Structure-based Discovery and Development of a Series of

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Structure-based Discovery and Development of a Series of Potent and Selective Bromodomain and Extra-Terminal Protein Inhibitors Jianping Hu, Changqing Tian, Mohammadali Soleimani Damaneh, Yanlian Li, Danyan Cao, kaikai Lv, Ting Yu, Tao Meng, Danqi Chen, Xin Wang, lin Chen, jian li, Shanshan Song, Xiajuan Huan, Lihuai Qin, Jingkang Shen, Yingqing Wang, Ze-Hong Miao, and Bing Xiong J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b01094 • Publication Date (Web): 06 Sep 2019 Downloaded from pubs.acs.org on September 6, 2019

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Structure-based Discovery and Development of a Series of Potent and Selective Bromodomain and Extra-Terminal Protein Inhibitors Jianping Hu1,4,#, Changqing Tian2,4,#, Mohammadali Soleimani Damaneh2,4, Yanlian Li1, Danyan Cao1, Kaikai Lv1,4, Ting Yu1, Tao Meng1, Danqi Chen1, Xin Wang1, Lin Chen1, Jian Li,1 Shanshan Song2, Xiajuan Huan2, lihuai Qin3, Jingkang Shen1, Yingqing Wang2,4,*, Zehong Miao2,4,*, Bing Xiong1,4,* 1Department

of Medicinal Chemistry, Shanghai Institute of Materia Medica, Chinese Academy of

Sciences, 555 Zuchongzhi Road, Shanghai 201203, China 2Division

of Anti-tumor Pharmacology, State Key Laboratory of Drug Research, Shanghai

Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203, China 3Center

for Chemical Biology and Drug Discovery, Department of Pharmacological Sciences,

Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY10029, United States 4University

#These

of Chinese Academy of Sciences, NO.19A Yuquan Road, Beijing 100049, China

authors contributed equally

*Corresponding

authors. Tel: +86 21 50806600 ext. 5412 fax: +86 21 50807088. Email: (B. X.)

[email protected]; (Z. M.) [email protected]; (Y. W.) [email protected]


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ABSTRACT BRD4 has recently emerged as a promising drug target. Therefore, identifying novel inhibitors with distinct properties could enrich their use in anticancer treatment. Guided by the cocrystal structure of hit compound 4 harboring a five-member-ring linker motif, we quickly identified lead compound 7, which exhibited good anti-tumor effects in a MM.1S xenograft model by oral administration. Encouraged by its high potency and interesting scaffold, we performed further lead optimization to generate a novel potent series of bromodomain and extraterminal (BET) inhibitors with a (1,2,4-triazol-5-yl)-3,4-dihydroquinoxalin-2(1H)-one

structure.

Among

them,

compound 19 was found to have the best balance of activity, stability, and anti-tumor efficacy. After confirming its low brain penetration, we conducted comprehensive preclinical studies, including a multiple species pharmacokinetics profile, extensive cellular mechanism studies, hERG assay, and in vivo anti-tumor growth effect testing, and we found that compound 19 is a potential BET protein drug candidate for the treatment of cancer.

Keywords: Bromodomain; (1,2,4-triazol-5-yl)-3,4-dihydroquinoxalin-2(1H)-one; blood-brain barrier penetration; five-membered-ring linker



INTRODUCTION Bromodomain-containing proteins (BCPs) can decipher the acetylation code of proteins via binding to their acetylated lysine (KAc) residues, which further modulates acetylation signal transduction and produces diverse physiological functions.1-3 In many diseases, and best studied in cancers, BCPs are found to be aberrantly expressed, and they further enhanced the expression of oncogenes and anti-apoptotic proteins.4,

5

Thus, BCPs have emerged as attractive epigenetic

therapeutic targets for cancer.6, 7 There are 61 bromodomains distributed in 46 distinct proteins encoded in the human genome, and these bromodomains can be divided into 8 subfamilies.8 Among them, the most studied is the Bromodomain and Extra-Terminal domain (BET) subfamily, which includes the following four members: BRD2, BRD3, BRD4, and BRDT.9 Since the first BET inhibitor, (+)-JQ1 (1),10 was discovered, diverse BET inhibitors have arisen, with some of them entering into clinical trials, including OTX-015 (2),11, 12 I-BET762 (3),13, 14 CPI061015 and ABBV-075,16, 17 to name a few. However, the cellular functions of BET proteins, specifically BRD4, are more complex than previously thought.18 As an epigenetic reader, BRD4 was initially believed to recognize the acetylated lysine in H4 with its two N-terminal bromodomains, further recruiting the transcription elongation factor b (p-TEFb) cyclin T1/CDK9 complex to phosphorylate RNA polymerase II (RNA pol II) to activate gene transcription.19 The profound downstream effect of this event was initially attributed to a reduction in the expression of the oncogene Myc, which


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encodes the multipurpose Myc protein related to cell growth and differentiation.20 Since directly targeting Myc protein was very difficult, targeting BRD4 to interrupt p-TEFb binding to histones was considered as a surrogate for Myc-related cancer treatment. Recently, with the advance of sequencing technologies, many other cellular roles of BRD4 have been revealed from transcriptome and epigenome studies. In addition to binding to acetylated H4, BRD4 can also act as a critical component of the superenhancer complex by binding to many transcription factors, including Myc, TWIST,21 CEBPA, and CEBPB,22 and the transcription-related protein RelA.23 Meanwhile, new downstream effects have been discovered; for example, BET protein inhibition can promote anti-tumor immunity by suppressing PD-L1 expression.24 As nontranscriptional roles, BRD4 has also been shown to control DNA damage checkpoint activation and repair as well as telomere maintenance.25 Using (+)-JQ1 as a chemical probe, BRD4 was identified to have an essential cognitive function in the brain, and BRD4 inhibition in the brain could impair memory abilities.26 These recent studies on the different functions of BRD4 reinforced the argument of Andrieu et al. that clinical trials for BET inhibitors should design with caution, as currently we are still in the primary phase of understanding the roles of BRD4.27 N N

S

N

N

O O

N

S

N

O NH

N

N

OH

Cl (+)JQ-1, 1

Cl OTX-015, 2

Figure 1. Structures of typical BET bromodomain inhibitors including (+)-JQ1



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(1), OTX-015 (2), I-BET762 (3), and hit compound 4. Nevertheless, the important role of BRD4 in cancer biology and partial successes of clinical trials with BET inhibitors28,

29

have spurred researchers to pursue new

inhibitors with novel scaffolds, as they may bring distinct values with different pharmacological effects or tissue distribution. Researchers recently discovered previous patents that described a series of dihydroquinoxalin-2(1H)-ones with substituted or unsubstituted nitrogen or oxygen linkers as BET inhibitors.30,

31

Moreover, researchers such as Conway et al. revealed a series of potent CREBBP bromodomain inhibitors with a dihydroquinoxalinone scaffold, and they emphasized the importance of the internal hydrogen bond, which is formed from the scaffold amine NH with the side-chain carbonyl.32 Meanwhile, we previously incrementally mapped

out

some

novel

BRD4-selective

inhibitors

with

a

dihydroquinoxalin-2(1H)-one scaffold that were derived from the dual PLK1-BRD4 inhibitor BI-2536.33-35 However, they did not possess comprehensive drug-like properties. Furthermore, we found that others had also tried to tune the selectivity and potency of BI-2536; for example, Watts et al. designed a series of anaplastic lymphoma kinase (ALK) and BRD4 dual inhibitors from BI-253636 and Liu et al. described a series of compounds with different selectivity between PLK1 and BRD4 using BI-2536 as starting point.37 Now, in this research, by preserving the core dihydroquinoxalin-2(1H)-ones modified from BI2536, we explored new side-chains and discovered novel hit compound 4 (Figure 1). Guided by crystal structures, we were able to discover a


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potent and selective inhibitor (19) as a potential drug candidate. The extensive profile of this compound showed that it displayed high selectivity for the BET subfamily over other bromodomains and more than twenty kinases from different families, possessed favorable metabolic stability in multiple species, showed a low blood-brain barrier penetration ratio and good PK properties in rodents and dog, and demonstrated excellent in vivo antitumor efficacy. Hereinafter, we report on the optimization of this potential drug candidate as well as comprehensive profiles of its in vitro and in vivo properties.

RESULTS AND DISCUSSION Hit to Lead Based on the BET-selective inhibitors reported in our previous work,33,

34

we

wanted to further improve their in vitro and in vivo properties to develop a drug candidate

for

clinical

research.

We

initially

preserved

the

core

dihydroquinoxalin-2(1H)-one and screened for new side chains, finding that compound 4 with a pyrrole ring had moderate activity (IC50 = 303.0 nM) and a low molecular weight that could be used as a starting point. Superimposition of the cocrystal structures of a reported BET inhibitor (5)38 in BRD4-BD2 to our crystal structure of 4 bound to BRD4-BD1 (Figure 2) found that there was a large space for modification around the pyrrole ring. From our previous study,39 we believed that the ortho position could be extended to reach the WPF subpocket to improve the potency and selectivity. Hence, we added a hydrophobic group (e.g., phenyl or p-methyl



phenyl) to this position and synthesized different five-membered ring linkers to generate a series of dihydroquinoxalin-2(1H)-one compounds (6-12, Table 1). The FP binding assay showed that they all had excellent potency, indicating this hydrophobic group may enter the WPF subpocket.

Figure 2. Superimposition of the cocrystal structure of BRD4 BD1 bound with 4 (PDB entry: 6JI3, green color) and BRD4-BD2 bound with compound 5 (PDB entry: 4Z93, gray color).

As listed in Table 1, we kept R1 as a racemic methyl or (R)-methyl, R2 as cyclopropyl or cyclopentyl and R3 as methyl or hydrogen to explore the five-membered ring linker. A comparison of compound 6 with 7 found that the addition of a methyl group to the triazole could enhance the molecular and cellular activity by 1.5-fold and 2.0-fold, respectively. By substituting the triazole group with a tetrazole ring, we found that the molecular binding activities were only slightly decreased (6 versus 8 and 10 versus 11); however, the cellular anti-proliferative activities were dramatically reduced, indicating that the more hydrophilic tetrazole


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may decrease the membrane permeability of the compounds. Replacement of the triazole with thiazolidinone or methylisoxazole resulted in a significant loss of activity (6 versus 9 or 10 versus 12). These results indicated that the methyl position of the five-member ring may be surrounded by small hydrophobic residues and could not tolerate the polar nitrogen or carbonyl group. Only by changing R2 cyclopropyl group to a cyclopentyl group could maintain the molecular activity; in contrast, the cellular activity was dependent on the overall molecule properties and could be retained (7 and 10) or weakened (8 and 11). These results indicated that the nature of the five-member ring linker and R2 group significantly affected their activities; in the future, perhaps we can change the linker to a pyrazole, pyrrole, or six-member linker such as phenyl or pyridine to explore more potent compounds. In short, compounds 6, 7 and 10 were found to have good molecular and cellular activities with IC50 values below 10 nM, which were worth further exploration. Table 1. Inhibitory activity and MM.1S cell proliferation inhibitory activity results of compounds 6-12 on BRD4 (I).

Linker

N

O

N R2

R1

R3

Cpd

(+)-JQ-1

R1

-

R2

-

R3

-

Linker

-


BRD4(I)

MM.1S

IC50 (nM)a

IC50 (nM)b

28.5 ± 1.5

29.8 ± 16.6


OTX-015

-

6

Me

7

Me

8

Me

9

Me

10

11

12

a



Me



-















-

-

Page 10 of 88

34.3 ± 6.5

6.3 ± 13.5

Me

8.6 ± 1.0

6.7 ± 0.3

Me

5.7 ± 0.2

3.3 ± 1.2

Me

14.4 ± 0.9

47.7 ± 25.1

Me

142.0 ± 3.0

N.D.c

Me

5.2 ± 1.6

3.9 ± 3.0

Me

9.5 ± 1.2

1228.7 ± 376.0

H

100.4 ± 6.4

N.D.c

The IC50 values are the mean of two independent experiments performed in duplicate with

standard error of the mean (SEM) values.

b

The IC50 values are the mean of three independent

experiments performed in triplicate with standard error of the mean (SEM) values. c N.D. means not determined.

According to the molecular and cellular activities, compounds 6, 7 and 10 were selected for in vitro metabolic stability evaluation (Table S2). We found that compounds 6 and 7 had better metabolic stabilities than compound 10 in both human and mouse liver microsomes. At 10 μM, compound 6 showed evident inhibition of most of the tested CYP450 enzymes; in contrast, compounds 7 and 10 exhibited a


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weak inhibitory effect on the CYP450 enzymes, except that compound 10 showed a more than 50% inhibition ratio for the 2C9 enzyme. Additionally, none of the compounds exhibited time-dependent inhibition of CYP450 enzymes, indicating less vulnerability of these compounds during drug-drug interactions. Considering the liver microsomal metabolic stability, we chose compounds 6 and 7 for in vivo pharmacokinetics (PK) study (Table 2). Pharmacokinetic tests demonstrated that compound 7 indeed possessed better plasma exposure after oral administration at a 3 mg/kg dosage than compound 6 at a 10 mg/kg dosage, suggesting that compound 7 could be advanced to an in vivo pharmacological study. Table 2. In vivo PK data for compounds 6 and 7 in male ICR mice.a Route of

Dose

Tmax

Cmax

AUC0-t

AUC0-inf

T1/2

administration

(mg/kg)

(h)

(ng/mL)

(ng·h/mL)

(ng·h/mL)

(h)

6

p.o.

10

0.3

163

161

164

1.5

7

p.o.

3

0.3

265

243

244

0.7

Cpd.

a1%

DMSO/0.5% HPMC was used as the vehicle.

The in vivo therapeutic effects of compound 7 were evaluated in a human MM.1S xenograft model. Tumor-bearing SCID mice were treated with 7 or the phase II BET inhibitor I-BET762 by oral administration at a dosage of 20 mg/kg daily. The results showed that treating with compound 7 demonstrated good efficacy on tumor growth inhibition with a T/C value of 67.4% (Figure S1). These encouraging results demonstrated that this new series of compounds bearing a novel triazole linker



deserved further optimization.

Lead to Candidate The good in vivo antitumor efficacy of lead compound 7 indicated that this class of compounds should be advanced for further exploitation. However, the moderate plasma exposure of compound 7 and its instability in mouse liver microsomes limited its subsequent development. To confirm the binding mode and guide further optimization, we solved the cocrystal structure of compound 7 bound to BRD4-BD1. As illustrated in Figure 3, the methyl-benzene group indeed stretched into the WPF shelf and contributed favorable VDW interactions to increase the binding affinity. Meanwhile, it was also found that the position adjacent to the methyl group on the triazole ring pointed to the solvent-accessible part, which could be utilized for improving the drug-like properties. Considering its fast metabolic rate and bad solubility (Table S3), we intended to introduce a hydrophilic group into this position to reduce the binding potential to metabolism enzymes and improve the solubility.

Figure 3. The crystal structure of compound 7 bound to BRD4-BD1 (PDB entry: 6JI4) is shown in cartoon mode and surface mode.


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We preserved the core structure of compound 7 or compound 10, which also displayed excellent molecular and cellular potency, and added a hydrophilic piperazine group to the five-member ring linker to generate compounds 13 and 14 (Table 3), respectively, finding that their molecular and cellular activities could be retained. Hence, we evaluated the drug-like properties of compounds 13 and 14. Table 3. Inhibitory activity and MM.1S cell proliferation inhibitory activity results of compounds 13 and 14 on BRD4 (I).

R1

Cpd

R2

R3

BRD4(I)

MM.1S

IC50 (nM)a

IC50 (nM)b

(+)-JQ-1

-

-

-

28.5 ± 1.5

29.8 ± 16.6

OTX-015

-

-

-

34.3 ± 6.5

6.3 ± 13.5

13

Me



9.4 ± 0.6

8.0 ± 6.1



5.4 ± 1.2

26.1 ± 12.3

14

aThe



IC50 values are the mean of two independent experiments performed in duplicate with

standard error of the mean (SEM) values. bThe IC50 values are the mean of three independent experiments performed in triplicate with standard error of the mean (SEM) values.



Page 14 of 88

We first evaluated the in vitro metabolic stabilities of compounds 13 and 14 (Table 4). Comparison of compound 13 or 14 with compound 7 demonstrated that compounds 13 and 14 had remarkably improved metabolic stability in mouse liver microsomes, indicating that the addition of a hydrophilic group could decrease the hydrophobic properties of the compounds to reduce their binding probability with metabolic enzymes. Moreover, they did not show evident direct or time-dependent inhibition on five selected CYP450 enzymes, revealing the potential defect in the drug-drug interaction. Table 4. In vitro liver microsome intrinsic clearance data and cytochrome P450 enzyme inhibition data. HLMa Cpd

mLMb

DI (%)c / TDI (kobs*10-4)d

Clearance

T1/2

Clearance

T1/2

(μL/min/mg)

(min)

(μL/min/mg)

(min)

7

44

31.5

460

13

22

62

14

61

23

aHLM

3A4

2D6

2C9

1A2

2C19

3.0

3/no

no/no

10/no

no/no

41/no

13

107

12/19

no/14

6/19

2/41

17/no

34

41

31/55

3/no

30/74

no/51

19/141

represents the human liver microsome. bmLM represents the mouse liver microsome. cDI,

which indicated direct inhibition and is sometimes referred to reversible inhibition, is assessed by measuring enzyme (CYP) activity in the presence of increasing concentrations of inhibitor without a preincubation step. DI < 20% means no direct inhibition, 20% < DI < 50% means weak direct inhibition, 50% < DI < 70% means moderate direct inhibition, and DI > 70% means strong direct


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inhibition, dTDI stands for the time-dependent inhibition, which refers to a change in enzyme inhibition during in vitro incubation and means irreversible inactivation of CYPs. The unit of TDI is 10-4/min, and TDI < 200 means no time-dependent inhibition. When the TDI value was close to zero, it was considered as none.

Encouraged by this result, we further investigated the in vivo pharmacokinetics properties of compounds 13 and 14 (Table 5). Pharmacokinetic tests showed that compound 14 had much better plasma exposure than compound 13 at the same 10 mg/kg dosage; when compared to compound 7, both compounds improved the plasma exposure considerably, indicating that the addition of a hydrophilic group to the triazole ring could enhance the solubility to increase oral absorption. Table 5. In vivo PK data for compounds 13 and 14 in male ICR mice.a Route of

Dose

Tmax

Cmax

AUC0-t

AUC0-inf

T1/2

administration

(mg/kg)

(h)

(ng/mL)

(ng·h/mL)

(ng·h/mL)

(h)

13

p.o.

10

0.3

1231

2742

2764

1.7

14

p.o.

10

0.3

42904

58132

58146

0.6

Cpd.

a1%DMSO/0.5%HPMC

was used as the vehicle.

