FR2721931A1 - CONJUGATES OF DIPHOSPHATE OR THREE-PHOSPHATE ANALOGS WITH ENZYMES OR ANTIGENIC PROTEINS AND IMMUNOLOGICAL ASSAYS USING SUCH CONJUGATES - Google Patents

Abstract

The invention relates to conjugates of analogues of diphosphates or triphosphates with enzymes or antigenic carriers and to an immunoassay using such conjugates.In these conjugates, the analog of diphosphate or triphosphate has the formula: (CF DRAWING IN BOPI ) where n is 0 or 1, X ** 1 and X ** 2 which can be CH2, CHF, CF2, CC12, CHC1 or NR ** 6, R ** 1, R ** 2, R ** 3 and R ** 4 represent a hydrogen atom, NH4 +, a quaternary ammonium ion or M ** + 1 / v, and R ** 5 is a group derived from a nucleoside, and it is coupled to the enzyme or the antigenic carrier, either through the R ** 5 group derived from nucleoside, or through one of R ** 1, R ** 2, R ** 3 and R * * 4.Such conjugated compounds can be used for the assay of phosphorylated metabolites of anti-viral agents such as AZT.

Description

CONJUGATES OF ANALOGUES OF DIPHOSPHATES OR TRIPHOSPHATES

  WITH ENZYMES OR ANTIGENIC CARRIERS AND DOSAGE

  IMMUNOLOGY USING SUCH CONJUGATES.

DESCRIPTION

  The subject of the invention is conjugated compounds formed of a diphosphate or triphosphate analog and a molecule selected from the group consisting of

  antigenic carriers and enzymes.

  More specifically, it relates to conjugated compounds wherein 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).

  Indeed, living cells capture, store, and transport energy in chemical form, mainly 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 ATP thanks to a solar energy input into the photosynthetic cells , or chemical energy in

animal cells.

  ATP is the main connecting link between two large sets of enzyme catalyzed reactions in cells. One of these sets conserves the chemical energy from the environment by causing phosphorylation of energy-poor ADP, energy-rich ATP. The other set uses ATP energy to biosynthesize cell components from simple precursors, to do the mechanical work necessary for contraction and locomotion, or to perform osmotic membrane transport work. These sets of enzyme catalyzed reactions are virtually identical in all living species. Also, ATP or ADP can be used in many techniques, for example to immobilize, guide or transport molecules.

having a biological interest.

  However, these techniques are limited because of the low chemical stability of ATP and

  ADP (hydrolysis of phosphoric anhydride linkages).

  It would therefore be of great interest to have analogs of ATP or ADP, not hydrolyzable

to fulfill these functions.

  Other interesting analogs of diphosphates and triphosphates are the diphosphates and triphosphates of nucleoside anti-viral agents such as 3'-azido-3'-deoxythymidine (AZT), dideoxyinosine (ddI), dideoxycytidine (ddC) and 2 ', 3'-dehydro-3'-deoxy-thymidine (d4T), which are

anti-HIV drugs.

  Indeed, conjugated compounds obtained by coupling analogs of diphosphates or triphosphates of nucleoside antiviral agents with biological molecules would be of great interest to carry out immunological assays of the phosphorylated metabolites of these antiviral agents in patients subjected to a

treatment by such agents.

  One of the major problems encountered in the therapeutic treatment of AIDS is the extreme variability of response from one patient to another, this being further complicated by the appearance of drug resistance phenomena. For example, one of the overriding goals of the last Berlin meeting on AIDS (9th International AIDS Conference, Berlin, 7-11 June 1993) is to quantify the active metabolites of

  different antivirals administered.

  For almost all these nucleoside molecules, the mechanism of action involves the phosphorylation of nucleotides 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 viral DNA strands. in the course of growth, playing thus a role of terminator of chain. Aside from side effect problems, virus strains that have become resistant to AZT have also recently been isolated. This led to the placing on the market of other drugs such as ddI or ddC

  the 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 rate of phosphorylation and their affinity for reverse transcriptase, but also with their possible catabolism (under the action of phosphorylases, phosphatases, deamidases ...). Given this, it is clear that from one patient to another, we will observe

  a great variability in the response to antivirals.

  Thus, the possibility of measuring the concentrations of phosphorylated metabolites rapidly and effectively in each treated individual should allow a better adjustment of the dosage and a follow-up.

more effective therapeutic.

  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) (Molema, G. et al., J. Chromatogra (1992), 579, 107-114). Dosages

  radioimmunoassays (RIA) have been described (S.

  Tedepalli and R. Quinn in Clin. Chem. (1989), 35, 1187) and RIA assay kits have been commercially available ("The Immunoassay Kit Directory", Part 4 (1992), E. Naysmith, Kluwer Academic Publishers, Lankaster, UK). other immunoassay techniques based on the use of non-radioactive markers such as FPIA

  Fluorescence polarization immunoassay) (G.G.

  Granich et al. - Antimicrob. Agents Chemother. (1989) 33, 1275-1279 or the ELISA (Immunoabsorbent Assay Linked to

  an enzyme) (S. Tedepally, R. Quinn - J. Acq Imm.

  Syndr. (1990) 3, 19-27). Finally, a microbiological technique has been described (H. Etesse-Carsenti and

al. AIDS (1990) 4, 265).

  HPLC techniques are the most used. They have the advantage of allowing a

  quantitative phosphorylated metabolites of AZT.

  However, they are limited in terms of sensitivity (-lng / ml) (large blood sample to have a sufficient number of cells) and they require the use of extraction processes (problems related to plasma interference). Immunoanalytical techniques are more sensitive (-O, lng / ml), simpler to implement, but those described so far do not discriminate AZT phosphorylated metabolites for reasons related to the strategy of obtaining the antibodies necessary for the assay. The research was therefore continued to develop a method for assaying metabolites of antiviral agents, satisfying the following requirements: - sensitive (to avoid taking too much test and minimizing non-specific interference by dilution), - specific (ability to specifically measure AZT and its phosphorylated metabolites particularly 5'triphosphate, - simple (be able to avoid purification steps, be adapted to large batch assays, be able to transfer technology without difficulty), and - little costly (with the exception of the investment to develop the tools of the method, it must subsequently require

simple and inexpensive).

  In this regard, immuno-analytical techniques are and remain unambiguously the most appropriate. They have not so far been satisfactory for the determination of phosphorylated metabolites because the immuno-analysts have been faced with the difficulty of obtaining antibodies specific for these metabolites. In fact, AZT-5'-triphosphate of formula: ## STR2 ##

OH OH H

  N3 which will be taken as an example, belongs to the category

haptens.

  It is necessary to covalently couple the molecule to an antigenic carrier (for example bovine serum albumin) and to administer the conjugate to an animal (rabbit or mouse, for example) to elicit an immune response likely to induce synthesis. antibodies specific for the hapten. If we want to direct the specificity towards the triphosphate chain, it will be necessary to couple with albumin another part of the molecule in order to present the immune system the desired epitope (together triphosphate chain + sugar). As the acid anhydride bonds of POPs involved in the triphosphate chain are particularly unstable, it is quite uncertain (given the short half-life of this compound), to hope to induce an immune response against a molecule of this type. type no

transformed.

