Design and optimization of orally spleen tyrosine kinase (SYK) inhibitors for treatment of solid tumor

Article abstract of DOI:10.1016/j.bioorg.2019.103547

As the aim to discover orally SYK inhibitors for solid tumor treatment, a series of novel derivatives based on imidazo[1,2-a]pyrazine scaffold were designed, synthesized and evaluated. Structure-activity relationship study of both enzymatic and cellular assays led to the identification of compound 12f. The novel SYK inhibitor 12f showed potent antitumor activity against solid tumors with favorable drug-like properties of lipophilicity and solubility. 12f could induce cell apoptosis of ovarian and lung cancer cell lines. In SKOV3 xenograft mouse model, oral administration of 12f led to significant tumour regression without obvious toxicity. 12f improved the limited response of traditional SYK inhibitors in solid tumors in vitro and in vivo. Taken together, this compound may act as a promising lead compound for further development of new SYK inhibitors for solid tumor therapy.

Full text of DOI:10.1016/j.bioorg.2019.103547

BioorganicChemistry95(2020)103547
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Design and optimization of orally spleen tyrosine kinase (SYK) inhibitors for
treatment of solid tumor
Cheng Wanga,b,1, Xin Wanga,b,1, Yao Lia, Tianqi Wanga, Zhi Huanga, Zhongxiang Qina,
⁎
⁎
Shengyong Yangd, Rong Xianga,b,c, , Yan Fana,b,
a Department of Medicinal Chemistry, School of Medicine, Nankai University, 94 Weijin Road, Tianjin 300071, China
b 2011 Project Collaborative Innovation Center for Biotherapy of Ministry of Education, 94 Weijin Road, Tianjin 300071, China
c State Key Laboratory of Medicinal Chemical Biology, 94 Weijin Road, Tianjin 300071, China
d Medical Oncology, Cancer Center, State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
SYK
As the aim to discover orally SYK inhibitors for solid tumor treatment, a series of novel derivatives based on
imidazo[1,2-a]pyrazine scaffold were designed, synthesized and evaluated. Structure-activity relationship study
of both enzymatic and cellular assays led to the identification of compound 12f. The novel SYK inhibitor 12f
showed potent antitumor activity against solid tumors with favorable drug-like properties of lipophilicity and
solubility. 12f could induce cell apoptosis of ovarian and lung cancer cell lines. In SKOV3 xenograft mouse
model, oral administration of 12f led to significant tumour regression without obvious toxicity. 12f improved the
limited response of traditional SYK inhibitors in solid tumors in vitro and in vivo. Taken together, this compound
may act as a promising lead compound for further development of new SYK inhibitors for solid tumor therapy.
Inhibitor
Cancer
1. Introduction
malignancies [16,18]. SYK is also revealed as a potential oncogenic
driver in lung cancer [19,20]. Inhibition of SYK markedly protected
Protein kinases regulate many signaling processes by appending
phosphate groups to proteins, which are important cellular targets for
the development of novel chemical drugs in cancer therapeutic area
[1–4]. Abnormal kinase activity has been implicated in cancers. Small
molecule inhibitors of protein kinases are widely used in cancer treat-
ment. These inhibitors are not only useful for investigation of cell sig-
naling processes but also significant for the treatment of kinase-medi-
ated disease processes.
against toxin-induced hepatic fibrosis, associated hepatocellular injury
and intra-hepatic inflammation, and hepatocarcinogenesis [21]. Col-
lectively, SYK pinpoints an important survival function and its inhibi-
tion frequently leads to cell apoptosis [22,23], which is considered as a
promising therapeutic target for the treatment of malignancies.
The discovery of safe SYK inhibitors has attracted much attention in
a number of therapeutic areas. SYK as a therapeutic target possesses a
high potential for the treatment of inflammatory diseases, which led to
many pharmaceutical companies and academic institutions involving in
the development of inhibitors of SYK. In recent years, a number of SYK
inhibitors have advanced to clinical trials. Fostamatinib (Fig. 1) was
once considered to be one of the most promising SYK small molecule
inhibitors to treat RA, but failed due to little efficacy observed in Phase
II studies and too many adverse reactions in Phase III clinical trials
[24–28]. PRT062607 (Fig. 1) is currently in clinical phase II and no
further data is reported [29]. Entospletinib (GS9973, Fig. 1) mono-
therapy has been identified clinical activity for patients with chronic
lymphocytic leukemia (CLL) who have relapsed following therapy with
BCR inhibitor in phase II study [30–33]. It is also developed in com-
bination with Idelalisib, a PI3K delta inhibitor. TAK-659 (Fig. 1), a dual
Spleen tyrosine kinase (SYK) plays an important role of survival
function in mediating signal transduction via multiple receptors. SYK is
a non-receptor cytoplasmic tyrosine kinase, which is a key mediator for
a variety of inflammatory cells and immunology signaling pathways
[5–7]. Researches have identified SYK is considered to be an attractive
target for the treatment of immunological diseases, such as rheumatoid
arthritis, asthma/rhinitis, malignancies and so on [7–12]. Recent stu-
dies have implicated abnormal SYK activation in several malignancies
[13–16]. In leukemia, SYK shows a cancer-modulating role as oncogene
and tumor promoter [17]. SYK overexpresses in recurrent ovarian
cancers and SYK signaling regulates ovarian cancer cell motility and
invasiveness, which provides a target strategy to suppress ovarian
⁎ Corresponding authors at: Department of Medicinal Chemistry, School of Medicine, Nankai University, 94 Weijin Road, Tianjin 300071, China.
E-mail addresses: rxiang@nankai.edu.cn (R. Xiang), yanfan@nankai.edu.cn (Y. Fan).
1 Cheng Wang and Xin Wang are contributed equally to this work.
https://doi.org/10.1016/j.bioorg.2019.103547
Received 10 October 2019; Received in revised form 18 December 2019; Accepted 21 December 2019
Availableonline24December2019
0045-2068/©2019ElsevierInc.Allrightsreserved.

C. Wang, et al.
BioorganicChemistry95(2020)103547
Fig. 1. Literature reported SYK inhibitors.
SYK and FLT3 inhibitor, is currently in clinical trials of advanced solid
tumors and lymphomas [34–40].
corresponding haloalkane to give the intermediates 4b–4d except 4a.
While 4a was obtained via Borch reductive amination from for-
maldehyde in the presence of sodium cyanoborohydride. Subsequently,
4a–4d and 2c were reduced to 5a–5e using the method depicted of the
synthesis of 3a–3c. Aim to explore the effect of the substituted group of
the 6-position on activity, we synthesized a series of derivatives in
Scheme 2. Commercial 6,8-dibromoimidazo[1,2-a]pyrazine (6) reacted
with 3a using DIEA as a base under conventional heating conditions to
give the key intermediate 7a, following Suzuki-Miyaura coupling per-
formed with 3-aminophenylboronic acid pinacol ester catalyzed by
PdCl2(dppf) to provide the derivative 8a. In order to modify the size of
the substituent at this position, we then condensed 8a with diversity
carboxylic acid to obtain 9a–9h. 9i was acylated with 1-(chlor-
ocarbonyl)cyclopropane-1-carboxylic acid prepared using SOCl2. Given
the N-Boc protection group, 9a–9c were deprotected by the solution of
4 M HCl in 1, 4-dioxane to lead to the derivatives 10a–10c. Carbonyl in
compound 9d was reduced absolutely by LiAlH4 to their aminoalcohol
product 10d. In Scheme 3, 12a–12k were accomplished via substitution
and Suzuki-Miyaura coupling in succession, 12a–12b of which need to
be removed the protecting group resulting in 13a–13b.
In the past, generation of selective SYK inhibitors has been chal-
lenging and advanced to clinical trials. Significant efforts have been
done for finding SYK inhibitors in decades, while none of them is put
into the market. Meanwhile, most of the inhibitors are applied for in-
flammatory disease indications, such as asthma, arthritis and allergic
diseases and few SYK inhibitors used to treat solid tumors. As men-
tioned above, SYK inhibition provides a desirable mechanistic inter-
vention for potential treatment of solid tumor. Thus, it is highly de-
sirable to find SYK inhibitors for solid tumor treatment.
In an effort to discover optimized SYK inhibitors, we performed a
structure activity relationship (SAR) study on imidazo[1,2-a]pyrazine
scaffold of entospletinib. Entospletinib produced only modest activity
against solid tumors with low lipophilicity (logP < 2) and low to
moderate solubility. In an effort to improve these properties and anti-
tumor activity, we have subsequently performed structural optimiza-
tion, designed and synthesized a series of compounds. Based on enzy-
matic and cellular activities, compound 12f was discovered. In vitro
and in vivo assays, 12f could effectively led tumor regression with fa-
vourable lipophilicity and solubility. This study provided valuable leads
for further structural optimization of SYK inhibitor for solid tumor
treatment.
2.2. SAR analyses of imidazo[1,2-a]pyrazine derivatives
Since our goal here is to discover SYK inhibitors with high potency
against solid tumor, both enzymatic and cellular assays were utilized to
evaluate the bioactivity of synthesized compounds in the SAR studies.
For the cellular assays, ovarian and lung cancer cell lines were chosen.
As reported, SYK is a potential oncogene in ovarian and lung cancer
[16,18–20].
2. Results and discussion
2.1. Chemistry
The synthetic routes for all compounds were shown in Schemes 1–3.
As shown in Scheme 1, the fluorine atom on 1-fluoro-4-nitrobenzene (1)
was substituted by morpholine, N-Boc-piperazine or polycyclic piper-
azine in the different conditions to give intermediates 2a–2d. 2a and 2b
underwent reduction reaction with hydrazine hydrate catalyzed by Pd/
C to afford intermediates 3a and 3b. After deprotection reaction, the
secondary amine (3d or 3e) was exposed to continue next step without
further purification. Various alkyl group were introduced on the sec-
ondary amine through nucleophilic substitution reaction with
As shown in Table 1, we explored the effects of different sub-
stituents at the 6-position in the first step. Compound 8a containing an
aniline group at the 6-position (Table 1) was first to be tested the en-
zyme inhibitory activity and showed acceptable potency against SYK.
We continued to add a long-chain alkyl group on free amino based on
compound 8a. Compared with 8a, substitution of 6-position by long
chain with a neutral ester group (9d and 9e, Table 1) decreased the
enzymatic activity. Subsequently, we adopted the acidic carboxyl
2

C. Wang, et al.
BioorganicChemistry95(2020)103547
Scheme 1. Synthesis of intermediates 3a–3c, 5a–5e.a. aReagents and conditions: (A) morpholine, 80 °C (2a); (B) mono-Boc-piperazine derivatives, DIEA, acet-
onitrile (2b–2d); (C) Pd/C, Hydrazine hydrate, ethanol, 75 °C; (D) CF3COOH, DCM, 0 °C; (E) haloalkane, K2CO3, acetonitrile, 85 °C (4b–4d); (F) NaCNBH3,
formaldehyde, Methanol, r.t. (4a).
functional group (9g–9i, Table 1). It was a pity that the enzyme ac-
tivities were still obvious reduction. After that, we used an amino as a
basic and strong electron-donating group (10a–10c, Table 1). Excit-
ingly, compound 10a displayed a slight improvement in enzyme ac-
tivity. While, as the carbon chain extended, the enzyme activity of the
compounds 10b–10c decreased obviously. Replacement of amino by a
weaker electron-donating hydroxyl functional group (10d, Table 1) led
to a slight drop in bioactivity against SYK. Introduction of a relative
rigid phenyl cyanide group (9f, Table 1) did not improve the bio-
chemical potency in enzymatic assay. Therefore, the results indicated
that modifications of 8a at R1 position had almost no effect on the
improvement of bioactivity.
Further optimization was proceeding. As shown in Table 2, the
hydrophilic piperazine ring replacement at R2 position and keeping 1H-
indazole at the R3 position (12g and 13b), showed notable enzyme
inhibitory activity (inhibition over 90% at 1 μM). As reported that the
introduction of the bridging ring might improve the drug-like proper-
ties of a compound, we attempted to adopt bridging piperazines [41].
However, introduction of bridging ring at R2 (13a) slightly decreased
inhibitory efficiency on the enzyme (Table 2). We tried to synthesize a
Scheme 2. Synthesis of compounds 9a–9l and 10a–10i.a. aReagents and conditions: (A) DIEA, isopropanol, 85 °C; (B) heteroaryl-boronic ester, PdCl2(dppf),
Na2CO3 (1 M aqueous solution), 1,4-dioxane, 100 °C; (C) substituted acid, EDCI, HOBt, DIEA, DMF (9a–9h); (D) 1,1-cyclopropanedicarboxylic acid, SOCl2, DIEA,
isopropyl acetate, r.t. (9l); (E) the solution of HCl in 1,4-dioxane (4 M), 0 °C (10a–10c); (F) LiAlH4, THF, 0 °C (10d).
3