Hence, we further explored the structure-activity relationships of the hydrophilic group based on the core structure of 14. Initially, we explored the SAR of the hydrophilic piperazine position. As elucidated in Table 6, opening the



N-methylpiperazine

to

Page 16 of 88

N1,N1,N2-trimethylethane-1,2-diamine

an

group

(15);

replacing the N-methylpiperazine methyl group with ethyl, isopropyl or cyclopropyl (16-18); or substituting the N-methylpiperazine with (2S,6R)-2,6-dimethylpiperazine (19) or larger groups, such as 1-methyl-1,4-diazepane (20) or a bridged ring, spiro ring or fused ring (21-27), could maintain the molecular activity, suggesting that the hydrophilic N-methylpiperazine group might be exposed to the solvent moiety of the protein and did not make direct interactions with the protein. We attempted to resolve the crystal structures of compounds 14, 15, 19 and 22 with BRD4-BD1 protein to verify this conjecture but had no success. However, a later crystal structure study did confirm this hypothesis (discussed in below part). Although their molecular activities were similar, compounds 15, 16 and 19-23 had better activities in the MM.1S cell viability test, which may be due to the subtle structural changes that affect compound permeability. Table 6. Inhibitory activity and MM.1S cell proliferation inhibitory activity results of compounds 14-27 on BRD4 (I). N O R

Cpd

(+)-JQ-1

R4

-

4

N

O

N

N N

BRD4(I)

MM.1S

IC50 (nM)a

IC50 (nM)b

28.5 ± 1.5

29.8 ± 16.6

Cpd

20


R4

BRD4(I)

MM.1S

IC50 (nM)a

IC50 (nM)b

4.6 ± 0.1

8.0 ± 5.1

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OTX-015

34.3 ± 6.5

6.3 ± 13.5

21

4.6 ± 0.2

10.1 ± 3.9

14

5.4 ± 1.2

26.1 ± 12.3

22

6.6 ± 0.2

7.9 ± 1.8

15

7.0 ± 1.3

16.2 ± 3.5

23

8.7 ± 0.6

6.4 ± 2.1

16

5.8 ± 0.8

25.1 ± 7.6

24

27.2 ± 1.8

2403 ± 1940

17

4.8 ± 0.6

125.0 ± 70.3

25

7.9 ± 0.8

484.8 ± 803.4

18

5.9 ± 0.9

87.5 ± 21.8

26

9.6 ± 0.5

484.6 ± 171.3

19

5.3 ± 0.4

2.3 ± 0.9

27

6.1 ± 0.8

43.5 ± 8.8

aThe

-


standard error of the mean (SEM) values. bThe IC50 values are the mean of three independent experiments performed in triplicate with standard error of the mean (SEM) values. c N.D. means not determined.

We also explored the SAR of the side-chain benzene ring (Table 7). By comparing compounds 28 and 31 with compound 14, we found that removal of the para-methyl group or replacement of the methyl with a chlorine substituent slightly reduced the molecular activity slightly. However, the introduction of a methyl group



Page 18 of 88

at the ortho position or a fluorine substituent at the meta position could slightly improve the molecular and cellular activities, indicating that the addition of another small hydrophobic group such as a methyl or fluoro group could enhance the VDW interaction of the side-chain with the WPF pocket. Table 7. Inhibitory activity and MM.1S cell proliferation inhibitory activity results of compounds 28-31 on BRD4(I). N O

N

N

N N

O

N

N R3

Cpd

R3

BRD4(I)

MM.1S

IC50 (nM)a

IC50 (nM)b

Cpd

R3

BRD4(I)

MM.1S

IC50 (nM)a

IC50 (nM)b

(+)-JQ-1

-

28.5 ± 1.5

29.8 ± 16.6

29

2,4-dimethyl

4.5 ± 0.4

17.8 ± 7.3

OTX-015

-

34.3 ± 6.5

6.3 ± 13.5

30

3-fluoro-4

4.2 ± 0.2

13.0 ± 1.4

6.8 ± 0.6

48.5 ± 10.4

-methyl 14

Me

5.4 ± 1.2

26.1 ± 12.3

28

H

7.1 ± 0.6

60.1 ± 18.0

aThe

31

4-chloro


standard error of the mean (SEM) values. bThe IC50 values are the mean of three independent experiments performed in triplicate with standard error of the mean (SEM) values. c N.D. means not determined.

We attempted to solve the crystal complex structures of these compounds with the


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BRD4-BD1 protein, and successfully obtained the crystal structure of compound 29 with BRD4-BD1 protein (Figure 4). As shown in Figure 4, the N-methyl piperazine group reached into the solvent region around the protein and the amide group oxygen formed a hydrogen bond with the residue Lys91. This binding mode was consistent with our presumption that this N-methyl piperazine position could be utilized for drug-like optimization and will not significantly affect the inhibitors’ binding affinity.

Figure 4. The crystal structure of BRD4-BD1 bound with compound 29 (PDB entry: 6JI5).

Based on the molecular and cellular activities, compounds 14, 15, 19-23 and 30 were selected to assess their in vivo pharmacokinetics properties in mice (Table 8 lists the PK properties for compounds 14, 19 and 30; less promising compounds 15 and 20-23 are listed in Table S4). In addition to compound 14, compounds 19 and 30 were also found to have good PK properties. When compared to compound 14, compounds 19 and 30 had lower plasma exposure but a longer half-life; in particular, compound



Page 20 of 88

19 had a half-life of 2.1 hours. Moreover, compound 19 had superior cellular activity compared with compounds 14 and 30. Considering the cellular activities and PK properties, we selected compound 19 as a preclinical candidate for subsequent comprehensive evaluation. Table 8. In vivo PK data for compounds 14, 19 and 30 in male ICR mice.a Route of

Dose

Tmax

Cmax

AUC0-t

AUC0-inf

T1/2

administration

(mg/kg)

(h)

(ng/mL)

(ng·h/mL)

(ng·h/mL)

(h)

14

p.o.

10

0.3

42904

58132

58146

0.6

19

p.o.

10

1.1

1898

7383

7386

2.1

30

p.o.

10

0.3

4967

8154

8365

1.5

Cpd.

a1%


Comprehensive Profile of Compound 19 To comprehensively understand the drug-like properties of compound 19, we performed a series of evaluations on compound 19, including examining its selectivity and cellular activities, cellular mechanism studies, a hERG study, in vitro metabolic stability studies, in vivo pharmacokinetics studies, and an in vivo antitumor study. Bromodomains Selectivity Bromodomains are evolutionarily conserved protein-protein interaction modules that are found in a wide range of proteins with diverse catalytic and scaffolding functions. They recognize acetylated lysine by forming several hydrogen bonds with


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the conserved asparagine (ASN140 in BRD4-BD1) residue at the top of bromodomains. Given that selectivity is critical for the success of drug discovery, we screened the binding specificities of compound 19 across 32 bromodomains using the DiscoveRx BROMOScan platform of Eurofins company at 1 μM concentration. As depicted in Figure 5, 19 exhibited inherent selectivity for BET family members over most tested bromodomains, except for moderate inhibitory effect on TAF1(2) (Details are provided in Table S5). We also examined the inhibition of compound 19 as well as other potent compounds, including 7, 10, 14-15, 20-23, and 30 on BRD4-BD2, BRD2-BD1, BRD2-BD2, and EP300, to examine the impact of the core structure on selectivity (Table S6). The results showed that these compounds also demonstrated good potency on other BET members (BRD4-BD2, BRD2-BD1, and BRD2-BD2), though not as good as on BRD4-BD1. Importantly, none of them displayed any potency on EP300, indicating that the scaffold may be the dominant factor for the BET selectivity. Moreover, we utilized isothermal titration calorimetry (ITC) as another binding assay method to test the binding of compound 19 to BRD4-BD1, and the results confirmed that compound 19 had excellent binding affinity (Kd = 15.2 nM) (Figure S2 and Table S7).



Figure 5. Selectivity assessment of compound 19 against the BRD family members using a BROMOscan panel. The 32 screened targets are labeled in black on the BRD family phylogenetic tree. BRDs that were not part of the screening panel are in gray. Compound 19 was screened at 1 μM concentration, and the results for primary screen binding interactions are reported as the ‘Percent Control’, with lower numbers indicating stronger hits in the matrix.

Kinase Selectivity As the original core structure stems from the PLK1-BRD4 dual inhibitor BI-2536, we also tested the inhibition of compound 19 on the PLK1 kinase and 24 other kinases distributed in different subfamilies in the kinome (Table S8). We found that BI-2536 displayed a significant inhibitory effect on PLK1 kinase at 1 μM; however, compound 19 only demonstrated negligible inhibitory activity for PLK1 kinase at 10 μM and 1 μM (less than 10% inhibition ratio). This is consistent with our previous


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findings that changing the pyrimidine nitrogen atoms in BI-2536 to carbon atoms will eliminate the essential hydrogen bonding with the PLK1 kinase hinge part. Moreover, compound 19 did not show obvious inhibition of other tested kinases, though it displayed moderate inhibition of Src, Abl, and IGF1R at 10 μM. Given the nanomolar activity of BET proteins, these results reinforced that compound 19 is a selective BET inhibitor. Cellular Activities and Mechanism Studies To further evaluate the therapeutic potential of compound 19, we tested its anti-proliferative activities against seventeen different cell lines, including leukemia, multiple myeloma, thymic carcinoma, hepatocellular carcinoma, lung cancer, breast cancer, colorectal cancer, gastric adenocarcinoma, ovarian cancer, etc. (Figure 6 and Table S9). As shown in Figure 6, compound 19 was found to exhibit better inhibitory activities on all of these cell lines than (+)-JQ-1, and show better inhibitory activities than OTX-015 in several sensitive cell lines, such as MV4-11 and Ty-82. Overall, the MV4-11, MM.1S, Ty-82, KG-1 and NCI-H1299 cell lines were sensitive to compound 19 treatment, implying that, besides leukemia, it may also provide potential therapeutic opportunities for treating multiple myeloma, thymic carcinoma, and lung cancer, which is worth further exploration.



Figure 6. The inhibitory activities of compound 19 against seventeen cell lines.

According to the above results, among the tested cell lines, MV4-11 is the most sensitive to compound 19. Therefore, we selected it to explore the anti-proliferative activity mechanism. As we know, Myc is an oncogene and its deregulation is frequently involved in human oncogenesis.40 It was reported that Myc expression is reduced after BRD4 inhibition, which leads to the inhibition of cell proliferation.41 Hence, we performed Western blotting and real-time quantitative PCR (RT-qPCR) assays to study the inhibitory effects of compound 19 on c-Myc mRNA and protein expression. Figure 7A shows that compound 19 displayed profound inhibitory effects on c-Myc protein expression, even at the 10 nM, while OTX-015 did not show obvious inhibition at that concentration. Moreover, Figure 7B showed that compound 19 demonstrated a better inhibitory effect on c-Myc mRNA expression at 10 nM and 100 nM than OTX-015, which was consistent with changes in protein levels (Figure


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7A). Therefore, we concluded that the inhibitory effects of compound 19 on MV4-11 cell proliferation is partially related to the downregulation of c-Myc expression.

Figure 7. (A) MV4-11 cells were treated with compound 19 or OTX-015 at the indicated doses for 24 h and examined for c-Myc and GAPDH protein levels by Western blotting. (B) Similar to (A), but examining c-Myc mRNA levels by RT-qPCR. p < 0.05, *. Error bars represent the SD from three independent experiments.

Previous studies suggest that BET inhibitors can induce apoptosis in tumor cells.42 Thus, we examined the effect of compound 19 on apoptosis in MV4-11 cells. The results showed that compound 19 could induce apoptosis in a dose-dependent manner (Figure 8A). At the same dose, compound 19 could induce more apoptosis than OTX-015 (Figure 8A). The changes in cleaved PARP, an apoptotic marker, were consistent with the above apoptotic phenomena (Figure 8B). Next, we found that compound 19 and OTX-015 caused a dose-dependent increase in apoptotic initiators (cleaved caspase-8 and cleaved caspase-9), accompanied by a decrease in full-length caspase-8 (Figure 8C). Additionally, apoptotic executors (cleaved caspase-3 and cleaved caspase-7) also showed a dose-dependent increase (Figure 8C).



Overexpression of pro-survival BCL-2 family regulator proteins inhibits apoptosis, so we further investigated whether compound 19 induced apoptosis by reducing BCL-2 family levels. The results showed that compound 19 and OTX-015 reduced the levels of BCL-2, p-BCL2 (Thr56), p-BCL2 (Ser70) and BCL-xL in a dose-dependent manner, as well as the pro-apoptotic proteins PUMA, BID and BIM (Figure 8D). Moreover, compound 19 and OTX-015 reduced the protein levels of the inhibitors of apoptosis family proteins (IAP) XIAP, c-IAP1 and c-IAP2 in a dose-dependent manner (Figure 8D). RT-qPCR results showed that changes in BCL-2 mRNA expression were in line with that of BCL-2 at the protein level (Figures 8D and 8E). Based on the above results, we concluded that compound 19 reduced the level of many anti-apoptotic proteins and apoptotic inhibitors, thereby activating caspase family proteins and inducing more apoptosis than OTX-015 at the same doses. BRD4 is also necessary for cell cycle progression, and inhibition of BRD4 leads to G1 arrest.43 Our results showed that at the same dose, compound 19 caused more G1 phase arrest than OTX-015 (Figure S3), which was consistent with the increased proliferation inhibitory activity of compound 19.


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Figure 8. (A) MV4-11 cells were treated with compound 19 or OTX-015 at the indicated doses for 24 h, and apoptosis was measured by Annexin V Apoptosis assay. p < 0.05, *; 0.01, **. (B) Similar to (A), but examined for cleaved PARP and GAPDH protein expression by Western blotting. (C) Similar to (A), but examined for caspase-3, cleaved caspase-3, caspase-7, cleaved caspase-7, caspase-8, cleaved caspase-8, caspase-9, cleaved caspase-9, and GAPDH protein expression by Western



Page 28 of 88

blotting. (D) Similar to (A), but examined for BCL-2, p-BCL-2(Thr56), p-BCL-2(Ser70), BCL-xL, MCL-1, XIAP, c-IAP1, c-IAP2, BAK, BAX, PUMA, BID, BIM and GAPDH protein expression by Western blotting. (E) Similar to (A), but examined for BCL-2 mRNA expression by RT-qPCR. p < 0.05, *; 0.01, **. Error bars represent the SD of three independent experiments.

In Vitro Metabolic Stability Studies Next, we examined the metabolic stability of 19 in liver microsomes from four different species, including human, mouse, rat, and dog. As depicted in Table 9, compound 19 showed good stability in these liver microsome assay. We also evaluated the effects of compound 19 on five common cytochrome P450 enzymes, finding that compound 19 displayed negligible direct or time-dependent inhibition on these enzymes, which indicated that it has less risk of drug-drug interactions (Table 9). Table 9. In vitro liver microsome intrinsic clearance data and cytochrome P450 enzyme inhibition data. CYP450

Direct Inhibition (%)e

Liver

Clearance

T1/2

TDI

Microsome

(μL/min/mg)

(min)

HLMa

52

27

3A4

23/28

8

mLMb

7

193

2D6

4

no

RLMc

28

49

2C9

28

no

DLMd

77

18

1A2

no

no

kobsf


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2C19 aHLM

12

no

represents the human liver microsome; bmLM represents the mouse liver microsome; cRLM

represents the rat liver microsome; and dDLM represents the dog liver microsome. eCYP direct inhibition (DI), which is sometimes referred to as reversible inhibition, is assessed by measuring enzyme (CYP) activity in the presence of increasing concentrations of inhibitor without a preincubation step. DI < 20% means no direct inhibition, 20% < DI < 50% means weak direct inhibition, 50% < DI < 70% means moderate direct inhibition, and DI > 70% means strong direct inhibition; fTDI stands for time-dependent inhibition, which refers to a change in enzyme inhibition during in vitro incubation and indicates irreversible CYP inactivation. The unit of TDI is 10-4/min. TDI < 200 means no time-dependent inhibition. When the TDI value was close to zero, it was considered as none.

hERG Study In cardiomyocytes, human ether-a-go-go related gene (hERG) encodes a potassium channel that mediates a delayed rectifier potassium current (IKr), and the inhibition of IKr is the most important mechanism for drug-induced prolongation of QT.44 Thus, we wanted to examine the effects of compound 19 and the positive control Cisapride on hERG before we advance the compound into in vivo studies. As illustrated in Table S10, compound 19 had no evident effects on hERG, even at 40 μM, indicating that it has a low potential for side effects on the heart.

In Vivo PK Study (+)-JQ1 was reported to have a high rate of brain penetration, which could cause neurological side effects.26 To investigate the distribution of compound 19 in the



Page 30 of 88

brain, we evaluated its concentration in the brain and plasma. As illustrated in Table 10, the ratio of the brain and plasma concentrations of compound 19 in mice or rats at 50 mg/kg dosage was 13.9% or 8.7%, respectively. As reported, (+)-JQ1 has a nearly 1:1 ratio of brain and plasma concentrations. Therefore, we thought compound 19 may have less potential neurological toxicity issues than (+)-JQ1. Table 10. In vivo pharmacokinetics data of compound 19 in multiple species.a

species

Dosage

Tmax

Cmax

AUC0-t

AUC0-inf

T1/2

(mg/kg)

(h)

(ng/mL)

(ng·h/mL)

(ng·h/mL)

(h)

Tissue

mouse

Plasma

50 (p.o.)

1.0

4019

16716

16790

2.8

mouse

Brain

50 (p.o.)

1.0

377

2321

2335

3.0

rat

plasma

50 (p.o.)

0.4

1251

3820

4309

6.3

rat

Brain

50 (p.o.)

0.9

87.6

331

399

9.3

mice

plasma

10 (p.o.)

1.1

1898

7383

7386

2.1

mice

plasma

50 (p.o.)

1.0

4019

16716

16790

2.8

mice

plasma

100 (p.o.)

1.5

13773

80595

82440

4.4

rat

plasma

10 (p.o.)

2.3

182

1215

1268

5.0

rat

plasma

50 (p.o.)

0.4

1251

3820

4309

6.3

rat

plasma

3 (i.v.)

/

/

2008

2085

1.8

dog

plasma

3 (p.o.)

0.5

216

747

798

6.6

dog

plasma

1 (i.v.)

