Design, synthesis and biological evaluation of novel 2-phenyl pyrimidine derivatives as potent Bruton's tyrosine kinase (BTK) inhibitors

Article abstract of DOI:10.1039/c8md00413g

BTK is an effective target for the treatment of B-cell malignant tumors and autoimmune diseases. In this work, a series of 2-phenyl pyrimidine derivatives were prepared and their preliminary in vitro activities on B-cell leukemia cells as well as the BTK enzyme were determined. The results showed that compound 11g displayed the best inhibitory activity on BTK with an inhibition rate of 82.76% at 100 nM and excellent anti-proliferation activity on three B-cell leukemia lines (IC₅₀ = 3.66 μM, 6.98 μM, and 5.39 μM against HL60, Raji and Ramos, respectively). Besides, the flow cytometry analysis results indicated that 11g inhibited the proliferation of the Raji cells in a dose- and time-dependent manner, and blocked the Ramos cells at the G0/G1 phase, which is in accordance with the positive control ibrutinib. The mechanism investigation demonstrated that 11g could inhibit the phosphorylation of BTK and its downstream substrate phospholipase γ2 (PLCγ2). All these results showed that 11g was a promising lead compound that merited further optimization as a novel class of BTK inhibitor for the treatment of B-cell lymphoblastic leukemia.

Full text of DOI:10.1039/c8md00413g

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RESEARCH ARTICLE
Design, synthesis and biological evaluation of
novel 2-phenyl pyrimidine derivatives as potent
Cite this: DOI: 10.1039/c8md00413g
Bruton's tyrosine kinase (BTK) inhibitors†
Xinyu Li,‡ Binyu Shi,‡ Yu Teng, Yu Cheng, Huizhu Yang, Jiurong Li, Lianjian Wang,
*
Siying He, Qidong You and Hua Xiang
BTK is an effective target for the treatment of B-cell malignant tumors and autoimmune diseases. In this
work, a series of 2-phenyl pyrimidine derivatives were prepared and their preliminary in vitro activities on
B-cell leukemia cells as well as the BTK enzyme were determined. The results showed that compound 11g
displayed the best inhibitory activity on BTK with an inhibition rate of 82.76% at 100 nM and excellent anti-
proliferation activity on three B-cell leukemia lines (IC50 = 3.66 μM, 6.98 μM, and 5.39 μM against HL60,
Raji and Ramos, respectively). Besides, the flow cytometry analysis results indicated that 11g inhibited the
proliferation of the Raji cells in a dose- and time-dependent manner, and blocked the Ramos cells at the
G0/G1 phase, which is in accordance with the positive control ibrutinib. The mechanism investigation dem-
onstrated that 11g could inhibit the phosphorylation of BTK and its downstream substrate phospholipase γ2
(PLCγ2). All these results showed that 11g was a promising lead compound that merited further optimiza-
tion as a novel class of BTK inhibitor for the treatment of B-cell lymphoblastic leukemia.
Received 29th August 2018,
Accepted 23rd December 2018
DOI: 10.1039/c8md00413g
rsc.li/medchemcomm
first-generation BTK inhibitor. It was approved by the FDA
for the treatment of mantle cell lymphoma (MCL) in 2013,
Introduction
Bruton's tyrosine kinase (BTK) is a member of the TEC family
of non-receptor tyrosine kinase which is predominantly
expressed in all hematopoietic cells except plasma and T
cells.1 It is a crucial kinase in the B-cell receptor (BCR) signal-
ing pathway.2,3 The overexpression of BTK usually causes se-
vere leukemia and B-cell-related lymphoma. Therefore, the de-
velopment of drug molecules targeting BTK has become an
attractive therapeutic approach.
