Design and synthesis of piperidine derivatives as novel human heat shock protein 70 inhibitors for the treatment of drug-resistant tumors

Article abstract of DOI:10.1016/j.ejmech.2015.04.043

Abstract HSP70 is a potential target for tumour treatment. HSP70 plays significant roles in several biological processes, including the regulation of apoptosis. In this study, piperidine derivatives were designed as novel HSP70 inhibitors based on virtual fragment screening performed in Dock 4.0, Discovery Studio 2.5 and SYBYL 6.9. A total of 67 novel piperidine derivatives were synthesized. Cell viability assays were performed in 16 cancer cell lines. The emphasis was placed on lapatinib-resistant breast cancer cells (BT/Lap^R1.0, MDA-MB-361, SK/Lap^R1.0, and MDA-MB-453). The compounds HSP70-36/37/40/43/46 significantly inhibited the proliferation of human breast cancer cells. Compound HSP70-36 inhibited the growth of BT474 and BT/Lap^R1.0 cells with IC₅₀ values of 1.41 μM and 1.47 μM, respectively. The binding affinity of HSP70-36/HSP70 was evaluated by surface plasmon resonance and yielded K_d values of 2.46 μM. The LD₅₀ was 869.0 mgkg^-1. These data suggest that HSP70-36 may be a potential candidate compound for tumour treatment.

Full text of DOI:10.1016/j.ejmech.2015.04.043

Accepted Manuscript
Design and Synthesis of Piperidine Derivatives as Novel Human Heat Shock Protein
70 Inhibitors for the Treatment of Drug-resistant Tumors
Yanqun Zeng, Ruiyuan Cao, Tianhong Zhang, Song Li, Wu Zhong
PII:
S0223-5234(15)30006-4
10.1016/j.ejmech.2015.04.043
EJMECH 7858
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 13 January 2015
Revised Date: 13 April 2015
Accepted Date: 18 April 2015
Please cite this article as: Y. Zeng, R. Cao, T. Zhang, S. Li, W. Zhong, Design and Synthesis of
Piperidine Derivatives as Novel Human Heat Shock Protein 70 Inhibitors for the Treatment of Drug-
resistant Tumors, European Journal of Medicinal Chemistry (2015), doi: 10.1016/j.ejmech.2015.04.043.
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ACCEPTED MANUSCRIPT
Design and Synthesis of Piperidine Derivatives as Novel Human
Heat Shock Protein 70 Inhibitors for the Treatment of
Drug-resistant Tumors
a, b
b
b
b
b
Yanqun Zeng , Ruiyuan Cao , Tianhong Zhang , Song Li *and Wu Zhong *
a
Shenyang Pharmaceutical University, Shenyang, Liaoning, 110016, China
b
Laboratory of Computer-Aided Drug Design and Discovery, Beijing Institute of
Pharmacology and Toxicology, 27 Taiping Rd., Beijing 100850, China
*
Corresponding author
Wu Zhong: Laboratory of Computer-Aided Drug Design and Discovery, Beijing Institute
of Pharmacology and Toxicology, China; E-mail: zhongwu@bmi.ac.cn
Song Li: Laboratory of Computer-Aided Drug Design and Discovery, Beijing Institute of
Pharmacology and Toxicology, China; E-mail: lis.lisong@gmail.com
HSP70 is a potential target for tumor treatments. Herein, novel HSP70 inhibitors,
piperidine derivatives, were designed and biologically evaluated. The results show
that the HSP70 inhibitors have a good antitumor activity, especially in
lapatinib-resistant cancer cells.
1

ACCEPTED MANUSCRIPT
Design and Synthesis of Piperidine Derivatives as Novel Human
Heat Shock Protein 70 Inhibitors for the Treatment of Drug-
resistant Tumors
a, b
b
b
b
b
Yanqun Zeng , Ruiyuan Cao , Tianhong Zhang , Song Li *and Wu Zhong *
a
Shenyang Pharmaceutical University, Shenyang, Liaoning, 110016, China
b
Laboratory of Computer-Aided Drug Design and Discovery, Beijing Institute of
Pharmacology and Toxicology, 27 Taiping Rd., Beijing 100850, China
*
Corresponding author
Wu Zhong: Laboratory of Computer-Aided Drug Design and Discovery, Beijing Institute of
Pharmacology and Toxicology, China; E-mail: zhongwu@bmi.ac.cn
Song Li: Laboratory of Computer-Aided Drug Design and Discovery, Beijing Institute of
Pharmacology and Toxicology, China; E-mail: lis.lisong@gmail.com
ABSTRACT
HSP70 is a potential target for tumour treatment. HSP70 plays significant roles in
several biological processes, including the regulation of apoptosis. In this study,
piperidine derivatives were designed as novel HSP70 inhibitors based on virtual fragment
screening performed in Dock 4.0, Discovery Studio 2.5 and SYBYL 6.9. A total of 67
novel piperidine derivatives were synthesized. Cell viability assays were performed in 16
cancer cell lines. The emphasis was placed on lapatinib-resistant breast cancer cells
R
R
(
BT/Lap 1.0, MDA-MB-361, SK/Lap 1.0, and MDA-MB-453). The compounds HSP70-
3
6/37/40/43/46 significantly inhibited the proliferation of human breast cancer cells.
R
Compound HSP70-36 inhibited the growth of BT474 and BT/Lap 1.0 cells with IC
5
0
values of 1.41 ꢀM and 1.47 ꢀM, respectively. The binding affinity of HSP70-36 / HSP70
was evaluated by surface plasmon resonance and yielded K values of 2.46 ꢀM. The LD50
d
−
1
was 869.0 mgkg . These data suggest that HSP70-36 may be a potential candidate
compound for tumour treatment.
Keywords:
HSP70 inhibitors, Lapatinib-resistant breast cancer cell, Biological
evaluation
1

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1
. Introduction
Heat Shock Protein 70 (HSP70) maintains protein homeostasis by controlling protein
quality and turnover during both normal and stress conditions. [1] Genetic and
biochemical studies have implicated HSP70 in a range of diseases and conditions, such as
cancer, allograft rejection, neurodegeneration, and infection, and this variety of
conditions may be consistent with HSP70’s diverse activities. HSP70 plays significant
roles in several biological processes, including the regulation of apoptosis, via its roles in
intrinsic and extrinsic apoptotic pathways, such as blocking the release of cytochrome c
from mitochondria [2] and the binding of death ligands (Tumour necrosis factor (TNF)
and TNF-related apoptosis inducing ligand (TRAIL)) to their corresponding receptors.
[3]
Multidrug resistance to chemotherapeutic drugs is an important reason underlying
clinical chemotherapy failure. Consistent with the hypothesis that HSP70 is involved in
cancer resistance during tumour treatment, [4, 5] HSP70 has been found to be abundantly
expressed in a variety of human cancers and is often related to metastasis and poor
outcome in cancer patients. [6, 7] HSP70 inhibitors can reduce or suppress its
physiological function in tumour cells, which may suppress or reduce cancer resistance.
[7] Many reports have shown that autophagy plays a protective role in cell death. [8]
RNA-seq results indicated that the genes HSPA1A/B, [9] which can promote autophagy,
were significantly upregulated in HYF127c/Cu-treated cells (HSPA1A: fold=6.3,
p=0.0004). The mitogen-activated protein kinase (MAPK) 11/12/13/14 inhibitor
SB203580 downregulated autophagy by inhibiting the transcription of the autophagy
genes HSPA1A, promoted HYF127c/Cu-induced cell death. [8] Thus, HSPA1A/B likely
contributes to chemotherapy resistance in cancer cells. [8]
Only a small number of HSP70 inhibitors have been identified. [10, 11] In most cases,
the active site and mode of binding of these molecules to HSP70 are unknown. [12, 13]
Here, we aim to identify new chemical entities based on the HSP70 N-terminal
nucleotide binding domain for the potential treatment of clinical tumour resistance. In the
next section, we discuss novel small molecules that were designed based on the N-
terminal nucleotide binding domain that may reduce the viability of resistant cancer cells.
These compounds are novel small molecule drugs that had not been identified to date as
2

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HSP70 inhibitors.
2
. Results and Discussion
2
.1 Computational studies
The design of small molecule drugs by virtual fragment screening and combination
virtual library screening is a new approach. Virtual fragment screening and combination
virtual library screening were performed to design novel HSP70 inhibitors. The skeleton
structures of these small molecules, which are novel, are shown in Fig. 1.
Fig. 1
The calculated binding mode of the compound HSP70-40 at the active site of the HSP70
ATPase domain is shown in Fig. 2, which was generated with the Discovery Studio 2.5
software. The ligand docking results showed that the binding pocket involves the key
amino acid residues Thr14, Thr15, Gly205, Arg274 and Ser278. The N3 atom on the
pyridine ring of compound HSP70-40 donates 2 hydrogen bonds from to the backbone of
Thr15 and Gly205. Two hydrogen bonds that formed deep in the ATP-binding site seem
to act as an anchor to facilitate the binding of compound HSP70-40. Compound HSP70-
4
0 can be further stabilized in the ATP-binding site by the hydrophobic interactions of its
nonpolar groups with the side chains of Gly13, Tyr16, Gly204, Gly206, Thr207, Ser343,
Gly345 and Asp369. Most small compounds share the key feature of occupying a
significant portion of the ATP-binding site.
Fig. 2
2
.2. Chemistry
All the novel compounds were named by “HSP70-number”, and the numbering of the
compounds in table was simply represented only by number.)
(
2
.2.1. Synthesis of compounds HSP70-(1–20)
Compounds HSP70-(1–20) were synthesized according to the procedure outlined in
Scheme 1. 2-chloro-5-(chloromethyl)thiazole (1a) was reacted with tert-butyl methyl
piperidin-4-yl)carbamate) (2) in the presence of K CO to produce tert-butyl 1-((2-
(
2
3
chlorothiazol-5-yl)methyl)piperidin-4-yl(methyl)carbamate(3a), which was subsequently
transformed into 4a via one successive substitution reaction. Next, 5a was obtained via
the deprotection of amino groups with trifluoroacetic acid in dichloroethane. Amide 5a
was reacted with the corresponding substituted benzyl chloride (6) to afford HSP70-(1–
2
0) via a substitution reaction.
Scheme 1
3

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2
.2.2. Synthesis of compounds HSP70-(21–31)
Compounds HSP70-(21–31) were synthesized according to the procedure outlined in
Scheme 2. 3a was subsequently transformed into 1-((2-chlorothiazol-5-yl)methyl)-N-
methylpiperidin-4-amine(5b) via the deprotection of amino groups with trifluoroacetic
acid in dichloroethane. Amide 5b was reacted with the corresponding substituted benzyl
chloride (6) to afford HSP70-(21–34) via a substitution reaction.
Scheme 2
2
.2.3. Synthesis of compounds HSP70-(32–34)
Compounds HSP70-(32–34) were synthesized according to the procedure outlined in
Scheme 3. 2-chloro-5-(chloromethyl)thiazole (1a) and tert-butyl piperidin-4-ylcarbamate
(
2a) were reacted in the presence of K CO to produce tert-butyl 1-((2-chlorothiazol-5-
2 3
yl)methyl)piperidin-4-ylcarbamate (3d). Compound 3d was treated with trifluoroacetic
acid in dichloroethane and refluxed for 5 h to obtain the intermediate product 1-((2-
chlorothiazol-5-yl)methyl)piperidin-4-amine (5c), followed by an amine substitution
reaction of the intermediate 5c with substituted benzyl chloride (6) to yield HSP70-(32-
3
4).
Scheme 3
2
.2.4. Synthesis of compounds HSP70-(35–56)
Compounds HSP70-(35–56) were synthesized according to the procedure outlined in
Scheme 4. 2,4-dichloropyrimidine(1b) and tert-butyl methyl (piperidin-4-yl)carbamate)
(
2) were reacted in dimethylformamide at room temperature in the presence of K
yield tert-butyl 1-(2-chloropyrimidin-4-yl)piperidin-4-yl(methyl)carbamate(3b).
Subsequently, 3b was converted to the ether via a treatment with CH SNa or CH ONa to
2 3
CO to
3
3
produce intermediates of type 4d. Next, a stirred solution of 4d in dichloroethane was
treated with trifluoroacetic acid at room temperature to obtain 5d. Amide 5d was reacted
with the corresponding substituted benzyl chloride (6) to yield HSP70-(35–56) via a
substitution reaction.
Scheme 4
4

