Design, synthesis, biological activities, and dynamic simulation study of novel thiourea derivatives with gibberellin activity towards Arabidopsis thaliana

Article abstract of DOI:10.1016/j.bmc.2019.06.032

Computer-aided drug design has advanced by leaps and bounds, and has been widely used in various fields, and especially in the field of drug discovery. Although the crystal structure of the gibberellin (GA) receptor GID1A had been reported in previous studies, there is still a lack of designs of gibberellin functional analogue based GID1A. In the present study, a series of 30 thiourea derivatives were designed, synthesized and biologically assayed. The results suggested that the synthetic compounds had good GA-like activities. Furthermore, the structure-activity relationship of the synthetic compounds was discussed, and the dynamic simulation and docking study revealed the binding properties of the GID1A receptor and compounds Y1, Y11, and Y21.

Full text of DOI:10.1016/j.bmc.2019.06.032

Accepted Manuscript
Design, Synthesis, Biological Activities, and Dynamic Simulation Study of
Novel Thiourea Derivatives with Gibberellin Activity towards Arabidopsis
thaliana
Zhikun Yang, Jine Wang, Hao Tian, Yan He, Hongxia Duan, Liusheng Duan,
Weiming Tan
PII:
S0968-0896(19)30402-X
https://doi.org/10.1016/j.bmc.2019.06.032
BMC 14969
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
11 March 2019
9 June 2019
19 June 2019
Please cite this article as: Yang, Z., Wang, J., Tian, H., He, Y., Duan, H., Duan, L., Tan, W., Design, Synthesis,
Biological Activities, and Dynamic Simulation Study of Novel Thiourea Derivatives with Gibberellin Activity
towards Arabidopsis thaliana, Bioorganic & Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.bmc.
2019.06.032
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1
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Design, Synthesis, Biological Activities, and Dynamic Simulation
Study of Novel Thiourea Derivatives with Gibberellin Activity
towards Arabidopsis thaliana
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4
Zhikun Yanga, 1, Jine Wanga, 1, Hao Tiana, Yan Hea, Hongxia Duanb, Liusheng Duana,
Weiming Tana*
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a College of Agronomy and Biotechnology, China Agricultural University, Beijing,
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100193, P. R. China
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b Department of Applied Chemistry, China Agricultural University, Beijing, 100193, P.
R. China
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13 Abbreviations:
14 * Corresponding author.
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E-mail address: tanwm@cau.edu.cn (W. M. Tan)
16 1 These authors contributed equally.
17
18 ABSTRACT
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Computer-aided drug design has advanced by leaps and bounds, and has been
widely used in various fields, and especially in the field of drug discovery. Although
the crystal structure of the gibberellin (GA) receptor GID1A had been reported in
previous studies, there is still a lack of designs of gibberellin functional analogue

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based GID1A. In the present study, a series of 30 thiourea derivatives were designed,
synthesized and biologically assayed. The results suggested that the synthetic
compounds had good GA-like activities. Furthermore, the structure-activity
relationship of the synthetic compounds was discussed, and the dynamic simulation
and docking study revealed the binding properties of the GID1A receptor and
compounds Y1, Y11, and Y21.
Keywords: GID1A receptor; Gibberellin activity; Thiourea derivatives;
Arabidopsis

32 1. Introduction
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Plant hormones are widespread in nature and play an important regulatory role in
growth and development as an indispensable signal substance in plants.1 Gibberellins
(GAs) are an important group of isoprenoid phytohormones that occur in minute
amounts. In 1938, the GA3 crystal was firstly extracted from the Gibberella causing
rice seedling disease. In 1958, MacMillan purified gibberellin (GA1) from immature
bean seeds, and since then, more than 130 GAs have been identified.2 Gibberellins,
found in higher plants, algae, fungi, and bacteria, are widely used as plant growth
regulators of many developmental processes, such as root and shoot elongation,
dormancy break, promotion of seed germination, leaf stretching, and flower regulation
in agricultural production, including flower preservation, and beer brewing.3-5
However, GAs are a class of tetracyclic diterpenoid hormones (Fig. 1), which have
136 different complex structures. From these structures, only a few, such as GA1, GA3,
GA4, and GA7, are bioactive, which poses a rather more formidable challenge to
organic synthesis and qualitative and quantitative analysis based on chromatographic
techniques.6,7 It can be seen that the discovery of a novel molecule with GA
bioactivities is of enormous significance to the study of GA signaling mechanisms and
practical applications.
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GA3
GA4
GA7
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pIC50=5.30
pIC50=7.30
pIC50=7.30

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AC94377
AC006
AC007
AC015
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pIC50=4.24
pIC50=4.12
pIC50=4.40
pIC50=4.47
Fig. 1. Chemical structure of gibberellic acids (GA3, GA4, GA7) and the reported N-substituted
phthalimide functional analogues.
In the past two decades, most studies were focused on GA receptors, with further
research being focused on the mechanism of GA action, which revealed the GA signal
transduction mechanism. In 2005, Ueguchi-Tanaka et al. cloned and encoded
GA-related genes in rice, and discovered that GIBBERELLIN INSENSITIVE
DWARF1 (GID1A) is a GA receptor, which was subsequently verified by Nakajim et
al., who identified and characterized Arabidopsis gibberellin receptors.8,9 In 2008,
Murase et al. and Shimada et al. reported the crystal structure of GID1A-GA-DELLA
complexes in Arabidopsis and rice, respectively.10,11 It was found that
GID1A-GA3-DELLA was formed in plants and degraded after GA3 was recognized
by the GID1A protein, which caused the gibberellin effect and promoted hypocotyl
elongation. These studies have provided a biological basis for the design of novel
target-specific GA functional analogs. Until today, only one gibberellin functional
analogue, named AC-94377, has been reported by the American Cyanamid
Corporation in 1977.12 It is a phthalimide-like substance that can break seed dormancy,
stimulate hypocotyl elongation, and be recognized by GID1A in plants to form the

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AtGID1A-AC94377-DELLA complex, inducing the degradation of DELLA protein,
similar to the action mechanism of GA3. 13,14
Nowadays, structure-based drug design based on receptor modeling has been
widely applied in the research and discovery of new drugs, benefited from the rapid
growth of computing power and the current level of theories and algorithms for
studying macromolecular systems.15,16 In addition, the screening technology plays an
increasingly important role in drug design with its highly efficiency, fast, low cost,
and high selectivity.17 For example, the discovery of Indinavir based on HIV-1
protease, and the E2020 drug development based on acetylcholinesterase have been
achieved using this technology. 18,19 However, few studies have focused on the design
of GA functional analogues with GA receptor protein GID1A as the target.
In the present study, 1-(2,3-dimethoxyphenethyl)-3-(3-(trifluoromethyl)
phenyl)thiourea, named Y1 in the MayBridge Screening Collection, was screened
using the SYBYL software based on the GID1A protein, and 30 compounds were
designed based on the isostere principle and active group splicing. Then, 30
compounds were synthesized and characterized by 1H-NMR, 13C-NMR and
high-resolution mass spectrometry (HRMS). At the same time, the biological
activities of the synthetic compounds were assayed and analyzed on Arabidopsis
thaliana, and the structure-activity relationship was calculated and discussed. Finally,
the growth situation of gibberellin-deficient dwarf mutant ga1-1, dynamical analysis,
and molecular docking were used to verify that the produced compounds have similar
function as GA3.

