Design and synthesis of phosphoryl-substituted diphenylpyrimidines (Pho-DPPYs) as potent Bruton's tyrosine kinase (BTK) inhibitors: Targeted treatment of B lymphoblastic leukemia cell lines

Article abstract of DOI:10.1016/j.bmc.2016.11.054

A family of phosphoryl-substituted diphenylpyrimidine derivatives (Pho-DPPYs) were synthesized and biologically evaluated as potent BTK inhibitors in this study. Compound 7b was found to markedly inhibit BTK activity at concentrations of 0.82?nmol/L, as well as to suppress the proliferations of B-cell leukemia cell lines (Ramos and Raji) expressing high levels of BTK at concentrations of 3.17?μM and 6.69?μM. Moreover, flow cytometry analysis results further indicated that 7b promoted cell apoptosis to a substantial degree. In a word, compound 7b is a promising BTK inhibitor for the treatment of B-cell lymphoblastic leukemia.

Full text of DOI:10.1016/j.bmc.2016.11.054

Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design and synthesis of phosphoryl-substituted diphenylpyrimidines
(Pho-DPPYs) as potent Bruton’s tyrosine kinase (BTK) inhibitors:
Targeted treatment of B lymphoblastic leukemia cell lines
Yang Ge a,1, Haijun Yang a,1, Changyuan Wang a, Qiang Meng a, Lei Li a, Huijun Sun a, Yuhong Zhen a,
Kexin Liu a, Yanxia Li b, Xiaodong Ma a,
⇑
a College of Pharmacy, Dalian Medical University, Dalian 116044, PR China
b Department of Respiratory, The First Affiliated Hospital of Dalian Medical University, Dalian 116011, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A family of phosphoryl-substituted diphenylpyrimidine derivatives (Pho-DPPYs) were synthesized and
biologically evaluated as potent BTK inhibitors in this study. Compound 7b was found to markedly inhibit
BTK activity at concentrations of 0.82 nmol/L, as well as to suppress the proliferations of B-cell leukemia
Received 16 September 2016
Revised 1 November 2016
Accepted 28 November 2016
Available online xxxx
cell lines (Ramos and Raji) expressing high levels of BTK at concentrations of 3.17 lM and 6.69 lM.
Moreover, flow cytometry analysis results further indicated that 7b promoted cell apoptosis to a substan-
tial degree. In a word, compound 7b is a promising BTK inhibitor for the treatment of B-cell lymphoblastic
leukemia.
Keywords:
Leukemia
BTK
Ó 2016 Elsevier Ltd. All rights reserved.
DPPY
Phosphoryl
Inhibitor
1. Introduction
(ClinicalTrials.gov identifier: NCT02717611)11–13
, GS-4059 (3)
(ClinicalTrials.gov identifier: NCT02457559),14,15 spebrutinib
(4) (ClinicalTrials.gov identifier: NCT01744626)16, HM71224 (5)
(ClinicalTrials.gov identifier: NCT01765478)17, and PLS-123 (6).18
(Fig. 1). Generally, a pyrimidine core accompanied by an acry-
lamide group can irreversibly inhibit BTK by using a Michael
acceptor to bind to the target Cys481. This is essential for these
novel BTK inhibitors to function properly, as depicted in
Fig. 1.8,19–21 The phosphoryl group, which has well-known biolog-
ical functionality, is often used to improve the aqueous solubility
and biological activity of anticancer alkylating agents that require
activation in vivo.22–24 In this study, with the primary goal of
improving these biological properties, a series of phosphoryl-sub-
stituted diphenylpyrimidine derivatives (Pho-DPPYs) were synthe-
sized as potent BTK inhibitors for B-cell lymphoblastic leukemia
(Fig. 2). The newly introduced phosphoryl group was observed to
form an additional hydrogen bond according to molecular simula-
tions, suggesting that they are effective structural modifiers.
