US3074930A - 6-mercaptopurine nucleosides and method of preparing them - Google Patents

Description

United States Patent 3,il741,93tl -MERCAETGEURHNE NUCLEOSEDES AND METHUD 9h PREPARHQG THEM George H. Hitchings, Yonkers, and Irving Goodman,

White Plains, N.Y., assignors to Burroughs Wellcome 31 60. (USA) inc, Tuclsahoe, N.Y., a corporation of New York No Drawing. Filed Jan. 5, 1959, Ser. No. 784,864 Qlairns priority, application Great liritain Aug. 11, W55 22 Claims. (6!. 26tl--211.5)

The present invention relates to 9-glycosido-6-mercaptopurines and to a process for preparing the same.

The ability of 6-rnercaptopurine to interfere with cell divisionhas found a useful expression in the production of remissions of acute leukemia. The mechanism of action of -rnercaptopurine is believed to be as an antagonist of the natural 6-substituted purines, which exist in cellular compositions combined through glycoside linkages.

The biological activity of G-mercaptopurine is modiiied, and becomes more selective in the new compounds with the result that the range of usefulness of the growth inhibitor is extended.

The usual method of preparing purine glycosides is by the reaction of a heavy metal salt of the purine with a glycosyl halide. In the present instance this method is inoperable presumably because the metal salts are attached to the sulfur of the mercaptan group. However, it has been found that -benzylrnercaptopurine will form a suitable silver salt, and that debenzylation of the eventual 9-glyccsido--benzylmercaptopurines can be effected by treating with sodium in liquid ammonia.

In our copending application Serial No. 574,575, now abandoned, we have described a method of preparing nucleosides of 6-marcaptopurines which consists of preparing first an acylated (thereby protected) nucleoside of a o-chloropurine and converting that to the acylated nucleoside of a 6-mercaptopurine.

The synthesis of nucleosides of 6-chloropurines being inconvenient, though feasible, it is often preferable to start with preformed purine nucleosides available commercially. These are particularly inosine (la) and guanosine (lb). These are first protected by acetylation or benzoylation of the sugar hydroriyls, and then converted to acylated nucleosides of 6-chloropurines by reaction with phosphorous oxychloride in the presence of a hydrogen halide acceptor. Considering the sensitivity of these nucleosides it is not surprising that this procedure is feasible only under special conditions. As the hydrogen halide-acceptor, weak tertiary organic bases are essential, and dimethyl and diethylaniline are preferred. Pyridine, picolines and lutidines can also be employed, but result in more tarformation.

The chloropurineacylnucleosides is then reacted with a sulfur-bearing reagent. For this purpose thiourea is satisfactory but a salt of a thiocarboxylic acid is even better. in both cases it is to be presumed that unstable sulfurcontaining intermeriates (thio-uronium salts and thiocarboxylates) are formed and these are hydrolyzed repidly under the conditions of the reaction:

The choice of the salt of a thiocarboxylic acid is one Efilldfifib Patented Jan. 22, 1%53 ice dithio-oxalic acid are readily available. Potassium dithiooxalate is probably the most convenient as being inexpensive and available commercially in pure form.

Finally as in other lines of synthesis, the protective acyl groups are removed, conveniently by the action of ammonia in alcoholic solution.

The 6-chloropurine acylnucleosides can also be reacted with other reagents. Of particular interest is the reaction with thiohexoses whereby are obtained 6,9-diglycosides of mercaptopurine (or of thioguanine). These compounds (Vii) and their esters (VI) are of therapeutic value in their own right.

These reactions are illustrated in the chart of reactions with sodium-l-thioglucose leading to the 6-(1'-thioglu cosides) (VI and Vii). It is believed that these particular compounds have the ti -configuration and are 1- thio-fl-d-glucopyranosides. However, neither the pyranose form nor the B-configuration have yet been proved beyond question. Therefore, at this point, we refrain from writing the steric form of this portion of these compounds.

