Cytotoxic and insecticidal activities of derivatives of harmine, a natural insecticidal component isolated from peganum harmala

Article abstract of DOI:10.3390/molecules15117775

In a continuing effort to develop novel β-carbolines endowed with better insecticidal activity, a simple high-yielding method for the synthesis of harmine compounds starting from L-tryptophan has been developed and a series of 1,3-substituted β-carboline derivatives have been synthesized and evaluated for their cytotoxicity against insect cultured Sf9 cell line in vitro and insecticidal activities against 4th instar larvae of mosquitos, Culex pipiens quinquefasciatus and mustard aphid, Lipaphis erysimi. The results demonstrated that 1-phenyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid (compound 2) and methyl 1-phenyl-β-carboline-3-carboxylate (compound 13) represented the best potential compounds, with Sf9 cells inhibition rates of 71.55% and 60.21% after 24 h treatment at concentrations of 50-200 mg/L, respectively. Both compounds 2 and 13 also showed strong insecticidal activity towards 4th instar larvae of mosquitos with LC₅₀ values of 20.82 mg/L and 23.98 mg/L, and their LC₉₀ values were 88.29 mg/L and 295.13 mg/L, respectively. Furthermore, the LC₅₀ values of compounds 2 and 13 against mustard aphids were 53.16 mg/L and 68.05 mg/L, and their LC ₉₀ values were 240.10 mg/L and 418.63 mg/L after 48 h treatment. The in vitro cytotoxicity of these compounds was consistent with the insecticidal activity in vivo. The results indicated that the 1- and 3-positions of the β-carboline ring deserve further investigation to develop biorational insecticides based on the natural compound harmine as a lead compound.

Full text of DOI:10.3390/molecules15117775

Molecules 2010, 15, 7775-7791; doi:10.3390/molecules15117775
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Cytotoxic and Insecticidal Activities of Derivatives of Harmine, a
Natural Insecticidal Component Isolated from Peganum harmala
Yong Zeng 1, Yaomou Zhang 2, Qunfang Weng 1, Meiying Hu 1 and Guohua Zhong 1,*
1
Laboratory of Insect Toxicology, South China Agricultural University, Guangzhou 510642, China;
E-Mails: yongz800@163.com (Y.Z.); huabao@scau.edu.cn (Q.F.W); humy@scau.edu.cn (M.Y.H.)
2
College of Science, South China Agricultural University, Guangzhou 510642, China;
E-Mail: zhangyaom@163.com (Y.Z.)
* Author to whom correspondence should be addressed; E-Mail: guohuazhong@scau.edu.cn;
Tel.: +86-20-85280308; Fax: +86-20-85280292.
Received: 23 August 2010; in revised form: 25 October 2010 / Accepted: 27 October 2010 /
Published: 2 November 2010
Abstract: In a continuing effort to develop novel β-carbolines endowed with better
insecticidal activity, a simple high-yielding method for the synthesis of harmine
compounds starting from L-tryptophan has been developed and a series of 1,3-substituted
β-carboline derivatives have been synthesized and evaluated for their cytotoxicity against
insect cultured Sf9 cell line in vitro and insecticidal activities against 4th instar larvae of
mosquitos, Culex pipiens quinquefasciatus and mustard aphid, Lipaphis erysimi. The
results demonstrated that 1-phenyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid
(compound 2) and methyl 1-phenyl-β-carboline-3-carboxylate (compound 13) represented
the best potential compounds, with Sf9 cells inhibition rates of 71.55% and 60.21% after
24 h treatment at concentrations of 50–200 mg/L, respectively. Both compounds 2 and 13
also showed strong insecticidal activity towards 4th instar larvae of mosquitos with LC50
values of 20.82 mg/L and 23.98 mg/L, and their LC90 values were 88.29 mg/L and
295.13 mg/L, respectively. Furthermore, the LC50 values of compounds 2 and 13 against
mustard aphids were 53.16 mg/L and 68.05 mg/L, and their LC90 values were 240.10 mg/L
and 418.63 mg/L after 48 h treatment. The in vitro cytotoxicity of these compounds was
consistent with the insecticidal activity in vivo. The results indicated that the 1- and
3-positions of the β-carboline ring deserve further investigation to develop biorational
insecticides based on the natural compound harmine as a lead compound.

