Discovery of thieno[2,3-d]pyrimidin-4(3H)-one derivatives as a new class of ROCK inhibitors

Article abstract of DOI:10.1016/j.bmcl.2020.126966

Herein, we report the discovery of a series of thieno[2,3-d]pyrimidin-4(3H)-one derivatives as a new class of ROCK inhibitors. Structure-activity relationship studies of these compounds led to the identification of the most potent compound, 3-(3-methoxybenzyl)-6-(1H-pyrrolo[2,3-b]pyridin-4-yl)thieno[2,3-d]pyrimidin-4(3H)-one (8k), which showed IC₅₀ values of 0.004 μM and 0.001 μM against ROCK Ⅰ and ROCK Ⅱ, respectively. In vitro, 8k significantly reduced the phosphorylation level of ROCK downstream signaling protein and induce changes in cell morphology and migration. Overall, this study provides a promising lead compound for drug discovery targeting ROCKs.

Full text of DOI:10.1016/j.bmcl.2020.126966

Journal Pre-proofs
Discovery of Thieno[2,3-d]pyrimidin-4(3H)-one Derivatives as a New Class of
ROCK Inhibitors
Zhuang Miao, Yu-meng Sun, Lan-ying Zhao, Yue-shan Li, Yi-fei Wang, Jin-
shan Nan, Ze-en Qiao, Lin-li Li, Sheng-yong Yang
PII:
S0960-894X(20)30019-6
DOI:
https://doi.org/10.1016/j.bmcl.2020.126966
BMCL 126966
Reference:
Bioorganic & Medicinal Chemistry Letters
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Accepted Date:
21 November 2019
3 January 2020
11 January 2020
Please cite this article as: Miao, Z., Sun, Y-m., Zhao, L-y., Li, Y-s., Wang, Y-f., Nan, J-s., Qiao, Z-e., Li, L-l., Yang,
S-y., Discovery of Thieno[2,3-d]pyrimidin-4(3H)-one Derivatives as a New Class of ROCK Inhibitors, Bioorganic
& Medicinal Chemistry Letters (2020), doi: https://doi.org/10.1016/j.bmcl.2020.126966
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Discovery of Thieno[2,3-d]pyrimidin-4(3H)-one Derivatives
as a New Class of ROCK Inhibitors
Zhuang Miaoa,#, Yu-meng Sunb,#, Lan-ying Zhaoa, Yue-shan Lia, Yi-fei Wangb, Jin-
shan Nana, Ze-en Qiaoa, Lin-li Lib,*, Sheng-yong Yanga,*
aState Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan
University, Chengdu, Sichuan 610041, China.
bKey Laboratory of Drug Targeting and Drug Delivery System of Ministry of Education,
West China School of Pharmacy, Sichuan University, Chengdu, Sichuan 610041, China
# These authors contributed to this work equally
* Corresponding author. Tel.: +86-28-85164063; fax: +86-28-85164060
E-mail address: (Sheng-yong Yang) yangsy@scu.edu.cn;
(Lin-li Li) ysylilinli@sina.com
1

Abstract
Herein, we report the discovery of a series of thieno[2,3-d]pyrimidin-4(3H)-one
derivatives as a new class of ROCK inhibitors. Structure-activity relationship studies
of these compounds led to the identification of the most potent compound, 3-(3-
methoxybenzyl)-6-(1H-pyrrolo[2,3-b]pyridin-4-yl)thieno[2,3-d]pyrimidin-4(3H)-one
(8k), which showed IC50 values of 0.004 μM and 0.001 μM against ROCK Ⅰ and ROCK
Ⅱ, respectively. In vitro, 8k significantly reduced the phosphorylation level of ROCK
downstream signaling protein and induce changes in cell morphology and migration.
Overall, this study provides a promising lead compound for drug discovery targeting
ROCK.
Keywords
ROCK, kinase inhibitor, structure-activity relationship, cell migration, cell morphology
2

