COVER STORY
MULTIPLICITY Combinatorial chemist performs parallel fractionation at Albany Molecular Research.
COMBINATORIAL CHEMISTRY Advances in synthesis, purification, and analysis further refine the combinatorial approach, now a mainstream tool in drug discovery STU BORMAN, C&EN WASHINGTON
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OMBINATORIAL CHEMISTRY—A TECHNOLOGY FOR SYN-
thesizing and characterizing collections of compounds and screening them for useful properties— was conceived about 20 years ago. Initially, the field focused primarily on the synthesis of peptide and oligonucleotide libraries. In the 1990s, the focus of the field changed predominantly to the synthesis of small, druglike organic compounds. And many HTTP://PUBS.ACS.ORG/CEN
pharmaceutical companies and biotechnology firms now use it in their drug discovery efforts. Questions arisefromtime to time about whether combinatorial chemistry has proved its mettle. Has it done any good? Have drugs been discovered with it that perhaps couldn't have beenfoundany other way? And if combinatorial chemistry is such a great idea, why hasn't the concept been recognized with a Nobel Prize? Many compounds discovered combinatorially, including a couple mentioned in this article, have at least entered preC&EN / NOVEMBER 1 1 , 2002
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COVER STORY
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BIODIVERSITY Porco, coprincipal investigator James Panek, and coworkers at Boston University's new CMLD facility used a distannoxane transesterification catalyst to cyclodimerize in a single step an amino acid- and polyketidecontaining hydroxy ester monomer to a 22-member macrodioUde—a compound that approaches the complexity of macrolide and macrodioUde antibiotics.
clinical or clinical trials. That's some proof of the value of combinatorial chemistry. But the bottom line is that many researchers in academia, industry, and government already recognize it as an integral component of the drug discovery repertoire. Not that there's no point in scouring the rainforest for interesting natural products or designing drugs by "rational" means. Both are still done. It's just that the com-
including the advent of carbohydratebased libraries. • An increased focus on "phase trafficking" techniques aimed at integrating synthesis with purification. • Novel strategies for purification and analysis, such as the combinatorial use of supercritical fluid chromatography • And the application of combinatorial chemistry to new targets, such as nuclear receptors. These and other developments were discussed at "Combinatorial Chemistry: Conventional Tools from RevolutionaryTechnology" an American Chemical Society ProSpectives conference held in Leesburg, Va., in September. The ACS ProSpectives program is a series of small conferences oriented toward industry scientists. The conference cochairs were Andrew P. C 0 C H AIR S Combs (left) and Hodges. Combs, head of a directed parallel synthesis group and binatorial approach is now used routinely a central nervous system medicinal chemto modify compounds from the rainforest istry project at Bristol-Myers Squibb, or as a complement to rational design. Wilmington, Del., and John C. (Jack) Combinatorial chemistry has become Hodges, senior director of cardiovascular established. It has changed drug discovery chemistry at Pfizer Global R&D, Ann ArIt's a mainstream tool many drug compa- bor, Mich. nies would not want to do without. And as far as the Nobel Prize goes—it could DIVERSITY-ORIENTED SYNTHESIS. Rehappen. searchers today are showing "how one Meanwhile, researchers continue to find might consider combining natural prodways to further enhance the capabilities of uct synthesis with combichem," Hodges combinatorial chemistry, including these said. "In the past, combichem has largely developments: focused on simple tried-and-true synthet• Agrowing trend toward the synthesis ic sequences, whereas the natural prodof complex natural-product-like libraries, uct route tends to be infinitely more
complicated and creative. Combichem is now growing into those more complex realms"—an approach called diversity-oriented synthesis. "Diversity-oriented synthesis is taking on a new level," Combs added. "People are advancing it to the point where theyVe going after much more complex syntheses than in the initial years of combinatorial chemistry. They're tackling compounds with multiple stereocenters and very complex natural-product-like libraries, and that just wasn't happening three years ago." Efforts to advance complex highthroughput synthesis got a boost this year from grants for two new Centers of Excellence in Chemical Methodologies & Library Development (CMLDs). At the ACS ProSpectives conference, the two principal investigators on those grants—chemistry professor Peter Wipf of the University of Pittsburgh and assistant professor of chemistry John A. Porco Jr. of Boston University—described current studies that exemplify the type of diversity-oriented synthesis projects that in the future will be carried out at their two centers. Wipf and coworkers at Pitt have been synthesizing analogs of the complex natural product curacin A, an antimitotic agent from blue-green algae, as potential leads for anticancer drugs. They have identified library compounds that exceed the potency and selectivity of the natural product. And Porco and coworkers at BU recently used combinatorial synthesis to rapidly synthesize highly functionalized angular structures from chiral epoxyquinol dienes. They also developed a reaction sequence in which polyketide- and amino acid-containing hydroxy esters were cy-
Researchers are showing how one might consider combining natural product synthesis with combichem.
