NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS, 20(4~7), 621-628 (2001)
`
`PRACTICAL SYNTHESIS, SEPARATION,
`_. AND STEREOCHEMICAL ASSIGNMENT
`OF THE PMPA 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, Califomia 94404
`
`ABSTRACT
`
`The practical synthesis of a mixed phenoxy-amidate derivative of PMPAwith
`high oral bioavailability and favorable pharmacokinetics is described. The non-
`stereoselective synthetic route produces a J:1 mixture of two diastereomers at
`phosphorous. Simulated moving bed chromatographyusing Chiralpak AS en-
`abled kilo-scale isolation of the more potent diastereomer (GS-7340). The GS-
`7340 phosphorouschiral center was found to be (S) by X-ray crystallography,
`
`. The nucleotide analog, 9-(2-(R)-phosphonomethoxypropy]Jadenine (PMPA,
`1) (1) has shownpotent activity against human immunodeficiency virus in vitro
`(2). The lipophilic diester pro-drug, tenofovir disoproxil fumarate (2),
`is cur-
`‘rently in advanced clinical evaluation as an oral AIDS therapy (3). Continuing
`research into novel PMPA pro-moieties has recently led to the-identification of the
`mixed phenoxy-amidate derivative ofPMPA (3) which was designated as GS-7171
`(Fig. 1). Due to the asymmetric centeratphosphorous and non:stereoselective syn-
`thetic route, GS-7171 was composed of a 1:1 mixture of two diastereomers (the
`(R)-PMPA side-chain and L-amino acid ester were homochiralstarting materials).
`Thehighoral bioavailabilityand favorable tissue-selective distribution of GS-7171
`madeit a promising candidate for further development. To ascertain the properties
`of the individual diastereomers, -a method to separate them was needed.
`
`“Corresponding author.
`
`62)
`
`Copyright © 200] by Marcel Dekker,Inc.
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`622
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`CHAPMAN ET AL.
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`rao 2 SY
`9
`.
`9
`
`
`Ap >P-on Phengl a 9 p-o-{-)$0Ck ° A 07 Poo
`
`
`
`OH
`LS
`DCC
`/
`OH
`J
`NH
`CH,
`CH,
`Ch é \e
`
`oN
`
`Phenol
`
`oo
`
`1
`
`4
`
`NH,
`
`5
`
`"
`
`°
`
`3)
`
`3
`
`°
`
`A
`.
`.
`Simulated Moving Bed
`J
`Chromatography
`a =
`
`oY
`oOBuo’
`a
`NH
`cioweYY
`
`NH’?
`
`OH
`
`ox’ >9
`J
`HO =H Ne NOTPHO
`L/
`4
`oO
`NH
`°
`. Hid é v
`
`N
`eN
`1!~~
`
`SN
`
`NH,
`
`ae
`
`Scheme I.
`
`:
`
`McGuigan and coworkers have recently reported related phenoxy-amidate
`pro-drugs of the monophosphates of antiviral nucleoside analogs (4). Although
`these compoundsare also P-chiral diastereomeric mixtures, the issues of diastere-
`omerseparation and differential activity have not been addressed. This communi-
`cation discloses a solution to the diastereomer “problem” that may be generally
`applicable.
`Theadditional pre-clinical testing required akilogram-scale preparation ofthe
`pure diastereomers. Esterification ofthe solubletriethylamine salt of PMPA (1) with
`phenol using dicyclohexylcarbodiimide (DCC) in hot 1-methyl-2-pyrrolidinone
`(NMP)afforded 251% yield of PMPA monopheny]ester (4). Activation of (4) with
`thiony] chloridein dichloromethane gave the phosphonochloridate, which smoothly
`coupled with an excess of isopropyl L-alanine (5) to give the GS-7171 mixture (3)
`in 47% yield (unoptimjzed). Both steps werereadily performed on multi-kilogram
`scale in standard pilot plant equipment.
`Initially, the component diastereomers of amidate prodrug analogs were sepa-
`rated with repeated HPLCpurifications on a preparative C18 column. The marginal
`resolution ofthe 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 HYV assay showedthat the less retained isomer (GS-7340, 6) was more
`potent by a factor of ~10 and wasselected as the candidate for additional testing.