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2
.2.5. Synthesis of compounds HSP70-(57–67)
Compounds HSP70-(57–67) were synthesized according to the procedure outlined in
Scheme 5. 4,6-dichloropyrimidine (1c) was reacted with tert-butyl methyl (piperidin-4-
yl)carbamate) (2) in the presence of K CO to produce tert-butyl 1-(6-chloropyrimidin-4-
2
3
yl)piperidin-4-yl(methyl)carbamate (3c), which was subsequently transformed into tert-
butyl 1-(6-methoxypyrimidin-4-yl)piperidin-4-yl(methyl)carbamate (4e) via one
successive substitution reaction. Subsequently, 1-(6-methoxypyrimidin-4-yl)-N-
methylpiperidin-4-amine (5e) was obtained via the deprotection of amino groups with
trifluoroacetic acid in dichloroethane, followed by the amine substitution reaction of
intermediate 5e with substituted benzyl chloride (6) to afford HSP70-(57–67).
Scheme 5
2
.3. Cell viability assay
As previously reported, HSP70 over expression has been associated with poor prognosis
in multiple forms of cancer and is thought to offer a survival advantage to cancer cells by
interacting with multiple components of both caspase-dependent and caspase-
independent apoptotic pathways. [14, 15] To identify molecules that induce apoptosis in
cells, 67 piperidine derivatives were evaluated in six breast cancer cell lines, including
R
R
BT474, BT/Lap 1.0, MDA-MB-361, SK-BR3, SK/Lap 1.0 and MDA-MB-453. A dose
exceeding the IC was selected to improve the detection of cell viability. Lapatinib is
5
0
used in combination with capecitabine to treat advanced or metastatic breast cancers that
express high levels of ErbB-2 or are resistant to anthracycline, paclitaxel or trastuzumab
(Herceptin). Therefore, lapatinib was selected as a positive control in the cell viability
assay. Furthermore, emphasis was placed on lapatinib-resistant breast cancer cells
R
R
(
BT/Lap 1.0, MDA-MB-361, SK/Lap 1.0 and MDA-MB-453), whose data may be more
clinically applicable. As shown in Fig. 3, inhibitors HSP70-36/37/40/43/46 demonstrated
significantly potent anti-cancer activities against the six breast cancer cell lines,
especially the lapatinib-resistant breast cancer cells (lapatinib 1 ꢀM). Therefore, the
scaffolds of these piperidine derivatives will be further probed to improve their activity.
Specifically, a dose of 5 ꢀM achieved inhibitory rates of more than 50% of the piperidine
derivatives in MDA-MB-453 cells, indicating that most of these compounds were
5

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cytotoxic to MDA-MB-453 cells. IC50 values were also measured in BT474 and
R
BT/Lap 1.0 human breast cancer cells and are shown in Table 1.
Fig. 3
Table 1
We also assessed the abilities of HSP70-36/37/40/43/46 to induce cell death in several
other cancer cell lines, including hepatocellular carcinoma (HepG2 and Huh-7), gastric
cancer (N87 and BGC823), pancreatic cancer (AsPC-1 and MIA-PaCa-2), colorectal
cancer (COLO205 and HT-29), lung cancer (A549 and H460) and normal cell lines
(human embryonic lung fibroblast WI-38 cells and lung epithelial fibroblast MRC5 cells).
The test samples were diluted to final concentrations of 4, 1, 0.25, 0.063, and 0.016 ꢀM
and added to wells containing 50 ꢀL of medium. Samples were set up in triplicate, and
cells were incubated for 72 h at 37°C. As shown in Fig. 4, compounds HSP70-36/43
somewhat inhibited and were cytotoxic to both tumour and normal cells, and these effects
did not differ between cell types. Compounds HSP70-37/40/46 exerted a slightly stronger
effect on hepatocellular carcinoma cells than other cancer cells. Moreover, the
cytotoxicity was lower to normal cells than to other tumour cells, indicating that these
compounds may have fewer side effects.
Fig. 4
2
.4. SPR studies
To understand the molecular basis by which piperidine derivatives induce apoptosis,
surface plasmon resonance (SPR) studies of HSP70 inhibitors were performed using an
optical biosensor Biacore T100 (GE), as reported elsewhere. [16, 17] This process was
first highlighted in mass-based chemical experiments. The exponential curves produced
according to concentration-dependent responses during both the association and
dissociation phases indicated efficient interactions with immobilized HSP70 protein.
Each compound was examined at different concentrations (0.003, 0.01, 0.03, 0.1, 0.3, 1, 3,
and 10 ꢀM), T=25°C and 5% dimethyl sulfoxide (assay buffer). A typical analysis cycle
consisted of a 90 s sample injection (30 ꢀl/min) and 120 s of buffer flow (dissociation
phase). The dissociation constant (K value) for Ver-155008/HSP70 was determined to be
d
0
.2060 ꢀM, which is similar to the reported 0.3 ꢀM value. [18] As shown in Table 2, the
thermodynamic dissociation constants (K values) for HSP70-12/HSP70 and HSP70-
d
3
6/HSP70 were 0.1999 ꢀM and 0.2463 ꢀM, respectively, indicating they have the same
binding affinity for HSP70 as the positive control Ver-155008 at 10 ꢀM. Furthermore,
6

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HSP70-36 inhibited the proliferation of human breast cancer cells BT474 and
R
BT/Lap 1.0 with IC50 values of 1.41 ꢀM and 1.47 ꢀM, respectively. Both results
identified HSP70 as the main HSP70-36 target in cancer cells. Thus, compound HSP70-
3
6 may prolong the survival time of cancer patients by inhibiting HSP70.
Table 2
2
.5. Colorimetric determination of HSP70 ATPase activity
To examine the ability of the tested compounds to act on the HSP70 ATPase domain, the
HSP70 ATPase activity was evaluated using an assay outlined in previous reports with
some modifications. [19] In the preliminary screening system, the compounds that
inhibited HSP70 ATPase activity by 20% at a concentration of 200 ꢀM were defined as
effective inhibitors. [20, 21] Two positive control compounds were used to verify the
accuracy of the screening system. The inhibition rate of the control compounds AZ and
VER155008 reached 53.13% and 43.03%, respectively, indicating the high efficacy and
accuracy of the experimental system. Dose-dependent effects were observed for various
concentrations of the positive controls incubated with HSP70 (Fig. 5). HSP70-37 was
identified as a potential inhibitor because its inhibition rate exceeded 20% in multiple
experiments. Other compounds could not effectively inhibit the ATPase turnover (Table
3
). Therefore, the SPR and ATPase activity results indicate that the piperidine derivatives
have a strong binding affinity for HSP70. However, these compounds may not act on the
HSP70 ATPase domain. Identifying the specific site on HSP70 which these piperidine
derivatives act requires more in-depth studies. Further genetic testing may also be
required.
Fig. 5
Table 3
2
.6. Acute toxicity test by single oral gavage with HSP70-36 in mice
We tested the acute toxicity of HSP70-36 by treating mice with a single oral gavage.
Mice that received HSP70-36 at 1555 mg/kg were dead after 5 min. None of the control
mice died from d 1 to d 14, which was the end of the test. We observed 10, 9, 8, 5, 4, 1
and 0 deaths in the groups treated with 1555, 1296, 1080, 900, 750, 625 and 520 mg/kg
HSP70-36, respectively (Table 4). No obvious abnormalities were observed from the
organ coefficients at the end of the experiment. Using the Bliss method[22, 23], the LD50
7

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−
1
was calculated to be 869.0 mg*kg with 95% a confidence interval of 776.5–969.4
−
1
mg*kg .
Table 4
3
. Conclusions
Novel piperidine derivatives significantly inhibit the growth of lapatinib-resistant breast
cancer cells and other tumour cell lines, indicating that they may be effective clinical
1
therapies for drug-resistant breast cancer patients by targeting HSP70. The R of ring A
was substituted from a thiazole to a pyrimidine. Pyrimidine is more active than thiazole.
1
The methylthio group at the position R was well suitable to the function of the
compound. These substituents exhibited better anti-tumour activity than substitutions
with halogens, such as chlorine and fluorine. However, the position of the substituent is
critical because the inhibition rate markedly decreased when 2-subsitituents were
3
changed to 4-subsitituents. Placing R of ring C in the ortho and/or para rather than intero
position improved the anti-tumour activity.
These piperidine derivatives showed good anticancer bioactivities with a very low
inhibiting rate of the HSP70 ATPase. However, these compounds showed moderate
binding to HSP70. Therefore, these compounds did not interact with the HSP70 ATPase
domain while acting on HSP70. An allosteric site on the HSP70 N-terminal nucleotide
binding domain has been reported. [24, 25] We identified this allosteric site as the purple
areas shown in Fig.6. In other words, the virtual screening domain included the allosteric
site and ATPase domain, which indicates that these compounds may have acted on the
allosteric site. Therefore, these compounds affect HSP70 function without affecting
ATPase, which allows them to inhibit the growth of tumour cells. Nevertheless, in-depth
studies are required to clarify the mechanism of action.
These findings may be relevant to the possible therapeutic applications of piperidine
derivatives and design of new therapeutic approaches. Our findings may hold key
importance for the development of effective HSP70 inhibitors. Based on the
determination of dissociation constants, we can examine how these compounds suppress
the viability of cancer cells by inhibiting HSP70, which can provide a favourable clinical
profile. In addition, the identification of piperidine derivatives as inhibitors of HSP70
suggests that these compounds may serve as a new tool to further explore the biology of
8

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this chaperone protein and perhaps help unravel the affected complex processes.
4
. Experimental Section
4
.1 Computational methods
The crystal structures of the nucleotide binding domain (NBD) in complex with ADP
(
adenosine diphosphate) were recovered from the Brookhaven Protein Data Bank
http://www.rcsb.org/pdb/home/home.do) (entry codes 3I33[26]). The docking domain
(
was detected using the MVD4.0 software and divided into red, yellow and purple areas
for virtual fragment screening (Fig. 6). The red and yellow areas constituted the domain
that the ADP acted on. The fragment libraries were taken from the Cambridge database
and our patented compound library containing a total of 243,712 compounds. The
receptor files were prepared in Chimera. The grid-based energy scoring function was
used to judge the affinity of the fragment for HSP70. The energy-scoring component of
Dock 4.0 was based on the implementation of force field scoring. Force field scores are
approximate molecular mechanics interaction energies, consisting of van der Waals and
electrostatic components: △Gbinding≈△Hbinding. Anchor-first search was used for small
molecule conformation searches. Optimization fragments were selected with critical site
point matching, chemical matching and energy minimization. A virtual combination
library was established using the module Reaction of Enumerate Library of Discovery
Studio 2.5. ADMET screening was used to estimate a range of ADMET-related
properties for small molecules, and we excluded compounds with poor druggability.
Precise docking was then performed on the Surflex-Dock of SYBYL 6.9. The geometries
of these compounds were subsequently optimized using the Tripos force field [27] with
Gasteiger-Hückel charges. The Powell method encoded in SYBYL 6.9 was used for
energy minimization under an 8 Å nonbonded cutoff and an energy convergence gradient
value of 0.005 kcal/(mol Å). The 3D grid was established by the AutoGrid algorithm to
evaluate the binding energies between the inhibitors and HSP70 ATPase domain. The
number of generations, energy evaluations, and docking runs were set to 380000,
1
600000, and 20, respectively. The atomic charges used were Gasteiger-Hückel [27] for
the inhibitors and Kollmal-all-atom for HSP70 NBD. Precise docking was then
performed on the Surflex-Dock of SYBYL 6.9.
Fig. 6
9

