Leprbar exeat
`130 TITLE
`Work continued from Page___
`
`PROJECT NO.
`BOOK NO.
`
`ANNs2!
`
`
`
`_ eo :
`
`
`
`Starting materials, reagents, solvents
`
`a (1 R,25)-2-(((2,4-Dimethy!)pyrimidin-5-yl)oxy)methyl)-2-(3-fluorophenyl)cyclopropanecarboxylic acid,
`Eisai Lot 13XP0234 “HND110", “The acid”.
`~~~
`2-Amino-5-fluoropyridine, Ambeed Lot A303477-015 “The aminopyridine’, “The amine’.
`O-(7-Azabenzotriazol-1-yi)-N,N,N',N'-tetramethyluronium hexafluorophosphate,
`Ambeed Lot A633512-026, 98%, “HATU”
`1-Propeanephosphonic anhydride, 50% in Ethyl acetate, AK Scientific Lot LC62588 &b664799, “T3P”
`_N,N-Diisopropylethylamine: TCI Lot JD262N-UK, >99.0%, "DIPEA’, “iPr2NEt’
`Dimethylformamide, Sigma-Aldrich, Anhydrous, 99.8% “DMF”
`Ethyl acetate, Acros, 99.5%+ “EtOAc”
`Heptane, Sigma-Aldrich, ReagentPlus, 99%
`tert-Butyl methyl ether, TCi, >99.0% “TBME", “MTBE”
`Sodium hydrogen carbonate, Alfa Aesar, 99%, “Sodium bicarbonate, NaHCO3”
`~~“ Celite® 545 Filter agent Sigma-Aldrich
`Molecular Sieves 3A, 8 - 12 mesh, Acros, Lot A015993701
`“TLC Silica G, Sorbtech withUV254—
`
`—
`
`:
`ee
`:
`we!
`
`ee
`
`——
`
`:
`
`a
`
`a
`
`oe
`
`5
`
`——
`
`~~~
`
`~-~
`
`10
`“~~
`
`/
`
`
`
`The acid was received on 12/15/21 in a sealed brownbottle inside a plastic bag with packing peanuts, inside a
`anotherbag, inside a cardboard packing cylinder wrappedin plastic, inside a stytofoam insulating chest packed
`~
`with dry ice, inside a polypropylene shipping container. On 12/16/21, the packaging was opened on our exterior
`~-----~
`18leading dock. Onthe loading dock, the bottle was wiped with a paper towel and storedin the lab at -20°C.It
`was warmed briefly to RT and wiped again with a peper towel before using. The paper towels andall packing a
`materials were discarded outside of the lab.
`.
`2-Amino-5-fluoropyridine, HATU, and T3P were stored at 5"C. Other reagents were stored at ambient
`temperature.
`0
`
`----~
`
`EtOAc (99.5%+) was stored overactivated 3A molecular seives.
`DMFwasfreshly distilled at 1 atm, and stored over activated 3A molecularseives.
`
`+
`
`Pitt!Pealngjonore
`ee
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`kDalg
`WTCoratine~rpPE-/20°
`Themee+mee138-6ASLA
`PoVARSaye 1uPanltat!
`
`www.scientificbindery88yrs.com _
`FEArea
`1A
`
`You)
`
`
`
`[
`| Ve, 2022.
`
`_ DATE
`
`— WorkcontinuedtoPage >) a
`(A)
`:
`
`|
`
`Page 1 of 15
`
`EISAI EXHIBIT 1055
`Eisai v. Crystal Pharm.
`PGR2021-00047
`
`Page 1 of 15
`
`EISAI EXHIBIT 1055
`Eisai v. Crystal Pharm.
`PGR2021-00047
`
`

`

` TITLE
`
`PROJECT No.LyyndyKEP131
`Work continued from Page
`BOOK NO:
`<2 4/0, 33
`CPI APRS oe pesehY Onl Anes OK(Deesde)
`coeee HOF EgongaGi,Be rent
`ff
`
`'~
`
`
`
`
`
`
`
`
`
`*109 Patent‘Example
`
`G. Rogers Protocol
`
`
`Weigh 12.80 g ofthe acid and add to a 500 mL 3-neck round bottom flask’
`(RBF), equipped with a thermometer, a 1” Teflon magnetic stirbar, a glass
`
`
`stopper, and a rubber septum, Weigh 4.76 g ofthe aminopyridine and add to
`
`
`the RBF. Using a Schlenk line, evacuate the flask and refill with N23 times,
`Flush flask thoroughly with N2.
`
`
`
`Connect N2line with ail
`Add 102.4 mL anhydrous EtOAc using a 60 mL syringe with stainless steel
`
`
`
`bubbler.
