J. CARBOHYDRATES -NUCLEOSIDES “NUCLEOTIDES, 4(5)}, 281-299 (1977)
`
`CHEMISTRY OF ANTITUMOR TRIAZINE NUCLEOSIDES.
`AN IMPROVED SYNTHESIS OF DIHYDRO-5-AZACYTIDINE.
`
`John A. Beisler?, Mohamed M. Abbasi”,
`
`James A. Kelley and John S. Driscoll
`
`Drug Design and Chemistry Section
`Laboratory of Medicinal Chemistry and Biology
`Developmental Therapeutics Program
`Division of Cancer Treatment
`National Cancer Institute
`National Institutes of Health
`Bethesda, Maryland 20014
`
`5-Azacytidine” (7, aza-C) is a nitrogen bioisostere of
`cytidine which has been used effectively for the clinical
`treatment of leukemia’. The severe gastrointestinal toxicity
`of 7 is best minimized by prolonged continuous infusion,°?®
`but working in opposition to this mode of administration is
`the instability of aza-C in aqueous formulations * which
`
`results in a continuously decreasing aza-C concentration and
`
`the production of increasing concentrations of hydrolysis
`
`products of unknown biological effects.
`
`A reduced analog,
`
`5,6-dihydro-5-azacytidine hydrochloride (6, DHaza-C), was
`
`281
`
`Copyright © 1977 by Marcel Dekker, Inc. AN Rights Reserved. Neither this work nor any part
`may be reproduced or transmitted in any form or by any meahs, electronic or mechanical, including
`photocopying, microfilming, and recording, or by any information storage and: retrieval system,
`without permission in writing from the publisher.
`CELGENE 2004
`CELGENE 2004
`APOTEX v. CELGENE
`APOTEX v. CELGENE
`IPR2023-00512
`IPR2023-00512
`ith
`
`:
`
`‘
`Supplied by the British Library 08 Feb 2017, 11:07 (Gt)
`
`
`
`CHEMISTRY OF ANTITUMOR TRIAZINE NUCLEOSIDES.
`AN IMPROVED SYNTHESIS OF DIHYDRO-5-AZACYTIDINE.
`
`John A. Beisler?, Mohamed M. Abbasi”,
`
`James A. Kelley and John S. Driscoll
`
`Drug Design and Chemistry Section
`Laboratory of Medicinal Chemistry and Biology
`Developmental Therapeutics Program
`Division of Cancer Treatment
`National Cancer Institute
`National Institutes of Health
`Bethesda, Maryland 20014
`
`5-Azacytidine” (7, aza-C) is a nitrogen bioisostere of
`cytidine which has been used effectively for the clinical
`treatment of leukemia’. The severe gastrointestinal toxicity
`of 7 is best minimized by prolonged continuous infusion,°?®
`but working in opposition to this mode of administration is
`the instability of aza-C in aqueous formulations * which
`
`results in a continuously decreasing aza-C concentration and
`
`the production of increasing concentrations of hydrolysis
`
`products of unknown biological effects.
`
`A reduced analog,
`
`5,6-dihydro-5-azacytidine hydrochloride (6, DHaza-C), was
`
`281
`
`Copyright © 1977 by Marcel Dekker, Inc. AN Rights Reserved. Neither this work nor any part
`may be reproduced or transmitted in any form or by any meahs, electronic or mechanical, including
`photocopying, microfilming, and recording, or by any information storage and: retrieval system,
`without permission in writing from the publisher.
`CELGENE 2004
`CELGENE 2004
`APOTEX v. CELGENE
`APOTEX v. CELGENE
`IPR2023-00512
`IPR2023-00512
`ith
`
`:
`
`‘
`Supplied by the British Library 08 Feb 2017, 11:07 (Gt)
`
`
`
`-4
`
`+ 1iz
`
`.ProcessssnTSSEPTATEEVEESVUSaterROemeNOreerersemeEeremairanasaDoren
`
`282
`
`BEISLER ET AL.
`
`recently synthesized by reduction of aza-C with sodium boro~
`
`hydride with the goal of eliminating the solution instability
`while retaining the antitumor activity of the parent drug’.
`
`Relative to the parent drug, DHaza-C proved to have a greatly
`
`enhanced aqueous stability over a broad pH range. DHaza-C also
`
`possessed substantial antitumor activity in mouse leukemia test
`systems, ’*8»9
`In order to ensure sufficient quantities of
`
`DHaza-C to initiate preclinical pharmacology studies, it was
`
`necessary to develop a more direct, economical synthesis.
`
`Accordingly, an improved synthesis of DHaza-C is described in
`
`this report, and in addition,
`
`the results of chemical investi-
`
`gations directed toward reactions at the critical 6-position of
`
`the aza-C triazine ring, the vulnerable locus for hydrolytic
`
`attack, are presented.
`
`RESULTS
`
`Synthesis of Dihydro-2-azacytidine Hydrochloride (6).
`The earlier? synthesis of DHaza-C (6) via a borohydride
`reduction required the use of aza-C (7) as a starting material.
