Discovery of 2-((3-Amino-4-methylphenyl)amino)-N-(2-methyl-5-(3-(trifluoromethyl)benzamido)phenyl)-4-(methylamino)pyrimidine-5-carboxamide (CHMFL-ABL-053) as a Potent, Selective, and Orally Available BCR-ABL/SRC/p38 Kinase Inhibitor for Chronic Myeloid Leukemia

Article abstract of DOI:10.1021/acs.jmedchem.5b01618

Starting from a dihydropyrimidopyrimidine core scaffold based compound 27 (GNF-7), we discovered a highly potent (ABL1: IC₅₀ of 70 nM) and selective (S score (1) = 0.02) BCR-ABL inhibitor 18a (CHMFL-ABL-053). Compound 18a did not exhibit apparent inhibitory activity against c-KIT kinase, which is the common target of currently clinically used BCR-ABL inhibitors. Through significant suppression of the BCR-ABL autophosphorylation (EC₅₀ about 100 nM) and downstream mediators such as STAT5, Crkl, and ERK's phosphorylation, 18a inhibited the proliferation of CML cell lines K562 (GI₅₀ = 14 nM), KU812 (GI₅₀ = 25 nM), and MEG-01 (GI₅₀ = 16 nM). A pharmacokinetic study revealed that 18a had over 4 h of half-life and 24% bioavailability in rats. A 50 mg/kg/day dosage treatment could almost completely suppress tumor progression in the K562 cells inoculated xenograft mouse model. As a potential useful drug candidate for CML, 18a is under extensive preclinical safety evaluation now.

Full text of DOI:10.1021/acs.jmedchem.5b01618

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Article
Discovery of 2-((3-amino-4-methylphenyl)amino)-N-(2-methyl-5-(3-
trifluoromethyl)benzamido)phenyl)-4-(methylamino)pyrimidine-5-
(
carboxamide (CHMFL-ABL-053) as a potent, selective and orally available
BCR-ABL/SRC/p38 kinase inhibitor for Chronic Myeloid Leukemia
Xiaofei Liang, Xiaochuan Liu, Beilei Wang, Fengming Zou, Aoli Wang,
Shuang Qi, Cheng Chen, Zheng Zhao, Wenchao Wang, Ziping Qi, Fengchao
Lv, Zhenquan Hu, Li Wang, Shanchun Zhang, Qingsong Liu, and Jing Liu
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01618 • Publication Date (Web): 20 Jan 2016
Downloaded from http://pubs.acs.org on January 21, 2016
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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

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Discovery of 2ꢀ((3ꢀaminoꢀ4ꢀmethylphenyl)amino)ꢀ
Nꢀ(2ꢀmethylꢀ5ꢀ(3ꢀ
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(trifluoromethyl)benzamido)phenyl)ꢀ4ꢀ
(
methylamino)pyrimidineꢀ5ꢀcarboxamide (CHMFLꢀ
ABLꢀ053) as a potent, selective and orally available
BCRꢀABL/SRC/p38 kinase inhibitor for Chronic
Myeloid Leukemia
1,2,7
1,3,7
1,2,7
1,2,7
1,4
Xiaofei Liang , Xiaochuan Liu , Beilei Wang , Fengming Zou , Aoli Wang , Shuang
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,2
1,2
1,2
1,2
1,2
1,4
Qi , Cheng Chen , Zheng Zhao , Wenchao Wang , Ziping Qi , Fengchao Lv , Zhenquan
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,2
1,2
2,5
1,2,4,6*
1,2*
Hu , Li Wang , Shanchun Zhang , Qingsong Liu
, Jing Liu
1. High Magnetic Field Laboratory, Chinese Academy of Sciences, Mailbox 1110, 350
Shushanhu Road, Hefei, Anhui, P. R. China, 230031
2. CHMFLꢀHCMTC Target Therapy Joint Laboratory, 350 Shushanhu Road, Hefei, Anhui, P.
R. China, 230031
3. Department of Chemistry, University of Science and Technology of China, Hefei, Anhui, P.
R. China, 230036
4. University of Science and Technology of China, P. R. China, Anhui, Hefei, P. R. China,
2
30036
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. Hefei Cosource Medicine Technology Co. LTD., 358 Ganquan Road, Hefei, Anhui, P. R.
China, 230031
6
. Hefei Science Center, Chinese Academy of Sciences, 350 Shushanhu Road, Hefei, Anhui, P.
R. China, 230031
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7. These authors contribute equally
ABSTRACT
Starting from a dihydropyrimidopyrimidine core scaffold based compound 27 (GNFꢀ7), we
discovered a highly potent (ABL1: IC of 70 nM) and selective (S score (1) = 0.02) BCRꢀABL
5
0
inhibitor 18a (CHMFLꢀABLꢀ053). 18a did not exhibit apparent inhibitory activity against cꢀKIT
kinase, which is the common target of currently clinically used BCRꢀABL inhibitors. Through
significant suppression of the BCRꢀABL autoꢀphosphorylation (EC50 about 100 nM) and
downstream mediators such as STAT5, Crkl and ERK’s phosphorylation, 18a inhibited the
proliferation of CML cell lines K562 (GI = 14 nM), KU812 (GI = 25 nM) and MEGꢀ01 (GI
50
5
0
50
=
16 nM). Pharmacokinetic study revealed that 18a had over 4 hours halfꢀlife and 24%
bioavailability in rats. 50mg/kg/day dosage treatment could almost completely suppress the
tumor progression in the K562 cells inoculated xenograft mouse model. As a potential useful
drug candidate for CML, 18a is under extensive preclinical safety evaluation now.
INTRODUCTION
Chronic myeloid leukemia (CML), a hematological cancer of bone marrow white blood cells,
constitutes about 15% of adult leukemia and usually 1ꢀ2 patient is diagnosed with CML per
1
100,000 people/per year in US. It is characterized by a reciprocal chromosomal translocation
between chromosome 9 and 22 of the break point cluster region (BCR) gene with the Abelson
(ABL) gene for ABL1 kinase, which leads to a shortened chromosome 22 (i.e. Philadelphia
2
, 3
chromosome).
The fusion tyrosine kinase BCRꢀABL is constitutively active and leads to
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uncontrolled myeloid cell proliferation through downstream mediators such as Stat5 and ERK .
