Design, synthesis and bioevaluation of inhibitors targeting HSP90-CDC37 protein-protein interaction based on a hydrophobic core

Article abstract of DOI:10.1016/j.ejmech.2020.112959

HSP90-CDC37 protein-protein interaction (PPI) works as a kinase specific-molecular chaperone system to regulate the maturation of kinases. Currently, selectively disrupting HSP90-CDC37 PPI, rather than the direct inhibition of the ATPase function of HSP90, is emerging as a promising strategy for cancer therapy by specifically blocking the maturation of kinases. However, due to the limited understanding of HSP90-CDC37 binding interface, design of small molecule inhibitors targeting HSP90-CDC37 PPI is challenging. In this work, based on the binding mode of compound 11 (previously reported by our group), we discovered a hydrophobic pocket centered on Phe213, which was previously unknown, contributing to the binding affinity of HSP90-CDC37 PPI inhibitors. A series of hydrophobic substituted inhibitors were utilized to confirm the importance of Phe213 hydrophobic core. Finally, we obtained an optimum compound DDO-5994 (exhibited an ideal binding pattern on hydrophobic core) with improved binding affinity (K_D = 5.52 μM) and antiproliferative activity (IC₅₀ = 6.34 μM). Both in vitro and in vivo assays confirmed DDO-5994 as a promising inhibitor to exhibit ideal antitumor efficacy through blocking HSP90-CDC37 PPI.

Full text of DOI:10.1016/j.ejmech.2020.112959

European Journal of Medicinal Chemistry xxx (xxxx) xxx
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Design, synthesis and bioevaluation of inhibitors targeting HSP90-
CDC37 protein-protein interaction based on a hydrophobic core
,
b
,
,
b
,
,
,
, b
Qiuyue Zhang a 1, Xuexuan Wu a 1, Jianrui Zhou a b, Lixiao Zhang a b, Xiaoli Xu a
,
Lianshan Zhang c **, Qidong You a ***, Lei Wang a
,
,
b,
, b, *
a State Key Laboratory of Natural Medicines and Jiang Su Key Laboratory of Drug Design and Optimization, China Pharmaceutical University, Nanjing,
210009, China
b Department of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical University, Nanjing, 210009, China
c Shanghai Hengrui Pharmaceutical Co., Ltd., Shanghai, 200245, China
a r t i c l e i n f o
a b s t r a c t
Article history:
HSP90-CDC37 protein-protein interaction (PPI) works as a kinase specific-molecular chaperone system to
regulate the maturation of kinases. Currently, selectively disrupting HSP90-CDC37 PPI, rather than the
direct inhibition of the ATPase function of HSP90, is emerging as a promising strategy for cancer therapy
by specifically blocking the maturation of kinases. However, due to the limited understanding of HSP90-
CDC37 binding interface, design of small molecule inhibitors targeting HSP90-CDC37 PPI is challenging.
In this work, based on the binding mode of compound 11 (previously reported by our group), we
discovered a hydrophobic pocket centered on Phe213, which was previously unknown, contributing to
the binding affinity of HSP90-CDC37 PPI inhibitors. A series of hydrophobic substituted inhibitors were
utilized to confirm the importance of Phe213 hydrophobic core. Finally, we obtained an optimum
compound DDO-5994 (exhibited an ideal binding pattern on hydrophobic core) with improved binding
Received 24 September 2020
Received in revised form
15 October 2020
Accepted 19 October 2020
Available online xxx
Keywords:
HSP90-CDC37
Protein-protein interaction inhibitors
Anti-proliferation activity
Kinase maturation
affinity (KD ¼ 5.52
m
M) and antiproliferative activity (IC50 ¼ 6.34
mM). Both in vitro and in vivo assays
confirmed DDO-5994 as a promising inhibitor to exhibit ideal antitumor efficacy through blocking
HSP90-CDC37 PPI.
© 2020 Elsevier Masson SAS. All rights reserved.
1. Introduction
HSP40 [12], HSP70 [12,13], AHA1 (activator of HSP90 ATPase pro-
tein 1) and p23 [14]. Among them, CDC37 is a kinase-specific co-
Molecular chaperone heat shock protein 90 (HSP90) is a mem-
ber of heat shock protein family which plays a pivotal role in
physiological function of cells, including cell growth, differentiation
and survival [1e3]. At present, more than 200 proteins are found to
be client proteins of HSP90, which are maturated with the help of
HSP90 [4]. The maturation of kinases is a huge project that
completed by HSP90 and many co-chaperones, including CDC37
(cell division cycle 37) [5e8], PP5 (protein phosphatase 5) [9e11],
chaperone, playing as a hunter in HSP90-CDC37-kinase chaperone
cycle for kinase’s recruitment and assisting HSP90 to realize the
chaperone function [15e17]. In the HSP90-CDC37-kinase chap-
erone cycle, CDC37 firstly distinguishes and binds to unfolded ki-
nases after phosphorylated by CK2 (casein kinase 2), and
subsequently conjugates with HSP90 (Fig. 1A). Then, HSP90 un-
dergoes
a conformational transformation after ATP binding.
Maturated kinase is released finally after PP5 combines with the
above complex and dephosphorylates p-CDC37. For the past
decade, considering the fundamental role of HSP90 in cancer,
HSP90 was regarded as a target for cancer therapy and the main
attention was focused on the inhibition of ATPase activity of HSP90
to hinder the kinase maturation [18].
Inhibitors targeting HSP90 ATPase were designed by imitating
ATP to occupy the ATP pocket on HSP90, impairing the chaperone
function of HSP90 totally [19]. The study of HSP90 ATPase inhibitors
kicked off since the discovery of geldanamycin (GA), the first
* Corresponding author. State Key Laboratory of Natural Medicines and Jiang Su
Key Laboratory of Drug Design and Optimization, China Pharmaceutical University,
Nanjing, 210009, China.
** Corresponding author.
*** Corresponding author. State Key Laboratory of Natural Medicines and Jiang Su
Key Laboratory of Drug Design and Optimization, China Pharmaceutical University,
Nanjing, 210009, China.
E-mail addresses: youqd@163.com (Q. You), leiwang.91@cpu.edu.cn (L. Wang).
These authors contributed equally.
1
https://doi.org/10.1016/j.ejmech.2020.112959
0223-5234/© 2020 Elsevier Masson SAS. All rights reserved.
Please cite this article as: Q. Zhang, X. Wu, J. Zhou et al., Design, synthesis and bioevaluation of inhibitors targeting HSP90-CDC37 protein-
protein interaction based on a hydrophobic core, European Journal of Medicinal Chemistry, https://doi.org/10.1016/j.ejmech.2020.112959

Q. Zhang, X. Wu, J. Zhou et al.
European Journal of Medicinal Chemistry xxx (xxxx) xxx
Fig. 1. CDC37-HSP90 PPI plays an important role in kinase maturation and targeting CDC37-HSP90 PPI is a promising strategy for cancer therapy. (A) HSP90-CDC37-kinase
chaperone cycle. CDC37 firstly distinguishes and binds to unfolded kinases after phosphorylated by CK2, and subsequently conjugates with HSP90. Then, HSP90 undergoes a
conformational transformation after ATP binding. Maturated kinase is released finally after that PP5 combined with the above complex and dephosphorylated CDC37. (B)
Representative compounds involving HSP90 ATPase inhibition and HSP90-CDC37 inhibition.
natural product of HSP90 ATPase inhibitor [20]. Up to date, more
than 30 HSP90 ATPase inhibitors (including IP-504, onalespib, PU-
H71) were entered into clinical trials and most of them were
stagnant due to the various inevitable side effects or unsatisfied
efficacy, including liver toxicity, heat shock response, high mor-
tality rate, low bioavailability and poor solubility [18,21e23]. Due to
multiple downstream pathways (such as CDK4/6 (cyclin dependent
kinase4/6), FGFR1 (fibroblast growth factor receptor 1), JAK1 (janus
kinase 1), CAMKV (caM kinase like vesicle associated), AKT2 (AKT
serine/threonine kinase 2) and TK(tyrosine kinase)) were found to
be depended on the function of HSP90, targeting HSP90 ATPase lack
of specificity which might induce the degradation of all HSP90
clients, contributing to the main reason for the unsatisfied clinical
results of HSP90 ATPase inhibitors. Because of the limited devel-
opment progress of HSP90 ATPase inhibitors, alternative inhibition
strategies to target HSP90 chaperone cycle were emerging. In order
to achieve the specific inhibition of HSP90 clients, researchers
began to develop HSP90-CDC37 PPI inhibitor with no ATPase in-
hibition for selectively blocking the kinase maturation in recent
years [24e28].