`
`However,
`
`the synthetic sequence leading to 6 can be compressed
`
`and the overall yield increased by reducing the appropriate
`
`blocked nucleosides (4 or 5) directly with sodium borohydride.
`
`The nucleosides (4 and 5) were prepared essentially according
`to literature? procedures utilizing the Friedel-Crafts
`
`catalyzed N-ribosylation of trimethylsilylated 5-azacytosine (1)
`
`with per-Q-acylribofuranose intermediates (2 or 3). Treatment
`
`Supplied by the British Library 08 Feb 2017, 14:07 (GMT}
`
`ekame
`
`
`
`IMPROVED SYNTHESIS OF DIHYDRO-5-AZACYTIDINE
`
`283
`
`of the tri-O-benzoyl nucleoside (5) with sodium borohydride in
`
`hexamethylphosphoramide (HMPA) solution led to the reduction
`
`of the imine linkage accompanied by the removal of the benzoyl
`
`Ac
`*|SSESEEe
`
`AO
`
`o
`
`Snti,
`
`AO
`
`sd
`o
`
`HNSiMey
`we ie
`asin
`
`1
`
`ne
`
`o
`
`RO
`
`oR
`
`a
`3 Amececed
`<
`EB
`
`NH
`ae
`
`\y
`
`o
`
`NH4/MoOH
`
`RO
`
`OR
`
`4, A= CHCO
`5, A= Cah,CO
`;
`
`NaBHy
`
`W/PdiHCl
`
`“NHy
`
`i+r
`HO |= |
`
`aSTFA
`
`.SpinaMeHgCOT “
`AO
`=
`
`rercree
`Ha /PafHcr
`
`SERGEramceerernmerorearrrer
`
`
`
`od hes
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`HO
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`o
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`19
`
`:
`
`8
`
`Supplied by the British Library 08 Feb 2017, 11:07 (GMT)
`
`
`
`284
`
`BEESLER ET AL.
`
`protecting groups to provide the reduced and deblocked nucleoside
`
`in the form of a boron complex. Without further purification
`the boren-containing product was hydrolyzed with aqueous acid?
`
`to give 6 in 72% yield (58% overall yield based on 3). Similarly,
`
`the tri-O-acetyl nucleoside (4) was reduced with borohydride to
`
`afford an almost identical
`
`(73%) yield of 6 (50% overall yield
`
`based on 2). Therefore, of the borohydride reductions the former
`
`sequence is the methed of choice.
`
`It is also possible to reduce and deblock the tri-O-acetyl
`
`derivative (4) by catalytic hydrogenation in ethanol under acidic
`
`conditions to give 6 in 92% yield (63% overall yield based on 2).
`However, similar hydrogenation of the tri-O-benzoyl derivative
`(5) resulted in reduction of the imino group, but the reduction
`
`unfortunately was net also accompanied by ethanolysis of the
`
`blocking groups.
`
`We have found that the hydrolysis of 5 with methanolic
`ammonia to give 7 proceeds in 62% yield. Since we demon-
`strated earlier® that 7 can be converted to 6 in 72% yield
`
`using a borohydride procedure, a 36% overall yield of 6 based
`
`on 3 can be realized with our original synthesis requiring
`
`three synthetic steps (3+5+7>+6). Altematively,
`
`the reduc-
`
`tion of 7 by hydrogenation in ethanol-hydrochloric acid gave 6
`
`in a similar (39%) overall yield based on 3.
`
`The present syn-
`
`
`
`thesis of DHaza~C requires two steps using the borohydride
`
`method (3+5+6) or the hydrogenation method (2+4+6) with
`
`Similar overall yields of 58% and 63%, respectively.
`
`tserrsnenreceunanscemerecereuNoueYeHPCUUPONNONLAURAESEA
`
`ce
`
`Supplied by the British Library 08 Feb 2017, 1:07 (GMT)
`
`_ereemNatloreuaLeMNMRRTE
`
`
`
`IMPROVED SYNTHESIS OF DIHYDRO-5-AZACYTIDINE
`
`285
`
`The Oxidation of 5-Azacytidine (7)
`
`to the Oxo-analog (9).
`
`As part of our effort to synthesize hydrolytically stable
`
`analogs of 7, we also investigated the conversion of 7 to a
`derivative having a higher oxidation state at the 6-position
`
`than aza-C.
`
`It was found that 7 was ‘susceptible to selective
`
`oxidation when treated with 303 hydrogen peroxide in acetic
`
`acid solution to give the high-melting oxo-S-azacytidine (9).
`
`The NMR spectrum of 9 lacked the singlet due to the C-6 aromatic
`
`proton characteristic of 7 and the uv spectrum differed con-
`
`The mass spectra of both the trimethyl-
`siderably from that of 7.