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AUTO CARBS Seeberger's automated oligosaccharide synthesizer (shown here in close-up) is a modified peptide synthesizer. clodimerized catalytically to form 22member ring compounds that approach the complexity of macrolide and macrodiolide antibiotics. "We are fundamentally interested in
transformations where molecular com plexity is generated rapidly," Porco said. "Future efforts will involve manipula tion of variables such as ring size and stereochemistry to produce large num
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bers of complex macrocycles using cyclooligomerization reactions." Natural-product-like libraries are also being constructed by postdoc Glenn C. Micalizio and coworkers in chemistry and chemical biology professor Stuart L. Schreiber's group at Harvard University The researchers vary the structure ofscaf folds—central structures on which a vari ety of functional groups are added or modified—to create diverse libraries. Combinatorial chemists have more typi cally varied the functional-group "decora tions" on scaffolds, rather than the scaf folds themselves, to generate libraries. Micalizio, grad studentJohn P. Rearick, and coworkers recently created a complex natural-product-like library by using a branched network of boronic ester annulation reactions on a varied set of aldehyde scaffolds. The boronic ester annulations produced a range of boronic acid inter mediates that were subsequendy modified into functionalized enones, trisubstituted allenes, enediols, tetraols, polycyclic heterocycles, and 1,3-diols. 'There's been a gradual paradigm shift from the original idea of combichem,
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where you would take a single scaffold and make 10,000 or 20,000 compounds around it," Hodges said. "If you have lots of scaffolds with lots of functional groups on each one, diversity goes a lot further." Associate professor Peter H. Seeberger and coworkers at Massachusetts Institute of Technology are bringing difficult-tosynthesize carbohydrates into the combinatorial fold. The researchers use automated solid-phase oligosaccharide synthesis to create large collections ofcomplex carbohydrates from monosaccharide building blocks. Seeberger's automated carbohydrate synthesizer delivers solvents and reagents to a reaction chamber where sugar building blocks are combined with supportbound carbohydrates in a programmed manner. After several cycles, oligosaccharide products are born. Seeberger described a strategy for using the synthesizer to make libraries of heparin-like glycosaminoglycans. Glycosaminoglycans are involved in growth factor and receptor interactions, blood coagulation, and plaque formation. New glycosaminoglycan agents created combinatorially might therefore prove useful as medications for cancer, viral infections, heart disease, and neurodegenerative diseases, among other conditions. Up to now, Seeberger and coworkers have validated their synthetic approach by constructing only a small number of glycosaminoglycan variants in solution phase. But their first automated runs are under way and the researchers are simultaneously pursuing a second research goal—the solid-phase synthesis of heparin chips (heparin combinatorial arrays). "Ofall the talks I saw at the conference, I would say that Seeberger's wowed me the most," Hodges said. "To have simplified and automated carbohydrate chemistry as well as he has is absolutely remarkable.,, The work could eventually lead to a "machine that would make different carbohydrates in different wells" of a combinatorial array, he said. "Seeberger has done what nobody had been able to accomplish to date," Combs said. "By automating the synthesis of oligosaccharides, he took a job that once required years of a chemist's time and reduced it to days on a solid-phase synthesizer. That's quite an accomplishment. It's the carbohydrate equivalent of the solidphase synthesis of peptides"—the technique developed in the 1960s by biochemistry professor and Nobel Prize winner R. Bruce Merrifield of Rockefeller University HTTP://PUBS.ACS.ORG/CEN
