Identification of a potent and selective phosphatidylinositol 3-kinase δ inhibitor for the treatment of non-Hodgkin's lymphoma

Article abstract of DOI:10.1016/j.bioorg.2020.104344

PI3Kδ has proved to be an effective target for anti-lymphoma drugs. However, the application of current approved PI3Kδ inhibitors has been greatly limited due to their specific immune-mediated toxicity and increased risk of infection, it is necessary to develop more PI3Kδ inhibitors with new scaffold. In this study, SAR study with respect to piperazinone-containing purine derivatives led to the discovery of a potent and selective PI3Kδ inhibitor, 4-(cyclobutanecarbonyl)-1-((2-(2-ethyl-1H-benzo[d]imidazol-1-yl)-9-methyl-6-morpholino-9H-purin-8-yl)methyl)piperazin-2-one (WNY1613). WNY1613 exhibits good antiproliferative activity against a panel of non-Hodgkin's lymphoma (NHL) cell lines by inducing cancer cell apoptosis and inhibiting the phosphorylation of PI3K and MAPK downstream components. In addition, it can also prevent the tumor growth in both SU-DHL-6 and JEKO-1 xenograft models without observable toxicity. WNY1613 thus could be developed as a promising candidate for the treatment of NHL after subsequent extensive pharmacodynamics and pharmacokinetics investigation.

Full text of DOI:10.1016/j.bioorg.2020.104344

Bioorganic Chemistry 105 (2020) 104344
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Identification of a potent and selective phosphatidylinositol 3-kinase δ
inhibitor for the treatment of non-Hodgkin’s lymphoma
,
b
,
,
Wei-Qiong Zuoa 1, Rong Hua 1, Wan-Li Wanga, Yong-Xia Zhub, Ying Xub, Luo-Ting Yub,
Zhi-Hao Liub *, Ning-Yu Wanga
,
,*
a School of Life Science and Engineering, Southwest Jiaotong University, Chengdu, China
b Lab of Medicinal Chemistry, Cancer Center, West China Hospital, Sichuan University and Collaborative Innovation Center, Chengdu, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
PI3Kδ has proved to be an effective target for anti-lymphoma drugs. However, the application of current
approved PI3Kδ inhibitors has been greatly limited due to their specific immune-mediated toxicity and increased
risk of infection, it is necessary to develop more PI3Kδ inhibitors with new scaffold. In this study, SAR study with
respect to piperazinone-containing purine derivatives led to the discovery of a potent and selective PI3Kδ in-
hibitor, 4-(cyclobutanecarbonyl)-1-((2-(2-ethyl-1H-benzo[d]imidazol-1-yl)-9-methyl-6-morpholino-9H-purin-8-
yl)methyl)piperazin-2-one (WNY1613). WNY1613 exhibits good antiproliferative activity against a panel of
non-Hodgkin’s lymphoma (NHL) cell lines by inducing cancer cell apoptosis and inhibiting the phosphorylation
of PI3K and MAPK downstream components. In addition, it can also prevent the tumor growth in both SU-DHL-6
and JEKO-1 xenograft models without observable toxicity. WNY1613 thus could be developed as a promising
candidate for the treatment of NHL after subsequent extensive pharmacodynamics and pharmacokinetics
investigation.
Non Hodgkin’s lymphoma
PI3Kδ inhibitor
Antiproliferative
Apoptosis
Piperazinone
Purine
1. Introduction
exhibited impressive therapeutic efficiency, specific immune-mediated
toxicity and increased risk of infection prevented their widespread
Members of the phosphoinositide 3-kinase (PI3K) family catalyze the
phosphorylation of phosphatidylinositol 4,5-diphosphate (PIP2) to
produce phosphatidylinositol 3,4,5-triphosphate (PIP3), which acts as a
second messenger to transduce extracellular signals into cells by inter-
acting with a variety of pH domain-containing proteins [1–2]. PI3Kδ is
an isoform of PI3K family which mainly expresses in leukocytes and
regulates many crucial physiological processes of leukocytes as well as
malignant lymphoma cells [3–5]. It is therefore considered as a potential
therapeutic target for several kinds of lymphomas, including chronic
lymphocytic leukemia (CLL) and non Hodgkin’s lymphomas (NHLs) [6].
In the last decade, a number of PI3Kδ inhibitors have been developed
and entered into clinical trials [7–9]. Several promising candidates,
including the selective PI3Kδ inhibitor Idelalisib as well as the partial
isoforms selective PI3Kδ inhibitors Duvelisib and Copanlisib (Fig. 1)
[10–12], have been approved for the treatment of follicular B-cell non-
Hodgkin lymphoma (FL), small lymphocytic lymphoma (SLL) and
chronic lymphocytic leukemia (CLL). Although these PI3Kδ inhibitors
application [8,13]. As most PI3Kδ inhibitors that are approved or in
advanced clinical trials are similar in structure [9], it is difficult to
determine whether their adverse events observed in clinical are struc-
tural related or target related. More structurally diverse PI3Kδ inhibitors
thus need to be developed.
We recently discovered that piperazinone-containing thieno[3,2-d]
pyrimidine was a promising scaffold for PI3Kδ inhibitors, and the
piperazinone fragment in this series played important roles in improving
the PI3Kδ potency and the δ isoform selectivity [14]. The preliminary
SAR study has led to the discovery of several potent and selective PI3Kδ
inhibitors with single-digit nanomolar IC50 values and selectivity index
of over 100 folds against other three PI3K isoforms. Because previous
studies had demonstrated that purine ring would be a better scaffold for
this kind of PI3Kδ inhibitors with respect to their isoform selectivity as
well as pharmacokinetic properties (GNE-293, for example) [15–16],
we speculated that the replacement of thieno[3,2-d]pyrimidine core
with purine ring would result in a new series of PI3Kδ inhibitors with
* Corresponding authors.
E-mail addresses: liuzhihao@scu.edu.cn (Z.-H. Liu), wangny-swjtu@swjtu.edu.cn (N.-Y. Wang).
1
These authors contributed equally to this work.
https://doi.org/10.1016/j.bioorg.2020.104344
Received 21 June 2020; Received in revised form 8 September 2020; Accepted 1 October 2020
Available online 8 October 2020
0045-2068/© 2020 Elsevier Inc. All rights reserved.

W.-Q. Zuo et al.
Bioorganic Chemistry 105 (2020) 104344
improved potency and drug-like properties (Fig. 2).
selected to further evaluate their isoforms selectivity for PI3Kβ and
PI3Kγ as well as their antiproliferative activity against mantle cell
lymphoma (MCL) cell line JEKO-1 by MTT assay (Table 2). Most com-
pounds exhibited good δ/β and δ/γ selectivity and good antiproliferative
activity against JEKO-1 with submicromolar IC50 values. Of these
compounds, 8p (WNY1613) was selected for further biological evalu-
ation for its excellent potency and selectivity for PI3Kδ as well as good
antiproliferative activity against JEKO-1.
In this study, a series of 4-((9-methyl-9H-purin-8-yl)methyl)piper-
azin-2-ones and 1-((9-methyl-9H-purin-8-yl)methyl)piperazin-2-ones
were designed and synthesized as PI3Kδ inhibitors (Fig. 2). SAR study
led to the discovery of the most promising candidate WNY1613, which
exhibited good target affinity and selectivity. Here we report the dis-
covery, the biological activity evaluation as well as the mechanism of
action (MOA) studies with respect to this PI3Kδ inhibitor in several
NHLs models.
2.3. Biochemical characterization of WNY1613
2. Results and discussion
To further evaluate the target selectivity of WNY1613, we next
evaluated the inhibitory activity of WNY1613 on a panel of 300 kinases.
WNY1613 only exhibited potency (>50%) against the four PI3K iso-
forms at a concentration of 500 nM, while no inhibitory activity was
observed for other kinases (Fig. 3A, B). Cellular thermal shift assay
(CETSA) could evaluate the potential direct interaction between protein
and its ligand [20]. We next evaluated the effect of WNY1613 on the
thermal stability of PI3Kδ. The result showed that WNY1613 could
significantly improve the thermal stability of PI3Kδ at a concentration of
2.1. Chemistry
The title compounds mentioned in this study were prepared ac-
cording to a similar synthetic route for the preparation of piperazinone-
containing thieno[3,2-d]pyrimidine PI3Kδ inhibitors[14]. As shown in
Scheme 1, nucleophilic substitution and methylation of compound 1
provided 4-(2-chloro-9-methyl-9H-purin-6-yl)morpholine (2), which
underwent formylation with dimethylformamide and subsequent
reduction with sodium borohydride to afford intermediate 3. The
alcohol 3 then coupled with 2-ethylbenzo[d]imidazole to provide 4,
which was further chloridized with thionyl chloride to yield in-
termediates 5. The title compounds 7a-7d was prepared by nucleophilic
substitution of 5 with different 1-substituted piperazin-2-ones [14,17].
And the critical building block 6 was prepared by nucleophilic substi-
tution of 5 with tert-butyl 3-oxopiperazine-1-carboxylate and subse-
quent deprotection step of boc group with trifluoroacetic acid. Then, it
was reacted with 2,2,2-trifluoroethyl methanesulfonate to produce 8a or
underwent reduction amination with different ketones to produce 8b-8i.
Deprotecting ethylene glycol of 8i with concentrated hydrochloric acid
provided ketone 8j, which was further reduced by sodium borohydride
to provide 8k. Finally, the acyl derivatives 8l-8q were prepared by
acylation reaction of 6 with different acyl chlorides, and the sulfonyl
substituted derivatives 8r-8t were prepared by sulfonylation reaction of
6 with different sulfonyl chlorides.
10 μM, implying the direct interaction between WNY1613 and PI3Kδ
(Fig. 3C). Overall, these results indicated that WNY1613 was a potent
and highly selective PI3Kδ inhibitor.
2.4. Molecular docking
The binding modes of WNY1613 with PI3Kδ (PDB ID: 2WXP) [21]
and PI3Kγ (PDB ID: 3DBS) [22] were then investigated by molecular
docking. As shown in Fig. 4, the morpholine and benzimidazole motifs of
WNY1613 adopt highly similar conformations in both PI3Kδ and PI3Kγ,
and the morpholine O atom of WNY1613 forms a crucial hydrogen bond
with both Val828 in PI3Kδ and Val882 in PI3Kγ, respectively. However,
an additional hydrogen bond could be exclusively formed between the
piperazinone O atom of WNY1613 and Asn836 of PI3Kδ. Moreover, the
bulky cyclobutanecarbonyl motif of WNY1613 could stack on the
“tryptophan shelf” formed by Thr750 and Trp760 in PI3Kδ while it could
not be well accommodated in PI3Kγ in which the small threonine
(Thr750) is replaced by a larger, charged lysine (Lys802) [16]. These
results thus explained the excellent PI3Kδ potency of WNY1613 and its
well PI3Kδ selectivity over other PI3K isoforms.
2.2. The discovery of WNY1613
A series of 4-((9-methyl-9H-purin-8-yl)methyl)piperazin-2-ones and
1-((9-methyl-9H-purin-8-yl)methyl)piperazin-2-ones were synthesized
in this study. As shown in Table 1, both 4-((9-methyl-9H-purin-8-yl)
methyl)piperazin-2-one series (7a-7d) and 1-((9-methyl-9H-purin-8-yl)
methyl)piperazin-2-one series (8a-8t) exhibited excellent PI3Kδ inhibi-
tory activity with moderate to good δ isoform selectivity. An acyl
substituted at the 4-position of piperazinone (8l-8q) in the 1-((9-methyl-
9H-purin-8-yl)methyl)piperazin-2-one series seem to be conducive to
the improvement of both potency and selectivity, while the alkyls (8a-
8k) and sulfonyls (8r-8t) substituted series exhibited less selectivity for
2.5. Antiproliferative activity of WNY1613
We next selected 10 different types of NHL cell lines, including seven
diffuse large B-cell lymphoma (DLBCL) cell lines (SU-DHL-6, SU-DHL-4,
OCI-LY-3, OCI-LY-10, U2932, WSU-DLCL-2, DB), two mantle cell lym-
phoma (MCL) cell lines (JEKO-1, Granta-519) and one Burkitt’s lym-
phoma (NAMALVA) cell line, to investigate their sensitivity to
WNY1613 by MTS assay. As shown in Table 3, several NHL cell lines,
including SU-DHL-6, SU-DHL-4, JEKO-1, OCI-LY- 3, Granta-519 and
NAMALVA, were sensitive to both Idelalisib and WNY1613 with
micromolar to sub-micromolar IC50 values, and the antiproliferative
PI3Kα.
To identify a lead compound from the abovementioned compounds
for subsequent biological evaluation, several potent compounds were
Fig. 1. Structure of three approved PI3Kδ inhibitors.
2

