NUCLEIC ACIDS COMPONENTS AND THEIR ANALOGUES,LI.*
`
`SYNTHESIS OF 1-GLYCOSYL DERIVATIVES OF 5-AZAURACIL
`AND 5-AZACYTOSINE
`
`Institute of Organic Chemistry and Biochemistry, Czechoslovak Academy of Sciences, Prague
`
`A. PisKALA and F. Sorm
`
`Received January 2nd, 1964
`
`For the preparation of 1-glycosyl derivatives of 5-azauracil and 5-aza-
`cytosine a method is described starting from peracetylglycosyl isocyanates.
`Addition of 2-methylisourea affords 1-peracetylglycosyl-4-methylisobiurets
`which are condensed with ethyl orthoformate to form 1-peracetylglycosyl-4-
`methoxy-2-oxo-1 ,2-dihydro-1,3,5-triazines. Deacetylation and demethylation
`of the latter compoundsaffords 1-glycosyl-5-azauracils whereas deacetylation
`followed by amination yields 1-glycosyl-S-azacytosines. By this procedure,
`glucopyranosyl, ribopyranosyl and ribofuranosyl derivatives of 5-azauracil
`as well as of 5-azacytosine have been prepared.
`
`In the course of biochemical studies about cultivation of Escherichia coli in the
`presence of a subbacteriostatic concentration of 5-azauracil (2,4-dioxo-1,2,3,4-tetra-
`hydro-1,3,5-triazine),
`ribosylbiuret
`forming by decomposition of the unstable
`ribosyl-5-azauracil has been found in the medium. A detailed study of 5-azauracil
`anabolites has shown that cleavage of ribosyl-5-azauracil to ribosylbiuret proceeds
`in the culture of E. coli via N-formylribosylbiuret’, i.e., analogously to the cleavage
`of 5-azauracil to biuret?. Moreover, it has been demonstrated with the use of a cell-
`free extract of E. coli that 5-azauracil is a suitable substrate for microbial phospho-
`rylases as well as pyrophosphorylases which makespossible the synthesis of ribosyl-5-
`azauracil and ribosyl-5-azauracil 5’-phosphate*, resp. We have expected that ribosyl
`derivatives of 5-azauracil or 5-azacytosine (4-amino-2-oxo-1,2-dihydro-l,3,5-tri-
`azine) could exhibit marked biological effects similar to those of the corresponding
`6-azaanalogues.In fact, preliminary experiments seem to support this assumption*~ ’.
`In this paper, we have studied the possibility of a chemical preparation of 1-glyco-
`syl derivatives of 5-azauracil and 5-azacytosine which would represent the hitherto
`undescribed analogues of the appropriate pyrimidine nucleosides. Our attention
`has been focussed on the glucopyranosyl, ribopyranosyl and ribofuranosyl deriva-
`tives. The ribosyl derivatives were proposed for correlation with the above products
`of enzymic reactions.
`
`* Part L.: This Journal 29, 1736 (1964).
`
`2060
`
`Collection Czechoslov. Chem. Commun.
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`Nucleic Acids Components and their Analogues. LI.
`
`In a recent paper®, we have described a general method for the preparation of
`1-substituted 5-azauracils as well as 5-azacytosines which consists in the condensation
`of isobiurets with orthoesters followed by treatment with hydrogen chloride or am-
`monia. Since this method represents, according to our experience, the most suitable
`procedure for the preparation of this type of compounds, we have tried to synthesize
`also the required glycosyl derivatives in this manner. We have started from tetra-
`acetylglucopyranosyl isocyanate which was prepared by Fischer? in a 22° yield on
`treatment of 1-bromo-2,3,4,6-tetra-O-acetyl-o-p-glucopyranose(I) with silver cyanate.
`Later on, Johnson and Bergmann'® described formation of a second, lower-melting
`modification. In several experiments, we isolated merely the higher-melting form.
`Furthermore, we were able to increase the yield (70%) by a suitable arrangement
`of the reaction conditions. Treatment with ammonia results in a product? to which the
`structure of 1-B-p-glucopyranosylurea was ascribed'!1”. Consequently, the product
`of the treatment with silver cyanate may be considered as 2,3,4,6-tetra-O-acetyl-B-
`D-glucopyranosyl isocyanate(II).
`Addition of 2-methylisourea afforded a high yield of a chromatographically homo-
`geneous amorphous product analyzing in agreement with the expected 1-(2,3,4,6-
`
`Table I
`
`Ultraviolet Spectra
`
`Compounds
`
`Solvent
`
`1,4-Dimethylisobiuret
`Til
`xX
`XVI
`1-Methyl-4-methoxy-2-oxo-1,2-
`dihydro-1,3,5-triazine
`Iv
`XT
`XVIT
`1-Methyl-5-azauracil
`Vv"
`XG?