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`PMPA PRO-DRUGGS-7340
`
`623
`
`oO
`
`nH \_H
`NH
`N
`oO
`iSapeieeer
`Cr
`N7
`TN
`07
`~P-O7™
`“07
`*O
`Sw7 N07
`~P-OH
`LY on LYXK0.0
`
`Chy
`CHy
`0 a
`’
`
`.
`
`.
`
`N
`
`1, PMPA |
`
`NH
`
`2, Tenofovir Disoproxil Fumarate
`
`3, GS-7171
`
`Figure 1.
`
`Screening of commercially available chiral stationary phases revealed that
`Diacel’s Chiralpak AS with a mobile phase of 30% methanol in acetonitrile was
`a markedly more efficient chromatographic system (5) (Fig. 3). The remarkable
`resolution of the diastereomers (a = ~9) and the high solubility of GS-7171
`in the mobile phase (>300 g/L) allowed facile diastereomer separation. Using a
`
`et3n/Phos ph 6.5
`
`:
`
`PeakPro Report
`
`12 3 45 6 7
`Elution time
`
`8
`
`39
`
`10
`
`42
`
`14°
`
`16
`
`18
`
`20
`
`22
`
`24
`min’s
`
`Figure 2. HPLCseparation of the GS-717) component diastereomers on C18 columnpacking.
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`CHAPMANETAL.
`
`624
`
`,
`
`PeakPro Report /
`mvV's3050.7090
`
`Detectorresponse 10
`
`Elution time
`
`min's
`
`Figure 3. HPLC separation of the GS-7171 component diastereomers on Chiracel AS column
`packing.
`
`laboratory-scale simulated moving bed chromatography (6) system composed of
`ten Chiralpak AS (7) columnsin series allowed separation of > 1 kg/day of mixture
`(8). The desired isomer, GS-7340, (6), was recoveredin nearly quantitative yield
`and >98% diastereomeric purity.
`a,
`:
`After chromatographic purification, GS-7340 wasreadily crystallized as the
`free base or as the fumarate salt (7). Needles of GS-7340free base were grown
`from water.
`.
`Structure determination by X-ray crystallography (9) allowed definitive as-
`signment of the phosphorous chiral center as (S) (Fig. 4) in the more active isomer,
`‘GS-7340.
`"Jn summary,a practical kilo-scale process for synthesis and purification of the
`phenoxy-amidate pro-dmg GS-7340 has been developed. The first correlation of
`activity with phosphorous absolute configuration in a phenoxy-amidate pro-drug
`has been made. Researchinto a diastereoselective synthetic process for GS-7340
`is ongoing.
`
`2» EXPERIMENTAL
`
`[(R)-2-(Phenylphosphonomethoxy)propylJadenine 4
`
`A glass-lined reactor was charged with anhydrous PMPA, (1) (14.6 kg,
`50.8 mol), pheno) (9.6 kg, 102 mol),and 1-methy]l-2-pyrrolidinone (39 kg). The
`mixture washeated to 85°C and triethylamine (6.3 kg, 62.3 mol) added. A solution
`of 1,3-dicyclohexylcarbodiimide (17.1 kg, 82.9 mol) in ]-methy]-2-pyrrolidinone
`(1.6 kg) was then added over6 hoursat 100°C. Heating was continued for 16 hours.
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`PMPA PRO-DRUG GS-7340
`
`625
`
`Figure 4. Crystal Structure ofGS:7340, (6).
`
`The reaction was cooled to 45°C,diluted with water (29 kg), and cooled to 25°C.
`Solids were removed byfiltration and rinsed with water (15.3 kg). The combined
`filtrate and rinse was concentrated to’a tan slurry under reducedpressure, water
`(24.6 kg) was added, and adjusted to pH 11 with NaOH (25% in water). Suspended
`solids were removedbyfiltration through diatomaceousearth (2 kg) followed bya
`water-(4.4 kg) rinse. The combinedfiltrate and rinse was extracted with ethyl acetate
`(28kg).The aqueoussolution wasadjusted to pH 3.1 with HC] (37% in water) (4 kg),
`precipitating crude 4 which wasisolated by filtration and washed with methanol
`(12.7 kg). The wet product wasslurried in methanol (58kg),isolated byfiltration,
`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, D20,8): 1.2 (d, 3H), 3.45
`(q, 2H), 3.7 (q, 2H), 4 (m, 2H),4.2 (q, 2H), 4.35 (dd, 2H),6.6 (d, 2H), 7 (t, 1H), 7.15
`(t, 2H), 8.15 (s, 1H), 8.2 (s, 1H); >!P NMR (72 MHz, D,0, 8):15.0 (decoupled).