97 2. Experimental section
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2.1. Chemicals and seeds
All solvents were purchased from Tongguang (Beijing, China). All reagents were
purchased from J&K Chemicals, Beijing, China. The solvents were dried and purified
according to standard procedures. All commercially available reagents were used
without further purification. Column chromatography was conducted on a silica gel
plug (200–300 mesh), and the reaction progress was monitored by thin-layer
chromatography on silica gel GF-254 and detected under UV light.
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The plant Arabidopsis (A. thaliana ecotype Columbia-0, Col-0) and its
gibberellin-deficient mutant ga1-1 were provided by the State Key Lab of Plant
Physiology and Biochemistry, China Agricultural University, Beijing, China.
2.2. Instruments
Pharmacophore models were built using the GALAHAD module in the SYBYL 7.2
software, and multiple screening was performed by combining the Lipinski’s rule of
five, the pharmacophore model, and the Surflex-dock module. Dynamic simulation
1
and binding free energy decomposition were generated using AMBER14. H NMR
13
and C NMR spectra were obtained at 300 MHz using a Bruker AVANCE DPX300
spectrometer in CDCl3 or DMSO-d6 solution with tetramethylsilane as the internal
standard. HRMS were performed using an Agilent 6520 Accurate-Mass-Q-TOF
LC/MS system, equipped with an electrospray ionization (ESI) source in the positive
ionization mode. The melting points were determined on a Stuart SMP3 melting point
apparatus and were uncorrected. The Arabidopsis growth data was obtained using

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ImageJ software (https://imagej.nih.gov/ij/index.html).
2.3 Pharmacophore models and virtual screening
Three gibberellins (GA3, GA4, GA7) widely used in agricultural production and
four N-substituted phthalimide (NSP) functional analogues (Fig. 1) that have higher
than gibberellins activity and have a common target protein GID1A with gibberellin
were used as training set to generate a 3D pharmacophore model using the
GALAHAD module. Parameters including population size, max generations, mols
required to N hit, and keep best N models were set to 45, 45, 5, and 20, respectively.
In the end, twenty pharmacophore models were obtained and evaluated, and a
representative model was selected based on the enrichment factor for virtual screening.
Thiourea compounds were filtered out from the Maybridge database using the
Lipinski’s rule of five (M<450),20,21 LogS (>-4), CLogP (<5), polar surface area (<120
Å2), toxicity (irritant, tumorigenic, mutagenic, reproductive effective),
physicochemical properties, pharmacophore model, and GID1A.
2.4. General Synthesis
The synthetic routes of all compounds are given in Fig. 2. In brief, a substituted
aniline was employed to related aromatic isothiocyanate,22 and followed by reaction
with a substituted aromatic ethylamine to obtain a thiourea derivative, named Y1 to
Y30, containing two aromatic rings.23
2.4.1. The preparation of aromatic isothiocyanate
Taking the synthesis of 3-(trifluoromethyl)phenyl 1-isothiocyanate as an example,
3-(trifluoromethyl)aniline (2.38 g, 14.77mmol, 1 eqv) and dimethylamino

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thiocarbonyl chloride (1.92 g, 15.50 mmol, 1.05 eqv) were added in dry toluene (20
mL) at room temperature, and then the reaction mixture was refluxed for 5 h. After the
reaction mixture was cooled down to room temperature, the solid dimethylamine
hydrochloride was filtered off, and the collected toluene fraction was removed under
reduced pressure. Colorless oil (2.55 g, 85%) was obtained and used in the next step
without additional purification.
The other aromatic isothiocyanates were synthesized in the same way.
2.4.2. The preparation of thiourea derivatives
3-(trifluoromethyl)phenyl isothiocyanate (0.53 g, 2.60 mmol, 1 eqv) was dissolved
in dichloromethane (30 mL), 2-(2,3-dimethoxyphenyl)ethyl amine (0.47 g, 2.60 mmol,
1 eqv) was added, and then the reaction mixture was stirred for 2 h. The solvent was
evaporated off by vacuum, and subsequently the residue was recrystallized from
ethylacetate. A white solid (0.92 g, 92 %) was obtained as compound Y1. m.p. 91.4-
92.2°C. 1H NMR (300 MHz, Chloroform-d) δ 8.69 (s, 1H), 7.48 – 7.36 (m, 3H), 7.27
(d, J = 7.3 Hz, 1H), 6.76 – 6.62 (m, 3H), 6.15 (s, 1H), 3.87 (q, J = 6.2 Hz, 2H), 3.81 (s,
3H), 3.78 (s, 3H), 2.86 (t, J = 6.7 Hz, 2H). 13C NMR (75 MHz, CDCl3) δ 180.05,
148.80, 147.48, 136.93, 131.84 (q, J = 33.0 Hz), 130.35, 130.03, 127.50, 122.78 (q, J
= 7.2, 3.5 Hz), 122.49 (q, J =128.4 Hz), 120.95 (q, J = 3.8 Hz), 120.26, 55.48, 55.45,
45.86, 33.88. HRMS (ESI-): m/z: 385.1189 [M+H]+.

S
Cl
N
H2N
R1
SCN
R1
PhMe reflux
R3
NH2
CH2Cl2 rt
R2
R3
R2
S
R1
N
H
N
H
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Y1: R1=CF3, R2=OCH3, R3=OCH3
Y2: R1=CH3, R2=OCH3, R3=OCH3
Y11: R1=CF3, R2=H, R3=OCH3
Y12: R1=CH3, R2=H, R3=OCH3
Y21: R1=CF3, R2=H, R3=H
Y22: R1=CH3, R2=H, R3=H
Y23: R1=CH2CH3, R2=H, R3=H
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Y3: R1=CH2CH3, R2=OCH3, R3=OCH3 Y13: R1=CH2CH3, R2=H, R3=OCH3
Y4: R1=CH(CH3)2, R2=OCH3, R3=OCH3 Y14: R1=CH(CH3)2, R2=H, R3=OCH3 Y24: R1=CH(CH3)2, R2=H, R3=H
Y5: R1=C(CH3)3, R2=OCH3, R3=OCH3 Y15: R1=C(CH3)3, R2=H, R3=OCH3
Y25: R1=C(CH3)3, R2=H, R3=H3
Y26: R1=H, R2=OH, R3=H
Y 27: R1=F, R2=H, R3=H
Y28: R1=Cl, R2=H, R3=H
Y29: R1=Br, R2=H, R3=H
Y30: R1=I, R2=H, R3=H
Y6: R1=H, R2=OCH3, R3=OCH3
Y7: R1=F, R2=OCH3, R3=OCH3
Y8: R1=Cl, R2=OCH3, R3=OCH3
Y9: R1=Br, R2=OCH3, R3=OCH3
Y10: R1=I, R2=OCH3, R3=OCH3
Y16: R1=H, R2=H, R3=OCH3
Y17: R1=F, R2=H, R3=OCH3
Y18: R1=Cl, R2=H, R3=OCH3
Y19: R1=Br, R2=H, R3=OCH3
Y20: R1=I, R2=H, R3=OCH3
Fig. 2. The synthetic routes of compounds Y1-Y30
3-(Trifluoromethyl)phenyl isothiocyanate (0.57 mg, 2.82 mmol, 1 eqv) was
dissolved in dichloromethane (30 mL), 2-(3-methoxyphenyl)ethyl amine (0.43 mg,
2.82 mmol, 1 eqv) was added, and then the reaction mixture was stirred for 2 h. The