`1-Methyl-5-azacytosine
`vi‘
`XI"
`XXII?
`
`96% ethanol
`96% ethanol
`96% ethanol
`96% ethanol
`
`acetonitril
`acetonitril
`acetonitril
`acetonitril
`96% ethanol
`96% ethanol
`96% ethanol
`water, pH 5-0
`water, pH 5-0
`water, pH 5-0
`water, pH 5-0
`
`
`
`
`
`Armax
`my
`
`i
`
`Zag
`221
`221
`221
`
`254
`254
`253
`253
`245
`240
`240
`247
`243
`243
`246
`
`loge
`
`4-18
`4-26
`4:28
`4:27
`
`3:34
`3:40
`3:39
`3°36
`3°20
`3°25
`3:24
`3°74
`3°81
`3-80
`3-78
`
`“ Spectra of the 5-azauracil and 5-azacytosine glycosyl derivatives were measured immediately
`after dissolution because of the time-dependenceoftheir extinction coefficient’?>.
`
`Vol. 29 (1964)
`
`2061
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`

`Piskala, Sorm:
`
`tetra-O-acetyl-B-p-glucopyranosyl)-4-methylisobiuret (JIJ). Its ultraviolet spectrum
`is practically identical with that of the analogous 1,4-dimethylisobiuret (see Table I).
`The above product was condensed at 135°C with ethyl orthoformate in the stream
`of dry nitrogen to yield crystalline 1-(2,3,4,6-tetra-O-acetyl-B-p-glucopyranosyl)-4-
`methoxy-2-oxo-1,2-dihydro-1,3,5-triazine (IV). Its structure was established on com-
`parison of the ultraviolet spectrum with that of the analogous 1-methyl-4-methoxy-2-
`oxo-1,2-dihydro-1,3,5-triazine (Table 1).
`
`OCH,
`
`sont
`ROCH,
`KEY oy
`
`
`
`Oo N=C=o
`‘
`OR
`
`OR
`
`AgNCO
`
`SY
`H,N N
`Ss
`No
`
`ROCH,
`|
`oy
`~~~ KOR
`
`
`
`Oo
`
`:
`
`OR
`
`NH
`
`~
`
`/
`
`il
`
`IT
`
`Oo
`
`A‘
`
`NHle
`
`hy
`
`HOCH,
`
`|___o
`|
`OH
`HO N
`
`4
`OH
`
`V
`
`NH,
`\
`N N
`)
`|
`la Nes
`|
`Kk
`HO pa
`
`HOCH,
`
`
`Oo
`
`OH
`
`OH
`
`OCH,
`\
`N WN
`lyno
`
`;
`
`ROCH,
`
`
`HC(OC2Hs)s
`
`OR
`RO \N—V
`|
`OR
`
`IV
`
`1. CH3ONa/CH30H
`(2. Dowex 50 W (H*)
`
`|
`
`__NHs/CH30H
`
`R =CH,CO
`
`VI
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`Nucleic Acids Components and their Analogues. LI.
`
`Transformation of the glucoside IV into the required 1-f-p-glucopyranosyl-5-
`azauracil
`(_V) was accomplished with methanolic hydrogen chloride or Dowex
`50 W(H*) ion exchange resin in methanol or sodium methoxide followed by Dowex
`50 W (H*) resin. When crystallised from aqueous ethanol, the substances formed
`a solvate with ethanol and water. Water may be removed by heating in vacuo at
`130°C for 1 hour whereas removal of ethanol requires 10 hours at the same tem-
`perature. Its structure was verified on a comparison of the ultraviolet spectrum with
`that of the analogous 1-methyl-5-azauracil’*, and, furthermore, by an acidic hydro-
`lysis yielding 5-azauracil and glucose. Moreover, the formation of a stable adduct
`with ethanol in the case of the glucoside Vclosely resembles the behaviour of 1-methyl-
`5-azauracil!*. The structure of both these adducts will be discussed in detail in a sub-
`sequent paper of this Series!’.
`A short action of a methanolic solution of ammonia at room temperature on the
`glucoside IV afforded crystalline 1-B-p-glucopyranosyl-5-azacytosine (VI) in a good
`yield. Its structure was confirmed on comparison of the ultraviolet spectrum with
`that of the analogous 1-methyl-5-azacytosine (see Table I) and by acidic hydrolysis
`yielding 5-azacytosine and glucose. The prolonged action of the methanolic solution
`of ammonia led to a second product, namely, 1-B-p-glucopyranosyl-3-guanylurea
`(VII). This compoundis obviously formed by cleavage of the triazine ting of the
`glucosyl derivative VI.
