Design, synthesis and biological evaluation of pyrazol-furan carboxamide analogues as novel Akt kinase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2016.03.074

A series of novel pyrazol-furan carboxamide analogues were designed, synthesized and biologically evaluated for their Akt1 inhibitory activities, as well as anti-proliferative efficacies against HCT116 and OVCAR-8 cell lines. Most compounds exhibited mode

Full text of DOI:10.1016/j.ejmech.2016.03.074

Accepted Manuscript
Design, synthesis and biological evaluation of pyrazol-furan carboxamide analogues
as novel Akt kinase inhibitors
Wenhu Zhan, Lei Xu, Xiaowu Dong, Jun Dong, Yi Xiao, Xiaodong Ma, Ni Qiu, Jia Li,
Bo Yang, Yubo Zhou, Yongzhou Hu
PII:
S0223-5234(16)30257-4
10.1016/j.ejmech.2016.03.074
EJMECH 8499
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 15 January 2016
Revised Date: 12 March 2016
Accepted Date: 25 March 2016
Please cite this article as: W. Zhan, L. Xu, X. Dong, J. Dong, Y. Xiao, X. Ma, N. Qiu, J. Li, B. Yang,
Y. Zhou, Y. Hu, Design, synthesis and biological evaluation of pyrazol-furan carboxamide analogues
as novel Akt kinase inhibitors, European Journal of Medicinal Chemistry (2016), doi: 10.1016/
j.ejmech.2016.03.074.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
Graphic abstract:
A series of pyrazol-furan carboxamide analogues featuring more restricted
conformation in contrast to GSK2141795 were rationally designed, synthesized and
biologically evaluated.

ACCEPTED MANUSCRIPT
Design, Synthesis and Biological Evaluation of Pyrazol-furan Carboxamide Analogues as Novel
Akt Kinase Inhibitors
Wenhu Zhana, Lei Xub, Xiaowu Dong a,, Jun Donga, Xiao Yia, Xiaodong Maa, Ni Qiua, Jia Lib, Bo
Yangc, Yubo Zhoub,*, Yongzhou Hua,*
a
Zhejiang Province Key Laboratory of Anti-Cancer Drug Research, College of Pharmaceutical
Sciences, Zhejiang University, Hangzhou, 310058, China
b National Center for Drug Screening, State Key Laboratory of Drug Research, Shanghai Institute of
Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China
*Corresponding authors:
E-mail: ybzhou@mail.shcnc.ac.cn (Y. Z.); huyz@zju.edu.cn (Y. H.)
Abstract:
A series of novel pyrazol-furan carboxamide analogues were designed, synthesized and
biologically evaluated for their Akt1 inhibitory activities, as well as anti-proliferative efficacies
against HCT116 and OVCAR-8 cell lines. Most compounds exhibited moderate to excellent Akt1
inhibitory activities, together with favorable cytotoxicities. Further kinase selectivity assay of the
most promising compound 25e illustrated that it was also potent against the structurally related AGC
kinases, including Akt2, Akt3, ROCK1 and PKA, but was specific over kinases from other
subfamilies. In addition, the Western blot analysis indicated that 25e could significantly suppress the
phosphorylation level of Akt substrate GSK3β in PC-3 cell. Moreover, 25e demonstrated a
concentration-dependent inhibition of phosphorylation of PRAS40 in LNCaP cell, with IC50 value of
30.4 nM.
Keywords: Akt1 inhibitor; Synthesis; Cytotoxicity; Molecular docking; Cancer.

ACCEPTED MANUSCRIPT
1. Introduction
The serine/threonine kinase Akt, also called protein kinase B (PKB), acts as an essential node in
the PI3K-Akt-mTOR signaling pathway which plays a critical role in mediating a wide range of
cellular functions including survival, metabolism, proliferation, growth and apoptosis [1]. Akt,
belonging to the AGC kinase family, is comprised of three highly homologous isoforms in mammals,
termed Akt1 (PKB-α), Akt2 (PKB-β) and Akt3 (PKB-γ) [2]. Among them, Akt1 is widely expressed
in various tissues, while Akt2 and Akt3 are relatively distinctive, mainly locating in
insulin-responsive tissues (Akt2) and brain, lung and kidney (Akt3) [3]. Aberrant activation of Akt
has been found in a large number of human cancers, including breast, prostate, lung, ovarian,
pancreatic and hematologic malignancies [4-7]. In addition, overexpression of Akt is often associated
with poor prognosis and resistance to cancer treatment with chemotherapy or targeted therapies, such
as EGFR and BRAF kinase inhibitors [8-10]. Therefore, the inhibition of Akt presents great potential
in the treatment of cancers [11-13]. To date, three kinds of Akt inhibitors with different mechanisms
of action have been advanced into clinical trial (Fig. 1), including phosphatidylinositol analog
inhibitors (e.g. Perifosine), allosteric inhibitors (e.g. MK-2206) and ATP-competitive inhibitors (e.g.
GDC-0068 and AZD5363) [14-18]. More recently, the structures of novel Akt inhibitors
GSK2141795 and GSK2110183 were disclosed, and both of them have entered several advanced
clinical trials for the treatment of hematologic and solid malignancies [19-26]. To be of interest, they
share the similar skeletons with furan-pyrazole and thiophene-pyrazole moiety respectively,
demonstrating that both of these fragments would be promising for Akt1 inhibitors. In this context,
with aim to explain the potent Akt1 inhibitory activities, we proposed the possible binding mode
between them and the ATP-bound pocket of Akt1 (Fig. 2A), consisting of three essential interactions
(hinge region, acidic hole and hydrophobic pocket).
As a well-established drug design strategy, the conformational restriction has been frequently
introduced to some structurally flexible ligands for optimizing the biological activity, specificity and
metabolic stability [27-33]. Considering this, our modification of GSK2141795 prioritized replacing
its flexible 3,4-difluorophenylpropanamine with the 4,4-disubstituted-piperidine moiety, a privileged
fragment for Akt1 inhibitors bearing more conformational rigidity (e.g. AT-7867) [34, 35]. After
assembling this fragment to the furan-pyrazol skeleton, the conformation of the resultant compound
25e matched favorably to that of GSK2141795 (Fig. 2B), thereby testifying its potential as an

ACCEPTED MANUSCRIPT
alternative to the 3,4-difluorophenylpropanamine. Herein, we reported our continued efforts in the
field of Akt inhibitors that culminated in a novel structural series of pyrazol-furan analogues
featuring more conformational restriction in contrast to GSK2141795 (Fig. 3).
2. Results and discussion
2.1. Chemistry
The synthetic routes for pyrazol-furan carboxamide analogues are outlined in Scheme 1-3. Suzuki
coupling of thiophene-2-carbaldehyde 1 with borate in the presence of Pd(PPh3)4, afforded
pyrazol-thiophene-2-carbaldehyde 2, which was subsequently converted to 3a,b through
electrophilic substitution with NCS or NBS in THF. Oxidation of 2 or 3a,b with KMnO4 in acetone
furnished pyrazol-thiophene-2-carboxylic acids 4a-c in good yields (Scheme 1).
The pyrazol-furan-2-carboxylic acid and its analogues were prepared as follows (Scheme 2). The
bromides 11a,b (Synthetic methods could be found in the Supporting Information) were coupled
with 1-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole to provide 12a,b.
Treatment of compounds 12a,b with different proportions of NCS or NBS at different temperature in
THF/DMF, followed by hydrolysis reaction under basic conditions yielded the intermediate 13a-f.
Similarly, key acid fragments 16a-c and 19a,b could be obtained by using 14 and 17 as the starting
materials.
Alkylation
of
commercially
available
2-arylacetonitrile
20a,b
with
N,N-bis(2-chloroethyl)benzylamine hydrochloride under phase-transfer conditions furnished
piperidines 21a,b. Hydrolysis of the cyano group of 21a,b with KOH in 1,2-ethanediol provided
acids 22a,b, which subsequently experienced a Curtius rearrangement to give the aminopiperidine
derivatives 23a,b. Treatment of 23a,b with 4a-c, 13a-f, 16a-c, or 19a,b, followed by removal of the
benzyl group yielded the desired compounds 24a-d, 25a-e, 26a-c, 27a-c and 28a,b in excellent
yields (Scheme 3).
2.2. Akt1 inhibitory and anti-proliferative activities
The Akt1 inhibitory activities of all the target compounds 24a-d, 25a-e, 26a-c, 27a-c and 28a,b
were evaluated via a competitive fluorescence polarization kinase activity assay. AZD5363 and
GSK2141795, two promising pan-Akt inhibitors under clinical trial, were used as the positive control.
The results are summarized in Table 1.

ACCEPTED MANUSCRIPT
As shown in Table 1, most of the tested compounds demonstrated moderate to excellent Akt1
inhibitory activities with IC50 values ranging from low micromolar to nanomolar level, except for the
compounds 26a, 26c and 27b (＜50% inhibition at 1 µg/mL). In the series of thiophen ring as the
linker (24a-d), introduction of halogen (chloro or bromo) to the 4-position of the pyrazol
hinge-binder improved the potency, as exemplified by compounds 24b and 24c, which were about
3-fold more potent than 24a. Replacement of 4-F substitution pattern with 3,4-dichloro on terminal
phenyl ring (24d, Akt1 IC50 = 0.071 µM) resulted in an over 10-fold improvement in Akt1 potency,
which is consistent with the SAR of previously reported Akt inhibitors [36]. In the set of
furan-linked analogues 25a-e, most of the compounds showed similar Akt1 inhibitory effect to that
of 24a-d. Introduction of a chlorine substituent at the 5-position of furan linkage boosted the potency
(25d vs 25a). Further introduction of 3,4-dichloro substitution pattern into 25d gave 25e, the direct
analogue of GSK2141795, which was proved to be most potent throughout the series (IC50 = 0.061
µM) and pertained to the same order of magnitude as AZD5363 (IC50 = 0.034 µM) and GSK2141795
(IC50 = 0.018 µM). Translocation of pyrazol moieties to 5-position of furyl linkage gave 26a-c.
Unfortunately, this modification was proved to be detrimental to the inhibitory potency. Encouraged
by this, we subsequently probed SARs around a selection of linkages. N-methylpyrrol analogues
27a-c, showed somewhat reduced Akt1 inhibiting effect. In addition, a larger phenyl linkage was
also found to be of no benefit to Akt1inhibitory potency, as illustrated by compounds 28a,b.
Furthermore, all the above compounds were tested for their anti-proliferative activities in vitro
against two human cancer cell lines including OVCAR-8 (Ovarian Adenocarcinoma) and HCT116
(Human Colon Cancer) by Sulforhodamine B (SRB) assay. All of them exhibited moderate to potent
cytotoxicities against both cell lines as outlined in Table 1. Among them, 24d bearing a
2,4-disubstituted thiophen linker was the most remarkable with IC50 values of 1.61 µM and 3.92 µM
against OVCAR-8 and HCT-116 cell lines, respectively. It is noteworthy that compound 25e, the
most potent Akt1 inhibitor (Akt1 IC50 = 0.061 µM)) of this series, also displayed favorable
anti-proliferative activities against OVCAR-8 (IC50 = 9.76 µM) and HCT-116 (IC50 = 7.76 µM) cell
lines, which was comparable to AZD5363 with the IC50 values of 7.27 and 5.20 µM, respectively.
2.3. Kinase selectivity assay
In order to investigate the selectivity profile of target compounds, compound 25e was evaluated

