Discovery and optimization of 5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one derivatives as mTORC1/mTORC2 dual inhibitors

Article abstract of DOI:10.1016/j.bioorg.2019.103232

New potent mTORC1/mTORC2 dual inhibitors, 5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one derivatives, were obtained by optimizing functional groups on our previously reported PI3Kα inhibitor. All the target compounds were synthesized and structural optimization on the structure of the lead compound based on cytotoxic activity. The results showed that some of the target compounds exhibited moderate to high cytotoxic activity against cell line U87MG and PC-3. The activities against mTOR kinase were investigated and the compound 12q showed excellent activity with an IC₅₀ value of 54 nM in the same level of the positive control BEZ235 with IC₅₀ value of 55 nM under the same test conditions. The western blot and cell cycle results demonstrate that compound 12q is a candidate as an mTORC1/mTORC2 dual-target inhibitor. The theoretical calculations were also performed to better understanding the binding modes of the compound 12q in the mTOR active site.

Full text of DOI:10.1016/j.bioorg.2019.103232

BioorganicChemistry92(2019)103232
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Discovery and optimization of 5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-
one derivatives as mTORC1/mTORC2 dual inhibitors
Shengquan Hu, Zhichang Zhao, Hong Yan⁎
Beijing Key Laboratory of Environmental and Viral Oncology, College of Life Science and Bio-engineering, Beijing University of Technology, Beijing 100124, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
New potent mTORC1/mTORC2 dual inhibitors, 5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one derivatives,
were obtained by optimizing functional groups on our previously reported PI3Kα inhibitor. All the target
compounds were synthesized and structural optimization on the structure of the lead compound based on cy-
totoxic activity. The results showed that some of the target compounds exhibited moderate to high cytotoxic
activity against cell line U87MG and PC-3. The activities against mTOR kinase were investigated and the
compound 12q showed excellent activity with an IC50 value of 54 nM in the same level of the positive control
BEZ235 with IC50 value of 55 nM under the same test conditions. The western blot and cell cycle results de-
monstrate that compound 12q is a candidate as an mTORC1/mTORC2 dual-target inhibitor. The theoretical
calculations were also performed to better understanding the binding modes of the compound 12q in the mTOR
active site.
mTOR
mTORC1/mTORC2 dual-target
5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one
Synthesis
Biological study
Theoretical calculation
1. Introduction
used as an antifungal and immunosuppressive agent [11,12]. It was
discovered to have potent immunosuppressive and antiproliferative
The PI3K-Akt-mTOR signaling pathway, regulated by growth fac-
tors, is one of the most important intracellular network pathways which
regulating various cell life activities including survival, metabolism,
proliferation and growth [1–4]. In mammalian cells, mammalian target
of rapamycin (mTOR) is composed of two complexes respectively,
mTORC1 and mTORC2, which regulate different cellular processes with
a variety of related proteins. As a regulatory-associated protein of
mTOR (Raptor), mammalian lethal with SEC13 protein 8 (mLST8) and
the non-core components PRAS40 and DEPTOR, mTORC1 regulates cell
growth and proliferation by controlling protein synthesis and regula-
tion of ribosomal biosynthesis [5,6]. As a rapamycin-insensitive com-
panion of mTOR (RICTOR), mLST8 and mammalian stress-activated
protein kinase interacting protein 1 (mSIN1), mTORC2 regulates Akt
phosphorylation upstream of Akt [7]. Abnormal activation of the mTOR
signaling pathway may directly or indirectly lead to the occurrence,
development, and metastasis of tumors [8]. Over-activation mainly
characterized by excessive activation of mTOR signaling pathway may
cause loss of PTEN function and obstruction of autophagy pathway
[9,10]. Therefore, inhibiting the activity of mTOR can inhibit the pro-
liferation of tumor cells, induce apoptosis of tumor cells and reverse the
resistance of tumor cells to cytotoxic drugs.
properties as an anti-cancer agent due to its ability to inhibit mTOR.
The complex of Rapamycin and Rapamycin analogs (Rapalogs) with
FK506-binding protein-12 (FKBP12) binds to mTOR via FKBP12-Ra-
pamycin binding (FRB) domain. It is near to the tyrosine kinase domain
of mTORC1 and consequently inhibits the phosphorylation of mTOR
substrates such as S6K1 and 4EBP1 [13–15]. The US Food and Drug
Administration (FDA) has approved several Rapalogs inhibitors, such as
Everolimus [16,17] and Temsirolimus [18,19] for cancer treatments
(Fig. 1). However, only limited benefits from Rapalogs therapy have
been observed in clinical due to incomplete inhibition of mTORC1 and
the inability to effectively inhibit mTORC2 [20,21].
The small-molecule inhibitors of mTOR under development are di-
vided into two categories: PI3K/mTOR dual-target inhibitors and
mTORC1/mTORC2 dual-target inhibitors [20,22]. The mTORC1/
mTORC2 dual-target inhibitors, also known as ATP-competitive mTOR
inhibitors, are completely inhibiting the phosphorylation of the Rapa-
mycin-insensitive sites Thr37 and Thr46 in the mTORC1 substrate 4E-
BP1 and S6K1 [23]. Moreover, it is inhibiting phosphorylation of
mTORC2 and its substrate Akt at Ser473 [22,24]. For their smaller
molecular weight than that of the FKBP-12 Rapamycin complex,
mTORC1/mTORC2 dual-target inhibitors were easier to target the
mTOR binding site and result in stronger inhibition of protein synthesis,
Rapamycin (Fig. 1), a natural macrolide compound, was originally
⁎ Corresponding author.
E-mail address: hongyan@bjut.edu.cn (H. Yan).
https://doi.org/10.1016/j.bioorg.2019.103232
Received 18 March 2019; Received in revised form 26 August 2019; Accepted 28 August 2019
Availableonline04September2019
0045-2068/©2019ElsevierInc.Allrightsreserved.

S. Hu, et al.
BioorganicChemistry92(2019)103232
Fig. 1. Chemical structures of Rapamycin and Rapamycin analogs.
Fig. 2. Chemical structures of mTORC1/mTORC2 dual-target inhibitors.
cell growth and autophagy-inducing effect [25]. In addition, they have
strong selectivity for mTOR and little or no inhibition of the PITOR
kinase family with high mTOR homology (Fig. 2) [20,24].
In our previous work, the lead compound of 4-aryl-5,7-dihydro-6H-
pyrrolo[2,3-d]pyrimidin-6-one derivatives were designed as a PI3Kα
inhibitor by molecular hybridization and scaffold hopping [26]. It
showed a strong PI3Kα inhibitory activity with IC50 of 113 nM and
mTOR inhibitory activity with IC50 of 1094 nM. Here, based on cyto-
toxic activities, the synthesis and structural optimization design on the
three functional groups of the lead compound centered on pyrrolo[2,3-
d]pyrimidin-6-one were investigated (Fig. 3). All the target compounds
were evaluated in vitro for their anti-mTORC1/mTORC2 activity
against cell lines U87MG and PC-3. The enzyme inhibitory activity
against mTOR kinase of compounds was evaluated inferior to the po-
sitive control of BEZ235. The intracellular mTOR pathway inhibitory
activities of the promising compound was evaluated by western blot
and cell cycle. Their preliminary structure-activity relationships (SARs)
and theoretical calculations were described to better understanding the
possible binding modes in the mTOR active site.
Fig. 3. Optimization design of 5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one
derivatives.
2. Result and discussion
2.1. Synthesis and structural optimization based on cytotoxic activity
The initial optimization design was the dearylation of the C-4 po-
sition substituents of the lead compound. The target compounds 9a-e
2

S. Hu, et al.
BioorganicChemistry92(2019)103232
Scheme 1. Reagents and conditions: (a) EtONa, EtOH; (b) MeONa, MeOH; (c) DIPEA, POCl3; (d) DIPEA, DMF; (e) 4-DMAP, DMF; (f) TsOH, Toluene; (g) DIPEA, DMA.
were synthesized with the diethyl malonate (1) and ethyl chloroacetate
(2) as starting materials (Scheme 1). The intermediate 3 was prepared
by the nucleophilic substitution of 1 and 2 under basic conditions. The
intermediate 3 was reacted with urea in a solution of sodium methoxide
to give the corresponding barbituric acid 4. Intermediate 5 was ob-
tained by the chlorinated of 4 with phosphorus oxychloride. The in-
termediate 6 was obtained by the substitution of 5 with 4-methox-
aliphatic groups. The target compounds 12a-p were synthesized with
the methyl (2,4,6-trichloropyrimidin-5-yl) acetate (5) as starting ma-
terials (Scheme 2). The 10a-p were synthesized by a one-pot synthesis
strategy in two substitutions of the 5 by the G2-amine and morpholine
under alkaline conditions. The 11a-p were obtained by the in-
traamidation of 10a-p described above. Moreover, the 11a-p reacted
with the (6-aminopyridin-3-yl) boronic acid to give the target com-
pounds 12a-p under the palladium catalysis.
ybenzylamine. The intermediate
7 was obtained by the non-
nucleophilic substitution reactions of 6 with morpholine. The inter-
mediate 7 was intraamidation under acidic conditions to form the
corresponding product 8. Intermediate 8 reacts with the corresponding
secondary amine under the basic condition to give the target com-
pounds 9a-e respectively.
The results of cytotoxic activities shown that the 12a-p activities
were improved but not obvious. The activities of 12k, 12l and 12m
were close to that of the lead compound with cyclohexyl, cyclopropyl
and isopropyl groups as G2 (Table 2). That means the derivatization on
the N-7 position does not improved significantly the cytotoxic activity
of target compounds.
Cytotoxic activities of 9a-e were carried out as described previously
in vitro detected on mTOR-driven cancer cell lines human malignant
glioblastoma U87MG and human prostate cancer PC-3 by CCK-8
[27,28]. The multi-targeted pyridinylfuranopyrimidine inhibitor PI-103
served as the reference compound. The inhibition rates of compounds
(50 µM) and positive control (50 µM) were presented in Table 1, which
calculated by Formula (1) calculate for inhibition rates.
Since the optimization design on the dearylation of the C-4 position
and the derivatization at the N-7 position does not improved sig-
nificantly the cytotoxic activity, the 6-aminopyridin-3-yl substitute at
C-4 position may be one of the best choices. Therefore, the 12q-y were
designed as target compounds while the G2 was cyclohexyl, cyclo-
propyl, and isopropyl, the G1 was a pyrimidinylamine or arylamine. The
12q-y were synthesized by the substitution of 11k-m with the aromatic
amine, the 13q was the acetylation of 12q (Scheme 3). The cytotoxic
activities of 12q-y and 13q shown that the compound 12q displayed
the highest inhibition with the inhibition rate of 71.60% on U87MG and
61.98% on PC-3 (Table 3). The compound 12q and 13q have higher
inhibition than the PI-103 positive control on both cancer cell lines with
the inhibition rate of 62.34% and 53.36%. These two compounds
achieve the same level of activity as the lead compound with an in-
hibition rate of 70.84% and 67.03%.
ODNegative ODTreatment
Inhibition rates =
× 100%
ODNegative
(1)
As can be seen from Table 1, the compound 9a-e shown no sig-
nificant cytotoxicity, all the inhibition rates were far less than that of
the positive control. These results led us to abandon the idea of dear-
ylation of the C-4 position substituents and reserve original 6-amino-
pyridin-3-yl at the C-4 position of lead compound.
The further optimization design was derivatization on the N-7 po-
sition of the lead compound by the various substituted benzyl or
The finally optimization design was the analogues of morpholine on
3

S. Hu, et al.
BioorganicChemistry92(2019)103232
Table 1
Cytotoxic activities of compound 9a-e on U87MG and PC-3 cancer cell.
Entry
Comp. No.
G1
Log
Kow
U87MG
ODb
PC-3
ODb
a
Inhibition Rate (%)
2.80
Inhibition Rate (%)
1
2
3
4
9a
9b
9c
9d
2.33
4.08
3.18
2.30
1.356
0.034
0.019
0.024
0.028
1.261
0.030
0.023
0.016
0.019
2.52
2.79
3.96
2.79
1.303
1.384
1.360
6.86
2.74
2.79
1.267
1.237
1.267
5
9e
2.54
2.84
1.88
1.350
0.407
0.530
0.011
0.037
0.037
2.06
1.062
0.427
0.603
0.055
0.025
0.024
16.62
67.03
53.36
Lead compound
70.84
62.34
Positive control
a
PI-103
The value predicted by KOWWIN.
b
The mean value of five times measurements.
Scheme 2. Reagents and conditions: (a) I DIPEA, DMF, II 4-DMAP, DMF; (b) TsOH, Toluene; (c) (dppf)2PdCl2, Na2CO3, Dioxane/H2O.
the C-2 position substituents of the lead compound. The 16a-f were
designed as target compounds while the G1 was 3-aminophenyl and G2
was 4-methoxybenzyl. The 17a-f was the acetylated products of 16a-f.
The 16a-f and 17a-f were synthesized by the method previously de-
scribed (Scheme 4). The cytotoxic activities of 16a-f and 17a-f show
that compound 16d displayed the highest inhibition with the inhibition
rate of 65.40% of U87MG and 68.07% of PC-3 (Table 4). The 17d also
displayed strong inhibition with the inhibition rate of 65.21% of
U87MG and 14.66% of PC-3. The piperidinyl and 4-methylpiperidinyl
on C-2 position may have the same effect as morpholinyl and be key
powerful pharmacophore.
2.2. Biological study
For the compounds 12q, 13q, 16d and 17d with higher inhibition,
the further investigations were carried out to determine their IC50 va-
lues on cell line U87MG and cell line PC-3 by CCK-8. As can be seen
from Table 5, compound 12q, 13q, 16d and 17d exhibit stronger ac-
tivity than the lead compound. The IC50 value against the PC-3 cell line
was between 10 and 20 μM and the IC50 value against the U87MG cell
line was between 13 and 38 μM. The compound 12q has significantly
inhibited the proliferation of mTOR-driven cancer cell lines with an
IC50 value of 12.71 µM against the cell line U87MG and 10.31 µM
against the cell line PC-3. It was at the same level as the positive control
PI-103 with an IC50 value of 8.49 µM of U87MG and 7.55 µM of PC-3
under the same test conditions.
Through the structural optimization design on the C-4, N-7 and C-2
position of the lead compound, the compounds (12q, 13q, 16d and
17d) were found with the similar inhibition with the lead compound
against U87MG and PC-3. The dearylation of the C-4 position of lead
compound was lowering the inhibition rates. The derivatization on the
N-7 position by the substituted benzyl or aliphatic groups does not
improved the cytotoxic activity significantly. The 6-aminopyridin-3-yl
substitute at C-4 position may be one of the best choices. The piper-
idinyl and 4-methyl-piperidinyl on the C-2 position have the same effect
as morpholinyl. They share the features that the C-2 position was the
six-membered heterocycly, the C-4 position was the 3-aminophenyl or
corresponding acetylation product, and the N-7 position was the cy-
clohexyl or 4-methoxybenzyl substitutes.
The enzyme inhibitory activities were carried out as described
previously [28]. As shown in Table 5, the compound 12q has higher
mTOR inhibitory activity with IC50 of 54 nM than the lead compound
with IC50 of 1094 nM. It is also at the same level to the positive control
BEZ235with IC50 of 55 nM (reported IC50 of 9 nM) under the same test
conditions. All these indicated that structural optimization was effec-
tive based on cytotoxic activity.
In order to investigate the intracellular mTOR pathway inhibitory
activities of compound 12q, the western blot was carried on [29].
Cellular efficacy of mTORC1/mTORC2 inhibition by inhibitor com-
pounds was measured by changes in phosphorylation of P-S6K1 and p-
4

S. Hu, et al.
BioorganicChemistry92(2019)103232
Table 2
Cytotoxic activities of compound 12a-p on U87MG and PC-3 cancer cell.
Entry
Comp. No
G2
Log
U87MG
ODb
PC-3
ODb
Kowa
Inhibition Rate (%)
Inhibition Rate (%)
6
7
12a
12b
2.76
3.16
1.288
1.317
0.012
0.034
8.26
6.18
0.980
0.991
0.015
0.033
22.22
21.35
8
9
12c
12d
3.18
2.40
0.997
1.186
0.039
0.039
27.70
14.00
0.780
1.208
0.018
0.018
40.64
8.07
10
11
12
12e
12f
12g
2.96
2.30
3.40
1.252
1.243
1.244
0.054
0.043
0.052
9.21
9.88
9.82
1.194
1.235
1.184
0.056
0.066
0.102
9.13
6.01
9.86
13
14
15
12h
12i
12j
3.72
3.30
2.96
1.309
1.304
1.315
0.031
0.034
0.014
5.08
5.42
4.61
1.239
1.227
1.081
0.053
0.013
0.046
5.72
6.62
17.70
16
12k
3.32
1.316
0.022
6.28
0.790
0.042
37.30
17
18
19
12l
12m
12n
1.85
1.96
1.01
0.906
0.924
0.993
0.052
0.044
0.019
35.44
34.22
28.01
0.658
0.684
0.991
0.049
0.014
0.020
47.78
45.68
24.57
20
21
12o
12p
1.01
0.08
1.292
0.986
0.026
0.015
6.31
0.777
0.700
0.014
0.025
40.87
44.43
29.76
Lead compound
Positive control
a
2.84
1.88
0.407
0.530
0.037
0.037
70.84
62.34
0.427
0.603
0.025
0.024
67.03
53.36
PI-103
The value predicted by KOWWIN.
b
The mean value of five times measurements.
Akt, respectively. U87MG cells were treated with 12q at the indicated
concentrations and subjected to western blot analysis. It was observed
that compound 12q almost completely inhibits the phosphorylation of
S6K1 at 10 μM in U87MG cell lines, demonstrating the potent efficacy
of 12q in inhibiting the enzymatic activity of mTORC1 (Fig. 4). The
dose-dependent decrease of phosphorylation of Akt at Ser 473 indicates
that compound 12q has a certain inhibition of mTORC2, which corre-
lates well with the cells and kinase inhibition data.
cytometry (Fig. 5) [30]. Cells were treated with compound 12q with
varying concentrations and vehicle (DMSO) for 36 h. In the vehicle, the
percentage in the cells at G1-phase is 71.9%. Compared to the vehicle,
the treatment of compound 12q led to an increase of 2.5% for 1 μM,
4.6% for 3 μM and 23.8% for 10 μM in the cells at G1-phase. The
concomitant concentration of compound 12q increase, a loss of S-phase
accompanies with an increase in the percentage of cells in G1-phase
demonstrated that compound 12q blocked the tumor cells in G1-phase
progression which resulted in decreased S-phase populations. This is
consistent with the reported effect of Rapamycin [31].
The cell cycle is the significant regulatory mechanisms of cell
growth, development, and differentiation. The effect of compound 12q
treatment on breast cancer cell lines MCF-7 was investigated by flow
Scheme 3. Reagents and conditions: (a) (dppf)2PdCl2, Na2CO3, Dioxane/H2O; (b), DIPEA, DCM.
5

