Novel Nicotinic Acetylcholine Receptor Ligands Based

In vitro

Dissertation

Lenka Munoz

For Alex

Table of Contents

π π π π π

t 27

93e 103e 117e 123e 118e 124e 119e 125e 120e 121e 122e 126e In Vitro

In Vitro

27 t 76 t 81 t 82 t 83 93e 94e 95e 96e 97e 98e 99e 100e 103e 104e 105e 106e 107e 108e 109e 110e H 117e 118e 119e 120e 121e H 122e H 123e 124e 125e H 126e 128e 129e

136 137 138 139 140 141 144 145 In Vitro

1 1 Introduction

1.1 Nicotinic Acetylcholine Receptors

1.1.1 The Structure of nAChRs

Figure 1-1 α β γ δ

2

β β α ββ α α

Figure 1-2 β β

3

Torpedo californica α β δ γ ε γ ε

α ααα ββ α

β αβ α β α α αβ Xenopus α β

αα α

αα γε β) α

α α γ δ ε β α

Lymnaea stagnalis

4

α- π π

1.1.2 nAChRs in Human Pathology

Tourette’s syndrome,

Schizophrenia, α α

Epilepsy α β

5

Depression/anxiety

Alzheimer’s disease (AD) αβ β α αβ α

Parkinson’s disease (PD)

Pain

Tobacco smoking,

6

1.2 Nicotinic Acetylcholine Receptors Ligands 1 π

µ α

acetylcholine 1 π

Figure 1-3 1 π

7

1.2.1 Class A: Acyclic HBA/ π and Acyclic Cation

π Class A

1 αβ

α 1 1

αβ 2 2 2 2

αβ 2

3 1

α µ

3 αβ µ

2 3

(α4β2 , ) (α4β2 , ) (α7 ) (α7 )

Figure 1-4 2 3

8

1.2.2 Class B: Cyclic HBA/ π and Acyclic Cation

Class B π

4, 4 αβ α 4

5 α αβ

et al 3-(N-methyl-N-ethylaminomethyl)pyridine 6

trans 7 [E]-N-methyl-4-[3-pyridinyl]-3-butene-1- amine monofumarate 7

αβ αβ trans 7 trans 7

7 α trans 8 (S)-(E)-N-methyl-5-[3-(5- isopropoxypyridinyl)]-4-penten-2-amine αβ αβ α α

9

4 5 6 (α4β2 , ) (α4β2 , ) (α4β2 ) (α7 ) (α7 )

7 8

(α4β2 ) (α4β2 ) (α7 )

Figure 1-5 Class B

1.2.3 Class C: Cyclic HBA/ π and Cyclic Cation

Class C π Class C

9 N1-dimethyl-N4-phenylpiperazinium iodide 1.3 nAChR Pharmacophore Models 9

αβ α µ

8 9 9 αβ

10 (S)-3-(1-methyl-2-pyrrolidinyl)-pyridine αβ αβ

10 

11 et al 11 12

10 αβ α 11 1210 12 α 10 1 α 13 α in vivo

10 14 (S)-(-)-5- ethynyl-3-(1-methyl-2-pyrrolidinyl)-pyridine

10 14

10 αβ

αβ 10

15 15

11

S 10 11

(α4β2) (α4β2 )

(α7) (α7 ) 9

(α4β2 )

(α7 )

12 13 14

(α4β2 ) (α4β2 ) (α4β2 )

(α7 ) (α7 )

15 16 17 α4β2 (α4β2 ) (α4β2 ) α7 (α7 ) (α7 )

18 (α4β2 ) 19 20 α4β2 (α4β2 (α4β2 ) (α7 ) α4β2 α7 (α7 )

Figure 1-6 Class C π

12 

16 3-((1-methyl-2-(S)-pyrrolidinyl)-methoxy)-pyridine αβ 173-(2-(S)-azetidinylmethoxy)-pyridine

αβ α 1710 α in vivo 18 5-(2-(R)-azetidinylmethoxy)-2-chloropyridine 18 αβ

αβδγ 18 in vitro

Class C 19 exo-2-(6-chloro-3- pyridyl)-7-azabicyclo-[2.2.1]heptane 19 S S R 19 R R S αβ 19 20

13

1.2.4 Class D: Acyclic HBA/ π and Cyclic Cation

Class D π

21 R S Anabaena flos-aquae 21

αβ α 21 αβ

α 21 α β 21

21 22 Darlingia ferruginea darlingiana 22 22 αβ

α 21 22 Class C

α β

αβ α αβ α

23 2-[6-((S)-2-hydroxy-2-phenylethyl)-1-methyl- piperidin-2-yl]-1-phenylethanone Lobelia inflata 23 α β 10 10 23 23

14 

21 (α4β2 ) (α7 ) 22

(α4β2 ) (α7 ) 24

(α4β2 )

(α7 )

(α4β2 ) (α7 )

Figure 1-7 Class D

10 23 10 in vitro 2310 23 π 23

15

Class D 24 Delphinium brownii 24 α α α 24 α

1.2.5 Class E: HBA/ π and Cation in Fused Ring System

Class E π

β 25 π β 25 αβ

26 (-)-spiro[1-azabicyclo[2.2.2]octane-3,5’-oxazolidin-2’-one] α

β 25 26 27

(α4β2) (α4β2 ) (α4β2 )

(α7 ) (α7 )

Figure 1-8 Class E π

16 

α αβ

26 26 α α

17

1.3 nAChR Pharmacophore Models

1 10 1 10

Å Å

Figure 1-9 Figure 1-10

18 

a b Point a b a---b

Å Å

b Å Site Point b

a Å

Site Point a

Figure 1-11

19 αβ 17 19 1719 et al 17 19 17 30 31

19

Table 1-1 αβ

N --- N distance Ki α4β2 Ligand Reference [Å] [nM]

+ N N 9

N N 17*

N N 10

N N 19

N N 28

N N 17*

N HN 29

H N N 30

+ N 31

20 

29 αβ et al

28 itself 32

αβ

33 Figure 1-12

Figure 1-13 10

21 2 Objectives

αβ α

αβ α βγδ

27 3

Project I

27 αβ 27 27

αβ αβ α

22 

Figure 2-1

3 5

Figure 2-2

27 27 27

23

αβ

α αβ α βγδ

Project II

αβ α α

3 1 α

α µ 3 α 3 α

4 αβ α 5 α

24 

Project

Structure of target compounds

H O N R

N O

Figure 2-3 αβ

ααβ α βγδ in vitro

25

3 Project I: Development of Novel nAChR Ligands based on Cytisine

3.1 Cytisine as a Lead Compound

3.1.1 Introduction

27 Laburnum anagyroides Cytisus laburnum 27 Ulex europeus Baptisia tinctoria. 27

27((7R,9S)-1,2,3,4,5,6-hexahydro-1,5-methano-pyrido[1,2-a][1,5]diazocin-8-one) 27 27 27 R S S R 27 R S

12 3 A 13 4 1 2 9 11 2 5 7 8 B 10 8 7 3 6 1 9 C 6 11a 5 4 11 10

Figure 3-1 27 RS

26 

27 Laburnum, L. anagyroides alpinum Laburnum x watereri) Laburnum 27

Picture 3-1 Laburnum anagyroides

3.1.2 Pharmacological Characterisation of Cytisine

10 27 αβ et al 27 αβ Xenopus laevis 27 27 αβ

αβ α β

27

Table 3-1

α4β2 [nM] Tissue Reference

Xenopus laevis αβ α7 [nM] Tissue Reference

α α3β4 [nM] Tissue Reference

Xenopus laevis αβ

(α1)2β1γδ [nM] Tissue Reference

Torpedo californica

27 27 α

α α µ

Xenopus laevis µ

27 αβ

27 αβγδ et al α

α β Xenopus 27

28 

β 27 β β α α α β 27 1 α α α β 271

27 β αβ1 µαβ 27 αβ 271

Xenopus 27 β αβ αβ αβ β αβ αβ αβ27 α

27 19 αβ αβ19

27 27 10 et al 27 10 27

k 10 k 27

3.1.3 Radioligand [ 3H]Cytisine

27 αβ 27 27 αβ

29

27 27 27 et al 27 27

27 Ex vivo 27 27

3.1.4 Cytisine in Human Medicine

27 27

27 34

30 

27 

36 27, in vivo 36

34 35 36

Figure 3-2 3435 36

3.1.5 Total Synthesis of Cytisine

27 27 27

27 27

31

27 37 38

Scheme 3-1 27 37 37 38 27 27 37 et al 27

27 27 27

32 

3.1.6 Modification of the Cytisine Scaffold

27 46 27 et al 39 40 41 4243 4445 46 47 48

39/40/41 42/43/44 45/46/47

Scheme 3-2

39 – 47 t

27 27 27

33

27 27

27 49 50

Scheme 3-3 27

51 52 27 53 54 55 56

34 

51 52 53

54 55 56 Figure 3-3 51 – 53 5456 27

27 αβ 57 27

Scheme 3-4 57

35

3.1.7 Structure-Activity Relationship of Cytisine Derivatives

27 27

SAR for α4β2 nAChR subtype

27 αβ 2753

49 50

39 41 αβ 40

αβ 2719 αβ

42 44 45 47

40 43 47 αβ

43 µ 46 µ27

40 41 40 αβ 27

44 27 43

36 

27 58 59 αβ

51 27 51 αβ

α 27 αβ

Table 3-2 αβ

R1 R2 R3 X Ki [nM]

