Discovery of selective HDAC/BRD4 dual inhibitors as epigenetic probes

Graphical abstract

A series of pyrrolopyridone derivatives were designed and synthesized for their HDAC1/6 and

BRD4(BD1/BD2) inhibitory activity and selectivity. Lead compounds with potent selective

HDAC and BRD4 dual inhibitions were achieved based on SARs and the first

HDAC6/BRD4(BD2) dual inhibitor was discovered.

Title:

Discovery of Selective HDAC/BRD4 Dual Inhibitors as Epigenetic Probes

1

Graphical abstract

A series of pyrrolopyridone derivatives were designed and synthesized for their HDAC1/6 and

BRD4(BD1/BD2) inhibitory activity and selectivity. Lead compounds with potent selective

HDAC and BRD4 dual inhibitions were achieved based on SARs and the first

HDAC6/BRD4(BD2) dual inhibitor was discovered.

2

Highlights

ò

Novel pyrrolopyridone-based HDAC/BRD4 dual inhibitors were synthesized.

Lead compounds with both potent enzymatic and cellular activities were evaluated.

HDAC isoforms and BRD4 bromodomain of lead compounds were investigated.

The first HDAC6/BRD4(BD2) dual inhibitor was discovered.

3

Title:

Discovery of Selective HDAC/BRD4 Dual Inhibitors as Epigenetic Probes

†

, a, c

†, b

a, c

b

a

b

Jingjing Chen

, Yalei Li , Jie Zhang , Minmin Zhang , Aihuan Wei , Hongchun Liu ,

a, c

a

a, c

, b, c

, a,

Zhicheng Xie , Wenming Ren , Wenwen Duan , Zhuo Zhang , Aijun Shen* , Youhong Hu*

c,d

a

State Key Laboratory of Drug Research, Department of Medicinal Chemistry, Shanghai Institute

of Materia Medica, Chinese Academy of Sciences, 555 ZuChongZhi Road, Shanghai 201203,

China

b

Division of Anti-Tumor Pharmacology, State Key Laboratory of Drug Research, Shanghai

Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Rd, Shanghai 201203,

China

c

University of Chinese Academy of Sciences, 19 Yuquan Road, Beijing 110039, China

d

School of Pharmaceutical Science and Technology，Hangzhou Institute for Advanced Study,

UCAS, Hangzhou 310024, China.

*Corresponding author: E-mail: yhhu@simm.ac.cn; shenaj@simm.ac.cn

Jingjing Chen and Yalei Li equally contributed to this work.

Abstract:

According to the binding mode of ABBV-744 with bromodomains and the cape space of HDAC,

the novel selective HDAC/BRD4 dual inhibitors were designed and synthesized by the

pharmacophore fusion strategy. Evaluating the biomolecular activities through SARs exploration

identified three kinds of selective dual inhibitors 41c (HDAC1/BRD4), 43a (pan-HDAC/BRD4)

and 43d (HDAC6/BRD4(BD2))，whose target-related cellular activities in MV-4-11 cells were

also confirmed. Significantly, the selective dual inhibitor 41c (HDAC1/BRD4) exhibited

synergistic effects against MV-4-11 cells, which strongly induced G0/G1 cell cycle arrest and

apoptosis, and the first HDAC6/BRD4(BD2) dual inhibitor was found. This study provides

support for selective HDAC/BRD4 dual inhibitors as epigenetic probes based on pyrrolopyridone

core for the future biological evaluation in different cancer cell lines.

Key words:

HDAC/ BRD4/ selective dual inhibitors/ anticancer

1

. Introduction

Histone acetylation, which broadly affects chromatin organization and gene expression, has

been defined as a hallmark of epigenetics [1, 2]. Three types of protein families play important

roles in this process. Histone acetyltransferases (HATs) are the “writers” of histone acetylation

transferring acetyl group to histone tails and facilitating transcription, while histone deacetylases

(HDACs) belong to the “erasers”, which remove the acetyl group thereby promoting chromatin

compression and transcriptional silence[3]. Bromodomain and extra- terminal (BET), which act as

the “readers”, specifically recognize acetylated lysine (KAc) residues of histones and recruit

P-TEFb (Positive Transcription Elongation Factor b) complex to control downstream

transcriptional activation [4]. Recently, both HDAC and BET families attracted high concern as

potential anti-tumor targets for their important role of transcriptional regulation.

In the past years, a number of HDAC and BRD4 inhibitors with diverse chemotypes have

been reported, and to date five HDAC inhibitors have been approved for the treatment of

4

hematologic cancers, among which are the hydroxamic acid-derived compounds Vorinostat

SAHA), Belinostat and Panobinostat as HDAC pan-inhibitors (pan-HDACi) while the cyclic

(

peptid Romidepsin and the ortho-aminoanilide Chidamide (approved in China) selectively inhibit

only HDAC class I members. [3]. However, most of the known HDAC inhibitors have limited

clinical benefits in nearly all types of solid tumors used as single agents[5]. On the other hand,

several BRD4 inhibitors with similar affinities to both BD1 and BD2 such as OTX015, CPI-0610,

GS-5829, INCB-5429, ABBV-075, etc. have been enrolled into clinical trials for the treatment of

different cancers, but dose-limiting adverse events such as reduced number of thrombocytes in the

blood (thrombocytopenia) and symptoms of gastrointestinal toxicity were observed in early

clinical trials[6, 7].

Owing to the distinct functions of different HDAC isoforms and the two individual

bromodomains in BRD4, the development of selective inhibitors targeting HDACs subtypes or

inhibiting individual BRD4 bromodomains may yield highly efficacious compounds with reduced

toxicity [6, 8]. For example, a recently reported BD2 selective inhibitor ABBV-744 in phase I

trials for acute myeloid leukemia and prostate cancer demonstrated fewer platelet and

gastrointestinal toxicities as compared to its analogue ABBV-075 [6, 9].

Another strategy to improve efficacy is to combine HDAC and BRD4 inhibitors, based on the

interface of histone acetylation [2]. Preclinical data suggests strong synergy at reduced doses with

BRD4 and HDAC inhibitors, which has the potential to avoid the overlapping toxicities associated

with these two drug types [7]. A combination of BRD4 inhibitors ((+)-JQ1, OTX015, I-BET151)

and pan-HDAC inhibitors (Vorinostat, Panobinostat) synergistically induced proliferation

inhibition and apoptosis in diverse cancer types including cutaneous T cell lymphoma (CTCL)[10,

1

1], gallbladder cancer[12], pancreatic cancer[13], breast cancer[5], neuroblastoma[14] and

melanoma[15] with different mechanisms. For example, in gallbladder cancer, the anticancer

effect induced by (+)-JQ1 and Vorinostat was associated with the downregulation of BRD4 and

suppression of PI3K/AKT and MAPK/ERK pathways[12], and BRD4-mediated LIFR-JAK-STAT

signaling pathway decreased the efficacy of HDAC inhibitors in triple negative breast cancer [5].

The selective class I HDAC inhibitor ortho-aminoanilide 4SC-202 induced significant BRD4

recruitment in pancreatic ductal adenocarcinoma (PDAC) and the expression of the genes induced

by class I HDAC inhibitors rely on BRD4 and Myc[16]. Jennifer et al. also reported that BRD4

inhibitors upregulated the expression HDAC6 in multiple myeloma (MM) and the combination of

ACY1215 with (+)-JQ1 induced significant reduction of c-Myc and Bcl2 expression and

apoptosis[17]. On the other hand, anti-tumor effects associated with selective HDAC6 inhibitor

ACY1215 and BRD4 inhibitor (+)-JQ1 combination seem dependent on the presence of immune

cells. In non-small cell lung cancer (NSCLC), ACY1215 with (+)-JQ1 led to a robust

immune-mediated tumor growth arrest in a manner that favors improved T-cell function and

reduces inhibitory cellular mechanisms[18]. In addition, the same combination strategy provoked

NK cell-mediated immunity in small cell lung cancer (SCLC), suggesting the importance of these

innate lymphoid cells in the SCLC tumor treatment response [19].

Inspired by these synergistic effects with the combination of HDAC and BRD4 inhibitors,

previous HDAC/BRD4 dual inhibitors based on the known BRD4 inhibitor (Fig. 1, compounds

1

-5) were designed and synthesized[20-24]. However, no obvious synergistic effect was observed

comparing with the single inhibitor in vitro biological evaluation. Recently, Sheng’s group

reported the (+)-JQ1-based hydroxamic acid 6 as dual BRD4/HDAC inhibitor showed remarkable

antitumor activity against Capan-1 cell lines in vitro and in vivo as compared to (+)-JQ1,

Vorinostat and their combination[25]. Also, another (+)-JQ1-based ortho-aminoanilide TW09 (Fig.

1

, compound 7) was synthesized ,which could induce cell death and mitochondrial apoptosis as

(+)-JQ1/MS-275 co-treatment in rhabdomyosarcoma (RMS) and PDAC cells[26, 27]. In addition,

Pan et al. reported compound 8 based on the combination of RVX-208 and Vorinostat exhibited a

superior anti-proliferative capacity on colorectal cancer[28]. Herein, we report the design and

synthesis of novel selective HDAC/BRD4 dual inhibitors based on the selective BD2 inhibitor

ABBV-744 for the discovery of special biological functions.

Fig. 1.

2

. Results and discussion

2

.1. Design of dual inhibitors.

5

Based on the crystal structure of ABBV-744 in complex with BD2 of BRD2 (PDB ID 6e6j)[6],

we designed the dual inhibitors by a pharmacophore fusion strategy. As shown in Fig. 2A, the

solvent-oriented ethyl amide region could be extending for tethering the different linkers with a

zinc-binding group (ZBG, hydroxamic acid or ortho-aminobenzamide ， Fig. 2B) as the

pharmacophore of HDAC inhibitor without disturbing their bind with BRD4. In addition,

maintaining the pyrrolopyridone core in the BRD4 pharmacophore may retain hydrogen bonding

within the conserved Asn 429 residue. Variety within the WPF region may also be matched with a

flexible cap group during HDAC inhibition, which may contribute to the selectivity for HDAC1

and HDAC6[8, 29] and BD2[6] by the steric effect. The novel diversified pyrrolopyrione-based

selective HDAC/BRD4 dual inhibitors were designed in Fig. 2C.

Fig. 2.

2

.2. Chemistry

The key intermediate 15 with 2-position functionalized pyrrolopyridone was synthesized as

reported (scheme 1)[9]. To enable exploration of linkers and ZBGs, compound 16 was generated

by a Suzuki-Miyaura coupling between 15 with (2-phenoxyphenyl) boronic acid. After the

hydrolysis of methyl ester 16, the different linkers were introduced at 2-position of

pyrrolopyridone by the condensation reaction to yield the intermediates 17a-17e. Finally, two

kinds of ZBGs (hydroxamic acid or ortho-aminoanilide) were introduced to generate HDAC

function as HDAC /BRD4 dual inhibitors 18a-18e and 20a-20d.

To improve potency and selectivity toward HDAC and BRD4, the compounds with substituents on the

phenyl ring were synthesized as shown in scheme 2. The bromide 15 was reacted with commercial

mono-substituted phenylboric acids 21a-21q via the catalytic palladium to produce the intermediates

2

2a-22q. The steps reported in Scheme 1 synthesized the optimized linker products 25a-25q.

Intermediates 27a-27g containing a meta-dimethyl carbinol group on phenyl ring were

synthesized by an addition of methyl Grignard reagent from the corresponding esters or ketones

scheme 3). Borate 36 was transferred from 15 and then coupled with 27a-27g to give the key

intermediates 37a-37g (scheme 4). The final designed dual inhibitors 41a-41g, 42a-42d, and

(

4

3a-43d were obtained using the previous method as described in scheme 1.

Scheme 1.

Scheme 2.

Scheme 3.

Scheme 4.

2

.3. Preliminary SAR study of dual inhibitors toward HDAC and BRD4

Initially, the molecular inhibitory activities against HDAC1, HDAC6 and BRD4 BD1 and BD2

of the compounds 18a-18e and 20a-20d with the substitutions of ortho-phenyl ether substituted

benzene at 4-position of pyrrolopyridone and two kinds of ZBGs (hydroxamic acid and

ortho-aminoanilide) with the different linkers at solvent-oriented region were evaluated. As shown

in Table 1, all the designed compounds maintained comparable inhibition against BRD4 without

BD domain selection as the positive control OTX015. These results indicate that the 2-position of

pyrrolopyridone is suitable to connect the ZBGs without disturbing the key binding interactions

with BRD4 protein. The selective BD inhibition is critical at the substitution of ortho-phenyl ether

at WPF region[6]. The hydroxamic acids with longer aliphatic linkers (5 or 6 carbons) tend to

exhibit favorable activities towards both HDAC1 and HDAC6. Compounds 18d with a six-carbon

linker exhibited inhibition of HDAC1 (IC₅₀= 29.5 nM) and HDAC6 (IC₅₀= 4.7 nM), which is

comparable with the positive control SAHA. When aliphatic linkers were shortened or replaced

with aromatic linkers, favorable HDAC6 selective inhibition was observed (compound 18a, 18b

and 18e). After changing ZBG with the ortho-aminoanilide, only 20b (with five-carbons aliphatic

linker) and 20d (with the para-substituted benzyl linker) maintained moderate inhibitory activity

against HDAC1. All compounds showed good inhibitory activity against MV-4-11 cells. Specially,

the benzamide derivatives 20b and 20d showed stronger antitumor potency against MV-4-11 as

compared to the positive HDAC1 inhibitor MS-275 and BRD4 inhibitor OTX015, which suggests

that the dual inhibitors exhibited synergist effects.

6

Table 1.

2

.4. Western blot analysis of the optimized dual inhibitors in MV-4-11 cell lines

In order to confirm the inhibitory activity and selectivity of the dual inhibitors against

HDACs, MV-4-11 cells were incubated with compounds 18b-18e and 20d at a range of

concentrations to analyze the modulation of Ac-tubulin, Ac-histone H3 and c-Myc which

associated with intracellular HDAC and BRD4 inhibition (Fig 3.). Compound 18d at 1μM

increased acetylated tubulin (Ac-Tub) and acetylated histone 3 (Ac-H3) to exhibit pan-HDAC

inhibitory activity. Compound 18e induced more significant Ac-Tub accumulation as compared to

Ac-H3, which indicates the selective inhibition of HDAC6. Although compound 18b showed

HDAC6 specific inhibition at the molecular level, no obvious increase of Ac-Tub was observed in

MV-4-11 cells. Notably, compound 20d induced pronounced upregulation of Ac-H3 without

increasing Ac-Tub in a similar manner as the positive control MS-275, suggesting that 20d is a

selective class I HDAC inhibitor. All compounds downregulated c-Myc protein as BRD4

inhibitors. Especially, stronger antitumor potency of HDAC1/BRD4 dual inhibitor 20d was

reflected through c-Myc downregulation and P21 upregulation. As a result, we initially proceeded

with SAR exploration based on 20d.

2

.5. Focused SAR study for ortho-aminoanilide moiety toward HDAC1 and BRD4

The SAR exploration of the substitution on the phenyl ring was shown in Table 2. First, we

investigated the electronic and steric effects of the mono-substituted phenyl ring at ortho, meta

and para positions. Generally, the compounds with electron-donating substituents such as alkoxyl

group benefited enzymatic activity towards both HDAC1 and BRD4 than with

electron-withdrawing substituents. HDAC1 activity was greatly affected by steric effects on the

ortho position. 2-isopropoxyl (25g) and 2-phenoxyl (20d) decreased HDAC1 inhibitory activity

and maintained BRD4 inhibition. Since a meta-dimethyl carbinol moiety helped maintain

hydrogen bonding with a backbone amide in a cleft adjacent to the ZA-loop [6, 9], compound 41a

exhibited excellent enzymatic inhibitory activity towards both HDAC1 and BRD4. However, the

antiproliferative activity against MV-4-11 cell lines was weak. We hypothesized that the

substituents on phenyl ring might change the physical character to cross the cell membrane.