Seeberger's and Micalizio's work "shows how far diversity has come since the early days ofsynthesizing libraries," Combs said. This progression reflects the growing influence of major academic synthetic organic chemistry groups on the art of combinatorial synthesis, he noted. Studies by professor of medicinal chemistry Ron Grigg and coworkers at the University of Leeds, in England, also exemplify this trend. "From the perspective of a real hard-core synthetic organic
person, Ron has been able to show how different types of cascading cyclization reactions can be brought to bear on diversity-oriented synthesis," Combs said. A cascade is a series of reactions that occur spontaneously "Ron uses cascading cyclizations to make some very complex molecules in very few steps," Hodges noted. "In a parallel synthesis, the more steps there are, the harder it is to get all your reactions to work. He has this amazingly fun set of multi-
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COVER STORY It took 3 7 days to develop the library thercomponent and multiring-forming reac- microwave oven heating in combinatoritions that get a lot done very quickly " An al synthesis. One scientist used a mi- mally and two days to develop it with the miexample is a novel palladium-indium dia- crowave system to develop a combinato- crowave oven—about an 18-fold productivstereoselective cascade allylation reaction rial library, and another used conventional ity increase. Sarko and coworkers calculated developed recently by Grigg's group, in thermal heating. The two scientists were the return-on-investment time (break-even which imines were converted to a diverse not permitted to communicate on meth- point)foradopting microwave synthesis to set ofN-tosyl and N-aryl homoallyl amine ods and results. The goal of the experi- be 5.8 months. According to Sarko, major products [Chem. Commun., 2002,1372}. ment was the generation of three diverse vendors of laboratory microwave heating Microwave-assisted synthesis can also library compounds with a liquid chro- equipment include Milestone (Sorisole, improve combinatorial productivity Us- matography-mass spectrometric (LC-MS) Italy), Personal Chemistry (Uppsala, Sweden; C&EN, Feb. 11, page 17), and CEM ing a laboratory microwave oven, "reac- purity exceeding 85%. _ Corp.OM^tthews,N.C.). tions that took days can ï 'The nice thing about be done in minutes, usu1 microwave heating is that ally with higher yields and Grubbs you can do synthesis oppotentially easier workcatalyst timization really fast," ups," said senior principal Hodges commented. irVbu research scientist Christocan try, say, 20 different Monomer pher Sarko of Boehringer reagent ROMPgel reagent (soluble or insoluble) conditions for the same Ingelheim Pharmaceuti(soluble) reaction in a matter of cals, Ridgefield, Conn. hours, rather than weeks. "The days of the old oil COMBINATORIAL ROMP Ring-opening metathesis And then once you get the bath"—a traditional lab polymerization of a derivatized norbornene (shown) or other soluble right synthesis method, apparatus for heating remonomer is used to create a ROMPgel reagent, which can be soluble you can use the same inactions—"are behind us." or insoluble. The monomer can be polymerized after it's been used in strument and go kind of Sarko and coworkers a combinatorial reaction and then filtered from solution—a process one at a time to build a liconducted an experiment called impurity annihilation—or the monomer can be polymerized brary. Nobody yet has a to determine the time first and then used as a supported reagent in parallel synthesis. parallel-synthesis microthat can be saved by using
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he University of Pittsburgh and Boston University already had combinatorial chemistry research centers. But now they're going to have better ones, thanks to grants announced last month by the National Institute of General Medical Sciences, Bethesda, Md.
B U S CMLD Instrumentation in the new facility includes Quest 205 and 210 parallel synthesizers (in the vented hoods) and a Waters prep high-performance liquid chromatography system (right).
