Structural requirement and stereospecificity of tetrahydroquinolines as potent ecdysone agonists

Article abstract of DOI:10.1016/j.bmcl.2014.02.043

Tetrahydroquinoline (THQ)-type compounds are a class of potential larvicides against mosquitoes. The structure-activity relationships (SAR) of these compounds were previously investigated (Smith et al., Bioorg. Med. Chem. Lett. 2003, 13, 1943-1946), and o

Full text of DOI:10.1016/j.bmcl.2014.02.043

Bioorganic & Medicinal Chemistry Letters 24 (2014) 1715–1718
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Structural requirement and stereospecificity of tetrahydroquinolines
as potent ecdysone agonists
Seiya Kitamura a, Toshiyuki Harada a, Hajime Hiramatsu b, Ryo Shimizu b, Hisashi Miyagawa a,
Yoshiaki Nakagawa a,
⇑
a Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan
b Mitsubishi Tanabe Pharma Corporation, Kashima 3-16-89, Yodogawa, Osaka 532-8505, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Tetrahydroquinoline (THQ)-type compounds are a class of potential larvicides against mosquitoes. The
structure–activity relationships (SAR) of these compounds were previously investigated (Smith et al.,
Bioorg. Med. Chem. Lett. 2003, 13, 1943–1946), and one of cis-forms (with respect to the configurations
of 2-methyl and 4-anilino substitutions on the THQ basic structure) was stereoselectively synthesized.
However, the absolute configurations of C2 and C4 were not determined. In this study, four THQ-type
compounds with cis configurations were synthesized, and two were submitted for X-ray crystal structure
analysis. This analysis demonstrated that two enantiomers are packed into the crystal form. We synthe-
sized the cis-form of the fluorinated THQ compound, according to the published method, and the
enantiomers were separated via chiral HPLC. The absolute configurations of the enantiomers were deter-
mined by X-ray crystallography. Each of the enantiomers was tested for activity against mosquito larvae
in vivo and competitive binding to the ecdysone receptor in vitro. Compared to the (2S,4R) enantiomer,
the (2R,4S) enantiomer showed 55 times higher activity in the mosquito larvicidal assay, and 36 times
higher activity in the competitive receptor binding assay.
Received 25 November 2013
Revised 17 February 2014
Accepted 18 February 2014
Available online 27 February 2014
Keywords:
Tetrahydroquinoline
Ecdysone
Stereospecificity
Mosquito
Larvicide
Ó 2014 Elsevier Ltd. All rights reserved.
Two major classes of peripheral insect hormones, juvenile hor-
mones (JHs) and molting hormones regulate insect growth. The
principal molting hormone of insects is 20-hydroxyecdysone
(20E).1 20E and its agonist bind to ecdysone receptors (EcRs) in col-
laboration with the heterodimeric partner, ultraspiracle (USP), and
transactivate molting-related genes.2 In a few arthropods, other
steroid compounds such as ponasterone A (PonA), makisterone A,
and ecdysone act as molting hormones.3 To date, the primary se-
quences of EcRs and USPs have been identified in various insects.3,4
Three-dimensional structures of the ligand binding domains of
EcRs with ponasterone A as the ligand molecule were solved by
X-ray crystal analysis in three insects: Heliothis virescens,5 Bemicia
tabacii,6 and Tolibolium castaneum.7 The ligand-binding character-
istics of 20E, which are similar to that of PonA, were also solved
in H. virescens.8 The binding sites of non-steroidal ecdysone ago-
nists for diacylhydrazine (DAH; BYI06830)5 and an imidazole type
compound (BYI08346; PDB 3IXP),9 were also solved by X-ray anal-
ysis. However, these structures differ from those of PonA and 20E.