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Keywords: harmine; synthesis; derivatives; cytotoxicity; insecticidal activity
1. Introduction
Harmine compounds and structurally related compounds, belonging to the β-carboline alkaloids,
and found in medicinal plants such as Peganum harmala and Eurycoma longifolia, have recently
drawn increasing interest due to their diverse pharmacological, neurophysiologic and biochemical
activities [1-6]. Some plants which contain these compounds are used in traditional medicine in China,
Brazil and other parts of the world for their emmenagogue, abortifacient, anticancer, antispasmodic
and sedative properties [7,8].
As for pest management Peganum L. plant species extracts, whose main components include a
mixture of harmine, harmaline, norharman and their derivatives, have been long known to have
insecticidal, fungicidal and plant growth regulatory properties [9-15]. After treatment with harmaline,
an important component of P. harmala, Indian meal moth (Plodia interpunctella) larvae growth and
development was inhibited [16]. When incorporated into the diet, harmaline caused larval weight loss
with a reduction in protein and glycogen contents and an inhibition of α-amylase activity. Using
electron microscopy, harmaline was shown to cause severe cytotoxicity on the epithelial cells of the
midgut, resulting in marked vacuolization of the cytoplasm, the appearance of numerous autophagic
vesicles and lysosomic structures, fragmentation of rough endoplasmic reticulum cisternae, disruption
of microvilli, and rupture of the plasma membrane leading to shedding of the cytoplasm contents into
the midgut lumen. Another study revealed an elevated juvenile hormone epoxide hydrolase (JHEH)
activity stimulated by norharmane by accelerating juvenile hormone metabolism in Reticulitermes
speratus and forcing termite caste differentiation in workers, nymphs, and ergatoids [17]. Our
investigations showed that a methanolic extract of P. harmala at a concentration of 10,000 mg/L
caused mortality rates of 93.07% and 96.36%, respectively, against Lipaphis erysimi 24 h and 48 h
after treatment [18]. Nematocidal toxicity tests showed that harmine was one of the main nematocidal
components of P. harmala and its LC50 value against mixed instar B. xylophilus was 135.74 mg/L after
48 h [11]. Further research demonstrated that harmaline and other active substances of P. harmala
could disturb normal physiological function, for example, in the 4th instar larvae of Spodoptera litura,
the haemolymph protein content was significantly reduced and the total body sugar was obviously
reduced when treated with the 12th and 14th fractions at 500 mg/L [19]. The latest research showed
that after treating with harmaline several growth and development related enzymes of Noctuidae
Lepidoptera changed regularly [15]. All research showed that harmine compounds could be used as
novel insect growth and development inhibitors. The structural simplicity of β-carboline alkaloids
hides a multitude of in vitro and in vivo effects and makes these molecules interesting from both a
biophysical and a medicinal perspective. Some such compounds have already been synthesized,
however, no systematical pesticidal activity has been reported yet. This paper describes work aimed at
preparing a series of new carboline derivatives that might possesses cytotoxic and insecticidal activity.
A cultured Sf9 insect cell line from S. frugiperda was used for primary screening, followed by the

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Molecules 2010, 15
treatment of larvae and adults to determine insecticidal activity. The purpose of this study is to find the
new lead compounds and evaluate the prospects of these compounds for practical use in agriculture.
2 Results and Discussion
2.1. Preparation of compounds
In this paper, five tetrahydro-β-carboline carboxylic acids 1-5 were produced by Pictet–Spengler
reaction of L-tryptophan with five different aldehydes (Table 1). Esterification reactions of the
tetrahydro-β-carboline carboxylic acid group at position 3 with methanol led to the five desired methyl
ester 6-10 in reasonable yields (Table 1). Oxidative dehydrogenation of methyl tetrahydro-β-carbolines
with potassium permanganate as oxidant was also carried out to prepare five β-carboline carboxylates
11-15 (Table 2). The β-carboline carboxylate 11 was hydrolysed using sodium hydroxide in ethanol to
give 16 in good yield (Table 2). All compounds were characterised by 1H-NMR.
Table 1. Structure of tetrahydro-β-carboline compounds.
COOR2
4
5
8
3
2
6
7
9
N
NH
N
1
CH3O
N
H
H
R1
CH3
Tetrahydro-β-carboline compounds
Harmine [8]
Compound
R1
R2
1
methyl
-H
2
phenyl
-H
3
-H
-H
4
trichloromethyl
-H
5
3-methoxy-4-hydroxyphenyl
-H
6
7
8
9
methyl
phenyl
methyl
methyl
methyl
methyl
methyl
3-methoxy-4-hydroxyphenyl
trichloromethyl
-H
10
Table 2. Structure of β-carboline compounds.
COOR2
5
8
4
6
7
3
2
9
N
N
1
H
R1

