when process chemists meet
TRANSCRIPT
SCIENCE & TECHNOLOGY
WHEN PROCESS CHEMISTS MEET Pharmaceutical researchers compare notes, share experiences at ACS Prospectives conference STEPHEN K. RITTER, C&EN WASHINGTON
PROCESS CHEMISTRY HAS BEEN
described as the interface between organic chemistry and business: Pharmaceutical process chemists serve as the ar
chitects that make the connection between the two, building a bridge from drug discovery to large-scale production.
"The role of process chemists is to develop a safe and cost-effective process to prepare a drug substance that can ultimately be used for commercial production," commented Judith H. Cohen, a research scientist at Johnson & Johnson Pharmaceutical R&D in Spring House, Pa. Cohen spoke at the ACS Prospectives conference titled "Process Chemistry in the Pharmaceutical Industry," held last month in San Juan, PR.
"Process chemists generally begin work on a new process by evaluating an existing synthetic method—usually provided by drug discovery—that may or may not be suitable for scale-up," she said. "Our goal is to eliminate any hazardous reaction conditions, reagents, and solvents. We also have to look at the practicality of the process—overall yield, isolation, and purification. And we need to do all of this while minimizing costs."
During the past decade, process chem
istry has come to be identified as a sub-discipline within chemistry for this vital role that it plays. One sign of the growing recognition of process chemistry was the launch in 1997 of the American Chemical Society's journal Organic Process Research & Development. Another sign has been the organization of small conferences for process chemists. These include Gordon Conferences and, in San Juan, the second Prospectives conference on process chemistry; the first conference was held last year in Barcelona (C&EN, May 27, 2002, page 53).
Some 90 industrial chemists representing pharmaceutical companies and related firms came together in Puerto Rico to share their ideas and experiences on the drug development process. Because part of the success of process chemistry depends on a working relationship with organic chemists in academia, who train new process chemists and serve as a source for new reactions, about a dozen academic chemists attended the conference as well.
By design, the conference provided the process chemists an opportunity to network in an informal environment—and within earshot of the hotel's lively casino. The choice of Puerto Rico for the conference location allowed attendees to double
PRODUCTIVE WATERS Condado Beach in San Juan provided an ideal setting for process chemists to meet and discuss their role in drug development.
up their attendance with visits to colleagues at company manufacturing sites on the island. Many of the 16 invited speakers used a case-study approach to review nonproprietary chemical advances involved in developing active pharmaceutical ingredients, and the attendees appreciated the candor of the speakers in outlining successes and pitfalls in their work.
FOR EXAMPLE, one case study Cohen presented was the process development of elarofiban, a piperidinyl-substituted pyridinepropanoic acid \J. Med. Chem., 42, 5254 (1999)]. The drug is an oral fibrogen receptor antagonist now in clinical trials to treat thrombosis, which is formation of blood clots in vessels associated with heart attack or angina.
Columbia University chemistry professor Ronald Breslow delivered the conference's keynote address, which focused on his group's efforts to develop artificial enzymes to perform selective reactions. This process, which he termed biomimetic chemistry more than 30 years ago, has been broadly extended by the research community to describe all aspects of chemistry in which new processes are inspired by biological systems.
One of Breslow's goals in pursuing enzyme mimics is "to liberate chemistry from the tyranny of functional groups," he said. Wha t he means is to design new compounds that function as enzymes but have a specificity for a functional group, unsaturated carbon, or even a saturated carbon while leaving other, perhaps normally more reactive, functional groups alone.
In organic synthesis, this regioselectiv-ity is typically achieved by blocking a reactive position with a protecting group or by activating a nonreactive position by changing a functional group, Breslow noted. By contrast, in biochemistry regiose-lectivity is achieved as a result of the geom-etry of an enzyme-substrate complex, which can override the normal activity pattern of the substrate, he said. Because this geometric control is commonplace in enzymatic reactions, fermentation methods can be used in manufacturing once the appropriate enzyme is found.
One of the prospects described by Breslow was his group's development of a modified synthetic polymer as a transaminase
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mimic. Breslow, graduate student Lei Liu, and undergraduate Mary Rozenman have attached the coenzyme pyridoxamine to a modifiedpolyethylenimine [J.Am. Chem. Soc, 124,12660 (2002)}.
