Identification of acylthiourea derivatives as potent Plk1 PBD inhibitors

Article abstract of DOI:10.1016/j.ejmech.2016.08.043

Thiourea derivatives have drawn much attention for their latent capacities of biological activities. In this study, we designed acylthiourea compounds as polo-like kinase 1 (Plk1) polo-box domain (PBD) inhibitors. A series of acylthiourea derivatives without pan assay interference structure (PAINS) were synthesized. Four compounds with halogen substituents exhibited binding affinities to Plk1 PBD in low micromole range. The most potent compound (3v) showed selectivity over other subtypes of Plk PBDs and inhibited the kinase activity of full-length Plk1.

Full text of DOI:10.1016/j.ejmech.2016.08.043

Accepted Manuscript
Identification of acylthiourea derivatives as potent Plk1 PBD inhibitors
Taikangxiang Yun, Qin Tan, Ying Liu, Luhua Lai
PII:
S0223-5234(16)30686-9
10.1016/j.ejmech.2016.08.043
EJMECH 8839
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 28 June 2016
Revised Date: 18 August 2016
Accepted Date: 19 August 2016
Please cite this article as: T. Yun, Q. Tan, Y. Liu, L. Lai, Identification of acylthiourea derivatives
as potent Plk1 PBD inhibitors, European Journal of Medicinal Chemistry (2016), doi: 10.1016/
j.ejmech.2016.08.043.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Identification of acylthiourea derivatives as
potent Plk1 PBD inhibitors
Taikangxiang Yuna, Tan Qinb, Ying Liua, b*, Luhua Laia, b, c
*
a Center for Quantitative Biology, Academy for Advanced Interdisciplinary Studies,
Peking University, Beijing 100871, China
b BNLMS, State Key Laboratory for Structural Chemistry of Unstable and Stable
Species, College of Chemistry and Molecular Engineering, Peking University, Beijing
100871, China
c Peking−Tsinghua Center for Life Sciences, Peking University, Beijing 100871,
China
*To whom correspondence should be addressed. Fax: (86)10-62751725 E-mail:
liuying@pku.edu.cn (Y. Liu), lhlai@pku.edu.cn (L. Lai).
ABSTRACT: Thiourea derivatives have drawn much attention for their latent
capacities of biological activities. In this study, we designed acylthiourea compounds
as polo-like kinase 1 (Plk1) polo-box domain (PBD) inhibitors. A series of
acylthiourea derivatives without pan assay interference structure (PAINS) were
synthesized. Four compounds with halogen substituents exhibited binding affinities to
Plk1 PBD in low micromole range. The most potent compound (3v) showed
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selectivity over other subtypes of Plk PBDs and inhibited the kinase activity of
full-length Plk1.
Keywords: Polo-like kinase 1; Polo-box domain; Small molecular inhibitor;
Halogenosulfamoylphenyl acylthiourea; Binding affinity; In vitro assay
1. Introduction
Polo-like kinases (Plks) belong to a conserved serine/threonine protein kinases
family. Five Plks (Plk1-5) were found in Homo sapiens[1]. The structures and
functions of Plk4-5 are distinct from Plk1-3[2]. Though with similar structure, Plk1-3
have different roles in the cell cycle. Plk1 participates in mitosis and cytokinesis. Plk2
functions as a tumor suppressor and participates in centriole duplication, G1/S
transition[3, 4]. Plk3 is a tumor suppressor and plays an important role in stress
response, cell cycle regulation and Golgi disassembly[3, 5]. Among five Plks, Plk1 is
the most studied one. Its overexpression was found in a variety of cancer cells[6-9]
and correlated with poor prognosis[10]. Plk1 is considered as a promising anti-cancer
target and its selective inhibition over other Plks is necessary[11].
Plk1 consists of a highly conserved kinase domain (KD) and a polo-box domain
(PBD). KD is the catalytic domain and similar to other protein kinases, while PBD is
a unique phosphopeptide-binding module involved in Plk target binding. It also
blocks KD to regulate its kinase activity[12]. PBD inhibitors affect the function of
Plk1 through blocking the interaction between PBD and its partner[13-17]. According
to references[3, 18-23], inhibitors targeting PBD can be divided into two categories:
phosphopeptides and small molecules. Phosphopeptide inhibitors showed high
affinity and specificity. Their Kd values were ranged from 2.0 nM to 2.5 ꢀM, while
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the affinities against Plk2 or Plk3 PBD were two orders of magnitude less[19].
Phosphopeptide mimics were also developed[20]. The phosphate group plays an
important role in binding. But phosphate ester, which is with negative charges and
prone to be hydrolyzed by intracellular phosphatases, leads to poor cell permeability
and proteolytic stability[20]. Reported small molecular PBD inhibitors have different
structures (Fig. 1) and may avoid the conundrum of phosphopeptide inhibitors.
Thymoquinone and Poloxin were identified through high throughput screening with
quinones scaffold[21]; while (-)-epigallocatechin (EGC), containing polyphenol
skeleton, was got by combined virtual and experimental screenings[22]. In addition to
blocking PBD binding to its partner, PBD inhibitors with terphenyl moiety were
reported to inhibit the kinase activity of full-length Plk1 with an IC50 value of 151
ꢀM[23]. We also found several small molecule Plk1 PBD inhibitors with diverse
scaffolds and some of them could induce mitotic arrest of HeLa cells at low
micromolar concentrations[24].
Put Fig. 1. Here
Thiourea derivatives have diverse biological properties[25-32], like
anti-inflammatory[25], anticancer[26], antimalarial[27], antituberculosis and
antithyroid. And some of them are applied in clinical practice: thioacetazone is used
in the treatment of tuberculosis[28]; and propylthiouracil is applied to treat
hyperthyroidism by decreasing of thyroid hormone[29]. Acylthiourea derivatives have
been studied by several research groups. Saeed et al. synthesized hybrid molecules
containing sulfanilamide and acylthiourea templates as urease inhibitors[30]. Mahdavi
et al. reported 3-aroyl-1-(4-sulfamoylphenyl)thiourea derivatives as 15-lipoxygenase
inhibitors[31]. Singh et al. reported N-(phenylcarbamothioyl)benzamide as histone
deacetylase 8 activator[32]. Until now, no Plk1 inhibitors with thiourea sub-structure
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have been reported. Considering the druggability of thiourea, acylthiourea derivatives
may be valuable for developing PBD inhibitors.