Further the western bolt analysis (see Fig. S1) demonstrated weak effects of 41a to HDAC1 and

BRD4 related proteins. Then we combined the privileged substituents at 2,5-position providing

4

1c and 41d, which exhibited the most potent HDAC1/BRD4 inhibitions. And the strongest

inhibitory activity against MV-4-11 cells (IC = 0.04 μM) indicated the significant synergy effects

5

0

as comparing with the single drug treatment of MS-275 or ABBV-744. Since the steric effect of

the substitutions at ortho position of phenyl ring might change the bromodomain (BD1/BD2)

inhibition[6, 9], compounds 41e-41g were prepared. Although compound 41g with

4

-fluoro-2,6-dimethylphenoxyl substitution could selectively inhibit BRD4(BD2) as ABBV-744,

the HDAC1 inhibitory activity was lost completely.

Table 2.

2

.6. Focused SAR study for hydroxamic acid moiety toward HDAC and BRD4

Finally, we mainly explored the optimized linkers and substituent groups on phenyl ring for

hydroxamic acids moiety to find the selective dual inhibitors with HDAC isoform and BRD4

(

BD1/BD2). As shown in Table 3, 4-fluoro-2,6-dimethylphenoxyl substitution at ortho-position

compound 42d and 43d) could reflect on the selective BD2 inhibition and dominant HDAC6

inhibition due to the bulky cap group [8, 29]. Compound 43a with a 2-isopropoxyl and a

six-carbon aliphatic linker revealed strong enzymatic and cellular inhibitory activities against all

targets tested (HDAC1 IC₅₀= 19.4 nM, HDAC6 IC₅₀= 5.4 nM; BRD4(BD1) IC₅₀= 29.5 nM,

BRD4(BD2) IC = 6.0 nM).

5

0

Table 3.

2

.7. HDAC isoform profiling of compound 41c, 43a and 43d.

To confirm the related HDAC isoform selectivity of the dual inhibitors, three compounds 41c,

3a and 43d were tested. As shown in Table 4, ortho-aminoanilide 41c showed HDAC3 inhibitory

4

activity weakly as Class I HDAC inhibitor. For the hydroxamic acids, pan-HDAC inhibitor 43a

7

also inhibited other HDAC3/6/8 with an IC values ranging from 6.8 to 99 nM as positive control

5

0

SAHA. Compound 43d as relatively HDAC6 inhibition comparing HDAC1 activity exhibited

HDAC3 inhibitory activity with an IC values of 161.2 nM and inhibited HDAC8/11 with an IC

5

0

50

values of 583.0 /2754.5 nM. These three compounds didn’t show any inhibitory activities for

HDAC4.

Table 4.

2

.8. Analysis Ac-tubulin, Ac-histone H3 and c-Myc levels of selected lead compounds in MV-4-11

cells by Western Blot

Three important compounds 41c, 43a and 43d were chosen to evaluate the target and

selectivity by western blot in MV-4-11 cells. As depicted in Fig. 4, in agreement with their HDAC

subtype selectivity in enzymatic assays, 41c induced mainly increase of Ac-H3 as a selective

HDAC1 inhibitor. 43a upregulated both Ac-H3 and Ac-tubulin levels as a pan-HDAC inhibitor

and 43d upregulated Ac-tubulin levels predominantly as a selective HDAC6 inhibitor. Moreover,

compound treatment decreased c-Myc in a dose-dependent manner.

Fig. 4.

2

.9. Effects on cell cycle and apoptosis of lead compounds

MV-4-11 cells were treated with the lead compounds to evaluate cell cycle arrest and

apoptosis induction using flow cytometry. As shown in Fig. 5, both HDAC inhibitors (SAHA,

MS-275) and BRD4 inhibitors (OTX015, ABBV-744) induced G0/G1 arrest in MV-4-11 cells in a

dose dependent manner. Similarly, dual inhibitors 41c and 43a with good HDAC1 inhibitory

activity triggered obvious G0/G1 arrest dose-dependently in MV-4-11 cells. Meanwhile,

HDAC6/BD2 dual inhibitor 43d slightly induced G0/G1 cell cycle arrest even at the concentration

of 1000 nM, similar to the selective BD2 inhibitor ABBV-744. Furthermore, 41c (57.31%) and

4

3a (53.73%) were much more potent in inducing apoptosis at 1000 nM as compared to 43d

(19.29%). Notably, although dual inhibitors 41c and 43a have different ZBGs respectively, they

exhibited similar effects in cell cycle arrest and apoptosis which suggests that HDAC1 inhibitory

activity may play a more important role in these dual inhibitors.

Fig. 5.

3

. Conclusion

In summary, we designed and synthesized a novel series of selective HDAC and BRD4 dual

inhibitors bearing a pyrrolopyridone scaffold using a pharmacophore fusion strategy by analysis of

the binding mode with the proteins. The compounds were evaluated on the assays of HDAC1,

HDAC6 and BRD4(BD1/BD2). 2-position of pyrrolopyridone at the solvent region is suitable to

connect the ZBGs without disturbing the key binding interactions with BRD4 protein. ZBG group

as ortho-aminoanilide moiety exhibited the selective HDAC1 inhibition as usual. The substituents

on 2-position of phenyl ring at WPF region of BRD4 could control HDAC6 and BRD4 BD2

selectivity. Three kinds of selective dual inhibitors 41c (HDAC1/BRD4), 43a (pan-HDAC/BRD4)

and 43d (HDAC6/BRD4(BD2)) were confirmed by biological evaluation. Significantly, the

ortho-aminoanilide 41c exhibited strong synergy effects against MV-4-11 cell lines as compared to

the single HDAC1 or BRD4 inhibitor. Western analysis of the selected dual inhibitors indicated

that the biomolecular activities could regulate the corresponding pathway on MV-4-11 cells. The

compounds 41c and 43a could trigger clear G0/G1 arrest dose-dependently and promote cell

apoptosis. The HDAC6/BRD4(BD2) dual inhibitor 43d induced similar biological effects as

BRD4(BD2) inhibitor ABBV-744. Compounds 41c, 43a and 43d could be used as epigenetic

probes for further biological studies to explore the special function of dual selective HDAC and

BRD4 inhibition in different cells lines.

4

. Experimental section

Unless otherwise noted, all commercially available reagents and solvents were used without

further purification. Nonaqueous reactions were performed under a positive pressure of argon in

over-dried glassware. All reactions were monitored by thin-layer chromatography (TLC) on glass

8

plates (silica gel GF254, 0.2 ± 0.03 mm thickness) and visualized by UV light (254 nm or 365 nM)

or iodine vapor. All compounds were purified by flash chromatography using silica gel (200−300

1

13

1

mesh). H NMR and proton-decoupled C NMR spectra were obtained on Bruker 400 ( H: 400

1

3

1

13

1

MHz, C NMR: 101 MHz), Bruker 500 ( H: 500 MHz, C NMR: 126 MHz), or Bruker 600 ( H:

1

3

5

00 MHz, C NMR: 151 MHz) spectrometer and referenced to the deuterium solvent

(

chloroform-d, MeOH-d₄_Dor DMSO-d ). Chemical shifts were reported in parts per million (ppm)

6

and coupling constants were expressed in Hz. Multiplicity were reported as single (s), double (d),

triplet (t), quarter (q), and multiplet (m). Low-resolution mass spectral were recorded on an

Agilent 1200 series LC−MS spectrometer, and high-resolution mass spectral (HRMS) data were

measured on Micromass Ultra Q-Tof. Analytical HPLC purity was determined using an Agilent

1

4

200 series LC system (Agilent ChemStation Rev.B.03.01; column, ZORBAX Eclipse XD B-C18,

.6 mm × 150 mm, 5 μm; flowrate, 1.0 mL/min). All final compounds achieved a minimum of 95%

purity.

4

.1. The general procedure for preparation of the key intermediates 15, 36 and 27a-27g

4

.1.1. 4-bromo-7-methoxy-1H-pyrrolo [2,3-c ]pyridine (13)

To a solution of 5-bromo-2-methoxy-4-methyl-3-nitropyridine 12 (25g, 101.19 mmol) in

dimethylformamide (DMF) (50 mL) was added N, N-Dimethylformamide dimethyl acetal (34 mL,

53 mmol). The reaction mixture was heated at 90 ℃ overnight, and after cooled to rt the solvent

2

was removed under reduced pressure to give a red solid. Then the solid was dissolved in acetic

acid (400 mL) and reduced iron powder (40 g, 708.36 mmol) was added under magnetic stirring.

The mixture was heated at 90 ℃ for 4h. Then cooled to rt, filtered, washed with ethanol. The

filtrate was concentrated and the residues was dissolved in EtOAc (500 mL) and washed with

saturated NaHCO solution (100 mL × 3) and saturated NaCl solution (100 mL × 3), dried over

3

Na SO , concentrated in vacuo. The residue was recrystallized in hexane/EtOAc to give 13 as a

2

4

1

sage green solid. Yield 87% (20 g). H NMR (400 MHz, Chloroform-d) δ 8.74 (s, 1H), 7.84 (s,

H), 7.32 (d, J = 2.9 Hz, 1H), 6.56 (d, J = 2.7 Hz, 1H), 4.07 (s, 3H). MS (ESI): 226.9 [M+H] .

+

1

4

.1.2. methyl 4-bromo-7-methoxy-1-tosyl-1H-pyrrolo[2,3-c] pyridine-2-carboxylate (14)

A solution of 13 (10 g, 44.04 mmol) in DMF (100 mL) was added NaH (2.11 g, 60%

dispersion in mineral oil, 52.85 mmol) at 0℃ and the tosyl chloride was added after 0.5 h. The

mixture was stirred at rt for 2 h and quenched with saturated NH Cl solution at 0℃ . A brown solid

4

was precipitated out and filtered, washed with water (100 mL × 3) and dried under vacuum. Then

the

solid

was

purified

by

silica

gel

chromatography

to

afford

1

4

-bromo-7-methoxy-1-tosyl-1H-pyrrolo[2,3-c] pyridine as a white solid. Yield 95% (16 g). H

NMR (400 MHz, CDCl ) δ 8.00 (d, J = 3.6 Hz, 1H), 7.92 (s, 1H), 7.80 (d, J = 8.4 Hz, 2H), 7.32 (d,

3

+

J = 8.2 Hz, 2H), 6.71 (d, J = 3.6 Hz, 1H), 3.90 (s, 3H), 2.43 (s, 3H). MS (ESI): 380.9 [M+H] .

To a solution of diisopropylamine (3.19 g, 31.5 mmol) in anhydrous tetrahydrofuran (100 mL)

was added n-BuLi (2 M, 17.3 mL, 34.7 mmol) dropwise at -70 ℃ and the reaction mixture was

then stirred from about -70 ℃ to about 0 ℃ for 45 minutes. To the solution of

4

-bromo-7-methoxy-1-tosyl-1H-pyrrolo[2,3-c] pyridine (10 g, 26.2 mmol) in anhydrous

tetrahydrofuran (150 mL) was added the above lithium diispropylamide solution dropwise at -70 ℃

and the mixture was stirred for 1 h at -50 ℃ . Then methyl carbonochloridate (2.97 g, 31.5 mmol)

was added dropwise to the above mixture. The reaction mixture was stirred at -50 ℃ for another 1

hours and quenched with saturated NH Cl solution at 0℃ . The aqueous layer was extracted with

4

ether (100 mL × 3) and the combined organic layers were washed with saturated NaCl solution

(

100 mL × 3) and dried over anhydrous Na SO and concentrated under reduced pressure. The

2 4

residue was purified by flash column chromatography on silica gel to afford 14 as a white solid.

1

Yield 87% (10 g). H NMR (400 MHz, Chloroform-d) δ 8.25 (d, J = 8.1 Hz, 2H), 7.99 (s, 1H),

7

.39 (d, J = 8.1 Hz, 2H), 7.11 (s, 1H), 4.02 (s, 3H), 3.92 (s, 3H), 2.47 (s, 3H). MS (ESI) m/z: 438.9

+

[

M + H] .

.1.3.

pyridine-2-carboxylate (15)

4

methyl

4-bromo-6-methyl-7-oxo-1-tosyl-6,7-dihydro-1H-pyrrolo[2,3-c]

The mixture of compound 14 (3 g, 6.8 mmol) in 4M HCl in dioxane (24 mL, 136.60 mmol)

was stirred at 70 ℃ for 2 h and the solvent was removed under reduced pressure to give a white

solid. A solution of this solid in DMF (30 mL) was added NaH (0.32 g, 60% dispersion in mineral

oil, 8.2 mmol) at 0℃ and CH I (1.16 g, 8.2 mmol) was added after 15 mins. The reaction mixture

3

9

was stirred at rt for 2 h and quenched with saturated NH Cl solution at 0℃ . A white solid was

4

precipitated out and filtered, washed with water (100 mL × 3) and dried under vacuum to give the

1

title compound 15 as a white solid. Yield 93% (2.8 g). H NMR (400 MHz, Chloroform-d) δ 8.48 (d,

J = 8.2 Hz, 2H), 7.40 (d, J = 8.2 Hz, 2H), 7.27 (s, 1H), 6.92 (s, 1H), 4.00 (s, 3H), 3.57 (s, 3H), 2.45 (s,

+

3

H). MS (ESI) m/z: 438.9 [M + H] .

4

.1.4.

methyl-6-methyl-7-oxo-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1-tosyl-6,7-dihydro-1H-py

rrolo[2,3-c] pyridine-2-carboxylate (36)

A mixture of 15 (2 g, 4.55 mmol), 4,4,4',4',5,5,5',5'-octamethyl-2,2'-bi(1,3,2-dioxaborolane)

(

1.39 g, 5.46 mmol), (2-(2,4,6-triisopropylphenethyl) phenyl) dicyclohexylphosphine (X-Phos)

0.55 g, 0.91 mmol), Pd (dba) (0.42 g, 0.46 mmol), and potassium acetate (0.89 g, 9.10 mmol) in

2

3

dioxane (20 mL) was degassed and back-filled with argon six times. The reaction mixture was

heated at 90 ℃ for 2 h. The reaction mixture was partitioned between water (50 mL) and ethyl

acetate (30 mL×3). The combined organic layers were washed with saturated NaCl solution (100

mL × 3), dried over anhydrous Na SO , filtered, and concentrated. The residue was purified by

2

4

flash column chromatography on silica gel to give the title compound 36 as a gray solid. Yield 68%

1

(

1.5 g) H NMR (400 MHz, Chloroform-d) δ 8.45 (d, J = 8.4 Hz, 2H), 7.60 (s, 1H), 7.38 (d, J =

8

4

.6 Hz, 2H), 7.31 (s, 1H), 3.99 (s, 3H), 3.58 (s, 3H), 2.44 (s, 3H), 1.33 (s, 12H). MS (ESI) m/z:

87.1 [M + H] .

+

The general procedure for preparation of 27a-27g (exemplified by 27g)

4

.1.5. 4-fluoro-2, 6-dimethylphenol (35)

To a solution of 2-bromo-5-fluoro-l,3-dimethylbenzene 34 (2.5 g, 12.3 mmol) in anhydrous

tetrahydrofuran (30 mL) was added n-butyllithium (5.9 mL, 14.8 mmol) dropwise at -78 °C. The

mixture was stirred for 2 hours and then trimethylborate (1.7 mL, 14.8 mmol) was added and the

mixture stirred for 2 hours at -78 °C, then warmed to ambient temperature for 4 hours. Then the

mixture was cooled to -10 °C and a precooled solution of NaOH (0.74 g, 18.5 mmol) and 30 %

hydrogen peroxide (20 mL, 197 mmol) was added and the mixture was stirred at ambient

temperature overnight. The reaction mixture was partitioned between water (50 mL) and ether (30

mL×3) after adjusted the pH to 1 with 2M HCl (40 mL). The combined organic layers were

washed with saturated NaHCO (30 mL×3) then stirred with a saturated aqueous NaS O solution

3

2

3

(

20 mL) for 15 minutes. The organic phase was dried with anhydrous Na SO , filtered, and

2 4

concentrated. The residue was purified by flash column chromatography on silica gel to give the

1

title compound 35 as a white solid. Yield 87% (1.5 g). H NMR (400 MHz, Chloroform-d) δ 6.69

+

(

d, J = 8.9 Hz, 2H), 4.40 (s, 1H), 2.23 (s, 6H). MS (ESI) m/z: 141.0 [M + H] .