C. Wang, et al.
BioorganicChemistry95(2020)103547
Scheme 3. Synthesis of compounds 12a–12 k and 13a–13b.a. aReagents and conditions: (A) DIEA, isopropanol, 85 °C; (B) heteroaryl-boronic ester, PdCl2(dppf),
Na2CO3 (1 M aqueous solution), 1,4-dioxane, 100 °C; (C) the solution of HCl in 1,4-dioxane (4 M), 0 °C.
Considering the absolute configuration of one-carbon bridges ring for
the activity, we synthesized compound 12e (Table 2). 12e exhibited a
similar inhibitory activity to that of 12f. Similar results were found by
the comparison of the compound 12h vs 12j (Table 2). Finally, we fixed
R2 as one-carbon bridges ring group and varied R3. However, it was
regrettable that compounds 12h–12k showed poor enzyme inhibition
activity.
Table 1
Structure and enzymatic inhibition activity of 8a, 9d–9i and
10a–10d.
The observation above led us to further measure the half maximal
inhibitory concentration (IC50) values of selected compounds with the
enzyme inhibition rate greater than 90%, as shown in Table 3. The
inhibitory activity of these compounds slightly decreased compared to
entospletinib. We subsequently evaluated these compounds in cellular
levels.
Compounds
R1
H
Inhibition (%) at 1 μM
8a
9d
65.5
39.5
1.2
0.2.9
9e
26.0
0.8
The preliminary cell growth inhibition of all compounds with fixed
concentrations of 10 μM and 1 μM was carried out using standard CCK8
assay and the results are shown in Table 4. The best tumor cell potency
of these analogues was obtained for molecule 12f, which was also ra-
tional inhibitor of SYK with IC50 value of 0.08 μM. The compound was
next evaluated in a dose response study against ovarian cancer cell lines
(SKOV3, Hey, ES2, OVCAR5, Cao3 and SW626) and lung cancer cell
lines (H1299, A549 and H460) to test cytotoxic activity (Tables 5). IC50
values were further detected on compound 12f. Compound en-
tospletinib, was used as reference. 12f displayed higher inhibition po-
tency than positive control entospletinib. The SYK inhibitor en-
tospletinib, was not potent in these solid tumor cells. This result
suggested that our compound had more efficient inhibition at the cel-
lular level.
9f
31.0
0.8
9g
22.5
0.4
9h
9i
10.5
37.5
0.4
3.7
10a
10b
10c
10d
86.5
77.0
76.5
66.5
0.4
0.8
0.4
0.4
series of compounds 12c, 12d and 12f (Table 2). The enzymatic po-
tency of 12f remained compared with compound 13b. Structural in-
troduction of isopropyl groups might be compatible in protein cavities.
2.3. Lipophilicity
We infered that the introduction of isopropyl and one-carbon bridge
4

C. Wang, et al.
BioorganicChemistry95(2020)103547
Table 2
lipophilicity, and the results of enzymatic activity and antitumor ac-
tivity in cell lines showed 12f was a promising SYK inhibitor worthy for
further study. We performed in-depth in vitro and in vivo biological
assays.
Structure
and
enzymatic
inhibition
activity
of
12c–12k
and
13a–13b.
2.4. Molecular modeling of compound 12f
We docked compound 12f and SYK protein (PDB code: 4PUZ) to
study their binding mode through the Discovery Studio 3.1. The images
were created using PyMOL. As shown in Fig. 2, we can see 12f suitably
resides in the active pocket of SYK. The NH of 1H-indazole was directly
involved in hydrogen bonding with side chain of D512. Furthermore,
the hinge region A451 residue formed two key hydrogen bonds with the
N-1 of imidazopyrazine and the secondary amine, respectively.
Compounds
R2
R3
Inhibition (%) at 1 μM
12c
66.5
0.4
12d
12e
85.5
91.0
0.4
0.8
2.5. Effect of compound 12f on cell apoptosis
It was subjected to an apoptotic assay for further evaluation of our
compound against cancer cell lines. We chose ovarian cancer cell line
(SKOV3) and lung cancer cell line (A549) with malignant phenotypes.
Cells were treated with vehicle (DMSO), compound 12f, and en-
tospletinib as reference drug for 48 h, which were analyzed by annexin
V/PI staining (Fig. 3). The percentage of apoptotic cells was defined as
the sum of early and late apoptosis (annexin V-positive and PI-positive
cells). Compound 12f induced cell apoptosis in a dose-dependent
manner. Compared with the vehicle condition and entospletinib, com-
pound 12f was significantly more active in apoptosis induction as the
concentration increased. Taken together, compound 12f could induce
cell death via apoptosis.
12f
93.5
0.4
12g
13a
13b
12h
95.5
87.5
96.5
45.5
0.4
1.2
0.4
0.4
12i
37.0
46.5
7.5
3.3
0.4
2.6. In vivo anticancer activity of compound 12f
12j
To further evaluate whether compound 12f was effective in in-
hibiting tumor growth, xenograft model of nude mice bearing SKOV3
cells was constructed. When the tumor reached a size of 100–120 mm3,
the mice were randomly assigned to 5 groups with 5 mice in each
group. Subsequently, they were orally administered with 12f (10 mg/
kg, 20 mg/kg, 40 mg/kg), entospletinib (40 mg/kg) or vehicle for
21 days respectively.
12 k
2.0
Table 3
As shown in Fig. 4A and B, 12f has better antitumor activity than
entospletinib and vehicle. 12f treatment with 10 mg/kg or 20 mg/kg or
40 mg/kg substantially inhibited the tumor growth with inhibition
rates of 57.5% 13.1%, 61.3% 8.2% and 76.7% 7.7%, respec-
tively. In contrast, tumor growth inhibitions of 40.1% 14.7% were
observed at doses of 40 mg/kg of entospletinib as the positive control. A
decrease in tumor weight was also observed at the end-point of the
study (Fig. 4C). There was no significant decrease in body weight
during administration, indicating that the compound 12f was well-
tolerated in vivo (Fig. 4D). These data demonstrated that compound 12f
had good tumor suppressing effect and low toxicity.
IC50 value of compounds entospletinib, 12e, 12f, 12g and
13b against SYK.
Compounds
IC50 (μM)
Entospletinib
0.011
0.079
0.080
0.029
0.029
0.001
0.001
0.003
0.002
0.002
12e
12f
12g
13b
moieties may change the lipophilicity and solubility of 12f for cell
uptake. We detected the LogP and LogD7.4 values, which are very im-
portant biopharmaceutical parameters and commonly used to char-
acterize the lipophilicity of a compound. The experimental LogP and
LogD7.4 values of compounds 12f and entospletinib were shown in
Table 6 using the traditional shake flask method. The result indicated
that compound 12f had a more reasonable partition coefficient than
entospletinib. We also detected the solubility of two compounds. The
solubility of 12f in PBS at pH 7.4 is 216 μg/mL. The solubility of en-
tospletinib in PBS at pH 7.4 is 28 μg/mL. The lipophilicity and solubility
are key parameters determined the availability of the inhibitors to enter
the biological membranes. The reasonable lipophilicity and solubility
allow inhibitors to easily penetrate the phospholipid bilayer of the cells
and bind to the target protein. Therefore, it was probably due to
compound 12f with a comfortable and optimal lipophilicity and solu-
bility for cell uptake. The enhanced drug-like characteristics of
3. Conclusion
In summary, we have designed and synthesized a new class of
imidazo[1,2-a]pyrazine derivatives as novel SYK inhibitors based on
entospletinib. Activity cliffs are an important type of structure-activity
relationship where small structural changes result in unexpected dif-
ferences in biological activity [42]. In our study, structural optimization
improved the pharmaceutical lipophilicity of entospletinib and also
obviously enhanced inhibition activity of solid cancer. The lipophilicity
and solubility of 12f were more reasonable than that of entospletinib. In
in vitro cellar assays, 12f showed potent activities against human
ovarian and lung cancer cell lines. In cell apoptosis assays, it was evi-
dent that compound 12f could induce cell death via apoptosis in a dose-
dependent manner. In in vivo assay, a once-daily dose of compound 12f
at 10–40 mg/kg for 21 days led to obvious tumor regression without
5

C. Wang, et al.
BioorganicChemistry95(2020)103547
Table 4
In vitro cell growth inhibition (%) of compounds against cancer cell lines.a
Comps.
Inhibition %
SKOV3
Hey
ES2
H460
1 μM
10 μM
1 μM
10 μM
1 μM
10 μM
1 μM
10 μM
8a
4.8
5.8
4.5
50.8
18.5
7.9
5.5
3.4
15.7
23.7
30.2
−4.6
4.1
7.8
7.4
6.6
31.7
14.3
18.5
−4.3
62.6
35.6
42.3
71.4
30.4
70.5
79.5
77.4
102.6
98.9
103.7
100.8
80.3
57.8
77.4
76.2
96.9
104.2
15.9
5.3
10.7
4.7
12.2
5.4
8.7
1.8
14.2
2.5
6.5
7.2
0.7
1.9
2.4
0.3
6.4
6.4
1.1
9.1
5.1
0.1
27.7
0.4
10.5
7.5
1.6
3.6
6.6
7.3
0.8
22.7
5.7
3.1
3.4
3.4
1.3
3.8
5.5
13.1
−13.1
12.9
−6.9
−4.5
−8.0
10.4
18.2
12.7
26.0
10.4
17.3
65.2
65.9
73.4
39.2
8.8
4.3
4.4
2.5
31.2
4.9
4.4
3.6
9d
15.3
−0.8
6.1
−5.8
−10.1
33.7
79.5
12.8
19.7
72.0
43.8
78.6
85.3
54.4
101.0
95.6
99.6
98.16
62.9
42.5
70.8
66.8
88.5
102.5
9e
6.6
9.2
−6.5
5.7
15.2
13.3
12.8
−6.1
34.4
64.2
32.6
42.5
41.3
87.1
100.6
100.6
97.8
100.9
76.6
79.9
75.5
78.1
89.2
101.7
4.4
11.4
8.9
6.1
8.5
16.9
19.6
9.9
8.9
16.9
0.6
0.4
1.9
0.4
9.5
5.4
5.2
6.5
3.3
0.4
9f
6.2
46.1
71.6
55.1
73.5
59.8
63.6
60.2
81.7
82.8
101.7
101.3
102.1
100.7
77.6
83.2
77.2
88.4
86.1
102.0
4.9
7.8
9.3
9.4
3.2
5.9
3.7
1.8
9.8
1.5
18.4
9g
9.3
15.9
10.3
6.8
9.5
6.9
6.6
4.8
15.1
−5.9
21.0
−17.4
10.2
7.6
1.5
4.7
3.0
6.3
8.2
1.5
1.5
6.2
18.3
2.5
9h
28.3
32.0
20.9
35.5
7.8
15.4
20.2
41.3
15.2
15.2
31.8
−0.8
29.7
33.7
30.3
57.9
46.1
29.8
43.3
29.7
35.6
31.3
33.9
9i
8.6
4.9
10a
10b
10c
10d
12c
12d
12e
12f
12g
12h
12i
12j
12k
13a
13b
7.9
10.9
12.5
2.8
10.8
3.7
22.0
22.7
20.2
51.8
60.1
32.9
20.7
3.0
8.3
6.9
15.5
2.7
2.2
13.2
6.2
4.9
2.5
5.9
7.5
2.4
6.4
9.9
18.9
3.1
9.3
11.3
6.5
0.2
12.7
2.7
23.7
13.5
42.5
34.8
6.4
1.7
4.7
1.5
0.4
0.8
2.3
4.1
10.2
8.0
4.4
3.1
2.6
3.4
1.3
2.8
15.7
6.2
16.6
6.7
1.6
6.6
8.3
2.9
2.1
6.6
7.2
4.0
2.3
1.9
5.4
7.9
2.4
5.9
20.2
8.1
4.9
5.1
1.3
25.4
16.26
15.5
23.7
−6.0
21.3
18.5
24.4
3.9
1.9
2.9
2.0
14.1
9.9
4.7
4.3
2.8
8.5
5.6
8.7
11.4
0.2
12.2
1.1
a
The cytotoxic effect of compounds was assayed using CCK-8 assay with 72 h incubation. Data are from three independent experiments.
Table 5
IC50 values of 12f against cancer cell lines.a
Cell lines
IC50 (μM)
12f
Entospletinib
SKOV3
Hey
0.76
1.01
1.96
1.53
7.39
9.17
0.83
0.72
1.48
0.12
> 10
> 10
> 10
0.17
0.34
0.16
0.30
1.3
ES2
OVCAR5
Cao3
8.30
6.86
> 10
> 10
6.26
> 10
2.5
0.35
SW626
A549
0.04
0.19
0.15
H1299
H460
0.54
a
The cytotoxic effect of compounds was assayed using CCK-8 assay with
72 h incubation. Data are from three independent experiments.
Fig. 2. Representation of the predicted binding mode of compound 12f (pink)
with SYK (PDB code 4PUZ), SYK backbone is shown in cyan, the hydrogen
bonds are shown in red.
Table 6
Lipid-water partition coefficient of compounds entospletinib and 12f.
(400 MHz for 1H and 101 MHz for 13C) and chemical shifts were ex-
pressed in ppm downfield using tetramethylsilane as the internal
standard. High-resolution mass spectra (HRMS) were performed on a
VG ZAB-HS mass spectrometer under electron spray ionization (ESI).
The final products were purified to > 95% purity which was de-
termined by HPLC analysis on a Shimadzu Prominence-i LC-2030C 3D
system (column, InertSustain C18, 4.6 mm × 250 mm, 5 μM; mobile
phase, gradient elution of methanol/H2O; low rate, 1.0 mL/min; UV
wavelength, 190–800 nm; temperature, 40 °C; injection volume, 10 μL).
1H, 13C NMR and purity scan of 12f are in the Supporting
Information.
Compounds
LogP
LogD7.4
Entospletinib
12f
1.86
2.69
1.83
2.51
obvious toxicity. All of the studies suggest that this compound could act
as promising lead compound for further development of SYK inhibitors,
which may be useful for solid tumor therapy.
4. Experimental section
4.1. General methods for chemistry
4.1.1. 4-(4-Nitrophenyl)morpholine (2a)
Morpholine (17.81 mL, 205 mmol) was added to 1-fluoro-4-ni-
trobenzene (1, 3.8 mL, 35.44 mmol) and stirred at 80 °C for 5 h. Upon
completion of the reaction, the mixture was poured into ice water
(1000 mL) and filtered to obtain intermediate 2a in 99% yield as yellow
solid. 1H NMR (400 MHz, CDCl3) δ 8.14 (d, J = 9.4 Hz, 2H), 6.84 (d,
Purification and distillation of all reagents and solvents were per-
formed according to standard procedures. All starting materials are
commercially available and used directly unless otherwise stated. Thin-
layer chromatography (TLC) was used to monitor all reactions. The
NMR spectra were taken on a Bruker AV-400 MHz spectrometer
J = 9.4 Hz, 2H), 3.90–3.83 (m, 4H), 3.37 (dd, J = 5.9, 4.0 Hz, 4H). 13
C
6