In the present study, we tried to design acylthiourea derivatives as potent PBD
inhibitors. N-((4-sulfamoylphenyl)carbamothioyl)acetamide (3a), derived from
screening an in-house chemical library with modest binding affinity to Plk1 PBD in
vitro, was chosen as the starting molecule. Based on 3a, a series of acylthiourea
derivatives were synthesized. Their binding affinities against Plk1 PBD were tested.
Competitive experiments were carried to verify whether the compounds bind to the
phosphopeptide binding site. Ligand binding assays were applied versus different Plk
subtypes to assure the selectivity. And kinase assays were performed to identify the
inhibition against Plk1 kinase activity.
2. Results and discussion
2.1. Chemistry
As pan assay interference structure (PAINS) may interfere in screening
technologies and cause false positives[33], non PAINS ones were designed for more
effective research. To explore the structure activity relationships, the general formula
R1CONHCSNHR2 was used. Acylthiourea fragment was kept, R1 was substituted
with aliphatic, alicyclic or aromatic hydrocarbons, R2 was substituted with
sulfamoylphenyl or its bioisosteric groups. 24 compounds (3a-x) were designed and
synthesized. The synthetic route is illustrated in Scheme 1.
Carbonyl isothiocyanate derivatives 2 were got by nucleophilic substitution
reaction between commercially available acyl chloride 1 and potassium thiocyanate.
Then the title compounds 3 were prepared by addition-elimination reaction. Through
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effective combinations of eleven carbonyl isothiocyanate (2) and ten commercially
aromatic amines, title compounds (3a-t) were obtained. 3u-x contained halogen in the
phenyl ring of R2. Their intermediate aromatic amines (Iu-x) were got by different
methods (Scheme 2). For halogenated disubstituted intermediates, Iu and Iv were got
by reacting sulfanilamide with halogen acid in hydrogen peroxide. Iodinated
disubstituted sulfanilamide, 4-amino-3,5-diiodobenzenesulfonamide, was also
synthesized by reacting sulfanilamide with iodine monochloride in acetic acid[34].
Unfortunately, the corresponding product was not obtained due to steric hindrance
effects of iodine atoms. Halogenated monosubstituted sulfanilamides,
4-amino-3-bromobenzenesulfonamide (Iw) was got by reacting sulfanilamide with
N-bromosuccinimide; 4-amino-3-iodobenzenesulfonamide (Ix) was prepared by
substitution reaction between sulfanilamide and iodine[35].
The yields of 3a-x range from 10% to 90%. R1 with aromatic rings usually led to
good yields, such as 3e-k. 1H NMR spectra of 3a-x showed two single signals both at
11-13 ppm corresponding to the iminos of thiourea, and 13C NMR revealed two
signals more than 157 ppm, presented two carbon atoms of acylthiourea fragment,
formatting characteristics of this kind of compounds.
Among the title compounds, fourteen of them (3b-c, 3k-s, 3v-x) have not been
reported.
Put Scheme 1 here
Put Scheme 2 here
2.2. Biological evaluation
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2.2.1. Binding against Plk1
We used microscale thermophoresis (MST) to measure the binding affinities
between Plk1 PBD and small molecules. MST is a method developed recently to
measure protein-ligand binding by detecting the mobility of molecules in temperature
gradients[36]. Phosphopeptide PLHSpT was used as a positive reference according to
previous studies[20, 37, 38]. Poloxin was also chosen as another reference.
The modest binding affinity of 3a to Plk1 PBD is 73.7±6.3 ꢀM (Table 1),
indicating the potential of acylthiourea as Plk1 PBD inhibitors. Then synthesized
compounds (3b-x) have been tested for their interactions with PBD. Ten compounds
(3b-k) have difference at R1. Changing R1 from methyl in 3a to ethyl (3b) or phenyl
(3e) group did not alter the binding affinities significantly, while the other R1 groups
like cyclopropyl (3c) or benzyl (3d) deteriorated binding (data not shown). We then
studied the influence of R2 on binding. Moving the sulfonamide group from the
para-position to the meta-position (3l) destroyed binding and substituents on the
sulfonamide (3m-q) also deteriorated binding (data not shown). Starting from 3e, we
further tested halogen substituents in the sulfamoylphenyl group at R2 (3u-x). Binding
affinities of all the four compounds were significantly improved with one order of
magnitude. Mono-substitutions (3w-x) improved binding significantly and
di-substitutions (3u-v) were much stronger. The dissociation constants of the four
compounds are all at single-digit micromole, stronger than PLHSpT (Kd = 14.0±1.3
ꢀM) and Poloxin (Kd = 10.5 ± 0.9 ꢀM) under our assay condition. The
dibromosubstituted compound, 3v, showed the strongest binding affinity with a Kd of
2.3±0.1 ꢀM.
Put Table 1. here
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We further tested the inhibition of 3v to Plk1 PBD by fluorescence polarization
assay. 3v (IC50 = 39.8 ± 3.5 ꢀM) blocked the binding of fluorescein-labeled
phosphopeptide to Plk1 PBD (Fig. S2) and its inhibition is comparable to Poloxin
(IC50 = 29.4±5.8 ꢀM) under our assay condition.
To verify the binding site of the compounds, the binding affinity of 3v was
measured in presence of PLHSpT (with a concentration of 240 ꢀM) by MST. The
thermophoresis signal dropped greatly (Fig. S3), suggesting that 3v competes with the
phosphopeptide for the same binding site in PBD.
2.2.2. Selectivity of 3v against other Plk family members
Selective inhibiting Plk1 hopes to get anticancer drugs. We test the selectivity of
3v: ligand binding assays were applied against Plk2 and Plk3 PBDs. Primary amines
(Lys or Arg) of different Plk subtypes were labeled to form stable
dye-protein-conjugates for fluorescence detection with MST. No significant signal
changes and concentration dependence were observed, indicating that 3v is highly
selective and does not bind with Plk2 (Fig. S4A) or Plk3 PBD (Fig. S4B).