ACCEPTED MANUSCRIPT
against a panel of over 20 kinases at concentrations of 10 µM and 0.5 µM.
According to the biological data in Table 2, 25e exhibited favorable specificity over kinases form
subfamilies including TK, CAMK, TKL, CMGC. However, as for other two Akt isoforms or kinases
highly homologous to Akt1 in the AGC family, the selectivity was compromised to some extent. At
the 0.5µM, more than 50% inhibition was obtained against PKA, RSK1, P70S6K, ROCK1, SGK,
MSK1 and PRKG1.
2.4. Western blot analysis
Compound 25e was further assessed for its capability to downregulate the phosphorylation of
GSK3β (S9), a key downstream effector of Akt in PC-3 cell line using Western Blot assay. As
illustrated in Fig. 4, 25e exhibited a significant inhibition effect on the phosphorylated GSK3β (S9),
without decreasing the overall total GSK3β, Akt and GAPDH protein levels. In addition, a feedback
increase in phosphorylated Akt (S473) was observed at 1µM, whereas phosphorylation on Akt was
also inhibited at higher concentration (10 µM). This phenomenon was consistent with that observed
for a myriad of ATP-competitive Akt inhibitors [37-39].
2.5. Phosphorylated PRAS40 (pThr246) cellular ELISA assay
Subsequently, the IC50 value against Akt was determined for 25e at the cellular level via ELISA
assay. The PTEN-null LNCaP prostate cell line was used for this analysis due to its overactive Akt
pathway. As shown in Fig. 5, 25e demonstrated a pronounced effect on the inhibition of
phosphorylation of PRAS40, a known Akt substrate, with IC50 value of 30.4 nM, which was over 10-
and 2-fold more efficacious than AZD5363 and GSK2141795, respectively.
2.6. Molecular docking study
With an attempt to explore the possible binding modes of these pyrazol-furan analogues, 25e and
28a were docked into the Akt1 catalytic cleft on the basis of the co-crystal structure of AZD5363
complexed with Akt1.
As shown in Fig. 6A, compound 25e occupied the ATP binding site of Akt1 via conferring four
hydrogen bonds. Firstly, the pyrazol ring formed an essential hydrogen bond with residue Ala230.
Meanwhile, the hydrogen of amide attached to the furan linkage served as an H-bond donor for

ACCEPTED MANUSCRIPT
Asp292. Besides, the basic piperidine group interacted with the side chains of Asn279 and Glu278 in
the carbonyl-rich region via two hydrogen bonds. Beyond these H-bond interaction, the
3,4-dichlorophenyl group of 25e inserted into a small hydrophobic pocket under the P-loop and
generated substantial hydrophobic contacts with neighboring residues, thereby accounting for its
favorable binding affinity. As for 28a, however, the larger phenyl linkage had to move away from the
neighboring residue for obviating the unfavorable contact. Consequently, the H-bond interactions of
the piperidine group with Asn279 and Glu278 decreased due to the concerted movement of the
whole molecule, hence weakening the potency.
3. Conclusion
On the basis of the structures of GSK2141795, we rationally designed and synthesized a series of
pyrazol-furan carboxamide analogues featuring more restricted conformation. Most of them
exhibited moderate to excellent Akt1 inhibitory activities, as well as preferable anti-proliferative
efficacy against HCT116 and OVCAR-8 cell lines. Besides, kinase selectivity assay of the
representative 25e illustrated that it is also potent against the several structurally related AGC kinases,
but was specific over kinases from other subfamilies. In addition, Western blot analysis indicated
that 25e could markedly suppress the phosphorylation level of Akt substrate GSK3β in PC-3 cell.
Moreover, 25e significantly decreased the level of p-PRAS40 with IC50 value of 30.4 nM in LNCAP
cell, which is much more efficient than AZD5363. Finally, the binding mode between AKT1 with
compound 25e was proposed by molecular docking. In conclusion, compound 25e was identified as a
potent pan-Akt inhibitor with vigorous cellular activity, which merited further development as a
potential anticancer agent.
4. Experimental
4.1. Chemistry
1
Melting points were obtained on a B-540 Büchi melting-point apparatus and are uncorrected. H
13
NMR spectra were recorded on a 500 MHz, C NMR were recorded on a 125 MHz spectrometer,
respectively (chemical shifts are given in ppm (δ) relative to TMS as internal standard, coupling
constants (J) are in hertz (Hz), and signals are designated as follows: s, singlet; d, doublet; t, triplet;
m, multiplet; br s, broad singlet, etc.). Mass spectral data were obtained on an Esquire-LC-00075

ACCEPTED MANUSCRIPT
spectrometer. High resolution mass spectra were measured on an Agilent 1290 HPLC-6224 Time of
Fight Mass Spectrometer.
4.1.1 4-(1-Methyl-1H-pyrazol-5-yl)thiophene-2-carbaldehyde 2
To a solution of 4-bromothiophene-2-carbaldehyde 1 (3.82 g, 20 mmol) in 1,4-dioxane/H2O (v/v =
3/1,
60
mL)
was
added
K2CO3
(5.52
g,
40
mmol)
and
1-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (5.0 g, 24 mmol), followed by
tetrakistriphenylphosphane Pd (0) (1.16 g, 5 mol %). The reaction mixture was heated at reflux under
nitrogen for 6 h. After cooling to r.t., the reaction solution was poured into H2O (50 mL) and
extracted with ethyl acetate (15 mL × 3). The combined organic layers were dried over Na2SO4,
concentrated under vacuum and purified on silica gel to afford the title compound 2 as a pale yellow
solid (3.11 g, 81%), mp: 70-72 °C.
1H NMR (500 MHz, CDCl3) δ 9.98 (d, J = 1.1 Hz, 1H), 7.85 (d, J = 1.1 Hz, 1H), 7.77 (s, 1H), 7.53
(d, J = 1.9 Hz, 1H), 6.39 (d, J = 1.9 Hz, 1H), 3.97 (s, 3H). ESI-MS: m/z = 193 [M + 1] +.
4.1.2 General procedure for preparation of compound 3a and 3b
A mixture of 2 (960 mg, 5 mmol) and NCS or NBS in dry THF (15 mL) was stirred at r.t. for 3 h.
The reaction mixture was concentrated and the crude residue was extracted with ethyl acetate and
washed with saturated aqueous Na2CO3. The organic layer was further washed with brine, dried over
anhydrous Na2SO4, and then concentrated, recrystallized from ethyl acetate to give the title
compound.
4.1.2.1 4-(4-Chloro-1-methyl-1H-pyrazol-5-yl)thiophene-2-carbaldehyde 3a
Reagent: NCS (798 mg, 6 mmol). The product was obtained as an off-white solid (1.0 g, 90%), mp:
98-100 °C. 1H NMR (500 MHz, CDCl3) δ 10.01 (d, J = 1.1 Hz, 1H), 7.92 (d, J = 1.1 Hz, 1H), 7.90 (s,
1H), 7.52 (s, 1H), 3.91 (s, 3H). ESI-MS (m/z): 227 [M + 1]+.
4.1.2.2 4-(4-Bromo-1-methyl-1H-pyrazol-5-yl)thiophene-2-carbaldehyde 3b
Reagent: NBS (980 mg, 5.5 mmol). The product was obtained as a yellow solid (1.23 g, 92%), mp:
92-95 °C. 1H NMR (500 MHz, CDCl3) δ 10.00 (s, 1H), 7.91 (s, 1H), 7.90 (s, 1H), 7.54 (s, 1H), 3.90
(s, 3H). ESI-MS (m/z): 271 [M + 1]+.
5.1.3 General procedure for synthesis of compounds 4a-4c
To a solution of aldehyde in acetone (30 mL) at 0 °C was added potassium permanganate (380 mg,
2.4mmol) in one portion. The mixture was stirred at r.t. for 3 h, then the potassium salts were filtered

ACCEPTED MANUSCRIPT
off and the filtrate was concentrated and purified on silica gel to give the title compounds.
4.1.3.1 4-(1-Methyl-1H-pyrazol-5-yl)thiophene-2-carboxylic acid 4a
Reagent: 2 (385 mg, 2 mmol). The product was obtained as a white solid (345 mg, 83%). 1H NMR
(500 MHz, DMSO) δ 13.27 (s, 1H), 8.10 (d, J=1.4 Hz, 1H), 7.93 (d, J=1.4 Hz, 1H), 7.43 (d, J=1.8
Hz, 1H), 6.54 (d, J=1.8 Hz, 1H), 3.92 (s, 3H). ESI-MS (m/z): 207 [M − H]−.
4.1.3.2 4-(4-Chloro-1-methyl-1H-pyrazol-5-yl)thiophene-2-carboxylic acid 4b
Reagent: 3a (452 mg, 2 mmol). The product was obtained as a white solid (411 mg, 85%). 1H
NMR (500 MHz, DMSO) δ 13.35 (s, 1H), 8.20 (d, J = 1.5 Hz, 1H), 7.93 (d, J = 1.5 Hz, 1H), 7.64 (s,
1H), 3.84 (s, 3H). ESI-MS (m/z): 241 [M − H]−.
4.1.3.3 4-(4-Bromo-1-methyl-1H-pyrazol-5-yl)thiophene-2-carboxylic acid 4c
Reagent: 3b (540 mg, 2 mmol). The product was obtained as a white solid (510 mg, 89%). 1H
NMR (500 MHz, DMSO) δ 13.37 (s, 1H), 8.19 (d, J =1.5Hz, 1H), 7.92 (d, J =1.5Hz, 1H), 7.64 (s,
1H), 3.84 (s, 3H). ESI-MS (m/z): 285 [M − H]−.
4.1.4 General procedure of Suzuki coupling for compounds 12a, 12b, 15 and 18
Tetrakistriphenylphosphane Pd (0) (580 mg, 5 mol %) was added to a stirred suspension of
1-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole
(2.5
g,
12
mmol),
K3PO4·3H2O (4.8 g, 18 mmol) and the corresponding bromide in DMF (100 mL) at 0 °C under
nitrogen. The reaction mixture was heated at 80-120 °C for 8 h, then poured into H2O (100 mL) and
extracted with ethyl acetate (45 mL × 3). The combined organic layers were washed with brine (50
mL × 2), dried over Na2SO4, and concentrated under vacuum to afford an off-white semisolid. The
crude product was purified by flash silica chromatography to obtain the title compound.
4.1.4.1 Methyl 4-(1-methyl-1H-pyrazol-5-yl)furan-2-carboxylate 12a
Reagent: methyl 4-bromofuran-2-carboxylate 11a (2.05 g, 10 mmol, Supporting Information).
The product was obtained as a white solid (1.63 g, 79%). 1H NMR (500 MHz, CDCl3) δ 7.73 (d, J =
0.8 Hz, 1H), 7.48 (d, J = 1.9 Hz, 1H), 7.31 (d, J = 0.8 Hz, 1H), 6.34 (d, J = 1.9 Hz, 1H), 3.94 (s, 3H),
3.93 (s, 3H). ESI-MS (m/z): 207 [M + 1]+.
4.1.4.2 Methyl 1-methyl-4-(1-methyl-1H-pyrazol-5-yl)-1H-pyrrole-2-carboxylate 12b
Reagent: methyl 4-bromo-1-methyl-1H-pyrrole-2-carboxylate 11b (2.18 g, 10 mmol, Supporting
Information). The product was obtained as a white solid (1.49 g, 68%).1H NMR (500 MHz, CDCl3) δ
7.45 (d, J = 1.8 Hz, 1H), 7.04 (d, J = 2.0 Hz, 1H), 6.94 (d, J = 2.0 Hz, 1H), 6.23 (d, J = 1.8 Hz, 1H),