C. Wang, et al.
BioorganicChemistry95(2020)103547
Fig. 3. 12f induced cell apoptosis in both ovarian and lung cancer cell lines. Flow cytometry (A and C) and quantitative analysis of apoptotic cells (B and D).
NMR (101 MHz, CDCl3) δ 155.0, 138.9, 125.9, 112.6, 66.4, 47.1.
General Procedure for the Preparation of 2b–2d. Add 1-fluoro-4-
nitrobenzene (1, 1.6 mL, 14 mmol) to mono-Boc- piperazine derivatives
(16 mmol), DIEA (7.1 mL, 32 mmol) in acetonitrile (25 mL), and stirred
at 80 °C overnight. After that, the mixture was concentrated under re-
duced pressure to give crude product. Then purification by silica gel
column chromatography gave the desired intermediates 2b–2d.
4.1.2. Tert-butyl 4-(4-nitrophenyl)piperazine-1-carboxylate (2b)
Light yellow solid; 97% yield; 1H NMR (400 MHz, CDCl3) δ 8.12 (d,
J = 9.3 Hz, 1H), 6.81 (d, J = 9.4 Hz, 2H), 3.60 (t, J = 5.3 Hz, 4H),
3.41 (t, J = 5.3 Hz, 4H), 1.48 (s, 9H); 13C NMR (101 MHz, CDCl3) δ
154.6, 154.5, 138.8, 126.0, 112.9, 80.4, 46.9, 28.4.
4.1.3. Tert-butyl
(1R,4R)-5-(4-nitrophenyl)-2,5-diazabicyclo[2.2.1]
heptane-2-carboxylate (2c)
Light yellow solid; 95% yield; 1H NMR (400 MHz, CDCl3) δ 8.12 (d,
7

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BioorganicChemistry95(2020)103547
Fig. 4. In vivo anti-tumor activity of 12f against SKOV3 tumor xenograft models. (A) Tumor growth curve of nude mice bearing SKOV3 cell were treated with 12f (10,
20, 40 mg/kg), entospletinib (40 mg/kg) or solvent control orally once per day (N = 5 per group, mean
SD). (B) Photograph of excised tumors of mice in each
group. (C) The tumor weight of excised tumors at day 21. (D) Body weight curves of mice treated with different treatments in 21 days. bars represent SD; *p < 0.05,
**p < 0.01, ***p < 0.001.
J = 8.9 Hz, 2H), 6.49 (d, J = 9.1 Hz, 2H), 4.53 (s, 1H), 3.59 (dd,
J = 1.9, 9.3 Hz, 1H), 3.46 (d, J = 8.5 Hz, 2H), 3.34 (d, J = 19.5 Hz,
2H), 2.01 (s, 2H), 1.46 (s, 9H); 13C NMR (101 MHz, CDCl3) δ 154.0,
151.1, 137.4, 126.4, 110.7, 80.2, 57.4, 56.7, 52.3, 51.9, 37.3, 28.5,
28.4.
47.4.
4.1.7. 4-((1R,4R)-5-methyl-2,5-diazabicyclo[2.2.1]heptan-2-yl)aniline
(5a)
Brown solid; 98% yield; 1H NMR (400 MHz, DMSO‑d6) δ 6.49 (d,
J = 8.0 Hz, 2H), 6.36 (d, J = 8.1 Hz, 2H), 6.20–5.91 (m, 2H), 4.17 (s,
1H), 3.62 (s, 1H), 3.31 (d, J = 9.3 Hz, 1H), 3.09 (d, J = 9.5 Hz, 1H),
2.82 (d, J = 10.0 Hz, 1H), 2.72 (d, J = 9.9 Hz, 1H), 2.37 (s, 3H), 1.88
(s, 2H); 13C NMR (101 MHz, DMSO‑d6) δ 139.7, 1389.0, 116.0, 114.5,
63.7, 58.2, 57.6, 52.7, 35.3, 22.0.
4.1.4. Tert-butyl
(1S,4S)-5-(4-nitrophenyl)-2,5-diazabicyclo[2.2.1]
heptane-2-carboxylate (2d)
Light yellow solid; 85% yield; 1H NMR (400 MHz, CDCl3) δ 8.10 (d,
J = 8.9 Hz, 2H), 6.48 (d, J = 8.9 Hz, 2H), 4.64 (d, J = 50.9 Hz, 1H),
4.53 (s, 1H), 3.58 (d, J = 9.3 Hz, 1H), 3.48–3.26 (m, 3H), 2.01 (d,
J = 5.9 Hz, 2H), 1.45 (s, 9H); 13C NMR (101 MHz, CDCl3) δ 154.0,
151.1, 144.9, 126.5, 110.7, 80.3, 57.9, 57.4, 56.9, 56.1, 37.8, 28.5,
28.4.
4.1.8. 4-((1R,4R)-5-ethyl-2,5-diazabicyclo[2.2.1]heptan-2-yl)aniline (5b)
Brown solid; 98% yield; 1H NMR (400 MHz, DMSO‑d6) δ 6.52 (d,
J = 8.1 Hz, 2H), 6.39 (d, J = 8.1 Hz, 2H), 6.23–5.94 (m, 2H), 4.34 (s,
1H), 4.17 (s, 1H), 3.46 (s, 1H), 3.17–2.88 (m, 5H), 2.12–1.97 (m, 2H),
1.12–1.06 (m, 3H); 13C NMR (101 MHz, DMSO‑d6) δ 121.2, 116.0,
114.8, 113.3, 63.6, 57.3, 56.4, 52.5, 48.9, 34.6, 11.5.
General Procedure for the Preparation of 3a–3b, 5a–5e. 10% Pd/C
(1.43 g, 13.5 mmol), hydrazine (8 mL) and nitrobenzene derivatives
2a–2c and 4a–4d (19 mmol) were dissolved in ethanol (60 mL) and use
argon to remove the gas three times. The resulted mixture was heated to
75 °C for 8 h. After that, the reaction solution was filtered through
celite. Then the crude products were purified by column chromato-
graphy on silica gel to afford desired intermediates 3a–3b, 5a–5e.
4.1.9. 4-((1R,4R)-5-isopropyl-2,5-diazabicyclo[2.2.1]heptan-2-yl)aniline
(5c)
Black viscous liquid; 98% yield; 1H NMR (400 MHz, CDCl3) δ 6.63
(d, J = 8.1 Hz, 2H), 6.42 (d, J = 8.2 Hz, 2H), 4.09 (s, 2H), 3.83 (s, 2H),
3.35 (dd, J = 9.5, 2.2 Hz, 1H), 3.22 (d, J = 9.4 Hz, 2H), 2.61–2.49 (m,
2H), 2.03 (d, J = 9.7 Hz, 1H), 1.97 (d, J = 4.7 Hz, 1H), 1.07 (d,
J = 6.2 Hz, 3H), 1.03 (d, J = 6.1 Hz, 3H); 13C NMR (101 MHz, CDCl3) δ
140.3, 136.9, 117.1, 114.0, 59.0, 57.3, 55.6, 50.9, 50.4, 37.4, 22.2,
21.6.
4.1.5. 4-Morpholinoaniline (3a)
Silver grey solid; 95% yield; 1H NMR (400 MHz, DMSO‑d6) δ 6.68
(d, J = 8.8 Hz, 2H), 6.51 (d, J = 8.8 Hz, 2H), 4.58 (s, 2H), 3.73–3.65
(m, 4H), 2.88–2.85 (m, 4H); 13C NMR (101 MHz, DMSO‑d6) δ 142.8,
142.8, 118.0, 115.2, 66.8, 51.1.
4.1.6. Tert-butyl 4-(4-aminophenyl)piperazine-1-carboxylate (3b)
Black gray solid; 95% yield; 1H NMR (400 MHz, DMSO‑d6) δ 6.70
(d, J = 8.6 Hz, 2H), 6.49 (d, J = 8.6 Hz, 2H), 4.63 (s, 2H), 3.41 (t,
J = 5.0 Hz, 4H), 2.82 (t, J = 5.1 Hz, 4H), 1.41 (s, 9H); 13C NMR
(101 MHz, DMSO‑d6) δ 144.9, 132.9, 132.3, 122.1, 116.4, 111.3, 53.6,
4.1.10. Tert-butyl
(1R,4R)-5-(4-aminophenyl)-2,5-diazabicyclo[2.2.1]
heptane-2-carboxylate (5d)
Off-white solid; 98% yield; 1H NMR (400 MHz, CDCl3) δ 6.42 (d,
J = 48.8 Hz, 4H), 4.35 (d, J = 6.8 Hz, 2H), 4.32–4.15 (m, 2H), 3.46 (s,
1H), 3.21 (s, 2H), 2.79 (s, 1H), 1.85 (s, 2H), 1.34 (dd, J = 20.6, 6.7 Hz,
8