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solvent was evaporated off by vacuum, and subsequently the residue was
recrystallized from ethylacetate. A white solid (0.95 g, 95 %) was obtained as
compound Y11. m.p. 59.8-61.0°C. 1H NMR (300 MHz, Chloroform-d) δ 8.89 (s, 1H),
7.50 – 7.37 (m, 3H), 7.26 (d, J = 7.1 Hz, 1H), 7.21 – 7.14 (m, 1H), 6.81 – 6.64 (m,
3H), 6.16 (s, 1H), 3.87 (q, J = 6.5 Hz, 2H), 3.75 (s, 3H), 2.89 (t, J = 6.8 Hz, 2H). 13C
NMR (75 MHz, CDCl3) δ 179.99, 159.59, 139.48, 136.83, 131.84 (q, J = 32.9 Hz),
130.02 (d, J = 7.3 Hz), 129.47, 127.69, 124.89, 122.89 (q, J = 3.5 Hz), 122.52 (q, J
=127.4 Hz), 121.16 (q, J = 3.8 Hz), 114.12, 111.69, 54.76, 45.75, 34.34. HRMS (ESI-):
m/z: 355.1082 [M+H]+.
3-(Trifluoromethyl)phenyl isothiocyanate (0.63 mg, 3.08 mmol, 1 eqv) was
dissolved in dichloromethane (30 mL), 2-phenylethyl amine (0.37 mg, 3.08 mmol, 1
eqv) was added, and then the reaction mixture was stirred for 2 h. The solvent was
evaporated off by vacuum, and subsequently the residue was recrystallized from
ethylacetate. A white solid (0.94 g, 94 %) was obtained as compound Y21. m.p.
96.2-96.7°C. 1H NMR (300 MHz, Chloroform-d) δ 8.36 (s, 1H), 7.56 – 7.26 (m, 5H),
7.24 – 7.18 (m, 2H), 7.18 – 7.11 (m, 2H), 5.99 (s, 1H), 3.90 (q, J = 6.7 Hz, 2H), 2.93
(t, J = 6.7 Hz, 2H). 13C NMR (75 MHz, CDCl3) δ 180.19, 137.85, 136.56, 132.11 (q, J
= 32.8 Hz), 130.22, 128.47, 128.30, 127.80, 126.46, 124.80, 123.16 (q, J = 7.4, 3.5
Hz), 121.32 (q, J = 3.7 Hz), 45.96, 34.31. HRMS (ESI-): m/z: 325.0977 [M+H]+.
The rest target compounds were synthesized using the same methodology, and the
1
characterization data of H NMR, 13C NMR, HRMS, and melting point are shown
below. The 1H NMR and 13C NMR spectra images can be found in the appendix.

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Compound Y2, yellow liquid (0.93 g, 93 %), 1H NMR (300 MHz, Chloroform-d) δ
8.01 (s, 1H), 7.29 – 7.19 (m, 2H), 7.08 (d, J = 1.7 Hz, 1H), 6.85 – 6.60 (m, 5H), 6.05
(s, 1H), 3.88 (q, J = 6.8 Hz,, 2H), 3.83 (s, 3H), 3.79 (s, 3H), 2.84 (t, J = 6.8 Hz, 2H),
13
1.24 (s, 9H). C NMR (75 MHz, CDCl3) δ 180.10, 153.46, 148.79, 147.42, 135.34,
130.58, 129.23, 123.89, 121.98, 121.68, 120.21, 111.49, 111.08, 55.55, 55.48, 46.02,
34.42, 34.08, 30.77. HRMS (ESI-): m/z: 373.1944 [M+H]+.
Compound Y3, yellow solid (0.95g, 95 %), m.p. 78.9- 80.6°C. 1H NMR (300 MHz,
Chloroform-d) δ 7.87 (s, 1H), 7.23 (t, J = 7.8 Hz, 1H), 7.10 (d, J = 7.8 Hz, 1H), 6.91 –
6.62 (m, 5H), 6.05 (s, 1H), 3.90 (q, J = 6.8 Hz,, 2H), 3.84 (s, 3H), 3.81 (s, 3H), 2.82
(m, 3H), 1.19 (s, 3H), 1.17 (s, 3H). 13C NMR (75 MHz, CDCl3) δ 179.98, 150.98,
148.79, 147.42, 135.63, 130.63, 129.50, 124.86, 122.76, 121.91, 120.23, 111.51,
111.07, 55.54, 55.48, 45.97, 34.04, 33.53, 23.39. HRMS (ESI-): m/z: 359.1785
[M+H]+.
Compound Y4, white solid (0.94 g, 94%), m.p. 124.5-125.2°C. 1H NMR (300 MHz,
Chloroform-d) δ 7.76 (s, 1H), 7.23 (t, J = 7.7 Hz, 1H), 7.08 (d, J = 8.0 Hz, 1H), 6.87 –
6.63 (m, 5H), 6.04 (s, 1H), 3.92 – 3.86 (m, 2H), 3.85 (s, 3H), 3.81 (s, 3H), 2.86 (t, J =
13
6.8 Hz, 2H), 2.57 (q, J = 7.6 Hz, 2H), 1.17 (t, J = 7.6 Hz, 3H). C NMR (75 MHz,
CDCl3) δ 180.12, 148.83, 147.46, 146.39, 135.46, 130.62, 129.59, 126.48, 124.09,
121.86, 120.25, 111.51, 111.06, 55.57, 55.50, 46.06, 34.01, 28.21, 14.91. HRMS
(ESI-): m/z: 345.1628 [M+H]+.
Compound Y5, white solid (0.94 g, 94%), m.p. 121.1-121.2°C. 1H NMR (300 MHz,
Chloroform-d) δ 7.68 (s, 1H), 7.26 – 7.18 (m, 1H), 7.07 (d, J = 7.7 Hz, 1H), 6.84 –

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6.75 (m, 3H), 6.71 – 6.66 (m, 2H), 6.05 (s, 1H), 3.90 (q, J = 6.6 Hz,, 2H), 3.88 (s, 3H),
3.84 (s, 3H), 2.88 (t, J = 6.7 Hz, 2H), 2.29 (s, 3H). 13C NMR (75 MHz, CDCl3) δ
180.13, 148.85, 147.48, 140.04, 135.36, 130.63, 129.53, 127.69, 125.22, 121.65,
120.28, 111.52, 111.09, 55.59, 55.50, 46.06, 33.98, 20.86. HRMS (ESI-): m/z:
331.1475 [M+H]+.
Compound Y6, white solid (0.96 g, 96%), m.p. 128.3-128.4°C. 1H NMR (300 MHz,
Chloroform-d) δ 7.68 (s, 1H), 7.39 – 7.23 (m, 3H), 7.05 – 6.97 (m, 2H), 6.77 (d, J =
8.7 Hz, 1H), 6.71 – 6.64 (m, 2H), 6.02 (s, 1H), 3.90 (q, J = 6.7 Hz, 2H), 3.88 (s, 3H),
13
3.84 (s, 3H), 2.87 (t, J = 6.8 Hz, 2H). C NMR (75 MHz, CDCl3) δ 180.15, 148.85,
147.49, 135.43, 130.52, 129.78, 126.91, 124.72, 120.29, 111.47, 111.12, 55.64, 55.51,
46.11, 33.99. HRMS (ESI-): m/z: 317.1315 [M+H]+.
1
Compound Y7, white solid (0.95 g, 95%), m.p. 146.3 -147.1 °C. H NMR (300
MHz, Chloroform-d) δ 8.59 (s, 1H), 7.27 – 7.20 (m, 1H), 6.89 (m, 1H), 6.83 – 6.73
(m, 3H), 6.66 (d, J = 6.9 Hz, 2H), 6.17 (s, 1H), 3.88 (q, J = 6.9 Hz,, 2H), 3.83 (s, 3H),
3.80 (s, 3H), 2.86 (t, J = 6.7 Hz, 2H). 13C NMR (75 MHz, CDCl3) δ 179.67, 162.80 (d,
J = 248.9 Hz), 148.88, 147.54, 137.51 (d, J = 9.4 Hz), 130.82 (d, J = 9.3 Hz), 130.39,
120.31, 119.56 (d, J = 3.0 Hz), 113.21 (d, J = 20.9 Hz), 111.49, 111.42, 111.16 (d, J =
2.1 Hz), 55.56, 55.48, 45.93, 33.88. HRMS (ESI-): m/z: 335.1224 [M+H]+.
1
Compound Y8, yellow solid (0.92 g, 92%), m.p. 119.1-119.7°C. H NMR (300
MHz, Chloroform-d) δ 7.88 (s, 1H), 7.25 – 7.18 (m, 2H), 7.05 (d, J = 2.0 Hz, 1H),
6.88 (m, 1H), 6.81 – 6.75 (m, 1H), 6.67 (d, J = 6.6 Hz, 2H), 6.02 (s, 1H), 3.89 (q, J =
13
6.9 Hz, 2H), 3.86 (s, 3H), 3.83 (s, 3H), 2.88 (t, J = 6.7 Hz, 2H). C NMR (75 MHz,

