Discovery of novel 5-fluoro-N2,N4-diphenylpyrimidine-2,4-diamines as potent inhibitors against CDK2 and CDK9

Article abstract of DOI:10.1039/c4md00412d

Based on a 3D-QSAR pharmacophore derived from a diverse set of known cyclin-dependent kinase 9 (CDK9) inhibitors and a composite pharmacophore extracted from the complex structure of flavopiridol (FVP)-CDK9, thirty novel 5-fluoro-N²,N⁴-diphenylpyrimidine-2,4-diamine derivatives were designed and synthesized. Initial tests against four tumor cell lines with the sulforhodamine B (SRB) assay identified a series of potent compounds with GI₅₀ values at a lower micromolar or submicromolar level. Most of the highly cytotoxic compounds exhibited potent inhibitory activities against both CDK2/cyclin E1 and CDK9/cyclin T1. Notably, inhibitions against the two enzymes were generally correlated well with the cytotoxicity of these compounds. Appreciable inhibition was also observed for selected compounds in the anti-HIV-1 assay. Docking studies on compounds 6d and 9g provided conducive clues to further structural optimization. This journal is

Full text of DOI:10.1039/c4md00412d

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Discovery of novel 5-fluoro-N2,N4-diphenylpyrimidine-2,4-diamines as potent
inhibitors against CDK2 and CDK9
†
†
Jiadi Gaoa, , Cheng Fanga, , Zhiyan Xiaoa,*, Li Huangb, ChinꢀHo Chenb, LiꢀTing Wangc, KuoꢀHsiung
Leec,d
a Beijing Key Laboratory of Active Substance Discovery and Druggability Evaluation, Institute of
Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing
100050, China
b Department of Surgery, Duke University Medical Center, Durham, NC 27710, USA
c Natural Products Research Laboratories, UNC Eshelman School of Pharmacy, University of North
Carolina, Chapel Hill, North Carolina 27599ꢀ7568, USA
d
Chinese Medicine Research and Development Center, China Medical University and Hospital,
Taichung, Taiwan
* To whom correspondence should be addressed. Corresponding author phone: +86ꢀ10ꢀ63189228;
Eꢀmail: xiaoz@imm.ac.cn
† These authors contribute equally.
1

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Abstract:
Based on a 3DꢀQSAR pharmacophore derived from a diverse set of known cyclinꢀdependent kinase
9 (CDK9) inhibitors and a composite pharmacophore extracted from the complex structure of
flavopiridol (FVP)ꢀCDK9, thirty novel 5ꢀfluoroꢀN2,N4ꢀdiphenylpyrimidineꢀ2,4ꢀdiamine derivatives
were designed and synthesized. Initial tests against four tumor cell lines with the sulforhodamine B
(SRB) assay identified a series of potent compounds with GI50 values at lower micromolar or
submicromolar level. Most of the highly cytotoxic compounds exhibited potent inhibitory activities
against both CDK2/cyclin E1 and CDK9/cyclin T1. Notably, inhibitions against the two enzymes
were generally correlated well with the cytotoxicity of these compounds. Appreciable inhibition was
also observed for selected compounds in the antiꢀHIVꢀ1 assay. Docking studies on compounds 6d
and 9g provided conducive clues to further structural optimization.
Key
words:
5ꢀfluoroꢀN2,N4ꢀdiphenylpyrimidineꢀ2,4ꢀdiamines,
cyclinꢀdependent
kinases,
pharmacophore, cytotoxicity, antiꢀHIVꢀ1
2

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Graphical Abstract
3

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1. Introduction
Cyclinꢀdependent kinases (CDKs) are a family of serine/threonine protein kinases involved in
the control of cell cycle and the regulation of gene transcription. Once bound to the regulatory
subunit cyclins, CDKs can phosphorylate the downstream substrates and activate various cellular
processes. Due to their intense involvement in many physiological processes and diseases, CDKs
have attracted extensive research interests1. Several of the CDKs play critical roles in cell cycle
progression and a number of CDK inhibitors are currently under clinical trials for the treatment of
various cancers2. As a member of the CDK family, CDK9 plays a pivotal role in transcriptional
regulation and is regarded as a potential target in oncology, virology and cardiology3. CDK9
complexes with cyclin T to form transcription elongation factor b (PꢀTEFb) and regulate gene
transcription by phosphorylating the Cꢀterminus of the large subunit of RNA polymerase II. It has
been reported that differential effects on HIV replication and Tꢀcell activation were observed upon
direct inhibition of the PꢀTEFb kinase activity in primary human peripheral blood lymphocytes4.
Therefore, selective CDK9 inhibitors have been actively pursued5ꢀ9 and evaluated as potentially
effective agents to block HIVꢀ1 replication5ꢀ7. The Xꢀray crystal structures of these inhibitors
complexed with CDK9 as well as CDK2 have been resolved and analyzed to investigate the
structural basis for isotype selectivity10ꢀ12. Nevertheless, recent studies13,14 have also suggested
implication of CDK2 in HIVꢀ1 transcription regulation through functional link with CDK9 (for
example, by phosphorylating CDK9 14). Therefore, compounds with good potency against CDK2
and CDK9 might be therapeutically useful in the treatment of cancer and HIV infection.
To search for new CDK inhibitors, especially CDK9 inhibitors as antiꢀcancer and antiꢀHIV
agents, we have previously established quantitative pharmacophore models based on a diverse set of
CDK9 inhibitors15. The most representative model depicted a pharmacophore with two
hydrogenꢀbond acceptor features and two hydrophobic aromatic features. As illustrated in Figure 1a
,
such pharmacophore matches well with the chemical features of flavopiridol (FVP), a flavonoid
4

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panꢀCDK inhibitor and the most potent inhibitor against PꢀTEFb identified to date16. The interaction
mode of FVP with CDK9 has been revealed by the crystal structure of the FVPꢀCDK9 complex17. As
shown in Figure 1b, the FVP molecule occupies the ATPꢀbinding site of CDK9. The O4 oxygen and
the O5 hydroxyl of FVP form hydrogen bonds with the CDK9 hinge residues Cys106 and Asp104,
respectively. An additional hydrogen bond is observed between the O3 hydroxyl and the Asp167 side
chain. Moreover, the chloroꢀphenyl ring of FVP makes hydrophobic contacts with Ile25, and the
protonated N1 of the piperidinyl group presents electrostatic interaction with Asp167. Therefore, a
composite pharmacophore with three hydrogen bond forming atoms, one hydrophobic aromatic
center and one positively charged center was constructed (Figure 1c). Remarkably, the quantitative
and the composite pharmacophore models are in good consistency.
(a)
(b)
(c)
Figure 1. Pharmacophore models for CDK9 inhibitors. (a) a 3DꢀQSAR pharmacophore (with
hydrogenꢀbond acceptors represented in green and hydrophobic aromatic features represented in
cyan) derived from diverse CDK9 inhibitors and its mapping with FVP; (b) Key interactions between
FVP and CDK9 revealed by their coꢀcrystal structure; (c) a composite pharmacophore extracted from
the interaction mode shown in (b). The hydrogen bond forming atoms are represented in green, the
hydrophobic aromatic center is represented in cyan and the positively charged center is represented
in purple.