Rho-associated coiled-coil containing protein kinases (ROCKs) belong to the
AGC subfamily of the serine/threonine protein kinases. There are two isoforms for
ROCK, namely, ROCK Ⅰ and ROCK Ⅱ, which share 65% overall sequence homology
and are highly homologous in their ATP-binding pocket.1 After activated by GTP-
bound Rho protein, ROCKs trigger changes in cell morphology2 and migration2,3, and
regulates cytoskeleton through the phosphorylation of their numerous downstream
proteins. Myosin light chain (MLC) and the myosin phosphatase targeting subunit 1
(MYPT1) act as the main substrates of ROCKs, which are highly associated with
contractility of smooth muscle cells.1 Therefore, ROCK inhibition provides a potential
treatment where smooth muscle intervention is required, such as erectile dysfunction,4
glaucoma,5,6 pulmonary arterial hypertension,7 and cardiovascular disease.8,9 In
addition, symptoms including neurological disorders,10 tumorigenesis,11 and Bullous
Keratopathy12 have also been demonstrated to be linked with ROCKs. Currently, a
number of ROCK inhibitors have been reported13 and three of them (Fasudil, Ripasudil
and Netarsudil) have been approved to use clinically (Figure 1). Fasudil is prescribed
for the treatment of cerebral vasospasm. Ripasudil and Netarsudil are used for the
treatment of glaucoma by increasing aqueous humor outflow.14,15 Even so, discovering
more potent ROCK inhibitors with new scaffolds is still necessary, which is for
increasing additional options for coping with other indications and possible drug
resistance in future.
Figure 1. Chemical structures of clinically used ROCK inhibitors.
3

To achieve this goal, we conducted a molecular docking based virtual screening
(VS) against our in-house chemical database (Figure S1), which gave a hit compound,
3-(3-methoxybenzyl)-7-(1H-indazol-5-yl)quinazolin-4(3H)-one (1, Figure 2). This
compound displayed moderate inhibitory activity against ROCK (ROCK Ⅰ: 3.106 µM,
and ROCK Ⅱ: 0.088 µM). We then carried out a structural optimization towards 1 to
improve its potency. The structural modifications were focused on three regions,
quinazolin-4(3H)-one (region I), indazole (region Ⅱ), and methoxy benzene (region Ⅲ).
A total of twenty-four compounds (4a-c, 8a-l, 10a-e, and 11a-e) were synthesized and
the structure-activity relationship was further discussed.
Figure 2. The chemical structure of hit compound 1 obtained by virtual screening.
In the first step, we fixed region Ⅱ, region Ⅲ and varied region Ⅰ. Four compounds
(4a-c, 8a) were prepared. Scheme 1 depicts the synthetic routes of compounds 4a-c.
Briefly, commercially available reagents 2a-c reacted with 3-methoxybenzyl bromide
gave intermediates 3a-c. Suzuki-Miyaura coupling of 3a-c with 1H-indazole-5-boronic
acid pinacol ester generated compounds 4a-c.
4

Scheme 1. Synthetic routes of compounds 4a-c. Reagents and conditions: (i) DMF, 3-
methoxybenzyl bromide, NaH, 0 oC – room temperature (RT), 12 h, 35%-45%; (ii) 1,4-
dioxane/H2O (5/1), 1H-indazole-5-boronic acid pinacol ester, Pd(PPh3)4, Cs2CO3, N2,
95 oC, 12 h, 19%-24%.
Chemical structures and ROCK inhibitory activities of these compounds are listed
in Table 1. Removing N1 or shifting it to 2-position had no obvious enhancement in
terms of the potency (4a, 4b vs 1). A decline in inhibitory activity was observed when
isoquinolin-1(2H)-one in 4b was saturated (4c vs 4b). It is worth noting that compound
8a, which contains a thieno[2,3-d]pyrimidin-4(3H)-one moiety, showed a significant
improvement in ROCK inhibitory potency. Therefore, thieno[2,3-d]pyrimidin-4(3H)-
one was selected as the optimal fragment in region Ⅰ.
Table 1. Chemical structures and ROCK inhibitory activities of compounds 4a-c and
8a.
5