S. Hu, et al.
BioorganicChemistry92(2019)103232
Table 3
Cytotoxic activities of compound 12q-y and 13q on U87MG and PC-3 cancer cell.
Entry
Comp. No
G2
G1
Log
U87MG
ODb
PC-3
Kowa
Inhibition rate
71.60
ODb
Inhibition rate
61.98
22
23
12q
12r
3.87
3.87
0.391
0.039
0.015
0.484
0.013
0.027
1.189
0.881
1.316
0.388
13.73
36.10
6.28
0.788
0.794
0.790
0.475
38.15
37.68
37.30
62.72
24
16
25
12s
12k
13q
2.67
3.32
3.89
0.017
0.022
0.011
0.031
0.042
0.022
71.81
26
27
12t
2.39
2.39
1.328
1.314
0.031
0.041
5.40
6.44
1.211
0.919
0.043
0.025
3.86
12u
27.06
28
17
12v
12l
1.20
1.85
0.919
0.906
0.022
0.052
33.28
35.44
0.675
0.658
0.053
0.049
47.03
47.78
29
30
12w
12x
2.50
2.50
1.022
0.995
0.026
0.034
27.44
29.38
0.602
0.872
0.025
0.056
54.53
34.12
31
12y
1.31
1.96
2.84
1.88
1.015
0.924
0.407
0.530
0.010
0.044
0.037
0.037
27.95
34.22
70.84
62.34
0.691
0.684
0.427
0.603
0.038
0.014
0.025
0.024
47.78
45.68
67.03
53.36
18
12m
Lead compound
Positive control
a
PI-103
The value predicted by KOWWIN.
b
The mean value of five times measurements.
2.3. Theoretical calculation
PSA (TPSA) assumes that the contribution of each of the same atom or
group type in a molecule to a polar surface area is similar, and the TPSA
of the entire molecule is the sum of the contributions of all atoms or
groups. Molecules with a TPSA greater than 140 Å2 are often difficult to
penetrate the cell membrane. Oral absorption of the drug TPSA is no
higher than 120 Å2. For drugs acting on the central nervous system, the
best TPSA for the molecule to penetrate the blood-brain barrier is be-
tween 60 and 70 Å2. The maximum cannot exceed 90 Å2.
For the study, the physicochemical properties and better under-
standing the possible binding modes of the target compounds in the
mTOR active site, the theoretical calculation were also performed. The
Log Kow values of the target compounds were calculated using
KOWWIN, a part of the EPI suite environmental modeling program,
which was developed and was maintained by the US Environmental
Protection Agency (EPA) and the Syracuse Research Corporation (SRC)
[32]. The molecular docking analysis was carried out using the Auto-
Dock software package as implemented through the graphical user in-
terface AutoDockTools (ADT 1.5.2). The protein crystal structure of
mTOR (PDB 4jt6) was chosen as a receptor protein [4].
The TPSA values of the synthesized target compounds were pre-
dicted using the SwissADME online database to evaluate their bioa-
vailability properties. The specific results are shown in Table 5, The
TPSA of the compounds were less than 100 Å2, indicating that the
compound is well absorbed orally.
To understand the binding modes of the target compounds in the
mTOR active site, the PI-103 was firstly re-dock to the active site of
mTOR to verify the reliability of the docking method and parameters
(Fig. 6). The stacking map showed that the dominant conformation of
re-docked PI-103 and the original ligand conformation almost com-
pletely coincide. The morpholine ring of re-dock PI-103 (blue) binds to
the adenine pocket and makes hydrogen bond to the hinge, whereas the
phenol group binds to the inner pocket and makes two hydrogen bonds
to the Tyr 2225 and Asp 2195 side chains at the back of the cleft. These
hydrogen bonds are likely to be important for the high affinity of PI-103
for mTOR. The same interaction also occurs in the original ligand in-
dicating that the docking method is reliable and effective.
The range of predicted Log Kow value of the target compounds was
between 0.08 and 5.16 with a molecular weight between 351 and
489 g/mol (shown in Tables 1–5). The number of hydrogen bond do-
nors (the total number of nitrogen–hydrogen and oxygen-hydrogen
bonds) or hydrogen bond acceptors (all nitrogen or oxygen atoms) are
also satisfied Lipinski's rule of five which is a rule of thumb to evaluate
drug-likeness [33]. These results indicate that the synthesized target
compound has good drug-like properties.
Molecular polar surface area (PSA) is a concept that describes the
passive transport of molecules through membranes and predicts in-
testinal absorption, Caco-2 monolayer permeability, and blood-brain
barrier penetration. A fast calculation of the PSA called the topological
6

S. Hu, et al.
BioorganicChemistry92(2019)103232
Scheme 4. Reagents and conditions: (a) 4-DMAP, DMF; (b) TsOH, Toluene; (c) (dppf)2PdCl2, Na2CO3, Dioxane/H2O; (d) DIPEA, DCM.
Table 4
Cytotoxic activities of compound 16a-f and 17a-f on U87MG and PC-3 cancer cell.
Entry
Comp. No
R
G3
Log
U87MG
ODb
PC-3
Kowa
Inhibition rate
ODb
Inhibition rate
32
33
34
16a
17a
16b
H
Ac
H
4.64
4.67
4.24
1.386
1.292
1.354
0.023
0.035
0.030
2.63
9.21
2.98
1.253
1.012
1.214
0.023
0.030
0.028
2.69
21.44
5.58
35
36
37
38
39
40
17b
16c
17c
16d
17d
16e
Ac
H
4.26
5.13
5.16
3.36
3.38
3.59
1.366
1.366
1.394
0.492
0.495
1.383
0.023
0.009
0.034
0.032
0.018
0.014
2.12
2.34
2.01
65.40
65.21
2.83
1.138
1.269
0.502
0.411
1.099
0.98
0.067
0.021
0.030
0.030
0.034
0.019
11.48
2.59
Ac
H
61.01
68.07
14.66
23.93
Ac
H
41
17e
Ac
3.62
1.388
0.036
2.46
1.204
0.040
6.49
42
43
16f
17f
H
3.59
3.62
1.381
1.375
0.042
0.003
2.95
3.36
1.255
1.254
0.025
0.021
2.53
2.61
Ac
Leadcompound
Positivecontrol
2.84
1.88
0.407
0.530
0.037
0.037
70.84
62.34
0.427
0.603
0.025
0.024
67.03
53.36
PI-103
a
b
The value predicted by KOWWIN.
The mean value of five times measurements.
The same method was used to dock compound 12q to the active site
There are some interactions, such as pi-sigma at Ile 2356, Ile 2237,
alkyl or pi-alkyl at Ieu 2185, Trp 2239 and Van der Waals at Asp 2357,
Lys 2187, Met 2345, Pro 2169, Leu 2354, Gly 2238, also found in the
original ligand. The cyclohexyl group at the N-7 position is at the edge
of the active pocket, so it does not contribute much to the activity.
When these two hydrogen bond positions are fixed, most of their
of mTOR (Fig. 7). The results showed that the morpholine ring of
compound 12q binds to the adenine pocket and makes hydrogen bond
to Val 2240. The aniline group binds to the inner pocket. Although, the
amino of aniline did not form a hydrogen bond with Tyr 2225 and Asp
2195, hydrogen bonding with Glu 2190 compensated for this loss.
7

S. Hu, et al.
BioorganicChemistry92(2019)103232
Table 5
The TPSAs and IC50 of compounds against U87MG, PC-3, PI3Kα and mTOR.
Comp. No.
Chemical Structure
TPSA(Å2)a
cell IC50 (μM)b
U87MG
enzyme IC50 (nM)c
PC-3
PI3Kα
113
mTOR
1094
Lead compound
106.70
28.39
50.42
12q
13q
84.58
87.66
12.71
15.20
10.31
20.43
1057
NT
54
NT
16d
87.82
90.90
NT
20.74
37.71
8.49
NT
10.95
10.35
7.55
NT
63,000
NT
ND
NT
NT
55
17d
PI-103
BEZ235
NT
NT
45
NT: not tested.
ND: not detected.
a
b
c
TPSA was calculated by SwissADME.
Ten concentrations calculated results, the mean value of five times measurements.
Ten concentrations calculated results, the mean value of twice measurements.
conformations overlap with the original ligand. Compared to the
docking results of the PI-103, the higher inhibition on mTOR of 12q
was due to the effective combination model to the active site of mTOR.
In addition, it is consistent with the test data of enzyme inhibitory ac-
tivity (12q with IC50 of 54 nM and PI-103with NT).
IC50 values of the typical compounds (12q, 13q, 16d and 17d) against
cancer cell lines were obtained in the range from 10 to 50 μM and at the
same level as the positive control of PI-103 with IC50 of 8.49 μM.
Moreover, the IC50 of the compound 12q inhibited mTOR at 54 nM and
the lead compound at 1094 nM, which equal to the positive control of
BEZ235 with IC50 of 55 nM. By the western blot analysis, the compound
12q was observed that it almost completely inhibit the mTORC1 at
10 μM in U87MG cell lines, and has a certain inhibition of mTORC2 by
the dose-dependent phosphorylation of Akt at Ser 473. The results of
compound 12q treatment on breast cancer cell lines MCF-7 show that it
arrests the cells in G1-phase of the cell cycle as well. The binding modes
of the compound 12q in the mTOR active site indicate that the higher
inhibition on mTOR of 12q was due to the similarity effective combi-
nation model to the active site of mTOR with that of the PI-103. These
results show that the compound 12q is a hopeful candidate as an
mTORC1/mTORC2 dual-target inhibitor and provides valuable in-
formation for the further investigation of mTOR inhibitors.
In the molecular docking study, it was found that compared to 12q,
the C-2 aliphatic ring of 16d (N-methylpiperazinyl) did not penetrate
the adenine pocket to form a hydrogen bond with Val 2240, which is
the key to the inhibition of mTOR (Fig. 8). This may be the cause of 16d
inactivity in the mTOR kinase activity test. The cytotoxicity of com-
pound 16d on cell lines U87MG and PC-3 may act on other signaling
pathways or mechanisms, pending further study.
3. Conclusion
Based on a novel PI3Kα inhibitor as a lead compound, 5,7-dihydro-
6H-pyrrolo[2,3-d]pyrimidin-6-one derivatives were synthesized and
structural optimization on the structures of lead compound based on
cytotoxic activity. All the synthesized target compounds were char-
acterized by 1H NMR, 13C NMR and HRMS. Through the structural
optimization design on the C-4, N-7 and C-2 position of lead compound,
the compounds (12q, 13q, 16d and 17d) were found with the similar
inhibition to the lead compound on U87MG and PC-3. They share the
features that the C-2 position was replaced by six-membered hetero-
cycly, the C-4 position was substituted by 3-aminophenyl or corre-
sponding acetylation product, and the N-7 position was substituted by
cyclohexyl or 4-methoxybenzyl substitutes. It is noteworthy that the
4. Experimental section
Unless noted otherwise, all reagents were commercially available
and used without further purification. All air and moisture sensitive
reactions were carried out under an inert atmosphere of dry nitrogen.
Purification of the crude products was performed using flash column
chromatography on silica gel (300–400 mesh). Reactions were mon-
itored by TLC carried out on 0.25 mm SDS silica gel coated glass plates
(60F254) and compounds were detected with UV light and/or with
8

S. Hu, et al.
BioorganicChemistry92(2019)103232
Fig. 4. Effects of compound 12q on p-Akt, Akt, p-S6K1 and S6K1.
iodide. The melting point is determined by X-5 precision micro melting
point tester produced by Beijing Fukai Instrument Co., Ltd. NMR
spectra were recorded on Bruker DRX 400 instrument. The following
abbreviations were used to designate the multiplicities: s = singlet,
d = doublet, t = triplet, q = quartet, quint = quintuplet, m = multi-
plet, br = broad. High-resolution mass spectra (HRMS) were performed
on Thermo Scientific™ Exactive™ Plus Orbitrap system. Tumor cells
were cultured in MCO-15AC carbon dioxide incubator produced by
Japan Sanyo Co., Ltd. The Thermo MK3 microplate reader produced by
American Thermoelectric Corporation measured the OD. Abbreviations
of solvents are used as follows: DCC, carbodicyclohexylimide; 4-DMAP,
4-(dimethylamino)-pyridine; ACN, acetonitrile; DCM, dichloromethane;
EA, ethyl acetate; PE, petroleum ether; TFA, trifluoroacetic acid; MeOH,
methanol; DMSO‑d6, dimethyl sulfoxide-d6; CDCl3, deuterated chloro-
form; TEA, triethylamine; THF, tetrahydrofuran.
cooled to room temperature. After filter, the filter cake was dissolved in
water (300 mL). Large amount of solid production was generated when
the pH adjusted to 1 with 2N hydrochloric acid. After filter, the filter
cake was dried in vacuum drying oven to afford the title compound 4
(30 g, 30% yield) as a pale yellow solid. 1H NMR (400 MHz, DMSO‑d6) δ
11.31 (s, 2H), 4.00 (t, J = 4.2 Hz, 1H), 3.59 (s, 3H), 3.00 (d, J = 4.3 Hz,
2H). 13C NMR (101 MHz, DMSO‑d6) δ 171.78, 169.57, 150.81, 52.01,
44.60, 30.32.
Methyl 2-(2,4,6-trichloropyrimidin-5-yl)acetate (5): DIPEA
(10.1 mL, 0.1 mol) was added dropwise to a mixture of methyl 2-(2,4,6-
trioxohexahydropyrimidin-5-yl)acetate (4) (10 g, 0.05 mol) in phos-
phorous(V) oxychloride (50 mL), and the mixture was heated to reflux
for 4 h, then cooled to room temperature. All volatiles were removed by
rotary evaporation; the residue was carefully poured into ice water
(200 g). Large amount of dark solid production was collected by fil-
tering; the filter cake was dissolved in EtOAc (300 mL) and dried over
Na2SO4. Filtered and evaporating solvent under reduced pressure to
afford the title compound 5 (7.3 g, 57% crude yield) as brown solid
which used for next step without purification. 1H NMR (400 MHz,
DMSO‑d6) δ 4.02 (s, 2H), 3.69 (d, J = 1.2 Hz, 3H). 13C NMR (101 MHz,
DMSO‑d6) δ 168.03, 162.82, 156.50, 126.06, 52.59, 34.92.
4.1. General experimental procedure
4.1.1. Chemistry
Triethyl ethane-1,1,2-tricarboxylate (3): Diethyl malonate (1)
(80 g, 0.5 mol) was added dropwise to a solution of sodium ethoxide
(34 g, 0.5 mol) in EtOH (600 mL) at 0 °C and stirred for 30 min. Ethyl
chloroacetate (2) (62 g, 0.5 mol) was added dropwise; the mixture was
heated to reflux for 6 h and then cooled to room temperature. All vo-
latiles were removed by rotary evaporation, the residue was partitioned
between EtOAc (600 mL) and water (300 mL), and the layers were se-
parated. The organic layer was washed with brine, dried over Na2SO4,
filtered and evaporating solvent under reduced pressure to afford the
title compound 3 (119 g, 97% crude yield) as colorless oil which used
for next step without purification.
Methyl 2-(2,4-dichloro-6-(4-methoxybenzylamino)pyrimidin-5-
yl)acetate (6): DIPEA (0.5 mL) and 4-methoxybenzylamine (137 mg,
1 mmol) was added to a solution of methyl (2,4,6-trichloropyrimidin-5-
yl) acetate (5) (255 mg, 1 mmol) in DMF (5 mL). The resulting mixture
was stirred at room temperature for 1 h, quenched by water (50 mL)
and then extracted with EtOAc (2 × 50 mL). The organic phases were
combined, washed by brine, dried over Na2SO4, evaporated and pur-
ified by chromatography on silica gel to afford the title compound 6
(295 mg, 83% yield) as a pale yellow solid. 1H NMR (400 MHz,
DMSO‑d6) δ 8.56–8.33 (m, 1H), 7.33–7.13 (m, 2H), 6.98–6.81 (m, 2H),
4.49 (d, J = 5.8 Hz, 2H), 3.75 (s, 2H), 3.72 (s, 3H), 3.65 (s, 3H). 13C
NMR (101 MHz, DMSO‑d6) δ 168.86, 162.38, 158.11, 157.56, 156.64,
129.99, 128.27, 113.50, 107.43, 54.82, 51.92, 43.55, 31.42.
Methyl
2-(2,4,6-trioxohexahydropyrimidin-5-yl)acetate
(4):
Triethyl ethane-1,1,2-tricarboxylate (3) (123 g, 0.5 mol) and urea (30 g,
0.5 mol) was added to a solution of sodium ethoxide (51 g, 0.75 mol) in
MeOH (500 mL), and the mixture was heated to reflux for 10 h, then
9

S. Hu, et al.
BioorganicChemistry92(2019)103232
100%
80%
60%
40%
20%
0%
3.94%
23.52%
76.48%
24.23%
74.40%
26.61%
70.27%
28.11%
71.89%
95.67%
S
G2
G1
Fig. 5. Compound 12q induces cell cycle arrest in breast cancer MCF-7. A: DMSO; B: 12q at 0.1 μM; C: 12q at 1 μM; D: 12q at 3 μM; E: 12q at 10 μM.
Methyl 2-(4-chloro-6-(4-methoxybenzylamino)-2-morpholinopyr-
imidin-5-yl)acetate (7): DMAP (244 mg, 2 mmol) and morpholine
(348 mg, 4 mmol) was added to a solution of methyl 2-(2,4-dichloro-6-
((4-methoxybenzyl)amino)pyrimidin-5-yl)acetate (6) (712 mg, 2 mmol)
in DMF (10 mL). The resulting mixture was stirred at room temperature
overnight; more morpholine was added until the methyl acetate com-
plete consumption. Water (100 mL) was added to the reaction mixture
then extracted with EtOAc (2 × 100 mL). The organic phases were
combined, washed by brine, dried over Na2SO4, evaporated and pur-
ified by chromatography on silica gel to afford the title compound 7
(577 mg, 71% yield) as a white solid. 1H NMR (400 MHz, DMSO‑d6) δ
7.64 (t, J = 5.9 Hz, 1H), 7.22 (d, J = 8.3 Hz, 2H), 6.93–6.81 (m, 2H),
4.44 (d, J = 5.7 Hz, 2H), 3.71 (s, 3H), 3.63 (s, 3H), 3.61 (s, 2H), 3.56 (s,
8H). 13C NMR (101 MHz, DMSO‑d6) δ 170.39, 161.63, 158.93, 158.29,
158.04, 131.85, 128.42, 113.51, 97.09, 65.89, 55.00, 51.79, 43.86,
43.53, 31.36.
7-(4-methoxybenzyl)-2,4-dimorpholino-5,7-dihydro-6H-pyrrolo
[2,3-d]pyrimidin-6-one (9a): solution of 4-chloro-7-(4-methox-
A
ybenzyl)-2-morpholino-5,7-dihydro-6H-pyrrolo[2,3-d] pyrimidin-6-one
(8) (100 mg, 0.27 mmol), DIPEA (70 mg, 0.54 mmol), morpholine
(47 mg, 0.54 mmol) and DMA (3 mL) was heated to 80 °C until complete
consumption of raw materials. Water (50 mL) was added to the reaction
mixture then extracted with DCM (2 × 50 mL). The organic phases
were combined, washed by brine, dried over Na2SO4, evaporated and
purified by chromatography on silica gel to afford the title compound
9a (36 mg, 31% yield) as
a light yellow solid. Melting point:
209–212 °C, 1H NMR (400 MHz, DMSO‑d6) δ 7.28–7.25 (m, 2H), 6.85
(d, J = 8.5 Hz, 2H), 4.68 (s, 2H), 3.71 (d, J = 3.3 Hz, 3H), 3.67 (s, 2H),
3.62 (s, 13H), 3.55 (d, J = 4.6 Hz, 4H). 13C NMR (101 MHz, DMSO‑d6)
δ 174.31, 164.90, 159.80, 158.46, 157.12, 129.32, 129.27, 113.67,
84.02, 65.97, 65.91, 55.01, 44.91, 44.12, 41.25, 35.06. HRMS Calcd for
C
22H28O4N5, 426.21358, found m/z 426.21307 [(M+H)+].
4-chloro-7-(4-methoxybenzyl)-2-morpholino-5,7-dihydro-6H-pyr-
rolo[2,3-d]pyrimidin-6-one (8): p-toluenesulfonic acid monohydrate
(570 mg, 3 mmol) was added to a solution of methyl 2-(4-chloro-
6-((4-methoxybenzyl)amino)-2-morpholinopyrimidin-5-yl)acetate (7)
(1.21 g, 3 mmol) in toluene (30 mL). The resulting mixture was heated
at reflux until the reaction was complete. Water (200 mL) was added to
the reaction mixture then extracted with EtOAc (2 × 200 mL). The
organic phases were combined, washed by brine, dried over Na2SO4,
evaporated and purified by chromatography on silica gel to afford the
title compound 8 (731 mg, 65% yield) as a pink solid. 1H NMR
(400 MHz, DMSO‑d6) δ 7.28 (d, J = 8.6 Hz, 2H), 6.91–6.81 (m, 2H),
4.71 (s, 2H), 3.74–3.60 (m, 11H), 3.55 (s, 2H). 13C NMR (101 MHz,
DMSO‑d6) δ 173.72, 165.74, 160.18, 158.66, 151.57, 129.48, 128.31,
113.79, 102.00, 65.75, 55.04, 44.20, 41.84, 32.34.
7-(4-methoxybenzyl)-2-morpholino-4-(piperidin-1-yl)-5,7-di-
hydro-6H-pyrrolo[2,3-d]pyrimidin-6-one (9b): The title compound 9b
(27 mg) was prepared from 4-chloro-7-(4-methoxybenzyl)-2-morpho-
lino-5,7-dihydro-6H-pyrrolo[2,3-d] pyrimidin-6-one (8) (100 mg,
0.27 mmol) and piperidine (46 mg, 0.54 mmol) using the procedure
described for compound 9a in 23% yield as a light yellow solid. Melting
point: 183–185 °C, 1H NMR (400 MHz, Chloroform-d) δ 7.34 (d,
J = 8.2 Hz, 2H), 6.82 (d, J = 8.2 Hz, 2H), 4.82 (s, 2H), 4.09 (s, 1H),
3.97 (d, J = 4.9 Hz, 2H), 3.87 (s, 2H), 3.76 (d, J = 8.2 Hz, 10H), 1.63 (t,
J = 19.0 Hz, 8H). 13C NMR (101 MHz, Chloroform-d) δ 173.02, 172.01,
161.89, 160.96, 159.31, 156.82, 130.15, 128.91, 114.01, 86.85, 77.37,
66.95, 66.75, 55.42, 44.84, 42.18, 24.82. HRMS Calcd for C23H30O3N5,
424.23432, found m/z 424.23410 [(M+H)+].
7-(4-methoxybenzyl)-2-morpholino-4-thiomorpholino-5,7-
10