  The subject of the present invention is precisely conjugated compounds of diphosphate analogs and triphosphates of nucleosides coupled to antigenic carriers, which precisely make it possible to obtain antibodies specific for the metabolites of these anti-viral agents. These compounds must have the dual feature of: - possessing phosphate bonds (di or

sort) stable,

  - mimic the natural phosphate bonds as well as possible so that the induced antibodies recognize the natural nucleotides. It also relates to conjugate compounds of diphosphate analogues or triphosphates of nucleosides coupled to enzymes, which are useful as markers in assays.

  enzymo-immunological properties of these metabolites.

  According to the invention, the conjugated compound of a diphosphate or triphosphate analogue and of a molecule selected from antigenic carriers and enzymes, wherein the analog corresponds to the formula: R ## STR2 ## P-Ol

O O O

  ORO wherein n is 0 or 1, - X1 and X2 which may be the same or different, represent CH2, CHF, CF2, CC12 CHC1 or NR6 with R6 being hydrogen, alkyl, aryl or aralkyl , the two R6 may be identical or different when n = 1 or may together form a hydrocarbon chain having a phenyl ring, R1, R2, R3 and R4 which may be the same or different, represent a hydrogen atom, NH4 +, an ion quaternary ammonium or an ion of formula M + 1 / v with M representing a metal and v being the valence state of this metal, and - R5 is a group derived from a nucleoside, and wherein the analog is coupled to said molecule, either via the group R5 derived from nucleoside, or via one of the

groups R1, R2, R3 and R4.

  The use in this conjugated compound of an analog of diphosphates or triphosphates corresponding to the formula (I) given above, is very interesting because the replacement of the polyphosphate chain POP of the natural diphosphate or triphosphate by a P-chain X1-P or P-X1-P-X2-P, leads to

  a stable non-hydrolyzable compound.

  Thus, it is possible to induce the synthesis of anti-viral anti-triphosphate antibodies, for example anti-AZT-5'-triphosphate antibodies using this chemical analogue 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 analogue recognize AZT-5'triphosphate with high affinity. 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 diphosphate or

  Nucleoside triphosphate is an antigenic carrier.

  Generally, the coupling is carried out via a spacer arm grafted on the pyrimidine or purine nucleoside base, which comprises at its other end a group capable of reacting

  with the antigenic carrier, for example an NH 2 group.

  The grafting of the spacer arm on the nucleoside may be carried out either on an exocyclic NH 2 group in the case where the base of the nucleoside comprises such a group, or on a nitrogen atom present in the base ring. It can also be done on sugar or

on the phosphorus chain.

  The spacer arm is generally a saturated hydrocarbon chain. However, other spacers may be used, for example a

  polyoxyethylene (CH 2 -CH 2 -O) n or a polyol - (CHOH) n.

  In some cases, the coupling can also be carried out directly on the nucleoside via a spacer fixed on the

sugar part of the nucleoside.

  In all cases, the group capable of reacting with the antigenic carrier is an NH 2 group, but it is also possible to use other groups capable of reacting with the antigenic carrier, by

  example a COOH group, maleimide, thil, aniline ...

  When the group capable of reacting with the antigenic carrier is an NH 2 group, the conjugate compound comprising a diphosphate or triphosphate analog coupled to an antigenic carrier may correspond to the following formula:

O O O

HOlP- 1 il X2 7

HO-P-P X-P-OR (CH 2 -NH-Z (I)

OH OH OH

  where R7 is a divalent group derived from a nucleoside, n is 0 or 1, X1 and X2 have the meanings given above, p is 0 or is an integer from 1 to 6 and Z is an antigenic carrier. For example, R7- (CH2) p-NH- may be of the following formulas: o

H N NH

HO OH '

  o N I. oN. Preferably, in the analog of formula (I), R5 is derived from a nucleoside antiviral agent such as those of the formulas: ## STR2 ## Neither AZT-2dC d4T Antigenic carriers capable of coupling to the nucleoside diphosphate or triphosphate analog to form the conjugated immunogenic compound may be of different types. In particular, proteins such as bovine albumin, ovalbumin, polylysine and 0-lactoglobulin can be used, for example 11. Patelle's hemocyanin (Keyhole lympet haemocyanin, KLH). According to a second embodiment of the invention, the conjugated compound is an enzymatic marker. In this case, the molecule coupled to the diphosphate or triphosphate analog of

nucleoside is an enzyme.

  The enzymes that can be used are those with interesting properties (sensitivity,

  threshold of detection) for immunological assays.

  By way of examples of such enzymes, mention may be made of peroxidases, phosphatases and 1-galactosidase, in particular acetylcholinesterases such as acetylcholinesterase.

of Electrophorus electricus.

  This coupling can also be carried out by means of appropriate reagents on an NH 2 group of the nucleoside of the diphosphate or triphosphate analog. By way of example of such a conjugate, mention may be made of the conjugate of the triphosphate analog of formula CH 4 and OH 2 O 2

OH OH OH OOH

  coupled with acetylcholinesterase.

  The nucleoside diphosphate and triphosphate analogs used in the invention can be prepared by reacting a diphosphate or triphosphate of the formula ## STR2 ##

OR 'OR

  wherein X1, X2 and n have the meanings given above, and R8 is a hydrocarbon group, with a nucleoside derivative whose reactive functions are protected by appropriate groups except for the primary alcohol function of the part

nucleoside sugar.

  Diphosphates or triphosphates of formula III ## STR1 ##

OR8 OR OR8

  with n = 1 and X1 = X2 = CH2 can be prepared by reaction of a bis-

  (halomethylene) phophinate of formula: O

XCH2 P CH2X

  wherein X is a halogen atom, with a mixed phosphite of formula: R80 -P --oRi OR wherein R8 is a hydrocarbon group and R9 is a benzyl or allyl group to obtain a compound of formula (III) with X1 = X2 = CH2 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- (halo.ethylene) 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 R8OH, R9OH

on PCl3 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 subjected to violent stirring in order to eliminate continuously

the formed halide R9X.

  The triphosphateS of formula (III) in which X1 and X2 represent the NR6 group with the two R6 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 : O I-. R8ai P Ci OR8 with a diamine of formula:

H2 N-R10-NH2

  wherein R8 is a hydrocarbon group, and R10 is the hydrocarbon chain formed by 2R6, to form a bisphosphoramide of the formula:

O O

he 10 Ila

R O- P NH -R -NH -P - OR

  OR25 b) cyclizing the bis phosphoramide thus obtained on a dichlorophosphite of formula:

C12P-OR 8

  wherein R8 is a hydrocarbon group, and

  c) oxidizing the cyclized product thus obtained.

  The diamine used as 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 fast and it makes it possible to obtain diphosphoramide with a high yield. The diphosphoramide is then cyclized with dichlorophosphite in the presence of triethylamine and 4-dimethylaminopyridine, in a suitable organic solvent such as

tetrahydrofuran.

  The oxidation of the cyclized product can be

  then carried out with 4-chloroperoxybenzoic acid.

  After this reaction, a triphosphate analog of formula (I) is obtained in which X 1 and X 2 are NR 6 and R 1, R 2, R 3, R 4 and R 5 are benzyl groups. In order to prepare a triphosphate analogue of formula (III) in which X 1 and X 2 are respectively CH 2 and NR 6, a process may be used which comprises reacting a methyl phosphonate of the formula:

O P CH

  Ow P CH Ra with a diphosphate analog of the formula: II l 11 Cl P-N P-OR8

OR8 R8

  to form the compound of the formula: ## STR2 ##

R C-P C-H2 P-N - OR8

OR OR8 18

  OR 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 1 is a methylene group may be prepared by a process which comprises reacting a chlorophosphate of the formula:

R80 CP CI

  OR8 with a methylphosphenate of the formula:

O P -CH3

  The diphosphates of formula (III) in which X1 is the NH group may be prepared by a process which comprises reacting the compound of formula: Ci3P-N PP Cl

3 1

this

C13P N - N - C

  C1 with a compound of formula R8OH to form the compound of formula:

O O

R80 P NH -P OR8

I

0 R 'OR8

  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 diphosphates of formula (III) in which X 1 represents the NR 6 group, these can be prepared by reaction of a compound of formula:

0 R6 0

he 1 He CI-P -N -P -Cl

I I

CI Cl

with an alcohol of formula R80H.

