Synthesis, biological activities, and quantitative structure-Activity relationship (QSAR) Study of novel camptothecin analogues

Article abstract of DOI:10.3390/molecules20058634

In continuation of our program aimed at the development of natural product-based pesticidal agents, three series of novel camptothecin derivatives were designed, synthesized, and evaluated for their biological activities against T. Cinnabarinus, B. brassicae, and B. xylophilus. All of the derivatives showed good-To-excellent activity against three insect species tested, with LC50 values ranging from 0.00761 to 0.35496 mmol/L. Remarkably, all of the compounds were more potent than CPT against T. Cinnabarinus, and compounds 4d and 4c displayed superior activity (LC50 0.00761 mmol/L and 0.00942 mmol/L, respectively) compared with CPT (LC50 0.19719 mmol/L) against T. Cinnabarinus. Based on the observed bioactivities, preliminary structure activity relationship (SAR) correlations were also discussed. Furthermore, a three-dimensional quantitative structure activity relationship (3D-QSAR) model using comparative molecular field analysis (CoMFA) was built. The model gave statistically significant results with the cross-validated q2 values of 0.580 and correlation coefficient r2 of 0.991 and 2 pred r of 0.993. The QSAR analysis indicated that the size of the substituents play an important in the activity of 7-modified camptothecin derivatives. These findings will pave the way for further design, structural optimization, and development of camptothecin-derived compounds as pesticidal agents.

Full text of DOI:10.3390/molecules20058634

Molecules 2015, 20, 8634-8653; doi:10.3390/molecules20058634
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis, Biological Activities, and Quantitative
Structure–Activity Relationship (QSAR) Study of Novel
Camptothecin Analogues
Dan Wu 1,2,†, Shao-Yong Zhang 1,†, Ying-Qian Liu 1,2,*, Xiao-Bing Wu 1,2, Gao-Xiang Zhu 2,
Yan Zhang 2, Wei Wei 2, Huan-Xiang Liu 2,* and An-Liang Chen 1,*
1
Local and National Joint Engineering Laboratory of Biopesticide High-Efficient Preparation
Technology, Zhejiang A&F University, Lin’an 311300, China;
E-Mails: wud2012@lzu.edu.cn (D.W.); 1zhangshaoyong@163.com (S.-Y.Z.);
wuxb13@lzu.edu.cn (X.-B.W.)
2
School of Pharmacy, Lanzhou University, Lanzhou 730000, China;
E-Mails: zhugx13@lzu.edu.cn (G.-X.Z.); Zhangyan10@lzu.edu.cn (Y.Z.);
WeiW13@lzu.edu.cn (W.W.)
†
These authors contributed equally to this work.
* Authors to whom correspondence should be addressed; E-Mails: yqliu@lzu.edu.cn (Y.-Q.L.);
hxliu@lzu.edu.cn (H.-X.L.); anlchen@126.com (A.-L.C.); Tel.: +86-931-8618795 (Y.-Q.L.);
Fax: +86-931-8915686 (Y.-Q.L.).
Academic Editor: Joshua G. Pierce
Received: 7 April 2015 / Accepted: 27 April 2015 / Published: 13 May 2015
Abstract: In continuation of our program aimed at the development of natural product-based
pesticidal agents, three series of novel camptothecin derivatives were designed, synthesized,
and evaluated for their biological activities against T. Cinnabarinus, B. brassicae, and
B. xylophilus. All of the derivatives showed good-to-excellent activity against three insect
species tested, with LC50 values ranging from 0.00761 to 0.35496 mmol/L. Remarkably, all
of the compounds were more potent than CPT against T. Cinnabarinus, and compounds 4d
and 4c displayed superior activity (LC50 0.00761 mmol/L and 0.00942 mmol/L, respectively)
compared with CPT (LC50 0.19719 mmol/L) against T. Cinnabarinus. Based on the observed
bioactivities, preliminary structure–activity relationship (SAR) correlations were also
discussed. Furthermore, a three-dimensional quantitative structure–activity relationship
(3D-QSAR) model using comparative molecular field analysis (CoMFA) was built. The

Molecules 2015, 20
8635
model gave statistically significant results with the cross-validated q2 values of 0.580 and
correlation coefficient r2 of 0.991 and rp2red of 0.993. The QSAR analysis indicated that the
size of the substituents play an important in the activity of 7-modified camptothecin
derivatives. These findings will pave the way for further design, structural optimization, and
development of camptothecin-derived compounds as pesticidal agents.
Keywords: camptothecin; synthesis; biological activity; SAR analysis; CoMFA
1. Introduction
The application of chemical pesticides in agriculture worldwide is still the most widespread method
for today’s insect pest management. However, rising resistance to available agrochemicals, combined
with their adverse side effects on the environment and human health, is driving the search for new
alternative molecules to some existing commercial products [1–3]. It is well recognized that plant
secondary metabolites result from the interaction between plants and the environment during the long
period of evolution in plants, and pesticides produced from plant secondary metabolites may result in
less or slower resistance development and lower pollution [4–6]. Intriguingly, some botanical pesticides,
such as pyrethroids, rotenones, and neonicotinoids, which emerged as attractive alternatives to traditional
chemical pesticides and had been widely commercialized, now have their own prominence [7]. Hence,
the discovery of new pesticidal compounds directly from plant secondary metabolites or by using them
as the lead compounds for further modification and structure optimization has recently been important
in the research and development of new pesticides [8,9].
Camptothecin (CPT, 1, Figure 1), a naturally occurring quinoline alkaloid, is the main secondary
metabolite isolated from the wood and bark of the Chinese tree C. acuminate. Besides its use as the lead
compound for the preparation of potent anticancer drugs, such as topotecan (2) and irinotecan
(3) [10,11], it also received much research attention for its interesting pesticidal activities [12–14]. In
ancient China, the crude extract of C. acuminate containing CPT has been used traditionally to control
insect pests for centuries, and it was reported to be a potent chemosterilant against the housefly and
cabbage caterpillar [15,16]. It also exhibited significant toxic effect against Empoasca vitis,
Nilaparvata lugens, and Chilo suppressalis, which suggested its potential application as an insecticide
in the field [17]. Additionally, a recent study showed that CPT could cause visible changes in the midgut
of the lepidopteran pests Trichoplusia ni and Spodoptera exigua, such as losing the single layer of
epithelial cells and disrupting the peritrophic membrane [18]. Investigations by Zhong et al. also
demonstrated that CPT-induced apoptosis in SL-1 cells and midgut cells of S. litura [19]. Consistent
with these results, Zhang et al. recently revealed that CPT caused Sf21 and IOZCAS-Spex-II cell
apoptosis via a mitochondrial-dependent apoptosis signal transduction pathway [20], suggesting that its
mode of action may be related to apoptosis. Moreover, pretreatment with CPT led to reduction in both
the enzymatic activity and the steady accumulation of the Topo I protein in IOZCAS-Spex-II cells
despite up-regulation of its mRNA expression in response to the treatment [21]. In connection with these
efforts, in order to find new camptothecin-derived insecticides with improved profiles and to clarify the
structure–activity relationships of camptothecin analogues, a number of CPT derivatives modified in the

Molecules 2015, 20
8636
different positions have been synthesized and their insecticidal activity evaluated against some important
insect pests by our group [22–27]; among which, some compounsd exhibited insecticidal activity equal
to or higher than that of toosendanin, a commercial botanical insecticide isolated from Melia azedarach.
Especially, some C-7 substituted CPT derivatives showed promising insecticidal activity against B.
longissima, B. xylophilus, and T. cinnabarinus [25–27], suggesting the possibility of further optimizing
CPT through rational C-7 modifications.
N
O
N
O
HO
N
O
N
O
N
N
N
N
O
O
N
O
O
O
O
O
OH
OH
OH
topotecan (2)
Camptothecin (1)
irinotecan (3)
Figure 1. Chemical structures of camptothecin (1), topotecan (2), and irinotecan (3).
These encouraging results prompted us to further extend our investigation by incorporating three
functional fragments (i.e., urea, thiourea, and acylthiourea) into C-7 substituted CPT and synthesizing
three series of novel CPT derivatives. As is well known in crop protection and bioactive chemicals,
ureas, thioureas, and acylthioureas have been reported to display a variety of biological activities, such
as insecticidal, fungicidal, herbicidal, antimicrobial, antitumor, etc. [28–33], Furthermore, the hydrazone
group is identified as an important pharmacophore that is widely used in pesticide and drug molecular
design [34–37]. With this in mind, in this paper we incorporated three functional fragments into CPT at
C-7 position through hydrazone as linker and synthesized three series of novel CPT derivatives according
to Schemes 1–3. Their lethal activities against T. Cinnabarinus, B. brassicae, and B. xylophilus were tested,
and the median lethal concentrations (LC50) were calculated accordingly. Based on the observed
bioactivities, the structure–activity relationship (SAR) of these analogs was also discussed. Furthermore,
a three-dimensional quantitative structure–activity relationship (3D-QSAR) model using comparative
molecular field analysis (CoMFA) was built to understand the deep relationship between the biological
activity and molecular structure of CPT analogues.
2. Results and Discussion
2.1. Chemistry
The synthetic routes to target camptothecin derivatives 4a–l, 5a–l and 6a–d are outlined in
Schemes 1–3. Briefly, CPT was firstly converted into 7-hydroxymethyCPT (2) through the Minisci free
radical reaction promoted by the aqueous acidic FeSO4/H2O2/CH3OH reagent system, and then refluxed
with acetic acid to give the intermediate camptothecin-7-aldehyde 3. Next, intermediate 3 was coupled
with various carbonylhydrazones in CHCl3-EtOH to afford the desired compounds 4a–l, 5a–l and 6a–
d, respectively. All synthesized target compounds were purified by column chromatography, and their
structures were characterized by m.p., IR, 1H-NMR, and elemental analysis.

