Design and synthesis of Grp94 selective inhibitors based on Phe199 induced fit mechanism and their anti-inflammatory effects

European Journal of Medicinal Chemistry 223 (2021) 1 13604

Contents lists available at ScienceDirect

European Journal of Medicinal Chemistry

journal homepage: h ttp://www.elsevier.com/locate/ejmech

Design and synthesis of Grp94 selective inhibitors based on Phe199

induced ﬁt mechanism and their anti-inﬂammatory effects

,

b

,

b

,

, b

Shicheng Xu ^{a 1}, Anping Guo ^{a 1}, Nan-nan Chen ^{a b}, Wei Dai ^{a b}, Huan-aoyu Yang ^a

,

Wenqin Xie ^{a b}, Mengjie Wang ^{a b}, Qi-Dong You ^{a **}, Xiao-Li Xu ^a

^aState Key Laboratory of Natural Medicines, Jiangsu Key Laboratory of Drug Design and Optimization, China Pharmaceutical University, Nanjing, 210009,

,

b,

, b, *

China

^bDepartment of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical University, Nanjing, 210009, China

a r t i c l e i n f o

a b s t r a c t

Article history:

Glucose-regulated protein 94 (Grp94), a member of the Heat shock protein 90 (Hsp90) family, is

implicated in many human diseases, including cancer, neurodegeneration, inﬂammatory, and infectious

diseases. Here, we describe our effort to design and develop a new series of Grp94 inhibitors based on

Phe199 induced ﬁt mechanism. Using an alkynyl-containing inhibitor as a starting point, we developed

compound 4, which showed potent inhibitory activity toward Grp94 in a ﬂuorescence polarization-based

assay. With improved physicochemical properties and suitable pharmacokinetic properties, compound 4

was advanced into in vivo bioactivity evaluation. In a dextran sulfate sodium (DSS)-induced mouse

model of ulcerative colitis (UC), compound 4 showed anti-inﬂammatory property and reduced the levels

Received 23 March 2021

Received in revised form

17 May 2021

Accepted 1 June 2021

Available online 9 June 2021

Keywords:

Grp94 inhibitors

2-Amino-5-ethynylbenzamide derivatives

Ulcerative colitis

of pro-inﬂammatory cytokines (TNF-

approach may be promising for further Grp94 drug development efforts.

a and IL-6). Together, these ﬁndings provide evidence that this

1. Introduction

to two aspects: on the one hand, the inhibition of Hsp90 can induce

the activation of heat shock transcription factor 1 (HSF1). After the

activation of HSF1, Hsp 70 and Hsp 27 will be overexpressed to

protect cells from apoptosis and offset the activity of Hsp90 in-

hibitors. On the other hand, Hsp90 is also highly expressed in

normal cells. It is perceived that Hsp90 pan-inhibition might

generate unaccepted toxicity and off-target effects [4,10e12]. With

increasing comprehension of Hsp90 chaperone system and the

modes of Hsp90-cochaperone-client complex interaction, novel

regulation strategies have emerged, including inhibiting the

Hsp90 C-terminal [13e16], inhibiting the interaction of Hsp90-

cochaperone-client complex [1 7e20], and targeting the isoforms

of Hsp90 (Fig. 1) [10,21e25].

Heat shock protein 90 (Hsp90) is a kind of widely expressed and

highly conventional molecule chaperone protein, which plays a

pivotal role in the maintenance of protein homeostasis by regu-

lating the maturation and secretion of client proteins [1,2]. The

dysregulation of Hsp90 is associated with kinds of human diseases,

including cancer, neurodegeneration, inﬂammatory and infectious

diseases [3e5]. Although more than 15 N-terminal Hsp90 inhibitors

have entered clinical tests for the treatment of multiple malignant

tumors, none of Hsp90 inhibitors have been approved for listing

through clinical trials for the lack of efﬁcacy, toxicity, and off-target

effects [6,7]. The most common adverse events are diarrhea, fa-

tigue, nausea, and anorexia [8,9]. The main reason can be attributed

Mammalian Hsp90 has four isoforms: Hsp90a and Hsp90b in

the cytosol, tumor necrosis factor receptor-associated protein-1

(Trap-1) in the mitochondria, and glucose-regulated protein 94

(Grp94) in the endoplasmic reticulum [26]. Considering that each

Hsp90 isoform is responsible for chaperoning speciﬁc client pro-

teins, selective inhibition of Hsp90 isoform might obtain better

efﬁcacy and fewer toxicities [27]. Grp94, serving as the chaperone

protein of Toll-like receptors (TLRs), integrins, human epidermal

growth factor receptor-2 (HER2), and Wnt coreceptor low-density

lipoprotein receptor-related protein 6 (LRP6), correlates closely

* Corresponding author. State Key Laboratory of Natural Medicines, Jiangsu Key

Laboratory of Drug Design and Optimization, China Pharmaceutical University,

Nanjing, 210009, China.

** Corresponding author. State Key Laboratory of Natural Medicines, Jiangsu Key

Laboratory of Drug Design and Optimization, China Pharmaceutical University,

Nanjing, 210009, China.

E-mail addresses: youqd@163.com (Q.-D. You), xuxiao_li@126.com (X.-L. Xu).

The two authors contribute equally.

1

https://doi.org/10.1016/j.ejmech.2021.113604

S. Xu, A. Guo, N.-n. Chen et al.

European Journal of Medicinal Chemistry 223 (2021) 113604

Fig. 1. Regulation strategies of Hsp90 chaperone system.

with the growth and metastasis of aberrant cancers, including

breast cancer, multiple myeloma, and inﬂammation-associated

colon cancer [28,29]. Grp94 is also involved in regulating insulin

like growth factor I/II (IGF-I/II) and channeling immune and in-

ﬂammatory signals [30]. As a potential therapeutic target for the

diseases, Grp94-selective inhibitors have been pursued by medic-

inal chemists in recent years.

The homology of the N-terminal ATP binding pocket of each

Hsp90 isoform is as high as 85%, which brings great difﬁculties to

the development of selective inhibitors [31e33]. Compared with

adenosine receptor agonist, its further application is limited. The

Gabriela Chiosis research group of the Kettering Cancer Center

studied the reported Hsp90 inhibitors and found that some purine-

scaffold inhibitors represented by PU-H54 in the backward bent

conformation have shown selective Grp94 inhibitory activity [23].

Through structure activity relationship (SAR) study, the compound

PU-WS13 (Grp94, K_d¼ 0.22

mM; Hsp90a, Kd＞250 mM) with better

activity and higher selectivity to Grp94 was obtained. Purine in-

hibitors showed different binding modes when binding to Hsp90

and Grp94. When the 8-aryl group of PU-H54 is rotated by 80^ꢀand

adopts the backward bent conformation, it can induce the rear-

rangement of the lid region of Grp94 by displacing Phe199, then a

deep and new hydrophobic pocket 2 (by Leu 104, Leu 163, Phe199,

Ala 202, Phe 203, Val211, Ile 247, and Leu 249) is exposed, which is

different from the site 3 pocket. The 8-aryl group can occupy this

Hsp90a/b, a 5-amino acid sequence of QEDGQ inserted at amino

acid 182 in Grp94, just in the 1,4,5-helix substructure of the ATP

binding pocket, causing the ATP pocket to undergo conformational

changes under the induction of small ligand, thereby exposing new

hydrophobic site 2 and 3 pockets next to the ATP pocket [23,34,35].

Hsp90

a

/b

cannot undergo conformational changes induced by

new hydrophobic pocket. However, site 2 in Hsp90a/b is blocked by

small molecules, which provides a structural basis for the devel-

opment of inhibitors that selectively target Grp94 [36e38].

Phe 138 [23,36]. So, this class of inhibitors shows a 100-fold

selectivity to Grp94 and has an excellent anti-tumor effect.

Blagg's group discovered a series of selective inhibitors of Grp94

based on resorcinol fragments. The compound BnIm (Grp94

Several Grp94 selective inhibitors that have been reported so far

ﬁrstly occupy the hinge region in the Grp94 ATP pocket, and then

fragments occupy the exposed speciﬁc hydrophobic pockets to

achieve selectivity (Fig. 2) [36]. NECA, as an adenosine receptor

agonist, was found to bind to Grp94 with a K_dvalue of 200 nM,

becoming the ﬁrst selective inhibitor of Grp94 [39,40]. The 5⁰-

ethylcarbamoyl group of NECA can occupy the newly exposed hy-

drophobic site 3 near the ATP pocket. Because NECA is also a potent

K_d¼ 1.14

radicicol-geldanamycin chimera Radamide (RDA), which has a

selectivity of 12 folds for Grp94/Hsp90 [35,41]. The binding mode

of BnIm is different from PU-WS13. KUNG29 (Grp94 K_d¼ 0.2 M;

Hsp90 M) was obtained by optimization, and the

K_d¼ 8.2

selectivity was increased to 41 times [41]. KUNG94 is a resorcinol

m

M; Hsp90

a

K_d¼ 13.1

mM) was obtained by optimizing the

a

m

a

m

Fig. 2. Representative Grp94 inhibitors and their selective proﬁles.

2

S. Xu, A. Guo, N.-n. Chen et al.

European Journal of Medicinal Chemistry 223 (2021) 113604

Grp94 selective inhibitor containing an isoindoline core, with K_dof

8 nM and 77 nM for Grp94 and Hsp90a, respectively [42].

effects, including tert-butyl, isobutyl, butyl, 1-chlorobutyl, and

isopentyl, were introduced as seen in compounds 2e6, respec-

tively. Interestingly, signiﬁcant improvements were observed in

their inhibitory activities against Grp94; compound 4 with butyl-

Gewirth group reported that the residue Phe199, as a gate-

keeper, is key to exposing site 2 in Grp94 for the design of the

paralog-selective inhibitors [36]. Our group also proved that the

Phe199 shift and rigid moieties in ligands are important structural

bases for the design of Grp94 selective inhibitors. A new series of

benzamide Grp94 selective inhibitors were developed by

combining an aryl group with a benzamide scaffold [43]. Among all

the Grp94 selective inhibitors, DDO-5813 was the most potent

compound with an IC₅₀value of 2 nM. However, the poor physi-

cochemical property of compound DDO-5813 restricts its further

druggability research. Here, we report the discovery of a novel se-

ries of alkynyl-containing Grp94 inhibitors by replacing the rigid

biphenyl core in DDO-5813. Moreover, its druggability and bio-

logical activity were also evaluated.

substituted

(IC₅₀¼ 0.060

showed

0.014

the

highest

inhibitory

activity

mM). We then introduced cyclopropyl,

cyclopentyl, and cyclohexyl groups, affording compounds 7e9,

which maintained the inhibitory activities. We further introduced

hydrophilic polar groups containing nitrogen such as N,N-

dimethylaminomethyl, N,N-diethylaminomethyl as seen in com-

pounds 10e11, respectively. The activities signiﬁcantly reduced

compared with the lead compound DDO-5813, and the inhibitory

activity towards Grp94 was almost lost. It was veriﬁed that the site

2 pocket was enormously hydrophobic and not suitable for hy-

drophilic polar groups. The results of SAR optimization revealed

that the butyl moiety is more desirable for Grp94 inhibitory activity

than rigid and bulky substituents (Table 1).

2. Results and discussion

Subsequent SAR optimization mainly focused on the cyclo-

hexanol group of 4. Substituents with different oxygen-bearing

chains, including hydroxyethyl, methoxy ethyl, ethoxy ethyl,

hydroxypropyl, methoxy propyl, ethoxy propyl, and acetamino

ethyl, as in 12e18, were introduced. It was found there was a sig-

niﬁcant decrease in activity in all the cases (Table 2). The intro-

duction of oxygen-containing ﬁve-or six-membered ring (19e22)

seemed to be beneﬁcial for activity, and the IC₅₀values of these

2.1. Design and SAR optimization of 2-amino-5-ethynylbenzamide

derivatives by in vitro Grp94 inhibitory activity

Inspired by the conclusion that ligand-induced “Phe199 shift”

effect is the structural basis of Grp94-selective inhibition, a series of

benzamide Grp94 selective inhibitors with biphenyl moiety were

developed, among which DDO-5813 manifested the most potent

Grp94 inhibitory activity with an IC₅₀value of 2 nM [43]. However,

the high fat solubility and low water solubility of compound DDO-

compounds ranged from 0.234 mM to 0.947 mM, showing the six-

membered ring structure is more suitable compared to ﬁve-

membered ring. However, introducing a nitrogen-containing ring

such as piperidyl group (24e26) brought about signiﬁcantly

decreased potency. The reduced activity may be due to the fact that

they cannot form a hydrogen bond with Lys 114. So, compounds 23

and 27e34 with different acids introduced on the piperidine were

designed to serve as hydrogen acceptors. However, there was no

signiﬁcant increase in activity. What is more, it was found that

compound 26 was more potent than compound 24. Through

docking analysis, we found that compound 26 with NeH of

piperidine could form hydrogen bond interaction with Glu 153.

5813 (solubility in PBS of 3.93 mg/mL, Log D7.4 of 4.59, permeability

of 99.18

2.61 ꢁ 10^ꢂ6cm/s) restrict its further druggability

research. The low water solubility may be related to the high crystal

lattice stacking energy of biphenyl structure, which seriously af-

fects the drug-like properties. Therefore, it is necessary to further

expand the compound design around this compound in the guid-

ance of this induced ﬁt strategy to get more druggable molecules. In

terms of spatial structure and electron cloud density, acetylene

group and phenyl group are considered to be isosterisms each other

in a broad sense, so replacement of the phenyl group with alkynyl

groups may be useful for ﬁnding novel inhibitors with improved

property. Here, we present a series of alkynyl-containing Grp94

inhibitors designed through the scaffold hopping approach by

replacing the biphenyl core in DDO-5813 with a rigid and linear

alkynyl-phenyl core, which may keep the potent inhibitory potency

and improve the physicochemical properties with lower crystal

lattice stacking energy than biphenyl structure (Fig. 3). We per-

formed the molecular docking studies to predict the binding modes

of DDO-5813 and compound 1 with Grp94. As for DDO-5813, the

isopropylbenzene moiety at interposition of benzene moiety ﬁt

deeply into the hydrophobic site 2 pocket. The occupation of site 2

plays a decisive role in the improvement of inhibitory activity.