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, 6
The seminal discovery of small molecule inhibitor Imatinib has validated BCRꢀABL as the
drug discovery target for CML. Since then, several ABL kinase inhibitors have been approved
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for clinical use such as Nilotinib , Dasatinib , Bosutinib and Ponatinib and a few are in the
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clinical trials now including Bafetinib , Danusertib and Rebastinib , etc. In addition, a
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number of newly discovered inhibitors are in the extensive preclinical study such as GZD824
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5
and allosteric inhibitor GNF2, GNF5 etc. Despite of the great clinical success, the FDA
approved drugs such as Imatinib, Nilotinib, Bosutinib and Dasatinib all potently inhibit other
targets such as DDR1/2, cꢀKIT and so on besides ABL1 kinase. Although the role of offꢀtargets
inhibition is not very clear in the clinical aspect, the highly selective BCRꢀABL inhibitor is still
highly demanded from both the preclinical pathological and clinical side effects mechanistic
study point of view. Here we describe our medicinal chemistry effort from a
1
6, 17
dihydropyrimidopyrimidine scaffold based multiple targets BCRꢀABL inhibitor GNFꢀ7
(
compound 27) to a pyrimidine scaffold based highly potent and selective BCRꢀABL inhibitor
compound 18a (CHMFLꢀABLꢀ053), which completely abolished the cꢀKIT kinase activity
(Figure 1).
Figure 1. Schematic illustration of discovery of 18a.
RESULTS AND DISCUSSTION
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Chemistry
The synthesis of 12 (Scheme 1) began with amide coupling of 4ꢀmethylꢀ3ꢀnitroaniline with
appropriate carbonyl chloride. The nitrobenzene was then converted to the corresponding aniline
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3
by reduction with hydrogen under 10% Pd/C condition. Intermediate pyrimidine aldehyde 7
was obtained from ethyl 4ꢀchloroꢀ2ꢀ(methylthio)pyrimidineꢀ5ꢀcarboxylate 4. Methylamine
pyrimidine 5 was prepared with methylamine in almost quantitative yield. Subsequent reduction
of 5 in the presence of LAH in anhydrous THF afforded desired alcohol 6, which was then
oxidized into aldehyde 7. Reductive amination with 7 and 3 afforded diamine derivative 8. The
diamine was then converted into the dihydropyrimidopyrimidine 9 with triphosgene, which was
subsequently oxidized to the corresponding sulfone 10. Alcohol or aniline derivatives were
readily reacted with this sulfone under etherification with base or simple thermal amination
condition to provide the compound 11. Finally, SnCl mediated reduction of the nitrobenzene or
2
NꢀBoc deprotection furnished compound 12.
a
Scheme 1. Synthetic route to dihydropyrimidopyrimidine 12
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a
Reagents and conditions: (a) R COCl, THF, DIPEA, 0 °C; (b) H , 10% Pd/C, MeOH, rt; (c)
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THF, MeNH , TEA, 0 °C; (d) LAH, THF, 0 °C to reflux; (e) DCM, MnO , rt; (f) 3, Na(CN)BH ,
MeOH, AcOH, rt; (g) triphosgene, DCM, DIPEA, 0 °C to rt; (h) mꢀCPBA, DCM, 0 °C; (i)
R NH , dioxane, TFA, 120 °C or R OH, dioxane, K CO , rt; (j) SnCl .2H O, MeOH, reflux or 4
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M HCl, dioxane, rt.
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Hydrolysis of ethyl ester of pyrimidine 13 followed by amideꢀcoupling conditions furnished
amide 15 (Scheme 2). The sulfide of 15 was then subjected to oxidation, amination and
subsequent reduction to provide aniline compounds 18.
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a
Scheme 2. Synthetic route to compound 18
O
R
O
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R
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R
b
CF3
a
N
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OEt
N
OH
H
H
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NO2
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CF3
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N
N
H
H
e
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N
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3a-18a, R=NHMe
3b-18b, R=N(Me)2
14c-18c, R=H
NH2
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8
a
Reagents and conditions: (a) for synthesis of 14a-b: NaOH, MeOH, H O, rt; (b) 3a, HATU,
DIPEA, DMF, rt; (c) mꢀCPBA, DCM, 0 °C; (d) 4ꢀmethylꢀ3ꢀnitroaniline, dioxane, TFA, 120 °C;
e) SnCl .2H O, MeOH, reflux.
2
(
2
2
As shown in Scheme 3, compound 26 was prepared starting from 4ꢀmethylꢀ3ꢀnitroaniline 1.
After Boc protection (19) and hydrogenation (20), amide 21 was obtained under the standard
amide coupling condition with compound 14a. Removal of the Boc protection (22) followed by
acylation with appropriate acyl chloride formed the amide 23, which was then subjected to
oxidation (24), amination (25) and reduction to provide aniline compound 26.
a
Scheme 3. Synthetic route to compound 26
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NO2
NO2
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NH2
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NHBoc
H
NHBoc
S
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NHMe
NH2
NHBoc
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NH2
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H
H
H
H
H
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N
NHMe
S
NHMe
S
N
NHMe
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O
O
O
O
2
3a-26a, R=2-OCH3-benzene
23b-26b, R=3-OCH3-benzene
23c-26c, R=4-OCH3-benzene
3d-26d, R=3,4-OCH3-benzene
3e-26e, R=3,5-OCH3-benzene
3f-26f, R=3,4,5-OCH3-benzene
3g-26g, R=[1,3]dioxolebenzene
23h-26h, R=[1,4]dioxinebenzene
N
N
N
H
R
h
N
N
N
N
R
g
H
H
H
HN
N
NHMe
HN
NHMe
2
2
2
2
NO2
NH2
26
2
5
a
Reagents and conditions: (a) (Boc) O, THF, DMAP, reflux; (b) H , 10% Pd/C, MeOH, rt; (c)
14a, HATU, DMF, DIPEA, rt; (d) 4 M HCl, MeOH, rt; (e) RCOCl, THF, DIPEA, 0 °C; (f) mꢀ
CPBA, DCM, 0 °C; (g) 4ꢀmethylꢀ3ꢀnitroaniline, dioxane, TFA, 120 °C; (h) SnCl .2H O, MeOH,
2
2
2
2
reflux.