4

.1.6. 1-(3-bromo-4-(4-fluoro-2,6-dimethylphenoxy) phenyl) ethan-1-one (33d)

To a solution of 35 (2 g, 9.22 mmol) in dimethyl sulphoxide (20 mL) was added cesium

carbonate (3.6 g, 11.06 mmol) and 1-(3-bromo-4-fluorophenyl) ethan-1-one (1.55 g, 11.06 mmol).

The mixture was stirred at 80 °C for 2h. After cooled to rt, the The reaction mixture was

partitioned between water (50 mL) and ether (30 mL×3). The combined organic layers were

washed with water (30 mL×3) and saturated NaCl solution (30 mL×3). The organic phase was

dried with anhydrous Na SO , filtered, and concentrated. The residue was purified by flash

2

4

column chromatography on silica gel to give the title compound 33d as a colorless oil. Yield 96%

1

(

3 g). H NMR (400 MHz, Chloroform-d) δ 8.32 (d, J = 2.1 Hz, 1H), 7.81 (dd, J = 8.6, 2.1 Hz,

1

H), 6.83 (d, J = 8.8 Hz, 1H), 6.38 (d, J = 8.6 Hz, 1H), 3.89 (s, 3H), 2.10 (d, J = 0.7 Hz, 6H). MS

+

(ESI) m/z: 337.0 [M + H] .

4

.1.7. 2-(3-bromo-4-(4-fluoro-2,6-dimethylphenoxy) phenyl) propan-2-ol (27g)

To a solution of 33d (2.5 g, 7.41 mmol) in anhydrous tetrahydrofuran (30 mL) was added 3M

methyl magnesium bromide (7.41 mL, 22.24 mmol) dropwise at 0 °C. The mixture was stirred for

hours at rt and quenched with saturated NH Cl solution at 0℃ . The reaction mixture was

4

partitioned between water (50 mL) and ether (30 mL×3). The organic phase was dried with

anhydrous Na SO , filtered, and concentrated. The residue was purified by flash column

2

4

chromatography on silica gel to give the title compound 27g as a colorless oil. Yield 95% (2.5 g).

1

H NMR (400 MHz, Chloroform-d) δ 7.74 (d, J = 2.3 Hz, 1H), 7.20 (dd, J = 8.6, 2.3 Hz, 1H), 6.81

(

d, J = 8.8 Hz, 2H), 6.30 (d, J = 8.6 Hz, 1H), 2.12 (s, 6H), 1.55 (s, 6H). MS (ESI) m/z: 375.0 [M +

+

Na] .

1

0

4

.2. The general procedure for preparation of dual inhibitors 18a-18e, 20a-20d, 25a-25q.

(exemplified by 18e and 20d).

4

.2.1. methyl 6-methyl-7-oxo-4-(2-phenoxyphenyl)-1-tosyl-6,7-dihydro-1H-pyrrolo[2,3-c]

pyridine-2-carboxylate (16)

A mixture of 15 (2 g, 4.55 mmol), (2-phenoxyphenyl) boronic acid (1.17 g, 5.46 mmol),

1

,3,5,7-tetramethyl-6-phenyl-2,4,8-trioxa-6-phosphaadamantane (0.20 g, 0.91 mmol), Pd (dba)

2 3

(0.42 g, 0.46 mmol), and cesium fluoride (1.38 g, 9.10 mmol) in dimethoxyethane (20 mL) and

water (4 mL) was degassed and back-filled with argon six times. The reaction mixture was heated

at 60 ℃ for 2 h. The reaction mixture was partitioned between water (50 mL) and ethyl acetate (30

mL×3). The combined organic layers were washed with saturated aqueous NaCl solution (100 mL

×

3), dried over anhydrous Na SO , filtered, and concentrated. The residue was purified by flash

2 4

column chromatography on silica gel to give the title compound 16 as a white solid. Yield 83% (2

1

g). H NMR (400 MHz, Chloroform-d) δ 8.49 (d, J = 8.5 Hz, 2H), 7.41 – 7.32 (m, 4H), 7.24 –

7

3

.19 (m, 3H), 7.15 (s, 1H), 7.06 – 7.01 (m, 2H), 6.89 (s, 1H), 6.81 (d, J = 7.7 Hz, 2H), 3.95 (s, 3H),

.54 (s, 3H), 2.44 (s, 3H). MS (ESI) m/z: 529.0 [M + H] .

+

4

.2.2. methyl 4-((6-methyl-7-oxo-4-(2-phenoxyphenyl)-6,7-dihydro-1H-pyrrolo[2,3-c]

pyridine-2-carboxamido) methyl) benzoate (17e)

To a solution of 16 (0.2 g, 0.38 mmol) in dioxane (3 mL) was added 2 M NaOH solution

(0.75 mL, 1.51 mmol). The mixture was stirred at 90 ℃ for 2 h and after cooled to ambient

temperature the reaction mixture was adjusted the pH to 3 with 2 M HCl (0.8 mL), extracted with

ethyl acetate (10 mL × 3). The combined organic layers were dried over anhydrous Na SO ,

2

4

filtered, and concentrated to give an acid as white solid. Then the acid was dissolved in anhydrous

dimethylformamide (2 mL) and 1-hydroxy-7-azabenzotriazole (0.072 g, 0.53 mmol),

N-(3-(dimethylamino) propyl) propionimidamidehydrochloride (0.101 g, 0.53 mmol) and N,

N-diisopropyl-ethylamin (0.195 g, 1.51 mmol) were added. After 0.5 h the methyl 4-(aminomethyl)

benzoate hydrochloride (0.092 g, 0.45 mmol) was added and the reaction mixture was stirred at

5

0 ℃ for 2 h. After cooled to rt, the The reaction mixture was partitioned between water (5 mL)

and ether (5 mL × 3). The combined organic layers were washed with water (5 mL × 3) and

saturated NaCl solution (5 mL × 3). The organic phase was dried with anhydrous Na SO , filtered,

2

4

and concentrated. The residue was purified by flash column chromatography on silica gel to give

1

the title compound 17e as a white solid. Yield 78% (0.15 g). H NMR (400 MHz, CDCl ) δ 12.18

3

(s, 1H), 8.37 (s, 1H), 8.00 (d, J = 8.3 Hz, 2H), 7.53 (dd, J = 7.6, 1.5 Hz, 1H), 7.41 (d, J = 8.3 Hz,

2

H), 7.39 – 7.34 (m, 1H), 7.24 (t, J = 8.0 Hz, 3H), 7.12 (d, J = 2.2 Hz, 1H), 7.06 (d, J = 8.2 Hz,

1

H), 7.04 (s, 1H), 7.01 (t, J = 7.4 Hz, 1H), 6.86 (d, J = 7.7 Hz, 2H), 4.81 (d, J = 5.9 Hz, 2H), 3.91

+

(s, 3H), 3.40 (s, 3H). MS (ESI) m/z: 508.1 [M + H] .

4

.2.3. N-(4-(hydroxycarbamoyl)

benzyl)-6-methyl-7-oxo-4-(2-phenoxyphenyl)-6,7-dihydro-1H-pyrrolo[2,3-c]

pyridine-2-carboxamide (18e)

To a solution of 17e (0.1 g, 0.197 mmol) in methanol (1 mL) and dichloromethane (1 mL)

was added 50% hydroxylamine in water (0.26 mL) and sodium hydroxide (0.157 g, 3.94 mmol) at

0

℃ and stirred for 2h. The reaction mixture was adjusted the pH to 3 with 0.5 M HCl (8 mL),

extracted with ethyl acetate (10 mL × 3). The combined organic layers were dried over anhydrous

Na SO , filtered, and concentrated. The residue was recrystallized in hexane/ dichloromethane to

2

4

1

give 18e as a white solid. Yield 70% (0.07 g). H NMR (400 MHz, DMSO) δ 12.29 (s, 1H), 11.19

s, 1H), 8.95 (t, J = 5.6 Hz, 1H), 7.72 (d, J = 8.1 Hz, 2H), 7.52 (d, J = 6.5 Hz, 1H), 7.45 – 7.35 (dd,

J = 17.4, 7.5 Hz, 3H), 7.33 – 7.25 (m, 4H), 7.08 – 6.98 (m, 2H), 6.95 (s, 1H), 6.87 (d, J = 7.5 Hz,

(

1

3

2

1

H), 4.49 (d, J = 5.7 Hz, 2H), 3.50 (s, 3H). C NMR (126 MHz, DMSO-d ) δ 164.44, 160.14,

6

57.09, 154.52, 154.12, 142.86, 133.46, 131.98, 131.83, 130.31, 130.22, 129.69, 129.55, 128.48,

27.67, 127.36, 124.79, 124.48, 123.39, 119.84, 118.29, 111.24, 105.77, 42.50, 36.15. HRMS

+

(

AP-ESI) m/z calcd for C H N O [M + H] 509.1820, found 509.1821. Purity: 95%.

2

9

25

4

5

4

.2.4. N-(4-((2-aminophenyl) carbamoyl)

benzyl)-6-methyl-7-oxo-4-(2-phenoxyphenyl)-6,7-dihydro-1H-pyrrolo[2,3-c]

pyridine-2-carboxamide (20d)

To a solution of 17e (0.1 g, 0.197 mmol) in dioxane (1.5 mL) was added 2 M NaOH solution

(0.39 mL, 0.788 mmol). The mixture was stirred at 90 ℃ for 2 h and after cooled to ambient

1

temperature the reaction mixture was adjusted the pH to 3 with 2 M HCl (0.4 mL), extracted with

ethyl acetate (10 mL × 3). The combined organic layers were dried over anhydrous Na SO ,

2

4

filtered, and concentrated to give the acid 19d as a white solid. Then the acid was dissolved in

anhydrous dimethylformamide (2 mL) and 1-hydroxy-7-azabenzotriazole (0.037 g, 0.275 mmol),

N-(3-(dmethylamino) propyl) propionimidamidehydrochloride (0.053 g, 0.275 mmol) and N,

N-diisopropyl-ethylamin (0.090 g, 0.788 mmol) was added. After 0.5 h the benzene-1,2-diamine

(0.025 g, 0.236 mmol) was added and the reaction mixture was stirred at 50 ℃ for 2 h. After

cooled to rt, the The reaction mixture was partitioned between water (5 mL) and ether (5 mL × 3).

The combined organic layers were washed with water (5 mL × 3) and saturated NaCl solution (5

mL × 3). The organic phase was dried with anhydrous Na SO , filtered, and concentrated. The

2

4

residue was purified by flash column chromatography on silica gel to give the title compound 20d

1

as a white solid. Yield 79% (0.09 g). H NMR (400 MHz, DMSO-d ) δ 12.34 (s, 1H), 9.66 (s, 1H),

6

7

.96 (d, J = 7.9 Hz, 2H), 7.54 (d, J = 7.5 Hz, 1H), 7.49 – 7.39 (m, 3H), 7.35 – 7.25 (m, 4H), 7.17

(d, J = 7.9 Hz, 1H), 7.04 (dd, J = 8.2, 4.9 Hz, 2H), 7.02 – 6.94 (m, 2H), 6.88 (d, J = 8.0 Hz, 2H),

6

3

1

.79 (d, J = 7.9 Hz, 1H), 6.61 (t, J = 7.6 Hz, 1H), 4.91 (s, 2H), 4.54 (d, J = 5.9 Hz, 2H), 3.52 (s,

H). C NMR (126 MHz, DMSO) δ 165.49, 160.17, 157.10, 154.53, 154.13, 143.53, 143.18,

33.68, 133.50, 131.99, 130.32, 130.23, 129.70, 129.56, 128.49, 128.28, 127.56, 127.08, 126.87,

13

24.81, 124.49, 123.73, 123.40, 119.85, 118.30, 116.66, 116.53, 111.25, 105.79, 42.53, 36.17.

+

HRMS (AP-ESI) m/z calcd for C H N O [M + H] 584.2292, found 584.2297. Purity: 95%.

3

5

30

5

4

.2.5.

N-(4-(hydroxyamino)-4-oxobutyl)-6-methyl-7-oxo-4-(2-phenoxyphenyl)-6,7-dihydro-1H-pyrrolo[2

,

3-c] pyridine-2-carboxamide (18a)

1

White solid (79%). H NMR (400 MHz, DMSO-d ) δ 12.20 (s, 1H), 10.38 (s, 1H), 8.71 (s,

6

1

7

2

1

H), 8.37 (t, J = 5.4 Hz, 1H), 7.52 (dd, J = 7.6, 1.7 Hz, 1H), 7.41 (td, J = 8.1, 1.8 Hz, 1H), 7.32 –

.23 (m, 4H), 7.07 – 6.97 (m, 2H), 6.89 – 6.82 (m, 3H), 3.50 (s, 3H), 3.23 (q, J = 6.8 Hz, 2H),

1

3

.01 (t, J = 7.5 Hz, 2H), 1.74 (q, J = 7.4 Hz, 2H). C NMR (126 MHz, DMSO) δ 169.21, 160.08,

57.21, 154.59, 154.14, 133.87, 132.03, 130.33, 130.27, 129.70, 129.60, 128.61, 124.68, 124.58,

23.42, 120.01, 118.30, 111.28, 105.65, 39.01, 36.19, 30.36, 25.60. HRMS (ESI) m/z calcd for

+

C H N O [M + H] 461.1820, found, 461.1824.

25

4

5

4

.2.6.

N-(5-(hydroxyamino)-5-oxopentyl)-6-methyl-7-oxo-4-(2-phenoxyphenyl)-6,7-dihydro-1H-pyrrolo[

2

,3-c] pyridine-2-carboxamide (18b)

1

White solid (73%). H NMR (400 MHz, DMSO) δ 12.20 (s, 1H), 10.35 (s, 1H), 8.76 – 8.59

(br, 1H), 8.35 (t, J = 5.4 Hz, 1H), 7.52 (dd, J = 7.5, 1.7 Hz, 1H), 7.41 (td, J = 7.8, 1.7 Hz, 1H),

7

.32 – 7.24 (m, 4H), 7.05 – 6.99 (m, 2H), 6.88 – 6.83 (m, 3H), 3.50 (s, 3H), 3.22 (dd, J = 12.3, 6.2

1

3

Hz, 2H), 1.97 (t, J = 7.0 Hz, 2H), 1.58 – 1.44 (m, 4H). C NMR (126 MHz, DMSO) δ 169.41,

1

59.98, 157.20, 154.60, 154.15, 133.92, 132.03, 130.28, 129.70, 129.59, 128.60, 124.66, 124.57,

23.42, 119.98, 118.31, 111.30, 105.66, 39.03, 36.19, 32.43, 29.04, 23.17. HRMS (AP-ESI) m/z

+

calcd for C H N O [M + H] 475.1976, found 475.1981. Purity: 97%.

2

6

27

4

5

4

.2.7.

N-(6-(hydroxyamino)-6-oxohexyl)-6-methyl-7-oxo-4-(2-phenoxyphenyl)-6,7-dihydro-1H-pyrrolo[2

,

3-c] pyridine-2-carboxamide (18c)

1

White solid (77%). H NMR (400 MHz, DMSO) δ 12.22 (s, 1H), 10.34 (s, 1H), 8.67 (s, 1lH),

8

7

.34 (s, 1H), 7.52 (d, J = 7.6 Hz, 1H), 7.42 (t, J = 7.9 Hz, 1H), 7.25 – 7.33 (m, 6.8 Hz, 4H), 7.06 –

.00 (m, 2H), 6.90 – 6.84 (d, J = 5.6 Hz, 3H), 3.51 (s, 3H), 3.23 (dd, J = 12.8, 6.7 Hz, 2H), 1.95 (t,

1

3

J = 6.7 Hz, 2H), 1.58 – 1.43 (m, 4H), 1.33 – 1.26 (m, 2H). C NMR (126 MHz, DMSO-d ) δ

6

1

68.98, 159.45, 156.69, 154.08, 153.63, 133.43, 131.52, 129.76, 129.18, 129.09, 128.09, 124.14,

24.06, 122.91, 119.48, 117.79, 110.78, 105.15, 38.70, 35.68, 32.19, 28.66, 26.06, 24.85. HRMS

+

(AP-ESI) m/z calcd for C H N O [M + H] 489.2133, found 489.2137. Purity: 97%.

