is stirred for another 2 hours under a pressure of 1 atmosphere of hydrogen. The mixture is then filtered on paper. The filtrate is washed twice with 10 ml of ethyl acetate and the aqueous phase is then lyophilized. 29 mg of a hygroscopic white solid are thus obtained (yield of 64%).

The characteristics of this compound are as follows 44

1 H NMR (D2O) 200 MHz: δ (ppm) 7.47 (s, 1H, Ha); 6.02

(m, 1H, Hl ¹ ); 4.62 (m, 1H, H3 ^" ); 4.19 (m, 1H, H4 ');

4.01 (m, 2H, H5 '); 2.65-2.25 (m, 6H, Hc, Hd, H2 '); 1.69

(s, 3H, Hb). Example 8: Preparation of a compound conjugate of compound No. 12 with keyhole limpet hemocyanin (KLH).

The coupling between compound n ^c 12 and keyhole limpet hemocyanin (KLH) is carried out via glutaraldehyde which reacts simultaneously with the primary amine functions carried by compound n ° 12 and the protein (lysine residues).

Practically:

To 2 ml of a solution of KLH (Keyhole Lympet Haemocyanin) at 5 mg / ml in phosphate buffer

10 ^~ 1M pH 7.4, 2 ml of a solution of compound no. 12 at 1.05 mg / ml are added successively, then 16 μl of a glutaraldehyde solution at

25%. After 2 hours of reaction at room temperature, the preparation is divided into 4 volumes of 1 ml and stored at -20 ° C until use.

The efficiency of the coupling is evaluated after filtration through a G25 column, by measuring the absorbance in the ultraviolet. These measurements showed that approximately 12 molecules of compound No. 12 were fixed by molecules of KLH (for an assumed molecular mass of 100,000 kDa).

Example 9: Preparation of anti-compound antibody No. 12 For this preparation, three 2.5 kg white Bouscat rabbits are used, which are treated as follows.

An emulsion is prepared by mixing 1 ml of the immunogen solution, ie 2.5 mg of the conjugate compound No. 12-KLH of Example 8, with 1 ml of distilled water and 2 ml of complete Freund's adjuvant . To the day 45

D0, each rabbit receives an injection of 1 ml of the above-mentioned emulsion intradermally, on the back at 50 points. On day D = 42, a first booster is performed under the same conditions as immunization, then the rabbits are bled once a week from Day D = 49.

Two reminders are made two months apart, under the same conditions.

In this way, collected in the blood serum collected antibodies specific for compound No. 12 are recovered.

Example 10: Preparation of a compound conjugate No. 12-acetyl cholinesterase from Electrophorus Electricus. The coupling between compound No. 12 and the acetylcholinesterase (AChE) of Electrophorus electricus (form G4) is obtained by means of a heterobifunctional reagent N-succinimidyl-4- (maleimidomethyl) cyclohexane-1-carboxylate ( CMCC). This method involves the reaction of a thiol group (previously introduced into compound No. 12) at the level of the primary amine function) with maleimide groups introduced on the surface of AChE by treatments with SMCC. a) Preparation of AChE-SMCC: at this stage, the active succinimidyl-ester function of the SMCC reacts with the primary amine functions of the enzyme. AChE is first treated with an excess of N-ethyl-maleimide so as to block all the thiol functions possibly present on the surface of the enzyme. The reaction with the SMCC is carried out in a 0.1M phosphate buffer, pH 7.4. The SMCC dissolved in anhydrous dimethyl formamide is added to the enzyme solution in a molecular ratio SMCC / AChE = 300. Practically: 46