Structure-Activity Relationship
The dihydropyrimidopyrimidine compound 27 (GNFꢀ7) has been reported as a type II BCRꢀABL
inhibitor which could also overcome “gatekeeper” T315I mutation. However, it also potently
1
7
inhibited other kinases such as JAK1, 2, 3, FGFR3, FLT3, PDGFR, TRKC, etc . After careful
analysis of the molecular modeling results, we envisioned that the methyl pyridine occupied
hinge binding area (so called “tail’ part), the dihydropyrimidopyrimidine moiety occupied
“
gatekeeper” residue Thr315 adjacent area
(so called “center” part) and the
trifluoromethylbenzene occupied DFG shifting created hydrophobic area (so called “tail” part)
still have medicinal exploration space for better selectivity (Figure 1). We then chose to
systematically optimize the “head”, “center” and “tail” parts of 27 to obtain a full spectrum of
structureꢀactivity relationship (SAR) as illustrated in Figure 2 with cell based assays using ABL
transfused isogenic BaF3 cells and intact CML cancer cell line K562 as primary readout.
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Figure 2. Illustration of SAR exploration rationale. ABL kinase was shown in white. GNFꢀ7 was
labeled in color by atoms (Carbon in green, Nitrogen in blue, Oxygen in red). The hydrogen
bonds were labeled as red dashed lines. The key amino acid residues for the binding were labeled
as Carbon in yellow, Nitrogen in blue and Oxygen in red. The gatekeeper residue Thr315 was
labeled as Carbon in magenta, Nitrogen in blue and Oxygen in red. The “head”, “Center”, “Tail”,
DFGꢀmotif, Activation loop were pointed out with red arrows.
The “head” moiety occupied the hydrophobic pocket located proximal to the hinge binding
region in Abl kinase. Removal of the hydrogen bond acceptor nitrogen atom in 2ꢀmethylpyridine
of 27 by replacement with a 2ꢀmethylbenzene (11g) led to significant loss of activity against
parental BaF3 cells (GI : 8.0 versus 0.12 ꢁM) but not in K562 cells (GI : 0.018 ꢁM versus
5
0
50
0.009 ꢁM) (Table 1) which suggested a better selectivity profile and potent onꢀtarget (ABL) antiꢀ
proliferation effect in the TEL and BCR transfused isogenic BaF3 cells whose growth were
dependent on the constitutively activated ABL kinase (GI : 0.050 ꢁM and 0.019 ꢁM,
50
respectively). Increasing the size of this moiety (11f, 11h, 11n, and 11o) retained the potency
and selectivity suggesting a large size pocket residing in this region. However, when aniline
analog was introduced (12a) it started to potently inhibit parental BaF3 cells again (GI : 0.44
50
ꢁ
M). Interestingly, replacement of the aromatic substituents with cyclic aliphatic rings such as
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piperidine (12g), NꢀBoc piperidine (11l) or the aliphatic chain (11j) did not affect the potency.
This suggested that the hydrophobic pocket in this area was flexible. Since the modeling study
revealed that the 2ꢀaminopyrimidine part of 27 formed two hydrogen bonds in the hingeꢀbinding
region with Met318, we then tried to replace the –NH to oxygen atom to see if the activity was
remained. Interestingly, compound 12i still retained the activities against the K562 cells, Telꢀ
ABLꢀBaF3 cells and P210ꢀBaF3 cells (GI : 0.013 ꢁM, 0.015 ꢁM and 0.032 ꢁM, respectively)
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meanwhile still exhibited good selectivity over parental BaF3 cells (GI : >10 ꢁM).
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Unfortunately, when the “head” moiety was replaced by aliphatic rings with the Oꢀbridged hinge
binding such as (S)ꢀNꢀBoc 3ꢀhydroxypyrrolidine (11k), NꢀBoc 4ꢀhydroxypiperidine (11m), (S)ꢀ
pyrrolidinꢀ3ꢀol (12f) and piperidinꢀ4ꢀol (12h), significant activity loss were observed in K562
cells, TelꢀABLꢀBaF3 cells and P210ꢀBaF3 cells. This suggested that the hinge binding factors
and “head” hydrophobic binding factors are dependent on each other to contribute to the binding.
We next explored the “tail” moiety, which occupied the hydrophobic pocket formed by the
“
DFG” motif shift in the inactive conformation. Keeping the “head” and “center” moiety as in
2a, while switching the trifluoromethyl group in the “tail” to a methyl group (12e) resulted in
about 23ꢀfold potency loss against K562 cells (GI : 0.069 ꢁM versus 0.003 ꢁM), though the
1
5
0
activities against TELꢀAblꢀBaF3 cells (GI : 0.006 ꢁM) and P210ꢀBaF3 cells (GI : 0.014 ꢁM)
5
0
50
and selectivity against parental BaF3 cells (GI : >10 ꢁM) were remained. In addition,
5
0
replacement of the trifluoromethyl group with a bulky aromatic group (12b, 12c) did not result in
significant activity loss in K562 cells (GI : 0.023 ꢁM and 0.026 ꢁM ), TelꢀAblꢀBaF3 cells (GI :
5
0
50
0
.039 ꢁM and 0.031 ꢁM) and P210ꢀBaF3 cells (GI : 0.046 ꢁM and 0.042 ꢁM). However,
50
modification of the 3, 5ꢀposition of the tail group with two tertꢀbutyl groups (12d) significantly
lowered the activity against K562 cells (GI : 1.3 ꢁM versus 0.003 ꢁM), which suggested that
5
0
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the “DFG” motif shifting created hydrophobic pocket in Abl kinase could only tolerate medium
size of hydrophobic moiety.
