Discovery of N-((3 S,4 S)-4-(3,4-Difluorophenyl)piperidin-3-yl)-2-fluoro-4-(1-methyl-1 H-pyrazol-5-yl)benzamide (Hu7691), a Potent and Selective Akt Inhibitor That Enables Decrease of Cutaneous Toxicity

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Discovery of N‑((3S,4S)‑4-(3,4-Diﬂuorophenyl)piperidin-3-yl)-2-

ﬂuoro-4-(1-methyl‑1H‑pyrazol-5-yl)benzamide (Hu7691), a Potent

and Selective Akt Inhibitor That Enables Decrease of Cutaneous

Toxicity

Jinxin Che, Xiaoyang Dai, Jian Gao, Haichao Sheng, Wenhu Zhan, Yang Lu, Dan Li, Zizheng Gao,

Zegao Jin, Binhui Chen, Peihua Luo, Bo Yang, Yongzhou Hu, Qiaojun He, Qinjie Weng,*

and Xiaowu Dong *

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ABSTRACT: Rash is one of the primary dose-limiting toxicities of Akt (protein kinase B) inhibitors in clinical trials. Here, we

demonstrate the inhibition of Akt2 isozyme may be a driver for keratinocyte apoptosis, which promotes us to search for new selective

Akt inhibitors with an improved cutaneous safety property. According to our previous research, compound 2 is selected for further

optimization for overcoming the disadvantages of compound 1, including high Akt2 inhibition and high toxicity against HaCaT

keratinocytes. The dihedral angle-based design and molecular dynamics simulation lead to the identiﬁcation of Hu7691 (B5) that

achieves a 24-fold selectivity between Akt1 and Akt2. Hu7691 exhibits low activity in inducing HaCaT apoptosis, promising kinase

selectivity, and excellent anticancer cell proliferation potencies. Based on the superior results of safety property, pharmacokinetic

proﬁle, and in vivo eﬃcacy, the National Medical Products Administration (NMPA) approved the investigational new drug (IND)

application of Hu7691.

GSK2141795, GDC-0068, and MK-2206.⁷⁻⁹The rash is a

1. INTRODUCTION

frequently seen side eﬀect of Akt inhibitors with an incidence of

The aberrant activation of Akt (protein kinase B) is one of the

13−52%. This cutaneous reaction has a negative impact because

it relates to the quality of life, secondary infections, and even may

aﬀect clinical outcome.^10,11It is essential to ﬁgure out the

underlying mechanism of Akt inhibition-induced rash for

facilitating the development of new Akt inhibitors with

improved safety proﬁles.

common reasons for the growth, survival, and drug resistance of

human cancers.¹⁻³Several leading Akt inhibitors, such as GDC-

0068, AZD5363, GSK2141795, and MK-2206, have been

applied in clinical trials worldwide to evaluate their therapeutic

eﬀect against diﬀerent types of cancer, including triple-negative

breast cancer (TNBC), melanoma, and prostate cancer (Figure

1).⁴These ongoing clinical trials demonstrate the promising

prospect of Akt inhibitors as anticancer agents.

As the clinical trials progressed, the safety, tolerability, and

pharmacokinetic data of Akt inhibitors from Phase I or II were

disclosed. The result of a phase I open-label study of AZD5363

indicated that rash, diarrhea, and hyperglycemia were the main

dose-limiting toxicities (DLT).^5,6Moreover, we notice that the

rash adverse event was reported in other clinical trials of

Received: May 5, 2021

Published: August 10, 2021

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Figure 1. Chemical structures of representative Akt inhibitors.

Figure 2. Akt2 is an essential knot for the survival of HaCaT cells. (A) Western blotting analysis of Akt1, Akt2, and Akt3 in HaCaT cells transfected

with shAkt1, shAkt2, and shAkt3, respectively. AZD5363 induces apoptosis of transfected HaCaT cells as indicated by Western blotting of cleaved

caspase 3 (B) and ﬂow cytometry (C).

The apoptosis of keratinocytes is a common histopathological

feature of the drug-induced rash.^12,13Blockade of PI3K/Akt/

mTOR may lead to abnormal dermatologic reactions.¹⁴Akt

contains three isozymes such as Akt1, Akt2, and Akt3, which

play complicated roles in participating the diﬀerentiation and

proliferation of various issues. However, the Akt isozymes

related to cutaneous toxicity is still unclear.¹⁵The Akt inhibitors

in clinical trials are pan-Akt inhibitors, so we ask whether we can

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Figure 3. MD simulation and analysis of the interaction of pyrazole phenyl derivatives with Akt1 protein (PDB ID: 4GV1). Binding mode (A), distance

from Glu278 (B), and dihedral angle change (C) of compound 2 plotted after simulation of 5 ns. (D) Structures of compounds 3−5. Binding mode

(E), distance from Glu278 (F), the contribution of inhibitor−residue interaction (G), and dihedral angle change (H) of compounds 3−5 plotted after

simulation of 5 ns. The two planes deﬁning the angles were marked as purple and orange, respectively.

identify the speciﬁc keratinocytes apoptosis-related Akt

isozymes and develop new selective Akt inhibitors, thereby

lowering the cutaneous toxicity, despite the Akt isozymes share

high homologies.

In our previous study, a series of work led to the identiﬁcation

of 3,4,6-trisubstituted piperidine derivative compound 1 that

displayed a promising pharmacokinetic proﬁle, low human

ether-a-go-go-related gene (hERG) blockade, and excellent in

vivo antitumor eﬃcacy (Figure 1).¹⁶⁻²¹However, its compli-

cated synthetic accessibility and high toxicity against HaCaT

keratinocytes (IC₅₀= 3.58 μM) did not make it a promising drug

candidate. On the basis of our understanding of the

pharmacology of 1, we turned our attention to the 3,4-

disubstituted piperidine derivative compound 2 (Figure 1).

Although its Akt1 inhibitory activity was less potent than that of

compound 1, it exhibited low hERG inhibition and better

synthetic accessibility, making compound 2 a promising starting

point. Here, we report further optimization of compound 2, with

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a focus on Akt1 inhibitory potency, pharmacokinetic properties,

and Akt isozymes selectivity suitable for an orally available agent

with low cutaneous toxicity. This work led to the discovery of a

selective Akt inhibitor B5 (also known as Hu7691) that

overcomes the shortcomings of compound 1 and displays low

inhibition against keratinocytes. Combined with other pre-

clinical assessments, Hu7691 is now approved for clinical trials

by National Medical Products Administration.

critical hydrogen-bond interaction between Glu278 and the

nitrogen on the piperidine ring was missing in the complex

(Figures 3A and S1B) compared to that in the compound 1−

Akt1 complex previously reported.¹⁶The distance between

Glu278 and the nitrogen was too long to form a hydrogen bond

during the whole dynamics simulation (Figure 3B). After

investigating the binding pose of compound 2, we assumed that

hydrogen-bond interaction might be formed through deﬂecting

the piperidine by a certain angle. We turned our attention to the

dihedral angle that formed by the phenyl ﬂat and the amide ﬂat.

The dihedral angle almost didn’t change during the simulation

and resulted in a −9.0° at the end point (Figure 3C). Dihedral

angle-based drug design is a commonly used approach for

improving the selectivity or activity of compounds.³⁰⁻³²

Therefore, combining with the docking result of compound 2,

we hypothesized whether the dihedral angle would be expanded

through introducing halogen groups on the phenyl, and thereby

deﬂecting the amide ﬂat, bringing the piperidine close to Glu278

to form strong hydrogen-bond interaction and improving the

Akt1 inhibitory potencies of compounds.

Considering the introduction of halogen groups may facilitate

the inhibitory activity of compound against Akt1, compounds 3

and 4 bearing 3-F- and 2,5-diF-substituted phenyl linkage,

respectively, were designed to investigate the eﬀects of diﬀerent

substituted sites of halogens on the dihedral angle and the

compounds’ activities. Moreover, compound 5 with 2-F, 6-Cl-

substituted phenyl linkage was designed for evaluating the

dihedral angle change that is inﬂuenced by diﬀerent halogens

(Figure 3D). The compounds were docked with Akt1 protein

and performed a 10 ns dynamics simulation. After the RMSD

values became stable (Figure S2), the compound−Akt1

complexes displayed similar binding patterns, Ala230 formed a

strong hydrogen bond with the pyrazole of the compounds, and

the 4-phenyl group on the piperidine inserted into the

hydrophobic pocket formed by the Gly159, Thr160, Phe161,

and Val164 residues. Moreover, the −NH on piperidine of the

compounds also formed hydrogen bonds with Glu278. The

phenyl and the amide of the compounds exhibited dihedral

angles of 24.3, 49.6, and −54.9°, respectively (Figure 3E). The

distances between the nitrogen on the piperidine ring and

Glu278 of the compounds were closer than that of compound 2

(Figure 3F). We then evaluated the Akt1 inhibitory activities of

the compounds. Compounds 3 and 4 displayed enhanced

activities than compound 2 as expected, with IC₅₀values of 2.7

and 9.2 nM, respectively (Table 1). However, compound 5

exhibited lower Akt1 inhibitory potency than compound 2. In

addition, all of the compounds displayed promising antiprolif-

eration activities. Compounds 3 and 4 showed low hERG

inhibition rate at 3 μM (10.7 and 27.2%, respectively).

2. RESULTS AND DISCUSSION

2.1. Inhibition of Akt2 Contributes to Cutaneous

Toxicity. The self-renewal process of the human epidermis is

a permanent turnover throughout life, which involves the

proliferation of keratinocytes to some extent. The apoptosis of

keratinocytes is often closely related to many dermatological

disorders, including rash. The HaCaT cell line used in our

studies is the most widely studied keratinocyte cell line. It is

capable of proliferating and diﬀerentiating and often used for the

mechanism studies of skin toxicity.²²⁻²⁴As a continuation of our

interest in cutaneous toxicity mechanism study,²⁵⁻²⁸we sought

to investigate the direct eﬀect of diﬀerent Akt subtypes on

keratinocytes by employing HaCaT cells. Stable clones of Akt1,

Akt2 or Akt3 silenced HaCaT cells were generated with short

hairpin RNA (shRNA). The levels of Akt protein decreased in

the corresponding cell lines (Figure 2A). We thus started

seeking for the Akt subtype that possibly drive apoptosis of

HaCaT cells. Cleaved caspase 3 was employed as an apoptosis

marker. After treating the cells with AZD5363 that was a pan-

Akt inhibitor,²⁹we found that the Akt2 silenced HaCaT cells

showed the lowest level of cleaved caspase 3 (Figure 2B),

indicating that AZD5363 can’t induce the apoptosis of the Akt2

silenced cells. The result was further conﬁrmed by ﬂow

cytometry, the survival of the cell was almost not inﬂuenced

by treating with AZD5363 (Figure 2C). Therefore, we deduced

that Akt2 is essential in mediating the HaCaT keratinocytes

apoptosis induced by Akt inhibitors. Akt inhibitors that show

low inhibitory potency against Akt2 may display low cutaneous

toxicity.

According to the above results, we further compared the Akt2

inhibitory potency between compounds 1 and 2. Compound 1

exhibited high inhibitory activity against Akt2, with an IC₅₀value

of 0.2 nM. The IC₅₀of compound 2 against Akt2 was 6.0 nM.

Moreover, compound 2 exhibited an IC₅₀value of 5.44 μM

against HaCaT cells, which was only 1.5-fold lower than that of

compound 1. Although the Akt2 inhibitory activity of

compound 2 was 30-fold less potent than that of compound 1,

it was not low enough to decrease cutaneous toxicity. Two

aspects should be paid more attention during the following

optimization of compound 2, one was how to improve the Akt1

inhibitory activity, the other was low down the compounds’

inhibitory potency against Akt2.