and was further used as a monotherapy to treat chronic lym-
phocytic leukaemia (CLL) in 2014.7 In 2015 and 2017,
ibrutinib was approved for the treatment of Waldenstrom's
macroglobulinaemia (WM) and chronic graft-versus-host dis-
ease (cGVHD), respectively.8 Despite the great success
achieved by ibrutinib, its off-target effect also leads to the
clinically observed adverse effects including arthralgias, rash,
atrial fibrillation, diarrhea, ecchymosis and massive hemor-
rhage due to ibrutinib's irreversible binding with other ki-
nases such as epidermal growth factor receptor (EGFR), ITK,
and TEC.9 Moreover, most of the BTK inhibitors mentioned
above have been reported to have limited efficacy and strong
side effects. Hence, there is still an increasing need to dis-
cover novel BTK inhibitors which could be developed into
therapeutic candidates for the treatment of B-cell
malignancies.10
Since BTK was discovered in the 1990s,4 many small-
molecule BTK inhibitors have been developed. Based on their
binding mode with BTK, researchers found that the non-
catalytic cysteine (Cys481) residue in the extended hinge re-
gion is the key binding site for the irreversible BTK inhibitors
such as ibrutinib (approved), acalabrutinib (approved), ONO-
4059 (phase II), spebrutinib (phase II) and HM71224 (phase
II) (Fig. 1). They generally contain a pyrimidine core with an
acrylamide group which can bind to the Cys481 residue cova-
lently as a Michael acceptor and irreversibly inhibit BTK.5,6
Ibrutinib, a highly potent oral small-molecule inhibitor, is a
Previous structure and activity relationship (SAR) explora-
tions showed that
a pyrimidine core and a typical
N-phenylacrylamide pharmacophore are important for novel
inhibitors to bind with BTK. CGI-560 (Fig. 1) was obtained by
screening from compound libraries and exhibited modest
BTK potency (half-maximal inhibitory concentration (IC50) =
400 nM) but promising selectivity (>ten-fold) over an initial
panel of 16 kinases.11 Based on this, we hope to splice some
pharmacophores on this skeleton to improve the selectivity
as well as inhibition for BTK, and then obtain the novel BTK
Jiangsu Key Laboratory of Drug Design and Optimization, China Pharmaceutical
University, Nanjing 210009, China. E-mail: xianghua@cpu.edu.cn;
Fax: +86 025 83271096; Tel: +86 025 83271096
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
c8md00413g
‡ These authors have the same contribution to this paper.
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Fig. 1 Chemical structures of the reported BTK inhibitors.
inhibitor. First, we introduced the key acrylamide group and
acyl side chain of the reported BTK ligand PLS-123 to substi-
tute the side chains at the two ends of CGI-560. Moreover,
Dan Zhao et al. reported the structural optimizations of
diphenylpyrimidine derivatives based on spebrutinib, which
showed that the chloro substituent at the C-5 position of the
pyrimidine core is more favorable than the fluoro atom due
to stronger hydrophobicity in the binding site.12 So we intro-
duced the chloro atom at the C-6 position after the skeleton
transition. Finally, we design a series of 2-phenyl pyrimidine
derivatives as novel potent BTK inhibitors which retain the
pyrimidine core and typical N-phenylacrylamide functionality
(Fig. 2).
The syntheses of novel 2-phenyl pyrimidine 11a–11i are
summarized in Scheme 1. 4-Nitrobenzaldehyde as the
starting material was condensed with hydroxylamine hy-
drochloride under reflux to afford benzonitrile 2 which
was transformed to compound 3 by reacting with ammo-
nium chloride. The pyrimidine nucleus 4 was prepared
through the condensation reaction of compound 3 and
diethyl malonate, and then it was reacted with POCl3 to
produce the key intermediate 5 which was reduced by Fe
Fig. 2 Rational design strategy of our compounds.
