Design, synthesis and biological evaluation of novel 3,4,5-trisubstituted aminothiophenes as inhibitors of p53-MDM2 interaction. Part 2

Article abstract of DOI:10.1016/j.bmc.2013.03.070

Five series of novel 3,4,5-trisubstituted aminothiophene derivatives and analogs were designed and synthesized based on our previous studies. All target compounds were evaluated for their p53-MDM2 binding inhibitory activities and anti-proliferation activities against A549 and PC3 tumor cell lines. Twelve compounds displayed comparable p53-MDM2 binding inhibitory activities to that of Nutlin-3. Among them, compound 7a exhibited marked binding affinity (IC ₅₀ = 0.086 μM). In addition, most target compounds showed potent anti-proliferation activities with IC₅₀ values at low micromolar level. A good selective profile for wild-type p53 expression cell line was also observed. Molecular docking analysis was performed as well to predict possible binding modes of target compounds with MDM2.

Full text of DOI:10.1016/j.bmc.2013.03.070

Accepted Manuscript
Design, synthesis and biological evaluation of novel 3,4,5-trisubstituted ami‐
nothiophenes as inhibitors of p53-MDM2 interaction. Part 2
Weisi Wang, Dan Lv, Ni Qiu, Lei Zhang, Chunqi Hu, Yongzhou Hu
PII:
S0968-0896(13)00292-7
DOI:
http://dx.doi.org/10.1016/j.bmc.2013.03.070
BMC 10727
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Please cite this article as: Wang, W., Lv, D., Qiu, N., Zhang, L., Hu, C., Hu, Y., Design, synthesis and biological
evaluation of novel 3,4,5-trisubstituted aminothiophenes as inhibitors of p53-MDM2 interaction. Part 2, Bioorganic
& Medicinal Chemistry (2013), doi: http://dx.doi.org/10.1016/j.bmc.2013.03.070
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Design, synthesis and biological evaluation of novel
3,4,5-trisubstituted aminothiophenes as inhibitors of p53-MDM2
interaction. Part 2
Weisi Wanga, Dan Lva, Ni Qiub, Lei Zhangb, Chunqi Hua, Yongzhou Hua*
aZJU-ENS Joint Laboratory of Medicinal Chemistry, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China
bInstitute of Pharmacology and Toxicology, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China.
*E-mail: huyz@zju.edu.cn; Fax: +86 571 8820 8460; Tel: +086 571 8820 8460
Abstract:
Five series of novel 3,4,5-trisubstituted aminothiophene derivatives and analogs were
designed and synthesized based on our previous studies. All target compounds were evaluated for
their p53-MDM2 binding inhibitory activities and anti-proliferation activities against A549 and
PC3 tumor cell lines. Twelve compounds displayed comparable p53-MDM2 binding inhibitory
activities to that of Nutlin-3. Among them, compound 7a exhibited marked binding affinity (IC50 =
0.086μM). In addition, most target compounds showed potent anti-proliferation activities with
IC50 values at low micromolar level. A good selective profile for wild-type p53 expression cell line
was also observed. Molecular docking analysis was performed as well to predict possible binding
modes of target compounds with MDM2.
Keywords: 3,4,5-trisubstituted aminothiophenes, P53-MDM2 interaction, Anti-cancer, SARs.
1

1. Introduction
P53, the “guardian of the genome”, is a transcriptional regulated protein which is activated by
a number of genotoxic factors1. In response to stress, p53 promotes the transcription of target
genes responsible for cell cycle arrest, DNA repair and apoptosis2, 3. However, in approximately
50% of all human tumors, p53 is inactivated by mutation or deletion4. The remaining half of
human tumors expresses wild-type p53, which is often down-regulated by other mechanisms. One
of the main reasons involves the oncoprotein MDM2 (murine double minute 2, also frequently
referred to as HDM2 in human), a master regulator of p535-7. In the last decade, the identification
of several kinds of potent small molecule p53-MDM2 binding inhibitors has demonstrated that
reactivation of p53 by disrupting the p53-MDM2 interaction is a promising anticancer therapeutic
strategy8-10
.
We have reported the identification of a 3,4,5-trisubstituted aminothiophene derivative
MCL0527 as a lead compound (Fig. 1)11. And the previous chemical modification and
structure-activity relationship studies of MCL0527, focusing on the 2-amino and 3-carboxy groups
of thiophene ring, have yielded several inhibitors with improved potency12. Herein, with the
attempt to find better p53-MDM2 binding inhibitors, the continuous chemical modification and
optimization of MCL0527 was carried out. Since the 4’-chlorophenyl at the 4-position of
thiophene ring, serving as a mimic of Trp23 residue, is a privileged structure available in many
kinds of p53-MDM2 binding inhibitors, our modification is focused on the optimization of
5-substituent, 3-ester on thiophene ring and core structure. Firstly, retaining the 3,4,5-trisubstituted
aminothiophene scaffold, the chlorine atom on the 4’-chlorophenyl at the 5-position of thiophene
ring was replaced. Secondly, in view of the wide application of amide or thioamide in drug
discovery, the amide or thioamide derivatives were prepared based on the principle of
bioisosterism. Thirdly, the core structure thiophene ring was changed into pyrrole ring or fused
rings to investigate corresponding impacts on potency. All synthesized compounds were evaluated
in vitro for their p53-MDM2 binding inhibitory activities and anti-proliferation activities against
two human cancer cell lines. Besides, molecular docking analysis was performed to determine
possible binding modes of target compounds with MDM2.
2. Results and discussion
2.1. Chemistry
3,4,5-trisubstituted aminothiophenes were prepared using the method described previously11.
The appropriate phenylacetic acids were converted into acyl chlorides (1a-e), which reacted with
chlorobenzene to give corresponding Friedel–Crafts acylation products (2a-e). Condensation of
acylation compounds (2a-e) with methyl cyanoacetate or 2-cyanoacetamide derivatives in the
presence of TiCl4 afforded olefin intermediates (3)13. Cyclization of olefin intermediates (3) with
elemental sulfur and diethylamine yielded target compounds (4a-y, Scheme 1). Some of the potent
compounds (4f, 4g, 5 and 6)11,12 were treated with Lawesson’s Reagent to afford thioamide
derivatives (7a-d, Scheme 2).
The ketone 2b was brominated to give bromoketone 814, which subsequently reacted with
ethyl 3-amino-3-iminopropanoate or 3-amino-N-cyclopropyl-3-iminopropanamide15 to yield the
3,4,5-trisubstituted aminopyrroles (10a, b, Scheme 3).
Hydrolysis of 4e with 2N NaOH aqueous solution afforded the carboxylic acid derivative 11,
which subsequently reacted with Meldrum's acid and corresponding aldehydes to give
2

