·'
`
`NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS, 20(4-7), 621-628 (2001)
`
`PRACTICAL SYNTHESIS, SEPARATION,
`AND STEREOCHEMICAL ASSIGNMENT
`OF THEPMPA PRO-DRUG GS-7340
`
`H. Chapman: M. Kernan, E. Prisbe, J, Rohloff, M. Sparacino,
`T. Terhorst, and R. Yu
`
`Gilead Sciences, 333 Lakeside Drive, Foster City, California 94404
`
`ABSTRACT
`
`Tiie practical synthesis of a mixed phenoxy-amid ate derivative of PMPA with
`high oral bioavailability and favorable pharmacokinetics is described. The non(cid:173)
`stereoselective synthetic route produces a I: I mixture of two diastereomers at
`phosphorous. Simulated moving bed chromatography using Ch~ralp'* AS en(cid:173)
`abled kilo-scale isolation of the more potent dia:stereomer (GS-7340). Th·e GS- ·
`7340 phosphorous chiral center was found to be (S) by X-ray crystallography.
`
`.. The nucleotide analog, 9-[2-(R),-phosphonomethoxypropyl]adeni~e (PMPA,
`I) (1) has shown potent activity against human immunodeficiency virus in vitro
`(2). The lipophilic diester pro-drug, tenofovir' disoproxil fumarate (2), is cur(cid:173)
`. rently in advanced clinical evaluation as an oral AIDS therapy (3). Continuing
`research into novel ;f>MPA pro-moieties has recently led to the-identification of the
`mixed phenoxy-amidate derivative of PMPA (3) which was designated as GS-7171
`(Fig. 1 ). Due to the asymmetric center at phosphorous and .~o·9~stereoselective syn~
`thetic route, GS-7 1 71 was composed of a I: 1 mixture of two diastereomers (the
`(R)-PMPA side-chain ~nd L-amino acid ester were homochiral starting materials).
`The high oral bioavailab:ility' and favorab_Je ~~ssue-selective distribution of GS-7171
`made it a promising candidate for further development. To ascertain the properties
`of the individual diastereomers, .a method to separate them was needed .
`
`. ·corresponding author.
`
`Copy~ght 0 2001 by Marcel DeJ?cer, Inc.
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`Simulated Moving Bed
`Chromatography
`
`A= ~ .. JL,
`N.J:_N
`
`..J....
`
`N
`
`Scheme 1.
`
`McGuigan and coworkers have recently reported related phenoxy-amidate
`pro-drugs of the m~nophosphates of antiviral nucleoside analogs (4). Although
`these compounds are also P-chiral diastereomeric mixtures, the issues of diastere(cid:173)
`omer separation and differential activity have not been addressed. This communi(cid:173)
`cation discloses a solution to the dia:stereomer "problem" that may be ge~erally
`applicable.
`The additional pre-clinical testing required a kilogram-scale preparation of the
`pure diastereomers. Esterification of the soluble triethylamine salt ofPMPA (1) with
`phenol using dicyclohexyJcarbodiimide (DCC) in hot 1-methyl-2-pyrrolidinone
`(NMP) afforded a 51% yield of PMPA monophenyl ester ( 4). Activation of ( 4) with
`thionyl chloride in dichloromethane gave the phosphonochloriaate, which smoothly
`coupled with an excess of isopropyl L-al'anine (5) to give the GS-7171 mixture (3)
`in 47% yield (unoptim,jzed). Both steps were readily perfonned on multi-kilogram
`scale in standard pilot plant equipment,
`·
`-
`Initially, the component diastereomers of ami date prodrug analogs were sepa-
`·
`rated with repeated HPLC purifications on a preparative CIS column. The marginal
`resoiution of the diastereomers on this system (Fig. 2) necessitated multiple passes
`but culminated in isolation of -100 mg of each isomer enriched to >95:5 purity.
`In vitro HIV assay showed that the less retained isomer (GS-7340, 6) was more
`potent by a factor of -10 and was selected as the candidate for additional testing.
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`PMPA PRO-DRUG GS-7340
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`623
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`1, PMPA
`
`2, Tenofovir Disoproxil Fumarate
`
`3, Gs-7171
`
`Figure 1.
`
`Screening of commercially available chiral stationary phases revealed that
`Diacel's Chiralpalc AS with a mobile phase of 30% methanol in acetonitrile was
`a markedly more efficient chromatographic ~ystem (5). (Fig. 3). The remarkable
`resolution of the diastereomers (a = -9) a'nd the high solubility of GS-7171
`in the mobile phase (>300 giL) allow~d facile diastereomer separation. Using a
`
`PeakPro Report
`
`1 2 3
`Elution time
`
`"
`
`5
`
`6 . .7
`
`8 9 10
`
`12
`
`14.