ACCEPTED MANUSCRIPT
3.98 (s, 3H), 3.94 (s, 3H), 3.85 (s, 3H). ESI-MS (m/z): 220 [M + 1]+.
4.1.4.3 Methyl 4-bromo-5-(1-methyl-1H-pyrazol-5-yl)furan-2-carboxylate 15
Reagent: methyl 4,5-dibromofuran-2-carboxylate 14 (2.84 g, 10 mmol). The product was obtained
as a white solid (2.36 g, 83%). 1H NMR (500 MHz, CDCl3) δ 7.54 (d, J = 2.1 Hz, 1H), 7.29 (s, 1H),
6.92 (d, J = 2.1 Hz, 1H), 4.14 (s, 3H), 3.92 (s, 3H). ESI-MS (m/z): 285 [M + 1]+.
4.1.4.4 Methyl 4-(1-methyl-1H-pyrazol-5-yl)benzoate 18
Reagent: methyl 4-bromobenzoate 17 (2.15 g, 10 mmol). The product was obtained as a white
solid (2.05 g, 95%). 1H NMR (500 MHz, CDCl3) δ 8.12 (d, J = 8.3 Hz, 2H), 7.54 (d, J = 1.9 Hz, 1H),
7.51 (d, J = 8.3 Hz, 2H), 6.38 (d, J = 1.9 Hz, 1H), 3.95 (s, 3H), 3.93 (s, 3H). ESI-MS (m/z): 217 [M +
1]+.
4.1.5 General procedure for preparation of compounds 13a-f, 16a-c, 19a and 19b
To a solution of the above methyl carboxylate (5 mmol) in dry THF/DMF at 0 °C was added NCS
or NBS (6−20 mmol) in one portion. The reaction mixture was stirred at r.t.−80 °C for 0.5–6 h, then
was diluted with ethyl acetate and washed with saturated aqueous NaHCO3. The organic layer was
further washed well with brine, concentrated under vacuum to afford the halogenated product, which
was subsequently dissolved in THF, and sodium hydroxide aqueous solution (2 N, 10 mL) was
added. The mixture was allowed to stir at r.t. for 3–10 h, then the solvent was removed and the
resulting crude mixture was dissolved in 20 mL of water and acidified with concentrated
hydrochloric acid until pH 1, and extracted with ethyl acetate (10 mL × 3), the combined organic
layers was further washed with brine, dried over anhydrous Na2SO4, and then concentrated to
dryness to yield the title acid.
4.1.5.1 4-(4-Chloro-1-methyl-1H-pyrazol-5-yl)furan-2-carboxylic acid 13a
Reagent: 12a (1.03 g, 5 mmol) and NCS (800 mg, 6 mmol), r.t., THF (15 mL). The product was
obtained as a white solid (788 mg, 70%). 1H NMR (500 MHz, DMSO) δ 13.48 (s, 1H), 8.38 (s, 1H),
7.64 (s, 1H), 7.58 (s, 1H), 3.87 (s, 3H). ESI-MS (m/z): 225 [M − H]−.
4.1.5.2 5-Chloro-4-(4-chloro-1-methyl-1H-pyrazol-5-yl)furan-2-carboxylic acid 13b
Reagent: 12a (1.03 g, 5 mmol) and NCS (2.67 g, 20 mmol), 80 °C, THF/DMF (v/v = 1/1, 20 mL).
1
The product was obtained as a white solid (820 mg, 63%). H NMR (500 MHz, DMSO) δ 13.75 (s,
1H), 7.70 (s, 1H), 7.69 (s, 1H), 3.77 (s, 3H). ESI-MS (m/z): 259 [M − H]−.
4.1.5.3 4-(4-Bromo-1-methyl-1H-pyrazol-5-yl)furan-2-carboxylic acid 13c

ACCEPTED MANUSCRIPT
Reagent: 12a (1.03 g, 5 mmol) and NBS (1.07 g, 6 mmol), r.t., THF (15 mL). The product was
1
obtained as a white solid (1.11 g, 82%). H NMR (500 MHz, DMSO) δ 12.16 (s, 1H), 8.36 (s, 1H),
7.65 (s, 1H), 7.58 (s, 1H), 3.87 (s, 3H). ESI-MS (m/z): 269 [M − H]−.
4.1.5.4 4-(4-Bromo-1-methyl-1H-pyrazol-5-yl)-1-methyl-1H-pyrrole-2-carboxylic acid 13d
Reagent: 12b (1.10 g, 5 mmol) and NBS (908 mg, 5.1 mmol), r.t., THF (15 mL). The product was
obtained as an off-white solid (1.03 g, 73%). 1H NMR (500 MHz, CDCl3) δ 7.51 (s, 1H), 7.24 (d, J =
1.6 Hz, 1H), 7.17 (d, J = 1.6 Hz, 1H), 4.02 (s, 3H), 3.92 (s, 3H). ESI-MS (m/z): 282 [M − H]−.
4.1.5.5 5-Bromo-4-(4-bromo-1-methyl-1H-pyrazol-5-yl)-1-methyl-1H-pyrrole-2-carb- oxylic acid
13e
Reagent: 12b (1.10 g, 5 mmol) and NBS (1.96 g, 11 mmol), 40 °C, THF (20 mL). The product
was obtained as an off-white solid (1.21 g, 67%). 1H NMR (500 MHz, CDCl3) δ 7.57 (s, 1H), 7.19 (s,
1H), 4.05 (s, 3H), 3.78 (s, 3H). ESI-MS (m/z): 362 [M − H]−.
4.1.5.6 3,5-Dichloro-4-(4-chloro-1-methyl-1H-pyrazol-5-yl)-1-methyl-1H-pyrrole-2- carboxylic acid
13f
Reagent: 12b (1.10 g, 5 mmol) and NCS (2.13 g, 16 mmol), 70 °C, THF/DMF (v/v = 2/1, 20 mL).
The product was obtained as an off-white solid (982 mg, 64%). 1H NMR (500 MHz, DMSO) δ 12.92
(s, 1H), 7.70 (s, 1H), 3.92 (s, 3H), 3.67 (s, 3H). ESI-MS (m/z): 306 [M − H]−.
4.1.5.7 4-Bromo-5-(1-methyl-1H-pyrazol-5-yl)furan-2-carboxylic acid 16a
Reagent: 15 (1.42 g, 5 mmol), treated with sodium hydroxide aqueous solution directly. The
product was obtained as a white solid (1.28 g, 95%). 1H NMR (500 MHz, CDCl3) δ 7.54 (d, J = 2.3
Hz, 1H), 7.41 (s, 1H), 6.97 (s, 1H), 6.68 (d, J = 2.3 Hz, 1H), 4.17 (s, 3H). ESI-MS (m/z): 269 [M −
H]−.
4.1.5.8 4-Bromo-5-(4-chloro-1-methyl-1H-pyrazol-5-yl)furan-2-carboxylic acid 16b
Reagent: 15 (1.42 g, 5 mmol), and NCS (800 mg, 6 mmol), r.t., THF (15 mL). The product was
1
obtained as a white solid (1.35 g, 89%). H NMR (500 MHz, CDCl3) δ 7.55 (s, 1H), 7.45 (s, 1H),
3.89 (s, 3H). ESI-MS (m/z): 305 [M − H]−.
4.1.5.9 4-Bromo-5-(4-bromo-1-methyl-1H-pyrazol-5-yl)furan-2-carboxylic acid 16c
Reagent: 15 (1.42 g, 5 mmol), and NBS (908 mg, 5.1 mmol), r.t., THF (15 mL). The product was
obtained as a white solid (1.53 g, 88%). 1H NMR (500 MHz, CDCl3) δ 7.59 (s, 1H), 7.46 (s, 1H),
3.90 (s, 3H). ESI-MS (m/z): 349 [M − H]−.

ACCEPTED MANUSCRIPT
4.1.5.10 4-(4-Chloro-1-methyl-1H-pyrazol-5-yl)benzoic acid 19a
Reagent: 18 (1.08 g, 5 mmol), and NCS (800 mg, 6 mmol), r.t., THF (15 mL). The product was
obtained as a white solid (1.07 g, 91%). 1H NMR (500 MHz, DMSO) δ 8.11 – 8.08 (m, 2H), 7.70 (s,
1H), 7.68 – 7.64 (m, 2H), 3.80 (s, 3H). ESI-MS (m/z): 235 [M − H]−.
4.1.5.11 4-(4-Bromo-1-methyl-1H-pyrazol-5-yl)benzoic acid 19b
Reagent: 18 (1.08 g, 5 mmol), and NBS (908 mg, 5.1 mmol), r.t., THF (15 mL). The product was
obtained as a white solid (1.17 g, 84%). 1H NMR (500 MHz, CDCl3) δ 8.26 (d, J = 8.3 Hz, 2H), 7.59
(s, 1H), 7.56 (d, J = 8.3 Hz, 2H), 3.87 (s, 3H). ESI-MS (m/z): 279 [M − H]−.
4.1.6 General procedure for preparation of compound 21a and 21b
To a well stirred suspension of tetrabutylammonium hydrogen sulfate (0.34 g, 1 mmol),
N,N-Bis(2-chloroethyl)benzylamine hydrochloride (2.69 g, 10 mmol), and 2-arylacetonitrile in
toluene (10 mL) was added dropwise 5 g of 50% (w/w) aqueous NaOH solution. The reaction
mixture was heated at 85 °C for 4 h, then poured into ice-water (30 mL) and extracted with ethyl
ether (15 mL × 3). The combined organic layers were dried over Na2SO4, concentrated under
vacuum, and the crude product was purified by flash silica chromatography to obtain the title
compound.
4.1.6.1 1-Benzyl-4-(4-fluorophenyl)piperidine-4-carbonitrile 21a
Reagent: 2-(4-fluorophenyl)acetonitrile 20a (1.35g, 10 mmol). The product was obtained as a
yellow oil (2.06 g, 70%). 1H NMR (500 MHz, CDCl3) δ 7.51 – 7.44 (m, 2H), 7.34 (m, 4H), 7.28 (m,
1H), 7.11 – 7.05 (m, 2H), 3.61 (s, 2H), 3.01 (d, J = 12.2 Hz, 2H), 2.56 – 2.48 (m, 2H), 2.10 (t, J = 9.5
Hz, 4H). ESI-MS (m/z): 295 [M+1]+.
4.1.6.2 1-Benzyl-4-(3,4-dichlorophenyl)piperidine-4-carbonitrile 21b
Reagent: 2-(3,4-dichlorophenyl)acetonitrile 20b (1.85g, 10 mmol). The product was obtained as a
1
yellow oil (2.23 g, 65%). H NMR (500 MHz, CDCl3) δ 7.59 (d, J = 2.3 Hz, 1H), 7.48 – 7.46 (m,
1H), 7.37 – 7.32 (m, 5H), 7.31 – 7.26 (m, 1H), 3.60 (s, 2H), 3.01 (d, J = 12.5 Hz, 2H), 2.56 – 2.42 (m,
2H), 2.09 – 2.05 (m, 4H). ESI-MS (m/z): 345 [M+1]+.
4.1.7 General procedure for preparation of compound 22a and 22b
To a suspension of 21 in 1,2-ethanediol (20 mL) was add KOH (5.6g, 100 mmol), and the mixture
was allowed to stir at 180°C for 20 h. After cooling to room temperature, the solution was poured
into ice-water (100 mL), and washed with ethyl ether (50 mL × 2), then acidified with concentrated