2.2.3. Inhibition of full-length Plk1
Whether 3v can inhibit the kinase activity of full-length Plk1 or not was tested,
with in vitro kinase assay by detecting the formation of ADP. The IC50 value of 3v is
95±21 ꢀM (Fig. 2), which is better than the most effective PBD inhibitor reported by
Mita et al. (IC50 = 151 ꢀM)[23]. This suggests that 3v not only binds to PBD affecting
its interaction with phosphopeptide, but also inhibits the kinase activity of full-length
Plk1. Molecular mechanism of these dual activities needs to be further explored.
Put Fig. 2. Here
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2.3. Molecular docking study
In the complex structure of Plk1 PBD with PLHSpT (PDB code: 3HIK)[39], the
minimal phosphopeptide occupies two pockets of PBD: its SpT motif acts as an
anchor to occupy the phosphate-binding pocket (His538/Lys540), and the Pro residue
occupies the pyrrolidine-binding pocket (Trp414/Arg516/Phe535)[20, 39].
Compound 3a occupies the same binding sites, both phosphate-binding pocket
and pyrrolidine-binding pocket, as PLHSpT does. In the binding mode of 3a,
acylthiourea fragment plays an important role. The imino groups of thiourea form
hydrogen bonds to the main-chain oxygen atom of Leu491. And the sulfur atom of
thiourea forms a hydrogen bond to the side-chain of the positively charged residue
Lys540. For the sulfanilamide moiety, its amino group of R2 forms a hydrogen bond
to the main-chain oxygen atom of Trp414 (Fig. 3A). There is still unoccupied space
between phosphate-binding pocket and pyrrolidine-binding pocket (Fig. 3B).
Elongation of compound to occupy this space may improve its binding to PBD.
The binding poses of our synthesized compounds are similar in modelling.
Compounds 3e and 3v are analyzed as examples (Fig. 3C). Their hydrogen-bonding
interactions are similar: both compounds contact to Trp414, Leu491 and Lys540; and
their phenyl ring of sulfonamide moiety at R2 both forms a T-shape π-π stacking with
Trp414.
However, in experiment, the binding affinities of our compounds are different:
the ones with halogen in R2 (3u-x) are higher than the others (such as 3a and 3e). For
the unique properties of 3v, one bromine atom might form a halogen bond with the
side-chain indole of Trp414, meanwhile the halogen atom occupies the unoccupied
space between phosphate-binding pocket and pyrrolidine-binding pocket. The other
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bromine atom might form another halogen bond with the main-chain amide hydrogen
atom of Leu491 (Fig. 3D). It is concluded that halogen atoms have crucial effects on
binding abilities. Chemical properties of halogen atoms have been reported to render
halogen bonding which is useful in drug design[40]. Sometimes halogen bonding
plays an important role in molecular recognition with comparable energetic
contribution as hydrogen bonding[39].
Put Fig. 3. Here
3. Conclusion
In this study, we explored the possibility of using acylthiourea derivatives as
potent
Plk1
PBD
inhibitors.
The
starting
compound,
N-((4-sulfamoylphenyl)carbamothioyl)acetamide (3a) showed moderate binding
strength to Plk1 PBD. Based on 3a, we designed a series of derivatives and tested
their binding affinities to Plk1 PBD. The important optimizations were the addition of
functional group in the phenyl ring of R2 to occupy the vacant space between
phosphate-binding pocket and pyrrolidine-binding pocket, and introducing halogen
atoms to form the halogen bonds. Both lead to the binding affinities increased. The
best compound 3v binds to Plk1 PBD with 30-fold higher affinity than 3a and 5-fold
higher affinity than PLHSpT. 3v is proved with selectivity over Plk2 and Plk3 PBDs
and also inhibited full-length Plk1 kinase activity. Being a dual functional compound,
3v can be further optimized for its activities and used as a molecular probe to study
the regulatory mechanism of Plk1.
4. Experimental section
4.1. Synthesis
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The reagents and solvents were commercially available and purified according to
conventional methods. Melting points were determined on an X-4 microscopic
1
melting point apparatus and uncorrected. H spectra were recorded at 400 MHz and
13C spectra were recorded at 100 MHz on a Bruker Ascend spectrometer. The
chemical shift values (δ) are reported in parts per million (ppm) relative to
1
tetramethylsilane as internal standard in DMSO-d6. H spectra in methanol-d4 were
recorded furthermore to assign active hydrogens of some compounds. High resolution
mass spectra (HRMS) were obtained on a Bruker Apex IV FTMS mass spectrometer
using electrospray ionization (ESI). Elemental analyses were performed on an
Elementar Vario EL CUBE instrument. Analyses of representative compounds
indicated within ± 0.4 % of the theoretical values (C, H, N).
4.1.1. General procedure for the synthesis of compounds Iu-x
4.1.1.1. 4-amino-3,5-dichlorobenzenesulfonamide (Iu).
Sulfanilamide (0.43 g, 0.0025 mol) was stirred in 15 mL of 6 N halogen acid (0.08
mol) at room temperature, and 2 ml of 30% hydrogen peroxide (0.02 mol) was slowly
added. After 5 h, the product was filtered and recrystallized from ethanol. Pink solid.
1
Yield: 70%. H NMR (DMSO-d6, 400MHz): δ 7.61 (s, 2H, ArH), 7.23 (s, 2H,
SO2NH2), 6.29 (s, 2H, NH2). 13C NMR (DMSO-d6, 100MHz): δ 144.2, 131.3, 125.7,
117.0.
4.1.1.2. 4-amino-3,5-dibromobenzenesulfonamide (Iv).
Sulfanilamide (0.43 g, 0.0025 mol) was stirred in 15 mL of 6 N halogen acid (0.08
mol) at room temperature, and 2 ml of 30% hydrogen peroxide (0.02 mol) was slowly
added. After 5 h, the product was filtered and recrystallized from ethanol. White solid.
