Design, synthesis of 1,3-dimethylpyrimidine-2,4-diones as potent and selective aldehyde dehydrogenase 1A1 (ALDH1A1) inhibitors with glucose consumption improving activity

Article abstract of DOI:10.1016/j.bioorg.2020.103971

LDH1A1, one of 19 NAD(P)⁺-dependent aldehyde dehydrogenases, participates in multiple metabolic pathways and has been indicated to play an important role in obesity and diabetes. In this study, a series of 1,3-dimethylpyrimidine-2,4-diones were designed, synthesized and evaluated as novel selective aldehyde dehydrogenase 1A1 inhibitors. Among them, compounds 46, 50, 53, 56 and 57 exhibited excellent inhibitory activity against ALDH1A1 with IC₅₀ values in the low nanomolar range and high selectivity over ALDH1A2, ALDH1A3, ALDH2 and ALDH3A1. Furthermore, in vitro study demonstrated that compound 57 effectively improved glucose consumption in HepG2 cells compared to compound 1 (CM026).

Full text of DOI:10.1016/j.bioorg.2020.103971

Journal Pre-proofs
Design, synthesis of 1,3-dimethylpyrimidine-2,4-diones as potent and selec-
tive aldehyde dehydrogenase 1A1 (ALDH1A1) inhibitors with glucose con-
sumption improving activity
Zonghui Ma, Ling Jiang, Guoxin Li, Dailin Liang, Luanfeng Li, Li Liu,
Cheng Jiang
PII:
S0045-2068(20)30392-8
DOI:
https://doi.org/10.1016/j.bioorg.2020.103971
YBIOO 103971
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
19 February 2020
13 May 2020
22 May 2020
Please cite this article as: Z. Ma, L. Jiang, G. Li, D. Liang, L. Li, L. Liu, C. Jiang, Design, synthesis of 1,3-
dimethylpyrimidine-2,4-diones as potent and selective aldehyde dehydrogenase 1A1 (ALDH1A1) inhibitors with
glucose consumption improving activity, Bioorganic Chemistry (2020), doi: https://doi.org/10.1016/j.bioorg.
2
020.103971
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Design, synthesis of 1,3-dimethylpyrimidine-2,4-diones as potent and
selective aldehyde dehydrogenase 1A1 (ALDH1A1) inhibitors with
glucose consumption improving activity
a,b
c
a,b
a,b
b
c
Zonghui Ma , Ling Jiang , Guoxin Li , Dailin Liang , Luanfeng Li , Li Liu **,
a,b
Cheng Jiang *
aJiang Su Key Laboratory of Drug Design and Optimization, China Pharmaceutical
University, Tongjiaxiang 24, Nanjing 210009, China
bDepartment of Medicinal Chemistry, China Pharmaceutical University,
Tongjiaxiang 24, Nanjing 210009, China
cKey Laboratory of Drug Metabolism and Pharmacokinetics, China Pharmaceutical
University, Tongjiaxiang 24, Nanjing 210009, China
AUTHOR INFORMATION
Corresponding Authors
*
*
*E-mail for L.L.: liulee@cpu.edu.cn
E-mail for C.J.: jc@cpu.edu.cn

+
Abstract: ALDH1A1, one of 19 NAD(P) -dependent aldehyde dehydrogenases,
participates in multiple metabolic pathways and has been indicated to play an important
role in obesity and diabetes. In this study, a series of 1,3-dimethylpyrimidine-2,4-diones
were designed, synthesized and evaluated as novel selective aldehyde dehydrogenase
1
A1 inhibitors. Among them, compounds 46, 50, 53, 56 and 57 exhibited excellent
inhibitory activity against ALDH1A1 with IC values in the low nanomolar range and
5
0
high selectivity over ALDH1A2, ALDH1A3, ALDH2 and ALDH3A1. Furthermore, in
vitro study demonstrated that compound 57 effectively improved glucose consumption
in HepG2 cells compared to compound 1 (CM026).
Key words: ALDH1A1; enzyme inhibition; selective; synthesis; glucose consumption
improvement

1
. Introduction
Human aldehyde dehydrogenases (ALDHs) comprise a superfamily of at least 19
+
isozymes, most of which catalyze the NAD (P) - dependent oxidation of both
endogenous and exogenous aldehydes to their respective carboxylic acid derivatives
[1]. ALDHs act as a key regulator in multiple metabolic pathways and have been
indicated to possess important physiological and toxicological functions in several
areas such as cancers, metabolic disorders and other diseases [2,3]. Mutations in
ALDHs genes can lead to aberrant aldehyde oxidation and have been proved to be
associated with the occurrence and development of a series of human diseases,
including Parkinson’s disease, alcoholism and diabetes [4-7].
Up-regulation of ALDH1A1, also known as retinaldehyde dehydrogenase
1
(RALDH1), has been implicated in obesity and diabetes [7-9]. Several studies have
demonstrated that ALDH1A1 knockout mice were able to resist diet-induced obesity
as well as insulin resistance [10,11]. In addition, treatment with exogenous
retinaldehyde can reduce hepatic gluconeogenesis and lipid accumulation in ob/ob
mice [11,12]. These results indicate that ALDH1A1 plays a crucial role in obesity and
adipogenesis. Previous researches in our lab have revealed that ALDH1A1 inhibitor
citral is capable of significantly improving glucose metabolism through inhibiting
ALDH1A1 activity [13]. These findings suggest that inhibition of ALDH1A1
enzymatic activity may provide a new therapeutic option for related diseases.
Several small molecule ALDH1A1 inhibitors identified by high throughput
screening (HTS) have been reported (Fig. 1). Previous studies by Hurley and
colleagues demonstrated that theophylline-based 1 (CM026) and substituted tricyclic
pyrimidinone 2 (CM37) displayed effective ALDH1A1 inhibitory activity (0.8 and
0
.02 μM for 1 and 2, respectively) and showed excellent selectivity against other

ALDH isozymes, such as ALDH2 and ALDH3A1 [14]. Quinoline-based compound
(NCT-505) disclosed by Yang et al. exhibited potent and highly selective ALDH1A1
inhibition (1A1 IC = 0.007 μM, 1A2, 1A3, ALDH2, ALDH3A1 IC values > 1 μM)
3
5
0
50
and displayed efficacious cellular activities in MIA PaCa-2 cells, which preferentially
express ALDH1A1 [15].
Most recently, we reported a series of coumarin-based derivatives (e.g., 4a and
4
b) with potent inhibitory activity to ALDH1A1 and high selectivity against other
ALDH isozymes. Among them, 4a was the most potent compound with an IC value
5
0
of 1.96 μM. Furthermore, Compound 4b can dramatically alleviate palmitic acid-
induced impairment of glucose consumption in HepG2 cells [16].
O
O
N
R1
N
O
O
N
CN
N
N
N
O
O
N
N
S
N
N
O
N
N
N
O
O
S
F
N
N
O
O
COOEt
R1=
R1=
4a
N
S
O
4b
O
1
(CM026)
2 (CM37)
3
(NCT-505)
4
Fig. 1. Representative reported ALDH1A1 inhibitors.
Compound 1 has a unique bicyclic core attached with two adjacent arms. By
analyzing the crystal structure of ALDH1A1 complexed with compound 1 (PDB code
4
WPN), we found that the xanthine ring interacts with His293, Cys302, and Gly458
7
through crucial hydrogen bond respectively (Fig. 2). However, N atom in the
imidazole ring exhibit no interaction with any amino residues of ALDH1A1. It has
been considered that theophylline was inactive to inhibit ALDH1A1 at concentrations
up to 250 μM, suggesting that two adjacent arms attached to the theophylline perform
an important role in ALDH1A1 inhibitory activity [14].