Eight g. of chloromercuri-6-chloropurine was added to a solution of 7 g. l-chloro-Z,3,5-triacetylribofuranose in 100 ml. anhydrous xylene. The solution was refluxed for 48 hours, filtered and the filtrate was concentrated to a syrup. The product was an impure oil.

EXAMPLE 2 9-Rz'bofuranosyl-d-Mercaptopurine Va) Two g. of the syrup (III) was refluxed with 0.2 g. thiourea and 0.2. g. sodium acetate in absolute ethanol for 18 hours. The resulting solution of the thiouronium salt TV was filtered, treated with H 8 to remove any remaining Hg, decolorized with Darco, concentrated to a syrup and taken up in absolute ethanol. The ethanolic solution was saturated with NH; and left for 18 hours at room temperature. A very small amount (50 mg.)

of product was obtained.

A max.=225, 321 at pH 1; 234, 311 at pH 11; the product gives a positive Mohlisch test and was shown by paper chromatography to have a mobility resembling that of the corresponding gluccsyl derivative.

EXAMPLE 3 6 -H yzfroxy -9- (2, 3,5 -Trl' -O-A cetylribofuranosyl) -Purine (II ac) 15 g. of inosine (I) is suspended in 200 ml. pyridine. To the suspension is added 100 ml. acetic anhydride. After stirring until homogeneous, the mixture is kept on the steam bath for 2 hours. The resulting clear ambercolored solution is concentrated to semi-solid state in vacuo. 250 ml. of ice Water is added to the residue, whereupon the solid dissolves with the evolution of heat. Upon cooling for minutes in an ice bath the white crystalline product appears. After keeping at 4 C. for one hour, the crystals are collected by filtration and washed with ice water. Yield, 19.5 g.; (89% of theory). M.P. 230. A max.=245 mu (in absolute EtOH).

EXAMPLE 4 6-C1ti0r0-9- (2,3,5-Tri-O-A cetylribofuranosyl) -Purz'ne (Illac) 16.0 g. of triacetylinosine (H) is intimately mixed with 10 m1. of dimethylaniline. To the mixture is added 100 ml. of POCI The suspension is heated on the steam bath for 4 hours (anhydrous conditions). The homogeneous dark brown solution is then concentrated in vacuo on the steam bath to remove the excess POCl The remaining brown syrup is dissolved in 250 ml. CHCl The CHCl solution is washed 5 times with 75 ml. portions of H 0 in a separatory funnel, after which the water washings are at pH 5 to 6. The CHCl layer is dried over anhydrous Na SO and is concentrated to a syrup.

.Yield, 800 mg. (62% of theory).

v 6 chloro 9 (2,3,5-tri (Hlczc).

- ethanol saturated centratedin vacuo to about 5 bath. 40 mg. of pure 6-MP Mp. at pH 11.

- and kept at room temperature for 18 Final traces of solvent are removed under high vacuum. The yield of crude product isolated as a syrup is 14.8 g. (89% of theory). A max.=265 m (in H O).

EXAMPLE 5 6-]l 1'ercapl'0-9- 2,3, 5 -Tri-O-A celylribofw'anosyl) -Purilz e (IVac) 1.3 g. of (lilac) is dissolved in 10 ml. of absolute ethanol. To this solution is added a solution containing 0.6 g. of sodium thioacetate in 10 ml. of absolute ethanol. The clear solution was refluxed on the steam bath for 1 hour. The Warm solution was filtered, and upon cooling the product crystallized in the form of white plates. max.=325 my in absolute ethanol.