W.-Q. Zuo et al.
Bioorganic Chemistry 105 (2020) 104344
O
N
O
N
O
N
S
S
N
N
O
R
or
N
N
N
N
R'
N
R'
N
N
O
O
N
N
N
R
R'
N
N
R
Piperazinone-containing thieno[3,2-d]pyrimidines
O
O
N
N
N
N
N
N
N
N
N
O
N
R
R'
O
N
N
N
N
N
Piperazinone-containing purines
S
GNE-293
O
O
Fig. 2. Design of piperazinone-containing purines as PI3Kδ inhibitors, GNE-293 is a potent and selective PI3Kδ inhibitor with purine core [16].
Scheme 1. Synthesis of purine derivatives 7a-7d and 8a-8 t. Reagents and conditions: (a).
morpholine, MeOH, rt; ② methyl iodide, MeCN, Cs2CO3, rt. (b).
n-
BuLi, DMF, THF, ꢀ 78℃; ② NaBH4, MeOH. (c). 2-ethyl-1H-benzo[d]imidazole, Pd2(dba)3, XPhos, Cs2CO3, 1,4-dioxane, 110℃. (d). thionyl chloride, DCM. (e). 1-
substituted piperazin-2-ones, DIPEA, i-PrOH, reflux. (f).
N-boc-3-oxopiperazine, t-BuOK, THF, 0℃; ② TFA, DCM, rt. (g). 2,2,2-trifluoroethyl methanesulfonate,
K2CO3, MeCN, reflux. (h). aldehydes or ketones, DCM, sodium triacetoxyborohydride. (i). DCM, THF, concentrated HCl(aq). (j). NaBH4, MeOH, 0℃- r.t. (k). L-lactic
acid, HOBT, EDCI, TEA, DCM. (l). acyl chlorides or sulfonyl chlorides, TEA, DCM.
3

W.-Q. Zuo et al.
Bioorganic Chemistry 105 (2020) 104344
Table 1
SAR study of piperazinone-containing purine PI3Kδ inhibitors.
Cpd
R
PI3Kδa
Selectivity fold over PI3Kαb
Cpd
8j
R
PI3Kδ
Selectivity fold over PI3Kα
IC50(nM)
IC50(nM)
7a
7b
5.1 ± 1.7
77
6.6 ± 3.1
63
3.4 ± 1.5
96
8k
3.3 ± 0.6
68
7c
7d
4.4 ± 2.1
8.6 ± 2.4
81
43
8l
2.7 ± 1.7
1.6 ± 0.8
83
8m
148
8a
8b
5.1 ± 0.7
4.8 ± 1.3
68
66
8n
8o
2.1 ± 1.1
1.8 ± 0.9
134
126
8c
8d
11 ± 3.8
4.5 ± 2.2
44
65
8p
8q
1.2 ± 0.7
4.6 ± 1.9
219
86
8e
4.2 ± 0.6
35
8r
2.7 ± 1.3
52
8f
2.7 ± 1.1
2.7 ± 1.4
2.0 ± 0.8
91
86
47
8s
6.9 ± 0.7
3.2 ± 1.4
6.2 ± 2.7
37
50
1.6
8 g
8 h
8t
PI-103c
8i
2.3 ± 0.4
58
Idelalisibd
4.7 ± 2.6
>106
a
b
c
IC50 values for PI3Kδ are the mean of three independent measurements and that for PI3K
α
are the mean of at least two independent measurements.
was calculated as the ratio of the IC50 values of a compound to PI3K and PI3Kδ.
PI-103 is a pan-PI3K inhibitor with reported IC50s of 2 nM and 3 nM against PI3K and PI3Kδ, respectively [18].
, β, δ and γ were 820 nM, 565 nM, 2.5 nM and 89 nM, respectively [19].
The selectivity fold over PI3K
α
α
α
d
The reported IC50s for Idelalisib against PI3K
α
activity of WNY1613 was better than Idelalisib to different extent across
all these sensitive cell lines, which is consistent with its better PI3Kδ
potency than Idelalisib.
apoptosis induction effects. Moreover, the apoptosis induction effects of
WNY1613 and Idelalisib as well as their influence on the protein level of
the apoptosis cascade in NAMALVA cells were significantly weaker than
that in other three cell lines, which could explain the weaker anti-
proliferative activity of both compounds on NAMALVA cells as
compared with other three cell lines.
2.6. Apoptosis induction activity of WNY1613
Apoptosis induction is a common mechanism to mediate the anti-
proliferative activity of kinase inhibitors, including PI3K inhibitors
[23–24]. We next assessed the apoptosis induction effect of WNY1613
on four NHL cell lines, including SU-DHL-6, SU-DHL-4, JEKO-1, and
NAMALVA. As shown in Fig. 5, WNY1613 could induce cancer cell
apoptosis (Annexin V-positive) across all four NHL cell lines in a
concentration-dependent manner. And the apoptosis induction effect of
WNY1613 was also more obvious than that for Idelalisib. We next
evaluated the protein levels of the apoptosis cascade, and the results
showed that WNY1613 could reduce the expression of anti-apoptotic
protein BCL-2 and increase the expression of pro-apoptotic protein
BAX as well as cleaved caspase-3 in a concentration-dependent manner
(Fig. 5C). And the effects of WNY1613 on the protein level of the
apoptosis cascade was also more significant than that for Idelalisib
across all four NHL cell lines, which was consistent with its better
2.7. PI3K pathway inhibition by WNY1613
The PI3K downstream components, AKT, S6, 4EBP1, as well as the
MAPK downstream component ERK have been reported to be phos-
phorylated by PI3K activation [25], which could promote the prolifer-
ation of lymphoma cells. We next examined the effects of Idelalisib and
WNY1613 on the expression of these proteins by western blotting. Both
Idelalisib and WNY1613 could inhibit the phosphorylation of AKT, S6,
4EBP1 and ERK in a dose- and time-dependent manners without
affecting their total protein levels (Fig. 6). And the inhibitory effects of
WNY1613 on the phosphorylation of these proteins were more signifi-
cant than Idelalisib in all four cell lines. In addition, consistent with the
results in antiproliferative assay and apoptosis induction test, the
phosphorylation of PI3K pathway-related proteins in NAMALVA cells
4

W.-Q. Zuo et al.
Bioorganic Chemistry 105 (2020) 104344
Increasing the dosage of WNY1613 to 50 mg/kg only slightly increased
the TGI in the JEKO-1 model, which might be attributed to the poor
pharmacokinetic properties of WNY1613 in its current dosage form.
Moreover, no apparent body weight loss or abnormal behavior was
observed during the treatment period (Fig. 7C), implying the good safety
profile of WNY1613.
Table 2
The PI3K isoforms selectivity and antiproliferative activity of piperazinone-
containing purine PI3Kδ inhibitors a
Cmpds
.
PI3Kδ
PI3K
α
PI3Kβ
PI3Kγ
JEKO-
1
(IC50
:
selectivity
selectivity
selectivity
nM)
(IC50
:
μ
M)
To investigate the underlying mechanisms of the in vivo antitumor
effects of WNY1613, immunohistochemical analysis was performed for
tumor tissues resected from both tumor models after drug withdrawal.
As shown in Fig. 7D and E, a significant reduction of p-AKT, p-P70S6K,
p-4EBP1 and p-ERK was observed in the Idelalisib and WNY1613 groups
than the vehicle group, indicating that WNY1613 could inhibit PI3K
pathway in vivo. Overall, the in vivo pharmacodynamics study further
confirmed the solid anticancer activity of WNY1613 via its inhibition on
PI3K.
7b
8f
3.4 ±
1.5
96
>147
133
10
>147
96
0.20 ±
0.08
2.7 ±
1.1
91
0.37 ±
0.06
8l
2.7 ±
1.7
83
96
0.28 ±
0.13
8m
8n
8o
8p
1.6 ±
0.8
148
134
126
219
>106
27
169
207
60
0.28 ±
0.10
2.1 ±
1.1
238
77
0.23 ±
0.14
1.8 ±
0.9
0.18 ±
0.05
1.2 ±
0.7
120
>106
250
52
0.14 ±
0.07
2.9. Conclusion
(WNY1613)
Idelalisib
4.7 ±
2.6
0.93 ±
0.54
A potent PI3Kδ inhibitor WNY1613 based on piperazinone-
containing purine scaffold was discovered from our SAR study. This
compound exhibited excellent target affinity and isoforms selectivity
with IC50 of 1.2 nM against PI3Kδ and selectivity index of over 100 folds
for other PI3K isoforms. The direct interaction between WNY1613 and
PI3Kδ was further validated by cellular thermal shift assay. WNY1613
could inhibit the proliferation of several NHL cell lines with micromolar
to sub-micromolar IC50 values. The MOA study demonstrated that it
could induce apoptosis of lymphoma cells by reducing the expression of
anti-apoptotic proteins and increasing the expression of pro-apoptotic
proteins. Moreover, as a PI3Kδ inhibitor, WNY1613 can inhibit the
phosphorylation of PI3K downstream components AKT, S6, 4EBP1 and
MAPK downstream component ERK in a concentration- and time-
dependent manners. Finally, WNY1613 demonstrated solid antitumor
activity in both SU-DHL-6 and JEKO-1 xenograft models without
observable toxicity. Overall, our study provided a potent PI3Kδ inhibitor
WNY1613 as a promising candidate for lymphoma. The optimization of
its dosage form would further improve its effectiveness in vivo and
further pharmacodynamic studies in more lymphoma models would
promote its development as a preclinical candidate.
a
IC50 values for PI3Kδ isoforms and antiproliferative activity against JEKO-1
are the mean of at least three independent measurements.
was also minimally affected by both compounds across all four cell lines,
implying that the antiproliferative effect of WNY1613 is mainly medi-
ated by its inhibition on PI3K pathway.
2.8. In vivo efficacy of WNY1613
To evaluate the in vivo anti-tumor activity of WNY1613, SU-DHL-6
and JEKO-1 cells were subcutaneously engrafted into NOD-SCID mice to
establish xenograft models. As shown in Fig. 7, tumor-bearing mice were
oral administration of WNY1613 with dosages of 25 and 50 mg/kg,
Idelalisib with dosage of 25 mg/kg, or the vehicle for 18 days. The re-
sults showed that both Idelalisib and WNY1613 demonstrated solid
antitumor activity in both xenograft models at a dose of 25 mg/kg, with
tumor growth inhibition (TGI) rates of 47.69% and 51.38% for SU-DHL-
6 model and 45.92% and 55.52% for JEKO-1 model, respectively.
Fig. 3. Biochemical characterization of WNY1613. (A) Heat map showing inhibitory rate of WNY1613 against a panel of 300 kinases. The diamond in the red frame
represents PI3Kδ. (B) Potency of WNY1613 against PI3Kα, PI3Kβ, PI3Kγ and PI3Kδ. (C) Cellular thermal shift assay from 40℃ to 100℃ of SU-DHL-6 and JEKO-1
lysates with or without WNY1613 incubation. Data represent the average inhibitory rate of two replicates for (A) and average ± SD of three replicates for (B).
(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
5

