Synthesis and insecticidal activities of novel spin-labeled derivatives of camptothecin

Article abstract of DOI:10.1002/hc.20734

Continuing our search for natural product based compounds for the control of Brontispa longissima larvae, eight spin-labeled camptothecin derivatives 7a-h and the intermediate 2 were first tested for their insecticidal activities against fifth-instar larv

Full text of DOI:10.1002/hc.20734

Heteroatom Chemistry
Volume 22, Number 6, 2011
Synthesis and Insecticidal Activities of Novel
Spin-Labeled Derivatives of Camptothecin
Ying-Qian Liu,1 Wei Dai,1 Jing Tian,1 Liu Yang,2 Gang Feng,3
Xing-Wen Zhou,1 Liang Kou,1 Yong-Long Zhao,1 Wen-Qun Li,1
Lin-Hai Li,1 and Hong-Yu Li1
1School of Pharmacy, Lanzhou University, Lanzhou 730000, People’s Republic of China
2Environmental and Municipal Engineering School, Lanzhou Jiaotong University,
Lanzhou 730000, People’s Republic of China
3Environment and Plant Protection Institute, Chinese Academy of Tropical Agricultural Science,
Danzhou 571737, People’s Republic of China
Received 2 March 2011; revised 16 April 2011
INTRODUCTION
ABSTRACT: Continuing our search for natural prod-
uct based compounds for the control of Brontispa
longissima larvae, eight spin-labeled camptothecin
derivatives 7a–h and the intermediate 2 were first
tested for their insecticidal activities against fifth-
instar larvae of Brontispa longissima. Among all the
tested compounds, especially compounds 7a and 2
showed promising insecticidal activities with the cor-
rected mortality rates of 69.55% and 74.07% against
fifth-instar larvae of B. longissima, respectively. The
different insecticidal activity ranges of these com-
pounds indicated that the variation of the structures
of L-amino acids in these compounds markedly af-
fected the activity profiles of this compound class, and
some important SAR information has been revealed
Brontispa longissima (Gestro) is one of the most
serious insect pests of palm plants [1,2]. Control of
the B. longissima larvae is primarily dependent on
repeated applications of conventional insecticides
such as organochlorine, organophosphorus, carba-
mate, and formamidines insecticides. Although ef-
fective, their extensive use for decades has produced
risks in the development of insect resistance and
residues to humans and to the environment [3,4].
These problems have highlighted the need for the
development of new strategies for their selective
control.
Plants may be an alternative source of insectici-
dal agents because they constitute a rich source of
bioactive chemicals. Much effort has been focused
on plant secondary metabolites as potential sources
of commercial insect control agents or as lead com-
pounds. Especially, the discovery of new insecticidal
leads from plant sources, followed by using them as
the useful prototypes for further modification and
structure optimization, has recently been one of the
important ways for the research and development of
new insecticides [5–8].
ꢀ
C
from it. 2011 Wiley Periodicals, Inc. Heteroatom Chem
22:687–691, 2011; View this article online at wileyonlineli-
brary.com. DOI 10.1002/hc.20734
Correspondence to: Y. Q. Liu; e-mail: yqliu@lzu.edu.cn.
Contract grant sponsor: National Natural Science Foundation
of China.
Contract grant number: 30800720.
Contract grant sponsor: Post-Doctor Research Foundation.
Contract grant number: 20090450142.
Camptothecin (1), a naturally occurring indole
alkaloid, besides its use as the molecular precur-
sor for the development of potent antineoplastic
drugs and antiviral agents [9–11], exhibited the
Contract grant sponsor: Fundamental Research Funds for the
Central Universities.
Contract grant number: 860120.
2011 Wiley Periodicals, Inc.
c
ꢀ
687