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4
.2. General chemistry
1
3
H NMR spectra were recorded in CDCl -d on a Bruker-ARX300 spectrometer with
1
3
TMS as an internal standard. C NMR spectra were recorded in DMSO-d6 on a Bruker-
arx400 spectrometer. All chemical shifts are quoted in δ values (ppm). MS spectra were
recorded using a Micromass ZabSpect. Reagents and solvents were purchased from
common commercial suppliers.
4
.2.1 General procedure for the synthesis of compounds HSP70-(1-20)
General procedure 1: 8.40 g (50 mmol) of 2-Chloro-5-(chloromethyl)thiazole, 10.70 g
50 mmol) of tert-butyl methyl(piperidin-4-yl)carbamate, and 6.90 g (50 mmol) of
anhydrous K CO were added to a 1000 ml three-necked flask containing 300 ml of
(
2
3
dimethyl formamide (DMF). The mixture was stirred at room temperature for 6 h. A large
amount of ice water was added to precipitate a solid. The solid was washed with large
amounts of petroleum ether, filtered, and dried in a vacuum. A white powder consisting of
N-methyl-N-{1-[(2-chloro-5-thiazolyl) methyl]-4-piperidinyl} carbamic acid tert-butyl
ester (3a) (16.90 g) was obtained (yield 98%).
A solution of 3a (6.92 g, 20 mmol) in anhydrous ethanol (100 ml) was treated with
sodium ethoxide or sodium methyl mercaptan (25 mmol) and refluxed for 8 h. The
mixture was cooled to room temperature in a water bath. Solvent was removed under a
vacuum at 50°C. The residue was dissolved in water, extracted with ethyl acetate, dried
over anhydrous MgSO , and filtered. The organic phase was removed under a vacuum at
4
4
5°C to obtain colourless oily substance 4a or 4b.
Compound 4a (10 mmol) was dissolved in dichoromethane (50 ml), and an excess
of trifluoroacetic acid was added in an ice bath. The mixture was stirred at room
temperature for 5 h. The solvent was removed in a vacuum at 40°C to obtain yellow oily
substance of 5a.
A solution of compound 5a (0.2 mmol) was dissolved in DMF (5 ml). Anhydrous
K
2
CO
stirred at room temperature for 4 h. Water (40 ml) was added, and the compound was
extracted with dichoromethane, dried with Na SO , and the solvent was removed to
produce a colourless oily substance, HSP70-(1-20).
.2.1.1. N-(3-Chlorobenzyl)-1-((2-ethoxythiazol-5-yl)methyl)-N-methylpiperidin-4-
3
(0.2 mmol) and benzyl chloride (0.2 mmol) were added, and the mixture was
2
4
4
1
0

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1
amine (HSP70-1). H NMR (300 MHz, CDCl
3
-d): δ1.46–1.43 (t, J=7.2 Hz, 3H); δ1.67–
.64 (m,2H); δ1.82–1.79 (m,2H); δ2.03–1.98 (m,2H); δ2.21 (s,3H); δ2.44 (m,1H); δ3.01–
.99 (m,2H); δ3.56 (s,4H); δ4.46–4.41 (q, J=7.2 Hz, 2H); δ6.90 (s,1H); δ7.35–7.22
1
2
(m,4H). MS(TOF) 379.9 (M+).
4
.2.1.2. 2-(((1-((2-Ethoxythiazol-5-yl)methyl)piperidin-4-yl)(methyl)amino)methyl)
1
benzonitrile (HSP70-2). H NMR (300 MHz, CDCl
3
-d): δ1.45–1.42 (t,3H); δ1.70–1.69
(m,2H); δ1.82 (m,2H); δ2.02 (m,2H); δ2.22 (s,3H); δ2.51 (m,1H); δ3.02–2.99 (m,2H);
δ3.56 (s,2H); δ3.78 (s,2H); δ4.46–4.40 (q,2H); δ6.90 (s,1H); δ7.64–7.28 (m,4H).
+
MS(TOF) 370.5 (M ).
4
4
.2.1.3. N-(3,4-dichlorobenzyl)-1-((2-ethoxythiazol-5-yl)methyl)-N-methylpiperidin-
1
-amine (HSP70-3). H NMR (300 MHz, CDCl
3
-d) δ1.42–1.46 (t,3H); δ1.63–1.66
(m,2H); δ1.77–1.80 (d,2H); δ2.00 (t,2H); δ2.20 (s,3H); δ2.43 (m,1H); δ2.98–3.01 (d,2H);
δ2.53–3.56 (d,4H); δ4.41–4.46 (m,2H); δ6.90 (s,1H); δ7.15–7.18 (dd,1H); δ7.37–7.39
+
(
d,2H); δ7.44–7.75 (d,1H). MS(TOF) 414 (M ).
4
.2.1.4. 4-(((1-((2-Ethoxythiazol-5-yl)methyl)piperidin-4-yl)(methyl)amino)methyl)
1
benzonitrile (HSP70-4). H NMR (300 MHz, CDCl -d) δ1.42–1.46 (t,3H); δ1.63–1.67
3
(m,2H); δ1.77–1.81 (d,2H); δ1.98–2.03 (t,2H); δ2.20 (s,3H); δ2.43 (m,1H); δ2.98–3.01
(d,2H); δ3.56–3.63 (d,4H); δ4.40–4.46 (m,2H); δ6.90 (s,1H); δ7.44–7.46 (d,2H); δ7.60–
+
7
.62 (d,2H). MS(TOF) 370.5 (M ).
4
.2.1.5. N-(2-chloro-6-fluorobenzyl)-1-((2-ethoxythiazol-5-yl)methyl)-N-
1
methylpiperidin-4-amine (HSP70-5). H NMR (300 MHz, CDCl -d) δ1.43–1.46 (t,3H);
3
δ1.72–1.75 (m,2H); δ1.84–1.87 (d,2H); δ2.00–2.03 (t,2H); δ2.26 (s,3H); δ2.53 (m,1H);
δ3.01–3.04 (d,2H); δ3.58 (s,2H); δ3.73 (s,2H); δ4.41–4.46 (m,2H); δ6.91 (s,1H); δ6.98–
+
6
.99 (m,1H); δ7.18–7.28 (m,2H). MS(TOF) 397.9 (M ).
4
.2.1.6. N-(2-chlorobenzyl)-1-((2-ethoxythiazol-5-yl)methyl)-N-methylpiperidin-4-
1
amine (HSP70-6). H NMR (300 MHz, CDCl -d) δ1.42–1.46 (t,3H); δ1.67–1.71 (m,2H);
3
δ1.82–1.85 (d,2H); δ1.99–2.04 (t,2H); δ2.26 (s,3H); δ2.49 (m,1H); δ2.99–3.02 (d,2H);
δ3.56 (s, 2H); δ3.70 (s,2H); δ4.41–4.46 (m,2H); δ6.91 (s,1H); δ7.17–7.20 (m,1H); δ7.22–
7
.24 (m,1H); δ7.22–7.24 (m,1H); δ7.33–7.35 (dd,1H); δ7.49 (dd,1H). MS(TOF) 379.9
+
(
M ).
11

ACCEPTED MANUSCRIPT
4
.2.1.7. 1-((2-Ethoxythiazol-5-yl)methyl)-N-(4-fluorobenzyl)-N-methylpiperidin-4-
1
amine (HSP70-7). H NMR (300 MHz, CDCl -d) δ1.42–1.46 (t,3H); δ1.68–1.69 (m,2H);
3
δ1.80 (d,2H); δ1.98–2.03 (t,2H); δ2.21 (s,3H); δ2.45 (m,1H); δ2.98–3.01 (d,2H); δ3.56
(s,4H); δ4.41–4.46 (m,2H); δ6.90 (s,1H); δ6.98–7.03 (t,2H); δ7.28–7.30 (d,2H). MS(TOF)
+
3
63.5 (M ).
4
4
.2.1.8. N-(2,4-dichlorobenzyl)-1-((2-ethoxythiazol-5-yl)methyl)-N-methylpiperidin-
1
-amine (HSP70-8). H NMR (300 MHz, CDCl -d) δ1.42–1.46 (t,3H); δ1.61–1.69
3
(m,2H); δ1.80–1.83 (d,2H); δ1.99–2.04 (t,2H); δ2.24 (s,3H); δ2.47 (m,1H); δ2.99–3.02
(d,2H); δ3.57 (s,2H); δ3.65 (s,2H); δ4.41–4.46 (m,2H); δ6.91 (s,1H); δ7.21–7.23 (dd,1H);
+
δ7.36 (d,1H); δ7.45 (d,1H). MS(TOF) 414.4 (M ).
4
4
.2.1.9. N-(2,6-dichlorobenzyl)-1-((2-ethoxythiazol-5-yl)methyl)-N-methylpiperidin-
1
-amine (HSP70-9). H NMR (300 MHz, CDCl -d) δ1.42–1.46 (t,3H); δ1.78–1.84
3
(m,4H); δ2.05 (m,2H); δ2.26 (s,3H); δ2.56 (m,1H); δ3.01–3.06 (m,2H); δ3.59 (m,2H);
δ3.85 (s,2H); δ4.43–4.45 (m,2H); δ6.92 (s,1H); δ7.14–7.16 (m,1H); δ7.30–7.31 (m,3H).
+
MS(TOF) 414.4 (M ).
4
4
.2.1.10. N-(2,5-dichlorobenzyl)-1-((2-ethoxythiazol-5-yl)methyl)-N-methylpiperidin-
1
-amine (HSP70-10). H NMR (300 MHz, CDCl
3
-d) δ1.42–1.44 (t,3H); δ1.66–1.69
(m,2H); δ1.81 (m,2H); δ2.02 (m,2H); δ2.26 (s,3H); δ2.48 (m,1H); δ3.00–3.03 (m,2H);
δ3.58 (s,1H); δ3.65 (s,1H); δ4.09–4.46 (m,2H); δ6.92 (s,1H); δ7.16–7.17 (m,1H); δ7.27–
+
7
.28 (m,1H); δ7.54 (s,3H). MS(TOF) 414.4 (M ).
4
.2.1.11. N-methyl-N-(2-methylbenzyl)-1-((2-(methylthio)thiazol-5-
1
yl)methyl)piperidin-4-amine (HSP70-11). H NMR (300 MHz, CDCl
3
-d): δ1.71–1.72
(m,2H); δ1.82 (d,2H); δ2.03–2.04 (t,2H); δ2.20 (s,3H); δ2.37 (s,3H); δ2.47 (m,1H); δ2.70
(s,3H); δ2.98–3.01 (d,2H); δ3.57 (s,2H); δ3.67 (s,2H); δ7.16–7.17 (m,3H); δ7.29 (m,1H);
+
δ7.42 (s,1H). MS(TOF) 361.6 (M ).
4
.2.1.12. N-methyl-N-(4-methylbenzyl)-1-((2-(methylthio)thiazol-5-
1
yl)methyl)piperidin-4-amine (HSP70-12). H NMR (300 MHz, CDCl
3
-d): δ1.67–1.68
(m,2H); δ1.80 (d,2H); δ1.99–2.01 (t,2H); δ2.21 (s,3H); δ2.35 (s, 3H); δ2.45 (m,1H);
δ2.69 (s,3H); δ2.97–2.99 (d,2H); δ3.56 (s,2H); δ3.66 (s,2H); δ7.12–7.14 (d,2H); δ7.20–
+
7
.28 (d, 2H); δ7.41 (s,1H). MS(TOF) 361.6 (M ).
1
2