`silylated (TMS) and trifluoroacetylated (TFA) derivatives of 9
`were consistent with the addition of one oxygen atom to the
`
`triazine ring of 7. Acid hydrolysis of the glycosidic bond
`
`of 9 gave ammelide (10) which was identified by elemental
`analysis and the mass spectrum of its TMS derivative. Although
`
`9, which has the novelty of a higher degree of symmetry in the
`triazine moiety than 7,
`ig stable in aqueous solution in accord
`
`with our expectations, it is devoid of antitumor activity in the
`L1210 leukemia assay!“ wherein aza-C exhibits high activity.
`Since it has been postulated®’9 that DHaza-C is a pro-drug
`i of aza-C, it was of interest to investigate the chemical
`
`form
`
`conversion of [Haza-C (6)
`
`to aza-C (7)
`
`in order to probe the
`
`likelihood of this conversion occurring as a result of in vivo
`
`An acetic acid solution of 6 when treated with
`metabolism.
`hydrogen peroxide at room temperature contained 7 after three
`days as shown by TLC. At the end of twelve days, the oxidized
`
`Supplied by the British Library 08 Feb 2017, 11:07 (GMT)
`
`
`
`2B6
`
`BELSLER ET AL.
`
`nucleoside (9) was also detectable in the reaction solution
`suggesting a sequential oxidation process:
`6+7~+9. After
`fifteen days,
`the reaction was worked-up and the products were
`
`derivatized with trifluoroacetic anhydride for MS analysis.
`
`Probe introduction of the TFA mixture into the spectrometer
`
`gave a composite spectrum of 6° TFA,» os TFA, , a TFA, and
`10: TFA. When a few crystals of ferrous sulphate were added
`
`as a catalyst to the oxidation system,
`
`the reaction rate in-
`
`creased considerably but only 9 and 10 were found (TLC, MS)
`
`as products, with 10 as the major product. As well as lending
`
`support
`
`to the pro-drug hypothesis of 6,
`
`these experiments
`
`Suggest the possibility that the oxidation of 7 to 9 (or 6 to
`9) could serve as a detoxification mechanism in vivo although 9
`
`has not been identified and reported as a metabolite of 7.
`
`The Dehydrogenation of 6 to Give 5-Azacytidine (7).
`
`Treatment of 6 with bis-(trimethylsily1) -trifluoroacetamide
`
`(BSTFA)
`
`in acetonitrile solution at room temperature led smoothly
`
`to the introduction of five TMS groups into the nucleoside.
`
`The
`
`TMS, derivative gave a single peak in the GC, which produced a
`mass spectrum consistent with the proposed structure upon sub-
`sequent GC-MS analysis’. However, if the silylation solution
`
`was heated, a second peak began to appear in the chromatogram
`
`due to tetrakis - (trimethylsilyl) -5-azacytidine (7: TMS4).
`This materia] was identified by MS and was identical to the
`
`product of 7 with this silylation reagent. Continued heating
`
`ultimately resulted in a complete conversion of 6° TMS, into
`
`f
`
`Supplied by the British Library 08 Feb 2017, 11:07 (GMT)
`
`
`
`IMPROVED SYNTHESIS OF DIHYDRO-5-AZACYTIDINE
`
`287
`
`Le TMS, with the formal loss of the elements of (CH,) SiH. This
`unexpected event seemed sufficiently unusual to warrant further
`
`investigation to determine its synthetic potential for the pre-
`
`paration of triazine nucleosides which might otherwise be
`
`in-
`
`accessible and thereby further extend the use of the trialkyl-
`
`silyl function in synthetic organic chemistry. Therefore, the
`
`reaction of 6 with the silylation mixture was repeated on a
`
`preparative scale and its progress monitored with GC. When the
`
`the product, 7° TMS, 5 was de-trimethyl-
`reaction was complete,
`silylated by methanolysis to give a 76% yield of 7. The aza-C
`synthesized in this way was identical to an authentic Sangte’>:
`
`Applying the silylation reaction to 6 containing a deuterium
`atom at the 6-position” of the triazine resulted in a 2.9:1
`
`mixture (GC-MS) of 6-deutero-5-azacytidine-TMS, and 7* TMS4
`apparently due to a primary deuterium isotope effect. Our
`earlier” positional assignment for the deuterium atom based on
`
`NMR spectral characteristics is,
`
`therefore, confirmed.
`
` Addi-
`
`tional synthetic work making use of the silylation-dehydrogena-
`
`tion reaction to prepare nucleosides of biological interest will
`
`be reported at a later date.
`
`Preparation of the 2',3'-O-isopropylidene derivative of
`
`7 obtained from the silylation-dehydrogenation procedure was
`
`accomplished in acetone Solution containing 2,2-dimethyoxy-
`propane and catalyzed by perchloric acia!®,
`A good yield of 8
`was obtained which was identical in melting point and spectral
`
`properties with the isopropylidene derivative prepared in the
`
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`
`
`
`286
`
`BEISLER ET AL.
`
`The difference in chemical shift
`same way from authentic! 7.
`{Aé) of the pair of singlets due to the isopropylidene methyl
`
`groups of 8 was observed to be 20 Hz.