50 μl of a 12.5 mg / ml solution of N-ethyl maleimide in 0.1M phosphate buffer pH 7.4 are added to 564 μl of an AChE solution (8.8 mg / ml) in the same stamp. After 30 minutes of reaction at 30 ° ^C. , 50 μl of a solution of SMCC (31.2 mg / ml) in anhydrous dimethyl formamide is added. After 30 minutes of reaction at 30 ° C., the AChE-SMCC is purified by filtration on a 1.5 m Biogel column. AChE-SMCC is stored at -80 ° C until use. b) Thiolation of Compound No. 12: Compound No. 12 is thiolated by reaction with the ester of S-acetyl-thioglycolic acid and N-hydroxysuccinimide (SATA). At this stage, the active succinimidyl ester function of SATA reacts with the primary amine function of compound No. 12. At 500 μl of a 2 mg / ml solution of compound No. 12 in 0.1M borate buffer pH 9 are added 50 μl of a 75 mg / ml SATA solution in anhydrous dimethyl formamide. After 30 minutes of reaction at 30 ° C, the reaction product (compound No. 12-SATA) is purified on a cartridge of Seppak Cl8. The methanolic eluate is lyophilized and stored at -20 ° C until use. c) Coupling between thiolated compound No. 12 and AChE-SMCC: the lyophilisate of compound No. 12-SATA is redissolved in a phosphate buffer 0, IM pH 6 then treated with hydroxylamine to unblock the thioester function of SATA . The released thiol functions are measured after reaction with 5-5'-dithiobis-nitrobenzoic acid (DTNB, Ellman method). The coupling between the thiolated compound No. 12 and the AChE-SMCC is obtained by mixing the two reagents in a thiol / AChE ratio of 10. Practically:

The lyophilisate of compound No. 12-SATA is taken up in 1.6 ml of phosphate buffer 0, IM pH 6 before 47

addition of 400 μl of hydroxylamine IM. After 10 minutes of reaction at room temperature, the thiols are measured by reaction of 150 μl of DTNB 10 ^{~ 3} M with 50 μl of thiol-containing compound No. 12. At this stage, the concentration of thiol groups is 4.4. 10 ^~ . 11.5 μl of thiol-containing compound No. 5 (5 nmol) are then added to 350 μl of a G4-SMCC solution (0.5 nmol). After 3 hours of reaction at 30 ° C, the conjugate is purified by filtration on a 0.5 m Biogel column and stored at -20 ° C until use.

Example 11: Enzyme-immunological determination of adenosine triphosphate (ATP) of formula:

Adenosine 5'-triphosphate

For this assay, micro-titration plates are used, the wells of which are coated with a mouse anti-rabbit IgG monoclonal antibody. 50 μl of a diluted antiserum solution obtained in Example 9, 50 μl of the conjugate obtained in Example 10 at a concentration of 1 unit / ml, and 50 μl of a solution are introduced into each of the wells Compound No. 12 or ATP standard. After overnight immunological reaction at + 4 ° C, the plates are washed with a 48

phosphate buffer, containing 0.05% Tween 20, then 200 μl of Ellan reagent used as substrate is added. After one hour of enzymatic reaction, the absorbance of each of the wells is measured at 414 n.

The results obtained are represented by curves 1 and 2 in FIG. 1.

Curve 1 relates to compound No. 12 and curve 2 relates to ATP.

These curves illustrate the variations in B / B0 (in%) as a function of the dose (in pmol / ml) of compound No. 12 or of assayed ATP. The two curves are very close and thus confirm that it is possible to assay ATP using the antibody directed against the ATP analog (compound No. 12) and the enzyme conjugate also produced from this analogue of ATP. EXAMPLE 12 Determination of Adenosine 5 Diphosphate (ADP)

Adenosine 5'-diphosphate

The same procedure is followed as in Example 11 to carry out this assay. The results obtained are illustrated by curve 3 in the figure. In view of this curve, it can be seen that the ADP can also be assayed with the antiserum and the enzyme conjugate, but the sensitivity is less good. Example 13: Determination of A-tetra-P of formula: 49

Adenosine 5'-tέtraphosphate The same procedure is followed as in Example 11 to carry out this assay using the same antiserum and the same enzymatic conjugate. The results obtained are illustrated by curve 4 in the figure.

Example 14: Determination of adenosine monophosphate AMP of formula:

Adenosine 5'-monophosphate The same procedure is followed as in Example 11 to carry out this assay using the same antiserum and the same enzymatic conjugate. The 50

results obtained are illustrated by curve 5 of the figure.

Examples 15 to 17

The same procedure is followed as in Example 11 for determining the adenosine, the dideoxy-ATP and the azido-ATP of formula:

Dideoxy ATP

Azido-ATP 51

using in each case the anti-serum and the enzyme conjugate of Examples 7 and 8. The results obtained are illustrated by curves 6 (adenosine), 7 (dideoxy-ATP) and 8 (azido-ATP) in the figure.