`needle, Using a magneticstirring hotplate, stir the solution until all solids
`
`dissolve. Thestirring speed shouldbe set to where a small vortex is formed for
`
`
`
`mixing without splashing on the RBFwalls.
`
` Prepare an ice bath and place on the stirring hotplate. Place the RBFin the ice
`bath and coolto 0-5 °C with stirring (samerate as above). Oncethe
`
`
`temperature is between 0-5 °C,add via 20 and 1 mL syringes with stainless
`
`
`
`.|steel needles, with stirring (samestirring speed as above), 14.10 mL DIPEA
`.|
`(Grade: Purified by redistillation, 99.5%) while monitoring the temperature to -
`
`
`ensure it does not increase to more than 15 °C, nor go below 0 °C,
`
`Ensure the temperature is between 0-10 °C and stir (stirring speed as above)
`
`
`for 25 min while keeping the flask in the ice bath and maintaining the solution
`temperature between 0-10 °C.
`
`
`
`Add 36.1 g (33.77 mL) T3P (50% w/w solution in ethyl acetate) via 60 mL
`
`
`syringe with stainless stee] needle with stirring (stirring speed as above) while
`
`monitoring the temperature to ensure it does not increase to more than 15 °C,
`hor go below 0 °C,
`
`
`
`Removefrom theice bath, place in a warmed water bath on a stirring hotplate,
`
`andstir until the solution has warmed to room temperature between 20-25 °C.
`
`
`Removefrom the water bath, place on the stirring hotplate andstir (same rate
`.|as above) for 20h(this time can be extended ifneeded to accommodate Stir at least 20-24 h,
`
`| Working schedules) ensuring the temperature remains 20-25 °C. After 20h
`‘
`
`(this time can be extended ifneeded to accommodate working schedules)
`Follow reaction progress by
`
`
`checkwhetherreaction is complete by TLC. TLC will use reagent grade
`TLC and HPLC(calculate % of
`
`
`~~
`:
`EtOAc and n-heptanein a 1:1 volumeratio. When thete is no changein the
`remaining acid),
`reaction progress(i.e., reaction is complete) quantify with HPLCand proceed
`to the nextstep, Save TLC plates and tape to the notebook;take pictures, Save
`Photograph TLCplates; save
`Sramatm
`
`
`the HPLC data.
`|
`images
`
`
`
`
`
`
`
`
`
`
`aD bay REF aovat mSEREIBincrihewe.5n
`
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`
`www.scientificbindery88yrs.com
`“SIGNATURE oo
`| DISCLOSEDTOANDUNDERSTOODB /
`[6/2022
`
`: DATE
`
`"WITNESS
`
`}
`
`Page 2 of 15
`
`Page 2 of 15
`
`

`

`
`PROJECT NO.
`132 TITLE
`|
`|
`BOOK NO.
`Work continuedfrom PageLZ1 me
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`
`Page 3 of 15
`
`Page 3 of 15
`
`

`

`PROJECT NO.
`Lo
`- Work continuedfrom Page(D2 JE Brent.Pe CnRE “NO.
`
`Whenthe reaction is complete(i.e., it has stoppedreacting), place the flaskin ~
`an ice bath on a stirring hotplate and cool to 0-5.°C with stirring (samerate as
`above). When the temperature reaches 0-5 °C, add 64.0 mL ofdistilled water
`
`
`
`via 60 mL syringe with stainless steel needle with stirring (samerate as
`
`
`
`above), while ensuring the solution temperature remains below 10 °C,
`
` Add water slowly,
`
`Add the solution to a 500 mL separatory funnel,slightly shake/invert the
`
`
`funnel, and then allow the layers to sepatate. Drain the bottom aqueous layer
`
`
`into a 250 mL Erlenmeyer flask andtransfer it into a separate 250 mL
`
`
`separatory funnel. Save the organic (EtOAc) layer in the original separatory
`
`
`
`
`funnel. To the aqueous layer add 76.8 mL ofteagent grade MTBE,gently
`shake/invert, and allow the two phases to separate. Drain the bottom aqueous
`
`
`
`layer into a 250 mL Erlenmeyer flask and discard,
`
`
` Save aqueous layer for possible
`
`133
`
`
`
`
`
`
`
`future analysis.
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`.
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Combinethe organic (MTBE)layer from above with the original organic layer
`Use freshly prepared NaHCO3
`
`solution.
`(EtOAc)in a 500 mL separatory funnel. Add 38.4 mL ofsaturated aqueous —
`
`
`
`NaHCO3(prepared using distilled water) shake/invert, and allow the two
`
`
`phasesto separate. Drain the bottom aqueous layer and discard. Then add 38.4 .
` [A] Vigorously shake.
`
`
`‘BPStakesinvert--—
`mL ofdistilled water to the separatory funnel with the organic layer,
`
`
`shake/invert, and allow the two phases to separate, Drain the bottom aqueous
`
`
`
`layer and discard.