C302
SH
.0 Å
W178
3
HN
O
N
N
N
2
O
.9 Å
N7
N
N
O
O
3
.4 Å
O
3
.8 Å
H
N
C
H293
G458
Fig. 2. Structure analysis of human ALDH1A1 with compound 1 (CM026) (PDB code 4WPN, the
key hydrogen bonds, illustrated with red dashed lines)
Thus a series of 1,3-dimethylpyrimidine-2,4-diones were designed through
opening the imidazole ring of compound 1 (Fig. 3), two substituted groups were
5
introduced to N to mimic the two adjacent arms of compound 1.
O
O
N
R1
N5
N
N
N
N
n
N7
N
R2
O
N
O
N
O
O
N
O
O
n=1,2,3
Target compounds
1
(CM026)
Fig. 3. Design of target compounds
2
. Results and discussion
2
.1. Chemistry
The designed compounds were synthesized following the synthetic route shown
in Scheme 1. The starting material 5-nitropyrimidine-2,4(1H,3H)-dione (5) was
allowed to react with iodomethane and sodium hydride to give intermediate 6.

Reduction of the nitro group of 6 gave the amine 7. Reaction of 7 with various halides
or aldehydes and sodium cyanoborohydride led to derivatives 8a-8z. 8a-8z reacted with
different acyl chlorides to afford intermediates 9a-9ab and then 9a-9ab reacted with
piperazine to generate compounds 10a-10ab. Condensation of 10a-10ab with different
commercial acids afforded target compounds 11-59 with good yields.
R1
O
O
O
O
NO2
NO2
NH2
NH
a
b
c
d
HN
N
N
N
O
N
H
O
e
N
O
N
O
N
5
6
7
8a-8z
O
R1
N
O
N
R1
N
R1
O
N
N
Cl
N
O
N
f
N
N
n
n
n
O
O
NH
R2
O
N
O
N
O
N
O
n=2 9a-9z
n=1 9aa
n=3 9ab
n=2 10a-10z
n=1 10aa
n=3 10ab
target compounds 11-59
Scheme 1. Reagents and conditions: (a) CH I, NaH, DMF, 0-25℃, 20 h; (b) H , 10% Pd/C, MeOH,
3
2
1
1
2
5℃, 2 h; (c) For 8a, 8l-8z, R -X (X=Br or I), K CO , DMF, 25℃, 2-3 h; For 8b-8f, R -X (X=Br
or I), K CO , DMF, 60℃, 5-10 h; For 8g-8k, R CHO, CH COOH, NaBH CN, DCM, 25℃, 3-7 h;
2 3
1
2
3
3
3
(
d) For 9a-9z, 3-Chloropropionyl chloride, Et N, DCM, 25℃, 2-3 h; For 9aa, Chloroacetyl chloride,
3
Et N, DCM, 25℃, 2-3 h; For 9ab, 4-Chlorobutyryl chloride, Et N, DCM, 25℃, 2-3 h; (e) piperazine,
3
3
2
K CO , KI, CH CN, 25℃, overnight; (f) R COOH, EDCI, Et N, DCM, 25℃, overnight.
2
3
3
3
2
.2. Biological evaluation
2
.2.1. Inhibitory activity against ALDH1A1
The inhibitory activities of the synthesized 1,3-dimethylpyrimidine-2,4-diones
were initially screened at a relatively high single concentration (10 µM) to identify the
most potent antagonist. Compound 1 (CM026), which was synthesized according to the
literature, was used as a control [3]. The inhibition rate of compound 1 was 75.22% at
2
the concentration of 10 µM. The influence of the terminal substituted group (R ) on
inhibitory activity for ALDH1A1 was initially explored as shown in Table 1. In these

1
compounds, the R group was fixed as isopentenyl and the length of linker between the
5
N atom attached to the core structure 1,3-dimethylpyrimidine-2,4-diones and
piperazine was kept as three-carbon atoms, same as compound 4a. Compound 11 with
unsubstituted phenyl and compound 12 with para-methyl phenyl substitution showed
decreased activity compared to compound 1. Compounds 13-15 containing an electron-
donating group at the para position of phenyl showed maintained inhibitory activity
(68.27%, 77.15 % and 60.51 % respectively at 10 µM). However, decreased activities
were observed when electron-withdrawing substituent attached to terminal phenyl
group (compounds 16-21). Other compounds, including five membered aromatic
heterocyclic (22), nitrogen-containing aromatic heterocycles (23 and 24) and aliphatic
ring (26) substitution exhibited poor inhibition to ALDH1A1. All results of inhibitory
2
activity against ALDH1A1 demonstrated that R group was critical to inhibitory
activity and phenyl bearing an electron-rich substituent was the optimal group. Among
2
this series of compounds, the preferred R group was 4-isopropylphenyl.
2
Table 1. Structure-activity relationships for R group.
O
O
N
N
R2
N
N
N
O
O
hALDH1A1
Compounds
(CM026)
R2
%
inhibition at 10 µMa
75.22 ± 5.28
1
1
1
1
1
2
3
39.23 ± 2.97
47.31 ± 3.69
68.27 ± 5.41
1
4
77.15 ± 7.02

1
1
5
6
O
60.51 ± 5.78
31.19 ± 3.18
F
1
1
1
2
2
2
2
2
7
8
9
0
1
2
3
4
Cl
37.43 ± 3.20
24.42 ± 2.56
3.86 ± 0.43
28.33 ± 2.12
24.52 ± 2.09
9.44 ± 1.01
30.95 ± 2.66
36.65 ± 3.03
Br
CF3
NO2
CN
O
N
N
O
2
2
5
6
35.93 ± 3.32
0.05 ± 0.18
O
a
Values are the mean ± SD of three independent experiments (n = 3)
5
To investigate the effect of the length of linker between N atom and piperazine,
compounds 27-34 were then designed and synthesized. In these compounds, the R1
2
group was kept as isopentenyl and R group was 4-methylphenyl, 4-ethylphenyl, 4-
isopropylphenyl and 4-methoxyphenyl, respectively. It was observed that compounds
5
2
7-34, with the linker between N and piperazine of 2 or 4 atoms (n=1 or 3), showed
significantly decreased inhibitory activities compared to compounds 12-15. This
indicates that the length of the linker exerted a crucial influence on the inhibitory
activity against ALDH1A1 and the optimal length is three carbon atoms (n=2 in Table
2
).
5
Table 2. Structure-activity relationships for the length of linker between N and piperazine.

O
N
N
N
N
n
O
N
R2
O
O
hALDH1A1
Compounds
n
R2
%
inhibition at 10 µMa
7
5.22 ± 5.28
1
(CM026)
1
1
1
1
2
2
2
3
3
3
3
3
2
3
4
5
7
8
9
0
1
2
3
4
2
2
2
2
1
1
1
1
3
3
3
3
47.31 ± 3.69
68.27 ± 5.41
77.15 ± 7.02
60.51 ± 5.78
8.50 ± 0.92
16.79 ± 1.23
14.47 ± 1.18
29.37 ± 2.78
32.19 ± 2.56
27.23 ± 1.64
33.01 ± 2.82
13.10 ± 0.77
O
O
O
a
Values are the mean ± SD of three independent experiments (n = 3)
Then compounds 35-59 were designed and synthesized in order to explore the
1
2
effect of R group on enzyme inhibitory activity. In these compounds, the R group was
5
4
-isopropylphenyl, the length of linker between N and piperazine was kept as three
1
atoms and various substituted groups R were introduced to replace the isopentenyl of
compound 14. As shown in Table 3, dramatically decreased inhibitory activities were
1
observed for compounds 35-39 with R group of an alkyl chain and the inhibitory
activity showed increased tendency accordingly with the extension of the chain.