Celastrol was the first natural derived product to exhibit HSP90-
CDC37 PPI inhibition activity by a covalently binding mechanism to
CDC37 [27]. Afterward, many other natural derived products were
discovered with covalent binding mechanism (including With-
aferin A (WA), FW-04-806 and Kongensin A (KA)) [29e31]. With
the help of crystal structure of HSP90-CDC37, firstly reported in
2004, the rational design of small molecular inhibitors targeting
HSP90-CDC37 PPI became feasible [32]. However, the research of
HSP90 PPI inhibitor stays in a primary stage and few small mo-
lecular inhibitors were reported. In 2018, DCZ3112 was reported as
2

Q. Zhang, X. Wu, J. Zhou et al.
European Journal of Medicinal Chemistry xxx (xxxx) xxx
a HSP90-CDC37 PPI inhibitor with ideal antitumor effects, while the
unknown binding site of DCZ3112 limited its further development
and optimization [33]. In 2019, our group firstly discovered a lead
compound (compound 11) by a multiple screening strategy tar-
geting the key interaction region between HSP90 and CDC37 (the
most critical polar interaction site for HSP90-CDC37 PPI was
confirmed as Glu47). Its structural optimization product, DDO-
5936, was identified as the first small molecule inhibitor of HSP90-
CDC37 PPI with distinct binding site (Glu47 on Hsp90) and mo-
lecular mechanism [34]. The discovery of DDO-5936 demonstrated
the feasibility of HSP90-CDC37 PPI inhibition as an effective way to
block the maturation of kinases. Then, we conducted a preliminary
structure-activity relationship study on DDO-5936 and obtained an
optimized compound 18h as previously reported [35]. In this work,
we firstly discovered a hydrophobic pocket centered on Phe213 on
HSP90 which was previously unknown and contributed to the
binding affinity of HSP90-CDC37 PPI inhibitors. We synthesized a
series of hydrophobic substituted inhibitors based on the binding
mode of hit compound 11 to occupy this hydrophobic pocket,
which further confirmed the importance of Phe213. Through a
structure-based optimization of inhibitors targeting this hydro-
phobic region, we have found out the main rules in the design of
compounds to interact with Phe213 on the hydrophobic core.
Finally, our efforts resulted in an optimum compound DDO-5994
2.2. Occupation of hydrophobic pocket lead to optimum compound
DDO-5994 with improved binding affinity and antiproliferative
activity
We introduced different kinds of hydrophobic substitutions on
the A ring to explore the optimal chemical scaffold for the occu-
pation of hydrophobic pocket centered on Phe213, including het-
erocycles, heteroatoms, aliphatic chains and aromatic rings
(Scheme 1). All compounds were determined by BLI (biolayer
interferometry assay) and antiproliferation assay firstly. As shown
in Table 1, DDO-5974 and DDO-5975 loss activity completely,
indicating that heterocycles like imidazole ring and thiophene ring
are not suitable for this pocket. DDO-5976~DDO-5980 exhibited no
binding affinity by BLI assay, indicating that halogen heteroatom
substitutions and cyano group are not suitable for this region either.
Subsequent result revealed DDO-5981 (KD
¼
18.4
mM,
IC50 ¼ 13.7 M) and DDO-5982 (KD ¼ 11.5 M, IC50 ¼ 19.3
m
m
mM) with
moderate binding affinity and antiproliferative activity. The binding
mode of DDO-5981(Koff ¼ 0.112 sꢀ1) showed that biphenyl substi-
tution could not entirely enter the hydrophobic pocket because of
the structural rigidity, accounting for the more rapid dissociation
rate comparing with DDO-5982 (Koff ¼ 0.0366 sꢀ1, Fig. 3A and D).
MD simulation revealed that the non-polar interaction with Phe213
was enhanced but the polar interactions between DDO-5981 and
HSP90 were reduced sharply (interaction with Glu47, Ser50,
Asn51), which accounted for the limitation of the KD value (Fig. 3B,
C, 3D). DDO-5982 with propyl substitution exhibited similar
binding mode with the above compound (Fig. 3A). MD simulation
shows that DDO-5982 exhibited no strong interaction with key
residue (Glu47, Ser50, Asn51, Phe213), which implied that there is
still capacity to be occupied (Fig. 3B and C).
with improved binding affinity (KD ¼ 5.52
activity (IC50 ¼ 6.34 M) by cyclizing linear aliphatic chain and
introducing an oxygen atom (compared to hit compound 11,
D > 20 M and IC50 > 50 M). Both in vitro and in vivo experiments
mM) and antiproliferative
m
K
m
m
were carried out to confirm the efficacy of DDO-5994 as a prom-
ising HSP90-CDC37 PPI inhibitor. In addition, our results also pro-
vided a novel binding site on Phe213 as a hydrophobic core for the
further design of HSP90-CDC37 PPI inhibitors.
Based on the above results, we subsequently changed benzene
rings into phenyl ether (DDO-5983) or benzyl ether (DDO-5984) to
reduce the structural rigidity for enhanced binding affinity. Disap-
pointingly, DDO-5983 and DDO-5984 lost binding affinity
completely as shown in Table 2. Above results suggested that it
might be too large for biphenyl substitution to enter the hydro-
phobic pocket properly. Next, we explored how long and which
type of aliphatic chain could match the hydrophobic pocket
appropriately. As shown in Table 2, substitutions with 1e5 carbon
aliphatic chain were synthesized and C4 (DDO-5988) showed
better binding affinity and antiproliferative activity. Compared the
compounds with branched chains (DDO-5987, DDO-5991,
2. Results and discussion
2.1. Molecular docking and MD simulation revealed a hydrophobic
pocket involving Phe213 on HSP90-compound 11 interaction surface
Our previous study discovered hit compound 11, which dis-
rupted HSP90-CDC37 PPI moderately through a direct binding
manner on HSP90 (KD ¼ 21.1
mM, inhibition rate ~56% at 100 mM)
[34]. Based on the binding mode of HSP90-compound 11, we
performed molecular docking and molecular dynamic (MD) simu-
lation to explore the characters of binding modes during the dy-
namic trajectory. Besides Glu47, Ser50 and Asn51, which were
previously reported as important polar sites for the binding of
HSP90-CDC37, energy decomposition revealed a hydrophobic res-
idue, Phe213, also contributed to the main driving force for the
interaction between HSP90 and compound 11, which was previ-
ously unknown (Fig. 2B). This hydrophobic pocket contributed to
the majority of hydrophobic forces adjacent to the binding surface
of compound 11 which might be an ideal site for small molecule
design. We supposed that occupation of this pocket would enhance
the binding affinity between HSP90 and inhibitors by introducing
hydrophobic groups. Based on this hypothesis, we divided the
structure of compound 11 into three parts, including A ring
(adjacent hydrophobic pocket), sulfamide part, and pyrimidine
substituents (forms hydrophilic interaction). In previous studies, by
changing pyrrole ring to piperazine ring in pyrimidine substituents
(18h) and forming hydrochloride, the hydrophilic interaction is
greatly improved and the physical properties were also well opti-
mized [35]. Here, our efforts are committed to increase the hy-
drophobic interactions. Thus, we conserved piperazine ring and
focused on A ring by employing hydrophobic substituted groups to
occupy the pocket involving Phe213 (Fig. 2C).
KD > 100
with linear chain (DDO-5982) exhibited advantages on binding
affinity and antiproliferative activity (KD ¼ 11.5 M, IC50 ¼ 19.3 M).
mM or NA, IC50 > 20 or NA), the compound substituted
m
m
Thus, we introduced an ester group with four atoms in the main
chain (DDO-5990, KD ¼ 5.13
m
M,Kon ¼ 3.55 ꢁ 104 Msꢀ1) and it
exhibited improved binding affinity compared to DDO-5988
(KD ¼ 11.6
m
M,Kon ¼ 1.04 ꢁ 104 Msꢀ1) with more rapid association
rate. Molecular docking and MD simulation show that the ester
group enters the hydrophobic pocket deeply and forms a strong
hydrophobic interaction with Phe213. However, the binding
pattern of DDO-5990 changed the conformation of skeleton,
resulting in the loss of binding energy with Glu47 (Fig. 3A, B, 3C). So
far, all the results show that the four atoms length is the most
suitable for the hydrophobic pocket and the ester group may be
beneficial to enhance the binding affinity.