ACCEPTED MANUSCRIPT
hydrochloric acid until pH 5. The precipitate was filtrated, rinsed with cooled ethyl ether and dried to
afford the title compound.
4.1.7.1 1-Benzyl-4-(4-fluorophenyl)piperidine-4-carboxylic acid 22a
1
Reagent: 21a (2.95g, 10 mmol). H NMR (500 MHz, DMSO) δ 7.41 (dd, J = 8.9, 5.4 Hz, 2H),
7.34 – 7.26 (m, 4H), 7.24 (dd, J = 10.7, 4.2 Hz, 1H), 7.16 (t, J = 8.9 Hz, 2H), 3.44 (s, 2H), 2.70 (d, J
= 9.6 Hz, 2H), 2.40 (d, J = 12.8 Hz, 2H), 2.14 (t, J = 10.9 Hz, 2H), 1.78 (t, J = 10.6 Hz, 2H). The
product was obtained as a white solid (2.23 g, 71%). ESI-MS (m/z): 314 [M+1]+.
4.1.7.2 1-Benzyl-4-(3,4-dichlorophenyl)piperidine-4-carboxylic acid 22b
Reagent: 21b (3.44g, 10 mmol). The product was obtained as an off-white solid (2.72 g, 75%). 1H
NMR (500 MHz, CDCl3) δ 7.59 (d, J = 2.3 Hz, 1H), 7.48 – 7.45 (m, 1H), 7.38 – 7.31 (m, 5H), 7.31 –
7.25 (m, 1H), 3.60 (s, 2H), 3.01 (m , 2H), 2.57 – 2.45 (m, 2H), 2.11 – 2.05 (m, 4H). ESI-MS (m/z):
364 [M+1]+.
4.1.8 General procedure for preparation of compounds 23a,b
To a solution of 22 in anhydrous toluene (15 mL) was added triethylamine (0.28 mL, 2.0 mmol),
followed by the addition of diphenyl phosphoryl azide (0.32 mL, 1.5 mmol). The mixture was stirred
at room temperature under nitrogen for 1 hour and then heated at 90 °C for 3 h. After cooling down
to room temperature, the solution was poured into a hydrochloric aqueous solution (6 N, 20 mL), and
the resulting mixture was heated at reflux for 3h. The solvent was concentrated and the residue was
dissolved in a saturated solution of NaHCO3 in water (20 mL), and extracted with ethyl acetate (10
mL × 3). The combined organic layers were dried over Na2SO4, concentrated under vacuum and
purified on silica gel to afford the title amine.
4.1.8.1 1-Benzyl-4-(4-fluorophenyl)piperidin-4-amine 23a
Reagent: 22a (313 mg, 1.0 mmol). The product was obtained as a yellow solid (207 mg, 73%). 1H
NMR (500 MHz, DMSO) δ 7.58 – 7.53 (m, 2H), 7.30 (dd, J = 8.7, 4.2 Hz, 4H), 7.23 (dt, J = 5.6, 4.2
Hz, 1H), 7.12 – 7.06 (m, 2H), 3.48 (s, 2H), 3.35 (s, 2H), 2.48 – 2.44 (m, 2H), 1.96 – 1.89 (m, 2H),
1.72 (s, 2H), 1.54 (d, J = 12.2 Hz, 2H). ESI-MS (m/z): 285 [M+1]+.
4.1.8.2 1-Benzyl-4-(3,4-dichlorophenyl)piperidin-4-amine 23b
Reagent: 22b (364 mg, 1.0 mmol). The product was obtained as a pale yellow solid (227 mg,
68%). ESI-MS (m/z): 335 [M+1]+.
4.1.9 General procedure for synthesis of the target compounds 24a-d, 25a-e, 26a-c, 27a-c, and

ACCEPTED MANUSCRIPT
28a,b.
To a solution of carboxylic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride
(77 mg, 0.4 mmol) and 1-hydroxybenzotriazole (54 mg, 0.4 mmol) in DCM (5 mL) at 0 °C was
added triethylamine (0.11 mL, 0.6 mmol), followed by the addition of corresponding amine. The
mixture was stirred at r.t. overnight, and then partitioned between ethyl acetate (20 mL) and brine
(15 mL × 3). The organics were dried over Na2SO4, concentrated under vacuum. The resulting white
solid was dissolved in 1,2-dichloroethane (5 mL) and 1-chloroethyl chloroformate (43 mg, 0.3 mmol)
was added. The resulting mixture was stirred at r.t. for 3 h, evaporated in vacuo, and then treated
with methanol (3 mL) at reflux for 1 h. After cooling to r.t., the mixture was basified with saturated
aqueous NaHCO3 (10 mL) and extracted with ethyl acetate (5 mL × 3). The combined organic layers
was further washed well with brine, dried over Na2SO4, concentrated under vacuum and purified on
silica gel to afford the title compound.
4.1.9.1 N-(4-(4-fluorophenyl)piperidin-4-yl)-4-(1-methyl-1H-pyrazol-5-yl)thiophene-2-carboxamide
24a
Reagent: 4a (42 mg, 0.2 mmol) and 23a (57 mg, 0.2 mmol). The product was obtained as a white
solid (44 mg, 57%), mp: 159-161 °C. 1H NMR (500 MHz, CDCl3) δ 7.64 (s, 1H), 7.49 (d, J = 1.6 Hz,
1H), 7.47 (s, 1H), 7.45 – 7.40 (m, 2H), 7.03 (t, J = 8.6 Hz, 2H), 6.35 (d, J = 1.6 Hz, 1H), 6.31 (s, 1H),
3.94 (s, 3H), 3.11 – 2.98 (m, 4H), 2.51 (d, J = 13.4 Hz, 2H), 2.16 – 2.09 (m, 2H). ESI-MS (m/z): 385
[M+1]+. HRMS (ESI): m/z calcd for (C20H21FN4OS+H)+: 385.1493; found: 385.1507.
4.1.9.2
4-(4-Chloro-1-methyl-1H-pyrazol-5-yl)-N-(4-(4-fluorophenyl)piperidin-4-yl)-thiophene-2-carboxami
de 24b
Reagent: 4b (49 mg, 0.2 mmol) and 23a (57 mg, 0.2 mmol). The product was obtained as a white
solid (55 mg, 66%), mp: 170-172 °C. 1H NMR (500 MHz, CDCl3) δ 7.69 (d, J = 1.4 Hz, 1H), 7.60 (d,
J = 1.4 Hz, 1H), 7.49 (s, 1H), 7.46 – 7.41 (m, 2H), 6.32 (s, 1H), 3.86 (d, J = 5.8 Hz, 3H), 3.09 – 2.99
(m, 4H), 2.52 (d, J = 13.0 Hz, 2H), 2.19 – 2.10 (m, 2H). ESI-MS (m/z): 419 [M+1]+.
4.1.9.3
4-(4-Bromo-1-methyl-1H-pyrazol-5-yl)-N-(4-(4-fluorophenyl)piperidin-4-yl)-thiophene-2-carboxami
de 24c
Reagent: 4c (57 mg, 0.2 mmol) and 23a (57 mg, 0.2 mmol). The product was obtained as a white

ACCEPTED MANUSCRIPT
solid (55 mg, 47%), mp: 170-172 °C. 1H NMR (500 MHz, CDCl3) δ 7.70 (d, J = 1.2 Hz, 1H), 7.60 (d,
J = 1.2 Hz, 1H), 7.52 (s, 1H), 7.46 – 7.41 (m, 2H), 7.05 – 7.00 (m, 2H), 6.35 (s, 1H), 3.87 (s, 3H),
3.12 – 2.99 (m, 4H), 2.53 (d, J = 13.1 Hz, 2H), 2.20 – 2.11 (m, 2H). 13C NMR (125 MHz, CDCl3) δ
161.90 (d, JC-F = 245.0 Hz), 161.79, 140.55, 139.21, 136.23, 131.78, 129.34, 128.73, 128.59, 127.02
(d, JC-F = 7.3 Hz), 115.67 (d, JC-F = 23.8 Hz), 94.00, 55.77, 40.82, 38.90, 32.10. ESI-MS (m/z): 463
[M+1]+.
4.1.9.4
N-(4-(3,4-dichlorophenyl)piperidin-4-yl)-4-(1-methyl-1H-pyrazol-5-yl)thiophene-2-carboxamide 24d
Reagent: 4a (42 mg, 0.2 mmol) and 23b (67 mg, 0.2 mmol). The product was obtained as a white
solid (44 mg, 74%), mp: 208-210 °C. 1H NMR (500 MHz, CDCl3) δ 7.60 (d, J = 1.4 Hz, 1H), 7.53 (d,
J = 2.3 Hz, 1H), 7.49 (m, 2H), 7.42 – 7.39 (m, 1H), 7.31 (dd, J = 8.5, 2.3 Hz, 1H), 6.34 (d, J = 1.9
Hz, 1H), 6.29 (s, 1H), 3.93 (s, 3H), 3.06 (dd, J = 9.3, 3.4 Hz, 2H), 3.01 – 2.94 (m, 2H), 2.44 (d, J =
12.6 Hz, 2H), 2.10 – 2.03 (m, 2H). ESI-MS (m/z): 435 [M+1]+. HRMS (ESI): m/z calcd for
(C20H20Cl2N4OS+H)+: 435.0808; found: 435.0818.
4.1.9.5
4-(4-Chloro-1-methyl-1H-pyrazol-5-yl)-N-(4-(4-fluorophenyl)piperidin-4-yl)-furan-2-carboxamide
25a
Reagent: 13a (45 mg, 0.2 mmol) and 23a (57 mg, 0.2 mmol). The product was obtained as an
off-white solid (39 mg, 49%), mp: 144-145 °C. 1H NMR (500 MHz, CDCl3) δ 7.79 (s, 1H), 7.47 (s,
1H), 7.44 (dd, J = 8.5, 5.3 Hz, 2H), 7.26 (s, 1H), 7.03 (t, J = 8.6 Hz, 2H), 6.61 (s, 1H), 3.88 (s, 3H),
3.11 – 2.97 (m, 4H), 2.51 (d, J = 13.4 Hz, 2H), 2.12 (dd, J = 17.7, 7.2 Hz, 2H). ESI-MS (m/z): 403
[M+1]+.
4.1.9.6
4-(4-Bromo-1-methyl-1H-pyrazol-5-yl)-N-(4-(4-fluorophenyl)piperidin-4-yl)-furan-2-carboxamide
25b
Reagent: 13c (57 mg, 0.2 mmol) and 23a (57 mg, 0.2 mmol). The product was obtained as an
off-white solid (56 mg, 63%), mp: 193-194 °C. 1H NMR (500 MHz, CDCl3) δ 7.79 (s, 1H), 7.51 (s,
1H), 7.45 (dd, J = 8.8, 5.2 Hz, 2H), 7.24 (s, 1H), 7.04 (t, J = 8.7 Hz, 2H), 6.59 (s, 1H), 3.88 (s, 3H),
3.12 – 2.98 (m, 4H), 2.51 (d, J = 13.0 Hz, 2H), 2.17 – 2.10 (m, 2H). ESI-MS (m/z): 447 [M+1]+.
4.1.9.7