1
Compound 40 (R = isopentyl) displayed comparable inhibitory activity to compound
1
4. Compounds 41-43 displayed slightly decreased potency for which the isopentenyl
1
was replaced by a small alicyclic group. Compound 44 (R = cyclohexylmethyl)
possessed a preferable inhibitory activity compared to compound 14. In these four
compounds, the inhibitory activity increased accordingly with the enlargement of the
1
ring, indicating that bulky group at this portion was favorable. Compound 45 (R =
1
benzyl) showed outstanding potency and the data obtained from R modifications
suggested that aromatic group was beneficial for improvement of the inhibitory activity.
Compounds 46-59 were thus designed and evaluated for their inhibitory activities.
Introduction of a halo atom to the para position of phenyl group displayed slightly
decreased inhibition (compounds 52 and 55 vs 45), indicating that a substituted group
on the para position was negative to inhibitory activity. Different from para
substitutions, introduction a halo atom to the ortho and meta position exhibited more
outstanding inhibitory activity. For example, compounds 53 and 54 which have a chloro
atom on the ortho and meta position of the phenyl respectively showed more potent
inhibitory activities compared to para position. The IC values for compounds 53 and
5
0
5
4 were 0.126 and 0.385 µM respectively, indicating that compared to meta position of
phenyl, introducing a substituted group to ortho position could be more beneficial to
improve inhibitory activity. Similarly, introduction of other hydrophobic groups, such
as methyl, methoxyl and trifluoromethyl groups (compounds 46-48 and 56-59) to
different positions of phenyl ring showed the same trend. These results illustrated that
the position of substituents had significant influence on inhibitory activity. Results of
inhibitory activities against ALDH1A1 obtained from compounds 35-59 showed that
1
the preferred R groups are 2-methylphenyl (46, 0.149 µM), 2-fluorophenyl (50, 0.429

µM), 2-chlorophenyl (53, 0.126 µM), 3-chlorophenyl (54, 0.385 µM), 2-
trifluoromethylphenyl (56, 0.059 µM), and 3-trifluoromethylphenyl (57, 0.379 µM).
1
Table 3. Structure-activity relationships for R group.
O
O
N
R1
N
N
N
N
O
O
hALDH1A1
inhibition at 10 µM
Compounds
R1
a
IC50 ± SD [µM] a
%
1
(CM026)
75.22 ± 5.28
1.21 ± 0.18
ND
2
7.18 ± 2.09
3
3
5
6
31.75 ± 3.11
20.00 ± 1.66
ND
ND
3
3
3
4
7
8
9
0
53.33 ± 4.57
38.67 ± 3.34
74.49 ± 6.76
77.15 ± 7.02
41.33 ± 3.35
57.33 ± 4.89
69.33 ± 6.69
82.67 ± 7.34
86.41 ± 8.12
ND
ND
ND
ND
ND
ND
ND
ND
1
4
4
4
4
4
4
1
2
3
4
5
1
.135 ± 0.26
4
6
98.67 ± 8.91
0.149 ± 0.11
4
4
7
8
95.15 ± 9.36
89.32 ± 9.01
0.715 ± 0.19
1.144 ± 0.31

4
9
74.03 ± 7.59
ND
F
5
5
0
1
90.04 ± 8.98
93.23 ± 9.01
0.429 ± 0.08
1.075 ± 0.35
F
5
2
74.03 ± 6.86
ND
F
Cl
5
5
3
4
100 ± 9.71
100 ± 8.98
0.126 ± 0.08
0.385 ± 0.10
Cl
5
5
80.89 ± 8.12
ND
Cl
F3C
5
5
5
5
6
7
8
9
100 ± 10.09
100 ± 9.57
0.059 ± 0.02
0.379 ± 0.07
0.872 ± 0.23
0.966 ± 0.13
CF3
H CO
3
94.94 ± 9.22
93.50 ± 9.24
OCH3
a
Values are the mean ± SD of three independent experiments (n = 3)
2
.2.2. Selectivity evaluation of selected analogs against other ALDH isozymes.
Structural research demonstrated that ALDH1A1 shares more than 70 % sequence
similarity with both ALDH1A2 and ALDH1A3, nearly 70 % with the mitochondrial
ALDH2 enzyme and less than 50 % with ALDH3A1 [17]. In order to verify the enzyme
selectivity of the compounds we designed, compounds with excellent ALDH1A1
inhibitory activity (46, 47, 50, 51, 53, 54 and 56-59) were selected to screen against a
panel of enzymes including ALDH1A subfamily (ALDH1A2, ALDH1A3) and ALDH

isozymes (ALDH2, ALDH3A1). As shown in Table 4, all tested compounds exhibited
almost no inhibition and showed excellent selectivity toward other ALDH isozymes.
Table 4. Selectivity against other ALDH isozymes.
O
O
N
R1
N
N
N
N
O
O
%
inhibition at 10 µM a
hALDH1A1 IC50
µM] a
Compounds
R1
[
ALDH1A2
ALDH1A3
ALDH2
ALDH3A1
1
(CM026)
1.21 ± 0.18
0.11 ± 0.07
0.13 ± 0.05
0.09 ± 0.03
0.15 ± 0.01
0.13 ± 0.04
0.05 ± 0.01
0.97 ± 0.14
5.26 ± 0.92
4
4
6
7
0.149 ± 0.11
0.715 ± 0.19
0.429 ± 0.08
0.90 ± 0.17
0.25 ± 0.08
0.34 ± 0.05
0.35 ± 0.02
0.09 ± 0.01
4.79 ± 0.95
9.09 ± 1.75
4.07 ± 0.72
F
5
5
5
5
5
5
5
0
1
3
4
6
7
8
F
1.075 ± 0.35
0.126 ± 0.08
0.385 ± 0.10
0.059 ± 0.02
0.379 ± 0.07
0.872 ± 0.23
0.38 ± 0.07
0.82 ± 0.08
0.76 ± 0.09
1.57 ± 0.31
2.68 ± 0.65
1.65 ± 0.26
0.45 ± 0.04
0.32 ± 0.04
0.21 ± 0.02
0.39 ± 0.03
6.25 ± 1.01
0.68 ± 0.08
3.78 ± 0.77
2.77 ± 0.32
8.06 ± 2.12
0.06 ± 0.01
0.08 ± 0.03
0.19 ± 0.05
2.63 ± 0.48
6.05 ± 0.88
1.77 ± 0.28
5.74 ± 1.18
3.11 ± 0.59
0.19 ± 0.03
Cl
Cl
F3C
CF3
H CO
3

OCH3
5
9
0.966 ± 0.13
1.87 ± 0.17
0.48 ± 0.12
0.21 ± 0.06
0.32 ± 0.07
a
Values are the mean ± SD of three independent experiments (n=3).
2
.2.3. Cell viability assay of representative ALDH1A1 inhibitors.
In order to evaluate the cytotoxicity of our synthesized compounds, the viability
of HepG2 cells that treated with representative ALDH1A1 inhibitors (46, 50, 53, 56
and 57) at different concentrations was determined. As shown in Figure 4, all tested
compounds showed negligible cytotoxic effect on HepG2 cells at concentrations of 0.1
µM, 1 µM and 10 µM, and the morphology of HepG2 cells was not affected, which
indicated that these inhibitors have low cytotoxicity to HepG2 cells.
Fig. 4. Cell viability assay of compound 1 and representative compounds. Data were expressed in
mean ± S.D. (n = 4).
2
.2.4. Improvement of glucose consumption in HepG2 cells.
To demonstrate the potential utility in the improvement of glucose consumption,
these selective ALDH1A1 inhibitors (46, 50, 53, 56 and 57) and positive control
Metformin were evaluated in HepG2 cells according to the reported method [18].
Results showed that compared to the blank control, compound 1 exhibited no effect on
glucose metabolism in HepG2 cells at concentrations of 1 µM and 10 µM. Among all

tested five compounds, 57 displayed about 20% improvement on glucose metabolism
in HepG2 cells at 1 µM. All these five compounds can significantly improve glucose
metabolism in HepG2 cells at 10 µM. The glucose consumption of compounds 46, 50,
5
3, 56 and 57 increased by 10%, 10%, 15%, 20% and 30% respectively (Fig. 5).
Moreover, compound 57 at 10 µM produced equivalent glucose consumption
improvement in HepG2 cells to positive control Metformin (Met) at 1 mM.
Fig. 5. Effects of compound 1 and representative compounds on glucose consumption in HepG2
cells. Data were expressed in mean ± S.D. (n = 4). *p < 0.05, **p < 0.01, ***p < 0.001 versus
untreated control cells (Con).
2
.2.5 Metabolic stability assay in human liver microsomal of optimized compounds.
The potent compounds 53, 56 and 57 were then chosen to evaluate their metabolic
stability in human microsomes. As shown in Table 5, the tested three compounds
showed microsomal stability with T1/2 values of 3, 10 and 12 min respectively. Thus,
improving the metabolic stability of these compounds through further structural
modifications has become our primary object next step.
Table 5. Metabolic parameters in human liver microsomal.
Parameters
53
56
57
-
1
k(min )
0.32
0.072
0.059