  By way of examples, the reaction scheme for the preparation of a non-hydrolyzable analogue of ATP equipped with a spacer arm is described below with a view to

  its coupling with an antigenic carrier or an enzyme.

Cl

1,, N ,,, N .. N

08 08 08n1

H2N, NN, NH

  ## STR2 ##

CH, -O I -CI I Y "N

I'l N '* n-C2PH--

OH5nOHn OH 08n OB n O08n

HO OH 0O0

  H2 N i6SH The coupling between the triphosphate analog and the nucosidemodifi is carried out by activation of the alcohol at the 5 'position on the sugar to form the nucleoside triphosphate. The chlorine on the nuclHoside base is then displaced by nuclear substitution with a diamine precursor. The molcule

  final is obtained by catalytic hydrogenation.

  In the case where a non-hydrolyzable coupling of AMT triphosphate is desired, the nucleoside-modified is accomplished by activation of the 5'-alcohol on the sugar to form the nucleoside triphosphate. The chlorine on the nucleoside basis is then displaced by nucleophilic substitution with a diamine precursor. The final molecule is obtained by catalytic hydrogenation. In the case where it is desired to prepare a non-hydrolyzable analogue of AMT triphosphate, the following reaction scheme can be followed:

_11NH N

  ## EQU1 ## In OBn OB n OB,

III N N

O

N3 N.]

o

0 0 O

It is 0 to HO-P 012-P CH2 P-j

OH OH OIH

  NH2 The coupling between the nucleoside and the triphosphate analogue is achieved by activation of the primary alcohol of the sugar which reacts

  then with the triphosphate analogue. The final deprotection is carried out as above

  catalytic hydrogenation.

  In the case where it is desired to prepare a non-hydrolyzable analogue of AZT triphosphate, the following reaction scheme may be used: ## STR2 ## 1 C

OHOHO _

OH N] N1

o o oo 0 00 N 0k 0

o, o o 'M M.l-

  l he N O BnO-P CH, P CH, P-0 Oan OBn OSn 1

At N 1 N:

V- N N 'NH,

  0-0 0.-CH.-oi HO - CHZ P CH2 Pl y0O

OH OH OH

  NS In this case, the nucleoside is prepared in two steps from a thymidine derivative described in the literature. The coupling with the triphosphate analogue is carried out as above. The final deprotection is carried out with trimethylsilyl bromide (TMSBr) followed by a passage in basic medium. To couple these analogs of nucleoside diphosphates or triphosphates with an enzyme or an antigenic carrier, conventional covalent coupling techniques are used between a reactive group carried by the nucleoside and a reactive group borne by the enzyme or carrier. antigenic carrier such as lysyl, tyrosyl, histidyl, tryptophanyl or glutamyl groups. The nucleoside reactive groups can be constituted by

  functions acid, amine, phenol, hydroxyl or ketone.

  Coupling reactions can be performed using reagents such as carbodiimides, gluraraldehyde, benzidine, mixed anhydrides, activated hapten esters and acid.

para-aminophenyl acetic acid.

  The conjugated compounds of the invention can be used either as immunogens or

as enzymatic markers.

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

O O O

he he <5 Il 11 11

HO P- 0 P O (V)

OH OH OH

  wherein n = O or 1 and R5 represents a nucleoside derivative, which comprises injecting into a mammal a conjugate compound of a nucleoside diphosphate or triphosphate analog of the formula:

0 0 0

1 o to iP xl - P2P-il-R (0

OR3 O

  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 a carrier antigenic, and - to subsequently take blood from the mammal

  immunized to isolate the antibodies produced.

  It also relates to a process for preparing monoclonal antibodies of nucleoside diphosphate or triphosphate analogs starting from the conjugate compound described above, by the technique

Kohler and Milstein.

  The invention further relates to a method for the immunological determination of a nucleoside diphosphate or triphosphate of the formula:

HO P - O P -O TO P O (V)

OOH OH

  in which n = 0 or 1 and R5 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 conjugated with an enzyme and a diphosphate or triphosphate of formula:

0 O O

R ilo, l X_

R1 O-P-X'-X2P - OR (D

R2 1 R4

  OR3 n wherein R5 and n have the meanings given for formula (V) and R1, R2, R3, R4, X1 and X2 have the meanings given for formula (I), for limited sites of a specific antibody of said analogous, and then to determine the amount of conjugated

attached to the antibody.

  This assay procedure can be applied to the determination of adenosine triphosphate (ATP) or the assay of a metabolite of a nucleoside anti-viral agent such as

  than anti-HIV drugs like AZT.

  Other features and advantages of the invention will appear better on reading the following examples given of course for illustrative and non-limiting purposes, with reference to FIG.

single annexed.

  The attached figure is a diagram illustrating the result of immunoassays performed according to the invention, that is to say the variation of the B / B0 ratio in which B represents the enzymatic activity bound to the antibody in the presence of a substance to be determined, and B0 represents the enzymatic activity bound to the antibody in the absence of the substance to be assayed, as a function of the concentration (in

pmol / ml) of the substance to be assayed.

  EXAMPLE 1 Preparation of Benzyl (dibenzyl phosphonomethyl) (benzyl hydrogenophosphono-methyl) phosphinate (Compound No. 1) C3OHa3OP3 MW: 614.50 a) Preparation of tribenzyl phosphite (compound No. 2) of formula: o

C21H2103P MW: 352.37

  To a solution of 25 ml (286.5 mmol, 1 eq) of phosphorus trichloride in 1.5 liters of anhydrous ether, cooled to -78 ° C, 123.8 ml (888, 3 mmol, 3.1 eq.) Of 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 off, the solvent is evaporated under vacuum and the residue is chromatographed on a silica column (ethyl ether / hexane / triethyl).

  amine: 40/60/1); 90 g of an oil are thus obtained

colorless (89%).

  The characteristics of the product obtained are as follows: TLC

  Rf = 0.5 (Et2O / hexane / Et3N: 50/50/1).

  1 H NMR: (CDCl 3) 200 MHz 6 (ppm): 7.37 (m, 15H, aromatic protons); 4.94 (d,

3H-P = 8 Hz, 6H, Ha).

  13 C NMR: (CDCl 3) 50 MHz 6 (ppm): 138.21 (3Cb); 128.37 (6 Cd); 127.67 (3Ce); 127.49

  (6cc); 64.47 (d, 2 C-P = 11.1 Hz, 3Ca).

31 P NMR: (CDCl 3 / H 3 PO 4) 81.015 MHz

6 (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. 2) of Formula B: ## STR2 ## PM: 181, 39 25 g (284.1 mmol, 1 eq.) of

  NaH2PO2 and 17.05 g (568.3 mmol, about 2 eq.

  depolymerized) of paraformaldehyde in 140 ml of distilled water. 50 ml of concentrated hydrochloric acid are added and the mixture is refluxed for 44 hours. After distilling off the water and then filtering the salts, the filtrate is taken up in 50 ml of acetone and the salts formed are filtered off again. The filtrate is concentrated and then 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 and then refluxed, while 72.53 ml (994.4 mmol, 3.5 eq) of thionyl chloride (large evolution of gas) is added dropwise over 3 hours. . After that, the reaction medium is allowed to reflux again for 12 hours. After evaporation of the solvent under vacuum, an oil is obtained which is purified by distillation in vacuo to give 44.4 g of bis (chloromethylene) chlorophosphinate under

  form of a colorless oil (86% yield).