Molecules 2015, 20
8637
OH
O
O
H2O2/H2SO4/FeSO4
MeOH-H2O
O
O
N
AcOH
reflux
N
N
N
N
N
O
O
O
OH
O
OH O
OH O
1
2
3
S
R1
N
R1
S
N
N
H
H
H2N
R1
4a
OCH3
4e
4i
N
N
O
H
H
N
N
10%HCl
CH3
F
4b
4c
4f
4j
4k
4l
O
4g
Cl
OH
O
OCH3
CH3
Cl
4a-l
Br 4h
4d
Scheme 1. Synthesis of 7-(N-substituted-thioureidohydrazono)-formyl-camptothecin (4a–l).
O
R1
O
H
N
R1
O
N
N
OCH3
CH3
5i
5j
H
H
5a
5e
5f
H2N
R1
O
N
N
O
N
H
H
N
F
F
N
N
5b
5c
10%HCl
O
O
Cl
Cl
5g
5k
5l
OH
O
OH O
5a-l
OCH3
Br
3
Br
5h
5d
Cl
Scheme 2. Synthesis of 7-(N-substituted-carbamidohydrazono)-formyl-camptothecin (5a–l).
S
O
O
H
N
S
O
N
N
R
H
H
H2N
O
O
N
N
R
R
O
N
H
H
N
Cl
6c
6a
6b
N
N
10%HCl
O
CH3
6d
OH O
OH
O
3
6a-d
Scheme 3. Synthesis of 7-(N-acylsubstituted-thioureidohydrazono)-formyl-camptothecin (6a–d).
2.2. Biological Activity
Based on the methodology in Schemes 1–3, three series of new camptothecin derivatives (4a–l,
5a–l and 6a–d) were obtained and examined for their lethal activity against T. Cinnabarinus,
B. xylophilus, and B. brassicae, and the median lethal concentrations (LC50) were calculated accordingly.

Molecules 2015, 20
8638
CPT was included as control and the results are summarized in Table 1. In preliminary bioassays, all of
the derivatives showed good-to-excellent activities against three insect species tested, with LC50 values
ranging from 0.00761 to 0.35496 mM. Notably, all new compounds exhibited promising acaricidal
activity against T. Cinnabarinus, with LC50 values ranging from 0.00761 to 0.16309 mM, and were more
potent than CPT (LC50: 0.19719 mM), but they displayed less potency than avermectin (0.013 μM).
Among them, compounds 4d (LC50: 0.00761 mM) and 4c (LC50: 0.00942 mM) exhibited the greatest
acaricidal activity against T. Cinnabarinus in all derivatives we prepared, suggesting that C-7 modified
camptothecin derivatives were more lethal against T. Cinnabarinus and this position may be amenable
to further synthetic modification for lethal potency. Moreover, except for compound 6a (LC50: 0.03649
mM), all other compounds showed similar or less potent nematicidal activities against B. xylophilus with
LC50 values of 0.07944–0.42748 mM compared to that of CPT (LC50: 0.06861 mM). Meanwhile, against
B. brassicae, most compounds were somewhat more active than that of CPT. In particular, 4b and 4d
displayed the best promising lethal activity, with LC50 values of 0.05939 and 0.05943 mM, respectively.
Table 1. Biological activity of compounds 4a–l, 5a–l and 6a–d against T. Cinnabarinus,
B. xylophilus, and B. brassicae (Linnaeus).
LC50 (mM)
Compd
Tetranychus cinnabarinus Bursaphelenchus xylophilus Brevicoryne brassicae
4a
4b
4c
4d
4e
4f
4g
4h
4i
0.09611
0.02384
0.00942
0.00761
0.07299
0.09457
0.13990
0.01156
0.01841
0.08835
0.06152
0.08218
0.04029
0.05929
0.08796
0.01707
0.06648
0.16309
0.04171
0.05408
0.05913
0.06063
0.08516
0.02745
0.09915
0.05079
0.03930
0.04877
0.19719
0.26150
0.16486
0.11331
0.27035
0.24100
0.24809
0.22729
0.24162
0.13670
0.28470
0.09212
0.26553
0.29812
0.07944
0.36781
0.20740
0.12162
0.42748
0.20948
0.18991
0.24922
0.10277
0.11697
0.21472
0.03649
0.32122
0.30762
0.27567
0.06861
0.20589
0.05939
0.15621
0.05943
0.35496
0.17584
0.11500
0.08253
0.20292
0.12832
0.15807
0.18599
0.11830
0.20210
0.21269
0.16198
0.10958
0.40716
0.17947
0.15555
0.12422
0.19429
0.15819
0.08071
0.11815
0.11710
0.07464
0.11019
0.14593
4j
4k
4l
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
6a
6b
6c
6d
1

Molecules 2015, 20
8639
As shown in Table 1, against T. cinnabarinus, within thiourea side chains, active compounds with a
p-bromophenyl moiety (4d, LC50: 0.00761 mM) were generally slightly more potent than corresponding
analogs with a p-chlorophenyl or p-fluorophenyl group (4c and 4j). Changing the position of the chloro
substituent from C-4 in phenyl ring to C-3 in 4c and 4k affected the potency. Lethal potency for
compounds 4a–l was slightly affected by the electronegativity or positions of substituents on the phenyl
ring, but the differences in potency were often not large. In contrast, investigation of different side chains
5a–l and 6a–d gave similar interesting results, and the order of potency was somehow changed; further
investigation is needed. When compounds 4c and 4d are compared to compounds 5c (LC50: 0.08796)
and 5d (LC50: 0.01707), we could find that one atom difference between thiourea and urea can cause a
change in their lethal activity.
In addition, the LC50 rates of the target compounds against T. Cinnabarinus formed a sharp contrast
to that of the activities against the other two insect species, but gave similar structure–activity
relationships for each species. Taken together, this study showed that three series of camptothecin
derivatives could exert significant insecticidal/acaricidal activity and changes in the substituents could
lead to remarkable changes in potency. Simultaneously, these results clearly underlined that the lethal
differences could be ascribed to a combination of factors, like the nature of the substitutes (which may
depend on the size of substitutes, electronic characteristics of substitutes, or other factors) or a different
interaction at the site.
2.3. CoMFA Analysis
The results of CoMFA analysis are listed in Tables 2 and 3. The model gave a cross-validated q2 of
0.580 with six components and non-cross-validated r2 of 0.991 with SEE of 0.041, showing that the built
model had a good conventional statistical correlation and high predictive ability. The correlation between
the predicted and experimental activities was plotted in Figure 2. To further validate the predictive ability
of the obtained model, five compounds not included in the construction of CoMFA model were used as
the test set. The predicted results of the test set were also listed in Table 3 (asterisk labeled) and shown
in Figure 2. From Figure 2, it can be seen clearly that the predicted pLC50 values of the test set
compounds are in good agreement with the experimental data, with rp2red of 0.993, indicating that the
CoMFA model could be reliably used to predict the activity of new compounds and design novel
inhibitors. The final non-cross-validated partial least squares analysis was further used to generate 3D
contour maps for CoMFA using the field type stdev*coeff. The contour maps (shown in Figure 3) can
provide a more detailed understanding of the key structural features required for the biological activity.
Figure 3a,b shows the contours of the steric and electrostatic maps, respectively. Here, the contributions
of the steric field (0.709) are twice those of the electrostatic field (0.291), indicating that the effects of the
steric field on the model are larger than the electrostatic field.
Table 2. PLS statistics of the CoMFA.
Cross-validation
No cross-validation
q2
component
r2
SEE
0.580
6
0.991
0.041

Molecules 2015, 20
8640
Table 3. Results of experimental and predicted pLC50.
Compd Act.
Pred.
Res.
Compd Act.
Pred.
4.128
4.794
4.104 −0.013
3.852 0.064
4.397 −0.059
4.287 0.020
4.225 −0.003
4.170 −0.047
4.017 −0.053
4.527 −0.034
Res.
0.072
0.026
*4a
4b
4c
4d
4e
4.017 3.936 −0.081
4.623 4.562 −0.061
5.026 5.032
5.119 5.161
4.137 4.121 −0.016
4.024 4.022 −0.002
5c
5d
5e
5f
4.056
4.768
4.117
3.788
4.380
4.267
4.228
4.217
4.070
4.561
4.004
4.294
4.406
4.312
0.006
0.042
*5g
5h
5i
4f
4g
3.854 3.885
4.937 4.920 −0.017
4.735 4.791 0.056
4.017 4.005 −0.012
0.031
4h
*4i
4j
5j
5k
5l
4k
*4l
5a
4.211 4.222
4.085 4.089
4.395 4.432
0.011
0.004
0.037
6a
6b
6c
6d
4.005
0.001
4.260 −0.034
4.393 −0.013
4.305 −0.007
*5b
4.227 4.206 −0.021
5.4
Training Set
Test Set
5.2
5.0
4.8
4.6
4.4
4.2
4.0
3.8
3.8
4.0
4.2
4.4
4.6
4.8
5.0
5.2
5.4
Experimental pLC50
Figure 2. The correlation plot of the experimental activities versus predicted activities of the
studied compounds based on the model of COMFA.
Figure 3. CoMFA StDev * Coeff contour maps based on compounds 4d. (a) Steric field:
green contours (80% contribution), yellow contours (20% contribution). (b) Electrostatic
field: blue contours (80% contribution), red contours (20% contribution).

