Novel 4-(2-pyrimidinylamino)benzamide derivatives as potent hedgehog signaling pathway inhibitors

Article abstract of DOI:10.1016/j.bmc.2018.08.037

A series of novel hedgehog signaling pathway inhibitors have been designed and synthesized based on our previously reported scaffold of 4-(2-pyrimidinylamino)benzamide. The Hh signaling pathway inhibitory activities were evaluated by Gli-luciferase reporter method and most compounds showed more potent inhibitory activities than vismodegib. Three compounds were picked out to evaluated in vivo for their PK properties, and compound 23b bearing a 2-pyridyl A-ring and (morpholin-4-yl)methylene at 3-position of D-ring demonstrated satisfactory PK properties. This study suggested the 4-(2-pyrimidinylamino)benzamides were a series of potent Hh signaling pathway inhibitors, deserving to further structural optimization.

Full text of DOI:10.1016/j.bmc.2018.08.037

Accepted Manuscript
Novel 4-(2-Pyrimidinylamino)benzamide Derivatives as Potent Hedgehog Sig-
naling Pathway Inhibitors
Minhang Xin, Liandi Zhang, Chongxing Tu, Feng Tang, Jun Wen
PII:
S0968-0896(18)31121-0
https://doi.org/10.1016/j.bmc.2018.08.037
BMC 14524
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
15 June 2018
28 August 2018
29 August 2018
Please cite this article as: Xin, M., Zhang, L., Tu, C., Tang, F., Wen, J., Novel 4-(2-Pyrimidinylamino)benzamide
Derivatives as Potent Hedgehog Signaling Pathway Inhibitors, Bioorganic & Medicinal Chemistry (2018), doi:
https://doi.org/10.1016/j.bmc.2018.08.037
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Novel 4-(2-Pyrimidinylamino)benzamide Derivatives as Potent Hedgehog Signaling
Pathway Inhibitors
Minhang Xin a, *, Liandi Zhang b, Chongxing Tu b, Feng Tang b, Jun Wen b
a Department of Medicinal Chemistry, School of Pharmacy, Health Science Center, Xi’an
Jiaotong University, No 76, Yanta West Road, Xi’an 710061, P.R. China
b
Jiangsu Simcere Pharmaceutical Co. Ltd., No 699-18, Xuan Wu District, Nanjing
210042, P.R. China
Footnotes
* Correspondence author: Minhang Xin, E-mail: xmhcpu@163.com
1

Abstract: A series of novel hedgehog signaling pathway inhibitors have been designed
and synthesized based on our previously reported scaffold of
4-(2-pyrimidinylamino)benzamide. The Hh signaling pathway inhibitory activities were
evaluated by Gli-luciferase reporter method and most compounds showed more potent
inhibitory activities than vismodegib. Three compounds were picked out to evaluated in
vivo for their PK properties, and compound 23b bearing a 2-pyridyl A-ring and
(morpholin-4-yl)methylene at 3-position of D-ring demonstrated satisfactory PK
properties. This study suggested the 4-(2-pyrimidinylamino)benzamides were a series of
potent Hh signaling pathway inhibitors, deserving to further structural optimization.
Keywords: hedgehog signaling pathway inhibitors, 4-(2-pyrimidinylamino)benzamide,
SAR, in vivo.
2

1. Introduction
Hedgehog (Hh) signaling plays an essential role in embryonic morphogenesis and
tissue patterning, and is generally silent in the adult. However, aberrantly constitutive
activation of Hh signaling was identified in the development of many tumors, such as
basal cell carcinoma (BCC), medulloblastoma (MB) and rhabdomysarcoma (RMS), and
hematological and solid tumors such as leukemia, lymphoma, glioblastoma, melanoma,
esophageal, lung, gastric, pancreatic, hepatocellular, colorectal, prostate, and ovarian
1, 2
cancer.
Thus, Hh signaling pathway emerged as an attractively therapeutic target for
treatment of cancers, and many small molecules were designed to block this Hh signaling
pathway. Vismodegib (GDC-0449, 1), the first-in-class FDA approved Hh inhibitor, was
very clinically efficient in treating locally advanced or metastatic BCC. Sonidegib
(LDE-225, 2), another FDA approved Hh inhibitor, also showed significant median
progress-free survival in the BCC clinical therapy. Other Hh inhibitors such as taladegib
(LY-2940680, 3), itraconazole (4), arsenic trioxide, were currently studies in clinical
trials (Figure 1). Although the proof-of-concept of Hh inhibitors were confirmed in BCC,
it is still believed Hh inhibitors have huge therapeutic potential against MB and RMS,
either by single or combination therapy. 3-5 Consequently, it is imperative to develop new
chemical structures of Hh inhibitors to further enable the medicinal investigation.
Figure 1. Representative chemical structures of reported Hh signaling inhibitors.
Our group was interested in developing new chemotypes of Hh inhibitors, and
3

recently a new series of 4-(2-pyrimidinylamino)benzamide derivatives were firstly
identified as potent Hh inhibitors, exemplified by compound A which inhibited the Hh
signaling pathway with IC50 value of 1.3 nM.6 Based on this skeleton of
4-(2-pyrimidinylamino)benzamide, a subsequent structure activity relationship was
7, 8, 9, 10
extensively investigated,
and several new scaffolds such as
11
pyrrolo[2,1-f][1,2,4]triazine, thieno[2,3-d]pyrimidines, furo[3,2-d]pyrimidines, purines,
12
13
and others were derivatized
.
In particular, in the series of
4-(2-pyrimidinylamino)benzamides, some representative analogues displayed potent Hh
inhibition and antitumor efficacy in vitro and in vivo, as well as satisfactory PK profiles
14
in rats. On the basis of these previous encouraging results, we intend to continuously
investigate 4-(2-pyrimidinylamino)benzamide core. In particular, it was previously
concluded that different substituents on A-ring, B-ring and D-ring could remarkably
affect the inhibitory activity of Hedgheg signaling pathway. Thus, a new series of
derivatives bearing the 4-(2-pyrimidinylamino)benzamide skeleton were synthesized,
mainly structural modification on A-ring, B-ring and D-ring. (Figure 2). Herein, we
reported
the
preparation
and
biological
evaluation
of
new
4-(2-pyrimidinylamino)benzamides as potent Hh inhibitors.
Figure 2. Current design of new 4-(2-pyrimidinylamino)benzamide derivatives
2. Chemistry
The title 4-(2-pyrimidinylamino)benzamides were prepared through the routes as
shown in Scheme 1, 2 and 3.
As depicted in Scheme 1, the intermediates 6a-6e were constructed by Suzuki coupling
reaction of the commercially available 5-methyl-2, 4-dichloropyrimidine (5a) and
4-trifluoromethoxyphenylboronic acid or phenylboronic acid, or 3-pyridylboronic acid
4

