Attila Cs. Bényei

Polymorphism of Pharmaceuticals

With Case Studies

Lecture Book

TÁMOP-4.1.2.D-12/1/KONV-2012-0008

Debrecen

2014

2

Contents

Preface............................................................................................................................................................. 5

Chapter 1 Introduction ............................................................................................................................... 7

1.1. Polymorphism definition ........................................................................................................................ 7

1.2. Polymorphism in everyday life and in pharmaceutical industry.............................................................. 10

1.2.1. Suggested project work: Polymorphism in everyday life ................................................................. 11

1.3. Overview of analytical methods ............................................................................................................ 11

1.4. Problem oriented analytical approach .................................................................................................... 12

1.5. The Ritonavir case ................................................................................................................................ 13

1.5.1. Suggested project work: The Ritonavir case, literature highlights. ................................................... 15

Chapter 2 Thermodynamics ................................................................................................................. 16

2.1. Basics of thermoanalytical methods and their application in polymorphism research.............................. 16

2.1.1. Hot stage microscopy ..................................................................................................................... 16

2.1.2. Differential Thermal Analysis (DTA) ............................................................................................. 17

2.1.3. Differential Scanning Calorimetry (DSC) ....................................................................................... 17

2.1.4. Thermogravimetry Analysis (TGA) ................................................................................................ 18

2.2. Energy-temperature diagrams. Monotrope and enantiotrope systems ..................................................... 19

2.3. The Burger-Ramberger rules................................................................................................................. 21

2.3.1. Heat of transition rules ................................................................................................................... 21

2.3.2. Heat of fusion rules ........................................................................................................................ 22

2.3.3. Entropy of fusion rule .................................................................................................................... 22

2.3.4. Heat capacity rule .......................................................................................................................... 23

2.3.5. Density rule ................................................................................................................................... 23

2.3.6. Infrared rule ................................................................................................................................... 23

2.4. Polymorphism of caffeine and chocolate ............................................................................................... 24

2.4.1. Suggested project work: The polymorphism of cocoa butter. Pharmaceutical consequences ............ 24

2.5. The polymorphism of nimodipine and sulfamethoxidiazine ................................................................... 24

2.5.1. Suggested project work: Search the literature for compounds forming enantiotropic or monotropic

systems ................................................................................................................................................... 25

Chapter 3 Patents ...................................................................................................................................... 26

3.1. Patent literature basics .......................................................................................................................... 26

3.2. The art of constructing claims ............................................................................................................... 28

3.3. Polymorphs and patents, IP issues ......................................................................................................... 29

3.4. The ranitidine hydrochloride, paroxetine hydrochloride and aspartame cases ......................................... 30

3.4.1. Suggested project work: A novel patent case of polymorphism. Pharmaceutical consequences. ....... 35

Chapter 4 Crystallization .......................................................................................................................... 36

4.1. Crystals show long range order ............................................................................................................. 36

4.2. The thermodynamics of phase transitions and crystal forming ............................................................... 36

4.3. Kinetics of crystallization ..................................................................................................................... 39

Chapter 5 Polymorphism prediction......................................................................................................... 41

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5.1. Computational approach, polymorphism prediction ............................................................................... 41

5.2. Blind tests for polymorphism prediction ............................................................................................... 41

5.3. Difficulties of QM calculations of van der Waals interactions. ............................................................... 42

5.4. Remarks on chemical bonds ................................................................................................................. 43

5.5. Structural motives of polymorphic forms .............................................................................................. 44

Chapter 6 X-ray diffraction basics, X-ray powder diffraction ................................................................. 48

6.1. Properties of X-ray radiation. ................................................................................................................ 48

6.2. Crystal systems, space groups, Miller indices and distance of lattice planes ........................................... 49

6.3. The Bragg’s law ................................................................................................................................... 53

6.4. Powder diffraction methods .................................................................................................................. 54

6.5. Quantitative X-ray Powder Diffraction (XRPD) of polymorphs ............................................................. 57

Chapter 7 Single crystal X-ray diffraction................................................................................................ 59

7.1. Single crystal X-ray diffraction in polymorphism research. All or none method ..................................... 59

7.1.1. The structure factor as Fourier transform of the electron density in the unit cell ............................... 59

7.1.2. Single crystal diffraction for the complete determination of solid state structure .............................. 62

7.1.3. Measurement of single crystal diffraction data ................................................................................ 63

7.2. The hydrogen bond ............................................................................................................................... 64

7.3. Co-crystals ........................................................................................................................................... 65

7.4. Structural motifs of polymorphs, case studies ........................................................................................ 66

7.4.1. Sulphonamide polymorphic forms .................................................................................................. 66

7.4.2. The carbamazepine case ................................................................................................................. 67

7.4.3. Suggested project work: Search of CSD for polymorphic forms of an API and identification of

structural motives on the basis of single crystal data ................................................................................. 68

7.5. Assignment of chirality using single crystal X-ray diffraction ................................................................ 69

7.5.1. Chirality and its pharmaceutical consequences ............................................................................... 69

7.5.2. Methods for assignment of chirality centres .................................................................................... 70

7.5.3. Assignment of chirality relative to known stereogenic centre .......................................................... 71

7.5.4. Assignment of chirality on the basis of anomalous scattering .......................................................... 71

7.6. The role of chirality in polymorphism screening ................................................................................... 73

7.7. Formation of racemic conglomerates .................................................................................................... 74

Chapter 8 Structure determination from powder diffraction data .......................................................... 77

8.1. Ab initio structure determination from powder diffraction ..................................................................... 77

8.2. Rietveld refinement .............................................................................................................................. 78

8.3. The cimetidine ..................................................................................................................................... 81

8.4. The structure of aspartame anhydrate from powder diffraction data ....................................................... 82

Chapter 9 Solid state NMR ....................................................................................................................... 85

9.1. Basics of solid state NMR..................................................................................................................... 85

9.2. Solid state NMR in polymorphism research .......................................................................................... 87

9.3. Polymorphism of aspartame, solid state NMR studies ........................................................................... 88

9.3.1. Suggested project work: Application of solid state NMR in the study of steroid compounds ............ 89

Chapter 10 IR and Raman spectroscopy/microscopy ............................................................................ 90

10.1. FT-IR and Raman spectroscopy .......................................................................................................... 90

10.2. FT-IR and Raman microscopy and their application in polymorphism research .................................... 94

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10.2.1. Chemical imaging ........................................................................................................................ 95

10.3. ATR techniques .................................................................................................................................. 95

10.4. Polymorphism of dyes and explosives ................................................................................................. 96

10.4.1. Suggested project work: Search for polymorphic forms of energetic materials............................... 99

Chapter 11 The use of Cambridge Structural Database in polymorphism research ........................... 100

11.1 The early history of compilation of single crystal data ........................................................................ 100

11.2. The method of depositing small molecule single crystal data ............................................................. 102

11.3. The Crystallographic Information File (CIF) ..................................................................................... 104

Chapter 12 Regulatory and quality control issues................................................................................ 106

12.1. Polymorphism - quality control issues ............................................................................................... 106

12.2. Regulatory questions of polymorphism: rules of U.S. Food and Drug Administration ........................ 108

12.3. Q6A decision trees ........................................................................................................................... 109

12.4. The Process Analytical Technology concept...................................................................................... 113

12.4.1. Example for PAT approach in monitoring and controlling polymorph compositions .................... 115

Chapter 13 Technological aspects of polymorphism research: Controlling polymorph compositions 116

13.1. The effect of contaminants and seeding ............................................................................................. 116

13.2. Scale-up ........................................................................................................................................... 117

13.3. The effect of ultrasound on crystallization ......................................................................................... 118

13.4. Micronization and nanonization ........................................................................................................ 118

Chapter 14 Summary and outlook. Concluding remarks .................................................................... 120

Index ........................................................................................................................................................... 124

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Preface

Polymorphism is known in several fields such as genetics, information technology or material

science with slightly different meanings. In our case polymorphism is the ability of chemical

compounds to form multiple crystal structures. The phenomenon has been known for

centuries and we can meet polymorphic forms in everyday life frequently. However,

polymorphism reached considerable research interest only in the last few decades. The

exponentially growing attention is fuelled mainly by pharmaceutical applications and

consequences as different crystal forms have slightly or dramatically different properties such

as solubility, rate of dissolution, biological availability etc. when the solids interact with

solutions or biological systems.

At the University of Debrecen, Faculty of Pharmacy an elective course of Polymorphism of

pharmaceuticals has been organized since 2008 and it is in the curriculum of students of

pharmacy studying in both Hungarian and English languages with code numbers

GYGPO0108 and GYGPO0208, respectively. This topic and the course offer unique

opportunity to give the most recent results for the students through case studies and in this

way strengthen the connection between higher education teaching/studies and industry which

represents a more and more urgent demand for Universities all over the world.

The TÁMOP-4.1.2.D-12/1/KONV-2012-0008 project gave the financial background to

summarize the teaching experiences of the last years and this book could be published. The

project is co-financed by the European Union and the European Social Fund.

Polymorphism has consequences in various points of pharmaceutical and material science

such as analytics, regulatory issues, structural research and intellectual property.

The course points out the most basic problems of all these areas of polymorphism. Students

get not only up-to-date information but also enforced for project- and problem-oriented

approach and establish analytical applications of multiple dimensions.

The limit of the book-pages and time does not allow the comprehensive description of all

aspects of polymorphism and excellent handbooks are also available.1 This book although

covers recent scientific achievements intended to be a hand out for university course to help

students of MSc level. Hopefully researchers can find valuable information in it, too.

We tried to apply a novel technique by giving the definitions, pinpoint the weak points of the

paradigms in connection with polymorphism and show cases when these questions became

1 a) H. G. Brittain: Polymorphism in Pharmaceutical Solids, Marcel Dekker, New York, Basel, 1999.

b) Polymorphism in pharmaceutical industry. Ed. R. Hilfiker, Wiley-VCH, Weinheim, 2006.

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important. In the course students get information not only the basics of various analytical and

other technologies but also gain skills to choose the right method for solving the given

structural, analytical, IP or other problems. The readers are directed to books, scientific papers

and web sources to get more information. However, these references are far not considered to

be comprehensive. Wikipedia is referenced in several cases but because of the open nature of

Wikipedia the information content should be handled with precaution. Several project works

are suggested for the students to search the scientific literature and newspapers. The

completed and submitted project work is part of the evaluation process of the course and helps

for 3rd year students of pharmacy to learn how to write a paper, too.

The main sources of this book are the work by Joel Bernstein2 and lecture notes and video

recordings of the course Diversity Amidst Similarity, 35th

Crystallography Course at Erice,

Italy.3 Recently more and more universities include dedicated polymorphism courses into the

curriculum of PhD or gradual pharmacy, chemist or chemical engineering students for

example at the University of Jyväskylä.4

Comments and help from the Lector Várkonyi-Schlovicskó Erika, colleagues, students of the

course and financial support from the TÁMOP-4.1.2.D-12/1/KONV-2012-0008 project are

gratefully acknowledged. Special thank for the members of the Crystallization and Drug

Formulation Group of the Hungarian Chemical Society for fruitful round table discussions

among industrial and academic researchers of polymorphism. The compilation of the book is

dedicated as an activity of the International Year of Crystallography, 2014.5

2 Joel Bernstein: Polymorphism in Molecular Crystals, IUCr Monographs on Crystallography No. 14, Calderon

Press, Oxford, 2002. 3 http://erice2004.docking.org/vcourse/polymorph/ 4 https://www.jyu.fi/science/muut_yksikot/summerschool/en/history/JSS20/courses/CH/main#ch1-organo-and-

hydrogelators 5 The logo is used with permission of International Union of Crystallography, http://www.iycr2014.org/

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Chapter 1 Introduction

Polymorphism in the sense of different solid forms of a single chemical compound is a very

interesting phenomenon. Although it is known for centuries the last decade gave the real

reload of the topic in the field of both academic and applied research. Fuelled by Intellectual

Property issues pharmaceutical companies are the main leaders of polymorphism research.

Our everyday life is also knotted with polymorphic forms from minerals to pigments and other

solids. Polymorphism is also often mentioned in the media. State of the art analytical methods

are used to distinguish the different polymorphic forms.

1.1. Polymorphism definition

Polymorphism from Greek πολύς (polys=many, much) and μορφή (morphē=form, shape) is

used in computer science,6

biology7

and other branches of sciences. In this book

polymorphism is used as a term of material science. The most accepted definition given by

McCrone8 is: “polymorph is a solid crystalline phase of a given compound resulting from the

possibility of at least two crystalline arrangements of the molecules of that compound in the

solid state”. This definition is a result of long development but still has scientific weak

points.9

As pharmaceutically important compounds are often produced or utilized in

amorphous form more and more attention is given to solid forms which have no long range

order i.e. they are not crystalline, but amorphous phases. However, these forms of the very

same compound may also have different pharmaceutically important characteristics.

Occurrence of multiple solid amorphous forms of a compound can be named poly-

amorphism10

but it can (and according to the author it should) be also considered as a case of

polymorphism. The phenomenon of polymorphism is known from the beginning of modern

chemistry research when analytical results proved the same chemical composition of different

solid crystals such as calcite and aragonite or in case of several other minerals. The allotropy

6 http://en.wikipedia.org/wiki/Polymorphism_(computer_science) 7 http://en.wikipedia.org/wiki/Polymorphism_(biology) 8 W.C. McCrone: Polymorphism. In Physics and chemistry of the organic solid state, (eds. D. Fox, M. M. Labes

and A. Weissberger), Interscience Publishers, London, 1965, Vol 2, pp. 725–67. 9 J. Bernstein: Polymorphism in Molecular Crystals, Calderon Press, Oxford, 2002. IUCr Monographs on

Crystallography No. 14, page 2-8. 10 L. Yu: Amorphous pharmaceutical solids: preparation, characterization and stabilization, Adv. Drug. Deliv.

2001, 48 27-42.

8

of the elements which basically means the same phenomenon is well known. A shorter

definition of polymorphism is the ability of compounds to form different crystalline phases.

The word ability clearly describes, that formation of polymorphic forms is a possibility.

Polymorphism is basically a kinetic phenomenon and to reach local minima at the energy

landscape highly depends on rather unpredictable experimental parameters. McCrone stated:

“It is at least this author’s opinion that every compound has different polymorphic forms and

that, in general, the number of forms known for a given compound is proportional to the time

and money spent in research on that compound.” This has been proven numerous times when

all of the sudden new polymorphic forms have been emerged. However, the opposite is also

true. In spite of repeated crystallization thousands of times in the last centuries polymorphic

forms of sucrose, which molecule contains several O-H groups capable for hydrogen bonds in

various orientation, have been reported only recently either at high pressure11

or with

disorder.12

Another well-known compound being crystallized frequently without sign of its

polymorphism is naphthalene having modification at high pressure.13

The unpredictable

number of polymorphic forms can be the reason for a rather chaotic nomenclature. The

literature uses Arabic or Roman numerals, Greek or Latin letters to denote the different forms

but frequently occurs that the assignment and naming of the forms in one comprehensive

study had to be changed in another one by inserting more polymorphs or detecting the identity

of solid forms. It is rather time consuming but it is essential to use the nomenclature similar to

patent claims representing the solid forms by its identical X-ray powder diffraction (XRPD)

pattern, melting point or other parameters.

In some cases it is also challenging to decide what does it mean the same compound or

different crystalline phases? By McCrone’s definition it is implied that molecules of the

compound can be clearly distinguished. In chemistry compounds are held together by

chemical bonds. In supramolecular chemistry the whole solid is considered to be a giant

supermolecule held together by primary (ionic or covalent) or secondary (van der Waals,

dispersion, hydrogen bond and other) interactions. In this context during the supramolecular

analysis of the solid state structure the traditional chemical definition of molecules is losing

its meaning. In case of molecular crystals the repeating asymmetric unit (Chapter 7.1.1.),

11 E. Patyk, J. Skumiel, M. Podsiadło, A. Katrusiak: High-Pressure (+)-Sucrose Polymorph Angew. Chem. Int.

Ed., 2012, 51, 2146–2150. 12 T. Lee and G. D. Chang: Sucrose Conformational Polymorphism: A Jigsaw Puzzle with Multiple Routes to a

Unique Solution, Cryst. Growth Des., 2009, 9, pp 3551–3561. 13 S. Block, C. E. Weir, G. J. Piermarini: Polymorphism in Benzene, Naphthalene, and Anthracene at High

Pressure, Science, 1970, 169, pp. 586-587.

9

which is the supramolecular building block, can be identical with the molecule of the

compound. Here we use the term molecule in chemical sense when the molecules are held

together with covalent bonds. But the asymmetric unit can also consist of a fraction of the

molecule and the symmetry of the lattice gives the whole molecule. Of course, in this case the

molecule itself should bear the appropriate symmetry element and the term molecule as

chemically sensible part of the crystal is only fraction of the molecule of the chemical

compound. In case of ion pairs the cation and the anion together – or their symmetry allowed

part – should be considered as the molecule i.e. chemically distinct portion of the crystal

structure. Moreover, in frequent cases there is more than one molecule of the compound in

the asymmetric unit. It is also difficult to decide whether to consider restricted rotation

rotamers14

as the same compound or different one. One can give an arbitrary energy level in

the given system but solvation or temperature effects may override it. This is not only a

theoretical question as e.g. a restricted conformation of a drug could be important to fit into an

enzyme pocket. These few arguments mentioned support another definition of polymorphism

given by Dunitz15

: if a crystal is looked at as a giant supermolecule, polymorphs are the

corresponding supramolecular isomers. However, classification of isomerism should be

related to polymorphism as supramolecular isomerism.

Altogether one can speak about the same compound and its different solid forms if these

forms (polymorphs) give the same vapour phase or the same solution in any solvent.

One of the governing principles of science is the integrated nature of structure and properties.

Approaching the phenomenon of polymorphism from the direction of properties the different

features of solid phase modifications, either crystalline or amorphous, can be utilized in

several fields. These differences are emerging when properties of the solid phase are studied

or when the solid interacts with a solvent or solution. The differences among solid state

modifications are especially important when the solid forms interact with biological systems.

The polymorphism can alter

- Mechanical properties: hardness, manufacturing characteristics such as drying and

filtration, crystal habit, particle size and its distribution

14 http://goldbook.iupac.org/F02520.html 15 J. D. Dunitz: Phase transitions in molecular crystals from a chemical viewpoint, Pure Applied Chem. 1991, 63,

177-185.

10

- Physical properties: density, melting point, conductivity, hygroscopicity, thermal

stability, vapour pressure, enthalpy of phase transitions such as melting or solid-solid

transition

- Kinetic properties: rate of dissolution, kinetic stability, solid state reactions

- Spectroscopic properties: colour, electronic, vibration (IR, Raman), solid state NMR

spectra

- Biological properties: availability, taste,

- Diffraction properties (X-ray, neutron or electron diffraction)

In practice and especially from the point of view of intellectual property rights differentiation

between forms is crucial and the altered properties mentioned above are the basis of this.

However, the enormous development of analytical methods and their availability cause

emergence of ‘new’ polymorphs as today for example using synchrotron radiation one can

differentiate among forms which are the same by all means using an in house diffractometer

or considering other parameters.

In our modern life beside the scientific definition of things the definition by the law is also

important. Concerning polymorphism the most important is the definition given by

pharmaceutical regulatory agencies.16

The Q6A specification gives the definition as

polymorphism includes single entity polymorphs, molecular adducts (solvates or hydrates)

and amorphous forms. The term pseudo polymorphism was introduced for different salts or

solvates of active pharmaceutical ingredients (API) but this nomenclature should be avoided.

1.2. Polymorphism in everyday life and in pharmaceutical industry

Polymorphism could be observed very frequently by everybody although little attention is

given to it. Just to mention a few cases: chocolate, pigments or minerals are examples for this

widespread phenomenon. The two different crystalline forms of carbon are diamond and

graphite. Modifications of minerals, avoiding the contaminants, are very important in

jewellery industry. The “right” polymorph i.e. form V of cocoa butter17

can be found in

16 L. X. Yu, M. S. Furness, A. Raw, K.P Outlaw, N. E. Nashed, E. Ramos, S. P. Miller, R. C. Adams, F. Fang, R. M. Patel, F. O. Holcombe, Y.Y. Chiu, A. S. Hussain: Scientific considerations of pharmaceutical solid

polymorphism in abbreviated new drug applications, Pharm Res., 2003, 20, 531-536. 17

Food Lipids: Chemistry, Nutrition, and Biotechnology, Second Edition, Ed. Casimir C. Akoh, David B. Min,

Marcel Dekker, New York, 2002.

11

chocolate bars while form VI and other forms may have stability or storing problems at higher

temperature by having lower melting point18

or even different colour or taste. Copper

phthalocyanine is used for colouring our cars and the colour is determined by the distance of

these large conjugated π systems. The absorbance maximum can be calculated by solving

Schrödinger’s equation for the one dimensional particle in a box19

case.

Polymorphism is especially important in pharmaceutical industry as it can have influence on

manufacturing, formulation, storage i.e. quality control of drug substances and drug products.

The regulatory agencies give the framework20

for all manufacturers to evaluate polymorphism

of drug substances and components of drug products.

1.2.1. Suggested project work: Polymorphism in everyday life

1.3. Overview of analytical methods

As polymorphism is related to the solid state all methods investigating the bulk solid or the

surface, in principle, can be used for detecting and characterizing polymorphs. The techniques

can be grouped according to their capability of giving or not giving structural information on

the polymorphic forms.

Complete solid state structural information is given by

- Single crystal diffraction studies using X-ray or neutron source (Chapter 7)

- Ab initio structure determination from powder diffraction (Chapter 8)

- In silico methods of polymorphism prediction (Chapter 5)

Partial or indirect structural information is given by

- IR and Raman spectroscopy and microscopy (Chapter 10)

- Solid state NMR (Chapter 9)

18 K. Roth: Chocolate – The Noblest Polymorphism I-III, Chemistry Views, 2010,

http://www.chemistryviews.org/details/ezine/745325/Chocolate_-_The_Noblest_Polymorphism_I.html

http://www.chemistryviews.org/details/ezine/808827/Chocolate__The_Noblest_Polymorphism_II.html

http://www.chemistryviews.org/details/ezine/854777/Chocolate__The_Noblest_Polymorphism_III.html 19 http://en.wikipedia.org/wiki/Particle_in_a_box 20 International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for

Human Use, ICH Harmonised Tripartite Guidline Specifications: Test Procedures and Acceptance Criteria for

New Drug Substances and New Drfug Products: Chemical Substances, 62901 Federal Register, 1997, Vol. 62,

No. 227.

12

- UV-VIS spectroscopy

- X-ray Powder Diffraction (XRPD, Chapter 6)

Analytical methods giving no or very limited structural information about the structure of

polymorphs (but thermoanalytical and microscopic methods can serve important primary

information in distinguishing the polymorphs)

- Thermochemical and thermoanalytical methods, Thermogravimetry (TG), Differential

Scanning Calorimetry (DSC), melting point (Chapter 2)

- Microscopic investigation of habits of different modification in polarized light, hot

stage microscopy

- Wet or solid state methods for determining material characteristics: solubility, electric

or heat conductance, mechanical properties such as compressibility, filtration

characteristics, electrostatic properties, drying properties

The most appropriate method for detecting or distinguish polymorphic forms is powder

diffraction techniques (X-ray or neutron) as they give intimate information on the crystal

lattice.

The art of polymorphism in all cases is the preparation of the desired form and keep it for the

required time in spite of thermodynamic force which directs the system towards more stable

forms, eventually to the thermodynamically stable form. For example the car paint should

have the same colour for decades in spite of 80-90 oC temperature close to the engine.

Similarly, a drug substance (API) goes through various formulation steps of significant

temperature and/or a solvent effect till it reaches the drug product state. The polymorphic

form of the API in the drug product has to be unchanged during formulation or extended

storage time because of pharmaceutical or Intellectual Property reasons. Preparation and

stabilization of different solid forms could be reached by very well controlled crystallization

and/or using appropriate additives. The ratio and nature of these additives could be a

manufacturing or know-how secret. Note: in several cases (for example chloramphenicol) the

polymorph transformation during formulation/storage could not be prevented.

1.4. Problem oriented analytical approach

13

It is a very strong requirement for the educational system worldwide to give the most recent

and at the same time useful information for the graduating students. This can be reached by

fostering the connection between academy and industry. In our case a novel discussion

methodology is applied throughout this course. The basic features of different analytical

techniques are summarized horizontally as it has been taught in analytical, structural, physical

chemistry and other courses. However, in a vertical dimension the applicability of the given

technique in polymorphism research is also discussed through recent case studies. This

problem oriented multidimensional teaching scheme hopefully helps students to choose the

right method to solve the emerging structural or manufacturing problem after graduation.

