Discovery of 3,4,6-Trisubstituted Piperidine Derivatives as Orally

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Discovery of 3,4,6-Trisubstituted Piperidine Derivatives as Orally Active, Low hERG Blocking Akt Inhibitors via Conformational Restriction and Structure-based Design Xiaowu Dong, Wenhu Zhan, Mengting Zhao, Jinxin Che, Xiaoyang Dai, Yizhe Wu, Lei Xu, Yu-Bo Zhou, Yanmei Zhao, Tian Tian, Gang Cheng, Zegao Jin, Jia Li, Yanfei Shao, Qiaojun He, Bo Yang, Qinjie Weng, and Yongzhou Hu J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00891 • Publication Date (Web): 12 Jul 2019 Downloaded from pubs.acs.org on July 16, 2019

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Discovery of 3,4,6-Trisubstituted Piperidine Derivatives as Orally Active, Low hERG Blocking Akt Inhibitors via Conformational Restriction and Structure-based Design Xiaowu Dong,a, ‡ Wenhu Zhan,a,#, ‡ Mengting Zhao,b, ‡ Jinxin Che,a Xiaoyang Dai,b Yizhe Wu,a Lei Xu,c Yubo Zhou,c Yanmei Zhao,a Tian Tian,a Gang Cheng,d Zegao Jin,a Jia Li,c Yanfei Shao,e Qiaojun He,b Bo Yang,b Qinjie Weng,b,* Yongzhou Hua,*

a

ZJU-ENS Joint Laboratory of Medicinal Chemistry, Hangzhou Institute of Innovative

Medicine, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, 310058, China b

Institute of Pharmacology & Toxicology, Zhejiang Province Key Laboratory of Anti-

Cancer Drug Research, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, 310058, China


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c National

Center for Drug Screening, State Key Laboratory of Drug Research, Shanghai

Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China d College

of Pharmaceutical Science, Zhejiang Chinese Medical University, Hangzhou,

311402, China e

Department of Pharmacy, Zhejiang Provincial People's Hospital, Hangzhou, 310014,

China

#

Present Address: Department of Microbiology & Immunology, Weill Cornell Medicine,

1300 York Avenue, New York, NY 10065, United States.

‡

These authors contributed equally to this work.

*

Corresponding authors: Q. Weng: [email protected]; Y. Hu: [email protected]

ABSTRACT: A series of 3,4-disubstituted piperidine derivatives were obtained based on a conformational restriction strategy and a lead compound, A12, that exhibited potent in

vitro and in vivo anti-tumor efficacy; however, obvious safety issues limited its further development. Thus, systematic exploration of the structure-activity relationship of


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compound A12, involving the phenyl group, hinge-linkage and piperidine moiety, led to the discovery of the superior 3,4,6-trisubstituted piperidine derivative E22. E22 showed increased potency in Akt1 and cancer cell inhibition, remarkably reduced hERG blockage, and significantly improved safety profiles. Compound E22 also exhibited good kinase selectivity, had a good pharmacokinetic profile, and displayed very potent in vivo anti-tumor efficacy, with over 90% tumor growth inhibition in the SKOV3 xenograft model. Further mechanistic studies were conducted to demonstrate that compound E22 could significantly inhibit the phosphorylation of proteins downstream of Akt kinase in cells and tumor tissue from the xenograft model.

KEYWORDS: Akt inhibitors; Conformational restriction; Structure-based design; hERG blockage; Maximum tolerated dose.

1. Introduction


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Akt, also known as protein kinase B or PKB, is a serine/threonine kinase that belongs to the AGC family of kinases, sharing high homology with PKA and PKC. Akt can be classified into three different isozymes, including Akt1, Akt2 and Akt3; all three share a high degree of overall homology and similar downstream targets.1, 2 It is very common that the PI3K/Akt signaling pathway is overactivated in various cancer cells,3 and tumor growth can be effectively decreased via the inhibition of Akt alone or in combination with other chemotherapeutics.4-9 Currently, the efficacy of a number of Akt inhibitors is evaluated in clinical trials (Figure 1). In particular, a phase II clinical trial of GDC0068 was completed10, and a phase III clinical trial is currently investigating the effectiveness of GDC0068 in the treatment of triple-negative breast cancer (TNBC) (NCT03337724) and metastatic prostate cancer (NCT03072238). In addition, the results of clinical studies on the use of AZD5363 in combination with paclitaxel for the treatment of TNBC have also been disclosed, indicating that this therapy resulted in a positive outcome of significantly longer progression-free survival and overall survival,11 thus fully demonstrating the promising prospect of Akt as an anti-cancer target.12-18 Additionally, GSK2141795 (also known as


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GSK-795 or Uprosertib) is another ATP-competitive Akt inhibitor developed by GlaxoSmithKline, which is currently in early clinical trials for evaluation of its therapeutic effect against different types of cancer, such as multiple myeloma (NCT01989598), breast cancer (NCT01964924), and others. In the discovery stage of AZD5363 and GDC0068,18,19 the druggability involving in vitro and in vivo anti-cancer efficacy, safety issues (hERG), PK profiles and kinase selectivity were primarily explored and optimized. In comparison with the preclinical data, some of their advantages are clear, such as low hERG blockage of AZD5363,18 high selectivity profiles of GDC0068,19 low effective dose and excellent PK profiles of GSK-795,20 the data pertaining to which have also provided a criterion for searching for novel Akt inhibitors.

OH

NH

F

O Cl

N

F

O Cl

NH2

N H

O Cl

N

O

N N

N HO GDC-0068

N N

N N AZD5363

NH2 N H

NH2

N O

Cl

N HN N

N H GSK2141795

MK-2206

Figure 1. Chemical structures of representative Akt inhibitors.


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Conformational restriction is one of the most effective methodologies for drug design, since it can help improve the PK profiles of compounds and stabilize a favorable binding pattern in order to improve the binding affinities or kinase isoform selectivity.21,22 This strategy has been widely applied in drug discovery.22,23 In fact, some FDA approved drugs were discovered and developed by using conformational restriction, such as Dolutegravir.24 In our previous study, the discovery of a series of 4,4-disubstituted piperidine derivatives featuring more restricted conformations in contrast to GSK-795 were successfully achieved,25-29 which proved that conformational restriction strategy could be used for discovery of new Akt inhibitors with novel skeletons. As a continuation of this earlier work, herein, the structural modifications of 3,4-disubstituted piperidine derivatives were reported, which exhibit excellent potency and good PK profiles. Further structure-based drug design was used to introduce a side chain, leading to the discovery of a series of novel and highly potent 3,4,6-trisubstituted derivatives of E22 with good PK profiles, lower hERG blockage and improved safety profiles. Furthermore, the molecular mechanisms of E22 were studied, confirming the blockage of the PI3KAkt pathway in cancer cells and tumor tissue.


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2. Results and Discussion 2.1 Discovery of 3,4-disubstituted piperidine derivatives as Akt inhibitors On the basis of multiple previous research projects,27-32 most of the ATP-competitive Akt inhibitors were found to share common pharmacophore characteristics, including a hinge-binding group, a basic center and a hydrophobic group. These three pharmacophores generate a “Y” shape through a linkage. Although the cocrystal structure of GSK-795 with Akt1 has not been reported, the docking results (Figure S1) disclosed that the amino and pyrazole groups were hydrogen-bonded to the amino acid residues of Glu278 and Ala230. The 3,4-difluorophenyl group inserted into the hydrophobic pocket and π-π interacted with Phe161. Therefore, we hypothesized that cyclization of the amino group with the methylene moiety of the benzyl group based on the conformational restriction strategy may facilitate conformational stability of the phenyl group and basic center, and therefore would improve the Akt inhibitory potency (Figure 2).


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Figure 2. Design rationale based on the structures of GSK-795.

In the present study, the racemic 3,4-disubstituted piperidine or pyrrolidine derivatives were synthesized for further exploration. The linkage and hinge groups selected were the privileged furan moiety and N-methyl pyrazole, respectively, according to the structure of GSK-795. As shown in Table 1, when R1, R2 and R3 substituents were all hydrogens, compound A1 resulted in an IC50 value of 709.6 nM. After introducing a halogen to A1, the Akt1 inhibitory potency was reinforced, as shown in compounds A2, A3 and A4, demonstrating the importance of halogens in maintaining the compound activity. Notably, when R3 was 4-CF3 or 3,4-difluoro, compounds A5 and A6 both exhibited comparable Akt1 inhibitory potency to that of GSK-795. However, after the replacement of piperidine with pyrrolidine, the activities of compounds A7-A9 were


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dramatically reduced. Therefore, highly potent 3,4-disubstituted compounds A5 and A6 were selected for further chiral resolution to determine the optimal configuration. Table 1. Akt1 inhibitory activities of compounds A1-A9

R1 N

R3 O

N

H N O

R2

R1 N

N H A1~A6

O

N

O

R2

R3

H N NH

A7~A9

Compd.

R1

R2

R3

Akt1 IC50 (nM)

A1

H

H

H

709.6

A2

H

H

3,4-difluoro

191.0

A3

Cl

Cl

H

17.6

A4

Cl

Cl

4-Cl

17.3

A5

Cl

Cl

4-CF3

7.2

A6

Cl

Cl

3,4-difluoro

6.4

A7

H

Cl

3,4-difluoro

1566

A8

H

Br

3,4-difluoro

3646

A9

Cl

Cl

4-CF3

538

GSK-795

-

-

-

4.7


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According to the asymmetric synthetic method for constructing 3,4-disubstituted piperidine derivatives,33 the trans-conformations (3S,4S or 3R,4R) of 3,4-disubstituted piperidine were the main products resulting from using (S)- or (R)-Jorgensen-Hayashi reagent catalysts, respectively. As shown in Table 2, the activity of the (3S,4S)enantiomer (compounds A10 and A12) was significantly better than that of the (3R,4R)enantiomer (compounds A11 and A13), including their Akt1 inhibitory activities and antiproliferative activities against different cancer cell lines, such as OVCAR-8 and HCT116. In addition, one of the cis-conformation products, the (3R,4S)-enantiomer, was further separated during the asymmetric synthesis and afforded the compound (3R,4S)A6, exhibiting a good IC50 value of 2.5 nM (Figure S2). Considering that the product yield of the cis-conformation of 3,4-disubstituted piperidine was minimal and difficult to obtain, we only focused on the (3S,4S)-enantiomer of 3,4-disubstituted piperidine derivatives in the following studies. Table 2. Akt1 enzymatic and cancer cell line activities for compounds A10-A13


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Cl N

Cl N

A5 R=4-CF3 A6 R=3,4-diF

(3S, 4S)-A5 R=4-CF3 (3S, 4S)-A6 R=3,4-diF N H

(3S, 4S)

H N O

Cl

H N O

Cl

R O

N

R O

N

Cl

N H

N

R O

N

Cl

H N

(3R, 4R)-A5 R=4-CF3 (3R, 4R)-A6 R=3,4-diF

O

N H

(3R, 4R)

Compd.

Akt1 IC50 (nM)

OVCAR-8 (μM)

HCT116 (μM)

(3S, 4S)-A5 (A10)

9.3

0.12

0.28

(3R, 4R)-A5 (A11)

127.4

0.90

1.05

(3S, 4S)-A6 (A12)

3.0

0.04

0.21

(3R, 4R)-A6 (A13)

51.2

0.49

1.09

GSK-795

8.3

0.24

0.72

As shown in Figure 3, compound A12 exhibited good pharmacokinetics and long t1/2 (10.7 hours) and excellent bioavailability (F = 97.9%) in mice. This compound was slightly unstable in the liver microsomes of monkeys (39.9% remaining after incubating for 1.5 hours) and had a high hERG inhibition rate (62.1% inhibition at 3 μM). Next, the safety profile of A12 in mice was assessed. After 7 days of intragastric administration of A12 (10 mg/kg, 20 mg/kg and 40 mg/kg), toxic effects can be obviously observed in moderate and high dose groups (Figure S3 and Table S1): three of five and two of five


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mice died at the dosages of 40 mg/kg and 20 mg/kg, respectively. Some of the serum hematological and biochemical parameters such as RBC, HGB, HCT and CREA were obviously downregulated at the high dose (Figure S3 B and C). Thus, we reasoned that 10 mg/kg may be a tolerated dose for an in vivo study. The effect of A12 in vivo was first characterized by measuring its anti-tumor activity in an MM1S multiple myeloma xenograft model. Following single oral doses of 10 mg/kg, the impact on tumor growth was assessed over 22 days. In total, 87.5% inhibition of tumor growth was observed, indicating that the in vivo efficacy of A12 was strong (Figure S4 A and B). However, significant body weight loss (up to 25%) was observed in the mice (Figure S4 C), and one of the four mice died during the experiment (Figure S4 D); the death may be attributed to lower tolerance to the dose in nude mouse. It is demonstrated that the MTD of A12 was lower than 10 mg/kg, and dose escalation of A12 was highly limited, suggesting that potential safety issues must be further resolved.


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Figure 3. The profiles of in vitro and in vivo biological activities and safety issues of compound A12.

2.2 Discovery of Akt inhibitors with improved safety profiles After careful evaluation of the safety profile, we found that obvious deficiencies of compound A12 existed: 1) the MTD dose of A12 was quite low; 2) some toxicity was indicated

by

serum

hematological

and

biochemical

parameters;

3)

heavy

gastrointestinal hemorrhage caused the death of mice in autopsy analysis; 4) high hERG inhibition of A12 was observed (62.1% inhibition at 3 μM). To address these issues, subsequent investigation primarily focused on structure optimization, including the hinge-linkage moiety and phenyl substituents. In particular, on the basis of the docking results of compound A12 to the ATP bound pocket of Akt1 kinase, it was found


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that the 5- or 6-position of the piperidine ring may be oriented to the solvent area, providing us with a suitable point for exploration (Figure 4).

Figure 4. Strategies for further optimization of compound A12.

Modification of the hinge-linkage moiety and phenyl substituents. As shown in Table 3, compounds B1 and B2, but not compound B3, showed IC50 values below 10 nM when the phenyl group was substituted with different halogens. Furthermore, different hinge binders and linkages were evaluated. The 4-position of the N-methyl pyrazole hinge binder linked to the 2- or 3-position of the furan linkage (compounds C1 and C3) lead to a dramatic loss of enzyme activity. Removal of the methyl group of C1 to yield


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compound C2 resulted in an increase in potency. Other hinge binders and linkages were also tested, such as 3-amino-pyrimidine (compound C4) and cyclopentanepyrimidine (compound C5), and the activities of both of these compounds were dramatically reduced. Furthermore, the anti-proliferation potencies of compounds against OVCAR-8 and HCT116 cells were determined in accordance with the Akt IC50 values. Compounds A12, B1 and B2 exhibited good Akt IC50 values of 1000

4.41

15.07

C2

N HN

O

50.3

5.00

21.78

C3

N N

>1000

10.01

23.65

529.5

12.27

37.11

>1000

24.05

22.96

8.3

1.32

1.27

C4

O

HN N

O

N

HO

C5 GSK-795 a

N

N

-

S

The Inhibition% or IC50 values are an average of three independent determinations.

Since we cannot identify better hinge-linkage fragments, N-methyl pyrazole was chosen as hinge binder and the linkage was further investigated. Different fivemembered ring linkages were first evaluated, including furan, thiophene and N-methyl pyrrole (Table 4). In the case of the furan linkage, removal of the chloro group on Nmethyl pyrazole yielded the unsubstituted compound D1 or D2, which resulted in a


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slight reduction in potency (D1 vs. B3; D2 vs. A12). In addition, introducing a bromo group to the pyrazole did not favor activity improvement (D3 vs. D1). After changing from the 2,4-disubstituted to 2,5-substituted furan linkage, the activities of the obtained compounds were reduced (compounds D4-D9). When the linkage was between thiophene and N-methyl pyrrole, the IC50 values of the resulting compounds (D10-D18) were over 100 nM. In contrast to the structure-activity relationship (SAR) when the linkage included furan, introduction of a chloro group to the 5-position of the thiophene linkage was unfavorable to the compound activity, which even suffered a significant loss of potency (D10 vs. D11). After introducing a chloro or bromo group to the N-methyl pyrrole, the inhibitory potency of compounds improved (D15 vs. D17; D16 vs. D18). The six-membered ring linkage was also evaluated, including substituted phenyl and pyridyl. Although the activities of compounds with six-membered ring linkages were not better than that of compounds with furan linkages, some of the IC50 values were still lower than 100 nM. Compound D19 showed the most potent IC50 value of 21.6 nM. The


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activities of compounds with pyridine linkages were inferior to those of compounds with phenyl linkages (D19 vs. D23; D20 vs. D24; D21 vs. D25; and D22 vs. D27). The cellular potencies of the compounds against OVCAR-8 and HCT-116 cells were relatively insensitive, with most of the compounds exhibiting IC50 values of greater than 1 μM. Compound D2, exhibiting an Akt1 IC50 value of 5.1 nM, only showed approximately 1 μM anti-proliferative activity, which is comparable to that of GSK-795. Compound D16, with lower Akt1 inhibitory activity, also exhibited high hERG inhibition, demonstrating no relationship between Akt1 and hERG inhibition. Despite the low hERG inhibition observed for compounds D19 and D22, in which each linkage consisted of phenyl groups, the Akt1 inhibitory potency was unsatisfactory. Table 4. Akt1 enzymatic and cancer cell line activities for compounds D1-D28

R2 N

N

H N

Linkage R1

O D1~D28

Compd.

Linkage

R1

R2

hERG inhibition at 3 μM a

N H

Akt1 IC50 (nM) a

IC50 (µM)a OVCAR8

HCT116


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H

4-Cl

N.T. b

29.4

3.50

1.56

Cl

3,4-diF

N.T.

5.1

1.40

0.99

D3

Br

4-Cl

N.T.

35.6

9.01

2.31

D4

H

4-Cl

N.T.

230.2

6.09

9.56

Cl

4-Cl

N.T.

13.7

4.28

2.56

Br

4-Cl

N.T.

31.2

3.68

3.76

H

4-Cl

N.T.

162.5

15.00

13.66

Cl

4-Cl

N.T.

40.0

6.05

11.68

Br

4-Cl

N.T.

29.8

4.54

10.80

H

4-Cl

N.T.

187.9

12.62

8.51

Cl

4-Cl

N.T.

448.9

2.51

2.44

Cl

3-F

N.T.

101.6

8.99

9.43

D13

Cl

3,4-diF

N.T.

222.2

3.51

2.81

D14

Cl

4-Cl-3-CF3

N.T.

321.6

1.15

1.75

Cl

4-Cl

N.T.

211.9

6.20

3.04

Br

4-Cl

63.8%

255.3

3.03

8.70

N

Cl

4-Cl

N.T.

149.3

12.36

7.33

N

Br

4-Cl

N.T.

199.1

4.92

7.42

D19

H

3,4-diF

25.0%

21.6

5.33

3.72

D20

Cl

3,4-diF

N.T.

27.3

2.18

9.88

D21

Br

3,4-diF

N.T.

73.5

2.90

22.17

D22

Cl

3-F

10.7%

72.3

5.82

-

D23

H

3,4-diF

N.T.

57.7

2.11

24.16

D1 O

D2

D5

O

D6 D7

Br

D8 O

D9

S

D10 D11 D12

Cl S

D15

N

D16 Cl

D17 Br

D18

N


19

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D24

Cl

3,4-diF

N.T.

215.0

6.12

34.66

D25

Br

3,4-diF

N.T.

269.8

6.15

26.93

D26

H

3-F

N.T.

76.6

7.40

5.63

Cl

3-F

N.T.

104.1

3.46

4.72

Br

3-F

N.T.

181.8

1.92

6.25

-

-

50.2%

8.3

1.32

1.27

D27

N

D28 GSK795

-

a

The Inhibition% or IC50 values are an average of three independent determinations.

b

N.T. = Not Tested.

3,4,6-Trisubstituted piperidine derivatives. Following the previous SAR studies of 3,4disubstituted piperidine derivatives, we found that the Akt and hERG inhibitory activities cannot be well balanced. Since the 5- or 6-position of the piperidine may be oriented to the solvent area, this may offer an overall balance, particularly with respect to Akt inhibitory potency. As shown in Table 5, a range of introduced side chains could be well tolerated

in

both

synthesized

(3S,4S,6S)-trisubstituted

piperidine

derivatives.

Additionally, the length of the side chains showed minor influence on the Akt1 inhibitory activity of compounds, and the IC50 values of most compounds were below 10 nM. Compounds with hydrophilic side chains performed better in terms of Akt1 inhibitory


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potency compared with those with hydrophobic side chains (compounds E1 and E2) and the positive control compounds GSK-795 and AZD5363. As for cellular potency, most of the compounds showed good anti-proliferation activity against LNCaP, OVCAR-8 and HCT116 cells. Comparing these cell lines, LNCaP cells were more sensitive to the inhibitors than the OVCAR-8 or HCT116 cells. The IC50 values of several compounds against LNCaP cells were below 0.1 μM (compounds A12, B1, E5, E12, E15, E17, E18, E20, E22 and E23). The hERG inhibition of compounds with lipophilic side chains still exhibited high potency: for example, compound E1. Introduction of a hydroxyl group to increase the hydrophilicity of the side chain (compound E3) led to a decrease in hERG inhibition. Furthermore, the removal of one carbon to obtain a shortened side chain (compound E4) resulted in lower hERG activity. Introduction of two hydroxyls to continue increasing the hydrophilicity of the side chain (compound E5) resulted in significantly reduced hERG activity. Further exploration of the SAR around the basic group analogues, including N,Ndimethyl (E7), piperidine (E9) and morpholine (E10), led to compounds with increased


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hERG affinity. By contrast, introducing a hydroxyl group to the piperidine analogue side chain (compound E11) led to an obvious reduction in hERG inhibition. When the side chains were analogues with carboxyl (compound E13) and carbamoyl (compound E14) substitution, hERG inhibition was significantly lowered, exhibiting only 9.0% and 15.7% of the activity, respectively. When the phenyl group on piperidine was 3,4-difluoro substituted, the hERG affinity values of compounds were lower than those of 3,4-dichloro substituted compounds (E20 vs. E4; E21 vs. E5; and E22 vs. E15). The hERG inhibition rates of compounds E20-E24 were all below 10%, which was comparable to the value for AZD5363 but much lower than the values of GSK-795 and A12. Following the exploration of the compounds’ reduced hERG inhibition, other ion channels were also evaluated to exclude potential off-target effects. The inhibition rates of compound E22 against 16 different ion channels, including chloride (CFTR), sodium (Nav1.7) and potassium ion channels (Kir1.1, 2.1; Kv1.2~1.8; HCN 1~4), were quite low at a concentration of 3 μM (Table S2); these results suggest a safer ion channel profile and significant advancement over the disubstituted piperidine lead compound, A12.