Molecules 2015, 20
8641
In the following analysis of CoMFA contour maps, compound 4d was selected as the reference
molecule. In the contour map of the steric field, the green contours indicate regions where large
substituents are favorable for the inhibitory activity, whereas the yellow contours indicate regions where
large substituents are unfavorable for activity. In the contour map of the electrostatic field, the blue
contours indicate regions where electropositive substituents increase activity, whereas the red contours
indicate regions where electronegative substituents increase activity. As shown in Figure 4a, a large
green contour around the p-bromophenyl group of compound 4d was recognized, indicating that large
substituents in the position are favorable for the inhibitory activity. For example, the order of the
activities of 7-modified camptothecin derivatives containing thiourea moieties is 4d > 4c > 4j. In a series
of derivatives containing urea moieties, the activity of compound 5d are higher than compound 5c. In a
series of 7-modified camptothecin derivatives containing acylthiourea moieties, compound 6c is more
favorable than compound 6a. A large yellow contour is also observed around the A ring of compound
4d. The observation is in accord with the experimental determinations such as 5e < 5b < 5a.
In the contour map of the electrostatic field (Figure 3b), a large blue contour and a large red contour
appear near the sulfur atoms of compound 4d, indicating that electrostatic contributions of thiourea, urea,
and acylthiourea moieties in the position have little influence on activity. Another small blue contour is
also observed around the E ring, which indicates that the introduction of electropositive substituents was
favorable for activity. The developed QSAR model based on the above analysis was helpful in
understanding the key structural factors affecting the bioactivity of this series of compounds.
3. Experimental Section
3.1. General
All reagents and solvents were of reagent grade or purified according to standard methods before use.
Analytical thin-layer chromatography (TLC) and preparative thin-layer chromatography (PTLC) were
performed with silica gel plates using silica gel 60 GF254 (Qingdao Haiyang Chemical Co., Ltd.,
Qingdao city, China). Melting points were determined in Kofler apparatus and were uncorrected. IR
spectra were obtained on NIC-5DX spectra photometer, 1H-NMR spectra were recorded at 400 MHz on
a Bruker AM-400 spectrometer using TMS as reference (Bruker Company, Boston, MA, USA).
Elemental analyses were determined on a Vario El Gmbh elemental analyzer. The starting camptothecin
was isolated from a Chinese medicinal plant C. acuminata and was purified before being used. A key
intermediate for the synthesis of three series of target compounds was camptothecin-7-aldehyde (3),
which was prepared according to Sawada et al. [38].
3.2. General Synthetic Procedure for Target Compounds 4a–l
To a solution of 7-formyl camptothecin (0.1 mmol) in chloroform (10 mL) and methanol (10 mL), an
appropriate N-substituent-thioureidohydrazine (0.12 mmol) and 10% hydrochloric (0.5 mL) were added.
The reaction mixture was stirred for 2 h at room temperature. A large amount of precipitate was
producted. After filteration, the precipitate was washed with chloroform to give a yellow solid.
Recrystallization from N, N-dimethyl formamine gave compounds 4a–l.

Molecules 2015, 20
8642
7-(N-phenylthioureidoiminomethyl)-camptothecin (4a). Yield: 85%; mp: 235 °C (decomp.); IR(KBr) cm−1:
3444 (OH), 3324, 3287 (N-H), 1737 (γ-lactone), 1655 (C=N), 1253 (C=S); 1H-NMR (400 MHz, DMSO-d6)
δ: 0.86–0.90 (m, 3H, 19-H), 1.82–1.93 (m, 2H, 18-H), 5.44 (s, 2H, 5-H), 5.66–5.78 (m, 2H, 17-H), 6.54
(s, 1H, 20-OH), 7.22–7.26 (m, 1H, 4'-H), 7.36 (s, 1H, 14-H), 7.40–7.44 (m, 2H, 2', 6'-H), 7.82 (t, 1H,
11-H), 7.85–7.89 (m, 2H, 3', 5'-H), 7.92 (t, 1H, 10-H), 8.24 (d, J = 8.0 Hz, 1H, 12-H), 8.33 (d, J = 8.0 Hz,
1H, 9-H), 9.19 (s, 1H, 7-CH), 10.01 (s, 1H, CSNHC), 12.32 (s, 1H, NNH). Anal. calc. for C28H23N5O4S:
C 63.99%, H 4.41%, N 13.33%. Found: C 63.98%, H 4.42%, N 13.34%.
7-(N-benzylthioureidoiminomethyl)-camptothecin (4b). Yield: 89%; mp: 243 °C (decomp.); IR(KBr) cm−1:
3436 (OH), 3387, 3266 (N-H), 1747 (γ-lactone), 1659 (C=N), 1258 (C=S); 1H-NMR (400 MHz, DMSO-d6)
δ: 0.86–0.89 (m, 3H, 19-H), 1.84–1.91 (m, 2H, 18-H), 4.89 (d, J = 5.6 Hz, 2H, benzyl-CH2), 5.45 (s, 2H,
5-H), 5.57–5.73 (m, 2H, 17-H), 6.53 (s, 1H, 20-OH), 7.23–7.27 (m, 1H, 4'-H), 7.35 (s, 1H, 14-H), 7.38
(d, J = 7.2 Hz, 2H, 2', 6'-H), 7.45 (d, J = 7.2 Hz, 2H, 3', 5'-H), 7.79 (t, 1H, 11-H), 7.90 (t, 1H, 10-H),
8.21 (d, J = 8.4 Hz, 1H, 12-H), 8.32 (d, J = 8.4 Hz, 1H, 9-H), 9.07 (s, 1H, 7-CH), 9.08 (s, 1H, CSNHC),
12.05 (s, 1H, NNH). Anal. calc. for C29H25N5O4S: C 64.55%, H 4.67%, N 12.98%. Found: C 64.56%,
H 4.66%, N 12.98%.
7-[N-(4-chlorinephenyl)thioureidoiminomethyl]-camptothecin (4c). Yield: 76%; mp: 265 °C (decomp.);
IR(KBr) cm−1: 3437 (OH), 3321, 3273 (N-H), 1730 (γ-lactone), 1651(C=N), 1249(C=S); 1H-NMR (400
MHz, DMSO-d6): δ 0.86–0.89 (m, 3H, 19-H), 1.82–1.93 (m, 2H, 18-H), 5.44 (s, 2H,
5-H), 5.69–5.81 (m, 2H, 17-H), 6.55 (s, 1H, 20-OH), 7.37 (s, 1H, 14-H), 7.47 (d, J = 8.8Hz, 2H, 2',
6'-H), 7.83–7.87 (m, 1H, 11-H), 7.91 (d, J = 8.8Hz, 2H, 3', 5'-H), 7.94–7.96 (m, 1H, 10-H), 8.26 (d,
J = 8.0 Hz, 1H, 12-H), 8.35 (d, J = 8.0 Hz, 1H, 9-H), 9.20 (s, 1H, 7-CH), 10.11 (s, 1H, CSNHC), 12.42
(s, 1H, NNH). Anal. calc. for C28H22N5O4SCl: C 60.05%, H 3.96%, N 12.51%. Found: C 60.07%, H
3.94%, N 12.51%.
7-[N-(4-bromophenyl)-thioureidoiminomethyl]-camptothecin (4d). Yield: 83%; mp: 253 °C (decomp.);
1
IR(KBr) cm−1: 3436 (OH), 3321, 3266 (N-H), 1729 (γ-lactone), 1650 (C=N), 1248 (C=S); H-NMR
(400 MHz, DMSO-d6) δ: 0.86–0.89 (m, 3H, 19-H), 1.82–1.93 (m, 2H, 18-H), 2.33 (s, 3H, 4'-CH3), 5.44
(s, 2H, 5-H), 5.70–5.81 (m, 2H, 17-H), 6.54 (s, 1H, 20-OH), 7.37 (s, 1H, 14-H), 7.60 (d, J = 8.8Hz, 2H,
2', 6'-H), 7.84 (d, J = 8.8Hz, 2H, 3', 5'-H), 7.85 (t, 1H, 11-H), 7.95 (t, 1H, 10-H), 8.26 (d, J = 8.4 Hz,
1H, 12-H), 8.35 (d, J = 8.4 Hz, 1H, 9-H), 9.20 (s, 1H, 7-CH), 10.10 (s, 1H, CSNHC), 12.43 (s, 1H, NNH).
Anal. calc. for C28H22N5O4SBr: C 55.64%, H 3.67%, N 11.59%. Found: C 55.64%, H 3.65%, N 11.61%.
7-[N-(4-methoxylphenyl)-thioureidoiminomethyl]-camptothecin (4e). Yield: 82%; mp: 234 °C
(decomp.); IR(KBr) cm−1: 3437 (OH), 3325, 3262 (N-H), 1728 (γ-lactone), 1653 (C=N), 1251 (C=S);
1H-NMR (400 MHz, DMSO-d6) δ: 0.86–0.89 (m, 3H, 19-H), 1.82–1.92 (m, 2H, 18-H), 3.78 (s, 3H,
4'-OCH3), 5.43 (s, 2H, 5-H), 5.64–5.76 (m, 2H, 17-H), 6.54 (s, 1H, 20-OH), 6.97 (d, J = 8.8Hz, 1H, 2',
6'-H), 7.36 (s, 1H, 14-H), 7.67 (d, J = 8.8Hz, 1H, 3', 5'-H), 7.82 (t, 1H, 11-H), 7.92 (t, 1H, 10-H), 8.23
(d, J = 8.4 Hz, 1H, 12-H), 8.32 (d, J = 8.4 Hz, 1H, 9-H), 9.15 (s, 1H, 7-CH), 9.91 (s, 1H, CSNHC),
12.22 (s, 1H, NNH). Anal. calc. for C29H25N5O5S: C 62.69%, H 4.54%, N 12.60%. Found: C 62.67%,
H 4.56%, N 12.60%.

