CHAPTER 3

RESULTS AND DISCUSSION

The previously reported,8,17 it was demonstrated that an aromatic ring

containing at least one ether function, carbonyl group8,17 and conjugated double

bonds17 containing in side chain of piperine derivatives is essential for high activity in

inhibition of CYP,8 stimulating melanocyte proliferation activity.17 This research we

focused on the preparation of ester and amide derivatives of piperine.

3.1 Isolation of piperine (1) from black pepper

A simple and efficient method has been developed for the isolation of piperine

from the fruits of Piper nigrum. Four methods and two kinds of solvents were used to

study the extraction of piperine from the black pepper. The structure of piperine was

confirmed by its melting point, comparison with IR, and 1H-NMR spectral data with

those from a reference standard, and co-chromatography with the reference standard

using thin-layer chromatography (TLC). The conditions for isolation of piperine from

black peper were shown in Table 20.

O

O

N

O

Piperine

60

Table 20 The reaction conditions for isolation of pipirine (1)

Entry Conditions % yield

1 95% EtOH, 100 g black pepper (reflux 24 h), rt 1.13

2 95% EtOH, 100 g black pepper, CaCO3 (reflux 24 h), rt 0.98

3 Isopropanol, 100 g black pepper (reflux 24 h), rt 1.60

4 Isopropanol, 100 g black pepper, CaCO3 (reflux 24 h), rt 1.42

The results shown that the best method for isolation of piperine from black

pepper was refluxed with isopropanol to obtain crude piperine in 1.6 % yield

3.2 Preparation of piperic acid (12) from piperine (1)

Piperic acid (12) was used as a precursor for preparation of the piperine

derivatives. Piperic acid (12) was obtained by alkaline hydrolysis of piperine (1). The

conditions for alkaline hydrolysis of piperic acid from piperine were shown in Table

21.

O

ON

OO

OOH

O1)10%ethanolic KOH

2) 35%HCl1 12

61

Table 21 The reaction conditions for alkaline hydrolysis of piperic acid (12) from

piperine (1)

Entry Conditions Refluxed

time (h)

% yield

1 Piperine (2.853 g), ethanolic KOH (2 N,10 ml) 2 35.45

2 Piperine (2.853 g), ethanolic KOH (2 N,10 ml) 5 48.50

3 Piperine (2.853 g), ethanolic KOH (2 N,10 ml) 10 52.82

4 Piperine (2.853 g), ethanolic KOH (2 N,10 ml) 15 62.54

5 Piperine (2.853 g), ethanolic KOH (2 N,10 ml) 20 84.85

6 Piperine (2.853 g), ethanolic KOH (2 N,10 ml) 25 98.98

The results shown that the most effective method for alkaline hydrolysis of

piperine (1) was refluxed with ethanolic KOH for 25 h to obtain piperic acid (12) in

98.98 % yield.

62

The reaction mechanism48 for preparation of piperic acid (12) from piperine

(1) by addition of hydroxide ion to the carbonyl group of piperine (1), followed by

elimination of the piperidine, after that abstraction of a proton from hydrochloric acid

to obtain piperic acid (12), as shown in Scheme 12.

Scheme 12 The reaction mechanism for preparation of piperic acid (12) from

piperine (1)

O

ON

OOH O

ON

O

OH

O

OO

O

H HN

O

OO

OH ClO

OOH

O

OH

1

12

63

3.3 Preparation of piperic acid chloride (13)

Piperic acid (12) was dissolved in dried THF and kept under nitrogen

atmosphere. Oxalyl chloride was added dropwise in to the solution. The reaction

mixture was stirred at room temperature for 6 h. Then the excess oxalyl chloride was

removed under reduced pressure to give acid chloride as an orange residue.

