Chiral Analysis in Drug Discovery and Development

IPT 35 2010 26/11/10 13:40 Page 18

Discovery Technology

By Yanan He With the number of single-enantiomer drugs expected to increase rapidly at BioTools, Inc in the coming years, it is crucial to have state-of-the-art tools available for chiral analysis to keep up with the demands of high-throughput screening in the current R&D environment.

Since Louis Pasteur discovered chirality in 1847, it has together (racemate) for a chiral drug (2). It was become an integral and important part of life. Chiral subsequently discovered, however, that often only one drugs have molecular structures that lack an internal of the two enantiomers was active and the other one plane of symmetry and are non-superimposable with was either non-active or even harmful. This is because their mirror images. This property of a molecule is also the interaction between a drug molecule and its target called ‘handedness’. A chiral molecule and its mirror is very much dependent on the 3D environment image are called a pair of enantiomers. Most molecules around them. For a pair of enantiomers, their found in nature – such as amino acids, sugars, interactions with the target in the 3D environment proteins, DNAs and RNAs – are chiral and usually are different. The most famous example is the only one of the two enantiomers is formed naturally. thalidomide molecule sold in the 1950s; the drug was For example, virtually all the amino acids found in introduced as a racemic mixture for use as a sedative, nature are L-(left-handed). In the pharmaceutical but was later withdrawn from the market following industry, currently more than two thirds of the drugs the occurrence of birth defects in the children of on the global market are chiral drugs serving in mothers who took it to treat morning sickness. It was myriads of therapeutic areas such as anxiety, later found that the inactive enantiomer was the cause indigestion, heartburn, arthritis, AIDS, cancer and of the teratogenicity. allergies (1). Several top-selling blockbuster drugs – such as Lipitor, Nexium and Plavix – are all chiral. In the US in 1992, the FDA recommended using With the recent rapid developments in biotechnology, stereochemically pure drugs; this required that the chiral drugs will play an even more important role in drug labelling should include a unique, established saving patients’ lives because almost all name and a chemical name with the appropriate biopharmaceuticals are chiral. stereochemical descriptors that should specify identity, strength, quality and purity from a stereochemical Originally, pharmaceutical companies were allowed by viewpoint (3). Since then, for pharmaceutical the US FDA to test and develop both enantiomers companies, single enantiomer drugs have become the standard when working with compounds featuring asymmetric centres.

Shortening timelines for chiral drug discovery and development usually depends on the efficiency of asymmetric synthesis, enantiomeric separation and determination of absolute configuration (AC). Therefore, the availability of powerful tools for chiral analysis is crucial in streamlining the discovery and development of a chiral drug. This article will review the tools available for chiral analysis, including techniques for generating single enantiomers and techniques for their AC determination. IPT 35 2010 26/11/10 13:40 Page 20

Figure 1: the column at different times and be separated. The IR (lower frame) and VCD (upper frame) 2 introduction of automated SFC instrumentation with spectra of (R)-(+)- and stacked injection produces much faster separations per (S)-(-)-Camphor in unit time, as well as fractions with higher purities CCl4 (0.8M for both 1 enantiomers); 100-µm compared with HPLC. Most of the enantiomer 4 path-length cell with separations in the drug discovery stage are carried out

BaF2 windows; 20

A x 10 using chiral chromatography (5).

minutes collection for Δ 0 both enantiomers and solvent; instrument Asymmetric Synthesis optimised at 1,400cm-1. This technique introduces chirality during the synthetic Solvent-subtracted IR -1 VCD sequence, and requires either asymmetric catalysis or and enantiomer- subtracted VCD auxiliary chiral synthesis. Ideally asymmetric synthesis spectra are shown should be the most cost-effective method for producing 0.8 (1R)-(+)-camphor single-enantiomer products because all the precursors (1S)-(-)-camphor are converted to the desired enantiomer. However, in

0.6 practice the implementation of this approach can be limited by several factors such as the efficiency of the

IR catalyst, the availability of the catalyst and the reaction 0.4 conditions (a very low temperature or high pressure, and

Absorbance reaction kinetics) (6). 0.2 DETERMINATION OF ABSOLUTE CONFIGURATION 0.0

