`
`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. Keman, E. Prisbe, J. Rohlofl, M- Sparacino,
`T. Terhorst, and R. Yu
`
`Gilead Sciences. 333 lakeside Drive. Foster City, California 94404
`
`ABSTRACT
`
`The practical synthesis of a mixed phenoxy-amidate derivative of PMPA with
`high oral bioavailability and favorable pharmacokinetics is described. The non—
`stereoselective synthetic route produces a 1:1 mixture of two diastereomers at
`phosphorous. Simulated moving bed chromatography using Chiralpalt AS en-
`abled kilo-scale isolation of the more potent dia‘stereorner (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]adenine (PMPA,
`1) (1) has shown potent 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-amidat'c derivative of PMPA (3) which was designated as GS-7l7l
`(Fig. 1). Due to the asymmetric center at phosphorous and _no'ii3-stereoseleetive syn'-
`thetic route, GS-7l71 was composed of a 1:1 mixture of two diastereomers (thc'
`(R)-PMPA side-chain and L-amino acid ester were homochiral starting materials).
`The high oral bioavailability and favorable tissue-selective distribution of GS-7 1 71
`made it a promising candidate for further development. To ascertain the properties
`of the individual diastereomers, -a method to separate them was needed.
`
`fCorrcsponding author.
`
`62]
`
`Copyright 0 2001 by Marcel Delgker. Inc.
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`'9
`9
`o/‘f-OH Phenol A 0A,?“ 0 soon,
`A
`OH
`CH
`L/
`959
`5H:
`1
`
`6H:
`4
`
`NH:
`
`Hac‘“ 07/
`0
`
`5
`
`A8 Q
`[Tao
`NH
`
`°r
`
`O
`
`o
`
`A
`L]
`5H:
`3
`
`0
`s‘ lth'
`Bed A oAii'o
`Imuaa
`OVII'I
`Chromatograpgy U NH
`——__—.—
`=
`”’14,ch Y
`c
`5
`O
`°
`
`5
`
`o
`Ho — OH
`O
`————h
`--
`
`0
`
`NH).
`N’ I
`R"
`
`0H
`o 9 Q
`N‘>
`oA'f'o
`N
`U NH
`:
`ch Y
`CH:
`.
`O
`0
`
`7
`
`N”:
`I
`\
`A= N
`
`1
`
`N
`
`N
`\>
`l“
`
`Scheme]
`
`McGuigan and coworkers have recently reported related phenoxy—amidate
`pro-drugs of the monophosphates of antiviral nucleoside analogs (4). Although
`these compounds are also P-chiral diastereomen'c mixtures, the issues of diastere-
`omer separation and differential activity haVe not been addressed. This communi-
`cation discloses a solution to the diastereomer “problem” that may be generally
`applicable.
`The additional pre-clinical testing required a kilogram-scale preparation of the-
`pure diastereomers. Esterification of the soluble tn'ethylamine salt of PMPA (1) with
`phenol using dicyclobexylcarbodiimide (DCC) in hot 1-methyl-2-pyrrolidinone
`(NNIP) afforded a 51% yield of PMPA monophenyl ester- (4). Activation of (4) with
`thionyl chloride in dichloromethane gave the phosphonochlorirlate, which smoothly
`coupled with an excess of isopropyl L—al'anine (5) to give the GS—717l mixture (3)
`in 47% yield (unoptimjzed). Both steps were readily performed on multi-kilogram
`scale in standard pilot plant equipment:
`'
`‘-
`Initially, the component diastereomers of amidate prodrug analogs were sepa-
`rated with repeated HPLC purifications on a preparative C18 column. The marginal
`resolution of the diastereomers on this system (Fig. 2) necessitated multiple passes
`but culminated in isolation of ~100 mg of each isomer enriched to >9S: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
`
`it A
`"(l ”\> 9.91) .9
`I
`N CAP-0A0 o
`Alf-OH
`u H
`\‘./ O\/O\|r°
`o
`CH3
`'
`CH3
`\I/
`0
`1. PMPA '
`2,TenofovirDisoproxilFumaiate
`
`NH2
`
`N
`
`\
`
`t.’ Q
`(I ‘>
`/\ p...
`I O
`N
`NL/O .
`NH
`_
`H36
`‘3
`o
`Yo
`.
`
`3.65—7171
`
`Figure].