2

7

29

4

5

4

.2.8.

N-(7-(hydroxyamino)-7-oxoheptyl)-6-methyl-7-oxo-4-(2-phenoxyphenyl)-6,7-dihydro-1H-pyrrolo[

2

,3-c] pyridine-2-carboxamide(18d)

1

White solid (72%). H NMR (600 MHz, DMSO) δ 12.20 (s, 1H), 10.33 (s, 1H), 8.66 (s, 1H),

8

.33 (t, J = 5.4 Hz, 1H), 7.52 (dd, J = 7.6, 1.6 Hz, 1H), 7.42 (td, J = 8.1, 1.7 Hz, 1H), 7.32 – 7.25

1

2

(

1

m, 4H), 7.05 – 7.01 (m, 2H), 6.88 – 6.84 (m, 3H), 3.51 (s, 3H), 3.23 (dd, J = 12.7, 6.7 Hz, 2H),

.94 (t, J = 7.4 Hz, 2H), 1.53 – 1.46 (m, 4H), 1.32 – 1.25 (m, 4H). C NMR (126 MHz, DMSO-d )

1

3

6

δ 169.53, 159.95, 157.17, 154.59, 154.15, 133.94, 132.02, 130.26, 129.69, 129.59, 128.58, 124.55,

23.42, 119.94, 118.31, 111.31, 105.64, 39.25, 36.18, 32.71, 29.29, 28.79, 26.65, 25.54. HRMS

1

+

(AP-ESI) m/z calcd for C H N O [M + H] 503.2289, found 503.2293. Purity: 97%.

28 31 4 5

4

.2.9.

N-(5-((2-aminophenyl)

amino)-5-oxopentyl)-6-methyl-7-oxo-4-(2-phenoxyphenyl)-6,7-dihydro-1H-pyrrolo[2,3-c]

pyridine-2-carboxamide(20a)

1

White solid (85%). H NMR (400 MHz, DMSO) δ 12.24 (s, 1H), 9.12 (s, 1H), 8.40 (t, J = 5.4

Hz, 1H), 7.52 (dd, J = 7.5, 1.2 Hz, 1H), 7.41 (td, J = 7.9, 1.4 Hz, 1H), 7.34 – 7.24 (m, 4H), 7.15 (d,

J = 7.4 Hz, 1H), 7.06 – 6.98 (m, 2H), 6.92 – 6.83 (m, 4H), 6.71 (d, J = 7.2 Hz, 1H), 6.53 (t, J = 7.6

Hz, 1H), 4.83 (s, 2H), 3.50 (s, 3H), 3.28 (dd, J = 11.0, 5.1 Hz, 2H), 2.35 (t, J = 7.3 Hz, 2H), 1.71 –

+

1

.60 (m, 2H), 1.60 – 1.50 (m, 2H). HRMS (AP-ESI) m/z calcd for C H N O [M + H]

32 32 5 4

5

50.2449, found 550.2451. Purity: 99%.

4

.2.10.

N-(6-((2-aminophenyl)

amino)-6-oxohexyl)-6-methyl-7-oxo-4-(2-phenoxyphenyl)-6,7-dihydro-1H-pyrrolo[2,3-c]

pyridine-2-carboxamide(20b)

1

White solid (90%). H NMR (400 MHz, DMSO) δ 12.24 (s, 1H), 9.13 (s, 1H), 8.38 (t, J = 5.4

Hz, 1H), 7.52 (dd, J = 7.6, 1.5 Hz, 1H), 7.42 (td, J = 8.0, 1.7 Hz, 1H), 7.33 – 7.24 (m, 3H), 7.14

(dd, J = 7.9, 1.1 Hz, 1H), 7.06 – 7.00 (m, 2H), 6.93 – 6.83 (m, 4H), 6.72 (dd, J = 8.0, 1.1 Hz, 1H),

6

1

.53 (t, J = 7.6 Hz, 1H), 4.97 (s, 2H), 3.51 (s, 3H), 3.30 – 3.22 (m, 2H), 2.32 (t, J = 7.5 Hz, 2H),

1

3

.68 – 1.60 (m, 2H), 1.59 – 1.51 (m, 3H), 1.42 – 1.32 (m, 2H).; C NMR (126 MHz, DMSO-d ) δ

6

1

71.04, 159.48, 156.69, 154.09, 153.64, 141.83, 133.45, 131.52, 129.76, 129.19, 129.09, 128.10,

1

25.63, 125.25, 124.15, 124.06, 123.52, 122.91, 119.47, 117.80, 116.11, 115.82, 110.80, 105.16,

+

3

8.72, 35.69, 28.75, 26.15, 25.03. HRMS (AP-ESI) m/z calcd for C H N O [M + H] 564.2605,

3

34

5

4

found 564.2603. Purity: 99%.

4

.2.11.

N-(7-((2-aminophenyl)

amino)-7-oxoheptyl)-6-methyl-7-oxo-4-(2-phenoxyphenyl)-6,7-dihydro-1H-pyrrolo[2,3-c]

pyridine-2-carboxamide(20c)

1

White solid (92%). H NMR (400 MHz, DMSO) δ 12.22 (s, 1H), 9.10 (s, 1H), 8.35 (t, J = 5.6

Hz, 1H), 7.52 (dd, J = 7.5, 1.6 Hz, 1H), 7.44 – 7.39 (m, 1H), 7.33 – 7.24 (m, 4H), 7.14 (d, J = 6.9

Hz, 1H), 7.05 – 7.00 (m, 2H), 6.84 – 6.91 (m, 4H), 6.70 (d, J = 6.8 Hz, 1H), 6.53 (td, J = 7.9, 1.4

Hz, 1H), 4.82 (s, 2H), 3.50 (s, 3H), 3.24 (dd, J = 11.5, 5.9 Hz, 2H), 2.31 (t, J = 7.4 Hz, 2H), 1.65 –

1

.56 (m, 2H), 1.55 – 1.47 (m, 2H), 1.42 – 1.30 (m, 4H). HRMS (AP-ESI) m/z calcd for

+

C H N O [M + H] 578.2762, found 578.2763. Purity: 99%.

34

36

5

4

.2.12. N-(4-((2-aminophenyl) carbamoyl)

benzyl)-6-methyl-7-oxo-4-phenyl-6,7-dihydro-1H-pyrrolo[2,3-c] pyridine-2-carboxamide(25a)

1

White solid (90%). H NMR (400 MHz, DMSO-d ) δ 12.47 (s, 1H), 9.64 (s, 1H), 9.06 (t, J = 5.9

6

Hz, 1H), 7.96 (d, J = 7.9 Hz, 2H), 7.61 (d, J = 6.8 Hz, 2H), 7.52 – 7.43 (m, 5H), 7.37 (t, J = 7.2 Hz,

1

4

1

H), 7.16 (d, J = 7.5 Hz, 2H), 6.97 (t, J = 6.8 Hz, 1H), 6.78 (d, J = 6.6 Hz, 1H), 6.59 (t, J = 7.5 Hz, 1H),

1

3

.89 (s, 2H), 4.56 (d, J = 5.9 Hz, 2H), 3.59 (s, 3H). C NMR (126 MHz, DMSO-d ) δ 165.49,

6

60.17, 154.58, 143.52, 143.14, 137.03, 133.84, 133.71, 129.25, 129.14, 128.48, 128.30, 127.80,

27.59, 127.40, 127.08, 126.87, 125.31, 123.72, 116.65, 116.52, 115.02, 105.16, 42.55, 36.18.

+

HRMS (AP-ESI) m/z calcd for C H N O [M + H] 492.2030, found 492.2032. Purity: 99%.

2

9

26

5

3

4

.2.13.

N-(4-((2-aminophenyl)

carbamoyl)

benzyl)-4-(2-fluorophenyl)-6-methyl-7-oxo-6,7-dihydro-1H-pyrrolo[2,3-c]

pyridine-2-carboxamide(25b)

1

White solid (93%). H NMR (400 MHz, DMSO) δ 12.47 (s, 1H), 9.65 (s, 1H), 9.04 (s, 1H),

.96 (d, J = 8.1 Hz, 2H), 7.55 (t, J = 6.8 Hz, 1H), 7.50 – 7.41 (m, 4H), 7.40 – 7.30 (m, 2H), 7.16 (d,

7

J = 7.3 Hz, 1H), 6.97 (t, J = 7.6 Hz, 1H), 6.89 (s, 1H), 6.78 (d, J = 8.0 Hz, 1H), 6.60 (t, J = 7.5 Hz,

H), 4.90 (s, 2H), 4.54 (d, J = 5.7 Hz, 2H), 3.59 (s, 3H). HRMS (AP-ESI) m/z calcd for

1

+

C H FN O [M + H] 510.1936, found 510.1941. Purity: 95%.

29

25

5

3

1

3

4

.2.14.

N-(4-((2-aminophenyl)

carbamoyl)

benzyl)-4-(2-chlorophenyl)-6-methyl-7-oxo-6,7-dihydro-1H-pyrrolo[2,3-c]

pyridine-2-carboxamide(25c)

1

White solid (92%). H NMR (400 MHz, DMSO-d ) δ 12.44 (s, 1H), 9.64 (s, 1H), 9.00 (t, J =

6

5

.9 Hz, 1H), 7.94 (d, J = 7.9 Hz, 2H), 7.65 – 7.58 (m, 1H), 7.52 – 7.39 (m, 5H), 7.34 (s, 1H), 7.16

(

=

d, J = 7.8 Hz, 1H), 6.97 (dd, J = 8.2, 6.6 Hz, 1H), 6.78 (d, J = 7.1 Hz, 1H), 6.71 (s, 1H), 6.59 (t, J

7.7 Hz, 1H), 4.90 (s, 2H), 4.51 (d, J = 5.9 Hz, 2H), 3.57 (s, 3H). MS (ESI) m/z: 526.1 [M + H]

+

4

.2.15. N-(4-((2-aminophenyl) carbamoyl) benzyl)-6-methyl-7-oxo-4-(2-(trifluoromethyl)

phenyl)-6,7-dihydro-1H-pyrrolo[2,3-c] pyridine-2-carboxamide (25d)

1

White solid (95%). H NMR (400 MHz, DMSO-d ) δ 12.49 (s, 1H), 9.64 (s, 1H), 8.96 (t, J =

6

7

1

.2 Hz, 1H), 7.94 (d, J = 8.0 Hz, 2H), 7.89 (d, J = 7.9 Hz, 1H), 7.78 (t, J = 7.5 Hz, 1H), 7.67 (t, J =

.7 Hz, 1H), 7.54 (d, J = 7.6 Hz, 1H), 7.42 (d, J = 8.0 Hz, 2H), 7.22 (s, 1H), 7.15 (d, J = 7.9 Hz,

H), 6.97 (t, J = 7.8 Hz, 1H), 6.78 (d, J = 8.0 Hz, 1H), 6.59 (t, J = 7.7 Hz, 1H), 6.57 (d, J = 2.0 Hz,

+

H), 4.90 (s, 2H), 4.50 (d, J = 5.9 Hz, 2H), 3.55 (s, 3H). MS (ESI) m/z: 560.1 [M + H]

4

.2.16.

N-(4-((2-aminophenyl)

carbamoyl)

benzyl)-6-methyl-7-oxo-4-(o-tolyl)-6,7-dihydro-1H-pyrrolo[2,3-c] pyridine-2-carboxamide(25e)

1

White solid (95%). H NMR (400 MHz, DMSO-d ) δ 12.40 (s, 1H), 9.64 (s, 1H), 8.99 (t, J =

6

5

(

.9 Hz, 1H), 7.94 (d, J = 7.9 Hz, 2H), 7.43 (d, J = 8.1 Hz, 2H), 7.37 – 7.29 (m, 2H), 7.30 – 7.26

m, 2H), 7.21 (s, 1H), 7.16 (d, J = 7.8 Hz, 1H), 6.97 (td, J = 7.6, 1.5 Hz, 1H), 6.78 (dd, J = 8.0, 1.4

Hz, 1H), 6.66 (s, 1H), 6.59 (td, J = 7.6, 1.4 Hz, 1H), 4.89 (s, 2H), 4.51 (d, J = 5.9 Hz, 2H), 3.57 (s,

+

3

H), 2.21 (s, 3H). HRMS (AP-ESI) m/z calcd for C H N O [M + H] 506.2187, found

30 28 5 3

5

06.2185. Purity: 99%.

4

.2.17.

N-(4-((2-aminophenyl)

carbamoyl)

benzyl)-4-(2-methoxyphenyl)-6-methyl-7-oxo-6,7-dihydro-1H-pyrrolo[2,3-c]

pyridine-2-carboxamide(25f)

1

White solid (90%). H NMR (400 MHz, DMSO-d ) δ 12.29 (d, J = 2.1 Hz, 1H), 9.65 (s, 1H),

6

8

7

.98 (t, J = 6.0 Hz, 1H), 7.95 (d, J = 8.0 Hz, 2H), 7.44 (d, J = 8.0 Hz, 2H), 7.39 (t, J = 7.9 Hz, 1H),

.32 (dd, J = 7.5, 1.7 Hz, 1H), 7.24 (s, 1H), 7.15 (d, J = 8.0 Hz, 2H), 7.04 (t, J = 7.5 Hz, 1H), 6.97

(

t, J = 7.6 Hz, 1H), 6.78 (d, J = 7.7 Hz, 1H), 6.74 (d, J = 2.1 Hz, 1H), 6.60 (t, J = 7.9 Hz, 1H), 4.90

1

3

(s, 2H), 4.53 (d, J = 5.9 Hz, 2H), 3.75 (s, 3H), 3.56 (s, 3H). C NMR (126 MHz, DMSO-d ) δ

6

1

65.48, 160.23, 157.22, 154.64, 143.52, 143.18, 133.68, 133.35, 131.15, 130.16, 129.88, 129.37,

1

28.28, 127.56, 127.07, 126.86, 125.43, 124.79, 123.72, 120.91, 116.65, 116.52, 112.38, 111.93,

+

06.01, 55.76, 42.52, 36.13. HRMS (AP-ESI) m/z calcd for C H N O [M + H] 522.2136,

3

0

28

5

4

found 522.2141. Purity: 95%.

4

.2.18.

N-(4-((2-aminophenyl)

carbamoyl)

benzyl)-4-(2-isopropoxyphenyl)-6-methyl-7-oxo-6,7-dihydro-1H-pyrrolo[2,3-c]

pyridine-2-carboxamide(25g)

1

White solid (89%). H NMR (600 MHz, DMSO-d ) δ 12.21 (s, 1H), 9.61 (s, 1H), 8.95 (t, J =

6

5

7

1

6

.9 Hz, 1H), 7.93 (d, J = 8.0 Hz, 2H), 7.42 (d, J = 8.2 Hz, 2H), 7.34 – 7.30 (m, 2H), 7.23 (s, 1H),

.14 (d, J = 7.6 Hz, 1H), 7.10 (d, J = 8.4 Hz, 1H), 7.00 (t, 1H), 6.95 (t, 1H), 6.82 (d, J = 1.6 Hz,

H), 6.76 (dd, J = 8.0, 1.2 Hz, 1H), 6.57 (td, J = 7.5, 1.4 Hz, 1H), 4.86 (s, 2H), 4.54 (p, J = 12.2,

1

3

.1 Hz, 1H), 4.51 (d, J = 5.8 Hz, 2H), 3.55 (s, 3H), 1.14 (d, J = 6.0 Hz, 6H). C NMR (126 MHz,

DMSO-d ) δ 165.47, 160.27, 155.31, 154.63, 143.53, 143.21, 133.66, 133.14, 131.40, 129.94,

6

1

5

29.14, 128.26, 127.50, 127.09, 126.87, 126.45, 124.84, 123.72, 120.82, 116.65, 116.53, 114.53,

+

12.64, 106.61, 70.13, 42.49, 36.12, 22.20. HRMS (AP-ESI) m/z calcd for C H N O [M + H]

3

2

32

5

4

50.2449, found 550.2454. Purity: 96%.