Chronic lymphocytic leukemia (CLL), the most common adult
leukemia in Western countries, is a neoplastic disease of mono-
clonal CD5+ B cells which accumulate in the blood, marrow, and
secondary lymphoid tissues.1,2 Bruton’s tyrosine kinase (BTK) is
an important member of the Tec non-receptor protein kinase fam-
ily that is always expressed in B cells and myeloid cells. The func-
tion of BTK in signaling pathways activated by the engagement of
the B cell receptor (BCR) and FCER on mast cells has been well
established.3,4 The unique structure of BTK, which is characterized
by a cysteine (Cys481) within the ATP-binding pocket, makes it an
attractive therapeutic target.5,6 Ibrutinib (1, Fig. 1) is a first-in-
class, irreversible small-molecule inhibitor of BTK that has the abil-
ity to covalently bind to Cys4817–10 and has displayed considerable
single-agent activity in patients with relapsed CLL and in previ-
ously untreated patients. Disease progression during ibrutinib
treatment is uncommon in patients with previously untreated
CLL or in patients with low-risk genomic abnormalities.
Until now, several novel preclinical or clinical candidates for the
treatment of CLL have been discovered, including acalabrutinib (2),
2. Results and discussion
2.1. Chemistry
⇑
Corresponding author.
E-mail address: xiaodong.ma@139.com (X. Ma).
These authors contributed equally to this work.
The synthetic route for the title Pho-DPPYs 7a–f is illustrated in
Scheme 1.25–28 Commercially available chlorine-substituted
1
http://dx.doi.org/10.1016/j.bmc.2016.11.054
0968-0896/Ó 2016 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Ge Y., et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.11.054

2
Y. Ge et al. / Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
O
N
HN
O
O
O
N
N
N
O
O
N
O
N
N
N
N
N
H2N
H2N
N
H2N
N
N
N
N
1
2
3
Ibrutinib
Acalabrutinib
GS-4059
H
N
N
F
N
S
N
N
NH
O
HN
O
N
NH
HN
N
O
O
NH
NH
NH
NH
O
NH
O
O
N
N
O
O
F3C
PLS-123
Fig. 1. Structures of the novel BTK inhibitors.
4
6
5
Spebrutinib
HM71224
ATP-binding pocket
Phe413/Ile472/Gly414
cycle and apoptosis rate were analyzed via flow cytometry. For
comparison, two novel BTK inhibitors, spebrutinib and ibrutinib,
were also tested as reference compounds. The results are provided
in Table 1.
Covalent bond
Cys481
O
O
O
N
P
O
O
Anticancer functionality
O
O
According to the kinase-based test results, the Pho-DPPYs effec-
tively inhibited BTK enzyme activity within concentrations of
112.0 nmol/L. Compound 7b (IC50 = 0.82 nM), bearing a 3-mor-
pholin-4-ylpropoxy substituent at the C-4 aniline moiety of its
pyrimidine core, was the most active inhibitor of BTK. When reduc-
ing the linker with a methylene group, the produced molecule 7a
reduced approximately 136-fold anti-BTK activity. The
diethoxyphosphinyl-substituted 7c was also notable, as it likewise
displayed strong capacity to inhibit BTK at concentrations of
1.23 nM. Replacing the linker with a long propoxy group was not
successful. For instance, inhibitor 7d reduced about 9-times anti-
BTK activity. Installing a fluorine atom at the C-5 position of pyrim-
idine core was less beneficial than the chlorine substituent. The
examples 7e and 7f with fluorine substituents lost approximately
18 and 29-fold the potency against BTK, respectively.
HN
Linker = -CH2- or -O(CH2)3-
HN
HN
N
NH
HN
N
NH
N
F
N
R
spebrutinib
BTK Inhibitor
Phase 䊢
Met477
Pho-DPPYs 7a-f
Improved activity
Fig. 2. Designed strategy of Pho-DPPYs as BTK inhibitors.
nitrobenzene 8a,b was reacted with triethylphosphate to provide
the diethoxyphosphinyl nitrobenzene 9a,b, which was reduced
under Fe-NH4Cl conditions to form the aniline 10a,b. Compounds
9a,b were treated with thionyl chloride and reacted with morpho-
line in dichloromethane solvent to produce the amidophospho-
nates 13a,b. To prepare the 4-chlorine pyrimidines 16a–b,
reagent 3-nitroaniline was utilized to create an acylation reaction
with acryloyl chlorine under base reagent NaHCO3. Aniline 15,
which was obtained by reducing the nitro group in N-acryloyl-4-
nitrobenzamide, was regioselectively coupled to the 4-position of
2,4-dichloropyrimidine to create the key intermediate 16a–b. With
these intermediates in hand, title molecules 7a–f were synthesized
via coupling reaction of 10a,b or 13a,b with 16a–b in the presence
of trifluoroacetic acid (TFA) under reflux temperature.