Table 1. Antiꢀproliferation efficacies against intact and isogenic cancer cell lines of
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
a
dihydropyrimidopyrimidine derivatives
BaF3
ꢁM)
TelꢀAblꢀBaF3
(ꢁM)
P210ꢀBaF3
(ꢁM)
K562
(ꢁM)
Compd
Structure
(
O
CF
3
N
N
N
N
N
N
H
HN
HN
HN
HN
N
N
N
N
N
N
O
O
O
O
2
7
0.12±0.0004 0.005±0.0003 0.014±0.0005 0.009±0.0006
O
CF
3
N
N
H
11f
3.76±0.781
8.00±1.136
>10
0.061±0.001
0.050±0.007
0.185±0.001
0.066±0.013
0.019±0.004
0.067±0.032
0.015±0.002
0.126±0.003
0.055±0.044
0.132±0.009
0.017±0.014
0.018±0.005
0.029±0.006
0.007±0.0002
0.030±0.0009
0.018±0.003
0.029±0.010
O
CF
3
N
N
H
11g
O
CF
3
N
N
N
H
N
N
11h
CF
3
O
CF
3
N
N
N
H
5.60±0.0002 0.009±0.0004
11j
N
N
N
N
O
H
O
O
CF
3
N
N
N
H
11k
O
N
N
O
8.38±2.569
7.52±0.778
>10
0.292±0.0014
0.105±0.000
0.288±0.0027
NBoc
O
CF
3
N
N
N
H
HN
N
N
O
11l
N
Boc
O
CF
3
N
N
N
H
O
N
N
O
11m
N
Boc
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
O
CF
3
N
N
N
N
N
N
N
H
HN
HN
HN
N
N
N
N
O
O
O
1
1n
>10
>10
0.020±0.001
0.042±0.005
0.005±0.000
0.039±0.007
0.031±0.0062
0.096±0.0062
0.006±0.0001
1.380±0.021
0.100±0.1
0.014±0.006
0.030±0.004
0.019±0.001
0.028±0.008
0.003±0.000
0.023±0.001
0.026±0.0021
1.3±0.236
NH
O
CF
3
N
H
N
N
11o
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
CF
3
N
H
0.005±±0.000
2
N
1
2a
2b
0.44±0.093
>10
NH
2
O
N
N
N
H
HN
N
N
O
1
0.046±0.013
0.042±0.006
0.12±0.006
0.014±0.005
1.26±0.03
NH
2
O
N
N
N
H
HN
N
N
O
1
2c
>10
NH
2
O
N
N
N
H
HN
N
N
O
12d
>10
NH
2
O
N
N
N
H
0
.0688±0.001
2
HN
N
N
O
1
2e
>10
NH
2
O
CF
3
N
N
N
H
1
2f
O
N
N
O
>10
0.45±0.0022
0.019±0.0007
0.220±0.038
0.013±0.001
NH
O
CF
3
3
3
N
N
N
H
HN
N
N
O
1
2g
2h
3ꢀ10
>10
0.15±0.005
2.705±0.327
0.032±0.003
N
H
O
CF
N
N
N
H
O
N
N
O
1
2.043±0.035
0.015±0.0003
N
H
O
CF
N
N
N
H
O
N
N
O
1
2i
>10
NH
2
a
all GI values are presented as mean±SEM (n=3).
50
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Despite the similar potencies of 12a against the model cell lines as 27, the narrow selectivity
window between the parental BaF3 and isogenic BaF3 cell lines led us to explore more of the
“center” moiety by opening the cyclic urea ring of 12a, which presumably would increase the
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
flexibility and improve the solubility. Removal of the carbonyl group offered 18a, which
displayed potent antiꢀproliferation efficacy against K562 cells (GI : 0.014 ꢁM) and P210ꢀBaF3
5
0
cells (GI : 0.007 ꢁM) meanwhile exhibited good selectivity over parental BaF3 cells (GI : >10
5
0
50
ꢁ
M) (Table 2). Either introduction of one more methyl group at this position (18b) or removal of
the 4ꢀmethylamino group (18c) resulted in significant activity loss against K562 cells (GI :
5
0
1.211 ꢁM and 0.33 ꢁM). Comparing to 18a-c, the nitro group bearing compounds 17a-c all
resulted in losing their potency slightly or significantly. We then turned our attention to the “tail”
part of 18a scaffold. Replacement of the 3ꢀtrifluoromethyl group with 3ꢀmethoxy group in the
tail phenyl ring (25b and 26b) dramatically decreased their potency against K562 cells (GI :
5
0
0
.48 ꢁM and 0.33 ꢁM, respectively). Shifting the 3ꢀmethoxy group to 2ꢀpostion (25a, 26a), 4ꢀ
position (25c, 26c) or bearing dimethoxy group (25d, 25e, 26d, 26e), trimethoxy group (25f, 26f),
1,3]dioxo (25g, 26g) and [1,4]dioxine (25h, 26h) substituents in the tail phenyl ring all led to
[
the significant activity loss.
Table 2. Antiꢀproliferation efficacies against intact and isogenic cancer cell lines of compound
a
18a and its analogs
BaF3
ꢁM)
TelꢀABL ꢀBaF3
(ꢁM)
P210ꢀBaF3
(ꢁM)
K562
(ꢁM)
Compd
Structure
(
O
O
CF
3
N
N
N
H
H
HN
N
NHMe
17a
>10
0.087±0.006
0.036±0.006
0.052±0.011
NO
2
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
O
O
CF
3
N
N
N
N
N
N
N
H
H
HN
HN
HN
HN
HN
N
NMe
2
1
7b
>10
8.70±3.655
>10
>10
>10
>10
NO
2
O
O
CF
3
N
H
N
H
N
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
17c
1.400±0.059
0.053±0.0012
1.030±0.200
0.590±0.247
>10
1.20±0.096
0.007±0.002
1.849±0.196
0.47±0.055
>10
0.38±0.027
0.014±0.001
1.211±0.031
0.33±0.025
>10
NO
2
O
O
O
O
CF
3
N
H
N
H
N
NHMe
1
8a
NH
2
O
CF
3
N
H
N
H
N
NMe
2
18b
>10
NH
2
O
CF
3
N
H
N
H
N
18c
>10
NH
2
O
O
OMe
N
N
N
H
H
HN
N
NHMe
2
5a
>10
NO
2
O
O
OMe
N
N
N
N
N
N
H
N
H
HN
HN
HN
HN
HN
N
NHMe
25b
6.31±0.1
>10
0.983±0.067
2.900±0.188
1.400±0.176
2.034±0.112
0.931±0.08
1.1±0.154
3.6±0.399
1.6±0.100
0.294±0.083
1.5±0.171
0.48±0.021
1.53±0.110
0.68±0.045
1.07±0.061
0.43±0.063
NO
2
O
O
O
O
N
H
N
H
N
NHMe
OMe
25c
NO
2
O
OMe
OMe
N
H
N
H
N
NHMe
25d
7.30±1.292
>10
NO
2
O
OMe
N
H
N
H
N
NHMe
2
5e
OMe
NO
2
O
O
OMe
OMe
N
H
N
H
N
NHMe
2
5f
>10
OMe
NO
2
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5
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7
8
9
O
O
O
O
N
N
N
H
H
HN
N
NHMe
2
5g
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
1.290±0.040
4.240±0.0763
5.20±0.202
0.524±0.003
1.300±0.017
1.100±0.259
0.628±0.004
0.445±0.111
0.570±0.010
0.787±0.129
0.214±0.095
4.1±0.617
6.4±0.212
0.57±0.097
2.1±0.181
1.6±0.174
0.311±0.084
0.67±0.117
0.124±0.020
1.1±0.153
0.499±0.030
0.32±0.017
1.98±0.351
0.33±0.155
0.61±0.079
0.38±0.078
0.230±0.151
0.25±0.047
0.353±0.006
0.32±0.086
NO
2
O
O
O
N
N
H
N
H
HN
N
NHMe
O
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
25h
NO
2
O
O
OMe
N
N
H
N
H
HN
N
NHMe
26a
NH
2
O
O
OMe
N
N
N
N
N
N
N
N
H
N
H
HN
HN
HN
HN
HN
HN
HN
N
NHMe
26b
NH
2
O
O
O
O
O
N
H
N
H
N
NHMe
OMe
26c
NH
2
O
OMe
OMe
N
H
N
H
N
NHMe
26d
NH
2
O
OMe
N
H
N
H
N
NHMe
2
6e
OMe
NH
2
O
OMe
OMe
N
H
N
H
N
NHMe
26f
OMe
NH
2
O
O
O
O
N
H
N
H
N
NHMe
26g
NH
2
O
O
O
O
N
H
N
H
N
NHMe
26h
NH
2
a
all GI values are presented as mean±SEM (n=3).