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CDCl3) δ 180.01, 148.96, 147.61, 136.90, 135.25, 130.65, 130.34, 126.78, 124.52,
122.48, 120.29, 111.42, 111.15, 55.59, 55.53, 46.08, 33.89. HRMS (ESI-): m/z:
351.0927 [M+H]+.
Compound Y9, white solid (0.95 g, 95%), m.p. 136.5-137.6°C. 1H NMR (300 MHz,
Chloroform-d) δ 7.98 (s, 1H), 7.36 (m, 1H), 7.24 – 7.13 (m, 2H), 6.94 (d, J = 8.1 Hz,
1H), 6.82 – 6.75 (m, 1H), 6.71 – 6.63 (m, 2H), 6.02 (s, 1H), 3.88 (q, J = 6.8 Hz, 2H),
3.86 (s, 3H), 3.83 (s, 3H), 2.87 (t, J = 6.7 Hz, 2H). 13C NMR (75 MHz, CDCl3) δ
180.00, 148.95, 147.59, 137.04, 130.88, 130.34, 129.72, 127.45, 123.09, 123.01,
120.29, 55.60, 55.54, 46.06, 33.89. HRMS (ESI-): m/z: 395.0422 [M+H]+.
1
Compound Y10, yellow solid (0.97 g, 97%), m.p. 146.5- 147.1°C. H NMR (300
MHz, Chloroform-d) δ 7.91 (s, 1H), 7.57 (d, J = 7.6 Hz, 1H), 7.42 (s, 1H), 7.00 (m,
2H), 6.79 (d, J = 8.6 Hz, 1H), 6.71 – 6.64 (m, 2H), 5.99 (s, 1H), 3.89 (q, J = 6.7 Hz,,
2H), 3.86 (s, 3H), 3.83 (s, 3H), 2.87 (t, J = 6.7 Hz, 2H). 13C NMR (75 MHz, CDCl3) δ
179.99, 148.92, 147.56, 136.87, 135.77, 133.37, 130.99, 130.33, 123.79, 120.28,
111.39, 111.15, 94.56, 55.63, 55.56, 46.07, 33.90. HRMS (ESI-): m/z: 443.0280
[M+H]+.
1
Compound Y12, yellow liquid (0.94 g, 94%), H NMR (300 MHz, DMSO-d6) δ
8.08 (s, 1H), 7.31 – 7.08 (m, 4H), 6.87 – 6.66 (m, 4H), 6.04 (s, 1H), 3.89 (q, J = 6.6
Hz, 2H), 3.75 (s, 3H), 2.88 (t, J = 6.8 Hz, 2H), 1.26 (s, 9H). 13C NMR (75 MHz,
CDCl3) δ 180.16, 159.51, 153.46, 139.71, 135.27, 129.33, 129.25, 123.99, 122.09,
121.85, 120.59, 113.99, 111.70, 54.78, 45.88, 34.58, 34.44, 30.81. HRMS (ESI-): m/z:
343.1835 [M+H]+.

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Compound Y13, brown solid (0.91 g, 91%), m.p. 59.8-61.0°C. 1H NMR (300 MHz,
DMSO-d6) δ 8.06 (s, 1H), 7.26 – 7.07 (m, 3H), 6.91 (t, J = 2.0 Hz, 1H), 6.83 (d, J =
7.8 Hz, 1H), 6.77 – 6.68 (m, 3H), 6.05 (s, 1H), 3.89 (q, J = 6.7 Hz, 2H), 3.76 (s, 3H),
2.89 (t, J = 6.7 Hz, 2H), 2.85 – 2.77 (m, 1H), 1.20 (s, 3H), 1.18 (s, 3H). 13C NMR (75
MHz, CDCl3) δ 180.08, 159.53, 151.02, 139.76, 135.52, 129.56, 129.33, 124.97,
122.92, 122.09, 120.62, 114.02, 111.70, 54.78, 45.86, 34.54, 33.54, 23.43. HRMS
(ESI-): m/z: 329.1679 [M+H]+.
Compound Y14, yellow solid (0.98 g, 98%), m.p. 58.9-60.1°C. 1H NMR (300 MHz,
DMSO-d6) δ 8.10 (s, 1H), 7.26 – 7.02 (m, 3H), 6.90 – 6.67 (m, 5H), 6.06 (s, 1H), 3.88
(q, J = 6.6 Hz, 2H), 3.75 (s, 3H), 2.88 (t, J = 6.7 Hz, 2H), 2.57 (q, J = 7.6 Hz, 2H),
1.17 (t, J = 7.6 Hz, 3H). 13C NMR (75 MHz, CDCl3) δ 180.04, 159.53, 146.29, 139.79,
135.55, 129.56, 129.32, 126.39, 124.15, 121.94, 120.65, 114.04, 111.70, 54.79, 45.84,
34.52, 28.22, 14.95. HRMS (ESI-): m/z: 315.1520 [M+H]+.
1
Compound Y15, yellow solid (0.93 g, 93%), m.p. 94.6 -94.7°C. H NMR (300
MHz, DMSO-d6) δ 7.73 (s, 1H), 7.19 (m, 2H), 7.05 (d, J = 7.6 Hz, 1H), 6.85 – 6.67
(m, 5H), 6.02 (s, 1H), 3.90 (q, J = 6.6 Hz, 2H), 3.77 (s, 3H), 2.90 (t, J = 6.7 Hz, 2H),
13
2.28 (s, 3H). C NMR (75 MHz, CDCl3) δ 180.15, 159.55, 140.01, 139.79, 135.33,
129.54, 129.35, 127.71, 125.31, 121.75, 120.66, 114.05, 111.72, 54.79, 45.91, 34.49,
20.88. HRMS (ESI-): m/z: 301.1363 [M+H]+.
1
Compound Y16, yellow solid (0.92 g, 92%), m.p. 112.5-113.6°C. H NMR (300
MHz, DMSO-d6) δ 8.27 (s, 1H), 7.35 – 7.13 (m, 5H), 7.01 (d, J = 7.6 Hz, 1H), 6.78 –
6.66 (m, 3H), 6.04 (s, 1H), 3.88 (q, J = 6.8 Hz, 2H), 3.76 (s, 3H), 2.87 (t, J = 6.8 Hz,