  The characteristics of this compound are as follows:

Teb = 85-88 ° C (1 mmHg).

  1 H NMR: (CDCl 3) 200 MHz 6 (ppm): 4.09 (AB part, ABX system, JAB = 14.2 Hz, JAX = 7.4 Hz, JBX = 7.5 Hz, vA = 4.13, vB = 4 , 03, 4H). 13 C NMR (CDCl3) 50MHz

6 (ppm): 36.99 (d, 1 C-P = 88.5Hz, 2C).

  31 P NMR: (CDCl 3 / H 3 PO 4) 81.015MHz 6 (ppm): 49.3 (s) IR (pure, film) cm-1:

  3020; 2998; 2942; 2870; 1490; 1384; 1258;

1200; 1120; 990.

(c) Preparation of bis (chlorinated

  benzyl methylene) phosphinate (compound 4) of the formula: ## STR2 ## 20 g (110.3 mmol, leq) of bis (chloromethylene) chlorophosphinate (compound 2) dissolved in 400 ml of ether Anhydrous at 0 ° C., 16.9 ml (121.3 mmol, 1 .mu.q) of anhydrous triethylamine are added. 13.1 g (121.3 mmol, 1.1 eq) of benzyl alcohol are then added over 15 minutes 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 off, the filtrate is evaporated in vacuo and chromatographed (ethyl acetate / hexane: 60/40 to 100/0); we thus obtain

* 24.6g of a colorless oil (88% yield).

  The characteristics of compound 4 are as follows: TLC: Rf = 0.5 (Et2O) 1H NMR: (CDCl3) 200 MHz 6 (ppm): 7.45-7.37 (m, 5H, aromatic protons); 5.19 (d, 3 HHp = 9.3 Hz, 2H, Ha); 3, 66 (AB part, ABX system, JAB = 12.5 Hz, JAX = 7.5 Hz, JBX = 8.1 Hz, VA = 3.72, VB = 3.60, 4H, Hf) 13 C NMR: (CDCl3) 50MHz 6 (ppm): 134.93 (d, 31PC = 5.1Hz, 1Cb); 128.63 (CEL); 128.40 (2Cd); 127.97 (2cc); 67, 73 (d, 2 Jcp = 6.5 Hz, 1Ca); 32.63 (d, 1Jcp = 104.7Hz, 2Cf). 31 P NMR: (CDCl 3 / H 3 PO 4) 81.015 MHz 6 (ppm): 41.25 (s). IR (CH2Cl2) cm-1:

  3091; 3050; 3000; 2950; 2895; 1498; 1456;

1393; 1262; 1200; 1110; 1007; 847.

  d) Preparation of the bis (dibenzylphosphono-

  benzyl methyl (phosphonyl) compound (compound 5) of the formula:

C37H3908P3 MW: 704.63

  To 8 g (31.61 mmol, eq of the 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 at 140 ° C. under 4 mm of mercury. And subjected to violent stirring to continuously remove benzyl chloride. After stirring for 10 hours, the reaction mixture is chromatographed on a silica (Et20 / ethyl acetate / Ethanol: 100/0/0 to 0/80/20) column; 15.8 g of the compound n 4 under

  the form of a colorless oil (71% yield).

  The characteristics of compound N 4 are as follows: TLC: Rf = 0.4 (AcOEt) 1 H NMR: (CDCl 3) 200 MHz 6 (ppm): 7.32-7.28 (m, 25H, aromatic protons); 5.13-4.95 (m, 3H-p = 8.5 Hz, 10H, Ha and

  Ha'); 2.84 (dd, 2H-pp = 20.3 Hz, 2H-Pa = 18.2Hz, 4Hf).

  13 C NMR: (CDCl 3) 50 MHz (ppm): 135.77-135.57 (m, 3 CI-P = 7.5 Hz, 4 Cb, and 1 Cb,); 128.75-127.53 (8Cd, 2Cd, 8Cc, 2Cc, 4Ce, 1Ce); 67.95 and 67.72 (2d, 2C-P = 6.2 Hz, 4Ca); 66.79 (d, 2C-P = 6.5 Hz, 1Ca,); 28, 65 (dd, 1C-P = 130 Hz,

1C-P = 87.5 Hz, 2Cf).

  31 P NMR: (CDCl 3 / H 3 PO 4) 121.44 MHz 6 (ppm): 38.47 (t, 2 Papp = 4.5 Hz, PCa) and 20.90 (d, 2 Pap = 4.5 Hz, 2 O).

MS: (CI / NH3)

m / z: 21.8 (MNH +)

IR (pure, film) cm-

  3088; 3C'3; 3033; 2954; 2894; 1497; .1468;

1380; 1250; 1182; P93.

  e) Preparation of (dibenzyl phosphonomethyl) (benzyl hydrogenphosphono-methyl)

  benzyl phosphinate (compound n 1).

  To 0.796 g of 1,4-diazabicyclo [2.2.2] octane (DABCO) (7.09 mmol, 1 eq), 5 g of compound No. 4 (7.09 mmol, leq) in solution in 70 ml are added. anhydrous toluene. The reaction mixture is refluxed for 2 hours. The solution is reduced under vacuum and the salt obtained is dissolved in 100 ml of a water / methanol mixture (30/70). To the above solution is added an ion exchange resin charged with H30 + ions (Dowex 50X8) and then stirred for 12 hours. The resin is recovered by filtration, the concentrate is concentrated

  filtrate and taken 3 times per 100 ml of toluene.

  There is thus obtained 4.18 g of compound n 12 in the form of a yellow oil which can be used without any

purification (96% yield).

  The characteristics of the compound n 1 are the following: 1 H NMR: (DMSO d 6) 200 MHz: 6 (ppm): 7.37-7.31 (m, 20H, aromatic protons); 5.07 (d, 3HHp = 7.2 Hz, 2H, benzyl H); 4.97 (d, 3HHp = 7.4Hz, 6H, benzyl H);

2.77 (t, 2 Hp = 20, LHz, 4H, Hf).

  13 C NMR: (CD 30 D) 50 MHz 6 (ppm): 137.82-137.38 (2 Cb, 1 Cb ', 1 Cb -1); 129.60-128.85 (4 Cd, 2 Cd', 2 Cd ', 2 Ce, 1 Ce, 1 Ce ", 4Cc, 2Cc ', 2Cc"); 69.45 (d, 2 Jcp = 5.7Hz) -68.75kd, 2 Jcp = 5.7Hz) 68.33 (d, 2 Jcp = 6.3Hz) (2Ca, 1Ca 'and ICa "); 29.62 (dd , 1Jcp = 87, 1Hz and 1Jcp = 130.6Hz, 2Cf) .31P NMR: (CDCl3 / H3Pz4) 81, O15MHz 6 (ppmU): 4I, 21 (d, 2 Jpp = 4.7Hz, Pa) and

18.59 (d, 2 Pp = 5.3: Hz, P? And Po ').