IC50 (µM)a
Compound
Region Ⅰ
ROCK Ⅰ
ROCK Ⅱ
3.106
0.088
1
> 10
1.877
2.062
0.020
0.398
0.107
0.632
0.002
4a
4b
4c
8a
aIC50 values were determined from Kinase Profiler of Eurofins. [ATP] = 10 µM.
In the second step, we optimized region Ⅱ, with region Ⅰ fixed as the optimal
fragment thieno[2,3-d]pyrimidin-4(3H)-one and region Ⅲ fixed as its original group.
To this end, various heterocyclic rings were utilized to replace the indazole moiety in
region Ⅱ. A total of 11 new compounds (8b-l) were synthesized. Scheme 2 shows the
synthesis routes of compounds 8a-l. Bromination of thieno[2,3-d]pyrimidin-4(3H)-one
(5) under acetic acid produced intermediate 6. Substitution reaction of intermediate 6
yielded 7. Then the desired products were obtained through Suzuki-Miyaura coupling
of 7 with different boric acid or boric acid ester groups.
6

Scheme 2. Synthesis routes of compounds 8a-l. Reagents and conditions: (i) AcOH,
Br2, RT, 4 h, 90%; (ii) DMF, 3-methoxybenzyl bromide, K2CO3, RT, 12 h, 47%; (iii)
1,4-dioxane/H2O (5/1), Pd(PPh3)4, Cs2CO3 (8a) or K2CO3 (8b-l), N2, 95 oC, 12 h, 21%-
57%.
Table 2 summarizes the chemical structures and ROCK inhibitory activities of
these compounds. Compound 8b, which has a pyridine group, displayed considerable
potency against ROCK. Lengthening pyridine by introducing an extra benzene ring, or
changing the position of nitrogen atom in pyridine led to an obvious loss in ROCK
inhibitory activity (8c, 8d vs 8b), indicating that the nitrogen atom should be fixed at
para-position. We then investigated the possible impact of different substituents on
pyridine. The generated compounds all exhibited obviously decreased potencies (8e-h
vs 8b) except 8i, which contains an amino substituent (2-aminopyridine). The
inhibitory activity almost disappeared when changing the position of nitrogen atom and
the amino group (8j vs 8i). A ring-closed analog (8k) of 8i, which possesses a 7-
azaindole fragment, displayed a higher ROCK Ⅰ activity (IC50 = 0.004 µM) and
7

comparable ROCK Ⅱ inhibition activity (IC50 = 0.001 µM). Replacing azaindole by
indole (8l vs 8k) significantly reduced the potency, indicating again the importance of
nitrogen atom at para-position. Collectively, 2-aminopyridine or 7-azaindole may be
the best choice for region II.
Table 2. Chemical structures and ROCK inhibitory activities of 8b-l.
IC50 (µM)a
Compound
Region Ⅰ
ROCK Ⅰ
ROCK Ⅱ
0.020
0.002
8a
0.045
> 10
0.006
3.475
8b
8c
> 10
> 10
8d
8e
1.050
0.083
8

> 10
2.515
> 10
0.501
0.042
6.915
0.001
8f
8g
8h
8i
0.018
> 10
> 10
8j
0.004
> 10
0.001
0.583
8k
8l
aIC50 values were determined from Kinase Profiler of Eurofins. [ATP] = 10 µM.
In the last step, we optimized region Ⅲ with region I and II fixed as their optimal
groups. A total of ten compounds (10a-e and 11a-e) with different substituted phenyl
groups at region III were synthesized. According to the fragments at region II, the
prepared compounds were classified into two categories: A (7-azaindole at region II)
and B (2-aminopyridine in region II). The synthetic routes for these compounds are
given in Scheme 3. Substitution reaction of 6 with various substitutional benzyl
9

bromide afforded intermediates 9a-e. Suzuki-Miyaura coupling of 9a-e with 7-
azaindole-4-boronic acid pinacol ester or 2-aminopyridine-4-boronic acid pinacol ester
provided final compounds 10a-e or 11a-e.
Scheme 3. Synthesis routes of compounds 10a-e and 11a-e. Reagents and conditions:
(i) DMF, K2CO3, RT, 12 h, 41%-53%; (ii) 1,4-dioxane/H2O (5/1), Pd(PPh3)4, K2CO3,
N2, 95 oC, 12 h, 37%-55%.
Chemical structures and ROCK inhibitory activities of these compounds are
shown in Table 3. It can be seen that compounds 8i and 8k are still the best one in their
corresponding categories. Therefore, region Ⅲ was fixed as its original form. Because
8k was the most potent one among all the synthesized compounds, further studies were
carried out only on this compound.
Table 3. Chemical structures and ROCK inhibitory activities of compounds 10a-e and
11a-e.
10