4

.2.19. N-(4-((2-aminophenyl)

carbamoyl)

benzyl)-4-(3-fluorophenyl)-6-methyl-7-oxo-6,7-dihydro-1H-pyrrolo[2,3-c]

pyridine-2-carboxamide(25h)

1

White solid (87%). H NMR (400 MHz, DMSO) δ 12.52 (s, 1H), 9.65 (s, 1H), 9.08 (t, J = 6.0

Hz, 1H), 7.96 (d, J = 8.0 Hz, 2H), 7.57 – 7.50 (m, 2H), 7.51 – 7.39 (m, 4H), 7.24 – 7.12 (m, 3H),

6

=

.97 (t, J = 7.6 Hz, 1H), 6.78 (d, J = 7.8 Hz, 1H), 6.59 (t, J = 7.4 Hz, 1H), 4.90 (s, 2H), 4.56 (d, J

+

5.5 Hz, 2H), 3.59 (s, 3H). HRMS (AP-ESI) m/z calcd for C H FN O [M + H] 510.1936,

29 25 5 3

found 510.1940. Purity: 97%.

1

4

.2.20.

N-(4-((2-aminophenyl)

carbamoyl)

benzyl)-4-(3-chlorophenyl)-6-methyl-7-oxo-6,7-dihydro-1H-pyrrolo[2,3-c]

pyridine-2-carboxamide(25i)

1

White solid (90%). H NMR (400 MHz, DMSO-d ) δ 12.53 (s, 1H), 9.65 (s, 1H), 9.09 (t, J =

6

5

.7 Hz, 1H), 7.96 (d, J = 7.9 Hz, 2H), 7.64 (s, 1H), 7.60 (d, J = 7.9 Hz, 1H), 7.56 (s, 1H), 7.52 (t, J

=

7.8 Hz, 1H), 7.45 (t, J = 9.4 Hz, 3H), 7.19 – 7.14 (m, 2H), 6.97 (t, J = 7.6 Hz, 1H), 6.78 (d, J =

8

.0 Hz, 1H), 6.60 (t, J = 7.6 Hz, 1H), 4.94 (s, 2H), 4.56 (d, J = 5.8 Hz, 2H), 3.59 (s, 3H). HRMS

+

(AP-ESI) m/z calcd for C H ClN O [M + H] 526.1641, found 526.1645.

2

9

25

5

3

4

.2.21.

N-(4-((2-aminophenyl)

carbamoyl)

benzyl)-6-methyl-7-oxo-4-(3-(trifluoromethyl)

phenyl)-6,7-dihydro-1H-pyrrolo[2,3-c] pyridine-2-carboxamide (25j)

1

White solid (85%). H NMR (400 MHz, DMSO-d ) δ 12.56 (s, 1H), 9.64 (s, 1H), 9.07 (s, 1H),

6

7

2

1

.95 (d, J = 7.9 Hz, 4H), 7.89 (s, 1H), 7.74 (d, J = 4.6 Hz, 2H), 7.62 (s, 1H), 7.46 (d, J = 8.0 Hz,

H), 7.18 – 7.12 (m, 2H), 6.96 (t, J = 7.7 Hz, 1H), 6.77 (d, J = 7.9 Hz, 1H), 6.59 (t, J = 7.4 Hz,

+

H), 4.89 (s, 2H), 4.56 (d, J = 5.8 Hz, 2H), 3.60 (s, 3H). MS (ESI) m/z: 560.1 [M + H] .

4

.2.22. N-(4-((2-aminophenyl) carbamoyl)

benzyl)-6-methyl-7-oxo-4-(m-tolyl)-6,7-dihydro-1H-pyrrolo[2,3-c] pyridine-2-carboxamide(25k)

1

White solid (90%). H NMR (400 MHz, DMSO-d ) δ 12.45 (s, 1H), 9.64 (s, 1H), 9.06 (t, J =

6

5

7

4

.8 Hz, 1H), 7.96 (d, J = 7.9 Hz, 2H), 7.46 (d, J = 8.0 Hz, 2H), 7.40 (dd, J = 13.9, 6.7 Hz, 4H),

.22 – 7.12 (m, 3H), 6.96 (t, J = 7.6 Hz, 1H), 6.77 (d, J = 8.0 Hz, 1H), 6.59 (t, J = 7.5 Hz, 1H),

+

.89 (s, 2H), 4.56 (d, J = 5.8 Hz, 2H), 3.59 (s, 3H), 2.39 (s, 3H). MS (ESI) m/z: 506.2 [M + H] .

4

.2.23.

N-(4-((2-aminophenyl)

carbamoyl)

benzyl)-4-(3-methoxyphenyl)-6-methyl-7-oxo-6,7-dihydro-1H-pyrrolo[2,3-c]

pyridine-2-carboxamide(25l)

1

White solid (92%). H NMR (400 MHz, DMSO-d ) δ 12.46 (d, J = 2.1 Hz, 1H), 9.65 (s, 1H),

6

9

.08 (t, J = 5.9 Hz, 1H), 7.95 (d, J = 8.0 Hz, 2H), 7.47 (s, 1H), 7.45 (d, J = 8.1 Hz, 2H), 7.40 (t, J

=

8.0 Hz, 1H), 7.22 – 7.10 (m, 4H), 7.01 – 6.91 (m, 2H), 6.77 (dd, J = 8.0, 1.5 Hz, 1H), 6.59 (t, J =

1

3

7

.7 Hz, 1H), 4.91 (s, 2H), 4.55 (d, J = 5.8 Hz, 2H), 3.83 (s, 3H), 3.59 (s, 3H). C NMR (126 MHz,

DMSO-d ) δ 165.49, 160.17, 160.02, 154.58, 143.52, 143.16, 138.42, 133.82, 133.70, 130.28,

6

1

29.25, 128.45, 128.30, 127.57, 127.08, 126.86, 125.28, 123.73, 120.14, 116.65, 116.53, 114.88,

1

13.39, 112.81, 105.09, 55.54, 42.55, 36.18. HRMS (AP-ESI) m/z calcd for C H N O [M + H]

3

0

28

5

4

+

522.2136, found 522.2137. Purity: 95%.

4

.2.24. N-(4-((2-aminophenyl)

carbamoyl)

benzyl)-4-(4-fluorophenyl)-6-methyl-7-oxo-6,7-dihydro-1H-pyrrolo[2,3-c]

pyridine-2-carboxamide(25m)

1

White solid (87%). H NMR (400 MHz, DMSO-d ) δ 12.50 (s, 1H), 9.65 (s, 1H), 9.06 (t, J =

6

5

.9 Hz, 1H), 7.96 (d, J = 7.9 Hz, 2H), 7.64 (dd, J = 8.1, 5.8 Hz, 2H), 7.50 – 7.42 (m, 3H), 7.34 (t, J

=

8.6 Hz, 2H), 7.17 (d, J = 8.0 Hz, 1H), 7.14 (s, 1H), 6.97 (t, J = 7.7 Hz, 1H), 6.78 (d, J = 7.9 Hz,

1

H), 6.60 (t, J = 7.6 Hz, 1H), 4.93 (s, 2H), 4.56 (d, J = 5.7 Hz, 2H), 3.59 (s, 3H). HRMS (AP-ESI)

+

m/z calcd for C H FN O [M + H] 510.1936, found 510.1938. Purity: 98%.

2

9

25

5

3

4

.2.25.

N-(4-((2-aminophenyl)

carbamoyl)

benzyl)-4-(4-chlorophenyl)-6-methyl-7-oxo-6,7-dihydro-1H-pyrrolo[2,3-c]

pyridine-2-carboxamide(25n)

1

White solid (90%). H NMR (400 MHz, DMSO-d ) δ 12.53 (s, 1H), 9.66 (s, 1H), 9.06 (t, J =

6

5

1

5

.9 Hz, 1H), 7.97 (d, J = 7.9 Hz, 2H), 7.64 (d, J = 8.6 Hz, 2H), 7.56 (d, J = 8.6 Hz, 2H), 7.50 (s,

H), 7.47 (d, J = 8.2 Hz, 2H), 7.21 – 7.13 (m, 2H), 6.98 (t, J = 7.6 Hz, 1H), 6.79 (d, J = 8.0 Hz,

H), 6.60 (t, J = 7.3 Hz, 1H), 4.90 (s, 2H), 4.57 (d, J = 5.8 Hz, 2H), 3.60 (s, 3H). MS (ESI) m/z:

+

26.1 [M + H]

4

.2.26.

N-(4-((2-aminophenyl)

carbamoyl)

benzyl)-6-methyl-7-oxo-4-(4-(trifluoromethyl)

phenyl)-6,7-dihydro-1H-pyrrolo[2,3-c] pyridine-2-carboxamide(25o)

1

White solid (90%). H NMR (400 MHz, DMSO-d ) δ 12.58 (s, 1H), 9.66 (s, 1H), 9.06 (t, J =

6

5

1

.7 Hz, 1H), 7.96 (d, J = 8.0 Hz, 2H), 7.85 (s, 4H), 7.62 (s, 1H), 7.47 (d, J = 8.0 Hz, 2H), 7.21 (s,

H), 7.16 (d, J = 7.8 Hz, 1H), 6.98 (t, J = 7.6 Hz, 1H), 6.79 (d, J = 8.0 Hz, 1H), 6.61 (t, J = 7.8 Hz,

+

H), 4.94 (s, 2H), 4.57 (d, J = 5.8 Hz, 2H), 3.61 (s, 3H). MS (ESI) m/z: 560.1 [M + H]

1

5

4

.2.27 N-(4-((2-aminophenyl) carbamoyl)

benzyl)-6-methyl-7-oxo-4-(p-tolyl)-6,7-dihydro-1H-pyrrolo[2,3-c] pyridine-2-carboxamide(25p)

1

White solid (85%). H NMR (400 MHz, DMSO-d ) δ 12.46 (s, 1H), 9.66 (s, 1H), 9.06 (t, J =

6

5

1

.7 Hz, 1H), 7.97 (d, J = 8.0 Hz, 2H), 7.51 (d, J = 7.9 Hz, 2H), 7.47 (d, J = 7.9 Hz, 2H), 7.41 (s,

H), 7.31 (d, J = 8.0 Hz, 2H), 7.20 – 7.13 (m, 2H), 6.98 (t, J = 7.5 Hz, 1H), 6.79 (d, J = 8.0 Hz,

H), 6.60 (t, J = 7.2 Hz, 1H), 4.91 (s, 2H), 4.56 (d, J = 6.0 Hz, 2H), 3.59 (s, 3H), 2.37 (s, 3H). MS

+

(ESI) m/z: 506.2 [M + H]

4

.2.28. N-(4-((2-aminophenyl) carbamoyl)

benzyl)-4-(4-methoxyphenyl)-6-methyl-7-oxo-6,7-dihydro-1H-pyrrolo[2,3-c]

pyridine-2-carboxamide(25q)

1

White solid (92%). H NMR (400 MHz, DMSO-d ) δ 12.43 (s, 1H), 9.65 (s, 1H), 9.06 (t, J =

6

5

1

6

3

1

.9 Hz, 1H), 7.97 (d, J = 7.9 Hz, 2H), 7.53 (d, J = 8.7 Hz, 2H), 7.47 (d, J = 8.1 Hz, 2H), 7.36 (s,

H), 7.17 (d, J = 7.6 Hz, 1H), 7.14 (s, 1H), 7.06 (d, J = 8.7 Hz, 2H), 6.97 (td, J = 7.7, 1.6 Hz, 1H),

.78 (dd, J = 8.1, 1.4 Hz, 1H), 6.60 (td, J = 7.5, 1.4 Hz, 1H), 4.90 (s, 2H), 4.56 (d, J = 5.8 Hz, 2H),

13

.81 (s, 3H), 3.59 (s, 3H). C NMR (126 MHz, DMSO-d ) δ 165.49, 160.18, 158.85, 154.51,

6

43.52, 133.74, 129.34, 128.96, 128.71, 128.36, 128.30, 127.60, 127.08, 126.87, 125.30, 123.73,

16.66, 116.53, 114.82, 114.68, 105.27, 55.61, 42.56, 36.13. HRMS (AP-ESI) m/z calcd for

+

C H N O [M + H] 522.2136, found 522.2139. Purity: 99%.

30

28

5

4

.3. The general procedure for preparation of 41a-41g, 42a-42d and 43a-43d.

The title dual inhibitors were synthesized following the procedure similar to 18e and 20d using the

intermediates 36 and 27a-27g as starting materials.

4

.3.1.

N-(4-((2-aminophenyl)

carbamoyl)

benzyl)-4-(3-(2-hydroxypropan-2-yl)

phenyl)-6-methyl-7-oxo-6,7-dihydro-1H-pyrrolo[2,3-c] pyridine-2-carboxamide(41a)

1

White solid (83%). H NMR (400 MHz, DMSO-d ) δ 12.47 (s, 1H), 9.66 (s, 1H), 9.05 (t, J =

6

5

7

5

.8 Hz, 1H), 7.96 (d, J = 8.0 Hz, 2H), 7.68 (s, 1H), 7.52 – 7.38 (m, 6H), 7.16 (d, J = 7.9 Hz, 1H),

.12 (d, J = 1.8 Hz, 1H), 6.97 (t, J = 7.6 Hz, 1H), 6.78 (d, J = 8.0 Hz, 1H), 6.60 (t, J = 7.6 Hz, 1H),

1

3

.11 (s, 1H), 4.92 (s, 2H), 4.56 (d, J = 5.8 Hz, 2H), 4.12 (s, 1H), 3.61 (s, 3H), 1.49 (s, 6H).

C

NMR (126 MHz, DMSO-d ) δ 165.50, 160.17, 154.60, 151.63, 143.44, 143.11, 136.40, 133.81,

6

1

33.71, 128.92, 128.62, 128.31, 127.60, 127.09, 126.87, 125.51, 125.32, 124.09, 123.77, 116.72,

16.58, 115.62, 105.50, 71.16, 42.58, 36.14, 32.45. HRMS (AP-ESI) m/z calcd for C H N O

4

3

2

32

5

+

[

M + H] 550.2449, found 550.2449. Purity: 98%.

.3.2. N-(4-((2-aminophenyl)

benzyl)-4-(5-(2-hydroxypropan-2-yl)-2-methoxyphenyl)-6-methyl-7-oxo-6,7-dihydro-1H-pyrrolo[2

4

carbamoyl)

,

3-c] pyridine-2-carboxamide(41b)

1

White solid (80%). H NMR (400 MHz, DMSO-d ) δ 12.25 (s, 1H), 9.64 (s, 1H), 8.96 (t, J =

6

5

(

.9 Hz, 1H), 7.95 (d, J = 7.8 Hz, 2H), 7.44 (dd, J = 8.6, 2.2 Hz, 3H), 7.38 (d, J = 2.4 Hz, 1H), 7.20

s, 1H), 7.16 (d, J = 7.8 Hz, 1H), 7.05 (d, J = 8.6 Hz, 1H), 6.96 (t, J = 7.4 Hz, 1H), 6.77 (d, J = 6.6

Hz, 1H), 6.71 (s, 1H), 6.59 (t, J = 7.4 Hz, 1H), 4.97 (s, 1H), 4.89 (s, 2H), 4.52 (d, J = 5.9 Hz, 2H),

1

3

1

3

6

.71 (s, 3H), 3.57 (s, 3H), 1.44 (s, 6H). C NMR (126 MHz, DMSO-d ) δ 165.08, 159.84, 155.07,

6

54.26，143.11, 142.76, 142.67, 133.27, 132.91, 129.82, 129.24, 127.87, 127.19, 127.15, 126.67,

26.46, 124.99, 124.35, 124.09, 123.32, 116.25, 116.12, 110.81, 105.88, 70.34, 55.43, 42.13,

+

5.69, 32.14. HRMS (AP-ESI) m/z calcd for C H N NaO [M + Na] 602.2374, found

3

33

5

02.2377. Purity: 95%.

4

.3.3.