As shown in Table 1, the Pho-DPPYs were able to inhibit B leu-
kemia cell lines at micromolar concentrations. Inhibitor 7b, which
displayed especially strong potency against BTK kinase, was the
most active inhibitor in regards to interfering with the Ramos
and Raji cell lines with IC50 values of 3.17 lM, and 6.69 lM, respec-
tively. To this effect, compound 7b exhibited superior biological
properties to the references spebrutinib and ibrutinib. Interest-
ingly, molecules 7b–d displayed higher potency in cell viability
despite their lower potency against BTK than the references. The
introduced phosphinyl substituent was likely the major reason
for the improved capability against B leukemia cells.
Inhibitor 7b was also tested in regards to its effects on the cell
cycle and apoptosis of the Ramos cell line via flow cytometry anal-
ysis. Again, for comparison, spebrutinib and ibrutinib were also
tested. Fig. 3 shows where molecule 7b, even at a low concentra-
2.2. Biological activity
The title molecules were evaluated in regards to their activity
against BTK kinase with an ADP-GloTM Kinase Assay.29–31 Their
potency for repressing the proliferations of B leukemia cells was
observed according to the CCK-8 assay method.19 Two typical B-
cell lymphoblastic leukemia cell lines, Ramos and Raji, which over-
expressed BTK enzyme were used for this purpose. In addition, cell
tion (5
brutinib (41.5%) and ibrutinib (47.9%) at high concentrations of
10 M. Cell cycle analysis via flow cytometry indicated that mole-
lM), could induced similar apoptosis rates (45.1%) as spe-
l
cule 7b significantly locked Ramos cells at the G2/M phase. Com-
pared to control group, the percentages of the G2/M phase
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Y. Ge et al. / Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
3
NO2
L
NO2
NH2
d
c
O
O
L
O
L
P
P
P
O
Cl
O
N
O
N
R1
O
O
O
O
L
O
P
13a,b
11a,b
12a,b
HN
NH2
b
NO2
L
O2N
L
h
O
O
a
d
HN
N
NH
P
O
O
L
N
P
R
Cl
8a,b
O
O
7a-f
10a,b
9a,b
R = Cl, F
R1 =
N
O
OCH2CH
3 or
R
NH2
R
N
N
NO2
L = -CH2- or -O(CH2)3-
Cl
N
Cl
e
Cl
N
NH
g
f
NH
NH2
O
NH
O
14
15
16a-b
Scheme 1. Synthetic route of the title compounds 7a–f. Reagents and conditions: (a) P(OEt)3, 5 h, 130 °C, 80–91%; (b) (COCl)2, DMF, 60 °C, 2 h; (c) morpholine, THF, 0 °C, 2 h,
92% (d) Fe-NH4Cl, MeOH-H2O, 2 h, 70 °C, 65–78%; (e) acryloyl chloride, NaHCO3, CH3CN, 0 °C, 0.5 h, 95%; (f) Fe-NH4Cl, MeOH-H2O, 2 h, 70 °C, 92%; (g) DIPEA, 1,4-dioxane,
60 °C, 2 h, 91%; (h) trifluoroacetic acid, 2-BuOH, 100 °C, 12 h, 6–20%.
increased from 4.98% to 11.22%, and those of the S phase decreased
from 55.1% to 50.8% via treatment with 7b at concentrations from
between the amino atom of acryl amide in 7c with Asp539; 3)
hydrophobic contacts generated by the C-5 chlorine atom of the
1
l
M, 5
lM, and 10
lM for 48 h. However, the percentages of the
pyrimidine core with the amino acid Met477; and 4) p-p contacts
G0/M phase had only minor changes. Apparently, both the new
synthesized molecule 7b and spebrutinib produced similar inhibi-
tion results in terms of arresting the cell cycle at the G2/M phase.
By contrast, inhibitor ibrutinib arrested the cell cycle at the S phase
(Fig. 4).
produced by the phenyl ring of the C-2 aniline moiety with Lys430.
Conversely, for the moderately active inhibitor 7f (Fig. 5c), only the
covalent bond and
p-p contacts remained at the end of the test.