et al αβ

37

Table 3-3 57 αβ

Compound R1 R2 R3 R4 Ki [nM] 27 57a 57b 57c 57d 57e

57f

57g

57h 57i 57j

αβ 57b 57i

27 57c 57d 57i

Figure 3-3 27 αβ

38 

SAR for α7 nAChR subtype

27 α αβ

α39 40 41 42 – 44 αβ 27 α 42 α 43 44 27 45 46

α 47

et al α α 40 43 46 α

46 µ 43 µ 27 µ 40 41

27 44 α 27 µ

α 58 59

α51 α 51 αβ α 51 51 27

39

57 57c 57d 57i α

27 57e α

α 27

27 α

Figure 3-4 27 α

SAR for α3β4 nAChR subtype 27 αβ 53vs.

27 39 40 41 42 – 44 27

42 43 44

58 27

αβ 59 27

40 

52 51

αβ 27

57 αβ 27

57f 57d 57e 57i αβ 27

27 αβ

Figure 3-5 27 αβ

SAR for (α1)2β1γδ nAChRs

27 27

40 41 27

41

3.2 Syntheses of Novel nAChRs based on Cytisine

3.2.1 Suzuki Cross-Coupling Reaction

42 

Scheme 3-5

3.2.1.1 Mechanism

61 62

trans 60

60 63 61

43

60 61

(B)

Scheme 3-6 60

61 62 cis trans trans cis cis

44 

3.2.1.2 Reaction Conditions

3.2.1.2.1 Palladium Catalyst

in situ

3.2.1.2.2 Base

45

60 63 61

Scheme 3-7

3.2.1.2.3 Organoboron Coupling Partner

Preparation and Coupling of Aryl- and Alkenylboron Derivatives

46 

C Scheme 3-8

Preparation and Coupling of Alkynylborane Derivatives

64 in situ 65 66

65 64 B

66 Scheme 3-9 64 66

47

Preparation and Coupling of Alkylboron Derivatives

67 68

67 68 Scheme 3-10 68 β

69 70 cis 71

69 71

Scheme 3-11

48 

3.2.1.2.4 Organic Halides / Pseudohalides as Coupling Partners

Coupling of Aryl Halides

Scheme 3-12

Coupling of Alkenyl Halides

49

Coupling of Alkyl Halides

Coupling of Triflates

3.2.2 Suzuki vs. Stille Cross-Coupling Reaction

Scheme 3-13

50 

72 19

72 19 Scheme 3-14 19

3.2.3 Suzuki Reaction in Microwave Assisted Organic Synthesis

51

3.2.3.1 Microwave

3.2.3.2 Microwaves as a Heating Source in Organic Synthesis All the chemistry operations could be reduced to decomposition and combination; hence, the fire appears as an universal agent in chemistry as in nature

52 

Figure 3-6

ε´ ε´´ tan δ

δ ε´´ ε´ tan δ

53

Table 3-4 δ Solvent tan δ Solvent tan δ tan δ tan δ tan δ

54 

3.2.3.3 Microwaves in Suzuki Cross-coupling Reaction 7374

73 74 Scheme 3-15 δ

Scheme 3-16

55

75 74

75 74 Scheme 3-17

56 

3.2.4 Isolation of Cytisine

3.2.4.1 Introduction

27 Laburnum anagyroides medicus Fabaceae) 27

27 L. anagyroides 27 L. anagyroides Laburnum watereri 27 27

Table 3-5 27 Laburnum anagyroides

Bojadshiewa et Marriére et al. Klaperski al.

seeds dissolved in

alkalization

acidification

alkalization

extraction

flash chromatography recrystallization

3.2.4.2 Method / Results

27 Laburnum anagyroides watereri in vacuo

57

3.2.4.3 Discussion

et al

27 27 27

3.2.5 Protection of the Secondary Amino Group of Cytisine

3.2.5.1 Introduction

tert t tert t t t 27

58 

t 76 76 tert 27

3.2.5.2 Method / Results

t 76 27 tert

27 76

Scheme 3-18 t 76

27 tert 76

27 t 76 27

59

Table 3-6 27 t 76 27 Experiment t 76 27 Yields 1 2 3 4 5

3.2.5.3 Discussion

27 76

27 tert 76 2727 t 76

3.2.6 2-Pyridone Scaffold 27

77 Camptotheca acuminata (Nyssaceae) 78

60 

7980

77 78

79 80

Figure 3-7

61

3.2.7 Bromination of Cytisine

3.2.7.1 Introduction

t76 27

27 t

et al

27 46 40 43 27 46

2776

62 

3.2.7.2 Method / Results

t 81 t 82

76 81 82

Scheme 3-19 81 82

81 82

83 81 82

Table 3-7 81 83

Cpd. R1 R2 Yields 81 82 83

63

3.2.7.3 Discussion

t 81 t 82

81 82

81 82 81 82

81 82 t t

t82

Table 3-8 3-bromo 5-bromo 3,5-dibromo Protecting 3- / 5- Solvent isomer isomer analogue group ratio [%] [%] [%] t

64 

83 81 82 83 t83

3.2.8 3-Phenyl Analogues of Cytisine

3.2.8.1 Introduction

t 84 81 85 86

86 85 81 84

Scheme 3-20 87

65

88 89 90 88

89 90

Scheme 3-21

91 91 92

66 

91 92

Scheme 3-22 91

3.2.8.2 Method / Results

∗

81 74 t 93 – 100

81 93 94 95 96

97 98 99 100 Scheme 3-23 t 81

∗ t 93 t 99

67

3.2.8.3 Discussion

27 t 81 t

93 – 100 84

68 

73 74 t 81

3.2.9 5-Phenyl Analogues of Cytisine

3.2.9.1 Introduction

t t 82

Scheme 3-24 t82

101

102

69

101 102

Scheme 3-25 101

3.2.9.2 Method / Results

103 100 108

t 108,

103 104 105 106 107 108 109 110

Scheme 3-26 t 82

70 

3.2.9.3 Discussion

t 82 t108

Table 3-9

t * Yields** t * Yields** Compound r R Compound r [min] [%] [min] [%] 93 103 94 104 95 105 96 106 97 107 98 108 99 109 100 110

71

t 95 105 µ

109t 99

72 

3.2.10 Heterocyclic Derivatives of Cytisine

3.2.10.1 Introduction

t 84 t 81 tert 111

81 111

Scheme 3-27 111 112 113 114 115 116

113 115 112 5

114 116 112

Scheme 3-28 t 112

73

3.2.10.2 Method / Results

81 82 117 123 118 t 82

124

t 119 125 H

122 126

120 121

81 82

74 

117 119 121

118 120 122

Scheme 3-29

117 126

t 81 82

75

123 124 125 126

Scheme 3-30

76 

Table 3-10 117 126

t * t * Yields** Compound r Yields** R Compound r [min] [%] [min] [%]

