Synthesis and biological evaluation of (3/4-(pyrimidin-2-ylamino)benzoyl)-based hydrazine-1-carboxamide/carbothioamide derivatives as novel RXRα antagonists

Journal of Enzyme Inhibition and Medicinal Chemistry

ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20

Synthesis and biological evaluation of (3/4-

(pyrimidin-2-ylamino)benzoyl)-based hydrazine-1-

carboxamide/carbothioamide derivatives as novel

RXRα antagonists

Jingbo Qin, Jie Liu, Chunxiao Wu, Jianwen Xu, Bowen Tang, Kaiqiang Guo,

Xiaohui Chen, Weihao Liu, Tong Wu, Hu Zhou, Meijuan Fang & Zhen Wu

To cite this article: Jingbo Qin, Jie Liu, Chunxiao Wu, Jianwen Xu, Bowen Tang, Kaiqiang Guo,

Xiaohui Chen, Weihao Liu, Tong Wu, Hu Zhou, Meijuan Fang & Zhen Wu (2020) Synthesis and

biological evaluation of (3/4-(pyrimidin-2-ylamino)benzoyl)-based hydrazine-1-carboxamide/

carbothioamide derivatives as novel RXRα antagonists, Journal of Enzyme Inhibition and Medicinal

Chemistry, 35:1, 880-896, DOI: 10.1080/14756366.2020.1740692

To link to this article: https://doi.org/10.1080/14756366.2020.1740692

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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY

2020, VOL. 35, NO. 1, 880–896

https://doi.org/10.1080/14756366.2020.1740692

RESEARCH PAPER

Synthesis and biological evaluation of (3/4-(pyrimidin-2-ylamino)benzoyl)-based

hydrazine-1-carboxamide/carbothioamide derivatives as novel RXRa antagonists

ꢀ

Jingbo Qin , Jie Liu , Chunxiao Wu, Jianwen Xu, Bowen Tang, Kaiqiang Guo, Xiaohui Chen, Weihao Liu, Tong Wu,

Hu Zhou, Meijuan Fang and Zhen Wu

Fujian Provincial Key Laboratory of Innovative Drug Target Research and State Key Laboratory of Cellular Stress Biology, School of

Pharmaceutical Sciences, Xiamen University, Xiamen, China

ABSTRACT

ARTICLE HISTORY

Received 11 November 2019

Revised 7 February 2020

Accepted 1 March 2020

Abnormal alterations in the expression and biological function of retinoid X receptor alpha (RXRa) have a

key role in the development of cancer. Potential modulators of RXRa as anticancer agents are explored in

growing numbers of studies. A series of (4/3-(pyrimidin-2-ylamino)benzoyl)hydrazine-1-carboxamide/carbo-

thioamide derivatives are synthesised and evaluated for anticancer activity as RXRa antagonists in this

study. Among all synthesised compounds, 6A shows strong antagonist activity (half maximal effective con-

centration (EC₅₀) ¼ 1.68 0.22 mM), potent anti-proliferative activity against human cancer cell lines HepG2

and A549 cells (50% inhibition of cell viability (IC₅₀) values < 10 mM), and low cytotoxic property in normal

cells such as LO2 and MRC-5 cells (IC₅₀values > 100mM). Further bioassays indicate that 6A inhibits 9-cis-

RA-induced activity in a dose-dependent manner, and selectively binds to RXRa-¼LBD with submicromolar

affinity (Kd ¼ 1.20 ꢁ 10^ꢂ7M). 6A induces time-and dose-dependent cleavage of poly ADP-ribose polymer-

ase, and significantly stimulates caspase-3 activity, leading to RXRa-dependent apoptosis. Finally, molecular

docking studies predict the binding modes for RXRa-LBD and 6A.

KEYWORDS

Hydrazine carbothioamide;

RXRa antagonist; selectivity;

anticancer activity;

apoptosis

GRAPHIC ABSTRACT

Compound 6A with low cytotoxic property in normal cells acts as a selective RXR alpha ligand to promote

TNF alpha-mediated apoptosis of cancer cells.

1. Introduction

domain (LBD)^1–3. RXRa forms dimers with itself or other nuclear

receptors such as the thyroid hormone receptor (TR), retinoic acid

receptor (RAR), vitamin D receptor (VDR), liver X receptor (LXR),

and peroxisome-proliferator-activated receptor (PPAR), to exert its

transcriptional activities in the nucleus and act biologically in the

Retinoid X receptor alpha (RXRa) is a ligand-dependent transcrip-

tion factor of the nuclear receptor superfamily, and it is composed

of A/B region (modulating N-terminal domain or AF-1 domain),

DNA-binding domain (DBD), hinge region, and ligand-binding

CONTACT Meijuan Fang

fangmj@xmu.edu.cn; Zhen Wu

wuzhen@xmu.edu.cn; Hu Zhou

huzhou@xmu.edu.cn

Fujian Provincial Key Laboratory of

Innovative Drug Target Research and State Key Laboratory of Cellular Stress Biology, School of Pharmaceutical Sciences, Xiamen University, Xiamen, 361102, China

Supplemental data for this article can be accessed here.

These authors contributed equally to this work.

ß 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.

ꢀ

This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits

unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY

881

Figure 1. Design and modification strategies of target compounds.

cytoplasm^4–7. It’s worth noting that RXRa dimers are activated by group to the nitrogen atom of carboxamide (R²), and synthesising

endogenous ligand binding to its LBD, and then regulates gene

expression in various biological processes^8,9. Besides, dysregula-

tions of RXRa expression and function are implicated in the initi-

ation and development of many cancers^1,10–12. So exogenous

RXRa modulators may obstruct the development of various cancer

and diseases. Experiments have shown that agonist can activate

RXRa and upregulated the expression of CDKN1A (a target gene

of RXR), resulting in p21^WAF1/CIP1-dependent cell apoptosis and

suppressing cell proliferation^13,14, while synthetic antagonists can

promote mitochondria-mediated apoptosis and inflamma-

tion^10,15,16, or induce TNFa-dependent apoptosis through sup-

pressing PI3K/AKT-mediated cell survival pathway^17,18. In a word,

previous studies have suggested a potential role for RXRa in can-

cer initiation and cancer therapy.

carboxamide or carbothioamide derivatives (X). Next, we synthes-

ised target compounds and evaluated their biological activities

such as in vitro antiproliferative activity and modulating RXRa

activity. Then, we attempt to establish the preliminary structur-

e ꢂ activity relationships, and the selected compound 6 A as a

new high potent RXRa antagonist was further investigated for the

RXRa-dependent apoptosis induction including inhibition of RXRa

transactivation, physical binding of 6A to RXRa-LBD, and induction

of cell apoptosis, and checking the level of cleaved poly ADP-

ribose polymerase (c-PARP) and caspase-3. At last, a molecular

docking study was performed to explore the binding nature of 6A

to the ligand-binding pocket (LBP) of RXRa with antagonistic con-

formation (PDB: 3A9E).

In recent years, increasing RXRa modulators including many

agonists and few antagonists were identified based on virtual

screening and structure-based design^19–21. Among them, only

Bexarotene (an RXR full agonist) has been clinically used for the

treatment of cancers such as Cutaneous T cell lymphoma²².

However, synthetic RXRa ligands reported are associated with

unacceptable toxicity or undesirable side effects²³. For example,

Bexarotene has adverse effects such as elevation of blood trigly-

ceride, weight gain, and hepatomegaly^24,25. So, the current chal-

lenge is to develop a variety of novel RXRa modulator with more

potent, high specific and low cytotoxic properties.

2. Results and discussion

2.1. Chemistry

The synthetic strategy of target compounds (Series A andB) is

depicted in Scheme 1. Commercially available ketones 1a–1e were

reacted with N,N-dimethylformamide dimethyl acetal (DMF-DMA)

in dry toluene at 110 ^ꢃC for 18 h to give corresponding compounds

3a–3e. Ethyl 4-aminobenzoate (2a) and ethyl 3-aminobenzoate

(2b) were refluxed with cyanamide in the presence of concentrated

HCl for 24 h in ethanol, and then treated with ammonium nitrate to

afford 4-guanidino benzoic acid ethyl ester nitrate (4a) and 3-gua-

nidino benzoic acid ethyl ester nitrate (4b), respectively. Reactions

of 4a with 3-(dimethylamino)-1-arylprop-2-en-1-ones (3a–3e) in the

presence of sodium hydroxide were performed under reflux condi-

tions in ethanol for 48 h to produce 4-((4-arylpyrimidin-2-yl)amino)-

benzoic acids, followed by refluxing with hydrazine hydrate in

ethanol to get corresponding aroylhydrzides 5a–5e. Finally, target

compounds of series A were prepared via reactions between aroyl-

hydrzides 5a–5e with different isocyanates or isothiocyanates. On

the other hand, target compounds of series B were synthesised

In this work, to explored novel RXRa antagonists, a series of

new (3/4-(pyrimidin-2-ylamino)benzoyl)-based hydrazine-1-carbox-

amide/carbothioamide derivatives were designed based on the

structures of compounds NSC-640358 and 24 (Figure 1). First, we

fused 4-substituted pyrimidine (replacing 3-phenyl-1H-pyrazole

moiety in NSC-640358) to 2-benzoylhydrazine-1-carboxamide (as a

substitute for (E)-N’-benzylideneacetohydrazide moiety in com-

pound 24) to build the scaffold structure of target compounds.

Second, to obtain potent RXRa antagonists, we modified the two

moiety structures such as introducing the aryl group to the C-4

position (R¹) of pyrimidine ring, introducing the alkyl/aryl/aralkyl with 4b instead of 4a in a similar method.

882

J. QIN ET AL.

Scheme 1. Synthesis of target compounds of Series A and B. Reaction conditions: (a) DMF-DMA, 110 ^ꢃC; (b) Cyanamide, concentrated HCl, EtOH, reflux, 24 h and

NH₄NO₃, 0 ^ꢃC; (c) NaOH, EtOH, reflux; (d) Hydrazine monohydrate, EtOH, reflux; and (e) EtOH, reflux.

Table 1. In vitro antiproliferative activities of 2–(4-((-pyrimidin-2-yl)amino)benzoyl) hydrazine-1-carboxamide derivatives (Series A)

on two selected cancer cell lines.^a

IC50[lM]

Compounds

R1

R2

X

HepG2

A549

6a

6b

6c

6d

6e

6f

6g

6h

6i

6j

6k

6l

6m

6n

6o

6p

6q

6r

4-Pyridyl

3-Pyridyl

2-Pryidyl

n-Butyl

t- Butyl

Cyclohexyl

Cyclopentyl

p-Tolyl

2,4-difluorophenyl

3-chloro-4-methylphenyl

Phenethyl

n-Butyl

Cyclopentyl

p-Tolyl

Phenethyl

n- Butyl

t-Butyl

Cyclopentyl

m-Tolyl

3,5-dimethylphenyl

t- Butyl

Ethyl

Cyclohexyl

Phenyl

Benzyl

Phenyl

O

S

5.32 0.07

5.39 0.11

2.17 0.08

2.71 0.13

1.28 0.09

8.10 0.12

>40

3.89 0.04

>40

5.73 0.26

>40

21.69 1.33

18.09 1.25

8.39 0.92

16.26 1.21

11.27 1.05

26.38 1.42

>40

13.11 1.11

38.93 1.59

17.09 1.23

8.385 0.92

23.71 1.37

>40

5.48 0.21

>40

15.71 0.11

>40

23.31 0.07

20.32 0.14

4.48 0.09

20.34 0.34

13.08 0.46

5.13 0.14

0.61 0.07

1.47 0.42

6.74 0.05

4.59 0.11

>40

17.24 0.07

1.29 0.09

16.89 1.22

2-Pryidyl

4-Chlorophenyl

4-Pyridyl

3-Pyridyl

2-Pryidyl

Phenyl

19.49 1.29

>40

6s

6t

>40

6A

6B

6C

6D

6E

6F

6G

6H

6I

5.09 0.70

15.95 1.20

17.57 1.24

38.51 1.58

>40

30.79 1.48

20.68 1.31

22.27 1.34

28.32 1.45

12.76 1.10

>40

S

Benzyl

Cyclohexyl

Phenyl

Benzyl

Phenyl

4-Chlorophenyl

6J

Benzyl

S

Sorafenib

^aData are means SD of triplicate experiments.

2.2. Biology activity

antiproliferative activities against two human cancer cell lines

(HepG2 and A549) by MTT method with Sorafenib as a reference.

The concentrations of compounds required for 50% inhibition of

cell viability (IC₅₀) were determined and listed in Tables 1 and 2.

2.2.1. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

(MTT) assay and structure activity relationships (SARs) of the anti-

proliferative activity

To explore the antitumor activity of the target compounds, all According to these data, the preliminary SARs of these novel 2–(3/

synthesised target compounds were first assayed for in vitro 4-((-pyrimidin-2-yl)amino)benzoyl)hydrazine-1-carboxamide

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY

883

derivatives were summarised in Figure 2: (1) First, the R¹substitu- (6A–6J), compounds with one aralkyl group (benzyl) at R²-position

ent is crucial to the antitumor activities. As shown in Table 1, urea

derivatives with 4-pyridyl at the R¹-position showed stronger anti-

proliferative activity than those with 3-pyridyl and 2-pyridyl (6a vs.

6i and 6m, 6d vs. 6j and 6o, 6e vs. 6k, 6h vs. 6l, and 6b vs. 6n).

The synthesised thiourea derivatives 6A–6J possessed a similar

SAR, with the 4-pyridyl group to the C-4 position (R¹) of pyrimi-

dine ring being the better substitution. 4-Pyridyl substitution at R¹

position was also better than 4-chlorophenyl and phenyl substitu-

tions (6b vs. 6t, 6A vs. 6I, 6A vs. 6G, and 6B vs. 6H). (2) For the

substituent R²of compounds (6aꢄ6h) which contained 4-pyridyl

group at R¹position, it was found that both compounds 6a–6d

bearing alkyl groups (n-Butyl, t-Butyl, Cyclohexyl, and Cyclopentyl)

and 6h having an aralkyl group (phenethyl) generally had good

anti-proliferative properties, while the R²analogues 6e (p-Tolyl), 6f

(2,4-Difluorophenyl), and 6g (3-Chloro-4-methylphenyl) exhibited

different cytotoxicity due to their different substituted phenyl ring

at the R²-position. A comparison of the activities of compounds

6e and 6f shows that the p-Tolyl substitution is better than

2,4-Difluorophenyl, but when 3-Chloro-4-methylphenyl was intro-

duced into the molecule, the biological activity of 6g was signifi-

cantly reduced. Among the synthesised thiourea derivatives

displayed the better cytotoxicity against HepG2 cells than those

with one aryl group (phenyl) such as 6B vs. 6A, 6E vs. 6D, and 6J

vs 6I. (3) The various scaffold had a certain influence on antitumor

activity. As shown in Tables 1 and 2, replacement of the 4-(pyrimi-

din-2-ylamino)benzohydrazide scaffold with the 3-(pyrimidin-2-yla-

mino)benzohydrazide tended to decrease the activity (7a vs. 6b,

7b vs. 6d, 7d vs. 6i, and 7f vs. 6m).