`
`Since it has been demon-
`
`strated that a Aéd > 15 Hz is proof of thé 8-configuration of the
`aglycone at C), carbon of ribofuranosyl nucleosides!’ , spectral
`confirmation is now at hand for the 8-configuration originally
`assignea!§ to aza-C as well as all of the nucleoside derivatives
`
`synthesized during the course of the present investigation.
`DISCUSSION
`
`With the synthesis of 9 we have added a third member in a
`
`series of triazine nucleosides differing only in the oxidation
`
`state of a carbon atom in the heterocyclic moiety. Thus,
`
`DHaza-C (6) represents the lowest oxidation state, aza-C (7)
`
`is intermediate, and 9 is at the highest oxidation state in
`
`the series.
`
`Ina structure-activity relationship context it is
`
`interesting to note that the intermediate oxidation state, 7,
`
`has the greatest antitumor potency against murine L1210 Leukemia,
`
`the lowest oxidation state, 6, has good activity but at a lesser
`
`potency, and 9, the highest oxidation state, is completely in-
`
`active in the L1210 leukemia test system.
`
`EXPERIMENTAL
`
`
`
`“THEFTEDPAERYNPOUNCERESETeeEEESEEREEREREREEERAPEUROSEREAASFESCENES
`
`
`
`Electron impact mass spectra were obtained on a GC-MS system
`
`consisting of a Varian Aerograph 2740 gas chromatograph coupled
`to a DuPont 21-492 mass spectroneter by a glass transfer line
`
`and single-stage jet separator; operating conditions were as
`
`Supplied by the British Library 08 Feb 2017, 11:07 (GMT)
`
`
`
`IMPROVED SYNTHESIS OF DIHYDRO-5-AZACYTIDINE
`
`289
`
`previously described’. Gas chromatographic separation of
`
`mixtures and determination of isothermal retention indices
`crt) 29 were accomplished on a 1.83m x 2mm i.d. glass column
`
`packed with 3% SE-30 on 100/120 mesh Gas-Chrom Q and operated
`
`at 220°. Nucleosides and their aglycones were derivatized for
`GC or MS analysis by either per-trifluoroacetylation® ’-°
`(direct probe MS) or by per-trimethylsilylation® (GC-MS) by
`
`using previously reported procedures unless otherwise indicated.
`
`Proton NMR spectra were recorded with a Varian T-60 or a Varian
`
`HA-100D spectrometer using tetramethylsilane as an internal
`
`standard.
`
`A Cary Model 15 spectrophotometer was used to obtain
`
`UV spectra and a Perkin-Elmer Model 621 was used to record
`
`infrared spectra. Optical rotations were measured in a 1-dm
`
`cell with a Perkin-Elmer Model 141 polarimeter. Elemental
`
`analyses were performed by the Section on Microanalytical
`
`Services and Instrumentation, NIAMDD, NIH and by Galbraith
`
`Laboratories, Inc., Knoxville, Tenn.
`
`Compound purity was
`
`routinely checked by TLC using 5 x 20 cm plates coated with
`Baker 1B2-F silica gel.
`Four solvent systems were employed:
`
`butanol-ethanol-water (40:11:19), butanol-acetic acid-water
`(55253);
`isopropanol-ammonia-water (7:1:2),
`isebutyric acid-
`
`anmonia-water (66:33:1.5). Melting points were determined with
`
`a Thomas-Hoover capillary apparatus and are uncorrected.
`
`5-Azacytosine, 2 and3 were purchased from the Aldrich Chemical
`
`Co., Milwaukee, Wisc. and were used without further purification.
`
`Supplied by the British Library 08 Feb 2017, 11:07 (GMT)
`
`
`
`290
`
`BEISLER ET AL,
`
`
`2,3,5~tri-0-acetyl-8-D-ribofuranosy1)1,3,5-
`
`triazin-2(1H)-one (4). With some modifications, which led to
`
`an improved yield,
`
`by Niedballa and Vorbruggen
`
`and 2 {5.0 g, 15.7 mmol)
`
`the procedure followed was that described
`10.
`To a solution of y= (21.4 mmol}
`in dry acetonitrile (150 ml), cooled
`
`to 0°, was added a solution of stannic chloride (3.2 ml, 27.8
`
`mmol} in acetonitrile (80 m1) under anhydrous conditions. The
`
`solution temperature was slowly allowed to increase from 0° to
`22° over 2 h, then maintained at 22° for 30 min. After work-up? ;0
`
`concluded by treatment with charcoal and crystallization fran
`
`ethyl acetate (75 ml), 3.92 g (68%) of 4 was obtained mp 160-
`161° (1it. 1 mp 160-161°). Using 1,2-dichloroethane as the
`0
`
`reaction solvent gave identical results.
`
` 4-Amino-1- (2 ,3,5-tri-0-benzoy1-8-D-ribofuranosy]) -1,3,5-
`
`The condensation of 1 and 3according
`triazin~2(1H)-one (5).
`to the procedure of Niedballa and Vorbruggen!? gave the
`
`O-benzoylated S-azacytidine (5)
`cuit. 2mp 186-187).