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BioorganicChemistry95(2020)103547
4.1.16. (1R,4R)-2-isopropyl-5-(4-nitrophenyl)-2,5-diazabicyclo[2.2.1]
9H); 13C NMR (101 MHz, DMSO‑d6) δ 153.9, 139.7, 137.9, 116.0,
114.4, 79.0, 57.5, 56.5, 55.4, 50.0, 36.9, 28.6,28.5.
heptane (4c)
Yellow solid; 94% yield; 1H NMR (400 MHz, CDCl3) δ 8.07 (d,
J = 9.2 Hz, 2H), 6.46–6.42 (m, 2H), 4.34 (s, 1H), 3.87 (s, 1H), 3.45 (dd,
J = 9.8, 1.5 Hz, 1H), 3.37 (dd, J = 9.8, 2.1 Hz, 1H), 3.20 (dd, J = 9.6,
2.1 Hz, 1H), 2.58–2.51 (m, 1H), 2.48 (dd, J = 9.6, 1.7 Hz, 1H), 2.06
(ddt, J = 9.9, 2.6, 1.4 Hz, 1H), 1.93–1.89 (m, 1H), 1.07 (d, J = 6.2 Hz,
3H), 1.03 (d, J = 6.1 Hz, 3H); 13C NMR (101 MHz, CDCl3) δ 151.0,
136.8, 126.5, 110.5, 58.2, 58.0, 57.1, 51.1, 50.9, 37.3, 22.5, 21.9.
4.1.11. 4-((1S,4S)-5-isopropyl-2,5-diazabicyclo[2.2.1]heptan-2-yl)aniline
(5e)
Reddish brown oil; 98% yield; 1H NMR (400 MHz, CDCl3) δ 6.64 (d,
J = 8.1 Hz, 2H), 6.43 (d, J = 8.1 Hz, 2H), 4.08 (s, 1H), 3.74 (s, 1H),
3.36–3.20 (m, 4H), 3.13 (d, J = 9.5 Hz, 1H), 2.53–2.45 (m, 2H), 1.98
(d, J = 9.6 Hz, 2H), 1.03 (d, J = 6.3 Hz, 3H), 0.99 (d, J = 6.0 Hz, 3H);
13C NMR (101 MHz, CDCl3) δ 140.6, 136.6, 117.1, 114.0, 58.7, 57.5,
55.6, 50.6, 50.4, 37.6, 22.7, 22.0.
4.1.17. (1S,4S)-2-isopropyl-5-(4-nitrophenyl)-2,5-diazabicyclo[2.2.1]
heptane (4d)
General Procedure for the Preparation of 3d–3e. 2c, 2d (12 mmol)
was dissolved in DCM (60 mL) at 0 °C, then slowly added trifluoroacetic
acid (25 mL) with a constant pressure dropping funnel. After 4 h, the
organic phase was spun off and the solid products were obtained for
3d–3e.
Yellow solid; 94% yield; 1H NMR (400 MHz, CDCl3) δ 8.09 (d,
J = 9.4 Hz, 2H), 6.46 (d, J = 9.4 Hz, 2H), 4.34 (s, 1H), 3.86 (d,
J = 2.7 Hz, 1H), 3.48–3.35 (m, 2H), 3.20 (dd, J = 9.6, 2.1 Hz, 1H),
2.57–2.46 (m, 2H), 2.10–2.03 (m, 1H), 1.96–1.88 (m, 1H), 1.07 (d,
J = 6.2 Hz, 3H), 1.03 (d, J = 6.1 Hz, 3H); 13C NMR (101 MHz, CDCl3) δ
151.04, 136.86, 126.47, 110.54, 58.16, 58.03, 57.12, 51.06, 50.98,
37.32, 22.60, 21.94.
4.1.12. (1R,4R)-2-(4-nitrophenyl)-2,5-diazabicyclo[2.2.1]heptane (3d)
Yellow-green solid; 94% yield; 1H NMR (400 MHz, DMSO‑d6) δ 8.09
(d, J = 8.9 Hz, 2H), 6.78 (d, J = 8.9 Hz, 2H), 4.89 (s, 1H), 4.56 (s, 1H),
3.68 (dd, J = 11.1, 2.5 Hz, 1H), 3.49 (d, J = 11.0 Hz, 1H), 3.42–3.30
(m, 2H), 3.24–3.17 (m, 1H), 2.17 (d, J = 11.0 Hz, 1H), 2.04–2.00 (m,
1H); 13C NMR (101 MHz, DMSO‑d6) δ 151.2, 137.2, 126.4, 112.2, 57.8,
56.1, 52.6, 51.0, 36.1.
General Procedure for the Preparation of 7a, 11a–11 g. 6,8-di-
bromoimidazo[1,2-a]pyrazine (6, 1.86 g, 6.72 mmol) was added to a
solution of 3a–3c, 5a–5e (5.6 mmol), DIPEA (1.4 mL, 8.4 mmol) in
isopropanol (60 mL), stirred at 85 °C for 8 h, and then the solvent was
evaporated to dryness. The crude product was purified by column
chromatography to obtain the desired intermediates 7a, 11a–11 g.
4.1.13. (1S,4S)-2-(4-nitrophenyl)-2,5-diazabicyclo[2.2.1]heptane (3e)
Brown solid; 91%; 1H NMR (400 MHz, DMSO‑d6) δ 8.02 (d,
J = 9.0 Hz, 2H), 6.61 (d, J = 9.0 Hz, 2H), 4.82 (d, J = 26.9 Hz, 1H),
4.63 (s, 1H), 3.49 (d, J = 9.4 Hz, 1H), 3.37 (s, 3H), 3.25 (d, J = 9.0 Hz,
1H), 2.04–1.89 (m, 1H), 1.85 (s, 1H); 13C NMR (101 MHz, DMSO‑d6) δ
150.1, 136.2, 125.1, 111.0, 56.4, 55.8, 50.4, 48.8, 35.6.
4.1.18. 6-Bromo-N-(4-morpholinophenyl)imidazo[1,2-a]pyrazin-8-amine
(7a)
White solid; 85% yield; 1H NMR (400 MHz, DMSO‑d6) δ 9.81 (s,
1H), 8.18 (d, J = 5.7 Hz, 1H), 7.91 (d, J = 3.1 Hz, 1H), 7.80 (d,
J = 9.3 Hz, 2H), 7.60 (d, J = 2.4 Hz, 1H), 6.92 (d, J = 8.3 Hz, 2H),
3.73 (d, J = 3.1 Hz, 4H), 3.06 (dt, J = 8.3, 4.6 Hz, 4H); 13C NMR
(101 MHz, DMSO‑d6) δ 147.58, 144.9, 132.9, 132.1, 131.8, 122.0,
121.3, 116.7, 115.7, 111.2, 66.6, 49.4.
4.1.14. (1R,4R)-2-methyl-5-(4-nitrophenyl)-2,5-diazabicyclo[2.2.1]
heptane (4a)
4.1.19. Tert-butyl
(1R,4R)-5-(4-((6-bromoimidazo[1,2-a]pyrazin-8-yl)
Formaldehyde (0.51 mL, 37% in water) was added to the for-
maldehyde (20 mL) solution of 3d (2.28 mmol), then sodium cyano-
borohydride (573 mg) was added in portions and stirred at room
temperature overnight. The reaction mixture was evaporated in va-
cuum and the crude product was obtained. The product was dissolved
in a small amount of DCM, and then a large amount of PE was added to
precipitate a solid and filtered to obtain the desired intermediate 4a in
81% yield as yellow solid. 1H NMR (400 MHz, DMSO‑d6) δ 8.02 (d,
J = 9.7 Hz, 2H), 6.65 (d, J = 9.7 Hz, 2H), 4.56 (s, 1H), 3.49 (s, 1H),
2.81 (dd, J = 9.5, 2.1 Hz, 1H), 2.74 (s, 1H), 2.43 (dd, J = 9.5, 1.5 Hz,
1H), 2.27 (d, J = 1.6 Hz, 4H), 1.91 (dd, J = 9.8, 2.4 Hz, 1H), 1.77 (d,
J = 9.6 Hz, 1H); 13C NMR (101 MHz, DMSO‑d6) δ 151.8, 135.9, 126.5,
111.4, 62.5, 60.0, 58.9, 53.0, 41.3, 35.7.
amino)phenyl)-2,5-diazabicyclo[2.2.1]heptane-2-carboxylate (11a)
Light yellow solid; 84% yield; 1H NMR (400 MHz, DMSO‑d6) δ 9.72
(d, J = 2.4 Hz, 1H), 8.15 (s, 1H), 7.90 (d, J = 1.1 Hz, 1H), 7.73 (d,
J = 8.6 Hz, 2H), 7.59 (d, J = 1.0 Hz, 1H), 6.62 (d, J = 9.0 Hz, 2H),
4.49 (d, J = 7.3 Hz, 1H), 3.55 (dd, J = 10.8, 8.6 Hz, 1H), 3.33 (d,
J = 6.7 Hz, 1H), 3.27–3.19 (m, 2H), 2.96 (t, J = 7.9 Hz, 1H), 1.93–1.87
(m, 2H), 1.36 (d, J = 21.9 Hz, 9H); 13C NMR (101 MHz, DMSO‑d6) δ
145.0, 143.7, 132.8, 132.2, 122.7, 122.7, 121.5, 116.7, 112.9, 112.9,
110.8, 79.1, 57.9, 56.7, 56.5, 51.2, 37.6, 28.6, 28.5.
4.1.20. Tert-butyl
4-(4-((6-bromoimidazo[1,2-a]pyrazin-8-yl)amino)
phenyl)piperazine-1-carboxylate (11b)
White solid; 84% yield; 1H NMR (400 MHz, CDCl3) δ 9.83 (s, 1H),
8.19 (s, 1H), 7.92 (s, 1H), 7.80 (d, J = 8.8 Hz, 2H), 7.61 (s, 1H), 6.96
(d, J = 8.8 Hz, 2H), 3.46 (t, J = 5.0 Hz, 4H), 3.06 (t, J = 5.1 Hz, 4H),
1.42 (s, 9H); 13C NMR (101 MHz, CDCl3) δ 155.1, 148.2, 145.7, 133.7,
132.9, 122.9, 122.7, 122.0, 117.5, 112.0, 80.2, 50.6, 29.3, 19.9.
General Procedure for the Preparation of 4b–4d. Halogenated al-
kane (6.8 mmol) was added to the nitrobenzene derivatives 3d and 3e
(4.5 mmol) and potassium carbonate (3.1 g, 22.5 mmol) in acetonitrile
(40 mL) solution at 85 °C. After 8 h, the reaction mixture was evapo-
rated to dryness and purified by silica gel column chromatography to
give desired intermediates 4b–4d.
4.1.21. 6-Bromo-N-(4-((1R,4R)-5-methyl-2,5-diazabicyclo[2.2.1]heptan-
2-yl)phenyl)imidazo[1,2-a]pyrazin-8-amine (11c)
4.1.15. (1R,4R)-2-ethyl-5-(4-nitrophenyl)-2,5-diazabicyclo[2.2.1]heptane
(4b)
White solid; 85% yield; 1H NMR (400 MHz, DMSO‑d6) δ 9.71 (s,
1H), 8.15 (s, 1H), 7.90 (s, 1H), 7.70 (d, J = 8.8 Hz, 2H), 7.59 (d,
J = 1.1 Hz, 1H), 6.59 (d, J = 8.7 Hz, 2H), 4.31 (s, 1H), 3.55 (s, 1H),
3.35 (dd, J = 9.5, 2.3 Hz, 1H), 3.18 (d, J = 9.3 Hz, 1H), 2.83 (dd,
J = 9.8, 2.0 Hz, 1H), 2.62 (d, J = 9.7 Hz, 1H), 2.33 (s, 3H), 1.90 (s,
1H), 1.81 (d, J = 9.5 Hz, 1H); 13C NMR (101 MHz, DMSO‑d6) δ 145.0,
143.8, 132.8, 132.2, 128.9, 122.8, 121.5, 116.7, 112.9, 110.8, 63.3,
58.9, 57.8, 52.8, 35.6, 21.7.
Yellow solid; 84% yield. 1H NMR (400 MHz, CDCl3) δ 8.09 (d,
J = 9.3 Hz, 2H), 6.46 (d, J = 9.4 Hz, 2H), 4.36 (s, 1H), 3.69 (d,
J = 2.4 Hz, 1H), 3.46–3.38 (m, 2H), 3.03 (dd, J = 9.7, 2.1 Hz, 1H),
2.61–2.51 (m, 3H), 2.05 (ddt, J = 9.9, 2.6, 1.3 Hz, 1H), 1.91 (dt,
J = 2.5, 1.2 Hz, 1H), 1.07 (t, J = 7.2 Hz, 3H); 13C NMR (101 MHz,
DMSO‑d6) δ 146.4, 132.1, 121.7, 105.8, 55.8, 53.3, 53.2, 47.4, 43.2,
31.6, 9.4.
9

