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MASARYK UNIVERSITY

FACULTY OF SCIENCE

National Center for Biomolecular Research

Ph.D. THESIS

RNDr. Lenka Grycová Brno 2010

MASARYK UNIVERSITY

FACULTY OF SCIENCE

National Center for Biomolecular Research

Lenka GRYCOVÁ

APPLICATIONS OF NMR TO STUDY STRUCTURES OF NATURAL COMPOUNDS

PhD. THESIS

SUPERVISOR:

Doc. RNDr. Radek MAREK, Ph.D.

Brno 2010

Bibliographical identification

Name and family name of author: Lenka Grycová

Title of doctoral thesis: Applications of NMR to study structures of natural compounds

Title of doctoral thesis in Czech: Aplikace NMR spektroskopie při studiu struktur přírodních látek

Degree programme: Biochemistry

Degree field (specialization), combination of fields: Biomolecular chemistry

Supervisor: Doc. RNDr. Radek Marek, Ph.D.

Year of graduation: 2010

Keywords: NMR spectroscopy, plant source, flavonoids, quaternary protoberberine alkaloids, nucleophilic addition, X-ray diffraction, cryptolepines, cytotoxicity, DNA binding

Keywords in Czech: NMR spektroskopie, rostlinné zdroje, flavonoidy, kvartérní protoberberinové alkaloidy, nukleofilní adice, rentgenová difrakční analýza, kryptolepiny, cytotoxicita, vazba na DNA

Acknowledgments

I would like to express my gratitude to my supervisor, Doc. RNDr. Radek Marek, Ph.D., whose expertise, understanding, and patience added considerably to my graduate experience.

I must also acknowledge the specialists from the cooperating faculties and universities who participated in the publications resulting from these projects: Doc. Jiří Dostál, Prof. Luc Pieters, Prof. Roger Dommisse, Prof. Filip Lemiere, Karel Šmejkal, PhD, and not least my friend and language consultant Frank Thomas Campbell, PhD.

I would also like to thank my family for the support they have provided me through my entire life and, in particular, I must acknowledge my parents, my husband Michal, and my brother Martin, my friends from the NMR group, and my Belgian “family” Fons, Orfa, Slávka, Anita, and Luc, without whose support I would not have finished this thesis.

In conclusion, this research would not have been possible without the financial support of Masaryk University Brno, the University of Veterinary and Pharmaceutical Sciences Brno, the University of Antwerp, and a Flemish grant for young scientists.

Intercidit eorum quae didiceris scientia nisi

continuetur

Seneca

To my dearest parents Jarmila Baráková and Zdeněk Barák

Declaration of student:

I declare that I have worked on this thesis independently, using only the primary and secondary sources listed in the references.

Lenka Grycová

.......................................

9

Contents

Abstract 17

Abstrakt 19

Chapter 1

Introduction 21

Chapter 2

NMR assay of natural products 24

2.1. Historical notes …………………………………………… 25

2.2. References ………………………………………………… 26

Chapter 3

Return to nature – new inspirations 27

3.1. Biologically active compounds …………………………… 27

3.2. Important or dispensable? ……..………………………… 27

3.3. New solutions for old problems ……………………….. 28

3.4. Historical notes ………………………………………………… 29

3.5. References ………………………………………….…………… 30

Contents ____________________________________________________________________________

10

Chapter 4

Quaternary protoberberine alkaloids: current knowledge 32

4.1. Biological activity ……………………………………………… 32

4.2. Occurrence and structural diversity ……………………...… 33

4.3. Chemical properties …………………………………………… 34

4.4. Historical notes and pictures ………………………………… 35

4.5. References …………………………………………….………… 37

Chapter 5

Cryptolepine derivatives: potential treatment of malaria 38


5.2. Malaria...............………………………………………………… 38

5.3. Occurrence and structural diversity .………………………… 39

5.4. Chemical properties …………………………………………… 40

5.5. Historical notes and pictures ………………………………… 41

5.6. References ……………………………………………….……… 41

Chapter 6

Flavonoids: the known compounds obtained from Paulownia tomentosa 43

6.1. Flavonoids ……………………………………………………… 43

6.2. Paulownia tree ………………………………………………… 43

6.3. Structural diversity of flavonoids ……………………….…… 44

6.4. Flavonoid content ……………………………………..……… 46

6.5. Biological activity ………………………………………..…… 47

6.6. Historical notes and pictures ……………………………..… 48

6.7. References ……………………………………………………… 49

Contents ____________________________________________________________________________

11

Chapter 7

Experimental part 51

7.1. Protoberberine alkaloids ……………………………………… 51

7.2. Cryptolepine alkaloids ………………………………………… 53

7.3. Flavonoids ……………………………………………………… 54

7.4. References…………………………………………………. 55

Chapter 8

Protoberberines: nucleophilic addition 56

8.1. Biological activity and interactions ………………………… 56

8.2. Useful or dangerous? ………………………………………… 56

8.3. NMR analysis and structural information ………………… 57

8.4. Results ...........................……………………………………… 58

8.5. References ……………………………………………………… 58

Appendix A

Quaternary protoberberine alkaloids 59

Appendix B

Covalent bonding of azoles to quaternary protoberberine alkaloids 87

Chapter 9

Cryptolepine derivatives: importance of pKa constants 97


9.2. NMR analytical possibilities and barriers for the project … 97

Contents

____________________________________________________________________________

12

9.3. Results …………………………………………………………… 98

9.4. References ……………………………………………………… 100

Appendix C

NMR determination of pKa values of indoloquinoline alkaloids 101

Chapter 10

Flavonoids of Paulownia tomentosa: determination of structures

109

10.1. Plant material …………………………………………………… 109

10.2. Isolation process ………………………………………………… 109


10.4. Determined structures ………………………………………… 110

10.5. Results …………………………………………………………… 114

10.6. References ……………………………………………………… 114

Appendix D

C-geranyl compounds from Paulownia tomentosa fruits 117

Chapter 11

Conclusions 125

Appendix E

Curriculum vitae 126

Appendix F

List of publications 128

13

List of Figures

Fig 4/1 Basic skeleton of QPA (5,6-dihydrodibenzo[a,g]quinolizinium).

Fig 4/2 Equilibrium between base and salt of QPA.

Fig 4/3 Possible mechanism of photooxidation of DNA with QPA.

Fig 4/4 Pictures of Chelidonium majus and Berberis vulgaris.

Fig 5/1 Structures of indoloquinoline alkaloids cryptolepine,

neocryptolepine, isocryptolepine, and isoneocryptolepine.

Fig 5/2 Standard “proton transfer” typical for indoloquinoline alkaloids.

Fig 5/3 Microscopic pictures of P.vivax, P.ovale, P.malarie, and

P.falciparum.

Fig 6/1 Paulownia tomentosa tree.

Fig 6/2 General flavonoid system C6 – C3 – C6.

Fig 6/3 General structures of flavonoid systems.

Fig 6/4 Apigenin, Mimulone and Diplacone.

Fig 6/5 Paulownia tomentosa tree.

Fig 10/1 Methodology of isolation processes.

List of Figures ____________________________________________________________________________

14

Fig 10/2 Backbones of flavanone and flavanonole.

Fig 10/3 Structures of diplacone and 3’-O-methyldiplacone.

Fig 10/4 Side chains of flavonoid structures.

Fig 10/5 Four compounds newly isolated from plant material: 3’-O-methyl-

5’-methoxydiplacol, tomentodiplacol, 6-isopentenyl-3’-O-

methyltaxifolin, and dihydrotricin.

15

List of Tables

Tab 9/1 Potential relationship between the pKa and the inhibition activity.

Tab 10/1 Biological effects of the flavonoid study.

16

List of Abbreviations

COSY Correlation Spectroscopy

DMSO Dimethylsulfoxide

DNA Deoxyribonucleic Acid

HMBC Heteronuclear Multiple-Bond Correlation

HMQC Heteronuclear Multiple-Quantum Coherence

HPLC High-Pressure Liquid Chromatography

HSQC Heteronuclear Single-Quantum Correlation spectroscopy

IR Infrared spectroscopy

MS Mass Spectrometry

NMR Nuclear Magnetic Resonance

NOE Nuclear Overhauser Effect

QPA Quaternary Protoberberine Alkaloid

THF Tetrahydrofuran

TLC Tin Layer Chromatography

UV Ultraviolet spectroscopy

WHO World Health Organization

17

Abstract

The evolution of diseases in Europe, and in other parts of the world

constrains scientists to cooperate more efficiently with nature.

Analysis of new structures and interactions constitutes one of the general

steps on the long path to the discovery of new pharmacological compounds

that are important for identification and treatment of diseases. Such analysis

includes the identification of the newly obtained compound, determination

of its chemical properties, and the characterization of its interactions with

small molecules as well as with macromolecules.

NMR spectroscopy can be used to identify the constitution and

configuration of natural products, and to characterize their various physical

and chemical properties. Complementary information can be obtained from

X-ray diffraction and mass spectrometry.

The NMR chemical shift is very sensitive to the electron distribution

around the nucleus of an atom, is an important indicator of changes in the

structural arrangement of molecules, for example, in protonation-

deprotonation equilibrium, tautomeric processes, conformation studies, and

the determination of pK values. Characterization of intermolecular

Abstract ____________________________________________________________________________

18

interactions is highly important in pharmacological and biochemical studies

of medicinal drugs.

Nucleophilic addition to a quaternary protoberberine alkaloid gives

a product with a new covalent bond between the alkaloid and a heterocyclic

structure, containing one, two, or three nitrogen atoms. The products have

been characterized by NMR spectroscopy, mass spectrometry, and, in some

cases, X-ray diffraction.

The pKa values of cryptolepine derivatives, which are potentially useful in

the treatment of malaria, are very important. NMR titration

of indoloquinolines showed the dependence of the NMR chemical shifts

on the pH. Analysis of these dependences gave the pKa values.

The structures of several flavonoids isolated from the fruit of the

Paulownia tree were determined by NMR spectroscopy. Some of these were

new and some had been isolated previously but from sources other than the

fruit of Paulownia tomentosa.

19

Abstrakt

Posun v evoluci nemocí nejen v Evropě, ale také na jiných místech světa

nutí vědce více se vracet k přírodě, možná proto, že v některých případech

může být takovýto přístup efektivnější.

Analýza nových látek a vzájemných interakcí je jedním ze základních

kroků vedoucích ke konečné sloučenině, zásadní pro včasnou identifikaci či

léčbu onemocnění. Analýzy zahrnují identifikaci nově izolovaných látek,

chemických vlastností, struktur s modelovými sloučeninami i chování látky

v prostředí makromolekul.

NMR lze použít k identifikaci přírodních látek zahrnující jak základní

určení struktury izolované látky, tak složitější studium různých vlastností.

Srovnatelné nebo doplňující informace může poskytnout například

rentgenová analýza nebo hmotnostní spektrometrie.

Chemický posun, který je velmi citlivý na rozložení elektronové hustoty,

je důležitým nástrojem pro určení široké škály chemických a fyzikálních

vlastností. Lze studovat rovnováhu protonačních dějů, tautomerii látek,

konformační procesy nebo například stanovovat pK hodnoty. Charakterizace

mezimolekulárních interakcí je jedním z velmi důležitých kroků při studiu

Abstrakt ____________________________________________________________________________

20

farmakologie a biochemie nových léků. 2D experimenty lze pak uplatnit

například při určení konstituce alkaloidních a flavonoidních látek.

Nukleofilní adice studovaná na kvartérních protoberberinových

alkaloidech poskytla produkty s kovalentní vazbou mezi studovaným

alkaloidem a modelovou heterocyklickou strukturou s jedním, dvěma nebo

třemi dusíkovými atomy. NMR spektroskopie byla použita k identifikaci

vznikajících produktů.

pKa hodnoty kryptolepinových alkaloidů, potenciálně aktivních látek pro

léčbu malárie, představují důležitou charakteristiku těchto struktur. Stanovení

pKa hodnot je možné několika spektroskopickými metodami. NMR titrace

indoloquinolinů poskytla závislost chemického posunu na pH prostředí.

Z těchto závislostí byly získány hodnoty pKa několika derivátů.

Pomocí NMR spektroskopie byla stanovena struktura několika derivátů

odvozených od flavonoidů isolovaných z okrasného stromu pavlownie

senekalské. Některé struktury jsou nové, některé byly již dříve popsány,

nicméně poprvé z tohoto studovaného stromu.

21

Introduction

The known vegetable kingdom includes around 400 000 – 500 000

varieties of plant and only a very small portion of these have been analyzed to

determine what compounds they contain. Secondary metabolites, in turn

represent only a fraction of the compounds contained in plant material. The

concept of a secondary metabolite and the term used for it has been known

since 1891[1]. They were brought into the microbial kingdom around 1961 by

John Desmond Bu`Lock[2]. The current interest in secondary metabolites

obtained from the plant kingdom was probably stimulated by the hope of

obtaining new sources of compounds potentially useful in therapeutic

programs. This interest was supported by the rapid development of analytical

methods for determining complicated molecular structures. The structural

methods providing resolution at the atomic level comprise especially nuclear

magnetic resonance (NMR) and X-ray diffraction.

This PhD thesis is focused on applications of modern NMR methods to the

study of the structures of several classes of natural compounds. It includes

two large groups of secondary metabolites; alkaloids and flavonoids. The first

project is focused on the structure and reactivity of several protoberberine

Introduction ____________________________________________________________________________

22

alkaloids and is followed by a project dealing with the pKa values of

cryptolepine derivatives and finally a structural study of some flavonoids.

All three projects were carried out in cooperation with researchers in other

laboratories in the Czech Republic and in Belgium:

• Department of Biochemistry, Faculty of Medicine, Masaryk

University, Brno, Czech Republic

• Department of Chemistry, University of Antwerp, Antwerp, Belgium

• Department of Pharmaceutical Sciences, Faculty of Pharmaceutical,

Biomedical and Veterinary Sciences, University of Antwerp, Antwerp,

Belgium

• Department of Natural Drugs and Department of Chemical Drugs,

University of Veterinary and Pharmaceutical Sciences Brno, Brno,

Czech Republic

Current knowledge about natural sources, structures, and the chemical

behavior of quaternary protoberberines has been summarized and published

in the journal Phytochemistry and the results of individual projects have been

published in journals focused on magnetic resonance or natural products.

Lists of publications are attached at the end of each individual chapter.

The thesis is divided into the three major parts:

• Current knowledge about plants, methods of analysis and the three

groups of compounds studied

• Experimental part

• Comments on results and published articles

Introduction ____________________________________________________________________________

23

References

[1] Karlovsky P., 2008, Secondary Metabolites in Soil Ecology. Vol. 14,

Springer, Berlin.

[2] DOI: 10.1007/BF00130753

24

NMR assay of natural products

NMR spectroscopy has been an important analytical tool for investigating

natural compounds for many years. It is an excellent alternative to X-ray

diffraction for compounds that are difficult to crystallize like to X-ray

diffraction, NMR analysis gives good quality information (e.g., composition,

conformation) about the structures of simple natural compounds obtained

from plants.

In the 1980s, multidimensional NMR methods capable of characterizing

the conformations of natural compounds, such as terpenes, were developed.

Nowadays NMR is not limited to small biomolecules; current research

includes the study of larger compounds and biomacromolecules, such as

proteins, nucleic acids, protein – nucleic acids complexes.

Combination of NMR with other experimental methods, such as HPLC,

enables the control of precursors and products, the determination of

compounds with biological activity, the study of the pharmacokinetics of

drugs or their metabolism in body fluids, and the fast analysis of plant

extracts.

NMR assay of natural compounds ____________________________________________________________________________

25

Recently, NMR analysis has been approved in the “European

Pharmacopeia” of the European Union as a one of the methods for

characterizing compounds. NMR identification of compounds has also been

included in recent editions of the “Czech Pharmacopeia”, In addition to

structural information about a natural compounds, NMR drug research

generally includes characterization of a range of its physical and chemical

properties and the possibility of studying its interactions with

biomacromolecules. The rapid development of NMR techniques has offered

many new opportunities for more effective research on natural products.

2.1 Historical notes[1-4]

1924 Prediction of magnetic moment (Wolfgang Pauli),

experimentally confirmed (Stern and Gerlach)

1938 First NMR experiment (Isidor Isaac Rabi)

1944 Nobel Prize in physics – Discovery of NMR (Isidor Isaac

Rabi)

1945 NMR signal in macroscopic material (Bloch with Stanford

team and Purcell with Harvard team)

1952 Nobel Prize in physics – NMR research (Felix Bloch and

Edward Mills Purcell)

1949-50 Definition of chemical shift (Knight, Proctor, and Dickinson)

1951 1H NMR spectrum of ethanol (Arnold, Dharmati and Packard)

1957 First 13C NMR spectrum (Lauterbur and Holm), relationship of

NMR - Fourier transformation (Lowe, Norberg)

1958 Magic angle spinning – solid phase (Andrew and Law)

NMR assay of natural compounds ____________________________________________________________________________

26

1965 Implementation of Fourier transformation in NMR (Ernst and

Anderson)

1971 Concept of 2D NMR (Jeener)

1982 Analysis of a small protein (Wagner and K. Wüthrich)

1983 First 3D structure of a protein determined (K. Wüthrich)

1988-90 NMR study of proteins with isotopic labeling (Bax)

1991 Nobel Prize in chemistry – development of NMR (Richard R.

Ernst)

2002 Nobel Prize in chemistry – development of NMR methods for

analysis of biological macromolecules (K. Wüthrich)

2003 Nobel Prize in physiology or medicine – discoveries

concerning magnetic resonance imaging (P. C. Lauterbur and

P. Mansfield)

2.2 References

[1] Sodomka L., Sodomková M., Sodomková M., 2004. Kronika

nobelových cen, Euromedia group, Praha, pp 68-72, 206.

[2] Buděšínský M., Pelnař J., 2000. Fyzikálně chemické metody

(NMR), Institute of Organic Chemistry and Biochemistry AS CR,

Praha, pp 1-6.

[3] Brus J., 2005. Solid phase NMR, Institute of Macromolecular

Chemistry AS CR, Praha

[4] http://nobelprize.org/nobel_prizes/

27

Return to nature – new inspirations

3.1. Biologically active compounds

Alkaloids and flavonoids are two large groups of secondary metabolites

with highly diverse biological functions. An array of their biological effects

is used in the production of pharmaceuticals. However, a lot of them are used

only in traditional medicine. Some forgotten compounds from traditional

medicine has been rediscovered, and studied for new possible medicinal

effects. [1 - 3]

3.2. Important or dispensable?

Alkaloids and flavonoids are members of a large group of natural

compounds – the secondary metabolites. Currently, their function in plants is

not clearly understood. Various kinds of secondary metabolites probably

have different functions in the plant. The alkaloids are assumed to have

a protective mechanism against herbovire animals and possibly some

Return to nature – new inspirations ____________________________________________________________________________

28

parasites.[4] Flavonoid glycosides and free aglycones are involved in the

interactions of plants with microorganisms, both pathogenic and symbiotic.

They also act as UV protectants in plant cells and pigment sources for flower

colouring compounds, and they play an important role in interactions with

insects.[5,6]

These plant metabolites also affect human and animal health. Flavonoids

have a significance in the diet, ascribed to their antioxidant properties,

estrogenic action,[7] and a wide spectrum of antimicrobial

and pharmacological activities.[5,6] Alkaloids are used in low concentrations

as efficacious remedies serving as cardiotonics, potential oncological

medicines, etc.

3.3. New solutions for old problems

The appearance of new and more complicated diseases in recent years,

along with persistence of old ones, constrains scientists to seek new and more

effective methods of treatment.

The resistance of hosts is one example of the big problems of current

medicaments, predominantly for antibiotics and other medicines used against

infectious diseases. The growing population of the world and the migration of

people increase the spread of very specific and dangerous kinds of diseases,

for example, malaria. If the pathogen that causes a disease becomes resistant

to the medicine used to treat it the number of patients increases and so does

the risk that the disease will spread to other countries.

Another very important concern for specialists is the problem of human

cancer. This is one of WHO`s (World Health Organisation) current

campaigns. The following citation taken is from the WHO project outline:


29

“Cancer affects everyone – the young and old, the rich and poor, men,

women and children – and represents a tremendous burden on patients,

families and societies. Cancer is one of the leading causes of death in the

world, particularly in developing countries.” [8]

In addition to prevention, it is essential to develop of more effective

methods to diagnose illnesses in their early stages, when treatment can be

more effective. Belated diagnosis can be a mortal danger because of the

absence of effective medicines to advanced disease.

Nature offers resources that include compounds which can potentially

solve many of these problems. Investigation of natural products obtained

from plants – the isolation of compounds and their modification, and the

evaluation of their biological activities – represents an important field of

biochemical and pharmaceutical research.

3.4. Historical notes

Traditional medicine has long used plants containing secondary

metabolites to treat different health problems. Natural compounds in

modified forms are currently used as “correct” medicaments.

1785 Withering first described the use of Digitalis purpurea extract

containing cardiac glycosides to treat of heart diseases[9]

1819 Wilhelm Meissner, a German pharmacist, introduced the term

“alkaloid”[10]

1841 Homole and Quevenne isolated digitalin[11]

1846 Rutin was isolated and identified

1928 Windaus isolated digitoxin and digoxin[11]


30

1930 Until this year, using of Quinine was the only effective treating

malaria[12]

1946 Discovery of the phytoestrogens activity of flavonoids

1952 Origins of species flavonoids, a group of plant polyphenols

with flavone structure[13]

1983 A simple general definition of alkaloids was suggested by

Pelletier[14]

1999 Harborne and Baxter published “The Handbook of Natural

Flavonoids”, listing 6467 known flavonoid structures, with

formulae, references, and information on biological

activities[15]

3.5. References

[1] Wollenweber, E., 1988. Occurrence of flavonoid aglycones in

medicinal plants in: Plant Flavonoids in Biology and Medicine II:

Biochemical Cellular and Medicinal Properties. Allan, J., Riss Inc.

pp.45-55, London.

[2] Weidenbörner, M., Hindorf, H., Jha, H. C., Tsotsonos, P. and Egge,

H., Phytochemistry, 1990, 29, 801.

[3] Dixon R.A., Steele c.L., Trends in Plant Science, 1999, 4, 394.

[4] Francois G., Timperman G., Eling W., Assi L.A., Holenz J.,

Bringmann G., Antimicrobial agents and chemotherapy, 1997, 41,

2533.

[5] Stobiecki M., Phytochemistry, 2000, 54, 237.

[6] Schijlen E.G.W.M., Ric de Vos C.H., Van Tunen A.J., Bovy A.G.,

Phytochemistry, 2004, 65, 2631.


31

[7] Miksicek, R.J., Molecular Pharmacology, 1993, 44, 37.

[8] http://www.who.int/en/ official web page of WHO

[9] Wilkins, M.R.; Kendall, M.J.; Wade, O.L., British Med. J., 1985,

290, 7.

[10] Dostál J., J. Chem. Edu., 2000, 77, 993.

[11] Havránek P., Aplikovaná botanika 3, 2008, Department of Botany,

Faculty of Science, Palacký University, Olomouc.

[12] Hobhouse, H. 1985, Seeds of Change, Harper and Row, New york.

[13] Watzl B., Rechkemmer G., Flavonoide. Ernähr. – Umsch., 2001,

48, 499

[14] Roberts, M.F., Wink, M., 1998. Biochemistry, ecology, and

medicinal applications. In: Roberts, M.F., Wink, M. (Eds.),

Alkaloids. Plenum Press, New York, London, pp. 1–7.

[15] Harborne J.B., Williams C.A., Phytochemistry, 2000, 55, 481.

32

Quaternary protoberberine alkaloids: current knowledge

4.1. Biological activity

Projects studying the biological activity of quaternary protoberberine

alkaloids (OPA) as well as the related structures from the isoquinoline group

play an important role in the discovery of potential new pharmaceuticals. The

relationship between structure and activity shows that some substituents may

increase the activity of the backbone structure.

Structural studies of the interaction of QPA with biomacromolecules can

afford answers about the mechanism of action for each individual compound

from a natural source. A recent NMR study of the interaction berberine (OPA

alkaloid) with DNA excluded the intercalation of berberine into the double

helix and confirmed that berberine binds into a minor groove of DNA.[1]

Quaternary protoberberine alkaloids: current knowledge ____________________________________________________________________________

33

4.2. Occurrence and structural diversity

The protoberberine alkaloids are the most widely distributed alkaloids of

the isoquinoline group. Berberine is probably the most widely known

alkaloid.

Most protoberberines exist in plants as tetrahydroprotoberberines,

approximately one quarter are quaternary protoberberine alkaloids, and a few

dihydroprotoberberines have also been described. Plant species that typically

contain QPA are from the families Berberidaceae, Fumariaceae,

Menispermaceae, Papaveraceae, and others. Alkaloids can be present in all

species of a family or only one representative species.

The basic skeleton of QPA is 5,6-dihydrodibenzo[a,g]quinolizinium

(C17H14N+) (Figure 4/1)

NA B

C

D

12

3

4 56

8

9

10

11

12

13

Figure 4/1

Substituents are most frequently present at positions 2,3,9 and 10 of the

QPA or at 2,3,10 and 11, for which the prefix “pseudo” is often used. The

typical substituents are hydroxy groups, methoxy groups, and O-CH2-O

groups. Compounds with substituents at carbon atoms C-1, C-4, C-5, and C-

13 have also been isolated from natural sources. The prefix “retro“ is used for

protoberberines characterized by the presence of an extra substituent as a side

chain of ring D.


34

The pathway for biosynthesis of QPA has been known in detail since the

1960s. This biosynthesis includes the precursor tyrosine and individual

intermediates such as dopamine or adrenaline. A few steps of the pathway

copy those found in the human body. The roles of several enzymes that

participate in the biosynthetic processes have been described.[2 – 9]

4.3. Chemical properties

QPA can exist in two different forms, salt and base. The equilibrium

between these two forms is affected by the pH of the medium (Figure 4/2).

However, the base is rather unstable. A change in pH results in a change in

color.[10] QPA are brightly colored solids with colors ranging from yellow to

orange.

N N OH

X

OH

HX

Figure 4/2

The general reaction that characterizes these compounds is nucleophilic

addition. The reaction of protoberberine with an O-nucleophile (OR-) is

analogous to the reaction of a protoberberine salt in alkali medium and the

formation of its base. A dimeric structure of the alkaloid can form as a minor

component in nonpolar solvents. Other nucleophiles such as N-nucleophiles,

S-nucleophiles, and C-nucleophiles can react with QPA as well.


35

The reactions of QPA also include oxidation and reduction, alkylation, and

disproportionation. A photochemical reaction involving QPA can be one of

the ways of transforming a biological system.[11,12] A possible mechanism for

the photooxidation of DNA has recently been published (Figure 4/3).[13]

Figure 4/3

4.4. Historical notes and pictures

1824 Hüttenschmid found a yellow coloring matter in what he

believed to be Geoffroya inermis, the Jamaica cabbage-tree,

and named the berberine as jamaicine

1826 Chevallier and Pelletan found a yellow alkaloid in the bark of

Xanthoxylum Clava Herculis and named the berberine as

xanthopicrite

1830 Buchner and Herberger obtained berberine, as a yellow extract

from Berberis vulgaris

19th century investigators looked towards general research, the

discovery of compounds from natural sources, and the

general roles of QPA

DNA

berberine DNA - berberine komplex

3O2

1O2

guanine specific oxydation


36

20th century the study of reactivity, analytical methods, and general

research into the biological aspects were emphasized

21st century biological activity and interactions with biological

materials are now thought to be most important

Two plants with containing QPA are typical for central Europe –

Chelidonium majus (a) and Berberis vulgaris (b, c) (Figure 4/4).

a b c

Figure 4/4

The current knowledge of the natural sources, structures, and chemical

behavior of quaternary protoberberines has been summarized in a review

published in the journal Phytochemistry. This review is enclosed as

Appendix A.

4.5. References

[1] Mazzini S., Belluci M.C., Mondelli R.: Bioorg. Med. Chem., 2003,

11, 505.

[2] Beecher C.W.W., Kelleher W.J., Tetrahedron Lett., 1984, 25, 4595.


37

[3] Kobayashi M., Frenzel T., Lee J.P., Zenk M.H., Floss H.G., J. Am.

Chem. Soc., 1987, 109, 6184.

[4] Amann M., Nagakura N. Zenk M.H., Eur. J. Biochem., 1988, 175,

17.

[5] Jenderzejewski S., Phytochemistry, 1990, 29, 135.

[6] Schneider B., Zenk M.H., Phytochemistry, 1993, 32, 897.

[7] Frenzel T., Beale J.M., Kobayashi M., Zenk M.H., Floss H.G.,

J. Am. Chem. Soc., 1988, 110, 7878.

[8] Rueffer M., Zenk M.H., Phytochemistry, 1994, 36, 1219.

[9] Kutchan T.M., Ditrich H., J. Biol. Chem., 1995, 270, 24475.

[10] Shamma M., 1972. The protoberberines and retroprotoberberines In:

Shamma M. (Ed.), The isoquinoline alkaloids. Academic Press,

New York, London, pp 268-314.

[11] Morrison H., 1990. Bioorganic biochemistry: Photochemistry and

the Nucleic Acids, vol. 1. John Wiley and Sons, New York.

[12] Contreras M.L., Rivas S. Rozas R., Heterocycles. 1984. 22, 101.

[13] Hirikawa K., Kawanishi S., Hirano S.: Chem. Res. Toxicol., 2005,

18, 1545-1552.

38

Cryptolepine derivatives: potential treatment of malaria


Cryptolepine and neocryptolepine, indoloquinoline alkaloids obtained

from the plant Cryptolepis sanguinolenta (Lindl.) Schlechter found in central

and western Africa, are known for their antiplasmodial properties and have

been used as the staring compounds for new antimalarial agents.[1 – 4]

5.2. Malaria

The global impact of malaria, one of the most dangerous parasitic diseases

transmitted to human and animal hosts by the Anopheles mosquito, confronts

both developing and industrialized nations. It is estimated that there are

between 200 and 300 million cases every year, and more than 2 million of

these are fatal.[3,5,6] Malaria is one of the problems addressed by programs of

WHO (the World Health Organization).

Cryptolepine derivatives: potential treatment of malaria ____________________________________________________________________________

39

Four different Plasmodium species are responsible for human infection,

P.vivax (a), P.ovale (b), P.malarie (c), and P.falciparum (d). The last one,

Plasmodium falciparum, leads to the most severe illness.[6]

A most serious problem in the prevention and the treatment of malaria is

represented by the increasing resistance of Plasmodium against the drugs

currently in use e.g., chloroquine,[7] hydrochloroquine, and amodiaquine.

These drugs are typically modified forms of backbone structures found in

nature. Current development employs this same strategy.

Artemisinin could represent a possible solution to the problem of

developing resistance.[8] This compound was isolated from Artemisia annua,

a plant used in traditional Chinese medicine.

Another prospective class of compounds is represented by the alkaloids

of the indoloquinoline group, which have shown some antiplasmodial

activity.

Along with the development of possible new drugs for use against

malaria, the implementation of protective tools, e.g., repellents and

insecticides, is also growing. Since the discovery of the repellent compound

DEET (N,N-Diethyl-m-toluamide) during World War II, chemists have been

working on innovative chemical protection, trying to find new ways to

protect against insects.

5.3. Occurence and structural diversity

Cryptolepine (1) is the major alkaloid of the plant Cryptolepis

sanguinolenta, found in central and western –Africa. Neocryptolepine (2), is

one of the minor alkaloids in this same plant. Three isomers of

indoloquinoline: cryptolepine (1), neocryptolepine (2), and isocryptolepine


40

(3), can been isolated from Cryptolepis species.[9] The fourth isomer

isoneocryptolepine (4) has never been found in nature (Figure 5/1).

N

N

CH3

NN

N

N

NN

CH3

CH3

CH3

1 2

3 4

Figure 5/1

5.4. Chemical properties

The chemical constitutions of indoloquinolines and protoberberines are

similar and the reactions are likewise similar. However, the base-salt

equilibrium has different mechanisms for QPA and indoloquinolines. While

the QPA are characterized by the “transport” of a hydroxyl group,

indoloquinolines are typical alkaloids characterized by a standard “proton

transfer.” The behavior of indoloquinolines in media with different levels of

pH is shown in Figure 5/2. Although the pKa value is an important

characteristic of a potential antimalarial agent, the only pKa values reported

up to now have been those for cryptolepine (pKa = 11.2-11.8).[5,10] No data

for the pKa values of the other derivatives mentioned have been found in the

literature.


41

N

N

CH3

N

N

CH3

H

+H

-H

Figure 5/2

5.5. Pictures

P.vivax (a), P.ovale (b), P.malarie (c) and P.falciparum (d). (Figure 5/3)

a b c d

Figure 5/3

5.6. References

[1] Arzel E., Rocca P., Grellier P., Labaeïd M., Frappier F., Guéritte F.,

Gaspard C., Marsais F., Godard A., Quéguiner G., J. Med. Chem.

2001, 44, 949.

[2] Jonckers T.H.M., Van Miert S., Cimanga K., C.Bailly, P.Colson,

M.-C.De Pauw-Gillet, H.Van den Heuvel, M.Claeys, F.Lemière,

E.L.Esmans, J.Rozenski, L.Quirijnen, L.Maes, R.Dommisse,

G.L.F.Lemière, A.Vlietinck, L.Pieters, J. Med. Chem., 2002, 45,

3497.


42

[3] Van Miert S., Jonckers T., Cimanga K., Maes L., Maes B.,

G.Lemière, Dommisse R., Vlietinck A., Pieters L., Experimental

Parasitology, 2004, 108, 163.

[4] Dhanabal T., Sangeetha R., Mohan P.S., Tetrahedron 2006, 62,

6258.

[5] Onyeibor O., Drift S.L., Dodson H.I., Fez-Haddad M., Kendrick H.,

Millington N.J., Parapini S., Philips R.M., Seville S., Shnyder S.D.,

Taramelli D., Wright C.W., J.Med.Chem. 2005, 48, 2701.

[6] Van Miert S., Hostyn S., Maes B.U.W., Cimanga K., Brun R.,

Kaiser M., Mátyus P., Dommisse R., Lemière G., Vlietinck A.,

Pieters L., J. Nat. Prod., 2005, 68, 674.

[7] Warhurst D.C., Craig J.C., Adagu I.S., Guy R.K., Madrid P.B.,

Fivelman Q.L., Biochem. Pharm., 2007, 73, 1910.

[8] Cazzeles J., Robert., A., Meunier B., C. R. Acad. Sci. Paris, Chimie

2001, 4, 85.

[9] Cooper M.M., Lovell J.M., Joule J.A., Tetrahedron Let. 1996, 37,

4283.

[10] Dwuma-Badu D., Fitoterapia 1987, LVIII, 5.

43

Flavonoids: The known compounds obtained from Paulownia tomentosa

6.1. Flavonoids

The flavonoids are distributed in a major part of plant material. They have

not been found only in algae and a few varieties of plants. The flavonoids are

one of the most structurally diverse groups of compounds. For example,

about thirty flavonoid types have been identified in Asteraceae.[1] We

focused on the flavonoid content of the mature fruit of the Paulownia tree.

6.2. Paulownia tree

The perennial tree Paulownia tometosa (Thunb) Steud. (Figure 6/1), also

known as the Empress Tree, Princess Tree, or Foxglove Tree; pao tong 泡桐

in Chinese, is a member of the Scrophulariaceae family, native to central and

western China,.It is widely distributed in Korea, Japan, and China.[2,3] This

Flavonoids: The known compounds obtained from Paulownia tomentosa ____________________________________________________________________________

44

deciduous tree grows up to 10-25 meters tall, with large, heart-shaped to five-

lobed leaves 15-40 cm across.

Flowers appear before leaves in the early spring, on panicles 10-30 cm

long, with a tubular purple corolla 4-6 cm long, resembling a foxglove

flower. The fruit is a dry, egg-shaped capsule 3-4 cm long, containing

numerous tiny seeds.

Paulownia tomentosa can survive wildfire because the roots can

regenerate new, very fast-growing stems. It is tolerant of pollution and not

fussy about soil type. For these reasons it functions ecologically as a pioneer

plant. Its nitrogen-rich leaves provide good fodder, and its roots prevent soil

erosion.[4]

Parts of the plant P. tomentosa

(leaves, wood, and fruits) have been

used in traditional Chinese herbal

medicine for the treatment of tonsillitis,

bronchitis, asthmatic attack,

and bacterial infections such as enteritis

or dysentery. The flower is the most

important material used in folk

medicine herbs.[2,5]

Figure 6/1

6.3. Structure diversity of flavonoids

The basic flavonoid structure is derived from a diphenylpropane system

C6-C3-C6 (Figure 6/2).


45

OH

A

B

Figure 6/2

The flavonoids can be classified into nine general structures (Figure 6/3):

flavone (1), flavonole (2), flavanone (3), dihydroflavonole (4), flavan-3-ole

(5), flavan-3,4-diole (6), chalkone (a structure with one opening ring),

aurone, and anthocyaninidine (with a positive charge on oxygen O-1), except

these nine basic structures, the flavonoid exist in biflavonoid and the

glycosidic form.