N-(4-((2-aminophenyl)

carbamoyl)

benzyl)-4-(5-(2-hydroxypropan-2-yl)-2-isopropoxyphenyl

6-methyl-7-oxo-6,7-dihydro-1H-pyrrolo[2,3-c] pyridine-2 carboxamide (41c)

-

1

White solid (83%). H NMR (400 MHz, DMSO) δ 12.24 (s, 1H), 9.64 (s, 1H), 8.97 (t, J = 5.7

Hz, 1H), 7.94 (d, J = 8.0 Hz, 2H), 7.41 (dd, J = 14.1, 5.2 Hz, 4H), 7.21 (s, 1H), 7.15 (d, J = 7.5 Hz,

H), 7.03 (d, J = 9.2 Hz, 1H), 6.97 (t, J = 7.6 Hz, 1H), 6.81 (s, 1H), 6.77 (d, J = 7.8 Hz, 1H), 6.59

t, J = 7.5 Hz, 1H), 4.96 (s, 1H), 4.89 (s, 2H), 4.57 – 4.46 (m, 3H), 3.57 (s, 3H), 1.44 (s, 6H), 1.14

1

(

1

3

d, J = 6.0 Hz, 6H). C NMR (126 MHz, DMSO-d ) δ 165.46, 160.27, 154.64, 153.51, 143.53,

6

1

43.20, 142.99, 133.66, 133.09, 130.03, 129.67, 128.26, 127.82, 127.48, 127.09, 126.87, 125.56,

25.24, 124.80, 123.72, 116.65, 116.52, 113.94, 113.35, 106.93, 70.74, 70.19, 42.50, 36.10, 32.52,

1

6

+

2

2.27. HRMS (AP-ESI) m/z calcd for C H N NaO [M + Na] 630.2692, found 630.2688.

35 37 5 5

Purity: 96%.

4

.3.4. N-(4-((2-aminophenyl) carbamoyl) benzyl)-4-(2-cyclopropoxy-5-(2-hydroxypropan-2-yl)

phenyl)-6-methyl-7-oxo-6,7-dihydro-1H-pyrrolo[2,3-c] pyridine-2-carboxamide(41d)

1

White solid (85%). H NMR (400 MHz, DMSO) δ 12.26 (s, 1H), 9.65 (s, 1H), 8.95 (t, J = 5.4

Hz, 1H), 7.95 (d, J = 7.7 Hz, 2H), 7.49 – 7.40 (m, 3H), 7.39 (s, 1H), 7.33 (d, J = 8.7 Hz, 1H), 7.19

–

1

0

1

3

6

7.12 (m, 2H), 6.97 (t, J = 7.7 Hz, 1H), 6.78 (d, J = 7.8 Hz, 1H), 6.69 (s, 1H), 6.60 (t, J = 8.1 Hz,

H), 4.98 (s, 1H), 4.89 (s, 2H), 4.52 (s, 2H), 3.84 – 3.75 (m, 1H), 3.56 (s, 3H), 1.45 (s, 6H), 0.77 –

1

3

.70 (m, 2H), 0.62 – 0.54 (m, 2H). C NMR (126 MHz, DMSO-d ) δ 165.08, 159.85, 154.24,

6

43.12, 143.03, 142.80, 133.26, 132.80, 129.63, 129.26, 127.86, 127.13, 127.06, 126.68, 126.46,

24.87, 124.36, 124.02, 123.33, 116.25, 116.13, 112.57, 112.48, 106.00, 70.36, 50.72, 42.08,

+

5.68, 32.15, 6.04. HRMS (AP-ESI) m/z calcd for C H N NaO [M + Na] 628.2536, found

3

5

35

5

28.2528. Purity: 96%.

4

.3.5.

N-(4-((2-aminophenyl)

carbamoyl)

benzyl)-4-(5-(2-hydroxypropan-2-yl)-2-phenoxyphenyl)-6-methyl-7-oxo-6,7-dihydro-1H-pyrrolo[2

,

3-c] pyridine-2-carboxamide(41e)

1

White solid (86%). H NMR (400 MHz, DMSO-d ) δ 12.30 (s, 1H), 9.65 (s, 1H), 8.99 (t, J =

6

5

.9 Hz, 1H), 7.95 (d, J = 7.9 Hz, 2H), 7.58 (d, J = 2.3 Hz, 1H), 7.48 (dd, J = 8.5, 2.4 Hz, 1H), 7.44

(d, J = 8.0 Hz, 2H), 7.29 – 7.22 (m, 3H), 7.16 (d, J = 7.8 Hz, 1H), 7.03 – 6.92 (m, 4H), 6.84 (d, J =

7

=

.4 Hz, 2H), 6.78 (d, J = 6.7 Hz, 1H), 6.59 (t, J = 7.2 Hz, 1H), 5.10 (s, 1H), 4.89 (s, 2H), 4.53 (d, J

13

5.8 Hz, 2H), 3.51 (s, 3H), 1.48 (s, 6H). C NMR (126 MHz, DMSO-d ) δ 165.08, 159.77,

6

1

56.97, 154.14, 151.73, 146.41, 143.12, 142.76, 133.28, 133.05, 129.75, 129.70, 129.33, 127.87,

1

27.78, 127.14, 126.67, 126.46, 125.40, 124.38, 123.33, 122.74, 118.89, 117.66, 116.25, 116.12,

+

111.50, 105.67, 70.49, 42.13, 35.72, 32.06. HRMS (AP-ESI) m/z calcd for C H N O [M + H]

3

8

36

5

6

42.2711, found 642.2707. Purity: 97%.

4

.3.6. N-(4-((2-aminophenyl)

carbamoyl)

benzyl)-4-(2-(2,4-difluorophenoxy)-5-(2-hydroxypropan-2-yl)

phenyl)-6-methyl-7-oxo-6,7-dihydro-1H-pyrrolo[2,3-c] pyridine-2-carboxamide(41f)

1

White solid (83%). H NMR (400 MHz, DMSO-d ) δ 12.36 (s, 1H), 9.65 (s, 1H), 8.98 (t, J =

6

5

7

6

1

.8 Hz, 1H), 7.95 (d, J = 7.9 Hz, 2H), 7.56 (d, J = 2.3 Hz, 1H), 7.49 – 7.35 (m, 4H), 7.33 (s, 1H),

.16 (d, J = 8.2 Hz, 1H), 7.16 – 7.06 (m, 1H), 7.03 (t, J = 9.0 Hz, 1H), 6.97 (t, J = 7.6 Hz, 1H),

.92 (s, 1H), 6.83 (d, J = 8.6 Hz, 1H), 6.78 (d, J = 8.0 Hz, 1H), 6.60 (t, J = 7.6 Hz, 1H), 5.10 (s,

1

3

H), 4.90 (s, 2H), 4.54 (d, J = 5.8 Hz, 2H), 3.56 (s, 3H), 1.47 (s, 6H). C NMR (126 MHz,

DMSO-d ) δ 165.55, 160.19, 158.24 (dd, J = 243.0, 10.2 Hz), 154.66, 153.52 (dd, J = 249.5, 12.7

6

Hz), 152.78, 146.55, 143.57, 143.18, 140.11 (dd, J = 11.4, 3.6 Hz), 133.76, 133.58, 130.22,

1

4

6

29.78, 128.32, 127.60, 127.12, 126.92, 126.15, 125.86, 124.83, 123.80, 122.59 (d, J = 9.5 Hz),

16.71, 116.59, 116.39, 112.30 (d, J = 22.4 Hz), 111.71, 106.19, 105.90 (d, J = 25.0 Hz), 70.90,

+

2.61, 36.20, 32.50. HRMS (AP-ESI) m/z calcd for C H F N O [M + H] 678.2523, found

38

34

2

5

78.2515. Purity: 96%.

4

.3.7.

N-(4-((2-aminophenyl)

carbamoyl)

benzyl)-4-(2-(4-fluoro-2,6-dimethylphenoxy)-5-(2-hydroxypropan-2-yl)

phenyl)-6-methyl-7-oxo-6,7-dihydro-1H-pyrrolo[2,3-c] pyridine-2-carboxamide(41g)

1

White solid (80%). H NMR (400 MHz, DMSO-d ) δ 12.38 (s, 1H), 9.64 (s, 1H), 8.99 (t, J =

6

5

1

6

.9 Hz, 1H), 7.95 (d, J = 7.9 Hz, 2H), 7.52 (d, J = 2.4 Hz, 1H), 7.44 (d, J = 8.0 Hz, 2H), 7.35 (s,

H), 7.32 (dd, J = 8.6, 2.4 Hz, 1H), 7.16 (d, J = 7.2 Hz, 1H), 7.02 – 6.94 (m, 3H), 6.93 (s, 1H),

.78 (dd, J = 8.0, 1.4 Hz, 1H), 6.59 (t, J = 7.0 Hz, 1H), 6.31 (d, J = 8.6 Hz, 1H), 5.01 (s, 1H), 4.89

1

3

(s, 2H), 4.53 (d, J = 5.8 Hz, 2H), 3.60 (s, 3H), 2.02 (s, 6H), 1.44 (s, 6H). C NMR (126 MHz,

DMSO-d ) δ 165.05, 159.75, 158.61 (d, J = 240.2 Hz), 154.31, 152.72, 146.97 (d, J = 1.9 Hz),

6

1

43.88, 143.13, 142.72, 133.28, 133.09, 132.84 (d, J = 9.1 Hz), 129.85, 129.50, 127.93, 127.87,

27.11, 126.68, 126.47, 125.22, 124.43, 123.81, 123.31, 116.25, 116.12, 115.16 (d, J = 22.6 Hz),

11.85, 111.55, 106.00, 70.33, 42.13, 40.45, 35.88, 32.06, 16.22. HRMS (AP-ESI) m/z calcd for

+

C H FN NaO [M + Na] 710.2749, found 710.2752, Purity: 98%.

40

38

5

4

.3.8. N-(4-(hydroxycarbamoyl)

benzyl)-4-(5-(2-hydroxypropan-2-yl)-2-isopropoxyphenyl)-6-methyl-7-oxo-6,7-dihydro-1H-pyrrol

o[2,3-c] pyridine-2-carboxamide(42a)

1

7

1

White solid (85%). H NMR (400 MHz, DMSO-d ) δ 12.23 (s, 1H), 11.20 (s, 1H), 9.01 (s,

6

1

H), 8.92 (t, J = 5.8 Hz, 1H), 7.72 (d, J = 8.3 Hz, 2H), 7.43 – 7.34 (m, 4H), 7.21 (s, 1H), 7.03 (d, J

=

9.3 Hz, 1H), 6.80 (d, J = 2.0 Hz, 1H), 4.95 (s, 1H), 4.55 – 4.45 (m, 3H), 3.57 (s, 3H), 1.44 (s,

1

3

6

1

H), 1.14 (d, J = 6.0 Hz, 6H). C NMR (126 MHz, DMSO-d ) δ 160.31, 154.69, 153.56, 143.06,

42.92, 133.13, 131.87, 130.08, 129.73, 127.87, 127.66, 127.38, 125.63, 125.28, 124.85, 114.03,

13.39, 106.99, 70.79, 70.27, 42.54, 36.14, 32.57, 22.32. HRMS (AP-ESI) m/z calcd for

6

+

C H N O [M + H] 533.2395, found 533.2398. Purity: 96%.

29

33

4

6

4

.3.9.

4-(2-cyclopropoxy-5-(2-hydroxypropan-2-yl)

phenyl)-N-(4-(hydroxycarbamoyl)

benzyl)-6-methyl-7-oxo-6,7-dihydro-1H-pyrrolo[2,3-c] pyridine-2-carboxamide(42b)

1

White solid (83%). H NMR (400 MHz, DMSO-d ) δ 12.23 (s, 1H), 11.20 (s, 1H), 9.01 (s,

6

1

H), 8.91 (t, J = 5.9 Hz, 1H), 7.72 (d, J = 8.3 Hz, 2H), 7.45 (dd, J = 8.6, 2.4 Hz, 1H), 7.41 – 7.36

(

3

m, 3H), 7.33 (d, J = 8.6 Hz, 1H), 7.17 (s, 1H), 6.68 (s, 1H), 4.97 (s, 1H), 4.49 (d, J = 5.8 Hz, 2H),

1

3

.82 – 3.76 (m, 1H), 3.56 (s, 3H), 1.45 (s, 6H), 0.77 – 0.68 (m, 2H), 0.62 – 0.54 (m, 2H).

C

NMR (126 MHz, DMSO-d ) δ 159.77, 154.18, 142.98, 142.38, 132.73, 131.40, 129.56, 129.20,

6

1

3

5

27.11, 127.08, 126.87, 124.80, 124.30, 123.97, 112.50, 112.44, 105.95, 70.31, 50.68, 42.01,

+

5.62, 32.10, 5.97. HRMS (AP-ESI) m/z calcd for C H N NaO [M + Na] 553.20630, found

2

9

30

4

6

53.2062. Purity: 98%.

4

.3.10. 4-(2-(2,4-difluorophenoxy)-5-(2-hydroxypropan-2-yl) phenyl)-N-(4-(hydroxycarbamoyl)

benzyl)-6-methyl-7-oxo-6,7-dihydro-1H-pyrrolo[2,3-c] pyridine-2-carboxamide(42c)

1

White solid (80%). H NMR (400 MHz, DMSO-d ) δ 12.33 (s, 1H), 11.20 (s, 1H), 9.02 (s,

6

1

3

1

H), 8.93 (s, 1H), 7.72 (d, J = 7.9 Hz, 2H), 7.56 (s, 1H), 7.45 (d, J = 7.7 Hz, 1H), 7.42 – 7.35 (m,

H), 7.32 (s, 1H), 7.17 – 7.06 (m, 1H), 7.02 (t, J = 8.9 Hz, 1H), 6.91 (s, 1H), 6.83 (d, J = 8.6 Hz,

13

H), 5.09 (s, 1H), 4.49 (d, J = 5.8 Hz, 2H), 3.56 (s, 3H), 1.47 (s, 6H). C NMR (126 MHz,

DMSO-d ) δ 160.17, 158.19 (dd, J = 232.8, 12.0 Hz), 154.65, 153.52 (dd, J = 249.5, 12.0 Hz), 152.77,

6

1

2

6

46.54, 142.84, 140.10 (d, J = 7.9 Hz), 133.55, 131.91, 130.22, 129.76, 128.34, 127.71, 127.40, 126.14,

25.85, 124.82, 122.59 (d, J = 8.9 Hz), 116.38, 112.29 (d, J = 25.7 Hz), 111.70, 106.18, 105.88 (d, J =

+

2.0 Hz), 70.89, 42.58, 36.19, 32.50. HRMS (AP-ESI) m/z calcd for C H F N O [M + H]

32

29

2

4

6

03.2050, found 603.2050. Purity: 98%.

4

.3.11.