We report herein the design, synthesis and biological evaluation of a series of novel
5

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5ꢀfluoroꢀN2 N4ꢀdiphenylpyrimidineꢀ2,4ꢀdiamine derivatives. This novel class of compounds were
,
designed by following a pharmacophoreꢀoriented scaffoldꢀhopping strategy. The hybrid
pharmacophore combining key features in the 3DꢀQSAR and composite models demonstrated in
Figure 1 was used to guide the molecular design (Figure 2). To ensure kinase inhibition and convey
structural novelty, the 2ꢀaminopyrimidine core structure, which is a privileged scaffold for CDK
inhibitors8,18,19, was utilized for the design. 5ꢀFluoro substitution was introduced according to
previous
structureꢀactivity
relationship
studies
on
CDK9
inhibitors
with
the
2ꢀanilinoꢀ4ꢀheteroarylpyrimidine scaffold8,18. The general formula and the pharmacophoric features
of the target compounds are shown in Figure 2.
Figure 2. General formula of target compounds and their pharmacophoric features.
R1 contains a hydrogen bond forming atom and/or a positively charged center. Various R2 and R3
substituents are incorporated on the two phenyl rings to meet the pharmacophoric requirements.
2. Results and discussion
2.1 Chemistry
The general synthetic route for compounds 6aꢀ6f was shown in Scheme 1. Compound 2a was
prepared through a crossꢀcoupling reaction between 1ꢀbromoꢀ3ꢀnitrobenzene (1a) and morpholine20,
and then followed by catalytic hydrogenation to afford compound 3a. Compounds 3b and 3c were
synthetized similarly. Compounds 5aꢀ5d were prepared by regioꢀselective substitution of the
appropriate amines to 2,4ꢀdichloroꢀ5ꢀfluoropyrimidine21. Further substitutions between 3aꢀ3c and
5aꢀ5d in alcoholic solvent afforded the target compounds 6aꢀ6f 22.
6

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R3
R3
R3
NO2
NO2
NH2
a
b
R3
Br
R1
R1
H
N
N
d
2
3
1
N
F
1a R3=H
2a,3a
R1=morpholino; R3=H
R1
HN
R2
1b
R3=OCH3
2b,3b R1=morpholino; R3=OCH3
2c,3c
R1=4-methylpiperazin-1-yl; R3=OCH3
6
Cl
N
Cl
N
c
6a-6d R1=morpholino; R3=H
N
N
6e
6f
R1=morpholino; R3=OCH3
R1=4-methylpiperazin-1-yl; R3=OCH3
F
F
Cl
HN
R2
R2 see Table 1
4
5
5a R2=3,5-dichlorophenyl
5b
-
R2= p fluorophenyl
5c R2=3,4-dichlorophenyl
5d
R2=2,6-difluorophenyl
H
H
N
H
N
N
N
N
N
f
b
N
N
N
F
F
F
HN
O
HN
NH2
HN
NO2
HN
Cl
N
R1
F
N
e
F
F
9
7
8
F
HN
9a-9h see Table 2
H
N
H
N
N
F
N
f
5b
N
HN
N
F
F
COOH HN
O
R1
F
F
10
11
11a-11p see Table 2
Scheme 1. Reagents and conditions: a. morpholine or
Nꢀmethyl piperazine, CuI, Lꢀproline, K2CO3,
DMSO, N2, 100ꢀ110oC; b. Pd/C, H2, CH3OH or THF; c. the appropriate amine, Et3N, EtOH; d.
CF3COOH,
CF3COOH,
i
ꢀPrOH or 2ꢀBuOH, reflux; e. 3ꢀNitroaniline (for 7) or 3ꢀaminobenzoic acid (for 10),
iꢀPrOH or 2ꢀBuOH, reflux; f. the appropriate acid or amine, DIEA, HATU, DMF.
The preparation of compounds 9aꢀ9h and 11aꢀ11p was also illustrated in Scheme 1. Compound
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5b was reacted with
respectively. Catalytic hydrogenation of compound 7 in the presence of Pd/C produced compound 8.
Both and 10 were subjected to condensation reactions under the treatment of
2ꢀ(1 ꢀ7ꢀazabenzotriazolꢀ1ꢀyl)ꢀ1,1,3,3ꢀtetramethylꢀuronium hexafluorophosphate (HATU) and
mꢀnitroaniline and mꢀaminobenzoic acid to give compounds 7 and 10,
8
H
N,Nꢀdiisopropylethylamine (DIEA) to afford the target amide compounds 9aꢀ9h and 11aꢀ11p 23.
2.2 Biological evaluation
All the target compounds were initially tested against the tumor cell lines of A594, DU145, KB
and KBvin with the sulforhodamine B (SRB) assay. The inhibitory activities of the compounds in
terms of GI50 values were shown in Tables 1, 2 and 3.
The cytotoxic results suggested that fluoroꢀsubstitution on 4ꢀanilino might be important for the
antiꢀproliferative effects (6a and 6b versus 6c and 6d). Therefore, pꢀfluoroanilino was retained at R2,
while methoxyl substitution was introduced at R3 (6e), and morpholino was further replaced with
4ꢀmethylpiperazinꢀ1ꢀyl (6f) to explore their effects on cytotoxicity. Although the introduction of
methoxyl group at R3 decreased the inhibitory activity (6c versus 6e), the presence of
4ꢀmethylpiperazinꢀ1ꢀyl instead of morpholino at R1 significantly increased potency (6e versus 6f).
Such observation implicated that the electrostatic interaction between the residue Asp167 and the
protonated nitrogen in the piperidinyl group is more advantageous over the hydrogen bonding
interaction between the oxygen atom in the morpholino moiety and Asp167. Therefore, positively
charged atoms should be preferred in the close proximity to R1.
Compounds 9aꢀ9h and 11aꢀ11p retained the pꢀfluoroanilino at R2, while incorporating diverse
R1 substituents with different sizes, electronic effects and hydrogen bond forming capability to
further investigated the structureꢀactivity relationships at this molecular area. For both compounds 9
and 11, aliphatic substituents, either linear or cyclic, were generally favored over aromatic
substituents at R1. The results implied that the R1 substituents might serve as a counterpart of the
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sugar moiety in the ATP structure, therefore, aliphatic substituents might be more likely to meet the
requirements for optimal pharmacophoric orientation and molecular shape of CDK inhibitors. In
addition, compounds with a positively charged center were more potent than their congeners with
only hydrogen bond forming atoms (9g versus 9h; 11a versus 11b; 11f versus 11g), which suggests
the importance of electrostatic interactions in this molecular area. The series 9 were generally more
potent than the series 11. For compounds with aromatic substituents at R1 of 9, electronꢀwithdrawing
groups might be preferred over electronꢀdonating moieties for cytotoxicity (9b versus 9c; 9a versus
9d). However, electrostatic properties showed no definite effects on the cytotoxicity of compounds in
series 11.
Table 1. Inhibitory activities of compounds 6a-6f against tumor cell growth.
a
GI50 (ꢀM)
Compd.
6a
R2
A549 b
DU145
KB
KBvin
11.59 ± 1.03
> 10 c
14.80 ± 1.02
22.47 ± 2.46
6b
6c
12.02 ± 0.90
3.83 ± 1.18
> 10
> 10
> 10
> 10
> 10
> 10
1.09 ± 0.46
4.24 ± 1.05
> 10
> 10
> 10
> 10
6d
6e
F
18.28 ± 3.34
> 10
6f
1.13 ± 0.61
0.91 ± 0.12
0.96 ± 0.21
0.91 ± 0.30
ꢀ
0.14 ± 0.007
0.15 ± 0.007
0.16 ± 0.007
0.18 ± 0.005
FVP
a The GI50 values are the average of three independent measurements;
b
Cell lines: A549 (lung cancer), DUꢀ145 (prostate cancer), KB (nasopharyngeal carcinoma) and
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KBvin (vincristineꢀresistant KB subline);
c No significant inhibition against tumor cell growth was observed under the concentration of 10
ꢀM.
