Discovery of a potent, selective, and covalent ZAP-70 kinase inhibitor

Article abstract of DOI:10.1016/j.ejmech.2021.113393

ZAP-70 (zeta-chain associated protein kinase 70 kDa) signaling pathway and its functions have been involved in the development and adaptive immune signaling of T cell. It thus represents a promising target for autoimmune diseases. Although reversible ZAP-70 kinase domain inhibitors have been developed, they are either weak or nonselective. We report herein the structure-guided development of the first potent and covalent inhibitor of ZAP-70 kinase domain. In particular, compound 18 (RDN009) showed good selectivity for ZAP-70 over structurally related Syk, and displayed potent inhibitory effects on T cell proliferation, activation, and inflammatory cytokine production. A mass spectrometry analysis further confirmed the covalent linkage between the inhibitor and ZAP-70 protein at C346. Overall, the covalent inhibitor RDN009 represents a potent and selective probe of ZAP-70 for further development for treatment of autoimmune diseases.

Full text of DOI:10.1016/j.ejmech.2021.113393

European Journal of Medicinal Chemistry 219 (2021) 113393
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Discovery of a potent, selective, and covalent ZAP-70 kinase inhibitor
,
e
,
,
Danni Rao a 1, Heng Li b 1, Xuelian Ren c, Yaoliang Sun a, Cuiyun Wen a,
Mingyue Zheng d e, He Huang c e, Wei Tang b **, Shilin Xu a
a Department of Medicinal Chemistry, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, #555 Zu Chong Zhi Road, Shanghai, 201203,
,
,
,
e
,
, e, *
China
b Laboratory of Anti-inflammation and Immunopharmacology, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, 201203,
China
c Center for Chemical Biology, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, 201203, China
d Drug Discovery and Design Center, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai,
201203, China
e University of Chinese Academy of Science, 19 Yuquan Road, Beijing, 110039, China
a r t i c l e i n f o
a b s t r a c t
Article history:
ZAP-70 (zeta-chain associated protein kinase 70 kDa) signaling pathway and its functions have been
involved in the development and adaptive immune signaling of T cell. It thus represents a promising
target for autoimmune diseases. Although reversible ZAP-70 kinase domain inhibitors have been
developed, they are either weak or nonselective. We report herein the structure-guided development of
the first potent and covalent inhibitor of ZAP-70 kinase domain. In particular, compound 18 (RDN009)
showed good selectivity for ZAP-70 over structurally related Syk, and displayed potent inhibitory effects
on T cell proliferation, activation, and inflammatory cytokine production. A mass spectrometry analysis
further confirmed the covalent linkage between the inhibitor and ZAP-70 protein at C346. Overall, the
covalent inhibitor RDN009 represents a potent and selective probe of ZAP-70 for further development for
treatment of autoimmune diseases.
Received 18 January 2021
Received in revised form
15 March 2021
Accepted 18 March 2021
Available online 30 March 2021
Keywords:
Zeta-chain associated protein kinase 70 kDa
(ZAP-70)
Covalent inhibitor
T cell
© 2021 Elsevier Masson SAS. All rights reserved.
Autoimmune diseases
1. Introduction
ZAP-70 is a non-receptor tyrosine kinase that belongs to the Syk
(spleen tyrosine kinase) kinase family, and plays a crucial role in
Autoimmune diseases are caused by imbalance of immune
system; there are nearly 4% of world’s populations living with one
of more than 80 different autoimmune diseases [1e3]. Despite
much progress in therapies for autoimmune diseases, many of
these agents have serious side effects [3], which accelerates the
need for the development of more specific strategies that lower the
risk of systemic immune suppression [3,4]. Given the important
function of T cells in immune system, fine-tuning of T cell activation
is required to prevent autoimmune attacks, avoiding inhibition of
immune response against infectious agents [5].
development and adaptive immune signaling of T cells [6,7]. Upon
activation of TCR (T cell receptor) by antigens, ZAP-70 is then
recruited, via its tandem-SH2 (Src-homology domain 2) domains,
to the intracellular CD3 z-chain of the TCR, where ZAP-70 is acti-
vated and phosphorylated. The activated ZAP-70 in turn phos-
phorylates its substrates, the scaffolding proteins, LAT (linker for
activation of T cell) and SLP-76 (SH2-domian-containing leukocyte
protein of 76 kDa) [8,9]. The tyrosine phosphorylation cascades will
eventually leads to T cell immune response, including T cell pro-
liferation and differentiation, and inflammatory cytokine produc-
tion [10]. The significant early involvement of ZAP-70 in T cell
activation and immune response makes it an attractive target for
the treatment of autoimmune diseases [11,12].
A specific and potent ZAP-70 kinase inhibitor could potentially
block signaling downstream of the kinase leading to T cell activa-
tion [11,13]. Since ZAP-70 expression is relatively restricted to T
cells, natural killer (NK) cells and a subset of B cells [14], effects of
specific ZAP-70 inhibitor would be limited to these cell types while
* Corresponding author. Department of Medicinal Chemistry, Shanghai Institute
of Materia Medica, Chinese Academy of Sciences, #555 Zu Chong Zhi Road,
Shanghai, 201203, China.
** Corresponding author. Laboratory of Anti-inflammation and Immunopharma-
cology, Shanghai Institute of Materia Medica, Chinese Academy of Sciences,
Shanghai, 201203, China.
E-mail addresses: tangwei@simm.ac.cn (W. Tang), slxu@simm.ac.cn (S. Xu).
These authors contributed equally to this research.
1
https://doi.org/10.1016/j.ejmech.2021.113393
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

D. Rao, H. Li, X. Ren et al.
European Journal of Medicinal Chemistry 219 (2021) 113393
avoiding unfavorable adverse effects [12]. To date, a cell-permeable
small-molecular inhibitor that specifically target the active site of
wild-type ZAP-70 have not yet been found [11,15e20]. Although
several classes of small-molecule ZAP-70 kinase domain inhibitors
have been reported (Fig. 1), they are either weak (1) [21], nonse-
lective (2) [22] or are inhibitors (3) [11,23] selectively targeting a
gene-edited mutant ZAP-70 rather than wild-type ZAP-70. There-
fore, there is an urgent need to develop small-molecule inhibitors
of wild-type ZAP-70 kinase with on-target activity in T cell.
High sequence homology within the ATP-binding pocket creates
a distinct challenge in achieving selectivity among Syk kinase
family. In this case, ZAP-70 and Syk share 21 out of 24 identical
residues within 5 Å of ligand (Supporting information, Fig. S1).
Analysis of X-ray crystal structures of ZAP-70 and Syk reveals that
ZAP-70 has a cysteine (C346) in the glycine-rich loop region
whereas Syk has a serine (S379) at this position (Supporting in-
formation, Fig. S1). The cysteine in the front pocket of kinases is
potentially able to form a covalent bond with a warhead group in
covalent ligands, which allows them to bind to kinases in an irre-
versible manner [24e26]. In addition, covalent inhibitors have
several advantages including prolonged drug residence time, high
potency, and suitability for rational design [24,27,28]. Therefore, we
hypothesized that covalent inhibitor of ZAP-70 kinase domain may
achieve much stronger potency and higher selectivity than non-
covalent ZAP-70 inhibitor. Herein, we report the design and char-
acterization of the first-in-class, potent and selective covalent
inhibitors targeting the ZAP-70 kinase domain. This most potent
inhibitor displays potent inhibitory activity against ZAP-70 kinase
with a low IC50 value and has good selectivity over Syk protein and
a small panel of inflammation-related kinases, and prevents T cell
activation and function in vitro.
information, Fig. S2). We thus replaced the ethyl acrylate substit-
uent on the para-position of the benzylamino group in compound 1
with an acrylamide group, a Michael acceptor. The resulting com-
pound 4 had weak and similar inhibitory activities against ZAP-70
and Syk kinases with 7% and 4% inhibition, respectively, at con-
centration of 1 mM in the caliper mobility shift assay (Table 1). To
examine the effect of the linker length, we inserted one more
methylene group between the NH group and acrylamide in 4,
yielding compounds 5 and 6. However, these two compounds also
displayed weak inhibitory activities against ZAP-70. The fails to
obtain potent ZAP-70 inhibitors suggested that the acrylamide on
the para-position of the benzyl amide may not extend directly to-
ward the C346 residue in ZAP-70 to form covalent bond. We,
therefore, moved the acrylamide warhead of 4 to the meta-
position, resulting in compound 7. Gratifyingly, 7 displayed signif-
icant increase in activity for ZAP-70 with 58% inhibition compared
to 4. Of note is that 7 did not inhibit closely related Syk at con-
centration of 1 mM. Next, we also added one more methylene group
to the linker of 7, however, the resulting compounds 8 and 9 had
weaker inhibitory activity against ZAP-70 than 7. These findings
indicate that the linker type has a great impact on the potency,
which inspired us to explore more types of linkers, such as piper-
idin-4-yl (10), piperidin-3-yl (11), pyrrolidine (12) and ethylamino
(13). However, none of these compounds showed stronger activity
than 7. They suppressed ZAP-70 activity with from 26% to 48% in-
hibition, and they exhibited poor selectivity for ZAP-70 over Syk.
Given the moderate activity and good selectivity of compound 7,
we used this compound as the template for further optimization. To
improve the physicochemical properties of compound 7, we
incorporated a dimethylaminomethyl group to the acrylamide
group, yielding compound 14 (Table 2). Compound 14 has the
inhibitory rate of 32% to ZAP-70, which is weaker than 7, but still
retained selectivity over Syk. Interestingly, an introduction of a
2. Results and discussion
methyl (15) to the
a-position of acrylamide of 7 increased the
inhibitory rate to Syk, but caused complete loss in potency for ZAP-
70. We also replaced the acrylamide in 7 with propynamide and 2-
butynamide, yielding compounds 16 and 17, respectively. However,
these two compounds showed much less inhibitory activities
against ZAP-70. Then, we replaced the acrylamide with 2-
chloroacetamide, a more reactive Michael acceptor [29]. Excit-
ingly, the resulting compound 18 had significantly enhanced ZAP-
70 inhibitory activity with 99% inhibition at concentration of
The inhibitory rates to ZAP-70 and Syk of our synthesized
compounds were initially evaluated using a caliper mobility shift
assay (Tables 1e3). For potent compounds, the apparent enzyme
IC50 values were further determined. A reported promiscuous ki-
nase inhibitor, staurosporine (2), was used as a control. Under the
experimental conditions, compound 2 inhibited ZAP-70 and Syk
with apparent enzyme IC50 values of 1.1 and 1.3 nM, respectively,
exhibiting poor selectivity between ZAP-70 and Syk.
In order to design covalent ligand for ZAP-70, we used com-
pound 1, a reported noncovalent inhibitor of ZAP-70, as the starting
point. Our modeled structure of compound 1 in a complex with
ZAP-70 shows that the C346 residue in the glycine-rich loop region
of ZAP-70 is close to the para-substituted benzylamino group of
compound 1. The distance between C346 and the reversible in-
hibitor 1 in the ATP binding site is less than 10 Å (Supporting
1
m
M (IC50 ¼ 55 nM) while exhibiting no inhibition of Syk at the
same concentration (IC50 > 1 M). Moreover, we moved the 2-
m
chloroacetamide to the para-position, the resulting compound 19
showed complete loss in potency for ZAP-70, supporting the
importance of the geometry of covalent warhead for potency. We
also prepared compound 20 as a non-covalent inhibitor control by
replacing the acrylamide warhead with propanamide. Compound
Fig. 1. Previously reported ZAP-70 inhibitors.
2