Regulatory and Intellectual Property issues are also reviewed to enhance the applicability of

the information gathered from the course.

1.5. The Ritonavir case

The Abbott Laboratories got approval from the Food and Drug Adminsitration, U.S. for a new

drug, Ritonavir against HIV on March 1st, 1996. This protease inhibitor as the main ingredient

of a ‘cocktail’ made it possible to regulate the virus rather than to kill it. Having this drug in

the preclinical stage the NBA star Magic Johnson could continue his sport career and together

with thousands of infected people got the chance for life of normal quality. However, on July

27th, 1998 one of the batches gave unsatisfactory results for the solubility test. As it is

prescribed by the corresponding FDA regulations the test was repeated but without

improvement. It turned out, that a new solid form of the drug emerged of very high stability

and at the same time low solubility ensuring not enough blood concentration of the drug for

satisfactory therapy. The following month gave wide range of publications and debates in

politics, business, tabloids and other media. The manufacturer tried several ways to solve the

problem, built new manufacturing lines and even a new plant with no success. The essence of

the problem turned to be a more stable polymorph with different hydrogen bond pattern21

and

crystal morphology than the earlier marketed drug. Finally, after one year of hysteric research

and trials the solution of the problem was a new soft gelatine capsule formulation. However,

because of stability reasons at high temperature, and precipitation of the low solubility form at

low temperature the drug product should be stored and transported all the time in a very

21 S. R. Chemburkar, J. Bauer, K. Deming, H. Spiwek, K. Patel, J. Morris, R. Henry, S. Spanton, W. Dziki, W.

Porter, J. Quick, P. Bauer, J. Donaubauer, B. A. Narayanan, M. Soldani, D. Riley, and K. McFarland: Dealing

with the Impact of Ritonavir Polymorphs on the Late Stages of Bulk Drug Process Developmentet, Org. Process

Res. Dev., 2000, 4, 413-417.

14

narrow temperature range of 2-8 oC, which gave a real challenge for manufacturers, traders,

pharmacies and the patients. As a scientific result more forms22

of the API were published.

22 S. L. Morissette, S. Soukasene, D. Levinson, M. J. Cima, and O. Almarsson: Elucidation of crystal form

diversity of the HIV protease inhibitor ritonavir by high-throughput crystallization, Proc. Natl Acad. Sci., USA,

2003, 100, 2180-2184.

15

1.5.1. Suggested project work: The Ritonavir case, literature highlights.

Figure 1.5.1.1

The Ritonavir case got intense publicity in the media

16

Chapter 2 Thermodynamics

Polymorphism is a kinetic phenomenon but thermodynamic considerations are very important

to understand the stability of the given form and also pinpoint possible polymorph

transformations in applications. The final goal is to draw energy-temperature diagrams for

all forms. To reach this goal thermal analytic techniques are applied. Unfortunately these

methods provide structural information in very rare cases but they are essential to conclude

kinetic and structural data, too.

2.1. Basics of thermoanalytical methods and their application in polymorphism research

In all thermoanalytical investigations effect of heat on the sample i.e. polymorphic forms are

studied. The main advantage of thermoanalytical methods is that sample preparation

requirements are rather simple, solid or liquid samples can be used to measure

thermodynamic quantities such as enthalpy change and transition temperature directly and

they can be simultaneous providing chemical and thermodynamic information in the same

measurement. However, in spite of tremendous efforts by the instrument manufacturers the

calibration is often cumbersome, experimental effects are difficult or cannot be eliminated and

in this way reproducibility is always a question. These techniques do not give structural

information.

The most frequently applied thermoanalytical methods:

2.1.1. Hot stage microscopy

Sample is heated or cooled (hot stage) and studied by means of a polarized light microscope.

It can be used to study23

- Compound morphology

- Solid-solid transformations

- Interaction between different compounds

- Dissolution of one compound in another

- Sublimation and/or evaporation

- Melting or liquefaction upon heating (solid-liquid transformations)

23 http://particle.dk/methods-analytical-laboratory/hot-stage-microscopy/

17

- Solidification upon cooling (liquid-solid transformations)

- Crystal growth and rate thereof

2.1.2. Differential Thermal Analysis (DTA)

The sample and an inert reference material are subjected to the same heat treatments (heating,

maybe in repeated cycles) and temperature difference between the sample and the reference is

recorded. The area under a DTA peak is the enthalpy change and is not affected by the heat

capacity of the sample. DTA curve provides data on the following transformations24

- Crystallization

- Melting

- Sublimation

- Glass transition

- Decomposition

- Evaporation etc.

2.1.3. Differential Scanning Calorimetry (DSC)

In this technique25

the heat required to increase the temperature of a sample and the reference

material is measured as a function of temperature. The method can be power compensated or

heat flux DSC. The required temperature can be modulated with periodic function (Modulated

DSC, MDSC). Calibration is a very important issue and the measured curve may strongly

depend on the heating rate (Figure 2.1.3.1). Energy changes of transitions and heat capacity

can be measured during

- Crystallization

- Melting

- Sublimation

- Glass transition

- Decomposition

- Evaporation etc.

24 http://en.wikipedia.org/wiki/Differential_thermal_analysis 25 http://en.wikipedia.org/wiki/Differential_scanning_calorimetry

18

Figure 2.1.3.1.

Effect of heating rate on melting (left) and a glass transition (right)

2.1.4. Thermogravimetry Analysis (TGA)

The mass of the sample is recorded during thermal treatment.26

The method can be coupled

with other techniques (Mass Spectrometry, Gas Chromatography or FTIR spectroscopy) to

analyse the decomposition products. The first commercial TG/DTA instrument, the

Derivatograph was developed by Hungarian researchers27

in the 1950s (Figure 2.1.4.1). The

method gives stoichiometry information on

- Vaporization

- Second order phase transition

- Absorption

- Adsorption

- Desorption

- Desolvation (dehydration)

- Sublimation

- Decomposition

- Solid-gas reactions28

etc.

26 http://en.wikipedia.org/wiki/Thermogravimetric_analysis 27 F. Paulik, J. Paulik and L. Erdey: Der Derivatograph I, Z. Anal. Chem., 1958, 160, 241. 28 a) D. Braga and F. Grepioni: Polymorphism, Crystal Transformations and Gas-Solid Reactions Design in

Crystal Design: Structure and Function Perspectives in Supramolecular ChemistryVolume 7, Ed. G. Desiraju,

John Wiley & Sons, Chichester, 2003, pp. 325-374.

19

Figure 2.1.4.1.

Schematic drawing of the Paulik-Paulik-Erdei Derivatograph. 1: Sample 2: Reference 3: Oven

4: Thermocouples 5: Corrund tubes 6: Flexible junction 7: Coil 8: Magnet 9: Optics 10:

Galvanometers 11: Photopaper

2.2. Energy-temperature diagrams. Monotrope and enantiotrope systems

In all cases the following questions should be answered on the basics of thermal analysis:

- How many polymorphs are formed under which conditions and what is their stability

relationship?

- Which polymorph of the API is the stable one?

- If the forms are enantiotropically related (see later) what is the transition temperature?

What is the enthalpy effect of the transition?

b) M. D. Meijer, R. J. M. Klein Gebbink and G. van Koten: Solid-Gas Interactions Between Small Gaseous

Molecules and Transition Metals in the Solid State. Toward Sensor Applications, in Crystal Design: Structure

and Function Perspectives in Supramolecular ChemistryVolume 7, Ed. G. Desiraju, John Wiley & Sons,

Chichester, 2003, pp. 375-386.

20

- Which forms are metastable ones and could they be transformed into the stable form

under the effect of formulation and/or storage? Can the metastable form(s) be used for

pharmaceutical purposes?

The answers can be deducted from thermoanalytical measurements and solubility studies.

Preparing energy-temperature diagrams29

gives valuable information. The energy-temperature

diagram is the graphic representation of the Gibbs-Helmholtz equation

(Figure 2.2.1).

Figure 2.2.1

Schematic energy-temperature diagram for enantiotrope and monotrope system, respectively.

For explanation see the text.

For the sake of simplicity we discuss systems where only two polymorphic forms exist

(dimorphic system). According to the nomenclature suggested form I has the higher melting

point. At Figure 2.2.1 the blue line represents the enthalpy (H) and the free energy (G) curve

of the liquid. The enthalpy (H) curves of the solid forms cannot intersect each other i.e. they

are close to parallel. From DSC measurements the heat capacity of the different forms can be

determined at various temperatures and using the formula (

) the H-T curves of the

forms can be constructed. The enthalpy of transition can be determined, too. The G isobars of

two forms can never intersect twice. If there is no intersection of the G curves one of the

forms is more stable than the other one at any temperature (stable and metastable forms,

29 A. Grunenberg, J. O. Henck and H. W. Siesler: Theroretical and practical application of energy/temperature

diagrams as an instrument in preformulation studies of polymorphic drug substances, Int. J. Pharm., 1996, 129,

147-158.

21

respectively) and the system is called monotropic or monotrope. Monotropic forms can be

interconverted only through the gaseous or liquid phase i.e. the form should be melted or

dissolved or pass to the gaseous state. However, it is also possible that two solid forms are in

thermodynamic equilibrium at a given temperature i.e. their G curves intersect as in

equilibrium

. These systems called enantiotropic or enantiotrope and below the

transition temperature the phase of the lower melting point is the stable form while above the

transition temperature the phase of the higher melting point is the stable one. It is very

important that the transformation is reversible and the transformation does not go through

gaseous or liquid phase The spontaneous solid-solid transformation can occur during storage

time. The G curves can be constructed from solubility measurements as

where and are the equilibrium solubility of form II and form I in a given solvent,

respectively. The less soluble form with the lowest vapour pressure is the more stable one.

The simultaneously appearing polymorphs are called concomitant polymorphs.30

2.3. The Burger-Ramberger rules

The energy-temperature diagram for a real system is more complicated than it is shown at

Figure 2.2.1. Several forms can exist and experimental errors make it difficult to see minute

differences. DSC measurements are prone to experimental and instrumental errors. Solubility

studies are also difficult and time consuming, especially if the solubility is very high or very

low. Burger and Ramberger performed comprehensive thermoanalytical studies31

on hundreds

of pharmaceutical compounds and their findings can be summarized into simple rules, these

are the Burger-Ramberger rules. The heat of transition rules and the heat of fusion rules can

be easily interpreted on Figure 2.2.1 and these are consequences of thermodynamic

considerations. The density and the infrared rules contain structural considerations but there

are several systems for which they are not valid.

2.3.1. Heat of transition rules

30 J. Bernstein, R. J. Davey and J.O. Henck: Concomitant Polymorphs, Angew. Chem. Int. Ed. Engl., 1999, 38,

3440-3461. 31 a) A. Burger and R. Ramberger: On the polymorphism of pharmaceuticals and other molecular crystals. I:

Theory of Thermodynamic Rules. Mikrochim. Acta II, 1979, 259-271.

b) A. Burger and R. Ramberger: On the polymorhism of pharmaceuticals and other molecular crystals. II:

Applicability of Thermodynamic Rules. Mikrochim. Acta II, 1979, 273-316.

22

No. 1: If an endothermic peak is observed at a given temperature on the DSC curve it

may be assumed, that there is a transition point below it and the two forms are related

enantiotropically.

In enantiotrope systems the green-red transition is endothermic,

. The red G-curve

represents the stable form (Figure 2.2.1, left) at any temperature higher than the transition

temperature. At lower temperature the other form is the thermodynamically stable one.

No. 2: If an exothermic peak is observed at given temperature on the DSC curve it may

be assumed, that there is no transition point below it and the two forms are related

monotropically.

In monotrope systems the red-green transition is always exothermic,

(Figure

2.2.1, right). The green G-curve represents the stable form at any temperature; it is of the

higher melting form, the other(s) are metastable one(s).

2.3.2. Heat of fusion rules

No. 1: If the higher melting form has lower heat of fusion the two forms are related

enantiotropically.

No. 2: If the higher melting form has higher heat of fusion the two forms are related

monotropically.

These rules are obvious from Figure 2.2.1.

2.3.3. Entropy of fusion rule

The entropy of fusion is

. This rule is the consequence of the two above rules.

In enantiotrope systems the higher melting form has the lower entropy of fusion. In

monotrope system the lower melting form has the lower entropy of fusion.

23

2.3.4. Heat capacity rule

The heat capacity is the first derivative of the H-T curve.

In enantiotrope systems the higher melting form has higher heat capacity at a given

temperature and the opposite is true for monotrope systems.

There are exemptions from this rule.32

2.3.5. Density rule

If a modification has lower density than another one, then it may be assumed that at

absolute zero temperature this crystal form is metastable (less stable).

This rule is valid if van der Waals interactions determine the solid state structure. Voids can

be in the lattice if hydrogen bonds are dominant to stabilize the structure resulting lower

density for the more stable form. Moreover, the accuracy of density measurement of different

forms is much lower than calculating their density on the basis of single crystal data. Unlike

the earlier Burger-Ramberger rules (Chapters 2.3.1-2.3.4) this rule is not a consequence of

thermodynamics.

2.3.6. Infrared rule

If the first absorption band in the infrared spectrum of a hydrogen-bonded molecular

crystal is at higher wavenumber for a modification than for the other one, that form

may be assumed to have the larger entropy.

This rule has also experimental difficulties to detect as the exact wavelength of a broad O-H

band is problematic to determine. Moreover, there are numerous exemptions because of

structural peculiarities

32 R. Hilfiker: Polymorphism in Crystalline Systems in Crystallization: Basic Concepts and Industrial

Applications. Ed. W. Beckmann, Wiley-VCH, Weinheim, 2013, p 91.

24

2.4. Polymorphism of caffeine and chocolate

Chocolate is a popular topic33

for polymorphism. In spite of detailed studies and numerous

practical as well as technological advances there are still open questions on the structure of

various forms. At the moment six polymorphic forms of cocoa butter are known having

melting points between 16oC

and 36

oC. It is advantageous if there is the beta2 polymorph

(some authors cite as polymorph V) in the final chocolate product the melting point of which

is 32-34oC. Moreover, cocoa butter is an ingredient of various drug products. From a more

general point of view crystal structure of triacylglycerols has paramount importance in food

industry, cosmetics, biology etc. Polymorphism of caffeine is also studied34

in details

including forms with disordered structures.35

The Cambridge Structural Database (Ver. 5.35

Update February, 2014) contains six polymorphic forms of anhydrous caffeine. Anhydrates

and hydrates of caffeine are subject36

of polymorphism studies frequently.

2.4.1. Suggested project work: The polymorphism of cocoa butter. Pharmaceutical

consequences

2.5. The polymorphism of nimodipine and sulfamethoxidiazine

The nimodipine case37

represents a successful application of thermodynamic principles to

predict38

the temperature of transition point. Two forms of nimodipine are forming an

enantriotrope system and the transition point could be calculated on the basis of heat of fusion

and heat capacity data of the liquid and the two forms as well as melting temperatures all

33 G. Tannenbaum: Chololate: A Marvelous Natural Product of Chemistry. J. Chem. Ed., 2004, 81, 1131-1135. 34 A. Hédoux, Y. Guinet, L. Paccou, F. Danède, and P. Derollez: Polymorphic transformation of anhydrous

caffeine upon grinding and hydrostatic pressurizing analyzed by low-frequency Raman spectroscopy. J. Pharm.

Sci., 2013, 102, 162–170. 35 A. Hédoux, A. A. Decroix, Y. Guinet, L. Paccou, P. Derollez and M. Descamps: Low- and High-Frequency

Raman Investigations on Caffeine: Polymorphism, Disorder and Phase Transformation, J. Phys. Chem. B, 2011,

115, 5746–5753. 36 E. Dichi, B. Legendre, M. Sghaier: Physico-chemical characterisation of a new polymorph of caffeine, J

Therm. Anal. Calorim., 2014, 115, 1551–1561. 37 A. Grunenberg, B. Keil and J. O. Henck: Polymorphism in binary mixture, as exemplified by nimodipine, Int.

J. Pharmaceutics, 1995, 118, 11-21. 38 L. Yu: Inferring thermodynamic stability relationship of polymorphs from melting data, J. Pharm. Sci., 1995,

84, 966-974.

25

derived from DSC measurements. Sulfamethoxidiazine has also several polymorphic forms,

six of them reported39

by Burger et al.

2.5.1. Suggested project work: Search the literature for compounds forming

enantiotropic or monotropic systems

39 A. Burger, R. Ramberger and K. Schulte: Analyse des polymorphen Systems von Sulfamethoxidiazin, Arch.

Pharm., 1980, 313, 1020-1028.

26

Chapter 3 Patents

The main driving force of polymorphism research is usually an IP issue. New forms can be

patented and huge financial advantage can be reached on the basis of polymorphism in

pharmaceutical industry. Public hearings of polymorphism cases reveal the difficulty to

sentence the true verdict in scientific questions by the law.

3.1. Patent literature basics

The patent is basically a contract between the inventor (patentee) and the government. The

inventor makes his invention public and if this invention is good for the general public, the

government grants exclusive rights for the inventor for a certain time and protects the rights of

the inventor with its power.

The first patent-like royal or governmental decrees were issued in Europe in the XVth

century. The first English patent was granted by King Henry VI to John Utynam for his

invention by which he made the stained glass windows of King College. However, the first

general patent law was issued in Venice in 1474 to provide exclusive rights for craftsmen who

disclose their technology. Today all nations have their own patent laws and the World

Intellectual Property Organization40

(WIPO) is the main board for handling international

registration systems of intellectual property treaties. As technology and life change these

treaties are modified but it is rather difficult to reach consensus as these negotiations imply

high volume financial consequences. Beside patent laws other issues connected to intellectual

property are handled by WIPO including

- Copyright and related rights

- Patents

- Trademarks

- Industrial design

- Geographical indicators

- Laws and regulations against unfair competition

- Protection of new varieties of plants

40 www.wipo.int

27

Title 35 of the United States Code (U.S.C.)41

will be reviewed here.

A few important concepts, for definitions, see relevant web pages:

- Patent

- Patent fee

- Royalty fee

- Invention

- Claim

- Specification

- Patent infringement

According to 35 U.S.C. requirements of patentability, (underlined text for this book):

- Utility, 35 U.S.C. § 101

“Whoever invents or discovers any new and useful process, manufacture, or composition of

matter, or any useful improvement thereof, may obtain a patent thereof, subject to the

conditions and requirements of this title.”

- Novelty , 35 U.S.C. § 102

“A person shall be entitled to a patent unless-

(a) the invention was known or used by others in this country, or patented or described in a

printed publication in this or a foreign country, before the invention thereof by the

applicant.”

(b) the invention was patented or described in a printed publication in this or a foreign

country or in public use or on sale in this country, more than one year prior to the date the

application for patent in the United States.”

- Non obviousness, 35 U.S.C. § 103

“A patent may not be obtained…if the differences between the subject matter as a whole

would have been obvious at the time the invention was made to a person having ordinary skill

in the art to which said subject matter pertains.”

41 http://en.wikipedia.org/wiki/Title_35_of_the_United_States_Code

28

Patentable inventions include:

– Processes

– Machines

– Manufacture

– Compositions of matter (Polymorphs)

Non patentable inventions, examples:

– Products naturally occurring in nature

– Scientific principles

– Laws of nature

– Mental processes

However, as in all cases the law gives only the frame and there are ample possibilities

especially to debate on the utility (Figure 3.1.1) and non-obvious nature of the invention.

Novelty is somewhat simpler to detect Applications are checked for novelty during the one

year examination period by the USPTO (United States Patent and Trademark Office) and

other National Patent Offices.

Figure 3.1.1.

Examples for ‘useful’ inventions which got patents.42

3.2. The art of constructing claims

42 http://erice2004.docking.org/vcourse/polymorph/18fri/1000-Levine/Levine.ppt

29

The 35 U.S.C. § 112 details the requirements of patent applications. Specification is basically

the disclosure of the invention and using the specification a person skilled in the art should be

able to reproduce the invention. According to the law the specification should cover the best

method, which is sometimes a rather debatable issue. The patent file “shall conclude with one

or more claims particularly pointing out and distinctly claiming the subject matter which the

applicant regards as his invention.” Claims are basically intended to clearly distinguish what

belongs to the invention and what is not. This is a very important question as having too

detailed claims the competitors may easily find alternative methods to prepare the invention

while too broad claims are also weak to protect the invention. Writing the appropriate claims

can be considered even as an art (see later the ranitidine hydrochloride case).

Nowadays existing patent laws are challenged by new scientific developments and changes in

the economy as well as in the society

- Questions on patentability of genetic and other -omics (proteomics, transcriptomics

etc.) inventions including patenting large gene sequence varieties.

- Ethical problems related to gene manipulated organizations and genetics of humans.

- Questions regarding high royalty fees which are unacceptable for underdeveloped

countries. They try to reach exemptions of patent laws on the basis of humanitarian

and/or economic grounds.

3.3. Polymorphs and patents, IP issues

A new solid form e.g. a new polymorph of the API usually can be patented and this opens

large playing field for both innovative and generic pharmaceutical companies. Polymorphism

screening is an essential part of the development of any innovative drug. As the Ritonavir

case (Chapter 1.5) shows polymorphism may cause serious problem, even after several years

of launching. Manufacturers of generic drugs also apply significant efforts to find new

polymorphs which can be patented and if the usefulness can be proved exclusivity can be

reached on the market. The real usefulness of a new solid modification for example the

advantages in production process are really difficult to challenge. However, when the

infringement of the patent is cited to court the case has serious danger for (pharmaceutical)

companies. A public trial may lead to disclose other important issues of company

management and this may cause more harm than the profit on that particular product. In other

30

areas, recently the Apple vs. Samsung trial gave an example for this as Apple reached a

victory of Pyrrhus. Although Samsung was sentenced to pay several billions of dollars but

Apple had to show publicly the exact methodology of its design/planning pipeline. It is not

surprising that agreements are reached between companies before the public trial of

polymorph cases, too.

3.4. The ranitidine hydrochloride, paroxetine hydrochloride and aspartame cases

These cases represent classic examples when large amount of money triggered pharmaceutical

giants to reach exclusivity on production of polymorphic forms.

The Glaxo had an annual sale of more than $3 billion in 1992 on Zantac, which was the trade

name of its drug product containing ranitidine hydrochloride. After expiry of the patent for the

original form (USP 4.128.658) in 1995 other companies including Novopharm tried to

manufacture it but found, that the product of the synthesis is Form 2 for which Glaxo had a

living patent (USP 4.521.431).43

“Glaxo filed a patent application covering Form 2 ranitidine hydrochloride in the United

Kingdom on October 1, 1980. It filed a United States application thereon the next year, which

eventually issued as the '431 patent in suit. When George Graham Brereton, Glaxo's patent

officer initially charged with pursuing the United States application, learned of the azeotropic

granulation process and Glaxo's desire to keep that process secret, he recommended that

Glaxo not claim pharmaceutical compositions of the Form 2 salt for fear of violating the best

mode requirement. Brereton apparently believed that disclosure of the azeotroping process

would be necessary because it was the best way to make the Form 2 salt for use in preparing

pharmaceutical compositions. He later moved to another position at Glaxo. The U.S.

application was eventually amended to include pharmaceutical composition claims, but Glaxo

did not amend the specification to disclose the azeotropic process.”

”On August 9, 1991, Novopharm Ltd. filed an Abbreviated New Drug Application (ANDA)

with the Food and Drug Administration (FDA), seeking FDA approval to manufacture and

sell a generic version of Form 2 ranitidine hydrochloride beginning December 5, 1995, the

expiration date of the '658 patent, well before the expiration date of the '431 patent in 2002.

43 http://openjurist.org/52/f3d/1043/glaxo-inc-v-novopharm-ltd

31

Glaxo filed this suit for patent infringement on November 13, 1991, alleging technical

infringement of claims 1 and 2 of the '431 patent by the ANDA filing as provided in 35 U.S.C.

Sec. 271(e)(2) (1988). Novopharm admitted infringement of the claims, but contended that the

'431 patent was invalid because it was anticipated by the disclosure of the '658 patent.”