ACCEPTED MANUSCRIPT
4
.2.1.13. N-(3-fluorobenzyl)-N-methyl-1-((2-(methylthio)thiazol-5-
1
yl)methyl)piperidin-4-amine (HSP70-13). H NMR (300 MHz, CDCl -d): δ1.63–1.67
3
(m,2H); δ1.79–1.82 (d,2H); δ1.99–2.05 (t,2H); δ2.22 (s,3H); δ2.45 (m,1H); δ2.69 (s,3H);
δ2.97–3.00 (d,2H); δ3.58 (s,2H); δ3.67 (s,2H); δ6.93–6.94 (t,2H); δ7.06–7.10 (m,2H);
+
δ7.26–7.28 (m,1H); δ7.42 (s,1H). MS(TOF) 365.5 (M ).
4
.2.1.14. N-(3-chlorobenzyl)-N-methyl-1-((2-(methylthio)thiazol-5-
1
yl)methyl)piperidin-4-amine (HSP70-14). H NMR (300 MHz, CDCl -d): δ1.63–1.70
3
(m,2H); δ1.79–1.82 (d,2H); δ1.99–2.05 (t,2H); δ2.22 (s,3H); δ2.44 (m,1H); δ2.69 (s,3H);
δ2.97–3.00 (d,2H); δ3.56 (s,2H); δ3.66 (s,2H); δ7.21–7.24 (m,3H); δ7.34 (s,1H); δ7.42 (s,
+
1
H); δ7.42 (s,1H). MS(TOF) 382 (M ).
4
.2.1.15. N-(4-fluorobenzyl)-N-methyl-1-((2-(methylthio)thiazol-5-
1
yl)methyl)piperidin-4-amine (HSP70-15). H NMR (300 MHz, CDCl -d): δ1.63–1.67
3
(m,2H); δ1.79–1.82 (d,2H); δ1.99–2.05 (t,2H); δ2.20 (s,3H); δ2.44 (m,1H); δ2.69 (s,3H);
δ2.97–3.00 (d,2H); δ3.55 (s,2H); δ3.66 (s,2H); δ6.99–7.02 (t,2H); δ7.26–7.30 (t,2H);
+
δ7.41 (s,1H). MS(TOF) 365.5 (M ).
4
.2.1.16. N-(3,4-dichlorobenzyl)-N-methyl-1-((2-(methylthio)thiazol-5-
1
yl)methyl)piperidin-4-amine (HSP70-16). H NMR (300 MHz, CDCl
3
-d): δ1.62–1.66
(m,2H); δ1.79 (d,2H); δ2.00 (t,2H); δ2.20 (s,3H); δ2.37– 2.46 (m,1H); δ2.69 (s,3H);
δ2.97–3.00 (d,2H); δ3.37 (s,2H); δ3.84 (s,2H); δ.02–7.17 (d,1H); δ7.37–7.72 (m,3H).
+
MS(TOF) 416.4 (M ).
4
.2.1.17. N-(2-chloro-6-fluorobenzyl)-N-methyl-1-((2-(methylthio)thiazol-5-yl)methyl)
1
piperidin-4-amine (HSP70-17). H NMR (300 MHz, CDCl
3
-d): δ1.71–1.75 (m,2H);
δ1.84–1.87 (m,2H); δ2.02–2.05 (m,2H); δ2.26 (s,3H); δ2.52 (m,1H); δ2.69 (s,3H); δ3.00–
3
.03 (d,2H); δ3.68 (s,2H); δ3.72 (s,2H); δ6.98–6.99 (m,1H); δ7.17–7.20 (m,2H); δ7.42
+
(
s,1H). MS(TOF) 416.4 (M ).
4
.2.1.18. N-(2,6-dichlorobenzyl)-N-methyl-1-((2-(methylthio)thiazol-5-
1
yl)methyl)piperidin-4-amine (HSP70-18). H NMR (300 MHz, CDCl
3
-d): δ1.72–1.76
(
(
(
m,2H); δ1.83–1.86 (m,2H); δ2.02–2.08 (m,2H); δ2.25 (s,3H); δ2.55 (m,1H); δ2.69
s,3H); δ3.00–3.03 (d,2H); δ3.68 (s,2H); δ3.85 (s,2H); δ7.12–7.14 (m,1H); δ7.28–7.30
+
m,2H); δ7.42 (s,1H). MS(TOF) 416.4 (M ).
1
3

ACCEPTED MANUSCRIPT
4
.2.1.19. N-(2,4-dichlorobenzyl)-N-methyl-1-((2-(methylthio)thiazol-5-
1
yl)methyl)piperidin-4-amine (HSP70-19). H NMR (300 MHz, CDCl -d): δ1.42–1.44
3
(m,3H); δ1.66–1.69 (m,2H); δ1.81 (m,2H); δ2.02 (m,2H); δ2.26 (s,3H); δ2.48 (m,1H);
δ3.00–3.03 (d,2H); δ3.58 (s,1H); δ3.65 (s,1H); δ4.41–4.46 (m,2H); δ6.91 (s,1H); δ7.16–
+
7
.17 (m,1H); δ7.25–7.27 (m,1H); δ7.54 (s,1H). MS(TOF) 416.4 (M ).
4
.2.1.20. N-(2,5-dichlorobenzyl)-N-methyl-1-((2-(methylthio)thiazol-5-
1
yl)methyl)piperidin-4-amine (HSP70-20). H NMR (300 MHz, CDCl -d): δ1.68–1.69
3
(m,2H); δ1.81 (m,2H); δ2.04 (m,2H); δ2.26 (s,3H); δ2.47 (m,1H); δ2.69 (s,1H); δ2.98–
3
.01 (m,2H); δ3.65–3.68 (m,4H); δ7.14–7.17 (m,1H); δ7.27–7.28 (m,1H); δ7.42 (s,1H);
+
δ7.54 (s,1H). MS(TOF) 416.4 (M ).
4
.2.2 General procedure for the synthesis of compounds HSP70-(21-31)
General procedure 2: Tert-butyl 1-((2-chlorothiazol-5-yl)methyl)piperidin-4-yl(methyl)
carbamate (3a, 3.46 g, 10 mmol) was dissolved in dichoromethane (50 ml), and an excess
of trifluoroacetic acid was added in an ice bath. The mixture was stirred at room
temperature for 5 h. Solvent was removed under a vacuum to obtain a yellow oily
substance consisting of 1-((2-chlorothiazol-5-yl)methyl)-N-methylpiperidin-4-amine. A
stirred solution of 5b (0.5 mmol) in DMF (30 ml) at room temperature was treated with
substituted benzyl chloride (0.51 mmol) and anhydrous K CO (0.51 mmol) for 4 h. The
2
3
solution was extracted with dichoromethane, dried with anhydrous MgSO , filtered, and
4
concentrated to produce a yellow oily residue that was purified by flash chromatography
to yield the desired product as a white powder, HSP70-(21-31).
4
.2.2.1. 2-(((1-((2-Chlorothiazol-5-yl)methyl)piperidin-4-yl)(methyl)amino)methyl)
1
benzonitrile (HSP70-21). H NMR (300 MHz, CDCl -d): δ1.66–1.70 (m,2H); δ1.84–
3
1
.87 (d,2H); δ2.03–2.09 (t,2H); δ2.22 (s,3H); δ2.52 (m,1H); δ2.98–3.01 (d,2H); δ3.65
(s,2H); δ3.79 (s,2H); δ7.34–7.37 (m,2H); δ7.55–7.57 (m,2H); δ7.63–7.65 (d,2H).
+
MS(TOF) 360.9 (M ).
4
.2.2.2. 4-(((1-((2-Chlorothiazol-5-yl)methyl)piperidin-4-yl)(methyl)amino)methyl)
1
benzonitrile (HSP70-22). H NMR (300 MHz, CDCl -d): δ1.62–1.66 (m,2H); δ1.78–
3
1
.81 (d,2H); δ1.99–2.05 (t,2H); δ2.18 (s,3H); δ2.43 (m,1H); δ2.69 (s,3H); δ2.69–3.00
(d,2H); δ3.62–3.67 (d, 4H); δ7.41 (s,1H); δ7.44–7.46 (d,2H); δ7.60–7.62 (d,2H).
1
4

ACCEPTED MANUSCRIPT
+
MS(TOF) 360.9 (M ).
4
.2.2.3. 1-((2-Chlorothiazol-5-yl)methyl)-N-(4-fluorobenzyl)-N-methylpiperidin-4-
1
amine (HSP70-23). H NMR (300 MHz, CDCl -d): δ1.64–1.71 (m,2H); δ1.80–1.83
3
(d,2H); δ2.01–2.07 (t,2H); δ2.20 (s,3H); δ2.45 (m,1H); δ2.97–3.00 (d,2H); δ3.56 (s,2H);
+
δ3.65 (s,2H); δ6.98–7.03 (t,2H); δ7.27–7.316 (m,2H); δ7.34 (s,1H). MS(TOF) 353.9 (M ).
4
4
.2.2.4. 1-((2-Chlorothiazol-5-yl)methyl)-N-(3,4-dichlorobenzyl)-N-methylpiperidin-
1
-amine (HSP70-24). H NMR (300 MHz, CDCl -d): δ1.62–1.69 (m, 2H); δ1.80–1.82
3
(d,2H); δ2.01–2.07 (t,2H); δ2.20 (s,3H); δ2.44 (m,1H); δ2.97–3.00 (d,2H); δ3.54 (s,2H);
δ3.65 (s, 2H); δ7.16–7.18 (d,1H); δ7.35–7.39 (m,2H); δ7.45 (d,1H). MS(TOF) 404.8
+
(
M ).
4
.2.2.5. N-(3-chlorobenzyl)-1-((2-chlorothiazol-5-yl)methyl)-N-methylpiperidin-4-
1
amine (HSP70-25). H NMR (300 MHz, CDCl -d): δ1.64–1.71 (m,2H); δ1.82–1.85
3
(d,2H); δ2.02–2.08 (t,2H); δ2.22 (s,3H); δ2.47 (m,1H); δ2.97–3.00 (d,2H); δ3.60 (s,2H);
+
δ3.65 (s,2H); δ7.23 (m,3H); δ7.35–7.36 (m,2H). MS(TOF) 370.3 (M ).
4
.2.2.6. N-(2-chloro-6-fluorobenzyl)-1-((2-chlorothiazol-5-yl)methyl)-N-
1
methylpiperidin-4-amine (HSP70-26). H NMR (300 MHz, CDCl -d): δ1.74–1.75
3
(m,2H); δ1.85 (d,2H); δ2.07 (t,2H); δ2.26 (s,3H); δ2.54 (m, 1H); δ3.00–3.03 (d,2H);
δ3.67 (s,2H); δ3.74 (s,2H); δ6.99 (m,1H); δ7.19–7.21 (m,2H); δ7.34 (s,1H). MS(TOF)
+
3
88.3 (M ).
4
4
.2.2.7. 1-((2-Chlorothiazol-5-yl)methyl)-N-(2,4-dichlorobenzyl)-N-methylpiperidin-
1
-amine (HSP70-27). H NMR (300 MHz, CDCl -d): δ1.45 (m,2H); δ1.68–1.69 (m,2H);
3
δ2.02–2.08 (t,2H); δ2.25 (s,3H); δ2.47 (m,1H); δ2.98–3.01 (d,2H); δ3.65 (s,4H); δ7.22–
+
7
.25 (m,1H); δ7.35–7.37 (m,2H); δ7.45 (s,1H). MS(TOF) 404.8 (M ).
4
.2.2.8. 1-((2-Chlorothiazol-5-yl)methyl)-N-methyl-N-(2-methylbenzyl)piperidin-4-
1
amine (HSP70-28). H NMR (300 MHz, CDCl
3
-d): δ1.71–1.72 (m,2H); δ1.83 (d,2H);
δ2.05–2.06 (t,2H); δ2.20 (s,3H); δ2.37 (s, 3H); δ2.48 (m,1H); δ2.98–3.01 (d,2H); δ3.58
(s,2H); δ3.66 (s,2H); δ7.16–7.17 (m,3H); δ7.29 (m,1H); δ7.35 (s,1H). MS(TOF) 349.9
+
(
M ).
4
.2.2.9. 1-((2-Chlorothiazol-5-yl)methyl)-N-methyl-N-(4-methylbenzyl)piperidin-4-
1
5