`
`in 81% yield, mp 184-186°
`
` 4-Amino-5 ,6-dihydro-1-8-D-ribofuranosyl1-1,3,5-triazin-2 (iH)
`
`-
`
`one Hydrochloride (6). Method A. Borohydride Reduction of
`
`Compound 4.
`
`A solution of 4 (1.11 g, 3.0 mmol)
`
`in 7 ml HMPA
`
`was stirred while 300 mg (7.8 mmol) of sodium borohydride was
`
`added.
`
`The reaction mixture was heated at 50° for 1h then
`
`left at room temperature for 3h. Excess borohydride was de-
`
`composed with water (10 m1) and methanol
`
`(10 m1) and the solvent
`
`was removed under reduced pressure (bath 30°) after standing
`
`Supplied by the British Library 08 Feb 2017, 11:07 (GMT)
`
`srenuavranumersarisnimneeeyianett
`
`SRSRTESSEEEAESRPSERUREESBNEoPEASEareeatease
`
`
`
`successively with ether (50 ml) and acetone (50 ml) to give a
`
`white solidwhichwas washed thoroughlywith ether and dried.
`
`After scirring the solid for 4 h at room temperature with 10 ml
`
`|
`
`IMPROVED SYNTHESIS OF DIHYDRO-5-AZACYTIDLINE
`
`291
`
`for 1 h at room temperature. The resulting syrup was triturated
`
`6N hydrochloric acid,
`
`the solution was concentrated under vacuum
`
`to one third its initial volume and diluted with 50 ml of absolute
`
`ethanol. The precipitated inorganic materials were removed by
`
`filtration and the filtrate stored overnight at 0° from which
`
`0.48 g of 6 was obtained as white crystals, mp 180-181° dec.
`
`The mother liquor, when evaporated and the residue crystallized
`
`from methanol-ethanol (4:1), provided an additional 0.14 g of 6
`(total yield, 73%), mp 180-181° dec; [a] -29° (c 1.0, H,0);
`1it.° mp 180-181° dec, [0], -29° (c 1.0, H,0).
`Method B. Borohydride Reduction of Compound 5.
`A solution
`of 5 (557 ri 1.0 mmol)
`in 3 ml HMPA was stirred and treated
`with sodium borohydride (120 mg, 3.1 mmol). The reaction mixture
`
`was heated at 50° for 6 h then cooled to room temperature and
`
`combined with water (10 ml) and methanol
`
`(10 ml}. The reaction
`
`was worked-up as described in Method A to give 202 mg (72%) of
`6, mp 180-181° dec; [a], -29.0° (c 1.0, H,0). The NMR spectrum
`was identical to that of an authentic” sample and the mmp was
`
`undepressed.
`
`Method C. Hydrogenation of Compound
`
`4.
`
`A solution of 4
`
`(1.0 g, 2.7 mmol)
`
`in 160 ml absolute ethanol containing 10 ml
`
`of ethanolic hydrogen chloride (saturated at 0°) was slurried
`
`Supplied by the British Library 08 Feb 2017; 11:07 (GMT)
`
`with 1.0 g of 10% palladium on charcoal catalyst and hydrogenated
`
`
`
`292
`
`BEISLER ET AL.
`
`at 50 psi for 16h. After removing the catalyst the reaction
`
`solution was evaporated under vacuum.
`
`The resulting syrup,
`
`when triturated with ether, gave a solid which was recrystallized
`from ethanol to give 0.7 g (928) of 6, mp 180-181° dec;
`[a]°®,
`-29° (c 1.0, H,0). The product was identical with an authentic
`sample? in mp, mp, NMR and UV.
`
`Method D. Hydrogenation of Compound 7 (aza-C}.
`
`A solution
`
`of 7 (S.0 g, 20.4 mmol)
`
`in a mixture of 6N hydrochloric acid
`
`(S50 m1) and ethanol
`
`(50 ml) was combined with 5.0 g of 10%
`
`palladium on charcoal and hydrogenated at an initial pressure
`
`of 50 psi for 8h.
`
`The catalyst was removed by filtration
`
`through a Celite pad which was subsequently washed with ethanol.
`
`The filtrate and washings were evaporated under vacuum (30°) and
`
`the residue was crystallized from methanol-ethanol (1:1) to
`give 4.43 g (77%) of 6, mp and mp 180-181° dec;
`[aJ”°, -28°
`{c 1.0, H,0). NMR and UV spectra were identical to an authentic
`sample” of 6.
`
`Hydrolysis of 5.
`
`4-Amino-1-8-D-ribofuranosy1-1,3,5-
`
`triazin-2(1H)-one (7). At room temperature 3 ml of methanolic
`
`ammonia (saturated at 0°) was added to 557 mg (1.0 mmol) of 5
`
`giving a complete solution from which crystals began to deposit
`
`after a few minutes. After maintaining the temperature at 21°
`
`for 90 min in a stoppered flask the solution was stored at -16°
`
`overnight. The crystals of 7 (132 mg, mp 230-232° dec) were
`
`collected by filtration and washed with methanol. Concentration
`
`Supplied by the British Library 08 Feb 2047, 11:07 (GMT)
`
`
`
`IMPROVED SYNTHESIS OF DIHYDRO-5-AZACYTIDINE
`
`293
`
`of the mother liquor gave a further 18 mg bringing the total
`yield to 62%.