We further analyzed the contribution of amino acid residues

and the dynamics change of dihedral angle of the compound−

Akt1 complex. It was found that Val164, Tyr229, Glu278, and

Met281 contributed mainly to the binding. The ΔG energy of

the key residues interacted with compound 5 was lower than

those of compounds 3 and 4 (Figure 3G), indicating a weak

interaction between compound 5 and Akt1. Moreover, a large

ﬂuctuation of the dihedral angle change of compound 5 was

observed during the dynamics simulation (Figure 3H), which

suggested an unstable binding pose of compound 5. Therefore,

the introduction of halogen to the phenyl had a great inﬂuence

on the dihedral angle and aﬀected the activity of the compounds.

We further investigated the SAR of the compounds containing

halogen-substituted phenyl linkage.

2.2. Molecular Dynamics (MD) Study of Compound 2−

Akt1 Complex and Design Rationale. For improving the

inhibitory activity of compound 2 against Akt1, we ﬁrst explored

the binding mode of compound 2−Akt1 complex by employing

molecular docking and dynamics study. The complex resulted

from docking was further simulated for 10 ns. After the root-

mean-square deviation (RMSD) became stable (Figure S1A),

indicating that dynamics equilibrium was attained. Based on the

relatively stable conformation, the pyrazole of compound 2 was

hydrogen-bonded with Ala230. Moreover, the 4-phenyl group

on the piperidine hydrophobically interacted with the Gly159,

Thr160, Phe161, and Val164 residues. However, we found a

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when the phenyl on 4-position of piperidine ring was substituted

with 4-F, the activities of which were dramatically reduced (A1

vs A8, 4 vs A9). The cellular potencies of the compounds against

HGC-27, 786-O, and KHOS were relatively insensitive, with

most of the compounds exhibiting IC₅₀values greater than 1

μM. Therefore, the dihalogen substitution on the phenyl linkage

cannot facilitate forming proper dihedral angles and thereby

improving the Akt inhibitory activities of compounds.

Table 1. Akt1 Enzymatic and Cancer Cell Line Activities for

Compounds A1−A9

According to the above results, we turned our attention to the

compounds with monohalogen-substituted phenyl linkage. It

could be observed that the Akt inhibitory and antiproliferative

activity of the B series compounds were generally better than

that of the A series compounds (Table 2). When the phenyl on

4-position of piperidine ring was substituted with 3,5-diF, the

compounds displayed comparable Akt1 inhibitory activities to

that of compounds with 3,4-diF substitution (B1 vs B4, B2, vs

B5). After moving 2-F to 3-F substitution, an improvement of

inhibitory activity was observed (B3 vs B2). However, removing

the methyl or changing the methyl to ethyl on pyrazole led to a

20- to 45-fold decrease of compounds’ potencies (B6, B7 vs B5).

The compounds exhibiting better Akt1 inhibitory potencies

such as B2, B4, and B5 performed better antiproliferation

activity against HGC-27, 786-O, and KHOS cells. However,

KHOS cells showed less sensitive to the Akt inhibitors than the

other two cell lines. As previously reported,^33,34Akt is high

expressed in KHOS cells. Its heterogeneity characteristics may

cause the insensitivity. Complicated compensatory signal

pathways may exist in the cells and regulate the KOHS cells’

proliferation, thereby leading to the KHOS cells not being as

sensitive to the treatment of the compound in comparison with

HGC-27 and 786-O. However, the IC₅₀’s of compounds against

KHOS cells were in acceptable ranges, so we thought that the

compounds were eﬀective for inhibiting the proliferation of

KHOS cells.

a

IC₅₀(μM)

Akt1 IC

a⁵⁰

compd.

R₁

3-F

R₂

(nM)

HGC-27 786-O KHOS

3

4

5

H

Cl

Br

Cl

Br

Cl

Br

H

2.7

9.2

0.18

1.45

1.7

0.95

5.05

2.2

7.99

4.28

3.56

NT

7.43

18.38

3.89

15.4

4.46

5.01

NT

3.06

3.67

3.61

29.1

17.57

2,5-diF

2-F,6-Cl

2,6-diF

2,5-diF

2-F,6-Cl

2,6-diF

2,5-diF

47.0

26.3

97.1

95.7

48.9

66.2

142.5

136.8

107.2

35.3

1.3

A1

A2

A3

A4

A5

A6

A7

A8

A9

4.59

4.86

2.9

b

NT

1.79

3.06

3.28

7.27

3.36

1.9

3.17

3.21

25.51

7.81

H

GDC-

0068

a

The IC₅₀values or hERG inhibitions are an average of three

b

independent determinations. NT, not tested.

2.3. In Vitro Activities Evaluation of Compounds. On

the basis of the above molecular dynamics research, we ﬁrst

explored the SAR of the dihalogen substitution on the phenyl

linkage (Table 1). After introducing halogen substitution to

pyrazolyl of the compounds, the Akt1 inhibitory potency was

decreased (A1 vs A2 and A3; 4 vs A4 and A5; 5 vs A6 and A7).

The activities of compounds with 2,5-diF substituent were

enhanced compared to that of compounds with 2,6-diF or 2-F,

6-Cl substituents (4, A4, A5 vs A1-A3 or 5, A6, A7). In addition,

Furthermore, we evaluated the hERG inhibition of the three

most potent compounds for preliminary evaluating their safety

proﬁle. The hERG inhibition of B2 performed lower than those

of B3 and B5, but the inhibitions of the compounds were within

acceptable limits. Considering that the inhibition of Akt2

contributed to cutaneous toxicity, the inhibitory activities of the

Table 2. Akt1 and Akt2 Enzymatic, hERG Inhibitory, and Cancer Cell Line Activities for Compounds B1−B7

a

IC₅₀(μM)

a

compd.

B1

B2

B3

B4

B5

B6

B7

GDC-0068

R₁

R₂

Akt1 IC₅₀(nM)

Akt2 IC₅₀(nM)

hERG inhibition at 3 μM

HGC-27

786-O

KHOS

b

2-Cl

2-F

3-F

2-Cl

2-F

15.0

4.6

1.8

24.0

4.0

83.0

176.0

1.3

NT

8.8

7.1

NT

97.5

NT

3.8

NT

1.24

0.51

0.15

1.4

0.68

4.34

3.57

3.79

2.22

1.01

4.07

2.5

11.84

17.14

7.27

11.68

14.86

10.72

5.46

6.4%

26.4%

NT

15.9%

NT

methyl

H

7.15

5.2

ethyl

NT

a

b

The IC₅₀values or hERG inhibition % are an average of three independent determinations. NT, not tested.

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Figure 4. RMSD value (A), distance from Glu278 (B), dihedral angle change (C), binding mode (D), and contribution of inhibitor−residue

interaction (E) of the B5−Akt1 complex plotted after simulation of 5 ns.

compounds against Akt2 were also investigated. Compound B5

showed much lower Akt2 inhibition than B2 and B3. The IC₅₀

value of B5 against Akt2 was about 24-fold lower than that

against Akt1. Therefore, the combined results suggested that

compound B5 (Hu7691) was suitable for further investigation.

2.4. Molecular Dynamics Investigation of Compound

B5 (Hu7691). The interaction mode of B5 with Akt protein was

further explored. After computational docking, a 10 ns dynamics

simulation was performed and a dynamics equilibrium was

attained as indicated by RMSD value (Figure 4A). Minor

ﬂuctuations of the distance and dihedral angle during the

simulation were observed, indicating a stable binding pattern of

B5−Akt1 complex (Figure 4B,C). A 43.2° dihedral angle was

observed between the phenyl ﬂat and amide ﬂat, leading to the

nitrogen on piperidine ring close to Glu278 and forming a stable

hydrogen bond (Figure 4D). Moreover, B5 showed a similar

binding pattern to that of other compounds, the pyrazole of B5

was hydrogen-bonded with Ala230, and the 4-phenyl group on

the piperidine hydrophobically interacted with the amino acid

residues such as Gly159, Thr160, Phe161, and Val164. As

indicated by the ΔG energy of the key residues, Gly159, Val164,

Tyr229, ALA230 Glu278, and Met281 contributed mainly to

the binding (Figure 4E).

For exploring how B5 achieves selectivity between Akt1 and

Akt2, after computational docking of B5 with Akt2 protein, a 10

ns dynamics simulation was performed and a dynamics

equilibrium was attained (Figure S3A). The distances between

the −NH on piperidine ring with Glu279 and the dihedral angle

showed minor changes during the simulation (Figure S3B,C).

The binding pose and interaction forces of B5−Akt2 complex

were similar to that of B5−Akt1 complex (Figure S3D). Amino

acid residues such as Val166, Glu279, Asn280, and Met282 also

contributed to the strong binding (Figure S3E). Although the

IC₅₀value of B5 against Akt2 was about 24-fold lower than that

against Akt1, the selectivity was relatively low. Moreover, Akt1

and Akt2 are highly homologous. Therefore, it is hard to explain

the selectivity using molecular docking and dynamics simu-

lation.

2.5. Assessment of Kinase Selectivity Proﬁle of B5

(Hu7691). To understand the selectivity proﬁle of compound

B5, the inhibition was assessed against four families of kinases

(AGC, TK, TKL, Lipid/Atypical; total number: 218) at a single-

dose concentration of 0.5 μM. The result indicated that B5

displayed low inhibitions against most of the kinases in the four

families (Table S1; 7/218, % Ctrl < 10). Some kinases

experienced marked inhibition, including PKA, PKG, and

PRKG2. For further verifying the selectivity of B5, the IC₅₀’s

of B5 against a panel of 19 kinases were tested (Table 3). B5

displayed high IC₅₀’s against Akt3 and PKA, while no high IC₅₀’s

were observed against other kinases. The selectivity of B5

against Akt3 was lower than that of B5 against Akt2. The

combined results suggested that B5 exhibited a favorable

selectivity proﬁle.

2.6. Compound B5 (Hu7691) Shows Low Activity in

Inducing HaCaT Apoptosis. Since compound B5 displayed

relatively low Akt2 inhibition and we have demonstrated the

critical role of Akt2 in maintaining the survival of cutaneous

cells, the activity of B5 against HaCaT cells was tested. As shown

in Figure 5A, B5 exhibited an IC₅₀value of 15.2 μM that was 3.6-

fold lower than that of AZD5363. The decrease of the

phosphorylation level of Akt (S473) induced by B5 was more

eﬀective than that induced by AZD5363. However, B5 showed

lower activity in inducing apoptosis against HaCaT than

AZD5363 as indicated by the level of cleaved caspase 3 (Figure

5B). These results conﬁrmed our hypothesis focused on

lowering Akt2 inhibition. Therefore, B5 may show low

cutaneous toxicity in subsequent development.