Although the less potent inhibitor 7a (Fig. 5d) did bind in the
ATP-binding pocket, all these important interaction forces were
lost during the test process. In a word, these modes were in accor-
dance with their biological activity.
2.3. Molecular simulations
To elucidate the binding mechanism of the phosphoryl-substi-
tuted pyrimidine derivatives, four typical inhibitors, 7b,c (most
active), 7f (moderately active), and 7a (less potent), were docked
individually into the ATP binding pocket of BTK (PDB: 3GEN).32
The program AutoDock 4.2 (default parameters) was used for this
test33–35; the docking modes are depicted in Fig. 5.
Apparently, two potent inhibitors, 7b (Fig. 5a) and 7c (Fig. 5b),
formed several important binding forces with the BTK enzyme,
including: 1) the expected covalent bond between the acryl amide
group with the amino acid Cys481; 2) hydrogen bond between the
oxygen atom of the phosphamide group in compound 7b with the
amino acids Phe413 and Gly414 through a water molecule, or
3. Conclusion
A series of phosphoryl-substituted diphenylpyrimidines were
synthesized in this exploration with the aim to improve their activ-
ity against B-cell leukemia cell lines. Among these novel com-
pounds, 7b was able to inhibit BTK activity at very low
concentrations of 0.82 nmol/L. Moreover, inhibitor 7b could sup-
press the proliferations of Ramos cells at concentrations of
3.17 lM and Raji cells at 6.69 lM. Flow cytometry results indicated
that 7b remarkably promoted cell apoptosis, and molecular simu-
lation results showed that 7b binds tightly with BTK enzyme. Over-
all, these results suggest that 7b is a promising BTK inhibitor which
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4
Y. Ge et al. / Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
Table 1
Biological activity of the newly synthesized compounds 7a–f.a
R1
O
O
L
O
P
HN
HN
N
NH
N
R
7a-f
Compound
R
R1
L
Enzymatic activity (IC50, nM)b
Antiproliferative activity
(IC50 M)
,
l
BTK
Ramos
Raji
7a
Cl
ACH2A
112.0
27.4
7.71
N
O
N
7b
Cl
AO(CH2)3A
0.82
3.17
6.69
O
7c
7d
7e
7f
Cl
Cl
F
ACH2A
1.23
10.6
32.1
21.6
11.7
5.40
9.36
11.5
13.5
6.07
34.9
16.4
O
AO(CH2)3A
ACH2A
O
O
O
F
AO(CH2)3A
Spebrutinib
Ibrutinib
0.72
0.34
15.1
5.14
25.3
19.5
a
Data represent the mean of at least three separate experiments.
The IC50 values are the concentrations in micromolar needed to inhibit cell growth by 50% as, as calculated using GraphPad Prim version 5.0.
b
may merit further development as a B lymphoblastic leukemia
treatment.
chloro-5-substitutedpyrimidin-4-yl)oxy)phenyl)acrylamides 16a,b
were synthesized using the procedures reported in Refs. 21,22. All
these intermediates were directly used without any purification
and structural characterization. With these intermediates in hand,
the newly obtained compounds were synthesized as described
below.
4. Experiential section
4.1. General methods and chemistry
A flask was charged with 2-chlorine-substituted pyrimidine
16a,b and aniline 10a,b (0.70 mmol), or 13a,b (0.70 mmol), TFA
(0.08 mL, 1.05 mmol), and 2-BuOH (10 mL).The slurry was heated
to 100 °C for 10 h. The reaction mixture was allowed to cool to
room temperature and was neutralized with a saturated aqueous
sodium bicarbonate solution. The aqueous mixture was then
extracted with CH2Cl2 (20 mL) three times. The crude product
was purified using flash chromatography with dichloromethane/
methanol (v/v, 30:1) as eluents.