`The mixture melting point of 7 with an authentic!”
`sample of 5-azacytidine showed no depression and the NMR spectra
`
`were superimposable.
`
`6-Amino-3-8-D-ribofuranosyl-1,3,5-triazine-2,4(1H, 3H)
`
`-
`
`
`dione (9).
`
`A mixture of 30% hydrogen peroxide (80 ml) and
`
`glacial acetic acid (80 ml) was stirred with 7 (4.88 g, 20 mmol)
`for 8 days at 25°. The reaction mixture was concentrated to
`
`about one third under vacuum (bath 30-35°), diluted with water
`
`($0 m1), andthe resulting precipitate removed by filtration.
`
`The precipitate was washed with water and methanol and dried to
`
`give 2.60 g (50%) of 9 which was recrystallized from water,
`mp > 360°; UV ne (H,0) 221 nm (€ 16300); NMR (D,0 exchanged,
`Me,SO-d_) 6 5.94 (d, J = 4Hz, 1, C,H), 4.57-3.30 (m, 5,
`ribosyl protons); MS (tetrakis - trifluoroacetyl derivative) m/e
`
`(rel intensity) 625 (M-F, 0.3), 575 (M-CF,, 1.1), 530 (M-CF,CO,H,
`2.7), 461 (M-TFAGH-CF,,43), 208 (8.9), 155 (8.9), 112 (8.7),
`69 (100); MS (pentakis -
`trimethylsilyl derivative) m/e (rel
`intensity) 605 (-ch., 1.2), 403 (4.1), 348 (10), 345 (4.5),
`344 (4.4), 273 (5.4), 272 (3.1), 245 (29), 217 (21), 73 (100).
`
`Anal. Calcd for CoH,oN06 (260.2): C, 36.92; H, 4.64;
`N, 21.53.
`Found: C, 36.66: H, 4.38; N, 21.73.
`
`6-Amino-1,3,5-triazine-2,4(1H,3H)-dione (10). A solution
`
`of 9 (600 mg, 2.3 mmol) in 6N hydrochloric acid (15 ml) was
`heated on a steam bath for 3h, cooled, and the dark precipitate
`
`removed by filtration and washed thoroughly with hot water.
`
`The
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`
`
`294
`
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`BEISLER ET AL.
`
`filtrate and washings were combined, decolorized with charcoal,
`
`and concentrated under vacuum (bath 35-40°) to yield 60 mp
`
`(20%) of ammelide (10) as a white powder. The product in 1N
`
`_ ammonium hydroxide (10 ml) solution was treated with 1N hydro-
`
`chloric acid to pH 7 which gave analytically pure 10, mp > 360°;
`
`IRI 1730) m/e (rel
`GC-MS (tris - trimethylsilyl derivative,
`intensity) 344 (M", 53), 329 (M-Ci,, 23), 171 (38), 100 (17),
`73 (100).
`
`Anal. Calcd for C,H,N,O, (128.1): C, 28.13; H, 3.15;
`N, 43.72.
`Found: C, 28.44; H, 3.18; N, 43.63.
`
`The Peroxide Oxidation of 6. Method A. Without Catalyst.
`
`A solution of 283 mg (1.0 mmol) of 6 in a mixture of 30% hydrogen
`peroxide {4 ml) and glacial acetic acid (4 ml) was stirred at
`
`room temperature. At daily intervals samples were removed,
`
`spotted without work-up on TLC plates, and analyzed using four
`
`different solvent systems. After 3 days the reaction solution
`
`contained 7 as well as starting material, and after 12 days the
`
`2-oxo nucleoside (9} was evident as a third spot. Spot identi-
`
`fications were made by comparison with authentic samples mm on
`
`the same plate. After a total of 15 days the white precipitate
`
`(10 mg) which had accumulated was removed by filtration, washed
`
`with water, and dried, mp > 300°. Mass spectroscopic analysis
`
`(direct probe) of the product following trifluoroacetyl deriva-
`tization showed the product to be a mixture of 6+ TFA,
`(M’,
`m/e 630), 7° TFA, (trace, M-19, m/e 609), 9: TFA,
`(M-19, m/e
`625) and 10° TFA (M, m/e 224).
`
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`IMPROVED SYNTHESIS OF DIHYDRO-5-AZACYTIDINE
`
`295
`
`Method B. Ferrous Catalyst.
`
`To a stirred solution of 400 mg
`
`(1.4 mmol) of 6 in 2 ml water containing a few crystals of ferrous
`
`sulphate was added 2 ml of 30% hydrogen peroxide dropwise at 10°.
`
`After the addition the reaction solution was maintained at 10°
`
`for 10 min then allowed to come to room temperature for 2 h.