ACCEPTED MANUSCRIPT
4-(4-Bromo-1-methyl-1H-pyrazol-5-yl)-N-(4-(3,4-dichlorophenyl)piperidin-4-yl)furan-2-carboxamid
e 25c
Reagent: 13c (57 mg, 0.2 mmol) and 23b (67 mg, 0.2 mmol). The product was obtained as a
yellow solid (73 mg, 74%), mp: 201-203 °C. 1H NMR (500 MHz, CDCl3) δ 7.81 (s, 1H), 7.54 (d, J =
2.2 Hz, 1H), 7.51 (s, 1H), 7.42 (d, J = 8.5 Hz, 1H), 7.32 (dd, J = 8.4, 2.3 Hz, 2H), 6.63 (s, 1H), 3.89
(s, 3H), 3.09 (d, J = 12.7 Hz, 2H), 3.01 (t, J = 11.3 Hz, 2H), 2.47 (d, J = 12.9 Hz, 2H), 2.13 – 2.05 (m,
2H). 13C NMR (125 MHz, CDCl3) δ 157.60, 147.67, 143.06, 139.48, 133.02, 132.13, 130.85, 130.48,
127.39, 124.73, 115.92, 115.37, 114.58, 94.11, 55.34, 40.47, 38.81, 32.11. ESI-MS (m/z): 497
[M+1]+.
4.1.9.8
5-Chloro-4-(4-chloro-1-methyl-1H-pyrazol-5-yl)-N-(4-(4-fluorophenyl)piperidin-4-yl)furan-2-carbo
xamide 25d
Reagent: 13b (52 mg, 0.2 mmol) and 23a (57 mg, 0.2 mmol). The product was obtained as a white
solid (52 mg, 60%), mp: 170-172 °C. 1H NMR (500 MHz, CDCl3) δ 7.50 (s, 1H), 7.47 – 7.42 (m,
2H), 7.13 (s, 1H), 7.07 – 7.01 (m, 2H), 6.47 (s, 1H), 3.79 (s, 3H), 3.10 (dd, J = 9.1, 3.6 Hz, 2H), 3.06
– 3.00 (m, 2H), 2.52 (d, J = 12.9 Hz, 2H), 2.19 – 2.12 (m, 2H). 13C NMR (125 MHz, CDCl3) δ
162.19 (d, JC-F = 247.9 Hz), 156.42, 146.46, 138.40, 137.42, 129.01 (d, JC-F = 9.9 Hz), 126.97,
126.90, 118.14, 115.88 (d, JC-F = 21.5 Hz), 111.60 111.17, 55.79, 40.47, 38.46, 32.49. ESI-MS (m/z):
437 [M+1]+.
4.1.9.9
5-Chloro-4-(4-chloro-1-methyl-1H-pyrazol-5-yl)-N-(4-(3,4-dichlorophenyl)-piperidin-4-yl)furan-2-c
arboxamide 25e
Reagent: 13b (52 mg, 0.2 mmol) and 23b (67 mg, 0.2 mmol). The product was obtained as a white
solid (69 mg, 71%), mp: 130-133 °C. 1H NMR (500 MHz, CDCl3) δ 7.54 (s, 1H), 7.50 (s, 1H), 7.43
(d, J = 8.4 Hz, 1H), 7.35 – 7.28 (m, 2H), 6.92 (s, 1H), 3.80 (s, 3H), 3.38 (t, J = 11.1 Hz, 2H), 3.28 (t,
J = 11.9 Hz, 2H), 2.79 (d, J = 13.9 Hz, 2H), 2.48 (t, J = 11.5 Hz, 2H). 13C NMR (125 MHz, CDCl3) δ
156.35, 146.53, 143.66, 138.49, 137.39, 133.00, 132.01, 130.77, 129.01, 127.45, 124.75, 118.10,
111.47, 111.14, 55.72, 40.76, 38.44, 32.87. ESI-MS (m/z): 489 [M+1]+. HRMS (ESI): m/z calcd for
(C20H18Cl4N4O2+H)+: 489.0227; found: 489.0239.
4.1.9.10

ACCEPTED MANUSCRIPT
4-Bromo-N-(4-(3,4-dichlorophenyl)piperidin-4-yl)-5-(1-methyl-1H-pyrazol-5-yl)furan-2-carboxamid
e 26a
Reagent: 16a (54 mg, 0.2 mmol) and 23b (67 mg, 0.2 mmol). The product was obtained as an
off-white solid (76 mg, 77%), mp: 118-120 °C. 1H NMR (500 MHz, CDCl3) δ 7.54 (d, J = 2.0 Hz,
1H), 7.50 (d, J = 2.2 Hz, 1H), 7.38 (d, J = 8.1 Hz, 1H), 7.30 – 7.26 (m, 2H), 6.82 (s, 1H), 6.78 (d, J =
2.0 Hz, 1H), 4.05 (s, 3H), 3.15 (d, J = 12.8 Hz, 2H), 3.03 (dd, J = 17.8, 10.1 Hz, 2H), 2.55 (d, J =
13
13.3 Hz, 2H), 2.21 – 2.12 (m, 2H). C NMR (125 MHz, CDCl3) δ 156.32, 147.15, 145.16, 143.21,
138.74, 132.74, 131.38, 130.54, 130.37, 127.47, 124.73, 119.14, 108.76, 101.66, 56.30, 41.84, 38.92,
35.05. ESI-MS (m/z): 497 [M+1]+.
4.1.9.11
4-Bromo-5-(4-chloro-1-methyl-1H-pyrazol-5-yl)-N-(4-(3,4-dichlorophenyl)-piperidin-4-yl)furan-2-c
arboxamide 26b
Reagent: 16b (61 mg, 0.2 mmol) and 23b (67 mg, 0.2 mmol). The product was obtained as a white
solid (74 mg, 70%), mp: 109-112 °C. 1H NMR (500 MHz, CDCl3) δ 7.55 (s, 1H), 7.50 (d, J = 2.2 Hz,
1H), 7.44 (d, J = 8.5 Hz, 1H), 7.33 – 7.27 (m, 2H), 6.66 (s, 1H), 3.90 (s, 3H), 3.40 – 3.30 (m, 2H),
3.20 (t, J = 12.6 Hz, 2H), 2.72 (d, J = 14.2 Hz, 2H), 2.49 (m, 2H). ESI-MS (m/z): 533 [M+1]+.
HRMS (ESI): m/z calcd for (C20H18BrCl3N4O2 +H)+: 532.9731; found: 532.9741.
4.1.9.12
4-Bromo-5-(4-bromo-1-methyl-1H-pyrazol-5-yl)-N-(4-(3,4-dichlorophenyl)-piperidin-4-yl)furan-2-c
arboxamide 26c
Reagent: 16c (70 mg, 0.2 mmol) and 23b (67 mg, 0.2 mmol). The product was obtained as a
pale-yellow solid (93 mg, 81%), mp: 127-130 °C. 1H NMR (500 MHz, CDCl3) δ 7.58 (s, 1H), 7.50
(d, J = 2.1 Hz, 1H), 7.42 (d, J = 8.5 Hz, 1H), 7.31 – 7.27 (m, 2H), 6.72 (s, 1H), 3.91 (s, 3H), 3.28 –
3.21 (m, 2H), 3.17 – 3.09 (m, 2H), 2.63 (d, J = 13.9 Hz, 2H), 2.35 (t, J = 11.6 Hz, 2H). ESI-MS (m/z):
577 [M+1]+. HRMS (ESI): m/z calcd for (C20H18Br2Cl2N4O2 +H)+: 576.9226; found: 576.9235.
4.1.9.13
4-(4-Bromo-1-methyl-1H-pyrazol-5-yl)-N-(4-(3,4-dichlorophenyl)piperidin-4-yl)-1-methyl-1H-pyrrol
e-2-carboxamide 27a
Reagent: 13d (57 mg, 0.2 mmol) and 23b (67 mg, 0.2 mmol). The product was obtained as an
1
off-white solid (48 mg, 47%), mp: 178-181 °C. H NMR (500 MHz, CDCl3) δ 7.53 (d, J = 2.2 Hz,