As for compound 1, it binds to Grp94 with the critical pharma-

cophoric benzamide moiety and bears a rigid alkynyl substitution

at interposition of benzene moiety. The alkynyl orients directly into

site 2 and allows for the extension of isopropyl group to induce and

occupy the site 2 pocket.

2.2. Synthesis of target compounds 1-34

The synthesis of compounds 1e11 is described in Scheme 1.

Different alkynes were coupled with commercial reagent 35 in THF

using (PPh₃)₂Cl₂Pd and CuI as the catalysts and Et₃N as the base to

afford intermediates 36e44. The intermediates were subsequently

treated with trans-4-aminocyclohexanol and DIPEA in DMSO at

120 ^ꢀC for 16 h. The ﬁnal compounds 1e9 were obtained by

hydrolyzation of the cyano group into a carbamoyl group. 35

reacted with the trans-4-aminocyclohexanol and hydrolyzed into

intermediate 45. Then 45 was treated with various alkynes by a

Sonogashira reaction to afford compounds 10e11 [43].

Compounds 12e34 were synthesized as shown in Scheme 2.

Different amines reacted with the critical intermediate 39 and

DIPEA in DMSO at 120 ^ꢀC for 16 h followed by hydrolyzation of the

cyano group to afford compounds 12e23 and intermediates

46e48.46e48 were reacted with silica gel in toluene at 100 ^ꢀC for

48 h to afford the compounds 24e26, respectively. Finally, 24e26

were condensed with different acids to generate desired com-

pounds 27e31, 32e33, and 34, respectively [44,45].

Our work started from compound 1 with an isopropyl group,

which is the most potent group of biphenyl inhibitors. Compound 1

was

a

moderately

potent

inhibitor

toward

Grp94

(IC₅₀¼ 0.914 0.034

m

M), which was revealed by the FP-based

assays in vitro. Based on the moderate potency, we conducted

further optimization on this new scaffold to develop more potent

Grp94 inhibitors.

2.3. Molecular modeling

Molecular docking of compound 4 and Grp94 was conducted

utilizing the software Discovery Studio (DS) 4.0 to obtain deeper

insight into the mutual interactions between these alkynyl-

containing compounds and Grp94. We selected the X-ray

Initial SAR optimization mainly focused on the ethynyl group of

1. In light of the fact that the isopropyl group in 1 is oriented to the

induced hydrophobic pocket, substituents with different steric

3

S. Xu, A. Guo, N.-n. Chen et al.

European Journal of Medicinal Chemistry 223 (2021) 113604

Fig. 3. Chemical structures of compound DDO-5813, the alkynyl-containing compound 1 and predicted binding poses with Grp94 (PDB ID: 3O2F). The hydrophobic surface of Grp94

is shown as blue. The active site of Grp94 is shown as purple. Compounds are shown as blue sticks. The surrounded residues are shown as grey sticks. Conserved water molecules

are shown as red spheres. The H-bonds are depicted as green dashes.

crystallographic complex of Grp94 and PU-H54 (PDB ID: 3O2F) to

establish the optimized settings for docking.

selective inhibitor, compound 4, can't activate HSF-1 and induce

the up-regulation of other heat shock proteins in cells, which may

reduce the side-effects of pan-isoform Hsp90 inhibition.

The carbamoyl moiety of compound 4 formed a hydrogen bond

network with Asp 149 and Thr 245, which anchored compound 4

into the ATP binding pocket. The alkynyl moiety entered into site 2

and formed interactions with the surrounding hydrophobic resi-

dues of Leu 104, Phe 195, Phe 199, Val 209, Val 211, and ILE 247.

Additionally, the trans-4-aminocyclohexanol moiety extended to

the solvent area and showed another hydrogen bond interaction

with the polar residue Lys 114 (Fig. 4).

2.5. Evaluation of physicochemical properties of representative

compounds

The physicochemical properties of compounds have essential

effects on their absorption, distribution, metabolism, and excretion

in vivo. The synthesis of this series of alkynyl-containing com-

pounds is to improve the physicochemical properties of biphenyl

inhibitors. So, the partition coefﬁcient (log D, pH 7.4), solubility, and

permeability coefﬁcients (Pe) of representative compounds were

determined. As shown in Table 3, compared with DDO-5813,

compounds 4 and 23 have similar membrane permeability and can

penetrate artiﬁcial membranes better. The water solubility of 4 and

23 is improved by 3e4 times, and log D_7.4is between 3e4, which

gets a litter lower. Therefore, the results suggest that the structural

optimization has improved the physical and chemical properties of

the biphenyl compounds. Although the water solubility of 23 is

slightly better than that of 4, its inhibitory activity on Grp94 is

reduced by 3 times. Considering the improved physicochemical

properties and activity, we selected compound 4 for further phar-

macokinetic and pharmacodynamic research.

2.4. Selectivity of compound 4 to Grp94 and Hsp90

a

On the basis of the above SAR optimization, compound 4 was

selected to reconﬁrm whether the alkynyl-containing Grp94 in-

hibitors represented by compound 4 selectively inhibited Grp94.

BLI assay was applied to determine its potency against Grp94 and

Hsp90

binding afﬁnities for Grp94 and Hsp90

9.37 M, respectively), which suggested that compound 4 has a

a

. As shown in Fig. 5, compound 4 displayed different

a

(K_D¼ 0.111 M and

m

remarkable Grp94 afﬁnity and selectivity. The high Grp94 selec-

tivity exhibited in both FP assay and BLI assay indicated that

compound 4 can be a probe to study the Grp94-speciﬁc biological

functions.

Moreover, different from Hsp90a inhibition, Grp94-selective

inhibition does not induce heat shock response and has no inﬂu-

ence on the Hsp70 expression level. To characterize the Grp94-

selective inhibitory activity of compound 4 in cells, Western blot

analysis was conducted to evaluate the expression levels of the

selected biomarkers in compound 4 treated HCT116 cells. As shown

in Fig. 3 (see supplementary data), no modulation of Hsp70 and Akt

(Hsp90a speciﬁc client) was observed. In contrast, the pan-Hsp90

inhibitor AT13387 obviously induced upregulation of Hsp70 and

degradation of Akt. These results conﬁrmed that the Grp94-

2.6. Evaluation of pharmacokinetic proﬁles and liver microsome

stability of compound 4

As shown in Table 4, acceptable T_1/2(4.69 h) was observed in

rats with intragastrical administration. Compound 4 exhibited

suitable bioavailability in rats (15.73%), which veriﬁed that com-

pound 4 was worthy of further studies on efﬁcacy in vivo. Besides,

we further assessed the liver microsome stability of compound 4 in

human and mouse liver microsomes in vitro, compared with

4

S. Xu, A. Guo, N.-n. Chen et al.

European Journal of Medicinal Chemistry 223 (2021) 113604

Table 1

maintenance of gut homeostasis and the induction of regulatory T

cells [48]. It has been reported that macrophage-speciﬁc Grp94 KO

mice were more resistive to DSS-induced colitis, which indicated

that Grp94 has detrimental roles in the exacerbation of intestinal

inﬂammation [49]. However, the function of Grp94 in gastrointes-

tinal homeostasis is not well understood, and there is an urgent

need for Grp94-speciﬁc modulators to explore whether inhibition

of Grp94 has some efﬁcacy. Considering both the suitable phar-

macokinetic characteristics and potent inhibitory activity of com-

pound 4, the efﬁcacy of 4 was further investigated in a mouse UC

model induced by DSS.

Inhibitory activity of compounds 1-11 against Grp94.

C57BL/6 mice were divided into the disease control group,

treatment groups, and the normal control group. The mice in dis-

ease control group and treatment groups were given 3% DSS

drinking water from day 1 to day 7. We established the UC model

successfully after the observation of typical symptoms, such as

weight loss, diarrhea, and rectal bleeding. Meanwhile, the treat-

ment groups were given compound 4 with 10 mg/kg, 30 mg/kg, and

50 mg/kg by intraperitoneal injection.

Compd.

Structure R¹¼ Grp94 FP activity ^a

Inhibition percentage (100 mM) IC₅₀(mM)

1

2

3

100 %

0.914 0.034

0.325 0.019

0.313 0.021

As shown in Fig. 6A, the body weights of all experimental mice

changed slightly in the ﬁrst four days. After day 4, the body weights

of normal group mice increased continuously. Conversely, the

weight loss was evident in the disease control group. Compound 4

could alleviate the weight loss of the mice in three treatment

groups. Based on the fact that inﬂammation often results in

enlargement of the spleen, we also investigated the changes in

spleen weight index. Mice in treatment groups presented a signif-

icant decrease of spleen weight index compared to that in the

disease control group, which indicated that compound 4 could

ameliorate the inﬂammation. The liver weight index of mice in

treatment groups was not signiﬁcantly reduced, conﬁrming that

compound 4 was safe enough to be a potential UC treatment agent

(Fig. 6B). Scoring changes in weight loss, stool consistency, and

stool blood were calculated and averaged to afford disease activity

index (DAI) scores. As shown in Fig. 6C, compound 4 signiﬁcantly

decreased DAI scores in a dose-dependent manner on day 8. On day

8, mice from the disease control group showed hair brightness loss,

ataxia, leanness, and severe rectal bleeding (Fig. 6D). The mice in

treatment groups showed varying degrees of improvements in

their appearances in the following order: 50 mg/kg, 30 mg/kg,

10 mg/kg.

4

5

6

100 %

0.060 0.014

0.205 0.007

0.519 0.042

7

8

100 %

0.251 0.017

0.100 0.004

9

100 %

0.210 0.008

10

11

63.9 %

28.0 %

62.627 9.028

> 100

DDO-5813

AT13387

100 %

0.020 0.004

0.013 0.004

a

IC₅₀values are the mean SD from three independent experiments. Compound

Colon shortening is another symptom, which is often used to

evaluate the colitis induced by DSS. However, compound 4 could

not signiﬁcantly prevent colon shortening induced by DSS (Fig. 7A).

Similarly, administration of compound 4 could not obviously

attenuate the severe colonic tissue damage induced by DSS

(Fig. 7B). The reason may be that the damage induced by DSS was so

substantial that compound 4 could not repair the damage within a

short time. Pro-inﬂammatory cytokines, such as tumor necrosis

DDO-5813 and AT13387 were selected as positive controls. Almost all compounds

exhibited 100% inhibition of Hsp90a at 100 mM (data not shown).

compound DDO-5813. As shown in Table 5, compound 4 exhibited

preferable metabolic stability in human and mouse liver micro-

somes (t_1/2¼ 346.5 min and 108.3 min, respectively), compared

with that of DDO-5813 (t_1/2¼ 256.7 min and 62.4 min, respec-

tively). Meanwhile, the clearance rates of compound 4 were lower.

Thus, compound 4 exhibited good stability and deserved further

evaluation.

factor-alpha (TNF-a) and interleukin-6 (IL-6), play essential roles in

the pathogenesis of UC and have been implicated as important

mediators of the inﬂammatory reaction in UC patients. To better

understand the anti-inﬂammatory mechanism of compound 4,

enzyme-linked immunosorbent assay (ELISA) and immunohisto-

chemical assay (IHC) were conducted to determine the levels of

2.7. Evaluation of anti-inﬂammatory efﬁcacy of compound 4 in a

DSS-Induced UC model

TNF-

tissues was observed in DSS-treated mice by the immunohisto-

chemical assay. Compound could signiﬁcantly and dose-

dependently reduce the levels of TNF- and IL-6 (Fig. 7C). We

also observed compound 4 could reduce the levels of TNF- and IL-

6 in ELISA (Fig. 7D). Thus, these results demonstrated that com-

pound 4 potentially possesses promising anti-inﬂammatory effects

by inhibiting the pro-inﬂammatory cytokines, including TNF-a and

IL-6.

a and IL-6. The obvious elevation of TNF-a and IL-6 in colonic

Ulcerative colitis (UC) is a typical bowel disease, the morbidity of

which has been increasing in recent years. Human UC symptoms

include the loss of weight, severe diarrhea, the bleeding of rectal,

the damage of intestinal mucosal, and the elevation of pro-

inﬂammatory cytokines in colonic tissues [46]. The multifaceted

roles of Grp94 in the development of UC have been reported in

various papers. Zihai Li's group reported that Grp94 played an

essential role in gut homeostasis via regulating the canonical Wnt

pathway [47]. Grp94 is important for CD11c^þcells in the

4

a

5

S. Xu, A. Guo, N.-n. Chen et al.

European Journal of Medicinal Chemistry 223 (2021) 113604

Table 2

Inhibitory activity of compounds 12-34 against Grp94.

Compd.

Structure R²

¼

Grp94 FP activity ^a

Inhibition percentage (100

97.2 %

m

M)

IC₅₀(mM)

12

13

14

15

16

6.839 0.323

10.898 1.969

7.544 0.481

2.769 0.455

2.061 0.234

98.9 %

100 %

17

18

100 %

3.312 0.169

2.017 0.336

19

100 %

0.310 0.081

20

21

100 %

0.947 0.080

0.234 0.030

22

23

100 %

0.393 0.065

0.203 0.026

24

100 %

1.607 0.243

25

26

65.2 %

100 %

16.599 1.141

0.627 0.007

27

28

29

100 %

97.8 %

0.528 0.024

0.797 0.060

1.108 0.122

6

S. Xu, A. Guo, N.-n. Chen et al.

European Journal of Medicinal Chemistry 223 (2021) 113604

Table 2 (continued)

a

Compd.