`
`Isopropyl L-alanine 5 -
`
`A glass-lined reactor was charged with L-alanine (7.1 kg, 80 mol) and iso-
`propanol (35.6 kg). The slurry was heated to reflux and chlorotrimethylsilane
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`CHAPMANETAL.
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`(14.6 kg, 134 mol) added over 69 minutes. Reflux was continued for 3 hours at
`which timethe reaction was homogeneous. Volatiles were removedby 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 vigorousstirring and
`further cooling to 21°C. Vigorous agitation was continued until crystals formed.
`The.slurry was cooled to about 0°C. Thesolid isopropyl L-alanine hydrochloride
`was isolated by filtration, rinsed with diethy! ether (5 kg), and dried to 12.35 kg
`off-white deliquescentcrystals. 1H NMR (300 MHz, CDCls, 5) :1.2 (m, 6H),1.7 (d,
`3H), 4.2 (m, 1H), 5.05 (m,1H), 8.6 (m, 3H). The free base isopropyl L-alanine was
`prepared by combininga solution of isopropyl! L-alanine hydrochloride, (9.12 kg,
`54 mol), in tetrahydrofuran (34 kg), with asolution of1 ,4-diazabicyclo[2.2.2]octane
`(5.48 kg, 49 mol) in tetrahydrofuran (15.5 kg) in a glass-lined reactor. Solid 1,4-
`diazabicyclo[2.2.2Joctane hydrochloride precipitated and was removed byfiltra-
`tion. Tetrahydrofuran was removedfrom the isopropy! L-alanine in the filtrate by
`carefuldistillation under reduced pressure. The productisopropyl L-alanine (5) was
`isolated as an oi) for use immediately in the next step withoutfurther processing or
`characterization.
`,
`
`GS-7171 3
`
`.
`
`‘
`
`A glass-lined reactor was charged with [(R)-2-(phenylphosphonomethoxy)-
`propylJadenine(4), (9.12 kg, 25.1 mol) and acetonitrile (30.7 kg). Thiony] chloride
`(6.57 kg, 56.7 mol) was added while maintaining the mixture below 50°C. The
`mixture washeated at 75°C until solids dissolved, then the reaction temperature
`was increased to 80°C andvolatiles (1 1.4 kg) collected by atmospheric distillation
`undernitrogen. Thepot residue was cooled to 25°C,diluted with dichloromethane
`(41 kg), and cooled to —29°C. A solution of isopropyl L-alanine (5) (ca. 7.1 kg,
`54 mol) in dichloromethane (36 kg) was added over 60 minutes at —1 8°C followed
`by triethylamine (7.66 kg, 75.7 mol) over 30 minutes at —18 to —11°C. The reac-
`tion mixture was warmed to room temperature and washed with 10% aq. sodium
`' dihydrogenphosphatesolution (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 under reduced pressure. Acetone (20 kg) was charged
`to the oil and the mixture concentrated under reduced pressure. Acetone (18.8 kg)
`was again chargedto theresulting oil. Half the product solution was purified by
`chromatography over a 38 x 38 cm 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 ofthe oil using’fresh silica gel and acetone. Clean productbearing
`fractions were concentrated under reduced pressure to an oil. Acetonitrile (19.6 kg)
`was charged to theoil and the mixture concentrated under reduced pressure. Ace-
`tonitrile (66.4 kg) was again charged andthe solution chilled to 0 to —5°C for
`16 hours. Solids were removed by filtration and thefiltrate concentrated underre-
`duced pressure affording 5.6 kg of 3 asa darkoil. 'H NMR (300 MHz, CDChh, 4):
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`PMPA PRO-DRUG GS-7340
`
`627
`
`1.1 (m, 12H), 3.7 (m, 1H), 4.0 (m, 5H), 4.2 (m, 1H), 5.0 (m, 1H), 6.2 (s, 2H),
`7.05 (m, 5H), 8.0 (s, 1H), 8.25 (d, 1H); 3!P NMR (72 MHz, CDCl, 6): 21.0, 22.5
`(decoupled).