`
`16
`
`18
`
`20
`
`22
`
`2o4
`min's
`
`Figure 2: HPLC separation of the qS-7171 component diaslereomers on CIB column packing.
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`624
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`~ 0 '"'
`" 0
`fi
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`tl
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`" 0
`
`Ul 7171
`
`CHAPMAN ET AL.
`
`PeakPro Rc.port
`
`m
`(')
`(')
`r-
`Ih
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`2
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`3
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`4
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`6
`
`7
`
`s·
`
`9
`
`10
`
`II
`
`12
`
`13
`
`14
`min's
`
`Elution time
`
`Figure 3. HPLC separation of the GS-7171 component diaslereomers on Chiracel AS column
`packing.
`
`laboratory-scale simulated moving bed chromatography (6) system composed of
`ten Chiralpak AS (7) columns in series allowed separation of> 1 kg/day of mixture
`(8). The desired isomer, GS-7340, (6), was recovered in nearly quantitative yield
`and >98% diastereomeric purity.
`After chromatographic purification, GS-7340 was readily crystallized as the
`free base or as the fumarate salt (7). Needles of GS-7340 free base were grown
`from water.
`Structure determination by X-ray crystallography (9) allowed definitive as(cid:173)
`signment of the phosphorous chiral center as .(S) (Fig. 4) in the more active isomer,
`"GS-7340.
`· In summary, a practical kilo-scale process for synthesis and purification of the
`phenoxy-amidate pro-drug GS-7340 has been developed. The first correlation of
`activity with phosphorous absolute c~nfiguration in a ph"enoxy-amidate pro-drug
`has been made. Research. into a diastereoselective synthetic process for GS-7340
`is ongoing.
`
`[(R)-2-(Phenylphosphonomethoxy)propyl)adenine 4
`
`·~ EXPERIMENTAL
`
`A glass-lined re~ctor was charged with anhydrous PMPA, (1) (14.6 kg,
`50.8 mol), phenol (9.6 kg, 102 mol),. and 1-methyl-2-pyrrolidinone (39 kg). The
`mixture was heated to 85°C and triethylamine (6.3 kg, 62.3 mol) added. A solution
`of 1,3-dicyclohexylcarbodiimide (17.1 kg, 82.9 If!Ol) in 1-methyl-2-pyrrolidinone
`(1.6 kg) was then a~ded over 6 hours at lOO~C. Heating was continued for 16 hours.
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`PMPA PRO-DRUG GS-7340
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`Figure 4. Crystal Suucture of GS"7340, (6).
`
`The reaction was cooled to 45°C, diluted with water (29 kg), and cooled to 25°C.
`Solids were removed by filtration and rinsed with water (15.3 kg). The combined
`filtrate and rinse was concentrated to ·a tan slurry under reduced pressure, water
`(24.6 kg) was added, and adjusted to pH 11 ~ith NaOH (25% in water). Suspended
`solids were removed by filtration through diatomaceous earth (2 kg) followed by a
`water-( 4.4 kg) rinse. The combined filtrate and rinse was extracted with ethyl acetate
`(28 kg). The aqueous solution was adjusted to pH 3.1 with HCI (37% in water) (4 kg),
`precipitating crude 4 which was isolated by filtration and washed with methanol
`(12.7 kg). The wet product was slurried in methanol (58 kg), isolated by filtration,
`washed with methanol (8.5 kg), and dried under reduced pressure to yield 4 as a
`white powder {9.33 kg, 51% yield). 1H NMR (300 MHz, 020, o): 1.2 (d, 3H), 3.45
`(q, 2H), 3.7 (q, 2H), 4 (m, 2H}I)4.2 (q, 2H), 4.35 (dd, 2H), 6.6 (d, 2~), 7 (t, lH), 7.15
`(t, 2H), 8.15 (s, lH), 8.2 (s, lH); 31 P NMR (72 MHz, 0 20, o):l5.0 (decoupled).
`
`Isopropyl L-alan.ine 5 · ·
`
`A glass-lined reactor was charged with L-alanine (7.1 kg, 80 mol) and iso(cid:173)
`propanol (35.6 kg). The slurry was heated to reflux and chlorotrimethylsilane
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`(14.6 kg, 134 mol) ad~ed over 69 minutes. Reflux was continued for 3 hours at
`which time the reaction was homogeneous. Volatiles were removed by distillation
`at atmospheric pressure until the pot temperature reached 125°C. The pot residue
`was cooled to 34°C and diethyl ether (50 kg) added with vigorous stirring and
`further cooling to 21°C. Vigorous agitation was continued until crystals formed.