`
`Save aqueous layer for possible
` future analysis. °
`
`
`
`
`
`
`Fill a 150 mL fritted funnel (medium porosity 10-15 wm) with Celite 545 to 4
`the height ofthe funnel. Add EtOAcuntilall the Celite is covered and gently
`cecenen
` [A] Fill to 5 mm height.
`
`
`tap the funnelon the side to remove any air gaps, Before all the EtOAcdrains,
`{B}-Fil-to-4¢-the-height-ofthe..
`
`
`
`start adding slowly the combined organic layers (from above step) under
`
`
`
`vacuum; collect the filtrate into a 500 ml. filtration flask. Wash the Celite filter
`
`
`aid with 12.8 mL MTBEandcollectinto the samefiltration flask,
`Photograph.
`
`Save Celite for possible future
`
`analysis.
`
`Transfer thefiltrate into a 300 mL RBF and concentrate the filtrate using a
`Totovap until no more solvent is removed andit becomes viscous like an oil,
`
`
` Weigh product
`being careful not to reach dryness. Atthis point, the RBF can be evacuated and
`sealed ifnecessary
`before continuing to the next step.
`
`Transfer the reaction mixture to a 500 mL 3-neck RBF equipped with a
`
`thermometer, a reflux condenser, and a glass stopper. Add a 1” Teflon coated
`
`
`magnetic stir bar. Place the flask on a stirring/hotplate equipped with a mineral
`
`
`oil bath and add 60,8 mL of reagent grade EtOAc, Begin stirring (samestir
`
`
`tate as above) the mixture andstart heating (usingtheoil bath); heatuntil a
`
`
`clear solution is obtained while ensuring thatthe internal temperature does not
` Maintain temperature below
`tise above 50 °C.
`
`
`50°C.
`Whena clear solution has been obtained, removeflask from the oi] bath and
`
`
`place on a hotplate/stirrer. Continuestirring (samerate as above) and add 86.3
`
`
`mL of reagent grade n-heptane using a 125 mL addition funnelover ca. 2-3
`min.
`
`
`
`
`
`
`
`
`
`
`
`
`hg ena geAoCe-212VENorkcontinuedtoPage~7Y__
`
`
`|DISCLOSEDTO AND UNDERSTOODBY
`
`Then) Mawr
`
`
`
`Page 4 of 15
`
`
`[DATE —SSsWWITTINESS
`UGf2028
`
`| DATE
`
`Page 4 of 15
`
`

`

`itardhennaoeaveelaa
`134 TITLE
`2.|Workconcontinued from rage
`/2fai2.
`
`aiidniaisietilbieiiiatatJs
`plate Continuestirring Game
`Tate as aloo.andallowthereactionmixture to coolto ambienttemperature
`(20-25 °C), When the reaction mixtureis between 20-25 °C (room
`temperature), removefrom the ioe bath, place the flask on the stirring hotplate,
`andstir (same rate as above)for 1 h. Ensure the temperature 20-25 °C is
`maintained, After 1 h, place the reaction mixture in an ice bath on thestirring
`
`hadieckttateacnMassasbetikatinasStatue
`hotplate with stirring (samerate as above) and allow the reaction mixture to
`cool to 0-5 °C, When the internal temperature is between 0-5 °C,stir for 1 h,
`ensuring
`the temperature remains between 0-5 °C,
`Pour the reaction mixture into a Buchner funnel (186 mL, 70 mm diameter)
`and collect the solids on Whatman filter paper (grade 1) using vacuum, Wash .
`the filtered solids twice with 12.8 mL of$:1 reagent grade n-heptane:EtOAc
`by adding the solvent mixtureonthefiltered solid and allowing the liquid to
`drain,
`
`
`
`
`
`
`
`PROJECT NO.
`
`BOOK NO,
`
`ifno precipitate forms in 1 h,
`continuestirring at 0-5 °C.
`Photographflask.
`Filter after precipitation(if any)
`appears complete.
`4
`
`Evaporate solventfrom filtrate,
`Weigh residue. Obtain TLC,
`HPLC, NMR.
`
`Dry solid to constant weight. —
`
`
`
`
`
`
`CATA)a-/?4-]leeeaieAEEeneaaaaaAERelstSatonrenchicaneretin-aonctbnatingnipc
`
`-|
`
`Exposethesolid cake to high vacuum to remove excess solvent,
`Note the color.
`Determine mass product,
`Take PXRD
`Take NMR
`
`15
`
`20
`
`25
`
`
`
`
`
`_
`cng
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`
`1/6 /2oz2.
`
`
`
`Page 5 of 15
`
`Page 5 of 15
`
`

`

`
`
`
`
`fateattare nee
`
`
`
`
`
`
`
`
`
`
`135
`PROJECT NO.