2.2.2. Identification of novel RXRa modulators from tar-

get compounds

RXRa is an attractive target for the treatment of cancer and we’d

like to discover new potential modulators of RXRa as anticancer

agents. So, all target compounds were evaluated for their effect

on regulating RXRa transcriptional function using a luciferase tran-

scription assay in HEK293T cells with the Gal4- RXRa/LBD chimaera

and Gal4 reporter system. When tested at 1.0 lM concentration

with absence 9-cis-RA in this system, no compound exhibited

increase transcriptional activity, suggesting no agonist effect for

them. However, 11 compounds (6c, 6e, 6k, 6A, 6B, 6C, 6D, 6E,

6F, 6G, and 6H) of series A showed good antagonist effect on

9cis-RA-induced RXRa transactivation with an inhibition of >50%

(Figure 3), while target compounds of series B exhibited little

antagonist effect on 9-cis-RA-induced reporter activity, indicating

4-(pyrimidin-2-ylamino)benzohydrazide scaffold being crucial to

the transcriptional activity of RXRa. Among these candidate com-

pounds, compound 6A showed the strongest inhibition activity 9-

cis-RA-induced Gal4-DBD-RXRa-LBD transactivation at the tested

concentration (1.0 lM). To confirm the antagonist effect of these

11 potential antagonists of RXRa, we next used a retinoid X recep-

tor response element (RXRE)-luciferase reporter-based assay to

evaluate their half maximal effective concentration (EC₅₀) values of

the inhibition on 9-cis-RA-induced RXRE-luciferase reporter activa-

tion. As shown in Table 3, many of them (6k, 6A, 6B, 6C, 6G, and

6H) showed good antagonism with EC₅₀less than 10.0 lM, while

the EC₅₀values of several compounds (6c, 6e, 6D, and 6E) could

not be calculated. Furthermore, we evaluated the cytoxic effect of

the six potent RXRa antagonists (EC₅₀< 10.0 lM) on two normal

cell lines to determine their safety (Table 3). Among the six tested

compounds, 6A showed low cytotoxic property to both LO2 and

MRC-5 with both IC₅₀values more than 100 lM.

Table 2. In vitro antiproliferative activities of 2–(3-((-pyrimidin-2-yl)amino)ben-

zoyl) hydrazine-1-carboxamide derivatives (Series B) on two selected cancer cell

a

lines.

IC50[lM]

Compounds

R1

R2

X

HepG2

A549

7a

7b

7c

7d

7e

7f

7A

7B

7C

7D

4-Pyridyl t-Buty

4-Pyridyl Cyclopentyl

3-Pyridyl t-Butyl

3-Pyridyl n-Butyl

3-Pyridyl 2,4-dimethoxyphenyl

2-Pyridyl n-Butyl

4-Pyridyl Benzyl

3-Pyridyl Benzyl

2-Pyridyl Phenyl

2-Pyridyl Benzyl

O

S

14.37 0.26 31.63 1.50

>40

31.45 0.44

>40

30.24 1.48

>40

30.59 1.48

14.96 0.22 34.43 1.53

17.86 0.28

17.45 0.34

20.23 0.08

>40

^aData are means SD of triplicate experiments.

Figure 2. Summarised SARs of synthesised compounds (Series A and B) in Scheme 1.

884

J. QIN ET AL.

Figure 3. Identification of RXRa antagonists via a luciferase transcription assay in HEK293T cells with the Gal4-RXRa/LBD chimaera and Gal4 reporter system. The inhib-

ition of target compounds at 1 lM on 9-cis-RA activating Gal4-DBD-RXRa-LBD transcriptional activity in HEK293T cells transfected with p-BIND-RXRa-LBD (50ng) and

pG5-luc (50ng) were tested. Luciferase activities were measured 12 h after treatment using the dual-luciferase assay system kit and Renilla luciferase values were nor-

malised to firefly luciferase activity and plotted as relative luciferase activity.

Table 3. The antagonist effect on RXRa (EC₅₀) and cytotoxic effect on two nor-

showed the dose-dependent effect on inhibiting the 9-cis-RA-

induced Gal4-DBD-RXRa-LBD transactivation and 9-cis-RA-induced

RXRE-luciferase reporter activation, and it at 2.0 lM displayed a

similar inhibitory effect when compared with UVI3003 (1.0 lM), a

known RXRa classical antagonist. In order to confirm compound

6A as RXRa ligand, we then performed a surface plasmon reson-

ance (SPR) technology-based experiment to directly measure the

physical binding of compound 6A to RXRa-LBD protein. As shown

in Figure 4(C), we found that 6 A dose dependently bound to

RXRa-LBD with a Kd value of 1.20 ꢁ 10^ꢂ7M. Furthermore, we

measured the physical binding of compound 6A to other nuclear

receptors (ERa-LBD, PPARc-LBD, and TR3-LBD). The results showed

that compound 6A could not bound to these tested nuclear

receptors well (Figure 3(D–F)). This implied that compound 6A

was able to selectively interact with RXRa.

mal cell lines (IC₅₀) of the selected compounds.^a

IC₅₀(lM)

EC₅₀(lM)

Compounds

RXRa

LO2

MRC-5

6c

ND

24.94 1.40

>100

14.27 1.16

32.09 1.51

ND

21.1 1.32

16.89 1.22

ND

6e

6k

6A

6B

6C

6D

6E

6F

6G

6H

1.05 0.18

1.68 0.22

7.23 0.32

1.75 0.35

ND

>100

ND

27.69 1.35

0.56 0.14

0.54 0.06

>100

^aData are means SD of triplicate experiments.

2.2.4. 6A induces RXRa-dependent apoptosis of cancer cells

Previous studies showed RXRa antagonists with anticancer activity

could induce apoptotic cell death. So, we studied whether the

2.2.3. 6A binds to RXRa and acts as an antagonist

Compound 6A displayed low cytotoxic property, potent antiproli-

ferative (IC₅₀< 5.30 lM) and high antagonistic (EC₅₀

¼

1.68 0.22 lM) effects, so compound 6A was selected for further inhibitory effect of 6A on the growth of cancer cells was caused

biological study. Compound 6A was assessed in a dose-dependent by cellular apoptosis. First, we examined the expression of c-PARP

reporter gene assay. As shown in Figure 4(A,B), compound 6A (an apoptosis-related protein) in several cancer cell lines treated

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY

885

Figure 4. The antagonist effect of 6A by reporter gene assay (A,B) and the physical binding of 6A to RXRa-LBD (C), ERa-LBD (D), PPARc-LBD (E), and TR3-LBD (F) by

SPR assay. (A) Dose-dependent effect of 6A on inhibiting RXRa transactivation. HEK293T cells transfected with p-BIND-RXRa-LBD and pG5-luc were treated with indi-

cated concentrations of 6A in the presence of 9-cis-RA (0.10 lM). (B) Inhibition of 9-cis-RA-induced RXRE-luciferase reporter activation by 6A. HEK293T cells transfected

with RXRE (50ng), full-length RXRa (50ng), Renilla (1 ng) were treated with 9-cis-RA in the presence of indicated concentrations of 6A or UVI3003 (1lM). (C) Surface

plasmon resonance of 6A binding to RXRa-LBD. D. Surface plasmon resonance of 6A binding to ERa-LBD. E. Surface plasmon resonance of 6A binding to PPARc-LBD.

F. Surface plasmon resonance of 6A binding to TR3-LBD. (C–F) Gradient concentrations of 6A were respectively injected through chips immobilised with RXRa-LBD,

ERa-LBD, PPARc-LBD, and TR3-LBD.

Figure 5. 6A-induced PARP cleavage in cancer cells. (A) SW620, LS174T, HepG2, and Hela cells were treated with 6A for 12 h and cell lysates prepared were analysed

by Western blotting. (B) A549 cells were time- and dose- dependently treated with 6A for 12 h and cell lysates prepared were analysed by Western blotting for PARP

cleavage. (C) A549 and RXRa-/- A549 cells were treated with 6A for 12 h and cell lysates prepared were analysed by Western blotting for PARP cleavage.

with or without 6A. As shown in Figure 5(A), the c-PARP increased effect on PARP cleavage in SW620 and LS174T cells. A time- and

markedly in HepG2 and Hela cells after treatment with 6A at dose- dependent increase of c-PARP, was also observed in A549

3.0 lM for 12 h, whereas 6A at the same concentration has a weak cells after treatment with 6A (Figure 5(B)). To address the role of

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J. QIN ET AL.

Figure 6. 6A-induced apoptotic effect. (A) The images of A549 and RXRa-/- A549 cells after treatment with or without 6A for 12 h; (B) Flow cytometry analysis of

A549 and RXRa-/- A549 cells after treatment with or without 6A for 12 h.

Figure 7. The effect of 6A on caspase-3 activation. A549 and RXRa-/- A549 cells were treated with or without 6A for 12 h, then stained with cleaved-caspase 3 anti-

body and DAPI and assessed by immunofluorescence.

RXRa, we then evaluated the effect of 6A on cleaved PARP in KO; Figure 5(C)). Thus, 6A could induce RXRa-dependent PARP

RXRa knockout A549 cells (PX330-KO). Comparison of the obvious cleavage in cancer cells.

effect of 6A in normal A549 cells (PX330), 6A showed little effect

To further confirm the role of RXRa in the apoptosis induction

on inducing PARP cleavage in RXRa knockout A549 cells (PX330- by 6A, we evaluated the apoptosis effect of compound 6A in

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY

887

Figure 8. Predicted binding modes of 6A with RXRa-LBP under antagonistic conformation (PDB ID: 3A9E). (A) Close-up view depiction (3 D model) of the superposition

of RXRa LBP bound to 6A. The RXRa binding site is presented with a grey ribbon. The 6A is shown in stick with orange carbon, blue nitrogen, and yellow sulphur.

The figure was prepared using PyMol (http://www.pymol.org/). (B) Two-dimensional diagram of interactions between compound 6A and RXRa. The p–p interactions

are displayed as green solid line. Colour lines around 6A stand for the binding pocket and the residues in colours nearby established the pocket. The green colour

denotes the hydrophobic nature of amino acids, the red colour denotes the acid amino acids, the purple denotes the alkalinity of amino acids, the cyan denotes the

polar amino acids, and the grey points of ligand atoms denotes the solvent accessibility.

A549 and RXRa knockout A549 cells. As observed in Figure 6(A), structure locates at the polar region involving HID440. Third, the

6A-treated A549 cells exhibited obvious apoptosis characteristics left phenyl group of Compound 6A is directed to the protein-solv-

with the accumulation of the nuclear convolution and fragmenta-

tion in membrane-enclosed bodies, but little apoptotic bodies

were detected in treated RXRa knockout A549 cells. Meanwhile,

Figure 6(B) showed that when A549 cells were incubated with 6A

at 10.0 lM, the percentage of apoptotic cells was strikingly ele-

vated to 30.8% from the control group (5.96%). However, the per-

centages of apoptotic cells in RXRa knockout A549 cells without

and with 6A treatment were 2.07 and 2.72%, respectively. These

results demonstrated that apoptosis cells increased significantly

after the treatment of 6A in A549 cells, and the apoptosis induc-

tion of 6A was mainly through RXRa-dependent pathways. It indi-

cates the expression of RXRa plays a role in the apoptosis

induction by 6A.

Finally, to find out whether the apoptotic induction of 6A is

dependent on caspase pathway, we next used immunofluores-

cence assay to evaluate the effect of 6A on the activation of cas-

pase-3 a major executioner caspase known to cleave PARP. As

shown in Figure 7, 6A treatment of A549 cells resulted in the

strong activation of caspase-3 and nuclear shrinkage compared

with control cells, whereas there is no significant difference

between treated and untreated RXR-/- A549 cells. This result was

consistent with c-PARP. It suggests that compound 6A induces

cell death in A549 cells via caspase-dependent pathway and RXRa

dependent pathway.

ent interface and makes hydrophobic interactions with surround-

ing lipophilic amino acids such as VAL347 and PHE443. These

findings may provide a direction for the further modifications of

this new class of RXRa regulator.

3. Conclusion

In summary, 2-(4-(pyrimidin-2-yl)amino)benzoyl)hydrazine-1-car-

boxamide/carbothioamide (Series A) and 2-(3-(pyrimidin-2-yl)ami-

no)benzoyl) hydrazine-1-carboxamide/carbothioamide (Series B)

derivatives were first synthesised and evaluated as potential anti-

tumour agents targeting RXRa. Compounds 6c, 6e, 6h, 6A, and 6J

exhibited potent antiproliferative activity with IC₅₀values of lower

than 15 lM against two human tumour cell lines, while com-

pounds 6k, 6A, 6B, 6C, 6G, and 6H were identified as good

antagonists of RXRa with EC₅₀values of lower than 10.0 lM at

RXRE-luciferase reporter-based assay. Compound 6A with the low

cytotoxity of normal cells was identified as the most potent anti-

cancer agent targeting RXRa. Moreover, it was found that the rep-

resentative compound 6A effectively induced RXRa-dependent

apoptosis of cancer cells in a time- and concentration- dependent

manner. SPR technology-based experiment confirmed that com-

pound 6A could selectively bind to RXRa-LBD with a Kd value of

1.20 ꢁ 10^ꢂ7M, and molecular docking analysis showed that com-

pound 6A could bind to the LBP of RXRa like the classical RXRa

antagonists. Taken together, this class of synthetic compounds

may be served as a good starting point to design and synthesise

new RXRa antagonists, and further studies of compound 6A on

activity in vivo would be considered in the future.

2.3. Molecular docking studies

Based on the antagonist effect of 6A on RXRa, the molecular

docking of 6A with antagonistic conformation of RXRa (PDB:

3A9E) was performed using Schrodinger 2015.1 Glide protocol to

explore their binding mode. The docking study showed that 6A

was well accommodated into LBP of RXRa (see Figure 8(A) and

Supplementary movie). The docking results predict the binding

features of 6A with RXRa. First, the right 4-(pyridin-4-yl)pyrimidine

portion of compound 6A occupies the sub-pocket formed by

hydrophobic aromatic amino acid residues LEU438, LEU331,

ALA276, PHE318, and TRP310, the pyridine ring forming p–p inter-

4. Experimental section

4.1. Chemistry

All reagents and solvents were purchased from commercial sour-

ces and were used without further purification. Oxygen or water

sensitive reactions, which required the use of nitrogen atmos-

action with PHE318. Second, the hydrazine-1-carbothioamide phere. All reactions were magnetically stirred and monitored by

888

J. QIN ET AL.

thin-layer chromatography (TLC) on (Qingdao Haiyang Chemical, (d, J ¼ 12.3 Hz, 1 H), 7.46–7.50 (m, 1 H), 7.41–7.45 (m, 2 H), 5.82 (d,

China) silica gel 60 F-254 by fluorescence. ¹H-NMR and ¹³C-NMR

J ¼ 12.3 Hz, 1 H), 3.14 (s, 3 H), 2.91 (s, 3 H); ¹³C NMR (150 MHz,

spectra were obtained using a Bruker AV2 600 Ultra shield spec-

trometer at 600 and 150 MHz, respectively. Chemical shifts were

given in ppm (d) relative to tetramethylsilane as the internal

standard. Spin multiplicities were described as s (singlet), d (doub-

let), dd (double doublet), t (triplet), br (broad signal), and orm

(multiplet). High-resolution mass spectra (HRMS) data were

acquired using a quadrupole-orbitrap (Q-Exactive) mass spectrom-

eter (Thermo Scientific, Hemel Hempstead, UK) equipped with a

heated electrospray ionisation source (HESI-II). Melting points were

measured on SGW X-4 micro-melting point spectrometer and

were uncorrected. The high-performance liquid chromatography

(HPLC) analysis was performed on an Agilent Technologies 1100

Series HPLC system (Palo Alto, CA). The mobile phase was (A)

methanol and (B) water in a linear gradient mode as follows: (A)

from 5 to 100% and (B) from 95 to 0% during 0–30 min. The flow

rate was 1 ml/min, and the detection wavelengths were 254

and 280 nm.