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2H). C NMR (75 MHz, CDCl3) δ 179.98, 159.55, 139.70, 135.62, 129.70, 129.40,
126.71, 124.70, 120.66, 114.07, 111.72, 54.82, 45.85, 34.50. HRMS (ESI-): m/z:
287.1209 [M+H]+.
1
Compound Y17, yellowish green solid (0.92 g, 92%), m.p. 74.2-75.0°C. H NMR
(300 MHz, DMSO-d6) δ 8.26 (s, 1H), 7.31 – 7.15 (m, 2H), 6.92 (m, 1H), 6.74 (m, 5H),
6.09 (s, 1H), 3.90 (q, J = 6.9 Hz, 2H), 3.77 (s, 3H), 2.90 (t, J = 6.7 Hz, 2H). 13C NMR
(75 MHz, CDCl3) δ 179.86, 162.88 (d, J = 249.1 Hz), 159.63, 139.54, 137.28 (d, J =
9.7 Hz), 130.92 (d, J = 9.1 Hz), 129.49, 120.61, 119.79 (d, J = 3.0 Hz), 114.15, 113.46
(d, J = 21.3 Hz), 111.72, 111.41, 54.80, 45.88, 34.33. HRMS (ESI-): m/z: 305.1115
[M+H]+.
1
Compound Y18, yellow liquid (0.97 g, 97%), H NMR (300 MHz, DMSO-d6) δ
8.23 (s, 1H), 7.25 – 7.16 (m, 3H), 7.08 (d, J = 2.1 Hz, 1H), 6.92 (m, 1H), 6.79 – 6.68
(m, 3H), 6.10 (s, 1H), 3.88 (q, J = 6.7 Hz, 2H), 3.77 (s, 3H), 2.90 (t, J = 6.7 Hz, 2H).
13C NMR (75 MHz, CDCl3) δ 180.00, 159.60, 139.56, 137.07, 135.15, 130.55, 129.48,
126.64, 124.52, 122.51, 120.63, 114.09, 111.78, 54.82, 45.89, 34.38. HRMS (ESI-):
m/z: 321.0821 [M+H]+.
1
Compound Y19, yellow liquid (0.95 g, 95%), H NMR (300 MHz, DMSO-d6) δ
8.16 (s, 1H), 7.41 – 7.31 (m, 1H), 7.25 – 7.13 (m, 3H), 6.95 (d, J = 8.0 Hz, 1H), 6.81
– 6.68 (m, 3H), 6.02 (s, 1H), 3.89 (q, J = 6.5 Hz, 2H), 3.77 (s, 3H), 2.90 (t, J = 6.7 Hz,
13
2H). C NMR (75 MHz, CDCl3) δ 179.99, 159.61, 139.53, 137.06, 130.84, 129.72,
129.51, 127.53, 123.11, 123.09, 120.62, 114.07, 111.83, 54.84, 45.92, 34.37. HRMS
(ESI-): m/z: 365.0316 [M+H]+.

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Compound Y20, yellow liquid (0.96 g, 96%), H NMR (300 MHz, DMSO-d6) δ
8.06 (s, 1H), 7.57 (m, 1H), 7.43 (d, J = 1.5 Hz, 1H), 7.25 – 7.18 (m, 1H), 7.11 – 6.94
(m, 2H), 6.81 – 6.68 (m, 3H), 5.99 (s, 1H), 3.88 (q, J = 6.7 Hz, 2H), 3.78 (s, 3H), 2.90
(t, J = 6.7 Hz, 2H). 13C NMR (75 MHz, CDCl3) δ 180.02, 159.59, 139.54, 136.92,
135.75, 133.44, 130.94, 129.52, 123.91, 120.63, 114.05, 111.87, 94.57, 54.87, 45.92,
34.39. HRMS (ESI-): m/z: 413.0172 [M+H]+.
1
Compound Y22, white solid (0.93 g, 93%), m.p. 126.5 -127.0°C. H NMR (300
MHz, Chloroform-d) δ 7.89 (s, 1H), 7.36 – 7.05 (m, 8H), 6.82 (d, J = 7.2 Hz, 1H),
6.00 (s, 1H), 3.90 (q, J = 6.5 Hz, 2H), 2.91 (t, J = 7.0 Hz, 2H), 1.26 (s, 9H). 13C NMR
(75 MHz, CDCl3) δ 180.32, 153.53, 138.10, 135.19, 129.30, 128.34, 126.25, 124.12,
122.22, 121.98, 46.00, 34.54, 34.45, 30.82. HRMS (ESI-): m/z: 313.1727 [M+H]+.
1
Compound Y23, yellow solid (0.91 g, 92%), m.p. 77.5- 78.6°C. H NMR (300
MHz, Chloroform-d) δ 7.86 (s, 1H), 7.30 – 7.08 (m, 7H), 6.90 (t, J = 2.0 Hz, 1H),
6.81 (m, 1H), 6.01 (s, 1H), 3.88 (q, J = 6.7 Hz, 2H), 2.91 (t, J = 6.8 Hz, 2H), 2.99 –
2.70 (m, 1H), 1.20 (s, 3H), 1.18 (s, 3H). 13C NMR (75 MHz, CDCl3) δ 180.22, 151.09,
138.15, 135.44, 129.60, 128.37, 128.34, 126.25, 125.09, 123.04, 122.20, 45.98, 34.49,
33.55, 23.43. HRMS (ESI-): m/z: 299.1573 [M+H]+.
1
Compound Y24, yellow solid (0.95 g, 95%), m.p. 71.0 -71.8°C. H NMR (300
MHz, Chloroform-d) δ 8.10 (s, 1H), 7.30 – 7.10 (m, 6H), 7.07 (d, J = 7.7 Hz, 1H),
6.88 – 6.77 (m, 2H), 6.03 (s, 1H), 3.88 (q, J = 6.8 Hz, 2H), 2.91 (t, J = 6.8 Hz, 2H),
2.57 (q, J = 7.6 Hz, 2H), 1.17 (t, J = 7.6 Hz, 3H). 13C NMR (75 MHz, CDCl3) δ

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180.05, 146.27, 138.20, 135.56, 129.58, 128.39, 128.34, 126.42, 126.26, 124.22,
122.00, 45.92, 34.48, 28.24, 14.98. HRMS (ESI-): m/z: 285.1415 [M+H]+.
Compound Y25, yellow solid (0.96 g, 96%), m.p. 92.4-93.3°C. 1H NMR (300 MHz,
Chloroform-d) δ 8.02 (s, 1H), 7.30 – 7.11 (m, 6H), 7.04 (d, J = 7.5 Hz, 1H), 6.80 (d, J
= 7.6 Hz, 2H), 6.02 (s, 1H), 3.88 (q, J = 6.7 Hz, 2H), 2.91 (t, J = 6.8 Hz, 2H), 2.27 (s,
13
3H). C NMR (75 MHz, CDCl3) δ 180.08, 139.92, 138.20, 135.45, 129.52, 128.41,
128.35, 127.64, 126.27, 125.33, 121.77, 45.93, 34.45, 20.95. HRMS (ESI-): m/z:
271.1259 [M+H]+.
1
Compound Y26, yellow solid (0.94 g, 94%), m.p. 103.1- 109.7°C. H NMR (300
MHz, Chloroform-d) δ 8.37 (s, 1H), 7.35 – 7.27 (m, 3H), 7.26 – 7.17 (m, 3H), 7.13
(m, 2H), 7.05 – 6.98 (m, 2H), 6.04 (s, 1H), 3.85 (q, J = 6.8 Hz, 2H), 2.90 (t, J = 6.8
Hz, 2H). 13C NMR (75 MHz, CDCl3) δ 179.96, 138.12, 135.72, 129.70, 128.41,
128.38, 126.67, 126.28, 124.71, 45.88, 34.47. HRMS (ESI-): m/z: 257.1103 [M+H]+.
1
Compound Y27, yellow solid (0.97 g, 97%), m.p. 79.9- 80. 8°C. H NMR (300
MHz, Chloroform-d) δ 7.85 (s, 1H), 7.33 – 7.20 (m, 4H), 7.16 (m, 2H), 6.94 (m, 1H),
13
6.74 (m, 2H), 6.04 (s, 1H), 3.88 (q, J = 6.5 Hz, 2H), 2.94 (t, J = 6.8 Hz, 2H). C
NMR (75 MHz, CDCl3) δ 179.87, 162.88 (d, J = 249.2 Hz), 137.94, 137.30 (d, J = 9.5
Hz), 130.94 (d, J = 9.3 Hz), 128.47, 128.37, 126.45, 119.79 (d, J = 3.0 Hz), 113.47 (d,
J = 21.0 Hz), 111.60 (d, J = 23.4 Hz), 45.98, 34.33. HRMS (ESI-): m/z: 275.1008
[M+H]+.
1
Compound Y28, brownish yellow liquid (0.93 g, 93%), H NMR (300 MHz,
Chloroform-d) δ 8.15 (s, 1H), 7.36 – 7.29 (m, 2H), 7.26 – 7.06 (m, 7H), 6.92 (d, J=