D. Rao, H. Li, X. Ren et al.
European Journal of Medicinal Chemistry 219 (2021) 113393
Table 1
In Vitro Enzymatic inhibitory activities for compounds 2 and 4e13.
Compd
Linker
ZAP-70
Syk
Inhibition @ 1
102.1 1.8
m
M (%)
IC50 (nM)
1.1 0.4
Inhibition @ 1
97.3 2.3
m
M (%)
IC50 (nM)
1.3 0.2
2
(Staurosporine)
4
7.1
2
3.9 0.6
5
6
7
5.7 2.9
7.7 2.6
58 8.2
35.3 0.2
ꢀ11.5 1.0
ꢀ1.4
0
8
9
25 4.9
ꢀ1.6 0.4
0
5.3 2.5
10
11
42.6 4.8
26 1.8
28.9 10.0
9.5 3.7
12
13
48.0 0.6
33 3.9
63.7 1.0
34.4 1.1
20 was much less potent for ZAP-70 than 18. Taken together, these
data suggests that the increased potency of 18 probably results
from contribution of formation of the covalent bond.
information, Table. S1). Therefore, compound 18 showed good
selectivity for ZAP-70 within the small panel of inflammation-
related kinases.
To further explore the effects of substitutions of left aniline in
compound 18 on potency and selectivity, we removed the dime-
thoxy groups and installed various hydrophilic groups at 4-position
of aniline, such as morpholino (21), dimethylamine (22), N-meth-
ylpiperazine (23) and N-methylhomopiperazine (24) (Table 3).
Most of these compounds showed comparable inhibitory activity
against ZAP-70, except for compound 22 with a slight decrease in
potency. However, these four compounds exhibited lower selec-
tivity for ZAP-70 over Syk. Overall, compound 18 with dimethoxy
groups showed the best potency and selectivity over Syk in these
series of compounds.
In order to confirm whether 18 formed a covalent bond with
ZAP-70 on the C346 residue, mass spectrometry was conducted to
analyze the peptide mass change from ZAP-70 protein treated with
compound following by trypsin protease. As expected, the LC-MS/
MS
spectrum
of
an
identified
peptide
[DNLLIA-
DIELGC(C24H23O4N7)GNFGSVR] demonstrated that the specific
modification of compound 18 on the C346 of ZAP-70 (Fig. 2).
Additionally, to understand how 18 binds to ZAP-70, we modeled
its binding mode with ZAP-70 kinase domain, based on the previ-
ously reported co-crystal structure of compound 2 with ZAP-70
(PDB: 1U59). Our modeling study predicted that compound 18
forms two hydrogen bonds with the E415 and A417 residues in the
hinge region, which is known to form an anchoring point for kinase
inhibitor binding (Fig. 3). We also observed an additional hydrogen
binding interaction between the acrylamide NH of compound 18
and the carbonyl oxygen of C346. Consistent with our mass
We also performed the selectivity assays for compound 18 in a
small panel of non-receptor tyrosine kinases that play roles in
inflammation, including ABL, BTK, HCK, ITK, JAK1, JAK2, JAK3, LCK,
SRC and TYK2. Compound 18 exhibited IC50 > 1
mM against most of
these kinases, except for JAK3 (IC50 ¼ 562 nM) (Supporting
3

D. Rao, H. Li, X. Ren et al.
European Journal of Medicinal Chemistry 219 (2021) 113393
Table 2
In Vitro Enzymatic inhibitory activities for compounds 14e20.
Compd
R1
R2
H
ZAP-70
Syk
Inhibition @ 1
32 4.2
m
M (%)
IC50 (nM)
Inhibition @ 1
mM (%)
IC50 (nM)
14
ꢀ10 5.2
15
H
ꢀ1.3 0.5
29.3 4.7
16
H
H
H
15.0 8.7
3.1 3.7
99 3.1
29.6 6.3
ꢀ2.2 1.2
ꢀ1.4 1.2
ꢀ8.8 1.2
34.7 1.9
17
18 (RDN009)
55
>1000
19
20
H
4.8 0.2
19.3 9.1
H
spectrometry result, the 2-chloroacetamide warhead of 18 forms a
covalent bond with C346, which may contribute to the selectivity
for ZAP-70 over Syk.
inhibitory properties of compound 18 to another maker of CD4þ
T
cell activation, CD25 (Supporting information, Fig. S3). These re-
sults supported that our compound 18 could inhibit the CD4þ T cell
activation.
ZAP-70 kinase catalytic activity has been shown to be essential
for T cell proliferation and division [11]. In addition, compound 18
showed potent kinase inhibitory activity and good selectivity over
Syk. Therefore, we investigated the inhibitory effects of 18 on the
proliferation and division of CD4þ T cells. The results were sum-
marized in Fig. 4. In response to stimulation with anti-CD3/CD28
antibodies in the presence of vehicle alone, the increased prolifer-
ation of CD4þ T cells was observed as measured by uptake of [3H]
thymidine after 48 h. In line with the inhibitory effects on ZAP-70,
compound 18 suppressed the proliferation of CD4þ Tcells in a dose-
dependent manner. Compound 18 was able to almost completely
inhibit CD4þ T cell proliferation at a concentration of 400 nM
(Fig. 4A). Moreover, we also observed similar dose-dependent
Next, we also assessed the effects of compound 18 on the
function of CD4þ T cells by measuring the production of effector
cytokines, such as interferon-g (IFN-g) and interleukin 4 (IL-4), in
the cultures of stimulated CD4þ T cells. Enzyme-linked immuno-
sorbent assay was conducted using CD4þ T cells treated with
increasing concentrations of compound 18 for 48 h. The results
demonstrated that compound 18 potently inhibited the production
of both IFN-g and IL-4 cytokines (Fig. 6).
3. Chemistry
inhibitory properties of compound 18 to the division of CD4þ
T
The syntheses of compounds 4e20 are outlined in Scheme 1.
Starting from commercially available 2,4,6-trichloropyrimidine
acid, the 2-carboxylic acid of 2,4,6-trichloropyrimidine acid was
converted to acyl chloride with oxalyl chloride in tetrahydrofuran.
The resulting acyl chloride crude was reacted with ammonium
hydroxide to afford intermediate 26. Compound 26 was further
substituted to give compound 27 in presence of 3,5-
dimethoxyaniline and diisopropylethylamine in 1,4-dioxane. The
key intermediate 28 was prepared by direct cyclization using
chloroacetaldehyde with NaHCO3 and NaI in t-BuOH under 80 ꢁC.
Compound 28 coupled with different substituted amines to give
compounds 29. After removal of the Boc protecting group, the
resulting primary amine intermediates 30 reacted with a variety of
cells, as analyzed by the cytosolic dye CFSE labeling CD4þ T cells
(Fig. 4B and C). Compound 18 is therefore potent and effective in
suppressing the proliferation and division of CD4þ T cells.
To further characterize the effect of compound 18 on CD4þ T cell
activation, we examined the ability of compound 18 to down-
regulate the expression of CD69, a maker for CD4þ T cell activa-
tion. As shown in Fig. 5, compound 18 could significantly suppress
the expression of CD69 on CD4þ Tcells stimulated by the antibodies
anti-CD3 plus anti-CD28. For example, the frequency of CD4þ/
CD69þ T cells was reduced from 14.9% to 3.64% with treatment of
18 at concentration of 400 nM for 48 h. We also observed similar
4