DMSO-d₆) d 154.6, 140.7, 131.2, 128.6 (2C), 127.6 (2C), 91.4, 45.0,

37.6 (2C).

4.1.1.5. (E)-1-(4-chlorophenyl)-3-(dimethylamino)prop-2-en-1-one

(3e). According to the general procedure, compound 3e was

obtained by using 4-chloroacetophenone, yellow solid, 2.6 g, yield:

56%. mp: 101–102 ^ꢃC. ¹H NMR (600 MHz, DMSO-d₆) d 7.89–7.94

(m, 2H), 7.73 (d, J ¼ 12.1 Hz, 1H), 7.49–7.51 (m, 1H), 7.47–7.48 (m,

1H), 5.82 (d, J ¼ 12.1 Hz, 1H), 3.15 (s, 3H), 2.92 (s, 3H); ¹³C NMR

(150 MHz, DMSO-d₆) d 184.7, 155.0, 139.4, 136.0, 129.5 (2C), 128.6

(2C), 91.1, 45.0, 37.6.

4.1.2. 4-Guanidinobenzoate ethyl ester nitrate (4a) and 3-guanidi-

nobenzoate ethyl ester nitrate (4A)

4.1.2.1. 4-guanidinobenzoate ethyl ester nitrate (4a). To a solution

of ethyl p-aminobenzoate (2a; 5.0 g, 0.030 mol) in ethanol (20 ml),

cyanamide (50.0% w/w, 7.6 g, 0.091 mol) was added, Concentrated

hydrochloric acid (3.8 ml, 0.045 mol) was added dropwise with stir-

ring at 0 ^ꢃC. After the completion of the dropwise addition, the

4.1.1. General procedure for the synthesis of compounds 3a–3e

To a solution of appropriate 1a–1e (0.041 mol) in toluene (50 ml),

DMF-DMA (0.082 mol) was added, the reaction mixture was stirred temperature was raised to reflux for 24 h. The reaction mixture

at 110 ^ꢃC for 18 h. Then, the mixture was cooled to room tempera-

was concentrated under reduced pressure. The residue was

ture, the solvent was removed by reduced pressure distillation.

Then 20 ml ethyl acetate was added. The resulting solution was

stirred at room temperature for 1 h, and filtered under suction,

washed with (ethyl acetate: n-hexane ¼ 3:1) to afford the com-

pounds 3a–3e.

diluted with 50 ml of water, and then an aqueous solution of

ammonium nitrate (4.8 g, 0.061 mol) was added dropwise at 0 ^ꢃC.

After the completion of the dropwise addition, the mixture was

kept for 1 h, then filtered to give 4a.

White solid, 6.3 g, yield: 78%. mp: 175–176 ^ꢃC. ¹H NMR

(600 MHz, DMSO-d₆): d 9.96 (br s, 1H), 8.00 (d, J ¼ 8.6 Hz, 2H), 7.65

(s, 3H), 7.36 (d, J ¼ 8.6 Hz, 2H), 4.32 (q, J ¼ 7.1 Hz, 2H), 1.30–1.34

(m, 3H); ¹³C NMR (150 MHz, DMSO-d₆): d 165.6, 155.8, 140.9,

4.1.1.1. (E)-3-(dimethylamino)-1-(pyridin-4-yl)prop-2-en-1-one (3a).

According to the general procedure, compound 3a was obtained

by using 4-acetylpyridine, yellow solid, 4.2 g, yield: 58.3%. Melting 131.2(2C), 127.2, 123.4(2C), 61.2, 14.6.

1

point (mp): 84–85 ^ꢃC. H NMR (600 MHz, DMSO-d₆) d 8.67–8.69 (m,

2H), 7.79 (d, J ¼ 12.1 Hz, 1H), 7.76 (d, J ¼ 5.5 Hz, 2H), 5.84 (d,

4.1.2.2. 3-guanidinobenzoate ethyl ester nitrate (4A). According to

J ¼ 12.1 Hz, 1H), 3.18 (s, 3H), 2.95 (s, 3H); ¹³C NMR (150 MHz,

DMSO-d₆) d 155.7, 150.6 (2C), 147.2, 121.5 (2C), 91.2, 45.2,

37.8 (2C).

the synthetic procedure of 4a, compound 4A was obtained by

using ethyl 3-aminobenzoate (2b).

White solid, 6.0 g, yield: 74%. mp: 177–178 ^ꢃC. ¹H NMR

(600 MHz, DMSO-d₆): d 7.85 (m, 1H), 7.78 (t, J ¼ 1.8 Hz, 1H),

7.58–7.62 (m, 1H), 7.51–7.56 (m, 4H), 4.34 (q, J ¼ 7.0 Hz, 2H), 1.33

(t, J ¼ 7.0 Hz, 3H); ¹³C NMR (150 MHz, DMSO-d₆): d 165.6, 156.3,

136.5, 131.7, 130.7, 129.6, 127.3, 125.3, 61.5, 14.6.

4.1.1.2. (E)-3-(dimethylamino)-1-(pyridin-3-yl)prop-2-en-1-one (3b).

According to the general procedure, compound 3b was obtained

by using 3-acetylpyridine, yellow solid, 3.2 g, yield: 49.5%. mp:

83–85 ^ꢃC. ¹H NMR (600 MHz, DMSO-d₆) d 9.07 (d, J ¼ 1.7 Hz, 1H),

8.66 (dd, J ¼ 4.8, 1.7 Hz, 1H), 8.22 (td, J ¼ 8.0, 1.7 Hz, 1H), 7.77 (d,

J ¼ 12.1 Hz, 1H), 7.47–7.51 (m, 1H), 5.87 (d, J ¼ 12.1 Hz, 1H), 3.17 (s,

3H), 2.94 (s, 3H); ¹³C NMR (150 MHz, DMSO-d₆) d 155.1, 151.8,

149.0, 135.6, 135.1, 123.9, 91.4, 45.1, 37.7 (2C).

4.1.3. General procedure for the synthesis of compounds 5a–5e

To a solution of (E)-3-(dimethylamino)-1-(pyridin-4-yl)prop-2-en-1-

one (1.3 g, 7.4 mmol) and ethyl 4-guanidinobenzoate nitrate

(4a;2.0 g, 7.4 mmol), sodium hydroxide (355 mg, 8.9 mmol) was

added, and the mixture was heated to reflux for 48 h. After the

completion of the reaction, the mixture was concentrated and

4.1.1.3. (E)-3-(dimethylamino)-1-(pyridin-2-yl)prop-2-en-1-one (3c).

According to the general procedure, compound 3c was obtained

by using 2-acetylpyridine, Yellow solid, 3.5 g, yield: 53%. mp: purified by column chromatography using appropriate mixtures of

78–79 ^ꢃC. ¹H NMR (600 MHz, DMSO-d₆) d ppm 8.62ꢂ8.64 (m, 1 H)

dichloromethane and methanol (MeOH) to afford ethyl 4-((4-(pyri-

7.99 (d, J ¼ 7.7 Hz, 1 H) 7.91 (td, J ¼ 7.7, 1.7 Hz, 1 H) 7.80 (d,

din-4-yl)pyrimidin-2-yl)amino)benzoate, then to

a solution of

J ¼ 12.0 Hz, 1 H) 7.50 (m, 1 H) 6.38 (d, J ¼ 12.0 Hz, 1 H) 3.18 (s, 3 H)

2.92 (s, 3 H); ¹³C NMR (150 MHz, DMSO-d₆) d 156.2, 154.8, 148.9,

137.5, 126.1, 121.6, 90.5, 45.1, 37.6 (2C).

appropriate compound. To a solution of ethyl 4-((4-(pyridin-4-

yl)pyrimidin-2-yl)amino)benzoate (1.00 eq) in ethanol, hydrazine

hydrate (1.00 eq) was added, and the reaction mixture was stirred

at 87 ^ꢃC for 20 h. The reaction mixture was cooled to room tem-

perature, filtered and the concentrated filter cake was dried to

4.1.1.4.

(E)-3-(dimethylamino)-1-phenylprop-2-en-1-one

(3d).

According to the general procedure, compound 3d was obtained obtain 5a. Yield: 84.3%.

Compounds 5b–5e were synthesised following the general

by using acetophenone, Yellow solid, 2.6 g, yield: 60%. mp:

95–96 ^ꢃC. ¹H NMR (600 MHz, DMSO-d₆) d 7.87–7.89 (m, 2 H) 7.71 procedure as described above with the yield of 52–68%.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY

889

4.1.3.1.

4-((4-(pyridin-4-yl)pyrimidin-2-yl)amino)benzohydrazide 4.1.4.2.

3-((4-(pyridin-3-yl)pyrimidin-2-yl)amino)benzohydrazide

(5B). Yellow solid, 1.2 g, yield: 79%. mp: 306–308 ^ꢃC. ¹H NMR

(600 MHz, DMSO-d₆): d 9.92 (s, 1H), 9.72 (s, 1H), 9.38 (dd, J ¼ 2.2,

0.7 Hz, 1H), 8.74 (dd, J ¼ 4.7, 1.6, Hz, 1H), 8.64 (d, J ¼ 5.1 Hz, 1H),

8.57 (dt, J ¼ 8.1, 1.9 Hz, 1H), 8.41 (s, 1H), 7.89–7.92 (m, 1H), 7.60

(m, 1H), 7.55 (d, J ¼ 5.1 Hz, 1H), 7.41–7.45 (m, 1H), 7.34–7.40 (m,

1H), 4.49–4.54 (m, 2H); ¹³C NMR (150 MHz, DMSO-d₆): d 166.8,

162.1, 160.6, 159.9, 152.0, 148.7, 141.0, 134.9, 134.5, 132.6, 128.9,

124.4, 121.9, 120.1, 118.7, 108.9.

(5a). Yellow solid 1.1 g, yield: 58%. mp: 298–300 ^ꢃC. ¹H NMR

(600 MHz, DMSO-d₆): d 10.11 (s, 1H), 9.62 (s, 1H), 8.81 (dd, J ¼ 4.4,

1.6 Hz, 2H), 8.72 (d, J ¼ 5.1 Hz, 1 H), 8.11 (dd, J ¼ 4.4, 1.6 Hz, 2H),

7.92 (dt, J ¼ 8.0, 1.8 Hz, 2H), 7.83 (dt, J ¼ 8.0, 1.8 Hz, 2H), 7.60 (d,

J ¼ 5.1 Hz, 1H), 4.46 (br s, 2H); ¹³C NMR (150 MHz, DMSO-d₆): d

166.3, 162.0, 160.5, 160.4, 151.1, 144.2(2C), 143.4, 128.2 (2C), 126.5,

121.4 (2C), 118.4 (2C), 109.7.

4.1.3.2.

4-((4-(pyridin-3-yl)pyrimidin-2-yl)amino)benzohydrazide

4.1.4.3.

3-((4-(Pyridine-2-yl)pyrimidin-2-yl)amino)benzohydrazide

(5b). White solid, 1.0 g, yield: 53%. mp: 285–287 ^ꢃC. ¹H NMR

(600 MHz, DMSO-d₆): d 10.05 (s, 1H), 9.61 (s, 1H), 9.36–9.37 (m,

1H), 8.75 (m, 1H), 8.66 (d, J ¼ 5.1 Hz, 1H), 8.52 (dt, J ¼ 8.01.8 Hz,

1H), 7.90 (dt, J ¼ 8.0, 1.8 Hz, 2H), 7.82 (dt, J ¼ 8.0, 1.8 Hz, 2H),

7.59–7.63 (m, 1 H), 7.58 (d, J ¼ 5.1 Hz, 1H), 4.45 (br s, 2H); ¹³C NMR

(150 MHz, DMSO-d₆): d 166.3, 162.2, 160.4, 159.9, 152.1, 148.7,

143.5, 134.9, 132.6, 128.2, 126.4, 124.4 (2C), 118.3 (2C), 109.4.

(5C). Yellow solid, 1.1 g, yield: 68%. Mp: 311–312 ^ꢃC. ¹H NMR

(600 MHz, DMSO-d₆): d 9.92 (s, 1H), 9.72 (s, 1H), 8.76 (td, J ¼ 4.6,

1.1 Hz, 1H), 8.67 (d, J ¼ 5.1 Hz, 1H), 8.51 (d, J ¼ 7.7 Hz, 1H), 8.48 (s,

1H), 8.05 (dt, J ¼ 7.7, 1.7, Hz, 1H), 7.90 (td, J ¼ 7.7, 1.7 Hz, 1H), 7.76

(d, J ¼ 5.1 Hz, 1H), 7.58 (ddd, J ¼ 7.7, 4.6, 1.1 Hz, 1H), 7.41–7.43 (m,

1H), 7.37–7.41 (m, 1H), 4.52 (br s, 2H); ¹³C NMR (150 MHz, DMSO-

d₆): d 166.9, 163.3, 160.5, 160.1, 154.0, 150.1, 141.1, 138.1, 134.5,

128.9, 126.2, 121.8, 121.7, 120.0, 118.6, 108.7.

4.1.3.3.

4-((4-(pyridin-2-yl)pyrimidin-2-yl)amino)benzohydrazide

(5c). White solid, 0.8 g, yield: 68%. mp: 286–288 ^ꢃC. ¹H NMR

(600 MHz, DMSO-d₆): d 10.06 (s, 1H), 9.62 (s, 1H), 8.76–8.78 (m,

1 H), 8.70 (d, J ¼ 5.1 Hz, 1H), 8.43 (d, J ¼ 7.9 Hz, 1H), 8.07 (td,

J ¼ 7.9, 1.8 Hz, 1H), 7.92 (dt, J ¼ 8.0, 1.8 Hz, 2H), 7.84 (dt, J ¼ 8.0,

1.8 Hz, 2H), 7.79 (d, J ¼ 5.1 Hz, 1H), 7.59 (m, 1H), 4.46 (br s, 2 H);

¹³C NMR (150 MHz, DMSO-d₆): d 166.3, 163.4, 160.3, 160.1, 154.0,

150.1, 143.6, 138.1, 128.2, 126.3, 126.3, 121.6 (2C), 118.3

(2C), 109.1.

4.1.5. General procedure for the synthesis of target compounds

6a–6t and 6A–6J (Series A)

Compounds 5a–5e (1.00 eq) were separately used to react with

an appropriate isocyanate or isothiocyanate (1.00 eq) in ethanol at

70 ^ꢃC for 4 h, then cooled to room temperature, filtered to get

crude products of compounds 6a-6t and 6A-6J, which were fur-

ther purified by silica gel column chromatography (DCM:MeOH

¼ 50:1).

4.1.3.4. 4-((4-phenylpyrimidin-2-yl)amino)benzohydrazide (5d).

White solid, 0.6 g, yield: 67%. mp: 303–305 ^ꢃC. ¹H NMR (600 MHz,

DMSO-d₆): d 9.98 (s, 1H), 9.60 (s, 1 H), 8.59–8.63 (m, 1H), 8.18–8.20

(m, 2H), 7.93 (d, J ¼ 8.8 Hz, 2H), 7.82 (d, J ¼ 8.8 Hz, 2H), 7.56–7.59

(m, 3H), 7.49 (d, J ¼ 5.1 Hz, 1H), 4.44 (s, 2H); ¹³C NMR (150 MHz,

DMSO-d₆): d 166.3, 164.2, 160.4, 159.6, 143.7, 137.0, 131.5 (2C),

129.4 (2C), 128.2, 127.4(2C), 126.2, 118.2 (2C), 109.1

4.1.5.1.