Figure 6/3

The prefix “iso” is used for flavonoids characterized with a ring B bonded

at position C-3 the isoflavonoids.

O

O

O

OOH

O

O

O

OOH

1 2 3

4 5 6

O

OH

OH OH OH

OH OH

O

OHOH OH

HO HO HO

HO HO HO

1

2

345

6

78

A

B1`

2`

3`

4`

5`

6`


46

Flavonoids can be bonded together through their hydroxy groups at

position C-6 and C-8, the resulting dimeric structure is known as

a biflavonoid and has an oxygen bridge between these positions. The other

alternative is a bond between C-3` and C-8. The hydroxyl groups of the

flavonoid structure can be free or methylated. A substituent side chain at

position C-6 is typical for flavonoids.

The sugar part of glycosylflavonoids may be a chain of mono-, di-, or tri-

saccharides, such as D-glucose, D-galactose, or D-allose, some pentoses, D-

glucuronic acid or D-galacturonic acid. This sugar side chain is bonded to the

flavonoid structure through a hydroxyl group at position C-3 or C-5.

The C-glycosylflavonoids with a side chain at C-6 or C-8 and without an

oxygen bridge form a group of special case. Mono-C-glucosylflavones are

the most common C-glycosylflasvonoids.

6.4. Flavonoid content

Flavonoids with a diphenylpropane structure C6-C3-C6 are contained in

extracts of P. tomentosa. Three of these Apigenin (AP) (7), Mimulone (MI)

(8), and Diplacone (DI) (9), were described in 2006 (Figure 6/4).


47

O

O

OH

O

O

OH

OH

O

O

OH

HO

OH

HO

OH

HO

OH

7 8

9

Figure 6/4

Apigenin which can be obtained from the Paulownia species is probably

the most widely known and most frequently reported flavonoid. The benzene

ring at position C2 of the flavone structure (if it was at C3, the structure

would be an isoflavone) is characteristic of all three structures (7, 8, 9).

Another common feature is the presence of the three hydroxyl groups at C5,

C7, and C4`. The geranyl side chain of mimulone and diplacone is typical of

the diverse segments of the flavonoid structure.


Apigenin has been found to show a variety of pharmacological activities,

including anti-inflammatory,[6,7] antispasmotic,[8] antidiarrhoea,[9]

vasorelaxant,[10] and antiproliferative,[10 – 12] as well as an alleopathic activity,

anantibacterial effect on mutant streptococci.[13,14]


48

Recent data demonstrate that apigenin may exert its anti-tumorigenic

effect in vivo not only via the inhibition of tumor cell proliferation, but also

via the impairment of the invasive potential of tumor cells.[15] Apigenin may

also induce apoptosis,[16] significantly in early and late stages.

Both mimulone and diplacone have structures characterized by a geranyl

side chain at position C-8. The possibility of applying their antiradical

activity in pharmacology has been reported.[17]

6.6. Historical notes and pictures

1834 The Paulownia tree was imported to Europe from Japan by

Philipp Franz von Siebold and named in honour of the Anna

Pavlowna Romanov, daughter of Czar Paul I. Petrovic

Romanov, Duchess of Russia, Princess of The Netherland.[18]

Figure 6/5

The “princess” Paulownia tree is a truly beautiful representative of

world flora (Figure 6/5), probably because it is an

interesting subject for many professional

photographers. Some collections of visual studies of


49

the Paulownia tree can be found at these web

addresses:

http://www.bomengids.nl/soorten/Anna_Paulownaboo

m__Paulownia_tomentosa__Empress_tree.html

http://www.cas.vanderbilt.edu/bioimages/species/pato2

.htm

6.7. References

[1] Bruneton, J.: Pharmacognosy, Phytochemistry, Medicinal Plants;

Springer-Verlag: New York, 1995.

[2] Jiang T.-F., Du X., Shi Y.-P., Chromatographia, 2004, 59, 255.

[3] Kang K.H., Huh H., Kim B.-K., Lee C.-K., Phytother. Res., 1999.

13, 624.

[4] http://en.wikipedia.org/wiki/Paulownia_tomentosa

[5] Šmejkal K., Grycová L., Marek R., Lemiére F., Jankovská D.,

Forejtníková H., Vančo J., Suchý V., J. Nat. Prod., 2007, 70, 1244.

[6] Ko H.-H., Weng J.-R., Tsao L.-T., Yen M.-H., JWang.-P., Lin C.-

N., Bioorg. Med. Chem. Lett., 2004, 14, 1011.

[7] Gerritsen M.E., Carley W.W., Ranges G.E., Shen C.P., Phan S.A.,

Ligon G.F., Perry C.A., Am. J. Pathol., 1995, 147, 278.

[8] Capasso A., Pinto A., Sorrentino R., Capasso F. J.

Ethnopharmacol., 1991, 34, 279.

[9] Carlo G.D., Autore G., Izzo A.A., Maiolino P., Mascolo N., Viola

P., Diurno M.V., Capasso F. J Pharm. Pharmacol., 1993, 45, 1054.


50

[10] Zhang Y.-H., Park Y.-S., Kim T.-J., Fang L.-H., Ahn H.-Y., Hong

J.T., Kim Y., Lee C.-K., Yun Y.-P., General Pharmacology, 2000,

35, 341.

[11] Comalada M., Ballester I., Bailón E., Sierra S., Xaus J., Gálvez J.,

Sánchez de Medina F., Zarzuelo A., Biochemical pharmacology,

2006, 72, 1010.

[12] Kim D.-I., Lee T.-K., Lim I.-S., Kim H., Lee Y.-C., Kim C.-H.,

Toxicology and Applied Pharmacology, 2005, 205, 213.

[13] Koo H., Pearson S.K., Scott-Anne K., Abranches J., Cury J.A.,

Rosalen P.L., Park Y.K., Marquis R.E., Bowen W.H., Oral

Microbiology Immunology, 2003, 17, 337.

[14] Basile A., Sorbo S., Giordano S., Ricciardi L., Ferrara S.,

Montesano D., Conbianchi R.C., Vuotto M.L., Ferrara L..

Fitoterapia, 2000, 71, 110.

[15] Czyz J., Madeja Z., Irmer U., Korohoda W., Hülser D.F., Int. J.

Cancer., 2004, 114, 12.

[16] Vargo M.A., Voss O.H., Poustka F., Cardounel A.J., Grotewold E.,

Doseff A.I., Biochemical pharmacology, 2006, 72, 681.

[17] Šmejkal K., Holubová P., Zima A., Muselik J., Dvorska M.,

Molecules, 2007, 12, 1210.

[18] http://www.neerlandstuin.nl/bomen/paulownia.html - history

51

Experimental part

7.1. Protoberberine alkaloids

Commercial reagents

Berberine chloride dihydrate (1, X = Cl), (Mrel = 407.86; m.p. 204-

206 °C), palmatine chloride hydrate (2, X = Cl) (Mrel = 387.86; m.p. 206-207

°C), pyrrole (Mrel = 67.09; b.p. 129-131 °C), pyrazole (Mrel = 68.08; m.p. 66

°C), methylpyrazole (Mrel = 82.10; b.p. 204 °C), imidazole (Mrel = 68.08;

m.p. 88-91 °C), and 1,2,4- triazole (Mrel 69.07, m.p. 116-120 °C) were

obtained in high purity from Sigma-Aldrich.

Berberine derivatives (4a, 4b, 4c, 4d)

The preparation of samples: 8-(Pyrrol-1-yl)-7,8-dihydroberberine (4a), 8-

(Pyrazol-1-yl)-7,8-dihydroberberine (4b), 8-(Imidazol-1-yl)-7,8-

dihydroberberine (4c), and 8-(1,2,4-Triazol-1-yl)-7,8-dihydroberberine (4d)

has been presented in our article in Magnetic Resonance in Chemistry

(Appendix B).

Experimental part ____________________________________________________________________________

52

8-(methyl-pyrazol-1-yl)-7,8-dihydroberberine (4e)

This, like 4a, was prepared using sodium hydride (36 mg), methylpyrazole

(59.9 mg), and berberine chloride (266.2 mg). The mixture was stirred at

laboratory temperature (303 K) for 5 h. A brown oil was isolated in 5% yield, 1H NMR: 7.19 (H-1) 6.60 (H-4) 2.69 (H-5a) 2.90 (H-5b) 3.31 (H-6a) 3.62

(H-6b) 7.02 (H-8, H-11, H-12) 6.17 (H-13) 5.96 (2,3 CH2) 5.89 (H-4´) 6.95

(H-5´) 2.24 (Me). 13C NMR: 104.80 (C-1), 147.4 (C-2), X (C-148.19), 108.31

(C-4), 129.97 (C-4a), 30.46 (C-5), 47.54 (C-6), 72.20 (C-8), 145.86 (C-9),

150.69 (C-10), 114.82 (C-11), 120.10 (C-12), 128.32 (C-12a), 95.73 (C-13),

137.49 (C-13a), 125.59 (C-13b), 101.86 (2,3-OCH2O), 60.81 (9-OCH3),

56.61 (10-OCH3), 147.74 (C-3`), 105.89 (C-4`), 129.27 (C-5`), 13.81 (Me). 15N NMR: 82.4 (N-7), 226.9 (N-1`), 303.1 (N-2`).

Palmatine derivatives (5a, 5b, 5c, 5d)

Preparation of these samples: 8-(Pyrrol-1-yl)-7,8-dihydropalmatine (5a),

8-(Pyrazol-1-yl)-7,8-dihydropalmatine (5b), 8-(Imidazol-1-yl)-7,8-

dihydropalmatine (5c), and 8-(1,2,4-Triazol-1-yl)-7,8-dihydropalmatine (5d)

has been described in our article in Magnetic Resonance in Chemistry

(Appendix B).

NMR analysis

NMR spectra were recorded using a Bruker Avance 300 spectrometer

operating at frequencies of 300.13 MHz (1H), 75.48 MHz (13C), and 30.42

MHz (15N) and a Bruker Avance 500 operating at frequencies of 500.13 MHz

(1H), 125.77 MHz (13C), and 50.67 MHz (15N). The temperature of the

measurements was 303 K. NMR samples were prepared by dissolving the

compounds in benzene-d6, dichloromethane-d2, or dimethylsulfoxide-d6. The


53

1H and 13C NMR chemical shifts were referenced to the signals of the

solvents (C6D6 [7.16 (1H) and 128.00 (13C)]; CD2Cl2 [5.31 (1H) and 53.80

(13C)]; DMSO [2.50 (1H) and 39.51 (13C)]). The 15N NMR chemical shifts

were referenced to liquid CH3NO2 (381.7 ppm) and are reported relative to

liquid NH3. The 1H-13C coupling constants were determined from the coupled

gs-HMQC, gs-HSQC (1JH,C = 160 Hz), and GSQMBC (nJH,C = 7.5 Hz)

spectra with an accuracy of ± 0.3 Hz. The 1H-15N GSQMBC and gs-HMBC

experiments were adjusted for the couplings of 3.2 or 5 Hz.

X-ray analysis

Diffraction data for some of the products were collected on a KM4CCD

four-circle area detector diffractometer by Docent Dr. Marek Nečas. The

details of the measurements can be found in Appendix B.

7.2. Cryptolepine alkaloids

NaOH, HCl, DMSO-d6, MeOH, and buffers (pH 4 and 7) were obtained

commercially. All samples were prepared in the laboratory of the Department

of Pharmaceutical Sciences, University of Antwerp. Three cryptolepine

alkaloids – cryptolepine·HCl, isocryptolepine·HCl. and neocryptolepine·HCl

were isloated from plant material (Cryptolepis), isoneocryptolepine·HI and

derivatives of neocryptolepine: 2-bromoneocryptolepine, 3-

bromoneocryptolepine·HCl, 3-chloroneocryptolepine·HCl, and 2-

methoxyneocryptolepine were prepared synthetically.


54

Determination of pKa

All alkaloid samples and reagents (NaOH, HCl) were prepared in the

solvent mixture (H2O:MeOH:DMSO-d6 - 4:4:2 by volume). The samples

were prepared by dissolving 1-3 mg of the cryptolepine in 2 ml of solvent

mixture. The concentrations of the NaOH solutions were 0.01 M, 0.025 M

and 0.04 M. The concentration of the HCl solution was 0.005 M.

The pH values of the solutions were measured at all points using a glass

electrode and a contact-drop electrode at a temperature 298 K. The same

temperature was used for the NMR experiments.

NMR analysis

NMR spectra were recorded using an Oxford 400 spectrometer operating

at a frequency of 400.00 MHz (1H) and a Bruker Avance 500 operating at

a frequency of 500.13 MHz (1H). The temperature of the measurements was

298 K. NMR samples were prepared by dissolving the compounds in the

mixture of H2O, MeOH, and DMSO-d6 as described previously. The 1H NMR chemical shifts were referenced to the signals of TMS (0 ppm).

7.3. Flavonoids

All samples were obtained from the Department of Natural Drugs after

separation and mass-spectrometric analysis. The structures of the six

flavonoid compounds isolated from the fruit of Paulownia tometosa were

determined.


55

NMR analysis

NMR spectra were recorded using a Bruker Avance DRX 500. All NMR

spectra were measured in DMSO-d6 at 303 K. The 1H and 13C NMR chemical

shifts (δ in ppm) were referenced to the resonance of the solvent [2.50 (1H)

for DMSO-d6 and 39.50 (13C) for DMSO-d6]. The 2D NMR experiments,

gradient-selected COSY, gs-HSQC, and gs-HMBC were used to assign the

individual 1H and 13C resonances.[1,2] The gradient ratio for the 1H-13C

HMBC experiment was 30:18:24 G cm-1, and the experiment was adjusted

for long-range coupling of 7.5 Hz.

7.4. References

[1] W. Willker, D. Leibfritz, R. Kerssebaum, W. Bermel, Magn. Reson.

Chem. 1993, 31, 287.

[2] Davis, A. L.; Laue, E. D.; Keeler, J.; Moskau, D.; Lohman, J.: J. Magn.

Reson. 1991, 94, 637-644.

56

Protoberberines: nucleophilic addition

8.1. Biological activity and interactions with biomacromolecules

Knowledge of the interaction mechanisms of alkaloids (as potential agents

for use in treatment) with biological materials is important for predicting

their biological and pharmacological activities. For example, a recent study

excluded the intercalation of berberine into the double helix of DNA[1] –

mentioned in section 4.1.

8.2. Useful or dangerous?

What is interesting about the interaction of these compounds with

biological targets? Their potential for pharmaceutical applications seems to

be very broad.[2]. But is their application safe? The alkaloids of a related

group – the benzo[c]phenanthridines - were used in stomatology. Many

studies during the last ten years have scrutinized just the safety of their

application in the oral hygiene of human beings. And the result? The alkaloid

Protoberberines: nucleophilic addition ____________________________________________________________________________

57

component has been removed from the composition of commercial products.

It was not safe.

The interactions of protoberberines with biological materials have not

been studied from the point of view of the stability of the eventual products.

Why is it important to know, whether or not they can form stable covalently

bonded adducts with biological molecules? A stable covalent bond to

a nucleic acid = a mutation of the genetic information = a carcinogenic or

teratogenic effect. The statistical probability of creating such adducts in

aqueous media is low, but just one badly behaving cell can kill you.

8.3. NMR analysis and structural information

The formation of products of QPA covalently bonded with nitrogen

heterocycles has been characterized by NMR spectroscopy. Due to the

differences in chemical shift between the quaternary form and the

dihydroberberine skeleton, the NMR data confirmed the formation of

a covalent bond between the reactants. The most significant changes in

chemical shift were observed for H-8 and H-13. The value of the 1H NMR

chemical shift of H-8 in the quaternary form is in the range 9-10 ppm. In

adducts, the value for this same atom ranges between 6 and 7 ppm. The same

tendency has been observed for the 13C NMR chemical shifts of the C-8 and

C-13 atoms.

A few adducts have been characterized by X-ray diffraction. See

Appendix B

Protoberberines: nucleophilic addition ____________________________________________________________________________

58

8.4. Results

All results of this study have been published in scientific journals.

A review article about QPA has appeared in Phytochemistry (Appendix A)

and the results of an experimental study have been published in Magnetic

Resonance in Chemistry (Appendix B).

8.5. References

[1] Mazzini S., Belluci M.C., Mondelli R.: Bioorg. Med. Chem., 2003,

11, 505.

[2] Grycová L., Dostál J., Marek R., Phytochemistry. 2007, 68, 150.

Appendix A

Review

Quaternary protoberberine alkaloids

Lenka Grycova a,b, Jirı Dostal c, Radek Marek a,b,*

a National Center for Biomolecular Research, Faculty of Science, Masaryk University, Kamenice 5/A4, CZ-625 00 Brno, Czech Republicb Department of Organic Chemistry, Faculty of Science, Masaryk University, Kotlarska 2, CZ-611 37 Brno, Czech Republic

c Department of Biochemistry, Faculty of Medicine, Masaryk University, Komenskeho nam. 2, CZ-662 43 Brno, Czech Republic

Received 30 June 2006; received in revised form 18 September 2006Available online 15 November 2006

Dedicated to Professor Jirı Slavık on the occasion of his 85th birthday.

Abstract

This contribution reviews some general aspects of the quaternary iminium protoberberine alkaloids. The alkaloids represent a veryextensive group of secondary metabolites with diverse structures, distribution in nature, and biological effects. The quaternary proto-berberine alkaloids (QPA), derived from the 5,6-dihydrodibenzo[a,g]quinolizinium system, belong to a large class of isoquinolinealkaloids.

Following a general introduction, the plant sources of QPA, their biosynthesis, and procedures for their isolation are discussed. Ana-lytical methods and spectral data are summarized with emphasis on NMR spectroscopy. The reactivity of QPA is characterized by thesensitivity of the iminium bond C@N+ to nucleophilic attack. The addition of various nucleophiles to the protoberberine skeleton is dis-cussed. An extended discussion of the principal chemical reactivity is included since this governs interactions with biological targets.

Quaternary protoberberine alkaloids and some related compounds exhibit considerable biological activities. Recently reported struc-tural studies indicate that the QPA interact with nucleic acids predominantly as intercalators or minor groove binders. Currently, inves-tigations in many laboratories worldwide are focused on the antibacterial and antimalarial activity, cytotoxicity, and potentialgenotoxicity of QPA.� 2006 Elsevier Ltd. All rights reserved.

Keywords: Berberidaceae; Quaternary protoberberine alkaloid; Plant source; Nucleophilic addition; NMR spectroscopy; X-ray diffraction; Cytotoxicity;DNA binding

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1511.1. General aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1511.2. Protoberberine skeleton . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152

2. Plant sources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1533. Biosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1534. Isolation and separation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1555. Identification and analytical methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 156

5.1. Mass spectrometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1575.2. UV–VIS spectroscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1575.3. NMR spectroscopy. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158

0031-9422/$ - see front matter � 2006 Elsevier Ltd. All rights reserved.

doi:10.1016/j.phytochem.2006.10.004

* Corresponding author. Tel.: +420 549 495 748; fax: +420 549 492 556.E-mail address: [email protected] (R. Marek).

www.elsevier.com/locate/phytochem

Phytochemistry 68 (2007) 150–175

PHYTOCHEMISTRY

mailto:[email protected]

5.4. X-ray diffraction analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1596. Properties and reactivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161

6.1. Nucleophilic addition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1616.1.1. O-Nucleophiles. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1616.1.2. N-Nucleophiles and S-nucleophiles. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1616.1.3. C-Nucleophiles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163

6.2. Disproportionation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1646.3. Oxidation and reduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1656.4. Alkylation at position C-13 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1676.5. Photochemical reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1676.6. Transformation of 2,3,9,10-substituted QPA to 2,3,10,11-substituted QPA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167

7. Biological activities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1687.1. Interactions with biomacromolecules – structural studies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1687.2. Cytotoxic activity and apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1697.3. Antimicrobial activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1697.4. Anti-inflammatory activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1707.5. Antimalarial activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1707.6. Other effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170

8. Conclusions and perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170

1. Introduction

1.1. General aspects

The alkaloids represent a very extensive group of second-ary metabolites, with diverse structures, distribution in nat-ure, and important biological activities. A simple generaldefinition (Roberts and Wink, 1998) of an alkaloid was firstsuggested by Pelletier in 1983: ‘‘An alkaloid is a cyclic com-pound containing nitrogen in a negative oxidation statewhich is of limited distribution in living organisms’’. Thisdefinition includes both alkaloids with nitrogen as part ofa heterocyclic system and many exceptions with exocyclicnitrogen, such as colchicines or capsaicin.

Generally, the function of secondary metabolites inplants is not unequivocally determined. Concerning thealkaloids, their protective mechanism against herbivoreanimals and possibly some parasites is assumed. In con-trast, these plants can become the targets for insects that

specialize in the alkaloid-producing plants (e.g., bark tree).However, strong physiological effects and the selectivity ofsome alkaloids present opportunities for utilizing the alka-loids in human medicine.

The isoquinolines are one of the largest groups of alka-loids. The isoquinoline skeleton is a basic building block ofvarious types of alkaloids including benzylisoquinolines,protopines, benzo[c]phenanthridines, protoberberines,and many others. The protoberberine alkaloids are bioge-netically derived from tyrosine. The quaternary protober-berine alkaloids (QPA) represent approximately 25% ofall the currently known alkaloids with a protoberberineskeleton isolated from natural sources. This article isfocused mainly on the iminium QPA because of their inter-esting chemical reactivity and biological activities.

Berberine is probably the most widely distributed alka-loid of all. In 1824, Huttenschmid found a yellow coloringmatter in what he believed to be Geoffroya inermis, theJamaica cabbage-tree, and gave it the name jamaicine. In

Table 12,3,9,10-Tetrasubstituted QPAs

Alkaloid 2 3 9 10

Berberine O–CH2–O OCH3 OCH3

Coptisine O–CH2–O O–CH2–OPalmatine OCH3 OCH3 OCH3 OCH3

Jatrorrhizine OCH3 OH OCH3 OCH3

Columbamine OH OCH3 OCH3 OCH3

Thalifendine O–CH2–O OCH3 OHStepharanine OH OCH3 OCH3 OHGroendlandicine OCH3 OH O–CH2–ODehydrocorydalmine OCH3 OCH3 OCH3 OHDehydrodiscretamine OCH3 OH OCH3 OHDehydrocheilanthifoline OH OCH3 O–CH2–ODemethylenberberine OH OH OCH3 OCH3

Epiberberine (Dehydrosinactine)a OCH3 OCH3 O–CH2–O

a Synonym is given in the parentheses.

L. Grycova et al. / Phytochemistry 68 (2007) 150–175 151

1826, Chevallier and Pelletan found a rich yellow alkaloidin the bark of Xanthoxylum clava herculis, and named itxanthopicrite. Both of these substances were subsequentlyproved to be identical with berberine, obtained by Buchnerand Herberger as a yellow extract from Berberis vulgaris in1830 (Felter and Lloyd, 1898).

Other representatives of the QPA, including the well-known alkaloids palmatine, jatrorrhizine, columbamine,and coptisine, are summarized in Tables 1–4.

1.2. Protoberberine skeleton

Most protoberberine alkaloids exist in plants either astetrahydroprotoberberines or as quaternary protoberberinesalts, although a few examples of dihydroprotoberberineshave also been described. 5,6-Dihydrodibenzo[a,g]quino-lizinium (C17H14N+) is the basic skeleton of the quaternaryprotoberberine alkaloids (Scheme 1).

Substituents are usually present at positions 2,3,9,10 or2,3,10,11 and the prefix pseudo- is often used for the lattersubstitution pattern. For example, pseudopalmatine,2,3,10,11-tetramethoxy-5,6-dihydrodibenzo[a,g]quinolizi-nium, is a regioisomer of palmatine, 2,3,9,10-tetramethoxy-5,6-dihydrodibenzo[a,g]quinolizinium. 2,3,9,10-Tetrasub-stituted and 2,3,10,11-tetrasubstituted QPA are summa-rized in Tables 1 and 2, respectively.

Additionally, compounds with substituents at carbonatoms C-1, C-4, C-5 and C-13 have been isolated fromnatural sources. The typical substituents are hydroxygroups, methoxy groups, and O–CH2–O groups. The prefixretro- is used for protoberberines characterized by the

Table 31,2,x,y-Tetrasubstituted, pentasubstituted, and hexasubstituted QPAs

Alkaloid 1 2 3 4 5 8 9 10 11 12 13

Corysamine (Worenine)a O–CH2–O O–CH2–O CH3

Dehydrothalictrifoline OCH3 OCH3 O–CH2–O CH3

Dehydrothalictricavine O–CH2–O OCH3 OCH3 CH3

Dehydrocorydaline OCH3 OCH3 OCH3 OCH3 OCH3

Dehydroapocavidine OH OCH3 O–CH2–O CH3

Berberastine O–CH2–O OH OCH3 OCH3

Thalidastine O–CH2–O OH OCH3 OHFissisaine OCH3 OCH3 OH OCH3 OHLincagenine OCH3 OCH3 OH OCH3 OCH3

Stephabine OH OCH3 OCH3 OCH3 OCH3

Dehydrocapaurimine OH OCH3 OCH3 OCH3 OH13-Me-Pseudoepiberberine OCH3 OCH3 O–CH2–O CH3

PO-4 (Dehydroorientalidine)a OCH3 O–CH2–O OCH3 O–CH2–O–CH2

PO-5 (Alborine) OCH3 O–CH2–O OCH3 OCH3 CH2OHMequinine OCH3 OCH3 OH OCH3

Caseadine OH OCH3 OCH3 OCH3


Table 4Synthetic protoberberines and isolation artifacts

Alkaloid 2 3 4 8 9 10 11 13 Ref.

Berberrubine(Chileninone)a

O–CH2–O OH OCH3 Shamma and Rahimizadech (1986)

Palmatrubine OCH3 OCH3 OH OCH3 Shamma et al. (1977) and Siwon et al. (1981)Dehydroscoulerine OH OCH3 OH OCH3 Schneider and Zenk (1993b) and Bjorklund

et al. (1995)MDD-coralyne O–CH2–O CH3 OCH3 OCH3 Makhey et al. (2000)DM/II/33 O–CH2–O OCH3 OCH3 Goldman et al. (1997)5,6-Dihydrocoralyne OCH3 OCH3 OCH3 OCH3 OCH3 Wang et al. (1996)Glabrine OCH3 OCH3 OCH3 OH OH Bhakuni and Jain (1986)Glabrinine OCH3 OCH3 OCH3 OCH3 OCH3 Bhakuni and Jain (1986)Dehydrocorybulbine OCH3 OH OCH3 OCH3 CH3 Santavy (1979)13-Aminopalmatine OCH3 OCH3 OCH3 OCH3 NH2 McCalla et al. (1994)


Table 22,3,10,11-Tetrasubstituted QPAs

Alkaloid 2 3 10 11

Pseudocoptisine O–CH2–O O–CH2–OPseudopalmatine OCH3 OCH3 OCH3 OCH3

Pseudojatrorrhizine OCH3 OH OCH3 OCH3

Pseudoepiberberine OCH3 OCH3 O–CH2–OPseudocolumbamine OH OCH3 OCH3 OCH3

Dehydrodiscretine OCH3 OH OCH3 OCH3

Dehydrocoreximine OH OCH3 OCH3 OHThalifaurine OCH3 OH O–CH2–O

152 L. Grycova et al. / Phytochemistry 68 (2007) 150–175

presence of an extra substituent as a side chain of ring D –position C-12 (PO-4, PO-5, mecambridine, and orientali-dine fall among the retroprotoberberines). In the past,the prefix epi- was used for interchanged 2,3 and 9,10 sub-stitution patterns (see, for example: berberine vs. epiberber-ine in Table 1). Various 1,2,x,y-tetrasubstituted,pentasubstituted, and hexasubstituted QPA are summa-rized in Table 3.

Two compounds were recently published as new quater-nary protoberberine alkaloids. However, their structuresare identical with alkaloids that have been known for along time. Burasaine (Kluza et al., 2003) is identical withpalmatine and pangrenine (Siddikov et al., 2005) is identi-cal with alborine (PO-5). Bisjatrorrhizine, formed by anortho-oxidative coupling of the phenolic group of jatror-rhizine (Fajardo et al., 1996), should also be included inthis review. Table 4 contains a small part of the quaternaryprotoberberine alkaloids which are not natural, but theywere synthesized from their natural precursors or formedartificially during the isolation processes. Although palma-trubine has been described as a natural product (Shammaet al., 1977; Siwon et al., 1981), it is assumed to originatefrom the rearrangement of palmatine during the isolationprocess (cf. berberrubine (Shamma and Rahimizadech,1986)).

In addition to the semi-synthetic protoberberine alka-loids summarized in Table 4, coralyne, 8-methoxy-5,6-dehydropseudopalmatine with a fully aromatic skeletonhas been described (Wang et al., 1996; Goldman et al.,1997; Makhey et al., 2000). The other aromatic compoundsare deoxythalidastine and dehydroberberrubine (Shammaand Dudock, 1965). Deoxythalidastine (dehydrothalifen-dine) is probably an artifact formed by dehydration of

the known 5-hydroxyprotoberberine alkaloid thalidastine(Ikuta and Itokawa, 1982). The structures of compoundswith a completely aromatic core are shown in Scheme 2.

2. Plant sources

The protoberberines are distributed in such plant fami-lies as Papaveraceae, Berberidaceae, Fumariaceae, Meni-spermaceae, Ranunculaceae, Rutaceae, Annonaceae, aswell as a few examples in Magnoliaceae and Convolvula-ceae (Bentley, 1997, 1998a,b, 1999, 2000, 2001, 2002,2003, 2004, 2005, 2006). The occurrence of a few represen-tatives of the QPA is restricted to only the one plant spe-cies. The distributions of QPA in plant families andindividual species are summarized in Table 5.

The protoberberine alkaloids are rather widely distrib-uted in at least six plant families. There are more thanone hundred of these compounds (tetrahydroprotoberbe-rines, quaternary protoberberines) (Bentley, 1997,1998a,b, 1999, 2000, 2001, 2002, 2003, 2004, 2005, 2006).However, the number of known quaternary iminium alka-loids is significantly smaller (see Tables 1–3). In plant tis-sues, the positive charge of the QPA is compensated forby chloride anions or the anions of organic acids, such assuccinic acid (Pervushkin et al., 1999). Generally, anionsare exchanged during the isolation process and the QPAare frequently obtained in the form of chlorides, iodides,or perchlorates.

Quantitative and qualitative differences in the alkaloidcontent of the plant tissues are affected by several factors,e.g., climate, environment, and soil composition. The con-tent of the individual alkaloids is also somewhat influencedby the vegetative season (Tome and Colombo, 1995). Theoccurrence of several alkaloids is restricted to a specificpart of the plant body, whereas other compounds are dis-tributed in the whole body with varying rates in differenttissues.

3. Biosynthesis

It has been shown (investigation of Hydrastis canaden-

sis) (Gear and Spenser, 1963) that tyrosine is a very efficientprecursor of berberine and it is incorporated into both the

N2

3

9

10

13

1

4 56

8

11

12

A B

C

D

Scheme 1.

N

MeO

MeO

OMeOMe

OMe N

OH

OMe

O

O N

OMe

OH

O

O

Coralyne Deoxythalidastine (dehydrothalifendine) Dehydroberberrubine

Scheme 2.


top (ring A and part of ring B) and the bottom (ring D andpart of ring C) parts of the alkaloid (Gear and Spenser,1963). However, if labeled dopamine is fed in, only onemolecule of this species is incorporated into the alkaloid.Tyrosine must, therefore, give rise to two different interme-diates during the biosynthetic process, one of them beingdopamine. Although not all of the individual steps haveyet been completely established, the general sequenceshown in Scheme 3 seems to prevail in the biosynthesis ofberberine (Shamma, 1972; Frick and Kutchan, 1999; Hara

et al., 1994). Several enzymes (Beecher and Kelleher, 1984;Kobayashi et al., 1987; Amann et al., 1988; Jendrzejewski,1990; Schneider and Zenk, 1993a) participate in the trans-formation pathway from reticuline to berberine (Kametaniet al., 1976; Jeffs and Scharver, 1976; Bhakuni et al., 1983),one of them is berberine bridge enzyme (Frenzel et al.,1988; Rueffer and Zenk, 1994; Kutchan and Ditrich,1995). Tetrahydroprotoberberine oxidase is employed inthe conversion of tetrahydroprotoberberine to the quater-nary form (Amann et al., 1984, 1988). Subsequently, the

Table 5Selected plant sources of QPAs

Alkaloid Typical plant species/family Refs.

Berberine Berberidaceae and others 1, 2, 3, 4, 33, 34, 39, 43, 44, 52, 53Coptisine Fumariaceae, Papaveraceae 2, 4, 48, 49, 52Palmatine Berberidaceae and others 2, 3, 4, 5, 37, 38, 39, 41, 42, 51, 54Jatrorrhizine Berberidaceae and others 1, 2, 3, 5, 33, 37, 39, 41, 50, 51, 54Columbamine Berberis vulgaris, Enantia chlorantha 3, 6, 7, 37, 38, 39, 45, 51Thalifendine Thalictrum sp. 1, 3, 6, 27, 30, 33, 37Stepharanine Xylopia parviflora, Stephania glabra 5, 8, 9Groendlandicine Coptis groendlandicum 10Dehydrocorydalmine Stephania glabra 9, 11Palmatrubine Thalictrum polygamum 12, 13Dehydrodiscretamine Fissistigma balansae, Thalictrum foliolosum 3, 7Dehydrocheilanthifoline Berberis cordata 10Demethylenberberine Thalictrum javanicum 1, 14, 32, 33Epiberberine (Dehydrosinactine)a Coptis chinensis, D. floribunda (aristata)a 15, 35, 36Pseudocoptisine Isopyrum thalictroides 2, 16Pseudopalmatine Stephania suberosa, Penianthus zenkeri 2, 21, 26, 34Pseudojatrorrhizine Fibraurea chloroleuca 13Pseudoepiberberine –b 2, 6Pseudocolumbamine Enanthia chlorantha, Fibraurea chloroleuca 13, 17Dehydrodiscretine Heptacylum zenkeri, Thalictrum fauriei 5, 27, 28Dehydrocoreximine Xylopia pauriflora 5Thalifaurine Thalictrum fauriei 1Caseadine Ceratocapnos heterocarpa 29Corysamine (Worenine)a Chelidonium majus 1, 2Dehydrothalictrifoline –b 6Dehydrothalictricavine –b 6Dehydrocorydaline Corydalis bulbosa 18Dehydroapocavidine Corydalis cava 1Berberastine Hydrastis canadensis 6, 19, 40, 46Thalidastine Thalictrum sp. 3, 6, 30, 31Fissisaine Fissistigma balansae 7Lincagenine Guatteria schomburgiana, G. sessilis 20Stephabine Stephania suberosa 21Dehydrocapaurimine Coptis platicarpa 1013-Methyl pseudoepiberberine –b 6PO-4 Papaver orientale 10, 22, 23, 24, 47Alborine (PO-5)a Papaver orientale 1, 10, 22, 23, 24, 47Mequinine Meconopsis quintuplinerva 25

1. Bhakuni and Jain (1986), 2. Pavelka and Smekal (1976), 3. Chattopadhyay et al. (1983), 4. Ro et al. (2001), 5. Nishiysma et al. (2004), 6. Shamma et al.(1969), 7. Chia et al. (1998), 8. Cava et al. (1968), 9. Doskotch et al. (1967), 10. Santavy (1979), 11. Kaneko and Naruto (1969), 12. Shamma et al. (1977),13. Siwon et al. (1981), 14. Bahadur and Shukla (1983), 15. Yang et al. (1998), 16. Moulis et al. (1977), 17. Bourdat-Deschamps et al. (2004), 18. Miyazawaet al. (1998), 19. Jeffs (1967), 20. Orfila et al. (2000), 21. Patra et al. (1987), 22. Shamma (1972), 23. Preininger et al. (1970), 24. Simanek et al. (1970), 25.Bentley (1997), 26. Tane et al. (1997), 27. Wright et al. (2000), 28. Duah et al. (1983), 29. Suau et al. (1993), 30. Ikuta and Itokawa (1982), 31. Gao et al.(1987), 32. Cava and Reed (1967), 33. Pan et al. (2002), 34. Suau et al. (1991), 35. Bauer and Zenk (1991), 36. Drost et al. (1974), 37. Lopez et al. (1988), 38.Bonora et al. (1990), 39. Hsieh et al. (2004), 40. Weber et al. (2003), 41. Suau et al. (1998), 42. Nishiysma et al. (2004), 43. Kost’alova et al. (1982), 44.Valencia et al. (1984), 45. Svendsen et al. (1984), 46. Iwasa et al. (1982), 47. Preininger (1986), 48. Henry (1949), 49. Murugesan and Shamma (1980), 50.Lloyd (1898), 51. Rueffer and Zenk (1986), 52. Barreto et al. (2003), 53. Kumar et al. (2003), 54. Slavık and Slavıkova (1995).

a Synonym is given in the parentheses.b Source not described in the reference.


berberine can be converted into other protoberberines, e.g.,jatrorrhizine (Rueffer et al., 1983).