D. Rao, H. Li, X. Ren et al.
European Journal of Medicinal Chemistry 219 (2021) 113393
Table 3
In Vitro Enzymatic inhibitory activities for compounds 21e24.
Compd
R3
ZAP-70
Syk
Inhibition @ 1
98.0 2.3
m
M (%)
Inhibition @ 1
49.1 0.3
mM (%)
21
Fig. 3. Predicted binding mode of compound 18 (gray carbons) in the ATP pocket of
ZAP-70 (PDB: 1U59). The ligand and the key residues are shown in stick model, and the
H-bonds are displayed in black dashed lines.
22
23
76.9 3.8
96.0 0.4
26.1 1.9
63.1 1.9
the ZAP-70 kinase domain up to date through structure-based and
medicinal chemistry optimization. The mass spectrometry of the
protein treated compound and trypsin protease was analyzed to
display the formation of a covalent modification on C346 of ZAP-70.
The covalent inhibitor 18 showed a potent activity for ZAP-70. It is
worth noting that 18 exhibited over 18-fold selectivity for ZAP-70
over structurally related Syk and also showed high selectivity
over a small panel of inflammation-related kinases. Moreover,
24
98.3 1.3
49.1 1.8
compound 18 also displayed potent inhibitory properties to CD4þ
T
cell proliferation, division, activation and function. Covalent in-
hibitor 18 represents a promising starting point for a variety of
applications to further study of ZAP-70 pharmacology. Further
optimization of 18 to improve potency and pharmacokinetic
properties is underway in our laboratory to demonstrate the
advantage for promising therapeutic strategy for autoimmune
diseases.
acyl chloride or carboxylic acid to produce the final compounds
4e20. The syntheses of compounds 21e24 are outlined in Scheme 2
and they were synthesized using the similar procedures described
for compounds 4e20.
4. Conclusion
5. Experimental section
In the present study, we described the first covalent inhibitor of
General Methods for Chemistry. Unless otherwise noted,
Fig. 2. MS/MS spectra of peptide DNLLIADIELGC(C24H23O4N7)GNFGSVR demonstrates the covalent modification on the C346 of ZAP-70. The b ion refers to the N-terminal parts of
the peptide, and the y ion refers to the C-terminal parts of the peptide.
5

D. Rao, H. Li, X. Ren et al.
European Journal of Medicinal Chemistry 219 (2021) 113393
Fig. 4. Compound 18 suppressed the proliferation and division of CD4þ T cells. (A) The proliferation capacity of CD4þ T cells was determined by the uptake of [3H]thymidine. (B)
CFSE-labeled CD4þ T cells were incubated and subjected to flow cytometry analysis of the division of CD4þ T cells. (C) Quantification of the division of CD4þ T cells by flow
cytometry. Data were shown as Mean SD. ***p < 0.001, **p < 0.01, *p < 0.05.
commercial solvents and reagents were used without further pu-
carboxamide (5). To a solution of compound 29b (140 mg,
0.25 mmol) in DCM (2 mL) was added trifluoroacetic acid (2 mL).
The mixture was stirred at room temperature for 1 h and then was
concentrated. Subsequently, the residue was dissolved in DCM,
rification. Proton nuclear magnetic resonance (1H NMR) was
recorded on a Bruker Advance 500 MHz/600 MHz spectrometer. 13
C
NMR was recorded on Varian Mercury 600 M NMR spectrometer.
1H NMR spectra were reported in parts per million (ppm) down-
field from tetramethylsilane (TMS). In the spectral data reported,
cooled to 0 ꢁC, and treated with DIPEA (133
lowed by acryloyl chloride (25
mL, 0.75 mmol), fol-
mL, 0.31 mmol). The mixture was
the format (
d
) chemical shift (multiplicity, J values in Hz, integra-
stirred at room temperature for 30 min. After concentrated under
vacuum, the residue was purified by prep-HPLC to give tri-
fluoroacetate salt 5 as white solid (80 mg, 62% yield). 1H NMR
tion) was used with the following abbreviations: s ¼ singlet,
d ¼ doublet, t ¼ triplet, q ¼ quartet, m ¼ multiplet, brs ¼ broad
singlet. Low resolution mass spectra (MS) was recorded on a
Thermo Fisher Finnigan LTQ and high-resolution mass spectra
(HRMS) was recorded on Agilent G6230 TOF LC/MS spectrometer.
Column chromatography was carried out on silica gel (200e300
mesh). The final compounds were all purified by C18 reverse phase
preparative Shim-pack GIS column (5 mm C18, 30*250 mm) with
solvent A (0.1% TFA in H2O) and solvent B (0.1% TFA in MeCN) as
eluents. The purity of all the final compounds was confirmed to be
(500 MHz, DMSO‑d6):
d
12.33 (s,1H), 8.99 (t, J ¼ 5.55 Hz,1H), 8.56 (t,
J ¼ 5.90 Hz, 1H), 7.92 (d, J ¼ 1.75 Hz, 1H), 7.39 (d, J ¼ 1.65 Hz, 2H),
7.34 (d, J ¼ 8.10 Hz, 2H), 7.21 (d, J ¼ 8.10 Hz, 1H), 6.80 (d, J ¼ 2.20 Hz,
2H), 6.24 (dd, J ¼ 17.10, 10.15 Hz, 1H), 6.16 (t, J ¼ 2.20 Hz, 1H), 6.10
(dd, J ¼ 17.05, 2.10 Hz, 1H), 5.60 (dd, J ¼ 10.15, 2.15 Hz, 1H), 4.78 (d,
J ¼ 5.65 Hz, 2H), 4.30 (d, J ¼ 5.95 Hz, 2H), 3.63 (s, 6H). HRMS (ESI)
for C26H28N7O4 [M þ H]þ, calcd: 502.2125, found:502.0402.
5-((4-Acrylamidophenethyl)amino)-7-((3,5-
>95% by HPLC analysis (a Shim-pack GIST column, 2
m
m C18,
dimethoxyphenyl)amino)imidazo[1,2-c]pyrimidine-8-
2.1*50 mm; 10%e100% MeCN in H2O containing 0.1% TFA in 15 min;
flow rate was 0.4 mL/min).
carboxamide (6). This compound was prepared from compound 28
by following a similar procedure as that for compound 29b and 5.
5-((4-Acrylamidobenzyl)amino)-7-((3,5-dimethoxyphenyl)
amino)imidazo[1,2-c]pyrimidine-8-carboxamide (4). This com-
pound was prepared from compound 28 by following a similar
procedure as that for compound 29b and 5. White solid (10 mg, 45%
White solid (8 mg, 30% yield). 1H NMR (500 MHz, DMSO‑d6):
d 12.45
(s, 1H), 10.11 (s,1H), 9.54 (d, J ¼ 3.30 Hz, 1H), 8.56 (t, J ¼ 5.25 Hz, 1H),
7.86 (s, 1H), 7.58 (d, J ¼ 8.30 Hz, 2H), 7.41 (d, J ¼ 3.20 Hz, 1H), 7.34 (d,
J ¼ 1.40 Hz, 1H), 7.19 (d, J ¼ 8.40 Hz, 2H), 6.86 (d, J ¼ 2.10 Hz, 2H),
6.44 (d, J ¼ 10.10 Hz, 1H), 6.43 (dd, J ¼ 17.00, 10.10 Hz, 1H), 6.24 (dd,
J ¼ 17.00,1.80 Hz,1H), 5.74 (dd, J ¼ 10.10,1.80 Hz,1H), 3.76e3.69 (m,
2H), 3.68 (s, 6H), 2.94e2.90 (m, 2H). HRMS (ESI) for C26H28N7O4
[M þ H]þ, calcd: 502.2125, found: 502.0399.
yield). 1H NMR (500 MHz, DMSO‑d6):
d 12.46 (s, 1H), 10.14 (s, 1H),
8.94 (t, J ¼ 5.45 Hz, 1H), 7.91 (d, J ¼ 1.35 Hz, 1H), 7.61 (d, J ¼ 8.40 Hz,
2H), 7.37 (d, J ¼ 1.35 Hz, 1H), 7.34 (d, J ¼ 8.45 Hz, 2H), 6.81 (d,
J ¼ 2.05 Hz, 2H), 6.41 (d, J ¼ 16.95, 10.15 Hz, 1H), 6.41 (d, J ¼ 16.95,
10.15 Hz, 1H), 6.23 (d, J ¼ 16.95, 1.80 Hz, 1H), 6.15 (s, 3H), 6.23 (d,
J ¼ 10.15, 1.80 Hz, 1H), 4.77 (d, J ¼ 5.55 Hz, 2H), 3.64 (s, 6H). HRMS
(ESI) for C25H26N7O4 [M þ H]þ, calcd: 488.1968, found: 488.0143.
5-((4-(Acrylaminomethyl)benzyl)amino)-7-((3,5-
5-((3-Acrylamidobenzyl)amino)-7-((3,5-dimethoxyphenyl)
amino)imidazo[1,2-c]pyrimidine-8-carboxamide (7). This com-
pound was prepared from 30d by following a similar procedure as
that for compound 18. White solid (34 mg, 55% yield). 1H NMR
dimethoxyphenyl)amino)imidazo[1,2-c]pyrimidine-8-
(500 MHz, DMSO‑d6): d 12.44 (s, 1H), 10.11 (s, 1H), 9.02 (brs, 1H),
6