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Table 5. Akt1 enzymatic and cancer cell line activities for compounds E1-E25

F

Cl

F

Cl Cl

Cl N

O

N

Cl

N

H N O

B1, E1~E19

N H

R1

O

N

Cl

O

N H A12, E20~E25

R1

Akt1 IC50 (nM) a

hERG inhibition at 3 μM a

A12

H

3.0

B1

H

Compd.

H N R1

IC50 (μM)a LNCaP

OVCAR8

HCT116

62%

0.06

0.04

0.21

2.9

87%

0.10

0.11

0.28

E1

10.44

85.3%

N.T. b

1.62

1.02

E2

12.41

N.T.

0.16

0.99

0.78

2.56

72.0%

0.21

0.60

0.51

2.74

51.8%

0.16

0.61

0.34

3.79

17.0%

0.07

1.61

1.11

7.01

N.T.

0.25

1.06

0.47

3.40

73.4%

1.93

1.51

1.71

E3

OH

E4 E5

OH

OH OH

E6

O

E7

N

E8

N

6.41

N.T.

1.92

1.84

1.94

E9

N

5.65

93.1%

1.78

1.66

1.02

E10

N

8.78

82.3%

0.41

1.76

1.56

5.99

12.0%

1.14

15.86

13.68

4.47

50.1%

0.06

1.56

0.56

E11 E12

O

N OH

N

N N


23

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O

E13

OH

O

E14

NH2 O

E15

N H O

E16

N H O

E17

N H O

E18

OH

N H

1.94

9.1%

0.64

5.12

4.02

3.24

15.7%

0.16

1.00

0.65

2.64

45.9%

0.04

1.13

0.45

3.95

70.4%

0.19

0.58

0.74

7.98

N.T.

0.08

0.61

0.43

1.41

6.7%

0.04

1.45

0.67

3.13

N.T.

0.66

5.90

7.09

0.98

9.0%

0.06

0.46

0.36

3.31

4.7%

0.10

0.69

0.80

1.37

4.0%

0.05

0.80

1.10

3.29

8.0%

0.05

1.53

0.92

2.49

7.4%

0.13

0.88

0.92

3.40

N.T.

N.T.

4.96

0.69

O

E19

N H

OH OH

E20 E21 E22

OH

OH OH

O N H O

E23

S N H O

O

E24

N H

O

E25

O

GSK-795

/

7.64

50.0%

0.07

1.32

1.27

AZD5363

/

25.43

7.1

0.178

7.27

5.20

a

The IC50 values or Inhibition% are an average of three independent determinations.

b

N.T. = Not Tested.

For preliminary assessment of the selectivity profiles of compounds, the IC50 values or inhibition rates of compounds A12, E20 and E22 were assayed against a panel of


24


Page 26 of 130

representative kinases (20), including AGC kinase family and tyrosine kinases (TK), among others (Table 6). Both compounds exhibited IC50 values of < 10 nM against Akt1-3. In addition, the tested compounds also exhibited good inhibitory activity against other AGC family kinases, such as PKA, PKC, ROCK1, RSK1, P70S6K and SGK, while no significant inhibition was observed against other non-AGC family kinases. Table 6. Results of preliminary assessment of compounds’ kinase selectivity profiles

IC50 (nM) / Inhibition (%, 0.5 μM) a

Kinases

AGC kinase family

TK

A12

E20

E22

GSK-795

AKT1

3.0

0.98

1.4

9.6

AKT2

< 0.64

0.54

1.2

1.0

AKT3

0.1

0.12

1.7

1.3

PKA

7

0.2

0.3

0.6

PKC

156

16

94

753

ROCK1

20

38

64

125

RSK1

48

32

131

339

P70S6K

7.0

7.1

8.9

60

SGK

59

102

145

211

MSK1

88.8%

4.2%

-1.0%

87.4%

PDK1

3.7%

4.0%

12.9%

-6.1%

TSSK1

-3.9%

-1.8%

-10.4%

6.2%

ABL

-3.0%

6.1%

-0.8%

2.4%


25

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TKL CMGC CAMK Atypical

JAK-2

N.I. b

N.I.b

N.I.b

N.I.b

ALK

20.8%

13.8%

8.5%

5.8%

JNK2

12.7%

6.3%

4.3%

8.4%

CDK2

N.I.b

N.I.b

N.I.b

N.I.b

CHK1

1.3%

-2.0%

-26.8%

-7.9%

PI3Ka

2.2%

6.3%

5.9%

0.8%

mTOR

-9.6%

-5.7%

-15.7%

NT b

a

The IC50 values or Inhibition% are an average of three independent determinations.

b

N.I. = No Inhibition.

To fully understand the selectivity profile of compound E22, inhibition was further assessed using the KINOMEscan methodology against a full panel of kinases (nonmutant, mutant, lipid, atypical and pathogen kinases; total number: 468) at a single dose concentration of 1 μM. The KINOMEscan selectivity score of S1 was 0.032 (13/468; S1 = (number of nonmutant kinases with %Ctrl 99, 89, 96, 84

>99, 69, 96, 18

Plasma stability (%, remaining percent @ 1 h) [mouse, human]

> 99, > 99

>99, >99

2.4 Compound E22 induces apoptosis and inhibits metastasis of cancer cells Compound E22 was very effective at inhibiting the phosphorylation of downstream Akt substrates in the LNCaP cell line, and it exhibited potent inhibition against phosphorylated PRAS40, which is a direct marker of Akt cell activity, with an observed IC50 value of 44 nM. To examine the influence of compound E22 on ovarian cancer cell survival prior to conducting an in vivo study, SKOV3, A2780 and OVCAR8 cells were incubated with different concentrations of E22 for 72 h. A concentration-dependent


28


Page 30 of 130

inhibitory effect of compound E22 was observed on the growth of ovarian cancer cells, which was similar to that of the positive control GSK-795 (Figure 5A). In addition, Western blot analysis was further performed to test the Akt inhibitory ability of compound E22 in tumor cells. The results demonstrated that upon treatment with the inhibitor E22 in vitro, a dose-dependent significant reduction in the phosphorylation of GSK3β, PRAS40 and FOX3a was observed in SKOV3 cells. Consistent with previously reported studies,20,

34, 35

E22 showed a concentration-dependent feedback increase in

Akt phosphorylation, similar to other ATP-competitive Akt kinase inhibitors (Figure 5B). To determine whether the antiproliferative activity of E22 against ovarian cancer cell proliferation was accompanied by enhanced cancer cell apoptosis, annexin V-PI staining plus fluorescence activated cell sorting (FACS) analysis was carried out, and the percentages of apoptotic cells were tested by conducting a flow cytometry assay. The results indicated that E22 had an apoptosis-inducing effect in a dose-dependent manner, with 79.9% of cells presenting as apoptotic at the concentration of 10 μM (Figure 5C and D).


29

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Figure 5. Compound E22 inhibits cell growth and induces apoptosis in vitro. (A) Cells (SKOV3, OVCAR8, and A2780) were exposed to E22 and GSK-795 for 72 h, and cell viability was then determined by SRB assay. (B) Western blot analysis of total AKT, pAKT, PRAS40, p-PRAS40 and p-FOX3a was conducted in the ovarian cancer cell line SKOV3. (C) Apoptosis was assessed upon treatment with the indicated concentrations (0.4-10 μM) by flow cytometry using the FL1-H channel (Annexin-V) and FL2-H channel (PI) of a Becton Dickinson FACSCalibur. Representative dot plots are shown, and the results are summarized in (D). The results are presented as the mean ± SD from three independent experiments. *p < 0.05, **p < 0.01 and ***p < 0.005 versus control.


30


Page 32 of 130

In addition, the effect of E22 on the metastasis of ovarian cancer cells was investigated in the present study through transwell and scratch assays. As shown in Figure 6A and B, the motility of the cells in the E22-treated group was significantly lower than that of the cells in the control group, indicating that compound E22 may inhibit the migratory capacity of ovarian cells. Similarly, the transwell migration assays showed that E22-exposed cells traversed less efficiently into the lower portion of the transwell chamber compared with the control cells (Figure 6C and D). As a consequence, it is evident that compound E22 can inhibit the metastasis of ovarian cells.


31

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Figure 6. Compound E22 suppresses the migratory capacity of ovarian cancer cells. (A) HUVECs were treated with E22 (0-2.5 μM) for 36 h. Afterwards, images were captured, and cell migration was determined using image analysis software; these results are summarized in (B). (C) HUVECs were placed in the upper Matrigel-coated invasion chamber, with E22 (0-2.5 μM) in the bottom chamber for 36 h. Invaded cells were stained with crystal violet and quantified by measuring the absorbance of dissolved crystals in 0.1% SDS at 540 nm; these results are summarized in (D). The results are presented as the mean ± SD from three independent experiments. *p < 0.05, **p < 0.01 and ***p < 0.005 versus control. Scale bars represent 200 μm.

2.5 Compound E22 exhibited improved safety and good in vivo efficacy The safety profile of E22 was evaluated before in vivo efficacy study. After 7 days of intragastric administration of E22 (80 mg/kg and 160 mg/kg), minimal body weight loss was observed (Figure S3 A). With lower hERG inhibition, we found that the MTD of E22 was increased to ≥80 mg/kg (two of five mice in the 160 mg/kg group died), and the serum hematological and biochemical parameters remained normal (Figure S3 B and


32


Page 34 of 130

C). Although the organ-to-body/brain weight ratio of thymus in E22-treated mice decreased (Table S1) and intestinal hemorrhage in mice could also be observed, the therapeutic safety window was obviously improved in comparison with compound A12. The in vivo effect of compound E22 was characterized by measuring its anti-tumor activity in the SKOV3 ovarian cancer xenograft model in nude mice. After 21 days of treatment with three doses of E22 (60 and 80 mg/kg, 40 mg/kg bid), remarkable tumor growth inhibition was observed. The E22 dose of 40 mg/kg twice per day appeared to be less effective than the dose of 80 mg/kg once per day (82.5% vs. 90% inhibition for 40 and 80 mg/kg, respectively), whereas it was comparable to the dose of 60 mg/kg once per day (82.5% vs. 79.2% for doses of 40 and 60 mg/kg, respectively); notably, the effect of all of these doses of E22 was better than that of GSK-795 at 60 mg/kg (63.3% inhibition; Figure 7A). In addition, the body weights of mice were not affected, and no mice died during treatment with the different doses (Figure 7B), demonstrating the improved safety profile of this compound in comparison with that of compound A12, which had an MTD of only approximately 10 mg/kg. After dosing at 50 mg/kg/d for 7 consecutive days, 560 ng/mL of E22 could be detected in the tumor, suggesting that


33

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E22 can effectively penetrate into the tumor tissue. After the completion of in vivo testing, Western blot analysis of the tumor tissues indicated that E22 showed a dosedependent inhibition of the levels of Akt substrate phosphorylation, including Fox3a and PRAS40 (Figure 7C and D).

Figure 7. Compound E22 develops an anti-tumor effect through inhibiting the kinase activity of Akt. (A, B and C) The mice transplanted with SKOV3 human xenografts were randomly divided into five groups (N = 5 mice/group) and were administered injections


34


Page 36 of 130

of E22 at different doses (60, 40+40 or 80 mg/kg, i.g.), GSK-795 (60 mg/kg, i.g.) or vehicle daily for a period of 21 days. The relative tumor volume, tumor weight, tumor growth inhibition and T/C values were measured after the mice were sacrificed. Data represent the mean ± SD. (D) Western blot analysis of total AKT, p-AKT, PRAS40, pPRAS40 and p-FOX3a in tumor tissues. The results are presented as the mean ± SD from three independent experiments. *p < 0.05, **p < 0.01, and ***p < 0.005 versus control.

2.6 Docking study and dynamic simulation To explore the possible binding mode of compound E22 with Akt protein, computational docking and molecular dynamics were performed. First, the complex of Akt1 protein (PDB code: 4GV1)18 with E22 was simulated for 4,000 ps. The RMSD value of the protein backbone was calculated in a trajectory from 0 to 4,000 ps. As shown in Figure 8A, after approximately 200 ps of simulation, the RMSD became stable, indicating that dynamic equilibrium was attained. The distances between E22 and Glu234 or Glu278 indicated that the system reached equilibrium conditions (Figure


35

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8B and 8C). Based on the relatively stable conformation, E22 was hydrogen bonded with the carboxyl group of Akt residue Glu 278. In addition, the 6-position side chain on the piperidine extended to the solvent area, while the 4-phenyl group was hydrophobic and interacted with the Gly159, Thr160, Phe161 and Val164 residues (Figure 8D).

Figure 8. MD simulations and analysis of the interaction of compound E22 with Akt1 protein (PDB ID: 4GV1). (A) RMSD of Akt backbone during the 4,000-ps simulation time; (B) Distance plot of Glu234 and Glu278 with E22; (C) Plot of the inhibitor-residue interaction spectrum; (D) 3D plot of the binding pattern.


36


Page 38 of 130

2.7 Chemistry Inhibitors of Akt described in this study were generally synthesized from readily available starting materials. Target compounds can be obtained by the condensation of the hinge-linkage acid fragments with the amine fragments. The synthetic route of acid fragments 1a-q is similar to the conditions in the literature (patent: WO2008098104), using coupling reactions to yield biaryl esters (compounds 4, 6, 9, 11, 14, 18, 20, 22 and 24) or aldehyde (16), and further introducing halogen substituents with NBS or NCS and subsequent hydrolysis or oxidation afforded the acid intermediates (see Supporting Information Figure S6). The synthetic routes of amines are outlined in Schemes 1 and 2. Knoevenagel condensation reaction was performed to convert the aryl aldehydes 25a-d to cinnamic acids 26a-d, with subsequent esterification providing the methyl cinnamates 27a-d. Further additive reaction with nitromethane yielded the compounds 28a-d. Next, cyclization (29a-d) and reduction afforded the benzyl-protected 3,4-disubstituted piperidine derivatives 30a-d, followed by Zn/AcOH-mediated reduction to obtain the corresponding amines 2a-d. Compounds 31a-b were prepared by condensation of


37

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substituted benzaldehyde with nitromethane, followed by cyclization to obtain 32a-b. Subsequent reduction with stannous chloride afforded amines 2e-f.

R1

a

H

R1

OH

25a~25d

O

e

f N

O 2N

29a~29d

g H

h

R1

28a~28d

R1 H 2N

31a, 31b

25a, 26a, 27a, 28a, 29a, 30a, 2a 25b, 26b, 27b, 28b, 29b, 30b, 2b 25c, 26c, 27c, 28c, 29c, 30c, 2c 25d, 26d, 27d, 28d, 29d, 30d, 2d

N

R1 =H R1 =3,4-diF R1 =4-Cl R1 =4-CF3

2a~2d

i

R1

NO2

d

O

O 2N

30a~30d

O

O

27a~27d

R1

N

O 2N

R1

c

O O

26a~26d

R1

25b, 25d

R1

O

O

R1

b

R1 N

N H 2N

O 2N 32a, 32b

2e, 2f

31a, 32a, 2e R1 =3,4-diF 31b, 32b, 2f R1 =4-CF3

Scheme 1. Synthetic route of amine fragments. a) malonic acid, piperidine, pyridine; b) dimethyl sulfate, K2CO3, acetone; c) nitromethane, DBU, MeCN; d) paraformaldehyde, benzylamine, EtOH; e) borane-methyl sulfide complex in THF; f) Zn, AcOH, EtOH; g) nitromethane, ammonium acetate, AcOH; h) CF3COOH, N-(Methoxymethyl)-N(trimethylsilylmethyl)benzylamine, DCM; i) SnCl2, EtOAc; According to a previously reported method,33 compounds 33a-f were subjected to R or S Jorgensen-Hayashi reagent-catalyzed cyclization with compound 34 to provide the (3S,4S) compounds 37a-f and (3R,4R) compounds 35a-b. Subsequent treatment of compounds 35a-b and 37a-f with Et3SiH, followed by reduction with Fe/NH4Cl, provided


38


Page 40 of 130

the amines 2g-o. As reported,36 BF3·OEt2 mediated allylation with Et3SiH can deliver 3,4,6trisubstituted piperidine derivatives 39a or 39b as a single product (2R,4S,5S). Further, the configuration of compound 39b was confirmed by NMR (NMR Spectra supporting information). The intermediate with the other configuration (2S,4S,5S) was impure and difficult to purify (Figure S7). Compound 39a was oxidized with borane-methyl sulfide complex/H2O2 to afford compound 40a. The desired amines 2p-r were finally acquired by reduction with the Fe/NH4Cl system.


39

Page 41 of 130 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


R1 NO2

a

Boc

a

CHO

Boc

33a~33f 33a 33b 33c 33d 33e 33f 33g

34

R1 =4-CF3 R1 =3,4-diF R1 =3,4-diCl R1 =3-CF3 R1 =4-Cl R1 =3-F R1 =4-Cl-3-CF3

Boc 2i, 2j

R1

R1 b

NO2

HN

35a, 36a, 2i R1 =4-CF3 35b, 36b, 2j R1 =3,4-diF

N

Boc 36a, 36b

R1

NO2

NH2

c

N

35a, 35b

+

NO2

b

N

R1

R1

R1

NO2

N

N

Boc

Boc

37a~37f

38a~38f

c

37a, 38a, 2g R1 =4-CF3 37b, 38b, 2h R1 =3,4-diF 37c, 38c, 2k R1 =3,4-diCl 37d, 38d, 2l R1 =3-CF3 37e, 38e, 2m R1 =4-Cl 37f, 38f, 2n R1 =3-F 37g, 38g, 2o R1 =4-Cl-3-CF3

NH2 N Boc 2g, 2h, 2k~2o

Cl

Cl

Cl

Cl

R1 d

NO2

e HO

N Boc 39a, 39b

NO2

c NH2 HO

N Boc

N Boc

40a

2r

R1 NH2 c

39a, 2p 39b, 2q

R1 =3,4-diCl R1 =3,4-diF

N Boc 2p, 2q

Scheme 2. Synthetic route of amine fragments. a) (S) or (R)-2-(diphenyl((trimethylsilyl) oxy)methyl)pyrrolidine, dichloromethane; ii. TFA, dichloromethane; b) i. Et3SiH, TFA; ii. Boc2O,

TEA,

dichloromethane;

c)

Fe,

NH4Cl,

EtOH/H2O;

d)

i.

(S)-2-

(diphenyl((trimethylsilyl)oxy) methyl)pyrrolidine, DCM; ii. allyltrimethylsilane, boron trifluoride etherate, DCM; e) borane-methyl sulfide complex, NaOH, H2O2.


40


Page 42 of 130

The synthesis of 3,4-disubstituted piperidine Akt inhibitors (A1-A13, B1-B3, C1-C5, D1-D28) described in this study is outlined in Schemes 3 and 4. Different acid fragments 1a-q and amines 2a-o were condensed to obtain the Boc- or Bn-protected intermediates, which were subsequently deprotected to yield the target compounds. In addition, some oily liquid target compounds were treated with L-tartaric acid to generate solid compounds.


41

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R1 R3

a, b, c N

H 2N

N

R3 O

N

Bn

O

R2

2a~2d

HOOC N H

HO A1 A2 A3 A4 A5 A6

A1~A6

2a R3 =H 2b R3 =3,4-diF 2c R3 =4-Cl 2d R1 =4-CF3

R3 H 2N

H N

R1 a, b

O

N

N

N Bn

R3

H N O

R2

OH COOH R1 =H, R2 =H, R3 =H R1 =H, R2 =H, R3 =3,4-diF R1 =Cl, R2 =Cl, R3 =H R1 =Cl, R2 =Cl, R3 =4-Cl R1 =Cl, R2 =Cl, R3 =4-CF3 R1 =Cl, R2 =Cl, R3 =3,4-diF

A7 R1 =H, R2 =Cl, R3 =3,4-diF A8 R1 =H, R2 =Br, R3 =3,4-diF A9 R1 =Cl, R2 =Cl, R3 =4-CF3

NH

A7~A9 2e~2f O

R2

2e R3 =3,4-diF 2f R3 =4-CF3 OH

N N

O R1

+

R

1a~1d 1a 1b 1c 1d

R1 =H, R2 =H R1 =Cl, R2 =Cl R1 =H, R2 =Cl R1 =H, R2 =Br

NH2 N Boc

Cl a, d, c

N

R N

OH

2g, A10 R =4-CF3 2h, A12 R=3,4-diF

COOH

R O

N

H N O

Cl

(3R, 4R)

HOOC N H

HO

OH COOH

2i, A11 R =4-CF3 2j, A13 R=3,4-diF

A11, A13

R

N Boc 2k~2m

HO

N H

Cl a, d, c

N (3R, 4R) Boc 2i~2j

NH2

HOOC

A10, A12

2g~2h

NH2

H N

O (3S, 4S)

Cl

(3S, 4S)

R O

N

Cl a, d

N

R O

N

Cl

H N O

B1~B3

N H

2k, B1 R =3,4-diCl 2l, B2 R =3-CF3 2m,B3 R =4-Cl

Scheme 3. Synthetic route of target compounds. a) EDCI, HOBT, DIPEA, DCM; b) chloroethyl chloroformate, 1,2-dichloroethane; c) L-tartrate; d) HCl/EA.