It is noted that these curves do not allow a satisfactory assay to be carried out, the affinity of these nucleotides for the antiserum not being sufficient.

Similarly, it has been found that adenine or the cyclic AMP of formula:

Adenosine 3'-5'-cycϋc monophosphate did not react with antiserum at doses greater than 10,000 pmol / ml. The results of the assays of Examples 9 to

15 show that it is possible to carry out a reliable assay of a nucleoside diphosphate or triphosphate, using antibodies directed against an analogue of this nucleoside diphosphate or triphosphate, as well as an enzyme conjugate prepared from this analogue.

In addition, it is noted that such antibodies do not recognize the azide nucleotides, the dideoxy nucleotides and the bases alone. 52

Thus, the recognition of the antibodies involves both the phosphate chain and the sugar, which guarantees the absence of cross-reaction with the natural nucleotides and nucleosides, in the case of the assay of phosphorylated metabolites of antiviral agents such as AZT .

Example 18: Preparation of 3'-deoxy-3'-azido-5'- (g, β: β, γ-dimethylene triphosphate) Nl- [5- (2,2,2- trifluoro-acetylamino) -pentyl] thymidine (compound no.15) of formula:

a) Preparation of 3'-deoxy-3'-azido-5'- p-methoxybenzyloxy- Nl- [5- (2,2,2-trifluoroacetylamino) -pentyl] thymidine (compound No. 16) of formula :

To a mixture of 3'-deoxy-3'-azido-5'-p-methoxybenzyloxy thymidine (2.33 g, 5.82 mmol, 1.0 eq.), Diisopropyl azodicarboxylate (2.86 ml, 53

14.54 mmol, 2.5 eq.) And 5- (2,2,2-trifluoroacetylamino) -1-pentanol (1.62 g, 8.15 mmol, 1.4 eq.) In 10 ml of anhydrous THF, triphenyl phosphine (3.81 g, 14.54 mmol, 2.5 eq.) is added in solution in 10 ml of THF, dropwise, at room temperature, in 10 min. The solvent is evaporated under vacuum and the residue is chromatographed on silica (diethyl ether / hexane: 25/75 to 100/0). 3.21 g of a white solid are obtained (yield: 94%).

The characteristics of compound 16 are as follows:

CCM: Rf = 0.45 (Et ₂ O)

¹ H NMR (CDC1 ₃ ) 200 MHz: δ (ppm) 7.99 (d, J = 3.4 Hz, 2H);

7.21 (s, 1H); 6.94 (d, 0 = 3.4 Hz, 2H); 6.78 (m, 1H);

6.19 (t, J = 6.3 Hz, 1H); 4.59 (qd, J = 6.2 Hz, J = 1.7 Hz,

2H); 4.39-4.30 (m, 1H); 4.21 (q, J = 2.0 Hz, 1H);

3.96-3.89 (m, 2H); 3.87 (s, 3H); 3.36 (q, J = 3.2 Hz, 2H); 2.61-2.28 (m, 2H); 1.79-1.59 (m, 7H); 1.42-1.35

(m, 2H).

IR (CHCl ₃ ) cm ^_1 : 3320.1; 2,916.0; 2105.2; 1713.1; 1639.2;

1469.1; 1258.8; 1168.2.

b) Preparation of 3'-deoxy-3'-azido-N-

1-5- (2,2,2-trifluoro-acetylamino) -pentyl] thymidine

(compound n ° 17) of formula:

# ₃ 54

Compound No. 16 obtained in a) (3.21 g 5.51 mmol, 1.0 eq.) Is stirred at room temperature with potassium carbonate (0.46 g, 3.30 mmol, 0.6 eq. .) in 40 ml of methanol. The reaction is followed in TLC. After 80 min, the solvent is evaporated, the residue is taken up in 50 ml of chloroform and is washed with a saturated aqueous NaCl solution. The organic phase is dried, evaporated and the residue is chromatographed on silica (Et ₂ 0). 1.90 g of a glassy solid are obtained (yield: 77%).

The characteristics of compound 17 are as follows:

TLC: Rf = 0.45 (Et ₂ O).