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Table 3. Results of Assessment of B5’s Kinase Selectivity

Proﬁles

Table 4. Activities of B5 against Diﬀerent Cancer Cell Lines

a

IC₅₀(μM)

a

kinases

IC₅₀(nM)

cancer

glioma

cell line

U87-MG

U251

95D

NCI-H1975

A549

SMMC-7721

HepG2

BEL7402

HT-29

769-P

786-O

OSRC-2

KHOS

MDA-MB-231

LnCap

PC3

SKOV3

OVCAR8

B5

AZD5363

AGC

kinase

family

AKT3

PKA

28

11

>2000

629

354

756

9.26 4.07

6.53 2.95

1.03 0.41

7.56 2.35

6.56 2.83

19.76 8.31

3.60 2.86

26.62 1.93

20.29 13.73

7.76 4.79

8.60 3.37

9.90 7.15

7.47 6.24

16.37 10.72

0.61 0.33

8.74 6.55

4.71 2.14

3.52 1.52

50.06 14.77

14.22 9.23

2.32 0.85

PKCθ

PKCη

ROCK1

RSK1

P70S6K

SGK

lung

liver

8.23 2.05

23.67 3.79

84.12 17.14

6.70 0.81

229

1009

49.6 28.41

36.92 7.76

10.75 8.14

23.28 8.10

53.43 9.59

40.42 25.15

60.52 22.34

0.46 0.27

MSK1

PDK1

TSSK1

ABL

>10 000

bowel

kidney

TK

TKL

CMGC

CAMK

Atypical

ALK

osteosarcoma

breast

prostate

JNK2

CHK1

PI3Ka

CDK2

AruraA

JAK-2

82.57 54.41

6.32 1.78

4.48 1.91

ovaries

a

The IC₅₀values are an average of three independent determinations.

a

The IC₅₀values are an average of three independent determinations.

2.7. Compound B5 (Hu7691) Exhibits Antiprolifera-

inhibition of cytochrome P450 enzymes (CYPs) was inves-

tigated by determining the activity of B5 against CYPs 1A2, 2C8,

2C9, 2C19, 2D6, and 3A4/5. The result indicated that B5

showed low inhibitory activities against the CYPs, with IC₅₀

values > 100 μM, except that against 2C19, exhibiting an average

inhibition of 65.0% at 100 μM (Table S2). Moreover, B5 was

incubated with liver microsomes from diﬀerent species. B5

showed decrease remaining percentage when incubating with rat

or monkey liver microsomes, while it exhibited excellent stability

during the incubation in human liver microsomes (Figure 6A).

The microsome stability parameters are summarized in Table 5,

B5 possessed longer T_1/2and lower clearance in human liver

microsomes than that in other species’ liver microsomes. In

addition, the plasma protein binding rates of B5 were found to

be generally high in diﬀerent species (Figure 6B), thus indicating

a tendency for long maintenance time and stable eﬀect of B5 in

vivo. The combined results demonstrated that B5 might have

promising pharmacokinetic properties in human.

tion Activity against Wide Cancer Types. Based on its

prominent AKT1 kinase inhibition, low hERG inhibition, and

high selectivity, we used sulforhodamine B (SRB) method to

evaluate B5’s cellular potency. Although compound B5

exhibited low Akt2 inhibition, we found that it has a signiﬁcant

inhibitory eﬀect on the growth of 18 kinds of human tumor cells

derived from diﬀerent tissues, with the IC₅₀range of 0.6−27 μM

that was comparable to or better than the IC₅₀’s of AZD5363

(Table 4). Especially, the IC₅₀values of B5 against several kinds

of cells were below 5 μM, including 95D, HepG2, LnCap,

SKOV3, and OVCAR8. We further tested the cytotoxicity of B5

against two noncancer cell lines such as HL7702 (human normal

liver cells) and HPDE6-C7 (human normal pancreatic duct

epithelial cells). The result indicated that B5 showed low

antiproliferation activities against the HL7702 and HPDE6-C7

normal cells, exhibiting IC₅₀values of 5.4 and 16.1 μM,

respectively (Figure S4).

2.8. Hu7691 Shows Good Pharmacokinetic Proﬁles.

These promising Akt inhibitory and anticancer cell proliferation

potencies promoted us to investigate the pharmacokinetic

proﬁles of compound B5 further. B5 was evaluated for its ability

to inhibit the major drug-metabolizing enzymes at ﬁrst. The

As shown in Table 6, after 15 mg/kg oral dosing in rats, we

found that the average T_maxand T_1/2of B5 were 1.67 and 8.68 h,

respectively, indicating a fast absorption and a long clearance

time. Furthermore, B5 showed a good bioavailability value of

66.2% in rats. Moreover, high plasma exposure concentration

Figure 5. B5 exhibits low toxicity against HaCaT cells. (A) IC₅₀curves of AZD5363 and B5 against HaCaT cells; (B) Western blotting analysis of

cleaved poly ADP-ribose polymerase (c-PARP), cleaved caspase 3 (c-c-3), and pAkt-Ser473 in HaCaT cells.

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Figure 6. Stability in liver microsomes (A) and plasma protein binding rate (B) of compound B5.

Information). There were six phase I metabolites, including

oxygenation + desaturation (M1), oxygenation (M2), deme-

thylation (M3), oxygenation + double desaturation (M4),

double oxygenation + desaturation (M5), and double oxygen-

ation (M6). Moreover, hydroxyl glucuronidated phase II

metabolite (M7) was detected. In the urine, the main

metabolites were M1−M4. The M1, M2, and M5 metabolites

were contained in the excrement. The bile contained the widest

variety of metabolites; M1−M5 were included. The primary

metabolites in the blood were M1 and M3. These results

conﬁrmed a regular metabolic pathway of compound B5.

Table 5. Microsome Stability Parameters of B5

species

_1/2(min)

Cl_int(μL/(min mg))

human monkey

dog

rat

mouse

T

972.7

1.4

101.8

13.6

336.2

4.1

69.4

20.0

403.7

3.4

and long clearance time were also detected in Beagle dogs after

an oral dose of 20 mg/kg.

To elucidate the metabolic pathway of B5, major metabolites

collected from the rat plasma, urine, excrement, and bile after

oral administration of B5 were characterized by UPLC/TOF/

MS/MS. Seven metabolites were detected, which from phase I

and II metabolisms (Figure 7, for MS results, see the Supporting

Table 6. Pharmacokinetic Properties of B5 in SD Rats (n = 6)

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Figure 7. Metabolic pathway of compound B5.

Figure 8. Eﬀect of B5 on weight change and serum parameters during a 28-day repeated dose toxicity study. Body weight (A), serum hematological

parameters (B), and serum biochemistry parameters (C) are recorded (n = 6).

2.9. Toxicity Studies of Compound B5 (Hu7691). As the

toxicity is vital for Akt inhibitors, a detailed safety evaluation was

performed. Single-dose toxicity was evaluated against SD rats

and Beagle dogs. The maximum toxicity dose (MTD) for mice

was 150 mg/kg, and the main clinical symptoms were reduced

activity, prone posture, brownish watery stools, and weight loss.

When the dosage increased to 250 mg/kg, the mice started to

die, and the target organ of toxicity was the intestine. The MTD

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Figure 9. Compound B5 shows an antitumor eﬀect in vivo. The mice transplanted with 786-O (A) and KHOS (B) xenograft were randomly divided

and administered drugs for a period of 22 days. The mice body weight (left), tumor volume (middle), and tumor weight (right) were measured. The

results are presented as mean (n = 6) SD. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 vs vehicle.

Scheme 1. Synthetic Route for Compounds 10a−c; (a) (i) (S)-2-(Diphenyl((trimethylsilyl)oxy)methyl)pyrrolidine, Benzoic

Acid, Dichloromethane; (ii) Triﬂuoroacetic Acid (TFA), Dichloromethane; (b) (i) Et₃SiH, TFA; (ii) Boc₂O, Triethylamine

(TEA), Dichloromethane; (c) Fe, NH₄Cl, EtOH/H₂O

for Beagle dog was 100 mg/kg, and thymic atrophy can be

observed.

were almost unchanged, and the biochemical parameters

remained normal (Figure 8B,C), indicating an acceptable safety

proﬁle of B5. In addition, there were slight histopathological

changes observed in the stomach, jejunum, spleen, thymus,

uterus, ovaries, thyroid, and bone marrow. However, these

changes were reversed after an additional 4-week period in the

absence of B5.

Compound B5 lacks genetic toxicity as assessed by

chromosome aberration assay (Table S3) and mice micro-

nucleus assay (Table S4). Therefore, the above combined data

indicated that B5 exhibits a promising safety proﬁle.

Further, the repeated dose toxicity was conducted at daily

doses of 0, 25, 50, and 75 mg/kg for 4 weeks. The rat body

weight increased during the evaluation except in the 75 mg/kg

group (Figure 8A). When the drug administration stopped, the

rats regained weight. Moreover, the clinical symptom of scab on

rat back skin that can be caused by keratinocytes apoptosis was

set as an indicator when performing the repeated dose toxicity

evaluation. During the whole treatment (4 weeks), the scab on

rat back skin was not observed in all dosing groups (25, 50, and

75 mg/kg), and no other skin lesions were observed, indicating

low cutaneous toxicity of B5.

2.10. Inhibitory Eﬃcacy of B5 in Tumor Xenograft

Mice. Given the potent inhibition of Akt kinase and many kinds

cell growth, together with the promising pharmacokinetic

properties, the in vivo antitumor eﬃcacy of B5 was evaluated

At the end of the repeated dose, the serum hematological and

biochemical parameters of rats were tested. The cell populations

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Scheme 2. Synthetic Route for Compounds 14a−k and 17a−b; (a) Corresponding Boron Pyrazole, Pd(PPh₃)₄, K₃PO₄,

Dimethylformamide (DMF); (b) NBS or NCS, THF/DMF; (c) 3N NaOH, THF/EtOH; and (d) Iodoethane, NaH

Scheme 3. Synthetic Route for Target Compounds 3−5, A1−A9, and B1−B7; (a) EDCI, HOBt, DIPEA; (b) EA, HCl

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using human 786-O and KHOS xenograft mouse model. After

22 days of treatment with three doses of B5, the mice body

weights were not aﬀected (Figure 9A,B), demonstrating a safety

proﬁle of B5. Dose-dependent tumor growth inhibition of B5 in

both models were observed (Figures 9A,B and S5). For KHOS

xenograft tumor, the maximum eﬀect of tumor growth inhibition

may have reached at the 25 mg/kg dosage, so the 50 mg/kg

dosage showed a similar tumor inhibition rate to that of 25 mg/

kg dosage. Notably, the tumor inhibition rate of B5 was better

than that of AZD5363 at an equal or lower dosage (70.0 vs

47.1% in 786-O xenograft model; 55.7 vs 33.9% in KHOS

xenograft model). Importantly, B5 was found well tolerated,

with no mortality being observed at each dose level.

2.11. Chemistry. The synthetic route to obtain a series of

disubstituted piperidine compounds 10a−c is shown in Scheme

1. Compounds 6a−c and compound 7 were cyclized under the

catalysis of (S)-2-(diphenyl((trimethylsilyl)oxy)methyl)-

pyrrolidine to provide the (3S,4S) compounds 8a−c. After the

double bond of compounds 8a−c was reduced by Et₃SiH, the

nitro group was reduced by Fe/NH₄Cl to obtain compounds

10a−c.

The synthetic route to obtain a series of biaryl carboxylic acids

14a−k and 17a−b is shown in Scheme 2. Corresponding boron

pyrazole and compounds 11a−f were coupled under the

catalysis of tetrakis(triphenylphosphine)palladium to obtain

compounds 12a−f/15. After the treatment of N-bromo

succinimide (NBS) or N-chloro succinimide (NCS) and

hydrolysis, the compounds 14a−k/17a were obtained. After

the treatment of NaH, iodoethane, and hydrolysis, compound

17b was obtained.

Diﬀerent acid fragments 14a−k, 17a−b, and amines 10a−c

were condensed to obtain the Boc-protected intermediates

(Scheme 3), which were subsequently deprotected to give the

target compounds A1−A9 and B1−B7.

candidate for the treatment of tumor and is now approved for

clinical trials by National Medical Products Administration

(NMPA), and ready for Phase I clinical trial.