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1
Yield: 62%. H NMR (DMSO-d6, 400MHz): δ 7.79 (s, 2H, ArH), 7.26 (s, 2H,
SO2NH2), 6.11 (s, 2H, NH2). 13C NMR (DMSO-d6, 100MHz): δ 145.8, 132.7, 129.5,
106.1.
4.1.1.3. 4-amino-3-bromobenzenesulfonamide (Iw).
o
4-amino-benzenesulfonamide (1 g, 5.81 mmol) was stirred in DMF (5 mL) at 0 C,
and 1.03 g NBS (5.81 mmol) was added. Stirred for 1 h, the mixture was quenched by
water. The product was filtered and recrystallized from ethanol. White solid. Yield:
74%. 1H NMR (DMSO-d6, 400MHz): δ 7.76 (m, 1H, ArH), 7.74 (dd, J = 8.5, 2.1 Hz,
1H, ArH), 7.07 (s, 2H, SO2NH2), 6.82 (d, J = 8.5 Hz, 1H, ArH), 6.04 (s, 2H, NH2).
13C NMR (DMSO-d6, 100MHz): δ 148.8, 131.7, 130.2, 126.3, 114.0, 105.5.
4.1.1.4. 4-amino-3-iodobenzenesulfonamide (Ix).
4-amino-benzenesulfonamide (1 g, 5.81 mmol) was dissolved in glacial acetic acid (5
o
mL), and 0.65 g iodine (2.56 mmol) was added. After being stirred at 85 C for 1 h,
sodium bisulfite (0.16 g, 1.54 mmol) was added to destroy the excess iodine. 10 mL
water was added. The product was filtered and recrystallized from ethanol. Silver
1
solid. Yield: 54%. H NMR (DMSO-d6, 400 MHz): δ 7.94 (d, J = 2.1 Hz, 1H, ArH),
7.48 (dd, J = 8.5, 2.1 Hz, 1H, ArH), 7.05 (s, 2H, SO2NH2), 6.77 (d, J = 8.5 Hz, 1H,
ArH), 5.93 (s, 2H, NH2). 13C NMR (DMSO-d6, 100MHz): δ 151.5, 136.5, 132.2,
127.0, 112.8, 80.7.
4.1.2. General procedure for the synthesis of compounds 3a-x
A solution of acyl chloride (5 mmol) and potassium thiocyanate (5.5 mmol) in
acetone (20 mL) was reacted under reflux for 1 h. Aromatic amine (5 mmol) was
added and the mixture was stirred under reflux for 2 h. The reaction mixture was
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cooled to room temperature. The precipitate was filtered, washed with water and
recrystallized from ethanol.
4.1.2.1. N-((4-sulfamoylphenyl)carbamothioyl)acetamide (3a). White solid. Yield:
1
60%. m.p. 238-240 °C. H NMR (DMSO-d6, 400MHz): δ 12.63 (s, 1H, NH), 11.61
13
(s, 1H, NH), 7.84 (m, 4H, ArH), 7.40 (s, 2H, SO2NH2), 2.17 (s, 3H, CH3). C NMR
(DMSO-d6, 100MHz): δ 179.1, 172.9, 141.0, 140.6, 126.2, 124.3, 23.8. HRMS (ESI):
calcd for C9H11N3O3S2, [(M+H)+], 274.0315, found 274.0314.
4.1.2.2. N-((4-sulfamoylphenyl)carbamothioyl)propionamide (3b). White solid. Yield:
1
67%. m.p. 206-208 °C. H NMR (DMSO-d6, 400MHz): δ 12.66 (s, 1H, NH), 11.56
(s, 1H, NH), 7.84 (m, 4H, ArH), 7.38 (s, 2H, SO2NH2), 2.48 (m, 2H, CH2), 1.06 (t, J =
7.5 Hz, 3H, CH3). 13C NMR (DMSO-d6, 100MHz): δ 179.1, 176.3, 141.0, 140.7,
126.2, 124.3, 29.2, 8.5. HRMS (ESI): calcd for C10H13N3O3S2, [(M+H)+], 288.0471,
found 288.0474.
4.1.2.3. N-((4-sulfamoylphenyl)carbamothioyl)cyclopropanecarboxamide (3c). White
1
solid. Yield: 45%. m.p. 229-231 °C. H NMR (DMSO-d6, 400MHz): δ 12.66 (s, 1H,
NH), 11.91 (s, 1H, NH), 7.84 (m, 4H, ArH), 7.37 (s, 2H, SO2NH2), 2.12 (td, J = 7.9,
4.0 Hz, 1H, CH), 0.97 (m, 4H, CH2). 13C NMR (DMSO-d6, 100MHz): δ 178.7, 175.9,
141.2, 140.7, 126.2, 124.1, 14.2, 9.4. HRMS (ESI): calcd for C11H13N3O3S2,
[(M+H)+], 300.0471, found 300.0468.
4.1.2.4. 2-phenyl-N-((4-sulfamoylphenyl)carbamothioyl)acetamide (3d). Pale yellow
1
solid. Yield: 71%. m.p. 222-224 °C. H NMR (DMSO-d6, 400MHz): δ 12.53 (s, 1H,
NH), 11.82 (s, 1H, NH), 7.82 (s, 4H, ArH), 7.33 (m, 7H, ArH and SO2NH2), 3.82 (s,
2H, CH2). 13C NMR (DMSO-d6, 100MHz): δ 179.1, 173.2, 141.3, 140.6, 134.1,
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129.5, 128.4, 127.0, 126.2, 124.3, 42.4. HRMS (ESI): calcd for C15H15N3O3S2,
[(M+H)+], 350.0628, found 350.0629.
4.1.2.5. N-((4-sulfamoylphenyl)carbamothioyl)benzamide (3e). Pale yellow solid.
1
Yield: 56%. m.p. 218-220 °C. H NMR (DMSO-d6, 400MHz): δ 12.71 (s, 1H, NH),
11.70 (s, 1H, NH), 7.99 (m, 2H, ArH), 7.92 (m, 2H, ArH), 7.85 (m, 2H, ArH), 7.67
(m, 1H, ArH), 7.55 (m, 2H, ArH), 7.40 (s, 2H, SO2NH2). 13C NMR (DMSO-d6,
100MHz): δ 179.4, 168.2, 141.3, 140.9, 133.2, 132.1, 128.7, 128.5, 126.3, 124.3.