dihydrothieno[2,3-b]pyridones (12a-c). Alkylation of 12a, b with bromochloropropane, followed
by reaction with morpholine or piperidine, yielded the target compounds 13a, b (Scheme 4).
Reaction of 14 with formic acid and acetic anhydride gave thieno[2,3-d]pyrimidone (15).
Treatment of 15 with phosphorus oxychloride yielded chlorinated intermediate 16, which
subsequently reacted with corresponding amines to afford thieno[2,3-d]pyrimidine derivatives
(17a-d, Scheme 5).
2.2. p53-MDM2 binding inhibitory activities
All synthesized target compounds were evaluated for their p53-MDM2 binding inhibitory
activities by fluorescence-polarization based binding assay (FP assay)16, 17. Nutlin-3 was used as
positive control. The results are summarized in Table 1-3.
As shown in Table 1, in the series of 3,4,5-trisubstituted aminothiophenes, 4’-chloro (4e-k),
4’-fluoro (4l-o), 4’-bromo (4p-t) or no substituents (4a-d) on the phenyl at 5-position of thiophene
ring generally imparted equally positive effect to the binding affinities. Introducing nitro group
(4u-y) to the 4’-position of the phenyl led to a sudden drop of potency. Retaining the
4’-chlorophenyl group at the 5-position of thiophene ring, replacing 3-carboxamide with
thioamide yielded an additive effect on potency (7a-d). For example, the thioamide derivative 7a
(Ki = 0.086 μM) is 6 times more potent than parent compound 4f (Ki = 0.54 μM). Replacing the
thiophene ring of compound 4f with pyrrole ring, compound 10b (Ki = 0.53 μM) still possessed
significant binding inhibitory activities. However, most of dihydrothieno[2,3-b]pyridone and
thieno[2,3-d]pyrimidine derivatives showed poor p53-MDM2 binding inhibitory activities (Table
2 and 3).
2.3. Tumor cell growth inhibition studies
The obtained target compounds were tested for their tumor cell growth inhibitory activity in
vitro against two human cancer cell lines, including human lung adenocarcinoma epithelial cells
A549 (wild-type p53) and human pancreatic carcinoma cells PC3 (p53 null). The results are
summarized in Table 1-3.
In general, most of the tested compounds showed good inhibitory selectivity for wild-type
p53 expression cell line A549 over p53 null cell line PC3. Particularly, compound 4l, 4r, 4s and
10a displayed more than 5-fold inhibitory selectivity for A549 over PC3, better than that of
Nutlin-3. Over 10 compounds exhibited comparable anti-proliferation activities to that of Nutlin-3
against A549 cell line. Especially, compound 10a showed the best growth inhibitory activity (IC50
= 2.53 μM). Most of the thioamide derivatives (7a-d) did not exhibit satisfactory anti-proliferation
activities, possibly due to the poor solubility. Replacing amide linkage with thioamide led to an
increase in liposolubility. For instance, the CloP value of thioamide 7a (CloP = 6.19) is much
higher than that of amide 4f (CloP = 5.38). On the other hand, dihydrothieno[2,3-b]pyridones
12a-c and thieno[2,3-d]pyrimidines 17a-d displayed weak in vitro anti-proliferation activities,
which were consistent with the MDM2 binding assay results. Interestingly, 3,4,5-trisubstituted
aminopyrroles (10a, b) showed good performance in not only p53-MDM2 binding inhibition but
also tumor cell anti-proliferation . Therefore, they can be utilized as novel leads for further
investigation.
2.4. Molecular docking studies and structural alignment analysis
3

To examine possible binding modes of target compounds (e.g., 7a and 10b) with MDM2,
molecular docking studies were conducted utilizing CDOCKER programme within Discovery
Studio 2.1 software package. The published X-ray crystal structure of MDM2 (PDB ID:1YCR)
was used for docking calculation. The docking models (Fig. 2) demonstrated that thioamide
derivative 7a and 3,4,5-trisubstituted aminopyrrole derivative 10b could mimic the p53 peptide to
interact with MDM2 protein. Generally, the interactions are nonspecific van der Waals contacts.
The binding modes involved three hydrophorbic pockets (Phe19, Trp23 and Leu26) that were
filled by one cyclopropyl and two 4’-chlorophenyl groups of 7a and 10b, respectively. The pyrrole
ring served as a core structure, just like the thiophene ring, which played the role of projecting
three hydrophobic substituents into the MDM2 binding cleft at proper directions. The docking
model also illustrated that the thioamide moiety, as a bioisostere of amide, was easier to be
exposed to the solvents, which may explain the fact that the binding affinity of compound 7a is
more potent than that of 4f. In addition, the NH of pyrrol ring (10b) was found exposed to the
solvents in the molecular docking results, where hydrosoluble side chains could be attached to
adjust the molecular drug-like properties.
With the attempt to explain the sudden drop of potency observed in 2,3,4-trisubstituted
dihydrothieno[2,3-b]pyridones and 4,5,6-trisubstituted thieno[2,3-d]pyrimidines, molecular
alignment was carried out. Structural energy minimization of thioamide derivative 7a,
dihydrothieno[2,3-b]pyridone derivative 12c (S and R isomers) and thieno[2,3-d]pyrimidine
derivative 17a was performed. The minimized conformers of 12c and 17a were aligned with the
most potent compound 7a, respectively, by fitting with thiophene ring skeleton as a common
structure. As shown in Fig. 3, although the bis-chloro-phenyl structures could match well, the
furane of (S)-12c and (R)-12c and the cyclopropyl of 17a displayed dramatically different spatial
orientation compared to the cyclopropyl group of 7a. It means that both core structures of 12c and
17a may not be able to project the furane ring or cyclopropyl group into the MDM2 binding
pocket at proper direction, even leading to surface contact with MDM2, which are responsible for
the loss of potency.
3. Conclusion
Five series of novel 3,4,5-trisubstituted aminothiophene derivatives and analogs were
designed and synthesized as novel p53-MDM2 binding inhibitors. In vitro biological evaluation
for their p53-MDM2 binding inhibitory activities and anti-proliferation activities revealed that
twelve compounds displayed comparable p53-MDM2 binding inhibitory activities to that of
Nutlin-3 and most tested compounds showed potent tumor cell anti-proliferation activities with
IC50 values at low micromolar level. Meanwhile, a good selective profile for wild-type p53
expression cell line was observed. Compound 7a, a thioamide derivative, exhibited the most
potent MDM2 binding affinity with a Ki value of 0.086 μM. Interestingly, 3,4,5-trisubstituted
aminopyrroles (10a, b) showed good performance not only in p53-MDM2 binding inhibition but
also in tumor cell anti-proliferation. Therefore, they can be utilized as novel leads for further
investigation. Molecular docking analysis further suggested the binding modes of 7a and 10b with
MDM2. This study might be useful in future development of novel p53-MDM2 binding inhibitors.
4. Experimental
4