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BioorganicChemistry95(2020)103547
4.1.22. 6-Bromo-N-(4-((1R,4R)-5-ethyl-2,5-diazabicyclo[2.2.1]heptan-2-
yl)phenyl)imidazo[1,2-a]pyrazin-8-amine (11d)
7.98 (s, 1H), 7.85 (d, J = 8.5 Hz, 1H), 7.74 (d, J = 8.6 Hz, 1H), 7.66 (s,
1H), 6.68 (d, J = 8.5 Hz, 2H), 4.55–4.46 (m, 2H), 3.30 (d, J = 9.3 Hz,
3H), 3.02 (s, 1H), 1.93 (d, J = 17.9 Hz, 2H), 1.37 (d, J = 25.1 Hz, 9H);
13C NMR (101 MHz, DMSO‑d6) δ 153.2, 144.8, 142.7, 140.6, 136.6,
136.3, 133.6, 132.0, 129.8, 122.7, 121.7, 120.6, 118.4, 116.5, 112.5,
108.4, 107.1, 102.10 99.5, 78.6, 57.4, 56.3, 50.7, 40.00, 36.6, 28.1,
28.0.
White solid; 80% yield; 1H NMR (400 MHz, CDCl3) δ 8.05–8.00 (m,
1H), 7.68–7.63 (m, 3H), 7.51–7.49 (m, 1H), 6.59 (d, J = 2.3 Hz, 1H),
6.57 (d, J = 2.2 Hz, 1H), 4.24 (s, 1H), 3.84 (s, 1H), 3.47–3.43 (m, 1H),
3.38 (d, J = 9.7 Hz, 1H), 3.24–3.20 (m, 1H), 2.66 (dtd, J = 14.3, 6.9,
5.6, 3.5 Hz, 3H), 2.14 (d, J = 10.0 Hz, 1H), 2.04–2.00 (m, 2H),
1.14–1.10 (m, 3H); 13C NMR (101 MHz, CDCl3) δ 144.3, 143.2, 132.6,
132.3, 128.4, 122.4, 121.6, 115.1, 113.0, 110.1, 61.2, 57.0, 56.5, 50.8,
46.9, 36.7, 13.3.
4.1.28. Tert-butyl 4-(4-((6-(1H-indazol-6-yl)imidazo[1,2-a]pyrazin-8-yl)
amino)phenyl)piperazine-1-carboxylate (12b)
White solid; 24% yield; 1H NMR (400 MHz, DMSO‑d6) δ 13.20 (s,
1H), 9.58–9.56 (m, 1H), 8.67 (d, J = 1.6 Hz, 1H), 8.18 (s, 1H),
8.10–8.08 (m, 1H), 8.05–8.02 (m, 2H), 7.99 (d, J = 1.5 Hz, 1H), 7.83
(d, J = 8.5 Hz, 1H), 7.72 (dt, J = 8.5, 1.6 Hz, 1H), 7.64 (s, 1H),
7.04–7.00 (m, 2H), 3.49 (t, J = 4.8 Hz, 4H), 3.10–3.06 (m, 4H), 1.43
(d, J = 1.6 Hz, 9H); 13C NMR (101 MHz, DMSO‑d6) δ 154.33, 146.89,
145.29, 140.95, 136.87, 135.63, 133.92, 133.27, 132.79, 132.59,
123.03, 121.60, 121.13, 118.75, 116.92, 109.19, 107.56, 79.45, 50.00,
49.64, 28.54.
4.1.23. 6-Bromo-N-(4-((1S,4S)-5-isopropyl-2,5-diazabicyclo[2.2.1]
heptan-2-yl)phenyl)imidazo[1,2-a]pyrazin-8-amine (11e)
White solid; 94% yield; 1H NMR (400 MHz, CDCl3) δ 7.96 (s, 1H),
7.69–7.60 (m, 3H), 7.50 (d, J = 4.4 Hz, 2H), 6.57 (d, J = 8.9 Hz, 2H),
4.32 (d, J = 13.6 Hz, 2H), 3.73–3.54 (m, 2H), 3.42 (d, J = 10.7 Hz,
1H), 3.10 (q, J = 7.4 Hz, 1H), 2.86 (d, J = 10.5 Hz, 1H), 2.59 (d,
J = 10.8 Hz, 1H), 2.23–2.14 (m, 1H), 1.37 (t, J = 5.7 Hz, 6H); 13C
NMR (101 MHz, CDCl3) δ 144.2, 141.6, 132.9, 132.2, 129.9, 122.0,
121.7, 115.2, 113.3, 110.6, 61.9, 56.3, 55.8, 5.74, 36.6, 19.5, 19.3.
4.1.29. 6-(1H-indazol-6-yl)-N-(4-((1R,4R)-5-methyl-2,5-diazabicyclo
[2.2.1]heptan-2-yl)phenyl)imidazo[1,2-a]pyrazin-8-amine (12c)
4.1.24. 6-Bromo-N-(4-((1R,4R)-5-isopropyl-2,5-diazabicyclo[2.2.1]
heptan-2-yl)phenyl)imidazo[1,2-a]pyrazin-8-amine (11f)
White solid; 35% yield; 1H NMR (400 MHz, DMSO‑d6) δ 13.24 (s,
1H), 9.43 (s, 1H), 8.64 (s, 1H), 8.18 (s, 1H), 8.09 (s, 1H), 7.97 (d,
J = 8.7 Hz, 3H), 7.83 (d, J = 8.5 Hz, 1H), 7.72 (d, J = 8.6 Hz, 1H),
7.63 (s, 1H), 6.67 (d, J = 8.5 Hz, 2H), 4.39 (s, 1H), 3.73 (s, 1H), 3.45
(d, J = 9.5 Hz, 1H), 3.26 (d, J = 9.7 Hz, 1H), 2.91 (d, J = 9.6 Hz, 2H),
2.45 (s, 3H), 2.03 (d, J = 10.0 Hz, 1H), 1.90 (d, J = 9.6 Hz, 1H); 13C
NMR (101 MHz, DMSO‑d6) δ 145.4, 143.0, 141.0, 137.0, 135.7, 133.9,
132.7, 130.3, 123.0, 122.2, 121.1, 118.8, 116.8, 113.2, 108.8, 107.6,
White solid; 94% yield; 1H NMR (400 MHz, CDCl3) δ 7.99 (s, 1H),
7.67 (d, J = 4.5 Hz, 3H), 7.51 (d, J = 2.5 Hz, 2H), 6.57 (d, J = 8.8 Hz,
2H), 4.46 (s, 1H), 4.39 (s, 1H), 3.75 (d, J = 10.7 Hz, 1H), 3.68 (dd,
J = 11.2, 2.5 Hz, 1H), 3.45 (q, J = 7.1, 6.1 Hz, 1H), 3.14 (q, J = 5.0,
3.5 Hz, 1H), 2.98 (d, J = 10.4 Hz, 1H), 2.76 (d, J = 10.6 Hz, 1H),
2.28–2.24 (m, 1H), 1.48 (d, J = 6.3 Hz, 6H); 13C NMR (101 MHz,
CDCl3) δ 144.3, 141.5, 132.8, 132.2, 129.9, 122.1, 121.8, 115.3, 113.3,
110.6, 62.0, 56.3, 55.8, 48.8, 36.6, 19.5, 19.3.
64.0, 59.3, 57.6, 52.9, 35.3, 29.5. HRMS (ESI, m/z) calcd for
+
C
25H25N8 [M+H]+, 437.2197, found 437.2192. HPLC purity 97%.
4.1.25. 6-Bromo-N-(4-(4-methylpiperazin-1-yl)phenyl)imidazo[1,2-a]
pyrazin-8-amine (11g)
4.1.30. N-(4-((1R,4R)-5-ethyl-2,5-diazabicyclo[2.2.1]heptan-2-yl)
phenyl)-6-(1H-indazol-6-yl)imidazo[1,2-a]pyrazin-8-amine (12d)
White solid; 24% yield; 1H NMR (400 MHz, DMSO‑d6) δ 13.25 (s,
1H), 9.42 (s, 1H), 8.64 (s, 1H), 8.19 (s, 1H), 8.10 (s, 1H), 7.99–7.96 (m,
3H), 7.83 (d, J = 8.5 Hz, 1H), 7.72 (dd, J = 8.6, 1.5 Hz, 1H), 7.63 (s,
1H), 6.66 (d, J = 8.6 Hz, 2H), 4.37 (s, 1H), 3.81 (s, 1H), 3.43 (d,
J = 9.5 Hz, 1H), 3.22 (d, J = 9.6 Hz, 1H), 2.96 (d, J = 9.7 Hz, 1H),
2.64 (s, 3H), 1.91 (t, J = 13.8 Hz, 2H), 1.00 (t, J = 7.2 Hz, 3H); 13C
NMR (101 MHz, DMSO‑d6) δ 145.4, 143.1, 141.0, 137.1, 135.8, 133.9,
132.6, 130.3, 123.1, 122.2, 121.1, 118.8, 116.9, 113.1, 108.8, 107.6,
White solid; 80% yield; 1H NMR (400 MHz, DMSO‑d6) δ 9.83 (s,
1H), 8.21 (d, J = 1.4 Hz, 1H), 7.94 (s, 1H), 7.83–7.79 (m, 2H), 7.61 (s,
1H), 7.00–6.97 (m, 2H), 3.29 (s, 4H), 3.02 (t, J = 5.0 Hz, 4H), 2.62 (s,
3H); 13C NMR (101 MHz, DMSO‑d6) δ 154.3, 143.1, 142.7, 142.0,
138.5, 121.1, 119.2, 116.5, 115.2, 109.7, 79.3, 51.2, 28.5.
General Procedure for the Preparation of 8a, 12a–12k. Heteroaryl-
boronic ester (1.2 mmol), 7a, 11a–11g (1 mmol), PdCl2(dppf) (82 mg,
0.1 mmol), sodium carbonate(1.5 mL, 1.5 mmol, 1 M aqueous solution),
dissolved in 1,4-dioxane (15 mL) and the mixture was heated at 100 °C
under argon atmosphere overnight. After that, the flask was cooled to
room temperature and the reaction solution was filtered with celite
while washing with dichloromethane. The solvent was then evaporated
and the residue was purified by flash column chromatography to give
the key intermediates 12a–12b and the final products 8a, 12c–12k.
62.2, 57.3, 57.0, 52.9, 48.3, 35.5, 29.5, 13.4. HRMS (ESI, m/z) calcd for
+
C
26H27N8 [M+H]+, 451.2353, found 451.2350. HPLC purity 96%.
4.1.31. 6-(1H-indazol-6-yl)-N-(4-((1S,4S)-5-isopropyl-2,5-diazabicyclo
[2.2.1]heptan-2-yl)phenyl)imidazo[1,2-a]pyrazin-8-amine (12e)
White solid; 25% yield; 1H NMR (400 MHz, DMSO‑d6) δ 13.22 (s,
1H), 9.39 (s, 1H), 8.63 (s, 1H), 8.20 (s, 1H), 8.10 (s, 1H), 7.96 (d,
J = 9.2 Hz, 3H), 7.84 (d, J = 8.5 Hz, 1H), 7.73 (d, J = 8.6 Hz, 1H),
7.63 (s, 1H), 6.63 (d, J = 8.5 Hz, 2H), 4.25 (s, 1H), 3.68 (s, 1H), 3.32
(d, J = 8.9 Hz, 1H), 3.18 (d, J = 9.1 Hz, 1H), 2.99 (d, J = 9.3 Hz, 1H),
2.39 (t, J = 7.0 Hz, 2H), 1.80 (s, 2H), 0.95 (d, J = 6.1 Hz, 3H), 0.91 (d,
J = 6.0 Hz, 3H); 13C NMR (101 MHz, DMSO‑d6) δ 144.89, 142.96,
140.49, 136.60, 135.28, 133.40, 132.18, 132.13, 129.33, 122.53,
121.70, 120.58, 118.33, 116.31, 112.46, 108.23, 107.08, 57.8, 57.0,
4.1.26. 6-(3-Aminophenyl)-N-(4-morpholinophenyl)imidazo[1,2-a]
pyrazin-8-amine (8a)
Off-white solid; 54% yield; 1H NMR (400 MHz, DMSO‑d6) δ 9.45 (s,
1H), 8.39 (s, 1H), 8.05 (d, J = 8.6 Hz, 2H), 7.98 (s, 1H), 7.61 (s, 1H),
7.22 (d, J = 2.2 Hz, 1H), 7.12 (d, J = 4.7 Hz, 2H), 6.99 (d, J = 8.6 Hz,
2H), 6.59 (dt, J = 6.1, 2.7 Hz, 1H), 5.42–5.13 (m, 2H), 3.76–3.72 (m,
4H), 3.07 (t, J = 4.7 Hz, 4H); 13C NMR (101 MHz, DMSO‑d6) δ 149.4,
146.8, 145.1, 138.4, 137.6, 133.2, 132.6, 132.6, 129.6, 121.2, 116.7,
116.0, 114.3, 113.8, 111.9, 108.3, 66.6, 49.6. HRMS (ESI, m/z) calcd
for C22H23N6O+ [M+H]+, 387.1928, found 387.1930. HPLC purity
96%.
55.4, 51.2, 50.2, 36.6, 22.6, 21.9. HRMS (ESI, m/z) calcd for
+
C
27H29N8 [M+H]+, 465.2510, found 465.2511. HPLC purity 97%.
4.1.32. 6-(1H-indazol-6-yl)-N-(4-((1R,4R)-5-isopropyl-2,5-diazabicyclo
[2.2.1]heptan-2-yl)phenyl)imidazo[1,2-a]pyrazin-8-amine (12f)
4.1.27. Tert-butyl
(1R,4R)-5-(4-((6-(1H-indazol-6-yl)imidazo[1,2-a]
pyrazin-8-yl)amino)phenyl)-2,5-diazabicyclo[2.2.1]heptane-2-carboxylate
(12a)
White solid; 25% yield; 1H NMR (400 MHz, DMSO‑d6) δ 13.23 (s,
1H), 9.40 (s, 1H), 8.64 (s, 1H), 8.15 (d, J = 39.4 Hz, 2H), 7.97 (d,
J = 9.5 Hz, 3H), 7.87–7.61 (m, 3H), 6.64 (d, J = 8.5 Hz, 2H), 4.28 (s,
1H), 3.73 (s, 1H) , 3.36 (s, 2H), 3.26–3.00 (m, 2H), 2.43 (s, 1H), 1.83 (s,
White solid; 50% yield; 1H NMR (400 MHz, DMSO‑d6) δ 13.23 (s,
1H), 9.44 (s, 1H), 8.66 (s, 1H), 8.20 (s, 1H), 8.12 (s, 1H), 8.00 (s, 2H),
10