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6.5 Hz, 1H), 3.90 (t, J = 6.6 Hz, 2H), 2.94 (t, J = 6.5 Hz, 2H). C NMR (75 MHz,
CDCl3) δ 137.97, 137.06, 135.15, 130.58, 128.48, 128.37, 126.69, 126.44, 124.61,
122.57, 46.15, 34.40. HRMS (ESI-): m/z: 291.0714 [M+H]+.
1
Compound Y29, yellow solid (0.95 g, 95%), m.p. 89.6- 90.0°C. H NMR (300
MHz, Chloroform-d) δ 8.57 (s, 1H), 7.39 – 7.10 (m, 8H), 6.95 (m, 1H), 6.05 (s, 1H),
3.87 (q, J = 6.7 Hz, 2H), 2.91 (t, J = 6.8 Hz, 2H). 13C NMR (75 MHz, CDCl3) δ
179.85, 137.97, 137.23, 130.84, 129.60, 128.50, 128.38, 127.48, 126.46, 123.10,
123.00, 45.97, 34.39. HRMS (ESI-): m/z: 335.0212 [M+H]+.
1
Compound Y30, yellow solid (0.94 g, 94%), m.p. 86.7- 88.0°C. H NMR (300
MHz, Chloroform-d) δ 8.38 (s, 1H), 7.62 – 7.41 (m, 2H), 7.34 – 6.94 (m, 7H), 6.00 (s,
1H), 3.87 (q, J = 6.5 Hz, 2H), 2.91 (t, J = 6.8 Hz, 2H). 13C NMR (75 MHz, CDCl3) δ
179.91, 137.97, 137.05, 135.67, 133.41, 130.95, 128.52, 128.38, 126.47, 123.93,
94.59, 46.00, 34.40. HRMS (ESI-): m/z: 383.0073 [M+H]+.
2.5. Biological assay
The biological activities of the synthesized compounds were assayed with the
following procedures. 24, 25
1/2 Murashige-Skoog (MS) culture medium, containing 0.8% agar, 1% sucrose, and
the synthetic compounds with indicated concentration, was prepared and sterilized.
Arabidopsis seeds including gibberellin-deficient dwarf mutant (ga1-1) and wild type
Arabidopsis (Columbia-0, Col-0) were sterilized in 70% ethanol for 1 min and in 1%
sodium hypochlorite solution for another 15 min, then were washed five times with
sterile water, and grown on the 1/2 MS medium. Subsequently, the media were placed

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in the illumination box at 22°C, after the Arabidopsis seeds were incubated at 4°C for
3 days. Hypocotyl length, taproot length, and root number of 7-day-old Arabidopsis
seedlings were measured using ImageJ after image acquisition.
The rate of hypocotyl elongation was calculated based on the following equation:
퐿 − 퐿0
푃 =
× 100%
퐿0
where P is the rate of hypocotyl elongation increase, and L and L0 are the average
lengths of the Arabidopsis hypocotyl in the presence of the target compounds and in
the control, respectively.
The inhibition rate of root growth was calculated according to the following
equation:
푑0 − d
퐼 =
× 100%
푑0
where I is the inhibition rate, and d0 and d are the average lengths of the
Arabidopsis root in the control and in the presence of the target compounds,
respectively.
2.6. Molecular dynamics simulation
Molecular dynamics simulation is a method for studying the interaction between
ligand and target. The protonation states of histidine residues in the protein were
manually inspected in order to ensure that all systems were optimized by the
AMBER14 software. The force field parameters for protein were generated using
AMBER ff99SB,26 while those for ligands were generated by general AMBER force
field (GAFF).27 A rectangular water box filled with TIP3P water molecules with an
edge of 12 Å was generated, and was used to fill the gaps between ligands and protein.

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In addition, Na+ ions were added to neutralize the system. This was followed by a 10
ns of production run of molecular dynamics simulations,28 and the binding free energy
was calculated using the MM/GBSA method.29
2.7. Molecular docking
Molecular docking was performed by the SYBYL 7.2 software using the
Surflex-dock algorithm,30,31 in which the crystal structure of GA3-GID1A-DELLA
from the RCSB protein data bank (PDB ID: 2ZSH) was used as the docking receptor.
The receptor model was prepared by Biopolymer module, comprise Add MMFF94
charge, atom types of AMBER7 FF99, addition of hydrogen atoms and protonation
states of amino acids were adjusted to pH 7.0. The optimal protocol in the active
domain of the receptor was obtained using a ligand docking mode to improve docking
accuracy. Unless otherwise indicated, all other parameters were defined as default
values.
409 3. Results and discussion
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3.1. Pharmacophore models and virtual screening
The GALAHAD module can accurately construct pharmacodynamics by using
external macro definition files to specify overlapping features.32 The pharmacophore
characteristics produced by ligands are consistent with the crystal structure, and the
additional pharmacophore characteristics help to enhance the affinity between ligands
and target.33 20 pharmacophore models were established using the GALAHAD
module and, a representative pharmacophore feature was selected from them (Fig. 3).

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The enrichment factor (EF) of the pharmacophore model was 14.29 (Table S1),
indicating that the model was reliable. The training set consisted of 200 active and
non-active compounds.34 The representative pharmacophore model comprised seven
pharmacodynamic characteristics, which were mainly hydrophobic centers and
acceptor atoms.
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Fig. 3. The molecular alignment of the N-substituted phthalimide molecule AC94377 with its
pharmacophore characteristics. Cyan spheres represent hydrophobic centers (HY), the magenta
sphere represents the donor atom (DA), and green spheres represent acceptor atoms (AA).
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Thiourea compounds were selected from the Maybridge database (Fig. S1). Initially,
7329 compounds were screened from the 56000 compounds of the Maybridge
database through the Lipinski’s rule of five and physicochemical properties.
Subsequently, the aforementioned pharmacophore model was used to screen 3078
compounds, while target-based screening was also performed. Finally,
1-(2,3-dimethoxyphenethyl)-3-(3-(trifluoromethyl)phenyl)thiourea was selected based
on their synthesizability, liposolubility, and water capacity as lead compounds, and
subsequent design and synthesis was performed.