In order to prevent the conformational change of DDO-5990, we
converted linear chain into loop to reduce the entropy loss. We
designed and synthesized DDO-5992~DDO-5994 by cyclization to
decrease the flexibility of n-butyl. As shown in Table 3, DDO-5994
showed the optimal results both in binding affinity and anti-
proliferation activity (KD ¼ 5.52
m
M, IC50 ¼ 6.34
mM). According to
the results of molecular docking and molecular dynamic simulation
3

Q. Zhang, X. Wu, J. Zhou et al.
European Journal of Medicinal Chemistry xxx (xxxx) xxx
Fig. 2. Binding mode analysis of HSP90-Compound 11 and design of hydrophobic substituted derivatives. (A) Binding mode of compound 11. (B) Energy decomposition of
Hsp90-compound 11 interaction. (C) Optimization strategies and compound design.
(Fig. 3), DDO-5994 occupied the hydrophobic pocket in an appro-
priate posture and formed strong interactions with key residues of
HSP90 (Glu47, Ser50, Asn51, Phe213). Comparing with aforemen-
tioned compounds, DDO-5994 interacted with HSP90 more
steadily (Fig. S1).
So far, some conclusions have been drawn from the process of
hydrophobic occupation. Halogen atoms and heterocycles (like
imidazole ring and thiophene ring) are taboos in this pocket, which
will lead to the complete loss of activity of compounds; biphenyl
substitution is too bulky to fit this pocket; four atoms are the op-
timum size to occupy the pocket, less than 4 atoms cannot form
strong interaction with Phe213, and more than 4 atoms cannot
enter the pocket in a proper posture; linear aliphatic chain can
Scheme 1. Synthetic route of compounds DDO-5974~DDO-5994. Reagents and con-
ditions: (a). 2,4-Dichloropyrimidine, i-PrOH, TEA, reflux; (b). TFA, DCM, rt.; (c). 1-Boc-
piperazine, DMF, K2CO3, 100 ꢂC; (d). Substituted benzenesulfonyl chloride, DCM, TEA,
rt; (e). 36% HCl aqueous, EA, rt. (f). 1 M isopropanol hydrochloride solution, EA, rt.
4

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European Journal of Medicinal Chemistry xxx (xxxx) xxx
Table 1
SARs of different types of hydrophobic substitutions on A ring.
Entry
1
Compound
R
KD
(m
M)<
Kon (1/Ms)
Koff (1/s)
IC50
NA
(mM)
DDO-5974
NA
NA
NA
2
3
DDO-5975
DDO-5976
NA
NA
NA
NA
NA
NA
>20
>20
4
5
DDO-5977
DDO-5978
NA
NA
NA
NA
NA
NA
>20
11.1 1.24
6
7
DDO-5979
DDO-5980
NA
NA
NA
NA
NA
NA
1.35 0.08
>20
8
9
DDO-5981
DDO-5982
18.4
11.5
6.10 ꢁ 103
3.17 ꢁ 103
0.112
13.7 1.86
19.3 4.85
0.0366
cause a great change in the conformation of compounds and reduce
the key polar interaction with Glu47 sharply. According to the
above process, we obtained the optimum compound DDO-5994
shown in Fig. 4C, kinase clients of HSP90 including CDK4 and CDK6,
were down-regulated significantly by DDO-5994 in
a
concentration-dependent manner, which led to the down-
regulation of cyclin D1 and the upregulation of p21/p27. Similar
with our previous results, HSP70 was remained on a constant level
which indicated to no risk of potential heat shock reaction (HSR).
All the above results indicated that DDO-5994 directly bound to
HSP90 and efficiently disrupted HSP90-CDC37 PPI in cells and
induced kinase degradation.
with moderate binding affinity (KD ¼ 5.52
mM) and antiproliferative
activity (IC50 ¼ 6.34 M).
m
2.3. DDO-5994 directly bound to Hsp90 and efficiently disrupted
HSP90-CDC37 PPI in cells and induced kinase degradation
To confirm the mechanism of DDO-5994, we firstly conducted a
cellular thermal shift assay (CESTA) to determine the binding affinity
2.4. DDO-5994-induced cell cycle arrest
in cells. As shown in Fig. 4A, with the treatment of 5 mM DDO-5994
The clients of Hsp90-Cdc37, CDK4 and CDK6, play important
roles in cell cycle process [36e38]. Based on the above results
(Fig. 4), we subsequently conducted a cell cycle distribution anal-
ysis following a standard flow cytometry method. HCT116 cells
were treated with DDO-5994 in different concentrations (0, 1, 5,
or DMSO for 12 h in HCT116 cells with temperature ranging from 35
to 65 ꢂC, DDO-5994 could stabilize HSP90 rather than CDC37 under
the high temperature environment by Western blot (WB) analysis,
indicating a direct binding manner of DDO-5994 to HSP90 in cells.
Subsequently, we operated a co-immunoprecipitation (co-IP) assay
to determine the ability of DDO-5994 as a HSP90-CDC37 PPI inhib-
itor. As shown in Fig. 4B, after the treatment with DDO-5994 (0, 1, 5,
and 10 mM) for 24 h and the DNA was stained with propidium io-
dide. As shown in Fig. 5, the proportion of HCT116 cells in G0/G1
phase was increased after the treatment of DDO-5994, which
indicating that DDO-5994 could inhibit cell cycle progression by
arresting in G0/G1 phase.
and 10 mM) in HCT116 cells, we immunoprecipitated Hsp90 to detect
the bait proteins. It was clearly observed that DDO-5994 disrupt
HSP90-CDC37 PPI in a concentration-dependent manner by quan-
titative analysis. Because of the block of HSP90-CDC37 PPI, CDK4, a
representative kinase client protein of HSP90 was decreased as ex-
pected (Fig. 4B).
2.5. DDO-5994 exhibits potency and safety in vivo
To further investigate the effect of DDO-5994 to the down-
stream kinases of HSP90, we carried out Western blot experiments
to determine the representative client proteins of HSP90-CDC37. As
As shown in Fig. 6, we evaluated the antitumor efficacy of DDO-
5994 in mice bearing HCT116 xenografts tumors. DDO-5994 and
18h were administrated daily for 37 days at 10 mg/kg and 20 mg/kg,
5

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European Journal of Medicinal Chemistry xxx (xxxx) xxx
Fig. 3. Overview of the interactions and binding affinities between key compounds and HSP90. (A) Predicted binding modes of key compounds by molecular docking. (B)
Timeline representation of the interactions and contacts (H-bonds, Hydrophobic, Ionic, Water bridges). (C) Simulation Interactions Diagram. Protein-ligand interactions are
categorized into four types: Hydrogen Bonds, Hydrophobic, Ionic and Water Bridges. (D) Binding affinity of key compounds with HSP90 protein, determined by binding kinetic assay
of biolayer interferometry (ForteBio Octet).
respectively. DDO-5994 and 18h were both tolerated with no
weight loss (Fig. 6A). DDO-5994 inhibited the growth of tumor in a
dose-dependent manner and exhibited increasing potency than
18h in the high dose group, which was characterized by tumor
volume and weight. The result of antitumor efficacy experiment
was consistence with the data reported previously [35].
Hematoxylin eosin (H&E) staining analysis showed consistent re-
sults and confirmed that DDO-5994 exhibited in vivo anti-
proliferation efficacy (Fig. 6D). Compared to the group of 18h, CDK4
was observably decreased in DDO-5994 group with a dose-
dependent manner indicated by immunohistochemical staining.
In brief, DDO-5994 was proved a better HSP90-CDC37 PPI inhibitor
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European Journal of Medicinal Chemistry xxx (xxxx) xxx
Table 2
SARs of different types of biphenyl and aliphatic chains substitutions on A ring.