D. Rao, H. Li, X. Ren et al.
European Journal of Medicinal Chemistry 219 (2021) 113393
Fig. 5. Compound 18 suppressed the activation of CD4þ T cells. (A) The expression of CD69 on CD4þ T cells was determined by flow cytometry analysis. (B) Quantification of the
expression of CD69 by flow cytometry. Data were shown as Mean SD. ***p < 0.001, **p < 0.01, *p < 0.05.
Fig. 6. Compound 18 suppressed the production of IFN-g g
and IL-4 from CD4þ T cells. The level of IFN- (A) and IL-4 (B) in the cultures of CD4þ T cells was determined by ELISA. Data
were shown as Mean SD. ***p < 0.001, **p < 0.01, *p < 0.05.
7.94 (s,1H), 7.64e7.61 (m, 2H), 7.39 (brs,1H), 7.28 (t, J ¼ 7.55 Hz,1H),
7.09 (d, J ¼ 7.80 Hz, 1H), 6.78 (d, J ¼ 2.20 Hz, 2H), 6.39 (dd, J ¼ 16.85,
10.10 Hz, 1H), 6.22 (dd, J ¼ 16.90, 1.90 Hz, 1H), 6.12 (t, J ¼ 2.10 Hz,
1H), 5.72 (dd, J ¼ 10.10,1.90 Hz,1H), 4.83 (d, J ¼ 5.65 Hz, 2H), 3.60 (s,
6H). HRMS (ESI) for C25H26N7O4 [M þ H]þ, calcd: 488.1968, found:
488.0339.
(s, 1H), 8.98 (s, 1H), 8.58e8.54 (m, 1H), 7.91 (s, 1H), 7.37 (s, 1H), 7.28
(d, J ¼ 2.50 Hz, 1H), 7.27 (s, 1H), 7.16e7.14 (m, 1H), 6.80 (d,
J ¼ 2.20 Hz, 2H), 6.22 (dd, J ¼ 17.10, 10.15 Hz, 1H), 6.14 (t, J ¼ 2.10 Hz,
1H), 6.07 (dd, J ¼ 17.05, 2.10 Hz,1H), 5.58 (dd, J ¼ 17.05, 2.20 Hz,1H),
4.80 (d, J ¼ 5.60 Hz, 2H), 4.30 (d, J ¼ 6.00 Hz, 2H), 3.62 (s, 6 H).
HRMS (ESI) for C26H28N7O4 [M þ H]þ, calcd: 502.2125, found:
502.0431.
5-((3-(Acrylamidomethyl)benzyl)amino)-7-((3,5-
dimethoxyphenyl)amino)imidazo[1,2-c]pyrimidine-8-
carboxamide (8). This compound was prepared from compound 28
by following a similar procedure as that for compound 29b and 5.
5-((3-Acrylamidophenethyl)amino)-7-((3,5-
dimethoxyphenyl)amino)imidazo[1,2-c]pyrimidine-8-
carboxamide (9). This compound was prepared from compound 28
by following a similar procedure as that for compound 29b and 5.
White solid (7 mg, 34% yield). 1H NMR (500 MHz, DMSO‑d6):
d 12.40
7

D. Rao, H. Li, X. Ren et al.
European Journal of Medicinal Chemistry 219 (2021) 113393
Scheme 1. Reagents and conditions: (a) (COCl)2, NH3$H2O, THF, 0 ꢁC; (b) 3,5-Dimethoxyaniline, 1,4-dioxane, DIPEA, 80 ꢁC; (c) ClCH2CHO, NaHCO3, NaI, t-BuOH, 80 ꢁC; (d)
NH2CH2XBoc, K2CO3, DMF, 80 ꢁC; (e) TFA, DCM, rt; (f) Corresponding acyl chlorides, DCM, DIPEA, rt (4e13, 15, 18e20); Corresponding
-unsaturated carboxylic acids, HATU, DIPEA,
DCM (14); Corresponding -unsaturated carboxylic acids, 4-DMAP, DCC, DCM (16e17).
a,b
a,b
Scheme 2. Reagents and conditions: (a) Corresponding aniline, 1,4-dioxane, DIPEA, 80 ꢁC; (b) ClCH2CHO, NaHCO3, NaI, t-BuOH, 80 ꢁC; (c) tert-Butyl (3-(aminomethyl)phenyl)
carbamate, K2CO3, DMF, 80 ꢁC; (d) TFA, DCM, rt; (e) Chloroacetyl chloride, DCM, DIPEA, rt.
White solid (6 mg, 33% yield). 1H NMR (500 MHz, DMSO‑d6):
28 by following a similar procedure as that for compound 29b and
5. White solid (7 mg, 45% yield). 1H NMR (500 MHz, DMSO‑d6):
d 12.23 (s, 1H), 8.57 (s,1H), 7.90 (s, 1H), 7.39 (s, 1H), 6.85e6.77 (m,
d
12.44 (s, 1H), 10.08 (s, 1H), 8.56e8.51 (m, 1H), 7.85 (d, J ¼ 1.75 Hz,
1H), 7.57e7.51 (m, 2H), 7.35 (d, J ¼ 1.60 Hz, 1H), 7.24 (t, J ¼ 7.70 Hz,
1H), 6.96 (d, J ¼ 7.55 Hz, 1H), 6.85 (d, J ¼ 2.20 Hz, 2H), 6.42 (dd,
J ¼ 16.95, 10.15 Hz, 1H), 6.24 (dd, J ¼ 16.95, 1.90 Hz, 1H), 6.16 (t,
J ¼ 2.20 Hz, 1H), 5.74 (dd, J ¼ 10.15, 1.80 Hz, 1H), 3.79e3.73 (m, 2H),
3.65 (s, 6H), 2.98e2.94 (m, 1H). HRMS (ESI) for C26H28N7O4 [M þ
H]þ, calcd: 502.2125, found: 502.0466.
3H), 6.21 (brs, 1H), 6.07 (dd, J ¼ 16.70, 2.40 Hz, 1H), 5,64 (dd,
J ¼ 10.40, 2.35 Hz, 1H), 4.43 (d, J ¼ 12.50 Hz, 1H), 4.07 (d,
J ¼ 13.20 Hz, 1H), 3.74 (s, 6H), 3.43 (q, J ¼ 5.65 Hz, 2H), 3.00 (t,
J ¼ 12.50 Hz, 1H), 2.59 (t, J ¼ 12.10 Hz, 1H), 2.10e1.98 (m, 1H), 1.84
(d, J ¼ 12.70 Hz, 2H), 1.18e1.03 (m, 2H). HRMS (ESI) for C24H30N7O4
[M þ H]þ, calcd: 480.2281, found: 480.0630.
5-(((1-Acryloylpiperidin-4-yl)methyl)amino)-7-((3,5-
dimethoxyphenyl)amino)imidazo[1,2-c]pyrimidine-8-
carboxamide (10). This compound was prepared from compound
5-(((1-Acryloylpiperidin-3-yl)methyl)amino)-7-((3,5-
dimethoxyphenyl)amino)imidazo[1,2-c]pyrimidine-8-
8