under the condition of Pd-catalyst and basic atmosphere in reflux dioxane, and 6f was
prepared by 5a and imidazole in the refluxing acetonitrile solution. Subsequently, 6a-6f
were treated with methyl 4-aminobenzoate by the Buchwald-Hartwig coupling condition
of Pd(OAc)2 catalyst and BINAP and Cs2CO3 to generate 7a-7f, which were hydrolyzed
by alkaline condition to afford free acids 8a-8f. Treatment of 8a with methyl
3-amino-4-methylbenzoate or 3-amino-4-methylphenol gave intermediates 9a-9b. 9a
o
directly reacted with 3-diethylaminopropylamine under 150 C microwave for 2h to
release compound 10a, whereas 9b was treated with 4-(2-chloroethyl)morpholine,
4-(3-chloropropyl)morpholine or 2-chloroethyl diethylamine to give compounds 10b-10d.
Similarly, acylation of 8b with 3-amino-4-methylbenzyl alcohol gave rise to 9e, which
was followed by substitution with 4-(2-chloroethyl)morpholine to produce 10e. (Scheme
1)
5

o
Scheme 1. Reagents and conditions: (a) Pd(PPh3)2Cl2, TEA, DMF/H2O, 80 C, 6-12h, 42-89 %, for
o
o
6a-6e; CH3CN, 65 C, 40h, 89 % for 6f; (b) Pd(OAc)2, BINAP, Cs2CO3, dioxane, reflux, 3-8h C,
43-94 %; (c) NaOH, MeOH/H2O, reflux overnight, 85-100 %; (d) SOCl2, TEA, DCM, overnight,
7-30 %; (e) Microwave, 150 oC, 2h, 51 %; (f) DMF, K2CO3, DMF, 2h, 80oC, 39-54 %.
The compounds 13a-13f were synthesized as shown in Scheme 2. The free acids 8b-8f
reacted with 3-amino-4-methylbenzyl alcohol to provide 11b-11f, which were
subsequently chlorinated by thionyl chloride to yield 12b-12f, followed by nucleophilic
substitution with piperidine, morpholine, N-methylpiperazine, or pyrrolidine to prepare
the desired product 13a-13f. (Scheme 2)
6

Scheme 2. Reagents and conditions: (a) HATU, DMF, DIPEA, rt, 16h, 45-80 %; (b) SOCl2, CH2Cl2, rt,
5h, 65-94 %; (c) CH3CN, K2CO3, reflux, 6h, 44-76 %.
As outlines in Scheme 3, the compounds 23a-23b were synthesized by alternative
route. Acylation of p-nitrobenzoic acid 14 with 3-amino-4-methylbenzyl alcohol
delivered intermediate 15. Subsequent chlorination of 15 provided 16, which was treated
with morpholine or N-methylpiperazine, followed by catalytic hydrogenation to afford
intermediate 18a-18b. Although 3-pyridyl substituted 6b was successfully prepared by
the Suzuki coupling reaction, it is difficult to construct 22 by similar Suzuki coupling
reaction of 2-pyridylboronic acid/ pinacol ester and 5a. After several reaction conditions
attempted, 22 was finally synthesized by Stille coupling reaction in 34 % yield based on
the firstly successful preparation of 2-(tributylstannyl)pyridine reagent in the presence of
n-BuLi. Accordingly, treatment of 22 with 18a-18b under Buchwald-Hartwig coupling
conditions produced the compounds 23a-23b. (Scheme 3)
7

Scheme 3. Reagents and conditions: (a) (1) SOCl2, reflux, 3h; (2) 3-amino-4-methylbenzyl alcohol,
THF, DIPEA, rt, 26h; 99 %; (b) SOCl2, CH2Cl2, rt, 2.5h, 100 %; (c) morpholine or 4-methylpiperazine,
DMF, K2CO3, 85 oC, 2.5h, 78-100 %; (d) 10 %Pd/C, H2, rt, 24h, 85-94 %; (e) n-BuLi, THF, -78 oC, 3h,
38%; (f)Toluene, Pd(PPh3)4, 150 oC, 2h, 34 %; (g) Pd(OAc)2, BINAP, Cs2CO3, dioxane, reflux, 2.5h ,
43-56%.
3. Result and discussion
3.1 Hh signaling inhibitory activity
The
Hh
signaling
inhibitory
activities
for
all
newly
prepared
4-(2-pyrimidinylamino)benzamides were evaluated by the luciferase reporter in NIH3T3
cell with a stably transfected Gli-reporter construct (Gli-luciferase reporter cell lines), and
vismodegib was used as positive control drugs. As shown in Table 1, all the
4-(2-pyrimidinylamino)benzamides displayed excellent inhibitory effects against Hh
signaling pathway with the IC50 values range from 0.62 nM to 28.99 nM, while
vismodegib showed an IC50 value of 7.17 nM. Compound 10a with an
N-(3-diethylamino)propylaminocarbonyl group at 3-postion of D-ring shown the most
8