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Molecules 2010, 15
Table 2. Cont.
Compound
R1
R2
11
12
13
14
15
16
methyl
4-chlorophenyl
phenyl
2-hydroxyphenyl
1-phenyl-3-methyl-5-chloropyrazol-4-yl
methyl
methyl
methyl
methyl
methyl
methyl
-H
Harmine compounds as multi-active chemicals could endogenously form from tryptophan and
tryptophan-derived indoleamines in alkaloid biosynthesis in mammals and plants [20]. Furthermore,
they can be produced from tryptophan residues during conventional high-temperature cooking [21]. In
present study, most of new structure β-carboline derivatives, tetrahydro-β-carboline and β-carboline
compounds were synthesized using the Pictet–Spengler and Bischler-Napieralski methods [22]. The
tetrahydro-β-carboline derivatives were obtained using different mole ratios of aldehydes to tryptophan
in the presence of a catalyst. As the literature reports, the yields of these reactions were determined by
the reaction conditions and the structures of the aldehydes used. Aldehydes with an
electron-withdrawing group underwent endo cyclization with tryptophan methyl ester in good yields in
water, while the aldehydes bearing electron-donating groups need a polar organic solvent [23].
β-Carboline derivatives were obtained by reaction of a tetrahydro-β-carboline with an oxidant such as
sulfur in refluxing xylene, KMnO4 in DMF, etc. [8]. There are also numerous reports concerning the
use of metal complexes as catalysts to generate dihydro-β-carboline or β-carboline alkaloids [24]. The
oxidation with sulfur in refluxing xylene would cause environmental pollution because of the organic
solvent. Other catalysts usually involve rare metals, which would increase costd. KMnO4 as oxidant is
very cheap and available, and the reaction can take place under mild conditions, so in this paper,
appropriate amounts of KMnO4 were added to DMF solutions of tetrahydro-β-carboline-3-carboxylate
to produce 1-substituted-β-carboline-3-carboxylates under mild conditions.
2.2. Biological activity
2.2.1. Cytotoxicity
The effects of the synthesized harmaline derivatives on the cytotoxicity of Sf9 cells were examined
first. Cells were treated with various concentrations for the indicated time periods (6, 12 and 24 h) and
cell cytotoxicity was then assessed using the MTT colorimetric assay. The differences of cytotoxicities
on Sf9 cells when treated with different derivatives were statistically significant at the concentrations
of 1, 50 and 200 mg/L. The compounds 2, 13 and 15 showed more cytotoxic effects and caused cell
growth inhibition rates of up to 71.55%, 53.42% and 60.86%, respectively, after 24 h treatment at the
concentration of 200 mg/L, while the maximum inhibition using harmine treatment as a positive
control was only 40.31% under the same conditions (Figure 1). Interestingly, harmine and some of its
derivatives exhibited significant differences between antitumor cytotoxicity and insect cell lethal
toxicity to some degree.

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Molecules 2010, 15
Figure 1. Cytotoxic effect of compounds 1-16 in insect cell line Sf9 determined by the
MTT method. A is the effect of 6 h treatment; B is the effect of 12 h treatment; C is the
effect of 24 h treatment. Treatments 1-16 are the synthesized compounds and treatment 17
is harmine, a positive control. The error bars represent mean±SEM for data derived from
three independent experiments. Mean values followed by the same letter above bars have
no significant difference (P ≤ 0.05) according to Duncan’s multiple-range test.
A
1 mg/L
100
50 mg/L
90
200 mg/L
80
a
70
a
a
a
60
a
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c d
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c
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c
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1
2
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5
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7
8
9
10 11 12 13 14 15 16 17
Treatment
1 mg/L
B
100
90
80
70
60
50
40
30
20
10
0
50 mg/L
200 mg/L
a
a
a
a
a
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a
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c
1
2
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8
9 10 11 12 13 14 15 16 17
Treatment

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Molecules 2010, 15
Figure 1. Cont.
C
100
90
80
70
60
50
40
30
20
10
0
1 mg/L
50 mg/L
200 mg/L
a
a
a
a
a
b
a
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a
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cd
b
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c
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c
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c
cd
d
d
d
1
2
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4
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6
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8
9 10 11 12 13 14 15 16 17
Treatment
For example, 1-trichloromethyl-1,2,3,4-tetrahydro-β-carboline has been reported as an inducer of
cell-free plasmid DNA damage [8,25], however, methyl 1-trichloromethyl-1,2,3,4-tetrahydro-
β-carboline-3-carboxylate (compound 9) did not show high cytotoxicity towards Sf9 cells in the
present tests, even though both compounds have similar chemical structures, which maybe results from
the existence of different targets.
2.2.2. Insecticidal activity
As shown in Figure 2, treatments of 4th instar mosquito larvae with compounds 1-16 resulted in a
marked dose-dependent toxicity. All the mortalities were less than 34.85% after treatment at a low
concentration of 1 mg/L for a short period of time (24 h), and were not statistically significant.
However, compounds 2 and 13 showed stronger toxicity to mosquitos than the other compounds after
treatments with both 50 mg/L and 100 mg/L. After 24 h of treatment with 100 mg/L, compounds 2 and
13 gave adjusted mortalities of 93.29% and 80.49%, respectively, against the tested mosquito larvae,
thus exhibiting excellent prospects for follow-up. The LC50 values of compounds 2 and 13 were
20.82 mg/L and 23.98 mg/L, and the LC90 values were 88.29 mg/L and 295.13 mg/L, respectively,
after 24 h treatment with the probit (mortality%)-log (concentration) line (Y = 2.3096 + 2.0404×) and
(Y = 3.3802 + 1.1740×).