After several rounds of trials and appeals it turned out, that the original patent really gives

Form 1 as it is proved by experiments of an independent laboratory. However exact know-

how from 20 years old lab notes should be given to them by Glaxo. Example 32 of the patent

(Figure 3.4.1), although seems to be extremely simple, contains such experimental nuances

which prevented competitors to prepare the product exactly on the same way in the same

quality. In this trial it was also the subject of the dispute what does it mean to disclose the best

mode of the invention:

“The best mode requirement arises from the first paragraph of 35 U.S.C. Sec. 112 (1988),

which provides that the patent specification "shall set forth the best mode contemplated by the

inventor of carrying out his invention." The best mode inquiry is twofold: first, did the

inventor know of a preferred mode or embodiment of the invention; and second, did the

inventor disclose that mode sufficiently to allow those skilled in the art to practice it.”

“Novopharm says Glaxo did not disclose the best mode of practicing the invention--the

azeotropic granulation process it used to formulate ranitidine hydrochloride into

pharmaceutical compositions. This defense became relevant only late in the game when, on

June 2, 1993, Glaxo produced some documents that indicated that it had withheld information

about the granulation process. Novopharm moved for summary judgment based on the best

mode violation, which the district court denied, Glaxo, Inc. v. Novopharm Ltd., 830 F.Supp.

869 (E.D.N.C.1993), but allowed Novopharm to present the best mode defense at trial.

Novopharm tried to take discovery on the issue, including a deposition of Crookes, the named

inventor of the '431 patent, and Collin, his immediate supervisor. After Glaxo resisted and

sought a protective order, the district court denied Novopharm any discovery, and the case

proceeded to trial on August 9, 1993. At the close of Novopharm's case-in-chief on its best

mode defense, the court decided to rule on that question as a matter of law, no factual issues

remaining. Glaxo presented no evidence on the issue. The court held that because Crookes

had no personal knowledge of the best mode, there was no requirement that it be disclosed.”

32

“But the statutory language demands that the patent disclose the best mode of "carrying out"

the claimed invention. As the district court recognized, this language encompasses not only

modes of making the invention, but of using it as well. See Christianson v. Colt Indus.

Operating Corp., 822 F.2d 1544, 1563, 3 USPQ2d 1241, 1255 (Fed.Cir.1987), vacated on

other grounds, 486 U.S. 800, 108 S.Ct. 2166, 100 L.Ed.2d 811 (1988). The azeotropic process

was the best way to formulate raw ranitidine hydrochloride into pharmaceutical compositions

suitable for use as a drug in human patients, the only use Glaxo contemplated for the

invention. Glaxo admits the process was not generally known to those skilled in the art.

Accordingly, the court could have found that disclosure of the process was required by

section 112, so long as the other, subjective, elements of the best mode test were met. Cf.

Chemcast, 913 F.2d at 930, 16 USPQ2d at 1038 ("Whether characterizable as

'manufacturing data,' 'customer requirements,' or even 'trade secrets,' information necessary

to practice the best mode simply must be disclosed.").”

Finally the same Court found ample support for the court’s factual finding that Glaxo failed to

prove infringement under a single peak analysis.44

This API and Example 32 caused headache

for pharmaceutical companies of the Soviet blocks at that time, too. The patent law was

different in these countries. Pharmaceutical companies tried to prepare the drug but they

failed to repeat Example 32 satisfactorily.

44

Philip Good: Applying Statistics in the Courtroom: A New Approach for Attorneys and Expert Witnesses

(Google e-book), CRC Press, 2001, p. 112.

33

Figure 3.4.1

The famous Example 32 to prepare ranitidine hydrochloride.

The next case, the paroxetine hydrochloride was also a billion dollar business in the 90’s.

Originally it was discovered as an anhydrate API but because of handling and formulation

reasons the marketed drug product contained the hemihydrate form. When the protection for

the anhydrate expired competitors developed the production of anhydrate. The originator,

SKB recognized the infringement of its patent rights. According to generic companies the

anhydrate transformed into hemihydrate form in spite of moisture excluding packing. Finally

in 2003 at the U.S. Federal Court in Chicago by Judge Richard Posner accepted that the

anhydrate formula does not infringe the patent protection of the hemihydrate form as the

quantity of the hemihydrate was very low.45

“To summarize: I construe claim 1 of SmithKline’s patent ‘723 to cover crystalline paroxetine

hydrochloride hemihydrate in any commercially significant quantity, and so construed the

45 http://openjurist.org/403/f3d/1331/smithkline-beecham-corporation-plc-v-apotex-corp

https://law.resource.org/pub/us/case/reporter/F3/403/403.F3d.1331.03-1313.03-1285.html

34

claim is valid against the various attacks on it made by Apotex but clearly will not be

infringed by Apotex’s anhydrate product.”

“Some conversion from anhydrate to hemihydrate is likely to occur in a seeded facility in

which the anhydrate is exposed to air; BCI’s plant is seeded; and the anhydrate manufactured

there is exposed to nondehumidified air before it leaves the plant. The evidence is sufficient to

support an inference that BCI will be making at least tiny amounts of the hemihydrate if it is

permitted to manufacture the anhydrate.”

“In sum, I am not persuaded that Apotex will produce an anhydrate that has sufficient

hemihydrate to be detectable by the methods in use in 1985.”

“The reason for excusing the alleged infringement in this case is not that Apotex stole only a

little hemihydrate from SmithKline. It stole nothing from Smithkline. It doesn’t want

hemihydrate, and it derives no value from the hemihydrate that it unavoidably creates and

“sells”. If it made hemihydrate deliberately, or if it took advantage of 100 percent conversion

to obtain a product that had hemihydrate’s superior handling characteristics, that would be

theft and it would be nonsense to point out that paroxetine is only 10 percent of the pill by

weight.“

However, after an appeal in 2004 the Federal Circuit reversed the claim construction of the

district court and in its Judgement infringement was declared. (Later Judge Richard Posner

was the jury in the Apple vs. Samsung case in 2012-2013.)

“In summary, this court reverses the claim construction of the district court and holds that

claim 1 covers any amount of crystalline paroxetine hydrochloride hemihydrate without

further limitation…affirms the district court’s finding that Apotex’s PHC anhydrate product

will infringe claim 1 under that broad construction…”

“…Notwithstanding that conclusion, this court holds, based on the undisputed facts, that

SmithKline’s clinical trials constituted a public use under §102(b) rendering claim 1 invalid.

Apotex is, therefore, not liable for infringing claim 1 of the ‘723 patent.”

The key compound of the third case, aspartame is a well-known sweetener. Originally

developed by Monsanto but to prevent liability cases, because of phenylalanine production

during the metabolism of aspartame, rights were transferred to a Japanese company

Ajinomoto. In this case it is interesting, that in Europe Ajinomoto applied for protection a

morphologically new form rather than a true polymorph. According to their point this form

35

could be prepared by slow addition of the precipitating agent and the result is a more

crystalline/single crystal like solid with advantageous filtration and drying properties. In this

case the X-ray powder pattern clearly shows, that the earlier and the new form has the same

lattice and only the crystallinity and the particle size differ. Finally experts of Ajinomoto

could prove the existence of the new form using X-ray powder diffraction (Figure 3.4.2) and

solid state NMR methods (Chapter 9.3). However, the European Patent Office issued new

guidelines and the morphology of the form cannot be patented any more.

Figure 3.4.2

Comparison of aspartame structures from the Cambridge Structural Database (calculated

powder patterns, up) with X-ray powder diffraction pattern of small crystals (wide peaks) and

powder pattern of larger single crystals (narrow peaks).

3.4.1. Suggested project work: A novel patent case of polymorphism. Pharmaceutical

consequences.

36

Chapter 4 Crystallization

Crystallization is highly non-linear process. It is very attractive in pharmaceutical industry

with wide spread applications as a purification and separation technique. Controlled

crystallization of the desired polymorph is a tempting challenge of research.

4.1. Crystals show long range order

Solid materials can be crystalline when long range order is observed or amorphous when only

short range order occurs in the solid state. Crystalline materials consist of unit cells as the

repeating unit and the whole macroscopic crystal can be built when these unit cells are shifted

in 3D in the direction of the cell axis vectors a, b and c. This model describes only perfect

crystals but in reality macroscopic crystals contain domains of perfect regions. The unit cell is

the smallest part which shows the symmetry of the lattice. It is well known that by breaking a

single crystal the small pieces are similar to the original one with the same angles of facets.

To fill the space or the plane continuously with uniform pieces can be reached only with

limited kind of unit cells given by the symmetry. For example trigonal, tetragonal or

hexagonal motives could be used to fill a plane but pentagonal or octagonal will always leave

empty regions behind. These and similar symmetry elements were thought to be forbidden in

3D lattices. However it turned out that it is not true and the Nobel Prize was awarded46

in

2011 to Dan Shechtman for the discovery of quasi-periodic crystals. In 1992 the International

Union of Crystallography had modified the definition of crystals as “a material is a crystal if

it has essentially a sharp diffraction pattern”.47

However, in this book we use the old

definition of crystal, mentioned above, which is based on filling direct space with motives

rather than the softer definition of IUCR based on reciprocal space.

4.2. The thermodynamics of phase transitions and crystal forming

Crystallization usually occurs in a non-ideal multicomponent system far from the

equilibrium.48

First the phase diagram49

of one component two phase systems is considered.50

46 http://www.nobelprize.org/nobel_prizes/chemistry/laureates/2011/press.html 47 http://reference.iucr.org/dictionary/Crystal 48 Encyclopedia of Supramolecular Chemistry, Eds. J.L: Atwood and J.W. Steed, Marcel Dekker/Taylor &

Francis Group: New York, 2004, page 365-370. 49 http://en.wikipedia.org/wiki/Phase_diagram

37

The phase diagram gives the combination of state parameters (pressure, temperature, molar

volume and in case of multicomponent systems concentration) in equilibrium. Gibbs’ phase

rule applied for such systems and the lever rule helps to calculate the amount of material in

the various phases. However, in real cases the system may reside in other points of the

parameter space even for very long time but minute disturbance causes the system to jump

into a point of the equilibrium curve. For example overheated water taken out from a

microwave oven51

is very dangerous in the kitchen as under the effect of vibration steam is

formed instantaneously.

The liquid/vapour phase transition can be described using the van der Waals equation. While

ideal gases obey the most simple state equation of

, van der Waals gases having the

(

)

state equation show a maximum-minimum curve at the pV plane (Figure

4.2.1). Here parameter b demonstrates that the molecules have finite volume and they are not

point while parameter a represents interaction among gas molecules.

Figure 4.2.1

50 For more detailed description: Nucleation Theory and Applications. Ed. J.W.P Schmelzer, Wiley, Weinheim,

2005. 51 http://www.youtube.com/watch?v=HKC37PoELbo

38

Isotherm of ideal gas (upper curve) and van der Waals gas (lower curve). For further

explanation see text.

Having our system as a van der Waals gas in A and increasing the pressure (Figure 4.2.1) to B

the liquid phase appears. Depending on the mass ratio of the liquid and vapour phase the

system is located at BE. In E we have liquid phase which is essentially uncompressible so the

system is at EF when higher pressure is applied. However, by careful pressurizing the system

can be somewhere at the BC (green) curve, too. This non equilibrium sate is very unstable and

can be stabilized when small vibration or other effects cause the system to jump to BE curve.

Similarly, the system can be at ED curve by careful expansion of the liquid. For real systems

similar phase diagrams can be detected experimentally. The CD curve (red) cannot be reached

with the homogeneous system by any means because of thermodynamic reasons. The free

energy curve has a negative curvature, its second derivative in respect the parameter

(temperature, concentration etc.) is negative. In this region spinodal decomposition52

occurs

into two phases i.e. no nucleation is necessary for the phase transition. The spinodal region

can be reached by means of very fast parameter change, for example quenching.

Similar phase diagrams can be constructed for multicomponent systems too, when a solute (S)

is dissolved in a good solvent. Solid material is formed (Figure 4.2.2) when a precipitating

agent (bad solvent or anti solvent) is added.

52 http://en.wikipedia.org/wiki/Spinodal_decomposition

39

icckdt

dN)(

Figure 4.2.2

Phase diagram of crystallization

The phase diagram of crystal forming can be constructed using experimental methods. When

the purpose is single crystal growing for example to perform single crystal X-ray diffraction

experiment the system should be kept in the metastable zone. However, in manufacturing

pharmaceutically active compound as solid material it is more important to keep controlled

and uniform particle size and crystallinity.

4.3. Kinetics of crystallization

The description of kinetics of crystal growing is also empirical. The first step of crystal

growing if the system is in the nucleation zone is the formation of crystal seeds i.e. nuclei of

crystals provided the system is not in the spinodal region of the phase diagram. Theoretically

these seeds can be formed from homogeneous solution by aggregation of several hundreds or

thousands of atoms. There are organometallic compounds which readily form clusters.

However, as total exclusion of dust or other solid contaminants of very small size is a very

challenging or even impossible task it is difficult to prove that the nucleation occurred really

from homogeneous solution. When the nucleus of a crystal is a small particle/contaminant

which has different composition than that of the bulk crystal we speak of heterogeneous

nucleation. In real systems secondary nucleation also occurs as nuclei are formed by breaking

the existing crystals into smaller pieces because of stirring or other mechanical effects. The

phenomenological description of nucleus formation reveals highly non-linear kinetics i.e.

differential equation of form

(4.3.1)

Here N is the number of crystal seeds, i > 1, c and c* is the concentration of the solute in the

supersaturated (crystallizing) system and in the system which is in thermodynamic

equilibrium, respectively. Finally k is the rate coefficient of this highly non-linear system

which depends on temperature and other effects but practically does not depend on the

concentration of the solute.

The next step is crystal growing. The mechanism can be controlled by diffusion or by

convection. The diffusion control is important when the purpose is growing single crystals.

40

)( ccAkdt

dM

Application of Fick’s first law of diffusion the change of crystal mass (M) in time can be

described by a linear differential equation:

(4.3.2)

Here k is the mass transfer coefficients which depends on the diffusion constant of the solute,

viscosity, temperature, density etc., while c and c* are the same concentrations as above and A

is the surface of the crystal. The surface is difficult to determine during crystal growing

nevertheless one can conclude that the rate of crystal growing is proportional with the

distance of the system from the equilibrium state. If the effect of convection is not negligible

the form of the differential equation which describes the growing of crystal mass in time is

rather similar:

(4.3.3)

Where k is similarly the surface rate coefficient and κ is the stirring rate dependent

coefficient. Combining these crystal growing models the rate of linear growing also depends

on the difference of concentrations

(4.3.4)

For small size crystals the Ostwald ripening53

occurs. As the solubility of small crystals is

higher than larger ones the small crystals dissolve while bigger crystals grow further. This

phenomenon can be modelled by calculating the surface energy of the crystal. The occurrence

of the Ostwald ripening depends on the actual system but generally it plays significant role

when the size of the crystals is in the μm range or smaller. The Ostwald ripening is very

important in micronization (Chapter 13.4).

53 http://en.wikipedia.org/wiki/Ostwald_ripening

)(11

1

ccA

k

dt

dM

)()(11

12

cckcc

k

dt

dlg

V

A

41

Chapter 5 Polymorphism prediction

Prediction of solid forms solely on the basis of in silico methods is a new and quickly

emerging field of research. Calculating the structure and stability of solid forms of small rigid

molecules can be achieved with considerable success. However, predicting the solid state

structure of flexible molecules, salts or cases when more than one molecule is present in the

asymmetric unit represent open questions for our present computational and theoretical tools.

5.1. Computational approach, polymorphism prediction

Having the difficulties in controlling the crystallization process and especially polymorph

composition of the product, outlined in Chapter 4, it was a plausible new direction of research

to predict crystal structures using computational methods. However, even in the early 90’s the

scientific community was rather reluctant to absorb the limited success in this field. One could

read rather disappointing comments i.e. ”No general solution for predicting crystal structures

is expected in the near future”54

or “Interactions in the lattice are too complicated to

handle”.55

Fortunately two decades of intense research and improvement in theory,

computational methods and hardware made this field a blooming area.56

5.2. Blind tests for polymorphism prediction

The Cambridge Structural Database periodically organizes blind tests57

for polymorphism

prediction by disclosing the chemical structure of various target molecules and researchers

give suggestions for the solid state structure. Of course, the single crystal X-ray diffraction

data are available for the targeted compounds – sometimes even more polymorphs – and it is

not allowed to prepare and measure crystals of the compounds.

There are approximately 20 research groups working in this field worldwide and fortunately

most of them regularly participate in the competition. These blind tests and especially their

results (Figure 5.2.1) are interesting to review the progress in the field.58

54 F.C. Hawthorne: Crystals from first principles, Nature, 1990, 345, 297. 55 P.J. Fagan and M.D. Ward: Building Molecular Crystals, Scientific American, 1992, 267, 48-54. 56 S.L. Price: Predicting crystal structures of organic compounds, Chem. Soc. Rev., 2014, 43, 2098-2111. 57 https://www.ccdc.cam.ac.uk/Community/Initiatives/Pages/CSPBlindTests.aspx 58 G.M. Day: Computational Crystal Structure Prediction: Towards In Silico Solid Form Screening in Organic

Crystal Engineering, Frontiers in Crystal Engineering, Eds. E. R. T. Tieknik, J. Vittal, M. Zaworotko, John

Wiley & Sons, Chichester, 2010, pp. 43-64.

42

Figure 5.2.1

Results59

of the first four blind tests for polymorph prediction organized by the Cambridge

Structural Database

As a result of these blind tests60

one can conclude, that solid state structure of rigid small

molecules can be predicted with promising accuracy.61

However flexible molecules with free

rotation around a few bonds represent tempting challenge. It is especially difficult to handle

cases using crystal structure predicting methods when more than one molecule are in the

asymmetric unit.

5.3. Difficulties of QM calculations of van der Waals interactions.

The classical basic approach in quantum mechanical calculations is the Hellmann-Feynman

Electrostatic Theory, i.e. in the Born-Oppenheimer approximation, where nuclei see a static,

electron cloud. The forces acting at nuclei are just Coulombic forces exerted by other nuclei

59 http://erice2004.docking.org/vcourse/polymorph/11fri/1700-Price/Price.ppt 60 G. M. Day et al.: Significant progress in predicting the crystal structures of small organic molecules – a report

on the fourth blind test, Acta Cryst., 2009, B65, 107-125. (open access) 61 D. A. Bardwell et al.: Towards crystal structure prediction of complex organic compounds – a report on the

fifth blind test, Acta Cryst., 2011, B67, 535–551. (open access)

43

and by the electron cloud. However, this approach is difficult to apply to predict solution or

solid state structures where the intramolecular interactions are very low in energy. In solution

the Polarizable Continuum Model (PCM) helps62

to describe solvation effects. To estimate the

energy differences of the predicted crystal structures we should consider the sublimation heat

as a function of molecular weight and as a rough estimate we can calculate that the average

energy of crystal stabilizing interactions is 6 kJ/atom. Compared with the energy of hydrogen

bonds of 30-100 kJ/mol it is obvious why it is difficult to distinguish among crystal structures

and find the thermodynamically stable polymorph. Additional problems emerge because of

the non-directed nature of secondary interactions. Hydrogen bonds have directional

preference as hydrogen bonds considered to be strong when the distance of the donor and

acceptor atoms is lower than 3 Å and the Donor-Hydrogen-Acceptor (DHA) angle is 160-

180o. Only limited directionality can exist in case of π-π interactions while electrostatic,

dispersion and repulsion interactions have spherical symmetry i.e. there are no preferred

directions when these interactions are significant in packing. It is also true that several low

energy interactions can override the effect of a very strong interaction such as hydrogen bond.

Such interactions are not oriented or have limited directionality and include

- Electrostatic (Coulombic)

- Van der Waals

- Dispersion (London)

- Repulsion.

5.4. Remarks on chemical bonds

To understand building up crystals and supramolecular forces it is interesting to think of what

is a chemical bond? Linus Pauling in his paramount book63

wrote about the definition of

chemical bond: “We shall say that there is a chemical bond between two atoms or groups of

atoms in case that the forces acting between them are such as to lead to the formation of an

aggregate with sufficient stability to make it convenient for the chemist (bolded by me, A.B.)

to consider it as an independent molecular species.”

62

http://en.wikipedia.org/wiki/Polarizable_continuum_model 63 L. Pauling: Tha nature of the chemical bond and the structure of molecules and crystals, Third Edition, Cornell

University Press, Ithaca, New York, 1960, p.6.

44

There have been enormous efforts to understand the nature of the chemical bond in the last

several decades on the basis of quantum mechanics, thermodynamics, structural studies etc.

Still, if the view of Pauling is accepted, as it has been done by chemists worldwide, the

chemical bond as a concept is merely a question of convenience of chemists and not

something dictated by mother nature. By the author’s opinion this should be kept in mind all

the time when supramolecular chemistry is considered as an analogue of synthetic chemistry

for examining and tailoring the weaker and reversible non-covalent interactions between

molecules.

5.5. Structural motives of polymorphic forms

The definition of polymorphism (Chapter 1.1) implies that the ability of the compound to

form different solid state structures is originated either from the possibility of different

orientation of the molecules in the lattice (packing polymorphism) and/or slight modification

of the conformation of the molecule (conformational polymorphism). The observed crystal

and molecular structure is always a minimum at the energy landscape, it can be either a local

or a global minimum. When there are several molecules in the asymmetric unit we have an

opportunity to guess the conformational freedom of the molecule under given crystal forming

conditions. Comparing these structures with the energy minimized conformation calculated on

the basis of quantum mechanics and also with the calculated energies of other conformers

some kind of energy (stability) scale of the polymorphic forms can be deducted from the point

of view of the individual molecule. However, as the crystalline state means the repetition of

the motif (asymmetric unit) the main target is to include secondary or packing interactions,

too, into the energy scale of the polymorphs. Comparison of the crystal structures of

chemically similar compounds or different crystal structures of the same molecule

(polymorphs) reveal what kind of motives are possible for the compound studied in terms of

both orientation and conformation.

Directionality of the hydrogen bond has been already discussed (Chapter 5.3). It is very

common that there are multiple hydrogen bonds between two molecules provided the donor

and hydrogen atoms can occupy favourable position in space. This is similar than the chelate

effect64

observed in transition metal complexes. For example carboxylic acids exist in dimer

form because of the two strong hydrogen bonds between them (Figure 5.5.1). The structure is

64 http://goldbook.iupac.org/C01012.html

45

R2

2

(8)

destroyed only by polar solvents and/or high temperature. Moreover, dimers of short chain

carboxylic acids can be detected even in the vapour phase at high temperature. Detecting and

investigating hydrogen bonds is one of the directions of supramolecular chemistry. Graph set

analysis is also useful to describe the hydrogen bond web. In this case the topology of the

hydrogen bond fixed association is considered without investigating the geometric parameters

such as donor-acceptor distance or donor-hydrogen-acceptor angle. Extending the hydrogen

bond network to multiple molecules or unit cells 1D, 2D and 3D patterns can be recognized,

for example chains or rings. According to the accepted nomenclature65

the topology of the

hydrogen atom network the graph set descriptor is ( ), in case of carboxylic acid dimers it

is ( ). We have an eight membered (N=8) ring (R) with two donor (subscript) and 2

acceptor (superscript) atoms. The designator (G) is C for chains, S for intramolecular

hydrogen bond and D for finite pattern.

Figure 5.5.1

Hydrogen bonds in carboxylic acid dimers and the designator as ( ).

When two different functional groups form the hydrogen bond network66

it is also possible

that there is a good steric match between them. For example guanidine cations as donors and

sulfonate anions as acceptors can be fixed in the lattice. The solid state structure of guanidine

salts of organic sulfonates67

are often subject of crystal engineering studies and they usually

crystallize very well. Moreover, a weaving hydrogen bond network is formed in 3D because

the sulfonate group is not planar (Figure 5.5.2). Similar oriented networks of the molecules

are also possible as a result of π-π interactions. Aromatic rings can form herringbone, parallel

65 M. C. Etter, J. C. MacDonald and J. Bernstein: Graph-set analysis of hydrogen-bond patterns in organic

crystals, Acta Cryst., 1990, B46, 256-262. 66 A. Nangia: Hydrogen Bonding and Molecular Packing in Multi-functional Crystal Structures in Organic

Crystal Engineering, Frontiers in Crystal Engineering, Eds. E. R. T. Tieknik, J. Vittal, M. Zaworotko, John

Wiley & Sons, Chichester, 2010, pp. 151-190. 67 J.A. Swift,A. M. Pivovar, A. M. Reynolds and M. D. Ward: Template Directed Architectural Isomerism of

Open Molecular Frameworks: Engineering of crystalline clathrates, J. Am. Chem. Soc., 1998, 120, 5887-5894.

46

R2

2

(8)

R6

3

(12)

or T-stacking, too. Recognition of these and similar patterns in the solid state structure is one

of the tools for the supramolecular description of the polymorphic structures, too.