S. Hu, et al.
BioorganicChemistry92(2019)103232
Fig. 6. A: Stacking map of re-docked PI-103 (blue) and the original ligand (red); B: the binding mode of original ligand (red) with mTOR (PDB:4jt6); C: the binding
mode of re-docked PI-103 (blue) with mTOR (PDB:4jt6).
dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one (9c): The title compound
9c (45 mg) was prepared from 4-chloro-7-(4-methoxybenzyl)-2-mor-
pholino-5,7-dihydro-6H-pyrrolo[2,3-d] pyrimidin-6-one (8) (100 mg,
0.27 mmol) and thiomorpholine (70 mg, 0.54 mmol) using the proce-
dure described for compound 9a in 37% yield as a black solid. Melting
point: 202–205 °C, 1H NMR (400 MHz, DMSO‑d6) δ 7.28 (d, J = 8.2 Hz,
2H), 6.85 (d, J = 8.0 Hz, 2H), 4.68 (s, 2H), 3.93–3.77 (m, 4H), 3.71 (s,
3H), 3.62 (s, 8H), 3.30 (s, 2H), 2.58 (t, J = 4.8 Hz, 4H). 13C NMR
35.13, 25.78. HRMS Calcd for C22H28O3N5S, 442.19074, found m/z
442.19031 [(M+H)+].
7-(4-methoxybenzyl)-4-(4-methylpiperazin-1-yl)-2-morpholino-
5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one (9d): The title com-
pound 9d (35 mg) was prepared from 4-chloro-7-(4-methoxybenzyl)-2-
morpholino-5,7-dihydro-6H-pyrrolo[2,3-d]
pyrimidin-6-one
(8)
(100 mg, 0.27 mmol) and 1-methylpiperazine (54 mg, 0.54 mmol) using
the procedure described for compound 9a in 30% yield as a yellow
solid. Melting point: 183–185 °C, 1H NMR (400 MHz, DMSO‑d6) δ 7.26
(d, J = 8.3 Hz, 2H), 6.85 (d, J = 8.4 Hz, 2H), 4.67 (s, 2H), 3.70 (s, 3H),
(101 MHz, DMSO‑d6)
δ 174.24, 164.94, 159.91, 158.48, 156.21,
129.42, 129.27, 113.66, 83.95, 65.90, 55.01, 47.56, 44.11, 41.27,
11

S. Hu, et al.
BioorganicChemistry92(2019)103232
Fig. 7. A: Stacking map of 12q (green) and the original ligand (red); B: the binding mode of 12q (green) with mTOR (PDB:4jt6).
3.63 (d, J = 15.1 Hz, 10H), 3.55 (t, J = 4.9 Hz, 4H), 2.31 (t, J = 4.9 Hz,
4H), 2.17 (s, 3H). 13C NMR (101 MHz, DMSO‑d6) δ 174.34, 164.86,
159.86, 158.48, 156.96, 129.36, 129.33, 113.70, 83.91, 65.95, 55.04,
54.46, 45.68, 44.40, 44.15, 41.27, 35.26. HRMS Calcd for C23H30O3N6,
439.24576, found m/z 439.24509 [(M+H)+].
Methyl 2-(4-benzylamino-6-chloro-2-morpholinopyrimidin-5-yl)
acetate (10a): DIPEA (1.0 mL) and benzylamine (620 mg, 5.8 mmol)
was added to a solution of methyl (2,4,6-trichloropyrimidin-5-yl)
acetate (5) (1.5 g, 5.8 mmol) in DMF (10 mL). The resulting mixture
was stirred at room temperature for 1 h, then DMAP (708 mg,
5.8 mmol) and morpholine (870 mg, 10 mmol) was added to the solu-
tion. The resulting mixture was stirred at room temperature overnight;
more morpholine was added until the intermediate complete con-
sumption. Water (200 mL) was added to the reaction mixture then ex-
tracted with EtOAc (2 × 200 mL). The organic phases were combined,
washed by brine, dried over Na2SO4, evaporated and purified by
chromatography on silica gel to afford the title compound 10a (911 mg,
42% yield) as a white solid. 1H NMR (400 MHz, DMSO‑d6) δ 7.73 (t,
J = 5.8 Hz, 1H), 7.29 (d, J = 4.4 Hz, 4H), 7.26–7.17 (m, 1H), 4.53 (d,
J = 5.8 Hz, 2H), 3.64 (s, 5H), 3.53 (s, 8H). 13C NMR (101 MHz,
DMSO‑d6) δ 170.43, 161.72, 158.94, 158.36, 140.02, 128.11, 127.08,
126.52, 97.14, 65.88, 51.83, 44.14, 43.86, 31.40.
4-(4-hydroxypiperidin-1-yl)-7-(4-methoxybenzyl)-2-morpholino-
5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one (9e): The title com-
pound 9e (15 mg) was prepared from 4-chloro-7-(4-methoxybenzyl)-2-
morpholino-5,7-dihydro-6H-pyrrolo[2,3-d] pyrimidin-6-one (8) (100 mg,
0.27 mmol) and 4-hydroxypiperidine (55 mg, 0.54 mmol) using the pro-
cedure described for compound 9a in 12% yield as a yellow solid. Melting
point:181-183 °C, 1H NMR (400 MHz, DMSO‑d6) δ 7.27 (d, J = 8.3 Hz,
2H), 6.84 (d, J = 8.8 Hz, 2H), 4.82–4.62 (m, 3H), 4.21 (dd, J = 12.4,
5.4 Hz, 2H), 3.96 (s, 1H), 3.71 (d, J = 2.9 Hz, 3H), 3.69–3.42 (m, 10H),
3.14 (ddd, J = 13.3, 10.0, 3.0 Hz, 2H), 1.73 (dd, J = 12.8, 4.1 Hz, 2H),
1.42–1.21 (m, 2H). 13C NMR (101 MHz, DMSO‑d6) δ 174.36, 164.97,
159.58, 158.47, 157.30, 129.39, 129.37, 113.65, 83.32, 66.34, 66.01,
55.03, 44.93, 41.42, 41.26, 35.10, 33.88. HRMS Calcd for C23H30O4N5,
440.22923, found m/z 440.22900 [(M+H)+].
Methyl 2-(4-chloro-6-(3,4-difluorobenzylamino)-2-morpholino-
pyrimidin-5-yl)acetate (10b): The title compound 10b (790 mg) was
Fig. 8. A: Stacking map of 16d (blue) and the original ligand (red); B: the binding mode of 16d (blue) with mTOR (PDB:4jt6).
12

S. Hu, et al.
BioorganicChemistry92(2019)103232
prepared from methyl (2,4,6-trichloropyrimidin-5-yl) acetate (5) (1.5 g,
5.8 mmol) and 3,4-difluorobenzylamine (829 mg, 5.8 mmol) using the
procedure described for compound 10a in 33% yield as a yellow solid.
1H NMR (400 MHz, DMSO‑d6) δ 7.70 (t, J = 5.7 Hz, 1H), 7.35 (td,
J = 8.7, 6.7 Hz, 1H), 7.18 (ddd, J = 10.5, 9.3, 2.6 Hz, 1H), 7.03 (tdd,
J = 8.5, 2.6, 1.0 Hz, 1H), 4.50 (d, J = 5.6 Hz, 2H), 3.63 (d, J = 5.0 Hz,
5H), 3.53 (p, J = 2.2 Hz, 8H). 13C NMR (101 MHz, DMSO‑d6) δ 170.37,
162.42, 162.30, 161.59, 161.26, 161.14, 159.99, 159.87, 158.87,
158.81, 158.69, 158.43, 130.43, 130.37, 130.34, 130.28, 123.00,
122.96, 122.85, 122.81, 111.33, 111.29, 111.12, 111.08, 103.66,
103.40, 103.14, 97.26, 65.85, 51.86, 43.78, 37.35, 37.31, 31.38.
5.8 mmol) and 4-(trifluoromethyl)benzylamine (1.0 g, 5.8 mmol) using
the procedure described for compound 10a in 29% yield as a light yellow
solid. 1H NMR (400 MHz, DMSO‑d6) δ 7.82 (t, J = 5.9 Hz, 1H), 7.66 (d,
J = 7.9 Hz, 2H), 7.49 (d, J = 8.0 Hz, 2H), 4.58 (d, J = 5.6 Hz, 2H), 3.64
(d, J = 3.6 Hz, 5H), 3.49 (s, 8H). 13C NMR (101 MHz, DMSO‑d6) δ
170.40, 161.67, 158.87, 158.43, 145.11, 128.22, 127.95, 127.68, 127.41,
127.10, 125.04, 125.00, 97.23, 65.84, 51.91, 43.90, 43.83, 31.42.
Methyl 2-(4-chloro-6-(4-methylbenzylamino)-2-morpholinopyr-
imidin-5-yl)acetate (10i): The title compound 10i (1.0 g) was prepared
from methyl (2,4,6-trichloropyrimidin-5-yl) acetate (5) (1.5 g,
5.8 mmol) and 4-methylbenzylamine (702 mg, 5.8 mmol) using the
procedure described for compound 10a in 44% yield as a white solid.
1H NMR (400 MHz, DMSO‑d6) δ 7.68 (t, J = 5.8 Hz, 1H), 7.20–7.15 (m,
2H), 7.09 (d, J = 7.8 Hz, 2H), 4.47 (d, J = 5.4 Hz, 2H), 3.69–3.60 (m,
5H), 3.60–3.48 (m, 8H), 2.26 (s, 3H). 13C NMR (101 MHz, DMSO‑d6) δ
170.41, 161.70, 158.94, 158.33, 136.93, 135.53, 128.65, 127.12,
97.12, 65.90, 51.81, 43.86, 31.39, 20.65.
Methyl
(R)-2-(4-chloro-2-morpholino-6-(1-phenylethylamino)
pyrimidin-5-yl)acetate (10c): The title compound 10c (1.1 g) was
prepared from methyl (2,4,6-trichloropyrimidin-5-yl) acetate (5) (1.5 g,
5.8 mmol) and (R)-α-methylbenzylamine (702 mg, 5.8 mmol) using the
procedure described for compound 10a in 46% yield as a white solid.
1H NMR (400 MHz, DMSO‑d6) δ 7.39–7.32 (m, 3H), 7.28 (dd, J = 8.5,
6.8 Hz, 2H), 7.20–7.13 (m, 1H), 5.19 (t, J = 7.1 Hz, 1H), 3.72 (s, 2H),
Methyl
2-(4-chloro-6-(3-fluorobenzylamino)-2-morpholinopyr-
3.66 (s, 3H), 3.51 (td, J = 8.7, 5.1 Hz, 8H), 1.45 (d, J = 7.1 Hz, 3H). 13
C
imidin-5-yl)acetate (10j): The title compound 10j (730 mg) was pre-
pared from methyl (2,4,6-trichloropyrimidin-5-yl) acetate (5) (1.5 g,
5.8 mmol) and 3-fluorobenzylamine (725 mg, 5.8 mmol) using the
procedure described for compound 10a in 32% yield as a white solid.
1H NMR (400 MHz, DMSO‑d6) δ 7.77 (t, J = 5.9 Hz, 1H), 7.33 (td,
J = 7.9, 6.1 Hz, 1H), 7.17–7.07 (m, 2H), 7.01 (td, J = 8.6, 2.7 Hz, 1H),
4.54 (d, J = 5.7 Hz, 2H), 3.65 (s, 5H), 3.53 (s, 8H). 13C NMR (101 MHz,
DMSO‑d6) δ 170.43, 163.45, 161.69, 161.04, 158.91, 158.42, 143.29,
143.22, 130.02, 129.94, 123.03, 123.00, 113.70, 113.49, 113.31,
113.10, 97.24, 65.88, 51.83, 43.86, 43.69, 31.43.
NMR (101 MHz, DMSO‑d6) δ 170.59, 161.05, 158.79, 158.51, 145.61,
128.07, 126.32, 125.91, 97.20, 65.89, 51.79, 50.47, 43.87, 31.48,
22.65.
Methyl 2-(4-chloro-6-(3,4-dimethoxybenzylamino)-2-morpholi-
nopyrimidin-5-yl)acetate (10d): The title compound 10d (1.2 g) was
prepared from methyl (2,4,6-trichloropyrimidin-5-yl) acetate (5) (1.5 g,
5.8 mmol) and veratrylamine (969 mg, 5.8 mmol) using the procedure
described for compound 10a in 47% yield as a white solid. 1H NMR
(400 MHz, DMSO‑d6) δ 7.65 (t, J = 5.8 Hz, 1H), 6.93 (d, J = 1.9 Hz,
1H), 6.90–6.79 (m, 2H), 4.44 (d, J = 5.8 Hz, 2H), 3.71 (d, J = 4.5 Hz,
6H), 3.63 (d, J = 3.1 Hz, 5H), 3.57 (s, 8H). 13C NMR (101 MHz,
DMSO‑d6) δ 170.46, 161.67, 158.97, 158.31, 148.53, 147.59, 132.44,
119.31, 111.63, 111.36, 97.15, 65.92, 55.54, 55.35, 51.80, 43.88,
43.88, 31.36.
Methyl 2-(4-chloro-6-(cyclohexylamino)-2-morpholinopyrimidin-
5-yl)acetate (10k): The title compound 10k (1.9 g) was prepared from
methyl (2,4,6-trichloropyrimidin-5-yl) acetate (5) (2.5 g, 10 mmol) and
cyclohexylamine (1.0 g, 10 mmol) using the procedure described for
compound 10a in 51% yield as a white solid. 1H NMR (400 MHz,
DMSO‑d6) δ 6.63 (d, J = 7.4 Hz, 1H), 3.89–3.78 (m, 1H), 3.60 (dt,
J = 11.8, 4.8 Hz, 13H), 1.84 (d, J = 10.0 Hz, 2H), 1.78–1.68 (m, 2H),
1.59 (d, J = 12.7 Hz, 1H), 1.28–1.21 (m, 4H), 1.16–1.08 (m, 1H). 13C
NMR (101 MHz, DMSO‑d6) δ 170.48, 161.05, 159.05, 158.39, 97.01,
65.94, 51.63, 49.94, 43.84, 32.04, 31.35, 25.38, 24.88.
Methyl
2-(4-chloro-6-(4-fluorobenzylamino)-2-morpholinopyr-
imidin-5-yl)acetate (10e): The title compound 10e (955 mg) was
prepared from methyl (2,4,6-trichloropyrimidin-5-yl) acetate (5) (1.5 g,
5.8 mmol) and 4-fluorobenzylamine (725 mg, 5.8 mmol) using the
procedure described for compound 10a in 42% yield as a white solid.
1H NMR (400 MHz, DMSO‑d6) δ 7.73 (t, J = 5.9 Hz, 1H), 7.37–7.28 (m,
2H), 7.19–7.08 (m, 2H), 4.49 (d, J = 5.7 Hz, 2H), 3.63 (d, J = 5.8 Hz,
5H), 3.59–3.47 (m, 8H). 13C NMR (101 MHz, DMSO‑d6) δ 170.40,
162.23, 161.64, 159.83, 158.92, 158.36, 136.19, 136.16, 129.01,
128.93, 114.92, 114.71, 97.15, 65.88, 51.85, 43.85, 43.43, 31.39.
Methyl 2-(4-chloro-6-(4-isocyanobenzylamino)-2-morpholinopyr-
imidin-5-yl)acetate (10f): The title compound 10f (630 mg) was pre-
pared from methyl (2,4,6-trichloropyrimidin-5-yl) acetate (5) (1.5 g,
5.8 mmol) and 4-(aminomethyl)benzonitrile (766 mg, 5.8 mmol) using
the procedure described for compound 10a in 27% yield as a white
solid. 1H NMR (600 MHz, DMSO‑d6) δ 7.84 (s, 1H), 7.80–7.75 (m, 2H),
7.46 (d, J = 8.3 Hz, 2H), 4.57 (d, J = 5.8 Hz, 2H), 3.65 (s, 3H), 3.64 (s,
2H), 3.49 (dd, J = 18.8, 5.1 Hz, 8H). 13C NMR (151 MHz, DMSO‑d6) δ
170.82, 162.08, 159.29, 158.88, 146.67, 132.57, 128.26, 119.42,
109.71, 97.69, 66.28, 52.36, 44.27, 34.36, 31.86.
Methyl
2-(4-chloro-6-(cyclopropylamino)-2-morpholinopyr-
imidin-5-yl)acetate (10l): The title compound 10l (1.3 g) was prepared
from methyl (2,4,6-trichloropyrimidin-5-yl) acetate (5) (2.5 g,
10 mmol) and cyclopropylamine (570 mg, 10 mmol) using the proce-
dure described for compound 10a in 40% yield as a white solid. 1H
NMR (400 MHz, DMSO‑d6) δ 7.09 (s, 1H), 3.58 (d, J = 34.7 Hz, 13H),
2.79 (s, 1H), 0.67 (s, 2H), 0.47 (s, 2H).
Methyl 2-(4-chloro-6-(isopropylamino)-2-morpholinopyrimidin-5-
yl)acetate (10m): The title compound 10m (1.4 g) was prepared from
methyl (2,4,6-trichloropyrimidin-5-yl) acetate (5) (2.5 g, 10 mmol) and
isopropylamine (590 mg, 10 mmol) using the procedure described for
compound 10a in 43% yield as a white solid. 1H NMR (400 MHz,
DMSO‑d6) δ 5.75 (s, 1H), 4.49 (p, J = 7.0 Hz, 1H), 3.66 (s, 8H), 3.47 (s,
5H), 1.40 (d, J = 6.9 Hz, 6H).
Methyl
(6-chloro-5-(2-methoxy-2-oxoethyl)-2-morpholinopyr-
Methyl
2-(4-chloro-6-(3-chlorobenzylamino)-2-morpholinopyr-
imidin-4-yl)-L-alaninate (10n): The title compound 10n (779 mg) was
prepared from methyl (2,4,6-trichloropyrimidin-5-yl) acetate (5) (1.5 g,
5.8 mmol) and L-alanine methyl ester hydrochloride (812 mg,
5.8 mmol) using the procedure described for compound 10a in 36%
yield as a white solid. 1H NMR (400 MHz, DMSO‑d6) δ 7.37 (d,
J = 5.6 Hz, 1H), 4.33–4.25 (m, 1H), 3.66 (d, J = 8.8 Hz, 2H), 3.64 (s,
3H), 3.61–3.57 (m, 7H), 3.56–3.51 (m, 4H), 1.38 (d, J = 7.3 Hz, 3H).
13C NMR (101 MHz, DMSO‑d6) δ 173.88, 170.44, 161.19, 158.95,
158.53, 97.26, 65.91, 51.80, 51.59, 50.43, 43.73, 31.26, 16.50.
imidin-5-yl)acetate (10g): The title compound 10g (1.0 g) was pre-
pared from methyl (2,4,6-trichloropyrimidin-5-yl) acetate (5) (1.5 g,
5.8 mmol) and 3-chlorobenzylamine (824 mg, 5.8 mmol) using the
procedure described for compound 10a in 42% yield as a light yellow
solid. 1H NMR (400 MHz, DMSO‑d6) δ 7.77 (t, J = 5.8 Hz, 1H),
7.36–7.30 (m, 2H), 7.28–7.23 (m, 2H), 4.51 (d, J = 5.8 Hz, 2H), 3.64
(d, J = 5.2 Hz, 5H), 3.53 (s, 8H). 13C NMR (101 MHz, DMSO‑d6) δ
170.39, 161.64, 158.89, 158.42, 142.78, 132.86, 129.97, 126.99,
126.46, 125.77, 97.25, 65.89, 51.87, 43.88, 43.69, 31.44.
Methyl
(6-chloro-5-(2-methoxy-2-oxoethyl)-2-morpholinopyr-
Methyl 2-(4-chloro-2-morpholino-6-(4-trifluoromethylbenzylamino)
pyrimidin-5-yl)acetate (10h): The title compound 10h (741 mg) was
prepared from methyl (2,4,6-trichloropyrimidin-5-yl) acetate (5) (1.5 g,
imidin-4-yl)-D-alaninate (10o): The title compound 10o (907 mg) was
prepared from methyl (2,4,6-trichloropyrimidin-5-yl) acetate (5) (1.5 g,
5.8 mmol) and D-alanine methyl ester hydrochloride (812 mg,
13