C. Wang, et al.
BioorganicChemistry95(2020)103547
2H), 1.07–0.85 (m, 6H); 13C NMR (101 MHz, DMSO‑d6) δ 145.4, 143.4,
141.0, 137.1, 135.8, 133.9, 132.7, 132.6, 129.9, 123.0, 122.2, 121.1,
118.8, 116.8, 113.0, 108.8, 107.6, 67.5, 57.4, 56.0, 51.6, 50.8, 37.1,
23.0, 22.0. HRMS (ESI, m/z) calcd for C27H29N8+ [M+H]+, 465.2510,
found 465.2506. HPLC purity 98%.
(1.56 mmol), EDCI (294 mg, 1.56 mmol), HOBt (298 mg, 1.56 mmol),
DIEA (466 μL, 2.08 mmol) were added to DMF (15 mL). After 20 min,
8a (400 mg, 1.04 mmol) was added to the reaction mixture and stirred
overnight at room temperature. Upon completion of the reaction, water
(40 mL) was added to the flask and the aqueous phase was extracted
with ethyl acetate (60 mL). Then the organic phase was separated and
dried over anhydrous sodium sulfate. The residue is purified by column
chromatography to obtain the key intermediates 9a–9c and final pro-
ducts 9d–9h.
4.1.33. 6-(1H-indazol-6-yl)-N-(4-(4-methylpiperazin-1-yl)phenyl)imidazo
[1,2-a]pyrazin-8-amine (12g)
White solid; 31% yield; 1H NMR (400 MHz, DMSO‑d6) δ 13.22 (s,
1H), 9.53 (s, 1H), 8.67 (s, 1H), 8.21 (s, 1H), 8.09 (s, 1H), 8.05–7.98 (m,
3H), 7.84 (d, J = 8.5 Hz, 1H), 7.73 (d, J = 8.5 Hz, 1H), 7.64 (s, 1H),
6.99 (d, J = 8.6 Hz, 2H), 3.15 (t, J = 4.8 Hz, 4H), 2.61–2.53 (m, 4H),
2.29 (s, 3H); 13C NMR (101 MHz, DMSO‑d6) δ 146.9, 145.3, 141.0,
136.9, 135.6, 133.9, 132.8, 132.7, 132.6, 123.0, 121.6, 121.1, 118.8,
4.1.38. Tert-butyl (3-((3-(8-((4-morpholinophenyl)amino)imidazo[1,2-a]
pyrazin-6-yl)phenyl)amino)-3-oxopropyl)carbamate (9a)
White solid; 99% yield; 1H NMR (400 MHz, DMSO‑d6) δ 10.10 (s,
1H), 9.62 (s, 1H), 8.55 (s, 1H), 8.52 (s, 1H), 8.10 (d, J = 8.6 Hz, 2H),
8.04 (d, J = 1.2 Hz, 1H), 7.70–7.68 (m, 1H), 7.63 (dt, J = 7.6, 1.5 Hz,
1H), 7.45 (d, J = 8.1 Hz, 1H), 7.39 (t, J = 7.8 Hz, 1H), 7.05 (d,
J = 8.6 Hz, 2H), 6.91 (t, J = 5.6 Hz, 1H), 3.77 (t, J = 4.8 Hz, 4H), 3.29
(q, J = 6.8 Hz, 2H), 3.11 (t, J = 4.8 Hz, 4H), 2.54 (t, J = 7.3 Hz, 2H),
1.38 (s, 9H); 13C NMR (101 MHz, DMSO‑d6) δ 170.0, 156.0, 144.9,
142.9, 140.2, 138.0, 137.2, 132.1, 132.0, 129.4, 121.3, 120.6, 119.2,
117.3, 117.1, 116.3, 108.7, 78.1, 66.6, 49.7, 37.2, 37.0, 28.7.
116.9, 116.2, 109.1, 107.6, 54.9, 48.9, 45.8. HRMS (ESI, m/z) calcd for
+
C
24H25N8 [M+H]+, 425.2197, found 425.2196. HPLC purity 97%.
4.1.34. 4-(8-((4-((1R,4R)-5-isopropyl-2,5-diazabicyclo[2.2.1]heptan-2-
yl)phenyl)amino)imidazo[1,2-a]pyrazin-6-yl)furan-2-carboxamide (12h)
Gray solid; 25% yield; 1H NMR (400 MHz, DMSO‑d6) δ 9.44 (s, 1H),
8.35 (s, 1H), 8.18 (s, 1H), 8.01–7.92 (m, 3H), 7.89 (s, 1H), 7.61 (s, 1H),
7.53 (s, 2H), 6.71 (d, J = 8.4 Hz, 2H), 4.56 (s, 2H), 3.60 (d,
J = 18.8 Hz, 2H), 3.42–3.23 (m, 2H), 2.11 (s, 2H), 1.98 (s, 1H), 1.20
(m, 6H); 13C NMR (101 MHz, DMSO‑d6) δ 159.2, 148.9, 145.1, 141.7,
132.0, 130.5, 130.2, 126.5, 121.5, 116.2, 112.9, 111.4, 107.5, 99.5,
59.7, 56.9, 56.4, 55.7, 54.6, 20.7, 14.1. HRMS (ESI, m/z) calcd for
C25H28N7O2+ [M+H]+, 458.2299, found 458.2295. HPLC purity 97%.
4.1.39. Tert-butyl (4-((3-(8-((4-morpholinophenyl)amino)imidazo[1,2-a]
pyrazin-6-yl)phenyl)amino)-4-oxobutyl)carbamate (9b)
White solid; 99% yield; 1H NMR (400 MHz, DMSO‑d6) δ 10.03 (s,
1H), 9.51 (s, 1H), 8.53 (s, 1H), 8.50 (s, 1H), 8.09 (d, J = 8.9 Hz, 2H),
8.01 (d, J = 1.1 Hz, 1H), 7.63–7.61 (m, 2H), 7.44–7.36 (m, 2H), 7.01
(d, J = 2.1 Hz, 1H), 7.00 (d, J = 2.2 Hz, 1H), 6.89 (t, J = 5.9 Hz, 1H),
3.76 (t, J = 4.7 Hz, 4H), 3.35 (s, 2H), 3.10–3.07 (m, 4H), 3.03–2.99 (m,
2H), 2.36 (t, J = 7.4 Hz, 2H), 1.77–1.72 (m, 2H), 1.38 (s, 9H); 13C NMR
(101 MHz, DMSO‑d6) δ 171.4, 156.1, 146.9, 145.2, 140.2, 138.16,
136.8, 133.1, 132.7, 132.6, 129.4, 121.4, 120.5, 119.1, 117.3, 117.0,
116.0, 108.6, 77.9, 66.7, 49.6, 34.2, 28.7, 26.0, 22.6.
4.1.35. N-(3-(8-((4-((1R,4R)-5-isopropyl-2,5-diazabicyclo[2.2.1]heptan-
2-yl)phenyl)amino)imidazo[1,2-a]pyrazin-6-yl)phenyl)acetamide (12i)
Gray solid; 26% yield; 1H NMR (400 MHz, DMSO‑d6) δ 10.10 (d,
J = 7.3 Hz, 1H), 9.44 (d, J = 7.1 Hz, 1H), 8.46 (d, J = 7.5 Hz, 1H),
8.38 (dd, J = 14.4, 7.2 Hz, 1H), 8.05 (q, J = 7.6 Hz, 3H), 7.63 (t,
J = 7.1 Hz, 2H), 7.49 (q, J = 9.0, 8.4 Hz, 1H), 7.39 (q, J = 7.9 Hz, 1H),
6.75 (t, J = 8.2 Hz, 2H), 4.63 (dd, J = 16.4, 7.1 Hz, 2H), 3.53–3.44 (m,
2H), 3.36 (d, J = 7.2 Hz, 3H), 2.28–2.15 (m, 2H), 2.11 (d, J = 7.4 Hz,
3H), 1.37–1.22 (m, 6H); 13C NMR (101 MHz, DMSO‑d6) δ 168.3, 144.8,
141.6, 139.8, 137.8, 136.4, 132.1, 132.0, 130.8, 128.9, 121.5, 120.3,
118.7, 116.6, 116.5, 113.1, 108.0, 61.9, 56.8, 56.1, 55.5, 55.3, 24.1,
4.1.40. Tert-butyl (5-((3-(8-((4-morpholinophenyl)amino)imidazo[1,2-a]
pyrazin-6-yl)phenyl)amino)-5-oxopentyl)carbamate (9c)
White solid; 93% yield; 1H NMR (400 MHz, DMSO‑d6) δ 10.03 (d,
J = 11.3 Hz, 1H), 9.58 (d, J = 11.1 Hz, 1H), 8.55–8.47 (m, 2H),
8.14–8.03 (m, 3H), 7.71–7.61 (m, 2H), 7.43 (td, J = 13.4, 6.2 Hz, 2H),
7.08–7.00 (m, 2H), 6.90–6.80 (m, 1H), 3.81–3.73 (m, 4H), 3.11 (dt,
J = 13.9, 4.8 Hz, 4H), 2.96 (dt, J = 13.4, 6.7 Hz, 2H), 2.51 (d,
J = 11.3 Hz, 2H), 2.38 (dt, J = 14.1, 7.2 Hz, 2H), 1.64 (dt, J = 14.5,
7.1 Hz, 2H), 1.46 (d, J = 7.7 Hz, 2H), 1.38 (d, J = 12.9 Hz, 9H); 13C
NMR (101 MHz, DMSO‑d6) δ 171.7, 156.1, 146.7, 145.0, 140.3, 138.1,
137.35, 137.0, 136.9, 132.3, 129.4, 121.4, 120.5, 119.2, 117.2, 117.0,
116.2, 108.7, 77.8, 66.6, 49.7, 40.1, 36.5, 29.6, 28.7, 22.9.
19.0, 17.4. HRMS (ESI, m/z) calcd for
C
28H32N7O+ [M+H]+
,
482.2663, found 482.2662. HPLC purity 97%.
4.1.36. 4-(8-((4-((1S,4S)-5-isopropyl-2,5-diazabicyclo[2.2.1]heptan-2-
yl)phenyl)amino)imidazo[1,2-a]pyrazin-6-yl)furan-2-carboxamide (12j)
Gray solid; 37% yield; 1H NMR (400 MHz, DMSO‑d6) δ 9.45 (s, 1H),
8.36 (s, 1H), 8.18 (s, 1H), 7.99–7.94 (m, 3H), 7.90 (s, 1H), 7.61 (s, 1H),
7.54 (s, 2H), 6.73 (d, J = 8.5 Hz, 2H), 4.61 (s, 1H), 3.61 (s, 1H), 3.46
(d, J = 11.6 Hz, 2H), 3.37 (s, 3H), 2.17 (d, J = 34.9 Hz, 2H), 1.24 (m,
6H); 13C NMR (101 MHz, DMSO‑d6) δ 159.2, 148.9, 145.1, 141.7,
132.0, 130.7, 130.2, 126.5, 121.5, 116.2, 113.0, 111.4, 107.6, 99.5,
61.7, 56.7, 55.5, 54.9, 53.0, 30.0, 19.0. HRMS (ESI, m/z) calcd for
4.1.41. Methyl
3-((3-(8-((4-morpholinophenyl)amino)imidazo[1,2-a]
pyrazin-6-yl)phenyl)amino)-3-oxopropanoate (9d)
White solid; 75% yield; 1H NMR (400 MHz, DMSO‑d6) δ 10.34 (s,
1H), 9.53 (s, 1H), 8.52 (s, 1H), 8.48 (t, J = 1.8 Hz, 1H), 8.08 (d,
J = 8.7 Hz, 2H), 8.01 (s, 1H), 7.67 (dt, J = 7.1, 1.7 Hz, 1H), 7.63 (s,
1H), 7.46–7.40 (m, 2H), 7.00 (d, J = 9.0 Hz, 2H), 3.75 (t, J = 4.7 Hz,
4H), 3.68 (s, 3H), 3.56 (s, 2H), 3.07 (t, J = 4.8 Hz, 4H); 13C NMR
(101 MHz, DMSO‑d6) δ 168.6, 164.6, 146.9, 145.3, 139.7, 138.3, 136.6,
133.1, 132.7, 132.6, 129.5, 121.4, 121.0, 119.2, 117.4, 117.0, 116.0,
C
25H28N7O2+ [M+H]+, 458.2299, found 458.2296. HPLC purity 97%.
4.1.37. N-(3-(8-((4-((1S,4S)-5-isopropyl-2,5-diazabicyclo[2.2.1]heptan-
2-yl)phenyl)amino)imidazo[1,2-a]pyrazin-6-yl)phenyl)acetamide (12k)
White solid; 31% yield; 1H NMR (400 MHz, DMSO‑d6) δ 10.09 (s,
1H), 9.37 (s, 1H), 8.43 (s, 1H), 8.35 (s, 1H), 8.03–7.95 (m, 3H),
7.66–7.59 (m, 2H), 7.52 (d, J = 8.0 Hz, 1H), 7.39 (t, J = 7.9 Hz, 1H),
6.64 (d, J = 8.4 Hz, 2H), 4.28 (s, 1H), 3.80 (s, 1H), 3.35 (d, J = 9.3 Hz,
1H), 3.19 (d, J = 9.5 Hz, 1H), 3.05 (d, J = 9.6 Hz, 1H), 2.52 (s, 2H),
2.10 (s, 3H), 1.84 (s, 2H), 1.04–0.91 (m, 6H); 13C NMR (101 MHz,
DMSO‑d6) δ 168.3, 144.8, 142.6, 139.7, 137.9, 136.5, 132.1, 132.1,
129.7, 128.9, 121.5, 120.3, 118.7, 116.6, 116.4, 112.7, 107.8, 58.5,
56.7, 55.6, 51.2, 51.0, 30.9, 26.3, 24.1. HRMS (ESI, m/z) calcd for
+
108.7, 66.7, 52.4, 49.6, 44.0. HRMS (ESI, m/z) calcd for C26H27N6O4
[M+H]+, 487.2088, found 487.2082. HPLC purity 96%.
4.1.42. Methyl
4-((3-(8-((4-morpholinophenyl)amino)imidazo[1,2-a]
pyrazin-6-yl)phenyl)amino)-4-oxobutanoate (9e)
White solid; 75% yield; 1H NMR (400 MHz, DMSO‑d6) δ 10.14 (s,
1H), 9.51 (s, 1H), 8.56 (s, 1H), 8.51 (s, 1H), 8.10 (d, J = 8.6 Hz, 2H),
8.01 (s, 1H), 7.63 (d, J = 6.4 Hz, 2H), 7.43–7.37 (m, 2H), 7.01 (d,
J = 8.5 Hz, 2H), 3.76 (t, J = 4.6 Hz, 4H), 3.61 (s, 3H), 3.08 (t,
J = 4.8 Hz, 4H), 2.67 (d, J = 3.6 Hz, 4H); 13C NMR (101 MHz,
C
28H32N7O+ [M+H]+, 482.2663, found 482.2663. HPLC purity 97%.
General Procedure for the Preparation of 9a–9h. Substituted acid
11