4. Isolation and separation

The whole plant or just a selected part such as the barkor the stem is used for isolating the principal constituentsor the minor components. Typically (Taborska et al.,1994), the part of the plant with the highest alkaloid con-tent is dried at room temperature. The extraction of alka-loids from the pulverized or finely cut dried plantmaterial is based on maceration and percolation, or is per-formed using a Soxhlet apparatus. The dried plant tissue isextracted for a long time (days) using methanol, with theend of the extraction indicated (Kost’alova et al., 1982)by Dragendorff’s reagent. Traditional methods such ascrystallization, column chromatography, and extractionare frequently used to obtain the individual alkaloids fromthe plant extract. Afterwards highly sensitive and sophisti-cated methods for separating (various chromatographicmethods including HPLC) and identifying (spectroscopicmethods) the constituents are used (see below).

Separation of the QPA from the crude plant extract con-taining various kinds of alkaloids can be performed usingseveral methods. A general isolation procedure wasreported by Slavıkova and Slavık (1966). The individualsteps of the published sequence are shown in Scheme 4.

The isolation procedure is based on the interconversionreaction between the protoberberine salt (soluble in water,stable in acidic and neutral media) and the base (soluble inorganic solvents). During the isolation and purificationprocess the protoberberine salts are converted into theircorresponding bases (Marek et al., 2003b) and thenextracted into an organic solvent. This principal reactivityis discussed in further detail in Chapter 6.

The individual components were subsequently separatedfrom fraction B by using chromatographic methods andrecrystalization. Column chromatography of palmatinechloride, berberine chloride, columbamine chloride, andjatrorrhizine chloride on acid-washed alumina was usedto separate the QPA from the crude extract.

Chloroform with increasing content of methanol wasused as the mobile phase (Cava and Reed, 1965). The paperchromatography RF values of the alkaloid chlorides for dif-ferent solvent systems (Cava and Reed, 1965) are summa-rized in Table 6.

In addition to the standard chromatography, severalmodern chromatographic techniques have been used forseparating and identifying the QPA. TLC analysis ofQPA can present some problems because these compoundsare highly polar and standard eluents are not suitable fortheir separation. Overpressure layer chromatography(OPLC) is one of the methods which resolve these compli-cations (Pothier et al., 1993). OPLC on silica gel plates withternary eluents was applied to the complete separation of

HO

COOH

NH2 HO

COOH

NH2

HO

HONH2 HO

COOH

O

HO

+

HO

NH

HO

HO

OH

OH

N

HO

HO

OH

OH

N

MeO

HO

OH

OMe

Me Me

Norlaudanosoline (+)-Laudanosoline (+)-Reticuline

N

MeO

HO

OH

OMe

N

OMe

OMe

N

O

O

OMe

OMe

Berberine

O

O

Tyrosine Dopa

Dopamine 3,4-Dihydroxyphenylpyruvic acid

Imonium ion (-)-Tetrahydroberberine

A

B

D

A

D

Scheme 3.


quaternary salts. This method can be used to separate thealkaloids from plant extracts and to determine the hRF val-ues of some authentic standards. Reversed-phase HPLCanalysis (Taborska et al., 1994) of the extract from Chelid-

onium majus was used to separate 27 alkaloids from a mix-ture of 28 compounds. However, with HPLC many peakscannot be assigned to the individual species (the sameproblem occurs with TLC). This complication can beresolved by using ion-pair HPLC and utilizing sodium per-chlorate for the extraction (Kato et al., 1996).

One of the problems connected with the isolation proce-dures for the QPA on chromatographic columns is a possi-ble structural rearrangement of the natural QPA leading to

unnatural rearranged compounds and isolation artifacts(see Chapter 6.1). Some isolation artifacts (Marek et al.,2003b) were classified as natural QPA for a long time.For example, berberrubine was described as a naturalproduct (Shamma et al., 1977; Peteu, 1965), but since1986 it has been known to be an isolation artifact (Shammaand Rahimizadech, 1986).

5. Identification and analytical methods

Currently, standard spectroscopic methods for investi-gating the structure of natural products comprise nuclear

plant material

crude extract

aqueous phase pH ~ 8-10

aqueous phase pH > 13

aqueous phase pH ~ 8-10

aqueous phase pH ~ 5.5-7

Acid + KI

NaOH

1% H2SO4

methanol

Na2CO3

Acid

ether

ether

ether citric acid

chloroform

chloroform

fraction L

fraction A

fraction B

fraction E

fraction I

Fraction L lipophilic compounds and non-basic alkaloids

Fraction A quaternary benzophenantridine alkaloids, protopine alkaloids, tertiary bases soluble in ether

Fraction B citrates of quaternary protoberberine alkaloids

Fraction E the rest of fraction A, non-polar compounds

Fraction I highly polar quaternary alkaloids (as iodides)

Scheme 4.


magnetic resonance (NMR), infrared spectroscopy (IR),and ultraviolet spectroscopy (UV), and these are oftencombined with mass spectrometry (MS). Single-crystal X-ray diffraction is a powerful technique used for determiningthe molecular topology. However, this technique suffersfrom the necessity of obtaining high-quality single crystals.Applications of the above mentioned methods to the struc-tural analysis and characterization of the QPA are dis-cussed in the following subchapters.

The quaternary protoberberine alkaloids are character-ized by bright colors. Their colors range from yellow (ber-berine) to orange (jatrorrhizine). The color of thecrystalline salts may vary depending on the method of crys-tallization or purification or both. The colors of their solu-tions may also vary slightly, depending on theconcentration, solvent, and pH. The melting points of theQPA salts usually range from 200 to 300 �C.

5.1. Mass spectrometry

Mass spectrometry has been a powerful tool for investi-gating the structures of complex molecules and naturalproducts for a long time. It is frequently used for character-izing the QPA in crude mixtures of alkaloids or in plantextracts. The base peaks produced by ESI-MS withoutCID (collision induced dissociation) of the abundantlyoccuring QPA are: berberine 336, palmatine 352, jatrorrhi-zine 338, columbamine 338, coptisine 320, epiberberine336, and berberastine 322. The ESI-MS spectra with CID(�30 V) contain 4–5 fragments (Chuang et al., 1996). Frag-mentations of the quaternary alkaloids berberine, jatrorrhi-zine, coptisine, palmatine, and 13-methylberberine using

electrospray ionization tandem mass spectrometry (ESI-MS) have been described recently (Wang et al., 2004a,b).For example, the fragmentation pathway of berberine(Mr!!MS5) has been described by the followingsequence: 336 (m/z, berberine)! 321 (–CH3

�)! 320(–H�)! 318 (–2H)! 290. The ESI-MS technique has beenused for the structural characterization and identificationof the alkaloids in Rhizoma Coptidis (for details, see Wanget al., 2004a,b).

9-Hydroxy-10-methoxy compounds can be differenti-ated from their 9-methoxy-10-hydroxy regioisomers(Shamma, 1972). Those with the former substitution pat-tern preferentially eliminate a methyl group from theequivalent of ion C (Scheme 5), yielding an ion at 135,whereas the latter lose a hydrogen atom from their ion Cequivalent, giving rise to a new ion at 149. Additionally,by careful analysis of peak intensities, it may even be pos-sible to differentiate between the C-9, C-10 and C-10, C-11substitution patterns (Habermehl et al., 1970; Schneiderand Zenk, 1993a).

The mass spectrum of berberine base shows a veryminor signal for the molecular ion due to the easy thermaldissociation of the C8–OH bond upon the formation of thequaternary alkaloid and to the disproportionation of ber-berine base into dihydroberberine and oxoberberine(Shamma, 1972; Dostal et al., 2004). Generally, the ion cor-responding to the quaternary alkaloid dominates the spec-trum of C8–X substituted dihydroprotoberberine, formedby the nucleophilic addition of X� to the carbon C-8 (Dos-tal et al., 2004). The formation of the quaternary ion is dueto the lability of the C8–X chemical bond and the thermalstability of the quaternary form.

5.2. UV–VIS spectroscopy

The pattern of the UV absorption spectra of the QPAbases is determined by the auxochromic groups bound toring D (Pavelka and Smekal, 1976), the methoxy groupson carbons C-9 and C-10 or C-10 and C-11. Quaternarysalts of protoberberine alkaloids in polar media exhibitfour well-defined absorption bands. The ultraviolet absorp-tion spectra of the pseudoberberine alkaloids are markedlydifferent from those of the protoberberine alkaloids(Shamma et al., 1969). The only difference between thepseudoberberines and the berberines is the position ofthe substituents on aromatic ring D; in pseudoberberinesthe second auxochromic group is bound to carbon C-11,

Table 6RF values of QPA chlorides in several solvent mixtures (paperchromatography)

System Berberine Palmatine Jatrorrhizine Columbamine

A 0.38 0.35 0.27 0.24B 0.77 0.75 0.74 0.73C 0.83 0.83 0.69 0.60D 0.68 0.66 0.73 0.69E 0.53 0.56 0.37 0.29F 0.53 0.53 0.38 0.37

(A) 100 ml butanol, 100 ml chloroform, 100 ml water; (B) 160 ml butanol,20 ml glacial acetic acid, 100 ml water; (C) 50 ml pentan-2-ol, 50 ml iso-amyl alcohol, 140 ml 28% formic acid, 36 ml chloroform; (D) 92 ml pyr-idine, 225 ml ethyl acetate, 200 ml water; (E) 200 ml butanol, 100 ml 7%(aq.) ammonia; (F) 160 ml propanol, 40 ml 7% (aq.) ammonia.

N

OMe

OMe

O

O NH

O

O NH

O

O

OMe

OMe++

Ion A Ion B Ion C

Scheme 5.


in berberines to C-9 (see Chapter 1.2). The protoberberinesalts show (Shamma et al., 1969) a minimum at 301–310 nm, while the pseudoberberine salts show strongabsorption in this region in the form of a peak or shoulder.According to one of the research groups (Preininger et al.,1970), it is the oxygenous electron-donating substituent onC-11 that has the decisive effect on the polarization of apseudoberberine molecule in the excited state.

The transition from the tetrahydroisoquinoline tertiarynitrogen in tetrahydroprotoberberine alkaloids to the qua-ternary nitrogen in the salts of protoberberine and pseudo-protoberberine bases manifests itself in the fluorescencespectra of these compounds by a characteristic bathochro-mic shift of the emission bands and by a considerableincrease in the intensity of fluorescence.

The oxygenous electron-donating groups (methoxy andhydroxy) in the quaternary protoberberine alkaloids appre-ciably reduce the energy of electron transfers between theground and the excited singlet state for these molecules,and, moreover, influence their absorbances and fluores-cence intensities. The significant bathochromic shifts ofthe absorption and the fluorescence bands upon the intro-duction of these auxochromic groups into a protoberberineor tetrahydroprotoberberine molecule originate in the pro-longed conjugated system of these molecules, as a result ofthe interaction of the free electron pairs of the oxygen withthe conjugated p-electron system of the aromatic rings(Pavelka and Smekal, 1976). The values of kmax of theQPA chlorides measured in ethanol are summarized inTable 7. However, the kmax values of a few QPA with other

anions measured in various solvents have also been pub-lished (Brezova et al., 2004).

5.3. NMR spectroscopy

Nuclear magnetic resonance spectroscopy is the mostpowerful method used for investigating the structure ofnatural products in the solution (Martin and Crouch,1994; Reynolds and Enriquez, 2002). Modern 1D and 2DNMR spectroscopy are capable of providing unequivocalinformation about the chemical bonding, i.e. the constitu-tion of the molecule. However, the magnitudes of the indi-rect spin-spin coupling constants are used to describe theconfigurational and conformational aspects. Nowadays,the widespread application of the Karplus equation indetermining the torsion angles represents a textbook exam-ple of the utilization of NMR to reconstruct 3D structuresof molecules (Marquez et al., 2001). The determination ofthe Nuclear Overhauser Effect (NOE) between two nuclei(predominantly 1H–1H) is a complementary approach usedto elucidate the molecular topology (Neuhaus and William-son, 1989). Currently, 1H–1H homonuclear (COSY, DQF-COSY) and 1H–13C heteronuclear (HMQC, HSQC,HMBC) correlation techniques are routinely applied inthe field of constitutional analysis. 1D or 2D NOE experi-ments are used predominantly for solving stereochemicalproblems.

The NMR chemical shift contains very detailed infor-mation about the electron distribution around the atomicnucleus. Structural modifications are strongly reflected inchanges in the chemical shift. The influence of the intercon-version between the salt and the base of the alkaloid ber-berine (Scheme 6) on the 1H and 13C chemical shifts isdemonstrated in Table 8 (Blasko et al., 1988; Jansenet al., 1989; Marek et al., 2003b).

Substantial changes in the chemical shieldings of H-8(Dd � 3.7 ppm) and especially of C-8 (Dd � 66 ppm), theatom at the direct center of the structural change, arenoticeable. However, the differences in the shieldings ofthe C-13 (Dd � 25 ppm) and N-7 (Dd � 106 ppm) atoms,which are components of the attacked heterocycle (ringC), reflect changes in the electronic structure to a largeextent. The 1H and 13C NMR chemical shifts of severalprotoberberines are summarized in Tables 9 and 10.

During the past few years, 15N NMR chemical shiftsand 1H–15N coupling constants have become entrenched

N

OMe

OMe

O

O

ClN

OMe

OMe

O

O OH+ NaOH (-NaCl)

+HCl (-H2O)

Berberine chloride Berberine base

Scheme 6.

Table 7kmax of QPA chlorides in ethanol (Pavelka and Smekal, 1976; Chia et al.,1998)

Alkaloid kmax (nm)

Berberine 230, 267, 344, 352, 432Coptisine 229, 241, 268, 354, 363, 467Palmatine 228, 240, 268, 276, 343, 350, 433Berberrubine 234, 275, 353–8, 455Jatrorrhizine 228, 241, 267, 352, 440Pseudocoptisine 220, 231, 266, 289, 317, 346, 380Pseudopalmatine 242, 265, 288, 310, 342, 380Pseudoepiberberine 220, 240, 264, 290, 310, 341, 380Columbaminea 206, 225, 265, 345Corysamine 230, 240, 268, 344, 352, 455Fissiaine 206, 230, 282, 337

a In methanol.


as important restraints used for elucidating the structuresof natural products (Martin and Hadden, 2000; Martinand Williams, 2005). Owing to the development of newNMR techniques and improvements in the spectrometerhardware, the detection of 15N NMR spectra at naturalabundance has become a routine task (Marek and Lycka,2002).

The 15N NMR chemical shifts of N-7 in four QPAare summarized in Table 11. It is evident from the tablethat the substitution patterns on rings A and D have noeffect on the shielding of the N-7 atom. However, thereduction of ring C (dihydroderivative) disturbs its aro-matic nature and has a crucial effect on the isotropicshielding of the nitrogen nucleus. Moreover, nitrogenshielding is influenced by the electronic properties ofthe substituent at position C-8 (see dihydroberberinesin Table 11).

5.4. X-ray diffraction analysis

Single-crystal X-ray diffraction is a precise method suit-able for studying the bond lengths, bond and torsionangles, intermolecular interactions, and complete topologyof crystal systems (Dostal et al., 2004; Man et al., 2001a,b;Marek et al., 2003a; Kariuki, 1995). Selected crystal data(bond lengths and valence angles) of the quaternary pro-toberberines and their 7,8-dihydroderivatives are summa-rized in Table 12. The table emphasizes the interatomicdistances and bond angles that are somehow related tothe iminium N7–C8 bond because this bond is the centerof the principal reactivity of the QPA.

Table 81H and 13C NMR chemical shifts (d in ppm) of berberine chloride(DMSO-d6) and berberine base (CD2Cl2) (Blasko et al., 1988; Mareket al., 2003b)

Atom dH dC

Berberinechloride

Berberinebase

Berberinechloride

Berberinebase

1 7.79 7.15 105.43 104.962 – – 147.63 147.293 – – 149.76 147.824 7.09 6.62 108.43 108.164a – – 130.64 129.735 3.22 2.86/2.89 26.35 30.486 4.95 3.51/3.72 55.19 46.668 9.91 6.18 145.42 79.508a – – 121.37 123.469 – – 143.61 146.1710 – – 150.36 150.3311 8.20 6.94 126.67 114.5812 8.01 6.90 123.53 119.7212a – – 132.94 127.6813 8.96 6.08 120.18 95.3413a – – 137.41 136.7213b – – 120.42 126.21OCH2O 6.17 5.93/5.95 102.07 100.999-OMe 4.10 3.91 61.95 61.0110-OMe 4.07 3.85 57.06 56.07

Tab

le9

1H

NM

Rch

emic

alsh

ifts

(din

pp

m)

of

sele

cted

QP

As

Ato

mB

erb

erin

eP

alm

atin

eJa

tro

rrh

izin

eP

seu

do

pal

mat

ine

Cas

ead

ine

Co

lum

bam

ine

Th

alif

end

ine

Fis

siai

ne

Deh

ydro

cory

dal

ine

H-1

7.79

7.75

7.33

7.45

–7.

527.

767.

297.

56H

-47.

097.

156.

506.

72–

6.80

7.07

–7.

06H

-53.

223.

253.

073.

153.

183.

303.

193.

233.

20H

-64.

954.

954.

795.

004.

744.

974.

894.

884.

94H

-89.

919.

959.

5010

.34

9.16

9.45

9.69

9.63

9.82

H-9

––

–7.

67–b

––

––

H-1

0–

8.10

––

––

––

–H

-11

8.20

8.15

7.96

––

7.58

7.84

7.77

8.05

H-1

28.

01–

7.85

7.45

–b8.

117.

847.

908.

17H

-13

8.96

9.15

8.44

8.40

9.08

8.45

8.83

8.77

–2-

OR

6.17

3.90

3.89

4.13

–b–a

6.16

4.01

–b

3-O

R3.

88–a

4.09

–b3.

983.

89–b

9-R

/10-

R4.

104.

154.

064.

06–b

4.08

4.06

4.15

–b

10-R

/11-

R4.

074.

004.

163.

98–b

4.08

–a–a

–b

13-O

Me

––

––

––

––

2.50

So

lven

tD

MS

O-d

6D

MS

O-d

6C

D3O

DC

DC

l 3C

DC

l 3C

D3O

DD

MS

O-d

6C

D3O

DD

MS

O-d

6

Ref

eren

ces

Bla

sko

etal

.(1

988)

Waf

oet

al.

(199

9)K

imet

al.

(200

0)P

atra

etal

.(1

987)

Su

auet

al.

(199

3)H

sieh

etal

.(2

004)

Ch

ud

ıket

al.

(200

6)C

hia

etal

.(1

998)

Miy

azaw

aet

al.

(199

8)

aN

ot

incl

ud

edin

ori

gin

alre

po

rt.

bN

MR

chem

ical

shif

tsh

ave

no

tb

een

assi

gned

toth

ein

div

idu

alat

om

s:C

asea

din

e7.

45,

7.30

,7.

00,

4.14

,4.

00;

Deh

ydro

cory

dal

ine

4.12

,4.

12,

4.07

,3.

91.


Tab

le10

13C

NM

Rch

emic

alsh

ifts

(din

pp

m)

of

QP

As

Ato

mB

erb

erin

eP

alm

atin

eP

seu

do

pal

mat

ine

Co

lum

bam

ine

Jatr

orr

hiz

ine

Ber

ber

rub

ine

Th

alif

end

ine

Deh

ydro

cory

dal

ine

C-1

105.

4310

9.70

105.

111

4.9

110.

910

5.1

105.

1811

1.6

C-2

147.

6315

0.80

152.

614

3.7

149.

4b

147.

5314

6.5

C-3

149.

7615

3.07

149.

215

0.4

146.

4b

149.

4015

0.1

C-4

108.

4311

2.00

111.

610

9.4

116.

310

8.6

108.

3011

2.4

C-4

a13

0.64

129.

8012

9.2

133.

613

0.2

b13

0.17

133.

3C

-526

.35

27.8

027

.226

.329

.127

.526

.33

26.0

C-6

55.1

956

.50

57.1

55.6

57.6

56.3

54.9

355

.6C

-814

5.42

146.

2014

4.1

144.

814

5.2

144.

414

3.55

145.

2C

-8a

121.

3712

4.30

123.

211

7.4

119.

2b

122.

0911

9.1

C-9

143.

6114

5.6

109.

314

8.0

151.

5b

141.

1115

0.8

C-1

015

0.36

151.

513

9.5

149.

914

5.1

b15

0.88

143.

8C

-11

126.

6712

8.00

153.

712

6.5

122.

9b

123.

3412

7.0

C-1

212

3.53

–a10

6.4

123.

312

5.7

b12

3.42

123.

4C

-12a

132.

9413

5.4

123.

112

8.2

128.

2b

131.

5812

8.5

C-1

312

0.18

121.

111

8.3

119.

811

9.3

b12

0.17

119.

5C

-13a

137.

4113

9.9

138.

013

8.3

134.

913

7.6

136.

0113

7.9

C-1

3b12

0.42

120.

411

9.3

121.

412

0.9

b12

0.62

121.

4C

2-R

102.

0757

.3c

–58

.310

2.4

101.

8956

.0C

3-R

56.8

c56

.9–

57.2

C9-

R/C

10-R

61.9

562

.4c

61.8

63.0

–60

.96

61.9

C10

-R/C

11-R

57.0

657

.4c

56.3

57.0

57.1

–56

.0C

13-O

Me

––

––

––

–39

.8

So

lven

tD

MS

O-d

6D

MS

O-d

6C

DC

l 3C

DC

l 3+

DM

SO

-d6

CD

Cl 3

+D

MS

O-d

6C

DC

l 3D

MS

O-d

6D

MS

O-d

6

Ref

eren

ces

Bla

sko

etal

.(1

988)

Waf

oet

al.

(199

9)S

uau

etal

.(1

991)

Ras

oan

aivo

etal

.(1

991)

Ras

oan

aivo

etal

.(1

991)

Su

auet

al.

(199

1)C

hu

dık

etal

.(2

006)

Miy

azaw

aet

al.

(199

8)

aN

ot

incl

ud

edin

ori

gin

alre

po

rt.

bN

MR

chem

ical

shif

tsh

ave

no

tb

een

assi

gned

toth

ein

div

idu

alat

om

s:14

3.2,

145.

4,14

9.0,

151.

2(C

-2,

C-3

,C

-9,

C-l

0),

119.

1,12

0.1,

125.

0(C

-11,

C-1

2,C

-13)

,11

7.1,

129.

6,13

2.7

(C-4

a,C

-8a,

C-1

2a,

C-1

3b).

cN

MR

chem

ical

shif

tsh

ave

no

tb

een

assi

gned

toth

ein

div

idu

alat

om

s:56

.9,

56.8

,56

.3,

55.8

.


The interatomic distance N7–C8 in quaternary protob-erberines lies in the range 132–134 pm. This bond is some-what longer in the 8-oxoderivatives (139 pm) and reaches144–145 pm in the dihydroderivatives (products of nucleo-philic addition to the QPA skeleton; see Chapter 6). Simi-larly, C8–C8a gets longer in the order QPA (138–140 pm),8-oxoderivatives (147 pm), dihydroderivatives (149–151 pm). In the same order of derivatives, the bond angleN7–C8–C8a is reduced (120–122�, 115–117�, 109–110�).

Quaternary protoberberine cations are relatively planarspecies. Their planarity is disturbed only in the partiallysaturated ring B, which adopts a twisted half-chair confor-mation with the atoms C5 and C6, deviating from the planeof rings A, C and D. In 8-substituted-7,8-dihydroderiva-tives (adducts) the ring C also becomes partially saturated,forming a shallow half-chair with the C-8 atom (in sp3

hybridization) deviating. The group attached at C-8 is usu-ally in the pseudoaxial position. The methoxy group at C-10 is positioned in-plane while the neighboring 9-OMe isalmost perpendicular to the plane of the D-ring. This is acommon feature of the tertiary 8-adducts, 8-oxoderiva-tives, and quaternary protoberberine salts. The interplanarangles between the aromatic rings of some QPA andderivatives are given in Table 13.

The crystal structures of the compounds studied pro-vided a number of interesting findings on non-covalentintermolecular interactions. In QPA salts, cations arepacked in columns with the spaces between occupied byanions (Kariuki, 1995). Berberine formate–succinic aciddisplayed non-conventional C–H� � �O hydrogen bonds(Marek et al., 2003a). Four molecules of 8-hydroxydihyd-roberberine were linked by four hydrogen bonds to makean eight-membered heterocycle with alternating H and Oatoms (Dostal et al., 2004). Also in 8-aminodihydrober-berine, four molecules were associated with six H-bonds(Man et al., 2001a).

6. Properties and reactivity

Quaternary protoberberine alkaloids are brightly col-ored solids. Phenolic QPA typically change the color uponchanges in the pH of their solution (Shamma, 1972). For

example, the alkaloid jatrorrhizine is yellow in a neutralsolution, but turns to deep red in an alkaline environment.Columbamine, which is also yellow in neutral solution,becomes only tan in hydroxide solution. The bright colorsof QPA can be employed advantageously in monitoring theprogress of a column-chromatography separation. Quater-nary protoberberines without a phenolic group changetheir bright colors from the typical yellow to very slightlycolored or even colorless substances.

As mentioned previously, quaternary protoberberinealkaloids are derivatives of 5,6-dihydrodibenzo[a,g]quino-lizinium. These iminium cations are characterized by thesensitivity of the polar C@N+ bond to a nucleophilic attackfollowed by the formation of adducts with substituents atposition C-8 (Marek et al., 2003b). Strong base is capableof converting the quaternary alkaloid to the correspondingbase (for pK values of QPA, see Table 14). In contrast tothe majority of classical alkaloids, however, the bases pos-sess a non-classical structure with an OH group covalentlybonded to carbon C-8 (Scheme 6).

Berberine chloride was converted to its base using a 10–20% solution of NaOH in water. When the product wasextracted into dichloromethane, 8-hydroxy-7,8-dihydrob-erberine, formed by the addition of OH� to the C-8 ofthe iminium moiety, was identified using NMR as a majorcomponent of the solution. In addition, the bimolecularaminoacetals (both diastereomers), formed by the conden-sation reaction between two molecules of the base, weredetected as minor forms in the organic solvent (Scheme7) (Dostal et al., 2004). However, these basic forms are veryunstable compounds and traces of acid immediately con-vert them back to the salts.

6.1. Nucleophilic addition

6.1.1. O-Nucleophiles

The nucleophilic addition of OR� to the berberine skel-eton is a simple analogy to the formation of a berberinebase in alkaline solution. The 8-methoxy and 8-ethoxy-7,8-dihydroderivatives prepared by the reaction of QPAwith NaOR (Scheme 8) are unstable compounds, easilyconverted back to the quaternary forms in an acidic envi-ronment. From time to time, 7,8-dihydroprotoberberineshave been reported as genuine natural products (Wafoet al., 1999; Li et al., 2001). However, due to the acidic nat-ure of the plant tissues, 8-hydroxy-, 8-methoxy- and 8-eth-oxy-7,8-dihydroprotoberberines must be classified asisolation artifacts. They are formed during the isolationor separation procedures in alkaline media in the presenceof common solvents (methanol, ethanol) (Marek et al.,2003b).

6.1.2. N-Nucleophiles and S-nucleophiles

In 2001, Man and co-workers (Man et al., 2001) triedto prepare 8-substituted-7,8-dihydroberberines by reactingthe quaternary berberine with azide and thiocyanate.

Table 1115N NMR chemical shifts (d in ppm)a of QPAs in DMSO-d6 (D) and 8-substituted-7,8-dihydroberberines in CD2Cl2 (C) at 303 K (Marek et al.,2003b, 1999, 2002)

Alkaloid/derivative Chemical shift

Berberine 194.8 (D)Coptisine 194.9 (D)Palmatine 194.5 (D)Jatrorrhizine 194.4 (D)8-Hydroxy-7,8-dihydroberberine 88.5 (C)8-Methoxy-7,8-dihydroberberine 83.6 (C)8-Trichloromethyl-7,8-dihydroberberine 64.9 (C)

a Referenced relative to liquid ammonia (Marek and Lycka, 2002).


Tab

le12

Bo

nd

len

gth

s(A

)an

db

on

dan

gles

(�)

of

sele

cted

QP

As

and

7,8-

dih

ydro

pro

tob

erb

erin

ed

eriv

ativ

es

Alk

alo

idR

-fac

tor

N7-

C6

N7-

C8

N7-

C13

aC

8-C

8aC

8-R

C6-

N7-

C8

N7-

C8-

C8a

C8-

N7-

C13

aC

DC

cod

eR

efs.

Ber

ber

ine

chlo

rid

ete

trah

ydra

te7.

011.

481

1.33

71.

378

1.40

3–

117.

6712

1.72

121.

67Y

UJH

AM

1B

erb

erin

ech

lori

de

eth

ano

lso

lvat

eh

emih

ydra

te5.

621.

491

1.31

61.

394

1.40

5–

118.

0912

2.08

122.

61Y

UJH

IU1

Ber

ber

ine

bro

mid

ed

ihyd

rate

4.71

1.48

61.

320

1.39

41.

396

–11

8.57

121.

0912

3.18

YU

JHO

A1

Ber

ber

ine

iod

ide

6.66

1.50

11.

316

1.38

11.

393

–11

7.58

121.

8112

3.61

YU

JHU

G1

Ber

ber

ine

hyd

roge

nsu

lfat

e6.

101.

499

1.32

41.

398

1.39

3–

118.

1712

2.00

122.

40C

ISR

EB

2b

is(B

erb

erin

e)su

lfat

eh

epta

hyd

rate

a7.

641.

472

1.34

11.

373

1.39

7–

119.

1412

3.22

122.

01Y

UJJ

AO

1B

erb

erin

eaz

ide

acet

on

itri

leso

lvat

e6.

291.

492

1.33

41.

390

1.39

8–

117.

5012

1.87

122.

29U

CA

CU

W3

Ber

ber

ine

thio

cyan

ate

8.29

1.48

41.

332

1.38

91.

401

–11

8.59

121.

4212

2.48

UC

AD

AD

3B

erb

erin

efo

rmat

esu

ccin

icac

id3.

401.

499

1.33

81.

399

1.40

4–

118.

1812

1.47

122.

34O

LO

FU

Q4

Pal

mat

ine

iod

ide

4.20

1.48

31.

340

1.40

21.

381

–11

7.93

122.

0012

1.81

QE

MY

UC

5P

alm

atin

ech

lori

de

ph

eno

lso

lvat

em

on

oh

ydra

te5.

501.

500

1.34

01.

385

1.39

9–

117.

3512

0.93

122.

38F

INP

UN

5

Jatr

orr

hiz

ine

chlo

rid

ed

ihyd

rate

5.80

1.49

91.

337

1.38

91.

407

–11

7.63

121.

3212

2.39

SO

HN

IM5

Jatr

orr

hiz

ine

chlo

rid

ed

ihyd

rate

5.70

1.49

51.

323

1.39

01.

398

–11

8.84

122.

2912

2.42

SO

HN

IM01

6D

ehyd

roco

ryd

alin

eh

ydro

chlo

rid

eh

ydra

te5.

301.

476

1.32

41.

404

1.40

1–

117.

2912

1.36

122.

90C

EX

NU

O5

8-H

ydro

xy-7

,8-d

ihyd

rob

erb

erin

e8.

911.

463

1.44

41.

400

1.49

41.

440

119.

4610

9.95

120.

55A

MIZ

IF7

8-M

eth

oxy

-7,8

-dih

ydro

ber

ber

ine

5.19

1.46

51.

437

1.41

01.

504

1.46

011

6.41

110.

6311

9.47

BA

PV

AQ

88-

Am

ino

-7,8

-dih

ydro

ber

ber

ine

8.75

1.46

41.

473

1.41

51.

512

1.45

411

3.34

109.

8711

7.40

/9

8-A

min

o-7

,8-d

ihyd

roco

pti

sin

e5.

721.

453

1.46

81.

394

1.50

21.

465

115.

3710

8.86

118.

98/

98-

Cya

no

dih

ydro

ber

ber

ine

4.36

1.46

21.

451

1.41

61.

515

1.50

811

4.13

110.

6511

5.94

UC

AD

EH

38-

(Tri

chlo

rom

eth

yl)-

7,8-

dih

ydro

ber

ber

ine

6.60

1.46

71.

441

1.38

81.

511

1.58

911

9.41

109.

9712

0.60

RE

NM

US

108-

(Tri

chlo

rom

eth

yl)-

7,8-

dih

ydro

ber

ber

ine

3.17

1.46

31.

443

1.39

81.

515

1.58

411

9.33

110.

0712

0.22

RE

NM

US

018

8-(T

rich

loro

met

hyl

)-7,

8-d

ihyd

rop

alm

atin

e3.

531.

464

1.45

01.

400

1.50

91.

577

118.

7011

2.31

119.

85B

AP

VE

U8

8-O

xob

erb

erin

e3.

371.

475

1.38

81.

395

1.47

41.

230

115.

9311

6.68

123.

99A

MIZ

UR

78-

Oxo

cop

tisi

ne

3.31

1.47

61.

388

1.40

41.

470

1.23

511

7.37

115.

4112

4.34

AM

IZO

L7

12,1

3-D

init

ro-o

xyb

erb

erin

ea4.

001.

473

1.39

21.

393

1.47

31.

221

115.

6211

6.49

123.

27S

IKG

IC11

1.K

ariu

ki

(199

5),

2.A

bad

iet

al.

(198

4),

3.M

anet

al.

(200

1b),

4.M

arek

etal

.(2

003a

),5.

CC

DC

(199

9)6.

Gh

osh

etal

.(1

993)

,7.

Do

stal

etal

.(2

004)

,8.

Mar

eket

al.

(200

3b),

9.M

anet

al.

(200

1a),

10.

Kh

amid

ov

etal

.(1

996)

,11

.C

ush

man

etal

.(1

990)

.a

Ave

rage

valu

esfo

rtw

osy

mm

etry

ind

epen

den

tm

ole

cule

s.


Under the experimental conditions described, however,the reaction did not afford the expected products. Instead,berberine azide and berberine thiocyanate were isolatedfrom the reaction mixture. In contrast, 8-aminoderivativeswere formed during the reaction of a QPA and a primaryamine in ethanol or methanol (Naruto et al., 1976). The

reaction of berberine and coptisine with liquid ammoniaproduced 8-aminoderivatives (Man et al., 2001). Further,the preparation of adducts with an exocyclic C8@NRdouble bond (Bhakuni and Jain, 1986) and the prepara-tion of 7,8-dihydroberberines with amine (–NH–R),hydrazine (–NH–NRR 0) or hydroxylamine (–NH–OR)groups attached to the C-8 has been published (Mohrleand Biegholdt, 1982).

To the best of our knowledge, the preparations or struc-tural characterizations of any covalent adduct with an S-nucleophile has not yet been reported.

6.1.3. C-NucleophilesThe sensitivity of atom C-8 in the polarized N7@C8

iminium bond to nucleophilic attack has allowed the con-densation of berberine with phenyl- or benzylmagnesiumbromide (Scheme 9).

Reacting berberine with benzylmagnesium bromide pro-duced 8-benzyl-7,8-dihydroberberine (Shamma, 1972).Reduction of the product with sodium borohydride yields8-benzylcanadine, which can undergo cleavage of ring Cthrough Hofmann degradation (Scheme 10).

Another possibility for incorporating a carbon atom atposition C-8 of a QPA is reaction with chloroform(Scheme 11). This reaction produces 8-trichlormethyl-7,8-dihydroberberine (Marek et al., 2003b; Shamma and Rah-imizadech, 1986; Miana, 1973; Slavık and Slavıkova,1989).

Generally, when chloroform is used for separating theQPA from plant material under alkaline conditions, 8-tri-chloromethyl derivates are easily formed. In the past, these8-trichloromethyl products have been incorrectly charac-terized as protoberberine bases (8-hydroxy derivatives)(Wafo et al., 1999; Li et al., 2001). Obviously, thesecompounds are isolation artifacts (Marek et al., 2003b).

Table 13Interplanar angles (�) in selected compounds derived from berberine

Compound A/C A/D C/D Refs.

Berberine formate 13.36 13.22 1.69 Marek et al. (2003a)Berberine azide 11.02 10.10 0.93 Man et al. (2001b)Berberine thiocyanate 11.02 10.10 0.93 Man et al. (2001b)8-Hydroxydihydroberberine 21.50 31.47 13.06 Dostal et al. (2004)8-Aminodihydroberberine 23.03 25.58 8.76 Man et al. (2001a)8-Cyanodihydroberberine 29.45 37.69 14.09 Man et al. (2001b)8-Trichloromethyldihydroberberine 39.58 45.26 14.64 Marek et al. (2003b)

Table 14pK values of QPAs (Simanek et al., 1976) obtained for methanolicsolutions

Alkaloid pK

Dihydrodibenzo[a,g]quinolizinium 14.3Palmatine 15.7Berberine 15.4Coptisine 13.8Corysamine 15.213-Methylberberine 16.513-Methoxyberberine 16.4

N

OMe

OMe

O

O

ClN

OMe

OMe

O

O-OH

OH

-H2O

N

OMe

OMe

O

ON

MeO

MeO

O

OO

Scheme 7.