117 123 118 124 119 125 120 121

122 126

3.2.10.3 Discussion

27 117 – 126

117126

77

t120 127

+ 127

+ + + + Scheme 3-31 120

127

3.2.11 3,5-Disubstituted Analogues of Cytisine

3.2.11.1 Introduction

45 45

78 

t 83 trans

3.2.11.2 Method / Results 128 129 t 83 74

74 128 83

129

Scheme 3-32 t83

3.2.11.3 Discussion

79

128 129

130 131

Scheme 3-33

3.2.12 Removal of the tBOC Protecting Group

3.2.12.1 Introduction

t t

t t t p

80 

3.2.12.2 Method/Results

t 93e 100e 103e 110e

117e 126e

93e 100e 103e 110e 117e 122e 123e 126e 128e 129e

Scheme 3-34 t

3.2.12.3 Discussion

93e 100e 103e110e

81

t 117e126e

t t t

82 

3.3 1H and 13 C NMR Chemical Shifts Assignment for the Novel nAChRs Ligands

3.3.1 Overview of used NMR Spectroscopy Methods 1H NMR 13 C NMR

Single-Frequency Decoupling

Distortionless Enhancement by Polarization Transfer

1D NMR 2D NMR

Co rrelated Spectroscopy

2D-Incredible Natural Abundance Double Qua ntum Transfer Experiment

83

heteronuclear correlation experiments Heteronuclear Single Quantum Coherence

Heteronuclear Multiple Bond Correlation

3.3.2 Project 27 27

27 27 27 27 27 27

81 82 δ δ

84 

Table 3-11 25

12 11 1 13 2 9 10 8 3 7 6 5 4

C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C13

27 27

93e 103e 117e 119e 121e 124e

85

3.3.3 Spectral Assignments of 1H and 13 C Chemical Shifts

3.3.3.1 Cytisine 27

27 27 sp 2 sp 3 sp 3 p2 δ δ

δ 27 δ δ δ δ δ δ δ

δ β

α δ δ δ

α β δ

δ δ δ δ δ δ

δ δ δ δ

86 

Figure 3-8 27

87

β α

α β α α β α

12 11

1 13 2 9 10 8 3 7 6 5 4

Figure 3-9 27

δ δ δ δ δ δ

88 

δ δ

δ δ δ δ δ δ δ

δδ δ δ δ δ δ

δ δ

89

12 11 1 13 2 9 10 8 3 7 6 5 4

Figure 3-10 27

90 

11 13 9 10 2 3 8 7 6 5 4

α β β α

Figure 3-11 27

91

Figure 3-12 27

92 

3 B 7 5

C 3 7 5

Figure 3-13 A B δ C 27

93

δ δ δ δ

∆ δ ∆ δ

27 ∆δ

Table 3-12 27

δ δ δ δ H3 C2 H4 C3 H5 C4 H7 C5

H8 A C6

H8 B C7 H9 C8 H10 α C9 H10 β C10 H11 C11 H13 C13

94 

27 et al

2.73 - 2.83 52.3 2.73 - 2.83 53.3 2.08 3.85/3.63 27.0 49.1 162.8

1.70 2.69 6.17 25.6 34.9 150.7 115.7

5.77 7.05 104.2 138.1 Figure 3-14 δ 27

95

3.3.3.2 3-Phenyl- and 5-Phenyl-cytisine 93e & 103e 93e 103e ∆δ 27

93e 103e δ 27 27

Table 3-13 27 93e 103e 27 93e 103e 27 93e 103e δ δ δ δ δ δ

H3 C2

H4 C3

H5 C4

H7 C5

H8 A C6

H8 B C7

H9 C8

H10 α C9

H10 β C10

H11 A C11

H11 B C13

H13 A C1’

H13 B C2’

H2’ C3’

H3’ C4’

H4’ C5’

H5’ C6’

H6’

96 

93e 103e δ δ

A B 8 8 δ 27

C D

8 8 5 3 δ δ δ

93e 103e

Figure 3-15 A 27 B 93e C 103e D

103e 27 δ 93e δ 103e δ δ δ δ

93e 103e 93e δ 103e δ

97

β α

α β

11 13 2 2' 3' 9 10 3 1' 8 7 4' 6

5 6' 5' 4

Figure 3-16 93e

93e

93e 103e

98 

α β

11 13 2 9 10 3 8 7 6

5 4 1' 2' 3' 6'

4' 5'

Figure 3-17 103e

∆δ ∆δ 27 ∆δ ∆δ93e 27 93e 103e

99

11 13 2 2' 3' 9 10 3 1' 8 7 4' 6

5 4 6' 5'

α β

Figure 3-18 93e

100 

11 13 2 9 10 3 8 7 6

5 4 1' 2' 3' 6'

4' 5'

Figure 3-19 103e

101

11 13 2 2' 3' 9 10 3 8 4' 7 6 1'

5 4 6' 5'

Figure 3-20 93e

102 

2 6 3 5 4 1' 2'

3' 6'

4' 5'

Figure 3-21 103e

103

93e 103e 103e 93e 93e

93e 103e

103e 93e 27,

δ 93e 103e δ

93e 27 δ δ 103e ∆δ δ

δ δ δ

δ δ

93e 103e

Figure 3-22 93e 103e

104 

3.3.3.3 3-Aryl Analogues of Cytisine

94e 100e 93e

94e 100e 94e 100e ∆δ 93e 94e 100e

93e 103e 94e100e δ δ

94e 100e 94e 100e 93e 94e 100e δ 93e δ

94e100e α δ α 99e α ∆ δ 94e 97e ∆δ ∆δ 98e 95e

1 Table 3-14 H chemical shifts of 3-aryl derivatives of cytisine 93e – 100e (CDCl3, 500 MHz)

11 NH O R 13 2 2' 9 10 N 3' 1' 8 7 4' 6 3 5 4 6' 5'

1H chemical shifts [ppm] R = H 93e NO2 94e CH3 95e CF3 96e OCF3 97e Cl 98e F 99e Ph 100e H4 7.46 (d) 7.54 (d) 7.43 (d) 7.49 (d) 7.45 (d) 7.44 (d) 7.45 (d) 7.51 (d) H5 6.09 (d) 6.13 (d) 6.06 (d) 6.11 (d) 6.07 (d) 6.08 (d) 6.08 (d) 6.09 (d) H7 2.91 (br s) 2.95 (br s) 2.91 (br s) 2.94 (br s) 2.91 (br s) 2.92 (br s) 2.92 (br s) 2.93 (br s) H8 1.96 (br s) 1.97 (br s) 1.97 (br s) 1.96 (br s) 1.95 (br s) 1.96 (br s) 1.96 (br s) 1.97 (br s) H9 2.34 (br s) 2.38 (br s) 2.34 (br s) 2.35 (br s) 2.35 (br s) 2.35 (br s) 2.36 (br s) 2.35 (br s) H10 3.96 (dd) 3.95 (dd) 3.94 (dd) 3.95 (dd) 3.93 (dd) 3.94 (dd) 3.93 (dd) 3.97 (dd) H10! 4.19 (d) 4.17 (d) 4.18 (d) 4.16 (d) 4.17 (d) 4.16 (d) 4.17 (d) 4.19 (d)

H11A 3.03 (d) 2.98 – 3.14 3.00 – 3.11 2.99 – 3.13 2.95 – 3.12 3.11 (d) 3.12 (d) 3.00 – 3.13

H11B 3.02 (d) (m) (m) (m) (m) 3.00 (d) 3.01(d) (m)

H13A 3.07 (dd) 2.98 – 3.14 3.00 – 3.11 2.99 – 3.13 2.95 – 3.12 3.06 (dd) 3.06 (dd) 3.00 – 3.13

H13B 3.02 (d) (m) (m) (m) (m) 3.00 (d) 2.98 (d) (m) H2´ 7.69 (dt) 8.53 (t) 7.52 (br s) 7.95 (s) 7.58 (br s) 7.69 (t) 7.43 (br d) 7.91 (t) H3´ 7.38 ------H4´ 7.29 (tt) 8.11 (m ovl) 7.09 (d) 7.52 (d) 7.11 (dqui) 7.58 (dt) 6.96 (tdd) 7.51 (dd) H5´ 7.38 (t) 7.52 (t) 7.26 (t) 7.47 (t) 7.38 (t) 7.28 (d) 7.32 (dt) 7.44 (t) H6’ 7.69 (dt) 8.11 (m ovl) 7.43 (d) 7.91 (d) 7.63 (ddd) 7.23 (dd) 7.43 (br d) 7.69 (ddd) 13 Table 3-15 C chemical shifts of 3-aryl derivatives of cytisine 93e – 100e (CDCl3,125 MHz)

11 NH O R 13 2 2' 9 10 N 3' 1' 8 7 4' 6 3 5 4 6' 5'

13C chemical shifts [ppm]