The purpose mechanism for preparation of piperic acid chloride (13) from

piperic acid (12) by addition of hydroxide ion of piperic acid (12) to the carbonyl

group of oxalyl chloride, followed by elimination of chloride ion to obtain the

intermediate 12b, then elimination of carbon monoxide and carbon dioxide to obtain

piperic acid chloride (13). The reaction mechanism was shown in Scheme 13.48

13 12

O

O

OH

O (COCl)2, THF, RT, 6 h O

O

O

Cl

64

Scheme 13 The reaction mechanism for preparation of piperic acid chloride (13)48

O

OOH

O C

O

Cl C

O

Cl

O

OO

O

Cl

O

H ClO

O

OO

O O

Cl

O

ClH

O

OO

O O

Cl

O

O

O

OCl

13

12

+ HCl + CO + CO2

12a

12b12c

65

3.4 Standard preparation of ester and amide derivatives of piperine

(compounds 76, 77, 41, 42 and 78)

Piperic acid chloride (13) was dissolved in dried THF under nitrogen

atmosphere and then added with vanillin, paracetamol, morpholine, benzylamine and

dibenzylamine solution, followed by triethylamine. The reaction mixture was stirred

at 60-70 ºC for 3-5 h. The solvent in the reaction mixture was then removed under

reduced pressure to give the yellow residue and was purified by column

chromatography on silica gel using ethyl acetate and hexane to obtain ester and amide

derivatives of piperine (compounds 76, 77, 41, 42 and 78) in 91.79 - 98.95 % yield.

The reaction conditions for preparation of compound 76 were showed in the

following table 22.

Table 22 The reaction conditions for preparation of compound 76

Entry Conditions Time (h) Results (%)

1 acid chloride, vanillin, triethylamine 60 ºC 3 43.35

2 acid chloride, vanillin, triethylamine 60 ºC 5 66.77

3 acid chloride, vanillin, triethylamine 70 ºC 5 97.55

The results shown that the most effective method for preparation of compound

76 was stirred at 70 ºC for 5 h to obtain compound 76 in 97.55 % yield. Thus, we

used this condition for preparation of compounds 77, 78, 41 and 42.

The synthetic route and the purpose mechanism for preparation of ester and

amide derivatives of piperine were shown in Scheme 14, 15, 16 and 17 respectively

66

Scheme 14 The synthetic route for preparation of ester derivatives of piperine

O

OCl

OO

OOR

O

ROH

=

OCH3

CHO

R

Et3N, THF

60-70 oC

O

NH

H3C

R =

(76)

(77)

13 76 and 77

67

The purpose mechanism for preparation of compounds (76, 77) from piperic

acid chloride (13) by addition of hydroxide ion of alcohol to the carbonyl group of

piperic acid chloride (13), followed by elimination of chloride ion to obtain the

intermediate 13b, then abstraction of proton by chloride ion to obtain ester 76, 77.

The reaction mechanism was shown in Scheme 15.

Scheme 15 The reaction mechanism for preparation of ester derivatives

of pipirine

O

OCl

O

O

OCl

O

OR

O

O

O

OR

ROHH

HO

O

O

OR

13 13a

13b76 and 77

68

Scheme 16 The synthetic route for preparation of amide derivatives of piperine

O

O

Cl

O

O

O

N

O

R2NH

=R2NH

Et3N, THF

60-70 oC

RNH2 =

HNO

H2N

HN

R2NH =

( 41)

( 42 )

( 78 )

13

R

R(H41, 42 and 78

or RNH2

69

The purpose mechanism for preparation of compounds (41, 42 and 78) from

piperic acid chloride (13) by addition of amine to the carbonyl group of piperic acid

chloride (13), followed by elimination of chloride ion to obtain the intermediate 13b,

then abstraction of proton by chloride ion to obtain amide 41, 42 and 78. The reaction

mechanism was shown in Scheme 17.

Scheme 17 The reaction mechanism for preparation of amide derivative

of piperine

O

OCl

O

O

OCl

O

NR

O

O

O

NR

R2NH

H

HClO

O

O

NR

R

RR

13 13a

13b41, 42 and 78

70

The structures of the ester and amide derivatives of piperine were elucidated

by their spectroscopic data, 1H-NMR, 13C-NMR, 2D-NMR, IR and MS.