Molecules 2015, 20
8643
7-[N-(4-methylphenyl)-thioureidoiminomethyl]-camptothecin (4f). Yield: 73%; mp: 246 °C (decomp.);
1
IR(KBr) cm−1: 3439 (OH), 3325, 3284 (N-H), 1731 (γ-lactone), 1650 (C=N), 1250 (C=S); H-NMR
(400 MHz, DMSO-d6) δ: 0.86–0.89 (m, 3H, 19-H), 1.82–1.93 (m, 2H, 18-H), 2.33 (s, 3H, 4'-CH3), 5.44
(s, 2H, 5-H), 5.69–5.81 (m, 2H, 17-H), 6.54 (s, 1H, 20-OH), 7.22 (d, J = 8.0Hz, 2H, 2', 6'-H), 7.37 (s,
1H, 14-H), 7.73 (d, J = 8.0Hz, 2H, 3', 5'-H), 7.85 (t, 1H, 11-H), 7.94 (t, 1H, 10-H), 8.25 (d, J = 8.4 Hz,
1H, 12-H), 8.34 (d, J = 8.4 Hz, 1H, 9-H), 9.20 (s, 1H, 7-CH), 9.97 (s, 1H, CSNHC), 12.29 (s, 1H, NNH).
Anal. calc. for C29H25N5O4S: C 64.55%, H 4.67%, N 12.98%. Found: C 64.56%, H 4.66%, N 12.98%.
7-[N-(2-methoxylphenyl)-thioureidoiminomethyl]-camptothecin (4g). Yield: 81%; mp: 272 °C
(decomp.); IR(KBr) cm−1: 3436 (OH), 3324 (N-H), 1741 (γ-lactone), 1657 (C=N), 1233 (C=S);
1H-NMR (400 MHz, DMSO-d6) δ: 0.86–0.90 (m, 3H, 19-H), 1.82–1.93 (m, 2H, 18-H), 3.88 (s, 3H,
2'-OCH3), 5.42 (s, 2H, 5-H), 5.53–5.64 (m, 2H, 17-H), 6.53 (s, 1H, 20-OH), 6.98–7.02 (m, 1H, 4'-H),
7.12–7.14 (m, 1H, 3'-H), 7.22–7.24 (m, 1H, 5'-H), 7.37 (s, 1H, 14-H), 7.85 (t, 1H, 11-H), 7.94 (t, 1H,
10-H), 8.25 (d, J = 8.4 Hz, 1H, 12-H), 8.38–8.41 (m, 2H, 9, 6' -H), 9.22 (s, 1H, 7-CH), 9.83 (s, 1H,
CSNHC), 12.35 (s, 1H, NNH). Anal. calc. for C29H25N5O5S: C 62.69%, H 4.54%, N 12.60%. Found: C
62.68%, H 4.55%, N 12.61%.
7-[N-(2-methylphenyl)-thioureidoiminomethyl]-camptothecin (4h). Yield: 77%; mp: 265 °C (decomp.);
1
IR(KBr) cm−1: 3471 (OH), 3279, 3126 (N-H), 1741 (γ-lactone), 1664 (C=N), 1257 (C=S); H-NMR
(400 MHz, DMSO-d6) δ: 0.85–0.88 (m, 3H, 19-H), 1.82–1.90 (m, 2H, 18-H), 2.38 (s, 3H, 2'-CH3), 5.41
(s, 2H, 5-H), 5.66–5.82 (m, 2H, 17-H), 6.53 (s, 1H, 20-OH), 7.22–7.42 (m, 4H, 3', 4', 5', 6'-H), 7.35 (s,
1H, 14-H), 7.83 (t, 1H, 11-H), 7.93 (t, 1H, 10-H), 8.24 (d, J = 8.4 Hz, 1H, 12-H), 8.35 (d, J = 8.4 Hz,
1H, 9-H), 9.19 (s, 1H, 7-CH), 9.82 (s, 1H, CSNHC), 12.25 (s, 1H, NNH). Anal. calc. for C29H25N5O4S:
C 64.55%, H 4.67%, N 12.98%. Found: C 64.56%, H 4.65%, N 12.99%.
7-[N-(1-naphthyl)-thioureidoiminomethyl]-camptothecin (4i). Yield: 87%; mp: 278 °C (decomp.);
1
IR(KBr) cm−1: 3436 (broad, OH, N-H), 1745 (γ-lactone), 1656 (C=N), 1227(C=S); H-NMR
(400 MHz, DMSO-d6) δ: 0.85–0.88 (m, 3H, 19-H), 1.80–1.91 (m, 2H, 18-H), 5.39 (s, 2H, 5-H),
5.74–5.90 (m, 2H, 17-H), 6.53 (s, 1H, 20-OH), 7.36 (s, 1H, 14-H), 7.55–7.66 (m, 4H, naphthyl-H), 7.85
(t, 1H, 11-H), 7.93 (t, 1H, 10-H), 7.94–8.01 (m, 3H, naphthyl-H), 8.25 (d, J = 8.4 Hz, 1H, 12-H), 8.42
(d, J = 8.4 Hz, 1H, 9-H), 9.23 (s, 1H, 7-CH), 10.37 (s, 1H, CSNHC), 12.37 (s, 1H, NNH). Anal. calc.
for C32H25N5O4S: C 66.77%, H 4.38%, N 12.17%. Found: C 66.76%, H 4.37%, N 12.19%.
7-[N-(4-fluorophenyl)-thioureidoiminomethyl]-camptothecin (4j). Yield: 74%; mp: 271 °C (decomp.);
1
IR(KBr) cm−1: 3438 (OH), 3321, 3266 (N-H), 1744 (γ-lactone), 1655 (C=N), 1236 (C=S); H-NMR
(400 MHz, DMSO-d6) δ: 0.87–0.91 (m, 3H, 19-H), 1.85–1.92 (m, 2H, 18-H), 5.45 (s, 2H, 5-H),
5.74–5.81 (m, 2H, 17-H), 6.56 (s, 1H, 20-OH), 7.25–7.29 (m, 2H, 2', 6'-H), 7.38 (s, 1H, 14-H),
7.81–7.85 (m, 2H, 3', 5'-H), 7.87 (s, 1H, 11-H), 7.95 (t, 1H, 10-H), 8.25 (d, J = 8.4 Hz, 1H, 12-H), 8.35
(d, J = 8.4 Hz, 1H, 9-H), 9.19 (s, 1H, 7-CH), 10.07 (s, 1H, CSNHC), 12.37 (s, 1H, NNH). Anal. calc.
for C28H22N5O4SF: C 61.87%, H 4.08%, N 12.88%. Found: C 61.88%, H 4.09%, N 12.86%.
7-[N-(3-chlorinephenyl)-thioureidoiminomethyl]-camptothecin (4k). Yield: 69%; mp: 269 °C
(decomp.); IR(KBr) cm−1: 3439 (OH), 3267, 3132 (N-H), 1741 (γ-lactone), 1651 (C=N), 1254 (C=S);

Molecules 2015, 20
8644
1H-NMR (400 MHz, DMSO-d6) δ: 0.87–0.91 (m, 3H, 19-H), 1.86–1.90 (m, 2H, 18-H), 5.45 (s, 2H,
5-H), 5.75–5.77 (m, 2H, 17-H), 6.56 (s, 1H, 20-OH), 7.31 (d, J = 7.6 Hz, 1H, 6'-H), 7.38 (s, 1H,
14-H), 7.46 (t, 1H, 5'-H), 7.80 (d, J = 9.6 Hz, 1H, 4'-H), 7.86 (t, 1H, 11-H), 7.95 (t, 1H, 10-H), 8.12 (s,
1H, 2'-H), 8.25 (d, J = 8.4 Hz, 1H, 12-H), 8.35 (d, J = 8.4 Hz, 1H, 9-H), 9.22 (s, 1H, 7-CH), 10.14 (s,
1H, CSNHC), 12.47 (s, 1H, NNH). Anal. calc. for C28H22N5O4SCl: C 60.05%, H 3.96%, N 12.51%.
Found: C 60.07%, H 3.93%, N 12.52%.
7-[N-cyclohexyl-thioureidoiminomethyl]-camptothecin (4l). Yield: 58%; mp: 263 °C (decomp.);
1
IR(KBr) cm−1: 3438 (OH), 3279, 3126 (N-H), 1746 (γ-lactone), 1652 (C=N), 1226 (C=S); H-NMR
(400 MHz, DMSO-d6) δ: 0.87–0.91 (m, 3H, 19-H), 1.21–1.27 (m, 1H, cyclohexane-H), 1.32–1.40 (m,
2H, cyclohexane-H), 1.47–1.55 (m, 2H, cyclohexane-H), 1.63–1.65 (m, 1H, cyclohexane-H), 1.77–1.80
(m, 2H, cyclohexane-H), 1.86–1.90 (m, 2H, 18-H), 2.01–2.03 (m, 2H, cyclohexane-H), 4.19 (s, 1H,
cyclohexane-H), 5.46 (s, 2H, 5-H), 5.53 (s, 2H, 17-H), 6.56 (s, 1H, 20-OH), 7.36 (s, 1H, 14-H), 7.83 (t,
1H, 11-H), 7.89 (d, J = 8.4 Hz, 1H, CSNHC), 7.94 (t, 1H, 10-H), 8.24 (d, J = 8.8 Hz, 1H, 12-H), 8.28
(d, J = 8.4 Hz, 1H, 9-H), 9.12 (s, 1H, 7-CH), 12.05 (s, 1H, NNH). Anal. calc. for C28H29N5O4S: C
63.26%, H 5.50%, N 13.17%. Found: C 63.25%, H 5.51%, N 13.17%.
3.3. General Synthetic Procedure for Target Compounds 5a–l
To a solution of 7-formyl camptothecin (0.1 mmol) in chloroform (10 mL) and methanol (10 mL), an
appropriate N-substituent-carbamidohydrazine (0.12 mmol) and 10% hydrochloric acid (0.5 mL) were
added. The reaction mixture was stirred for 2 h at room temperature. A large amount of precipitate was
produced. After filtration, the precipitate was washed with chloroform to give a pale yellow solid.
Recrystallization from N, N-dimethyl dimethylformamide gave compounds 5a–l.
7-(N-phenylaminocarbonylhydrazonomethyl)camptothecin (5a). Yield: 82%; mp: 232 °C (decomp.);
IR(KBr) cm−1: 3397 (broad, OH, N-H), 1751 (γ-lactone), 1689 (C=O) 1658(C=N); 1H-NMR (400 MHz,
DMSO-d6) δ: 0.87–0.91 (m, 3H, 19-H), 1.83–1.93 (m, 2H, 18-H), 5.45 (s, 2H, 5-H), 5.67 (s, 2H, 17-H),
6.53 (s, 1H, 20-OH), 7.05–7.08 (m, 1H, 4'-H), 7.33–7.35 (m, 2H, 2', 6'-H), 7.36 (s, 1H, 14-H),
7.75–7.77 (m, 2H, 3', 5'-H), 7.80 (t, 1H, 11-H), 7.90 (t, 1H, 10-H), 8.22 (d, J = 8.4 Hz, 1H, 12-H), 8.34
(d, J = 8.4 Hz, 1H, 9-H), 8.79 (s, 1H, 7-CH), 8.96 (s, 1H, CONHC), 11.37 (s, 1H, NNH). Anal. alc. for
C28H23N5O5: C 66.00%, H 4.55%, N 13.75%. Found: C 66.02%, H 4.53%, N 13.75%.
7-[N-(4-fluorophenyl)-carbonylhydrazonomethyl]-camptothecin (5b). Yield: 74%; mp: 236 °C
(decomp.); IR(KBr) cm−1: 3406 (broad, OH, N-H), 1742 (γ-lactone), 1706 (C=O), 1656 (C=N);
1H-NMR (400 MHz, DMSO-d6) δ: 0.87–0.90 (m, 3H, 19-H), 1.82–1.93 (m, 2H, 18-H), 5.46 (s, 2H,
5-H), 5.71 (s, 2H, 17-H), 6.55 (s, 1H, 20-OH), 7.17–7.21 (m, 2H, 2', 6'-H), 7.37 (s, 1H, 14-H),
7.75–7.78 (m, 2H, 3', 5'-H), 7.83 (t, 1H, 11-H), 7.93 (t, 1H, 10-H), 8.24 (d, J = 8.0 Hz, 1H, 12-H), 8.36 (d,
J = 8.0 Hz, 1H, 9-H), 8.92 (s, 1H, 7-CH), 8.98 (s, 1H, CONHC), 11.40 (s, 1H, NNH). Anal. calc. for
C28H22N5O5F: C 63.75%, H 4.20%, N 13.28%. Found: C 63.75%, H 4.21%, N 13.27%.
7-[N-(4-chlorophenyl)-carbonylhydrazonomethyl]-camptothecin (5c). Yield: 85%; mp: 225 °C
(decomp.); IR(KBr) cm−1: 3294–3401 (broad, OH, N-H), 1751 (γ-lactone), 1706 (C=O), 1668 (C=N);