`|
`ITLE
`
`WorcontinuedfomPage!37_ ExpirducatBE ROONN
`
`
`
`Cee ReposaG-) _ - a .
`
`
`ae
`. fF] 7109 Patent Example G. Rogers Protocol
`:
`BihovskyModifications
`
`
`
`
`
`-neck round bottom flask
`pene a
`Weigh 12.80 g ofthe acid and add to a 500 mL. 3
`.
`(RBF), equipped with a thermometer, a 1” Teflon magnetic stirbar, a glass
`
`
`
`
`Stopper, and a rubber septum. Weigh 4.76g ofthe aminopyridine and add to
`
`
`
`the RBF. Using a Schlenk line, evacuate the flask andrefill with N2 3 times,
`Flush flask thoroughly with N2.
`
`
`
`
`“| Add 102.4 mL anhydrous EtOAcusing a 60 mL syringe with stainlesssteel
`Connect N2 line with oil
`
`
`
`"| needle. Using a magneticstirring hotplate, stir the solution until all solids
`bubbler.
`.
`dissolve. Thestirring speed should be set to where a smal! vortex is formed for
`
`
`
`-mixing without splashing on the RBF walls.
`
`
`
`2|Prepare an ice bath and place on the stirring hotplate. Place the RBFin the ice
`mn sareat renee
`bath and coolto 0-5 °C with stirring (samerate as above). Once the
`sername sn
`
`a
`.
`temperature is between 0-5 °C, add via 20 and 1 mL syringes with stainless
`
`.|,.|Steel needles, with stirring (samestirring speed as above), 14.10 mI, DIPEA
`
`(Grade: Purified by redistillation, 99.5%) while monitoring the temperature to
`
`.|_2: | ensure itdoes not inorease to more than 15 °C, nor go below 0 °C. : Co
`
`
`
`vaeecenetne ‘| 3|Ensure the temperatureis between 0-10 °C andstir (stirring speed as above)
`
`a
`for 25 min while keepingthe flask in the ice bath and maintaining the solution
`temperature between 0-10 °C.
`
`
`my 44
`TAdd361 g (33.77 mL) T3P (50% w/w solution in ethyl acetate) via 60 mL
`.|Syringe with stainless steel needle with stirring (stirringspeed as above) while
`monitoring the temperature to ensureit does not increase to more than 15 °C,
`/
`
`nor go below 0 °C,
`Note any color change
`esewenntn
`5|Remove from the ice bath, place ina warmed water bath on a stirring hotplate,
`
`andstir until the solution has warmed to room temperature between 20-25 °C,
`:
`
`
`— .|Remove from the water bath, place on the stirring hotplate andstir (samerate oe
`
`
`as above) for 20h(this time can be extended ifneeded to accommodate Stir at least 20-24 h,
`
`'
`.| working schedules) ensuring the temperature remains 20-25 °C. After 20 h
`senseneeennscinens
`
`ores
`(this time can be extended ifneeded to aecommodate working schedules)
`Follow reaction progress by
`
`
`check whether reaction is complete by TLC. TLC will use reagent grade
`TLC and HPLC (calculate % of
`
`“EtOAcand n-heptane in a 1:1 volumeratio. When thereis no change in the
`remainingacid,
`reaction progress (i.e., reaction is complete) quantify with HPLC and proceed
`.
`to the nextstep, Save TLC plates and tape tothe notebook; take pictures. Save
`Photograph TLCplates; save
`
`
`na ~.|the HPLC data. images
`
`
`
`When thereaction is complete (Le., it has stopped reacting), place the flask in
`
`an ice bath on a stirring hotplate and coolto 0-5 °C with Stirring (samerate as
`
`above), When the temperature reaches 0-5 °C, add 64.0 mL, ofdistilled water
`
`
`via 60 mL syringe with stainless steel needle with stirring (samerate as
`
`
`
`
`-above), while ensuring the solution temperature remains below 10 °C,
`
`Add water slowly.
`
`Add the solution to a 500 mL separatory funnel, slightly shake/invert the —
`{A}-Aigoroush-shake:
`
`
`funnel, and then allow the layers to separate, Drain the bottom aqueous layer
`[B} Slightly shake/invert.
`
`
`
`into a 250 mL Erlenmeyerflask and transfer it into a separate 250 mL
`
`
`
`
`separatory funnel. Save the organic (EtOAc)layerin the original separatory
`
`
`funnel. To the aqueous layer add 76,8 mL ofTeagent grade MTBE,gently
`
`(A)Migoraush-shake.-
`
`
`shake/invert, and allow the two phasesto separate, Drain the bottom aqueous
`
`{B] Gently shake/invert,
`‘layer into a 250 mL Erlenmeyerflask and discard,
`
`
`
`
`
`
`
`
`
`Save aqueous layer for possible
`future analysis.