Figure 5.5.2.

Hydrogen bond network in guanidine salt of a sulfonate derivative with graph set designators

of the ring systems (left) and the weaving hydrogen bond pattern in 3D (right). The organic

side chain is omitted for clarity. Unpublished research results from the University of

Debrecen.

However, these patterns are obvious only for the human eyes and detailed thermoanalytical

studies and quantummechanical calculations coupled with spectroscopic (IR, Raman and solid

state NMR) investigations should reveal the energy gain reached by the system because of the

formation of such patterns. In the final example (Figure 5.5.3) not only polar but also apolar

frameworks are stabilizing the lattice and the observed crystal structure is formed with the

interpenetration of the two frameworks.

47

Figure 5.5.3.

Polar hydrogen bonded (left)

and apolar π-π stacking

(middle) framework in the

solid state structure of

guanidine salt of a sulfonated

triphenylphosphine derivative. The complete packing diagram (right) shows the

interpenetration of the two networks. Unpublished research results from the University of

Debrecen.

48

Chapter 6 X-ray diffraction basics, X-ray powder diffraction

X-ray is a form of electromagnetic radiation of very high energy and frequency. Powder

diffraction techniques are the most adequate methods to study crystalline solids and in this

way X-ray powder diffraction is the leading analytical tool to differentiate among polymorph

phases as it gives a fingerprint of the lattice. Other techniques should be validated against

powder diffraction results.

6.1. Properties of X-ray radiation.

At November 8th, 1895 Wilhelm Conrad Röntgen in Würzburg, Germany performed

experiments with Phillip von Lenard’s tube and discovered that a radiation is emitted by the

apparatus. He named it X-ray. A few weeks later his publication has been published and for

his discovery he got the Nobel Prize for physics in 1901. In very early stage of his research he

made a famous picture of his wife’s hand. The World War triggered medical application of X-

ray, too.

X-ray is a form of electromagnetic radiation with wavelength in the range of atomic distances

and with energy several order of magnitudes higher than energy of chemical reactions. The

frequency of X-rays is also several magnitudes higher than frequency of electron transitions in

molecules.

Properties of X-ray radiation:

- Wavelength (λ): 0.1-100 Å = 10 pm -10 nm.

- Energy (E): 100 eV – 100 keV = 104 – 10

7 kJ/mol

- Frequency (ν): 1016

– 1019

Hz

When the electromagnetic radiation interacts with any material68

it can be absorbed, scattered

by loss of energy (non-elastic or Compton scattering), scattered by no loss of energy (elastic

or Rayleigh scattering), the phase maintained (coherent) or not (incoherent) or the absorption

can cause subsequent fluorescence. Here we discuss only coherent (Rayleigh) scattering when

electrons start vibrating because of the electromagnetic field and they emit the same

wavelength radiation only with some change of direction.

68 http://www.xtal.iqfr.csic.es/Cristalografia/parte_05-en.html

49

Questions related to producing monochromatic X-ray radiation and safety requirements to

work with ionizing radiation are not covered in this course. Safety issues related to X-ray or

other ionizing radiations are not discussed either.

Several techniques are available for single molecule69

spectroscopy and fluorescence.

However, right now the scattering caused by single molecule70

can be measured only by the

most intense (brilliant) light sources such as free electron lasers. Fortunately we have an

easier way. If the atoms or the molecules are arranged into a lattice the electron density is

periodic. Crystalline materials show high degree of long range order as crystals are made of

unit cells as repeating building blocks in three dimensions (Chapter 4.1). The size of unit cells

in case of small molecules is approximately 10 Ǻ x 10 Ǻ x 10 Ǻ. In a macroscopic single

crystal dimensions of a few tenth of mm in all directions there are 1016

- 1019

unit cells. These

very large numbers together with the periodicity of the electron cloud in the lattice cause an

enormous amplification of the scattered coherent radiation.

6.2. Crystal systems, space groups, Miller indices and distance of lattice planes

From simple geometric consideration if we want to fill the three dimensional space

continuously with uniform bodies we are led to the theory of crystals well known for centuries

and the seven crystal systems71

can be deducted (Figure 6.2.1). The dimensions of the unit

cell can be described by minimum 1, maximum 6 parameters, depending on the crystal

system. These parameters are the length (a,b,c) and angle (,,) parameters. We use here a

right handed coordinate system as it is usual in the science. Based on the centricity of the unit

cell 14 Bravais lattices72

can be defined (Figure 6.2.2).

69 http://en.wikipedia.org/wiki/Single-molecule_experiment 70 http://www.ncbi.nlm.nih.gov/pubmed/11042456 71 http://www.materials.ac.uk/elearning/matter/Crystallography/3dCrystallography/7crystalsystems.html applet 72 http://en.wikipedia.org/wiki/Bravais_lattice

50

Figure 6.2.1

The seven crystal systems.

51

Figure 6.2.2

The 14 Bravais lattices in different crystal systems.

Considering the possible symmetry of the unit cells there are 230 space groups73

which can

be used to describe the symmetry of the crystal in 3D. Further discussion of symmetry and

symmetry elements are based on group theory and are out of the scope of this course. It is

impossible to consider all unit cells even in the smallest macroscopic crystal individually.

Fortunately we can use the Miller indices74

to denote the lattice planes (more accurately

lattice plane families) in the crystal. The (h,k,l) Miller indices (Figure 6.2.3) shows where the

plane intercepts the a, b, and c axis vectors i.e. in a/h, b/k, c/l or its multiple. Here if any of

the Miller indices is 0 it means that the plane intercepts the axis in infinity that it is parallel to

that axis. The Miller indices help us to define a reciprocal lattice75

as a geometric construct in

which all (h,k,l) lattice planes of the crystals are represented by a point. Further discussion of

73 http://en.wikipedia.org/wiki/Space_group 74 http://en.wikipedia.org/wiki/Miller_index 75 http://en.wikipedia.org/wiki/Reciprocal_lattice

52

reciprocal lattice and reciprocal space is not covered here (Notation: a,b,c are the axis vectors

in direct space while a*,b

*,c

* are the axis vectors in the reciprocal space). At Figure 6.2.3 it is

also shown for the cubic case, that the dhkl distance of the (100) and similarly that of the (010)

and (001) planes are equal to the a parameter of the unit cell (a1=a2=a3=a). In general, in any

crystal system the d distance of the (h,k,l) lattice planes can described using solely the (h,k,l)

plane and a,b,c,,, unit cell parameters. The formula is the most complicated one for

triclinic system i.e. when all a,b,c unit cell parameters are different ones and none of the α, β,

γ angles is 90o. V is the volume of the unit cell:

(6.2.1)

2

2

2

2

2

2

2

2222

2

2222

2

2222

2

)coscos(cos2

)coscos(cos2

)coscos(cos2

sinsinsin1

V

cablh

V

bcakl

V

abchk

V

bal

V

cak

V

cbh

dhkl

√

53

Figure 6.2.3

Miller indices in cubic lattice (a1=a2=a3=a).

6.3. The Bragg’s law

The properties of waves76

and especially the phenomenon of constructive and destructive

interference77

are supposed to be known here. First Sir William Henry Bragg and his son, Sir

William Lawrence Bragg recognized that interference of waves scattered by a crystal lattice

76 http://theory.uwinnipeg.ca/mod_tech/node120.html 77 http://theory.uwinnipeg.ca/mod_tech/node125.html

54

can be described by a simple equation called Bragg’s equation or Bragg’s law. For their

discovery they got the Nobel Prize in 1915. As it is shown at Figure 6.3.1, the incident

electromagnetic waves A and B are reflected by P and Q lattice planes which have a distance

of dhkl. An applet78

gives a demonstration of the phenomenon. The reflected E and D waves

has a constructive interference if nλ=2dsinθ where λ is the wavelength, θ is the angle of

incident beam to P or Q.

Figure 6.3.1

The Bragg’s equation as geometric model of diffraction.

6.4. Powder diffraction methods

The Bragg’s equation turned to be a very useful geometric model of the diffraction

phenomenon. Submitting the powder sample to X-ray radiation if large number of crystallites

is present in random orientation peaks can be found at cones which fulfil the Bragg equation.

The arrangement is called Debye-Scherrer or capillary geometry (Figure 6.4.1) and it is free

78 http://www.eserc.stonybrook.edu/ProjectJava/Bragg/ applet

55

from these systematic errors. In this case very high intensity X-ray source, preferably

synchrotron beam, is necessary for accurate measurements and peak shapes are often

asymmetric ones.

Figure 6.4.1

Debye Scherrer geometry to measure XRD of powder sample.

For in house powder diffractometers the Bragg-Brentano arrangement is also advantageous

(Figure 6.4.2.) where high counts can be reached in well-defined sample geometry. However,

preferred orientation is an important issue as it is difficult to achieve random orientation of

crystallites. Sample transparency can be a source of errors if the sample contains heavy

elements, especially metals.

Figure 6.4.2

Bragg-Brentano geometry for data collection on powder samples. Image is courtesy of Bruker

AXS, Germany.

56

Figure 6.4.3

Calculated X-ray powder pattern of ranitidine hydrochloride polymorphs on the basis of CSD,

compounds with Refcodes TADZAZ, TADZAZ01, TADZAZ02 and TADZAZ03.

The resulted powder pattern (Figure 6.4.3) gives a unique characterization of the lattice.

Location of the peaks i.e. 2θ angles depends solely on the lattice parameters and h,k,l indices

(Eq. 6.2.1). Because of symmetry consideration some reflections are extinct. The origin of

these systematic absences79

as well as their importance is not detailed here and the reader is

directed to crystallography text books and web sites.80

The width of the peak (FWHM, Full

Width at Half Maximum) depends on the size of the crystals. Particle size measurement can

be performed using various techniques such as light scattering, mechanical methods etc. and

powder X-ray diffraction gives one kind of particle size. The equation named Scherrer

equation gives the particle size (τ) calculated from line broadening:

where K is a

shape factor, usually 1, λ is the wavelength of the radiation θ is the Bragg angle while β is the

FWHM value (Δ2θ) for the given peak. The lines are broadened because of other factors such

as strain in the crystal, lattice imperfections, twining, stress etc., too, which are usually

covered by the term crystallinity. Nevertheless powder pattern gives a lower limit of particle

size for crystals approximately 100-200 nm of diameter. The smaller are the particles the

narrower are the peaks. The accuracy of size determination can be increased if the given peak

is not overlapped (Figure 3.4.2 for aspartame). The powder pattern is sometimes named

79 http://reference.iucr.org/dictionary/Systematic_absences 80 http://goodwin.chem.ox.ac.uk/goodwin/TEACHING_files/l7_handout.pdf

10 15 20 25

4998

2499

0

TADZAZ.search1 25.0%

8.28

9.44

11.65

12.80

13.59

14.09

14.43

15.21

15.74

16.36

16.61

17.59

17.98

18.42

18.74 18.94

19.24

19.65

20.15

20.22

20.81

21.06

21.23

22.62

22.78

23.41

23.99

24.25

24.57

24.72

25.02

25.42

TAD ZAZ01.search1 25.0%

8.04

8.54

14.53

15.39

16.04

17.13

18.18

19.91

20.14

20.54

21.90

21.95

23.89

25.06

TAD ZAZ02.search1 25.0%

TAD ZAZ03.search1 25.0%

9.43

11.64

19.71

20.14

23.99

57

spectrum. However, powder diffraction is not a spectroscopy method and the signal is

measured as a function of a geometric parameter (θ) rather than as a function of energy or

wavelength.

There are several well designed web sites81

for introduction into powder diffraction methods.

Altogether X-ray powder diffraction is suitable for

- Qualitative phase identification

- Quantitative phase analysis provided none or known amount of amorphous material is

present

- Peak shape analysis provides information on particle size, stress in the crystal, lattice

defects etc.

In certain cases (Chapter 8) powder pattern can be used to solve and refine the structure, too.

Neutron source and neutron powder diffraction technique is available only at very few places

but it can have application in polymorphism research in some cases. While X-ray radiation is

scattered by the electron cloud neutrons are scattered by the nuclei. The X-ray scattering

factor of atoms is proportional to the atomic numbers i.e. number of electrons. Heavy

elements such as transition metals have overwhelming effect on intensities of reflections in X-

ray diffraction while hydrogen atoms have small effect. In case of neutron diffraction there is

a sign difference in the scattering factors if hydrogen or deuterium is considered. Usually

neutron powder diffraction pattern of normal and deuterated samples are compared. The

theory of neutron diffraction is not covered here.

Powder patterns are also compiled in databases such as PDF by International Centre for

Diffraction Data (ICDD). Among the advantages of powder diffraction method it should be

noted that small or no sample preparation is needed for the measurement, it can be applied for

mixtures of unknown composition and can be applied as a variable temperature technique to

follow phase transformations, too. However, experimental difficulties such as preferred

orientation and absorption also occur. The Curiosity rover performed82

X-ray powder

diffraction measurement at the Mars.

6.5. Quantitative X-ray Powder Diffraction (XRPD) of polymorphs

81 http://epswww.unm.edu/xrd/xrd-course-info.htm 82 http://iucr2014.org/side_program/scientific_program/documents/BishIUCr2014_Abstract.pdf

58

X-ray powder pattern can be used to determine the amount of different phases83

i.e.

polymorphic forms in a mixture. Quantitative XRPD measurement requires careful sample

preparation, data collection and extensive modelling work to determine

instrumental/experimental/sample parameters affecting peak intensity. As amorphous material

does not give XRPD signal except very wide background peak it is always a challenging task

to determine the amount of crystalline phases present. The tricks of quantitative XRPD

technique are not covered here but while modern quantitative analytical methods work with at

least 0.1% accuracy in quantitative X-ray powder diffraction ±1-5% error is respectable.

In the case of a certain dimorphic API the two forms mixed in various ratios gave nice

patterns (Figure 6.5.1) and high accuracy could be reached in determination of composition of

unknown mixture. Amount of the amorphous phase was not studied in this case.

Figure 6.5.1

Quantification of polymorphic forms of API. Results from the University of Debrecen, with

permission of Alkaloida Research & Development Ltd, Tiszavasvári, Hungary.

83 http://epswww.unm.edu/xrd/xrdclass/09-Quant-intro.pdf

59

Chapter 7 Single crystal X-ray diffraction

Single crystal X-ray diffraction gives the final description of all solid state structures

provided close-to-perfect crystal (single crystal) of the material can be prepared. All

structural differences of polymorphic forms can be described and analysed by means of this

method and complete supramolecular description of the structures as well as calculating the

powder pattern is possible.

7.1. Single crystal X-ray diffraction in polymorphism research. All or none method

Powder diffraction pattern can be interpreted in high detail on the basis of the Bragg’s

equation and simple samples and experimental considerations such as particle size, stress, and

detector sensitivity for scaling etc. However, the scattering of X-ray by a crystal is a much

more complicated phenomenon. The theory of diffraction scattered by single crystals is

described here.

7.1.1. The structure factor as Fourier transform of the electron density in the unit cell

The X-ray beam as electromagnetic wave interacts with the periodic electron density of the

crystal. Note that the periodicity of the crystal is represented by the lattice. The smallest

repeating unit of the lattice is the unit cell. The whole macroscopic single crystal can be

assembled by shifting (translating) the unit cell parallel to it’s a, b and c axis vectors (Chapter

4.1). Here the crystal is considered to be perfect i.e. free from chemical contaminants, lattice

distortions or errors, dislocations etc. However, because of the symmetry of the unit cell there

are equivalent positions within the unit cell.

In 3D the symmetry of the lattice can be described using the 230 space groups (Chapter 6.2).

The smallest symmetry independent unit of the lattice is the asymmetric unit. The unit cell

can be built up by applying the space group symmetry onto the asymmetric unit and

translating the unit cell we can get the macroscopic crystal again. The group theory is a useful

tool to describe symmetry. While point groups are used to describe the symmetry of

individual molecules because of the translation in the lattice space groups should be used to

describe the symmetry in case of a 3D lattice.

The electromagnetic wave interacts with the electric cloud of the unit cell that is electron

density in the crystal. In case of elastic scattering or Rayleigh scattering no energy is absorbed

60

by the crystal However, as the wavelength of the applied X-ray radiation is comparable with

the periodicity of the crystal interference occurs between the waves scattered by the electron

cloud i.e. scattered from different points of the unit cell. Actually the X-ray radiation is

scattered by the respective (hkl) lattice planes which are in reflecting position i.e. fulfil the

Bragg’s law. In this way information originated from the specimen, in case of X-ray radiation

electron density distribution is encoded in the scattered radiation. If we have suitable lenses to

enforce another interference of the scattered waves and reconstruct the enlarged picture of the

specimen we have a microscope. In case of visible light glass or plastic can be used to slow

down photons i.e. they have an optical density higher than 1.00. In the range of IR radiation

other materials for example NaCl or ZnS are suitable to prepare optics. Combined electric and

magnetic field can be used to manipulate electron waves in an electron microscope.

Unfortunately, in the X-ray region of electromagnetic radiation all material has an optical

density of 1.0000 and no X-ray lenses can be prepared in this way. Another trick is needed to

figure out the encoded information from the scattered radiation: in a tiny macroscopic single

crystal dimensions of 0.1 - 0.3 mm in all three directions there are 1016

-1019

unit cells

resulting amplification of the information content. Mathematically speaking the scattered

radiation as a wave can be represented by a vector (called structure factor, F). The structure

factor84

is a complex number, F=A+Bi having a real (A) and imaginary (Bi) part, i is the

imaginary unit: √ . It is a very useful modell for decoding the information said about

the unit cell that the structure factor is the Fourier transform of the electron density ρcell in the

unit cell. As the size of the electrons is very small comparing to the size of an atom and the

size of an atom is small comparing to the unit cell we can say that electron density is localized

in the atoms and all atoms in the unit cell have an atomic scattering factor f. For any atom the

scattering factor at θ=0o i.e. for the direct beam basically is equal to the number of electrons,

but the scattering factor depends on θ. More accurately the scattering factor is a complex

number and as a result of this anomalous scattering occurs, which can be used to determine

the absolute configuration of the lattice and assign chirality centres in pure enantiomers

(Chapter 7.5).

The given (hkl) plane of the lattice can be placed into reflecting position by rotating the

crystal in respect the beam (not detailed here further) to fulfil the Bragg equation. The

structure factor for that particular reflection can be calculated using Fourier transformation.

This is the structure factor equation (in exponential form):

84 http://www.ruppweb.org/Xray/comp/strufac.htm

61

(7.1.1.1)

∑ [ ( )]

Here h,k and l are the Miller indices of the given reflections, fj is the atomic scattering factor

of the j-th atom and xj,yj,zj are the fractional coordinates of that atom in the unit cell. (We use

j for the index of the atom as i has different meaning.) For each (hkl) reflection there is

information coded by all atoms as their scattering factor and coordinates influence the

structure factor and vice versa. If the structure factor is known (both its amplitude and phase)

inverse Fourier transformation can be used to determine the electron density in the cell using

the electron density equation85

(Vcell is the volume of the unit cell, the required minimum and

maximum values of the h,k,l inidices depends on crystal quality (resolution) and space group:

(7.1.1.2)

( )

∑ ∑ ∑ [ ( )]

There is another difficulty, which is called the crystallographic phase problem. To describe a

wave and perform the inverse Fourier transformation we need to know both its amplitude and

its phase, its real and imaginary part. However, for electromagnetic waves in the X-ray region

the frequency is 1016

Hz, which makes it impossible to measure both the amplitude and the

phase at the same time which can be easily performed for sound waves. We can measure

instead the intensity (I) of the wave as a real number, the multiple of the F=A+Bi complex

number with its complex conjugate F*=A-Bi, I

2=A

2+B

2. However, the phase contains

significant amount of information about the structure but we lose this information in the

detector. Several methods were developed to circumvent this problem and solve the phase

problem. Fortunately in case of small molecules there are very robust mathematical and

computational techniques to solve the phase problem (not detailed here). These methods give

a starting set for the position of the atoms in the unit cell and the atomic coordinates can be

refined by minimizing the difference between the calculated and measured intensities

(structure factors). In case of small molecules the number of data points (measured intensities)

is very high (10x) compared to the number of parameters to be refined (3 coordinates and 6

85 http://www.ruppweb.org/Xray/101index.html

62

atomic displacement parameters for each atom, unit cell, orientation matrix and scaling

parameter). This highly over-determined least square fitting problem in a several hundred

dimensional parameter space together with the small residual86

difference (5-10%) between

the measured and calculated intensities make single crystal structure determination the final

unambiguous tool in structure verification. The final results of single crystal structure

determination are:

- Crystal structure: dimensions of unit cell and space group symmetry.

- Molecular structure: types of atoms and their coordinates within the unit cell.

7.1.2. Single crystal diffraction for the complete determination of solid state structure

Structure determination by single crystal X-ray diffraction contributed heavily to the

development of chemistry and biology in the last century. The method is independent and

unlike spectroscopic and all chemical methods it requires no or minimal information about the

synthesis or pre-treatment of the sample. The knowledge of the atomic coordinates in the unit

cell makes it possible to verify the connectivity within the molecule (chemical structure) and

also recognize the secondary interactions responsible for the stabilization of the crystal

(supramolecular structure). These secondary interactions include strong (sterically favoured

donor-H…acceptor) or weak (C-H…acceptor or sterically unfavoured donor-H…acceptor)

hydrogen bonds (Chapter 7.2), electrostatic interactions as well as van der Waals and π-π

interactions and also repulsions. As a result complete supramolecular structural comparison of

polymorphic forms can be performed. The technique investigates the bulk solid phase

although the measured sample is a single crystal of 0.1-0.3 mm dimensions in all three

directions and the weight of the crystal depending on the density is a few micrograms.

Electron microscopy or atomic force microscopy can give structural information of atomic

resolution but usually from the surface of the sample.

The single crystal diffraction is an all or none method. From the structure factor equation,

7.1.1.1 we can see that all atoms contribute to each reflection intensity. Moreover, if the

determination of the unit cell or the space group i.e. description of the lattice is wrong or we

fail to solve the phase problem the structure determination remains unsuccessful. This is in

sharp contrast with spectroscopic methods (NMR, IR UV-VIS) which can give valuable

86 http://en.wikipedia.org/wiki/R-factor_(crystallography)

63

information even if the whole spectrum could not been assigned. Single crystal X-ray

diffraction cannot answer for such requests: ‘I only need the ……. bond distance’. Either we

know everything about the structure or (almost) nothing.

7.1.3. Measurement of single crystal diffraction data

The single crystal sample is fixed onto a goniometer head and subjected to X-ray radiation

from a s

uitable X-ray source. The scattered radiation is measured with a point or preferably a CCD

detector (Figure 7.1.3.1). The intensity of different (hkl) reflections can be measured by

rotating the crystal and the detector around three or four axis (the source is fixed).

Figure 7.1.3.1

Agilent Technologies’ SuperNova Atlas S2 (Dual Cu, Mo) CCD area detector diffractometer

system, with cryo-attachment in place. (With permission, Agilent Technologies.)

64

The main steps of structure determination by means of single crystal diffraction

- Single crystal growing

- Selection of a suitable (perfect) crystal

- Measurement of unit cell parameters by collecting high intensity reflections randomly

(or a few frames with a CCD).

- Determination of orientation matrix and quality of the crystal as well as the space

group of the lattice

- Comprehensive data collection for high number of reflections

- Solution of the phase problem (direct methods, charge flipping etc.)

- Refinement of the structure

- Interpretation of the structure. Comparison with database search and powder

diffraction results

The very first step of single crystal structure determination is to prepare a single crystal. In

polymorphism research the main question is how the structure of this crystal is related to the

structure of the powder used for example in formulating the drug. Growing perfect crystals

usually requires dissolving and crystallization of the sample which may take days or weeks. It

is possible that other minimum is found at the crystal-energy landscape and another phase is

in our hand. Fortunately single crystal data contain the complete lattice as well as molecular

structure information. It is easy to calculate a simulated powder pattern: location and intensity

of all expected peaks of powder diffraction measurement. After scaling and considering

reasonable crystal size and crystallinity to simulate peak width and even preferred orientation

one can get a powder pattern very similar to the experimentally measured one.

7.2. The hydrogen bond

The strongest one of the secondary interactions which stabilizes the solid state structure is

hydrogen bond87

. This is the interaction of a hydrogen atom bonded to a donor atom (D-H)

with the lone electron pair(s) of an acceptor atom (A) with the right orientation and

appropriate distance of the donor and the acceptor atoms. The strength of the hydrogen bond

87 L. Brammer: Hydrogen Bonds in Inorganic Chemistry: Application to Crystal Design in Crystal Design:

Structure and Function Perspectives in Supramolecular ChemistryVolume 7, Ed. G. Desiraju, John Wiley &

Sons, Chichester, 2003, pp. 1-75.