`
`The white precipitate (81 mg, mp > 300°) which had separated was
`
`removed by filtration and washed with water. MS analysis (direct
`
`probe) of the precipitate following treatment with trifluoro-
`
`acetic anhydride indicated 10° TFA contaminated with some 9+ TFA, .
`TLC analysis of the filtrate from the reaction solution showed
`
`the absence of 6and 7.
`
` 1-methylethylidine) -8-D-ribofuranosyl1]-
`4-Amino-1-
`16
`
`1,3,5-triazin-2(1H)-one (8). The procedure of Zderic et al.
`
`was used to convert 244 mg (1.0 mmol) of 7 (obtained via the
`
`dehydrogenation reaction described below) to 240 mg (84%) of the
`
`isopropylidene derivative (8), mp 279-280°. Recrystallization
`
`from dimethylformamide gave 180 mg, mp 279-280°; IR (Nujol) 3410,
`3285, 3090, 1688, 1609, 1110, 854, 804 cm”; NMR (Me,SO-d,) & 8.39
`(s, 1, C,H), 7.56 (broad s, 2, NH), $.72 (d, Je2Hz, 1, C,H),
`1.37 (d, J=20Hz, 6, Me,C) ; GC-MS (bis-trimethylsily]l derivative,
`IRI 2580) m/e (rei intensity) 428 (M', 1.4), 413 (M-CH,, 6.5),
`370 (M-Me,CO), 1.5), 256 (3.6), 185 (22), 103 (24), 100 (91),
`73 (100).
`
`Anal. Calcd for C,,H,,N,0.
`11°16 °4°S
`
`(284.3): C, 46.47; H, 5.67;
`
`N, 19.71.
`
`Found: C, 46.66; H, 5.68; N, 19.76.
`
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`BEISLER ET AL.
`
`An authentic sample!” of 7 treated similarly gave an iso-
`
`propylidene derivative with identical spectral properties, mp
`
`and map as the compound described above.
`
`s-Azacytidine hydrochloride (7* HCl} was obtained from 8
`
`(100 mg) on treatment with 6N hydrochloric acid (2 ml) at 25°
`
`for 4h. Addition of absolute ethanol
`
`(5 ml)
`
`to the reaction
`
`solution caused the crystallization of 7° HCl (95 mg, mp. 179-
`
`181° dec) which was recrystallized from ethanol~benzene, mp 180-
`
`181°dec.
`
`Anal. Caled for CoH,N,0, » HCl (280.7): C, 34.23; H, 4.67;
`N, 19.96; Cl, 12.63.
`Found: C, 33.88; H, 4.89; N, 19.58;
`
`Gi, 12.359.
`
`A sample of 7* HCl prepared from authentic 7 gave the same
`
`UV, NMR, mp and mmp as the material described above.
`
`The Dehydrogenation of 6:
`
`4-Amino-1-8-D-ribofuranosyl-
`
`A mixture of BSTFA (17 m1) and dry
`1,3,5-triazin-2 (1H) -one (7).
`acetonitrile (30 ml) was refluxed gently with 566 mg (2 mmol) of
`
`6 under anhydrous conditions. As the reaction proceeded the
`
`single peak in the gas chromatogram due to 6° TMS. (IRI 2465)
`was accompanied by increasing concentrations of a second compound
`
`with a longer retention time.
`
`By GC-MS analysis it was shown
`
`the latter
`to be 7° TMS, (IRI 2620). After refluxing 18 h,
`campound was the only peak in the chromatogram indicating a
`
`complete conversion had occurred. The solvent was removed under
`
`vacuum and the syrupy residue was taken up in 100 ml of absolute
`
`[a
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`
`
`
`_svertinaynieningmrevtenentARHsuRSETERTECSREUSEAAOATUNIHSECTARIANUAERE
`
`
`
`
`
`
`
`IMPROVED SYNTHESIS OF DIHYDRO-5-AZACYTIDINE
`
`297
`
`methanol.
`
`Removal of the trimethylsilyl groups from the desired
`
`product was effected by slow co-distillation of volatile silicon-
`
`containing materials with methanol.
`
`The methanol volume was
`
`renewed twice over the course of the distillation (~~3 h) during
`
`which time the boiling point increased from 51° to 65° and
`
`crystals began to separate from the boiling solution. Crystalli-
`
`zation was allowed to continue at room temperature overnight to
`give 370 mg (76%) of 7, mp 232-234° dec (1it.1® mp 228-230° dec);
`(a}?6p + 24.4° (c 1.0, H,0) (1it.?* fay®D + 26.6° (c 1.0, H,0).
`Mixture melting point with an authentic!» sample of 7 showed no
`depression and the NMR and UV spectra were superimposable.
`
`The free base of 6 could also be dehydrogenated to give 7
`
`using the identical reaction conditions described above for the
`
`salt.
`
`SUMMARY
`
`Dihydro-5-azacytidine (6) is a hydrolysis-resistant,
`biologically-active analog of the clinical antitumor agent,
`S-azacytidine (7).