42


Cl

Cl

N N

1eg, C1 R1 =

R1

COOH

+

a, c, b

H 2N

1b, 1e~1i

N

H N

R1 O

N Boc 2m

Linkage R1

OH

1f, C2 R1 =

N HN

1g, C3 R1 =

N N

hydrochloride or L-tartrate

N H C1~C5

H 2N

a, c

O

N Boc

N

1i, C5 R1 =

N

S

N

Linkage R1

O D1~D28

N H HCl

Linkage Br

Linkage

N

1n, 2m, D18 R1 =Br, R2 =4-Cl

O 1k, 2m, D11 1k, 2n, D12 1k, 2h, D13 1k, 2o, D14

1ea, 2m, D4 R1 =H, R2 =4-Cl 1eb, 2m, D5 R1 =Cl, R2 =4-Cl 1ec, 2m, D6 R1 =Br, R2 =4-Cl

1j, 2m, D10 R1 =H, R2 =4-Cl

HO O

2h, 2m~2o

1a, 2m, D1 R1 =H, R2 =4-Cl 1c, 2h, D2 R1 =Cl, R2 =3,4-diF 1d, 2m, D3 R1 =Br, R2 =4-Cl

O

N

H N

Linkage

1ed, 2m, D7 R1 =H, R2 =4-Cl 1ee, 2m, D8 R1 =Cl, R2 =4-Cl 1ef, 2m, D9 R1 =Br, R2 =4-Cl

O

N

R2 N

+

1a, 1c, 1d, 1ea~1ef, 1j, 1ka~1kd, 1la~1lb, 1m, 1n, 1oa~1ob,1pa~1pc, 1qa~1qc

HN

O 1h, C4 R1 =

R2 N

Page 44 of 130

R1 =Cl, R2 =4-Cl R1 =Cl, R2 =3-F R1 =Cl, R2 =3,4-diF R1 =Cl, R2 =4-Cl-3-CF3

Cl S

O N

1la, 2m, D15 R1 =Cl, R2 =4-Cl 1lb, 2m, D16 R1 =Br, R2 =4-Cl

Br

O

Cl S

1m, 2m, D17 R1 =Cl, R2 =4-Cl

N

1oa, 2h, D19 1ob, 2h, D20 1oc, 2h, D21 1ob, 2n, D22

R1 =H, R2 =3,4-diF R1 =Cl, R2 =3,4-diF R1 =Br, R2 =3,4-diF R1 =Cl, R2 =3-F

1pa, 2h, D23 R1 =H, R2 =3,4-diF 1pb, 2h, D24 R1 =Cl, R2 =3,4-diF 1pc, 2h, D25 R1 =Br, R2 =3,4-diF 1qa, 2n, D26 R1 =H, R2 =3-F 1qb, 2n, D27 R1 =Cl, R2 =3-F 1qc, 2n, D28 R1 =Br, R2 =3-F

N

N

Scheme 4. Synthetic route of target compounds. a) EDCI, HOBT, DIPEA, DCM; b) Ltartrate; c) HCl/EA.

The synthesis of 3,4,6-trisubstituted piperidine Akt inhibitors described in this study is outlined in Scheme 5. Pyrazole-furan acid 1b was condensed with the amines 2p-q to yield the key allyl building blocks 41a-b, which were subsequently oxidized to afford compounds 42a-b and 43a-b. The aldehydes 43a-b were further reduced to alcohols


43

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44a-b, which then served as useful precursors to the amine 46a or were oxidized to acids 44c-d. Reduction of the allyl 41a and deprotection produced inhibitor E1. Similarly, removing the protecting groups of compounds 41b, 42a-b and 44a-c yielded the inhibitors E2, E4, E5, E13, E20 and E21. Reduction of compound 2r produced an amine, which was then condensed with acid 1b and deprotected to afford inhibitor E3. Nucleophilic reaction of compound 45a with different bases provided inhibitors E6-E12 after removal of the protecting groups. Furthermore, condensation of compounds 44c-d with various substituted amines afforded the inhibitors E14-E19 and E22 after deprotection. As before, capping 46a or 44b with mesyl or acetyl groups, followed by deprotection, yielded the inhibitors E23-E25.


44


Cl 1b + 2p or 1b + 2q

a

R1 O

N

N

O

Cl

Cl

H N

N

b

H N O

Cl

N Boc 41a~b

Cl

R1 O

N

Page 46 of 130

R1 O

N

N

c

H N

OH

O

O

Cl

OH

N Boc 42a~b

N Boc

H

43a~b

d

e Cl

Cl

R1 O

N

N

H N O

Cl

N

g, h N Boc

46a

H N

N

f

O

Cl

NH2

Cl

R1 O

N

45a~b

N Boc

H N O

Cl

OMs

Cl

R1 O

N

N

N Boc 44a~b

R1 O

N

H N O

Cl

OH

O N Boc 44c~d

OH

2p, 41a, 42a, 43a, 44a, 44c, 45a R1 = 3,4-diCl 2q, 41b, 42b, 43b, 44b, 44d, 45b, 46a R1 = 3,4-diF

R1 41a 41b, 42a~b, 44a~d

side chain

Cl

h

N

i

E1

O

N

H N

E2

O

Cl

N H

R1=3,4-diCl

E13 R1=3,4-diCl

E4 R1=3,4-diCl E20 R1=3,4-diF

Side Chain

side chain

OH

COOH OH

E5 R1=3,4-diCl E21 R1=3,4-diF

OH

E1, E2, E4, E5, E13, E20, E21

Cl

Cl

N

O

j, i

H N O

Cl

45a

E6

Cl

Cl N

side chain

Cl

Cl

O

N Boc

O

N

N

O S O

H N N H

E6~E12

Side Chain

E8

O

Cl

H N O 44c~d

N

j, a, i

O

Cl

OH

O

E14~E19, E22

Side Chain

+ H 2N N Boc 2r

E19 R1=3,4-diCl

N H

OH

N

F Cl

O

N

Cl

H N O

46a, 44b N H

N H

F Cl

a, i

k, i

N

OH

E3

E23 O

N

Cl

H N O

OH

N H O

O

Cl 1b

E18 R1=3,4-diCl

N H

E22 R1=3,4-diF

Cl

Cl

N H O

O

E16 R1=3,4-diCl

Cl

E17 R1=3,4-diCl

NH2

E15 R1=3,4-diCl N H

side chain O

side chain

H N

O N Boc

N N

E14 R1=3,4-diCl

O

N

OH N

N

R1 Cl

Cl

N

N E12

R1 N

O

N

E7

E9

N

N

E11

O

Cl

side chain E10

O

N H E23~E25

OH OH

side chain O S N H O O

E24 Side Chain

N H O

E25

O

Scheme 5. Synthetic route of target compounds. a) EDCI, HOBT, DIPEA, DCM; b) OsO4, NMO, THF/H2O; c) NaIO4, THF/H2O; d) NaClO2, KH2PO4, 2-methylbut-2-ene; e)


45

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NaBH4, THF/MeOH; f) MsCl, DIPEA, DCM; g) NaN3, DCM; h) Pd/C, H2, EtOAc; i) HCl/EA; j) amines or CH3ONa; k) MsCl or AcCl, DIPEA.

3. Conclusion Compound A12, designed based on the conformational restriction strategy, served as a promising lead Akt inhibitor with an acceptable pharmacokinetic profile and in vivo antitumor efficacy. Nevertheless, it exhibited unfavorable safety profile characteristics such as low MTD, high hERG inhibition and gastrointestinal hemorrhage. Further optimization of this compound was ultimately explored with a range of diverse hinge or linkage groups, as well as by introducing side chains suggested by the docking results of this compound bound to Akt1, leading to compound E22. This compound inhibited all Akt isoforms with a potency of < 2 nM, with good selectivity against other kinases, especially non-AGC family kinases. It also exhibited low hERG ion channel inhibition, outstanding pharmacokinetic properties and effective xenograft tumor inhibitory activity. The 3,4,6-trisubstituted piperidine derivative E22 is better tolerated than A12, with


46


Page 48 of 130

improved safety properties, demonstrating that E22 is a promising drug candidate for further preclinical study.

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-d6 solution, with tetramethylsilane (TMS) serving as 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) were obtained from Agilent Technologies 6224 TOF 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 a 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 oC, eluting with water (0.1% trifluoroacetic acid) as A phase and


47

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methanol as B phase (0 min, A phase: 90%, B phase: 10%; 8 min, A phase: 10%, B phase: 90%; 13 min, A phase: 10%, B phase: 90%; 15 min, A phase: 90%, B phase: 10%; 20 min, A phase: 90%, B phase: 10%). Unless otherwise noted, reagents and solvents were obtained from commercial suppliers and without further purification. General procedure I: To a suspension of the acid (0.2 mmol), EDCI (69 mg, 0.36 mmol), HOBT (49 mg, 0.36 mmol) and DIPEA (87 μL, 0.5 mmol) in dichloromethane was added the amine (0.2 mmol), then the mixture was reacted for 5 h at room temperature with mechanical stirring. After it was fully reacted, the mixture was concentrated under vacuum. The residue was dissolved in ethyl acetate (50 mL), washed by 1N HCl (20 mL × 2) and saturated brine (20 mL × 2) and dried over anhydrous sodium sulphate. After solvent removal, the residue was purified by column chromatography to afford the product. General procedure II: To the solution of disubstituted piperidine compound in 1,2dichloroethane was added 1-chloroethyl chloroformate (0.3mmol) dropwise. The resulting mixture was heated to reflux for 4h. After it was fully reacted, the mixture was concentrated under vacuum. The residue dissolved in MeOH (5mL) was heated to


48


Page 50 of 130

reflux for another 2h. After solvent removal, the residue was added saturated sodium bicarbonate and extracted with ethyl acetate. The combined organic extracts were washed with brine, dried over Na2SO4, and concentrated. The residue was purified by silica gel chromatography to afford the product. To a solution of above resulted product in MeOH was added L-tartaric acid, stirred in room temperature for 2 h. After the solvent removal, the residue was washed with diethyl ether, dried in vacuum to afford L-tartrate. General procedure III: To a solution of the Boc-protected target compound in ethyl acetate was added HCl saturated ethyl acetate, stirred overnight in room temperature. Then, the reaction was added saturated sodium bicarbonate, extracted with ethyl acetate. The combined organic extracts were washed with brine, dried over Na2SO4, concentrated, and dried in vacuum to afford white solid. General procedure IV: To a solution of the Boc-protected target compound in ethyl acetate was added HCl saturated ethyl acetate, stirred at room temperature for 6 h. White solid was precipitated, filtered, washed with diethyl ether and dried in vacuum to afford target product as white powder


49

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General procedure V: To a solution of substituted piperidine compound in ethyl acetate (0.5mL) was added HCl saturated ethyl acetate (2mL), stirred overnight in room temperature. Then, the reaction was added saturated sodium bicarbonate (5mL), extracted with ethyl acetate. The combined organic extracts were washed with brine, dried over Na2SO4, concentrated, and dried in vacuum to afford the product. Then a solution of above resulted product in MeOH (2mL) was added L-tartaric acid, stirred in room temperature for 2 h. After the solvent removal, the residue was washed with diethyl ether, dried in vacuum to afford the L-tartrate. General procedure VI: To the solution of compound 45a (71 mg, 0.1 mmol) in anhydrous DMF(5mL) was added amines (1.0 mmol) dropwise under N2 protection. The reaction was heated to 80 oC and stirred for 6h. After fully reaction, the mixture was poured into H2O(10 mL), extracted with DCM(5 mL × 3). The combined organic extracts were washed with brine, dried over Na2SO4, and concentrated. The residue was purified by silica gel chromatography to afford white solid. Then, a solution of above white solid in ethyl acetate (0.3 mL) was added HCl saturated ethyl acetate(4 mL) at ice-bath,


50


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stirred overnight in room temperature. White solid was precipitated, filtered, washed with diethyl ether and dried in vacuum to afford target product as white powder. General procedure VII: To the solution of trisubstituted piperidine (44c or 44d, 0.1mmol) and DIPEA (37 mg，0.3 mmol) in anhydrous DMF (5mL) was added HBTU (76 mg，0.2 mmol). After stirred at RT for 30min, the mixture was added amines and continue to stir overnight. Then, the mixture was poured into H2O(10 mL), extracted with ethyl acetate (5 mL × 3). The combined organic extracts were washed with brine (5 mL × 2), dried over Na2SO4, and concentrated. The residue was purified by silica gel chromatography to afford white solid. Then, a solution of above white solid in ethyl acetate (0.3mL) was added HCl saturated ethyl acetate(4mL) at ice-bath, stirred overnight in room temperature. White solid was precipitated, filtered, washed with diethyl ether and dried in vacuum to afford target product as white powder. General procedure VIII: To the solution of compound 46a (0.05 mmol) and DIPEA (26 μL, 0.15 mmol) in THF (5 mL) was added MsCl (10 μL, 0.10 mmol) in ice bath. The resulting mixture was stirred at RT for 4h. After fully reacted, the reaction was poured into saturated NaHCO3 (5 mL) extracted with ethyl acetate (5 mL × 3). The combined


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organic extracts were washed with brine (5 mL × 2), dried over Na2SO4, and concentrated. The residue was purified by silica gel chromatography to afford white solid. Then, a solution of above white solid in ethyl acetate (0.1mL) was added HCl saturated ethyl acetate(1 mL) at ice-bath, stirred overnight in room temperature. White solid was precipitated, filtered, washed with diethyl ether and dried in vacuum to afford target product as white powder. After the synthesize, 74 compounds were obtained in total, the compound information of the target compounds and the intermediates can be found in the following part and Supporting Information respectively. 4.1.1

4-(1-Methyl-1H-pyrazol-5-yl)-N-(4-phenylpiperidin-3-yl)furan-2-carboxamide

(2R,3R)-2,3-dihydroxysuccinate(A1) General procedure I and General procedure II, yield: 21.5%;1H NMR(500MHz, DMSO) δ 8.51-8.54 (d, J = 11.4 Hz, 1H), 8.17 (s, 1H), 7.40 (s, 1H), 7.17-7.33 (m, 6H), 6.45 (s, 1H), 4.55 (m, 1H), 4.21 (s,2H), 3.87 (s, 3H), 3.38 (m, 2H), 2.93-3.07 (m, 3H), 1.96 (m, 2H).

13C

NMR (500 MHz, DMSO) δ 173.46(×2), 156.66, 147.67, 141.89,

141.67, 137.86, 133.44, 128.34(×2), 127.29(×2), 126.71, 117.06, 113.58, 105.64,


52


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71.94(×2), 41.64, 46.60, 44.59, 43.36, 37.83, 30.22. HRMS: calculated (M+H): 351.1552; Found: 351.1808. 4.1.2

N-(4-(3,4-Difluorophenyl)piperidin-3-yl)-4-(1-methyl-1H-pyrazol-5-yl)furan-2-

carboxamide (2R,3R)-2,3-dihydroxysuccinate(A2) General procedure I and General procedure II, yield:22.5%; 1H NMR (500 MHz, DMSO) δ 8.61 (d, J =10.75 Hz, 1H), 8.20 (s, 1H), 7.92 (d, J=15.75Hz, 1H), 7.21-7.41 (m, 3H), 7.11 (m, 1H), 6.48 (s, 1H), 4.49 (m, 1H), 4.18 (s, 2H), 3.88 (s, 3H), 3.35 (m, 2H), 3.08 (m, 1H), 2.94 (m, 1H), 2.85 (m, 1H), 1.97 (m, 2H).

13C

NMR (500 MHz,

DMSO) δ 173.61(×2), 168.81, 156.72, 147.57, 141.98, 137.86, 133.44, 124.13, 117.36, 117.19, 117.08, 116.39, 116.23, 113.67, 105.65, 71.93(×2), 46.73, 46.53, 44.09, 43.18, 37.84, 29.69. HRMS: calculated (M+H): 387.1633; Found: 387.1642. 4.1.3

5-Chloro-4-(4-chloro-1-methyl-1H-pyrazol-5-yl)-N-(4-phenylpiperidin-3-yl)furan-

2-carboxamide (2R,3R)-2,3-dihydroxysuccinate(A3) General procedure I and General procedure II, yield: 21.2%;1H NMR (500 MHz, dDMSO) δ 8.77-8.79 (d, J = 11.15 Hz, 1H), 7.71 (s, 1H), 7.46 (s, 1H), 7.24-7.33 (m, 5H), 4.59 (m, 1H), 4.26 (s, 2H), 3.77 (s, 3H), 3.42 (m, 2H), 3.12 (m, 1H), 3.00 (m, 2H), 2.01


53

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(m, 2H).

13C

NMR (500 MHz, d-DMSO) δ 173.80(×2), 155.63, 146.75, 141.62, 137.22,

136.58, 128.87, 128.37(×2), 127.31(×2), 126.78, 116.83, 110.06, 109.26, 72.04(×2), 46.72, 46.32, 44.50, 43.24, 38.13, 30.02. HRMS: calculated (M+H): 419.1042; Found: 419.0971. 4.1.4 5-Chloro-4-(4-chloro-1-methyl-1H-pyrazol-5-yl)-N-(4-(4-chlorophenyl)piperidin-3yl)furan-2-carboxamide (2R,3R)-2,3-dihydroxysuccinate(A4) General procedure I and General procedure II, yield: 21.6%;1H NMR(500MHz, dDMSO) δ 8.75-8.77 (d, J=10.9 Hz, 1H), 7.68 (s, 1H), 7.46 (s, 1H), 7.35-7.37 (d, J =10.3 Hz, 2H), 7.28-7.30 (d, J=10.25Hz, 2H), 4.54 (m, 1H), 4.22 (s, 2H), 3.74 (s, 3H), 3.39 (m, 2H), 3.08 (m, 2H), 2.96 (m, 1H), 1.96 (m, 2H).

13C

NMR (500 MHz, DMSO) δ

173.69(×2), 155.62, 146.74, 140.69, 137.20, 136.58, 131.27, 129.24(×2), 128.87, 128.35(×2), 116.96, 110.12, 109.24, 72.04(×2), 46.73, 46.31, 43.98, 43.16, 38.17, 29.93. HRMS: calculated (M+H): 453.0651; Found: 453.0479. 4.1.5

5-Chloro-4-(4-chloro-1-methyl-1H-pyrazol-5-yl)-N-(4-(4-(trifluoromethyl)phenyl)

piperidin-3-yl)furan-2-carboxamide (2R,3R)-2,3-dihydroxysuccinate (A5)


54


Page 56 of 130

General procedure I and General procedure II, yield: 20.1%;1H NMR(500MHz, DMSO) δ 8.79-8.81 (d, J = 10.9 Hz, 1H), 7.68 (s, 1H), 7.49 (s, 1H), 7.13-7.38 (m, 4H), 4.49 (m, 1H), 4.21 (s, 4H), 3.74 (s, 3H), 3.36 (m, 2H), 3.10 (m, 1H), 2.95 (m, 2H), 1.98 (m, 2H).

13C

NMR (500 MHz, d-DMSO) δ 173.79(×2), 155.67, 146.72, 139.48, 137.23,

136.58, 128.87, 124.17, 117.41, 117.14, 116.99, 116.41, 116.24, 110.12, 109.24, 72.04(×2), 46.81, 46.29, 43.89, 43.11, 38.15, 29.76. MS: calculated (M+H): 487.09; Found: 487.09. 4.1.6 5-Chloro-4-(4-chloro-1-methyl-1H-pyrazol-5-yl)-N-(4-(3,4-difluorophenyl)piperidin -3-yl)furan-2-carboxamide (2R,3R)-2,3-dihydroxysuccinate (A6) General procedure I and General procedure II, yield: 25.9%;1H NMR (500 MHz, DMSO) δ 8.63-8.65 (d, J= 9.05 Hz, 1H), 8.23 (s, 1H), 7.62 (s, 1H), 7.36 (s, 1H), 7.107.34 (m, 3H), 4.46 (m, 1H), 4.21 (s, 2H), 3.82 (s, 3H), 3.36 (m, 3H), 2.90 (m, 2H), 2.85 (m, 1H), 1.98 (m, 1H) , 1.91 (m, 1H). HRMS: calculated (M+H): 455.0854; Found: 455.0840 4.1.7

5-Chloro-N-(4-(3,4-difluorophenyl)pyrrolidin-3-yl)-4-(1-methyl-1H-pyrazol-5-

yl)furan-2-carboxamide(A7)


55

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General procedure I and General procedure II, yield: 42.5%; 1H NMR (500 MHz, CDCl3 ) δ 7.75 (s, 1H), 7.57-7.59 (d, J=7.65 Hz, 1H), 7.43 (s, 1H), 7.39 (s, 1H), 7.077.20 (m, 3H), 4.69 (m, 1H), 3.86 (s, 3H), 3.68 (m, 1H), 3.59 (m, 1H),3.50 (m, 1H), 3.26 (s, 1H) 3.20 (m, 1H). ESI-MS: m/z = 407.68 [M+H]+. 4.1.8

5-Bromo-N-(4-(3,4-difluorophenyl)pyrrolidin-3-yl)-4-(1-methyl-1H-pyrazol-5-yl)

furan-2-carboxamide(A8) General procedure I and General procedure II, yield: 40.0%;1H NMR (500 MHz, CDCl3 ) δ 7.78 (s, 1H), 7.73-7.74 (d, J=6.75 Hz, 1H), 7.48 (s, 1H), 7.41 (s, 1H), 7.10-7.24 (m, 3H), 4.72 (m, 1H), 3.88 (s, 3H), 3.72 (m, 1H), 3.58 (m, 2H), 3.26-3.30 (m, 2H). ESI-MS: m/z = 453 [M+H]+. 4.1.9

5-Chloro-4-(4-chloro-1-methyl-1H-pyrazol-5-yl)-N-(4-(4-(trifluoromethyl)phenyl)

pyrrolidin-3-yl)furan-2-carboxamide(A9) General procedure I and General procedure II, yield: 40.5%;1H NMR (500 MHz, DMSO) δ 9.03 (d, J=8.1 Hz, 1H), 7.71-7.72 (d, J=8.1 Hz, 2H), 7.69 (s, 1H), 7.61-7.63 (d,

J=8.1 Hz, 2H), 7.53 (s, 1H),

4.71 (m, 1H), 3.74 (s, 3H), 3.65 (m, 2H), 3.52 (m, 1H),

3.19 (m, 1H), 3.09 (m, 1H). ESI-MS: m/z= 473 [M+H]+.


