Synthesis and insecticidal activity of novel 4beta-halogenated benzoylamino podophyllotoxins against Pieris rapae Linnaeus.

Article abstract of DOI:10.1248/cpb.50.399

Twelve new 4beta-halogenated benzoylamino compounds (7.1-7.12) of podophyllotoxin have been synthesized, and their structures were confirmed by IR, 1H-NMR, MS spectra as well as CHN elemental analysis. These compounds showed delayed insecticidal activity

Full text of DOI:10.1248/cpb.50.399

March 2002
Notes
Chem. Pharm. Bull. 50(3) 399—402 (2002)
399
b
Synthesis and Insecticidal Activity of Novel 4 -Halogenated Benzoylamino
Podophyllotoxins against Pieris rapae LINNAEUS
Hui XU,a,b Xing ZHANG,b Xuan TIAN,c Min LU,b and Yan-guang WANG*,a
Department of Chemistry, Zhejiang University,a Hangzhou, 310027, P.R. China, Research and Development Center of
Biorational Pesticide, Northwest Science and Technology University of Agriculture and Forest,b Yangling, Shaanxi,
712100, P.R. China, and National Laboratory of Applied Organic Chemistry, Lanzhou University,c Lanzhou, 730000, P.R.
China. Received August 14, 2001; accepted November 26, 2001
Twelve new 4b-halogenated benzoylamino compounds (7.1—7.12) of podophyllotoxin have been synthesized,
and their structures were confirmed by IR, 1H-NMR, MS spectra as well as CHN elemental analysis. These com-
pounds showed delayed insecticidal activity against 5th instar larvae of Pieris rapae LINNAEUS in vivo, when tested
by a leaf-dipping method at a concentration of 250 ppm. By preliminary qualitative structure–activity relation-
ship analysis, we found the following results: 1) Compounds 7.2, 7.5—7.9 were more potent than the nature par-
ent product in the mortality after 15 d against P. rapae in vivo. Especially compounds 7.5 and 7.6 bearing meta-
and para-chlorobenzene substituents respectively, were the most potent of these compounds; 2) Substitution on
the benzene ring moiety of 4b-benzoylamino podophyllotoxin (PPT) with Cl, Br, I at the para or at the meta posi-
tion yielded compounds which were as potent or more potent than those containing the corresponding substitut-
ing group at the ortho position. 3) Substitution on the benzene ring moiety of 4b-benzoylamino podophyllotoxin
with I either at the ortho, meta or para position yielded less potent compounds (7.10—7.12) when compared with
PPT.
Key words podophyllotoxin; botanical pesticide; synthesis; Pieris rapae; delayed insecticidal activity
Podophyllotoxin (PPT, 1) is a phenyltetralin-type of lignan alkylation of the target enzyme to occur and causing protein
which is widely distributed in higher plants. As a result of linked DNA breakage, so we herein, in order to find the more
the development of etoposide (VP-16, 2) and teniposide potent compounds than PPT, introduced halogenated benzoy-
(VM-26, 3) as anticancer drugs, semi-synthetic analogues of lamino moiety into PPT at the C-4b position and have syn-
the naturally occurring PPT have drawn much renewed inter- thesized twelve novel 4b-halogenated benzoylamino com-
est in recent years. It is believed that such analogues of 4Ј- pounds 7.1—7.12. The insecticidal activity of these new
demethylepipodophyllotoxin exert their cytotoxic, antitumor compounds was tested in vivo against the 5th instar larvae of
activity through stabilization of a cleavable complex between Pieris rapae LINNAEUS.