MS: (CI / NH3)

m / z: 63 ', 9?' MNH 4 +); 524.2 (M-Bn + H +) -

IR (pure, film) cm-1A

  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 No. 6) of formula: ## STR1 ## a) Preparation of 2 ', 3'-O-benzylidene inosine (Compound No. 7) Ès OH ON 20 g (74.5 mmol, 1 eq) of inosine are added to 56.7 g (372.5 mmol, 5 eq.) Of benzaldehyde dimethyl acetal in solution in 400 ml of bromine. anhydrous acetonitrile and cooled to 0 ° C. The mixture is treated dropwise with 13.9 ml (149 mmol, 2 eq) of freshly distilled phosphorus oxychloride, and the mixture is stirred for 1 hour at 0.degree. After 2 hours at room temperature, the solution becomes yellow and clear and then cloudy, The reaction mixture is then poured very slowly over a mixture of saturated NaHCO 3 / ice water and stirred for about 3/4 of an hour. A white precipitate appears, is filtered and then washed with water and with ether After drying under vacuum, 23.9 g of 2 ', 3'-O-benzylidene inosine are obtained.

(90% yield).

  The characteristics of this compound are the following: TLC: Rf = 0.3 (CH2Cl2 / EtOH: 90/10)

Mp 254-2550 ° C

  1 H NMR (CD 3 O) 200 MHz 6 (ppm): 8.33 (s, 1H, H 8), 8.08 (s, 11H, H 2), 7.54-7.46 (m, 5H, aromatic protons); 6.26 (d, 3HH, -H 2 '= 3Hz, 1H, H 1,); 6, 01 (s, 1H, Ha); 40 (dd, 3H2-H1 = 3HZ, 3H2 -H3, = 5HZ, 1H, H2,); , 12 (dd, 3HH3'-H2 '= 5HZ, 3H3H4 = 2.5Hz, 1H, H3,);

  4.60 (m, 3HH-H3 '= 2.5HZ, 1H, H4.); 3.75 (m, 2H, H5,).

  13 C NMR (DMSO-d 6) 50 MHz 6 (ppm): 156.42 (1 C 6); 147.75 (1C4); 146.02 (1C2); 138.81 (1C8); 136.12 (LCB); 129.71 (CEL); 128.37 (2Cd); 126.82 (2cc); 124.42 (1C5); 106.58 (LCA);

  89.55 (1C1 '); 86.53 (1C4,); 84.44 (1C2), 82.61 (1C3 ');

61.46 (1C5).

IR (KBr) cm-1:

  3392; 3048; 2876; 1700; 1589; 1550; 1508;

1419; 1214; 1098; 974.

b) Preparation of 2,3'-O-benzyl

  lidene (5'-acetyl) inosine (compound n 8) of formula: ## STR1 ##

C19H18N406MM: 398.37

  24 g (67.35 mol, leq) of the previously obtained 2,3'-O-benzylidene inosine, dissolved in 500 ml of anhydrous acetonitrile, 11.26 ml (80.82 mmol, 1.2 g) are added. eq.) of anhydrous triethylamine, 7 ml (74.0 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

  room. The solvent is evaporated to give an oil.

  The product is then precipitated using diethyl ether / CH 2 Cl 2: 90/10. The precipitate is filtered off, washed with ether and dried under vacuum. 23.34 g of 2 ', 3'-O-benzylidene (5'-acetyl) inosine are thus obtained in the form of

  a white powdery solid (yield 87%).

  The characteristics of this compound are as follows:

TLC: Rf = 0.5 (CH2C12 / EtOH: 90/10).

F = 170-171 ° C

  1 H NMR: (CDCl 3) 200 MHz 6 (ppm): 8.27 (s, 1H, H 8); 7.95 (s, H, H2); 7, 60-7.43 (m, 5H, aromatic protons); 6.27 (d, 3HH1-H2t = 1.9H2, 1H, H11); 6.21 (s, 1H, Ha); , (Dd3JH2, -H1i = 1.9Hz, 3H2f-H3 = 6.5HZ, 1H, H2,); 5.13 (dd, 3H3, -H4, = 3.2Hz, 3H3'-H2, = 6, 5Hz; 1H, H3,); 4.67 (part X of an ABX, 1H, H4,); 4.33 (AB system ABX part, Jab = 11.9 Hz, JAX = 4.3 Hz, JBX = 6.1 Hz, vA = 4.39, vB = 4.28, 2H, H5s); 2.02 (s, 3H, Hg). 13 C NMR (DMSO-d 6) 50 MHz 8 (ppm): 169.87 (1 CF); 156.46 (1C6); 147.65 (1C4); 146.02 (1C2); 139.02 (1C8); 135.88 (LCB); 129.78 (CEL); 128.37 (2Cd); 126.85 (2cc); 124.42 (1Cs); 106.85 (1Ca); 89.09 (1C1); 84.34 (1C4,); 83.64 (1C2,),

  81.99 (1C3 '); 63.56 (1C5,); 20.32 (1Cg).

IR (CH2Cl2) cm-1:

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

  1547; 1513; 1460; 1374; 1229; 1093; 977.

  c) Preparation of 6-chloro-2 ', 3'-O-benzylidene (5'-acetyl) purine-9H of formula: ## STR1 ## (50.20 mmol, leq) 2 ', 3'-O-benzylidene (5'-acetyl) inosine to a mixture of ml of phosphorus oxychloride and 6.37 ml (50.20 mmol, léq .) N, N'-dimethylaniline. An oil bath is preheated to 120 ° C. The mixture is stirred vigorously, heated for 1 minute and seconds under reflux, then cooled to 0 ° C. and immediately distilled under vacuum with a water jet ml. phosphorus oxychloride. The remaining solution is poured very slowly on a mixture of saturated water

  in NaHCO3 / ice, and stirred for about 30 minutes.

  The aqueous solution is extracted with methylene chloride. The organic phase is dried over sodium sulfate, reduced in vacuo and purified by chromatography on a silica column (ethyl ether / hexane: 80/20 to 100/0); 16.1 g of 6-chloro-2 ', 3'-O-benzylidene are thus obtained.

  (5'-acetyl) purine-9H (77% yield).

  The characteristics of this compound are as follows:

TLC: Rf = 0.5 (Et2O).

  1 H NMR: (CDCl 3) 200 MHz 6 (ppm): 8.78 (s, 1H, H 8); 8.26 (s, 1H, H2); 7.567.39 (m, 5H, aromatic protons); 6.32 (d, 3HH1, -H2, = 2, 1Hz, 1H, Hi,); 6.06 (s, 1H, Ha); 64 (dd, 3H2'-H1, = 2.1HZ, 3H2, -H3, = 6.5HZ, 1H, H2,); , 16 (dd, 3H3, -H2, = 6.5Hz, 3H3, -H4 '= 3.1Hz, 1H, H3,); 4.70 (part X of us ABX, 1H, H4 '); 4.30 (AB part ABAB system, JAB = 20Hz, a.J = 4.4Hz, JBx = 6Hz, vA = 4.37, vB = 4.25,

2H, H5 '); 1.97 (s, 3H, Hg).

  13 C NMR: (CDCl 3) 5.jMHz 6 (pprn): -, 94 (1Cf); 152.00 (1C6); 151.54 (1C2 or 1C4); 150.82 tC2 or 1C4); 144.23 (1C8); 135.36 (1Cb); 131.41 (1C5). 129, 35 (ICe); 128.46 (2Cd); 126.54 (2cc); 108.01 (1Ca); 90.85 (1C1); 84.60 (1C4 'and 1C2,);

  82.30 (1C3); 63.52 (1C5,); 20.37 (LCG).