50
Since compound 18a exhibited the best activity and selectivity profile, we then moved forward
to study the binding mode of 18a with ABL kinase via molecular modeling. The model
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
illustrated that it did prefer to adopt a type II binding mode as designed. The aminopyrimidine
formed two hydrogen bonds with Met318 in the hinge area (Figure 3A). The Glu286 residue in
the cꢀHelix and “DFG” residue Asp381 formed two typical hydrogen bonds with amide linkage
between the “center” moiety and the “tail” moiety. The gatekeeper residue Thr315 formed a
hydrogen bond with the carbonyl group linking the aminopyrimidine moiety and the “tail” part.
Furthermore, Tyr253 also formed a hydrogen bond with the amino group in the “head” moiety,
which provided the explanation for the activity difference between 17aꢀc and 18aꢀc. The
hydrophobic pocket in the hinge binding area is shallow and flat, which explained that why both
aromatic and aliphatic rings and chains could be tolerated (Figure 3B). 3ꢀ
Trifluoromethylbenzene in the “tail” occupied the hydrophobic pocket formed by “DFG” motif
through Van der Waals interactions (Figure 3C). In addition, Leu248, Val256 and Tyr253
formed a small hydrophobic pocket, which could accommodate the aminomethyl group in the
aminopyrimidine “center” moiety. And this could explain the reason that compound 18b and 18c
lost the activity (Figure 3D).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 3. Compound 18a was docked into Abl kinase (PDB ID: 2HYY). Hydrogen bonds are
indicated by red dashed lines to key amino acid residues. (A) Cartoon view of the binding mode
of 18a with ABL1 kinase. (B) Solid surface view of the shallow hydrophobic pocket located
adjacent to hinge binding area. (C) Solid surface view of the hydrophobic pocket formed by
D(out)FG inactive conformation. (D) Solid surface view of the small hydrophobic pocket formed
by Leu248, Val256 and Try253 to accommodate the methylamine moiety.
We next examined the kinome wide selectivity profile of 18a (1 ꢁM) on the DiscoveRx’s
KinomeScan profiling platform. The results demonstrated that 18a was highly selective among
468 kinases tested and exhibited an S score (1)=0.02 (Figure 4 and Supplemental Table 1). The
data also indicated that 18a might had strong binding (with percent control number less than 1)
against BLK, DDR1, DDR2, EPHA8, EphB6, HCK, LCK, p38α and SRC kinases. Further
confirmation with Invitrogen SelectScreen biochemical assay revealed that 18a exhibited an IC50
of 70 nM against ABL1 kinase, meanwhile also strongly inhibited p38α (IC : 62 nM) and SRC
50
kinase (IC : 90 nM) (Table 3). However it was less potent to DDR1 (IC : 292 nM) and DDR2
5
0
50
(
IC : 457 nM). Previous reports also showed that Dasatinib and Nilotinib had strong binding to
50
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
p38α (Kd: 27 nM and 460 nM respectively) (Supplemental Table 2). Bosutinib and Dasatinib
showed strong binding to SRC (Kd: 1 nM and 0.21 nM respectively). In addition, 18a did not
exhibit apparent potency against cꢀKIT kinase (IC : over 10000 nM), which is the common offꢀ
5
0
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
target for clinically used BCRꢀABL inhibitors Imatinib, Nilotinib, Botutinib and Dasatinib
(Table 3 and Supplemental Table 2). In the TELꢀfused isogenic BaF3 cells, 18a displayed great
selectivity between the BCRꢀABL and other potential offꢀtargets including SRC, DDR1, DDR2,
LCK, BLK and HCK (Table 4). We also tested 18a against a variety of clinically important
mutations of ABL in the p210 fused BaF3 assay system. Interestingly, it was sensitive against
ABL F317L, F317I and M351T mutants but was relatively resistant against E255K, Q252H,
Y253F, H369P mutants and completely lost the activity to the gatekeeper mutant T315I (Table
4).
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Figure 4. Kinome wide selectivity profiling of 18a with DiscoveRx KinomeScan technology.
