chem1008np_ch3 piperine
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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
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