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Molecules 2010, 15
Figure 2. Mosquitocidal effect of compounds 1-16 against 4th instar mosquito (Cx.
pipiens quinquefasciatus) larvae. A is the effect of 2 h treatment; B is the effect of 12 h
treatment; C is the effect of 24 h treatment. Treatments 1-16 are the synthesized
compounds and treatment 17 is harmine, a positive control. The error bars represent
mean±SEM for data derived from three independent experiments. Mean values followed
by the same letter above bars have no significant difference (P ≤ 0.05) according to
Duncan’s multiple-range test.
100
90
80
70
60
50
40
30
20
10
0
A
1 mg/L
50 mg/L
100 mg/L
a
a
a
a
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Treatment
100
B
1 mg/L
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100 mg/L
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Treatment

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Molecules 2010, 15
Figure 2. Cont.
100
C
a
1 mg/L
50 mg/L
100 mg/L
90
80
70
60
50
40
30
20
10
0
b
a
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cd
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cd
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b
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b
cd
b
d
b
b
b
d
b
1
2
3
4
5
6
7
8
9
10 11 12 13 14 15 16 17
Treatment
The mustard aphid mortality increased with oncentration after 24 h and 48 h treatment with 50, 100
and 200 mg/L of the synthesized harmine derivatives, as shown in Figure 3.
Figure 3. Toxic effect of compounds 1-16 on mustard aphids (L. erysimi). A is the effect of
24 h treatment; B is the effect of 48 h treatment. Treatments 1-16 are the synthesized
compounds and treatment 17 is harmine, a positive control. The error bars represent
mean±SEM for data derived from three independent experiments. Mean values followed
by the same letter above bars have no significant difference (P ≤ 0.05) according to
Duncan’s multiple-range test.
100
A
50 mg/L
90
100 mg/L
200 mg/L
a
80
70
60
50
40
30
20
10
0
b
a
b
b
b
a
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cd
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cd
d
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d
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c
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c
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c
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cd
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d
cd
d
d
1
2
3
4
5
6
7
8
9
10 11 12 13 14 15 16 17
Treatment

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Molecules 2010, 15
Figure 3. Cont.
50 mg/L
100
B
100 mg/L
200 mg/L
a
90
80
70
60
50
40
30
20
a
a
a
ab
b
b
b
a
bc
b
b
b
ab
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c
c
b
c
c
c
c
c
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c
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c c
c
bc
c
c
c
cd
c
d
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d
d
d
cd
c
c
c
c
cd
cd
d
d
d
10
0
1
2
3
4
5
6
7
8
9
10 11 12 13 14 15 16 17
Treatment
Both compounds 2 and 13 showed the strongest insecticidal activities. The mortalities were only
36.42% and 15.32% after 24 h treatment with compounds 2 and 13 at the concentration of 50 mg/L,
but they increased to 87.26% and 75.76%, respectively, after 48 h at 200 mg/L, with a significant
difference from treatment with harmine alone (49.62% adjusted mortality) and CK. The LC50 values
for compounds 2 and 13 were 53.16 mg/L and 68.05 mg/L, and their corresponding LC90 values
against mustard aphids were 240.10 mg/L and 418.63 mg/L, respectively, after 48 h treatment, as
determined in the following equation with probit (mortality%)-log (concentration) line:
Y = 1.6268 + 1.9548×, Y = 2.0266 + 1.6223×, The results were in line with the cytotoxicity and
mosquitocidal toxicity experiments, and suggest these compounds could be used can be used as
candidate compounds to guide further research.
3. Experimental
3.1. General procedures
Solvents and reagents were obtained from commercial sources and purified according to the usual
procedures described in the literature [26]. Progress of the reaction and purity of the compounds were
1
monitored by thin-layer chromatography (TLC). H-NMR spectra were recorded on a Bruker
Avance-600 superconducting nuclear magnetic resonance instrument, with chemical shift (δ) values
reported in ppm relative to tetramethylsilane (TMS) as internal standard. Melting points were
determined on an XT-4 binocular microscope (Beijing Tech Instrument Co., Beijing, China) and are
uncorrected. Bioassays were carried out under standard lab conditions.