`
`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
`
`tan/Phos an 6.5
`
`'
`
`PeakPro REDDrt
`
` 1
`
`12
`
`14‘
`
`16
`
`la
`
`20
`
`22
`
`24
`Mn's
`
`3 4'5 6'7 3 910
`Z
`Elution tint:
`
`Figure 2: HPLC separation of the QS-7l7l component diastereomers on C18 column packing.
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`\
`_
`
`_
`
`..
`
`N
`
`o
`
`‘N
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`CHAPMAN ET AL.
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`PeakPro Report
`
`mV's
`
` .
`3050.7.090
`Detectorresponse 10
`
`
`
`2
`
`1
`Elution lime
`
`3
`
`4
`
`5
`
`6
`
`7
`
`8'
`
`9
`
`10
`
`11
`
`12
`
`13
`
`_
`
`I4
`min’s
`
`Figure 3. HPLC separation of the 65-717) 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% diastereomen'c purity.
`,
`_
`,
`.
`,
`After chromatographic purification, 65—7340 was readily Crystallized as the
`free base or as the fumarate salt ('7). Needles of 08-7340 free 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.
`'
`'
`In summary, a practical kilo-scale process for synthesis and purification of the
`phenoxy»amidate pro-dmg 68—7340 has been developed. The first correlation of
`activity with phosphorous absolute configuration in a ph'enoxy-amidate pro-drug
`has been made. Research into a diastereoselective synthetic process for 05-7340
`is ongoing.
`
`5 EXPERIMENTAL
`
`[( R)-2-(Phenylphosphonomethoxy)propyl]adenine 4
`
`A glass-lined reactor 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 tn'ethylamine (6.3 kg. 62.3 mol) added. A solution
`of 1,3-dicyclohexy1carbodiimide (17.1 kg, 82.9 mol) in 1-methyl-2-pyrrolidinone
`(1.6 kg) was then added over 6 hours at lOOf'C. Heating was continued for 16 hours.
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`PMPA PRO-DRUG GS-7340
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`625
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`
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`Figure 4. Crystal Structure oermdo. (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 with 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
`(28kg).The aqueous solution was adjusted to pH 3.1 with HCl (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). ‘H NMR (300 MHz, D20, 6): 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, 11-1); 3’P NMR (72 MHz, D20, 5):]50 (decoupled).
`
`Isopropyl L—alan'ine 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 chloroln'methylsilane
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`, (14.6 kg, 134 mol) added 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.slurry was cooled to about 0°C. The solid isopropyl L—alanine hydrochloride
`was isolated by filtration, rinsed with diethyl ether (5 kg), and dried to 12.35 kg
`off-white deliquescent crystals. l1'1 NMR (300 MHz, CDC13. 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 combining a solution of isopropyl Llalanine hydrochloride, (9.12 kg,
`54 mol), in tetrahydrofuran (34 kg), with asolution of 1,4-diazabicyclo[2.2.2]octane
`(5.48 kg, 49 mol) in tetrahydrofuran (15.5 kg) in a glass-lined reactor. Solid 1,4-
`diazabicyclo[2.2.2]octane hydrochloride precipitated and was removed by filtra-
`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)-
`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 washeated at 75°C until solids diss’olved, then the reaction temperature
`was increased to 80°C and volatiles (1 1.4 kg) collected by atmospheric distillation
`under nitrogen. The pot 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 —18°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
`' 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 under 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 38cm 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 usingflfresh 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-
`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-
`duced pressure affording 5.6 kg of 3 as a dark oil. ll-l NMR (300 MHz, CDC13I 5):
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`PMPA PRO-DRUG 68-7340
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`'
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`627
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`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 (5, 11-1), 8.25 (d, 1H); 3"P NMR (72 MHz, CDC13, 6): 21.0, 22.5
`(decoupled).
`
`.
`
`'9-.[(B)~2.-[[(§)-[[(§)-1-(Isopropoxycarbonyl)ethyl]amino]—
`phenoxyphosphinyl)methoxy]propy1]adeninc 6
`
`GS-717l (3), 2.8 kg, was purified by continuous simulated moving bed chro-
`matography. Ten columns filled with 10 cm by 5 cm beds of packing, 20 micron
`Chiralp'ak'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
`acetonitn'le (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, 13 kg 6 , 98.7% diastereomeric purity by HPLC: mp 117—
`- 120°C; 'H NMR (300 MHz,CDC13. 8): _1.15 (111, 121-1), 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, 1H), 8.2 (s, 1H); 31P NMR
`(72 MHz, CDC13, 8): 21.0 (decoupled).
