IL44537A - 9-beta-d-arabinofuranosyl purine nucleotides and their preparation - Google Patents

Description

purine nucleotides and their preparation ICN C 42618 INVENTION During past many nucleoside analogs have been found to exhibit antitumor and antiviral the presently known synthetic nuclcosidic antiviral the more important arc considered to be deoxyuridine 9 a inofuranosvl adenine and 1 arabino uranosyIcytos ine Of these only is available speci ically as an antiviral and this compound has extremely low a maximum solubility of about weight and is also highly presently is undergoing clinical testing as an antiviral and while the reported evidence suggests that is an effective agent against a spectrum of virus its utility is severely limited by its low and by toxic symptoms which include mild and central nervous system involvement which causes illusions and nucleosidic analogs are used to inhibit viral or tumor the nucleosides are usually metabolized in vivo to their correspondin mono or poly phosphates which arc the actual inhibitors of such Λ major obstacle in the use of nucleoside analogs in chemotherapy lias been the emergence of cellular resistance whereby such compounds are degraded to a form where they may be less effective It is accordingly desirable to have nucleosidic analogs which are capable of effectively inhibiting the development of virus infections and which also possess superior solubility and less toxicity than presently known antiviral The production of such a is quite since relatively few nucleosidic compounds are known which have demonstrated antiviral even in to provide such a compound which acceptable activity and which is also capable of tacting the virus infection in effective concentrations makes this task exceedingly In Herman Patent is as a as an antiviral This as an and a precursor to an ATI1 analog is rapidly in the metabolic system to uric Since ATP levels in the system arc carefully maintained by the metabolic system at low levels the e ectiveness of such compounds as antiviral agents is accordingly In of the we thus sought to prepare additional nuclcosidic analogs in order to investigate the possibilities that certain such compounds might be capable of withstanding rapid metabolic degradation and also penetrating the cellular membrane contacting virus infections in effective As will be apparent from the cription which we have synthesized and found such compound to demonstrate marked activity against a spectrum of herpes and other Vie have also synthesized other uranosy 1 nucleotides having a pliosphorylatcd joined thereto by a glycoside linkage and found that many of them demonstrate activity against a spectrum of herpes and other SU MM R Y O P P I The present invention thus relates to compounds which wherein Z is H or and X is and one of Y or is H and the other is 0 II or Y and taken together are or Z is H and X is and one of Y and is H and the other is 0 II i is or and is OH or O and is substituted alkali or alkaline earth provided that when X is and is then is not OH or DETAILED DESCRIPTION OF THE INVENTION The purine nucleotides of this invention may be prepared by the methods set forth in Examples I VIII which nofuranosyladcnine nucleotides may be prepared by first reacting the appropriate furanosyl with a suitable phosphorus oxychlor for phosphorus oxychloride or its methyl phosphorodichloridate in a suitable tr ialkylpliospliate preferably to form the corresponding furanosyl The nucleoside is added to the phosphorus oxychloride solution with stirring and the reaction allowed to proceed to completion at from about to about which time is from about 3 hours to about 24 The nucleotide product of the first step is next treated with an alkaline as for example sodium bicarbonate or potassium bicarbonate until a stable pT1 of from about 5 to about 7 is The adenine nucleotide is then recovered for by chromatography and lyophil ine nucleotides may also be prepared by reacting the appropriate adenine nucleotide in water with glacial acetic acid and sodium The sodium nitrite is preferably added to a solution of the nucleotide glacial acetic acid and the is allowed to proceed to completion at from about to about preferably to The reaction period may be from about 15 hours to about 24 The nucleotide product is recovered and with an alkaline carbonate for potassium bicarbonate or sodium bicarbonate recovered as by cry In the following spectra were recorded on a spectrophotometer infrared spectra were determined on a Model All temperatures are in degrees centigrade and all parts by weigh I 9 Arab S Method 1 To an cooled suspension adenine in water and glacial acetic acid was added sodium nitrite The flask was loosely stoppered and stirred for liours in the ice The stirring continued overnight without adding ice to the ice Tic solvent N1 I 1 I2 indicated completion of the reaction The colorless solution was evaporated in vacuo to the residue was dissolved in water and was carefully neutralized with solid The neutral solution was applied to a column containing 75 ml of Dowex ion exchange The column was washed with water and the fractions containin material were pooled and concentrated vacuo to about 25 was added to the concentrated solution and chilled The solid that separated was washed with cold water and crystallized from water as colorless yield g calcd for 12 9 b o n t h c Method 2 arabino uranosyl