S. Hu, et al.
BioorganicChemistry92(2019)103232
5.8 mmol) using the procedure described for compound 10a in 42%
yield as a white solid. 1H NMR (600 MHz, DMSO‑d6) δ 7.35 (d,
J = 5.7 Hz, 1H), 4.34–4.26 (m, 1H), 3.65 (d, J = 6.8 Hz, 5H), 3.60 (q,
J = 5.2, 3.8 Hz, 7H), 3.55 (q, J = 5.1, 4.4 Hz, 4H), 1.38 (d, J = 7.3 Hz,
3H). 13C NMR (151 MHz, DMSO‑d6) δ 173.92, 170.47, 161.24, 159.00,
158.58, 97.30, 65.96, 51.80, 51.58, 50.47, 43.77, 31.30, 16.52.
7-benzyl-4-chloro-2-morpholino-5,7-dihydro-6H-pyrrolo[2,3-d]
pyrimidin-6-one (11a): The title compound 11a (511 mg) was pre-
pared from methyl 2-(4-(benzylamino)-6-chloro-2-morpholinopyr-
imidin-5-yl)acetate (10a) (890 mg, 2.4 mmol) using the procedure de-
scribed for compound 8 in 62% yield as a yellow solid. 1H NMR
(400 MHz, DMSO‑d6) δ 7.34–7.32 (m, 2H), 7.30 (dd, J = 7.9, 0.9 Hz,
2H), 7.28 (s, 1H), 4.78 (s, 2H), 3.68–3.61 (m, 8H), 3.52 (s, 2H). 13C
NMR (101 MHz, DMSO‑d6) δ 173.87, 165.79, 160.20, 151.64, 136.32,
128.46, 127.92, 127.49, 102.09, 65.78, 44.20, 42.41, 32.40.
4-chloro-7-(3-chlorobenzyl)-2-morpholino-5,7-dihydro-6H-pyr-
rolo[2,3-d]pyrimidin-6-one (11g): The title compound 11g (433 mg)
was prepared from methyl 2-(4-chloro-6-((3-chlorobenzyl)amino)-2-
morpholinopyrimidin-5-yl)acetate (10g) (980 mg, 2.4 mmol) using the
procedure described for compound 8 in 48% yield as a white solid. 1H
NMR (600 MHz, DMSO‑d6) δ 7.43 (d, J = 1.9 Hz, 1H), 7.37–7.32 (m,
2H), 7.30 (dt, J = 7.1, 1.8 Hz, 1H), 4.79 (s, 2H), 3.67 (q, J = 3.8,
3.0 Hz, 4H), 3.62 (dd, J = 5.4, 3.6 Hz, 4H), 3.59 (s, 2H). 13C NMR
(151 MHz, DMSO‑d6)
δ 173.87, 165.64, 160.12, 151.64, 138.67,
132.99, 130.37, 127.91, 127.49, 126.59, 102.18, 65.76, 44.20, 41.84,
32.44.
4-chloro-2-morpholino-7-(4-(trifluoromethyl)benzyl)-5,7-di-
hydro-6H-pyrrolo[2,3-d]pyrimidin-6-one (11h): The title compound
11h (271 mg) was prepared from methyl 2-(4-chloro-2-morpholino-6-
((4-(trifluoromethyl)benzyl)amino)pyrimidin-5-yl)acetate
(10h)
4-chloro-7-(3,4-difluorobenzyl)-2-morpholino-5,7-dihydro-6H-
pyrrolo[2,3-d]pyrimidin-6-one (11b): The title compound 11b
(325 mg) was prepared from methyl 2-(4-chloro-6-((3,4-difluorobenzyl)
amino)-2-morpholinopyrimidin-5-yl)acetate (10b) (750 mg, 1.8 mmol)
using the procedure described for compound 8 in 47% yield as a yellow
solid. 1H NMR (400 MHz, DMSO‑d6) δ 7.43 (td, J = 8.7, 6.6 Hz, 1H),
7.22 (ddd, J = 10.5, 9.4, 2.6 Hz, 1H), 7.04 (tdd, J = 8.6, 2.6, 1.0 Hz,
1H), 4.80 (s, 2H), 3.72–3.52 (m, 10H). 13C NMR (101 MHz, DMSO‑d6) δ
173.67, 165.57, 163.06, 162.94, 161.45, 161.45, 161.33, 161.32,
160.61, 160.49, 160.13, 158.98, 158.86, 151.64, 131.84, 131.79,
131.74, 131.69, 119.37, 119.22, 111.62, 111.59, 111.41, 111.38,
104.09, 103.84, 103.58, 102.07, 65.76, 44.17, 35.83, 35.79, 32.40.
(R)-4-chloro-2-morpholino-7-(1-phenylethyl)-5,7-dihydro-6H-
pyrrolo[2,3-d]pyrimidin-6-one (11c): The title compound 11c
(673 mg) was prepared from methyl (R)-2-(4-chloro-2-morpholino-6-
((1-phenylethyl)amino)pyrimidin-5-yl)acetate (10c) (1.0 g, 2.6 mmol)
using the procedure described for compound 8 in 74% yield as a white
solid. 1H NMR (400 MHz, DMSO‑d6) δ 7.43–7.37 (m, 2H), 7.34–7.29
(m, 2H), 7.28–7.22 (m, 1H), 5.56 (q, J = 7.2 Hz, 1H), 3.59 (ddd,
J = 13.8, 5.2, 2.5 Hz, 10H), 1.81 (d, J = 7.2 Hz, 3H). 13C NMR
(720 mg, 1.6 mmol) using the procedure described for compound 8 in
41% yield as a yellow solid. 1H NMR (600 MHz, DMSO‑d6) δ 7.69 (d,
J = 8.0 Hz, 2H), 7.56 (d, J = 8.0 Hz, 2H), 4.89 (s, 2H), 3.65 (dd,
J = 5.8, 3.9 Hz, 4H), 3.63–3.57 (m, 6H). 13C NMR (151 MHz, DMSO‑d6)
δ 173.86, 165.62, 160.14, 151.67, 140.97, 128.60, 128.21, 128.00,
125.36, 125.34, 125.31, 125.29, 125.06, 123.26, 102.12, 65.73, 44.16,
41.97, 32.43.
4-chloro-7-(4-methylbenzyl)-2-morpholino-5,7-dihydro-6H-pyr-
rolo[2,3-d]pyrimidin-6-one (11i): The title compound 11i (519 mg)
was prepared from methyl 2-(4-chloro-6-((4-methylbenzyl)amino)-2-
morpholinopyrimidin-5-yl)acetate (10i) (980 mg, 2.5 mmol) using the
procedure described for compound 8 in 58% yield as a white solid. 1H
NMR (600 MHz, DMSO‑d6)
δ 7.22 (d, J = 8.0 Hz, 2H), 7.11 (d,
J = 7.8 Hz, 2H), 4.74 (s, 2H), 3.67 (d, J = 4.8 Hz, 4H), 3.62 (td,
J = 5.7, 3.2 Hz, 6H), 2.25 (s, 3H). 13C NMR (151 MHz, DMSO‑d6) δ
173.83, 165.81, 160.20, 151.60, 136.71, 133.32, 128.98, 127.93,
102.08, 65.78, 44.20, 42.15, 32.38, 20.66.
4-chloro-7-(3-fluorobenzyl)-2-morpholino-5,7-dihydro-6H-pyr-
rolo[2,3-d]pyrimidin-6-one (11j): The title compound 11j (259 mg)
was prepared from methyl 2-(4-chloro-6-((3-fluorobenzyl)amino)-2-
morpholinopyrimidin-5-yl)acetate (10j) (700 mg, 1.8 mmol) using the
procedure described for compound 8 in 40% yield as a light yellow
solid. 1H NMR (600 MHz, DMSO‑d6) δ 7.36 (td, J = 8.1, 6.2 Hz, 1H),
7.22–7.14 (m, 2H), 7.09 (ddd, J = 10.6, 8.2, 2.6 Hz, 1H), 4.81 (s, 2H),
3.69–3.64 (m, 4H), 3.61 (dd, J = 5.6, 3.7 Hz, 4H), 3.59 (s, 2H). 13C
NMR (151 MHz, DMSO‑d6) δ 173.88, 165.65, 162.89, 161.27, 160.14,
151.63, 139.07, 139.02, 130.47, 130.42, 123.82, 123.80, 114.65,
114.51, 114.35, 114.21, 102.17, 65.74, 44.18, 41.86, 32.42.
(101 MHz, DMSO‑d6)
δ 173.73, 165.78, 160.04, 151.70, 140.06,
128.25, 127.30, 126.91, 102.05, 65.75, 49.69, 44.25, 32.49, 16.70.
4-chloro-7-(3,4-dimethoxybenzyl)-2-morpholino-5,7-dihydro-6H-
pyrrolo[2,3-d]pyrimidin-6-one (11d): The title compound 11d
(851 mg) was prepared from methyl 2-(4-chloro-6-((3,4-dimethox-
ybenzyl)amino)-2-morpholinopyrimidin-5-yl)acetate (10d) (1.2 g,
2.7 mmol) using the procedure described for compound 8 in 78% yield
as a white solid. 1H NMR (400 MHz, DMSO‑d6) δ 6.99 (s, 1H), 6.87 (d,
J = 1.1 Hz, 2H), 4.70 (s, 2H), 3.75–3.67 (m, 10H), 3.64 (dd, J = 5.7,
3.7 Hz, 4H), 3.56 (s, 2H). 13C NMR (101 MHz, DMSO‑d6) δ 173.79,
165.80, 160.17, 151.57, 148.52, 148.29, 128.74, 120.48, 112.19,
111.69, 102.08, 65.80, 55.50, 55.36, 44.20, 42.22, 32.37.
4-chloro-7-cyclohexyl-2-morpholino-5,7-dihydro-6H-pyrrolo[2,3-
d]pyrimidin-6-one (11k): The title compound 11k (1.1 g) was prepared
from methyl 2-(4-chloro-6-(cyclohexylamino)-2-morpholinopyrimidin-
5-yl)acetate (10k) (1.8 g, 4.9 mmol) using the procedure described for
compound 8 in 67% yield as a white solid. 1H NMR (400 MHz,
DMSO‑d6) δ 4.08 (td, J = 12.2, 10.4, 6.0 Hz, 1H), 3.66 (s, 8H), 3.47 (s,
2H), 2.27–2.10 (m, 2H), 1.79 (d, J = 13.0 Hz, 2H), 1.60 (d,
J = 13.5 Hz, 3H), 1.29 (t, J = 13.4 Hz, 2H), 1.15 (q, J = 12.6 Hz, 1H).
13C NMR (101 MHz, DMSO‑d6) δ 173.45, 166.09, 160.14, 151.57,
101.95, 65.77, 51.22, 44.19, 32.35, 28.55, 25.39, 24.99.
4-chloro-7-(4-fluorobenzyl)-2-morpholino-5,7-dihydro-6H-pyr-
rolo[2,3-d]pyrimidin-6-one (11e): The title compound 11e (569 mg)
was prepared from methyl 2-(4-chloro-6-((4-fluorobenzyl)amino)-2-
morpholinopyrimidin-5-yl)acetate (10e) (900 mg, 2.3 mmol) using the
procedure described for compound 8 in 68% yield as a light yellow
solid. 1H NMR (600 MHz, DMSO‑d6) δ 7.45–7.36 (m, 2H), 7.14 (t,
J = 8.9 Hz, 2H), 4.77 (s, 2H), 3.67 (t, J = 4.9 Hz, 4H), 3.64–3.61 (m,
4H), 3.57 (s, 2H). 13C NMR (151 MHz, DMSO‑d6) δ 173.81, 165.67,
162.31, 160.70, 160.17, 151.62, 132.54, 132.52, 130.13, 130.08,
115.27, 115.13, 102.09, 65.76, 44.19, 41.66, 32.39.
4-chloro-7-cyclopropyl-2-morpholino-5,7-dihydro-6H-pyrrolo
[2,3-d]pyrimidin-6-one (11l): The title compound 11l (750 mg) was
prepared from methyl 2-(4-chloro-6-(cyclopropylamino)-2-morpholi-
nopyrimidin-5-yl)acetate (10l) (1.3 g, 3.9 mmol) using the procedure
described for compound 8 in 64% yield as a white solid. 1H NMR
(400 MHz, DMSO‑d6) δ 3.66 (q, J = 4.0 Hz, 8H), 3.40 (s, 2H), 2.71 (dq,
J = 7.3, 4.9, 3.7 Hz, 1H), 0.92 (d, J = 3.8 Hz, 4H). 13C NMR (101 MHz,
DMSO‑d6) δ 174.40, 166.93, 160.32, 151.38, 101.84, 65.85, 44.19,
32.29, 22.33, 5.03.
4-((4-chloro-2-morpholino-6-oxo-5,6-dihydro-7H-pyrrolo[2,3-d]
pyrimidin-7-yl)methyl)benzonitrile (11f): The title compound 11f
(209 mg) was prepared from methyl 2-(4-chloro-6-((4-isocyanobenzyl)
amino)-2-morpholinopyrimidin-5-yl)acetate (10f) (610 mg, 1.5 mmol)
using the procedure described for compound 8 in 37% yield as a white
solid. 1H NMR (600 MHz, DMSO‑d6) δ 7.79 (d, J = 8.3 Hz, 2H), 7.52 (d,
J = 8.3 Hz, 2H), 4.88 (s, 2H), 3.65–3.58 (m, 10H). 13C NMR (151 MHz,
DMSO‑d6) δ 173.94, 165.60, 160.14, 151.70, 141.88, 132.43, 128.63,
110.29, 102.19, 65.76, 44.17, 42.09, 32.48.
4-chloro-7-isopropyl-2-morpholino-5,7-dihydro-6H-pyrrolo[2,3-
d]pyrimidin-6-one (11m): The title compound 11m (939 mg) was
prepared from methyl 2-(4-chloro-6-(isopropylamino)-2-morpholino-
pyrimidin-5-yl)acetate (10m) (1.3 g, 3.9 mmol) using the procedure
14