HRMS (ESI): calcd for C14H13N3O3S2, [(M+H)+], 336.0471, found 336.0473.
4.1.2.6. 4-methyl-N-((4-sulfamoylphenyl)carbamothioyl)benzamide (3f). White solid.
Yield: 54%. m.p. 227-229 °C. 1H NMR (400MHz, DMSO): 12.76 (s, 1H, NH), 11.59
(s, 1H, NH), 7.89 (m, 6H, ArH), 7.37 (m, 4H, ArH and SO2NH2), 2.40 (s, 3H, CH3).
13C NMR (DMSO-d6, 100MHz): δ 179.4, 168.1, 144.0, 141.1, 140.9, 129.2, 128.9,
128.6, 126.3, 124.4, 21.1. HRMS (ESI): calcd for C15H15N3O3S2, [(M+H)+],
350.0628, found 350.0626.
4.1.2.7. 3-methyl-N-((4-sulfamoylphenyl)carbamothioyl)benzamide (3g). Pale yellow
1
solid. Yield: 71%. m.p. 215-217 °C. H NMR (DMSO-d6, 400MHz): δ 12.73 (s, 1H,
NH), 11.61 (s, 1H, NH), 7.88 (m, 5H, ArH), 7.78 (d, J = 7.7 Hz, 1H, ArH), 7.47 (m,
2H, ArH), 7.39 (s, 2H, SO2NH2), 2.40 (s, 3H, CH3). 13C NMR (DMSO-d6, 100MHz):
δ 179.4, 168.4, 141.1, 140.9, 138.1, 133.9, 131.8, 128.9, 128.5, 126.3, 125.7, 124.5,
20.7. HRMS (ESI): calcd for C15H15N3O3S2, [(M+H)+], 350.0628, found 350.0627.
4.1.2.8. 4-nitro-N-((4-sulfamoylphenyl)carbamothioyl)benzamide (3h). Pale yellow
1
solid. Yield: 48%. m.p. 208-210 °C. H NMR (DMSO-d6, 400MHz): δ 12.48 (s, 1H,
NH), 12.05 (s, 1H, NH), 8.36 (d, J = 8.0 Hz, 2H, ArH), 8.18 (d, J = 8.2 Hz, 2H, ArH),
7.87 (m, 4H, ArH), 7.40 (s, 2H, SO2NH2). 13C NMR (DMSO-d6, 100MHz): δ 179.1,
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166.6, 149.8, 141.4, 140.8, 138.0, 130.3, 126.3, 124.4, 123.4. HRMS (ESI): calcd for
C14H12N4O5S2, [(M+H)+], 381.0322, found 381.0325.
4.1.2.9. 3-nitro-N-((4-sulfamoylphenyl)carbamothioyl)benzamide (3i). Pale yellow
1
solid. Yield: 50%. m.p. 214-216 °C. H NMR (DMSO-d6, 400MHz): δ 12.52 (s, 1H,
NH), 12.12 (s, 1H, NH), 8.79 (s, 1H, ArH), 8.50 (d, J = 8.3 Hz, 1H, ArH), 8.38 (d, J =
13
7.3 Hz, 1H, ArH), 7.87 (m, 5H, ArH), 7.40 (s, 2H, SO2NH2). C NMR (DMSO-d6,
100MHz): δ 179.2, 166.2, 147.4, 141.4, 140.8, 135.1, 133.8, 130.2, 127.4, 126.3,
124.4, 123.7. HRMS (ESI): calcd for C14H12N4O5S2, [(M+H)+], 381.0322, found
381.0326.
4.1.2.10. 4-[3-(Furan-2-carbonyl)-thioureido]-benzenesulfonamide (3j). White solid.
1
Yield: 56%. m.p. 208-210 °C. H NMR (DMSO-d6, 400MHz): δ 12.46 (s, 1H, NH),
11.44 (s, 1H, NH), 8.09 (s, 1H, ArH), 7.87 (m, 5H, ArH), 7.39 (s, 2H, SO2NH2), 6.78
(dd, J = 3.4, 1.4 Hz, 1H, ArH). 13C NMR (DMSO-d6, 100MHz): δ 179.0, 157.5,
148.6, 144.6, 141.3, 140.9, 126.2, 124.4, 118.8, 112.7. HRMS (ESI): calcd for
C12H11N3O4S2, [(M+H)+], 326.0264, found 326.0261.
4.1.2.11. N-((4-sulfamoylphenyl)carbamothioyl)-1-naphthamide (3k). White solid.
1
Yield: 86%. m.p. 219-221 °C. H NMR (DMSO-d6, 400MHz): δ 12.75 (s, 1H, NH),
12.10 (s, 1H, NH), 8.20 (dd, J = 43.9, 8.2 Hz, 2H, ArH), 7.94 (m, 6H, ArH), 7.64 (m,
3H, ArH), 7.42 (s, 2H, SO2NH2). 13C NMR (DMSO-d6, 100MHz): δ 179.3, 169.9,
141.3, 140.9, 133.0, 131.6, 131.3, 129.5, 128.5, 127.4, 127.2, 126.5, 126.3, 124.8,
124.4, 122.4. HRMS (ESI): calcd for C18H15N3O3S2, [(M+H)+], 386.0628, found
386.0630.
4.1.2.12. N-((3-sulfamoylphenyl)carbamothioyl)acetamide (3l). Pink solid. Yield:
1
33%. m.p. 207-209 °C. H NMR (DMSO-d6, 400MHz): δ 12.58 (s, 1H, NH), 11.60
14

ACCEPTED MANUSCRIPT
(s, 1H, NH), 8.11 (t, J = 2.0 Hz, 1H, ArH), 7.83 (m, 1H, ArH), 7.69 (m, 1H, ArH),
13
7.59 (t, J = 7.9 Hz, 1H, ArH), 7.44 (s, 2H, SO2NH2), 2.17 (s, 3H, CH3). C NMR
(DMSO-d6, 100MHz): δ 179.2, 172.8, 144.5, 138.2, 129.3, 127.6, 123.3, 121.4, 23.8.