R = H 93e NO2 94e CH3 95e CF3 96e OCF3 97e Cl 98e F 99e Ph 100e C2 162.1 161.7 162.2 161.9 161.8 161.8 161.9 162.1 C3 127.4 124.7 127.6 125.8 125.6 125.9 126.0 129.1 C4 137.4 137.6 137.0 137.4 137.3 137.3 137.3 137.6 C5 105.0 105.0 105.0 104.9 105.0 104.9 105.0 105.3 C6 150.3 152.0 150.0 151.4 151.1 151.0 150.7 150.4 C7 35.7 35.7 35.6 35.7 35.6 35.7 35.5 35.3 C8 26.3 26.1 26.3 26.2 26.1 26.2 26.1 26.3 C9 27.9 27.7 27.8 27.8 27.7 27.8 27.7 27.5 C10 50.2 50.3 50.1 50.3 50.2 50.2 50.1 50.2 C11 54.0 52.9 52.8 53.0 52.8 53.0 52.7 53.0 C13 53.0 53.8 53.9 54.0 53.7 53.9 53.6 53.9 C1’ 137.0 139.0 137.5 138.1 139.3 139.1 139.4 137.8 C2’ 128.6 123.3 129.3 123.8 119.5 128.6 114.0 ---* C3’ 128.0 148.2 137.3 130.4 149.0 133.9 163.6 141.4 C4’ 127.2 121.9 128.1 125.3 121.1 127.2 115.6 ---* C5’ 128.0 128.9 128.0 128.4 129.2 129.2 129.4 ---* C6’ 128.6 134.7 125.7 131.9 126.9 126.7 124.1 ---* *) the signals of the biphenyl moiety could not be unambiguously assigned Cytisine: NMR spectroscopy of novel ligands 107 phenyl attachment (entry 100e ) both caused downfield shifts ( ∆ δ 9.3 and 13.4 ppm, respectively), owing to the rule δ(C tert ) < δ(C quart ).

The large deshielding effect observed on the 13 C shift of a carbon atom directly attached to a halogen atom (e.g. fluorine) or to a substituent with negative inductive effect (e.g. nitro or trifluoromethoxy group) does not apply to the chemical shifts of the β- and γ-carbons. In the cases of the electronegative substitution, the 13 C chemical shifts of the corresponding γ- carbons are reported to move upfield. 286 Indeed, the 13 C chemical shift of C5’ in 3-(3’- fluorophenyl)-cytisine 99e showed an upfield shift of ∆ δ 1.4 ppm. The SCSs (i.e. the additivity increments) of various groups for the 13 C chemical shifts of the phenyl carbons in the series of 3-aryl-cytisine 93e – 99e are given in Table 3-16. The data found are in 286 agreement with the literature SCSs data. The additivity increments for OCF 3 substitution (97e ) are not listed, since the literature data for SCSs of a trifluoromethoxy group are still not available.

Table 3-16 Additivity increments [ppm] of various groups for the 13 C chemical shifts of the phenyl carbons in the series of 3-aryl cytisine derivatives ( 94e – 96e and 98e – 99e ), literature SCSs values shown in parentheses 286 NH O R 2' 3' N 1' 4'

6' 5'

Additivity Increments [ppm]

R = H 93e NO 2 94e CH 3 95e CF 3 96e Cl 98e F 99e [δ,ppm] C1’ 137.00 2.0 (0.8) 0.5 (0.0) 1.1 (-0.3) 2.1 (1.0) 2.4 (0.9)

C2’ 128.6 -5.3 (-5.3) 0.7 (0.6) -4.8 (-2.6) 0.0 (0.2) -14.6 ( -14.3)

C3’ 128.0 20.2 (19.6) 9.3 (9.3) 2.4 (2.6) 5.9 (6.4) 35.6 (35.1)

C4’ 127.2 -5.3 (-5.3) 0.9 (0.6) -1.9 (-2.6) 0.0 (0.2) -11.6 (-14.3)

C5’ 128.0 0.9 (0.6) 0.0 (0.0) 0.4 (-0.3) 1.2 (1.0) 1.4 (0.9)

C6’ 128.6 6.1 (6.0) -2.9 (-3.1) 3.3 (-3.2) -1.9 (-2.0) -4.5 (-4.4)

The 13 C chemical shift assignment of the biphenyl substituent in 100e remains incomplete, as nine tertiary carbons of the biphenyl moiety possess nearly identical chemical shifts in the range δ 125.5 ppm to δ 127.6 ppm. Only the assignments of the quaternary carbons C1’ ( δ 137.8 ppm), C3’ ( δ 141.4 ppm) and C1’’ ( δ 141.0 ppm) could be estimated, however, they might be reversed. Due to the small amount of the substance, detailed 2D NMR correlation experiments (e.g. HSQC or HMBC) required for a complete and final assignment were not measured.

108 Cytisine: NMR spectroscopy of novel ligands

In the 13 C NMR spectrum of 3-(3’-fluorophenyl)-cytisine 99e a doublet was observed at δ 1 163.6 ppm, corresponding to an aromatic C-F coupling, with a JC,F = 244.1 Hz. Generally, the long range coupling of fluorine with their neighbouring aromatic carbons greatly facilitate the assignment of the remaining 13 C chemical shifts. The fluorine-carbon couplings observed 2 2 here ranged over two bonds to C2’ ( JC,F = 21.2 Hz) and C4’ ( JC,F = 22.4 Hz), over three 3 3 bonds to C1’ ( JC,F = 8.2 Hz) and to C5’ ( JC,F = 8.5 Hz) as well as over four bonds to C6’ 4 4 286 ( JC,F = 2.7 Hz) and C3 ( JC,F = 2.3 Hz) and all of them agreed with literature citation (Table 3-17).

Table 3-17 13 C -19 F Coupling constants in 3-(3’-fluorophenyl)-cytisine 99e, the respective coupling carbon atom given in parentheses

13 C -19 F Coupling constant [Hz] literature 286 found NH O F 1 2' 3' JC,F 245 244.1 (C3’) N 1' 2 JC,F 21 21.2 (C2’) 4'

22.4 (C4’)

6' 5' 3 JC,F 8 8.2 (C1’)

99e 8.5 (C5’)

4 3 2.3 (C3) JC,F 2.7 (C6’)

Also in the 13 C NMR spectra of 3-(3’-trifluoromethyl-phenyl)-cytisine 96e and 3-(3’- trifluomethoxy-phenyl)-cytisine 97e, the 13 C-19 F couplings were apparent and raised several 1 2 3 quartets. The characteristic coupling constants ( JC,F = 272.5 Hz; JC,F = 31.7 Hz; JC,F = 3.7 Hz) found in the spectrum of 96e originate from the direct and the long range couplings between fluorine and its adjacent carbons. The constants are nearly identical with the literature values reported (Table 3-18). The quartet at δ 121.2 ppm in the 13 C chemical shifts 1 spectrum of 3-(3’-trifluoromethoxyphenyl)-cytisine 97e with coupling constant of JC,F = 257 Hz was assigned to the carbon of the trifluoromethoxy group. Additionally, in the spectrum of 97e a long range 13 C3’-19 F coupling was observed ( 3J = 1.5 Hz).

Table 3-18 13 C -19 F Coupling constants in 3-(3’-trifluoromethyl-phenyl)-cytisine 96e

NH 13 -19 O CF3 C F Coupling constant [Hz] 2' 3' N 286 1' literature found 4' 1 JC,F 272 272.5 (CF 3)

6' 5' 2 JC,F 32 31.7 (C3’)

96e 3 JC,F 4 3.7 (C2’)

3.7 (C4’)

Cytisine: NMR spectroscopy of novel ligands 109

3.3.3.4 5-Aryl Analogues of Cytisine

The 1H and 13 C chemical shifts of the 5-aryl derivatives of cytisine 103e – 110e are listed in Tables 3-20 and 3-21. Assignment of 1H and 13 C signals was carried out by comparison with the assignments for 5-phenyl-cytisine 103e as well as through the increment additivity rules.

The 1H chemical shifts of the cytisine moiety of the 5-aryl derivatives 104e – 110e compare greatly to those of the cytisine part in the 5-phenyl analogue 103e (Table 3-20). Protons H8 (assigned to a sharp methylene singlet in the 1H spectra of the 3-aryl derivatives 93e – 100e) appeared in the spectra of the 5-aryl derivatives as two separated broad singlets ( 104e , 107e 2 and 108e ) or as two broad doublets with geminal couplings JH8 α/H8 β between 12.6 and 13 Hz (105e , 106e and 109e ). The A and B doublets of the 11 and 13 methylene AB systems are all well separated and may be correlated to their geminal partners with the aid of their “roof” effect.