4. EXPERIMENTAL SECTION

4.1. Chemistry. ¹H NMR and ¹³C NMR spectra were recorded at

500 MHz using a Bruker AVANCE III spectrometer in CDCl₃, or

DMSO-d₆solution, with tetramethylsilane (TMS) serving as the

internal standard. Chemical shift values (δ) were reported in ppm.

Multiplicities are recorded by the following abbreviations: s, singlet; d,

double; t, triplet; q, quartet; m, multiplet; J, coupling constant (Hz).

High-resolution mass spectrum (HRMS) was obtained from Agilent

Technologies 6224 TOF liquid chromatography−mass spectrometry

(LC−MS). The purities of compounds for biological testing were

assessed by NMR and HPLC, and the purities were ≥95%. The

analytical HPLC was performed on an Agilent 1260 Inﬁnity II (LC03)

machine and a C18 reversed-phase column (Agilent Eclipse XDB-C18,

4.6 × 250 mm, 5 μm), with a ﬂow rate of 1.0 mL/min, the detection by

UV absorbance at a wavelength of 254 nm, the column temperature was

25 °C, eluting with water (0.1% formic acid) as A phase and methanol

as B phase (0 min, A phase: 90%, B phase: 10%; 15 min, A phase: 10%, B

phase: 90%; 25 min, A phase: 90%, B phase: 10%). Unless otherwise

noted, reagents and solvents were obtained from commercial suppliers

and without further puriﬁcation. The synthesis and compound

information of the intermediates can be found in the Supporting

Information.

4.1.1. General Procedure for the Synthesis of Target Compounds

3−5, A1−A9, and B1−B7. To a suspension of carboxylic acids 14a−k,

17a−b (0.4 mmol), EDCI (0.72 mmol), HOBT (0.72 mmol), and

DIPEA (1 mmol) in 5 mL of dichloromethane was added amine 10a−c

(0.4 mmol) at room temperature. The reaction was stirred for 5 h, and

then poured into 10 mL of water. The aqueous phase was extracted with

dichloromethane (10 mL × 3), the combined organic phase was washed

with saturated brine (10 mL × 2), and dried over anhydrous sodium

sulfate and concentrated. The white powder was directly subjected to

the next reaction step without further puriﬁcation. To a solution of Boc-

protected amide (crude) in 2 mL of ethyl acetate was slowly added

HCl-saturated ethyl acetate at room temperature. The reaction was

stirred overnight and concentrated under reduced pressure. Then, the

reaction was added with ether, stirred for 2 h, ﬁltered, and puriﬁed by

ﬂash column chromatography to obtain a white solid product.

4.1.1.1. N-((3S,4S)-4-(3,4-Diﬂuorophenyl)piperidin-3-yl)-3-ﬂuoro-

4-(1-methyl-1H-pyrazol-5-yl)benzamide Hydrochloride (3). General

procedure, yield: 63%; ¹H NMR (500 MHz, MeOD) δ 7.72 (s, 1H),

7.55 (dd, J = 24.2, 13.9 Hz, 3H), 7.30−7.23 (m, 1H), 7.16 (s, 2H), 6.52

(s, 1H), 4.61 (s, 1H), 3.79 (s, 3H), 3.61−3.57 (m, 1H), 3.51 (d, J = 20.3

Hz, 1H), 3.20 (s, 3H), 2.23−2.01 (m, 2H). ¹³C NMR (125 MHz,

MeOD) δ 167.26, 161.60, 159.61, 152.00 (d, J = 108.9 Hz), 150.09 (d, J

= 96.1 Hz), 139.44, 138.72, 138.37, 133.14, 125.44, 124.65, 121.65 (d, J

= 15.5 Hz), 118.40 (d, J = 17.2 Hz), 117.64 (d, J = 17.7 Hz), 116.26 (d, J

= 23.9 Hz), 109.41, 49.55, 48.07, 46.01, 45.33, 37.70, 31.21. HRMS: m/

z (ES⁺) [M + H]⁺= 415.1806 (theor 415.1746).

4.1.1.2. N-((3S,4S)-4-(3,4-Diﬂuorophenyl)piperidin-3-yl)-2,5-di-

ﬂuoro-4-(1-methyl-1H-pyrazol-5-yl)benzamide Hydrochloride (4).

General procedure, yield: 65%; ¹H NMR (400 MHz, MeOD) δ 7.95 (s,

1H), 7.47−7.31 (m, 3H), 7.26 (d, J = 17.3 Hz, 2H), 6.71 (s, 1H), 4.69

(t, J = 9.3 Hz, 1H), 3.93 (s, 3H), 3.71 (d, J = 10.0 Hz, 1H), 3.60 (d, J =

11.7 Hz, 1H), 3.30−3.16 (m, 3H), 2.19 (d, J = 18.5 Hz, 2H). ¹³C NMR

(101 MHz, MeOD) δ 162.94 (s), 156.43 (s), 153.94 (s), 151.27 (d, J =

12.7 Hz), 150.56 (d, J = 12.6 Hz), 148.82 (d, J = 12.7 Hz), 148.12 (d, J =

12.6 Hz), 138.09 (s), 137.88 (s), 136.70 (s), 125.67 (dd, J = 16.8, 7.8

Hz), 124.10 (s), 120.25 (dd, J = 16.9, 9.7 Hz), 119.03 (d, J = 27.6 Hz),

117.48−116.72 (m), 116.44 (d, J = 17.7 Hz), 108.64 (s), 46.36 (s),

44.59 (s), 43.71 (s), 36.29 (s), 29.64 (s). HRMS: m/z (ES⁺) [M + H]⁺

= 433.1703 (theor 433.1651).

3. CONCLUSIONS

Several clinical studies of Akt inhibitors have suggested that rash

is the main DLT. To characterize the cutaneous toxicity

mechanisms in patients, we investigated the eﬀect of diﬀerent

Akt isozymes on keratinocytes by employing HaCaT cells. The

result indicated that Akt2 inhibition was a driver for keratinocyte

apoptosis. Akt inhibitors with low Akt2 inhibition may confer

low cutaneous toxicity.

The complicated synthetic accessibility, high Akt2 inhibition

(IC₅₀= 0.2 nM), and toxicity against HaCaT keratinocytes (IC₅₀

= 3.58 μM) of compound 1 make us turn to our interest in

compound 2. The molecular dynamics analysis and dihedral

angle-based design led to the identiﬁcation of compound B5

(Hu7691), with an improved Akt1 inhibitory activity (IC₅₀

4.0 nM) that was 24-fold higher than Akt2 inhibition (IC₅₀

=

97.5 nM), achieving a moderate selectivity between these two

high homology proteins. Hu7691 showed low activity in

inducing HaCaT keratinocytes apoptosis (IC₅₀= 15.17 μM).

Although Hu7691 exhibited low Akt2 inhibition, it possesses

promising kinase selectivity, anticancer cell proliferation

potencies (IC₅₀= 0.6−27 μM), and a suitable pharmacokinetic

proﬁle. Concerns centered on the toxicity of Hu7691 were

demonstrated by a comprehensive safety evaluation, and the

scab clinical symptom on mice back skin was not observed,

suggesting low cutaneous toxicity of Hu7691 as expected. Oral

administration of Hu7691 displayed eﬃcient inhibitory

eﬃcacies against tumor growth in mice. On the basis of these

ﬁndings, Hu7691 was considered as a promising Akt inhibitor

4.1.1.3. 2-Chloro-N-((3S,4S)-4-(3,4-diﬂuorophenyl)piperidin-3-yl)-

6-ﬂuoro-4-(1-methyl-1H-pyrazol-5-yl)benzamide Hydrochloride

1

(5). General procedure, yield: 56%; H NMR (400 MHz, MeOD) δ

7.57 (d, J = 11.9 Hz, 1H), 7.44 (s, 1H), 7.33 (dd, J = 14.8, 8.1 Hz, 2H),

7.30−7.24 (m, 1H), 7.21 (s, 1H), 6.51 (s, 1H), 4.66 (t, J = 9.1 Hz, 1H),

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3.90 (s, 3H), 3.78−3.66 (m, 1H), 3.57 (d, J = 11.1 Hz, 1H), 3.23 (t, J =

12.2 Hz, 1H), 3.10 (t, J = 11.2 Hz, 2H), 2.23 (d, J = 13.6 Hz, 1H), 2.15−

1.99 (m, 2H). ¹³C NMR (101 MHz, MeOD) δ 162.69 (s), 160.08 (s),

157.58 (s), 156.14 (s), 137.86 (s), 133.98 (d, J = 11.2 Hz), 131.96 (d, J

= 7.0 Hz), 125.16 (s), 124.43 (d, J = 22.7 Hz), 123.97 (s), 117.12 (d, J =

17.4 Hz), 116.48 (d, J = 18.3 Hz), 114.49 (s), 114.25 (s), 45.49 (d, J =

Hydrochloride (A6). General procedure, yield: 53%; H NMR (400

MHz, CDCl3) δ 10.67 (d, J = 29.3 Hz, 1H), 10.07 (d, J = 8.5 Hz, 1H),

8.72 (d, J = 9.0 Hz, 1H), 8.53 (d, J = 7.2 Hz, 2H), 8.44 (dd, J = 19.0, 8.7

Hz, 1H), 8.35−8.20 (m, 1H), 8.15 (s, 1H), 5.60 (d, J = 3.8 Hz, 1H),

4.40 (t, J = 13.8 Hz, 2H), 3.98 (td, J = 11.7, 3.4 Hz, 2H), 3.86 (t, J = 11.3

Hz, 1H), 3.06 (dd, J = 30.1, 12.6 Hz, 2H), 2.25 (s, 1H). 13C NMR (101

MHz, CDCl3) δ 165.68 (d, J = 9.1 Hz), 164.63 (s), 162.15 (s), 155.55

(d, J = 12.5 Hz), 154.76 (d, J = 12.6 Hz), 153.11 (d, J = 12.4 Hz), 152.33

(d, J = 12.6 Hz), 144.33 (s), 141.69 (s), 141.18 (s), 136.41 (d, J = 6.8

Hz), 135.60 (d, J = 9.9 Hz), 131.56 (s), 131.01 (dd, J = 23.3, 5.2 Hz),

129.51 (s), 122.56 (d, J = 17.2 Hz), 121.90 (d, J = 17.0 Hz), 121.35 (s),

121.11 (s), 113.31 (s), 52.24 (d, J = 10.5 Hz), 49.11 (s), 48.03 (s).

HRMS: m/z (ES⁺) [M + H]⁺= 483.0991 (theor 483.0966).

195.5 Hz), 36.46 (s), 30.23 (s). HRMS: m/z (ES⁺) [M + H]⁺

=

449.1394 (theor 449.1356).

4.1.1.4. N-((3S,4S)-4-(3,4-Diﬂuorophenyl)piperidin-3-yl)-2,6-di-

ﬂuoro-4-(1-methyl-1H-pyrazol-5-yl)benzamide Hydrochloride

(A1). General procedure, yield: 63%;¹H NMR (400 MHz, MeOD) δ

7.72 (s, 1H), 7.31 (s, 1H), 7.27 (d, J = 7.6 Hz, 3H), 7.21 (s, 1H), 6.61 (s,

1H), 4.66 (s, 1H), 3.96 (s, 3H), 3.70 (d, J = 8.9 Hz, 1H), 3.58 (d, J = 9.8

Hz, 1H), 3.24 (s, 1H), 3.14 (s, 2H), 2.23 (d, J = 13.3 Hz, 1H), 2.14 (s,

1H). ¹³C NMR (101 MHz, MeOD) δ 160.44 (d, J = 12.6 Hz), 157.91

(d, J = 8.0 Hz), 151.31 (d, J = 12.9 Hz), 150.61 (d, J = 12.1 Hz), 148.86

(d, J = 12.5 Hz), 148.16 (d, J = 12.8 Hz), 141.83 (s), 137.81 (s), 137.43

(s), 133.28 (d, J = 10.5 Hz), 124.00 (s), 117.05 (d, J = 17.4 Hz), 116.46

(d, J = 18.0 Hz), 114.18 (s), 112.05 (d, J = 26.3 Hz), 107.18 (s), 46.49

(s), 44.65 (s), 43.76 (s), 36.57 (s), 29.97 (s). HRMS: m/z (ES⁺) [M +

H]⁺= 433.1671 (theor 433.1651).