EXAMPLE 6 6 -M ercapzo-9- 2,3,5 -Tri -O-A cetylrib 0 furanosyl -Purine (IVac) In ten ml. of absolute ethanol was dissolved 2.5 g. of O acetylribofuranosyl)-purine To this was added a suspension of 1 g. of potassium dithiooxalate (COSK) in 25 m1. of absolute ethanol. The mixture was heated at reflux for 1 hour. It was then filtered and the filtrate was concentrated in EXAMPLE 7 6-Merdapt0-9-Ribofuranosylpurine (Va) mg. of (IV) was dissolved in 25 ml. of absolute with NH;,. The solution was kept at room temperature for 18 hours after which it was conml. and cooled in an ice riboside (V) was isolated (60% of theory). A max.=225, 321 .at pH 1; 234, 311

EXAMPLE 8 d-Glucothiopyrahosyl-Q( 2,3,5 -Tri-O-A cetylribofurnosyl) -Purine Vlac) 13 g. of crystalline product (VIac) was isolated (66% of theory). A max.=280

mm (in absolute ethanol).

EXAMPLE 9 6-Glzzc0thiopyranos yl-9-Riboyuranosylpurine VIIa) 5 g. of (VIac) was dissolved in '70 ml. ethanolic NH hours. The solution was concentrated in vacuo to 10 ml. Upon adding 10 ml. anhydrous ether, the product crystallized. Yield, 2.8 g. (72% of theory). max.=280 m (in H O).

EXAMPLE 10 6-H ydr0xy-9- (2,3,5 -Tri-0-Benzoylribofuranosyl Purine (Had) Five g. of inosine was suspended in The mixture was cooled to 0 in cold suspension, 25 ml. of CHCI benzoyl chloride was added. The mixture was heated at for 2 hours, allowing the CHCl to distill on.

a The resultant syrup was dissolved in 100 ml. of CHCI 10 ml. of pyridine. an ice bath. To the followed by 10 g. of

derivative. Yield, 10 g. (92% of theory) N=9.61%. Theory N=9.68%. x max.=230 and 265 my in alcohol.

EXAMPLE l1 6-Chl0i'o-9-(2,3,5-Tri-0-Benzoylribofuranosyl) -Pz4rine (Iliad) Five g. of tribenzoylinosine (Example 10) was suspended in 5 ml. of dimethylaniline. To this suspension was added 61 g. of POCl whereupon the solid dissolved. The mixture was heated at 100 for 4 hours. The resulting light yellow solutionwas poured into 800 ml. of ice water-CHCl (1:1 by volume). The water layer was extracted twice with Cl-ICl The CHCl layer was washed twice with saturated NaHCO solution and then with water. After drying over Na SO the CHCL, layer was concentrated in vacuo to a syrup. The resulting syrup was dissolved in 100 ml. of absolute alcohol and poured into 600 ml. of water. Upon acidification to pH 2 with HCl, a precipitate was formed. Yield 6 g. of crude product. A max.=230 and 260 m in alcohol. The 230 mu peak is characteristic of the benzoyl derivatives while the 260 peak is similar to that of the parent purine, 6-chloropurine with a peak at 265 (aqueous solution, pH 1). The product contains gives a positive Mohlisch test.

EXAMPLE 12 6-5 1 ercapt-9- (2 ,3 ,5 -Tri-O-B enzoy lrib 0 furzmosyl Purine (IVad) EXAMPLE l3 6-6lucotltiopyranosyl-9-(2,3,5-Tri-Olie-rlz0yZribofuranosyl) -Purine (V1 ad) of the chloronucleoside of Example 11 was dissolved in 50 ml. of absolute ethanol. To this solution was added a solution containing 1 g. of sodium thioglucose dihydrate in ml. of water. The mixture was heated at 100 for 15 minutes, then filtered. From the fiiterate was isolated 0.8 g. of crude product with max.=228 and 280 me in 50% ethanol. The 280 mp.

Cl (organic) an Two g.

eak is characteristic of the 6-purinethoiglycosides. Upon hydrolysis this yields G-rnercaptopurine. The prodnot still contains the benzoyl groups as shown by the 228 mp peak.