4.1. Chemistry
Melting points were determined with a B-540 Büchi apparatus and are uncorrected. NMR
spectra were recorded on a Brüker 500 (500 MHz) spectrometer (chemical shifts are given in
ppm(δ) relative to TMS as internal standard, coupling constants (J) are in hertz (Hz), and signals
are using the following abbreviations: s, singlet; d, doublet; t, triplet; m, multiplet, etc. Mass
spectra (MS), ESI (positive) were recorded on an Esquire-LC-00075 spectrometer. Thin layer
chromatography was carried out using plate silica gel F254 Merck. Reagents and solvents were
purchased from common commercial suppliers and were used without further purification. All
yields are unoptimized and generally represent the result of a single experiment.
4.1.1. Synthesis of 1-(4-chlorophenyl)-2-phenylethanone derivatives (2a-e) and 2-cyanoacetamide
derivatives were exactly following the procedures of reference 18 and 1918, 19
.
4.1.2. General procedure for the synthesis of 3,4,5-trisubstituted aminothiophene derivatives
(4a-y)
A mixture of 1-(4-chlorophenyl)-2-phenylethanone (2, 1.1 mmol) and 2-cyanoacetamide (2.2
mmol) in THF (10 mL) was slowly added to a solution of TiCl4 (5.5 mmol) in dry THF (20 mL) at
0℃. After removing the ice bath, pyridine (0.2 mL) was added. And the reaction mixture was
stirred at room temperature overnight. The progress of the reaction was monitored by TLC. After
completion of the reaction, THF was removed under reduced pressure and the residue was
dissolved in ethyl acetate (30 mL), washed with water (2 x 30 mL) and brine (2 x 30 mL). The
organic layer was dried over anhydrous Na2SO4 and concentrated under reduced pressure. The
crude olefin intermediate (3) was used for the next step without purification. To a stirred solution
of the crude product (3) in THF (20 mL), elemental sulfur (2.2 mmol) and diethylamine (0.2 mL)
were added. The reaction mixture was stirred at room temperature for 2~4h. THF was removed
under reduced pressure and the residue was dissolved in ethyl acetate (20 mL), washed with water
(2 x 20 mL) and brine (2 x 20 mL). The organic layer was dried over anhydrous Na2SO4 and
concentrated under reduced pressure. The crude products were purified by column
chromatography in gradient elution to give the target compounds (4a-y).
4.1.2.1.
2-Amino-4-(4-chlorophenyl)-N-cyclopropyl-5-phenylthiophene-3-carboxamide
(4a).
1
Yellow solid (81%), mp: 183 ~ 185 °C. H NMR (500 MHz, CDCl3) δ 7.37 (d, J = 8.4 Hz, 2H,
Ar-H), 7.20 (d, J = 8.4 Hz, 2H, Ar-H), 7.16 (m, 3H, Ar-H), 7.00 (d, J = 8.6 Hz, 2H, Ar-H), 6.19 (br,
2H, NH2), 4.88 (s, 1H, NH), 2.60 (m, 1H, CH), 0.63 (m, 2H, CH2), 0.04 (m, 2H, CH2). ESI-MS:
m/z = 369 [M+1]+.
4.1.2.2. (2-Amino-4-(4-chlorophenyl)-5-phenylthiophen-3-yl)(pyrrolidin-1-yl)methanone (4b).
Yellow solid (72%), mp: 187 ~ 189 °C. 1H NMR (500 MHz, CDCl3) δ 7.21 (m, 5H, Ar-H), 7.16 (d,
J = 8.0 Hz, 2H, Ar-H), 7.10 (d, J = 8.0 Hz, 2H, Ar-H), 3.50 (m, 4H, 2CH2), 1.60 (m, 4H, 2CH2).
ESI-MS: m/z = 383 [M+1]+.
4.1.2.3. (2-Amino-4-(4-chlorophenyl)-5-phenylthiophen-3-yl)(morpholino)methanone (4c). Yellow
1
solid (70%), mp: 203 ~ 205 °C. H NMR (500 MHz, CDCl3) δ 7.24 (d,J = 8.5 Hz, 2H, Ar-H),
7.21(m, 3H, Ar-H), 7.10 (d,J = 8.4 Hz, 2H, Ar-H), 7.09 (d,J = 6.6 Hz, 2H, Ar-H), 3.36 (m, 8H,
4CH2). ESI-MS: m/z = 399 [M+1]+.
4.1.2.4. 2-Amino-N-benzyl-4-(4-chlorophenyl)-5-phenylthiophene-3-carboxamide (4d). Yellow
5

solid (66%), mp: 186 ~ 188 °C. 1H NMR (500 MHz, CDCl3) δ 7.28 (m, 4H, Ar-H), 7.16 (m, 6H,
Ar-H), 6.98 (dd, J = 7.6, 1.8 Hz, 2H, Ar-H), 6.94 (dd, J = 7.6, 1.8 Hz,, 2H, Ar-H), 6.34 (br, 2H,
NH2), 5.08 (t, J = 5.2 Hz, 1H, NH), 4.28 (d, J = 5.2 Hz, 2H, CH2). ESI-MS: m/z = 419 [M+1]+.
4.1.2.5. 2-Amino-4-(4-chlorophenyl)-N-cyclopropyl-5-(4-fluorophenyl)thiophene-3-carboxamide
(4l). White solid (63%), mp: 175 ~ 176 °C. 1H NMR (500 MHz, CDCl3) δ 7.38 (d, J = 8.4 Hz, 2H,
Ar-H), 7.18 (d, J = 6.4 Hz, 2H, Ar-H), 6.94 (d, J = 6.5 Hz, 2H, Ar-H), 6.87 (d, J = 8.4 Hz, 2H,
Ar-H), 6.11 (br, 2H, NH2), 4.88 (s, 1H, NH), 2.58 (dt,J = 10.4, 3.4 Hz, 1H, CH), 0.68 (m, 2H,
CH2), 0.06 (m, 2H, CH2). ESI-MS: m/z = 387 [M+1]+.
4.1.2.6. (2-Amino-4-(4-chlorophenyl)-5-(4-fluorophenyl)thiophen-3-yl)(pyrrolidin-1-yl)methanone
(4m). Yellow solid (61%), mp: 185 ~ 186 °C. 1H NMR (500 MHz, CDCl3) δ 7.20 (d, J = 8.5 Hz, 2H,
Ar-H), 7.11 (d, J = 8.5 Hz, 2H, Ar-H), 7.05 (d, J = 8.4 Hz, 2H, Ar-H), 6.90 (d, J = 8.4 Hz, 2H, Ar-H),
3.42 (m, 4H, 2CH2), 1.58 (m, 4H, 2CH2). ESI-MS: m/z = 401 [M+1]+.
4.1.2.7.
(2-Amino-4-(4-chlorophenyl)-5-(4-fluorophenyl)thiophen-3-yl)(morpholino)methanone
(4n). White solid (78%), mp: 190 ~ 191 °C. 1H NMR (500 MHz, CDCl3) δ 7.25 (d, J = 8.5 Hz, 2H,
Ar-H), 7.08 (d, J = 8.4 Hz, 2H, Ar-H), 7.03 (d, J = 8.4 Hz, 2H, Ar-H), 6.90 (d, J = 8.6 Hz, 2H,
Ar-H), 3.60 (m, 8H, 4CH2). ESI-MS: m/z = 417 [M+1]+.
4.1.2.8. 2-Amino-N-benzyl-4-(4-chlorophenyl)-5-(4-fluorophenyl)thiophene-3-carboxamide (4o).
White solid (81%), mp: 192 ~ 194 °C. 1H NMR (500 MHz, CDCl3) δ 7.25 (m, 3H, Ar-H), 7.18 (d, J
= 8.4 Hz, 2H, Ar-H), 7.12 (d, J = 8.4 Hz, 2H, Ar-H), 6.93 (m, 4H, Ar-H), 6.84 (m, 2H, Ar-H), 5.07 (t,
J = 5.1 Hz, 1H, NH), 4.27 (d, J = 5.2 Hz, 2H, CH2). ESI-MS: m/z = 437 [M+1]+.
4.1.2.9. 2-Amino-5-(4-bromophenyl)-4-(4-chlorophenyl)-N-cyclopropylthiophene-3-carboxamide
1
(4p). Yellow solid (56%), mp: 182 ~ 184 °C. H NMR (500 MHz, CDCl3) δ 7.55 (d, J = 8.4 Hz,
2H, Ar-H), 7.12 (d, J = 8.5 Hz, 2H, Ar-H), 7.11 (d, J = 8.4 Hz, 2H, Ar-H), 6.92(d, J = 8.5 Hz, 2H,
Ar-H), 5.97 (br, 2H, NH2), 4.87 (s, 1H, NH), 2.58 (dt,J = 10.4, 3.4 Hz, 1H, CH), 0.65 (m, 2H,
CH2), 0.04 (m, 2H, CH2). ESI-MS: m/z = 447 [M+1]+.
4.1.2.10. 2-Amino-5-(4-bromophenyl)-4-(4-chlorophenyl)-N-cyclohexylthiophene-3-carboxamide
(4q). Yellow solid (59%), mp: 192 ~ 194 °C. 1H NMR (500 MHz, CDCl3) δ 7.55 (d, J = 8.4 Hz, 2H,
Ar-H), 7.16 (d, J = 8.4 Hz, 2H, Ar-H), 7.12 (d, J = 8.6 Hz, 2H, Ar-H), 6.91 (d, J = 8.6 Hz, 2H, Ar-H),
4.80 (d, J = 7.5 Hz, 1H, NH), 3.72 (m, 1H, CH), 1.61 (m, 2H, CH2), 1.45 (m, 1H, CH2), 1.35 (m, 2H,
CH2), 1.25 (m, 2H, CH2), 1.09 (m, 1H, CH2), 0.75 (m, 2H, CH2). ESI-MS: m/z = 489 [M+1]+.
4.1.2.11.
(2-Amino-5-(4-bromophenyl)-4-(4-chlorophenyl)thiophen-3-yl)(pyrrolidin-1-yl)methanone (4r).
1
Yellow solid (50%), mp: 195 ~ 197 °C. H NMR (500 MHz, CDCl3) δ 7.36 (d,J = 8.4 Hz, 2H,
Ar-H), 7.17 (d,J = 8.5 Hz, 2H, Ar-H), 7.06 (d,J = 8.4 Hz, 2H, Ar-H), 6.99 (d,J = 8.5 Hz, 2H, Ar-H),
3.69 (m, 4H, 2CH2), 1.59 (m, 4H, 2CH2). ESI-MS: m/z = 461 [M+1]+.
4.1.2.12. (2-Amino-5-(4-bromophenyl)-4-(4-chlorophenyl)thiophen-3-yl)(morpholino)methanone
(4s). Yellow solid (61%), mp: 200 ~ 201 °C. 1H NMR (500 MHz, CDCl3) δ 7.42 (d,J = 8.1 Hz, 2H,
Ar-H), 7.17 (d,J = 8.2 Hz, 2H, Ar-H), 7.03 (d,J = 8.2 Hz, 2H, Ar-H), 6.98 (d,J = 8.2 Hz, 2H, Ar-H),
3.70 (m, 8H, 4CH2). ESI-MS: m/z = 477 [M+1]+.
4.1.2.13. 2-Amino-N-benzyl-5-(4-bromophenyl)-4-(4-chlorophenyl)thiophene-3-carboxamide (4t).
1
Yellow solid (55%), mp: 223 ~ 225 °C. H NMR (500 MHz, CDCl3) δ 7.35 (d, J = 8.3 Hz, 2H,
Ar-H), 7.31 (m, 3H, Ar-H), 7.10 (d, J = 8.6 Hz, 2H, Ar-H), 7.06 (d, J = 8.3 Hz, 2H, Ar-H), 6.93 (m,
2H, Ar-H), 6.88 (d, J = 8.5 Hz, 2H, Ar-H), 5.05 (t, J = 4.7 Hz, 1H, NH), 4.26 (d, J = 5.0 Hz, 2H,
CH2). ESI-MS: m/z = 497 [M+1]+.
6