Measurements were performed at a concentration of 1 ꢁM of the inhibitor. The affinity was
defined with respect to a DMSO control. (A). Treespot demonstration of 18a’s selectivity in 468
kinase targets. (B). Other targets that demonstrated strong binding to 18a with a percent control
number less than 1.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 3. Invitrogen SelectScreen biochemical characterization of 18a (values=mean±SEM, n=2)
Kinase
18a (IC : nM)
50
ABL1
DDR1
DDR2
cꢀKIT
P38
70±5
292±53
457±23
>10000
62±6
SRC
90±3
Table 4. Confirmation of target inhibition revealed in the KinomeScan with isogenic BaF3 cell
a
lines
1
8a
Imatinib
Nilotinib
Dasatinib
Cell line
(
ꢀM)
(ꢀM)
(ꢀM)
(ꢀM)
Parental BaF3
BaF3/p210
>10
6.7±0.2
0.38±0.03
2.1±0.05
3ꢀ10
2.1±0.05
0.004±0.0005
0.47±0.02
1.1±0.02
>10
0.007±0.004
0.2±0.005
3.3±0.08
>10
0.003±0.0001
<0.0003
TELꢀSRC
TELꢀDDR1ꢀBaF3
TELꢀDDR2ꢀBaF3
TELꢀLCKꢀBaF3
TELꢀBLKꢀBaF3
TELꢀHCKꢀBaF3
10ꢀ3
7.7±0.5
0.5±0.06
4.1±1.6
9.7±2
1.4±0.05
10ꢀ3
0.6±0.01
1.1±0.17
0.98±0.004
0.87±0.21
1.3±0.03
0.001±0.00009
0.005±0.00002
0.039±0.0001
4.1±0.2
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1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
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3
3
3
3
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4
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5
5
5
5
5
5
5
5
5
6
p210ꢀE255KꢀBaF3
p210ꢀF317LꢀBaF3
p210ꢀF317IꢀBaF3
p210ꢀM351TꢀBaF3
p210ꢀQ252HꢀBaF3
p210ꢀY253FꢀBaF3
p210ꢀH369PꢀBaF3
p210ꢀT315IꢀBaF3
0.313±0.032
1.93±0.253
2.169±0.039
0.855±0.081
0.625±0.253
0.659±0.072
>10
0.021±0.006
0.017±0.0004
0.014±0.0009
0.01±0.001
0.045±0.002
0.073±0.008
0.045±0.002
0.14±0.033
0.363±0.001
0.440±0.029
9.25±0.01
0.202±0.01
0.0546±0.004
0.017±0.001
0.023±0.001
1.093±0.029
0.025±0.0044
>10
0
1
2
3
4
5
6
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8
9
0
1
2
3
4
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9
0
1
2
3
4
5
6
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8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
0.003±0.0005
0.008±0.001
0.001±0.0006
0.004±0.0002
9.94±4.0
1.69±0.177
>10
a
all GI values are presented as mean±SEM (n=3).
5
0
It is intriguing that compound 18a uniquely abolished cꢀKIT kinase activity in comparison to
other ABL inhibitors. In order to understand this from the structure point of view, we then
docked compound 18a into cꢀKIT kinase (PDB ID: 1T46) (Figure 5). The modeling results
suggested that 18a might adopt a similar type II binding mode as it binds to ABL kinase (Figure
5
A). However, when cꢀKIT and ABL kinase were superimposed, we found that in ABL kinase
the Tyr253 located in the pꢀloop which could provide a key hydrogen bond with the methyl
aniline moiety in the “head” part of 18a was replaced by Gly596 residue in cꢀKIT kinase (Figure
5
B). Lacking of this key hydrogen bond may result in the loss of inhibitory potency of 18a
against cꢀKIT kinase.
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Figure 5. Comparison of the binding modes of compound 18a between ABL and cꢀKIT kinase.
(A). Compound 18a was docked into cꢀKIT kinase (PDB ID: 1T46). CꢀKIT protein was shown
in magenta. Compound 18a was displayed as Carbon in green, Nitrogen in blue and Oxygen in
red. The key binding amino acid residues from the protein were displayed as Carbon in magenta,
Nitrogen in blue and Oxygen in red. (B). Superimposition of cꢀKIT (in magenta) and ABL (in
gray, PDB ID: 2HYY) kinase in complex with compound 18a.
We then compared 18a with Imatinib and Nilotinib in a panel of intact leukemia cancer cell lines
including the CML and AML (Table 5). 18a exhibited potent antiꢀproliferation efficacies against
all of the three BCRꢀABL driven CML cell lines K562 (GI : 14 nM), KU812 (GI : 25 nM) and
5
0
50
MEGꢀ01 (GI : 16 nM) but not other AML cell lines, implying strong and selective onꢀtarget
5
0
effects. In addition, 18a did not display any apparent activity against CHL cell line, indicating a
good nonꢀspecific toxicity profile.
a
Table 5. Antiꢀproliferation effects of 18a against varieties of intact cancer cell lines
18a
Imatinib
Nilotinib
Dasatinib
Cell line
Cell type
(
ꢀM)
(ꢀM)
(ꢀM)
(ꢀM)
K562
KU812
MEGꢀ01
CML
CML
CML
0.014±0.006
0.14±0.001
0.16±0.005
0.002±0.0002
0.001±0.0002
0.016±0.0014
<0.0003
<0.0003
<0.0003
0.025±0.002
0.016±0.0056 0.24±0.0267
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MV4ꢀ11
MOLM14
U937
AML
AML
AML
AML
8±0.4
>10
>10
>10
>10
>10
3.6±0.1
2.3±0.09
>10
>10
>10
>10
0
1
2
3
4
5
6
7
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9
0
1
2
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9
0
1
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0
1
2
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9
0
1
2
3
4
5
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7
8
9
0
HEL
>10
5.3±0.2
3.9±0.05
5.3±0.4
Hamster
lung cell
CHL
>10
>10
4.2±0.6
0.27±0.1
a
all GI values are presented as mean±SEM (n=3).
50
We then investigated 18a’s effects on the BCRꢀABL mediated signals in BCRꢀABL driven CML
cell lines K562, KU812 and MEGꢀ01 (Figure 6). Compound 18a almost completely suppressed
BCRꢀABL kinase autoꢀphosphorylation at Y245 site in K562, KU812 and MEGꢀ01 at the
concentration of 300 nM. BCRꢀABL kinase downstream mediator Stat5, CrkL and ERK
phosphorylation was also significantly inhibited in a concentrationꢀdependent manner.
Interestingly, unlike Imatinib which had no effect on CrkL’s phosphorylation, both 18a and
Dasatinib could affect CrkL’s phosphorylation, which indicated a different pharmacology profile
among them. The results also showed that phosphorylation of SRC kinase was much less
potently inhibited than phosphorylation of BCRꢀABL by 18a, though they showed similar
sensitivity to 18a in the biochemical assay. This indicated that the BCRꢀABL inhibitory activity
of 18a majorly contributed to the antiꢀproliferative effect in these CML cell lines. P38α
phosphorylation was potently inhibited which is in accordance to its biochemical inhibitory
activities. In addition, even in the early 12 h, 18a could doseꢀdependently arrest the cell cycle
progression in the G0/G1 phase in these cells (Figure 7A). Upon 24 or 48 hours drug treatment,
100 nM concentration of 18a could significantly induce the apoptotic cell death (Figure 7B).