EXAMPLE l4 Fifty g. of guanosine (commercial grade) was suspended in 500 ml. of pyridine. T 0 this was added 250 ml. of acetic anhydride. The mixture was heated at 100 for 17 hours. The resulting solution was poured into 1600 ml. of water and extracted with CH'Cl (3- 200 ml. portions). The CHCl solution was washed twice with saturated NaHSO solution (2 x 200 ml.), twice with 200 ml. portions of H 0, dried over Na SO and concentrated to a syrup in vacuo. The syrup, on trituration with anhydrous ether formed a crude solid product (63 g.). A max.=:258 mp. at pH 6', 265 m at EXAMlLE l5 Twenty-five g. of the above ester was suspended in 35 ml. of dimethylaniline. To this was added 240 g. of POCl The mixture was heated at under reflux for 4 hours. The resulting solution was concentrated in vacuo on the steam bath to a syrup. The syrup was dissolved in 200 ml. of CHCl which solution was extracted several times with 75 ml. portions of H 0 until the pH of the washings was about 4. After drying over Na S0 the CHCl layer was concentrated in vacuo to a syrup. To this was added anhydrous ether. The resulting hygroscopic solid was dried in vacuo. Yield 12 -g. A max. 252 and 285 m, in alcohol. Contains chlorine (organic). Positive Mohlisch test.

EXAMPLE l6 Z-Amino-d-Mermp t0-9- (2 ,3 ,5 Tri-GAcetylrib ofurzmosyl) -Pztrine (ll be) One g. of the above chloro compound was dissolved in 25 ml. of absolute alcohol. To this was added a solution of 0.5 g. of sodium thioacetate in 5 ml. of absolute alcohol. The mixture was refluxed for 2 hours, filtered, and concentrated to dryness in vacuo. The solid residue was treated with 50 ml. of water. A precipitate formed with U.V. characteristics similar to the parent purine compound, thioguanine. U.V., )t max. at 250 and 340 m in absolute alcohol. The 340 peak is characteristic of the mercapto group of thioguanine. A positive Mohlisch test indicates the presence intact of the ribosyl group.

*XAMPLE l7 Z-Amino-d-Mercapto-fi-D rib0;furanosylpurine (l Vb) One-tenth g. of Z-aminoG-mercapto-9-triacetylribofuranosylpurine (Example 16) was dissolved in 2.5 cc. of alcohol saturated with ammonia. The solution was allowed to stand over-night at room temperature and was then evaporated in vacuo. The residue, after washing with absolute ethanol was a brown amorphous powder. it gave a positive ylohlisch test and had the ultra violet absorption of a thioguanine derivative. (r max. (H 0 260 and 34-0 my" At pH ll, 7\ max. 250 and 320 m The peaks at 340 and 320 m are very distinct and characteristic.)

EXAMPLE 1% Tribenzoyl Guanosine (Hbd) Benzoylation was accomplished by the method described for the benzoylation of inosine (Example 10). The product was isolated as a crystalline solid, Ml. 205-8". N=ll.36%. Calculated N=ll.75%. A :230, 270 in alcohol.

EXAMPLE 19 2-Amin0-6-Ch l0;'09(2,3 ,5 -Tri-O-Bez .z0y iri '00 fumnosyl) Farina (Hl'bd) 2Amin0-6-Gluc0thiopymnosyl-9-(2,3,5-Tri-O- Benzoylribofumnosyl)-Pzzriue (Vlbd) One-half g. of the d-chloro compound of Example 19 was treated with 0.25 g. of thioglucose dihydrate by the o-chloropuriue and reacting method of Examples 3 and 13. as a white crystalline solid. t max.=230 and 300 mu. Upon hydrolysis, the thioglucosyl linkage is split leaving a product, 2-amino-6-mercapto-9tribenzoylribofuranosylpurine, with A max. 230 and 340 at pH 1 and 230 and 312 at pH 11. The 230 peak indicates that the benzoyl ribose portion of the molecule is intact.