Compounds that regulate insect molting and metamorphosis
are known as insect growth regulators (IGRs) or more recently as
insect growth disruptors (IGDs), and some of these compounds
are used as insecticides in the agricultural field.10 IGDs can be clas-
sified into three major categories, juvenile hormone agonists, chi-
tin synthesis inhibitors and molting hormone agonists. Among
molting hormone agonists, five DAH-type compounds (tebufenoz-
ide, methoxyfenozide, chlomafenozide, fufenozide, and halofenoz-
ide) are used currently. Most of DAH-type compounds are
selectively toxic to Lepidoptera, except for halofenozide, which is
registered to control both Lepidoptera and Coleoptera. However,
the binding affinity of halofenozide to coleopteran receptors is
not stronger than that of other lepidopteran-specific DAHs.11
The high degree of specificity of DAHs against Lepidoptera trig-
gered a search for new ecdysone agonists, in both random and ra-
tional manners. Although novel non-steroidal ecdysone agonists
have been reported, none of these has been used commercially.12
Among new ecdysone agonists, tetrahydroquinoline (THQ)-type
compounds are reported to be dipteran-specific,13 particularly to
the mosquito EcR.14 These compounds have a unique specificity to-
ward mosquitos, which may offer a more selective and environ-
mentally friendly pest-management option. Therefore, THQs are
promising leads to develop into larvicides for mosquito control.
⇑
Corresponding author. Tel.: +81 75 753 6117; fax: +81 75 753 6128.
E-mail address: naka@kais.kyoto-u.ac.jp (Y. Nakagawa).
http://dx.doi.org/10.1016/j.bmcl.2014.02.043
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

1716
S. Kitamura et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1715–1718
Table 1
A previous structure–activity relationship (SAR) study of THQs fo-
cused on the optimization of the substituents X and Y, and showed
that a fluorinated THQ (X = F, Y = 4-Cl: THQ in Fig. 1) had the high-
est ecdysone agonistic activity for the Aedes aegypti EcR among the
35 compounds tested.13 We initially synthesized a few THQ ana-
logs according to the published method,13,15 and compared the
binding and insecticidal activity of the analogs with the reported
activities as shown in Table 1. We also reported that compounds
lacking the benzene ring of the quinoline structure and the aniline
moiety, as well as the trans isomer of the 2,4-positions of quinoline
moiety were inactive.15
X-ray crystallographic data for enantiomers 4a and 4b
Retention time (HPLC)
4.3 min
4a
5.8 min
4b
Empirical formula
Formula weight
Crystal system
Space group
a (Å)
C23H19BrF2N2O
457.32
C23H19BrF2N2O
457.32
Orthorhombic
P212121 (#19)
7.726 (1)
11.425 (1)
23.284 (2)
2055.4 (3)
4, 1.478
928.00
Orthorhombic
P212121 (#19)
7.7240 (2)
11.423 (3)
23.288 (5)
2054.8 (8)
4, 1.478
928.00
b (Å)
c (Å)
According to Smith and co-workers13, one of two possible cis-
stereoisomers (2R, 4S) was obtained as a final product by the Doeb-
ner-von Miller reaction. However, convincing data regarding the
stereochemistry of this compound was not reported. In the present
study, we observed that Smith’s synthesis method did not yield a
stereochemically pure intermediate. Instead, it yields a racemic
mixture via Doebner-von Miller reaction. We therefore hypothe-
sized that the THQ reaction mixture prepared by this method also
contained the (2S, 4R) cis enantiomer. In our preliminary study, we
analyzed the X-ray crystal structure to determine the absolute
configuration of the cis isomers of compounds 1 (CCDC 984528:
V (Å3)
Z, Dcalc (g/cm3)
F (000)
l
(Cu K
a
) (mmꢀ1
)
3.024
243
3506
265
3.025
243
3338
265
T (K)
No. obsd (I >2.00
No. parameters
R1, wR2
Flack parameter
GOF
r
(I))
0.040, 0.156
0.01(2)
0.043, 0.145
0.01(2)
1.300
1.031
Supplement Table S1) and
2 (CCDC 984529: Supplement
Table S1), in which the two enantiomers are packed, respectively.
Smith et al. reported the stereochemistry of THQs based on H
NMR analysis, which is difficult13,16 without using special chiral
analysis techniques such as chiral NMR solvent.