D. Rao, H. Li, X. Ren et al.
European Journal of Medicinal Chemistry 219 (2021) 113393
carboxamide (11). This compound was prepared from compound
28 by following a similar procedure as that for compound 29b and
5. White solid (12 mg, 45% yield). 1H NMR (500 MHz, DMSO‑d6):
mixture was stirred at room temperature overnight. After
concentrated under vacuum, the residue was purified by prep-
HPLC to give trifluoroacetate salt 16 as white solid (10 mg, 18%
d
12.20 (s, 1H), 8.58e8.52 (m, 1H), 7.91 (s, 1H), 7.41 (d, J ¼ 7.15 Hz,
yield). 1H NMR (500 MHz, DMSO‑d6):
d 12.41 (s, 1H), 10.77 (s,1H),
1H), 6.84 (d, J ¼ 10.85 Hz, 2H), 6.79e6.57 (m, 1H), 6.22 (d,
J ¼ 8.75 Hz, 1H), 6.02 (d, J ¼ 16.76 Hz, 1H), 5.63e5.53 (m, 1H), 4.37
(d, J ¼ 12.51 Hz, 1H), 3.75 (s, 6H), 3.57e3.52 (m, 1H), 3.51e3.43 (m,
1H), 3.42e3.36 (m, 1H), 3.07e2.92 (m, 1H), 2.80e2.53 (m, 1H), 1.89
(brs, 1H), 1.73e1.70 (m, 1H), 1.37e1.23 (m, 1H). HRMS (ESI) for
9.01 (brs, 1H), 7.92 (brs, 1H), 7.58 (brs, 1H), 7.52 (d, J ¼ 7.60 Hz, 1H),
7.40 (brs, 1H), 7.27 (t, J ¼ 7.40 Hz, 1H), 7.12 (d, J ¼ 6.95 Hz, 1H), 6.76
(brs, 2H), 6.12 (s, 1H), 4.80 (d, J ¼ 5.10 Hz, 1H), 4.36 (s, 1H), 3.60 (s,
6H). HRMS (ESI) for C25H23N7O4 [M þ H]þ, calcd: 486.1812, found:
486.0091.
C
24H30N7O4 [M þ H]þ, calcd: 480.2281, found: 480.0645.
5-((3-(but-2-Ynamido)benzyl)amino)-7-((3,5-
5-(((1-Acryloylpyrrolidin-3-yl)methyl)amino)-7-((3,5-
dimethoxyphenyl)amino)imidazo[1,2-c]pyrimidine-8-
carboxamide (17). This compound was prepared from compound
30d by following a similar procedure as that for compound 16.
White solid (10 mg, 35% yield). 1H NMR (500 MHz, DMSO‑d6):
dimethoxyphenyl)amino)imidazo[1,2-c]pyrimidine-8-
carboxamide (12). This compound was prepared from compound
28 by following a similar procedure as that for compound 29b and
5. White solid (10 mg, 40% yield). 1H NMR (500 MHz, DMSO‑d6):
d
12.43 (s, 1H), 10.55 (s, 1H), 8.99 (t, J ¼ 5.65 Hz, 1H), 7.93 (d,
d
12.25e12.17 (m, 1H), 8.69e8.59 (m, 1H), 7.89 (d, J ¼ 11.09 Hz, 1H),
J ¼ 1.60 Hz, 1H), 7.57 (s, 1H), 7.53 (d, J ¼ 8.00 Hz, 1H), 7.39 (d,
J ¼ 1.60 Hz,1H), 7.25 (t, J ¼ 7.85 Hz,1H), 7.09 (d, J ¼ 7.55 Hz,1H), 6.77
(d, J ¼ 2.15 Hz, 2H), 6.12 (t, J ¼ 2.15 Hz, 1H), 4.80 (d, J ¼ 5.65 Hz, 2H),
3.60 (s, 6H), 2.01 (s, 3H). HRMS (ESI) for C26H26N7O4 [M þ H]þ,
calcd: 500.1968, found: 500.0258.
7.42e7.40 (m, 1H), 6.84 (d, J ¼ 1.95 Hz, 2H), 6.56e6.48 (m, 1H),
6.22e6.20 (m, 1H), 6.14e6.09 (m, 1H), 5.65 (td, J ¼ 10.19, 2.37 Hz,
1H), 3.74 (s, 6H), 3.69e3.48 (m, 4H), 3.38e3.29 (m, 1H), 3.19 (dd,
J ¼ 12.30, 6.92 Hz, 1H), 2.79e2.59 (m, 1H), 2.14e1.98 (m, 1H),
1.80e1.61 (m, 1H). HRMS (ESI) for C23H28N7O4 [M þ H]þ, calcd:
466.2125, found: 466.0507.
5-((3-(2-Chloroacetamido)benzyl)amino)-7-((3,5-
dimethoxyphenyl)amino)imidazo[1,2-c]pyrimidine-8-
carboxamide (18). Compound 30d (100 mg, 0.23 mmol) was dis-
5-((3-Acrylamidopropyl)amino)-7-((3,5-dimethoxyphenyl)
amino)imidazo[1,2-c]pyrimidine-8-carboxamide
(13).
This
solved in DCM, cooled to 0 ꢁC, and treated with DIPEA (96
0.69 mmol), followed by 2-chloroacetyl chloride (22
m
m
L,
L,
compound was prepared from compound 28 by following a similar
procedure as that for compound 29b and 5. White solid (15 mg, 40%
0.28 mmol). The mixture was stirred at room temperature for
30 min. After concentrated under vacuum, the residue was purified
by prep-HPLC to give trifluoroacetate salt 18 as white solid (53 mg,
yield). 1H NMR (500 MHz, DMSO‑d6):
d 12.29 (s, 1H), 8.50 (brs, 1H),
8.13 (t, J ¼ 5.20 Hz, 1H), 7.87 (brs, 1H), 7.40 (brs, 1H), 6.87 (d,
J ¼ 2.20 Hz, 2H), 6.20e6.13 (m, 2H), 6.06 (dd, J ¼ 17.10, 2.25 Hz, 1H),
5.56 (dd, J ¼ 10.00, 2.25 Hz, 1H), 3.74 (s, 6H), 3.24 (q, J ¼ 6.70 Hz,
2H), 1.86 (m, 2H). HRMS (ESI) for C21H26N7O4 [M þ H]þ, calcd:
440.1968, found: 440.0438.
(E)-7-((3,5-Dimethoxyphenyl)amino)-5-((3-(4-(dimethyla-
mino)but-2-enamido)benzyl)amino)imidazo[1,2-c]pyrimidine-
8-carboxamide (14). To a solution of compound 30d (100 mg,
0.23 mmol), (E)-4-(dimethylamino) but-2-enoic acid (36 mg,
0.28 mmol), and HATU (106 mg, 0.28 mmol) in DCM (5 mL) was