All solvents and reagents were obtained from commercial sup-
plies and were used as received. High resolution ESI-MS were per-
formed on an AB Sciex TripleTOFÒ 4600 LC/MS/MS system. 1H NMR
and 13C NMR spectra on a Brucker AV 400 MHz spectrometer were
recorded in [d] DMSO. Coupling constants (J) are expressed in hertz
(Hz). Chemical shifts (d) of NMR are reported in parts per million
(ppm) units relative to internal control (TMS). 1H NMR spectra
are reported in the following order: multiplicity, approximate cou-
pling constant (J value) in hertz, and number of protons; signals are
characterized as s (singlet), d (doublet), dd (doublet of doublets), t
(triplet), dt (doublet of triplets), q (quartet), m (multiplet), and br s
(broad signal). All reactions were monitored by TLC, using silica gel
plates with fluorescence F254 (TLC Silica gel 60 F254, Merck) and
UV light visualization. Flash chromatography separations were
obtained on Silica Gel (300–400 mesh) using dichloromethane/
methanol as eluents.
4.2.1. N-(3-((5-Chloro-2-((4-(((1-morpholino)ethoxyphosphinyl)
methy))amino)phenylamino-4-pyrimidinyl)amino)phenyl)acrylamide
(7a)
Off-white solid (172 mg, 15.6%); 1H NMR (400 MHz, DMSO-d6):
d 10.18 (s, 1H), 9.29 (s, 1H), 8.95 (s, 1H), 8.14 (s, 1H), 7.86 (s, 1H),
7.63–7.41 (m, 3H), 7.40–7.25 (m, 2H), 7.03 (d, J = 7.0 Hz, 2H), 6.46
(dd, J = 16.9 10.1 Hz, 1H), 6.26 (d, J = 16.8 Hz, 1H), 5.75 (d,
J = 10.0 Hz, 1H), 4.00–3.76 (m, 2H), 3.40 (d, J = 3.1 Hz, 4H), 2.93
(dd, J = 55.4, 27.9 Hz, 6H), 1.20 (t, J = 7.0 Hz, 3H); 13C NMR
(400 MHz, DMSO-d6): d 163.1 157.6, 156.2, 154.8, 139.1, 138.8
(d, J = 38.8 Hz), 131.9, 129.7 (d, J = 25.2 Hz), 128.5, 126.9, 125.0,
124.9, 119.4, 118.7 (2C), 115.5, 115.2, 103.8, 66.4 (d, J = 18.8 Hz,
4.2. General procedure for the synthesis of 7a–f25–28
Phosphoryl-substituted anilines 10a,b and 13a,b were prepared
according to the literatures.23,24 While the intermediates N-(3-((2-
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Y. Ge et al. / Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
5
Fig. 3. Compound 7b induced Ramos cell apoptosis in vitro. The cells were incubated with the indicated concentrations of 7b for 48 h, and the cells were stained with annexin
V/FTIC, followed by flow cytometry analysis. One representative experiment is shown. p < 0.05.
2C), 59.1 (d, J = 27.6 Hz), 43.7 (2C), 32.6, 31.3, 16.3 (d, J = 24.8 Hz);
HRMS (ESI) m/z calcd for C26H30ClN6O4P [M+H]+ 557.1833, found
557.1790.
J = 22.4 Hz, 2C); HRMS (ESI) m/z calcd for C24H27ClN5O4P [M+H]+
516.1562, found 516.1488.
4.2.4. N-(3-((5-Chloro-2-((4-((diethoxyphosphoryl)propoxyl))amino)
phenylamino-4-pyrimidinyl)amino)phenyl)acrylamide (7d)
4.2.2. N-(3-((5-Chloro-2-((4-(((1-morpholino)ethoxyphosphinyl)
propoxyl))amino)phenylamino-4-pyrimidinyl)amino)phenyl)
acrylamide (7b)
Off-white solid (123 mg, 11.0%); 1H NMR (400 MHz, DMSO-d6):
d 10.18 (s, 1H), 9.15 (s, 1H), 8.90 (s, 1H), 8.10 (s, 1H), 7.88 (s, 1H),
7.57–7.40 (m, 3H), 7.31 (d, J = 5.2 Hz, 2H), 6.69 (d, J = 8.8 Hz, 2H),