^X H NMR (CDC1 ₃₎ 200 MHz: δ (ppm) 7.49 (s, IH); 7.25 (m, 1H); 6.08 (t, J = 6.4 Hz, 1 H); 4.37 (m, 1H); 3.95-3.75 (m, 5H); 3.35-3.27 (m, 3H); 2.52 (2.24 (m, 2H); 1.87 (s, 3H); 1.70-1.59 (m, 4H); 1.40-1.32 (m, 2H). SM ( IC / CH ₄ ) m / z: 449 (MH ⁺ ).

IR (CHCl ₃ ) cm ^-1 : 3435.7; 3315.7; 3096.3; 2,941.8; 2105.1; 1704.4; 1634.5; 1471.4; 1185.1.

c) Preparation of 3'-deoxy-3'-azido-5'- (tetrabenzyl-α, β: β, γ-dimethylene triphosphate) - ³ N-1- [5- (2,2,2-trifluoro- acetylamino) -pentyl] thymidine (compound n ° 18) of formula:

55

Compound No. 17 above (100 mg, 0.223 mmol, 1.0 eq.), Tetrabenzyl-α, β: β, γ di ethylene triphosphoric acid (165 mg, 0.268 mmol, 1.2 eq.) And diethyl azodicarboxylate (175 μl, 1.11 mmol, 5.0 eq.) are suspended in 2 ml of anhydrous THF. A solution of triphenyl phosphine (292 mg, 1.11 mmol, 5.0 eq., In 5 ml of THF) is added dropwise at room temperature. The solvent is evaporated in vacuo and the residue is chromatographed (AcOEt / EtOH: 100/0 to 85/15) to give 151 mg of an amorphous solid (yield: 65%).

The characteristics of compound 18 are as follows:

TLC: Rf = 0.8 (AcOEt / EtOH: 9 / l)

RM ^ H (CDCL ₃ ) 200 Mhz, mixture of diastereomers: δ (ppm) 7.41-7.26 (m, 21H); 6.80 (m, 1H); 6.14 (m, 1H); 5.16-4.91 (m, 8H); 4.32-4.16 (m, 4H); 3.96-3.90 (m, 2H); 3.35 (q, J = 6, 1 Hz; 2H); 2.92-2.64 (m, 4H); 2.36-2.26 (m, 2H); 1.91-1.80 (m, 3H); 1.69-1.65 (m, 4H); 1.38-1.22 (m, 2H). IR (CHCl ₃ ) cm ^_1 : 3236.5; 3060.3; 2,931.0; 2108.3; 1713.4; 1637.0; 1249.1; 1178.6; 998.5.

d) Preparation of compound No. 15

Compound 18 above (67mg, 63.9 μmol,

1.0 eq.) In solution in 3 ml of anhydrous dichloromethane is treated dropwise with TMSBr (42 μl,

320μmol, 5.0 eq.) At room temperature. After 6 hours, the reaction medium is decomposed by the addition of a 5% ammonia solution (300 μl). The mixture is evaporated to dryness and the residue is purified by TLC (HPTLC RP18 F 254s plates, acetonitrile / water: 7/3). 56

The acetamide obtained is stirred for 24 hours at room temperature and in a sealed tube, in 3 ml of methanol saturated with ammonia. The solvent is evaporated off and the residue is purified by chromatography on reverse phase silica (RP 18) (acetonitrile / water: 100/0 to 0/100). 32 mg of a hygroscopic white solid are obtained which corresponds to the ammonium salt of the desired compound (yield: 76%).

The characteristics of compound 15 are as follows:

TLC: Rf = 0.45 (Et ₂ O).

RMî ^ H) (D ₂ 0) 200 Mhz, mixture of diastereomers: δ

(ppm) 7.55 and 7.49 (2s, 1H); 6.08 (m, 1H); 4.61-4.36 (m, 2H); 4.05-3.72 (m, 4H); 2.79 (t, J = 7.8 Hz, 2H);

2.34-2.03 (m, 6H); 1.50 and 1.47 (2s, 3H); 1.55-1.38 (m,

4H); 1.25-1.13 (m, 2H).

IR (KBr) cm ^_1 : 3214.0 (wide); 2109.3; 1702.1; 1637.5;

1472.9; 1185.6; 1055.0.

Example 19: Preparation of a compound conjugate of compound No. 14 with keyhole limpet hemocyanin (KLH).