Entry
1
Compound
R
KD
(
mM)
Kon (1/Ms)
Koff (1/s)
IC50 (mM)
DDO-5983
NA
NA
NA
>20
2
DDO-5984
NA
NA
NA
7.05 1.20
3
4
5
DDO-5985
DDO-5986
DDO-5987
NA
NA
NA
NA
e
NA
NA
e
>20
>20
>20
>100
6
7
DDO-5988
DDO-5989
11.6
1.04 ꢁ 104
0.121
7.50 1.60
16.7 2.73
>100
e
e
8
9
DDO-5990
DDO-5991
5.13
3.55 ꢁ 104
0.182
16.4 3.15
NA
NA
NA
>20
with more potent antitumor efficacy and good tolerance by the
introduction of hydrophobic substituted groups.
introduced diverse type of hydrophobic groups to A ring of com-
pound 11 to enhance hydrophobic interaction between compound
and HSP90 and constantly adjusted the optimization strategy ac-
cording to real-time results. Some rules were revealed by adapting
the hydrophobic pockets with diverse hydrophobic groups. Halogen
atoms and heterocycles (like imidazole ring and thiophene ring) are
taboos in this pocket, which will lead to the complete loss of activity
of compounds; biphenyl substitution is too bulky to fit this pocket;
linear aliphatic chains are more adapted than branch aliphatic chains
and four atoms are the optimum size to occupy the pocket, less than
4 atoms cannot form strong interaction with Phe213, and more than
4 atoms cannot enter the pocket in a proper posture; linear aliphatic
chain can cause a great change in the conformation of compounds
and reduce the key polar interaction with Glu47 sharply; ester group
with four atoms in main chain is beneficial to binding affinity. In
order to prevent the conformational change of skeleton caused by
long linear aliphatic chain, we decreased the flexibility of linear
3. Conclusions
PPIs play important roles in the function of cells. Specific mod-
ulation of PPIs is a promising strategy to regulate the function of cells
preciously and achieve therapy goals. HSP90-CDC37 PPI plays a key
role in the maturation of kinases that related to cancer closely.
Comparing with HSP90 ATPase inhibitor, HSP90-CDC37 PPI inhibitor
has more specificity to avoid side effects like HSR. In this study, we
discovered a hydrophobic pocket centered on Phe213 based on
HSP90-compound 11 binding mode by molecular docking and MD
simulation. Phe213 was previously unknown and contributed to the
binding affinity of HSP90-CDC37 PPI inhibitors. We supposed the
hydrophobic pocket where Phe213 located was a breakthrough to
improve binding affinity and antiproliferative efficacy. We
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European Journal of Medicinal Chemistry xxx (xxxx) xxx
Table 3
SARs of different types of cyclic aliphatic chain substitutions on A ring.
Entry
1
Compound
R
KD
(
mM)
Kon (1/Ms)
3.02 ꢁ 103
Koff (1/s)
IC50 (mM)
DDO-5992
10.6
9.52
5.52
0.0321
5.82 1.08
13.4 3.78
6.34 2.67
2
3
DDO-5993
DDO-5994
3.54 ꢁ 104
2.03 ꢁ 104
0.337
0.112
4
Compound 11
>20
e
e
>50
Fig. 4. DDO-5994 disrupted the Hsp90-Cdc37 PPI in cells through directly binding to Hsp90, and down-regulated kinase clients of Hsp90 without HSR. (A). CETSA of
HCT116 cells treated with DMSO or 5 M of DDO-5994 for 12 h. Data are the representative of three independent experiments. (B). co-IP assay of DDO-5994 (concentrations of the
compound increasing from 0, 1, and 5e10 M) in HCT116 cells. DDO-5994 efficiently blocked Hsp90-Cdc37 PPI through a dose-dependent manner, leading to the dissociation of
CDK4 from the Hsp90-Cdc37 complex. Western blots were performed with anti-Hsp90, anti-Cdc37, or anti-CDK4 in each experiment. (C). Dose-dependent Western blot analysis of
m
m
Hsp90, Hsp70, Cdc37, CDK4, CDK6, cyclin D1, p21, and p27 protein expression levels in HCT116 cells after treatment with 0, 1, 5, and 10
loading control. Data are the representative of three independent experiments.
mM DDO-5994 for 12 h. b-Actin was used as a
aliphatic chain by cyclization strategy and introduction of oxygen
atom to obtain DDO-5994 finally. In vitro and in vivo assays
confirmed that DDO-5994 directly bound to HSP90 and disrupted
HSP90-CDC37 interaction, thus to block kinases maturation. DDO-
5994 exhibited antiproliferative activity by down-regulating CDK4/6
to cause cell cycle arrest. In vivo assay also revealed DDO-5994 with
improved antiproliferation activity and safety. In this study, it was
proved that Phe213 could be regarded as a suitable hydrophobic
binding site for the design of small molecule inhibitors targeting
HSP90-CDC37 PPI. In addition, DDO-5994 was achieved with
improved in vivo efficacy, providing new options for the further
design of HSP90-CDC37 inhibitors.
Comparing with HSP90 ATPase inhibitors, HSP90-CDC37 PPI
inhibitors exhibit significant advantages in the mechanism with
8

Q. Zhang, X. Wu, J. Zhou et al.
European Journal of Medicinal Chemistry xxx (xxxx) xxx
Fig. 5. DDO-5994 induced cell cycle arrest in HCT116 cells. (A) Cell cycle distribution determined by PI staining of HCT116 cells after treatment with 0, 1, 5, and 10 mM of DDO-5994
for 24 h, respectively. (B) Graphic histogram of cell cycle distribution.
specificity to kinase maturation. The study of HSP90-CDC37 PPI
inhibitors is in primary stage and further research may focused on
following directions: (1) discovering small molecules directly
binding with CDC37 to disrupt HSP90-CDC37 PPI; (2) improving the
druggability of existing compounds in terms of physical and
chemical properties; (3) HSP90-CDC37 PPI modulators play an
important role in the regulation of HSP90-CDC37-kinase cycle and
might be used as chemical probes to study the mechanism of
HSP90-CDC37 chaperone cycle for kinase recruitment, which help
to discovery more efficient HSP90-CDC37 PPI inhibitors for the
cancer therapy; (4) with the different mechanism to inhibit kinase,
HSP90-CDC37 PPI inhibitors might be used in combination with
other kinase inhibitors to achieve more potential cancer therapy
effects.
product. The crude product purified by silica gel chromatography
(PE-EA, 4:1), yielding tert-butyl (4-((2-chloropyrimidin-4-yl)
amino)phenyl)carbamate as light yellow solid (6.6 g, 85%). tert-
butyl (4-((2-chloropyrimidin-4-yl)amino)phenyl)carbamate (10 g,
31.2 mmol) was dissolved in DCM(60 ml) followed by TFA (20 ml).
The mixture was stirred at room temperature for 4 h. Then satu-
rated NaHCO3 aqueous was added to adjust the pH to 8e9.
Collected organic layer and dry over anhydrous Na2SO4 and evap-
orated under reduce pressure to give compound 2 as off-white solid
(6.8 g, 99%).1H NMR (300 MHz, DMSO‑d6)
d 9.63 (s, 1H), 8.02 (d,
J ¼ 5.9 Hz, 1H), 7.23e7.05 (m, 2H), 6.65 (d, J ¼ 8.7 Hz, 2H), 6.54 (d,
J ¼ 6.0 Hz, 1H). HRMS (ESI): found 221.05887 (C10H9ClN4 [M þ H]þ,
requires 221.05885).
4.2.2. Tert-butyl 4-(4-((4-aminophenyl)amino)pyrimidin-2-yl)
piperazine-1-carboxylate (3)
4. Experimental section
Compound 2 (3.5 g, 15.9 mmol), 1-Boc-piperazine (3.5 g,
18.8 mmol) were dissolved in dry DMF (20 ml) followed by dry
K2CO3 (4.38 g, 31.7 mmol). The reaction mixture was stirred at
100 ꢂC under nitrogen gas for overnight. After completion of the
reaction, the mixture was cool to room temperature and water
(230 ml) was added to the mixture by drops to precipitate the
product. Filtered and cake was dried in a vacuum desiccator before
washed with water (20 ml). Compound 3 was afforded as purple
4.1. Materials and methods
Unless otherwise mentioned, all of the commercial reagents
were reagent grade and directly used without further purification.