`
`6
`NON
`Sh
`CHO
`
`CH,
`XXI
`
`oO
`
`NH,
`|
`: =O
`
`NH
`
`—O
`
`|
`NH
`
`HOCH,
`
`os,
`oo
`—
`
`HO OH
`XIX
`
`NH,
`_an
`
`NH
`
`|C
`
`=O
`
`|
`NH
`
`HOCH,
`
`
`-—o,,
`Kon
`Ho N/V
`OH
`wa
`
`The above procedure was used also for the synthesis of 5-azauracil and 5-azacyto-
`sine ribopyranosyl derivatives. The starting 1-chloro-2,3,4-tri-O-acetyl-B-D-ribopyra-
`nose (VIII) wasreacted with silver cyanate undersimilar conditionsasin the preceding
`case. 2,3,4-Tri-O-acetyl-B-p-ribopyranosyl isocyanate (1X) was obtained as amorphous
`solid but its analysis as well as infrared spectrum, v(.N=C=O), 2252 cm~’, closely
`resembling that of peracetylglucosyl isocyanate IT, (WN=C=O), 2253 cm™~*, both
`speak in favour of the supposedstructure. Since in the reactions of halogenoses with
`silver cyanate the same steric course can be assumed as in the reactions with the
`
`Vol. 29 (1964)
`
`2063
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`

`Piskala, Sorm:
`
`silver or mercury salts of pyrimidine and purine bases, we haveused the rule of Baker!®
`for allotment of the configuration at the glycosidic centre of the molecule. Treatment
`of the isocyanate 1X with 2-methylisourea afforded a high yield of crystalline 1-(2,3,4-
`tri-O-acetyl-B-p-ribopyranosyl)-4-methylisobiuret
`(X)
`the structure of which was
`confirmed by the ultraviolet spectrum (see Table I). Condensation of this compound
`with ethyl orthoformate at 135°C resulted in crystalline 1-(2,3,4-tri-O-acetyl-B-p-
`ribopyranosyl)-4-methoxy-2-oxo-1,2-dihydro-1,3,5-triazine (XJ) the ultraviolet spec-
`trum of which corresponded to the proposed structure (Table 1).
`Successive treatment of the ribosyl derivative XJ with sodium methoxide and
`Dowex 50 W (H*) ion exchange resin yielded a product to which the structure of
`
`OCH,
`
`X
`H,N N
`
`|o
`
`N
`
`Oo
`
`ads
`
`—o Cc
`\|
`RO 7
`
`,
`
`OR OR
`
`Aenco
`
`0 N=C-0
`%‘
`RO +—/
`
`OR OR
`
`oe
`
`vu
`
`1x
`
`|—o 3"
`
`RO \—/
`
`OR OR
`
`x
`
`O |
`
`N NH
`lL
`N’\o
`
`1, CH3ONa/CH30H_,
`(2. Dowex 50 W (H*)
`
`/\
`
`e
`
`\
`
`HO ty
`\
`OH OH
`XI
`
`‘NH,
`A
`i
`N’No
`:
`%
`/\
`K
`nd NY
`
`——O
`
`OH OH
`XI
`
`NH3/CH30H =
`
`R = COCH,
`
`OCH,
`>
`¥ \
`{| )
`Bo
`
`S\
`
`io
`HC(OC2Hs)s K
`_
`-—
`
`XI
`
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`Nucleie Acids Components and their Analogues. LI.
`
`1-B-p-ribopyranosyl-S-azauracil (XII) was ascribed on the basis of the ultraviolet
`spectrum (see Table I). This compound wascleaved by the action of hydrochloric
`acid with the formation of ribose and 5-azauracil, and, furthermore, formed a sol-
`vate with ethanol.
`Treatment of the compound XJ with a methanolic solution of ammonia afforded
`the expected 1-B-p-ribopyranosyl-5-azacytosine (XIII) as crystals. The proposed
`structure was again confirmed bythe ultraviolet spectrum (Table I) and acidic hydro-
`lysis yielding ribose and 5-azacytosine.
`Finally, the above method was applied also for the preparation of 5-azauracil
`and 5-azacytosine ribofuranosyl derivatives. The reaction of 1-chloro-2,3,5-tri-O-
`acetyl-p-ribofuranose (XIV) with silver cyanate was performed under similar con-
`ditions as in the preceding examples. 2,3,5-Tri-O-acetyl-B-p-ribofuranosyl isocyanate
`(XV) was obtained as sirup and was characterised by the infrared spectrum,
`v(N=C=O), 2252 cm~'. The B-configurationat the glycosidic centre of the molecule
`was ascribed on the basis of Baker’s rule'®. The proof of validity of this assumption
`follows also from the structure of ribofuranosyl-5-azauracil (XX) (vide infra).