N-butyl-2-(4-((4-(pyridin-4-yl)pyrimidin-2-yl)amino)ben-

zoyl)hydrazine-1-carboxamide (6a). Yellow solid (94.52%), 0.056 g,

yield: 58%, mp: 213–215 ^ꢃC. ¹H NMR (600 MHz, DMSO-d₆): d 10.16

(s, 1H), 9.96 (br s, 1H), 8.82 (dd, J ¼ 4.4, 1.4 Hz, 2H), 8.73 (d,

J ¼ 5.1 Hz, 1H), 8.13 (dd, J ¼ 4.4, 1.4 Hz, 2H), 7.94 (d, J ¼ 9.0 Hz, 2H),

7.89 (d, J ¼ 9.0 Hz, 2H), 7.74 (s, 1H), 7.62 (d, J ¼ 5.1 Hz, 1H), 6.45 (br

s, 1H), 3.03 (q, J ¼ 6.7 Hz, 2H), 1.38 (quin, J ¼ 7.3 Hz, 2H), 1.28 (sxt,

J ¼ 7.3 Hz, 2H), 0.87 (t, J ¼ 7.3 Hz, 3H); ¹³C NMR (150 MHz, DMSO-

d₆): d 166.5, 161.9, 160.5, 159.0, 150.9 (2C), 144.4, 143.9 (2C), 128.9

(2C), 125.8, 121.5 (2C), 118.3 (2C), 109.8, _þ39.3, 32.5, 19.9, 14.2;

HRMS (ESI, m/z): calcd for C₂₁H₂₄N₇O₂[M þ H]^þ406.1986,

found 406.1985.

4.1.3.5. 4-((4-(4-chlorophenyl)pyrimidin-2-yl)amino)benzohydrazide

(5e). Yellow solid, 1.1 g, yield: 58%. mp: 300–302 ^ꢃC. ¹H NMR

(600 MHz, DMSO-d₆): d 9.98 (s, 1 H), 9.60 (s, 1H), 8.61 (d, J ¼ 5.1 Hz,

1H), 8.18–8.20 (m, 2H), 7.93 (d, J ¼ 8.8 Hz, 2H), 7.82 (d, J ¼ 8.8 Hz,

2H), 7.57–7.58 (m, 2H), 7.49 (d, J ¼ 5.1 Hz, 1H), 4.44 (s, 2H); ¹³C

NMR (150 MHz, DMSO-d₆): d 166.3, 163.0, 160.4, 159.8, 143.6,

136.3, 135.8, 129.5(2C), 129.2 (2C), 128.2 (2C), 126.3, 118.3

(2C), 109.0.

4.1.5.2. N-(tert-butyl)-2-(4-((4-(pyridin-4-yl)pyrimidin-2-yl)amino)-

benzoyl) hydrazine-1-carboxamide (6b). Yellow solid (99.24%),

0.054 g, yield: 60%, mp: 171–172 ^ꢃC. ¹H NMR (600 MHz, DMSO-d₆):

d 10.16 (s, 1H), 9.95 (s, 1H), 8.80 (dd, J ¼ 4.4, 1.4 Hz, 2H), 8.73 (d,

J ¼ 5.1 Hz, 1H), 8.11 (dd, J ¼ 4.4, 1.4 Hz, 2H), 7.93 (d, J ¼ 9.0 Hz, 2H),

7.88 (d, J ¼ 9.0 Hz, 2H), 7.61 (s, 1H), 7.60 (d, J ¼ 5.1 Hz, 1H)6.08 (br

s, 1H), 1.27 (s, 9H); ¹³C NMR (150 MHz, DMSO- d₆): d 166.3, 162.0,

160.5, 157.8, 151.1(2C), 144.2, 143.9 (2C), 128.7 (2C), 125.7, 121.4

4.1.4. General procedure for the synthesis of compounds 5A–5C

According to the synthesis of compounds 5a–5c, compounds

5A–5C were synthesised with 4A instead of 4a in a simi-

lar method.

(2C), 118.3 (2C), 109.8, 49.9, 29.6 (3 C); HRMS (ESI, m/z): calcd for

þ

C₂₁H₂₄N₇O₂

[M þ H]^þ406.1986, found 406.1983, calcd for

C₂₁H₂₃N₇O₂Na^þ[M þ Na]^þ428.1805, found 428.1803.

4.1.4.1.

3-((4-(pyridin-4-yl)pyrimidin-2-yl)amino)benzohydrazide

(5A). Yellow solid, 0.5 g, yield: 63%. mp: 302–304 ^ꢃC. ¹H NMR

(600 MHz, DMSO-d₆) d 9.98 (s, 1H), 9.73 (s, 1H), 8.79–8.80 (m, 1H),

8.79–8.79 (m, 1H), 8.69 (d, J ¼ 5.1 Hz, 1H), 8.45 (s, 1H), 8.13–8.17

(m, 2H), 7.87–7.91 (m, 1H), 7.58 (d, J ¼ 5.1 Hz, 1H), 7.41–7.44 (m,

1H), 7.38–7.41 (m, 1H), 4.54 (br s, 2H); ¹³C NMR (150 MHz, DMSO-

d₆) d 166.8, 161.8, 160.7, 160.4, 151.1 (2C), 144.2, 140.9, 134.5,

128.9, 122.0, 121.4 (2C), 120.2, 118.8, 109.2.

4.1.5.3.

N-cyclohexyl-2-(4-((4-(pyridin-4-yl)pyrimidin-2-yl)amino)-

benzoyl) hydrazine-1-carboxamide (6c). White solid (93.67%),

0.055 g, yield: 58%, mp: 181–183 ^ꢃC. ¹H NMR (600 MHz, DMSO-d₆):

d 10.15 (s, 1H), 9.96 (s, 1H), 8.80 (dd, J ¼ 4.4, 1.4 Hz, 2H), 8.72 (d,

J ¼ 5.1 Hz, 1H), 8.10 (dd, J ¼ 4.4, 1.4 Hz, 2H), 7.92 (d, J ¼ 9.0 Hz, 2H),

7.88 (d, J ¼ 9.0 Hz, 2H), 7.69 (s, 1H), 7.60 (d, J ¼ 5.1 Hz, 1H), 6.26 (d,

J ¼ 8.0 Hz, 1H), 3.38–3.43 (m, 1H), 1.75 (dd, J ¼ 9.3, 3.8 Hz, 2H), 1.64

890

J. QIN ET AL.

(dd, J ¼ 9.3, 3.8 Hz, 2H), 1.53 (dd, J ¼ 9.3, 3.8 Hz, 1H), 1.22–1.30 (m, 4.1.5.8.

2H), 1.11–1.20 (m, 3H); ¹³C NMR (150 MHz, DMSO-d₆): d 166.4,

N-phenethyl-2-(4-((4-(pyridin-4-yl)pyrimidin-2-yl)amino)-

benzoyl)hydrazine-1-carboxamide (6h). Yellow solid (96.95%),

0.072 g, yield: 75%, mp: 240–241 ^ꢃC. ¹H NMR (600 MHz, DMSO-d₆):

d 10.17 (s, 1H), 9.99 (s, 1H), 8.81 (dd, J ¼ 4.4, 1.4 Hz, 2H), 8.74 (d,

162.0, 160.4, 158.2, 151.1(2C), 144.2, 143.9 (2C), 128.8 (2C), 125.7,

121.4 (2C), 118.3(2C), 109.8, 48.6, 3_þ3.5 (2C), 25.7, 25.1 (2C); HRMS

(ESI, m/z): calcd for C₂₃H₂₆N₇O₂[M þ H]^þ432.2142, found J ¼ 5.1 Hz, 1H), 8.12 (dd, J ¼ 4.4, 1.4 Hz, 2H), 7.94 (d, J ¼ 9.0 Hz, 2H),

432.2141, calcd for C₂₃H₂₅N₇O₂Na^þ[M þ Na]^þ454.1962,

7.91 (d, J ¼ 9.0 Hz, 2H), 7.86 (s, 1H), 7.62 (d, J ¼ 5.1 Hz, 1H), 7.29 (t,

J ¼ 7.6 Hz, 3H), 7.20–7.24 (m, 3H), 7.17–7.20 (m, 1H), 6.51 (br s, 1H),

3.27 (d, J ¼ 7.3 Hz, 2H), 2.72 (t, J ¼ 7.3 Hz, 2H); ¹³C NMR (150 MHz,

DMSO-d₆): d 166.5, 162.0, 160.5, 158.9, 151.1(2C), 144.2, 144.0 (2C),

140.1, 129.1 (2C), 128.9 (2C), 128.8, 128.8, 126.5, 125.8, 121.4 (2C),

found 454.1960.

4.1.5.4. N-cyclopentyl-2-(4-((4-(pyridin-4-yl)pyrimidin-2-yl)amino)-

benzoyl) hydrazine-1-carboxamide (6d). Yellow solid (95.45%),

0.054 g, yield: 57%, mp: 238–239 ^ꢃC. ¹H NMR (600 MHz, DMSO-d₆):

d 10.15 (s, 1H), 9.95 (s, 1H), 8.80 (dd, J ¼ 4.4, 1.4 Hz Hz, 2H), 8.72

(d, J ¼ 5.1 Hz, 1H), 8.11 (dd, J ¼ 4.4, 1.4 Hz, 2H), 7.92–7.95 (m, 2H),

7.88 (d, J ¼ 9.0 Hz, 2H), 7.67 (s, 1H), 7.60 (d, J ¼ 5.1 Hz, 1H), 6.36 (d,

J ¼ 7.5 Hz, 1H), 3.90 (m, 1H), 1.76–1.83 (m, 2H), 1.58–1.65 (m, 2H),

1.45–1.53 (m, 2H), 1.33–1.41 (m, 2H); ¹³C NMR (150 MHz, DMSO-

d₆): d 166.4, 162.0, 160.4, 158.5, 151.1(2C), 144.2, 143.9 (2C), 128.8

(2C), 125.8, 121.4 (2C), 118.3 (2C), 109._þ8, 51.6, 33.1 (2C), 23.7 (2C);

HRMS (ESI, m/z): calcd for C₂₂H₂₄N₇O₂[M þ H]^þ418.1986, found

418.1984, calcd for C₂₂H₂₃N₇O₂Na^þ[M þ Na]^þ440.1805,

found 440.1804.

118.3 (2C), 109.8, 41.4, 36.5; HRMS (ESI, m/z): calcd for

þ

C₂₅H₂₄N₇O₂

[M þ H]^þ454.1986, found 454.1979, calcd for

C₂₅H₂₃N₇NaO₂[M þ Na]^þ476.1805, found 476.1799.

þ

4.1.5.9.

N-butyl-2-(4-((4-(pyridin-3-yl)pyrimidin-2-yl)amino)ben-

zoyl)hydrazine-1-carboxamide (6i). Yellow solid (99.63%), 0.076 g,

yield: 81%, mp: 179–181 ^ꢃC. ¹H NMR (600 MHz, DMSO-d₆): d 10.10

(s, 1H), 9.96 (br s, 1H), 9.36 (d, J ¼ 2.0 Hz, 1H), 8.75 (dd, J ¼ 4.7, 2.0,

Hz, 1H), 8.67 (d, J ¼ 5.1 Hz, 1H), 8.53 (dt, J ¼ 8.0, 2.0 Hz, 1H), 7.93

(d, J ¼ 9.0 Hz, 2H), 7.89 (d, J ¼ 9.0 Hz, 2H), 7.73 (s, 1H), 7.61 (dd,

J ¼ 8.0, 4.7, Hz, 1H), 7.59 (d, J ¼ 5.1 Hz, 1H), 6.45 (br s, 1H),

3.03–3.04 (m, 2H), 1.38 (quin, J ¼ 7.3 Hz, 2H), 1.28 (sxt, J ¼ 7.3 Hz,

2H), 0.87 (t, J ¼ 7.3 Hz, 3H); ¹³C NMR (150 MHz, DMSO- d₆): d 166.5,

162.2, 160.3, 160.0, 159.0, 152.1, 148.6, 144.0, 134.9, 132.5, 128.9

(2C), 125.7, 124.5, 118.2 (2C), 109.5, 39._þ3, 32.5, 19.9, 14.2; HRMS

4.1.5.5.

2-(4-((4-(pyridin-4-yl)pyrimidin-2-yl)amino)benzoyl)-N-(p-

tolyl)hydrazine-1-carboxamide (6e). Yellow solid (99.58%), 0.061 g,

yield: 69%, mp: 230–231 ^ꢃC. ¹H NMR (600 MHz, DMSO-d₆): d 10.17

(s, 1H), 10.14 (s, 1H), 8.80 (dd, J ¼ 4.4, 1.4 Hz, 2H), 8.76 (br s, 1H),

8.72 (d, J ¼ 5.1 Hz, 1H), 8.11 (dd, J ¼ 4.4, 1.4 Hz, 2H), 8.07 (s, 1H),

7.95 (d, J ¼ 9.0 Hz, 2H), 7.93 (d, J ¼ 9.0 Hz, 2H), 7.60 (d, J ¼ 5.1 Hz,

1H), 7.35 (d, J ¼ 8.2 Hz, 2H), 7.07 (d, J ¼ 8.2 Hz, 2H), 2.23 (s, 3H); ¹³C

NMR (150 MHz, DMSO-d₆): d 166.7, 162.0, 160.5, 156.3, 151.1 (2C),

144.2, 144.1 (2C), 137.5, 131.2, 129.5 (2C), 128.9 (2C), 125.6, 121.4

(ESI, m/z): calcd for C₂₁H₂₄N₇O₂

found 406.1980.

[M þ H]^þ406.1986,

4.1.5.10. N-cyclopentyl-2-(4-((4-(pyridin-3-yl)pyrimidin-2-yl)amino)-

benzoyl) hydrazine-1-carboxamide (6j). Yellow solid (95.47%),

0.071 g, yield: 75%, mp: 159–161 ^ꢃC. ¹H NMR (600 MHz, DMSO-d₆):

d 10.11 (s, 1H), 9.94 (s, 1H), 9.37 (d, J ¼ 1.6 Hz, 1H), 8.75 (dd,

J ¼ 4.7, 1.6, Hz, 1H), 8.68 (d, J ¼ 5.1 Hz, 1H), 8.53 (d, J ¼ 7.5 Hz, 1H),

7.92–7.95 (d, J ¼ 9.0 Hz, 2H), 7.88 (d, J ¼ 9.0 Hz, 2H), 7.66 (s, 1H),

7.61–7.63 (m, 1H), 7.59 (d, J ¼ 5.1 Hz, 1H), 6.35 (d, J ¼ 7.5 Hz, 1H),

3.91 (m, 1H), 1.76–1.83 (m, 2H), 1.58–1.65 (m, 2H), 1.45–1.53 (m,

2H), 1.33ꢂ1.41 (m, 2H); ¹³C NMR (150 MHz, DMSO-d₆): d 166.4,

162.2, 160.4, 159.9, 158.5, 152.1, 148.7, 144.0, 134.9, 132.5, 128.8

(2C), 125.7, 124.5, 118.2 (2C), 109.5, _þ51.5, 33.1 (2C), 23.7 (2C);

(2C), 119.1(2C), 118.3 (2C), 109.8, 20.8; HRMS (ESI, m/z): calcd for

þ

C₂₄H₂₂N₇O₂

[M þ H]^þ440.1829, found 440.1824, calcd for

C₂₄H₂₁N₇O₂Na^þ[M þ Na]^þ462.1649, found 462.1642.

4.1.5.6.