W.-Q. Zuo et al.
Bioorganic Chemistry 105 (2020) 104344
Fig. 4. Predicted binding modes of WNY1613 with PI3Kδ (A) and PI3Kγ (B). Hydrogen bonds are shown as green dashed lines. (For interpretation of the references
to colour in this figure legend, the reader is referred to the web version of this article.)
filtration, and the filter cake was washed by water (50 mL) and MeOH
Table 3
(30 mL), dried under reduced pressure to afford 4-(2-Chloro-9H-purin-6-
IC50 values of Idelalisib and WNY1613 against a panel of NHL cell lines.
yl)morpholine (21.412 g, 90%) as a white solid. ESI-MS [M + Na]+ (m/
Cell lines
Idelalisib
WNY1613
z): 262.2.
(IC50
:
μ
M)
(IC50: μM)
Cs2CO3 (24.437 g, 75 mmol) and MeI (4.7 mL, 75 mmol) was slowly
U2932
>10
>10
>10
>10
added to the acetonitrile solution of 4-(2-Chloro-9H-purin-6-yl)mor-
pholine (11.965 g, 50 mmol). The mixture was stirred under room
temperature for 4 h until the starting materials completely transformed.
The solvent was removed under reduced pressure and the residue was
suspended in dichloromethane. The suspension was stirred for 1 h and
separated by filtration. The filtrate was concentrated under reduced
pressure to afford the crude product, which was recrystallized by ethyl
acetate to afford 4-(2-chloro-9-methyl-9H-purin-6-yl)morpholine
(11.633 g, 91%) as a white solid. ESI-MS [M + Na]+ (m/z): 276.1.
OCI-LY-10
OCI-LY-3
DB
5.2 ± 2.3
>10
1.6 ± 0.2
>10
SU-DHL-4
WSU-DLCL-2
SU-DHL-6
JEKO-1
0.30 ± 0.13
>10
0.048 ± 0.029
5.0 ± 1.7
0.12 ± 0.03
0.12 ± 0.06
1.7 ± 0.8
9.2 ± 2.9
0.038 ± 0.011
0.077 ± 0.023
0.55 ± 0.15
2.0 ± 0.6
Granta-519
NAMALVA
a. IC50 values for the antiproliferative activity of Idelalisib and WNY1613 are the
mean of three independent measurements and represented as mean ± SD.
3.1.2. Synthesis of (2-chloro-9-methyl-6-morpholino-9H-purin-8-yl)
methanol (3) [26]
3. Experimental
2.5 M solution of n-butyl lithium (9.6 mL.24 mmol) in n-hexane was
slowly added to the solution of 2 (5.065 g, 20 mmol) in anhydrous
tetrahydrofuran (30 mL) at ꢀ 78 ℃. The resulting mixture was stirred
under ꢀ 78 ℃ for 1 h and anhydrous dimethylformamide (2.3 mL, 30
mmol) was added, followed by stirring for another 2 h under ꢀ 78 ℃.
Then it was warmed up to room temperature and the reaction mixture
was poured into cold saturated ammonium chloride solution (100 mL)
and stirred for 30 min. After filtration, the filter cake was washed with
ice water (20 mL) and dried under vacuum to afford 2-chloro-9-methyl-
6-morpholino-9H-purine-8-carbaldehyde as a yellowish solid (5.407 g,
95%). ESI-MS [M+H]+ (m/z): 284.0.
3.1. Chemistry
Unless otherwise noted, all materials were obtained from commer-
cial suppliers and used without further purification. The 1H and 13C
NMR spectra were collected on a Bruker Avance 400 spectrometer at 25
ꢀ using CDCl3 as the solvent. Chemical shifts (δ) are reported in ppm
relative to TMS (internal standard), coupling constants (J) are reported
in hertz, and peak multiplicity are reported as s (singlet), d (doublet), t
(triplet), q (quartet), m (multiplet), or br s (broad singlet). Mass spectra
(MS) were measured on a Micromass Q-TOF Premier mass spectrometer
with electron spray ionization (ESI). Thin layer chromatography (TLC)
was performed on 0.20 mm silica gel F-254 plates (Qingdao Haiyang
Chemical, China). Preparative thin layer chromatography was per-
formed on 1.00 mm silica gel F-254 plates (Qingdao Haiyang Chemical,
China). Visualization of TLC was accomplished with UV light and/or I2
in silica gel. Column chromatography was performed using silica gel of
300–400 mesh (Qingdao Haiyang Chemical, China). The purities of the
title compounds were determined on an UltiMate 3000 (Dionex, USA)
HPLC system and were of > 95% purity. (Phenomenex C18 reversed-
Sodium borohydride (0.756 g, 20 mmol) was slowly added to the
suspension of 2-chloro-9-methyl-6-morpholino-9H-purine-8-carbalde-
hyde (2.830 g.10 mmol) in MeOH (30 mL) at ꢀ 10 ℃. The resulting
mixture was stirred under ꢀ 10 ℃ for 1 h until the starting materials
completely transformed. Then 90 mL of ice water was added to the re-
action mixture and the mixture was stirred for another 10 min. After
filtration, the filter cake was washed with ice water (20 mL) and dried
under vacuum to afford the product 3 as a white solid (2.625 g.92%).
ESI-MS [M + Na]+ (m/z): 308.1.
column, 4.6 mm × 150 mm, 5 μm, isocratic elution with acetonitrile/
3.1.3. Synthesis of (2-(2-ethyl-1H-benzo[d]imidazol-1-yl)-9-methyl-6-
morpholino-9H-purin-8-yl)methanol (4) [26]
H2O = 60/40 or 65/35; flow rate, 1.0 mL/min; detection wavelength,
254 nm; temperature, 30 ℃).
Under nitrogen atmosphere, a 1,4-dioxane solution of (2-chloro-9-
methyl-6-morpholino-9H-purin-8-yl)methanol (3) (2.566 g, 9 mmol), 2-
ethyl-1H-benzo[d]imidazole, Pd2(dba)3 (0.9 mmol), Xphos (1.8 mmol)
and CsCO3 (18 mmol) was warmed up to 110 ℃ and stirred for 5 h until
the starting materials completely transformed. the reaction mixture was
filtered after cooled to room temperature, and the filtrate was purified
by column chromatography with dichloromethane/methanol (V/V =
100/1–50/1) to afford (2-(2-ethyl-1H-benzo[d]imidazol-1-yl)-9-methyl-
3.1.1. Synthesis of 4-(2-chloro-9-methyl-9H-purin-6-yl)morpholine (2)
[26]
Morpholine (150 mmol) was added dropwise to the MeOH (500 mL)
solution of 2,6-dichloropurine (18.900 g, 100 mmol) at 0 ℃. The
mixture was stirred under room temperature for 2 h until the starting
materials completely transformed. Then the precipitate was collected by
6

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Bioorganic Chemistry 105 (2020) 104344
Fig. 5. WNY1613 induced cancer cell apoptosis in NHL cell lines. (A) FCM analysis of the apoptosis cells in four NHL cell lines after treatment with Idelalisib and
WNY1613 with indicated concentrations for 72 h. (B) Quantification of the FCM analysis. Graphic data were run in triplicate and shown as the mean ± SD, *p < 0.05,
**p < 0.01, ***p < 0.001. (C) Western blotting analysis of apoptosis related protein levels, including BCL2, Bax and cleaved caspase-3, after treated with Idelalisib or
WNY1613 with the indicated concentrations for 72 h.
6-morpholino-9H-purin-8-yl)methanol (4) as a yellowish solid (1.874 g,
53%). ESI-MS [M + H]+ (m/z): 396.2.
oxopiperazine (1.005 g, 5 mmol) in anhydrous tetrahydrofuran (20
mL). The mixture was stirred for 20 min under room temperature after
by compound 5 (1.653 g, 4 mmol) added. Then the reaction mixture was
warmed up to room temperature and stirred for another 4 h until the
starting materials completely transformed. The solvent was removed
under reduced pressure and the residue was extracted with water and
ethyl acetate. The organic phase was washed with brine, dried over
Na2SO4, and concentrated under reduced pressure. The crude product
was purified by column chromatography with dichloromethane/meth-
anol (V/V = 100/1–50/1) as mobile phase to afford tert-butyl 4-((2-(2-
ethyl-1H-benzo[d]imidazol-1-yl)-9-methyl-6-morpholino-9H-purin-8-
yl)methyl)-3-oxopiperazine-1-carboxylate (1.829 g, 79%), which was
further deprotected of the boc group with trifluoroacetic acid in
dichloromethane to produce 6 with yield of 100%.
3.1.4. Synthesis of 4-(8-(chloromethyl)-2-(2-ethyl-1H-benzo[d]imidazol-
1-yl)-9-methyl-9H-purin-6-yl)morpholine (5) [26]
SOCl2 (9 mmol) was slowly added to the dichloromethane solution of
4 (1.778 g, 4.5 mmol) at 0 ℃ over 10 min. The reaction mixture was
stirred under room temperature for 2 h until the starting materials
completely transformed. After concentrated under reduced pressure, the
residue was suspended in water (20 mL) and stirred for 1 h. After
filtration, the filter cake was washed by cold water (10 mL) and dried
under vacuum to afford 4-(8-(chloromethyl)-2-(2-ethyl-1H-benzo[d]
imidazol-1-yl)-9-methyl-9H-purin-6-yl)morpholine (5) as a yellowish
solid (1.719 g, 92%). ESI-MS [M + H]+ (m/z): 414.3.
3.1.5. Synthesis of 1-((2-(2-ethyl-1H-benzo[d]imidazol-1-yl)-9-methyl-6-
morpholino-9H-purin-8-yl)methyl)piperazin-2-one trifluoroacetate (6)
t-BuOK (7.5 mmol) was added to the solution of 1-boc-3-
3.1.6. General procedure for the synthesis of 7a-7d
DIPEA (1.0 mmol) was added to the isopropanol (5 mL) solution of
different 1-substituted piperazin-2-ones (0.3 mmol) and 5 (0.083 g, 0.2
7