4.1.2.14. 2-Amino-4-(4-chlorophenyl)-N-cyclopropyl-5-(4-nitrophenyl)thiophene-3-carboxamide
(4u). Red solid (75%), mp: 203 ~ 204 °C.1H NMR (500 MHz, CDCl3) δ 7.98 (d, J = 8.7 Hz, 2H,
Ar-H), 7.44 (d, J = 8.2 Hz, 2H, Ar-H), 7.20 (d, J = 8.2 Hz, 2H, Ar-H), 7.05 (d, J = 8.7 Hz, 2H,
Ar-H), 4.84 (s, 1H, NH), 2.59 (m, 1H, CH), 0.63 (m, 2H, CH2), 0.02 (m, 2H, CH2). ESI-MS: m/z
= 414 [M+1]+.
4.1.2.15.
2-Amino-4-(4-chlorophenyl)-N-cyclohexyl-5-(4-nitrophenyl)thiophene-3-carboxamide
(4v). Red solid (71%), mp: 182 ~ 183 °C.1H NMR (500 MHz, CDCl3) δ 7.98 (d,J = 8.7 Hz, 2H,
Ar-H), 7.45 (d,J = 8.2 Hz, 2H, Ar-H), 7.25 (d,J = 8.3 Hz, 2H, Ar-H), 7.06 (d,J = 8.7 Hz, 2H, Ar-H),
6.51 (br, 2H, NH2), 4.77 (d,J = 6.8 Hz, 1H, NH), 3.72 (m, 1H, CH), 1.62 (m, 2H, CH2), 1.45 (m,
1H, CH2), 1.37 (m, 2H, CH2), 1.26 (m, 2H, CH2), 1.14 (m, 1H, CH2), 0.73 (m, 2H, CH2). ESI-MS:
m/z = 456 [M+1]+.
4.1.2.16. (2-Amino-4-(4-chlorophenyl)-5-(4-nitrophenyl)thiophen-3-yl)(pyrrolidin-1-yl)methanone
(4w). Red solid (78%), mp: 207 ~ 209 °C.1H NMR (500 MHz, CDCl3) δ 8.04 (d, J = 8.9 Hz, 2H,
Ar-H), 7.26 d, J = 8.9 Hz, 2H, Ar-H), 7.17 (m, 4H, Ar-H), 3.41 (m, 2H, CH2), 2.79 (m, 2H, CH2),
1.66 (m, 2H, CH2), 1.54 (m, 2H, CH2). ESI-MS: m/z = 428 [M+1]+.
4.1.2.17. (2-Amino-4-(4-chlorophenyl)-5-(4-nitrophenyl)thiophen-3-yl)(piperidin-1-yl)methanone
(4x). Red solid (69%), mp: 185 ~ 187 °C.1H NMR (500 MHz, CDCl3) 1H NMR (500 MHz, CDCl3)
δ 8.03 (d, J = 8.7 Hz, 2H, Ar-H), 7.28 (d, J = 8.3 Hz, 2H, Ar-H), 7.17 (d, J = 8.7 Hz, 2H, Ar-H),
7.13 (d, J = 8.3 Hz, 2H, Ar-H), 3.57 (m, 2H, CH2), 3.40 (m, 2H, CH2), 1.67 (m, 4H, 2CH2), 1.59
(m, 2H, CH2). ESI-MS: m/z = 442 [M+1]+.
4.1.2.18.
(2-Amino-4-(4-chlorophenyl)-5-(4-nitrophenyl)thiophen-3-yl)(morpholino)methanone
(4y). Red solid (81%), mp: 199 ~ 201 °C.1H NMR (500 MHz, CDCl3) δ 8.04 (d, J = 8.9 Hz, 2H,
Ar-H), 7.31 (d, J = 8.5 Hz, 2H, Ar-H), 7.17 (d, J = 8.9 Hz, 2H, Ar-H), 7.12 (d, J = 8.4 Hz, 2H,
Ar-H), 3.36 (m, 8H, 4CH2). ESI-MS: m/z = 444 [M+1]+.
4.1.3. General procedure for the synthesis of aminothiophene thioamide derivatives (7a-d)
Lawesson reagent (1.0 mmol) was added to a solution of 3,4,5-Trisubstituted
aminothiophene (4f, 4g, 5 and 6, 1.0 mmol) in toluene (15 mL), and then the mixture was stirred
for 1h at 80℃. After cooling to room temperature, the reaction mixture was evaporated under
reduced pressure. The residue was extracted with ethyl acetate (2 x 20 mL). The organic layer was
washed with water (2 x 20 mL) and brine (2 x 20 mL), dried over anhydrous Na2SO4, and
concentrated under reduced pressure. The crude products were purified by column
chromatography (PE: EtOAc=20:1, v/v) to give the target compounds (7a-d).
4.1.3.1. 2-Amino-4,5-bis(4-chlorophenyl)-N-cyclopropylthiophene-3-carbothioamide (7a). Yellow
1
solid (39%), mp: 190 ~ 192 °C. H NMR (500 MHz, CDCl3) δ 7.34 (d, J = 8.0 Hz, 2H, Ar-H),
7.15 (d, J = 10.0 Hz, 2H, Ar-H), 7.12 (d, J = 10.0 Hz, 2H, Ar-H), 6.92 (d, J = 8.0 Hz, 2H, Ar-H),
6.60 (br, 2H, NH2), 6.25 (s, 1H, NH), 3.02 (m, 1H, CH), 0.74 (m, 2H, CH2), 0.00 (m, 2H, CH2).
ESI-MS: m/z = 419 [M+1]+.
4.1.3.2. 2-Amino-4,5-bis(4-chlorophenyl)-N-cyclopentylthiophene-3-carbothioamide (7b). Yellow
1
solid (43%), mp: 204 ~ 206 °C. H NMR (500 MHz, CDCl3) δ 7.33 (d, J = 8.3 Hz, 2H, Ar-H),
7.17(m, 4H, Ar-H), 6.89 (d, J = 8.5 Hz, 2H, Ar-H), 6.38 (br, 2H, NH2), 6.32 (d, J = 6.8 Hz, 1H,
NH), 4.58 (m, 1H, CH), 1.85 (m, 2H, CH2), 1.49 (m, 2H, CH2), 1.30 (m, 2H, CH2), 0.95 (m, 2H,
CH2). ESI-MS: m/z = 447 [M+1]+.
4.1.3.3. 2-Amino-4,5-bis(4-chlorophenyl)-N-cyclohexylthiophene-3-carbothioamide (7c). Yellow
7