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3.2. Synthesis
In general, amines and thiophosgene as beginning materials are usually used in the
synthesis of isothiocyanates due to the rapid reaction, ease in operation, and high yield.
However, thiophosgene is virulent. In this study, commercial available solid
dimethylamino thiocarbonyl chloride instead of thiophosgene was used with the same
features of ease operation, rapid reaction, and high yield. At the same time, it was
found that the substituents of aniline had little effect on isothiocyanate formation.
Since isothiocyanate is a reactive compound, the target compounds Y1-Y30 required
were prepared by only the mixing of the isothiocyanate with the amine. All target
compounds were characterized by 1H NMR, 13C NMR, and HRMS.
3.3. Evalation of Biological activities of target compounds
3.3.1. Elongation eff ect of Arabidopsis hypocotyls
In the bioassay, Arabidopsis was treated by all compounds (Y1-Y30) at
concentrations of 0.1, 1, 5, 10, 30 and 100 μmol/L in order to explore the multiple
effects of the synthetic compounds on Arabidopsis growth. The biological activities
results of compounds Y1-Y30 on Arabidopsis hypocotyl length (Columbia-0, Col-0)
are listed in Table 1. In total, most of the tested compounds had a promoting
elongation effect on Arabidopsis hypocotyl growth at 0.1, 1, and 10 μmol/L, and the
elongation increased with the increase of the concentration. However, the promoting
effect decreased, or turned to inhibition at 100 μmol/L. Among 30 compounds,

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compound Y21 possesses the best promoting activity with hypocotyl elongation
increase by 86.6% at 1 μmol/L, while the promotion effect of GA3 was only 18.1% at
the concentration. Thus, the promotion of hypocotyl elongation of compound Y21 was
much better than that of gibberellin at low concentration. The overview of
Arabidopsis growth treated with or without compound Y21 was showed in Fig. 4.
Table 1. Effect of compounds Y1-Y30 to hypocotyl length, taproot length,
and root number of Arabidopsis (Columbia-0, Col-0)
Concn.
Concn.
Compd
Enlongation
rate (%)
Inhibition
rate (%)
Root
Promoting
rate (%)
Inhibition
rate (%)
Root
Compd.
Y17
.
number
number
(μmol/L)
(μmol/L)
Y1
0.1
1
6.6±0.2
8.0±0.6
0.5±0.1
28.3±1.6
50.3±3.3
94.7±2.5
2.8±0.4
1
1
2
1
1
1
3
1
1
1
4
1
1
1
3
1
1
1
1
1
1
1
1
1
1
1
1
1
0.1
1
1.0±0.2
16.9±0.2
16.3±0.2
93.5±0.2
0.0±0.1
-0.1±0.2
17.4±0.2
42.7±0.2
90.4±0.2
1.2±0.4
2
2
3
1
1
3
3
1
1
3
4
1
1
3
3
1
3
4
2
1
3
3
2
1
2
4
3
1
10
100
0.1
1
118.1±1.3
-21.8±0.3
1.5±0.7
10
100
0.1
1
Y2
Y3
Y4
Y5
Y6
Y7
Y18
Y19
Y20
Y21
Y22
Y23
6.1±1.1
22.6±1.7
51.2±1.9
92.0±4.3
7.4±0.6
7.4±0.5
22.5±2.0
48.9±3.8
90.3±6.1
4.2±1.0
10
100
0.1
1
68.1±3.2
40.0±1.2
1.9±3.1
10
100
0.1
1
22.7±1.3
118.1±2.6
3.6±0.7
29.2±0.9
99.6±2.4
67.9±0.2
1.0±0.4
21.1±0.5
66.3±2.4
91.4±3.8
10.7±1.4
38.2±2.5
38.6±0.9
92.5±0.8
2.7±0.4
28.5±2.9
110.3±3.0
96.8±5.1
0.9±0.3
31.6±2.3
50.2±2.5
90.7±4.6
14.2±1.4
56.5±0.9
67.8±1.7
90.2±2.3
-19.0±1.9
20.7±2.1
76.5±2.2
90.7±3.3
6.5±0.3
10
100
0.1
1
10
100
0.1
1
12.4±0.6
106.9±0.5
27.0±2.2
2.0±0.6
15.9±1.0
79.3±0.8
123.2±1.2
6.2±0.9
10
100
0.1
1
10
100
0.1
1
9.3±0.3
17.6±0.2
24.1±1.6
92.5±0.8
5.7±0.4
86.6±5.0
29.8±3.6
0.6±0.2
10
100
0.1
1
34.7±1.1
0.3±0.1
10
100
0.1
1
1.7±2.3
9.2±1.0
3.6±0.8
6.1±3.7
22.1±0.6
44.3±0.3
22.8±1.5
1.7±1.0
70.5±1.9
75.2±3.0
90.1±0.9
16.1±1.2
58.4±3.8
74.9±1.6
99.4±2.7
10
100
0.1
1
27.3±0.3
-21.2±0.4
9.0±1.1
7.2±1.2
10
100
0.1
1
92.0±4.0
10.2±0.0
11.8±0.7
16.6±3.4
91.4±2.6
20.2±1.0
33.7±2.8
22.9±2.1
14.6±2.6
45.2±4.0
-43.9±0.5
10
100
10
100

Y8
0.1
1
1.6±0.8
5.8±0.3
0.5±0.1
3.5±0.2
1
1
1
1
1
1
3
1
1
2
2
1
1
3
3
1
2
3
4
2
3
3
4
1
3
4
4
1
2
3
3
1
1
1
2
1
Y24
Y25
0.1
1
7.9±1.4
21.9±0.7
53.1±2.0
-46.2±3.8
1.1±0.5
-6.1±0.1
32.1±1.2
69.5±0.4
99.4±3.3
2.5±0.1
1
3
3
1
1
1
2
1
1
1
1
1
1
1
2
1
1
3
3
1
1
3
3
1
2
3
1
1
1
1
1
1
1
1
1
1
10
100
0.1
1
39.7±1.2
-31.4±1.4
0.0±0.3
48.7±2.4
97.2±5.2
-34.7±0.5
2.3±0.6
10
100
0.1
1
Y9
5.4±0.7
4.0±14
28.7±0.8
58.7±2.3
99.4±5.6
-1.0±0.2
12.3±0.3
34.7±0.7
90.4±3.2
13.4±0.8
14.8±0.6
35.4±2.8
92.8±0.6
0.4±0.1
10
100
0.1
1
18.2±1.0
-30.9±1.1
1.3±0.4
35.9±2.0
98.2±6.5
5.6±0.9
10
100
0.1
1
44.5±2.6
-39.3±0.8
4.9±1.1
Y10
Y11
Y12
Y13
Y14
Y15
Y16
Y26
26.5±1.6
50.7±2.3
-4.5±1.8
1.5±0.5
19.6±1.4
60.9±2.6
93.7±0.7
21.4±0.5
43.3±0.8
78.1±0.4
92.5±0.3
3.4±2.1
8.4±0.7
10
100
0.1
1
10
100
0.1
1
9.0±1.6
7.9±0.5
Y27
4.0±0.1
57.5±0.9
97.7±1.3
5.6±0.5
19.2±0.3
12.3±0.9
11.0±1.1
9.0±0.4
10
100
0.1
1
10
100
0.1
1
1.3±0.1
Y28
36.4±1.0
68.8±2.1
39.5±2.7
5.5±0.9
40.7±1.8
59.5±1.6
90.0±0.7
5.8±0.3
20.3±0.6
55.9±3.5
10.1±0.6
6.5±0.7
-2.5±0.3
34.9±2.4
90.5±2.9
-0.1±0.2
-2.5±0.8
72.3±4.0
99.4±2.5
26.1±0.2
60.6±1.4
80.0±0.9
99.4±1.7
-3.7±0.5
-1.6±0.1
0±0.2
10
100
0.1
1
10
100
0.1
1
Y29
20.7±1.5
88.4±1.6
18.0±0.2
0.2±0.3
56.6±0.4
65.9±0.9
90.4±3.0
-1.5±0.2
43.4±1.5
65.1±2.4
90.6±0.7
5.2±0.1
55.7±0.8
64.1±2.1
-44.0±2.0
0.4±0.1
10
100
0.1
1
10
100
0.1
1
Y30
42.0±2.9
77.4±2.6
50.1±3.1
1.2±0.4
51.0±0.9
68.0±3.5
-53.4±0.6
2.4±0.3
10
100
0.1
1
10
100
0.1
1
GA3
8.6±1.6
27.6±0.4
46.1±0.5
90.3±2.6
5.6±0.8
18.1±0.4
60.6±2.2
123.5±0.9
0.2±0.5
10
100
0.1
1
23.9±0.7
18.8±0.9
0.0±0.2
10
100
0.1
1
8.6±0.5
DMSO
0.0±0.1
9.6±0.6
16.4±2.9
43.6±1.4
91.6±2.5
0.2±0.4
0.7±0.2
10
100
33.8±3.4
93.1±2.1
10
100
-2.2±0.0
-3.6±0.6
1.9±0.7
12.7±0.4
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In detail, most of the tested compounds, as well as GA3, produced little effect on
the growth of Arabidopsis hypocotyl at 0.1 μmol/L. At 1 μmol/L, the
growth-promoting effect of several compounds, such as compounds Y3, Y10, Y11,
Y21, Y29, and Y30 on Arabidopsis hypocotyls was greater than that achieved by the
same concentration of GA3. In particular, the promotion of compound Y21 was 4.8
times that of GA3. At 10 μmol/L, the growth promoting effects of most compounds on
Arabidopsis hypocotyls exceeded that of GA3. For example, the hypocotyl length in
Arabidopsis treated with compound Y1 at 10 μmol/L was 118% longer than that of the
untreated ones, while the effect of GA3 at the same concentration led to an only 73%
elongation. However, when the concentration reached 100 μmol/L, the growth
promoting effect of the tested compounds on Arabidopsis hypocotyl decreased, or
turned to inhibition, which was consistent with the fact that plant growth regulators
inhibit plant growth at high concentrations, but stimulate growth at lower
concentrations.
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Fig. 4. The growth situation of 7-day-old Arabidopsis (Columbia-0, Col-0) seedlings, grown on
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1/2 MS, containing 1% sucrose and 0.8% agar, either without or supplemented with compound