S. Hu, et al.
BioorganicChemistry92(2019)103232
34.21. HRMS Calcd for C22H21O2N6F2, 439.16886, found m/z
described for compound 8 in 80% yield as a white solid. 1H NMR
(400 MHz, DMSO‑d6) δ 4.49 (p, J = 6.9 Hz, 1H), 3.66 (q, J = 2.4 Hz,
8H), 3.46 (s, 2H), 1.39 (d, J = 6.9 Hz, 6H). 13C NMR (101 MHz,
DMSO‑d6) δ 173.50, 166.09, 160.17, 151.55, 102.09, 65.80, 44.20,
43.40, 32.46, 19.13.
439.16818 [(M+H)+].
(R)-4-(6-aminopyridin-3-yl)-2-morpholino-7-(1-phenylethyl)-5,7-
dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one (12c): The title compound
12c (28 mg) was prepared from (R)-4-chloro-2-morpholino-7-(1-pheny-
lethyl)-5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one (11c) (100 mg,
0.28 mmol) and (6-aminopyridin-3-yl)boronic acid (46 mg, 0.33 mmol)
using the procedure described for compound 12a in 24% yield as a light
green solid. Melting point: 255–257 °C, 1H NMR (400 MHz, DMSO‑d6) δ
8.55 (d, J = 2.4 Hz, 1H), 8.00 (dd, J = 8.8, 2.5 Hz, 1H), 7.42 (d,
J = 7.3 Hz, 2H), 7.31 (dd, J = 8.3, 6.6 Hz, 2H), 7.28–7.20 (m, 1H), 6.51
(d, J = 8.8 Hz, 1H), 6.45 (s, 2H), 5.63 (q, J = 7.2 Hz, 1H), 3.83 (s, 2H),
3.74–3.66 (m, 4H), 3.62 (dd, J = 8.1, 3.5 Hz, 4H), 1.85 (d, J = 7.2 Hz,
3H). 13C NMR (101 MHz, DMSO‑d6) δ 174.63, 165.46, 160.65, 160.07,
154.48, 148.70, 140.58, 136.38, 128.18, 127.13, 126.90, 120.91, 107.39,
99.02, 65.94, 49.23, 44.23, 34.27, 16.84. HRMS Calcd for C23H25O2N6,
417.20335, found m/z 417.20273 [(M+H)+].
Methyl (S)-2-(4-chloro-2-morpholino-6-oxo-5,6-dihydro-7H-pyr-
rolo[2,3-d]pyrimidin-7-yl)propanoate (11n): The title compound 11n
(333 mg) was prepared from methyl (6-chloro-5-(2-methoxy-2-ox-
oethyl)-2-morpholinopyrimidin-4-yl)-L-alaninate
(10n)
(750 mg,
2.0 mmol) using the procedure described for compound 8 in 49% yield
as a white solid. 1H NMR (600 MHz, DMSO‑d6) δ 5.07 (q, J = 7.1 Hz,
1H), 3.62 (dd, J = 10.8, 8.7 Hz, 13H), 1.49 (d, J = 7.2 Hz, 3H). 13C
NMR (151 MHz, DMSO‑d6) δ 173.04, 169.76, 164.84, 159.96, 151.99,
101.69, 65.75, 52.42, 47.88, 44.13, 32.30, 14.26.
Methyl (R)-2-(4-chloro-2-morpholino-6-oxo-5,6-dihydro-7H-pyr-
rolo[2,3-d]pyrimidin-7-yl)propanoate (11o): The title compound 11o
(309 mg) was prepared from methyl (6-chloro-5-(2-methoxy-2-ox-
oethyl)-2-morpholinopyrimidin-4-yl)-D-alaninate
(10o)
(800 mg,
4-(6-aminopyridin-3-yl)-7-(3,4-dimethoxybenzyl)-2-morpholino-
5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one (12d): The title com-
pound 12d (40 mg) was prepared from 4-chloro-7-(3,4-dimethox-
ybenzyl)-2-morpholino-5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one
(11d) (100 mg, 0.25 mmol) and (6-aminopyridin-3-yl)boronic acid
(41 mg, 0.30 mmol) using the procedure described for compound 12a
in 35% yield as a light yellow solid. Melting point: 266–268 °C, 1H NMR
(400 MHz, DMSO‑d6) δ 8.56 (dd, J = 2.4, 0.7 Hz, 1H), 8.02 (dd,
J = 8.8, 2.5 Hz, 1H), 7.03 (d, J = 1.5 Hz, 1H), 6.88 (t, J = 1.3 Hz, 2H),
6.51 (dd, J = 8.7, 0.8 Hz, 1H), 6.46 (s, 2H), 4.74 (s, 2H), 3.83 (s, 2H),
3.77 (dd, J = 5.7, 3.7 Hz, 4H), 3.71 (d, J = 3.4 Hz, 6H), 3.67 (dd,
J = 5.7, 3.8 Hz, 4H). 13C NMR (101 MHz, DMSO‑d6) δ 174.70, 165.40,
160.68, 160.20, 154.38, 148.76, 148.49, 148.22, 136.41, 129.31,
120.83, 120.48, 112.29, 111.75, 107.40, 99.10, 66.02, 55.52, 55.37,
44.21, 41.80, 34.19. HRMS Calcd for C24H27O4N6, 463.20883, found
m/z 463.20786 [(M+H)+].
2.1 mmol) using the procedure described for compound 8 in 43% yield
as a white solid. 1H NMR (600 MHz, DMSO‑d6) δ 5.07 (q, J = 7.1 Hz,
1H), 3.65–3.59 (m, 13H), 1.49 (dd, J = 7.2, 1.7 Hz, 3H). 13C NMR
(151 MHz, DMSO‑d6)
δ 173.06, 169.78, 164.85, 159.97, 152.02,
101.69, 65.77, 52.44, 47.90, 44.15, 32.31, 14.26.
4-chloro-7-(2-hydroxyethyl)-2-morpholino-5,7-dihydro-6H-pyr-
rolo[2,3-d]pyrimidin-6-one (11p): The title compound 11p (300 mg)
was prepared from methyl (2,4,6-trichloropyrimidin-5-yl) acetate (5)
(1.5 g, 5.8 mmol) and ethanolamine (354 mg, 5.8 mmol) directly using
the procedure described for compound 10a in 17% yield as a white
solid. 1H NMR (400 MHz, DMSO‑d6) δ 4.75 (t, J = 5.9 Hz, 1H), 3.65 (dt,
J = 18.8, 9.5 Hz, 13H), 3.49 (s, 2H). 13C NMR (101 MHz, DMSO‑d6) δ
173.95, 166.33, 160.23, 151.33, 102.05, 65.78, 57.14, 44.13, 41.67,
32.33.
4-(6-aminopyridin-3-yl)-7-benzyl-2-morpholino-5,7-dihydro-6H-
pyrrolo[2,3-d]pyrimidin-6-one (12a): A solution of 7-benzyl-4-chloro-
4-(6-aminopyridin-3-yl)-7-(4-fluorobenzyl)-2-morpholino-5,7-di-
hydro-6H-pyrrolo[2,3-d]pyrimidin-6-one (12e): The title compound
12e (23 mg) was prepared from 4-chloro-7-(4-fluorobenzyl)-2-mor-
pholino-5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one (11e) (100 mg,
0.28 mmol) and (6-aminopyridin-3-yl)boronic acid (46 mg, 0.33 mmol)
using the procedure described for compound 12a in 20% yield as a light
yellow solid. Melting point: 281–283 °C, 1H NMR (400 MHz, DMSO‑d6)
δ 8.60–8.54 (m, 1H), 8.02 (dd, J = 8.8, 2.5 Hz, 1H), 7.47–7.36 (m, 2H),
7.21–7.11 (m, 2H), 6.51 (dd, J = 8.8, 0.8 Hz, 1H), 6.46 (s, 2H), 4.81 (s,
2H), 3.85 (s, 2H), 3.74 (dd, J = 5.5, 3.5 Hz, 4H), 3.66 (dd, J = 5.3,
3.4 Hz, 4H). 13C NMR (101 MHz, DMSO‑d6) δ 174.76, 165.27, 160.70,
160.22, 154.50, 148.77, 136.42, 133.11, 130.16, 130.08, 120.80,
115.33, 115.12, 107.42, 99.08, 66.00, 44.20, 41.26, 34.21. HRMS
Calcd for C22H22O2N6F, 421.17828, found m/z 421.17749 [(M+H)+].
4-((4-(6-aminopyridin-3-yl)-2-morpholino-6-oxo-5,6-dihydro-
7H-pyrrolo[2,3-d]pyrimidin-7-yl)methyl)benzonitrile (12f): The title
compound 12f (19 mg) was prepared from 4-((4-chloro-2-morpholino-
6-oxo-5,6-dihydro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)methyl)benzoni-
trile (11f) (100 mg, 0.27 mmol) and (6-aminopyridin-3-yl)boronic acid
(45 mg, 0.32 mmol) using the procedure described for compound 12a
in 16% yield as a light yellow solid. Melting point: 272–279 °C, 1H NMR
(400 MHz, DMSO‑d6) δ 8.62–8.54 (m, 1H), 8.02 (dd, J = 8.8, 2.5 Hz,
1H), 7.84–7.77 (m, 2H), 7.58–7.50 (m, 2H), 6.52 (dd, J = 8.7, 0.7 Hz,
1H), 6.48 (s, 2H), 4.91 (s, 2H), 3.88 (s, 2H), 3.70 (t, J = 4.8 Hz, 4H),
3.64 (dd, J = 5.6, 3.8 Hz, 4H). 13C NMR (101 MHz, DMSO‑d6) δ 174.81,
165.11, 160.72, 160.15, 154.56, 148.79, 142.43, 136.40, 132.43,
128.63, 120.74, 118.70, 110.17, 107.42, 99.07, 65.95, 44.15, 41.69,
34.25. HRMS Calcd for C23H22O2N7, 428.18295, found m/z 428.18237
[(M+H)+].
2-morpholino-5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one
(11a)
(100 mg, 0.29 mmol), (6-aminopyridin-3-yl)boronic acid (48 mg,
0.35 mmol), Pd(dppf)2Cl2 (10 mg, 0.014 mmol), 2 N Na2CO3 aqueous
solution (1.5 mL) and 1,4-dioxane (5 mL) was heated under 100 W of
microwave radiation for 30 min under nitrogen protection. Water
(50 mL) was added to the reaction mixture then extracted with DCM
(2 × 50 mL). The organic phases were combined, washed by brine,
dried over Na2SO4, evaporated and purified by chromatography on si-
lica gel to afford the title compound 12a (35 mg, 30% yield) as a light
yellow solid. Melting point: 278–280 °C, 1H NMR (400 MHz, DMSO‑d6)
δ 8.57 (dd, J = 2.4, 0.7 Hz, 1H), 8.02 (dd, J = 8.8, 2.5 Hz, 1H),
7.39–7.23 (m, 5H), 6.51 (dd, J = 8.8, 0.8 Hz, 1H), 6.46 (s, 2H), 4.82 (s,
2H), 3.86 (s, 2H), 3.73 (dd, J = 5.7, 3.5 Hz, 4H), 3.65 (dd, J = 5.5,
3.6 Hz, 4H). 13C NMR (101 MHz, DMSO‑d6) δ 174.78, 165.39, 160.69,
160.23, 154.46, 148.77, 136.86, 136.42, 128.44, 127.92, 127.38,
120.83, 107.42, 99.09, 65.99, 44.20, 41.99, 34.20. HRMS Calcd for
C
22H23O2N6, 403.18770, found m/z 403.18692 [(M+H)+].
4-(6-aminopyridin-3-yl)-7-(3,4-difluorobenzyl)-2-morpholino-
5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one (12b): The title com-
pound 12b (42 mg) was prepared from 4-chloro-7-(3,4-difluorobenzyl)-
2-morpholino-5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one
(11b)
(100 mg, 0.26 mmol) and (6-aminopyridin-3-yl)boronic acid (43 mg,
0.31 mmol) using the procedure described for compound 12a in 37%
yield as a yellow solid. Melting point: 267–269 °C, 1H NMR (400 MHz,
DMSO‑d6) δ 8.56 (dd, J = 2.4, 0.7 Hz, 1H), 8.01 (dd, J = 8.8, 2.5 Hz,
1H), 7.42 (td, J = 8.7, 6.6 Hz, 1H), 7.23 (ddd, J = 10.4, 9.3, 2.6 Hz,
1H), 7.04 (tdd, J = 8.6, 2.6, 1.0 Hz, 1H), 6.51 (dd, J = 8.8, 0.7 Hz, 1H),
6.47 (s, 2H), 4.84 (s, 2H), 3.86 (s, 2H), 3.70 (dd, J = 5.7, 3.6 Hz, 4H),
3.64 (dt, J = 5.8, 2.6 Hz, 4H). 13C NMR (101 MHz, DMSO‑d6) δ 174.61,
165.14, 160.71, 160.16, 154.50, 148.78, 136.42, 131.66, 131.60,
131.56, 131.51, 120.79, 119.95, 119.91, 119.80, 119.76, 111.63,
111.60, 111.42, 111.38, 107.44, 103.82, 99.01, 65.97, 44.15, 35.34,
4-(6-aminopyridin-3-yl)-7-(3-chlorobenzyl)-2-morpholino-5,7-di-
hydro-6H-pyrrolo[2,3-d]pyrimidin-6-one (12g): The title compound
12g (47 mg) was prepared from 4-chloro-7-(3-chlorobenzyl)-2-mor-
pholino-5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one (11g) (100 mg,
15

S. Hu, et al.
BioorganicChemistry92(2019)103232
0.26 mmol) and (6-aminopyridin-3-yl)boronic acid (44 mg, 0.31 mmol)
using the procedure described for compound 12a in 41% yield as a
yellow solid. Melting point: 286–288 °C, 1H NMR (400 MHz, DMSO‑d6)
δ 8.57 (dd, J = 2.5, 0.7 Hz, 1H), 8.02 (dd, J = 8.8, 2.5 Hz, 1H), 7.44 (d,
J = 2.0 Hz, 1H), 7.38–7.29 (m, 3H), 6.51 (dd, J = 8.8, 0.7 Hz, 1H), 6.47
(s, 2H), 4.83 (s, 2H), 3.87 (s, 2H), 3.74 (dd, J = 5.6, 3.6 Hz, 4H), 3.65
(dd, J = 5.5, 3.6 Hz, 4H). 13C NMR (101 MHz, DMSO‑d6) δ 174.79,
165.20, 160.71, 160.16, 154.54, 148.79, 139.24, 136.42, 132.97,
130.41, 127.96, 127.42, 126.64, 120.79, 107.42, 99.12, 65.99, 44.21,
41.47, 34.23. HRMS Calcd for C22H22O2N6Cl, 437.14873, found m/z
437.14853 [(M+H)+].
34.25, 28.76, 25.61, 25.18. HRMS Calcd for C21H27O2N6, 395.21900,
found m/z 395.21811 [(M+H)+].
4-(6-aminopyridin-3-yl)-7-cyclopropyl-2-morpholino-5,7-di-
hydro-6H-pyrrolo[2,3-d]pyrimidin-6-one (12l): The title compound
12l (44 mg) was prepared from 4-chloro-7-cyclopropyl-2-morpholino-
5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one
(11l)
(100 mg,
0.34 mmol) and (6-aminopyridin-3-yl)boronic acid (57 mg, 0.41 mmol)
using the procedure described for compound 12a in 37% yield as a light
green solid. Melting point: 276–278 °C, 1H NMR (400 MHz, DMSO‑d6) δ
8.55 (d, J = 2.4 Hz, 1H), 8.00 (dd, J = 8.8, 2.4 Hz, 1H), 6.51 (d,
J = 8.8 Hz, 1H), 6.44 (s, 2H), 3.75 (dd, J = 5.9, 3.9 Hz, 4H), 3.72–3.65
(m, 6H), 2.80–2.73 (m, 1H), 0.99–0.90 (m, 4H). 13C NMR (151 MHz,
DMSO‑d6) δ 175.27, 166.56, 160.60, 160.35, 154.05, 148.66, 136.37,
120.95, 107.38, 98.84, 66.03, 44.19, 34.15, 22.16, 4.98. HRMS Calcd
for C18H21O2N6, 353.17205, found m/z 353.17133 [(M+H)+].
4-(6-aminopyridin-3-yl)-2-morpholino-7-(4-(trifluoromethyl)
benzyl)-5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one (12h): The
title compound 12h (25 mg) was prepared from 4-chloro-2-morpholino-
7-(4-(trifluoromethyl)benzyl)-5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-
6-one (11h) (100 mg, 0.24 mmol) and (6-aminopyridin-3-yl)boronic
acid (40 mg, 0.29 mmol) using the procedure described for compound
12a in 22% yield as a yellow solid. Melting point: 244–246 °C, 1H NMR
(400 MHz, DMSO‑d6) δ 8.57 (dd, J = 2.5, 0.7 Hz, 1H), 8.03 (dd,
J = 8.8, 2.5 Hz, 1H), 7.72–7.68 (m, 2H), 7.57 (d, J = 8.1 Hz, 2H), 6.52
(dd, J = 8.8, 0.9 Hz, 1H), 6.47 (s, 2H), 4.92 (s, 2H), 3.88 (s, 2H), 3.71
(q, J = 3.7, 2.9 Hz, 4H), 3.64 (dd, J = 5.2, 3.4 Hz, 4H). HRMS Calcd for
4-(6-aminopyridin-3-yl)-7-isopropyl-2-morpholino-5,7-dihydro-
6H-pyrrolo[2,3-d]pyrimidin-6-one (12m): The title compound 12m
(32 mg) was prepared from 4-chloro-7-isopropyl-2-morpholino-5,7-di-
hydro-6H-pyrrolo[2,3-d]pyrimidin-6-one (11m) (100 mg, 0.34 mmol)
and (6-aminopyridin-3-yl)boronic acid (57 mg, 0.41 mmol) using the
procedure described for compound 12a in 27% yield as a light yellow
solid. Melting point: 243–245 °C, 1H NMR (400 MHz, DMSO‑d6) δ 8.56
(s, 1H), 8.00 (dd, J = 8.8, 2.4 Hz, 1H), 6.52 (d, J = 8.8 Hz, 1H), 6.44 (s,
2H), 4.55 (p, J = 6.9 Hz, 1H), 3.73 (s, 2H), 3.72 (d, J = 4.0 Hz, 4H),
3.68 (dd, J = 5.7, 3.6 Hz, 4H), 1.42 (d, J = 6.9 Hz, 6H). 13C NMR
C
23H22O2N6F, 471.17509, found m/z 471.17496 [(M+H)+].
4-(6-aminopyridin-3-yl)-7-(4-methylbenzyl)-2-morpholino-5,7-
dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one (12i): The title compound
12i (44 mg) was prepared from 4-chloro-7-(4-methylbenzyl)-2-mor-
pholino-5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one (11i) (100 mg,
0.28 mmol) and (6-aminopyridin-3-yl)boronic acid (46 mg, 0.33 mmol)
using the procedure described for compound 12a in 37% yield as a
yellow solid. Melting point: 288–290 °C, 1H NMR (400 MHz, DMSO‑d6)
δ 8.56 (t, J = 1.6 Hz, 1H), 8.01 (dt, J = 8.9, 1.8 Hz, 1H), 7.29–7.22 (m,
2H), 7.11 (d, J = 7.7 Hz, 2H), 6.51 (d, J = 8.7 Hz, 1H), 6.45 (s, 2H),
4.77 (s, 2H), 3.84 (s, 2H), 3.74 (t, J = 4.5 Hz, 4H), 3.69–3.64 (m, 4H),
2.25 (s, 3H). 13C NMR (101 MHz, DMSO‑d6) δ 174.66, 165.36, 160.63,
160.20, 154.41, 148.70, 136.51, 136.36, 133.81, 128.90, 127.89,
120.84, 107.36, 99.04, 65.95, 44.18, 41.69, 34.14, 20.61. HRMS Calcd
for C23H25O2N6, 417.20335, found m/z 417.20297 [(M+H)+].
(101 MHz, DMSO‑d6)
δ 174.42, 165.78, 160.58, 160.19, 154.26,
148.58, 136.38, 121.05, 107.44, 99.15, 65.99, 44.22, 42.91, 34.26,
19.25. HRMS Calcd for C18H23O2N6, 355.18770, found m/z 355.18713
[(M+H)+].
Methyl (S)-2-(4-(6-aminopyridin-3-yl)-2-morpholino-6-oxo-5,6-
dihydro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)propanoate (12n): The title
compound 12n (40 mg) was prepared from methyl (S)-2-(4-chloro-2-
morpholino-6-oxo-5,6-dihydro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)pro-
panoate (11n) (100 mg, 0.29 mmol) and (6-aminopyridin-3-yl)boronic
acid (49 mg, 0.35 mmol) using the procedure described for compound
12a in 35% yield as a light green solid. Melting point: 184–186 °C, 1H
NMR (400 MHz, DMSO‑d6) δ 8.58 (d, J = 2.4 Hz, 1H), 8.03 (dd,
J = 8.8, 2.5 Hz, 1H), 6.55–6.51 (m, 1H), 6.48 (s, 2H), 5.09 (q,
J = 7.1 Hz, 1H), 3.88 (d, J = 1.9 Hz, 2H), 3.68 (h, J = 3.9 Hz, 8H), 3.61
(s, 3H), 1.50 (d, J = 7.1 Hz, 3H). 13C NMR (101 MHz, DMSO‑d6) δ
173.93, 170.08, 164.42, 160.69, 159.93, 154.85, 148.76, 136.40,
120.77, 107.43, 98.66, 65.92, 52.28, 47.42, 44.08, 34.08, 14.43. HRMS
Calcd for C19H23O4N6, 399.17753, found m/z 399.17706 [(M+H)+].
Methyl (R)-2-(4-(6-aminopyridin-3-yl)-2-morpholino-6-oxo-5,6-
dihydro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)propanoate (12o): The title
compound 12o (27 mg) was prepared from methyl (R)-2-(4-chloro-2-
morpholino-6-oxo-5,6-dihydro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)pro-
panoate (11o) (100 mg, 0.29 mmol) and (6-aminopyridin-3-yl)boronic
acid (49 mg, 0.35 mmol) using the procedure described for compound
12a in 23% yield as a light black solid. Melting point: 167–169 °C, 1H
NMR (400 MHz, DMSO‑d6) δ 8.62–8.55 (m, 1H), 8.03 (dd, J = 8.8,
2.5 Hz, 1H), 6.53 (dd, J = 8.8, 0.7 Hz, 1H), 6.48 (s, 2H), 5.09 (q,
J = 7.1 Hz, 1H), 3.88 (d, J = 1.9 Hz, 2H), 3.69 (d, J = 3.1 Hz, 4H), 3.66
(d, J = 3.2 Hz, 4H), 3.61 (s, 3H), 1.50 (d, J = 7.1 Hz, 3H). 13C NMR
4-(6-aminopyridin-3-yl)-7-(3-fluorobenzyl)-2-morpholino-5,7-di-
hydro-6H-pyrrolo[2,3-d]pyrimidin-6-one (12j): The title compound
12j (30 mg) was prepared from 4-chloro-7-(3-fluorobenzyl)-2-morpho-
lino-5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one (11j) (100 mg,
0.28 mmol) and (6-aminopyridin-3-yl)boronic acid (46 mg, 0.33 mmol)
using the procedure described for compound 12a in 26% yield as a light
yellow solid. Melting point: 270–272 °C, 1H NMR (400 MHz, DMSO‑d6)
δ 8.62–8.56 (m, 1H), 8.02 (dd, J = 8.8, 2.5 Hz, 1H), 7.37 (td, J = 8.0,
6.0 Hz, 1H), 7.21–7.15 (m, 2H), 7.10 (tt, J = 8.8, 1.8 Hz, 1H), 6.52 (dd,
J = 8.8, 0.7 Hz, 1H), 6.47 (s, 2H), 4.85 (s, 2H), 3.87 (s, 2H), 3.73 (dd,
J = 5.6, 3.6 Hz, 4H), 3.65 (dd, J = 5.6, 3.5 Hz, 4H). 13C NMR
(101 MHz, DMSO‑d6)
δ 174.74, 165.19, 163.26, 160.83, 160.65,
160.16, 154.51, 148.71, 139.60, 139.53, 136.36, 130.44, 130.36,
123.81, 123.78, 120.80, 114.67, 114.45, 114.25, 114.04, 107.38,
99.09, 65.92, 44.16, 41.44, 34.18. HRMS Calcd for C22H22O2N6F,
421.17828, found m/z 421.17789 [(M+H)+].
4-(6-aminopyridin-3-yl)-7-cyclohexyl-2-morpholino-5,7-dihydro-
6H-pyrrolo[2,3-d]pyrimidin-6-one (12k): The title compound 12k
(19 mg) was prepared from 4-chloro-7-cyclohexyl-2-morpholino-5,7-
dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one (11k) (100 mg, 0.28 mmol)
and (6-aminopyridin-3-yl)boronic acid (46 mg, 0.33 mmol) using the
procedure described for compound 12a in 16% yield as a light green
solid. Melting point: 250–252 °C, 1H NMR (400 MHz, DMSO‑d6) δ 8.55
(d, J = 2.4 Hz, 1H), 8.00 (dd, J = 8.8, 2.5 Hz, 1H), 6.51 (d, J = 8.8 Hz,
1H), 6.43 (s, 2H), 4.13 (ddt, J = 12.1, 7.4, 3.8 Hz, 1H), 3.80–3.66 (m,
10H), 2.35–2.20 (m, 2H), 1.81 (d, J = 12.9 Hz, 2H), 1.62 (dd, J = 23.6,
12.3 Hz, 3H), 1.31 (dd, J = 9.9, 6.7 Hz, 2H), 1.16 (d, J = 12.8 Hz, 1H).
13C NMR (101 MHz, DMSO‑d6) δ 174.56, 165.90, 160.66, 160.26,
154.34, 148.69, 136.46, 121.06, 107.48, 99.15, 66.07, 50.89, 44.29,
(101 MHz, DMSO‑d6)
δ 173.93, 170.08, 164.42, 160.69, 159.93,
154.85, 148.75, 136.40, 120.77, 107.43, 98.66, 65.92, 52.28, 47.43,
44.08, 34.08, 14.43. HRMS Calcd for C19H23O4N6, 399.17753, found
m/z 399.17734 [(M+H)+].
4-(6-aminopyridin-3-yl)-7-(2-hydroxyethyl)-2-morpholino-5,7-
dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one (12p): The title compound
12p (25 mg) was prepared from 4-chloro-7-(2-hydroxyethyl)-2-mor-
pholino-5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one (11p) (100 mg,
0.34 mmol) and (6-aminopyridin-3-yl)boronic acid (57 mg, 0.41 mmol)
using the procedure described for compound 12a in 21% yield as a light
green solid. Melting point: 246–248 °C, 1H NMR (400 MHz, DMSO‑d6) δ
8.57 (d, J = 2.3 Hz, 1H), 8.02 (dd, J = 8.8, 2.5 Hz, 1H), 6.52 (d,
16