45% yield). 1H NMR (500 MHz, DMSO‑d6)
d 12.35 (s, 1H), 10.26 (s,
1H), 9.05 (brs, 1H), 7.95 (brs, 1H), 7.57e7.50 (m, 2H), 7.42 (brs, 1H),
7.29 (t, J ¼ 7.95 Hz, 1H), 7.12 (d, J ¼ 7.60 Hz, 1H), 6.77 (d, J ¼ 1.80 Hz,
2H), 6.13 (t, J ¼ 2.20 Hz, 1H), 4.83 (d, J ¼ 5.60 Hz, 2H), 4.20 (s, 2H).
3.60 (s, 6H). 13C NMR (151 MHz, DMSO‑d6)
d 169.10, 165.09, 161.00,
154.99, 147.55, 145.98, 141.73, 139.27, 139.24, 129.51, 123.14, 118.79,
118.05, 108.55, 98.89, 95.57, 82.78, 55.42, 44.65, 44.01. HRMS (ESI)
for C24H25ClN7O4 [M þ H]þ, calcd: 510.1578, found: 509.9892.
5-((4-(2-Chloroacetamido)benzyl)amino)-7-((3,5-
added DIPEA (96
mL, 0.69 mmol). The mixture was stirred at room
dimethoxyphenyl)amino)imidazo[1,2-c]pyrimidine-8-
temperature overnight. After concentrated under vacuum, the
residue was purified by prep-HPLC to give trifluoroacetate salt 14 as
white solid (20 mg, 62% yield). 1H NMR (500 MHz, DMSO‑d6):
carboxamide (19). This compound was prepared from compound
30a by following a similar procedure as that for compound 18.
White solid (63 mg, 57% yield). 1H NMR (500 MHz, DMSO‑d6):
d
12.51 (s, 1H), 10.03 (s, 1H), 9.56 (d, J ¼ 3.60 Hz, 1H), 8.99 (t,
d 12.43 (s, 1H), 10.29 (s, 1H), 8.96 (brs, 1H), 7.91 (s, 1H), 7.53 (d,
J ¼ 5.80 Hz, 1H), 7.94 (d, J ¼ 1.70 Hz, 1H), 7.62 (d, J ¼ 8.25 Hz, 1H),
7.60 (s, 1H), 7.44 (d, J ¼ 3.60 Hz, 1H), 7.38 (d, J ¼ 1.60 Hz, 1H), 7.27 (t,
J ¼ 7.80 Hz, 1H), 7.08 (d, J ¼ 7.70 Hz, 1H), 6.78 (d, J ¼ 2.20 Hz, 2H),
6.70 (dt, J ¼ 15.40, 6.95 Hz, 1H), 6.23 (d, J ¼ 15.40 Hz, 1H), 6.11 (t,
J ¼ 2.20 Hz, 1H), 4.83 (d, J ¼ 5.60 Hz, 2H), 3.60 (s, 6H), 3.07 (d,
J ¼ 5.60 Hz, 1H), 2.18 (s, 6H). HRMS (ESI) for C28H33N8O4 [M þ H]þ,
calcd: 545.2547, found: 545.1116.
J ¼ 8.35 Hz, 2H), 7.36 (t, J ¼ 4.15 Hz, 2H), 7.36 (d, J ¼ 8.20 Hz, 2H),
6.80 (brs, 2H), 6.15 (brs, 1H), 4.77 (d, J ¼ 5.05 Hz, 2H), 4.23 (s, 2H),
3.63 (s, 6H). HRMS (ESI) for C24H25ClN7O4 [M þ H]þ, calcd:
510.1578, found: 509.9869.
7-((3,5-Dimethoxyphenyl)amino)-5-((3-
propionamidobenzyl)amino)imidazo[1,2-c]pyrimidine-8-
carboxamide (20). This compound was prepared from compound
30d by following a similar procedure as that for compound 16.
White solid (10 mg, 34% yield). 1H NMR (500 MHz, DMSO‑d6):
7-((3,5-Dimethoxyphenyl)amino)-5-((3-
methacrylamidobenzyl)amino)imidazo[1,2-c]pyrimidine-8-
carboxamide (15). This compound was prepared from compound
30d by following a similar procedure as that for compound 18.
White solid (50 mg, 45% yield). 1H NMR (500 MHz, DMSO‑d6):
d
12.36 (brs, 1H), 9.84 (s, 1H), 9.05 (brs, 1H), 9.04 (brs, 1H), 7.95 (s,
1H), 7.56 (s, 1H), 7.54 (s, 1H), 7.42 (d, J ¼ 3.85 Hz, 1H), 7.24 (t,
J ¼ 7.85 Hz, 1H), 7.04 (d, J ¼ 7.65 Hz, 1H), 6.78 (d, J ¼ 2.20 Hz, 2H),
6.12 (s, 1H), 4.81 (d, J ¼ 5.60 Hz, 2H), 3.60 (s, 6H), 2.27 (q, J ¼ 7.55 Hz,
2H), 1.04 (t, J ¼ 7.55 Hz, 3H). HRMS (ESI) for C25H28N7O4 [M þ H]þ,
calcd: 490.2125, found: 490.0588.
d
12.45 (s, 1H), 9.76 (s,1H), 8.99 (brs, 1H), 7.94 (brs, 1H), 7.66 (brs,
1H), 7.60 (d, J ¼ 8.05 Hz, 1H), 7.39 (s, 1H), 7.26 (t, J ¼ 7.85 Hz, 1H),
7.09 (d, J ¼ 7.35 Hz, 1H), 6.79 (brs, 2H), 6.12 (d, J ¼ 1.95 Hz, 1H), 5,74
(brs, 1H), 5.48 (brs, 1H), 4.82 (d, J ¼ 5.00 Hz, 1H), 3.60 (s, 6H), 1.91 (s,
3H). HRMS (ESI) for C26H28N7O4 [M þ H]þ, calcd: 502.2125, found:
502.0396.
5-((3-(2-Chloroacetamido)benzyl)amino)-7-((4-
morpholinophenyl)amino)imidazo[1,2-c]pyrimidine-8-
carboxamide (21). This compound was prepared from 26 by
following a similar procedure as that for 5. White solid (15 mg, 40%
7-((3,5-Dimethoxyphenyl)amino)-5-((3-
propiolamidobenzyl)amino)imidazo[1,2-c]
pyrimidine-8-
yield). 1H NMR (600 MHz, CD3OD)
d 7.84e7.80 (m, 2H), 7.51 (d,
carboxamide (16). To a solution of compound 30d (50 mg,
0.12 mmol), propiolic acid (9 mg, 0.13 mmol) in DCM (5 mL) was
added DCC (27 mg, 0.13 mmol) and 4-DMAP (1 mg, 0.01 mmol). The
J ¼ 2.05 Hz, 1H), 7.39e7.40 (m, 4H), 7.10 (d, J ¼ 7.05 Hz, 1H), 7.04
(brs, 2H), 4.76 (s, 2H), 4.19 (s, 2H), 3.90 (t, J ¼ 4.55 Hz, 4H), 3.26 (brs,
4H). HRMS (ESI) for C26H28ClN8O3 [M þ H]þ, calcd: 535.1895, found:
9