We then asked which enantiomer was biologically active. To
determine this, the cis product of compound 4 (Y = 4-Br) containing
two enantiomers [4a (2S,4R) and 4b (2R,4S)] was prepared accord-
ing to the reported procedure21, and analyzed by chiral HPLC.22
A
baseline separation of the two enantiomers was achieved with an
amylose-based chiral HPLC column,17 packed with a silica gel
bound to tris (3,5-dimethylphenylcarbamate) derivatives of amy-
lose and eluted with hexane/ethanol (90/10, v/v). The enantiomer
excess was determined by chiral HPLC and found to be >99%. Each
fraction was recrystallized from hexane–ethyl acetate and the
absolute configurations were determined by X-ray crystallography
as shown in Figure 2 with their chemical structures.23
4a
4b
Crystal data and the data collection parameters for compounds
4a (CCDC 984530) and 4b (CCDC 984531) are listed in Table 1.
The SARs for compounds 1–3 were consistent with the previ-
ously reported SARs.13 The newly synthesized compound 4 is three
times more toxic against mosquitoes than compound 3, as shown
in Table 2. The cis-(2R,4S) enantiomer (4b) showed 55 times higher
larvicidal activity against mosquito Culex pipiens pallens than the
cis-(2S,4R) enantiomer (4a).24 The concentration–response rela-
tionships for the larvicidal activity of these enantiomers are shown
in Figure 3. Consistently, the active compound 4b had approxi-
mately two times higher larvicidal activity than the racemic com-
pound 4.
Figure 2. X-ray structures of enantiomer 4a and 4b.
The EcR–THQ interaction was then investigated to understand
the specific larvicidal activity of these enantiomers in vitro. We
Figure 1. Structures of ecdysone agonists.

S. Kitamura et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1715–1718
1717
Table 2
whereas the 2R methyl group and the hydrogen do not. The aniline
moiety in position 4 is essential for the larvicidal activity as re-
ported previously15 and the 4S chiral conformation is also critical
for activity. The phenyl group on tetrahydroquinoline ring is also
required for activity, but the substituent R3 is not essential.13 The
larvicidal potency was improved approximately 3-fold by changing
R4 from 4-chloro to 4-bromo, which can be further optimized in fu-
ture studies. This SAR summary provides valuable information for
the molecular design of novel ecdysone agonists.
In vivo and in vitro biological activities of ecdysone agonistsa
Compounds
No.
Larvicidal activity
pLC50 (M)
Binding activity
pIC50 (M)
(THQ)b
X
Y
1
2
3
4
4a
4b
CH3
CH3
F
F
F
4-CH3
4-Cl
4-Cl
4-Br
4-Br
4-Br
5.33
5.38
n.d.c
5.93
n.d.
6.52
6.92d
6.65 0.13 (n = 3)
5.70 0.16 (n = 2)
7.26 0.04 (n = 2)
9.01 0.01 (n = 2)
7.12 0.03 (n = 2)
5.65 0.42 (n = 2)
7.40 0.04 (n = 2)
F
Acknowledgments
Ponasterone A
Tebufenozide
n.d.
n.d.
We thank Dr. Qing X. Li of the University of Hawaii for review-
ing the manuscript. The Culex pipiens eggs were kindly provided by
Sumitomo Chemical Co. Aedes albopictus cells (NIAS-AeAl-2) were
obtained from the GeneBank of National Institute of Agrobiological
Sciences (NIAS). This study was supported in part by the Ministry
of Education, Culture, Sports, Science, and Technology of Japan
(No. 25450070) and the 21st century COE program for Innovative
Food and Environmental Studies Pioneered by Entomomimetic Sci-
ences. Dr. Toshiyuki Harada was a recipient of a Research Fellow-
ship of the Japan Society for the Promotion of Science for Young
Scientist.
a
b
c
Mean values with standard deviation. Number of experiments.
Basic structure is shown in Figure 1.
n.d. not determined.
d
The trans-type compound (2S,4S + 2R,4R) is inactive (<4.52).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.02.
043.