`In order to facilitate the acquisition of
`
`sufficient quantities of 6 for preclinical pharmacology studies,
`
`a shorter synthesis of 6 was devised giving a substantial
`improvement in overall yield. As part of a chemical investi-
`
`gation of the reactive 6-position of 7 (¢} an analog of 7
`
`bearing an oxygen atom at C-6 (9) was synthesized; (zt) the
`
`peroxide oxidation of 6 was studied and the results related to
`
`possible metabolic transformations of 6 and 7; (zit) via a per-
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`
`
`
`298
`
`BEISLER ET AL.
`
`trimethylsilylated derivative, 6 was converted to 7 utilizing
`
`a novel thermal elimination reaction.
`
`From an NMR analysis of
`
`an isopropylidene derivative (8)
`
`the 8-configuration for 7,
`
`and all the nucleoside derivatives described in this report,
`
`was confirmed.
`
`REFERENCES
`
`Address correspondence to this author at the National
`Institutes of Health, Building 37, Room 6D19, Bethesda,
`Maryland 20014,
`
`NIH Visiting Postdoctoral Fellow from the National Research
`Center, Dokki, Cairo, Egypt.
`
`J. Skoda in "Antineoplastic and Immmosuppressive Agents’,
`Part I], A. C. Sartorelli and D. G. Johns, Ed., Springer-
`Verlag, Berlin, 1975, p. 348.
`
`B. D. VonHoff, M. Slavik and F. M. Muggia, Ann. Intern. Med. ,
`85, 237 (1976).
`
`P. L. Lomen, L. H. Baker, G, L. Neil and M. K. Samson,
`Cancer Chemother. Rep., 59, 1123 (1975).
`
`A. H. Israili, W. R. Vogler, E. S. Mingioli, J. L. Pirkle,
`R. W. Smithwick and J. H. Goldstein, Cancer Res., 36, 1453
`(1976).
`
`J. A. Beisler, M. M. Abbasi, J. S. Driscoll and J. A. Kelley,
`Abstracts (MEDI 72), The 172nd National Meeting of the
`American Chemical Society, San Francisco, Calif., August 1976.
`
`J. A. Beisler, M. M. Abbasi and J. S. Driscoll, Cancer
`Treat. Rep., 60, 1671 (1976}.
`
`J. A. Beisler, M. M. Abbasi, J. A. Kelley and J. 5. Driscoll,
`J. Med. Chem., 20, 806 (1977).
`
`U. Niedballa and H. Vorbruggen, J. Org. Chem., 39, 3672
`(1974).
`
`This reaction was discussed by Niedballa and Vorbruggen
`(ref. 10) but experimental details and yield were not given.
`
`10,
`
`11.
`
`Supplied by the British Library 08 Feb 2017, 11:07 (GMT)
`
`STEEMECHHOHeEPSONFEFETEETE
`
`ssgLRRSPHABA
`
`
`
`IMPROVED SYNTHESLS OF DIHYDRO=-5-AZACYTIDINE
`
`299
`
`i
`
`12. Protocols! established by the Division of Cancer Treatment,
`National Cancer Institute were followed.
`The test compound
`(9) was administered by intraperitoneal (i.p.) injection
`to mice on days 1, 5 and 9 following i.p.
`implantation of 10
`L1210 leukemia cells. At dose levels of 200, 100, 50, 25,
`12.5, 6.25 and 3.12 mg/kg, no significant increase in survival
`time over untreated control animals was observed.
`
`5
`
`{
`‘
`
`13.
`
`14.
`
`1S.
`
`16.
`
`17.
`
`18.
`
`19.
`
`R. I. Geran, N. H. Greenberg, M. M. Macdonald, A. M.
`Schumacher and B. J. Abbott, Cancer Chemother. Rep., Part 3,
`3; 1 (1972).
`
`A. J. Repta in "'Pro-drugs as Novel Drug Delivery Systems",
`T. Higuchi and V. Stella, Ed. , American Chemical Society,
`Washington, D,
`€.,
`1975, pp. 196-223.
`
`S-Azacytidine was made available by Dr. H. B. Wood, National
`Cancer Institute, Bethesda, Md. whom we gratefully acknowledge.
`
`J. A. Zderic, J. G. Moffatt, D. Kan, K. Gerzon and W. E.
`Fitzgibbon, J. Med. Chem., 8, 275 (1965).
`
`J.-L.
`
`Imbach, Ann. N. Y. Acad. Sci., 255, 177 (1975).
`
`A. Piskala and F. Sorm, Coll. Czech. Chem. Commm., 29,
`2060 (1964).
`
`E. Kovats, Helv. Chim. Acta, 41, 1915 (1958).
`
`Received June 13, 1977
`
`20. W. A. Koenig, L. C. Smith, P. F. Crain and J. A. McCloskey,
`Biochemistry, 10, 3968 (1971).
`
`21. M. W. Winkley and R. K. Robins, J. Org. Chem., 35, 491 (1970).
`
`Supplied by the British Library 08 Feb 2017, 11:07 (GMT)
`
`
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