`AlFGCUDoe REE pes goer.+ Crd Be ET. BOAE oven 3h
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`www.sclentificbindery88yrs.com CZ): eo, t
`“a
`sé
`/
`
`*
`
`
`Page 6 of 15
`
`
`
`DATE
`| WITNESS
`. Véfwo2e
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`
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`
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`Page 6 of 15
`
`

`

`136 «TITLE
`PROJECT NO.
`Work continued from Page135 mf
`‘magPoe” ceECL .
`
`13¢zeke)
`
`
`
`Combinethe organic (MTBE)layer from above with the original organic layer
`
`
`Use freshly prepared NaHCO3
`solution.
`(EtOAc)in a 500 mL separatory funnel, Add 38.4 mL ofsaturated aqueous
`—
`
`
`[’,|NaHCO3(prepared usingdistilled water) shake/invert, and allow the two
`wwe on+,|phases to separate. Drain the bottom aqueous layer and discard. Then add 38.4
` mL ofdistilled water to the separatory funnel with the organic layer,
`[B] Shake/invert .
`
`_.
`shake/invert, and allow the two phases to separate, Drain the bottom aqueous
`Save aqueous layer for possible e anal
`— |
`layer and discard.
`
`
` sis.
`
` Fill a 150 ml fritted funnel (medium porosity 10-15 ym) with Celite 545 to 4
`
`5
`[ATFill-te-S-mum-height-
`the heightofthe fumnel, Add EtOAc until all the Celite is covered and gently
`
`
`ee tap the funnelon the side to removeany air gaps. Before all the EtOAc drains,|[B] Fill to % the heightof the
`start adding slowly the combined organic layers (from above step) under
`fannel.
`_
`:
`vacuum;collect thefiltrate into a 500 mL. filtration flask. Washthe Celite filter
`cnn aid with 12.8 mL MTBEandcollect into the samefiltration flask,
`Photograph.
`
`enveveceeeteneeee
`SaveCelite for possible future nee
`.
`Ls
`analysis.
`
`10|Transfer the filtrate into a $00 mL RBF and concentrate the filtrate using 2
`totovap until no more solvent is removed and it becomes viscous like an oil,
`
`
`being careftil not to reach dryness, Atthis point, the RBF can be evacuated and
`Weigh product
`“
`sealed ifnecessary
`before continuing to the nextstep.
`
`11|Transfer the reaction mixture to a $00 mL 3-neck RBF equipped with a
`
`
`
`
`thermometer, a reflux condenser,and a glass stopper, Add a 1” Teflon coated
`
`a
`magnetic stir bar. Place the flask on a stirring/hotplate equipped with a mineral
`-|oil bath and add 60.8 mL ofreagent grade EtOAc, Begin stirring (samestir :
`
`a Tate as above) the mixture and start heating (using the oi! bath); heat until a
`tere erie
`
`'.|Clear solution is obtained while ensuring that the internal temperature does not{Maintain temperature below .
`Tise above 50 °C,
`50°C,
`When a clear solution has been obtained, removeflask from the oil bath and
`oe
`place on ahotplate/stirrer. Continuestirring (samerate as above) and add 86.3
`erntTf
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`-{- [min,
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`seussts ATE Oe ere
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`| DISCLOSED TO ANDUI
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`PROJECT NO.
`‘TITLE
`Workcontinuedfrom Page/3¢ ro antec BFBQOKNO,
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`
`
`
`
`
`Place the reaction flask in an ice bath on the hotplate, Continue stirring (same
`rate as above) and allow the reaction mixture to cool to ambient temperature
`(20-25 °C), Whenthe reaction mixture is between 20-25 °C (room
`temperature), remove from theice bath, place the flask on the stirring hotplate,
`
`| and stir (samerate as above) for 1 h. Ensure the temperature 20-25 °C is
`
`
`maintained, After | h, place the reaction mixturein an ice bath on the stirring
`
`
`hotplate with stirring (samerate as above) and allow the reaction mixture to
`Ifno precipitate forms in | h,
`
`
`coolto 0-5 °C. When the internal temperature is between 0-5 °C,stir for 1 h,
`continuestirring at 0-5 °C,
`
`
`
`_| ensuring the temperature remains between 0-5 °C,
`Photographflask.
`Pour the reaction mixture into a Buchner funnel (186 mL, 70 mm diameter)
`Filter afterprecipitation (ifany)
`
`
`ee andcollect the solids on Whatman filter paper (grade 1) using vacuum, Wash
`appears complete, ee
`
`
`the filtered solids twice with 12.8 mL of5:1 Teagent grade n-heptane:EtOAc
`’
`‘
`
`
`by adding the solvent mixture onthefiltered solid and allowing the liquid to
`drain.