ACCEPTED MANUSCRIPT
1H), 7.48 (s, 1H), 7.42 (d, J = 8.5 Hz, 1H), 7.30 (dd, J = 8.5, 2.3 Hz, 1H), 6.98 (s, 1H), 6.91 (s, 1H),
6.34 (s, 1H), 3.91 (s, 3H), 3.89 (s, 3H), 3.22 – 3.05 (m, 4H), 2.53 (m, 2H), 2.24 (m, 2H). ESI-MS
(m/z): 510 [M+1]+.
4.1.9.14
5-Bromo-4-(4-bromo-1-methyl-1H-pyrazol-5-yl)-N-(4-(3,4-dichlorophenyl)piperidin-4-yl)-1-methyl-
1H-pyrrole-2-carboxamide 27b
Reagent: 13e (72 mg, 0.2 mmol) and 23b (67 mg, 0.2 mmol). The product was obtained as a
1
yellow solid (45 mg, 38%), mp: 149-151 °C. H NMR (500 MHz, CDCl3) δ 7.53 – 7.49 (m, 2H),
7.41 (dd, J = 8.4, 4.2 Hz, 1H), 7.29 (dd, J = 8.4, 2.3 Hz, 1H), 6.88 (s, 1H), 6.43 (s, 1H), 3.90 (s, 3H),
3.77 (s, 3H), 3.15 (d, J = 12.7 Hz, 2H), 3.06 (dd, J = 22.0, 10.4 Hz, 2H), 2.51 (d, J = 13.4 Hz, 2H),
2.21 – 2.12 (m, 2H). 13C NMR (125 MHz, CDCl3) δ 160.11, 145.66, 139.06, 135.11, 132.77, 131.23,
130.57, 127.90, 127.37, 124.56, 113.55, 112.71, 110.79, 95.38, 56.12, 41.77, 38.32, 35.26, 35.07.
ESI-MS (m/z): 590 [M+1]+.
4.1.9.15
3,5-Dichloro-4-(4-chloro-1-methyl-1H-pyrazol-5-yl)-N-(4-(3,4-dichlorophenyl)piperidin-4-yl)-1-met
hyl-1H-pyrrole-2-carboxamide 27c
Reagent: 13f (61 mg, 0.2 mmol) and 23b (67 mg, 0.2 mmol). The product was obtained as a white
solid (71 mg, 66%), mp: 131-134 °C. 1H NMR (500 MHz, CDCl3) δ 7.56 – 7.50 (m, 2H), 7.43 (d, J =
8.5 Hz, 1H), 7.31 (dd, J = 8.5, 2.1 Hz, 1H), 6.91 (s, 1H), 3.85 (s, 3H), 3.76 (s, 3H), 3.17 – 2.98 (m,
4H), 2.50 – 2.36 (m, 2H), 2.18 – 2.00 (m, 2H). 13C NMR (125 MHz, CDCl3) δ 158.35, 145.98,
137.15, 132.75, 131.06, 130.55, 129.79, 127.41, 124.56, 123.55, 122.81, 113.93, 111.99, 106.87,
56.90, 41.87, 38.11, 35.92, 34.45. ESI-MS (m/z): 536 [M+1]+.
4.1.9.16 4-(4-Chloro-1-methyl-1H-pyrazol-5-yl)-N-(4-(3,4-dichlorophenyl)piperidin-4-yl)benzamide
28a
Reagent: 19a (47 mg, 0.2 mmol) and 23b (67 mg, 0.2 mmol). The product was obtained as a white
solid (64 mg, 69%), mp: 99-102 °C. 1H NMR (500 MHz, CDCl3) δ 7.95 (d, J = 8.0 Hz, 2H), 7.51 (d,
J = 7.8 Hz, 2H), 7.47 (d, J = 8.0 Hz, 2H), 7.41 – 7.35 (m, 1H), 7.30 (d, J = 7.3 Hz, 1H), 7.20 (s, 1H),
13
3.79 (s, 3H), 3.34 – 3.20 (m, 4H), 2.76 (d, J = 13.8 Hz, 2H), 2.36 (m, 2H). C NMR (125 MHz,
CDCl3) δ 166.55, 144.62, 138.32, 137.29, 134.69, 132.84, 13158, 131.47, 130.66, 129.90, 127.69,
127.41, 124.70, 109.17, 55.87, 41.13, 38.45, 33.38. ESI-MS (m/z): 465 [M+1]+. HRMS (ESI): m/z

ACCEPTED MANUSCRIPT
calcd for (C22H21Cl3N4O +H)+: 465.0824; found: 465.0836.
4.1.9.17 4-(4-Bromo-1-methyl-1H-pyrazol-5-yl)-N-(4-(3,4-dichlorophenyl)piperidin-4-yl)benzamide
28b
Reagent: 19b (56 mg, 0.2 mmol) and 23b (67 mg, 0.2 mmol). The product was obtained as a white
solid (78 mg, 77%), mp: 148-150 °C. 1H NMR (500 MHz, CDCl3) δ 7.93 (d, J = 8.3 Hz, 2H), 7.55 (d,
J = 2.3 Hz, 1H), 7.53 (d, J = 2.3 Hz, 1H), 7.50 (d, J = 8.3 Hz, 2H), 7.42 (d, J = 8.5 Hz, 1H), 7.31 (dd,
J = 8.5, 2.3 Hz, 1H), 6.74 (s, 1H), 3.82 (s, 3H), 3.25 – 3.12 (m, 4H), 2.63 (d, J = 13.8 Hz, 2H), 2.32 –
2.23 (m, 2H). 13C NMR (125 MHz, CDCl3) δ 166.12, 145.05, 140.01, 139.45, 135.00, 132.84,
132.01, 131.43, 130.60, 130.20, 127.44, 127.42, 124.66, 93.86, 56.12, 41.23, 38.47, 34.39. ESI-MS
(m/z): 507 [M+1]+.
4.2. Pharmacological assay
4.2.1 Akt1 assay
The Akt1 inhibitory activities were determined using the homogeneous time-resolved fluorescence
(HTRF) KinEASE-STK S1 kit (Cat.#62ST2PEC, Cisbio) according to the manufacturer’s
instructions. Akt1 kinase was expressed and purified from Escherichia coli system in-house. In short,
the kinase reactions were carried out in a 384 ProxiPlate with 10 µL reaction volume per well
containing 0.25 µg/mL Akt1, 1 µM peptide substrate, test compound and 20 µM ATP in assay buffer
(50 mM HEPES pH 7.5, 0.01% BRIJ-35, 5 mM MgCl2, 1 mM EGTA). After incubation for 1 h at r.t.,
the reaction was stopped by the addition of 5µL Sa-XL665 and 5 µL STK Antibody-Eu(K) in EDTA.
The plate was sealed and incubated for 1 h at r.t., and the resulting TR-FRET signal was measured on
Envision-PerkinElmer. The fluorescence emission was measured at 615 nm (cryptate) and 665 nm
(XL665). An Emission Ratio was calculated (665/615) for each well and the Percent inhibitions were
expressed as follows: Percent inhibition = (max-sample Ratio)/ (max-min)*100 (“Min” means the
Ratio of no enzyme control and “max” means the Ratio of DMSO control). Compounds were
initially tested at a fixed concentration (1 µg/mL), and those displaying more than 50% inhibition
were further tested for dose–response IC50 values.
4.2.2 Kinase selectivity assay
To determine the kinase selectivity of the target compounds, the representative 25e was assayed

ACCEPTED MANUSCRIPT
against a panel of 22 protein kinases at SHANGHAI CHEMPARTNER CO.LTD. All the proteins
and kinase assay kits were mainly purchased from Carna, Invitrogen or Millipore. Compound 25e
was tested at concentrations of 0.5 and 10 µM over most kinases, including ABL, ALK, Akt2, Akt3,
JNK2, JAK2, ROCK1, RSK1, P70S6K, MSK1, SGK, PDK1 and PRKG1, using Mobility shift assay
with ATP concentration at Km. PI3Kα inhibitory activity was screened by Kinase-Glo Luminescent
Assay kit (Promege) with ATP concentration at 25µM. CHK1 and TSSK1 inhibitory activities were
determined using the Luminescent ADP-Glo assay kit (Promege). In addition, the inhibition of
mTOR activity was evaluated in a Lance Ultra assay. Moreover, PKA, GSK3β and CDK2/cyclin A
activities were evaluated at the concentration of 1 µg/mL by Z-LYTE Kinase Assay kits (Invitrogen).
Like Akt1 assay, the Aurora A and BRAF inhibitory activities of compound 25e were measured by
the HTRF Kinase Assay kits purchased from Cisbio. The protocols for these assays could be found in
the manufacturer’s instructions.
4.2.3 Anti-proliferative assay
All of the prepared target compounds were tested for their anti-proliferative activities in vitro
against two human cancer cell lines including OVCAR-8 (Ovarian Adenocarcinoma) and HCT116
(Human Colon Cancer) by sulforhodamin B (SRB) assay. OVCAR-8 and HCT-116 cells were seeded
into 96-well plates and cultured for 10 h. Subsequently, it was exposed to serial concentrations of
compound for 72 h. Cells were then washed with PBS and fixed with 10% (w/v) trichloroacetic acid
at 4 °C for 1 hour. After washing, the cells were stained for 30 min with 0.4% SRB dissolved in 1%
acetic acid and then washed by 1% acetic acid for 5 times. Finally, the protein-bound dye was
extracted using 10 mM unbuffered Tris base. The absorbance was obtained at 515 nm on a multiscan
spectrum (Thermo Fisher). The inhibition rate on cell proliferation of each well was calculated using
the formula of (A515 control cells - A515 treated cells)/A515 control cells × 100%. Data was
presented in MS Excel and the curves fitted by Graphpad Prism 5.0.
4.2.4 Western blot assay
PC-3 prostate cancer cells were exposed to compound 25e at 1uM and 10 uM for 3 h, and were
then trypsinized and wash once with 2ml PBS. Protein samples were prepared by adding 100 µL
RIPA lysis buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate,

ACCEPTED MANUSCRIPT
0.1% SDS) with protease and phosphatase inhibitor (Roche, Cat.#4693124001) onto cells and all cell
lysate was collected and then centrifuged at 4˚C. The supernatant was collected as protein sample,
and the total concentration was quantified using BCA kit (Pierce, Cat.#23225) and normalized.
Subsequently, Proteins were transferred from gel to a nitrocellulose membrane (Life Tech,
IB301002), and membrane was blocked in SuperBlock blocking buffer (Thermo Fisher, Cat. #37535)
for 1 h, then in primary antibody dilution overnight at 4˚C (Rabbit anti-pAKT(S473), anti-AKT and
anti-GSK3β(S9) were purchased from Cell signaling; Rabbit anti-GSK3β and mouse anti-GAPDH
were purchased from Abcam). After 5×5 minutes washes in TBST buffer (25mM Tris HCL pH7.6,
150mM NaCl, 0.1% Tween-20), membrane was incubated in diluted secondary antibody for 1 h at
r.t., and washed in TBST again. Membrane was imaged using LICOR Oddessy system.
4.2.5 ELISA assay
ELISA plates were prepared by coating with anti-PRAS40 antibody (R&D Systems), and blocked
with 5% Milk/0.1% Tween-20. LNCaP Cells were seeded at 25,000 cells/96-well overnight and
treated with DMSO or various concentrations of compounds for 1 h. Subsequently, cells were lysed
in 20 mM Tris-HCl (pH 8.0), 137 mM NaCl, 2 mM EDTA, 10% glycerol and 1% Triton X-100 and
lysates transferred to ELISA plates and incubated overnight. The plates were washed and incubated
with rabbit anti- PRAS40 (pThr246) antibody (R&D Systems) for 1 h. After washing, plates were
developed using HRP-linked anti-rabbit IgG, and 3,39,5,59-tetramethylbenzidine as substrate.
Absorption was measured in a microplate spectrophotometer at 450 nm, Dose−response curves were
generated using the four-parameter logistic model, and 50% inhibitory concentration (IC50) values
were determined from these curve fits.
4.3. Molecular docking
Docking analysis was carried out by using C-DOCKER module (Discovery Studio, version 2.5;
Accelrys, San Diego, CA, USA, 2008) to compare the binding modes between compound 25e and
28a bound to Akt1. The X-ray crystal structure of Akt1 (PDB ID: 4GV1) was used for the docking
calculation. After removing the ligand and water molecules, the CHARMm-force field was applied
to the protein. And the ATP binding pocket was chosen as the active site with a radius set as 8 Å. The
ligands were generated random conformations using CHARMm-based molecular dynamics (1000