potent inhibitory activity with an IC50 value of 0.62 nM, while compound 10b with a
(2-dimethylamino)ethoxy group showed retained inhibitory activity with an IC50 value of
1.07 nM. Replacement of the terminal diethylamino tail with morpholine group resulted
in compound 10c showing tolerable inhibition against Hh signaling pathway (IC50 = 2.45
nM). Extending the ethoxy linker to propoxy led to compound 10d also shown the
inhibitory effect remained (IC50 = 2.14 nM). The derivatives 10e and 13a with a phenyl
group in A-ring were investigated. Compound 10e tailed a 2-(morpholin-4-yl)ethoxy at
3-position of D-ring showing slightly lower inhibitory activity (IC50 = 4.28 nM)
compared to 10b, while compound 13a bearing a (piperidin-1-yl)methylene group
afforded reserved inhibitory activity (IC50 = 1.94 nM). Additionally, an attempt of
cyclopropyl or ethyl instead of the methyl group at 5-position of B ring led to the
preparation of representative compounds 13b, 13c and 13d. Although the cyclopropyl
analogue 13b linked the (morpholin-4-yl)methylene at D-ring showed descreased
inhibitory activity with an IC50 value of 11.95 nM, both the cyclopropyl analogue 13c and
the ethyl analogue 13d tailed the (4-methylpiperazin-1-yl)methylene demonstrated potent
activity with the IC50 value of 1.54 and 2.21 nM, respectively, compared with 13a.
Moreover, several representative heteroaryl groups as A-ring moiety were investigated,
exemplified by 13e-13f, and 23a-23b. Compound 13e with 3-pyridyl group and 13f with
1-imidazolyl group at A-ring showed lower inhibitory activity (IC50 = 28.99 and 21.11
nM,
respectively),
while
the
2-pyridyl
substituted
23a
attached
(4-methylpiperazin-1-yl)methylene group at 3-position of D-ring showed equivalent
potency compared with vismodegib and compound 23b attached the
(morpholin-4-yl)methylene group showed more potent inhibition. Building on the above
evaluation, this series of 4-(2-pyrimidinylamino)benzamide derivatives were certainly
potent Hh signaling pathway inhibitors, and most of them showed more potent inhibitory
activities than vismodegib.
Table 1. Hh signaling pathway inhibition of 4-(2-pyrimidinylamino)benzamide derivatives
9

R2
(A-ring)
Gli-luc reporter
IC50 (nM)
compds
R1
R3
Me
0.62±0.14
1.07±0.34
2.45±1.44
2.14±0.72
10a
Me
Me
Me
10b
10c
10d
Me
Me
4.28±1.60
10e
1.94±0.65
11.95±4.3
13a
13b
1.54±0.40
2.21±0.90
28.99±12.33
21.11±3.86
7.37±1.74
13c
13d
13e
13f
Et
H
Me
Me
23a
Me
-
1.89±0.50
7.17±2.18
23b
-
-
vismodegib
3.2 The calculated drug-like data for compounds
The rule of 5 is usually used to predict the drug-like properties of compounds.
Considering all the derivatives showed moderate to high potency, all compounds were
calculated for their molecular weights, numbers of H-donors, numbers of H-acceptors,
10

and ClogP values to examine their drug-like properties. The calculated data were shown
in Table 2. Obviously, only four compounds (including 13e, 13f, 23a and 23b) met the
rule of 5. In particularly, compound 13e and 23b showed the lowest ClogP values. (Table
2)
Table 2. The calculated data for compounds a
numbers of
H-donors
numbers of
H-acceptors
compds
10a
Molecular weight
ClogP
6.94
6.37
6.68
6.35
5.22
6.05
6.37
6.93
6.90
3.19
3.70
4.16
3.60
2.74
634
607
621
565
523
491
603
616
604
480
467
507
494
420
3
2
2
2
2
2
2
2
2
2
2
2
2
1
12
12
12
11
8
10b
10c
10d
10e
13a
6
13b
11
11
11
8
13c
13d
13e
13f
8
23a
8
23b
8
vismodegib
6
a The data were calculated by Chemdraw 14.0;
3.3 Pharmacokinetic (PK) profiles of selective compounds in vivo
Based on the Hh signaling pathway inhibitory activities and the calculated drug-like
datas of all the 4-(2-pyrimidinylamino)benzamide derivatives, compounds 13e and 23b
were selected to examine their pharmacokinetic (PK) profiles in vivo. In addition,
considering the structural diversity, compound 10e was also included for comparation.
The PK data were illustrated in Table 3, and three compounds showed different PK
11

parameters after administration to SD rats by intravenous injection (iv) with a dose of 1
mg/kg. compound 10e bearing a 2-(morpholin-4-yl)ethoxy substituent at 3- position of
D-ring showed a low peak-plasma-concentration (Cmax = 863 ng/mL) and low plasma
exposure (AUC = 787 hr×ng/mL) which means it may be administrated by a high dose as
so to afford in vivo antitumor efficacy, whereas compound 10e had a large volume of
distribution (Vz = 2745 mL/kg) because of the high lipophilicity (ClogP = 5.22). In
addition, compound 10e exhibited a clearance of 1288 mL/hr/kg and a half time of 1.44 h.
Unfortunately, although compound 13e possessed the lowest ClogP value, it showed the
lowest biggest area-under-curve (AUC = 521 hr×ng/mL) and half time (t1/2 = 0.25 h), and
higher clearance (Cl = 2016 mL/hr/kg) which means this compound may be easily
eliminated or metabolized. Compound 23b displayed satisfactory area-under-curve (AUC
= 1370 hr×ng/mL) and peak-plasma-concentration (Cmax = 1544 ng/mL), whose
clearance (Cl = 736 mL/hr/kg) and half time (t1/2 = 0.84 h) were moderate, although its
PK profiles were not better than vismodegib. In the light of the in vivo PK properties
evaluation, compound 23b was the preferable compound for further evaluation. (Table 3)
Table 3. Pharmacokinetic properties for represented compounds in SD rats a
AUC
(hr*ng/mL)
Vz
(mL/kg)
compds
10e
Cmax (ng/mL)
Cl (mL/hr/kg)
T1/2 (h)
863±166
2520±701
1544±211
1250±94
787±128
521±136
1370±174
2456±354
2745±1068
700±124
888±54
1288±230
2016±605
736±95
1.44±0.29
0.25±0.03
0.84±0.02
1.64±0.12
13e
23b
vismodegib
954±95
406±66
a compound (including GDC-0449) was formulated using 5%DMA+5% Tween-80, iv 1mg/kg;
4. Conclusion
A series of new 4-(2-pyrimidinylamino)benzamide derivatives have been designed and
synthesized. Their Hh signaling pathway inhibitory effects were evaluated and most
derivatives exhibited more potent Hh signaling inhibitory activity than vismodegib.
Subsequently, on base of the calculated drug-like data, three compounds were profiled in
vivo for their PK properties, and compound 23b bearing a 2-pyridyl A-ring and
(morpholin-4-yl)methylene at 3-position of D-ring afforded optimal PK properties. This
study suggested the 4-(2-pyrimidinylamino)benzamides were a series of potent Hh
12

