United States Patent Office
`
`3,350,388
`Patented Oct 31, 1967
`
`No Drawing Filed Dec. 14,1964, Ser. No. 418 273
`CIa,ms pnority application cWhosWakiaf
`DAc- .2 2'1 9 6 3' 7,093/63
`'
`t n
`10 Claims. (Cl. 260—211.5)
`f a c T u r e5^ ? ^1 1' !0,11 r e I a t eS t0 a p r 0 C e ss f or ** ™anu-
`tecture of l-gIycosyl-5-azacytosines of the general for-
`
`10
`
`V
`A
`
`E2-
`
`;=o
`
`R2-C(ORe)3
`
`v
`
`1 2-dihvdm i T /1 • • " " alkyJmercapto - 2 - oxo-
`1,2 dihydro-l,3,5-triaz,nes of the general formula
`SH«
`
`15
`
`wherein Ri represents the same as in the Formula T^
`
`•0
`
`J L
`\
`i.
`
`R3_NH—Ri
`
`V II
`
`wherem Ri represents a glycosyl or peracylglycosyl resi(cid:173)
`due R? representa a hydrogen atom or an alky group
`TA™
`a t 0 m S' R3 a nd R4 (d i f erent or i d e nS
`ItJelJ
`
`eventually substituted in L S ie
`
`ring *"**
`
`^
`
`^o^^Jj^ta^oLj^^
`
`tion of ammonia in methanol
`
`p r e t e raMy by the ac-
`
`30 with eompound, of t h e g S e r S p o Z T, m„
`
`,
`
`" ""
`
`quickly growing biological systems.
`The process for the manufachirp nf th~
`
`0f
`
`*
`
`STS?
`
`nriifaZscdisclosed
`
`in z^^
`
`reaCtVng Peracylglycosyl isocya^
`n " el with o T v ^1 8 68
`t h e r e by
`^ r0' ?6 3 8'
`f o r m i ng I-Peracyl-
`filvcosvT 4
`
`triazines. The latter are transform^ into 1 ™ n e i ^"
`glycosyl derivatives of 5 - azacytosine which o^ ako
`
`SSSiiSS?* •*"*the — ^ 4 °:
`
`Later on, our experiments have shown that 1 - slvcosvl
`
`ay way of a comprehensive statement, the nrocess of
`
`d o r n o r a S o t t e t o l1" ";? i n , e n' 'on ' " " "W »1><>™
`
`50 tJRgrJlil^tl^Itllt T^ ^
`
`55
`
`0f ?* ^ " t e ^ i ng
`h y t S ^ c i o rr
`the
`SPS • ^
`S' "e replacement of the residiiP
`^S
`t he compound
`50 of the general F o ^ V n^ ?r m i ng
`a
`simultaneou r e m o Xf tL nrL e, a C C O m p a n i ed ^
`If Present in thlt
`i
`^ Protecting groups aforesaid.
`be?emovedbv known t l ^- ** p r 0 t e c t i nS S^PS can
`c icmovea by known convenient methods.
`c l oS fnro
`t he i n V e nti on « Otfaer dis-
`Ch?fnn0Vided by
`- iIIUStrat-
`t
`^ n o t l i i S l I l I Z l e l l ^^ ^
`•^Pkl^lfM-tri-O^cety^D-ribofuranosyn.
`4-methyl-4-isothiobiuret
`
`
`
`60
`
`i s o ^ S T a ^ f . ^ ^ ^ - O - ^ ^ - D - r i b o f u r a n o s yl
`t-"1 ln a n h>'d r o us acetone (50 ml.) was
`r e a t ed Li
`dus on o? • C 0 0 l!ng .a nd mechanical stirring under ^x
`elusion of atmospheric moisture with a solutior
`Ifl
`S 0 1 u t I 0n of S-
`fl_ methylisothiourea (0 90 s) in nX^
`oa mi i Th
`,,..