wave device that would let you run, say, 96 microwave-irradiated reactions all at once. That may come someday But in the meanwhile, microwave ovens are so fast that you can run serial reactions and still get a lot done. It's a new and coming technology in combinatorial chemistry" Another emerging concept is the use of microreaction devices for miniaturizing combinatorial synthesis and screening. By reducing the space in which reactions take place, microreaction devices can make it fester and easier to optimize synthetic yield and selectivity evaluate new reaction pathways, and carry out screening procedures, according to Managing Director Wolfgang Ehrfeld of Ehrfeld Mikrotechnik, Wendelsheim, Germany Characteristic dimensions of microreaction technology range from the submillimeter to submicrometer range, compared with centimeters or more in conventional reactions. 'There's been lots of talk of microreaction technology," Combs said, "but I think Ehrfeld gave a convincing talk that this is actually going to be emerging soon." PHASE TRAFFICKING. The most familiar way to synthesize most organic compounds HTTP://PUBS.ACS.ORG/CEN
NIGMS has awarded a total of more than $5.5 million to the two institutions over the next year to convert their combinatorial facilities into Centers of Excellence in Chemical Methodologies & Library Development (CMLDs). The two grants together are expected to total more than $20 million over five years. The purpose of the grants is to facilitate research on "new, highly efficient methods for synthesis, separation, purification, and analysis that will accelerate the creation of libraries and enable a broader range of structures to be made," NIGMS Program Director John M. Schwab says. Pitt's new CMLD, directed by chemistry professor Peter Wipf, will focus on methods for peptidomimetic library synthesis, the use of fluoropolymer-based microreactors for nanoscale parallel organic synthesis, and library syntheses based on fluorous phase separation. "We're not just making small variations on chemistry that's already published," Wipf says. "We're trying to explore new techniques and new strategies to make molecules that look very different from
what you can purchase or find elsewhere." Assistant professor of chemistry John A. Porco Jr., director of BU's CMLD, says it will specialize in stereochemical and positional diversity in library synthesis and in the asymmetric synthesis of complex natural-product-like libraries. Porco and three other BU chemistry faculty members, each with a different subspecialty in the field of chemical synthesis, will carry out research in the center. "We expect that the BU CMLD will become an epicenter of chemical research and training in the Boston area and will provide molecules of unprecedented complexity for use in biological research," Porco says. The CMLD grants are "substantial," comments Andrew P. Combs, head of a directed parallel synthesis group and a central nervous system medicinal chemistry project at Bristol-Myers Squibb. Pitt and BU "will now become powerhouses in diversity-oriented synthesis and the development of new methods. It will be interesting to see what they can do with the technologies developed in their CMLD labs."
is with classical solution-phase reactions, I phase trafficking. With solid-phase organin which all the reagents, intermediates, ic synthesis, it's easy to purify products by and products remain in solution. "The filtration, it's possible to do mix-and-split beauty of pure solution-phase synthesis is synthesis (a technique used to make very the large lexicon of reactions one can per- large libraries), excess reagents can be used form," explained Daniel L. Flynn, presi- to drive reactions to completion, and syndent of Deciphera Pharmaceuticals, Nat- theses can be automated easily However, ick, Mass. However, purifying products relative to solution-phase synthesis, solidformed by solution synthesis can be diffi- phase synthesis often requires more develcult. "In solution-phase synthesis, typical- opment time (to adapt familiar solutionly an organic chemist will spend 10% of phase reactions to the solid-phase milieu) the time running a reaction and 90% of and additional linkage and cleavage steps are needed (to attach compounds to supthe time purifying it," Flynn said. That's why chemists are increasingly ex- ports and later detach them). In addition, ploring phase trafficking, an alternative to solid-phase reactions tend to be slower and conventional solution-phase synthesis in product yields are more limited. A new generation of phase-trafficking which synthesis and purification are carried out more or less simultaneously In phase techniques was introduced in the 1990s, trafficking, reagents, by-products, or prod- when Hodges and Flynn developed polyucts are directed into a separate phase so mer-supported scavenging techniques that the products can be isolated easily from can be used combinatorially With these the reaction mixture. Phase-trafficking techniques, you carry out a reaction in somethods "can be mixed and matched to lution and afterward use polymer-supportenable high-throughput synthesis of a wide ed affinity agents to scavenge reagents and by-products out ofsohitioa Filtering the soarray of compounds," Flynn noted. Solid-phase organic synthesis—an ap- lution to remove the scavenger leaves the proach in which reagents or products are at- product in solution phase. In many cases, tached to solid supports such as polystyrene this method eliminates the need to purify beads—is the most traditional form of I products chromatographically C & E N / NOVEMBER
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COVER STORY
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because they areflow-throughmaterials, whereas conventional solid supports only carry active groups on their surfaces [Chem. Rev., 102,3301 (2002)}. "Typically with polystyrene reagents, one would use three or four equivalents, and the reactions are slow," Barrett said. "With ROMPgels, we would typically use about 1.2 equivalents, and the reactions are fast." One way to carry out a combinatorial reaction with a ROMPgel is to synthesize a functionalized ROMP monomer, use it as a soluble reagent to carry out a transformation, and then polymerize it. Excess reagent, whether modified in the reaction or not, thus forms a polymer that falls out of solution, whereas the desired synthetic product stays in solution. This technique— which Barrett calls "impurity annihilation"—permits solution-phase parallel synthesis to be carried out with minimal need for additional purification. ROMPgels can also be used as Afunctional reagents and as sequestration-enabling reagents (SERs). A Afunctional reagent is a soluble monomeric reagent with both chemical reactivity and phasetrafficking components. And an SER reacts with an impurity that's difficult to remove and converts it to a sequesterable species. It's also possible to make relatively short (20-60 unit) ROMP reagents, which are soluble in many traditional organic solvents but can be readily precipitated from solution using methanol or ether. They therefore "retain favorable reaction kinetics associated with homogeneous solution-phase synthesis, yet require only precipitation and filteringfroma pertinent solvent as the sole purification protocol," said associate pro-
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COMBI CARBS Seeberger and coworkers are developing an automated parallel synthesis of heparin-like glycosaminoglycans. Each of four glucosamines is added to a glucuronic or iduronic acid, forming eight disaccharides. The disaccharides are modified and combined, and the resulting oligosaccharides are deprotected and sulfated.