56


4.1.10 (trifluoromethyl)

Page 58 of 130

5-Chloro-4-(4-chloro-1-methyl-1H-pyrazol-5-yl)-N-((3S,4S)-4-(4phenyl)

piperidin-3-yl)

furan-2-carboxamide

(2R,3R)-2,3-

dihydroxysuccinate (A10) General procedure I and General procedure V, yield: 49%; 1H NMR (500 MHz, DMSO) δ 8.81 (d, J = 8.9 Hz, 1H), 7.70 – 7.60 (m, 3H), 7.49 (d, J = 7.9 Hz, 2H), 7.43 (s, 1H), 4.66 – 4.53 (m, 1H), 4.20 (s, 2H), 3.71 (s, 3H), 3.45 – 3.34 (m, 2H), 3.25 – 3.17 (m, 1H), 3.05 – 2.92 (m, 2H), 2.05 – 1.93 (m, 2H).

13C

NMR (125 MHz, DMSO) δ 173.96,

155.78, 146.76, 146.65, 137.30, 136.65, 128.94, 128.36, 127.50 (q, J = 32.5 Hz), 125.35, 124.30 (q, J = 270.0 Hz), 117.10, 110.21, 109.31, 72.15, 46.68, 46.36, 44.53, 43.17, 38.23, 30.02. HRMS (ESI) (m/z): calcd for C21H20Cl2F3N4O2 [M + H]+ 487.0910, found 487.0923. 4.1.11 (trifluoromethyl)

5-Chloro-4-(4-chloro-1-methyl-1H-pyrazol-5-yl)-N-((3R,4R)-4-(4phenyl)

piperidin-3-yl)

furan-2-carboxamide(2R,3R)-2,3-

dihydroxysuccinate (A11) General procedure I and General procedure V, yield: 71%; Retention time: 10.014 min, purity: 98.57 %; ESI-MS: m/z = 487 [M+H]+.


57

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4.1.12

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

difluorophenyl) piperidin-3-yl)furan-2-carboxamide (2R,3R)-2,3-dihydroxysuccinate(A12) General procedure I and General procedure V, yield: 56%; Retention time: 10.214 min, purity: 98.49 %; 1H NMR (500 MHz, DMSO) δ 8.91 – 8.79 (m, 1H), 7.65 (s, 1H), 7.46 (d, J = 17.4 Hz, 1H), 7.36 – 7.27 (m, 2H), 7.12 (s, 1H), 4.50 (d, J = 6.8 Hz, 1H), 4.15 (s, 2H), 3.72 (s, 3H), 3.35 (d, J = 7.1 Hz, 2H), 3.07 (d, J = 24.7 Hz, 1H), 3.16 – 2.88 (m, 3H), 2.96 (d, J = 9.1 Hz, 2H), 1.97 (s, 3H).

13C

NMR (126 MHz, DMSO) δ 174.70,

155.76, 150.17, 150.08, 149.26, 149.16, 148.22, 148.11, 147.31, 147.22, 146.86, 139.68, 137.25, 136.62, 128.95, 124.27, 117.32, 117.19, 116.90, 116.48, 116.35, 110.14, 109.31, 72.26, 47.17, 46.45, 44.10, 43.15, 38.17, 29.95. ESI-MS: m/z = 455 [M+H]+. 4.1.13

5-Chloro-4-(4-chloro-1-methyl-1H-pyrazol-5-yl)-N-((3R,4R)-4-(3,4-

difluorophenyl) piperidin-3-yl)furan-2-carboxamide (2R,3R)-2,3-dihydroxysuccinate(A13) General procedure I and General procedure V, yield: 62%; Retention time: 10.014 min, purity: 98.57 %; ESI-MS: m/z = 455[M+H]+.


58


4.1.14

Page 60 of 130


dichlorophenyl) piperidin-3-yl)furan-2-carboxamide (2R,3R)-2,3-dihydroxysuccinate (B1) General procedure I and General procedure V, yield: 63%; Retention time: 10.746 min, purity: 97.38 %; HRMS (ESI) (m/z): calcd for C20H19Cl4N4O2 [M + H]+ 489.0227, found 489.0235. 4.1.15 (trifluoromethyl)

5-Chloro-4-(4-chloro-1-methyl-1H-pyrazol-5-yl)-N-((3S,4S)-4-(3phenyl)piperidin-3-yl)furan-2-carboxamide

(2R,3R)-2,3-

dihydroxysuccinate (B2) General procedure I and General procedure V, yield: 53%; 1H NMR (500 MHz, DMSO) δ 8.81 (d, J = 9.0 Hz, 1H), 7.66 (s, 1H), 7.64 – 7.51 (m, 4H), 7.43 (s, 1H), 4.59 – 4.49 (m, 1H), 4.16 (s, 2H), 3.71 (s, 3H), 3.44 – 3.34 (m, 2H), 3.25 – 3.16 (m, 1H), 2.96 (t, J = 11.4 Hz, 2H), 2.11 – 1.96 (m, 2H). 13C NMR (125 MHz, DMSO) δ 174.20, 155.72, 146.79, 143.05, 137.29, 136.66, 131.25, 129.55, 128.95, 128.92 (q, J = 30.0 Hz), 124.60 (q, J = 3.8 Hz), 124.19 (q, J = 271.3 Hz), 123.63 (q, J = 3.8 Hz), 116.97, 110.14, 109.32, 72.14, 47.05, 46.47, 44.51, 43.21, 38.15, 29.47. HRMS (ESI) (m/z): calcd for C21H20Cl2F3N4O2 [M + H]+ 487.0910, found 487.0933.


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4.1.16

5-Chloro-4-(4-chloro-1-methyl-1H-pyrazol-5-yl)-N-((3S,4S)-4-(4-chlorophenyl)

piperidin-3-yl)furan-2-carboxamide (2R,3R)-2,3-dihydroxysuccinate (B3) General procedure I and General procedure V, yield: 43%; 1H NMR (400 MHz, DMSO) δ 8.75 (d, J = 8.7 Hz, 1H), 7.67 (s, 1H), 7.44 (s, 1H), 7.35 (d, J = 8.2 Hz, 2H), 7.28 (d, J = 8.1 Hz, 2H), 4.59 – 4.44 (m, 1H), 4.21 (s, 2H), 3.72 (s, 3H), 3.40 – 3.33 (m, 2H), 3.15 – 2.89 (m, 3H), 2.01 – 1.88 (m, 2H).

13C

NMR (100 MHz, DMSO) δ 173.76,

155.69, 146.81, 140.76, 137.27, 136.65, 131.34, 129.31, 128.94, 128.42, 117.03, 110.19, 109.31, 72.11, 46.80, 46.38, 44.05, 43.23, 38.24, 30.00. ESI-MS: m/z = 453 [M + H]+. HRMS (ESI) (m/z): calcd for C20H20Cl3N4O2 [M+H]+ 453.0646, found 453.0679. 4.1.17 N-((3S,4S)-4-(4-Chlorophenyl)piperidin-3-yl)-5-(1-methyl-1H-pyrazol-4-yl)furan2-carboxamide hydrochloride (C1) General procedure I and General procedure IV, yield: 61%; Retention time: 8.803 min, purity: 95.23 %; 1H NMR (500 MHz, DMSO) δ 9.62 (s, 1H), 9.53 (s, 1H), 8.60 (d, J = 6.1 Hz, 1H), 8.16 (s, 1H), 7.84 (s, 1H), 7.35 – 7.26 (m, 4H), 7.12 – 7.08 (m, 1H), 6.55 (d, J = 3.3 Hz, 1H), 4.58 – 4.47 (m, 1H), 3.85 (s, 3H), 3.32 (t, J = 12.7 Hz, 2H), 3.22 (s, 1H), 2.99 (t, J = 15.7 Hz, 1H), 2.94 – 2.89 (m, 1H), 2.10 – 1.90 (m, 2H).

13C

NMR (125 MHz,


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DMSO) δ 157.08, 149.91, 144.74, 140.99, 136.09, 131.29, 129.46, 128.41, 128.34, 115.95, 112.70, 105.35, 46.55, 46.43, 44.03, 43.29, 38.81, 29.50. ESI-MS: m/z = 385 [M + H]+. HRMS (ESI) (m/z): calcd for C20H22ClN4O2 [M + H]+ 385.1426, found 385.1425. 4.1.18

N-((3S,4S)-4-(4-Chlorophenyl)piperidin-3-yl)-5-(1H-pyrazol-4-yl)furan-2-

carboxamide(C2) General procedure I and General procedure III, yield: 53%; Retention time: 8.771 min, purity: 95.54 %; 1H NMR (500 MHz, CDCl3) δ 7.79 (s, 2H), 7.27 – 7.18 (m, 4H), 7.01 (d,

J = 3.5 Hz, 1H), 6.36 (d, J = 3.5 Hz, 1H), 6.07 (d, J = 8.3 Hz, 1H), 4.29 – 4.22 (m, 1H), 3.57 – 3.44 (m, 2H), 3.17 (d, J = 12.5 Hz, 1H), 2.79 – 2.69 (m, 2H), 1.96 – 1.88 (m, 1H), 1.75 – 1.62 (m, 1H).

13C

NMR (125 MHz, CDCl3) δ 158.12, 149.98, 145.61, 141.04,

132.61, 131.31, 129.01, 128.87, 116.47, 112.99, 106.43, 52.10, 51.11, 48.50, 46.44, 36.16. ESI-MS: m/z = 371 [M + H]+. HRMS (ESI) (m/z): calcd for C19H20ClN4O2 [M + H]+ 371.1269, found 371.1292. 4.1.19 N-((3S,4S)-4-(4-Chlorophenyl)piperidin-3-yl)-4-(1-methyl-1H-pyrazol-4-yl)furan2-carboxamide hydrochloride (C3)


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General procedure I and General procedure IV, yield: 73%; Retention time: 8.966 min, purity: 95.13 %; 1H NMR (500 MHz, DMSO) δ 9.62 (d, J = 9.4 Hz, 1H), 9.52 (d, J = 10.4 Hz, 1H), 8.56 (d, J = 9.1 Hz, 1H), 7.98 (s, 1H), 7.92 (s, 1H), 7.65 (s, 1H), 7.32 – 7.26 (m, 3H), 7.24 (d, J = 8.5 Hz, 2H), 4.56 – 4.48 (m, 1H), 3.80 (s, 3H), 3.29 (t, J = 9.6 Hz, 2H), 3.14 – 3.06 (m, 1H), 2.94 – 2.83 (m, 2H), 2.08 – 1.96 (m, 1H), 1.95 – 1.89 (m, 1H).

13C

NMR (125 MHz, DMSO) δ 156.93, 147.51, 140.86, 139.59, 136.34, 131.27, 129.38, 128.33, 128.05, 119.56, 112.99, 112.22, 46.46, 46.29, 44.06, 43.20, 38.63, 29.50. ESIMS: m/z = 385 [M + H]+. HRMS (ESI) (m/z): calcd for C20H22ClN4O2 [M + H]+ 385.1426, found 385.1403. 4.1.20

N-((3S,4S)-4-(4-Chlorophenyl)piperidin-3-yl)-5-(6-(methylamino)pyrimidin-4-

yl)furan-2-carboxamide (C4) General procedure I and General procedure IV, yield: 41%; Retention time: 8.522 min, purity: 96.15 %; 1H NMR (500 MHz, DMSO) δ 9.71 (d, J = 9.1 Hz, 1H), 9.58 – 9.35 (m, 3H), 8.72 – 8.60 (m, 1H), 7.91 – 7.62 (m, 1H), 7.52 – 7.28 (m, 5H), 7.28 – 7.10 (m, 1H), 4.56 (s, 1H), 3.42 – 3.20 (m, 3H), 3.13 – 2.85 (m, 5H), 2.12 – 2.00 (m, 1H), 1.97 (d, J =


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9.4 Hz, 1H). ESI-MS: m/z = 412 [M + H]+. HRMS (ESI) (m/z): calcd for C21H23ClN5O2 [M + H]+ 412.1535, found 412.1539. 4.1.21

N-((3S,4S)-4-(4-Chlorophenyl)piperidin-3-yl)-5-(7-hydroxy-6,7-dihydro-5H-

cyclopenta[d] pyrimidin-4-yl)thiophene-2-carboxamide(C5) General procedure I and General procedure III, yield: 32%; Retention time: 8.838 min, purity: 95.08 %; 1H NMR (500 MHz, MeOD) δ 8.92 (s, 1H), 7.67 (dd, J = 4.1, 2.4 Hz, 1H), 7.54 (dd, J = 4.1, 1.0 Hz, 1H), 7.32 – 7.27 (m, 2H), 7.27 – 7.23 (m, 2H), 5.10 (dd, J = 7.6, 6.4 Hz, 1H), 4.27 (td, J = 11.1, 4.5 Hz, 1H), 3.29 – 3.22 (m, 2H), 3.12 (d, J = 12.8 Hz, 1H), 3.08 – 3.01 (m, 4.3 Hz, 1H), 2.88 (dd, J = 11.7, 3.8 Hz, 1H), 2.79 – 2.71 (m, 1H), 2.68 – 2.62 (m, 1H), 2.62 – 2.54 (m, 1H), 2.03 – 1.96 (m, 1H), 1.95 – 1.88 (m, 1H), 1.82 – 1.73 (m, 1H).

13C

NMR (125 MHz, MeOD) δ 173.10, 160.28, 155.06, 152.90,

144.09, 140.55, 139.95, 130.41, 128.30, 127.34, 127.25, 127.25, 126.54, 72.20, 49.64, 48.68, 45.57, 43.74, 32.57, 30.67, 25.70. ESI-MS: m/z = 455 [M + H]+. 4.1.22 N-((3S,4S)-4-(4-Chlorophenyl)piperidin-3-yl)-4-(1-methyl-1H-pyrazol-5-yl)furan2-carboxamide hydrochloride (D1)


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General procedure I and General procedure IV, yield: 76%; Retention time: 9.050 min, purity: 95.02 %; 1H NMR (500 MHz, DMSO) δ 9.54 (d, J = 9.9 Hz, 1H), 9.42 (d, J = 10.3 Hz, 1H), 8.73 (d, J = 9.1 Hz, 1H), 8.19 (d, J = 0.8 Hz, 1H), 7.46 (d, J = 0.8 Hz, 1H), 7.42 (d, J = 1.9 Hz, 1H), 7.33 (d, J = 8.5 Hz, 2H), 7.27 (d, J = 8.6 Hz, 2H), 6.46 (d, J = 1.9 Hz, 1H), 4.57 – 4.50 (m, 1H), 3.88 (s, 3H), 3.37 – 3.30 (m, 2H), 3.15 – 3.10 (m, 1H), 2.97 – 2.88 (m, 2H), 2.08 – 1.99 (m, 1H), 1.98 – 1.92 (m, 1H).

13C

NMR (125 MHz,

DMSO) δ 156.66, 147.64, 142.08, 140.74, 137.83, 133.61, 131.26, 129.33, 128.30, 116.99, 113.68, 105.68, 46.56, 46.26, 43.99, 43.19, 37.89, 29.47. ESI-MS: m/z = 385 [M + H]+. HRMS (ESI) (m/z): calcd for C20H22ClN4O2 [M + H]+ 385.1426, found 385.1399. 4.1.23

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

difluorophenyl)piperidin-3-yl)furan-2-carboxamide hydrochloride (D2) General procedure I and General procedure IV, yield: 71%; Retention time: 9.531 min, purity: 96.56 %; 1H NMR (400 MHz, MeOD) δ 8.04 (s, 1H), 7.50 (s, 1H), 7.37 (s, 1H), 7.29 – 7.16 (m, 3H), 4.61 – 4.53 (m, 1H), 3.86 (s, 3H), 3.61 – 3.49 (m, 2H), 3.16 – 3.04 (m, 3H), 2.19 (d, J = 14.4 Hz, 1H), 2.07 – 1.99 (m, 1H). ESI-MS: m/z = 421 [M + H]+.


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4.1.24

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4-(4-Bromo-1-methyl-1H-pyrazol-5-yl)-N-((3S,4S)-4-(4-chlorophenyl)piperidin-

3-yl)furan-2-carboxamide (2R,3R)-2,3-dihydroxysuccinate (D3) General procedure I and General procedure V, yield: 55%; 1H NMR (400 MHz, DMSO) δ 8.65 (d, J = 8.9 Hz, 1H), 8.22 (s, 1H), 7.61 (s, 1H), 7.38 (s, 1H), 7.34 (d, J = 8.2 Hz, 2H), 7.28 (d, J = 8.3 Hz, 2H), 4.58 – 4.46 (m, 1H), 4.21 (s, 2H), 3.83 (s, 3H), 3.43 – 3.29 (m, 2H), 3.14 – 3.04 (m, 1H), 3.02 – 2.87 (m, 2H), 2.03 – 1.89 (m, 2H).

13C

NMR (100 MHz, DMSO) δ 173.80, 156.67, 147.71, 144.30, 140.81, 138.64, 132.04, 131.32, 129.32, 129.32, 128.39, 128.25, 114.54, 113.88, 92.90, 72.11, 46.78, 46.45, 44.10, 43.24, 38.64, 29.93. HRMS (ESI) (m/z): calcd for C20H21BrClN4O2 [M + H]+ 463.0531, found 463.0509. 4.1.25 N-((3S,4S)-4-(4-Chlorophenyl)piperidin-3-yl)-5-(1-methyl-1H-pyrazol-5-yl)furan2-carboxamide hydrochloride (D4) General procedure I and General procedure IV, yield: 61%; 1H NMR (500 MHz, DMSO) δ 9.47 (d, J = 10.0 Hz, 1H), 9.36 (d, J = 10.0 Hz, 1H), 8.61 (d, J = 9.0 Hz, 1H), 7.47 (d, J = 2.0 Hz, 1H), 7.35 – 7.26 (m, 4H), 7.21 (d, J = 3.6 Hz, 1H), 6.90 (d, J = 3.6 Hz, 1H), 6.71 (d, J = 2.0 Hz, 1H), 4.54 – 4.46 (m, 1H), 3.97 (s, 3H), 3.37 – 3.31 (m, 2H),


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3.18 (td, J = 11.8, 3.9 Hz, 1H), 3.02 – 2.88 (m, 2H), 2.08 – 1.98 (m, 2H).13C NMR (125 MHz, DMSO) δ 156.78, 146.52, 145.61, 140.74, 138.05, 132.48, 131.29, 129.34, 128.31, 115.62, 110.16, 106.06, 46.83, 46.31, 43.91, 43.24, 38.54, 29.32. ESI-MS: m/z = 385 [M + H]+. HRMS (ESI) (m/z): calcd for C20H22ClN4O2 [M + H]+ 385.1426, found 385.1431. 4.1.26

5-(4-Chloro-1-methyl-1H-pyrazol-5-yl)-N-((3S,4S)-4-(4-chlorophenyl)piperidin-

3-yl)furan-2-carboxamide hydrochloride (D5) General procedure I and General procedure IV, yield: 69%; 1H NMR (500 MHz, DMSO) δ 9.50 (d, J = 10.1 Hz, 1H), 9.40 (d, J = 10.1 Hz, 1H), 8.64 (d, J = 9.0 Hz, 1H), 7.68 (s, 1H), 7.35 – 7.26 (m, 5H), 6.99 (d, J = 3.6 Hz, 1H), 4.53 – 4.44 (m, 1H), 3.97 (s, 3H), 3.37 – 3.32 (m, 2H), 3.17 (td, J = 11.9, 3.8 Hz, 1H), 3.01 – 2.88 (m, 2H), 2.12 – 1.93 (m, 2H).

13C

NMR (125 MHz, DMSO) δ 156.61, 147.35, 142.44, 140.67, 136.87,

131.29, 129.32, 128.97, 128.28, 115.22, 112.73, 108.41, 47.04, 46.24, 43.83, 43.20, 39.74, 29.14. ESI-MS: m/z = 419 [M + H]+. HRMS (ESI) (m/z): calcd for C20H22Cl2N4O2 [M + H]+ 419.1036, found 419.1060.


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4.1.27

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3-yl)furan-2-carboxamide hydrochloride (D6) General procedure I and General procedure IV, yield: 74%; 1H NMR (500 MHz, DMSO) δ 9.49 (d, J = 9.6 Hz, 1H), 9.39 (d, J = 10.3 Hz, 1H), 8.63 (d, J = 9.0 Hz, 1H), 7.73 – 7.61 (m, 1H), 7.36 – 7.26 (m, 5H), 7.01 (d, J = 3.6 Hz, 1H), 4.53 – 4.45 (m, 1H), 3.96 (s, 3H), 3.37 (dd, J = 14.0, 7.0 Hz, 2H), 3.17 (td, J = 11.9, 3.8 Hz, 1H), 2.99 – 2.88 (m, 2H), 2.11 – 1.93 (m, 2H).

13C

NMR (125 MHz, DMSO) δ 156.62, 147.42, 142.80,

140.67, 139.11, 131.31, 130.57, 129.34, 128.29, 115.17, 113.05, 93.50, 47.04, 46.25, 43.84, 43.21, 39.65, 29.15. ESI-MS: m/z = 463 [M + H]+. 4.1.28

4-Bromo-N-((3S,4S)-4-(4-chlorophenyl)piperidin-3-yl)-5-(1-methyl-1H-pyrazol-

5-yl)furan-2-carboxamide (2R,3R)-2,3-dihydroxysuccinate (D7) General procedure I and General procedure V, yield: 52%; 1H NMR (500 MHz, DMSO) δ 8.59 (d, J = 8.5 Hz, 1H), 7.56 (d, J = 1.8 Hz, 1H), 7.34 (d, J = 7.7 Hz, 2H), 7.31 – 7.23 (m,3H), 6.76 (d, J = 1.8 Hz, 1H), 4.41 (d, J = 7.4 Hz, 1H), 4.12 (s, 2H), 3.92 (s, 3H), 3.40 – 3.32 (m, 2H), 3.10 – 2.86 (m, 3H) , 2.03 – 1.90 (m, 2H).