4.1.1.5. 4-(4-Chloro-1-methyl-1H-pyrazol-5-yl)-N-((3S,4S)-4-(3,4-

diﬂuorophenyl)piperidin-3-yl)-2,6-diﬂuorobenzamide Hydrochlor-

ide (A2). General procedure, yield: 60%; HPLC: t_R13.562 min, purity

100.00%. ¹H NMR (400 MHz, MeOD) δ 7.57 (s, 1H), 7.29 (dd, J =

18.8, 10.5 Hz, 2H), 7.20 (d, J = 7.6 Hz, 3H), 4.65 (s, 1H), 3.82 (s, 3H),

3.69 (d, J = 9.7 Hz, 1H), 3.57 (d, J = 9.4 Hz, 1H), 3.23 (s, 1H), 3.12 (s,

2H), 2.22 (d, J = 13.9 Hz, 1H), 2.07 (dd, J = 29.4, 9.4 Hz, 1H). ¹³C

NMR (101 MHz, MeOD) δ 160.79−160.09 (m), 157.85 (d, J = 8.0

Hz), 151.33 (d, J = 12.3 Hz), 150.61 (d, J = 12.5 Hz), 148.94 (s), 148.17

(d, J = 12.5 Hz), 137.75 (s), 136.70 (s), 131.65 (d, J = 10.5 Hz), 123.96

(s), 117.06 (d, J = 17.5 Hz), 116.42 (d, J = 17.6 Hz), 114.53 (s), 113.18

(s), 112.92 (s), 109.11 (s), 46.49 (s), 44.65 (s), 43.77 (s), 37.41 (s),

29.98 (s). HRMS: m/z (ES⁺) [M + H]⁺= 467.1312 (theor 467.1262).

4.1.1.6. 4-(4-Bromo-1-methyl-1H-pyrazol-5-yl)-N-((3S,4S)-4-(3,4-

diﬂuorophenyl)piperidin-3-yl)-2,6-diﬂuorobenzamide Hydrochlor-

ide (A3). General procedure, yield: 62%; ¹H NMR (400 MHz,

MeOD) δ 7.55 (s, 1H), 7.31−7.20 (m, 2H), 7.16 (d, J = 7.6 Hz, 3H),

4.61 (t, J = 9.5 Hz, 1H), 3.80 (s, 3H), 3.67 (d, J = 8.7 Hz, 1H), 3.54 (d, J

= 11.6 Hz, 1H), 3.20 (t, J = 12.4 Hz, 1H), 3.09 (t, J = 11.1 Hz, 2H), 2.19

(d, J = 13.9 Hz, 1H), 2.13−1.96 (m, 1H). HRMS: m/z (ES⁺) [M + H]⁺

= 511.0782 (theor 511.0757).

4.1.1.7. 4-(4-Chloro-1-methyl-1H-pyrazol-5-yl)-N-((3S,4S)-4-(3,4-

diﬂuorophenyl)piperidin-3-yl)-2,5-diﬂuorobenzamide Hydrochlor-

ide (A4). General procedure, yield: 61%; ¹H NMR (500 MHz,

MeOD) δ 7.57 (s, 1H), 7.29 (qdd, J = 21.8, 14.5, 8.6 Hz, 4H), 7.16 (s,

1H), 4.58 (dd, J = 11.3, 7.8 Hz, 1H), 3.72 (d, J = 6.2 Hz, 3H), 3.67 (dd, J

= 11.9, 3.6 Hz, 1H), 3.54 (d, J = 11.4 Hz, 1H), 3.20−3.07 (m, 3H), 2.20

(d, J = 13.7 Hz, 1H), 2.07 (dd, J = 24.5, 12.7 Hz, 1H). ¹³C NMR (126

MHz, MeOD) δ 163.13 (s), 156.54 (s), 156.06 (s), 154.57 (s), 154.10

(s), 151.07 (s), 149.10 (s), 148.50 (s), 137.90 (s), 136.74 (s), 132.41

(s), 125.72 (s), 123.97 (s), 119.60 (s), 119.38 (s), 117.26−116.57 (m),

116.34 (d, J = 17.7 Hz), 110.31 (s), 46.41 (s), 44.66 (s), 43.73 (s),

37.15 (d, J = 3.0 Hz), 29.79 (s). HRMS: m/z (ES⁺) [M + H]⁺=

467.1292 (theor 467.1262).

4.1.1.8. 4-(4-Bromo-1-methyl-1H-pyrazol-5-yl)-N-((3S,4S)-4-(3,4-

diﬂuorophenyl)piperidin-3-yl)-2,5-diﬂuorobenzamide Hydrochlor-

ide (A5). General procedure, yield: 60%;¹H NMR (400 MHz,

MeOD) δ 7.63 (s, 1H), 7.39 (dd, J = 8.7, 5.7 Hz, 1H), 7.37−7.31

(m, 2H), 7.25 (dd, J = 18.8, 10.3 Hz, 2H), 4.65 (t, J = 9.4 Hz, 1H), 3.78

(s, 3H), 3.72 (d, J = 8.9 Hz, 1H), 3.59 (d, J = 10.8 Hz, 1H), 3.22 (dd, J =

27.5, 11.7 Hz, 3H), 2.28−2.10 (m, 2H). ¹³C NMR (101 MHz, MeOD)

δ 163.11 (s), 156.75 (s), 156.26 (s), 154.29 (s), 153.79 (s), 151.33 (d, J

= 12.8 Hz), 150.61 (d, J = 12.8 Hz), 148.87 (d, J = 12.6 Hz), 148.16 (d, J

= 12.3 Hz), 138.97 (s), 137.97 (s), 134.17 (s), 126.29−125.45 (m),

124.00 (s), 120.02 (dd, J = 24.6, 15.6 Hz), 119.53 (s), 117.34−116.64

(m), 116.37 (d, J = 17.6 Hz), 94.75 (s), 46.40 (s), 44.64 (s), 43.74 (s),

37.20 (s), 29.75 (s). HRMS: m/z (ES⁺) [M + H]⁺= 511.0744 (theor

511.0757).

4.1.1.10. 4-(4-Bromo-1-methyl-1H-pyrazol-5-yl)-2-chloro-N-

((3S,4S)-4-(3,4-diﬂuorophenyl)piperidin-3-yl)-6-ﬂuorobenzamide

1

Hydrochloride (A7). General procedure, yield: 54%; H NMR (400

MHz, MeOD) δ 7.58 (d, J = 2.0 Hz, 1H), 7.40 (s, 1H), 7.34−7.23 (m,

3H), 7.19 (s, 1H), 4.64 (s, 1H), 3.81 (d, J = 1.9 Hz, 3H), 3.71 (d, J =

11.7 Hz, 1H), 3.55 (d, J = 11.9 Hz, 1H), 3.23 (d, J = 13.0 Hz, 1H), 3.09

(d, J = 11.1 Hz, 2H), 2.22 (d, J = 12.9 Hz, 1H), 2.06 (d, J = 14.3 Hz,

1H). ¹³C NMR (101 MHz, MeOD) δ 162.51 (s), 160.01 (s), 157.52

(s), 138.85 (s), 138.25 (s), 137.83 (s), 132.25−131.71 (m), 126.57 (s),

125.41 (s), 125.18 (s), 123.98 (s), 117.12 (d, J = 17.7 Hz), 116.42 (d, J

= 17.9 Hz), 115.82 (d, J = 23.3 Hz), 93.59 (s), 44.51 (s), 43.60 (s),

37.27 (s), 30.19 (s), 29.32 (s). HRMS: m/z (ES⁺) [M + H]⁺

=

527.0479 (theor 527.0461).

4.1.1.11. 2,6-Diﬂuoro-N-((3S,4S)-4-(4-ﬂuorophenyl)piperidin-3-

yl)-4-(1-methyl-1H-pyrazol-5-yl)benzamide Hydrochloride (A8).

General procedure, yield: 56%; H NMR (400 MHz, DMSO) δ 9.59

1

(s, 1H), 8.96 (d, J = 8.7 Hz, 1H), 7.51 (d, J = 1.8 Hz, 1H), 7.38 (d, J =

8.1 Hz, 2H), 7.31 (dd, J = 8.5, 5.6 Hz, 2H), 7.18 (t, J = 8.8 Hz, 2H), 6.55

(d, J = 1.8 Hz, 1H), 4.64−4.51 (m, 1H), 3.90 (s, 3H), 3.43 (d, J = 4.0

Hz, 1H), 3.35 (s, 1H), 3.09−2.92 (m, 2H), 2.86 (t, J = 11.8 Hz, 1H),

2.08 (dd, J = 23.7, 11.5 Hz, 1H), 1.95 (d, J = 12.9 Hz, 1H). ¹³C NMR

(101 MHz, DMSO) δ 162.73 (s), 160.62−159.99 (m), 159.11 (d, J =

9.1 Hz), 157.67 (d, J = 9.1 Hz), 140.36 (s), 138.48 (s), 137.89 (s),

133.77 (s), 129.75 (d, J = 8.0 Hz), 115.54 (s), 115.33 (s), 114.68 (s),

112.15 (d, J = 25.3 Hz), 107.56 (s), 47.90 (s), 46.62 (s), 44.42 (s),

43.46 (s). HRMS: m/z (ES⁺) [M + H]⁺= 415.1825 (theor 415.1746).

4.1.1.12. 2,5-Diﬂuoro-N-((3S,4S)-4-(4-ﬂuorophenyl)piperidin-3-

yl)-4-(1-methyl-1H-pyrazol-5-yl)benzamide Hydrochloride (A9).

General procedure, yield: 59%; HPLC: t_R12.110 min, purity 99.48%.

¹H NMR (400 MHz, MeOD) δ 7.57 (d, J = 2.0 Hz, 1H), 7.41 (dd, J =

8.6, 5.3 Hz, 2H), 7.36−7.28 (m, 2H), 7.10 (t, J = 8.7 Hz, 2H), 6.46 (d, J

= 1.7 Hz, 1H), 4.65 (td, J = 11.5, 4.0 Hz, 1H), 3.80 (d, J = 1.1 Hz, 3H),

3.71 (dd, J = 12.0, 3.8 Hz, 1H), 3.58 (d, J = 12.4 Hz, 1H), 3.30−3.11 (m,

3H), 2.16 (dt, J = 23.6, 13.2 Hz, 2H). ¹³C NMR (101 MHz, MeOD) δ

163.21 (d, J = 13.5 Hz), 160.85 (s), 156.56−156.21 (m), 153.91 (d, J =

6.1 Hz), 138.33 (s), 136.38 (d, J = 3.1 Hz), 135.70 (s), 129.14 (d, J = 8.1

Hz), 124.61 (d, J = 7.6 Hz), 122.26 (dd, J = 18.0, 9.6 Hz), 118.87 (s),

118.59 (s), 116.83 (d, J = 26.1 Hz), 116.66−116.47 (m), 115.19 (s),

114.97 (d, J = 21.5 Hz), 107.73 (s), 46.47 (s), 44.63 (s), 43.81 (s),

36.21 (s), 30.02 (s). HRMS: m/z (ES⁺) [M + H]⁺= 415.1848 (theor

415.1746).