1,300 1,200 1,100 1,000 900 Techniques that are prevalent in the pharmaceutical Wavenumber (cm-1) industry for determination of the AC of chiral molecules include X-ray crystallography, nuclear magnetic SEPARATION OF ENANTIOMERS resonance (NMR) methods (Mosher’s method, a chiral liquid crystal NMR technique) and vibrational circular Chiral Salt Resolution dichroism (VCD). Chiral salt resolution (or diastereomeric crystallisation) is the classical method for the separation of racemic X-ray Crystallography mixtures by seeding with a pure enantiomer or a chiral X-ray is still considered the most reliable technique, but resolving agent during the crystallisation of the racemic usually requires at least one heavy atom (atomic number mixture. When successful, this is the best method for greater than 23) and a single crystal. The amount of the manufacture of single enantiomers in bulk. For material required for testing different crystallisation example, Merck has used this with great success in the conditions is significant because, at discovery stage, manufacture of α-methyl DOPA. Unfortunately, the usually only a small amount of compound is synthesised method has very limited application because it can only and it has to be used for multiple tests. The turnaround be applied to a conglomerate (a mixture of crystals of time (growth of single crystal plus AC determination) is the two enantiomers), and these account for only less usually slow on the discovery time scale. Very often, than 20 per cent of all known racemates (4). when the X-ray result is finally given to the chemist who synthesised the compound, he or she is no longer Chiral Chromatography interested because other test results that have come back Currently, the most popular technique for the in the mean time show that the compound is of very separation of a racemic mixture is either normal phase or little, if any, potency. Therefore X-ray crystallography is reverse phase high performance liquid chromatography typically used on a potential drug candidate at the late (HPLC) or supercritical fluid chromatography (SFC). discovery or early development stage (5). Chiral chromatography uses columns coated with polysaccharide-based chiral compounds as the NMR Methods stationary phase that has different affinities for a pair of NMR methods are used routinely by structure enantiomers in the mobile phase running through the elucidation teams in pharmaceutical R&D departments column. Therefore, the two enantiomers will elute from to resolve the relative stereochemistry of compounds. For

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Figure 2: Spectra of (S)-Binaphthol can be determined (6). This Absolute configuration Observed VCD 40.0 method generally requires a determination of (S)- 30.0

20.0 Δ OH A x 10 Binaphthol. On the left Structure of (S)-Binaphthol 10.0 larger sample size (40-50mg) and OH 0.0 5 are the structure and -10.0 -20.0 is experimentally complex. -30.0 optimised geometries 80.0 Calculated VCD: S of the lowest-energy Optimised geometries of the three calculated 60.0 Calculated VCD: R 40.0

conformers of the (S)- configuration 3 conformers, their 20.0 Vibrational Circular x 10 0.0 Boltzmann populations Δε -20.0 Dichroism (VCD) and relative energies. -40.0 Conformer1, 0% -60.0 VCD is one of the two forms On the right are the Observed IR RE = 7.7 kcal/mol 0.8

observed and calculated Absorbance of vibrational optical activity 0.4 IR (lower frame) and (VOA). VOA is the differential VCD spectra (upper 0.2 0.0 response of a chiral molecule frame) of (S)- Calculated IR 400 Binaphthol. The sample Conformer2, 0% to left versus right circularly RE = 4.2 kcal/mol 300 was measured in CDCl3 200 polarised infrared radiation 100 (8.7mg/0.18mL) in a Molar absorptivity during a vibrational transition 100-µm path-length cell 0 1,600 1,500 1,400 1,300 1,200 1,100 1,000 (7). The other form of VOA is with BaF2 windows; 10 Conformer3, 100% Frequency hour collection for both RE = 0 kcal/mol Raman optical activity (ROA). Scale = 0.997 sample and solvent; The IR spectra of a pair of instrument optimised at TNS (IR) = 95.2 TNS (VCD) = 82.1 SNS (S) = 82.9 SNS (R) = 15.0 ESI = 67.9 1,400cm-1. Solvent- enantiomers are the same, while subtracted IR and VCD The absolute configuration of (S)-Binaphthol is S their VCD spectra are equal in The confidence level is 100% spectra are shown intensity but opposite in sign (mirror images of each other AC determination, regular NMR experiments would not about the zero baseline), as shown in Figure 1 for (1R)- give the answer. However, with Mosher’s NMR method, (+)- and (1S)-(-)-Camphor. The VCD spectrum of a one can obtain derivatives of the unknown enantiomers chiral molecule can be calculated using density with Mosher’s reagent, which has a known chirality, and functional theory (DFT). The AC of a chiral molecule then determine the AC of the two enantiomers by can be determined by comparing the measured examining the different changes in chemical shifts in the VCD spectrum with the calculated spectrum. The two derivatives. Mosher’s method is used primarily on confidence level of such a comparison can be obtained secondary alcohols, amines and acids. The chiral liquid by the VOACompare algorithm recently developed crystal NMR method uses chiral liquid crystals as an by BioTools. NMR solvent; the two enantiomers then exhibit different coupling constants. By comparing the The VCD technique was discovered and developed in experimental data with the calculated results, the AC the 1970s and has become a powerful tool for the AC determination of small Figure 3: Observed VCD spectra of chiral molecules in the solution 25 (2R,3R)-phenylglycidol Phenylglycidols a state (7). The first commercial (2R, 3R)-phenylglycidol analogs. The samples c VCD instrument was brought were measured in 0

CDCl3 (0.17M) using b to market by BioTools in H a a Dual-PEM ChiralIR -25 d 1997, and since then many spectrometer with a O c -50 3-F pharmaceutical companies and 100-µm path-length cell with BaF windows; b academic research groups have 2 R OH -75 a 4-hour collection for d adopted this technique for their