Compound 76, yellow crystals and m.p. 155.8-156.4 °C (CH2Cl2/hexane)

The ESITOF MS shown an (M+Na)+ peak at m/z 375.0847, consistent with

a molecular formula of C20H16O6Na. The IR spectrum, appendix A5, shown the

absent of the broad peak of hydroxyl group, C-H stretch of methyl and methylene

groups at 2900, 2850 cm-1, carbonyl group of the aldehydric group at 1750 cm-1and

the ester at 1720 cm-1, a conjugated double bonds at 1640 cm-1 and benzene rings at

1600, 1590, 1500 cm-1. The 1H NMR spectrum indicated an aldehyde proton at 9.90

ppm, methoxy protons at 3.91 ppm and methylene protons at 6.00 ppm corresponding

with 13C NMR spectral data revealed an aldehydric carbon at 191.13 ppm, a methoxy

carbon at 56.4 ppm and methylene carbon at 101.46 ppm. 1H NMR and 13C NMR

spectra were shown in Table 23.

Compound 76

O

OO

O OCH3

O

H

71

Table 23 The 1H NMR and 13C NMR spectroscopic data (CDCl3) of compound 76

5-(3,4-Methylenedioxyphenyl)-penta-2E,4E-dienoic acid vanilinyl ester (76)

position 13C (ppm) 1H (ppm)

1

2

3

4

5

6

7

8, 9

10

11

12

2´

3´

4´

5´

6´

7´

8´

9´

164.40

118.21

147.49

123.03

124.17

130.28

141.75

148.34, 148.89

105.94

108.54

101.46

145.15

152.15

110.78

135.05

124.50

123.36

56.40

191.08

-

6.15 d (J=15.2 Hz)

7.63 dd (J= 10.8, 15.2 Hz)

6.95 dd (J=1.6, 8.1 Hz)

6.77 d (J=10.8 Hz)

-

6.88 (s)

-

7.03 d (J=1.6 Hz)

6.80 d (J= 8.1 Hz)

6.00 (s)

-

-

7.51 (s)

-

7.48 d (J=7.8 Hz)

7.29 d (J=7.8 Hz)

3.91 (s)

9.90 (s)

72

Compound 77, yellow crystals and m.p.215.6-216.7 °C (CH2Cl2/hexane)

The ESITOF MS shown an (M+Na)+ peak at m/z 374.1004, consistent with

a molecular formula of C22H20O5NNa. The IR spectrum, appendix A12, shown N-H

stretch of secondary amide at 3350 cm-1, C=O stretch of a the ester at 1720 cm-1and

the amide at 1680 cm-1 and C=C stretch of a conjugated double bonds at 1630 cm-1.

The 1H NMR spectrum indicated acetyl proton at 2.07 ppm, methylene protons at 6.05

ppm corresponding with 13C NMR spectral data revealed methyl carbon at 24.23 ppm

and methylene carbon at 102.58 ppm. The 1H NMR and 13C NMR spectra were

shown in Table 24.

Compound 77

O

O

O

OHN CH3

O

73

Table 24 The 1H NMR and 13C NMR spectroscopic data (CDCl3) of compound 77

5-(3,4-methylenedioxyphenyl)-penta-2E,4E-dienoic acid paracetamyl ester (77)

position 13C (ppm) 1H (ppm)

1

2

3

4, 7,11, 3´, 7´

5

6

8, 9

10

12

2´

4´, 6´

5´

8´

9´

168.86

120.10

147.61

122.72, 124.34, 125.49,142.27

109.34

131.70

149.47, 149.84

106.72

102.58

147.39

120.66

138.06

165.93

24.23

-

6.18 d (J= 15.2 Hz)

7.79 dd (J= 8.1, 15.2 Hz)

7.06-7.10 (m)

6.88 d (J= 8.1 Hz)

-

-

7.21 d (J= 1.6 Hz)

6.05 (s)

-

7.67 d (J= 8.9 Hz)

-

-

2.07 (s)

74

Compound 41, white crystals, m.p. 159.8-161.4 °C (CH2Cl2/hexane)

The ESITOF MS shown an (M+Na)+ peak at m/z 310.1055, consistent with a

molecular formula of C16H17NO4Na. The IR spectrum, appendix A19, shown the

absorption of C=O stretch of the amide group at 1650 cm-1, C=C stretch of the

conjugated double bonds at 1630 cm-1, a benzene ring indicate C=C stretch at 1600,

1500 cm.-1 The 1H NMR spectrum indicated C-12 methylene protons at 3.61 ppm and

the methylene protons of morpholine ring, C-2´, C-5´ and C-3´, C-4´ at 3.71, 3.61

ppm respectively, corresponding with 13C NMR spectrum shown the signals of

methylene carbons C-12, C-2´ and C-5´, C-3´ and C-4´ at 101.29, 66.80 and 44.24

ppm, respectively. The 1H NMR and 13C NMR spectra were shown in Table 25.