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Y21 at the different indicated concentrations, bar = 5 mm.
Furthermore，it was noteworthy that the substituents on the phenyl and the number
of methoxy groups on phenylethyl moiety of thiourea had a significant impact on
hypocotyl elongation. For example, hypocotyl elongation was increased when R1
(trifluoromethyl, chlorine, bromine or iodine) was an electron-withdrawing group and
the number decrease of methoxy groups, and the elongation increase was
tremendously improved from compound Y1 (8.0%, 1 μmol/L) to compound Y11
(57.5%, 1 μmol/L), and to compound Y21 (86.6%, 1 μmol/L). When R1 was an
electron-donating group, most of the compounds indicated that the reduction in the
amount of the methoxy groups enhanced the bioactivities, and the complete
disappearance of the methoxy groups weakened them.
At the same time, the electronic effect of substituents on the benzene ring affected
bioactivity. When R2, R3 were methoxy, the activities of the compounds containing R1
as an electron-donating group were much higher than those of the compounds
containing R1 as an electron-withdrawing group. For example, comparing compounds
Y3 (99.6%, 10 μmol/L), and Y4 (106.9%, 10 μmol/L) with compounds Y7 (33.7%, 10
μmol/L) and Y8 (39.7%, 10 μmol/L), when R2 was hydrogen and R3 was hydrogen or
methoxy, the activities of the compounds containing R1 as an electron-withdrawing
group were higher than those of the compounds containing R1 as an electron-donating
group.
Additionally, when the number of methoxy groups was constant, the promoting

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activity of the compounds on Arabidopsis hypocotyls increased with the decrease of
the electron donating ability and steric hindrance of group R1. For instance,
comparing compound Y2 (68.1%, 10 μmol/L) with compound Y3 (99.6%, 10 μmol/L),
with compound Y4 (106.9%, 10 μmol/L). However, when R1 was methyl or hydrogen,
the activities of the compounds were dramatically decreased, and the order of
activities of compounds substituted by electron-donating groups was obviously
ethyl > isopropyl > tert-butyl > methyl > hydrogen. When R1 was an
electron-withdrawing group, the promoting activity of the compounds on Arabidopsis
hypocotyl increased with the decrease of the electron-withdrawing ability. However,
the trifluoromethyl group with higher steric hindrance did not follow the above laws,
which led to higher activity. Thus, it was clear that the order of activities of
compounds substituted by electron-donating groups was trifluoromethyl > iodium >
bromo > chlorine > fluorine.
3.3.2. Evaluation of eff ect on Arabidopsis roots
While the synthetic compounds had an effect on Arabidopsis hypocotyl growth,
they also had a significant effect on the growth of Arabidopsis roots, the IC50 values
of Compounds Y1-Y30 to Arabidopsis taproot are shown in Table 2 and the numbers
of the roots are shown in the Table 1. The results demonstrated that the synthetic
compounds promoted or inhibited the growth of Arabidopsis taproots, and promoted
the generation of Arabidopsis lateral roots. For example, compound Y21 promoted a
taproot elongation of 19.0% at 0.1 μmol/L, while the IC50 values of compoundY21 to

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Arabidopsis taproot is 6.3 μmol/L. At the same time, compound Y21 could effectively
increase the number of lateral roots to 3-4. The effect diagram of compound Y21 can
be seen in Fig. 4 and the biological activities results of compounds Y1-Y30 on taproot
length and root number of Arabidopsis are listed in Table 1.
In detail, it was observed that compounds Y9 and Y21 had a certain promoting
effect on the growth of Arabidopsis taproots at 0.1 μmol/L, with compound Y9
demonstrating the best promoting effect, reaching 34.7%. However, most of the
compounds inhibited Arabidopsis taproot growth, and it was found that the half
maximal inhibitory concentration values (Table 2) of the compounds with the best
inhibitory effect, such as compounds Y30, was 1.3 μmol/L. When the concentration
was 100 μmol/L, the inhibition rate of the tested compounds on taproot growth was
over 90%.
Table 2. The IC50 Values of Compounds Y1-Y30 to Arabidopsis Taproot
Compd
Y1
IC50 (μmol/L)
21.6
Compd
Y11
Y12
Y13
Y14
Y15
Y16
Y17
Y18
Y19
Y20
IC50 (μmol/L)
6.5
Compd
Y21
Y22
Y23
Y24
Y25
Y26
Y27
Y28
Y29
Y30
IC50 (μmol/L)
6.3
15
Y2
25.4
18.8
Y3
17.4
10.1
3.3
8.2
18.2
49.2
33
Y4
23.7
20.2
Y5
27.9
31.3
Y6
43.6
33.4
Y7
35.6
30.5
Y8
16.9
33.6
38.5
4.6
1.3
Y9
17
27.2
Y10
14.8
10.7

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With the inhibition of taproot growth, the effect of the tested compounds on the
number of lateral roots was also apparent. The root number of Arabidopsis treated
with compounds Y13, Y14, Y21, and Y22 at 0.1 μmol/L was 3, and of the control was
only 1. The root number of Arabidopsis treated with compounds Y14, Y21, and Y23
at 1 μmol/L was 4, and several compounds led to a root number of 3 and 2. The root
number of Arabidopsis treated with compounds Y3, Y12, Y13, Y14, and Y19 at 10
μmol/L was 4, and several compounds led to a root number of 3 and 2. When the
concentration was 100 μmol/L, the inhibition was too strong, which made the
Arabidopsis roots to almost disappear and grow abnormally.
3.3.3. Evaluation of eff ect on Arabidopsis mutants
In addition, the gibberellin-deficient dwarf mutant (ga1-1) of Arabidopsis that
could not grow normally without the application of exogenous GA, was also cultured
in order to judge whether or not compound Y21 possessed a GA-like function of
restoring mutant growth. The growth condition of the mutants can be seen in Fig. 5A
and Fig. 5B. According to the results, it was observed that the 7-year-old mutant,
treated without GA3 or compound Y21, produced only short radicles and grew slowly,
but after treated with GA3 or compound Y21, it recovered its growth at different
extent. As shown in Fig. 5A, the mutants treated with GA3 at 10 and 100 μmol/L,
grew well and its hypocotyl elongation was obvious, however, a weak root inhibition
was observed at the concentration of 100 μmol/L. And the same biological activities