Table 2. Inhibitory activities of compounds 9a-9h against tumor cell growth
a
GI50
DU145
(ꢀM)
Compd.
R1
A549 b
KB
KBvin
> 10 c
> 10
> 10
9a
9b
37.82 ± 4.88
7.25 ± 0.96
12.62 ± 0.70
15.10 ± 1.16
11.35 ± 2.08
> 10
> 10
> 10
> 10
9c
9d
1.65 ± 0.67
10.29 ± 1.98
1.33 ± 0.10
1.18 ± 0.13
4.33 ± 2.39
26.38 ± 4.57
1.15 ± 0.33
1.21 ± 0.08
5.28 ± 3.49
23.55 ± 3.92
1.70 ± 0.80
1.26 ± 0.05
4.57 ± 2.58
20.10 ± 2.95
11.93 ± 1.92
1.81 ± 0.60
9e
9f
9g
9h
2.05 ± 0.52
4.46 ± 0.70
6.25 ± 1.38
3.94 ± 1.04
ꢀ
0.14 ± 0.007
0.15 ± 0.007
0.16 ± 0.007
0.18 ± 0.005
FVP
a The GI50 values are the average of three independent measurements.
b
Cell lines: A549 (lung cancer), DUꢀ145 (prostate cancer), KB (nasopharyngeal carcinoma) and
KBvin (vincristineꢀresistant KB subline);.
c No significant inhibition against tumor cell growth was observed under the concentration of 10
ꢀM.
Table 3. Inhibitory activities of compounds 11a-11p against tumor cell growth
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a
GI50
DU145
(ꢀM)
Compd.
R1
A549 b
KB
KBvin
11a
11b
11c
11d
11.21 ± 2.06
11.94 ± 0.81
10.89 ± 0.82
13.02 ± 0.65
17.91 ± 1.09
14.58 ± 2.93
12.58 ± 3.05
14.78 ± 2.53
20.29 ± 4.09
13.05 ± 2.24
9.07 ± 1.30
13.21 ± 1.45
12.22 ± 1.95
12.27 ± 1.90
13.81 ± 0.87
> 10 c
11e
11f
11g
9.96 ± 0.60
1.45 ± 0.07
15.65 ± 1.44
4.34 ± 1.40
14.88 ± 2.20
4.16 ± 0.69
16.68 ± 1.46
7.26 ± 3.65
11.85 ± 2.08
13.38 ± 2.60
12.39 ± 2.48
21.40 ± 2.38
> 10
> 10
> 10
> 10
11h
11i
11j
> 10
> 10
> 10
> 10
> 10
> 10
11.03 ± 0.52
15.71 ± 1.38
11k
13.85 ± 0.15
13.67 ± 1.72
15.47 ± 1.58
12.74 ± 1.65
12.86 ± 0.71
17.45 ± 2.49
11.58 ± 0.18
17.50 ± 3.03
14.08 ± 0.79
20.08 ± 1.06
12.01 ± 1.66
20.73 ± 3.14
11l
11m
15.30 ± 0.86
15.06 ± 1.54
20.03 ± 10.96
13.67 ± 1.59
11n
11o
> 10
> 10
> 10
> 10
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11p
10.12 ± 0.80
0.14 ± 0.007
15.10 ± 2.27
0.15 ± 0.007
14.57 ± 1.70
0.16 ± 0.007
13.12 ± 0.96
0.18 ± 0.005
ꢀ
FVP
a The GI50 values are the average of three independent measurements.
b
Cell lines: A549 (lung cancer), DUꢀ145 (prostate cancer), KB (nasopharyngeal carcinoma) and
KBvin (vincristineꢀresistant KB subline);.
c No significant inhibition against tumor cell growth was observed under the concentration of 10
ꢀM.
Nine of the most cytotoxic compounds were further evaluated against CDK2/cyclin E1 and
CDK9/cyclin T1. Most of these compounds exhibited potent inhibitory activities against both
systems (Table 4). Due to limited data, it is difficult to deduce informative SAR from the results.
However, for most compounds, the molecular potency generally correlated well with the cellular
cytotoxicity, which indicated that CDK inhibition might be responsible for the cytotoxicity of these
compounds. Compound 6f was the only exception. The discrepancy between cellular and molecular
activities of compound 6f implicated the involvement of alternative molecular mechanisms other
than CDK inhibition. For all the compounds tested at molecular level, only compounds 6d and 9d
showed moderate selectivity to CDK9 over CDK2.
Table 4. Inhibitory activities of selected compounds against CDK2/cyclin E1 and CDK9/cyclin T1
IC50 (ꢀM)*
Compd.
CDK2/CyclinE1
CDK9/CyclinT1
5.85
> 10 #
15.30
1.24
0.33
> 10
2.57
2.45
0.78
6d
6f
9d
9f
1.71
9g
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3.27
22.10
60.60
4.65
2.02
11.30
9h
11b
11e
11f
15.60
1.72
FVP
0.13
4.59 (nM)
* The IC50 value was interpolated from doseꢀresponse data with at least six different concentrations.
Each test was performed in duplicate. Data represent the average of duplicate measurements.
# No significant inhibition was observed at the concentration of 10
ꢀM.
To determine whether selective CDK9 inhibition correlates with antiꢀHIV activity, three
compounds (6d, 11e and 11f) with different level of selectivity to CDK9 were tested in an antiꢀHIVꢀ1
assay. Appreciable inhibition was observed for all three compounds. Notably, the results did reveal a
trend that higher selectivity to CDK9 might lead to better therapeutic index (Table 5). Further
validation with more CDK9 inhibitors is undergoing.
Table 5. AntiꢀHIVꢀ1 activities of selected compounds.
*
*
Compd.
EC50
IC50
Selectivity
TI#
CDK2/CDK9
(ꢀM)
17.7
3.9
7.45
2.18
1.08
4.47
1.37 ± 0.42
7.08 ± 1.54
3.15 ± 0.66
0.015 ± 0.005
10.22 ± 1.27
15.45 ± 1.72
3.40 ± 0.27
6d
11e
11f
2.7
28.3
FVP
0.067 ± 0.007
* The EC50 and IC50 values are the average of three independent measurements;
# TI, therapeutic index, the ratio of the IC50 value to the EC50 value.
2.3 Molecular modeling
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Docking studies on selected compounds were performed to recognize their interaction modes
with CDKs. As exemplified by the docking poses of compound 9g in the ATPꢀbinding sites of CDK9,
two alternative binding poses were suggested (Figure 3). For pose A (Figure 3a), the molecule
assumed an orientation similar to that of FVP. Besides the hydrogen bond between Nꢀ1 atom of the
pyrimidine ring and Cys106, additional hydrogen bonds were observed between the 2ꢀamino and
Asp104, as well as the carboxyl oxygen and Asp167. Electrostatic interaction between the nitrogen in
N,Nꢀdimethyl glycine and Asp167 were also monitored. Alternatively, compound 9g could also adopt
pose B (Figure 3b). It was a turnover of pose A, where the N,Nꢀdimethyl glycine moiety occupied
the hydrophobic subpocket engaged by the chloroꢀphenyl of FVP. The Nꢀ1 atom and 2ꢀamino moiety
on the pyrimidine ring interacted with the hinge residue Cys106 of CDK9 through hydrogen bonding,
while the 4ꢀanilino group protruded toward Asp167. The electronꢀwithdrawing fluoroꢀsubstitution on
N4ꢀphenyl made it electron deficient and presented electrostatic interaction with Asp 167. The
disclosure of alternative binding poses of this compound class might provide new clues for further
structural optimization.