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Scheme 1 Reagents and conditions: (a) hydroxylamine hydrochloride, DMSO, 100 °C, 0.5 h, 82.0%; (b) methanol, sodium methoxide, rt, 5 h;
ammonium chloride, rt, 4 h, 97.0%; (c) ethanol, diethyl malonate, sodium methoxide, N2, reflux, 4 h, 87.4%; (d) POCl3, DMF, 100 °C, 8 h, 46.3%; (e)
methanol : water (1 : 1), Fe, NH4Cl, 70 °C, 5 h, 60.7%; (f) DMF, acryloyl chloride, triethylamine, 0 °C, 1 h, 56.5%; (g) THF, triethylamine, different
amines or phenylamine, 0 °C, 0.5 h, 91.5%; (h) methanol : water (1 : 1), Fe, NH4Cl, 70 °C, 0.5 h, 69.2%; (i) anhydrous dioxane, PdIJPPh3)2Cl2, K2CO3,
100 °C, 8 h, 20.5–34.0%.
to yield compound 6. Substitution of compound 6 with
acryloyl chloride generated compound 7. On the other
hand, 4-nitrobenzoyl chloride was reacted with different
amines or phenylamine to provide compounds 9a–9i, fol-
lowing a reduction reaction by Fe to get the different side
chains (10a–10i). Finally, the Pd-catalyzed Buchwald–
3-methyl phenylcarbamoyl substituent at the C-4 aniline moi-
ety of the pyrimidine core, possessed the most promising
anti-proliferative activity on the three leukemia lines. Particu-
larly, it is higher than ibrutinib for its activity against the Raji
cells (IC50 = 6.98 μM and 14.5 μM, respectively), suggesting
that a new action mechanism for them to be produced inter-
feres with the B-cell lymphoblastic leukemia cells. Neverthe-
less, the fluorine-substituted and methoxy-substituted C-4 an-
iline moieties almost lost their activity against the B-cell
leukemia lines (IC50 not determined) in spite of their high ca-
pability to inhibit the BTK activity. We speculated that the
low water solubility might be the main reason for their low
anti-B lymphoma cell activity. In contrast, 11b, 11c, 11d and
11f, which only possessed moderate activity against BTK
(Inh% = 40–60%), were able to strongly inhibit the prolifera-
tion of the three leukemia lines with an IC50 value ranging
from 1 to 20 μmol L−1.
Additionally, the effect of treatment time and drug con-
centration was further explored for compound 11g on Raji
cell viability. As a result, all of these compounds inhibited
the proliferation of the Raji cells in a dose- and time-
dependent manner, and almost completely blocked the Raji
cells (90%) at a concentration of 40 μM after 72 h (Fig. 3).
Moreover, 11g displayed better anti-proliferation activity than
positive control ibrutinib in a time-dependent manner at a
concentration of 5 μM.
Hartwig amination of
7
with different side chains
(10a–10i) afford target compounds 11a–11i.
The activity against BTK of the synthesized molecules was
evaluated using an ADP-Glo™ kinase assay system.13–15 Three
B-cell leukemia lines (Raji, HL60 and Ramos) which over-
expressed the BTK enzyme were used for the cell proliferation
evaluations through the MTT method. For comparison,
ibrutinib was tested as the positive control. As shown in
Table 1, most of our designed compounds showed good inhi-
bition (>50%) against BTK. In particular, 11e, 11g and 11h
could strongly inhibit the BTK activity, which displayed 82.7–
83.9% inhibition in contrast with that of ibrutinib (99.4%).
The kinase-based test results confirmed that the large-size
substituent groups (analogues 11d, 11e, 11g and 11h) are
more favorable than the small-size groups (analogues
11a–11c). It was not beneficial for the inhibition with the di-
substituted arylamine as R (analogues 11f and 11i). As a
global observation, most of the target compounds inhibit the
proliferations of these leukemia lines at concentrations rang-
ing from 1 to 20 μmol L−1. Compound 11g, which has a
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Table 1 SAR of the synthesized compounds
HL60
Raji
Ramos
BTKa
Compound
R
IC50 (μM)
IC50 (μM)
IC50 (μM)
Inh% at 100 nm
11a
>50
9.34
15.8
5.94
ND
42.9
12.5
19.2
14.7
ND
>50
11.2
10.4
7.24
ND
NDb
40.3
59.8
60.7
83.9
11b
11c
11d
11e
11f
4.79
9.36
6.18
56.5
11g
11h
3.66
ND
6.98
ND
5.39
ND
82.7
83.9
11i
14.9
3.57
20.9
14.5
16.8
2.31
ND
Ibrutinib
99.4
a
Percentage inhibition rate of each compound on BTK at 100 nM was measured using the ADP-Glo™ kinase assay system, shown as means of
three experiments. b Not determined.