4.1.1.13. 2-Chloro-N-((3S,4S)-4-(3,5-diﬂuorophenyl)piperidin-3-

yl)-4-(1-methyl-1H-pyrazol-5-yl)benzamide Hydrochloride (B1).

General procedure, yield: 59%; H NMR (500 MHz, MeOD) δ 7.78

1

(d, J = 1.8 Hz, 1H), 7.58 (s, 1H), 7.48 (d, J = 7.4 Hz, 1H), 7.27 (d, J =

7.5 Hz, 1H), 7.00 (d, J = 6.3 Hz, 2H), 6.85 (t, J = 9.0 Hz, 1H), 6.58 (d, J

= 1.7 Hz, 1H), 4.63 (t, J = 9.6 Hz, 1H), 3.90 (s, 3H), 3.67 (d, J = 8.5 Hz,

1H), 3.52 (d, J = 11.3 Hz, 1H), 3.23−3.03 (m, 3H), 2.19 (d, J = 14.0 Hz,

1H), 2.06 (dd, J = 24.3, 12.2 Hz, 1H). ¹³C NMR (126 MHz, MeOD) δ

167.30 (s), 164.23 (d, J = 12.9 Hz), 162.27 (d, J = 12.9 Hz), 145.09 (s),

143.23 (s), 136.80 (s), 136.27 (s), 131.76 (s), 131.03 (s), 129.84 (s),

128.82 (s), 127.28 (s), 110.85−110.41 (m), 107.15 (s), 102.43 (s),

102.22 (s), 102.02 (s), 46.48 (s), 45.16 (s), 43.65 (s), 36.35 (s), 29.83

(s). HRMS: m/z (ES⁺) [M + H]⁺= 431.1483 (theor 431.1450).

4.1.1.14. N-((3S,4S)-4-(3,5-Diﬂuorophenyl)piperidin-3-yl)-2-ﬂuo-

ro-4-(1-methyl-1H-pyrazol-5-yl)benzamide Hydrochloride (B2).

4.1.1.9. 2-Chloro-4-(4-chloro-1-methyl-1H-pyrazol-5-yl)-N-

((3S,4S)-4-(3,4-diﬂuorophenyl)piperidin-3-yl)-6-ﬂuorobenzamide

1

General procedure, yield: 58%; H NMR (500 MHz, MeOD) δ 8.04

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(s, 1H), 7.60 (s, 1H), 7.50−7.43 (m, 2H), 7.02 (d, J = 6.6 Hz, 2H), 6.84

(t, J = 8.9 Hz, 1H), 6.78 (s, 1H), 4.66 (s, 1H), 4.02 (s, 3H), 3.66 (d, J =

9.2 Hz, 1H), 3.56 (d, J = 10.9 Hz, 1H), 3.18 (dd, J = 25.9, 12.9 Hz, 3H),

2.20 (t, J = 13.5 Hz, 1H), 2.13 (d, J = 12.0 Hz, 1H). ¹³C NMR (126

MHz, MeOD) δ 164.41−163.99 (m), 162.17 (d, J = 12.9 Hz), 160.37

(s), 158.36 (s), 145.04 (d, J = 8.8 Hz), 144.46 (s), 135.81 (s), 132.14 (d,

J = 8.7 Hz), 130.61 (s), 125.02 (s), 124.06 (d, J = 14.4 Hz), 116.89 (s),

116.69 (s), 110.76−110.31 (m), 107.78 (s), 102.38 (s), 102.18 (s),

101.97 (s), 46.43 (s), 45.10 (s), 43.71 (s), 36.51 (s), 29.49 (s). HRMS:

m/z (ES⁺) [M + H]⁺= 415.1788 (theor 415.1746).

J = 7.3 Hz, 2H), 3.73−3.63 (m, 1H), 3.58 (d, J = 12.1 Hz, 1H), 3.28−

3.11 (m, 3H), 2.26−2.09 (m, 2H), 1.54 (t, J = 7.3 Hz, 3H). ¹³C NMR

(101 MHz, MeOD) δ 164.85 (s), 161.18 (s), 158.69 (s), 151.31 (d, J =

12.7 Hz), 150.57 (d, J = 12.6 Hz), 148.86 (d, J = 12.8 Hz), 148.52−

147.95 (m), 138.11 (d, J = 4.2 Hz), 137.17 (d, J = 9.0 Hz), 131.89 (s),

130.26 (d, J = 2.6 Hz), 124.03 (s), 121.63 (s), 121.57−121.07 (m),

117.00 (d, J = 17.3 Hz), 116.35 (d, J = 17.8 Hz), 113.04−112.87 (m),

112.65 (d, J = 24.6 Hz), 103.73 (s), 46.46 (s), 44.62 (s), 43.72 (s),

29.70 (s), 14.47 (s). HRMS: m/z (ES⁺) [M + H]⁺= 429.1951 (theor

429.1902).

4.2. Molecular Docking and Dynamics Simulation. The

molecular docking was performed using Ligand Docking implanted

in Maestro 11.9. Parameters were maintained at the default

conﬁguration. The docked structure of the compounds complexed

within the active pocket of 4GV1 was used as the initial structures for

MD calculations using Amber. ﬀ99SB and gaﬀ2 force ﬁelds were

applied to the complex, and the resulting system was subjected to a

double-fold minimization (5000 cycles of steepest descent minimiza-

tion and 5000 cycles of conjugate gradient minimization) protocol

three times, with cartesian restraints imposed on heavy atoms, Cα

atoms of the complex and cartesian restraints released, respectively. The

system was heated from 0 to 300 K under cartesian restraints over a

period of 100 ps and subsequently equilibrated for 100 ps with cartesian

restraints and 100 ps without cartesian restraints. Starting from the last

frame of the equilibration, a production simulation was performed for

10 ns using the Berendsen barostat and Langevin thermostat under a

constant temperature of 300 K and pressure of 1 atm. Finally, the GBSA

binding free energy of the last 2 ns and the dihedral angle of the last 5 ns

were analyzed.

4.3. Akt1 and Akt2 Inhibitory Activity Assay. The Akt1 and

Akt2 inhibitory activities were determined by Sundia MediTech

Company using the mobility shift assay. Akt1 and Akt2 kinase were

purchased from Carna Biosciences. The kinase reactions were carried

out in a 384 OptiPlate (PerkinElmer) with 10 μL reaction volume per

well containing Akt1or Akt2 kinase, Caliper substrate 6 (GL), test

compound, and adenosine triphosphate (ATP) in assay buﬀer. After

incubation for 1 h at room temperature, the reaction was stopped by the

addition of 30 μL of reaction stop buﬀer. The plate was sealed and

incubated for 30 s at room temperature, and the resulting signal was

measured on Caliper EZ Reader II. The percent inhibitions were

calculated as follows: percent inhibition = (max − sample ratio)/(max

− min) × 100 (“min” means the ratio of no enzyme control and “max”

means the ratio of DMSO control).

4.4. Kinase Selectivity Assay. The representative compounds

were assayed against a panel of 19 protein kinases at Shanghai

Chempartner Co. Ltd. All of the proteins and kinase assay kits were

mainly purchased from Carna, Invitrogen, or Millipore. Compounds

were tested using mobility shift assay with ATP concentration at Km.

PI3Kα inhibitory activity was screened by Kinase-Glo Luminescent

Assay kit (Promege) with ATP concentration at 25 μM. CHK1

inhibitory activity was determined using the Luminescent ADP-Glo

assay kit (Promege). Moreover, PKA and CDK2/cyclin A activities

were evaluated at the concentration of 1 μg/mL by Z-LYTE Kinase

Assay kits (Invitrogen). Like Akt1 assay, the Aurora A inhibitory activity

was measured by the HTRF Kinase Assay kits purchased from Cisbio.

The protocols for these assays could be found in the manufacturer’s

instructions.

4.1.1.15. N-((3S,4S)-4-(3,5-Diﬂuorophenyl)piperidin-3-yl)-3-ﬂuo-

ro-4-(1-methyl-1H-pyrazol-5-yl)benzamide Hydrochloride (B3).

1

General procedure, yield: 60%; H NMR (500 MHz, MeOD) δ 7.94

(d, J = 10.9 Hz, 1H), 7.69−7.56 (m, 3H), 7.01 (d, J = 6.3 Hz, 2H), 6.81

(ddd, J = 9.1, 5.6, 2.1 Hz, 1H), 6.68 (s, 1H), 4.66 (td, J = 11.5, 3.9 Hz,

1H), 3.88 (s, 3H), 3.62 (dt, J = 6.5, 4.1 Hz, 1H), 3.59−3.53 (m, 1H),

3.28 (s, 1H), 3.26−3.15 (m, 2H), 2.21 (d, J = 13.1 Hz, 1H), 2.10 (dd, J

= 24.0, 12.2 Hz, 1H). ¹³C NMR (126 MHz, MeOD) δ 165.78 (s),

164.11 (d, J = 13.0 Hz), 162.14 (d, J = 13.0 Hz), 160.32 (s), 158.33 (s),

145.20 (s), 139.22 (s), 137.50 (d, J = 7.4 Hz), 136.46 (s), 131.78 (s),

123.34 (d, J = 3.5 Hz), 119.35 (d, J = 15.1 Hz), 115.08 (s), 114.89 (s),

110.44 (dd, J = 19.8, 5.9 Hz), 108.46 (s), 102.37 (s), 102.16 (s), 101.96

(s), 46.44 (s), 45.02 (s), 43.63 (s), 36.11 (d, J = 3.5 Hz), 29.53 (s).

HRMS: m/z (ES⁺) [M + H]⁺= 415.1792 (theor 415.1746).

4.1.1.16. 2-Chloro-N-((3S,4S)-4-(3,4-diﬂuorophenyl)piperidin-3-

yl)-4-(1-methyl-1H-pyrazol-5-yl)benzamide Hydrochloride (B4).

General procedure, yield: 62%; HPLC: t_R12.260 min, purity 99.22%.

¹H NMR (400 MHz, CDCl3) δ 7.50 (dd, J = 12.7, 4.5 Hz, 2H), 7.38 (s,

1H), 7.31 (dd, J = 5.7, 2.0 Hz, 2H), 7.14 (ddd, J = 10.2, 7.6, 2.9 Hz, 2H),

7.07 (s, 1H), 6.31 (d, J = 1.6 Hz, 1H), 4.32 (dd, J = 8.4, 3.8 Hz, 1H),

3.86 (s, 3H), 3.60 (dd, J = 12.1, 4.0 Hz, 1H), 3.21 (d, J = 12.6 Hz, 1H),

2.82−2.62 (m, 3H), 2.06 (s, 2H). ¹³C NMR (100 MHz, CDCl3) δ

165.53, 151.82−150.39 (m), 148.67 (dd, J = 108.3, 12.6 Hz), 141.17,

139.62, 138.86 (d, J = 4.1 Hz), 134.61, 133.96, 130.87, 130.40, 130.09,

127.21, 123.54, 117.51 (d, J = 16.9 Hz), 116.65 (d, J = 17.3 Hz), 106.83

(d, J = 3.6 Hz), 52.43, 51.71, 48.45, 46.30, 37.72, 29.80. HRMS: m/z

(ES⁺) [M + H]⁺= 431.1468 (theor 431.1450).