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Molecules 2010, 15
3.2. Chemical synthesis
1-Methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid (1): Acetaldehyde (40%, 1.1 mL) was
added to a solution of L-tryptophan (2.0 g, 10 mmol) in 0.1M aqueous hydrochloric acid (10 mL). This
reaction mixture was stirred at room temperature for 12 h. The resulting crystals were filtered,
dissolved in hot water and the hot solution was adjusted to pH 8 with aqueous sodium hydroxide
solution. Upon cooling, the product [27-29] was collected as white needle-like crystals in 90% yield,
mp, >200 °C; 1H-NMR (DMSO-d6) δ: 1.25 (3H, d, J = 7.8 Hz, CH3), 2.87 (1H, dd, J = 6.6 Hz, C(4)H,
c), 3.19 (1H, dd, J = 4.8 Hz, C(4)H, c), 3.51 (1H, bs, N(2)H, c), 4.31 (1H, q, J = 7.6 Hz, C(1)H, c),
4.95 (1H, q, J = 6.6 Hz, C(3)H, c), 7.08~7.72 (4H, m, C(5,6,7,8)H, c), 9.35 (1H, bs, N(9)H, c), 10.30
(1H, s, COOH).
1-Phenyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid (2): L-Tryptophan (2.0 g, 10 mmol) was
dissolved in methanol (10 mL) which contain hydrochloric acid (1 mL), and then benzaldehyde (2 mL)
was added to the solution. This reaction mixture was heated to reflux for 2 h. The resulting mixture
was cooled to room temperature, poured into water and adjusted to pH 8 with aqueous sodium
hydroxide solution [30]. The precipitate was filtered and dissolved in a hot mixture of 5mL methanol
and 8mL ethyl acetate. Upon cooling, the product was collected in 82.4% yield as a white powder, mp,
1
174–176 °C; H-NMR (DMSO-d6): δ 2.24 (1H, bs, N(2)H, c), 3.00 (1H, dd, J = 4.8 Hz, C(4)H, c),
3.23 (1H, dd, J = 8.4 Hz, C(4)H, c), 4.09 (1H, q, J = 7.6 Hz, C(3)H, c), 5.61 (1H, q, J = 5.2 Hz, C(1)H,
c), 6.99~7.49 (4H, m, C(5,6,7,8)H, c), 7.45 (4H, s, C(2,3,5,6)H, Ph), 9.24 (1H, bs, N(9)H, c), 10.60
(1H, s, COOH).
1,2,3,4-Tetrahydro-β-carboline-3-carboxylic acid (3): A mixture of methanal (30%, 1 mL) and
L-tryptophan (2.0 g, 10 mmol) in acetic acid (5 mL) was heated to reflux for 6 h or stirred at room
temperature for 24 h. The precipitate was filtered and recrystallized from hot methanol, in 40.1% yield,
1
mp, >200 °C. H-NMR (DMSO-d6): δ 1.88 (1H, bs, N(2)H, c), 2.76 (1H, dd, J = 5.2 Hz, C(4)H, c),
3.06 (1H, dd, J = 6.8 Hz, C(4)H, c), 4.23 (1H, d, J = 7.8 Hz, C(1)H, c), 4.38 (1H, d, J = 10.2 Hz,
C(1)H, c), 5.39 (1H, q, J = 8.4 Hz, C(3)H, c), 7.03 ~7.48 (4H, m, C(5,6,7,8)H, c), 8.80 (1H, bs, N(9)H,
c), 11.24 (1H, s, COOH).
1-(Trichloromethyl)-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid (4): The method used to prepare
this compound was as described in 3.2.3. L-Tryptophan (2.0 g) and trichloroacetaldehyde monohydrate
(1.65 g) were reacted at reflux temperature, and finally collected in crystalline form in 32.6% yield,
mp > 200 °C. The crystals readily changed color in air. 1H-NMR (DMSO-d6): δ 1.92 (1H, bs, N(2)H,
c), 2.96 (1H, dd, J = 6.8 Hz, C(4)H, c), 3.09 (1H, dd, J = 8.4 Hz, C(4)H, c), 4.29 (1H, q, J = 10.8Hz,
C(3)H, c), 5.11 (1H, q, J = 7.2 Hz, C(1)H, c), 6.99~7.48 (4H, m, C(5,6,7,8)H, c), 9.28 (1H, bs, N(9)H,
c), 10.78 (1H, s, COOH).
1-(4-Hydroxy-3-methoxyphenyl)-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid (5): The method
described in 3.2.3 was used to prepare the title compound. L-Tryptophan (2.0 g) and vanillic aldehyde
(1.5g) with 1:1 molar quantities were added to a round-bottom flask and stirred at room temperature
for 24 h. A large amount of a yellowish green solid was deposited on the bottom of the flask. The target