Fig. 3 The effects of treatment time and drug concentration for compound 11g on Raji cell viability.
Flow cytometry assays by incubating Ramos cells with 5
μM compound 11g for 48 h were also performed. Excitingly,
the result shown in Fig. 4 indicate that inhibitor 11g induced
Ramos cell apoptosis with 38.5% which is higher than the
reference agent ibrutinib (7.83%) at the concentration of 5
μM. In addition, to investigate the anti-proliferative mecha-
nism of our designed compounds on B-cell leukemia cells,
we further evaluated the effect of the most active inhibitor
11g on the distribution of the Ramos cells in the cell cycle
with ibrutinib as the positive control. As shown in Fig. 5, af-
ter incubation with 5 μmol L−1 inhibitor 11g and ibrutinib
for 48 h, the DNA contents of the Ramos cells were 36.27%
and 30.5% in the G0/G1 phase, respectively. Apparently, the
newly synthesized molecule 11g blocked the Ramos cells at
the G0/G1 phase, which is in accordance with the reference
compound ibrutinib.
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Fig. 4 Compound 11g induced Ramos cell apoptosis in vitro. The cells were incubated at the concentration of 5 μM for 48 h, and DMSO and
ibrutinib were treated as the blank and positive controls, respectively.
Fig. 5 Cell cycle analysis of the treatment of the Ramos cells with compound 11g at the concentration of 5 μM for 48 h, and DMSO and ibrutinib
were treated as the blank and positive controls, respectively.
The BCR signaling pathway is relevant to several severe
leukemias and lymphomas. The BCR is the first aggregate
with CD79 which contains immunoreceptor tyrosine-based
activation motifs (ITAM). After that, ITAM are phosphory-
lated primarily by Src-family tyrosine kinases, such as Lyn.
The Src-family kinases and phosphorylated ITAM serve as
a scaffolding platform for engaging and activating SH2 do-
mains containing kinases, including Syk, which leads to
BTK being recruited to the cell membrane and phosphory-
lated in its SH2 domain. The activated BTK then phos-
phorylates its substrate phospholipaseγ2 (PLCγ2), leading
to activation of the downstream signaling pathways, such
as NF-κB and STAT3.16 In fact, over-expression of BTK
may cause the disorder of the B-cell signaling pathway,
leading to B-cell malignancies and autoimmune diseases.17
Thus we performed the western blotting assay to detect
the phosphorylation of BTK and its substrate PLCγ2. The
result showed that 11g could inhibit their phosphoryla-
tion, though it is weaker than the positive control
ibrutinib (Fig. 6).
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Fig. 6 Effect of 11g on BTK and PLCγ2 phosphorylation in the Ramos
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Conclusions
In summary, we designed and synthesized a series of 2-phe-
nyl pyrimidine derivatives based on the skeleton of highly se-
lective but modestly potent BTK inhibitor CGI-560, then a va-
riety of pharmacophores were used to improve their BTK
inhibition. Most of them exhibited good inhibitory activities
in both cellular and enzymatic assays. In particular, com-
pound 11g with a 3-methyl phenylcarbamoyl substituent at
the C-4 aniline moiety of the pyrimidine core displayed stron-
ger activity than the positive control (ibrutinib) against Raji
cells. The further mechanism study showed that compound
11g could not only induce cell cycle arrest and apoptosis in
B-cell leukemia lines but also inhibit the phosphorylation of
BTK and its substrate PLCγ2. Overall, 11g could serve as a
promising highly selective lead candidate for further study.
Conflicts of interest
There are no conflicts to declare.
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Acknowledgements
We are grateful to the Program of Jiangsu Key Laboratory of
Drug Design and Optimization, China Pharmaceutical Univer-
sity, the postgraduate innovation fund of Huahai medical cor-
poration and the college students' innovation and entrepre-
neurship training program (201810316104) for the financial
support of this research.
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Med. Chem. Commun.
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