4-(2-(4-fluoro-2,6-dimethylphenoxy)-5-(2-hydroxypropan-2-yl)

benzyl)-6-methyl-7-oxo-6,7-dihydro-1H-pyrrolo[2,3-c]

phenyl)-N-(4-(hydroxycarbamoyl)

pyridine-2-carboxamide(42d)

1

White solid (80%). H NMR (400 MHz, DMSO-d ) δ 12.33 (s, 1H), 11.21 (s, 1H), 9.03 (s,

6

1

2

8

H), 8.96 (t, J = 5.9 Hz, 1H), 7.73 (d, J = 8.2 Hz, 2H), 7.53 (d, J = 2.4 Hz, 1H), 7.39 (d, J = 8.2 Hz,

H), 7.36 (s, 1H), 7.33 (dd, J = 8.6, 2.4 Hz, 1H), 7.00 (d, J = 9.1 Hz, 2H), 6.93 (s, 1H), 6.32 (d, J =

1

3

.6 Hz, 1H), 5.02 (s, 1H), 4.50 (d, J = 5.7 Hz, 2H), 3.61 (s, 3H), 2.02 (s, 6H), 1.45 (s, 6H). C

NMR (126 MHz, DMSO-d ) δ 164.50, 159.16 (d, J = 260.7 Hz), 154.77, 153.18, 147.43, 144.34,

6

1

42.87, 133.53, 133.30 (d, J = 8.8 Hz), 131.91, 130.30, 129.96, 128.38, 127.69, 127.42, 125.67,

1

24.88, 124.27, 115.61 (d, J = 22.4 Hz), 112.30, 112.01, 106.47, 70.78, 42.57, 36.33, 32.52, 16.67.

+

HRMS (AP-ESI) m/z calcd for C H FN O [M + H] 613.2457, found 613.2448. Purity: 95%.

3

4

34

4

6

4

.3.12.

N-(7-(hydroxyamino)-7-oxoheptyl)-4-(5-(2-hydroxypropan-2-yl)-2-isopropoxyphenyl)-6-methyl-7-

oxo-6,7-dihydro-1H-pyrrolo[2,3-c] pyridine-2-carboxamide(43a)

1

White solid (85%). H NMR (400 MHz, DMSO-d ) δ 12.13 (s, 1H), 10.34 (s, 1H), 8.67 (s,

6

1

H), 8.30 (t, J = 5.4 Hz, 1H), 7.42 – 7.36 (m, 2H), 7.20 (s, 1H), 7.02 (d, J = 9.4 Hz, 1H), 6.72 (s,

H), 4.95 (s, 1H), 4.50 (p, J = 6.0 Hz, 1H), 3.57 (s, 3H), 3.22 (q, J = 6.5 Hz, 2H), 1.94 (t, J = 7.3

1

3

Hz, 2H), 1.50 (q, J = 7.3 Hz, 4H), 1.44 (s, 6H), 1.37 – 1.25 (m, 4H), 1.13 (d, J = 6.0 Hz, 6H). C

NMR (126 MHz, DMSO-d ) δ 169.53, 160.06, 154.70, 153.54, 143.05, 133.55, 130.03, 129.68,

6

1

27.86, 125.65, 125.26, 124.63, 114.04, 113.38, 106.79, 70.79, 70.25, 39.24, 36.13, 32.71, 32.56,

+

2

9.26, 28.80, 26.65, 25.56, 22.30. HRMS (AP-ESI) m/z calcd for C H N O [M + H] 527.2864,

2

8

39

4

6

found 527.2861. Purity: 98%.

4

.3.13.

4-(2-cyclopropoxy-5-(2-hydroxypropan-2-yl)

phenyl)-N-(7-(hydroxyamino)-7-oxoheptyl)-6-methyl-7-oxo-6,7-dihydro-1H-pyrrolo[2,3-c]

pyridine-2-carboxamide(43b)

1

White solid (83%). H NMR (400 MHz, DMSO-d ) δ 12.15 (s, 1H), 10.34 (s, 1H), 8.67 (s,

6

1

H), 8.28 (s, 1H), 7.45 (d, J = 10.3 Hz, 1H), 7.38 (s, 1H), 7.33 (d, J = 8.5 Hz, 1H), 7.16 (s, 1H),

1

8

6

.60 (s, 1H), 4.97 (s, 1H), 3.85 – 3.74 (m, 1H), 3.56 (s, 3H), 3.22 (q, J = 6.4, 6.0 Hz, 2H), 1.94 (t,

J = 7.4 Hz, 2H), 1.53 – 1.47 (m, 4H), 1.45 (s, 6H), 1.34 – 1.25 (m, 4H), 0.75 – 0.66 (m, 2H), 0.61

1

3

–

1

3

5

0.53 (m, 2H). C NMR (126 MHz, DMSO-d ) δ 169.53, 160.03, 154.69, 143.47, 133.65, 130.02,

6

29.66, 127.58, 125.29, 124.58, 124.50, 113.00, 112.95, 106.25, 70.81, 51.16, 39.22, 36.12, 32.71,

2.60, 29.26, 28.80, 26.64, 25.56, 6.47. HRMS (AP-ESI) m/z calcd for C H N O [M + H]

25.2708, found 525.2712. Purity: 99%.

+

2

8

37

4

6

4

.3.14.

4-(2-(2,4-difluorophenoxy)-5-(2-hydroxypropan-2-yl)

phenyl)-N-(7-(hydroxyamino)-7-oxoheptyl)-6-methyl-7-oxo-6,7-dihydro-1H-pyrrolo[2,3-c]

pyridine-2-carboxamide(43c)

1

White solid (80%). H NMR (400 MHz, DMSO-d ) δ 12.23 (s, 1H), 10.35 (s, 1H), 8.68 (s,

6

1

5

H), 8.32 (t, J = 5.4 Hz, 1H), 7.55 (d, J = 2.4 Hz, 1H), 7.45 (dd, J = 8.6, 2.4 Hz, 1H), 7.39 (td, J =

1.5, 8.8, 2.9 Hz, 1H), 7.31 (s, 1H), 7.14 – 7.06 (m, 1H), 7.06 – 6.99 (m, 1H), 6.85 – 6.79 (m, 2H),

.10 (s, 1H), 3.56 (s, 3H), 3.22 (q, J = 6.5 Hz, 2H), 1.94 (t, J = 7.3 Hz, 2H), 1.55 – 1.39 (m, 10H),

1

3

1

.34 – 1.24 (m, 4H). C NMR (126 MHz, DMSO-d ) δ 169.54, 159.94, 158.23 (dd, J = 242.7,

6

1

2

0.4 Hz), 154.66, 153.52 (dd, J = 249.7, 12.7 Hz), 152.76, 146.52, 140.11 (d, J = 8.2 Hz), 133.98,

30.16, 129.73, 128.34, 126.16, 125.83, 124.61, 122.59 (d, J = 9.4 Hz), 116.39, 112.28 (d, J =

1.6 Hz), 111.71, 105.97, 105.90 (d, J = 27.3 Hz), 70.89, 39.27, 36.19, 32.71, 32.49, 29.25, 28.79,

+

6.65, 25.55. HRMS (AP-ESI) m/z calcd for C H F N O [M + H] 597.2519, found 597.2522.

3

1

35

2

4

6

Purity: 96%.

4

.3.15.

4-(2-(4-fluoro-2,6-dimethylphenoxy)-5-(2-hydroxypropan-2-yl)

phenyl)-N-(7-(hydroxyamino)-7-oxoheptyl)-6-methyl-7-oxo-6,7-dihydro-1H-pyrrolo[2,3-c]

pyridine-2-carboxamide(43d)

1

White solid (80%). H NMR (400 MHz, DMSO-d ) δ 12.25 (s, 1H), 10.34 (s, 1H), 8.67 (s,

6

1

7

H), 8.33 (t, J = 5.5 Hz, 1H), 7.52 (d, J = 2.4 Hz, 1H), 7.35 (s, 1H), 7.33 (dd, J = 8.7, 2.6 Hz, 1H),

.00 (d, J = 9.1 Hz, 2H), 6.85 (s, 1H), 6.31 (d, J = 8.5 Hz, 1H), 5.01 (s, 1H), 3.60 (s, 3H), 3.23 (q,

J = 6.5 Hz, 2H), 2.02 (s, 6H), 1.94 (t, J = 7.3 Hz, 2H), 1.53 – 1.47 (m, 4H), 1.44 (s, 6H), 1.33 –

1

3

1

2

6

.24 (m, 4H). C NMR (126 MHz, DMSO-d ) δ 169.53, 159.04 (d, J = 232.4 Hz), 154.78, 153.16,

6

47.42, 144.33, 133.94, 133.30 (d, J = 9.2 Hz), 130.24, 129.89, 128.38, 125.65, 124.67, 124.30,

15.61 (d, J = 22.9 Hz), 112.34, 112.00, 106.28, 70.78, 39.24, 36.32, 32.71, 32.51, 29.26, 28.79,

6.65, 25.56, 16.66. HRMS (AP-ESI) m/z calcd for C H FN NaO [M + Na] 629.2746, found

+

3

39

4

6

29.2746. Purity: 95%.

4

.4. Enzyme assays

For HDAC assays: HDAC enzyme was diluted to working concentration and added into the

84 reaction plate. For some no enzyme control wells, the same volume of buffer (50 mM tris-HCl,

3

pH 8.0, 137 mM NaCl, 2.7 mM KCl, and 1 mM MgCl ) was added instead. The compounds were

2

diluted to the desired working concentration with buffer as indicated above and added following

the platemap. Then the plate was spinned down and preincubated. Meanwhile, the substrate

mixture was prepared and added into all reaction wells to initiate the reaction. The plate was spun

and shaken. After 1h incubation at 37℃ with a seal, detection buffer with 10 μM SAHA was

added to stop the reaction. Equilibrium was reached after the reaction proceded for 30 minutes.

The signal of fluorescence was then detected on Envison (PerkinElmer) and analyzed in GraphPad

Prism.

For BRD4 assays: The compounds were diluted to the required concentrations with

EPIgenerous binding buffer. BRD4 HIS-tag or FLAG-tag (1/2) was added to all the wells

excepted for some negative control wells which added binding buffer instead. The compound and

BRD4(1/2) were mixed with a shaker, and then the [Lys (5,8,12,16) Ac] H4(1–21) biotinylated

peptide was added in the wells to initiate the reaction. The plate was spinned down and incubated

for 30 mins at 37℃ with seal. Detection buffer was added with streptavidin-acceptor and anti

HIS-donor Ab or FLAG-donor Ab into the wells. After 3 h incubation at room temperature, the

fluorescence signal was collected with Envison (PerkinElmer) and analyzed in GraphPad Prism.

4

.5. Cell proliferation inhibition assays

MV-4-11 cells were seeded in 96-well plate (5000 cells/well) overnight. The cells were

treated with selected compounds at various doses for 72 h. Cell activity was evaluated by CCK8

method. IC₅₀values were calculated by concentration–response curve fitting using a SoftMax

1

9

pro-based four-parameter method

.6. Immunoblotting

MV-4-11 cells were seeded into 12-well plates (0.5×10 cells/well) and treated with inhibitor

.

4

6

(0, 0.01, 0.1 and 1 μM) for 24 h. Then cells were collected by centrifugation at 450 g and washed

twice with PBS to remove the medium. After that, the cell pellet was lysed in SDS loading buffer

supplemented with 0.1M DTT. The lysate was heated at 100℃ for 15 mins and loaded into 12.5%

gel SDS-PAGE. Proteins were then transferred to PVDF membrane (Millipore) and blocked with

3

% BSA in TBST for 1 h. The following antibodies were used for detection: anti-acetylated

tubulin(Lys40) (#12152) (CST,), anti-acetylated H3 (Lys9/Lys14) (9677S) anti-c-Myc [Y69]

#32072, Abcam), anti-p21Waf1/Cip1(12D1) (#2947, CST).

(

4

.7. Cell cycle analysis

MV-4-11 cells were seeded into 12-well plates (0.3×10 cells/well) with complete medium

overnight. The selected compounds with indicated concentrations were added into each well. After

4 h incubation, cells were collected by centrifuging at 450 g and washed twice with ice-cold PBS,

6

2

then fixed with 75% cold ethanol at -20 ℃ overnight and stained for 30 mins with PI/RNase

staining buffer (BD Pharmingen). Finally, flow cytometry was performed by Calibur and analyzed

by FlowJo_V10.

4

.8. Cell apoptosis analysis

MV-4-11 cells were seeded into 6-well plates (0.4×10 cells/well) with complete medium

6

overnight. The selected compounds with indicated concentrations were then added into each well.

After 48 h incubation, cells were collected in 2 mL tubes separately and rinsed twice with PBS by

centrifugation at 450 g, which was followed by annexin V-FITC/PI staining for 10 mins using

Annexin V-FITC/PI Apoptosis Detection Kit (A211 Vazyme), Cell apoptosis was performed by

Calibur and analyzed by FlowJo_V10

.

Acknowledgments

This study was supported by a grant from the "Personalized Medicines——Molecular

Signature-based Drug Discovery and Development", Strategic Priority Research Program of the

Chinese Academy of Sciences (Grant No. XDA12020357) and the National Natural Science

Foundation of China (81703340).

References

[

1] D.S. Hewings, T.P.C. Rooney, L.E. Jennings, D.A. Hay, C.J. Schofield, P.E. Brennan, S. Knapp, S.J.

Conway, Progress in the development and application of small molecule inhibitors of

bromodomain–acetyl-lysine interactions, J. Med. Chem. 55 (2012) 9393-9413.

[

2] G. Manzotti, A. Ciarrocchi, V. Sancisi, Inhibition of BET proteins and histone deacetylase (HDACs):

crossing roads in cancer therapy, Cancers. 11 (2019), 304.

3] A. Wawruszak, J. Kalafut, E. Okon, J. Czapinski, M. Halasa, A. Przybyszewska, P. Miziak, K. Okla,

A. Rivero-Muller, A. Stepulak, Histone deacetylase inhibitors and phenotypical transformation of

cancer cells, Cancers. 11 (2019), 148.

[

4] A.G. Cochran, A.R. Conery, R.J. Sims, Bromodomains: a new target class for drug development,

Nat. Rev. Drug. Discov. 18 (2019) 609-628.

5] H. Zeng, J. Qu, N. Jin, J. Xu, C. Lin, Y. Chen, X. Yang, X. He, S. Tang, X. Lan, X. Yang, Z. Chen,

M. Huang, J. Ding, M. Geng, Feedback activation of leukemia inhibitory factor receptor limits

response to histone deacetylase inhibitors in breast cancer, Cancer Cell 30 (2016) 459-473.

6] E.J. Faivre, K.F. McDaniel, D.H. Albert, S.R. Mantena, J.P. Plotnik, D. Wilcox, L. Zhang, M.H. Bui,

G.S. Sheppard, L. Wang, V. Sehgal, X. Lin, X. Huang, X. Lu, T. Uziel, P. Hessler, L.T. Lam, R.J.

Bellin, G. Mehta, S. Fidanze, J.K. Pratt, D. Liu, L.A. Hasvold, C. Sun, S.C. Panchal, J.J. Nicolette,

S.L. Fossey, C.H. Park, K. Longenecker, L. Bigelow, M. Torrent, S.H. Rosenberg, W.M. Kati, Y.

Shen, Selective inhibition of the BD2 bromodomain of BET proteins in prostate cancer, Nature 578

[

(

2020) 306-310.

7] D.B. Doroshow, J.P. Eder, P.M. LoRusso, BET inhibitors: a novel epigenetic approach, Ann. Oncol.

8 (2017) 1776-1787.

8] K.J. Falkenberg, R.W. Johnstone, Histone deacetylases and their inhibitors in cancer, neurological

diseases and immune disorders, Nat. Rev. Drug. Discov. 13 (2014) 673-691.

[

2

0

[

9] G.S. Sheppard, L. Wang, S.D. Fidanze, L.A. Hasvold, D. Liu, J.K. Pratt, C.H. Park, K.L.

Longenecker, W. Qiu, M. Torrent, P. Kovar, M. Bui, E.J. Faivre, X. Huang, X. Lin, D. Wilcox, L.

Zhang, Y. Shen, D.H. Albert, T.J. Magoc, G. Rajaraman, W.M. Kati, K.F. McDaniel, Discovery of

N-ethyl-4-[2-(4-fluoro-2,6-dimethyl-phenoxy)-5-(1-hydroxy-1-methyl-ethyl)phenyl]-6-methyl-7-ox

o-1H-pyrrolo[2,3-c]pyridine-2-carboxamide (ABBV-744), a BET bromodomain inhibitor with

selectivity for the second bromodomain, J. Med. Chem. 63 (2020) 5585-5623

[

10] L. Zhao, J.P. Okhovat, E.K. Hong, Y.H. Kim, G.S. Wood, Preclinical studies support combined

inhibition of BET Family proteins and histone deacetylases as epigenetic therapy for cutaneous

T-cell lymphoma, Neoplasia 21 (2019) 82-92.

