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Chromatography as an Enabling Technology in Pharmaceutical Process Development:  Expedited Multikilogram Preparation of a Candidate HIV Protease Inhibitor

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Chromatography as an Enabling Technology in Pharmaceutical Process Development:  Expedited Multikilogram Preparation of a Candidate HIV Protease Inhibitor
  Chromatography as an Enabling Technology in Pharmaceutical ProcessDevelopment: Expedited Multikilogram Preparation of a Candidate HIVProtease Inhibitor Christopher J. Welch,* ,† Fred Fleitz,* ,† Firoz Antia,* ,‡ Pete Yehl, § Robert Waters, § Norihiro Ikemoto, † Joseph D. Armstrong, III, † and David J. Mathre †  Merck & Co., Inc., Rahway, New Jersey 07065, U.S.A. Abstract: Chromatography plays a vital role in supporting preclinicalpharmaceutical development, whether in providing assays forpurity determinations, preparative separation of small amountsof intermediates for route selection studies, or purification of bulk drug substances on multikilogram scale. All three ap-proaches are illustrated in the recent development of acandidate HIV protease inhibitor in these laboratories. Chiralsupercritical fluid chromatography (SFC) on the hundreds-of-milligrams scale afforded an enantiopure intermediate tofacilitate early synthetic studies, HPLC on the tens-of-gramsscale provided purified material for use in salt form investiga-tions, and HPLC using a 30 cm column was used to purify 5.6kg of a key intermediate to provide material for early preclinicalevaluations. Introduction Two decades after the emergence of HIV/AIDS, thisdisease remains a major threat to human health. 1 Despiteconsiderable progress, the emergence of resistant viral strainsand the limitations of current treatments mean that improvedtherapies are needed. 2 Candidate HIV protease inhibitors withstructural similarity to the commercial Crixivan/indinavirHIV protease inhibitor 3 but with an improved profile in thetreatment of resistant strains of HIV in preliminary screeninghave recently been reported. 4 We now describe the prepara-tion of several kilograms of a related candidate,  1 , using adevelopment strategy in which chromatographic separationplays a key role in the synthesis. The rapid development of a method to provide kilogram amounts of   1  illustrates thevital role that chromatography often plays in modernpharmaceutical process research. In addition to the traditionalrole as the preferred technique for purity and enantiopurityanalysis, chromatography is becoming increasingly utilizedfor rapid purification of bulk material, 5 especially during theearlier stages in pharmaceutical discovery and development.The use of preparative HPLC in support of synthesis hasa long history, dating at least to R. B. Woodward’s 1973prediction that preparative HPLC “...will be indispensablein the laboratory of every organic chemist in the very nearfuture”. 6 Recent improvements in equipment, materials, andtechniques have led to an increasing adoption of thetechnique in pharmaceutical development, driven largely bythe sheer speed and the minimal labor required to develop apreparative chromatographic method.We herein describe some of the ways in which chroma-tography has been utilized in the development of   1 , includingassaying purity and enantiopurity of intermediates, prepara-tive separation of small amounts of intermediates for routeselection studies, and larger-scale purification of bulk drugsubstances. Results and Discussion A number of routes to the preparation  1  were investigated.Several approaches to the enantioselective synthesis of a keyaminochromanol precursor have been described. 7 The centralfeature of the route that was ultimately selected for prepara-tion of   1  involves the coupling of substituted piperazine,  3 ,with epoxide,  4 , to give acetonide,  2 , which affords the target, 1 , upon acetonide hydrolysis.As with the synthesis of any complex target containingmultiple stereocenters, the development of a scaleable processfor the synthesis of   1  involved the investigation of a numberof routes and intermediates, each of which required timely † Process Research. ‡ Chemical Engineering Research and Development. § Analytical Research.