All the reactions were visualized using UV light at 254 nm and
monitored by thin layer chromatography (TLC) on 0.25 mm silica
gel plates (GF-2.5). 1H NMR and 13C NMR spectra were tested by
Bruker AV300 instrument with tetramethylsilane as the standard to
report chemical shifts. ESI-MS and high-resolution mass spectra
(HRMS) results of all the compounds were recorded on a Waters Q-
Tof micro mass spectrometer (all data were within 0.4% of the
theoretical values). The purity (ꢃ95%) of the compounds was
brown solid (5.8 g, 98%). 1H NMR (300 MHz, DMSO‑d6)
d 8.78 (s,
1H), 7.84 (d, J ¼ 5.7 Hz, 1H), 7.17 (d, J ¼ 8.1 Hz, 2H), 6.56 (dd,
J ¼ 8.7 Hz, 2H), 5.90 (d, J ¼ 5.8 Hz, 1H), 4.89 (s, 2H), 3.66 (dd, J ¼ 6.9,
3.7 Hz, 4H), 3.39 (dd, J ¼ 7.2 Hz, 4H), 1.45 (s, 9H). HRMS (ESI): found
371.22013 (C19H26N6O2 [M þ H]þ, requires 371.219).
determined by the HPLC on
(4.6 mm ꢁ 150 mm, 3.5 m) using a mixture of solvent containing
methanol/methanol with 1 TEA at a flow rate of 1.0 mL/min and
a
Waters C18 column
m
4.2.3. General procedure for the preparation of compounds DDO-
5974~DDO-5994
‰
the peaks were determined at 254 nm under UV. The HPLC chro-
matograms and 1H NMR, 13C NMR and HRMS spectra of the new
compounds are presented in supporting information.
Compound 3 (350 mg, 0.9 mmol), TEA (0.025 ml,1.8 mmol) were
dissolved in DCM (15 ml) followed adding substituted benzene-
sulfonyl chloride (0.8 mmol, dissolved in DCM, 3 ml) by drops at
room temperature. The mixture was stirred for overnight and then
evaporated under reduce pressure. The crude residue purified by
silica gel chromatography (PE-EA, 4:1). Compound with Boc group
was obtained as light yellow solid and then was dissolved in EA
(20 ml) followed by adding 36% HCl aqueous (4 ml). The mixture
was stirred at room temperature for 2 h and solvent was adjusted to
pH ¼ 7e8 by saturated sodium bicarbonate solution. Organic layer
was dried by anhydrous sodium sulfate and evaporated under
reduce press to get free compounds. Then free compound was
dissolved in EA followed 1 M isopropanol hydrochloride solution (1
eq) at room temperature for 1 h. The residue crystalized from EA to
give DDO-5974~DDO-5994 finally.
4.2. Chemistry
4.2.1. N1-(2-chloropyrimidin-4-yl)benzene-1,4-diamine (2)
Tert-butyl (4-aminophenyl)carbamate (5.0 g, 24.0 mmol), 2,4-
dichloropyrimidine (7.2 g, 48.3 mmol) were dissolved in i-PrOH
(80 ml), followed by TEA (13 ml, 93.8 mmol). The mixture was
reflux overnight. After completion of the reaction, the mixture was
cool to room temperature and solvent was removed by evaporation
under reduce pressure. Then ethyl acetate (50 ml) was added to
residues and stirred in room temperature for 30 min. Filtered and
the solvent was evaporated under reduce pressure to give crude
9

Q. Zhang, X. Wu, J. Zhou et al.
European Journal of Medicinal Chemistry xxx (xxxx) xxx
Fig. 6. DDO-5994 dose-dependently impaired the growth of xenografted HCT116 cells in immunocompromised mice and safety in vivo. (A) In vivo antitumor efficacy of DDO-
5994 and 18h in the HCT116 xenograft model. Mice were administered with DDO-5994 (0, 10, and 20 mg/kg, respectively) and 18h (20 mg/kg) daily by gavage starting 7 days after
cell implantation. Effects on body weight changes of the mice treated with DDO-5994 and 18h. (B) Tumor volumes were determined by calipers. The tumor volumes are plotted as
the mean SD. (*P < 0.05; **P < 0.01; ***P < 0.001, n ¼ 6). (E) H&E staining showed the tumor tissue morphology of different groups with DDO-5994 and 18h. Scale bars were
represented as 300 mm. The expression levels of CDK4 were analyzed in tumor tissues by immunohistochemistry.
4.2.4. Characterization data for final compounds DDO-5974~DDO-
5994
4.2.4.1. 1-Methyl-N-(4-((2-(piperazin-1-yl)pyrimidin-4-yl)amino)
phenyl)-1H-imidazole-4-sulfonamide hydrochloride (DDO-5974).
It was obtained in 35% yield as off-white solid; 1H NMR (300 MHz,
1H), 3.58 (d, J ¼ 5.0 Hz, 4H), 2.71 (t, J ¼ 5.0 Hz, 4H). HRMS (ESI):
found 503.18567 (C26H27N6O3S [M þ H]þ, requires 503.18599).
4.2.4.3. 4-Cyano-N-(4-((2-(piperazin-1-yl)pyrimidin-4-yl)amino)
phenyl) benzenesulfonamide hydrochloride (DDO-5976). It was ob-
tained in 33% yield as off-white solid;1H NMR (300 MHz, DMSO‑d6)
DMSO‑d6)
d
11.19 (s, 1H),10.27 (s,1H), 9.69 (s, 3H), 7.86 (d, J ¼ 7.1 Hz,
1H), 7.82 (s, 1H), 7.77 (s, 1H), 7.53 (d, J ¼ 7.0 Hz, 4H), 7.19 (d,
J ¼ 8.6 Hz, 3H), 6.54 (s, 1H), 4.03 (t, J ¼ 4.7 Hz, 4H), 3.66 (s, 4H), 3.25
(s, 8H). HRMS (ESI): found 415.16595 (C18H22N8O2S [M þ H]þ, re-
quires 415.16592).
d
9.16 (s, 1H), 8.01 (d, J ¼ 8.5 Hz, 2H), 7.93e7.77 (m, 3H), 7.46 (d,
J ¼ 8.9 Hz, 2H), 6.97 (d, J ¼ 8.9 Hz, 2H), 5.97 (d, J ¼ 5.7 Hz, 1H), 3.62
(t, J ¼ 5.0 Hz, 4H), 2.78 (d, J ¼ 5.0 Hz, 4H). HRMS (ESI): found
436.15469 (C21H21N7O2S [M þ H]þ, requires 436.15502).
4.2.4.2. N-(4-((2-(piperazin-1-yl)pyrimidin-4-yl)amino)phenyl)thio-
4.2.4.4. 4-Fluoro-N-(4-((2-(piperazin-1-yl)pyrimidin-4-yl)amino)
phenyl) benzenesulfonamide hydrochloride (DDO-5977). It was ob-
tained in 38% yield as off-white solid; 1H NMR (300 MHz, DMSO‑d6)
phene-2-sulfonamide hydrochloride (DDO-5975). It was obtained in
36% yield as off-white solid;1H NMR (300 MHz, DMSO‑d6)
d 9.15 (s,
1H), 7.87 (d, J ¼ 5.6 Hz, 1H), 7.70 (d, J ¼ 8.9 Hz, 2H), 7.53e7.37 (m,
d
11.04 (s, 1H), 10.41 (s, 1H), 9.59 (s, 2H), 7.89 (d, J ¼ 6.8 Hz, 1H),
4H), 7.23 (t, J ¼ 7.4 Hz, 1H), 7.11e6.97 (m, 6H), 5.95 (d, J ¼ 5.6 Hz,
7.86e7.74 (m, 2H), 7.55 (d, J ¼ 8.0 Hz, 3H), 7.47e7.36 (m, 2H), 7.15 (d,
10

Q. Zhang, X. Wu, J. Zhou et al.
European Journal of Medicinal Chemistry xxx (xxxx) xxx
J ¼ 8.9 Hz, 2H), 6.49 (d, J ¼ 6.8 Hz, 1H), 4.00 (t, J ¼ 5.2 Hz, 4H), 3.24
(s, 4H). HRMS (ESI): found 429.15058 (C20H21FN6O2S [M þ H]þ,
requires 429.15035).