N-(2,4-difluorophenyl)-2–(4-((4-(pyridin-4-yl)pyrimidin-2-

yl)amino)benzoyl) hydrazine-1-carboxamide (6f). Yellow solid

(93.67%), 0.065 g, yield: 71%, mp: 252–254 ^ꢃC. ¹H NMR (600 MHz,

DMSO-d₆): d 10.23 (br s, 1H), 10.17 (s, 1H), 8.79 (dd, J ¼ 4.4, 1.4 Hz,

2H), 8.72 (d, J ¼ 5.1 Hz, 1H), 8.65 (br s, 1H), 8.42 (s, 1H), 8.11 (dd,

J ¼ 4.4, 1.4 Hz, 2H), 7.95 (d, J ¼ 9.0 Hz, 2H), 7.90 (d, J ¼ 9.0 Hz, 2H),

7.61 (d, J ¼ 5.1 Hz, 1H), 7.26–7.31 (m, 1H), 6.99–7.08 (m, 1H); ¹³C

NMR (150 MHz, DMSO-d₆): d 166.6, 162.0, 160.5, 160.4, 156.1,

151.1(2C), 144.2, 144.1(2C), 128.9 (2C), 125.4, 124.2, 121.4, 118.4

(2C), 111.6, 111.4, 109.8, 104.5, 104.3, 104.3, 104.1; HRMS (ESI, m/

HRMS (ESI, m/z): calcd for C₂₂H₂₄N₇O₂[M þ H]^þ418.1986, found

[M þ Na]^þ440.1805,

þ

418.1980, calcd for C₂₂H₂₃N₇NaO₂

found 440.1799.

4.1.5.11. 2-(4-((4-(pyridin-3-yl)pyrimidin-2-yl)amino)benzoyl)-N-(p-

tolyl)hydrazine-1-carboxamide (6k). Yellow solid (96.10%), 0.056 g,

yield:59%, mp: 209–210 ^ꢃC. ¹H NMR (600 MHz, DMSO-d₆): d 10.13

(s, 2H), 9.37 (d, J ¼ 1.6 Hz, 1H), 8.76 (dd, J ¼ 4.7, 1.6 Hz, 1H), 8.74

(br s, 1H), 8.68 (d, J ¼ 5.1 Hz, 1H), 8.53 (dt, J ¼ 8.0, 1.6 Hz, 1H), 8.07

(s, 1H), 7.95 (d, J ¼ 9.0 Hz, 2H), 7.92 (d, J ¼ 9.0 Hz, 2H), 7.61–7.63

(m, 1H), 7.60 (d, J ¼ 5.1 Hz, 1H), 7.36 (d, J ¼ 8.2 Hz, 2H), 7.07 (d,

J ¼ 8.2 Hz, 2H), 2.24 (s, 3H); ¹³C NMR (150 MHz, DMSO-d₆): d 166.6,

162.2, 160.4, 160.0, 152.1, 148.7, 144.2, 137.6, 135.0, 132.5, 131.1,

129.6, 129.5 (2C), 128.9 (2C), 125.5, 124.5, 121.5 (2C),_þ118.3 (2C),

109.5, 20.8; HRMS (ESI, m/z): calcd for C₂₄H₂₂N₇O₂[M þ H]^þ

þ

z): calcd for C₂₃H₁₈F₂N₇O₂[M þ H]^þ462.1485, found 462.1476,

calcd for C₂₃H₁₇F₂N₇O₂Na^þ[M þ Na]^þ484.1304, found 484.1296.

4.1.5.7.

N-(3-chloro-4-methylphenyl)-2-(4-((4-(pyridin-4-yl)pyrimi-

din-2-yl)amino) benzoyl)hydrazine-1-carboxamide (6g). Yellow

solid (99.12%), 0.063 g, yield: 64%, mp: 209–210 ^ꢃC. ¹H NMR

(600 MHz, DMSO-d₆): d 10.18 (s, 1H), 10.15 (s, 1H), 8.97 (br s, 1H),

8.81 (dd, J ¼ 4.4, 1.4 Hz, 2H), 8.73 (d, J ¼ 5.1 Hz, 1H), 8.23 (br s, 1H),

8.12 (dd, J ¼ 4.4, 1.4 Hz, 2H), 7.96 (d, J ¼ 9.0 Hz, 2H), 7.93 (d,

J ¼ 9.0 Hz, 2H), 7.68 (d, J ¼ 1.4 Hz, 1H), 7.62 (d, J ¼ 5.1 Hz, 1H), 7.29

(br s, 1H), 7.22 (d, J ¼ 8.2 Hz, 1H), 2.25 (s, 3H); ¹³C NMR (150 MHz,

þ

440.1829, found 440.1823, calcd for C₂₄H₂₁N₇NaO₂[M þ Na]^þ

462.1649, found 462.1643.

DMSO-d₆): d 166.7, 162.0, 160.5, 160.4, 151.0(2C), 144.3, 144.1 (2C), 4.1.5.12. N-phenethyl-2-(4-((4-(pyridin-3-yl)pyrimidin-2-yl)amino)-

139.4, 133.4, 131.5, 128.9 (2C), 128.7, 125.6 (2C), 121.5 (2C), 118._þ3

benzoyl) hydrazine-1-carboxamide (6l). Yellow solid (95.57%),

0.054 g, yield: 56%, mp: 177–178 ^ꢃC. ¹H NMR (600 MHz, DMSO-d₆):

(2C), 109.8, 19.2; HRMS (ESI, m/z): calcd for C₂₄H₂₁ClN₇O₂

[M þ H]^þ474.1440, found 474.1440, calcd for C₂₄H₂₀ClN₇O₂Na^þ

d 10.09 (s, 1H), 9.99 (s, 1H), 9.35 (d, J ¼ 2.0 Hz, 1H), 8.74 (dd,

[M þ Na]^þ496.1259, found 496.1259.

J ¼ 4.7, 1.4 Hz, 1H), 8.66 (d, J ¼ 5.1 Hz, 1H), 8.52 (dt, J ¼ 8.0, 1.4 Hz,

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY

891

1H), 7.93 (d, J ¼ 9.0 Hz, 2H), 7.88–7.90 (d, J ¼ 9.0 Hz, 2H), 7.86 (s, 4.1.5.17. N-(3,5-dimethylphenyl)-2-(4-((4-(pyridin-2-yl)pyrimidin-2-

1H), 7.61 (dd, J ¼ 8.0, 4.7 Hz, 1H), 7.58 (d, J ¼ 5.1 Hz, 1H), 7.26ꢂ7.30

yl)amino) benzoyl)hydrazine-1-carboxamide (6q). Yellow solid

(98.69%), 0.073 g, yield: 79%, mp: 245–246 ^ꢃC. ¹H NMR (600 MHz,

(m, 2H), 7.20–7.23 (m, 2H), 7.17–7.19 (m, 1H), 6.53 (br s, 1H),

3.22–3.29 (m, 2H), 2.71 (t, J ¼ 7.3 Hz, 2H); ¹³C NMR (150 MHz,

DMSO-d₆): d 10.13 (s, 1H), 10.12 (s, 1H), 8.77 (d, J ¼ 4.0 Hz, 1H),

DMSO-d₆): d 166.6, 162.2, 160.3, 160.0, 159.0, 152.1, 148.6, 144.1,

8.72 (d, J ¼ 5.1 Hz, 1H), 8.68 (br s, 1H), 8.44 (d, J ¼ 7.8 Hz, 1H),

140.0, 135.0, 132.5, 129.1 (2C), 128.9 (2C), 128.8 (2C), 126.5, 125.6,

8.06–8.10 (m, 2H), 7.98 (d, J ¼ 8.6 Hz, 2H), 7.93 (d, J ¼ 8.6 Hz, 2H),

124.5, 118.2 (2C), 109.5, 41.4, 36.4； HRMS (ESI, m/z): calcd for

7.81 (d, J ¼ 5.1 Hz, 1H), 7.58–7.61 (m, 1H), 7.11 (s, 2H), 6.60 (s, 1H),

C₂₅H₂₄N₇O₂[M þ H]^þ454.1986, found 454.1981.

þ

2.22 (s, 6H); ¹³C NMR (150 MHz, DMSO-d₆): d 166.6, 163.4, 160.2,

160.1, 156.2, 153.9, 150.2, 144.2, 140.0, 138.1, 138.0 (2C), 128.9

(2C), 126.3, 125.5, 123.9 (2C), 121.6, 118.2 (2C), 116.7, 109.2, 21.6

4.1.5.13. N-(tert-butyl)-2-(4-((4-(pyridin-2-yl)pyrimidin-2-yl)amino)-

benzoyl) hydrazine-1-carboxamide (6m). Yellow solid (98.73%),

0.076 g, yield: 79%, mp: 213–214 ^ꢃC. ¹H NMR (600 MHz, DMSO-d₆):

d 10.10 (s, 1H), 9.95 (s, 1H), 8.76 (s, 1H), 8.71 (d, J ¼ 4.9 Hz, 1H),

8.43 (d, J ¼ 7.8 Hz, 1H), 8.07 (td, J ¼ 7.8, 1.6 Hz, 1H), 7.94 (d,

J ¼ 8.6 Hz, 2H), 7.88 (d, J ¼ 8.6 Hz, 2H), 7.80 (d, J ¼ 4.9 Hz, 1H),

7.58–7.61 (m, 2H), 6.08 (br s, 1H), 1.27 (s, 9H); ¹³C NMR (150 MHz,

DMSO-d₆): d 166.3, 163.4, 160.2, 160.1, 157.8, 153.9, 150.2, 144.1,

138.1, 128.7 (2C), 126.3, 125.6, 121.6, 118.2 (2C), 109.2, 49.9, 29.6

(2C); HRMS (ESI, m/z): calcd for C₂₅H₂₄N₇O₂[M þ H]^þ454.1986,

þ

found 454.1983.

4.1.5.18. N-(tert-butyl)-2-(4-((4–(4-chlorophenyl)pyrimidin-2-yl)ami-

no)benzoyl) hydrazine-1-carboxamide (6r). White solid (92.63%),

0.065 g, yield:76%, mp: 170–172 ^ꢃC. ¹H NMR (600 MHz, DMSO-d₆):

d 10.05 (s, 1H), 9.93 (s, 1H), 8.63 (d, J ¼ 5.1 Hz, 1H), 8.21 (d,

J ¼ 8.6 Hz, 2H), 7.92 (d, J ¼ 8.8 Hz, 2H), 7.86 (d, J ¼ 8.8 Hz, 2H), 7.64

(d, J ¼ 8.6 Hz, 2H), 7.59 (s, 1H), 7.51 (d, J ¼ 5.1 Hz, 1H), 6.07 (br s,

1H), 1.26 (s, 9H); ¹³C NMR (150 MHz, DMSO-d₆): d 166.3, 163.0,

160.3, 159.8, 157.8, 144.1, 136.3, 135.8, 129.5 (2C), 129. 2(2C), 128.7

(2C), 125.5, 118.2_þ(2C), 109.1, 49.9, 29.6 (3 C); HRMS (ESI, m/z): calcd

for C₂₂H₂₄ClN₆O₂[M þ H]^þ439.1644, found 439.1641, calcd for

þ

(3 C); HRMS (ESI, m/z): calcd for C₂₁H₂₄N₇O₂[M þ H]^þ406.1986,

found 406.1982.

4.1.5.14.

N-butyl-2-(4-((4-(pyridin-2-yl)pyrimidin-2-yl)amino)ben-

zoyl)hydrazine-1-carboxamide (6n). White solid (96.55%), 0.072 g,

yield: 78%, mp: 228–229 ^ꢃC. ¹H NMR (600 MHz, DMSO-d₆): d 10.10

(s, 1H), 9.96 (s, 1H), 8.77 (s, 1H), 8.71 (d, J ¼ 4.9 Hz, 1H), 8.43 (d,

J ¼ 7.8 Hz, 1H), 8.07 (td, J ¼ 7.8, 1.6 Hz, 1H), 7.95 (d, J ¼ 8.6 Hz, 2H),

7.90 (d, J ¼ 8.6 Hz, 2H), 7.80 (d, J ¼ 4.9 Hz, 1H), 7.74 (s, 1H),

7.57–7.62 (m, 1H), 6.44 (br s, 1H), 3.03 (m, 2H), 1.39 (quin,

J ¼ 7.3 Hz, 2H), 1.28 (sxt, J ¼ 7.3 Hz, 2H), 0.88 (t, J ¼ 7.3 Hz, 3H); ¹³C

NMR (150 MHz, DMSO-d₆): d 166.6, 163.4, 160.2, 160.1, 159.1,

153.9, 150.1, 144.1, 138.1, 128.9 (2C), 126.3, 125.6, 121.6, 118._þ2

(2C), 109.2, 32.4, 19.9, 14.2; HRMS (ESI, m/z): calcd for C₂₁H₂₄N₇O₂

[M þ H]^þ406.1986, found 406.1985, calcd for C₂₁H₂₃N₇O₂Na^þ

[M þ Na]^þ428.1805, found 428.1806.

þ

C₂₂H₂₃ClN₆NaO₂[M þ Na]^þ461.1463, found 461.1459.

4.1.5.19. 2-(4-((4-(4-chlorophenyl)pyrimidin-2-yl)amino)benzoyl)-N-

ethylhydrazine-1-carboxamide (6s). White solid (97.87%), 0.077 g,

yield: 82%, mp: 241–242 ^ꢃC. ¹H NMR (600 MHz, DMSO-d₆): d 10.06

(s, 1H), 9.95 (s, 1H), 8.64 (d, J ¼ 5.1 Hz, 1H), 8.22 (d, J ¼ 8.6 Hz, 2H),

7.93 (d, J ¼ 8.8 Hz, 2H), 7.88 (d, J ¼ 8.8 Hz, 2H), 7.76 (s, 1H),

7.62–7.68 (m, 2H), 7.52 (d, J ¼ 5.1 Hz, 1H), 6.45 (br s, 1H), 3.01–3.11

(m, 2H), 1.02 (t, J ¼ 7.0 Hz, 3H); ¹³C NMR (150 MHz, DMSO-d₆): d

166.6, 163.0, 160.3, 159.8, 158.9, 144.1, 136.3, 135.7, 129.5 (2C),

129.2 (2C), 128.9 (2C), 125.5, 118.2 _þ(2C), 109.1, 34.5, 16.0; HRMS

(ESI, m/z): calcd for C₂₀H₂₀ClN₆O₂[M þ H]^þ411.1331, found

þ

411.1330, calcd for C₂₀H₁₉ClN₆NaO₂

found 433.1149.