(1) Gallo, R. C.; Montagnier, L.  Science  2002 ,  298  , 1730.(2) Menendez-Arias, L.  TIPS   2002 ,  23 , 381.(3) (a) Dorsey, B. D.; Vacca, J. P.  Infect. Dis. Ther  .  2002 ,  25 , 65. (b) Askin,D. A.  Drug Disco V  ery De V  .  1998 ,  1 , 338.(4) (a) Chen, Y.; Zhang, F.; Rano, T. A.; Lu, Z.; Schleif, W. A.; Gabryelski,L.; Olsen, D. B.; Stahlhut, M.; Rutowski, C. A.; Lin, J. H.; Jin, L.; Emini,E. A.; Chapman, K. T.; Tata, J. R.  Bioorg. Med. Chem. Lett.  2002 ,  12 ,2419. (b) Duffy, J. L.; Kevin, N. J.; Kirk, B. A.; Chapman, K. T.; Schleif,W. A.; Olsen, D. B.; Stahlhut, M.; Rutkowski, C. A.; Kuo, L. C.; Jin, L.;Lin, J. H.; Emini, E. A.; Tata, J. R.  Bioorg. Med. Chem. Lett.  2002 ,  12 ,2423.(5) (a) Francotte, E. R.  J. Chromatogr  .  2001 ,  906  , 379. (b) Andersson, S.;Allenmark, S.  J. Biochem. Biophys. Methods  2002 ,  54 , 11.(6) Woodward, R. B.  Pure Appl. Chem .  1973 ,  33 , 145.(7) (a) Hansen, K. B.; Rabbat, P.; Springfield, S. A.; Devine, P.; Grabowski,E. J. J.; Reider, P. J.  Tetrahedron Lett  .  2001 ,  42 , 8743. (b) Davies, I. W.;Taylor, M.; Marcoux, J. F.; Matty, L.; Wu, J.; Hughes, D.; Reider, P. J. Tetrahedron Lett  .  2000 ,  41 , 8021. Organic Process Research & Development  2004 ,  8,  186 − 191 186  •  Vol. 8, No. 2, 2004 / Organic Process Research & Development 10.1021/op0300443 CCC: $27.50 © 2004 American Chemical SocietyPublished on Web 01/15/2004  analytical chiral chromatographic support. We employ astandardized approach for developing chromatographic meth-ods for enantiopurity determination for the potentially dozensof different intermediates evaluated during the developmentof a typical process. 8 A robotic screen of at least 12 differentchiral columns using supercritical fluid chromatography(SFC) with a standard gradient of 4 - 40% methanol in carbondioxide successfully elutes most pharmaceutical intermedi-ates. For basic compounds that are poorly eluted under theseconditions, we employ an identical gradient elution using25 mM isobutylamine in methanol as the polar modifier. Thescreening procedure requires only a few minutes to set up,is normally performed overnight, and often affords a methodfor enantiopurity analysis that can be utilized without anyfurther worker intervention. We have found that this auto-mated chiral method development capability is helpful inexpediting route investigation and process research onenantiopure drug candidates, allowing us to rapidly evaluatenew synthetic concepts with minimal assay developmentlabor. Furthermore, the use of SFC offers a number of inherent advantages over HPLC, including generally superiorefficiency and lower eluent viscosity, which together permitfaster separations.In addition to its great value in analytical determinationsof enantiopurity, we have found that SFC is very useful forrapid isolation of small amounts of intermediates or finaldrug candidates. 9 In addition to the previously mentionedadvantage of speed, preparative SFC offers the importantadvantage over HPLC of significant reductions in wastesolvent generation resulting from the replacement of petro-chemical hydrocarbon solvents with supercritical carbondioxide. Selecting a method for semi-preparative SFCseparation often requires only a straightforward extensionof the results obtained in analytical SFC screening. Thus, itis sometimes possible for a worker to screen a new racemateand resolve a small amount of the individual enantiomerswithin a day of sample receipt. We routinely use a semi-preparative SFC instrument with a 2-cm i.d. chiral columnoperating at a flow rate of 50 mL/min to purify up to 20-gquantities of selected intermediates. This timely availabilityof chromatographically derived enantiopure material allowsresearchers to rapidly conduct important studies aimed atdevising a crystallization, developing a final salt