/

/

726

856

9.4

a1%



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Furthermore, we determined compound 19’s dose-dependent PK properties in different species. Table 10 demonstrates that the plasma exposure of compound 19 continuously improved with increasing doses (oral administration of 10 mg/kg, 50 mg/kg or 100 mg/kg in mice or oral administration of 10 mg/kg or 50 mg/kg in rats). To assess the PK properties in large animals, we further examined its PK properties in dog (Table 10). In these three species, 19 displayed a reasonable plasma exposure and half-life. Additionally, intravenous injection of compound 19 at a 3 mg/kg dose in rat or 1 mg/kg dose in dog showed that it had moderate oral bioavailability at 18.2% in rat and 34.3% in dog. In Vivo PD Study To evaluate the in vivo therapeutic effects of compound 19, we established a human MV4-11 xenograft model. Tumor-bearing BALB/c nude mice were treated with different doses of compound 19 using OTX-015 as the positive control (Figure 9A). Treatment with compound 19 and OTX-015 by oral administration at the same dosage of 50 mg/kg daily showed that compound 19 had superior inhibitory effects on the tumor growth with a growth inhibition (GI) of 95.6% (OTX-015 had a GI of 71.5%). Compound 19 reduced tumor growth in a dose-dependent manner (Figure 9A). Furthermore, as depicted in Figure 9A, compound 19 even showed a little better anti-tumor effects at one fourth the dosage compared with OTX-015 (25 mg/kg daily of 19 with a GI of 89.7% vs. 100 mg/kg of OTX-015 daily with a GI of 87.5%). Moreover, compound 19 did not promote any obvious loss in body weight at multiple dosages and no animal death occurred during the experiment (Figure 9B), indicating



that it has a good safety window. We also examined the pharmakinetics-pharmcodynamics relationship by assessing the drug concentration in plasma and tumor tissue after administration of compound 19 at the above dosage for 1 hour and 8 hours. The data (Table S11) showed that plasma exposure was increased in a dose-dependent manner at 1 h and 8 h, but decreased with time at the three different concentrations, which is consistent with the previous PK study. However, the tumor concentration of compound 19 after oral administration remains higher than plasma, especially at the 8 h time point, indicating that 19 has a longer half-life in tumor tissue. This effect is more evident in the high dose groups (25 mg/kg and 50 mg/kg), which correlated well with the excellent antitumor efficacy in vivo at these two different dosages.

Figure 9. (A) Relative tumor volume (RTV) of human MV4-11 xenografts in Balb/c nude mice after treatments. OTX-015 or compound 19 group versus the vehicle group; p < 0.01, **; 0.001, ***. Formulation: 19, 0.5% Tween80 and 0.5% methylcellulose aqueous solution, and OTX-015, 5% dimethylacetamide and 0.5% hydroxypropyl methyl cellulose. (B) Body weight changes in mice. Chemistry


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The syntheses of novel dihydroquinoxalin-2(1H)-one derivatives were concluded in schemes 1-7. As depicted in Scheme 1, the synthetic route of compound 36 was described in our previous work,33 which was coupled with N-Boc-2-pyrroleboronic acid to generate compound 4. Scheme 1 Synthetic Route of Compound 4

Reagents and conditions: a) cyclopropylamine, ClCH2CH2Cl, 80 °C, 12 h; b) Fe, NH4Cl, EtOH, 80 °C, 1 h; c) 1. 2-bromopropanoyl bromide, DIPEA, DCM, 0 °C - rt, 2 h; 2. CH3CN, DIPEA, 80 °C, overnight; d) NaH, 0 °C, 30 min, iodomethane, rt, 2 h; e) N-Boc-2-pyrroleboronic acid, Pd(dppf)Cl2, K2CO3, DMF, 120 °C, 4 h.

In Scheme 2, condensation of commercially available 3-fluoro-4-nitrobenzoic acid (compound 37) with p-toluidine furnished compound 38, which was then reacted with cyclopropylamine or cyclopentylamine to afford corresponding compound 39 or 40. The reaction of compound 39 or 40 with Lawesson’s reagent acquired compound 41 or 42. Treatment of 41 with N2H4.H2O followed by adding trimethyl orthoformate gained compound 43. Cyclization of compound 41 or 42 with C3H9N3Si obtained compound 44 or 45. Reduction of compounds 43-45 with tin(II) chloride dihydrate



and con.HCl yielded compounds 46-48, which was converted to compounds 6, 8 and 11 through the similar reaction of scheme 1 c-d. Scheme 2 Synthetic Routes of Compounds 6, 8 and 11

Reagents and conditions: a) p-toluidine, HATU, DIPEA, DMF, rt, overnight; b) R2NH2, ClCH2CH2Cl, 80 °C, 12 h; c) Lawesson’s reagent, toluene, 110 ℃, overnight; d) 43: 1. N2H4.H2O, MeOH, rt, 2h; 2. Trimethylorthoformate, DMF, AcOH, rt, overnight; 44-45: Hg(OAc)2, C3H9N3Si, THF, 0 °C, 3 h; e) tin(II) chloride dihydrate, con.HCl, rt, 5 h; f) 1. 2-bromopropanoyl bromide, DIPEA, DCM, 0 °C - rt, 2 h; 2. CH3CN, DIPEA, 80 °C, overnight; g) NaH, 0 °C, 30 min, iodomethane, rt, 2 h.

In schemes 3, the reaction of compound 37 with SOCl2 and methanol afforded compound 52, which was then converted to compound 56 through the similar reaction of scheme 1 a-d. The reaction of compound 56 with hydrazine hydrate acquired compound 57, which was further transformed to compound 7 by reacting with N-(p-tolyl)ethanethioamide.


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Scheme 3 Synthetic Route of Compound 7 NO2 HO

NO2

a O

F O

NO2

b

O

F

O

N

O

53

N e

N O

O 55

54

N

O

N

f

O

NH O

52

H N

d

O

NH O

O 37

NH2

c

H2 N

H N

O g

N

N

N

O

N N

O 56

57

7

Reagents and conditions: a) SOCl2, MeOH, 60 °C, 24 h; b) cyclopropylamine, ClCH2CH2Cl, 80 °C, 12 h; c) Fe, NH4Cl, EtOH, 80 °C, 1 h; d) 1. 2-bromopropanoyl bromide, DIPEA, DCM, 0 °C - rt, 2 h; 2. CH3CN, DIPEA, 80 °C, overnight; e) NaH, 0 °C, 30 min, iodomethane, rt, 2 h; f) N2H4.H2O, EtOH, 80 °C, 12 h; g) N-(p-tolyl)ethanethioamide, Hg(OAc)2, AcOH, THF, 0 °C - rt, 24 h.

In schemes 4, the reaction of compound 58 with p-toluidine and thioglycolic acid received compound 59, which was then transformed to compound 9 through the similar reaction of scheme 1 a-d except for using the tin(II) chloride dehydrate as reduction reagent instead of Fe powder. Scheme 4 Synthetic Route of Compound 9



NO2 NO2

S

a H

F

S

O

59

S

61

60

O

N e

N

NH N

O

58 H N

S

c

N

O

d

NH

N

O

NH2

NO2

b

F

Page 36 of 88

S

O

N

N

N

O

O

9

62

Reagents and conditions: a) p-toluidine, THF, 0 ℃ - rt, 12 h; b) cyclopropylamine, ClCH2CH2Cl, 80 °C, 12 h; c) tin(II) chloride dihydrate, con.HCl, rt, 5 h; d) 1. 2-bromopropanoyl bromide, DIPEA, DCM, 0 °C - rt, 2 h; 2. CH3CN, DIPEA, 80 °C, overnight; e) NaH, 0 °C, 30 min, iodomethane, rt, 2 h.

In schemes 5, the reaction of compound 52 with D-alanine in the presence of K2CO3 obtained compound 63, which was then converted to compound 64 under the condition of K2CO3 and Na2S2O4. Treatment of compound 64 with cyclopentanone via reductive amination provided compound 65, which was switched to compound 10 through the similar reaction of scheme 3 e-g. Scheme 5 Synthetic Route of Compound 10 NO2

NO2 a

O

O

F

b

NH O

O

c

O

O

H N

O O

N H

O

N O

OH

52

63 N

d

H N

O

N e

O

N O

64

H 2N

H N

N

O f

N O

66

65

67


N

N

N N

10

O

O

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Reagents and conditions: a) D-alanine, K2CO3, EtOH, H2O, 80 ℃, 8 h; b) K2CO3, Na2S2O4, H2O, 60 °C, overnight; c) phenylsilane, cyclopentanone, dibutyltindichloride, THF, rt, 10 h; d) NaH, 0 °C, 30 min, iodomethane, rt, 2 h; e) N2H4.H2O, EtOH, 80 °C, 12 h; f) N-(p-tolyl)ethanethioamide, Hg(OAc)2, AcOH, THF, 0 °C - rt, 24 h.

In scheme 6, compound 71 was prepared in the same manner as compound 66. Treatment of compound 71 with bis(pinacolato)diboron obtained compound 72. Suzuki coupling of compound 72 with 4-bromo-5-methyl-3-phenylisoxazole (the synthetic route was described by Dong et.al45) resulted in compound 12. Similarly, suzuki

coupling

of

compound

72

with

ethyl

5-bromo-1-phenyl-1H-1,2,4-triazole-3-carboxylate derivatives (the synthetic route was described by Zibinsky et.al46), and subsequent hydrolysis yielded corresponding compounds 78-82, which was further converted to the desired derivatives 14-31 by condensation with different amines. Scheme 6 Synthetic Routes of Compounds 12, and 14-31

Reagents and conditions: a) D-alanine, K2CO3, EtOH, H2O, 80 ℃, 8 h; b) K2CO3, Na2S2O4, H2O,



60 °C, overnight; c) phenylsilane, cyclopentanone, dibutyltin dichloride, THF, rt, 10 h; d) NaH, 0 °C, 30 min, iodomethane, rt, 2 h; e) Pd(dppf)Cl2, bis(pinacolato)diboron, AcOK, DMSO, 80 °C, overnight; f) Pd(dppf)Cl2, NaHCO3, THF/H2O, 80 °C, overnight; g) Pd(dppf)Cl2, NaHCO3, THF/H2O, 80 °C, overnight; h) LiOH, THF/H2O, rt, overnight; i) HATU, DIPEA, DMF, rt, overnight.

In scheme 7, compound 13 was obtained through the similar reaction of scheme 6 e and g-I from compound 36. Scheme 7 Synthetic Route of Compound 13

Reagents and conditions: a) Pd(dppf)Cl2, bis(pinacolato)diboron, AcOK, DMSO, 80 °C, overnight; b) Pd(dppf)Cl2, NaHCO3, THF/H2O, 80 °C, overnight; c) LiOH, THF/H2O, rt, overnight; d) HATU, DIPEA, DMF, rt, overnight.

Conclusion Based on our previously reported novel BET-selective inhibitors, we wanted to discover a candidate for further clinical research. With the help of the cocrystal structure of hit compound 4 harboring a new rigid five-member ring, we found a lead compound 7 that showed moderate antitumor activity in a human MM.1S xenograft


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model in SCID mice by oral administration. Inspired by this result, we resolved the cocrystal structure of compound 7 with BRD4-BD1 for further optimization. Introduction of a hydrophilic group to the five-membered ring linker yielded a novel series of (1,2,4-triazol-5-yl)-3,4-dihydroquinoxalin-2(1H)-one compounds with excellent molecular and cellular activities. By evaluating the molecular and cellular activities, in vitro metabolic stability, and in vivo PK properties, compound 19 was discovered as a potential candidate for preclinical research. After a comprehensive evaluation, we confirmed that compound 19 displayed the following properties: high selectivity for BET over other bromodomains and tested kinases distributed in different subfamilies; excellent inhibitory activities on the MV4-11, MM.1S, Ty-82, KG-1, and NCI-H1299 cell lines; increased G1 cell cycle arrest and apoptosis than OTX-015; stable metabolic stability in multiple species; a low rate of blood-brain barrier penetration; good PK properties in mouse, rat and dog; and good antitumor efficacy in vivo. Overall, we demonstrated that compound 19 is a potential drug candidate for BET-relevant cancer treatment.

Experimental Section Chemistry General: 1H

NMR (400 MHz) spectra were recorded by using a Varian Mercury-400 High

Performance Digital FT-NMR spectrometer with tetramethylsilane (TMS) as an internal standard.

13C

NMR (126 MHz) spectra were recorded by using a Varian

Mercury-500 High Performance Digital FT-NMR spectrometer. Abbreviations for peak patterns in NMR spectra: br = broadened, s = singlet, d = doublet, t = triplet, dd



= doublet of doublets and m = multiplet. Low-resolution mass spectra were obtained with a Finnigan LCQ Deca XP mass spectrometer using a CAPCELL PAK C18 (50mm × 2.0mm, 5 µM) or an Agilent ZORBAX Eclipse XDB C18 (50mm × 2.1m, 5 µM) in positive or negative electrospray mode. High-resolution mass spectra were recorded by using a Finnigan MAT-95 mass spectrometer. Purity of all compounds was determined by analytical Gilson high-performance liquid chromatography (HPLC) using an YMC ODS3 column (50 mm × 4.6 mm, 5 µM) and confirmed to be more than 95%. Conditions were as follows: CH3CN/H2O eluent at 2.5 mLmin-1 flow [containing 0.1% trifluoroacetic acid (TFA)] at 35 °C, 8 min, gradient 5% CH3CN to 95% CH3CN, monitored by UV absorption at 214 nm and 254 nm. TLC analysis was carried out with glass precoated silica gel GF254 plates. TLC spots were visualized under UV light. Flash column chromatography was performed with a Teledyne ISCO CombiFlash Rf system. All solvents and reagents were used directly as obtained commercially unless otherwise noted. All air and moisture sensitive reactions were carried out under an atmosphere of dry argon with heat-dried glassware and standard syringe techniques. Melting points were determined using a SGW X-4 hot stage microscope and are uncorrected. (spectra data of the synthesized compounds were provided as supporting material) Synthetic Procedures: Compounds 32, 37 and 58 were purchased. Other compounds were prepared by one of seven schemes. 4-cyclopropyl-1,3-dimethyl-6-(1H-pyrrol-2-yl)-3,4-dihydroquinoxalin-2(1H)-one (4). To a


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solution of compound 36 (0.2 g, 0.7 mmol) in anhydrous DMF (10 mL) was added K2CO3 (0.5 g, 3.4 mmol). The mixture was purged with nitrogen for 2 minutes. Then Pd(dppf)Cl2.CH2Cl2 (0.1 g, 0.1 mmol) was added and the mixture was sealed in a microwave tube and heated to 120 °C for 4 h. The reaction was monitored by TLC. Upon completion, the reaction mixture was acidified with 1 N aq. HCl to pH 7 – 8, diluted with water and extracted with EtOAc (3 × 50 mL). The combined organic fractions were washed with brine, dried over Na2SO4, concentrated by evaporation under reduced pressure. Purification by silica gel column chromatography (gradient elution, gradient 0 to 25% EtOAc/60–90 °C petroleum ether) gave compound 4 as a white soild (0.09 g, 0.3 mmol, 43% yield). 1H NMR (400 MHz, CDCl3) δ 8.63 (s, 1H), 7.25 (d, J = 1.9 Hz, 1H), 7.00 (dd, J = 8.2, 2.0 Hz, 1H), 6.91 (d, J = 8.3 Hz, 1H), 6.86 (td, J = 2.6, 1.5 Hz, 1H), 6.51 – 6.47 (m, 1H), 6.31 (dd, J = 6.0, 2.7 Hz, 1H), 4.13 (q, J = 6.8 Hz, 1H), 3.35 (s, 3H), 2.49 – 2.39 (m, 1H), 1.19 (d, J = 6.9 Hz, 3H), 1.03 – 0.96 (m, 1H), 0.84 – 0.76 (m, 1H), 0.68 – 0.61 (m, 1H), 0.60 – 0.54 (m, 1H); HRMS (ESI) m/z [M + H]+ calcd for (C22H24N5O+) 374.1975, found 374.1969; retention time 3.21 min, > 99% pure. 4-cyclopropyl-1,3-dimethyl-6-(4-(p-tolyl)-4H-1,2,4-triazol-3-yl)-3,4-dihydroquinoxalin-2(1H) -one (6). To a solution of compound 49 (0.8 g, 2.2 mmol) in anhydrous DMF (2 mL) was added NaH (0.2 g, 6.7 mmol) at 0 °C, the mixture was stirred at 0 °C for 30 min, then iodomethane (0.2 mL, 3.4 mmol) was added and the mixture was stirred at room temperature for another 2 h. The reaction was monitored by TLC. Upon completion, the reaction mixture was acidified with 1 N aq. HCl to pH 7 – 8, diluted with water and extracted with EtOAc (3 × 50 mL). The combined organic fractions were washed with brine, dried over Na2SO4, and concentrated by evaporation under reduced pressure. Purification by silica gel column chromatography (gradient elution, gradient 0 to



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10% EtOAc/60–90 °C petroleum ether) gave compound 6 as a white soild (0.2 g, 0.6 mmol, 27% yield). 1H NMR (400 MHz, CDCl3) δ 8.28 (s, 1H), 7.29 – 7.24 (m, 2H), 7.17 – 7.14 (m, 2H), 7.14 – 7.12 (m, 1H), 7.04 (dd, J = 8.3, 1.9 Hz, 1H), 6.85 (d, J = 8.3 Hz, 1H), 4.05 (q, J = 6.9 Hz, 1H), 3.31 (s, 3H), 2.42 (s, 3H), 2.22 – 2.14 (m, 1H), 1.10 (d, J = 6.8 Hz, 3H), 0.72 – 0.64 (m, 1H), 0.58 – 0.47 (m, 2H), 0.21 – 0.13 (m, 1H);

13C

NMR (126 MHz, CDCl3) δ 168.80, 153.33, 144.87,

139.76, 135.83, 132.42, 130.86, 130.57 (2 × C), 125.73 (2 × C), 121.66, 120.07, 114.31, 114.30, 58.15, 29.08, 27.84, 21.27, 12.27, 9.10, 6.65; MS(ESI) [M+H] +: 374.31; retention time 2.93 min, > 96% pure.