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

(Compound No. 6)

  8 g (19.19 mmol) of the 6-chloro-2 ', 3'-O-benzylidene (5'-acetyl) purine-9H obtained above 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 0); thus obtaining 5.89 g of 6-chloro-2 ', 3'-O-benzylidene (5'-hydroxy) purine-9H under

  the shape of a white solid (82% yield).

  The characteristics of this compound are as follows: TLC: Rf = 0.4 (Et20)

F = 190-191 C.

  1 H NMR: (CDCl 3) 200 MHz 6 (ppm): 8.79 (s, 1H, H 8); 8.22 (s, 1H, H2); 7.60-7.46 (m, 5H, aromatic protons); 6.12 (d, 3H, -H2, = 4.6Hz, 1H, Hi,); 6.09 (s, 1H, Ha); 5.36 (dd, 3H2-H1 '= 4.6Hz, 3H2'-H3' = 6.2Hz, 1H, H2,); 5.23 (dd, 3H3, -H2, = 6.2Hz, 3H3, -H4, = 1.3 Hz, 1H, H3,); 5.09 (dd, 3HF-H5, = 2.7HZ, 3HFH5, = 9.8HZ, 1H, Hf);

  4.73 (m, 1H, H4,); 3.93 (m, 2H, H5,).

  RpMN13C: (DMSO-d6) 50MHz 68 (ppm): 151.58 (1C6); 151.29 (1C2 or 1C4); 149.29 (1C2 or 1C4); 145.74 (1C8); 136.03 (LCB); 131.38 (1C5); 129.66 (CEL); 128.31 (2Cd); 126.77 (2cc); 106.38 (1Ca); 90.32 (1C1l); 87.13 (1C4,); 84.29 (1C2 ');

82.59 (1C3 '); 61.33 (1C5 ').

MS: (CI / HN3)

m / z: 375 (MH +) IR (KBr) cm-1:

  3400; 3069; 2926; 2876; 1593; 1565; 1493;

1434; 1406; 1339; 1201; 1111; 1072.

  Example 3: Preparation of 6-chloro-2 ', 3'-O-benzylidene 5' [tetrabenzyl-a, Q: a, y

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

this

NOT

  ## STR16 ## To a solution of 1.19 g (1.93 mmol, 1 eq) of compound 1, 726 mg (1.93 mmol, 1 eq) of 6-chloro-2 ', 3 O-benzylidene (5'-hydroxy) purine-9H (Compound No. 6) and 1.27 g (4.82 mmol, 2.5 eq.) Of triphenylphosphine dissolved in 30 ml of anhydrous THF are added dropwise. drop 759 g (4.82 mmol, 2.5 eq.)

  ethyl azodicarboxylate at room temperature.

  After 1 hour, the solvent is evaporated under reduced pressure and the residue is chromatographed on a silica column (ethyl ether / ethyl acetate / ethanol: 100/0/0 to 0/90/10); 1.32 g of compound no. 10 is thus obtained in the form of a white powder (yield of%). The characteristics of this compound are as follows:

TLC:

Rf = 0.5 (AcOEt / EtOH 95/5).

Mp 49-50 ° C

  RNlH: (CDCl3) 200MHz, mixture of diastereoisomers: 6 (ppm): 8.75-8.46 (m, 2H, H8 and H2); 7.61-7.2 (m, 25H, aromatic protons); 6.41-6.27 (4d, 3H1, _H2, = 2.6Hz, 1H, Hi '); 6.01-5.94 (6s, 1H, Hf); 58-5.52 (m, 1H, H2 '); 5.40-5.33 (m, 1H, H3,); 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: (CDCl 3) 50 MHz, mixture of diastereoisomers: 6 (ppm): 151.58 (1 C 6); 151.15-150.96 (1C2 and 1C4); 144.44-144.35 (1C8); 135.66-135.18 (2Cb, 1Cb ', 1Cb "and 1Cg); 132.08-131.39 (1C5); 129.88-129.87 (2Ce); , 1Ce, 1Ce "and 1Cj), 128.52-127.92 (4Cd, 2Cd ', 2Cd", 4Cc, 2Cc', 2Cc ", 2Ch and 2Cj); 107.92 (1See);

, From 70 to 90.58 (1C1 '); 90.23 to 90.22 (1C4');

  , From 05 to 84.44 (1C2 '); 82.05 to 81.78 (1C3'); From 68.41 to 61.83 (2Ca,

  lCa ', lCa "and 1C5'); 30.76-25.7-0 (1Ck and 1Ck ').

MS: (CI / NH3)

  m / z: 633.1 (M-H ++ 357 + NH4 +); 614.9 (M-373 + H +

+ NH4 +) -

CI Cl

N N N

e / XJ </ J 0kON 0NN

N N

o / o \ / Ph Ph

M = 373 M = 357

IR (CC14) cm-1:

  3088; 3063; 3033; 2954; 2894; 1593; 1505;

1468; 1380; 1250; 1108; 1074.

  Example 4 Preparation of N6 - (4-azidobutyl) -2 ', 3'-O-

  benzylidene-5 '- (tetrabenzyl-a, 3: 0, 1-dimethyltriphosphate) adenosine (compound n 11) of the formula: ## STR1 ## ## STR1 ##

N_5 N

  ## STR00006 ## To a solution of 0.59 g (0.61 mmol, 1 eq) of compound no. 10 in 20 ml of anhydrous methanol was added 0.20 g (1.74 mmol, 2.8 eq. ) 4-azido-1-butylamine at room temperature. The reaction mixture is stirred for 36h, then it is reduced under vacuum and the residue is chromatographed on silica (ethyl acetate / ethanol: 0 to 90/10). 0.51 g of the compound n11 is thus obtained.

  in the form of a glassy solid (80% yield).

  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 (2H, H8, H2); 7.41-7.26 (m, 25H, aromatic protons); 6.31-5.92 (m, 3H, NH, H +, Hf); , 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, H1); 2.95-2.65 (m, 4H, Hk,

Hk '); 1.66 (m, 4H, Hm, Hn).

  IR (CHCl 3) cm-1: 3291, 2942, 2096, 1619, 1458, 1251,

998, 828.

  Example 5 Preparation of N6- (4-aminobutyl) -5'-

  (a, 0: 0, 3-yl dimethyl triphosphate) adenosine (compound 12) of formula:

HO N.HN NH2

b d cps n ad nma h HO HO HO 1i

3 2 '

HO OH

C16H29N6011P3 PM: 574

  To a solution of 76 mg (72.5umol, 1 eq) of compound 11 in 3 ml of a methanol / water mixture: 9/1, 76 mg of 10% Pd / C and 460 mg (7.2 mmol, 100 eq. ) ammonium formate. The mixture is heated at 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 the product is precipitated by the addition of ethanol. It is centrifuged, the supernatant is discarded and the pellet is redissolved in 2 ml of water and then lyophilized. 34 mg of the compound n 12 are thus obtained in the form of a white powder.

hygroscopic (82% yield).