N

OMe

OMe

N

O

O

OMe

OMe

OMeX

MeOH

MeONa

O

O

Berberine 8-Methoxy-7,8-dihydroberberine

Scheme 8.


However, compounds of this type must be characterizedcarefully because their identification is not always straight-forward due of the lack of protons in the –CCl3 substituent(1H NMR), the easy splitting of the C8–CCl3 bond (massspectrometry), and the greatly reduced intensity of the–CCl3 signal in 13C NMR spectra (long relaxation timeor inefficient NOE).

6.2. Disproportionation

In a protic medium, a dihydroprotoberberine derivativeis in equilibrium with its iminium form (Scheme 12). Thelatter species is unstable and undergoes rapid dispro-portionation to form a mixture of the quaternary proto-berberine salt and the tetrahydroprotoberberine.

N

OMe

OMe

O

O

XN

OMe

OMe

O

O RRMgBr

Berberine 8-Substituted dihydroberberine

R = Ph-, Ph-CH2-

Scheme 9.

N

OMe

OMe

O

O Ph N

OMe

OMe

O

O Ph N

OMe

OMe

O

O PhMe

1.CH3I2.HofmannNaBH4

8-Benzylcanadine

Scheme 10.

N

OMe

OMe

O

O

XN

OMe

OMe

O

O CCl3CHCl3

OH-

Berberine 8-Trichloromethyl-7,8-dihydroberberine

Scheme 11.

N

OR

RO

RO

OR

N

OR

RO

RO

OR

ROH or H

N

OR

RO

RO

OR

N

OR

RO

RO

OR

Dihydroprotoberberine

Disproportionation

Scheme 12.


Quaternary protoberberine salts are unstable in thepresence of concentrated alkali. The alkaloid bases formedunder basic conditions are subsequently transformed into

the 8-oxo-7,8-dihydro- and 7,8-dihydroderivatives (Scheme13) (Shamma, 1972).

6.3. Oxidation and reduction

The 8-oxoderivative can be obtained quantitatively fromthe QPA by oxidation with potassium ferricyanide (Scheme14).

Quaternary protoberberine salts can be reduced(Shamma, 1972) to the corresponding tetrahydroprotob-erberines with a variety of reducing agents (Scheme 15)(Bhakuni and Jain, 1986). When mixed metal hydride in

N

R

R

OHH

OHN

R

R

OH

Protoberberine base

N

R

RDihydroderivative

N

R

R

O

Oxo derivative

+

R

R

R

R

R

R

R

R

Scheme 13.

N

R

R

N

R

R

OK3Fe(CN)6

R

R

R

R

Scheme 14.

N

OMe

OMe

O

O

N

OMe

OMe

O

O

N

OMe

OMe

O

O

X

LiAlH4, etheror NaBH4, pyridine

I2, or Hg(OAc)2, or air

I2, or Hg(OAc)2, or air

NaBH4, ROH, or H2, Pt, or Zn, HCl

Berberine

Dihydroberberine

Tetrahydroberberine

Scheme 15.

N

OMe

OMe

O

O N

COO

O

OHNO3

COOH

Berberine Berberidic acid

Scheme 16.


a dry aprotic solvent is used for the reduction, the reactionis stopped at the dihydroprotoberberine stage. Re-oxida-tion of the dihydro- or tetrahydroprotoberberine to formthe quaternary salt can be accomplished with iodine (Cavaet al., 1968), mercuric acetate, or simply by standing in air(Meise, 1971). The redox reaction of berberine on a mer-cury electrode (Komorsky-Lovric, 2000) is totally irrevers-ible and occurs between �0.95 and �1.35 V, depending onthe berberine concentration and the pH of the solution.

Oxidation of a quaternary protoberberine salt with hotdilute nitric acid yields a golden betaine. In the case of ber-berine, the product is presumably the betaine berberidicacid, which has been formulated as indicated below(Scheme 16) (Shamma, 1972; Bhakuni and Jain, 1986).

N

OMe

OMe

MeO

HOHO

MeON

OMe

OMe

Scheme 17.

N

OMe

MeO

MeO

N

OMe

MeO

OMe

MeO

N

OMe

MeO

MeO

OMe

OMeI CH3

Me

N

OMe

MeO

MeO

OMe

N

OMe

MeO

MeO

OMeMe Me

HH

Corydaline Mesocorydaline

Scheme 18.

N

OMe

OMe

O

Ohν

O2

N

OMe

OMe

O

O OO

R N

OMe

OMe

O

O ROO

N

OMe

OMe

O

O R

O

R = OMe, OEt, SC6H13

Berberine

H

ODioxetane

Scheme 19.


Bisjatrorrhizine (Scheme 17), obtained from Jatrorrhiza

palmata, is formally produced by the ortho oxidative cou-pling of the phenolic group of jatrorrhizine (Bhakuni andJain, 1986; Fajardo et al., 1996).

6.4. Alkylation at position C-13

The conversion of palmatine to corydaline and mesoco-rydaline (Scheme 18) via an intermediate is an early exam-ple of the currently well-known C-alkylation of an enamine(Jeffs, 1967). The relative proportion of corydaline tomesocorydaline obtained from the intermediate salt appar-ently varies with the nature of the reducing agent; zinc–acidsystems afford predominantly corydaline while borohyd-ride reduction is claimed to give corydaline exclusively.

6.5. Photochemical reactions

Photochemical reaction (Morrison, 1990) has beenproved to be an important pathway for the transformationof myriad molecules in biological systems. The photochem-istry of protoberberines (Contreras et al., 1984) is currentlyof great interest to molecular biologists and biochemistsinvestigating biologically relevant samples containing pro-toberberines (see Chapter 7). Very recently, berberine hasbeen indicated as a photosensitizer in the oxidation ofguanine to the 8-oxoguanine (Hirakawa et al., 2005).

The Rose-Bengal sensitized photooxygenation of ber-berine and also dihydroberberine gives the correspondingberberinephenolbetaine in good yields. In the absence ofany sensitizer the photochemical oxidation of dihydroberb-erine produces mainly berberine. The reaction of berberinedoes not proceed under similarly unsensitized conditions

unless sodium methoxide or an other nucleophile is presentin the reaction mixture. Under those conditions the lac-tamic aldehyde is formed (Scheme 19).

8-Hydroxymethylberberines can be prepared from thecorresponding quaternary protoberberines by a photo-chemical route. The reaction mechanism includes two sub-sequent steps – chemical sensitization (acetone,benzophenone) and single electron transfer (Suau et al.,1999a,b). For other photochemical reactions, see thereview by Singh et al. (1980).

6.6. Transformation of 2,3,9,10-substituted QPA to

2,3,10,11-substituted QPA

The 2,3,10,11-tetrasubstituted QPA are sometimescalled pseudoberberines (see Chapter 1.2). Their formationfrom the 2,3,9,10-substituted analogs was published in1985 (Hanaoka et al., 1985). Naturally occurring2,3,10,11-substituted QPA were synthesized from the corre-sponding 2,3,9,10-substituted QPA through oxidative C8–C8a bond cleavage, photocyclization, and subsequentdeoxygenation.

Berberine was oxidized with m-chloroperbenzoic acid indry tetrahydrofuran in the presence of sodium hydride toafford an open intermediate in 76% yield (Scheme 20). Thisintermediate had previously been synthesized from berber-ine by a similar oxidation using sodium bicarbonate insteadof sodium hydride, however, in only 20% yield. The photo-cyclization reaction of the enamide intermediate in ethanolwas done under a stream of nitrogen using a high-pressuremercury lamp. Subsequent reduction using sodium borohy-dride produced 12-hydroxytetrahydropseudoberberine(yield 79%).

N

OMe

OMe

O

O NCHO

OMe

OMe

O

O N

OMe

O

OOH

OMeHO

Berberine 12-Hydroxytetrahydropseudoberberine

Scheme 20.

N

OMe

O

O N

OMe

O

O N

OMe

O

O

OMeOMeHO

OMe(EtO)2POO

12-Hydroxytetrahydropseudoberberine

Scheme 21.


Treatment of 12-hydroxytetrahydropseudoberberinewith diethyl chlorophosphate in the presence of sodiumhydride afforded the phosphate, hydrogenolysis of whichusing sodium in liquid ammonia (at �70 �C) produced tet-rahydropseudoberberine (53%) (Scheme 21).

Beside the transformation of 2,3,9,10-substituted QPAto 2,3,10,11-tetrasubstituted QPA, transformations ofQPA can produce structurally related benzo[c]phenanthri-dines (Iwasa et al., 1989; Iwasa and Kamigauchi, 1996;Jeffs and Scharver, 1976; Iwasa and Kim, 1997; Hanaokaet al., 1986). Conversions of berberine, (Moniot andShamma, 1979; Elango and Shamma, 1983), karachine(Blasko et al., 1982), and berberrubine (Iwasa and Kami-gauchi, 1996; Das and Srinivas, 2002) have beenpublished.

7. Biological activities

Phytomedicine (Tsai and Tsai, 2004), including naturalproducts from traditional herbal medicines used for medi-cal and health-fortifying purposes, is gaining internationalpopularity. Extracts of Berberis aristata and Coptis chinen-

sis have been used in traditional Oriental medicine for thetreatment of gastroenteritis and secretory diarrhea. It isreasonable to presume that the quaternary protoberberinesbelong to the biologically active constituents of theseplants.

Protoberberine alkaloids display a great variety of bio-logical and pharmacological activities. These activitiesinclude the inhibition of DNA synthesis (Schmelleret al., 1997), protein biosynthesis, the inhibition of mem-brane permeability, and the uncoupling of oxidative phos-phorylation. These processes likely contribute to theallelochemical and toxic effects observed against bacteria,fungi, other plants, insects, and vertebrates. Interactionwith DNA and inhibition of reverse transcription couldbe responsible for the inhibition of phages and otherviruses. The interactions with neuroreceptors, the inhibi-tion of ATPase, and the binding to microtubules shouldaffect mainly insects and vertebrates. However, only somerepresentative examples of the biological effects of QPAare mentioned briefly in the following paragraphs. Thenumber of biological studies amounts to hundreds ofpapers published annually.

7.1. Interactions with biomacromolecules – structural studies

The investigation of the interactions of QPA with bio-macromolecules at the atomic level and the structuraldescription of their complexes represent key steps in thereal understanding of the biological function of the QPA.This research field has been developing dynamically duringthe past few years as documented by the number of paperspublished annually. The development is to a large extentdriven by the increasing number of sophisticated instru-mental techniques, including nuclear magnetic resonance

spectroscopy (NMR), mass spectrometry (MS), capillaryzone electrophoresis, and many others.

The ability of the protoberberines to act as a poisonagainst Topoisomerase-I (Topo-I) and Topoisomerase-II(Topo-II), has been related to their antitumor activity(Kim et al., 1998; Mazzini et al., 2003). The inhibitors ofthe enzyme Topo-II have been studied more frequentlythan those of Topo-I and the principal mechanisms of inhi-bition of Topo-II have been discovered. However, the roleof the drug–DNA interactions in Topo-I inhibition is stillunclear, although the binding of the protoberberine toDNA has been considered to be responsible for thisactivity.

Three principal noncovalent modes of binding smallligands to oligonucleotides can be distinguished: (a) exter-nal binders, often polyamines that make nonspecific elec-trostatic contacts with the the DNA backbone; (b) minorgroove binders, the most sequence-selective class, thanksto a network of specific H-bonds to base-pairs and back-bone functions; and (c) base-pair intercalators which estab-lish extended and partly specific, van de Waals contactswith the floors of aromatic pairs (Micco et al., 2006). Theinteraction of berberine with several oligonucleotides, stud-ied by NMR spectroscopy, showed that berberine bindspreferentially to AT-rich sequences (Mazzini et al., 2003).2D NOE experiments enabled the detection of severalcontacts between protons of berberine and protons of theself-complementary oligomer d(AAGAATTCTT)2. The ber-berine molecule is located in the minor groove of the dou-ble helix of the nucleotide at the level of the A4–T7 and A5–T6 base pairs. It lies with the convex side on the helixgroove, thus presenting the positive nitrogen atom of thealkaloid close to the negative ionic surface of oligonucleo-tide. Ring A and the methylendioxy group are external tothe helix, while the aromatic protons H-11 and H-12 areclose to the ribose of cytidine C8.

The noncovalent complexes of several protoberberinealkaloids (berberine, palmatine, jatrorrhizine, and copti-sine) have been investigated by using electrospray ioniza-tion mass spectrometry (ESI-MS) (Chen et al., 2004). Theresults indicated that palmatine exhibited the greatest bind-ing affinity with the double-stranded DNA, while berberinehad the lowest affinity. The preliminary results indicatedthat the berberine showed some sequence selectivities.Other ESI-MS studies of the noncovalent complexes haveshown several new results (Chen et al., 2005b). Five cyto-toxic protoberberine alkaloids, berberine, palmatine, jat-rorrhizine, coptisine, and berberrubine were tested with afew double stranded oligonucleotides. The different oligo-nucleotides gave the different orders of the relative bindingaffinity. The results from ESI-MS and fluorescence titra-tion experiments indicated that sequence selectivity of thesefive alkaloids was not significant and no remarkable AT- orGC-rich DNA binding preference was obtained. This is inclear contrast to the above-mentioned reports (Chen et al.,2004; Mazzini et al., 2003) describing the preferential bind-ing of berberine to AT-rich DNA.


Characterization of the quaternary berberines modifiedat the position C-9 showed that the berberine derivatives,especially those with a primary amino group, strongly bindwith calf-thymus DNA, presumably via an intercalationmechanism (Pang et al., 2005). Structure–activity relation-ships of the protoberberine analogs demonstrate that sub-stitution at the C-9 position is an important determinant ofthe biological activity (Park et al., 2004).

Studies of bridged berberine derivatives (two berberineunits bridged at the position C9 with different linkerlengths) showed that compounds with a propyl chain exhi-bit the highest binding affinity to DNA (Chen et al., 2005a;Qin et al., 2006).

The results of binding sanguinarine and berberine to tri-ple and double helical DNA and RNA structures show thatboth alkaloids can bind and stabilize the DNA and RNAtriplexes more strongly than their respective parentduplexes (Das et al., 2003).

The interaction of QPA with DNA was investigatedusing capillary zone electrophoresis (Vlckova et al.,2005). In this experiment two QPA were studied. Ethidi-nium bromide, a classic DNA intercalator, was used as areference. The results indicated that these alkaloids donot attach covalently to DNA constituents.

7.2. Cytotoxic activity and apoptosis

Probably the most important and most frequently testedproperty of the QPA is cytotoxicity (Sanders et al., 1998;Orfila et al., 2000; Colombo et al., 2001; Chen et al.,2005). The cytotoxic activity of the QPA seems to bemainly due to apoptosis and inhibition of telomerase.

Berberine was tested for its potential inhibitory effectagainst telomerase activity (Meyerson, 2000) on a humanleukemia cell line (Naasani et al., 1999). It was identifiedas a moderate inhibitor with 50% inhibition at 35 lM con-centration. Results obtained on telomerase of Plasmodium

falciparum (Sriwilaijareon et al., 2002) showed, that the tel-omerase is sensitive to inhibition by the berberine in arange of 30–300 lM.

The high concentration of berberine (75 lg/ml) inducedan acute cytotoxic activity (Letasiova et al., 2005). Berber-ine derivatives were identified as potential inhibitors of cas-pase 3, a major apoptosis effector. The inhibitory effect wasdetermined at 20 lM concentration of ligand (Letasiovaet al., 2005; Kim et al., 2002).

A comparative study of the inhibitory and anti-leukemicactivities of some protoberberine and benzo[c]phenanthri-dine alkaloids showed that the activities of the QPA arelower than those of the benzophenanthridines (Sethi,1985).

The cytotoxic activity of protoberberine was tested pre-dominantly in vitro (Iwasa et al., 2001). Extensive experi-ments on twenty four quaternary protoberberinealkaloids were evaluated on thirty eight human cancer celllines. Six compounds were cytotoxic and some othersexhibited lower levels of cytotoxicity. From a structure–

activity point of view, some trends were observed. For ber-berine and palmatine derivatives bearing a 13-alkyl sidechain, the cytotoxicity increased parallel to the number ofCH2 units in the side chain. 12-Bromo-8-hexylberberinewas more cytotoxic than the corresponding 8-phenyl and8-butylderivatives, suggesting that the length of the carbonunit at C-8 also influences the cytotoxicity. Bromination atC-12 increased the cytotoxicity (Iwasa et al., 2001).

The 8-oxoderivatives of the protoberberine alkaloidswere tested for cytostatic activity in vitro using MDA-MB-231 mammary tumor cells (Weimar et al., 1991). Tet-ramethoxy-8-oxoberberine inhibited cell proliferation at aconcentration of 10�5 M, its cytostatic effect did notdepend on intercalation into DNA.

The antimutagenic potency of berberine and jatrorrhi-zine was evaluated against acridine orange (AO) by usingEuglena gracilis as a eucaryotic test model (Cernakovaet al., 2002). Both alkaloids showed significant concentra-tion-dependent inhibitory effect against the AO-inducedchloroplast mutagenesis of E. gracilis. The quaternarybenzo[c]phenanthridine alkaloids tested did not exhibitthe same effect.

Slaninova et al. investigated the effects of berberine, cop-tisine, and some benzo[c]phenanthridine alkaloids on theyeasts Saccharomyces cerevisiae and Schizosaccharomyces

japonicus. The protoberberine alkaloids exhibited someeffects; however, these effects were significantly smallerthan those of the benzophenanthridines (Slaninova et al.,2001).

7.3. Antimicrobial activity

Berberine, jatrorrhizine, and the crude extract of Maho-

nia aquifolium have shown strong activity against twentyclinical isolates of Propionibacterium acnes with minimalinhibitory concentration values (Slobodnıkova et al.,2004). Berberine seems to be more active than jatrorrhizineagainst coagulase-negative staphylococci. The antifungalactivity tested against Candida showed that only C. tropi-

calis (resistant to nyastin, miconazole, and econazole)was strongly inhibited by all of the agents tested.

Experiments with the 8-alkyl and 8-phenyl-substitutedberberines and their bromo derivatives showed that theintroduction of hydrocarbon groups at position C-8increased the antimicrobial activity (Iwasa et al., 1998).The 12-bromo derivatives of the 8-alkyl- and 8-phenyl-pro-toberberines showed higher activity against the microor-ganisms tested than did their non–brominated analogs.

Jatrorrhizine may serve as a leading compound for fur-ther studies to develop new antifungal agents with highlypotent antifungal activity and low host toxicity (Vollekovaet al., 2003). The side carbon chain at position C-13 of qua-ternary protoberberines was investigated for its potential toincrease the fungicidal and herbicidal activity (Iwasa et al.,2000). Parallel to the QPA, tetrahydroforms with methylgroup at position C-13 were tested for in vitro andin vivo activities. Whereas the tertiary forms of alkaloids


did not exhibit significant activity, a few of the quaternaryforms showed some activity against Leptosphaeria nodrum

and Puccinia recongita.13-Hexylberberine and 13-hexylpalmatine showed the

strong activity against Staphylococcus aureus, being moreactive than berberine and kanamycin sulfate. Both hexylde-rivatives possessed antifungal activity (Iwasa et al., 1997).Berberrubine, the isolation artifact, was found to be activeagainst Mycobacterium smegmatis (Gharbo et al., 1973).

7.4. Anti-inflammatory activity

Extracts of the roots of berberine–containing Berberida-ceae were tested in acute cases of inflammation (carra-geenan- and zymosan-induced paw oedema in mice). Thetotal ethanol extract showed a higher reducing effect ascompared to the three alkaloid fractions and the majorconstituents berberine and oxyacanthine (Ivanovska andPhilipov, 1996).

7.5. Antimalarial activity

It has been reported that berberine is a potent in vitroinhibitor of both nucleic acid and protein synthesis inhuman malaria Plasmodium falciparum FCR-3 (Elford,1986; Vennerstrom and Klayman, 1988). In addition, theinhibitor activity against telomerase of human malaria P.

falciparum has been presented (Sriwilaijareon et al., 2002).In vitro structure–activity relationship studies for anti-

malarial activity against P. falciparum have been published(Iwasa et al., 1998, 1999). The type of the oxygen substitu-ent on rings A, C, and D and the position of the oxygenfunctions on ring D influence the activity of the protoberb-erine alkaloids. Shifting the oxygen function from C-9 andC-10 to C-10 and C-11 results in a significant increase inactivity.

7.6. Other effects

Berberine possesses an anti-diabetic effect (Leng et al.,2004), which is related to the property of stimulating insu-lin secretion and modulating lipids. The anti-arrhythmicactivity (Yang and Wang, 2003) of a new derivate of ber-berine CPU-86017 (7-(4-chlorbezyl)-7,8,13,13a-tetrahyd-roberberine chloride) has been tested. Inhibitory effects ofberberine on potassium and calcium currents in isolatedrat hepatocytes may be involved in hepatoprotection (Tsaiand Tsai, 2004; Wang et al., 2004a,b). Kinetics of alkaloiduptake to vacuoles has been described (Sato et al., 1993).

8. Conclusions and perspectives

In conclusion, quaternary protoberberine alkaloids rep-resent a very interesting and significant group of naturalproducts with a broad range of biological activities. Owingto the presence of the polarized iminium bond, positive

charge, and relatively planar skeleton, QPA interact witha range of molecular and biological targets, includingnucleic acids and proteins.

The isolation and purification procedures of QPA repre-sent a relatively well established field. The classical isola-tion sequences are frequently being replaced by modernchromatographic techniques at micro-preparative and pre-parative scales. Parallel to the isolation of new structuresfrom natural sources, modified berberine skeletons are con-structed using modern synthetic methods in order to findhighly biologically active substances.

Structural studies of complexes between protoberber-ine alkaloids and oligonucleotides have been publishedvery recently. These studies, based mainly on NMRspectroscopy, mass spectrometry, and molecular model-ing, allow the explanation of the biological activities ofthe QPA at the molecular level and represent a pow-erful tool for designing new and more specificsubstances.

Acknowledgements

The authors would like to thank Dr. Jaromır Marek andDr. Marek Necas (Masaryk University, Brno) for theirhelp with compiling the crystallographic data. The finan-cial support of the Ministry of Education of the CzechRepublic (MSM0021622413 and LC06030 to R.M.) andthe Grant Agency of the Czech Republic (525/04/0017 toL.G.) is gratefully acknowledged.

References

Abadi, B.E.A., Moss, D.S., Palmer, R.A., 1984. Molecular-structure of anancient folk medicine berberine hydrogen sulfate. J. Crystallogr.Spectrosc. Res. 14, 269–281.

Amann, M., Nagakura, N., Zenk, M.H., 1984. (S)-Tetrahydroprotoberb-erine oxidase, the final enzyme in protoberberine biosynthesis. Tetra-hedron Lett. 25, 953–954.

Amann, M., Nagakura, N., Zenk, M.H., 1988. Purification and propertiesof (S)-tetrahydroprotoberberine oxidase from suspension-culturedcells of Berberis wilsoniae. Eur. J. Biochem. 175, 17–25.

Bahadur, S., Shukla, A.K., 1983. Studies on native medicinal plants, I.The quaternary alkaloid of Thalictrum javanicum. J. Nat. Prod. 46,454–457.

Barreto, M.C., Pinto, R.E., Arrabaca, J.D., Pavao, M.L., 2003. Inhibitionof mouse liver respiration by Chelidonium majus isoquinoline alkaloids.Toxicol. Lett. 146, 37–47.

Bauer, W., Zenk, M.H., 1991. Formation of (R) – configurated tetrahy-droprotoberberine alkaloids in vivo and in vitro. Tetrahedron Lett. 32,487–490.

Beecher, C.W.W., Kelleher, W.J., 1984. Enzymatic study of the late stagesof protoberberine alkaloid biosynthesis. Tetrahedron Lett. 25, 4595–4598.

Bentley, K.W., 1997. b-Phenylethylamines and the isoquinoline alkaloids.Nat. Prod. Rep. 14, 387–411.

Bentley, K.W., 1998a. The Isoquinoline Alkaloids. Harwood AcademicPublishers, Amsterdam, pp. 219–263.

Bentley, K.W., 1998b. b-Phenylethylamines and the isoquinoline alka-loids. Nat. Prod. Rep. 15, 341–362.










Bhakuni, D.S., Jain, S., 1986. Protoberberine alkaloids. In: Manske,R.H.F. (Ed.), The Alkaloids, 28. Academic Press, New York, pp. 95–181.

Bhakuni, D.S., Gusta, S., Jain, S., 1983. The biosynthesis of the alkaloidsof Stephania glabra. Tetrahedron 39, 4003–4010.

Bjorklund, J.A., Frenzel, T., Rueffer, M., Kobayashi, M., Mocek, U.,Fox, C., Beale, J.M., Groger, S., Zenk, M.H., Floss, H.G., 1995.Cryptic stereochemistry of berberine alkaloid biosynthesis. J. Am.Chem. Soc. 117, 1533–1545.

Blasko, G., Murugesan, N., Freyer, A.J., Shamma, M., 1982. Karachine:an unusual protoberberine alkaloid. J. Am. Chem. Soc. 104, 2039–2041.

Blasko, G., Cordell, G.A., Bhamarapravati, S., Beecher, Ch.W.W., 1988.Carbon-13 NMR assignments of berberine and sanguinarine. Hetero-cycles, 911–916.

Bonora, A., Tosi, B., Dall’Olio, G., Bruni, A., 1990. Quaternary alkaloidsin rhizomes of Ranunculus serbicus. Phytochemistry 29, 2389–2390.

Bourdat-Deschamps, M., Herrenknecht, C., Akendengue, B., Laurens, A.,Hocquemiller, R., 2004. Separation of protoberberine quaternaryalkaloids from a crude extract of Enantia chlorantha bycentrifugal partition chromatography. J. Chromatogr. A 1041, 143–152.

Brezova, V., Dvoranova, D., Kost’alova, D., 2004. Oxygen activation byphotoexcited protoberberinium alkaloids from Mahonia aquifolium.Phytother. Res. 18, 640–646.

Cambridge Structure Database. 1999. Cambridge Crystallographic DataCenter (CCDC), 12 Union Road, Cambridge CB2 1EZ, UK.

Cava, M.P., Reed, T.A., 1965. An efficient separation of the commonalkaloids of the berberine group; the isolation and characterization ofcolumbamine. Lloydia 28, 73–83.

Cava, M.P., Reed, T.A., 1967. A synthesis of columbamine fromberberine. J. Org. Chem. 32, 1640–1641.

Cava, M.P., Nomura, K., Talapatra, S.K., Mitchell, M.J., Schlessinger,R.H., Buck, K.T., Beal, J.L., Douglas, B., Raffauf, R.F., Weisbach,J.A., 1968. The alkaloids of Stephania glabra. A direct chemicalcorrelation of the absolute configuration of some benzyltetrahydro-isoquinoline, proaporphine, and aporphine alkaloids. A new protob-erberine alkaloid. J. Org. Chem. 33, 2785–2789.

Cernakova, M., Kost’alova, D., Kettmann, V., Plodova, M., Toth, J.,Drımal, J., 2002. Potential antimutagenic activity of berberine, aconstituent of Mahonia aquifolium. BMC Complementary Alter. Med.2, 1–6.

Chattopadhyay, S.K., Ray, A.B., Slatkin, D.J., Schiff, P.L., 1983.Quaternary alkaloids of Thalictrum foliolosum. Phytochemistry 22,2607–2610.

Chen, W.H., Chan, C.L., Cai, Z., Luo, G.A., Jiang, Z.H., 2004. Studyon noncovalent complexes of cytotoxic protoberberine alkaloids withdouble-stranded DNA by using electrospray ionization mass spec-trometry. Bioorg. Med. Chem. Lett. 14, 4955–4959.

Chen, W.H., Pang, J.Y., Qin, Y., Peng, Q., Cai, Z., Jiang, Z.H., 2005a.Synthesis of linked berberine dimers and their remarkably enhancedDNA-binding affinities. Bioorg. Med. Chem. Lett. 15, 2689–2692.

Chen, W.H., Qin, Y., Cai, Z., Chan, C.L., Luo, G.A., Jiang, Z.H., 2005b.Spectrometric studies of cytotoxic protoberberine alkaloids binding todouble-stranded DNA. Bioorg. Med. Chem. 13, 1859–1866.

Chia, Y.C., Chang, F.E., Li, C.M., Wu, Y.C., 1998. Protoberberinealkaloids from Fissistigma balansae. Phytochemistry 48, 367–369.

Chuang, W.C., Young, D.S., Liu, L.K., Sheu, S.J., 1996. Liquidchromatographic-electrospray mass spectrometric analysis of Coptidis

rhizoma. J. Chromatogr., A 755, 19–26.Chudık, S., Marek, R., Seckarova, P., Necas, M., Dostal, J., Slavık, J.,

2006. Revision of the structure of escholidine. J. Nat. Prod. 69, 954–956.

Colombo, M.L., Bugatti, C., Mossa, A., Pescalli, N., Piazzoni, L.,Pezzoni, G., Menta, E., Spinelli, S., Johnson, F., Gupta, R.C.,Dasaradhi, L., 2001. Cytotoxicity evaluation of natural coptisineand synthesis of coptisine from berberine. Il Farmaco 56, 403–409.

Contreras, M.L., Rivas, S., Rozas, R., 1984. An approach to themechanism of photooxidation of berberine under basic conditions.Heterocycles 22, 101–106.

Cushman, M., Chinnasamy, P., Patrick, D.A., McKenzie, A.T., Toma,P.H., 1990. A novel ring expansion observed during the lithiumaluminum hydride reduction of 13-nitrooxyberberine. J. Org. Chem.55, 5995–6000.

Das, B., Srinivas, K.V.N.S., 2002. Conversion of berberine into berb-errubine by selective demethylation under microwawe irradiation.Synth. Commun. 32, 3027–3029.

Das, J., Kumar, G.S., Ray, A., Maiti, M., 2003. Spectroscopic andthermodynamic studies on the binding of sanguinarine and berberineto triple and double helical DNA and RNA structures. Biomol. Struct.Dyn. 20, 703–714.

Doskotch, R.W., Malik, M.O., Beal, J.L., 1967. The identification ofdehydrocorydalmine and a new protoberberine alkaloid, stepharanine,in Stephnnia glabra tubers. J. Org. Chem. 32, 3253–3254.

Dostal, J., Man, S., Seckarova, P., Hulova, D., Necas, M., Potacek, M.,Tousek, J., Dommisse, R., Van Dongen, W., Marek, R., 2004.Berberine and coptisine free bases. J. Mol. Struct. 687, 135–142.

Drost, K., Szaufer, M., Kowalewski, Z., 1974. Alkaloidy rodzaju Berberis

L. i ich dzalanie farmakologiczne. Herba Pol. 20, 301–312.Duah, F.K., Owusu, P.D., Slatkin, D.J., Schiff, P.L., 1983. Dehydrodis-

cretine, an infrequently occuring quaternary protoberberine alkaloidfrom Heptacyclum zenkeri. Phytochemistry 22, 321–322.

Elango, V., Shamma, M., 1983. A pyrolytic route to the phthalide-isoquinolines. J. Org. Chem. 48, 4879–4881.

Elford, B.C., 1986. L-Glutamine influx in malaria-infected erythrocytes: atarget for antimalarials? Parasitol. Today 2, 309–312.

Fajardo, V., Caramo, C., Moreno, B., 1996. Ilicifoline: new berberinedimer alkaloid from Berberis ilicifolia. Heterocycles 43, 949–951.

Felter, H.W., Lloyd, J.U., 1898. King’s American Dispensatory. OhioValley Co., Cincinnatti http://www.ibiblio.org/herbmed/eclectic/kings/hydrastis.html.

Frenzel, T., Beale, J.M., Kobayashi, M., Zenk, M.H., Floss, H.G., 1988.Stereochemistry of enzymatic formation of the berberine bridge inprotoberberine alkaloids. J. Am. Chem. Soc. 110, 7878–7880.

Frick, S., Kutchan, T.M., 1999. Molecular cloning and functionalexpression of O-methyltransferases common to isoquinoline alkaloidand phenylpropanoid biosynthesis. Plant J. 17, 329–339.

Gao, C.Y., Lou, Z.C., Lin, F.T., Lin, M.C., Schiff, P.L., 1987. Quaternaryalkaloids of Thalictrum cultratum. Phytochemistry 26, 3003–4004.

Gear, J.R., Spenser, I.D., 1963. The biosynthesis of hydrastine andberberine. Can. J. Chem. 41, 783–803.

Gharbo, S.A., Beal, J.L., Doskotch, W., Mitscher, L.A., 1973. Alkaloidsof Thalictrum. XIV. Isolation of alkaloids having antimicrobialactivity from Thalictrum polygamum. Lloydia 36, 349–351.

Ghosh, R., Mukherjee, K., Roychowdhury, P., Klayman, D.L., 1993.Structure of an antimalarial alkaloid, jatrorrhizine. Acta Crystallogr.,Sect. C. 49, 1665–1667.

Goldman, G.H., Yu, C., Wu, H.Y., Sanders, M.M., LaVoie, E.J., Liu,L.F., 1997. Differential poisoning of human and Aspergillus nidulans


http://www.ibiblio.org/herbmed/eclectic/kings/hydrastis.html

http://www.ibiblio.org/herbmed/eclectic/kings/hydrastis.html

DNA topoisomerase I by bi- and terbenzimidazoles. Biochemistry 36,6488–6494.

Habermehl, G., Schunk, J., Schaden, G., 1970. Thermisches und mass-enspektroskopiesches verhalten von berberin und anderen pseuudo-basen. Liebigs Ann. Chem. 742, 138–144.

Hanaoka, M., Marutani, M., Saitoh, K., Mukai, C., 1985. Transforma-tion of 2,3,9,10-tetraoxygenated protoberberine alkaloids into2,3,10,11-tetraoxygenated protoberbeine alkaloids. Heterocycles 23,2927–2930.

Hanaoka, M., Motonishi, T., Mukai, C., 1986. Chemical transformationof protoberberines. Part 9. A biomimetic synthesis of oxychelerythrine,dihydrochelerythrine and chelerythrine from berberine. J. Chem. Soc.,Perkin Trans. I, 2253–2256.

Hara, M., Tanaka, S., Tabata, M., 1994. Induction of a specificmethyltransferase activity regulating berberine biosynthesis by cytoki-nin in Thalictrum minus cell cultures. Phytochemistry 36, 327–332.

Henry, T.A., 1949. The berberis and related alkaloids. In: Henry, T.A.(Ed.), The Plant Alkaloids. J.A. Churchill Ltd., London, pp. 328–346.

Hirakawa, K., Kawanishi, S., Hirano, T., 2005. The mechanism ofguanine specific photooxidation in the presence of berberine andpalmatine: activation of photosensitized singlet oxygen generationthrough DNA-binding interaction. Chem. Res. Toxicol. 18, 1545–1552.

Hsieh, T.J., Chia, Y.C., Wu, Y.C., Chen, C.Y., 2004. Chemical constit-uents from the stems of Mahonia japonica. J. Chin. Chem. Soc. 51,443–446.

Ikuta, A., Itokawa, H., 1982. Berberine and other protoberberinealkaloids in callus tissue of Thalictrum minus. Phytochemistry 21,1419–1421.

Ivanovska, N., Philipov, S., 1996. Study on the anti-inflammatory actionof Berberis vulgaris root extract, alkaloid fractions and pure alkaloids.Immunopharmacology 18, 553–561.

Iwasa, K., Kamigauchi, M., 1996. 13-hydroxylation of tetrahydroberber-ine in cell suspension cultures of some Corydalis species. Phytochem-istry 41, 1511–1515.

Iwasa, K., Kim, C.W., 1997. Biotransformations of protoberberine in cellcultures of Dicentra spectabilis. Phytochemistry 46, 1359–1363.

Iwasa, K., Sugiura, M., Takao, N., 1982. Stereochemistry of 13-hydroxy-protoberberines, their derivatives, and a protopine-type alkaloid. J.Org. Chem. 47, 4275–4280.

Iwasa, K., Kamigauchi, M., Takao, N., Cushman, M., Chen, J., Wong,W.C., McKenzie, A., 1989. Synthesis and evaluation of hypotheticalintermediates in the biosynthetic conversion of protoberberine tobenzo[c]phenanthridine alkaloids. Evidence for oxidative C–N bondfission followed by intramolecular recyclization in cell cultures ofCorydalis incise. J. Am. Chem. Soc. 111, 7925–7931.