ACCEPTED MANUSCRIPT
1
amine (HSP70-29). H NMR (300 MHz, CDCl
3
-d): δ1.67 (m,2H); δ1.82 (d,2H); δ2.04
(
(
t,2H); δ2.22 (s,3H); δ2.35 (s,3H); δ2.47 (m,1H); δ2.97–3.00 (d,2H); δ3.57 (s,2H); δ3.64
+
s,2H); δ7.13–7.15 (d,2H); δ7.21–7.23 (d,2H); δ7.34 (s,1H). MS(TOF) 349.9 (M ).
4
.2.2.10. 1-((2-Chlorothiazol-5-yl)methyl)-N-(3-fluorobenzyl)-N-methylpiperidin-4-
1
amine (HSP70-30). H NMR (300 MHz, CDCl -d): δ1.63–1.67 (m,2H); δ1.80–1.83
3
(d,2H); δ2.01–2.07 (t,2H); δ2.22 (s,3H); δ2.46 (m,1H); δ2.97–3.00 (d,2H); δ3.59 (s,2H);
δ3.65 (s,2H); δ6.94 (m,1H); δ7.07–7.10 (m,2H); δ7.24–7.28 (m,1H); δ7.34 (s,1H).
+
MS(TOF) 353.9 (M ).
4
.2.2.11. 1-((2-Chlorothiazol-5-yl)methyl)-N-(2,6-dichlorobenzyl)-N-
1
methylpiperidin-4-amine (HSP70-31). H NMR (300 MHz, CDCl -d): δ1.76–1.84
3
(m,2H); δ2.08 (m,2H); δ2.26 (m,2H); δ2.56 (s,3H); δ3.01–3.04 (m,1H); δ3.67 (m,2H);
δ3.86 (s,2H); δ7.15–1.17 (s,1H); δ7.28–7.30 (t,1H); δ7.32–7.36 (m,3H). MS(TOF) 404.8
+
(
M ).
4
.2.3 General procedure for the synthesis of compounds HSP70- (32-34)
General procedure 3: General procedures 1: 2-Chloro-5-(chloromethyl)thiazole (8.40 g,
5
5
0 mmol), tert-butyl piperidin-4-ylcarbamate (50 mmol), and anhydrous K
2 3
CO (6.90 g,
0 mmol) were to a 1000 ml three-necked flask containing 300 ml of dimethyl
formamide (DMF). The mixture was stirred at room temperature for 6 h. A large amount
of ice water was added to precipitate a solid. The solid was washed with large amounts of
petroleum ether, filtered, and dried in a vacuum. A white powder consisting of tert-butyl
1
-((2-chlorothiazol-5-yl)methyl)piperidin-4-ylcarbamate (3d, 16.90 g) was obtained
(
yield 98%).
One part F CCOOH was added to 10 mmol of tert-butyl 1-((2-chlorothiazol-5-
3
yl)methyl)piperidin-4-ylcarbamate in anhydrous dichloroethane (50 ml). The solution
was stirred at room temperature for 5 h. The solution was removed at 35°C under a
vacuum and recrystallized from ethanol-ethyl acetate to yield a white powder. A stirred
solution of white powder (5 mmol) in DMF (30 ml) at room temperature was treated with
substituted benzyl chloride (5.1 mmol) and anhydrous K CO (5.1 mmol) for 4 h. The
2
3
solution was extracted with dichoromethane, dried with anhydrous MgSO
4
, filtered, and
concentrated to provide yellow oily residue that was purified by flash chromatography to
1
6

ACCEPTED MANUSCRIPT
yield the desired product as a white powder, HSP70-(32-34).
4
.2.3.1. 1-((2-Chlorothiazol-5-yl)methyl)-N-(3,4-dichlorobenzyl)piperidin-4-amine
1
(
HSP70-32). H NMR (300 MHz, CDCl -d): δ1.44–1.45 (m,2H); δ1.871 (m,2H); δ2.09–
3
2
7
.10 (d,2H); δ2.51 (m,1H); δ2.86–2.89 (d,2H); δ3.64–3.65 (d,2H); δ3.78 (s,2H); δ7.17–
+
.19 (dd,1H); δ7.34 (s,1H); δ7.38–7.41 (d,1H); δ7.46 (d, 1H). MS(TOF) 390.8 (M ).
4
.2.3.2. 1-((2-chlorothiazol-5-yl)methyl)-N-(4-fluorobenzyl)piperidin-4-amine
1
(
HSP70-33). H NMR (300 MHz, CDCl -d): δ1.46–1.51 (m,2H); δ1.89–1.92 (m,2H);
3
δ2.05–2.12 (d,2H); δ2.54–2.56 (m,1H); δ2.87–2.90 (d,2H); δ3.64 (s,2H); δ3.80 (s,2H);
+
δ6.99–7.04 (m,2H); δ7.31–7.34 (d,3H). MS(TOF) 339.9 (M ).
4
.2.3.3. N-(3-chlorobenzyl)-1-((2-chlorothiazol-5-yl)methyl)piperidin-4-amine
1
(
HSP70-34). H NMR (300 MHz, CDCl CDCl -d): δ1.43–1.46 (m,2H); δ1.88–1.92
3
3
(m,2H); δ2.07–2.13 (m,2H); δ2.53 (m,1H); δ2.86–2.90 (d,2H); δ3.64 (s,2H); δ3.81 (s,2H);
+
δ7.21–7.26 (m,3H); δ7.34–7.35 (m,2H). MS(TOF) 356.3 (M ).
4
.2.4 General procedure for the synthesis of compounds HSP70-(35-56)
General procedure 4: 2, 4-dichloropyrimidine (40 mmol), tert-butyl methyl(piperidin-4-
2 3
yl)carbamate (41 mmol), and anhydrous K CO (41 mmol) were added to a 1000 ml
three-necked flask containing 300 ml of DMF. The mixture was stirred at room
temperature for 6 h. A large amount of ice water was added to precipitate a solid. The
solid was washed with large amounts of petroleum ether, filtered, and dried in a vacuum.
Flash chromatography (ethyl acetate-petroleum ether, 1:2) of the residue was performed
to obtain white powder 3b (tert-butyl 1-(2-chloropyrimidin-4-yl) piperidin-4-yl
(methyl)carbamate).
3
A solution of 3b (20 mmol) in anhydrous THF (100 ml) was treated with CH SNa or
CH ONa (30 mmol) and KI (20 mmol) and refluxed for 8 h. The mixture was cooled to
3
room temperature in a water bath. Solvent was removed under vacuum at 50°C. The
residue was dissolved in water, extracted with ethyl acetate, dried over anhydrous
Na SO , and filtered. The organic phase was removed under a vacuum at 45°C to obtain
2
4
colourless oily residue 4 d.
The colourless oily residue (10 mmol) was dissolved in dichoromethane (50 ml). An
1
7

ACCEPTED MANUSCRIPT
excess of trifluoroacetic acid was added in an ice bath. The mixture was stirred at room
temperature for 5 h. Solvent was removed under a vacuum to obtain yellow oily residue
5
d.
A solution of yellow oily residue 5d (0.3 mmol) was dissolved in DMF (5 ml).
Anhydrous K CO (0.31 mmol) and substituted benzyl chloride (0.31 mmol) were added,
2
3
and the solution was stirred at room temperature for 4 h. A total of 40 ml water was added,
and the compound was extracted with dichoromethane and dried with Na SO , and the
2
4
solvent was removed to produce a colourless oily substance, HSP70-(35-56)
4
.2.4.1. N-(4-fluorobenzyl)-N-methyl-1-(2-(methylthio)pyrimidin-4-yl)piperidin-4-
1
amine (HSP70-35). H NMR (300 MHz, CDCl -d): δ1.56–1.60 (m,2H); δ1.90–1.93
3
(d,2H); δ2.20 (s,3H); δ2.53 (s,3H); δ2.73 (m,1H); δ2.85–2.92 (m,2H); δ3.57 (s,2H);
δ4.47–4.49 (d,2H); δ6.21–6.23 (d,1H); δ6.99–7.03 (t,2H); δ7.28–7.31 (m,2H); δ8.01–
+
8
.03 (d,1H). MS(TOF) 346.5 (M ).
4
.2.4.2. 4-((Methyl(1-(2-(methylthio)pyrimidin-4-yl)piperidin-4-yl)amino)methyl)
1
benzonitrile (HSP70-36). H NMR (300 MHz, CDCl -d): δ1.55–1.59 (m,2H); δ1.91–
3
1
.94 (d,2H); δ2.21 (s,3H); δ2.50 (s,3H); δ2.75 (m,1H); δ2.84–2.90 (t,2H); δ3.65 (s,2H);
δ4.48 (d,2H); δ6.21–6.22 (d,1H); δ7.46–7.48 (d,3H); δ7.61–7.63 (d,2H); δ8.01–8.02
1
3
(
d,1H). C-NMR (DMSO, 100 MHz) δ13.90; 27.80; 37.97; 43.40; 57.29; 60.83; 99.58;
+
1
09.95; 119.52; 129.63; 132.63; 146.97; 156.06; 160.77; 170.48. MS(TOF) 353.5 (M ).
4
.2.4.3. N-(2-chloro-6-fluorobenzyl)-N-methyl-1-(2-(methylthio)pyrimidin-4-
1
yl)piperidin-4-amine (HSP70-37). H NMR (300 MHz, CDCl -d): δ1.62–1.69 (m,2H);
3
δ1.97–2.00 (d,2H); δ2.28 (s,3H); δ2.50 (s,3H); δ2.85–2.92 (t,3H); δ3.71 (s,2H); δ4.5
(s,2H); δ6.21–6.23 (d,1H); δ6.99–7.05 (m,1H); δ7.20–7.22 (m,2H); δ8.00–8.02 (d,1H).
1
3
C-NMR (DMSO, 100 MHz) δ13.90; 27.56; 37.19; 43.52; 48.32; 61.48; 99.56; 114.72;
1
25.42;126.08; 130.40; 135.96; 136.02; 156.06; 160.75; 163.29; 170.48.MS(TOF) 380.9
+
(
M ).
4
4
.2.4.4. N-(3,4-dichlorobenzyl)-N-methyl-1-(2-(methylthio)pyrimidin-4-yl)piperidin-
1
-amine (HSP70-38). H NMR (300 MHz, CDCl -d): δ1.55–1.57 (m,2H); δ1.90–1.93
3
(d,2H); δ2.20 (s,3H); δ2.50 (s,3H); δ2.74–2.90 (m,3H); δ3.55 (s,2H); δ4.48 (s,2H);
δ6.20–6.22 (d,1H); δ7.19 (s,1H); δ7.37–7.45 (m,2H); δ8.00–8.02 (d,1H). MS(TOF) 397.4
1
8