1
solid (35%), mp: 202 ~ 204 °C. H NMR (500 MHz, CDCl3) δ 7.33 (d, J = 8.4 Hz, 2H, Ar-H),
7.16 (d, J = 8.4 Hz, 2H, Ar-H), 7.13 (d, J = 8.6 Hz, 2H, Ar-H), 6.89 (d, J = 8.6 Hz, 2H, Ar-H), 6.24
(d, J = 7.4 Hz, 1H, NH), 4.27 (m, 1H, CH), 1.69 (m, 2H, CH2), 1.49 (m, 2H, CH2), 1.32 (m, 3H,
CH2), 1.06 (m, 1H, CH2), 0.68 (m, 2H, CH2). ESI-MS: m/z = 461 [M+1]+.
4.1.3.4. 2-Amino-N-(tert-butyl)-4,5-bis(4-chlorophenyl)thiophene-3-carbothioamide (7d). Yellow
1
solid (32%), mp: 201 ~ 204 °C. H NMR (500 MHz, CDCl3) δ 7.34 (d, J = 8.4 Hz, 2H, Ar-H),
7.18 (m, 4H, Ar-H), 6.89 (d, J = 8.5 Hz, 2H, Ar-H), 6.26 (s, 1H, NH), 6.14 (br, 2H, NH2), 1.19 (s,
9H, 3CH3). ESI-MS: m/z = 435 [M+1]+.
4.1.4.
3-amino-3-iminopropanoate/3-amino-N-cyclopropyl-3-iminopropanamide (9a-b) were exactly
following the procedures of reference 14 and 1514, 15
2-Bromo-2,3-bis(4-chlorophenyl)ethanon
(8)
and
ethyl
.
4.1.5 General procedure for the synthesis of 3,4,5-trisubstituted aminopyrrole derivatives (10a
and 10b)
A
mixture of 2-Bromo-2,3-bis(4-chlorophenyl)ethanon (8, 1.0mmol),
ethyl
3-amino-3-iminopropanoate/3-amino-N-cyclopropyl-3-iminopropanamide (9a-b, 1.0mmol) and
NaHCO3 (2.5 mmol) in 5 mL ethanol was refluxed for 1.5 h. After cooling to room temperature,
the reaction mixture was evaporated under reduced pressure. The residue was extracted with ethyl
acetate (2 x 20 mL). The organic layer was washed with water (2 x 20 mL) and brine (2 x 20 mL),
dried over anhydrous Na2SO4, and concentrated under reduced pressure. The crude products were
purified by column chromatography (PE: EtOAc=3:1, v/v) to give the target compounds (10a-b).
4.1.5.1. Ethyl 2-amino-4,5-bis(4-chlorophenyl)-1H-pyrrole-3-carboxylate (10a). White solid
(32%), mp: 253 ~ 255 °C. 1H NMR (500 MHz, DMSO) δ 10.83 (s, 1H, NH), 7.32 (d, J = 8.3 Hz,
2H, Ar-H), 7.24 (d, J = 8.5 Hz, 2H, Ar-H), 7.15 (d, J = 8.3 Hz, 2H, Ar-H), 7.00 (d, J = 8.5 Hz, 2H,
Ar-H), 5.83 (s, 2H, NH2), 3.89 (q, J = 7.0 Hz, 2H, CH2), 0.92 (t, J = 7.1 Hz, 3H, CH3). ESI-MS:
m/z = 375 [M+1]+.
4.1.5.2. 2-Amino-4,5-bis(4-chlorophenyl)-N-cyclopropyl-1H-pyrrole-3-carboxamide (10b). White
solid (18%), mp: 230 ~ 232 °C. 1H NMR (500 MHz, CDCl3) δ 8.88 (s, 1H, NH), 7.38 (d, J = 8.3
Hz, 2H, Ar-H), 7.22 (d, J = 8.2 Hz, 2H, Ar-H), 7.13 (d, J = 8.3 Hz, 2H, Ar-H), 6.92 (d, J = 8.1 Hz,
2H, Ar-H), 5.02 (s, 1H, NH-amide), 2.53 (m, 1H, CH), 0.55 (m, 2H, CH2), 0.08 (m, 2H, CH2).
ESI-MS: m/z = 386 [M+1]+.
4.1.6.
General
procedure
for
the
synthesis
of
2,3,4-trisubstituted
4,5-dihydrothieno[2,3-b]pyridin-6(7H)-ones (12a-c)
A mixture of 2-amino-4,5-bis(4-chlorophenyl)thiophene-3-carboxylic acid12 (11, 1.0 mmol),
malonic acid cyclic isopropylidene ester (1.1 mmol) and corresponding aldehydes (1.05 mmol) in
5 mL AcOH was refluxed for 2 h. After cooling to room temperature, the reaction mixture was
evaporated under reduced pressure. The residue was extracted with ethyl acetate (2 x 20 mL). The
organic layer was washed with water (2 x 20 mL) and brine (2 x 20 mL), dried over anhydrous
Na2SO4, and concentrated under reduced pressure. The crude products were purified by column
chromatography to give the target compounds (12a-c).
4.1.6.1. 2,3-Bis(4-chlorophenyl)-4-phenyl-4,5-dihydrothieno[2,3-b]pyridin-6(7H)-one (12a).
White solid (61%), mp: 247 ~ 248 °C. 1H NMR (500 MHz, CDCl3) δ 8.51 (s, 1H, NH), 7.28 (m,
3H, Ar-H), 7.18 (m, 4H, Ar-H), 7.03 (m, 4H, Ar-H), 6.82 (d, J = 8.1 Hz, 2H, Ar-H), 3.97 (d, J =
7.2 Hz, 1H, CH), 3.11 (dd, J = 16.1, 7.5 Hz, 1H, CH2), 2.78 (d, J = 16.1 Hz, 1H, CH2). ESI-MS:
m/z = 450 [M+1]+.
8