HRMS (ESI): calcd for C9H11N3O3S2, [(M+H)+], 274.0315, found 274.0320.
4.1.2.13. N-((4-(N-carbamimidoylsulfamoyl)phenyl)carbamothioyl)acetamide (3m).
1
White solid. Yield: 45%. m.p. 239-241 °C. H NMR (DMSO-d6, 400MHz): δ 12.61
(s, 1H, NH), 11.58 (s, 1H, NH), 7.78 (m, 4H, ArH), 6.73 (br, 4H, guanidylH), 2.16 (s,
3H, CH3). 13C NMR (DMSO-d6, 100MHz): δ 178.9, 172.8, 158.1, 141.8, 140.1,
126.1, 123.9, 23.8. HRMS (ESI): calcd for C10H13N5O3S2, [(M+H)+], 316.0533, found
316.0532.
4.1.2.14. N-((4-(N-acetylsulfamoyl)phenyl)carbamothioyl)acetamide (3n). White
1
solid. Yield: 20%. m.p. 219-221 °C. H NMR (DMSO-d6, 400MHz): δ 12.73 (s, 1H,
NH), 12.12 (s, 1H, NH), 11.64 (s, 1H, NH), 7.92 (m, 4H, ArH), 2.17 (s, 3H, CH3),
1.94 (s, 3H, CH3). 13C NMR (DMSO-d6, 100MHz): δ 179.0, 172.9, 169.2, 142.2,
135.9, 128.3, 124.0, 23.8, 23.1. HRMS (ESI): calcd for C11H13N3O4S2, [(M+H)+],
316.0420, found 316.0424.
4.1.2.15. N-((4-(N-(thiazol-2-yl)sulfamoyl)phenyl)carbamothioyl)acetamide (3o). Pale
1
yellow solid. Yield: 35%. m.p. 230-232 °C. H NMR (DMSO-d6, 400MHz): δ 12.79
(s, 1H, NH), 12.64 (s, 1H, NH), 11.59 (s, 1H, NH), 7.82 (m, 4H, ArH), 7.27 (d, J =
4.6 Hz, 1H, ArH), 6.85 (d, J = 4.6 Hz, 1H, ArH), 2.16 (s, 3H, CH3). 13C NMR
(DMSO-d6, 100MHz): δ 179.0, 172.9, 168.9, 140.9, 139.2, 126.4, 124.4, 124.3,
108.4, 23.8. HRMS (ESI): calcd for C12H12N4O3S3, [(M+H)+], 357.0144, found
357.0140.
15

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4.1.2.16. N-((4-(N-(pyrimidin-2-yl)sulfamoyl)phenyl)carbamothioyl)acetamide (3p).
1
White solid. Yield: 60%. m.p. 247-249 °C. H NMR (DMSO-d6, 400MHz): δ 12.69
(s, 1H, NH), 11.88 (s, 1H, NH), 11.61 (s, 1H, NH), 8.52 (d, J = 4.9 Hz, 2H, ArH),
7.95 (m, 4H, ArH), 7.07 (t, J = 4.9 Hz, 1H, ArH), 2.16 (s, 3H, CH3). 13C NMR
(DMSO-d6, 100MHz): δ 178.9, 172.9, 158.3, 156.6, 141.6, 137.2, 128.2, 123.8,
115.7, 23.7. HRMS (ESI): calcd for C13H13N5O3S2, [(M+H)+], 352.0533, found
352.0528.
4.1.2.17.
N-((4-(N-(3,4-dimethylisoxazol-5-yl)sulfamoyl)phenyl)carbamothioyl)acetamide (3q).
1
White solid. Yield: 54%. m.p. 211-213 °C. H NMR (DMSO-d6, 400MHz): δ 12.71
(s, 1H, NH), 11.64 (s, 1H, NH), 11.09 (s, 1H, NH), 7.94 (m, 2H, ArH), 7.77 (m, 2H,
ArH), 2.17 (s, 3H, CH3), 2.09 (s, 3H, CH3), 1.64 (s, 3H, CH3). 13C NMR (DMSO-d6,
100MHz): δ 179.0, 172.9, 161.7, 155.2, 142.0, 136.7, 127.3, 124.4, 105.4, 23.8, 10.2,
5.7. HRMS (ESI): calcd for C14H16N4O4S2, [(M+H)+], 369.0686, found 369.0686.
4.1.2.18. N-((4-hydroxyphenyl)carbamothioyl)acetamide (3r). Brown solid. Yield:
1
20%. m.p. 222-224 °C. H NMR (DMSO-d6, 400MHz): δ 12.26 (s, 1H, NH), 11.37
(s, 1H, NH), 9.55 (s, 1H, OH), 7.34 (m, 2H, ArH), 6.76 (m, 2H, ArH), 2.14 (s, 3H,
CH3). 13C NMR (DMSO-d6, 100MHz): δ 178.7, 172.7, 155.7, 129.1, 125.8, 115.1,
23.8. HRMS (ESI): calcd for C9H10N2O2S, [(M+H)+], 211.0536, found 211.0539.
4.1.2.19. 4-(3-acetylthioureido)benzamide (3s). Pale yellow solid. Yield: 10%. m.p.
240-242 °C. 1H NMR (DMSO-d6, 400MHz): δ 12.64 (s, 1H, NH), 11.56 (s, 1H, NH),
7.99 (s, 1H, CONH), 7.89 (m, 2H, ArH), 7.75 (m, 2H, ArH), 7.39 (s, 1H, CONH),
2.17 (s, 3H, CH3). 1H NMR (methanol-d4, 400MHz): δ 7.88 (d, J = 3.6 Hz, 4H, ArH),
2.17 (s, 3H, CH3). 13C NMR (DMSO-d6, 100MHz): δ 178.7, 172.9, 167.5, 140.3,
16

ACCEPTED MANUSCRIPT
131.5, 128.0, 123.4, 23.8. HRMS (ESI): calcd for C10H11N3O2S, [(M+H)+], 238.0645,
found 238.0645.