Structure R²

¼

Grp94 FP activity

Inhibition percentage (100

100 %

mM)

IC₅₀(mM)

30

31

1.488 0.281

100 %

0.985 0.044

32

33

34

90.2 %

94.3 %

98.6 %

4.779 0.330

2.405 0.264

2.045 0.269

a

IC₅₀values are the mean SD from three independent experiments. Almost all compounds exhibited 100% inhibition of Hsp90

a

at 100

mM (data not shown).

alleviated by compound 4, and the DAI scores in the treatment

group mice were obviously lower than those in the disease control

group mice. These results revealed that compound 4 is an orally

active anti-inﬂammatory agent for the treatment of UC.

4. Experimental section

4.1. General methods and materials

All reagents and solvents purchased from commercial suppliers

were used directly. Thin-layer chromatography (TLC) was carried

out on 0.25 mm silica gel plates (GF254) to monitor reactions, and

spots were visualized with UV light. Melting points were deter-

mined with a Melt-Temp II apparatus. The structures of synthesized

compounds were characterized by ¹H NMR, ¹³C NMR, MS, and

HRMS. The ¹H NMR and ¹³C NMR spectra were measured on a

Bruker AV-300 instrument using deuterated solvents with tetra-

methylsilane (TMS) as an internal standard. ESI-mass and high-

resolution mass spectra (HRMS) were recorded on a Water Q-Tof

micro mass spectrometer by electrospray ionization (ESI). The pu-

rity (ꢃ95%) of the compounds was veriﬁed by the high-

performance liquid chromatography (HPLC) performed on an Agi-

Scheme 1. Synthetic routes for compounds 1e11. Reagents and conditions: (a)

different alkynes, CuI, (PPh₃)₂Cl₂Pd, Et₃N, rt,

8 h; (b) trans-4-aminocyclohexanol,

DIPEA, DMSO, 120 ^ꢀC, 16 h; (c) 2 M NaOH, 30% H₂O₂, EtOH, DMSO, 30 ^ꢀC, 12 h.

lent C18 (4.6 mm ꢁ 150 mm, 3.5

mm) column using a mixture of

solvent methanol/water at a ﬂow rate of 0.5 mL/min and monitored

by UV absorption at 254 nm. The LC/MS analyses were carried out

on HPLC-MS system with a triple quadrupole mass spectrometer

(Shimadzu) using an Inertsil C18 column at a ﬂow rate of 0.4 mL/

min.

3. Conclusion

In this study, computational methods and medicinal chemistry

were used to design and synthesize more potent Grp94 inhibitors.

Based on the conclusion that the ligand induced “Phe199 shift”

effect contributes to the selective inhibition of Grp94, a novel series

of alkynyl-containing Grp94 inhibitors were developed by replac-

ing the phenyl ring at interposition of benzene moiety with rigid

and linear alkynyl substituents. Compound 4 with highest inhibi-

tory activity in vitro, preferable stability, and improved physical

properties was used for further investigations. In light of the

excellent PK properties in vivo, compound 4 was selected further to

conﬁrm the anti-inﬂammatory effects in the DSS-challenged mouse

model. The results showed that compound 4 could reduce the

4.2. Synthesis

4.2.1. General Procedure for intermediates 36e44. (PPh₃)₂Cl₂Pd

(0.01 equiv), CuI (0.02 equiv), and Et₃N (5.0 equiv) were added to a

mixture of 35 (1.0 equiv) and appropriate alkyne (2.0 equiv) in THF

and stirred at room temperature for 8 h under nitrogen protection.

After conﬁrming the progress of the reaction by thin-layer chro-

matography, the black reaction mixture was ﬁltered, and the ﬁltrate

was concentrated under vacuum. EA (20 mL) was added, and the

organic layer was washed with water and brine, dried over anhy-

drous sodium sulfate, and concentrated in vacuo. Puriﬁcation by

silica gel chromatography (petroleum ether) afforded

levels of circulating pro-inﬂammatory cytokines (TNF-a and IL-6) in

colonic tissues and improve the inﬂammatory responses of UC

mice. The loss of body weights in treatment group mice was

7

S. Xu, A. Guo, N.-n. Chen et al.

European Journal of Medicinal Chemistry 223 (2021) 113604

Scheme 2. Synthetic route for compounds 12e34. Reagents and conditions: (a) different amines, DIPEA, DMSO, 120 ^ꢀC, 16 h; (b) 2 M NaOH, 30% H₂O₂, EtOH, DMSO, 30 ^ꢀC, 12 h; (c)

silica gel, toluene, 100 ^ꢀC, 48 h; (d) different acids, BOP, DIPEA, DMF, rt, 4 h.

Fig. 4. Docking study of the binding mode between 4 and Grp94 (PDB ID: 3O2F). The surface of Grp94 is shown in the hydrophobic state. The carbon atoms of the ligands and Grp94

residues are colored grey. The hydrogen bonds are represented as green dashed lines.

Fig. 5. BLI doseꢂresponse curves of 4 binding to Grp94 and Hsp90

a.

8

S. Xu, A. Guo, N.-n. Chen et al.

European Journal of Medicinal Chemistry 223 (2021) 113604

procedures. Colorless oil (260 mg, 92.7%). ¹H NMR (300 MHz,

Table 3

Physicochemical properties of the compounds 4, 23, and DDO-5813.

chloroform-d)

d

7.66e7.53 (m, 2H), 7.14 (t, J ¼ 8.7 Hz,1H), 2.82e2.72

(m, 1H), 1.26 (d, J ¼ 6.9 Hz, 6H).

Compd.

Physicochemical properties

4.2.1.2.

2-ﬂuoro-5-(3,3-dimethylbut-1-yn-1-yl)benzonitrile

(10^ꢂ6cm/s)

7.4

a

Log D pH ¼ 7.4

Solubility (

m

g/mL)

Pe, pH

(37). Intermediate 37 was prepared from 35 (370 mg,1.5 mmol) and

3,3-dimethylbut-1-yne (246 mg, 3.0 mmol) according to the gen-

eral procedures. White solid (295 mg, 97.8%). ¹H NMR (300 MHz,

4

23

3.69

3.24

4.59

11

16

3.9

97.86 1.50

94.61 0.22

99.18 2.61

DDO-5813

chloroform-d)

9H).

d

7.68e7.57 (m, 2H), 7.15 (t, J ¼ 8.7 Hz, 1H), 1.32 (s,

a

Data are presented as mean SD of three separate experiments.

4.2.1.3. 2-ﬂuoro-5-(4-methylpent-1-yn-1-yl)benzonitrile (38).

Intermediate 38 was prepared from 35 (370 mg, 1.5 mmol) and 4-

methylpent-1-yne (246 mg, 3.0 mmol) according to the general

procedures. Light yellow oil (272 mg, 90.2%). ¹H NMR (300 MHz,

Table 4

Pharmacokinetic proﬁles of compound 4.

PK parameters

4

chloroform-d)

d

7.66e7.58 (m, 2H), 7.15 (t, J ¼ 8.6 Hz, 1H), 2.30 (d,

rat (iv, 10 mg/kg)

rat (po, 40 mg/kg)

J ¼ 6.6 Hz, 2H), 1.98e1.85 (m, 1H), 1.04 (d, J ¼ 6.7 Hz, 6H).

T_max(h)

T_1/2(h)

C_max(mg/mL)

AUC_0ꢂ∞(h$

Vz (L/kg)

CL (L/h/kg)

F (%)

0.05

15.25

2.41

1.51

1.47

6.66

0.38

4.69

0.60

0.95

2.81

4.37

15.73

4.2.1.4. 2-ﬂuoro-5-(hex-1-yn-1-yl)benzonitrile (39). Intermedi-

ate 39 was prepared from 35 (370 mg, 1.5 mmol) and hex-1-yne

(246 mg, 3.0 mmol) according to the general procedures. Color-

less oil (270 mg, 89.5%). ¹H NMR (300 MHz, chloroform-d)

mg/mL)

d

7.66e7.56 (m, 2H), 7.15 (t, J ¼ 8.6 Hz, 1H), 2.41 (t, J ¼ 6.9 Hz,

2H), 1.65e1.55 (m, 2H), 1.53e1.43 (m, 2H), 0.97 (t, J ¼ 7.3 Hz, 3H).

4.2.1.5. 2-ﬂuoro-5-(5-chloropent-1-yn-1-yl)benzonitrile (40).

Intermediate 40 was prepared from 35 (370 mg, 1.5 mmol) and 5-

chloropent-1-yne (306 mg, 3.0 mmol) according to the general

procedures. Light yellow oil (258 mg, 77.8%). ¹H NMR (300 MHz,

Table 5

In vitro metabolic stability of compound 4 and DDO-5813 in human and mouse liver

microsomes.

chloroform-d)

d

7.67e7.59 (m, 2H), 7.17 (t, J ¼ 8.6 Hz, 1H), 3.71 (t,

Compd

human liver microsomes

_{1/2 (}min₎CL ( L/min/mg)

mouse liver microsomes

J ¼ 6.3 Hz, 2H), 2.63 (t, J ¼ 6.9 Hz, 2H), 2.12e2.03 (m, 2H).

4.2.1.6. 2-ﬂuoro-5-(5-methylhex-1-yn-1-yl)benzonitrile (41).

Intermediate 41 was prepared from 35 (370 mg, 1.5 mmol) and 5-

methylhex-1-yne (288 mg, 3.0 mmol) according to the general

procedures. Light yellow oil (288 mg, 89.3%). ¹H NMR (300 MHz,

T

m

T_{1/2 (}min₎

CL (mL/min/mg)

4

346.5

256.7

11.0

13.5

108.3

62.4

12.8

25.5

DDO-5813

chloroform-d)

d

7.66e7.56 (m, 2H), 7.15 (t, J ¼ 8.7 Hz, 1H), 2.41 (t,

J ¼ 7.4 Hz, 2H), 1.79e1.68 (m, 1H), 1.51 (q, J ¼ 7.3 Hz, 2H), 0.95 (d,

intermediates 36e44.

J ¼ 6.6 Hz, 6H).

4.2.1.1. 2-ﬂuoro-5-(3-methylbut-1-yn-1-yl)benzonitrile (36).

Intermediate 36 was prepared from 35 (370 mg, 1.5 mmol) and 3-

methylbut-1-yne (204 mg, 3.0 mmol) according to the general

4.2.1.7. 2-ﬂuoro-5-(cyclopropylethynyl)benzonitrile (42). Inter-

mediate 42 was prepared from 35 (370 mg, 1.5 mmol) and ethy-

nylcyclopropane (198 mg, 3.0 mmol) according to the general

Fig. 6. Anti-inﬂammatory efﬁcacy of compound 4 in DSS-induced UC mice. (A) Body weight changes of each group during treatment. (B) The spleen/liver weight index of each

group. The spleen/liver weight index was calculated according to the following formula: spleen/liver index (mg/g) ¼ spleen/liver weight (mg)/animal body weight (g). (C) DAI scores

during treatment. Data are expressed as mean

and > 15% ¼ 4), stool consistency (normal ¼ 0; slightly soft ¼ 1; soft ¼ 2; very soft ¼ 3; diarrhea ¼ 4), and stool blood (none ¼ 0; occult blood ¼ 2; fecal blood ¼ 4). (D) The general

status of the UC mice at day 8. Compared with the disease control group: *P < 0.05, **P < 0.01, ***P < 0.001.

SEM. The detailed scoring criteria for DAI was as follows: body weight loss (<1% ¼ 0; 1e5% ¼ 1; 5e10% ¼ 2; 10e15% ¼ 3

9

S. Xu, A. Guo, N.-n. Chen et al.

European Journal of Medicinal Chemistry 223 (2021) 113604

Fig. 7. Efﬁcacy of compound 4 on colons from each group and the inhibitory effect of compound 4 on the up-regulated inﬂammatory cytokines in the colon tissues of DSS-induced

UC mice. (A) Colon length of each group on day 8. (B) Representative H&E images of the colon samples from each group. Scale bar, 200 m. (C) The levels of TNF- and IL-6 were

detected in IHC. (D) The levels of TNF- and IL-6 were detected in ELISA. Compared with the disease control group: *P < 0.05, **P < 0.01, ***P < 0.001.

m

a

procedures. Light yellow oil (270 mg, 97.3%). ¹H NMR (300 MHz,

chloroform-d)

(m, 1H), 0.96e0.86 (m, 2H), 0.85e0.79 (m, 2H).

4.2.1.8. 2-ﬂuoro-5-(cyclopentylethynyl)benzonitrile (43). Inter-

mediate 43 was prepared from 35 (370 mg, 1.5 mmol) and ethy-

nylcyclopentane (282 mg, 3.0 mmol) according to the general

procedures. Colorless oil (266 mg, 83.2%). ¹H NMR (300 MHz,

J ¼ 8.9 Hz, 1H), 4.58 (d, J ¼ 4.2 Hz, 1H), 3.50e3.42 (m, 1H), 3.31e3.24

(m, 1H), 2.80e2.71 (m, 1H), 1.96e1.91 (m, 2H), 1.83e1.78 (m, 2H),

1.36e1.23 (m, 4H), 1.18 (d, J ¼ 6.8 Hz, 6H). ¹³C NMR (75 MHz,

d

7.65e7.51 (m, 2H), 7.13 (t, J ¼ 8.7 Hz, 1H), 1.49e1.40

DMSO‑d₆) d ppm 171.23, 148.65, 135.56, 132.50, 113.71, 111.88,

107.94, 92.83, 80.35, 68.22, 49.6, 33.63, 30.39, 23.35, 20.67. HRMS

(ESI): calcd for C₁₈H₂₄N₂O₂[M þ H]^þ301.18, found 301.19. Purity:

96.54% by HPLC (MeOH/H₂O ¼ 90:10). R_f: 0.32 by TLC (PE/EA ¼ 1:1).

chloroform-d)

(m, 1H), 2.06e1.95 (m, 2H), 1.80e1.53 (m, 6H).

d

7.66e7.56 (m, 2H), 7.14 (t, J ¼ 8.7 Hz,1H), 2.87e2.77

[

a

]

²⁵D ¼ 0.00 (C 1.0, MeOH).