was added with stirring to a ice of ml trimethyl phosphate and g phosphorus lor ide After all the solid had dissolved it was stored for hours at solvent I indicated the reaction to be it was slowly into ice water containing g The ice water solution was allowed to stand for 1 hour to stabilize the at The solution was extracted with ether x 75 to the trimethyl The volume of the aqueous phase was duced vacuo until crystals began to was added to dissolve the and the solution was applied to the top of a 750 g Cheney charcoal The charcoal was washed with water to remove and then the product was eluted off with 50 aqueous containing The eluant was reduced to a small volume vacuo odor was and the was adjusted to was added until the solution became turbid and the solution was stored at Crystals were filtered and dried at under aspirator vacuum TIT f i 5 0 1 t c in freshly distilled phosphate was cooled to in an ice The ice bath was removed as g dried at or 5 was There was no noticeable initial rise in The temperature was monitored between After 2 hours a clear solution was which was stored overnight at The solution was then poured onto ice water containing g of Additional sodium bicarbonate was added periodically until the was stable at about an Trimethyl phosphate was removed by extraction with ether x 150 Dissolved ether and excess water were removed by evaporation under reduced pressure until salts began to Enough water was added to achieve solution and the was checked The solution was added carefully to the top of a Dowex column 300 The column was washed with water until no further absorbing species were detected in the Gradient to formic gave the product in a thick The appropriate fractio were evaporated in keeping the temperature below to about 100 The remaining solution was frozen and lyophilized to obtain fluffy solid weighing g 257 λ 25S nm nm ax calcd for 1 TV 9 0 n th 5 0 methylphosph te To an ice cooled solution of by the method of Hxainplc in water and glacial acetic acid was added sodium nitrite The flask was loosely stoppered and stirred for hours in an ice The stirring was continued overnight without adding ice to the ice Tic solvent indicated the completion of the The colorless solution was evaporated in vacuo to The residue was dissolved in water and carefully neutralized with solid potassium The neutral solution was applied to a column containing 80 ml of 50 x 8 ion exchange The column was washed with water and the fractions containing absorbing material were pooled and concentrated in vacuo to about 20 Hthanol was added to the concentrated aqueous solution and it was chilled The solid that separated was washed with a small volume of cold water and crystallized from aqueous ethanol to yield g uv 248 nra 10 251 max J max max calcd EXAMPLE V th 5 To an ice cooled suspension of as described in et of 721 in water containing glacial acetic acid was added sodium nitrite The flask was loosely stoppered and stirred for hours in an ice The reaction was allowed to proceed for a further After evaporation of the clear reaction mixture and neutralization with potassium it was treated in the same way as described in Example IV to yield g uv 255 n calcd for EXAMPLE rabinofura phosphate To an ice cooled suspension of ranosyl phosphate the method described in Patent in water containing glacial acetic acid was added sodium nitrite The flask was loosely stoppered and stirred for hours in an ice The reaction was allowed to proceed 20 After evaporation and neutralization it was treated in the way as described in Example IV to g 247 for VII A mixture of phosphorus oxychloride and freshly distilled phosphate was cooled to in an ice The ice bath was removed as finely powdered dried at according to the method of Renis et Che was The temperature was monitored between After 30 min a colorless solution was which was stored overnight at The reaction mixture was then poured onto ice water containing g of sodium Additional sodium bicarbonate was added periodically until the pli was stable at an Trimethy Iphosphate was removed by extraction with etlver x 75 Dissolved ether and excess of water was removed by evaporation in vacuo until salts began to Enough water was added to achieve solution and the was checked before placing the solution on the top of a Dowex 1 x 2 column 200 60 The column was washed with water until no absorbing material was present in the The nbove mixture of phosphates was in freshly prepared sodium methoxide in and the solution was allowed to stand at room temperature The reaction mixture was carefully neutralized with 50 x 8 The resin was removed and the filtrate concentrated to about 5 It was applied to a Dowex x 2 column 40 The column was washed with water until no absorbing material was present in the Upon gradient elution to formic emerged first followed by the The 3 fractions were lyophilized to yield 105 g of 257 258 nm 6 258 nm calcd for EXAMPLE VIII 9 phate To an ice cooled solution of 9 rab inofuranosyl in water and glacial acetic acid was added sodium nitrite The flask was loosely stoppered and stirred overnight at After evaporation and neutralization with potassiuin it was treated in the same way as described in Example IV to yield mg 252 The salts of purine indicated above may be in the conventional manner by reaction of the free acid with a such as to for the Reaction with other appropriate bases yield the