signaling pathway inhibitors, deserving to further structural optimization for develop new
chemotype drug-candidate.
5. Experimental
5.1 General methods
13
1H-NMR spectra (400 MHz) and C-NMR (100 MHz) spectra were recorded on a
Bruker BioSpin AG (Ultrashield Plus AV 400) spectrometer. Mass spectra (MS) were
recorded on an Agilent technologies 6120 quadrupole LC/MS spectrometer. The purity of
the compounds was verified by the HPLC study using a mixture of solvent
methanol/water or acetonitrile/water at the flow rate of 2 mL/min and peak detection at
254 nm under UV. Column chromatography was carried out on silica gel (200–300 mesh).
All the reactions were monitored using thin layer chromatography (TLC) on silica gel
plates. All the reagents were purchased from commercial sources and used without
further purification unless especially stated.
5.1.1 4-((5-Methyl-4-(4-(trifluoromethoxy)phenyl)pyrimidin-2-yl)amino)benzoic acid
(8a)
It was synthesized as we recently described,6 as white product (720 mg, 56 %).
5.1.2 4-((5-Methyl-4-phenyl)pyrimidin-2-yl)amino)benzoic acid (8b)
It was prepared according to the method we recently reported, 6 as white product (100 mg,
87 %).
5.1.3
4-((5-Cyclopropyl-4-(4-(trifluoromethoxy)phenyl)pyrimidin-2-yl)amino)benzoic
acid (8c)
It was synthesized as we recently described 6, as white product (350 mg, 68 %).
5.1.4 4-((5-Ethyl-4-(4-(trifluoromethoxy)phenyl)pyrimidin-2-yl)amino)benzoic acid
(8d)
It was synthesized as we recently described 6, as white product (75 mg, 74 %).
5.1.5 4-((4-(Pyridin-3-yl)pyrimidin-2-yl)amino)benzoic acid (8e)
It was synthesized as we recently described 6, as yellow product (126 mg, 84 %).
5.1.6 2-Chloro-4-(1H-imidazol-1-yl)-5-methylpyrimidine (6f)
The solution of 5a (300 mg, 1.85 mmol) and 1H-imidazole (151 mg, 2.22 mmol) in
13

MeCN (10 mL) was heated to 65oC and refluxed for 40h. Then the mixture was cooled to
room temperature and concentrated. The residue was purified by chromatography
(EtOAc/petroleum ether, 5:1) to give 6f (319 mg, 89 %), MS (ESI) m/z: [M+H]+ = 195.1.
1H NMR (400M, DMSO-d6) δ 8.83 (s, 1H, ArH), 8.35 (s, 1H, ArH), 7.84-7.80 (m, 1H,
ArH), 7.19 (s, 1H, ArH), 2.44 (s, 3H, ArCH3) ppm.
5.1.7 4-((4-(1H-imidazol-1-yl)-5-methylpyrimidin-2-yl)amino)benzoic acid (8f)
It was synthesized as we described 6, as yellow product (484 mg, 68 %).
5.1.8 Methyl 4-methyl-3-(4-((5-methyl-4-(4-(trifluoromethoxy)phenyl)pyrimidin-2-yl)
amino) benzamido)benzoate (9a)
A solution of 8a (200 mg, 0.51 mmol) in SOCl2 (5 mL) was refluxed for 30 min, then
the mixture was concentrated to afford acyl chloride intermediate. The residue was
dissolved in drug DCM (5 mL) in ice bath, and methyl 3-amino-4-mehtylbenzoate (128
mg, 0.75mmol) and TEA (150mg, 1.53 mmol) were added. The mixture was warmed to
room temperature, and stirred overnight. The reaction mixture was concentrated, and the
residue was purified by chromatography (DCM/ MeOH, 80:1) to give compound 9a (161
mg, 58 %) as white solid. MS (ESI) m/z: [M+H]+ = 537.2. 1H NMR (400M, DMSO-d6) δ
10.00 (s, 1H, NH), 9.79 (s, 1H, NH), 8.53 (s, 1H, ArH), 8.01 (s, 1H, ArH), 7.94 (s, 4H,
ArH), 7.88 (d, 2H, J = 8.4 Hz, ArH), 7.75 (d, 1H, J = 8.0Hz, ArH), 7.55 (d, 2H, J = 8.0
Hz, ArH), 7.43 (d, 1H, J = 8.0 Hz, ArH), 3.85 (s, 3H, OCH
(s, 3H, ArCH ) ppm.
3
), 2.32 (s, 3H, ArCH ), 2.26
3
3
5.1.9 N-(3-(Diethylamino)propyl)-4-methyl-3-(4-((5-methyl-4-(4-(trifluoromethoxy)
phenyl)pyrimidin-2-yl)amino)benzamido)benzamide (10a)
A mixture of 9a (160 mg, 0.3 mmol) and N, N-diethyl-1, 3-propyldiamine (153 mg, 1.5
o
mmol) in the tube were stirred at 150 C by microwave machine, then the mixture was
concentrated and purified by chromatography (DCM/ MeOH, 50:1) to give compound
1
10a (46 mg, 25 %) as white solid. MS (ESI) m/z: [M+H]+ = 635.2. H NMR (400M,
DMSO-d6) δ 9.99 (s, 1H, CONH), 9.79 (s, 1H, NH), 8.53 (s, 2H, ArH), 7.94 (s, 4H, ArH),
7.88 (d, 2H, J = 8.4 Hz, ArH), 7.82 (s, 1H, ArH), 7.64 (d, 2H, J = 8.0 Hz, ArH), 7.55 (d,
2H, J = 8.4 Hz, ArH), 7.36 (d, 1H, J = 7.6 Hz, ArH), 3.32-3.24 (m, 2H, NCH
(m, 6H, CH N(CH ), 2.27 (s, 3H, ArCH ), 2.26 (s, 3H, ArCH ), 1.63 (m, 2H, CH
0.99-0.89 (m, 6H, (CH ) ppm, HPLC: 94.39 %.
2
), 2.48-2.40
2
2
)
2
3
3
2
),
3)2
14