`Treatment of the isocyanate XVwith 2-methylisourea afforded crystalline 1-(2,3,5-tri-
`O-acetyl-B-p-ribofuranosyl)-4-methylisobiuret (XVI) the spectrum of which wassimi-
`lar to spectra of the preceding isobiurets (Table I). Condensation of this product
`with ethyl orthoformate at 135°C resulted in glassy 1-(2,3,5-tri-O-acetyl-B-p-ribofura-
`nosyl)-4-methoxy-2-oxo-1,2-dihydro-1,3,5-triazine (XVII) possessing the expected
`ultraviolet spectrum (Table 1).
`Deacetylation and demethylation of the ribofuranosyl derivative XVII was ex-
`pected, in analogy to the treatment of the compounds IV and XI, to yield 1-B-p-
`ribofuranosyl-5-azauracil (5-azauridine; XVIII). The reaction was again performed
`by the successive treatment with sodium methoxide and Dowex 50 W(H*) ion
`exchange resin. A crystalline product was obtained the elementary analysis of which
`corresponded to 5-azauridine. The substance, however, did not form any adduct
`with ethanol (in contrast to the analogous products V and XJJ) and, in a neutral
`medium, did not exhibit any maximum in the near ultraviolet region. At pH 5-0, the
`expected maximum(J,,,, 241 mp) was registered but the molar extinction coefficient
`was considerably lower(log ¢ 2-68) in comparison with other 1-substituted 5-azaura-
`cils. Moreover, the absorption disappeared rather quickly (no maximumcould be
`registered after one hour). The infrared spectrum (in dimethyl sulfoxide) showed
`a broad band at approx. 1535 cm™! (the triazine ring) and two carbonyl maxima
`at 1670 and 1715cm7?. No maximum at 1630 cm! (characteristic for the azo-
`methine linkage**) could be found (also in nujol suspension). Oxidation with potas-
`sium periodate at pH 7:0 and room temperature consumed 1:5 mole of periodic
`acid per 1 mole of the ribosyl derivative in the course of 24 hours as determined
`polarographically!7” (the theory requires 1 mole of periodic acid). The higher value
`is obviously due to a subsequent oxidation. During the paper electrophoresis in
`a borate buffer (pH 7-0) the substance migrated to the anodesimilar to uridine. The
`
`Vol. 29 (1904)
`
`2065
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`Piskala, Sorm:
`
`substance wasreadily cleaved with hydrochloric acid even at room temperature with
`the formation of ribose and 5-azauracil, and with dilute aqueous ammonia afforded
`1-B-p-ribofuranosylbiuret (XIX). Spectral data showed that in neutral
`solutions.
`and in the solid state the substance possessed no double bond in the position 5,6 of
`the azauracil moiety. This fact can be explained by the addition of a hydroxylic
`function to the azomethine linkage. Principally, any of the three hydroxylic functions
`in positions 2’, 3’ or 5’ can be involved intra- or intermolecularly. The possibility
`of a intermolecular addition can be precluded on the basis of the fact that both
`glucopyranosyl derivative V and ribopyranosyl derivative XII of 5-azauracil do not
`undergo such a reaction. Results of the oxidation with potassium periodate and the
`
`OCH,
`
`\
`
`ROCH,
`
`0
`
`Sshycl
`
`RO Ae
`
`AgNco
`
`ROCH,
`
`=o
`
`N=C=O
`
`0
`
`y
`
`OR OR
`
`ROCH,
`
`H,N i
`No
`
`HN
`
`c“
`
`on Co
`
`XIV
`
`XV
`
`XVI
`
`O |
`
`rh "
`H,oA
`
`_1.CHsONa/CH30H__
`2. Dowex 50 W (H*)
`
`nha
`|
`“x
`N WN
`
`|
`IL
`~ N“No
`»|
`OR to
`
`XVI
`
`HC(OC2Hs)3
`
`LCi
`XX
`IsN N
`l he
`
`HOCH,
`
`N No
`
`NH,
`
`NH3/CH30H
`
`OH OH
`
`XXII
`
`2066
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`Nucleic Acids Components and their Analogues. LI.
`
`electrophoretic behaviour are in agreement with a free cis-diol arrangement on the
`carbon atoms 2’ and 3’. The intramolecular addition to the azomethine linkageis,
`therefore, accomplished with the primary alcoholic function and the substance may
`be formulated as 5’,6-anhydro-6-hydroxy-5,6-dihydro-5-azauridine (XX). Theinfra-
`red spectrum of the substanceis similar to that of the related 1,3,5-trimethyl-6-meth-
`oxy-2,4-dioxohexahydro-1,3,5-triazine’® (XXI) measured in dimethyl
`sulfoxide
`(a broad band at 1520, 1680 and 1720 cm~*); this fact is a further support of the
`proposed structure.