4.2.2.2.

2-(((1R,4R)-4-hydroxycyclohexyl)amino)-5-(3,3-

4.2.1.9. 2-ﬂuoro-5-(cyclohexylethynyl)benzonitrile (44). Inter-

mediate 44 was prepared from 35 (370 mg, 1.5 mmol) and ethy-

nylcyclohexane (324 mg, 3.0 mmol) according to the general

procedures. Light yellow oil (322 mg, 94.5%). ¹H NMR (300 MHz,

dimethylbut-1-yn-1-yl)benzamide (2). Compound 2 was prepared

from intermediate 37 (295 mg, 1.47 mmol) according to the general

procedures. White solid (272 mg, 58.9%). Mp 207.1e208.8 ^ꢀC. ¹H

NMR (300 MHz, DMSO‑d₆)

d

8.28 (d, J ¼ 7.6 Hz, 1H), 7.91 (s, 1H), 7.59

chloroform-d)

d

7.66e7.58 (m, 2H), 7.15 (t, J ¼ 8.7 Hz,1H), 2.63e2.54

(s, 1H), 7.19 (d, J ¼ 8.5 Hz, 1H), 7.13 (s, 1H), 6.64 (d, J ¼ 8.8 Hz, 1H),

4.57 (d, J ¼ 4.0 Hz, 1H), 3.50e3.42 (m, 1H), 3.29e3.24 (m, 1H),

1.96e1.93 (m, 2H), 1.83e1.79 (m, 2H), 1.36e1.32 (m, 2H), 1.26 (s,

9H), 1.19e1.09 (m, 2H). HRMS (ESI): calcd for C₁₉H₂₆N₂O₂[M þ H]^þ

315.20, found 315.20. Purity: 99.05% by HPLC (MeOH/H₂O ¼ 90:10).

(m, 1H), 1.94e1.83 (m, 2H), 1.79e1.70 (m, 2H), 1.57e1.48 (m, 2H),

1.44e1.25 (m, 4H).

4.2.2. General Procedure for the Preparation of Compounds 1e9.

To a stirred solution of the appropriate intermediate 36e44 (1.0

equiv) in DMSO were added trans-4-aminocyclohexanol (4.5 equiv)

and DIPEA (9.0 equiv). The reaction mixture was reﬂuxed at 120 ^ꢀC

for 16 h. After the reaction was ﬁnished, the reaction mixture was

diluted with water, and the aqueous layer was extracted with EA.

The combined EA layers were dried over anhydrous Na₂SO₄,

concentrated under reduced pressure and dissolved in EtOH (15 ml)

and DMSO (1 ml). The reaction mixture which was added NaOH

(2 M, 2 ml), and 30% H₂O₂(2 ml) stirred at 30 ^ꢀC for 12 h. After

removing the EtOH by concentration under reduced pressure, the

residue was dissolved in EA (20 ml), washed with water (20 ml ꢁ 2)

and brine (30 mL ꢁ 3), dried over anhydrous Na₂SO₄, and

concentrated in vacuo. The residue was puriﬁed by silica gel column

chromatography (PE/EA, 2:1) to afford 1e9.

R_f: 0.33 by TLC (PE/EA ¼ 1:1). [

a

]

²⁵D ¼ 0.00 (C 1.0, MeOH).

4.2.2.3.

2-(((1R,4R)-4-hydroxycyclohexyl)amino)-5-(4-

methylpent-1-yn-1-yl)benzamide (3). Compound 3 was prepared

from intermediate 38 (272 mg, 1.35 mmol) according to the general

procedures. White solid (193 mg, 45.5%). Mp 157.3e158.5 ^ꢀC. ¹H

NMR (300 MHz, DMSO‑d₆)

d

8.31 (d, J ¼ 7.2 Hz, 1H), 7.91 (s, 1H), 7.65

(s, 1H), 7.25e7.17 (m, 2H), 6.65 (d, J ¼ 8.7 Hz, 1H), 4.59 (d, J ¼ 3.9 Hz,

1H), 3.50e3.41 (m, 1H), 3.32e3.21 (m, 1H), 2.28e2.26 (m, 2H),

1.96e1.93 (m, 2H), 1.83e1.79 (m, 3H), 1.33e1.15 (m, 4H), 0.99 (d,

J ¼ 6.5 Hz, 6H). HRMS (ESI): calcd for C₁₉H₂₆N₂O₂[M þ H]^þ315.20,

found 315.21. Purity: 96.87% by HPLC (MeOH/H₂O ¼ 90:10). R_f: 0.35

by TLC (PE/EA ¼ 1:1). [

a

]

²⁵D ¼ 0.00 (C 1.0, MeOH).

4.2.2.4. 2-(((1R,4R)-4-hydroxycyclohexyl)amino)-5-(hex-1-yn-

1-yl)benzamide (4). Compound 4 was prepared from intermediate

39 (270 mg, 1.34 mmol) according to the general procedures. White

solid (166 mg, 39.3%). Mp 173.3e175.1 ^ꢀC. ¹H NMR (300 MHz,

4.2.2.1.

2-(((1R,4R)-4-hydroxycyclohexyl)amino)-5-(3-

methylbut-1-yn-1-yl)benzamide (1). Compound 1 was prepared

from intermediate 36 (260 mg, 1.39 mmol) according to the general

procedures. White solid (160 mg, 38.3%). Mp 198.6e200.0 ^ꢀC. ¹H

DMSO‑d₆)

d

8.30 (d, J ¼ 7.6 Hz, 1H), 7.89 (s, 1H), 7.64 (s, 1H), 7.23 (d,

NMR (300 MHz, DMSO‑d₆)

(d, J ¼ 1.8 Hz, 1H), 7.21 (dd, J ¼ 8.8, 1.5 Hz, 1H), 7.16 (s, 1H), 6.65 (d,

d

8.30 (d, J ¼ 7.8 Hz,1H), 7.91 (s, 1H), 7.62

J ¼ 8.7 Hz, 1H), 7.13 (s, 1H), 6.65 (d, J ¼ 8.8 Hz, 1H), 4.55 (s, 1H),

3.49e3.43 (m, 1H), 3.28e3.23 (m, 1H), 2.37 (t, J ¼ 6.7 Hz, 2H),

10

S. Xu, A. Guo, N.-n. Chen et al.

European Journal of Medicinal Chemistry 223 (2021) 113604

1.97e1.93 (m, 2H), 1.83e1.79 (m, 2H), 1.55e1.38 (m, 4H), 1.36e1.25

(m, 2H),1.23e1.12 (m, 2H), 0.91 (t, J ¼ 6.9 Hz, 3H). ¹³C NMR (75 MHz,

(s, 2H), 1.50e1.10 (m, 10H). HRMS (ESI): calcd for C₂₁H₂₈N₂O₂[M þ

H]^þ341.22, found 341.22. Purity: 97.59% by HPLC (MeOH/

DMSO‑d₆)

d

170.97, 148.39, 135.29, 132.27, 113.42, 111.64, 107.84,

H₂O ¼ 90:10). R_f: 0.37 by TLC (PE/EA ¼ 1:1). [

a

]

²⁵D ¼ ꢂ0.20 (C 1.0,

87.13, 80.93, 67.96, 49.35, 33.36, 30.55, 30.12, 21.41, 18.37, 13.46.

HRMS (ESI): calcd for C₁₉H₂₆N₂O₂[M þ H]^þ315.20, found 315.21.

Purity: 98.86% by HPLC (MeOH/H₂O ¼ 90:10). R_f: 0.32 by TLC (PE/

MeOH).

4.2.3. General Procedure for the Intermediate 2-(((1R,4R)-4-

hydroxycyclohexyl)amino)-5-iodobenzamide (45). 35 (1 equiv)

was dissolved in DMSO, followed by addition of trans-4-

aminocyclohexanol (4.5 equiv) and DIPEA (9 equiv) and stirred

for 16 h at 120 ^ꢀC. After the reaction was ﬁnished, the mixture was

poured into water (60 ml) and extracted with ethyl acetate

(60 ml ꢁ 2). Finally, the EA layer was washed with brine (30 ml ꢁ 3),

dried over anhydrous sodium sulfate, and concentrated under

reduced pressure to afford the residue. The residue was dissolved in

EtOH (30 ml) and DMSO (5 ml). NaOH (2 M, 4 ml) and 30% H₂O₂

(3 ml) were added. Reaction was stirred at 30 ^ꢀC for 12 h. EtOH was

removed under reduced pressure after the reaction was completed.

The residue was dissolved in EA (50 ml) and washed with water

(50 ml ꢁ 2) and brine (30 mL ꢁ 3), dried over anhydrous Na₂SO₄

and concentrated in vacuo. The residue was puriﬁed by silica gel

column chromatography (PE/EA, 2:1) to afford intermediate 45.

Intermediate 45 was prepared from 35 (987.0 mg, 4.0 mmol) ac-

cording to the general procedures. Light yellow solid (731 mg,

EA ¼ 1:1). [

a

]

²⁵D ¼ - 0.20 (C 1.0, MeOH).

4.2.2.5.

2-(((1R,4R)-4-hydroxycyclohexyl)amino)-5-(5-

chloropent-1-yn-1-yl)benzamide (5). Compound 5 was prepared

from intermediate 40 (258 mg, 1.17 mmol) according to the general

procedures. White solid (75 mg,19.2%). Mp 184.0e185.7 ^ꢀC. ¹H NMR

(300 MHz, DMSO‑d₆)

d

8.32 (d, J ¼ 7.1 Hz, 1H), 7.89 (s, 1H), 7.66 (s,

1H), 7.24 (d, J ¼ 8.1 Hz, 1H), 7.15 (s, 1H), 6.65 (d, J ¼ 8.9 Hz, 1H), 4.56

(d, J ¼ 3.9 Hz, 1H), 3.76 (t, J ¼ 6.4 Hz, 2H), 3.50e3.42 (m, 1H),

3.28e3.24 (m, 1H), 2.55e2.52 (m, 2H), 1.98e1.91 (m, 4H), 1.82e1.78

(m, 2H), 1.36e1.27 (m, 2H), 1.19e1.11 (m, 2H). ¹³C NMR (75 MHz,

DMSO‑d₆)

d 170.92, 148.53, 135.32, 132.45, 113.34, 111.65, 107.36,

85.43, 81.64, 67.95, 49.32, 44.21, 33.36, 31.30, 30.11, 16.22. HRMS

(ESI): calcd for C₁₈H₂₃ClN₂O₂[M þ Na]^þ357.14, found 357.13. Pu-

rity: 99.90% by HPLC (MeOH/H₂O ¼ 90:10). R_f: 0.36 by TLC (PE/

EA ¼ 1:1). [

a

]

²⁵D ¼ 0.30 (C 1.0, MeOH).

4.2.2.6.

2-(((1R,4R)-4-hydroxycyclohexyl)amino)-5-(5-

methylhex-1-yn-1-yl)benzamide (6). Compound 6 was prepared

from intermediate 41 (288 mg, 1.34 mmol) according to the general

procedures. White solid (240 mg, 54.6%). Mp 156.1e157.6 ^ꢀC. ¹H

50.8%). ¹H NMR (300 MHz, DMSO‑d₆)

d

8.04 (d, J ¼ 7.7 Hz, 1H), 7.80

(s, 1H), 7.74 (d, J ¼ 2.2 Hz, 1H), 7.37 (dd, J ¼ 8.8, 2.1 Hz, 1H), 7.10 (s,

1H), 6.47 (d, J ¼ 9.0 Hz, 1H), 4.47 (d, J ¼ 4.3 Hz, 1H), 3.44e3.29 (m,

1H), 3.20e3.11 (s, 1H), 1.86e1.82 (m, 2H), 1.73e1.68 (m, 2H),

1.28e0.98 (m, 4H).

NMR (300 MHz, DMSO‑d₆)

d

8.29 (d, J ¼ 7.5 Hz,1H), 7.88 (s, 1H), 7.64

(s, 1H), 7.23 (d, J ¼ 8.6 Hz, 1H), 7.12 (s, 1H), 6.66 (d, J ¼ 8.8 Hz, 1H),

4.54 (s,1H), 3.52e3.43 (m,1H), 3.29e3.25 (m,1H), 2.38 (t, J ¼ 7.3 Hz,

2H),1.97e1.93 (m, 2H),1.84e1.80 (m, 2H),1.76e1.65 (m,1H),1.43 (q,

J ¼ 7.2 Hz, 2H), 1.34e1.13 (m, 4H), 0.91 (d, J ¼ 6.6 Hz, 6H). HRMS

(ESI): calcd for C₂₁H₃₀N₂O₂[M þ H]^þ329.22, found 329.22. Purity:

97.12% by HPLC (MeOH/H₂O ¼ 90:10). R_f: 0.25 by TLC (PE/EA ¼ 1:1).

4.2.4. General Procedure for the compounds 10e11. The method

used for the synthesis of compounds 10e11 is similar to the one

used for the synthesis of compounds 1e9.

4.2.4.1.2-(((1R,4R)-4-hydroxycyclohexyl)amino)-5-(3 (dimethy-

lamino)prop-1-yn-1-yl)benzamide (10). Compound 10 was pre-

pared from intermediate 45 (270 mg, 0.75 mmol) and N,N-

dimethylprop-2-yn-1-amine (125 mg, 1.5 mmol) according to the

general procedures. Light yellow solid (67 mg, 28.3%). Mp

[

a

]

²⁵D ¼ ꢂ0.10 (C 1.0, MeOH).