References and notes
1. Truman, J. W. Vitam. Horm. 2005, 73, 1.
2. Palli, S. R.; Hormann, R. E.; Schlattner, U.; Lezzi, M. Vitam. Horm. 2005, 73, 59.
3. Nakagawa, Y.; Henrich, V. C. FEBS J. 2009, 276, 6128.
4. Morishita, C., Minakuchi C., Yokoi T., Takimoto S., Hosoda A., Akamatsu M.,
Tamura H., Nakagawa Y. J. Pestic. Sci. in press. http://dx.doi.org/10.1584/
jpestics. D13–074.
Figure 3. Concentration–response relationships for 4a (s) and 4b (d) against the
survival of mosquito larvae.
5. Billas, I. M.; Iwema, T.; Garnier, J. M.; Mitschler, A.; Rochel, N.; Moras, D. Nature
2003, 426, 91.
6. Carmichael, J. A.; Lawrence, M. C.; Graham, L. D.; Pilling, P. A.; Epa, V. C.; Noyce,
L.; Lovrecz, G.; Winkler, D. A.; Pawlak-Skrzecz, A.; Eaton, R. E., et al J. Biol. Chem.
2005, 280, 22258.
7. Iwema, T.; Billas, I. M.; Beck, Y.; Bonneton, F.; Nierengarten, H.; Chaumot, A.;
Richards, G.; Laudet, V.; Moras, D. EMBO J. 2007, 26, 3770.
8. Browning, C.; Martin, E.; Loch, C.; Wurtz, J. M.; Moras, D.; Stote, R. H.;
Dejaegere, A. P.; Billas, I. M. J. Biol. Chem. 2007, 282, 32924.
performed a competitive binding assay using [3H]PonA as a radio-
active ligand for Aedes albopictus (AeAl) cell (NIAS-AeAl-2) bind-
ing.18,19,25 The results revealed that 4b has a binding affinity for
EcR that is 36 times more potent than 4a (Table 2). These data indi-
cate that differences in binding affinity toward EcR account for the
observed larvicidal activity.
Figure 4 shows a summary of the SARs of THQs. The methyl
group in position 2 is required to be in the 2R conformation, and
the methyl substituent can be replaced with hydrogen. Conversely,
the 2S methyl substituent leads to lower activity. This implies that
the 2S methyl group has a steric collision with the receptor,
9. HomePage
http://wwwncbinlmnihgov/Structure/mmdb/
mmdbsrvcgi?unid=3ixp [accessed on Nov 15, 2013].
10. Pener, M. P.; Dhadialla, T. S. Adv. Insect Physiol. 2012, 43, 1.
11. Ogura, T.; Minakuchi, C.; Nakagawa, Y.; Smagghe, G.; Miyagawa, H. FEBS J.
2005, 272, 4114.
12. Dinan, L.; Nakagawa, Y.; Hormann, R. E. Adv. Insect Physiol. 2012, 43, 251.
13. Smith, H. C.; Cavanaugh, C. K.; Friz, J. L.; Thompson, C. S.; Saggers, J. A.;
Michelotti, E. L.; Garcia, J.; Tice, C. M. Bioorg. Med. Chem. Lett. 2003, 13, 1943.
14. Palli, S. R.; Tice, C. M.; Margam, V. M.; Clark, A. M. Arch. Insect Biochem. Biophys.
2005, 58, 234.
R2
15. Soin, T.; Swevers, L.; Kotzia, G.; Iatrou, K.; Janssen, C. R.; Rouge, P.; Harada, T.;
Nakagawa, Y.; Smagghe, G. Pest Manag. Sci. 2010, 66, 1215.
16. Funabashi, M.; Iwakawa, M.; Yoshimura, J. Bull. Chem. Soc. Jpn. 1969, 42, 2885.
17. Enomoto, N.; Furukawa, S.; Ogasawara, Y.; Akano, H.; Kawamura, Y.; Yashima,
E.; Okamoto, Y. Anal. Chem. 1996, 68, 2798.