D. Rao, H. Li, X. Ren et al.
European Journal of Medicinal Chemistry 219 (2021) 113393
534.9447.
calcd for C15H15ClN5O3 [M þ H]þ, calcd: 348.1, found: 348.2.
5-((3-(2-Chloroacetamido)benzyl)amino)-7-((4-(dimethyla-
mino)phenyl)amino)imidazo[1,2-c]pyrimidine-8-carboxamide
(22). This compound was prepared from 26 by following a similar
procedure as that for 21. White solid (5 mg, 32% yield). 1H NMR
tert-Butyl
(4-(((8-carbamoyl-7-((3,5-dimethoxyphenyl)
amino)imidazo[1,2-c]pyrimidin-5-yl)amino)methyl)benzyl)
carbamate (29b). To solution of compound 28 (100 mg,
a
0.29 mmol) and tert-butyl (4-(aminomethyl) benzyl)carbamate
(136 mg, 0.58 mmol) in DMF (5 mL) was added K2CO3 (120 mg,
0.87 mmol). After stirring at 80 ꢁC for 1 h, the mixture was allowed
to cool to room temperature. Then, 2 mL water was added to
quench the reaction. The mixture was poured into water (30 mL)
and then extracted with EtOAc (3 ꢂ 15 mL). Then the organic phase
was concentrated under high vacuum and purified by a column
chromatography on silica gel to give compound 29b (MeOH:
DCM ¼ 0e1:20) as orange solid (140 mg, 92% yield). 1H NMR
(500 MHz, chloroform-d)
d 11.78 (s, 1H), 7.56 (s, 1H), 7.44 (d,
J ¼ 7.45 Hz, 1H), 7.36e7.29 (m, 4H), 7.21 (s, 1H), 7.11 (d, J ¼ 7.55 Hz,
1H), 6.77e6.75 (m, 2H), 4.70 (d, J ¼ 5.45 Hz, 2H), 4.20 (s, 2H), 3.49 (s,
2H), 2.94 (s, 6H). HRMS (ESI), for C24H26ClN8O2 [M þ H]þ, calcd:
493.1789, found: 493.1897.
5-((3-(2-Chloroacetamido)benzyl)amino)-7-((4-(4-
methylpiperazin-1-yl)phenyl)amino)imidazo[1,2-c]pyrimidine-
8-carboxamide (23). This compound was prepared from 26 by
following a similar procedure as that for 21. White solid (6 mg, 40%
(500 MHz, DMSO‑d6):
d
12.29 (s, 1H), 9.00 (t, J ¼ 5.35 Hz, 1H), 7.92
yield). 1H NMR (500 MHz, CD3OD)
d
7.77e7.38 (m, 2H), 7.42e7.38
(d, J ¼ 1.75 Hz, 1H), 7.40 (d, J ¼ 1.75 Hz, 1H), 7.33 (d, J ¼ 7.95 Hz, 2H),
7.18 (d, J ¼ 7.95 Hz, 2H), 6.80 (d, J ¼ 2.20 Hz, 1H), 6.15 (t, J ¼ 2.20 Hz,
1H), 4.77 (d, J ¼ 5.40 Hz, 2H), 4.07 (d, J ¼ 6.05 Hz, 2H), 3.62 (s, 6H),
1.36 (s, 9H). ESI-MS calcd for C28H34N7O5 [M þ H]þ, calcd: 548.3,
found: 548.3.
(m, 2H), 7.37e7.30 (m, 3H), 7.12 (d, J ¼ 7.40 Hz, 1H), 6.91 (d,
J ¼ 8.90 Hz, 2H), 4.77 (s, 2H), 4.19 (s, 2H), 3.80e3.77 (m, 2H),
3.62e3.59 (m, 2H), 3.28e3.25 (m, 2H), 3.01e2.95 (m, 5H). HRMS
(ESI), for C27H31ClN9O2 [M þ H]þ, calcd: 548.2211, found: 548.0922.
5-((3-(2-Chloroacetamido)benzyl)amino)-7-((4-(4-methyl-
1,4-diazepan-1-yl)phenyl)amino)imidazo[1,2-c]pyrimidine-8-
carboxamide (24). This compound was prepared from 26 by
following a similar procedure as that for 21. White solid (5 mg, 33%
5-((4-Aminobenzyl)amino)-7-((3,5-dimethoxyphenyl)
amino)imidazo[1,2-c]pyrimidine-8-carboxamide (30a). To a so-
lution of compound 28 (289 mg, 0.83 mmol) and 4-(aminomethyl)
aniline (204 mg, 1.67 mmol) in DMF (8 mL) was added K2CO3
(344 mg, 2.49 mmol) and the mixture was stirred at 80 ꢁC for 1 h.
Then, the mixture was allowed to cool to room temperature,
poured into water (50 mL) and then extracted with EtOAc
(3 ꢂ 20 mL). The organic phase was concentrated under high vac-
uum and purified by a column chromatography on silica gel to give
compound 30a (MeOH: DCM ¼ 0e1:20) as orange solid (1.4 g, 70%
yield). 1H NMR (600 MHz, CD3OD)
d 7.76e7.74 (m, 2H), 7.42e7.37
(m, 2H), 7.34e7.29 (m, 3H), 7.11 (d, J ¼ 7.85 Hz, 1H), 6.72e6.70 (m,
2H), 4.75 (s, 2H), 4.19 (s, 2H), 3.83e3.80 (m, 1H), 3.70e3.64 (m, 2H),
3.53e3.49 (m, 3H), 3.34e3.28 (m, 2H), 2.95 (s, 3H), 2.30e2.19 (m,
2H). HRMS (ESI) for C28H33ClN9O2 [M þ H]þ, calcd: 562.2367,
found: 562.0583.
4-Amino-2,6-dichloropyrimidine-5-carboxamide (26). To a
solution of 2,4,6-trichloropyrimidine-5-carboxylic acid (5.0 g,
22 mmol) in dry tetrahydrofuran (150 mL) was treated with 2 drops
of N, N-dimethylformamide at 0 ꢁC. Oxalyl chloride (4.5 mL,
44 mmol) was added dropwise over 15 min and stirring was
continued for 1 h. The solution was concentrated and dried under
vacuo to give the crude acid chloride. The crude acid chloride was
dissolved in dry tetrahydrofuran (50 mL) and added dropwise to a
solution of ammonium hydroxide (23 mL) in tetrahydrofuran at
yield). 1H NMR (500 MHz, DMSO‑d6):
d 12.38 (s, 1H), 9.03 (t,
J ¼ 5.80 Hz, 1H), 7.92 (d, J ¼ 1.75 Hz, 1H), 7.40e7.37 (m, 3H), 7.11 (d,
J ¼ 8.35 Hz, 2H), 6.78 (d, J ¼ 2.20 Hz, 2H), 6.15 (t, J ¼ 2.20 Hz, 1H),
4.78 (d, J ¼ 5.65 Hz, 1H), 3.64 (s, 6H). ESI-MS calcd for C22H24N7O3
[M þ H]þ, calcd: 434.2, found: 434.2.
5-((3-Aminobenzyl)amino)-7-((3,5-dimethoxyphenyl)
amino)imidazo[1,2-c]pyrimidine-8-carboxamide (30d). To a so-
lution of compound 28 (1.6 g, 4.66 mmol) and 3-(aminomethyl)
aniline (0.8 mg, 7.00 mmol) in DMF (50 mL) was added K2CO3 (1.9 g,
0.87 mmol) and the mixture was stirred at 80 ꢁC for 1 h. Then, the
mixture was allowed to cool to room temperature, poured into
water (300 mL) and then extracted with EtOAc (3 ꢂ 150 mL). The
organic phase was concentrated under high vacuum and purified
by a column chromatography on silica gel to give compound 30d
(MeOH: DCM ¼ 0e1: 20) as orange solid (1.4 g, 70% yield). 1H NMR
0
ꢁC. After stirring at room temperature for 2 h, the organic phase
was concentrated under high vacuum and purified by a column
chromatography on silica gel to give compound 26 (MeOH:
DCM ¼ 0e1:20) as white solid (3.97 g, 87% yield). ESI-MS for
C5H3Cl2N4O [M ꢀ H]-, calcd: 205.0, found:205.0.
4-Amino-2-chloro-6-((3,5-dimethoxyphenyl)amino)pyrimi-
dine-5-carboxamide (27). To a mixture of compound 26 (1.0 g,
4.83 mmol) and 3,5-dimethoxyaniline (0.89 g, 5.80 mmol) in 1,4-
dioxane (60 mL) was added diisopropylethylamine (1.50 mL,
9.66 mmol) and the mixture was stirred at 80 ꢁC overnight.
Resulting precipitates were collected by suction filtration, washed
with water and dried under reduced pressure to give compound 27
(500 MHz, DMSO‑d6):
d
12.52 (s, 1H), 9.55 (d, J ¼ 3.55 Hz, 1H), 8.88
(t, J ¼ 5.80 Hz, 1H), 7.94 (d, J ¼ 1.75 Hz, 1H), 7.42 (d, J ¼ 3.55 Hz, 1H),
7.37 (d, J ¼ 1.65 Hz, 1H), 6.96 (t, J ¼ 7.70 Hz, 1H), 6.82 (d, J ¼ 2.25 Hz,
2H), 6.54 (brs, 1H), 6.51 (d, J ¼ 7.45 Hz, 1H), 6.45 (dd, J ¼ 8.00,
1.30 Hz,1H), 6.12 (t, J ¼ 2.20 Hz,1H), 5.04 (s, 2H), 4.71 (d, J ¼ 5.65 Hz,
2H), 3.61 (s, 6H). ESI-MS calcd for C22H24N7O3 [M þ H]þ, calcd:
434.2, found: 434.3.
as white solid (0.6 g, 38% yield). 1H NMR (500 MHz, DMSO‑d6)
d 9.67
(s, 1H), 7.70 (s, 1H), 7.20 (s, 1H), 6.78 (d, J ¼ 2.15 Hz, 2H), 6.19 (t,
J ¼ 2.10 Hz,1H), 3.72 (s, 6H). HRMS (ESI) for C13H15ClN5O3 [M þ H]þ,
calcd: 324.0785, found: 323.9773.
In vitro kinase assay. The caliper mobility shift assay of all final
compounds against ZAP-70 and Syk was conducted by Shanghai
ChemPartner Co., Ltd. (China) with ATP concentration at Km. The
proteins ZAP-70 (Cat. No. 08e176) and Syk (Cat. No. 08e377) were
purchased from Carna. Peptide FAM-22 and staurosporine were
mainly purchased from GL Biochem and Selleckchem. Briefy, the
compounds and ZAP-70 or Syk protein were diluted in kinase buffer
and pre-incubated for 10 min prior to initiating the reaction by
adding peptide FAM-22 and ATP solution. The assay plate was
incubated for additional 30 min. Then, the reaction was stopped
and the data was collected on Caliper. Details on procedures are
provided in the supporting information.
5-Chloro-7-((3,5-dimethoxyphenyl)amino)imidazo[1,2-c]py-
rimidine-8-carboxamide (28). To a mixture of compound 27
(250 mg, 0.77 mmol) and NaHCO3 (65 mg, 0.77 mmol) in t-BuOH
(50 mL) was added NaI (5 mg) then, 40% chloroacetaldehyde
(0.6 mL, 2.31 mmol) and the mixture was stirred at 80 ꢁC overnight.
The mixture was allowed to cool to room temperature, concen-
trated under high vacuum and purified by a column chromatog-
raphy on silica gel to give compound 28 (MeOH: DCM ¼ 0e1:20) as
yellow solid (100 mg, 37% yield). 1H NMR (500 MHz, chloroform-d)
d
11.60 (s, 1H), 7.51 (d, J ¼ 1.65 Hz, 1H), 7.44 (d, J ¼ 1.65 Hz, 1H), 6.80
Mass spectrometry sample treatment. Kinase ZAP-70 (Carna,
(d, J ¼ 2.15 Hz, 2H), 6.28 (t, J ¼ 2.20 Hz, 1H), 3.82 (s, 6H). ESI-MS
Cat. No. 08e176) was added into 60
mL of assay buffer (0.01%
10