Molecules 2015, 20
8645
1H-NMR (400 MHz, DMSO-d6) δ: 0.87–0.91 (m, 3H, 19-H), 1.82–1.93 (m, 2H, 18-H), 5.46 (s, 2H, 5-H),
5.65 (s, 2H, 17-H), 6.53 (s, 1H, 20-OH), 7.36 (s, 1H, 14-H), 7.39 (d, J = 8.8Hz, 2H, 2', 6'-H), 7.79 (d,
J = 8.8Hz, 2H, 3', 5'-H), 7.80 (t, 1H, 11-H), 7.91 (t, 1H, 10-H), 8.22 (d, J = 8.4 Hz, 1H, 12-H), 8.34 (d,
J = 8.4 Hz, 1H, 9-H), 8.96 (s, 1H, 7-CH), 8.97 (s, 1H, CONHC), 11.43 (s, 1H, NNH). Anal. calc. for
C28H22N5O5Cl: C 61.82%, H 4.08%, N 12.87%. Found: C 61.81%, H 4.09%, N 12.87%.
7-[N-(4-bromophenyl)-carbonylhydrazonomethyl]-camptothecin (5d). Yield: 72%; mp: 237 °C
(decomp.); IR(KBr) cm−1: 3443 (OH), 3393 (N-H), 1733 (γ-lactone), 1709 (C=O), 1650 (C=N);
1H-NMR (400 MHz, DMSO-d6) δ: 0.87–0.91 (m, 3H, 19-H), 1.83–1.93 (m, 2H, 18-H), 5.45 (s, 2H,
5-H), 5.66 (s, 2H, 17-H), 6.54 (s, 1H, 20-OH), 7.36 (s, 1H, 14-H), 7.51 (d, J = 8.8Hz, 2H, 2', 6'-H), 7.74
(d, J = 8.8Hz, 2H, 3', 5'-H), 7.80 (t, 1H, 11-H), 7.91 (t, 1H, 10-H), 8.22 (d, J = 8.4 Hz, 1H, 12-H), 8.34
(d, J = 8.4 Hz, 1H, 9-H), 8.96 (s, 1H, 7-CH), 8.97 (s, 1H, CONHC), 11.45 (s, 1H, NNH). Anal. calc. for
C28H22N5O5Br: C 57.15%, H 3.77%, N 11.90%. Found: C 57.15%, H 3.76%, N 11.91%.
7-[N-(4-methoxyphenyl)-carbonylhydrazonomethyl]-camptothecin (5e). Yield: 76%; mp: 254 °C
(decomp.); IR(KBr) cm−1: 3259–3409 (broad, OH, N-H), 1739 (γ-lactone), 1687 (C=O), 1661 (C=N);
1H-NMR (400 MHz, DMSO-d6) δ: 0.87–0.90 (m, 3H, 19-H), 1.83–1.93 (m, 2H, 18-H), 3.75(s, 3H,
4'-OCH3), 5.46 (s, 2H, 5-H), 5.67–5.70 (m, 2H, 17-H), 6.54 (s, 1H, 20-OH), 6.92 (d, J = 8.8Hz, 2H, 2',
6'-H), 7.37 (s, 1H, 14-H), 7.65 (d, J = 8.8Hz, 2H, 3', 5'-H), 7.82 (t, 1H, 11-H), 7.92 (t, 1H, 10-H), 8.23
(d, J = 8.4 Hz, 1H, 12-H), 8.35 (d, J = 8.4 Hz, 1H, 9-H), 8.70 (s, 1H, 7-CH), 8.96 (s, 1H, CONHC),
11.31 (s, 1H, NNH). Anal. calc. for C29H25N5O6: C 64.56%, H 4.67%, N 12.98%. Found: C 64.56%, H
4.68%, N 12.97%.
7-[N-(4-methylphenyl)-carbonylhydrazonomethyl]-camptothecin (5f). Yield: 87%; mp: 239 °C
(decomp.); IR(KBr) cm−1: 3502 (OH), 3401, 3211(N-H), 1749 (γ-lactone), 1688 (C=O), 1659 (C=N);
1H-NMR (400 MHz, DMSO-d6) δ: 0.87–0.91 (m, 3H, 19-H), 1.83–1.93 (m, 2H, 18-H), 2.28(s, 3H,
4'-CH3), 5.46 (s, 2H, 5-H), 5.66 (s, 2H, 17-H), 6.54 (s, 1H, 20-OH), 7.15 (d, J = 8.4Hz, 2H, 2', 6'-H),
7.36 (s, 1H, 14-H), 7.64 (d, J = 8.4Hz, 2H, 3', 5'-H), 7.79–7.83 (m, 1H, 11-H), 7.89–7.93 (m, 1H,
10-H), 8.22 (d, J = 8.0 Hz, 1H, 12-H), 8.34 (d, J = 8.0 Hz, 1H, 9-H), 8.71 (s, 1H, 7-CH), 8.96 (s, 1H,
CONHC), 11.35 (s, 1H, NNH). Anal. calc. for C29H25N5O5: C 66.53%, H 4.81%, N 13.38%. Found: C
66.52%, H 4.81%, N 13.37%.
7-[N-(2-methoxyphenyl)-carbonylhydrazonomethyl]-camptothecin (5g). Yield: 78%; mp: 242 °C
(decomp.); IR(KBr) cm−1: 3469 (OH), 3384, 3298 (N-H), 1746 (γ-lactone), 1686 (C=O), 1660 (C=N);
1H-NMR (400 MHz, DMSO-d6) δ: 0.87–0.91 (m, 3H, 19-H), 1.86–1.90 (m, 2H, 18-H), 3.91(s, 3H,
2'-OCH3), 5.44 (s, 2H, 5-H), 5.51 (s, 2H, 17-H), 6.54 (s, 1H, 20-OH), 6.93–7.09 (m, 3H, 3', 4', 5'-H),
7.37 (s, 1H, 14-H), 7.83 (t, 1H, 11-H), 7.93 (t, 1H, 10-H), 8.17(dd, J = 8.0, 1.2Hz, 6'-H), 8.25 (d,
J = 8.4 Hz, 1H, 12-H), 8.42 (d, J = 8.4 Hz, 1H, 9-H), 8.69 (s, 1H, 7-CH), 9.00 (s, 1H, CONHC), 11.47
(s, 1H, NNH). Anal. calc. for C29H25N5O6: C 64.56%, H 4.67%, N 12.98%. Found: C 64.54%, H 4.68%,
N 12.98%.
7-[N-(3-bromophenyl)-carbonylhydrazonomethyl]-camptothecin (5h). Yield: 72%; mp: 253 °C
(decomp.); IR(KBr) cm−1: 3282–3396 (broad, OH, N-H), 1746 (γ-lactone), 1699 (C=O), 1665 (C=N);