ACCEPTED MANUSCRIPT
+
(
M ).
4
.2.4.5. N-(3-chlorobenzyl)-N-methyl-1-(2-(methylthio)pyrimidin-4-yl)piperidin-4-
1
amine (HSP70-39). H NMR (300 MHz, CDCl -d): δ1.57–1.60 (m,2H); δ1.90–1.93
3
(d,2H); δ2.22 (s,3H); δ2.52 (s,3H); δ2.74 (m,1H); δ2.89–2.90 (m,2H); δ3.58 (s,2H);
δ4.47–4.49 (d,2H); δ6.21–6.23 (d,1H); δ7.24–7.28 (m,3H); δ7.36 (s,1H); δ8.02–8.03
+
(
d,1H). MS(TOF) 362.9 (M ).
4
4
.2.4.6. N-(2,4-dichlorobenzyl)-N-methyl-1-(2-(methylthio)pyrimidin-4-yl)piperidin-
1
-amine (HSP70-40). H NMR (300 MHz, CDCl -d): δ1.64–1.68 (m,2H); δ1.96 (d,2H);
3
δ2.27 (s,3H); δ2.53 (s,3H); δ2.86–2.89 (t,3H); δ3.89 (s,2H); δ4.54 (m,2H); δ6.23–6.24
1
3
(
d,1H); δ7.17–7.19 (t,1H); δ7.32–7.34 (d,2H); δ8.02–8.04 (d,1H). C-NMR (DMSO,
1
1
00 MHz) δ13.39; 27.30; 37.21; 38.87; 42.95; 53.97; 60.57; 99.07; 127.18;
+
28.59;131.87; 133.91; 136.47; 155.55; 160.25; 169.97. MS(TOF) 397.4 (M ).
4
.2.4.7. 2-((Methyl(1-(2-(methylthio)pyrimidin-4-yl)piperidin-4-yl)amino)methyl)
1
benzonitrile (HSP70-41). H NMR (300 MHz, CDCl
3
-d): δ1.59–1.65 (m,2H); δ1.96–
1
.99 (d,2H); δ2.21 (s,3H); δ2.51 (s,3H); δ2.82–2.93 (t,3H); δ3.80 (s,2H); δ4.50–4.52
(m,2H); δ6.22–6.23 (d,1H); δ7.35–7.38 (m,1H); δ7.56–7.57 (m,2H); δ7.64–7.66 (d,1H);
+
δ8.01–8.02 (d,1H). MS(TOF) 353.5 (M ).
4
4
.2.4.8. N-(2,6-dichlorobenzyl)-N-methyl-1-(2-(methylthio)pyrimidin-4-yl)piperidin-
1
-amine (HSP70-43). H NMR (300 MHz, CDCl
3
-d): δ1.63–1.67 (m,2H); δ1.94–1.97
(d,2H); δ2.24 (s,3H); δ2.86–2.90 (m,3H); δ3.87 (s,2H); δ3.94 (s,3H); δ4.51–4.53 (m,2H);
1
3
δ6.21–6.22 (d,1H); δ7.13–7.17 (t,1H); δ7.30–7.33 (m,2H); δ8.02–8.03 (d,1H). C-NMR
(DMSO, 100 MHz) δ13.39; 26.93; 36.09; 38.87; 43.14; 52.22; 60.95; 99.05; 128.64;
+
1
28.59;129.72; 134.72; 136.09; 155.57; 160.23; 169.97. MS(TOF) 396.3(M ).
4
.2.4.9. N-methyl-N-(4-methylbenzyl)-1-(2-(methylthio)pyrimidin-4-yl)piperidin-4-
1
amine (HSP70-42). H NMR (300 MHz, CDCl
3
-d): δ1.58–1.62 (m,2H); δ1.92–1.95
(d,2H); δ2.23 (s,3H); δ2.36 (s,3H); δ2.52 (s,3H); δ2.76 (m,1H); δ2.85–2.92 (t,2H); δ3.59
(s,2H); δ4.47 (m,2H); δ6.21–6.23 (d,1H); δ7.14–7.16 (d,2H); δ7.22–7.24 (d,2H); δ8.02–
+
8
.03 (d,1H). MS(TOF) 342.5 (M ).
4
.2.4.10. N-(2,5-dichlorobenzyl)-N-methyl-1-(2-(methylthio)pyrimidin-4-
1
yl)piperidin-4-amine (HSP70-44). H NMR (300 MHz, CDCl3-d): δ1.58–1.62 (m,2H);
1
9

ACCEPTED MANUSCRIPT
δ1.92–1.95 (d,2H); δ2.25 (s,3H); δ2.52 (s,3H); δ2.86–2.87 (m,1H); δ2.89–2.93 (t,2H);
δ3.67 (s,2H); δ4.49–4.51 (m,2H); δ6.22–6.23 (d,1H); δ7.23–7.26 (dd,1H); δ7.38–7.45
+
(
s,1H); δ7.45–7.47 (d,1H); δ8.02–8.04 (d,1H). MS(TOF) 397.4 (M ).
4
.2.4.11. N-(3-fluorobenzyl)-N-methyl-1-(2-(methylthio)pyrimidin-4-yl)piperidin-4-
1
amine (HSP70-45). H NMR (300 MHz, CDCl -d): δ1.57–1.61 (m,2H); δ1.92–1.95
3
(d,2H); δ2.23 (s,3H); δ2.52 (s,3H); δ2.86–2.87 (m,1H); δ2.89–2.93 (t,2H); δ3.61 (s,2H);
δ4.48–4.50 (m,2H); δ6.22–6.23 (d,1H); δ6.95 (t,1H); δ7.09–7.11 (m,2H); δ7.29 (s,1H);
+
δ8.02–8.04 (d,1H). MS(TOF) 346.5 (M ).
4
.2.4.12. N-(2-chlorobenzyl)-N-methyl-1-(2-(methylthio)pyrimidin-4-yl)piperidin-4-
1
amine (HSP70-46). H NMR (300 MHz, CDCl -d): δ1.57–1.67 (m,2H); δ1.94–1.97
3
(d,2H); δ2.27 (s,3H); δ2.76–2.79 (m,1H); δ2.88–2.94 (t,2H); δ3.72 (s,2H); δ3.95 (s,3H);
δ4.48–4.50 (m,2H); δ6.21–6.22 (d,1H); δ7.18–7.26 (m,2H); δ7.35–7.37 (dd,1H); δ7.49–
1
3
7
4
1
.51 (d,1H); δ8.02–8.04 (d,1H). C-NMR (DMSO, 100 MHz) δ13.90; 27.80; 37.70;
3.48; 55.02; 61.10; 99.58; 127.50;128.93; 129.71; 131.20; 133.63; 137.66; 156.08;
+
60.77; 170.48.MS(TOF) 361.9 (M ).
4
.2.4.13. N-(2-chlorobenzyl)-1-(2-methoxypyrimidin-4-yl)-N-methylpiperidin-4-
1
amine (HSP70-47). H NMR (300 MHz, CDCl
3
-d): δ1.60–1.64 (m,2H); δ1.95–1.98
(d,2H); δ2.27 (s,3H); δ2.52 (s,3H); δ2.87–2.88 (m,1H); δ2.91–2.94 (t,2H); δ3.72 (s,2H);
δ4.49–4.50 (m,2H); δ6.21–6.24 (d,1H); δ7.20–7.26 (m,2H); δ7.35–7.37 (d,1H); δ7.49
+
(
d,1H); δ8.02–8.03 (d,1H). MS(TOF) 362.9 (M ).
4
.2.4.14. 4-(((1-(2-Methoxypyrimidin-4-yl)piperidin-4-yl)(methyl)amino)methyl)
1
benzonitrile (HSP70-48). H NMR (300 MHz, CDCl
3
-d): δ1.54–1.58 (m,2H); δ1.89–
1
.92 (d,2H); δ2.20 (s,3H); δ2.72 (m,1H); δ2.85–2.92 (t,2H); δ3.64 (s,2H); δ3.92 (s,3H);
δ4.45–4.48 (m,2H); δ6.19–6.20 (d,1H); δ7.44–7.46 (d,2H); δ7.59–7.61 (d,2H); δ7.49–
+
7
.51 (d,1H); δ8.01–8.02 (d,1H). MS(TOF)337.4 (M ).
4
.2.4.15. 2-(((1-(2-Methoxypyrimidin-4-yl)piperidin-4-yl)(methyl)amino)methyl)
1
benzonitrile (HSP70-49). H NMR (300 MHz, CDCl
3
-d): δ1.60–1.64 (m,2H); δ1.97–
2
.00 (d,2H); δ2.23 (s,3H); δ2.84–2.94 (m,3H); δ3.82 (s,2H); δ3.95 (s,3H); δ4.49–4.52
(m,2H); δ6.21–6.22 (d,1H); δ7.31–7.39 (m,1H); δ7.57–7.58 (m,2H); δ7.65–7.67 (d,1H);
+
δ8.03–8.04 (d,1H). MS(TOF) 337.4 (M ).
2
0

ACCEPTED MANUSCRIPT
4
.2.4.16. 1-(2-Methoxypyrimidin-4-yl)-N-methyl-N-(4-methylbenzyl)piperidin-4-
1
amine (HSP70-50). H NMR (300 MHz, CDCl -d): δ1.57–1.61 (m,2H); δ1.91–1.92
3
(d,2H); δ2.22 (s,3H); δ2.35 (s,3H); δ2.74 (m,1H); δ2.86–2.92 (t,2H); δ3.58 (s,2H); δ3.94
(s,3H); δ4.45–4.47 (m,2H); δ6.20–6.21 (d,1H); δ7.13–7.15 (dd,2H); δ7.21–7.23 (dd,2H);
+
δ8.02–8.03 (d,1H). MS(TOF) 326.4 (M ).
4
.2.4.17. N-(2,5-dichlorobenzyl)-1-(2-methoxypyrimidin-4-yl)-N-methylpiperidin-4-
1
amine (HSP70-51). H NMR (300 MHz, CDCl -d): δ1.57–1.61 (m,2H); δ1.92–1.95
3
(d,2H); δ2.24 (s,3H); δ2.76 (m,1H); δ2.86–2.93 (t,2H); δ3.67 (s,2H); δ3.93 (s,3H);
δ4.47–4.50 (m,2H); δ6.20–6.21 (d,1H); δ7.22–7.25 (dd,2H); δ7.37 (d,1H); δ7.45–7.47
+
(
d,1H); δ8.02–8.03 (d,1H). MS(TOF) 381.3 (M ).
4
.2.4.18. N-(3,4-dichlorobenzyl)-1-(2-methoxypyrimidin-4-yl)-N-methylpiperidin-4-
1
amine (HSP70-52). H NMR (300 MHz, CDCl -d): δ1.54–1.60 (m,2H); δ1.92–1.95
3
(d,2H); δ2.24 (s,3H); δ2.76 (m,1H); δ2.90–2.92 (t,2H); δ3.55 (s,2H); δ3.93 (s,3H); δ4.48
(s,2H); δ6.19–6.21 (d,1H); δ7.17–7.19 (d,2H); δ7.30–7.44 (m,1H); δ8.01–8.03 (d,1H).
+
MS(TOF) 381.3 (M ).
4
.2.4.19. N-(2-chloro-6-fluorobenzyl)-1-(2-methoxypyrimidin-4-yl)-N-
1
methylpiperidin-4-amine (HSP70-53). H NMR (300 MHz, CDCl
3
-d): δ1.63–1.66
(m,2H); δ1.95–1.98 (d,2H); δ2.26 (s,3H); δ2.78 (m,1H); δ2.86–2.92 (t,2H); δ3.74 (s,2H);
δ3.93 (s,3H); δ4. 51 (s,2H); δ6.20–6.22 (d,1H); δ6.99–7.01 (m,2H); δ7.19–7.21 (m,1H);
+
δ8.01–8.03 (d,1H). MS(TOF) 364.8 (M ).
4
.2.4.20. N-(3-chlorobenzyl)-1-(2-methoxypyrimidin-4-yl)-N-methylpiperidin-4-
1
amine (HSP70-54). H NMR (300 MHz, CDCl
3
-d): δ1.55–1.62 (m,2H); δ1.91–1.94
(d,2H); δ2.21 (s,3H); δ2.74 (s,1H); δ2.85–2.90 (t,2H); δ3.58 (s,2H); δ3.93 (s,3H); δ4.48
(m,2H); δ6.19–6.21 (d,1H); δ7.23 (m,2H); δ7.34 (s,2H); δ8.00–8.02 (d,1H). MS(TOF)
+
3
46.9 (M ).
4
.2.4.21. N-(3-fluorobenzyl)-1-(2-methoxypyrimidin-4-yl)-N-methylpiperidin-4-
1
amine (HSP70-55). H NMR (300 MHz, CDCl
3
-d): δ1.56–1.64 (m,2H); δ1.91–1.94
(d,2H); δ2.22 (s,3H); δ2.74–2.75 (m,1H); δ2.86–2.93 (t,2H); δ3.60 (s,2H); δ3.94 (s,3H);
δ4.46–4.49 (m,2H); δ6.20–6.21 (d,1H); δ6.93–6.97 (t,2H); δ7.08–7.11 (m,2H); δ7.25–
+
7
.30 (m,1H); δ8.02–8.04 (d,1H). MS(TOF) 330.4 (M ).
2
1