4.2.2.7.

2-(((1R,4R)-4-hydroxycyclohexyl)amino)-5-(cyclo-

propylethynyl)benzamide (7). Compound 7 was prepared from

intermediate 42 (270 mg, 1.46 mmol) according to the general

procedures. White solid (260 mg, 59.7%). Mp 218.8e220.4 ^ꢀC. ¹H

189.4e190.9 ^ꢀC. ¹H NMR (300 MHz, DMSO‑d₆)

d 8.30e8.25 (m, 1H),

7.84 (s,1H), 7.61 (s, 1H), 7.19e7.16 (m, 1H), 7.10 (s,1H), 6.61e6.56 (m,

1H), 4.50 (s, 1H), 3.42e3.30 (m, 3H), 3.21e3.14 (m, 1H), 2.16 (s, 6H),

1.87e1.83 (m, 2H), 1.73e1.69 (m, 2H), 1.23e1.06 (m, 4H). HRMS

(ESI): calcd for C₁₈H₂₅N₃O₂[M þ H]^þ316.19, found 316.20. Purity:

96.62% by HPLC (MeOH/H₂O ¼ 90:10). R_f: 0.34 by TLC (DCM/

NMR (300 MHz, DMSO‑d₆)

d

8.31 (d, J ¼ 7.6 Hz,1H), 7.87 (s, 1H), 7.63

(s, 1H), 7.21 (d, J ¼ 8.3 Hz, 1H), 7.14 (s, 1H), 6.64 (d, J ¼ 8.8 Hz, 1H),

4.56 (d, J ¼ 4.0 Hz, 1H), 3.50e3.42 (m, 1H), 3.29e3.22 (m, 1H),

1.95e1.91 (m, 2H), 1.82e1.78 (m, 2H), 1.52e1.44 (m, 1H), 1.36e1.24

(m, 2H), 1.23e1.11 (m, 2H), 0.86e0.80 (m, 2H), 0.68e0.63 (m, 2H).

HRMS (ESI): calcd for C₁₈H₂₂N₂O₂[M þ H]^þ299.17, found 299.18.

Purity: 96.62% by HPLC (MeOH/H₂O ¼ 90:10). R_f: 0.25 by TLC (PE/

MEOH ¼ 4:1). [

a

]

²⁵D ¼ 0.10 (C 1.0, MeOH).

4.2.4.2.2-(((1R,4R)-4-hydroxycyclohexyl)amino)-5-(3-(dieth-

ylamino)prop-1-yn-1-yl) benzamide (11). Compound 11 was pre-

pared from intermediate 45 (360 mg, 1 mmol) and N,N-

diethylprop-2-yn-1-amine (222 mg, 2 mmol) according to the

general procedures. Hazel solid (144 mg, 42.0%). Mp

EA ¼ 1:1). [

a

]

²⁵D ¼ 0.30 (C 1.0, MeOH).

4.2.2.8.

2-(((1R,4R)-4-hydroxycyclohexyl)amino)-5-(cyclo-

pentylethynyl)benzamide (8). Compound 8 was prepared from in-

termediate 43 (266 mg, 1.25 mmol) according to the general

procedures. White solid (227 mg, 55.7%). Mp 208.8e210.6 ^ꢀC. ¹H

196.9e197.9 ^ꢀC. ¹H NMR (300 MHz, DMSO‑d₆)

d

8.37 (d, J ¼ 8.0 Hz,

1H), 7.88 (s, 1H), 7.67 (s, 1H), 7.24 (d, J ¼ 9.2 Hz,1H), 7.18 (s, 1H), 6.62

(d, J ¼ 8.9 Hz, 1H), 4.55 (s, 1H), 4.03 (s, 2H), 2.97 (d, J ¼ 7.5 Hz, 4H),

2.41 (s, 2H), 1.84 (d, J ¼ 16.0 Hz, 2H), 1.71 (d, J ¼ 11.7 Hz, 2H),

1.31e1.15 (m, 4H), 1.12 (t, J ¼ 7.0 Hz, 6H). ¹³C NMR (75 MHz, DMSO)

NMR (300 MHz, DMSO‑d₆)

d

8.29 (d, J ¼ 7.6 Hz,1H), 7.90 (s, 1H), 7.62

(s, 1H), 7.21 (d, J ¼ 8.4 Hz, 1H), 7.14 (s, 1H), 6.64 (d, J ¼ 8.9 Hz, 1H),

4.57 (d, J ¼ 3.5 Hz, 1H), 3.49e3.43 (m, 1H), 3.29e3.24 (m, 1H),

2.85e2.75 (m, 1H), 1.96e1.92 (m, 4H), 1.82e1.79 (m, 2H), 1.70e1.66

(m, 2H), 1.61e1.55 (m, 4H), 1.36e1.25 (m, 2H), 1.23e1.11 (m, 2H).

HRMS (ESI): calcd for C₂₀H₂₆N₂O₂[M þ H]^þ327.20, found 327.21.

Purity: 97.66% by HPLC (MeOH/H₂O ¼ 90:10). R_f: 0.34 by TLC (PE/

d

170.91, 148.63, 135.49, 132.52, 113.37, 111.66, 107.07, 84.97, 81.87,

67.95, 49.30, 46.54, 41.02, 33.36, 30.08, 12.49. HRMS (ESI): calcd for

C

₂₀H₂₉N₃O₂[M þ H]^þ344.23, found 344.23. Purity: 98.00% by HPLC

(MeOH/H₂O ¼ 90:10). R_f: 0.32 by TLC (DCM/MEOH ¼ 4:1). [

a]

EA ¼ 1:1). [

a

]

²⁵D ¼ ꢂ0.10 (C 1.0, MeOH).

²⁵D ¼ 0.40 (C 1.0, MeOH).

4.2.2.9.

2-(((1R,4R)-4-hydroxycyclohexyl)amino)-5-(cyclo-

4.2.5. General Procedure for the compounds 12e23 and in-

termediates 46e48. The method used for the synthesis of com-

pounds 12e23 and intermediates 46e48 is similar to the one used

for the synthesis of compounds 1e9.

hexylethynyl)benzamide (9). Compound 9 was prepared from in-

termediate 44 (322 mg, 1.42 mmol) according to the general

procedures. White solid (236 mg, 48.8%). Mp 191.6e193.5 ^ꢀC. ¹H

NMR (300 MHz, DMSO‑d₆)

d

8.30e8.28 (m, 1H), 7.91 (s, 1H), 7.61 (s,

4.2.5.1.2-((2-hydroxyethyl)amino)-5-(hex-1-yn-1-yl)benzamide

(12). Compound 12 was prepared from intermediate 39 (201 mg,

1.0 mmol) and 2-aminoethan-1-ol (275 mg, 4.5 mmol) according to

the general procedures. White solid (92 mg, 35.4%). Mp

1H), 7.21 (d, J ¼ 8.6 Hz, 1H), 7.14 (s, 1H), 6.65e6.63 (m, 1H),

4.58e4.56 (m, 1H), 3.50e3.40 (m, 1H), 3.29e3.23 (m, 1H),

2.61e2.55 (m, 1H), 1.95e1.91 (m, 2H), 1.81e1.78 (m, 4H), 1.70e1.63

11

S. Xu, A. Guo, N.-n. Chen et al.

European Journal of Medicinal Chemistry 223 (2021) 113604

81.5e82.8 ^ꢀC. ¹H NMR (300 MHz, DMSO‑d₆)

d

8.26 (t, J ¼ 5.2 Hz, 1H),

found 325.19. Purity: 99.21% by HPLC (MeOH/H₂O ¼ 90:10). R_f: 0.4

by TLC (PE/EA ¼ 2:1).

7.81 (s, 1H), 7.54 (s, 1H), 7.15 (d, J ¼ 8.1 Hz, 1H), 7.06 (s, 1H), 6.52 (d,

J ¼ 8.3 Hz, 1H), 4.70 (t, J ¼ 4.8 Hz, 1H), 3.50e3.44 (m, 2H), 3.09e3.04

(m, 2H), 2.28 (t, J ¼ 6.2 Hz, 2H), 1.42e1.29 (m, 4H), 0.81 (t, J ¼ 7.0 Hz,

3H). HRMS (ESI): calcd for C₁₅H₂₀N₂O₂[M þ Na]^þ283.15, found

283.14. Purity: 99.26% by HPLC (MeOH/H₂O ¼ 90:10). R_f: 0.25 by

TLC (PE/EA ¼ 1:1).

4.2.5.7.2-((2-acetamidoethyl)amino)-5-(hex-1-yn-1-yl)benza-

mide (18). Compound 18 was prepared from intermediate 39

(201 mg, 1.0 mmol) and N-(2-aminoethyl)acetamide (459 mg,

4.5 mmol) according to the general procedures. White solid

(159 mg, 52.8%). Mp 141.2e143.6 ^ꢀC. ¹H NMR (300 MHz, DMSO‑d₆)

4.2.5.2.2-((2-methoxyethyl)amino)-5-(hex-1-yn-1-yl)benza-

mide (13). Compound 13 was prepared from intermediate 39

(201 mg, 1.0 mmol) and 2-methoxyethan-1-amine (337 mg,

4.5 mmol) according to the general procedures. White solid

(105 mg, 38.3%). Mp 83.6e85.1 ^ꢀC. ¹H NMR (300 MHz, chloroform-

d

8.25 (s, 1H), 7.92 (s, 1H), 7.82 (s, 1H), 7.56 (s, 1H), 7.16 (d, J ¼ 8.8 Hz,

1H), 7.10 (s, 1H), 6.60 (d, J ¼ 8.7 Hz, 1H), 3.14e3.04 (m, 4H), 2.28 (t,

J ¼ 6.5 Hz, 2H),1.71 (s, 3H),1.45e1.29 (m, 4H), 0.81 (t, J ¼ 7.0 Hz, 3H).

¹³C NMR (75 MHz, DMSO‑d₆)

d 170.79, 169.49, 149.04, 135.28,

132.13, 113.91, 111.10, 108.31, 87.32, 80.85, 41.33, 37.95, 30.52, 22.53,

21.42, 18.36, 13.49. HRMS (ESI): calcd for C₁₇H₂₃N₃O₂[M þ Na]^þ

324.19, found 324.17. Purity: 99.37% by HPLC (MeOH/H₂O ¼ 90:10).

R_f: 0.3 by TLC (PE/EA ¼ 2:1).

d)

d

8.09 (s, 1H), 7.47 (s, 1H), 7.36 (d, J ¼ 8.6 Hz, 1H), 6.64 (d,

J ¼ 8.6 Hz, 1H), 5.76 (brs, 2H), 3.64 (t, J ¼ 5.5 Hz, 2H), 3.42 (s, 3H),

3.38e3.37 (m, 2H), 2.40 (t, J ¼ 6.8 Hz, 2H), 1.63e1.54 (m, 2H),

1.52e1.44 (m, 2H), 0.96 (t, J ¼ 7.1 Hz, 3H). HRMS (ESI): calcd for

4.2.5.8.2-((tetrahydrofuran-3-yl)amino)-5-(hex-1-yn-1-yl)ben-

zamide (19). Compound 19 was prepared from intermediate 39

(201 mg, 1.0 mmol) and tetrahydrofuran-3-amine (392 mg,

4.5 mmol) according to the general procedures. White solid (98 mg,

34.2%). Mp 153.0e154.8 ^ꢀC. ¹H NMR (300 MHz, chloroform-d)

C

₁₆H₂₂N₂O₂[M þ Na]^þ297.17, found 297.16. Purity: 99.22% by HPLC

(MeOH/H₂O ¼ 90:10). R_f: 0.25 by TLC (PE/EA ¼ 2:1).

4.2.5.3.2-((2-ethoxyethyl)amino)-5-(hex-1-yn-1-yl)benzamide

(14). Compound 14 was prepared from intermediate 39 (201 mg,

1.0 mmol) and 2-ethoxyethan-1-amine (401 mg, 4.5 mmol) ac-

cording to the general procedures. White solid (105 mg, 38.3%).

White solid (218 mg, 75.6%). Mp 96.5e97.8 ^ꢀC. ¹H NMR (300 MHz,

d

8.14e8.12 (m, 1H), 7.47e7.46 (m, 1H), 7.37e7.34 (m, 1H), 6.58 (d,

J ¼ 8.7 Hz, 1H), 5.74 (brs, 2H), 4.13e4.01 (m, 2H), 3.99e3.86 (m, 2H),

3.72e3.68 (m, 1H), 2.39 (t, J ¼ 6.9 Hz, 2H), 2.33e2.24 (m, 1H),

1.96e1.87 (m, 1H), 1.63e1.42 (m, 4H), 0.94 (t, J ¼ 7.1 Hz, 3H). HRMS

(ESI): calcd for C₁₇H₂₂N₂O₂[M þ Na]^þ309.17, found 309.16. Purity:

98.23% by HPLC (MeOH/H₂O ¼ 90:10). R_f: 0.25 by TLC (PE/EA ¼ 2:1).

4.2.5.9.2-(((tetrahydrofuran-3-yl)methyl)amino)-5-(hex-1-yn-

1-yl)benzamide (20). Compound 20 was prepared from interme-

diate 39 (201 mg, 1.0 mmol) and (tetrahydrofuran-3-yl)methan-

amine (455 mg, 4.5 mmol) according to the general procedures.