4-cyclopropyl-1,3-dimethyl-6-(5-methyl-4-(p-tolyl)-4H-1,2,4-triazol-3-yl)-3,4-dihydroquinoxa lin-2(1H)-one (7). To a solution of compound 57 (0.6 g, 2.2 mmol) in THF (10 mL) and acetic acid (4 mL) were added N-(p-tolyl)ethanethioamide (0.4 g, 2.2 mmol) and Hg(OAc)2 (1.1 g, 3.3 mmol) at 0 °C, then the mixture was stirred at 0 °C for 3 h and further stirred at rt for 24 h. The reaction was monitored by TLC. Upon completion, the residue was basified with saturated NaHCO3 to pH 7 – 8, diluted with water and extracted with EtOAc (3 × 40 mL). Combined organic layers were washed with brine, and purification by silica gel column chromatography (gradient elution, gradient 0 to 10% MeOH/CH2Cl2) gave compound 7 as a red soild (0.3 g, 0.8 mmol, 35% yield). 1H NMR (400 MHz, CDCl3) δ 7.30 (d, J = 8.0 Hz, 2H), 7.15 (d, J = 1.9 Hz, 1H), 7.09 (d, J = 8.3 Hz, 2H), 6.98 (dd, J = 8.3, 1.9 Hz, 1H), 6.81 (d, J = 8.4 Hz, 1H), 4.04 (q, J = 6.8 Hz, 1H), 3.29 (s, 3H), 2.43 (s, 3H), 2.34 (s, 3H), 2.20 – 2.15 (m, 1H), 1.09 (d, J = 6.8 Hz, 3H), 0.73 – 0.65 (m, 1H), 0.64 – 0.56 (m, 1H), 0.55 – 0.48 (m, 1H), 0.23 – 0.14 (m, 1H);

13C

NMR

(126 MHz, CDCl3) δ 168.87, 154.08, 152.80, 139.96, 135.79, 132.76, 130.78 (2 × C), 130.62, 127.14 (2 × C), 122.47, 119.81, 114.24, 114.04, 58.21, 29.08, 27.87, 21.34, 12.23, 11.55, 9.23,


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6.68; HRMS (ESI) m/z [M + H]+ calcd for (C23H26N5O+) 388.2132, found 388.2123; retention time 2.90 min, > 99% pure. 4-cyclopropyl-1,3-dimethyl-6-(1-(p-tolyl)-1H-tetrazol-5-yl)-3,4-dihydroquinoxalin-2(1H)-one (8). To a solution of compound 50 (0.6 g, 1.7 mmol) in anhydrous DMF (2 mL) was added NaH (0.1 g, 5.0 mmol) at 0 °C, the mixture was stirred at 0 °C for 30 min, then iodomethane (0.2 mL, 3.0 mmol) was added and the mixture was stirred at room temperature for another 2 h. The reaction was monitored by TLC. Upon completion, the reaction mixture was acidified with 1 N aq. HCl to pH 7 – 8, diluted with water and extracted with EtOAc (3 × 50 mL). The combined organic fractions were washed with brine, dried over Na2SO4, and concentrated by evaporation under reduced pressure. Purification by silica gel column chromatography (gradient elution, gradient 0 to 10% EtOAc/60–90 °C petroleum ether) gave compound 8 (0.2 g, 0.5 mmol, 29% yield) as a white soild. 1H NMR (400 MHz, CDCl3) δ 7.36 – 7.28 (m, 4H), 7.23 (d, J = 1.7 Hz, 1H), 7.15 (dd, J = 8.3, 1.8 Hz, 1H), 6.91 (d, J = 8.3 Hz, 1H), 4.08 (q, J = 6.9 Hz, 1H), 3.33 (s, 3H), 2.45 (s, 3H), 2.25 – 2.15 (m, 1H), 1.13 (d, J = 6.8 Hz, 3H), 0.76 – 0.65 (m, 1H), 0.58 – 0.48 (m, 2H), 0.26 – 0.15 (m, 1H); 13C NMR (151 MHz, CDCl3) δ 168.71, 153.72, 140.85, 136.16, 132.47, 131.94, 130.52 (2 × C), 125.46 (2 × C), 120.45, 118.66, 114.53, 114.29, 58.08, 29.13, 27.88, 21.42, 12.49, 9.12, 6.74; MS(ESI) [M+H] +: 375.20; retention time 3.63 min, > 95% pure. 2-(4-cyclopropyl-1,3-dimethyl-2-oxo-1,2,3,4-tetrahydroquinoxalin-6-yl)-3-(p-tolyl)thiazolidin -4-one (9). To a solution of compound 62 (0.1 g, 0.3 mmol) in anhydrous DMF (5 mL) was added NaH (0.02 g, 0.8 mmol) at 0 °C, the mixture was stirred at 0 °C for 30 min, then iodomethane (0.03 mL, 0.5 mmol) was added and stirred at room temperature for another 2 h. The reaction was monitored by TLC. Upon completion, the reaction mixture was acidified with 1 N aq. HCl to pH 7



– 8, diluted with water and extracted with EtOAc (3 × 20 mL). The combined organic fractions were washed with brine, dried over Na2SO4, and concentrated by evaporation under reduced pressure. Purification by silica gel column chromatography (gradient elution, gradient 0 to 25% EtOAc/60–90 °C petroleum ether) gave compound 9 (15 mg, 0.04 mmol, 15% yield) as a white solid. 1H NMR (400 MHz, CDCl3) δ 7.11 – 7.04 (m, 3H), 7.02 (s, 1H), 7.01 – 6.97 (m, 1H), 6.86 – 6.79 (m, 1H), 6.78 – 6.75 (m, 1H), 6.04 (d, J = 34.1 Hz, 1H), 4.06 (q, J = 6.9 Hz, 1H), 3.96 (dd, J = 15.8, 9.5 Hz, 1H), 3.87 (d, J = 15.8 Hz, 1H), 3.27 (d, J = 4.1 Hz, 3H), 2.38 – 2.30 (m, 1H), 2.25 (d, J = 3.0 Hz, 3H), 1.11 (dd, J = 8.7, 7.1 Hz, 3H), 0.94 – 0.85 (m, 1H), 0.82 – 0.71 (m, 1H), 0.63 – 0.54 (m, 1H), 0.41 – 0.29 (m, 1H); MS (ESI) [M+H]+: 408.21; retention time 3.49 min, > 99% pure. (R)-4-cyclopentyl-1,3-dimethyl-6-(5-methyl-4-(p-tolyl)-4H-1,2,4-triazol-3-yl)-3,4-dihydroquin oxalin-2(1H)-one (10). To a solution of compound 67 (0.6 g, 2.0 mmol) in THF (10 mL) and acetic acid (4 mL) were added N-(p-tolyl)ethanethioamide (0.3 g, 2.0 mmol) and Hg(OAc)2 (1.0 g, 3.0 mmol) at 0 °C, then the mixture was stirred at 0 °C for 3 h and further stirred at rt for 24 h. The reaction was monitored by TLC. Upon completion, the residue was basified with saturated NaHCO3 to pH 7 – 8, diluted with water and extracted with EtOAc (3 × 40 mL). Combined organic layers were washed with brine, and purification by silica gel column chromatography (gradient elution, gradient 0 to 10% MeOH/CH2Cl2) gave compound 10 as a white soild (0.3 g, 0.8 mmol, 40% yield). 1H NMR (400 MHz, CDCl3) δ 7.30 (d, J = 8.1 Hz, 2H), 7.10 (d, J = 8.1 Hz, 2H), 7.00 (dd, J = 8.4, 1.5 Hz, 1H), 6.83 (d, J = 8.1 Hz, 2H), 4.10 (q, J = 6.8 Hz, 1H), 3.43 (dt, J = 14.6, 7.4 Hz, 1H), 3.30 (s, 3H), 2.42 (s, 3H), 2.33 (s, 3H), 1.87– 1.76 (m, 1H), 1.75 – 1.46 (m,


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7H), 0.96 (d, J = 6.8 Hz, 3H); HRMS (ESI) m/z [M + H]+ calcd for (C25H30N5O+) 416.2445, found 416.244; retention time 3.11 min, > 99% pure. Following the similar procedures as for compound 8 gave compound 11. 4-cyclopentyl-1,3-dimethyl-6-(1-(p-tolyl)-1H-tetrazol-5-yl)-3,4-dihydroquinoxalin-2(1H)-one (11). White soild, 35% yield. 1H NMR (400 MHz, CDCl3) δ 7.30 (q, J = 8.4 Hz, 4H), 7.13 (d, J = 8.3 Hz, 1H), 6.93 (d, J = 8.1 Hz, 2H), 4.15 (q, J = 6.7 Hz, 1H), 3.48 (dd, J = 14.9, 7.4 Hz, 1H), 3.34 (s, 3H), 2.43 (s, 3H), 1.83 (dd, J = 14.2, 8.8 Hz, 2H), 1.74 – 1.66 (m, 1H), 1.64 – 1.48 (m, 5H), 0.99 (d, J = 6.8 Hz, 3H);

13C

NMR (151 MHz, CDCl3) δ 168.95, 153.70, 140.85, 135.87,

132.89, 132.44, 130.53 (2 × C), 125.52 (2 × C), 119.91, 118.43, 115.60, 114.74, 58.97, 54.47, 30.76, 30.36, 29.22, 23.99, 23.50, 21.38, 14.22; MS(ESI)[M+H] +: 403.13; retention time 3.92 min, > 99% pure. (R)-4-cyclopentyl-1,3-dimethyl-6-(5-methyl-3-phenylisoxazol-4-yl)-3,4-dihydroquinoxalin-2( 1H)-one (12). To a solution of compound 72 (1 g, 2.7 mmol) and NaHCO3 (0.5 g, 5.4 mmol) in THF (10 mL) and H2O (3 mL) was added 4-bromo-5-methyl-3-phenylisoxazole (1.3 g, 5.4 mmol), and the mixture was bubbled with N2 for 5 min, then Pd(dppf)2Cl2.CH2Cl2 (0.2 g, 0.3 mmol) was added, the mixture was further bubbled with N2 for 5 min. Then the mixture was heated to 80 °C overnight. The reaction was monitored by TLC. Upon completion, the reaction mixture was diluted with water and extracted with EtOAc (3 × 50 mL). The combined organic fractions were washed with brine, dried over Na2SO4, and concentrated by evaporation under reduced pressure. Purification by silica gel column chromatography (gradient elution, gradient 0 to 20% EtOAc/60– 90 °C petroleum ether) gave compound 12 (0.7 g, 1.7 mmol, 56% yield) as a light yellow liquid. 1H

NMR (400 MHz, CDCl3) δ 7.58 – 7.55 (m, 2H), 7.36 – 7.30 (m, 3H), 7.00 (d, J = 8.2 Hz, 1H),



6.82 (dd, J = 8.1, 1.8 Hz, 1H), 6.66 (d, J = 1.8 Hz, 1H), 4.20 (q, J = 6.8 Hz, 1H), 3.66 – 3.56 (m, 1H), 3.41 (s, 3H), 2.29 (s, 3H), 1.90 – 1.82 (m, 1H), 1.81 – 1.67 (m, 2H), 1.62 – 1.46 (m, 5H), 1.08 (d, J = 6.8 Hz, 3H)；13C NMR (126 MHz, CDCl3) δ 169.21, 164.42, 160.19, 136.06, 130.61, 129.71, 128.70 (2 × C), 128.10, 127.08 (2 × C), 125.59, 120.31, 117.29, 116.33, 114.97, 58.99, 55.01, 30.87, 30.79, 29.19, 24.21, 23.74, 13.91, 10.87; MS (ESI) [M+H]+: 402.19; retention time 4.23 min, > 99% pure. 4-cyclopropyl-1,3-dimethyl-6-(3-(4-methylpiperazine-1-carbonyl)-1-(p-tolyl)-1H-1,2,4-triazol -5-yl)-3,4-dihydroquinoxalin-2(1H)-one (13). To a solution of compound 85 (0.7 g, 1.7 mmol) in DMF (10 mL) was added HATU (0.7 g, 1.9 mmol), and the mixture was stirred at rt for 30 min. Then N-methylpiperazine (0.2 g, 1.9 mmol) and DIPEA (0.3 mL, 1.9 mmol) were added and the mixture was stirred at rt overnight. The reaction was monitored by TLC. Upon completion, the reaction mixture was diluted with saturated NaHCO3 and extracted with EtOAc (3 × 50 mL). The combined organic fractions were washed with brine, dried over Na2SO4, and concentrated by evaporation under reduced pressure. Purification by silica gel column chromatography (gradient elution, gradient 0 to 20% EtOAc/60–90 °C petroleum ether) gave compound 13 (0.5 g, 1.0 mmol, 59% yield) as white solid. 1H NMR (400 MHz, CDCl3) δ 7.30 – 7.27 (m, 2H), 7.25 – 7.22 (m, 2H), 7.17 (dd, J = 8.2, 1.9 Hz, 1H), 7.14 (d, J = 1.8 Hz, 1H), 6.89 (d, J = 8.2 Hz, 1H), 4.06 (q, J = 6.8 Hz, 1H), 4.01 – 3.96 (m, 2H), 3.90 – 3.85 (m, 2H), 3.33 (s, 3H), 2.52 (dt, J = 14.2, 5.0 Hz, 4H), 2.41 (s, 3H), 2.34 (s, 3H), 2.20 – 2.12 (m, 1H), 1.11 (d, J = 6.8 Hz, 3H), 0.73 – 0.63 (m, 1H), 0.57 – 0.42 (m, 2H), 0.21 – 0.11 (m, 1H); MS (ESI) [M+H]+: 500.41; retention time 3.04 min, > 99% pure. (R)-4-cyclopentyl-1,3-dimethyl-6-(3-(4-methylpiperazine-1-carbonyl)-1-(p-tolyl)-1H-1,2,4-tri


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azol-5-yl)-3,4-dihydroquinoxalin-2(1H)-one (14). To a solution of compound 78 (0.7 g, 1.6 mmol) in DMF (10 mL) was added HATU (0.6 g, 1.7 mmol), and the mixture was stirred at rt for 30 min. Then N-methylpiperazine (0.2 g, 1.7 mmol) and DIPEA (0.3 mL, 1.7 mmol) were added and the mixture was stirred at rt overnight. The reaction was monitored by TLC. Upon completion, the reaction mixture was diluted with saturated NaHCO3 and extracted with EtOAc (3 × 50 mL). The combined organic fractions were washed with brine, dried over Na2SO4, and concentrated by evaporation under reduced pressure. Purification by silica gel column chromatography (gradient elution, gradient 0 to 20% EtOAc/60–90 °C petroleum ether) gave compound 14 (0.5 g, 1.0 mmol, 63% yield) as white solid. 1H NMR (400 MHz, CDCl3) δ 7.29 – 7.26 (m, 2H), 7.25 – 7.22 (m, 2H), 7.14 (dd, J = 8.3, 1.9 Hz, 1H), 6.90 (d, J = 8.4 Hz, 1H), 6.84 (d, J = 1.9 Hz, 1H), 4.12 (q, J = 6.8 Hz, 1H), 4.01 – 3.92 (m, 2H), 3.90 – 3.83 (m, 2H), 3.48 – 3.40 (m, 1H), 3.34 (s, 3H), 2.54 – 2.49 (m, 2H), 2.49 – 2.45 (m, 2H), 2.39 (s, 3H), 2.32 (s, 3H), 1.84 – 1.74 (m, 1H), 1.72 – 1.63 (m, 1H), 1.61 – 1.43 (m, 5H), 1.35 – 1.27 (m, 1H), 0.97 (d, J = 6.8 Hz, 3H);

13C

NMR (126 MHz, CDCl3) δ 169.08, 160.70, 156.86, 154.45, 139.61, 135.72, 135.52,

132.26, 130.15 (2 × C), 125.67 (2 × C), 122.18, 120.21, 116.16, 114.56, 59.05, 55.48, 54.71, 54.56, 47.10, 46.10, 42.43, 30.78, 30.35, 29.24, 24.01, 23.55, 21.30, 14.14; HRMS (ESI) m/z [M + H]+ calcd for (C30H38N7O2+) 528.3081, found 528.3081; retention time 2.88 min, > 99% pure. Following the similar procedures as for compound 14 gave compounds 15-31. (R)-5-(4-cyclopentyl-1,3-dimethyl-2-oxo-1,2,3,4-tetrahydroquinoxalin-6-yl)-N-(2-(dimethyla mino)ethyl)-N-methyl-1-(p-tolyl)-1H-1,2,4-triazole-3-carboxamide (15). White solid, 64% yield. 1H NMR (400 MHz, CDCl3) δ 7.26 – 7.19 (m, 4H), 7.13 (dt, J = 8.3, 2.1 Hz, 1H), 6.90 (dd, J = 8.4, 4.1 Hz, 1H), 6.77 (dd, J = 19.2, 1.6 Hz, 1H), 4.10 (q, J = 6.7 Hz, 1H), 3.85 – 3.79 (m, 1H),



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3.78 – 3.69 (m, 2H), 3.45 – 3.35 (m, 1H), 3.34 (s, 2H), 3.32 (s, 3H), 2.84 (t, J = 6.5 Hz, 1H), 2.74 – 2.66 (m, 1H), 2.49 (s, 3H), 2.37 (s, 3H), 2.24 (s, 3H), 1.81 – 1.70 (m, 1H), 1.69 – 1.59 (m, 1H), 1.60 – 1.40 (m, 5H), 1.31 – 1.24 (m, 1H), 0.95 (dd, J = 6.8, 2.7 Hz, 3H);

13C

NMR (126 MHz,

CDCl3) δ 168.97 (d, J = 3.3 Hz), 162.25 (d, J = 9.6 Hz), 156.83 (d, J = 12.6 Hz), 154.40 (d, J = 3.1 Hz), 139.64 (d, J = 14.7 Hz), 135.56 (d, J = 15.1 Hz), 135.46, 132.25 (d, J = 10.4 Hz), 130.15, 130.11, 125.57 (2 × C), 121.97 (d, J = 19.9 Hz), 120.14, 116.02 (d, J = 9.6 Hz), 114.60 (d, J = 10.3 Hz), 58.93, 57.63, 56.15, 54.45, 45.63, 45.16, 37.62, 30.71, 30.25, 29.16, 23.94, 23.48, 21.23, 14.07; HRMS (ESI) m/z [M + H]+ calcd for (C30H40N7O2+) 530.3238, found 530.3244; retention time 3.01 min, > 99% pure. (R)-4-cyclopentyl-6-(3-(4-ethylpiperazine-1-carbonyl)-1-(p-tolyl)-1H-1,2,4-triazol-5-yl)-1,3-di methyl-3,4-dihydroquinoxalin-2(1H)-one (16). White solid, 58% yield. 1H NMR (400 MHz, CDCl3) δ 7.30 – 7.26 (m, 2H), 7.25 – 7.22 (m, 2H), 7.15 (dd, J = 8.3, 1.8 Hz, 1H), 6.90 (d, J = 8.4 Hz, 1H), 6.86 (d, J = 1.8 Hz, 1H), 4.13 (q, J = 6.8 Hz, 1H), 4.05 – 3.95 (m, 2H), 3.94 – 3.84 (m, 2H), 3.50 – 3.41 (m, 1H), 3.35 (s, 3H), 2.59 – 2.51 (m, 4H), 2.47 (q, J = 7.2 Hz, 2H), 2.40 (s, 3H), 1.84 – 1.75 (m, 1H), 1.73 – 1.64 (m, 1H), 1.62 – 1.44 (m, 5H), 1.36 – 1.28 (m, 1H), 1.11 (t, J = 7.2 Hz, 3H), 0.98 (d, J = 6.8 Hz, 3H); MS (ESI) [M+H]+: 542.42; retention time 2.98 min, > 99% pure. (R)-4-cyclopentyl-6-(3-(4-isopropylpiperazine-1-carbonyl)-1-(p-tolyl)-1H-1,2,4-triazol-5-yl)-1 ,3-dimethyl-3,4-dihydroquinoxalin-2(1H)-one (17). White solid, 62% yield. 1H NMR (400 MHz, CDCl3) δ 7.30 – 7.26 (m, 2H), 7.26 – 7.22 (m, 2H), 7.15 (dd, J = 8.3, 1.9 Hz, 1H), 6.91 (d, J = 8.4 Hz, 1H), 6.84 (d, J = 1.8 Hz, 1H), 4.13 (q, J = 6.8 Hz, 1H), 4.01 – 3.94 (m, 2H), 3.91 – 3.83 (m, 2H), 3.48 – 3.40 (m, 1H), 3.34 (s, 3H), 2.83 – 2.70 (m, 1H), 2.69 – 2.57 (m, 4H), 2.39 (s, 3H),