H]þ, requires 517.20164).
4.2.4.12. 4-Methyl-N-(4-((2-(piperazin-1-yl)pyrimidin-4-yl)amino)
phenyl) benzenesulfonamide hydrochloride (DDO-5985). It was ob-
tained in 35% yield as off-white solid; 1H NMR (300 MHz, DMSO‑d6)
4.2.4.5. 4-Chloro-N-(4-((2-(piperazin-1-yl)pyrimidin-4-yl)amino)
phenyl) benzenesulfonamide hydrochloride (DDO-5978). It was ob-
tained in 36% yield as off-white solid; 1H NMR (300 MHz, DMSO‑d6)
d
11.02 (s, 1H), 10.31 (s, 1H), 9.59 (s, 2H), 7.88 (d, J ¼ 7.0 Hz, 1H),
7.71e7.56 (m, 2H), 7.52 (d, J ¼ 8.5 Hz, 2H), 7.34 (d, J ¼ 8.1 Hz, 2H),
7.22e7.03 (m, 2H), 6.48 (d, J ¼ 7.0 Hz, 1H), 4.00 (t, J ¼ 5.3 Hz, 4H),
3.24 (s, 4H), 2.33 (s, 3H). HRMS (ESI): found 425.17507
(C21H24N6O2S [M þ H]þ, requires 425.17542).
d
10.46 (s,1H), 9.56 (s, 2H), 7.89 (d, J ¼ 6.8 Hz,1H), 7.75 (d, J ¼ 8.7 Hz,
2H), 7.64 (d, J ¼ 8.7 Hz, 2H), 7.55 (d, J ¼ 8.5 Hz, 2H), 7.14 (d,
J ¼ 8.9 Hz, 2H), 6.47 (d, J ¼ 6.8 Hz,1H), 4.00 (t, J ¼ 5.1 Hz, 4H), 3.24 (s,
4H). HRMS (ESI): found 445.12065 (C20H21ClN6O2S [M þ H]þ, re-
quires 445.1208).
4.2.4.13. 4-Ethyl-N-(4-((2-(piperazin-1-yl)pyrimidin-4-yl)amino)
phenyl) benzenesulfonamide hydrochloride (DDO-5986). It was ob-
tained in 30% yield as off-white solid; 1H NMR (300 MHz, DMSO‑d6)
4.2.4.6. 4-Bromo-N-(4-((2-(piperazin-1-yl)pyrimidin-4-yl)amino)
phenyl) benzenesulfonamide hydrochloride (DDO-5979). It was ob-
tained in 38% yield as off-white solid; 1H NMR (300 MHz, DMSO‑d6)
d
11.06 (s, 1H), 10.34 (s, 1H), 9.61 (s, 2H), 7.88 (d, J ¼ 7.1 Hz, 1H), 7.68
(d, J ¼ 8.3 Hz, 2H), 7.54 (d, J ¼ 8.8 Hz, 2H), 7.38 (d, J ¼ 8.3 Hz, 2H),
7.15 (d, J ¼ 8.9 Hz, 2H), 6.49 (d, J ¼ 6.6 Hz, 1H), 4.01 (s, 4H), 3.24 (s,
4H), 2.64 (q, J ¼ 7.6 Hz, 2H), 1.15 (t, J ¼ 7.6 Hz, 3H). HRMS (ESI):
found 439.19062 (C22H26N6O2S [M þ H]þ, requires 439.19107).
d
10.48 (s, 1H), 9.61 (s, 2H), 7.89 (d, J ¼ 7.0 Hz,1H), 7.78 (d, J ¼ 8.7 Hz,
2H), 7.67 (d, J ¼ 8.7 Hz, 2H), 7.56 (d, J ¼ 8.4 Hz, 2H), 7.15 (d,
J ¼ 8.9 Hz, 2H), 6.50 (d, J ¼ 7.0 Hz,1H), 4.01 (t, J ¼ 5.5 Hz, 4H), 3.25 (s,
4H). HRMS (ESI): found 491.06796 (C20H21BrN6O2S [M þ H]þ, re-
quires 489.07028).
4.2.4.14. 4-Isopropyl-N-(4-((2-(piperazin-1-yl)pyrimidin-4-yl)
amino)phenyl) benzenesulfonamide hydrochloride (DDO-5987).
It was obtained in 37% yield as off-white solid; 1H NMR (300 MHz,
4.2.4.7. N-(4-((2-(piperazin-1-yl)pyrimidin-4-yl)amino)phenyl)-4-
(trifluoromethoxy) benzenesulfonamide hydrochloride (DDO-5980).
It was obtained in 41% yield as off-white solid; 1H NMR (300 MHz,
DMSO‑d6)
d 11.08 (s, 0H), 10.38 (s, 1H), 9.64 (s, 2H), 7.90 (d,
J ¼ 7.0 Hz, 1H), 7.72 (d, J ¼ 8.3 Hz, 2H), 7.57 (d, J ¼ 8.5 Hz, 2H), 7.44
(d, J ¼ 8.3 Hz, 2H), 7.19 (d, J ¼ 8.9 Hz, 2H), 6.51 (d, J ¼ 7.0 Hz, 1H),
4.04 (t, J ¼ 5.1 Hz, 4H), 3.27 (s, 4H), 2.96 (p, J ¼ 6.8 Hz, 1H), 1.20 (d,
J ¼ 6.9 Hz, 6H). HRMS (ESI): found 453.20629 (C23H28N6O2S [M þ
H]þ, requires 453.20672).
DMSO‑d6)
d 11.05 (s, 0H), 10.53 (s, 1H), 9.59 (s, 1H), 7.89 (d,
J ¼ 8.1 Hz, 2H), 7.57 (d, J ¼ 8.3 Hz, 3H), 7.16 (d, J ¼ 8.9 Hz, 1H), 6.49
(d, J ¼ 7.0 Hz, 1H), 4.01 (d, J ¼ 5.1 Hz, 4H), 3.24 (s, 4H). HRMS (ESI):
found 495.14170 (C21H21F3N6O3S [M þ H]þ, requires 495.14207).
4.2.4.8. N-(4-((2-(piperazin-1-yl)pyrimidin-4-yl)amino)phenyl)-
[1,10-biphenyl]-4-sulfonamide hydrochloride (DDO-5981). It was
obtained in 40% yield as off-white solid; 1H NMR (300 MHz,
4.2.4.15. 4-Butyl-N-(4-((2-(piperazin-1-yl)pyrimidin-4-yl)amino)
phenyl) benzenesulfonamide hydrochloride (DDO-5988). It was ob-
tained in 39% yield as off-white solid; 1H NMR (300 MHz, DMSO‑d6)
DMSO‑d6)
d
9.17 (s, 1H), 7.93 (d, J ¼ 5.6 Hz, 1H), 7.91e7.81 (m, 4H),
d
9.16 (s, 1H), 7.93 (d, J ¼ 5.6 Hz, 1H), 7.67 (d, J ¼ 8.1 Hz, 2H), 7.51 (d,
7.79e7.70 (m, 2H), 7.58e7.43 (m, 5H), 7.10 (d, J ¼ 8.7 Hz, 2H), 6.01
(d, J ¼ 5.7 Hz, 1H), 3.64 (t, J ¼ 5.1 Hz, 4H), 2.78 (t, J ¼ 5.0 Hz, 4H).
HRMS (ESI): found487.19082 (C26H26N6O2S [M þ H]þ, requires
487.19107).
J ¼ 8.8 Hz, 2H), 7.38 (d, J ¼ 8.1 Hz, 2H), 7.04 (d, J ¼ 8.8 Hz, 2H), 6.00
(d, J ¼ 5.6 Hz,1H), 3.63 (t, J ¼ 4.9 Hz, 4H), 2.76 (t, J ¼ 4.9 Hz, 4H), 2.65
(t, J ¼ 7.7 Hz, 2H), 1.66e1.49 (m, 2H), 1.36e1.26 (m, 2H), 0.91 (t,
J ¼ 7.3 Hz, 3H). HRMS (ESI): found 467.22212 (C24H30N6O2S [M þ
H]þ, requires 467.22237).