S. Hu, et al.
BioorganicChemistry92(2019)103232
J = 8.7 Hz, 1H), 6.45 (s, 2H), 4.80 (t, J = 5.8 Hz, 1H), 3.75 (s, 2H),
3.73 (d, J = 5.3 Hz, 4H), 3.71 (s, 2H), 3.69–3.66 (m, 4H), 3.63 (t,
J = 5.8 Hz, 2H). 13C NMR (101 MHz, DMSO‑d6) δ 174.85, 165.87,
160.55, 160.23, 154.05, 148.55, 136.37, 120.92, 107.42, 99.09, 65.98,
57.35, 44.14, 41.19, 34.15. HRMS Calcd for C17H21O3N6, 357.16697,
found m/z 357.16620 [(M+H)+].
and 4-aminophenylboronic acid pinacol ester (90 mg, 0.41 mmol) using
the procedure described for compound 12a in 23% yield as a light
yellow solid. Melting point: 265–268 °C, 1H NMR (400 MHz, DMSO‑d6)
δ 7.73 (d, J = 8.7 Hz, 2H), 6.62 (d, J = 8.7 Hz, 2H), 5.62 (s, 2H), 3.75
(dd, J = 6.0, 4.0 Hz, 4H), 3.71–3.62 (m, 6H), 2.75 (ddd, J = 7.9, 6.5,
3.6 Hz, 1H), 1.00–0.89 (m, 4H). 13C NMR (101 MHz, DMSO‑d6) δ
175.37, 166.52, 160.33, 155.68, 150.83, 129.34, 123.98, 113.20,
98.52, 66.06, 44.22, 34.52, 22.17, 4.97. HRMS Calcd for C19H22O2N5,
352.17680, found m/z 352.17648 [(M+H)+].
4-(3-aminophenyl)-7-cyclohexyl-2-morpholino-5,7-dihydro-6H-
pyrrolo[2,3-d]pyrimidin-6-one (12q): The title compound 12q
(28 mg) was prepared from 4-chloro-7-cyclohexyl-2-morpholino-5,7-
dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one (11k) (100 mg, 0.30 mmol)
and 3-aminobenzeneboronic acid (49 mg, 0.36 mmol) using the proce-
dure described for compound 12a in 24% yield as a light yellow solid.
Melting point: 167–169 °C, 1H NMR (400 MHz, DMSO‑d6) δ 7.30–7.18
(m, 1H), 7.11 (d, J = 5.8 Hz, 2H), 6.65 (dt, J = 5.9, 2.7 Hz, 1H), 5.20
(s, 2H), 4.14 (td, J = 10.2, 8.3, 6.0 Hz, 1H), 3.73 (tt, J = 9.3, 4.3 Hz,
10H), 2.29 (tt, J = 12.8, 7.1 Hz, 2H), 1.82 (d, J = 13.0 Hz, 2H),
1.73–1.55 (m, 3H), 1.39–1.25 (m, 2H), 1.19 (t, J = 12.9 Hz, 1H). 13C
NMR (101 MHz, DMSO‑d6) δ 174.41, 166.07, 160.23, 156.33, 148.75,
137.70, 128.84, 115.51, 115.42, 113.25, 100.95, 65.97, 50.84, 44.22,
34.34, 28.63, 25.51, 25.09. HRMS Calcd for C22H28O2N5, 394.22375,
found m/z 394.22360 [(M+H)+].
4-(2-aminopyrimidin-5-yl)-7-cyclopropyl-2-morpholino-5,7-di-
hydro-6H-pyrrolo[2,3-d]pyrimidin-6-one (12v): The title compound
12v (34 mg) was prepared from 4-chloro-7-cyclopropyl-2-morpholino-
5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one
(11l)
(100 mg,
0.34 mmol) and (2-Aminopyrimidin-5-yl)boronic acid pinacol ester
(91 mg, 0.41 mmol) using the procedure described for compound 12a
in 28% yield as a light yellow solid. Melting point: 281–283 °C, 1H NMR
(600 MHz, DMSO‑d6) δ 8.82 (s, 2H), 7.17 (s, 2H), 3.75 (d, J = 8.2 Hz,
6H), 3.68 (t, J = 4.8 Hz, 4H), 2.76 (tt, J = 7.2, 4.1 Hz, 1H), 0.97–0.90
(m, 4H). 13C NMR (151 MHz, DMSO‑d6) δ 175.21, 166.63, 163.61,
160.36, 157.79, 151.97, 119.31, 99.14, 66.02, 44.17, 33.69, 22.17,
4.98. HRMS Calcd for C17H20O2N7, 354.16730, found m/z 354.16660
[(M+H)+].
4-(4-aminophenyl)-7-cyclohexyl-2-morpholino-5,7-dihydro-6H-
pyrrolo[2,3-d]pyrimidin-6-one (12r): The title compound 12r (39 mg)
was prepared from 4-chloro-7-cyclohexyl-2-morpholino-5,7-dihydro-
6H-pyrrolo[2,3-d]pyrimidin-6-one (11k) (100 mg, 0.30 mmol) and 4-
aminophenylboronic acid pinacol ester (79 mg, 0.36 mmol) using the
procedure described for compound 12a in 33% yield as a white solid.
Melting point: 292–295 °C, 1H NMR (400 MHz, DMSO‑d6) δ 7.73 (d,
J = 8.7 Hz, 2H), 6.62 (d, J = 8.7 Hz, 2H), 5.62 (s, 2H), 4.13 (ddd,
J = 12.2, 8.4, 3.8 Hz, 1H), 3.71 (dd, J = 10.8, 4.6 Hz, 10H), 2.29 (qd,
J = 12.6, 11.5, 2.9 Hz, 2H), 1.81 (d, J = 13.0 Hz, 2H), 1.63 (dd,
J = 24.3, 12.3 Hz, 3H), 1.35–1.11 (m, 3H). 13C NMR (101 MHz,
DMSO‑d6) δ 174.62, 165.84, 160.21, 155.94, 150.88, 129.38, 124.05,
113.25, 98.80, 66.08, 50.81, 44.30, 34.61, 28.78, 25.61, 25.18. HRMS
Calcd for C22H28O2N5, 394.22375, found m/z 394.22336 [(M+H)+].
4-(2-aminopyrimidin-5-yl)-7-cyclohexyl-2-morpholino-5,7-di-
hydro-6H-pyrrolo[2,3-d]pyrimidin-6-one (12s): The title compound
12s (41 mg) was prepared from 4-chloro-7-cyclohexyl-2-morpholino-
4-(3-aminophenyl)-7-isopropyl-2-morpholino-5,7-dihydro-6H-
pyrrolo[2,3-d]pyrimidin-6-one (12w): The title compound 12w
(18 mg) was prepared from 4-chloro-7-isopropyl-2-morpholino-5,7-di-
hydro-6H-pyrrolo[2,3-d]pyrimidin-6-one (11m) (100 mg, 0.34 mmol)
and 3-aminobenzeneboronic acid (56 mg, 0.41 mmol) using the proce-
dure described for compound 12a in 15% yield as a green solid. Melting
point: 178–180 °C, 1H NMR (400 MHz, DMSO‑d6) δ 7.25–7.21 (m, 1H),
7.15–7.08 (m, 2H), 6.67–6.64 (m, 1H), 5.21 (s, 2H), 4.58 (q, J = 6.8 Hz,
1H), 3.75 (dd, J = 5.6, 3.5 Hz, 4H), 3.73–3.68 (m, 6H), 1.44 (d,
J = 6.9 Hz, 6H). 13C NMR (101 MHz, DMSO‑d6) δ 174.40, 166.03,
160.25, 156.33, 148.76, 137.73, 128.87, 124.86, 115.56, 113.28,
101.04, 65.97, 44.23, 42.93, 34.43, 19.22. HRMS Calcd for
C
19H24O2N5, 354.19245, found m/z 354.19226 [(M+H)+].
4-(4-aminophenyl)-7-isopropyl-2-morpholino-5,7-dihydro-6H-
pyrrolo[2,3-d]pyrimidin-6-one (12x): The title compound 12x
(35 mg) was prepared from 4-chloro-7-isopropyl-2-morpholino-5,7-di-
hydro-6H-pyrrolo[2,3-d]pyrimidin-6-one (11m) (100 mg, 0.34 mmol)
and 4-aminophenylboronic acid pinacol ester (90 mg, 0.41 mmol) using
the procedure described for compound 12a in 29% yield as a yellow
solid. Melting point: 260–262 °C, 1H NMR (400 MHz, DMSO‑d6) δ 7.74
(d, J = 8.7 Hz, 2H), 6.62 (d, J = 8.7 Hz, 2H), 5.62 (s, 2H), 4.56 (p,
J = 6.9 Hz, 1H), 3.73 (d, J = 5.4 Hz, 6H), 3.69 (dd, J = 5.3, 3.4 Hz,
4H), 1.43 (d, J = 6.9 Hz, 6H). 13C NMR (101 MHz, DMSO‑d6) δ 174.46,
165.71, 160.14, 155.87, 150.78, 129.28, 124.04, 113.20, 98.79, 65.98,
44.22, 42.79, 34.60, 19.27. HRMS Calcd for C19H24O2N5, 354.19245,
found m/z 354.19223 [(M+H)+].
5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one
(11k)
(100 mg,
0.30 mmol) and (2-Aminopyrimidin-5-yl)boronic acid pinacol ester
(80 mg, 0.36 mmol) using the procedure described for compound 12a
in 35% yield as a light yellow solid. Melting point: 280–282 °C, 1H NMR
(400 MHz, DMSO‑d6) δ 8.83 (s, 2H), 7.16 (s, 2H), 4.13 (ddd, J = 12.2,
8.4, 3.7 Hz, 1H), 3.81 (s, 2H), 3.78–3.59 (m, 8H), 2.27 (qd, J = 12.8,
3.6 Hz, 2H), 1.81 (d, J = 12.8 Hz, 2H), 1.70–1.56 (m, 3H), 1.36–1.13
(m, 3H). 13C NMR (101 MHz, DMSO‑d6) δ 174.43, 165.89, 163.65,
160.20, 157.78, 152.22, 119.34, 99.37, 66.00, 50.86, 44.20, 33.75,
28.66, 25.52, 25.10. HRMS Calcd for C20H26O2N7, 396.21425, found
m/z 396.21381 [(M+H)+].
4-(2-aminopyrimidin-5-yl)-7-isopropyl-2-morpholino-5,7-di-
hydro-6H-pyrrolo[2,3-d]pyrimidin-6-one (12y): The title compound
12y (38 mg) was prepared from 4-chloro-7-isopropyl-2-morpholino-
4-(3-aminophenyl)-7-cyclopropyl-2-morpholino-5,7-dihydro-6H-
pyrrolo[2,3-d]pyrimidin-6-one (12t): The title compound 12 t (36 mg)
was prepared from 4-chloro-7-cyclopropyl-2-morpholino-5,7-dihydro-
6H-pyrrolo[2,3-d]pyrimidin-6-one (11l) (100 mg, 0.34 mmol) and 3-
aminobenzeneboronic acid (56 mg, 0.41 mmol) using the procedure
described for compound 12a in 30% yield as a yellow solid. Melting
point: 212–215 °C, 1H NMR (400 MHz, DMSO‑d6) δ 7.22 (q, J = 1.4 Hz,
1H), 7.15–7.07 (m, 2H), 6.65 (dt, J = 6.3, 2.4 Hz, 1H), 5.23 (s, 2H),
3.77 (dd, J = 5.6, 3.5 Hz, 4H), 3.73–3.66 (m, 6H), 2.76 (tt, J = 7.4,
3.6 Hz, 1H), 0.99–0.90 (m, 4H). 13C NMR (101 MHz, DMSO‑d6) δ
175.31, 166.87, 160.43, 156.14, 148.86, 137.69, 128.90, 115.51,
115.45, 113.31, 100.80, 66.04, 44.22, 34.36, 22.18, 5.00. HRMS Calcd
for C19H22O2N5, 352.17680, found m/z 352.17621 [(M+H)+].
4-(4-aminophenyl)-7-cyclopropyl-2-morpholino-5,7-dihydro-6H-
pyrrolo[2,3-d]pyrimidin-6-one (12u): The title compound 12u
(28 mg) was prepared from 4-chloro-7-cyclopropyl-2-morpholino-5,7-
dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one (11l) (100 mg, 0.34 mmol)
5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one
(11m)
(100 mg,
0.34 mmol) and (2-Aminopyrimidin-5-yl)boronic acid pinacol ester
(91 mg, 0.41 mmol) using the procedure described for compound 12a
in 31% yield as a white solid. Melting point: 297–299 °C, 1H NMR
(400 MHz, DMSO‑d6) δ 8.83 (s, 2H), 7.16 (s, 2H), 4.60–4.52 (m, 1H),
3.80 (s, 2H), 3.74 (dd, J = 5.9, 3.6 Hz, 4H), 3.68 (dd, J = 5.7, 3.7 Hz,
4H), 1.43 (d, J = 6.9 Hz, 6H). 13C NMR (101 MHz, DMSO‑d6) δ 174.32,
165.80, 163.62, 160.17, 157.73, 152.18, 119.33, 99.41, 65.94, 44.17,
42.93, 33.78, 19.20. HRMS Calcd for C17H22O2N7, 356.18295, found
m/z 356.18250 [(M+H)+].
N-(3-(7-cyclohexyl-2-morpholino-6-oxo-6,7-dihydro-5H-pyrrolo
[2,3-d]pyrimidin-4-yl)phenyl)acetamide (13q): Acetyl chloride (5 mg,
0.06 mmol) was added to a solution of 4-(3-aminophenyl)-7-cyclohexyl-
2-morpholino-5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one
(12q)
(20 mg, 0.05 mmol) and DIPEA (8 mg, 0.06 mmol) in DCM (5 mL) in an
17