t (min)
3
10
12
0.5
1
/2
Pm(mg/mL)
0.25
1.28
0.25
0.288
C (μL/min/mg)
0.118
lint
3
. Conclusion
In summary, a series of 1,3-dimethylpyrimidine-2,4-diones were designed,
synthesized and evaluated as potent and selective ALDH1A1 inhibitors. Several of
them presented potent inhibitory activities against ALDH1A1 with IC values in the
5
0
low nanomolar range and a high degree of selectivity over other ALDH isozymes
ALDH1A2, ALDH1A3, ALDH2 and ALDH3A1). Compounds 46, 50, 53, 56 and 57
(
exhibited more potent activity in enzymatic assays comparing to compound 1. These
compounds also effectively improved glucose consumption in HepG2 cells and the
compound 57 was the most potent. Although these compounds exhibit excellent
activities in vitro, their metabolic stability is poor. So, further work has been focused
on the structural optimization of these compounds to improve their metabolic stability
and this work is proceeding.
4
. Experimental section
4
.1. Chemistry
Chemical reagents and solvents were obtained from commercial sources and used
without further purification. All reactions were monitored by thin-layer
chromatography (TLC) on silica gel plates and visualized under UV light. Silica gel
1
flash chromatography was performed using silica gel (300-400 mesh). H-NMR and
13C-NMR spectra were recorded on Bruker 300 MHz or 400 MHz spectrometer using
deuterated solventswith tetramethylsilane as an internal standard. Chemical shifts were

reported in parts per million (ppm, δ) downfield from tetramethylsilane. Proton
coupling patterns were described as singlet (s), doublet (d), doublet of doublets (dd),
triplet (t) and multiplet (m). The purity of the synthesized compounds were measured
by analytical high performance liquid chromatography (HPLC, Agilent) using the C18
reverse phase column. High-resolution mass spectroscopic (HRMS) measurements
were performed on an Agilent QTOF 6520 mass spectrometer with electron spray
ionization (ESI) as the ion source.
4
.1.1. 1,3-Dimethyl-5-nitropyrimidine-2,4(1H,3H)-dione (6).
To a solution of 5-nitropyrimidine-2,4(1H,3H)-dione (1.0 g, 6.37 mmol) in DMF
(10 ml) was added NaH (0.61 g, 15.28 mmol) and stirred at 0 ℃ for 2 h. The mixture
was added CH I (2.26 g, 15.91 mmol) and stirred at room temperature for 20 h. After
3
cooling to 0 ℃, the mixture was added ice water (30 ml) and then stirred for 30 min.
The solid was collected by filtration and washed successively with H O (2× 10 mL) to
2
1
give a pale yellow product (0.95 g, 81 % yield). Mp 155-157 °C. H NMR (300 MHz,
+
DMSO-d ) δ 9.28 (s, 1H), 3.40 (s, 3H), 3.13 (s, 3H). ESI-MS m/z: 186.1 [M+H] .
6
4
.1.2 5-Amino-1,3-dimethylpyrimidine-2,4(1H,3H)-dione (7).
To a mixture of 1,3-dimethyl-5-nitropyrimidine-2,4(1H,3H)-dione 6 (1 g, 5.4
mmol) in MeOH (20 mL) was added 10% Pd/C (100 mg). The air was removed and
refilled with H gas(3 times). The mixture was stirred at room temperature for 2 h under
2
H gas. The solvent was filtered through Celite and eluted with CH Cl . The filtrate was
2
2
2
concentrated in vacuo to give 5-amino-1,3-dimethylpyrimidine-2,4(1H,3H)-dione as a
1
white solid which was used without purification (0.75 g, 90 % yield). H NMR (300
MHz, DMSO) δ 6.84 (s, 1H), 4.16 (s, 2H), 3.21 (d, J = 10.6 Hz, 6H). ESI-MS m/z:
1
56.2 [M+H]+.

4
.1.3 General procedure for preparation of compounds (8a, 8l-8z).
To a solution of 5-amino-1,3-dimethylpyrimidine-2,4(1H,3H)-dione 7 (1 g, 6.45
mmol) in DMF (10 mL) was added potassium carbonate (1.78 g, 12.89 mmol) and
stirred at 0℃ for 0.5 h. Then different halides (7.09 mmol) was added sequentially and
stirred at room temperature for 1.5 h. The resulted solution was poured into water (50
mL) and extracted with EtOAc (45 mL × 2). The combined organic layer was dried
(
Na SO4) and filtered. After removal of solvent, the crude product was purified by
2
silica gel chromatography to give 8a, 8l-8z respectively.
4
.1.3.1. 5-((3-Methylbut-2-en-1-yl)amino)-1,3-dimethylpyrimidine-2,4(1H,3H)-dione
1
(
8a). A yellow oil. Yield 58 %. H NMR (300 MHz, CDCl ) δ 6.12 (s, 1H), 5.27 (t, J =
3
6
1
.6 Hz, 1H), 3.87 (s, 1H), 3.51 - 3.42 (m, 2H), 3.38 (d, J = 4.9 Hz, 6H), 1.74 (s, 3H),
.68 (s, 3H). ESI-MS m/z: 224.2 [M+H]+.
4
.1.3.2. 5-(Benzylamino)-1,3-dimethylpyrimidine-2,4(1H,3H)-dione (8l). A pale yellow
1
solid. Yield 61 %. H NMR (300 MHz, CDCl ) δ 7.39 - 7.27 (m, 5H), 6.07 (s, 1H), 4.16
3
(
s, 2H), 3.39 (s, 3H), 3.29 (s, 3H). ESI-MS m/z: 246.1 [M+H]+.
4
.1.3.3. 5-((2-Methylbenzyl)amino)-1,3-dimethylpyrimidine-2,4(1H,3H)-dione (8m). A
1
pale yellow solid. Yield 65 %. H NMR (300 MHz, CDCl ) δ 7.30 - 7.24 (m, 1H), 7.23
3
-
7.13 (m, 3H), 6.11 (s, 1H), 4.27 (s, 1H), 4.07 (s, 2H), 3.39 (d, J = 1.6 Hz, 3H), 3.33
d, J = 1.6 Hz, 3H), 2.34 (s, 3H). ESI-MS m/z: 260.3 [M+H]+.
(
4
.1.3.4. 5-((3-Methylbenzyl)amino)-1,3-dimethylpyrimidine-2,4(1H,3H)-dione (8n). A
1
pale yellow solid. Yield 60 %. H NMR (300 MHz, CDCl ) δ 7.23 (d, J = 7.4 Hz, 1H),
3
7
2
.16 - 7.08 (m, 3H), 6.07 (s, 1H), 4.42 (s, 1H), 4.11 (s, 2H), 3.40 (s, 3H), 3.30 (s, 3H),
.35 (s, 3H). ESI-MS m/z: 260.3 [M+H]+.