688 Liu et.al.
promising insecticidal activity [12]. Meanwhile, it
has been reported that the introduction of a sta-
ble nitroxyl radical to the bioactive molecule would
usually potentiate the biochemical or pharmaco-
logical properties of the original molecule [13].
More recently, the introduction of the L-amino acids
containing a nitroxyl radical moiety into camp-
tothecin by esterifying the 20-hydroxyl group as po-
tential insecticides has been reported by our group
and some compounds showed more promising in-
secticidal activity than camptothecin [14]. During
investigation of structure–insecticidal activity re-
lationships of 1, interestingly, 5-(2’-hydroxythoxy)-
20(S)-camptothecin (DRF-1042) 2 was found to dis-
play more potent insecticidal activity than 1 and
toosendanin, a commercial insecticide derived from
Melia azedarach. These encouraging results, there-
fore, prompted us in the present work to use DRF-
1042 as a lead model for further synthesis of novel
spin-labeled camptothecin derivatives as insecticidal
agents.
2,2,5,5-tetramethyl
pyrroline-3-carbonyl)-amino
acids 6a–h. Spectral data for 6a–h are identical to
those reported by Hankovszky and coworkers [17].
Camptothecin 1 was isolated from a Chinese
medicinal plant Camptotheca acuminata, and the
conversion of the isolated available camptothecin to
DRF-1042 (2) was accomplished in suitable yields
by using the combination of sulfuric acid and
FeCl3 in the presence of HOCH2CH2OH [18]. The
desired compounds 7a–h was achieved by treat-
ing DRF-1042 with the corresponding N-(1-oxyl-3-
carbonyl-2,2,5,5-tetramethylpyrroline) amino acids
6a–h in the presence of N,Nꢁ-dicyclohexyl carbodi-
imide (DCC) and 4-dimethylaminopyridine (DMAP).
Synthesized target compounds 7a–h were charac-
terized by melting point, electron spin resonance
(ESR), IR, and HRMS spectral analyses (Scheme 2).
Bioassay
On the basis of the methodology described in
Scheme 2, with eight spin-labeled camptothecin
derivatives 7a–h and the intermediate 2 in hand,
we next examined their insecticidal effects against
the fifth-instar larvae of B. longissima in vivo at the
concentration of 0.1 mg/mL. The results are summa-
rized in Table 1.
As shown in Table 1, the corresponding cor-
rected mortality rates caused by these compounds
after 9 days were far higher than those after 3 and 6
days. For example, the corrected mortality rate of 7a
against B. longissima after 3 days was only 35.93%,
after 9 days the corresponding mortality rate was in-
creased to 69.55%, which was nearly two times of
the mortality rate after 3 days. That is, these com-
pounds, different from those conventional neuro-
toxic insecticides, such as organophosphates, carba-
mates, and pyrethroids, showed delayed insecticidal
RESULTS AND DISCUSSION
Chemistry
As shown in Scheme 1, the reactive anhydride 4 was
prepared in almost quantitative yield by addition
of ethyl chloroformate in the presence of catalytic
amount of triethylamine to 2,2,5,5-tetramethyl-
pyrroline-3-carboxylic acid-1-oxyl 3. Compound 4
was further converted into the corresponding acid
azide 5 [15,16] when dissolved in an aqueous ace-
tone solution of sodium azide within a few minutes
at 0◦C, without further purification, the reaction of
the free-radical acid azide 5 with the correspond-
ing amino acids in the presence of magnesium
oxide at room temperature afforded N-(1-oxyl-
CON3
COOCOOC2H5
COOH
Ethyl chloroformate
NaN3/H2O
N
O
NEt3
N
O
N
O
5
4
3
R
R
O
OH
6e
6c CH2CH2SCH3
CH2Ph
6a
H
N
H
Amino acids/MgO
stir, 24 h
O
6f CH2CH(CH3)2
N
O
6d CH(CH3)CH2CH3
6b CH3
CH2
6h
6g CH(CH3)2
6a–h
N
H
SCHEME 1 Synthesis of N-(1-oxyl-2,2,5,5-tetramethylpyrroline-3-carb onyl)-amino acids 6a–h.
Heteroatom Chemistry DOI 10.1002/hc