S. Hu, et al.
BioorganicChemistry92(2019)103232
ice bath. The resulting mixture was stirred at room temperature until
complete consumption of raw materials. Water (50 mL) was added to
the reaction mixture then extracted with DCM (2 × 30 mL). The or-
ganic phases were combined, washed by brine, dried over Na2SO4,
evaporated and purified by chromatography on silica gel to afford the
title compound 13 (10 mg, 46% yield) as a green solid. Melting point:
183–185 °C, 1H NMR (400 MHz, Chloroform-d) δ 8.13 (d, J = 2.0 Hz,
1H), 7.78 (s, 1H), 7.65–7.54 (m, 2H), 7.38 (t, J = 7.9 Hz, 1H),
4.30–4.19 (m, 1H), 3.93–3.74 (m, 8H), 3.64 (s, 2H), 2.36 (dd, J = 12.5,
3.6 Hz, 2H), 2.19 (s, 3H), 1.90–1.83 (m, 2H), 1.74–1.64 (m, 3H), 1.37
(d, J = 13.4 Hz, 3H). HRMS Calcd for C24H30O3N5, 436.23432, found
m/z 436.23407 [(M+H)+].
4-chloro-7-(4-methoxybenzyl)-2-thiomorpholino-5,7-dihydro-6H-
pyrrolo[2,3-d]pyrimidin-6-one (15b): The title compound 15b
(115 mg) was prepared from methyl 2-(4-chloro-6-((4-methoxybenzyl)
amino)-2-thiomorpholinopyrimidin-5-yl)acetate
(14b)
(210 mg,
0.50 mmol) using the procedure described for compound 8 in 59% yield
as a yellow solid. 1H NMR (400 MHz, DMSO‑d6) δ 7.29 (d, J = 8.2 Hz,
2H), 6.87 (d, J = 8.2 Hz, 2H), 4.70 (s, 2H), 4.02 (t, J = 4.9 Hz, 4H),
3.71 (s, 4H), 3.54 (s, 2H), 2.62–2.55 (m, 4H).
4-chloro-7-(4-methoxybenzyl)-2-(piperidin-1-yl)-5,7-dihydro-6H-
pyrrolo[2,3-d]pyrimidin-6-one (15c): The title compound 15c (90 mg)
was prepared from methyl 2-(4-chloro-6-((4-methoxybenzyl)amino)-2-
(piperidin-1-yl)pyrimidin-5-yl)acetate (14c) (160 mg, 0.40 mmol) using
the procedure described for compound 8 in 60% yield as a light yellow
solid. 1H NMR (400 MHz, DMSO‑d6) δ 7.32–7.24 (m, 2H), 6.86 (d,
J = 8.5 Hz, 2H), 4.70 (s, 2H), 3.71 (d, J = 5.9 Hz, 7H), 3.52 (s, 2H),
1.62 (t, J = 6.0 Hz, 2H), 1.50 (q, J = 5.6 Hz, 4H). 13C NMR (101 MHz,
DMSO‑d6) δ 173.78, 165.71, 159.90, 158.66, 151.66, 129.51, 128.42,
113.78, 100.84, 55.06, 44.67, 41.81, 32.35, 25.13, 24.14.
Methyl 2-(4-chloro-6-(4-methoxybenzy)amino)-2-(pyrrolidin-1-
yl)pyrimidin-5-yl)acetate (14a): The title compound 14a (231 mg)
was prepared from methyl 2-(2,4-dichloro-6-((4-methoxybenzyl)
amino)pyrimidin-5-yl)acetate (6) (250 mg, 0.70 mmol) and tetrahydro
pyrrole (100 mg, 1.40 mmol) using the procedure described for com-
pound 7 in 85% yield as a white solid. 1H NMR (400 MHz, DMSO‑d6) δ
7.51 (t, J = 5.9 Hz, 1H), 7.26–7.20 (m, 2H), 6.87–6.82 (m, 2H), 4.44 (d,
J = 5.8 Hz, 2H), 3.71 (s, 3H), 3.62 (s, 3H), 3.58 (s, 2H), 3.35 (s, 4H),
1.88–1.80 (m, 4H). 13C NMR (101 MHz, DMSO‑d6) δ 170.56, 161.46,
158.10, 157.99, 157.75, 132.17, 128.56, 113.45, 95.82, 54.99, 51.72,
46.09, 43.43, 31.35, 24.89.
4-chloro-7-(4-methoxybenzyl)-2-(4-methylpiperazin-1-yl)-5,7-di-
hydro-6H-pyrrolo[2,3-d]pyrimidin-6-one (15d): The title compound
15d (113 mg) was prepared from methyl 2-(4-chloro-6-((4-methox-
ybenzyl)amino)-2-(4-methylpiperazin-1-yl)pyrimidin-5-yl)acetate
(14d) (210 mg, 0.50 mmol) using the procedure described for com-
pound 8 in 58% yield as a white solid. 1H NMR (400 MHz, DMSO‑d6) δ
7.28 (d, J = 8.2 Hz, 2H), 6.90–6.84 (m, 2H), 4.70 (s, 2H), 3.70 (d,
Methyl 2-(4-chloro-6-(4-methoxybenzylamino)-2-thiomorpholi-
nopyrimidin-5-yl)acetate (14b): The title compound 14b (222 mg) was
prepared from methyl 2-(2,4-dichloro-6-((4-methoxybenzyl)amino)
pyrimidin-5-yl)acetate (6) (250 mg, 0.70 mmol) and thiomorpholine
(144 mg, 1.40 mmol) using the procedure described for compound 7 in
75% yield as a yellow solid. 1H NMR (400 MHz, DMSO‑d6) δ 7.81 (s,
1H), 7.23 (d, J = 8.1 Hz, 2H), 6.86 (d, J = 8.1 Hz, 2H), 4.34 (d,
J = 6.1 Hz, 2H), 3.71 (s, 3H), 3.64 (s, 3H), 3.50 (s, 2H), 3.41 (d,
J = 4.9 Hz, 4H), 2.66 (d, J = 6.1 Hz, 4H). 13C NMR (101 MHz,
DMSO‑d6) δ 171.15, 161.43, 159.43, 158.06, 131.98, 128.46, 128.30,
113.53, 113.53, 54.96, 51.91, 51.14, 43.60, 33.92, 26.23.
J = 3.7 Hz, 7H), 3.53 (s, 2H), 2.33 (t, J = 5.1 Hz, 4H), 2.20 (s, 3H). 13
C
NMR (101 MHz, DMSO‑d6) δ 173.74, 165.72, 160.05, 158.65, 151.57,
129.48, 128.34, 113.77, 101.60, 55.04, 54.12, 45.67, 43.62, 41.82,
32.34.
4-chloro-2-(3-hydroxypiperidin-1-yl)-7-(4-methoxybenzyl)-5,7-
dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one (15e): The title compound
15e (98 mg) was prepared from methyl 2-(2,4-dichloro-6-((4-methox-
ybenzyl)amino)pyrimidin-5-yl)acetate (6) (250 mg, 0.70 mmol) and 3-
hydroxypiperidine (141 mg, 1.40 mmol) directly using the procedure
described for compound 7 in 35% yield as a white solid. 1H NMR
(400 MHz, DMSO‑d6) δ 7.30 (d, J = 8.6 Hz, 2H), 6.86 (d, J = 8.7 Hz,
2H), 4.94 (s, 1H), 4.70 (s, 2H), 4.38–4.30 (m, 1H), 4.20 (d, J = 12.4 Hz,
1H), 3.71 (s, 3H), 3.51 (s, 2H), 3.46 (s, 1H), 3.14–3.04 (m, 1H), 2.95 (t,
J = 10.9 Hz, 1H), 1.89 (d, J = 9.9 Hz, 1H), 1.73 (q, J = 6.3, 4.2 Hz,
1H), 1.43–1.31 (m, 2H). 13C NMR (101 MHz, DMSO‑d6) δ 173.74,
165.67, 160.05, 158.68, 151.62, 129.58, 128.40, 113.78, 100.98,
65.01, 55.04, 50.86, 43.76, 41.82, 33.12, 32.34, 22.51.
Methyl 2-(4-chloro-6-(4-methoxybenzylamino)-2-(piperidin-1-yl)
pyrimidin-5-yl)acetate (14c): The title compound 14c (181 mg) was
prepared from methyl 2-(2,4-dichloro-6-((4-methoxybenzyl)amino)
pyrimidin-5-yl)acetate (6) (250 mg, 0.70 mmol) and piperidine
(120 mg, 1.40 mmol) using the procedure described for compound 7 in
64% yield as a light yellow solid. 1H NMR (400 MHz, DMSO‑d6) δ 7.55
(s, 1H), 7.25–7.16 (m, 2H), 6.90–6.82 (m, 2H), 4.42 (d, J = 5.7 Hz, 2H),
3.71 (s, 3H), 3.62 (s, 3H), 3.59 (d, J = 5.2 Hz, 6H), 1.56 (d, J = 5.5 Hz,
2H), 1.43 (d, J = 10.0 Hz, 4H). 13C NMR (101 MHz, DMSO‑d6) δ
170.50, 161.61, 158.65, 158.35, 157.99, 132.00, 128.37, 113.48,
96.01, 55.00, 51.72, 44.12, 43.53, 31.33, 25.17, 24.25.
4-chloro-2-(4-hydroxypiperidin-1-yl)-7-(4-methoxybenzyl)-5,7-
dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one (15f): The title compound
15f (107 mg) was prepared from methyl 2-(2,4-dichloro-6-((4-meth-
oxybenzyl)amino)pyrimidin-5-yl)acetate (6) (250 mg, 0.70 mmol) and
4-hydroxypiperidine (141 mg, 1.40 mmol) directly using the procedure
described for compound 7 in 39% yield as a white solid. 1H NMR
(400 MHz, DMSO‑d6) δ 7.31–7.26 (m, 2H), 6.86 (d, J = 8.6 Hz, 2H),
4.74 (d, J = 4.1 Hz, 1H), 4.70 (s, 2H), 4.17 (d, J = 13.2 Hz, 2H), 3.71
(s, 4H), 3.53 (s, 2H), 1.80–1.72 (m, 2H), 1.31 (dtd, J = 12.8, 9.1,
3.8 Hz, 2H). 13C NMR (101 MHz, DMSO‑d6) δ 173.74, 165.73, 159.90,
158.64, 151.63, 129.49, 128.39, 113.76, 101.06, 65.73, 55.04, 41.80,
41.43, 33.70, 32.34.
Methyl 2-(4-chloro-6-(4-methoxybenzylamino)-2-(4-methylpiper-
azin-1-yl)pyrimidin-5-yl)acetate (14d): The title compound 14d
(232 mg) was prepared from methyl 2-(2,4-dichloro-6-((4-methox-
ybenzyl)amino)pyrimidin-5-yl)acetate (6) (250 mg, 0.70 mmol) and 1-
methylpiperazine (140 mg, 1.40 mmol) using the procedure described
for compound 7 in 79% yield as a light yellow solid. 1H NMR (400 MHz,
DMSO‑d6) δ 7.60 (s, 1H), 7.21 (d, J = 8.5 Hz, 2H), 6.93–6.77 (m, 2H),
4.43 (d, J = 5.8 Hz, 2H), 3.71 (s, 3H), 3.62 (s, 3H), 3.58 (dd, J = 9.6,
4.9 Hz, 6H), 2.25 (t, J = 5.0 Hz, 4H), 2.16 (s, 3H). 13C NMR (101 MHz,
DMSO‑d6) δ 170.41, 161.61, 158.82, 158.28, 158.02, 131.89, 128.40,
113.49, 96.68, 54.99, 54.28, 51.75, 45.75, 43.54, 43.21, 31.34.
4-chloro-7-(4-methoxybenzyl)-2-(pyrrolidin-1-yl)-5,7-dihydro-
6H-pyrrolo[2,3-d]pyrimidin-6-one (15a): The title compound 15a
(139 mg) was prepared from methyl 2-(4-chloro-6-((4-methoxybenzyl)
4-(3-aminophenyl)-7-(4-methoxybenzyl)-2-(pyrrolidin-1-yl)-5,7-
dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one (16a): The title com-
pound 16a (59 mg) was prepared from 4-chloro-7-(4-methoxybenzyl)-
2-(pyrrolidin-1-yl)-5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one
(15a) (115 mg, 0.32 mmol) and 3-aminobenzeneboronic acid (48 mg,
0.35 mmol) using the procedure described for compound 12a in 44%
yield as a purple solid. Melting point: 210–212 °C, 1H NMR (400 MHz,
DMSO‑d6) δ 7.39–7.33 (m, 2H), 7.24 (d, J = 2.4 Hz, 1H), 7.11 (d,
J = 6.7 Hz, 2H), 6.90–6.85 (m, 2H), 6.65 (dd, J = 6.8, 2.1 Hz, 1H), 5.17
(s, 2H), 4.75 (s, 2H), 3.76 (s, 2H), 3.71 (s, 3H), 3.57 (d, J = 6.5 Hz, 4H),
1.97–1.91 (m, 4H). 13C NMR (101 MHz, DMSO‑d6) δ 174.63, 165.27,
158.99, 158.57, 156.62, 148.65, 137.85, 129.67, 128.91, 128.76,
amino)-2-(pyrrolidin-1-yl)pyrimidin-5-yl)acetate
(14a)
(215 mg,
0.55 mmol) using the procedure described for compound 8 in 70% yield
as a white solid. 1H NMR (400 MHz, DMSO‑d6) δ 7.37–7.25 (m, 2H),
6.87 (d, J = 8.6 Hz, 2H), 4.70 (s, 2H), 3.70 (d, J = 3.2 Hz, 3H), 3.53 (s,
2H), 3.42 (s, 4H), 1.91 (d, J = 6.9 Hz, 4H). 13C NMR (101 MHz,
DMSO‑d6) δ 173.74, 165.38, 158.80, 158.65, 151.47, 129.64, 128.41,
113.78, 100.47, 55.03, 46.59, 41.79, 32.33, 24.79.
18

S. Hu, et al.
BioorganicChemistry92(2019)103232
115.56, 115.34, 113.75, 113.34, 99.43, 55.02, 46.33, 41.39, 34.32,
24.93. HRMS Calcd for C24H26O2N5, 416.20810, found m/z 416.20792
[(M+H)+].
4-(3-aminophenyl)-2-(4-hydroxypiperidin-1-yl)-7-(4-methox-
ybenzyl)-5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one (16f): The
title compound 16f (35 mg) was prepared from 4-chloro-2-(4-hydro-
xypiperidin-1-yl)-7-(4-methoxybenzyl)-5,7-dihydro-6H-pyrrolo[2,3-d]
pyrimidin-6-one (15f) (100 mg, 0.25 mmol) and 3-aminobenzene-
boronic acid (39 mg, 0.28 mmol) using the procedure described for
4-(3-aminophenyl)-7-(4-methoxybenzyl)-2-thiomorpholino-5,7-
dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one (16b): The title compound
16b (41 mg) was prepared from 4-chloro-7-(4-methoxybenzyl)-2-thio-
morpholino-5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one
(15b)
compound 12a in 31% yield as a yellow solid. Melting point:
(100 mg, 0.26 mmol) and 3-aminobenzeneboronic acid (39 mg,
0.28 mmol) using the procedure described for compound 12a in 35%
yield as a green solid. Melting point: 201–203 °C, 1H NMR (400 MHz,
DMSO‑d6) δ 7.33 (d, J = 8.2 Hz, 2H), 7.21 (s, 1H), 7.11 (d, J = 6.9 Hz,
2H), 6.88 (d, J = 8.2 Hz, 2H), 6.66 (d, J = 6.7 Hz, 1H), 5.20 (s, 2H),
4.84–4.71 (m, 2H), 4.14 (d, J = 5.0 Hz, 4H), 3.78 (s, 2H), 3.71 (s, 3H),
2.62 (d, J = 4.9 Hz, 4H). 13C NMR (101 MHz, DMSO‑d6) δ 174.58,
165.69, 159.61, 158.59, 156.61, 148.76, 137.60, 129.58, 128.87,
128.78, 115.52, 115.51, 113.76, 113.27, 100.56, 55.05, 46.24, 41.48,
34.30, 25.78. HRMS Calcd for C24H26O2N5S, 448.18017, found m/z
448.17953 [(M+H)+].
190–192 °C, 1H NMR (400 MHz, DMSO‑d6) δ 7.37–7.30 (m, 2H), 7.22
(d, J = 2.4 Hz, 1H), 7.10 (d, J = 6.9 Hz, 2H), 6.90–6.84 (m, 2H), 6.65
(dt, J = 7.1, 2.2 Hz, 1H), 5.20 (s, 2H), 4.75 (s, 2H), 4.71 (d, J = 4.3 Hz,
1H), 4.37 (d, J = 13.1 Hz, 2H), 3.74 (d, J = 23.4 Hz, 6H), 3.29 (d,
J = 13.6 Hz, 2H), 1.80 (d, J = 12.5 Hz, 2H), 1.33 (d, J = 9.6 Hz, 2H).
13C NMR (101 MHz, DMSO‑d6) δ 174.63, 165.59, 160.09, 158.57,
156.57, 148.74, 137.76, 129.52, 128.88, 128.84, 115.52, 115.43,
113.75, 113.25, 100.05, 66.27, 55.04, 41.45, 34.32, 33.91, 26.30.
HRMS Calcd for C25H28O3N5, 446.21867, found m/z 446.21820 [(M
+H)+].
N-(3-(7-(4-methoxybenzyl)-6-oxo-2-(pyrrolidin-1-yl)-6,7-di-
hydro-5H-pyrrolo[2,3-d]pyrimidin-4-yl)phenyl)acetamide (17a): The
title compound 17a (17 mg) was prepared from 4-(3-aminophenyl)-7-
(4-methoxybenzyl)-2-(pyrrolidin-1-yl)-5,7-dihydro-6H-pyrrolo[2,3-d]
pyrimidin-6-one (16a) (30 mg, 0.07 mmol) using the procedure de-
scribed for compound 13 in 53% yield as a brown solid. Melting point:
131–133 °C, 1H NMR (400 MHz, DMSO‑d6) δ 10.07 (s, 1H), 8.24–8.13
(m, 1H), 7.75 (d, J = 8.1 Hz, 1H), 7.67 (d, J = 7.8 Hz, 1H), 7.45–7.31
(m, 3H), 6.87 (d, J = 8.2 Hz, 2H), 4.74 (s, 2H), 3.78 (s, 2H), 3.70 (d,
J = 1.4 Hz, 3H), 3.58 (d, J = 6.7 Hz, 4H), 2.07 (s, 3H), 1.94 (d,
J = 6.9 Hz, 4H). 13C NMR (101 MHz, DMSO‑d6) δ 174.52, 168.37,
165.47, 158.97, 158.59, 155.42, 139.46, 137.55, 129.69, 128.83,
128.70, 122.34, 120.17, 118.44, 113.75, 99.66, 55.01, 46.34, 41.45,
34.23, 24.92, 24.01. HRMS Calcd for C26H28O3N5, 458.21867, found
m/z 458.21817 [(M+H)+].
4-(3-aminophenyl)-7-(4-methoxybenzyl)-2-(piperidin-1-yl)-5,7-
dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one (16c): The title compound
16c (35 mg) was prepared from 4-chloro-7-(4-methoxybenzyl)-2-(pi-
peridin-1-yl)-5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one
(15c)
(80 mg, 0.22 mmol) and 3-aminobenzeneboronic acid (32 mg,
0.24 mmol) using the procedure described for compound 12a in 37%
yield as a purple solid. Melting point: 197–200 °C, 1H NMR (400 MHz,
DMSO‑d6) δ 7.36–7.30 (m, 2H), 7.26–7.18 (m, 1H), 7.10 (d, J = 6.6 Hz,
2H), 6.90–6.85 (m, 2H), 6.65 (dd, J = 6.6, 2.5 Hz, 1H), 5.20 (s, 2H),
4.75 (s, 2H), 3.81 (t, J = 5.3 Hz, 4H), 3.77 (s, 2H), 3.71 (s, 3H), 1.64 (s,
2H), 1.53 (d, J = 7.2 Hz, 4H). 13C NMR (101 MHz, DMSO‑d6) δ 174.65,
165.55, 160.10, 158.57, 156.56, 148.74, 137.81, 129.50, 128.89,
128.83, 115.52, 115.41, 113.75, 113.24, 99.84, 55.04, 44.51, 41.41,
34.33, 25.21, 24.40. HRMS Calcd for C25H28O2N5, 430.22375, found
m/z 430.22366 [(M+H)+].
N-(3-(7-(4-methoxybenzyl)-6-oxo-2-thiomorpholino-6,7-dihydro-
5H-pyrrolo[2,3-d]pyrimidin-4-yl)phenyl)acetamide (17b): The title
compound 17b (10 mg) was prepared from 4-(3-aminophenyl)-7-(4-
methoxybenzyl)-2-thiomorpholino-5,7-dihydro-6H-pyrrolo[2,3-d]pyr-
imidin-6-one (16b) (20 mg, 0.04 mmol) using the procedure described
for compound 13 in 46% yield as a yellow solid. Melting point:
137–139 °C, 1H NMR (400 MHz, DMSO‑d6) δ 10.06 (s, 1H), 8.16 (s, 1H),
7.76 (d, J = 8.1 Hz, 1H), 7.64 (d, J = 7.8 Hz, 1H), 7.40 (t, J = 7.9 Hz,
1H), 7.33 (d, J = 8.2 Hz, 2H), 6.88 (d, J = 8.2 Hz, 2H), 4.76 (s, 2H),
4.15 (t, J = 4.7 Hz, 4H), 3.81 (s, 2H), 3.71 (s, 3H), 2.62 (t, J = 4.8 Hz,
4H), 2.07 (s, 3H). 13C NMR (101 MHz, DMSO‑d6) δ 174.46, 168.41,
165.88, 159.64, 158.61, 155.46, 139.52, 137.31, 129.59, 128.82,
128.70, 122.34, 120.32, 118.37, 113.76, 100.83, 55.04, 46.27, 41.54,
34.20, 25.78, 24.02. HRMS Calcd for C26H28O3N5S, 490.19074, found
m/z 490.19009 [(M+H)+].
4-(3-aminophenyl)-7-(4-methoxybenzyl)-2-(4-methylpiperazin-1-
yl)-5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one (16d): The title
compound 16d (48 mg) was prepared from 4-chloro-7-(4-methox-
ybenzyl)-2-(4-methylpiperazin-1-yl)-5,7-dihydro-6H-pyrrolo[2,3-d]pyr-
imidin-6-one (15d) (100 mg, 0.25 mmol) and 3-aminobenzeneboronic
acid (39 mg, 0.28 mmol) using the procedure described for compound
12a in 43% yield as a white solid. Melting point: 221–223 °C, 1H NMR
(400 MHz, DMSO‑d6) δ 7.38–7.30 (m, 2H), 7.23 (t, J = 1.6 Hz, 1H),
7.11 (d, J = 5.9 Hz, 2H), 6.90–6.84 (m, 2H), 6.66 (dt, J = 5.9, 2.6 Hz,
1H), 5.19 (s, 2H), 4.76 (s, 2H), 3.83–3.76 (m, 6H), 3.71 (s, 3H), 2.37 (t,
J = 5.0 Hz, 4H), 2.22 (s, 3H). 13C NMR (101 MHz, DMSO‑d6) δ 174.64,
165.58, 160.20, 158.58, 156.49, 148.76, 137.65, 129.48, 128.86,
128.83, 115.54, 115.48, 113.77, 113.26, 100.56, 55.04, 54.37, 45.82,
43.57, 41.44, 34.33. HRMS Calcd for C25H29O2N6, 445.23465, found
m/z 445.23407 [(M+H)+].
N-(3-(7-(4-methoxybenzyl)-6-oxo-2-(piperidin-1-yl)-6,7-dihydro-
5H-pyrrolo[2,3-d]pyrimidin-4-yl)phenyl)acetamide (17c): The title
compound 17c (14 mg) was prepared from 4-(3-aminophenyl)-7-(4-
methoxybenzyl)-2-(piperidin-1-yl)-5,7-dihydro-6H-pyrrolo[2,3-d]pyr-
imidin-6-one (16c) (20 mg, 0.05 mmol) using the procedure described
for compound 13 in 64% yield as a light yellow solid. Melting point:
166–168 °C, 1H NMR (400 MHz, DMSO‑d6) δ 10.07 (s, 1H), 8.16 (s, 1H),
7.77 (d, J = 8.1 Hz, 1H), 7.64 (d, J = 7.8 Hz, 1H), 7.39 (t, J = 7.9 Hz,
1H), 7.33 (d, J = 8.2 Hz, 2H), 6.87 (d, J = 8.2 Hz, 2H), 4.76 (s, 2H),
3.82 (dd, J = 11.1, 5.7 Hz, 6H), 3.71 (s, 3H), 2.07 (s, 3H), 1.64 (d,
J = 6.0 Hz, 2H), 1.54 (d, J = 7.5 Hz, 4H). 13C NMR (101 MHz,
DMSO‑d6) δ 174.57, 168.41, 165.76, 160.12, 158.59, 155.42, 139.52,
137.54, 129.52, 128.82, 128.82, 122.35, 120.25, 118.35, 113.76,
100.13, 55.04, 44.53, 41.48, 34.23, 25.21, 24.39, 24.04. HRMS Calcd
for C27H30O3N5, 472.23432, found m/z 472.23389 [(M+H)+].
N-(3-(7-(4-methoxybenzyl)-2-(4-methylpiperazin-1-yl)-6-oxo-
6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-4-yl)phenyl)acetamide
(17d): The title compound 17d (18 mg) was prepared from 4-(3-ami-
nophenyl)-7-(4-methoxybenzyl)-2-(4-methylpiperazin-1-yl)-5,7-
4-(3-aminophenyl)-2-(3-hydroxypiperidin-1-yl)-7-(4-methox-
ybenzyl)-5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one (16e): The
title compound 16e (42 mg) was prepared from 4-chloro-2-(3-hydro-
xypiperidin-1-yl)-7-(4-methoxybenzyl)-5,7-dihydro-6H-pyrrolo[2,3-d]
pyrimidin-6-one (15e) (100 mg, 0.25 mmol) and 3-aminobenzene-
boronic acid (39 mg, 0.28 mmol) using the procedure described for
compound 12a in 37% yield as
a yellow solid. Melting point:
204–206 °C, 1H NMR (400 MHz, DMSO‑d6) δ 7.40–7.31 (m, 2H),
7.26–7.18 (m, 1H), 7.17–7.05 (m, 2H), 6.92–6.84 (m, 2H), 6.66 (dt,
J = 6.1, 2.6 Hz, 1H), 5.20 (s, 2H), 4.90 (d, J = 4.5 Hz, 1H), 4.76 (s, 2H),
4.65–4.55 (m, 1H), 4.45 (d, J = 12.9 Hz, 1H), 3.76 (d, J = 2.0 Hz, 2H),
3.72 (s, 3H), 3.52–3.44 (m, 1H), 3.02 (d, J = 10.4 Hz, 1H), 2.89 (dd,
J = 12.5, 9.1 Hz, 1H), 1.93 (s, 1H), 1.75 (s, 1H), 1.45–1.35 (m, 2H). 13
C
NMR (101 MHz, DMSO‑d6) δ 175.14, 166.03, 160.65, 159.08, 157.11,
149.22, 138.29, 130.07, 129.37, 129.33, 116.04, 115.92, 114.26,
113.76, 100.50, 65.74, 55.53, 51.42, 44.04, 41.91, 34.80, 34.05, 23.34.
HRMS Calcd for C25H28O3N5, 446.21867, found m/z 446.21817 [(M
+H)+].
19