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Bioorganic Chemistry 105 (2020) 104344
Fig. 6. WNY1613 showed a dose- and time-dependent downregulation of PI3K pathway related proteins in all four NHL cell lines. (A) Western blotting analysis for
PI3K pathway related proteins, including p-AKT, AKT, p-S6, S6, p-4EBP1, 4EBP1, p-ERK and ERK, after treatment with Idelalisib or WNY1613 with indicated
concentrations (
μM) for 24 h. (B) Western blotting analysis for PI3K pathway related proteins after treatment with Idelalisib or WNY1613 (1 μM for SU-DHL-6 and
JEKO-1 cell lines, 3
μ
M for SU-DHL-4 cell line and 9 μM for NAMALVA cell line) for indicated times.
mmol). The mixture was stirred under 85℃ for 24 h until the starting
materials completely transformed. The solvent was removed under
reduced pressure and the residue was extracted with water/dichloro-
methane, the organic phase was washed with brine and dried over
MgSO4. The crude product was purified by preparative TLC with
dichloromethane/methanol (V/V = 10/1) as mobile phase to afford the
title compounds 7a-7d with yields of 40%-70%.
62%. 1H NMR (400 MHz, CDCl3) δ 8.05–7.94 (m, 1H), 7.75 (dd, J = 5.7,
3.3 Hz, 1H), 7.33–7.19 (m, 2H), 5.03–4.83 (m, 2H), 3.89–3.70 (m, 9H),
3.65–3.56 (m, 1H), 3.50 (d, J = 5.4 Hz, 1H), 3.42–3.12 (m, 7H),
2.86–2.71 (m, 2H), 1.92–1.76 (m, 4H), 1.74–1.57 (m, 4H), 1.45 (t, J =
7.5 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 166.14, 156.94, 153.70 (2C),
150.73, 146.61, 142.60, 134.62, 122.73, 122.65, 119.10, 116.99,
113.23, 67.02 (2C), 57.60, 54.22, 53.85, 49.66 (2C), 45.89, 40.92,
29.20, 27.87 (2C), 24.07 (2C), 23.77, 12.38. ESI-MS [M+H]+ (m/z):
544.3.
1-(cyclopropylmethyl)-4-((2-(2-ethyl-1H-benzo[d]imidazol-1-yl)-9-
methyl-6-morpholino-9H-purin-8-yl)methyl)piperazin-2-one (7a). White
solid, yield: 68%. 1H NMR (400 MHz, CDCl3) δ 8.11–7.97 (m, 1H),
7.87–7.72 (m, 1H), 7.41–7.22 (m, 2H), 4.36 (s br, 4H), 3.89 (d, J = 6.8
Hz, 6H), 3.84 (s, 3H), 3.45 (t, J = 5.6 Hz, 2H), 3.37 (q, J = 7.5 Hz, 2H),
3.31 (d, J = 8.9 Hz, 4H), 2.84 (t, J = 5.6 Hz, 2H), 1.46 (t, J = 7.5 Hz, 3H),
0.98 (d, J = 6.9 Hz, 1H), 0.54 (d, J = 7.6 Hz, 2H), 0.26 (d, J = 5.1 Hz,
2H). 13C NMR (101 MHz, CDCl3) δ 166.02, 156.93, 153.69 (2C), 150.69,
146.62, 142.49, 134.58, 122.84, 122.67, 119.04, 117.00, 113.22, 67.01
(2C), 57.35, 54.23, 50.50, 49.49, 46.40 (2C), 45.68, 29.21, 23.74, 12.39,
8.96, 3.55 (2C). ESI-MS [M+H]+ (m/z): 530.3.
4-((2-(2-ethyl-1H-benzo[d]imidazol-1-yl)-9-methyl-6-morpholino-9H-
purin-8-yl)methyl)-1-(1-methyl-1H-pyrazol-4-yl)piperazin-2-one (7c). Pale
solid, yield: 69%. 1H NMR (400 MHz, CDCl3) δ 8.03 (s, 1H), 8.02–7.97
(m, 1H), 7.80–7.71 (m, 1H), 7.46 (s, 1H), 7.34–7.22 (m, 2H), 4.36 (s,
4H), 4.01–3.77 (m, 12H), 3.71 (t, J = 5.5 Hz, 2H), 3.43 (s, 2H), 3.35 (q,
J = 7.5 Hz, 2H), 2.96 (t, J = 5.5 Hz, 2H), 1.45 (t, J = 7.5 Hz, 3H). 13
C
NMR (101 MHz, CDCl3) δ 164.10, 156.93, 153.72 (2C), 150.78, 146.31,
142.56, 134.59, 129.45, 124.84, 123.08, 122.84, 122.68, 119.08,
117.01, 113.22, 67.01 (2C), 57.76, 54.11, 49.10, 47.44 (2C), 45.89,
39.34, 29.22, 23.78, 12.38. ESI-MS [M+H]+ (m/z): 556.3.
1-cyclopentyl-4-((2-(2-ethyl-1H-benzo[d]imidazol-1-yl)-9-methyl-6-
morpholino-9H-purin-8-yl)methyl)piperazin-2-one (7b). White solid, yield:
2-(4-((2-(2-ethyl-1H-benzo[d]imidazol-1-yl)-9-methyl-6-morpholino-
8