Compound 41

O

O

O

N O

75

Table 25 The 1H NMR and 13C NMR spectroscopic data (CDCl3) of compound 41

1-E,E-piperinonyl-morpholine (41)

position 13C (ppm) 1H (ppm)

1

2

3

4,

5

6

7

8, 9

10

11

12

2´, 5´

3´,4´

165.60

119.23

143.71

123.73

124.90

130.33

139.24

148.19, 148.30

105.65

108.49

101.29

66.80

44.24

-

6.36 d (J = 14.6 Hz)

7.45 dd (J = 10.3, 14.6 Hz)

6.89 dd (J = 1.6, 8.0 Hz)

6.73 d (J = 10.3 Hz)

-

6.78 (s)

-

6.97 d (J = 1.6 Hz)

6.77 d (J = 2.3 Hz)

5.98 (s)

3.70 t (J = 4.0 Hz)

3.61 t (J = 4.0 Hz)

76

Compound 42, white crystals, m.p.188.2-189.4 °C (CH2Cl2/hexane)

The ESITOF MS shown an (M+Na)+ peak at m/z 330.1108, consistent with a

molecular formula of C19H17NO3Na. The IR spectrum, appendix A26, shown the

absorption of N-H stretch of secondary amide at 3300 cm-1, C-H stretch of methyl and

methylene groups at 2900, 2850 cm-1, C=O stretch of carbonyl group of the amide at

1650 cm-1. The 1H NMR spectrum indicated C-12 methylene protons at 5.93 ppm and

the methylene protons of benzylamine, C-2´, at 4.45 ppm respectively, corresponding

with 13C NMR spectral data shown the signals of methylene carbons C-12 and C-2´ at

101.07 and 43.58 ppm, respectively. The 1H NMR and 13C NMR spectra were shown

in Table 26.

Compound 42

O

O

O

NH

77

Table 26 The 1H NMR and 13C NMR spectroscopic data (CDCl3) of compound 42

1-E,E-piperinonyl- benzylamine (42)

position 13C (ppm) 1H (ppm)

1

2

3

4

5

6

7

8, 9

10

11

12

2´

3´

4´, 5´, 6´, 7´, 8´

166.57

122.24

140.96

122.40

124.36

130.45

138.67

147.84, 147.90

105.52

108.02

101.07

43.58

137.97

126.84, 127.30, 128.10

-

6.04 d (J = 15.0 Hz)

7.35 dd (J = 10.4, 15.0 Hz)

6.86 dd (J = 1.5, 8.0 Hz)

6.69 d (J = 10.4 Hz)

-

6.72 (s)

-

6.95 d (J = 1.5 Hz)

6.74 d (J = 5.1 Hz)

5.93 (s)

4.45 (s)

-

7.20-7.30 (m)

78

Compound 78, yellow crystals, m.p. 125.9-126.4 °C (CH2Cl2/hexane)

The ESITOF MS shown an (M+Na)+ peak at m/z 420.1578, consistent with a

molecular formula of C26H23NO3Na. The IR spectrum, appendix A32, shown the

absorption of C=O stretch of the amide group at 1650 cm-1, C=C stretch of the

conjugated double bonds at 1630 cm-1, a benzene rings indicate C=C stretch at 1600,

1500 cm.-1 The 1H NMR spectrum indicated C-12 methylene protons at 5.96 ppm and

the methylene protons of dibenzylamine, C-2´and C-2´´ at 4.55 ppm and 4.70 ppm,

corresponding with 13C NMR spectrum shown signals of methylene carbons C-12, C-

2´ and C-2´´, at 101.29, 49.91 and 48.76 ppm respectively. The 1H NMR and 13C

NMR spectra were shown in Table 27.