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Molecules 2010, 15
compound was isolated in 77.8% yield by filtering the mixture, mp, 184–186 °C; 1H-NMR (DMSO-d6):
δ 1.91 (1H, bs, N(2)H, c), 2.93 (1H, dd, J = 8.4 Hz, C(4)H, c), 3.11 (1H, q, J = 7.8 Hz, C(4)H, c), 3.65
(1H, q, J = 5.2 Hz, C(3)H, c), 3.74 (3H, s, Ar-OCH3), 5.47 (1H, q, J = 6.6 Hz, C(1)H, c), 6.50~6.71 (2H,
m, C(5,6)H, Ph), 7.01 (1H, s, C(2)H, Ph), 6.97~7.46 (4H, m, C(5,6,7,8)H, c), 9.32 (1H, bs, N(9)H, c),
10.69 (1H, s, COOH).
Methyl 1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylate (6): The synthesis of this compound
was accomplished by a two-step process. Methanol (15 mL) was cooled to 0 °C and thionyl chloride (1
mL) was added dropwise. Fifteen min later, the cooling bath was removed and compound 1 was added
to the solution. The mixture was stirred overnight followed by distillation under reduced pressure and
the residue was adjusted to pH 8 with aqueous sodium hydroxide solution [29]. The deposits were
filtered and re-crystallized from hot methanol (3 mL) with the addition of ethyl acetate (8 mL) to give
a 85.9% yield of the target compound, mp, 185–187 °C; 1H-NMR (DMSO-d6): δ 1.72 (3H, d, J = 6.6
Hz, CH3, c), 2.50 (1H, bs, N(2)H, c), 3.02 (1H, dd, J = 11.2 Hz, C(4)H, c), 3.26 (1H, dd, J = 6.4 Hz,
C(4)H, c), 3.86 (3H, s, COOCH3), 4.58 (1H, q, J= 8.2 Hz, C(3)H, c), 4.72 (1H, q, J = 6.0 Hz, C(1)H, c),
7.02~7.48 (4H, m, C(5,6,7,8)H, c), 11.36 (1H, bs, N(9)H, c).
Methyl 1-phenyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylate (7): In a round-bottomed, short-necked
flask crude methyl 2-amino-3-(1H)-indol-3-yl propanoate (2.55 g, 10 mmol) was dissolved in
methanol (10 mL) and benzaldehyde (1 g, 10 mmol) was added to the mixture with stirring. The
resulting solution was boiled for 6 h under reflux. The excess methanol was distilled off under reduced
pressure and the residue was adjusted to pH 8 with aqueous sodium hydroxide solution [30]. The
deposits were filtered and re-crystallized from hot methanol (1 mL) with the addition of ethyl acetate
1
(5 mL), to give a 87.2% yield of compound 7, mp, >200 °C; H-NMR (DMSO-d6): δ 2.25 (1H, bs,
N(2)H, c), 2.99 (1H, dd, J = 8.8 Hz, C(4)H, c), 3.21 (1H, dd, J = 4.6 Hz, C(4)H, c), 3.80 (3H, s,
COOCH3), 3.96 (1H, q, J = 10.2 Hz, C(3)H, c), 5.22 (1H, q, J = 8.2 Hz, C(1)H, c), 7.10~7.53 (4H, m,
C(5,6,7,8)H, c), 7.18~7.36 (5H, s, C(2,3,4,5,6)H, Ph), 11.13 (1H, s, N(9)H, c).
Methyl 1-(4-hydroxy-3-methoxyphenyl)-1,2,3,4-tetrahydro-β-carboline-3-carboxylate (8): The general
procedure described in section 3.2.6 was used and the residue was purified by re-crystallization from hot
methanol with addition of ethyl acetate (1+5) to give the desired compound in 70% yield, mp
170–172 °C; 1H-NMR (DMSO-d6): δ 1.96 (1H, bs, N(2)H, c), 2.80 (1H, dd, J = 10.2 Hz, C(4)H, c), 3.00
(1H, dd, J = 8.4 Hz, C(4)H, c), 3.63 (3H, s, COOCH3), 3.71 (3H, s, Ar-OCH3), 3.72 (1H, q, J = 8.6 Hz,
C(3)H, c), 3.84 (1H, s, Ar-OH), 5.10 (1H, q, J = 5.4 Hz, C(1)H, c), 6.76 (2H, s, C(5, 6)H, Ph), 6.90
(1H, s, C(2)H, Ph), 6.93~7.41 (4H, m, C(5,6,7,8)H, c), 10.25 (1H, bs, N(9)H, c).
Methyl 1-(trichloromethyl)-1,2,3,4-tetrahydro-β-carboline-3-carboxylate (9): The procedure described
in section 3.2.6 was used and the residue was purified by re-crystallization from ethyl acetate with
addition of petroleum ether (1+3) to give 9 in 60% yield, mp > 200 °C. 1H-NMR (DMSO-d6): δ 2.41 (1H,
bs, N(2)H, c), 3.14~3.47 (2H, m, C(4)H, c), 3.62 (3H, s, COOCH3), 4.34 (1H, q, J = 10.8 Hz, C(3)H, c),
5.16 (1H, q, J = 6.8 Hz, C(1)H, c), 7.00~7.45 (4H, m, C(5,6,7,8)H, c), 11.78 (1H, bs, N(9)H, c).