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Bioorganic Chemistry 105 (2020) 104344
Fig. 7. In vivo anticancer activity of WNY1613 in NHL xenograft models. Tumor bearing NOD-SCID mice were orally treated with vehicle, Idelalisib (25 mg/kg),
WNY1613(25 mg/kg) and WNY1613(50 mg/kg) once every day for indicated days. (A) Volume of tumors from sacrificed mice, data are presented as the mean ± SD.
(B) Weight of tumors from sacrificed mice, data are presented as the mean ± SD. (C) The average body weight changes of the vehicle, Idelalisib, WNY1613 treated
groups, data are presented as the mean ± SD. (D) Immunohistochemical analysis of p-AKT, p-S6, p-4EBP1and p-ERK levels in tumor tissues of SU-DHL-6 and JEKO-1
xenograft models from sacrificed mice. (E) Quantification of percentage of positive staining cells, data are presented as the mean ± SD, *p < 0.05, **p < 0.01, ***p <
0.001, ****p < 0.0001.
9H-purin-8-yl)methyl)-2-oxopiperazin-1-yl)acetamide (7d). Yellowish
solid, yield: 43%. 1H NMR (400 MHz, CDCl3) δ 8.08–7.96 (m, 1H),
7.82–7.69 (m, 1H), 7.27 (q, J = 3.7 Hz, 2H), 6.27 (s, 1H), 5.56 (s, 1H),
4.35 (s br, 4H), 4.03 (s, 2H), 3.99–3.80 (m, 9H), 3.50 (t, J = 5.5 Hz, 2H),
3.35 (q, J = 7.7 Hz, 2H), 3.34 (s, 2H), 2.88 (t, J = 5.5 Hz, 2H), 1.45 (t, J
= 7.5, 3H). 13C NMR (101 MHz, CDCl3) δ 170.22, 167.32, 156.93,
153.72, 153.67, 150.76, 146.28, 142.50, 134.57, 122.86, 122.70,
119.06, 117.00, 113.22, 67.00 (2C), 57.03, 54.01, 50.39, 49.33, 48.05
(2C), 45.79, 29.21, 23.75, 12.38. ESI-MS [M+H]+ (m/z): 533.3.
3.1.7. Synthesis of 1-((2-(2-ethyl-1H-benzo[d]imidazol-1-yl)-9-methyl-6-
morpholino-9H-purin-8-yl)methyl)-4-(2,2,2-trifluoroethyl)piperazin-2-one
(8a)
2,2,2-Trifluoroethyl methanesulfonate (0.3 mmol) and K2CO3 (0.3
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W.-Q. Zuo et al.
Bioorganic Chemistry 105 (2020) 104344
mmol) were added to the acetonitrile solution of 6 (0.089 g, 0.15 mmol).
The mixture was stirred under 85 ℃ for 2 h until the starting materials
completely transformed. The solvent was removed under reduced
pressure and the residue was extracted with water and ethyl acetate. The
organic phase was washed with brine, dried over Na2SO4, and concen-
trated under reduced pressure. The crude product was purified by pre-
parative TLC with dichloromethane/methanol (V/V = 10/1) as mobile
phase to afford 8a (0.042 g, 50%) as a white solid.1H NMR (400 MHz,
CDCl3) δ 8.07–7.95 (m, 1H), 7.82–7.70 (m, 1H), 7.33–7.21 (m, 2H), 4.87
(s, 2H), 4.35 (s br, 4H), 3.86 (t, J = 7.2 Hz, 4H), 3.84 (s, 3H), 3.50 (t, J =
5.8 Hz, 2H), 3.48 (s, 2H), 3.35 (q, J = 7.5 Hz, 2H), 3.08 (q, J = 9.2 Hz,
2H), 2.97 (t, J = 5.4 Hz, 2H), 1.44 (t, J = 7.5 Hz, 3H). 13C NMR (101
MHz, CDCl3) δ 166.02, 156.91, 153.68, 153.46, 150.83, 145.96, 142.48,
134.54, 124.90 (q, J = 279 Hz), 122.89, 122.72, 119.08, 117.27,
113.23, 66.99 (2C), 57.18, 57.04 (q, J = 29 Hz), 50.03, 46.48 (2C),
45.89, 42.03, 29.25, 23.78, 12.37. 19F NMR (376 MHz, CDCl3) δ ꢀ 69.17.
ESI-MS [M + H]+ (m/z): 558.2
7.74 (d, J = 4.5 Hz, 1H), 7.27 (dd, J = 8.8, 5.3 Hz, 2H), 4.86 (s, 2H),
3.95–3.75 (m, 7H), 3.44 (d, J = 13.1 Hz, 4H), 3.33 (d, J = 12.5 Hz, 4H),
3.00 (d, J = 10.9 Hz, 2H), 2.77 (q, J = 5.4, 2H), 2.35 (s, 5H), 2.12 (t, J =
10.3 Hz, 3H), 1.84 (d, J = 9.8 Hz, 2H), 1.68 (dd, J = 30.0, 19.6 Hz, 2H),
1.41 (t, J = 7.5 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 167.27, 156.94,
153.65, 153.43, 150.73, 146.20, 142.43, 134.52, 122.89, 122.79,
119.00, 117.26, 113.24, 66.99 (2C), 59.41, 54.42 (2C), 54.15, 46.79,
45.90, 45.53 (2C), 45.49, 41.94, 29.27, 27.28 (2C), 23.73, 12.38. ESI-
MS [M+H]+ (m/z): 573.3.
1-((2-(2-ethyl-1H-benzo[d]imidazol-1-yl)-9-methyl-6-morpholino-9H-
purin-8-yl)methyl)-4-(tetrahydro-2H-pyran-4-yl)piperazin-2-one
(8f).
White solid, yield: 45%. 1H NMR (400 MHz, CDCl3) δ 8.07–7.94 (m, 1H),
7.83–7.70 (m, 1H), 7.37–7.20 (m, 2H), 4.87 (s, 2H), 4.31 (s br, 4H), 4.03
(dd, J = 11.5, 2.9 Hz, 2H), 3.95–3.76 (m, 7H), 3.53–3.29 (m, 8H), 2.79
(q, J = 5.4, 2H), 2.48 (m, 1H), 1.91–1.70 (m, 2H), 1.55 (m, 2H), 1.44 (t,
J = 7.5 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 167.21, 156.92, 153.66,
153.45, 150.72, 146.21, 142.25, 134.47, 122.95, 122.87, 118.98,
117.28, 113.26, 66.99 (4C), 59.68 (2C), 53.83, 46.73, 45.89, 45.57 (2C),
41.96, 29.30, 29.24 (2C), 23.71, 12.39. ESI-MS [M + H]+ (m/z): 560.3.
1-((2-(2-ethyl-1H-benzo[d]imidazol-1-yl)-9-methyl-6-morpholino-9H-
purin-8-yl)methyl)-4-(tetrahydro-2H-thiopyran-4-yl)piperazin-2-one (8 g).
White solid, yield: 51%. 1H NMR (400 MHz, CDCl3) δ 8.00 (d, J = 6.8 Hz,
1H), 7.76 (d, J = 6.6 Hz, 1H), 7.32 (d, J = 32.2 Hz, 2H), 4.87 (s, 2H),
4.35 (s br, 4H), 3.95–3.75 (m, 7H), 3.45–3.30 (m, 6H), 2.80–2.57 (m,
6H), 2.47–2.32 (m, 1H), 2.21–2.02 (m, 2H), 1.72–1.64 (m, 2H), 1.44 (t,
J = 7.5 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 167.44, 156.90, 153.66,
153.44, 150.65, 146.29, 142.00, 134.40, 122.94, 122.86, 118.92,
117.31, 113.28, 67.00 (2C), 61.39, 53.23, 46.95, 45.64, 45.43 (2C),
42.01, 29.93 (2C), 29.29, 28.34 (2C), 23.65, 12.42. ESI-MS [M+H]+ (m/
z): 576.3.
3.1.8. General procedure for the synthesis of 8b-8i.
Sodium acetate (0.3 mmol) and sodium triacetoxyborohydride (0.4
mmol) was added to the dichloromethane (10 mL) solution of 6 (0.089 g,
0.15 mmol) and different aldehydes or ketones (0.25 mmol) at 0 ℃. The
mixture was stirred under room temperature for 12 h until the starting
materials completely transformed. Then saturated sodium bicarbonate
solution was added to the reaction mixture and the mixture was stirred
for another 30 min. After the organic phase separated by extraction, the
aqueous phase was reextracted with dichloromethane. The combined
organic phase was washed with brine, dried over MgSO4 and concen-
trated under reduced pressure. The crude product was further purified
by preparative TLC with dichloromethane/methanol (V/V = 10/1) to
afford 8b-8i with yields of 30–60%.
4-(1,1-dioxidotetrahydro-2H-thiopyran-4-yl)-1-((2-(2-ethyl-1H-benzo
[d]imidazol-1-yl)-9-methyl-6-morpholino-9H-purin-8-yl)methyl)piperazin-
2-one (8 h). White solid, yield: 33%. 1H NMR (400 MHz, CDCl3) δ
8.06–7.96 (m, 1H), 7.81–7.70 (m, 1H), 7.33–7.20 (m, 2H), 4.87 (s, 2H),
4.35 (s br, 4H), 3.99–3.78 (m, 7H), 3.48 (dd, J = 6.3, 4.4 Hz, 2H),
3.41–3.29 (m, 4H), 3.27–3.12 (m, 2H), 2.99–2.86 (m, 2H), 2.78 (t, J =
6.3 Hz, 2H), 2.58 (d, J = 3.2 Hz, 1H), 2.33–2.12 (m, 4H), 1.44 (t, J = 7.4
Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 166.30, 156.90, 153.70, 153.44,
150.87, 145.89, 142.50, 134.54, 122.89, 122.73, 119.10, 117.30,
113.25, 66.99 (2C), 57.10, 54.27, 48.64 (2C), 46.63, 45.75 (3C), 41.92,
29.30, 25.80 (2C), 23.81, 12.38. ESI-MS [M + H]+ (m/z): 608.3.
1-((2-(2-ethyl-1H-benzo[d]imidazol-1-yl)-9-methyl-6-morpholino-9H-
purin-8-yl)methyl)-4-(1,4-dioxaspiro[4.5]decan-8-yl)piperazin-2-one (8i).
8i was prepared from 6 (0.3 mmol) as a white solid, yield: 57%. 1H NMR
(400 MHz, CDCl3) δ 8.00 (d, J = 5.4 Hz, 1H), 7.82–7.69 (m, 1H), 7.26 (d,
J = 4.9 Hz, 2H), 4.86 (s, 2H), 4.35 (s br, 4H), 3.93 (s br, 4H), 3.87 (s,
4H), 3.83 (s, 3H), 3.46–3.24 (m, 6H), 2.77 (q, J = 5.4, 2H), 2.45–2.35
(m, 1H), 1.88–1.49 (m, 8H), 1.44 (t, J = 7.5, 3H). 13C NMR (101 MHz,
CDCl3) δ 167.55, 156.92, 153.66, 153.47, 150.74, 146.29, 142.45,
134.54, 122.87, 122.70, 119.04, 117.27, 113.24, 108.17, 67.00 (2C),
64.37, 64.29, 60.51, 54.12, 46.95, 45.91 (3C), 41.99, 33.18 (2C), 29.28,
25.36 (2C), 23.76, 12.38. ESI-MS [M + H]+ (m/z): 616.3.
4-cyclopentyl-1-((2-(2-ethyl-1H-benzo[d]imidazol-1-yl)-9-methyl-6-
morpholino-9H-purin-8-yl)methyl)piperazin-2-one (8b). White solid, yield:
1
54%. H NMR (400 MHz, CDCl3) δ 8.07–7.93 (m, 1H), 7.84–7.68 (m,
1H), 7.32–7.19 (m, 2H), 4.87 (s, 2H), 4.48–4.19 (s br, 4H), 3.92–3.79
(m, 7H), 3.49–3.24 (m, 6H), 2.75 (q, J = 5.4, 2H), 2.57 (t, J = 7.6 Hz,
1H), 1.98–1.78 (m, 2H), 1.78–1.65 (m, 2H), 1.63–1.51 (m, 2H),
1.50–1.35 (m, 5H). 13C NMR (101 MHz, CDCl3) δ167.15, 156.95,
153.67, 153.49, 150.78, 146.24, 142.47, 134.55, 122.89, 122.72,
119.08, 117.27, 113.24, 67.02 (2C), 66.60, 56.77, 48.66, 46.52 (2C),
45.89, 41.89, 30.37 (2C), 29.33, 24.05 (2C), 23.77, 12.39. ESI-MS [M +
H]+ (m/z): 544.3.
4-cyclohexyl-1-((2-(2-ethyl-1H-benzo[d]imidazol-1-yl)-9-methyl-6-
morpholino-9H-purin-8-yl)methyl)piperazin-2-one (8c). White solid, yield:
1
59%. H NMR (400 MHz, CDCl3) δ 8.04–7.96 (m, 1H), 7.80–7.72 (m,
1H), 7.32–7.22 (m, 2H), 4.87 (s, 2H), 4.30 (s br, 4H), 3.95–3.78 (m, 7H),
3.45–3.28 (m, 6H), 2.76 (q, J = 5.4, 2H), 2.32 (dd, J = 13.5, 6.1 Hz, 1H),
1.95–1.78 (m, 4H), 1.44 (t, J = 7.5 Hz, 3H), 1.35–1.05 (m, 6H). 13C NMR
(101 MHz, CDCl3) δ 167.66, 156.93, 153.58, 153.49, 150.74, 146.34,
142.39, 134.53, 122.91, 122.74, 119.05, 117.27, 113.25, 67.02 (2C),
62.36, 53.67, 46.86, 45.90 (3C), 41.98, 29.31, 28.60 (2C), 26.04, 25.43
(2C), 23.75, 12.39. ESI-MS [M+H]+ (m/z): 558.3.
4-(4,4-difluorocyclohexyl)-1-((2-(2-ethyl-1H-benzo[d]imidazol-1-yl)-
9-methyl-6-morpholino-9H-purin-8-yl)methyl)piperazin-2-one (8d). White
solid, yield: 43%. 1H NMR (400 MHz, CDCl3) δ 8.09–7.96 (m, 1H),
7.82–7.70 (m, 1H), 7.33–7.20 (m, 2H), 4.87 (s, 2H), 4.35 (s br, 4H),
3.99–3.78 (m, 7H), 3.43 (dd, J = 6.3, 4.4 Hz, 2H), 3.40–3.27 (m, 4H),
2.77 (t, J = 5.3 Hz, 2H), 2.42 (d, J = 9.7 Hz, 1H), 2.25–2.04 (m, 2H),
1.91–1.58 (m, 6H), 1.44 (t, J = 7.4 Hz, 3H). 13C NMR (101 MHz, CDCl3)
δ 167.12, 156.92, 153.67, 153.45, 150.79, 146.16, 142.48, 134.54,
122.87, 122.70, 122.68 (t, J = 239 Hz), 119.05, 117.26, 113.24, 66.99
(2C), 59.42, 54.17, 46.83, 45.96 (3C), 41.97, 31.86 (t, J = 24.5 Hz, 2C),
29.27, 24.35 (2C), 23.78, 12.38. ESI-MS [M+H]+ (m/z): 594.3.
1-((2-(2-ethyl-1H-benzo[d]imidazol-1-yl)-9-methyl-6-morpholino-9H-
purin-8-yl)methyl)-4-(1-methylpiperidin-4-yl)piperazin-2-one (8e). White
solid, yield: 39%. 1H NMR (400 MHz, CDCl3) δ 8.01 (d, J = 4.7 Hz, 1H),
3.1.9. Synthesis of 1-((2-(2-ethyl-1H-benzo[d]imidazol-1-yl)-9-methyl-6-
morpholino-9H-purin-8-yl)methyl)-4-(4-oxocyclohexyl)piperazin-2-one
(8j)
THF(10 mL) and concentrated hydrochloric acid (2 mL) was added to
a solution of 8i (0.092 g, 0.15 mmol) in dichloromethane (10 mL) under
0 ℃. The mixture was stirred under refluxing for 4 h. The solvent was
removed under reduced pressure and the residue was dissolved in
dichloromethane and purified by preparative TLC with dichloro-
methane/methanol (V/V = 10/1) as mobile phase to afford 8 l (0.069 g,
80%) as a pale solid. 1H NMR (400 MHz, CDCl3) δ 8.07–7.95 (m, 1H),
7.82–7.69 (m, 1H), 7.27 (q, J = 2.2 Hz, 2H), 4.88 (s, 2H), 4.35 (s br, 4H),
3.94–3.78 (m, 7H), 3.47 (t, J = 6.3 Hz, 2H), 3.43–3.27 (m, 4H), 2.82 (t, J
= 6.3 Hz, 2H), 2.77–2.67 (m, 1H), 2.55–2.45 (m, 2H), 2.38–2.27 (m,
10