White solid (176 mg, 58.6%). Mp 138.5e139.9 ^ꢀC. ¹H NMR (300 MHz,

chloroform-d)

d

8.09 (s, 1H), 7.47e7.46 (m, 1H), 7.35 (dd, J ¼ 8.8,

1.8 Hz, 1H), 6.64 (d, J ¼ 8.8 Hz, 1H), 5.71 (brs, 2H), 3.68 (t, J ¼ 5.7 Hz,

2H), 3.57 (q, J ¼ 7.0 Hz, 2H), 3.38 (t, J ¼ 5.5 Hz, 2H), 2.40 (t, J ¼ 6.9 Hz,

2H), 1.63e1.54 (m, 2H), 1.52e1.42 (m, 2H), 1.25 (t, J ¼ 7.0 Hz, 3H),

0.96 (t, J ¼ 7.2 Hz, 3H). HRMS (ESI): calcd for C₁₇H₂₄N₂O₂[M þ Na]^þ

311.18, found 311.17. Purity: 96.97% by HPLC (MeOH/H₂O ¼ 90:10).

R_f: 0.3 by TLC (PE/EA ¼ 2:1).

4.2.5.4.2-((3-hydroxypropyl)amino)-5-(hex-1-yn-1-yl)benza-

mide (15). Compound 15 was prepared from intermediate 39

(201 mg, 1.0 mmol) and 3-aminopropan-1-ol (338 mg, 4.5 mmol)

according to the general procedures. White solid (105 mg, 38.3%).

White solid (127 mg, 46.3%). Mp 124.7e126.5 ^ꢀC. ¹H NMR

DMSO‑d₆)

d 8.35 (s, 1H), 7.85 (s, 1H), 7.58e7.57 (m, 1H), 7.18e7.15

(m, 1H), 7.11 (s, 1H), 6.57e6.54 (m, 1H), 3.67e3.62 (m, 2H),

3.58e3.50 (m, 1H), 3.35e3.30 (m, 1H), 3.03e2.99 (m, 2H),

2.30e2.26 (m, 2H), 1.94e1.86 (m, 1H), 1.55e1.45 (m, 2H), 1.43e1.29

(m, 4H), 0.84e0.79 (m, 3H). HRMS (ESI): calcd for C₁₈H₂₄N₂O₂[M þ

Na]^þ323.18, found 323.17. Purity: 99.55% by HPLC (MeOH/

H₂O ¼ 90:10). R_f: 0.2 by TLC (PE/EA ¼ 2:1).

(300 MHz, DMSO‑d₆)

d

8.31 (t, J ¼ 5.2 Hz, 1H), 7.92 (s, 1H), 7.65e7.64

(m, 1H), 7.27e7.23 (m, 1H), 7.19 (s, 1H), 6.62 (d, J ¼ 8.7 Hz, 1H), 4.56

(t, J ¼ 5.1 Hz,1H), 3.48 (q, J ¼ 5.9 Hz, 2H), 3.16 (q, J ¼ 6.4 Hz, 2H), 2.37

(t, J ¼ 6.6 Hz, 2H), 1.73e1.64 (m, 2H), 1.54e1.36 (m, 4H), 0.91 (t,

J ¼ 7.0 Hz, 3H). HRMS (ESI): calcd for C₁₆H₂₂N₂O₂[M þ Na]^þ297.17,

found 297.16. Purity: 97.65% by HPLC (MeOH/H₂O ¼ 90:10). R_f: 0.5

by TLC (DCM/MEOH ¼ 20:1).

4.2.5.10.2-((tetrahydro-2H-pyran-4-yl)amino)-5-(hex-1-yn-1-

yl)benzamide (21). Compound 21 was prepared from intermediate

39 (201 mg, 1.0 mmol) and tetrahydro-2H-pyran-4-amine (455 mg,

4.5 mmol) according to the general procedures. White solid

(165 mg, 55%). Mp 182.1e183.9 ^ꢀC. ¹H NMR (300 MHz, DMSO‑d₆)

4.2.5.5.2-((3-methoxypropyl)amino)-5-(hex-1-yn-1-yl)benza-

mide (16). Compound 16 was prepared from intermediate 39

(201 mg, 1.0 mmol) and 3-methoxypropan-1-amine (401 mg,

4.5 mmol) according to the general procedures. Light yellow solid

(185 mg, 64.2%). Mp 86.8e88.5 ^ꢀC. ¹H NMR (300 MHz, chloroform-

d

8.32 (d, J ¼ 7.6 Hz, 1H), 7.84 (s, 1H), 7.57 (s, 1H), 7.14 (d, J ¼ 8.5 Hz,

1H), 7.10 (s, 1H), 6.62 (d, J ¼ 8.8 Hz, 1H), 3.73 (d, J ¼ 12.0 Hz, 2H),

3.54e3.45 (m, 1H), 3.36 (t, J ¼ 10.7 Hz, 2H), 2.28 (t, J ¼ 6.7 Hz, 2H),

1.83e1.79 (m, 2H), 1.45e1.21 (m, 6H), 0.81 (t, J ¼ 7.1 Hz, 3H). ¹³C

d)

d

8.00 (s, 1H), 7.46e7.45 (m, 1H), 7.37e7.33 (m, 1H), 6.65 (d,

NMR (75 MHz, DMSO‑d₆) d 170.93, 147.97, 135.29, 132.31, 113.60,

J ¼ 8.5 Hz, 1H), 5.67 (brs, 2H), 3.50 (t, J ¼ 5.9 Hz, 2H), 3.37 (s, 3H),

3.31e3.27 (m, 2H), 2.40 (t, J ¼ 6.7 Hz, 2H), 1.97e1.88 (m, 2H),

1.61e1.54 (m, 2H), 1.51e1.44 (m, 2H), 0.96 (t, J ¼ 7.1 Hz, 3H). HRMS

(ESI): calcd for C₁₇H₂₄N₂O₂[M þ Na]^þ311.18, found 311.17. Purity:

99.18% by HPLC (MeOH/H₂O ¼ 90:10). R_f: 0.3 by TLC (PE/EA ¼ 2:1).

4.2.5.6.2-((3-ethoxypropyl)amino)-5-(hex-1-yn-1-yl)benza-

mide (17). Compound 17 was prepared from intermediate 39

(201 mg, 1.0 mmol) and 3-ethoxypropan-1-amine (464 mg,

4.5 mmol) according to the general procedures. Light yellow solid

(159 mg, 52.6%). Mp 75.7e76.9 ^ꢀC. ¹H NMR (300 MHz, chloroform-

111.74, 108.20, 87.28, 80.84, 65.53, 46.95, 32.67, 30.52, 21.42, 18.36,

13.48. HRMS (ESI): calcd for C₁₇H₂₄N₂O₂[M þ H]^þ301.18, found

301.19. Purity: 96.51% by HPLC (MeOH/H₂O ¼ 90:10). R_f: 0.3 by TLC

(PE/EA ¼ 2:1).

4.2.5.11.2-(((tetrahydro-2H-pyran-4-yl)methyl)amino)-5-(hex-

1-yn-1-yl)benzamide (22). Compound 22 was prepared from in-

termediate 39 (201 mg, 1.0 mmol) and (tetrahydro-2H-pyran-4-yl)

methanamine (518 mg, 4.5 mmol) according to the general pro-

cedures. White solid (137 mg, 43.6%). Mp 150.3e151.9 ^ꢀC. ¹H NMR

(300 MHz, chloroform-d) d 8.04 (s, 1H), 7.37e7.36 (m, 1H), 7.26 (dd,

d)

d

7.99 (s, 1H), 7.46 (s, 1H), 7.35 (d, J ¼ 8.6 Hz, 1H), 6.66 (d,

J ¼ 8.7, 2.0 Hz, 1H), 6.52 (d, J ¼ 8.8 Hz, 1H), 5.60 (brs, 2H), 3.91 (dd,

J ¼ 11.1, 3.2 Hz, 2H), 3.31 (t, J ¼ 11.0 Hz, 2H), 2.99 (d, J ¼ 6.2 Hz, 2H),

2.31 (t, J ¼ 6.9 Hz, 2H), 1.84e1.76 (m, 1H), 1.67e1.62 (m, 2H),

1.54e1.23 (m, 6H), 0.87 (t, J ¼ 7.1 Hz, 3H). HRMS (ESI): calcd for

J ¼ 8.5 Hz,1H), 5.65 (brs, 2H), 3.56e3.47 (m, 4H), 3.33e3.28 (m, 2H),

2.40 (t, J ¼ 6.9 Hz, 2H), 1.97e1.89 (m, 2H), 1.64e1.55 (m, 2H),

1.52e1.45 (m, 2H), 1.23 (t, J ¼ 6.9 Hz, 3H), 0.96 (t, J ¼ 7.0 Hz, 3H). ¹³

C

NMR (75 MHz, DMSO‑d₆)

d

170.88, 149.20, 135.33, 132.10, 113.66,

C

₁₉H₂₆N₂O₂[M þ Na]^þ337.20, found 337.19. Purity: 99.34% by HPLC

111.03, 108.08, 87.22, 80.90, 67.28, 65.34, 30.54, 28.88, 21.41, 18.37,

(MeOH/H₂O ¼ 90:10). R_f: 0.25 by TLC (PE/EA ¼ 2:1).

15.04, 13.47. HRMS (ESI): calcd for C₁₈H₂₆N₂O₂[M þ Na]^þ325.20,

4.2.5.12.2-((1-acetylpiperidin-4-yl)amino)-5-(hex-1-yn-1-yl)

12

S. Xu, A. Guo, N.-n. Chen et al.

European Journal of Medicinal Chemistry 223 (2021) 113604

benzamide (23). Compound 23 was prepared from intermediate 39

(201 mg, 1.0 mmol) and 1-(4-aminopiperidin-1-yl)ethan-1-one

(639 mg, 4.5 mmol) according to the general procedures. White

solid (102 mg, 29.9%). Mp 165.1e166.4 ^ꢀC. ¹H NMR (300 MHz,

(d, J ¼ 9.5 Hz, 1H), 6.67 (d, J ¼ 8.9 Hz, 1H), 3.13e3.03 (m, 4H), 2.61 (t,

J ¼ 12.2 Hz, 2H), 2.38 (t, J ¼ 6.6 Hz, 2H), 1.85e1.81 (m, 3H), 1.61e1.46

(m, 4H), 1.28e1.18 (m, 2H), 0.97 (t, J ¼ 7.2 Hz, 3H). HRMS (ESI): calcd

for C₁₉H₂₇N₃O [M þ H]^þ314.22, found 314.22. Purity: 95.21% by

HPLC (MeOH/H₂O ¼ 90:10). R_f: 0.25 by TLC (PE/EA ¼ 1:1).

DMSO‑d₆)

d

8.34 (d, J ¼ 7.3 Hz, 1H), 7.83 (s, 1H), 7.58 (s, 1H), 7.16 (d,

J ¼ 7.7 Hz, 1H), 7.10 (s, 1H), 6.64 (d, J ¼ 8.3 Hz, 1H), 4.03e3.99 (m,

1H), 3.64e3.52 (m, 2H), 3.17e3.09 (m, 1H), 2.84e2.76 (m, 1H),

2.30e2.26 (m, 2H), 1.91 (s, 3H), 1.82e1.75 (m, 2H), 1.43e1.23 (m,

4H), 1.11e1.08 (m, 2H), 0.83e0.79 (m, 3H). HRMS (ESI): calcd for

4.2.6.3.2-(piperidin-3-ylamino)-5-(hex-1-yn-1-yl)-benzamide

(26). Compound 26 was prepared from intermediate 48 (713 mg,

1.79 mmol) and silica gel (1.08 g, 17.9 mmol) according to the

general procedures. Light yellow solid (271 mg, 50.6%). Mp

C

₁₈H₂₅N₃O [M þ H]^þ364.21, found 364.20. Purity: 95.35% by HPLC

106.1e108.4 ^ꢀC. ¹H NMR (300 MHz, Methanol-d₄)

d 7.62 (s, 1H), 7.28

(MeOH/H₂O ¼ 90:10). R_f: 0.3 by TLC (PE/EA ¼ 2:1).

(d, J ¼ 8.7 Hz, 1H), 6.74 (d, J ¼ 8.8 Hz, 1H), 3.52e3.42 (m, 1H),

3.23e3.19 (m, 1H), 2.96e2.92 (m, 1H), 2.61 (t, J ¼ 11.7 Hz, 1H),

2.44e2.36 (m, 3H), 2.13e2.09 (m, 1H), 1.82e1.77 (m, 1H), 1.67e1.41

(m, 6H), 0.97 (t, J ¼ 6.6 Hz, 3H). HRMS (ESI): calcd for C₁₈H₂₅N₃O

[M þ H]^þ300.20, found 300.21. Purity: 96.33% by HPLC (MeOH/

H₂O ¼ 90:10). R_f: 0.4 by TLC (PE/EA ¼ 2:1).

4.2.5.13. tert-butyl-4-((2-carbamoyl-4-(hex-1-yn-1-yl)phenyl)

amino)piperidine-1-carboxylate (46). Intermediate 46 was pre-

pared from intermediate 39 (2.41 g, 12.0 mmol) and tert-butyl 4-

aminopiperidine-1-carboxylate (10.81 g, 54 mmol) according to

the general procedures. White solid (3.0 g, 62.6%). ¹H NMR

(300 MHz, DMSO‑d₆)

d

8.42 (d, J ¼ 7.6 Hz, 1H), 7.94 (s, 1H), 7.67 (s,

4.2.7. General Procedure for the compounds 27e34. A mixture of

appropriate compound (24e26, 1 equiv), certain acid (1.5 equiv),

Bop (1.5 equiv), and DIPEA (3 equiv) in DMF were stirred at room

temperature for 4 h. After the completion of reaction, the residue

was obtained by concentration under reduced pressure and dis-

solved in EA (20 ml). The organic phase was washed with water

(20 ml ꢁ 2) and saturated NaCl solution (20 ml ꢁ 2). The organic

layer was dried over anhydrous Na₂SO₄, ﬁltered and the solvent

was evaporated to dryness under reduced pressure. Finally, the

compounds 27e34 were obtained by silica gel column chroma-

tography (PE/EA ¼ 2:1).