DNA and type II DNA topoisomerase. This leads ultimately
Experimental
to inhibition of DNA catenation activity and produces single
All melting points were taken on a Kofler melting point apparatus and un-
and double strand breaks.1—3) Meanwhile, It has been re-
1
corrected. IR spectra were obtained on a NIC-5DX spectrophotometer. H-
ported that deoxypodophyllotoxin (DPT, 4) has insectici-
dal,4—6) phytogrowth inhibitory and ichthytoxic activities.7)
As regards the insecticidal activity, it was confirmed that the
symptoms caused by DPT developed slowly; namely, DPT is
a delayed toxicant.4,8,9) This conclusion was supported by the
finding that the action of DPT on the 5th instar larvae of silk-
worm, Bombyx mori LINNE, involved severe damage to the
epidermal cells accompanied with coagulation of the chro-
matin.6) Meanwhile, it was reported that the trans-lactone and
the 4Ј-OCH3 groups in the PPT derivatives were essential to
NMR spectra were obtained by using either a Bruker AM-400 or AC-80
NMR spectrometer. In all cases, samples were dissolved in deuterochloro-
form and all chemical shifts were reported in ppm from tetramethylsilane
(TMS). The elemental analyses were determined on a Carlo Elba 1106 in-
strument, mass spectral analyses were taken on a VG ZAB-HS instrument at
70 eV and optical rotations were determined on a J-20C spectropolarimeter.
Podophyllotoxin (1), mp 175—176 °C, [a]D25 Ϫ109° (cϭ1.0, CHCl3), iso-
lated from a Chinese medicinal herb Podophyllum emodi. Wall var. Chinesis
sprague, was used as the starting material. The synthesis route of target com-
pounds was shown in Fig. 2.
4b-Azido Podophyllotoxin (5) PPT (2.0 g, 4.8 mmol) and hydrazoic
acid (4.8 ml, Cϭ1.04 M, benzene) were suspended in anhydrous dichloro-
keep the insecticidal activity.4,10) But up to the present, there methane (70 ml) and cooled to below Ϫ10 °C and stirred. BF3·Et2O (0.9 ml)
was added dropwise so as to keep temperature below Ϫ10 °C. After TLC
has been few reports that deal with insecticidal activity and
structure-insecticidal activity relationship studies of PPT
analogues.11—13) According to the known structure–activity
relationship on the medical research and our previous stud-
ies,14—20) we found that replacement of the 4b-O-glucosidic
substituent of compound 2 with 4b-halogenated anilino moi-
ety yielded a number of compounds which were as potent or
more potent than 2 in inhibiting the human DNA topoiso-
merase II and causing cellular protein-linked DNA breakage
because the basic nitrogen and the aromatic ring might cause
a bond delocalization to generate a charged 4b-N atom. Con-
sidering 4b-halogenated benzoylamino moiety might cause a
bond delocalization to generate a more stable benzyl onium
Fig. 1. The Chemical Structures of PPT and Its Analogues
ion than 4b-halogenated anilino moiety, thereby allowing the
To whom correspondence should be addressed. e-mail: orgwyg@css.zju.edu.cn
© 2002 Pharmaceutical Society of Japan

400
Vol. 50, No. 3
analysis showed that most of the starting material had been consumed, pyri-
General Procedure of Synthesizing Compounds (7.1—7.12) To a so-
dine (0.9 ml) and distilled water (20 ml) were added. The organic phase was lution of compound 6 (2.5 mmol) in dry dichloromethane (40 ml) was added
seperated from the mixture, washed by distilled water, 5% hydrochloric acid, corresponding acid (2.5 mmol) and dicyclohexylcarbodiimide (DCC,
distilled water, 2% sodium bicarbonate, distilled water, dried over anhydrous 0.65 g). The mixture was stirred at 0 °C until TLC indicated that the reaction
sodium sulfate, and then filtered and evaporated under reduced pressure. was complete. Several drops of glacial acetic acid was added. Then the mix-
The residue was recrystallized in acetone–methanol to produce a white ture was filtered, and the filtrate was washed successively by 5 N ammonia
solid.Yield 84.0%, mp 192—194 °C, [a]D25 Ϫ80.8° (cϭ0.5, CHCl3); MS liquor, distilled water twice, then dried over anhydrous sodium sulfate. The
(electron impact (EI)): 439 (Mϩ, 51%); 1H-NMR d: ppm: 6.82 (s, 1H, H-5), solvent was evaporated off in vacuum. The residue was purified by silica gel
6.60 (s, 1H, H-8), 6.28 (s, 2H, H-2Ј, H-6Ј), 6.02 (s, 2H, OCH2O), 4.78 (d, column chromatography using chloroform–methanol as eluent and recrystal-
Jϭ3.2 Hz, 1H, H-4), 4.60 (d, Jϭ4.6 Hz, 1H, H-1), 4.34 (d, Jϭ8.0 Hz, 2H, H-
lized in acetone to produce a pure solid. The analytical data and spectra data
11), 3.82 (s, 3H, 4Ј-OCH3), 3.70 (s, 6H, 3Ј, 5Ј-OCH3), 3.10 (m, 2H, H-2, H- were illustrated in Tables 1 and 2, respectively.