`
`
`Evaporate solvent from filtrate.
`Weigh residue. Obtain TLC,
`
`
`HPLC, NMR.
`
` Expose the solid cake to high vacuum to remove excess solvent,
`Dry solid to constant weight.
`
`
`Note thecolor.
`Determine mass product.
`
`Take PXRD
`:
`Take NMR
`
`
`
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`
`
`
`
`
`
`
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`www.scientificbindery88yrs.com
`SIGNATURE
`
`oa
`
`“DISCLOSED TO AND UNDERSTOODB
`
`
`
`
`SP MeLivulrinst,
`
`
`
`—— Workcontinued toPage Be
`‘DATE.
`.
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`ipa PWHNESS = :
`| DATE
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`Page 8 of 15
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`PROJECT NO.
`138 TITLE
`BOOK NO.
`.
`137°:
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`Work continued from Page_ =
`~ Ep etre
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`igOUT Pa BEcea,Raga|
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`
`
`
`
`
`#|Alternative Example G, Rogers Protocol Bihovsky Modifications
`
`
`“1 21|Weigh 12,80 § ofthe acid and add to a 500 mL 3-neck round bottom flask Distill DMF & store over
`(RBF), equipped with a thermometer, a 1” Teflon magnetic stirbar, a glass
`molecular sieves,
`stopper, and a rubber septum. Weigh 4.76 g of the aminopyridine and add to
`
`'.|the RBF, Weigh 16.16 g HATU andadd to the RBF. Using a Schlenk tine,
`omen | evacuate the flask and refill with N23 times. Add 64 mL anhydrous DMF
`Flush flask thoroughly with N2, eee
`
`- "|.|using a 60 mL syringe with stainless steel needle, Using a magnetic stirring ConnectN2 line with oil
`hotplate,stir the solution untilall solids dissolve, The stirring speed should
`bubbler.
`[TTT teens ceeceattention
`beset to where a small vortex is formed for mixing without splashing on the
`_| RBF walls,
`Prepare an ice bath and place on the stirring hotplate. Place the RBF in the
`ice bath and cool to 0-5 °C with stirring (samerate as above). Oncethe
`ob
`-
`ne | .:|temperature is between 0-5 °C, add via 20 and ] m1, syringes with stainless (mmm
`
`.
`steel needles, with stirring (samestirring speedas above), 14.10 mL DIPEA
`(Grade: Purified by redistillation, 99.5%) while monitoring the temperature
`SC ceeeeeeuie en
`sionertue em
`‘
`Noteany color change
`to ensure it remains below 10 °C.
`
`23|Removefrom theice bath, place in a warmed water bath on a stirring =
`hotplate, andstir until the solution has warmed to room temperature between
`20-25 °C, Removefrom thewaterbath, place onthe stirringhotplate andstir
`.
`:
`/
`
`
`
`
`oo +|(same rate as above) for 20 h (this time can be extendedifneeded to7 - ee
`accommodate warkingschedules) ensuring the temperatureremains 20-25
`15
`.°
`)
`77}
`:
`;
`
`- “| oC, After20h(this time can be extendedifneededto accommodate working| eee ean
`
`
`schedules) check whether reaction is complete by TLC, TLCwill use reagent
`
`_|gfade EtOAcand n-heptanein a 1:1 volumeratio. When there is no change Follow reaction progress by
`
`
`noes _|in the reaction progress (i.¢., reaction is complete) quantify with NMR and TLC, HPLC, and NMR 5
`La HPLC,Based on the amountofTemaining acid proceed to Alternate A,
`(calculate % ofremaining
`Alternate B,or the next step. Save TLC plates and tape to the notebook; take
`acid)..
`ert tenn ener
`pictures. Save the HPLC and NMR data, Iftheacid is Present at <2% and the
`conversionto thetitle compoundis 97% skip Alternate A and B and go to
`Photograph TLC plates; save
`
`ee a __|imagesthe nextstep,
`
`
`
`
`
`
`
`
`
`25
`
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`www.sclentificbindery88yrs.com
`SIGNATURE Ae.
`DISCLOSEDTO AND UNDERSTOODBY
`Iei
`i
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`Page 9 of 15
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`oPage_/77 1
`Work continuedt
`; DATE
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`Page 9 of 15
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`PROJECT NO.
`ITLE
`BOOKNO.
`ork continuedfrom Page pe
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`
`
`139
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`1/6/2022 a
`"5 -LOSED TO AND
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`IDERSTOODBY,
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`"DATE oo | WITNESS: eee ' DATE /
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`140 ‘TITLE
`Workcontinued from Page_/37
`
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`PROJECT NO.
`
`BOOK NO.