(a)
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(b)
Figure 3. Alternative interaction modes of 9g as suggested by the docking study.
Compound 6d, the inhibitor with the highest selectivity toward CDK9, was docked to both
CDK2 and CDK9 to provide insights on structural basis for selective CDK9 inhibition. As shown in
Figure 4, compound 6d bound to the ATP sites of CDK2 and CDK9 with an orientation different
from that of FVP. It adopted a pose similar to pose B in Figure 3b. The prevalence of pose B for 6d
could be attributed to its structural features in R1 and R2. Due to the absence of protonable nitrogen
in R1, the electrostatic interaction between R1 and Asp145 in CDK2 or Asp167 in CDK9 was missed.
On the other hand, the electronꢀwithdrawing difluoroꢀsubstitution (R2) on N4ꢀphenyl strengthened its
interaction with Asp145 (CDK2) or Asp167 (CDK9).
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(a)
(b)
Figure 4. Interaction modes of 6d with CDK2 (a) and CDK9 (b) as suggested by the docking study.
As indicated in Figure 4, the key interactions of 6d in CDK2 and CDK9 were similar. Hydrogen
bonds existed between Nꢀ1 atom/2ꢀamino moiety of 6d and the hinge residue (Leu83 for CDK2 and
Cys105 for CDK9). The difluoroꢀsubstituted N4ꢀphenyl ring extended to the proximity of Asp145
(CDK2) or Asp167 (CDK9). The most obvious difference between the interaction modes of 6d with
CDK2 and CDK9 was the orientation of the morpholino ring in R1. Compound 6d assumed a
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condensed Uꢀconformation in the ATPꢀbinding site of CDK2, and the morpholino ring was virtually
parallel to the N4ꢀphenyl ring (Figure 4a). While, it was more extended in the binding site of CDK9
and the morpholino ring was basically vertical to the N4ꢀphenyl ring. The changing of the Lys89 in
CDK2 to Gly122 in CDK9 was responsible for the discrepancy in the interaction modes. Due to such
a difference, the binding site of CDK9 is relatively larger and can accommodate more steric moieties
in R1. The lone pair interaction between the nitrogen atom in morpholino and the Lys89 in CDK2
might cause further shift of the morpholino ring toward the N4ꢀphenyl. The structural insights gained
from docking studies might guide further molecular design of selective CDK9 inhibitors.
3. Experimental
3.1 Materials and methods
All reactions were monitored by thinꢀlayer chromatography with preꢀcoated silica gel F254
plates purchased from Merck, Inc. All melting points were determined on Yanaco Melting point
apparatus and are uncorrected. Mass spectra (MS) were taken in HRꢀESI mode on Exactive PlusTM
1
LC/MSD Orbitrap from Thermo Fisher Scientific. HꢀNMR spectra were recorded on Varian
Mercury 300ꢀMHz spectrometer. Chemicals were obtained from local suppliers and were used
without further purification.
3.2 Chemistry
3.2.1 Preparation of compound 3
2ꢀNitroꢀ4ꢀbromoanisole (2.32 g, 10 mmol), morpholine (3.58 g, 40 mmol), CuI (0.19 g, 0.1 mmol),
Lꢀproline (0.23 g, 2 mmol) and K2CO3 (2.76 g, 20 mmol) were dissolved in 20 ml DMSO and stirred
under nitrogen for 48 h at 100 °C. After cooling to room temperature, the reaction mixture was
poured into water and extracted with ethyl acetate, the combined organic layers were successively
washed by water and saturated solution of Na2CO3. The organic layer was dried over Na2SO4, and
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filtered. The solvents were removed under reduced pressure and the crude material was purified by
column chromatography on silica gel, eluted with petroleum ether/ethyl acetate (10:1) to afford
compound 2b as a yellow oil (1 g, 42%).
Compound 2b (1 g, 4.8 mmol) and Pd/C (0.1 g) were added in methanol and stirred under
hydrogen atmosphere for 24 h at room temperature. Pd/C was filtered by diatomite and the solvents
were removed under reduced pressure. The crude material was purified by column chromatography
on silica gel, eluted with petroleum ether/ethyl acetate (2:1) to afford compound 3b as a white solid
(0.3 g, 34%).
Compounds 3a and 3c were synthetized from different start materials using the same procedure
as described.
3.2.2 Preparation of compound 5
2,4ꢀDichloroꢀ5ꢀfluoroꢀuricpyrimidine (4, 1.67 g, 10 mmol), pꢀfluoroꢀaniline (1.11 g, 10 mmol) and
trifluoroacetic acid (1.52 g, 15 mmol) were mixed in 10 ml ethanol and stirred for 24 h at room
temperature. The precipitation was filtered and then washed by ethanol, dried over Na2SO4, and
filtered. The solvents were removed under reduced pressure getting 5b as a white solid (2.4 g, 99%).
Compound 5a, 5c and 5d were synthetized from different starting materials with the same
procedure as described.
3.2.3 Preparation of compound 6
To a solution of compound 3a (122 mg, 0.687 mmol) and compound 5d (200 mg, 0.687 mmol)
in 10 ml isopropanol, one drop of acetic acid was added. The reaction mixture was stirred at 110
℃
and was refluxed for 24 h. After cooling to room temperature, the precipitation was filtered to get
compound 6d as a white solid (200 mg, 40%).
Compounds 6a-6c and 6e-6f were synthetized from different starting materials using the same
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procedure as described.
Compound 6a: yield: 67 %, mp: 189ꢀ190°C, 1HꢀNMR (300 MHz, DMSOꢀd6)
：δ 10.07 (s, 1H), 9.74
(s, 1H), 8.28 (d, J = 3.9 Hz, 1H), 7.87 (s, 2H), 7.31 (s, 1H), 7.15ꢀ7.18 (m, 3H), 6.74 (s, 1H), 3.74 (s,
4H), 3.08 (s, 4H). HRꢀESIꢀMS: m/z = 434.0942 [M+H]+, calcd for C20H19N5OFCl2: 434.0945.
Compound 6b: yield: 90%, mp: 196ꢀ198°C, 1HꢀNMR (300 MHz, DMSOꢀd6)
：δ 10.40 (s, 1H), 10.14
(s, 1H), 8.32 (d, J = 4.2 Hz, 1H), 8.04 (d, J = 1.8 Hz, 1H), 7.72 (d, J = 7.2 Hz, 1H), 7.56 (d, J = 8.7
Hz, 1H), 7.18ꢀ7.25 (m, 2H), 7.09 (d, J = 7.5 Hz, 1H), 6.81 (d, J = 6.6 Hz, 1H), 3.73 (s, 4H), 3.06 (s,
4H). HRꢀESIꢀMS: m/z = 434.0952 [M+H]+, calcd for C20H19N5OFCl2: 434.0945.