`S'''
`ln anhydrous acetone nn
`ated nnder diminished press,™. S ' S S ^ S IK
`
`R i — N = C =0
`
`SE5
`
`HsN—C=NH
`ni
`wherein Rs represents an alkyl group possessinp 1-4 ^,
`bon atoms of an aralkyl group therebv SS
`^
`glycosylisothiobiurets of tfe g^eral f o r L u ™'
`^^
`
`SE«
`
`E i N H - C O - N = C - N H2
`
`wherein Ri represents peracylglycosyl and R5 represents
`
`Apotex v. Cellgene - IPR2023-00512
`Petitioner Apotex Exhibit 1023-0001
`
`

`

`to stand in an open vessel for the period of three to four
`hours to deposit colourless needles of the product (0.4
`?. The lower syrupy layer was dissolved in anhydrous
`acetone (10 ml.) and the solution treated dropw.se with
`ti ing S
`light petroleum till t-bid On inocculation,
`tta crvstalline product began to separate. The next day,
`the c r S al were collected and the mother liquor concen-
`tiated STSford another crop of the product Tota yield
`of
`l-(2 3 5-tri-O-acetyl-p-D-ribofuranosyl) - 4 - methyl-4-
`isotiiobimet 3.31 g. (85%, based on S-methylisothio-
`urea), M.P. 138-139° C. (ethanol).
`Example 2 —l-(2,3,5-tri-0-acetyl-§-D-ribofumnosyl)-
`ZetJlmercJto-2-oxo-l,2-dihydro-l,3,5-triazine
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`3,350,388
`amount of absolute ether The mother liquors were con(cid:173)
`centrated under diminished pressure to one fourth^ or
`thdr original volume and tta concentrate was treated
`under sfeing with anhydrous light petroleu*i (50 m ^
`The mixture was allowed to stand for a short time and
`He slje Jltalt was decanted. The sirupous residue was
`t s ZH
`absolute ether (10ml.), the solution w*.in(cid:173)
`occulated and allowed to stand w ^ ™ ™ ? ^.
`Total yield 3.5 g. (71%) ^ ^ ' ^ i X t t S-
`S - D - ribofuranesyl)-4-methylmercapto-2-oxo-l,2-dihy
`dro-l,3,5-triazine, M.P. 201-202° C.
`Example 6.—2'-deoxy-5-azacytidine
`A suspension of finely Pondered l-(3,5-di-0-p-toluyl-
`2-deoxy-3-D-ribof uranesyl) -4-methylmercapto-2-oxo-l,2
`dihydro-13,5 - triazine (4.95 g.) in absolute methanol
`tsOO ml.) previously saturated at 0° C. with dry ammonia
`S
`aUoied to stand under occasional stirring ma c to d
`vessel at room temperature for the Pe r i o d,0^2!l tSn
`A small amount of precipitate was removed by filtration
`and the fiUrate was evaporated under diminished pres(cid:173)
`sure Tlie residue was triturated with three 50 ml. por-
`S£ I? absolute ether and then crystallised from anhy(cid:173)
`drous methanol to give 1.32 g. (58%) of 2-deoxy-5
`azacytidine, M.P. 198-199° C. (decomposition).
`Example 7.—1 - (2,3,4,6-tetra-O-acetyl-p-D-glucopyrano-
`sy^-methyM-isothiobiuret
`A solution of 2,3,4,6-tetra-O-acetyl-p-D-glucopyrariosyl-
`isocyanate (3.73 g.) in absolute acetone (5 nd. > was
`treated with cooling and mechanic stirring undei^exclu
`sion of atmospheric moisture with a solution of S-methyl
`isothiourea (0.90 g.) in absolute acetone (30 ml.), lhe
`T s u Zg solution was allowed to stand at room empera(cid:173)
`ture for the period of 20 minutes and evaporated under
`diminished pressure. The residual thick sirup wa dis(cid:173)
`solved in anhydrous benzene (10 ml.) and the solution
`wt precipitated by tte addition of anhydrous light
`petroleum (50 ml.). The precipitate was thoroughly tn-
`furated with a glass rod till a white amorphous powder
`was obtained. The solid was collected .thorough^ washed
`with light petroleum and dried under diminished pressure
`To give 4.2 g. (91%) of 1 - (2,3-4-6-tetra-O-acetyl-^-D-
`glucopyranosyl)-4-methyl-4-isothiobiuret an amorphous
`solid, melting unsharply between 90-100 C,
`X^oH 244 mp (log e 4.30)
`
`40
`
`A solution of l.(2,3,5-tri-0-acetyl-(3-D-ribofuranosyl)-
`4-methyl-4^othiobiu;et (3.91 g.) in ethyl orthoformate
`(50 mU was heated at 135° C. in a distillation apparatu
`in a slow stream of dry nitrogen for the period of eight
`hours On cooling, tte solution was precipitated with
`anhydrous light petroleum (250 ml.). The resulting
`S p o us preliXal was dissolved in anhydrous benzene
`(10 m l) and the solution was precipitated with light
`petroleum (50 ml.). This purification was repeated once
`more and the sirup was dried under diminished pressure.