65

is in the 1-40 kcal/mol range. The nature of the hydrogen bond can be covalent (strong H-

bond, 15-40 kcal/mol, for example dimers of strong acids/bases, H2F2 etc.), mixed covalent

and electrostatic (medium strong, 4-15 kcal/mol, such as O-H…acceptor, acids, amines etc.)

or electrostatic (weak hydrogen bond, below 4 kcal/mol, C-H…O, O-H….π etc.). The energy

gain by hydrogen bond formation is comparable with the energy difference of polymorphic

forms either calculated or determined by DSC (below 10 kcal/mol). Two important

consequences :

- Reorganization of the hydrogen bond network can easily lead to new polymorphic

forms.

- In the solid state structure almost all hydrogen atoms connected to a donor atom

should have an acceptor pair. Hydrogen bonds are favoured even if voids remain in the

structure.

The geometric parameters which characterise the hydrogen bond include the donor-acceptor

distance (2.2-3.2 Å) and the donor-hydrogen acceptor angle (160-180o). As hydrogen atoms

are difficult to locate by X-ray diffraction the hydrogen acceptor distance is less informative

also because hydrogen atoms often placed into geometric position even for alcohols or

amines. To describe the topology of the hydrogen bond web graph theory or graph set

analysis88

can be used (Chapter 5.5). Analysis of hydrogen bond network is a main tool for

the supramolecular comparison of the structure of polymorphic forms. Spectroscopic methods

(IR, Raman and solid state NMR) are also useful to detect hydrogen bond and identify

polymorphic forms on the basis of different hydrogen bond networks.

7.3. Co-crystals

The definition of co-crystals gained significant attention in the last few years.89

Basically if a

compound crystallizes by incorporating another solid compound into the lattice it is

considered as co-crystal. These compounds could not be considered as polymorphic forms as

they represent a thermodynamically different system when the co-crystal is dissolved. The

importance of co-crystals is originated from their ability to have higher solubility and/or

dissolution rate than the base molecule. Moreover, the scientific community tried to

88

M. C. Etter, J. C. MacDonald and J. Bernstein: Graph-set analysis of hydrogen-bond patterns in organic

crystals Acta Cryst., 1990, B46, 256-262. 89 S. Aitipamula et al.: Polymorphs, Salts, and Cocrystals: What’s in a Name? Cryst. Growth Des. 2012, 12,

2147−2152.

66

distinguish co-crystals from clathrates and solvates by assuming that no hydrogen bond

occurs in the solid state structure. However, as the definition of hydrogen bond also changed

and existence of weak hydrogen bonds such as C-H…O got acceptance90

definition of co-

crystals became rather fluid. Finally in 2013 the Food and Drug Administration gave the

definition91

for co-crystals from regulatory point of view, although in a non-binding

regulation, as “Solids that are crystalline materials composed of two or more molecules in the

same crystal lattice”. This definition has not been accepted by the scientific community yet

as this definition covers salts, solvates etc. under the same heading. Refinement of the

definition is foreseen.

7.4. Structural motifs of polymorphs, case studies

The supramolecular description of the solid state structure is the basis of comparison of

polymorphic forms. Supramolecular chemistry is a rapidly evolving science with strong

motivation by the pharmaceutical industry, too. Our goal is crystal engineering92

that is a

priori description of the structure and/or design and preparation of solid forms of desired

properties.93

We are very far from these targets. Combining X-ray diffraction, spectroscopic

methods and QM calculations are necessary for understanding why the observed structural

motifs are formed. However, the polymorphism is a kinetic phenomenon and to fulfil

engineering requirements mechanism of crystal growing should be understood, too. Here are

mentioned a few representative examples.

7.4.1. Sulphonamide polymorphic forms

Our research gave an interesting result in polymorphism screening. A sulphonamide

derivative API was targeted to prepare and all solution spectroscopy techniques verified the

right structure except melting point, which was 10oC different from the literature data. Single

90 J. Bernstein: “It isn’t”, Cryst. Growth Des., 2013, 13, 961−964. 91 Guidance for Industry Regulatory Classification of Pharmaceutical Co-Crystals,

http://www.fda.gov/downloads/Drugs/Guidances/UCM281764.pdf 92 a) M. L. Peterson, E. A. Collier, M. B. Hickey, H. Guzman and Ö. Almarson: Multi-component

Pharmaceutical Crystalline Phases: Engineering for Performance in Crystal Engineering, Frontiers in Crystal

Engineering, Eds. E. R. T. Tieknik, J. Vittal, M. Zaworotko, John Wiley & Sons, Chichester, 2010, pp. 67-100 b)

K. Birdaha and L. Rajput: Crystal Engineering with Molecules Containing Amide and Pyridine Functionalities,

ibid, pp. 215-238. 93 Crystal Design: Structure and Function in Perspectives in Supramolecular ChemistryVolume 7, Ed. G.

Desiraju, John Wiley & Sons, Chichester, 2003.

67

crystals could be grown from both forms and the single crystal X-ray diffraction study

revealed that the forms are conformational polymorphs (Figure 7.4.1.1). Rotation around the

C1-O1 bond results different hydrogen bond pattern which is responsible for the difference in

the melting points. Actually it is rather common94

that sulphonamides exist in several crystal

forms.

Figure 7.4.1.1

Structure of conformational polymorphs of an API. (University of Debrecen, unpublished

results, with permission of Alkaloida Research & Development Ltd, Tiszavasvári, Hungary.)

7.4.2. The carbamazepine case

Carbamazepine is the active substance of several drug products (Tegretol, Timonil, Telesmin,

Calespin, Carbama, Equetro, Epitol, Teril, Amizepin, Hermolepsin) used to treat epilepsy.

Carbamazepine (5H-dibenzo[b,f]azepine-5-carboxamide) contains a rather unusual seven

member ring. It has at least four anhydrous polymorphic forms with symmetry of triclinic,

monoclinic and trigonal crystal systems. Because of the intense therapeutic use the solid

94 S.S. Yang, J.K. Guillory: Polymorphism in Sulfonamides, J. Pharm. Sci., 1972, 61, 26-40.

68

forms are studied95

thoroughly using DSC,96

single crystal97

(Figure 7.4.2.1) and powder98

X-

ray diffraction as well as solid state NMR99

methods (Chapter 9).

Figure 7.4.2.1

Conformational polymorphism in carbamazepine structures (Based on data from the

Cambridge Structural Database).

7.4.3. Suggested project work: Search of CSD for polymorphic forms of an API and

identification of structural motives on the basis of single crystal data

Atomic coordinates of a drug substance is downloaded from the Cambridge Structural

Database. Structural motives should be identified. Geometric parameters of hydrogen bond

are calculated using the MERCURY program.100

95 C. Rustichelli, G. Gamberini, V. Ferioli, M. C. Gamberini, R. Ficarra and S. Tommasini: Solid-state study of polymorphic drugs: carbamazepine, J. Pharm. Biomed. Anal., 2000, 23, 41-54. 96 http://www.shimadzu.com/an/industry/pharmaceuticallifescience/n9j25k00000cdmf8.html 97 A. L. Grzesiak, L. Meidong, K. Kim and A. J. Matzger: Comparison of the Four Anhydrous Polymorphs of

Carbamazepine and the Crystal Structure of Form I, J. Pharm. Sci., 2003, 92, 2260-2271. 98 M. Otsuka, H. Hasegawa and Y. Matsuda: Effect of Polymorphic Forms of Bulk Powders on Pharmaceutical

Properties of Carbamazepine Granules, Chem. Pharm. Bull., 1999, 47, 852—856. 99 C. J. Bonin, A. Andrea and D. J. Pusiol: NQR frequencies of anhydrous carbamazepine polymorphic phases

http://worldwidescience.org/topicpages/a/anhydrous+carbamazepine+polymorphic.html 100 http://www.ccdc.cam.ac.uk/Solutions/CSDSystem/Pages/Mercury.aspx

69

7.5. Assignment of chirality using single crystal X-ray diffraction

Chirality is a very important phenomenon in chemistry. A carbon atom can be considering as

stereogenic centre if four different substituents are connected to it. Chirality can also be

associated with the shape of the molecule without occurrence of a stereogenic (chiral) atom.

The stereogenic centre can exists in two configurations which are one kind of stereoisomerism

as the isomers differ in the spatial arrangements of the constituent atoms. They are the mirror

images of each other i.e. they are not superimposable. In enantiomers the four substituents of

the central atom are not in one plane or in two perpendicular planes. Asymmetric tetrahedral

atoms represent an example of chiral centres.

7.5.1. Chirality and its pharmaceutical consequences

Enantiomers react on the same way with achiral reagents or have the same property when they

interact with achiral agent. For example they have the same melting point or boiling point,

enthalpy of fusion, hydrolytic reaction etc. However, their interaction with chiral molecules or

chiral agents can have different rates and the products can be diastereomers. Diastereomers

have different physical characteristics, such as melting point or solubility in achiral solvent.

Pure enantiomers or mixtures of enantiomers of different ratio rotates polarized light i.e. they

are optically active. Enantiomers may have completely different smell, for example R-(-)

carvone has a smell of spearmint while S-(+) carvone has a smell of caraway. Enzymes as all

natural proteins consist of only L-amino acids so they are chiral. Enantiomers of biologically

active molecules have different effects on biological systems. For example when living

organisms are fed with racemate (1:1 mixture) of amino acids only the natural L-amino acids

are used by the organism and the un-natural D-amino acids remain there. For the

nomenclature of stereoisomers, (the Cahn-Ingold-Prelog, CIP system) see relevant organic

chemistry textbooks.

In connection with chirality the drug Thalidomide101

gained high attention. It became over the

counter (OTC) drug in Germany in 1957. As the drug was administered as a racemate the

“wrong” enantiomer caused prenatal malformation of thousands of infants whose mother got

this drug to decrease morning sickness during pregnancy. Based on the experiences the whole

101 http://en.wikipedia.org/wiki/Thalidomide

70

system of regulations for chiral drugs was reviewed and new rules were laid down to ensure

safety of patients. According to the Food and Drug Administration regulations:102

“The

stereoisomeric composition of a drug with a chiral centre should be known and the

quantitative isomeric composition of the material used in pharmacologic, toxicologic, and

clinical studies known. Specifications for the final product should assure identity; strength,

quality, and purity from a stereochemical viewpoint. To evaluate the pharmacokinetics of a

single enantiomer or mixture of enantiomers, manufacturers should develop quantitative

assays for individual enantiomers in in vivo samples early in drug development. This will

allow assessment of the potential for interconversion and the absorption, distribution,

biotransformation, and excretion (ADBE) profile of the individual isomers. When the drug

product is a racemate and the pharmacokinetic profiles of the isomers are different,

manufacturers should monitor the enantiomers individually to determine such properties as

dose linearity and the effects of altered metabolic or excretory function and drug-drug

interactions. If the pharmacokinetic profile is the same for both isomers or a fixed ratio

between the plasma levels of enantiomers is demonstrated in the target population, an achiral

assay or an assay that monitors one of the stereoisomers should suffice for later evaluation.

In vivo measurement of individual enantiomers should be available to help assess toxicologic

findings, but if this cannot be achieved, it would be sufficient in some cases to establish the

kinetics of the isomers in humans.”

7.5.2. Methods for assignment of chirality centres

The assignment of chiral centres means to define the correct stereoisomerism i.e. R or S

configuration according to the CIP nomenclature. The only absolute and always applicable

method to assign chirality centres is based on single crystal X-ray diffraction structure

determination. Spectroscopic methods such as application of NMR shift reagents are all based

on comparison of the effect with that of observed in case of known chiral compounds.

Chemical methods i.e. degradation to product(s) of known configuration represent very time-,

chemical- and labour-intensive approach. Circular dicroism measurement coupled with

quantum mechanical calculations are applicable in certain cases. This later method is based on

explicit calculation of optical activity on various conformers of the compound for both

configuration. However, a suitable chromophore group is also necessary to achieve

102 http://www.fda.gov/drugs/GuidanceComplianceRegulatoryInformation/Guidances/ucm122883.htm

71

assignment. To perform reliable conformer distribution calculation it is advantageous to have

atomic coordinates deducted from single crystal diffraction data. Comparing the measured

Cotton effect with the calculated one for the given conformer population makes it possible to

give a guess for the chirality with significant probability. A very recent103

but probably

quickly expanding method is based on non-linear resonant phase-sensitive microwave

spectroscopy of gas phase samples in the presence of an adiabatically switched non-resonant

orthogonal electric field.

7.5.3. Assignment of chirality relative to known stereogenic centre

Single crystal X-ray diffraction gives unambiguous result for the constitution and three

dimensional connectivity of the atoms in the molecule. If the absolute configuration (R or S)

of at least one stereogenic centre is known in the molecule (or in the crystal) the unknown

chirality centre(s) can be assigned relative to this known chiral centre. It is a little strange but

correct term: “assignment of absolute chirality of C(5) as (S) relative to known C(10) as (R)”.

7.5.4. Assignment of chirality on the basis of anomalous scattering

There is a direct way to assign chirality centre(s) on the basis of single crystal X-ray

diffraction data. This method is based on the anomalous scattering or anomalous

dispersion.104

At the first order analysis of the diffraction pattern the structure factors (Chapter

7.1.1) for (h,k,l) and (-h,-k-l) reflections (so called Friedel pairs or Bijovet pairs) are equal:

F(h,k,l) = F(-h,-k,-l). That means the single crystal diffraction pattern has inversion

symmetry. However, more elaborated theory reveals that atomic scattering factors (fj) are

complex numbers rather than real numbers. Depending on the applied wavelength of the X-

ray radiation this eventually causes a small (a few %) difference in the intensities of Friedel

pair reflections according to the structure factor equation (Chapter 7.1.1.). In case of in house

data collection if the diffractometer works with Mo Kα radiation (λ=0.709 Å) the presence of

a heavy atoms (sulphur, phosphorous or even heavier element) is necessary to detect these

small differences in intensities. However, using Cu Kα radiation (λ=1.541 Å) absolute

configuration can be assigned for molecules containing only light atoms (C, H, N, O) and

103 D. Patterson, M. Schnell and J. M. Doyle: Enantiomer-specific detection of chiral molecules via microwave

spectroscopy, Nature, 2013, 497, 475–477. 104 Jack D. Dunitz: X-ray Analysis and the Structure of Organic Molecules, 2nd Ed., Wiley-VHC, Basel, 1995.

p.131.

72

even configuration of hydrocarbons can be assigned. The effect of anomalous dispersion is

widely used in protein crystallography for phase determination and this usually requires

synchrotron radiation to use. For assignment of chirality centres in optically pure compounds

actually the small difference in the intensities of Friedel pair reflections shows whether we

have the modelled or the opposite configuration in hand. Bijovet used this methodology105

to

assign chirality centres in sodium rubidium tartarate tetrahydrate using Zr radiation.106

The

most widely used method to verify the absolute configuration of our structure by means of

small molecule single crystal X-ray diffraction method is to determine the Flack parameter107

in the final stage of the refinement. This parameter should have a value of 0 for the right

structure which is in the model and 1 for the opposite configuration structure. Actually it is

supposed that the crystal consists of two domains which are mirror images of each other

(twinned by inversion) and the Flack parameter means the molar fraction of the domain

represented in the three dimensional model of the crystal structure. Fortunately this parameter

can be handled as any of other refining parameters provided Friedel pair intensity data are

present in the dataset in sufficient number. To have a reliable assignment of chiral centres the

error of the Flack parameter should not be higher than 0.3. The novel method to calculate the

probability of the assignment of chiral centres is based on Parsons’ quotient.108

In this case

Bayesian statistics is applied to give more reliable assumption for the right configuration

(Figure 7.5.4.1) and the method is incorporated into standard crystallographic packages

(SHELX, PLATON), too. The assignment of the chirality of the crystal structure also works

in systems where the molecules in the asymmetric unit are achiral but the whole crystal

structure is chiral as in the case of quartz.

105 J. M. Bijvoet, A. F. Peerdeman and A.J. van Bommel, A. J. : Determination of the Absolute Configuration of

Optically Active Compounds by Means of X-Rays, Nature, 1951, 168, 271–273. 106 M. Lutz and and A. M. M. Schreurs: Was Bijvoet right? Sodium rubidium (+)-tartrate tetrahydrate revisited,

Acta Cryst., 2008, C64, m296-m299. 107 a) H. D. Flack: On Enantiomorph-Polarity Estimation, Acta Crystallogr., 1983, A39, 876-881.

b) G. Bernardinelli and H. D. Flack: Least-squares absolute-structure refinement. Practical experience and

ancillary calculations, Acta Crystallogr., 1985, A41, 500-511. 108 S. Parsons and H. Flack: Precise absolute-structure determination in light-atom crystals, Acta Cryst., 2004,

A60, s61.

73

Figure 7.5.4.1

Assignment of chirality on the basis of Bayesian probability. Reflections marked with black

support the right configuration while reflections with red (grey) contradict and suggest the

opposite configuration.

7.6. The role of chirality in polymorphism screening

Chirality may influence the formation of different solid forms i.e. polymorphs in several

ways. Pharmaceutical companies are eager to discover new solid forms because of Intellectual

Property advantages. During the development of an innovative drug it is essential to have

suitable protection for all crystal forms. Also it is important to map the polymorphism of the

API to find the optimal formulation and the solid form which is good for this formulation.

Solubility, mechanical properties and stability issues may become very important. An

extensive polymorphism screening is necessary to find as many solid forms as it is possible.

This requires crystallization of the API in a wide parameter space of solvents, temperature,

cooling rate etc. which requires thousands of experiments. There are several robotic systems

on the market to facilitate polymorph screening. Still there is no guarantee that all forms have

been found. However, chirality offers a unique tool for us to open the parameter space even

74

wider and to find more solid forms. Crystallization of an achiral API from the two

enantiomers of a chiral solvent makes a perturbation on packing interaction which may lead to

new solid forms. Another approach could be the co-crystallization of the API with the

enantiomer pair of a co-crystal forming agent. This setup also influences the forces during the

crystal formation. Another important factor is the presence of contaminants. The effect of

contaminants on crystallization is very difficult to study as components of very small

concentration may influence the crystal growing dramatically (Chapter 13.1). However,

crystallizing the API with two enantiomers eventually represents different contaminant

profile. For example one of the co-crystal formers is derived from natural source while the

other one is synthetic or resolved product. In case of a chiral API similar assumptions can be

made and in this case the presence of a chiral co-crystal former may result diastereomeric

crystal forms, too. It has been proven several times:109

“Part per million levels of impurity

have been shown to profoundly influence the size, shape, and rate of growth of some

industrially important compounds. Entities as common as table salt are often modified

intentionally by producers seeking desirable properties.”

7.7. Formation of racemic conglomerates

To fulfil the regulatory requirements for chiral drug substances in vivo measurement of

biological effect of individual enantiomers should be available. Specification for the

stereochemical purity of the drug product should be given. Altogether the desired enantiomer

of the compound should be prepared (Chapter 7.5.1). This can be done via enantioselective

synthesis either using chiral reagents, in stereoselective homogeneous catalytic reaction or by

applying genetically modified microorganism to produce the desired enantiomer. However,

the traditional method to prepare the pure enantiomer is the resolution of the racemate. This

can be reached by adding an optically active agent to the solution of the racemate and this

resolving agent forms salt or adduct with both enantiomers of the compound. Supramolecular

chemistry tools help110

to choose the resolving agent. The adducts of the two enantiomers are

diastereomer pairs of each other and their solubility properties may differ significantly enough

to be separated by crystallization. Repeated crystallization results the pure diastereomer and

109 H. H. Tung, E. L. Paul, M. Midler and J. A. McCauley: Crystallization of Organic Compounds An Industrial

Perspective, John Wiley & Sons, Inc., Hoboken, New Jersey, 2009, p.89. 110 Z. Urbanczyk-Lipkowska and F. Toda: Inclusion Complexation as a Tool in Resolution of Racemates and

Separation of Isomers in Separations and Reactions in Organic Supramolecular Chemistry, Perspectives in

Supramolecular ChemistryVolume 8, Ed. F. Toda, John Wiley & Sons, Chichester, 2004, pp. 1-32.

75

removal of the resolving agent eventually will lead to the pure desired enantiomer. However,

in certain cases the crystals are equimolar mechanical mixture of the pure or almost pure

enantiomers. This is called racemic conglomerate111

and the process of its formation is

spontaneous resolution. It is a great opportunity for the pharmaceutical industry if the drug

substance can be prepared by spontaneous resolution. Unfortunately, spontaneous resolution

was observed in quite rare cases and it is very difficult to prove whether the resolution is

really spontaneous or a chiral impurity is causing the precipitation of crystals of pure

enantiomers. Such contaminants can be in the dust always around us serving as seeds of

crystallization. It is even more difficult to establish a reliable production process on the basis

of spontaneous resolution. It was our great surprise when single crystal X-ray diffraction

study of a potentially biologically active material112

indicated that spontaneous resolution had

occurred although the synthesis was achiral and as it was expected the racemate was formed.

The crystal structure showed a chiral space group and only one of the enantiomers was

present in the asymmetric unit. The molecule contained not one but two chiral centres (Figure

7.7.1.) and while the nitrogen atom can be inverted via the conformational change of the

seven member ring the chiral carbon atom cannot be inverted without breaking of C-H or C-C

bonds. Circular dichroism measurement was performed on single crystal samples and it had

independently proved that indeed we have optically active crystals with different sign of

rotation while the solution of the bulk material did not show any optical activity (Figure

7.7.1). Spontaneous resolution can give the answer for intriguing questions about the origin of

life. Living organisms contain almost exclusively L-amino acids although studies indicate113

that unnatural D-amino acids can be incorporated into proteins. The biological effect of D-

amino acid containing proteins is being explored.

111 http://goldbook.iupac.org/R05028.html 112 A. Polonka-Balint, C. Saraceno, K. Ludanyi, A. Benyei and P. Matyus: Novel Extensions of the tert-Amino

Effect: Formation of Phenanthridines and Diarene-Fused Azocines from ortho-ortho '-Functionalized Biaryls.

Synlett, 2008, 18, pp. 2846-2850. 113 L. M. Dedkova, N. E. Fahmi, S. Y. Golovine, and S. M Hecht: Construction of modified ribosomes for

incorporation of D-amino acids into proteins, Biochemistry, 2006, 45, 5541-51.

76

Figure 7.7.1

The structure of an azocine derivative which showed spontaneous resolution (left). Circular

dichroism study showed Cotton effect of different signs when two different single crystals

were measured (right). No optical activity was observed when the dissolved bulk was studied.

Unpublished results, University of Debrecen.

77

Chapter 8 Structure determination from powder diffraction data

Structure determination from powder diffraction data makes it possible to describe the solid

state structure of the crystalline API as it is used in subsequent formulation steps. Because of

experimental difficulties for high resolution data collection together with theoretical

restrictions such as indexing and integration of peaks the method is applicable only for rather

rigid small molecules of maximum 10-30 non hydrogen atoms.

8.1. Ab initio structure determination from powder diffraction data

Single crystal X-ray diffraction is applicable to describe the crystal and molecular structure of

the material in solid state provided single crystals could be grown. Powder diffraction pattern

gives a fingerprint of the crystal structure and comparison of polymorphic forms as crystalline

powder samples could be performed qualitatively and quantitatively.114

There is a huge

demand of the pharmaceutical industry for the combination of these two methods i.e.

determination of the solid state structure ab initio from the powder pattern. In some cases this

is possible but there are very strong limitations.

(i) Data collection in single crystal diffraction means collecting reflection intensities for

hundreds or tens of thousands of (hkl) reflections. As the crystal and the source as well as the

crystal and the detector distances are constant practically this means moving the detector on

the surface of a sphere around the crystal and collecting intensity data i.e. counts in 3D. In

case of modern CCD detectors the crystal to detector distance is variable but in this context it

is not important. As the crystal and the detector is rotated around three or four axis almost all

(hkl) reflections can be collected separately up to the detection limit (I > 2σI). The resolution

in diffraction methods means maximum θ angle (and d spacing) till measurable intensities (I >

2σI) can be detected. As all atoms contribute to all reflections according to the structure factor

equation(Chapter 7.1.1) this is different from the definition of resolution used in microscopy.