`
`. "9-[(R)-2-[[(S)-[[(S)-1-(sopropoxycarbonyl)ethylJamino}-
`phenoxyphosphinyl)methoxy]propyladenine 6
`
`GS-7171 (3), 2.8 kg, was purified by continuous simulated moving bed chro-
`matography. Ten columnsfilled with 10 cm by 5 cm 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 crystalline mass wet with ace-
`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-
`~ 120°C; '"H NMR (300 MHz, CDCI, 8): 1.15 (m, 12H),3.7 (t, 1H), 4.0 (m, 5H), 4.2
`(dd, 1H), 5.0 (m, 1H), 6.05 (s, 2H), 7.1 (m, 5H), 8.0 (s, 1), 8.2 (s, 1H); 7!P NMR
`(72 MHz, CDCls, 6): 21.0 (decoupled).
`
`9-[(R)-2-[[(S)-[[(S)-1-(sopropoxycarbony])ethylJamino]-
`phenoxyphosphinyl]methoxy]propyljadenine Fumarate (1:1) 7
`
`A glass-lined reactor was charged with 9-[(R)-2-[[(S)-[[(S)-1-(isopropoxycar-
`bonyl)ethy]]amino]phenoxyphosphinylJmethoxy]propyl] adenine (6),
`(1.29. kg,
`2.71 mol), fumaric acid (284 g, 2.44 mol), and acetonitrile (24.6 kg). The mix-
`ture washeated torefluxto dissolve the solids,filtered while-hot, and cooled to 5°C _
`for 16 hours. The productwas isolated byfiltration, rinsed with acetonitrile (9.2 kg),
`and dried to 1.33 kg 7 as a white powder: mp 119.7-121.1°C; [a]p29-41.7° (c 1.0,
`acetic acid),
`
`REFERENCES
`
`1. Schultze, 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.; Pero, C. F; Witvrouw, M.; Holy, A.; De> Clercq, E. Biochem.
`Biophy. Res. Commun., 1996, 219, 337.
`3. Schooley, R.; Myers, T. R.; Ruane, P.;Beall,G.; Lampiris, H.: McGowan, I. A Double-
`Blind, Pilacebo-Controlled Study ofTenofovir Disoproxil Fumarate (TDF) for the Treat-
`ment of HIV Infection. Presentedat the 39" Interscience Conference on Antimicrobial
`Agents and Chemotherapy, San Francisco, CA, September, 1999.
`a) Siddiqui, A. Q., McGuigan,C.; Ballatore, C.; Zuccotto, F.; Gilbert, I. H.; De Clercq,
`E.; Balzarini, J. J. Med. Chem. 1999, 42, 4122-4128. b) Siddiqui, A. Q.; Ballatore,
`
`4.
`
`.
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`u
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`CHAPMAN ET AL.
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`’
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`C.; McGuigan, C.; De Clercq, E.; Balzarini, J. J. Med. Chem. 1999, 42, 393-399.
`c) McGuigan, C.; Tsang, H-W.; Sutton, P. W.; De Clereq, E.; Balzarini, J. Antiviral
`Chemistry and Chemotherapy, 1998, 9, 109-115. d) McGuigan, C.; Tsang, H-W.;
`Cahard, D.; Turner, K.; Velaquez, S.; Salgado, A.; Bidois, L.; Naesens, L.; De Clercq,
`#B.; Balzarini; J. Antiviral Research, 1997, 35, 195-204. e) McGuigan, C.; Salgado,
`A.; Yamnold, 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.
`:
`.
`,
`Chapman, H.; Keman, M.; Rohloff, J.; Sparacino, M.; Terhorst, T. Purification of
`PMPAAmidate Prodrugs by SMB Chromatography and X-Ray Crystallographyofthe
`Diastereomerically Pure GS-7340. Submitted for publication in a supplementto Nucle-
`osides and Nucleotides on the XIV International Roundtable: Nucleosides, Nucleotides
`and their Biological Applications, San Francisco, CA, September, 2000.
`Francotte, E. R.; Richert, P. Journal of Chromatography A, 1997, 769, 101-107.
`Chiral Technologies Inc., amylose-tris(alpha-methy] benzyl! carbamate) coated onsilica
`gel.
`Continuous simulated moving bed chromatography was performed by Aerojet Fine
`’ Chemicals, Rancho Cordova, CA.
`Single-crystal x-ray structure determination was performed by F. J. Hollander at
`University of California, Berkeley.
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