4.1.1.17. N-((3S,4S)-4-(3,4-Diﬂuorophenyl)piperidin-3-yl)-2-ﬂuo-

ro-4-(1-methyl-1H-pyrazol-5-yl)benzamide Hydrochloride (B5).

General procedure, yield: 67%; HPLC: t_R12.312 min, purity

1

100.00%. H NMR (400 MHz, MeOD) δ 7.48 (d, J = 7.5 Hz, 1H),

7.45 (s, 1H), 7.33−7.23 (m, 3H), 7.18 (dd, J = 18.6, 8.5 Hz, 1H), 7.12

(s, 1H), 6.39 (s, 1H), 4.55 (t, J = 9.6 Hz, 1H), 3.82 (s, 3H), 3.61 (d, J =

9.4 Hz, 1H), 3.49 (d, J = 11.4 Hz, 1H), 3.18−3.00 (m, 3H), 2.14 (d, J =

13.6 Hz, 1H), 2.03 (dd, J = 24.8, 12.3 Hz, 1H). ¹³C NMR (101 MHz,

MeOD) δ 164.61 (s), 158.13 (s), 151.36 (s), 150.51 (s), 148.91 (s),

148.19 (s), 141.66 (s), 138.09 (d, J = 11.7 Hz), 135.00 (d, J = 9.1 Hz),

130.20 (s), 124.44 (s), 124.00 (s), 122.39 (d, J = 14.4 Hz), 117.04 (d, J

= 17.1 Hz), 116.64−116.01 (m), 115.89 (s), 106.51 (s), 46.46 (s),

44.66 (s), 36.50 (s), 29.76 (s). HRMS: m/z (ES⁺) [M + H]⁺

=

415.1785 (theor 415.1746).

4.1.1.18. N-((3S,4S)-4-(3,4-Diﬂuorophenyl)piperidin-3-yl)-2-ﬂuo-

ro-4-(1H-pyrazol-5-yl)benzamide Hydrochloride (B6). General pro-

cedure, yield: 58%; ¹H NMR (400 MHz, MeOD) δ 7.97 (s, 1H), 7.65

(dd, J = 18.7, 9.8 Hz, 2H), 7.53 (t, J = 7.5 Hz, 1H), 7.37−7.29 (m, 1H),

7.27−7.18 (m, 2H), 6.95 (s, 1H), 4.65 (t, J = 9.4 Hz, 1H), 3.73−3.66

(m, 1H), 3.62−3.54 (m, 1H), 3.28−3.10 (m, 3H), 2.18 (dd, J = 31.9,

12.9 Hz, 2H). ¹³C NMR (101 MHz, MeOD) δ 164.68 (s), 161.12 (s),

158.63 (s), 151.30 (d, J = 12.8 Hz), 150.57 (d, J = 12.5 Hz), 148.85 (d, J

= 12.7 Hz), 148.13 (d, J = 12.3 Hz), 147.14 (s), 138.07 (s), 135.48 (d, J

= 8.8 Hz), 132.97 (s), 130.47 (s), 124.03 (s), 122.35 (d, J = 14.4 Hz),

121.67 (s), 117.00 (d, J = 17.4 Hz), 116.37 (d, J = 17.7 Hz), 113.31 (s),

113.06 (s), 46.47 (s), 44.62 (s), 43.75 (s), 29.70 (s). HRMS: m/z (ES⁺)

[M + H]⁺= 401.1641 (theor 401.1589).

4.1.1.19. N-((3S,4S)-4-(3,4-Diﬂuorophenyl)piperidin-3-yl)-4-(1-

ethyl-1H-pyrazol-5-yl)-2-ﬂuorobenzamide Hydrochloride (B7). Gen-

eral procedure, yield: 55%; HPLC: t_R12.857 min, purity 100.00%. ¹H

NMR (400 MHz, MeOD) δ 7.84 (d, J = 2.1 Hz, 1H), 7.62 (dd, J = 22.0,

10.0 Hz, 2H), 7.50 (t, J = 7.7 Hz, 1H), 7.37−7.30 (m, 1H), 7.22 (dd, J =

13.7, 9.7 Hz, 2H), 6.80 (t, J = 6.8 Hz, 1H), 4.69−4.58 (m, 1H), 4.32 (q,

4.5. Cell Culture. The diﬀerent cancer cells and HaCaT cells were

obtained from the Cell Bank of the China Science Academy, Shanghai,

and were cultured in Dulbecco’s modiﬁed Eagle’s medium (DMEM),

F-12K or RPMI-1640 medium (Gibco Laboratories), supplemented

with 10% fetal bovine serum (Gibco Laboratories), 105 U/L penicillin,

and 100 mg/L streptomycin at 37 °C in an atmosphere containing 5%

CO₂. Cells were grown to 70−80% conﬂuency in dishes or cell culture

plates and treated under various conditions as indicated.

4.6. Antiproliferative Assay. The cancer cells or HaCaT cells

were treated with the compounds at various doses for 72 h, and cell

proliferation was tested by a sulforhodamine B (SRB) protein assay.

The cells (5000/well) were incubated with 10% trichloroacetic acid

(TCA) for 1 h (4 °C) and then stained with SRB for 20 min. The SRB

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was washed away with 1% glacial acetic acid, and 100 μL of 1% Tris-

base was added to each well. The optical density (OD) was determined

at 515 nm by a Multiskan Spectrum plate reader (Thermo Electron

Corporation, Marietta, OH).

dogs (male, 40 weeks old, 8−10 kg) were purchased from Nanjing

Chaimen Biological Technology Co. Ltd. These animals were bred at

the Center for Drug Safety Evaluation and Research, College of

Pharmaceutical Sciences, Zhejiang University. The animals were

maintained under a 12 h light−dark cycle with free access to water

and food. All animal studies were performed in strict accordance with

the institutional guidelines as deﬁned by the Institutional Animal Care

and Use Committee (IUCAC) and approved by the Animal Care and

Use Committee, Zhejiang University Laboratory Animal Center

(Hangzhou, China).

4.12. Pharmacokinetic and Metabolites Studies. SD rats or

Beagle dogs were administered Hu7691 by oral gavage in saline (5%

DMSO + 15% 0.1 g/mL Kolliphor HS) or in capsules, respectively.

Venous blood (100 μL) samples of rats were collected at 0, 0.25, 0.5, 1,

2, 4, 8, 24, 30, 48, 54, and 72 h for oral gavage or 0, 0.083, 0.25, 0.5, 0.75,

1, 2, 4, 6, 8, and 24 h for intravenous injection. Venous blood (100 μL)

samples of dogs were collected at 0, 0.25, 0.5, 1, 2, 4, 6, 10, 24, 30, 48, 72,

and 96 h. Plasma was separated from whole blood by centrifugation and

stored at −20 °C until analysis. The rat plasma (from 0.5, 1, 2, 4, 6 h),

urine (from 4 to 7, 24 to 48, 48 to 72 h), excrement (from 0 to 4, 4 to 7,

7 to 24 h), and bile (from 0 to 6, 6 to 12, 12 to 24 h) were collected for

metabolites study. For pharmacokinetic studies, an aliquot of 50 μL of

plasma was quenched using 300 μL of acetonitrile containing 5 ng/mL

internal standard compound (Loratadine, Aladdin). For metabolites

study, the samples were quenched using 300 μL of acetonitrile. After

quenching, the mixture was vortexed and centrifuged, the resulting

solution was injected into an AB Sciex Qtrap 5500 LC−MS/MS system

(pharmacokinetic study) or Waters XEVO G2-XS Q-TOF LC−MS/

MS system (metabolites study). The C_max, T_max, T_1/2, and AUC values

were evaluated using Winnolin 8.0.

4.13. Toxicology Evaluation. The rats were randomly divided

into four groups and received Hu7691 (25, 50, 75 mg/kg, i.g.) or

vehicle (0.5% MC, i.g.) once daily for 28 days. Hu7691 was dissolved in

0.5% MC. The body weight of the mice was recorded every 7 days.

During the experiment, the scab clinical symptom on rat back skin was

recorded. At the end of the experiment, the mice were sacriﬁced, and

the serum hematological parameters and serum biochemistry

parameters were recorded on Sysmex XT-2000i and Roche Cobas c

311, respectively.

4.14. In Vivo Evaluation of Tumor Inhibition. BALB/c (nu/nu)

mice were used for the experiments. When 786-O or KHOS xenograft

tumors reached 100−300 mm³, the mice were randomly divided into

ﬁve groups and received Hu7691 (12.5, 25, 50 mg/kg, i.g.), AZD5363

(50 or 100 mg/kg, i.g.), or vehicle (0.5% MC, i.g.) once daily for 22

days. Compounds were dissolved in 0.5% MC. The body weight of mice

and tumor volume were recorded every 3 days. At the end of the

experiment, the mice were sacriﬁced and the tumors were weighed and

photographed.

4.7. Western Blot Assay. Whole protein extracts from cultured

cells or tissues were prepared and subjected to western blotting. Protein

samples (40 μg) were loaded and run on 10% sodium dodecyl sulfate−

polyacrylamide gel electrophoresis (SDS-PAGE), then transferred to

poly(vinylidene diﬂuoride) (PVDF) membranes (Merck Millipore,

IPVH00010), and incubated with primary antibodies overnight at 4 °C.

After that, the membranes were washed three times by phosphate-

buﬀered saline (PBS) with 0.1% Tween-20 (T-PBS), and incubated

with secondary antibodies for 1 h at room temperature. After being

washed three times with T-PBS, membranes were incubated with

Western Lightning Plus-ECL reagent (PerkinElmer, NEL105001EA),

and then were exposed using Amersham Imager 600 (General Electric

Company). The primary antibodies were listed as follows: Akt1 (Cell

Signaling Technology, 75692s), Akt2 (Cell Signaling Technology,

2964S), Akt3 (Cell Signaling Technology, 14982S), pAKT-Ser473

(Santa Cruz Biotechnology, sc-514032), Cleaved Caspase 3 (HuaBio,

ET1602-47), c-PARP (Cell Signaling Technology, 5625s), GAPDH

(DiagBio, db106). Appropriate secondary antibodies (MultiSciences

Biotech, GAM007 and GAR007) and ECL (PerkinElmer, The

Netherlands) were used to visualize the protein signaling.

4.8. hERG Inhibition Assay. Currents were recorded from HEK-

293 cells, using the whole-cell patch-clamp technique. The cells were

transferred to a perfusion chamber, and the perfusion was performed

with extracellular ﬂuid. The extracellular ﬂuid (mM): KAspartate, 130;

MgCl₂, 5; EGTA, 5; HEPES, 10; Tris-ATP, 4; pH 7.2. Electrodes were

pulled using a dual-stage glass micropipette puller (Narishige PC-10,

Japan). Current traces of hERG channels were elicited by applying a

pulse from −80 to +40 mV for 4 s followed by a step to −40 mV for 2 s.

The procedure was repeated every 20 s. After the maximum current was

stabilized, the tested compounds (3 μM) were perfused. The inhibition

rate was calculated when the current was stable.

4.9. Liver Microsome Stability Assay. Microsomes from diﬀerent

species were purchased from Ruide Research Institute for Liver

Diseases (Shanghai) Co. Ltd. (Human: SUNK, Monkey: CYJC, Beagle

Dog: DMXD, Rat: JPXY, Mouse: STOM-2). A 1 mg/mL microsome

solution was mixed with 20 mL of 50 mM NADPH (Roche) solution to

prepare a microsome-NADPH solution. The microsome-NADPH

solution (500 μL) was prewarmed at 37 °C for 5 min. Hu7691 solution

was then added to a ﬁnal concentration of 50 μM to initiate the reaction.