The protons H3 and H4 of the 5-substituted pyridone moiety in the series of 5-aryl derivatives 3 103e – 110e , always representing an AB system, display cis coupling constants JH3,H4 = 9.1 3 – 9.3 Hz. The respective vicinal coupling constants JH3,H4 = 9.1 and 9.5 Hz were established in the 1H spectrum of cytisine 27 and 5-phenyl-cytisine 103e . These results suggest that the meta-substitution on the phenyl ring does not particularly influence the vicinal coupling of the H3 and H4 protons.

However, the meta-substituent changed the 1H chemical shifts of phenyl moiety in 104e – 110e when compared to the chemical shifts of 5-phenyl-cytisine 103e (Table 3-20). The assignment of the four protons H2’, H4’, H5’ and H6’ was feasible through their multiplicities and substituent chemical shifts. Significant differences between the chemical shifts of H2’ in 5-aryl and 3-aryl analogues were observed. The H2’ protons of 3-aryl substituted cytisines 94e – 100e were deshielded by the neighbouring carbonyl group while in the 5-aryl derivatives 103e – 110e this neighbour effect was lacking (Table 3-19).

Table 3-19 1H chemical shifts of H2’ protons in 3- and 5-aryl derivatives of cytisine (signed as 3- H2’ and 5-H2’) (CDCl 3, 500 MHz)

1H chemical shifts of H2’ [ppm]

R = H CH 3 Cl NO 2 CF 3 F OCF 3 3-H2’ 7.69 7.52 7.69 8.53 7.95 7.43 7.58 5-H2’ 7.21 7.00 7.32 8.10 7.46 6.90 7.08 ∆∆∆ δ 0.48 0.52 0.37 0.43 0.49 0.53 0.50 ∆∆∆ δ = 0.47 ± 0.06 ppm

1 Table 3-20 H chemical shifts of 5-phenyl derivatives of cytisine 103e – 110e (CDCl3, 500 MHz)

11 NH O 13 2 9 10 N 8 3 7 6

1' 5 4 2' 3' 6' R 4' 5'

1H chemical shifts [ppm]

R = H 103e NO2 104e CH3 105e CF3 106e OCF3 107e Cl 108e F 109e Ph 110e H3 6.49 (d) 6.52 (d) 6.47 (d) 6.49 (d) 6.50 (d) 6.49 (d) 6.47 (d) 6.51 (d) H4 7.21 (d) 7.18 (d) 7.19 (d) 7.17 (d) 7.19 (d) 7.17 (d) 7.16 (d) 7.26 (d) H7 3.04 (br s) 2.93 (ovl) 3.07 (br s) 2.93 (br s) 3.11 (br s) 3.04 (br s) 3.07 (br s) 3.09 (br s)

H8A 1.92 (d) 1.94 (br s) 1.92 (br d) 1.93 (br d) 1.91 (br s) 1.93 (br s) 1.91 (br s) 1.94 (br d)

H8B 1.84 (d) 1.87 (br s) 1.83 (br d) 1.82 (br d) 1.84 (br s) 1.81 (br s) 1.84 (d br) 1.84 (br d) H9 2.30 (br s) 2.35 (s) 2.29 (br s) 2.31 (br s) 2.46 (br s) 2.34 (br s) 2.31 (br s) 2.30 (br s) H10 3.95 (dd) 3.96 (dd) 3.93 (dd) 3.94 (dd) 3.95 (dd) 3.95 (dd) 3.92 (dd) 3.96 (dd) H10! 4.19 (d) 4.19 (d) 4.19 (d) 4.18 (d) 4.26 (d) 4.21 (d) 4.17 (d) 4.22 (d)

H11A 3.12 (d) 3.10 (d) 3.04 (d) 3.06 (d) 3.39 (d) 2.73 (d) 3.03 (d) 3.07 (d)

H11B 2.92 (d) 2.93 (ovl) 2.91 (d) 2.90 (d) 3.07 (d) 2.73 (d) 2.93 (br s) 2.91 (d)

H13A 2.81 (d) 2.74 (ovl) 2.81 (d) 2.75 (d) 2.92 (dd) 2.95 (d) 2.79 (br d) 2.85 (d)

H13B 2.69 (dd) 2.74 (ovl) 2.68 (dd) 2.69 (dd) 2.82 (d) 2.81 (dd) 2.71 (br d) 2.72 (d) H2’ 7.21 (d) 8.09 – 8.11 7.00 (s ovl) 7.46 (s) 7.08 (d) 7.32 (m ovl) 6.90 (dt) 7.55 – 7.58 (m) H3’ 7.37 (tt) ------H4’ 7.31 (tt) 8.16 – 8.21 7.12 (br d) 7.39 (d) 7.20 (ddd) 7.32 (m) 7.01 (tdd) 7.17 (dt) H5’ 7.37 (tt) 7.55 – 7.50 7.25 (t) 7.50 (t) 7.41 (t) 7.21 (t) 7.32 (td) 7.41 – 7.46 (m) H6’ 7.18 (tt) 7.55 – 7.50 6.98 (br d) 7.58 (d) 7.15 (dt) 7.09 (dd) 6.96 (dt) 7.41 – 7.46 (m) 13 Table 3-21 C chemical shifts of 5-phenyl derivatives of cytisine 103e – 108e and 109e – 110e (CDCl3, 125 MHz)

11 NH O 13 2 9 10 N 8 3 7 6

1' 5 4 2' 3' 6' R 4' 5'

13C chemical shifts [ppm]

R = H 103e NO2 104e CH3 105e CF3 106e OCF3 107e F 109e Ph 110e

C2 163.1 163.0 163.1 163.0 163.2 163.0 163.1 C3 116.1 116.3 116.0 116.4 117.0 116.2 116.1 C4 141.4 141.0 141.4 140.8 141.1 140.9 141.3 C5 119.3 117.2 119.5 117.6 116.4 118.0 119.1 C6 147.5 148.1 147.4 148.0 148.3 147.6 147.7 C7 31.6 32.1 31.6 31.7 30.5 31.6 31.4 C8 26.4 26.3 26.4 26.3 25.5 26.2 26.4 C9 27.4 27.7 27.4 27.3 26.4 27.3 27.4 C10 50.4 50.5 50.4 50.5 49.8 50.4 50.5 C11 52.2 52.0 52.2 52.1 50.4 52.1 52.3 C13 53.0 52.9 53.0 53.0 51.2 52.8 53.0 C1’ 138.5 140.0 138.4 139.3 140.0 140.6 139.0 C2’ 129.8 124.2 130.4 126.5 120.0 116.8 --- C3’ 128.6 148.4 138.3 130.9 149.3 163.6 140.6 C4’ 127.4 121.9 128.1 124.3 122.3 114.4 --- C5’ 128.6 129.2 128.4 129.2 130.2 130.1 --- C6’ 129.8 135.5 126.8 133.2 128.2 125.6 --- 112 Cytisine: NMR spectroscopy of novel ligands

13 C chemical shifts of the phenyl moieties in 103e – 110e are in agreement with 13 C chemical shifts of the corresponding carbons in their 3-aryl counterparts 93e – 100e as well as with the predicted values (i.e. 13 C chemical shifts of the phenyl ring in 103e ± SCS of the substitutent with regard to the position 286 ). Thus, the additivity increments calculated from the experimental data are in good accordance with literature SCSs data 286 (Table 3-22).

Table 3-22 Additivity increments [ppm] of various groups for the 13 C chemical shifts of the phenyl carbons in the series of 5-aryl cytisine derivatives ( 104e – 106e and 109e ), literature SCSs values shown in parentheses 286 NH O N

1' 2' 3' R 6' 4' 5'

Substituent chemical shifts [ppm]

R = H 103e NO 2 104e CH 3 105e CF 3 106e F 109e [δ, ppm] C1’ 138.5 1.5 (0.8) -0.1 (0.0) 0.8 (-0.3) 2.1 (0.9)

C2’ 129.8 -5.6 (-5.3) 0.6 (0.6) -3.3 (-2.6) -13.0 ( -14.3)

C3’ 128.6 19.8 (19.6) 9.7 (9.3) 2.3 (2.6) 35.0 (35.1)

C4’ 127.4 -5.5 (-5.3) 0.7 (0.6) -3.1 (-2.6) -13.0 (-14.3)

C5’ 128.6 0.6 (0.6) -0.2 (0.0) 0.6 (-0.3) 1.5 (0.9)

C6’ 129.8 5.7 (6.0) -3.0 (-3.1) 3.4 (-3.2) -4.2 (-4.4)

One of the “diagnostical tools” to distinguish the 3- and 5-substituted counterparts is the chemical shift difference of approximately δ 3 ppm between the carbons C6 and C7 in the 3- substituted 93e – 100e and 5-substituted analogues 103e – 110e . While in the 13 C NMR spectra of cytisine 27 and the 3-phenyl derivatives 93e – 100e the carbons C6 and C7 display signals around δ 150 ppm and δ 35 ppm, respectively (Table 3-15), the same carbons are shifted upfield in the 13 C spectra of the 5-substituted analogues 103e – 110e (Table 3-21). Hence, their carbons C6 are located between δ 147.4 – 148.1 ppm and their C7 between δ 30.5 – 32.1 ppm. Similar shift mutation, however in the opposite direction, was observed for C4 of the 5-substituted derivatives. While 3-substitution did not alter the 13 C chemical shift of C4 compared with cytisine 27 ( δ 137.0 – 137.6 ppm) (Table 3-15), a 5- substitution resulted in a medium downfield shift of ∆ δ 2.27 ± 0.23 ppm (n=6) and thereby, the C4 signals were found between δ 140.8 and 141.4 ppm (Table 3-21). The 13 C chemical shifts of the remaining carbons in the bispidine and pyridone ring maintained the values

Cytisine: NMR spectroscopy of novel ligands 113 found in the spectra of the unsubstituted alkaloid 27 and the 3-substituted analogues 93e – 100e.