form, ormeasuring racemization rates or reaction diastereoselectivi-ties. The availability of enantiopure intermediates frompreparative SFC allows the synthetic effort on a project tobe focused primarily on the key synthetic challenges, andnot on how to prepare the small amounts of enantiopurematerials that are essential for informed route exploration.An illustration of the utility of SFC in expediting processresearch is provided by an example involving the substitutedpiperazine  3 . Preliminary attempts at the opening of epoxide 4  with piperazine  3  afforded a number of minor impuritiesin addition to the desired acetonide,  2 . These impurities weresubsequently identified and brought under control, but duringthe initial stages of the project the question arose as towhether the formation of these impurities was related to thelow enantiopurity of the substituted piperazine  3  used in thereaction. This question was easily and quickly answered byproviding enantiopure  3  using SFC. Initial evaluation of an80% ee sample of   3  using the standard twelve column screenwith methanol as the polar modifier afforded poor peak-shapes, a not uncommon occurrence for basic compounds.Subsequent screening using 25 mM isobutylamine in metha-nol as the polar modifier showed baseline resolution of theenantiomers using the Chiralpak AS column, with partialresolution being obtained on the Chiralpak AD and Whelkocolumns. The separation using the Chiralpak AS column wasscaled up to a 2-cm i.d. column for semi-preparative SFCseparation, where 290 mg of   3  at only 80% ee was upgradedto afford 215 mg of material with  > 99% ee. A representativechromatogram illustrating this separation is shown in Figure1.Similarly, achiral preparative chromatography can beuseful for upgrading the purity of intermediates or finalproducts containing unsuitably high levels of offendingimpurities. Although medium-pressure chromatography on (8) (a) Welch, C. J.; Kress, M. H.; Beconi, M.; Mathre, D. J.  Chirality  2003 , 15 , 143. (b) Villeneuve, M. S.; Anderegg, R. J.  J. Chromatogr  .  1998 ,  826  ,217.(9) Berger, T. A.; Smith, J.; Fogelman, K.; Kruluts, K.  Am. Lab .  2002 ,  34 , 14. Figure 1.  Semipreparative SFC separation of an enrichedmixture of substituted piperazine, 3. Conditions: Chiralpak AS(20  ×  250 mm), 12% (25 mM isobutylamine in methanol)/ carbon dioxide, 100 bar, 35  ° C, 50 mL/min, UV 320 nm,injection of 1 mL @ 60 mg/mL. Vol. 8, No. 2, 2004 / Organic Process Research & Development  •  187  silica has been widely used in organic synthesis for a numberof years, recent widespread availability of preparative HPLCequipment has resulted in the increased use of this techniquein routine purifications in support of pharmaceutical processresearch. As a result, preparative HPLC is now often usedas a “safety net” to enable the rapid preparation of newcompounds for biological evaluation or clinical studies, animportant consideration when there is an urgent need to movea drug candidate through the development process with thegreatest possible speed.An illustration of this approach is given by the examplein Figure 2. In an early route to the preparation of   1 , thepresence of a problematic impurity that was not readilyrejected by recrystallization was noted. The presence of impurities in a free base oftentimes hinders the appropriateselection of a final salt form, and when possible, it isadvisable to conduct salt-selection studies with material of the highest possible purity. In this instance, preparative HPLCon silica easily provided several grams of highly purematerial that enabled the work on the final salt form of thedrug candidate to begin well before the complete syntheticroute had been developed.This same HPLC method was subsequently used to purifyadditional 20- and 15-g batches of   1  generated by differentroutes, and in each case having a different profile of offending impurities. In each instance, silica HPLC provedhighly productive and afforded material with  > 99% purity.These relatively small amounts of material supplied bypreparative HPLC enabled important initial developmentwork to proceed without delay. At this point we consideredthe possibility of using chromatography for the preparationof more substantial amounts of   1  to support advancedpreclinical testing and early clinical evaluations.Preparative HPLC on multikilogram scale is increasinglyused in pharmaceutical development. 