The incubation mixture was kept at 37 °C, and 200 μL aliquots were

taken at 0, 10, 30, and 60 min. In each aliquot, the reaction was

quenched using 400 μL of acetonitrile containing 5 ng/mL internal

standard compound (Verapamil, Aladdin). After quenching, the

mixtures were vortexed and centrifuged at 4 °C. The supernatant was

transferred, and 10 μL was injected into a XEVO TQ-S LC−MS/MS

system. The peak area ratio of a test article versus the internal standard

was used in the calculation of the rate of disappearance of a test article.

4.10. Plasma Protein Binding Rate Assay. The plasma from

diﬀerent species such as Beagle dog, monkey, and rat were provided by

Center for Drug Safety Evaluation and Research, College of

Pharmaceutical Sciences, Zhejiang University. The human plasma

was provided by the Second Aﬃliated Hospital Zhejiang University

School of Medicine. A 300 μL plasma containing 50 or 2500 ng/mL

Hu7691 was added to the sample chamber of Thermo Equilibrium

Dialysis Device (Red Device Insert), and 500 μL of PBS dialysate was

added to the buﬀer chamber. After sealing, the mixture was incubated at

37 °C for 8 h. An aliquot of 50 μL of each mixture was quenched using

500 μL of acetonitrile containing 5 ng/mL internal standard compound

(Loratadine, Aladdin). After quenching, the mixture was vortexed and

centrifuged and the resulting solution was injected into a Waters XEVO

TQ-S system. PPB % = (1 − concentration in PBS/concentration in

plasma) × 100.

4.15. Statistical Analysis. Statistical analysis was performed using

GraphPad Prism 5 software (GraphPad Software, Inc., La Jolla, CA).

Unless otherwise stated, the values for all samples in the diﬀerent

experimental conditions were averaged, and the standard deviation of

the mean was calculated. Statistical diﬀerences between two groups

were assessed by Student’s t-test. A P value of less than 0.05 was

considered statistically signiﬁcant.

ASSOCIATED CONTENT

* Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00815.

■

sı

RMSD value and contribution of inhibitor−residue

interaction of compound 2 (Figure S1); RMSD values

of compounds 3, 4, and 5 (Figure S2); molecular

dynamics result of B5−Akt2 complex (Figure S3); IC₅₀

curves of compound B5 against the normal cells (Figure

S4); images of 786-O and KHOS xenograft tumors in

mice that treated with compound B5 for 22 days (Figure

S5); selectivity proﬁle of B5 (Table S1); inhibition of

4.11. Animals. SD rats (male, 8 weeks old, 250−300 g) were

purchased from Zhejiang Vital River Laboratory Animal Technology

Co. Ltd. Balb/c mice (nu/nu, female, 3−4 weeks old, 20−25 g) were

purchased from Shanghai Sippr-BK Laboratory Animal Co. Ltd. Beagle

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Article

Research, College of Pharmaceutical Sciences, Zhejiang

University, Hangzhou 310058, P. R. China

Zegao Jin − Hangzhou Institute of Innovative Medicine,

Institute of Drug Discovery and Design, College of

Pharmaceutical Sciences, Zhejiang University, Hangzhou

310058, P. R. China

compounds against CYPs (Table S2); result of

chromosome aberration assay of compound B5 (Table

S3); NMR spectra and HPLC results for the target

compounds (Table S4); result of mice micronucleus assay

of compound B5; MS results of metabolites; compound

information for the intermediates (PDF)

Binhui Chen − Hangzhou Institute of Innovative Medicine,

Institute of Drug Discovery and Design, College of

Pharmaceutical Sciences, Zhejiang University, Hangzhou

310058, P. R. China

Molecular formula strings (CSV)

PDB complex of docking result (PDB)

Peihua Luo − Institute of Pharmacology & Toxicology, Zhejiang

Province Key Laboratory of Anti-Cancer Drug Research,

College of Pharmaceutical Sciences, Zhejiang University,

Hangzhou 310058, P. R. China

PDB complex of docking result (PDB)

Bo Yang − Hangzhou Institute of Innovative Medicine, Institute

of Drug Discovery and Design, College of Pharmaceutical

Sciences, Zhejiang University, Hangzhou 310058, P. R. China;

Institute of Pharmacology & Toxicology, Zhejiang Province

Key Laboratory of Anti-Cancer Drug Research, College of

Pharmaceutical Sciences, Zhejiang University, Hangzhou

310058, P. R. China

Yongzhou Hu − Hangzhou Institute of Innovative Medicine,

Institute of Drug Discovery and Design, College of

Pharmaceutical Sciences, Zhejiang University, Hangzhou

310058, P. R. China; orcid.org/0000-0001-7349-4062

Qiaojun He − Hangzhou Institute of Innovative Medicine,

Institute of Drug Discovery and Design, College of

Pharmaceutical Sciences, Zhejiang University, Hangzhou

310058, P. R. China; Center for Drug Safety Evaluation and

Research, College of Pharmaceutical Sciences, Institute of

Pharmacology & Toxicology, Zhejiang Province Key

Laboratory of Anti-Cancer Drug Research, College of

Pharmaceutical Sciences, and Cancer Center, Zhejiang

University, Hangzhou 310058, P. R. China; Innovation

Institute for Artiﬁcial Intelligence in Medicine of Zhejiang

University, Hangzhou 310018, P. R. China

AUTHOR INFORMATION

Corresponding Authors

■

Qinjie Weng − Center for Drug Safety Evaluation and Research,

College of Pharmaceutical Sciences and Institute of

Pharmacology & Toxicology, Zhejiang Province Key

Laboratory of Anti-Cancer Drug Research, College of

Pharmaceutical Sciences, Zhejiang University, Hangzhou

310058, P. R. China; Innovation Institute for Artiﬁcial

Intelligence in Medicine of Zhejiang University, Hangzhou

310018, P. R. China; Email: wengqinjie@zju.edu.cn

Xiaowu Dong − Hangzhou Institute of Innovative Medicine,

Institute of Drug Discovery and Design, College of

Pharmaceutical Sciences, Zhejiang University, Hangzhou

310058, P. R. China; Innovation Institute for Artiﬁcial

Intelligence in Medicine of Zhejiang University, Hangzhou

310018, P. R. China; Cancer Center, Zhejiang University,

Hangzhou 310058, P. R. China; orcid.org/0000-0002-

2178-4372; Email: dongxw@zju.edu.cn

Authors

Jinxin Che − Hangzhou Institute of Innovative Medicine,

Institute of Drug Discovery and Design, College of

Pharmaceutical Sciences, Zhejiang University, Hangzhou

310058, P. R. China

Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.jmedchem.1c00815

Xiaoyang Dai − Center for Drug Safety Evaluation and

Research, College of Pharmaceutical Sciences, Zhejiang

University, Hangzhou 310058, P. R. China

Jian Gao − Hangzhou Institute of Innovative Medicine, Institute

of Drug Discovery and Design, College of Pharmaceutical

Sciences, Zhejiang University, Hangzhou 310058, P. R. China

Haichao Sheng − Hangzhou Institute of Innovative Medicine,

Institute of Drug Discovery and Design, College of

Pharmaceutical Sciences, Zhejiang University, Hangzhou

310058, P. R. China

Wenhu Zhan − Hangzhou Institute of Innovative Medicine,

Institute of Drug Discovery and Design, College of

Pharmaceutical Sciences, Zhejiang University, Hangzhou

310058, P. R. China; Present Address: Department of

Microbiology & Immunology, Weill Cornell Medicine,

1300 York Avenue, New York, New York 10065, United

States; orcid.org/0000-0002-7095-7081

Author Contributions

J.C. synthesized the compounds, analyzed the data, and drafted

the manuscript. X.D. performed the anticancer activity

evaluation of the compounds. Z.G. performed the cutaneous

related experiments. J.G. performed the molecule dynamics

simulation. W.Z. contributed to the conceptualization, inves-

tigation, and synthetic methodology construction of the Akt

inhibitors. H.S., Y.L, D.L., and Z.J. synthesized the compounds.

B.C., P.L., B.Y., Y.H., Q.H., Q.W., and X.D. conceived the study.

J.C. and X.D. contributed equally to this work.

Funding

National Natural Science Foundation of China; Key R&D

Program of Zhejiang Province; Natural Science Foundation of

Zhejiang Province; National Major Scientiﬁc and Technological

Special Project for “Signiﬁcant New Drugs Development”.

Yang Lu − Hangzhou Institute of Innovative Medicine, Institute

of Drug Discovery and Design, College of Pharmaceutical

Sciences, Zhejiang University, Hangzhou 310058, P. R. China

Dan Li − Hangzhou Institute of Innovative Medicine, Institute of

Drug Discovery and Design, College of Pharmaceutical

Sciences, Zhejiang University, Hangzhou 310058, P. R. China

Zizheng Gao − Institute of Pharmacology & Toxicology,

Zhejiang Province Key Laboratory of Anti-Cancer Drug

Notes

The authors declare the following competing ﬁnancial

interest(s): The authors declare the following competing

ﬁnancial interest(s): Zhejiang University has ﬁled a number of

patent applications on this class of Akt inhibitors, which have

been licensed to Guangzhou LiXin Pharmaceuticals, X. Dong,

Q. Weng, Y. Hu, Q. He, B. Yang, B. Chen, W. Zhan, X. Dai, and J.

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Article

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ACKNOWLEDGMENTS

■

The authors thank Jianyang Pan (Research and Service Center,

College of Pharmaceutical Sciences, Zhejiang University) for

performing NMR spectrometry for structure elucidation. This

work was supported by grants from the National Natural Science

Foundation of China (81973172 and 82003579), Key R&D

Program of Zhejiang Province (2020C03010), Natural Science

Foundation of Zhejiang Province (LR21H300003 and

LQ21H300005), and National Major Scientiﬁc and Techno-

logical Special Project for “Signiﬁcant New Drugs Develop-

ment” (2018ZX09711002-007 and 2018ZX09711002-011-

023).

ABBREVIATIONS

■

AST, aspartate aminotransferase; ALP, alkaline phosphatase; c-

c-3, cleaved caspase 3; c-PARP, cleaved poly ADP-ribose

polymerase; CYPs, cytochrome P450 enzymes; DIPEA, N,N-

diisopropylethylamine; DLT, dose-limiting toxicities; DMF,

dimethylformamide; EA, ethyl acetate; EDCI, 1-ethyl-3-(3′-

dimethylaminopropyl) carbodiimide hydrochloride; hERG,

human ether-a-go-go related gene; HOBt, 1-hydroxybenzotria-

zole; MC, methyl cellulose; MTD, maximum tolerated dose;

mTOR, mammalian target of rapamycin; NBS, N-bromo

succinimide; NCS, N-chloro succinimide; PKA, protein kinase

A; PKG, protein kinase G; PI3K, phosphatidylinositol 3-kinase;

RMSD, root-mean-square deviation; shRNA, short hairpin

RNA; TFA, triﬂuoroacetic acid; TEA, triethylamine; THF,

tetrahydrofuran; UPLC, ultraperformance liquid chromatog-

raphy

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Article Doi

Discovery of N-((3 S,4 S)-4-(3,4-Difluorophenyl)piperidin-3-yl)-2-fluoro-4-(1-methyl-1 H-pyrazol-5-yl)benzamide (Hu7691), a Potent and Selective Akt Inhibitor That Enables Decrease of Cutaneous Toxicity

DOI: 10.1021/acs.jmedchem.1c00815

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