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1.84 – 1.75 (m, 1H), 1.72 – 1.64 (m, 1H), 1.63 – 1.44 (m, 5H), 1.34 – 1.27 (m, 1H), 1.06 (d, J = 6.6 Hz, 6H), 0.98 (d, J = 6.8 Hz, 3H);

13C

NMR (151 MHz, CDCl3) δ 169.10, 160.58, 156.87,

154.47, 139.62, 135.70, 135.53, 132.26, 130.17 (2 × C), 125.69 (2 × C), 122.18, 120.22, 116.16, 114.58, 59.01, 54.84, 54.58, 49.31, 48.32, 47.45, 42.75, 30.80, 30.35, 29.25, 24.03, 23.56, 21.32, 18.45 (2 × C), 14.11; MS (ESI) [M+H]+: 556.44; retention time 3.04 min, > 99% pure. (R)-4-cyclopentyl-6-(3-(4-cyclopropylpiperazine-1-carbonyl)-1-(p-tolyl)-1H-1,2,4-triazol-5-yl )-1,3-dimethyl-3,4-dihydroquinoxalin-2(1H)-one (18). White solid, 63% yield. 1H NMR (400 MHz, CDCl3) δ 7.30 – 7.21 (m, 4H), 7.15 (d, J = 7.2 Hz, 1H), 6.90 (d, J = 8.4 Hz, 1H), 6.85 (s, 1H), 4.21 – 4.04 (m, 1H), 3.93 – 3.86 (m, 2H), 3.84 – 3.76 (m, 2H), 3.50 – 3.39 (m, 1H), 3.34 (s, 3H), 2.75 – 2.62 (m, 4H), 2.39 (s, 3H), 2.28 – 2.15 (m, 1H), 1.82 – 1.74 (m, 1H), 1.69 – 1.63 (m, 2H), 1.58 – 1.46 (m, 5H), 0.97 (d, J = 6.7 Hz, 3H), 0.87 – 0.82 (m, 2H), 0.49 – 0.43 (m, 2H); 13C NMR (126 MHz, CDCl3) δ 169.05, 160.68, 156.92, 154.41, 139.56, 135.72, 135.50, 132.24, 130.13 (2 × C), 125.66 (2 × C), 122.19, 120.20, 116.16, 114.53, 59.03, 54.54, 53.77, 52.97, 47.21, 42.53, 38.41, 30.77, 30.34, 29.77, 29.21, 24.00, 23.53, 21.28, 14.12, 5.99; MS (ESI) [M+H]+: 554.44; retention time 3.00 min, > 99% pure. (R)-4-cyclopentyl-6-(3-((3S,5R)-3,5-dimethylpiperazine-1-carbonyl)-1-(p-tolyl)-1H-1,2,4-tria zol-5-yl)-1,3-dimethyl-3,4-dihydroquinoxalin-2(1H)-one (19). White solid, 59% yield. 1H NMR (400 MHz, CDCl3) δ 7.30 –7.21 (m, 4H), 7.16 – 7.11 (m, 1H), 6.90 (dd, J = 8.3, 2.7 Hz, 1H), 6.85 (d, J = 7.3 Hz, 1H), 4.68 (d, J = 12.9 Hz, 1H), 4.44 (d, J = 11.9 Hz, 1H), 4.12 (q, J = 6.8 Hz, 1H), 3.49 – 3.39 (m, 1H), 3.33 (s, 3H), 3.04 – 2.89 (m, 2H), 2.89 – 2.78 (m, 1H), 2.48 – 2.40 (m, 1H), 2.38 (s, 3H), 2.34 (s, 1H), 1.82 – 1.74 (m, 1H), 1.72 – 1.63 (m, 1H), 1.62 – 1.42 (m, 6H), 1.14 (d, J = 6.2 Hz, 3H), 1.07 (d, J = 5.8 Hz, 3H), 0.97 (d, J = 6.6 Hz, 3H)；13C NMR (151 MHz, CDCl3)



δ 169.04, 160.50(d, J = 5.1 Hz), 156.88, 154.42, 139.60, 135.63, 135.50, 132.22, 130.13 (2 × C), 125.63 (2 × C), 122.10, 120.15, 116.07, 114.55 (d, J = 4.2 Hz), 58.95, 54.52, 53.49 (d, J = 4.0 Hz), 51.62, 50.79, 48.70, 30.77, 30.32(d, J = 4.3 Hz), 29.21, 23.98, 23.51, 21.28, 19.38, 19.19, 14.07(d, J = 5.5 Hz) ; MS (ESI) [M+H]+: 542.42; retention time 2.99 min, > 98% pure. (R)-4-cyclopentyl-1,3-dimethyl-6-(3-(4-methyl-1,4-diazepane-1-carbonyl)-1-(p-tolyl)-1H-1,2,4 -triazol-5-yl)-3,4-dihydroquinoxalin-2(1H)-one (20). White solid, 62% yield. 1H NMR (400 MHz, CDCl3) δ 7.30 – 7.22 (m, 4H), 7.14 (ddd, J = 8.1, 6.2, 1.8 Hz, 1H), 6.91 (d, J = 8.4 Hz, 1H), 6.86 (dd, J = 3.9, 1.8 Hz, 1H), 4.18 – 4.10 (m, 1H), 4.00 – 3.95 (m, 1H), 3.91 (t, J = 6.4 Hz, 1H), 3.89 – 3.86 (m, 1H), 3.83 (t, J = 6.4 Hz, 1H), 3.51 – 3.41 (m, 1H), 3.35 (s, 3H), 2.86 – 2.78 (m, 2H), 2.73 – 2.64 (m, 2H), 2.44 – 2.38 (m, 6H), 2.09 – 1.98 (m, 2H), 1.85 – 1.76 (m, 1H), 1.73 – 1.65 (m, 1H), 1.62 – 1.46 (m, 5H), 1.34 – 1.28 (m, 1H), 0.99 (dd, J = 6.8, 1.3 Hz, 3H); 13C NMR (126 MHz, CDCl3) δ 169.08, 162.31, 157.40, 154.38 (d, J = 6.4 Hz), 139.56, 135.80, 135.57, 132.28, 130.17 (2 × C), 125.68 (2 × C), 122.29, 120.21 (d, J = 4.5 Hz), 116.19 (d, J = 3.3 Hz), 114.56, 59.98, 59.08, 57.53 (d, J = 22.2 Hz), 56.64, 54.59, 48.48, 46.69 (d, J = 13.2 Hz), 46.15, 30.84, 30.41, 29.26, 24.06, 23.58, 21.33, 14.19; HRMS (ESI) m/z [M + H]+ calcd for (C31H40N7O2+) 542.3238, found 542.323; retention time 2.97 min, > 99% pure. (3R)-4-cyclopentyl-1,3-dimethyl-6-(3-(8-methyl-3,8-diazabicyclo[3.2.1]octane-3-carbonyl)-1-( p-tolyl)-1H-1,2,4-triazol-5-yl)-3,4-dihydroquinoxalin-2(1H)-one (21). White solid, 67% yield. 1H

NMR (400 MHz, CDCl3) δ 7.30– 7.20 (m, 4H), 7.14 (d, J = 8.2 Hz, 1H), 6.89 (d, J = 8.4 Hz,

1H), 6.85 (s, 1H), 5.15 (s, 1H), 4.87 (d, J = 5.9 Hz, 1H), 4.12 (q, J = 6.7 Hz, 1H), 3.51 – 3.39 (m, 1H), 3.33 (s, 3H), 2.74 (d, J = 10.1 Hz, 2H), 2.50 – 2.42 (m, 2H), 2.38 (s, 3H), 2.25 (s, 3H), 2.03 – 1.91 (m, 4H), 1.83 – 1.74 (m, 1H), 1.71 – 1.63 (m, 1H), 1.61 – 1.42 (m, 6H), 0.97 (d, J = 6.8 Hz,


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3H);

13C

NMR (126 MHz, CDCl3) δ 169.03, 157.24, 157.01, 154.41, 139.52, 135.80, 135.44,

132.18, 130.08 (2 × C), 125.67 (2 × C), 122.27, 120.20, 116.18, 114.49, 61.95, 60.28, 59.03, 56.44, 54.57, 52.85, 45.18, 30.74, 30.32, 29.20, 28.66, 26.78, 23.97, 23.52, 21.28, 14.11; MS (ESI) [M+H]+: 554.44; retention time 2.98 min, > 99% pure. (3R)-4-cyclopentyl-1,3-dimethyl-6-(3-(3-methyl-3,8-diazabicyclo[3.2.1]octane-8-carbonyl)-1-( p-tolyl)-1H-1,2,4-triazol-5-yl)-3,4-dihydroquinoxalin-2(1H)-one (22). White solid, 60% yield. 1H

NMR (400 MHz, CDCl3) δ 7.29 – 7.21 (m, 4H), 7.15 (d, J = 8.3 Hz, 1H), 6.90 (d, J = 8.4 Hz,

1H), 6.83 (t, J = 2.0 Hz, 1H), 5.21 (s, 1H), 4.88 (d, J = 6.2 Hz, 1H), 4.12 (q, J = 6.8 Hz, 1H), 3.47 – 3.38 (m, 1H), 3.33 (s, 3H), 2.85 – 2.77 (m, 2H), 2.50 (dd, J = 15.8, 11.1 Hz, 2H), 2.38 (s, 3H), 2.29 (d, J = 2.2 Hz, 3H), 2.06 – 1.86 (m, 4H), 1.82 – 1.73 (m, 1H), 1.73 – 1.63 (m, 1H), 1.60 – 1.42 (m, 6H), 0.96 (d, J = 6.8 Hz, 3H)； 13C NMR (151 MHz, CDCl3) δ 169.03, 157.06, 156.97, 154.50, 139.59, 135.69, 135.43, 132.20, 130.11 (2 × C), 125.65 (2 × C), 122.14, 120.19, 116.12, 114.54, 61.83, 60.20, 58.96, 56.33, 54.56, 52.81, 45.12, 30.73, 30.28, 29.19, 28.52, 26.59, 23.96, 23.50, 21.27, 14.06 (d, J = 2.9 Hz); MS (ESI) [M+H]+: 554.42; retention time 3.01 min, > 99% pure. (3R)-4-cyclopentyl-1,3-dimethyl-6-(3-(octahydropyrrolo[1,2-a]pyrazine-2-carbonyl)-1-(p-toly l)-1H-1,2,4-triazol-5-yl)-3,4-dihydroquinoxalin-2(1H)-one (23). White solid, 62% yield. 1H NMR (400 MHz, CDCl3) δ 7.31 – 7.20 (m, 4H), 7.14 (d, J = 8.1 Hz, 1H), 6.90 (d, J = 8.4 Hz, 1H), 6.88 – 6.83 (m, 1H), 4.84 (dd, J = 54.8, 12.9 Hz, 1H), 4.56 (dd, J = 26.1, 13.0 Hz, 1H), 4.12 (q, J = 6.9 Hz, 1H), 3.49 – 3.39 (m, 1H), 3.34 (s, 3H), 3.11 (t, J = 8.5 Hz, 2H), 3.07 – 2.98 (m, 1H), 2.70 – 2.61 (m, 1H), 2.39 (s, 3H), 2.30 (t, J = 11.1 Hz, 1H), 2.23 – 2.14 (m, 1H), 2.12 – 2.01 (m, 1H), 1.95 – 1.63 (m, 6H), 1.62 – 1.41 (m, 6H), 0.97 (d, J = 6.7 Hz, 3H); MS (ESI) [M+H]+:



554.46; retention time 3.09 min, > 99% pure. (R)-6-(3-(8-azaspiro[4.5]decane-8-carbonyl)-1-(p-tolyl)-1H-1,2,4-triazol-5-yl)-4-cyclopentyl-1 ,3-dimethyl-3,4-dihydroquinoxalin-2(1H)-one (24). White solid, 63% yield. 1H NMR (400 MHz, CDCl3) δ 7.28 – 7.24 (m, 2H), 7.21 (d, J = 8.3 Hz, 2H), 7.13 (dd, J = 8.3, 1.9 Hz, 1H), 6.88 (d, J = 8.4 Hz, 1H), 6.84 (d, J = 1.8 Hz, 1H), 4.11 (q, J = 6.8 Hz, 1H), 3.80 – 3.70 (m, 4H), 3.48 – 3.38 (m, 1H), 3.32 (s, 3H), 2.37 (s, 3H), 1.83 – 1.73 (m, 1H), 1.69 – 1.58 (m, 5H), 1.56 – 1.43 (m, 13H), 1.33 – 1.24 (m, 1H), 0.96 (d, J = 6.8 Hz, 3H) ； 13C NMR (126 MHz, CDCl3) δ 168.99, 160.82, 157.27, 154.26, 139.39, 135.73, 135.42, 132.12, 130.04 (2 × C), 125.60 (2 × C), 122.27, 120.15, 116.13, 114.46, 58.98, 54.49, 45.36, 41.48, 40.54, 38.21, 37.78, 37.73, 37.01, 30.72, 30.29, 29.16, 24.34 (2 × C), 23.95, 23.48, 21.23, 14.07; MS (ESI) [M+H]+: 567.70; retention time 4.75 min, > 95% pure. (R)-6-(3-(2-oxa-8-azaspiro[4.5]decane-8-carbonyl)-1-(p-tolyl)-1H-1,2,4-triazol-5-yl)-4-cyclop entyl-1,3-dimethyl-3,4-dihydroquinoxalin-2(1H)-one (25). White solid, 65% yield. 1H NMR (400 MHz, CDCl3) δ 7.31 – 7.26 (m, 2H), 7.23 (d, J = 8.3 Hz, 2H), 7.14 (dd, J = 8.3, 1.5 Hz, 1H), 6.90 (d, J = 8.4 Hz, 1H), 6.85 (s, 1H), 4.13 (q, J = 6.8 Hz, 1H), 3.89 (t, J = 7.1 Hz, 3H), 3.80 – 3.69 (m, 2H), 3.60 (d, J = 1.0 Hz, 2H), 3.47 – 3.39 (m, 1H), 3.34 (s, 3H), 2.39 (s, 3H), 1.86 – 1.76 (m, 4H), 1.72 – 1.65 (m, 5H), 1.60 – 1.45 (m, 6H), 0.98 (d, J = 6.8 Hz, 3H); 13C NMR (126 MHz, CDCl3) δ 169.06, 160.90, 157.08, 154.43, 139.56, 135.74, 135.52, 132.26, 130.14 (2 × C), 125.66 (2 × C), 122.23, 120.20, 116.16, 114.54, 67.42, 59.05, 54.55, 45.37, 42.38, 40.73, 37.33, 37.29, 35.78, 34.60, 30.79, 30.36, 29.23, 24.02, 23.55, 21.30, 14.15; MS (ESI) [M+H]+: 569.49; retention time 3.68 min, > 98% pure. tert-butyl((S)-5-(5-((R)-4-cyclopentyl-1,3-dimethyl-2-oxo-1,2,3,4-tetrahydroquinoxalin-6-yl)-


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1-(p-tolyl)-1H-1,2,4-triazole-3-carbonyl)-5-azaspiro[2.4]heptan-7-yl)carbamate (26). White solid, 58% yield. 1H NMR (400 MHz, CDCl3) δ 7.27 – 7.19 (m, 5H), 7.16 – 7.09 (m, 1H), 6.87 (dd, J = 8.4, 3.2 Hz, 1H), 6.82 (d, J = 3.5 Hz, 1H), 4.89 – 4.79 (m, 1H), 4.29 (dd, J = 12.1, 5.3 Hz, 1H), 4.18 – 4.07 (m, 2H), 4.00 (dd, J = 12.9, 5.7 Hz, 1H), 3.92 – 3.85 (m, 1H), 3.83 – 3.69 (m, 2H), 3.53 – 3.38 (m, 2H), 3.31 (d, J = 2.7 Hz, 3H), 2.37 (s, 3H), 1.81 – 1.72 (m, 1H), 1.69 – 1.61 (m, 1H), 1.59 – 1.44 (m, 6H), 1.42 – 1.36 (m, 10H), 0.95 (dd, J = 6.8, 1.4 Hz, 3H); 13C NMR (126 MHz, CDCl3) δ 168.99 (d, J = 5.5 Hz), 159.83 (d, J = 33.6 Hz), 157.03 (d, J = 11.9 Hz), 155.48 (d, J = 9.6 Hz), 154.48, 139.53 (d, J = 9.1 Hz), 135.71, 135.41, 132.16, 130.07, 130.05, 125.59 (2 × C), 122.13 (d, J = 6.2 Hz), 120.20 (d, J = 12.2 Hz), 116.14 (d, J = 7.5 Hz), 114.46, 58.96, 56.70, 55.71, 54.75, 54.47, 52.71, 49.46, 30.73, 30.72, 30.29, 29.17, 28.36 (3 × C), 27.01, 23.95, 23.48, 21.24, 14.07 (d, J = 3.0 Hz); MS (ESI) [M+H]+: 640.31; retention time 4.10 min, > 99% pure. (R)-6-(3-((S)-7-amino-5-azaspiro[2.4]heptane-5-carbonyl)-1-(p-tolyl)-1H-1,2,4-triazol-5-yl)-4 -cyclopentyl-1,3-dimethyl-3,4-dihydroquinoxalin-2(1H)-one (27). White solid, 67% yield. 1H NMR (400 MHz, CDCl3) δ 7.30 – 7.22 (m, 4H), 7.20 – 7.11 (m, 1H), 6.93 – 6.88 (m, 1H), 6.87 – 6.84 (m, 1H), 4.36 – 4.19 (m, 1H), 4.13 (q, J = 6.8 Hz, 1H), 4.04 – 3.94 (m, 1H), 3.87 (dd, J = 37.4, 12.0 Hz, 1H), 3.66 (dd, J = 56.3, 13.2 Hz, 1H), 3.50 – 3.40 (m, 1H), 3.34 (d, J = 2.3 Hz, 3H), 3.18 – 3.11 (m, 1H), 2.40 (s, 3H), 2.00 (s, 2H), 1.85 – 1.74 (m, 1H), 1.73 – 1.64 (m, 1H), 1.61 – 1.43 (m, 6H), 0.98 (d, J = 6.8 Hz, 3H), 0.83 – 0.76 (m, 1H), 0.70 – 0.54 (m, 3H); 13C NMR (126 MHz, CDCl3) δ 169.11, 159.90, 157.29 (d, J = 6.4 Hz), 154.51 (d, J = 6.1 Hz), 139.59, 135.86, 135.52, 132.25, 130.14 (2 × C), 125.71 (2 × C), 122.30, 120.30 (d, J = 10.3 Hz), 116.23, 114.56, 59.06, 57.23, 56.68, 55.29, 54.86, 54.55, 52.90, 30.82, 30.38, 29.81, 29.25, 24.03, 23.57, 21.33, 14.17; MS (ESI) [M+H]+: 540.55; retention time 2.66 min, > 99% pure.