6.46 (dd, J = 17.0, 10.1 Hz, 1H), 6.26 (dd, J = 17.0, 2.0 Hz, 1H), 5.76
(dd, J = 10.2, 2.0 Hz, 1H), 4.05–3.88 (m, 6H), 1.92–1.79 (m, 4H),
1.23 (t, J = 7.0 Hz, 6H); 13C NMR (400 MHz, DMSO-d6): d 163.3,
158.0, 156.3, 155.0, 153.3, 139.2, 139.1, 133.9, 132.1, 128.8,
127.1, 120.7 (2C), 119.4, 115.6 (d, J = 57.2 Hz), 114.3 (2C), 103.4,
67.3 (d, J = 68.0 Hz), 61.2 (d, J = 24.8 Hz, 2C), 22.5 (d, J = 18.0 Hz),
Off-white solid (50 mg, 6.8%); 1H NMR (400 MHz, DMSO-d6): d
10.21 (s, 1H), 9.16 (s, 1H), 8.89 (s, 1H), 8.10 (s, 1H), 7.89 (s, 1H),
7.52 (dd, J = 34.2, 8.8 Hz, 3H), 7.30 (d, J = 4.9 Hz, 2H), 6.69 (d,
J = 8.3 Hz, 2H), 6.46 (dd, J = 16.9, 10.1 Hz, 1H), 6.25 (d, J = 16.8 Hz,
1H), 5.76 (d, J = 10.0 Hz, 1H), 4.10–3.76 (m, 4H), 3.56 (d,
J = 35.6 Hz, 4H), 2.94 (t, J = 38.4 Hz, 4H), 2.05–1.68 (m, 4H), 1.21
(t, J = 7.0 Hz, 3H); 13C NMR (400 MHz, DMSO-d6): d 163.1, 157.8,
156.1, 154.8, 153.1, 139.0, 138.9(d, J = 60.8 Hz), 133.6, 131.9,
128.6, 126.9, 120.5 (2C), 119.1, 115.4 (d, J = 57.2 Hz), 114.1 (2C),
103.2, 67.3 (d, J = 67.6 Hz), 66.6 (d, J = 19.6 Hz, 2C), 58.8 (d,
J = 26.4 Hz), 43.5 (2C), 22.5 (d, J = 18.0 Hz), 21.9, 20.5, 16.3 (d,
J = 24.4 Hz); HRMS (ESI) m/z calcd for C28H34ClN6O5P [M+H]+
601.2090, found 601.2044.
22.1, 20.7, 16.5 (d, J = 22.8 Hz, 2C); HRMS (ESI) m/z calcd for C26
-
H
31ClN5O5P [M+H]+ 560.1824, found 560.1731.
4.2.5. N-(3-((5-Fluorine-2-((4-((diethoxyphosphoryl)methy))amino)
phenylamino-4-pyrimidinyl)amino)phenyl)acrylamide (7e)
Off-white solid (136 mg, 12.7%); 1H NMR (400 MHz, DMSO-d6):
d 10.16 (s, 1H), 9.44 (s, 1H), 9.17 (s, 1H), 8.11 (d, J = 2.7 Hz, 1H),
7.93 (s, 1H), 7.57 (dd, J = 16.8, 7.9 Hz, 3H), 7.44 (d, J = 7.5 Hz, 1H),
7.29 (t, J = 7.9 Hz, 1H), 7.06 (d, J = 6.8 Hz, 2H), 6.47 (dd, J = 16.8,
10.2 Hz, 1H), 6.26 (d, J = 16.8 Hz, 1H), 5.76 (d, J = 10.0 Hz, 1H),
4.09–3.77 (m, 4H), 3.08 (d, J = 21.0 Hz, 2H), 1.16 (t, J = 6.8 Hz,
4.2.3. N-(3-((5-Chloro-2-((4-((diethoxyphosphoryl)methy))amino)
phenylamino-4-pyrimidinyl)amino)phenyl)acrylamide (7c)
Off-white solid (190 mg, 18.9%); 1H NMR (400 MHz, DMSO-d6):
d 10.19 (s, 1H), 9.31 (s, 1H), 8.95 (s, 1H), 8.14 (s, 1H), 7.86 (s, 1H),
7.61–7.41 (m, 3H), 7.40–7.22 (m, 2H), 7.01 (d, J = 6.7 Hz, 2H), 6.46
(dd, J = 17.0, 10.1 Hz, 1H), 6.26 (dd, J = 17.0, 1.8 Hz, 1H), 5.75 (dd,
J = 10.2, 1.8 Hz, 1H), 3.99–3.81 (m, 4H), 3.06 (d, J = 21.1 Hz, 2H),
1.15 (t, J = 7.0 Hz, 6H); 13C NMR (400 MHz, DMSO-d6): d 163.4,
157.9, 156.4, 155.0, 139.4, 139.1 (d, J = 20.8 Hz), 132.1, 129.8 (d,
J = 26.0 Hz), 128.7, 127.1, 124.7, 124.6, 119.6, 118.9 (2C), 115.7,
115.4, 104.0, 61.5 (d, J = 25.6 Hz, 2C), 32.5, 31.1, 16.5 (d,
6H); 13C NMR (400 MHz, DMSO-d6):
d
163.1, 155.5 (d,
J = 10.4 Hz), 149.8 (d, J = 43.6 Hz), 141.8, 140.8 (d, J = 76.8 Hz),
139.4 (d, J = 12.8 Hz), 139.1 (d, J = 28.0 Hz), 132.0, 129.6 (d,
J = 25.2 Hz), 128.6, 126.9, 124.2, 124.1, 118.4 (2C), 117.5, 114.8,
113.3, 61.3 (d, J = 25.6 Hz, 2C), 32.3, 30.9, 16.3 (d, J = 22.4 Hz, 2C);
HRMS (ESI) m/z calcd for C24H27FN5O4P [M+H]+ 500.1857, found
500.1786.