10 While relativelynonproductive chromatographic methods may be suitable forsmaller-scale separations, a highly productive method isessential for larger-scale implementation. Both high chro-matographic selectivity ( R  ) and excellent solubility in themobile phase are required for outstanding productivity. Afterconsideration of a number of options, we selected the crudeacetonide,  2 , formed by coupling of the substituted piper-azine,  3 , with epoxide,  4 , to be the most suitable species forlarger-scale HPLC purification. We observed the presenceof several low-level impurities in the crude material, one of which, the double addition product,  5 , proved especiallydifficult to reject by crystallization as the pharmaceuticallydesirable HCl salt. Certainly, impurity rejection with othersalts may have been possible, but we chose in this instanceto proceed rapidly with the HPLC purification approach,rather than to devote time and material to exploring alterna-tive strategies.HPLC analysis using the conditions previously developedfor the purification of   1  also afforded acceptable purificationof crude acetonide,  2 . However, a modified method employ-ing an eluent of 10% methanol in isopropyl acetate affordedbetter productivity and was used in a pilot separationconducted on 40-g scale using a 6-cm i.d. dynamic axialcompression (DAC) column packed with 600 g of silica.Excellent solubility of crude  2  in the eluent was observed,and a feed concentration of 38% was used. Injection of increasing volumes of feed with collection of the centralportion of the main peak revealed excellent purity andrecovery, even with injection of 40 mL of the 38% feedsolution (Figure 3). With the potential for a cycle time of less than 10 min if overlapping injections are used, thiscorresponds to a productivity of nearly 4 kkd (kilograms of desired component per kilogram of stationary phase per day),suggesting relatively facile access to multikilogram quantitiesof   2 .Following satisfactory evaluation at the pilot level, wedeemed that purification of   2  on multikilogram scale wouldbe feasible. We opted to carry out the separation using a 30 (10) (a) Nicoud, R. M.; Majors, R. E.  LC-GC   2000 ,  18  , 683. (b) Blehaut, J.;Ludemann-Hombourger, O.; Perrin, S. R.  Chim. Oggi  2001 ,  19 , 24. Figure 2.  Preparative HPLC upgrade of purity of 1 to prepare material for use in salt form studies. 188  •  Vol. 8, No. 2, 2004 / Organic Process Research & Development  cm i.d. DAC column without overlapping injections. Ac-cordingly, the column was packed with 14 kg of silica andused to purify 15.7 kg of a 35.1 assay wt % solution of crudeacetonide,  2 , (total of 5.51 assay kg) in IPAc. The feedmaterial contained about 1% (by LC area) of bis-adduct  5 ,which needed to be reduced below 0.5 A%. Crude acetonide 2  was chromatographed in 19 runs, each 16 min long, withan eluent of 10% MeOH in IPAc and a flow rate of 7.5L/min, with detection at 320 nm. This corresponds to ademonstrated productivity of 1.9 kkd and a specific solventconsumption of about 400 L/kg purified product. A repre-sentative chromatogram is illustrated in Figure 4.Although no further attempts were made to optimize themethod, it is evident from the chromatogram in Figure 4that with overlapping injections, injection cycle time couldhave been reduced to about 11 min, corresponding to aproductivity of 2.7 kkd and a specific solvent consumptionof 280 L/kg.Following HPLC analysis to verify purity, fractions werecombined and concentrated, and the solvent was switchedto methanol under vacuum in a 500-gal cone-bottomedstainless steel still. The methanol concentrate (42.2 kg)contained 5.65 assay kg of the