Iwasa, K., Kamigauchi, M., Sugiura, M., Nanba, H., 1997. Antimicrobialactivity of some 13-alkyl substituted protoberberinium salts. PlantaMed. 63, 196–198.

Iwasa, K., Hye-Sook, K., Wataya, Y., Dong-Ung, L., 1998a. Antimalarialactivity and structure–activity relationships of protoberberine alka-loids. Eur. J. Med. Chem. 33, 65–69.

Iwasa, K., Lee, D.U., Kang, S.I., Wiegrebe, W., 1998b. Antimicrobialactivity of 8-alkyl- and 8-phenyl-substituted berberines and their 12-bromo derivatives. J. Nat. Prod. 61, 1150–1153.

Iwasa, K., Nishiyama, Y., Ichimaru, M., Moriyasu, M., Kim, H.S.,Wataya, Y., Yamori, T., Takashi, T., Lee, D.U., 1999.Structure-activity relationships of quaternary protoberberine alkaloidshaving an antimalarial activity. Eur. J. Med. Chem. 34, 1077–1083.

Iwasa, K., Moriyasu, M., Nader, B., 2000. Fungicidal and herbicidalactivities of berberine related alkaloids. Biosci. Biotechnol. Biochem.64, 1998–2000.

Iwasa, K., Moriyasu, M., Yamori, T., Turuo, T., Lee, D.U., Wiegrebe,W., 2001. In Vitro cytotoxicity of the protoberberine-type alkaloids. J.Nat. Prod. 64, 896–898.

Jansen, R.H.A.M., Lousberg, J.J.C., Wijkens, P., Kruk, C., Theuns, H.G.,1989. Assigment of 1H and 13C NMR resonances of some isoquinolinealkaloids. Phytochemistry 28, 2833–2839.

Jeffs, P.W., 1967. The protoberberine alkaloids. In: Manske, R.H.F. (Ed.),The Alkaloids, vol. 9. Academic Press, New York, pp. 41–115.

Jeffs, P.W., Scharver, J.D., 1976. Biosynthesis of ophiocarpine. Introduc-tion of a stereospecific label through transannular cyclization. J. Am.Chem. Soc. 98, 4301–4310.

Jendrzejewski, S., 1990. Degradation of reticuline in berberis suspensioncultures. Phytochemistry 29, 135–139.

Kametani, T., Takemura, M., Ihara, M., Takahashi, K., Fukumoto, K.,1976. Biotransformation of 1-benzyl-1,2,3,4-tetrahydro-2-methyliso-quinolines into tetrahydroprotoberberines with rat liver enzymes. J.Am. Chem. Soc. 98, 1956–1959.

Kaneko, H., Naruto, S., 1969. Studies on the constituents of Corydalis sp.VI. Alkaloids from chinese corydalis and the identity of d-corydalminewith d-corybulbine. J. Org. Chem. 34, 2803–2805.

Kariuki, B.M., 1995. Five salts of berberine. Acta Crystallogr., Sect. C 51,1234–1240.

Kato, A., Moriyasu, M., Ichimaru, M., Nishiyama, Y., 1996. Isolation ofalkaloidal constituents of Zanthoxylum usambarense and Zanthoxylum

chalybeum using ion-pair HPLC. J. Nat. Prod. 59, 316–318.Khamidov, I.I., Tashkhodzhaev, B., Aripova, S.F., Telezhenetskaya,

M.V., Karimov, A.K., 1996. Berberis alkaloids. XXXVII. Investiga-tion of the alkaloids of B. oblongata and B. integerrima. Crystalstructure of 8-trichloromethyldihydroberberine. Khim. Prir. Soedin.(Chem. Nat. Compnd.), 889–893 (in Russian), 889–893.

Kim, S.A., Kwon, Y., Muller, M.T., Chung, I.K., 1998. Induction oftopoisomerase II-mediated DNA cleavage by a protoberberine alka-loid, berberrubine. Biochemistry 37, 16316–16324.

Kim, J.P., Jung, M.Y., Kim, J.P., Kim, S.Y., 2000. Antiphotooxidativeactivity of protoberberines derived from Coptis japonica Makino in thechlorophyll-sensitized photooxidation of oil. J. Agric. Food Chem. 48,1058–1063.

Kim, E., Yoo, S., Yi, K.Y., Lee, S., Noh, J., Jung, Y.S., Kom, E., Neony,N., 2002. Design, synthese and biological evaluations of nonpeptidiccaspase 3 inhibitors. Bull. Kor. Chem. Soc. 23, 1003–1010.

Kluza, J., Baldeyrou, B., Colson, P., Rasoanaivo, P., Mambu, L.,Frappier, F., Bailly, C., 2003. Cytotoxicity and DNA bindingproperties of the plant alkaloid burasaine. Eur. J. Pharm. Sci. 20,383–391.

Kobayashi, M., Frenzel, T., Lee, J.P., Zenk, M.H., Floss, H.G., 1987.Stereochemical fate of O-methyl groups in the biosynthesis ofprotoberberine alkaloids. J. Am. Chem. Soc. 109, 6184–6185.

Komorsky-Lovric, S., 2000. Adsorption and reduction of berberine at amercury electrode. Electroanalysis 12, 599–604.

Kost’alova, D., Brazdovicova, B., Jin, H.Y., 1982. Alkaloids from theabove-ground parts of Berberis coreana palib. Farmaceuticky Obzor,213–216.

Kumar, G.S., Das, S., Bhadra, K., Maiti, M., 2003. Protonated forms ofpoly[d(G-C)] and poly(dG).poly(dC) and their interaction withberberine. Bioorg. Med. Chem. 11, 4861–4870.

Kutchan, T.M., Ditrich, H., 1995. Characterization and mechanism of theberberine bridge enzyme, a covalently flavinylated oxidase of benz-ophenanthridine alkaloid biosynthesis in plants. J. Biol. Chem. 270,24475–24481.

Leng, S.H., Lu, F.E., Xu, L.J., 2004. Therapeutic effects of berberine inimpaired glucose tolerance rats and its influence on insulin secretion.Acta Pharmacol. Sin. 25, 496–502.

Letasiova, S., Jantova, S., Horvathova, M., Lakatos, B., 2005. Toxicityand apoptosis induced by berberine – a potential anticancer drug.Biologia 60, 97–100.

Li, M., Chen, X., Tang, Q.M., Zhang, J.S., 2001. Isoquinoline alkaloidsfrom Thalictrum delevayi. Planta Med. 67, 189–190.

Lloyd, J.U., 1898. Jateorhiza calumba. Western Druggist, XX., 8-11.http://www.swsbm.com/ManualsOther/Jateorhiza_Lloyd.pdf.

Lopez, J.A., Lin, M.C., Schiff, P.L., 1988. Alkaloids of Thalictrum

lankesteri. Phytochemistry 27, 3335–3336.Makhey, D., Yu, C., Liu, A., Liu, L.F., LaVoie, E.J., 2000. Substituted

benz[a]acridines and benz[c]acridines as mammalian topoisomerasepoisons. Bioorg. Med. Chem. 8, 1171–1182.


http://www.swsbm.com/ManualsOther/Jateorhiza_Lloyd.pdf

Man, S., Dostal, J., Necas, M., Zak, Z., Potacek, M., 2001a. Berberineand coptisine in liquid ammonia. Heterocyclic Commun. 7, 243–248.

Man, S., Potacek, M., Necas, M., Zak, Z., Dostal, J., 2001b. Molecularand crystal structures of three berberine derivatives. Molecules 6, 433–441.

Marek, R., Lycka, A., 2002. 15N NMR spectroscopy in structural analysis.Curr. Org. Chem. 6, 35–66.

Marek, R., Humpa, O., Dostal, J., Slavık, J., Sklenar, V., 1999. 15N NMRstudy of isoquinoline alkaloids. Magn. Reson. Chem. 37, 195–202.

Marek, R., Marek, J., Dostal, J., Taborska, E., Slavık, J., Dommisse, R.,2002. Isoquinoline alkaloids: a 15N NMR and X-ray study. Part 2.Magn. Reson. Chem. 40, 687–692.

Marek, J., Hulova, D., Dostal, J., Marek, R., 2003a. Berberine formate–succinic acid (1/1). Acta Crystallogr., Sect. C. 59, 583–585.

Marek, R., Seckarova, P., Hulova, D., Marek, J., Dostal, J., Sklenar, V.,2003b. Palmatine and berberine isolation artifacts. J. Nat. Prod. 66,481–486.

Marquez, B.L., Gerwick, W.H., Williamson, R.T., 2001. Survey of NMRexperiments for the determination of nJ(C,H) heteronuclear couplingconstants in small molecules. Magn. Reson. Chem. 39, 499–530.

Martin, G.E., Crouch, R.C., 1994. Two-dimensional NMR in naturalproducts and pharmaceutical chemistry. In: Croasmun, W.R., Carl-son, R.M.K. (Eds.), Two-Dimensional NMR Spectroscopy, Applica-tions for Chemists and Biochemists. VCH Publishers., New York, pp.873–914.

Martin, G.E., Hadden, C.E., 2000. Long-range 1H–15N heteronuclearshift correlation at natural abundance. J. Nat. Prod. 63, 543–585.

Martin, G.E., Williams, A.J., 2005. Long-range 1H–15N heteronuclearshift correlation. Annu. Rep. NMR Spectrosc. 55, 1–119.

Mazzini, S., Belluci, M.C., Mondelli, R., 2003. Mode of binding of thecytotoxic alkaloid berberine with the Double Helix oligonucleotideD(AAGAATTCTT)2. Bioorg. Med. Chem. 11, 505–514.

McCalla, D.L.C., Alexanderb, J., Barbera, J., Jaouharia, R.G., Satoskarb,A., Waigh, R.D., 1994. The first protoberberine alkaloid analouguewith in vivo antimalarial activity. Bioorg. Med. Chem. Lett. 4, 1663–1666.

Meise, W., 1971. 2,3-Dimethoxy-13,13 a dehydro-berbin: Herstellung undspektroskopische Charakterisierung. Liebigs Ann. Chem. 748, 55–58.

Meyerson, M., 2000. Role of telomerase in normal and cancer cells. J.Clin. Oncol. 18, 2626–2634.

Miana, G.A., 1973. Tertiary and dihydroprotoberberine alkaloids ofBerberis lyceum. Phytochemistry 12, 1822–1823.

Micco, S.D., Bassarello, C., Bifulco, G., Riccio, R., Gomez-Paloma, L.,2006. Differential-frequency saturation transfer difference NMR spec-troscopy allows the detection of different ligand–DNA binding modes.Angew. Chem. Int. Ed. 45, 224–228.

Miyazawa, M., Yoshio, K., Ishikawa, Y., Kameoka, H., 1998. Insecticidalalkaloids from Corydalis bulbosa against Drosophila melanogaster. J.Agric. Food Chem. 46, 1914–1919.

Mohrle, H., Biegholdt, M., 1982. Die Struktur des ‘‘Berberinoxims’’ nachGadamer. Arch. Pharm. 315, 919–925.

Moniot, J.L., Shamma, M., 1979. Conversion of berberine into phtha-lideisoquinolines. J. Org. Chem. 44, 4337–4342.

Morrison, H., 1990. Bioorganic Photochemistry: Photochemistry and theNucleic Acids, vol. 1. John Wiley & Sons, New York.

Moulis, C., Gleye, J., Stanislas, E., 1977. Alcaloıdes de l’ Isopyrum

thalictroıdes—bases quaternaires des feuilles—isolement et identifica-tion de quatre pseudoprotoberberines. Phytochemistry 16, 1283–1287.

Murugesan, N., Shamma, M., 1980. The synthesis of rugosinone andledecorine. Heterocycles 14, 585–587.

Naasani, I., Seimiya, H., Yamori, T., Tsuruo, T., 1999. FJ002: A potenttelomerase inhibitor identified by exploiting the disease-orientedscreening program with COMPARE analysis. Cancer Res. 59, 4004–4011.

Naruto, S., Mizuta, H., Nishimura, H., 1976. A novel amination ofisoquinolinium salts via nucleophilic substitution reaction. Tetrahe-dron Lett. 19, 1597–1600.

Neuhaus, D., Williamson, M., 1989. The Nuclear Overhauser Effect inStructural and Conformational Analysis. VCH Publishers, New York.

Nishiysma, Y., Moriyasu, M., Ichimaru, M., Iwasa, K., Kato, A.,Mathenge, S.G., Mutiso, P.B.C., Juma, F.D., 2004. Quaternaryisoquinoline alkaloids from Xylopia parviflora. Phytochemistry 65,939–944.

Orfila, L., Rodrıguez, M., Colman, T., Hasegawa, M., Merentes, E.,Arvelo, F., 2000. Structural modification of berberine alkaloids inrelation to cytotoxic activity in vitro. J. Ethnopharmacol. 71, 449–456.

Pan, J.F., Yu, C., Zhu, D.Y., Zhang, H., Zeng, J.F., Jiang, S.H., Ren,J.Y., 2002. Identification of three sulfate-conjugated metabolites ofberberine chloride in healthy volunteers’ urine after oral administra-tion. Acta Pharmacol. Sin. 1, 77–82.

Pang, J.Y., Qin, Y., Chen, W.H., Luo, G.A., Jiang, Z.H., 2005. Synthesisand DNA-binding affinities of monomodified berberines. Bioorg. Med.Chem. 13, 5835–5840.

Park, H.S., Kim, E.H., Kang, M.R., Chung, I.K., Cheong, C., Lee, W.,2004. Spectroscopic studies on interaction of protoberberines with thedeoxyoligonucleotide d(GCCGTCGTTTTACA)2. Bull. Kor. Chem.Soc. 25, 1559–1563.

Patra, A., Montgomery, C.T., Freyer, A.J., Guinaudeau, H., Shamma,M., Taivtbewie, B., Pharadai, K., 1987. The protoberberine alkaloidsof Stephania suberosa. Phytochemistry 26, 547–549.

Pavelka, S., Smekal, E., 1976. The fluorescence properties of protoberb-erine and tetrahydroprotoberberine alkaloids. Collect. Czech. Chem.Commun. 41, 3157–3169.

Pervushkin, S.V., Sokhina, A.A., Kurkin, V.A., Zapesochnaya, G.G.,Astrakhanova, M.M., Alekseev, K.V., Ivanova, I.P., 1999. Quantita-itve determination of alkaloids sum content in medicinal preparation‘‘tincture chelidonii’’. Rastit. Resursy 35, 123–128.

Peteu, P., 1965. Phytochemische Untersuchungen an Berberis hauniensis

Zab. Arch. Pharm. 298, 73–77.Pothier, J., Galand, N., Tivollier, P., Viel, C., 1993. Separation of

quaternary alkaloids in plant extracts by overpressured layer chroma-tography. J. Planar Chromatogr. 6, 220–222.

Preininger, V., 1986. Chemotaxonomy of Papaveraceae and Fumariaceae.In: Manske, R.H.F. (Ed.), The Alkaloids, vol. 29. Academic Press,New York, pp. 1–95.

Preininger, V., Hruban, L., Simanek, V., Santavy, F., 1970. Isolation andchemistry of alkaloids from the plants of the genus Papaver XLVI. Thestructure of the alkaloids PO-4, PO-5 and of the dehydrogenated a-coralydine. Collect. Czech. Chem. Commun. 35, 124–135.

Qin, Y., Pang, J.Y., Chen, W.H., Cai, Z., Jiang, Z.H., 2006. Synthesis,DNA-binding affinities, and binding mode of berberine dimers.Bioorg. Med. Chem. 14, 25–32.

Rasoanaivo, P., Ratsimamanga-Urverg, S., Rakoto-Ratsimamanga, A.,Raharisololalao, A., 1991. Constituants chimiques de trois especes deBurasaia (Menispermacees) endemiques de Madagascar. Biochem.Syst. Ecol. 19, 433–437.

Reynolds, W.F., Enriquez, R.G., 2002. Choosing the best pulse sequences,acquisition parameters, postacquisition processing strategies, andprobes for natural product structure elucidation by NMR spectros-copy. J. Nat. Prod. 65, 221–244.

Ro, J.S., Lee, S.S., Lee, K.S., Lee, M.K., 2001. Inhibition of type Amonoamine oxidase by coptisine in mouse brain. Life Sci. 70, 639–645.

Roberts, M.F., Wink, M., 1998. Biochemistry, ecology, and medicinalapplications. In: Roberts, M.F., Wink, M. (Eds.), Alkaloids. PlenumPress, New York, London, pp. 1–7.

Rueffer, M., Zenk, M.H., 1986. Columbamine, the central intermediate inthe late stages of protoberberine biosynthesis. Tetrahedron Lett. 27,923–924.

Rueffer, M., Zenk, M.H., 1994. Canadine synthase from Thalictrum

tuberosum cell cultures catalyses the formation of the methylenedioxybridge in berberine synthesis. Phytochemistry 36, 1219–1223.

Rueffer, M., Ekundayo, O., Nagakura, N., Zenk, M.H., 1983. Biosyn-thesis of the protoberberine alkaloids jatrorrhizine. Tetrahedron Lett.24, 2643–2644.


Sanders, M.M., Liu, A.A., Li, T.K., Wu, H.Y., Desai, S.D., Mao, Y.,Rubin, E.H., LaVoie, E.J., Makhey, D., Liu, L.F., 1998. Selectivecytotoxicity of topoisomerase-directed protoberberines against Glio-

blastoma cells. Biochem. Pharmacol. 56, 1157–1166.Santavy, F., 1979. Berbine (dibenzo[a,g]quinolizidine) (protoberberine,

pseudoprotoberberine, corydaline and corytenchirine types) group. In:Manske, R.H.F. (Ed.), The Alkaloids, vol. 17. Academic Press, NewYork, pp. 439–461.

Sato, H., Tanaka, S., Tabata, M., 1993. Kinetics of alkaloid uptake bycultured cells of Coptis japonica. Phytochemistry 34, 693–701.

Schmeller, T., Latz-Bruning, B., Wink, M., 1997. Biochemical activitiesof berberine, palmatine and sanguinarine mediating chemicaldefence against microorganisms and herbovires. Phytochemistry44, 257–266.

Schneider, B., Zenk, M.H., 1993a. Usual methyl transfer in the biosyn-thesis of aporphine and protoberberine alkaloids. Phytochemistry 32,897–903.

Schneider, B., Zenk, M.H., 1993b. Metabolism of (S)-reticuline in analkaloid non-producing cell culture strain of Thalictrum tuberosum.Phytochemistry 33, 1431–1435.

Sethi, M.L., 1985. Comparison of inhibition of reverse transcriptase andantileucemic activities exhibited by protoberberine and benzophenan-thridine alkaloids and structure–activity relationships. Phytochemistry24, 447–454.

Shamma, M., 1972. The protoberberines and retroprotoberberines. In:Shamma, M. (Ed.), The Isoquinoline Alkaloids. Academic Press, NewYork, London, pp. 268–314.

Shamma, M., Dudock, S.S., 1965. Thalictrum alkaloids IX. Thalidastine.Tetrahedron Lett. 43, 3825–3828.

Shamma, M., Rahimizadech, M., 1986. The identity of chileninone withberberrubine. The problem of true natural products vs. artifacts ofisolation. J. Nat. Prod. 49, 398–405.

Shamma, M., Hillman, M.J., Jones, C.D., 1969. The UV spectra ofprotoberberines. Chem. Rev. 69, 779–784.

Shamma, M., Moniot, J.L., Chinnasamy, P., 1977. Thalipine, a newaporphine-benzylisoquinoline alkaloid. Heterocycles 6, 399–403.

Siddikov, D., Zhalolov, I.Z., Tashkhodzhaev, B., Turgunov, K.K.,Aripova, S.F., Khuzhaev, V.U., 2005. Alkaloids from Papaver

angrenicum. II. Crystal and molecular structure of the new diisoquin-oline alkaloid pangrenine. Chem. Nat. Comp. 41, 442–445.

Simanek, V., Preininger, V., Sedmera, P., Santavy, F., 1970. Isolation andchemistry of alkaloids from the plants of the genus Papaver XLVIII.Studies in exhaustive methylation of tetrahydroprotoberberine alka-loids and the structure of the alkaloids PO-4, PO-5, orientalidine andmecambridine. Collect. Czech. Chem. Commun. 35, 1440–1455.

Simanek, V., Preininger, V., Lasovsky, J., 1976. The pseudobase forma-tion in some isoquinoline alkaloids. Collect. Czech. Chem. Commun.41, 1050–1055.

Singh, S.P., Stenberg, V.I., Parmar, S.S., 1980. Photochemistry ofalkaloids. Chem. Rev. 80, 269–282.

Siwon, J., Verpoorte, R., Svendsen, A.B., 1981. Studies on Indonesianmedicinal plants VI. Further alkaloids from Fibraurea chloroleuca.Planta Med. 41, 65–68.

Slaninova, I., Taborska, E., Bochorakova, H., Slanina, J., 2001. Interac-tion of benzo[c]phenanthridine and protoberberine alkaloids withanimal and yeast cells. Cell Biol. Toxicol. 17, 51–63.

Slavık, J., Slavıkova, L., 1989. Alkaloids from Corydalis nobilis and C.

intermedia.: Collect. Czech. Chem. Commun. 54, 2009–2020.Slavık, J., Slavıkova, L., 1995. Quaternary isoquinoline alkaloids and

some diterpenoid alkaloids in plant of the Czech Republic. Collect.Czech. Chem. Commun. 60, 1034–1041.

Slavıkova, L., Slavık, J., 1966. Alkaloide der Mohngewachse. Uber dieAlkaloide von drei Arten der Gattung Eschscholtzia und uber dieKonstitution des neuen Alkaloids Escholamin. Collect. Czech. Chem.Commun. 31, 3362–3371.

Slobodnıkova, L., Kost’alova, D., Labudova, D., Kotulova, D., Kett-mann, V., 2004. Antimicrobial activity of Mahonia aquifolium crudeextract and its major isolated alkaloids. Phytother. Res. 18, 674–676.

Sriwilaijareon, N., Petmitr, S., Mutirangura, A., Ponglikitmogkol, M.,Wilairat, P., 2002. Stage specificity of Plasmodium falciparum

telomerase and its inhibition by berberine. Parasitol. Int. 51, 99–103.

Suau, R., Silva, M.V., Valpuesta, M., 1991. A novel oxidation stage in thechemistry of protoberberine alkaloids. Synthesis of 7,8-dehydrober-bines. Tetrahedron 30, 5841–5846.

Suau, R., Silva, M.V., Valpuesta, M., 1993. Quaternary protoberberinealkaloids from Ceratocapnos heterocarpa. Phytochemistry 34, 559–561.

Suau, R., Rico, R., Lopez-Romero, J.M., Najera, F., Cuevas, A., 1998.Isoquinoline alkaloids from Berberis vulgaris subsp. Australis. Phyto-chemistry 49, 2545–2549.

Suau, R., Najera, F., Rico, R., 1999a. Hydroxymethylation of protob-erberine alkaloids by photoinduced SET. The total synthesis of (+)-solidaline. Tetrahedron 55, 4019–4028.

Suau, R., Najera, F., Rico, R., 1999b. Photochemical hydroxymethylationof protoberberine alkaloids. Total synthesis of (±)-solidaline. Tetra-hedron Lett. 37, 3575–3578.

Svendsen, A.B., Van Kempen-Verleun, A.M., Verpoorte, R., 1984.Circular thin-layer chromatography of quaternary alkaloids. Quater-nary prototberberine alkaaloids in plant material. J. Chromatogr. 291,389–391.

Taborska, E., Bochorakova, H., Paulova, H., Dostal, J., 1994. Separationof alkaloids in Chelidonium majus by reversed phase HPLC. PlantaMed. 60, 380–381.

Tane, P., Wabo, H.K., Ayafor, J.F., Sterner, O., 1997. Peniankerine, an18-norclerodane diterpenoid from stem bark of Penianthus zenkeri.Phytochemistry 46, 165–167.

Tome, F., Colombo, M.L., 1995. Distribution of alkaloids in Chelidonium

majus and factors affecting their accumulation. Phytochemistry 40, 37–39.

Tsai, P.L., Tsai, T.H., 2004. Hepatobiliary excretion of berberine. DrugMetab. Dispos. 32, 405–412.

Valencia, E., Weiss, I., Firdous, S., Treter, A.J., Shamma, M., 1984. Theisoindolobenzazepine alkaloids. Tetrahedron 40, 3957–3962.

Vennerstrom, J.L., Klayman, D.L., 1988. Protoberberine alkaloids asantimalarials. J. Med. Chem. 31, 1084–1087.

Vlckova, M., Kuban, V., Vicar, J., Simanek, V., 2005. Capillary zoneelectrophoretic studies of interactions of some quaternary isoquinolinealkaloids with DNA constituents and DNA. Electrophoresis 26, 1673–1679.

Vollekova, A., Kost’alova, D., Kettmann, V., Toth, J., 2003. Antifungalsctivity of Mahonia aquifolium extract and its major protoberberinealkaloids. Phytother. Res. 17, 834–837.

Wafo, P., Nyasse, B., Fontaine, C., 1999. A 7,8-dihydro-8-hydroxypalm-atine from Enantia chlorantha. Phytochemistry 50, 279–281.

Wang, L.K., Rogers, B.D., Hecht, S.M., 1996. Inhibition of topoisomer-ase I function by coralyne and 5,6-dihydrocoralyne. Chem. Res.Toxicol. 9, 75–83.

Wang, D., Liu, Z., Guo, M., Liu, S., 2004a. Structural elucidation andidentification of alkaloids in Rhizoma Coptidis by electrosprayionization tandem mass spectrometry. J. Mass Spectrom. 39, 1356–1365.

Wang, F., Zhou, H.Y., Zhao, G., Fu, L.Y., Cheng, L., Chen, J.G., Yao,W.X., 2004b. Inhibitory effects of berberine on ion channels of rathepatocytes. World J. Gastroenterol. 10, 2842–2845.

Weber, H.A., Zart, M.K., Hodges, A.E., Molloy, H.M., O’Brien, B.M.,Moody, L.A., Clark, A.P., Harris, R.K., Overstreet, J.D., Smith, C.S.,2003. Chemical comparison of goldenseal (Hydrastis canadensis L.)root powder from three commercial suppliers. J. Agric. Food Chem.51, 7352–7358.

Weimar, C., Angerer, S., Wiegrebe, W., 1991. Methoxy- and acetoxy-8-oxoberbines – synthesis, antitumor activity, and interaction withDNA. Arch. Pharm. 324, 509–518.

Wright, C.W., Marshall, S.J., Russell, P.F., Anderson, M.M., Phillipson,J.D., Kirby, G.C., Warhurst, D.C., Schiff, P.L., 2000. In vitro

antiplasmodial, antiamoebic, and cytotoxic activities of some mono-meric isoquinoline alkaloids. J. Nat. Prod. 63, 1638–1640.


Yang, H.T., Wang, G.J., 2003. Transport and uptake characteristics of anew derivative of berberine (CPU-86017) by human intestinal epithe-lial cell line: Caco-2. Acta Pharmacol. Sin. 24, 1185–1191.

Yang, F., Zhang, T., Zhang, R., Yoichiro, I., 1998. Application ofanalytical and preparative high-speed counter-current chromatogra-phy for separation of alkaloids from Coptis chinensis Franch. J.Chromatogr. A 829, 137–141.

Lenka Grycova graduated from theMasaryk University, Brno, CzechRepublic, in 2003. Her M.Sc. was focusedon the organic photochemistry. Duringher studies, she spent several monthsworking in the phytochemistry laboratoryof Professor Luc Pieters at the Univer-sity of Antwerp, Belgium, under theguidance of Slavka Baronikova, Ph.D.She is currently completing her Ph.D.research on structural investigations ofquaternary protoberberine and benzophe-nanthridine alkaloids, under the supervi-sion of associate professor Radek Marek.

Jirı Dostal obtained his M.Sc. (1982) andPh.D. (1993) in organic chemistry atMasaryk University, Brno, CzechRepublic. He teaches medical chemistryand biochemistry at the Faculty of Medi-cine, Masaryk University, Brno, as anassociate professor. His primary researchinterest is the chemistry of isoquinolinealkaloids, especially quaternary benz-ophenanthridines, protoberberines, andrelated natural products.

Radek Marek is an associate professor atthe Masaryk University, Brno, CzechRepublic. He graduated from theMasaryk University in 1991 and obtainedhis Ph.D. in organic chemistry in 1995.After graduating he joined NMR group ofProfessor Vladimır Sklenar at theNational Center for BiomolecularResearch, Masaryk University, Brno, as aresearch associate. He received the Prix de

Chimie (J.-M. Lehn Prize) in 1996 and theAlfred Bader Prize in bioorganic chemis-try in 2002. In 1993, 1999, and 2002 hespent several months as a research fellow,

working with Professor Roger Dommisse at the University of Antwerp,Belgium. In 2004, he received his Habilitation for work on NMR spec-troscopy of nitrogen heterocycles. His main research areas are the struc-ture of natural compounds, NMR spectroscopy, and intermolecularinteractions. His research group is involved in the study of isoquinolinealkaloids and purine derivatives.


Appendix B

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27

Research ArticleReceived: 2 June 2008 Revised: 4 August 2008 Accepted: 5 August 2008 Published online in Wiley Interscience: 10 September 2008

(www.interscience.com) DOI 10.1002/mrc.2325

Covalent bonding of azoles to quaternaryprotoberberine alkaloidsLenka Grycova,a Dagmar Hulova,a Lukas Maier,a Stanislav Standara,a

Marek Necas,b Filip Lemiere,c Radovan Kares,b Jirí Dostald andRadek Mareka∗

Adducts of the quaternary protoberberine alkaloids (QPA) berberine, palmatine, and coptisine were prepared with nucleophilesderived from pyrrole, pyrazole, imidazole, and 1,2,4-triazole. The products, 8-substituted 7,8-dihydroprotoberberines, wereidentified by mass spectrometry and 1D and 2D NMR spectroscopy, including 1H– 15N shift correlations at natural abundance.In addition, two adducts of QPA with chloroform and methanethiolate were characterized by using NMR data. Single-crystalX-ray structures of 8-pyrrolyl-7,8-dihydroberberine, 8-pyrazolyl-7,8-dihydroberberine, and 8-imidazolyl-7,8-dihydroberberineare also presented. Copyright c© 2008 John Wiley & Sons, Ltd.

Supporting information may be found in the online version of this article.

Keywords: NMR; 1H; 13C; 15N; berberine; palmatine; coptisine; protoberberine alkaloid; nucleophilic addition; X-ray diffraction

Introduction

Protoberberines are widely distributed isoquinoline alkaloids,and berberine is probably the most widely distributed alkaloidof all.[1,2] Quaternary protoberberine alkaloids (QPA) are typicalfor many species of the families Berberidaceae, Fumariaceae,Menispermaceae, Papaveraceae, and others.[2,3] The salts ofQPA have typical colors, mostly yellow. In plants, the positivecharge of the quaternary protoberberines is balanced by anumber of different physiological anions.[4] Berberine and somerelated compounds exhibit considerable biological activities,such as antimicrobial,[5,6] antifungal,[7,8] anti-inflammatory,[9]

antimalarial,[5,10] and cytotoxic.[11 – 13]

The interactions of QPA with nucleic acids and proteins areimportant as they relate to the biological activities of thesealkaloids. The complexation of berberine with the double helixhas been discussed in a large number of studies[14] which reportedan intercalative binding mode. However, recent studies haveindicated that the mechanism of interaction could be different.NMR (1H, 31P, NOE) and fluorescence studies have shown berberineto be localized in a minor groove of the adenine-thymine (AT)-richsequences.[15] Generally, it is of great importance to investigatethe capacity of a QPA to form covalent bonds with nucleic acidbases, nucleic acids (cytotoxicity, mutagenic effects),[13,16] or thefunctional groups of proteins.

The basic skeleton of QPA is 5,6-dihydrodibenzo[a,g]quinolizinium. Berberine (1), palmatine (2), and coptisine (3) arerepresentatives of 2,3,9,10-tetrasubstituted QPA.[3] The reactivityof a QPA is characterized by the sensitivity of the iminium bondC N+ to nucleophilic attack followed by the formation of an8-substituted 7,8-dihydroprotoberberine.[1]

Several 8-substituted adducts have been prepared, e.g. withliquid ammonia,[17] chloroform,[18 – 20] cyanide,[21] sodium methox-ide, and sodium ethoxide,[20,22] and characterized by NMR spec-troscopy and X-ray diffraction. However, we report here the first

study of the nucleophilic addition of some azoles to the protober-berine skeleton.

∗ Correspondence to: Radek Marek, National Center for Biomolecular Research,Faculty of Science, Masaryk University, Kamenice 5/A4, CZ - 625 00 Brno, CzechRepublic. E-mail: [email protected]

a National Center for Biomolecular Research, Faculty of Science, MasarykUniversity, Kamenice 5/A4, CZ-625 00 Brno, Czech Republic

b Department of Chemistry, Faculty of Science, Masaryk University, Kamenice5/A12, CZ-625 00 Brno, Czech Republic

c Department of Chemistry, University of Antwerp, Groenenborgerlaan 171,B-2020, Antwerp, Belgium

d Department of Biochemistry, Faculty of Medicine, Masaryk University, Kamenice5/A16, CZ-625 00 Brno, Czech Republic

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Results and Discussion

The reaction between a QPA and the sodium salt of an azoleproduced an 8-substituted 7,8-dihydroprotoberberine (Scheme 1)in moderate yield. The sodium salt (Na+Nu−) was prepared by

reacting the corresponding nitrogen base with NaH under an

argon atmosphere.

Mass spectrometry (MS), NMR spectroscopy, and X-ray diffrac-

tion analysis were used to characterize the structures of the

products. The peaks m/z corresponding to the molecular ions were

Scheme 1. The nucleophilic addition to the protoberberine skeleton.

Table 1. 1H NMR chemical shifts (δ in ppm) for berberine chloride (1, X = Cl), palmatine chloride (2, X = Cl), coptisine chloride (3, X = Cl), and8-substituted 7,8-dihydroprotoberberines (4a–6f) at 303 K

Compound 1 4a 4b 4c 4d a 4e 2 5a 5b 5cc 5d 3 6a 6b 6f 6f

Solvent d D C C C C B D C C C C D C C C CH

H-1 7.79 7.17 7.15 7.17 7.11 7.12 7.73 7.20 7.17 7.15 7.25 7.78 7.18 7.15 7.15 7.15

H-4 7.08 6.56 6.57 6.57 6.58 6.35 7.09 6.60 6.60 6.60 6.62 7.08 5.95 6.57 6.62 6.60

H-5 3.21 2.66 2.64 2.70 2.67 2.27 3.23 2.70 2.66 2.73 2.84 3.20 2.68 2.63 2.69 2.71

– 2.89 2.86 2.92 2.86 2.51 – 2.94 2.90 2.96 2.84 – 2.89 2.86 3.33 3.34

H-6 4.95 3.05 3.22 3.09 3.15 2.75 4.96 3.08 3.22 3.06 3.53 4.89 3.04 3.17 3.67 3.83

– 3.43 3.59 3.44 3.52 3.51 – 3.46 3.60 3.45 3.53 – 3.39 3.54 3.78 3.70

H-8 9.91 6.68 7.08 6.84 7.13 6.34 9.89 6.71 7.09 6.86 7.17 9.95 6.58 6.92 5.41 5.40

H-11 8.09 6.95b 6.99b 6.97b 7.00b 6.62 8.20 6.98 7.00b 7.00 7.05b 8.03 6.81 6.85 6.85 6.85

H-12 7.91 6.95b 6.99b 6.97b 7.00b 6.74 8.05 6.97 7.00b 6.97 7.05b 7.83 6.72 6.76 6.67 6.67

H-13 8.98 6.11 6.14 6.16 6.19 5.92 9.07 6.17 6.19 6.23 6.31 8.97 6.15 6.18 6.15 6.14

2,3-OCH2O/2-OCH3 6.17 5.93 5.93 5.93 5.95 5.34 3.94 3.84 3.88 3.88 3.88 6.17 5.92 5.94 5.94 5.94

3-OCH3 – – – – – – 3.87 3.90 3.85 3.82 3.86 – 5.92 5.94 5.95 5.95

9,10-OCH2O/9-OCH3 4.10 3.48 3.50 3.57 3.55 4.05 4.11 3.50 3.50 3.53 3.64 6.53 6.17 5.92 5.90 5.90

10-OCH3 4.07 3.83 3.85 3.82 3.82 3.40 4.07 3.82 3.81 3.81 3.82 – 6.17 5.96 6.01 6.02

H-2′ – 6.65 – 7.48 – – – 6.67 – 7.51 – – 6.68 – – –

H-3′ – 5.95 7.39 – 7.69 – – 5.97 7.39 – 7.84 – 5.98 7.39 – –

H-4′ – 5.95 6.07 6.82 – – – 5.97 6.08 6.82 – – 5.98 6.12 – –

H-5′ – 6.65 7.07 6.86 7.78 – – 6.67 7.09 6.87 7.84 – 6.68 7.16 – –

8-SCH3 – – – – – 2.04 – – – – – – – – – –

a Measured at 233 K.b Overlapped signals.c Measured at 263 K.d B = C6D6; C = CD2Cl2; D = DMSO-d6. CH = CDCl3.