Essential
Essential
HN
18. Nakagawa, Y.; Minakuchi, C.; Takahashi, K.; Ueno, T. Insect Biochem. Mol. Biol.
2002, 32, 175.
4S chirality critical
19. Nakagawa, Y.; Minakuchi, C.; Ueno, T. Steroids 2000, 65, 537.
20. Sheldrick, G. M. Acta Crystallogr. A 2008, 64, 112.
R3
R1: 2R Me or H essential
21. Compounds 1–4 were synthesized according to the reported methods.13
Briefly, ice-cold acetaldehyde (5 ml, 157 mmol) was added dropwise to a
mixture of p-toluidine (9.57 g, 89.3 mmol) and benzotriazole (2.13 g,
17.9 mmol) in 90 ml of EtOH, and the mixture was stirred at room
temperature for 4 days. After evaporating the solvent, the residue was
purified by column chromatography (hexane/ethyl acetate = 4:1) to give 2,6-
N
R1
(not 2S Me)
Not required
O
R4
dimethyl-4-(4-methylphenylamino)-1,2,3,4-tetrahydroquinoline
(3.49 g,
13.1 mmol, 29%) as cis/trans mixture. 2,6-dimethyl-4-(4-
methylphenylamino)-1,2,3,4-tetrahydroquinoline in THF and anhydrous
pyridine were added dropwise to a solution of 4-methylbenzoyl chloride
a
Important to improve potency
Figure 4. Structural requirements of THQs for ecdysone agonist activity.

1718
S. Kitamura et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1715–1718
(1.14 ml, 7.39 mmol) in anhydrous THF at 0 °C. The reaction mixture was
parameters. Hydrogen atoms were placed in geometrically idealized positions
and standard riding atoms. Crystal data, data collection parameters, and results
of the analyses for compound 4a and 4b are deposited under the designations
984530 (4a) and 984531 (4b). Crystal data for compounds 1 and 2 were also
deposited under designations 984528 and 984529, respectively.
warmed up to room temperature and stirred overnight. After adding 240 ml of
THF, the solution was washed with a saturated NaHCO3 aqueous solution and
brine. The organic layer was dried over MgSO4 and concentrated. The residue
was purified by column chromatography (hexane/ethyl acetate = 4:1) to give
cis isomer compound 1 (270 mg (0.702 mmol), 19%). Mp: 185 °C. Anal. Calcd
for C26H28N2O C, 81.21; H, 7.34. Found: C, 81.32; H, 7.56. Compound 2. Mp:
183 °C. Anal. Calcd for C25H25ClN2O: C, 74.15; H, 6.22. Found C, 74.27; H, 6.36.
Compound 3 mp: 178 °C. Anal. Calcd for C23H19ClF2N2O: C, 66.91; N, 6.79; H,
4.64. Found: C, 66.80; N, 6.82; H, 4.76. Compound 4: 1H NMR (CDCl3,
400 MHz); d (ppm) 1.26 (3H, d, J = 6.4 Hz), 1.35 (1H, m), 2.81 (1H, m), 3.72 (1H,
br), 4.33 (1H, dd, J = 4.6, 12.0 Hz), 4.88 (1H, m), 6.48 (1H, br), 6.61 (2H, m), 6.68