4.1.2.20. N-((4-acetamidophenyl)carbamothioyl)benzamide (3t). White solid. Yield:
1
90%. m.p. 211-213 °C. H NMR (DMSO-d6, 400MHz): δ 12.53 (s, 1H, NH), 11.53
(s, 1H, NH), 10.04 (s, 1H, NH), 7.98 (d, J = 7.0 Hz, 2H, ArH), 7.57 (m, 7H, ArH),
2.06 (s, 3H, CH3). 13C NMR (DMSO-d6, 100MHz): δ 178.9, 168.3, 168.2, 137.5,
133.1, 132.8, 132.2, 128.6, 128.4, 124.8, 119.0, 24.0. HRMS (ESI): calcd for
C16H15N3O2S, [(M+H)+], 314.0958, found 314.0959.
4.1.2.21. N-((2,6-dichloro-4-sulfamoylphenyl)carbamothioyl)benzamide (3u). White
1
solid. Yield: 52%. m.p. 222-224 °C. H NMR (DMSO-d6, 400MHz): δ 12.10 (s, 1H,
NH), 11.98 (s, 1H, NH), 8.03 (d, J = 7.4 Hz, 2H, ArH), 7.92 (s, 2H, ArH), 7.70 (m,
1
3H, ArH and SO2NH2), 7.56 (t, J = 7.7 Hz, 2H, ArH). H NMR (methanol-d4,
400MHz): δ 7.99 (m, 4H, ArH), 7.68 (t, J = 7.4 Hz, 1H, ArH), 7.56 (t, J = 7.7 Hz, 2H,
ArH). 13C NMR (DMSO-d6, 100MHz): δ 180.9, 168.0, 144.6, 137.8, 134.6, 133.4,
131.8, 128.8, 128.5, 125.4. HRMS (ESI): calcd for C14H11Cl2N3O3S2, [(M+H)+],
403.9692, found 403.9693.
4.1.2.22. N-((2,6-dibromo-4-sulfamoylphenyl)carbamothioyl)benzamide (3v). White
1
solid. Yield: 35%. m.p. 232-234 °C. H NMR (DMSO-d6, 400MHz): δ 12.16 (s, 1H,
NH), 11.98 (s, 1H, NH), 8.05 (m, 4H, ArH), 7.70 (m, 3H, ArH and SO2NH2), 7.56 (t,
J = 7.8 Hz, 2H, ArH). 13C NMR (DMSO-d6, 100MHz): δ 180.5, 168.0, 145.0, 140.4,
133.4, 131.7, 128.9, 128.8, 128.5, 124.8. HRMS (ESI): calcd for C14H11Br2N3O3S2,
[(M+H)+], 491.8681, found 491.8673.
4.1.2.23. N-((2-bromo-4-sulfamoylphenyl)carbamothioyl)benzamide (3w). White
1
solid. Yield: 74%. m.p. 226-227 °C. H NMR (DMSO-d6, 400MHz): δ 12.70 (s, 1H,
17

ACCEPTED MANUSCRIPT
NH), 11.96 (s, 1H, NH), 8.12 (m, 2H, ArH), 8.01 (d, J = 7.6 Hz, 2H, ArH), 7.86 (dd, J
= 8.4, 2.1 Hz, 1H, ArH), 7.69 (t, J = 7.8 Hz, 1H, ArH), 7.56 (m, 4H, ArH and
SO2NH2). 1H NMR (methanol-d4, 400MHz): δ 8.46 (d, J = 8.6 Hz, 1H, ArH), 8.20 (d,
J = 2.0 Hz, 1H, ArH), 7.99 (m, 2H, ArH), 7.90 (dd, J = 8.6, 2.0 Hz, 1H, ArH), 7.68 (t,
J = 7.5 Hz, 1H, ArH), 7.57 (t, J = 7.7 Hz, 2H, ArH). 13C NMR (DMSO-d6, 100MHz):
δ 180.4, 168.5, 143.3, 140.0, 133.3, 131.8, 129.8, 129.0, 128.8, 128.5, 125.1, 119.4.
HRMS (ESI): calcd for C14H12BrN3O3S2, [(M+H)+], 413.9576, found 413.9575.
4.1.2.24. N-((2-iodo-4-sulfamoylphenyl)carbamothioyl)benzamide (3x). White solid.
1
Yield: 72%. m.p. 230-232 °C. H NMR (DMSO-d6, 400MHz): δ 12.52 (s, 1H, NH),
11.92 (s, 1H, NH), 8.31 (s, 1H, ArH), 8.02 (d, J = 7.5 Hz, 2H, ArH), 7.87 (s, 2H,
ArH), 7.69 (t, J = 7.4 Hz, 1H, ArH), 7.59 (m, 4H, ArH and SO2NH2). 13C NMR
(DMSO-d6, 100MHz): δ 180.6, 168.4, 143.5, 143.4, 135.8, 133.4, 131.8, 129.0,
128.8, 128.5, 125.7, 97.7. HRMS (ESI): calcd for C14H12IN3O3S2, [(M+H)+],
461.9438, found 461.9433.
4.2. Biology
4.2.1. Expression and purification of PBDs
Protein expression and purification were performed similar as previously
described[24]. Escherichia coli strain Rosetta (DE3) carried plasmids were grown
until the OD600 reached 0.6, and introduced with IPTG (final concentration 0.4 mM).
The cells were grown overnight at 20 °C.
Cells were resuspended and then lysed in buffer contained 20 mM HEPES, pH
7.4, 300 mM NaCl, 0.1 mM PMSF and 1 mM DDT. The cell lysates were clarified.
The supernatants of Plk1 PBD were filtered and then applied to nickel column (GE
18

ACCEPTED MANUSCRIPT
Healthcare) and further purified by Sephacryl S200 gel-filtration column (GE
Healthcare) that had been equilibrated with buffer contained 20 mM HEPES, pH 7.4,
300 mM NaCl. The supernatants of Plk2 PBD, Plk3 PBD were filtered, applied to
GSTrap column (GE Healthcare) and further purified by Sephacryl S200 gel-filtration
column (GE Healthcare) respectively. The final purities of PBDs were more than
90%.