Another phase-trafficking technique currently being developed by several research groups is the use of ring-opening metathesis polymerization (ROMP) to create ROMPgels—soluble or insoluble oligpmeric or polymeric reagents and scavengers that can be used in combinatorial synthesis. ROMPgels are made by using Grubbs catalysts to carry out ROMP reactions on strained alkenes such as norbornene and 7-oxanorbornene. Functional groups on the alkene monomers are the active agents used to mediate combinatorial reactions or cleanups. "I can make compounds on a ROMPgel that you can't make on polystyrene supports," said organic chemistry professor Anthony G. M. Barrett of Imperial College of Science, London. And die activity and reaction rate of ROMPgels are high 50
C & E N / NOVEMBER
1 1 . 2002
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fessor of chemistry Paul R. Hanson of the cessible by this route. But the technique libraries of 100 to 1,000 compounds by University of Kansas. has now been adapted to moderate-size Curran; Wei Zhang of Fluorous TechIn fact, "there's a whole myriad of applications" of ROMPgel technology, said Flynn, who recently reviewed its various permutations [Curr Opin. Drug Discovery Dev., 5,571 (2002)}. "It's a liberal system and a very forgiving one," he said. "One simply makes the choice—When do you want to introduce the reagent or scavenger, and when do you want to get rid of it?* Soluble ROMP reagents are related to a liquid-phase combinatorial synthesis strategy developed in 1995 by chemistry professor Kim D. Janda and coworkers at Scripps Research Institute. In that technique, synthesis is carried out on a soluble polyethylene glycol (PEG) support, and the polymer is precipitated out of solution at the end of the reaction by changing the solvent. 'Although PEG is a very powerful and effective soluble support, its limitations, including low loading [low density of active groups}, have driven efforts toward the development of new designer polymer systems," Hanson said. ROMP reagents are higher loading than PEG reagents because each monomer unit is derivatized with a "At the Buck Institute, wefocuson the study of aging mechanisms, functional group, whereas each PEG polyand In doing so, we are looking towards the development of mer is only derivatized at its end, Hanson strategies to detect, prevent and treat disease such as noted. Ater^rner's, Parklnsorfs, cancer and heart disease. Using StereoGraphlcs* CrystalEyes®3 enables me to accurately Another promising phase-trafficking visualize molecular mechanisms as they truly exist approach is fluorous mixture synthesis, expediting my research and allowing me to get one step a technique developed by chemistry procloser tofindinga cure." fessor Dennis P. Curran and coworkers at Gabriel del Rio, Ph.D. the University of Pittsburgh. Fluorous Staff Scientist, Buck Institute mixture synthesis makes it possible to run reactions on a number of fluoroustagged starting materials and then easiFor over 20 years, scientists have relied upon Stereo3D™ products from StereoGraphlcs, me leader In stereoscopic visualization technologies. ly separate and identify the reaction For desktop and projected stereo Images and animations, rely on the products. professional's choice - CrystalEyes and ZScreens*. First, fluorous tags of different chain lengths are added to a series of starting materials. The tagged substrates are mixed ^SCREEN' and taken through a multistep reaction sequence. Fluorous chromatography is then used to separate the tagged product mixtures, based on the fluorine content of the For More Information: different tags. Finally, compounds in the call toll free 866-455-1490, separate solutions are "detagged" to release visit www.stereographics.com/sm/ the final products. or email
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COVER STORY
CASE S T U D Y
'A Pharmaceutical Company Would Never Do This'
A