3); IR (KBr) cmϪ1: 2102 (N3), 1768 (g-lactone), 1580, 1507 and 1484 (aro-
matic, CϭC).
Bioassay Leaf-dipping method was used.21) The 5th instar larvae of P.
rapae of the same size, health and starvation for 2 h were used, which were
4b-NH2-podophyllotoxin (6) To a solution of the 4b-azido-podophyllo- gathered from field and reared in room. For each compound, 30 larvae (10
toxin (2.0 g, 4.6 mmol) in EtOAC (40 ml) was added 10% palladium on car-
bon (400 mg). This mixture was shaken under 5 atm of H2 and at 40 °C for
larvae per group) were used. Acetone solutions of compounds 7.1—7.12,
PPT, 6 and Parathion (used as a standard) were prepared at a concentration
24 h. The reaction mixture was filtered and the filtrate evaporated in vacuum. of 250 ppm. Fresh wild cabbage leaves were dipped into the solution for 3 s,
The residue was chromatographed on a silica gel column and eluted with then taken out and dried in a room. Leaves treated with acetone alone were
acetone–petroleum ether to get a white powder. Yield 64.4%, mp 174— used as a control group. Several treated leaves were kept in each dish, and
1
176 °C, [a]D25 Ϫ18.6° (cϭ0.5, CHCl3); MS (EI): 413 (Mϩ, 79%); H-NMR every 10 larvae was raised in it. If the treated leaves were consumed, the cor-
d: ppm: 6.86 (s, 1H, H-5), 6.52 (s, 1H, H-8), 6.28 (s, 2H, H-2Ј, H-6Ј), 5.98 responding ones were added to the dish. The entire experiment was repri-
(s, 2H, OCH2O), 4.60 (d, Jϭ4.0 Hz, 1H, H-4), 4.32 (m, 3H, H-11, H-1), 3.85 cated three times. Every experiment was carried out at 25Ϯ2 °C, relative hu-
(s, 3H, 4Ј-OCH3), 3.75 (s, 6H, 3Ј, 5Ј-OCH3), 3.40 (dd, 1H, H-2), 2.88 (m, midity (RH) 65—80% and on 12 h/12 h (light/dark) photoperiod.
1H, H-3): IR (KBr) cmϪ1: 3323 (NH), 1771 (g-lactone), 1587, 1508 and
1483 (aromatic, CϭC).
The number of deaths was observed at intervals of 5 d for 15 d until
emerging adults of P. rapae. The final mortality of these compounds against
P. rapae and control group examined after 5, 10 and 15 d were shown in
Table 3.
Discussion and Conclusion
The assignment of the configuration at C-4 position for
compounds 5, 6 and 7.1—7.12 was based on J3,4 coupling
constants. According to Karplus dihedral angle rule in six-
membered rings, the C-4 b-substituted compounds have
J3,4Ϸ4.0 Hz, due to a cis relationship between H-3 and H-4.