Compound 6c: yield: 47%, mp: 220ꢀ222°C, 1HꢀNMR (300 MHz, DMSOꢀd6)
：δ 9.36 (s, 1H), 9.03 (s,
1H), 8.08 (d, J = 3.6 Hz, 1H), 7.78 (dd, J = 5.1, 9.3 Hz, 2H), 7.21 (s, 1H), 7.12 (t, J = 6.6 Hz, 3H),
7.05 (t, J = 8.1 Hz, 1H), 6.50 (d, J = 7.5 Hz, 1H), 3.68 (t, J = 4.8 Hz, 4H), 2.96 (t, J = 4.8 Hz, 4H).
HRꢀESIꢀMS: m/z = 384.1625 [M+H]+, calcd for C20H20N5OF2: 384.1630.
1
Compound 6d: yield: 40%, mp: 181ꢀ183°C, HꢀNMR ( 300 MHz, DMSOꢀd6)
：
δ 9.19 (s, 1H), 8.92
(s, 1H), 8.10 (d, J = 3.3 Hz, 1H), 7.36ꢀ7.43 (m, 1H), 7.22 (t, J = 8.1 Hz, 2H), 7.02 (t, J = 8.4 Hz, 2H),
6.85 (t, J = 8.1 Hz, 1H), 6.41 (d, J = 6.9 Hz, 1H), 3.66 (t, J = 4.8 Hz, 4H), 2.90 (t, J = 4.8 Hz, 4H).
HRꢀESIꢀMS: m/z = 402.1534 [M+H]+, calcd for C20H19N5OF3: 402.1536.
Compound 6e: yield: 23%, mp: 195ꢀ198°C, 1H NMR (400MHz, DMSOꢀd6)
：δ 9.40 (s,1H), 8.09(d, J
= 3.6 Hz,1H), 7.70ꢀ7.67 (m,4H), 7.12 (t, J = 8.8 Hz, 2H), 6.88 (d, J = 8.8 Hz, 1H), 6.51 (dd, J = 2.8,
8.8 Hz, 1H), 3.76 (s, 3H), 3.64 (t, J = 4.4 Hz, 4H), 2.82 (t, J = 4.4 Hz, 4H). HRꢀESIꢀMS: m/z =
414.1728 [M+H]+, calcd for C20H21N2O5: 414.1736.
Compound 6f: yield: 21%, mp: 71ꢀ73°C, 1HꢀNMR ( 300 MHz, DMSOꢀd6)
：δ 9.40 (s, 1H), 8.09 (d, J
= 5.4 Hz, 1H), 7.71ꢀ7.64 (m, 4H), 7.11 (t, J = 8.5 Hz, 2H), 6.86 (d, J = 9.0 Hz, 1H), 6.52 (dd, J= 3.9,
5.4 Hz, 1H), 3.75 (s, 3H), 2.87 (m, 4H), 2.38 (m, 4H), 2.20 (s, 3H). HRꢀESIꢀMS: m/z = 427.2040
[M+H]+, calcd for C22H25N6O F2: 427.2052.
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3.2.4 Preparation compound 8
Compound 5b (2.42 g, 10 mmol), mꢀnitroaniline (1.38 g, 10 mmol) and trifluoroacetic acid
(1.14 g, 10 mmol) were mixed in 10 ml isopropanol and stirred under reflux for 24 h. After cooling
to room temperature, the precipitation was filtered and recrystallized in CH2Cl2 to afford a light
yellow solid 2.5 g (7).
Compound 7 (2.5 g, 7.2 mmol) and Pd/C (0.25 g) were added in a mixture of methanol and THF,
and stirred under hydrogen atmosphere for 24 h at room temperature. Pd/C was filtered by diatomite
and the solvents were removed under reduced pressure and the crude material was recrystallized in
CH2Cl2 to afford compound 8 as a light yellow solid (2.0 g, yield for two steps: 64%).
3.2.5 Preparation compound 9
Compound 8 (150 mg, 0.479 mmol) and pyrazineꢀ2ꢀcarboxylic acid (65.3 mg, 0.527 mmol)
were dissolved in a mixture of CH2Cl2 and THF. HATU (218.5 mg, 0.576 mmol) and DIEA (379 mg,
2.874 mmol) were then added. The reaction mixture was stirred at room temperature for 24 h, and
was then poured into icy water. The precipitation was filtered and washed by dry methanol for
several times to afford compound 9e as a white solid (101 mg, 51%).
Compounds 9a-9d and 9f-9h were synthetized from different start materials using similar
procedure as described.
Compound 9a: yield: 43%, mp: 253ꢀ255°C, 1HꢀNMR (300 MHz, DMSOꢀd6)
：δ 10.16 (s, 1H), 9.37
(s, 1H), 9.25 (s, 1H), 8.10 (d, J = 3.3 Hz, 1H), 8.04 (s, 1H), 7.93 (d, J = 6.9 Hz, 2H), 7.85 (dd, J = 5.7,
8.7 Hz, 2H), 7.49ꢀ7.58 (m,3H), 7.41 (d, J = 8.1Hz, 1H), 7.26 (t, J = 7.5 Hz, 1H), 7.19 (t, J = 7.8 Hz,
1H), 7.08 (t, J = 9.0 Hz, 2H). HRꢀESIꢀMS: m/z = 418.1478 [M+H]+, calcd for C23H18N5OF2:
418.1474.
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Compound 9b: yield: 33%, mp: 242ꢀ243°C, 1HꢀNMR (300 MHz, DMSOꢀd6)
：
δ 10.50 (s, 1H), 9.39
(s, 1H), 9.30 (s, 1H), 8.77 (s, 1H), 8.41 (dd, J = 9.0, 12.9 Hz, 2H), 8.09 (d, J = 10.2 Hz, 2H), 7.84 (t,
J = 6.9 Hz, 3H), 7.42 (d, J = 7.2 Hz, 1H), 7.20ꢀ7.30 (m, 2H), 7.07 (t, J = 8.4 Hz, 3H). HRꢀESIꢀMS:
m/z = 463.1329 [M+H]+, calcd for C23H17N6O3F2: 463.1325.
1
Compound 9c: yield: 99%, mp: > 285°C, H NMR (300MHz, DMSOꢀd6)
：
δ 9.77 (s, 1H), 9.34 (s,
1H), 9.18 (s, 1H), 8.08 (d, J = 3.6 Hz, 2H), 7.98 (s, 1H), 7.86ꢀ7.81 (m, 3H), 7.35 (d, J = 8.1 Hz, 1H),
7.24 (d, J = 8.4 Hz, 1H), 7.17ꢀ7.04 (m, 3H), 6.73 (d, J = 9 Hz, 2H), 2.98 (s, 6H). HRꢀESIꢀMS: m/z =
461.1885[M+H]+, calcd for C20H21N2O5: 461.1896.
Compound 9d: yield: 48%, mp: 280°C (dec.), 1H NMR (400MHz, DMSOꢀd6)
：δ 10.40 (s, 1H), 9.38
(s, 1H), 9.28 (s, 1H), 8.77 (d, J = 5.2 Hz, 2H), 8.10 (d, J = 3.6 Hz, 1H), 8.04 (s, 1H), 7.84ꢀ7.81 (m,
4H), 7.43 (d, J = 8 Hz, 1H), 7.28ꢀ7.19 (m, 2H), 7.10ꢀ7.06 (m, 2H). HRꢀESIꢀMS: m/z =
419.1414[M+H]+, calcd for C20H21N2O5: 419.1426.