D. Rao, H. Li, X. Ren et al.
European Journal of Medicinal Chemistry 219 (2021) 113393
Triton ꢂ 100, 50 mM HEPES, 5 mM MgCl2, 1 mM MnCl2) with a final
concentration of 75 nM. Compound 18 was then added to the
protein solution with a final concentration of 55 nM, and the re-
action mixture was incubated at 28 ꢁC for 5 min. Next, dithiothreitol
was added to the mixture with a final concentration of 2 mM at
room temperature for 10 min. Then, the protein was digested with
streptomycin (100
m
g/mL) in a humidified, 37 ꢁC, 5% CO2-containing
incubator. Compounds were dissolved with 100% dimethylsulf-
oxide (DMSO, Sinopharm, China) and then diluted with RPMI 1640
media containing 10% FBS. After incubation for 24 h or 48 h, the cell
cultures were pulsed with 0.5 m
Ci/well [3H] thymidine to determine
the proliferation activity of CD4þ T cells. The incorporated radio-
activity was counted using a Beta Scintillation Counter (MicroBeta
Trilux, PerkinElmer Life Sciences, Boston, MA). The cell superna-
tants were collected to determine the cytokine levels, meanwhile,
the activation of T cells was detected by flow cytometry.
0.1 m
g of trypsin at 37 ꢁC for 18 h, and the digestion was terminated
by adding trifluoroacetic acid with a final volume of 2%. The peptide
mixture was desalted by C18 Tips.
Drug conjugate sites analysis. The desalted peptides were
dissolved in HPLC buffer A (0.1% formic acid, v/v) and separated by
EASY-nLC 1200 UHPLC system (ThermoFisher Scientific) with a
30 min gradient of 5e90% HPLC buffer B (0.1% formic acid in 80%
acetonitrile, v/v). The eluted peptides were analyzed by a Q Exactive
HF-X mass spectrometer (ThermoFisher Scientific) using a nano-
spray source. Mass spectrometry was performed in data depen-
dent acquisition mode with a full-scan (350e1350 m/z) mass res-
olution of 60,000. The 9 most intensive ions were fragmented with
28% normalized collision energy and tandem mass spectra were
acquired with a mass resolution of 30,000. Charge states of the
precursors other than 2e5 were filtered out for MS2 analysis. The
AGC numbers were 1 ꢂ 10ꢀ6 and 2 ꢂ 10ꢀ5 for MS1 and MS2,
respectively. The maximum injection times of MS1 and MS2 were
set to 45 ms and 100 ms, respectively. Dynamic exclusion is set to
5 s. The resulted data was searched according to the ZAP-70
sequence using Mascot 2.3. The drug conjugation on Cysteine
(þ473.1812 Da) was designated as a variable modification.
CFSE labeling. Primary CD4þ T cells were labeled with 5-
carboxyfluorescein diacetate succinimide ester (CFSE,
4 mM,
Sigma-Aldrich, St. Louis, MO, USA) at 37 ꢁC for 5 min and then
washed for three times with the RPMI 1640 media containing 20%
FBS. Labeled cells were then cultured with anti-CD3 antibodies
(5 mg/ml) and anti-CD28 antibodies (2 mg/ml) in the RPMI 1640
media containing 10% FBS for 72 h. The fluorescence intensity of
CFSE was detected by flow cytometry.
Flow cytometry analysis. CD4þ T Cells were blocked with anti-
CD16/CD32 mAb (eBioscience) and stained with fluorescein iso-
thiocyanate (FITC)-conjugated CD3, phycoerythrin (PE)-conjugated
CD25, allophycocyanin (APC)-conjugated CD4, and brilliant violet
(BV421)-conjugated CD69. All immunofluorescent mAbs used in
this research were obtained from BD Biosciences (Franklin Lakes,
NJ, USA). The data were analyzed using FlowJo software (Tree Star,
Ashland, OR, USA).
Enzyme-linked immunosorbent assay (ELISA). Cytokines in
culture supernatants were determined by using mouse IL-4 and
IFN-g ELISA kits (BD Pharmingen) according to the manufacturer’s
instructions. The OD values were obtained at 450 nm by a micro-
plate reader (Molecular Devices, Sunnyvale, CA, USA).
Docking procedures. The published crystal structure of ZAP-70
in complex with compound 2 (PDB: 1U59) was used for the
modelling of binding modes. The protein structure was prepared
€
using the protein preparation wizard in Maestro 9.1 (Schrodinger,
LLC, New York, NY, 2010), which assigns bond order, adds hydro-
gens, and deletes waters. Compounds 2 and 18 were built by Lig-
Prep module using OPLS_2005 force field. The grid box was
centered on the cocrystallized ligand. Compound 2 was docked in
Ligand Docking module using extra precision (XP) approach, and
during docking, H-bond constraints were imposed in the hinge
region. The docking program for compound 18 was performed in
Covalent Docking module. The reaction type of nucleophilic sub-
stitution was selected. Pose prediction (Thorough) approach was
applied with other setting by default parameters. The final poses
with low energy conformations and good hydrogen-bond geome-
tries were selected.
Statistical analysis. Data in the present research were shown as
mean
SD. Statistical significance was determined by one-way
ANOVA with Dunnet’s multiple comparisons test by GraphPad
Prism 8.0 (La Jolla, CA, USA) with no significant variance in-
homogeneity in groups of more than two. p < 0.05 was considered
statistically significant.
Declaration of competing interest
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.
Mice. Inbred female BALB/c mice (6-8-week-old, 18e20 g) were
obtained from Shanghai Laboratory Animal Center of Chinese
Academy of Sciences and housed under specific pathogen-free
(SPF) conditions with a stabilized condition (12 h of light/12 h of
Acknowledgment
We would like to thank the financial support from the Natural
Science Foundation of Shanghai (20ZR1468400), the Science &
Technology Commission of Shanghai Municipality, China (No.
18431907100), and Young Elite Scientists Sponsorship Program by
CAST (2019-2021QNRC001), National Natural Science Foundation
of China (81973164), Shanghai Pujiang Program (19PJ1411200), and
Shanghai Institute of Materia Medica.
dark cycle, 24
2
ꢁC, 55 5% relative humidity). All experiments
were carried out according to the Bioethics Committee of the
Shanghai Institute of Materia Medica.
CD4þ T cells purification and in vitro stimulation. Mono-
nuclear cell suspensions from murine spleens were prepared from
BALB/c mice. Briefly, the erythrocytes from splenocytes were
depleted with ammonium chloride buffer solution and then cell
debris and clumps were removed. Upon mononuclear splenocytes
preparation, the polyclonal CD4þ T cells were purified using Easy-
Sep™ mouse CD4þ T Cell isolation kit (Stemcell, Vancouver, BC,
Canada) according to the manufacturer’s instructions by immuno-
magnetic negative selection. The purity of the CD4þ T cells was
consistently >98%, analyzed by flow cytometry. Subsequently, pu-
rified CD4þ T cells were incubated with medium alone or com-
pounds at the indicated concentrations in the presence of anti-CD3
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2021.113393.
Abbreviations
4-DMAP 4-Dimethylaminopyridine
antibodies (5
and anti-CD28 antibodies (2
RPMI 1640 media containing 10% FBS, penicillin (100 U/mL), and
m
g/ml, Thermo Fisher Scientific, Waltham, MA, USA)
AGC
APC
BV
automatic gain control
allophycocyanin
brilliant violet
mg/ml, Thermo Fisher Scientific) in the
11

D. Rao, H. Li, X. Ren et al.
European Journal of Medicinal Chemistry 219 (2021) 113393
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CFSE
DCC
5-carboxyfluorescein diacetate succinimide ester
Dicyclohexylcarbodiimide
dichloromethane
N,N-Diisopropylethylamine
N,N-Dimethylformamide
Dimethyl sulfoxide
electrospray ionization mass spectrometry
Foetal Bovine Serum
fluorescein isothiocyanate
High performance liquid chromatography
interferon-g
interleukin 4
linker for activation of T cell
natural killer cell
phycoerythrin
SH2-domian-containing leukocyte protein of 76 kDa
specific pathogen-free
spleen tyrosine kinase
T cell receptor
trifluoroacetic acid
tetrahydrofuran
Ultra performance liquid chromatography
zeta-chain associated protein kinase 70 kDa
DCM
DIPEA
DMF
DMSO
ESI-MS
FBS
FITC
HPLC
IFN-
IL-4
LAT
NK
g
,
PE
SLP-76
SPF
Syk
TCR
TFA
THF
UPLC
ZAP-70
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12