  The characteristics of this compound are as follows: 1 H NMR (D 2 O) 200 MHz: 6 (ppm) 8.07 (s, 1H, H 2 or H 8); 7.78 (s, 1H, H2 or H8); 5.92 (d, 3HH-H2, = 4.6Hz, H1 '); 4.50 (m, 1H, H2 '); 4.34 (m, 1H, H3 '); 3.98 (m, 2H, H5 '); 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-c, 4:, y-dimethyl triphosphate) thymidine

(Compound No. 13) of formula:

h o o o NH 0 0 o

C40H43N5011P3 PM: 862

  To a solution of 165 mg (0.62 mmol, lq) of AZT in 5 ml of anhydrous toluene was added 380 mg (0.62 mmol, léq) of the compound n l. The solution is evaporated to dryness, taken up with 5 ml of anhydrous toluene and 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 are added. To this solution was slowly added dropwise 648 mg (2.47 mmol, 4 eq) of triphenylphosphine dissolved in 2 ml of anhydrous THF. The addition is carried out in lh and the solution is stirred for 3h 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

(32% yield).

  The characteristics of this compound are as follows:

TLC: Rf = 0.2 (ethyl acetate).

  200 MHz NMR (CDCl3), mixture of diastereoisomers:

  6 (ppm) 7.30 (m, 21H, aromatic protons, Ha); 6,22-

  6.01 (m, 1H, H1 '); 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).

  Example 7 Preparation of 3'-deoxy-3'-amino-5'-

  (α, β, γ-Dimethyl triphosphate) thymidine (Compound No. 14) of formula:

0

NH _- _ P u =. 'N O

C12H22N3011P3 PM: 477

  To a solution of 82 mg (95.11mol, 1 eq) of compound 100 in 10 ml of ethanol is added 3 ml of THF and 69 mg of 10% Pd / C. The mixture is thoroughly purged with hydrogen and stirred at room temperature for 1.5 h, under a pressure of 1 atmosphere of hydrogen. 5 ml of water are then added and the mixture is reduced by half in a vacuum. The medium is purged with hydrogen and stirred for a further 2 hours under a pressure of 1 atmosphere of hydrogen. The mixture is then filtered on paper. The fLitrat is washed twice with 10 ml of ethyl acetate and the aqueous phase is then lyophilized. We thus obtain 29 mg of a white solid

hygroscopic (64% yield).

  The characteristics of this compound are as follows: RIMN1H (D20) 200 MHz: (ppm) 7.47 (s, 1H, Ha); 6.02 (m, 1H, Hi '); 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 Conjugate Compound

  compound No. 12 with keyhole limpet hemocyanin (KLH).

  The coupling between compound 12 and keyhole limpet hemocyanin (KLH) is carried out via glutaraldehyde which simultaneously reacts with the primary amine functions carried by compound 12 and the protein (lysine residues). Practically: To 2 ml of a solution of KLH (Keyhole Lympet Haemocyanin) at 5 mg / ml in -1M phosphate buffer pH 7.4 is successively added 2 ml of a solution of compound No. 12 to 1.05 mg / ml in the same buffer then 16 g of a solution of glutaraldehyde at%. After 2 hours of reaction at ambient temperature, the preparation is divided into 4 volumes

  of 1 ml and stored at -20 C until use.

  The efficiency of coupling after G25 column filtration is evaluated by measuring the absorbance in the ultraviolet. These measurements showed that about 12 molecules of compound 12 were fixed by KLH molecules (for a supposed molecular mass

100,000 kDa).

  EXAMPLE 9 Preparation of Anti-Compound Antibodies 12 For this preparation, three 2.5 kg white Bouscat rabbits are used.

treats as follows.

  An emulsion is prepared by mixing 1 ml of the immunogen solution, ie 2.5 mg of the compound compound 12-KLH of Example 8, with 1 ml of distilled water and 2 ml of complete Freund's adjuvant. On day 0, each rabbit receives an injection of 1 ml of the aforementioned emulsion intradermally, on the back in 50 points. At day D = 42, a first booster is made under the same conditions as the immunization, and then the rabbits are bled once a week at

from the day D = 49.

  Two reminders are made at two months

  interval, under the same conditions.

  Thus, in the serum, blood is recovered

  collected antibodies specific for compound No. 12.

  Example 10 Preparation of a compound conjugate

  n 12-acetyl cholinesterase from Electrophorus Electricus.

  The coupling between compound No. 12 and acetylcholinesterase (AChE) of Electrophorus electricus (form G4) is obtained via a heterobifunctional reagent N-succinimidyl-4- (maleimidomethyl) cyclohexane-1-carboxylate (SMCC). ). This method involves the reaction of a thiol group (previously introduced in the compound n 12) at the level of the primary amine function) with maleimide groups introduced on the surface of the AChE

by treatments with the CMCC.

  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-methyl maleimide so as to block all the thiol functions that may be present on the surface of the enzyme. The reaction with the SMCC is carried out in a 0.1M phosphate buffer pH 7.4. SMCC dissolved in anhydrous dimethylformamide is added to the enzyme solution

  in a molecular ratio SMCC / AChE = 300.

  Practically: a solution of 12.5 mg / ml of N-ethyl maleimide in 0.1M phosphate buffer pH 7.4 is added to 564 μl of a solution of AChE (8.8 mg / ml) in the same buffer. After 30 minutes of reaction at 30 ° C., 50 μl of a solution of SMCC (31.2 mg / ml) in anhydrous dimethylformamide is added. After 30 minutes of reaction at 30 ° C., the AChE-SMCC is purified by filtration on a 1.5 m Biogel A 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-acetylthioglycolic acid and N-hydroxysuccinimide (SATA). At this stage, the succinimidyl active ester function of SATA reacts with the primary amine function of compound No. 12. To 500 μl of a 2 mg / ml solution of compound No. 12 in 0.1M borate buffer pH 9 are added to of a 75 mg / ml SATA solution in anhydrous dimethylformamide. After 30 minutes of reaction at 30 ° C., the product of the reaction (compound

  12-SATA) is purified on a Seppak C18 cartridge.

  The methanolic eluate is lyophilized and stored at -20 ° C.

until use.

  c) Coupling Between Thiol Compound 12 and AChE-SMCC: The lyophilizate of compound 12-SATA is redissolved in 0.1M phosphate buffer pH 6 and then treated with hydroxylamine to release the thioester function of SATA. The liberated thiol functions are measured after reaction with 5'-dithiobis-nitrobenzoic acid (DTNB, Ellman's method). The coupling between thiol compound 12 and AChE-SMCC is obtained by mixing the two reagents

in a thiol / AChE ratio of 10.

  Practically: The lyophilizate of compound 12-SATA is taken up in 1.6 ml of 0.1M phosphate buffer pH 6 before addition of 400 g of 1M hydroxylamine. After 10 minutes of reaction at room temperature, the thiols are measured by reaction of 150 μl of 10-3 M DTNB with 50 μl of the thiol compound n 12. At this stage, the concentration of thiol groups is 4.1-4M. 11.5 μl of the thiolated compound (5 nmol) is then added to 350 μl of a solution of G4-SMCC (0.5 nmol). After 3 hours of reaction at 30 ° C., the conjugate is purified by filtration on a 0.5 m Biogel A column and stored at room temperature.

-20 C until use.

  Example 11 Enzymo-Immunological Assay of Adenosine Triphosphate (ATP) of Formula: NH2

N N

N% N

O O O

[l He tl

HO-P-O - P-O-P-OCH

OH OH OH

  Adenosine 5'-triphosphate For this assay, microtiter plates are used, the wells of which are coated with a mouse monoclonal anti-rabbit IgG antibody. 50 μl of a diluted anti-serum 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. standard of compound 12 or ATP. After overnight immunological reaction at +4 ° C., the plates were washed with phosphate buffer, containing 0.05% Tween 20, and then 200 g of Ellman reagent used as substrate was added. After an hour of enzymatic reaction,

  measures the absorbance of each well at 414 nm.