13C

NMR (125

MHz, DMSO) δ 174.01, 156.01, 146.88, 142.44, 140.69, 138.31, 131.35, 129.80,


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129.33, 128.37, 117.88, 108.06, 100.35, 71.96, 47.39, 46.59, 44.19, 43.30, 38.58, 29.75. ESI-MS: m/z = 465 [M + H]+. HRMS (ESI) (m/z): calcd for C20H21BrClN4O2 [M + H]+ 465.0510, found 465.0526. 4.1.29

4-Bromo-5-(4-chloro-1-methyl-1H-pyrazol-5-yl)-N-((3S,4S)-4-(4-chlorophenyl)

piperidin-3-yl)furan-2-carboxamide (2R,3R)-2,3-dihydroxysuccinate (D8) General procedure I and General procedure V, yield: 69%; 1H NMR (500 MHz, DMSO) δ 8.72 (d, J = 8.4 Hz, 1H), 7.77 (s, 1H), 7.41 (s, 1H), 7.36 – 7.22 (m, 4H), 4.42 – 4.33 (m, 1H), 4.10 (s, 2H), 3.76 (s, 3H), 3.40 – 3.28 (m, 2H), 3.12 – 2.89 (m, 3H), 2.12 – 1.89 (m, 2H).

13C

NMR (125 MHz, DMSO) δ 174.26, 155.76, 148.93, 140.71, 139.22,

137.08, 131.32, 129.34, 128.31, 127.48, 117.57, 111.56, 104.51, 71.98, 47.42, 46.59, 44.20, 43.28, 38.68, 29.74. ESI-MS: m/z = 497 [M + H]+. HRMS (ESI) (m/z): calcd for C20H20BrCl2N4O2 [M + H]+ 499.0121, found 499.0127. 4.1.30

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

chlorophenyl)piperidin-3-yl)furan-2-carboxamide (2R,3R)-2,3-dihydroxysuccinate (D9) General procedure I and General procedure V, yield: 52%; 1H NMR (500 MHz, DMSO) δ 8.69 (d, J = 8.7 Hz, 1H), 7.77 (s, 1H), 7.40 (s, 1H), 7.36 – 7.21 (m, 4H), 4.44 –


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4.34 (m, 1H), 4.09 (s, 2H), 3.76 (s, 3H), 3.40 – 3.27 (m, 2H), 3.08 – 2.97 (m, 1H), 2.97 – 2.81 (m, 2H), 2.03 – 1.87 (m, 2H).

13C

NMR (125 MHz, DMSO) δ 174.08, 155.74,

148.86, 140.68, 139.74, 139.26, 131.30, 129.32, 129.29, 128.31, 117.51, 104.56, 96.82, 71.87, 47.42, 46.67, 44.20, 43.34, 38.60, 29.76. ESI-MS: m/z = 543 [M + H]+. HRMS (ESI) (m/z): calcd for C20H20Br2ClN4O2 [M + H]+ 542.9616, found 542.9629. 4.1.31

N-((3S,4S)-4-(4-Chlorophenyl)piperidin-3-yl)-4-(1-methyl-1H-pyrazol-5-yl)

thiophene-2-carboxamide (2R,3R)-2,3-dihydroxysuccinate (D10) General procedure I and General procedure V, yield: 39%; 1H NMR (400 MHz, DMSO) δ 8.70 (d, J = 8.4 Hz, 1H), 7.95 (s, 1H), 7.89 (s, 1H), 7.43 (s, 1H), 7.38 – 7.25 (m, 4H), 6.41 (s, 1H), 4.57 – 4.39 (m, 1H), 4.25 (s, 2H), 3.89 (s, 3H), 3.60 – 3.49 (m, 1H), 3.44 – 3.34 (m, 1H), 3.11 – 3.02 (m, 2H), 3.01 – 2.86 (m, 1H), 2.06 – 1.90 (m, 2H). 13C

NMR (100 MHz, DMSO) δ 173.37, 160.17, 140.66, 139.63, 137.79, 137.24, 131.29,

130.63, 129.29, 128.42, 128.34, 113.88, 105.65, 72.07, 47.45, 46.58, 44.15, 43.29, 37.88, 29.67. HRMS (ESI) (m/z): calcd for C20H22ClN4OS [M + H]+ 401.1197, found 401.1179.


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4.1.32


piperidin-3-yl)thiophene-2-carboxamide hydrochloride (D11) General procedure I and General procedure IV, yield: 74%; Retention time: 10.372 min, purity: 97.31 %; 1H NMR (500 MHz, DMSO) δ 9.36 (s, 1H), 9.29 (s, 1H), 9.06 (d, J = 8.9 Hz, 1H), 8.11 (s, 1H), 8.09 (s, 1H), 7.36 – 7.27 (m, 4H), 4.52 – 4.44 (m, 1H), 3.82 (s, 3H), 3.42 – 3.36 (m, 2H), 3.21 – 3.15 (m, 1H), 2.96 (d, J = 9.1 Hz, 2H), 2.08 – 1.93 (m, 2H). 13C NMR (125 MHz, DMSO) δ 159.89, 140.65, 140.24, 136.14, 134.71, 132.38, 131.24, 129.31, 128.40, 128.26, 126.52, 105.18, 47.44, 46.37, 43.84, 43.22, 39.02, 29.24. ESI-MS: m/z = 471 [M + H]+. HRMS (ESI) (m/z): calcd for C20H20Cl3N4OS [M + H]+ 471.0388, found 471.0412. 4.1.33

5-Chloro-4-(4-chloro-1-methyl-1H-pyrazol-5-yl)-N-((3S,4S)-4-(3-fluorophenyl)

piperidin-3-yl)thiophene-2-carboxamide hydrochloride (D12) General procedure I and General procedure IV, yield: 68%; Retention time: 10.007 min, purity: 99.27 %; 1H NMR (500 MHz, MeOD) δ 7.93 (d, J = 1.2 Hz, 1H), 7.79 (s, 1H), 7.30 (td, J = 7.9, 6.2 Hz, 1H), 7.16 (d, J = 7.8 Hz, 1H), 7.11 (dd, J = 10.1, 2.0 Hz, 1H), 6.93 (td, J = 8.5, 2.3 Hz, 1H), 4.58 (td, J = 11.6, 4.3 Hz, 1H), 3.80 (s, 3H), 3.65 – 3.59


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(m, 1H), 3.54 (d, J = 12.8 Hz, 1H), 3.25 – 3.10 (m, 3H), 2.23 – 2.15 (m, 1H), 2.14 – 2.04 (m, 1H).

13C

NMR (125 MHz, MeOD) δ 164.31 (d, J = 243.8 Hz), 162.95, 144.73 (d, J =

6.3 Hz), 140.70, 137.65, 137.28, 133.52, 131.47 (d, J = 7.5 Hz), 129.83, 128.68, 124.60 (d, J = 2.5 Hz), 115.44 (d, J = 22.5 Hz), 115.13 (d, J = 21.3 Hz), 107.68, 49.49, 48.04, 46.39, 45.14, 39.20, 31.18. ESI-MS: m/z = 453 [M + H]+. 4.1.34


difluorophenyl) piperidin-3-yl)thiophene-2-carboxamide hydrochloride (D13) General procedure I and General procedure IV, yield: 57%; Retention time: 9.432 min, purity: 98.90 %; 1H NMR (500 MHz, DMSO) δ 9.18 (s, 2H), 9.01 (d, J = 8.9 Hz, 1H), 8.13 (d, J = 1.3 Hz, 1H), 8.08 (s, 1H), 7.37 – 7.29 (m, 2H), 7.15 – 7.10 (m, 1H), 4.49 – 4.43 (m, 1H), 3.82 (s, 3H), 3.42 – 3.35 (m, 2H), 3.21 – 3.13 (m, 1H), 3.00 – 2.90 (m, 2H), 2.08 – 1.97 (m, 2H).

13C

NMR (125 MHz, DMSO) δ 159.97, 149.12 (dd, J = 242.5, 12.5

Hz), 148.32 (dd, J = 242.5, 12.5 Hz), 140.12, 139.41, 136.14, 134.73, 132.47, 128.41, 126.55, 124.16, 117.32 (d, J =17.5 Hz), 116.50 (d, J =17.5 Hz), 105.17, 47.51, 46.38, 43.73, 43.19, 38.97, 29.07. ESI-MS: m/z = 471 [M + H]+.


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4.1.35

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

(trifluoromethyl)phenyl)piperidin-3-yl)thiophene-2-carboxamide hydrochloride (D14) General procedure I and General procedure IV, yield: 71%; Retention time: 10.942 min, purity: 96.05 %; 1H NMR (500 MHz, DMSO) δ 9.31 (s, 1H), 9.18 (s, 1H), 9.12 (d, J = 9.0 Hz, 1H), 8.12 (d, J = 1.2 Hz, 1H), 8.10 (s, 1H), 7.79 (d, J = 1.6 Hz, 1H), 7.66 (d, J = 8.3 Hz, 1H), 7.58 (dd, J = 8.3, 1.5 Hz, 1H), 4.52 – 4.44 (m, 1H), 3.82 (s, 3H), 3.35 – 3.25 (m, 3H), 2.97 (q, J = 11.1 Hz, 2H), 2.13 – 2.01 (m, 2H).

13C

NMR (125 MHz,

DMSO) δ 160.00, 141.59, 140.01, 136.15, 134.74, 132.75, 132.53, 131.69, 128.93, 128.44, 127.48 (q, J = 5.3 Hz), 126.54, 126.23 (q, J = 30.1 Hz), 122.78 (q, J = 271.1 Hz), 105.20, 56.01, 47.44, 46.37, 43.78, 43.17, 28.54. ESI-MS: m/z = 537 [M + H]+. 4.1.36

4-(4-Chloro-1-methyl-1H-pyrazol-5-yl)-N-((3S,4S)-4-(4-chlorophenyl)piperidin-

3-yl)-1-methyl-1H-pyrrole-2-carboxamide(2R,3R)-2,3-dihydroxysuccinate (D15) General procedure I and General procedure V, yield: 40%; Retention time: 10.058 min, purity: 95.54 %; 1H NMR (500 MHz, DMSO) δ 8.15 (d, J = 7.0 Hz, 1H), 7.52 (s, 1H), 7.38 – 7.24 (m, 5H), 6.86 (s, 1H), 4.40 –4.31 (m, 1H), 4.00 (s, 2H), 3.78 (s, 3H), 3.76 (s, 3H), 3.42 – 3.28 (m, 2H), 3.01 – 2.83 (m, 3H), 2.02 –1.87 (m, 2H).

13C

NMR


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(125 MHz, DMSO) δ 174.53, 160.16, 141.13, 136.23, 133.88, 131.15, 129.43, 128.23, 127.46, 125.89, 111.82, 107.78, 105.94, 71.85, 47.25, 47.09, 44.66, 43.38, 38.60, 36.23, 30.07. ESI-MS: m/z = 432 [M + H]+. HRMS (ESI) (m/z): calcd for C21H24Cl2N5O [M + H]+ 432.1352, found 432.1363. 4.1.37


3-yl)-1-methyl-1H-pyrrole-2-carboxamide (2R,3R)-2,3-dihydroxysuccinate (D16) General procedure I and General procedure V, yield: 65%; 1H NMR (500 MHz, DMSO) δ 8.15 (d, J = 8.8 Hz, 1H), 7.53 (s, 1H), 7.38 – 7.23 (m, 5H), 6.85 (d, J = 1.4 Hz, 1H), 4.45 – 4.33 (m, 1H), 4.11 (s, 2H), 3.80 (s, 3H), 3.74 (s, 3H), 3.39– 3.30 (m, 2H), 3.01 – 2.86 (m, 3H), 2.01 – 1.90 (m, 2H). 13C NMR (125 MHz, DMSO) δ 174.19, 160.16, 140.96, 138.38, 135.45, 131.19, 129.40, 128.25, 127.70, 125.77, 112.23, 108.18, 91.28, 71.98, 47.01, 46.80, 44.45, 43.23, 38.49, 36.20, 29.71. ESI-MS: m/z = 478 [M + H]+. HRMS (ESI) (m/z): calcd for C21H24BrClN5O [M + H]+ 478.0853, found 478.0847. 4.1.38


piperidin-3-yl)-1-methyl-1H-pyrrole-2-carboxamide

(2R,3R)-2,3-dihydroxysuccinate

(D17)


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General procedure I and General procedure V, yield: 39%; Retention time: 10.367 min, purity: 95.05 %; 1H NMR (500 MHz, DMSO) δ 8.29 (d, J = 7.9 Hz, 1H), 7.61 (s, 1H), 7.34 (d, J = 7.8 Hz, 2H), 7.28 (d, J = 7.8 Hz, 2H), 6.76 (s, 1H), 4.47 – 4.35 (m, 1H), 4.18 (s, 2H), 3.73 (s, 3H), 3.65 (s, 3H), 3.41 – 3.30 (m, 2H), 3.06 – 2.80 (m, 3H), 2.01– 1.90 (m, 2H).

13C

NMR (125 MHz, DMSO) δ 173.82, 159.54, 140.82, 136.39, 132.29,

131.27, 129.42, 128.29, 125.92, 121.39, 112.98, 108.68, 105.71, 72.06, 47.07, 46.66, 44.44, 43.20, 37.88, 32.90, 29.60. ESI-MS: m/z = 466 [M + H]+. HRMS (ESI) (m/z): calcd for C21H23Cl3N5O [M + H]+ 466.0963, found 466.0987. 4.1.39

5-Bromo-4-(4-bromo-1-methyl-1H-pyrazol-5-yl)-N-((3S,4S)-4-(4-chlorophenyl)

piperidin-3-yl)-1-methyl-1H-pyrrole-2-carboxamide

(2R,3R)-2,3-dihydroxysuccinate

(D18) General procedure I and General procedure V, yield: 43%; Retention time: 9.742 min, purity: 95.19 %;

1H

NMR (500 MHz, DMSO) δ 8.27 (d, J = 8.1 Hz, 1H), 7.61 (s, 1H),

7.34 (d, J = 8.1 Hz, 2H), 7.28 (d, J = 8.1 Hz, 2H), 6.74 (s, 1H), 4.39 (d, J = 8.4 Hz, 1H), 4.09 (s, 2H), 3.75 (s, 3H), 3.64 (s, 3H), 3.40 – 3.31 (m, 2H), 3.02 – 2.80 (m, 3H), 2.07 – 1.89 (m, 2H).

13C

NMR (125 MHz, DMSO) δ 174.29, 159.56, 140.90, 138.43, 134.88,


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131.23, 129.42, 128.28, 127.57, 113.87, 111.15, 109.60, 94.22, 71.97, 47.20, 46.76, 44.53, 43.21, 37.92, 34.59, 29.74. ESI-MS: m/z = 556 [M + H]+. 4.1.40

N-((3S,4S)-4-(3,4-Difluorophenyl)piperidin-3-yl)-4-(1-methyl-1H-pyrazol-5-yl)

benzamide (2R,3R)-2,3-dihydroxysuccinate (D19) General procedure I and General procedure V, yield: 51%; Retention time: 9.318 min, purity: 95.52 %; 1H NMR (400 MHz, DMSO) δ 8.60 (d, J = 8.8 Hz, 1H), 7.75 (d, J = 8.3 Hz, 2H), 7.58 (d, J = 8.3 Hz, 2H), 7.47 (d, J = 1.9 Hz, 1H), 7.37 – 7.27 (m, 2H), 7.14 (s, 1H), 6.44 (d, J = 1.9 Hz, 1H), 4.56 – 4.46 (m, 1H), 4.14 (s, 2H), 3.84 (s, 3H), 3.42 – 3.36 (m, 2H), 3.14 – 3.04 (m, 1H), 3.03 – 2.86 (m, 2H), 2.01 – 1.95 (m, 2H).

13C

NMR (100

MHz, DMSO) δ 173.92, 165.36, 149.13 (dd, J = 244.0, 12.0 Hz), 148.38 (dd, J = 244.0, 12.0 Hz), 141.78, 139.70, 138.00, 133.39, 132.94, 128.20, 127.54, 124.27, 117.25 (d, J =16.0 Hz), 116.41 (d, J =16.0 Hz), 106.31, 71.94, 47.62, 46.63, 44.23, 43.24, 37.66, 29.73. ESI-MS: m/z = 397 [M + H]+. 4.1.41


difluorophenyl)piperidin-3-yl) benzamide hydrochloride (D20)


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General procedure I and General procedure V, yield: 70%; Retention time: 9.842 min, purity: 98.26 %; 1H NMR (400 MHz, DMSO) δ 8.65 (d, J = 8.3 Hz, 1H), 7.80 (d, J = 8.0 Hz, 2H), 7.67 (s, 1H), 7.57 (d, J = 8.0 Hz, 2H), 7.39 – 7.27 (m, 2H), 7.14 (s, 1H), 4.53 – 4.42 (m, 1H), 4.10 (s, 2H), 3.76 (s, 3H), 3.43 – 3.27 (m, 2H), 3.12 – 3.02 (m, 1H), 2.97 – 2.83 (m, 2H), 2.02 – 1.89 (m, 2H).

13C

NMR (100 MHz, DMSO) δ 174.07, 165.35,

149.07 (dd, J = 234.0, 11.0 Hz), 148.17(dd, J = 234.0, 11.0 Hz), 139.75, 138.03, 136.48, 134.44, 130.07, 129.50, 127.54, 124.30, 117.27 (d, J =17.0 Hz), 116.39 (d, J =18.0 Hz), 107.51, 71.88, 47.69, 46.71, 44.29, 43.29, 38.35, 29.90. ESI-MS: m/z = 431 [M + H]+. 4.1.42

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

difluorophenyl)piperidin-3-yl)benzamide (2R,3R)-2,3-dihydroxysuccinate (D21) General procedure I and General procedure V, yield: 73%; Retention time: 9.980 min, purity: 95.07 %; 1H NMR (400 MHz, DMSO) δ 8.64 (d, J = 8.3 Hz, 1H), 7.78 (d, J = 7.8 Hz, 2H), 7.66 (s, 1H), 7.55 (d, J = 7.9 Hz, 2H), 7.41 – 7.27 (m, 2H), 7.14 (s, 1H), 4.51 – 4.42 (m, 1H), 4.07 (s, 2H), 3.75 (s, 3H), 3.43 – 3.28 (m, 2H), 3.13 – 3.02 (m, 1H), 3.00 – 2.82 (m, 2H), 2.08 – 1.89 (m, 2H).

13C

NMR (100 MHz, DMSO) δ 174.22, 165.36,


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149.15 (dd, J = 243.0, 11.0 Hz), 148.58 (dd, J = 243.0, 11.0 Hz), 139.82, 139.64, 138.58, 134.48, 130.65, 129.67, 127.47, 124.29, 117.25 (d, J =16.0 Hz), 116.39 (d, J =17.0 Hz), 92.68, 71.84, 47.81, 46.84, 44.37, 43.34, 38.35, 30.07. ESI-MS: m/z = 475 [M + H]+. 4.1.43 4-(4-Chloro-1-methyl-1H-pyrazol-5-yl)-N-((3S,4S)-4-(3-fluorophenyl)piperidin-3yl)benzamide hydrochloride (D22) General procedure I and General procedure IV, yield: 81%; Retention time: 9.661 min, purity: 99.19 %;; 1H NMR (500 MHz, MeOD) δ 7.75 (d, J = 8.4 Hz, 2H), 7.56 (s, 1H), 7.51 (d, J = 8.4 Hz, 2H), 7.36 – 7.30 (m, 1H), 7.19 (d, J = 7.8 Hz, 1H), 7.15 – 7.11 (m, 1H), 6.99 – 6.94 (m, 1H), 4.70 – 4.62 (m, 1H), 3.77 (s, 3H), 3.66 – 3.62 (m, 1H), 3.55 (d,

J = 12.8 Hz, 1H), 3.26 – 3.17 (m, 2H), 3.14 (t, J = 12.0 Hz, 1H), 2.21 (dd, J = 14.5, 2.3 Hz, 1H), 2.15 – 2.05 (m, 1H).

13C

NMR (125 MHz, MeOD) δ 169.03, 164.34 (d, J =

243.4 Hz), 144.86 (d, J = 7.1 Hz), 140.19, 137.93, 135.72, 132.23, 131.49 (d, J = 8.3 Hz), 130.96, 128.74, 124.68 (d, J = 2.6 Hz), 115.42 (d, J = 21.8 Hz), 115.17 (d, J = 22.4 Hz), 110.15, 49.47, 48.01, 46.51, 45.16, 38.61, 31.28. ESI-MS: m/z = 413 [M + H]+.


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4.1.44

N-((3S,4S)-4-(3,4-Difluorophenyl)piperidin-3-yl)-6-(1-methyl-1H-pyrazol-5-

yl)nicotinamide hydrochloride (D23) General procedure I and General procedure IV, yield: 47%; Retention time: 9.166 min, purity: 98.84 %; 1H NMR (500 MHz, DMSO) δ 9.74 – 9.58 (m, 2H), 9.25 (d, J = 8.9 Hz, 1H), 8.97 (d, J = 1.9 Hz, 1H), 8.24 (dd, J = 8.3, 2.1 Hz, 1H), 7.85 (d, J = 8.3 Hz, 1H), 7.51 (d, J = 1.9 Hz, 1H), 7.35 – 7.29 (m, 2H), 7.14 (d, J = 4.5 Hz, 1H), 6.87 (d, J = 1.9 Hz, 1H), 4.63 – 4.55 (m, 1H), 4.10 (s, 3H), 3.41 – 3.33 (m, 2H), 3.32 – 3.25 (m, 1H), 3.06 – 2.89 (m, 2H), 2.18 – 2.09 (m, 1H), 2.03 – 1.96 (m, 1H).

13C

NMR (125 MHz,

DMSO) δ 163.76, 150.86, 149.16 (dd, J = 243.8, 12.5 Hz), 148.31 (dd, J = 242.5, 12.5 Hz), 147.89, 139.75, 139.70, 137.90, 136.57, 127.71, 124.46, 122.39, 117.33 (d, J =16.3 Hz), 116.69 (d, J =16.3 Hz), 107.93, 47.64, 46.33, 43.89, 43.25, 39.65, 29.00. ESI-MS: m/z = 398 [M + H]+. 4.1.45


difluorophenyl)piperidin-3-yl)nicotinamide hydrochloride (D24) General procedure I and General procedure IV, yield: 54%; Retention time: 9.801 min, purity: 98.68 %; 1H NMR (500 MHz, DMSO) δ 9.62 – 9.49 (m, 2H), 9.23 (d, J = 8.9 Hz,


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1H), 9.03 (d, J = 1.8 Hz, 1H), 8.30 (dd, J = 8.2, 2.2 Hz, 1H), 7.78 (d, J = 8.2 Hz, 1H), 7.69 (s, 1H), 7.36 – 7.29 (m, 2H), 7.15 (d, J = 4.7 Hz, 1H), 4.65 – 4.57 (m, 1H), 3.92 (s, 3H), 3.43 – 3.34 (m, 2H), 3.27 – 3.21 (m, 1H), 3.04 – 2.91 (m, 2H), 2.16 – 2.07 (m, 1H), 2.00 (d, J = 12.6 Hz, 1H).