11] S.R. Kim, J.M. Lewis, B.M. Cyrenne, P.F. Monico, F.N. Mirza, K.R. Carlson, F.M. Foss, M.

Girardi, BET inhibition in advanced cutaneous T cell lymphoma is synergistically potentiated by

BCL2 inhibition or HDAC inhibition, Oncotarget 9 (2018) 29193-29207.

12] S. Liu, F. Li, L. Pan, Z. Yang, Y. Shu, W. Lv, P. Dong, W. Gong, BRD4 inhibitor and histone

deacetylase inhibitor synergistically inhibit the proliferation of gallbladder cancer in vitro and in

vivo, Cancer Sci. 110 (2019) 2493-2506.

13] P.K. Mazur, A. Herner, S.S. Mello, M. Wirth, S. Hausmann, F.J. Sanchez-Rivera, S.M. Lofgren, T.

Kuschma, S.A. Hahn, D. Vangala, M. Trajkovic-Arsic, A. Gupta, I. Heid, P.B. Noel, R. Braren, M.

Erkan, J. Kleeff, B. Sipos, L.C. Sayles, M. Heikenwalder, E. Hessmann, V. Ellenrieder, I. Esposito,

T. Jacks, J.E. Bradner, P. Khatri, E.A. Sweet-Cordero, L.D. Attardi, R.M. Schmid, G. Schneider, J.

Sage, J.T. Siveke, Combined inhibition of BET family proteins and histone deacetylases as a

potential epigenetics-based therapy for pancreatic ductal adenocarcinoma, Nat Med. 21 (2015)

1163-1171.

[

14] J. Shahbazi, P.Y. Liu, B. Atmadibrata, J.E. Bradner, G.M. Marshall, R.B. Lock, T. Liu, The

bromodomain inhibitor JQ1 and the histone deacetylase inhibitor panobinostat synergistically

reduce N-Myc expression and induce anticancer effects, Clin. Cancer Res. 22 (2016) 2534-2544.

15] A. Heinemann, C. Cullinane, R.D. Paoli-Iseppi, J.S. Wilmott, D. Gunatilake, J Madore, D Strbenac,

J.Y. Yang,, K Gowrishankar, J.C. Tiffen, R.K. Prinjha, N. Smithers, G.A. McArthur, S.J. Gallagher,

Combining BET and HDAC inhibitors synergistically induces apoptosis of melanoma and

suppresses AKT and YAP signaling, Oncotarget 6 (2015) 21507- 21521.

[

16] V.K. Mishra, F. Wegwitz, R.L. Kosinsky, M. Sen, R. Baumgartner, T. Wulff, J.T. Siveke, H.U.

Schildhaus, Z. Najafova, V. Kari, H. Kohlhof, E. Hessmann, S.A. Johnsen, Histone deacetylase

class-I inhibition promotes epithelial gene expression in pancreatic cancer cells in a BRD4- and

MYC-dependent manner, Nucleic Acids Res. 45 (2017) 6334-6349.

[

17] J.S. Carew, W. Zhao, V. Visconte, F. Anwer, K.R. Kelly, and S. T. Nawrocki, Rational cotargeting

of HDAC6 and BET proteins yields synergistic antimyeloma activity, Blood Adv. 3 (2019) 1318–

1

329.

18] D.O. Adeegbe, Y. Liu, P.H. Lizotte, Y. Kamihara, A.R. Aref, C. Almonte, R. Dries, Y. Li, S. Liu, X.

Wang, T. Warner-Hatten, J. Castrillon, G.C. Yuan, N. Poudel-Neupane, H. Zhang, J.L. Guerriero, S.

Han, M.M. Awad, D.A. Barbie, J. Ritz, S.S. Jones, P.S. Hammerman, J. Bradner, S.N. Quayle,

K.K. Wong, Synergistic Immunostimulatory effects and therapeutic benefit of combined histone

deacetylase and bromodomain inhibition in non-small cell lung cancer, Cancer Discov. 7 (2017)

8

52-867.

[

19] Y. Liu, Y. Li, S. Liu, D.O. Adeegbe, C.L. Christensen, M.M. Quinn, R. Dries, S. Han, K.

Buczkowski, X. Wang, T. Chen, P. Gao, H. Zhang, F. Li, P.S. Hammerman, J.E. Bradner, S.N.

Quayle, K.K. Wong, NK cells mediate synergistic antitumor effects of combined inhibition of

HDAC6 and BET in a SCLC preclinical model, Cancer Res. 78 (2018) 3709-3717.

20] S.J. Atkinson, P.E. Soden, D.C. Angell, M. Bantscheff, C.-w. Chung, K.A. Giblin, N. Smithers,

R.C. Furze, L. Gordon, G. Drewes, I. Rioja, J. Witherington, N.J. Parr, R.K. Prinjha, The structure

based design of dual HDAC/BET inhibitors as novel epigenetic probes, Med. Chem. Comm. 5

(

2014) 342-351.

21] Z. Zhang, S. Hou, H. Chen, T. Ran, F. Jiang, Y. Bian, D. Zhang, Y. Zhi, L. Wang, L. Zhang, H. Li,

Y. Zhang, W. Tang, T. Lu, Y. Chen, Targeting epigenetic reader and eraser: Rational design,

synthesis and in vitro evaluation of dimethylisoxazoles derivatives as BRD4/HDAC dual

inhibitors, Bioorg. Med. Chem. Lett. 26 (2016) 2931-2935.

[

22] S. Amemiya, T. Yamaguchi, Y. Hashimoto, T. Noguchi-Yachide, Synthesis and evaluation of novel

dual BRD4/HDAC inhibitors, Bioorg. Med. Chem. 25 (2017) 3677-3684.

23] M. Shao, L. He, L. Zheng, L. Huang, Y. Zhou, T. Wang, Y. Chen, M. Shen, F. Wang, Z. Yang, L.

Chen, Structure-based design, synthesis and in vitro antiproliferative effects studies of novel dual

BRD4/HDAC inhibitors, Bioorg. Med. Chem. Lett. 27 (2017) 4051-4055.

[

24] G. Cheng, Z. Wang, J. Yang, Y. Bao, Q. Xu, L. Zhao, D. Liu, Design, synthesis and biological

2

1

evaluation of novel indole derivatives as potential HDAC/BRD4 dual inhibitors and anti-leukemia

agents, Bioorg. Chem. 84 (2019) 410-417.

[

25] S. He, G. Dong, Y. Li, S. Wu, W. Wang, C. Sheng, Potent dual BET/HDAC inhibitors for efficient

treatment of pancreatic cancer, Angew. Chem. 132 (2020) 3052-3056.

26] S. Laszig, C. Boedicker, T. Weiser, S. Knapp, S. Fulda, The novel dual BET/HDAC inhibitor

TW09 mediates cell death by mitochondrial apoptosis in rhabdomyosarcoma cells, Cancer Lett.

4

86 (2020) 46-57.

[

27] X. Zhang, T. Zegar, T. Weiser, F.H. Hamdan, B.T. Berger, R. Lucas, D.I. Balourdas, S. Ladigan,

P.F. Cheung, S.T. Liffers, M. Trajkovic-Arsic, B. Scheffler, A.C. Joerger, S.A. Hahn, S.A. Johnsen,

S. Knapp, J.T. Siveke, Characterization of a dual BET/HDAC inhibitor for treatment of pancreatic

ductal adenocarcinoma, Int. J. Cancer, (2020), https://doi.org/10.1002/ijc.33137.

28] Z. Pan, X. Li, Y. Wang, Q. Jiang, L. Jiang, M. Zhang, N. Zhang, F. Wu, B. Liu, G. He, Discovery

of thieno[2,3-d]pyrimidine-based hydroxamic acid derivatives as bromodomain-containing protein

4

/histone deacetylase dual inhibitors induce autophagic cell death in colorectal carcinoma cells, J.

Med. Chem. 63 (2020) 3678-3700.

29] N.J. Porter, J.D. Osko, D. Diedrich, T. Kurz, J.M. Hooker, F.K. Hansen, D.W. Christianson,

Histone deacetylase 6-selective inhibitors and the influence of capping groups on

hydroxamate-zinc denticity, J. Med. Chem. 61 (2018) 8054-8060.

2

Table, Figure and Scheme Captions

Table 1. SAR exploration of linkers and ZBGs

Table 2. SAR study for ortho-aminoanilide moiety

Table 3. SAR of hydroxamic acid moiety

Table 4: HDAC isoform profiling of compound 41c, 43a and 43d.

Fig. 1. Structures of published HDAC/BRD4 dual inhibitors

Fig. 2. (A) The binding mode of ABBV-744 in complex with BD2 of BRD2 (PDB ID 6e6j); (B)

The pharmacophore model of HDACi; (C) Design strategy of the pyrrolopyridone-based selective

HDAC/BRD4 dual inhibitors.

Fig. 3. Western blotting analysis of selected compounds from Table 1 in MV-4-11 cell lines.

Fig. 4. Western blotting analysis of selected compounds in MV-4-11 cell lines.

Fig. 5. (A) Cell cycle and (B) apoptosis analysis of selected dual inhibitors with different HDAC

selectivity in MV-4-11 cell lines.

α

Scheme 1. Synthesis of the compounds 18a-18e and 20a-20d.

α

Scheme 2. Synthesis of the compounds 25a-25q.

α

Scheme 3. Synthesis of the intermediates 27a-27g.

α

Scheme 4. Synthesis of the compounds 41a-41g, 42a-42d and 43a-43d..

2

3

Table 1. SAR exploration of linkers and ZBGs

a

IC (nM) ±SD

IC (μM) ±SD

5

0

50

Comp.

R

HDAC1 HDAC6

>500.0 143.5±51.0

BRD4(1)

BRD4(2)

10.3±0.1

MV-4-11

1

8a

8b

14.2±1.1

13.1±0.8

0.38±0.01

1

142.0±19.4 4.0±1.4

4.8±0.7

0.18±0.03

18c

18d

18e

20a

44.4±8.8 3.4±0.0

29.5±2.4 4.7±1.2

168.4±46.8 15.3±4.3

21.4±0.2

38.6±2.0

42.1±3.7

73.0±1.3

3.8±0.7

5.3±0.2

0.61±0.08

0.18±0.01

0.57±0.09

0.24±0.04

11.3±1.2

11.3±3.5

>500.0

20b

20c

20d

176.1±16.1 >500.0

58.3±3.1

139.9±18.2

31.7±3.4

8.4±0.1

15.4±3.3

18.7±3.5

0.07±0.01

0.13±0.04

0.08±0.01

>500.0

126.5±12.5 >500.0

SAHA

ACY1215

MS-275

-

54.6±15.7 17.2±2.9

126.8±0.7 4.4±1.0

178.3±50.2 >10000

>10000

15.4±0.5

3.3±0.3

0.26±0.01

0.89±0.03

0.24±0.01

0.20±0.03

0.25±0.05

>10000

OTX015

ABBV-744

>10000

9.2±0.2

327.5±14.9

2

4

a

The IC values are shown as the average values from two separate experiments.

50

2

5

Table 2. SAR study for ortho-aminoanilide moitey

a

IC (nM) ±SD

IC (uM) ±SD

5

0

50

Comp.

R₁

R₂

R₃

HDAC1 BRD4(1) BRD4(2)

52.0±12.9 47.8±3.1 75.8±16.6

20.8±0.4 85.9±16.9 55.3±12.8

48.8±0.1 101.6±22.5 206.3±15.8

209.7±17.6 371.0±36.7 571.3±210.8

63.9±1.1 117.4±12.9 148.8±23.0

MV-4-11

0.23±0.01

2

5a

5b

5c

5d

5e

5f

H

F

H

2

b

2

Cl

N.T.

2

CF₃

Me

OMe

N.T.

2

0.31±0.04

0.20±0.02

2

57.6±16.5 22.6±3.3

116.2±28.6 22.8±3.9

47.6±8.2

15.3±0.3

2

5g

H

0.13±0.03

0.09±0.00

2

0d

5h

260.8±0.8 32.9±2.9

53.1±13.8 50.9±4.8

12.3±2.5

75.9±8.3

H

F

H

0.21±0.02

0.19±0.02

N.T.

2

5i

Cl

134.1±18.7 65.5±0.9 158.4±31.3

>500.0 82.3±3.5 313.0±15.7

5j

CF₃

Me

OMe

2

5k

5l

1a

5m

54.9±6.9 52.8±3.4 104.9±8.2

54.5±7.4 30.1±4.8 116.1±43.2

N.T.

2

0.25±0.02

4

H

28.1±4.2 20.0±6.4

47.3±11.8 44.1±7.3

30.0±3.3

79.9±4.1

2.2±0.11

2

H

F

0.22±0.02

N.T.

2

5n

5o

5p

5q

Cl

59.3±5.9 62.4±13.5 278.3±44.7

2

CF₃239.9±7.7 146.0±15.2 363.6±12.9

Me 94.0±2.3 61.0±24.6 121.9±3.1

OMe 46.8±13.7 40.5±0.8

33.3±1.6 3.9±2.2

N.T.

2

N.T.

61.2±2.6

7.0±3.7

0.32±0.05

4

1b

OMe

H

0.42±0.06

2

6

41c

41d

41e

H

59.7±15.4 5.6±1.2

68.8±16.3 7.7±1.2

229.0±36.8 13.4±0.8

2.1±0.8

2.1±0.6

3.0±0.5

0.04±0.00

0.10±0.01

4

1f

H

164.0±55.3 18.1±2.8

1.5±0.1

0.09±0.01

0.24±0.01

4

1g

>500.0 228.8±17.0 36.6±0.9

MS-275

ABBV-744

OTX015

-

102.6±14.7 >10000

>10000 261.0±21.9 4.1±0.2

>10000 8.4±0.5 10.7±3.8

>10000

0.26±0.01

0.30±0.03

0.15±0.00

a

The IC values are shown as the average values from two separate experiments.

50

b

The N.T. means not tested.

2

7

Table 3. SAR of hydroxamic acid moitey

a

IC (nM) ±SD

IC (uM)

50

5

0

Compd.

R₁

R₂linker

HDAC1

36.0±1.3

HDAC6

5.2±1.6

BRD4(1)

31.3±1.7

BRD4(2) MV-4-11

10.0±1.0 0.35±0.00

4

2a

4

2b

20.8±6.2

7.9±0.2

25.2±1.3

76.7±4.1

6.9±0.4 0.42±0.00

12.4±1.1 0.19±0.01

4

2c

74.0±19.6 15.6±0.1

4

2d

292.8±67.2 39.3±8.8 305.0±20.3 1.5±0.6 0.44±0.18

4

3a

-(CH ) - 19.4±1.1

5.4±0.6

5.5±1.5

29.5±3.4

26.2±0.2

6.0±1.4 0.16±0.03

5.8±1.6 0.22±0.02

2

6

4

3b

-(CH ) - 29.8±3.3

2 6

4

3c

-(CH ) - 52.0±10.8 8.7±0.5

54.0±1.7

7.2±0.3 0.25±0.02

2

6

4

3d

-(CH ) - 228.3±13.8 17.2±0.8 368.7±56.2 1.2±0.1 0.30±0.02

2 6

SAHA

46.1±5.7 19.9±3.2

>10000

312.9±5.0

10.1±2.1

>10000 0.58±0.02

1.8±0.4 0.26±0.02

9.0±1.4 0.17±0.01

ABBV-744

>10000

OTX015

a

The IC values are shown as the average values from two separate experiments.

50

2

8

Article Doi

Discovery of selective HDAC/BRD4 dual inhibitors as epigenetic probes

DOI: 10.1016/j.ejmech.2020.112868

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Article abstract of DOI:10.1016/j.ejmech.2020.112868

Full text of DOI:10.1016/j.ejmech.2020.112868

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