C. Wang, et al.
BioorganicChemistry95(2020)103547
DMSO‑d6) δ 173.4, 170.4, 146.9, 145.2, 140.2, 138.2, 136.7, 133.13,
4.1.47. 3-Amino-N-(3-(8-((4-morpholinophenyl)amino)imidazo[1,2-a]
132.7, 132.6, 129.4, 121.3, 120.4, 118.9, 117.1, 117.0, 116.1, 108.7,
pyrazin-6-yl)phenyl)propanamide (10a)
+
66.7, 51.9, 49.6, 31.3, 28.9. HRMS (ESI, m/z) calcd for C27H29N6O4
White solid; 96% yield; 1H NMR (400 MHz, DMSO‑d6) δ 10.89 (s,
1H), 10.57 (s, 1H), 8.78 (s, 1H), 8.67 (s, 1H), 8.30 (d, J = 1.7 Hz, 1H),
8.28–8.22 (m, 3H), 8.08 (s, 3H), 7.67 (d, J = 7.7 Hz, 1H), 7.54 (d,
J = 8.3 Hz, 2H), 7.47 (t, J = 7.8 Hz, 1H), 3.96 (s, 4H), 3.38 (s, 4H),
3.19–3.12 (m, 2H), 2.85 (t, J = 6.9 Hz, 2H); 13C NMR (101 MHz,
DMSO‑d6) δ 169.1, 141.9, 140.4, 140.2, 136.4, 136.4, 129.8, 127.8,
[M+H]+, 501.2245, found 501.2247. HPLC purity 97%.
4.1.43. 4-Isocyano-N-(3-(8-((4-morpholinophenyl)amino)imidazo[1,2-a]
pyrazin-6-yl)phenyl)benzamide (9f)
Light green solid; 88% yield; 1H NMR (400 MHz, DMSO‑d6) δ 10.63
(s, 1H), 9.55 (s, 1H), 8.66 (s, 1H), 8.54 (s, 1H), 8.18 (d, J = 8.0 Hz, 2H),
8.07 (t, J = 8.1 Hz, 4H), 8.03 (s, 1H), 7.75 (d, J = 7.8 Hz, 1H), 7.65 (d,
J = 6.4 Hz, 2H), 7.48 (t, J = 7.9 Hz, 1H), 7.00 (d, J = 8.6 Hz, 2H),
3.73 (t, J = 4.5 Hz, 4H), 3.05 (d, J = 4.8 Hz, 4H); 13C NMR (101 MHz,
DMSO‑d6) δ 164.6, 146.8, 145.2, 139.6, 139.4, 138.2, 136.7, 133.1,
133.0, 132.6, 129.4, 129.0, 121.7, 121.6, 120.7, 118.8, 118.6, 117.0,
116.0, 114.4, 108.8, 66.6, 49.6. HRMS (ESI, m/z) calcd for
124.7, 121.1, 120.9, 120.7, 120.3, 120.2, 117.8, 117.6, 109.3, 66.8,
+
65.0, 35.4, 33.8. HRMS (ESI, m/z) calcd for C25H28N7O2 [M+H]+
,
458.2299, found 458.2296. HPLC purity 98%.
4.1.48. 4-Amino-N-(3-(8-((4-morpholinophenyl)amino)imidazo[1,2-a]
pyrazin-6-yl)phenyl)butanamide (10b)
White solid; 98% yield; 1H NMR (400 MHz, DMSO‑d6) δ 11.02 (s,
1H), 10.49 (s, 1H), 8.80 (s, 1H), 8.65 (s, 1H), 8.34 (d, J = 1.8 Hz, 1H),
8.29–8.24 (m, 3H), 8.17 (s, 3H), 7.65 (d, J = 7.8 Hz, 2H), 7.54 (dd,
J = 7.9, 2.0 Hz, 1H), 7.45 (t, J = 7.9 Hz, 1H), 4.00 (t, J = 4.4 Hz, 4H),
3.44 (s, 4H), 2.89 (q, J = 6.7 Hz, 2H), 2.55 (t, J = 7.2 Hz, 2H),
2.01–1.94 (m, 2H); 13C NMR (101 MHz, DMSO‑d6) δ 171.1, 142.5,
142.0, 140.4, 136.5,136.4, 129.7, 127.9, 124.8, 121.0, 120.9, 120.7,
C
30H26N7O2+ [M+H]+, 516.2142, found 516.2142. HPLC purity 96%.
4.1.44. 4-((3-(8-((4-Morpholinophenyl)amino)imidazo[1,2-a]pyrazin-6-
yl)phenyl)amino)-4-oxobutanoic acid (9g)
White solid; 60% yield; 1H NMR (400 MHz, DMSO‑d6) δ 12.17 (s,
1H), 10.11 (s, 1H), 9.50 (s, 1H), 8.59 (s, 1H), 8.50 (s, 1H), 8.11 (d,
J = 8.7 Hz, 2H), 8.01 (s, 1H), 7.63 (d, J = 5.9 Hz, 2H), 7.43–7.37 (m,
2H), 7.01 (d, J = 8.8 Hz, 2H), 3.75 (t, J = 4.7 Hz, 4H), 3.08 (t,
J = 4.8 Hz, 4H), 2.62 (dt, J = 11.7, 5.2 Hz, 4H); 13C NMR (101 MHz,
DMSO‑d6) δ 174.4, 170.7, 146.9, 145.2, 140.3, 138.2, 136.7, 133.1,
132.7, 132.6, 129.4, 121.3, 120.4, 118.9, 117.1, 117.0, 116.0, 108.6,
120.2, 117.8, 117.5, 115.2, 109.2, 65.0, 52.5, 38.9, 33.5, 23.5. HRMS
+
(ESI, m/z) calcd for
C
26H30N7O2
[M+H]+
, 472.2455, found
472.2457. HPLC purity 99%.
+
66.7, 49.6, 31.5, 29.2. HRMS (ESI, m/z) calcd for C26H27N6O4 [M
4.1.49. 5-Amino-N-(3-(8-((4-morpholinophenyl)amino)imidazo[1,2-a]
pyrazin-6-yl)phenyl)pentanamide (10c)
+H]+, 487.2088, found 487.2084. HPLC purity 96%.
White solid; 98% yield; 1H NMR (400 MHz, DMSO‑d6) δ 11.16 (s,
1H), 10.47 (s, 1H), 8.85 (s, 1H), 8.67 (s, 1H), 8.39 (d, J = 1.8 Hz, 1H),
8.33 (d, J = 1.7 Hz, 1H), 8.28 (d, J = 8.7 Hz, 2H), 8.15 (d, J = 6.0 Hz,
2H), 7.83–7.70 (m, 2H), 7.64 (d, J = 7.7 Hz, 1H), 7.58–7.53 (m, 1H),
7.45 (t, J = 7.8 Hz, 1H), 4.05 (t, J = 4.6 Hz, 4H), 3.50 (t, J = 4.6 Hz,
4H), 2.85 (h, J = 7.0, 6.1 Hz, 2H), 2.47 (t, J = 7.0 Hz, 2H), 1.71 (dq,
J = 23.3, 8.0 Hz, 4H); 13C NMR (101 MHz, DMSO‑d6) δ 171.7, 141.7,
140.8, 140.6, 139.2, 136.3, 129.7, 127.5, 124.2, 120.92, 120.7, 120.6,
120.3, 120.2, 117.9, 117.4, 109.3, 66.8, 64.7, 39.0, 36.20 27.04 22.5.
4.1.45. 5-((3-(8-((4-Morpholinophenyl)amino)imidazo[1,2-a]pyrazin-6-
yl)phenyl)amino)-5-oxopentanoic acid (9h)
White solid; 60% yield; 1H NMR (400 MHz, DMSO‑d6) δ 12.14 (s,
1H), 10.05 (s, 1H), 9.52 (s, 1H), 8.59 (s, 1H), 8.51 (s, 1H), 8.11 (d,
J = 8.4 Hz, 2H), 8.01 (s, 1H), 7.63 (s, 2H), 7.39 (d, J = 7.7 Hz, 2H),
7.01 (d, J = 8.5 Hz, 2H), 3.75 (t, J = 4.5 Hz, 4H), 3.07 (t, J = 4.6 Hz,
4H), 2.43 (t, J = 7.4 Hz, 2H), 2.33 (t, J = 7.4 Hz, 2H), 1.86 (q,
J = 7.4 Hz, 2H); 13C NMR (101 MHz, DMSO‑d6) δ 174.7, 171.3, 146.9,
145.2, 140.2, 138.2, 136.8, 133.1, 132.7, 132.6, 129.4, 121.4, 120.4,
HRMS (ESI, m/z) calcd for C27H32N7O2 [M+H]+, 486.2612, found
+
119.1, 117.4, 117.0, 116.0, 108.6, 66.6, 49.6, 35.8, 33.5, 20.9. HRMS
486.2610. HPLC purity 97%.
+
(ESI, m/z) calcd for
C27H29N6O4
[M+H]+
, 501.2245, found
501.2236. HPLC purity 96%.
4.1.50. N-(4-((1R,4R)-2,5-diazabicyclo[2.2.1]heptan-2-yl)phenyl)-6-
(1H-indazol-6-yl)imidazo[1,2-a]pyrazin-8-amine (13a)
4.1.46. 1-((3-(8-((4-Morpholinophenyl)amino)imidazo[1,2-a]pyrazin-6-
yl)phenyl)carbamoyl)cyclopropane-1-carboxylic acid (9i)
White solid; 91% yield; 1H NMR (400 MHz, DMSO‑d6) δ 10.85 (s,
1H), 9.91 (s, 1H), 9.18 (s, 1H), 8.88 (s, 1H), 8.32 (d, J = 4.0 Hz, 2H),
8.21 (s, 1H), 8.13 (s, 1H), 7.98 (d, J = 8.2 Hz, 2H), 7.88 (d, J = 8.3 Hz,
1H), 7.71 (d, J = 8.3 Hz, 1H), 6.78 (d, J = 8.4 Hz, 2H), 4.63 (s, 1H),
4.43 (s, 1H), 3.61 (d, J = 10.0 Hz, 1H), 3.40 (d, J = 10.1 Hz, 1H), 3.22
(d, J = 21.1 Hz, 2H), 2.13 (d, J = 10.3 Hz, 1H), 1.98 (d, J = 10.3 Hz,
1H); 13C NMR (101 MHz, DMSO‑d6) δ146.5, 142.4, 141.0, 140.3, 136.3,
133.4, 129.4, 126.6, 123.1, 122.9, 121.2, 121.0, 118.3, 117.2, 113.3,
Dichlorosulfoxide (40 μL, 0.52 mmol) was added dropwise to a
solution of 1,1-cyclopropanedicarboxylic acid (68 mg, 0.52 mmol) in
isopropyl acetate at 0 °C, and after stirring for 6 h, DIEA (130 μL,
0.78 mmol), 8a (100 mg, 0.26 mmol) was added and reacted at room
temperature for two hours. Thereafter, water (20 mL) was added to the
reaction mixture to quench the reaction and extracted with ethyl
acetate (35 mL). The organic phase is dried over anhydrous sodium
sulfate and purified by silica gel column chromatography to give the
final product 9i in 61% yield as white solid. 1H NMR (400 MHz,
DMSO‑d6) δ 11.00 (s, 1H), 10.83 (s, 1H), 8.81 (s, 1H), 8.57 (s, 1H), 8.32
(s, 1H), 8.27 (s, 1H), 8.22 (d, J = 10.6 Hz, 1H), 8.19 (s, 2H), 7.70 (d,
J = 7.5 Hz, 1H), 7.50 (dt, J = 15.5, 8.0 Hz, 4H), 3.98–3.92 (m, 4H),
3.38 (s, 4H), 1.55–1.51 (m, 2H), 1.49 (d, J = 3.8 Hz, 2H); 13C NMR
(101 MHz, DMSO‑d6) δ 173.9, 171.9, 167.0, 161.2, 157.5, 141.5, 139.8,
139.4, 136.1, 129.4, 127.4, 124.3, 120.8, 120.3, 120.0, 117.3, 117.1,
108.0, 57.3, 55.1, 52.2, 48.7, 35.6. HRMS (ESI, m/z) calcd for
+
C
24H23N8 [M+H]+, 423.2040, found 423.2040. HPLC purity 96%.
4.1.51. 6-(1H-indazol-6-yl)-N-(4-(piperazin-1-yl)phenyl)imidazo[1,2-a]
pyrazin-8-amine (13b)
White solid; 95% yield; 1H NMR (400 MHz, DMSO‑d6) δ 11.03 (s,
1H), 9.58 (s, 2H), 8.96 (s, 1H), 8.34 (d, J = 4.5 Hz, 2H), 8.24 (s, 1H),
8.14 (s, 1H), 8.08 (d, J = 8.4 Hz, 2H), 7.89 (d, J = 8.3 Hz, 1H), 7.73 (d,
J = 8.4 Hz, 1H), 7.20 (d, J = 8.5 Hz, 2H), 3.46 (t, J = 4.6 Hz, 4H),
3.28 (s, 4H); 13C NMR (101 MHz, DMSO‑d6) δ 145.74, 141.52, 141.41,
140.82, 133.83, 133.74, 133.24, 127.16, 123.62, 123.46, 121.61,
120.99, 118.77, 117.79, 117.61, 109.00, 108.55, 46.74, 42.80. HRMS
+
108.6, 64.8, 51.5, 28.5, 17.5. HRMS (ESI, m/z) calcd for C27H27N6O4
[M+H]+, 499.2088, found 499.2089. HPLC purity 96%.
General Procedure for the Preparation of 10a–10c, 13a–13b.
Compounds 9a–9c and 12a–12b (0.2 mmol) were placed in a 100 mL
flask and then a 1,4-dioxane solution (4 M HCl gas, 10 mL) was slowly
added dropwise at 0 °C. After reacting for 3 h, it was filtered to give a
white solid and dried to give the final products 10a–10c, 13a–13b.
(ESI, m/z) calcd for C23H23N8 [M+H]+, 411.2040, found 411.2040.
+
HPLC purity 95%.
12