The commercially available polymer has been shown to be an effective hydrolysis catalyst on its own, Breslow noted, while pyridoxamine is known to catalyze transaminations between a keto acid and an amino acid. The polyediylenimine-pyri-doxamine combination was used to convert pyruvic acid to alanine with a 10,000-fold rate enhancement compared with pyridoxamine alone, and to convert in-dolepyruvic acid to tryptophan with a 240,000-fold rate enhancement, Breslow said.
THE GEOMETRIC BENEFIT of the polymer is that it surrounds the coenzyme with strongly acidic (protonated nitrogen) and strongly basic (unprotonated nitrogen) sites, which perform the catalytic proton transfers in the transamination process, he explained. The polymer also provides a hydrophobic pocket in which the chemistry can occur, even if water is the solvent. The additional boost in rate for indolepyruvic acid comes from it being hydrophobic and binding in the hydrophobic pocket, whereas pyruvic acid does not, Breslow pointed out.
"This is a potential approach to try to do chemistry in a slightly different way," Breslow concluded. "I don't know if it will be practical or not, but we will see if it can perform other enzymelike functions. However, I think we still have a long way to go before we can routinely make artificial enzymes."
Breslow's subject was timely for the pharmaceutical process chemists assembled in San Juan. Processes involving microbial fermentation and mammalian cell cultures currently are only being used to a small extent to prepare active ingredients for pharmaceutical and agricultural chemicals (C&EN, Feb. 17, page 55). Yet one of the challenges for process chemists is to design production routes that are economically and environmentally sound. If a single-step reaction or a route that requires only a few steps using an enzyme or enzyme mimic could replace a multistep reaction or a total synthesis, that could be a boon for the company in terms of time and costs saved.
Most companies, however, are still in the wait-and-see mode when it comes to biomanufacturing because of initial infrastructure costs. Several attendees at the conference commented that most companies will want to gain more knowledge and experience before committing to large-scale bioprocessing.
The issue of awareness of the benefits of biocatalysis was addressed in a presentation by Mark J. Burk, vice president for chemical product development at Diver-sa. Burk has been speaking on the fine and specialty chemicals conference circuit during the past two years in an effort to educate chemists on the usefulness of bio-
Polyethylenimine-pyridoxamine
ENZYME COPYCAT Attaching the coenzyme pyridoxamine to a modified polyethylenimine provides an enzyme mimic that catalyzes transamination of keto acids to amino acids at an orders-of-magnitude faster rate than pyridoxamine alone.
catalysis and what's required to take advantage of the biodiversity that nature has to offer.
Less than 1% of microorganisms can be coaxed to grow well when taken out of their natural environment, Burk said, be it a hot spring, a coral reef, or the gut of an insect. Diversa scientists instead remove DNA samples from microbes and use recombinant technology to clone the enzymes in easy-to-culture microorganisms, he explained. The enzymes are then screened for their ability to provide the desired
chemical endpoint, and the promising candidates are further developed by directed evolution techniques.
The result is a set of libraries that companies can peruse when looking for an enzyme to catalyze a specific chemical transformation, Burk noted. Literally millions of enzymes are available, he said, and many will be needed since each substrate encountered will likely need a different enzyme.
"There is no sense in trying to introduce biotechnology in areas where chemistry already performs well," Burk said. "Diver-sa's approach is to try to implement biotechnology in areas where traditional chemistry struggles." Target areas for biocatalysis outlined by Burk include some types of oxidations, hydroxylations, and reductions; racemizations and isomeriza-tions; carbon-carbon bond formation; and carbon-hydrogen bond functionalization.
Burk went on to describe Diversa's work in developing some 200 nitrilases for producing enantiomerically pure intermediates in high yields, such as mandelic acid frommandelonitrile [J.Am. Chem. Soc, 124, 9024 (2002)]. Another example he presented was the enantioselective preparation of ethyl (2£)-3-hydroxy-4-cyanobu-tyrate, an intermediate in the synthesis of Pfizer's cholesterol-lowering drug Lipitor (atorvastatin).
