Identification and structure–activity relationship exploration of uracil-based benzoic acid and ester derivatives as novel dipeptidyl Peptidase-4 inhibitors for the treatment of type 2 diabetes mellitus

European Journal of Medicinal Chemistry 225 (2021) 113765

Contents lists available at ScienceDirect

European Journal of Medicinal Chemistry

journal homepage: http://www.elsevier.com/locate/ejmech

Identiﬁcation and structureeactivity relationship exploration of

uracil-based benzoic acid and ester derivatives as novel dipeptidyl

Peptidase-4 inhibitors for the treatment of type 2 diabetes mellitus

Qing Li ^a, Xiaoyan Deng ¹, Neng Jiang ^d, Liuwei Meng , Junhao Xing ,

,

*

,

1

b

,

1

b

c

b

a, **

Weizhe Jiang , Yanjun Xu

College of Chemistry and Materials Science, Sichuan Normal University, Chengdu, 610068, China

Pharmaceutical College, Guangxi Medical University, Nanning, 530021, China

Department of Organic Chemistry and State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing, 210009, China

Department of Pharmacy, Afﬁliated Tumor Hospital of Guangxi Medical University, Nanning, 530021, China

a

b

c

d

a r t i c l e i n f o

a b s t r a c t

Article history:

Received 28 May 2021

Received in revised form

Our previously reported carboxyl-containing DPP-4 inhibitors were highly potent but were poorly

bioavailable. Esters of the carboxyl analogs exhibited a signiﬁcant DPP-4 potency loss albeit with

enhanced oral absorption. Herein, we described identiﬁcation and structureeactivity relationship (SAR)

exploration of a novel series of benzoic acid and ester derivatives as low single-digit nanomolar DPP-4

inhibitors. Importantly, the esters displayed comparable activities to the acids counterparts. Molecular

simulation revealed that ester adopts a similar binding mode to acid. Moreover, the selected esters and

acids demonstrated high selectivity and low cytotoxicity, as well as good metabolic stability. And more

importantly, the esters possessed excellent pharmacokinetic proﬁles for oral administration. The best

compound ester 19b demonstrated long DPP-4 inhibition in vivo, and robustly improved the glucose

tolerance in normal and db/db mice while ensuring glucose-lowering potency in chronic treatment. Our

results supported that the compound 19b can be served as a potential candidate for the treatment of type

2 diabetes.

23 July 2021

Accepted 6 August 2021

Available online 10 August 2021

Keywords:

Benzoic acid

DPP-4 inhibitor

Ester

SAR exploration

Type 2 diabetes

©

1

. Introduction

These agents are associated with side effects such as increased risk

of hypoglycemia and weight gain, which have limited their use [5].

Several novel glucose-lowering agents such as DPP-4 inhibitors [6],

glucagon-like peptide-1 (GLP-1) receptor agonists [7] and sodium-

glucose co-transporter 2 (SGLT2) inhibitors [8] are developed and

currently under clinical investigation. These novel agents have

demonstrated their overall good safety proﬁle and tolerability

[9,10]. Nevertheless, patients are usually more compliant to oral

agents than to injectable GLP-1 receptor agonists [10], whereas

SGLT2 inhibitors are associated with an annoying increase in uri-

nation frequency and an increased risk of urinary tract infection [9].

DPP-4 inhibitors have drawn great attention in recent years

[6,11]. The efﬁcacy of DPP-4 inhibitors is related directly to their

ability to inhibit dipeptidyl peptidase-4 (DPP-4) activity [11,12].

This serine protease is involved in the enzymatic degradation of

incretins such as GLP-1 and GIP [6,12]. Both of them are secreted by

the gut upon nutrient consumption to enhance glucose-dependent

insulin secretion [12,13]. Approved DPP-4 inhibitors such as sita-

gliptin, vildagliptin, saxagliptin, linagliptin, and alogliptin (Fig. S1 in

Diabetes mellitus (DM) has reached epidemic proportions. Ac-

cording to the International Diabetes Federation (IDF), about 463

million of adults are currently diagnosed with diabetes, and the

number is estimated to increase to 700 million by 2045 [1]. Type 2

diabetes mellitus (T2DM) accounts for 90e95 % of all diabetes

cases, which can lead to severe complications such as retinopathy,

neuropathy, and nephropathy [2]. In addition, T2DM is a leading

cause of cardiovascular disease. Thus DM is a key global health

priority [1,3].

Traditionally used anti-diabetic drugs include metformin, sul-

fonylureas, thiazolidinediones and

a-glucosidase inhibitors [4].

*

Corresponding author.

*

* Corresponding author.

E-mail addresses: qingli2021@sicnu.edu.cn (Q. Li), xuyj@sicnu.edu.cn (Y. Xu).

Those authors contributed equally to this works.