(R)-4-cyclopentyl-1,3-dimethyl-6-(3-(4-methylpiperazine-1-carbonyl)-1-phenyl-1H-1,2,4-tria zol-5-yl)-3,4-dihydroquinoxalin-2(1H)-one (28). White solid, 63% yield. 1H NMR (400 MHz, CDCl3) δ 7.47 – 7.36 (m, 5H), 7.11 (dd, J = 8.3, 1.5 Hz, 1H), 6.89 (d, J = 8.3 Hz, 1H), 6.84 (s, 1H), 4.11 (q, J = 6.7 Hz, 1H), 3.99 – 3.92 (m, 2H), 3.89 – 3.82 (m, 2H), 3.47 – 3.37 (m, 1H), 3.33 (s, 3H), 2.54 – 2.44 (m, 4H), 2.31 (s, 3H), 1.82 – 1.72 (m, 1H), 1.70 – 1.61 (m, 1H), 1.60 – 1.42 (m, 5H), 1.34 – 1.25 (m, 1H), 0.96 (d, J = 6.8 Hz, 3H); 13C NMR (126 MHz, CDCl3) δ 169.01, 160.59, 156.96, 154.52, 138.14, 135.53, 132.29, 129.57(2 × C), 129.29, 125.78(2 × C), 122.03, 120.14, 116.13, 114.54, 59.02, 55.43, 54.65, 54.49, 47.06, 46.05, 42.40, 30.72, 30.32, 29.20, 23.96, 23.50, 14.16; MS (ESI)[M+H]+: 514.44; retention time 2.83 min, > 99% pure. (R)-4-cyclopentyl-6-(1-(2,4-dimethylphenyl)-3-(4-methylpiperazine-1-carbonyl)-1H-1,2,4-tria zol-5-yl)-1,3-dimethyl-3,4-dihydroquinoxalin-2(1H)-one (29). White solid, 59% yield. 1H NMR (400 MHz, CDCl3) δ 7.20 – 7.15 (m, 2H), 7.15 – 7.13 (m, 1H), 7.13 – 7.09 (m, 1H), 6.89 (d, J = 1.9 Hz, 1H), 6.86 (d, J = 8.5 Hz, 1H), 4.11 (q, J = 6.8 Hz, 1H), 4.00 – 3.94 (m, 2H), 3.89 – 3.82 (m, 2H), 3.40 – 3.33 (m, 1H), 3.31 (s, 3H), 2.54 – 2.50 (m, 2H), 2.50 – 2.46 (m, 2H), 2.38 (s, 3H), 2.32 (s, 3H), 1.98 (s, 3H), 1.83 – 1.75 (m, 1H), 1.74 – 1.66 (m, 1H), 1.66 – 1.55 (m, 2H), 1.55 – 1.44 (m, 3H), 1.36 – 1.27 (m, 1H), 0.95 (d, J = 6.8 Hz, 3H) ； 13C NMR (151 MHz, CDCl3) δ 169.04, 160.67, 156.77, 155.01, 140.48, 135.57, 135.13, 134.96, 132.22, 132.15, 127.98, 127.45, 122.09, 119.66, 115.09, 114.58, 58.95, 55.44, 54.69, 54.23, 47.08, 46.06, 42.43, 30.79, 30.35, 29.19, 23.94, 23.43, 21.33, 17.64, 14.17; MS (ESI) [M+H]+: 542.45; retention time 2.98 min, > 99% pure. (R)-4-cyclopentyl-6-(1-(3-fluoro-4-methylphenyl)-3-(4-methylpiperazine-1-carbonyl)-1H-1,2, 4-triazol-5-yl)-1,3-dimethyl-3,4-dihydroquinoxalin-2(1H)-one (30). White solid, 56% yield. 1H


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NMR (400 MHz, CDCl3) δ 7.22 (t, J= 8.0 Hz, 1H), 7.11 – 7.03 (m, 3H), 6.89 (d, J = 8.4 Hz, 1H), 6.83 (d, J = 1.7 Hz, 1H), 4.11 (q, J = 6.8 Hz, 1H), 3.94 – 3.88 (m, 2H), 3.87 – 3.79 (m, 2H), 3.53 – 3.43(m, 1H), 3.32 (s, 3H), 2.52 – 2.42 (m, 4H), 2.29 (s, 3H), 2.28 (d, J = 1.5 Hz, 3H), 1.84 – 1.75 (m, 1H), 1.72 – 1.62 (m, 1H), 1.60 – 1.43 (m, 5H), 1.37 – 1.24 (m, 1H), 0.96 (d, J = 6.8 Hz, 3H); 13C

NMR (151 MHz, CDCl3) δ 168.98, 160.39, 156.94, 154.52, 136.69 (d, J = 9.8 Hz), 135.54,

132.40, 132.05 (d, J = 5.8 Hz), 126.48 (d, J = 17.1 Hz), 121.73, 121.13 (d, J = 3.3 Hz), 120.11, 116.03, 114.58, 112.93, 112.76, 58.95, 55.37, 54.59, 54.54, 47.01, 46.01, 42.36, 30.68, 30.29, 29.17, 23.95, 23.46, 14.43 (d, J = 2.9 Hz), 14.02; HRMS (ESI) m/z [M + H]+ calcd for (C30H37FN7O2+) 546.2987, found 546.2984; retention time 2.97 min, > 99% pure. (R)-6-(1-(4-chlorophenyl)-3-(4-methylpiperazine-1-carbonyl)-1H-1,2,4-triazol-5-yl)-4-cyclope ntyl-1,3-dimethyl-3,4-dihydroquinoxalin-2(1H)-one (31). White solid, 66% yield. 1H NMR (400 MHz, CDCl3) δ 7.44 – 7.39 (m, 2H), 7.37 – 7.32 (m, 2H), 7.08 (dd, J = 8.3, 1.7 Hz, 1H), 6.91 (d, J = 8.3 Hz, 1H), 6.82 (d, J = 1.7 Hz, 1H), 4.14 (q, J = 6.8 Hz, 1H), 4.00 – 3.90 (m, 2H), 3.90 – 3.79 (m, 2H), 3.55 – 3.43 (m, 1H), 3.34 (s, 3H), 2.55 – 2.50 (m, 2H), 2.50 – 2.45 (m, 2H), 2.32 (s, 3H), 1.87 – 1.78 (m, 1H), 1.74 – 1.66 (m, 1H), 1.63 – 1.46 (m, 5H), 1.37 – 1.28 (m, 1H), 0.99 (d, J = 6.8 Hz, 3H);

13C

NMR (126 MHz, CDCl3) δ 169.00, 160.42, 157.21, 154.67, 136.56, 135.71,

135.35, 132.52, 129.80 (2 × C), 127.00 (2 × C), 121.74, 120.14, 116.06, 114.67, 59.11, 55.46, 54.68, 54.55, 47.07, 46.08, 42.45, 30.81, 30.43, 29.25, 24.05, 23.57, 14.22; MS (ESI) [M+H]+: 548.38; retention time 3.01 min, > 99% pure. 3-fluoro-4-nitro-N-(p-tolyl)benzamide (38). To a solution of compound 37 (2 g, 10.8 mmol) in anhydrous DMF (10 mL) was added HATU (4.1 g, 10.8 mmol), the mixture was stirred at rt for 30 min, then p-methylaniline (1.2 g, 10.8 mmol) and DIPEA (1.9 mL, 10.8 mmol) were added and



the mixture was stirred at room temperature overnight. The reaction was monitored by TLC. Upon completion, the reaction mixture was diluted with saturated NaHCO3 and extracted with EtOAc (3 × 40 mL). The combined organic extracts were washed with brine, dried (Na2SO4), and concentrated under reduced pressure. Purification by silica gel column chromatography (gradient elution, gradient 0 to 25% EtOAc/60–90 °C petroleum ether) gave compound 38 as a light yellow soild (2.2 g, 8.0 mmol, 74% yield). 1H NMR (400 MHz, DMSO-d6) δ 10.49 (s, 1H), 8.31 (t, J = 8.1 Hz, 1H), 8.10 (d, J = 12.0 Hz, 1H), 7.96 (d, J = 8.5 Hz, 1H), 7.65 (d, J = 8.4 Hz, 2H), 7.19 (d, J = 8.3 Hz, 2H), 2.29 (s, 3H); retention time 3.29 min, > 95% pure. 3-(cyclopropylamino)-4-nitro-N-(p-tolyl)benzamide (39). To a solution of compound 38 (2 g, 7.3 mmol) in 1,2-dichloroethane (10 mL) was added cyclopropylamine (1.0 mL, 14.6 mmol), then the mixture was heated to 80 ℃ for 12 h. The reaction was monitored by TLC. Upon completion, the solvent was evaporated, diluted with water and extracted with CH2Cl2 (3× 30 mL). The combined organic extracts were washed with brine, dried (Na2SO4), and concentrated under reduced pressure to give compound 39 as a red soild (2.1g, 6.7 mmol, 92% yield). 1H NMR (400 MHz, CDCl3) δ 8.23 (d, J = 8.8 Hz, 1H), 8.12 (s, 1H), 7.86 (s, 1H), 7.80 (s, 1H), 7.53 (d, J = 8.1 Hz, 2H), 7.19 (d, J = 8.3 Hz, 2H), 7.01 (d, J = 8.7 Hz, 1H), 2.69 – 2.62 (m, 1H), 2.35 (s, 3H), 1.00 – 0.94 (m, 2H), 0.71– 0.66 (m, 2H); retention time 3.45 min, > 95% pure. 3-(cyclopropylamino)-4-nitro-N-(p-tolyl)benzothioamide (41). To a solution of compound 39 (2 g, 6.4 mmol) in toluene (10 mL) was added Lawesson’s reagent (1.4 g, 3.4 mmol), then the mixture was heated to 110 ℃ overnight. The reaction was monitored by TLC. Upon completion, the solvent was evaporated and the residue was dissolved with CH2Cl2. Purification by silica gel column chromatography (gradient elution, gradient 0 to 15% EtOAc/60–90 °C petroleum ether)


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gave compound 41 as a red soild (1.8 g, 5.5 mmol, 86% yield). 1H NMR (400 MHz, DMSO-d6) δ 11.97 (s, 1H), 8.12 (d, J = 8.8 Hz, 2H), 7.73 (d, J = 8.2 Hz, 2H), 7.65 (s, 1H), 7.26 (d, J = 8.2 Hz, 2H), 7.07 (d, J = 8.8 Hz, 1H), 2.71 (s, 1H), 2.33 (s, 3H), 0.89 (d, J = 5.0 Hz, 2H), 0.68 (s, 2H). MS (ESI) [M+H]+: 328.11; LCMS m/z (ESI, positive) found [M + H]+ 328.09; retention time 3.68 min, > 95% pure. N-cyclopropyl-2-nitro-5-(4-(p-tolyl)-4H-1,2,4-triazol-3-yl)aniline (43). To a solution of compound 41 (1.8 g, 5.5 mmol) in methol (10 mL) and THF (20 mL) was added hydrazine hydrate (2.7 mL, 55.0 mmol), then the mixture was stirred at rt for 4 h. The reaction was monitored by TLC. Upon completion, the solvent was evaporated and the residue was dissolved with DMF (5 mL), then addition of acetic acid (4 mL) and trimethyl orthoformate (1.6 mL, 14.8 mmol). The reaction mixture was stirred at rt overnight. The reaction was monitored by TLC. Upon completion, the residue was basified with saturated NaHCO3 to pH 7 – 8, diluted with water and extracted with EtOAc (3 × 40 mL). Combined organic layers were washed with brine, and purification by silica gel column chromatography (gradient elution, gradient 0 to 30% EtOAc/60– 90 °C petroleum ether) gave compound 43 as a red soild (1.5 g, 4.5 mmol, 82% yield). 1H NMR (400 MHz, CDCl3) δ 8.34 (s, 1H), 8.10 (d, J = 8.8 Hz, 1H), 8.03 (s, 1H), 7.45 (s, 1H), 7.31 (d, J = 7.9 Hz, 2H), 7.17 (d, J = 8.2 Hz, 2H), 6.81 (d, J = 8.8 Hz, 1H), 2.44 (s, 3H), 2.36 – 2.29 (m, 1H), 0.74 – 0.68 (m, 2H), 0.51 – 0.46 (m, 2H); retention time 3.41 min, > 95% pure. N-cyclopropyl-2-nitro-5-(1-(p-tolyl)-1H-tetrazol-5-yl)aniline (44). To a solution of compound 41 (2 g, 6.1 mmol) in THF (10 mL) were added Hg(OAc)2 (3.9 g, 12.2 mmol) and azidotrimethylsilane (8.0 mL, 61.2 mmol ) at 0 °C, then the mixture was stirred at 0 °C for 4 h. The reaction was monitored by TLC. Upon completion, the mixture was diluted with water and



extracted with EtOAc (3 × 40 mL). Combined organic layers were washed with brine, and purification by silica gel column chromatography (gradient elution, gradient 0 to 10% EtOAc/60– 90 °C petroleum ether) gave compound 44 as a red soild (1.8 g, 5.4 mmol, 88% yield). 1H NMR (400 MHz, CDCl3) δ 8.14 (d, J = 8.9 Hz, 1H), 8.03 (s, 1H), 7.53 (d, J = 1.8 Hz, 1H), 7.35 (d, J = 8.2 Hz, 2H), 7.32 – 7.27 (m, 2H), 6.86 (dd, J = 8.9, 1.9 Hz, 1H), 2.46 (s, 3H), 2.39 – 2.31 (m, 1H), 0.76 – 0.68 (m, 2H), 0.55 – 0.44 (m, 2H); MS(ESI)[M+H] +: 337.09; retention time 3.43 min, > 95% pure. Compound 45 was prepared from compound 40 using the same manner as compound 44. N1-cyclopropyl-5-(4-(p-tolyl)-4H-1,2,4-triazol-3-yl)benzene-1,2-diamine (46). To a solution of compound 43 (1.5 g, 4.5 mmol) in con. HCl (8 mL) was added tin chloride dihydrate (5.0 g, 22.3 mmol), then the mixture was stirred at rt for 3 h. The reaction was monitored by TLC. Upon completion, the solvent was evaporated and the residue was basified with saturated NaHCO3 to pH 7 – 8, diluted with water and extracted with EtOAc (3 × 20 mL). The combined organic extracts were washed with brine, dried (Na2SO4), and concentrated under reduced pressure to give compound 46 as yellow powder (1.2 g, 3.9 mmol, 87% yield). retention time 3.44 min, > 95% pure. Compound 47 was prepared from compound 44 using the same manner as compound 46 and compound 48 was prepared from compound 45 using the same manner as compound 46. 4-cyclopropyl-3-methyl-6-(4-(p-tolyl)-4H-1,2,4-triazol-3-yl)-3,4-dihydroquinoxalin-2(1H)-one (49). To a stirred solution of compound 46 (1.2 g, 3.9 mmol) in anhydrous dichloromethane (8 mL) at 0 °C were slowly added DIPEA (1.4 mL, 7.9 mmol) and 2-bromopropanoyl bromide (0.6 mL, 5.9 mmol). After the addition was completed the cooling bath was removed and the reaction


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was stirred for 2 h at rt. The reaction mixture was cooled again to 0 °C, diluted with water, and extracted with EtOAc (3 × 50 mL). The combined organic fractions were washed with brine, dried (Na2SO4), and concentrated by evaporation under reduced pressure. The residue and DIPEA (8 mL) in acetonitrile (20 mL) were heated to 80 °C overnight. The reaction was monitored by TLC. Upon completion, the reaction mixture was diluted with water, and extracted with EtOAc (3 × 40 mL). The combined organic extracts were washed with brine, dried (Na2SO4), and concentrated under reduced pressure. Purification by silica gel column chromatography (gradient elution, gradient 0 to 25% EtOAc/60–90 °C petroleum ether) gave compound 49 as a white soild (0.8 g, 2.2 mmol, 56% yield, 2 steps). retention time 3.39 min, > 95% pure. Compound 50 was prepared from compound 47 using the same manner as compound 49 and compound 51 was prepared from compound 48 using the same manner as compound 49. methyl 3-fluoro-4-nitrobenzoate (52). To a solution of compound 37 (5.0 g, 27.0 mmol) in MeOH (12.9 mL) was added SOCl2 (4.1 mL, 56.7 mmol) at 0 °C. The mixture was heated to 60 °C for 24 hours and monitored by TLC. Upon completion, the solvent was evaporated. The residue was basified with saturated NaHCO3 to pH 7 – 8, diluted with water and extracted with EtOAc (3 × 40 mL). Combined organic layers were washed with brine, dried with Na2SO4 and evaporated to afford compound 52 (5.1 g, 25.7 mmol, 95% yield) as a light yellow solid. 1H NMR (400 MHz, CDCl3) δ 8.11 (t, J = 7.8 Hz, 1H), 7.97 (s, 1H), 7.94 (d, J = 3.0 Hz, 1H), 3.98 (s, 3H); MS m/z (EI) found [M]+ 199; retention time 3.28 min, > 95% pure. methyl 3-(cyclopropylamino)-4-nitrobenzoate (53). A solution of compound 52 (5.0 g, 25.1 mmol) and cyclopropanamine (3.5 mL, 50.3 mmol) in 1,2-dichloroethane (20 mL) were heated to 80 °C for 12 h. The reaction was monitored by TLC. Upon completion, the solvent was



evaporated, diluted with water and extracted with dichloromethane (3 × 50 mL). The combined organic layers were washed with brine, dried over sodium sulfate, filtered and excess solvent was removed via rotary evaporator to give compound 53 (5.4 g, 22.9 mmol, 91% yield) as a red solid. 1H

NMR (400 MHz, DMSO-d6) δ 8.15 (d, J = 8.8 Hz, 1H), 8.03 (s, 1H), 7.92 (d, J = 1.7 Hz, 1H),

7.22 (dd, J = 8.8, 1.8 Hz, 1H), 3.89 (s, 3H), 2.72 – 2.65 (m, 1H), 0.94 – 0.84 (m, 2H), 0.70 – 0.60 (m, 2H); MS m/z (EI) found [M]+ 236; retention time 3.80 min, > 99 % pure. methyl 4-amino-3-(cyclopropylamino)benzoate (54). To a solution of compound 53 (5.4 g, 22.9 mmol) and iron (5.1 g, 91.8 mmol) in EtOH (20 mL) was added ammonium chloride (4.9 g, 91.8 mmol) in 5 mL water at 50 – 55 °C. The reaction mixture was heated to 80 °C for 1 hour, then cooled to room temperature, and filtered through Celite. The filtrate was basified with saturated NaHCO3 solution to pH 7 – 8, diluted with water, and extracted with EtOAc (3 × 40 mL). The combined organic extracts were washed with brine, dried (Na2SO4), and concentrated under reduced pressure. The crude proudct was purified by flash column chromatography (gradient elution, gradient 0 to 20% EtOAc/60–90 °C petroleum ether) to give compound 54 (3.5 g, 17.0 mmol, 74% yield) as brown liquid; LCMS m/z (ESI, positive) found [M + H]+ 207.61; retention time 2.36 min, > 95% pure. methyl 4-cyclopropyl-3-methyl-2-oxo-1,2,3,4-tetrahydroquinoxaline-6-carboxylate (55). To a stirred solution of compound 54 (3.5 g, 17.0 mmol) in anhydrous dichloromethane (15 mL) at 0 °C were slowly added DIPEA (5.9 mL, 33.9 mmol) and 2-bromopropanoyl bromide (2.1 mL, 20.4 mmol). After the addition was completed the cooling bath was removed and the reaction stirred for 2 h at rt. The reaction mixture was cooled again to 0 °C, diluted with water, and extracted with EtOAc (3 × 30 mL). The combined organic fractions were washed with brine, dried