In the 13 C spectra of the compounds 106e , 107e and 109e containing fluorine, characteristic 13 C-19 F couplings were observed (Table 3-23). C-F doublets in the 5-(3’-fluoro)-phenyl analogue 109e and C-F3 quartets in the spectrum of 106e supported the carbon assignment when literature data 286 were considered. The C-F doublet in 109e was found at δ 163.6 ppm

286 (literature: δ 163.6 ppm) and the chemical shift of the CF 3 – quartet in 106e ( δ 124.9 ppm)

286 also definitely agreed with the value published ( δ 124.5 ppm) . The presence of a CF 3- group gave hints towards the C3’, C2’ and C4’ signals (δ 130.9; 126.5 and 124.3 ppm, respectively) which were split into quartets, their shifts matching the literature ppm values ( δ 130.8 ppm for C3’ and δ 125.4 ppm for ortho C2’ and C4’). 286

Table 3-23 13 C-19 F Coupling constants in 5-(3’-trifluoromethylphenyl)-cytisine 106e (A) and 5-(3’- fluorophenyl)-cytisine 109e (B), coupling carbon given in parentheses

A NH 13 C -19 F Coupling constant [Hz] O 286 N literature found

1 JC,F 272 272.5 (CF3)

2' 1' 2 JC,F 32 32.3 (C3’) 3' CF 6' 3 3 JC,F 4 3.8 (C2’)

4' 5' 3.8 (C4’)

106e

B NH 13 C -19 F Coupling constant [Hz] O 286 N literature found 1 JC,F 245 247.6 (C3’)

2' 1' 2 JC,F 21 21.0 (C2’)

3' F 6' 21.0 (C4’)

3 J 8 7.7 (C1’) 4' 5' C,F

8.5 (C5’) 109e 4 JC,F 3 2.7 (C6’)

1.7 (C5)

114 Cytisine: NMR spectroscopy of novel ligands

3.3.3.5 Heterocyclic Analogues of Cytisine

1H and 13 C chemical shifts of the cytisine derivatives 117e – 126e , substituted in 3- and 5- position with heterocycle are listed in the Tables 3-24, 3-25 and 3-26.

In general it may be concluded that a 3-substitution with a heterocyclic moiety moved all the 1H chemical shifts of the cytisine moiety downfield when compared to the unsubstituted alkaloid 27 (Table 3-24). The examination of the 13 C data obtained from cytisine 27 and its 3- heterocyclic derivatives 117e – 122e revealed only a difference in the chemical shift of C3, whereas the chemical shifts of other cytisine carbons (C2-C13) were largely unaffected (Table 3-25).

The substitution of the position 5 with a heterocycle ( 123e – 126e ) also caused a downfield shift of all 1H signals for cytisine moiety (Table 3-26). The 13 C chemical shifts of the bispidine and pyridone moieties in the structures of the 5-substituted analogues 123e – 126e were consistent with the trends reported for 5-aryl derivatives 103e – 110e (i.e. upfield shift of C6 and C7 signals) (Table 3-26).

Structures and assignment of NMR signals of the heterocyclic analogues 117e – 126e is discussed separately for each set of corresponding 3- and 5-isomer.

3.3.3.5.1 3- and 5-(5’-Indolyl)-cytisine 117e & 123e

The 1H chemical shifts of 3-(5’-indolyl)-cytisine 117e are listed in Table 3-24. The doublet at δ 6.06 ppm ( 3J = 7.0 Hz) was assigned to H5. This signal provided the starting point for assignments of the remaining aromatic protons located in the region δ 8.3 – 6.0 ppm. In the COSY 2D spectrum of 117e (Figure 3-24), H5 displayed a cross peak to a doublet at δ 7.47 ppm ( 3J = 7.0 Hz), which therefore was assigned to H4. A broad singlet at δ 8.28 ppm appeared as the most downfield shifted signal, it was assigned to the indolic NH group. Another singlet at δ 7.92 ppm was recognised as H2’ and it showed only one correlation towards the doublet at δ 7.52 ppm. This doublet had an additional cross peak with the doublet at δ 7.38 ppm, therefore these two signals were identified as H7’ (coupling with H2’ and H6’) and H6’ (coupling with H7’). A pseudotriplet at δ 7.16 ppm produced a cross peak with the NH group at δ 8.28 ppm, thus it was assigned to the proton H4’. Finally, the cross signal between δ 7.16 ppm and δ 6.54 ppm could only arise from a coupling between H4’ ( δ 7.16 ppm) and H3’ ( δ 6.54 ppm).

1 Table 3-24 H chemical shifts of 3-heterocyclic derivatives of cytisine 117e – 122e (CDCl3, 500 MHz)

11 NH O 13 2 9 10 N 3 8 7 6 R 5 4

2' 1' 3' 2' 2' 3' 2' 3' 8' 2' 3' 2'a 1' 3' 1' CH3 O N 8'a N H 1' 1' 2' 7' 1' N 4' CH 4' N 4' R 7' 6' 2 N 27 5'a N 5' 4' O 3' 6' 4' cytisine 5' 6' 5' 6' 5' 4'a 5' 6' H 4' 5' 117e 118e 119e 120e 121e 122e H3 6.10 ------H4 7.00 7.47 7.38 7.49 7.57 7.58 7.56 H5 5.72 6.06 6.05 6.11 6.13 6.16 6.05 H7 2.62 2.89 2.90 2.95 2.94 2.95 2.90 H8 1.65 1.96 1.95 1.96 1.96 1.96 1.94 H9 2.03 2.34 2.34 2.36 2.36 2.33 2.33 H10 3.57 3.96 3.93 3.94 3.95 3.96 3.94 H10! 3.77 4.22 4.16 4.16 4.16 4.21 4.18 H11A 3.12 3.11 3.11 3.11 3.09 2.70 – 2.75 3.08 H11B 3.00 – 3.06 3.00 2.99 – 3.03 3.00 2.96 H13A 3.06 3.06 3.07 3.02 3.03 2.70 – 2.75 3.00 – 3.06 H13B 3.00 2.99 – 3.03 3.04 3.13 2.99 H2’ --- 7.92 7.26 8.78 7.67 7.85 7.80 H3’ --- 6.54 ------8.57 7.56 --- H4’ --- 7.16 --- 8.49 --- 7.78 --- H5’ --- 8.28 (NH) 6.81 7.29 8.57 8.15 8.29 H6’ --- 7.38 7.12 8.16 7.67 7.35 --- H7’ --- 7.52 ------8.86 ---

5.94 (CH2) 3.90 (CH3) 13 Table 3-25 C chemical shifts of 3-heterocyclic derivatives of cytisine 117e – 122e (CDCl3, 125 MHz)