Molecules 2015, 20
8646
1H-NMR (400 MHz, DMSO-d6) δ: 0.87–0.90 (m, 3H, 19-H), 1.82–1.93 (m, 2H, 18-H), 5.46 (s, 2H,
5-H), 5.70 (s, 2H, 17-H), 6.55 (s, 1H, 20-OH), 7.11–7.33 (m, 3H, 4', 5', 6'-H), 7.36 (s, 1H, 14-H), 7.70
(d, J = 8.4Hz, 1H, 2'-H), 7.81–7.86 (m, 1H, 11-H), 7.93 (t, 1H, 10-H), 8.24 (d, J = 8.4 Hz, 1H, 12-H),
8.36 (d, J = 8.4 Hz, 1H, 9-H), 8.99 (s, 1H, 7-CH), 9.04 (s, 1H, CONHC), 11.49 (s, 1H, NNH). Anal.
calc. for C28H22N5O5Br: C 57.15%, H 3.77%, N 11.90%. Found: C 57.16%, H 3.76%, N 11.92%.
7-[N-(2-fluorophenyl)-carbonylhydrazonomethyl]-camptothecin (5i). Yield: 67%; mp: 232 °C
(decomp.); IR(KBr) cm−1: 3276–3398 (broad, OH, N-H), 1732 (γ-lactone), 1702 (C=O), 1655 (C=N);
1H-NMR (400 MHz, DMSO-d6) δ: 0.86–0.90 (m, 3H, 19-H), 1.85–1.89 (m, 2H, 18-H), 5.43 (s, 2H,
5-H), 5.54 (s, 2H, 17-H), 6.54 (s, 1H, 20-OH), 7.15–7.34 (m, 3H, 3', 4', 5'-H), 7.35 (s, 1H, 14-H),
7.80–7.84 (m, 1H, 6'-H), 7.90–7.92 (m, 1H, 11-H), 7.94–8.00 (m, 1H, 10-H), 8.23 (d, J = 8.0 Hz, 1H,
12-H), 8.43 (d, J = 8.0 Hz, 1H, 9-H), 8.68 (s, 1H, 7-CH), 8.97 (s, 1H, CONHC), 11.45 (s, 1H, NNH).
Anal. calc. for C28H22N5O5F: C 63.75%, H 4.20%, N 13.28%. Found: C 63.74%, H 4.23%, N 13.26%.
7-[N-(3-chlorophenyl)-carbonylhydrazonomethyl]-camptothecin (5j). Yield: 83%; mp: 248 °C
(decomp.); IR(KBr) cm−1: 3631 (OH), 3398, 3292 (N-H), 1751 (γ-lactone), 1691(C=O), 1659 (C=N);
1H-NMR (400 MHz, DMSO-d6) δ: 0.87–0.90 (m, 3H, 19-H), 1.82–1.93 (m, 2H, 18-H), 5.45 (s, 2H,
5-H), 5.69 (s, 2H, 17-H), 6.55 (s, 1H, 20-OH), 7.11(dd, J = 8.0, 1.2Hz, 1H, 6'-H), 7.34–7.38 (m, 1H, 5'-H),
7.37 (s, 1H, 14-H), 7.65–7.67 (m, 1H, 4'-H), 7.82 (t, 1H, 11-H), 7.89 (t, 1H, 10-H), 7.94–7.96 (m, 1H,
2'-H), 8.23 (d, J = 8.4 Hz, 1H, 12-H), 8.35 (d, J = 8.4 Hz, 1H, 9-H), 8.98 (s, 1H, 7-CH), 9.03 (s, 1H,
CONHC), 11.49 (s, 1H, NNH). Anal. calc. for C28H22N5O5Cl: C 61.82%, H 4.08%, N 12.87%. Found:
C 61.84%, H 4.07%, N 12.85%.
7-[N-(1-naphthyl)-carbonylhydrazonomethyl]-camptothecin (5k). Yield: 84%; mp: 275 °C (decomp.);
1
IR(KBr) cm−1: 3402 (broad, OH, N-H), 1741 (γ-lactone), 1694 (C=O), 1658 (C=N); H-NMR
(400 MHz, DMSO-d6) δ: 0.88–0.91 (m, 3H, 19-H), 1.84–1.98 (m, 2H, 18-H), 5.50 (s, 2H, 5-H),
5.70–5.80 (m, 2H, 17-H), 6.53 (s, 1H, 20-OH), 7.38 (s, 1H, 14-H), 7.38–7.50 (m, 2H, naphthyl-H), 7.83
(t, 1H, 11-H), 7.85–7.90 (m, 4H, naphthyl-H), 7.93 (t, 1H, 10-H), 8.24 (d, J = 8.0 Hz, 1H, 12-H), 8.36
(d, J = 8.4 Hz, 1H, 9-H), 9.01 (s, 1H, 7-CH), 9.03 (s, 1H, CONHC), 11.54 (s, 1H, NNH). Anal. calc. for
C32H25N5O5: C 68.69%, H 4.50%, N 12.52%. Found: C 68.69%, H 4.51%, N 12.54%.
7-[N-(2-chlorophenyl)-carbonylhydrazonomethyl]-camptothecin (5l). Yield: 69%; mp: 251 °C
(decomp.); IR(KBr) cm−1: 3281–3397 (broad, OH, N-H), 1748 (γ-lactone), 1689 (C=O), 1664 (C=N);
1H-NMR (400 MHz, DMSO-d6) δ: 0.88–0.92 (m, 3H, 19-H), 1.87–1.90 (m, 2H, 18-H), 5.45 (s, 2H,
5-H), 5.56 (s, 2H, 17-H), 6.55 (s, 1H, 20-OH), 7.13 (t, 1H, 4'-H), 7.37 (s, 1H, 14-H), 7.38 (t, 1H, 5'-H),
7.56 (d, J = 8.0 Hz, 1H, 3'-H), 7.83 (t, 1H, 11-H), 7.93 (t, 1H, 10-H), 8.27(dd, J = 8.0, 18.0 Hz, 6'-H),
8.32 (d, J = 8.4 Hz, 1H, 12-H), 8.40 (d, J = 8.4 Hz, 1H, 9-H), 8.73 (s, 1H, 7-CH), 9.08 (s, 1H, CONHC),
11.58 (s, 1H, NNH). Anal. calc. for C28H22N5O5Cl: C 61.82%, H 4.08%, N 12.87%. Found: C 61.83%,
H 4.07%, N 12.86%.

Molecules 2015, 20
8647
3.4. General Synthetic Procedure for Target Compounds 6a–d
To a solution of 7-formyl camptothecin (0.1 mmol) in chloroform (10 mL) and methanol (10 mL), an
appropriate N-acylsubstituent-thioureidohydrazine (0.12 mmol) and 10% hydrochloric acid (0.5 mL) were
added. The reaction mixture was stirred for 2 h at room temperature. A large amount of precipitate was
produced. After filtration, the precipitate was washed with chloroform to give a deep yellow solid.
Recrystallization from N, N-dimethyl dimethylformamide gave compounds 6a–d.
7-(N-benzoylthioureidoiminomethyl)camptothecin (6a). Yield: 91%; mp: 277 °C (decomp.); IR(KBr)
1
cm−1: 3435 (broad, OH, N-H), 1745 (γ-lactone), 1660 (C=O), 1602 (C=N), 1229 (C=S); H-NMR
(400 MHz, DMSO-d6) δ: 0.87–0.91 (m, 3H, 19-H), 1.84–1.91 (m, 2H, 18-H), 4.89 (d, J = 5.6 Hz, 2H,
benzyl-CH2), 5.44 (s, 2H, 5-H), 5.54 (s, 2H, 17-H), 6.54 (s, 1H, 20-OH), 7.36 (s, 1H, 14-H), 7.55–7.59
(m, 2H, 2', 6'-H), 7.67–7.71 (m, 1H, 4'-H), 7.83 (t, 1H, 11-H), 7.92 (t, 1H, 10-H), 8.03–8.05 (m, 2H, 3',
5'-H), 8.26 (d, J = 8.4 Hz, 1H, 12-H), 8.76 (d, J = 8.4 Hz, 1H, 9-H), 9.81 (s, 1H, 7-CH), 11.94 (s, 1H,
CSNHCO), 13.99(s, 1H, NNH). Anal. calc. cor C29H23N5O5S: C 62.92%, H 4.19%, N 12.65%. Found:
C 62.91%, H 4.18%, N 12.67%.
7-[N-(4-methylbenzoyl)-thioureidoiminomethyl]-camptothecin (6b). Yield: 84%; mp: 282 °C
(decomp.); IR(KBr) cm−1: 3437 (broad, OH, N-H), 1744 (γ-lactone), 1658 (C=O), 1598 (C=N), 1229
(C=S); 1H-NMR (400 MHz, DMSO-d6) δ: 0.88–0.92 (m, 3H, 19-H), 1.84–1.95 (m, 2H, 18-H), 2.35(s,
3H, 4'-CH3), 5.46 (s, 2H, 5-H), 5.69–5.83 (m, 2H, 17-H), 6.56 (s, 1H, 20-OH), 7.24 (d, J = 8.0Hz, 2H,
2', 6'-H), 7.39 (s, 1H, 14-H), 7.75 (d, J = 8.0Hz, 2H, 3', 5'-H), 7.87 (t, 1H, 11-H), 7.96 (t, 1H, 10-H),
8.27 (d, J = 8.4 Hz, 1H, 12-H), 8.36 (d, J = 8.4 Hz, 1H, 9-H), 9.22 (s, 1H, 7-CH), 9.99 (s, 1H, CSNHCO),
12.31 (s, 1H, NNH). Anal. calc. for C30H25N5O5S: C 63.48%, H 4.44%, N 12.34%. Found: C 63.47%,
H 4.43%, N 12.35%.
7-[N-(4chlorobenzoyl)-thioureidoiminomethyl]-camptothecin (6c). Yield: 76%; mp: 269 °C (decomp.);
IR(KBr) cm−1: 3438 (broad, OH, N-H), 1746 (γ-lactone), 1658 (C=O), 1599 (C=N), 1248 (C=S);
1H-NMR (400 MHz, DMSO-d6) δ: 0.87–0.91 (m, 3H, 19-H), 1.82–1.94 (m, 2H, 18-H), 5.51 (s, 2H,
5-H), 5.76–5.77 (m, 2H, 17-H), 6.56 (s, 1H, 20-OH), 7.38 (s, 1H, 14-H), 7.48 (d, J = 8.8Hz, 2H, 2',
6'-H), 7.84–7.88 (m, 1H, 11-H), 7.92 (d, J = 8.8Hz, 2H, 3', 5'-H), 7.95–7.97 (m, 1H, 10-H), 8.27 (d,
J = 8.0 Hz, 1H, 12-H), 8.36 (d, J = 8.0 Hz, 1H, 9-H), 9.21 (s, 1H, 7-CH), 10.12 (s, 1H, CSNHCO), 12.43
(s, 1H, NNH). Anal. calc. for C29H22N5O5SCl: C 59.23%, H 3.77%, N 11.91%. Found: C 59.25%, H
3.76%, N 11.93%.
7-[N-(2-naphthoyl)-thioureidoiminomethyl] -camptothecin (6d). Yield: 77%; mp: 275 °C (decomp.);
IR(KBr) cm−1: 3439 (broad, OH, N-H), 1747 (γ-lactone), 1658 (C=O), 1598 (C=N), 1228 (C=S);
1H-NMR (400 MHz, DMSO-d6) δ: 0.85–0.89 (m, 3H, 19-H), 1.81–1.92 (m, 2H, 18-H), 5.40 (s, 2H,
5-H), 5.75–5.91 (m, 2H, 17-H), 6.54 (s, 1H, 20-OH), 7.37 (s, 1H, 14-H), 7.56–7.67 (m, 4H,
naphthyl-H), 7.86 (t, 1H, 11-H), 7.94 (t, 1H, 10-H), 7.95–8.02 (m, 3H, naphthyl-H), 8.26 (d,
J = 8.4 Hz, 1H, 12-H), 8.43 (d, J = 8.4 Hz, 1H, 9-H), 9.24 (s, 1H, 7-CH), 10.38 (s, 1H, CSNHCO), 12.38
(s, 1H, NNH). Anal. calc. for C33H25N5O5S: C 65.66%, H 4.17%, N 11.60%. Found: C 65.67%, H 4.16%,
N 11.61%.