4.2.4.9. N-(4-((2-(piperazin-1-yl)pyrimidin-4-yl)amino)phenyl)-4-
propylbenzenesulfonamide hydrochloride (DDO-5982). It was ob-
tained in 39% yield as off-white solid; 1H NMR (300 MHz, DMSO‑d6)
4.2.4.16. 4-Pentyl-N-(4-((2-(piperazin-1-yl)pyrimidin-4-yl)amino)
phenyl) benzenesulfonamide hydrochloride (DDO-5989). It was ob-
tained in 32% yield as off-white solid; 1H NMR (300 MHz, DMSO‑d6)
d
9.14 (s, 1H), 7.88 (d, J ¼ 5.7 Hz, 1H), 7.62 (d, J ¼ 8.1 Hz, 2H), 7.47 (d,
J ¼ 8.9 Hz, 2H), 7.33 (d, J ¼ 8.1 Hz, 2H), 6.99 (d, J ¼ 8.9 Hz, 2H), 5.96
(d, J ¼ 5.7 Hz,1H), 3.58 (t, J ¼ 4.8 Hz, 4H), 2.72 (t, J ¼ 4.9 Hz, 4H), 2.57
(t, J ¼ 7.6 Hz, 2H), 1.56 (h, J ¼ 7.4 Hz, 2H), 0.84 (t, J ¼ 7.3 Hz, 3H).
HRMS (ESI): found 453.20688 (C23H28N6O2S [M þ H]þ, requires
453.20672).
d
11.02 (s, 1H), 10.32 (s, 1H), 9.60 (s, 2H), 7.88 (d, J ¼ 7.0 Hz, 1H),
7.69e7.63 (m, 2H), 7.53 (d, J ¼ 8.6 Hz, 2H), 7.39e7.33 (m, 2H),
7.18e7.10 (m, 2H), 6.48 (d, J ¼ 6.9 Hz, 1H), 4.01 (t, J ¼ 4.9 Hz, 4H),
3.23 (t, J ¼ 8.2 Hz, 4H), 2.60 (t, 2H), 1.66e1.44 (m, 2H), 1.29e1.19 (m,
4H), 0.83 (t, J ¼ 6.9 Hz, 3H). HRMS (ESI): found 481.23766
(C25H32N6O2S [M þ H]þ, requires 481.23802).
4.2.4.10. 4-Phenoxy-N-(4-((2-(piperazin-1-yl)pyrimidin-4-yl)amino)
phenyl) benzenesulfonamide hydrochloride (DDO-5983). It was ob-
tained in 33% yield as off-white solid; 1H NMR (300 MHz, DMSO‑d6)
4.2.4.17. Ethyl
4-(N-(4-((2-(piperazin-1-yl)pyrimidin-4-yl)amino)
phenyl)sulfamoyl) benzoate hydrochloride (DDO-5990). It was ob-
d
9.15 (s, 1H), 7.87 (d, J ¼ 5.6 Hz, 1H), 7.70 (d, J ¼ 8.9 Hz, 2H),
tained in 34% yield as off-white solid; 1H NMR (300 MHz, DMSO‑d6)
7.53e7.37 (m, 4H), 7.23 (t, J ¼ 7.4 Hz, 1H), 7.11e6.97 (m, 6H), 5.95 (d,
J ¼ 5.6 Hz, 1H), 3.58 (t, J ¼ 4.9 Hz, 4H), 2.71 (t, J ¼ 5.0 Hz, 4H). HRMS
d
9.17 (s, 1H), 8.08 (d, J ¼ 8.0 Hz, 2H), 7.89 (d, J ¼ 5.7 Hz, 1H), 7.84 (d,
J ¼ 8.1 Hz, 2H), 7.47 (d, J ¼ 8.5 Hz, 2H), 6.99 (d, J ¼ 8.4 Hz, 2H), 5.98
(d, J ¼ 5.7 Hz, 1H), 4.33 (q, J ¼ 7.1 Hz, 2H), 3.62 (t, J ¼ 5.0 Hz, 4H),
2.78 (t, J ¼ 4.9 Hz, 4H), 1.32 (t, J ¼ 7.2 Hz, 3H). HRMS (ESI): found
483.18073 (C23H26N6O4S [M þ H]þ, requires 483.18090).
(ESI): found 221.05887 (C26H26N6O3S [M
221.05885).
þ
H]þ, requires
4.2.4.11. 4-(benzyloxy)-N-(4-((2-(piperazin-1-yl)pyrimidin-4-yl)
amino)phenyl) benzenesulfonamide hydrochloride (DDO-5984).
It was obtained in 34% yield as off-white solid; 1H NMR (300 MHz,
4.2.4.18. 2,4,6-Triisopropyl-N-(4-((2-(piperazin-1-yl)pyrimidin-4-yl)
amino)phenyl) benzenesulfonamide hydrochloride (DDO-5991).
It was obtained in 40% yield as off-white solid; 1H NMR (300 MHz,
DMSO‑d6)
d
9.13 (s, 1H), 7.88 (d, J ¼ 5.6 Hz, 1H), 7.64 (d, J ¼ 8.9 Hz,
2H), 7.53e7.28 (m, 7H), 7.12 (d, J ¼ 8.9 Hz, 2H), 6.99 (d, J ¼ 8.9 Hz,
2H), 5.95 (d, J ¼ 5.7 Hz, 1H), 5.13 (s, 2H), 3.58 (t, J ¼ 5.0 Hz, 4H), 2.71
(t, J ¼ 5.0 Hz, 4H). HRMS (ESI): found 517.20129(C27H28N6O3S [M þ
DMSO‑d6)
d
9.15 (s, 1H), 7.87 (d, J ¼ 5.6 Hz, 1H), 7.48 (d, J ¼ 8.7 Hz,
2H), 7.19 (s, 2H), 6.95 (d, J ¼ 8.6 Hz, 2H), 5.94 (d, J ¼ 5.7 Hz, 1H), 3.57
(t, J ¼ 4.9 Hz, 4H), 2.70 (t, J ¼ 5.3 Hz, 4H), 1.17 (d, J ¼ 6.9 Hz, 6H), 1.12
11

Q. Zhang, X. Wu, J. Zhou et al.
European Journal of Medicinal Chemistry xxx (xxxx) xxx
(d, J ¼ 6.7 Hz, 12H). HRMS (ESI): found 537.30023 (C29H40N6O2S
[M þ H]þ, requires 537.30062).
thawed three times in liquid nitrogen before being centrifuged
and analyzed by Western blot.
4.2.4.19. N-(4-((2-(piperazin-1-yl)pyrimidin-4-yl)amino)phenyl)-
2,3-dihydrobenzofuran-5-sulfonamide hydrochloride (DDO-5992).
It was obtained in 37% yield as off-white solid; 1H NMR (300 MHz,
4.3.5. Co-IP assay
HCT116 cells were seeded in 10 cm cell culture dishes to reach
~90% confluence. Then, the cells and the indicated concentrations
of compounds or the same volume of DMSO as controls were
incubated together for 12 h. After being washed twice with ice cold
PBS, the cells were lysed by radio-immunoprecipitation assay
(RIPA) buffer (50 mM Tris, 150 mM NaCl, 1% NP-40, and protease
inhibitor cocktail, Roche, pH ¼ 7.4) for 1 h on ice, whose lysates
were centrifuged at 12,000 rpm at 4 ꢂC for 15 min. Afterward, 1 mg
DMSO‑d6)
d
9.10 (s, 1H), 7.88 (d, J ¼ 5.6 Hz, 1H), 7.58 (s, 1H), 7.49 (d,
J ¼ 2.3 Hz, 1H), 7.48e7.42 (m, 2H), 7.01 (d, J ¼ 9.0 Hz, 2H), 6.85 (d,
J ¼ 8.4 Hz,1H), 5.96 (d, J ¼ 5.6 Hz,1H), 4.60 (t, J ¼ 8.8 Hz, 2H), 3.60 (t,
J ¼ 5.0 Hz, 4H), 3.20 (t, J ¼ 8.7 Hz, 2H), 2.73 (t, 4H). HRMS (ESI):
found 453.17072 (C22H24N6O3S [M þ H]þ, requires 453.17034).