`The spontaneous intramolecular addition of the primary hydroxylic function to the
`azomethine linkage represents simultaneously a proof of the B-configuration on the
`glycosidic centre of the molecule. Since glucosyl-5-azauracil V which also possesses.
`a free primary alcoholic function does not nudergothis reaction, it may be assumed
`that the addition will be probably limited to furanosyl derivatives with B-configura-
`tion and a free hydroxylic function in the position 5’.
`Both 5’,6-anhydro-6-hydroxy-5,6-dihydro-5-azauridine (XX) and ribosylbiuret
`XIX were identical with products forming in the medium in the course of cultivation
`of E. coli in the presence of 5-azauracil’. The identification was carried out by means.
`of paper chromatography.
`
`From the ultraviolet spectrum of the ribosyl derivative XX in a weakly acidic medium it could
`be assumed that the compoundis partly transformed into 5-azauridine (XVIII) and that some
`equilibrium exists depending on the pH of the solution. This problem will be discussed in full
`detail elsewhere! >.
`
`——eae
`
`O
`
`N
`/
`HN NH
`Hs)
`
`H,C——O”“N’\y
`a,
`=
`
`OH OH
`
`oO
`
`HOCH,
`
`|
`N NH
`a
`NNXo
`O%.
`IN{
`
`OH OH
`
`XX
`
`XVII
`
`Treatment of the ribofurancsyl derivative XVII with a methanolic solution of
`ammoniaaffordedreadily crystalline 1-B-p-ribofuranosyl-5-azacytosine (5-azacytidine
`XXII). Structure of this compound was confirmed on comparison of ultraviolet
`(see Table I) and infrared (Fig. 1) spectra with those of the analogous 1-methyl-5-
`azacytosine. By means of hydrochloric acid, the product was cleaved into ribose
`and 5-azacytosine.
`In summaryit may be noted that the S-azaanalogues of pyrimidine nucleosides differ
`in their properties very markedly from the pyrimidine as well as 6-azapyrimidine
`nucleosides, especially by the ease of cleavage of the N-glycosidic bond by the action
`
`Vol. 29
`
`(1964)
`
`2067
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`Piskala, Sorm:
`
`of mineral acids and by theinstability of the triazine moiety in alkaline media. As
`quite exceptional may be regarded also the behaviour of the 5-azauracil ribofuranosyl
`derivative where an intramolecular addition of the primary alcoholic function occurs
`to the azomethine linkage.
`
`1
`
`2
`
`0
`
`%(T)
`
`50
`
`100
`0
`
`50
`
`100
`
`1500
`
`cm:!
`
`1700
`
`Fig. 1
`Infrared Spectra of 1-Methyl-5-azacytosine (1) and 5-Azacytidine (2)
`(Measured in Nujol Suspension)
`
`Experimental
`
`Melting points were determined on aheated microscope stage and are corrected. Unless otherwise stated, the analytical
`samples were dried at 0-3 mm Hg and room temperature for ten hours, Paper chromatography was performed on paper
`Whatman No | by the descending technique (without previous saturations) in solvent systems (S;) 1-butanol—acetic
`acid—water (4: 1: 1) and (S2) 1-butanol-ethanol-water (40: 11: 19), Detection was carried out by viewing in ultraviolet
`light or according to Reindel and Hoppe!®. Thevicina }cis-diol arrangement ofthe ribosy! derivatives was detected with
`potassium periodate and benzidine?°.
`
`2,3,4,6-Tetra-O-acetyl-8-p-glucopyranosyl isocyanate (//)
`
`a«-Acetobromoglucose (4:11 g; 0-01 mole) was heated in dry xylene (20 ml) with thoroughly
`dried silver cyanate?! (4-5 g; 0-03 mole) under efficient stirring and with exclusion of atmospheric
`moisture. The bath temperature was gradually increased to 100°C in the course of 30 minutes
`and the mixture was kept at this temperature for further 30 minutes. Additional two portions of
`silver cyanate (1-5 g; 0-01 mole) were then addedin intervals of 30 minutes, the mixture was heated
`for 30 minutes, filtered off while still hot and the precipitate washed twice with 10-ml portions
`of hot xylene. The combinedfiltrates were cooled and precipitated with light petroleum (40 ml).
`
`2068
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`Nucleic Acids Components and their Analogues. LI.
`
`The supernatant was decanted (the residual precipitate was discarded), treated with additional
`20 ml of light petroleum anda little active charcoal and the mixture was quickly filtered through
`a French filter. The clear filtrate deposited almost instantaneously the crystalline isocyanate.
`The next day, the product wascollected, yield 2-3 g; m.p. 119—120°C (literature records'® m.p.