Molecules 2015, 20
8648
3.5. Biological Assay
All bioassays were performed on representative test organisms reared in the laboratory. The bioassay
was repeated at 25 ± 2 °C according to statistical requirements. Assessments were made on a dead/alive
basis, and mortality rates were corrected using Abbott’s formula [39]. Evaluations are based on a
percentage scale of 0~100, in which 0 = no activity and 100 = total kill. The deviation of values was
±5%. Probit analysis was used to determine lethal concentrations of 50% (LC50) by using the SPSS
program, version 13.0. For comparative purposes, CPT was tested as a reference. All bioassay results
are summarized in Tables 1–3.
3.5.1. Lethal Activity against Tetranychus cinnabarinus
The acaricidal activity of compounds 4a–l, 5a–l, 6a–d, and CPT (positive control) was evaluated
using the slide immersion method recommended by FAO [40]. Thirty adult spider mites were fixed
dorsally to a strip of double-sided tape attached to the slide by using a small brush. The slide was
immersed and shaken for 3 s in the diluted solution of the test compound. After the excess solution was
removed, the treated slides with the mites were kept at 25 ± 2 °C in a Petri dish with moist filter paper.
Mortality rates were calculated 24 h after treatment. Each treatment was replicated with triplicate
experiments and each replicate involved 30 adult mites. Control groups were tested with acetone only.
3.5.2. Lethal Activity against Brevicoryne brassicae
The insecticidal activity of compounds 4a–l, 5a–l, 6a–d, and CPT (positive control) against B. brassicae
was evaluated according to the reported procedure [41]. Thirty healthy adult aphids were dipped into the
diluted solutions of tested compound for 5 s, superfluous fluid was removed, and aphids were placed in
an air-conditioned room. Mortality rates were calculated 24 h after treatment. Each treatment was
performed in triplicate. Control groups were tested with acetone only.
3.5.3. Lethal Activity against Bursaphelenchus xylophilus
Acetone solutions of compounds 4a–l, 5a–l, 6a–d, and CPT (positive control) were first prepared at
different concentrations. Then 10 μL of the above solutions was added to the aqueous suspension (90 μL)
containing approximately 2500 living nematodes (third-instar and fourth-instar larvae of B. xylophilus)
per milliliter. The blank control group was prepared in the same way but lacked the tested compound.
Three replicates in each trial were made and kept at 25 °C for 24 h. Finally, the activities of five
concentrations of the tested compounds were monitored under a microscope by recording the death rate
of the tested nematodes. Nematodes that did not move when prodded with a needle were considered to be
dead. The LC50 values of tested compounds were calculated using the probit method [42].
3.6. CoMFA Analysis
To gain insights into the key structural factors affecting bioactivity, a 3D-QSAR model using
comparative molecular field analysis (CoMFA) was performed using the SYBYL 6.9 software
package [43]. Firstly, all compounds were sketched. Then, these compounds were minimized using the

Molecules 2015, 20
8649
Tripos force field with a distance-dependent dielectric and Powell conjugate gradient algorithm with a
convergence criterion of 0.05 kcal/mol. Partial atomic charges were calculated using the
Gasteiger–Hückel charge. Further conformational search was performed with the multisearch routine.
All the molecules were aligned using compound 4d (the most active compound) as the template. The
DATABASE ALIGNMENT method was used to align these molecules to the template with common
atoms (in total, 28 atoms) in compound 4d (Figure 4a). The resulting alignment is shown in Figure 4b.
To build and validate the CoMFA model, the dataset was split into training and test sets by considering
the distribution of activity of compounds. The training set contained 23 compounds. The test set was
composed of five compounds (labeled in Table 3).
Br
S
∗
N
∗
∗
N
N
（a）
∗
∗
H∗
H
∗
∗
∗
∗
O
∗
N
∗
∗
∗
∗
∗
N
∗
∗
∗
O
∗
∗
∗
∗
OH
∗
O
∗
∗
Figure 4. (a) The common scaffold (marked atoms with star) of camptothecin derivatives in
the alignment shown in compound 4d; (b) the alignment of camptothecin derivatives using
the low energy conformers by atom fit.
After the alignment of compounds, the molecular fields including steric (Lennard–Jones potentials)
and electrostatic (Coulomb potentials) field energies were calculated using sp3 carbon as a probe atom
with a grid step size of 2 Å. The cutoff value of 30 kcal/mol was adopted.
To build the relationship between the molecular fields and the biological activities
(Tetranychus cinnabarinus), partial least squares (PLS) regression analyses were used to develop
CoMFA models using the standard implementation in the SYBYL package. The CoMFA descriptors
were used as independent variables, and pLC50 values were used as the target variables.
Cross-validation in PLS was carried out using the leave-one-out method to obtain the optimal number
of components. The final model was constructed with the optimum number of components equal to that
yielding the highest q2. In addition, a test set of molecules with known biological activities that were not
included in the model generation was used to further validate the obtained models.
4. Conclusions
In summary, three novel series of 7-modified camptothecin derivatives containing thiourea, urea, and
acylthiourea moieties were synthesized, and their lethal activity against T. Cinnabarinus,
B. xylophilus, and B. brassicae (Linnaeus) was evaluated. Most of the target compounds possessed good-
to-excellent activities against three insect species, some of which were much better in comparison with
CPT. As we envisioned, SAR analysis indicated an important role of various C-7 modified derivatives
in the activity profiles of 1 analogs, and the size, electron density, and distribution of the substituents

Molecules 2015, 20
8650
within the different side chain are critical to the derivatives’ activity. Furthermore, 3D-QSAR model of
these derivatives is developed to understand the key structural factors affecting the inhibitory activity.
The high predictive ability of the model indicates that it can be reliably used to predict activity of the
inhibitors and design novel compounds. In addition, the QSAR analysis indicates that the size of the
substituents plays an important role in the activity of 7-modified camptothecin derivatives. This finding
indicated that the incorporation of the active fragment into the C-7 position could improve the lethal
activity of CPTs as expected and warrant further studies for designing new molecules with good
biological activity. Continuing studies to substantiate and improve activity profiles are underway in our
laboratory and will be reported in due course.
Acknowledgments
This work was financially supported by the National Natural Science Foundation of China (31371975,
30800720) and the Fundamental Research Funds for the Central Universities (lzujbky-2013-69), as well
as the Foundation of Priority forestry disciplines in Zhejiang A&F University (KF201325).
Author Contributions
Y.Q. Liu and H.X. Liu designed the research; D. Wu, X.B. Wu, S.Y. Zhang, Y. Zhang, W. Wei, and
G.X. Zhu performed the research and analyzed the data; Y.Q. Liu and H.X. Liu wrote the paper; and
A.L. Chen polished the paper. All authors read and approved the final manuscript.
Conflicts of Interest
The authors declare no conflict of interest.
References
1. Dively, G.P.; Kamel, A. Insecticide residues in pollen and nectar of a cucurbit crop and their
potential exposure to pollinators. J. Agric. Food Chem. 2012, 60, 4449–4456.
2. Hollomon, D.W. Do we have the tools to manage resistance in the future? Pest. Manag. Sci. 2012,
68, 149–154.
3. Wood, R.J.; Bishop, J.A. Insecticide resistance: populations and evolution. Genet. Conseq. Man
Made Chang. 1981, 97–127, 343–400.
4. Isman, M.B. Botanical insecticides, deterrents, and repellents in modern agriculture and an
increasingly regulated world. Annu. Rev. Entomol. 2006, 51, 45–66.
5. Petroski, R.J.; Stanley, D.W. Natural Compounds for Pest and Weed Control. J. Agric. Food Chem.
2009, 57, 8171–8179.
6. Dayan, F.E.; Cantrell, C.L.; Duke, S.O. Natural products in crop protection. Bioorg. Med. Chem.
2009, 17, 4022–4034.
7. Cantrell, C.L.; Dayan, F.E.; Duke, S.O. Natural Products as Sources for New Pesticides. J. Nat. Prod.
2012, 75, 1231–1242.
8. Pillmoor, J.B.; Wright, K.; Terry, A.S. Natural Products as a Source of Agrochemicals and Leads
for Chemical Synthesis. Pestic. Sci. 1993, 39, 131–140.