4.1.6.2.
2,3-Bis(4-chlorophenyl)-4-(thiophen-2-yl)-4,5-dihydrothieno[2,3-b]pyridin-6(7H)-one
1
(12b). Yellow solid (65%), mp: 240 ~ 241 °C. H NMR (500 MHz, CDCl3) δ 8.50 (s, 1H, NH),
7.25 (d, J = 8.4 Hz, 2H, Ar-H), 7.19 (d, J = 8.5 Hz, 2H, Ar-H), 7.17 (d, J = 5.0 Hz 1H, Ar-H), 7.05
(d, J = 8.5 Hz, 2H, Ar-H), 6.99 (d, J = 8.4 Hz, 2H, Ar-H), 6.90 (dd, J = 5.0, 3.6 Hz, 1H, Ar-H),
6.70 (d, J = 3.4 Hz, 1H, Ar-H), 4.26 (d, J = 6.3 Hz, 1H, CH), 3.10 (dd, J = 16.1, 6.9 Hz, 1H, CH2),
2.91 (d, J = 16.0 Hz, 1H, CH2). ESI-MS: m/z = 456 [M+1]+.
4.1.6.3. 2,3-Bis(4-chlorophenyl)-4-(furan-2-yl)-4,5-dihydrothieno[2,3-b]pyridin-6(7H)-one (12c).
Yellow solid (45%), mp: 238 ~ 239 °C. 1H NMR (500 MHz, CDCl3) δ 8.67 (s, 1H, NH), 7.31 (d, J
= 1.5 Hz, 1H, Ar-H), 7.29 (d, J = 8.5 Hz, 2H, Ar-H), 7.20 (d, J = 8.3 Hz, 2H, Ar-H), 7.07 (m, 4H,
Ar-H), 6.24 (dd, J = 3.1, 1.9 Hz, 1H, Ar-H), 5.89 (d, J = 3.2 Hz, 1H, Ar-H), 4.04 (t, J = 4.5 Hz, 1H,
CH), 2.97 (d, J = 4.5 Hz, 2H, CH2). ESI-MS: m/z = 440 [M+1]+.
4.1.7.
General
procedure
for
the
synthesis
of
2,3,4-trisubstituted
4,5-dihydrothieno[2,3-b]pyridin-6(7H)-ones (13a-b)
Compound 12a, b (1.0 mmol) and K2CO3 (1.5 mmol) were dissolved in DMF (5 mL) and
stirred for 30 min. Then 1-bromo-3-chloropropane (1.2 mmol) was slowly added with vigorous
stirring at 80℃ for 45 min. After cooling to room temperature, the reaction mixture was washed
with water (2 x 10 mL) and extracted with ethyl acetate (2 x 10 mL). The organic layer was dried
over anhydrous Na2SO4, and concentrated under reduced pressure. Then, the obtained residue was
dissolved in 5 mL dioxane, to which was added triethylamine (5.0 mmol) and correspongding
amines (10.0 mmol), refluxed for 24h. After cooling to room temperature, the reaction mixture
was evaporated under reduced pressure. The residue was extracted with ethyl acetate (2 x 10 mL).
The organic phase was washed with water (2 x 10 mL) and brine (2 x 10 mL), dried over
anhydrous Na2SO4, and concentrated under reduced pressure. The obtained residue was purified
by silica gel column chromatography (petroleum ether/ethyl acetate = 1:1, v/v) to give the target
compounds 13a-b.
4.1.7.1.
2,3-Bis(4-chlorophenyl)-7-(3-morpholinopropyl)-4-phenyl-4,5-dihydrothieno[2,3-b]pyridin-6(7H)
-one (13a). White solid (31%), mp: 105 ~ 107 °C. 1H NMR (500 MHz, CDCl3) δ 7.28 (d, J = 7.1 Hz,
2H, Ar-H), 7.22 (m, 1H, Ar-H), 7.17 (m, 4H, Ar-H), 7.04 (d, J = 8.4 Hz, 2H, Ar-H), 6.99 (d, J = 7.4
Hz, 2H, Ar-H), 6.82 (d, J = 8.0 Hz, 2H, Ar-H), 4.16 (m, 1H, CH), 3.90 (d, J = 7.0 Hz, 1H, CH2), 3.74
(m, 4H, 2CH2), 3.66 (dt, J = 13.9, 6.8 Hz, 1H, CH2), 3.09 (dd, J = 15.9, 7.5 Hz, 1H, CH2), 2.82 (d, J =
15.9 Hz, 1H, CH2), 2.45 (m, 6H, 3CH2), 1.98 (d, J = 32.0 Hz, 2H, CH2). ESI-MS: m/z = 577 [M+1]+.
4.1.7.2.
2,3-Bis(4-chlorophenyl)-4-(furan-2-yl)-7-(3-(pyrrolidin-1-yl)propyl)-4,5-dihydrothieno[2,3-b]pyri
din-6(7H)-one (13b). Yellow oil (35%). 1H NMR (500 MHz, CDCl3) δ 7.30 (m, 1H, Ar-H), 7.25 (m,
2H, Ar-H), 7.18 (dd, J = 8.6, 2.5 Hz, 2H, Ar-H), 7.05 (dd, J = 8.5, 2.2 Hz, 4H, Ar-H), 6.23 (m, 1H,
Ar-H), 5.89 (m, 1H, Ar-H), 4.25 (m, 1H, CH), 3.95 (m, 1H, CH2), 3.57 (m, 1H, CH2), 3.49 (m, 1H,
CH2), 3.42 (m, 1H, CH2), 2.96 (m, 2H, CH2), 2.54 (m, 4H, 2CH2), 1.91 (m, 2H, CH2), 1.81 (m, 4H,
2CH2). ESI-MS: m/z = 581 [M+1]+.
4.1.8. Synthesis of 2-amino-4,5-bis(4-chlorophenyl)thiophene-3-carbonitrile (14)
Same as 4a-y above, except with malononitrile. Target compound 14 was obtained as a
1
yellow solid (53%). H NMR (500 MHz, CDCl3) δ 7.69 (d, J = 8.4 Hz, 2H, Ar-H), 7.26 (m, 4H,
Ar-H), 7.06 (d,J = 8.3 Hz, 2H, Ar-H), 6.11 (br, 2H, NH2). ESI-MS: m/z = 345 [M+1]+.
9