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Bonding of azoles to QPA

found in the Electron impact MS (EI-MS) spectra with direct inser-tion. However, the intensity of these peaks was very low in somecases (<0.2%) and the spectra were dominated by the fragmentscorresponding to the quaternary ions and heterocyclic bases. Thisobservation is in accordance with the fragmentation patterns ob-tained for other 8-substituted 7,8-dihydroprotoberberines.[20,23]

Due to the lability of the C8–N1′ bond and the extraordinarystability of the quaternary form, quaternary ions and heterocyclicbases were formed spontaneously during the ionization process.The EI–MS data for individual compounds are summarized in theExperimental section.

In an effort to obtain accurate masses for compounds 4–6and thus confirm their elemental compositions, electrosprayionization (ESI) mass spectra were recorded. Although all of themeasurements were carefully optimized to avoid degradation ofthe samples (vide infra), only for 5a could be the protonatedmolecule (m/z 419.1971) detected in the mass spectrum. Noprotonated molecules were detected for any other compound,and the spectra were dominated by the quaternary cations 1 and2 at m/z 336 (4a–4d) and m/z 352 (5a–5d), respectively. Since it

was expected that these ions would arise from the elimination ofthe five-member heterocyclic rings, all spectrometer parametersthat could induce this elimination were adjusted; the cone voltagewas reduced from 25 to 5 V, and the collision cell energy wasreduced from 10 to 2 V. Unfortunately, neither of these measuresallowed for detecting the protonated molecule in the acquiredspectra. The compounds are apparently so susceptible to theelimination of the heterocycle that even the minimal energydelivered to the molecule to bring about protonation in theESI source and the transfer through the mass spectrometer isenough to fragment the molecule. However, the structures of thesecompounds were unequivocally confirmed by NMR spectroscopyand X-ray diffraction, as discussed in the following paragraphs.

The 1H and 13C NMR chemical shifts of compounds 4a–6f andQPA 1–3 are summarized in Tables 1 and 2. Although 1H and13C NMR data for several QPA have already been published,[24,25]

many contradictory conclusions and discussions can be found inthe literature. For instance, a recent publication[26] inexplicablycompares NMR data obtained for quaternary berberine in sev-eral different solvents with published data for a berberine base

Table 2. 13C NMR chemical shifts (δ in ppm) for berberine chloride (1, X = Cl), palmatine chloride (2, X = Cl), coptisine chloride (3, X = Cl), and8-substituted 7,8-dihydroprotoberberines (4a–6f) at 303 K

Compound 1 4a 4b 4c 4da 4e 2 5a 5b 5cb 5d 3 6ac 6b 6f 6f

Solvent e D C C C C B D C C C C D C C C CH

C-1 105.37 104.63 104.81 104.62 104.18 104.98 108.83 108.19 108.20 107.10 108.24 105.27 104.79 104.80 104.08 104.02

C-2 147.50 147.28 147.45 147.35 146.70 147.85 148.67 148.73 148.72 148.16 148.86 147.65 147.51 147.26 147.20 146.80

C-3 149.72 148.02 148.25 148.18 147.63 147.26 150.14 150.11 150.15 149.55 150.39 149.74 148.26 148.47 147.99 147.54

C-4 108.32 108.05 108.35 108.12 107.90 108.03 111.24 111.43 111.44 110.63 111.41 108.37 108.30 108.37 108.49 108.16

C-4a 130.57 129.53 129.97 129.17 129.03 129.33 128.48 128.23 128.45 127.55 128.40 130.51 129.67 129.94 129.25 128.69

C-5 26.26 30.25 30.48 30.14 29.71 29.93 25.93 29.92 29.93 29.49 29.83 26.23 30.38 30.43 30.76 30.52

C-6 55.10 46.74 47.51 46.82 47.00 47.06 55.28 47.03 47.59 46.73 48.35 55.05 46.86 47.40 52.31 51.93

C-8 145.37 70.14 72.71 68.86 70.96 67.85 145.31 70.43 72.82 68.74 68.35 144.50 70.11 72.51 74.18 73.94

C-8a 121.32 121.20 120.08 119.89 117.73 121.93 121.26 121.33 120.03 119.70 118.57 111.61 109.88 106.37 105.17 104.85

C-9 143.62 145.49 145.88 145.27 144.58 144.55 143.58 145.72 145.91 144.86 145.64 143.79 143.94 144.23 145.32 144.84

C-10 150.29 150.51 150.75 150.51 150.16 150.83 151.42 150.61 150.65 150.31 150.70 147.02 145.93 146.03 146.05 145.61

C-11 126.70 114.31 114.94 114.70 113.88 113.56 126.67 114.52 114.96 114.17 115.94 120.92 109.71 110.12 109.37 109.63

C-12 123.43 119.89 120.19 120.04 119.99 118.71 123.39 119.92 120.03 119.90 120.25 121.01 117.46 117.61 116.96 116.73

C-12a 132.94 127.73 128.26 127.35 126.77 129.00 133.09 128.05 128.37 127.05 128.26 132.31 128.40 128.81 130.50 130.07

C-13 120.14 95.50 95.81 95.95 95.94 97.45 119.94 95.18 95.30 95.38 97.13 121.71 96.14 96.33 98.31 98.08

C-13a 137.38 137.64 137.49 137.41 136.34 138.86 137.58 137.82 137.47 137.21 137.31 136.79 137.66 137.34 137.76 137.40

C-13b 120.36 125.48 125.54 125.13 124.51 125.77 118.87 124.18 124.05 123.30 123.59 120.45 125.57 125.50 125.23 125.06

2,3-OCH2O/2-OCH3

101.99 101.70 101.90 101.77 101.64 101.00 56.19 56.57 56.63 56.15 56.64 102.03 101.22 101.94 101.70 101.08

3-OCH3 – – – – – – 55.80 56.62 56.64 56.15 56.73 – – – – –

9,10-OCH2O/9-OCH3

61.86 60.44 60.79 60.59 60.55 60.23 61.86 60.63 60.79 60.56 61.10 104.43 101.91 102.38 101.47 100.86

10-OCH3 57.02 56.36 56.65 56.38 55.84 55.82 57.01 56.41 56.41 56.08 56.43 – – – – –

C-2′ – 119.61 – 135.92 – – – 119.81 – 135.86 – – 119.48 – – –

C-3′ – 108.05 138.61 – 142.58 – – 108.27 138.63 – 142.69d – 108.62 138.82 – –

C-4′ – 108.05 106.17 129.36 – – – 108.27 106.12 128.98 – – 108.62 106.61 – –

C-5′ – 119.61 128.52 117.79 150.82 – – 119.81 128.53 117.85 150.81d – 119.48 128.28 – –

8-SCH3/8-CCl3

– – – – – 13.66 – – – – – – – – 106.09 105.66

a Measured at 233 K.b Measured at 263 K.c Measured at 298 K.d Not obtained at 303 K. Values extracted from the 2D spectra measured at 203 K.e B = C6D6; C = CD2Cl2; D = DMSO-d6. CH = CDCl3.

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(8-hydroxy-7,8-dihydroberberine) measured in CD2Cl2 andC6D6.[20] Large differences in chemical shifts found in 1H NMR(up to 3.44 ppm) and 13C NMR (up to 67.67 ppm) and summarizedin the tables in this paper[26] are attributed to the effects of thedifferent solvents. However, it should be pointed out that quater-nary berberine (e.g. berberine chloride) is insoluble in CD2Cl2 andC6D6 and has not been tested in these solvents. The differencesin the NMR chemical shifts result unequivocally from significantdifferences between the chemical structures of berberine chlorideand the berberine base.

As expected, large differences in chemical shifts were obtainedbetween atoms at the same position in the 8-substituted 7,8-dihydroberberines and the quaternary forms of these alkaloids.Significant changes in the chemical shifts of H-8 and H-13were observed, due to the transformation of the QPA into the7,8-dihydroprotoberberine skeleton. The chemical shift of H-8 in a 2,3,9,10-substituted QPA is found in the approximaterange between 9.45 and 9.95 ppm[1,27]; for all products a–dδH8 = 6.3–7.2 ppm. An analogous tendency was observed forthe 1H NMR chemical shift of H-13. For example, the chemicalshift of H-13 in berberine chloride (1, X = Cl) is 8.96 ppm; for theproducts: δ = 6.11 ppm (4a), δ = 6.14 ppm (4b), δ = 6.16 ppm(4c), andδ = 6.19 ppm (4d). The same trends were obtained for the13C NMR spectra. The chemical shifts of C-8 were: δ = 145.37 ppm(1), δ = 70.14 ppm (4a), δ = 72.71 ppm (4b), δ = 68.86 ppm (4c),and δ = 70.96 ppm (4d); and of C-13: 120.14 ppm (1), 95.50 ppm(4a), 95.81 ppm (4b), 95.95 ppm (4c), and 95.94 ppm (4d). Theresonance of C-8 was easily assigned by detecting the correlationsignal between C-8 and H-6 in an 1H–13C gs-HMBC spectrum.Further, interunit long-range interactions of the H-8 atom withcarbons of the five-member rings (e.g. C3′ –C5′ for 4b) wereobtained.

The one-bond 1H–13C coupling constants (1JH,C) were extractedfrom the coupled 1H–13C HMQC or HSQC spectra. The 1JH,C

for berberine and its derivatives a–e are collected in Table S1and those of palmatine in Table S2 (Supporting Information).Representative long-range 1H–13C coupling constants for 4b arelisted in Table S3 (Supporting Information). A 2D 1H–1H NOESYspectrum showed the interactions of H-4 with H-5, H-6 with H-8,H-1 with H-13, and, most important for this study, the correlationof H-8 with protons of the attached heterocycle.

The 15N chemical shifts of N-7 and the nitrogen atoms ofthe attached heterocycle were obtained, and these data aresummarized in Table 3. In some cases the1H–15N Gradient-enhanced single quantum multiple bond correlation (GSQMBC)[28]

and gs-HMBC[29] experiments showed an interaction between H-8and N-1′, which gave additional support for the presence of acovalent C8–N1′ bond. Further, interaction between H-8 and N-2′

was obtained for the b compounds. Selected 1H–15N couplingconstants are presented in Table S4 (Supporting Information).

In three cases, crystallization of the products from tetrahydrofu-rane (THF) and CD2Cl2 afforded crystals suitable for single-crystalX-ray diffraction analysis (Crystallographic data for the structuresreported in this paper have been deposited with the CambridgeCrystallographic Data Centre. Copies of the data can be ob-tained, free of charge, on application to CCDC, 12 Union Road,Cambridge CB2 1EZ, UK (fax: +44-(0)1223–336 033 or e-mail: [email protected]).), and selected crystallographic data arecompiled in Table 4. A perspective view of the molecular structureof compound 4a as determined by X-ray analysis is shown in Fig. 1and a stereodrawing of the molecular structure of 4b in Fig. 2. X-rayanalysis confirmed the constitution of the molecules drawn on the

Figure 1. Perspective view of the X-ray structure of compound 4a.

basis of the NMR measurements and unambiguously confirmedthe formation of the covalent bond C8–N1′.

The interatomic distance C8–N1′ was 1.481(18) Å for 4a,1.478(3) Å for 4b, and 1.490(19) Å for 4c. Due to the changein the hybridization of C-8 (sp2 → sp3), the berberine partsof compounds 4a–4c deviate significantly from planar. Theinterplanar angle between rings A and D is 12.4◦ for the quaternaryberberine iodide[30] and 13.2◦ for berberine formate,[31] whereas itis 24.99(0.06)◦ for 4a, 39.02(0.06)◦ for 4b, and 25.94(0.08)◦ for 4c.

However, the products 4–6 are quite unstable and decomposevery easily in the presence of traces of water or acid. In additionto the nitrogen nucleophiles, selected S- and C-nucleophileswere also investigated. 8-(Methylsulfanyl)-7,8-dihydroberberine(4e) was prepared by reacting berberine chloride with sodiummethanethiolate. The chemical shifts of H-8 (δ = 6.34 ppm) andC-8 (δ = 67.85 ppm) indicated the presence of a covalentlyattached –SCH3 group at position C-8. This bonding was confirmedby a 2D NOESY experiment. The NOE was observed between H-4and H-5, H-6 and H-8, and H-8 and the –SCH3 group. To the bestof our knowledge, this is the first unequivocal characterization ofa covalent C8–S bond in 7,8-dihydroprotoberberines.

Recently, the base of 1-hydroxycoptisine (1,8-dihydroxy-7,8-dihydrocoptisine) has reportedly been isolated from Thalictrumdelavayi Franch.[32] Significant discrepancies in the chemical shiftswere identified when the published data were compared withthose of the bases of the alkaloids palmatine and berberine.[20]

Whereas for O-adducts the resonance of H-8 generally rangesbetween 6.0 and 6.2 ppm and that of C-8 is found around 85 ppm,the published values are δH8 = 5.38 ppm and δC8 = 74.00 ppm.[32]

These reported values are remarkably close to the chemical shifts of

Table 3. 15N NMR chemical shifts (δ in ppm)a for compounds 4 and 5in CD2Cl2 at 303 K

Compound 4a 5a 4b 5b 4c 5cb

N-7 82.4 82.6 82.0 82.0 79.0 80.4

N-1′ 183.9 184.2 232.6 232.7 193.2 194.2

N-2′ – – 306.5 306.4 – –

N-3′ – – – – 257.3 256.2

a Referenced to nitromethane and reported relative to liquid ammonia.b Measured at 268 K.4d: 249.7 (N-4′); 5d: 249.0 (N-4′).


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Table 4. Crystal data for compounds 4a, 4b, and 4c

Parameter 4a 4b 4c

CCDC reference number 686 512 686 513 686 514

Empirical formula C24H22N2O4 C23H21N3O4 C23H21N3O4

Formula weight 402.44 403.43 403.43

Crystallized from THF THF CD2Cl2Temperature (K) 120(2) 120(2) 120(2)

Wavelength (Å) 0.71073 0.71073 0.71073

Crystal system Monoclinic Orthorhombic Monoclinic

Space group P2(1)/C P2(1)2(1)2(1) P2(1)/C

a (Å) 14.035(6) 7.508(3) 14.078(12)

b (Å) 8.102(6) 11.222(4) 8.139(9)

c (Å) 17.730(17) 23.098(8) 17.208(7)

α (deg) 90.0 90.0 90

β (deg) 109.57(6) 90.0 108.48(6)

γ (deg) 90.0 90.0 90

Volume (Å3

) 1 900(2) 1 946.0(12) 1 870(3)

Z 4 4 4

Calculated density (mg m−3) 1.407 1.377 1.433

Absorption coefficient (mm−1) 0.097 0.096 0.100

F (000) 848 848 848

Crystal size (mm) 0.50 × 0.50 × 0.40 0.35 × 0.15 × 0.15 0.40 × 0.30 × 0.30

Crystal color Light yellow Light yellow Brown

θ range (deg) 2.79–25.00 3.24–25.00 2.80–25.00

Index range, h −16 → 12 −8 → 8 −16 → 16

Index range, k −9 → 9 −12 → 13 −9 → 9

Index range, l −20 → 21 −23 → 27 −20 → 17

Reflections collected/unique 12 164/3 343 9 445/3 413 15 197/3 291

Data/restraints/parameters 3 343/0/271 3 413/0/271 3 291/0/271

GOF 1.050 0.713 0.927

Final R/wR2 (I > 2σ/I) 0.0313/0.0848 0.0354/0.0393 0.0301/0.0703

Final R/wR2 (all data) 0.0425/0.0881 0.0893/0.0435 0.0443/0.0728

Figure 2. Stereodrawing of the molecular structure of compound 4b as determined by single-crystal X-ray diffraction.

8-trichloromethyl-7,8-dihydroprotoberberines.[20] Therefore, wedecided to prepare 8-trichloromethyl-7,8-dihydrocoptisine (6f)in order to determine its NMR parameters. The hydroxy groupat position C-1 was expected to affect the chemical shifts ofH-8 and C-8 marginally. This assumption would justify studyingthe available coptisine chloride (3, X = Cl) rather than the un-

available 1-hydroxycoptisine. The chemical shifts obtained for8-trichloromethyl-7,8-dihydrocoptisine (δH8 = 5.40 ppm; δC8 =73.94 ppm) are very similar to those reported for 1,8-dihydroxy-7,8-dihydrocoptisine. Thus the reported compound is probably1-hydroxy-8-trichloromethyl-7,8-dihydrocoptisine, and its struc-ture has been incorrectly determined. This compound could


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have been formed during the process of isolation (CHCl3/NaOH)from the plant material.[20,32] Very recently, an analogous reac-tion employing CHBr3 to expand the heterocyclic portion of theisoquinoline skeleton during the formation of benzazepin-2-onederivatives has been reported.[33]

Conclusions

To summarize our results, 8-heteroaryl-7,8-dihydro-protoberberine were prepared, and their structures wereunequivocally characterized by NMR spectroscopy. In threecases, molecular structures were determined by single-crystalX-ray diffraction analysis. The present findings indicate that QPAcould be capable of forming covalent bonds with deprotonatednitrogen bases. A covalently bonded S-alkyl group at C-8 wasunambiguously characterized for the first time. These factspoint to the theoretical possibility of forming a covalent bondbetween C-8 of the QPA and the N–H or S–H groups of biologicalmacromolecules. However, such bonds would be rather unstable,and under biological conditions the system would immediatelyhydrolyze to the starting compounds. Stable C8–X covalentbonding of QPA to a biomacromolecule would require differentconditions or subsequent ring transformations, e.g. in thepresence of molecular oxygen combined with ultraviolet (UV)light.[22,34] Further studies in this direction will be conducted.

Experimental

General procedures

Melting points were determined on a Kofler block WagentechnikRAPIDO, Franz Kuestner Nacht. KG Dresden HMK 66/1565 and aBuchi 535, melting point temperatures are uncorrected. Electronionization (EI) mass spectra were measured on a TRIO 1000quadrupole mass spectrometer, Finnigan MAT (Fisons Instrument,San Jose, CA, USA); a direct insertion probe (DIP) was used in theEI+ ionization mode, applying electron energies of 30 or 70 eV.ESI mass spectra were obtained from a Quadrupole time-of-flight(QTOF) II system (Waters, Manchester, UK) using a Nanomateelectrospray source (Advion Biosystems, Ithaca, NY, USA) in chip-based infusion mode. Pressure (approx. 0.4 bar) and voltage(approx. 1.5 kV) were optimized to obtain a stable spray current.The samples were dissolved in acetonitrile or THF to avoid proticconditions and analyzed immediately.

NMR spectroscopy

NMR spectra were recorded using a Bruker Avance 300 spec-trometer operating at frequencies of 300.13 MHz (1H), 75.48 MHz(13C), and 30.42 MHz (15N) and a Bruker Avance 500 operating atfrequencies of 500.13 MHz (1H), 125.77 MHz (13C), and 50.67 MHz(15N). The temperature of the measurements was 303 K. NMRsamples were prepared by dissolving the compounds in benzene-d6, dichloromethane-d2, or dimethylsulfoxide-d6. The 1H and 13CNMR chemical shifts were referenced to the signals of the solvents(C6D6 [7.16 (1H) and 128.00 (13C)]; CD2Cl2 [5.31 (1H) and 53.80(13C)]; DMSO [2.50 (1H) and 39.51 (13C)]). The 15N NMR chemicalshifts were referenced to liquid CH3NO2 (381.7 ppm) and are re-ported relative to liquid NH3.[35 – 38] No susceptibility correctionwas applied. The temperature was calibrated using methanolsample.

A 5-mm QNP [13C/19F/31P{1H}] or a 5-mm multinuclear inverseBBI [1H{BB}] probe with a self-shielded z-gradient coil was used tomeasure the 1H, 13C, and heteronuclear shift correlation spectra.1H NMR spectra: pulse 90◦, relaxation delay 10 s, number ofscans 4–16, and resolution <0.005 ppm per point. 13C NMRspectra: pulse 45◦, relaxation 3 s, number of scans 1024–32 768,and resolution <0.01 ppm per point. The 1H–13C couplingconstants were determined from the coupled gs-HMQC, gs-HSQC (1JH,C = 160 Hz), and GSQMBC (nJH,C = 7.5 Hz) spectrawith an accuracy of ±0.3 Hz. The 1H–15N GSQMBC[28] and gs-HMBC[29] experiments were adjusted for the couplings of 3.2 or5 Hz. Computer processing was performed with Bruker TopSpinsoftware.

X-ray diffraction analysis

Diffraction data were collected on a KM4CCD four-circle area de-tector diffractometer (Oxford Diffraction, Abingdon, UK) equippedwith an Oxford Cryostream Cooler (Oxford Cryosystems, Oxford,UK). We performed the ω-scan technique with different κ and ϕ

offsets in order to cover the entire independent part of the reflec-tion set up to 25◦

θ . The crystallographic package ShelXTL[39] wasused to solve and refine the structures and to prepare the figures.

Synthesis

Berberine chloride dihydrate (1, X = Cl), (Mrel = 407.86; mp204–206 ◦C), palmatine chloride hydrate (2, X = Cl) (Mrel =387.86; mp 206–207 ◦C), pyrrole (Mrel = 67.09; bp 129–131 ◦C),pyrazole (Mrel = 68.08; mp 66 ◦C), imidazole (Mrel = 68.08; mp88–91 ◦C), and 1,2,4-triazole (Mrel = 69.07; mp 116–120 ◦C) wereobtained in high purity from Sigma-Aldrich; coptisine chloride (3,X = Cl) (Mrel = 320.33) was isolated from natural sources.[40]

8-(Pyrrol-1-yl)-7,8-dihydroberberine (4a)

A mixture of sodium hydride (15.0 mg) and pyrrole (18.0 mg) indry THF was sonicated in an argon atmosphere. After 15 min, flashdried berberine chloride (1, X = Cl) was added, and the mixturewas stirred under an argon atmosphere at laboratory temperaturefor 5 h. The organic phase was separated, evaporated to half itsvolume, and left to crystallize in the freezer. Yellow crystals formedin 59% yield, mp 171–175 ◦C; for 1H, 13C, and 15N NMR data, seeTables 1, 2 and 3, respectively; EIMS (30 eV) m/z 402 [M]+ (8.7%),336 [M-pyrr.]+ (100), 320 (21); for crystallographic data, see Table 4.

8-(Pyrazol-1-yl)-7,8-dihydroberberine (4b)

This was prepared like 4a, using sodium hydride (39.7 mg),pyrazole (50.5 mg), and berberine chloride (215.7 mg). The mixturewas stirred for 3 h. Yellow crystals formed in 29% yield, mp165–175 ◦C (dec); for 1H, 13C, and 15N NMR data, see Tables 1,2 and 3, respectively; EIMS (70 eV) m/z 403 [M]+ (2.5%), 336[M-pyraz.]+ (28), 320 (17), 68 (100); for crystallographic data, seeTable 4.

8-(Imidazol-1-yl)-7,8-dihydroberberine (4c)

This was prepared like 4a, using sodium hydride (12.0 mg),imidazole (18.3 mg), and berberine chloride (100.0 mg). Themixture was stirred at 50–55 ◦C for 4 h. Yellow crystals formedin 58% yield, mp 172–173.5 ◦C; for 1H, 13C, and 15N NMR data, seeTables 1, 2 and 3, respectively; EIMS (70 eV) m/z 403 [M]+ (10.1%),336 [M-imid.]+ (75), 335 (100), 320 (98), 68 (18); for crystallographicdata, see Table 4.


11

33


8-(1,2,4-Triazol-1-yl)-7,8-dihydroberberine (4d)

This was prepared like 4a, using sodium hydride (42.7 mg), triazole(54.2 mg), and berberine chloride (246.4 mg). The mixture wasstirred at laboratory temperature for 5 h. Yellow powder wasisolated in 5% yield, mp 166–170 ◦C; for 1H, 13C, and 15N NMRdata, see Tables 1, 2 and 3, respectively; EIMS (30 eV) m/z 404 [M]+

(0.1%), 336 [M-triaz.]+ (23), 335 (11), 320 (11), 69 (100).

8-(Methylsulfanyl)-7,8-dihydroberberine (4e)

Berberine chloride (1, X = Cl, 100.0 mg), sodium methanethiolate(60.0 mg), and the catalyst cetyltrimethylammonium bromidewere mixed in 20 ml of dioxane and sonicated under an argonatmosphere. After 3 h silicagel was added, and the phases of thesuspension were separated. Evaporation of the solvent gave theproduct in 55% yield, mp 142–145 ◦C; for 1H and 13C NMR data,see Tables 1 and 2, respectively; EIMS (30 eV) m/z 383 [M]+ (0.5%),337 (33), 336 [M–SMe]+ (42), 47 (100).

8-(Pyrrol-1-yl)-7,8-dihydropalmatine (5a)

This was prepared like 4a, using sodium hydride (9.8 mg), pyrrole(10.3 µl), and palmatine chloride (2, X = Cl) (44.0 mg). The mixturewas stirred at laboratory temperature for 5 h. Yellow crystalsformed in 98% yield, mp 145–150 ◦C (dec); for 1H, 13C, and 15NNMR data, see Tables 1, 2 and 3, respectively; EIMS (30 eV) m/z418 [M]+ (2.0%), 352 [M-pyrr.]+ (55), 336 (15), 335 (2), 320 (5),67 (100); High-resolution MS (HR-MS) Time-of-flight electrospray(TOF-ES) TOF–ES+ [M + H]+ m/z 419.1971 (calculated mass forC25H27N2O4

+ m/z 419.1967).

8-(Pyrazol-1-yl)-7,8-dihydropalmatine (5b)

This was prepared like 4a, using sodium hydride (9.6 mg), pyrazole(10.2 mg), and palmatine chloride (43.8 mg). The mixture wasstirred at laboratory temperature for 3 h. Yellow crystals formed in63% yield, mp 151–166 ◦C (dec); for 1H, 13C, and 15N NMR data, seeTables 1, 2 and 3, respectively; EIMS (30 eV) m/z 419 [M]+ (0.2%),352 [M-pyraz.]+ (42), 336 (100), 320 (31), 68 (3).

8-(Imidazol-1-yl)-7,8-dihydropalmatine (5c)

This was prepared like 4a, using sodium hydride (7.2 mg), imidazole(11.2 mg), and palmatine chloride (44.6 mg). The mixture wasstirred at laboratory temperature for 17.5 h. Yellow crystals formedin 88% yield, mp 170.5–176.5 ◦C (dec); for 1H, 13C, and 15N NMRdata, see Tables 1, 2 and 3, respectively; EIMS (30 eV) m/z 419 [M]+

(20.6%), 352 [M-imidaz]+ (98), 351 (100), 336 (98), 320 (19), 68 (37).

8-(1,2,4-Triazol-1-yl)-7,8-dihydropalmatine (5d)

This was prepared like 4a, using sodium hydride (7.2 mg), triazole(11.8 mg), and palmatine chloride (46.4 mg). The mixture wasstirred at laboratory temperature for 17.5 h. Yellow product formedin 74% yield, mp 143–156 ◦C (dec); for 1H, 13C, and 15N NMR data,see Tables 1, 2 and 3, respectively; EIMS (30 eV) m/z 420 [M]+

(0.1%), 352 [M-triaz.]+ (51), 351 (100), 336 (85), 320 (14), 69 (25).

8-(Pyrrol-1-yl)-7,8-dihydrocoptisine (6a)

This was prepared like 4a, using sodium hydride (10.6 mg), pyrrole(13.1 µl), and coptisine chloride (3, X = Cl) (67.6 mg). The mixturewas stirred at laboratory temperature for 16 h. The yellow-brownproduct formed in 39% yield, mp 157–164 ◦C (dec), for 1H and 13CNMR data, see Tables 1 and 2, respectively; EIMS (30 eV) m/z 386[M+] (6.8%), 335 (34), 320 (97), 67 (100).

8-(Pyrazol-1-yl)-7,8-dihydrocoptisine (6b)

This was prepared like 4a, using sodium hydride (8.3 mg), pyrazole(13.4 mg), and coptisine chloride (3, X = Cl) (69.8 mg). The mixturewas stirred at laboratory temperature for 16 h. Product formed in35% yield, for 1H and 13C NMR data, see Tables 1 and 2, respectively;EIMS (30 eV) m/z 335s (6.7%), 320 (15), 69 (100), 68 (45).

8-Trichloromethyl-7,8-dihydrocoptisine (6f)

Coptisine chloride (3, X = Cl, 50.0 mg), chloroform (3.0 ml), andconcentrated aqueous ammonia (0.1 ml) were mixed and stirred.After 24 h the phases were separated, and the organic phase wasdried with a mixture of NaOH and anhydrous Na2SO4. Evaporationof the solvent gave yellow crystals in 80% yield, for 1H and 13CNMR data, see Tables 1 and 2, respectively.

Supporting information

Supporting information may be found in the online version of thisarticle.

Acknowledgements

The authors would like to thank Lubomír Prokes for his initial helpwith measuring the EI mass spectra. The financial support from theMinistry of Education of the Czech Republic (MSM0021622413 andLC06030 to LM, RM) and the Grant Agency of the Czech Republic(525/04/0017 to LG) is gratefully acknowledged.

References

[1] L. Grycova, J. Dostal, R. Marek, Phytochemistry 2007, 68, 150.[2] K. W. Bentley, The Isoquinoline Alkaloids, Harwood Academic

Publishers: Amsterdam, 1998 pp. 219, Chapt. 10.[3] M. Shamma, The protoberberines and retroprotoberberines, The

Isoquinoline Alkaloids, Academic Press: New York, London, 1972.[4] S. V. Pervushkin, A. A. Sokhina, V. A. Kurkin, G. G. Zapesochnaya,

M. M. Astrakhanova, K. V. Alekseev, I. P. Ivanova, Rastit. Resursy1999, 35, 123.

[5] K. Iwasa, D. U. Lee, S. I. Kang, W. Wiegrebe, J. Nat. Prod. 1998, 61,1150.

[6] L. Slobodníkova, D. Kost’alova, D. Labudova, D. Kotulova,V. Kettmann, Phytother. Res. 2004, 18, 674.

[7] A. Vollekova, D. Kost’alova, V. Kettmann, J. Toth, Phytother. Res.2003, 17, 834.

[8] K. Iwasa, M. Moriyasu, B. Nader, Biosci. Biotechnol. Biochem. 2000,64, 1998.

[9] N. Ivanovska, S. Philipov, Immunopharmacology 1996, 18, 553.[10] K. Iwasa, Y. Nishiyama, M. Ichimaru, M. Moriyasu, H. S. Kim,

Y. Wataya, T. Yamori, T. Takashi, D. U. Lee, Eur. J. Med. Chem. 1999,34, 1077.

[11] M. L. Sethi, Phytochemistry 1985, 24, 447.[12] K. Iwasa, M. Moriyasu, T. Yamori, T. Turuo, D. U. Lee, W. Wiegrebe,

J. Nat. Prod. 2001, 64, 896.[13] I. Slaninova, E. Taborska, H. Bochorakova, J. Slanina, Cell Biol. Toxicol.

2001, 17, 51.[14] M. Maiti, G. S. Kumar, Med. Res. Rev. 2007, 27, 649.


11

34

L. Grycova et al.

[15] S. Mazzini, M. C. Belluci, R. Mondelli, Bioorg. Med. Chem. 2003, 11,505.

[16] M. Stiborova, V. Simanek, E. Frei, P. Hobza, J. Ulrichova, Chem. Biol.Interact. 2002, 140, 231.

[17] S. Man, J. Dostal, M. Necas, Z. Zak, M. Potacek, Heterocycl. Commun.2001, 7, 243.

[18] M. Shamma, M. Rahimizadech, J. Nat. Prod. 1986, 49, 398.[19] J. Slavík, L. Slavíkova, Collect. Czech. Chem. Commun. 1989, 54, 2009.[20] R. Marek, P. Seckarova, D. Hulova, J. Marek, J. Dostal, V. Sklenar,

J. Nat. Prod. 2003, 66, 481.[21] S. Man, M. Potacek, M. Necas, Z. Zak, J. Dostal, Molecules 2001, 6,

433.[22] M. L. Contreras, S. Rivas, R. Rozas, Heterocycles 1984, 22, 101.[23] J. Dostal, S. Man, P. Seckarova, D. Hulova, M. Necas, M. Potacek,

J. Tousek, R. Dommisse, W. Van Dongen, R. Marek, J. Mol. Struct.2004, 687, 135.

[24] G. Blasko, G. A. Cordell, S. Bhamarapravati, C. W. W. Beecher,Heterocycles 1988, 27, 911.

[25] Y. R. Wu, Y. B. Ma, Y. X. Zhao, S. Y. Yao, J. Zhou, Y. Zhou, J. J. Chen,Planta Med. 2007, 73, 787.

[26] A. N. Tripathi, L. Chauhan, P. P. Thankachan, R. Barthwal, Magn.Reson. Chem. 2007, 45, 647.

[27] S. Chudík, R. Marek, P. Seckarova, M. Necas, J. Dostal, J. Slavík, J. Nat.Prod. 2006, 69, 954.

[28] R. Marek, L. Kralík, V. Sklenar, Tetrahedron Lett. 1997, 38, 665.[29] W. Willker, D. Leibfritz, R. Kerssebaum, W. Bermel, Magn. Reson.

Chem. 1993, 31, 287.[30] B. M. Kariuki, W. Jones, Acta Crystallogr., Sect. C 1995, 51, 1234.[31] J. Marek, D. Hulova, J. Dostal, R. Marek, Acta Crystallogr., Sect. C 2003,

59, 583.[32] M. Li, X. Chen, Q. M. Tang, J. S. Zhang, Planta Med. 2001, 67, 189.[33] L. Jean-Gerard, M. Pauvert, S. Collet, A. Guingant, M. Evain,

Tetrahedron 2007, 63, 11 250.[34] K. Hirakawa, S. Kawanishi, T. Hirano, Chem. Res. Toxicol. 2005, 18,

1545.[35] G. E. Martin, C. E. Hadden, J. Nat. Prod. 2000, 63, 543.[36] R. Marek, A. Lycka, Curr. Org. Chem. 2002, 6, 35.[37] G. E. Martin, A. J. Williams, Annu. Rep. NMR Spectrosc. 2005, 55, 1.[38] R. Marek, A. Lycka, E. Kolehmainen, E. Sievanen, J. Tousek, Curr. Org.

Chem. 2007, 11, 1154.[39] G. M. Sheldrick, SHELXTL, Version 5.10, Bruker AXS: Madison, 1997.[40] J. Slavík, V. Hanus, L. Slavíkova, Collect. Czech. Chem. Commun. 1991,

56, 1116.


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Cryptolepine derivatives: importance of pKa constants


Indoloquinolines have been investigated as potential new drugs for use

against malaria. This group of alkaloids has shown some antiplasmodial

activity through a mechanism similar to that of chloroquine, one of the most

frequently used drugs for treating malaria.

The search for a mechanism shows that cryptolepine, neocryptolepine, and

their derivatives are able to inhibit the formation of β-haematin in the process

by which the malaria parasite infects the human blood cells.[1] Because the

process of inhibition is located in an acid food vacuole, the pKa value of an

antimalarial agent is important.

9.2. NMR analytical possibilities and barriers for the project

Methods employing NMR for determination of pKa have been reported

very frequently. Most often a set of buffers is used for the NMR experiments,

Cryptolepine derivatives: importance of pKa constants ____________________________________________________________________________

98

with the result that the NMR chemical shifts depend of on the pH of the

medium.

The aim of our study was to determine the pKa values of the

indoloquinoline alkaloid cryptolepine and seven of its derivatives.

At the beginning we set two requirements for the experiment: aqueous

media and magnetic resonance.

Some other barriers arose during execution:

• Very small amounts of the alkaloid samples

• Limited solubility in pure water

• Relatively high melting points of the solvent mixtures

• Volume of liquid sample insufficient for measuring the pH

These barriers forced us to use just one sample for each alkaloid in

a mixed solvent (H2O:MeOH:DMSO-d6 - 4:4:2 by volume). The selection of

solvent ratios was limited by solubility of the samples and the concentration

limit for DMSO (no correction is needed up to 27 vol. %).[2] Correction for

mixture water-methanol (1:1) has been applied.[3]

9.3. Results

NMR analysis has been used to determine the pKa values of eight

indoloquinoline alkaloids as described in our paper in Magnetic Resonance in

Chemistry (Appendix C). The pKa value obtained the NMR experiment for

each compound can be correlated with its potential antiplasmodial activity.

Cryptolepine derivatives: importance of pKa constants

____________________________________________________________________________

99

Antiplasmodial agents such as quinine or chloroquine are characterized by

antiplasmodial activity against Plasmodium falciparum. The pKa value of

an agent corresponds with the ability of that agent to accumulate in a vacuole

(The pH of blood plasma is 7.4 and that of the vacuole medium 4.8). Other

characteristics[1 - 5] influenced by the structure include β-haematin inhibitory

activity and the interaction with DNA. All these data see in Table 9/1.