BUT WHY, he asked rhetorically, is biocatalysis still underutilized in manufacturing despite these successes? The range of reasons he ticked off included high enzyme costs, unreliable long-term supply sources, limited number of commercial off-the-shelf enzymes that can be tested in early process development, and enzyme instability under industrial reaction conditions.
Perhaps the most important reason he cited, however, is simply lack of general awareness by chemists. "Chemists need to gain a comfort level in working with bio-catalysts and have the confidence that they are going to work," Burk said.
Echoing some of the thoughts presented by Burk, Martin R. Owen, an investigator at GlaxoSmithKline in Stevenage, England, discussed implementing effective experimental design as part of a company's development of strategic technologies, such as automated parallel experimentation systems.
A challenge for process chemists is to design production routes that are economically and environmentally sound. 34 C&EN / MARCH 1 7, 2003 HTTP:/ /WWW.CEN-ONLINE.ORG
Pyridoxamin
"When we evaluate the newer tools now available in the chemists' tool kit, the key question is: Are they any good?" Owen began. 'And the answer is: Even if they are, the only way they are going to be α useful is if you actually take them t out of the tool kit and start to g use them." Owen's thesis that developing new technolo- ™ gies won't be beneficial unless I the researchers who could use them can do so effectively
"In order to do that, we have to make change happen," Owen continued. "We have to change the way we have worked in the past." For example, the classical approach to optimizing a reaction or a set of reactions is to change one factor at a t ime (OFAT)—such as temperature,
ing multiple factors simultaneously in a structured way, studies can be designed to produce optimum processes quickly, he suggested.
reaction time, mole ratios, and ARCH ITECTS Breslow (left) and Confalone. amount of starting material— and find the best value for that factor before moving on to the next factor. But this method is not very effective, Owen noted, because it does not examine interactions between factors. However, by chang
As an example, Owen used the coordinates of a cube to represent different factors. He showed that by choosing low, medium, and high values for each factor along the axis coordinates, a set of exper
iments could be designed in which combinations of factors are assessed for their impact on a critical output, typically reaction yield and purity [Org. Process Res. Dev.,
5, 308 and 324 (2001)]. Analysis of the impact of the factors on the results provides much more information than the same number of experiments in the OFAT method, he said.
Because process chemists aren't traditionally taught concepts such as experimental design during their formal education, a number of companies are developing in-house training programs for their researchers. Owen described a workshop used in GlaxoSmithKline's modular training program in which process chemists determine the optimal head of foam on a poured pint of beer.
In some countries or regions, he noted, it's optimal to have the head as large as possible, while in other places it's desirable to have little or no head. For the teams of process chemists involved in the study the
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idea is for them to learn how to design experiments to find the optimal way to pour beer to achieve the type of head desired.
To the amusement of the audience, Owen demonstrated some of the pouring techniques on stage with cans of beer and glasses. Some of the factors considered by the training groups included operating procedure (tilting or rotating the glass), batch input variability (can-to-can, manufacturer-to-manufacturer), equipment variability (glass-to-glass), and operator and measurement variability The experiments generated a lot of data, he related, which were analyzed by statistical software.
The results: One training group found that the important factors are the type of beer used and a one-stage or two-stage pouring process. Unimportant factors included whether a woman or a man was pouring. "From a purely statistical point of view, there is no difference between males and females," he said, tongue in cheek. Afterward, the beer Owen poured was enjoyed by a few members of the audience.
Owen presented several case studies to demonstrate the impact of experimental design in early R&D, technology transfer,
HEADS UP GlaxoSmithKline process chemists participate in an experimental design workshop to devise optimal methods to pour different beers and achieve the desired head of foam.
already on the market or in clinical trials. For example, Pat N. Confalone, executive director of Bristol-Myers Squibb's Pharmaceutical Research Institute in Deep-water, N.J., described the development of the commercial synthesis of HIV-1 protease inhibitors and nonnucleoside reverse transcriptase inhibitors (NNRTIs). These
FRUIT OF THE LAB Process chemists turn original syntheses into safe and economic production-scale routes to commercial products
Elarofiban fibrogen receptor antagonist to treat thrombosis
Efavirenz reverse transcriptase inhibitor to combat AIDS
Sumanirole dopamine agonist to treat Parkinson's disease
and troubleshooting of a manufacturing process. "We use experimental design to explore many aspects of our operations, including reducing costs of goods, mapping impurity formation, controlling quality, improving process throughput, and reducing waste," he concluded.