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Bioorganic Chemistry 105 (2020) 104344
2H), 2.14–1.98 (m, 2H), 1.94–1.82 (m, 2H), 1.44 (t, J = 7.5 Hz, 3H). 13
C
purin-8-yl)methyl)-4-isobutyrylpiperazin-2-one (8n). White solid, yield:
1
NMR (101 MHz, CDCl3) δ 209.89, 166.97, 156.91, 153.68, 153.48,
150.84, 146.10, 142.52, 134.55, 122.87, 122.72, 119.11, 117.27,
113.23, 67.00 (2C), 59.03, 54.37, 46.76, 46.20 (2C), 45.80, 41.95, 38.43
(2C), 29.29, 27.74 (2C), 23.80, 12.38. ESI-MS [M + H]+ (m/z): 572.3.
54%. H NMR (400 MHz, CDCl3) δ 8.05–7.95 (m, 1H), 7.82–7.73 (m,
1H), 7.27 (q, J = 4.7 Hz, 2H), 4.88 (s, 2H), 4.30 (s br, 6H), 3.92–3.72 (m,
9H), 3.56 (s br, 2H), 3.35 (q, J = 7.5 Hz, 2H), 2.82–2.69 (m, 1H), 1.44 (t,
J = 7.5 Hz, 3H), 1.15 (d, J = 6.8 Hz, 6H). 13C NMR (101 MHz, CDCl3) δ
170.08, 165.96, 156.89, 153.68, 153.40, 150.82, 145.09, 142.20,
134.44, 122.98, 122.83, 119.01, 117.31, 113.23, 66.97 (2C), 49.11,
46.45, 46.13 (2C), 42.43, 38.86, 30.55, 29.24, 23.70, 19.17 (2C), 12.38.
ESI-MS [M+H]+ (m/z): 546.3.
3.1.10. Synthesis of 1-((2-(2-ethyl-1H-benzo[d]imidazol-1-yl)-9-methyl-
6-morpholino-9H-purin-8-yl)methyl)-4-(4-hydroxycyclohexyl)piperazin-2-
one (8k)
Sodium borohydride (0.2 mmol) was slowly added to the suspension
of 8 l (0.052 g, 0.09 mmol) in MeOH (5 mL) at 0 ℃. The resulting
mixture was stirred under room temperature for 2 h until the starting
materials completely transformed. Then the solvent was removed under
reduced pressure and the residue was dissolved in dichloromethane and
purified by preparative TLC with dichloromethane/methanol (V/V =
10/1) as mobile phase to afford 8k (0.039 g, 76%) as a pale solid.. 1H
NMR (400 MHz, CDCl3) δ 8.09–7.94 (m, 1H), 7.84–7.69 (m, 1H),
7.41–7.18 (m, 2H), 4.87 (d, J = 3.3 Hz, 2H), 4.35 (s br, 4H), 3.92–3.73
(m, 7H), 3.66–3.52 (m, 1H), 3.52–3.23 (m, 6H), 2.77 (t, J = 5.5 Hz, 2H),
2.42–2.26 (m, 1H), 2.16–1.96 (m, 2H), 1.96–1.50 (m, 4H), 1.44 (t, J =
7.4 Hz, 3H), 1.37–1.21 (m, 2H). 13C NMR (101 MHz, CDCl3) δ 167.42,
156.92, 153.66, 153.48, 150.79, 146.25, 142.52, 134.56, 122.86,
122.70, 119.08, 117.26, 113.22, 70.09, 67.01 (2C), 61.35, 53.96, 46.87,
46.25 (2C), 45.77, 41.97, 34.08 (2C), 29.29, 26.25 (2C), 23.78, 12.38.
ESI-MS [M+H]+ (m/z): 574.3.
4-(cyclopropanecarbonyl)-1-((2-(2-ethyl-1H-benzo[d]imidazol-1-yl)-9-
methyl-6-morpholino-9H-purin-8-yl)methyl)piperazin-2-one (8o). White
solid, yield: 53%. 1H NMR (400 MHz, CDCl3) δ 8.05–7.95 (m, 1H),
7.81–7.69 (m, 1H), 7.31–7.21 (m, 2H), 4.89 (s, 2H), 4.35 (s br, 6H),
3.95–3.82 (m, 9H), 3.57 (s, 2H), 3.35 (q, J = 7.5 Hz, 2H), 1.74–1.58 (m,
1H), 1.44 (t, J = 7.5 Hz, 3H), 1.04–1.00 (m, 2H), 0.91–0.77 (m, 2H). 13
C
NMR (101 MHz, CDCl3) δ172.0, 165.1, 156.93, 153.68, 153.41, 150.84,
145.15, 142.38, 134.49, 122.94, 122.77, 119.02, 117.30, 113.24, 66.98
(2C), 48.97, 46.63, 46.02 (2C), 42.48, 39.04, 29.27, 23.75, 12.38, 11.12,
8.23. ESI-MS [M+H]+ (m/z): 544.3.
4-(cyclobutanecarbonyl)-1-((2-(2-ethyl-1H-benzo[d]imidazol-1-yl)-9-
methyl-6-morpholino-9H-purin-8-yl)methyl)piperazin-2-one (8p). White
solid, yield: 65%. 1H NMR (400 MHz, CDCl3) δ 8.04–7.96 (m, 1H),
7.80–7.72 (m, 1H), 7.32–7.22 (m, 2H), 4.87 (s, 2H), 4.35 (s br, 4H), 4.11
(s, 1H), 3.95–3.78 (m, 9H), 3.61 (s, 1H), 3.54 (t, J = 6.5 Hz, 2H), 3.35 (q,
J = 7.5 Hz, 2H), 3.29–3.19 (m, 1H), 2.41–1.95 (m, 6H), 1.44 (t, J = 7.5
Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 173.24, 164.90, 156.90, 153.67,
153.40, 150.84, 145.58, 142.36, 134.48, 122.94, 122.78, 119.03,
117.28, 113.22, 66.97 (2C), 48.55, 46.64, 46.06 (2C), 42.47, 38.80,
37.11, 29.22, 24.89 (2C), 23.75, 17.91, 12.37. ESI-MS [M+H]+ (m/z):
558.3. HRMS m/z calculated for C29H36N9O+3 [M + H]+: 574.2936,
found 558.2948.
3.1.11. Synthesis of (S)-1-((2-(2-ethyl-1H-benzo[d]imidazol-1-yl)-9-
methyl-6-morpholino-9H-purin-8-yl)methyl)-4-(2-hydroxypropanoyl)
piperazin-2-one (8l)
Triethylamine (0.3 mmol), HOBT (0.25 mmol) and EDCI (0.3 mmol)
was added to the dichloromethane (5 mL) solution of 6 (0.089 g, 0.15
mmol) and L-lactic acid (0.3 mmol). The mixture was stirred under room
temperature for 12 h. Then the mixture was extracted with dichloro-
methane/water. The organic phase was washed with brine, dried over
MgSO4 and concentrated under reduced pressure. The crude product
was further purified by preparative TLC with dichloromethane/meth-
anol (V/V = 10/1) to afford 8l (0.053 g, 65%) as a white solid. 1H NMR
(400 MHz, CDCl3) δ 8.06–7.95 (m, 1H), 7.80–7.71 (m, 1H), 7.27 (q, J =
3.9 Hz, 2H), 4.87 (s, 2H), 4.51–4.09 (m, 8H), 3.92–3.72 (m, 7H),
3.79–3.55 (m, 4H), 3.34 (q, J = 7.5 Hz, 2H), 1.44 (t, J = 7.4 Hz, 3H),
1.36 (d, J = 6.7 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 169.49, 165.01,
156.91, 153.70, 153.40, 150.93, 145.41, 142.47, 134.50, 122.92,
122.77, 119.08, 117.28, 113.22, 66.96 (2C), 64.51, 48.39, 46.03, 45.84
(2C), 42.49, 39.72, 29.26, 23.79, 21.06, 12.37. ESI-MS [M+H]+ (m/z):
548.3.
4-(cyclopentanecarbonyl)-1-((2-(2-ethyl-1H-benzo[d]imidazol-1-yl)-9-
methyl-6-morpholino-9H-purin-8-yl)methyl)piperazin-2-one (8q). White
solid, yield: 49%. 1H NMR (400 MHz, CDCl3) δ 7.99 (dd, J = 5.7, 3.2 Hz,
1H), 7.81–7.71 (m, 1H), 7.35–7.20 (m, 2H), 4.88 (s, 2H), 4.47–4.16 (m,
6H), 3.90–3.83 (m, 9H), 3.63–3.49 (m, 2H), 3.35 (q, J = 7.5 Hz, 2H),
2.96–2.70 (m, 1H), 1.89–1.69 (m, 6H), 1.65–1.53 (m, 2H), 1.43 (t, J =
7.5 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ174.70, 165.0, 156.96,
153.69, 153.41, 150.83, 145.60, 142.29, 134.46, 122.96, 122.80,
119.03, 117.30, 113.22, 66.98 (2C), 49.13, 46.52, 46.10 (2C), 42.48,
41.28, 38.94, 30.00 (2C), 29.23, 26.04 (2C), 23.69, 12.40. ESI-MS [M +
H]+ (m/z): 572.3.
1-((2-(2-ethyl-1H-benzo[d]imidazol-1-yl)-9-methyl-6-morpholino-9H-
purin-8-yl)methyl)-4-(methylsulfonyl)piperazin-2-one (8r). White solid,
yield: 57%. 1H NMR (400 MHz, CDCl3) δ 8.08–7.95 (m, 1H), 7.84–7.69
(m, 1H), 7.28 (q, J = 5.8 Hz, 2H), 4.88 (s, 2H), 4.35 (s br, 4H), 4.00 (s,
2H), 3.92–3.72 (m, 7H), 3.66 (d, J = 5.4 Hz, 2H), 3.55 (t, J = 5.4 Hz,
3.1.12. General procedure for the synthesis of 8m-8t
Different acyl chloride or sulfonyl chloride (0.3 mmol) was slowly
added to the dichloromethane (5 mL) solution of 6 (0.089 g, 0.15 mmol)
and triethylamine (0.3 mmol) at 0 ℃. The mixture was stirred under
room temperature for 3–12 h until the starting materials completely
transformed. Then the mixture was extracted with dichloromethane/
water. The organic phase was washed with brine, dried over MgSO4 and
concentrated under reduced pressure. The crude product was further
purified by preparative TLC with dichloromethane/methanol (V/V =
10/1) to afford 8 m-8 t with yields of 40%-70%.
2H), 3.35 (q, J = 7.5 Hz, 2H), 2.89 (s, 3H), 1.44 (t, J = 7.5 Hz, 3H). 13
C
NMR (101 MHz, CDCl3) δ 163.99, 156.92, 153.71, 153.41, 150.93,
145.45, 142.54, 134.55, 122.90, 122.73, 119.10, 117.27, 113.23, 66.98
(2C), 48.63, 46.78 (2C), 45.83, 42.66, 42.40, 36.41, 29.25, 23.81, 12.37.
ESI-MS [M+H]+ (m/z): 554.2.
1-((2-(2-ethyl-1H-benzo[d]imidazol-1-yl)-9-methyl-6-morpholino-9H-
purin-8-yl)methyl)-4-(propylsulfonyl)piperazin-2-one (8 s). White solid,
yield: 51%. 1H NMR (400 MHz, CDCl3) δ 8.00 (dd, J = 7.5, 2.1 Hz, 1H),
7.82–7.70 (m, 1H), 7.32–7.21 (m, 2H), 4.88 (s, 2H), 4.34 (s br, 4H), 4.04
(s, 2H), 3.86 (d, J = 8.1 Hz, 7H), 3.70–3.52 (m, 4H), 3.35 (q, J = 7.4 Hz,
2H), 3.04–2.91 (m, 2H), 1.92–1.76 (m, 2H), 1.44 (t, J = 7.4 Hz, 3H),
1.07 (t, J = 7.4 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 164.26, 156.91,
153.70, 153.43, 150.89, 145.50, 142.46, 134.52, 122.91, 122.75,
119.08, 117.28, 113.23, 66.99 (2C), 52.68, 48.59, 47.09 (2C), 45.89,
42.73, 42.47, 29.24, 23.78, 16.98, 13.00, 12.37. ESI-MS [M + H]+ (m/
z): 582.3.
4-acetyl-1-((2-(2-ethyl-1H-benzo[d]imidazol-1-yl)-9-methyl-6-morpho-
lino-9H-purin-8-yl)methyl)piperazin-2-one (8m). White solid, yield: 62%.
1H NMR (400 MHz, CDCl3) δ 8.06–7.95 (m, 1H), 7.81–7.70 (m, 1H),
7.33–7.22 (m, 2H), 4.88 (d, J = 4.6 Hz, 2H), 4.33 (s br, 6H), 3.92–3.72
(m, 9H), 3.63–3.53 (m, 2H), 3.34 (q, J = 7.5 Hz, 2H), 2.13 (s, 3H), 1.44
(t, J = 7.5 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 168.91, 165.64,
164.68, 156.91, 153.70, 150.91, 145.53, 142.52, 134.53, 122.90,
122.74, 119.10, 117.29, 113.22, 66.97 (2C), 49.68, 46.39, 46.05 (2C),
42.46, 38.54, 29.23, 23.80, 21.34, 12.37. ESI-MS [M+H]+ (m/z): 518.3.
1-((2-(2-ethyl-1H-benzo[d]imidazol-1-yl)-9-methyl-6-morpholino-9H-
4-(cyclopropylsulfonyl)-1-((2-(2-ethyl-1H-benzo[d]imidazol-1-yl)-9-
methyl-6-morpholino-9H-purin-8-yl)methyl)piperazin-2-one (8 t). White
solid, yield: 62%. 1H NMR (400 MHz, CDCl3) δ 8.08–7.96 (m, 1H),
11