Synthesis and Insecticidal Activities of Novel Spin-Labeled Derivatives of Camptothecin 689
O
H
N
O
OH
N
R
O
O
O
O
6a–h
O
O
O
HO(CH2)2OH
FeCl3/H2SO4
N
N
N
DCC/DMAP
O
N
N
N
O
O
O
OH
O
O
OH
OH
1
7a–h
2
SCHEME 2 Synthesis of target compounds 7a–h.
activity, which coincided very well with our previous
studies [14], Thus it further showed that the delayed
insecticidal activity would be a common property of
camptothecins’ derivatives.
other factors) or by a different interaction at the site.
Hence, a systemic, predictable correlation could be
made between the nature of amino acids and insecti-
cidal activities. As can be seen, as a whole, the intro-
duction of a stable nitroxyl radical into the molecule
of 2 with L-amino acids led to potentiate their insec-
ticidal activity, which also indicated that the design
and synthesis of these compounds might be bene-
ficial for camptothecin as insecticides and further
studies would be taken to reveal the mode of insecti-
cides of these interesting compounds and to survey
quantitative structure–activity relationship as to find
the biorational pesticide.
In conclusion, eight novel spin-labeled camp-
tothecin derivatives 7a–h and the intermediate
2 have been synthesized and evaluated for their
insecticidal Activity against the fifth-instar larva
B. longissima in vivo at the concentration of 0.1
mg/mL. The bioactivity assay for these analogues
showed that some of them are either similar or bet-
ter than camptothecin itself. Especially, 7a and 2
exhibited the most potent insecticidal activity com-
pared with camptothecin. The results suggested that
the design and synthesis of these compounds may
be beneficial for the insecticidal activity of camp-
tothecin and related analogues, and this study will be
of assistance to investigators involved in the design
and preparation of biologically useful camptothecin
congeners of this class as insecticidal agents in the
future.
From the screening, all these compounds exhib-
ited less potent than the intermediate 2, whereas the
corrected mortality rates of these compounds were
either similar or better than those of the prototypical
compound camptothecin 1. Among all the test com-
pounds, compounds 7a and 2 possessed the highest
overall insecticidal potency, with the corrected mor-
tality rates of 69.55% and 74.07% against fifth-instar
larvae of B. longissima, respectively. From insecti-
cidal activity values, it emerged that the relative re-
lationship between bioactivity and substituents at
α-carbon of amino acid, for example, compound
7a containing the L-glycine group exhibited more
promising and pronounced insecticidal activity than
1; whereas compounds 7d and 7h bearing the
L-leucine and L-proline groups would be reduced as
compared to 1. The results also clearly underlined
the insecticidal difference that could be ascribed to
a combination of factors, such as the nature of the
substitutes (which may depend on the size of sub-
stitutes, electronic characteristics of substitutes, and
TABLE 1 Insecticidal Activity of Compounds 7a–h against
Fifth-Instar Larvae of B. Longissima in vivo at the Concentra-
tion of 0.1 mg/mL
Corrected Mortality Rate (%)
Entry
3 Days
6 Days
9 Days
EXPERIMENTAL
General
7a
7b
7c
7d
7e
7f
35.93 7.56
13.70 5.48
17.41 6.51
6.67 5.77
19.01 7.46
14.07 12.24
14.07 7.06
13.33 11.55
20.74 1.28
25.93 6.42
37.57 7.25
31.85 8.98
17.78 5.88
7.41 6.42
31.40 6.29
14.07 5.13
21.11 9.49
24.81 4.49
42.96 2.57
27.16 5.66
69.55 12.18
48.15 6.42
37.04 6.42
25.93 6.42
44.44 6.17
29.63 6.42
22.22 11.11
44.44 11.11
74.07 6.42
51.85 14.24
Melting points were taken on a Kofler melt-
ing point apparatus and were uncorrected. Mass
spectra were recorded on a ZAB-HS and Bruker
Daltonics APEXII49e instrument, and the infrared
spectra were recorded on a NIC-5DX spectropho-
tometer. The ESR spectra were obtained with a
Bruker A300 X-band EPR spectrometer. IR spectra
7g
7h
2
1
Heteroatom Chemistry DOI 10.1002/hc