ACCEPTED MANUSCRIPT
steps), and then docked into the defined Akt1 binding site. The other parameters were set as default.
The final binding conformation of 25e and 28a was determined based on the calculated CDOCKING
ENERAGE. The most stable binding modes among the top 10 docking poses of 25e and 28a were
presented in Fig. 6A and 6B, respectively.
Acknowledgement
We thank Mrs. Jianyang Pan (Pharmaceutical Informatics Institute, Zhejiang University) for
performing the NMR spectrometry, the support from Zhejiang province Provincial Natural Science
Foundation of China (LY13H300002) and Program for Zhejiang Leading Team of S&T Innovation
(2011R50014).
Reference:
[1] P.X. Liu, H.L. Cheng, T.M. Roberts, J.J. Zhao, Targeting the phosphoinositide 3-kinase pathway
in cancer, Nat. Rev. Drug Discov. 8 (2009) 627-644.
[2] B.D. Manning, L.C. Cantley, AKT/PKB signaling: navigating downstream, Cell 129 (2007)
1261-1274.
[3] C.S. Mitsiades, N. Mitsiades, M. Koutsilieris, The Akt pathway: Molecular targets for anti-cancer
drug development, Curr. Cancer Drug Tar. 4 (2004) 235-256.
[4] Bellacosa, C.C. Kumar, A. Di Cristofano, J.R. Testa, Activation of AKT kinases in cancer:
implications for therapeutic targeting, Adv. Cancer Res. 94 (2005) 29-86.
[5] J.Q. Cheng, B. Ruggeri, W.M. Klein, G. Sonoda, D.A. Altomare, D.K. Watson, J.R. Testa,
Amplification of AKT2 in human pancreatic cancer cells and inhibition of ATK2 expression and
tumorigenicity by antisense RNA, Proc. Natl. Acad. Sci. U. S. A. 93 (1996) 3636-3641.
[6] J.R. Testa, A. Bellacosa, AKT plays a central role in tumorigenesis, Proc. Natl. Acad. Sci. U. S.
A. 98 (2001) 10983-10985.
[7] S.P. Staal, Molecular-Cloning of the Akt Oncogene and Its Human Homologs Akt1 and Akt2 -
Amplification of Akt1 in a Primary Human Gastric Adenocarcinoma, Proc. Natl. Acad. Sci. U. S. A.
84 (1987) 5034-5037.
[8] A.S. Clark, K. West, S. Streicher, P.A. Dennis, Constitutive and inducible Akt activity promotes
resistance to chemotherapy, trastuzumab, or tamoxifen in breast cancer cells, Mol. Cancer Ther. 1
(2002) 707-717.
[9] A. Lassen, M. Atefi, L. Robert, D.J. Wong, M. Cerniglia, B. Comin-Anduix, A. Ribas, Effects of
AKT inhibitor therapy in response and resistance to BRAF inhibition in melanoma, Mol. Cancer 13
(2014) 83.
[10] D. Kim, H.C. Dan, S. Park, L. Yang, Q. Liu, S. Kaneko, J. Ning, L. He, H. Yang, M. Sun, S.V.
Nicosia, J.Q. Cheng, AKT/PKB signaling mechanisms in cancer and chemoresistance, Front Biosci.
10 (2005) 975-987.
[11] M.E. Mattmann, S.L. Stoops, C.W. Lindsley, Inhibition of Akt with small molecules and

ACCEPTED MANUSCRIPT
biologics: historical perspective and current status of the patent landscape, Expert Opin. Ther. Pat. 21
(2011) 1309-1338.
[12] S. Sestito, G. Nesi, S. Daniele, A. Martelli, M. Digiacomo, A. Borghini, D. Pietra, V. Calderone,
A. Lapucci, M. Falasca, P. Parrella, A. Notarangelo, M.C. Breschi, M. Macchia, C. Martini, S.
Rapposelli, Design and synthesis of 2-oxindole based multi-targeted inhibitors of PDK1/Akt
signaling pathway for the treatment of glioblastoma multiforme, Eur. J. Med. Chem. 105 (2015)
274-288.
[13] S. Jangir, V. Bala, N. Lal, L. Kumar, A. Sarswat, A. Kumar, Hamidullah, K.S. Saini, V. Sharma,
V. Verma, J.P. Maikhuri, R. Konwar, G. Gupta, V.L. Sharma, Novel alkylphospholipid-DTC hybrids
as promising agents against endocrine related cancers acting via modulation of Akt-pathway, Eur. J.
Med. Chem. 85 (2014) 638-647.
[14] S.K. Pal, K. Reckamp, H. Yu, R.A. Figlin, Akt inhibitors in clinical development for the
treatment of cancer, Expert Opin. Investig. Drugs 19 (2010) 1355-1366.
[15] T. Ippolito, N. Czuczman, C. Mavis, D. Rolland, M. Lim, A. Tiwari, M.S. Cairo, R.R. Miles,
M.J. Barth, MK-2206, an Akt inhibitor, decreases proliferation, induces cell cycle arrest and
increases responsiveness to chemotherapy in therapy-sensitive and -resistant Burkitt lymphoma cell
line models, Br. J. Haematol. 171 (2015) 25-25.
[16] H. Hirai, H. Sootome, Y. Nakatsuru, K. Miyama, S. Taguchi, K. Tsujioka, Y. Ueno, H. Hatch,
P.K. Majumder, B.S. Pan, H. Kotani, MK-2206, an allosteric Akt inhibitor, enhances antitumor
efficacy by standard chemotherapeutic agents or molecular targeted drugs in vitro and in vivo, Mol.
Cancer Ther. 9 (2010) 1956-1967.
[17] J.F. Blake, R. Xu, J.R. Bencsik, D. Xiao, N.C. Kallan, S. Schlachter, I.S. Mitchell, K.L. Spencer,
A.L. Banka, E.M. Wallace, S.L. Gloor, M. Martinson, R.D. Woessner, G.P. Vigers, B.J. Brandhuber,
J. Liang, B.S. Safina, J. Li, B. Zhang, C. Chabot, S. Do, L. Lee, J. Oeh, D. Sampath, B.B. Lee, K.
Lin, B.M. Liederer, N.J. Skelton, Discovery and preclinical pharmacology of a selective
ATP-competitive Akt inhibitor (GDC-0068) for the treatment of human tumors, J. Med. Chem. 55
(2012) 8110-8127.
[18] M. Addie, P. Ballard, D. Buttar, C. Crafter, G. Currie, B.R. Davies, J. Debreczeni, H. Dry, P.
Dudley, R. Greenwood, P.D. Johnson, J.G. Kettle, C. Lane, G. Lamont, A. Leach, R.W.A. Luke, J.
Morris, D. Ogilvie, K. Page, M. Pass, S. Pearson, L. Ruston, Discovery of
4-Amino-N-[(1S)-1-(4-chlorophenyl)-3-hydroxypropyl]1-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidi
ne-4-carboxamide (AZD5363), an Orally Bioavailable, Potent Inhibitor of Akt Kinases, J. Med.
Chem. 56 (2013) 2059-2073.
[19] M. Dumble, M.C. Crouthamel, S.Y. Zhang, M. Schaber, D. Levy, K. Robell, Q. Liu, D.J.
Figueroa, E.A. Minthorn, M.A. Seefeld, M.B. Rouse, S.K. Rabindran, D.A. Heerding, R. Kumar,
Discovery of novel AKT inhibitors with enhanced anti-tumor effects in combination with the MEK
inhibitor, PLoS One 9 (2014) e100880.
[20] S. Blagden, A. Hamilton, L. Mileshkin, M. Hall, T. Meniawy, S. Wong, S. Anandra, M. Buck,
D. McAleer, B.A. Reedy, R.B. Noble, D.A. Smith, S.R. Morris, H. Gabra, Afuresertib
(GSK2110183), an oral AKT kinase inhibitor, in combination with carboplatin and paclitaxel in
recurrent ovarian cancer, Eur. J. Cancer 50 (2014) 7-7.
[21] R. Kurzrock, A. Patnaik, L. Rosenstein, S. Fu, K.P. Papadopoulos, D.A. Smith, G.S. Falchook,
G. Chambers, J.L. Gauvin, A. Naing, L.S. Smith, T. Gonzalez, A.M. Tsimberidou, T.A. Mays, D.S.
Cox, D.S. Hong, D.J. DeMarini, N.T. Le, S.R. Morris, A.W. Tolcher, Phase I dose-escalation of the