[M þ Na]^þ433.1150,

4.1.5.15. N-cyclopentyl-2-(4-((4-(pyridin-2-yl)pyrimidin-2-yl)amino)-

benzoyl) hydrazine-1-carboxamide (6o). White solid (95.44%),

0.063 g, yield: 73%, mp: 156–158 ^ꢃC. ¹H NMR (600 MHz, DMSO-d₆):

d 10.10 (s, 1H), 9.94 (s, 1H), 8.77 (d, J ¼ 4.0 Hz, 1H), 8.71 (d,

J ¼ 4.9 Hz, 1H), 8.43 (d, J ¼ 7.8 Hz, 1H), 8.07 (td, J ¼ 7.8, 1.6 Hz, 1H),

7.94–7.97 (d, J ¼ 8.6 Hz, 2H), 7.90 (d, J ¼ 8.6 Hz, 2H), 7.80 (d,

J ¼ 4.9 Hz, 1H), 7.66 (s, 1H), 7.59 (m, 1H), 6.35 (d, J ¼ 7.5 Hz, 1H),

3.91 (m, 1H), 1.76–1.84 (m, 2H), 1.58–1.66 (m, 2H), 1.46–1.54 (m,

2H), 1.34–1.41 (m, 2H); ¹³C NMR (150 MHz, DMSO-d₆): d 166.4,

163.4, 160.2, 160.1, 158.5, 153.9, 150.2, 144.1, 138.1, 128.8 (2C),

126.3, 125.6, 121.6, 118.2 (2C), 109.2, 51.5, 33.1 (2C), 23.7 (2C);

4.1.5.20. 2-(4-((4-(4-chlorophenyl)pyrimidin-2-yl)amino)benzoyl)-N-

cyclohexyl-hydrazine-1-carboxamide (6t). White solid (97.94%),

0.046 g, yield: 48%, mp: 165–167 ^ꢃC. ¹H NMR (600 MHz, DMSO-d₆):

d 10.06 (s, 1H), 9.94 (s, 1H), 8.64 (d, J ¼ 5.1 Hz, 1H), 8.20–8.23 (m,

2H), 7.92 (d, J ¼ 8.8 Hz, 2H), 7.88 (d, J ¼ 8.8 Hz, 2H), 7.69 (s, 1H),

7.63–7.66 (m, 2H), 7.52 (d, J ¼ 5.1 Hz, 1H), 6.24 (d, J ¼ 8.0 Hz, 1H),

3.38–3.47 (m, 1H), 1.73–1.78 (m, 2H), 1.66 (m, 2H), 1.51–1.56 (m,

1H), 1.22–1.31 (m, 2H), 1.10–1.21 (m, 3H); ¹³C NMR (150 MHz,

DMSO-d₆): d 166.4, 163.0, 160.3, 159.8, 158.1, 144.1, 136.3, 135.8,

129.5 (2C), 129.2 (2C), 128.8 (2C), 125.6, 118.1 (2C), 109.1, 48.6,

HRMS (ESI, m/z): calcd for C₂₂H₂₄N₇O₂[M þ H]^þ418.1986,

þ

found 418.1980.

þ

33.5 (2C), 25.7, 25.1 (2C); HRMS (ESI, m/z): calcd for C₂₄H₂₆ClN₆O₂_þ

[M þ H]^þ465.1800, found 465.1799, calcd for C₂₄H₂₅ClN₆NaO₂

[M þ Na]^þ487.1620, found 487.1619.

4.1.5.16. 2-(4-((4-(pyridin-2-yl)pyrimidin-2-yl)amino)benzoyl)-N-(m-

tolyl) hydrazine-1-carboxamide (6p). Yellow solid (97.54%),

0.054 g, yield: 67%, mp: 229–230 ^ꢃC. ¹H NMR (600 MHz, DMSO-d₆):

d 10.13 (s, 1H), 8.77 (d, J ¼ 4.0 Hz, 2H), 8.72 (d, J ¼ 5.1 Hz, 1H), 8.44

(d, J ¼ 7.7 Hz, 1H), 8.11 (s, 1H), 8.07 (dt, J ¼ 7.7, 1.6 Hz, 1H), 7.98 (d,

4.1.5.21. N-phenyl-2-(4-((4-(pyridin-4-yl)pyrimidin-2-yl)amino)ben-

zoyl)hydrazine-1-carbothioamide (6A). Yellow solid (97.57%),

J ¼ 8.6 Hz, 2H), 7.93 (d, J ¼ 8.6 Hz, 2H), 7.81 (d, J ¼ 5.1 Hz, 1H), 0.054 g, yield: 66%, mp: 267–269 ^ꢃC. ¹H NMR (600 MHz, DMSO-d₆):

7.58–7.61 (m, 1H), 7.31 (s, 1H), 7.29 (d, J ¼ 7.7 Hz, 1H), 7.14 (t, d 10.40 (s, 1H), 10.19 (s, 1H), 9.79 (br s, 1H), 9.68 (br s, 1H), 8.80

J ¼ 7.7 Hz, 1H), 6.78 (d, J ¼ 7.7 Hz, 1H), 2.26 (s, 3H); ¹³C NMR (dd, J ¼ 4.4, 1.4 Hz, 2H), 8.74 (d, J ¼ 4.9 Hz, 1H), 8.11 (dd, J ¼ 4.4,

(150 MHz, DMSO-d₆): d 166.6, 163.4, 160.3, 160.1, 156.3, 153.9, 1.4 Hz, 2H), 7.96 (br s, 4H), 7.63 (d, J ¼ 4.9 Hz, 1H), 7.46 (br s, 2H),

150.2, 144.2, 140.1, 138.2, 138.1, 128.9 (2C), 128.9, 126.3, 125.5, 7.33 (t, J ¼ 7.7 Hz, 2H), 7.16 (t, J ¼ 7.7 Hz, 1H); ¹³C NMR (150 MHz,

123.1, 121.6, 119.5, 118.2 (2C), 116.1, 109.2, 21.7; HRMS (ESI, m/z): DMSO-d₆): d 166.1, 162.0, 160.5, 160.4, 151.1(2C), 144.2, 139.8 (2C),

þ

calcd for C₂₄H₂₂N₇O₂[M þ H]^þ440.1829, found 440.1826, calcd 129.3 (2C), 128.4 (2C), 128.2, 126.4 (2C), 125.5(2C), 121.4, 118.4,

þ

for C₂₄H₂₁N₇NaO₂[M þ Na]^þ462.1649, found 462.1646.

118.1 (2C), 109.8; HRMS (ESI, m/z): calcd for C₂₃H₂₀N₇OS^þ[M þ H]^þ

892

J. QIN ET AL.

442.1445, found 442.1440, calcd for C₂₃H₁₉N₇NaOS^þ[M þ Na]^þ2H), 1.57 (d, J ¼ 13.0 Hz, 1H), 1.28–1.33 (m, 2H), 1.22–1.27 (m, 2H),

464.1264, found 464.1261.

1.01–1.12 (m, 1H); ¹³C NMR (150 MHz, DMSO-d₆): d 166.0, 164.2,

160.3, 159.6, 144.4, 137.0, 131.5 (2C), 129.5 (2C), 129.2, 127.4 (2C),

125.2, 118.0 (2C), 109.3, 53.4, 32.3 (2C), 25.6, 25.4 (2C); HRMS (ESI,

m/z): calcd for C₂₄H₂₇N₆OS^þ[M þ H]^þ447.1962, found 447.1959,

calcd for C₂₄H₂₆N₆NaOS^þ[M þ Na]^þ469.1781, found 469.1780.

4.1.5.22. N-benzyl-2-(4-((4-(pyridin-4-yl)pyrimidin-2-yl)amino)ben-

zoyl)hydrazine-1-carbothioamide (6B). Yellow solid (98.33%),

0.065 g, yield: 73%, mp: 240–241 ^ꢃC. ¹H NMR (600 MHz, DMSO-d₆):

d 10.27 (s, 1H), 10.18 (s, 1H), 9.42 (s, 1H), 8.80 (dd, J ¼ 4.4, 1.4 Hz,

2H), 8.73 (d, J ¼ 5.1 Hz, 1H), 8.65 (br s, 1H), 8.11 (dd, J ¼ 4.4, 1.4 Hz,

2H), 7.91–7.97 (m, 4H), 7.62 (d, J ¼ 5.1 Hz, 1H), 7.29– 7.33 (m, 4H),

7.20–7.24 (m, 1H), 4.75 (d, J ¼ 6.0 Hz, 2H); ¹³C NMR (150 MHz,

DMSO-d₆): d 166.1, 162.0, 160.5, 160.4, 151.1(2C), 144.1, 140.0 (2C),

129.2 (2C), 128.5 (2C), 127.5, 127.0 (2C), 125.5, 121.4(2C), 118.1(2C),

109.8, 47.2; HRMS (ESI, m/z): calcd for C₂₄H₂₂N₇OS^þ[M þ H]^þ

456.1601, found 456.1595, calcd for C₂₄H₂₁N₇OSNa^þ[M þ Na]^þ

478.1421, found 478.1415.

4.1.5.27. N-phenyl-2-(4-((4-phenylpyrimidin-2-yl)amino)benzoyl)hy-

drazine-1-carbothioamide (6G). White solid (97.80%), 0.034 g, yield:

50%, mp: 210–212 ^ꢃC. ¹H NMR (600 MHz, DMSO-d₆): d 10.38 (br s,

1H), 10.06 (s, 1H), 9.79 (br s, 1H), 9.67 (br s, 1H), 8.63 (d, J ¼ 5.3 Hz,

1H), 8.19ꢂ8.21 (m, 2H), 7.98 (d, J ¼ 8.8 Hz, 2H), 7.95 (d, J ¼ 8.8 Hz,

2H), 7.57–7.59 (m, 3H), 7.51 (d, J ¼ 5.3 Hz, 1H), 7.43–7.49 (m, 2H),

7.33 (t, J ¼ 7.8 Hz, 2H), 7.13–7.19 (m, 1H); ¹³C NMR (150 MHz,

DMSO-d₆): d 166.2, 164.2, 160.3, 159.7, 144.5, 139.8, 137.0, 131.5

(2C), 129.5 (2C), 129.3 (2C), 128.4, 127.4 (2C), 126.5 (2C), 125.5,

125.3, 118.0 (2C), 109.3; HRMS (ESI, m/z): calcd for C₂₄H₂₁N₆OS^þ

[M þ H]^þ441.1492, found 441.1490, calcd for C₂₄H₂₀N₆NaOS^þ

[M þ Na]^þ463.1312, found 463.1309.

4.1.5.23. N-benzyl-2-(4-((4-(pyridin-3-yl)pyrimidin-2-yl)amino)ben-

zoyl)hydrazine-1-carbothioamide (6C). Yellow solid (98.60%),

1

0.072 g, yield: 75%, mp: 159–160 ^ꢃC. H NMR (600 MHz, DMSO-d₆): d

10.27 (s, 1H), 10.12 (s, 1H), 9.42 (s, 1H), 9.35–9.38 (s, 1H), 8.75 (dd,

J ¼ 4.9, 1.8 Hz, 1H), 8.68 (d, J ¼ 5.1 Hz, 1H), 8.66 (d, J ¼ 4.9 Hz, 1H),

8.52 (td, J ¼ 8.0, 1.8 Hz, 1H), 7.92–7.96 (br s, 4H), 7.61–7.63 (m, 1H),

7.60 (d, J ¼ 5.1 Hz, 1H), 7.28–7.34 (m, 4H), 7.20–7.24 (m, 1H), 4.75 (d,

J ¼ 6.0 Hz, 2H); ¹³C NMR (150 MHz, DMSO- d₆): d 166.2, 162.2, 160.3,

160.0, 152.1, 148.7, 144.2, 140.0, 134.9, 132.5, 129.2 (2C), 128.5 (2C),

127.5, 127.0 (2C), 125.4, 124.5, 118.1 (2C), 109.5, 47.2; HRMS (ESI, m/

z): calcd for C₂₄H₂₂N₇OS^þ[M þ H]^þ456.1601, found 456.1599, calcd

for C₂₄H₂₁N₇NaOS^þ[M þ Na]^þ478.1421, found 478.1419.

4.1.5.28. N-benzyl-2-(4-((4-phenylpyrimidin-2-yl)amino)benzoyl)hy-

drazine-1-carbothioamide (6H). White solid (99.08%), 0.054 g, yield:

62%, mp: 289–291 ^ꢃC. ¹H NMR (600 MHz, DMSO-d₆): d 10.26 (s,

1H), 10.05 (s, 1H), 9.41 (s, 1H), 8.65 (br s, 1H), 8.62 (d, J ¼ 5.1 Hz,

1H), 8.17–8.22 (m, 2H), 7.97 (d, J ¼ 8.8 Hz, 2H), 7.93 (d, J ¼ 8.8 Hz,

2H), 7.57 (m, 3H), 7.51 (dd, J ¼ 5.1, 1.6 Hz, 1H), 7.28–7.34 (m, 4H),

7.22 (d, J ¼ 5.5 Hz, 1H), 4.75 (d, J ¼ 5.5 Hz, 2H); ¹³C NMR (150 MHz,

DMSO-d₆): d 166.2, 164.2, 160.3, 159.7, 144.4, 140.0, 136.9, 131.6

(2C), 129.5 (2C), 129.2, 128.5 (2C), 127.5, 127.4 (2C), 127.0 (3 C),

125.2, 117.9 (2C), 109.3, 47.2; HRMS (ESI, m/z): calcd for

C₂₅H₂₃N₆OS^þ[M þ H]^þ455.1649, found 455.1644, calcd for

C₂₅H₂₂N₆NaOS^þ[M þ Na]^þ477.1468, found 477.1465.

4.1.5.24. N-phenyl-2-(4-((4-(pyridin-2-yl)pyrimidin-2-yl)amino)ben-

zoyl)hydrazine-1-carbothioamide (6D). Yellow solid (99.24%),

1

0.046 g, yield: 48%, mp: 200–202 ^ꢃC. H NMR (600 MHz, DMSO-d₆): d

10.39 (s, 1H), 10.13 (s, 1H), 9.80 (br s, 1H), 9.68 (s, 1H), 8.76–8.78 (d,

J ¼ 4.0 Hz, 1H), 8.72 (d, J ¼ 5.1 Hz, 1H), 8.43 (d, J ¼ 7.7 Hz, 1H), 8.07

(td, J ¼ 7.7, 1.8 Hz, 1H), 7.98 (s, 4H), 7.81 (d, J ¼ 5.1Hz, 1H), 7.59 (m,

1H), 7.46 (d, J ¼ 1.8Hz, 2H), 7.34 (t, J ¼ 7.7 Hz, 2H), 7.13–7.19 (m,

1H); ¹³C NMR (150 MHz, DMSO-d₆): d 166.2, 163.4, 160.2, 160.1,

153.9, 150.2, 144.3, 139.8, 138.1, 129.3 (2C), 128.4 (2C), 126.5, 126.3

(2C), 125.4, 121.6, 118.0 (2C), 109.3, 40.5; HRMS (ESI, m/z): calcd for

C₂₃H₂₀N₇OS^þ[M þ H]^þ442.1445, found 442.1444, calcd for

C₂₃H₁₉N₇OSNa^þ[Mþ Na]^þ464.1264, found 464.1265.

4.1.5.29. 2-(4-((4–(4-chlorophenyl)pyrimidin-2-yl)amino)benzoyl)-N-

phenylhydrazine-1-carbothioamide (6I). White solid (98.95%),

0.045 g, yield: 48%, mp: 275–277 ^ꢃC. ¹H NMR (600 MHz, DMSO-d₆):

d 10.38 (s, 1H), 10.09 (s, 1H), 9.79 (br s, 1H), 9.68 (s, 1H), 8.65 ((d,

J ¼ 5.1, 1H), 8.22 (d, J ¼ 8.6 Hz, 2H), 7.95 (br s, 4H), 7.65 (d,

J ¼ 8.6 Hz, 2H), 7.52 (d, J ¼ 5.1 Hz, 1H), 7.46 (br s, 2H), 7.33 (m, 2H),

7.13–7.19 (m, 1H). ¹³C NMR (150 MHz, DMSO-d₆): d 163.0, 160.3,

159.9, 144.3, 139.8, 136.3, 135.8, 129.5 (2C), 129.3 (2C), 129.2 (2C),

128.4 (2C), 126.5 (2C), 125.5, 125.4, 118.0 (2C), 109.2; HRMS (ESI,

m/z): calcd for C₂₄H₂₀ClN₆OS^þ[M þ H]^þ475.1102, found 475.1100,

calcd for C₂₄H₁₉ClN₆NaOS^þ[M þ Na]^þ497.0922, found 497.0920.