What we claim is:

1. The method of preparing nucleosides selectedv from the class consisting of nucleosides of G-mercaptopurine, 2-arnino-6-mercaptopurine, purine-6-thioglycoside and 2- aminopurine-o-thioglycoside which comprises converting consisting of inosine and guanosine to its O-acyl derivative, reacting the acylated nucleoside with phosphorus oxychloride in the presence of a hydrogen halide acceptor, so as to form the acylated nucleoside of the corresponding 6-chloropurine, reacting the latter with a reagent selecte from the class consisting of thiourea, the alkali thiohexoses and the salts of aliphatic thiocarhoxylic acids and deacylating the product with alcoholic ammonia to yield the desired nucleoside.

, 2. The method of preparing nucleosides selected from the class consisting of nucleosides of side and 2-aminopurine-G-thioglycoside which comprises The product was isolated the product with alcoholic ammonia.

3. The method of preparing nucleosides selected from the class consisting of nucleosides of 6-mercaptopurine and 2-amino-6-mercaptopurine which comprises converthydrogen halide acceptor, so as to form the acylated nucleoside of the corresponding 6-chloropurine, reacting the latter compound with thiourea and dcacylating the product with alcoholic ammonia.

4. The method of preparing nucleosides selected from the class consisting of nucleosides of 6-mercaptopurine and 2-amino-6-mercaptopurine which com rises convertlug a reagent of the class consisting of inosine and guanoderivative, reacting the acylated nucleoammonia.

5. The method of preparing nucleosides selected from the class consisting of nucleosides of 6-mercaptopurine and Z-amino-6-mercaptopurine which comprises converting a reagent of the class consisting of inosine and guanosiue to its O-acyl derivative, reacting the acylated nucleoside with phosphorus oxychloride in the presence of a hydrogen halide acceptor, so as to form the acylated nucleoside of the corresponding 6-ch1oropurine, reacting the latter compound with an alkali salt and deacylating the product with alcoholic ammonia.

6. The method of preparing an O-acylated nucleoside which comprises converting a reagent of the class consisting of inosine and guanosine to its O-acyl derivative, reacting the acylated nucleoside with phosphorus oxychloride in the presence of a hydrogen halide acceptor, so as to form the acylated nucleoside of the corresponding the latter compound with an alkali thiohexose.

7. The method or" preparing an O-acylated nucleoside which comprises converting a reagent of the class consisting of inosine and guanosine to its O-acyl derivative, reacting the acylated nucleoside with phosphorus oxychloride in the presence of a hydrogen halide acceptor, so as .to form the acylated nuclcoside of the corresponding 6- of dithiooxalic acid I sisting of inosine and chloropurine and reacting the latter compound with thiourea.

8. The method of preparing an O-acylated nucleoside which comprises converting a reagent of the class consisting of inosine and guanosine to its G-acyl derivative, reacting the acylated nucleoside with phosphorus oxychloride in the presence of a hydrogen halide acceptor, so as to form the acylated nucleoside of the corresponding 6- chloropurine and reacting the latter compound with an alakali salt of thioacetic acid.

9. The methodjof preparing an O-acylated nucleoside which comprises converting a reagent of the class conguanosine to its O-acyl derivative, reacting the acylated nucleoside with phosphorus oxychloride in the presence of a hydrogen halide acceptor, so as to form the acylated nucleoside of the corresponding 6-chloropurine and reacting the latter compound with an alkali salt of dithiooxalic acid.

10. The method of preparing nucleosides selected from the class consisting of nucleosides of 6-thioglycosido purine and 2-amino-6-thioglycosido purine which comprises converting a reagent of the class consisting of O-triacylinosine and O-tn'acylguanosine into its 6-chloro derivative by reaction With phosphorus oxychloride in the presence of a hydrogen halide acceptor, reacting the 6-chloro compound with an alkali thiohexose to form the 6-thi0- glycoside and deacylating this product with alcoholic ammonia.