While the C-4 a-substituted compounds have J3,4м10.0 Hz,
as H-3 is trans to H-4.14)
The insecticidal activity of compounds 7.1—7.12, PPT
and 6 against P. rapae in vivo at a concentration of 250 ppm
was examined by the leaf-dipping method. As shown in
Table 3, we found that, in contrast to parathion, the mortali-
ties caused by these compounds were far higher after 15 d
than those after 10 and 5 d. That is, these compounds showed
delayed insecticidal activity. Meanwhile, we noticed that
spontaneous movement in the insects treated by these com-
pounds was little different from that of control insects in 24 h
after treatment. But after 48 h some of the treated groups
were paralyzed, lost body liquid and were becoming immobi-
lized. Immobilization was increased with the passage of
Fig. 2. The Synthesis Route of Compounds 7.1—7.12
Table 1. The Analytical Data of Compounds 7.1—7.12
[a]D25 (°)
Calculated/Found (%)
Formula
Compd.
Yield (%)
mp (°C)
(cϭ0.5, CHCl3)
C
H
N
7.1
7.2
7.3
7.4
7.5
7.6
7.7
7.8
7.9
38.6
36.7
38.6
41.2
43.1
57.9
52.0
52.0
43.3
59.4
72.3
56.2
120—124
124—127
138—140
132—136
118—120
152—154
158—160
148—150
158—160
118—120
138—140
161—164
Ϫ29.2
Ϫ82
Ϫ38
Ϫ83.6
Ϫ48
Ϫ24.8
Ϫ96.4
Ϫ48
Ϫ176.8
Ϫ65.6
Ϫ56.4
Ϫ106.8
C29H26O8NF·1/2H2O
C29H26O8NF·H2O
C29H26O8NF·H2O
C29H26O8NCl·1/2H2O
C29H26O8NCl·H2O
C29H26O8NCl·H2O
C29H26O8NBr·1/2H2O
C29H26O8NBr
63.97/64.13
62.93/63.14
62.93/62.86
62.14/62.47
61.16/61.29
61.16/61.25
57.61/57.31
58.49/58.24
58.49/58.41
53.37/53.15
53.37/53.24
53.37/53.18
4.96/5.08
5.06/5.18
5.06/4.93
4.82/4.71
4.92/4.77
4.92/4.74
4.47/4.28
4.37/4.21
4.37/4.19
4.14/4.10
4.14/4.01
4.14/4.09
2.57/2.82
2.53/2.72
2.53/2.31
2.50/2.78
2.46/2.66
2.46/2.71
2.32/2.54
2.35/2.58
2.35/2.48
2.15/2.11
2.15/2.19
2.15/2.12
C29H26O8NBr
7.10
7.11
7.12
C29H26O8NI·1/2H2O
C29H26O8NI·1/2H2O
C29H26O8NI·1/2H2O

March 2002
401
Table 2. Spectral Data of Compounds 7.1—7.12
Compd.
7.1
IR (KBr)/C cmϪ1
MS (EI)/m/z
1H-NMR d ppm
3306 (NH), 1777 (g-lactone), 1655 (NHCO),
1505, 1483 (Aromatic, CϭC)
3298 (NH), 1776 (g-lactone), 1651 (NHCO),
1587, 1506, 1483 (Aromatic, CϭC)
3327 (NH), 1777 (g-lactone), 1657 (NHCO),
1590, 1532, 1500 (Aromatic, CϭC)
3284 (NH), 1776 (g-lactone), 1649 (NHCO),
1590, 1505, 1483 (Aromatic, CϭC)
3226 (NH), 1777 (g-lactone), 1640 (NHCO),
1590, 1505, 1483 (Aromatic, CϭC)
3324 (NH), 1776 (g-lactone), 1658 (NHCO),
1591, 1505, 1482 (Aromatic, CϭC)
3260 (NH), 1776 (g-lactone), 1649 (NHCO),
1588, 1505, 1482 (Aromatic, CϭC)
3325 (NH), 1776 (g-lactone), 1659 (NHCO),
1588, 1504, 1484 (Aromatic, CϭC)
3328 (NH), 1777 (g-lactone), 1657 (NHCO),
1589, 1504, 1482 (Aromatic, CϭC)
3271 (NH), 1775 (g-lactone), 1642 (NHCO),
1585, 1504, 1483 (Aromatic, CϭC)
3326 (NH), 1775 (g-lactone), 1628 (NHCO),
1586, 1504, 1484 (Aromatic, CϭC)
3323 (NH), 1776 (g-lactone), 1658 (NHCO),
1586, 1504, 1481 (Aromatic, CϭC)
535 (Mϩ, 8%), 396 (44%), 168 (16%)