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Molecules 2010, 15
Methyl 1,2,3,4-tetrahydro-β-carboline-3-carboxylate (10): The method described in section 3.2.6 was
used. The residue was purified by column chromatography using methanol+ethyl acetate (1+3) as
1
eluent to produce the desired compound in 50.6% yield, mp > 200 °C; H-NMR (DMSO-d6): δ 3.04
(1H, bs, N(2)H, c), 3.21 (3H, s, COOCH3), 3.42 (1H, dd, J = 10.5 Hz, C(4)H, c), 3.97 (1H, dd,
J = 7.8 Hz, C(4)H, c), 4.44 (1H, d, J = 7.8 Hz, C(1)H, c), 4.56 (1H, d, J = 7.5 Hz, C(1)H, c), 5.47 (1H,
q, J = 9.4 Hz, C(3)H, c), 7.11~7.53 (4H, m, C(5,6,7,8)H, c), 11.34 (1H, bs, N(9)H, c).
Methyl 1-methyl-β-carboline-3-carboxylate (11):
Methyl 1-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]-indole-3-carboxylate (6, 1.3 g) was dissolved in
DMF (10 mL) in a round-bottomed, short-necked flask. The solution was cooled to 0 °C and potassium
permanganate (0.4 g) was added to the mixture that was then left stirring overnight. The mixture was
poured into water to collect the top deposit which was re-crystallized from hot methanol with addition
of ethyl acetate(1+1) to give the title compound in 71.3% yield [22], mp > 200 °C; 1H-NMR
(DMSO-d6): δ 2.85 (3H, bs, CH3), 4.03 (3H, s, COOCH3), 7.37~8.79 (4H, m, C(5,6,7,8)H, c), 9.17
(1H, s, C(4)H, c), 11.62 (1H, bs, N(9)H, c).
Methyl 1-(4-chlorophenyl)-β-carboline-3-carboxylate (12): The procedure described in section 3.2.6
was used and the reaction product was further purified by re-crystallization from hot methanol to give
compound 12 in 60.2% yield, mp > 200 °C; 1H-NMR (DMSO-d6): δ 3.94 (3H, s, COOCH3), 7.34~8.44
(4H, m, C(5,6,7,8)H, c), 7.70~8.05 (4H, m, C(2,3,5,6)H, Ph), 8.96 (1H, s, C(4)H, c), 9.95 (1H, bs,
N(9)H, c).
Methyl 1-phenyl-β-carboline-3-carboxylate (13): The methodology as described in section 3.2.6 was
used and the reaction product was further purified by re-crystallization from hot methanol to give the
1
target molecule in 82.7% yield, mp > 200 °C; H-NMR（DMSO-d6): δ 4.02 (3H, s, COOCH3),
7.33~7.60 (5H, m, C(2,3,4,5,6)H, Ph), 7.65~8.27 (4H, m, C(5,6,7,8)H, c), 8.90 (1H, s, C(4)H, c), 9.04
(1H, bs, N(9)H, c).
Methyl 1-(2-hydroxyphenyl)-β-carboline-3-carboxylate (14): The procedure described in section 3.2.6
was used. The reaction product was further purified by re-crystallization from hot methanol to give 14
in 35.5% yield, TLC: Rf: 0.65 (petroleum ether + ethyl acetate, 5 + 1 by volume), mp > 200 °C;
1H-NMR (DMSO-d6): δ 3.98 (3H, s, COOCH3), 5.71 (1H, m, Ar-OH), 7.08~7.41 (4H, m, C(3,4,5,6)H,
Ph), 7.63 (4H, m, C(5,6,7,8)H, c), 8.98 (1H, s, C(4)H, c), 10.27 (1H, bs, N(9)H, c).
Methyl
1-(5-chloro-3-methyl-1-phenyl-1H-pyrazol-4-yl)-β-carboline-3-carboxylate
(15):
The
procedure described in section 3.2.6 was followed and the reaction product was further purified by
re-crystallization from hot methanol to give the title compound in 76.9% yield, mp > 200 °C; 1H-NMR
(DMSO-d6): δ 2.34 (3H, s, CH3), 7.32~8.44 (4H, m, C(5,6,7,8)H, c), 7.53~7.60 (5H, m, C(2,3,4,5,6)H,
Ph), 78.86 (1H, s, C(4)H, c), 11.84 (1H, bs, N(9)H, c).
1-Methyl-β-carboline-3-carboxylic acid (16): Methyl 1-methyl-β-carboline-3-carboxylate (11, 2.3 g,
10 mmol) was dissolved in methanol (3 mL) and heated to reflux in 2 M aqueous sodium hydroxide