Compound 9e: yield: 51%, mp: 265ꢀ266°C, 1HꢀNMR (300 MHz, DMSOꢀd6)
：δ 10.47 (s,1H), 9.39 (s,
1H), 9.28 (d, J = 10.2 Hz, 2H), 8.92 (s, 1H), 8.79 (s, 1H), 8.12 (dd, J = 3.6, 10.2 Hz, 2H), 7.09 (m,
2H), 7.38 (d, J = 7.5 Hz, 2H), 7.22 (t, J = 8.1 Hz, 1H), 7.07 (t, J = 8.7 Hz, 2H). HRꢀESIꢀMS: m/z =
420.1381 [M+H]+, calcd for C21H16N7OF2: 420.1379.
Compound 9f: yield: 35%, mp: 272°C (dec.), 1HꢀNMR (300 MHz, DMSOꢀd6)
：δ 9.73 (s, 1H), 9.36
(s, 1H), 9.17 (s, 1H), 8.08 (d, J = 3.6 Hz, 1H), 7.81ꢀ7.87 (m, 3H), 7.32 (d, J = 8.4 Hz, 1H), 7.07ꢀ7.18
(m, 4H), 2.82 (d, J = 11.1 Hz, 2H), 2.17 (s, 3H), 1.88 (t, J = 9.6 Hz, 2H), 1.60ꢀ1.69 (m, 4H).
HRꢀESIꢀMS: m/z = 439.2055 [M+H]+, calcd for C23H25N6OF2: 439.2052.
Compound 9g: yield: 48%, mp: 206ꢀ208°C, 1HꢀNMR (300 MHz, DMSOꢀd6)
：δ 9.50 (s, 1H), 9.36 (s,
1H), 9.19 (s, 1H), 8.08 (d, J = 3.6 Hz, 1H), 7.85ꢀ7.82 (m, 3H), 7.36 (d, J = 8.4 Hz, 1H), 7.19 (d, J =
8.4 Hz, 1H), 7.20ꢀ7.10 (m, 3H), 3.04 (s, 2H), 2.26 (s, 6H). HRꢀESIꢀMS: m/z = 399.1743 [M+H]+,
calcd for C20H21N6OF2: 399.1739.
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Compound 9h: yield: 24%, mp: 211ꢀ214°C, 1H NMR (300MHz, DMSOꢀd6)
：δ 9.57 (s, 1H), 9.37 (s,
1H), 9.21 (s, 1H), 8.08 (d, J = 3.9 Hz, 1H), 7.85ꢀ7.81 (m, 3H), 7.38ꢀ7.35 (m, 1H), 7.16ꢀ7.11 (m, 4H),
3.96 (s, 2H), 3.36 (s, 3H). HRꢀESIꢀMS: m/z = 386.1421[M+H]+, calcd for C20H21N2O5: 386.1423.
3.2.6 Preparation compound 10
Compound 5d (2.42 g, 10 mmol), mꢀaminobenzoic acid (1.37 g, 10 mmol) and trifluoroacetic
acid (1.14 g, 10 mmol) were mixed in 15 mL isopropanol and was stirred under reflux for 24 h. After
cooling to room temperature, the precipitation was filtered and washed by CH2Cl2 for several times
to afford compound 10 as a white solid (2.5 g, 73%).
3.2.7 Preparation compound 11
Compound 10 (200 mg, 0.585 mmol) and morpholine (62 mg, 0.702 mmol) were dissolved in
DMF, HATU (267 mg, 0.702 mmol) and DIEA (302 mg, 2.34 mmol) were then added. The reaction
mixture was stirred at room temperature for 24 h and then was poured into icy water. The
precipitation was filtered to afford compound 11a as a white solid (188 mg, 78%).
Compound 11b-11p were synthetized from different start materials using similar procedure as
described.
Compound 11a: yield: 78%, mp: 174ꢀ176°C, 1H NMR (300MHz, DMSOꢀd6)
：δ 9.42 (s, 1H), 9.37 (s,
1H), 8.12 (d, J = 3.6 Hz, 1H), 7.78ꢀ7.74 (m, 3H), 7.65 (d, J = 7.5 Hz, 1H), 7.27 (t, J = 7.8 Hz, 1H),
7.15 (t, J = 8.7 Hz, 2H), 6.89 (d, J = 7.8 Hz, 1H), 3.55 (m, 6H), 3.31 (m, 2H). HRꢀESIꢀMS: m/z =
412.1574[M+H]+, calcd for C20H21N2O5: 412.1580.
Compound 11b: yield: 24%, mp: 184ꢀ185°C, 1H NMR (300MHz, DMSOꢀd6)
：δ 9.42 (s, 1H), 9.37 (s,
1H), 8.12 (d, J = 3.6 Hz, 1H), 7.78ꢀ7.74 (m, 3H), 7.65 (d, J = 8.1 Hz, 1H), 7.26 (t, J = 7.8 Hz, 1H),
7.15 (t, J = 8.7 Hz, 2H), 6.86 (d, J = 7.8 Hz, 1H), 3.57 (s, 2H), 3.31 (s, 2H), 2.26 (s, 4H), 2.16 (s, 3H).
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HRꢀESIꢀMS: m/z = 425.1883[M+H]+, calcd for C20H21N2O5: 425.1896.
Compound 11c: yield: 73%, mp: 197ꢀ199°C, 1HꢀNMR (300 MHz, DMSOꢀd6)
：
δ 9.41 (s, 1H), 9.35
(s, 1H), 8.12 (d, J = 3.6 Hz, 1H), 7.74ꢀ7.79 (m, 3H), 7.64 (d, J = 8.1 Hz, 1H), 7.25 (t, J = 8.1 Hz, 1H),
7.15 (t, J = 8.4 Hz, 2H), 6.84 (d, J = 7.5 Hz, 1H), 3.54 (brs, 2H), 3.24 (brs, 2H), 1.39ꢀ1.58 (brs, 6H).
HRꢀESIꢀMS: m/z = 410.1794 [M+H]+, calcd for C22H22N5OF2: 410.1787.
Compound 11d: yield: 78%, mp: 263ꢀ265°C, 1HꢀNMR (300 MHz, DMSOꢀd6)
：δ 9.42 (s, 1H), 9.38
(s, 1H), 8.12 (d, J = 3.6 Hz, 1H), 7.76 (t, J = 5.1 Hz, 3H), 7.68 (d, J = 8.4 Hz, 1H), 7.28 (t, J = 8.1 Hz,
1H), 7.16 (t, J = 8.7 Hz, 2H), 6.92 (d, J = 7.2 Hz, 1H), 3.55ꢀ3.80 (brs, 2H), 3.35ꢀ3.50 (brs, 2H),
3.00ꢀ3.20 (brs, 4H), 2.89(s, 3H). HRꢀESIꢀMS: m/z = 489.1518 [M+H]+, calcd for C22H23N6O3F2S:
489.1515.