acetonide,  2 , with bis-adductimpurity  5  reduced to 0.30 area %.Following successful purification of acetonide  2 , removalof the acetonide protecting group and crystallization of   1  asthe HCl salt were required to complete the synthesis. Anumber of conditions were evaluated for carrying out therequired deprotection of acetonide  2 . Trifluoroacetic acid inTHF/H 2 O afforded no deprotection, even upon heating to40  ° C, while the use of concentrated HCl in IPA gave onlyslow deprotection. Changing the solvent to methanol sig-nificantly shortened the reaction time, but even in methanol,30 equiv of concentrated HCl were required for completereaction. However, treatment of acetonide  2  with a solutionof gaseous HCl in methanol allowed the reaction to be run Figure 3.  Pilot study of HPLC purification of 2 using 6-cm i.d. DAC HPLC column. Preparative conditions: 6-cm i.d. ProChromDAC column packed with 600 g of Amicon grade 631 silica (18  µ m, irregular); 90:10 IPAc/MeOH; 300 mL/min; UV 320 nm;injection 40 mL of 38% crude 2 in IPAc. A productivity as great as 4 kkd may be possible with overlapping injections. Figure 4.  Large-scale preparative HPLC purification of 2 using 30-cm i.d. DAC HPLC column, showing typical location of fractionscollected during the run. Purified 2 was recovered in fraction F1, while bis-adduct 5 was separated in fraction B. Preparativeconditions: 30-cm i.d. ProChrom DAC column packed with 14 kg of Amicon Grade 631 silica (18  µ m, irregular). 90:10 IPAc/ MeOH, 7.5 L/min; UV 320 nm; 0.9 kkd with specific solvent consumption of about 400 L/kg. Vol. 8, No. 2, 2004 / Organic Process Research & Development  •  189  at lower temperature with reduced reaction time, affordingthe deprotected target  1  as the free base in 90% yieldfollowing extractive workup and charcoal treatment.After deprotection, target molecule  1  was isolated as thepharmaceutically desirable hydrochloride salt by first solventswitching the MeOH/IPAc free base extracts to IPAc, thenadding IPA, and heating to 60  ° C, followed by addition of a solution of HCl in IPA. Crystallization was induced bythe addition of seed crystals (obtained from workup of theearlier pilot-scale chromatographic separation) and then agingfor 2 h at 60 ° C with overnight cooling to room temperatureto complete the crystallization. Isolation by filtration afforded2.71 kg of   1 ‚ HCl as a white solid (needles) in which theoffending impurity  5  was reduced to an acceptable 0.27HPLC area percent. Following analytical testing, this materialwas delivered for use in preclinical evaluation. Conclusions Process research on emerging drug candidates must strikea balance between synthetic quality and speed. On one hand,it is important to produce material quickly, so that preclinicalevaluation may proceed with the fastest possible pace. Onthe other hand, it is important to develop a synthesis whichwill be suitable for production at larger scale, should thecompound survive the intense scrutiny of the drug selectionprocess. In the development of the candidate HIV proteaseinhibitor described herein, a middle ground approach provedto be the best strategy. We employed a synthesis with realscale-up potential, but used the enabling tool of chromatog-raphy to allow fast early-stage production at the multi-kilogram level. As a result, we were able to prepare theseveral kilograms of investigational compound required forpreclinical development with the greatest possible speed. Experimental Section General.  