Many of the talks presented in San Juan focused on process development of drugs
Atorvastatin reductase inhibitor to lower cholesterol
classes of drugs are being used in combination therapy against HIV-1 to delay the progression to AIDS.
"The number of people living with AIDS on the planet, as referred to in President Bush's State of the Union address, continues to be a real human tragedy," Confalone said, putting the importance of the work in perspective. "The United
Nations places the AIDS epidemic on a scale equal to nuclear war and worldwide famine in terms of a catastrophic threat to humanity"
The first topic he addressed was synthesis of symmetrical and unsymmetrical cyclic urea diols, a class of HIV-1 protease inhibitors developed by DuPont-Merck Pharmaceutical, later DuPont Pharmaceuticals, which was acquired by Bristol-Myers Squibb in 2001. The cyclic ureas are seven-membered rings that have four chi-ral carbons and are nanomolar inhibitors of HIV-1 protease. He described how the desired RSSR isomers are made from inexpensive L-tartaric acid in a stereospecif-ic fashion [J. Med. Chem., 41,5113 (1998)}.
Confalone next outlined Merck's discovery of trifluoromethyl-substituted di-hydroquinazolinones as HIV-1 specific NNRTI drug candidates. One of the candidates, efavirenz, was identified as the most promising and was licensed to DuPont-Merck for development. "The work at this point became a project for process chemists to find a safe and economical way to make it, since the initial costs of production were prohibitively expensive," he said. Confalone discussed the successes and failures in determining the best choice of starting materials, reagents, key intermediates, and reaction conditions for efavirenz.
A collaborative effort between Merck and DuPont-Merck chemists resulted initially in a seven-step enantioselective route to make up to kilogram quantities of efavirenz from 4-cWoroariiline with 62% overall yield \J. Org. Chem., 63 , 8536 (1998)]. Further refinements are nowused in production of multiton quantities of the drug, which is being marketed as Sustiva by
36 C&EN / MARCH 1 7, 2003 H T T P : / / W W W . C E N - O N L I N E . O R G
Bristol-Myers Squibb and as Stocrin by Merck.
Confalone wrapped up his discussion by outlining chiral routes to second-generation NNRTIs that are being developed to combat resistant strains of HIV {7· Org Chem., 68,754 (2003)}. These include compounds in which a nitrogen is incorporated into one of the rings of efavirenz in place of an oxygen heteroatom. Economic routes have been developed for these compounds, and they also are being made in multiton quantities, he noted.
A SAM PLI NG of other presentations in San Juan includes the following:
• Ben L. Feringa, professor of chemistry at the University of Groningen, the Netherlands, discussed his group's work on asymmetric catalysis based on mono-dentate chiral phosphoramidite ligands, which contradicts the common thought that bidentate chiral ligands are needed to reach high enantioselectivity {Org. Lett., 5, 681 (2003); J. Am. Chem. Soc, 124,14552 (2002)}.
• Ambarish K. Singh, an associate director in the process research and development department at Bristol-Myers Squibb, New Brunswick, N.J., described the science and techniques for optimizing crystallization methods and screening for polymorphic crystal forms, which can be applied toward developing more efficient, environmentally friendlier, and cost-effective processes {C&EN, April 22,2002, page 30; Org. Process Res. Dev., 5, 508 (2001)}.
• Peter Wipf, professor of chemistry at the University of Pittsburgh, discussed alkenylzirconocene-mediated carbon-carbon bond-forming reactions to prepare al-lylic alcohols and amines, cyclopropyl-alkylamines, and other compounds [Chem. Eur. J., 8,1778 (2002); J. Am. Chem. Soc, 125,761(2003)}.