Compound 11e: yield: 39%, mp:161ꢀ163°C, 1HꢀNMR (300 MHz, DMSOꢀd6)
：δ 9.42 (s, 1H), 9.37 (s,
1H), 8.12 (d, J = 3.0 Hz, 1H), 7.74ꢀ7.79 (m, 3H), 7.63 (d, J = 7.2 Hz, 1H), 7.26 (t, J = 7.5 Hz, 1H),
7.13 (t, J = 4.2 Hz, 2H), 6.76ꢀ6.83 (m, 1H), 3.50ꢀ3.61 (m, 3H), 3.40ꢀ3.45 (m, 3H), 1.97ꢀ2.02 (m, 3H),
1.79ꢀ1.85 (m, 2H), 1.63 (s, 1H), 1.49 (s, 1H). HRꢀESIꢀMS: m/z = 467.2015 [M+H]+, calcd for
C24H25N6O2F2: 467.2002.
1
Compound 11f: yield: 34%, mp: 174ꢀ176°C, HꢀNMR (300 MHz, DMSOꢀd6)
：
δ 9.39 (s, 1H), 9.32
(s, 1H), 8.20 (m, 1H), 8.10 (d, J = 3.6 Hz, 1H), 8.02 (s, 1H), 7.83ꢀ7.75 (m, 3H), 7.34ꢀ7.25 (m, 2H),
7.11 (t, J = 8.7 Hz, 2H), 2.36 (t, J = 7.2 Hz, 4H), 2.16 (m, 6H). HRꢀESIꢀMS: m/z = 413.1899 [M+H]+,
calcd for C21H23N6OF2: 413.1896.
Compound 11g: yield: 67%, mp: 212ꢀ214°C, 1HꢀNMR (300 MHz, DMSOꢀd6)
：δ 9.39 (s, 1H), 9.32
(s, 1H), 8.26 (t, J = 5.4 Hz, 1H), 8.11 (d, J = 3.9 Hz, 1H), 8.08 (s, 1H), 7.79ꢀ8.83 (m, 2H), 7.34 (t, J =
7.8 Hz, 1H), 7.29 (t, J = 7.8 Hz, 1H), 7.11 (t, J = 8.7 Hz, 2H), 4.69 (t, J = 5.7 Hz, 1H), 3.48 (dd, J =
6.3, 9.0 Hz, 2H), 3.29 (t, J = 6.0 Hz, 1H). HRꢀESIꢀMS: m/z = 386.1431 [M+H]+, calcd for
C19H18N5O2F2: 386.1423.
Compound 11h: yield: 5.7%, mp:247ꢀ248°C, 1HꢀNMR (300 MHz, DMSOꢀd6)
：δ 10.51 (s, 1H), 9.42
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(d, J = 5.7 Hz, 1H), 9.41 (s, 1H), 8.13ꢀ8.17 (dd, J = 3.9, 12.0 Hz, 2H), 7.98 (d, J = 8.4 Hz, 2H), 7.85
(d, J = 6.9 Hz, 1H), 7.79 (dd, J = 4.8, 9.0 Hz, 2H), 7.71 (d, J = 9.3 Hz, 2H), 7.47 (d, J = 8.1 Hz, 1H),
7.38 (t, J = 7.5 Hz, 1H), 7.07 (t, J = 8.7 Hz, 2H). HRꢀESIꢀMS: m/z = 486.1362 [M+H]+, calcd for
C24H17N5OF5: 486.1348.
Compound 11i: yield: 50%, mp: 269ꢀ271°C, 1HꢀNMR (300 MHz, DMSOꢀd6)
：δ 9.98 (s, 1H), 9.37 (s,
1H), 8.11 (t, J = 3.6 Hz, 2H), 7.81 (t, J = 8.7 Hz, 3H), 7.61 (d, J = 8.7 Hz, 2H), 7.44 (d, J = 8.4 Hz,
1H), 7.34 (t, J = 7.5 Hz, 1H), 7.09 (t, J = 8.7 Hz, 2H), 6.92 (d, J = 8.7 Hz, 2H), 3.72 (t, J = 4.8 Hz,
4H), 3.06 (t, J = 4.8 Hz, 4H). HRꢀESIꢀMS: m/z = 503.2010 [M+H]+, calcd for C27H25N6O2F2:
503.2002.
Compound 11j: yield: 69%, mp: 227ꢀ228°C, 1HꢀNMR (300 MHz, DMSOꢀd6)
：δ 10.05 (s, 1H), 9.39
(d, J = 3.6 Hz, 1H), 8.13 (d, J = 3.6 Hz, 2H), 7.78ꢀ7.83 (m, 3H), 7.65 (d, J = 8.4 Hz, 2H), 7.44 (d, J =
7.5 Hz, 1H), 7.34 (t, J = 7.8 Hz, 1H), 7.09 (t, J = 8.7 Hz, 2H), 6.90 (d, J = 8.7 Hz, 2H). HRꢀESIꢀMS:
m/z = 448.1591 [M+H]+, calcd for C24H20N5O2F2: 448.1580.
Compound 11k: yield:47 %, mp:214ꢀ216°C, 1HꢀNMR (300 MHz, DMSOꢀd6)
：δ 10.09 (s, 1H), 9.40
(s, 1H), 8.13 (s, 2H), 7.78ꢀ7.81 (m, 3H), 7.42 (t, J = 7.5 Hz, 1H), 7.36 (t, J = 7.5 Hz, 1H), 7.07ꢀ7.13
(m, 4H), 6.25 (s, 1H), 3.72 (s, 6H). HRꢀESIꢀMS: m/z = 478.1697 [M+H]+, calcd for C25H22N5O3F2:
478.1685.
Compound 11l: yield: 45%, mp: 224ꢀ226°C, 1HꢀNMR ( 300 MHz, DMSOꢀd6)
：δ 10.07 (s, 1H), 9.39
(s, 2H), 8.12 (t, J = 3.9 Hz, 2H), 7.78ꢀ7.82 (m, 3H), 7.41 (t, J = 1.8 Hz, 2H), 7.35 (t, J = 7.8 Hz, 1H),
7.16 (dd, J = 2.1, 8.4 Hz, 1H), 7.09 (t, J = 9.0 Hz, 2H), 6.87 (d, J = 8.4 Hz, 1H), 5.99 (s, 2H).
HRꢀESIꢀMS: m/z = 462.1383 [M+H]+, calcd for C24H18N5O3F2: 462.1372.
Compound 11m: yield: 25%, mp: 224ꢀ226°C, 1HꢀNMR (300 MHz, CD3OD)
：δ 8.13 (s, 1H), 7.87 (d,
J = 3.0 Hz, 1H), 7.65 (d, J = 1.5 Hz, 2H), 7.56ꢀ7.60 (m, 3H), 7.38 (d, J = 7.2 Hz, 1H), 7.28 (t, J = 7.5
Hz, 1H), 7.10 (s, 1H), 6.88 (t, J = 8.7 Hz, 2H). HRꢀESIꢀMS: m/z = 486.0710 [M+H]+, calcd for
C23H16N5OF2Cl2: 486.0695.
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1
Compound 11n: yield: 26%, mp: 131ꢀ133°C, HꢀNMR ( 300 MHz, DMSOꢀd6)
：
δ 10.58 (s, 1H),
9.39 (d, J = 5.7 Hz, 2H), 8.36 (d, J = 3.6 Hz, 1H), 8.12ꢀ8.19 (m, 3H), 7.77ꢀ7.86 (m, 4H), 7.74 (d, J =
7.8 Hz, 1H), 7.30 (t, J = 8.1 Hz, 1H), 7.05ꢀ7.16 (m, 3H). HRꢀESIꢀMS: m/z = 419.1426 [M+H]+,
calcd for C22H17N6OF2: 419.1426.