3 c both enantiomers; H 3-Cl -100 AC determination needs (8). instrument optimised at x 10

-1 Δε b VCD measurements require 1,400cm . Enantiomer- R= o-, m-, p- -125 subtracted VCD a d only five to 10mg of pure 3-CF3 c spectra are shown (9) -150 sample in solution form. The F b fast development of computer Cl -175 a c d CF3 processors and memories and 3-OMe OCH3 -200 the implementation of VCD b calculations into Gaussian -225 d 1,800 1,600 1,400 1,200 1,000 programs have made it possible Wavenumber (cm-1) to carry out DFT-level VCD calculations on PCs. An example

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of the AC determination of (S)-Binaphthol is shown in In drug discovery and development, Figure 2. Determining the AC by VCD analysis is chiral analysis will become more and more typically more rapid for small chiral molecules (less than 60 atoms) when compared with analysis by X-ray important. Over the past two decades, the crystallography. number of single-enantiomer drugs has been

Due to its relatively low sample requirement and short increasing steadily – approaching 70 per cent analysis time, VCD is especially useful for AC of all APIs – and is expected to increase more determination in the early exploratory and discovery rapidly in the coming years. stages when a project is engaged in structure-activity relationship (SAR) and/or structure-property relationship (SPR) studies. SAR and SPR studies References usually involve synthesising a series of analogs of the 1. Stinson SC, Chiral Pharmaceuticals, C&EN, 79 (40): lead compounds; the analogs are then tested for their pp79-97, 2001 biological activities and other important properties 2. Federsel H, Facing Chirality in the 21st Century: such as solubility, stability and permeability. The results Approaching the Challenges in the Pharmaceutical of the SAR/SPR studies then direct the synthesis Industry, Chirality, 15, S128-S142, 2003 towards more potent activity, greater target selectivity 3. FDA’s Policy Statement for the Development of New and improved physicochemical properties of the lead Stereoisomeric Drugs, Chirality, 4: pp338-340, 1992 compounds. Therefore, VCD analysis in the 4. Andrew C, Encyclopedia of Separation, Vol 5: p2,326 exploratory and discovery stages usually involves 5. McConnell OJ, Bach II AC, Balibar C et al, analysing a series of analogs with the same core Enantiomeric Separation and Determination of structure but with different substituents. Such series of Absolute Stereochemistry of Asymmetric Molecules analogs often exhibit similar observed and calculated in Drug Discovery – Building Chiral Technology VCD features as shown in Figure 3 for phenylglycidal Toolboxes, Chirality, 19(9): pp658-682, 2007 compounds (9). Since the rate-limiting step in VCD 6. Marathias VM, Tawa GJ, Goljer I and Bach II AC, analysis is most often the VCD calculations, it would Sterochemical Identification of (R)- and (S)- be beneficial to carry out VCD calculations for only Ibuprofen Using Residual Dipolar Couplings, NMR one compound in a series of analogs and use it as a and Modeling, Chirality, 19: pp741-750, 2007 reference for AC determination of the other analogs in 7. Feedman TB, Cao X, Dukor RK and Nafie LA. the same series by comparing the observed VCD of the Absolute Configuration Determination of other compounds with that of the reference. This Chiral Molecules in the Solution State Using would significantly reduce the analysis time for AC Vibrational Circular Dichroism, Chirality, 15: determination by VCD for analogs. pp743-758, 2003 8. Polavarapu PL, New spectroscopic tool: Absolute CONCLUSION configuration determination of pharmaceutical compounds by vibrational circular dichroism, In drug discovery and development, chiral analysis will Spectroscopy, 9(9): pp48-55, 1994 become more and more important. Over the past two 9. McConnell OJ, He Y, Nogle L and Sarkahian A, decades, the number of single-enantiomer drugs has Application of Chiral Technology in a Pharmaceutical been increasing steadily – approaching 70 per cent of Company. Enantiomeric Separation and all APIs – and is expected to increase more rapidly in Spectroscopic Studies of Key Asymmetric the coming years. It is crucial to have state-of-the-art Intermidiates Using a Combination of Techniques, techniques for chiral analysis to keep up with the Chirality, 19(9): pp716-730, 2007 demands of high-throughput screening of drug candidates in the current pharmaceutical R&D Yanan He is a Senior Research Scientist at BioTools, Inc environment. The various types of technique have (Jupiter, FL, US). She obtained her PhD in Analytical and their own advantages and shortcomings, and Physical Chemistry from Syracuse University (New York, US). are complementary to one another. No single Prior to joining BioTools, she was a Senior Research Scientist technique can do it all. In practice, it is often a at the Department of Chemical and Screening Sciences, symphony of several techniques that can provide the Wyeth Research. Her primary research interests are chiral analysis of molecules with pharmaceutical interests and best way forward for a particular discovery and/or computational chemistry. Email: [email protected] development project.