535 (Mϩ, 12%), 396 (62%), 168 (32%)
535 (Mϩ, 5%), 396 (31%), 168 (17%)
7.24—8.06 (4H, aromatic ring H), 6.82
(1H, H-5), 6.58 (1H, H-8), 5.42 (1H, NH)
7.20—7.56 (4H, aromatic ring H), 6.82
(1H, H-5), 6.58 (1H, H-8), 5.42 (1H, NH)
7.06—7.84 (4H, aromatic ring H), 6.84
(1H, H-5), 6.58 (1H, H-8), 5.42 (1H, NH)
7.24—7.72 (4H, aromatic ring H), 6.90
(1H, H-5), 6.52 (1H, H-8), 5.46 (1H, NH)
7.28—8.06 (4H, aromatic ring H), 6.82
(1H, H-5), 6.52 (1H, H-8), 5.46 (1H, NH)
7.36—7.82 (4H, aromatic ring H), 6.82
(1H, H-5), 6.52 (1H, H-8), 5.40 (1H, NH)
7.22—7.59 (4H, aromatic ring H), 6.92
(1H, H-5), 6.56 (1H, H-8), 5.44 (1H, NH)
7.2
7.3
7.4
553 (Mϩ, 2%), 551 (Mϩ, 6%), 396 (75%),
168 (19%)
7.5
553 (Mϩ, 2%), 551 (Mϩ, 7%), 396 (80%),
168 (35%)
7.6
553 (Mϩ, 2%), 551 (Mϩ, 5%), 396 (52%),
168 (27%)
7.7
597 (Mϩ, 5%), 595 (Mϩ, 5%), 396 (100%),
168 (40%)
7.8
597 (Mϩ, 10%), 595 (Mϩ, 10%), 396 (96%), 7.24—7.96 (4H, aromatic ring H), 6.84
168 (46%)
(1H, H-5), 6.60 (1H, H-8), 5.44 (1H, NH)
7.38—7.64 (4H, aromatic ring H), 6.84
(1H, H-5), 6.68 (1H, H-8), 5.44 (1H, NH)
7.10—7.94 (4H, aromatic ring H), 6.96
(1H, H-5), 6.56 (1H, H-8), 5.42 (1H, NH)
7.20—8.10 (4H, aromatic ring H), 6.80
(1H, H-5), 6.48 (1H, H-8), 5.42 (1H, NH)
7.44—7.86 (4H, aromatic ring H), 6.82
(1H, H-5), 6.52 (1H, H-8), 5.42 (1H, NH)
7.9
597 (Mϩ, 11%), 595 (Mϩ, 11%), 396
(100%), 168 (43%)
7.10
7.11
7.12
643 (Mϩ, 7%), 396 (70%), 168 (23%)
643 (Mϩ, 13%), 396 (100%), 168 (47%)
643 (Mϩ, 12%), 396 (72%), 168 (34%)
tality after 15 d against P. rapae. Especially compounds 7.5
and 7.6, whose final mortalities to P. rapae were 94.73% and
94.95% respectively at the concentration of 250 ppm, were
the most active of these compounds, i.e. The introduction of
Cl at the meta or para position on the benzene ring of 4b-
benzoylamino podophyllotoxin will enhance the insecticidal
activity to a great extent. We also found that substitution on
the benzene ring of 4b-benzoylamino podophyllotoxin with
Cl, Br, I at the para or at the meta position yielded com-
pounds which were as potent or more potent than those con-
taining the corresponding substituting group at the ortho po-
sition, substitution with F, Cl, I at the ortho position of 4b-
benzoylamino podophyllotoxin afforded less active com-
pounds (7.1, 7.4, 7.10), and substitution on the benzene ring
moiety of 4b-benzoylamino podophyllotoxin with I either at
the ortho, meta or para position yielded less potent com-
pounds (7.10—7.12) when compared with PPT. These results
demonstrate the possibility of further elaboration of the 4b-
amino substitutent to optimize the structure of this class of
insecticidal compounds. The nature of the difference in
mechanism of action between PPT derivatives and conven-
tional insecticide is not clear at present. Further study for in-
secticidal activity of these synthesized compounds is in
progress.