11 NH O 13 2 9 10 N 3 8 7 6 R 5 4

2' 3' 2' 2' 3' 2' 3' 8' 2' 3' 1' 2'a 1' 3' 1' CH3 O N 8'a N 1' 1' 2' 7' 1' N H 4' CH 4' N 4' R 7' 6' 2 N 27 5'a N 5' 4' O 3' 6' 4' cytisine 6' 5' 6' 5' 6' 5' 4'a 5' H 4' 5' 117e 118e 119e 120e 121e 122e C2 163.7 162.6 162.1 161.9 161.5 162.5 161.2 C3 116.8 128.9 127.1 123.9 122.8 136.5 119.8 C4 138.8 136.7 136.4 137.2 137.8 139.8 132.4 C5 105.1 105.1 104.9 105.0 104.9 104.8 105.1 C6 150.9 148.9 149.9 151.5 152.6 150.4 148.0 C7 35.5 35.6 35.7 35.7 35.8 35.8 35.5 C8 26.2 26.4 26.4 26.2 26.2 26.4 26.4 C9 27.7 27.9 27.9 27.8 27.8 27.9 27.9 C10 49.7 50.1 50.2 50.3 50.3 50.1 50.1 C11 53.8 52.9 53.0 53.0 53.0 53.0 52.9 C13 52.8 53.8 54.0 53.9 53.9 53.9 53.9 C1’ --- 129.1 131.4 133.2 144.9 139.6 117.8 C2’ --- 120.9 109.4 149.1 123.9 131.0 136.8 C3’ --- 103.0 147.3 --- 149.6 126.2 --- C4’ --- 124.3 146.8 148.2 --- 127.7 --- C5’ ------108.0 122.8 149.6 136.4 129.9 C6’ --- 110.5 122.1 136.1 123.9 120.8 --- C7’ --- 123.2 ------149.9 ---

127.8 (C2’a) 100.9 (CH2) 146.5 (C8’a) 135.3 (C5’a) 128.6 (C4’a) 

α β



δ δ δ δ δ δ δ

, δ δ δ δ δ



α β



δ δ δ δ δ

δ ∆δ δ δ



δ δ δ δ

δ δ δ δ δ ∆ δ



β α

δ δ

δ δ δ δ



δ δ δ δ δ

δ δ δ δ δ δ δ

δ δ H δ δ



β α

H ∆ δ ∆δ



δ δ δ δ

δ δ



δ δ δ δ



In Vitro

αβ α αβ Torpedo californica

αβγδ

αβ

αβ αβ



αβ α

αβ α βγδ

(α βγδ αβ α αβ



αβ

αβ αβ αβ

(αβγδ

(α βγδ

αβ α



αβ

et al

αβ

α4β2

α4β2 α4β2

α4β2

αβ →

→



αβ

α4β2 α4β2 α4β2

αβ

αβ αβ

α βγδ

Torpedo californica



αβ

(α βγδ

Torpedo californica



αβ α αβ α βγδ

(α βγδ αβ α αβ



αβ

αβ π αβ

α4β2 α4β2

α4β2



αβ

H αβ

αβ αβ H

(α βγδ



αβ α αβ α βγδ

(α βγδ αβ α αβ



α4β

αβ

(α βγδ

Torpedo californica

α β



αβ α αβ α βγδ

(α βγδ αβ α αβ



αβ

αβ ππ

H αβ

αβ



αβ

αβ αβ

αβ αβ ≈

αβ αβ≈ αβ H αβ αβ αβ ≈

α4β

αβ



αβ α αβ α βγδ

(α βγδ αβ α αβ

(α βγδ



αβ •

•

• H

•

α4β2



αβ

•

• H

• αβ

, ,, ,

α α βγδ



αβ µ α

et al α

αβ



α4β2 (α7 )

(α4β2 ) (α4β2 ) (α7 )

(α7 ) (α7 ) (α4β2 ) (α4β2 )



1,3-di-m-tolyl-urea



In Vitro

αβ α αβ

Torpedo californica αβγδ

αβ α

α µ αβ αβ

αβ αβ α ≈

α αβ



αβ α αβ α βγδ

(α βγδ αβ α αβ



αβ α αβ α βγδ (α βγδ αβ α αβ

αβ

α βγδ



αβ α αβ αβ α

αβ α

α π αβ π α α α αβ



in vivo

αβ



Chemistry

• Laburnum anagyroides watereri, Fabaceae

• t

•

• t



• t t

•

δ δ δ

δ δ



Pharmacology

αβ α αβ α βγδ

•

αβ

≈

• αβ αβ

≈



•

αβ αβ

• αβ

αβ αβαβα α βγδ

• αβ αβ

•

αβαβα α βγδ

•

αβ ππ

• α

α βγδ

• αβ α αβ

αβ



αβ αβ αβ

αβ

αβ α α

αβ µ α

α α α



Chemistry

•

Pharmacology

• α αβ



• αβ α

• α αβ

≈

αβ α π α π α

α4β2 α

α7

α4β2



αβ

α π π α αβ α π



ULTRA-TURRAX T50 DPX CEM BAKERBOND

Knauer Knauer Knauer Knauer Knauer µ ×

Alpha 1-4 LSC Vacuubrand



Avance 500

δ δ MS-50 A.E.I. MAT 95 XL, Thermoquest Perkin-Elmer VarioEL



t t t t



Laburnum anagyroides Laburnum watereri

in vacuo



, δ β α , δ , δ α β

, δ



t t



, δ β α

t , δ t

t t

in vacuo

t t



t t t t , δ β α t

, δ t

t t , δ β α t



, δ t

in vacuo

, δ β α



t , δ t

Suzuki cross-coupling reaction t

in vacuo t

HPLC method

t in vacuo



Deprotection - Method A t

in vacuo. Deprotection - Method B t

, δ β



, δ

, δ β α



, δ

, δ β α



, δ

, δ β α



, δ

, δ β α



, δ

, δ β α



, δ

, δ β



, δ



β α , δ



, δ β

, δ



, δ β α

, δ



, δ β

α , δ



, δ β

α , δ



, δ β α , δ



, δ β

α ,



, δ β α , δ



, δ β α



, δ ∗

∗



, δ β

α , δ



, δ β

α , δ



, δ β

, δ



, δ β α

, δ



, δ β α , δ



, δ β

, δ

H-



, δ β

, δ



, δ β



, δ

, δ β α



, δ



, δ β

α , δ



, δ β α , δ



, δ β

, δ



ααα

, δ



α , δ

, δ



, δ



, δ



→

, δ



, δ



,d6 δ



,d6 δ



Instruments   Chemicals



≤ ≤ ≤ ≤ Radioligands Tissues Torpedo californica



Buffer Solutions

Preparation of rat brains

× ×



Preparation of calf adrenals

Preparation of Torpedo californica electroplax Torpedo californica ×

Competition assay using (±)-[3H]epibatidine ([ 3H]Epi) and rat brain P2-fraction (α4β2* nAChR) µ µ µ µ µ µ µ



Competition assay using [ 3H]methyllycaconitine ([ 3H]MLA) and rat brain P2-fraction (α7* nAChR) µ µ µ µ µ µ

Competition assay using (±)-[3H]epibatidine ([ 3H]Epi) and calf adrenals membrane fraction (α3β4* nAChR)

µ µ µ µ µ µ µ



Competition assay using (±)-[3H]epibatidine ([ 3H]Epi) and Torpedo californica electroplax

(( α1) 2β1γδ nAChR) µ µ µ µ µ µ



α α t tert

β β

γ α α



Tocris Reviews JPET Nature Reviews Neuroscience J Mol Evol Rev Physiol Biochem Pharmacol Torpedo californica Cold Spring Harb Symp Quant Biol Nature In: J Biol Chem Br J Pharmacol Prog Neurobiol Biochemistry FEBS Lett Biochemistry J Protein Eng Biochemistry J Biol Chem J Biol Chem



Nature Science Proc Natl Acad Sci Psychol Med . Biol Psychiatry Biomed Pharmacother J Clin Psychiatry Progress Neurobiol Schizophr Res Am J Psychiatry Biol Psychiatry Neuronal Nicotinic receptors: Pharmacology and Therapeutic Opportunities Neurology Epilepsia Nat Genet Pflugers Arch J Comp Neurol Behav Genet



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

The Society for Neuroscience Drug Disc Today Eur J Pharmacol Curr Drug Targets – CNS & Neurological Disorders J Neural Transm Curr Topics Med Chem Curr Opin Inv Drugs Curr Topics Med Chem Expert Opin Ther Patents Expert Opin Ther Targets Expert Opin Ther Patents Expert Opin Emerg Drugs Curr Topics Med Chem Pharm Acta Helv Curr Top Med Chem Curr Med Chem Neurosci Lett α Mol Pharmacol Mol Pharmacol Eur J Pharmacol Biochem Pharmacol Eur J Neurosci α Synapse



J Biol Chem Drugs Fut. Eur J Med Chem CNS Drug Rev WO96/36637 WO96/20929 CNS Drug Rev . Mol Pharmacol Eur J Pharmacol Psychopharmacology Bioorg Med Chem J Med Chem Drug Dev Res Med Chem Res Med Chem Res J Med Chem Bioorg Med Chem Lett J Phar Exp Ther