J ¼ 2.1 Hz, 1H), 7.24 (d, J ¼ 9.0 Hz, 1H), 7.18 (s, 1H), 6.71 (d, J ¼ 8.7 Hz,

1H), 3.80e3.76 (m, 2H), 3.09e2.92 (m, 2H), 2.37 (t, J ¼ 6.8 Hz, 2H),

1.91e1.87 (m, 2H), 1.55e1.41 (m, 4H), 1.40 (s, 9H), 1.26e1.19 (m, 2H),

0.91 (t, J ¼ 7.0 Hz, 3H).

4.2.5.14. tert-butyl 4-(((2-carbamoyl-4-(hex-1-yn-1-yl)phenyl)

amino)methyl)piperidine-1-carboxylate (47). Intermediate 47 was

prepared from intermediate 39 (402 mg, 2.0 mmol) and tert-butyl

4-(aminomethyl)piperidine-1-carboxylate (1.93 g, 9.0 mmol) ac-

cording to the general procedures. Light yellow solid (673 mg,

81.4%). ¹H NMR (300 MHz, DMSO‑d₆)

d

8.44 (t, J ¼ 5.5 Hz, 1H), 7.91

(s, 1H), 7.64 (s, 1H), 7.22 (d, J ¼ 8.5 Hz, 1H), 7.15 (s, 1H), 6.62 (d,

J ¼ 8.8 Hz, 1H), 3.95e3.91 (m, 2H), 3.03e2.99 (m, 2H), 2.77e2.59

(m, 2H), 2.35 (t, J ¼ 6.7 Hz, 2H), 1.67e1.64 (m, 3H), 1.56e1.41 (m,

4H), 1.37 (s, 9H), 1.11e0.98 (s, 2H), 0.89 (t, J ¼ 7.0 Hz, 3H).

4.2.7.1 2-((1-(cyclopropanecarbonyl)piperidin-4-yl)amino)-5-

(hex-1-yn-1-yl)benzamide (27). Compound 27 was prepared from

24 (150 mg, 0.5 mmol) and cyclopropanecarboxylic acid (86 mg,

1.0 mmol) according to the general procedures. White solid

(121 mg, 65.9%). Mp 144.5e145.8 ^ꢀC. ¹H NMR (300 MHz, DMSO‑d₆)

4.2.5.15. tert-butyl-3-((2-carbamoyl-4-(hex-1-yn-1-yl)phenyl)

amino)piperidine-1-carboxylate (48). Intermediate 48 was pre-

pared from intermediate 39 (402 mg, 2.0 mmol) and tert-butyl 3-

aminopiperidine-1-carboxylate (1.80 g, 9 mmol) according to the

general procedures. Light yellow oil (713 mg, 89.3%). ¹H NMR

d

8.43 (d, J ¼ 7.2 Hz, 1H), 7.92 (s, 1H), 7.65 (s, 1H), 7.24 (d, J ¼ 9.7 Hz,

1H), 7.19 (s, 1H), 6.73 (d, J ¼ 9.1 Hz, 1H), 4.12e4.07 (m, 2H),

3.68e3.59 (m, 1H), 3.39e3.35 (m, 1H), 2.95e2.88 (m, 1H), 2.36 (t,

J ¼ 6.6 Hz, 2H), 2.01e1.92 (m, 3H), 1.53e1.37 (m, 4H), 1.22e1.13 (m,

2H), 0.89 (t, J ¼ 6.9 Hz, 3H), 0.68e0.66 (m, J ¼ 7.8 Hz, 4H). HRMS

(ESI): calcd for C₂₂H₂₉N₃O₂[M þ Na]^þ390.23, found 390.22. Purity:

97.97% by HPLC (MeOH/H₂O ¼ 90:10). R_f: 0.2 by TLC (PE/EA ¼ 2:1).

4.2.7.2.2-((1-(2-cyclopropylacetyl)piperidin-4-yl)amino)-5-

(300 MHz, chloroform-d)

d

8.04 (d, J ¼ 7.0 Hz, 1H), 7.46 (s, 1H), 7.34

(dd, J ¼ 8.6, 2.0 Hz, 1H), 6.74 (d, J ¼ 8.8 Hz, 1H), 5.72 (brs, 2H),

3.88e3.83 (m, 1H), 3.45e3.34 (m, 1H), 2.98e2.90 (m, 1H),

2.83e2.66 (m, 1H), 2.39 (t, J ¼ 6.9 Hz, 2H), 2.14e2.08 (m, 1H),

1.83e1.48 (m, 8H), 1.46 (s, 9H), 0.95 (t, J ¼ 7.1 Hz, 3H).

(hex-1-yn-1-yl)benzamide (28). Compound 28 was prepared from

24 (150 mg, 0.5 mmol) and 2-cyclopropylacetic acid (100 mg,

1.0 mmol) according to the general procedures. White solid

(133 mg, 69.8%). Mp 133.4e135.3 ^ꢀC. 1H NMR (300 MHz, DMSO‑d₆)

4.2.6. General Procedure for the compounds 24e26. Silica gel

(10 equiv) was added to a mixture of appropriate intermediate

46e48 (1 equiv) in toluene and stirred at 100 ^ꢀC for 48 h. After the

reaction was completed, it was concentrated and puriﬁed directly

by silica gel column chromatography (DCM/MeOH ¼ 30:1e10:1) to

give title compounds 24e26.

d

8.41 (d, J ¼ 7.7 Hz, 1H), 7.92 (s, 1H), 7.65 (s, 1H), 7.23 (d, J ¼ 8.1 Hz,

1H), 7.18 (s, 1H), 6.72 (d, J ¼ 8.9 Hz, 1H), 4.14e4.10 (m, 1H),

3.74e3.70 (m, 1H), 3.64e3.57 (m, 1H), 3.19 (t, J ¼ 12.1 Hz, 1H), 2.89

(t, J ¼ 11.7 Hz, 1H), 2.35 (t, J ¼ 6.7 Hz, 2H), 2.24 (d, J ¼ 6.8 Hz, 2H),

1.96e1.86 (m, 2H), 1.48e1.37 (m, 4H), 1.28e1.21 (m, 3H), 0.89 (t,

J ¼ 6.9 Hz, 3H), 0.42 (d, J ¼ 7.7 Hz, 2H), 0.09 (d, J ¼ 4.1 Hz, 2H). HRMS

(ESI): calcd for C₂₃H₃₁N₃O₂[M þ Na]^þ404.24, found 404.23. Purity:

96.39% by HPLC (MeOH/H₂O ¼ 90:10). R_f: 0.25 by TLC (PE/EA ¼ 1:1).

4.2.7.3.2-((1-(dimethylglycyl)piperidin-4-yl)amino)-5-(hex-1-

yn-1-yl)benzamide (29). Compound 29 was prepared from 24

(150 mg, 0.5 mmol) and dimethylglycine (103.1 mg, 1.0 mmol) ac-

cording to the general procedures. White solid (65 mg, 33.8%). Mp

4.2.6.1.2-(piperidin-4-ylamino)-5-(hex-1-yn-1-yl)benzamide

(24). Compound 24 was prepared from intermediate 46 (3 g,

7.5 mmol) and silica gel (4.51 g, 75 mmol) according to the general

procedures. White solid (2.1 g, 93.6%). Mp 183.2e183.9 ^ꢀC. ¹H NMR

(300 MHz, DMSO‑d₆)

d

8.39 (d, J ¼ 7.5 Hz, 1H), 7.92 (s, 1H), 7.65 (s,

1H), 7.22 (d, J ¼ 8.4 Hz, 1H), 7.18 (s, 1H), 6.67 (d, J ¼ 8.7 Hz, 1H),

3.47e3.38 (m, 1H), 2.95e2.90 (m, 2H), 2.59 (t, J ¼ 11.0 Hz, 2H), 2.37

(t, J ¼ 6.5 Hz, 2H), 1.88e1.84 (m, 2H), 1.52e1.38 (m, 4H), 1.26e1.22

(m, 2H), 0.91 (t, J ¼ 7.0 Hz, 3H). ¹³C NMR (75 MHz, DMSO‑d₆)

d

170.96, 148.08, 135.28, 132.31, 113.43, 111.63, 107.87, 87.17, 80.90,

153.9e155.6 ^ꢀC. ¹H NMR (300 MHz, DMSO‑d₆)

d

8.43 (d, J ¼ 7.6 Hz,

48.45, 44.46, 33.01, 30.54, 21.42, 18.36, 13.48. HRMS (ESI): calcd for

1H), 7.93 (s, 1H), 7.67 (s, 1H), 7.25 (d, J ¼ 8.9 Hz, 1H), 7.19 (s, 1H), 6.74

(d, J ¼ 8.8 Hz, 1H), 4.13e4.08 (m, 1H), 3.93e3.89 (m, 1H), 3.66e3.60

(m, 1H), 3.22e3.03 (m, 3H), 2.95e2.87 (m, 1H), 2.54 (s, 3H), 2.37 (t,

J ¼ 6.5 Hz, 2H), 2.19 (s, 3H), 1.96e1.92 (m, 2H), 1.55e1.39 (m, 4H),

1.24e1.16 (m, 2H), 0.91 (t, J ¼ 6.9 Hz, 3H). HRMS (ESI): calcd for

C

₁₈H₂₅N₃O [M þ H]^þ300.20, found 300.21. Purity: 95.35% by HPLC

(MeOH/H₂O ¼ 90:10). R_f: 0.3 by TLC (PE/EA ¼ 1:1).

4.2.6.2.2-((piperidin-4-ylmethyl)amino)-5-(hex-1-yn-1-yl)ben-

zamide (25). Compound 25 was prepared from intermediate 47

(673 mg,1.63 mmol) and silica gel (979 mg,16.3 mmol) according to

the general procedures. White solid (356 mg, 69.7%). Mp

C

₂₂H₃₂N₄O₂[M þ H]^þ385.25, found 385.26. Purity: 97.73% by HPLC

(MeOH/H₂O ¼ 90:10). R_f: 0.4 by TLC (PE/EA ¼ 2:1).

159.0e160.8 ^ꢀC. ¹H NMR (300 MHz, Methanol-d₄)

d

7.62 (s, 1H), 7.29

4.2.7.4.2-((1-(2-morpholinoacetyl)piperidin-4-yl)amino)-5-

13

S. Xu, A. Guo, N.-n. Chen et al.

European Journal of Medicinal Chemistry 223 (2021) 113604

(hex-1-yn-1-yl)benzamide (30). Compound 30 was prepared from

24 (150 mg, 0.5 mmol) and 2-morpholinoacetic acid (145.1 mg,

1.0 mmol) according to the general procedures. White solid

(137 mg, 64.3%). Mp 91.2e92.7 ^ꢀC. ¹H NMR (300 MHz, DMSO‑d₆)

5. Biology

5.1. Protein expression and puriﬁcation

D41

d

8.43 (d, J ¼ 7.6 Hz, 1H), 7.93 (s, 1H), 7.68 (s, 1H), 7.26 (d, J ¼ 8.6 Hz,

Canine Grp94^N

(residues 69e337; residues 286e328 from

1H), 7.17 (s, 1H), 6.74 (d, J ¼ 8.8 Hz, 1H), 4.12e4.07 (m, 1H),

3.95e3.91 (m,1H), 3.72e3.61 (m,1H), 3.61e3.53 (m, 4H), 3.25e3.20

(m, 2H), 3.11e3.06 (m, 1H), 2.97e2.89 (m, 1H), 2.42e2.36 (m, 6H),

1.99e1.91 (m, 2H), 1.54e1.39 (m, 4H), 1.28e1.19 (m, 2H), 0.91 (t,

J ¼ 7.1 Hz, 3H). HRMS (ESI): calcd for C₂₄H₃₄N₄O₃[M þ H]^þ427.26,

found 427.27. Purity: 99.35% by HPLC (MeOH/H₂O ¼ 90:10). R_f: 0.3

by TLC (DCM/MEOH ¼ 20:1).

the charged linker were replaced by four glycine residues) was

overexpressed in E. coli strain BL21 star (DE3) as GST fusions. The

puriﬁcation was in accordance with the protocol using GSTrap HP

column (GE Healthcare, Sweden, 17528202). The N-terminal

domain of human Hsp90a (residues 1e236) was also expressed in

E. coli strain BL21 star (DE3) as His fusions. The puriﬁcation of His-

tagged Hsp90a^Nwas as described using HisTrap HP column (GE

Healthcare, Sweden, 17528202).

4.2.7.5.2-((1-(3-morpholinopropanoyl)piperidin-4-yl)amino)-

5-(hex-1-yn-1-yl)benzamide (31). Compound 31 was prepared

from 24 (120 mg, 0.4 mmol) and 3-morpholinopropanoic acid

(127.3 mg, 0.8 mmol) according to the general procedures. Light

yellow solid (102 mg, 57.9%). Mp 65.5e66.9 ^ꢀC. ¹H NMR (300 MHz,

5.2. Fluorescent polarization assay

All ﬂuorescent polarization (FP) experiments were performed

on the SpectraMax multimode microplate reader (Molecular De-

vices) with excitation and emission wavelength at 485 and 535 nm,

respectively. The assay was performed in 384-well format in black,

DMSO‑d₆)

d

8.44 (d, J ¼ 7.7 Hz, 1H), 7.93 (s, 1H), 7.68 (s, 1H), 7.26 (d,

J ¼ 8.7 Hz,1H), 7.17 (s,1H), 6.75 (d, J ¼ 8.8 Hz,1H), 4.16e4.11 (m,1H),

3.80e3.66 (m, 6H), 3.25e3.20 (m, 2H), 2.99e2.91 (m, 2H),

2.73e2.60 (m, 4H), 2.38 (t, J ¼ 6.6 Hz, 2H), 1.99e1.92 (m, 2H),

1.53e1.34 (m, 4H), 1.25e1.23 (m, 2H), 0.92 (t, J ¼ 7.0 Hz, 3H). HRMS

(ESI): calcd for C₂₅H₃₆N₄O₃[M þ H]^þ441.28, found 441.29. Purity:

99.34% by HPLC (MeOH/H₂O ¼ 90:10). R_f: 0.2 by TLC (DCM/

MEOH ¼ 20:1).