• Peter G. M. Wilts, a senior fellow at Pharmacia, Kalamazoo, Mich., described the synthesis and process development of sumanirole, a tricyclic imidazoquinoline compound in Phase II clinical trials for treatment of Parkinson's disease [Pure Appl. Chem., 74,1359 (2002)}.
The conference culminated with a roundtable discussion on the Food & Drug Administration's drug approval process, led byjohn E. Simmons, director of FDAs Division of New Drug Chemistry 1. Although FDA has had more open communication with companies since the Prescription Drug User Fee Act was initiated in 1992, this was the first time that Simmons knew of that a representative from
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FDAhad made a presentation at a confer- I has been rocky— there's still uncertain-ence that focused on process chemistry ty as to how FDA does things and why"
"The pharmaceutical industry has be- This uncertainty is particularly true for come global," he commented. "This has process chemists, he added, since they been good for the industry, but the road I are in the no-man's-land between drug
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discovery and bringing products online. Although process chemists come up
with the final synthesis procedure and specifications for the active ingredient in a new drug application, Simmons said, they rarely interact with FDA staff. Instead, company regulatory affairs personnel are generally FDA's points of contact. This additional layer sometimes can lead to confusion when discussing what a company can and can't do under FDA regulations, he noted.
Simmons at tempted to "demystify" FDA operations by reviewing how the agency is organized and regulated. He included a brief history of U.S. laws that govern FDA activities, and gave a broad-brush overview of the pathway from drug discovery to drug approval (C&EN, Dec. 2, 2002, page 58).
The large number of mergers in the pharmaceutical industry has made relationships with FDA more interesting in recent years, Sirnmons said. "Every company is unique, with its own personality," he explained. "When two companies with sometimes vastly different corporate philosophies are forced to come together,
it doesn't make the drug approval process easy"
But being knowledgeable about FDA and the approval process is essential for scientists at pharmaceutical companies to help overcome any potential difficulties, Simmons said. He encouraged the chemists to regularly read the Code of Federal Regulations for food and drugs (CFR 21) and the Federal Register to observe key rules generated by or pertaining to FDA. He also suggested that process chemists access the agency's website and review the "ICH Quality" guidance documents, which complement and expand what is in the regulations.
These guidance documents, such as Q6A, provide information to help understand how measurements for impurities in a new drug should be made, what ranges of impurities might be acceptable, how often measurements should be made, the re-
"At FDA, we are in a fishbowl. Everything we write, every determination we make is open to evaluation by the larger community."
Organic Synthesis for Early Discovery
producibility of measurements, and in what form the data should be presented. They have been developed through consensus by FDA, pharmaceutical companies, regulatory agencies in other countries, academicians, and nongovernmental
organizations to promote international h a r m o n i z a t i o n of technical procedures, he said.
Simmons' descriptions of some of the specifications in the guidance documents spa rked r e q u e s t s from the audience for clarification. "If you follow the guid
ances, we aren't going to ask too many questions," he said. "If you want to do something different from the guidances, we're going to ask questions. But if a testing protocol is set up based on sound scientific principles, and these are discussed with us, then these types of options probably would be allowed."
One message Simmons heard, and was sympathetic to, was that pharmaceutical companies are often leery of trying to change a production process once a drug has been approved. "Sometimes it isn't necessary to have all the process option i's dotted and t's crossed at the time of approval," he said. "Sometimes it's wise to handle changes postapproval if it makes sense scientifically and intermediates have well-defined specifications."
As an example, he said selection of the starting material in a synthesis needs to be a well-defined compound that is known in the literature and has a set of guidance-based specifications for it. There may also be intermediates in a multistep pathway that could have their own sets of specifications. In this way, companies seeking to lower the cost of a process could change a synthesis to prepare an intermediate by a different method or even outsource production of the intermediate rather than make it themselves.
'At FDA, we are in a fishbowl," Simmons said. "Everything we write, every determination we make is open to evaluation by the larger community. That's a powerful incentive to ensure that we do things right, but in a reasonable way" He emphasized that process chemists should try to get a seat at the table when their company has meetings with FDA reviewers concerning a new drug application. "Don't be afraid to come in and ask questions," he concluded. •
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