t the University of Pittsburgh, researchers have been working on a combinatorial project that drug firms would consider too much of a gamble, according to the project's principal investigator, chemistry professor Peter Wipf. He described the project—the pursuit of analogs of the natural product curacin A as potential anticancer lead compounds—at the recent ACS ProSpectives conference on combinatorial chemistry. Curacin A—first isolated by professor of natural products chemistry William H. Gerwick and coworkers at Oregon State University, Corvatlis—is an antimitotic algal natural product that acts by inhibiting tubulin polymerization in cells. It has an impressive antiproliferative profile, Wipf said, but its lability (it has a readily oxidized thiazoline heterocycle) and its high lipophiticity have been strong deterrents to its use as a lead compound for drug discovery. In addition, "the target of that natural product is the colchicine site on tubulin, which is not a validated site—that is, there's no clinically used drug that acts at that site," Wipf said. Nevertheless, Wipf, grad student Jonathan T. Reeves, and coworkers used combichem and rational design to identify analogs that exceed the potency and selectivity of curacin A. "A pharmaceutical company would never do this," Wipf said, "because it's way too risky. The question for us was, could we actually demonstrate the utility of doing something
because the syntheses were long," he said. So the researchers decided to use an efficient mixture synthesis technique to make second-generation analogs. They used a fluorous scavenging approach to streamline purification of the mix£ tures and then used liquid chromatography-nuclear magnetic reso\ 0CH, nance spectroscopy "to ensure we £ had made what we claimed and to s evaluate the purity," Wipf said. Over£ alt, the mixture synthesis approach i increased their productivity sixfold ° relative to the first-generation (+)-Curacin A process. One second-generation analog turned out to be an interesting lead compound. In fact, "it was one of the most potent synthetic curacin A analogs identified to date, and it also had a simpler structure, greater water solubility, and better chemical stability" than the natural product, Wipf said. Starting with that lead compound, Second-generation he said, "we played around with the analogs central part in a combinatorial sense by changing motifs and making a He = heterocycle number of derivatives." This resulted in a bunch of inactive compounds. But additional rational changes in the scaffold eventually led them to a hot prospect. Its activity in a tubulin polymerization assay "was remarkCHoO able in that its IC50 [the concentration Second-generation at which it inhibited 50% of tubulin lead compound 0CH3 polymerization activity] was 170 nM," Wipf said. "This was clearly superior to that of curacin A, which is 520 nM. It was the most attractive analog of curacin A reported to date." Mouse studies unfortunately ONLY ACADEMIC? Wipf and CH30 ^ ^ OCH3 Analog that's more active showed that the compound was coworkers have created several OCH3 than curacin A but metabolically unstable metabolically unstable. However, "we generations of curacin A analogs in an effort to develop potential were able to get useful information anticancer agents. Shown are the on what the first metabolites are," Wipf natural product and two sites (blue) said, "and we think we've identified the enWipf and coworkers began by synthethe researchers modified when they sizing a first-generation library of curacin zyme that causes the degradation. Now synthesized a first-generation analog A analogs U Med Chem., *5,1901 (2002)]. we're going to build that information back library, a second generation library "We tried three different approaches to into a new library to address the issue of they created by mixture synthesis, a make those—solution, solid support, and metabolic instability." lead compound from that library that fluorous synthesis," Wipf said. They Once they solve that problem, perhaps showed good activity, and an analog screened about 100 of the analogs for ac- a company might want to take over the derived from that lead that exhibited tivity, and found that most were complete- project, Wipf said. But ironically, at that better activity than curacin A on a ly inactive. "Also, in the first-generation point "we would kind of lose interest in it. tubulin polymerization assay but was approach, we were not able to produce the It would be completed for us. It's a very metabolically unstable. quantities of materials we realty wanted to academic approach."
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that companies would totally reject out of hand? We are trying to show there is value in a type of combinatorial project that companies do not consider at all valuable."