ACCEPTED MANUSCRIPT
4
.2.4.22. N-(4-fluorobenzyl)-1-(2-methoxypyrimidin-4-yl)-N-methylpiperidin-4-
1
amine (HSP70-56). H NMR (300 MHz, CDCl -d): δ1.56–1.60 (m,2H); δ1.91–1.94
3
(d,2H); δ2.20 (s,3H); δ2.74 (s,1H); δ2.84–2.90 (t,2H); δ3.58 (s,2H); δ3.93 (s,3H); δ4.47
(s,2H); δ6.18–6.20 (d,1H) δ6.98–7.02 (t,2H); δ7.30 (m,2H); δ7.25–7.30 (m,1H); δ8.00–
+
8
.02 (d,1H). MS(TOF) 330.4 (M ).
4
.2.5 General procedure for the synthesis of compounds HSP70-(57-67)
General procedure 5: 4, 6-dichloropyrimidine was processed instead of 2, 4-
dichloropyrimidine as described in General procedure 4 to provide the product HSP70-
57-67).
(
4
.2.5.1. N-(2-chlorobenzyl)-1-(6-methoxypyrimidin-4-yl)-N-methylpiperidin-4-amine
1
(
HSP70-57). H NMR (300 MHz, CDCl -d): δ1.60–1.67 (m,2H); δ1.93–1.96 (d,2H);
3
δ2.27 (s,3H); δ2.77 (m,1H); δ2.85–2.92 (t,2H); δ3.72 (s,2H); δ3.93 (s,3H); δ4.40–4.44
(m,2H); δ5.86 (s,1H); δ7.20–7.26 (m,2H); δ7.35–7.37 (dd,1H); δ7.49–7.51 (d,1H); δ8.34
+
(
s,1H). MS(TOF) 346.9 (M ).
4
.2.5.2. 1-(6-Methoxypyrimidin-4-yl)-N-methyl-N-(4-methylbenzyl)piperidin-4-
1
amine (HSP70-58). H NMR (300 MHz, CDCl -d): δ1.58–1.62 (m,2H); δ1.91–1.94
3
(
(
(
d,2H); δ2.22 (s,3H); δ2.35 (s,3H); δ2.74 (m,2H); δ2.83–2.90 (t,2H); δ3.58 (s,2H); δ3.93
s,3H); δ4.38–4.42 (m,2H); δ5.85 (s,1H); δ7.13–7.15 (d,2H); δ7.22–7.24 (d,2H); δ8.34
+
s,1H). MS(TOF) 326.4 (M ).
4
.2.5.3. N-(2,6-dichlorobenzyl)-1-(6-methoxypyrimidin-4-yl)-N-methylpiperidin-4-
1
amine (HSP70-59). H NMR (300 MHz, CDCl -d): δ1.63–1.67 (m,2H); δ1.93–1.96
3
(d,2H); δ2.24 (s,3H); δ2.83–2.90 (m,3H); δ3.87 (s,2H); δ3.93 (s,3H); δ4.43–4.46 (m,2H);
+
δ5.86 (s,1H); δ7.15–7.17 (t,1H); δ7.29–7.32 (m,2H); δ8.34 (s,1H). MS(TOF) 381.3 (M ).
4
.2.5.4. N-(3,4-dichlorobenzyl)-1-(6-methoxypyrimidin-4-yl)-N-methylpiperidin-4-
1
amine (HSP70-60). H NMR (300 MHz, CDCl
3
-d): δ1.43–1.58 (m,2H); δ1.88–1.90
(m,2H); δ2.19 (s,3H); δ2.72 (m,1H); δ2.82–2.89 (m,2H); δ3.53 (s,2H); δ3.91 (s,3H);
δ4.38–4.42 (m,2H); δ5.84 (s,1H); δ7.16 (s,1H); δ7.37–7.44 (m,2H); δ8.32 (s,1H).
+
MS(TOF) 381.3 (M ).
4
.2.5.5. N-(4-fluorobenzyl)-1-(6-methoxypyrimidin-4-yl)-N-methylpiperidin-4-amine
2
2

ACCEPTED MANUSCRIPT
1
(
HSP70-61). H NMR (300 MHz, CDCl
3
-d): δ1.56–1.62 (m,2H); δ1.89–1.92 (m,2H);
δ2.19 (s,3H); δ2.72 (s,1H); δ2.82–2.89 (m,2H); δ3.56 (s,2H); δ3.88 (s,3H); δ4.38–4.41
(m,2H); δ5.84 (s,1H); δ6.98–7.02 (t,1H); δ7.27–7.29 (m,2H); δ8.32 (s,1H). MS(TOF)
+
3
30.4 (M ).
4
.2.5.6. N-(3-chlorobenzyl)-1-(6-methoxypyrimidin-4-yl)-N-methylpiperidin-4-amine
1
(
(
(
HSP70-62). H NMR (300 MHz, CDCl
3
): δ1.55–1.62 (m,2H); δ1.89–1.92 (m,2H); δ2.20
s,3H); δ2.72 (m,1H); δ2.83–2.89 (m,2H); δ3.57 (m,2H); δ3.91 (s,3H); δ4.38–4.41
+
m,2H); δ5.83 (s,1H); δ7.23 (m,3H); δ7.34 (s,1H); δ8.32 (s,1H). MS(TOF) 346.9 (M ).
4
.2.5.7. N-(2-chloro-6-fluorobenzyl)-1-(6-methoxypyrimidin-4-yl)-N-
1
methylpiperidin-4-amine (HSP70-63). H NMR (300 MHz, CDCl -d): δ1.59–1.68
3
(
(
(
m,2H); δ1.94–1.96 (m,2H); δ2.25 (s,3H); δ2.75 (m,1H); δ2.84–2.87 (m,2H); δ3.73
s,2H); δ3.91 (s,3H); δ4.42–4.45 (m,2H); δ5.84 (s,1H); δ6.99 (t,1H); δ7.19 (d,2H); δ8.32
+
s,1H). MS(TOF) 364.8 (M ).
4
.2.5.8. 2-(((1-(6-Methoxypyrimidin-4-yl)piperidin-4-yl)(methyl)amino)methyl)
1
benzonitrile (HSP70-64). H NMR (300 MHz, CDCl ): δ1.58–1.62 (m,2H); δ1.93–1.96
3
(m,2H); δ2.20 (s,3H); δ2.72 (m,1H); δ2.83–2.87 (m,2H); δ3.78 (s,2H); δ3.91 (s,3H);
δ4.41–4.44 (m,2H); δ5.84 (s,1H); δ7.35 (s,3H); δ7.54 (s,1H); δ7.63–7.65 (d,1H); δ8.32
+
(
s,1H). MS(TOF) 337.4 (M ).
4
.2.5.9. 4-(((1-(6-Methoxypyrimidin-4-yl)piperidin-4-yl)(methyl)amino)methyl)
1
benzonitrile (HSP70-65). H NMR (300 MHz, CDCl ): δ1.55–1.61 (m,2H); δ1.88–1.91
3
(m,2H); δ2.22 (s,3H); δ2.71 (m,1H); δ2.80–2.89 (m,2H); δ3.63 (s,2H); δ3.93 (s,3H);
δ4.39–4.41 (d,2H); δ5.8 (s,1H); δ7.26 (s,3H); δ7.44–7.46 (d,1H); δ7.60–7.62 (d,1H);
+
δ8.32 (s,1H). MS(TOF) 337.4 (M ).
4
.2.5.10. N-(2,5-dichlorobenzyl)-1-(6-methoxypyrimidin-4-yl)-N-methylpiperidin-4-
1
amine (HSP70-66). H NMR (300 MHz, CDCl
3
): δ1.59–1.62 (m,2H); δ1.91–1.94
(m,2H); δ2.25 (s,3H); δ2.75 (m,1H); δ2.85–2.92 (m,2H); δ3.66 (s,2H); δ3.93 (s,3H);
δ4.41–4.44 (m,2H); δ5.86 (s,1H); δ7.23–7.25 (t,1H); δ7.38 (m,1H); δ7.45–7.47 (m,1H);
+
δ8.34 (s,1H). MS(TOF) 381.3 (M ).
4
.2.5.11. N-(2,5-dichlorobenzyl)-1-(6-methoxypyrimidin-4-yl)-N-methylpiperidin-4-
1
amine (HSP70-67). H NMR (300 MHz, CDCl
3
-d): δ1.58–1.64 (m,2H); δ1.91–1.94
2
3

ACCEPTED MANUSCRIPT
(m,2H); δ2.21 (s,3H); δ2.74 (m,1H); δ2.84–2.91 (m,2H); δ3.61 (m,2H); δ3.93 (s,3H);
δ4.40–4.43 (m,2H); δ5.86 (s,1H); δ6.95 (t,1H); δ7.11 (m,2H); δ7.28–7.30 (s,2H); δ8.34
+
(
s,1H). MS(TOF) 330.4 (M ).
4
.3 Biological evaluation
4
.3.1 Cell viability assay
A luminescent signal indicating the ATP levels was used as a marker of cell viability. The
R
R
breast cancer cell lines BT474, BT/Lap 1.0, MDA-MB-361, SK-BR3, SK/Lap 1.0 and
MDA-MB-453 were selected for the cell viability assays. The cell lines were obtained
from the American Type Culture Collection (ATCC). The epidermal growth factor (EGFR:
ErbB-1, ErbB-2) tyrosine kinase inhibitor lapatinib (10 mM in DMSO, BioVision, Cat:
1
624-100, Lot: 50324) was used as the positive control. The amount of ATP was
estimated with the use of an ATPlite kit (CellTiter-Glo Substrate, Promega, Part: G755B,
Lot: 32513501, EXP: 2014-05) according to the manufacturer’s protocol. Briefly, the
cells were trypsinized in 1 mL of 0.25% trypsin (Gibco) supplemented with 2 mL of
5
medium (containing 10% FBS, Gibco) and seeded (1×10 cells/mL, 50 ꢀL/well, 5000
cells/well) into 96-well plates. The plates were incubated for 24 h at 37°C. The
compounds were diluted to 10, 5, 2.5, 1.25, 0.625, 0.31, 0.16, 0.08, 0.04 and 0 µM. The
serially diluted compounds or control (50 µL) were added to each well in triplicate. The
cells were incubated for 72 h at 37°C. A volume of the CellTiter-Glo Reagent (50 µL)
equal to the volume of the cell culture medium present in each well was added. The plates
were shaken for 3 min on an orbital shaker to induce cell lysis. To stabilize the
luminescent signal, the plate was incubated at room temperature for 10 min. The liquid
supernatant (100ꢀL/well) was aspirated, and the luminescent signal was measured. The
cell viability was determined based on the luminescence: cell viability (%) =
RLUexperimental group/RLUblank group×100%.[28]
4
.3.2 Surface plasmon resonance (SPR) analyses
SPR studies of HSP70 inhibitors were performed using optical biosensor Biacore T100
(GE Company) as reported elsewhere. [16, 17] Briefly, human HSP70 (ADI-ESP-550-D,
Enzo Life Sciences) was immobilized on a CM5 sensor chip (GE) using 10 mmol/L
phosphate-buffered saline (PBS), pH 5.0, RU=11209. Binding experiments were
−
1
performed at 25°C using a flow rate of 30 ꢀlml , with 90 s of monitoring for association
2
4