C. Wang, et al.
BioorganicChemistry95(2020)103547
4.1.52. 3-((3-(8-((4-Morpholinophenyl)amino)imidazo[1,2-a]pyrazin-6-
yl)phenyl)amino)propan-1-ol (10d)
according to federal and institutional guidelines and regulations.
Inhibition rate of tumor growth was calculated using the following
formula: 100 × {1 − [(tumor volumefinal − tumor volumeinitial) for
12f-treated group]/[(tumor volumefinal − tumor volumeinitial) for the
vehicle-treated group]}.
9 g (200 mg, 0.41 mmol) and LiAlH4 (32 mg, 0.82 mmol) were
placed in a two-necked flask and degassed with argon for 5 min, then
slowly added to the anhydrous tetrahydrofuran (8 mL) solution with a
syringe at 0 °C. After 10 h, the reaction was quenched by water (10 mL)
and extracted with ethyl acetate (20 mL). The upper liquid was dried
over anhydrous sodium sulfate and then purified by chromatography to
give the final product 10d in 36% yield as white solid. 1H NMR
(400 MHz, DMSO‑d6) δ 9.44 (s, 1H), 8.46 (s, 1H), 8.05 (d, J = 8.8 Hz,
2H), 7.97 (s, 1H), 7.61 (s, 1H), 7.26 (s, 1H), 7.16–7.10 (m, 2H), 6.96 (d,
J = 8.7 Hz, 2H), 6.60 (dd, J = 7.4, 2.1 Hz, 1H), 5.70 (t, J = 5.5 Hz,
1H), 4.46 (t, J = 5.1 Hz, 1H), 3.75 (t, J = 4.7 Hz, 4H), 3.45 (t,
J = 5.8 Hz, 2H), 3.12 (t, J = 6.2 Hz, 2H), 3.07 (t, J = 4.8 Hz, 4H), 1.65
(dd, J = 10.2, 4.9 Hz, 2H), 1.57 (dd, J = 8.6, 5.8 Hz, 2H); 13C NMR
(101 MHz, DMSO‑d6) δ 149.3, 146.4, 144.6, 137.8, 137.0, 132.7, 132.1,
127.0, 120.8, 116.2, 115.4, 112.7, 112.6, 108.6, 107.8, 66.1, 60.6,
4.2.6. Procedure for lipophilicity determination
LogP and LogD7.4 were determined by using the well-known “shake-
flask” method. The concentrations of the test compound in the two
phases were determined by HPLC (Shimadzu Prominence-i LC-2030C
3D system; column, InertSustain C18, 4.6 mm × 250 mm, 5 μm; mobile
phase, gradient elution of methanol/H2O; low rate, 1.0 mL/min; UV
wavelength, 190–800 nm; temperature, 40 °C; injection volume, 10 μL).
The LogP/D7.4 was calculated by employing the following equation:
COCT CH O
2
LogP = (
)
CH O
2
+
49.1, 42.9, 30.3, 25.4. HRMS (ESI, m/z) calcd for C26H31N6O2 [M
where COCTis the concentration of the compounds initially in n-octanol
and CH O is the compound’s concentration in aqueous phase after
mixing.2
+H]+, 459.2503, found 459.2499. HPLC purity 96%.
4.2. Biology
COCT CPBS
LogD7.4 = (
)
CPBS
4.2.1. Materials and methods
where COCTis the concentration of the compounds initially in n-octanol
and CPBS is the compound’s concentration in buffered liquid phase after
mixing.
Cancer cell lines were purchased from ATCC. All the cell lines were
recently authenticated by cellular morphology and short tandem repeat
analysis at Microread Inc. (Beijing, China; May 2014) according to the
guideline from ATCC. DMSO as vehicle, entospletinib are purchased
from commercial supplier Solarbio Science & Technology and Ark
Pharmaceutical Co., Ltd.
4.2.7. Aqueous solubility determination
One milligram of 12f or entospletinib was dissolved in 250 μL
DMSO and then diluted into different concentrations of sample with a
large volume of PBS in pH 7.4. The samples were sonicated, vortexed,
and equilibrated by stirring at 100 rpm for 24 h and subsequently left
standing for an additional 24 h at room temperature. After the equili-
bration was completed, the entire sample volume was filtered manually
through a 2.5 μm glass fiber syringe filter. Finally, the filtrate was
analyzed by HPLC for the solubility of compounds, and the solubility of
the samples was determined in triplicate.
4.2.2. Cytotoxicity assays
Cells were seeded at a cell density of 3 × 103 cells/0.1 mL/well in a
96-well plate. The compounds were added 24 h postplating at the in-
dicated concentrations. Cell counts were determined from duplicate
wells 72 h post-treatment. The total number of viable cells was de-
termined using the CCK-8 assay (Promega, WI) in conjunction with the
Microplate reader (Promega, WI).
4.2.8. Statistical analysis
4.2.3. Kinase inhibition assays
Statistical analysis Data were represented as the mean
s.d. All
Kinase inhibition profiles were determined using KinaseProfiler
services provided by Eurofins, and ATP concentrations used are the Km
of corresponding kinases.
the statistical analysis was performed using GraphPad Prism 5 software
(GraphPad Software, Inc., La Jolla, CA). The unpaired Student’s t-test
was used for the comparisons between two different groups. p < 0.05
was considered as significant. * represented p < 0.05; ** represented
p < 0.01; *** represented p < 0.001.
4.2.4. Apoptosis assay
Cells (3 × 105 cells/mL) were seeded in six-well plates and treated
with compounds at different concentration for 48 h. The cells were then
harvested by trypsinization and washed twice with cold PBS. After
centrifugation and removal of the supernatants, cells were resuspended
in 500 μL of 1 × binding buffer, which was then added to 5 μL of
annexin V-FITC and incubated at room temperature for 15 min. After
adding 10 μL of PI, the cells were incubated at room temperature for
another 15 min in dark. The stained cells were analyzed by a flow
cytometer. All experiments were performed three times.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgments
This work was supported by the National Key Research and
Development Program of China (2018YFE0114300) and the National
Natural Science Foundation of China (81972882 and 81903099).
4.2.5. In vivo assay
The experimental procedures of the animal study were proved by
the Animal Care and Use Committee at Nankai University. SKOV3
ovarian cancer cells (1 × 106) were injected bilaterally in the mam-
mary fat pads of 6–8 weeks old female BALB/c nude mice. Once the
tumors grew to a volume of approximately 100 mm3 they were placed
into five treatment groups (n = 5, with a total tumor number of 25).
The mice were treated daily for 21 days via oral gavage. Body weights
and tumor size were determined every other day. Tumor measurements
were found using a digital vernier caliper, and the volumes were de-
termined using the following calculation: (short2) × long × 0.5.
Experiments were performed under an approved IACUC protocol
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bioorg.2019.103547.
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