690 Liu et.al.
were measured on a Nicolet 5DX-FT-IR spectrom-
eter on neat samples placed between KBr plates.
The synthetic compounds were purified by flash
chromatography on Merck silica gel (70–230 mesh).
Thin-layer chromatography (TLC) involved the use
of silica gel plates with a fluorescent indicator
(Merck Silica Gel 60 F254 0.25 mm thick). N-(1-Oxyl-
2, 2, 5, 5-tetramethyl pyrroline-3-carbonyl)-amino
acids 6a–h were synthesized by employing previous
procedures [15,16]. The intermediate 3 was prepared
from 1 by a modified previous procedure [15].
(ArH), 1664 (C O), 1113, 1153, 1234 (C O), 1361
(NO•); MS m/z : 722 [M + H]+; HRMS: m/z calcd for
C40H41N4O9: 722.2944 [M + H]+, Found: 722.2946
[M + H]+; ESR: g0 = 2.0055, AN = 14.62Gs (triplet
peak in 1 × 10−4 M, DMF).
Compound 7f: yield: 68%; mp 117–119◦C; IR
(KBr) υ (cm−1): 3418 (NH), 1747 (NHCO), 3065,
1621, 1562 (ArH), 1664 (C O), 1114, 1157, 1227
(C O), 1359 (NO•); MS m/z: 688 [M + H]+; HRMS:
m/z calcd for C37H43N4O9: 688.3110 [M + H]+,
Found: 688.3103 [M + H]+; ESR: g0 = 2.0055,
AN = 14.62Gs (triplet peak in 1 × 10−4 M, DMF).
Compound 7g: yield: 76%; mp 120–122◦C; IR
(KBr) υ (cm−1): 3416 (NH), 1750 (NHCO), 3092,
1620, 1538 (ArH), 1665 (C O), 1106, 1157, 1231
(C O), 1359 (NO•); MS m/z: 674 [M + H]+; HRMS:
m/z calcd for C36H41N4O9: 674.2956 [M + H]+,
Found: 674.2946 [M + H]+; ESR: g0 = 2.0055,
AN = 14.62Gs (triplet peak in 1 × 10−4 M, DMF).
Compound 7h: yield: 54%; mp 127–129◦C; IR
(KBr) υ (cm−1): 3413 (NH), 1745 (NHCO), 3061,
1619, 1522 (ArH), 1663 (C O), 1086, 1158, 1229
(C O), 1356 (NO•); MS m/z: 761 [M + 1]+; HRMS:
m/z calcd for C42H42N5O9: 761.3040 [M + H]+,
Found: 761.3055 [M + H]+; ESR: g0 = 2.0055,
AN = 14.62Gs (triplet peak in 1 × 10−4 M, DMF).
General Procedure of Synthesis of 7a–h
A mixture of the corresponding N-(1-oxyl-2,2,5,5-
tetramethyl pyrroline-3-carbonyl)-amino acids 6a–
h (0.5 mmol), compound 2 (0.5 mmol), and DMAP
(20 mg) was stirred in dry CH2Cl2 (10 mL) for
5 min at room temperature under nitrogen. N,N-
Dicyclohexylcarbodiimide (DCC, 106 mg, 0.5 mmol)
was added, and the mixture was stirred for 4 h. The
reaction mixture was filtered, and the filtrate was
evaporated. The residue was separated by column
chromatography on silica gel with CH2Cl2–acetone
to afford compounds7a–h.
Compound 7a: yield: 90%; mp 115–117◦C; IR
(KBr) υ (cm−1): 3346 (NH), 1715 (NHCO), 3062,
1626, 1490 (ArH), 1662 (C O), 1242, 1155,1090
.
(C O), 1364(NO ); MS m/z: 632 [M + H]+; HRMS:
Biological Assay
m/z calcd for C33H35N4O9: 632.2481 [M + H]+,
Found: 632.2477 [M + H]+; ESR: g0 = 2.0055,
AN = 14.62Gs (triplet peak in 1 × 10−4 M, DMF).
Compound 7b: yield: 86%; mp 120–122◦C; IR
(KBr) υ (cm−1): 3429 (NH), 1743 (NHCO), 3056,
1624, 1552 (ArH), 1662 (C O), 1087, 1156, 1231
(C O), 1362 (NO•); MS m/z : 646 [M + H]+; HRMS:
m/z calcd for C34H37N4O9: 646.2625 [M + H]+,
Found: 646.2633[M + H]+; ESR: g0 = 2.0058, AN =
14.62Gs (triplet peak in 1× 10−4 M, DMF).
The insecticidal activity of compounds 7a–h against
the fifth-instar larvae of B. longissima was assessed
by the leaf-dipping method as described previously
[19]. For each compound, 30 larvae (10 larvae per
group) were used. Acetone solutions of 7a–h, camp-
tothecin (used as a positive control), were prepared
at the concentration of 0.1 mg/mL. Fresh coconut
leaves were dipped into the corresponding solu-
tion for 3 s, then taken out, and dried in a room.
Leaves treated with acetone alone were used as a
control group. Several treated leaves were kept in
each dish, where every 10 larvae were raised. If the
treated leaves were consumed, corresponding ones
were added to the dish. After 48 h, untreated fresh
leaves were added to all dishes until the adult emer-
gence. The experiment was carried out at 25 2◦C
and relative humidity (RH) 65%–80% on a 12 h/12 h
(light/dark) photoperiod. The insecticidal activity of
the tested compounds against the fifth-instar larvae
of B. longissima was calculated by the formula
Compound 7c: yield: 82%; mp 155–157◦C; IR
(KBr) υ (cm−1): 3423 (NH), 1711 (NHCO), 3060,
1625, 1563 (ArH), 1663 (C O), 1088, 1153, 1236
(C O), 1362 (NO•); MS m/z: 706 [M + H]+; HRMS:
m/z calcd for C36H41N4O9S: 706.2656 [M + H]+.
Found: 706.2267 [M + H]+; ESR: g0 = 2.0055,
AN = 14.62Gs (triplet peak in 1× 10−4 M, DMF).
Compound 7d: yield: 75%; mp 160–162◦C; IR
(KBr) υ (cm−1): 3423 (NH), 1746 (NHCO), 3091,
1621, 1562 (ArH), 1664 (C O), 1114, 1155, 1228
.
(C O), 1359 (NO ); MS m/z : 688 [M + H]+;
Corrected mortality rate (%)
HRMS: m/z calcd for C37H43N4O9: 688.3098 [M +
H]+; Found: 688.3103 [M + H]+; ESR: g0 = 2.0055,
AN = 14.62Gs (triplet peak in 1 × 10−4 M, DMF).
Compound7e: yield: 87%; 142–144◦C; IR (KBr)
υ (cm−1): 3425 (NH),1744 (NHCO), 3093, 1623, 1564
= (T − C) × 100/(1 − C)
where T is the mortality rate in the treated group
expressed as a percentage and C is the mortality rate
Heteroatom Chemistry DOI 10.1002/hc

Synthesis and Insecticidal Activities of Novel Spin-Labeled Derivatives of Camptothecin 691
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and the results are presented in Table 1.
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