1

https://doi.org/10.1016/j.ejmech.2021.113765

0

Q. Li, X. Deng, N. Jiang et al.

European Journal of Medicinal Chemistry 225 (2021) 113765

Supporting Information) have demonstrated their efﬁcacy in

improving glycaemic control [14], and major advantages including

low risk of hypoglycemia and beneﬁcial effect on weight [12].

Nevertheless, adverse effects associated with DPP4 inhibitors have

been reported, these include a small increased risk of acute

pancreatitis, hospitalization for heart failure and and joint pain

pharmacokinetic properties, as well as potent in vivo blood

glucose-lowering effects in both normal and db/db mice.

2. Results and discussion

2.1. Chemistry

[

15,16]. New two antidiabetic drugs with the once weekly dosing

regimen, omarigliptin and trelagliptin (Fig. S1 in Supporting In-

formation), were approved in Japan to improve patient satisfaction

and compliance [17,18].

In our previous researches, several fragments were introduced

2

to explore more structure types of DPP-4 inhibitors [19e21]. The S ’

subsite of DPP-4 active site confers molecular recognition in-

teractions that contribute to the highest afﬁnity [22], and this

subsite has not been fully explored [23], we, therefore, have ratio-

nally designed 2-substituted benzoic acid to interact with both

Lys554 and Trp629 in this subsite [20], and the resulting com-

pounds 1a and 2a exhibited potent DPP-4 inhibitory activities

The target compounds were prepared according to the our

previously published procedure [20]. The general protocols are

outlined in Schemes 1 and 2. 6-Chlorouracil was selective alkylated

with 1-bromo-but-2-yne or 2-(bromomethyl)benzonitrile to give

products 4a-b. Further alkylation of 4a-b with benzyl bromide or

methyl (bromomethyl)benzoate provided intermediates 5a-g. The

chlorines of 5a-b were substituted with (R)-3-N-Boc-amino-

piperidine to afford compounds 6a-g, which were deprotected to

provide the desired target compounds 7a-b and 7d-g. Hydrolysis of

esters 6c-g with LiOH offered benzoic acids 8c-g. Compound 8c was

condensed with a variety of amino acid esters to obtain compounds

(

Fig. 1), however, compounds 1a and 2a suffered from poor oral

1

0a-d. Finally, deprotection of 8d-g and 10a-d provided target

compounds 9d-g and 11a-d, respectively. Hydrolysis of 10a-d, fol-

lowed by deprotection of Boc group, gave the target compounds

2a-d.

bioavailability [19,20]. Esters of the carboxyl analogs (such as 1b

and 2b) showed a signiﬁcant DPP-4 potency loss albeit with

enhanced oral absorption (Fig.1) [19,20]. Structure optimization via

replacement of 2-substituted benzoic acid with 3-substituted

benzoic acid led to identiﬁcation of compound 3a and its ester

1

As shown in Scheme 2, the target compounds 18a-h and 19a-h

were prepared starting from substituted methyl 3-

methylbenzoates 13a-h. Bromination of 13a-h with N-bromo-

succinimide (NBS) provided 3-bromomethylbenzoates 14a-h.

Treatment of compounds 14a-h with compound 4a, followed by

substitution with (R)-3-N-Boc-aminopiperidine, gave compounds

6a-h, which were subsequently hydrolyzed with LiOH to afford

compounds 17a-h. Compounds 17a-h and 16a-h underwent

deprotection to furnish the target compounds 18a-h and 19a-h.

3

b, which displayed potent DPP-4 activities [24]. Most importantly,

the ester 3b is more potent than acid 3a (Fig. 1). This is markedly

different from the 2-substituted esters 1b and 2b, suggesting 3-

substituted ester 3b can be a promising lead compound that

potentially has both improved potency and physicochemical

properties for oral administration. Given the potential conversion

of ester to acid in vivo, further SAR exploration around both acid 3a

and its ester 3b was performed in order to dissect in more detail the

structural elements required to exert biological activity. Herein we

reported our efforts to identify substituted benzoic acid and ester

derivatives as novel DPP-4 inhibitors, which revealed higher po-

tency than alogliptin and better selectivity over other DPP family

members such as DPP-8 and DPP-9. Furthermore, the optimal

compound 19b showed good metabolic stability, excellent oral

1

2

.2. In vitro activities and SAR exploration

The DPP-4 inhibitory activities of the target compounds were

evaluated using a previously reported ﬂuorescent based assay [25],

and alogliptin was used as a positive control. We ﬁrstly synthesized

compound 7a without carboxyl moiety. As shown in Table 1,

Fig. 1. The structures of compounds 1a-b, 2a-b, 3a-b and the design of target compounds.