Molecules 2015, 20
8651
9. Crombie, L. Natural product chemistry and its part in the defense against insects and fungi in
agriculture. Pestic. Sci. 1999, 55, 761–774.
10. Wall, M.E.; Wani, M.C.; Cook, C.E.; Palmer, K.H. Plant antitumor agents. I. The isolation and
structure of camptothecin, a novel alkaloidal leukemia and tumor inhibitor from Camptotheca
acuminate. J. Am. Chem. Soc. 1966, 88, 3888–3890.
11. Zunino, F.; Dallavalle, S.; Laccabue, D.; Berettaa, G.; Merlini, L.; Pratesi, G. Current Status and
Perspectives in the Development of Camptothecins. Curr. Pharm. Des. 2002, 8, 2505–2520.
12. Hu, Q.; Han, B.Y.; Ma, J.Y.; Tang, D.; Yang, J. Field efficiency trials on Empoasca vitis Gothe
with extractive from the leaf of Camptotheca acuminate. China Tea 2009, 31, 32–33.
13. Tong, S.M.; Wang, P.W.; Sun, Y.Z.; Zhang, L.Q.; Ma, J.Y.; Sheng, X.Q. Insecticidal effect of
camptothecin against Nilaparvata lugens, Brevicoryne brassicae and Chilo suppressalis Walker.
Acta Agric. Zhejiangensis 2009, 21, 288–292.
14. Jiang, H.Y. Study on the Insecticidal Activity and Mechanism of Bioactive Ingredients from the
Seeds of Camptotheca acuminate Decne and Their Derivatives. Ph.D. Thesis, China Agricultural
University, Beijing, China, 20 June 2008.
15. DeMilo, A.B.; Borkovec, A.B. Camptothecin, a potent chemosterilant against the house fly.
J. Econ. Entomol. 1974, 67, 457–458.
16. Borkovec, A.B. Control and management of insect populations by chemosterilants.
Environ. Health Perspect. 1976, 14, 103–107.
17. Ma, J.Y.; Tong, S.M.; Wang, P.W.; Liao, W.L.; Liu, H.B.; Zhang, L.Q. Insecticidal Activity of
Camptothecin against Nilaparvata lugens, Brevicoryne brassicae, and Chilo suppressalis.
J. Econ. Entomol. 2010, 103, 492–496.
18. Sun, S.F.; Cheng, Z.S.; Fan, J.; Cheng, X.H.; Pang, Y. The utility of camptothecin as a synergist of
Bacillus thuringiensis var. kurstaki and nucleopolyhedroviruses against Trichoplusiani and
Spodoptera exigua. J. Econ. Entomol. 2012, 105, 1164–1170.
19. Wang, W.X.; Zhong, G.H.; Hu, M.Y.; Huang, J.F.; Ge, C.C. Induced apoptosis of camptothecin in
Spodoptera frugiperda Sf9 cells. Acta Entomol. Sin. 2011, 54, 894–901.
20. Zhang, L.; Zhang, Y.N.; He, W.Z.; Ma, D.J.; Jiang, H.Y. Effects of camptothecin and
hydroxycamptothecin on insect cell lines Sf21 and IOZCAS-Spex-II. Pest. Manag. Sci. 2012, 68,
652–657.
21. Zhang, L.; Ma, D.J.; Zhang, Y.N.; He, W.Z.; Yang, J.J.; Li, C.R.; Jiang, H.Y. Characterization of
DNA Topoisomerase-1 in Spodoptera exigua for Toxicity Evaluation of Camptothecin and
Hydoxy-Camptothecin. PLoS ONE 2013, 8, e56458.
22. Liu, Y.Q.; Yang, L.; Zhao, Y.L.; Li, H.Y. Synthesis of novel derivatives of camptothecin as
potential insecticides. Pest. Biochem. Physiol. 2010, 98, 219–223.
23. Liu, Y.Q.; Dai, W.; Tian, J.; Yang, L.; Feng, G.; Zhou, X.W.; Kou, L.; Zhao, Y.L.; Li, W.Q.;
Li, L.H.; et al. Synthesis and insecticidal activities of novel spin-labeled derivatives of camptothecin.
Heteroat. Chem. 2011, 22, 687–691.
24. Li, W.Q.; Liu, Y.Q.; Zhao, Y.L.; Zhou, X.W.; Yang, L.; Feng, G.; Kou, L. Antifeedant Activity of
Camptothecin and its Semisynthetic Derivatives Against Spodoptera litura (Fabricius)
(Lepidoptera: Noctuidae) Larvae. Curr. Bioact. Compd. 2012, 8, 291–295.

Molecules 2015, 20
8652
25. Yang, L.; Zhang, Z.J.; Liu, Y.Q.; Zhao, C.Y.; Wang, M.J.; Nan, X.; Feng, G. Evaluation of
insecticidal activity of camptothecin analogs against Brontispa longissima. Med. Chem. Res. 2014,
23, 980–986.
26. Zhang, S.Y. Structure-Insecticidal Activity Relationships of Camptothecin Derivatives.
MD Dissertation, Zhejiang A&F University, Lin’an, China, 5 June 2011.
27. Zhang, S.Y.; Chen, A.L.; Zhang, L.Q. Nematocidal activity against pine wood nematode
(Bursaphelenchus xylophilus) of 7-C-substituted 20-(S)-camptothecins. Chin. J. Pestic. Sci. 2011,
13, 127–132.
28. Sun, R.F.; Zhang, Y.L.; Chen, L.; Li, Y.Q.; Li, Q.S.; Song, H.B.; Huang, R.Q.; Bi, F.C.; Wang, Q.M.
Design, synthesis, bioactivity, and structure–activity relationship (SAR) studies of novel
benzoylphenylureas containing oxime ether group. J. Agric. Food Chem. 2008, 56, 11376–11391.
29. Zhang, J.F.; Xu, J.Y.; Wang, B.L.; Li, Y.X.; Xiong, L.X.; Li, Y.Q.; Ma, Y.; Li, Z.M. Synthesis and
Insecticidal Activities of Novel Anthranilic Diamides Containing Acylthiourea and Acylurea.
J. Agric. Food Chem. 2012, 60, 7565–7572.
30. Rao, X.P.; Wu, Y.; Song, Z.Q.; Shang, S.B.; Wang, Z.D. Synthesis and antitumor activities of
unsymmetrically disubstitutedacylthioureas fused with hydrophenanthrene structure. Med. Chem. Res.
2011, 20, 333–338.
31. Sun, C.W.; Huang, H.; Feng, M.Q.; Shi, X.L.; Zhang, X.D.; Zhou, P. A novel class of potent
influenza virus inhibitors: Polysubstitutedacylthiourea and its fused heterocycle derivatives.
Bioorg. Med. Chem. Lett. 2006, 16, 162–166.
32. Hallur, G.; Jimeno, A.; Dalrymple, S.; Zhu, T.; Jung, M.K.; Hidalgo, M.; Isaacs, J.T.; Sukumar, S.;
Hamel, E.; Khan, S.R. Benzoylphenylurea sulfur analogues with potent antitumor activity.
J. Med. Chem. 2006, 49, 2357–2360.
33. Li, W.Q.; Wang, X.L.; Qian, K.D.; Liu, Y.Q.; Wang, C.Y.; Yang, L.; Tian, J.; Morris-Natschke, S.L.;
Zhou, X.W.; Lee, K.H. Design, synthesis and potent cytotoxic activity of novel podophyllotoxin
derivatives. Bioorg. Med. Chem. 2013, 21, 2363–2369.
34. Boger, M.; Durr, D.; Gsell, L.; Hall, R.G.; Karrer, F.; Kristiansen, O. Synthesis and structure–activity
relationships of benzophenone hydrazone derivatives with insecticidal activity. Pest. Manag. Sci.
2001, 57, 191–202.
35. Hollingshaus, J.G. Inhibition of mitochondrial electron transport by hydramethylnon: A new
amidinohydrazone insecticide. Pest. Biochem. Physiol. 1987, 27, 61–70.
36. Taki, T.; Kisida, H.; Saito, S.; Isayama, S.; Salto, S. New Bicyclic Amino Alkylidene Hydrazone
Derivatives—Used as Broad Spectrum Insecticides and Acaricides Active against Resistant Strains.
E.P. Patent 567138, 19 September 1994.
37. Mio, S.; Okui, E.; Ito, M.; Ichinose, R.; Kodama, S.; Iwabuchi, A. Heteroaryl Hydrazone
Derivatives or Their Salts, Useful for Controlling Insects, Mites, Nematodes in Agriculture and
Horticulture. JP Patent 2000297086, 8 February 2000.
38. Sawada, S.; Nokata, K.; Furuta, T.; Yokokura, T.; Miyasaka, T. Chemical Modification of an
Antitumor Alkaloid Camptothecin: Synthesis and Antitumor Activity of 7-C-Substitued Camptothecins.
Chem. Pharm. Bull. 1991, 39, 2574–2580.
39. Abbott, W.S. A method of computing the effectiveness of an insecticide. J. Econ. Entomol. 1925,
18, 265–267.

Molecules 2015, 20
8653
40. Wang, Y.J.; Ou, X.M.; Pei, H.; Lin, X.M.; Yu, K. Toxicities of novel insecticide chlorfenpyr against
several insects in lab. Res. Appl. 2006, 10, 20–23.
41. Zhao, Q.Q.; Li, Y.Q.; Xiong, L.X.; Wang, Q.M. Design, synthesis and insecticidal activity of novel
phenylpyrazoles containing a 2,2,2-trichloro-1-alkoxyethyl moiety. J. Agric. Food Chem. 2010, 58,
4992–4998.
42. Kohno, T.; Togashi, K.; Fukamiya, N. The nematocidal activity and the structure–activity
relationships of stilbenes. Nat. Prod. Res. 2007, 21, 606–615.
43. Morris, G. SYBYL Software, version 6.9; Tripos Associates: St. Louis, MO, USA, 2002.
Sample Availability: All synthetic Samples are available from the authors.
© 2015 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/4.0/).