4
.1.3.5. 5-((4-Methylbenzyl)amino)-1,3-dimethylpyrimidine-2,4(1H,3H)-dione (8o). A
1
pale yellow solid. Yield 62 %. H NMR (300 MHz, CDCl ) δ 7.20 (d, J = 7.8 Hz, 2H),
3
7
3
.14 (d, J = 7.8 Hz, 2H), 6.07 (s, 1H), 4.41 (s, 1H), 4.09 (s, 2H), 3.38 (s, 3H), 3.28 (s,
H), 2.33 (s, 3H). ESI-MS m/z: 260.3 [M+H]+.
4
.1.3.6.
5-((3,5-Dimethylbenzyl)amino)-1,3-dimethylpyrimidine-2,4(1H,3H)-dione
1
(
8p). A pale yellow solid. Yield 55%. H NMR (300 MHz, CDCl ) δ 6.96 - 6.87 (m,
3
3
H), 6.08 (s, 1H), 4.39 (s, 1H), 4.05 (s, 2H), 3.38 (s, 3H), 3.29 (s, 3H), 2.29 (s, 6H).
ESI-MS m/z: 274.3 [M+H]+.
4
.1.3.7. 5-((2-Fluorobenzyl)amino)-1,3-dimethylpyrimidine-2,4(1H,3H)-dione (8q). A
1
pink solid. Yield 51%. H NMR (300 MHz, CDCl ) δ 7.40 - 7.20 (m, 2H), 7.18 - 6.99
3
(m, 2H), 6.16 (s, 1H), 4.45 (s, 1H), 4.21 (s, 2H), 3.38 (s, 3H), 3.31 (s, 3H). ESI-MS m/z:
2
64.2 [M+H]+.
4
.1.3.8. 5-((3-Fluorobenzyl)amino)-1,3-dimethylpyrimidine-2,4(1H,3H)-dione (8r). A
1
pink solid. Yield 54%. H NMR (300 MHz, CDCl ) δ 7.38 - 7.24 (m, 1H), 7.15 - 6.90
3
(m, 3H), 6.03 (s, 1H), 4.55 (s, 1H), 4.18 (s, 2H), 3.39 (s, 3H), 3.29 (s, 3H). ESI-MS m/z:
2
64.2 [M+H]+.
4
.1.3.9. 5-((4-Fluorobenzyl)amino)-1,3-dimethylpyrimidine-2,4(1H,3H)-dione (8s). A
1
pink solid. Yield 57%. H NMR (300 MHz, CDCl ) δ 7.36 - 7.24 (m, 2H), 7.10 - 6.97
3
(m, 2H), 6.05 (s, 1H), 4.41 (s, 1H), 4.12 (s, 2H), 3.40 (s, 3H), 3.30 (s, 3H). ESI-MS m/z:
2
64.2 [M+H]+.
4
.1.3.10. 5-((2-Chlorobenzyl)amino)-1,3-dimethylpyrimidine-2,4(1H,3H)-dione (8t). A
1
yellow solid. Yield 52%. H NMR (300 MHz, CDCl ) δ 7.40 - 7.34 (m, 2H), 7.27 - 7.22
3

(m, 2H), 6.08 (s, 1H), 4.54 (s, 1H), 4.26 (s, 2H), 3.40 (s, 3H), 3.30 (s, 3H). ESI-MS m/z:
2
80.2 [M+H]+.
4
.1.3.11. 5-((3-Chlorobenzyl)amino)-1,3-dimethylpyrimidine-2,4(1H,3H)-dione (8u).
1
A yellow solid. Yield 56%. H NMR (300 MHz, CDCl ) δ 7.32 (s, 1H), 7.30 - 7.24 (m,
3
2
H), 7.21 (d, J = 6.2 Hz, 1H), 6.02 (s, 1H), 4.50 (s, 1H), 4.16 (s, 2H), 3.40 (s, 3H), 3.30
(
s, 3H). ESI-MS m/z: 280.2 [M+H]+.
4
.1.3.12. 5-((4-Chlorobenzyl)amino)-1,3-dimethylpyrimidine-2,4(1H,3H)-dione (8v).
1
A yellow solid. Yield 59%. H NMR (300 MHz, CDCl ) δ 7.29 (q, J = 8.5 Hz, 4H),
3
6
.02 (s, 1H), 4.47 (s, 1H), 4.14 (s, 2H), 3.40 (s, 3H), 3.29 (s, 3H). ESI-MS m/z: 280.2
[
M+H]+.
4
.1.3.13. 5-((2-(Trifluoromethyl)benzyl)amino)-1,3-dimethylpyrimidine-2,4(1H,3H)-
1
dione (8w). A yellow solid. Yield 51%. H NMR (300 MHz, CDCl ) δ 7.62 - 7.41 (m,
3
3
3
H), 7.35 - 7.27 (m, 1H), 5.95 (s, 1H), 4.62 (s, 1H), 4.31 (s, 2H), 3.30 (s, 3H), 3.19 (s,
H). ESI-MS m/z: 314.2 [M+H]+.
4
.1.3.14. 5-((3-(Trifluoromethyl)benzyl)amino)-1,3-dimethylpyrimidine-2,4(1H,3H)-
1
dione (8x). A yellow solid. Yield 57%. H NMR (300 MHz, CDCl ) δ 7.60 (s, 1H), 7.58
3
-
7.44 (m, 3H), 6.05 (s, 1H), 4.56 (s, 1H), 4.24 (s, 2H), 3.40 (s, 3H), 3.30 (s, 3H). ESI-
MS m/z: 314.2 [M+H]+.
4
.1.3.15. 5-((2-Methoxybenzyl)amino)-1,3-dimethylpyrimidine-2,4(1H,3H)-dione (8y).
1
A white solid. Yield 59%. H NMR (300 MHz, CDCl ) δ 7.25 - 7.20 (m, 2H), 6.97 -
3
6
3
.90 (m, 2H), 6.89 - 6.86 (m, 1H), 6.16 (s, 1H), 4.44 (s, 1H), 4.14 (s, 2H), 3.85 (s, 3H),
.38 (s, 3H), 3.30 (s, 3H). ESI-MS m/z: 276.1 [M+H]+.

4
.1.3.16. 5-((3-Methoxybenzyl)amino)-1,3-dimethylpyrimidine-2,4(1H,3H)-dione (8z).
1
A white solid. Yield 61%. H NMR (300 MHz, CDCl ) δ 7.28 - 7.22 (m, 1H), 6.94 -
3
6
3
.77 (m, 3H), 6.06 (s, 1H), 4.49 (s, 1H), 4.14 (s, 2H), 3.80 (s, 3H), 3.39 (s, 3H), 3.29 (s,
H). ESI-MS m/z: 276.1 [M+H]+.
4
.1.4. General procedure for preparation of compounds (8b-8f).
To a solution of 5-amino-1,3-dimethylpyrimidine-2,4(1H,3H)-dione 7 (1 g, 6.45
mmol) in DMF (10 mL) was added potassium carbonate (1.78 g, 12.89 mmol) and
stirred at 0℃ for 0.5 h. Then different halides (7.09 mmol) was added sequentially and
stirred at 60 °C for 5-10 h until the reaction was completed. After cooling to room
temperature, the resulted solution was poured into water (50 mL) and extracted with
EtOAc (45 mL × 2). The combined organic layer was dried (Na SO ) and filtered. After
2
4
removal of solvent, the crude product was purified by silica gel chromatography to give
8
b-8f respectively.
4
.1.4.1. 5-(Ethylamino)-1,3-dimethylpyrimidine-2,4(1H,3H)-dione (8b). A yellow oil.
1
Yield 45%. H NMR (300 MHz, CDCl ) δ 6.10 (s, 1H), 3.89 (s, 1H), 3.34 (s, 3H), 3.30
3
(
s, 3H), 2.92 - 2.83 (m, 2H), 1.23 (t, J = 7.1 Hz, 3H). ESI-MS m/z: 184.2 [M+H]+.
4
.1.4.2. 5-(Propylamino)-1,3-dimethylpyrimidine-2,4(1H,3H)-dione (8c). A yellow oil.
1
Yield 50%. H NMR (300 MHz, CDCl ) δ 6.09 (s, 1H), 3.85 (s, 1H), 3.35 (d, J = 4.8
3
Hz, 6H), 2.87 - 2.76 (m, 2H), 1.71 - 1.56 (m, 2H), 0.97 (t, J = 7.5 Hz, 3H). ESI-MS m/z:
1
98.3 [M+H]+.
4
.1.4.3. 5-(Isopropylamino)-1,3-dimethylpyrimidine-2,4(1H,3H)-dione (8d). A yellow
1
oil. Yield 51%. H NMR (300 MHz, CDCl ) δ 6.10 (s, 1H), 3.32 (d, J = 1.6 Hz, 6H),
3
3
.23 - 3.12 (m, 1H), 1.12 (d, J = 6.2 Hz, 6H). ESI-MS m/z: 198.3 [M+H]+.