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

In vitro Neuropharmacology J Med Chem In vitro J Pharm Exp Ther Drug Dev Res Drug Dev Res J Med Chem J Am Chem Soc Bioorg Med Chem Lett Bioorg Med Chem Lett N J Med Chem J Med Chem J Med Chem J Med Chem J Med Chem



Eur J Pharmacol Bioorg Med Chem Lett syn syn Tetrahedron J Chem Soc Perkin Trans R R S R S S Tetrahedron Lett . J Med Chem Tetrahedron Lett c J Org Chem Science Mol Pharmacol J Pharmacol Exp Ther Bioorg Med Chem J Neurochem Drug Dev Res J Org Chem Bioorg Med Chem Neuropharmacology



Life Science J Phar Exp Ther Neuropharmacology Br J Pharmacol J Med Chem Med Chem Res Neuropharmaco Bioorg Med Chem FEBS Tetrahedron Lett FEBS Lett α Neuropharmacol α J Med Chem J Pharmacol Exp Ther J Med Chem Behav Pharmacol



Behav Brain Res Nature J Med Chem J Med Chem ø J Med Chem ø J Comp-Aided Mol Des ø Curr Med Chem Med Chem Res J Med Chem Bioorg Med Chem Lett J Med Chem . αβ Curr Topics Med Chem Pharm Acta Helv Eur J Med Chem Mol Pharmacol J Med Chem Farmaco Farmaco



Farmaco Bioorg Med Chem Chem Ber Chem Berichte Chem Berichte Chem Berichte Archiv der Pharm J Chem Soc Chem. Ind. Chem Pharm Bull Chem Pharm Bull Phytochemistry Phytochemistry Phytochemistry Pharmazie . Deutscher Apoth Ztg Neuroreport αβ Synapse



β β Mol Pharmacol Neuropharmacology J Neurochem J Neurosci Bioorg Med Chem Lett J Pharmacol Exp Ther Psychopharmacology Neuropharmacol Mol Pharmacol Eur J Pharmacol J Pharmacol Exp Ther Brain Research Life Sci Nuclear Med Comm Dtsche Gesundheitswesen Exp Clin Psychopharmacol αβ J Pharmacol Exp Ther dl J Am Chem Soc J Am Chem Soc J Am Chem Soc



Chem Ber J Chem Soc Org Lett Org Lett Tetrahedron Lett Org Biomol Chem Org Lett J Org Chem Archiv der Pharmazie Archiv der Pharmazie Archiv der Pharmazie Chemistry of Natural Compounds Chemistry of Natural Products Chemistry of Natural Products Chemistry of Natural Products Univ., Diss. Bonn, Univ., Diss. Org Lett Bioorg Med Chem Lett Tetrahedron Lett



E J Chem Soc Chem Com 19, 866 - 867 (1979) Synth Comm Pure & Appl Chem Metal-catalyzed cross-coupling reactions Metal-catalyzed cross-coupling reactions J Am Chem Soc J Am Chem Soc Inorg Synth J Organomet Chem J Am Chem Soc Synlett J Am Chem Soc J Am Chem Soc



Tetrahedron Lett Metal-catalyzed cross-coupling reactions J Org Chem J Org Chem Tetrahedron J Am Chem Soc J Am Chem Soc trans J Am Chem Soc Tetrahedron Lett Synlett J Am Chem Soc Angew Chem Int Ed J Am Chem Soc Org Lett Angew Chem Int Ed J Am Chem Soc



. Tetrahedron Lett J Chem Soc Chem Commun J Org Chem Chem Commun J Am Chem Soc . J Am Chem Soc . Z E Org Synth Tetrahedron Lett . Ang Chem Int Ed J Am Chem Soc Synthesis Synthesis Tetrahedron Lett . J Am Chem Soc Metal-catalyzed cross-coupling reactions J Am Chem Soc J Am Chem Soc Org Lett Heterocycles J Am Chem Soc α Tetrahedron Lett



Tetrahedron Lett Tetrahedron Lett Angew Chem Int Ed Acc Chem Rev Curr Topics Med Chem Microwaves in Organic Synthesis Chem Soc Rev J Chem Soc Chem Commun Chem Soc Rev Ang Chem Int Ed Tetrahedron Microwaves in organic synthesis Can J Chem Tetrahedron Angew Chem Int Ed Angew Chem Int Ed J Org Chem Tetrahedron Lett J Org Chem Org Lett Adv Synth Catal



Angew Chem Int Ed Angew Chem Int Ed Pharmazie J Chem Soc Perkin Trans 1 . Pure Appl. Chem . Synthesis In Chem Pharm Buletin J Am Chem Soc J Org Chem H H J Org Chem J Org Chem J Chem Soc Perkin Trans I Tetrahedron J Med Chem Bioorg Med Chem Lett Bioorg Med Chem Lett Bioorg Med Chem Lett



Tetrahedron J Org Chem J Org Chem H QSAR & Comb Sci Synlett Tetrahedron Lett Drug Discovery Today J Med Chem Tetrahedron Lett Heterocycles α J Org Chem Org Lett Tetrahedron Lett Synlett J Am Chem Soc Metal-catalyzed cross-coupling reactions J Org Chem J Organomet Chem J Org Chem Int J Pept Protein Res

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J Org Chem Ang Chem Int Ed Tetrahedron Lett . Tetrahedron Lett Tetrahedron Lett Tetrahedron Lett Ntert Tetrahedron Lett Synlett Synth Commun Magn Reson Chem Magn Reson Chem ChemDraw Ultra 7.0 J Alzheim Disease Bioorg Med Chem Lett . Bonn, Univ, Diss Bioorg Med Chem Brit J Pharmacol Br J Pharmacol U.S. Patent



Synthesis J Chem Soc Perkin Trans 2 J Org Chem Tetrahedron Lett J Org Chem USSR J Med Chem N J Med Chem J Med Chem Synthesis p J Chem Soc Perkin Trans 2 . J Org Chem Chem Phar Bulletin αBioorg Med Chem Lett α J Med Chem α J Neurosci



Design ynthesis and Biological Evaluation of Novel Cytisine Derivatives as Ligands for Nicotinic Acetylcholine Receptors Synthesis and Evaluation of Phenylcarbamate Derivatives as Ligands for Nicotinic Acetylcholine Receptors Study of local anaesthetics, part 156: Some physicochemical and lipophilic properties of o-, m-, p- alkoxysubstituted pyrrolidinoethylesters of phenylcarbamic acid Permeability profiles of m-alkoxysubstituted pyrrolidinoethylesters of phenylcarbamic acid across Caco-2 monolayers and human skin Novel cytisine analogs as potent nicotinic acetylcholine receptor (nAChR) ligands. Novel Cytisine Analogues: Synthesis and Biological Activity Novel ligands for nicotinic acetylcholine receptors (nAChRs) based on choline and cytisine: Synthesis and in vitro evaluation for different subtypes and tissues. , Novel Cytisine Analogues: Synthesis and Biological Activity , Synthesis of the receptorligand [ 131 I]-3-Iodo-cytisine for in vivo imaging of the nAChReceptor



, Synthesis of the receptorligand [ 131 I]-3-Iodo-cytisine for in vivo imaging of the nAChReceptor , Synthesis of the receptorligand [ 131 I]-3-Iodo-cytisine for in vivo imaging of the nAChReceptor Synthesis and In Vitro Evaluation of Phenylcarbamates and Choline Phenyl Ether Derivatives for Nicotinic Acetylcholine Receptors (nAChRs) ,,, , Synthesis and in vitro evaluation of structural variants of choline, cytisine, ferruginine, anatoxin-a, diazabicyclononane- and quinuclidin-2- ene based ligands for nicotinic acetylcholine receptors , Synthesis and In Vitro Evaluation of Novel Ligands for Nicotinic Acetylcholine receptors (nAChRs): Structural Variants of Choline and Alkaloidal Toxins and 3,9-Diazabicyclo[4.2.1]nonane and Quinuclidin-2- ene based Derivatives



Chefin

Trudi, ďakujem za všetko vďaka za podporu there is always a light at the end of the tunnel

, Sicherheit die Einweisung für den Umgang mit Lenca meine Lieblingsdoktorandin

Semesterleiter

☺

im Haus Die Chemikalien sind da szepség Sekretariat, Danke schön

süss



die Jungs

Ordnungsamt put the pizza in the oven