After solving the phase problem we have a ten times or more over determined least square

problem (in case of small molecules) so accurate structure could be determined. However, in

case of powder diffraction we collect data only in one dimension. This results significant

overlap of peaks even at moderate resolution and peaks are highly overlap at high resolution

114 Powder Diffraction Theory and Practice, Eds. R. E. Dinnebier and S. J. L. Billinger, RSC Publishing,

Cambridge, 2008.

78

(Figure 8.1.1). This inherent property of powder diffraction method makes it impossible to

determine intensities of higher angle (hkl) reflections even in case of rather small molecules

(usually the number of atoms is not larger than 10-30).

Figure 8.1.1

Indexed calculated powder pattern of ranitidine hydrochloride (CSD Refcode TADZAZ01) in

the range of 2θ=3-9o (left) and 2θ=20-25

o (right), Mo Kα radiation.

(ii) In spectroscopy the shape of the peaks is Gaussian as a result of theoretical

considerations as the energy levels are occupied according to the Boltzmann distribution. So

in NMR, UV-VIS, IR or Raman spectroscopy overlapping peaks can be de-convolved into

Gaussian curves and in this way number of energy transition and integrals can be calculated

with high accuracy. However, in diffraction the shape of the peak is not Gaussian by theory!

Peak width is influenced by the crystallinity of the sample. Moreover, peaks’ shapes vary with

θ, too. Again, even if we could overcome geometrical difficulties to measure the θ angle with

very high accuracy and eliminate detector limit to have high counts it is impossible to

determine the intensity of reflection even at moderate θ angles. In single crystal X-ray

diffraction it is expected to measure intensities up to θ=25o in case of Mo-Kα radiation and

this requirement can be fulfilled in case of organic molecules easily.

(iii) Symmetry elements may cause systematic absences. Also from the electron density

equation it can be seen that if we cannot determine the unit cell and the space group

accurately structure determination fails even if high resolution data are available. Detection of

systematic absences is even more difficult if we have a powder pattern so unit cell or space

group information is ambiguous.

8.2. Rietveld refinement of powder data.

3 4 5 6 7 8 9

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79

The difficulties of ab initio structure determination can be somewhat relaxed. The number of

parameters to be refined can be significantly decreased by carefully choosing the coordinate

system. Instead of using the fractional coordinates of each atom in the unit cell we can use a

coordinate system fixed to the internal coordinates within the molecule (bond length and bond

angles). Moreover, we can apply strong constrains on these coordinates. For example C-C

distances and C-C-C bond angles in aromatic systems have almost no variability. Ethyl

benzene contains 8 non hydrogen atoms giving 24 coordinate parameters to be determined

and refined but they are not independent from each other. Assuming the known bond length

and bond angle data for the phenyl ring and sp3-sp

3 C-C bonds we need 3 coordinate of the

mass centre, 3 coordinates for the direction of the molecule and one variable bond angle

parameter for the methyl group that is 7 parameters to describe the structure of the molecule.

The breakthrough in ab initio structure refinement was suggested by Hugo M. Rietveld.115

A

whole profile refinement can be performed using all measured data points by considering all

factors influencing peak intensities, their dependence of θ, peak multiplicity, preferred

orientation, extinction, having a reasonable model including space group and good starting set

of atomic positions etc. Using either brutal force of computational power or careful relaxation

of variable parameters calculated and measured intensities i.e. counts can be fitted to the

various parameters (Figure 8.2.1). Of course, data collection has paramount importance to

have success in structure solution and whole-profile Rietveld refinement116

, too. Figure 8.2.2

shows the labyrinth of ab initio structure determination.117

In the best case using synchrotron source solid state structure of small (10-30 atoms) and rigid

molecules with isotropic refinement can be achieved using powder diffraction data although

there are reports for success with tetracycline hydrochloride and even a 117 atom zeolite

structure.118

This is rather intriguing compared that for single crystal X-ray diffraction

structure determination. There is no limit for the size of the molecule and complete crystal

and molecular structure can be calculated including the atomic displacement parameters for

115 a) H. M. Rietveld: Line profiles of neutron powder-diffraction peaks for structure refinement, Acta Cryst.,

1967, 22, 151-152.

b) H.M. Rietveld: A profile refinement method for nuclear and magnetic structures, J. Appl. Cryst., 1969, 2, 65-

71. 116 L. B. McCusker, R. B. Von Dreele, D. E. Cox, D. Louër and P. Scardi: Rietveld refinement guidelines, J.

Appl. Cryst., 1999, 32, 36-50. 117 http://pd.chem.ucl.ac.uk/pdnn/solve1/strategy.htm 118 http://www.cristal.org/SDPDRR/sdpdrr-1-cpd25.pdf

80

all or at least for non hydrogen atoms. The atomic displacement parameters represent the

movement of the atoms around the equilibrium position. This movement can be described as a

tensor which means six further parameters for each atom. In single crystal structure

determination the data/parameter ratio is above 10 even if these parameters are refined, too.

However, as it has benn mentioned earlier in powder diffraction pattern it is not possible to

separate peaks even at moderate 2θ angles. In this way the integration i.e. determination of

peak intensities is also cumbersome giving very low data/parameter ratio. Other limitations of

powder X-ray diffraction measurement119

include:

- Issues related to available equipment (laboratory diffractometer and limited detector

sensitivity).

- Expertise of the scientist performing the experiment and that of the end user.

- Stating the right questions and find the correct answers.

- Careful analysis of the results.

- Necessity to use complementary methods.

- Avoiding pitfalls and misconceptions.

Figure 8.2.1

Rietveld refinement parameter groups.

119 L. M. D. Cranswick: An Overview of Powder Diffraction, in Principles and Applications of Powder

Diffraction, Eds. A. Clearfield, J. H. Reibenspies and N. Bhuvanesh, John Wiley & Sons, Chichester, 2008, pp.

17.27.

81

Figure 8.2.2

Flow chart of structure determination from powder diffraction data.120

With permission of

Rigaku Corporation.

8.3. The cimetidine case

Cimetidine is a histamine H2-receptor antagonist that inhibits the production of acid in the

stomach. It is largely used in the treatment of heartburn and peptic ulcers. It is marketed by

GlaxoSmithKline under the trade name Tagamet (sometimes Tagamet HB or Tagamet

HB200). Cimetidine was approved in the UK in 1976 and was approved in the US by the

Food & Drug Administration for prescriptions starting January 1, 1979.

Histamine released by ECL cells in the stomach is blocked from binding on parietal cell H2

receptors, which stimulate acid secretion; therefore, other substances that promote acid

secretion (such as gastrin and acetylcholine) have a reduced effect on parietal cells when the

H2 receptors are blocked. Like the H1-antihistamines, the H2 antagonists are inverse agonists

rather than true receptor antagonists. Cimetidine is a known inhibitor of many isozymes of the

120 A. Sasaki, A. Himeda, H. Konaka, and N. Muroyama: Ab initio structure analysis based on powder diffraction

data using PDXL, The Rigaku Journal, 2010, 26, 10-14.

82

cytochrome P450 enzyme system121

(specifically CYP1A2, CYP2C9, CYP2C19, CYP2D6,

CYP2E1, and CYP3A4). This inhibition forms the basis of the numerous drug interactions

that occur between cimetidine and other drugs. For example, cimetidine may decrease

metabolism of some drugs, such as those used in hormonal contraception. Cimetidine

interferes with the metabolism of the hormone estrogenic, enhancing estrogenic activity. In

women, this can lead to galactorrhea, whereas in men, gynecomastia has been reported;122

during postmarketing surveillance in the 1980s, cases of male sexual dysfunction were also

reported.123

Cimetidine also affects the metabolism of methadone, sometimes resulting in

higher blood levels and a higher incidence of side effects, and may interact with the

antimalarial medication hydroxychloroquine. The development of longer-acting H2-receptor

antagonists with reduced adverse effects such as ranitidine proved to be the downfall of

cimetidine and, though it is still used, it is no longer among the more widely used H2-receptor

antagonists. Side effects can include dizziness, and more rarely, headache.124

The main powder X-ray peaks of cimetidine form A (anhydrate) and monohydrate, can be

observed at (16.7°, 17.8°, 23.5°, 26.0°, 27.2°) and (13.8°, 15.1°, 15.8°, 20.7°, 26.0°) angles

(2θ, Cu-Kα radiation, on the basis of CSD data), respectively while other forms were

amorphous with no diffraction peak or a halo pattern, respectively. The DSC curves of form

A had one endothermic peak at 143°C due to melting.

8.4. The structure of aspartame anhydrate from powder diffraction data

The structure determination of aspartame anhydrate is reviewed on the basis of literature

data125

to illustrate the method following Figure 8.2.2. Using DSC measurement it was

verified that really the anhydrate form was prepared. A laboratory diffractometer and not a

synchrotron source was used to collect sufficiently accurate powder diffraction data. The

peaks were indexed successfully and the unit cell parameters as well as the space group were

121 M. Levine, E. Y. Law, S. M. Bandiera, T. K. Chang and G. D. Bellward: In vivo cimetidine inhibits hepatic CYP2C6 and CYP2C11 but not CYP1A1 in adult male rats, J. Pharm. Exp. Ther., 1998, 284, 493–499. 122 J. J. Michnovicz and R. A. Galbraith: Cimetidine inhibits catechol estrogen metabolism in women.

Metabolism: clinical and experimental, 1991, 40, 170–174. 123 D. Sawyer, C. S. Conner and R. Scalley: Cimetidine: adverse reactions and acute toxicity. Am. J Hosp

Pharm., 1981, 38, 188–197. 124 D. E. Furst: Pharmacokinetics of hydroxychloroquine and chloroquine during treatment of rheumatic

diseases. Lupus, 1996, 5 Suppl 1: S11–5. 125 C. Guguta, H. Meekes, and R. de Gelder: Crystal Structure of Aspartame Anhydrate from Powder Diffraction

Data. Structural Aspects of the Dehydration Process of Aspartame, Cryst. Growth Des. 2006, 6, 2686-2692.

83

determined. Figure 8.4.1 shows the comparison of indexed, observed and all possible126

peaks.

126

https://docs.google.com/spreadsheet/ccc?key=0Ao5e1nqD3GGvdDhpaGoybGhfa3RtX1VfUGdTNEtlU2c#gid=

0

84

Figure 8.4.1

Indexed powder pattern of Aspartame anhydrate (up), powder pattern (middle, calculated with

error and indeed it is very similar to the observed pattern) and maximum number of peaks

calculated from unit cell parameters without considering structure (down). (On the basis of

data from the Cambridge Structural Database.)

After integration of peaks 327 reflections were identified (cf. approx. 1500 unique reflections

would be measurable for single crystal data). Comparison of unit cell volume, space group

and molecular formula suggests that there are two independent aspartame molecules in the

asymmetric unit and this finding was verified using 13

C solid state NMR study. Based on

known aspartame hemihydrate and hydrate structures a model was constructed for the already

known conformations of aspartame molecules in solid state. The solution of the phase

problem by direct methods usually fails for such low number of data. Simulated annealing

method127

was used to solve the structure. Finally Rietweld refinement (Figure 8.2.1) was

performed for all 5408 data points of powder diffraction data collection.

127 http://en.wikipedia.org/wiki/Simulated_annealing

5 10 15 20 25 30 35 40 45

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85

Chapter 9 Solid state NMR

The Magic Angle Spinning Solid State NMR with Cross-Polarization to enhance sensitivity

(CP-MAS NMR) is the perfect supplement of powder diffraction techniques. Amorphous

materials can also be studied and structural information can be gathered using 2D and 3D

NMR methods with appropriate pulse sequences implemented for solid state NMR, too.

9.1. Basics of solid state NMR

Nuclear Magnetic Resonance (NMR) spectroscopy is the most powerful method to determine

the structure of organic molecules and from small to medium size peptides in solution and in

solid state as well as study the kinetics of chemical reactions on a wide time range. The

method is based on the Zeeman effect128

or Zeeman splitting. Nuclei of certain isotopes (for

example 1H,

13C,

15N etc.) have a magnetic spin of

(or 1,

,

) that is they are small

magnetic dipoles and in (very strong) external magnetic field (B0) there is a measurable split

of energy levels due to the interactions of the nuclei with the magnetic field. The stronger is

the magnetic field the bigger is the difference between the energy levels. The energy levels

can be excited by photons of a few hundred MHz frequencies, i.e. radio frequency (RF) pulse

if the applied magnetic field is several thousand times stronger than the magnetic field of the

Earth. Moreover, valence bond electrons also interact with the magnetic moment of the nuclei

The actual magnetic field at the nuclei will be different than the B0 so the energy levels are

modified by the chemical environment. The shielding is called chemical shift (δ) and

measured in parts per million (ppm). The mathematical description of the interaction between

the magnetic field and the magnetic moment of the nuclei suggest a picture: the nuclear spins

precess around the magnetic field (Figure 9.1.1). The precession rate changes when the

system is irradiated with RF pulse i.e. it absorbs electromagnetic radiation of suitable

wavelength. When the external field is switched off the magnetization vector relaxes and the

oscillating magnetic field causes a signal called Free Induction Decay (FID). We use

appropriate RF pulses to perform various changes in the magnetism of the nuclei and with

another pulse we can read out the actual change in magnetism. The Fourier transform of the

FID signal gives information on the chemical environment (Figure 9.1.2) and the integrals are

proportional with the number of interacting nuclei, for example protons (hydrogen atoms).

128 http://en.wikipedia.org/wiki/Zeeman_effect

86

Changing the elapsed time between the excitation and the reading pulse and/or temperature of

the sample information on thermodynamics and kinetics of molecular movements and/or

chemical reactions can be gathered.

Figure 9.1.1

Model of the NMR experiment, nuclear spins precess around the magnetic field.

Figure 9.1.2

1H NMR spectrum of ethanol. There are three groups of peaks for the three chemically

different hydrogen atoms.

The interaction between the valence electrons and the external magnetic field can be

described as a tensor and it is anisotropic, i.e. direction dependent. Apart from the shielding

which is measured in chemical shift there are coupling among the magnetizations of the

87

nuclei and coupling constants (J) can be determined. We have scalar (J), dipolar and if the

spin is higher than ½ quadruple couplings. In all cases the interaction is proportional with

( ), here is the angle between the external magnetic field and the appropriate

molecule-determined direction. In solution the molecules rotate easily and all directions are

possible. In solid state the rotation is highly restricted and due to the anisotropy the signal is

very weak. However, if we can assure that the direction of the crystallites to the external field

is than and these interactions highly diminish or vanish. This is

the ‘magic angle’ and the sample is quickly (with up to a few 10 kHz frequency) rotated

mechanically around the axis 54.7o to the external field in magic-angle spinning (MAS)

NMR. With appropriate pulse sequence (cross polarization, CP) the signal-to-noise ratio can

be further improved (CPMAS NMR). In this experimental setup useful information

characteristic for the chemical environment and also for the lattice can be extracted. The main

advance of NMR technique is that the experiment i.e. controlled interaction of external

magnetic field and the chemical system can be performed several (usually hundreds or

thousands) times always starting from the (chemically) same system. In solid state NMR the

rotation may cause warming up sample or building up of the electrostatic charge but usually

these problems can be handled. Because of the natural abundance of some isotopes (13

C:1%,

15N:0.3%) enriched samples should be measured for better signal-to-noise ratio and higher

fields are also advantageous, similarly to solution NMR. Basically all pulse sequences and

NMR experiments (2D, 3D, HETCOR, etc., acronyms are widely used for abbreviation of

pulse sequences in NMR) can be adapted to solid samples, too.

9.2. Solid state NMR in polymorphism research

The NMR in both solution and solid phase applicable to map short range order by detecting

distances of nuclei and in this way intra- and intermolecular structure can be deducted. The

solid state NMR can be used for crystalline or amorphous samples. It can be applicable to

distinguish polymorphic forms and in some extent quantitative information on their ratio can

be gained. Using solid state NMR structural information of the asymmetric unit can be

reached and multicomponent systems such as formulated drugs, can also be measured. The

term NMR crystallography is readily used129

and molecular modelling is essential to

elucidate the structural results. Solid state NMR can facilitate the structure determination from

129 http://en.wikipedia.org/wiki/Nuclear_magnetic_resonance_crystallography

88

powder diffraction data as we can conclude distance constraints for flexible parts of the

molecule applicable to the given structure (Chapter 8.4).

The solid sate NMR in polymorphism research can be used for:

- Quantification (drug substance and drug product)

- Study molecular movements, intramolecular mobility

- Detect disorder in polymorphic forms

- Determine distances of nuclei

- Locate hydrogen bonds

- Give information on molecular symmetry, distinguish enantiomers and racemates

- Measure amorphous materials

- Study formation of host-guest complexes

- Investigate API-formulation interactions

- Detect effect of humidity

- Define transitions between polymorphs (variable temperature)

9.3. Polymorphism of aspartame, solid state NMR studies

Even the calculated powder pattern of aspartame shows little difference for the various

solvato-morphic forms (anhydrate and hemihydrate, Figure 3.4.2) Variable temperature X-ray

powder diffraction also can be used130

for studying the transformation between hemihydrate

and anhydrate. Although the structure of aspartame hemihydrate has already been known

from single crystal X-ray diffraction studies but as the calculated powder pattern was different

from the observed pattern it was suggested that a new polymorph of the hemihydrate could be

prepared by ball-milling or by exposing to high temperature and steam. A comprehensive

study131

using combination of methods of solid state NMR, X-ray powder diffraction, in silico

modelling, thermoanalytical studies (Differential Scanning Calorimetry and

Thermogravimetry) combined with chemical method for the determination of water content

(Karl Fischer), Scanning Electron Microscopy etc. gave proof for the existence of the new

130 S. Rastogi, M. Zakrzewski and R. Suryanarayanan: Investigation of Solid-State Reactions Using Variable

Temperature X-Ray Powder Diffractrometry. I. Aspartame Hemihydrate, Pharmaceutical Research, 2001, 18,

267-273.

131 S. S. Leung, B. E. Padden, E. J. Munson amd A. J. W. Grant: Solid-State Characterization of Two

Polymorphs of Aspartame Hemihydrate, J. Pharm. Sci, 1998, 87, 501-507.

89

polymorphic form. The authors also described132

the nature of transformations between

hemihydrate and anhydrate from structural and kinetic point of view, too.

9.3.1. Suggested project work: Application of solid state NMR in the study of steroid

compounds

There are several drug products which contain steroids. Literature search will give examples

for the application of solid state NMR in polymorphism research of this class of compounds.

132 S. S. Leung, B. E. Padden, E. J. Munson amd A. J. W. Grant: Hydration and Dehydration Behavior of

Aspartame Hemihydrate, J. Pharm. Sci, 1998, 87, 508-513.

90

Chapter 10 IR and Raman spectroscopy/microscopy

Vibration spectroscopy methods give indirect information on the solid state structure. Raman

and IR as microscopy techniques can be used for chemical mapping and imaging of drug

products at μm resolution to distinguish among crystallites of polymorphic forms of an API.

10.1. FT-IR and Raman spectroscopy

Several spectroscopic methods are based on the interaction between electromagnetic radiation

(light) and matter. Light has dual nature, it can be considered both as a wave and as a particle,

named photon. The wave nature of electromagnetic radiation leads to phenomena as

interference and diffraction (Chapter 6). When the energy of the radiation corresponds to the

energy difference between two energy levels of the material light is absorbed and the material

is in excited state. The reverse is also true: excited system emits radiation of appropriate

wavelength. The visible and ultraviolet light is capable to excite electron transitions and

according to the appropriate selection133

or transition rules134

vibrations may be excited, too.

However, photons of smaller energy that is infrared radiation can excite only vibrational135

degrees of freedom. Absorption of infrared light136

is studied by infrared spectroscopy.137

The

symmetry properties of the molecule i.e. group theory can be used to determine which types

of vibrations can be excited and/or what is the minimal number of vibrational modes which

are necessary to describe complicated vibration movement. The basic vibration normal modes

are related to the irreducible symmetry representations and not discussed further.

Traditionally in IR (and Raman, see later) spectroscopy the wave number ( ) is used as a

measure of frequency (energy) and its unit is cm-1

. The main difference between IR and

Raman spectroscopy that vibrations altering the dipole moment of the molecule are appearing

in the IR spectrum while vibrations causing change of polarizability are Raman active.

However, symmetry forbidden vibrations result peaks (bands), too, of low intensity. The

vibrations of molecules can be described using the simple spring model and Hooke’s law. For

133 http://wwwchem.uwimona.edu.jm/courses/selrules.html 134 http://en.wikipedia.org/wiki/Selection_rule 135

http://chemwiki.ucdavis.edu/Physical_Chemistry/Spectroscopy/Vibrational_Spectroscopy/Vibrational_Modes/N

umber_of_vibrational_modes_for_a_molecule 136 http://en.wikipedia.org/wiki/Infrared_spectroscopy 137 Infrared and Raman Spectroscopy. Methods and Applications Ed. Bernhard Schrader, Wiley-VCH,

Weinheim, 1995.

91

a diatomic molecule if atomic mass units are used and the force constant (f) is in

the

frequency of vibration is

(10.1.1)

√ (

)

where m1 and m2 are the masses of the two atoms while the f force constant is basically the

strength of the bond between them. The energy transitions can be described using the

harmonic potential model at the first approximation and for more complete description of

energy transitions anharmonicity is considered, too. As both the strength of the bond(s) and

the mass of a functional group is approximately independent from that of the other part of the

molecule there are characteristic138

IR bands (Table 10.1.1.) in the spectrum which are highly

representative of the given functional group. These features made IR spectroscopy as

workhorse of structural analytical methods to help chemical synthesis. Chromatographic

methods represent another utility to contribute the development of chemical synthesis of the

last few decades.

Table 10.1.1

Selected characteristic IR absorption frequencies of organic functional groups.

Functional

Group

Type of

Vibration

Characteristic

Absorptions (cm-1

) Intensity

Alcohol

O-H (stretch,

H-bonded) 3200-3600 strong, broad

O-H (stretch, free) 3500-3700 strong, sharp

C-O (stretch) 1050-1150 strong

Alkyl Halide

C-F stretch 1000-1400 strong

C-Cl stretch 600-800 strong

C-Br stretch 500-600 strong

138 http://www2.ups.edu/faculty/hanson/Spectroscopy/IR/IRfrequencies.html

92

Functional

Group

Type of

Vibration

Characteristic

Absorptions (cm-1

) Intensity

C-I stretch 500 strong

Amine

N-H stretch 3300-3500

medium (primary amines: two

bands; secondary: one band, very

weak)

C-N stretch 1080-1360 medium-weak

N-H bending 1600 medium

Aromatic

C-H stretch 3000-3100 medium

C=C stretch 1400-1600 medium-weak,

multiple bands

Analysis of C-H out-of-plane bending can often distinguish substitution patterns

Carbonyl

C=O stretch 1670-1820 strong

(conjugation moves absorptions to lower wave numbers)

Acid

C=O stretch 1700-1725 strong

O-H stretch 2500-3300 strong, very broad

C-O stretch 1210-1320 strong

Aldehyde

2210-2260 medium

C=O stretch 1740-1720 strong

=C-H stretch 2820-2850 & 2720-2750 medium, two peaks

Amide

C=O stretch 1640-1690 strong

N-H stretch 3100-3500 unsubstituted have two bands

N-H bending 1550-1640

93

Functional

Group

Type of

Vibration

Characteristic

Absorptions (cm-1

) Intensity

Anhydride

C=O stretch 1800-1830 & 1740-1775 two bands

Ester

C=O stretch 1735-1750 strong

C-O stretch 1000-1300 two bands or more

Vibrational spectroscopic studies give valuable structural information for gaseous, liquid or

solid samples and mixtures equally. Reaction media can be analysed effectively, too. While

the characteristic bands are informative for the functional groups present in the sample the

fingerprint region139

of 1500-500 cm-1

helps to identify pure compounds unambiguously.

Polarization occurs also in the infrared region and Vibrational Circular Dichroism (VCD)

spectroscopy140

is a novel technique to study chirality and helps to assign stereogenic centres.

One of the main difficulties in handling IR radiation is that special optics are needed made

of141

rock salt (NaCl) or other highly vapour sensitive materials (caesium salts) which require

dry air all the way in the beam path. Water causes similar etching on these materials as H2F2

does on glassware as it dissolves SiO2. Sensitivity of detectors in the IR region is low even if

the detector is cooled using liquid nitrogen or by other techniques142

such as Peltier cooling.

The sensitivity issue can be handled by applying143

Fourier Transform Infrared Spectroscopy

(FT-IR) where the time domain is measured several times and the spectrum is obtained after

mathematical treatment of time domain data (Fourier transformation). While qualitative

information of IR spectra is enormous quantitative information is difficult to gather and has

much higher error than concentration determination using UV-VIS spectroscopy.