4
.1.4.4. 5-(Butylamino)-1,3-dimethylpyrimidine-2,4(1H,3H)-dione (8e). A white oil.
1
Yield 48%. H NMR (300 MHz, CDCl ) δ 6.08 (s, 1H), 3.33 (d, J = 3.1 Hz, 6H), 2.88
3
-
2.76 (m, 2H), 1.64 - 1.50 (m, 2H), 1.45 - 1.33 (m, 2H), 0.90 (t, J = 7.5 Hz, 3H). ESI-
MS m/z: 212.2 [M+H]+.
4
.1.4.5. 5-(Isobutylamino)-1,3-dimethylpyrimidine-2,4(1H,3H)-dione (8f). A white oil.
1
Yield 49%. H NMR (300 MHz, CDCl ) δ 6.09 (s, 1H), 3.94 (s, 1H), 3.39 (s, 3H), 3.37
3
(s, 3H), 2.72 - 2.65 (m, 2H), 1.95 - 1.83 (m, 1H), 0.99 (s, 3H), 0.98 (s, 3H). ESI-MS
m/z: 212.2 [M+H]+.
4
.1.5. General procedure for preparation of compounds (8g-8k).
To a solution of 5-amino-1,3-dimethylpyrimidine-2,4(1H,3H)-dione 7 (1 g, 6.45
mmol) in DCM (20 mL) was added acetic acid (0.04 g, 0.65 mmol) and different
aliphatic aldehydes (7.73 mmol). The mixture was stirred at room temperature for 1 h.
Then sodium cyanoborohydride (0.81 g, 12.89 mmol) was added sequentially and
stirred at room temperature for 3-7 h until the reaction was completed. The reaction was
quenched with a saturated aqueous solution (50 mL) and the mixture was extracted with
DCM (50 mL × 3). The combined organic layer was dried over anhydrous sodium
sulfate and filtered. After removal of solvent, the crude product was purified by silica
gel chromatography to give 8g-8k respectively.
4
.1.5.1. 5-(Isopentylamino)-1,3-dimethylpyrimidine-2,4(1H,3H)-dione (8g). A yellow
1
oil. Yield 45%. H NMR (300 MHz, CDCl ) δ 6.04 (s, 1H), 3.73 (s, 1H), 3.29 - 3.19
3
(m, 6H), 2.81 - 2.70 (m, 2H), 1.66 - 1.51 (m, 1H), 1.46 - 1.33 (m, 2H), 0.81 (d, J = 6.5
Hz, 6H). ESI-MS m/z: 226.3 [M+H]+.
4
.1.5.2. 5-((Cyclopropylmethyl)amino)-1,3-dimethylpyrimidine-2,4(1H,3H)-dione (8h).
1
A yellow oil. Yield 49%. H NMR (300 MHz, CDCl ) δ 6.06 (s, 1H), 3.30 (d, J = 6.1
3

Hz, 6H), 2.69 - 2.60 (m, 2H), 1.06 - 0.97 (m, 1H), 0.54 - 0.45 (m, 2H), 0.20 - 0.09 (m,
2
H). ESI-MS m/z: 210.3 [M+H]+.
4
.1.5.3. 5-((Cyclobutylmethyl)amino)-1,3-dimethylpyrimidine-2,4(1H,3H)-dione (8i).
1
A yellow oil. Yield 47%. H NMR (300 MHz, CDCl ) δ 6.10 (s, 1H), 3.80 (s, 1H), 3.37
3
(s, 3H), 3.36 (s, 3H), 2.87 (d, J = 7.2 Hz, 2H), 2.63 - 2.52 (m, 1H), 2.15 - 2.06 (m, 2H),
1
.98 - 1.82 (m, 2H), 1.76 - 1.66 (m, 2H). ESI-MS m/z: 224.3 [M+H]+.
4
.1.5.4. 5-((Cyclopentylmethyl)amino)-1,3-dimethylpyrimidine-2,4(1H,3H)-dione (8j).
1
A yellow oil. Yield 41%. H NMR (300 MHz, CDCl ) δ 6.08 (s, 1H), 3.89 (s, 1H), 3.34
3
(
d, J = 5.2 Hz, 6H), 2.80 - 2.69 (m, 2H), 2.20 - 2.06 (m, 1H), 1.87 - 1.73 (m, 2H), 1.68
1.48 (m, 4H), 1.30 - 1.13 (m, 2H). ESI-MS m/z: 238.2 [M+H]+.
-
4
.1.5.5. 5-((Cyclopentylmethyl)amino)-1,3-dimethylpyrimidine-2,4(1H,3H)-dione (8k).
1
A yellow oil. Yield 49%. H NMR (300 MHz, CDCl ) δ 6.05 (s, 1H), 3.89 (s, 1H), 3.32
3
(
d, J = 3.8 Hz, 6H), 2.69 - 2.62 (m, 2H), 1.79 - 1.60 (m, 5H), 1.58 - 1.42 (m, 1H), 1.22
1.12 (m, 3H), 0.94 (t, J = 13.3 Hz, 2H). ESI-MS m/z: 252.2 [M+H]+.
-
4
.1.6. General procedure for preparation of compounds (9a-9z).
To a solution of 8a-8z (2.24 mmol) in DCM (5 mL) was added triethylamine (0.34
g, 3.36 mmol) and stirred at 0 ℃ for 15 min. 3-Chloropropanoyl chloride (0.34 g, 2.69
mmol) was added dropwise and stirred at room temperature for 2-3 h until the reaction
was completed. The resulted solution was poured into water (15 mL). The aqueous
layer was extracted with DCM (10 mL × 3). The combined organic layer was dried
(
Na SO ) and filtered. After removal of solvent, the crude product was purified by silica
2 4
gel chromatography to give 9a-9z respectively.

4
.1.6.1. 3-Chloro-N-(1,3-dimethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-5-yl)-N-(3-
1
methylbut-2-en-1-yl)propanamide (9a). A white solid. Yield 79%. H NMR (300 MHz,
CDCl ) δ 7.93 (s, 1H), 5.15-5.06 (m, 1H), 4.41 - 4.27 (m, 1H), 3.81 - 3.63 (m, 3H), 3.33
3
(s, 3H), 3.20 (s, 3H), 2.87 - 2.70 (m, 1H), 2.50 - 2.35 (m, 1H), 1.65 (s, 3H), 1.52 (s,
3
H). ESI-MS m/z: 314.2 [M+H]+.
4
.1.6.2.
3-Chloro-N-(1,3-dimethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-5-yl)-N-
1
ethylpropanamide (9b). A yellow oil. Yield 80 %. H NMR (300 MHz, CDCl ) δ 7.37
3
(s, 1H), 3.94 - 3.76 (m, 2H), 3.48 (s, 3H), 3.45 - 3.29 (m, 5H), 2.70 (dt, J = 15.5, 7.6
Hz, 1H), 2.53 (dt, J = 16.2, 6.1 Hz, 1H), 1.11 (t, J = 7.2 Hz, 3H). ESI-MS m/z: 274.2
M+H]+.
[
4
.1.6.3.
3-Chloro-N-(1,3-dimethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-5-yl)-N-
1
propylpropanamide (9c). A yellow oil. Yield 78 %. H NMR (300 MHz, CDCl ) δ 7.29
3
(s, 1H), 3.93 - 3.81 (m, 1H), 3.79 - 3.63 (m, 3H), 3.46 (s, 3H), 3.38 (s, 3H), 2.76 - 2.62
(m, 1H), 2.60 - 2.46 (m, 1H), 1.56 - 1.44 (m, 2H), 0.89 (t, J = 7.5 Hz, 3H). ESI-MS m/z:
2
88.3 [M+H]+.
4
.1.6.4.
3-Chloro-N-(1,3-dimethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-5-yl)-N-
1
isopropylpropanamide (9d). A yellow oil. Yield 78 %. H NMR (300 MHz, CDCl ) δ
3
7
2
.30 (s, 1H), 3.93 - 3.81 (m, 2H), 3.79 - 3.63 (m, 1H), 3.46 (s, 3H), 3.35 (s, 3H), 2.72 -
.58 (m, 1H), 2.55 - 2.43 (m, 1H), 1.15 - 1.10 (m, 6H). ESI-MS m/z: 288.3 [M+H]+.
4
.1.6.5.
3-Chloro-N-(1,3-dimethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-5-yl)-N-
1
butylpropanamide (9e). A yellow oil. Yield 71 %. H NMR (300 MHz, CDCl ) δ 7.31
3
(s, 1H), 3.90 - 3.79 (m, 3H), 3.45 (s, 3H), 3.38 (s, 3H), 3.29 - 3.19 (m, 1H), 2.73 - 2.60