Table 9/1 Potential relationship between pKa and inhibition activity.

Compound pKa

Inhibition of ββββ-haematin formation DNA

interaction

IC50 (µµµµM)

MRC-5

IC50 (µµµµM) Yes/No BHIAa assay IC50 (Meq)b

Cryptolepine 11.0 + 1.72 65.7 1.5

Neocryptolepine 7.1 + 5.97 92.8 11.0

Isocryptolepine 9.8 + 7.59 119

Isoneocryptolepine 10.8 + 5.24 124

2-Br-neocryptolepine 6.6 + 1.77 >400 >32

2-MeO-

neocryptolepine 7.7 - - 77.9 4.0

3-Cl-neocryptolepine 6.7 + 2.35 >700 15.5

3-Br-neocryptolepine 6.6 + 2.56 >600 18.5

Chloroquine 8.4 + 2.56 / n.t.

Quinine 8.7 + 7.40 >360 n.t.

a BHIA – β-haematin inhibitory activity b Meq – molar equivalents of test compounds relative to haemin

The relationship between pKa and inhibition activity cannot be simply

deduced from the results. The cytotoxicity of an agent to the human cell line

Cryptolepine derivatives: importance of pKa constants ____________________________________________________________________________

100

MRC-5 correlates with its therapeutical properties. As the best structure was

identified the 2-Br-neocryptolepine, with the best rate between the

therapeutical activity and cytotoxicity.

9.5. References

[1] Van Miert S., Jonckers T., Cimanga K., Maes L., Maes B.,

G.Lemière, Dommisse R., Vlietinck A., Pieters L., Experimental

Parasitology 2004, 108, 163.

[2] Holmes D.R., Lightner D.A., Tetrahedon, 1995, 51, 1607.

[3] De Nogales V., Ruiz R., Rosés M., Ráfols C., Canals I., Bosch E., J.

Chromatogr. A, 2006, 1123, 113.

[4] Jonkers T.H.M., Van Miert S., Cimanga K., Bailly C., Colson P.,

De Pauw-Gillet M.-C., Van den Heuvel H., Claeys M., Lemière F.,

Esmans E.L., Rozenski J., Quirijnen L., Maes L., Dommisse R.,

Lemière G.L.F., Vlietinck A., Pieters L.: J. Med. Chem, 2002, 45,

3497.

[5] Van Miert S., Hostyn S., Maes B., Cimanga K., Brun R., Kaiser

M., Mátyus P., Dommisse R., Lemière G., Vlietinck A., Pieters L.,

J. Nat. Prod. 2005, 68, 674.

Appendix C

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NoteReceived: 9 April 2009 Revised: 30 June 2009 Accepted: 2 July 2009 Published online in Wiley Interscience: 3 August 2009

(www.interscience.com) DOI 10.1002/mrc.2494

NMR determination of pKa valuesof indoloquinoline alkaloidsLenka Grycova,a∗,† Roger Dommisse,b Luc Pietersc and Radek Mareka∗

Malaria is one of the most serious global health problems. Isolating new therapeutic agents with potential antimalarial activityfrom natural sources or preparing such agents either semisynthetically or synthetically is one strategy for solving the problemof resistance constantly evolving to the drugs currently in use.

For alkaloids, the acid–base dissociation constant, pKa, is an important characteristic, thought to be associated withbiological activity. In this contribution, pKa values for several indoloquinoline alkaloids were determined by using 1H NMRspectroscopy in a mixture of solvents. The data were recalculated for water solutions using the correction factors reportedpreviously. The structural dependence of the pKa values for cryptolepine and its isomers neocryptolepine, isocryptolepine andisoneocryptolepine as well as some substituted neocryptolepine derivatives is discussed. Copyright c© 2009 John Wiley & Sons,Ltd.

Supporting information may be found in the online version of this article.

Keywords: 1H NMR; chemical shift; cryptolepine; isocryptolepine; neocryptolepine; isoneocryptolepine; malaria; pH; determination ofpKa

Introduction

The global impact of malaria, transmitted by the Anophelesmosquito and one of the world’s most dangerous parasiticdiseases, is felt mainly in developing countries. It is estimatedthat there are between 200 and 300 million human infectionsevery year, with more than 2 million fatal cases.[1,2] The treatmentof malaria is currently a priority of the World Health Organization(WHO). Four different Plasmodium species cause human infection:P. vivax, P. ovale, P. malariae, and P. falciparum – which leads tothe most dangerous form of the disease.[3] Resistance to thedrugs currently in use, e.g. chloroquine,[4] is an increasinglyserious problem. Artemisinin, isolated from Artemisia annua, aplant used in traditional Chinese medicine, represents a possiblesolution.[5]

Another prospective class of compounds for treatment ofmalaria is represented by indoloquinoline alkaloids such ascryptolepine. Cryptolepine (1), the major alkaloid containedin the Central- and West-African plant Cryptolepis sanguino-lenta (Lindl.) Schlechter, and neocryptolepine (2), one ofthe minor alkaloids in the same plant,[6] are known fortheir antiplasmodial properties and have been used as start-ing compounds for the development of new antimalarialagents.[2,7 – 9]

It has been reported that the desmethyl analogs of thesealkaloids (the desmethyl analog of cryptolepine called quindoline)are not active as antimalarial agents.[8] At least part of theantiplasmodial activity of cryptolepines is due to inhibiting theformation of hemozoin, a process that takes place in the acidfood vacuole of the parasite. Aminoquinoline antimalarials, suchas chloroquine, have the same target. In order to be active,these drugs have to accumulate in the acid food vacuole, andtherefore the molecule must contain a basic functionality, suchas a basic nitrogen atom (pH trapping). The pH-dependent

∗ Correspondence to: Lenka Grycova and Radek Marek National Centerfor Biomolecular Research, Faculty of Science, Masaryk University, Ka-menice 5/A4, CZ-625 00 Brno, Czech Republic. E-mail: [email protected];[email protected]

† Current address: AROMATICA CZ Ltd., Masarykovo namestí 101/3, CZ 664 51Slapanice, Czech Republic.

a National Center for Biomolecular Research, Faculty of Science, MasarykUniversity, CZ-625 00 Brno, Czech Republic

b Department of Chemistry, University of Antwerp, Groenenborgerlaan 171,B-2020 Antwerp, Belgium

c Department of Pharmaceutical Sciences, University of Antwerp, Universiteit-splein 1, B-2610 Antwerp, Belgium

Magn. Reson. Chem. 2009, 47, 977–981 Copyright c© 2009 John Wiley & Sons, Ltd.

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L. Grycova et al.

Scheme 1. Equilibrium between cryptolepine (1) and its salt.

equilibrium between the base and the corresponding salt ofcryptolepine[10,11] is shown in Scheme 1. Since the methylatedindole-N in cryptolepine and neocryptolepine is basic, in contrastto the non-methylated analogs, this basicity may explain why themethylated forms are biologically active and their non-methylatedanalogs are not.

In order to study the potential relationship between theacid–base properties of a compound and its antiplasmodialactivity, it is necessary to determine its acid–base ionizationconstants (pKa). Onyeibor has used a spectrophotometric methodto measure the pKa value of cryptolepine.[1]

In this work, we have used 1H NMR spectroscopy to de-termine the pKa values of a series of indoloquinoline isomersand substituted neocryptolepine derivatives. Three isomers of in-doloquinoline, namely cryptolepine (1), neocryptolepine (2), andisocryptolepine (3), have been isolated from Cryptolepis species.[12]

Surprisingly, the fourth isomer, isoneocryptolepine (4), has notbeen found in nature. However, isoneocryptolepine (4), some2- and 3-substituted derivatives of neocryptolepine, and an arrayof other derivatives have been synthesized and tested for theirbiological activities.[1,3,8,13,14]

Although the pKa value is an important characteristic ofany potential antimalarial agent that targets the formation ofhemozoin in the acid food vacuole, pKa values have been reportedonly for cryptolepine (pKa = 11.2 and 11.8).[1,15] No other pKa

values for indoloquinoline derivatives have been found in theliterature.

Various methods can be used to determine pKa values. Changesin physical, chemical, or spectral properties can be determined atseveral different pH values by using capillary zone electrophoresis,potentiometric titrations,[16 – 18] UV-Vis spectrophotometry,[17] orNMR spectroscopy, to name just a few methods. Alternatively, the-oretical calculations[19,20] may be combined with X-ray analysis.[21]

NMR spectroscopy has been selected for the current study.Several NMR approaches have previously been used to

determine pKa values in a broad range of organic samplescontaining carbonyl groups or amino acids (e.g. proteins).[22 – 25]

Temperature-dependent 1H NMR spectroscopy[26] and deuteriumNMR spectroscopy[27] have also been employed. However, one ofthe most widely used approaches is the determination of the pHdependence of 1H NMR chemical shifts.[28] This is the method usedin the present study.

Generally, NMR spectroscopy suffers from its inherently lowsensitivity and, as a consequence, relatively highly concentratedsamples must be used. This represents a severe problem forsamples that are of limited solubility in an appropriate solvent,e.g., in water. Binary or ternary solvent systems are used to achieveconcentrations that are adequate for NMR analysis. A correctionfactor must subsequently be applied to calculate the pKa inwater, based on the value determined in the mixed solvent. Manystudies for various solvent mixtures have been published,[29 – 31]

with a large portion of these describing a binary methanol–watersystem. It should be noted that the relationship between the pKa

values for H2O and D2O has also been published.[32]


Several indoloquinoline-type compounds with established orpotential biological activities against malaria were studied. Thisset comprised four basic isomers of indoloquinoline (compounds1–4), two 2-substituted neocryptolepines (5, 6), and two3-substituted neocryptolepines (7, 8). Since the water solubilityof these compounds was insufficient for NMR measurements, a1 : 1 mixture of water and methanol was used. To further increasethe solubility of the samples, dimethylsulfoxide (DMSO, 20%) wasadded to the water–methanol mixture to form the ternary solventH2O:MeOH:DMSO in the proportion 2 : 2 : 1 (DMSO-d6 was used toprepare the samples suitable for NMR experiments). A previouspublication[33] showed that the difference in pKa is less than ±0.2for solutions containing up to 27% of DMSO.

All the compounds investigated can exist in two primary forms,the base and the salt, with different NMR characteristics.[11] NMRtitration of the samples with aqueous solutions of NaOH or HClwas used to determine the pKa values. The bases were titratedwith a dilute acid solution (5–20 mM) and the hydrochloride saltswith an aqueous solution of sodium hydroxide (10–40 mM).

In each step, the pH of the solution (alkaloid in solvent mixture)was modified by the dropwise addition (12.5–50 µl) of acid or baseand measured at constant temperature. A portion (550 µl) of theresultant solution was then transferred into an NMR tube and the1H NMR spectrum obtained. 1H chemical shifts were referencedto the signal of TMS as internal standard. Subsequently, samplefrom the NMR tube was transferred back to the parent stocksolution (because of limited sample availability). By repeating thisprocess several times, we obtained the dependence of the 1H NMRchemical shifts on pH.

The dependence of the chemical shift of H-11 in 2-Br-neocryptolepine (5) on the pH of the solution is shown in Fig. 1. Theobserved 1H NMR chemical shift dependencies for all compoundswere analyzed by two approaches. Firstly, a graphical estimationof the inflection point gave the values presented in Table 1.

Secondly, the data were analyzed using the Hender-son–Hasselbalch (H–H) method.[34 – 36] This logarithmic analysisgives the dependence of the chemical shift on the pH of thesample in a linear form. The H–H relationship between the pH andthe 1H chemical shift is represented in Eqn 1.

pH = pKa + log[δ(max) − δ]

[δ − δ(min)](1)

Figure 2 shows an example, graphic representation of the H–Hanalysis for H-11 in 2-Br-neocryptolepine (5). This type of analysis isexpected to give reasonable data for concentrations of 2–11 mMused in this study, with an activity coefficient close to unity.[37,38]

The possible error introduced into the pKa value by neglecting theeffects of dilution during the NMR titration experiment has beencalculated to be <3%.

In order to calculate pKa values for substances dissolved inpure water, the correction factor reported previously for the 1 : 1H2O : MeOH mixtures[39] was used in the following form:

pKa (H2O) = [pKa (H2O/MeOH) + B]

A(2)

where A = 0.97 and B = 0.33.[39] This calculation resultedin the individual pKa values for water solutions presented inTable 1. As expected, the two types of analysis employed in thisstudy provided very similar data with the differences between


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pKa values of indoloquinolines

Figure 1. pH dependence of 1H NMR chemical shifts for 2-Br-neocryptolepine.

Table 1. The pKa values for compounds 1–8 determined at 298 K

H–Hb Mean Correctedc

Compound Reagent Proton Inflectiona MeOH–H2O H2O

1 Cryptolepine NaOH H-11 10.26 10.29 10.3 11.0

2 Neocryptolepine NaOH H-6 6.60 6.61 6.6 7.1

3 Isocryptolepine NaOH H-6 9.17 9.17 9.2 9.8

4 Isoneocryptolepine NaOH H-11 10.06 10.10 10.1 10.8

5 2-Br-neocryptolepine HCl H-11 6.10 6.11 6.1 6.6

6 2-MeO-neocryptolepine HCl H-11 7.11 7.10 7.1 7.7

7 3-Cl-neocryptolepine NaOH H-11 6.16 6.17 6.2 6.7

8 3-Br-neocryptolepine NaOH H-11 6.11 6.14 6.1 6.6

a Determined from the inflection point.b Determined by Henderson–Hasselbalch analysis.c Using Eqn 2.

related values being less than 0.1. The only pKa values foundin the literature for any indoloquinoline alkaloid are those forcryptolepine (1). Two values have been published: pKa = 11.2(applied method not reported)[15] and pKa = 11.8 (1H NMRexperiment),[1] with the former being relatively close to the valueof pKa = 11.0 determined in this work.

The values determined for cryptolepine (1) (pKa = 11.0) andisoneocryptolepine (4) (pKa = 10.8) are notably similar. Thiscould be expected based on their similar structural features.The two nitrogen atoms are separated by three chemical bondsin both compounds. The pKa value is somewhat reduced forisocryptolepine (3) (pKa = 9.8), with nitrogen atoms four chemicalbonds distant from each other. The smallest pKa value wasobtained for neocryptolepine (2) (pKa = 7.1) with nitrogens onlytwo bonds apart. Substitution of the neocryptolepine skeleton(see compounds 5–8) has only a minor effect on the pKa values(6.6–7.7).

Conclusions

The pKa values for several indoloquinoline derivatives havebeen determined by pH-dependent 1H NMR spectroscopy.The experimental chemical shifts were measured in solventmixtures and analyzed by the Henderson–Hasselbalch approach.

Subsequently, a correction factor was applied to calculate thevalues for aqueous solutions. The pKa values are substantiallydependent on the structure, and a dependence of the pKa on thebond separation between two nitrogen atoms in a molecule hasbeen observed.

Experimental

Chemicals

NaOH, HCl, DMSO-d6, MeOH, and standard reference bufferswere obtained commercially. Cryptolepine (1) was isolated fromCryptolepis sanguinolenta, as reported previously;[40] isocryp-tolepine (2), neocryptolepine (3), isoneocryptolepine (4), andthe neocryptolepine derivatives 2-bromoneocryptolepine (5),2-methoxyneocryptolepine (6), 3-bromoneocryptolepine (7) and3-chloroneocryptolepine (8) were prepared synthetically.[8,41]

Compounds 1–4, 7, and 8 were used as hydrochlorides andcompounds 5 and 6 as bases.

Preparation of samples and pH measurements

All samples (alkaloids and their derivatives) and reagents(NaOH, HCl) were prepared as solutions in the solvent mixture(H2O : MeOH : DMSO-d6 = 2:2 : 1). The samples were prepared by


98

0

L. Grycova et al.

Figure 2. Graphic representation of the Henderson–Hasselbalch analysis for 2-Br-neocryptolepine.

dissolving 1–3 mg of alkaloid in 2 ml of solvent. The concentrationsof the NaOH solutions were 10, 25 and 40 mM. The concentrationsof the HCl solutions were 5 and 10 mM.

The pH values of the solutions at 298 K were measured usinga glass electrode (Hamilton polyplast probe; Eutech instrumentCyberscan 510, Singapore) and a contact drop electrode (SENTRONmanual hot-line probe with instrument Mettler Toledo DELTA 320).Exactly the same temperature was used for the NMR experiments.

NMR spectroscopy

NMR spectra were recorded using a Bruker DRX-400 spectrometeroperating at a frequency of 400.13 MHz (1H), and a Bruker Avance500 instrument operating at a frequency of 500.13 MHz (1H),always at 298 K. The NMR samples were prepared by dissolvingthe compounds in the mixture of H2O, MeOH and DMSO-d6 asdescribed above, and the 1H NMR chemical shifts were referencedto the signal of internal TMS (0 ppm). The following parameterswere used to record the 1H NMR spectra: pulse 90◦ , relaxation delay

5 s, number of scans 64–256, resolution <0.003 ppm per point.Computer processing was performed with the Bruker TopSpinsoftware.

The pH dependence of selected 1H NMR chemical shifts wasused for analysis. The selected hydrogen atoms were H-11 forcryptolepine and for neocryptolepine and its derivatives and H-6for isocryptolepine and isoneocryptolepine. Selected 1H chemicalshifts for the base and salt of each compound are summarized inTable 2.

Acknowledgement

The financial support of this work given to LG by the Flemishgovernment is gratefully acknowledged. This work was alsopartially supported by the Ministry of Education of the CzechRepublic (grants MSM0021622413 and LC06030 to LG and RM).The Special Research Fund of the University of Antwerp (ConcertedAction) is acknowledged for financial support.

Table 2. 1H NMR chemical shifts for relevant signals of compounds 1–8 in the solvent mixture (H2O : MeOH : DMSO = 2 : 2 : 1) at 298 K and previouslypublished values

Salt Base

Alkaloid Proton δ δ (pH) δ δ (pH)

1 Cryptolepine H-11 9.11a,b 9.15 (8.60) 8.93a,c 8.92 (11.56)

2 Isocryptolepine H-6 9.90a,c 9.83 (7.84) 8.90a,b 9.21 (12.54)

3 Isoneocryptolepine H-6 9.98a,c 9.70 (7.29) 9.55a,c 9.37 (11.66)

4 Neocryptolepine H-11 9.37a,b 9.47 (5.46) 8.54a,d 8.96 (8.63)

5 2-Br-neocryptolepine H-11 – 9.41 (4.58) 8.37d,e 8.89 (7.60)

6 2-MeO-neocryptolepine H-11 – 9.43 (4.21) 8.47d,e 8.96 (7.88)

7 3-Br-neocryptolepine H-11 – 9.36 (5.11) 8.56d,e 8.91 (8.50)

8 3-Cl-neocryptolepine H-11 – 9.43 (5.19) 8.49d,e 8.90 (8.51)

a Data from Ref. [11].b Measured in CD3OD.c Measured in DMSO-d6.d Measured in CDCl3.e Data from Ref. [8].


98

1

pKa values of indoloquinolines

Supporting information

Supporting information may be found in the online version of thisarticle.

References

[1] O. Onyeibor, S. L. Drift, H. I. Dodson, M. Fez-Haddad, H. Kendrick,N. J. Millington, S. Parapini, R. M. Philips, S. Seville, S. D. Shnyder,D. Taramelli, C. W. Wright, J. Med. Chem. 2005, 48, 2701.

[2] S. Van Miert, T. Jonckers, K. Cimanga, L. Maes, B. Maes, G. Lemiere,R. Dommisse, A. Vlietinck, L. Pieters, Exp. Parasitol. 2004, 108, 163.

[3] S. Van Miert, S. Hostyn, B. U. W. Maes, K. Cimanga, R. Brun, M. Kaiser,P. Matyus, R. Dommisse, G. Lemiere, A. Vlietinck, L. Pieters, J. Nat.Prod. 2005, 68, 674.

[4] D. C. Warhurst, J. C. Craig, I. S. Adagu, R. K. Guy, P. B. Madrid,Q. L. Fivelman, Biochem. Pharmacol. 2007, 73, 1910.

[5] J. Cazzeles, A. Robert, B. Meunier, C. R. Acad. Sci. Paris, Chimie. 2001,4, 85.

[6] K. Cimanga, T. De Bruyne, L. Pieters, M. Claeys, A. Vlietinck,Tetrahedron Lett. 1996, 37, 1703.

[7] E. Arzel, P. Rocca, P. Grellier, M. Labaeï d, F. Frappier, F. Gueritte,C. Gaspard, F. Marsais, A. Godard, G. Queguiner, J. Med. Chem. 2001,44, 949.

[8] T. H. M. Jonckers, S. Van Miert, K. Cimanga, C. Bailly, P. Colson,M.-C. De Pauw-Gillet, H. Van den Heuvel, M. Claeys, F. Lemiere,E. L. Esmans, J. Rozenski, L. Quirijnen, L. Maes, R. Dommisse,G. L. F. Lemiere, A. Vlietinck, L. Pieters, J. Med. Chem. 2002, 45, 3497.

[9] T. Dhanabal, R. Sangeetha, P. S. Mohan, Tetrahedron. 2006, 62,6258.

[10] E. Gellert, R. Hamet, E. Schlittler, Helv. Chim. Acta. 1951, 34, 642.[11] J. Tousek, S. Van Miert, L. Pieters, G. Van Baelen, S. Hostyn,

B. U. W. Maes, G. Lemiere, R. Dommisse, R. Marek, Magn. Reson.Chem. 2008, 46, 42.

[12] M. M. Cooper, J. M. Lovell, J. A. Joule, Tetrahedron Lett. 1996, 37,4283.

[13] S. Hostyn, B. U. W. Maes, L. Pieters, G. L. F. Lemiere, P. Matyus,G. Hajos, R. A. Dommisse, Tetrahedron. 2005, 61, 1571.

[14] S. Seville, R. M. Phillips, S. D. Shnyder, C. W. Wright, Bioorg. Med.Chem. 2007, 15, 6353.

[15] D. Dwuma-Badu, Fitoterapia. 1987, 58, 348.

[l6] J. J. Van Luppen, J. A. Lepoivre, R. A. Dommisse, F. C. Alderweireldt,Org. Magn. Reson. 1979, 12, 399.

[17] M. Andrasi, P. Buglyo, L. Zekany, A. Gaspar, J. Pharm. Biomed. Anal.2007, 44, 1040.

[18] H. Yuksek, M. Alkan, S. Bahceci, I. Cakmak, Z. Ocak, H. Baykara,O. Aktas, E. Agyel, J. Mol. Struct. 2008, 873, 142.

[19] M. N. Davies, C. P. Toseland, D. S. Moss, D. R. Flower, BMC Biochem.2006, 7, 18.

[20] E. Soriano, S. Cerdan, P. Ballesteros, THEOCHEM. 2004, 684, 121.[21] J. E. Nielsen, A. McCammon, Protein Sci. 2003, 12, 1894.[22] M. Zhang, H. J. Vogel, J. Biol. Chem. 1993, 268, 22420.[23] D. L. Holmes, D. A. Lightner, Tetrahedron. 1996, 52, 5319.[24] A. Blasko, C. A. Bunton, S. Bunel, C. Ibarra, E. Moraga, Carbohydr. Res.

1997, 298, 163.[25] J. Quijada, G. Lopez, R. Versace, L. Ramírez, M. L. Tasayco, Biophys.

Chem. 2007, 129, 242.[26] A. Kahyaoglu, F. Jordan, Protein Sci. 2002, 11, 965.[27] B. Lau, P. M. Macdonald, Biochim. Biophys. Acta. 1995, 1237, 37.[28] (a) Z. Szakacs, G. Hagele, Talanta 2004, 62, 819; (b) F. Reniero,

C. Guillou, C. Frassineti, S. Ghelli, Anal. Biochem. 2003, 319,179; (c) L. Orfi, C. K. Larive, S. M. LeVine, Lipids 1997, 32, 1035;(d) C. S. Handlose, M. R. Chakraba, M. W. Mosher, J. Chem. Educ.1973, 50, 510; (e) A. Kahyaoglu, F. Jordan, Protein Sci. 2002, 11,965.

[29] A. Niazi, A. Yazdanipour, M. Ramezani, Chin. Chem. Lett. 2007, 18,989.

[30] F. Z. Erdemgil, S. Sanli, N. Sanli, G. Ozkan, J. Barbosa, J. Guiteras,J. L. Beltran, Talanta. 2007, 72, 489.

[31] R. Wrobel, L. Chmurzynski, Anal. Chim. Acta. 2000, 405, 303.[32] A. Krezel, W. Bal, J. Inorg. Biochem. 2004, 98, 161.[33] D. L. Holmes, D. A. Lightner, Tetrahedron. 1995, 51, 1607.[34] E. Breitmaier, K. H. Spohn, Tetrahedron. 1973, 29, 1145.[35] L. J. Henderson, Am. J. Physiol. 1908, 21, 173.[36] K. A. Hasselbalch, Biochem. Z. 1917, 78, 112.[37] H. N. Po, N. M. Senozan, J. Chem. Educ. 2001, 78, 1499.[38] R. de Levie, J. Chem. Educ. 2003, 80, 146.[39] (a) V. de Nogales, R. Ruiz, M. Roses, C. Rafols, E. Bosch, J. Chromatogr.

A 2006, 1123, 113; (b) F. Rived, I. Canals, E. Bosch, M. Roses, Anal.Chim. Acta 2001, 439, 315.

[40] K. Cimanga, T. De Bruyne, L. Pieters, A. Vlietinck, C. A. Turger, J. Nat.Prod. 1997, 60, 688.

[41] T. H. M. Jonckers, B. U. W. Maes, G. L. F. Lemiere, G. Rombouts,L. Pieters, A. Haemers, R. A. Dommisse, Synlett 2003, 615.


109

Flavonoids of Paulownia tomentosa: cytotoxic structures

10.1 Plant material

Fruits of Paulownia tomentosa, collected in Brno during October of 2000,

were used to isolate the flavonoid compounds. The plant material was

processed at the Department of Natural Drugs, Faculty of Pharmacy,

University of Veterinary and Pharmaceutical Sciences Brno, Brno, Czech

Republic.

10.2 Isolation process

The flavonoid compounds were at Department of Natural Drugs isolated

using ethanol and chloroform, followed by the column chromatography. The

five fractions (I-V) obtained were than subjected to further isolation

processes as depicted in Figure 10/1.[1]

Flavonoids of Paulownia tomentosa: determination of structures ____________________________________________________________________________

110

Figure 10/1

All isolated samples (yellow powders) were characterized for preliminary

purposes by mass spectrometry.

10.3 Biological activity

In parallel with the determination of the structures, a study of the

biological activities proceeded at the Department of Natural Drugs. The

compounds were studied for their potential antiradical and cytotoxic

activities at a sample concentration of 10 µM. Diplacone was found to be the

strongest antioxidant of the compounds tested, and it also had the most

cytotoxic activity.

10.4 Structures determined

The structures of the six flavonoid compounds isolated from Paulownia

tomentosa fruit were determined. The flavanone or flavanonole backbones


111

(Figure 10/2) were found to be substituted at position C-6 with a prenyl,

geranyl, or modified geranyl side chain. The phenyl ring bonded at position

C-2 is substituted with one or more hydroxy or methoxy groups.

O

O

O

O

OH

flavanone flavanole

Figure 10/2

Diplacone (1),[2] the first structure characterized, was already known and

the compound has been previously isolated from Paulownia tomentosa.[3] Its

backbone flavanone structure has a substitutions at positions C-6 (geranyl

side chain) and C-3’ (hydroxy group), see Figure 10/3.

The second structure characterized, 3’-O-Methyldiplacone (2), was also

known before,[4 - 6] but it has never been isolated from a Paulownia species

(Figure 10/3) before our study.

O

OH

HO

O

OH

OH 1


112

O

OH

HO

O

OMe

OH 2

Figure 10/3

The chemical shifts of both structures were compared with published

values[2,4,6] (for 1H and 13C NMR data in Appendix D, characteristic NMR

shifts have been discussed there).

Geranyl side chain

The geranyl (a) chain (C10H17) is one of the structural blocks which can be

found in the flavonoid compounds. Geranyl is most frequently bonded at the

position C-6,[2,4,8] but other positions are “allowed,” e.g., C-3 or C-8of

flavone structure.[9,10] The current knowledge about prenylation patterns in

flavonoids was summarized in a review article in 1996.

The prenyl (b) side chain (C5H9) is the frequently occurring fragment in

flavonoids. Like geranyl, it can be bonded to C-6 or C-8.[8,9,11,12,13]

A prenyl side chain can be present at C-3’ and C-5’of the phenyl group

(ring B) of a flavonoid.[14] prenylation at positions C-2’ and C-6’ of the

isoflavone structures has also been described.[15] For the structures, see

Figure 10/4.

X X

a b

Figure 10/4


113

The remaining four compounds (Figure 10/5) have not been previously

isolated from plant material: 3’-O-methyl-5’-methoxydiplacol (3),

tomentodiplacol (4),[7] 6-isopentenyl-3’-O-methyltaxifolin (5), and

dihydrotricin (6).

O

OH

HO

OH O

OMe

OH

OMe

3

O

OH

HO

OH O

OMe

OH

OH

4

O

OH

HO

OH O

OMe

OH

5

O

OH

HO

OH O

OMe

OMe

6

Figure 10/5


114

The results are summarized in a paper published in the Journal of Natural

Products (Appendix D).

10.5 Results

Analysis of the compounds isolated from the fruits of Paulownia

tomentosa yielded four new flavonoid compounds with interesting structural

modifications. These compounds have been tested for their antioxidant

activity; partial data are presented in Table 10/1. Compound 2 showed

antibacterial activity against gram-positive bacteria. A cytotoxic effect has

been observed for some of the compounds.

Table 10/1 Biological effects of the flavonoid study.

C6 C3` C4` C5` TEACa

Gram+

bacteriab

IC 50

[µµµµM] c

1 geranyl OH OH H 5.2 14.3

2 geranyl OMe OH H 0.8 + 30.2

3 geranyl H OH H 0.4

4

OH

OMe OH H 2.0

a Trolox equivalent antioxidant capacity. b Antibacterial activity against 6 gram-positive bacteria with MICs from 2 to 8 µg/mL. c Cytotoxicity of compound.

10.6 References

[1] Šmejkal K., Grycová L., Marek R., Lemiére F., Jankovská D.,

Forejtníková H., Vančo J., Suchý V., J. Nat. Prod., 2007, 70, 1244.


115

[2] Philips, W.R.; Bay, N.J.; Gunatilaka, A.A.L.; Kingston, D.G.I., J.

Nat. Prod., 1996, 59, 495.

[3] Jiang T.-F., Du X., Shi Y.-P., Chromatographia, 2004,. 59, 255.

[4] Yang X., Sun Y., Xu Q., Guo Z., Org. Biomol. Chem., 2006, 4,

2483.

[5] Hare J.D., Biochemical Systematics and Ecology, 2002, 30, 709.

[6] Hare, J.D.; Borchardt, D. B., Phytochemistry, 2002, 59, 375.

[7] Barron D., Ibrahim R.K., Phytochemistry, 1996, 43, 921.

[8] Meragelman K.M., McKee T. C., Boyd M.R., J. Nat. Prod., 2001,

64, 546.

[9] Chan S.-C., Ko H.-H., Lin C.-N., J. Nat. Prod., 2003, 66, 427.

[10] Akihisa T., Tokuda H., Hasegawa D., Ukiya M., Kimura Y., Enjo

F., Suzuki T., Nishino H., J. Nat. Prod., 2006, 69, 38.

[11] Stevens J.F., Page J.E., Phytochemistry, 2004, 65, 1317.

[12] Ito C., Itoigawa M., Kumagaya M., Okamoto Y., Ueda K.,

Nishihara T., Kojima N., Furukawa H., J. Nat. Prod., 2006, 69, 138.

[13] Hayashi K.-I., Nakanishi Y., Bastow K.F., Cragg G., Nozaki H.,

Lee K.-H., J. Nat. Prod., 2003, 66, 125.

[14] El-Masry S., Amer M.E., Abdel-Kader M.S., Zaatout H.H.,

Phytochemistry, 2002, 60, 783.

[15] Tahara S., Ibrahim R.K., Phytochemistry, 1995, 38, 1073.

Appendix D

C-Geranyl Compounds from Paulownia tomentosaFruits

Karel Smejkal,*,† Lenka Grycova´,‡ Radek Marek,*,‡ Filip Lemiere,§ Dagmar Jankovska´,† Hana Forejtnı´kova,† Jan Vanco,⊥ andVaclav Suchy´†

Department of Natural Drugs and Department of Chemical Drugs, UniVersity of Veterinary and Pharmaceutical Sciences Brno, Palacke´ho 1-3,CZ-612 42 Brno, Czech Republic, National Center for Biomolecular Research, Faculty of Science, Masaryk UniVersity, Kamenice 5/A4,CZ-625 00 Brno, Czech Republic, and Mass Spectrometry Research Unit, Department of Chemistry, UniVersity of Antwerp,Groenenborgerlaan 171, B-2020 Antwerp, Belgium

ReceiVed February 9, 2007

Five geranylflavonoids, one prenylated flavonoid, and a simple flavanone were isolated from an ethanolic extract ofPaulownia tomentosafruit. Tomentodiplacol (1), 3′-O-methyl-5′-methoxydiplacol (2), 6-isopentenyl-3′-O-methyltaxifolin(3), and dihydrotricin (4) are reported from a natural source for the first time and 3′-O-methyldiplacone (6) for the firsttime from the genusPaulownia. The structures of the compounds were determined by mass spectrometry, includingHRMS, and by 1D and 2D NMR spectroscopy. The cytotoxicity and DPPH (2,2-diphenyl-1-picrylhydrazyl)-quenchingactivity of some of these compounds were tested, with diplacone proving to be the best antioxidant, although the mostcytotoxic compound.

Paulownia tomentosaSteud. (Scrophulariaceae) is a largedeciduous tree, usually 10-20 m tall, with strong branches thatare dark felted when they are small. Paulownia trees bloom in Mayand June, before the leaves appear. The flowers are colored whiteto light purple. The fruit is a double-capsuled, red-brown ligneousseed ball, approximately 4 cm long. The fruit ripens in autumnand then bursts, and many winged seeds fly away.

Extracts from P. tomentosaare used in traditional Chinesemedicine. Extracts from the fruit, leaves, and wood are used inadjuvant therapy for bronchitis, and fruit extracts decrease thefrequency of asthmatic attacks.1 An aqueous extract of the fruitand leaves regenerates hair and stimulates the scalp.2 Extracts fromthe fruit show a hypotensive effect, and extracts from the woodare used to treat some bacterial infections.2

Previous publications have reported polyphenolic substances,such as iridoids, phenolic glycosides, flavonoids, and phenyletha-noids in the MeOH and EtOH extracts ofP. tomentosa. In fact,most of the substances that have been isolated so far are polar,usually glycosides.3-14

This work is focused on the isolation and identification of partlypolar and nonpolar compounds from the European paulownia treeand aims to find potentially bioactive substances with antiradicalor cytotoxic activities. The screening test of an EtOH extract ofP.tomentosabased on the DPPH (diphenylpicrylhydrazyl) assayshowed significant antiradical activity (EC50 7.928 mg/L). In thispaper, we report the isolation and structural elucidation of sevencompounds from an EtOH extract ofP. tomentosafruit and thecytotoxic and antiradical activities of some of them.


The EtOH extract ofP. tomentosafruit was subjected to liquid-liquid fractionation, and the antiradical-active CHCl3-soluble frac-tion was repeatedly separated by column chromatography on silica.The fractions, selected by TLC analysis, were further separated bypreparative RP-HPLC. This extensive separation process resultedin the isolation of tomentodiplacol (1), 3′-O-methyl-5′-methoxy-

diplacol (2), 6-isopentenyl-3′-O-methyltaxifolin (3), and dihydro-tricin (4). 3′-O-Methyldiplacone (6) is reported from the genusPaulowniafor the first time. Mimulone (5) and diplacone (7) havebeen isolated previously.14

The UV spectra of the compounds showed similar behavior, withmaxima atλ 229-242 (sh), 289-296, and 335-345 (sh) nm,corresponding to theπ f π* and n f π* electronic transitionsmatched to flavanone skeletons.15 The IR spectra of the isolatedcompounds showed a similar series of absorption bands atνmax

3500-3400 cm-1, corresponding to OH vibrations; 2950-2850cm-1, corresponding to CH vibrations, which are not usually soapparent in flavonoid spectra; and 1639-1622 cm-1, assigned tothe CdO vibration of the carbonyl group.15

The molecular formula of tomentodiplacol (1) was determinedon the basis of HRMS Q-TOF measurements that gave thepseudomolecular ion [M- H]- at m/z 469.1851 (calcd massm/z469.1862) as C26H30O8. ESIMS spectra in the negative ionization

* To whom correspondence should be addressed. Tel:+420/541562839.E-mail: [email protected]; [email protected].

† Department of Natural Drugs, University of Veterinary and Pharma-ceutical Sciences Brno.