5.1.10
N-(5-Hydroxy-2-methylphenyl)-4-((5-methyl-4-(4-(trifluoromethoxy)phenyl)pyrimid
in-2-yl)amino)benzamide (9b)
9b was synthesized by the similar preparation of 9a, as white solid (45 mg, 14 %). MS
(ESI) m/z: [M+H]+ = 495.2. 1H NMR (400M, DMSO-d6) δ 9.98 (s, 1H, CONH), 9.49 (s,
1H, NH), 9.25 (s, 1H, PhOH), 8.52 (s, 1H, ArH), 7.91 (s, 4H, ArH), 7.86 (d, 2H, J = 8.4
Hz, ArH), 7.54 (d, 2H, J = 8.4 Hz, ArH), 7.02 (d, 1H, J = 8.4 Hz, ArH), 6.84 (d, 1H, J =
2.0 Hz, ArH), 6.56 (dd, 1H, J = 8.0, 2.4 Hz, ArH), 2.25 (s, 3H, ArCH ), 2.21 (s, 3H,
3
ArCH ) ppm.
3
5.1.11
N-(5-(2-(Dimethylamino)ethoxy)-2-methylphenyl)-4-((5-methyl-4-(4-(trifluorometho
xy)phenyl)pyrimidin-2-yl)amino)benzamide (10b)
To a solution of 8a (58 mg, 0.12 mmol) in DMF (5 mL), K2CO3 (24 mg, 1.8 mmol)
and N, N-dimethyl-2-chloroethylamine hydrochloride (17 mg, 0.12 mmol) were added.
The mixture was refluxed at 80 oC for 2h. The reaction mixture was concentrated and
purified by chromatography (DCM/ MeOH, 50:1) to give compound 10b (31 mg, 54 %)
1
as white solid. MS (ESI) m/z: [M+H]+ = 566.3. H NMR (400M, DMSO-d6) δ 9.99 (s,
1H, CONH), 9.58 (s, 1H, NH), 8.52 (s, 1H, ArH), 7.92 (s, 4H, ArH), 7.87 (d, 2H, J = 8.0
Hz, ArH), 7.55 (d, 2H, J = 8.0 Hz, ArH), 7.15 (d, 1H, J = 8.4 Hz, ArH), 7.00 (s, 1H,
ArH), 6.76 (d, 1H, J = 8.0 Hz, ArH), 4.03 (s, 2H, J = 9.2 Hz, OCH
2
), 2.70-2.64 (m, 2H, J
= 5.2 Hz, NCH
2
), 2.26 (s, 9H, N(CH
3)2
+ArCH
3
), 2.16 (s, 3H, ArCH ) ppm. HPLC:
3
92.51 %.
5.1.12
4-((5-Methyl-4-(4-(trifluoromethoxy)phenyl)pyrimidin-2-yl)amino)-N-(2-methyl-5-(2
-morpholinoethoxy)phenyl)benzamide (10c)
10c was synthesized by the similar preparation of 10a, as white solid (17 mg, 47 %).
1
MS (ESI) m/z: [M+H]+ = 608.3. H NMR (400M, DMSO-d6) δ 10.00 (s, 1H, CONH),
9.65 (s, 1H, NH), 8.52 (s, 1H, ArH), 7.96 (m, 4H, ArH), 7.87 (d, 2H, J = 8.4 Hz, ArH),
7.55 (d, 2H, J = 8.4 Hz, ArH), 7.15 (d, 1H, J = 8.4 Hz, ArH), 7.00 (s, 1H, ArH), 6.76 (d,
1H, J = 8.0 Hz, ArH), 4.09-4.04 (m, 2H, OCH ), 3.93-3.84 (m, 4H, morpholine-H), 2.71
2
(m, 2H, NCH
2
), 2.54-2.47 (m, 4H, morpholine-H), 2.26 (s, 3H, ArCH ), 2.16 (s, 3H,
3
15

ArCH3) ppm. HPLC: 95.04%.
5.1.13
4-((5-Methyl-4-(4-(trifluoromethoxy)phenyl)pyrimidin-2-yl)amino)-N-(2-methyl-5-(3
-morpholinopropoxy)phenyl)benzamide (10d)
10d was synthesized by the similar preparation of 10a, as white solid (27 mg, 42 %).
1
MS (ESI) m/z: [M+H]+ = 622.3. H NMR (400M, MeOD) δ 8.43 (s, 1H, ArH), 7.92 (s,
4H, ArH), 7.84 (d, 2H, J = 8.4 Hz, ArH), 7.44 (d, 2H, J = 8.4 Hz, ArH), 7.17 (d, 1H, J =
8.4 Hz, ArH), 7.00 (d, 1H, J = 2.4 Hz, ArH), 6.78 (dd, 1H, J = 8.4, 2.4 Hz, J = 6.0 Hz,
ArH), 4.03 (t, 2H, J = 4.4 Hz, OCH
2
), 3.73 (t, 4H, J = 4.4 Hz, CH
2
OCH
2
), 2.68-2.60 (m,
) ppm.
6H, N(CH ), 2.31 (s, 3H, ArCH ), 2.22 (s, 3H, ArCH
2
)3
3
3), 2.04-1.96 (m, 2H, CH
2
HPLC: 97.06%.
5.1.14
4-((5-Methyl-4-phenylpyrimidin-2-yl)amino)-N-(2-methyl-5-(2-morpholinoethoxy)p
henyl)benzamide (10e)
10e was synthesized by the similar preparation of 10a, as white solid (50 mg, 39 %).
1
MS (ESI) m/z: [M+H]+ = 524.3. H NMR (400M, DMSO-d6) δ 9.95 (s, 1H, CONH),
9.55 (s, 1H, NH), 8.50 (s, 1H, ArH), 7.95 (m, 4H, ArH), 7.72 (d, 2H, J = 7.2 Hz, ArH),
7.59-7.53 (m, 3H, ArH), 7.15 (d, 1H, J = 8.4 Hz, ArH), 7.01 (d, 1H, J = 2.4 Hz, ArH),
6.76 (dd, 1H, J = 8.4, 2.4 Hz, ArH), 4.07 (t, 2H, J = 5.2 Hz, OCH
Hz, morpholine-H), 2.69 (t, 2H, J = 5.2 Hz, NCH ), 2.54-2.46 (m, 4H, morpholine-H),
) ppm. HPLC: 96.28 %.
2), 3.59 (t, 4H, J = 4.4
2
2.26 (s, 3H, ArCH
3
), 2.16 (s, 3H, ArCH
3
5.1.15
4-((5-Methyl-4-phenylpyrimidin-2-yl)amino)-N-(2-methyl-5-(piperidin-1-ylmethyl)p
henyl)benzamide (13a)
13a was synthesized by the similar preparation of 13b, as white solid (22 mg, 76 %).
MS (ESI) m/z: [M+H]+ = 492.3. 1H NMR (400M, DMSO-d6) δ 9.95 (s, 1H, CONH), 9.64
(s, 1H, NH), 8.50 (s, 1H, ArH), 7.96-7.90 (m, 4H, ArH), 7.72 (d, 2H, J = 7.2 Hz, ArH),
7.59-7.55 (m, 3H, ArH), 7.29-7.23 (m, 2H, ArH), 7.13-7.07 (m, 1H, ArH), 3.34 (s, 2H,
PhCH
2
N), 2.37-2.30 (m, 4H, piperidine-H), 2.26 (s, 3H, ArCH
3
), 2.21 (s, 3H, ArCH ),
3
1.54-1.47 (m, 4H, piperidine-H), 1.28-1.20 (m, 2H, piperidine-H) ppm. HPLC: 97.79 %.
5.1.16
16