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(Na2SO4), and concentrated by evaporation under reduced pressure to give the intermediate. The intermediate and DIPEA (5.9 mL, 33.9 mmol) in acetonitrile (15 mL) were heated to 80 °C overnight. The reaction was monitored by TLC. Upon completion, the reaction mixture was diluted with water, and extracted with EtOAc (3 × 40 mL). The combined organic extracts were washed with brine, dried (Na2SO4), and concentrated under reduced pressure. Purification by silica gel column chromatography (gradient elution, gradient 0 to 25% EtOAc/60–90 °C petroleum ether) gave compound 55 (1.8 g, 6.9 mmol, 41% yield, 2 steps) as a white solid. 1H NMR (400 MHz, CDCl3) δ 8.61 (s, 1H), 7.75 (d, J = 1.6 Hz, 1H), 7.53 (dd, J = 8.1, 1.8 Hz, 1H), 6.78 (d, J = 8.1 Hz, 1H), 4.13 – 4.05 (m, 1H), 3.90 (s, 3H), 2.52 – 2.45 (m, 1H), 1.24 (d, J = 6.9 Hz, 3H), 1.10 – 1.02 (m, 1H), 0.86 – 0.78 (m, 1H), 0.68 – 0.60 (m, 1H), 0.60 – 0.51 (m, 1H); MS m/z (ESI, positive) found [M + H]+ 261.20; retention time 3.01 min, > 99% pure. methyl 4-cyclopropyl-1,3-dimethyl-2-oxo-1,2,3,4-tetrahydroquinoxaline-6-carboxylate (56). To a solution of compound 55 (1.8 g, 6.9 mmol) in anhydrous DMF (10 mL) was added NaH (0.8 g, 20.8 mmol) at 0 °C, the mixture was stirred at 0 °C for 30 min, then iodomethane (0.7 mL, 10.4 mmol) was added and stirred at room temperature for another 2 h. The reaction was monitored by TLC. Upon completion, the reaction mixture was acidified with 1 N aq. HCl to pH 7 – 8, diluted with water and extracted with EtOAc (3 × 20 mL). The combined organic fractions were washed with brine, dried over Na2SO4, and concentrated by evaporation under reduced pressure. Purification by silica gel column chromatography (gradient elution, gradient 0 to 25% EtOAc/60– 90 °C petroleum ether) gave compound 56 (1.5 g, 5.5 mmol, 79% yield) as a white solid. 1H NMR (400 MHz, CDCl3) δ 7.75 (d, J = 1.9 Hz, 1H), 7.59 (dd, J = 8.3, 1.9 Hz, 1H), 6.93 (d, J = 8.4 Hz, 1H), 4.12 (q, J = 6.9 Hz, 1H), 3.89 (s, 3H), 3.35 (s, 3H), 2.48 – 2.42 (m, 1H), 1.15 (d, J = 6.9 Hz,



Page 62 of 88

3H), 1.07 – 0.99 (m, 1H), 0.84 – 0.76 (m, 1H), 0.66 – 0.58 (m, 1H), 0.56 – 0.48 (m, 1H); LCMS m/z (ESI, positive) found [M + H]+ 275.18; retention time 3.38 min, > 99% pure. 4-cyclopropyl-1,3-dimethyl-2-oxo-1,2,3,4-tetrahydroquinoxaline-6-carbohydrazide (57). To a solution of compound 56 (1.5 g, 5.5 mmol) in EtOH (10 mL) was added hydrazine hydrate (1.3 mL, 27.4 mmol) at 0 °C, then the mixture was heated to 80 °C for 24 h. The reaction was monitored by TLC. Upon completion, the solvent was evaporated to give compound 57 (1.3 g, 4.7 mmol, 87% yield) as a white solid. retention time 3.32 min, > 95% pure. 2-(3-fluoro-4-nitrophenyl)-3-(p-tolyl)thiazolidin-4-one

(59).

To

a

solution

of

3-fluoro-4-nitrobenzaldehyde (6.3 g, 37.4 mmol) in THF (20 mL) was added p-toluidine (2 g, 18.7 mmol) at 0 °C. The reaction mixture was stirred at 0 °C for 10 min, then thioglycolic acid (3.9 mL, 56.1 mmol) was added, and the mixture further stirred at 0 °C for 10 min. Then N,N'-dicyclohexylcarbodiimide (4.6 g, 22.4 mmol) was added and the mixture was stirred at rt overnight. The reaction was monitored by TLC. Upon completion, the mixture was filtered through Celite. The filtrate was concentrated under reduced pressure. The crude proudct was purified by flash column chromatography (gradient elution, gradient 0 to 10% EtOAc/60–90 °C petroleum ether) to give compound 59 (3.5 g, 10.5 mmol, 56 % yield) as light yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 8.06 (t, J = 8.1 Hz, 1H), 7.70 (d, J = 12.0 Hz, 1H), 7.47 (d, J = 8.5 Hz, 1H), 7.24 (d, J = 8.2 Hz, 2H), 7.12 (d, J = 8.2 Hz, 2H), 6.61 (s, 1H), 4.11 (d, J = 15.6 Hz, 1H), 3.89 (d, J = 15.6 Hz, 1H), 2.21 (s, 3H). retention time 3.24 min, > 95% pure. 2-(3-(cyclopropylamino)-4-nitrophenyl)-3-(p-tolyl)thiazolidin-4-one

(60).

A

solution

of

compound 59 (2 g, 6.0 mmol) and cyclopropanamine (0.8 mL, 12.0 mmol) in 1,2-dichloroethane (10 mL) were heated to 80 °C for 12 h. The reaction was monitored by TLC. Upon completion, the solvent was evaporated, diluted with water and extracted with dichloromethane (3 × 50 mL). The combined organic layers were washed with brine, dried over sodium sulfate, filtered and excess solvent was removed via rotary evaporator to give compound 60 (1.8 g, 4.9 mmol, 81%


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yield) as a red solid. 1H NMR (400 MHz, CDCl3) δ 8.11 (d, J = 8.9 Hz, 1H), 8.07 (s, 1H), 7.13 – 7.11 (m, 4H), 6.66 (dd, J = 8.9, 1.9 Hz, 1H), 6.05 (d, J = 1.0 Hz, 1H), 3.99 (dd, J = 15.8, 1.6 Hz, 1H), 3.87 (d, J = 15.8 Hz, 1H), 2.54– 2.47(m, 1H), 2.28 (s, 3H), 0.92 – 0.84 (m, 2H), 0.60 – 0.54 (m, 2H). retention time 3.31 min, > 95% pure. 2-(4-amino-3-(cyclopropylamino)phenyl)-3-(p-tolyl)thiazolidin-4-one (61). To a solution of compound 60 (1.5 g, 4.1 mmol) in con.HCl (4 mL) was added tin chloride dihydrate (4.4 g, 19.3 mmol), then the mixture was stirred at rt for 3 h. The reaction was monitored by TLC. Upon completion, the solvent was evaporated and the residue was basified with saturated NaHCO3 to pH 7 – 8, diluted with water and extracted with EtOAc (3 × 20 mL). The combined organic extracts were washed with brine, dried (Na2SO4), and concentrated under reduced pressure to give compound 61 as yellow powder (1.1 g, 3.2 mmol, 80 % yield). retention time 3.21 min, > 95% pure. 2-(4-cyclopropyl-3-methyl-2-oxo-1,2,3,4-tetrahydroquinoxalin-6-yl)-3-(p-tolyl)thiazolidin-4-o ne (62). To a stirred solution of compound 61 (1.1 g, 3.2 mmol) in anhydrous dichloromethane (10 mL) at 0 °C were slowly added DIPEA (1.1 mL, 6.5 mmol) and 2-bromopropanoyl bromide (0.4 mL, 3.9 mmol). After the addition was completed the cooling bath was removed and the reaction was stirred for 2 h at rt. The reaction was monitored by TLC. Upon completion, the reaction mixture was diluted with water, and extracted with EtOAc (3 × 20 mL). The combined organic fractions were washed with brine, dried (Na2SO4), and concentrated by evaporation under reduced pressure to give the intermediate. The intermediate and DIPEA (1.1 mL, 6.5 mmol) in acetonitrile (10 mL) were heated to 80 °C overnight. The reaction was monitored by TLC. Upon completion, the reaction mixture was diluted with water, and extracted with EtOAc (3 × 30 mL). The



combined organic extracts were washed with brine, dried (Na2SO4), and concentrated under reduced pressure. Purification by silica gel column chromatography (gradient elution, gradient 0 to 25% EtOAc/60–90 °C petroleum ether) gave compound 62 (0.1 g, 0.3 mmol, 9% yield, 2 steps) as a white solid. retention time 3.20 min, > 95% pure. (5-bromo-2-nitrophenyl)-D-alanine (68). To a solution of compound 32 (25 g, 113.6 mmol) in EtOH (150 mL) and H2O (50 mL) were added D-alanine (11.1 g, 125.0 mmol) and K2CO3 (17.3 g, 125.0 mmol). The reaction mixture was heated to 80 °C for 8 h. The reaction was monitored by TLC. Upon completion, the reaction mixture was acidified with 1 N aq. HCl to pH 2 – 3. Then the mixture was filtered and the solid was dried to give compound 68 (28.7 g, 99.3 mmol, 87 % yield) as yellow solid. 1H NMR (400 MHz, CDCl3) δ 8.35 (d, J = 6.9 Hz, 1H), 8.06 (d, J = 9.1 Hz, 1H), 6.90 (s, 1H), 6.85 (d, J = 9.2 Hz, 1H), 4.33 (p, J = 7.0 Hz, 1H), 1.67 (d, J = 7.0 Hz, 3H); retention time 3.08 min, > 95% pure. (R)-6-bromo-3-methyl-3,4-dihydroquinoxalin-2(1H)-one (69). To a solution of compound 68 (28.7 g, 99.3 mmol) and K2CO3 (27.4 g, 198.6 mmol) in H2O (500 mL) was added Na2S2O4 (86.5 g, 496.6 mmol). The reaction mixture was heated to 60 °C overnight. The reaction was monitored by TLC. Upon completion, the reaction mixture was acidified with 1 N aq. HCl to pH 7 – 8. Then the mixture was filtered and the solid was dried to give compound 69 (9 g, 37.3 mmol, 38 % yield) as yellow solid. 1H NMR (400 MHz, CDCl3) δ 9.42 (s, 1H), 6.89 – 6.83 (dd, J = 8.2, 2.0 Hz, 1H), 6.81 (s, 1H), 6.68 – 6.59 (dd, J = 13.1, 7.6 Hz, 1H), 4.02 (q, J = 6.7 Hz, 1H), 3.92 (s, 1H), 1.45 (d, J = 6.7 Hz, 3H); retention time 3.01 min, > 95% pure. (R)-6-bromo-4-cyclopentyl-3-methyl-3,4-dihydroquinoxalin-2(1H)-one (70). A solution of compound 69 (9 g, 37.3 mmol), phenylsilane (11.9 g, 113.1 mmol), cyclopentanone (10.0 mL,


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113.1 mmol) and dibutyltin dichloride (17.0 g, 56.0 mmol) in THF (100 mL) were stirred at rt for 10 h. The reaction was monitored by TLC. Upon completion, the solvent was evaporated, diluted with water and extracted with dichloromethane (3 × 100 mL). The combined organic layers were washed with brine, dried over sodium sulfate, and concentrated by evaporation under reduced pressure. Purification by silica gel column chromatography (gradient elution, gradient 0 to 25% EtOAc/60–90 °C petroleum ether) gave compound 70 (10.4 g, 33.6 mmol, 90% yield) as a white solid. 1H NMR (400 MHz, CDCl3) δ 9.68 (s, 1H), 6.92 (d, J = 1.9 Hz, 1H), 6.88 (dd, J = 8.2, 2.0 Hz, 1H), 6.69 (d, J = 8.2 Hz, 1H), 4.10 (q, J = 6.8 Hz, 1H), 3.88 – 3.75 (m, 1H), 2.08 – 1.94 (m, 2H), 1.78 – 1.55 (m, 6H), 1.14 (d, J = 6.8 Hz, 3H); MS (ESI) [M+H]+: 309.03; retention time 3.05 min, > 95% pure. (R)-6-bromo-4-cyclopentyl-1,3-dimethyl-3,4-dihydroquinoxalin-2(1H)-one (71). To a solution of compound 70 (10.4g, 33.6 mmol) in anhydrous DMF (50 mL) was added NaH (1.6 g, 67.3 mmol) at 0 °C, the mixture was stirred at 0 °C for 30 min, then iodomethane (3.1 mL, 50.4 mmol) was added and stirred at room temperature for another 2 h. The reaction was monitored by TLC. Upon completion, the reaction mixture was acidified with 1 N aq. HCl to pH 7 – 8, diluted with water and extracted with EtOAc (3 × 200 mL). The combined organic fractions were washed with brine, dried over Na2SO4, and concentrated by evaporation under reduced pressure. Purification by silica gel column chromatography (gradient elution, gradient 0 to 25% EtOAc/60–90 °C petroleum ether) gave compound 71 (10 g, 30.9 mmol, 92% yield) as a white solid. 1H NMR (400 MHz, CDCl3) δ 6.97 (dd, J = 8.4, 2.1 Hz, 1H), 6.92 (d, J = 2.1 Hz, 1H), 6.78 (d, J = 8.5 Hz, 1H), 4.17 (q, J = 6.8 Hz, 1H), 3.81 – 3.72 (m, 1H), 3.33 (s, 3H), 2.08 – 1.96 (m, 2H), 1.84 – 1.74 (m, 1H), 1.72 – 1.58 (m, 5H), 1.05 (d, J = 6.8 Hz, 3H); retention time 3.06 min, > 95% pure.



Page 66 of 88

(R)-4-cyclopentyl-1,3-dimethyl-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,4-dihydroq uinoxalin-2(1H)-one (72). To a solution of compound 71 (10 g, 31.0 mmol) and AcOK (6.1 g, 61.9 mmol) in DMSO (20 mL) was added bis(pinacolato)diboron (8.7 g, 34.1 mmol), and the mixture was bubbled with N2 for 5 min, then Pd(dppf)2Cl2.CH2Cl2 (1.3 g, 1.6 mmol) was added, the mixture was further bubbled with N2 for 5 min. Then the mixture was heated to 80 °C overnight. The reaction was monitored by TLC. Upon completion, the reaction mixture was diluted with water and extracted with EtOAc (3 × 100 mL). The combined organic fractions were washed with brine, dried over Na2SO4, and concentrated by evaporation under reduced pressure. Purification by silica gel column chromatography (gradient elution, gradient 0 to 15% EtOAc/60– 90 °C petroleum ether) gave compound 72 (9.5 g, 25.7 mmol, 83% yield) as a light yellow liquid. 1H

NMR (400 MHz, CDCl3) δ 7.36 (d, J = 8.3 Hz, 1H), 7.29 (s, 1H), 6.95 (d, J = 7.9 Hz, 1H),

4.17 (q, J = 6.8 Hz, 1H), 3.94 – 3.85 (m, 1H), 3.37 (s, 3H), 2.09 – 1.98 (m, 2H), 1.83 – 1.74 (m, 1H), 1.73 – 1.58 (m, 5H), 1.35 (s, 12H), 1.03 (d, J = 6.9 Hz, 3H); MS (ESI) [M+H]+: 371.14; retention time 3.07 min, > 95% pure. ethyl(R)-5-(4-cyclopentyl-1,3-dimethyl-2-oxo-1,2,3,4-tetrahydroquinoxalin-6-yl)-1-(p-tolyl)-1 H-1,2,4-triazole-3-carboxylate (73). To a solution of compound 72 (1 g, 2.7 mmol) and NaHCO3 (0.5

g,

5.4

mmol)

in

THF

(10

mL)

and

H2O

(3

mL)

was

added

ethyl

5-bromo-1-(p-tolyl)-1H-1,2,4-triazole-3-carboxylate (1.2 g, 3.8 mmol), and the mixture was bubbled with N2 for 5 min, then Pd(dppf)2Cl2.CH2Cl2 (0.2 g, 0.3 mmol) was added, the mixture was further bubbled with N2 for 5 min. Then the mixture was heated to 80 °C overnight. The reaction was monitored by TLC. Upon completion, the reaction mixture was diluted with water and extracted with EtOAc (3 × 50 mL). The combined organic fractions were washed with brine,


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dried over Na2SO4, and concentrated by evaporation under reduced pressure. Purification by silica gel column chromatography (gradient elution, gradient 0 to 20% EtOAc/60–90 °C petroleum ether) gave compound 73 (0.8 g, 1.7 mmol, 63% yield) as a light yellow liquid. 1H NMR (400 MHz, CDCl3) δ 7.29 (d, J = 8.4 Hz, 2H), 7.24 (d, J = 8.3 Hz, 2H), 7.20 – 7.16 (m, 1H), 6.90 (d, J = 8.4 Hz, 1H), 6.85 (s, 1H), 4.53 (q, J = 7.3 Hz, 2H), 3.52 – 3.42 (m, 1H), 3.35 (s, 3H), 2.40 (s, 3H), 1.84 – 1.77 (m, 1H), 1.74 – 1.64 (m, 3H), 1.61 – 1.48 (m, 5H), 1.45 (t, J = 7.1 Hz, 3H), 0.97 (d, J = 6.8 Hz, 3H); MS (ESI) [M+H]+: 474.35; retention time 3.88 min, > 99% pure. (R)-5-(4-cyclopentyl-1,3-dimethyl-2-oxo-1,2,3,4-tetrahydroquinoxalin-6-yl)-1-(p-tolyl)-1H-1, 2,4-triazole-3-carboxylic acid (78). To a solution of compound 73 (0.8 g, 1.7 mmol) in THF (10 mL) and H2O (5 mL) was added LiOH (0.3 g, 6.8 mmol), and the mixture was stirred at rt overnight. The reaction was monitored by TLC. Upon completion, the reaction mixture was acidified with 1 N aq. HCl to pH 2 – 3, diluted with water and extracted with EtOAc (3 × 20 mL). The combined organic fractions were washed with brine, dried over Na2SO4, and concentrated by evaporation under reduced pressure to give compound 78 (0.7 g, 1.6 mmol, 94% yield) as a white solid. 1H NMR (400 MHz, CDCl3) δ 8.81 (s, 1H), 7.30 (d, J = 8.2 Hz, 2H), 7.26 – 7.19 (m, 3H), 6.93 (d, J = 8.2 Hz, 1H), 6.84 (s, 1H), 4.19 (q, J = 6.7 Hz, 1H), 3.50 – 3.40 (m, 1H), 3.35 (s, 3H), 2.39 (s, 3H), 1.84 – 1.74 (m, 1H), 1.71 – 1.62 (m, 1H), 1.61 – 1.42 (m, 5H), 1.36 – 1.24 (m, 1H), 0.98 (d, J = 6.8 Hz, 3H); MS (ESI) [M+H]+: 446.30; retention time 3.27 min, > 99% pure.