`The.slurty was cooled to about 0°C. The solid isopropyl L-alanine hydroc;hloride
`was isolated by filtration, rinsed with diethyl ether (5 kg), and dried to 12.35 kg
`off-white deliquescent crystals. 1 H NMR (300 MHz, CDC~), o) :1.2 (m: 6H), 1 ;7 (d,
`3H), 4.2 (m, lH), 5.05 (m, lH), 8.6 (m, 3H). The free base isopropyl L-alanine was
`prepared by combining a solution of isopropyl Calanine hydrochloride, (9.12 kg,
`54 mol), in tetratiydrofuran (34 kg), with a solution of 1,4-qiazabicyclo[2.2.2)octane
`(5.48 kg, 49 mol) in tetrahydrofuran (15.5 kg) in a glass-lined reactor. Solid 1,4-
`diazabicyclo[2.2.2]octane hydrochloride precipitated and was removed by filtra(cid:173)
`tion. Tetrahydrofuran was removed from the isopropyl L-alanine in the filtrate by
`careful distillation under reduced pressure. The product isopropyl L-alanine (5) was
`isolated as an oil for use immediately in the next step without further processing or
`characterization.
`·
`
`GS-71713
`
`A glass-lined reactor was charged with [(R)-2-{phenylphosphonomethoxy)(cid:173)
`propyl]adenine ( 4), (9.12 kg, 25.1 mol) and acetonitrile (30. 7 kg). Thionyl chloride
`(6.57 kg, 56.7 mol) was added while maintaining the mixture below 50°C. The
`mixture was heated at 75°C until solids dissolved, then the reaction temperature
`was increased to 80°C and volatiles (11.4 kg) collected by atmospheric distillation
`under nitrogen. The pot residue was cooled to 25°C, diluted with dichloromelhane
`(41 kg), and cooled to· -29°C. A solution of isopropyl L-alanine (5) (ca. 7.1 kg,
`54 ~ol) in dichloromethane (36 kg) was added over 60 minutes at -l8°C followed
`by triethylamine (7.66 kg, 75.7 mol) over 30 minutes at -18 to -11 °C. The reac(cid:173)
`tion mixture was warmed to room temperature and washed with 10% aq. sodium
`· dihydrogenphosphate solution (5 x 15.7 kg). The organic solution was dried with
`anhydrous sodium sulfate (18.2 kg), filtered, rinsed with dichloromethane (28 kg),
`and concentrated· to an oil und_er reduced pressure. Acetone (20 kg) was charged
`to the oil and the mixture concentrated under reduced pressure. Acetone ( 18.8 kg)
`was again charged to the resulting oil. Half the product solution was purified by
`chromatography over a 38 x 38 em bed of 22 kg silica gel 60 (230 to 400) mesh.
`The column was eluted with 480 kg acetone. The purification was repeated on
`the second half of the oil using'fresh silica gel and acetone. Clean product bearing
`fractions were concentrated under reduced pressure to an oil. Acetonitrile (19.6 kg)
`was charged to the oil and the mixture concentrated under reduced pressure. Ace(cid:173)
`tonitrile (66.4 kg) was· again charged and the solution chilled to 0 to -5°C for
`16 hours. Solids were removed by filtration and the filtrate concentrated under re(cid:173)
`duced pressure affording 5.6 kg of 3 as a dark oil. 1 H NMR (300 MHz, CDCJ3, o):
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`1.1 (m, i2H), 3.7 (m, lH), 4.0 (m, SH), 4.2 (m, 1H), 5.0 {m, IH), 6.2 {s, 2H),
`7.05 (m, 5H), 8.0 (s, lH), 8.2? (d, lH); 31 P NMR (72 MHz, CDCJ3 , c5): 21.0, 22.5
`(decoupled).
`
`9-:[@)-2~[[@-[[@-1-(lsopropoxycarbonyl)ethyl)amino)­
`phenoxyphosphiny l]methoxy ]propyl)adenine 6
`
`GS-7171 (3), 2.8 kg, was purified by continuous simulated moving bed chro(cid:173)
`matography. Ten columns filled .with 10 em by 5 em beds of packing, 20 micron
`Chiralpak AS (1.2 kg) were used. The columns were eluted with 30% methanol
`in acetonitrile.· Product bearing fractions were concentrated to a solution of 6 in
`acetonitrile (2.48 kg). The solution solidified to a crystalliile mass wet with ace(cid:173)
`tonitrile on standing. The crystalline mass was dried under reduced pressure to a
`tan crystalline powder, 1.3 kg 6, 98.7% diastereomeric purity by HPLC: mp 117-
`1200C; 1HNMR(300MHz, CDC~), ~):_1.15 (m, 12H), 3.7 (t, lH), 4.0(m, 5H), 4.2
`(dd, 1H), 5.0 (m, IH), 6.05 (s, 2H), 7.1 (in, 5H), 8.0 (s, lH), 8.2 (s, lH); 31 P NMR
`(72 MHz, CDCJ3, ~): 21.0 (decoupled).