4.1.9. Synthesis of 5,6-bis(4-chlorophenyl)thieno[2,3-d]pyrimidin-4(3H)-one (15)
A mixture of 2-amino-4,5-bis(4-chlorophenyl)thiophene-3-carbonitrile 14 (1.0 mmol), formic
acid (4 mL) and acetic anhydride (4 mL) was heated at 110 ℃ for 16 h. The solvent was
removed under reduced pressure and the residue was extracted with ethyl acetate (2 x 20 mL) and
organic layer was washed with water (2 x 20 mL) and brine (2 x 20 mL), dried over anhydrous
Na2SO4, and concentrated under reduced pressure. The crude product was purified by column
chromatography (petroleum ether/ethyl acetate = 10:1, v/v) to give the target compounds (15).
1
White solid (86%), mp: > 250 °C. H NMR (500 MHz, CDCl3) δ 8.58 (s, 1H, Ar-H), 8.01 (s, 1H,
NH), 7.45 (d, J = 8.4 Hz, 2H, Ar-H), 7.30 (d, J = 8.4 Hz, 2H, Ar-H), 7.17 (d, J = 8.3 Hz, 2H, Ar-H),
7.00 (d, J = 8.3 Hz, 2H, Ar-H). ESI-MS: m/z = 373 [M+1]+.
4.1.10. Synthesis of 4-chloro-5,6-bis(4-chlorophenyl)thieno[2,3-d]pyrimidine (16)
A solution of 5,6-bis(4-chlorophenyl)thieno[2,3-d]pyrimidin-4(3H)-one (15, 1.0 mmol) in
POCl3 (3 mL) was heated at 70℃ for 5 h. After cooling to room temperature, the reaction mixture
was quenched with ice water, and then extracted with ethyl acetate (2 x 20 mL). The organic layer
was washed with water (2 x 20 mL) and brine (2 x 20 mL), dried over anhydrous Na2SO4, and
concentrated under reduced pressure. The crude product was purified by column chromatography
(petroleum ether/ethyl acetate = 15:1, v/v) to give the target compounds (16). Yellow solid (95%),
mp: 200 ~ 202 °C. 1H NMR (500 MHz, DMSO) δ 8.89 (s, 1H, Ar-H), 7.62 (d, J = 8.5 Hz, 2H, Ar-H),
7.56 (d, J = 8.8 Hz, 2H, Ar-H), 7.51 (m, 4H, Ar-H). ESI-MS: m/z = 391 [M+1]+.
4.1.11. General procedure for the synthesis of 4,5,6-trisubstituted thieno[2,3-d]pyrimidine
derivatives (17a-d).
A mixture of 4-chloro-5,6-bis(4-chlorophenyl)thieno[2,3-d]pyrimidine (16, 1.0 mmol) and
corresponding amines (3.0 mmol) in n-butanol (4ml) was refluxed for 1h. After cooling to room
temperature, the solvent was removed under reduced pressure and the residue was extracted with
ethyl acetate (2 x 20 mL). The organic layer was washed with water (2 x 20 mL) and brine (2 x 20
mL), dried over anhydrous Na2SO4, and concentrated under reduced pressure. The crude product
was purified by column chromatography to give the target compounds (17a-d).
4.1.11.1. 5,6-Bis(4-chlorophenyl)-N-cyclopropylthieno[2,3-d]pyrimidin-4-amine (17a). White
solid (61%), mp: 152 ~ 153 °C. 1H NMR (500 MHz, CDCl3) δ 8.88 (s, 1H, Ar-H), 7.38 (d, J = 8.3
Hz, 2H, Ar-H), 7.22 (d, J = 8.2 Hz, 2H, Ar-H), 7.09 (m, 2H, Ar-H), 6.89 (m, 2H, Ar-H), 5.02 (s,
1H, NH), 1.26 (m, 1H, CH), 0.55 (m, 2H, CH2), 0.09 (m, 2H, CH2). ESI-MS: m/z = 412 [M+1]+.
4.1.11.2. 5,6-Bis(4-chlorophenyl)-N-cyclohexylthieno[2,3-d]pyrimidin-4-amine (17b). White solid
(69%), mp: 153 ~ 154 °C. 1H NMR (500 MHz, CDCl3) δ 8.40 (s, 1H, Ar-H), 7.55 (d, J = 8.0 Hz, 2H,
Ar-H), 7.43 (d, J = 8.3 Hz, 2H, Ar-H), 7.42 (d, J = 8.1 Hz, 2H, Ar-H), 7.26 (d, J = 8.2 Hz, 2H, Ar-H),
4.58 (s, 1H, NH), 4.07 (m, 1H, CH), 1.88 (m, 2H, CH2), 1.52 (m, 2H, CH2), 1.40 (m, 2H, CH2), 1.23 (m,
2H, CH2), 1.03 (m, 2H, CH2). ES I-MS: m/z = 454 [M+1]+.
4.1.11.3. 5,6-Bis(4-chlorophenyl)-4-(piperidin-1-yl)thieno[2,3-d]pyrimidine (17c). White solid
(49%), mp: 173 ~ 175 °C. 1H NMR (500 MHz, CDCl3) δ 8.47 (s, 1H, Ar-H), 7.46 (d, J = 8.6 Hz,
2H, Ar-H), 7.36 (d, J = 8.8 Hz, 2H, Ar-H), 7.33 (d, J = 8.6 Hz, 2H, Ar-H), 7.26 (d, J = 8.8 Hz, 2H,
Ar-H), 3.23 (m, 4H, 2CH2), 1.47 (m, 2H, CH2), 1.33 (m, 4H, 2CH2). ESI-MS: m/z = 440 [M+1]+.
4.1.11.4. 4-(5,6-Bis(4-chlorophenyl)thieno[2,3-d]pyrimidin-4-yl)morpholine (17d). White solid
(53%), mp: 203 ~ 205 °C. 1H NMR (500 MHz, CDCl3) δ 8.52 (s, 1H, Ar-H), 7.48 (d, J = 8.1 Hz, 2H,
10

Ar-H), 7.37 (dd, J = 14.3, 8.4 Hz, 4H, Ar-H), 7.28 (d, J = 8.9 Hz, 2H, Ar-H), 3.43 (m, 4H, 2CH2), 3.22
(m, 4H, 2CH2). ESI-MS: m/z = 442 [M+1]+.
4.2. Biological evaluation
4.2.1. MDM2 protein expression and purification
MDM2 (1-118) plasmid was provided by Dr. Shaomeng Wang's group, and transformed into
Escherichia coli BL-21 (DE3). Cultures were grown at 37℃ in TB medium, and induced by 0.4
mM IPTG at an OD600 of 0.6 at 18℃ for 20 h. Cells were lysed in 50 mM Tries, pH 7.5 buffer
containing 500 mM NaCl and 10% glycerol. MDM2 (1-118) was purified from the soluble
fraction using Ni-NTA resin, and desalted in PBS buffer pH 7.5, 150 mM NaCl and 10% glycerol.
The protein was purified to >95% as judged SDS–PAGE.
4.2.2. Fluorescence polarization competitive binding assay
Measurements were made with a Beckmancoulter DTX880 Multilabel Plate Reader using a
485 nM excitation filter and a 535 nM emission filter. Assays were performed in Microtiter
96-Well black and round bottom plates. Nutlin-3 was used as a positive control, while DMSO was
used as a negative control. Assays were performed in duplicate and repeated at least three times on
separate days. Competition experiments were carried out in a total volume of 20 μL 40 mM Tris–
HCl, pH 7.5, 150 mM NaCl, and 1 mM DTT, 4% DMSO. Probe peptide was present at a final
concentration of 1 nM, and MDM2 was present at a final concentration of 10nM. Plates were
allowed to incubate at room temperature for 1 h prior to measurement. The Ki values for inhibitors
were calculated by a web-based computer program20.
4.2.3. Cell proliferation assay
Cell proliferation was assessed by sulforhodamin B (SRB) assay. Briefly, A549 and PC3 cells
were seeded into 96-well plates and cultured overnight; exposed to serial concentrations of
compounds for 72 h. Cells were then washed with PBS and fixed with 10% (w/v) trichloroacetic
acid at 4 oC for an hour. After washing, the cells were stained for 30 min with 0.4% SRB
dissolved in 1% acetic acid. Then the cells were washed by 1% acetic acid for 5 times, and
protein-bound dye was extracted with 10 mmol unbuffered Tris base. The absorbance was
measured at 515 nm using a multiscan spectrum (Thermo Electron Co., Vantaa, Finland). The
inhibition rate on cell proliferation of each well was calculated as (A515 control cells – A515
treated cells )/ A515 control cells×100%. The average IC50 values were determined by Logit
method from at least two independent tests.
4.3. Molecular docking and structural alignment
Docking simulations were carried out by using CDOCKER module (Discovery Studio,
version 2.1; Accelrys, San Diego, CA, USA, 2008). The X-ray crystal structure of MDM2 bound
to the transactivation domain of p53 (PDB ID:1YCR) was used for the docking calculation. After
removing the ligand and solvent molecules, the CHARMm-force field was applied to the protein.
11