The coupling between compound No. 14 and keyhole limpet hemocyanin is obtained via glutaraldehyde as in Example No. 8.

To 5 ml of a solution of KLH (Keyhole Lympet Haemocyanin) at 4 mg / ml in 10 ~ 1M phosphate buffer pH 7.4, 2.8 mg of compound n "14" are added successively then 26 μl of a 25% glutaraldehyde solution. After 2 hours of reaction at room temperature, the preparation is divided into 5 volumes of 1 ml and stored at -20 ° C until use. 57

The efficiency of the coupling is evaluated after filtration through a G25 column, by measuring the absorbance in the ultraviolet. These measurements showed that approximately 11 molecules of compound No. 14 were ^" fixed by molecules of KLH (for an assumed molecular mass of 100,000 kDa).

Example 20 Preparation of Anti-Compound Antibody No. 14

Three 2.5 kg white Bouscat rabbits are used for this preparation, which are treated as follows.

An emulsion is prepared by mixing 1 ml of the immunogen solution, ie 4 mg of the conjugate compound No. 14-KLH of Example 19, with 1 ml of distilled water and 2 ml of complete Freund's adjuvant. On day D0, each rabbit receives an injection of 1 ml of the abovementioned emulsion by the intradermal route, on the back at 50 points. On day D = 42, a first booster is performed under the same conditions as immunization, then the rabbits are bled once a week from Day D = 49.

Two reminders are made two months apart, under the same conditions. In this way, collected in the serum of the blood collected, antibodies specific for compound No. 14 are recovered.

Example 21: Preparation of a compound conjugate No. 14-acetyl cholinesterase from Electrophorus Electricus.

The coupling between compound n ° 14 and the acetylcholinesterase (AChE) of Electrophorus electricus (form G4) is obtained by means of a heterobifunctional reagent N-succinimidyl-4- (maleimidomethyl) 58

cyclohexane-1-carboxylate (SMCC). This method involves the reaction of a thiol group (previously introduced into compound no. 14) at the level of the primary amine function) with maleimide groups introduced onto the surface of AChE by treatments with the SMCC as described in example 10.

a) The preparation of AChE-SMCC is the same as that described in example n ° 10.

Practically: b) Thiolation of Compound No. 14: Compound No. 14 is thiolated by reaction with the ester of S-acetyl-thioglycolic acid and N-hydroxysuccinimide (SATA). At this stage, the active succinimidyl-ester function of SATA reacts with the primary amine function of compound No. 14. At 300 μl of a 1.6 mg / ml solution of compound No. 14 in 0.1M borate buffer pH 9 are added 38 μl of a SATA solution at 115 mg / ml in anhydrous dimethyl formamide. After 30 minutes of reaction at 30 ° C, the reaction product (compound No. 14-SATA) is purified on a cartridge of Seppak C18. The methanolic eluate is lyophilized and stored at -20 ° C until use. c) Coupling between the thiolated compound No. 14 and the AChE-SMCC: the lyophilisate of the compound No. 14-SATA is redissolved in a 0.1M phosphate buffer pH 6 then treated with hydroxylamine to unblock the thioester function of the SATA . The released thiol functions are measured after reaction with 5-5'-dithiobis-nitrobenzoic acid (DTNB, Ellman method). The coupling between thiol-containing compound No. 14 and AChE-SMCC is obtained by mixing the two reagents in a thiol / AChE ratio of 10. Practically: 59

The lyophilisate of compound No. 14-SATA is taken up in 0.8 ml of 0.1M phosphate buffer pH 6 before adding 200 μl of 1M hydroxylamine. After 10 minutes of reaction at room temperature, the thiols are measured by reaction of 100 μl of 10 ^" 3M DTNB with 100 μl of thiol-containing compound No. 14. At this stage, the concentration of thiol groups is 1.3 × 10 ^{~ 3} M. 1.9 μl of thiol-containing compound No. 14 (5 nmol) are then added to 175 μl of a G4-SMCC solution (0.25 nmol). After 3 hours of reaction at 30 ° C., the conjugate is purified by filtration on a 0.5 m Biogel column and stored at -20 ° C until use.

Example No. 22: Preparation of a compound conjugate of compound No. 15 with keyhole limpet hemocyanin (KLH).