4-((5-Cyclopropyl-4-(4-(trifluoromethoxy)phenyl)pyrimidin-2-yl)amino)-N-(5-(hydr
oxymethyl)-2-methylphenyl)benzamide (11c)
To a solution of 8c (150 mg, 0.36 mmol) and 3-amino-4-methylbenzyl alcohol (85 mg,
0.62 mmol) in DMF (10mL), HATU (274 mg, 0.72 mmol) and DIPEA (130 mg, 1.1
o
mmol) were added, and then the mixture was warmed at 80 C and stirred for 15 h. The
reaction mixture was poured into ice water (50 mL) followed by extraction with EtOAc.
The organic layer was dried by Na2SO4 and concentrated. The residue was purified by
chromatography (DCM/ MeOH, 80:1) to generate 11c (160 mg, 82 %) as a white solid,
MS (ESI) m/z: [M+H]+ = 535.2. 1H-NMR (400 M, DMSO-d6) δ 10.02 (s, 1H, CONH),
9.66 (s, 1H, NH), 8.40 (s, 1H, ArH), 8.01 (d, 2H, J = 8.4 Hz, ArH), 7.94 (s, 4H, ArH),
7.55 (d, 2H, J = 8.4 Hz, ArH), 7.30 (s, 1H, ArH), 7.22 (d, 1H, J = 8.4 Hz, ArH), 7.11 (d,
1H, J = 8.4 Hz, ArH), 5.21-5.15 (m, 1H, OH), 4.49 (s, 2H, ArCH2O), 2.21 (s, 3H, ArCH
3
),
1.94-1.88 (m, 1H, ArCH), 0.94-0.85 (m, 2H, CH
2
), 0.75-0.67 (m, 2H, CH ) ppm.
2
5.1.17
4-((5-Cyclopropyl-4-(4-(trifluoromethoxy)phenyl)pyrimidin-2-yl)amino)-N-(5-(chlor
omethyl)-2-methylphenyl)benzamide (12c)
To a solution of 11c (110 mg, 0.2 mmol) in anhydrous DCM (20 mL), SOCl2 (60 mg,
0.4 mmol) was added, and then the mixture was stirred at room temperature for 15 h. The
reaction mixture was concentrated and purified by chromatography (DCM/ MeOH, 80:1)
to generate 11c (86 mg, 76 %) as a yellow solid, MS (ESI) m/z: [M+H]+ = 553.2.
1H-NMR (400 M, DMSO-d6) δ 10.04 (s, 1H, CONH), 9.73 (s, 1H, NH), 8.40 (s, 1H,
ArH), 8.01 (d, 2H, J = 8.4 Hz, ArH), 7.94 (s, 4H, ArH), 7.57 (d, 2H, J = 8.4 Hz, ArH),
7.44 (s, 1H, ArH), 7.30 (d, 1H, J = 8.4 Hz, ArH), 7.24 (d, 1H, J = 8.4 Hz, ArH), 4.77 (s,
2H, ArCH
2
Cl), 2.24 (s, 3H, ArCH
3
), 1.94-1.88 (m, 1H, ArCH), 0.94-0.85 (m, 2H, CH ),
2
0.75-0.67 (m, 2H, CH ) ppm.
2
5.1.18
4-((5-Cyclopropyl-4-(4-(trifluoromethoxy)phenyl)pyrimidin-2-yl)amino)-N-(2-methy
l-5-(morpholinomethyl)phenyl)benzamide (13b)
To a solution of 12c (86 mg, 0.15 mmol) in anhydrous MeCN (20 mL), morpholine (20
mg, 0.22 mmol) and K2CO3 were added, and then the mixture was refluxed for 6 h. The
reaction mixture was filtered, concentrated and purified by chromatography (DCM/
17