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Figure 6. Compound 18a’s effects on BCRꢀAbl kinase mediated signaling pathway in KU812,
K562 and MEGꢀ01 cancer cell lines. Cells were treated with 18a at the indicated concentrations
for 1 h, and whole cell lysates were then subjected to Western blot analyses.
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0
Figure 7. (A) 18a arrested the cell cycle progression. (B) 18a induced apoptosis in K562,
KU812 and MEGꢀ01 cell lines.
In the study of PK profiling in rats, compound 18a exhibited good systemic exposure
(AUC=1715.51 ng/mL*hr, Cmax=367.61 ng/mL), favorable oral bioavailability (F=24.19%) and
acceptable halfꢀlife (T =4.33 hr) following oral administration of a single dose of 10 mg/kg
1
/2
(Table 6).
a
Table 6. Pharmacokinetic study of 18a on Sprague Dawley rats
MRT
t1/2
hr
Tmax
hr
Cmax
AUC(0ꢀt)
AUC(0ꢀ∞)
Vz
CLz
F%
(0ꢀ∞)
ng/mL ng/mL*hr ng/mL*hr
mL/kg mL/hr/kg
hr
IV
1
mg/kg
Mean
2.82
0.02 2395.22
635.98
208.84
720.69
256.99
6025.13 1538.39
3.1
NA
NA
SD (n=3) 0.53
0.00
449.56
1632.22
642.39
0.69
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PO
10 mg/kg 4.33
Mean
1.00
0.87
367.61
202.21
1715.51
1083.42
1743.43
1091.08
NA
NA
NA
NA
5.51
0.50
24.19
NA
SD (n=3) 1.11
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a
Compound 18a was formulated as a clear solution in 5% DMSO, 40% PEG400 and 55% of 20% HPꢀβꢀCD in water
for intravenous and oral administration.
The in vivo antiꢀtumor study of 18a was performed in the K562 cell inoculated xenograft mouse
model. After 16 days continuous treatment, compound 18a doseꢀdependently inhibited the
growth of the K562 tumor and a 50mg/kg/day dosage could almost completely suppress the
tumor progression (Figure 8A). All doses of 18a were well tolerated, with no mortality and no
significant body weight loss observed (Figure 8B). Compound 18a displayed obvious antitumor
efficacy (TGI=48.3%) at 50 mg/kg/day dosage (Figure 8C, D). In addition, the
immunohistochemistry (IHC) stain revealed that the proliferation was effectively inhibited (Kiꢀ
6
7 lane) and significant apoptosis was induced (TUNEL lane) in the tumor (Figure 8E).
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Figure 8. Compound 18a’s antiꢀtumor efficacy in K562 xenograft model. Female nu/nu mice
bearing established control group, K562 tumor xenografts were treated with 18a at 25.0, 50.0
mg/kg/d, or vehicle. Daily oral administration was initiated when K562 tumors had reached a
3
size of 200 to 400 mm . Each group contained 5 animals. Data=mean ± SEM. (A) Body weight
measurement from K562 xenograft mice after 18a administration. Initial body weight was set as
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00%. (B) Tumor size measurements from K562 xenograft mice after 18a administration. Initial
tumor size was set as 100%. (C) Representative photographs of tumors in each group after 25.0
or 50.0 mg/kg/d 18a or vehicle treatment. (D) Comparison of the final tumor weight in each
group after 16ꢀday treatment period. (E) Representative micrographs of hematoxylin and eosin
(HE), Kiꢀ67 and TUNEL staining of tumor tissues with 18a treatment compared to the vehicle
group. Note the specific nuclear staining of cells with morphology consistent with proliferation
and apoptosis (E, red arrow).
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CONCLUSION
Starting from a multiple targets dihydropyrimidopyrimidine scaffold based compound 27 (GNFꢀ
7
) bearing high BCRꢀABL potency, we used a focused medicinal chemistry approach guided by
computerꢀaided drug design to obtain an aminopyrimidine scaffold based compound 18a
CHMFLꢀABLꢀ053) via 7 steps of chemical syntheses (11% overall yield), which possessed
(
highly potent antiꢀproliferative efficacy against BCRꢀABL driven CML cell lines and exhibited
good safety window against other leukemia cell lines such as AML. Compound 18a displayed a
high selectivity profile. Besides the ABL kinase, it also inhibited structurally related SRC kinase
and p38α kinase, which is desired and might contribute positively to exert synergistic antiꢀ
leukemic effect of 18a since SRC kinase is downstream of BCRꢀABL and contributes to the
proliferation and survival of myeloid cell line and p38α kinase is involved in the BCRꢀABL
18, 19
inhibitor induced apoptosis pathway.
Comparing to clinically used BCRꢀABL inhibitors
Imatinb, Nilotinib, Bosutinib and Dasatinib, compound 18a completely abolished the cꢀKIT
kinase inhibitory activity and exhibited better selectivity against DDR1/2 kinases. In addition,
1
8a also showed suitable PK profile and potent in vivo antiꢀtumor efficacy. It is worthy to note
that although compound 18a exhibits a good selectivity among kinome, we cannot exclude the
possibility that it may also affect other effective targets that may also contribute to its antiꢀ
leukemia effect, like Imatinib has been reported to inhibit the quinone oxidoreductase2
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(
NQO2). Currently 18a is under extensive preclinical safety evaluation and it might be a
potential useful pharmacological candidate supplementary to the current BCRꢀABL target
therapies for the treatment of CML.
0
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Experimental Procedures
Chemical Synthesis. All reagents and solvents were purchased from commercial sources and
were used as received, unless specified otherwise, or prepared as described in the literature. All
moistureꢀsensitive reactions were carried out using dry solvents under ultra pure argon
protection. Glassware was dried in an oven at 140 °C for at least 12 h prior to use, and then
assembled quickly while hot, sealed with rubber septa, and allowed to cool under a stream of
argon. Reactions were stirred magnetically using Teflonꢀcoated magnetic stirring bars.
Commercially available disposable syringes were used for transferring the reagents and solvents.