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nologies, Rttsburgh; and coworkers. 10,000-member AGONIST AND ECSTASY Collins and They recently used fluorous carboxylic acid library coworkers identified the peroxisome mixture synthesis to prepare a proliferator-activated receptor (PPAR) Screen against 560-member library of analogs of PPAR8 agonist GW501516 by screening a 10,000the natural product mappicine member library against PPAR8 and \J. Am. Chem. Soc, 124, 10443 synthesizing analogs of an active compound (2002)}. The procedure required (GW8547) found in the screen. Molecular 90 reactions, compared with 630 model shows how GW501516 interacts for a corresponding parallel synwith three key PPAR8 residues (His = thesis. And the separation phase histidine, Thr = threonine, and Tyr = of the procedure required only 80 tyrosine). chromatographic steps, whereas GW85A7 560 would have been needed with Structure-based parallel synthesis. analog synthesis A new medium for solid-phase organic synthesis was also dis0 cussed at the conference. Combinatorial solid-phase organic HO synthesis requires the use of multiple aliquots of loose resin beads, CF3 which can be difficult to handle and keep track of. A number of 6W501516 technologies have been designed to make it easier to manipulate and Pines InstituteforMolecular Studies, San International, which uses "kan" microretrack solid-support beads, including the Diego, in which small mesh bags are used actors containing resin and miniature encoding devices. to hold aliquots of resin. following: • SynPhase Lanterns, offered commer• Tea bag synthesis, developed by • Directed sorting technology, marketRichard A. Houghten, president ofTorrey ed by the Irori unit of Discovery Partners cially by Mimotopes, in Clayton, Australia,
O
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The Seventh Annual GlaxoSmithKline Chemistry Scholars Award The US Chemistry Division of GlaxoSmithKline is pleased to announce the 2003 Award Winners: Professor Justin Du Bois Stanford University
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Professor Tomislav Rovis Colorado State University
Professor Brian Stoltz California Institute of Technology
Past award winners: 2002 Peter Seeberger Matt Shair Ross Widenhoefer
2001 2000 David MacMillan Jim Leighton James Morken Scott Miller Fraydoon Rastinejad Yian Shi
1999 P. Andrew Evans David Gin Marc Snapper
1998 1997 Carolyn Bertozzi Gregory Fu Kevan Shokat David VanVranken Keith Woerpel John Wood
The GlaxoSmithKline Chemistry Scholars Award is a two year unrestricted research grant awarded to exceptional young faculty in the fields of organic, bioorganic, medicinal, or structural chemistry.
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C&EN / NOVEMBER 11, 2002
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COVER STORY in which reactive sites are grafted on solid polymer surfaces. Now, Polymer Laboratories, Amherst, Mass., has introduced StratoSpheres, rigid foamlike plugs in which resin beads are immobilized within an inert porous polyethylene matrix. The plugs "entrap a predetermined quantity ofresin in such away that it is free to react but is otherwise constrained inside the porous matrix," explained Polymer Laboratories Vice President Aubrey Mendonca. "They generally have significantly higher levels of functional group loading compared with equivalent-sized grafted or container devices." A future generation of StratoSpheres will have a broader range of shapes and dimensions and the capacity for encoding, he noted.
binatorial purification and analysis continue to advance. By screening compounds for activity as mixtures, researchers can cover a lot of ground quickly In fact, screening of mixtures is the traditional method used to evaluate compounds extracted from natural sources, such as microbes and plants. However, when compounds are synthesized and screened as mixtures, "usually, not only are you screening the things you intended to make, but you're screening a lot of debris that's in there with it," Hodges said. "That really muddies the waters as to what's active at the end of the day That's one of the things that gives screening of mixtures a bad name." At the conference, associate professor Michael G. Organ of "York University, Toronto, proposed one solution to this probPURIFICATION AND ANALYSIS. Combi- lem—the use of frontal affinity chronatorial chemists already have in hand ef- matography-mass spectrometry (FACfective techniques for purifying many types MS) to screen mixtures. of compound collections and for quantiFAC separates a mixture of compounds tating their purity, but some gaps still need in a mobile phase by the compunds' affinto be filled. Speakers at the ACS ProSpec- ity for an immobilized enzyme or other tives conference addressed some of these target, and electrospray MS monitors ions needs and discussed ways in which com- generated from each separated compo-