MeOH, 80:1) to generate 13b (18 mg, 21 %) as a white solid, MS (ESI) m/z: [M+H]+ =
604.3. 1H-NMR (400 M, DMSO-d6) δ 10.01 (s, 1H, CONH), 9.66 (s, 1H, NH), 8.40 (s,
1H, ArH), 8.00 (d, 2H, J = 8.4 Hz, ArH), 7.93 (s, 1H, ArH), 7.57 (d, 2H, J = 8.0 Hz,
ArH), 7.29 (s, 1H, ArH), 7.23 (d, 1H, J = 7.6 Hz, ArH), 7.10 (d, 1H, J = 7.2 Hz, ArH),
3.59-3.54 (m, 4H, morpholine-H), 3.37 (s, 2H, ArCH
2
N), 2.39-2.34 (m, 4H,
),
morpholine-H), 2.24 (s, 3H, ArCH3), 1.94-1.88 (m, 1H, CH), 0.94-0.85 (m, 2H, CH
2
0.75-0.67 (m, 2H, CH ) ppm. HPLC: 96.79 %.
2
5.1.19
4-((5-Cyclopropyl-4-(4-(trifluoromethoxy)phenyl)pyrimidin-2-yl)amino)-N-(2-methy
l-5-((4-methylpiperazin-1-yl)methyl)phenyl)benzamide (13c)
13c was synthesized by the similar prepareation of 13b (45 mg, 53 %) as a yellow
solid, MS (ESI) m/z: [M+H]+ = 617.3. 1H-NMR (400 M, DMSO-d6) δ 10.00 (s, 1H,
CONH), 9.63 (s, 1H, NH), 8.39 (s, 1H, ArH), 7.99 (d, 2H, J = 7.6 Hz, ArH), 7.92 (s, 1H,
ArH), 7.56 (d, 2H, J = 8.4 Hz, ArH), 7.28 (s, 1H, ArH), 7.21 (d, 1H, J = 8.0 Hz, ArH),
7.08 (d, 1H, J = 7.6 Hz, ArH), 3.45 (s, 2H, ArCH
2.20 (s, 3H, ArCH3), 1.94-1.87 (m, 1H, CH), 0.92-0.88 (m, 2H, CH
CH ) ppm. HPLC: 96.19 %.
5.1.20
2
N), 2.44-2.36 (m, 8H, piperazine-H),
2), 0.74-0.67 (m, 2H,
2
4-((5-Ethyl-4-(4-(trifluoromethoxy)phenyl)pyrimidin-2-yl)amino)-N-(2-methyl-5-((4-
methylpiperazin-1-yl)methyl)phenyl)benzamide (13d)
13d was synthesized by the similar prepareation of 13b (28 mg, 25 %) as a yellow
1
solid, MS (ESI) m/z: [M+H]+ = 605.3. H-NMR (400 M, DMSO-d6) δ 10.01 (s, 1H,
CONH), 9.63 (s, 1H, NH), 8.57 (s, 1H, ArH), 7.94 (d, 2H, J = 7.6 Hz, ArH), 7.78 (s, 1H,
ArH), 7.55 (d, 2H, J = 8.4 Hz, ArH), 7.26 (s, 1H, ArH), 7.20 (d, 1H, J = 8.0 Hz, ArH),
7.07 (d, 1H, J = 7.6 Hz, ArH), 3.57 (s, 2H, ArCH
2.54-2.48 (m, 8H, piperazine-H), 2.35 (s, 3H, CH3), 2.16 (s, 3H, CH3), 1.18-1.12 (m, 2H,
CH ) ppm. HPLC: 90.76 %.
2
N), 2.63-2.58 (m, 2H, ArCH ),
2
2
5.1.21
N-(2-Methyl-5-(morpholinomethyl)phenyl)-4-((4-(pyridin-3-yl)pyrimidin-2-yl)amino
)benzamide (13e)
13e was synthesized by the similar prepareation of 13b (20 mg, 44 %) as a yellow
18

1
solid, MS (ESI) m/z: [M+H]+ = 481.2. H-NMR (400 M, DMSO-d6) δ 10.16 (s, 1H,
CONH), 9.73 (s, 1H, NH), 9.39 (d, 1H, J = 2.0 Hz, ArH), 8.76 (dd, 1H, J = 8.8, 1.6 Hz,
ArH), 8.70 (d, 1H, J = 5.2 Hz, ArH), 8.56 (d, 1H, J = 8.4 Hz, ArH), 7.98 (s, 4H, ArH),
7.67-7.62 (m, 2H, ArH), 7.29 (s, 1H, ArH), 7.23 (d, 1H, J = 7.6 Hz, ArH), 7.13-7.07 (m,
1H, J = 7.6 Hz, ArH), 3.59-3.55 (m, 4H, morpholine-H), 3.44 (s, 2H, ArCH2), 2.39-2.33
(m, 4H, morpholine-H), 2.22 (s, 3H, ArCH3) ppm. HPLC: 98.30 %.
5.1.22
4-((4-(1H-imidazol-1-yl)-5-methylpyrimidin-2-yl)amino)-N-(2-methyl-5-(pyrrolidin-
1-ylmethyl)phenyl)benzamide (13f)
Similar procedure of 13b was performed to give compound 13f (62 mg, 46 %) as a
1
yellow solid, MS (ESI) m/z: [M+H]+ = 468.3. H-NMR (400 M, DMSO-d6) δ 10.14 (s,
1H, CONH), 9.68 (s, 1H, NH), 8.61 (s, 1H, ArH), 8.35 (s, 1H, ArH), 7.96 (d, 2H, J = 8.4
Hz, ArH), 7.87 (d, 2H, J = 8.8 Hz, ArH), 7.82 (s, 1H, ArH), 7.28 (s, 1H, ArH), 7.19 (d,
2H, J = 6.8 Hz, ArH), 7.07 (d, 1H, J = 7.6 Hz, ArH), 3.54 (s, 2H, ArCH2N), 2.42 (s, 4H,
N(CH2)2), 2.33 (s, 3H, ArCH3), 2.21 (s, 3H, ArCH3), 1.69 (s, 4H, (CH2)2) ppm. HPLC:
98.62 %.
5.1.234-Amino-N-(2-methyl-5-((4-methylpiperazin-1-yl)methyl)phenyl)benzamide
(18a)
11
It was synthesized as we recently described , as white product (200 mg, 85 %), MS
(ESI) m/z: [M+H]+ = 339.2. 1H-NMR (400 M, DMSO-d6) δ 9.34 (s, 1H, NH), 7.70 (d,
2H, J = 8.4 Hz, ArH), 7.24 (s, 1H, ArH), 7.15 (d, 1H, J = 7.6 Hz, ArH), 7.04 (d, 1H, J =
7.6 Hz, ArH), 6.58 (d, 2H, J = 8.4 Hz, ArH), 5.70 (s, 2H, NH2), 3.40 (s, 2H, CH2),
2.36-2.30 (m, 8H, piperazine-H), 2.17 (s, 3H, CH3), 2.14 (s, 3H, CH3) ppm.
5.1.24 4-Amino-N-(2-methyl-5-(morpholinomethyl)phenyl)benzamide (18b)
Similar procedure of 18a was performed to give compound 18b (78 mg, 94 %), MS
(ESI) m/z: [M+H]+ = 326.2. 1H-NMR (400 M, DMSO-d6) δ 9.35 (s, 1H, NH), 7.71 (d,
2H, J = 8.4 Hz, ArH), 7.26 (s, 1H, ArH), 7.18 (d, 1H, J = 7.6 Hz, ArH), 7.05 (d, 1H, J =
7.6 Hz, ArH), 6.60 (d, 2H, J = 8.4 Hz, ArH), 5.71 (s, 2H, NH2), 3.59-3.53 (m, 4H,
CH2*2), 3.41 (s, 2H, CH2), 2.35 (s, 4H, CH2*2), 2.18 (s, 3H, CH3) ppm.
5.1.25 2-Chloro-5-methyl-4-(pyridin-2-yl)pyrimidine (22)
It was synthesized similar to the procedure as we recently described 11, as yellow solid
19