Please cite this article in press as: Ge Y., et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.11.054

6
Y. Ge et al. / Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
Fig. 4. Effects of spebrutinib, ibrutinib and 7b on Ramos cell cycle arrest detected by flow cytometry assay. Results are representative of three separate experiments, dates are
expressed as the mean standard deviation, p < 0.05.
4.2.6. N-(3-((5-Fluorine-2-((4-((diethoxyphosphoryl)propoxyl))
amino)phenylamino-4-pyrimidinyl)amino)phenyl)acrylamide (7f)
Off-white solid (52 mg, 7.0%); 1H NMR (400 MHz, DMSO-d6): d
10.13 (s, 1H), 9.38 (s, 1H), 8.98 (s, 1H), 8.06 (d, J = 3.7 Hz, 1H),
7.93 (s, 1H), 7.51 (dd, J = 13.1, 9.2 Hz, 3H), 7.42 (d, J = 8.1 Hz, 1H),
7.28 (t, J = 8.1 Hz, 1H), 6.75 (d, J = 9.0 Hz, 2H), 6.46 (dd, J = 16.9,
10.1 Hz, 1H), 6.26 (dd, J = 17.0, 1.9 Hz, 1H), 5.76 (dd, J = 10.2,
1.8 Hz, 1H), 4.22–3.67 (m, 6H), 1.92–1.79 (m, 4H), 1.22 (t,
J = 7.0 Hz, 6H); 13C NMR (400 MHz, DMSO-d6): d 163.3, 155.9 (d,
J = 10.4 Hz), 153.2, 150.0 (d, J = 42.8 Hz), 141.8, 141.1 (d,
J = 74.8 Hz), 139.3 (d, J = 28.0 Hz), 134.4, 132.2, 128.9, 127.1,
120.5 (2C), 117.6, 115.0, 114.4 (2C), 113.6, 67.4 (d, J = 68.4 Hz),
61.2 (d, J = 25.2 Hz, 2C), 22.5 (d, J = 18.4 Hz), 22.1, 20.7, 16.5 (d,
J = 22.8 Hz, 2C); HRMS (ESI) m/z calcd for C26H31FN5O5P [M+H]+
544.2120, found 544.2019.
instructions of the manufacturer. The more detailed and complete
protocols, see the ADP-GloTM kinase Assay Technical Manual avail-
able at: http://cn.promega.com/resources/protocols/product-infor-
mation-sheets/n/btk-kinase-enzyme-system-protocol/. The test
was performed in a 384-well plate, and includes the major steps
below: (1) perform a 5
(e.g., 1Â reaction buffer A), (2) incubate at room temperature for
60 min, (3) add 5
L of ADP-GloTM Reagent to stop the kinase reac-
lL kinase reaction using 1Â kinase buffer
l
tion and deplete the unconsumed ATP, leaving only ADP and a very
low background of ATP, (4) incubate at room temperature for
40 min, (5) add 10 lL of Kinase Detection, (6) reagent to convert
ADP to ATP and introduce luciferase and luciferin to detect ATP,
(7) incubate at room temperature for 30 min, (8) plate was mea-
sured on TriStarÒ LB942 Multimode Microplate Reader (BERT-
HOLD) to detect the luminescence (Integration time 0.5–1 s).