13C

NMR (125 MHz, DMSO) δ 163.78, 149.16 (dd, J = 243.8,

12.5 Hz), 148.78, 148.59, 148.30 (dd, J = 243.8, 12.5 Hz), 139.62, 136.73, 136.47, 136.19, 128.64, 124.42, 124.17, 117.34 (d, J =16.3 Hz), 116.51 (d, J =16.3 Hz), 108.59, 47.56, 46.27, 43.88, 43.19, 39.19, 29.08. ESI-MS: m/z = 432 [M + H]+. 4.1.46

6-(4-Bromo-1-methyl-1H-pyrazol-5-yl)-N-((3S,4S)-4-(3,4-

difluorophenyl)piperidin-3-yl)nicotinamide (D25) General procedure I and General procedure III, yield: 54%; Retention time: 9.831 min, purity: 98.46 %; 1H NMR (500 MHz, DMSO) δ 9.68 – 9.53 (m, 2H), 9.27 (d, J = 8.9 Hz, 1H), 9.03 (d, J = 1.7 Hz, 1H), 8.31 (dd, J = 8.2, 2.2 Hz, 1H), 7.78 (d, J = 8.2 Hz, 1H), 7.68 (s, 1H), 7.37 – 7.30 (m, 2H), 7.15 (d, J = 4.5 Hz, 1H), 4.66 – 4.58 (m, 1H), 3.90 (s, 3H), 3.42 – 3.34 (m, 2H), 3.28 – 3.22 (m, 1H), 3.05 – 2.91 (m, 2H), 2.18 – 2.06 (m, 1H), 2.04 – 1.97 (m, 1H).

13C

NMR (125 MHz, DMSO) δ 163.77, 149.16 (dd, J = 243.8, 12.5

Hz), 149.23, 148.60, 148.30 (dd, J = 243.8, 12.5 Hz), 139.65, 138.89, 138.12, 136.12,


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128.72, 124.55, 124.43, 117.34 (d, J =17.5 Hz), 116.52 (d, J =17.5 Hz), 93.53, 47.57, 46.27, 43.87, 43.20, 39.19, 29.09. ESI-MS: m/z = 476 [M + H]+. 4.1.47

N-((3S,4S)-4-(3-Fluorophenyl)piperidin-3-yl)-5-(1-methyl-1H-pyrazol-5-

yl)picolinamide hydrochloride (D26) General procedure I and General procedure IV, yield: 74%; Retention time: 8.960 min, purity: 97.65 %; 1H NMR (500 MHz, DMSO) δ 9.71 (s, 1H), 9.58 (s, 1H), 8.98 (d, J = 9.5 Hz, 1H), 8.74 (d, J = 1.7 Hz, 1H), 8.12 (dd, J = 8.1, 2.1 Hz, 1H), 7.99 (d, J = 8.1 Hz, 1H), 7.53 (d, J = 1.9 Hz, 1H), 7.28 (dd, J = 14.3, 7.9 Hz, 1H), 7.09 (d, J = 7.8 Hz, 1H), 7.06 (d, J = 10.2 Hz, 1H), 6.97 (td, J = 8.6, 2.3 Hz, 1H), 6.60 (d, J = 1.9 Hz, 1H), 4.72 – 4.63 (m, 1H), 3.87 (s, 3H), 3.35 – 3.22 (m, 3H), 3.04 (q, J = 11.1 Hz, 1H), 2.88 (dd, J = 23.6, 11.9 Hz, 1H), 2.14 – 1.97 (m, 2H). 13C NMR (125 MHz, DMSO) δ 162.97, 162.03 (d, J = 246.0 Hz), 148.51, 147.52, 144.87 (d, J = 7.0 Hz), 138.82, 138.20, 137.19, 130.27 (d, J = 8.0 Hz), 128.78, 123.63 (d, J = 2.1 Hz), 122.08, 114.13 (d, J = 21.0 Hz), 113.57 (d, J = 20.8 Hz), 107.22, 46.77, 46.05, 44.27, 43.19, 37.74, 29.66. ESI-MS: m/z = 380 [M + H]+. 4.1.48 5-(4-Chloro-1-methyl-1H-pyrazol-5-yl)-N-((3S,4S)-4-(3-fluorophenyl)piperidin-3yl) picolinamide hydrochloride (D27)


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General procedure I and General procedure IV, yield: 74%; Retention time: 9.675 min, purity: 97.71 %; 1H NMR (500 MHz, DMSO) δ 9.61 (d, J = 9.7 Hz, 1H), 9.48 (d, J = 10.3 Hz, 1H), 9.04 (d, J = 9.5 Hz, 1H), 8.73 (d, J = 1.5 Hz, 1H), 8.13 (dd, J = 8.1, 2.1 Hz, 1H), 8.06 (d, J = 8.0 Hz, 1H), 7.73 (s, 1H), 7.33 – 7.26 (m, 1H), 7.10 (d, J = 7.8 Hz, 1H), 7.07 (d, J = 10.2 Hz, 1H), 6.98 (td, J = 8.5, 2.2 Hz, 1H), 4.72 – 4.65 (m, 1H), 3.79 (s, 3H), 3.38 – 3.23 (m, 3H), 3.04 (q, J = 11.2 Hz, 1H), 2.93 – 2.83 (m, 1H), 2.10 – 1.99 (m, 2H). 13C

NMR (125 MHz, DMSO) δ 162.88, 162.00 (d, J = 241.5 Hz), 149.34, 148.55, 144.83

(d, J = 7.0 Hz), 138.79, 136.65, 135.31, 130.27 (d, J = 8.0 Hz), 126.23, 123.59, 122.15, 114.11(d, J = 21.1 Hz), 113.58 (d, J = 20.5 Hz), 108.37, 46.74, 46.04, 44.24, 43.20, 38.50, 29.73. ESI-MS: m/z = 414 [M + H]+. 4.1.49 5-(4-Bromo-1-methyl-1H-pyrazol-5-yl)-N-((3S,4S)-4-(3-fluorophenyl)piperidin-3yl)picolinamide hydrochloride (D28) General procedure I and General procedure IV, yield: 80%; Retention time: 9.598 min, purity: 97.58 %; 1H NMR (500 MHz, DMSO) δ 9.64 (d, J = 9.7 Hz, 1H), 9.52 (d, J = 10.1 Hz, 1H), 9.04 (d, J = 9.5 Hz, 1H), 8.72 (d, J = 1.5 Hz, 1H), 8.12 (dd, J = 8.1, 2.0 Hz, 1H), 8.06 (d, J = 8.0 Hz, 1H), 7.73 (s, 1H), 7.30 (dd, J = 14.3, 7.8 Hz, 1H), 7.09 (dd, J = 18.3,


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9.0 Hz, 2H), 6.98 (td, J = 8.6, 2.2 Hz, 1H), 4.71 – 4.64 (m, 1H), 3.80 (s, 3H), 3.40 – 3.23 (m, 3H), 3.04 (q, J = 11.3 Hz, 1H), 2.89 (dd, J = 22.6, 11.3 Hz, 1H), 2.10 – 1.97 (m, 2H). 13C

NMR (125 MHz, DMSO) δ 162.87, 162.00 (d, J = 241.4 Hz), 149.35, 148.74, 144.84

(d, J = 7.0 Hz), 138.96, 138.76, 136.92, 130.27(d, J = 8.3 Hz), 126.80, 123.59, 122.08, 114.11 (d, J = 20.9 Hz), 113.58 (d, J = 20.8 Hz), 93.58, 46.72, 46.03, 44.23, 43.18, 38.51, 29.73. ESI-MS: m/z = 458 [M + H]+. 4.1.50

5-Chloro-4-(4-chloro-1-methyl-1H-pyrazol-5-yl)-N-((3S,4S,6R)-4-(3,4-

dichlorophenyl)-6-propylpiperidin-3-yl)furan-2-carboxamide hydrochloride (E1) General procedure IV , yield: 67%; Retention time: 10.865 min, purity: 96.10 %; 1H NMR (400 MHz, MeOD) δ 7.58 – 7.51 (m, 2H), 7.46 (d, J = 8.3 Hz, 1H), 7.33 – 7.25 (m, 2H), 4.58 – 4.51 (m, 1H), 3.81 – 3.66 (m, 4H), 3.45 – 3.33 (m, 2H), 2.23 – 2.08 (m, 2H), 2.07 – 1.96 (m, 1H), 1.89 – 1.80 (m, 1H), 1.58 – 1.44 (m, 2H), 1.07 (t, J = 7.3 Hz, 3H). 13C

NMR (100 MHz, MeOD) δ 158.39, 147.83, 142.48, 140.20, 138.12, 133.54, 132.34,

131.86, 131.05, 130.78, 128.62, 118.78, 112.07, 112.00, 53.66, 48.36, 42.35, 40.55, 38.55, 33.60, 30.89, 20.19, 14.03. ESI-MS: m/z = 531 [M + H]+. HRMS (ESI) (m/z): calcd for C23H25Cl4N4O2 [M + H]+ 531.0697, found 531.0709.


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4.1.51 N-((3S,4S,6R)-6-allyl-4-(3,4-dichlorophenyl)piperidin-3-yl)-5-chloro-4-(4-chloro1-methyl-1H-pyrazol-5-yl)furan-2-carboxamide hydrochloride (E2) General procedure IV, yield: 82%; Retention time: 10.981 min, purity: 97.54 %; 1H NMR (500 MHz, MeOD) δ 7.53 (s, 1H), 7.51 (d, J = 2.0 Hz, 1H), 7.46 (d, J = 8.3 Hz, 1H), 7.30 (s, 1H), 7.27 (dd, J = 8.3, 2.0 Hz, 1H), 5.90 – 5.81 (m, 1H), 5.41 (dd, J = 17.0, 1.1 Hz, 1H), 5.30 (d, J = 10.3 Hz, 1H), 4.61 – 4.55 (m, 1H), 3.83 – 3.77 (m, 1H), 3.75 (s, 3H), 3.44 – 3.32 (m, 3H), 2.82 – 2.69 (m, 2H), 2.21 – 2.09 (m, 2H).

13C

NMR (125 MHz,

MeOD) δ 158.33, 147.80, 142.43, 140.19, 138.11, 133.53, 133.24, 132.34, 131.88, 130.99, 130.77, 128.61, 120.78, 118.80, 112.06, 111.99, 52.88, 48.77, 42.18, 40.37, 38.57, 33.41, 33.33. ESI-MS: m/z = 529 [M + H]+. HRMS (ESI) (m/z): calcd for C23H23Cl4N4O2 [M + H]+ 529.0540, found 529.0550 4.1.52

5-Chloro-4-(4-chloro-1-methyl-1H-pyrazol-5-yl)-N-((3S,4S,6R)-4-(3,4-

dichlorophenyl)-6-(3-hydroxypropyl)piperidin-3-yl)furan-2-carboxamide (E3) General procedure I and General procedure III, yield: 47%; Retention time: 9.621 min, purity: 96.32 %; 1H NMR (500 MHz, CDCl3) δ 7.54 – 7.46 (m, 2H), 7.44 (s, 1H), 7.40 (d,

J = 8.2 Hz, 1H), 7.25 – 7.18 (m, 2H), 4.49 – 4.44 (m, 1H), 3.81 – 3.70 (m, 4H), 3.68 –


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3.59 (m, 1H), 3.31 – 3.12 (m, 4H), 2.18 – 2.09 (m, 1H), 2.04 – 1.94 (m, 2H), 1.89 – 1.66 (m, 3H).

13C

NMR (125 MHz, CDCl3) δ 156.78, 146.88, 141.55, 138.42, 137.51, 132.98,

131.26, 130.97, 129.83, 129.34, 127.06, 117.60, 111.23, 111.13, 62.50, 52.35, 49.74, 43.81, 40.80, 38.52, 34.62, 30.37, 29.95. ESI-MS: m/z = 547 [M + H]+. HRMS (ESI) (m/z): calcd for C23H25Cl4N4O3 [M + H]+ 547.0646, found 546.9982. 4.1.53

5-Chloro-4-(4-chloro-1-methyl-1H-pyrazol-5-yl)-N-((3S,4S,6S)-4-(3,4-

dichlorophenyl) -6-(2-hydroxyethyl)piperidin-3-yl)furan-2-carboxamide (E4) General procedure III, yield: 77%; Retention time: 10.753 min, purity: 95.41 %; 1H NMR (500 MHz, MeOD) δ 7.53 (s, 1H), 7.48 (dd, J = 12.9, 1.9 Hz, 1H), 7.41 (t, J = 7.0 Hz, 1H), 7.28 – 7.22 (m, 1H), 7.21 (d, J = 6.2 Hz, 1H), 4.37 – 4.31 (m, 1H), 3.79 – 3.71 (m, 5H), 3.47 – 3.40 (m, 1H), 3.23 – 3.15 (m, 1H), 3.13 – 2.99 (m, 2H), 2.16 – 2.10 (m, 1H), 2.09 – 1.99 (m, 1H), 1.97 – 1.91 (m, 1H), 1.89 – 1.77 (m, 1H).

13C

NMR (125 MHz,

MeOD) δ 158.39, 148.31, 144.73, 139.81, 138.11, 133.21, 131.57, 131.51, 131.06, 130.91, 128.59, 118.16, 111.95, 111.92, 61.16, 51.82, 50.91, 44.75, 42.61, 38.54, 37.95, 33.01. ESI-MS: m/z = 533 [M + H]+. HRMS (ESI) (m/z): calcd for C22H23Cl4N4O3 [M + H]+ 533.0489, found 532.8913.


84


4.1.54

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dichlorophenyl) -6-(2,3-dihydroxypropyl)piperidin-3-yl)furan-2-carboxamide (E5) General procedure III, yield: 72%; Retention time: 10.722 min, purity: 95.31 %; 1H NMR (500 MHz, MeOD) δ 7.53 (s, 1H), 7.52 (d, J = 2.0 Hz, 1H), 7.46 (dd, J = 8.3, 1.4 Hz, 1H), 7.27 (dd, J = 5.9, 2.6 Hz, 2H), 4.59 – 4.52 (m, 1H), 4.03 – 3.94 (m, 1H), 3.94 – 3.84 (m, 1H), 3.75 (s, 3H), 3.64 – 3.55 (m, 2H), 3.45 – 3.34 (m, 3H), 2.37 – 2.09 (m, 3H), 2.05 – 1.90 (m, 1H). 13C NMR (125 MHz, MeOD, 1:1 ratio due to epimers) δ 158.41 and 158.38, 147.81, 142.58 and 142.50, 140.21, 138.12, 133.52 and 133.51, 132.32 and 132.27, 131.86 and 131.84, 131.07 and 131.06, 130.79, 128.55, 118.79, 112.07, 112.00, 72.00 and 70.39, 67.17 and 66.62, 53.17, 51.87, 42.40 and 42.19, 40.90 and 40.78, 38.56, 35.65 and 35.28, 32.20 and 31.36. ESI-MS: m/z = 563 [M + H]+. 4.1.55 dichlorophenyl)

5-Chloro-4-(4-chloro-1-methyl-1H-pyrazol-5-yl)-N-((3S,4S,6S)-4-(3,4-6-(2-methoxyethyl)piperidin-3-yl)furan-2-carboxamide

hydrochloride

(E6) To the solution of compound 45a (71 mg, 0.1 mmol) in methanol (5mL) was added sodium methylate (11 mg, 0.2 mmol) under N2 atmosphere. The reaction was stirred at


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45 oC overnight. After fully reacted, it was poured into H2O and extracted with ethyl ecetate (5 mL× 3). The combined organic extracts were washed with brine, dried over Na2SO4, and concentrated. The residue was purified by silica gel chromatography to afford white solid. To a solution of above white solid in ethyl acetate (0.3mL) was added HCl saturated ethyl acetate at ice-bath, stirred overnight in room temperature. Then, the reaction was added saturated sodium bicarbonate, extracted with ethyl acetate. The combined organic extracts were washed with brine, dried over Na2SO4, concentrated. The residue was purified by silica gel chromatography to afford white solid (18 mg, 0.033mmol). Yield: 33%; Retention time: 10.791 min, purity: 95.16 %; 1H NMR (500 MHz, MeOD) δ 7.54 (s, 1H), 7.51 (s, 1H), 7.47 (d, J = 8.3 Hz, 1H), 7.29 – 7.24 (m, 2H), 4.57 – 4.48 (m, 1H), 3.92 – 3.83 (m, 1H), 3.75 (s, 3H), 3.68 – 3.62 (m, 2H), 3.45 – 3.32 (m, 6H), 2.40 – 2.31 (m, 1H), 2.23 – 2.10 (m, 2H), 2.11 – 2.03 (m, 1H).

13C

NMR (125 MHz, MeOD) δ

158.40, 147.84, 142.47, 140.21, 138.14, 133.56, 132.35, 131.88, 131.04, 130.78, 128.53, 118.82, 112.12, 112.02, 71.09, 59.34, 53.36, 47.95, 42.39, 40.84, 38.55, 34.95,


86


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28.84. ESI-MS: m/z = 547 [M + H]+. HRMS (ESI) (m/z): calcd for C23H25Cl4N4O3 [M + H]+ 547.0646, found 547.0654. 4.1.56 dichlorophenyl)

5-Chloro-4-(4-chloro-1-methyl-1H-pyrazol-5-yl)-N-((3S,4S,6S)-4-(3,4-6-(2-(dimethylamino)ethyl)piperidin-3-yl)furan-2-carboxamide

hydrochloride (E7) To the solution of compound 45a (71 mg, 0.1 mmol) and DIPEA (260 μL,1.5 mmol) in anhydrous DMF(5mL) was added dimethylamine hydrochloride (80 mg, 1.0 mmol) dropwise.The reaction was heated to 80 oC and stirred for 6h. After fully reaction, the mixture was poured into H2O(10 mL), extracted with DCM(5 mL × 3). The combined organic extracts were washed with brine, dried over Na2SO4, and concentrated. The residue was purified by silica gel chromatography to afford white solid. To a solution of above white solid in ethyl acetate (0.3 mL) was added HCl saturated ethyl acetate (4 mL) at ice-bath, stirred overnight in room temperature. White solid was precipitated, filtered, washed with diethyl ether and dried in vacuum to afford target product as white powder (33 mg, 0.052mmol).Yield: 52%; Retention time: 10.101 min, purity: 95.62 %; 1H NMR (500 MHz, DMSO) δ 10.86 (s, 1H), 9.99 (s, 1H), 9.58 (d, J =


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9.7 Hz, 1H), 9.03 (d, J = 9.2 Hz, 1H), 7.69 (s, 1H), 7.63 (s, 1H), 7.61 – 7.57 (m, 2H), 7.31 (dd, J = 8.4, 1.9 Hz, 1H), 4.62 – 4.51 (m, 1H), 3.81 – 3.71 (m, 4H), 3.51 – 3.44 (m, 1H), 3.30 – 3.17 (m, 4H), 2.81 (d, J = 4.1 Hz, 3H), 2.78 (d, J = 4.1 Hz, 3H), 2.37 – 2.27 (m, 2H), 2.21 – 2.14 (m, 1H), 1.99 – 1.92 (m, 1H).

13C

NMR (125 MHz, DMSO) δ

155.51, 146.72, 142.64, 137.40, 136.68, 130.78, 130.60, 130.16, 129.40, 128.93, 127.79, 117.14, 110.13, 109.32, 52.73, 48.46, 46.20, 42.07, 41.76, 40.61, 38.35, 38.26, 31.59, 22.22. ESI-MS: m/z = 560 [M + H]+. HRMS (ESI) (m/z): calcd for C24H28Cl4N5O2 [M + H]+ 560.0962, found 559.9503. 4.1.57


dichlorophenyl)-6-(2-(pyrrolidin-1-yl)ethyl)piperidin-3-yl)furan-2-carboxamide hydrochloride (E8) General procedure VI, yield: 67%; Retention time: 10.162 min, purity: 95.00 %; 1H NMR (500 MHz, DMSO) δ 11.10 (s, 1H), 9.93 (s, 1H), 9.53 (d, J = 10.4 Hz, 1H), 8.99 (d,

J = 9.2 Hz, 1H), 7.68 (s, 1H), 7.62 – 7.56 (m, 3H), 7.31 (dd, J = 8.4, 2.0 Hz, 1H), 4.60 – 4.50 (m, 1H), 3.84 – 3.77 (m, 1H), 3.74 (s, 3H), 3.48 – 3.26 (m, 4H), 3.25 – 3.12 (m, 2H), 3.06 – 2.99 (m, 2H), 2.35 – 2.28 (m, 2H), 2.20 – 2.13 (m, 1H), 2.07 – 1.87 (m, 6H).

13C


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NMR (125 MHz, DMSO) δ 155.50, 146.70, 142.54, 137.35, 136.63, 130.76, 130.56, 130.10, 129.38, 128.89, 127.76, 117.11, 110.12, 109.29, 55.99, 52.91, 52.68, 50.07, 48.51, 46.25, 40.64, 38.33, 38.21, 31.54, 23.55, 22.68. ESI-MS: m/z = 586 [M + H]+. HRMS (ESI) (m/z): calcd for C26H30Cl4N5O2 [M + H]+ 586.1119, found 586.1138. 4.1.58


dichlorophenyl)-6-(2-(piperidin-1-yl)ethyl)piperidin-3-yl)furan-2-carboxamide hydrochloride (E9) General procedure VI, yield: 46%; Retention time: 10.226 min, purity: 95.03 %; 1H NMR (500 MHz, DMSO) δ 10.75 (s, 1H), 9.94 (s, 1H), 9.55 (s, 1H), 8.97 (d, J = 5.5 Hz, 1H), 7.68 (s, 1H), 7.61 (d, J = 2.0 Hz, 2H), 7.58 (d, J = 8.3 Hz, 1H), 7.32 (dd, J = 8.4, 2.0 Hz, 1H), 4.61 – 4.51 (m, 1H), 3.79 – 3.71 (m, 4H), 3.58 – 3.47 (m, 3H), 3.30 – 3.13 (m, 4H), 2.95 – 2.84 (m, 2H), 2.41 –2.31 (m, 2H), 2.16 (t, J = 12.6 Hz, 1H), 1.97 (d, J = 11.9 Hz, 1H), 1.90 –1.77 (m, 4H), 1.77 – 1.68 (m, 1H), 1.49 – 1.34 (m, 1H).