11. The method of preparing nucleosides selected from the class consisting of nucleosides of o-mercaptopurine and 2-amino-6-mercaptopurine which comprises converting a compound of the class consisting of O-triacylinosine and O-triacylguanosine' to its 6-chloro derivative by reaction with phosphorus oxychloride in the presence of a hydrogen halide acceptor, reacting the 6-chl0ro compound With thiourea to give the 6-mercapto derivative and deacylating the latter with alcoholic ammonia.

12. The method of preparing nucleosides selected from the class consisting of nucleosides of G-mercaptopurine and 2-amino-6-mercaptopurine which comprises convertcapto derivative and deacylating the latter with alcoholic ammonia.

13. The method of preparing nucleosides selected from the class consisting of nucleosides of 6-mercaptopurine and 2-amino-6-mercaptopurine which comprises converting a compound of the class consisting of O-triacylinosine and O-triacylguanosine to its 6-chloro derivative by resides of 2 -amino-6-chloropurine with an acetic acid to alkali salt of-thiogive the 6-mercapto derivative and deacylating the latter with alcoholic ammonia.

16. The method of preparing nucleosides selected from the class consisting of nucleosides of G-mercaptopurine and 2-amino-6-mercaptopurine which comprises reacting 18. Purine-9-fi-d-ribcfuranoside-6-thioglucoside. a compound selected from the class consisting of the tY'll9. Purine-9 6-d-triaeetylriboside-6-thioglucoside. acylnucleosides of 6-chloropurine and the triacylnucleo- 29. 2-aminopurine B t -cl-ribofutancside-6-thioglucosides of 2-amino-6-cl1loropurine with an alkali salt of (11- side. thiooxalic acid to give the G-mercapto derivative and 5 21. 2-aminopurine 9 p-d-triacetylribofuranosicle-6- deacylating the latter with alcoholic ammonia. tnloglucoside.

17. A compound represented by the formula: 22. 2 amino-6-chloropurine-9-fl-cl-triacetylribofurano- Sl-glucose side.

\1, A/ m Reierences Cited in the tile of this patent l X L UNlTED STATES PATENTS I N 2,746,961 Hitchings et a1 May 22, 1956 R 2,852,505 Baker Sept. 16, 1958 q RCPCHQ 2,852,596 Goldman et a1 cept. 16, 1958 H G 7 OTHER REFERENCES Friedkin: Biochem ct Biophys Am 18, pp. 447-448 63 (1955). (Cited in Chem. Abstracts 40, 3516, 1956.)

. Eohnson, In, tal.: lAm/hemSo .78,3863 1956 wherein R is selected from the class conslstmg of CH CO, 20 e b c C H CO and hydrogen and R is selected from the class consisting of the amino group and hydrogen.

Claims (2)

1. THE METHOD OF PREPRING NUCLEOSIDES SELECTED FROM THE CLASS CONSISTING OF NUCLEOSIDES OF 6-MERCAPTOPURINE, 2-AMINO-6-MERCAPTOPURINE, PURINE-6-THIOGLYCOSIDE AND 2AMINOPURINE-6-THIOGLYCOSIDE WHICH COMPRISES CONVERTING A REAGENT OF THE CLASS CONSISTING OF INOSINE AND GUANOSINE TO ITS O-ACYL DERIVATIVE, REACTING THE ACYLATED NUCLEOSIDE WITH PHOSPHORUS OXYCHLORIDE IN THE PRESENCE OF A HYDROGEN HALIDE ACCEPTOR, SO AS TO FORM THE ACYLATED NUCLEOSIDE OF THE CORRESPONDING 6-CHLOROPURINE, REACTING THE LATTER WITH A REAGENT SELECTED FROM THE CLASS CONSISTING OF THIOUREA, THE ALKALI THIOHEXOSES AND THE SALTS OF ALIPHATIC THIOCARBOXYLIC ACIDS AND DEACYLATING THE PRODUCT WITH

17. A COMPOUND REPRESENTED BY THE FORMULA: