Piperlongumine B and analogs are promising and selective inhibitors for acetylcholinesterase

ACCEPTED MANUSCRIPT

Piperlongumine B and analogs are promising and selective inhibitors for

acetylcholinesterase

a

b

Jana Wiemann , Julia Karasch , Anne Loesche , Lucie Heller , Wolfgang Brandt , René

a*

Csuk

a

Martin-Luther University Halle-Wittenberg, Organic Chemistry, Kurt-Mothes Str. 2, D-

0

6120 Halle (Saale), Germany

b

Leibniz Institute of Plant Biochemistry, Bioorganic Chemistry, Weinberg 3, D-06120 Halle

(Saale), Germany

Abstract

Piperlongumine B (19), an alkaloid previously isolated from long pepper (Piper longum) has

been synthesized for the first time in a short sequence and in good yield together with 19

analogs. Screening of these compounds in Ellman’s assays showed several of them to be good

inhibitors of acetylcholinesterase while being less active for butyrylcholinesterase. Activity of

the compounds increased with the ring size of the heterocycle, and a maximum of activity was

observed for an analog holding 12 methylene groups in the aliphatic side chain. These

compounds may be regarded as promising candidates for the development of efficient

inhibitors of acetylcholinesterase being useful for the treatment of Alzheimer’s disease.

1

. Introduction

Piper Longum, also known as long pepper or Indian pepper is a small plant native to the

tropical rain forests of the Indo-Malaya region. Its fruits have been used for many centuries

not only in the traditional Indian medicine Ayurveda but also as a spice. Most important

secondary metabolites of this plant are the alkaloids piperine, and piperlongumine A (Fig. 1).

While piperine adds to the hot/spicy taste of the fruits, piperlongumine A (= piplartine)

moved into the focus of scientific interest [1-11] due to its anticancer activity [12-17] and as a

senolytic agent [18].

1

ACCEPTED MANUSCRIPT

Fig. 1. Structures of most important alkaloids from Piper longum.

Parallel to the discovery of these compounds, extraction of the fruits yielded another

compound, piperlongumine B, and preliminary studies showed some anti-leishmanial activity

[

19, 20] combined with a weak cytotoxicity [7] for HL-60 human leukemia cells.

Piperlongumine B has been accessed by extraction of the fruits only. Generally, extraction

processes are convenient, but not so for the isolation of piperlongumine B. Thus, extraction

[

21-23] of 2.5 kg of air-dried fruits finally gave 6 mg of piperlongumine B. To allow extended

biological screening the preparation of larger amounts of this compound (and of analogs

thereof) was called for.

The cholinergic system plays an important role in the regulation of learning and memory

processes. As a consequence, it has been a target for the design and development of anti-

Alzheimer’s drugs. Alzheimer’s disease (AD) is characterized by deficits in the cholinergic

system. Inhibitors of cholinesterases are regarded as enhancers of cholinergic transmission,

and several of them (for example, galantamine, donezepil, and rivastigmine) are already on

the market to help Ad patients do deal with her progressive neurodegenerative disorder and to

increase their quality of life.[24]

Recent investigations showed two enzymes being involved in AD: acetylcholinesterase

(AChE) and butyrylcholinesterase (BChE), the latter of which seems to serve as a co-

regulator of the cholinergic transmission. BChE, however, is mainly localized in the

peripheral tissues, and only a very small amount is present in the brain region. As a

consequence, the selective inhibition of AChE over BChE may result in drugs showing a

lesser degree of side effects while being equally effective.[24-26] Thus, we became interested

in the investigation of piperlongumine B and analogs as potential inhibitors of

cholinesterases.

2

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2

. Results and Discussion

α,β-Unsaturated acids 1-4 are ideal starting materials for these syntheses (Scheme 1); they

were either bought or easily prepared from aldehydes by Wittig reaction using (tert-

butoxycarbonylmethylene)triphenylphosphorane [27, 28] followed by a hydrolysis of

intermediary tert-butyl esters with TFA. Reaction of the acids 1-4 with cyclic amines 5-9 in

the presence of EDC/HOBt [29] for several hours, or by the reaction of the acids with oxalyl

chloride [30] followed by the addition of the amines in the presence of DMAP and NEt in

3

DCM furnished the target compounds 10-29 in good yields. The latter method failed to give

fair yields of azetidine derived 10-13. Modification of the conditions following a procedure

previously reported by Liu [31], however, gave better results and higher yields of 10-13 were

obtained.

Scheme 1. Synthesis of piperlogumine B (19) and analogs 13-18 and 20-29: a) EDC, HOBt,

DCM, r.t., 12 h, 67-99%; b) oxalyl chloride, DCM, 2 h, then K CO , Et O, H O, 71-79%.

2

3

2

Furthermore, compounds 15 and 16 have previously been extracted from Piper nigrum and

Piper amalgo [32, 33] , 17 also from Piper reticulatum [34]; compound 18 from Piper

longum and muntok pepper [22, 35]. Compound 20 [36-40] is an inhibitor of cytochrome

P450 2D6 but has also been discussed as an antibacterial and antiinsectal compound; it has

also been extracted from the underground parts of Achillea lyaaconica and A.

chamaemelifora. There have been no syntheses for compounds 10-26 so far.

Compounds 10-29 were subjected to Ellman’s assays [41] using acetylcholinesterase (from

Electrophorus electricus, electric eel) and butyrylcholinesterase (from equine serum) to

3

ACCEPTED MANUSCRIPT

evaluate their potential as inhibitors. The results of these investigations are summarized in

Table 1 and in Fig. 2.

Table 1. Inhibition of acetylcholinesterase (AChE from Electrophorus electricus) and

butyrylcholinesterase (BChE from equine serum) from Ellman’s assays in % (final substrate

concentration 50 µM); single experiments were each repeated ten times; standard: GH

galantamine hydrobromide. Compounds 12, 13, 16, 17, 20, 21, 25 and 29 were not soluble.

a

Inhibition [%]

AChE

BChE

GH 97.77 ± 0.38 85.97 ± 0.81

1

2

0

1

4

5

8

9

2

3

4

6

7

8

1.28 ± 0.29

38.91 ± 0.96

12.09 ± 1.81 9.82 ± 1.51

10.14 ± 0.64 32.63 ± 0.45

23.16 ± 1.99 10.95 ± 0.95

44.20 ± 0.95 18.79 ± 0.21

51.86 ± 1.86 17.76 ± 0.54

61.21 ± 0.36 28.56 ± 3.27

62.25 ± 0.75 18.54 ± 0.48

61.28 ± 1.00 22.16 ± 2.23

71.00 ± 0.64 28.72 ± 2.31

74.28 ± 0.42 16.76 ± 0.60

73.12 ± 0.07 18.72 ± 1.80

2

a

Mean ± SEM (standard mean error)

100

90

80

70

60

50

40

30

20

10

0

GH

10

11

14

15

18

19

22

23

24

26

27

28

AChE BChE

4

ACCEPTED MANUSCRIPT

Fig. 2. Graphical representation: Inhibition of acetylcholinesterase (AChE from Electrophorus

electricus) and butyrylcholinesterase (BChE, equine serum) from Ellman’s assays in %;

experiments were performed ten times; standard: GH galantamine hydrobromide. Compounds

1

2, 13, 16, 17, 20, 21, 25 and 29 were not soluble.

For the most active compounds of this series kinetic data were measured, and the inhibition

constants Ki were determined (Table 2). This data showed compounds 19, 22-24, and 26-28

as competitive inhibitors of AChE.

Table 2. Inhibition of AChE (Electrophorus electricus); data from Ellman’s assays;

individual experiments were repeated ten times; standard galantamine hydrobromide (GH); K_i

in µM.

Compound

GH

Inhibition constant K , K ’ [in µM]

i i

K

= 0.37 ± 0.14

i

19

22

23

24

26

27

28

= 7.45 ± 0.64; K

= 9.99 ± 0.52; K

= 3.41 ± 0.42; K

= 3.16 ± 0.15; K

= 6.83 ± 0.07; K

= 2.04 ± 0.03; K

= 5.09 ± 0.54; K

i

’ > 50

’ = 45.41 ± 0.42

’ = 81.85 ± 1.11

’ = 41.48 ± 0.75

’ = 22.99 ± 0.04

’ = 23.32 ± 4.30

’ > 50

i

As a result, compounds holding larger rings are stronger inhibitors for AChE. For smaller

rings, chain extension, too, led to analogs of higher activity for this enzyme. These

compounds are – by and large – only weak inhibitors for BChE. Interestingly enough, while

all compounds of this study are stronger inhibitors for AChE than for BChE, compounds 10

and 14 are an exception.

Molecular modeling studies were performed, and the results help at least partially to

understand the results ofvthe biological screening. These calculations showed the interaction

energy of the compounds being much higher for AChE than for BChE. This is mainly caused

by the narrower and even more hydrophobic gorge of the entrance to the active site in AChE

as compared to BChE. Thus, the hydrophobic interactions of the lipophilic aliphatic moiety

are much more favored to interact with AChE than with BChE. For example, the docking

arrangements of compound 27 are displayed in Fig. 3 and Fig. 4.

5

ACCEPTED MANUSCRIPT

Compound 27 exhibited hydrophobic interactions with the side chains of the amino acids

Y72, Y124, W286, F295, F297, F338, and Y341. Furthermore, the hydrophobic ring of this

compound interacts with the side chains of W86 and Y337. Obviously, these hydrophobic

interactions increase with the ring size getting larger. This corresponds well with the results

from the biological screening.

Fig. 3. Docking arrangement of 27 to AChE. Grey shaded surfaces represent lipophilic and

green shaded surfaces hydrophilic areas. Left: View from to top the gorge of the ligand

binding site. Right: Side view for better visualization of the hydrophobic interaction between

the side chain of W286 and the hydrophobic ring system of the ligand.

6

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Fig. 4. Docking arrangement of 27 to BChE. Grey shaded surfaces represent lipohilic and

green shaded surfaces hydrophilic areas.

A length of the side chain m = 12 seems to be optimal for the hydrophobic interactions in the

gorge. As a consequence, the very end of longer aliphatic residues is located at the water

accessible surface, and residues of diminished chain length (m < 12) exhibit reduced

hydrophobic interactions. These results from the docking, however, must be critically review

since these compounds are very flexible.

Penetration of the blood brain barrier (BBB) is crucial for AChE inhibitors intended to be of

any help in fighting AD. Hence, some calculations were performed to predict BBB

penetration using molecular dynamics simulations.[42] Thus, for the most active compound

2

7 of this series a log BBB = 1.4 was calculated; compounds with log BBB > 0.3 are usually

regarded to readily cross the BBB.[43]

In addition, the compounds were subjected to a cytotoxicity screening using sulforhodamine

B assays. As a result, highest cytotoxicity (cf. Supplementary data) was observed for 27 and

the human malignant cell line HT29 (colorectal adenocarcinoma). The cytotoxicity of this

compound was significant lower for the non-malignant cell line NIH 3T3 (mouse fibroblasts),

Piperlongumine B (19) showed only weak cytotoxicity for human breast adenocarcinoma cell

line MCF-7 even at a concentration of 60 µM.

3

. Conclusion

7

ACCEPTED MANUSCRIPT

Piperlongumine B and 19 analogs were synthesized and screened in Ellman’s assays. Several

of them were good inhibitors of acetylcholinesterase while being less active for

butyrylcholinesterase. Activity of the compounds for acetylcholinesterase increased with the

ring size of the heterocycle, and a maximum of activity was observed for an analog holding

1

2 methylene groups in the aliphatic side chain.

4

. Experimental

General

Detailed procedures for the screening and modelling as well as a description of the equipment

1

13

is given in the Supplementary material; H and C NMR spectra are depicted.

4

.1. (2E) Tetradecenoic acid (1)

A solution of dodecanal (700 mg, 3.797 mmol) and (tert-butoxycarbonylmethylene)-

triphenylphosphorane (1400 mg, 3.72 mmol) in dry DCM (2.5 mL) was stirred for 12 h at

room temperature. The solvent was removed under diminished pressure, and the residue was

suspended in Et O/n-hexane (1:1, 20 mL). The filtrate was evaporated, and the residue

2

subjected to chromatography (SiO , n-hexane/ethyl acetate, 95:5) to yield tert-butyl-

2

tetradecenoate (861 mg, 80.3%) as a colorless oil. An analytical sample showed: R = 0.29

F

(

SiO , n-hexane/ethyl acetate, 95:5); IR (KBr): ν� = 3456m, 2926vs, 2854s, 1718vs, 1654m,

2

-

1

466m, 1458m, 1392w, 1368m, 1290m, 1256m, 1156s, 1126m, 980m cm ; UV-Vis (CHCl ):

3

1

λ_m_a_x(log ε) = 238 (2.86) nm; H NMR (400 MHz, CDCl ): δ = 6.85 (ddd, J = 15.5, 6.9, 6.9

3

Hz, 1H, CH (3)), 5.73 (ddd, J = 15.5, 1.6, 1.6 Hz, 1H, CH (2)), 2.16 (dddd, J = 7.2, 7.2, 7.0,

1

0

1

.5 Hz, 2H, CH (4)), 1.48 (s, 9H, CH (16)), 1.46 – 1.39 (m, 2H, CH (5)), 1.34 – 1.23 (m,

2 3 2

6H, CH (6) + CH (7) + CH (8) + CH (9) + CH (10) + CH (11) + CH (12) + CH (13)),

2

13

.88 (t, J = 6.8 Hz, 3H, CH (14)) ppm; C NMR (100 MHz, CDCl ): δ = 166.0 (C=O, C1),

3

48.0 (CH, C3), 122.7 (CH, C2), 79.8 (C_q_u_a_r_t, C15), 31.9 (CH , C4), 31.8 (CH , C12), 29.5

2

(

[

CH ), 29.5 (CH ), 29.4 (CH ), 29.3 (CH ), 29.2 (CH ), 29.0 (CH ), 28.0 (CH , C16), 27.9

2 2 2 2 2 2 3

CH , C5), 22.5 (CH , C13), 13.9 (CH , C14) ppm; MS (ESI, MeOH): m/z = 227.1 (34%,

2

3

+

M+H−iso-butene] ), 305.1 (100%, [M+Na] ), 337.1 (36%, [M+Na+MeOH] ), 587.1 (50%,

+

2M+Na] ). A solution of this ester (486 mg, 1.72 mmol) in TFA (1.5 mL) was stirred at

room temperature for 12 h, the solvent was removed under diminished pressure, and 1

386 mg, 99.0%) was obtained as a colorless solid; an analytical sample showed: m.p.: 33 °C;

R = 0.31 (SiO , n-hexane/ethyl acetate/HCOOH, 7:3:0.05); IR (KBr): ν� = 3450vs, 2960m,

(

F

2

8

ACCEPTED MANUSCRIPT

-1

2

920s, 2850s, 1718m, 1694m, 1656m, 1468w, 1420w, 1312w, 1304w, 1286w cm ; UV-Vis

1

(

acetone): λ_m_a_x(log ε) = 209 (4.11) nm; H NMR (400 MHz, CDCl ): δ = 7.09 (ddd, J = 15.6,

3

7

.0, 7.0 Hz, 1H, CH (3)), 5.82 (ddd, J = 15.6, 1.6, 1.6 Hz, 1H, CH (2)), 2.23 (dddd, J = 7.1,

.1, 7.1, 1.6 Hz, 2H, CH (4)), 1.47 (quint, J = 7.2 Hz, 2H, CH (5)), 1.36 – 1.22 (m, 16H,

7

2

CH (6) + CH (7) + CH (8) + CH (9) + CH (10) + CH (11) + CH (12) + CH (13)), 0.88

2

1

3

(

t, J = 7.0 Hz, 3H, CH (14)) ppm; C NMR (100 MHz, CDCl ): δ = 171.8 (C=O, C1), 152.5

3

CH, C3), 120.5 (CH, C2), 32.3 (CH , C4), 31.9 (CH , C12), 29.6 (CH ), 29.6 (CH ), 29.5

2

CH ), 29.3 (CH ), 29.3 (CH ), 29.1 (CH , C6), 27.9 (CH , C5), 22.7 (CH , C13), 14.1 (CH ,

2

3

−

C14) ppm; MS (ESI, MeOH): m/z = 225.3 (34%, [M−H] ), 271.0 (6%, [M+HCO ] ), 467.1

2

−

(

20%, [2M−H] ), 473.5 (100%, [2M−2H+Na] ); analysis calcd for C H O (226.36): C

14 26 2

7

4.29, H 11.58; found: C 74.02, H 11.83.

4

.2. (2E) Hexadecenoic acid (2)

Commercially obtained.

4

.3. (2E) Octadecenoic acid (3)

Following the procedure given for the synthesis of 1, from hexadecanal (510 mg,

2

9

.121 mmol)

and

(tert-butoxycarbonylmethylene)triphenylphosphorane

(974 mg,

.587 mmol) in dry DCM (5 mL) followed by chromatography (SiO , n-hexane/ethyl acetate,

2

5:5) tert-butyl octadecenoate (710 mg, 98.8%) was obtained as a colorless oil; an analytical

sample showed: R = 0.27 (SiO , n-hexane/ethyl acetate, 95:5); IR (KBr): ν� = 3438m,

F

2

-1

2

924vs, 2852s, 1716s, 1654m, 1470m, 1390w, 1366m, 1322m, 1292m, 1158s, 1128m cm ;

1

UV-Vis (acetone): λ_m_a_x(log ε) = 213 (2.58) nm; H NMR (400 MHz, CDCl ): δ = 6.86 (ddd, J

3

=

15.5, 6.9, 6.9 Hz, 1H, CH (3)), 5.73 (ddd, J = 15.6, 1.5, 1.5 Hz, 1H, CH (2)), 2.15 (dddd, J

7.1, 7.1, 7.1, 1.6 Hz, 2H, CH (4)), 1.48 (s, 9H, CH (20)), 1.46 – 1.39 (m, 2H, CH (5)),

2

3

2

1

.35 – 1.21 (m, 24H, CH (6) + CH (7) + CH (8) + CH (9) + CH (10) + CH (11) + CH

2 2 2 2 2 2

2

(

12) + CH (13) + CH (14) + CH (15) + CH (16) + CH (17)), 0.88 (t, J = 6.9 Hz, 3H, CH

2 2 2 2 2

3

1

3

(

18)) ppm; C NMR (100 MHz, CDCl ): δ = 166.4 (C=O, C1), 148.3 (CH, C3), 123.0 (CH,

3

C2), 80.1 (C_q_u_a_r_t, C19), 32.2 (CH , C4), 32.1 (CH , C16), 29.9 (CH ), 29.8 (CH ), 29.8 (CH ),

2

9.8 (CH ), 29.8 (CH ), 29.7 (CH ), 29.6 (CH ), 29.5 (CH ), 29.4 (CH , C6), 28.3 (CH ,

2 2 2 2 2 2 3

C20), 28.3 (CH , C5), 22.9 (CH , C17), 14.3 (CH , C18) ppm; MS (ESI, MeOH): m/z = 283.2

2

3

+

(

16%, [M+H−iso-butene] ), 361.2 (98%, [M+Na] ), 677.1 (42%, [2M+H] ), 696.4 (36%,

2

+

[

4M+K+H] ), 699.1 (100%, [2M+Na] ). From the reaction of this ester (205 mg,

0

.605 mmol) in dry DCM (6 mL) with TFA (1.8 mL) for 1 h at room temperature 3 (169 mg,

9

ACCEPTED MANUSCRIPT

9

8.8%) was obtained as a colorless solid. An analytical sample showed: m.p.: 56 °C (lit.: 58

°

C [44]) R = 0.80 (SiO , n-hexane/ethyl acetate, 1:1); IR (KBr): ν� = 3448m, 2960m, 2920vs,

F 2

-1

2

848s, 1720m, 1692m, 1656m, 1468m, 1420w, 1312w, 1288w cm ; UV-Vis (acetone): λ

max

1

(

log ε) = 209 (4.10) nm; H NMR (400 MHz, CDCl ): δ = 7.09 (ddd, J = 15.6, 7.0, 7.0 Hz,

3

1

H, CH (3)), 5.82 (ddd, J = 15.6, 1.6, 1.6 Hz, 1H, CH (2)), 2.23 (dddd, J = 7.1, 7.1, 7.1, 1.6

Hz, 2H, CH (4)), 1.53 – 1.41 (m, 2H, CH (5)), 1.35 – 1.22 (m, 24H, CH (6) + CH (7) +

2

CH (8) + CH (9) + CH (10) + CH (11) + CH (12) + CH (13) + CH (14) + CH (15) +

2

1

3

CH (16) + CH (17)), 0.88 (t, J = 6.5 Hz, 3H, CH (18)) ppm; C NMR (100 MHz, CDCl ):

2

3

δ = 172.0 (C=O, C1), 152.8 (CH, C3), 120.6 (CH, C2), 32.5 (CH , C4), 32.1 (CH , C16), 29.9

2

(

CH ), 29.9 (CH ), 29.8 (CH ), 29.8 (CH ), 29.8 (CH ), 29.7 (CH ), 29.5 (CH ), 29.5 (CH ),

2 2 2 2 2 2 2 2

2

9.3 (CH , C6), 28.0 (CH , C5), 22.9 (CH , C17), 14.3 (CH , C18) ppm; MS (ESI, MeOH):

2 2 2 3

−

m/z = 281.3 (12%, [M−H] ), 327.0 (12%, [M+HCO ] ), 563.1 (100%, [2M−H] ); analysis

2

calcd for C H O (282.47): C 76.54, H 12.13; found: C 76.37, H 12.30.

1

8

34

2

4

.4. (2E) Eicosenoic acid (4)

Following the precedure given for the synthesis of 1, from octadecanal (500 mg, 1.862 mmol)

and (tert-butoxycarbonylmethylen)triphenylphosphorane (873 mg, 2.319 mmol) followed by

chromatography (SiO , n-nexan/ethyl acetate, 95:5) tert-butyl eicosenoate (558 mg, 81.7%)

2

was obtained as a colorless solid; analytical data: m.p.: 34 °C; R = 0.32 (SiO , n-hexane/ethyl

F

2

acetate, 95:5); IR (KBr): ν� = 3444s, 2920vs, 2850s, 1710s, 1652m, 1472m, 1366m, 1284w,

-1

1

250m, 1166m, 1128w, 1060w cm ; UV-Vis (acetone): λ

(log ε) = 213 (2.96) nm; H

max

NMR (400 MHz, CDCl ): δ = 6.86 (ddd, J = 15.6, 6.9, 6.9 Hz, 1H, CH (3)), 5.73 (ddd,

3

J = 15.6, 1.6, 1.6 Hz, 1H, CH (2)), 2.16 (dddd, J = 7.1, 7.1, 7.1, 1.6 Hz, 2H, CH (4)), 1.48 (s,

2

9

H, CH (22)), 1.46 – 1.39 (m, 2H, CH (5)), 1.34 – 1.23 (m, 28H, CH (6) + CH (7) + CH

3 2 2 2 2

(

8) + CH (9) + CH (10) + CH (11) + CH (12) + CH (13) + CH (14) + CH (15) + CH (16)

2 2 2 2 2 2 2 2

1

3

+

CH (17) + CH (18) + CH (19)), 0.87 (t, J = 7.0 Hz, 3H, CH (20)) ppm; C NMR (100

2 2 2 3

MHz, CDCl ): δ = 166.4 (C=O, C1), 148.4 (CH, C3), 123.0 (CH, C2), 80.1 (C_q_u_a_r_t, C21), 32.2

3

(

CH , C4), 32.1 (CH , C18), 29.9 (CH ), 29.8 (CH ), 29.8 (CH ), 29.8 (CH ), 29.7 (CH ),

2 2 2 2 2 2 2

2

1

9.6 (CH ), 29.5 (CH ), 29.4 (CH , C6), 28.3 (CH , C22), 28.3 (CH , C5), 22.9 (CH , C19),

2 2 2 3 2 2

+

4.3 (CH , C20) ppm; MS (ESI, MeOH): m/z = 311.2 (26%, [M+H-iso-butene] ), 332.6 (4%,

3

+

[

(

M+Na−iso-butene] ), 342.9 (40%, [M+H−iso-butene+MeOH] ), 267.2 (8%, [M+H] ), 389.2

+

100%, [M+Na] ); analysis calcd for C H O (366.63): C 78.63, H 12.65; found: C 78.49, H

2

4

48

2

10

ACCEPTED MANUSCRIPT

1

2.75. To a solution of this ester (330 mg, 0.90 mmol) in dry DCM (8 mL) at 0 °C TFA (2.7

mL) was added; stirring at room temperature was continued for another 45 min, and the

solvents were removed under reduced pressure to yield 4 (259 mg, 92.7%) as a colorless

solid; an analytical sample showed: m.p.: 63 °C; R = 0.32 (SiO , n-hexane/ethyl acetate, 9:1);

F

2

-1

IR (KBr): ν� = 3452s, 2960m, 2920vs, 2848s, 1718m, 1654m, 1470m, 1420w, 1284w cm ;

1

UV-Vis (acetone): λ_m_a_x(log ε) = 211 (4.09) nm; H NMR (400 MHz, CDCl ): δ = 7.09 (ddd, J

3

=

15.5, 6.9, 6.9 Hz, 1H, CH (3)), 5.82 (ddd, J = 15.6, 1.6, 1.6 Hz, 1H, CH (2)), 2.23 (dddd, J

7.1, 7.1, 7.1, 1.5 Hz, 2H, CH (4)), 1.47 (quint, J = 7.1 Hz, 2H, CH (5)), 1.34 – 1.23 (m,

2

8H, CH (6) + CH (7) + CH (8) + CH (9) + CH (10) + CH (11) + CH (12) + CH (13) +

2 2 2 2 2 2 2 2

CH (14) + CH (15) + CH (16) + CH (17) + CH (18) + CH (19)), 0.88 (t, J = 6.7 Hz, 3H,

2

1

3

CH (20)) ppm; C NMR (100 MHz, CDCl ): δ = 171.9 (C=O, C1), 152.7 (CH, C3), 120.6

3

(

CH, C2), 32.5 (CH , C4), 32.1 (CH , C18), 29.9 (CH ), 29.8 (CH ), 29.8 (CH ), 29.8 (CH ),

2 2 2 2 2 2

2

9.8 (CH ), 29.7 (CH ), 29.5 (CH ), 29.5 (CH ), 29.3 (CH , C6), 28.0 (CH , C5), 22.9 (CH ,

2 2 2 2 2 2 2

−

C19), 14.3 (CH , C20) ppm; MS (ESI, MeOH): m/z = 309.3 (100%, [M−H] ), 354.9 (36%,

3

−

[

M+HCO ] ); analysis calcd for C H O (310.52): C 77.36, H 12.34; found: C 77.26, H

2

20 38

2

1

2.51.

4

.5. (2E) 1-(Azetidin-1-yl)-tetradec-2-en-1-one (10)

To a solution of 1 (150 mg, 0.663 mmol) in dry DCM (5 mL) at 0 °C oxalyl chloride

0.14 mL, 1.657 mmol) was slowly added, and stirring was continued for another 2 h. The

(

volatiles were removed under reduced pressure, and the residue was added slowly to a

mixture of potassium carbonate (106 mg, 0.763 mmol) and azetidine hydrochloride (5.HCl,

7

1 mg, 0.763 mmol) in Et O (1 mL) and water (1 mL). Stirring at 0 °C was continued

2

overnight, and the mixture was diluted with ether (100 mL). Usual aq. workup followed by

chromatography (SiO , ethyl acetate) yielded 10 (137 mg, 77.8%) as a colorless solid; m.p.:

2

5

1

7 °C; R = 0.31 (SiO , ethyl acetate); IR (KBr): ν� = 3448m, 2950m, 2916vs, 2872m, 2848s,

F 2

668s, 1618vs, 1490w, 1470s, 1442s, 1382w, 1324w, 1298w, 1242w, 1158w, 1022w, 990m

-1

1

cm ; UV-Vis (acetone): λ (log ε) = 213 (4.14) nm; H NMR (400 MHz, CDCl ): δ = 6.85

max

3

(ddd, J = 15.4, 7.0, 7.0 Hz, 1H, CH (3)), 5.81 (ddd, J = 15.3, 1.6, 1.6 Hz, 1H, CH (2)), 4.13 (t,

J = 7.7 Hz, 4H, CH (1') + CH (3')), 2.32 – 2.23 (m, 2H, CH (2')), 2.17 (dddd, J = 7.2, 7.2,

2

7

.2, 1.5 Hz, 2H, CH (4)), 1.42 (quint, J = 7.2 Hz, 2H, CH (5)), 1.33 – 1.21 (m, 16H, CH (6)

2 2 2

+

CH (7) + CH (8) + CH (9)+ CH (10) + CH (11) + CH (12) + CH (13)), 0.87 (t, J = 6.9

2 2 2 2 2 2 2

1

3

Hz, 3H, CH (14)) ppm; C NMR (100 MHz, CDCl ): δ = 166.4 (C=O, C1), 145.8 (CH, C3),

3

1

18.5 (CH, C2), 49.0 (CH , C1' + C3'), 32.5 (CH , C4), 32.1 (CH , C12), 29.8 (CH ), 29.8

2 2 2 2

11

ACCEPTED MANUSCRIPT

(

CH ), 29.7 (CH ), 29.6 (CH ), 29.5 (CH ), 29.3 (CH , C6), 28.5 (CH , C5), 22.8 (CH , C13),

2

+

1

5.5 (CH , C2'), 14.2 (CH , C14) ppm; MS (ESI, MeOH): m/z = 266.3 (44%, [M+H] ), 288.2

2 3

+

(

8%, [M+Na] ), 531.13 (20%, [2M+H] ), 553.2 (100%, [2M+Na] ); analysis calcd for

C H NO (265.44): C 76.92, H 11.77, N 5.28; found: C 76.73, H 11.97, N 5.04.

17

31

4

.6. (2E) 1-(Azetidin-1-yl)-hexadec-2-en-1-one (11)

Following the procedure given for the synthesis of 10, from 2 (200 mg, 0.786 mmol) oxalyl

chloride (0.17 mL, 1.965 mmol) azetidine hydrochlorid (5.HCl, 88 mg, 0.943 mmol) and

K CO (130 mg, 0.943 mmol) followed by chromatography (SiO , n-hexane/ethyl acetate,

2

3

2

1

:1) 11 (173 mg, 75.0%) was obtained as a colorless solid; m.p.: 66 °C; R = 0.23 (SiO , n-

F 2

hexane/ethyl acetate, 1:1); IR (KBr): ν� = 3448vs, 2950m, 2916vs, 2848s, 1668m, 1618s,

-1

1

470m, 1442m, 1384w, 1298w, 1242w, 1160w cm ; UV-Vis (acetone): λ

(log ε) = 212

max

1

(

4.34) nm; H NMR (400 MHz, CDCl ): δ = 6.85 (ddd, J = 15.1, 7.1, 7.1 Hz, 1H, CH (3))

3

5

2

.81 (ddd, J = 15.3, 1.5, 1.5 Hz, 1H, CH (2)), 4.13 (t, J = 7.7 Hz, 4H, CH (1') + CH (3')),

2 2

.32 – 2.23 (m, 2H, CH (2')), 2.17 (dddd, J = 7.2, 7.2, 7.2, 1.5 Hz, 2H, CH (4)), 1.43 (quint,

2

J = 7.2 Hz, 2H, CH (5)), 1.34 – 1.21 (m, 20H, CH (6) + CH (7) + CH (8) + CH (9) + CH

2

(

10) + CH (11) + CH (12) + CH (13) + CH (14) + CH (15)), 0.87 (t, J = 6.9 Hz, 3H, CH

2 2 2 2 2

3

1

3

(

16)) ppm; C NMR (100 MHz, CDCl ): δ = 166.4 (C=O, C1), 145.8 (CH, C3), 118.5 (CH,

3

C2), 48.9 (br, CH , C1' + C3'), 32.5 (CH , C4), 32.1 (CH , C14), 29.8 (CH ), 29.8 (CH ), 29.8

2

(

CH ), 29.7 (CH ), 29.6 (CH ), 29.5 (CH ), 29.3 (CH , C6), 28.5 (CH , C5), 22.8 (CH , C15),

2 2 2 2 2 2 2

+

1

5.5 (CH , C2'), 14.3 (CH , C16) ppm; MS (ESI, MeOH): m/z = 294.4 (100%, [M+H] ),

2 3

+

3

16.2 (16%, [M+Na] ); analysis calcd for C H NO (293.50): C 77.76, H 12.07, N 4.77;

19 35

found: C 77.46, H 12.18, N 4.46.

4

.7. (2E) 1-(Azetidin-1-yl)-octadec-2-en-1-one (12)

Following the procedure given for the synthesis of 10, from 3 (150 mg, 0.531 mmol), oxalyl

chloride (0.12 mL, 1.33 mmol), potassium carbonate (88 mg, 0.637 mmol) and 5.HCl (60 mg,

0

.637 mmol) followed by chromatography (SiO , ethyl acetate) 12 (134 mg, 78.5%) was

2

obtained as a colorless solid; m.p.: 72 °C; R = 0.31 (SiO , ethyl acetate); IR (KBr): ν� =

F

2

-1

3

448s, 2916vs, 2848s, 1668m, 1618s, 1470m, 1442m, 1384w cm ; UV-Vis (acetone): λ

max

1

(

log ε) = 214 (4.18) nm; H NMR (400 MHz, CDCl ): δ = 6.86 (ddd, J = 15.2, 7.0, 7.0 Hz,

3

1

H, CH (3)), 5.81 (ddd, J = 15.3, 1.6, 1.6 Hz, 1H, CH (2)), 4.13 (t, J = 7.3 Hz, 4H, CH (1') +

2

CH (3')), 2.32 – 2.23 (m, 2H, CH (2')), 2.17 (dddd, J = 7.2, 7.2, 7.2, 1.5 Hz, 2H, CH (4)),

2

1

.43 (quint, J = 7.1 Hz, 2H, CH (5)), 1.34 – 1.21 (m, 24H, CH (6) + CH (7) + CH (8) +

2 2 2 2

12

ACCEPTED MANUSCRIPT

CH (9) + CH (10) + CH (11) + CH (12) + CH (13) + CH (14) + CH (15) + CH (16) +

2

1

3

CH (17)), 0.87 (t, J = 6.8 Hz, 3H, CH (18)) ppm; C NMR (100 MHz, CDCl ): δ = 166.4

2

3

(

C=O, C1), 145.8 (CH, C3), 118.5 (CH, C2), 49.1 (br, CH , C1' + C3'), 32.5 (CH , C4), 32.1

2 2

CH , C16), 29.8 (CH ), 29.8 (CH ), 29.8 (CH ), 29.8 (CH ), 29.7 (CH ), 29.6 (CH ), 29.5

2

CH ), 29.3 (CH , C6), 28.5 (CH , C5), 22.8 (CH , C17), 15.5 (CH , C2'), 14.3 (CH , C18)

2

3

+

ppm; MS (ESI, MeOH): m/z = 322.4 (36%, [M+H] ), 344.2 (4%, [M+Na] ), 643.3 (54%,

+

[

2M+H] ), 665.3 (100%, [2M+Na] ); analysis calcd for C H NO (321.55): C 78.44, H

21 39

1

2.23, N 4.36; found: C 78.19, H 12.41, N 4.25.

4

.8. (2E) 1-(Azetidin-1-yl)-eicos-2-en-1-one (13)

Following the procedure given for the synthesis of 10, from 4 (150 mg, 0.483 mmol), oxalyl

chloride (153 mg, 1.208 mmol) and 5.HCl (54 mg, 0.579 mmol) followed by chromatography

(

SiO , ethyl acetate) 13 (120 mg, 71.1%) was obtained as a colorless solid; m.p.: 74 °C; R =

2

F

0

.36 (SiO , ethyl acetate); IR (KBr): ν� = 3450s, 2916vs, 2848s, 1668m, 1618s, 1470m,

2

-1

1

442m, 1384w, 1298w, 1240vw, 1160w cm ; UV-Vis (acetone): λ (log ε) = 212 (4.15) nm;

max

1

H NMR (400 MHz, CDCl ): δ = 6.85 (ddd, J = 14.6, 7.0, 7.0 Hz, 1H, CH (3)), 5.82 (ddd,

3

J = 15.3, 1.7, 1.7 Hz, 1H, CH (2)), 4.13 (br, 4H, CH (1') + CH (3')), 2.28 (quint, J = 7.7 Hz,

2

H, CH (2')), 2.17 (dddd, J = 7.2, 7.2, 7.2, 1.4 Hz, 2H, CH (4)), 1.43 (quint, J = 7.2 Hz, 2H,

2 2

CH (5)), 1.33 – 1.21 (m, 28H, CH (6) + CH (7) + CH (8) + CH (9) + CH (10) + CH (11)

2

+

CH (12) + CH (13) + CH (14) + CH (15) + CH (16) + CH (17) + CH (18) + CH (19)),

2 2 2 2 2 2 2 2

1

3

0

.87 (t, J = 6.7 Hz, 3H, CH (20)) ppm; C NMR (100 MHz, CDCl ): δ = 166.4 (C=O, C1),

3 3

1

45.8 (CH, C3), 118.5 (CH, C2), 49.9 (CH , C3'), 48.1 (CH , C1'), 32.5 (CH , C4), 32.1

2

(CH , C18), 29.8 (CH ), 29.8 (CH ), 29.8 (CH ), 29.8 (CH ), 29.7 (CH ), 29.6 (CH ), 29.5

2 2 2 2 2 2 2

(

CH ), 29.3 (CH , C6), 28.5 (CH , C5), 22.8 (CH , C19), 15.5 (CH , C2'), 14.3 (CH , C20)

2

3

+

ppm; MS (ESI, MeOH): m/z = 350.5 (34%, [M+H] ), 372.3 (4%, [M+Na] ), 699.3 (52%,

+

[

2M+H] ), 721.3 (100%, [2M+Na] ); analysis calcd for C H NO (349.60): C 79.02, H

23 43

1

2.40, N 4.01; found: C 78.87, H 12.53, N 3.81.

4

.9. (2E) 1-(Pyrrolidin-1-yl)-tetradec-2-en-1-one (14)

To an ice-cold solution of 1 (144 mg, 0.636 mmol) in dry DCM (5 mL) EDC (134 mg,

.699 mmol) and HOBt (127 mg, 0.827 mmol) were added, and stirring was continued for 15

0

min. Freshly distilled pyrrolidine (6, 0.06 mL, 0.763 mmol) was added, the reaction allowed

to warm to room temperature, and stirring was continued overnight. The solvent was removed

under reduced pressure, and the residue subjected to chromatography (SiO , n-hexane/ethyl

2

13

ACCEPTED MANUSCRIPT

acetate, 1:1) to yield 14 (135 mg, 75.9%) as a colorless solid; m.p.: 45 °C; R = 0.29 (SiO , n-

F

2

hexane/ethyl acetate, 1:1); IR (KBr): ν� = 3452s, 2956s, 2918vs, 2852s, 1670m, 1614s,

-1

1

472m, 1448m, 1318w, 1276vw, 986w cm ; UV-Vis (acetone): λ (log ε) = 213 (4.11) nm;

max

1

H NMR (400 MHz, CDCl ): δ = 6.91 (ddd, J = 15.0, 7.0, 7.0 Hz, 1H, CH (3)), 6.08 (ddd,

3

J = 15.1, 1.6, 1.6 Hz, 1H, CH (2)), 3.51 (t, J = 6.7 Hz, 3H, CH (1') + CH (4')), 2.19 (dddd,

2

J = 7.2, 7.2, 7.2, 1.5 Hz, 2H, CH (4)), 1.90 (br, 4H, CH (2') + CH (3')), 1.44 (quint, J = 7.2

2

Hz, 2H, CH (5)), 1.34 – 1.21 (m, 16H, CH (6) + CH (7) + CH (8) + CH (9) + CH (10) +

2

13

CH (11) + CH (12) + CH (13)), 0.87 (t, J = 6.7 Hz, 3H, CH (14)) ppm; C NMR (100

2

3

MHz, CDCl ): δ = 165.1 (C=O, C1), 146.2 (CH, C3), 121.6 (CH, C2), 46.3 (br, CH , C1' +

3

2

C4'), 32.6 (CH , C4), 32.1 (CH , C12), 29.8 (CH ), 29.8 (CH ), 29.7 (CH ), 29.6 (CH ), 29.5

2

(

CH ), 29.4 (CH , C6), 28.6 (CH , C5), 26.2 (br, CH , C3'), 24.5 (br, CH , C2'), 22.8 (CH ,

2 2 2 2 2 2

+

C13), 14.3 (CH , C14) ppm; MS (ESI, MeOH): m/z = 280.3 (30%, [M+H] ), 302.3 (6%,

3

+

[

(

M+Na] ), 559.1 (36%, [2M+H] ), 581.2 (100%, [2M+Na] ); analysis calcd for C H NO

18 33

279.47): C 77.36, H 11.90, N 5.01; found: C 77.15, H 12.04, N 4.86.

4

.10.

To a solution of 2 (300 mg, 1.179 mmol) in dry DCM (10 mL) at 0 °C EDC (249 mg,

.299 mmol) and DMAP (144 mg, 1.179 mmol) were added. After 5 min of stirring,

(2E) 1-(Pyrrolidin-1-yl)-hexadec-2-en-1-one (15)

1

pyrrolidine (6, 0.10 mL, 1.299 mmol) was slowly added. The mixture was allowed to warm to

room tempetrature, and stirring was continued overnight. The solvent was removed under

reduced pressure, and the residue subjected to chromatography (SiO , n-hexane/ethyl acetate,

2

6

:4) to yield 15 (243 mg, 66.9%) as a colorless solid; m.p.: 53 °C; R = 0.23 (SiO , n-

F 2

hexane/ethyl acetate, 6:4); IR (KBr): ν� = 3447vs, 2920s, 2852m, 1668m, 1616m, 1472w,

-

1

6

3

448w cm ; UV-Vis (CHCl ): λ (log ε) = 232 (3.93) nm; H NMR (500 MHz, CDCl ): δ =

3 max 3

.91 (ddd, J = 15.1, 7.0, 7.0 Hz, 1H, CH (3)), 6.08 (ddd, J = 15.1, 1.6, 1.6 Hz, 1H, CH (2)),

.51 (t, J = 6.7, Hz, 4H, CH (1') + CH (4')), 2.19 (dddd, J = 7.2, 7.2, 7.2, 1.5 Hz, 2H, CH

2

(

4)), 1.93 (br, 2H, CH (3')), 1.87 (br, 2H, CH (2')), 1.44 (quint, J = 7.3 Hz, 2H, CH (5)),

2 2 2

1

.34 – 1.21 (m, 20H, CH (6) + CH (7) + CH (8) + CH (9) + CH (10) + CH (11) + CH

2 2 2 2 2 2 2

1

3

(

12) + CH (13) + CH (14) + CH (15)), 0.87 (t, J = 6.9 Hz, 3H, CH (16)) ppm; C NMR

2 2 2 3

(

100 MHz, CDCl ): δ = 165.1 (C=O, C1), 146.1 (CH, C3), 121.7 (CH, C2), 46.6 (br, CH ,

3

2

C4'), 46.0 (br, CH , C1'), 32.6 (CH , C4), 32.1 (CH , C14), 29.8 (CH ), 29.8 (CH ), 29.8

2

(

CH ), 29.7 (CH ), 29.6 (CH ), 29.5 (CH ), 29.4 (CH , C6), 28.6 (CH , C5), 26.3 (br, CH ,

2 2 2 2 2 2 2

14

ACCEPTED MANUSCRIPT

C3'), 24.5 (br, CH , C2'), 22.8 (CH , C15), 14.3 (CH , C16) ppm; MS (ESI, MeOH): m/z =

2

+

3

08.4 (80%, [M+H] ), 315.2 (18%, [2M+H] ), 330.3 (12%, [M+Na] ), 480.9 (5%,

2

+

[

3M+K+H] ), 637.3 (100%, [2M+Na] ); analysis calcd for C H NO (307.52): C 78.11, H

20 37

1

2.13 N 4.55; found: C 77.90, H 12.32, N 4.21.

4

.11.

To an ice-cold solution of 3 (158 mg, 0.559 mmol) in dry DCM (5 mL) EDC (118 mg,

.615 mmol) and HOBt (111 mg, 0.727 mmol) were added, and stirring was continued for

(2E) 1-(Pyrrolidin-1-yl)-octadec-2-en-1-one (16)

0

another 15 min; pyrrolidine (6, 0.06 mL, 0.671 mmol) was slowly added, the mixture was

allowed to warm to room temperature, and stirring was continued for 12 h. The solvent was

removed under reduced pressure, and the residue was subjected to chromatography (SiO₂,

n-hexane/ethyl acetate, 1:1) to yield 16 (159 mg, 0.474 mmol, 84.8%) as a colorless solid;

m.p.: 63 °C; R = 0.41 (SiO , n-hexane/ethyl acetate, 1:1); IR (KBr): ν� = 3448s, 2956m,

F

2

-1

2

918vs, 2850s, 1670m, 1614m, 1472m, 1448m, 1384w, 1338vw cm ; UV-Vis (acetone): λ

max

1

(

log ε) = 212 (4.19) nm; H NMR (400 MHz, CDCl ): δ = 6.92 (ddd, J = 15.2, 7.0, 7.0 Hz,

3

1

H, CH (3)), 6.08 (ddd, J = 15.1, 1.5, 1.5 Hz, 1H, CH (2)), 3.56 – 3.47 (m, 4H, CH (1') +

2

CH (4')), 2.19 (dddd, J = 7.2, 7.2, 7.2, 1.5 Hz, 2H, CH (4)), 1.90 (br, 4H, CH (2') + CH

2

(

3')), 1.44 (quint, J = 7.1 Hz, 2H, CH (5)), 1.34 – 1.22 (m, 24H, CH (6) + CH (7) + CH (8)

2 2 2 2

+

CH (9)+ CH (10) + CH (11) + CH (12) + CH (13) + CH (14) + CH (15) + CH (16) +

2 2 2 2 2 2 2 2

13

CH (17)), 0.87 (t, J = 6.9 Hz, 3H, CH (18)) ppm; C NMR (100 MHz, CDCl ): δ = 165.1

2

3

(

C=O, C1), 146.2 (CH, C3), 121.6 (CH, C2), 46.3 (br, CH , C1' + C4'), 32.6 (CH , C4), 32.1

2 2

CH , C16), 29.8 (CH ), 29.8 (CH ), 29.8 (CH ), 29.8 (CH ), 29.7 (CH ), 29.6 (CH ), 29.5

2

CH ), 29.4 (CH , C6), 28.6 (CH , C5), 26.0 (br, CH , C3'), 24.7 (br, CH , C2'), 22.8 (CH ,

2

+

C17), 14.3 (CH , C18) ppm; MS (ESI, MeOH): m/z = 366.4 (82%, [M+H] ), 358.3 (4%,

3

+

[

(

M+Na] ), 671.3 (80%, [2M+H] ), 693.3 (100%, [2M+Na] ); analysis calcd for C H NO

22 41

335.58): C 78.74, H 12.32, N 4.17; found; C 78.51, H 12.33, N 4.02.

4

.12.

To an ice-cold solution of 4 (150 mg, 0.483 mmol) in dry DCM (5 mL) EDC (101 mg,

.528 mmol) and HOBt (96 mg, 0.624 mmol) werde added, and stirring was continued for 15

(2E) 1-(Pyrrolidin-1-yl)-eicos-2-en-1-one (17)

0

min. Pyrrolidine (6, 0.05 mL, 0.624 mmol) was slowly added, the mixture was allowed to

warm to room temperature, and stirred for another 12 h. The solvent was removed, and the

residue was subjected to chromatography (SiO , n-hexane/ethyl acetate, 1:1) to yield 17

2

(

161 mg, 91.7%) as a colorless solid; m.p.: 70 °C; R = 0.38 (SiO , n-hexane/ethyl acetate,

F 2

15

ACCEPTED MANUSCRIPT

1

:1); IR (KBr): ν� = 3448m, 2954m, 2916vs, 2870m, 2850s, 1670m, 1614s, 1472m, 1448m

-1

1

cm ; UV-Vis (acetone): λ (log ε) = 212 (4.21) nm; H NMR (500 MHz, CDCl ): δ = 6.91

max

3

(ddd, J = 15.1, 7.0, 7.0 Hz, 1H, CH (3)), 6.08 (ddd, J = 15.1, 1.6, 1.6 Hz, 1H, CH (2)), 3.51 (t,

J = 6.7 Hz, 4H, CH (1') + CH (4')), 2.19 (dddd, J = 7.2, 7.2, 7.2, 1.5 Hz, 2H, CH (4)), 1.90

2

(br, 4H, CH (2') + CH (3')), 1.44 (quint, J = 7.3 Hz, 2H, CH (5)), 1.33 – 1.22 (m, 28H, CH

2 2 2 2

(

6) + CH (7) + CH (8) + CH (9) + CH (10) + CH (11) + CH (12) + CH (13) + CH (14) +

2

CH (15) + CH (16) + CH (17) + CH (18) + CH (19)), 0.87 (t, J = 6.9 Hz, 3H, CH (20))

2

3

1

3

ppm; C NMR (100 MHz, CDCl ): δ = 165.1 (C=O, C1), 146.2 (CH, C3), 121.6 (CH, C2),

3

4

6.3 (br, CH , C1' + C4'), 32.6 (CH , C4), 32.1 (CH , C18), 29.8 (CH ), 29.8 (CH ), 29.8

2 2 2 2 2

(CH ), 29.8 (CH ), 29.7 (CH ), 29.6 (CH ), 29.5 (CH ), 29.4 (CH , C6), 28.6 (CH , C5), 26.2

2 2 2 2 2 2 2

(

br, CH , C3'), 24.7 (br, CH , C2'), 22.8 (CH , C19), 14.3 (CH , C20) ppm; MS (ESI,

2

3

+

MeOH): m/z = 364.5 (68%, [M+H] ), 386.3 (4%, [M+Na] ), 727.3 (64%, [2M+H] ), 749.4

+

(

100%, [2M+Na] ); analysis calcd for C H NO (363.63): C 79.27, H 12.47, N 3.85; found:

2

4

45

C 79.02, H 12.60, N 3.58.

4

.13.

To an ice-cold solution of 1 (270 mg, 1.193 mmol) in dry DCM (10 mL) EDC (247 mg,

.299 mmol) and HOBt (235 mg, 1.534 mmol) were added, and stirring was continued for 15

(2E) 1-(Piperidin-1-yl)-tetradec-2-en-1-one (18)

1

min. zugegeben. Piperidine (7, 0.14 mL, 1.416 mmol) was added, the mixture was allowed to

warm to room temperature, and stirring was continued overnight. The solvent was removed

under reduced pressure, and the residue was subjected to chromatography (SiO , n-

2

hexane/ethyl acetate, 7:3 ) to yield 18 (331 mg, 99.2%) as a colorless solid; m.p.: 34 °C; R =

F

0

1

.37 (SiO , n-hexane/ethyl acetate, 7:3); IR (KBr): ν� = 3448s, 2918vs, 2850s, 1660m, 1612s,

2

-1

466m, 1442m, 1300vw, 1278w, 1258w, 1230w, 1220w, 1136w, 1122w cm ; UV-Vis

1

(

acetone): λ_m_a_x(log ε) = 212 (4.53) nm; H NMR (400 MHz, CDCl ): δ = 6.82 (ddd, J = 15.0,

3

7

.0, 7.0 Hz, 1H, CH (3)), 6.22 (ddd, J = 15.0, 1.5, 1.5 Hz, 1H, CH (2)), 3.53 (br, 4H, CH (1')

2

+

CH (5')), 2.18 (dddd, J = 7.1, 7.1, 7.1, 1.5 Hz, 2H, CH (4)), 1.68 – 1.60 (m, 2H, CH (3')),

2 2 2

1

.59 – 1.51 (m, 4H, CH (2') + CH (4')), 1.44 (quint, J = 7.2 Hz, 2H, CH (5)), 1.34 – 1.21

2 2 2

(m, 16H, CH (6) + CH (7) + CH (8) + CH (9) + CH (10) + CH (11) + CH (12) + CH

2 2 2 2 2 2 2 2

1

3

(

13)), 0.87 (t, J = 6.8 Hz, 3H, CH (14)) ppm; C NMR (100 MHz, CDCl ): δ = 165.8 (C=O,

3

C1), 146.2 (CH, C3), 120.4 (CH, C2), 45.7 (br, CH , C5'), 43.9 (br, CH , C1'), 32.7 (CH ,

2

C4), 32.1 (CH , C12), 29.8 (CH ), 29.8 (CH ), 29.7 (CH ), 29.6 (CH ), 29.5 (CH ), 29.4

2

(CH , C6), 28.6 (CH , C5), 26.2 (br, CH , C2' + C4'), 24.8 (CH , C3'), 22.8 (CH , C13), 14.2

2 2 2 2 2

+

(

CH , C14) ppm; MS (ESI, MeOH): m/z = 294.4 (100%, [M+H] ), 316.3 (16%, [M+Na] );

3

16

ACCEPTED MANUSCRIPT

analysis calcd for C H NO (293.50): C 77.76, H 12.02, N 4.77; found: C 77.49, H 12.31, N

1

9

35

4

.56.

4

.14.

(2E) 1-(Pyrrolidin-1-yl)-hexadec-2-en-1-one (19), Piperlongumine B

To an ice-cold solution of 2 (100 mg, 0.393 mmol) in dry DCM (3 mL), oxalyl chloride

0.18 mL, 0.983 mmol) and one drop of DMF were added; stirring at 0 °C was continued for

h, and the volatiles were removed under diminished pressure. The residue was dissolved in

(

2

dry DCM (5 mL) containing catalytic amounts of DMAP and TEA. Slow addition of

piperidine (7, 0.43 mL, 0.589 mmol) followed by stirring at room temperature overnight and

chromatography (SiO , n-hexane/ethyl acetate, 7:3) gave 19 (74 mg, 58.5%) as a colorless

2

solid; m.p.: 42 °C; R = 0.45 (SiO , n-hexane/ethyl acetate, 7:3); IR (KBr): ν� = 3448vs,

F

2

920s, 2850s, 1660m, 1636m, 1614m, 1470w, 1454w, 1442w, 1254w, 1226w, 1136w, 1120w

-1

1

cm ; UV-Vis (CHCl ): λmax (log ε) = 232 (3.78) nm; H NMR (500 MHz, CDCl ): δ = 6.82

3

(ddd, J = 15.1, 7.0, 7.0 Hz, 1H, CH (3)), 6.22 (ddd, J = 15.1, 1.6, 1.6 Hz, 1H, CH (2)), 3.53

(

br, 4H, CH (1‘) + CH (5‘)), 2.18 (dddd, J = 7.1, 7.1, 7.1, 1.5 Hz, 2H, CH (4)), 1.69 – 1.59

2

(

m, 2H, CH (3‘)), 1.60 – 1.51 (m, 4H, CH (2‘) + CH (4‘)), 1.48 – 1.37 (m, 2H, CH (C5)),

2 2 2 2

1

.33 – 1.23 (m, 20H, CH (6) + CH (7) + CH (8) + CH (9) + CH (10) + CH (11) +

2 2 2 2 2 2

1

3

CH (12) + CH (13) + CH (14) + CH (15)), 0.87 (t, J = 6.8 Hz, 3H, CH (16)) ppm; C

2

3

NMR (100 MHz, CDCl ): δ = 165.8 (C=O, C1), 146.1 (CH, C3), 120.4 (CH, C2), 47.0 (br,

3

CH , C5‘), 43.3 (br, CH , C1‘), 32.7 (CH , C4), 32.1 (CH , C14), 29.8 (CH ), 29.8 (CH ),

2

9.8 (CH ), 29.7 (CH ), 29.6 (CH ), 29.5 (CH ), 29.4 (CH , C6), 28.6 (CH , C5), 26.6 (br,

2 2 2 2 2 2

CH , C4‘), 26.1 (br, CH , C2‘), 24.8 (CH , C3‘), 22.8 (CH , C15), 14.2 (CH , C16) ppm; MS

2

3

+

(

ESI, MeOH): m/z = 322.4 (97%, [M+H] ), 344.3 (28%, [M+Na] ), 643.3 (8%, [2M+H] ),

+

6

65.3 (100%, [2M+Na] ); analysis calcd for C H NO (321.55): C 78.44, H 12.23, N 4.36;

2

1

39

found: C 74.21, H 12.42, N 4.17.

4

.15.

To an ice-cold solution of 3 (150 mg, 0.531 mmol) in dry DCM (5 mL) EDC (112 mg,

.584 mmol) and HOBt (106 mg, 0.690 mmol) were added, and stirring was continued for

(2E) 1-(Pyrrolidin-1-yl)-octadec-2-en-1-one (20)

0

another 5 min. Piperidine (7, 0.07 mL, 0.690 mmol) was slowly added, the mixture was

allowed to warm to room temperature, and stirring was continued overnight. The solvent was

removed, and the residue was subjected to chromatography (SiO , n-hexane/ethyl acetate, 7:3)

2

to yield 20 (174 mg, 93.7%) as a colorless solid; m.p.: 48 °C; R = 0.38 (SiO , n-hexane/ethyl

F

2

17

ACCEPTED MANUSCRIPT

acetate, 7:3); IR (KBr): ν� = 3446s, 2920vs, 2850s, 2362w, 2344w, 1660m, 1614s, 1472m,

-1

1

454m, 1442m, 1384w, 1274w, 1222w, 1120w, 1020w cm ; UV-Vis (acetone): λ (log ε) =

max

1

2

11 (4.24) nm; H NMR (400 MHz, CDCl ): δ = 6.83 (ddd, J = 15.0, 7.0, 7.0 Hz, 1H, CH

3

(

3)), 6.22 (ddd, J = 15.0, 1.5, 1.5 Hz, 1H, CH (2)), 3.54 (t, J = 5.4 Hz, 4H, CH (1‘) + CH₂

2

(

5‘)), 2.18 (dddd, J = 7.1, 7.1, 7.1, 1.5 Hz, 2H, CH (4)), 1.68 – 1.61 (m, 2H, CH (3')), 1.60 –

2

1

.52 (m, 4H, CH (2') + CH (4')), 1.48 – 1.38 (m, 2H, CH (5)), 1.34 – 1.19 (m, 24H, CH (6)

2 2 2 2

+

CH (7) + CH (8) + CH (9) + CH (10) + CH (11) + CH (12) + CH (13) + CH (14) +

2

13

CH (15) + CH (16) + CH (17)), 0.87 (t, J = 6.6 Hz, 3H, CH (18)) ppm; C NMR (100

2

3

MHz, CDCl ): δ = 165.8 (C=O, C1), 146.2 (CH, C3), 120.4 (CH, C2), 45.6 (br, CH , C5'),

3

2

4

2

4.3 (br, CH , C1'), 32.7 (CH , C4), 32.1 (CH , C16), 29.8 (CH ), 29.8 (CH ), 29.8 (CH ),

2 2 2 2 2 2

9.8 (CH ), 29.8 (CH ), 29.7 (CH ), 29.6 (CH ), 29.5 (CH ), 29.4 (CH , C6), 28.6 (CH , C5),

2

6.3 (br, CH , C2' + C4'), 24.8 (CH , C3'), 22.8 (CH , C17), 14.3 (CH , C18) ppm; MS (ESI,

2

3

+

MeOH): m/z = 350.5 (46%, [M+H] ), 372.3 (2%, [M+Na] ), 699.3 (100%, [2M+H] ), 721.3

+

(

98%, [2M+Na] ); analysis calc dor C23H43NO (349.60): C 79.02, H 12.40, N 4.01; found:

C 78.82, H 12.51, N 3.86.

4

.16.

(2E) 1-(Pyrrolidin-1-yl)-eicos-2-en-1-one (21)

Reaction of 4 (150 mg, 0.483 mmol) in dry DCM (5 mL) with EDC (102 mg, 0.531 mmol),

HOBt (96 mg, 0.628 mmol) and piperidine (7, 0.06 mL, 0.628 mmol) for 12 h as described

above, followed by chromatography (SiO , n-hexane/ethyl acetate, 7:3) gave 21 (160 mg,

2

8

7.7%) as a colorless solid; m.p.: 57 °C; R = 0.36 (SiO , n-hexane/ethyl acetate, 7:3); IR

F 2

(KBr): ν� = 3450m, 2918vs, 2850s, 1660m, 1612s, 1472m, 1454m, 1442m, 1278w, 1252w,

-1

1

218w, 1020w cm ; UV-Vis (acetone): λ

(log ε) = 212 (4.26) nm; H NMR (400 MHz,

max

CDCl ): δ = 6.82 (ddd, J = 15.1, 7.0, 7.0 Hz, 1H, CH (3)), 6.22 (ddd, J = 15.1, 1.6, 1.6 Hz,

3

1

H, CH (2)), 3.53 (br, 4H, CH (1') + CH (5')), 2.18 (dddd, J = 7.1, 7.1, 7.1, 1.5 Hz, 2H, CH

2 2 2

(

4)), 1.68 – 1.61 (m, 2H, CH (3')), 1.60 – 1.51 (m, 4H, CH (2') + CH (4')), 1.44 (quint, J =

2 2 2

7

.3 Hz, 2H, CH (5)), 1.34 – 1.20 (m, 28H, CH (6) + CH (7) + CH (8) + CH (9) + CH (10)

2 2 2 2 2 2

+

CH (11) + CH (12) + CH (13) + CH (14) + CH (15) + CH (16) + CH (17) + CH (18) +

2

13

CH (19)), 0.87 (t, J = 6.7 Hz, 3H, CH (20)) ppm; C NMR (100 MHz, CDCl ): δ = 165.8

2

3

(

C=O, C1), 146.2 (CH, C3), 120.4 (CH, C2), 45.8 (br, CH , C5'), 44.6 (br, CH , C1'), 32.7

2 2

(

CH , C4), 32.1 (CH , C18), 29.8 (CH ), 29.8 (CH ), 29.8 (CH ), 29.8 (CH ), 29.8 (CH ),

2 2 2 2 2 2 2

2

9.7 (CH ), 29.6 (CH ), 29.5 (CH ), 29.4 (CH , C6), 28.6 (CH , C5), 26.3 (br, CH , C2' +

2 2 2 2 2 2

C4'), 24.8 (CH , C3'), 22.8 (CH , C19), 14.3 (CH , C20) ppm; MS (ESI, MeOH): m/z = 378.5

2

3

+

(

64%, [M+H] ), 400.3 (4%, [M+Na] ), 755.3 (56%, [2M+H] ), 777.4 (100%, [2M+Na] );

18

ACCEPTED MANUSCRIPT

analysis calcd for C H NO (377.66): C 79.51, H 12.54, N 3.71; found C 79.39, H 12.72, N

2

5

47

3

.51.

4

.17.

(2E) 1-(Azepan-1-yl)-tetradec-2-en-1-one (22)

Reaction of 1 (126 mg, 0.557 mmol) in dry DCM (5 mL) with EDC (117 mg, 0.613 mmol),

HOBt (111 mg, 0.724 mmol) and hexamethylenimine (8, 0.08 mL, 0.668 mmol) for 12 h as

described above, followed by chromatography (SiO , n-hexane/ethyl acetate, 7:3) yielded 22

2

(

156 mg, 91.1%) as a colorless oil; R = 0.36 (SiO , n-hexane/ethyl acetate, 7:3); IR

F 2

(KBr): ν� = 3448vs, 2926s, 2854m, 1660m, 1618m, 1458w, 1448w, 1426w, 1374w, 1356vw,

-1

1

290w, 1128vw, 1102vw cm ; UV-Vis (acetone): λ (log ε) = 212 (3.81) nm; H NMR (400

max

MHz, CDCl ): δ = 6.89 (ddd, J = 15.0, 7.0, 7.0 Hz, 1H, CH (3)), 6.21 (ddd, J = 15.0, 1.5, 1.5

3

Hz, 1H, CH (2)), 3.57 (br, 2H, CH (1')), 3.50 (br, 2H, CH (6')), 2.19 (dddd, J = 7.1, 7.1, 7.1,

2

1

.5 Hz, 2H, CH (4)), 1.72 (br, 4H, CH (3') + CH (4')), 1.56 (br, 4H, CH (2') + CH (5')),

2 2 2 2 2

.44 (quint, J = 7.3 Hz, 2H, CH (5)), 1.36 – 1.22 (m, 16H, CH (6) + CH (7) + CH (8) +

2

CH (9) + CH (10) + CH (11) + CH (12) + CH (13)), 0.87 (t, J = 7.0 Hz, 3H,

2

13

CH (14)) ppm; C NMR (100 MHz, CDCl ): δ = 166.7 (C=O, C1), 146.5 (CH, C3), 120.5

3

(

CH, C2), 48.0 (CH , C6'), 46.4 (CH , C1'), 32.7 (CH , C4), 32.1 (CH , C12), 29.8 (CH ),

2 2 2 2 2

2

9.8 (CH ), 29.7 (CH ), 29.6 (CH ), 29.5 (CH ), 29.4 (br, CH , C3'/C4'), 29.4 (CH , C6), 28.6

2 2 2 2 2 2

(

CH , C5), 27.8 (br, CH , C3' or C4'), 27.1 (br, CH , C2' or C5'), 26.8 (br, CH , C2' or C5'),

2 2 2 2

+

2

2.8 (CH , C13), 14.3 (CH , C14) ppm; MS (ESI, MeOH): m/z = 308.4 (44%, [M+H] ), 330.3

2 3

+

(

8%, [M+Na] ), 615.1 (30%, [2M+H] ), 637.3 (100%, [2M+Na] ); analysis calc for

C H NO (307.52): C 78.11, H 12.13, N 4.55; found: C 78.01, H 12.34, N 4.39.

20

37

4

.18.

(2E) 1-(Azepan-1-yl)-hexadec-2-en-1-one (23)

Reaction of 2 (300 mg, 1.179 mmol) in dry DCM (10 mL) with EDC (247 mg, 1.299 mmol),

HOBt (235 mg, 1.534 mmol) and hexamethylenimine (8, 0.16 mL, 1.416 mmol) for 12 h as

described above followed by chromatography (SiO , n-hexane/ethyl acetate, 7:3) gave 23

2

(

370 mg, 93.4%) as a colorless oil; R = 0.46 (SiO , n-hexane/ethyl acetate, 7:3); IR (KBr):

F 2

ν� = 3450m, 2924vs, 2854s, 1660m, 1618m, 1450m, 1426m, 1374w, 1354w, 1286w, 1192w,

-1

1

170w cm ; UV-Vis (acetone): λ (log ε) = 212 (4.04) nm; H NMR (400 MHz, CDCl ): δ

max 3

=

6.89 (ddd, J = 15.2, 7.0, 7.0 Hz, 1H, CH (3)), 6.20 (ddd, J = 15.0, 1.6, 1.6 Hz, 1H, CH (2)),

3

.56 (br, 2H, CH (1')), 3.50 (br, 2H, CH (6')), 2.19 (dddd, J = 7.2, 7.2, 7.2, 1.5 Hz, 2H, CH

2 2 2

(

4)), 1.72 (br, 4H, CH (3') + CH (4')), 1.56 (br, 4H, CH (2') + CH (5')), 1.49 – 1.38 (m, 2H,

2 2 2 2

CH (5)), 1.35 – 1.20 (m, 20H, CH (6) + CH (7) + CH (8) + CH (9) + CH (10) + CH (11)

2

19

ACCEPTED MANUSCRIPT

13

+

CH (12) + CH (13) + CH (14) + CH (15)), 0.87 (t, J = 7.2 Hz, 3H, CH (16)) ppm; C

2 2 2 2 3

NMR (100 MHz, CDCl ): δ = 166.7 (C=O, C1), 146.4 (CH, C3), 120.5 (CH, C2), 48.0 (CH ,

3

2

C6'), 46.4 (CH , C1'), 32.7 (CH , C4), 32.1 (CH , C14), 29.8 (CH ), 29.8 (CH ), 29.8 (CH ),

2

9.7 (CH ), 29.6 (CH ), 29.5 (CH ), 29.4 (br, CH , C3' or C4’), 29.4 (CH , C6), 28.6 (CH ,

2 2 2 2 2 2

C5), 27.8 (br, CH , C3' or C4’), 27.1 (br, CH , C2' or C5'), 26.8 (br, CH , C2' or C5'), 22.8

2

+

(

CH , C15), 14.3 (CH , C16) ppm; MS (ESI, MeOH): m/z = 336.4 (58%, [M+H] ), 358.3

2 3

+

2+

+

(

6%, [M+Na] ), 523.1 (4%, [3M+K+H] ), 671.3 (8%, [2M+H] ), 693.3 (100%, [2M+Na] );

analysis calcd for C H NO (335.58): C 78.74, H 12.32, N 4.17; found: C 78.58, H 12.51, N

2

41

4

.03.

4

.19.

(2E) 1-(Azepan-1-yl)-octadec-2-en-1-one (24)

Reaction of 3 (130 mg, 0.460 mmol) in dry DCM (5 mL) with EDC (97 mg, 0.506 mmol),

HOBt (92 mg, 0.598 mmol) and hexamethylenimine (8, 0.07 mL, 0.598 mmol) for 12 h as

described above followed by chromatography (SiO , n-hexane/ethyl acetate, 7:3) gave 25

2

(

141 mg, 84.2%) as a colorless solid; m.p.: 32 °C; R = 0.38 (SiO , n-hexane/ethyl acetate,

F 2

7

1

:3); IR (KBr): ν� = 3346s, 2916vs, 2850s, 1660m, 1612m, 1472m, 1456w, 1428m, 1370w,

-1

1

196w, 1170w, 1126vw cm ; UV-Vis (acetone): λ (log ε) = 212 (4.12) nm; H NMR (400

max

MHz, CDCl ): δ = 6.90 (ddd, J = 15.0, 7.0, 7.0 Hz, 1H, CH (3)), 6.21 (ddd, J = 15.0, 1.6, 1.6

3

Hz, 1H, CH (2)), 3.58 (br, 2H, CH (1')), 3.50 (br, 2H, CH (6')), 2.19 (dddd, J = 7.2, 7.2, 7.2,

2

1

.5 Hz, 2H, CH (4)), 1.73 (br, 4H, CH (3') + CH (4')), 1.57 (br, 4H, CH (2') + CH (5')),

2 2 2 2 2

.44 (quint, J = 7.2 Hz, 2H, CH (5)), 1.35 – 1.21 (m, 24H, CH (6) + CH (7) + CH (8) +

2

CH (9) + CH (10) + CH (11) + CH (12) + CH (13) + CH (14) + CH (15) + CH (16) +

2

1

3

CH (17)), 0.87 (t, J = 7.0 Hz, 3H, CH (18)) ppm; C NMR (125 MHz, CDCl ): δ = 166.7

2

3

(

C=O, C1), 146.5 (CH, C3), 120.5 (CH, C2), 48.0 (CH , C6'), 46.5 (CH , C1'), 32.7 (CH ,

2 2 2

C4), 32.1 (CH , C16), 29.8 (CH ), 29.8 (CH ), 29.8 (CH ), 29.8 (CH ), 29.8 (CH ), 29.7

2

(

CH ), 29.6 (CH ), 29.5 (CH ), 29.4 (br, CH , C3' or C4'), 29.4 (CH , C6), 28.6 (CH , C5),

2 2 2 2 2 2

2

7.8 (br, CH , C3' or C4'), 27.1 (br, CH , C2' or C5'), 26.8 (br, CH , C2' or C5'), 22.8 (CH ,

2 2 2 2

+

C17), 14.3 (CH , C18) ppm; MS (ESI, MeOH): m/z = 364.5 (54%, [M+H] ), 386.33 (4%,

3

+

[

(

M+Na] ), 727.3 (42%, [2M+H] ), 749.4 (100%, [2M+Na] ); analysis calc for C H NO

24 45

363,63): C 79.27, H 12.47, N 3.85; found: C 79.03, H 12.57, N 3.70.

4

.20.

(2E) 1-(Azepan-1-yl)-eicos-2-en-1-one (25)

Reaction of 4 (150 mg, 0.483 mmol) in dry DCM (5 mL) with EDC (102 mg, 0.531 mmol),

HOBt (96 mg, 0.628 mmol) and hexamethylenimine (8, 0.07 mL, 0.628 mmol) for 12 h as

20

ACCEPTED MANUSCRIPT

described above followed by chromatography (SiO , n-hexane/ethyl acetate, 7:3) gave 25

2

(

174 mg, 92.0%) as a colorless solid; m.p.: 40 °C; R = 0.34 (SiO , n-Hexan/Ethylacetat 7:3);

F 2

IR (KBr): ν = 3448m, 2916vs, 2850s, 1660m, 1612s, 1472m, 1428m, 1370w, 1252w, 1194w,

-1

1

170w cm ; UV-Vis (Aceton): λ (log ε) = 212 (4.21) nm; H NMR (400 MHz, CDCl ): δ

max 3

=

6.89 (ddd, J = 14.7, 7.0, 7.0 Hz, 1H, CH (3)), 6.21 (ddd, J = 15.1, 1.5, 1.5 Hz, 1H, CH (2)),

3

.57 (t, J = 6.2 Hz, 2H, CH (1')), 3.49 (t, J = 6.0 Hz, 2H, CH (6')), 2.19 (dddd, J = 7.2, 7.2,

2 2

7

.2, 1.5 Hz, 2H, CH (4)), 1.79 – 1.66 (m, 4H, CH (3') + CH (4')), 1.61 – 1.52 (m, 4H, CH

2

(

2') + CH (5')), 1.44 (quint, J = 7.1 Hz, 2H, CH (5)), 1.34 – 1.21 (m, 28H, CH (6) + CH (7)

2 2 2 2

+

CH (8) + CH (9) + CH (10) + CH (11) + CH (12) + CH (13) + CH (14) + CH (15) +

2 2 2 2 2 2 2 2

13

CH (16) + CH (17) + CH (18) + CH (19)), 0.88 (t, J = 6.7 Hz, 3H, CH (20)) ppm; C

2

3

NMR (100 MHz, CDCl ): δ = 166.7 (C=O, C1), 146.4 (CH, C3), 120.6 (CH, C2), 48.0 (CH ,

3

2

C6'), 46.4 (CH , C1'), 32.7 (CH , C4), 32.1 (CH , C18), 29.8 (CH ), 29.8 (CH ), 29.8 (CH ),

2

9.8 (CH ), 29.7 (CH ), 29.6 (CH ), 29.5 (CH ), 29.5 (br, CH , C4'), 29.4 (CH , C6), 28.6

2 2 2 2 2 2

(

CH , C5), 27.8 (CH , C3'), 27.2 (CH , C5'), 26.8 (CH , C2'), 22.8 (CH , C19), 14.3 (CH ,

2 2 2 2 2 3

+

C20) ppm; MS (ESI, MeOH): m/z = 392.5 (36%, [M+H] ), 414.3 (2%, [M+Na] ), 783.4

+

(

50%, [2M+H] ), 805.5 (100%, [2M+Na] ); analysis calcd for C H NO (391.68): C 79.73,

26 49

H 12.61, N 3.58; found: C 79.54, H 12.89, N 3.32.

4

.21.

(2E) 1-(Azocan-1-yl)-tetradec-2-en-1-one (26)

Reaction of 1 (158 mg, 0.698 mmol) in dry DCM (5 mL) with EDC (147 mg, 0.768 mmol),

HOBt (139 mg, 0.907 mmol) and heptamethylenimine (9, 0.11 mL, 0.838 mmol) for 12 h as

described above followed by chromatography (SiO , n-hexane/ethyl acetate, 7:3) gave 26

2

(

188 mg, 83.8%) as a colorless oil; R = 0.42 (SiO , n-hexane/ethyl acetate, 7:3); IR (KBr):

F 2

-1

ν� = 3348vs, 2926s, 2854m, 2360m, 2342w, 1658m, 1618m, 1466w, 1424w, 1376w cm ; UV-

1

Vis (acetone): λ_m_a_x(log ε) = 210 (4.49) nm; H NMR (400 MHz, CDCl ): δ = 6.90 (ddd, J =

3

1

4.4, 7.0, 7.0 Hz, 1H, CH (3)), 6.20 (ddd, J = 15.0, 1.5, 1.5 Hz, 1H, CH (2)), 3.53 (t, J = 6.0

Hz, 2H, CH (1')), 3.47 (t, J = 5.7 Hz, 2H, CH (7')), 2.19 (dddd, J = 7.2, 7.2, 7.2, 1.5 Hz, 2H,

2

CH (4)), 1.81 – 1.66 (m, 4H, CH (2') + CH (6')), 1.62 – 1.49 (m, 6H, CH (3') + CH (4') +

2

CH (5’)), 1.44 (quint, J = 7.1 Hz, 2H, CH (5)), 1.35 – 1.21 (m, 16H, CH (6) + CH (7) +

2

CH (8) + CH (9)+ CH (10) + CH (11) + CH (12) + CH (13)), 0.87 (t, J = 6.4 Hz, 3H,

2

1

3

CH (14)) ppm; C NMR (100 MHz, CDCl ): δ = 166.7 (C=O, C1), 146.3 (CH, C3), 120.9

3

(

CH, C2), 48.9 (CH , C7'), 48.1 (CH , C1'), 32.7 (CH , C4), 32.1 (CH , C12), 29.8 (CH ),

2 2 2 2 2

2

9.8 (CH ), 29.7 (CH ), 29.6 (CH ), 29.5 (CH ), 29.4 (CH , C6), 28.8 (CH , C6'), 28.6 (CH ,

2 2 2 2 2 2 2

C5), 27.1 (CH , C4'), 26.1 (CH , C2' + C3'), 25.4 (CH , C5'), 22.8 (CH , C13), 14.3 (CH ,

2

3

21

ACCEPTED MANUSCRIPT

+

C14) ppm; MS (ESI, MeOH): m/z = 322.4 (54%, [M+H] ), 344.2 (6%, [M+Na] ), 643.3

+

(

28%, [2M+H] ), 665.3 (100%, [2M+Na] ); analysis calcd for C H NO (321.55): C 78.44,

21 39

H 12.23, N 4.36; found: C 78.21, H 12.50, N 4.17.

4

.22.

(2E) 1-(Azocan-1-yl)-hexadec-2-en-1-one (27)

Reaction of 2 (300 mg, 1.179 mmol) in dry DCM (10 mL) with EDC (247 mg, 1.299 mmol),

HOBt (235 mg, 1.534 mmol) and heptamethylenimine (9, 0.19 mL, 1.416 mmol) for 12 h as

described above followed by chromatography (SiO , n-hexane/ethyl acetate, 6:4) gave 27

2

(

335 mg, 81.2%) as a colorless solid; m.p.: 26 °C; R = 0.48 (SiO , n-hexane/ethyl acetate,

F 2

7

1

:3); IR (KBr): ν� = 3448s, 2924vs, 2854s, 1660m, 1620m, 1446w, 1422w, 1374w, 1356w,

-1

1

202w, 1120w cm ; UV-Vis (CHCl ): λ

(log ε) = 230 (4.37) nm; H NMR (400 MHz,

3

max

CDCl ): δ = 6.91 (ddd, J = 15.0, 7.0, 7.0 Hz, 1H, CH (3)), 6.20 (ddd, J = 15.0, 1.5, 1.5 Hz,

3

1

H, CH (2)), 3.53 (t, J = 5.9 Hz, 2H, CH (1')), 3.47 (t, J = 5.9 Hz, 2H, CH (7')), 2.19 (dddd,

2 2

J = 7.2, 7.2, 7.2, 1.5 Hz, 2H, CH (4)), 1.81 – 1.67 (m, 4H, CH (2') + CH (6')), 1.62 – 1.50

2

(

m, 6H, CH (3') + CH (4') + CH (5’)), 1.44 (quint, J = 7.0 Hz, 2H, CH (5)), 1.34 – 1.21 (m,

2 2 2 2

2

0H, CH (6) + CH (7) + CH (8) + CH (9) + CH (10) + CH (11) + CH (12) + CH (13) +

2 2 2 2 2 2 2 2

13

CH (14) + CH (15)), 0.87 (t, J = 6.9 Hz, 3H, CH (16)) ppm; C NMR (100 MHz, CDCl ):

2

3

δ = 166.7 (C=O, C1), 146.3 (CH, C3), 120.8 (CH, C2), 48.9 (CH , C7’), 48.1 (CH , C1'), 32.7

2

(

CH , C4), 32.1 (CH , C14), 29.8 (CH ), 29.8 (CH ), 29.8 (CH ), 29.7 (CH ), 29.6 (CH ),

2 2 2 2 2 2 2

2

9.5 (CH ), 29.4 (CH , C6), 28.8 (CH , C6'), 28.6 (CH , C5), 27.1 (CH , C4'), 26.1 (CH , C2'

2 2 2 2 2 2

+

C3'), 25.4 (CH , C5'), 22.8 (CH , C15), 14.3 (CH , C16) ppm; MS (ESI, MeOH): m/z =

2

3

+

2+

3

50.5 (82%, [M+H] ), 372.3 (8%, [M+Na] ), 544.5 (4%, [3M+K+H] ), 699.1 (5%,

+

[

2M+H] ), 721.3 (100%, [2M+Na] ); analysis calc for C H NO (349.60): C 79.02, H 12.40,

23 43

N 4.01; found: C 78.88, H 12.57, N 3.79.

4

.23.

(2E) 1-(Azocan-1-yl)-octadec-2-en-1-one (28)

Reaction of 3 (143 mg, 0.506 mmol) in dry DCM (5 mL) with EDC (107 mg, 0.557 mmol),

HOBt (101 mg, 0.658 mmol) and heptamethylenimine (9, 0.09 mL, 0.658 mmol) for 12 h as

described above followed by chromatography (SiO , n-hexane/ethyl acetate, 7:3) gave 28

2

(

161 mg, 84.3%) as a colorless solid; m.p.: 29 °C; R = 0.42 (SiO , n-hexane/ethyl acetate,

F 2

7

1

:3); IR (KBr): ν� = 3448s, 2922vs, 2850s, 1658m, 1608s, 1490w, 1466m, 1452m, 1426m,

-1

1

378w, 1290w, 1212w, 1148w cm ; UV-Vis (acetone): λ (log ε) = 214 (4.27) nm; H NMR

max

(

400 MHz, CDCl ): δ = 6.91 (ddd, J = 14.9, 7.0, 7.0 Hz, 1H, CH (3)), 6.20 (ddd, J = 15.0, 1.5,

3

1

.5 Hz, 1H, CH (2)), 3.53 (t, J = 6.1, 2H, CH (1')), 3.48 (t, J = 6.2, Hz, 2H, CH (7')), 2.19

2 2

22

ACCEPTED MANUSCRIPT

(

dddd, J = 7.2, 7.2, 7.2, 1.5 Hz, 2H, CH (4)), 1.81 – 1.65 (m, 4H, CH (2') + CH (6')), 1.62 –

2

1

2

.50 (m, 6H, CH (3') + CH (4') + CH (5')), 1.54 – 1.37 (m, 2H, CH (5)), 1.36 – 1.20 (m,

2 2 2 2

4H, CH (6) + CH (7) + CH (8) + CH (9) + CH (10) + CH (11) + CH (12) + CH (13) +

2

1

3

CH (14) + CH (15) + CH (16) + CH (17)), 0.88 (t, J = 6.5 Hz, 3H, CH (18)) ppm; C

2

3

NMR (125 MHz, CDCl ): δ = 166.7 (C=O, C1), 146.4 (CH, C3), 120.8 (CH, C2), 48.9 (CH ,

3

2

C7'), 48.1 (CH , C1'), 32.7 (CH , C4), 32.1 (CH , C16), 29.8 (CH ), 29.8 (CH ), 29.8 (CH ),

2

1

7

9.8 (CH ), 29.8 (CH ), 29.7 (CH ), 29.6 (CH ), 29.5 (CH ), 29.4 (CH , C6), 28.8 (CH , C6'),

2 2 2 2 2 2 2

8.6 (CH , C5), 27.1 (CH , C4'), 26.1 (CH , C2' + C3'), 25.4 (CH , C5'), 22.8 (CH , C17),

2

+

4.3 (CH , C18) ppm; MS (ESI, MeOH): m/z = 378.5 (58%, [M+H] ), 400.3 (4%, [M+Na] ),

3

+

55.3 (78%, [2M+H] ), 777.4 (100%, [2M+Na] ); analysis calcd for C H NO (377.66): C

2

5

47

9.51, H 12.54, N 3.71; found: C 79.38, H 12.74, N 3.50

4

.24.

(2E) 1-(Azocan-1-yl)-eicos-2-en-1-one (29)

Reaction of 4 (150 mg, 0.483 mmol) in dry DCM (5 mL) with EDC (102 mg, 0.531 mmol),

HOBt (96 mg, 0.628 mmol) and heptamethylenimine (9, 0.08 mL, 0.628 mmol) for 12 h as

described above followed by chromatography (SiO , n-hexane/ethyl acetate, 7:3) gave 29

2

(

181 mg, 92.3%) as a colorless solid; m.p.: 38 °C; R = 0.40 (SiO , n-hexane/ethyl acetate,

F 2

-1

7

:3); IR (KBr): ν� = 3448vs, 2920s, 2850m, 1658m, 1610m, 1466w, 1424w, 1380w cm ; UV-

1

Vis (acetone): λ_m_a_x(log ε) = 212 (4.24) nm; H-NMR (400 MHz, CDCl ): δ = 6.90 (ddd, J =

3

1

4.8, 7.0, 7.0 Hz, 1H, CH (3)), 6.20 (ddd, J = 15.0, 1.5, 1.5 Hz, 1H, CH (2)), 3.53 (t, J = 5.9

Hz, 2H, CH (1')), 3.48 (t, J = 5.7 Hz, 2H, CH (7')), 2.19 (dddd, J = 7.2, 7.2, 7.2, 1.5 Hz, 2H,

2

CH (4)), 1.81 – 1.67 (m, 4H, CH (2') + CH (6')), 1.62 − 1.50 (m, 6H, CH (3') + CH (4') +

2

CH (5')), 1.48 – 1.40 (m, 2H, CH (5)), 1.36 – 1.20 (m, 28H, CH (6) + CH (7) + CH (8) +

2

CH (9) + CH (10) + CH (11) + CH (12) + CH (13) + CH (14) + CH (15) + CH (16) +

2

1

3

CH (17) + CH (18) + CH (19)), 0.88 (t, J = 6.9 Hz, 3H, CH (20)) ppm; C-NMR (100

2

3

MHz, CDCl ): δ = 166.7 (C=O, C1), 146.3 (CH, C3), 120.9 (CH, C2), 48.9 (CH , C7'), 48.1

3

2

(

CH , C1'), 32.7 (CH , C4), 32.1 (CH , C18), 29.9 (CH ), 29.8 (CH ), 29.8 (CH ), 29.8 (CH ),

2 2 2 2 2 2 2

2

9.7 (CH ), 29.6 (CH ), 29.5 (CH ), 29.4 (CH , C6), 28.8 (CH , C6'), 28.6 (CH , C5), 27.1

2 2 2 2 2 2

(

CH , C4'), 26.1 (CH , C2' + C3'), 25.4 (CH , C5'), 22.8 (CH , C19), 14.3 (CH , C20) ppm;

2 2 2 2 3

+

MS (ESI, MeOH): m/z = 406.5 (50%, [M+H] ), 428.4 (4%, [M+Na] ), 811.4 (70%,

+

[

2M+H] ), 833.4 (100%, [2M+Na] ); analysis calcd for C H NO (405.71): C 79.93, H

27 51

1

2.67, N 3.45; found: C 79.86, H 12.76, N 3.16.

Acknowledgements

23

ACCEPTED MANUSCRIPT

We like to thank Dr. D. Ströhl and his team for the NMR spectra, and Dr. R. Kluge for

measuring the MS spectra. The IR und UV-vis spectra were recorded by Ms V. Simon, the

micro-analyses were measured by Mrs J. Pech. The cell lines were kindly provided by Dr Th.

Müller (Dep. of Haematology/Oncology, Martin-Luther Universität Halle-Wittenberg). The

authors declare no conflict of interests.

Appendix A. Supplementary data

Supplemenatry data related to this article can be found at

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28

Article Doi

Piperlongumine B and analogs are promising and selective inhibitors for acetylcholinesterase

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