(1H, m), 6.96 (2H, m), 7.06 (1H, m), 7.12 (2H, d, J = 6.6 Hz), 7.41 (2H, d,
J = 6.8 Hz). Mp: 187 °C. Anal. Calcd for C23H19BrF2N2O: C, 60.41; N, 6.13; H,
4.19. Found: C, 60.21; N, 6.17; H, 4.23.
24. The eggs of the mosquito Culex pipiens pallens were kindly provided by
Sumitomo Chemical Company (Hyogo, Japan), and reared to the 2nd instar in
the laboratory using a 300 cm2 (approx. 5 cm water depth) tank. Three tablets
of Ebios (Asahi Food and Health) were added under conditions of a long-day
photo period (16 h light: 8 h dark). Twenty larvae grown for two days after
hatching and were transferred to a paper cup containing 30 ml of water and a
small amount of Ebios. To this was added 10 ll of DMSO solution with varying
concentrations of the test compounds. After rearing for three additional days,
the mortality of the mosquitos was evaluated. In each experiment, DMSO was
used as a negative control, and tebufenozide treatment (33 lM) was used as a
22. Separation of enantiomers of compound 4 by chiral HPLC: The enantiomers were
separated on a chiral HPLC column containing a silica gel bound to tris(3,5-
dimethylphenylcarbamate) derivatives of amylose (ADMPC) at a 2.0 ml/min
flow rate at 40 °C using hexane/ethanol (90/10). The analytes were detected by
UV absorption at 254 nm. An ADMPC-bound silica gel column was prepared
according to the published method (Method-II in the report by Enomoto and
positive control (100% mortality). The median lethal concentration (LC50) was
evaluated from the concentration-response curves, and the reciprocal
logarithm of LC50 (pLC50) was used as the index of larvicidal activity.
25. The binding assay procedure is the same as that reported previously18,19. Insect
cells of the forest day mosquito Aedes albopictus (NIAS-AeAl-2) were obtained
from the National Institute of Agrobiological Sciences (NIAS) GeneBank (http://
www.gene.affrc.go.jp/index_en.php). AeAl cells were cultured at 25 °C in 25-
cm3 tissue culture flasks containing approximately 5 ml of the culture medium
EX-Cell 401 (SAFC Biosciences) supplemented with 10% fetal bovine serum
coworkers17). The particle size of the column material was 5
lm and was
packed in a stainless steel tube (250 mm ꢁ 4.6 mm). The retention times for
each fraction are 4.3 min (4a) and 5.8 min (4b), respectively. This HPLC
fractionation was repeated 200 times, and all fractions were combined.
Enantiomer excess (ee) was determined by the same method and found to
be > 99%. After evaporating the solvent, each compound was recrystallized
from hexane-ethyl acetate to obtain 4a and 4b crystals. Melting points of these
compounds were both 201 °C. The HRMS were 456.0647, 458.0631 (4a) and
456.0655, 458.0636 (4b), (calcd 456.0649, 458.0628). Optical rotations were
(FBS). Cell suspensions (400
(12 mm ꢁ 75 mm) containing 1
l
l
l) were added to disposable glass tubes
l of the test compound in a DMSO solution
at the bottom of the glass tube. Two microliters (ca. 60,000 dpm) of [3H] PonA
(140 Ci/mmol; American Radiolabeled Chemicals, Inc., St Louis, MO, USA)
solution diluted with 70% ethanol was then added at 1-min intervals and
incubated at 25 °C. After 30 min of incubation, 3 ml of water was added to the
tubes. The contents were immediately filtered through glass filters (GF-75, /
25 mm; ADVANTEC, Tokyo, Japan) and washed two times with 3 ml of water.
The filters were dried under an infrared lamp, and placed into LSC vials. The
amount of radioactivity collected on the filters was measured in 3 ml of
Aquasol-2 (Packard Instrument Co., Meriden, CT, USA) using an Aloka LSC-5000
counter (Aloka Co., Ltd, Tokyo, Japan). The concentration required to give 50%
inhibition of the binding of [3H]PonA (IC50, M) was determined from the
concentration-response curve, and the reciprocal logarithm of IC50 (pIC50) was
used as the index of binding activity.
½
a 2D9
ꢂ
+443 (c = 0.040, ethanol) for 4a, and ½a D29
ꢀ441 (c = 0.037, ethanol) for 4b.
ꢂ
23. The crystals were mounted on a glass fiber, and measurements were made on a
Rigaku RINT RAPID/R with graphite monochromated Cu
Ka radiation
(k = 1.5418 Å) at 243 K. All data were processed and corrected for Lorentz
and polarization effects. Intensity data within 2h 6 136.4° were measured
using an imaging plate area detector. The structure was solved by direct
method using the SHELXS-97 program.20 Positional parameters of non-H
atoms were refined by full-matrix least squares using the SHELXL-97
program20. All non-hydrogen atoms were refined with anisotropic thermal