`S
`of
`the glassy
`l-(2,3,5-tri-0-acetyl-|3-D-ribofur-
`anosyl)-4-methylmercapto-2-oxo-l,2-dihydro-l,3,5-triazine
`3.05 g. (75%),
`A £ SCN 275 mM (log e 4.10)
`Example 3.—5-azacytidine
`l-(2 3 5-tri-0-acetyl-/3-D - ribofuranosyl) - 4 - methyl-
`mercapto-2-oxo-l,2-dihydro-l,3,5-triazine (401 g.) was
`dissolved in anhydrous methanol (30 ml.) previously
`saturated at 0° C. with dry ammonia and the resulting
`solution was allowed to stand in a sealed vessel at room
`temperature for the period of twelve hours The pre(cid:173)
`cipitate was collected, washed with methanol and dried
`under diminished pressure. Yield 1.65 g. (68%) of 5-
`azacytidine, M.P. 230-231° C. (decomposition).
`Example 4.—1-(3,5-di-0-p-toluyl-2-deoxy-p-D-
`ribof uranosyl) -4-isothiobiuret
`A solution of crude 3,5-di-0-p-toludyl-l,2-deoxy-/3-D-
`ribofuranosyl isocyanate (3.95 g.) in anhydrous ether
`(50 ml.) was treated with cooling and stirrmg under
`exclusion of atmospheric moisture with a solution of t>-
`methylisothiourea in anhydrous acetone (30 ml.), ine
`resulting solution was allowed to stand at room tem(cid:173)
`perature for the period of 20 minutes and evaporated
`under diminished pressure. The residual sirup was dis(cid:173)
`solved in absolute ether (10 ml.) and the solution was
`treated under stirring with small portions of light petro(cid:173)
`leum till turbid. On trituration with a glass rod the solu(cid:173)
`tion began to deposit crystals. The next day, the product
`was collected and washed with a small amount of ether
`The mother liquors were treated with additional light
`petroleum and the mixture containing the partly pre(cid:173)
`cipitated sirup was inocculated. After two days of stand(cid:173)
`ing at room temperature under occasional stirrmg, the
`sirup solidified and the supernatant deposited another
`crop of crystals. Total yield of l-p.S-di-O-p-to uyW-
`deoxy-^-D-ribofuranosyl)-4-methyl-4-isothiobiuret 3.90 g.
`(80%), M.P. 169-171° C. (ethanol).
`
`45
`
`50
`
`55
`
`- (2,3,4,6 - tetra - O-acetylI - P^f
`"fu(cid:173)
`Example 8.-1
`ranosyl) - 5 - methylmercapto-2-oxo-l,2-dihydro-1,3,5-
`triazine
`A solution of 1 - (2,3,4,6-tetra-O-acetyM-D-glucopy-
`ranosyl)-4-methyl-4-isothiobiuret (4.63 g.) in ethyl ortho(cid:173)
`formate (50 ml.) was heated in a distillation apparatus
`at 135° C. in a slow stream of dry nitrogen for the period
`of eight hours. After thirty minutes of heating, the solu(cid:173)
`tion began to deposit the product. TTie next day, the sohd
`was collected and washed with a small amount of absolute
`etherto give 3.21 g. (68%) of l-(2,3,4,6-tetra-0-acetyl-
`,9 - D-glucopyranosyl)-4-methylmercapto-2-oxo-l,2-dihy-
`dro-l,3,5-triazine, M.P. 269° C.