  The results obtained are represented by

curves 1 and 2 of FIG.

  Curve 1 refers to compound No. 12 and

curve 2 refers to ATP.

  These curves illustrate the B / B0 variations (in%) as a function of the dose (in pmol / ml) of compound 12 or ATP assayed. The two curves are very close and confirm that the ATP can be assayed using the antibody directed against the ATP analogue (compound 12) and the enzyme conjugate.

  also produced from this analog of ATP.

  Example 12: Determination of adenosine 5'diphosphate (ADP) of formula: NH, I3

NOT

Ol O

HO-P-O - P - OCH |

J I -

OH OH

  Adenosine 5'-diphosphate OH OH The same procedure as in Example 11 is followed to perform this assay. The results obtained are illustrated by curve 3 of the figure. In view of this curve, it is noted that the ADP can also be assayed with the antiserum and the conjugate.

  enzymatic, but the sensitivity is less good.

  Example 13 Assay of A-tetra-P of Formula: NH, N

0 0 0 0

  The same procedure as in Example 11 is followed to perform this assay using the same procedure as in Example 11. anti-serum and the same enzymatic conjugate. The results obtained are illustrated by curve 4 of the figure. EXAMPLE 14 Assay of Adenosine Monophosphate AMP of Formula: N2 or O Il

HO-P - OCH2

  Adenosine 5'-monophosphate The same procedure as in Example 11 is followed to carry out this assay using the same antiserum and the same enzyme conjugate. The results obtained are illustrated by the curve 5 of the figure. Examples 15 to 17 The same procedure as in Example 11 is followed for the determination of adenosine, dideoxy-ATP and azido-ATP of formula: NH 2

NOT

KNN

O O O

He he He

HO-P-O - P - O-P-OCH 2

OH OH OH

H H

Dideoxv ATP

NH2 NH2

NOT

0 0 0

KN N K1 N

O O O

He He He HOCH2

HO-P-O-P-O-P-OCH, I

OH OH OH

N3OH H OH

  N3Azido-ATP Adeosine Azido-ATPAdnse using in each case the anti-serum and the enzymatic conjugate of Examples 7 and 8. The results obtained are illustrated by the curves 6 (adenosine),

  7 (dideoxy-ATP) and 8 (azido-ATP) of FIG.

  Note that these curves do not allow to achieve a satisfactory dosage, the affinity of these nucleotides for anti-serum is not sufficient. Similarly, it has been found that adenine or cyclic AMP of formula: NH 2 NK s O It

HO-P-OCH 2

-0

O OH

  Adenosine 3'-5'-cyclopyrophosphate did not give rise to a reaction with the antiserum to

  doses greater than 10,000 pmol / ml.

  The results of the assays of Examples 9 to show that it is possible to perform a reliable assay of a nucleoside diphosphate or triphosphate, using antibodies directed against an analog of this nucleoside diphosphate or triphosphate, as well as an enzymatic conjugate. prepared from this analog. In addition, it is noted that such antibodies do not recognize azide nucleotides,

  dideoxy nucleotides and bases alone.

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

such as AZT.

Claims (16)

  A conjugated compound of a diphosphate or triphosphate analogue and a molecule selected from antigenic carriers and enzymes, wherein the analog is of the formula: ## STR2 ## OR 5 ( O OO   Where n is 0 or 1, X 1 and X 2, which may be the same or different, represent CH 2, CHF, CF 2, CCl 2, CHCl or NR 6 with R 6 being an atom of hydrogen, an alkyl, aryl or aralkyl group, the two R 6 may be identical or different when n = 1 or may together form a hydrocarbon chain containing a phenyl ring, - R 1, R 2, R 3 and R 4 which may be identical or different , represent a hydrogen atom, NH4 +, a quaternary ammonium ion or an ion of formula M + 1 / v with M representing a metal and v being the valence state of this metal, and - R5 is a group derived from a nucleoside, and wherein the analog is coupled to said molecule, either via the R5 group derived from nucleoside, or via one of the groups R1, R2, R3 and R4.   2. Conjugate compound according to claim 1, characterized in that R5 is derived from an agent antiviral nucleoside.   3. Conjugate compound according to claim 1 or 2, characterized in that R5 corresponds to one of the following formulas: oo NHZ H-N MN N OHM N3. N N OHN o o AZT A NMT d d MN '- "NH2 NOT N < ES = N EN z HO OH   4. Conjugate compound according to any one of claims 1 to 3, characterized in that said   molecule is an antigenic carrier. 5. Conjugate compound according to claim 4, characterized in that the antigenic carrier is keyhole limpet hemocyanin.   6. Conjugate compound according to claim 4 or 5, 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 of a saturated hydrocarbon chain, terminated by a group NH2 grafted on the purine base or Pyrimidine of R5.   Conjugate compound according to claim 4 or 5, characterized in that the coupling of the diphosphate or triphosphate analogue with the antigenic carrier is carried out by means of a polyoxyethylene (CH 2 -CH 2 -O) n or polylol spacer arm. hn 8. Conjugated compound corresponding to the formula: 0 0 0 I o he X 1 _ he __ ilp 7   HO-P-X -P -A --P - OR - (CH2 -NH -Z (II) OH OH OH   wherein n, X1 and X2 have the meanings given in claim 1, R7 is a divalent group derived from a nucleoside, p is 0 or an integer from 1 to 6, and Z is an antigenic carrier . 9. Conjugate compound according to claim 8, characterized in that R7 (CH2) pNH corresponds to one of the following formulas H N NH \ -K o N NOT HOOH   10. Conjugate compound according to any one of claims 1 to 3, characterized in that said   molecule is an enzyme. Conjugate compound according to claim 10, characterized in that said enzyme is an acetylcholinesterase. 12. Conjugate compound of the triphosphate analog of formula: HHQ '- "H2 Oo Oi HO-O-P'CH2.'PCH2P. O OH Oh CH- HO.OH   and acetylcholinesterase from Electrophorus electricus.   13. Process for the preparation of antibodies specific for a nucleoside diphosphate or triphosphate of formula: HO-P-O -O 11 11 Ho - P O- -PO0 PPOR5 (V) I II OH OH OH   wherein n = 0 or 1 and R5 represents a nucleoside derivative, which comprises injecting into a mammal a conjugate compound of a nucleoside diphosphate or triphosphate analog of the formula: 0 0 0   he [1 he R10 p-X-XI * P-P-R5 21 (14 R 2 OR3 OR4   wherein R1, R2, R3, R4, X1 and X2 have the meanings given in claim 1), and n and R5 have the meanings given above for the formula (V), coupled to an antigenic carrier, and to then take the blood of the mammal   immunized to isolate the antibodies produced.   14. A method for immunologically assaying a nucleoside diphosphate or triphosphate of formula: O O O IJ IF He there 11 - HO P - O 'P - OR (V) OH OH OH   in which n = 0 or 1 and R5 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 compound conjugated with an enzyme and a diphosphate or triphosphate analogue of the formula: ## STR2 ## O OR 'OR   wherein R 5 and n have the meanings 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 analogue, and in that the amount of   conjugate compound attached to the antibody.   15. The method of claim 14, characterized in that the diphosphate or triphosphate to be assayed is adenosine triphosphate (ATP) or a   metabolite of a nucleoside antiviral agent.   16. The method of claim 15, characterized in that the antiviral agent is 3'-azido-3'-deoxythymidine (AZT)