`120°C and 125°C (see”). Concentration of the mother liquors followed by crystallisation from the
`ethyl acetate—-light petroleum solvent mixture afforded additional 0-37 g of the product. Total
`yield 2-55 g (70%). Infrared spectrum (chloroform): »(N—C—O), 2253 cm.
`
`1-(2,3,4,6-Tetra-O-acetyl-8- p-glucopyranosyl)-4-methylisobiuret (IJ)
`
`A stirred soiution of 2,3,4,6-tetra-O-acetyl-6-p-glucopyranosyl isocyanate (3-73 g; 0-01 mole)
`in dry chloroform (50 ml) was slowly treated at —15°C under exclusion of atmospheric moisture
`with a solution of freshly prepared 2-methylisourea?? (0-7 g; 9-5 millimoles) in chloroform (10 ml).
`The resulting solution was allowed to stand at room temperature for one hour and the chloroform
`was evaporated under diminished pressure (bath temperature 30°C). The residual sirup was dis-
`solved in benzene (10 ml) and the solution treated with light petroleum (50 ml). The supernatant
`was decanted and discarded. The glassy residue was dissolved in hot benzene (10 ml), the solution
`allowed to cool and treated with light petroleum (50 ml). The resulting solid precipitate was
`repeatedly triturated underlight petroleum and decanted. Yield 4-01 g (94%, based on 2-methyl-
`isourea), m.p.90—100°C. Attempts to crystallise this product from various solvents failed. The
`Rr value 0°84 (the solvent system S,), detected according to Reindel and Hoppe?!®. The ultra-
`violet spectrum is given in Table I. For C,7H,5N30,, (447:3) calculated: 45-65% C, 5-64% H,
`9-39°% N; found: 45-48% C, 5-889 H, 906% N.
`
`1-(2,3,4,6-Tetra-O-acetyl-8-p-glucopyranosyl)-4-methoxy-2-oxo-1 ,2-dihydro-1,3,5-triazine
`(TV)
`
`A solution of the isobiuret derivative I/I (4-47 g; 0-01 mole) in ethyl orthoformate (40 ml)
`was heated (bath temperature, 135°C) in distillation apparatus in a stream of pure dry nitrogen
`under exclusion of atmospheric moisture (potassium hydroxide tube) for eight hours. After
`five hours of heating, the crystalline condensation product began to deposit and clog the nitrogen
`inlet tube. For the remaining period of heating nitrogen was introduced to the surface of the
`mixture. After standing at —15°C overnight, the crystals were collected and washed with a small
`amount of ether. Yield 2-28 g (50%); m. p. 235—237°C. The melting point did not change on
`recrystallisation of the product from absolute ethanol. The ultraviolet spectrum is shown in
`Table I. For C,gH,3N30,, (457-3) calculated: 47-27% C, 507% H, 9-19% N; found: 47-40% C,
`5-09 H, 9:12% N.
`
`1-8-p-Glucopyranosyl-5-azauracil (V)
`
`A suspension of the methoxytriazine JV (4°57 g; 0-01 mole) in absolute methanol (300 ml)
`was allowed to stand at room temperature in a stoppered vessel with 50 ml of 0-5N sodium
`methoxide in methanol for 30 minutes. The resulting solution was treated with moist Dowex
`50 W (H*) ion exchangeresin (100 g) previously washed with water and methanol. The mixture
`was kept at room temperature under occasionalstirring for two hours, filtered off and the resin
`washed thoroughly with methanol. The combinedfiltrates were evaporated under diminished pres-
`sure (bath temperature 30°C) and the glassy residue was dissolved without any heating in 1000 ml
`of the ethanol. The resulting solution was concentrated under diminished pressure (bath tem-
`perature 30°C) to a small volume (30 ml). A solid was deposited in the course of the evaporation.
`The mixture was allowed to stand for three days at room temperature and for one day at +3°C.
`The crystalline product was collected and washed with a small amount of ethanol. Yield 2-41 g
`
`Vol 29 (1964)
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`2069
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`Apotex v. Cellgene - IPR2023-00512
`Petitioner Apotex Exhibit 1024-0010
`
`Apotex v. Cellgene - IPR2023-00512
`Petitioner Apotex Exhibit 1024-0010
`
`

`

`Piskala, Sorm:
`
`(71%) of a chromatographically homogeneous product; the detection was performed by viewing
`in ultraviolet light and according to Reindel and Hoppe!® (treatment of the paper with chlorine
`was prolonged for 30 minutes); the Ry value 0-11 (the solvent system S,) and 0-08 (the solvent
`system S,). The substance melted at 175—180°C (softening from 165°C). The analysis showed
`that glucosyl-5-azauracil V was solvated with ethanol and water. For C,H,3;N3,07-C,H,O.H,O
`(339-3) calculated: 38-94% C, 6-24% H, 12-3994 N; found: 39-05% C, 620% H, 12-65% N. —
`When dried over P,O; for
`1 hour at 130°C/0-05mm Hg,
`the substance loses the water.