IC50 (µM)a
Compound
series
Region Ⅲ
ROCK Ⅰ
ROCK Ⅱ
0.004
0.013
0.001
8k
0.001
10a
0.110
0.013
0.025
0.007
0.002
0.004
10b
10c
10d
A
0.074
0.004
10e
0.018
0.034
0.001
0.005
8i
11a
B
0.490
0.025
11b
11

1.445
> 10
0.046
0.219
11c
11d
> 10
0.058
11e
aIC50 values were determined from Kinase Profiler of Eurofins. [ATP] = 10 µM.
Western blot assays were used to examine the activity of compound 8k in intact
cells. Here 293T cells were used, and the phosphorylation level of ROCK direct
downstream protein MYPT1 was measured. Phosphatase inhibitor Cocktail was
utilized to stabilize phosphorylated MYPT1 (p-MYPT1) because p-MYPT1 is easy to
be dephosphorylated at the time of cell disruption. As shown in Figure 3, under the
protection of Cocktail, we could observe the phosphorylated MYPT1, and 8k
significantly reduced the phosphorylation level of MYPT1. We also noticed that 8k did
not influence the level of MYPT1. Because MYPT1 is one of the common substrates
of ROCK I and II 13,16 and 8k has inhibitory activity against both ROCK I and II, it is
expected that the decrease in the phosphorylation level of MYPT1 is due to the co-
inhibitory effects of 8k against ROCK I and ROCK II. Overall, these results
demonstrated that 8k inhibited the ROCK activity in intact cells.
Scratch wound assay was carried out to investigate whether 8k can induce changes
in cell migration. Here, MDA-MB-231 cells were used. As exhibited in Figure 4, 8k
12

significantly prevented cell migration compared with the control group. We also tracked
the cell shape, and observed that most of the MDA-MB-231 cells displayed spindle-
shaped morphology on the edges upon 8k treatment (see Figure 5). These results
indicate that 8k is able to inhibit cell migration in vitro and has a potential to regulate
cell morphology.
Finally, we examined the selectivity of compound 8k. To this end, 100
representative kinases covering all kinase families were selected. Inhibition rates of 8k
against these kinases were measured at 1 µM (Table S1). The human kinome
dendrogram based on the measured inhibition rates is shown in Figure 6. In addition to
ROCKs, 8k also displays considerable activity against PKA, LIMK2, and Flt1. Of note
is that 8k did not show obvious inhibitory activity against all other tested kinases,
indicating a good selectivity. The calculated scores, S(5), S(10) and S(20), are 0.029,
0.049 and 0.137, respectively.
Figure 3. (A) 8k reduced p-MYPT1 level in the 293T cell line. (B) The quantification of the
immunoblot (* P < 0.05, t test).
13

Figure 4. 8k inhibited MDA-MB-231 cells migration in vitro.
Figure 5. MDA-MB-231 cells morphology changes at 60 min after treating with 1 µM 8k.
14

Figure 6. Kinase selectivity of 8k shown on the human kinome dendrogram determined by the
Kinase Profiler of Eurofins.
Collectively, we obtained a new series of thieno[2,3-d]pyrimidin-4(3H)-one
derivatives as ROCK inhibitors. The most potent compound corresponds to 8k, which
showed IC50 values of 0.004 µM and 0.001 µM against ROCK I and ROCK II,
respectively. In vitro, 8k could efficiently inhibit ROCK activity in intact cells, alter
cell morphology, and inhibit cell migration. 8k could be a promising lead compound
for drug discovery targeting ROCK and deserves further studies.
Acknowledgment
This work was supported by the National Natural Science Foundation of China
(81773633, 21772130, and 81930125), National Science and Technology Major Project
15

(2018ZX09711002-014-002,
2018ZX09711002-011-019,
2018ZX09201018,
2018ZX09711003-003-006, and 2019ZX09301-135), and 1.3.5 project for disciplines
of excellence, West China Hospital, Sichuan University.
Supporting Information
Supplementary data associated with this article can be found in the online version.
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