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nent. Together, they make it possible to separate and identify compounds in the mixture that bind strongly to a target. "Thus, the speed advantage of assaying mixtures can be realized while still treating compounds individually," Organ noted. 1 A big trade-off with mixture screening is that you usually have to deconvolute— go back and remake each active compound individually" Combs said. "With FAC-MS, you don't." Researchers at ChemRx, South San Francisco, a unit of Discovery Partners International, have developed an Accelerated Retention Window (ARW) method that rapidly cleans up large combinatorial libraries to 90% purity or greater. ARW calculates the LC solvent gradient composition that would be needed to elute a compound by preparative LC at a predetermined accelerated retention time. Using this setting makes it possible to collect the compound efficiently as a clean individual fraction. 'The ARWprocess has been used in the purification of more than 180,000 druglike compounds in the last 24 months," said ChemRx Vice President and General
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COVER STORY Manager Nathan Collins. "Compounds were produced and purified to meet client specifications in 2- to 50-mg quantities at greater than 90% purities." Another group has developed a set of techniques that make it easier to use LCMS to purify parallel-synthesis libraries. Effective LC-MS purification conditions can be hard to work out, and generic LCMS system settings are frequently problematic. This is particularly evident in labs where synthetic chemists carry out their own separations without assistance from chromatographic specialists. Senior research scientist Karl F. Blom and coworkers at Bristol-Myers Squibb have now developed an LC sample-loading technique called "at-column dilution" that aids preparative LC ofparallel libraries and a method-optimization protocol that facilitates LC-MS analysis of the library compounds \J. Comb. Chem., 4, 295 (2002)}. The at-column dilution technique was adapted in part from an approach reported earlier by Thomas E. Wheat of Waters Corp., Milford, Mass.The two techniques establish specific LC conditions and MS fraction collection parameters for each sam-
RESIN PLUG In Stratosphere plugs (left), synthesis beads are immobilized in a flow-through polyethylene matrix (shown here at 50X magnification).
pie in an automated way, using customized software. Together, they improve chromatographic resolution by about a factor of three, compared to generic or universal prep LC methods, and increase the relia-
bility and effectiveness of the MS analyses. An emerging technology for purification of combinatorial libraries is supercriticalfluidchromatography (SFC). 'The lore that SFC can only analyze a small per-
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centage of the structural types HPLC can handle has proven to be totally invalid for druglike molecules," said LarryTruesdale, director of combinatorial chemistry technologies for Pfizer, San Diego. "SFC is generally superior to HPLC due to its milder chromatographic environment and the greater number of theoretical plates available." The low cost of SFC solvents (such as CO2) and waste disposal is also advantageous. "We estimate the cost of running SFC is about half the cost of HPLC," Truesdale said. "This difference is very important when one is doing hundreds of thousands of separations a year." SFC instrumentation is expensive, but Truesdale noted that costs could plummet as the technology matures. "SFC is obviously a direction that a lot of pharmaceutical companies are going to want to take," Combs commented.
drome X[Proc. Natl. Acad. Set. USA, 98, 5306(2001)}. 'The utilization oftarget-biased library design and solid-phase parallel array synthesis were critical in accelerating the discovery of GW501516 as a potent, selective PPAR8 agonist," Collins said. The work "exemplifies the potential utility of combinatorial chemistry in accelerating drug discovery" According to Combs: "Collins gave one ofthe most beautiful presentations on how
APPLICATIONS. One combinatorial application discussed at the ACS ProSpectives conference was the discovery of an inhibitor for NS3-4A protease, an enzyme produced by the infectious hepatitis C virus. Scott Harbeson and fellow medicinal chemists at Vertex Pharmaceuticals, Cambridge, Mass., used solidphase mix-and-split synthesis to prepare a combinatorial library of peptidyl aldehydes for rapid mapping of binding subsites on the protease. "We then prepared small, focused libraries of discrete compounds to elaborate the structure-activity relationships for subsites of particular interest," Harbeson noted. This led to the identification of VX-950, an orally dosable candidate for treatment of hepatitis C viral infection, which is now in preclinical development. Recent combinatorial studies at GlaxoSmithKline, ResearchTriangle Park, N.C., have focused on peroxisome proliferatoractivated receptors (PPARs) and other ligand-activated transcription factors in the nuclear receptor protein superfamily Jon L. Collins and coworkers at GlaxoSmithKline made a 10,000-member carboxylic acid library of potential PPARtargeted agents. The library was screened against PPAR8, and an active pool ofcompounds was deconvoluted to find an active agent, called GW8547. Optimization of that compound using structure-based analog synthesis led to the identification of GW501516 as a potent, selective PPAR8 agonist. The compound is now a clinical candidate for treatment of cardiovascular problems associated with an "orphan disease" called metabolic syn-
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