And the area around the p53 peptide was chosen as the active site with a radius set as 10 Å. Each
compound was generated random conformations using CHARMm-based molecular dynamics
(1000 steps), and then docked into the defined MDM2 binding site. The other parameters were set
as default. The final binding conformation of 7a and 10b was determined based on the calculated
CDOCKING ENERAGE. The most stable binding mode among the top 10 docking poses of each
Structural alignment analysis was carried out by using Fit atoms module in SYBYL 6.9
molecular modeling software. Structural energy minimization was performed using the standard
Tripos molecular mechanics force field and Gasteiger-Hückel charge, the max iterations for the
minimization was set to 2000. The minimization was terminated when the energy gradient
convergence criterion of 0.01 kcal/mol Å was reached. The minimized conformers of (S)-12c,
(R)-12c and 17a were aligned with the most potent compound 7a, respectively, by fitting with
thiophene ring skeleton as a common structure. The aligned molecules were shown in Fig. 3.
Acknowledgments
This study was financially supported by the National Natural Science Foundation of China
(30873163). The authors also thank Dr. Shaomeng Wang for providing the plasmid of MDM2
(1-118) and the tracer, and Dr. Jia Li for helping on the protein purification experiment.
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12

18. Lutjens, H.; Zickgraf, A.; Figler, H.; Linden, J.; Olsson, R. A.; Scammells, P. J. J. Med. Chem.
2003, 46, 1870.
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20. Nikolovska-Coleska, Z.; Wang, R.; Fang, X.; Pan, H.; Tomita, Y.; Li, P.; Roller, P. P.; Krajewski,
K.; Saito, N. G.; Stuckey, J. A.; Wang, S. Anal. Biochem. 2004, 332, 261.
Figure captions.
Fig. 1. Structure of the lead compound MCL0527.
Fig. 2. Molecular docking analysis of 7a and 10b with MDM2. (A) Stereoview of 7a bound to the
p53-binding site of MDM2, displayed with selected residues. (B) Stereoview of 10b bound to the
p53-binding site of MDM2, displayed with selected residues. (C) Overlay of 7a (green) with the
p53’s 9-mer peptide backbone and side chains Phe 19, Trp 23, and Leu 26 (pink) in a surface show
of MDM2. (D) Overlay of 10b (yellow) with the p53’s 9-mer peptide backbone and side chains
Phe 19, Trp 23, and Leu 26 (pink) in a surface show of MDM2.
Fig. 3. Structural alignment analysis of (S)-12c, (R)-12c, 17a with 7a. (A) Superposition of
(S)-12c (green) and 7a (white). (B) Superposition of (R)-12c (magenta) and 7a (white). (C)
Superposition of 17a (orange) and 7a (white).
Scheme 1. Synthetic route of 3,4,5-trisubstituted aminothiophenes (4a-y). Reagents and
conditions: (a) oxalyl chloride, CH2Cl2, rt; (b) AlCl3, CH2Cl2, rt; (c) methyl cyanoacetate or
2-cyanoacetamide derivatives, TiCl4, pyridine, THF, rt; (d) sulfur, NHEt2, THF, rt.
Scheme 2. Synthetic route of 3,4,5-trisubstituted aminothiophene thioamides (7a-d). Reagents and
conditions: (a) Lawesson’s Reagent, toluene, 80℃.
Scheme 3. Synthetic route of 3,4,5-trisubstituted aminopyrroles (10a, b). Reagents and conditions:
(a) Br2, CHCl3, Et2O, reflux; (b) NaHCO3, EtOH, reflux.
Scheme 4. Synthetic route of 2,3,4-trisubstituted dihydrothieno[2,3-b]pyridones (12a-c, 13a, b).
Reagents and conditions: (a) 2N NaOH aqueous solution, EtOH, reflux; (b) Meldrum's acid,
aldehydes, AcOH, reflux; (c) K2CO3, DMF, 1-bromo-3-chloropropane, 80℃; TEA, amines,
dioxane, reflux.
Scheme 5. Synthetic route of 4,5,6-trisubstituted thieno[2,3-d]pyrimidines (17a-d). Reagents and
conditions: (a) HCOOH, Ac2O, reflux; (b) PO3Cl, 70℃; (c) amines, n-butanol, reflux.
13

Table 1
P53–MDM2 binding inhibitory activities and in vitro tumor cell anti-proliferation activities of
3,4,5-trisubstituted aminothiophenes (4a-y, 5, 6 and 7a-d) and 3,4,5-trisubstituted aminopyrroles
(10a, b).
Kia
SRB IC50 (μM)
Compd.
R1
H
R2
X
S
S
Y
O
O
A549a
PC3a
(μM)
0.63
4a
4b
4.3
17.09
H
1.96
0.28
1.14
5.8
14.23
8.51
22.24
29.27
22.76
4c
H
H
S
S
O
O
4d
4ed
4fd
Cl
Cl
OCH3
S
S
O
O
1.52
0.54
5.04
1.95
2.78
3.68
4gd
4hd
4ie
Cl
Cl
Cl
Cl
S
S
S
S
O
O
O
O
0.23
0.74
1.21
0.43
2.41
0.89
4.62
1.87
7.3
6.22
14.33
11.69
4je
4ke
4l
Cl
F
S
S
S
O
O
O
0.15
0.34
1.22
31.81
27.95
20.39
16.30
3.00
18.81
4m
F
12.72
4n
F
S
O
0.22
25.13
9.95
>50
9.59
4o
4p
4q
F
S
S
S
O
O
O
0.60
2.18
1.06
25.79
37.31
28.37
Br
Br
7.46
4r
Br
S
O
0.75
37.45
1

7.49
18.67
NTc
4s
4t
Br
Br
S
S
S
S
S
S
S
S
O
O
O
O
O
O
O
O
1.48
0.76
NAb
NAb
NAb
NAb
NAb
0.54
>50
>50
4u
4v
4w
4x
4y
5e
NO2
NO2
NO2
NO2
NO2
Cl
NTc
15.03
10.70
NTc
25.87
42.57
NTc
16.72
6.79
>50
21.24
6e
7a
7b
Cl
Cl
Cl
S
S
S
O
S
S
0.81
0.086
0.17
15.14
22.05
29.04
8.85
8.28
22.05
7.41
>50
7c
Cl
Cl
S
S
S
S
0.32
0.58
27.74
34.02
7d
2.53
4.62
4.62
10a
10b
Cl
Cl
—
OCH2CH3
—
NH
NH
—
O
O
1.24
0.53
16.14
19.33
20.04
Nutlin-3
—
0.055
a Values are means of two experiments.
b NA, no activity.
c NT, not tested.
d These compounds were reported in our previous studies11.
e These compounds were reported in our another previous studies12.
Table 2
P53–MDM2 binding inhibitory activities and in vitro tumor cell anti-proliferation activities of
2,3,4-trisubstituted dihydrothieno[2,3-b]pyridone derivatives (12a-c, 13a, b).
2

SRB IC50 (μM)
A549a PC3a
Inhibition at
10μM (%)
Compd.
12a
R1
R2
H
H
H
17.22
20.21
25.44
NTb
6.07
0
>50
>50
>50
NTb
12b
12c
0
13a
23.75
13b
0
NTb
4.62
NTb
20.04
Nutlin-3
—
—
100
a Values are means of two experiments.
b NT, not tested.
Table 3
P53–MDM2 binding inhibitory activities and in vitro tumor cell anti-proliferation activities of
4,5,6-trisubstituted thieno[2,3-d]pyrimidines (17a-d).
SRB IC50 (μM)
A549a PC3a
Inhibition at
10μM (%)
30.5
Compd.
17a
R1
42.75
>50
>50
17b
12.5
16.8
>50
17c
>50
>50
17d
18.9
100
>50
>50
4.62
20.04
Nutlin-3
—
a Values are means of two experiments.
3

Figure 1

Figure 2

Figure 3

Scheme 1

Scheme 2

Scheme 3

Scheme 4

Scheme 5

Graphical abstract
Five series of novel 3,4,5-trisubstituted aminothiophene derivatives were designed and
synthesized as p53–MDM2 binding inhibitors.
1