`
`
`
`242/21—
`
`
`
`
`
`
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`
`
`24] Alternate A:Ifthe acid is present at >2% (HPLC), add xxx HATU atroam
`temperature (20-25 °C) with stirring, (The HATU additionis calculated
`based on the amount ofunreacted acid andtheinitial stoichiometry of 1.05
`equiv ofHATU to 1 equiv ofacid, Once the number ofequiv. of unreacted
`acid is known,1.05 times that number of equivalents of HATU will be
`added.) Place the RBFin the ice bath and cool to 0-3 °C with stirring (same
`tate as above). Oncethe temperatureis below 10 °C, add via 20 mL syringe
`with stainless steel needle yyy DIPEA with stirring ensuring the reaction
`temperature remains below 10 °C, (The DIPEA addition is caleulated based
`on the amount ofunreacted acid andtheinitial stoichiometry of2 equiv of
`DIPEA to I equiv of acid. Once the number ofequiv, ofunreacted acid is
`known,2 times that number ofequivalents ofDIPEA will be added.)
`Removefiom the ice bath, place in a warmed water bath on a stirring
`| hotplate, andstir until the solution has warmed toroom temperature between
`20-25 °C. Remove from the water bath, place on the stirring hotplate and stir
`(samerate as above) ensuring the temperature remains 20-25 °C, Check
`periodically whetherreaction is complete by TLC. TLC will use reagent
`grade EtOAc and n-heptane in a 1:1 volumeratio. Whenthere is no change
`in the reaction progress(i.e., reaction is complete) quantify with NMR and
`
`HPLC.Proceed to the next step.
`
`
`
`
`
`
`
`
` Follow reaction progress by
`
`
`
`
`TLC, HPLC, and NMR
`(calculate % ofremaining
`acid)..
`.
`
`Photograph TLC plates; save
`images
`
`
`
`Follow reaction progress by
`TLC, HPLC, and NMR
`(calculate % ofremaining
`acid).
`
` q
`
`compound is <97%, add zzz aminopyridine andstir (same rate as above)
`ensuring the temperature remains 20-25 °C, (The aminopyridine addition is
`calculated based on the amountofunreacted acid and theinitial
`stoichiometry of1.05 equiv of aminopyridine to 1 equiv ofacid, Oncethe
`number ofequiv, ofunreacted acid is known, 1.05 times that numberof
`equivalents of aminopyridine will be added.) Check periodically whether
`reaction is complete by TLC. TLC will use reagent grade EtOAcand nheptane
`in a 1:1 volumeratio, Whenthere is no changein the reaction
`progress (i.¢., reaction is complete) quantify with NMR. and HPLC, Proceed
`to the nextstep.
`
`Photograph TLCplates; save
`images
` 26|Using a 60 and | mL. syringe with stainless steel needles, add 51.2 mL of
`
`anhydrous MTBE andplace the RBFin the ice bath on the stirring hotplate
`
`
`to cool to 0-5 °C with stirring (same speed as above). When the reaction
`
`temperature reaches 0-5 °C, add 64.0 mL ofdistilled water via 60 mL
`syringe with stainless steel needle with stirring, while ensuring the solution
`
`temperature remains below 10 °C,
`
`
`27|Removefrom the ice bath, place in a warmed water bath ona stirring
`hotplate, andstir until the reaction mixture has warmed to room temperature
`
`between 20-25 °C, Remove from the water bath and place on the stirring
`
`
`
`hotplate (samestirring rate as above). Add 76.8 mL of reagent grade MTBE.
`
`
` Ph.
`
`BOLEHeofBeIh
`
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`
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`
`
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`|
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`deouterten:an
`
`RSTOOD BY i
`| DISCLOSED TO AND UND:
`"DATE
`| WITNESS
`V6feoz:
`
`www.scientificbindery88yrs.com
`
`Page 11 of 15
`
`Page 11 of 15
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`

`

`
`
`TITLE
`
`
`Work continued from PageEe...140
`
`
`
`-, coe
`
`PROJECT NO.
`
`
`.
`
`141
`
`_apalel—_—.
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Add the solution to a 500 mL separatory funnel, slightly shake/invert the
`funnel, and then allow the layers to separate. Drain the bottom aqueous layer
`into a 250 mL Erlenmeyer flask and transfer it in a separate 500 mL
`separatory funnel. Save the organic (DMF/MTBE)layer in the original
`separatory funnel. To the aqueous layer add 128.0 mL ofreagent grade
`MTBE,gently shake/invert, and allow the two phases to separate, Drain the
`bottom aqueous layer into a 250 mL Erlenmeyerflask. Drain the top organic
`layer into a separate 250 mL Erlenmeyerflask, Add the aqueous layer back
`into the 500 mL separatory funnel and add 102.4 mL ofreagent grade
`
`
`toluene, gently shake/invert, and allow the two phases to separate. Drain the
`bottom aqueous layer into a 250 mL Erlenmeyerflask and discard.