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solution (20 mL) for 2 h. After completion of the reaction the pH of the mixture was adjusted to 8. The
precipitate was filtered off and the residue was re-crystallized from hot methanol as a yellow floc,
obtained in 97% yield [29], mp > 200 °C; 1H- NMR (DMSO-d6): δ 2.83 (3H, s, -CH3), 7.29~8.35 (4H,
m, C(5,6,7,8)H, c), 8.76 (1H, s, C(4)H, c), 10.81 (1H, bs, N(9)H, c), 12.02 (1H, s, COOH).
3.3. Bioassays
To evaluate the biological activities of the synthesized compounds, cytotoxic assays were carried
out with insect cultured cell line Sf9 from Spodoptera frugiperda, insecticidal assays were conducted
with the reared colonies of 4 th instar larvae of Culex pipiens quinquefasciatus and apterous adults of
mustard aphids [Lipaphis erysimi (Kaltenbach)]. The cytotoxic and insecticidal activity of harmine
(95%, purity, Sigma) was also evaluated against the aforementioned species as positive control.
3.3.1. Cytotoxicity
Sf9 cells were maintained at 27 °C in TC-199-MK medium supplemented with 10% FCS (vol/vol),
1% L-glutamine 200 mmol/L and penicillin–streptomycin–neomycin solutions (vol/vol) described in
Zhong et al. [31]. The cell growth inhibition was measured by the MTT method described by Mosman
[32]. Cells were seeded in 96-well microtitration plates at the exponential growth phase. Different
concentrations of compounds diluted with medium were added at their log-phase growth stage. And
0.2 μL solvent (DMSO) only was added as control (CK). The final concentration of solvent in the
cultures assayed was 1%. Compound was solubilized with dimethyl sulfoxide and the first
concentration was 1,000 mg/L. The harmine was used as positive control. After 6 h, 12 h and 24 h of
treatment, 5 mg/mL MTT was dissolved in PBS and 20 μL of this stock solution was added to the
culture cells. After additional 3 h of incubation, the medium was discarded and the 96-well plates were
dried in the air. Then 100 μL DMSO were added to dissolve the formazan crystals, and the absorbance
was measured at 570 nm by a microplate reader (Spectramex, 190 Molecular Devices Inc., USA) [31].
The cytotoxic effect was expressed as a relative percentage of inhibition calculated as follows:
Cell growth inhibition rate (%) = [(A control –A treatment) / A control] ×100
3.3.2. Insecticidal activity
3.3.2.1. Mosquito
Mosquito (4th instar larvae of Culex pipiens quinquefasciatus) used in the experiments were
obtained from cultures at the Guangdong Center for Disease Control and Prevention. Bioactivities of
compounds were tested by the mixed pesticide method by the procedure as recommended in the
literature [33,34]. The compounds were first dissolved in a mixture of DMSO and acetone at a
concentration of 10 g/L and suspended in distilled water with the addition of 0.5% of the commercial
surfactant Tween-80 to obtain final concentrations of 1, 10, 50 and 100 mg/L. Twenty early 4th instar
Cx. pipiens quinquefasciatus were placed into a 20 mL bioassay bowl containing 10 mL of the test
solution and incubated at 27 °C. The number of dead larvae was determined at the start of the
experiment (0 h), and after 2 h, 12 h and 24 h, respectively. Larvae were considered dead when they

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were unable to reach the surface of the solution on shaking the cups. Mortality was scored and
compared with controls that had been exposed to a diet treated with solvent only. The cumulative
mortality data were corrected by Abbott’s formula.
3.3.2.2. Aphis
Mustard aphids (Lipaphis erysimi (Kaltenbach)) were reared on mustard leaves. Apterous adult
aphids fed on equally vigorous leaves were selected for the experiments. The compounds were first
dissolved in DMSO and acetone mixture solvent at a concentration of 10 g/L and suspended in
distilled water with 0.5% of emulsifier to obtain final concentrations of 50, 100 and 200 mg/L. The
leaf discs with about 30–50 apterous adult aphids were dipped in compound solutions, shaken gently
for 3 s, and then taken out. The excess solution was immediately absorbed with a filter paper. Then
treated leaf discs were held at 25 ± 1 °C, 50–60% relative humidity. Solvent only was used as the
control. The mortality was recorded at 24 and 48 h after the treatment. The mortality was corrected
with reference to the control and assessed statistically [33].
3.4. Data analyses
Experiments were carried out in triplicate and data analyses were run on the SAS 8.01 (2000)
software and P ≤ 0.05 was considered to be significant. Data were subjected to one-way ANOVA and
mean values were compared using Duncan’s multiple-range test [35]. Mean values followed by the
same letter have no significant difference (P ≤ 0.05) according to Duncan’s multiple-range test.
4. Conclusions
Recently, a number of investigations have demonstrated that harmine derivatives not only possessed
antifungal activity, but that they were also highly cytotoxic against several kinds of cancer cell lines.
However, the insecticidal activity of harmine derivatives has not been previously reported. Due to their
diverse biological activities and the feasibility of their simple structure modification, researchers have
been strongly interested in the synthesis and the structure-activity relationships of a series of harmine
derivatives, especially for the purpose of developing anticancer drugs. In this paper, the preparation of
16 harmine derivatives was reported and their cytotoxicities and insecticidal activities were tested. The
compounds 2 and 13 showed the strongest activities both in vitro and in vivo. They both caused 71%
mortality to Sf9 cells after 24 h treatment at a concentration of 200 mg/L. Furthermore, the LC50
values for compounds 2 and 13 were 20.82 mg/L and 23.98 mg/L against 4th instar larvae mosquito
(Cx. pipiens quinquefasciatus) after 24 h treatment, and 53.16 mg/L and 68.05 mg/L against mustard
aphids (L. erysimi) after 48 h treatment, respectively. The insecticidal activities in vivo were in line
with the in vitro cytotoxicity. These results may provide a basis for the production of novel potential
pesticides based on harmine and norharman natural products. Therefore, further in-depth studies to
identify the insecticidal mechanism of compounds 2 and 13 are warranted for their development as
biorational pesticides.

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Acknowledgment
The work was supported by grants from the China National Nature Science Foundation (No.
30971944) and the Foundation for the Author of National Excellent Doctoral Dissertation of China
(FANEDD, No.200461).
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© 2010 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/3.0/).