(150 mg, 34 %), MS (ESI) m/z: [M+H]+ = 206. 1H-NMR (400 M, CDCl3) δ 8.72 (d, 1H,
J = 4.0 Hz, ArH), 8.55 (s, 1H, ArH), 8.10 (d, 2H, J = 8.0 Hz, ArH), 7.90-7.86 (m, 1H,
ArH), 7.42-7.39 (m, 1H, ArH), 2.60 (s, 3H, CH3) ppm.
5.1.26
4-((5-methyl-4-(pyridin-2-yl)pyrimidin-2-yl)amino)-N-(2-methyl-5-(4-methylpiperaz
in-1-ylmethyl)phenyl)benzamide (23a)
6
It was synthesized similar to the procedure as we recently described , as yellow solid
(23 mg, 72 %), MS (ESI) m/z: [M+H]+ = 508.3.1H-NMR (400M, CDCl3) δ 8.75 (d, 1H, J
= 4.0 Hz, ArH), 8.43 (s, 1H, CONH), 8.00-7.90 (m, 2H, ArH), 7.88 (d, 2H, J = 8.0 Hz,
ArH), 7.82 (d, 2H, J = 8.0 Hz, ArH), 7.64 (s, 1H, NH), 7.41-7.39 (m, 2H, ArH), 7.19 (d,
1H, J = 8.0 Hz, ArH), 7.08 (d, 1H, J = 8.0 Hz, ArH), 3.55 (s, 2H, NCH2Ph), 2.80-2.60 (m,
8H, piperazine-H), 2.48 (s, 3H, ArCH3), 2.39(s, 3H, NCH3), 2.33 (s, 3H, ArCH3) ppm,
HPLC：98 %.
5.1.27
4-((5-methyl-4-(pyridin-2-yl)pyrimidin-2-yl)amino)-N-(2-methyl-5-(morpholinometh
yl)phenyl)benzamide (23b)
It was synthesized as we recently described 6, as yellow solid (60 mg, 43 %), MS (ESI)
m/z: [M+H]+ = 495.3.1H-NMR (400M,CDCl3) δ 8.75 (d, 1H, J = 4.0 Hz, ArH), 8.44 (s,
1H, CONH), 7.99 (d 1H, J = 8.0 Hz, ArH), 7.95 (s, 1H, NH), 7.92-7.87 (m, 3H, ArH),
7.82 (d, 2H, J = 8.0 Hz, ArH), 7.63 (s, 1H, ArH), 7.41-7.38 (m, 2H, ArH), 7.19 (d, 1H, J
= 8.0 Hz, ArH), 7.09 (d, 1H, J = 8.0 Hz, ArH), 3.72-3.70 (m, 4H, morpholine-H), 3.50 (s,
2H, NCH2Ph), 2.50-2.43 (s, 7H, morpholine-H+ArCH3), 2.33 (s, 3H, ArCH3) ppm,
HPLC: 97 %.
5.2 In vitro Gli-luciferase reporter assay to evaluate the Hh signaling pathway
inhibitory activity
The Hh signaling pathway inhibitory activity for all the target compounds were used a
luciferase reporter assay in NIH3T3 cells carrying a stably transfected Gli-reporter
construct (Gli-luc reporter cell line), which were similar to our previous manuscript.6
Firstly, the NIH3T3/Gli-luc cells were treated with DMEM and 10% FBS and 1μg/mL
Puromycin. Secondly, The cells were seeded onto 96-well plates at 2×104cells/well and
cultured in the condition of 5% CO2 and 37 oC overnight. Thirdly, after incubation, all the
20

prepared compounds (including GDC-0449, as an internal standard control) diluted in a
serial 8×solution (0.05-300nM) containing 0.5% FBS and 0.7μg/mL Sonic Hh agonist
were added to each well (n = 4 wells per concentration). The cells were incubated for an
additional 48 h. Cells were incubated in media containing 0.1% DMSO with or without
Sonic Hh (0% or 100% inhibition control) respectively. Lastly, cells were then harvested
and lysed in reporter lysis buffer, and luciferase activities were measured using a
Dual-Luciferase® Reporter Assay System (Promega E1910). The activity of the Gli
reporter was defined as the ratio of Firefly/Renilla luciferase activities.
5.3 Pharmacokinetic Studies of the selected compounds in SD rats
The pharmacokinetic studies of the selected compounds were evaluated according to
the procedure of our previous report. 6 Compounds were administered to 3 male SD rats
(weight ranging from 180 g to 240 g) by a dose of 1 mg/kg in iv administration, and the
dosing volume was 5 mL/kg. After administration, blood samples were collected at the
point including 2 min, 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, and 24 h for
analyses, the collected blood samples were centrifuged at 4000 rpm for 5 min at 4 oC, and
then analyzed after protein precipitation. LC/MS/MS analysis of compounds were
performed under optimized conditions to obtain the best sensitivity and selectivity of the
analyte in selected reaction monitoring mode (SRM) containing an internal standard.
Plasma concentration-time data were measured by a noncompartmental approach using
the software WinNonlin Enterprise, version 5.2.
Acknowledgements
The authors thank the excellent works from the scientists in the Drug Discovery
Institute of Jiangsu Simcere Pharmaceutical Co. Ltd. This work was partly supported by
Shaanxi Province Postdoctoral Science Foundation.
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2013;23:6777-6783.
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Novel 4-(2-Pyrimidinylamino)benzamide Derivatives as Potent Hedgehog Signaling
Pathway Inhibitors
Minhang Xin a, *, Liandi Zhang b, Chongxing Tu b, Feng Tang b, Jun Wen b
a Department of Medicinal Chemistry, School of Pharmacy, Health Science Center, Xi’an
Jiaotong University, No 76, Yanta West Road, Xi’an 710061, P.R. China
b
Jiangsu Simcere Pharmaceutical Co. Ltd., No 699-18, Xuan Wu District, Nanjing
210042, P.R. China
Graphical Abstract
22

Footnotes
* Correspondence author: E-mail: xmhcpu@163.com
23