(m, 1H), 2.58 - 2.46 (m, 1H), 1.60 - 1.48 (m, 2H), 1.42 - 1.30 (m, 2H), 0.89 (t, J = 7.5
Hz, 3H). ESI-MS m/z: 302.4 [M+H]+.
4
.1.6.6.
3-Chloro-N-(1,3-dimethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-5-yl)-N-
1
isobutylpropanamide (9f). A yellow oil. Yield 73 %. H NMR (300 MHz, CDCl ) δ
3
7
2
.28 (s, 1H), 3.91 - 3.80 (m, 3H), 3.46 (s, 3H), 3.38 (s, 3H), 3.30 - 3.19 (m, 1H), 2.77 -
.60 (m, 1H), 2.57 - 2.45 (m, 1H), 1.90 - 1.80 (m, 1H), 0.94 (s, 3H), 0.92 (s, 3H). ESI-
MS m/z: 302.4 [M+H]+.
4
.1.6.7.
3-Chloro-N-(1,3-dimethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-5-yl)-N-
1
isopentylpropanamide (9g). A yellow oil. Yield 70 %. H NMR (300 MHz, CDCl ) δ
3
7
2
.30 (s, 1H), 3.88 - 3.59 (m, 3H), 3.44 (s, 3H), 3.35 (s, 3H), 3.29 - 3.17 (m, 1H), 2.72 -
.57 (m, 1H), 2.55 - 2.41 (m, 1H), 1.60 - 1.47 (m, 1H), 1.40 - 1.23 (m, 2H), 0.90 - 0.82
(
m, 6H). ESI-MS m/z: 316.3 [M+H]+.
4
.1.6.8.
3-Chloro-N-(1,3-dimethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-5-yl)-N-
1
cyclopropylmethylpropanamide (9h). A yellow oil. Yield 79 %. H NMR (300 MHz,
CDCl ) δ 7.40 (s, 1H), 3.89 - 3.63 (m, 4H), 3.46 (s, 3H), 3.37 (s, 3H), 2.77 - 2.63 (m,
3
1
H), 2.59 - 2.47 (m, 1H), 0.92 - 0.82 (m, 1H), 0.50 - 0.39 (m, 2H), 0.17 - 0.12 (m, 2H).
ESI-MS m/z: 300.3 [M+H]+.
4
.1.6.9.
3-Chloro-N-(1,3-dimethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-5-yl)-N-
1
cyclobutylmethylpropanamide (9i). A yellow oil. Yield 81 %. H NMR (300 MHz,
CDCl ) δ 7.22 (s, 1H), 3.95 - 3.80 (m, 2H), 3.45 (s, 3H), 3.40 - 3.28 (m, 5H), 2.75 -
3
2
.60 (m, 1H), 2.58 - 2.37 (m, 2H), 2.04 - 1.91 (m, 2H), 1.89 - 1.78 (m, 2H), 1.73 - 1.65
(
m, 2H). ESI-MS m/z: 314.3 [M+H]+.

4
.1.6.10.
3-Chloro-N-(1,3-dimethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-5-yl)-N-
1
cyclopentylmethylpropanamide (9j). A yellow oil. Yield 80 %. H NMR (300 MHz,
CDCl ) δ 7.35 (s, 1H), 4.00 - 3.83 (m, 2H), 3.82 - 3.70 (m, 1H), 3.53 (s, 3H), 3.45 (s,
3
3
1
H), 3.33 - 3.18 (m, 1H), 2.83 - 2.70 (m, 1H), 2.65 - 2.52 (m, 1H), 2.14 - 1.97 (m, 1H),
.77 - 1.64 (m, 4H), 1.63 - 1.48 (m, 2H), 1.39 - 1.21 (m, 2H). ESI-MS m/z: 328.4
[
M+H]+.
4
.1.6.11.
3-Chloro-N-(1,3-dimethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-5-yl)-N-
1
cyclohexylmethylpropanamide (9k). A yellow oil. Yield 76 %. H NMR (300 MHz,
CDCl ) δ 7.36 (s, 1H), 3.99 - 3.88 (m, 1H), 3.87 - 3.69 (m, 3H), 3.54 (s, 3H), 3.45 (s,
3
3
1
H), 2.84 - 2.70 (m, 1H), 2.66 - 2.53 (m, 1H), 1.82 - 1.65 (m, 5H), 1.53 - 1.41 (m, 1H),
.27 - 1.15 (m, 3H), 1.14 - 0.95 (m, 2H). ESI-MS m/z: 342.3 [M+H]+.
4
.1.6.12.
3-Chloro-N-(1,3-dimethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-5-yl)-N-
1
benzylpropanamide (9l). A yellow solid. Yield 83 %. H NMR (300 MHz, CDCl ) δ
3
7
3
2
.37 - 7.29 (m, 3H), 7.27 - 7.16 (m, 2H), 6.72 (s, 1H), 5.63 (d, J = 14.5 Hz, 1H), 4.02 -
.86 (m, 1H), 3.84 (d, J = 14.5 Hz, 1H), 3.81 - 3.67 (m, 1H), 3.38 (s, 3H), 3.23 (s, 3H),
.84 - 2.68(m, 1H), 2.67 - 2.52 (m, 1H). ESI-MS m/z: 336.2 [M+H]+.
4
.1.6.13. 3-Chloro-N-(1,3-dimethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-5-yl)-N-(2-
1
methylbenzl)propanamide (9m). A yellow solid. Yield 81 %. H NMR (300 MHz,
CDCl ) δ 7.24 - 7.15 (m, 2H), 7.15 - 7.05 (m, 1H), 7.04 - 6.96 (m, 1H), 6.49 (s, 1H),
3
5
.62 (d, J = 14.5 Hz, 1H), 4.02 - 3.86 (m, 2H), 3.80 - 3.64 (m, 1H), 3.36 (s, 3H), 3.16
(s, 3H), 2.84 - 2.67 (m, 1H), 2.65 - 2.53 (m, 1H), 2.25 (s, 3H). ESI-MS m/z: 350.3
[
M+H]+.