ACCEPTED MANUSCRIPT
oral MEK1/2 inhibitor GSK1120212 (GSK212) dosed in combination with the oral AKT inhibitor
GSK2141795 (GSK795), J. Clin. Oncol. 29 (2011) Abstr 3085.
[22] A. Spencer, S.S. Yoon, S.J. Harrison, S.R. Morris, D.A. Smith, R.A. Brigandi, J. Gauvin, R.
Kumar, J.B. Opalinska, C. Chen, The novel AKT inhibitor afuresertib shows favorable safety,
pharmacokinetics, and clinical activity in multiple myeloma, Blood 124 (2014) 2190-2195.
[23] A.W. Tolcher, A. Patnaik, K.P. Papadopoulos, D.W. Rasco, C.R. Becerra, A.J. Allred, K.
Orford, G. Aktan, G. Ferron-Brady, N. Ibrahim, J. Gauvin, M. Motwani, M. Cornfeld, Phase I study
of the MEK inhibitor trametinib in combination with the AKT inhibitor afuresertib in patients with
solid tumors and multiple myeloma, Cancer Chemother. Pharmacol. 75 (2015) 183-189.
[24] H. Gungor, A. Saleem, R. Agarwal, S.P. Blagden, A. Michael, E.A. Stronach, M. Chen, E.
Pickford, N.R. Rama, Y.L. Lewis, S.C. Carme, C. Salinas, D.A. Smith, E. Krachey, A.
Santiago-Walker, R.N. Gunn, M. El-Bahrawy, S.A. Babar, S.R. Morris, H. Gabra, Pharmacokinetic
(PK)/pharmacodynamic (PD) analysis of escalating repeat doses of the AKT inhibitor GSK2141795
(GSK795) in patients (pts) with ovarian cancer, J. Clin. Oncol. 29 (2011) Abstr 5046.
[25] U. Matulonis, A.A. Wright, S.A. Collins, S.M. Campos, P. Konstantinopoulos, K. MacNeill, S.
Morrissey, V. Patterson, C. Whalen, J. Liu, A single arm, single stage phase II trial of trametinib
(GSK1120212) and GSK2141795 in persistent or recurrent cervical cancer, J. Clin. Oncol. 33 (2015)
Abstr TPS5618.
[26] A. Spencer, S.S. Yoon, S.J. Harrison, S. Morris, D. Smith, S.J. Freedman, R. Brigandi, A. Oliff,
J.B. Opalinska, C. Chen, Novel AKT Inhibitor GSK2110183 Shows Favorable Safety,
Pharmacokinetics, and Clinical Activity in Multiple Myeloma. Preliminary Results From a Phase I
First-Time-In-Human Study, Blood 118 (2011) 808-808.
[27] Z.J. Fang, Y.N. Song, P. Zhan, Q.Z. Zhang, X.Y. Liu, Conformational restriction: an effective
tactic in 'follow-on'-based drug discovery, Future Med. Chem. 6 (2014) 885-901.
[28] S. Hanessian, L. Auzzas, The Practice of Ring Constraint in Peptidomimetics Using Bicyclic
and Polycyclic Amino Acids, Acc. Chem. Res. 41 (2008) 1241-1251.
[29] I.J. Kang, L.W. Wang, C.C. Lee, Y.C. Lee, Y.S. Chao, T.A. Hsu, J.H. Chern, Design, synthesis,
and anti-HCV activity of thiourea compounds, Bioorg. Med. Chem. Lett. 19 (2009) 1950-1955.
[30] I.J. Kang, L.W. Wang, T.K. Yeh, C.C. Lee, Y.C. Lee, S.J. Hsu, Y.S. Wu, J.C. Wang, Y.S. Chao,
A. Yueh, J.H. Chern, Synthesis, activity, and pharmacokinetic properties of a series of
conformationally-restricted thiourea analogs as novel hepatitis C virus inhibitors, Bioorg. Med.
Chem. 18 (2010) 6414-6421.
[31] A.E. Kummerle, M.M. Vieira, M. Schmitt, A.L.P. Miranda, C.A.M. Fraga, J.J. Bourguignon,
E.J. Barreiro, Design, synthesis and analgesic properties of novel conformationally-restricted
N-acylhydrazones (NAH), Bioorg. Med. Chem. Lett. 19 (2009) 4963-4966.
[32] R.K. Tiwari, K. Parang, Conformationally Constrained Peptides as Protein Tyrosine Kinase
Inhibitors, Curr. Pharm. Des. 18 (2012) 2852-2866.
[33] G. Valle, W.M. Kazmierski, M. Crisma, G.M. Bonora, C. Toniolo, V.J. Hruby, Constrained
Phenylalanine
Analogs
-
Preferred
Conformation
Of
the
1,2,3,4-Tetrahydroisoquinoline-3-Carboxylic Acid (Tic) Residue, Int. J. Pept. Protein Res. 40 (1992)
222-232.
[34] G. Saxty, S.J. Woodhead, V. Berdini, T.G. Davies, M.L. Verdonk, P.G. Wyatt, R.G. Boyle, D.
Barford, R. Downham, M.D. Garrett, R.A. Carr, Identification of inhibitors of protein kinase B using
fragment-based lead discovery, J. Med. Chem. 50 (2007) 2293-2296.

ACCEPTED MANUSCRIPT
[35] K.M. Grimshaw, L.J. Hunter, T.A. Yap, S.P. Heaton, M.I. Walton, S.J. Woodhead, L. Fazal, M.
Reule, T.G. Davies, L.C. Seavers, V. Lock, J.F. Lyons, N.T. Thompson, P. Workman, M.D. Garrett,
AT7867 is a potent and oral inhibitor of AKT and p70 S6 kinase that induces pharmacodynamic
changes and inhibits human tumor xenograft growth, Mol. Cancer Ther. 9 (2010) 1100-1110.
[36] K.S. Ashton, D.J. St Jean, S.F. Poon, M.R. Lee, J.G. Allen, S.W. Zhang, J.A. Lofgren, X.L.
Zhang, C. Fotsch, R. Hungate, Design and synthesis of novel amide AKT1 inhibitors with selectivity
over CDK2, Bioorg. Med. Chem. Lett. 21 (2011) 5191-5196.
[37] B.R. Davies, H. Greenwood, P. Dudley, C. Crafter, D.H. Yu, J. Zhang, J. Li, B. Gao, Q. Ji, J.
Maynard, S.A. Ricketts, D. Cross, S. Cosulich, C.C. Chresta, K. Page, J. Yates, C. Lane, R. Watson,
R. Luke, D. Ogilvie, M. Pass, Preclinical pharmacology of AZD5363, an inhibitor of AKT:
pharmacodynamics, antitumor activity, and correlation of monotherapy activity with genetic
background, Mol. Cancer Ther. 11 (2012) 873-887.
[38] N. Rhodes, D.A. Heerding, D.R. Duckett, D.J. Eberwein, V.B. Knick, T.J. Lansing, R.T.
McConnell, T.M. Gilmer, S.Y. Zhang, K. Robell, J.A. Kahana, R.S. Geske, E.V. Kleymenova, A.E.
Choudhry, Z. Lai, J.D. Leber, E.A. Minthorn, S.L. Strum, E.R. Wood, P.S. Huang, R.A. Copeland, R.
Kumar, Characterization of an Akt kinase inhibitor with potent pharmacodynamic and antitumor
activity, Cancer Res. 68 (2008) 2366-2374.
[39] J. Lin, D. Sampath, M.A. Nannini, B.B. Lee, M. Degtyarev, J. Oeh, H. Savage, Z. Guan, R.
Hong, R. Kassees, L.B. Lee, T. Risom, S. Gross, B.M. Liederer, H. Koeppen, N.J. Skelton, J.J.
Wallin, M. Belvin, E. Punnoose, L.S. Friedman, K. Lin, Targeting activated Akt with GDC-0068, a
novel selective Akt inhibitor that is efficacious in multiple tumor models, Clin. Cancer Res. 19 (2013)
1760-1772.

ACCEPTED MANUSCRIPT
Figures Caption:
Figure 1 Structures of representative Akt inhibitors in clinical trials.
Figure 2 A) The proposed rough binding model for GSK2141795 (or GSK2110183) with Akt1; B)
Overlay of the energy-minimized conformations of GSK2141795 (pink) and 25e (yellow).
Figure 3 The design concept based on the strutures of GSK2141795 and AT-7867.
Figure 4 Effect of Compound 25e on levels of phosphorylated and total Akt and GSK3β in PC-3
cells.
Figure 5 Effect of compound 25e (A), AZD5363 (B) and GSK2141795 (C) on inhibiting the
phosphorylation of PRAS40 in LNCaP cell line, as a direct marker of Akt cell activity.
Figure 6 Proposed molecular binding modes of compound 25e (A) and 28a (B) at the ATP binding
site of Akt1 (PDB ID: 4GV1). The coloring is described as follow: Gray, Akt1 kinase domain; Yellow,
compound 25e; Orange, compound 28a; Red, oxygen atom; Blue, nitrogen atom; Light green,
chlorine atom. Green dashed lines indicate hydrogen bonds, and all graphical pictures were made
using Pymol.
Scheme 1 Reagents and conditions: (a) 1-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)
-1H-pyrazole, Pd(PPh3)4, K2CO3, 1,4-dioxane-H2O, 80 °C, 6 h, 81%; (b) NBS or NCS, r.t., 3 h,
90-92% ; (c) KMnO4, acetone, r.t., 3-4 h, 83-89%.
Scheme 2 Reagents and conditions: (a) 1-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)
-1H-pyrazole, Pd(PPh3)4, K3PO4·3H2O, DMF, 80-120 °C, 5–10 h, 68-95%; (b) (i) NBS or NCS,
THF-DMF, r.t.–80 °C, 0.5–6 h, 70–97% ; (ii) NaOH, THF–H2O, r.t., 3–10 h, 89–100% .
Scheme 3 The synthetic route for the target compounds 24a-d, 25a-e, 26a-c, 27a-c and 28a,b.
Reagents
and
conditions:
(a)
N,N-Bis(2-chloroethyl)benzylamine
hydrochloride,
tetrabutylammonium hydrogen sulfate, 50% NaOH (aq.), toluene, 85°C, 4 h, 65–70%; (b) KOH,
1,2-ethanediol, 180°C, 20 h, 71–75%; (c) diphenyl phosphoryl azide, Et3N, anhydrous toluene, 90 °C,
3 h, then 6 N HCl, reflux, 3 h, 68–73%; (d) (i) 4a-c, 13a-f, 16a-c or 19a,b, EDCI, HOBt, TEA, DCM,
0 °C-r.t., overnight, 73-87% ; (ii) 1-chloroethyl chloroformate, 1,2-dichloroethane, r.t., 3 h, then
MeOH, reflux, 1h, 87-99%.

ACCEPTED MANUSCRIPT
Table 1
The AKT1 inhibitory and in vitro anti-proliferative activities of compounds 24a-d,
25a-e, 26a-c, 27a-c and 28a,b.
Akt1inhibitory activity
Anti-proliferative activity (IC50, µM)
a
Compd.
IC50 , µM
(inhibition, %)b
0.92
OVCAR-8
HCT116
14.30
8.19
7.78
1.61
6.46
20.14
3.77
4.81
9.76
6.87
5.95
5.81
2.83
4.70
1.77
5.21
1.96
7.27
/
24.82
18.26
17.52
3.92
10.08
18.11
4.00
24a
24b
24c
24d
25a
25b
25c
25d
25e
26a
26b
26c
27a
27b
27c
28a
28b
0.32
0.36
0.071
0.92
2.02
0.44
0.39
6.47
7.76
0.061
(49.4%) b
1.48
24.77
6.97
16.40
12.87
7.75
(43.5%) b
1.26
(30.3%) b
0.85
9.28
1.91
1.40
0.034
0.018
11.88
11.03
5.20
AZD5363
GSK2141795
/
a All IC50 data are the mean of three experiments.
b The inhibition rate of Akt1 activities were determined at a concentration of 1 µg/mL.

ACCEPTED MANUSCRIPT
Table 2
Kinase selectivity profile of 25e in comparison with that of Staurosporine
% inhibition
Staurosporine
Kinase family
Kinases
( IC50, nM)
10 µM 0.5 µM
98.2 a
90.6 a
17.6
16
10
Akt1
Akt2
Akt3
/
/
98.6
99.1
8.2
PKA
/
/
/
88.5
83.5
94.4
3.7
64.7
88.8
85.6
-3.9
-3.0
-4.7
1.2
1.0
0.68
5.2
14
RSK1
P70S6K
ROCK1
PDK1
AGC
24.9
69.2
98.3
87.9
38.5
9.6
/
SGK
1.4
5.0
MSK1
PRKG1
TSSK1
ABL
JAK2
BRAF
ALK
CHK1
PI3Kα
mTOR
JNK2
0.33
339
0.31
5.03
7.1
TK
7.2 a
TKL
65.9
50.7
17.8
13.8
55.8
20.8
1.3
2.2
/
1.2
CAMK
Atypical
8.7b
8.8b
2270
5.8
12.7
5.1 a
6.6 a
CDK2/cyclin A
GSK3β
Aurora A
CMGC
Other
51.6
7.63
0.43 a
a The inhibition rate was determined at a concentration of 1 µg/mL.
b IC50 value was determined used PI103 as control.

ACCEPTED MANUSCRIPT