`
`
`
`
`[D] Vigorously shake.
`[B}-Shighty-shakefnvert.
`
`Date
`
`[D] Vigorously shake.
`
`[D] Vigorously shake.
`[E}-Genthy-shakedavert.
`
`Save aqueous layer for possible
`future analysis.
`
`
`
`Use freshly prepared NaHCO3
`solution.
`
`[D] Vigorausly shake,
`fE}-Shighdy-shakeApvert-t
`
`
`
`orem me
`
`ee
`
`a
`
`
`
`
`
`_
`
`Photograph.
`
`
`
`Combinethe organic MTBElayer and organic toluene layer from above with”
`
`
`the saved DMF/MTBEorganic layer in the 500 mL separatory funnel. Add
`
`38.4 mL ofsaturated aqueous NaHCO3(prepared using distilled water)
`
`
`shake/invert, and allow the two phases to separate. Drain the bottom aqueous
`
`
`layer and discard, Then add 32,0 mL of 18% aq NaC!in distilled water to the
`
`
`separatory funnel shake/invert, and allow the two phasesto separate. Drain
`
`
`[D] Vigorously shake each’
`the bottom aqueous layer and discard. Add another 32,0 mL of 18% aq NaCl
`
`
`cette
`time.
`in distilled water to the separatory funnel shake/invert, and allow the two
`HE}-ShakeAnvert-each-time—
`phases to separate. Drain the bottom aqueous layer and discard.
`
`
`
`
`Check the HATU concentration in the washed organic layer by NMR. If the
`Check concentration of HATU
`HATU is <0.2%,proceedto the next step. If the HATU is >0.2%, add
`and HOAt (HATU
`
`
`another 32.0 mL of 18% NaClin distilled water to the separatory funnel
`decomposition product) by
`
`
`shake/invert, and allow the two phases to separate. Drain the bottom aqueous
`HPLC.
`
`
`layer and discard, Check the HATU concentration in the washed organic.
`
`
`layer by NMR and HPLC,Repeat the 18% aq NaCl washesuntil the HATU
`
`
`concentration is <0.2%.
`
`
`[D] Fill to 5 mm height.
`
`
`Fill a 150 mL fitted funnel (medium porosity 10-15 ym) with Celite 545 to “1en1 i
`¥% the height of the funnel, Add MTBEuntil all the Celité is covered and
`
`
`funnel.
`gently tap the funnel on the side to remove any air gaps, Beforeall the
`
`
`
`MTBEdrains,start adding slowly the combined organic layers (from above
`step) under vacuum;collect the filtrate into a 500 mLfiltration flask. Wash
`
`
`the Celite filter aid with 12.8 mL MTBEandcollect into the samefiltration
`flask.
`Save Celite for possible future ee
`
`analysis,
`
`
`
`Transfer the filtrate into a 1 L RBF and concentrate thefiltrate using a
`rotovap until no more solvent is removed and it becomesviscous like an oil,
`being careful not to reach dryness. At this point, the RBF can be evacuated
`Weigh product
`and sealed ifnecessary before continuing to the nextstep.
`
`Determine toluene content by qNMR and HPLCandif more than 10%
`
`Determine toluene content by
`HPLC or NMR.
`enone
`repeat step above. If S 10%, continue to next step. At this point, the RBF can
`
`
`be evacuated and sealedifnecessarybefore-continuingtothenext sten._
`
`. ce wo DpH
`MeATY.OroAipHe
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`| |
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`| DATE
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`{ WITNESS
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`{ DATE
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`Wb[222
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`Page 12 of 15
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`142 TITLE
`
`PROJECT NO.
`
`
`
`
`
`Work continued from Page+O sgl St 5 Bok SS
`
`SeDS,fm ee_|Addthesolutionto@500mLseparatoryfunnel,slightlyshake/invertthe i
`DD}Vigoreusly-shaker
`funnel, and then allow the layers to separate, Drain the bottom aqueous layer
`[E] Slightly shake/invert.
`into a 250 mL Erlenmeyerflask and transfer it in a separate 500 mL
`separatory funnel, Save the organic (DMF/MTBE)layer in the original
`separatory funnel. To the aqueous layer add 128.0 mL ofreagent grade
`MTBE,gently shake/invert, and allow the two phasesto separate. Drain the
`bottom aqueous layer into a 250 ml, Erlenmeyerflask. Drain the top organic
`layer into a separate 250 ml. Erlenmeyerflask, Add the aqueous layer back
`into the 500 mL separatory funnel and add 102.4 mL ofreagent grade
`5
`toluene, gently shake/invert, and allow the two phases to separate. Drain the
`fP}-Yeersuslyshake:
`
`ne bottom aqueous layer into a 250 mL Erlenmeyerflask and discard.
`[E] Gently shake/inver