The phenomenon of Raman scattering helps to circumvent problems associated with the

power of optical devices in the IR region. When electromagnetic radiation is scattered by the

material it could be elastic scattering or Rayleigh scattering. In that case no energy is

absorbed by the material and the phenomenon is used among others in diffraction techniques

139 http://www.chemguide.co.uk/analysis/ir/fingerprint.html 140 http://www.gaussian.com/g_whitepap/vcd.htm 141 http://www.photonics.com/EDU/Handbook.aspx?AID=25495 142 http://en.wikipedia.org/wiki/Thermoelectric_cooling 143

http://chemwiki.ucdavis.edu/Physical_Chemistry/Spectroscopy/Vibrational_Spectroscopy/Infrared_Spectroscop

y/How_an_FTIR_Spectrometer_Operates

94

(Chapter 6). However, when electrons are excited by UV-VIS light the small energy

difference of vibrational energy levels is superponated on the electron transition spectrum.

That means the material excited by the absorbed light of ν0 frequency emits photons not only

of the same energy but higher and lower ones, ( ). At ambient temperature the

molecules are usually in their vibrational ground states, so some of the energy of the absorbed

photon is transformed into vibrational energy of the molecule (the sample warms up) and the

emitted photon has lower energy ( ). These are the so called Stokes lines and νs

values are the frequencies or wave numbers of photons associated with vibrational transitions

so they give the vibrational spectrum. The Raman-effect was discovered by Indian scientist

Chandrasekhara Venkata Raman144

who got the Nobel Prize of Physics in 1930. According to

the Boltzman distribution a small portion of the molecules is in vibrational excited states even

at room temperature and eventually this leads to the appearance of anti-Stokes lines when the

emitted photon has higher energy than that of the absorbed one on the expense of thermal

energy of the system. The intensity of anti-Stokes lines is always low and in the Raman

spectrum usually the Stokes lines are measured. As transition rules dictate not all vibrational

energy transitions are allowed in the Raman spectrum only those which change the

polarizability of the molecule. Nevertheless as symmetry rules are not always fulfilled

forbidden bands appear with low intensity. The basis of Raman spectroscopy is to irradiate the

sample with high intensity visible light (for example HeNe laser, λ=633 nm) and applying the

appropriate glass optics to separate Stokes lines and detect them as a vibrational spectrum.

The Raman spectrum gives similar information than the IR one i.e. characteristic bands give

intramolecular structural information, which can be perturbed by intermolecular interactions

such as different solid state structure. Again, both solid and liquid samples and mixtures of

different components in different phases can be studied by Raman spectroscopy. In theory

Raman spectroscopy is a non-destructive method. However, as the light intensity of the laser

is very high photochemical reaction can occur. In the presence of transition metal complexes

as contaminants and/or local warming of the sample can result sample decomposition or burn

in the practice.

10.2. FT-IR and Raman microscopy and their application in polymorphism research

144 http://en.wikipedia.org/wiki/C._V._Raman

95

Vibrational spectroscopy gives information on molecular structure. Intramolecular effects

such as differences in solid state structures are detected only indirectly as they cause minor

shifts of characteristic bands. However, using the appropriate optics a microscope can be

constructed in the IR region, too. In case of multicomponent samples such as a drug product

even μm size regions of the sample can be studied. The same is true for Raman microscopy.

These techniques are extensively used in forensic science, too.

10.2.1. Chemical imaging

A novel method of polymorphism research utilizes both the high resolution of IR or Raman

microscopy and the enormous amount of structural information given by vibrational

spectroscopy. Moving the sample in one or two dimensions in very small steps the vibrational

spectrum of that small area can be collected and detailed information can be deducted on the

chemical composition distribution of the sample by mapping the spectra. Strictly speaking

chemical imaging means projecting the image of the sample onto an array detector which

resolves the spectra. The final result is approximately the same and detailed distribution of

excipients and API in the drug product can be determined (Figure 10.2.1.1) distinguishing

among the solid forms of the API.

Figure 10.2.1.1

Tablet imaging showing the distribution of different chemical compounds. Credit to HORIBA

Jobin Yvon.

10.3. ATR techniques

96

The main problem with classic KBr pastille sample preparation for IR spectroscopy is that the

sample is warmed up and the temperature increase accelerates polymorph transformation into

another solid form which is stable at higher temperature. Similar transformation between

different solid forms may occur during formulation of drug substances because of heat effect

and presence of solvent (water). The phenomenon of total internal reflections is used by the

Attenuated Total Reflection145

(ATR) methods. In this case the pure sample is pressed onto

the surface of a cell made of diamond or other suitable material146

of appropriate shape. As

the heat conductivity of diamond is very high (approximately 10x higher than that of Al) and

no ballast material is pressed there is no temperature change of the sample (Figure 10.3.1).

Figure 10.3.1

ATR measurement of IR spectra shows clear differences of two solid forms of a certain API

both in terms of wave number ( ) and intensity differences while in

KBr the two spectra are identical ( ) . Results from the University of

Debrecen, with permission of Alkaloida Research & Development Ltd, Tiszavasvári,

Hungary.

10.4. Polymorphism of dyes and explosives

145 http://en.wikipedia.org/wiki/Attenuated_total_reflectance 146 www.utsc.utoronto.ca/~traceslab/ATR_FTIR.pdf

97

Polymorphism of dyes has wide spread applications in everyday life. The colour of solid

materials depends not only on the possible energy transitions and absorption of light of

different wavelength. Other factors such as particle shape and size as well as interference of

light greatly influence the observed colour (for example in case of the colourful wing of a

butterfly). Even neglecting these later effects the different solid state structures have influence

on the light absorption of the material. A good example can be the copper-phthalocyanine

dyes147

(CPC) which are the basic ingredients of car paints. The very high thermal and UV

stability of such materials together with the wide range of available colours make CPC ideal

for this purpose. The distance and orientation of the large delocalized π electron systems

differ in the various solid forms resulting different colours in the range of blue and Ferrari red.

Some of the polymorphic forms could be prepared as single crystals, too, and the structures

clearly show these effects (Figure 10.4.1). Having the data of the size of the electron system

even the colour can be calculated solving the Schrödinger equation for one dimensional

particle in a box case.148

Several solid forms of copper-phthalocyanine dyes are only known

as powder.

Figure 10.4.1

Single crystal structure of different solid forms of copper phthalocyanine. (Based on data

from the Cambridge Structural Database.)

The material was prepared by BASF researchers in 1935 and the main question is the trick as

all the time: how to stabilize the desired solid form against the thermodynamic tendency

towards the stable form. For example the temperature of the car paint can reach near 100 oC at

the engine and the high temperature may facilitate the polymorph transformation into the

147 http://www.pcimag.com/articles/copper-phthalocyanines 148 http://hyperphysics.phy-astr.gsu.edu/hbase/quantum/pbox.html

98

stable form. However, appropriate additives help to prevent form transition and keep the

desired colour for a long time.

Quinacridone (Pigment violet 19) also shows polymorphism. The colour is determined149

by

the structure of the solid form (Figure 10.4.2) and the hydrogen bond pattern apart from

substituents on the ring system. The different solid forms of quinacridone and its derivatives

are extensively used in paints and in inkjet toners.

Figure 10.4.2

Hydrogen bond pattern in quinacridone polymorphs. (Based on data from the Cambridge

Structural Database.)

Polymorphism has paramount importance on the production and use of energetic materials150

such as explosives, too. Here we consider chemical explosion which is usually a solid-gas

reaction. In this way the structure of the solid phase influences the chemical reaction, heat

generation, shock wave etc. The polymorphism of 2-4-6-trinitrotoluene, TNT151

is a famous

example. The metastable solid form which was the product of the synthesis caused

unexpected explosions152

in TNT factories during the World War II. Military had to accept

that in spite of the urgently high demand for munition an appropriate storage time was needed

for the freshly prepared explosive to have the expected solid form which could be processed

into bullets. Explosives are used not only in the military or industry but also in fireworks or in

149 http://en.wikipedia.org/wiki/Quinacridone 150 www.nrl.navy.mil/content_images/08FA1.pdf 151 http://pubs.acs.org/doi/abs/10.1021/cg0340704 152 http://images.sciencesource.com/preview/SD9969.html

99

air bags. A very common fertilizer is ammonium nitrate and its polymorphism153

is especially

important as it can be explosive, too.

10.4.1. Suggested project work: Search for polymorphic forms of energetic materials

153 H. B. Wu, M. N. Chan and C. K. Chan: FTIR Characterization of Polymorphic Transformation of

Ammonium Nitrate, Aerosol Science and Technology, 2007, 41, 581-588.

100

Chapter 11 The use of Cambridge Structural Database in polymorphism research

The vast amount of structural data provided by single crystal structure determination is

collected in the Cambridge Structural Database (CSD). These data help to understand the

structural features of polymorphic forms. The database contains practically all crystal

structures of organic and organometallic molecules published in scientific journals but

because of Intellectual Property reasons the structures of several drug substances are

missing. In other cases the database contains information for derivatives or salts different

from that of actually used in the drug product. Together with databases of powder diffraction

patterns and protein structures (PDB) the CSD is a very useful tool in polymorphism

research.154

11.1 The early history of compilation of single crystal data

In 1914 the first crystal structure was determined by W.H. Bragg and W. L. Bragg and the

atomic structure of sodium chloride crystal was revealed. In the following years the method

quickly spread and the structures of several minerals and inorganic compounds were

determined. The first organic molecule structure was determined in 1923 and this molecule

was the hexamethylenetetramine. The single crystal X-ray diffraction method started to

supply enormous amount of experimental data which helped the development of the concept

of chemical bond and also to that of the non-covalent interactions. The structural data gave

unambiguous support for the existence of ionic bond as well as tetrahedral arrangement of

covalent bonds around the sp3 carbon atoms. Synthetic chemists became also extensive end-

user of the results of single crystal X-ray diffraction data. One of the biggest breakthrough

was that in 1951 Bijvoet determined the absolute configuration of (+) sodium rubidium

tartarate as (R,R) on the basis of anomalous dispersion data using single crystal X-ray

diffraction measurement (Chapter 7.5.4). These results had proved that the earlier assumption

for the chirality of (+)-glyceraldehyde was indeed correct. In the 1950s and 1960s the fast

growth of the number of solved crystal structures was handled similarly as other material

data: ambitious projects were initiated for the comprehensive and complete collection of

154 A. D. Bond: The Role of the Cambridge Structural Database in Crystal Engineering in Organic Crystal

Engineering, Frontiers in Crystal Engineering, Eds. E. R. T. Tieknik, J. Vittal, M. Zaworotko, John Wiley &

Sons, Chichester, 2010, pp. 1-42.

101

crystal structures.155

These book series156

worked well for several years but it became obvious

that collection of data, their analysis and organization into books by traditional methods

require too much resources. Numerical data had to be handled using computers! In 1965 the

Cambridge Structural Database was established by O. Kennard and J.D. Bernal. The system

established by them is so well developed that still works fine. The CSD is maintained by the

Cambridge Crystallographic Data Centre (CCDC) “dedicated to the advancement of

chemistry and crystallography for the public benefit through providing high quality

information, software and services”.157

“The CCDC compiles and distributes the Cambridge

Structural Database (CSD), the world's repository of experimentally determined organic and

metal-organic crystal structures. It also develops knowledge bases and applications which

enable users quickly and efficiently to derive huge value from this unique resource.” In 2014

the CSD contains more than 750000 crystal structures and the growth is exponential (Figure

11.1.1) as the number of deposited structures is doubled in every 6 - 7 years.

155 Landolt-Börnstein numerical data and functional relationships in science and technology. Group 3, Crystal

and solid state physics. Vol. 7, Crystal structure data of inorganic compounds by H Landolt R Börnstein O Madelung; K H Hellwege; Wolfgang Pies; Alarich Weiss; Gerhard Pieper; Springer, Berlin, 1985. 156 R. W. G. Wyckoff: Crystal Structures Vol 1, Interscience Publishers, New York, 1963. Vol. 2, Inorganic

compounds RXn, RnMX2, RnMX3, John Wiley Interscience Publishers, New York, 1964. Vol. 3, Inorganic

compounds Rx(MX4)y’ Rx(MnXp) y’ hydrates and ammoniates. Second edition, John Wiley Interscience

Publishers, New York, 1965. Vol. 4, Miscellaneous inorganic compounds, silicates, and basic structural

information. Second edition. John Wiley Interscience Publishers, New York, 1968. Vol. 5, The Structure of

Aliphatic Compounds, John Wiley, New York, 1965. Vol. 6, The Structure of Benzene Derivatives Part 1,

Interscience Publishers, New York, 1966. 157 https://www.ccdc.cam.ac.uk/pages/Home.aspx

102

Figure 11.1.1

The number of deposited structures in Cambridge Structural Database between 1970 and 2014

suggests exponential growth. Data from the CCDC website:

http://www.ccdc.cam.ac.uk/Solutions/CSDSystem/Pages/CSD.aspx

11.2. The method of depositing small molecule single crystal data

Developments in instrumentation and theory of structure determination together with the

increase of power and number of computers made it possible to maintain an exponential

growth (Figure 11.1.1) of deposited data in the last 40 years and the trend probably will

continue. Earlier scientific journals of chemistry and structural biology employed

crystallographers to check crystal structure data. Today practically all publishers and journals

accept the recommendations of the International Union of Crystallography. The main work is

done by experts working at the Cambridge Crystallographic Data Centre (CCDC). This is a

non-profit organization which maintains the Cambridge Structural Database. “Each crystal

structure undergoes extensive validation and cross-checking by expert chemists and

crystallographers to ensure that the CSD is maintained to the highest possible standards.

Also, each database entry is enriched with bibliographic, chemical and physical property

information, adding further value to the raw structural data. These editorial processes are

vital for enabling scientists to interpret structures in a chemically meaningful way.”158

158 http://www.ccdc.cam.ac.uk/Solutions/CSDSystem/Pages/CSD.aspx

103

The method was developed for the specialized crystallographic journals (Acta

Crystallographica) as a result of enthusiastic work of highly experienced crystallographers to

make an accurate and reliable method for the validation of crystal structures together with

continuous feedback from the research community. The main steps of structure deposition

are:

- Researchers prepare new compounds, grow single crystals and determine the structure

by means of diffraction methods

- The Crystallographic Information File (CIF, Chapter 11.3) is constructed and the

structure is validated using freely available in house software resources (PLATON159

)

or at web sites maintained by IUCR or other organizations.

- The results of validation are error messages of different levels (A to G). Level A

means serious errors to be considered and answers for ambiguous data should be

entered into the CIF file.

- Prior to the submission of the article to the scientific journal the crystallographer sends

the CIF file to the CSD to be deposited into the database.

- After further tests for uniqueness of the structure and validation the CSD issues the

depository number and makes the structural data available for peer review referees of

the journal. The depository numbers are included in the paper. The IUCR and CSD

encourage authors to deposit not only the coordinates but also the measured and

calculated structure factors to have more chance for scientists perform other

calculations and refinement on the whole dataset.

- The publication is reviewed and accepted, the paper is available in electronic and/or

printed form and the structure gets its unique name (number) called Refcode (five

letters and two number digits) in the CSD and the data are included in the next

upgrade or issue of CSD.

- If the authors perform further refinement of the structure as a result of referee’s

comments or submit a new CIF file because of other reasons the deposited data are

overwritten.

- If the same structure or a polymorph of the compound is deposited into the database

but the journal accepts as new scientific result the numbers at the end of the Refcode

are changed.

159 http://www.cryst.chem.uu.nl/spek/platon/pl000000.html

104

- If the deposited structure is not published within 3 years the data became part of the

database as personal communication.

The database is available for a yearly fee to browse and extract new information by

comparing the deposited structures and applying new theories, concepts and methodology. In

this way the CSD is the data source of the supramolecular analysis which is one of the main

tools of polymorphism research.

11.3. The Crystallographic Information File (CIF)

Crystallographers have been extensive users of computers for a long time and they always

inspire the increasing demand for higher and higher calculation capacity. The primary result

of data collection is a few MB data (point detector) but with modern CCD detector the dataset

is several hundred MB. In case of macromolecular data collection on multiple crystals and

datasets the amount of primary data could be several GB. However, the final result of the

structure determination with all atomic coordinates and ADP parameters is only 1 kB for a

small molecule and even for macromolecules it is less than 100 kB. To ensure the portability

of data between computers, operation systems and software packages is an essential

requirement in crystallography. Under the auspices of the International Union of

Crystallography the International Tables for Crystallography160

have been published. Volume

G of this series: Definition and Exchange of Crystallographic Data and it deals with these

issues. The dictionary of Crystallographic Information File (CIF) ensures compatibility of

data between softwares and it is part of a wider protocol. According to the IUCR:161

“The

acronym CIF is used both for the Crystallographic Information File, the data exchange

standard file format … and for the Crystallographic Information Framework, a broader

system of exchange protocols based on data dictionaries and relational rules expressible in

different machine-readable manifestations, including, but not restricted to, Crystallographic

Information File and XML.”

Crystallographic program packages use extensively the CIF format to communicate results.

After completion of structure solution and refinement the data harvesting is very

straightforward using CIF and validation can be an automatic process. Recently

160 http://it.iucr.org/ 161 http://www.iucr.org/resources/cif

105

macromolecular, powder diffraction and small angle scattering versions of CIF have been

developed. The CIF file (extension: .cif) is basically a text file and can be checked in any

reader. The lines usually start with a descriptor which is easy to understand and it is followed

by the corresponding data. The dictionaries of the CIF language are regularly updated162

and

developed further. Using appropriate (free) softwares such as MERCURY163

or PLATON164

not only crystallographic calculations (bond length and bond angle data) but graphical

representation of the structure can be achieved easily. It is worth to note that other formats are

also used for transferring structural data. In crystallography the crystal structure i.e. the unit

cell parameters (a,b,c,,,) and space group symmetry are explicitly described and atomic

coordinates are given as fractional coordinates. In computational chemistry the main subject

of calculation is usually one molecule, so coordinates are expressed as Cartesian xyz

coordinates. The pdb format is extensively used in protein crystallography.

It can be anticipated, that standardized description of scientific results will be the requirement

in other areas of science, too. Actually, in the field of characterization of new compounds

(elemental analysis, spectroscopic data) this process has already been initiated by the Royal

Society of Chemistry, the American Chemical Society and other leading organizations. In my

opinion both publishers and fellow scientist accept the rules if these rules are flexible enough

to incorporate variability of the field but strict enough to be reliable and highly transferable.

These requirements can be achieved if a world wide body (association or organization) leads

the project and highly experienced researchers actively take part in the development of the

system.

162 http://www.iucr.org/resources/cif/dictionaries 163 http://www.ccdc.cam.ac.uk/Solutions/CSDSystem/Pages/Mercury.aspx 164164 http://www.cryst.chem.uu.nl/platon/

106

Chapter 12 Regulatory and quality control issues

National regulatory agencies and international bodies give strict recommendations and

guidance in all aspects of pharmaceutical industry. The Q6A specification outlines the basic

decision trees be applied in controlling polymorphic forms of drug substances and in drug

products.

12.1. Polymorphism - quality control issues

Drug products represent a very special type of merchandise. Their production, trade, use and

even price are strictly controlled. The end user, the patient has little or no influence which

drug to use as he/she has to follow the medical doctors’ recommendation and prescription.

The price of the drug product is heavily influenced by governmental decisions via health

plans and the role of the market is restricted. Pharmaceutical companies are profit-oriented

and there is a strong tendency for globalization in this industry, too. Moreover, shares of

pharmaceutical companies are valuable and safe long time investments which make them a

good target for retirement funds worldwide. All these complicated relations of economy,

society and politics had to be handled with keeping safety in mind all the time as drugs

represent significant health hazard for patients. Nowadays environment protection, sustainable

industry as well as safety of employees working in the production of drugs are also important

issues. To keep pharmaceutical production and products in line with safety requirements very

strong, strict and global regulatory principles and rules had to be enforced. The most

influencing agencies are the U.S. Food and Drug Administration165

(FDA in the USA),

European Medicines Agency166

(EMA in Europe, together with national agencies) and

Pharmaceuticals and Medical Devices Agency167

(PMDA in Japan). The global organization

launched in 1990 is The International Conference on Harmonisation of Technical

Requirements for Registration of Pharmaceuticals for Human Use (ICH). The “ICH’s mission

is to make recommendations towards achieving greater harmonisation in the interpretation

and application of technical guidelines and requirements for pharmaceutical product

registration, thereby reducing or obviating duplication of testing carried out during the

165 http://www.fda.gov/ 166 http://www.ema.europa.eu/ema/ 167 http://www.pmda.go.jp/english/

107

research and development of new human medicines”.168

The strongest national agency is

inevitably the U.S. Food and Drug Administration and its recommendations are usually

followed by global pharmaceutical giants and small companies equally. Requirements for

New Drug Applications and eventually human tests for new (innovative or brand name) drugs

are more and more strict. Also the price of new drugs is very high to recover the initial

investment during the relatively short 5-10 years of patent protection. The protection period

left after launching is usually 20 years from the submission of the patent application for a new

compound but development and testing of a new drug product take several years. The volume

of the initial investment is around 1 billion (109) US Dollars. These factors result a strong

tendency in the pharmaceutical industry to move towards generic drugs (Figure 12.1.1) or

combination drugs the affectivity and risk (e.g. side effects) of which were proven.

The definition of Generic Drug: A drug product that is comparable to a brand/reference listed

drug product in dosage form, strength, route of administration, quality and performance

characteristics, and intended use.

Figure 12.1.1

Trend in Brand/Generic mix of retail prescription drug sales in the US.169

168 http://www.ich.org/about/vision.html 169 http://www.uspharmacist.com/content/s/253/c/41309/

0

10

20

30

40

50

60

70

80

2005 2006 2007 2008 2009 2010

Generic 49.4 55.2 59.2 64.5 67.4 71.2

Brand 50.6 44.8 40.8 35.5 32.6 28.8

%

Trend in Retail Prescription Drug Sales, US

108

12.2. Regulatory questions of polymorphism: rules of U.S. Food and Drug

Administration

The regulations issued by the Food and Drug Administration in connection polymorphism are

reviewed here and ICH recommendations are also mentioned.

To market a new innovative drug the pharmaceutical company has to submit a New Drug

Application (NDA) to the appropriate Agency170

and provide all necessary information.

According to the regulations “goals of the NDA are to provide enough information to permit

FDA reviewer to reach the following key decisions:

- Whether the drug is safe and effective in its proposed use(s), and whether the benefits

of the drug outweigh the risks.

- Whether the drugs’ proposed labelling (package insert) is appropriate, and what it

should contain.

- Whether the methods used in manufacturing the drug and the controls used to

maintain the drug's quality are adequate to preserve the drug's identity, strength,

quality, and purity.”

However, after the patent protection expires Abbreviated New Drug Application can be

submitted for a generic drug with the following requirements:

- Same active ingredient(s)

- Same route of administration

- Same dosage form

- Same strength

- Same conditions of use

- Inactive ingredients already approved in a similar NDA

The polymorphism has paramount importance in the competition between pharmaceutical

companies. According to the Food and Drug Administration it is a requirement, that ”ANDA

applicants investigate whether the drug substance in question can exist in polymorphic forms.

170

http://www.fda.gov/Drugs/DevelopmentApprovalProcess/HowDrugsareDevelopedandApproved/ApprovalAppli

cations/NewDrugApplicationNDA/default.htm

109

Polymorphic forms in the context of this guidance refer to crystalline and amorphous forms

as well as solvate and hydrate forms. …. Solvates are crystal forms containing either

stoichiometric or nonstoichiometric amounts of a solvent.

If the incorporated solvent is water,

the solvate is commonly known as a hydrate.

- Crystalline forms have different arrangements and/or conformations of the molecules in

the crystal lattice.

- Amorphous forms consist of disordered arrangements of molecules that do not possess a

distinguishable crystal lattice.

12.3. Q6A decision trees

The Q6A specification171

of ICH deals with test procedures and acceptance criteria for new

drug substances (Figure 12.3.1) and drug products (Figure 12.3.2). Following the decision

tree it can be decided:

- Do multiple polymorphic forms exist?

- Is routine polymorph testing of the drug substance necessary?

- Is routine polymorph testing of the drug product necessary?