Compound 11o: yield: 100%, mp: > 285°C, 1H NMR(300MHz, DMSOꢀd6)
：δ 9.32 (s, 1H), 9.18 (s,
1H), 8.23 (s, 1H), 8.09 (d, J = 3.6 Hz, 1H), 7.88ꢀ7.84 (m, 2H), 7.67 (d, J = 7.5 Hz, 2H), 7.20ꢀ7.18 (m,
2H), 7.15ꢀ7.07 (m, 3H), 6.60 (s, 1H). HRꢀESIꢀMS: m/z = 425.0984[M+H]+, calcd for C20H21N2O5:
425.0991.
Compound 11p:yield: 48%, mp: 221ꢀ223°C, 1HꢀNMR ( 300 MHz, DMSOꢀd6)
：δ 10.06 (s, 1H), 9.36
(s, 1H), 9.23 (s, 1H), 8.10 (d, J = 3.6 Hz, 1H), 8.03 (s, 1H), 7.82 (dd, J = 4.8, 9.0 Hz, 2H), 7.07ꢀ7.43
(m, 7H), 3.96 (s, 3H). HRꢀESIꢀMS: m/z = 422.1538 [M+H]+, calcd for C21H18N7OF2: 422.1535.
3.3 Biological evaluation
3.3.1 Cytotoxic activity assay
The following human tumor cell lines were used in the assay: A549 (lung cancer), DUꢀ145 (prostate
cancer), KB (nasopharyngeal carcinoma) and KBvin (vincristineꢀresistant KB subline). All cell lines
were obtained from the Lineberger Comprehensive Cancer Center (UNCꢀCH) or from ATCC
(Rockville, MD) and were cultured in RPMIꢀ1640 medium supplemented with 25 mM HEPES, 0.25%
sodium bicarbonate, 10% fetal bovine serum, and 100 mg/mL kanamycin.
The cytotoxicity assay was performed by following experimental protocols described in the
reference24. Freshly trypsinized cell suspensions were seeded in 96ꢀwell microtiter plates at densities
of 1500ꢀ7500 cells per well with compounds added from DMSOꢀdiluted stock. After 3 days in
culture, attached cells were fixed with cold 50% trichloroacetic acid and then stained with 0.4%
sulforhodamine B (SRB). The absorbance at 562 nm was measured using a microplate reader after
solubilizing the bound dye. The mean IC50 is the concentration of agents that reduces cell growth by
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50% under the experimental conditions and it is the average from three independent determinations
that are reproducible and statistically significant.
3.3.2 CDK2/Cyclin E1 kinase assay
The kinase assay against CDK2/cyclinE1 was performed by following experimental protocols
described in the reference25. The compounds were mixed with the enzyme (2.28 ng) in a buffer
containing 40 mM Hepes/Tris(pH 7.4), 0.8 mM EGTA/Tris, 8 mM MgCl2, 1.6 mM DTT and 0.008%
Tween 20. Thereafter, the reaction was initiated by adding 100 nM of the substrate
UlightꢀCFFKNIVTPRTPPPSQGKꢀamide (MBP) and 10 µM ATP, and the mixture was incubated
for 15 minutes at room temperature. For control basal measurements, the enzyme was omitted from
the reaction mixture. Following incubation, the reaction was stopped by adding 13 mM EDTA. After
5 minutes, the antiꢀphosphoꢀMBP antibody labeled with europium chelate was added. After 60 more
minutes, the fluorescence transfer was measured at λex=337 nm, λem=620 nm and λem=665 nm
using a microplate reader (Envision, Perkin Elmer). The enzyme activity was determined by dividing
the signal measured at 665 nm by that measured at 620 nm (ratio). The results were expressed as a
percent inhibition of the control enzyme activity. The compounds were tested in each experiment at
several concentrations to obtain an inhibition curve from which the IC50 values were calculated.
3.3.3 CDK9/Cyclin T1 kinase assay
The kinase assay against CDK9/cyclinT1 was performed by following experimental protocols
described in the reference26. The compounds were mixed with the enzyme (21.72 ng) in a buffer
containing 40 mM Hepes/Tris(pH 7.4), 0.8 mM EGTA/Tris, 8 mM MgCl2, 1.6 mM DTT and 0.008%
Tween 20. Thereafter, the reaction was initiated by adding 100 nM of the substrate
UlightꢀCFFKNIVTPRTPPPSQGKꢀamide (MBP) and 10 µM ATP, and the mixture was incubated
for 90 minutes at room temperature. For control basal measurements, the enzyme was omitted from
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the reaction mixture. Following incubation, the reaction was stopped by adding 13 mM EDTA. After
5 minutes, the antiꢀphosphoꢀMBP antibody labeled with europium chelate was added. After 60 more
minutes, the fluorescence transfer was measured at λex=337 nm, λem=620 nm and λem=665 nm
using a microplate reader (Envision, Perkin Elmer). The enzyme activity was determined by dividing
the signal measured at 665 nm by that measured at 620 nm (ratio). The results were expressed as a
percent inhibition of the control enzyme activity. The compounds were tested in each experiment at
several concentrations to obtain an inhibition curve from which the IC50 values were calculated.
3.3.4 Anti-HIV-1 assay
The antiꢀHIVꢀ1 assay was performed with NL4ꢀ3 virus by following experimental protocols
described in the reference27. A 96ꢀwell microtiter plate was used to set up the HIVꢀ1 NL4ꢀ3
replication assay. HIVꢀ1 NL4ꢀ3 at a multiplicity of infection (MOI) of 0.01 was used to infect MT4
cells. Culture supernatants were collected on day 4 post infection for the p24 assay using an ELISA
kit from ZeptoMetrix Corporation (Buffalo, New York).
3.4 Molecular modeling
The structures of compounds 6d and 9g were generated and molecular docking was performed with
the Discovery Studio 2.5 software package (Accelrys, San Diego, USA). The structures of CDK2
and CDK9 were obtained from the Protein Data Bank (PDB codes: 4BCP and 4BCG). The docking
calculation was carried out with the CDOCK protocol. Default settings were used. All calculations
were performed on a DELL Precision T5500 workstation.
4. Conclusions
Thirty novel 5ꢀfluoroꢀN2,N4ꢀdiphenylpyrimidineꢀ2,4ꢀdiamine derivatives were designed as potential
CDK, especially CDK9 inhibitors. Cytotoxicity for all the target compounds was measured with the
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SRB assay. Compounds with GI50 values against tumor cells at lower micromolar to submicromolar
level were identified. Some most cytotoxic compounds exhibited potent inhibitory activities against
both CDK2/cyclin E1 and CDK9/cyclin T1. Good correlation between cellular and molecular
activities was observed. Noticeable inhibition against HIVꢀ1 replication was also detected in an
antiꢀHIVꢀ1 assay, and there appeared to be a correlation between selective CDK9 inhibition and the
antiꢀHIVꢀ1 therapeutic index. Docking studies on this compound class hinted the existence of
alternative binding poses and explained the realization of isotype selectivity, which provided new
clues for further structural optimization.
Acknowledgements
This investigation was supported by the National Natural Science Foundation of China (81172985
and 81261120391) awarded to Z. Xiao. Partial support was also provided by NIH grants CA177584
from the National Cancer Institute and AI 033066 from the National Institute of Allergy and
Infectious Disease awarded to K.H. Lee.
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