The coupling between compound n ° 15 and keyhole limpet hemocyanin is obtained by means of glutaradéhyde as in example n ° 8 and example n ° 19.

To 3.5 ml of a solution of KLH (Keyhole Lympet Haemocyanin) at 4 mg / ml in 10 ^-1 M phosphate buffer pH 7.4, 4.3 mg of compound No. 15 is added successively then 26 μl d '' 25% glutaraldehyde solution. After 2 hours of reaction at room temperature, the preparation is divided into 5 volumes of 1 ml and stored at -20 ° C until use.

The efficiency of the coupling is evaluated after filtration through a G25 column, by measuring the absorbance in the ultraviolet. These measurements showed that approximately 13 molecules of compound No. 15 were fixed by molecules of KLH (for an assumed molecular mass of 100,000 kDa). 60

Example 23 Preparation of Anti-Compound Antibody No. 14

An emulsion is prepared by mixing 1 ml of the immunogen solution, ie 4 mg of the conjugate compound No. 15-KLH of Example 22, with 1 ml of distilled water and 2 ml of complete Freund's adjuvant. On day D0, each rabbit receives an injection of 1 ml of the abovementioned emulsion by the intradermal route, on the back at 50 points. On day D = 42, a first booster is performed under the same conditions as immunization, then the rabbits are bled once a week from Day D = 49.

Two reminders are made two months apart, under the same conditions.

In this way, collected in the serum of the blood collected antibodies specific for compound No. 15 are recovered.

Example 24: Preparation of a compound conjugate No. 14-acetyl cholinesterase from Electrophorus Electricus.

The coupling between compound No. 15 and the acetylcholinesterase (AChE) of Electrophorus electricus (form G4) is obtained by means of a heterobifunctional reagent the

N-succinimidyl-4- (maleimidomethyl) cyclohexane-1-carboxylate (SMCC). This method involves the reaction of a thiol group (previously introduced into compound No. 15) at the level of the primary amine function) with maleimide groups introduced on the surface of AChE by treatments with SMCC as described in example 10. 61

a) The preparation of AChE-SMCC is the same as that described in example n ° 10. b) Thiolation of Compound No. 15: Compound No. 15 is thiolated by reaction with the ester of S-acetyl-thioglycolic acid and N-hydroxysuccinimide (SATA). At this stage, the active succinimidyl-ester function of SATA reacts with the primary amine function of compound No. 14. At 300 μl of a solution at 2.2 mg / ml of compound No. 15 in 0.1M borate buffer pH 9 are added 38 μl of a SATA solution at 115 mg / ml in anhydrous dimethyl formamide. After 30 minutes of reaction at 30 ° C, the reaction product (compound No. 15-SATA) is purified on a cartridge of Seppak C18. The methanolic eluate is lyophilized and stored at -20 ° C until use. c) Coupling between thiol-containing compound No. 15 and AChE-SMCC: the lyophilisate of compound No. 15-SATA is redissolved in a 0.1M phosphate buffer pH 6 then treated with hydroxylamine to unblock the thioester function of SATA . The released thiol functions are measured after reaction with 5-5'-dithiobis-nitrobenzoic acid (DTNB, Ellman method). The coupling between thiol-containing compound No. 15 and AChE-SMCC is obtained by mixing the two reagents in a thiol / AChE ratio of 10. Practically:

The lyophilisate of compound No. 15-SATA is taken up in 0.8 ml of 0.1M phosphate buffer pH 6 before adding 200 μl of 1M hydroxylamine. After 10 minutes of reaction at room temperature, the thiols are measured by reaction of 100 μl of DTNB 10 ^{~ 3} M with 100 μl of thiol-containing compound No. 15. At this stage, the concentration of thiol groups is 9.10 ^-4 M. Then add 2.8 μl of thiol-containing compound No. 14 (5 nmol) to 175 μl of a G4-SMCC solution (0.25 nmol) . After 3 hours of 62

reaction at 30 ° C, the conjugate is purified by filtration through a 0.5 m Biogel column and stored at -20 ° C until use.