Curve fitting and data presentations were performed using Graph-
Pad Prism version 5.0.29–31
4.3. In vitro kinase enzymatic assay
The BTK kinase enzyme system (Catalog. V9071) was purchased
from Promega Corporation (USA). Concentrations consisting of
suitable levels from 0.1 to 100 nM were used for all of the tested
compounds. The experiments were performed according to the
4.4. Cellular activity assay
All the cell viability assays were performed according to the
CCK-8 method. The cells were seeded in 96-well plates at a density
Please cite this article in press as: Ge Y., et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.11.054

Y. Ge et al. / Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
7
Fig. 5. a) Putative binding mode of inhibitor 7b within BTK (PDB code: 3GEN). b) Putative binding mode of inhibitor 7c within BTK (PDB code: 3GEN). c) Putative binding
mode of inhibitor 7f within BTK (PDB code: 3GEN). d) Putative binding mode of inhibitor 7a within BTK (PDB code: 3GEN).
of 2000–3000 cells/well and were maintained at 37 °C in a 5% CO2
incubator in RPMI1640 containing 10% fetal bovine serum (FBS,
Gibco) for one day. Cells were exposed to treatment for 48 h, and
the number of cells used per experiment for each cell lines was
adjusted to obtain an absorbance of 0.5–1.2 at 450 nm with a
microplate reader (Thermo, USA). Compounds were tested at
appropriate concentrations (1.25–40 lM), with each concentration
duplicated five times. The IC50 values were calculated using Graph-
Pad Prim version 5.0.
4.6. Molecular docking study
The crystal structure (PDB: 3GEN) of the kinase domain of BTK
bound to
a
pyrrolopyrimidine-containing compound.32 The
enzyme preparation and the hydrogen atoms adding was per-
formed in the prepared process. The whole BTK enzyme was
defined as a receptor and the site sphere was selected on the basis
of the binding location of pyrrolopyrimidine inhibitor. By moving
the inhibitor and the irrelevant water, molecules 7a–c, 7f were
placed, respectively. The binding interaction energy was calculated
to include van der Waals, electrostatic, and torsional energy terms
defined in the tripos force field. Ten docking poses were generated
and clustered, and compounds with predicted binding affinities
better than 7.0 kcal/mol in each of the three docking runs were
selected for further analysis and visual inspection. The structure
optimization was performed using a genetic algorithm, and only
the best-scoring ligand protein complexes were kept for analy-
ses.33–35 The WWW site also includes many resources for use of
AutoDock, including detailed Tutorials that guide users through
basic AutoDock usage, docking with flexible rings, and virtual
screening with AutoDock. Tutorials may be found at: http://auto-
dock.scripps.edu/faqs-help/tutorial.
4.5. Cell apoptosis assay
The Ramo cells (1–5 Â 105 cells/well) incubated in 6-well plates
were treated with solvent control (DMSO), spebrutinib, ibrutinib,
or compound 7b in medium containing 5% FBS for 48 h. Then, col-
lected and fixed with 70% ethanol at 4 °C overnight. After being
fixed with 75% ethanol at 4 °C for 24 h, the cells were stained with
Annexin V-FITC (5 lL)/propidium iodide (5 lL), and analyzed by
flow cytometry assay (Becton-Dickinson, USA). For cell cycle anal-
ysis, Ramos cells at a density of approximately 2 Â 105 cells/well
were incubated in 6-well plates, treated with different concentra-
tions of inhibitors for 48 h, collected and fixed with 70% ethanol at
4 °C overnight. After fixation, the cells were washed with PBS and
stained with propidium iodide (PI) for 10 min under subdued light.
Stained cells were analyzed flow cytometry assay (Becton-Dickin-
son, NJ, USA), and the results were performed using FCS Express
flow cytometry analysis software (ModFit LT 3.1).
Acknowledgments
We are grateful to the National Natural Science Foundation of
China (No. 81402788), the PhD Start-up Fund of Natural Science
Please cite this article in press as: Ge Y., et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.11.054

8
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Cultivation Project of Youth Techstars, Dalian (No. 2016RQ043)
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