LC/MS were performed on an Agilent 6224 TOF using an ESI source coupled to an Agilent
1
260 Infinity HPLC system operating in reverse mode with an Agilent XDBꢀC18 column
(
4.6×50 mm, 1.8 ꢀm) using a water/acetonitrile (each with 0.2% (v/v) formic acid) gradient at a
1
13
flow rate at 0.4 mL/min. H and C spectra were recorded on a Bruker 400 MHz NMR
spectrometer. Chemical shifts are expressed in ppm. In the NMR tabulation, s indicates singlet;
d, doublet; t, triplet; q, quartet; and m, multiplet. Flash column chromatography was conducted
using silica gel (Silicycle 40−64 ꢁm). The purities of all compounds were determined to be
>
95% by HPLC.
General Method A:
N-(3-amino-4-methylphenyl)-3-(trifluoromethyl)benzamide (3a) To a solution of 4ꢀmethylꢀ3ꢀ
nitroaniline (2.82 g, 18.5 mmol, 1.00 equiv) in DCM (30 mL) was added TEA (2.84 mL, 20.35
mmol, 1.10 equiv). Then a solution of 3ꢀ(trifluoromethyl)benzoyl chloride (5.0 g, 19.5 mmol,
1.05 equiv) in DCM (15 mL) was dropwise added in 30 min at 0 °C under argon. The reaction
mixture was stirred at 0 °C for 2 hours, then was allowed to warm to room temperature for
overnight (10 h). The resulting solution was diluted with DCM (100 mL) and EtOAc (20 mL),
washed with 1 M HCl (2×100 mL), 1 M NaOH (2×100 mL), brine (100 mL) and dried over
anhydrous MgSO . Evaporation of the solvent afforded the crude product 2a, which was used in
4
the next step without further purification. To a solution of 2a (18.5 mmol, 1.00 equiv) in
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methanol (20 mL) was added 10% Pd/C (0.2 g) at room temperature under argon. Then the
reaction mixture was stirred under a balloon of hydrogen for 20 h. The resulting mixture was
filtered through a pad of celite and washed with methanol. Evaporation of the filtrate provided
the crude product, which was purified by silica gel flash chromatography (eluting with MeOH in
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
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46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
DCM 0ꢀ4%) to give 3a as a white solid (2.83 g, two steps yield 54%). H NMR (400 MHz,
DMSOꢀd ) δ 9.97 (s, 1H), 8.30 (d, J = 14.1 Hz, 2H), 7.95 (d, J = 7.1 Hz, 1H), 7.78 (t, J = 7.2 Hz,
6
1H), 6.93 (d, J = 7.7 Hz, 1H), 6.64 (s, 1H), 6.47 (d, J = 7.7 Hz, 1H), 4.97 (s, 2H), 2.09 (s, 3H).
1
3
C NMR (101 MHz, DMSOꢀd ) δ 164.12, 147.36, 136.66, 136.15, 132.09, 130.95, 130.15,
6
1
29.90, 129.58, 128.39, 124.68, 120.93, 112.92, 112.85, 17.39. LCꢀMS (ESI, m/z): 295.0992
+
[
M+H] .
1
N-(3-amino-4-methylphenyl)biphenyl-3-carboxamide (3b) (Method A) yield 75%. H NMR
(400 MHz, DMSOꢀd ) δ 10.09 (s, 1H), 8.24 (s, 1H), 7.96 (d, J = 7.4 Hz, 1H), 7.85 (d, J = 7.4 Hz,
6
1
6
1
H), 7.78 (d, J = 7.1 Hz, 2H), 7.62 (d, J = 7.5 Hz, 1H), 7.43 (d, J = 7.1 Hz, 1H), 7.22 (s, 1H),
13
.92 (s, 2H), 4.92 (s, 2H), 2.08 (s, 3H). C NMR (101 MHz, DMSOꢀd ) δ 165.48, 147.02,
6
40.69, 140.10, 138.07, 136.52, 130.20, 129.92, 129.47, 128.26, 127.40, 127.28, 126.23, 117.41,
+
109.47, 107.08, 17.50. LCꢀMS (ESI, m/z): 303.1425 [M+H] .
1
N-(3-amino-4-methylphenyl)-3,5-dimethylbenzamide (3c) (Method A) yield 72%. H NMR
(
400 MHz, DMSOꢀd ) δ 9.86 (s, 1H), 7.57 (s, 2H), 7.20 (s, 1H), 7.17 (s, 1H), 6.88 (s, 2H), 4.86
6
13
(
s, 2H), 2.36 (s, 6H), 2.06 (s, 3H). C NMR (101 MHz, DMSOꢀd ) δ 165.84, 146.92, 138.20,
6
1
37.86, 135.90, 132.95, 130.14, 125.78, 117.20, 109.37, 106.95, 21.34, 17.46. LCꢀMS (ESI, m/z):
+
255.1425 [M+H] .
1
N-(3-amino-4-methylphenyl)-3,5-di-tert-butylbenzamide (3d) (Method A) yield 71%. H
NMR (400 MHz, DMSOꢀd ) δ 9.92 (s, 1H), 7.77 (s, 2H), 7.59 (s, 1H), 7.11 (s, 1H), 6.89 (s, 2H),
6
13
2
.07 (s, 3H), 1.34 (d, J = 13.6 Hz, 18H). C NMR (101 MHz, DMSOꢀd ) δ 166.31, 150.80,
6
146.92, 138.04, 135.41, 130.08, 125.26, 122.13, 117.31, 109.82, 107.46, 35.16, 31.68, 17.48.
+
LCꢀMS (ESI, m/z): 339.2365 [M+H] .
1
N-(3-amino-4-methylphenyl)-3-methylbenzamide (3e) (Method A) yield 65%. H NMR (400
MHz, DMSOꢀd ) δ 10.50 (s, 1H), 8.07 (s, 1H), 7.79 (dd, J = 8.2, 4.8 Hz, 2H), 7.65 (d, J = 8.2 Hz,
6
1
3
1
H), 7.41 (d, J = 4.5 Hz, 2H), 7.29 (d, J = 8.3 Hz, 1H), 2.40 (s, 3H), 2.36 (s, 3H). C NMR (101
MHz, DMSOꢀd ) δ 166.22, 138.45, 138.17, 135.05, 132.76, 131.70, 130.93, 128.75, 128.70,
6
+
1
27.37, 125.41, 120.43, 115.90, 21.43, 17.20. LCꢀMS (ESI, m/z): 241.1272 [M+H] .
28
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