`Example 9.—l-p-D-glucopyranosyl-5-azacytosine
`
`60
`
`65
`
`A solution of 1 - (2,3,4,6-tetra-D-acetyl-i3-D-glucopy-
`ranosyl) - 4 - methylmercapto - 2-oxo-l,2-dihydro-l)3,5-
`triazine (4.73 g.) in absolute methanol (40 ml.) previ(cid:173)
`ously saturated at 0° C. with dried ammonia was allowed
`to stand at room temperature in a sealed vessel for the
`period of eight hours. The solution was then evaporated
`under diminished pressure and the residual thick sirup
`was triturated with three 50 ml. portions of absolute
`ether The white amorphous solid was dissolved in a
`minimum amount of absolute methanol and the resulting
`solution was allowed to stand at room temperature over(cid:173)
`night. The next day, the product was collected, wasted
`75 with methanol and dried under diminished pressure to
`
`70
`
`- di - O-p-toluyl-2-deoxy-p-D-ribo-
`Example 5.-l-(3,5
`juranosyl)-4-methyl-mercapto-2-oxo-l,2-dihydro-l,3,5-
`triazine
`A solution of 1 - (3,5-di-0-p-toluyl-2-deoxy-p-D-ribo-
`furanosyl) - 4 - methyl-4-isothiobiuret (4.85 g.) m ethyl
`orthoformate (40 ml.) was heated in a distillation ap(cid:173)
`paratus at 135° C. in a slow stream of dry nitrogen for
`the period of eight hours. After 60 minutes of heating
`the reaction mixture began to deposit crystals. The next
`day tta product was collected and washed with a small
`
`Apotex v. Cellgene - IPR2023-00512
`Petitioner Apotex Exhibit 1023-0002
`
`

`

`3,350,388
`
`st: is
`
`f6(;56r)co^ld^D"gIuc•o^ranosyl-5-azac^-
`
`W h a t w e c l a i m i s -C-( d e C O m P O S I t l 0 n )-
`thlgtnllauJJlj^1^
`
`^ ^ - ^ - a c y t o s i n es of
`
`Es
`
`E*
`
`V
`I
`/^
`N
`
`N
`
`E4J=O
`
`R"ere~srthPeesents the same as in the F°^ n
`S e ^ S T i? h e T o r m r m ^ r13 ^
`^^
`with a c o m p o u n d e rs r r m r^ ^
`
`R3—NH—R4
`
`^
`
`VI
`
`10
`
`lar ratio and in J
`
`inert s oS
`
`^ ^
`
`e^m o l e-
`
`wherein Ri represents glycosyl or peracylglycosyl R*
`
`the ^ertriotllaVTjZlI
`lower than the boiling ^
`
`t l ^I "** 0f
`UToZlsTlIId
`**
`
`20
`
`R i — N = C =0
`
`ffistssysasss*-*
`
`^ c o s ^ L y t
`
`t ^ g e r ^ L u r8
`S Ei
`
`•"'•»-»»«-
`
`II
`
`^^
`
`H J N - C = NH
`III
`r ^6 1 1 1. ?6 ^ Pr e s e nts an alkyl group having 1-4 carbon
`
`SR>
`
`" ^ "^ " C l a im ^ w h e r e in - id aralkyi
`25 is b e n tT
`6. A process as defined in claim 1, wherein the thn,
`
`rdicoehdoS.pound of the 8enerai Fo;muia '"-S-S
`
`30
`
`7 A process as defined in claim 6, wherein said alco
`holysis is carried out by the action of methanol it
`Z
`presence of sodium methylate.
`metnanol m the
`8. A process as defined in claim 1 wherein thP th,,.
`Produced compo
`to ammonolysis.
`Lit-im™. ffined " Claim 8' w h e r e i° ^id am
`
`E i N H - C O - N = ( ! 3 - . N H2
`Iv
`wherein Ri represents peracylglycosyl and R5 represents
`* SOTmma
`" I; condensing the latter
`with Zth
`t
`with orthoesters of aliphatic acids of the general formula 35
`R2-C(OR6)3
`v
`wherein R^ represents the same as in the Formula I and
`R6 represents a methyl or an ethyl group therebv form
`mg1[ - peracylglycosyl - 4 - alkylmercapto - 2 oxo -Tl
`'
`4ihydro-l;3,5-triazincs of the general formula
`
`40 CM10' I- P r 0i: e SS aS d e f i n ed in c l a im 2> therein said inert
`
`SEi A
`
`N
`
`N
`B2-
`
`>=o
`
`I
`
`No references cited,
`
`LEWIS GOTTS, P / Wy ^a m /„^
`
`J. R. BROWN, Assistant Examiner,
`
`45
`
`v?
`
`Apotex v. Cellgene - IPR2023-00512
`Petitioner Apotex Exhibit 1023-0003
`
`