ACCEPTED MANUSCRIPT
and 120 s of monitoring for dissociation. The complete binding study was performed
using an eight-point concentration series (0.003, 0.01, 0.03, 0.1, 0.3, 1, 3, and 10 ꢀM),
which were prepared with 5% DMSO in PBS. Different concentrations of compounds
were typically injected over the sensor chip, and 5% DMSO in PBS was used as a
running buffer and blank control. Ver-155008 was used as a positive control. The data
d
were processed using an equilibrium dissociation constant (K value), which was
calculated with the steady state model: Response= Conc * Rmax / [conc + K ] + offset.
d
4
.3.3 Colorimetric determination of ATPase activity
The assay procedure was adopted from previous reports [19] with some modifications
where indicated. Stock solutions of malachite green (0.1% w/v), polyvinyl alcohol (2.3%
w/v), and ammonium molybdate tetrahydrate (1% w/v in 1 M HCl) were prepared and
mixed with water at a ratio of 2:1:1:2 to prepare the malachite green reagent, which is
stable at room temperature for at least 2 h. The reagent should be filtered through a 0.22-
ꢀ
M membrane prior to use when its colour changes from dark brown to yellow. ATP,
malachite green, polyvinyl alcohol and ammonium molybdate tetrahydrate were
purchased from Sigma and used without further purification.
For compound screening, an aliquot of a master mixture of DnaK:DnaJ (2.0 ꢀM:3.5 ꢀM)
prepared in assay buffer (0.017% Triton X-100, 100 mM Tris-HCl, 20 mM KCl, and 6
mM MgCl , pH 7.4) was added to each well of a 96-well plate (14 ꢀL per well). To this
2
solution, 1 ꢀL of either one of the test compounds (5 mM) or DMSO was added, and the
plate was shaken for 10 min and incubated for 30 min at 37°C. Ten microliters of 2.5 mM
ATP was added to start the reaction. The final reaction volume was 25 ꢀL, and the
concentration of the tested compounds was 200 ꢀM. After 3 h of incubation at 37°C, 80
ꢀ
L of the malachite green reagent was added to each well, and the plates were gently
shaken. Immediately following this step, 10 ꢀL of 34% sodium citrate was added to
terminate the nonenzymatic hydrolysis of ATP. After the samples were thoroughly mixed
and incubated at 37°C for 15 min, the OD620 was measured using a SpectraMax M5
(Molecular Devices, Sunnyvale, CA, USA).
4
.3.4 Acute toxicity test by single oral gavage with HSP70-36 in mice
2
5

ACCEPTED MANUSCRIPT
Seventy mice (KM) were randomly divided into seven groups, with 10 mice per group
5 males and 5 females). HSP70-36 was diluted to concentrations of 155.5, 129.6, 108.0,
(
−
1
9
0.0, 75.0, 62.5, and 52.0 mg*ml with 0.5% sodium carboxymethyl cellulose. The
fixed-dose procedure was used for this test. According to the acute toxicity test results,
−
1
the mice received 1555, 1296, 1080, 900, 750, 625 and 520 mg*kg HSP70-36 via oral
gavage and were continuously observed for 14 days. Death and toxicity were recorded
after 5 min, 10 min, 20 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, 18 h, 1 d, 2 d, 3 d, 7 d
and 14 d. The LD50 was calculated based on a statistical analysis with SAS.
5
. Acknowledgments
This work was supported by Grant No. 81473139 to Wu Zhong from the National
Natural Science Foundation of China and the State Key Laboratory of Toxicology and
Medical Countermeasures.
6
. References
[
1] E. Meimaridou, S.B. Gooljar, J.P. Chapple, From hatching to dispatching: the multiple cellular
roles of the Hsp70 molecular chaperone machinery, J Mol Endocrinol. 42 (2009) 1-9.
[
2] E. Schmitt, M. Gehrmann, M. Brunet, G. Multhoff, C. Garrido, Intracellular and extracellular
functions of heat shock proteins: repercussions in cancer therapy, J Leukoc Biol. 81 (2007) 15-27.
[
3] J.S. Guo, J.F. Chau, X.Z. Shen, C.H. Cho, J.M. Luk, M.W. Koo, Over-expression of inducible heat
shock protein 70 in the gastric mucosa of partially sleep-deprived rats, Scand J Gastroenterol. 39
(
[
2004) 510-515.
4] M. Pocaly, V. Lagarde, G. Etienne, J.A. Ribeil, S. Claverol, M. Bonneu, F. Moreau-Gaudry, V.
Guyonnet-Duperat, O. Hermine, J.V. Melo, M. Dupouy, B. Turcq, F.X. Mahon, J.M. Pasquet,
Overexpression of the heat-shock protein 70 is associated to imatinib resistance in chronic myeloid
leukemia, Leukemia. 21 (2007) 93-101.
[
5] X. Yang, J. Wang, Y. Zhou, Y. Wang, S. Wang, W. Zhang, Hsp70 promotes chemoresistance by
blocking Bax mitochondrial translocation in ovarian cancer cells, Cancer Lett. 321 (2012) 137-143.
[
6] M. Daugaard, M. Rohde, M. Jaattela, The heat shock protein 70 family: Highly homologous
proteins with overlapping and distinct functions, FEBS Lett. 581 (2007) 3702-3710.
2
6

ACCEPTED MANUSCRIPT
[
7] S. Patury, Y. Miyata, J.E. Gestwicki, Pharmacological targeting of the Hsp70 chaperone, Curr Top
Med Chem. 9 (2009) 1337-1351.
[
8] W. Zhong, H. Zhu, F. Sheng, Y. Tian, J. Zhou, Y. Chen, S. Li, J. Lin, Activation of the
MAPK11/12/13/14 (p38 MAPK) pathway regulates the transcription of autophagy genes in response
to oxidative stress induced by a novel copper complex in HeLa cells, Autophagy. 10 (2014) 1285-
1
300.
[
9] Y. Yang, W. Fiskus, B. Yong, P. Atadja, Y. Takahashi, T.K. Pandita, H.G. Wang, K.N. Bhalla,
Acetylated hsp70 and KAP1-mediated Vps34 SUMOylation is required for autophagosome creation in
autophagy, Proc Natl Acad Sci U S A. 110 (2013) 6841-6846.
[
10] M.V. Powers, K. Jones, C. Barillari, I. Westwood, R.L. van Montfort, P. Workman, Targeting
HSP70: the second potentially druggable heat shock protein and molecular chaperone?, Cell Cycle. 9
(
[
2010) 1542-1550.
11] J.I. Leu, J. Pimkina, A. Frank, M.E. Murphy, D.L. George, A small molecule inhibitor of
inducible heat shock protein 70, Mol Cell. 36 (2009) 15-27.
[
12] D.R. Williams, S.K. Ko, S. Park, M.R. Lee, I. Shin, An apoptosis-inducing small molecule that
binds to heat shock protein 70, Angew Chem Int Ed Engl. 47 (2008) 7466-7469.
[
13] M.V. Powers, P.A. Clarke, P. Workman, Dual targeting of HSC70 and HSP72 inhibits HSP90
function and induces tumor-specific apoptosis, Cancer Cell. 14 (2008) 250-262.
[
(
14] M.P. Hatfield, S. Lovas, Role of Hsp70 in cancer growth and survival, Protein Pept Lett. 19
2012) 616-624.
[
15] A.L. Rerole, G. Jego, C. Garrido, Hsp70: anti-apoptotic and tumorigenic protein, Methods Mol
Biol. 787 (2011) 205-230.
[
16] F. Dal Piaz, A. Vassallo, L. Lepore, A. Tosco, A. Bader, N. De Tommasi, Sesterterpenes as tubulin
tyrosine ligase inhibitors. First insight of structure-activity relationships and discovery of new lead, J
Med Chem. 52 (2009) 3814-3828.
[
17] F. Dal Piaz, A. Tosco, D. Eletto, A.L. Piccinelli, O. Moltedo, S. Franceschelli, G. Sbardella, P.
Remondelli, L. Rastrelli, L. Vesci, C. Pisano, N. De Tommasi, The identification of a novel natural
activator of p300 histone acetyltranferase provides new insights into the modulation mechanism of
this enzyme, Chembiochem. 11 (2010) 818-827.
2
7

ACCEPTED MANUSCRIPT
[
18] A.T. Macias, D.S. Williamson, N. Allen, J. Borgognoni, A. Clay, Z. Daniels, P. Dokurno, M.J.
Drysdale, G.L. Francis, C.J. Graham, R. Howes, N. Matassova, J.B. Murray, R. Parsons, T. Shaw, A.E.
Surgenor, L. Terry, Y. Wang, M. Wood, A.J. Massey, Adenosine-derived inhibitors of 78 kDa glucose
regulated protein (Grp78) ATPase: insights into isoform selectivity, J Med Chem. 54 (2011) 4034-
4
041.
[
19] L. Chang, E.B. Bertelsen, S. Wisen, E.M. Larsen, E.R. Zuiderweg, J.E. Gestwicki, High-
throughput screen for small molecules that modulate the ATPase activity of the molecular chaperone
DnaK, Anal Biochem. 372 (2008) 167-176.
[
20] H.J. Cho, H.Y. Gee, K.H. Baek, S.K. Ko, J.M. Park, H. Lee, N.D. Kim, M.G. Lee, I. Shin, A
small molecule that binds to an ATPase domain of Hsc70 promotes membrane trafficking of mutant
cystic fibrosis transmembrane conductance regulator, J Am Chem Soc. 133 (2011) 20267-20276.
[
21] A.J. Massey, D.S. Williamson, H. Browne, J.B. Murray, P. Dokurno, T. Shaw, A.T. Macias, Z.
Daniels, S. Geoffroy, M. Dopson, P. Lavan, N. Matassova, G.L. Francis, C.J. Graham, R. Parsons, Y.
Wang, A. Padfield, M. Comer, M.J. Drysdale, M. Wood, A novel, small molecule inhibitor of
Hsc70/Hsp70 potentiates Hsp90 inhibitor induced apoptosis in HCT116 colon carcinoma cells, Cancer
Chemother Pharmacol. 66 (2010) 535-545.
[
22] C.I. Bliss, The determination of the dosage-mortality curve from small numbers, Quart J Pharm
and Pharmacol. 11 (1938) 192-216.
[
23] A. S. El-Nockrashy, C. M. Lyman, J.W. Dollahite, The acute oral toxicity of cottonseed pigment
glands and intraglandular pigments, Journal of the American Oil Chemists Society. 40 (1963) 14-17.
[
24] T. Taldone, Y. Kang, H.J. Patel, M.R. Patel, P.D. Patel, A. Rodina, Y. Patel, A. Gozman, R.
Maharaj, C.C. Clement, A. Lu, J.C. Young, G. Chiosis, Heat shock protein 70 inhibitors. 2. 2,5'-
thiodipyrimidines, 5-(phenylthio)pyrimidines, 2-(pyridin-3-ylthio)pyrimidines, and 3-
phenylthio)pyridines as reversible binders to an allosteric site on heat shock protein 70, J Med Chem.
7 (2014) 1208-1224.
(
5
[
25] Y. Kang, T. Taldone, H.J. Patel, P.D. Patel, A. Rodina, A. Gozman, R. Maharaj, C.C. Clement,
M.R. Patel, J.L. Brodsky, J.C. Young, G. Chiosis, Heat shock protein 70 inhibitors. 1. 2,5'-
thiodipyrimidine and 5-(phenylthio)pyrimidine acrylamides as irreversible binders to an allosteric site
on heat shock protein 70, J Med Chem. 57 (2014) 1188-1207.
2
8