`·
`
`·9-[@)-2-[[@-[[@-1-(Isopropoxycarbony l)ethyl]amino ](cid:173)
`phenoxyphosphinyl)methoxy]propyl]adenine Fumarate (1:1) 7
`
`A glass-lined reactor was charged with 9-[(R)-2-[[ (ID-[[~)-1-(isopropoxycar­
`bonyl)ethyl]amino]phenoxyphosphinyl]methoxy]propyl] adenine (6), (1.29 kg,
`2.71 mol), fumaric acid (284 g, 2.44 mol), and acetonitrile (24.6 kg). The mix(cid:173)
`ture was heated to reflux. to dissolve the solids, filtered while hot, and cooled to 5°C
`for 16 hours. The product was isolated by filtration, rinsed with acetonitrile (9 .2 kg), .
`20-41.7° (c 1.0,
`and dried to 1.33 kg 7 as a white powder: mp 119.7-121.1°C; [a]0
`acetic acid).
`·
`
`REFERENCES
`
`1. Sc}Jultze, L. M.; Chapman, H. H.; Dubree, N.J.; Jones, R. J.; Kent, K. M.; Lee, T. T.;
`Louie, M.S.; Postich, M. J.; Prisbe, E. J.; Rohloff, J. C.; Yu, R. H. Tetrahedron Letters, .
`1998,39, 1853-1856.
`2. Balzarini, J.; Aquaro, S.; Perno, C. F.; Witvrouw, M.; Holy, A.; DeClercq, E. Biochem.
`Biophy. Res. Commun.,1996, 219,337.
`.
`.
`3. Schooley, R.; Myers, T. R.; Ruane, P.; Beall, ·a.; Lampiris, H.; McGowan, I. A Double(cid:173)
`Blind, Placebo-Controlled Study of.Tenofovir Disoproxil Fumarate (IDF) for the Treat(cid:173)
`ment of HlV Infection. Presented at the 39th lnterscience Conference on Antimicrobial
`Agents and Chemotherapy, San Francisco, CA, September, 1999.
`4. a) Siddiqui, A. Q.; McGuigan, C.; Ballatore, C.; Zuccotto, F.; Gilbert, I. H.; DeClercq,
`E.; Balzarini, J. 1. Med. Chern. 1999, 42, 4122-4128. b) Siddiqui, A. Q.; Ballatore,
`
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`C.; McGuigan, C.; De Clercq, E.; Balzarini, J. J. Med. Chern. 1999, 42, 393-399.
`c) McGuigan, C.; Tsang, H-W.; Sutton, P. W.; DeClercq, E.; Balzarini, J. Antiviral
`Chemistry and Chemotherapy, 1998, 9, 109-) 15. d) McGuigan, C.; Tsang, H-W.;
`Cahard, D.; Turner, K.; Velaquez, S.; Salgado·, A.; Bidois, L.; Naesens, L.; DeClercq,
`E.; Balzarini; J. A~tiviral Research, 1997, 35, 195-204. e) McGuigan, C.; Salgado,
`A.; Yamold, C.; Harries, T.Y.; De Clercq, E.; Balzarini, J. Antiviral Chemistry and
`·Chemotherapy, 1996, 7, 184-188. ·f) Balzarini, J.; Karlsson, A., Aquaro, S.; Perno,
`C.-F.; Cahard, D.; Naesens, L.; DeClercq, -E.; McGuigan, C. Proc. Natl. Acad. Sci.
`USA,1996,93, 7295-7299.
`.
`5. Chapman,. H.; Kernan, M.; Rohloff, J.;. Sparacino, M.; Terhorst, T. Purification of
`PMPA Arilidate Prodrugs by SMB Chromatography an~ X-Ray Crystallography of the
`Diastereomerically Pure GS-7340. Submitted for publication in a supplement to Nucle(cid:173)
`osides and Nucleotides on the XIV International Roundtable: Nucleosides, Nucleotides
`and their Biological Applications, San Francisco, CA, September, 2000.
`6. Francone, E. R.; Richert, P. Journal ofChromatographyA, 1997,769, 101-107.
`· 7. Chiral Technologies Inc., amylose-tris(alpha~methyl benzyl carbamate) coated on silica
`gel.
`8. Continuous simulated moving bed chromatography was performed by Aerojet Fine
`·
`Chemicals, Rancho Cordova, CA.
`9. Single-cryst~ x-ray structure determination was performed by F. J. Hollander at
`University of California, Berkeley.
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