Claims

63

CLAIMS 1. Compound conjugate of a diphosphate or triphosphate analog and a molecule chosen from antigenic carriers and enzymes, in

where n is 0 or 1,

- χl and X ² which may be identical or different, represent CH ₂ , CHF, CF ₂ , CC1 ₂ , CHCl or NR ⁶ with R ⁶ being a hydrogen atom, an alkyl, aryl or aralkyl group, the two R ^ which may be identical or different when n = 1 or which may together form a hydrocarbon chain comprising a phenyl ring,

- R ¹ , R ² , R ³ and R ⁴ which may be the same or different, represent a hydrogen atom, NH4 ^" , a quaternary ammonium ion or an ion of formula M ⁺ / _v with M representing a metal and v being the valence state of this metal, and

- R ⁵ is a group derived from a nucleoside, and in which the analog is coupled to said molecule, either via the group R ^ derived from nucleoside, or via one of the groups R- '-, R ² , R ³ and R ^ provided that XI does not represent CH ₂ when n is equal to 0.

2. A conjugate compound according to claim 1, characterized in that n is equal to 1.

3. A conjugate compound according to claim 1 or 2, characterized in that R ^ is derived from an nucleoside antiviral agent. 64

4. A conjugate compound according to any one of claims 1 to 3, characterized in that R ^ corresponds to one of the following formulas:

AZT AMT ddl ddC d4T

5. A conjugate compound according to any one of claims 1 to 4, characterized in that said molecule is an antigenic carrier.

6. A conjugate compound according to claim 5, characterized in that the antigenic carrier is keyhole limpet hemocyanin. 7. Conjugate compound according to claim

5 or 6, characterized in that the coupling of the diphosphate or triphosphate analog with the antigenic carrier is carried out by means of a spacer arm formed by a saturated hydrocarbon chain, terminated by 65

an NH2 group, grafted on the purine or pyrimidine base of R ^.

8. A conjugate compound according to claim 5 or 6, characterized in that the coupling of the diphosphate or triphosphate analog with the antigenic carrier is carried out by means of a spacer arm made of polyoxyethylene (CH2-CH2 ~ 0) _n or polylol

Conjugate compound meeting the

OR ⁷ - (CH 2 ^y _P —NH — Z (D)

in which n, X * and X ² have the meanings given in claim 1, R ⁷ is a bivalent group derived from '' a nucleoside, p is equal to 0 or is an integer ranging from 1 to 6, and Z is a antigen carrier.

10. Conjugate compound according to claim 9, characterized in that R ⁷ (CH2) _p NH corresponds to one of the following formulas

NH- 66

11. A conjugate compound according to any one of claims 1 to 4, characterized in that said molecule is an enzyme.

12. A conjugate compound according to claim 11, characterized in that said enzyme is an acetylcholinesterase.

13. Conjugate compound of the triphosphate analog of formula:

and Electrophorus electricus acetylcholinesterase.

14. Process for the preparation of antibodies specific for a nucleoside diphosphate or triphosphate of formula:

67

in which n = 0 or 1 and R represents a nucleoside derivative, which consists

to inject a mammal with a conjugate compound of a nucleoside diphosphate or triphosphate analog of formula:

in which R ¹ , R ² , R ³ , R ⁴ , X ¹ and X ² have the meanings given in claim 1), and n and R ⁵ have the meanings given above for the formula (V), coupled to an antigenic carrier, and

- then to draw the blood of the immunized mammal to isolate the antibodies produced.

15. Immunological assay method for a nucleoside diphosphate or triphosphate of formula:

68

in which n = 0 or 1 and R ′ ⁵ represents a nucleoside derivative, characterized in that a competition reaction is carried out between the diphosphate or triphosphate of formula (V) to be assayed and a conjugated compound of an enzyme and a diphosphate or triphosphate analog of formula:

wherein R ⁵ is a nucleoside derivative identical or different from R ' ⁵ , has the meaning given above, and R ¹ , R ² , R ³ , R ⁴ , X ¹ and X ² have the meanings given in claim 1, for limited sites of an antibody specific for said analog, and in that the quantity of conjugated compound fixed on the antibody is determined. 16. Method according to claim 15, characterized in that the diphosphate or triphosphate to be assayed is adenosine triphosphate (ATP) or a metabolite of a nucleosic antiviral agent.

17. Method according to claim 1, characterized in that the antiviral agent is 3'-azido-3'-deoxythymidine (AZT).