potassium and ammonium or substituted ammonium as will be appreciated readily by the art I iii Several compounds of this invention were tested for activity by virus rating method of Sidwell ct described in Applied The compound is dissolved in a cell culture medium consisting of amino streptomycin and indicator dye in The virus suspended in the cell culture medium was added to an monolayer of KB or RK 13 and an equal volume of compound was then added within 15 The infected treated cells were incubated three days and the degree of viral pathogenic effect on the cells was graded following microscopic Controls for each experiment included cell controls and cell culture medium virus controls and virus and cell culture and toxicity controls and chemical and cell culture Of the viruses employed in the antiviral herpes type 1 is implicated in labialis herpes keratitis and herpes The herpes virus is also implicated in infectious lymphoma and cervical Vaccinia is an avirulent form of pox virus employed for smallpox which occasionally results in undesired side Myxoma causes death in domestic and wild preceded by respiratory illness and severe Pscudorabies causes infectious bulbar paralysis also referred to as the disease in dogs and The results of the o experiments are shown in Table I which The virus rating system of Sidwell ct al described in was used to the degree of signi of C A VR greater than is indicative of definite antiviral TABLE J ANTIVIRAL ACTIVITY NUCLEOTIDES IN CELL CULTURE SYSTEMS Type 1 2 Herpes Simplex Herpes Simplex Vaccini Name Virus Virus Virus 3 inofuranosy1 5 phosphate phosphate 3 phosphate 9 inofuranosy1 The results clearly show that the above compounds exhibit significant in vitro activity against some or all of the listed EXAMPLE The following experiment was carried out using against herpes virus in In this the compound was dissolved in saline and inoculated intraperitoneally 4 hours after the virus continuing twice daily thereafter for 8 The results in Table II show that was less effective and that treatment prevented up to 50 of the mice from dying This compound had an activity of against myxoma and against pseudorabies Encephalitis Activity of g Male Swiss Mice Drug Intra Herpes strain 123 Treatment Virus Dose and intracerebral Treatment frequen Observation 21 days for 8 days post Survivors Name Dose Perc Numb 250 125 adenTne value square The effect of 5 and on herpes encephalitis of mice was Tn these young adult Swiss mice were inoculated intracerebrally with a moderately lethal dose of killing of the of type 1 herpes Six hours later the animals were jected intracerebrally with one or more concentrations of either drug in saline solution or with saline only The highest dose used of each drug was the maximum tolerated dose the highest dose not lethally toxic to the The animals were then observed for 21 days and deaths recorded as In each was more effective than when the percent increase in survivors to virus control was plotted against the relative MTD dose of each Up to 5 experiments were run with the various relative drug and the average percent survivor increase was The data is summarized in figure 1 to have a therapeutic advantage over 1 Effect of intracerebral treatment with arab 5 and 9 rab inofuranosyl adenine on type 1 herpes virus induced encephalitis in mice of for each point XII Tn this the compound was evaluated against herpes keratitis in both eyes in New Zealand white rabbits were anesthetized with proparacainc and the corneal epithelium was then uniformly Λ pension of type 1 herpes simplex virus was added to each in sufficient quantity to cause a unifrom keratitis to develop within 3 Four animals were treated topically drop per with or or of in polyvinyl alcohol or PVA only in the case of virus was hourly to with each drug in Lacrilube ophthalmic ointment applied at daily for 7 days beginning 24 hours after virus The eyes on days and 9 for corneal lesion size and swelling and Λ score of 0 to 4 was given for The person examining the eyes did not know which eyes were treated with drug or The opacity and lesion scores were multiplied by 10 and the other parameter scores were multiplied by then added together for a lesion which was plotted vs day of This graphic display of the effect of each drug is seen in Figure at either concentration used was more effective than in inhibiting the development of the 20 it lif ect of optical treatment with and on type 1 nduced keratitis i rabbit DAYS XIII The effect of and on equine abortion induced hepatitis mortality in liamsters was Young adult hamsters were inoculatetl intraperitoneally with a lethal dose equine abortion Each dissolved or suspended in or saline only for virus was administered intrapcritoneally to the animals twice daily for 4 beginning 1 hour The animals were observed 21 days and deaths recorded as they each experiment proved more effective and less toxic than keeping up to of the infected animals alive the duration of the The results are summarized in Table III Effect of and 9 on hepatitis associated mortality in hamsters infected with female Syrian golden hamsters Treatme Virus 10 Treatme Virus Inoculation days Observa Toxicity Su Control Treated In r 250 125 250 125 Virus control r 250 250 Virus control P Probability exact Animals dying on or before day P Probability Insufficient animals died for accurate statistical insufficientOCRQuality