Details concerning preparation of   2 ,  3 , and  4 will be described in a separate publication. Chiral SFCscreening was carried out using a pair of Berger Instrumentsanalytical supercritical fluid chromatographs fitted with six-position column selection valves and Agilent model 1100diode array UV - visible detectors. Chiral HPLC screeningwas carried out using an Agilent model 1100 HPLCinstrument. Loading studies were performed with an Agilent1100 HPLC instrument fitted with a preparative autosamplerand a well plate fraction collector. Semi-preparative SFCpurification was carried out using a Berger InstrumentsMultiGram Preparative SFC Instrument. Intermediate-scalepreparative HPLC was carried out using a system containingdual Varian SD-1 pumps (800 mL/min), Varian 215 injectorpump 100 mL/min, Varian 320 variable wavelength UV/visdetector, R&S Techologies/Varian LC ReSonator liquidhandling module and control software, with a Prochrom/ Novasep dynamic axial compression (DAC) column (6 or 8cm i.d.). Larger-scale preparative HPLC was carried outusing a system consisting of a KP3000 pumping skid(Biotage, Charlottesville, VA), consisting of a main eluentpump (capable of 13000 mL/min flow) with a smaller-feedpump with a 3000 mL/min capacity, and a variable-wavelength detector with a full-flow flow cell and a 30-cmi.d. ProChrom DAC column (NovaSep, Inc., Boothwyn, PA). Preparative SFC Upgrade of enantiopUrity of 3.  ChiralSFC screening was carried out using a pair of BergerInstruments analytical supercritical fluid chromatographsfitted with six position column selection valves and Agilentmodel 1100 diode array UV - visible detectors. Chiralstationary phases evaluated included Chiralpak AD and AS,Chiralcel OD, OJ, OF, and OB (Chiral Technologies),Whelko (Regis Technologies), Chirobiotic V, R and T(Astec), and TBB (Eka-Nobel). An achiral silica column(Kromasil, Eka-Nobel) was also included in the SFCscreening system as a means of identifying the presence of achiral impurities. All screening columns were of a standard25-cm length and 4.6-mm inner diameter. The two systemsare run in parallel, and employ a standard gradient methodwith a flow rate of 1.5 mL/min, an outlet pressure of 200bar, an oven temperature of 35  ° C, UV detection at 215 nm,and a mobile phase of 4% MeOH in CO 2  for 4 min,increasing to 40% MeOH over 18 min with a hold at 40%MeOH for 3 min and a 5 min post time. Initial screening of  3  revealed very poor chromatographic peakshapes; thus, anidentical screen utilizing a 25 mM solution of isobutylaminein MeOH as the polar modifier was performed. The resultsindicated that Chiralpak AS afforded the best separation of the enantiomers of   3 . Semi-preparative SFC enantiopurityupgrade of 290 mg of an enriched mixture of substitutedpiperazine,  3 , was performed using a Berger Instrumentsmultigram preparative SFC instrument. Conditions: Chiral-pak AS (20 mm  ×  250 mm), 12% (25 mM isobutylaminein methanol)/carbon dioxide, 100 bar, 35 - C, 50 mL/min,UV 320 nm, with a typical injection of 1 mL @ 60 mg/mL. Preparative HPLC Purification of 1.  Preparative HPLCupgrade of purity of   1  was carried out using a Varianpreparative HPLC system with an 8-cm i.d. ProChrom DACcolumn packed with Kromasil 10  µ m silica operating at aflow rate of 300 mL/min with an eluent of 88.5:10:1.5 IPAc/ MeOH/H 2 O and using UV detection at 290 nm. A typicalinjection consisted of 10 mL@180 mg/mL (1.8 g) with arun time of about 12 min. Analytical HPLC assay condi-tions: Kromasil 5  µ m, (4.6 mm  ×  250 mm); 2 mL/min;88.5:10:1.5 IPAc/MeOH/H 2 O, UV 254 nm. Pilot-Scale HPLC Purification of 2.  Pilot-scale HPLCupgrade of the purity of   2  was carried out using a Varianpreparative HPLC system with a 6-cm i.d. NovaSep ProChromDAC column packed with 600 g of Amicon grade 631 silica(18  µ m, irregular); operating at a flow rate of 300 mL/minwith an eluent of 90:10 IPAc/MeOH and using UV detectionat 320 nm. A typical injection consisted of 40 mL@ 38%crude  2  in IPAc with a run time of about 10 min. Large-Scale HPLC Purification of 2.  Large-scale HPLCupgrade of the purity of   2  was carried out using a Biotagepreparative HPLC pumping skid with a 30-cm i.d. NovaSepProChrom DAC column packed with 14 kg of Amicon grade631 silica (18  µ m, irregular; packed column bed length ca.42 cm) operating at a flow rate of 7500 mL/min with aneluent of 90:10 IPAc/MeOH and using UV detection at 320nm. A typical injection of crude feed solution contained about 190  •  Vol. 8, No. 2, 2004 / Organic Process Research & Development
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