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Bioorganic Chemistry 105 (2020) 104344
7.81–7.71 (m, 1H), 7.35–7.22 (m, 2H), 4.88 (s, 2H), 4.35 (s, 4H), 4.06 (s,
2H), 3.94–3.78 (m, 7H), 3.72–3.55 (m, 4H), 3.35 (q, J = 7.5 Hz, 2H),
2.37–2.25 (m, 1H), 1.44 (t, J = 7.5 Hz, 3H), 1.25–1.19 (m, 2H),
1.10–0.99 (m, 2H). 13C NMR (101 MHz, CDCl3) δ 164.25, 156.91,
153.71, 153.42, 150.91, 145.52, 142.46, 134.52, 122.91, 122.75,
119.09, 117.27, 113.24, 66.98 (2C), 49.08, 46.83 (2C), 45.88, 43.02,
42.36, 29.27, 26.58, 23.79, 12.38, 4.79 (2C). ESI-MS [M+H]+ (m/z):
580.3.
non-radioactive cell proliferation MTT (Promega, Madison, WI, USA)
assay [29] for the SAR study in Section 2.2 or the CellTiter 96®
AQueous MTS (Promega, Madison, WI, USA) assay [30] for the anti-
proliferative activity study in Section 2.5.
3.7. Cell apoptosis analysis
The cell apoptosis assay was conducted on flow cytometry (FCM)
[31], and before analysis, cells were treated with Idelalisib or WNY1613
for 72 h. Next, the cells were harvested and stained by an Annexin V-
fluorescein isothiocyanate (FITC)/PI apoptosis detection kit (Roche,
Indianapolis, IN, USA) according to the manufacturer’s protocol. A
minimum of 1 × 104 cells was analyzed using BD FACSCanto™ II (BD
Biosciences, San Jose, CA USA), the data was analyzed by FlowJo soft-
ware (V10.4, Ashland, OR, USA).
3.2. Kinase assay
The inhibitory activity of WNY1613 against 300 diverse human
protein kinases including PI3K family was assessed by Reaction Biology
Corp. Activity of individual kinases was evaluated by the Hot Spot assay
platform, which contains specific kinase/substrate pairs and required
cofactors [27]. The reaction mixture, which containing the compound
and 33P-ATP, was incubated at 25℃ for 2 h and spotted onto P81 ion
exchange paper and the kinase activity was measured as 33P intensity of
the spots. The extent of the kinase activity was indicated as a percentage
of the kinase activity obtained without the compound. The inhibition
rates were defined as the percentage of the kinase activity decreasing in
the presence of the compound. The inhibition rate of WNY1613 on all
the tested kinases were listed in the Supplementary material.
3.8. Immunoblotting analysis
Cells were treated by Idelalisib or WNY1613 for indicated time and
then were harvested and washed with PBS. The harvested cells were
lysed in RIPA buffer (Beyotime, Beijing, China) contained phosphatase
inhibitors (Roche, Basel, CH) and cocktail (1:1000) for 30 min and
equalized before loading. The samples were separated by SDS-PAGE gel
and transferred onto nitrocellulose (NC) filter membranes (Merck Mil-
lipore, MS, USA). Then the membranes were incubated with appropriate
primary antibody overnight at 4℃, and then corresponding secondary
antibody [32]. Chemiluminescence detection were used to detect spe-
cific protein bands. The antibodies used in this article are listed in the
Supplementary material.
3.3. Thermal shift assay
Thermal shift assay was concluded to evaluate whether WNY1613
binds with PI3Kδ protein [20]. SU-DHL-6 and JEKO-1 cells were har-
vested, washed with PBS, and lysed by RIPA lysis buffer containing
cocktail (1:1000). Next, centrifugation at 13000 rpm at 4 ℃ for 20 min
and separated the soluble fraction (lysate) from the cell debris, which
were then divided into two aliquots, with one aliquot being treated with
3.9. Xenograft mouse model
WNY1613 (10
μM) and the other aliquot with control. After 30 min of
All animal experiments have been approved by the Institutional
Animal Care and Treatment Committee of Sichuan University in China
(Permit Number: 20170135) and were carried out in accordance with
the approved guidelines. NOD-SCID (6- to 9-week-old) mice [33] used in
this study were purchased from Beijing HFK bioscience Co. LTD, Beijing,
China and were performed in a specific-pathogen-free (SPF) condition
facility. Mice injected subcutaneously with ~1 × 107 SU-DHL-6 or
JEKO-1 cells were randomly divided into four groups: (i) Vehicle
(Vehicle, n = 4); (ii) Idelalisib (25 mg/kg, n = 4); (iii) WNY1613 (25
mg/kg, n = 4); (iiii) WNY1613 (50 mg/kg, n = 4). Treatment group or
the Vehicle consisting of 10% castor oilin normal saline (NS) solution,
2.5% ethyl alcohol, 2.5% DMSO was administered at the indicated doses
once a day for 18 or 21 days by oral gavage. Two diameters of tumors
measured by electronic slide caliper every three days and tumor volumes
were calculated using the following formula: tumor volume (mm3) =
0.52 × length × width2. The TGI values were calculated with the
following formula: TGI = [1ꢀ (Tn ꢀ T0)/(Cn ꢀ C0)] × 100%, T0 and Tn
represent average tumor volume before treatment and that of n days
after treatment. C0 and Cn represent average tumor volume before
treatment and that of n days after treatment in vehicle group.
incubation at 25℃, the respective lysates were divided into individual
50 μL aliquots and heated at different temperatures (35, 40, 45, 50, 55,
60, 70, 80, 90, 100 ℃) for 6 min and then cooled at 25℃ for 3 min. Then
the heated lysates were centrifuged at 20000 rpm at 4 ℃ for 20 min and
removed the denatured proteins, transfering 20 μL of supernatants into
new microtubes and analyzed by immunoblotting analysis to determine
the degradation temperature of PI3Kδ protein under WNY1613 or
control treatment.
3.4. Molecular modelling
The 3D structures of the PI3Kδ and PI3Kγ were downloaded from the
PDB (http://www.rcsb.org/, PDB ID: 2WXP, 3DBS). The protein was
prepared with Discovery Studio 3.1 and the ligand WNY1613 was
prepared with ChemBio3D and optimized at molecular mechanical
level. Then WNY1613 was docked to the hinge and affinity pocket of
PI3Kδ and PI3Kγ using Autodock 4.2,[28] and the results were visual-
ized by Discovery Studio 3.1.
3.5. Cell culture and reagents
3.10. Immunohistochemistry (IHC) analysis
All cell lines were purchased from the Cell Bank of the Chinese
Academy of Sciences (Shanghai, China) and were cultured in Rosewell
Park Memorial Institute (RPMI) 1640 media supplemented with 20%
FBS and Penicillin-Streptomycin under humidified conditions with 5%
CO2 at 37 ℃.
Tumor tissues were fixed in 4% paraformaldehyde overnight and
embedded in paraffin. Sections were subjected to immunohistochemical
staining with antibody after cut into 5 μm sections [32]. The antibodies
used in this article are listed in the Supplementary material.
3.6. Cell proliferation assay
3.11. Statistical analysis
Cells were seeded in 96-well plates at 6 ~ 8 × 103 cells per well with
a total volume of 100
μ
L media and incubated for 24 h. Then different
The Student’s t-test was used to examine statistically significant
differences and all quantitative results are expressed as mean values ±
SD. P < 0.05 indicates statistically significant.
doses of compounds in 100
μL media were added to the cells. After
indicated times, cell viability was determined using the CellTiter 96®
12