13C

NMR (125

MHz, DMSO) δ 155.50, 146.70, 142.61, 137.34, 136.63, 130.76, 130.55, 130.11, 129.36, 128.89, 127.75, 117.10, 110.12, 109.29, 52.00, 51.82, 48.63, 46.15, 40.61,


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38.34, 38.21, 31.73, 22.17, 21.74, 21.41. ESI-MS: m/z = 600 [M + H]+. HRMS (ESI) (m/z): calcd for C27H32Cl4N5O2 [M + H]+ 600.1275, found 600.1285. 4.1.59


dichlorophenyl)-6-(2-morpholinoethyl)piperidin-3-yl)furan-2-carboxamide

hydrochloride

(E10) General procedure VI, yield: 55%; Retention time: 10.178 min, purity: 96.14 %; 1H NMR (500 MHz, DMSO) δ 11.48 (s, 1H), 9.89 (s, 1H), 9.50 (s, 1H), 8.94 (s, 1H), 7.68 (s, 1H), 7.62 – 7.56 (m, 3H), 7.32 (dd, J = 8.4, 1.9 Hz, 1H), 4.62 – 4.52 (m, 1H), 4.04 – 3.96 (m, 2H), 3.92 – 3.78 (m, 3H), 3.74 (s, 3H), 3.53 (d, J = 12.0 Hz, 1H), 3.50 – 3.39 (m, 2H), 3.35 – 3.06 (m, 6H), 2.42 – 2.29 (m, 2H), 2.21 – 2.10 (m, 1H), 2.04 – 1.96 (m, 1H). 13C

NMR (125 MHz, DMSO) δ 155.51, 146.69, 142.56, 137.35, 136.63, 130.78, 130.57,

130.13, 129.39, 128.88, 127.73, 117.13, 110.15, 109.29, 63.06, 55.99, 52.10, 50.99, 50.75, 48.51, 46.14, 40.63, 38.33, 38.21, 31.67, 21.31. ESI-MS: m/z = 602 [M + H]+. HRMS (ESI) (m/z): calcd for C26H30Cl4N5O3 [M + H]+ 602.1068, found 602.1105.


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4.1.60

Page 92 of 130


dichlorophenyl)-6-(2-(4-hydroxypiperidin-1-yl)ethyl)piperidin-3-yl)furan-2-carboxamide hydrochloride (E11) General procedure VI, yield: 41%; Retention time: 10.082 min, purity: 95.92 %; 1H NMR (500 MHz, MeOD) δ 7.63 (d, J = 1.5 Hz, 1H), 7.54 (s, 1H), 7.46 (d, J = 8.3 Hz, 1H), 7.38 – 7.34 (m, 1H), 7.33 (s, 1H), 4.64 – 4.56 (m, 1H), 4.17 – 4.10 (m, 1H), 3.90 – 3.83 (m, 1H), 3.75 (s, 3H), 3.74 – 3.68 (m, 1H), 3.57 – 3.48 (m, 3H), 3.46 – 3.33 (m, 4H), 3.22 – 3.11 (m, 1H), 2.68 – 2.56 (m, 1H), 2.47 – 2.36 (m, 1H), 2.27 – 2.11 (m, 4H), 1.99 – 1.81 (m, 2H).

13C

NMR (125 MHz, MeOD) δ 158.35, 147.75, 142.24, 140.30, 138.10,

133.43, 132.33, 131.78, 131.39, 130.80, 128.72, 118.79, 112.03, 111.99, 66.89, 54.39, 52.79, 51.32, 49.63, 42.71, 40.32, 38.57, 33.38, 32.83, 30.98. ESI-MS: m/z = 616 [M + H]+. HRMS (ESI) (m/z): calcd for C27H32Cl4N5O3 [M + H]+ 616.1224, found 616.1239. 4.1.61 N-((3S,4S,6S)-6-(2-(1H-1,2,4-triazol-1-yl)ethyl)-4-(3,4-dichlorophenyl)piperidin3-yl)-5-chloro-4-(4-chloro-1-methyl-1H-pyrazol-5-yl)furan-2-carboxamide

hydrochloride

(E12)


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General procedure VI, yield: 37%; 1H NMR (500 MHz, MeOD) δ 9.51 (s, 1H), 8.63 (s, 1H), 7.59 (d, J = 1.9 Hz, 1H), 7.53 (s, 1H), 7.45 (d, J = 8.3 Hz, 1H), 7.33 (dd, J = 8.3, 1.9 Hz, 1H), 7.32 (s, 1H), 4.76 – 4.69 (m, 1H), 4.67 – 4.57 (m, 2H), 3.96 – 3.90 (m, 1H), 3.75 (s, 3H), 3.50 – 3.38 (m, 3H), 2.82 – 2.70 (m, 1H), 2.65 – 2.56 (m, 1H), 2.32 – 2.22 (m, 1H), 2.22 – 2.14 (m, 1H).

13C

NMR (125 MHz, MeOD) δ 158.39, 151.26, 147.89,

147.74, 142.26, 140.28, 138.11, 133.45, 132.32, 131.80, 131.25, 130.82, 128.78, 118.77, 111.99, 111.97, 51.24, 49.63, 49.28, 42.75, 40.46, 38.60, 33.53, 28.85. ESIMS: m/z = 584 [M + H]+. HRMS (ESI) (m/z): calcd for C24H24Cl4N7O2 [M + H]+ 584.0711, found 584.0722. 4.1.62

2-((2S,4S,5S)-5-(5-Chloro-4-(4-chloro-1-methyl-1H-pyrazol-5-yl)furan-2-

carboxamido)-4-(3,4-dichlorophenyl)piperidin-2-yl)acetic acid hydrochloride (E13) General procedure IV, Yield: 92%; Retention time: 10.688 min, purity: 97.32 %; 1H NMR (500 MHz, MeOD) δ 7.54 (s, 1H), 7.53 (d, J = 1.2 Hz, 1H), 7.47 – 7.42 (m, 1H), 7.29 (d, J = 8.2 Hz, 2H), 4.64 – 4.54 (m, 1H), 4.21 – 4.10 (m, 1H), 3.74 (s, 3H), 3.49 – 3.42 (m, 1H), 3.40 – 3.32 (m, 2H), 3.21 – 3.10 (m, 1H), 3.01 – 2.92 (m, 1H), 2.35 – 2.22 (m, 1H), 2.18 – 2.09 (m, 1H).

13C

NMR (125 MHz, MeOD) δ 173.53, 158.39, 147.77,


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142.22, 140.22, 138.11, 133.52, 132.36, 131.85, 131.10, 130.78, 128.62, 118.81, 112.03, 111.99, 50.47, 49.28, 42.51, 40.77, 38.58, 34.52, 32.93. ESI-MS: m/z = 547 [M + H]+. HRMS (ESI) (m/z): calcd for C22H21Cl4N4O4 [M + H]+ 547.0282, found 547.0296. 4.1.63

N-((3S,4S,6S)-6-(2-Amino-2-oxoethyl)-4-(3,4-dichlorophenyl)piperidin-3-yl)-5-

chloro-4-(4-chloro-1-methyl-1H-pyrazol-5-yl)furan-2-carboxamide hydrochloride (E14) General procedure VII, yield: 79%; Retention time: 10.574 min, purity: 95.65 %; 1H NMR (500 MHz, MeOD) δ 7.55 (s, 1H), 7.53 (s, 1H), 7.46 (dd, J = 8.1, 2.3 Hz, 1H), 7.34 – 7.28 (m, 2H), 4.61 – 4.51 (m, 1H), 4.16 – 4.08 (m, 1H), 3.75 (s, 3H), 3.49 – 3.33 (m, 3H), 3.13 – 3.05 (m, 1H), 2.86 – 2.77 (m, 1H), 2.31 – 2.22 (m, 1H), 2.12 – 2.05 (m, 1H). 13C

NMR (126 MHz, MeOD) δ 174.54, 158.41, 147.78, 142.27, 140.27, 138.11, 133.53,

132.36, 131.85, 131.12, 130.80, 128.60, 118.79, 111.98, 112.00, 50.86, 49.28, 42.22, 40.75, 38.56, 34.66, 32.79. ESI-MS: m/z = 546 [M + H]+. HRMS (ESI) (m/z): calcd for C22H22Cl4N5O3 [M + H]+ 546.0442, found 546.0452. 4.1.64


dichlorophenyl)-6-(2-(methylamino)-2-oxoethyl)piperidin-3-yl)furan-2-carboxamide hydrochloride (E15)


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General procedure VII, yield: 81%; Retention time: 10.683 min, purity: 97.19 %; 1H NMR (500 MHz, MeOD) δ 7.55 (s, 1H), 7.53 (s, 1H), 7.48 – 7.44 (m, 1H), 7.33 – 7.27 (m, 2H), 4.61 – 4.50 (m, 1H), 4.07 – 4.13 (m, 1H), 3.75 (s, 3H), 3.48 – 3.35 (m, 3H), 3.10 – 3.02 (m 1H), 2.83 – 2.83 (m, 4H), 2.33 – 2.22 (s, 1H), 2.09 – 2.03 (m, 1H).

13C

NMR

(125 MHz, MeOD) δ 172.28, 158.41, 147.77, 142.27, 140.28, 138.11, 133.51, 132.35, 131.84, 131.13, 130.81, 128.63, 118.78, 112.03, 112.00, 51.03, 49.29, 42.25, 40.71, 38.57, 34.63, 33.27, 26.35. ESI-MS: m/z = 560 [M + H]+. HRMS (ESI) (m/z): calcd for C23H24Cl4N5O3 [M + H]+ 560.0598, found 560.0612. 4.1.65

5-Chloro-4-(4-chloro-1-methyl-1H-pyrazol-5-yl)-N-((3S,4S,6S)-6-(2-

(cyclopropylamino)-2-oxoethyl)-4-(3,4-dichlorophenyl)piperidin-3-yl)furan-2carboxamide hydrochloride (E16) General procedure VII, yield: 67%; Retention time: 10.727 min, purity: 97.67 %; 1H NMR (500 MHz, MeOD) δ 7.57 – 7.52 (m, 2H), 7.46 (d, J = 7.1 Hz, 1H), 7.34 – 7.26 (m, 2H), 4.60 – 4.51 (m, 1H), 4.13 – 4.08 (m, 1H), 3.75 (s, 3H), 3.48 – 3.33 (m, 3H), 3.05 – 2.97 (m, 1H), 2.77 – 2.70 (m, 2H), 2.32 – 2.20 (m, 1H), 2.09 – 2.02 (m, 1H), 0.79 – 0.70 (m, 2H), 0.61 – 0.52 (m, 2H).

13C

NMR (125 MHz, MeOD) δ 173.19, 158.42, 147.79,


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142.25, 140.26, 138.12, 133.54, 132.39, 131.87, 131.12, 130.79, 128.57, 118.81, 112.04, 112.01, 50.95, 49.29, 42.28, 40.71, 38.56, 34.68, 33.26, 23.45, 6.43, 6.30. ESIMS: m/z = 586 [M + H]+. HRMS (ESI) (m/z): calcd for C25H26Cl4N5O3 [M + H]+ 586.0755, found 586.0768. 4.1.66

5-Chloro-4-(4-chloro-1-methyl-1H-pyrazol-5-yl)-N-((3S,4S,6S)-6-(2-

(cyclobutylamino)-2-oxoethyl)-4-(3,4-dichlorophenyl)piperidin-3-yl)furan-2-carboxamide hydrochloride (E17) General procedure VII, yield: 75%; Retention time: 10.920 min, purity: 98.81 %; 1H NMR (500 MHz, MeOD) δ 7.56 – 7.52 (m, 2H), 7.47 (d, J = 8.2 Hz, 1H), 7.31 (s, 1H), 7.28 (d, J = 8.3 Hz, 1H), 4.56 – 4.50 (m, 1H), 4.39 – 4.32 (m, 1H), 4.11 – 4.05 (m, 1H), 3.75 (s, 3H), 3.46 – 3.33 (m, 3H), 3.05 – 2.97 (m, 1H), 2.75 – 2.68 (m, 1H), 2.34 – 2.19 (m, 3H), 2.07 – 1.96 (m, 3H), 1.81 – 1.69 (m, 2H). 13C NMR (125 MHz, MeOD) δ 170.71, 158.42, 147.80, 142.22, 140.27, 138.13, 133.57, 132.41, 131.89, 131.11, 130.79, 128.55, 118.83, 112.07, 112.02, 50.98, 49.28, 46.00, 42.22, 40.74, 38.56, 34.75, 33.14, 31.43, 31.29, 16.01. ESI-MS: m/z = 600 [M + H]+. HRMS (ESI) (m/z): calcd for C26H28Cl4N5O3 [M + H]+ 600.0911, found 600.0922.


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4.1.67


dichlorophenyl)-6-(2-((2-hydroxyethyl)amino)-2-oxoethyl)piperidin-3-yl)furan-2carboxamide hydrochloride (E18) General procedure VII, yield: 69%; Retention time: 10.758 min, purity: 95.27 %; 1H NMR (500 MHz, MeOD) δ 7.57 – 7.54 (m, 2H), 7.47 (d, J = 8.3 Hz, 1H), 7.31 – 7.28 (m, 2H), 4.49 – 4.43 (m, 1H), 3.99 – 3.93 (m, 1H), 3.75 (s, 3H), 3.64 (t, J = 5.6 Hz, 2H), 3.36 (t, J = 5.6 Hz, 2H), 3.33 – 3.28 (m, 3H), 3.00 – 2.95 (m, 1H), 2.72 – 2.67 (m, 1H), 2.24 – 2.15 (m, 1H), 2.06 – 2.00 (m, 1H).

13C

NMR (125 MHz, MeOD) δ 172.81, 158.41,

147.97, 143.00, 140.13, 138.12, 133.43, 132.11, 131.77, 131.13, 130.84, 128.59, 118.61, 112.01, 112.00, 61.44, 50.82, 49.83, 43.08, 42.99, 41.34, 38.55, 35.70, 34.66. ESI-MS: m/z = 590 [M + H]+. HRMS (ESI) (m/z): calcd for C24H26Cl4N5O4 [M + H]+ 590.0704, found 590.0717. 4.1.68


dichlorophenyl)-6-(2-((2,3-dihydroxypropyl)amino)-2-oxoethyl)piperidin-3-yl)furan-2carboxamide hydrochloride (E19)


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General procedure VII, yield: 47%; Retention time: 10.509 min, purity: 96.16 %; 1H NMR (500 MHz, MeOD) δ 7.53 (s, 1H), 7.50 (d, J = 1.9 Hz, 1H), 7.42 (d, J = 8.3 Hz, 1H), 7.26 (dd, J = 8.3, 1.9 Hz, 1H), 7.22 (s, 1H), 4.32 – 4.26 (m, 1H), 3.75 (s, 3H), 3.73 – 3.69 (m, 1H), 3.64 – 3.58 (m, 1H), 3.50 (d, J = 5.7 Hz, 2H), 3.47 – 3.34 (m, 2H), 3.27 – 3.21 (m, 1H), 3.08 – 3.02 (m, 1H), 3.00 – 2.94 (m, 1H), 2.85 – 2.79 (m, 1H), 2.58 – 2.53 (m, 1H), 2.07 – 2.01 (m, 1H), 1.90 – 1.84 (m, 1H). 13C NMR (125 MHz, MeOD) δ 174.44, 158.41, 148.32, 144.39, 139.85, 138.12, 133.26, 133.21, 131.61, 131.11, 130.91, 128.59, 118.24, 111.98, 111.96, 71.86, 65.09, 51.74, 50.47, 47.93, 44.69, 43.39, 42.50, 38.54, 37.13. ESI-MS: m/z = 620 [M + H]+. HRMS (ESI) (m/z): calcd for C25H28Cl4N5O5 [M + H]+ 620.0810, found 620.0819. 4.1.69


difluorophenyl)-6-(2-hydroxyethyl)piperidin-3-yl)furan-2-carboxamide (E20) General procedure III , yield: 80%; Retention time: 9.945 min, purity: 98.15 %; 1H NMR (400 MHz, MeOD) δ 7.53 (s, 1H), 7.32 – 7.06 (m, 4H), 4.30 – 4.22 (m, 1H), 3.82 – 3.69 (m, 5H), 3.35 (s, 1H), 3.28 – 3.23 (m, 1H), 3.18 – 3.09 (m, 1H), 3.02 – 2.86 (m, 2H), 2.14 – 2.05 (m, 1H), 2.00 – 1.91 (m, 1H), 1.91 – 1.75 (m, 2H).

13C

NMR (100 MHz,


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MeOD) δ 158.36, 151.50 (dd, J = 245.0, 13.0 Hz), 150.38 (dd, J = 244.0, 13.0 Hz), 148.43, 141.78, 139.71, 138.11, 130.92, 125.17, 118.08 (d, J = 16.0 Hz), 118.00, 117.48 (d, J = 17.0 Hz), 111.95, 111.88, 61.31, 52.39, 50.62, 45.16, 42.86, 38.68, 38.52, 33.44. ESI-MS: m/z = 499 [M + H]+. HRMS (ESI) (m/z): calcd for C22H23Cl2F2N4O3 [M + H]+ 499.1110, found 499.1123. 4.1.70


difluorophenyl)-6-(2,3-dihydroxypropyl)piperidin-3-yl)furan-2-carboxamide (E21) General procedure III, yield: 47%; Retention time: 9.935 min, purity: 98.07 %; 1H NMR (500 MHz, MeOD) δ 7.54 (s, 1H), 7.29 (s, 1H), 7.28 – 7.24 (m, 1H), 7.24 – 7.16 (m, 1H), 7.16 – 7.11 (m, 1H), 4.59 – 4.52 (m, 1H), 4.03 – 3.97 (m, 1H), 3.90 – 3.86 (m, 1H), 3.75 (s, 3H), 3.66 – 3.57 (m, 2H), 3.45 – 3.33 (m, 3H), 2.36 – 2.28 (m, 1H), 2.25 – 2.15 (m, 2H), 1.99 – 1.92 (m, 1H).

13C

NMR (125 MHz, MeOD) δ 158.39, 151.60 (dd, J = 245.0,

12.5 Hz), 150.84 (dd, J = 245.0, 12.5 Hz), 147.87, 140.18, 139.30, 138.13, 130.78, 125.33, 118.72, 118.50 (d, J = 16.3 Hz), 117.59 (d, J = 17.5 Hz), 112.06, 112.00, 70.38, 66.62, 51.89, 49.28, 42.41, 40.77, 38.54, 35.90, 32.26. ESI-MS: m/z = 529 [M + H]+. HRMS (ESI) (m/z): calcd for C22H25Cl2F2N4O4 [M + H]+ 529.1215, found 529.1229.


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difluorophenyl)-6-(2-(methylamino)-2-oxoethyl)piperidin-3-yl)furan-2-carboxamide hydrochloride (E22) General procedure VII, yield: 81%; Retention time: 9.842 min, purity: 97.73 %; 1H NMR (400 MHz, MeOD) δ 7.53 (s, 1H), 7.38 – 7.26 (m, 2H), 7.22 – 7.12 (m, 2H), 4.61 – 4.51 (m, 1H), 4.17 – 4.07 (m, 1H), 3.75 (s, 3H), 3.50 – 3.33 (m, 3H), 3.09 – 2.99 (m, 1H), 2.84 – 2.70 (m, 4H), 2.31 – 2.22 (m, 1H), 2.11 – 2.00 (m, 1H).

13C

NMR (100 MHz,

MeOD) δ 172.25, 158.33, 151.52 (dd, J = 245.0, 12.0 Hz), 150.34 (dd, J = 245.0, 13.0 Hz), 147.83, 140.20, 139.04, 138.08, 130.79, 125.45, 118.70, 118.48 (d, J =17.0 Hz), 117.67 (d, J =18.0 Hz), 112.03, 111.97, 51.04, 48.90, 42.27, 40.70, 38.55, 34.87, 33.33, 26.34. ESI-MS: m/z = 526 [M + H]+. HRMS (ESI) (m/z): calcd for C23H24Cl2F2N5O3 [M + H]+ 526.1219, found 526.1229. 4.1.72


difluorophenyl)-6-(2-(methylsulfonamido)ethyl)piperidin-3-yl)furan-2-carboxamide hydrochloride (E23)


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General procedure VIII, yield: 86%; Retention time: 9.939 min, purity: 96.04 %; 1H NMR (400 MHz, MeOD) δ 7.54 (s, 1H), 7.34 – 7.26 (m, 2H), 7.24 – 7.13 (m, 3H), 4.58 – 4.48 (m, 1H), 3.93 – 3.84 (m, 1H), 3.75 (s, 3H), 3.55 – 3.34 (m, 3H), 3.30 – 3.23 (m, 2H), 2.99 (s, 3H), 2.32 – 2.08 (m, 4H). 13C NMR (100 MHz, MeOD) δ 158.36, 151.53 (dd, J = 245.0, 13.0 Hz), 150.47 (dd, J = 245.0, 13.0 Hz), 149.65, 149.52, 147.85, 140.18, 139.09, 138.12, 130.77, 125.40, 118.74, 118.51 (d, J =18.0 Hz), 117.67 (d, J =18.0 Hz), 112.06, 112.00, 51.45, 42.67, 40.60, 40.38, 39.59, 38.54, 34.15, 34.01, 29.82. ESI-MS:

m/z = 576 [M + H]+. HRMS (ESI) (m/z): calcd for C23H26Cl2F2N5O4S [M + H]+ 576.1045, found 576.1050. 4.1.73

N-((3S,4S,6S)-6-(2-Acetamidoethyl)-4-(3,4-difluorophenyl)piperidin-3-yl)-5-

chloro-4-(4-chloro-1-methyl-1H-pyrazol-5-yl)furan-2-carboxamide hydrochloride (E24) General procedure VIII, yield: 78%; Retention time: 10.043 min, purity: 95.35 %; 1H NMR (500 MHz, DMSO) δ 9.67 (t, J = 10.3 Hz, 1H), 9.06 (d, J = 11.0 Hz, 1H), 8.80 (d, J = 9.2 Hz, 1H), 8.18 (t, J = 5.7 Hz, 1H), 7.68 (s, 1H), 7.53 (s, 1H), 7.38 (dd, J = 19.2, 8.6 Hz, 1H), 7.34 – 7.27 (m, 1H), 7.15 – 7.10 (m, 1H), 4.58 – 4.49 (m, 1H), 3.73 (s, 3H), 3.61 – 3.52 (m, 1H), 3.34 – 3.27 (m, 1H), 3.25 – 3.03 (m, 4H), 2.17 – 2.09 (m, 1H), 2.02


100


– 1.92 (m, 3H), 1.84 (s, 3H).