Claims (16)

44537/3 CLAIMS:

1. A compound of the structure: wherein Z is H or OH; and X is =0, and one of Y or Y1 is H and the other is — P— R, or Y and Y1 taken together are 0=P— I or Z is H and X is =NH and one of Y and Y ' is H and the other is is OH, C^-C^-O-alk l or OM and is OH or OM; and M is ammonium, substituted ammonium, alkali metal, or alkaline earth metal; provided that when X is =NH and Y" is H, then is not OH or OM. 44537/2

2. > Compounds of Claim 1 wherein Z is H, X is =0, and Y' is H of the structure: and R- is OH, C, -Cc -0-alkyl or OM and R_ is OH or OM and M is ammonium, 1 l b — i. substituted ammonium, alkali metal, or alkaline earth metal.

3. Compounds of Claim 1 wherein Z is H, X is =0 or =NH, and Y is H of the structure:, and Rj^ is OH, 0χ -C^O-alkyl or OM and R2 is OH or OM and M is ammonium, substituted ammonium, alkali metal, or alkaline earth metal. 44537/2

4. Compounds of Claim 1 wherein Z is H and X is =0 of the structure: and R- is OH, C. -C, -0-alkyl, or OM and M is ammonium, substituted 1 1 D — ammonium, alkali metal, or alkaline earth metal.

5. 9-β -D-Arabinofuranos lhypoxanth i.nc- 5 ' -phos ate

6. 9-3-n-Arabinofuranosyl ypoxanthine-3 ' , 5 ' -cvcli phosphat c .

7. 9 - P>- Π- Arab i nnfuranosy lhynox n thine- 5 ' -O-riethv] - phosphate .

8. 9 - B- U-Arab i iio uranosyl -M^ -hydroxyhypoxantli nc-5 phosphate .

9. 9- B-l_)-Arabinofuranosylhypoxanthinc-3 * -phosphate

10. 9-P- -Arabinofuranosyladenine-5 ' -0-mcthyl - phospiiate.

11. 9-6-Q-Arabinofuranosyladeninc-3 ' -phosphate . 44537/2

12. A process for preparing 0-f3-T)-arabinofuranosyl- 4 adenine nucleotides comprising the steps of: reacting a corresponding 9-B-l)-furanosyl nucleoside with a phosphorus oxychloride compound in t lie presence of a trialkyl-phospliate solvent for from about 3 hours to about 24 hours at from about 0°C to about 15°C to form a corresponding arabino-furanosyladenine nucleotide; and recovering the product.

13. The process of claim 12 wherein the recovery step includes the steps of treating the nucleotide with an alkaline carbonate to a pH of from about 5 to about 7 and separating the product by chromatography and lyophil zat ion .

14. A process for preparing 9-6- -arabinofuranosyl-hypoxanthine nucleotides of Claim 1 comprising the steps of : reacting a corresponding 9-2 -D-arabinofuranosyl- adenine nucleotide with glacial acetic acid and sodium nitrite in the presence of water for from about 15 hours to about 24 hours, at from about 10°C to about 30°C to form a corresponding arabinofuranosylliypoxantnine nucleotide product; and recovering said nucleotide product.

15. ' The process of claim 1 wherein said recovery step includes the steps of treating the nucleotide product with an alkaline carbonate and crystallizing the nucleotide product. 44537/2

16. The process of Claim 14 for preparing 9-B-D-arabino- furanosylhypoxanthine-5 ' -phosphate comprising the steps of : reacting 9-6-n-arabinofuranosyladeninc-5 ' -phosphate with glacial acetic acid and sodium nitrite in the presence of water for from about 15 hours to about 24 hours , at from about 10°C to about 30°C to form said hypoxanthine- 51 -phosphate as a product; and recovering said product by treating it with an alkaline carbonate and then crystallizing it. For the Applicants DR. REINHy0DQ COH AND PARTNERS By :