The main starting point of all regulatory rules is the safety of the patients and to prevent

similar tragedies observed by Thalidomide (Chapter 7.5.1). According to the Abbreviated

New Drug Application the sameness has to be demonstrated. The applicant has to

demonstrate that the possible polymorphic forms of the API how affect bioequivalence (BE)

and bioavailability (BA). If all the existing forms have high solubility and / or high

dissolution rate differences in the bioavailability of the solid forms are unexpected.

“In this context, concepts from the biopharmaceutical classification system (BCS) provide a

scientific framework for regulatory decisions regarding drug polymorphism. For drugs

exhibiting poor aqueous solubility and high intestinal permeability (BCS Class II), it would be

anticipated that dissolution would be the rate-limiting step to drug absorption and it may even

be possible to establish an in vivo–in vitro correlation. Hence, for such BCS Class II drugs

171 http://www.ich.org/products/guidelines/quality/quality-single/article/specifications-test-procedures-and-

acceptance-criteria-for-new-drug-substances-and-new-drug-produc.html

110

such as carbamazepine, one would anticipate that differences in the solubilities of the various

polymorphic forms have the potential to affect drug product BA/BE.”172

Figure 12.3.1

Decision tree for investigating the need to set acceptance criteria for polymorphism in drug

substances. From Q6A specification with added notes.

The first part of the decision tree defines the existence of different solid forms and sets the

identification methods for the polymorphs. The second part is related to the polymorphic

behaviour of the drug substance (API) and defines when acceptance criteria should be set.

However, “drug product stability depends upon not only the intrinsic chemical reactivity of

the drug substance polymorphic form but also on other factors, including formulation,

manufacturing process, and packaging, many of these facets should be incorporated into the

172 S. P. F. Miller, A. S. Raw and L. X. Yu: Scientific Considerations of Pharmaceutical Solid Polymorphism in

Regulatory Applications in Polymorphism, Ed. R. Hilfiker, Wiley-VCH, Weinheim, 2006, p. 386.

111

scheme of making a rational determination as to what the relative risk a change in

polymorphic form would have upon drug product stability. … In conclusion, the differing

chemical and physical properties of polymorphic forms can certainly impact drug product

manufacturability, bioavailability and stability, and hence impact drug product safety,

efficacy, and quality. Under these circumstances it is important to have suitable controls on

polymorphic forms. However, one must be cognizant of the instances in which polymorphism

has little or no effect upon the critical drug product performance quality attributes. In this

situation, there is clearly no rational reason to have controls on polymorphs forms. Hence,

controls on polymorphs should be incorporated only when appropriate. …. Generally, when

the drug substance manufacturing process routinely gives a single polymorphic form,

preparation and testing of drug product from other forms of the

drug substance would not be necessary.”173

The Ritonavir case (Chapter 1.5.) demonstrates that in spite of a substantial polymorphism

screening a new form was emerging after several years of production, though the

manufacturing process was believed to be in hand.

173 S. P. F. Miller, A. S. Raw and L. X. Yu: Scientific Considerations of Pharmaceutical Solid Polymorphism in

Regulatory Applications in Polymorphism, Ed. R. Hilfiker, Wiley-VCH, Weinheim, 2006. pp. 387-398.

112

Figure 12.3.2

Decision tree for investigating the need to set acceptance criteria for polymorphism in drug

products. From Q6A specification with added notes.

“When drug product samples made from different solid-state forms of the drug substance are

studied to set appropriate controls for the drug substance, these data may show that

dissolution testing is sensitive to the solid-state form of the input drug substance. However,

when no change in dissolution is seen, it may be that both forms were converted into a similar

solid state composition during drug product manufacturing, or that the dissolution procedure

is not adequately sensitive…The outcome of monitoring polymorph during stability of drug

product will be reached in Part 3 of Q6A’s Decision Tree #4 for situations where (a) there

are multiple relevant polymorphic forms with properties that are sufficiently different that

effects on drug product performance could reasonably be anticipated, and, in addition, (b) it

113

has not been possible to show that an in vitro test such as dissolution is sensitive to the

polymorphic composition within the drug product.”

12.4. The Process Analytical Technology concept

In 2002 the U.S. Food and Drug Administration initiated a new approach, the Process

Analytical Technology (PAT) for further increase the safety of drug products and issued

guidance in 2004. The European regulation followed this tendency. According to the Food

and Drug Administration174

”The goal of PAT is to understand and control the manufacturing

process, which is consistent with our current drug quality system: quality cannot be tested

into products; it should be built-in or should be by design.”

In the name of Process Analytical Technology the analytical and technology terms are used in

slightly different sense than in chemistry. „The PAT is a system for designing, analysing, and

controlling manufacturing through timely measurements (i.e., during processing) of critical

quality and performance attributes of raw and in-process materials and processes with the

goal of ensuring final product quality. It is important to note that the term analytical in PAT

is viewed broadly to include chemical, physical, microbiological, mathematical, and risk

analysis conducted in an integrated manner.

Process Analytical Technology tools:

There are many current and new tools available that enable scientific, risk-managed

pharmaceutical development, manufacture, and quality assurance. These tools, when used

within a system can provide effective and efficient means for acquiring information to

facilitate process understanding, develop risk-mitigation strategies, achieve continuous

improvement, and share information and knowledge. In the PAT framework, these tools can

be categorized as:

- Multivariate data acquisition and analysis tools

- Modern process analysers or process analytical chemistry tools

- Process and endpoint monitoring and control tools

174 http://www.fda.gov/aboutfda/centresoffices/officeofmedicalproductsandtobacco/cder/ucm088828.htm

114

- Continuous improvement and knowledge management tools

An appropriate combination of some, or all, of these tools may be applicable to a single-unit

operation, or to an entire manufacturing process and its quality assurance.

A desired goal of the PAT framework is to design and develop processes that can consistently

ensure a predefined quality at the end of the manufacturing process. Such procedures would

be consistent with the basic tenet of quality by design and could reduce risks to quality and

regulatory concerns while improving efficiency. Gains in quality, safety and/or efficiency will

vary depending on the product and are likely to come from:

- Reducing production cycle times by using on-, in-, and/or at-line measurements and

controls.

- Preventing rejects, scrap, and re-processing.

- Considering the possibility of real time release.

- Increasing automation to improve operator safety and reduce human error.

- Facilitating continuous processing to improve efficiency and manage variability

- Using small-scale equipment (to eliminate certain scale-up issues) and dedicated

manufacturing facilities.

- Improving energy and material use and increasing capacity. ”

On the basis of PAT new requirements open for controlling polymorph composition of an API

during manufacturing and also formulating the drug products. Application of these regulations

is the task for pharmaceutical companies for the near future. In the field of polymorphism

research scientists may and should provide further information in understanding the

phenomenon of polymorphism and provide further theoretical background of the field for the

industry.

“Within this PAT framework, and in the particular context of polymorphism, real-time control

provides continuous quality assurance that the final product possesses the polymorphic forms

that may be critical to product performance. More importantly, real-time measurements with

the aid of chemometric tools and appropriate experimental design enable one to quickly

identify the critical sources of polymorphic variability that may impact product performance.

Identification of critical variables can help design and develop a process, which not only

115

controls polymorphic form variability but also reliably predicts such variability over the

design space established for raw materials, process parameters, and other conditions.”175

12.4.1. Example for PAT approach in monitoring and controlling polymorph

compositions

On the basis of the PAT initiative efforts are taken176

for the in-line controlling of polymorph

composition. A fast development in this field is anticipated in the following years. As an

example, it was possible to monitor crystallization of carvedilol, a non-selective β blocker

using Raman spectroscopy method in real time.177

Based on the feedback of the Raman signal

even controlling of polymorphic form178

could be achieved by applying suitable PLC

(Programable Logic Controller) and appropriate algorithm.

175 S. P. F. Miller, A. S. Raw and L. X. Yu: Scientific Considerations of Pharmaceutical Solid Polymorphism in Regulatory Applications in Polymorphism, Ed. R. Hilfiker, Wiley-VCH, Weinheim, 2006. p. 399. 176 P. Barrett, B. Smith, J. Worlitschek, V. Bracken, B. O’Sullivan and D. O’Grady: A Review of the Use of

Process Analytical Technology for the Understanding and Optimization of Production Batch Crystallization

Processes, Org. Process Res. Dev., 2005, 9, 348-355. 177 H. Pataki, I. Markovits, B. Vajna, Z. K. Nagy and G. Marosi: In-Line Monitoring of Carvedilol

Crystallization Using Raman Spectroscopy, Cryst. Growth Des., 2012, 12, 5621−5628. 178 H. Pataki, I. Csontos, Z. K. Nagy, B. Vajna,M. Molnar, L. Katona and G. Marosi: Implementation of Raman

Signal Feedback to Perform Controlled Crystallization of Carvedilol, Org. Process Res. Dev., 2012, 2013, 17, pp

493–499.

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Chapter 13 Technological aspects of polymorphism research: Controlling polymorph

compositions

The total control of chemical reactions and also crystallization is more and more important in

pharmaceutical industry. The Process Analytical Technology (PAT, Chapter 12) enforced by

regulatory agencies, too, represents significant steps towards this target. However, controlling

crystallization i.e. nucleation, crystal growth and morphology is very challenging. The

theoretical description of the metastable region (Chapter 4) is rather straightforward.

Description of the kinetics of nucleation is much more difficult because of both experimental

and theoretical reasons. Efforts to handle homogeneous nucleation gave promising results in

understanding the nature of the nanoscale world and nucleation.179

However, in real examples

there are several indications that nucleation occurs on heterogeneous manner. See for example

the ranitidine hydrochloride and the ritonavir cases when it was proved that unintentional

seeding can cause formation of the undesired or patent protected solid form.

13.1. The effect of contaminants and seeding

Dust is around us and can contain particles of pharmaceutical compounds, too. These particles

can serve as seeds of crystallization. Removal of solids from air is question of technology and

money. These “seeds” can be chemicals, remains of living organisms such as spore of

bacteria, viruses etc. Decreasing their number is essential in medicine, surgery, semiconductor

industry, biology or pharmaceutical industry. The size of visible particles is around 10 μm

while sterile filters of 0.2 μm are also available180

for different applications. However, even in

clean rooms181

the possibility of unintentional seeding cannot be excluded. From

crystallization and polymorphism control aspect trace of contaminants makes an additional

difficulty. At atomic level every chemical system is multicomponent one because of the

magnitude of the Avogadro’s number (6.02x1023

mol-1

). Analytical tools and methods make it

possible to analyse contaminants to the ppb or ppt (parts per billion or parts per trillion, 10-9

,

10-12

) level. Regulations of U.S. Food and Drug Administration, US Pharmacopeia, EMEA

etc. give threshold of this order of magnitude for example for heavy metal contaminants in

drug substances or drug products but this means that we have millions of contaminant

179 V. G. Dubrovskii: Nucleation Theory and Growth of Nanostructures, Springer Heidelberg New York

Dordrecht London, 2014. 180 http://microsite.sartorius.com/platinum/application-matrix.html 181 http://www.cemag.us/topics/contamination-control-and-out-cleanroom

117

molecules in the smallest sample. At the same time contaminant profile of Active

Pharmaceutical Ingredients is routinely checked only to the ppm (parts per million, 10-6

) level.

Components of lower concentration are very difficult to analyse and identify. Still these

contaminants can alter the crystallization process as their activity i.e. partial molar Gibbs

energy should be considered in the thermodynamic description of multicomponent multiphase

systems such as crystallization. “It has long been recognized that the presence of even minute

amounts of impurities substantially affects the kinetics of crystal nucleation, growth, and

dissolution. Because of the complexity of the process, however, the exact mode of operation of

these impurities on the molecular level is still, by and large, obscure. Any theory trying to

explain the role played by these molecules must take into consideration structural parameters

of the growing crystals, their morphologies, and the stereochemistry of the impurities. … An

understanding of these stereochemical aspects provides a powerful tool for the design of

useful auxiliary molecules which can deliberately be added to the solution to improve the

crystallization process.”182

13.2. Scale-up

One of the central problems of chemical engineering is scale-up. Courses such as

Pharmaceutical Technology give more details but from the point of view of polymorphism

research the dependence of mass and heat transport on the size or even the shape of the

reactors is obvious. Here we do not consider examples for crystallization of inorganic

compounds, metal alloys or silicon, although they are also very important from industrial

aspect and are performed on very large scale. In connection with polymorphism the target is

controlling the structure of the solid form completely. A few g of material is needed for

laboratory experiments to reach a reasonable map of the metastable zone of crystallization in

terms of cooling rate, additives or salt effect. However in manufacturing scale even small

number of experiments can have unacceptable cost. According to the literature183

a new form

of ranitidine hydrochloride (Form II) was discovered by Glaxo during a scale up. The new

polymorph had IR spectrum and X-ray powder diffraction pattern markedly different from the

previous batches.

182 I. Weissbuch, L. Leiserowity and M. Lahav: ‘‘Tailor-Made’’ Additives and Impurities in Crystallization

Technology Handbook, 2nd Edition, Ed. A. Mersmann, Marcel Dekker, New York, 2001, p. 525. 183 J. Bernstein: Polymorphism in Molecular Crystals, Calderon Press, Oxford, 2002. IUCr Monographs on

Crystallography, p. 299.

118

13.3. The effect of ultrasound on crystallization

As an example for emerging new technologies application of ultrasound is a relatively new

tool in polymorphism research. Measuring the velocity of ultrasound the metastable zone can

be mapped as a function of parameters such as antisolvent concentration, agitation or cooling.

It is a good supplement of optical methods when reliable detection of crystal formation is

difficult. We have a wide range of electromagnetic waves from UV-Vis through NIR, IR and

Terahertz radiation to detect solid material in the solution. However, application of ultrasound

makes it possible to develop a conceptionally different method. Ultrasound can be used

beneficially in several key areas of crystallization, such as184

- Initiation of primary nucleation, narrowing the metastable zone width

- Secondary nucleation

- Crystal habit and perfection

- Crystal size distribution

- Reduced agglomeration

- Improved product handling

13.4. Micronization and nanonization

Solubility of the Active Pharmaceutical Ingredient (API) can be the key factor to reach the

desired biological effect of the drug. Several methods and tools are available to increase

solubility in the physical form’s research such as co-crystal forming. The most frequently

applied method in pharmaceutical industry maybe the production of amorphous material.

Crystal forming can occur from the amorphous form of very high energy in the formulation

step or during storage. It is an acceptable reason to prepare crystalline material of very small

particle size rather than amorphous material. Micronization185

i.e. the reduction of particles to

a region of 5-10 μm can be reached traditionally by grinding. There are different methods of

nanonization for preparing even smaller particles bellow , for example by using supercritical

fluids, etc. to increase the surface-to-volume ratio resulting

184 H. H. Tung, E. L. Paul, M. Midler and J. A. McCauley: Crystallization of Organic Compounds An Industrial

Perspective, John Wiley & Sons, Inc., Hoboken, New Jersey, 2009, p.237. 185 http://en.wikipedia.org/wiki/Micronization

119

120

Chapter 14 Summary and outlook. Concluding remarks

Polymorphism is a well known feature of the solid state. However, the last 3 decades brought

an explosively growing interest of that field. Because of the interdisciplinary nature of

polymorphism widespread application of several branches of science is a must to understand

this complicated phenomenon. However, the other direction of scientific development is also

vivid. Polymorphism research is not simply the clever application of theoretical, analytical

process development etc methods. The questions emerging when polymorphism is studied

fertilize both academic and industrial research fields. Manufacturers of scientific instruments

are also keen on polymorphism as there is a quickly expanding demand for coupled

techniques, further improvement of the existing methods and even opens new market for well

established instrumentation. For example the market of PAT-ready analytical instruments i.e.

in-line or on-line monitoring of chemical systems is also booming. Polymorphism research

gives ample opportunity for academic research, too. Very elaborated theories and application

of first principles are needed for the description of complicated observation such as

crystallization of the different solid forms, transformations between phases or structural

background of different solid forms existing. All these research topics are fuelled by the high

demand of the pharmaceutical companies. Generic drugs occupy larger and larger market

share. New polymorphs can give exclusivity and market advantage financial background of

polymorphism research is very strong. The investment in this field is much lower in volume

and risk than the cost needed developing an innovative drug. However, we should add that the

number of new small-molecule drugs significantly shrank in the last few years. The whole

pharmaceutical industry moves towards protein based medication, monoclonal antibodies etc.

and eventually towards biologically engineered drug products. This tendency will probably

continue still solid state polymorphism studies will further expand in the coming decades.

Polymorphism of genes and the concept of polymorphism as used in proteomics are not

subject of our discussion.

In the next decades significant improvement in the following fields are foreseen and will give

job for thousands of researchers:

(i) Theoretical description of nucleation, crystal growth and solid state

transformation.

(ii) Predicting polymorph structures on the basis of quantum mechanical calculations

and first principles will become more reliable as a result of improvement in both

121

theory and availability of computational power which leads to smaller error in

energy calculation. Eventually mapping the energy landscape of different solid

forms with high accuracy becomes possible for complicated flexible molecules,

salts, co-crystals etc.

(iii) Supramolecular description of solid state structure moves towards real crystal

engineering i.e. understanding the property-structure relation of the solid state and

intentionally influencing the properties of solid material. The structural motives

determined by single crystal X-ray diffraction methods get their real weights in

determining the solid state structures as more and more techniques such as solid

state NMR, IR and Raman spectroscopy and computational tools are coupled with

the concept of supramolecular chemistry.

(iv) Analytical techniques had to be applied at the edge of their capacity and

applicability for the identification and unambiguous characterisation of solid

forms. This gives a strong impetus and demand for instrument and method

development and application of new technologies.

(v) Regulatory consequences of polymorphism are also under improvement. The

newest trend is to prepare deuterated derivatives of compounds and interesting

polymorphic forms have been found.186

Similar polymorphism among drug

substances is also possible and new rules and guidelines in this respect are

foreseen. The new definition of co-crystals by Food and Drug Administration

made significant turmoil in the scientific community. The definition will change

probably to accommodate better to scientific principles, still the questions of co-

crystals and polymorphism had to be handled in terms of regulatory, academic,

application and engineering point of view.

(vi) The approach of Process Analytical Technology (PAT) gains more and more

acceptance by regulatory authorities. The concept will encourage academic

research to understand the exact mechanism of complicated synthetic reactions.

Moreover, new analytical instruments are being developed for example in-line

probes for monitoring chemical composition, solid form etc. during the

manufacturing process. Of course process development and manufacturing drug

186 R. Chitra, R.R. Choudhury, F. Capet, P. Roussel and H. Bhatt: Effect of deuteration: A new isotopic

polymorph of glycine silver nitrate, J. Mol. Struct., 2013, 104, 27–35.

122

substances have to be re-designed in several cases by observing the methodology

and guidelines of PAT.

(vii) Protection of intellectual property rights is one of the main issues in polymorphism

research. The sophisticated scientific approach is essential in every case.

Nevertheless very different and surprising projections of the phenomenon of

polymorphism can be observed in the decisions of the jury as in some cases the

court deals with questions which are really surprising from chemical point of view.

Moreover, public hearing and trials are not necessarily represent the interest of

pharmaceutical giants as information highly confidential for the companies may

get publicity.

Polymorphism research is the science of this and the next century. Our understanding of

nature will be highly improved by studying the structure and properties of solid forms.

Successful research in the field of polymorphism requires mastering physical chemistry,

several analytical and structure determination method as well as careful balance in intellectual

property issues and regulatory guidelines. It is a really tempting intellectual challenge!

Moreover, the fight for money among pharmaceutical companies peaks in polymorphism

research. The columnist of Economist in 2003 (March 6) wrote a summarizing article (Figure

14.1) on interesting patent issues and let us finish this book with a citation from there: “Forget

horse-racing, says one patent lawyer: Patent litigation is the true sport of kings.”

123

Figure 14.1.

Economist, March 6th, 2003, http://www.economist.com/node/1622601 .

124

Index

A

allotropy · 8 amorphous · 7, 9, 10, 36, 58, 59, 85, 89, 90, 111, 120 anomalous scattering · 61, 73 aspartame · 30, 34, 35, 58, 85, 86, 90 assignment of chiral centres · 72, 74 asymmetric unit · 9, 42, 43, 60, 74, 86, 89

B

Bragg’s law · 54, 55, 61 Bragg-Brentano · 56 Bragg-Brentano geometry · 56 Burger-Ramberger rules · 21

density rule · 23 entropy of fusion rule · 23 heat capacity rule · 23 heat of fusion rules · 22 heat of transition rules · 22 Infrared rule · 23

C

caffeine · 24 Cambridge Crystallographic Data Centre · 103, 104 Cambridge Structural Database · 24, 35, 42, 43, 70, 86,

99, 100, 102, 103, 104 chemical bond · 44, 45, 102 chemical imaging · 97 chirality · 61, 70, 71, 72, 73, 75, 95, 102 chocolate · 11, 24 cimetidine · 84, 85 cocoa butter · 11, 24 co-crystals · 67, 123 conformational polymorphism · 45 copper phthalocyanine · 11 crystal lattice · 12, 54, 68, 111 crystallographic information file · 105, 106, 107

D

Debye-Scherrer · 55 Differential Scanning Calorimetry · 12, 17, 91 Differential Thermal Analysis · 17 dissolution · 16 dust · 40

E

electromagnetic radiation · 49, 61, 87, 92, 95 enantiotrope · 19, 20, 21, 22, 23

energy-temperature diagrams · 16, 19, 20 enthalpy · 10, 16, 17, 20, 71 enthalpy of transition · 21 European Medicines Agency · 108

F

Food and Drug Adminsitration · 13

G

generic drugs · 29, 109, 122

H

heat flux DSC · 17 hemihydrate · 33, 34, 86, 90 heterogeneous nucleation · 40 hydrogen bond · 8, 14, 44, 66, 67, 68, 70, 100

I

ICH · 11, 108, 110, 111 impurity · 77 Intellectual Property · 7, 13, 26, 75, 102 International Centre for Diffraction Data · 58 International Year of Crystallography · 6 inventions · 28, 29

L

lattice planes · 50, 52, 55, 61

M

magnetic spin · 87 metastable zone · 39, 119, 120 Miller indices · 50, 52, 54, 62 monotrope · 19, 20, 21, 22, 23 morphology · 14, 16, 35, 118 multicomponent system · 37

N

New Drug Application · 31, 110, 111 nimodipine · 24, 25 nucleation · 38, 39, 118, 119, 120, 122

125

P

packing polymorphism · 45 paroxetine hydrochloride · 30, 33, 34 particle size · 10, 35, 39, 57, 58, 60 patent · 26, 27, 28, 35

claims · 28, 29, 31 non obviousness · 27 novelty · 27, 28 utility · 27

Pharmaceuticals and Medical Devices Agency · 108 phase diagram · 37, 39 polymorph

nomenclature · 8 polymorph prediction · 12, 43 polymorph screening · 29 polymorph transformation · 13, 98 polymorphism

definition · 7 polymorphism can alter · 10 polymorphism prediction · 42 powder diffraction · 12, 35, 49, 58, 59, 66, 79, 80, 82, 83,

85, 86, 87, 90, 102, 107, 120 powder pattern · 35, 57, 58, 59, 60, 66, 79, 80, 81, 86, 90 Process Analytical Technology · 115, 117, 118, 123

Q

Q6A specification · 10, 108, 111, 112, 114

R

racemic conglomerates · 76 Raman spectroscopy · 12, 24, 80, 92, 96, 117, 123 ranitidine hydrochloride · 29, 30, 31, 32, 33, 57, 80, 118,

119 rate of dissolution · 5, 10

Rayleigh scattering · 49, 61, 96 regulatory agencies · 10, 11, 108, 118 Rietveld refinement · 81, 83 ritonavir · 13, 14, 15, 118

S

single · 63 single crystal · 60, 64, 65, 73, 79, 99 single crystal data · 23, 66, 70, 86, 102, 104 solid state NMR · 12, 87, 89, 90 spinodal · 38, 39 spontaneous resolution · 77, 78 Stokes lines · 96 structure determination

single crystal · 12 structure factor equation · 62, 64, 73, 79 supramolecular · 8, 44, 45, 60, 63, 67, 68, 106, 123 supramolecular chemistry · 8, 45, 123 symmetry · 9, 36, 44, 52, 57, 60, 63, 69, 73, 90, 92, 96,

107 symmetry element · 9

T

Thalidomide · 71, 111 thermoanalytical methods · 12, 16 thermogravimetry · 12, 18, 91

X

X-ray · 10, 12, 35, 39, 42, 49, 50, 55, 57, 58, 59, 60, 61, 62, 63, 64, 67, 68, 69, 70, 72, 73, 79, 80, 82, 85, 90, 102, 120, 123

X-ray powder diffraction · 12, 59