4
.1.6.14. 3-Chloro-N-(1,3-dimethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-5-yl)-N-(3-
1
methylbenzyl)propanamide (9n). A yellow solid. Yield 84 %. H NMR (300 MHz,
CDCl ) δ 7.15 (t, J = 7.5 Hz, 1H), 7.04 (d, J = 7.7 Hz, 1H), 7.01 (s, 1H), 6.95 (d, J =
3
7
.8 Hz, 1H), 6.71 (s, 1H), 5.55 (d, J = 14.5 Hz, 1H), 3.94 - 3.81 (m, 1H), 3.78 - 3.64
(m, 2H), 3.33 (s, 3H), 3.20 (s, 3H), 2.78 - 2.65 (m, 1H), 2.62 - 2.50 (m, 1H), 2.28 (s,
3
H). ESI-MS m/z: 350.3 [M+H]+.
4
.1.6.15. 3-Chloro-N-(1,3-dimethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-5-yl)-N-(4-
1
methylbenzyl)propanamide (9o). A yellow solid. Yield 81 %. H NMR (300 MHz,
CDCl ) δ 7.25 -7.19 (m, 2H), 7.14 -7.08 (m, 2H), 6.51 (s, 1H), 6.95 (d, J = 7.8 Hz, 1H),
3
6
.71 (s, 1H), 5.57 (d, J = 14.6 Hz, 1H), 3.91 - 3.79 (m, 1H), 3.73 - 3.60 (m, 2H), 3.34
(s, 3H), 3.20 (s, 3H), 2.80 - 2.68 (m, 1H), 2.65 - 2.53 (m, 1H), 2.27 (s, 3H). ESI-MS
m/z: 350.3 [M+H]+.
4
.1.6.16.
3-Chloro-N-(1,3-dimethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-5-yl)-N-
1
(
3,5-dimethylbenzyl)propanamide (9p). A yellow solid. Yield 81 %. H NMR (300
MHz, CDCl ) δ 6.89 (s, 1H), 6.83 - 6.77 (m, 2H), 6.70 (s, 1H), 5.57 (d, J = 14.4 Hz,
3
1
1
H), 3.97 - 3.82 (m, 1H), 3.77 - 3.63 (m, 2H), 3.36 (s, 3H), 3.22 (s, 3H), 2.80 - 2.66 (m,
H), 2.64 - 2.52 (m, 1H), 2.26 (s, 6H). ESI-MS m/z: 364.2 [M+H]+.
4
.1.6.17. 3-Chloro-N-(1,3-dimethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-5-yl)-N-(2-
1
fluorobenzyl)propanamide (9q). A yellow solid. Yield 80 %. H NMR (300 MHz,
CDCl ) δ 7.45 - 7.36 (m, 1H), 7.35 - 7.21 (m, 1H), 7.16 - 7.08 (m, 1H), 7.05 - 6.94 (m,
3
2
3
H), 5.31 (d, J = 14.5 Hz, 1H), 4.28 (d, J = 14.5 Hz, 1H), 3.95 - 3.80 (m, 1H), 3.79 -
.64 (m, 1H), 3.36 (s, 3H), 3.31 (s, 3H), 2.84 - 2.69 (m, 1H), 2.66 - 2.51 (m, 1H). ESI-
MS m/z: 354.2 [M+H]+.

4
.1.6.18. 3-Chloro-N-(1,3-dimethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-5-yl)-N-(3-
1
fluorobenzyl)propanamide (9r). A yellow solid. Yield 78 %. H NMR (300 MHz,
CDCl ) δ 7.37 - 7.22 (m, 1H), 7.06 - 6.93 (m, 3H), 6.88 (s, 1H), 5.56 (d, J = 14.7 Hz,
3
1
1
H), 4.02 - 3.83 (m, 2H), 3.81 - 3.66 (m, 1H), 3.38 (s, 3H), 3.29 (s, 3H), 2.86 - 2.69 (m,
H), 2.69 - 2.52 (m, 1H). ESI-MS m/z: 354.2 [M+H]+.
4
.1.6.19. 3-Chloro-N-(1,3-dimethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-5-yl)-N-(4-
1
fluorobenzyl)propanamide (9s). A yellow solid. Yield 74 %. H NMR (300 MHz,
CDCl ) δ 7.28 - 7.17 (m, 2H), 7.07 - 6.94 (m, 2H), 6.77 (s, 1H), 5.53 (d, J = 14.7 Hz,
3
1
1
H), 4.02 - 3.82 (m, 2H), 3.80 - 3.66 (m, 1H), 3.38 (s, 3H), 3.28 (s, 3H), 2.84 - 2.68 (m,
H), 2.66 - 2.51 (m, 1H). ESI-MS m/z: 354.2 [M+H]+.
4
.1.6.20. 3-Chloro-N-(1,3-dimethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-5-yl)-N-(2-
1
chlorobenzyl)propanamide (9t). A yellow solid. Yield 75 %. H NMR (300 MHz,
CDCl ) δ 7.40 - 7.36 (m, 1H), 7.33 - 7.29 (m, 1H), 7.24 - 7.17 (m, 2H), 6.92 (s, 1H),
3
5
.39 (d, J = 14.7 Hz, 1H), 4.35 (d, J = 14.7 Hz, 1H), 3.95 - 3.80 (m, 1H), 3.78 - 3.63
(m, 1H), 3.33 (s, 3H), 3.25 (s, 3H), 2.84 - 2.67 (m, 1H), 2.67 - 2.51 (m, 1H). ESI-MS
m/z: 370.1 [M+H]+.
4
.1.6.21. 3-Chloro-N-(1,3-dimethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-5-yl)-N-(3-
1
chlorobenzyl)propanamide (9u). A yellow solid. Yield 77 %. H NMR (300 MHz,
CDCl ) δ 7.29 - 7.22 (m, 3H), 7.18 - 7.10 (m, 1H), 6.94 (s, 1H), 5.50 (d, J = 14.8 Hz,
3
1
1
H), 3.96 - 3.86 (m, 2H), 3.80 - 3.66 (m, 1H), 3.37 (s, 3H), 3.30 (s, 3H), 2.87 - 2.69 (m,
H), 2.68 - 2.52 (m, 1H). ESI-MS m/z: 370.1 [M+H]+.
4
.1.6.22. 3-Chloro-N-(1,3-dimethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-5-yl)-N-(4-
1
chlorobenzyl)propanamide (9v). A yellow solid. Yield 76 %. H NMR (300 MHz,

CDCl ) δ 7.32 - 7.26 (m, 2H), 7.23 - 7.15 (m, 2H), 6.85 (s, 1H), 5.50 (d, J = 14.7 Hz,
3
1
1
H), 3.99 - 3.85 (m, 2H), 3.79 - 3.68 (m, 1H), 3.38 (s, 3H), 3.29 (s, 3H), 2.84 - 2.70 (m,
H), 2.66 - 2.54 (m, 1H). ESI-MS m/z: 370.1 [M+H]+.
4
.1.6.23. 3-Chloro-N-(1,3-dimethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-5-yl)-N-(2-
1
(
trifluoromethyl)benzyl)propanamide (9w). A yellow solid. Yield 80 %. H NMR (300
MHz, CDCl ) δ 7.61 - 7.44 (m, 3H), 7.36 - 7.30 (m, 1H), 6.90 (s, 1H), 5.41 (d, J = 15.7
3
Hz, 1H), 4.43 (d, J = 15.6 Hz, 1H), 3.96 - 3.81 (m, 1H), 3.74 - 3.64 (m, 1H), 3.29 (s,
3
H), 3.21 (s, 3H), 2.85 - 2.68 (m, 1H), 2.68 - 2.52 (m, 1H). ESI-MS m/z: 404.2 [M+H]+.
4
.1.6.24. 3-Chloro-N-(1,3-dimethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-5-yl)-N-(3-
1
(
trifluoromethyl)benzyl)propanamide (9x). A yellow solid. Yield 79 %. H NMR (300
MHz, CDCl ) δ 7.59 - 7.51 (m, 1H), 7.50 - 7.43 (m, 3H), 6.83 (s, 1H), 5.54 (d, J = 14.8
3
Hz, 1H), 4.07 - 3.85 (m, 2H), 3.79 - 3.64 (m, 1H), 3.36 (s, 3H), 3.27 (s, 3H), 2.87 - 2.67
(
m, 1H), 2.66 - 2.51 (m, 1H). ESI-MS m/z: 404.2 [M+H]+.
4
.1.6.25. 3-Chloro-N-(1,3-dimethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-5-yl)-N-(2-
1
methoxybenzyl)propanamide (9y). A yellow solid. Yield 75 %. H NMR (300 MHz,
CDCl ) δ 7.26 - 7.14 (m, 2H), 6.90 - 6.83 (m, 1H), 6.79 - 6.73 (m, 2H), 5.21 (d, J =
3
1
4.3 Hz, 1H), 4.20 (d, J = 14.3 Hz, 1H), 3.89 - 3.80 (m, 1H), 3.70 - 3.62 (m, 4H), 3.30
(s, 3H), 3.18 (s, 3H), 2.75 - 2.62 (m, 1H), 2.60 - 2.46 (m, 1H). ESI-MS m/z: 365.3
[
M+H]+.
4
.1.6.26. 3-Chloro-N-(1,3-dimethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-5-yl)-N-(3-
1
methoxybenzyl)propanamide (9z). A yellow solid. Yield 79 %. H NMR (300 MHz,
CDCl ) δ 7.31 - 7.18 (m, 1H), 6.86 - 6.73 (m, 4H), 5.63 (d, J = 14.5 Hz, 1H), 4.03 -
3