4.2.2. Expression and purification of full-length Plk1
Protein expression and purification were performed similar as previously
described[24]. Escherichia coli strain Rosetta (DE3) carried plasmids were grown
until the OD600 reached 0.6, and introduced with IPTG (final concentration 0.6 mM).
The cells were grown overnight at 25 °C for 8 h.
Cells were resuspended and then lysed in buffer contained 40 mM MOPS, pH
7.4, 200 mM NaCl, 0.1 mM PMSF and 1 mM DDT. The cell lysates were clarified.
The supernatants were filtered and then applied to nickel column (GE Healthcare) and
further purified by Sephacryl S200 gel-filtration column (GE Healthcare) that had
been equilibrated with buffer contained 40 mM MOPS, pH 7.4, 200 mM NaCl. The
final purity of full-length Plk1 was more than 90%.
4.2.3. Microscale thermophoresis based ligand binding assay
The peptide of PLHSpT (purity judged by HPLC > 99%) was from GL Biochem
(Shanghai) Ltd. Poloxin (purity > 98%) was purchased from MedChem Express.
According to the manufacturer’s protocol the PBD of Plks was labeled with the
red fluorescent dye NT-647. Measurements were carried out in the solution contained
20 mM HEPES, pH 7.4, 300 mM NaCl, 0.05% tween-20, 5% DMSO, by using 20%
19

ACCEPTED MANUSCRIPT
LED power and 20% MST power. The concentration of PBD was adjusted by the
fluorescence and was about 0.2 ꢀM. DMSO was used as negative control.
Nanotemper Analysis software V1.5.41 was applied to analyze data.
4.2.4. Fluorescence polarization assay
The fluorescein-labeled peptide of 5-FAM-DPPLHSpTAI-OH (purity judged by
HPLC > 96%) was from GL Biochem (Shanghai) Ltd.
The FP assay procedure was based on the reference with slight
modifications[37]. Fluorescein-labeled peptide (final concentration: 20 nM) was
incubated with Plk1 PBD (final concentration: 300 nM). Final concentrations of the
buffer were as follows: 10 mM Tris, pH 8.0, 1 mM EDTA, 50 mM NaCl, 0.1%
Nonidet P-40 and 5% DMSO. Fluorescence polarization was analyzed 1 h after
mixing all components in the 96-well format at 25 °C. Assays were run as triplicates.
Results were monitored with micro-plate reader Synergy4 (BIOTEK).
4.2.5. Kinase assay
Kinase assay was performed as previously described[41]. AmpliteTM Universal
Fluorimetric Kinase Assay Kit from AAT Bioquest Inc. was applied to measure
kinase activity according to the manufacturer’s protocols. The kit monitors kinase
activity based on ADP formation. 2 ꢀL peptide of ALMDASFADQ was mixed with 2
ꢀL Plk1, then 16 ꢀL kinase buffer containing ATP and compound was added. The
final concentration of buffer components was 0.34 ꢀM Plk1, 83 ꢀM ALMDASFADQ,
200 ꢀM ATP and 5% DMSO. The mixture was incubated at 30 °C for 40ꢁmin. For
detection of kinase activity of Plk1, 20 ꢀL of ADP Sensor Buffer and 10 ꢀL of ADP
Sensor were added and incubated at 30 oC for 40ꢁmin. Finally, fluorescence was read
20

ACCEPTED MANUSCRIPT
with excitation (Ex)/emission (Em) = 540ꢁnm/590ꢁnm. Assays were run as triplicates.
IC50 values were fitted by Hill equation with OriginPro 8.
4.3. Molecular modeling
The crystal structure of Plk1 PBD-PLHSpT complex (PDB code: 3HIK[39]) was
used for the docking study. The peptide was removed from the structure first. Cavity
V1.1[42, 43] was used to analysis the binding sites and pharmacophores of Plk1 PBD.
The structures of the compounds were prepared using Ligprep with the OPLS_2005
force field in Schrödinger software package. Docking calculations were performed
using Glide[44, 45] XP mode with default settings.
Acknowledgments
This work was supported in part by the National Natural Science Foundation of
China (Grant Nos. 21573012 and 91313302) and the Ministry of Science and
Technology of China (2015CB910300). We thank Prof. Chuanmao Zhang for
engaging in scientific discussions during the course of this project.
Appendix A. Supplementary data
Supplementary data associated with this article can be found.
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CAPTION FOR FIGURES
Fig. 1. Structures of known small molecule PBD inhibitors.
Fig. 2. Dose-response curve of 3v against full-length Plk1.
Fig.3. The predicted binding models. Figures are prepared by Pymol. Hydrogen bonds
are shown with green dashed lines. A, Binding mode of 3a. B, Binding modes of
PLHSpT and 3a. Phosphopeptide is colored by magenta and 3a is colored by orange.
C, Binding modes of 3e and 3v. 3e is colored by green and 3v is colored by yellow. D,
Binding mode of 3v. Halogen bonds are shown with magenta dashed lines.
CAPTION FOR SCHEMES
Synthesis of compounds
. (i), KSCN, acetone; (ii), the amine
3a-x
Scheme 1.
derivative of R2, acetone.
Synthesis of intermediates
. (i), haloid acid, hydrogen peroxide; (ii),
Iu-x
Scheme 2.
NBS, DMF; (iii), iodine, glacial acetic acid.
CAPTION FOR TABLES
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Table 1. Dissociation constants measured by MST for synthesized compounds with
significant binding signals. Detailed results are shown in Fig. S1.
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Table 1.
Compds
R1
-
R2
-
Kd (µM)
14.0±1.3
10.5±0.9
PLHSpT
Poloxin
-
-
3a
3b
3e
73.7±6.3
64.3±3.8
68.8±3.7
5.1±0.3
2.3±0.1
6.9±0.6
5.6±0.3
3u
3v
3w
3x

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