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Bioorganic Chemistry 105 (2020) 104344
inhibitors exhibiting synergistic effect with other direct-acting antiviral agents,
J. Med. Chem. 58 (6) (2015) 2764–2778.
Declaration of Competing Interest
[18] F.I. Raynaud, S.A. Eccles, S. Patel, S. Alix, G. Box, I. Chuckowree, A. Folkes,
S. Gowan, A.D.H. Brandon, F.D. Stefano, A. Hayes, A.T. Henley, L. Lensun,
G. Pergl-Wilson, A. Robson, N. Saghir, A. Zhyvoloup, E. McDonald, P. Sheldrake,
S. Shuttleworth, M. Valenti, N.C. Wan, P.A. Clarke, P. Workman, Biological
properties of potent inhibitors of class I phosphatidylinositide 3-kinases: from PI-
103 through PI-540, PI-620 to the oral agent GDC-0941, Mol. Cancer Ther. 8 (7)
(2009) 1725–1738.
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgements
[19] B.J. Lannutti, S.A. Meadows, S.E.M. Herman, A. Kashishian, B. Steiner, A.
J. Johnson, J.C. Byrd, J.W. Tyner, M.M. Loriaux, M. Deininger, B.J. Druker, K.
D. Puri, R.G. Ulrich, N.A. Giese, CAL-101, a p110δ selective phosphatidylinositol-3-
kinase inhibitor for the treatment of B-cell malignancies, inhibits PI3K signaling
and cellular viability, Blood 117 (2) (2011) 591–594.
This work was supported by the Fundamental Research Funds for the
Central Universities (No. 2682018CX37).
[20] D.M. Molina, R. Jafari, M. Ignatushchenko, T. Seki, E.A. Larsson, C. Dan,
L. Sreekumar, Y. Cao, P. Nordlund, Monitoring drug target engagement in cells and
tissues using the cellular thermal shift assay, Science 341 (6141) (2013) 84–87.
[21] A.J. Folkes, K. Ahmadi, W.K. Alderton, S. Alix, S.J. Baker, G. Box, I.S. Chuckowree,
P.A. Clarke, P. Depledge, S.A. Eccles, L.S. Friedman, A. Hayes, T.C. Hancox,
A. Kugendradas, L. Lensun, P. Moore, A.G. Olivero, J. Pang, S. Patel, G.H. Pergl-
Wilson, F.I. Raynaud, A. Robson, N. Saghir, L. Salphati, S. Sohal, M.H. Ultsch,
M. Valenti, H.J.A. Wallweber, N.C. Wan, C. Wiesmann, P. Workman, A. Zhyvoloup,
M.J. Zvelebil, S.J. Shuttleworth, The identification of 2-(1H-indazol-4-yl)-6-(4-
methanesulfonyl-piperazin-1-ylmethyl)-4-morpholin-4-yl-thieno[3,2-d]pyrimidine
(GDC-0941) as a potent, selective, orally bioavailable inhibitor of class I PI3 kinase
for the treatment of cancer, J. Med. Chem. 51 (18) (2008) 5522–5532.
[22] A. Berndt, S. Miller, O. Williams, D.D. Le, B.T. Housema, J.I. Pacold, F. Gorrec, W.-
C. Hon, P. Ren, Y. Liu, C. Rommel, P. Gaillard, T. Rückle, M.K. Schwarz, K.
M. Shokat, J.P. Shaw, R.L. Williams, The p110δ structure: mechanisms for
selectivity and potency of new PI(3)K inhibitors, Nat. Chem. Biol. 6 (2) (2010)
117–124.
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bioorg.2020.104344.
References:
[1] L.C. Cantley, The phosphoinositide 3-kinase pathway, Science 296 (5573) (2002)
1655–1657.
[2] R. Fritsch, J. Downward, SnapShot: class I PI3K isoform signaling, Cell 154 (4)
(2013) 940.
[3] D. Chantry, A. Vojtek, A. Kashishian, D.A. Holtzman, C. Wood, P.W. Gray, J.
A. Cooper, M.F. Hoekstra, p110δ, a novel phosphatidylinositol 3-kinase catalytic
subunit that associates with p85 and is expressed predominantly in leukocytes,
J. Biol. Chem. 272 (31) (1997) 19236–19241.
[23] S. Fulda, Modulation of mitochondrial apoptosis by PI3K inhibitors, Mitochondrion
13 (3) (2013) 195–198.
[4] B. Vanhaesebroeck, M.J. Welham, K. Kotani, R. Stein, P.H. Warne, M.J. Zvelebil,
K. Higashi, S. Volinia, J. Downward, M.D. Waterfield, p110δ, a novel
phosphoinositide 3-kinase in leukocytes, Proc. Natl. Acad. Sci. USA 94 (9) (1997)
4330–4335.
[24] M. Will, A.C.R. Qin, W. Toy, Z. Yao, V. Rodrik-Outmezguine, C. Schneider,
X. Huang, P. Monian, X. Jiang, E.de Stanchina, J. Baselga, N. Liu, S. Chandarlapaty,
N. Rosen, Rapid induction of apoptosis by PI3K inhibitors is dependent upon their
transient inhibition of RAS-ERK signaling, Cancer Discov. 4 (3) (2014) 334–347.
[25] H. Ebi, C. Costa, A.C. Faber, M. Nishtala, H. Kotani, D. Juric, P.D. Pelle, Y. Song,
S. Yano, M. Mino-Kenudson, C.H. Benes, J.A. Engelman, PI3K regulates MEK/ERK
signaling in breast cancer via the Rac-GEF, P-Rex1, Proc. Natl. Acad. Sci. USA 110
(52) (2013) 21124–21129.
[5] S.T. Jou, N. Carpino, Y. Takahashi, R. Piekorz, J.R. Chao, N. Carpino, D. Wang, J.
N. Ihle, Essential, nonredundant role for the phosphoinositide 3-kinase p110δ in
signaling by the B-cell receptor complex, Mol. Cell Biol. 22 (24) (2002) 8580–8591.
[6] K. Blajecka, A. Borgstrom, A. Arcaro, Phosphatidylinositol 3-kinase isoforms as
novel drug targets, Curr. Drug Targets 12 (7) (2011) 1056–1081.
[7] D.A. Fruman, C. Rommel, PI3Kδ inhibitors in cancer: rationale and serendipity
merge in the clinic, Can. Discov. 1 (7) (2011) 562–572.
[26] G. Castanedo, B. Chan, D. M. Goldstein, R. K. Kondru, M. C. Lucas, W. S. Palmer, S.
Price, B. Safina, P. P. A. Savy, E. M. Seward, D. P. Sutherlin, Z. K. Sweeney,
Preparation of bicyclic pyrimidines as selective inhibitors of the p110 delta isoform
of PI3K for treating inflammation, immune diseases and cancers, WO 2010138589,
May 26 (2010).
[8] D.L. Kienle, S. Stilgenbauer, Approved and emerging PI3K inhibitors for the
treatment of chronic lymphocytic leukemia and non-Hodgkin lymphoma, Expert
Opin. Pharmaco. 21 (8) (2020) 1–13.
[9] Y. Feng, X. Cu, M. Xin, PI3Kδ inhibitors for the treatment of cancer: a patent review
(2015-present), Expert Opin. Ther. Pat. 29 (12) (2019) 925–941.
[10] C.Y. Cheah, N.H. Fowler, Idelalisib in the management of lymphoma, Blood 128 (3)
(2016) 331–336.
[27] T. Anastassiadis, S.W. Deacon, K. Devarajan, H. Ma, J.R. Peterson, Comprehensive
assay of kinase catalytic activity reveals features of kinase inhibitor selectivity, Nat.
Biotechnol. 29 (11) (2011) 1039–1045.
[28] G.M. Morris, R. Huey, W. Lindstrom, M.F. Sanner, R.K. Belew, D.S. Goodsell, A.
J. Olson, AutoDock4 and AutoDockTools4: automated docking with selective
receptor flexibility, J. Comput. Chem. 30 (16) (2009) 2785–2791.
[11] I.W. Flinn, S. O’Brien, B. Kahl, M. Patel, Y. Oki, F.F. Foss, P. Porcu, J. Jones, J.
A. Burger, N. Jain, Duvelisib, a novel oral dual inhibitor of PI3K-δ, γ, is clinically
active in advanced hematologic malignancies, Blood 131 (8) (2018) 877–887.
[29] T. Hideshima, D. Chauhan, T. Hayashi, K. Podar, M. Akiyama, C. Mitsiades,
N. MItsiades, B. Gong, L. Bonham, P. de Vries, N. Munshi, P.G. Richardson, J. W.
Singer,K. C. Anderson,, Antitumor activity of lysophosphatidic acid
acyltransferase-beta inhibitors, a novel class of agents, in multiple myeloma, Can.
Res. 63 (23) (2003) 8428–8436.
¨
[12] M. Dreyling, A. Santoro, L. Mollica, S. Leppa, G.A. Follows, G. Lenz, W.S. Kim,
A. Nagler, P. Panayiotidis, J. Demeter, Phosphatidylinositol 3-Kinase Inhibition by
Copanlisib in Relapsed or Refractory Indolent Lymphoma, J. Clin. Oncol. 35 (35)
(2017) 3898–3905.
[13] C.L. Lucas, A. Chandra, S. Nejentsev, A.M. Condliffe, K. Okkenhaug, PI3Kδ and
primary immunodeficiencies, Nat. Rev. Immunol. 16 (11) (2016) 702–714.
[14] N.Y. Wang, W.Q. Zuo, R. Hu, W.L. Wang, Y.X. Zhu, Y. Xu, L.T. Yu, Z.H. Liu, Design,
synthesis and structure-activity relationship study of piperazinone-containing
thieno[3,2-d]pyrimidine derivatives as new PI3Kδ inhibitors, Bioorg. Med. Chem.
Lett. 30 (20) (2020), 127479.
[30] L.A.M. Grinera, R. Guhaa, P. Shinna, R.M. Young, J.M. Keller, D. Liu, I.S. Goldlust,
A. Yasgar, C. McKnight, M.B. Boxer, D.Y. Duveau, J.-K. Jiang, S. Michael,
T. Mierzwa, W. Huang, M.J. Walsh, B.T. Mott, P. Patel, W. Leister, D.J. Maloney, C.
A. Leclair, G. Rai, A. Jadhav, B.D. Peyser, C.P. Austin, S.E. Martin, A. Simeonov,
M. Ferrer, L.M. Staudt, C.J. Thomas, High-throughput combinatorial screening
identifies drugs that cooperate with ibrutinib to kill activated B-cell–like diffuse
large B-cell lymphoma cells, Proc. Natl. Acad. Sci. USA 111 (6) (2014) 2349–2354.
[31] L.S. Chen, S. Redkar, D. Bearss, W.G. Wierda, V. Gandhi, Pim kinase inhibitor, SGI-
1776, induces apoptosis in chronic lymphocytic leukemia cells, Blood 114 (19)
(2009) 4150–4157.
[15] J.M. Murray, Z.K. Sweeney, B.K. Chan, M. Balazs, E. Bradley, G. Castanedo,
C. Chabot, D. Chantry, M. Flagella, D.M. Goldstein, R. Kondru, J. Lesnick, J. Li, M.
C. Lucas, J. Nonomiya, J. Pang, S. Price, L. Salphati, B. Safina, P.P.A. Savy, E.
M. Seward, M. Ultsch, D.P. Sutherlin, Potent and highly selective benzimidazole
inhibitors of PI3-kinase delta, J. Med. Chem. 55 (17) (2012) 7686–7695.
[16] B.S. Safina, Z.K. Sweeney, J. Li, Br.K. Chan, A. Bisconte, D. Carrera, G. Castanedo,
M. Flagella, R. Heald, C. Lewis, J.M. Murray, J. Nonomiya, J. Pang, S. Price,
K. Reif, L. Salphati, E.M. Seward, B. Wei, D.P. Sutherlin, Identification of GNE-293,
a potent and selective PI3Kδ inhibitor: navigating in vitro genotoxicity while
improving potency and selectivity, Bioorg. Med. Chem. Lett. 23 (17) (2013)
4953–4959.
[32] Y. Xu, Q. Wang, K. Xiao, Z. Liu, L. Zhao, X. Song, X. Hu, Z. Feng, T. Gao, W. Zuo,
J. Zeng, N. Wang, L, Yu, novel dual BET and PLK1 inhibitor WNY0824 exerts
potent antitumor effects in CRPC by inhibiting transcription factor function and
inducing mitotic abnormality, Mol. Cancer. Ther. 19 (6) (2020) 1221–1231.
[33] L.D. Shultz, P.A. Schweitzer, S.W. Christianson, B. Gott, I.B. Schweitzer,
B. Tennent, S. McKenna, L. Mobraaten, T.V. Rajan, D.L. Greiner, Multiple defects in
innate and adaptive immunologic function in NOD/LtSz-scid mice, J. Immunol.
154 (1) (1995) 180–191.
[17] N.Y. Wang, Y. Xu, W.Q. Zuo, K.J. Xiao, L. Liu, X.X. Zeng, X.Y. You, L.D. Zhang,
C. Gao, Z.H. Liu, T.H. Ye, Y. Xia, Y. Xiong, X.J. Song, Q. Lei, C.T. Peng, H. Tang, S.
Y. Yang, Y.Q. Wei, L.T. Yu, Discovery of imidazo[2,1-b]thiazole HCV NS4B
13