`For CyH,3N3;07.C,H,O (321-3) calculated: 41-12% C, 5-96% H, 13-08% N; found: 41-30% C,
`5:94% H, 13-28% N. Ethanol was removed by drying over phosphorus pentoxide at 130°C/0-:05mm
`Hg for ten hours. For CgH,3;N307 (275-2) calculated: 39-27% C, 4-76% H, 15-27% N; found:
`38-94% C, 501% H, 15-03% N. The substance is sparingly soluble in ethanol and readily soluble
`in water. The recrystallisation was performed by dissolving the substance in a minimum amount
`water without any heating, addition of a tenfold volume of ethanol, inocculation and standing at
`+3°C overnight. The substance separated in the form of colorless fine needles solvated with
`ethanol and water; m.p. 183—185°C. The ultraviolet spectrum is shown in Table I.
`
`The preparation of glucosyl-5-azauracil V from the methoxytriazine J V was accomplished also
`by the action of moist Dowex 50 W (H*) ion exchangeresin for 4 hours (without the previous
`removalof the protecting acetyl groups with sodium methoxide) or by treatment with an 1% solution
`of dry hydrogen chloride in absolute methanol for 4 hours (the hydrogen chloride was removed
`with silver carbonate). In both these procedures, the yield was lower than in the preparation
`described in the preceding paragraph. The action of methanolic hydrogen chloride was accompa-
`nied by the formation of 5-azauracil (Rp value 0-31) as shown by paper chromatography in the
`solvent system S,.
`
`Hydrolysis. Glucosyl-5-azauracil V (275 mg; 1 millimole) was heated in a steam bath with 1 ml
`of 6N-HCI for five minutes. The mixture was evaporated under diminished pressure, the residue
`was treated with ethanol (10 ml) and evaporated again. The residue was refluxed shortly with
`ethanol (2 ml) and the solution allowed to stand at +3°C overnight. The crystalline solvate of
`5-azauracil with ethanol was collected and washed with ethanol. Drying over phosphorus pento-
`xide at 130°C/0-05 mm Hgfor ten hours afforded 92 mg (81%) of S-azauracil, m.p. 284—285°C
`(decomposition; sealed capillary) undepressed on admixture’ with an authentic specimen. The
`identification was further performed by comparison of the ultraviolet spectra (4,,,, 237 mu,
`log e 3-5) and by paper chromatography. In the mother liquors, glucose was detected by means
`of paper chromatography. The cleavage with hydrochloric acid was found to proceed even at
`room temperature, though very slowly.
`
`1-8-p-Glucopyranosyl-5-azacytosine (VJ)
`
`The methoxytriazine JV (4:57 g; 0-01 mole) was kept at room temperature for 90 minutes
`in a sealed tube with 50 ml of absolute methanol previously saturated at 0°C with dry ammonia
`gas. The mixture was shaken occasionally. In the course of 40 minutes, a clear solution was
`obtained. The solution was concentrated under diminished pressure (bath temperature 25°C)
`to a small volume (the amorphous product partly separated). The residue was triturated with
`absolute ether (300 ml) and the resulting powder was repeatedly decanted with additional crops
`of absolute ether (three 100-ml portions). The amorphous product was then collected, washed with
`ether and triturated with 80 mlabs. methanol. Thesubstance gradually dissolved and separated
`again in the form of crystals. The mixture was allowed to stand at room temperature overnight
`and thenfiltered off. The product was washed with methanol and dried under diminished pressure.
`Yield 1-65 g (60°) of glucosyl-5-azacytosine VJ, m.p. 258—260°C (decomposition). The ultra-
`violet spectrum is shown in Table I. The substance was chromatographically homogeneous and
`was detected in ultraviolet light or according to Reindel and Hoppe’?; the Ry values 0-07 (the
`
`2070
`
`Collection Czechoslov. Chem. Commun.
`
`Apotex v. Cellgene - IPR2023-00512
`Petitioner Apotex Exhibit 1024-0011
`
`Apotex v. Cellgene - IPR2023-00512
`Petitioner Apotex Exhibit 1024-0011
`
`

`

`Nucleic Acids Components and their Analogues. LI.
`
`solvent system S,) and 0-05 (the solvent system S.). The recrystallisation was performed by dis-
`solving in a minimum amount of water (without heating), addition of a tenfold volume of metha-
`nol, inocculation and standing at +3°C overnight. The recrystallised substance melted at 260 to
`262°C (decomposition). For CgH,4N4O,¢ (274-2) calculated: 39-42% C, 5-15% H, 20-43% N;
`found: 39-33% C, 5-19% H, 20-15% N.