4.2.4.20. N-(4-((2-(piperazin-1-yl)pyrimidin-4-yl)amino)phenyl)-
2,3-dihydro-1H-indene-5-sulfonamide hydrochloride (DDO-5993).
It was obtained in 35% yield as off-white solid; 1H NMR (300 MHz,
of cell lysates was incubated with 5 mg of anti-Hsp90 (sc-13119,
Santa Cruz Biotechnology), anti-Cdc37 (4793S, Cell Signaling
Technology), anti-CDK4 (ab199728, Abcam), or normal rabbit
immunoglobulin G (IgG) (sc-2027, Santa Cruz Biotechnology)
separately overnight at 4 ꢂC on a vertical roller. Another 3 h incu-
bation was continued after adding protein A/G Magnetic Agarose
Beads (78609, Thermo Fisher Scientific) to the lysates. Lastly, the
beads were washed with RIPA buffer 5 times and then subjected to
SDS-PAGE, followed by Western blot analysis.
DMSO‑d6)
d
9.10 (s, 1H), 7.87 (d, J ¼ 5.6 Hz, 1H), 7.57 (s, 1H), 7.47 (t,
J ¼ 9.5 Hz, 3H), 7.34 (d, J ¼ 7.9 Hz, 1H), 7.01 (d, J ¼ 8.8 Hz, 2H), 5.95
(d, J ¼ 5.7 Hz,1H), 3.58 (t, J ¼ 5.0 Hz, 4H), 2.87 (t, J ¼ 7.4 Hz, 4H), 2.72
(t, J ¼ 5.1 Hz, 4H), 2.09e1.96 (m, 2H). HRMS (ESI): found 451.19085
(C23H26N6O2S [M þ H]þ, requires 451.19107).
4.2.4.21. 2,2,4,6,7-Pentamethyl-N-(4-((2-(piperazin-1-yl)pyrimidin-
4-yl)amino)phenyl)-2,3-dihydrobenzofuran-5-sulfonamide
chloride (DDO-5994). It was obtained in 42% yield as off-white
solid; 1H NMR (300 MHz, DMSO‑d6)
11.10 (s, 0H), 10.10 (s, 1H),
9.65 (s, 2H), 7.87 (d, J ¼ 7.0 Hz,1H), 7.52 (d, J ¼ 8.4 Hz, 2H), 7.21e6.99
(m, 2H), 6.50 (d, J ¼ 6.8 Hz, 1H), 4.02 (s, 4H), 3.24 (s, 4H), 2.98 (s,
2H), 2.47 (s, 3H), 2.40 (s, 3H), 2.01 (s, 3H), 1.40 (s, 6H). 13C NMR
hydro-
4.3.6. Cell cycle analysis
Cells treated with the indicated concentrations of DDO-5994
were incubated at 37 ꢂC for 12 h before being harvested and fixed
with 75% ethanol overnight at 4 ꢂC. Cells were incubated with
d
ribonuclease I (50
PI (50 g/mL) for 15 min. Cell cycle assays were performed on Flow
m
g/mL) in PBS at 37 ꢂC for 30 min, and stained by
m
(75 MHz, DMSO‑d6)
d
170.85, 161.25, 160.75, 159.22, 156.28, 139.29,
cytometry and FACSDiva software, v6.2 (BD Biosciences). FlowJo
software was used to analyze the cell cycle phases.
136.90, 134.12, 132.12, 129.37, 125.57, 121.77, 120.65, 117.35, 97.83,
87.38, 60.25, 49.05, 43.60, 42.69, 41.93, 28.62, 21.23, 19.67, 17.93,
14.53, 12.80. HRMS (ESI): found 523.24844 (C27H34N6O3S [M þ H]þ,
requires 523.24859).
4.3.7. Cell culture
HCT116 cell lines were obtained from the Cell Resource Center of
Shanghai Institute for Biological Sciences, Chinese Academy of
Sciences. The cell lines used in this study were maintained in RPMI
1640 medium supplemented with 10% fetal bovine serum. All the
cells were authenticated by short tandem repeat profiling, exam-
ined for mycoplasma contamination, and cultured at 37 ꢂC in a
humidified, 5% CO2 containing atmosphere incubator (Thermo
Fisher Scientific).
4.3. Biological assays and computational studies
4.3.1. Cloning, expression, and purification of recombinant Hsp90
and Cdc37
The process of cloning, expression, and purification of recom-
binant Hsp90 and Cdc37 was performed as a similar procedure as
previous reported [35].
4.3.8. Tumor xenograft experiments
4.3.2. Biolayer interferometry assay
The process of biolayer interferometry assay was performed as a
similar procedure as previous reported [35].
HCT116 cells (1 ꢁ 107) and Matrigel (BD Biosciences) were
mixed together in a 3:1 vol on ice and injected into the flanks of 6-
week-old BALB/c nude mice which were randomly divided into
three groups to generate xenografts. The treatment began when the
mean tumor volumes reached 125 mm3, and continued for 37 days,
giving an oral administration of 0.9% saline solution containing 5%
DMSO (vehicle) and compounds (DDO-5994 and 18h, 10 or 20 mg/
kg) daily. By measuring the two perpendicular diameters of the
tumors, the tumor volume was determined everyday (V ¼ length
(mm) ꢁ width (mm)2/2), and the body weights were recorded
every 1e4 days. After 37 days, the mice were sacrificed and
dissected, whose tumor tissues and experimental organs were
weighed and collected for further study. All animals used in this
study were handled according to federal and institutional guide-
lines under a protocol approved by the Institutional Animal Care
and Use Committee at China Pharmaceutical University.
4.3.3. In vitro antiproliferative assay
Antiproliferative activities of all the compounds against
HCT116 cell line were determined using an MTT assay kit. First, cells
were seeded into 96-well plates at 3000e5000 cells per well and
incubated for 24 h. Subsequently, serial dilutions of the compounds
were added in the plates for 72 h. Then, the cell proliferation was
determined by the MTT kit according to the standard protocol
(Beyotime, Jiangsu, China).
4.3.4. Cellular thermal shift assay
CETSAs were performed according to previously published
standard protocols [39]. HCT116 cells were seeded in 10 cm cell
culture dishes to reach ~90% confluence. HCT116 cells were treated
with 5
mM DDO-5994 or the same amount of DMSO before they
4.3.9. H&E staining and immunohistochemistry
were collected and washed with PBS buffer three times. The
collected cells were isolated and resuspended in PBS buffer, which
The process of H&E staining and immunohistochemistry was
performed as a similar procedure as previous reported [35].
were equally distributed into 200 mL PCR tubes. The tubes were
heated at the indicated temperature (37e75 ꢂC) to denature the
4.3.10. Statistics
samples for 3 min. Subsequently, the denatured cells were freeze-
GraphPad Prism 8.2.1 software was used to process
12

Q. Zhang, X. Wu, J. Zhou et al.
European Journal of Medicinal Chemistry xxx (xxxx) xxx
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experimental data. Values are expressed in the form of mean SD.
The data were analyzed from at least three parallel experiments.
Significant differences between two groups were indicated as
*P < 0.05, **P < 0.01, and ***P < 0.001.
4.3.11. Molecular docking and molecular dynamics (MD) simulation
The crystal structure of Hsp90-Cdc37 (PDB ID: 1US7) was ob-
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molecular docking. Conformations’ generation of protein and
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Author contributions
The manuscript was written through contributions of all au-
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manuscript.
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yet? Clin. Canc. Res. 18 (2012) 64e76.
Declaration of competing interest
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The authors declare that they have no known competing
financial interests or personal relationships that could have
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Acknowledgments
This study was supported by projects 82003582, 81872737,
81773639 and 81930100 of the National Natural Science Founda-
tion of China; BK20190559 of the Natural Science Foundation of
Jiangsu Province of China; CPU2018GY02 of the Double First Class
Innovation Team of China Pharmaceutical University; the National
Science & Technology Major Project “Key New Drug Creation and
Manufacturing
Program”,
China
(No:
2018ZX09711002,
2017ZX09302003 and 2015ZX09101032); the Project Program of
State Key Laboratory of Natural Medicines, China Pharmaceutical
University(No. SKLNMZZ202003 and No. SKLNMZZ202003); the
Priority Academic Program Development of Jiangsu Higher Edu-
cation Institutions.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2020.112959.
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