‡ Masaryk University.§ University of Antwerp.⊥ Department of Chemical Drugs, University of Veterinary and Phar-

maceutical Sciences Brno.

1244 J. Nat. Prod.2007,70, 1244-1248

10.1021/np070063w CCC: $37.00 © 2007 American Chemical Society and American Society of PharmacognosyPublished on Web 07/11/2007

mode showed the presence of a pseudomolecular ion [M- H]- atm/z 469. The constitution of compound1 was determined by1Hand 13C NMR spectroscopy (Tables 1 and 2). COSY,17 HSQC,18

and HMBC18 experiments were used to assign the observedresonances to individual atoms. The long-range1H-13C interactionsobserved in gs-HMBC (adjusted for a long-range coupling of 7.5Hz) are summarized in Table 2. The two singlets atδ 3.78 (3H)and 5.94 (1H) in the1H NMR spectrum of1 were unequivocallyassigned to the methyl group and H-8, respectively. Resonances at

δ 6.78, 6.89, and 7.08 and their coupling patterns indicated a 3,4-disubstituted phenyl ring. The1H-13C interactions observed inHMBC (Table 2) identified the connection of this substituted phenylto C-2 (δC ) 83.11 ppm). A modified geranyl-type side chain isbonded at C-6 (long-range interactions H1′′-C6 and H2′′-C6 weredetected in the HMBC). A structure with a similar, unusual part ofthe side chain has been published recently.19 The configuration atC2-C3 was determined to betrans on the basis of the magnitudeof 3JH2,H3 ) 11.5 Hz. Generally, coupling constants for thetrans

Table 1. 1H NMR Chemical Shifts (δ in ppm) and Indirect Spin-Spin Coupling Constants (J in Hz) of Compounds1-4 in DMSO-d6at 303 K

1 2 3 4

position δH (J in Hz) δH (J in Hz) δH (J in Hz) δH (J in Hz)

2 5.00 d (11.5) 4.94 d (11.4) 4.99 d (11.1) 5.41 dd (2.9; 13.0)3 4.62 d (11.5) 4.61 d (11.4) 4.47 d (11.1) 2.68 dd (2.9; 17.1)d

3.34 dd (13.0; 17.1)e

6 5.89 d8 5.94 s 5.92 s 5.95 s 5.91 d2′ 7.08 d (1.8) 6.75 s 6.92c 6.79 s5′ 6.78 d (8.1) 6.93 d (8.2)6′ 6.89 dd (1.8; 8.1) 6.75 s 6.88 dd (1.8; 8.2) 6.79 s1′′ 3.13 d 3.14 d 3.12 d2′′ 5.13 t 5.09 t 5.13 t4′′ 1.70 s 1.66 s 1.62 s5′′ 1.85 m 1.87 t 1.70 s

1.94 m6′′ 1.45 m 1.96 t7′′ 3.80 q 5.00 t9′′ 1.61 s 1.56 s10′′ 4.70 s 1.49 s

4.81 s3′-OMe 3.78 s 3.73 s 3.78 s 3.77 s5′-OMe 3.73 s 3.77 s3-OH 5.71 d 5.71 s 5.74 d5-OH 12.19 s 12.16 s 12.16 s 12.15 s7-OH 10.75 bsa 8.98a b b4′-OH 9.07 s 8.47 bs 9.02 s 8.47 bs7′′-OH 4.60 s

a Broad resonance.bNot obtained.cJ not determined due to the overlap.dcis relative to H-2.etrans relative to H-2.

Table 2. 13C NMR Chemical Shifts (δC in ppm) of Compounds1-4 in DMSO-d6 at 303 K

1 2 3 4

position δC, mult. HMBCa δC, mult. HMBCa δC, mult. HMBCa δC, mult. HMBCa

2 83.1, CH 3, 2′, 6′ 83.5, CH 3, 2′, 6′ 82.7, CH 3, 5′, 6′ 78.9, CH 3a, 2′, 6′3 71.4, CH 2 71.5, CH 2 71.5, CH 2 42.2, CH2

4 198.0, Cq 2, 3, 8 198.0, Cq 2, 8 197.6, Cq 2, 3 196.3, Cq 2, 3a, 3b, 64a 100.2, Cq 8 100.3, Cq 8 100.1, Cq 8 101.6, Cq 65 160.3, Cq 1′′ 160.5, Cq 1′′ 160.3, Cq 1′′ 163.2, Cq 66 107.8, Cq 8, 1′′, 2′′ 107.9, Cq 8, 1′′ 107.7, Cq 8, 1′′ 95.8, CH 87 164.4, Cq 8, 1′′ 164.5, Cq 8, 1′′ 164.3, Cq 8, 1′′ 163.5, Cq 6, 88 94.4, CH 94.5, CH 94.3, CH 95.0, CH 68a 160.1, Cq 8 160.2, Cq 2, 8 160.0, Cq 2, 8 162.9, Cq 21′ 128.2, Cq 2, 3, 2′, 5′ 127.3, Cq 2, 3, 2′, 6′ 129.8, Cq 2, 3, 2′ 128.5, Cq 2, 3a, 2′, 6′2′ 112.2, CH 2, 5′, 6′ 106.0, CH 2 111.8, CH 104.7, CH 2, 3b, 6′3′ 147.3, Cq 2′, 5′ 147.7, Cq 2′, 6′ 147.9, Cq 5′, 6′ 147.8, Cq 2′, 6′4′ 147.0, Cq 6′ 136.1, Cq 2′, 6′ 146.1, Cq 2′ 135.9, Cq 2′, 6′5′ 114.9, CH 2′ 147.7, Cq 2′, 6′ 114.9, CH 2, 6′ 147.8, Cq 2′, 6′6′ 121.1, CH 2, 2′ 106.0, CH 2 119.1,CH 2, 5′ 104.7, CH 2, 2′1′′ 20.5, CH2 2′′ 20.6, CH2 2′′ 20.6, CH2 8, 2′′2′′ 122.0, CH 1′′, 4′′, 5′′ 122.4, CH 1′′, 4′′, 5′′ 122.5, CH 1′′, 4′′, 5′′3′′ 134.1, Cq 1′′, 4′′, 5′′, 6′′ 133.9, Cq 1′′, 4′′, 5′′ 130.3, Cq 1′′, 4′′, 5′′4′′ 16.0, CH3 2′′, 5′′ 15.9, CH3 2′′, 5′′ 25.4, CH3 2′′, 5′′5′′ 35.2, CH2 2′′, 4′′, 6′′, 7′′ 39.3, CH2 2′′, 4′′, 6′′, 7′′ 17.6, CH3 2′′, 4′′6′′ 33.4, CH2 5′′, 7′′ 26.3, CH2 5′′7′′ 73.4, CH 5′′, 6′′, 9′′, 10′′ 124.2, CH 5′′, 6′′, 9′′, 10′′8′′ 148.2, Cq 6′′, 7′′, 9′′, 10′′ 130.7, Cq 6′′, 9′′, 10′′9′′ 17.5, CH3 7′′, 10′′ 25.5, CH3 7′′, 10′′10′′ 109.8, CH3 7′′, 9′′ 17.5, CH3 7′′, 9′′3′-OMe 55.7, CH3 56.1, CH3 55.7, CH3 56.1, CH3

5′-OMe 56.1, CH3 56.1, CH3

a 1H-13C HMBC (adjusted to 7.5 Hz) correlations are from the carbon(s) specified to the protons indicated.

C-Geranyl Compounds from Paulownia tomentosa Fruits Journal of Natural Products, 2007, Vol. 70, No. 81245

arrangement20,21 range between 11 and 12 Hz, whereas couplingsfor the cis configuration22 are typically found in the range 1-3Hz. A low amount of1 prevented definition of the C-7′′ configu-ration.

The molecular formula of 3′-O-methyl-5′-methoxydiplacol (2)was determined to be C27H32O8 by HRMS Q-TOF and proved bythe presence of the pseudomolecular ion [M- H]- atm/z483.2015(calcd massm/z 483.2019). The ESIMS in the negative modeshowed the presence of a pseudomolecular ion [M- H]- at m/z483. The structure of compound2 was determined by using 1Dand 2D NMR spectroscopy (Tables 1 and 2) and the samemethodology as for1. The 1H NMR spectrum indicates a 3,4,5-trisubstituted phenyl group connected to carbon C-2. The1H and13C chemical shifts of the geranyl side chain attached to C-6 aresimilar to data of the known compounds mimulone (5)23 anddiplacone (7).24 The trans configuration at C2-C3 was deducedfrom the coupling constant between H-2 and H-3. All observedlong-range1H-13C interactions are summarized in Table 2.

HRMS Q-TOF of 6-isopentenyl-3′-O-methyltaxifolin (3) gavea pseudomolecular ion [M- H]- at m/z 385.1297 (calcd massm/z385.1287). These data, together with the1H and 13C NMR data,established the molecular formula of3 as C21H22O7. The ESIMSspectrum in the negative mode showed the presence of a pseudo-molecular ion [M- H]- at m/z 385, which confirmed the deducedmolecular weight of3, Mr ) 386. The structure of compound3was determined by using 1D and 2D NMR (Tables 1 and 2). Theimportant long-range1H-13C interactions are summarized in Table2. The substitution pattern of the phenyl ring of this compound isidentical with that of compound1, as proved by the1H NMRspectrum. The prenyl chain is connected to C-6. The acquired datamatched those found in the literature.25 The configuration at C2-C3 was determined to betrans by the H2-H3 coupling constant.

The molecular formula of4 was determined to be C17H16O7 asproved by the HRMS Q-TOF and the pseudomolecular ion [M-H]- measured atm/z331.0811 (calcd mass 331.0818). The ESIMSspectrum in the negative mode showed the presence of thepseudomolecular ion [M- H]- at m/z 331. Major fragments atm/z 177, 165, and 161 were obtained by MS/MS fragmentation ofthe parent ionm/z 331. All three of these fragments are linked tothe cleavage of ring C of the flavonoid backbone.16 The structureof compound4 was elucidated by 1D and 2D NMR spectroscopy(Tables 1 and 2). The important long-range1H-13C interactionsare summarized in Table 2. The1H NMR spectrum shows that thesubstitution pattern of the phenyl ring is identical with that of2.Compound4 was identified as dihydrotricin, a simple derivativeof the flavone tricin, which is widely distributed in many plantspecies.26

Compounds5, 6, and7 were identified as the 6-geranylflavanonesmimulone, 3′-O-methyldiplacone, and diplacone. These are major

flavonoid constituents of the leaf resin ofMimulus auranticus,23

and they have also been isolated fromM. cleVelandii.24 The physicaland spectroscopic data used to identify5, 6, and 7 agreed withthose reported previously.23,24

The absolute configurations at stereogenic centers C-2 and C-3of the substances were determined by analyzing their circulardichroism spectra. A positive Cotton effect for the nf π* electronictransition at 320-360 nm and negative Cotton effect for theπ fπ* electronic transition at 280-310 nm were observed forcompounds1 and 3-7. A 2R,3R-configuration was assigned tocompounds1 and 3 by comparison of CD and NMR data withthose of flavanones.27 The configuration for compounds4-7 wasdetermined as 2S.27 No Cotton effect was observed in the CDspectrum of2. Since the arrangement at C-2/C-3 was determinedastransby NMR, compound2 is a racemic mixture of 2R,3R and2S,3S enantiomers.

The antiradical activity and cytotoxicity of some of the isolatedcompounds were tested using the DPPH and the NR cell-viabilityassays. The compounds tested showed significantly differentantiradical activities at the concentration of 10µM. The antioxi-dative capacity was expressed as Trolox equivalent antioxidantcapacity, TEAC. The most effective scavenger was diplacone (7)(TEAC 5.2( 0.001). According to postulated theories, its antiradi-cal activity corresponds with anortho-dihydroxy functionality ofthe B ring, where the 4′-OH is the site of the donation of an electronand a proton to reduce diphenylpicrylhydrazil (DPPH•) radical todiphenylpicrylhydrazine (DPPH-H) and the 3′-OH assists in theformation of a stable flavonoid radical or in the termination reactionstep.28,29 In comparison, single 4′-OH substitution (5) (TEAC 0.4( 0.004) and 4′-OH substitution combined with 3′-OCH3 (1 and6) (TEAC 2.0 ( 0.007 and 0.8( 0.006) show significantlydiminished activities. The geranyl side chain does not affect activityin a significant way, but it could modify the solubility of diplaconeand eventually affects the reaction kinetics. The DPPH assay wasperformed using the modified method of Braca et al.;30 the timedependence of the DPPH radical quenching, which effectivelycharacterizes the antioxidants, was monitored. The compoundstested were found to be “slow scavengers”, since the effectivenessof the DPPH quenching increased slowly during the 30 min of theassay. The time dependence of the quenching effect is shown inFigure 1.

The cytotoxicity of compounds5, 6, and7 was determined byusing the NR cell-viability assay and by measuring of the capacityof cells (epithelioid cell line WB 344) to actively take up neutralred.31 The calculated values of IC50 and the results are summarizedin Table 3. A previous study (using HL-60 cells) showed that 4′-methoxy substitution or the loss of the 4′-hydroxy group signifi-cantly decreased the cytotoxicity of 4′-hydroxyflavanone,32 whileanother study on flavanones showed no activity of taxifolin

Figure 1. Time dependence of the antiradical activity of the compounds tested at a concentration of 10µM.

1246 Journal of Natural Products, 2007, Vol. 70, No. 8 Smejkal et al.

(3,3′,4′,5,7-pentahydroxyflavanone) on colorectal carcinoma cells.33

This agreed in part with our cytotoxicity data: diplacone (7) >mimulone (5) > tomentodiplacol (1). Theortho-dihydroxy substitu-tion of ring B (6) enhances the cytotoxic effect, and 4′-methoxysubstitution lowers the cytotoxicity on the WB 344 cell line.Insufficient amounts precluded testing of the other compounds.

The facts show thatP. tomentosais a medicinal plant with ahigh polyphenol secondary metabolism and with a terpenoid sidechain incorporation into the flavonoid skeleton. The products ofthis metabolic pathway are liposoluble compounds with a highactivity against UV radiation, which can be excreted onto the surfaceof the P. tomentosafruits, where these flavonoids probably serveas antifeedants and as protection against excessive UV radiation.

Experimental Section

General Experimental Procedures.Melting points were determinedon a Kofler hot-stage apparatus and are not corrected. UV spectra ofMeOH solutions were recorded on a Hewlett-Packard 8453 spectro-photometer. CD spectra were recorded on a Jasco J-810 spectrometer(MeOH; molar elipticity Θλ values are presented). IR spectra weredetermined using the KBr disc method or ATR on a Nicolet Impact400D FT-IR spectrophotometer.

NMR spectra were recorded using a Bruker Avance DRX 500spectrometer operating at frequencies of 500.13 MHz (1H) and 125.77MHz (13C). All NMR spectra were measured in DMSO-d6 at 303 K.The 1H and 13C NMR chemical shifts (δ in ppm) were referenced tothe resonance of the solvent [2.50 (1H) for DMSO-d5 and 39.50 (13C)for DMSO-d6]. The 2D NMR experiments, gradient-selected COSY,gs-HSQC, and gs-HMBC were used to assign the individual13C and1H resonances.16,17The gradient ratio for the1H-13C HMBC experimentwas 30:18:24 G cm-1, and the experiment was adjusted for long-rangecouplings of 7.5 Hz.

ESIMS was done using an Agilent HP 1100 LC/MSD Trap VLSeries and directly infusing MeOH solutions at a flow rate of 300µL/min with a linear pump (kd Electronics). The spectra were collected inthe negative mode; the nebulizing and drying gas was N2 (t ) 300°C)flowing at a rate of 10 L/min; the nebulizer pressure was 80 psi andthe capillary voltage 3.5 kV. The full mass scan covered the rangefrom m/z 200 to 1500. Collision fragmentation experiments wereperformed in an ion trap, using helium as the collision gas. Full scanmass spectra, MS/MS of the selected pseudomolecular ion, and (MS)n

(n ) up to 3) were all collected in the negative mode.The Q-TOF 2 instrument (Micromass/Waters, Manchester, UK) was

calibrated in the mass range fromm/z 80 to 600 using a 0.1% aqueousH3PO4/MeOH (50/50) solution. The same solution was added to themobile phase at a flow rate of 0.5µL/min through a T-piece justupstream from the ES interface. The phosphate cluster ions were usedfor internal mass reference in the acquired spectra. MeOH containing

0.1% HCOOH and flowing at a rate of 100µL/min was used as themobile phase. The precise masses of the compounds were obtained inthe negative-ion mode by injecting 5µL of a MeOH solution of theanalyte and recording the spectra. The spectra were locked on a selectedphosphate cluster ion near the deprotonated molecule. The accuratemasses of the deprotonated molecules were compared with theircalculated masses.

Column chromatography was performed using silica gel LachemaL 400/100. Flash chromatography was performed on silica gel Merck60 (particle size 0.040-0.063 mm). Precoated silica gel 60 F254(Merck) was used for the TLC analyses, with detection using UV lightat 254 and 366 nm after spraying with Neu’s reagent (1% dipheny-laminoethylborate in MeOH) and heating to 110°C for 10 min.Analytical and preparative HPLC were carried out on an Agilent 1100instrument equipped with DAD. The columns used for analysis werefilled with the stationary phase Supelcosil ABZ+Plus (column length250× 10 mm i.d., particle size 5µm for the semipreparative analyses;column length 150× 4.6 mm i.d., particle size 3µm for the analyticalHPLC). The mobile phase for the gradient elution was a mixture ofMeCN and 40 mM HCOOH.

Plant Material. The fruits ofP. tomentosawere collected in Brno(Czech Republic) during October of 2000. A voucher specimen (PT-02O) was deposited at the herbarium of the Department of NaturalDrugs, Faculty of Pharmacy, University of Veterinary and Pharmaceuti-cal Sciences Brno, Czech Republic.

Extraction and Isolation. The plant material (9.3 kg) was extractedwith EtOH (4 L). The EtOH extract (500 g) was diluted to about 60%with H2O (300 mL) and extracted with CHCl3 (500 mL × 3). TheCHCl3 extract (84 g) was evaporated to drynessin Vacuoat 45°C andsubjected to column chromatography on silica. It was eluted withbenzene/acetone (95:5, 12 L) for fractions 1-83, benzene/acetone (9:1, 9 L) for fractions 84-147, benzene/acetone (8:2, 6.5 L) for fractions148-193, benzene/acetone (7:3, 7 L) for fractions 194-240, and finallybenzene/acetone (1:1, 5 L) for fractions 241-271. Fractions of about150 mL were collected. Based on TLC analyses, the combined fractionsI (26-30), II (55-70), III (109-112), IV (123-151), and V (180-189) were chosen for further work. Fractions III and IV were separatedby FC on silica with benzene/CHCl3/MeOH (7.4:2.5:0.1, 5 L), andsubfractions of about 150 mL were collected. On the basis of the TLCanalyses, the combined fractions III/1 and IV/15-17 were chosen forfurther work. Subfraction IV/15-17 (320 mg) was then separated bysemipreparative RP-HPLC, using the stationary phase SupelcosilABZ+Plus. The mobile phase consisted of (A) MeCN and (B) 40 mmolof HCOOH/MeCN (9:1). The gradient elution started with a mobilephase composition of A:B (65:35, v/v) and finished with a compositionof A (100%). It required 15 min at a flow rate of 14.7 mL/min. Theinjection volume was 1 mL, and UV detection atλ 280 nm was used.Fractions were collected according to the detector response. Purecompound1 (11 mg) was obtained from the subfraction IV/15-17.Subfraction III/1 (330 mg) was separated by the same method asdescribed above for the subfraction IV/15-17 to give 5 (25 mg).Fraction II was repeatedly separated by flash chromatography withbenzene/EtOAc (9:1, 1 L× 2) and benzene/CHCl3/MeOH (7.4:2.5:0.1, 1 L× 2). The major compound6 (120 mg) was then purified bypreparative HPLC, using the method descibed above. Fraction V wasseparated by flash chromatography on silica with benzene/CHCl3/MeOH(7.4:2.5:0.1, 2 L× 2, fractions of about 150 mL). Pure compound7(58 mg) precipitated from the subfraction V/36-50. Fraction I wasseparated directly by semipreparative chromatography, using a mobilephase consisting of (A) MeCN and (B) 40 mmol of HCOOH. Thegradient elution started with a mobile phase composition of A:B (50:50, v/v) and finished with pure A (100%) after 20 min, at a flow rate

Scheme 1

Table 3. Cytotoxicity of the Compounds Tested after a 24 hTreatment, as Determined by a Neutral Red Assay (expressed as% of the control- DMSO)a

5 µM 25 µM 100 µM IC50[µM]

diplacone (7) 98 ( 0.03 57( 0.30 2( 0.00 14.33′-O-methyldiplacone (6) 100( 0.02 73( 0.05 2( 0.10 30.2mimulone (5) 97 ( 0.03 95( 0.04 9( 0.01 b

a Each result is expressed as the mean( SD of three independentmeasurements.b The IC50 could not be estimated because the cytotox-icity was less than 50% of that of the control.

C-Geranyl Compounds from Paulownia tomentosa Fruits Journal of Natural Products, 2007, Vol. 70, No. 81247

of 5 mL/min. The injection volume was 100µL, and UV detection atλ 280 nm was used. Subfractions were collected according to thedetector response, and compounds2 (21 mg),3 (15 mg), and4 (12mg) were obtained.

Tomentodiplacol (1):yellow powder; UV (MeOH)λmax (log ε) 215(4.12), 236 (sh), 295 (3.52), 341 (sh) nm; CD (MeOH)Θ332.5+16 264,Θ296 -64 821.8,Θ224.560 815.8; IR (KBr)νmax 3419, 2929, 2853, 1636,1517, 1463, 1276, 1164, 1118 cm-1; for 1H and 13C NMR data, seeTables 1 and 2; HRMS Q-TOF [M- H]- m/z 469.1851 (calcd forC26H29O8

- 469.1862); ESIMS [M- H]- m/z 469.3′-O-Methyl-5′-methoxydiplacol (2):yellow powder; UV (MeOH)

λmax (log ε) 214 (4.42), 229 (sh), 289 (4.23), 335 (sh) nm; IR (ATR)νmax 3444-3328, 2922, 2909, 1634, 1604, 1515, 1457, 1381, 1330,1157, 1002 cm-1; for 1H and13C NMR data, see Tables 1 and 2; HRMSQ-TOF [M - H]- m/z 483.2015 (calcd for C27H31O8

- 483.2019);ESIMS [M - H]- m/z 483.

6-Isopentenyl-3′-O-methyltaxifoline (3): yellow powder; UV (MeOH)(log ε) λmax 212 (4.23), 229(sh), 289 (4.01), 335(sh) nm; CD (MeOH)Θ332.5 +8813.5,Θ296 -33 711.5,Θ224.5 +30 223.1; IR (ATR)νmax

3444-3328, 2966, 2909, 1622, 1496, 1440, 1323, 1285, 1244, 1177,1096 cm-1; for 1H and 13C NMR data, see Tables 1 and 2; HRMSQ-TOF [M - H]- m/z 385.1297, (calcd for C21H21O7

- 385.1287);ESIMS [M - H]- m/z 385.

Dihydrotricin (4): yellow powder; UV (MeOH)λmax (log ε) 218(4.28), 242(sh), 296 (3.89), 345(sh) nm; CD (MeOH)Θ335 +2673.7,Θ295.5 -16 103.2,Θ224.5 +13 478.8; IR (ATR)νmax 3361, 2919, 2850,1625, 1515, 1452, 1338, 1270, 1211, 1155, 1109 cm-1; for 1H and13CNMR data, see Tables 1 and 2.; HRMS Q-TOF [M- H]- m/z331.0811(calcd for C17H15O7

- 331.0818); ESIMS [M- H]- m/z 331.Mimulone (5): yellow needles from MeOH; mp 120-122 °C; UV

(MeOH) λmax (log ε) 207 (4.38), 229 (sh), 295 (4.07), 336 (sh) nm;CD (MeOH)Θ331.5+9065.3,Θ295 -30458.7,Θ221 +31649.7; IR (KBr)νmax 3401, 2917, 2853, 1636, 1599, 1450, 1343, 1307, 1155, 1082 cm-1;1H and 13C NMR data in agreement with that published;22,23 ESIMS[M - H]- m/z 407.

3′-O-Methyldiplacone (6): yellow powder; UV (MeOH)λmax (logε) 218 (4.26), 235 (sh), 290 (3.88), 345 (sh) nm; CD (MeOH)Θ331.5

+9132.7,Θ292.5 -43801.7,Θ220 +38376; IR (KBr)νmax 3546, 2973,2915, 1639, 1597, 1516, 1492, 1451, 1342, 1274, 1158, 1086 cm-1;1H and 13C NMR data in agreement with that published;22,23 ESIMS[M - H]- m/z 437.

Diplacone (7): light brown powder; UV (MeOH)λmax (log ε) 211(4.20), 234 (sh), 290 (3.99), 345 (sh) nm; CD (MeOH)Θ331 +8058.3,Θ294 -29 856.6,Θ219 +32 587.5; IR (KBr)νmax3530, 3189, 2966, 2913,1637, 1602, 1450, 1388, 1227, 1145, 1180 cm-1; 1H and 13C NMRdata in agreement with that published;22,23 ESIMS [M - H]- m/z 423.

Cytotoxicity Assay. One hundred microliters of cells (BG/F) inDulbecco’s modified Eagle’s medium (DMEM) (Sigma-Aldrich, Pra-gue, Czech Republic) was cultured in 96-well culture plates forapproximately 24 h. The compounds to be tested were dissolved inDMSO and added to the wells (100µL). Following a 24 h incubation,the solution was removed from all the plates and replaced with freshDMEM containing neutral red (NR) at a concentration of 40 g/L. Theviable cells were allowed to take up the supravital dye into lysosomesfor 3 h. The cells were then washed twice with PBS, followed byfixation with 0.5% (v/v) formaldehyde supplemented with 1% (w/v)CaCl2. After 15 min, the fixing solution was removed and the cellswere washed with PBS and transferred to a 1% (v/v) HOAc, 50% (v/v) EtOH solution to extract the dye from the cells. The plates werethen shaken gently on a plate shaker for 20 min at room temperature.The absorbance of the extracted dye was measured at 540 nm by meansof a spectrophotometric microplate reader (Labsystems iEMS Reader,Labsystems, Turku, Finland). The percentage inhibition of cell growthwas calculated as cell growth inhibition (%)) (1 - T/C) × 100, whereC was the OD540 value of the control andT was the OD540 value of thechemical tested. The 50% inhibitory concentrations (IC50 values) wereobtained from dose-response curves.

DPPH-Quenching Assay.The modified method of Braca et al. wasused to quantify the DPPH-quenching activity.30 The MeOH DPPH(Sigma) solution was prepared at a concentration of 22 mg/L. Thecompounds to be tested were dissolved in MeOH at a concentration of10 µM. A volume of 0.2 mL of the test solution was mixed with 1.8mL of DPPH solution, and the absorbance of the mixture at 517 nm

was measured every minute for the first 5 min of the experiment andthen every 5 min for the next 25 min. The recorded data were used todraw the course of the increasing activity as a function of time, andthe differences between the compounds tested were then compared.Results were expressed as Trolox equivalents.

Acknowledgment. Financial support of this work by the IGA VFU(grant No. 23/2004 to K.Sˇ .), the Ministry of Education of the CzechRepublic (MSM0021622413 and LC06030 to R.M. and L.G.), and theMinistry of Health (1A8666 to V.S.) is gratefully acknowledged.

References and Notes

(1) Zhao-Hua, Z.; Ching-Ju, C.; Xin-Yu, L.; Yao Gao, X. Asian networkfor biological science and international development research centre,Chinese Academy of Forestry, Beijing, 1986. http://idrinfo.idrc.ca/archive/corpdocs/071235/index_e.html (cited 2005-03-1).

(2) Duke J. A.; Ayensu J. S.Medicinal Plants of China; ReferencePublications Inc., 1985.

(3) Kyoung, H. K.; Seong, K. J.; Bank-Kwang, K.; Man, K. P.Arch.Pharm. Res. 1994, 17, 470-475.

(4) Kawada, T.; Asano, R.; Makino, K.; Sakuno, T.J. Wood Sci. 2002,48, 512-515.

(5) Wysokinska, H.; Rozga, M.J. Plant Physiol.1998, 152, 78-83.(6) Schilling, G.; Hugel, M.; Mayer, W.Z. Naturforsch. B1982, 37,

1633-1635.(7) Ota, M.; Azuma, T.; Onodera, S.; Taneda, K.Mokuzai Gakkaishi

1993, 39, 479-485.(8) Oprea, E.; Radulescu, V.; Chiliment, S.ReV. Chem.2004, 55, 410-

412.(9) Franzyk, H.; Jensen, S. R.; Thale, Z.; Olsen, C. E.J. Nat. Prod.

1999, 62, 275-278.(10) Adriani, C.; Bonini, C.; Iavarone, C.; Trogolo, C.J. Nat. Prod. 1981,

44, 739-744.(11) Damtoft, S.; Jensen, S. R.Phytochemistry1993, 34, 1636-1638.(12) Damtoft, S.Phytochemistry1994, 35, 1187-1189.(13) Kang, K. H.; Huh, H.; Kim, B. K.; Lee, C. K.Phytother. Res. 1999,

13, 624-626.(14) Jiang, T. F.; Du, X.; Shi, Y. P.Chromatographia2004, 59, 255-

258.(15) Mabry, T. J.; Markham, K. R.; Thomas, M. B.The Systematic

Identification of FlaVonoids; Springer-Verlag: New York, 1969.(16) Fabre, N.; Rustan, I.; De Hoffmann, E.; Quentin-Leclercq, J.J. Am.

Soc. Mass Spectrom.2001, 12, 707-715.(17) Davis, A. L.; Laue, E. D.; Keeler, J.; Moskau, D.; Lohman, J.J.

Magn. Reson. 1991, 94, 637-644.(18) Willker, W.; Leibfritz, D.; Kerssebaum, R.; Bermel, W.Magn. Reson.

Chem. 1993, 31, 287-292.(19) Wang, Y.; Xu, K.; Lin, L.; Pan, Y.; Zheng, X.Phytochemistry2007,

68, 1300-1306.(20) Mbwambo, Z. H.; Kapingu, M. C.; Moshi, M. J.; Machumi, F.; Apers,

S.; Cos, P.; Ferreira, D.; Marais, J. P. J.; Vanden Berghe, D.; Maes,L.; Vlietinck, A.; Pieters, L.J. Nat. Prod. 2006, 69, 369-372.

(21) Ferrari, J.; Terreaux, C.; Kurta´n, T.; Szikzai-Kiss, A.; Antus, S.;Msonthi, J. D.; Hostettmann, K.HelV. Chim. Acta2003, 86, 2768-2778.

(22) Yang, X.; Sun, Y.; Xu, Q.; Guo, Z.Org. Biomol. Chem.2006, 4,2483-2491.

(23) Hare, J. D.; Borchardt, D. B.Phytochemistry2002, 59, 375-378.(24) Philips, W. R.; Bay, N. J.; Gunatilaka, A. A. L.; Kingston, D. G. I.

J. Nat. Prod.1996, 59, 495-497.(25) Narvaez-Mustache, M. C.; Gardun˜o-Ramı´rez, M. L.; Alvarez, L.;

Delgado, G.J. Nat. Prod.2006, 69, 1687-1691.(26) Toth, L.; Bulyaki, M.; Bujtas, G. Y.Pharmazie1981, 36, 577.(27) Slade, D.; Ferreira, D.; Marais, J. P. J.Phytochemistry2005, 66,

2177-2215.(28) Seyoum, A.; Asres, K.; El-Fiky Kandeel, F.Phytochemistry2006,

67, 2058-2070.(29) Cao, G.; Sofic, E.; Prior, R. L.Free Radical Biol. Med. 1997, 22,

749-760.(30) Braca, A.; De Tommasi, N.; Di Bari, L.; Pizza, C.; Politi, M.; Morelli,

I. J. Nat. Prod.2001, 64, 892-895.(31) Babich, H.; Goldstein, S. H.; Borenfreund, E.Toxicol. Lett.1990,

50, 143-149.(32) Ko, C. H.; Shen, S. C.; Chen, Y. C.Free Radical Biol. Med.2004,

36, 897-910.(33) Shen, S. C.; Ko, C. H.; Tseng, S. W.; Tsai, S. H.; Chen, Y. C.Toxicol.

Appl. Pharmacol.2004, 197, 84-95.

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1248 Journal of Natural Products, 2007, Vol. 70, No. 8 Smejkal et al.

125

Conclusions

Results of all three projects described in this thesis have been summarized

in papers published in journals Phytochemistry, Magnetic Resonance

in Chemistry, and Journal of Natural Products.

Our current knowledge about natural occurrence, chemical reactions,

structural parameters, and biological activities of quaternary protoberberine

alkaloids has been collected in a review article. An experimental study

of nucleophilic additions of azoles to the QPA brought new information

about chemical behavior of these alkaloids and estimated the capability of the

QPA to form a covalent bonding with nucleic acids.

NMR determination of pKa values for cryptolepines provided one of the

important characteristics in the study of their antimalarial properties. New

natural compounds from Pavlownia tomentosa have been isolated,

characterized, and tested for various biological effects.

Studies of natural products still represent a very promising way to

discover pharmaceutically interesting substances important for the potential

treatment of a wide variety of diseases. If even an imperceptible part of the

results presented in this thesis contributes to saving a life of just one person

around the world, the work on these projects was not useless.

126

Curriculum vitae

Date and place of birth: February 26, 1978, Brno, Czech Republic Married (nee Baráková)

Current position: Product manager, AROMATICA CZ s.r.o., Masarykovo nám. 101/3, CZ-664 51 Šlapanice

and PhD student, National Center for Biomolecular Research, Institute of Biochemistry, Faculty of Science, Masaryk University, Kamenice 5/A4, CZ-62500 Brno, Czech Republic

and

External author of Czech pharmaceutical journal Pharmanews Education: Mgr. 2003 Organic Chemistry, Masaryk University, Brno, Czech Republic “Photochemistry of organic compounds in polycrystalic ice” RNDr. 2007 Organic Chemistry, Masaryk University, Brno, Czech Republic “Quaternary protoberberine alkaloids”

Professional interest:

Antitumor agens, isolations, natural products, alkaloids

Curriculum vitae

____________________________________________________________________________

127

Stays abroad:

September 2002-January 2003 SOCRATES ERASMUS program grant Study and research stay, University of Antwerp (Antwerp, B) “Bioassay guided isolation of anti-HIV active

compounds from the methanol extract of Aleurites

moluccana husk” September - December 2007 Flemish fellowship program grant

Research stay, University of Antwerp (Antwerp, B) project

“Using of NMR for determination of pKa values

of indoloquinoline alkaloids“

Scientific Publications:

R. Růžička, Baráková L., Klán P., 2005: Photodecarbonylation of Dibenzyl Ketones and Trapping of Radical Intermediates by Copper(II) Chloride in Frozen Aqueous Solutions, J. Phys. Chem. B., 109, 9346-9353. Grycová L., Dostál J., Marek R. Quaternary protoberberine alkaloids. Phytochemistry, 2007, 68, 150-175. Šmejkal K., Grycová L., Marek R., Lemière F., Jankovská D., Forejtníková H., Vančo J., Suchý V., C-geranyl compounds from Paulownia tomentosa (Scrophulariaceae) fruits. J. Nat. Prod., 2007, 70, 1244-1248. Grycová L., Hulová D. Maier L., Standara S., Nečas M., Lemiére F., Kareš R., Dostál J., Marek R., Covalent bonding of azoles to quaternary protoberberine alkaloids. Magn. Res. Chem., 2008, 46, 1127-1134. Grycová L., Dommisse R., Pieters L., Marek R., NMR determination of pKa values of indoloquinoline alkaloids, Magn. Res. Chem., 2009, 47, 977-981. Barák M., Merta J., Merta O., Grycová L., Experimentální tavby železa ve Staré

huti u Adamova v sezónách 2008 a 2009. ISBN 978-80-86413-69-3, Archeologia

technica, 2010, 21, 5-24.

128

List of publications

Grycová L., Dostál J., Marek R. Quaternary protoberberine alkaloids. Phytochemistry, 2007, 68, 150-175. Šmejkal K., Grycová L., Marek R., Lemière F., Jankovská D., Forejtníková H., Vančo J., Suchý V., C-geranyl compounds from Paulownia tomentosa (Scrophulariaceae) fruits. J. Nat. Prod., 2007, 70, 1244-1248. Grycová L., Hulová D. Maier L., Standara S., Nečas M., Lemiére F., Kareš R., Dostál J., Marek R., Covalent bonding of azoles to quaternary protoberberine alkaloids. Magn. Res. Chem., 2008, 46, 1127-1134. Grycová L., Dommisse R., Pieters L., Marek R., NMR determination of pKa values of indoloquinoline alkaloids, Magn. Res. Chem., 2009, 47, 977-981.

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