`
`‘9—[@)—2-[[(§)-[[(§)—1-(1sopropoxycarbonyl)et11yl)amino]-
`phenoxyphosphinyl]methoxy]propyl]adenine Fumarate (1:1) 7
`
`A glass-lined reactor was charged with 9—[®-2-[[@-[[@)—1-(isopropoxycar—
`bonyl)ethyl]amino]phenoxyphosphirtyl]methoxy]propy1] adenine (6), (1.29 kg,
`2. 71 mol), fumaric acid (284 g, 244 mol), and acetonitrile (24.6 kg) The mix-
`ture was heated to reflux to dissolve the solids, filtered whilc-hot, and cooled to 5°C
`for 16 hours The product was isolated by filtration, rinsed with acetonitrile (92 kg).
`and dried to 1.33 kg 7 as a white powder: mp 119.7—121.1°C; [ohm—41.7” (c 1.0,
`acetic acid).
`
`REFERENCES
`
`1. Schultzc.L. M.; ChapmanJi. 11.; Dubree, N. 1.; Jones, R. 1.; Kent K. M‘; Lee T. 1:;
`Louie, M. S.; Postich, M. J.; Pnsbe, E. J.; Rohloff, J. C.', Yu, R. H. Tetrahedron Letters,
`1998, 39, 1853—1856.
`2. Balzarini,1.,,Aquaro S.; Pemo, C. F.; Wilvrouw, M. Holy, A,De C1ercq,E. Biochem.
`Biophy. Res. Commun” 1996. 219. 337.
`3. Schooley, R.“Myers T. R.; Ruane, P.B,eall,G.; Lampiris,H.;McGowan,I.ADoub1e-
`Blind, Placebo—Controlled Study of.Tenofovir Disoproxil Furnarate (TDF) for the Treat—
`ment of HIV infection. Presented at the 39lh Interscience Conference on Antimicrobial
`Agents and Chemotherapy, San Francisco, CA, September, 1999.
`a) Siddiqui, A. Q.; McGuigan, C.; Ballatore, C.; Zuccotto, F.; Gilbert, 1. H.: De Clercq,
`E; Balzarini, J. J. Med. Chem. 1999, 42, 4122—4128. b) Siddiqui, A. Q.; Ballatore,
`
`4.
`
`-
`
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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 Clercq, E.; Balzarini, J. Antiviral
`Chemistry and Chemotherapy, 1998, 9. 109—115. (J) McGuigan, C.; Tsang, H-W.;
`Cahard, D.; Turner, K.; Velaquez, 8.; Salgado‘, A.; Bidois, L; Naesens, L; De Clercq,
`' E.; Balzarini; J. Antiviral Research, 1997, 35, 195—204. C) McGuigan, C.; Salgado,
`A.; Yamold, C.; Harries. T.Y.; De Clercq, E.; Balzarini. J. Antiviral Chemistry and
`Chemotherapy, 1996, 7 184—188. D Balzarini, J.; Karlsson, A.. Aquaro, S.; Pemo
`C.-F.; Cahard. D.; Naesens, L.; DeClercq, -.;E McGuigan, C Pmc. Natl Acari. Sci.
`USA 1996, 93 7295—7299.
`Chapman, H.; Keman, M.; RohJoff. J.;_Sparacino, M4 Terhorst, T. Purification of
`PMPA Amidate Prodrugs by SMB Chromatography and X—Ray Crystallography of the
`Diastereomen'cally Pure 68-7340. Submitted for publication in a supplement to Nucle-
`asides and Nucleotides on the XIV lntemational Roundtable: Nucleosides Nucleotides
`and their Biological Applications, San Francisco, CA, September 2000.
`Francotte E R.; Richen P. Joumal of ChromatographyA 1997 769, 101— 107.
`Chiral Technologies Inc. amylose-tris(alpha-Jmethyl benzyl carbamate) coated on silica
`gel.
`Continuous simulated moving bed chromatography was performed by Aerojet Fine
`' Chemicals. Rancho Cordova. CA.
`Single-crystal x-my structure determination was performed by F. J. Hollander at
`University of California. Berkeley.
`
`F"
`>‘
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