4.1.6.25. N-benzyl-2-(4-((4-(pyridin-2-yl)pyrimidin-2-yl)amino)ben-

zoyl)hydrazine-1-carbothioamide (6E). White solid (99.14%),

0.045 g, yield: 67%, mp: 222–223 ^ꢃC. ¹H NMR (600 MHz, DMSO-d₆):

d 10.27 (s, 1H), 10.12 (s, 1H), 9.42 (s, 1H), 8.76–8.78 (m, 1H), 8.71

(d, J ¼ 4.9 Hz, 1H), 8.65 (br s, 1H), 8.42 (d, J ¼ 7.7 Hz, 1H), 8.07 (td,

J ¼ 7.7, 1.8 Hz, 1H), 7.92–7.98 (br s, 4H), 7.80 (d, J ¼ 4.9 Hz, 1H),

7.59 (m, 1H), 7.28–7.34 (m, 4H), 7.20–7.24 (m, 1H), 4.75 (m, 2H);

¹³C NMR (150 MHz, DMSO-d₆): d 166.2, 163.4, 160.2, 160.2, 153.9,

150.2, 144.3, 140.0, 138.1, 129.3 (2C), 128.5 (2C), 127.5, 127.0 (2C),

126.3, 125.3, 121.6, 118.0 (2C), 109.3, 70.2, 47.2; HRMS (ESI, m/z):

calcd for C₂₄H₂₂N₇OS^þ[M þ H]^þ456.1601, found 456.1599, calcd

for C₂₄H₂₁N₇NaOS^þ[M þ Na]^þ478.1421, found 478.1419.

4.1.5.30. N-benzyl-2-(4-((4–(4-chlorophenyl)pyrimidin-2-yl)amino)-

benzoyl) hydrazine-1-carbothioamide (6J). White solid (95.91%),

1

0.035 g, yield: 56%, mp: 235–237 ^ꢃC. H NMR (600 MHz, DMSO-d₆): d

10.26 (s, 1H), 10.08 (s, 1H), 9.42 (s, 1H), 8.63 (d, J ¼ 5.1 Hz, 2H), 8.20

(d, J ¼ 8.6 Hz, 2H), 7.91ꢂ 7.95 (br s, 4H), 7.65 (d, J ¼ 8.6 Hz, 2H), 7.52

(d, J ¼ 5.1 Hz, 1H), 7.29–7.33 (m, 4H), 7.20– 7.24 (m, 1H), 4.75 (d,

J ¼ 6.0 Hz, 2H); ¹³C NMR (150 MHz, DMSO-d₆): d 166.2, 163.0, 160.3,

159.9, 144.3, 140.0, 136.3, 135.8, 129.5 (2C), 129.2 (2C), 128.5 (2C),

127.5 (2C), 127.0 (2C), 125.3, 118.0 (2C), 109.2, 47.2; HRMS (ESI, m/z):

calcd for C₂₅H₂₂ClN₆OS^þ[M þ H]^þ489.1259, found 489.1255, calcd

for C₂₅H₂₁ClN₆NaOS^þ[M þ Na]^þ511.1078, found 511.1075.

4.1.6.26.

N-cyclohexyl-2-(4-((4-phenylpyrimidin-2-yl)amino)ben-

zoyl)hydrazine-1-carbothioamide (6F). White solid (98.99%),

0.032 g, yield:59%, mp: 230–232 ^ꢃC. ¹H NMR (600 MHz, DMSO-d₆):

d 10.10 (br s, 1H), 10.05 (s, 1H), 9.16 (br s, 1H), 8.63 (d, J ¼ 5.1 Hz,

1H), 8.20–8.21 (m, 1H), 8.18–8.20 (m, 1H), 7.96 (d, J ¼ 8.8 Hz, 2H),

4.1.6. General procedure for the synthesis of compounds 7a–7f

and 7A–7D (Series B)

7.92 (d, J ¼ 8.8 Hz, 2H), 7.68 (d, J ¼ 3.8 Hz, 1H), 7.56–7.60 (m, 3H), Compounds 5A–5C (1.00 eq) were separately used to react with

7.51 (d, J ¼ 5.1 Hz, 1H), 4.15 (br s, 1H), 1.79 (br s, 2H), 1.64–1.72 (m, an appropriate isocyanate or isothiocyanate (1.00 eq) in ethanol at

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY

893

70 ^ꢃC for 4 h, then cooling to room temperature, filtered to get (94.02%), 0.032 g, yield: 55%, mp: 231–232 ^ꢃC. ¹H NMR (600 MHz,

crude products of compounds 7a–7f and 7A–7D, which were fur- DMSO- d₆): d 10.28 (br s, 1H), 9.97 (s, 1H), 9.38 (d, J ¼ 1.8 Hz, 1H),

ther purified by silica gel column chromatography (DCM:MeOH).

8.73 (dd, J ¼ 4.7, 1.4 Hz, 1H), 8.64 (d, J ¼ 5.1 Hz, 1H), 8.61 (s, 1H), 8.57

(dt, J ¼ 8.0, 1.8 Hz, 1H), 8.48 (s, 1H), 8.03 (br s, 1H), 7.95 (dd, J ¼ 8.0,

1.4 Hz, 1H), 7.88 (d, J ¼ 8.8 Hz, 1H), 7.57–7.59 (m, 1H), 7.55–7.57 (m,

4.1.6.1. N-(tert-butyl)-2-(3-((4-(pyridin-4-yl)pyrimidin-2-yl)amino)-

1H), 7.49–7.52 (m, 1H), 7.43–7.47 (m, 1H), 6.62 (d, J ¼ 2.5 Hz, 1H),

benzoyl) hydrazine-1-carboxamide (7a). Yellow solid (98.13%),

6.48 (dd, J ¼ 8.8, 2.5 Hz, 1H), 3.84 (s, 3H), 3.73 (s, 3H); ¹³C NMR

0.076 g, yield: 83%, mp: 161–162 ^ꢃC. ¹H NMR (600 MHz, DMSO-d₆):

(150 MHz, DMSO- d₆): d 167.3, 162.1, 160.6, 160.0, 156.0, 155.4,

d 10.09 (br s, 1H), 10.06 (s, 1H), 8.84 (d, J ¼ 6.0 Hz, 2H), 8.75 (d,

152.0, 149.6, 148.7, 141.1, 135.0, 133.7, 132.6, 129.0, 124.4, 122.6,

J ¼ 5.1 Hz, 1H), 8.56 (s, 1H), 8.20 (d, J ¼ 6.0 Hz, 2H), 7.94 (dd, J ¼ 8.0,

122.2, 120.6, 120.0, 118.9, 1_þ09.0, 104.6, 99.2, 56.3, 55.7; HRMS (ESI,

m/z): calcd for C₂₅H₂₄N₇O₄[M þ H]^þ486.1884, found 486.1883,

calcd for C₂₅H₂₃N₇NaO₄^þ[M þ Na]^þ508.1704, found 508.1703.

1.4 Hz, 1H), 7.72 (s, 1H), 7.64 (d, J ¼ 5.1 Hz, 1H), 7.54 (d, J ¼ 7.7 Hz,

1H), 7.48 (t, J ¼ 7.7 Hz, 1H), 6.04–6.20 (m, 1H), 1.33 (s, 9H); ¹³C NMR

(150 MHz, DMSO-d₆): d 167.0, 161.8, 160.7, 160.5, 157.7, 151.1,

144.2 (2C), 140.9, 133.9, 129.0, 122.5, 121.5 (2C), 120.7, 119.0,

þ

4.1.6.6.

N-butyl-2-(3-((4-(pyridin-4-yl)pyrimidin-2-yl)amino)ben-

109.3, 50.0, 29.6 (3 C); HRMS (ESI, m/z): calcd for C₂₁H₂₄N₇O₂_þ

[M þ H]^þ406.1986, found 406.1982, calcd for C₂₁H₂₃N₇NaO₂

[M þ Na]^þ428.1805, found 428.1801.

zoyl)hydrazine-1-carboxamide (7f). Yellow solid (98.48%), 0.043 g,

yield: 80%, mp: 183–184 ^ꢃC. ¹H NMR (600 MHz, DMSO-d₆): d 10.05

(s, 1H), 9.94 (s, 1H), 8.74–8.77 (m, 1H), 8.68 (d, J ¼ 4.9 Hz, 1H), 8.53

(s, 1H), 8.51 (d, J ¼ 7.9 Hz, 1H), 8.04 (td, J ¼ 7.9, 1.7 Hz, 1H), 7.92

(dd, J ¼ 7.9, 1.7 Hz, 1H), 7.83 (s, 1H), 7.77 (d, J ¼ 5.1 Hz, 1H), 7.58

(m, 1H), 7.52 (d, J ¼ 7.9 Hz, 1H), 7.42 (t, J ¼ 7.9 Hz, 1H), 6.43 (t,

J ¼ 5.1 Hz, 1H), 3.05 (q, J ¼ 6.8 Hz, 2H), 1.39 (quin, J ¼ 7.3 Hz, 2H),

1.29 (sxt, J ¼ 7.3 Hz, 2H), 0.88 (t, J ¼ 7.3 Hz, 3H); ¹³C NMR (150 MHz,

DMSO-d₆): d 167.2, 163.3, 160.5, 160.1, 158.8, 154.0, 150.1, 141.1,

138.1, 133.9, 128.9, 126.2, 122.4, 121.8, 120.7, 119.0, 108.7, 39.4,

4.1.6.2. N-cyclopentyl-2-(3-((4-(pyridin-4-yl)pyrimidin-2-yl)amino)-

benzoyl) hydrazine-1-carboxamide (7b). Yellow solid (99.30%),

1

0.076 g, Yield: 79%, mp: 221–223 ^ꢃC. H NMR (600 MHz, DMSO-d₆):

d 10.05 (s, 1H), 10.01 (s, 1H), 8.79 (d, J ¼ 6.0 Hz, 2H), 8.70 (d,

J ¼ 5.1 Hz, 1H), 8.50 (s, 1H), 8.15 (d, J ¼ 6.0 Hz, 2H), 7.91 (dd, J ¼ 7.7,

1.5 Hz, 1H), 7.75 (s, 1H), 7.58 (d, J ¼ 5.1 Hz, 1H), 7.51 (d, J ¼ 7.7 Hz,

1H), 7.40–7.45 (J ¼ 7.7 Hz, 1H), 6.36 (m, 1H), 3.93 (m, 1H), 1.78–1.85

(m, 2H), 1.58–1.67 (m, 2H), 1.47–1.55 (m, 2H), 1.35–1.43 (m, 2H);

¹³C NMR (150 MHz, DMSO-d₆): d 167.2, 161.8, 160.6, 160.5, 158.4,

151.0, 144.2 (2C), 140.9, 133.9, 129.0, 122.5, 121.4 (2C), 120.8,

þ

32.5, 19.9, 14.2；HRMS (ESI, m/z): calcd for C₂₁H₂₄N₇O₂[M þ H]^þ

þ

406.1986, found 406.1980, calcd for C₂₁H₂₃N₇NaO₂[M þ Na]^þ

428.1805, found 428.1802.

119.0, 109.3, 51.6, 33.1 (2C), 23.7 (2C); HRMS (ESI, m/z): calcd for

[M þ H]^þ418.1986, found 418.1983, calcd for

þ

C₂₂H₂₄N₇O₂

4.1.6.7.

N-benzyl-2-(3-((4-(pyridin-4-yl)pyrimidin-2-yl)amino)ben-

C₂₂H₁₃N₇NaO^þ[M þ Na]^þ440.1805, found 440.1803.

zoyl)hydrazine-1-carbothioamide (7A). Yellow solid (98.66%),

1

0.042 g, yield: 53%, mp: 218–220 ^ꢃC. H NMR (600 MHz, DMSO- d₆):

d 10.40 (s, 1H), 10.01 (s, 1H), 9.51 (s, 1H), 8.79 (d, J ¼ 6.0 Hz, 2H),

8.69 (d, J ¼ 5.1 Hz, 2H), 8.54 (br s, 1H), 8.15 (d, J ¼ 6.0 Hz, 2H), 7.94

(dd, J ¼ 1.2, 8.0 Hz, 1H), 7.57–7.61 (m, 2H), 7.44 (t, J ¼ 7.8 Hz, 1H),

7.31–7.34 (m, 2H), 7.27– 7.31 (m, 2H), 7.19–7.23 (m, 1H), 4.76 (d,

J ¼ 5.8 Hz, 2H); ¹³C NMR (150 MHz, DMSO- d₆): d 166.8, 161.8, 160.6,

160.5, 151.0, 144.2 (2C), 140.9, 139.9, 133.5, 128.8 (2C), 128.5, 127.5,

127.0 (2C), 122.8, 121.4, 121.3 (2C), 119.4, 109.3, 47.2; HRMS (ESI, m/

z): calcd for C₂₄H₂₂N₇OS^þ[M þ H]^þ456.1601, found 456.1599, calcd

for C₂₄H₂₁N₇NaOS^þ[M þ Na]^þ478.1421, found 478.1418.

4.1.6.3. N-(tert-butyl)-2-(3-((4-(pyridin-3-yl)pyrimidin-2-yl)amino)-

benzoyl) hydrazine-1-carboxamide (7c). White solid (97.12%),

0.079 g, yield: 81%, mp: 206–207 ^ꢃC. ¹H NMR (600 MHz, DMSO-d₆):

d 10.04 (br s 1H), 9.94 (s, 1H), 9.38 (d, J ¼ 1.6 Hz, 1H), 8.74 (dd,

J ¼ 4.8, 1.8 Hz, 1H), 8.64 (d, J ¼ 5.1 Hz, 1H), 8.57 (dt, J ¼ 7.9, 1.8 Hz,

1H), 8.45 (s, 1H), 7.93 (dd, J ¼ 7.9, 1.8 Hz, 1H), 7.66 (s, 1H), 7.59 (dd,

J ¼ 7.9, 4.8 Hz, 1H), 7.56 (d, J ¼ 5.1 Hz, 1H), 7.49 (d, J ¼ 7.7 Hz, 1H),

7.40–7.43 (t, J ¼ 7.7 Hz 1H), 6.08 (br s, 1H), 1.28 (s, 9H); ¹³C NMR

(150 MHz, DMSO-d₆): d 166.9, 162.1, 160.6, 159.9, 157.7, 152.0,

148.7, 141.0, 135.0, 133.8, 132.6, 128.9, 124.4, 122.4, 120.6, 119.0,

þ

108.9, 49.9, 29.6 (3 C); HRMS (ESI, m/z): calcd for C₂₁H₂₄N₇O₂_þ

4.1.6.8.

N-benzyl-2-(3-((4-(pyridin-3-yl)pyrimidin-2-yl)amino)ben-

[M þ H]^þ406.1986, found 406.1982, calcd for C₂₁H₂₃N₇NaO₂

[M þ Na]^þ428.1805, found 428.1801.

zoyl)hydrazine-1-carbothioamide (7B). White solid (96.84%),

0.055 g, yield: 58%; mp: 212–213 ^ꢃC . ¹H NMR (600 MHz, DMSO-

d₆): d 10.38 (s, 1H), 9.95 (s, 1H), 9.49 (s, 1H), 9.38 (d, J ¼ 1.8 Hz, 1H),

4.1.6.4.