S. Hu, et al.
BioorganicChemistry92(2019)103232
dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one (16d) (30 mg, 0.07 mmol)
using the procedure described for compound 13 in 55% yield as a green
solid. Melting point: 159–161 °C, 1H NMR (400 MHz, DMSO‑d6) δ 10.13
(s, 1H), 8.18 (t, J = 2.0 Hz, 1H), 7.78 (dd, J = 8.0, 2.1 Hz, 1H), 7.65 (d,
J = 7.8 Hz, 1H), 7.39 (t, J = 7.9 Hz, 1H), 7.36–7.27 (m, 2H), 6.91–6.82
(m, 2H), 4.76 (s, 2H), 3.86 (s, 4H), 3.81 (s, 2H), 3.70 (s, 3H), 2.54 (s,
4H), 2.32 (s, 3H), 2.07 (s, 3H). 13C NMR (101 MHz, DMSO‑d6) δ 174.54,
168.49, 165.85, 160.12, 158.62, 155.40, 139.57, 137.29, 129.55,
128.85, 128.74, 122.40, 120.38, 118.41, 113.79, 101.13, 55.06, 53.82,
45.03, 43.01, 41.54, 34.23, 24.05. HRMS Calcd for C27H31O3N6,
487.24576, found m/z 487.24506 [(M+H)+].
4.2.2. Kinase enzyme assay
These assays were carried out as described previously. All of the
enzymatic reactions were conducted at 30 °C for 40 min. The 50 μL
reaction mixture contains 40 mM Tris, pH 7.4, 10 mM MgCl2, 0.1 mg/
mL BSA, 1 mM DTT, 10 μM ATP, 0.2 ug/mL PI3 Kinase and 100 uM
lipid substrate. The compounds were diluted in 10% DMSO and 5 μL of
reactions. The assay was performed using Kinase-Glo Plus luminescence
kinase assay kit. It measures kinase activity by quantitating the amount
of ATP remaining in solution following a kinase reaction. The lumi-
nescenct signal from the assay was correlated with the amount of ATP
present and is inversely correlated with the amount of kinase activity.
The IC50 values were calculated using nonlinear regression with nor-
malized dose-response fit using Prism GraphPad software.
N-(3-(2-(3-hydroxypiperidin-1-yl)-7-(4-methoxybenzyl)-6-oxo-
6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-4-yl)phenyl)acetamide
(17e): The title compound 17e (12 mg) was prepared from 4-(3-ami-
nophenyl)-2-(3-hydroxypiperidin-1-yl)-7-(4-methoxybenzyl)-5,7-di-
hydro-6H-pyrrolo[2,3-d]pyrimidin-6-one (16e) (20 mg, 0.04 mmol)
using the procedure described for compound 13 in 56% yield as a
yellow solid. Melting point: 168–170 °C, 1H NMR (400 MHz, DMSO‑d6)
δ 10.08 (s, 1H), 8.13 (s, 1H), 7.77 (d, J = 8.0 Hz, 1H), 7.66 (d,
J = 7.8 Hz, 1H), 7.40 (t, J = 8.0 Hz, 1H), 7.35 (d, J = 8.6 Hz, 2H), 6.88
(d, J = 8.6 Hz, 2H), 4.91 (d, J = 4.5 Hz, 1H), 4.76 (s, 2H), 4.59 (d,
J = 12.0 Hz, 1H), 4.44 (d, J = 13.0 Hz, 1H), 3.79 (d, J = 2.4 Hz, 2H),
3.71 (s, 3H), 3.48 (d, J = 4.4 Hz, 1H), 3.06 (t, J = 11.2 Hz, 1H), 2.92
(dd, J = 12.5, 9.1 Hz, 1H), 2.07 (s, 3H), 1.93 (s, 1H), 1.75 (s, 1H), 1.40
(d, J = 8.3 Hz, 2H). 13C NMR (101 MHz, DMSO‑d6) δ 174.54, 168.49,
165.85, 160.12, 158.62, 155.40, 139.57, 137.29, 129.55, 128.85,
128.74, 122.40, 120.38, 118.41, 113.79, 101.13, 55.06, 53.82, 53.82,
45.03, 45.02, 43.01, 41.54, 34.23, 24.05. HRMS Calcd for C27H30O4N5,
488.22923, found m/z 488.22876 [(M+H)+].
4.2.3. Western blotting
After drug treatment, cells were washed twice with ice-cold PBS
(137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 1.8 mM KH2PO4, pH
7.4) and lysed in SDS sample buffer. Cell lysates containing equal
amounts of protein were separated by SDS polyacrylamide gel elec-
trophoresis and transferred to polyvinylidene difluoride membranes.
After being blocked in 5% nonfat milk in Tris-buffered saline with 0.1%
Tween 20, pH 7.6, the membranes were incubated with the appropriate
primary antibodies at 4 °C overnight and then exposed to secondary
antibodies for 2 h at room temperature. Immunoreactive proteins were
visualized using the enhanced chemiluminescence system from Pierce
Chemical.
4.2.4. Cell cycle assay
MCF-7 cells (5 × 105 cells/mL) were seeded in six-well plates and
treated with compounds at different concentrations for 24 h. The cells
were then harvested by trypsinization and washed twice with cold PBS.
After centrifugation and removal of the supernatants, cells were re-
suspended in 400 μL of 1 × PBS buffer. After adding 10 μL of PI the cells
were incubated at room temperature for 15 min in the dark. The stained
cells were analyzed by a flow cytometer (BD Accuri C6).
N-(3-(2-(4-hydroxypiperidin-1-yl)-7-(4-methoxybenzyl)-6-oxo-
6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-4-yl)phenyl)acetamide
(17f): The title compound 17f (10 mg) was prepared from 4-(3-ami-
nophenyl)-2-(4-hydroxypiperidin-1-yl)-7-(4-methoxybenzyl)-5,7-di-
hydro-6H-pyrrolo[2,3-d]pyrimidin-6-one (16f) (20 mg, 0.04 mmol)
using the procedure described for compound 13 in 47% yield as a
yellow solid. Melting point: 181–183 °C, 1H NMR (400 MHz, DMSO‑d6)
δ 10.07 (s, 1H), 8.16 (s, 1H), 7.84–7.73 (m, 1H), 7.64 (d, J = 7.8 Hz,
1H), 7.40 (t, J = 7.9 Hz, 1H), 7.33 (d, J = 8.3 Hz, 2H), 6.87 (d,
J = 8.6 Hz, 2H), 4.76 (s, 2H), 4.72 (d, J = 4.3 Hz, 1H), 4.38 (d,
J = 12.8 Hz, 2H), 3.80 (s, 2H), 3.71 (s, 4H), 3.29 (s, 2H), 2.07 (s, 3H),
1.80 (d, J = 12.2 Hz, 2H), 1.34 (d, J = 9.9 Hz, 2H). 13C NMR (101 MHz,
DMSO‑d6) δ 174.55, 168.42, 165.81, 160.13, 158.59, 155.45, 139.52,
137.49, 129.54, 128.81, 128.81, 122.36, 120.28, 118.37, 113.76,
100.35, 66.23, 55.05, 41.47, 41.46, 34.22, 33.90, 24.04. HRMS Calcd
for C27H30O4N5, 488.22923, found m/z 488.22867 [(M+H)+].
Acknowledgment
This study was supported by Beijing Natural Science Foundation
(No. 2192004). Thanks to Xiuqing Song for the NMR test. The services
of cytotoxic activity and cell IC50 were kindly provided by Nanjing
Ogpharmaceutical Co., Ltd. (Nanjing, China) The service of enzyme
IC50, WB and cell cycle was kindly provided by Huawei Pharmaceutical
Co., Ltd. (Shandong, China)
Appendix A. Supplementary material
4.2. Biological assay
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bioorg.2019.103232.
4.2.1. Cytotoxicity assay
The tests were carried out under the condition that the ratio of
living cells was greater than 90%. The cell proliferation inhibition assay
was performed using the EnoGeneCell™ Counting Kit-8 (CCK-8) cell
viability assay kit. The cells were digested, counted, and prepared into a
cell suspension at a concentration of 1 × 105 cells/mL. 100 μL of cell
suspension (1 × 104 cells per well) was added to each well of a 96-well
plate. The 96-well plates were incubated at 37 °C for 24 h under 5%
CO2. 100 μL of the corresponding drug-containing medium was added
to each well at a concentration of 50 μM and set up a negative control
group, vehicle control group, positive control group, 5 replicate wells
per group. The 96 well plates were incubated at 37 °C for 72 h under 5%
CO2. 10 μL of CCK-8 solution was added to each well, and the 96 well
plates were incubated in an incubator for 4 h. The OD value at 450 nm
was measured with a microplate reader. The inhibition rates of 28
compounds and positive control against human malignant glioblastoma
cell line U87MG and human prostate cancer cell line PC-3 were cal-
culated.
References
[1] A.M. Martelli, C. Evangelisti, F. Chiarini, C. Grimaldi, A. Cappellini, A. Ognibene,
J.A. McCubrey, The emerging role of the phosphatidylinositol 3-kinase/Akt/mam-
malian target of rapamycin signaling network in normal myelopoiesis and leuke-
mogenesis, Cancers 2010 (1803) 991–1002, https://doi.org/10.3390/
cancers2031576.
[2] J. Yu, W. Cui, Proliferation, survival and metabolism: the role of PI3K/AKT/mTOR
signalling in pluripotency and cell fate determination, Development 143 (2016)
3050–3060, https://doi.org/10.1242/dev.137075.
[3] R.A. Saxton, D.M. Sabatini, mTOR signaling in growth, metabolism, and disease,
Cell 168 (2017) 960–976, https://doi.org/10.1016/j.cell.2017.03.035.
[4] H. Yang, D.G. Rudge, J.D. Koos, B. Vaidialingam, H.J. Yang, N.P. Pavletich, mTOR
kinase structure, mechanism and regulation, Nature 497 (2013) 217–224, https://
doi.org/10.1038/nature12122.
[5] D.-H. Kim, D.D. Sarbassov, S.M. Ali, J.E. King, R.R. Latek, H. Erdjument-Bromage,
P. Tempst, D.M. Sabatini, mTOR interacts with raptor to form a nutrient-sensitive
complex that signals to the cell growth machinery, Cell 110 (2002) 163–175,
https://doi.org/10.1016/S0092-8674(02)00808-5.
[6] D.-H. Kim, D.D. Sarbassov, S.M. Ali, R.R. Latek, K.V.P. Guntur, H. Erdjument-
20

S. Hu, et al.
BioorganicChemistry92(2019)103232
Bromage, P. Tempst, D.M. Sabatini, GβL, a Positive Regulator of the Rapamycin-
[21] D.P. Sutherlin, L. Bao, M. Berry, G. Castanedo, I. Chuckowree, J. Dotson, A. Folks,
L. Friedman, R. Goldsmith, J. Gunzner, et al., Discovery of a potent, selective, and
orally available class I phosphatidylinositol 3-kinase (PI3K)/mammalian target of
rapamycin (mTOR) kinase inhibitor (GDC-0980) for the treatment of cancer, J.
Med. Chem. 54 (2011) 7579–7587, https://doi.org/10.1021/jm2009327.
[22] S. Schenone, C. Brullo, F. Musumeci, M. Radi, M. Botta, ATP-competitive inhibitors
of mTOR: an update, Curr. Med. Chem. 18 (2011) 2995–3014, https://doi.org/10.
2174/092986711796391651.
Sensitive Pathway Required for the Nutrient-Sensitive Interaction between Raptor
and mTOR, Mol. Cell 11 (2003) 895–904, https://doi.org/10.1016/S1097-
2765(03)00114-X.
[7] M.A. Frias, C.C. Thoreen, J.D. Jaffe, W. Schroder, T. Sculley, S.A. Carr,
D.M. Sabatini, mSin1 is necessary for Akt/PKB phosphorylation, and its isoforms
define three distinct mTORC2s, Curr. Biol. 16 (2006) 1865–1870, https://doi.org/
10.1016/j.cub.2006.08.001.
[8] D.A. Fruman, H. Chiu, B.D. Hopkins, S. Bagrodia, L.C. Cantley, R.T. Abraham, The
PI3K pathway in human disease, Cell 170 (2017) 605–635, https://doi.org/10.
1016/j.cell.2017.07.029.
[23] C.C. Thoreen, S.A. Kang, J.W. Chang, Q. Liu, J. Zhang, Y. Gao, L.J. Reichling,
T. Sim, D.M. Sabatini, N.S. Gray, An ATP-competitive mTOR inhibitor reveals ra-
pamycin-insensitive functions of mTORC1, J. Biol. Chem. 284 (2009) 8023–8032,
https://doi.org/10.1074/jbc.M900301200.
[9] M. Laplante, D.M. Sabatini, mTOR signaling in growth control and disease, Cell 149
(2012) 274–293, https://doi.org/10.1016/j.cell.2012.03.017.
[24] F. Chiarini, C. Evangelisti, J.A. McCubrey, A.M. Martelli, Current treatment stra-
tegies for inhibiting mTOR in cancer, Trends Pharmacol. Sci. 36 (2015) 124–135,
https://doi.org/10.1016/j.tips.2014.11.004.
[10] I. Sansal, W.R. Sellers, The biology and clinical relevance of the PTEN tumor sup-
pressor pathway, J. Clin. Oncol. 22 (2004) 2954–2963, https://doi.org/10.1200/
JCO.2004.02.141.
[25] M.E. Feldman, B. Apsel, A. Uotila, R. Loewith, Z.A. Knight, D. Ruggero,
K.M. Shokat, Active-site inhibitors of mTOR target rapamycin-resistant outputs of
mTORC1 and mTORC2, PLOS Biol. 7 (2009) e1000038, https://doi.org/10.1371/
journal.pbio.1000038.
[11] H. Takeuchi, Y. Kondo, K. Fujiwara, T. Kanzawa, H. Aoki, G.B. Mills, S. Kondo,
Synergistic augmentation of rapamycin-induced autophagy in malignant glioma
cells by phosphatidylinositol 3-kinase/protein kinase B inhibitors, Cancer Res. 65
(2005) 3336–3346, https://doi.org/10.1158/0008-5472.CAN-04-3640.
[12] S.R. Edwards, T.J. Wandless, The rapamycin-binding domain of the protein kinase
mammalian target of rapamycin is a destabilizing domain, J. Biol. Chem. 282
(2007) 13395–13401, https://doi.org/10.1074/jbc.M700498200.
[13] S.I. Arriola Apelo, D.W. Lamming, Rapamycin: an InhibiTOR of aging emerges from
the soil of Easter Island, J. Gerontol A-Biol. 71 (2016) 841–849, https://doi.org/10.
1093/gerona/glw090.
[26] S. Hu, Z. Zhao, Y. Ni, H. Xin, H. Yan, X. Song, Design, synthesis and biological
evaluation of 4-Aryl-5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one derivatives as a
PI3Kα inhibitor, Biol. Pharm. Bull. 42 (2019) 1013–1018, https://doi.org/10.1248/
bpb.b19-00080.
[27] M. Sugiyama, H. Takahashi, K. Hosono, H. Endo, S. Kato, K. Yoneda, Y. Nozaki,
K. Fujita, M. Yoneda, K. Wada, H. Nakagama, A. Nakajima, Adiponectin inhibits
colorectal cancer cell growth through the AMPK/mTOR pathway, Int. J. Oncol. 34
(2009) 339–344, https://doi.org/10.3892/ijo_00000156.
[14] Q. Li, R. Rao, J. Vazzana, P. Goedegebuure, K. Odunsi, W. Gillanders, P.A. Shrikant,
Regulating mammalian target of rapamycin to tune vaccination-induced CD8+ T
cell responses for tumor immunity, J. Immunol. 188 (2012) 3080–3087, https://
doi.org/10.4049/jimmunol.1103365.
[28] M.A. Kashem, R.M. Nelson, J.D. Yingling, S.S. Pullen, A.S. Prokopowicz III,
J.W. Jones, J.P. Wolak, G.R. Rogers, M.M. Morelock, R.J. Snow, C.A. Homon,
S. Jakes, Three mechanistically distinct kinase assays compared: Measurement of
intrinsic ATPase activity identified the most comprehensive set of ITK inhibitors, J.
Biomol. Screen 12 (2007) 70–83, https://doi.org/10.1177/1087057106296047.
[29] M. Zhan, Y. Deng, L. Zhao, G. Yan, F. Wang, Y. Tian, L. Zhang, H. Jiang, Y. Chen,
Design, synthesis, and biological evaluation of dimorpholine substituted thieno-
pyrimidines as potential class I PI3K/mTOR dual inhibitors, J. Med. Chem. 60
(2017) 4023–4035, https://doi.org/10.1021/acs.jmedchem.7b00357.
[30] Q. Guo, C. Yu, C. Zhang, Y. Li, T. Wang, Z. Huang, X. Wang, W. Zhou, Y. Li, Z. Qin,
C. Wang, R. Gao, Y. Nie, Y. Ma, Y. Shi, J. Zheng, S. Yang, Y. Fan, R. Xiang, Highly
selective, potent, and oral mTOR inhibitor for treatment of cancer as autophagy
inducer, J. Med. Chem. 61 (2018) 881–904, https://doi.org/10.1021/acs.
jmedchem.7b01402.
[15] J.B. Easton, P.J. Houghton, mTOR and cancer therapy, Oncogene 25 (2006)
6436–6446, https://doi.org/10.12688/f1000research.9207.1.
[16] H.A. Lane, J.M. Wood, P.M. McSheehy, P.R. Allegrini, A. Boulay, J. Brueggen,
A. Littlewood-Evans, S.-M. Maira, G. Martiny-Baron, C.R. Schnell, P. Sini,
T. O'Reilly, mTOR inhibitor RAD001 (everolimus) has antiangiogenic/vascular
properties distinct from a VEGFR tyrosine kinase inhibitor, Clin. Cancer Res. 15
(2009) 1612–1622, https://doi.org/10.1158/1078-0432.CCR-08-2057.
[17] Y. Zhu, X. Zhang, Y. Liu, S. Zhang, J. Liu, Y. Ma, J. Zhang, Antitumor effect of the
mTOR inhibitor everolimus in combination with trastuzumab on human breast
cancer stem cells in vitro and in vivo, Tumor Biol. 33 (2012) 1349–1362, https://
doi.org/10.1007/s13277-012-0383-6.
[18] L. Wu, D.C. Birle, I.F. Tannock, Effects of the mammalian target of rapamycin in-
hibitor CCI-779 used alone or with chemotherapy on human prostate cancer cells
and xenografts, Cancer Res. 65 (2005) 2825–2831, https://doi.org/10.1158/0008-
5472.CAN-04-3137.
[31] J. Kunz, R. Henriquez, U. Schneider, M. Deuter-Reinhard, N.R. Movva, M.N. Hall,
Target of rapamycin in yeast, TOR2, is an essential phosphatidylinositol kinase
homolog required for G1 progression, Cell 73 (1993) 585–596, https://doi.org/10.
1016/0092-8674(93)90144-F.
[19] B. Geoerger, K. Kerr, C.-B. Tang, K.-M. Fung, B. Powell, L.N. Sutton, P.C. Phillips,
A.J. Janss, Antitumor activity of the rapamycin analog CCI-779 in human primitive
neuroectodermal tumor/medulloblastoma models as single agent and in combina-
tion chemotherapy, Cancer Res. 61 (2001) 1527–1532.
[32] S. Rayne, K. Forest, Performance of the ALOGPS 2.1 program for octanol-water
partition coefficient prediction with organic chemicals on the Canadian Domestic
Substances List, Nat. Preced. (2009), https://doi.org/10.1038/npre.2009.3882.1.
[33] M.D. Shultz, Two decades under the influence of the rule of five and the changing
properties of approved oral drugs, J. Med. Chem. (2018), https://doi.org/10.1021/
acs.jmedchem.8b00686.
[20] Y.-J. Zhang, Y. Duan, X.F.S. Zheng, Targeting the mTOR kinase domain: the second
generation of mTOR inhibitors, Drug Discov. Today 16 (2011) 325–331, https://
doi.org/10.1016/j.drudis.2011.02.008.
21