Compound 78

O

O

O

N

79

Table 27 The 1H NMR and 13C NMR spectroscopic data (CDCl3) of compound 78

1-E,E-piperinonyl- dibenzylamine (78)

position 13C (ppm) 1H (ppm)

1

2

3

4

5

6

7

8, 9

10

11

12

2´, 2´´

3´, 3´´

4´, 5´, 6´, 7´, 8´

4´´, 5´´, 6´´, 7´´, 8´´

167.47

119.26

144.34

122.72

125.09

130.80

139.34

148.19, 148.30

105.95

108.32

101.29

48.76, 49.91

136.63, 137.33

126.49, 127.41, 127.63,

128.33, 128.59, 128.92

-

6.41 d (J = 14.6 Hz)

7.62 dd (J = 10.9, 14.6 Hz)

6.89 dd (J = 1.6, 8.0 Hz)

6.71 d (J = 10.9 Hz)

-

6.83 (s)

-

6.95 d (J = 1.6 Hz)

6.77 d (J = 8.0 Hz)

5.96 (s)

4.55, 4.70 (s)

-

7.29-7.39 (m)

80

3.5 Biological activities

Cytotoxicity was determined by Green Fluorescent Protein (GFP)-bassed assay

method. The reference compound, elipticine, exhibited activity towards KB cell line

with respective IC50 values of 1.56 µg/ml, all of the synthetic compounds 76, 77, 41,

42 and 78 exhibited non-cytotoxicity.

Antimararial was determined by microculture radioisotope technique method.

The reference compound, dihydroartemisinine, exhibited IC50 values of 4.5 nM, all of

the synthetic compounds 76, 77, 41, 42 and 78 exhibited inactive.

Anti-Mycobacterium tuberculosis H37Ra strain was determined by Green

Fluorescent Protein microplate assay (GFPMA). The reference compound, rifampicin,

stetomycin, isoniazid and ofloxacin, exhibited IC50 values of 0.012, 0.313, 0.046 and

0.781 µg/ml respectively, the compound 76 demonstrated anti mycobacterium

tuberculosis exhibited IC50 values 50 µg/ml. For the synthetic compounds 77, 41, 42

and 78 exhibited inactive.

Antibacterial was determined by paper disc diffusion method. The reference

compound, gentamicin, exhibited clear zone values 2.4 cm, the synthetic compound,

76, exhibited clear zone values 0.7 mm. For the synthetic compounds 77, 41, 42 and

78 exhibited inactive.

Antifungal activity was assessed against A.niger, C.albicans. and Cand.krusei

was determine by paper disc diffusion method. 100% DMSO and 100% acetone were

used as a positive and a negative control respectively, the synthetic compounds 76,

77, 41, 42 and 78 exhibited inactive.

From the previously reported the compounds 41 and 42 were demonstrated

bioactivities in stimulation of melanocyte proliferation activity17 and against

81

epimastigotes activity,18 compound 41 exhibited increase in highly dendritic of

melanocytes and increase the total length of the dendrites,17 therefore it shown

inactive against epimastigotes.18 Compound 42 was demonstrated that inactive in

melanocyte proliferation stimulatory activity17 and against epimastigotes.18

The biological activities of compounds 1, 12, 76, 77, 41, 42 and 78 were shown

in Table 28.

Table 28 The biological activities of compounds 1, 12, 76, 77, 41, 42 and 78

Bioactive

Compound Anti-TB Anti-fungal Anti-

malarial

Anti-bacterial

(clear zone, mm)

Cytotoxcity

(vero cell)

1 Inactive Inactive Inactive 0.7 Non-cytotoxic

12 Inactive Inactive Inactive Inactive Non-cytotoxic

76 50* Inactive Inactive 0.7 Non-cytotoxic

77 Inactive Inactive Inactive Inactive Non-cytotoxic

41 Inactive Inactive Inactive Inactive Non-cytotoxic

42 Inactive Inactive Inactive Inactive Non-cytotoxic

78 Inactive Inactive Inactive Inactive Non-cytotoxic

Note

* IC50:µg/ml