N-butyl-2-(3-((4-(pyridin-3-yl)pyrimidin-2-yl)amino)ben- 8.74 (dd, J ¼ 4.7, 1.6 Hz, 1H), 8.66 (br s, 1H), 8.63 (d, J ¼ 5.1 Hz, 1H),

8.57 (dt, J ¼ 8.0, 1.8 Hz, 1H), 8.50 (s, 1H), 7.95 (dd, J ¼ 8.0, 1.6 Hz,

1H), 7.57–7.60 (m, 2H), 7.56 (d, J ¼ 5.1 Hz, 1H), 7.43 (t, J ¼ 8.0 Hz,

1H), 7.27–7.34 (m, 4H), 7.19–7.23 (m, 1H), 4.75 (d, J ¼ 5.8 Hz, 2H);

¹³C NMR (150 MHz, DMSO- d₆): d 166.7, 162.1, 160.6, 160.0, 152.1,

148.7, 141.0, 139.9, 135.0, 133.5, 132.5, 128.7, 128.5, 127.5 (2C),

127.0 (2C), 124.4, 122.7, 121.1, 119.3, 108.9, 47.2; HRMS (ESI, m/z):

calcd for C₂₄H₂₂N₇OS^þ[M þ H] þ 456.1601, found 456.1599, calcd

for C₂₄H₂₁N₇NaOS^þ[M þ Na]^þ478.1421, found 478.1418.

zoyl)hydrazine-1-carboxamide (7d). Yellow solid (99.36%), 0.066 g,

1

Yield: 79%, mp: 196–198 ^ꢃC. H NMR (600 MHz, DMSO-d₆): d 10.05

(s, 1H), 9.94 (s, 1H), 9.38 (d, J ¼ 1.9 Hz, 1H), 8.74 (dd, J ¼ 4.7, 1.4 Hz,

1H), 8.64 (d, J ¼ 5.1 Hz, 1H), 8.57 (dt, J ¼ 8.0, 1.9 Hz, 1H), 8.45 (s,

1H), 7.94 (dd, J ¼ 8.0, 1.4 Hz, 1H), 7.82 (s, 1H), 7.59 (dd, J ¼ 8.0,

4.7 Hz, 1H), 7.56 (d, J ¼ 5.1 Hz, 1H), 7.52 (d, J ¼ 7.7 Hz, 1H), 7.42 (t,

J ¼ 7.7 Hz, 1H), 6.42 (t, J ¼ 7.7 Hz, 1H), 3.04 (q, J ¼ 6.7 Hz, 2H), 1.39

(quin, J ¼ 7.3 Hz, 2H), 1.28 (sxt, J ¼ 7.3 Hz, 2H), 0.88 (t, J ¼ 7.3 Hz,

3H); ¹³C NMR (150 MHz, DMSO-d₆): d 167.1, 162.1, 160.6, 159.9,

158.8, 152.0, 148.7, 141.0, 135.0, 133.9, 132.6, 128.8, 124.4, 122.5,

4.1.6.9.

N-phenyl-2-(3-((4-(pyridin-2-yl)pyrimidin-2-yl)amino)ben-

120.8, 119.1, 108.9, 39.4, 32.5, 19.9, 14.2; HRMS (ESI, m/z): calcd for

zoyl)hydrazine-1-carbothioamide (7C). Yellow solid (97.88%),

þ

1

C₂₁H₂₄N₇O₂

[M þ H]^þ406.1986, found 406.1980, calcd for

0.043 g, Yield: 59%, mp: 202–203 ^ꢃC . H NMR (600 MHz, DMSO-d₆):

C₂₁H₂₃N₇NaO₂[M þ Na]^þ428.1805, found 428.1802.

þ

d 10.51 (s, 1H), 9.93–10.00 (s, 1H), 9.83 (br s, 1H), 9.75 (br s, 1H),

8.75 (m, 1H), 8.68 (d, J ¼ 5.0 Hz, 1H), 8.60 (br s, 1H), 8.51 (d,

J ¼ 7.8 Hz, 1H), 8.00 (t, J ¼ 7.8 Hz, 1H), 7.94 (d, J ¼ 8.0 Hz, 1H), 7.77

4.1.6.5. N-(2,4-dimethoxyphenyl)-2-(3-((4-(pyridin-3-yl)pyrimidin-2-

yl)amino) benzoyl)hydrazine-1-carboxamide (7e). White solid (d, J ¼ 5.0 Hz, 1H), 7.62 (d, J ¼ 6.9 Hz, 1H), 7.56 (dd, J ¼ 6.9, 5.0 Hz,

894

J. QIN ET AL.

1H), 7.42–7.50 (m, 3H), 7.34 (t, J ¼ 7.3 Hz, 2H), 7.14–7.19 (t, 4.2.4. RXRE-mediated transcription assay

J ¼ 7.3 Hz, 1H); ¹³C NMR (150 MHz, DMSO-d₆): d 166.7, 163.3, 160.5,

HEK293T cells were seeded into 48-well plates at 40% confluence

160.2, 154.0, 150.1, 141.1, 139.8, 138.1, 133.6, 128.8, 128.4 (2C),

and cotransfected with RXRE luciferase reporter gene (100.0 ng),

126.2, 125.5 (2C), 122.6, 121.8, 121.2, 119.2, 108.7; HRMS (ESI, m/z):

Myc-RXRa (50.0 ng), and Renilla control plasmid (1.0 ng). After 24 h

calcd for C₂₃H₂₀N₇OS^þ[M þ H]^þ442.1445, found 442.1445, calcd post transfection, the compounds or vehicle were added. Cells

for C₂₃H₁₉N₇NaOS^þ[M þ Na]^þ464.1264, found 464.1261.

were harvested after 12 h of incubation with ligands and then

quantitated using the Dual-Luciferase Reporter Assay System

(Promega). Luminescence of firefly luciferase values was normal-

ised to the Renilla luciferase activity.

4.1.6.10. N-benzyl-2-(3-((4-(pyridin-2-yl)pyrimidin-2-yl)amino)ben-

zoyl)hydrazine-1-carbothioamide (7D). Yellow solid (98.95%),

1

0.052 g, yield: 56%, mp: 203–205 ^ꢃC . H NMR (600 MHz, DMSO-d₆):

d 10.40 (s, 1H), 9.95 (s, 1H), 9.50 (s, 1H), 8.75 (d, J ¼ 4.5 Hz, 1H),

8.68 (t, J ¼ 5.1 Hz, 1H), 8.67 (d, J ¼ 5.1 Hz, 1H), 8.59 (br s, 1H), 8.52

(d, J ¼ 7.8 Hz, 1H), 8.00 (dt, J ¼ 8.0, 1.6 Hz, 1H), 7.93 (dd, J ¼ 8.0,

1.6 Hz, 1H), 7.77 (d, J ¼ 5.1 Hz, 1H), 7.56–7.60 (m, 2H), 7.44 (t,

J ¼ 7.8 Hz, 1H), 7.32 (t, J ¼ 7.3 Hz, 2H), 7.29 (t, J ¼ 7.3 Hz, 2H), 7.22

(t, J ¼ 7.3 Hz, 1H), 4.76 (d, J ¼ 5.8 Hz, 2H); ¹³C NMR (150 MHz,

DMSO-d₆): d 166.8, 163.3, 160.4, 160.2, 154.0, 150.1, 141.1, 139.9,

138.1, 133.5, 128.8, 128.5 (2C), 127.5, 127.0 (3 C), 126.3, 122.6,

121.8, 121.1, 119.2, 108.7, 47.2; HRMS (ESI, m/z): calcd for

C₂₄H₂₂N₇OS^þ[M þ H]^þ456.1601, found 456.1599, calcd for

C₂₄H₂₁N₇NaOS^þ[M þ Na]^þ478.1421, found 478.1418.

4.2.5. EC₅₀determination

The EC₅₀values were determined from full dose-response curves

for compounds tested ranging from 0.10 to 20.0 lM. HEK293T cells

were plated, transfected, and treated as above using Luciferase

Reporter Gene Assay. Then cells were treated with DMSO vehicle

(0.1%) or compounds tested (final concentration of 0.1, 0.3125,

0.625, 1.25, 2.5, 5.0, 10.0, and 20.0 lM) for 24 h. The amount of

rexinoid activity at each concentration was measured using the

same luciferase assay described above and EC₅₀values were

derived from dose-response curves of ligand concentration versus

normalised luciferase activity using Graph Pad Prism 5 software.

4.2. Bioassay

4.2.6. Western blot analysis

All the human cancer cell lines were purchased from the

American type culture collection (ATCC, Shanghai, China). Goat

antirabbit and anti-mouse secondary antibody conjugated to

horseradish peroxidase (HRP) was obtained from Thermo Fisher

Scientific (Waltham, MA), anti-rabbit IgG conjugated with Cy3

from Chemicon International (Temecula, CA), anti-RXRa (D-20, sc-

553) from Santa Cruz Biotechnology (Santa Cruz, CA), anti-PARP

(46D11, 9542) and anti-Clevage Caspase 3 (Asp175, 9661S) from

Cell Signalling Technology (Boston, MA), anti-a-tublin antibody

from Proteintech (Rosemont, IL), and JC-1 Probe (Cat. T3168) from

Thermo Fisher Scientific.

Equal amounts of cell lysates were boiled in sodium dodecyl sul-

phate (SDS) sample loading buffer, then separated through 10%

SDS-polyacrylamide gel electrophoresis and electro transferred to

nitrocellulose membranes. The membranes were blocked with 5%

non-fat milk in 10 mM TriseHCl (pH 8.0), 150 mM NaCl, 0.05%

Tween 20 (TBST) buffer for 1 h at room temperature. The mem-

branes were incubated overnight at 4 ^ꢃC, with specific primary anti-

bodies, after washing, followed by incubated with HRP-conjugated

anti-mouse or anti-rabbit secondary antibodies, immunoreactive

products were visualised with a chemiluminescence solution.

4.2.7. Generation of RXRa knock-out cell by CRISPR/Cas9 system

For RXRa genes, sgRNAs were designed based on the original Cas9

approach. Then RXRa sgRNA were colonised into gRNA cloning vec-

tor PX330 (add gene 71707) and confirmed by sequencing. The

cells were infected with control vector and gRNA expression vec-

tors for 24 h and selected single colonies using G418 (0.5 mg/mL).

The knockout cell was determined using western blot analysis

4.2.1. Cell culture and transfection

SW620, LS174T, HepG2, and A549 were maintained in Dulbecco’s

Modified Eagles Medium supplemented with 10% foetal bovine

serum (FBS), HeLa was maintained in Modified Eagles Medium

supplemented with 10% FBS and were cultured at 37 ^ꢃC, under

90% humidity with 5% carbon-di-oxide (CO₂).

4.2.2. Antiproliferative activity assay

4.2.8. SPR

The logarithmic phase of HepG2 and A549 cells were seeded in 96-

well plates at about 1 ꢁ 10⁴cells per well. treated with various con-

centrations of compounds for 24 h. then 15 lL MTT was added and

incubated for 4 h at 37^ꢃC and 5% CO₂. the light intensity was meas-

ured by microplate reader (PerkinElmer, Waltham, MA) at a wave-

length of 490 nm. Experiments were repeated three times. The

results were analysed by GraphPad Prism 5.01 software, La Jolla, CA.

BIAcore-T200 machine (GE Healthcare) was used to measure the

binding kinetics of compound 6A with RXRa-LBD, ERa-LBD, PPARc-

LBD, and TR3-LBD. Purified RXRa-LBD, ERa-LBD, PPARc-LBD, and

TR3-LBD were respectively immobilised on the CM5 chips accord-

ing to the manufacturer’s instructions. Then various concentrations

of 6A was injected into the flow cells in running buffer (phos-

phate buffer saline (PBS), 0.4% DMSO) at a flow rate of 30.0 lL/

min, when all cycle were collected, The results were analysed

using Biacore T200 Evaluation Software.

4.2.3. Luciferase reporter gene assay

HEK293T cells were cotransfected with pG5-luc reporter (50.0 ng)

and Pbind-RXRa-LBD (50.0 ng). One day after transfection, cells 4.2.9. Analysis of mitochondrial membrane potential

were treated with DMSO, compounds (1 lM) or 9-cis-RA (0.1 lM). For detection of apoptosis by FACS, A549 cells were incubated

After 12 h, cells were lysed and quantitated using the Dual- with 6A at 3.0 lM for 12 h. After treatment, the cells were har-

Luciferase Reporter Assay System (Thermo Fisher Scientific). vested and washed with PBS followed by stained with JC-1 for

Transfection and expression efficiency were normalised to Renilla 30 min at room temperature in the dark, Flow-cytometric analysis

luciferase activity.

was performed using FACS.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY

895

4.2.10. Immunofluorescence microscopy

7. Cesario RM, Stone J, Yen WC, et al. Differentiation and

growth inhibition mediated via the rxr: PPAR-gamma hetero-

dimer in colon cancer. Cancer Lett 2006;240:225–33.

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ploring their (patho)physiological functions. Cell Death Differ

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A549 cells were seeded on glass cover slips, incubated for 12 h in

the presence of compound 6A at a final concentration of 3.0 lM.

Cells mounted on glass slides were fixed in 4% paraformaldehyde

and permeabilised with PBS containing 0.1% Triton X-100 and

0.10 mol/l glycine for 10 min, and then blocked with 1% bovine

serum albumin in PBS for 30 min at room temperature, followed

with incubation with cleaved-caspase 3 antibodies (1:100) at room

temperature for 2 h, and detected by anti-rabbit IgG conjugated

with Cy3 (1:200) at room temperature for 2 h. Cells were contained

with 4’6⁰-diamidino-2-phenylindole (DAPI) (1:10,000) to visualise

nuclei. The images were analysed by using MRC-1024 MP laser-

scanning confocal microscope (Leica).

4.3. Molecular docking studies

The molecular docking studies were performed with the

Schrodinger 2015.1 Glide²⁶. The crystal structure of RXRa (PDB

code: 3A9E) were downloaded from the RCSB protein data bank

and prepared with Protein Preparation Wizard panel as imple-

mented in Maestro 10.5: water molecules were removed, missing

hydrogens and residues were added. The glide grid centre was

setting according the geometrical centre of original inhibitor

(LG100754) and the grid size was 20 ꢁ 20 ꢁ 20 Å³. Compound 6A

were prepared with LigPrep panel with OPLS2005 force field, gen-

erating the protonation and tautomeric states at 7 2 pH units²⁷.

Compound 6A was docked into the RXRa binding site using Glide

in XP mode. All of docking results were processed maestro 10.1

and PyMOL software.

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common transcriptional target of retinoid receptors:

Pleiotropic regulatory mechanism through retinoic acid

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p21WAF1/CIP1 by indenoisoquinolines in MCF7 cells. Cancer

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Disclosure statement

No potential conflict of interest was reported by the author(s).

16. Tunctan B, Kucukkavruk SP, Temiz-Resitoglu M, et al.

Bexarotene,

a

selective RXR alpha agonist, reverses

hypotension associated with inflammation and tissue injury

in a rat model of septic shock. Inflammation 2018;41:

337–55.

Funding

This work was supported by the National Natural Science

Foundation of China [No. 81773600], the Natural Science

Foundation of Fujian Province of China [No. 2018J01132], and the

Fundamental Research Funds for the Central Universities [No.

20720180051].

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bind RXR alpha and inhibit RXR alpha-dependent AKT sig-

naling. Cancer Cell 2010;17:560–73.

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and biological evaluation of potential retinoid X receptor

(RXR)-selective agonists: analogues of 4-[1-(3,5,5,8,8-pentam-

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Article Doi

Synthesis and biological evaluation of (3/4-(pyrimidin-2-ylamino)benzoyl)-based hydrazine-1-carboxamide/carbothioamide derivatives as novel RXRα antagonists

DOI: 10.1080/14756366.2020.1740692

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Full text of DOI:10.1080/14756366.2020.1740692

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