Table 3. Insecticidal Activity of PPT and Its Derivatives against Pieris
rapae (%)
Mortality
Entry
5 d
10 d
15 d
Control
PPT
6
7.1
7.2
7.3
7.4
7.5
7.6
7.7
7.8
7.9
7.10
7.11
7.12
Parathion
0
6.67Ϯ1.56
53.36Ϯ2.83
36.43Ϯ4.17
46.41Ϯ1.66
49.46Ϯ3.65
44.74Ϯ2.55
36.36Ϯ3.18
54.25Ϯ2.75
72.08Ϯ4.48
44.78Ϯ1.77
69.23Ϯ3.15
51.85Ϯ2.43
48.36Ϯ1.99
40.71Ϯ1.64
48.28Ϯ2.08
—
10Ϯ1.22
74.74Ϯ3.23
49.48Ϯ2.86
68.42Ϯ2.66
86.70Ϯ2.30
66.76Ϯ1.70
65.56Ϯ1.28
94.73Ϯ1.48
94.95Ϯ2.86
74.74Ϯ1.56
85.42Ϯ0.98
85.97Ϯ1.98
69.69Ϯ2.26
69.69Ϯ3.62
73.87Ϯ1.88
—
34.02Ϯ2.44
21.69Ϯ2.36
22.68Ϯ3.47
18.57Ϯ2.59
11.11Ϯ1.92
4.54Ϯ1.33
16.56Ϯ1.87
36.84Ϯ2.53
4.35Ϯ1.02
46.15Ϯ3.74
14.81Ϯ2.15
12.62Ϯ1.95
8.35Ϯ2.65
20.73Ϯ3.61
100Ϯ0
a) Temperature: 25Ϯ2 °C; RH: 65—80%; photoperiod: light/darkϭ12 h/12 h. b)
Experimental size: 10 insects per group, 3 groups. c) Concentration: 250 ppm. d)
Bioassay method: Leaf-dipping method. e) Each value represents the meanϮS.D.
time. We also found that normal adults emerged from pupae
However, there are much work to be done in the future.
in the control group and some adultoids (i.e. adults retaining For example, to gain insight into the mechanism of delayed
pupal characteristics) resulting from the PPT and its deriva- insecticidal action, compounds 7.2, 7.5, 7.6, 7.8 and 7.9,
tives treatment failed to reproduce. What is more, the stage which were more active than PPT against P. rapae, should be
of P. rapae treated by PPT and its derivatives from the larvae further studied on toxicology. Moreover, as we all know that
till it reached adulthood was longer than that of control the activities of compounds are also determined by different
group. In this work, preliminary qualitative analysis showed insect pests, so other important agricultural insect pests
the relative relationship between the bioactivity and the sub- should be added in bioassay later to extend the scope of
stituting group. It was shown that hydroxy group at C-4 of study. Meanwhile, many compounds of PPT analogues with
PPT merely substituted by amino group (compound 6) can- appropriately substituted group should be synthesized for
not lead to increasing the final mortality. Compounds 7.2, screening and surveying quantitative structure-activity rela-
7.5, 7.6, 7.8 and 7.9 were more potent than PPT in the mor- tionship as to find the biorational pesticide, the latter is being

402
Vol. 50, No. 3
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