`1 millimole) was heated in a steam bath with
`Hydrolysis. Glucosy]-5-azacytosine VJ (274 mg;
`1 ml of 6N-HCI for five minutes. The mixture was allowed to cool, diluted with acetone (2 ml) and
`kept at room temperature overnight. The crystalline material was collected, washed with acetone
`a dried under diminished pressure to give 104 mg (70%) of 5-azacytosine hydrochloride. The
`identification was performed by comparison of the ultraviolet spectra (in 4n-HCI, 4,,,, 250 my,
`log ¢ 2:95) and by paper chromatography. The cleavage proceeds readily even at room temperature.
`Glucose was detected in the mother liquors.
`
`1-8-p-Glucopyranosyl-3-guanylurea (VIJ)
`
`The methoxytriazine JV (0-457 g; 1 mmole) was kept at room temperature with 5 ml of metha-
`nolic ammonia for 48 hours (cf. the preceding preparation). The solution was evaporated under
`diminished pressure (bath temperature 30°C), the residue was dissolved in methanol (50 ml)
`andthe resulting solution evaporated again. The residue was triturated with methanol (4 ml) and
`allowed to stand overnight. The obtained crystals were collected, washed with methanol and dried
`under diminished pressure to give 0:12 g (41%) of the solvate of glucosylguanylurea VII with
`methanol as indicated by analysis; m.p. 201—203°C (decomposition). For CgH,6N40,-CH,O
`(296-2) calculated: 36-49% C, 6-81% H, 18-92% N; found: 36-33% C, 6-60% H, 18-94% N. The
`recrystallisation was performed by dissolving the substance in a minimum amount of water and
`treating the solution with a tenfold volume of hot ethanol. The resulting crystals melted at
`207—209°C (decomposition) and represented the hydrate of glucosylguanylurea VJJ. For
`CgH,6N4O0,-H,0O (282-2) calculated: 34-03% C, 6-42% H, 19-85% N; found: 34-04% C, 6-13% H,
`20-00% N. The solvated water was removed on heating over phosphorus pentoxide at 100°C/0-05
`mm Hgfor 8 hours. For CgH,;,N40, (264-2) calculated: 36-36% C, 6:11% H, 21:20% N; found:
`36-48% C, 6:35%4 H, 20-90% N. The substance exhibited no absorption in the near ultraviolet
`region. The paper chromatograms were detected according to Reindel and Hoppe??. The R--
`values; 0-14 (the solvent system S,) and 0-11 (the solvent system S,). Some glucosyl-5-azacytosine
`VI was detected in the mother liquors by means of paper chromatography.
`
`2,3,4-Tri-O-acetyl--p-ribopyranosyl isocyanate (LY)
`
`Dry hydrogen chloride was introduced with cooling and under exclusion of atmospheric
`moisture into a suspension of finely ground 1,2,3,4-tetra-O-acetyl-8-p-ribopyranose (6:36 g; 0-02
`mole) in absolute ether (100 ml) and acetyl chloride (1 ml) for two hours. The stoppered flask was
`allowed to stand at +3°C for five days. The solution was evaporated under diminished pressure
`(bath temperature 20°C) to the consistence of a thick sirup. The residual hydrogen chloride and
`acetic acid were removed by repeated codistillations (i vacuo) with five 40-ml portions of dry
`toluene (bath temperature 30°C). The colorless sirup was diluted with absolute ether (10 ml) to
`deposit immediately the crystalline 1-chloro-2,3,4-tri-O-acetyl-@-p-ribopyranosyl chloride (VII).
`The mixture was allowed to stand at — 15°C overnight,filtered off, the product washed with a small
`amountof absolute ether and dried in a desiccator over potassium hydroxide. Yield 4-9 g (83%);
`m.p. 93—95°C (theliterature records”? m.p. 95°C).
`The above halogenose VIII (2:95 g; 0-01 mole) was heated with the thoroughly dried silver
`cyanate? (4-5 g; 0-03 mole) in 20 ml of dry xylene under similar conditions as in the case of glu-
`cosyl isocyanate JJ. When the reaction was finished, the silver salts were removed byfiltration
`and washed with two 10-ml portions of hot xylene. The combined filtrates were cooled and
`
`Vol. 29 (1964)
`
`2071
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`Apotex v. Cellgene - IPR2023-00512
`Petitioner Apotex Exhibit 1024-0012
`
`Apotex v. Cellgene - IPR2023-00512
`Petitioner Apotex Exhibit 1024-0012
`
`

`

`Piskala, Sorm:
`
`precipitated with dry light petroleum (80 ml). The mixture was allowed to stand in a stoppered
`vessel at +3°C overnight. The clear colorless supernatant was decanted (the residual glassy
`precipitate was discarded) and evaporated under diminished pressure (bath temperature