Protein expression The BRD4 (I) protein expression followed the protocol of Filippakopoulos et al. Colonies from freshly transformed plasmid DNA in E. coli BL21(DE3)-condon plus-RIL cells, were grown overnight at 37 ºC in 50 mL of Terrific Broth medium



with 50 μg/mL kanamycin and 34 μg/mL chloramphenicol (start-up culture). Then start-up culture was diluted 100 fold in 1 L of fresh TB medium and cell was growth at 37 ºC to an optical density of about 0.8 at OD600 before the temperature was decreased to 16 ºC. When the system equilibrated at 16 ºC the optical density was about 1.2 at OD600 and protein expression was induced over night at 16 ºC with 0.2 mmol isopropyl-β-D-thiogalactopyranoside (IPTG). The bacteria were harvested by centrifugation (4000 × g for 20 min at 4 ºC) and were frozen at -80 ºC as pellets for storage. Cells expressing His6-tagged proteins were re-suspended in lysis buffer (50 mmol HEPES, pH 7.5 at 25 ºC, 500 mmol NaCl, 10 mmol imidazole, 5% glycerol with freshly added 0.5 mmol TCEP (Tris(2-carboxyethyl)phosphine hydrochloride) and 1 mmol PMSF (phenylmethanesulfonyl fluoride) and lysed with an JN 3000 PLUS high pressure homogenizer (JNBIO - Guangzhou, China) at 4 ºC. The lysate was cleared by centrifugation (12,000 × g for 1 h at 4 ºC) and was applied to a nickel-nitrilotiacetic acid agarose column. The column was washed once with 50 mL of wash buffer containing 30 mmol imidazole. The protein was eluted using a step elution of imidazole in elution buffer (100-250 mmol imidazole in 50 mmol HEPES, pH 7.5 at 25 ºC, 500 mmol NaCl, 5% glycerol). All fractions were collected and monitored by SDS-polyacrylamide gel electrophoresis (Bio-Rad Criterion™ Precast Gels, 4-12% Bis-Tris, 1.0 mm, from Bio-Rad, CA.). After the addition of 1 mmol dithiothreitol (DTT), the eluted protein was treated overnight at 4 ºC with Tobacco Etch Virus (TEV) protease to remove the His6 tag. The protein was concentrated and further purified with size exclusion chromatography on a Superdex 75 16/60 HiLoad


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gel filtration column. Samples were monitored by SDS-polyacrylamide gel electrophoresis and concentrated to 8-10 mg/mL in the gel filtration buffer, 10 mmol Hepes pH 7.5, 500 mmol NaCl, 1 mmol DTT and were used for protein binding assay and crystallization. Crystallization and Data collection Aliquots of the purified proteins were set up for crystallization using the vapour diffusion method. The complex structure of BRD4 (I) and ligand was grown at 18 °C in 1 μL protein with an equal volume of reservoir solution containing 1.8-3.8 M sodium formate and 10% glycerol. Crystals grew to diffracting quality within 1−3 weeks in all cases. Data were collected at 100 K on beam Line BL17U at the Shanghai Synchrotron Radiation Facility (SSRF) (Shanghai, China) for the co-crystallized structures. The data were processed with the HKL2000,47 software packages, and the structures were then solved by molecular replacement, using the CCP4 program MOLREP.48 The search model used for the crystals was the BRD4-JQ1 complex structure (PDB code 3MXF). The structures were refined using the CCP4 program REFMAC5 combined with the simulated-annealing protocol implemented in the program PHENIX.49 With the aid of the program Coot,50 compound, water molecules, and others were fitted into to the initial Fo-Fc maps. Fluorescence anisotropy binding assay The binding of compounds to BRD4 was assessed using a Fluorescence Anisotropy Binding Assay. The fluorescent ligand was prepared by attaching a fluorescent



fragment (Fluorescein isothiocyanate isomer I, 5-FITC) to the (+)-JQ1. Generally the method involves incubating the Bromodomain protein BRD4, fluorescent ligand and a variable concentration of test compound together to reach thermodynamic equilibrium under conditions such that in the absence of test compound the fluorescent ligand is significantly (> 50%) bound and in the presence of a sufficient concentration of a potent inhibitor the anisotropy of the unbound fluorescent ligand is measurably different from the bound value. Detailedly, all components were dissolved in buffer of composition 50 mmol HEPES pH 7.4, 150 mmol NaCl and 0.5 mmol CHAPS with final concentrations of BRD4 (I) 10 nM, fluorescent ligand 5 nM. This reaction mixture was added various concentrations of test compound or DMSO vehicle (2 ‰ final) in Corning 384 well Black low volume plate (CLS3575) and equilibrated in dark for 4 hours at room temperature. Fluorescence anisotropy was read on BioTek Synergy2 Multi-Mode Microplate Reader (ex= 485 nm, EM = 530 nm; Dichroic -505 nM). In Vitro Metabolic Stability Study Microsomes (Human microsome, Xenotech, Lot No. H0610; Mouse microsome, Xenotech, Lot No. M1000) (0.5 mg/mL) were preincubated with 1 μM test compound for 5 min at 37 °C in 0.1 M phosphate buffer (pH 7.4) with 1 mmol of EDTA and 5 mmol of MgCl2. The reactions were initiated by adding prewarmed cofactors (1 mmol of NADPH). After 0, 5, 10, and 30 min incubations at 37 °C, the reactions were stopped by adding an equal volume of cold acetonitrile. The samples were vortexed for 10 min and then centrifuged at 15,000 rmp for 10 min. Supernatants were


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analyzed by LC/MS/MS for the amount of parent compound remaining, and the corresponding loss of parent compound was also determined by LC/MS/MS. The CYP enzymatic activities were characterized based on their probe reactions: CYP3A4 (midazolam or testosterone), CYP2D6 (dextromethorphan), CYP2C9 (Diclofenac), CYP1A2 (phenacetin), and CYP2C19 (Mephenytoin). Incubation mixtures were prepared in a total volume of 100 μL as follows: 0.2 mg/mL microsome (Human microsome, Xenotech, Lot No. H0610), 1 mmol of NADPH, 100 mmol of phosphate buffer (pH 7.4), probe substrates cocktail (10 μM midazolam, 100 μM testosterone, 10 μM dextromethophan, 20 μM diclofenac, 100 μM phenacetin, and 100 μM Mephenytoin) and 10 μM tested compound or positive control cocktail (10 μM ketoconazole, 10 μM quinidine, 100 μM sulfaphenazole, 10 μM naphthoflavone, and 1000 μM tranylcypromine) or negative control (PBS). The final concentration of organic reagent in incubation mixtures was less than 1% v/v. There was a 5 min preincubation period at 37 °C before the reaction was initiated by adding a NADPH generating system. Reactions were conducted for 20 min for CYPs. For each probe drug, the percentage of metabolite conversion was less than 20% of substrate added. The inhibition rate was calculated as (the formation of the metabolite of probe substrates with 10 μM tested compound) / (the formation of the metabolite of probe substrates with PBS) × 100%. The CYP enzymatic activities were characterized based on their probe reactions: CYP3A4 (midazolam or testosterone), CYP2D6 (dextromethorphan), CYP2C9 (Diclofenac), CYP1A2 (phenacetin), and CYP2C19 (S-(+)-mephenytoin). Incubation



Page 72 of 88

mixtures were prepared in a total volume of 200 μL as follows: 0.2 mg/mL microsome (Human microsome, Xenotech, Lot No. H0610), 1 mmol of NADPH, 100 mmol of phosphate buffer (pH 7.4), 10 μM tested compound, positive control cocktail (10 μM troleandomycin, 10 μM paroxetine, 10 μM tienilic Acid, 10 μM furafylline) or negative control (PRO). The final concentration of organic reagent in incubation mixtures was less than 1% v/v. There was a 0min, 5min, 10min and 30min preincubation period at 37 °C before the reaction was initiated by adding a NADPH and probe substrates cocktail (5 μM midazolam, 50 μM testosterone, 5 μM dextromethophan,

10

μM

diclofenac,

50

μM

phenacetin,

and

50

μM

S-(+)-mephenytoin) generating system. Reactions were conducted for 10 min for CYPs. The CYP2C19 enzyme was made alone, and the concentration of positive control cocktail S - (+) - fluoxetine was 100 μM. For each probe drug, the percentage of metabolite conversion was less than 20% of substrate added. The inhibition rate was calculated as (the formation of the metabolite of probe substrates with 10 μM tested compound)/(the formation of the metabolite of probe substrates with PRO) × 100%. BROMOscan Assay T7 phage strains display bromodomains 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 (5,000 x g) and filtered (0.2μm) to remove cell debris. Streptavidin-coated


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magnetic beads were treated with biotinylated small molecule or acetylated peptide ligands for 30 minutes at room temperature to generate affinity resins for bromodomain 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 non-specific phage binding. Binding reactions were assembled by combining bromodomains, liganded affinity beads, and test compounds in 1x binding buffer (16 % SeaBlock, 0.32x PBS, 0.02%BSA, 0.04 % Tween 20, 0.004% Sodium azide, 7.9 mM DTT). Compound 19 were prepared as 1000X stocks in 100% DMSO and subsequently diluted 1:25 in monoethylene glycol (MEG). The compound 19 were then diluted directly into the assays such that the final concentrations of DMSO and MEG were 0.1% and 2.4%, respectively. 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% Tween20). The beads were then re-suspended in elution buffer (1x PBS, 0.05% Tween 20, 2 μM non-biotinylated affinity ligand) and incubated at room temperature with shaking for 30 minutes. The bromodomain concentration in the eluates was measured by qPCR. Compound 19 was screened at the 1 μM concentration, and results for primary screen binding interactions are reported as '% Ctrl', where lower numbers indicate stronger hits in the matrix. PLK1 Kinase Assay: Z´-LYTE™ Kinase Assay Kinase Reaction was performed with assay buffer (50 mM Hepes pH 7.5, 1 mM



EGTA, 10 mM MgCl2, 0.01% Brij-35), and the PLK1 protein concentration was set to 10 nM and the substrate peptide and ATP concentration was set to 2 mM Ser/Thr 16 Peptide, 13 mM ATP, and the reaction was carried out about 60 minutes at 23 ℃ . The detection Equipment is Envision (PerkinElmer # 2104). Cell Culture Ty-82 cell line was purchased from the Japanese Collection of Research Bioresources Cell Bank (Osaka, Japan). MV4-11, MM.1S, KG-1, RKO, Hep3B, NCI-H522, MDA-MB-468, HT-29, AGS, BT-549, SKOV3, MDA-MB-436, HuTu-80 and A549 cell lines were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). NCI-H1299 and SW620 cell lines were purchased from the Cell Bank of the Chinese Academy of Science Type Culture Collection (Shanghai, China). All cell culture conditions follow the supplier's recommendation. Cell Viability Assays Different cells were cultured in the corresponding medium conditions recommended by the suppliers. Different cells were seeded onto 96-well plates at a suitable density in a volume of 100 μL medium. After incubation overnight, compounds dissolved in DMSO stock solutions were thawed at room temperature and diluted to the desired concentrations with normal saline. The compounds were added to the assay plate and after 72 h of incubation, the IC50 was measured with Cell Counting Kit-8 (CCK-8) assay (Dojindo, Japan) for suspended cells or sulforhodamine B (SRB) (Sigma; MO, USA) for adherent cells. Western Blotting


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MV4-11 and RKO cells were plated into 6-well plates and incubated for 24 h. Then, cells were harvested after 24 h treatment with compounds at the indicated doses. Western blotting was performed as previously described method.33 Real-time Quantitative PCR MV4-11 and RKO cells were seeded into 6-well plates and incubated for 24 h. Compounds were added at the indicated doses and treated for 24 h. Then total RNA were extracted with TRIzol (Life Technologies; CA, USA) and turned into cDNA with PrimeScript™ RTMaster Mix (Perfect Real Time) (RR036A) (TaKaRa; Kusatsu, Shiga, Japan). Real-time quantitative PCR were performed with SYBR® Premix Ex Taq™ (Tli RNaseH Plus) (Takara). The primers were used as follow, c-Myc: forward 5’-GCTGCCAGGACCCGCTTCTC-3’, 5’-ACGTTGAGGGGCATCGTCGC-3’;

reverse BCL-2:

5’-CCTGTGGATGACTGAGTACCTG-3’, 5’-CAGAGGCCGCATGCTGGG-3’;

forward reverse

β-actin:

5’-ATCGTGCGTGACATTAAGGAGAAG-3’,

forward reverse

5’-AGGAAGGAAGGCTGGAA GAGTG-3’. Cell Cycle Assay RKO cells were seeded into 6-well plates and incubated for 24 h. Then treated cells with compounds at the indicated concentrations for 24 h. Cells were digested, collected and washed. Next fixed cells with 70% ethanol for overnight. Samples were digested with RNAase (Beyotime; Shanghai, China) then treated with propidium iodide (Beyotime; Shanghai, China). Finally, samples were detected by flow



cytometry and analyzed by ModFit LT software. Apoptosis Assay MV4-11 cells were plated into 96-well plates at 10000 cells/well and incubated for 24 h. Compounds were added and treated for 24 h. Then apoptosis was measured by RealTime-GloTM Annexin V Apoptosis and Necrosis Assay (Promega; Madison, WI, USA) according to the manufacturer’s protocol. The apoptotic signal refers to Annexin V signal corrected by the number of cells. hERG Assay Qpatch automatic patch clamp method was used to measure the hERG inhibition of compound 19. To prepare the compound, we diluted the compound mother liquor (10 μL) with extracellular fluid (4990 μL). Then we continuously diluted 3 times in the extracellular fluid containing 0.2% DMSO to give the final concentration to be tested. The highest concentration of the compound was 40 μM, followed by 40, 13.33, 4.44, 1.48, 0.49 and 0.16 μM, respectively. The highest concentration of positive compound Cisapride was 1 μM, followed by 0.333, 0.111, 0.037, 0.012, 0.004 μM, respectively. The final test concentration of DMSO content of not more than 0.2%, this concentration of DMSO on the hERG potassium channel has no effect. In Vivo Study All the animal experiments were performed according to the institutional ethical guide-lines of animal care. In Vivo PK Study Compounds dissolved in 1%DMSO/0.5%HPMC to a concentration of 1 mg/mL, and


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was given to ICR mice (Male, 18 – 22 g, n = 3) by gavage administration. Blood samples were collected at 0.25, 0.5, 1, 2, 4, 8, and 24 h after administration (anticoagulant: EDTA-Na2). 100 μL of solvent of methanol: acetonitrile (1:1, v/v) with internal standard was added to 10 μL of plasma and vortexed thoroughly. It was centrifuged for 5 min, then 20 μL of the supernatant was mixed with 20 μL of water for analysis. Samples were analyzed by Xevo TQ-S triple quadrupole mass spectrometer (Waters, USA). The ACQUITY UPLC BEH C18 (1.7 μm, 2.0 mm × 50 mm, Waters, USA) was used for the analysis. Gradient elution was applied consisting of 5 mM ammonium acetate aqueous solution containing 0.1% formic acid and acetonitrile containing 0.1% formic acid. After analyzing the concentrations of these compounds, the value of AUClast, AUCINF_obs and MRTINF_obs was calculated from time - concentration curves in each animal using Phoenix WinNonlin (CERTARA, USA). Cmax was determined as the maximum plasma concentration, and Tmax was the time to reach the maximum concentration. In Vivo pharmacological Study Four to five weeks old female SCID nude mice were obtained from shanghai lingchang Biotechnology Co., Ltd (Shanghai, China), and were acclimated one week prior to tumor cell inoculation. A total of 5×106 human multiple myeloma cells MM.1S were injected subcutaneously. Thirteen days after inoculation, mice with established xenografts were randomized into different groups for treatments of daily I-BET762 (From Pharmacodia) at 20 mg/kg PO, vehicle and compound 7 at 20 mg/kg PO, respectively (vehicle n =12, other groups n = 6).



Page 78 of 88

Six week old female Balb/c nude mice were obtained from Beijing HFK Bioscience Co., Ltd (Beijing, China), and were acclimated one week prior to tumor cell inoculation. A total of 8×106 human multiple myeloma cells MV4-11 were injected subcutaneously. Thirteen days after inoculation, mice with established xenografts were randomized into different groups for treatments of daily OTX-015 (From Pharmacodia) at 50 mg/kg PO and 100 mg/kg PO, vehicle and compound 19 at 10 mg/kg PO, 25 mg/kg PO and 50 mg/kg PO, respectively (vehicle n =12, other groups n = 6). The maximum width (X) and length (Y) of the subcutaneous xenograft were measured with a caliper twice a week and the volume (V) was calculated using the formula: V = (X2Y)/2. Then, relative tumor volume (RTV) was calculated as follows: RTV = Vt/V0; growth inhibition rate (GI) was calculated as: GI = [1 － (TVt － TV0)/(CVt－CV0)]×100%, where V0 was the tumor volume at the beginning of the treatment, and Vt was the tumor volume at the end of treatments, TV was the treatment, CV was the control group. The animal body weight was also measured at the same time.

ASSOCIATED CONTENT Supporting Information The table of Molecular Formula Strings is submitted and available for download. And the supplemental materials include the crystallography information for the complex structures, in vitro PK profiles, the in vivo data of compound 7, the solubility data of compounds 7 and 19, and in vivo PK properties of compound 15 and 20-23, the bromodomain


and kinase selectivity data,

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ITC binding affinity data of 19, hERG inhibition data of 19 and spectra data of representative compounds.

AUTHOR INFORMATION

Corresponding Authors

*(B.X.) E-mail: [email protected]; *(Z. M.) [email protected]; * (Y. W.) [email protected].

Author Contributions

J.H. and C.T. contribute equally to this manuscript.

ACKNOWLEDGEMENTS We are grateful for financial supports from the National Science & Technology Major Project “Key New Drug Creation and Manufacturing Program” of China (Grant No. 2018ZX09711002-011-018) and the National Natural Science Foundation of China (Grant No. 81330076).

PDB entry codes: 6JI3, 6JI4 and 6JI5.

Authors will release the atomic coordinates and experimental data upon article publication.

ABBREVIATIONS USED ALK, anaplastic lymphoma kinase; BCPs, Bromodomain-containing proteins; BET,



bromodomain and extra-terminal; BETi, bromodomain and extra-terminal inhibitor; BRD4-BD1, first bromodomain of BRD4; FA, fluorescence anisotropy; GI, growth inhibition; ITC, isothermal titration calorimetry; KAc, acetylated lysine; PLK1, Polo-like Kinase 1; P-TEFb, positive transcription elongation factor B; RT-qPCR, real-time quantitative PCR; SARs, structure- activity relationships; SSRF, Shanghai Synchrotron Radiation Facility.

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