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NMR (125 MHz, DMSO) δ 169.76, 155.52, 149.22 (dd,

J = 243.8, 12.5 Hz), 148.34 (dd, J = 243.8, 13.8 Hz), 146.74, 139.25, 137.30, 136.65, 128.90, 124.29, 117.48 (d, J =17.5 Hz), 117.17, 116.45 (d, J =17.5 Hz), 110.23, 109.31, 49.15, 46.47, 40.77, 38.57, 38.24, 34.98, 32.11, 27.57, 22.60. ESI-MS: m/z = 540 [M + H]+. HRMS (ESI) (m/z): calcd for C24H26Cl2F2N5O3 [M + H]+ 540.1375, found 540.1395. 4.1.74

2-((2S,4S,5S)-5-(5-Chloro-4-(4-chloro-1-methyl-1H-pyrazol-5-yl)furan-2-

carboxamido)-4-(3,4-difluorophenyl)piperidin-2-yl)ethyl acetate hydrochloride (E25) To the solution of compound 44b (60 mg, 0.1 mmol) and DIPEA (62 μL,0.3 mmol) in THF(5 mL) was added acetylchloride (23 μL, 0.20 mmol) at ice bath.The mixture was stired at RT for 4h. After fully reacted, it was poured into saturated NaHCO3 (5 mL) and extracted with ethyl acetate (5 mL × 3). The combined organic extracts were washed with brine, dried over Na2SO4, and concentrated. The residue was purified by silica gel chromatography to afford white solid. To a solution of above solid in ethyl acetate(0.3mL) was added HCl saturated ethyl acetate(3mL), stirred overnight in room temperature. Then, the reaction was added saturated sodium bicarbonate (8mL), extracted with ethyl acetate (5mL×3). The


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combined organic extracts were washed with brine, dried over Na2SO4, concentrated to afford white solid ( 38.4 mg, 0.071mmol). Yield: 71%; Retention time: 10.802 min, purity: 95.31 %; 1H NMR (400 MHz, MeOD) δ 7.52 (s, 1H), 7.35 – 7.05 (m, 4H), 4.42 – 4.08 (m, 3H), 3.74 (s, 3H), 3.25 – 3.08 (m, 2H), 3.03 – 2.94 (m, 1H), 2.92 – 2.81 (m, 1H), 2.20 – 2.12 (m, 1H), 2.04 (s, 3H), 2.00 – 1.89 (m, 2H), 1.88 – 1.80 (m, 1H).

13C

NMR (100

MHz, MeOD) δ 172.91, 158.34, 151.50 (dd, J = 244.0, 12.0 Hz), 150.40 (dd, J = 243.0, 13.0 Hz), 148.44, 141.67, 139.69, 138.11, 130.91, 125.16, 118.09 (d, J = 18.0 Hz), 118.00, 117.48 (d, J = 17.0 Hz), 111.94, 111.89, 63.49, 52.41, 49.79, 45.15, 42.75, 38.52, 38.30, 30.36, 20.83. ESI-MS: m/z = 541 [M + H]+. HRMS (ESI) (m/z): calcd for C24H25Cl2F2N4O4 [M + H]+ 541.1215, found 541.1223. 4.2 Akt1 inhibitory activity assay The Akt1 inhibitory activities were determined using the homogeneous time-resolved fluorescence (HTRF) KinEASE-STK S1 kit (Cat.#62ST2PEC, Cisbio) according to the manufacturer’s instructions. Akt1 kinase was expressed and purified from Escherichia coli system in-house. In short, the kinase reactions were carried out in a 384 ProxiPlate with 10 µL reaction volume per well containing 0.25 µg/mL Akt1, 1 µM peptide


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substrate, test compound and 20 µM ATP in assay buffer (50 mM HEPES pH 7.5, 0.01% BRIJ-35, 5 mM MgCl2, 1 mM EGTA). After incubation for 1 h at r.t., the reaction was stopped by the addition of 5µL Sa-XL665 and 5 µL STK Antibody-Eu(K) in EDTA. The plate was sealed and incubated for 1 h at r.t., and the resulting TR-FRET signal was measured on Envision-PerkinElmer. The fluorescence emission was measured at 615 nm (cryptate) and 665 nm (XL665). An Emission Ratio was calculated (665/615) for each well and the Percent inhibitions were expressed as follows: Percent inhibition = (max-sample Ratio)/ (max-min)*100 (“Min” means the Ratio of no enzyme control and “max” means the Ratio of DMSO control). Compounds were initially tested at a fixed concentration (1 µg/mL), and those displaying more than 50% inhibition were further tested for dose–response IC50 values. 4.3 Kinase selectivity assay The representative compounds were assayed against a panel of 22 protein kinases at SHANGHAI CHEMPARTNER CO.LTD. All the proteins and kinase assay kits were mainly purchased from Carna, Invitrogen or Millipore. Compounds was tested at concentrations of 0.5 and 10 µM over most kinases, including ABL, ALK, Akt2, Akt3,


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JNK2, JAK2, ROCK1, RSK1, P70S6K, MSK1, SGK, PDK1 and PRKG1, using Mobility shift assay with ATP concentration at Km. PI3Kα inhibitory activity was screened by Kinase-Glo Luminescent Assay kit (Promege) with ATP concentration at 25µM. CHK1 and TSSK1 inhibitory activities were determined using the Luminescent ADP-Glo assay kit (Promege). In addition, the inhibition of mTOR activity was evaluated in a Lance Ultra assay. Moreover, PKA, GSK3β and CDK2/cyclin A activities were evaluated at the concentration of 1 µg/mL by Z-LYTE Kinase Assay kits (Invitrogen). Like Akt1 assay, the Aurora A and BRAF inhibitory activities of compounds were measured by the HTRF Kinase Assay kits purchased from Cisbio. The protocols for these assays could be found in the manufacturer’s instructions. Compound E22 was assayed against a panel of 468 protein kinases at DiscoverX Co. Kinase-tagged T7 phage strains were grown in parallel in 24-well blocks in an E. coli host derived from the BL21 strain. E. coli were grown to log-phase and infected with T7 phage from a frozen stock (multiplicity of infection = 0.4) and incubated with shaking at 32 °C until lysis (90-150 minutes). The lysates were centrifuged (6,000 x g) and filtered (0.2µm) to remove cell debris. The remaining kinases were produced in HEK-293 cells


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and subsequently tagged with DNA for qPCR detection. Streptavidin-coated magnetic beads were treated with biotinylated small molecule ligands for 30 minutes at room temperature to generate affinity resins for kinase assays. The liganded beads were blocked with excess biotin and washed with blocking buffer (SeaBlock (Pierce), 1 % BSA, 0.05 % Tween 20, 1 mM DTT) to remove unbound ligand and to reduce nonspecific phage binding. Binding reactions were assembled by combining kinases, liganded affinity beads, and test compounds in 1x binding buffer (20 % SeaBlock, 0.17x PBS, 0.05 % Tween 20, 6 mM DTT). E22 was prepared as 40x stocks in 100% DMSO and directly diluted into the assay (1000 nM). All reactions were performed in polypropylene 384-well plates in a final volume of 0.02 mL. The assay plates were incubated at room temperature with shaking for 1 hour and the affinity beads were washed with wash buffer (1x PBS, 0.05 % Tween 20). The beads were then resuspended in elution buffer (1x PBS, 0.05 % Tween 20, 0.5 µM non-biotinylated affinity ligand) and incubated at room temperature with shaking for 30 minutes. The kinase concentration in the eluates was measured by qPCR. The results for primary screen


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binding interactions are reported as % Ctrl = (test compound signal – positive control signal)/(negative control signal – positive control signal)×100. 4.4 Cell culture Human ovarian carcinoma cells (SKOV3, OVCAR8), Human prostate cancer cells (LnCap), human colon cancer cells (HCT 116) and Human umbilical vein endothelial cells (HUVEC) were obtained from the Cell Bank of the China Science Academy, Shanghai and were cultured in 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% to 80% confluency in dishes or cell culture plates and treated under various conditions as indicated. 4.5 Anti-proliferative assay SKOV3 and HCT-116 cells were treated with the compounds at various doses for 72h, and cell proliferation was tested by a sulforhodamine B (SRB) protein assay (Sigma, S1402). 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 was washed away with 1% glacial


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acetic acid, and 100 μ l 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, USA). 4.6 Western blot assay Whole protein extracts from cultured cells or tissues were prepared and subjected to western blotting. Protein samples were size-fractionated by 10% SDS-PAGE, analysed by immunoblotting and visualized by enhanced chemiluminescence (ECL, Amersham Biosciences, Castle Hill, Australia) and then exposed on X-ray film. Proteins in the lysates were equalized and then analyzed by specific antibodies. The primary antibodies were listed as follows: AKT (CST, 9272S), pAKT-Thr308 (Santa, sc-16646), pAKT-Ser473 (Santa, sc-7985), PRAS40 (CST, 2691P), p-PRAS40 (CST, 13175P), GSK-3β (Santa, sc-7291), p-GSK-3β (CST, 8566S), β-Actin (Santa, SC-1615). Appropriate secondary antibodies and ECL were used to visualize the protein signaling. 4.7 Flow cytometry SKOV3 cells were seeded in 6-well dish plates at 1×105 cells/well and exposed to different concentrations of E22. After 72 h, the cells were harvested and stained with


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Annexin-V/PI Solution using the FITC Annexin V Apoptosis Detection Kit I (BD Bioscience, 556547) for 10–15 min and resuspended in binding buffer. Then the fluorescence emission at 530 nm and 585 nm using 488 nm excitation were measured by flow cytometry (BD FACSCalibur). 4.8 ELISA assay ELISA plates were prepared by coating with anti-PRAS40 antibody (R&D Systems), and blocked with 5% Milk/0.1% Tween-20. LNCaP Cells were seeded at 25,000 cells/96-well overnight and treated with DMSO or various concentrations of compounds for 1 h. Subsequently, cells were lysed in 20 mM Tris-HCl (pH 8.0), 137 mM NaCl, 2 mM EDTA, 10% glycerol and 1% Triton X-100 and lysates transferred to ELISA plates and incubated overnight. The plates were washed and incubated with rabbit antiPRAS40 (pThr246) antibody (R&D Systems) for 1 h. After washing, plates were developed using HRP-linked anti-rabbit IgG, and 3,39,5,59-tetramethylbenzidine as substrate. Absorption was measured in a microplate spectrophotometer at 450 nm, Dose-response curves were generated using the four-parameter logistic model, and 50% inhibitory concentration (IC50) values were determined from these curve fits.


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4.9 Migration and Invasion Assay HUVEC cells were cultured in 6-well dish plates at a number of 3×105 cells/well and grown overnight to confluence. A wound was created by scratching a straight line in the monolayer with a 200 μL pipet tip. The cells were then incubated with E22 in serum-free medium for 36 h and the wound area was then photographed using a light microscope (Leica, DMI3000B). The rate of wound closure was assessed by measuring distances from 6 randomly selected fields. Transwell invasion assays were performed using BD BioCoat matrigel invasion chambers (BD Biosciences, 354480) in 24-well cell culture plates following the manufacturer’s instruction. After 36 h, cells that had migrated through the Matrigel to the underside of the transwell filters were fixed in 4% paraformaldehyde, stained with 0.5% crystal violet, and counted under a light microscope in 6 random fields. Stained cells were further quantified by measuring the absorbance of dissolved crystals in 0.1% SDS at 540 nm. 4.10 hERG inhibition assay


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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): K Aspartate, 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 mV to +40 mV for 4 s followed by a step to 40 mV for 2 s. The procedure was repeated every 20 seconds. After the maximum current was stabilized, the tested compounds (3 μM) were perfused. The inhibition rate was calculated when the current was stable. All data represent at least 3 independent experiments (n = 3). 4.11 Liver microsome stability assay 1 mg/mL microsome solution (purchased from Ruide Research Institute for Liver Diseases (Shanghai) Co. Ltd) was mixed with 20 mL of 50 mM NADPH (Aladdin) solution to prepare a microsome-NADPH solution. 500 µL of the microsome-NADPH solution was pre-warmed at 37 oC for 5 minutes. 5 µL of a 100 µg/mL test article solution was then added to initiate the reaction. The incubation mixture was kept at 37


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and 100 µL aliquots were taken at 0, 15, and 45 minutes. In each aliquot, the

reaction was quenched using 400 µL of methanol containing 1 µg/mL internal standard compound (from the in-house database). After quenching, the mixtures were vortexed and centrifuged. The supernatant was transferred and 10 µL was injected into an API4000 + LC/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. 4.12 Plasma stability assay A 500 µL aliquot of rat plasma or human plasma (purchased from Shanghai Yuduo Biotechnology Company) was added to 5 µL of a test article solution at 100 µg/mL to give a final solution. The mixture was incubated at 37 oC with gentle agitation. An aliquot of 100 µL of the reaction mixture was taken at 0, 15, and 45 minutes and quenched using 400 µL of methanol containing 1 µg/mL internal standard compound (from the in-house database). After quenching, the mixture was vortexed and centrifuged and 10 µL of the resulting solution was injected into an API4000 + LC/MS system. The percentage remaining of the test article at the incubation times of 15- and


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45-minutes relative to that at 0 minutes was calculated using the peak area ratio of the test article versus the internal standard. 4.13 Plasma protein binding rate assay A 300 μL mouse plasma containing 300 ng/mL E22 was added to the sample chamber of Thermo Equilibrium Dialysis Device (Red Device Insert), and 500 μL PBS dialysate was added to the buffer chamber. After sealing, the mixture was incubated at 37 oC for 8h. An aliquot of 50 µL of each mixture was quenched using 500 µL of acetonitrile containing 0.5 ng/mL internal standard compound (Loratadine). After quenching, the mixture was vortexed and centrifuged, the resulting solution was injected into a Waters XEVO TQ-S system. The PPB % = (1-concentration in PBS/concentration in plasma) ×100. The PPB rate was an average of three independent determinations. 4.14 Pharmacokinetic studies This study was performed in strict accordance with the Laboratory Animal Management Regulations (State Scientific and Technological Commission Publication No. 8-27 Rev. 2017) and was approved by Zhejiang University Laboratory Animal Center (Hangzhou, China). SD rats or ICR mice (purchased from Zhejiang Academy of


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Medical Sciences) were administered compound by oral gavage in saline. As for the evaluation of compound A12 and E22 in mice, venous blood (100 µL) samples were collected at 0, 0.167, 0.5, 1, 2, 3, 4, 8, 24 and 48 h for oral gavage or 0, 0.083, 0.25, 0.5, 1, 2, 4, 8, 24 and 48 h for intravenous injection. As for the evaluation of compound E20 and E22 in rats, venous blood (100 µL) samples were collected at 0, 0.5, 1, 2, 4, 8, 12 and 24 h. Plasma was separated from whole blood by centrifugation and stored at 20 oC until analysis. Compound levels were determined using a Waters Xevo TQ LCMS/MS system. The Cmax, Tmax, t1/2 and AUC were evaluated using Analyst 1.5.1. 4.15 Xenotransplantation BALB/c (nu/nu) mice (National Rodent Laboratory Animal Resource, Shanghai), 6-8 weeks of age, were used for all experiments. When MM1S xenograft tumours reached 100 mm3, mice were randomly divided into five groups and received A12 (10 mg/kg) orally once daily for 22 days. Other mice were orally administered normal saline as controls for 22 days. When SKOV3 xenograft tumours reached 100 mm3, mice received E22 (40,80,40+40 mg/kg) orally once daily for 21 days. Other mice were orally administered normal saline as controls for 21 days.


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Statements of Ethical Approval: The animal studies were approved by the Institutional Animal Care &Use Committee (IACUC) at Zhejiang University, and all protocols were conducted in accordance with institutional guidelines. 4.16 Toxicology evaluation ICR mice (National Rodent Laboratory Animal Resource, Shanghai), 6-8 weeks of age, were used for this experiment. Mice were randomly divided into six groups and received A12 (10 mg/kg, 20 mg/kg, 40 mg/kg, i.g.), E22 (80 mg/kg, 160 mg/kg, i.g.) or vehicle (saline, i.g.) once daily for 7 days. The body weight of mice was recorded once daily. At the end of the experiment, mice were sacrificed, the serum hematological parameters and serum biochemistry parameters were recorded on Sysmex XT-2000i and Roche Cobas c 311, respectively. Statements of Ethical Approval: The animal studies were approved by the Institutional Animal Care &Use Committee (IACUC) at Zhejiang University, and all protocols were conducted in accordance with institutional guidelines. 4.17 Molecular docking and dynamic simulation


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The molecular docking was performed using Ligand Docking implanted in Maestro. Parameters were maintained at the default configuration. The docked structure of compound E22 complexed within the active pocket of 4GV1 was used as the initial structures for MD calculations using Discovery Studio 2.5. A CHARMm force field was applied to the complex and the resulting system was subjected to double-fold minimization (10,000 cycles of steepest descent minimization and 10,000 cycles of conjugate gradient minimization). The system was heated from 50 K to 300 K over a period of 4 ps and subsequently equilibrated for 200 ps. Starting from the last frame of the equilibration, a production simulation was performed for 4000 ps using the NPT ensemble under a constant temperature of 300 K and pressure of 1 atm. Other parameters of MD simulation were maintained at the default Discovery Studio configuration.

ASSOCIATED CONTENT

Supporting Information. The Supporting Information is available free of charge on the ACS Publications website at DOI:


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Figure S1 showing the proposed binding mode of GSK-795 with Akt protein; Figure S2 showing the synthesis and chemical information of compound (3R,4S)-A6; Figure S3 showing the proposed binding mode of GSK-795 with Akt protein; Figure S4 showing the in vivo anti-tumor effect of compound A12; Figure S5 showing the comparison of the selectivity profile between compound E22 and GSK-795; Figure S6 showing the synthetic route of acid fragments. Figure S7 showing the analysis of the configurations. Table S1 listing the effect of A12 and E22 on organ-to-brain weight ratio during 7 days repeated dose toxicity study; Table S2 listing the inhibition rate of compound E22 against different ion channels; Table S3 listing the selectivity profile of compound E22; Compound information for the intermediates; NMR spectra and HPLC results for the target compounds. Molecular formula strings (CSV) Accession Codes. PDB ID code 4GV1 was used. The authors will release the atomic coordinates upon article publication for the models between compound E22 with Akt1, which was built on 4GV1. AUTHOR INFORMATION


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Corresponding Author *

Corresponding authors: Q. Weng: [email protected]; Y. Hu: [email protected]

Present Address: #

W. Zhan: Department of Microbiology & Immunology, Weill Cornell Medicine, 1300

York Avenue, New York, NY 10065, United States.

Author Contributions X.D. and W.Z. conceived the study, synthesized the compounds and analyzed the data. M.Z., X.D., Q.W., L.X., Y.Z. and Y.S. performed the biological experiments and analyzed the data, J.C. synthesized the compounds and drafted the manuscript, Y.W., Y.Z., T.T. and Z.J. synthesized the compounds, G.C. performed the docking calculation, J.L., B.Y., Q.H. and Y.H. conceived the study. ‡ X.D., W.Z. and M.Z. contributed equally to this work.

Funding Sources National Natural Science Foundation of China; Zhejiang Provincial Natural Science Foundation of China; the Health Bureau of Zhejiang Province; Zhejiang Provincial


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Natural Science Foundation of China; National Major Scientific and Technological Special Project for “Significant New Drugs Development”.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT We 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 grant from the National Natural Science Foundation of China (81673294, 81872878, 81741172), Zhejiang Provincial Natural Science Foundation of China (LGF18H310001, LGF19H310002), the Health Bureau of Zhejiang Province (2015RCB002), National Major Scientific and Technological Special Project for “Significant New Drugs Development” (2018ZX09711002-007, 2018ZX09711002-011-023).

ABBREVIATIONS

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hERG, human ether-a-go-go related gene; MTD, maximum tolerated dose; PKA, protein kinase A; TNBC, triple-negative breast cancer; PK, pharmacokinetic; WBC, white blood cell; RBC, red blood cell; HGB, Hemoglobin; HCT, Red blood cell specific volume; PLT, blood platelet; ALT, Alanine aminotransferase; AST, Aspartate aminotransferase; CREA, Creatinine; RMSD, root-mean-square deviation; NBS, NBS, N-Bromo Succinimide; NCS, N-Chloro Succinimide; TFA, Trifluoracetic acid; TEA, Triethylamine; HOBt, 1-hydroxybenzotriazole; EDCI, 1-ethyl-3-(3'-dimethylaminopropyl) carbodiimide hydrochloride;

DIPEA,

N,N-diisopropylethylamine;

EA,

Ethyl

acetate;

DCM,

dichloromethane; DBU, 1,8-diazabicyclo[5.4.0]undec-7-ene; TFA, trifluoroacetic acid; THF, tetrahydrofuran; NMO, N-methylmorpholine N-oxide; DMF, dimethylformamide.

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238x67mm (300 x 300 DPI)


Discovery of 3,4,6-Trisubstituted Piperidine Derivatives as Orally

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