ﬂat bottom plates (Corning no. 3575) with a ﬁnal volume of 60

mL.

20 L of assay buffer (20 mM HEPES, 50 mM KCl, 5 mM MgCl2, pH

m

7.4, 2 mM DTT, 0.1 mg/ml BGG, and 0.01% NP-40) containing three-

fold serial dilutions of compounds (the starting and ﬁnal concen-

trations were 60

buffer containing 60 nM of either puriﬁed Grp94^Nor Hsp90

20 L of assay buffer containing 18 nM GM-FITC (ﬂuorescent tracer,

mM and 0.33 nM, respectively), 20

m

L of assay

4.2.7.6.2-(((1-acetylpiperidin-4-yl)methyl)amino)-5-(hex-1-yn-

1-yl)benzamide (32). Compound 32 was prepared from 25

(156.6 mg, 0.5 mmol) and acetic acid (60 mg, 1.0 mmol) according

to the general procedures. Light yellow solid (102 mg, 57.9%). White

solid (87 mg, 45.6%). Mp 137.9e140.4 ^ꢀC. ¹H NMR (300 MHz,

a

^N, and

m

stock in DMSO and diluted in assay buffer) were added to each well.

For each assay, negative controls (GM-FITC only), positive controls

(GM-FITC in the presence of Grp94^Nor Hsp90 ^N), and test wells

a

DMSO‑d₆)

d

8.52e8.38 (m, 1H), 7.92 (s, 1H), 7.66 (s, 1H), 7.24 (d,

(inhibitors and GM-FITC in the presence of Grp94^Nor Hsp90a^N

)

J ¼ 9.6 Hz, 1H), 7.17 (s, 1H), 6.64 (d, J ¼ 8.9 Hz, 1H), 4.37 (d,

J ¼ 12.3 Hz, 1H), 3.81 (d, J ¼ 14.6 Hz, 1H), 3.07e2.88 (m, 3H), 2.43 (d,

J ¼ 5.8 Hz,1H), 2.37 (t, J ¼ 6.6 Hz, 2H), 1.97 (s, 3H), 1.83e1.63 (m, 3H),

1.45 (dq, J ¼ 20.2, 7.3, 6.5 Hz, 4H), 1.23 (s, 2H), 0.90 (t, J ¼ 7.0 Hz, 3H).

HRMS (ESI): calcd for C₂₃H₃₁N₃O₂[M þ Na]^þ378.23, found 378.22.

Purity: 97.57% by HPLC (MeOH/H₂O ¼ 90:10). R_f: 0.4 by TLC (DCM/

MEOH ¼ 20:1).

were included on each assay plate. Plates were incubated with

rocking for 4 h at 4 ^ꢀC in the dark. IC₅₀values were calculated using

GraphPad Prim 6.0 software.

5.3. BLI assay

As a mature biophysical assay, we chose biolayer interferometry

(BLI) as another method to revalidate candidates identiﬁed as in-

hibitors by the FP competition assay. Biolayer interferometry assays

4.2.7.7.2-(((1-(cyclopropanecarbonyl)piperidin-4-yl)methyl)

amino)-5-(hex-1-yn-1-yl)benzamide (33). Compound 33 was pre-

pared from 25 (156.6 mg, 0.5 mmol) and cyclopropanecarboxylic

acid (86 mg, 1.0 mmol) according to the general procedures. White

solid (126 mg, 70.9%). Mp 145.6e146.8 ^ꢀC. ¹H NMR (300 MHz,

ꢀ

on Octet Red 96 instrument (ForteBio, MenloPark, CA, USA) were

used to determine the interaction between the ligand and protein.

Proteins used in this assay were all biotinylated by EZ-link sul-

foeNHSeLC-biotinylation kit (no. 21340, Thermo Pierce) in 1 ꢁ PBS

DMSO‑d₆)

d 8.54e8.42 (m, 1H), 7.95 (s, 1H), 7.67 (s, 1H), 7.25 (d,

ꢀ

J ¼ 8.5 Hz, 1H), 7.19 (s, 1H), 6.65 (d, J ¼ 8.1 Hz, 1H), 4.43e4.22 (m,

2H), 3.15e2.92 (m, 3H), 2.58 (s, 1H), 2.43e2.31 (m, 2H), 2.02e1.91

(m, 1H), 1.86e1.63 (m, 3H), 1.58e1.37 (m, 4H), 1.30e1.21 (m, 2H),

0.91 (t, J ¼ 7.1 Hz, 3H), 0.67 (d, J ¼ 7.9 Hz, 4H). HRMS (ESI): calcd for

(pH 7.4). Super Streptavidin (SSA) biosensors tips (ForteBio, Inc.,

Menlo Park, CA) were prewetted by dipping them into kinetics

buffer (PBS, 0.05% BSA, 0.01% Tween-20) for 10 min before use, and

they were incubated with biotinylated Grp94^N(a ﬁnal concentra-

tion of 400 nM) or Hsp90a^N(a ﬁnal concentration of 400 nM) to

immobilize the protein on sensor tips. Different concentrations of

compound 4 were added to be immobilized onto SSA sensors. All

the data were collected at 30 ^ꢀC and analyzed on Octet data analysis

software (7.1). The signals were corrected each step and analyzed

using a double reference subtraction protocol to ﬁt globally to a 1:1

binding model. The equilibrium dissociation constant (KD) values

were calculated from the ratio of K_offto K_on(KD ¼ K_off/K_on).

C

₂₁H₂₉N₃O₂[M þ Na]^þ404.24, found 404.23. Purity: 97.57% by

HPLC (MeOH/H₂O ¼ 90:10). R_f: 0.25 by TLC (PE/EA ¼ 1:1).

4.2.7.8.2-((1-acetylpiperidin-3-yl)amino)-5-(hex-1-yn-1-yl)

benzamide (34). Compound 34 was prepared from 26 (120 mg,

0.4 mmol) and acetic acid (48 mg, 0.8 mmol) according to the

general procedures. White solid (112 mg, 82.1%). Mp 97.4e99.0 ^ꢀC.

¹H NMR (300 MHz, chloroform-d)

d

8.14 (dd, J ¼ 45.6, 7.1 Hz, 1H),

7.49 (d, J ¼ 11.3 Hz, 1H), 7.41e7.33 (m, 1H), 6.73 (dd, J ¼ 46.3, 8.6 Hz,

1H), 5.75 (s, 2H), 4.27 (dd, J ¼ 196.6, 12.8 Hz, 1H), 3.75 (dd, J ¼ 27.3,

12.8 Hz, 1H), 3.50 (d, J ¼ 33.8 Hz, 1H), 3.32e3.14 (m, 1H), 2.79e2.66

(m, 1H), 2.42 (t, J ¼ 6.4 Hz, 2H), 2.12 (d, J ¼ 24.8 Hz, 4H), 1.96e1.80

(m, 1H), 1.66e1.50 (m, 6H), 0.98 (t, J ¼ 7.0 Hz, 3H). HRMS (ESI): calcd

for C₂₀H₂₇N₃O₂[M þ Na]^þ364.21, found 364.20. Purity: 99.34% by

HPLC (MeOH/H₂O ¼ 90:10). R_f: 0.5 by TLC (DCM/MEOH ¼ 20:1).

5.4. Microsomal stability assay

10 mM compound 4 or DDO-5813 was preincubated with MgCl₂

(10 mM), human microsomes (0.2 mg/ml) or mouse microsomes

(0.5 mg/ml) for 5 min at 37 ^ꢀC in phosphate buffer (100 mM,

pH ¼ 7.4). The addition of 1 mM NADPH initiated the reactions.

After 0, 5, 10, 30, 60, 90, 120 min incubations at 37 ^ꢀC, the cold

acetonitrile with an internal standard (Reserpine, 10 ng/ml) was

14

S. Xu, A. Guo, N.-n. Chen et al.

European Journal of Medicinal Chemistry 223 (2021) 113604

utilized to precipitate the protein. Lastly, the supernatants were

used for analysis using a developed LC-MS/MS method.

levels of inﬂammatory cytokines (IL-6 and TNF-a). Mouse IL-6 ELISA

kit (Catalog #EK0411) and TNF-a ELISA kit (Catalog #EK0527) were

provided by Boster Biological Technology Co. Ltd. Plates were read

in a SpectraMax Plus microplate reader (Molecular Devices) at

492 nm.

5.5. Pharmacokinetic experiments

All animal care and in vivo procedures conducted were in

accordance with the guidelines of Institutional Animal Care and Use

Committee of China Pharmaceutical University. Compound 4 was

tested in pharmacokinetic studies on Sprague Dawley® （SD）rats

weighing 220e240 g, with six rats (male) in each group. Animals

were allowed ad libitum access to water and food. The test com-

pound was administered orally (po) at a dose of 40 mg/kg or

intravenously (iv) at a dose of 10 mg/kg. After dosing, serial blood

samples were collected via the retrobulbar vein at indicated time

points (0, 0.167, 0.25, 0.5 1, 2, 4, 8, 12, and 24 h). Blood samples in

heparinized Eppendorf tubes were centrifuged at 3000 rpm for

10 min to get plasma samples, which were stored at ꢂ20 ^ꢀC until

analysis by LCꢂMS/MS. The PK parameters were then calculated

5.10. Immunohistochemistry

Colon samples, ﬁxed in 4% formalin, were embedded in parafﬁn

and cut into 5

mm sections. Then the parafﬁn sections were

deparafﬁnized through a graded alcohol series to water and PBS.

The sections were incubated in 3% hydrogen peroxide (H₂O₂) to

quench the endogenous peroxidase. All the slides were incubated

with the primary antibody overnight at 4 ^ꢀC after they were

blocked with 10% normal goat serum in 2% PBS. Then, the sec-

ondary antibody was incubated with the slides at 37 ^ꢀC for 20 min.

DAB and hematoxylin were used to stain and retain the samples,

respectively. A light microscope (200 ꢁ , Nikon, Tokyo, Japan)

coupled with a charge-coupled device camera was used to obtain

all photomicrographs.

using

a non-atrioventricular model (Phoenix WinNonlin 7.0,

Pharsight).

5.6. Induction of UC model and compound 4 treatment

6. Docking

The complex structure of PU-H54 and Grp94^N(PDB ID: 3O2F)

was obtained from the Protein Data Bank (PDB). All compounds and

the protein structures needed for docking were prepared by Dis-

covery Studio (DS) 4.0 software. The water molecules HOH22,

HOH441, which are conserved in the binding site, are kept for

docking. The conformations of protein and compounds were

generated with the protocol “Prepare Protein” and “Prepare Li-

gands”, respectively. The residues of Grp94 around compound 4

(radius 11.0 Å) were deﬁned as the binding site, which completely

covered the binding site. The molecular docking using the

“CDOCKER” tool was performed, and other docking processes were

default.

Male C57BL/6 mice (20e22 g) were purchased from Jinan Pen-

gyue Laboratory Animal Breeding Co. Ltd. and randomly divided

into ﬁve groups (n ¼ 8 per group): normal control group, disease

control group, compound 4 group (10 mg/kg), compound 4 group

(30 mg/kg), compound 4 group (50 mg/kg). The studies were

conducted in accordance with the guidelines of Institutional Ani-

mal Care and Use Committee of China Pharmaceutical University.

The normal control group mice were given distilled water, and the

other four groups were given 3% DSS (36e50 KD) orally in drinking

water to induce UC for 7 days. All animals had free access to food

and water, which were changed every day. The compound 4-

treated groups were co-administrated (ip, q.d.) with DSS, while

normal control group and disease control group mice were treated

with vehicle. The changes in body weight, stool consistency, and

rectal bleeding of all mice were recorded once a day. The colons

were collected and measured when experimental mice were

sacriﬁced on day 8. The serum was used for the determination of

cytokines. Sections of distal colons were excised, ﬁxed in 4%

formaldehyde, and preserved for H&E staining analysis. The

remaining parts of the colons were immediately ﬂash-frozen in

liquid nitrogen for further examination.

Declaration of competing interest

The authors declare that they have no known competing

ﬁnancial interests or personal relationships that could have

appeared to inﬂuence the work reported in this paper.

Acknowledgments

This work was supported by Projects 82073766, 81872737, and

81930100 of the National Natural Science Foundation of China;

BK20201331 of Foundation Research Project of Jiangsu Province;

[GrantCPU2018GY02] of Double First Class Innovation Team of

China Pharmaceutical University; the Project Program of State Key

Laboratory of Natural Medicines, China Pharmaceutical University

(No. SKLNMZZ202003); the Priority Academic Program Develop-

ment of Jiangsu Higher Education Institutions; Qinglan Project of

Jiangsu Province.

5.7. Calculation of disease activity index (DAI) scores

DAI scores were calculated daily according to general clinical

symptoms, including body weight loss, stool consistency, and stool

blood. The mean of the scores of the three symptoms was calcu-

lated as DAI score.

5.8. Hematoxylin-eosin (H&E) staining

Colon tissues were ﬁxed in 4% formaldehyde solution, and

embedded in parafﬁn. Then, they were cut to make the thickness

Appendix A. Supplementary data

5 mm and stained with H&E. The sections were detected under an

optical microscope (Olympus CKX53, Tokyo, Japan).

Supplementary data to this article can be found online at

https://doi.org/10.1016/j.ejmech.2021.113604.

5.9. Enzyme-linked immunosorbent assay (ELISA)

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Design and synthesis of Grp94 selective inhibitors based on Phe199 induced fit mechanism and their anti-inflammatory effects

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