2

Q. Li, X. Deng, N. Jiang et al.

European Journal of Medicinal Chemistry 225 (2021) 113765

Scheme 1. Synthesis of target compounds 7a-b, 7d-g, 9d-g, 11a-d and 12a-d. Reagents and conditions: (a) 1-bromo-but-2-yne or 2-(bromomethyl)benzonitrile, DIPEA, DMF, r.t.,

ꢀ ꢀ

2 3 2 3

CO , DMF, r.t. 12 h; (c) (R)-3-N-Boc-aminopiperidine, K CO , DMF, 60 C, 10 h; (d) HCl gas, EA/ether, 0 C; (e) LiOH,

12 h; (b) Benzyl bromide or methyl (bromomethyl)benzoate, K

H

2

O/MeOH, r.t.; (f), NHR , EDCI, HOBt, DIPEA, CH CN, r.t.

4

R

5

3

ꢀ

2 2 2 2 2 3

Scheme 2. Synthesis of target compounds 18a-h and 19a-h. Reagents and conditions: (a) NBS, (PhCO ) , ClCH CH Cl, 80 C, 12 h; (b) compound 4a, K CO , DMF, r.t.; (c) (R)-3-N-Boc-

ꢀ

aminopiperidine, K

2

CO

3

, DMF, 60 C, 10 h; (d) HCl gas, EA/ether, 0 C; (e) LiOH, H

2

O/MeOH, r.t.

3

Q. Li, X. Deng, N. Jiang et al.

European Journal of Medicinal Chemistry 225 (2021) 113765

Table 1

In vitro DPP-4 inhibitory activities of compounds 7a-b, 7d-g and 9d-g.

Compounds

R

2

R

4

%Inhibition at 200 nM^a

%Inhibition at 40 nM^a

IC50 (nM)^a^,^b

106.3

7a

H

78.8 ± 3.1

46.6 ± 2.9

7b

H

84.2 ± 1.3

56.5 ± 3.1

18.2

0

7

d

e

4 -COOMe

85.0 ± 2.2

66.0 ± 3.0

57.7 ± 1.4

23.5 ± 0.4

9.2

NT

0

7

2 -COOMe

0

7

f

3 -COOMe

97.7 ± 3.3

86.4 ± 2.1

83.0 ± 1.1

64.1 ± 3.8

15.0

20.2

0

g

4 -COOMe

0

9

d

e

4 -COOH

11.7 ± 1.9

57.8 ± 1.6

0.6 ± 3.4

NT

0

2 -COOH

25.0 ± 5.3

0

9

f

3 -COOH

49.2 ± 2.9

58.7 ± 8.0

96.7 ± 1.8

22.4 ± 1.7

11.9 ± 2.5

85.1 ± 0.1

NT

3.1

0

g

4 -COOH

alogliptin

a

Measured in two independent experiments.

NT: not tested.

b

compound 7a exhibited an almost 15 times lower IC50 value

carboxyl handle of 3a. As shown in Table 2, conjugation of glycine

and its ester provided compounds 12a and 11a, which showed

similar inhibitory afﬁnity as 3a-b, suggesting that elongating

(

IC50 ¼ 106.3 nM) compared with carboxyl compound 3a. Com-

0

pound 9d with 4 -carboxyl and its ester 7d were then prepared.

0

However, the 4 -carboxyl 9d showed essentially a total loss of ac-

carboxyl did not improve DPP-4 potency. a-alanine (compound

tivity, while its ester 7d retained the DPP-4 activity (IC50 ¼ 9.2 nM).

11d, IC50 ¼ 9.1 nM) and its ester (compound 10d, IC50 ¼ 23.7 nM)

exhibited a slight decrease in potency. The introduction of annular

proline (both R and S conﬁguration) further decreased potency

(IC50 ¼ 19.1 and 34.6 nM), and their esters substantially reduced

DPP-4 inhibition. The results indicate that bulky acids and esters

were not tolerated. Previous data revealed that introduction of

some substituents on benzene ring, especially some halogens,

signiﬁcantly improved DPP-4 potency in 2-substituted benzoic acid

DPP-4 inhibitors [19,20]. We, therefore, focused on introduction of

halogen to benzene rings of 3-substituted benzoic acid 3a and ester

3b to improve DPP-4 potency.

As illustrated in Table 3, there was a signiﬁcant improvement in

DPP-4 potency. A ﬂuorine was ﬁrstly introduced to 2, 4 and 6-

position of 3a as it is the most common bioisosteric replacement

for the hydrogen atom [27]. The resulting acids 18a-c showed the

potency trend with the order of 4-F > 6-F > 2-F, in which 4-F

substituted compound 18b diplayed the IC50 value of 1.0 nM, be-

ing approximately 3 times more active than reference alogliptin

(IC50 ¼ 3.6 nM). The ﬂuoro group at 6-position also resulted in a

substantial improvement of DPP-4 activities (18c, IC50 ¼ 2.8 nM),

but the 2-ﬂuoro-acid 18a lost DPP-4 activity with the IC50

These results suggest carboxyl plays a marked different role in DPP-

4

binding at different positions. In addition, alogliptin has 2-

cyanobenzyl interacting the hydrophobic S pocket [26]. We syn-

1

thesized compounds with 2-cyanobenzyl coupled with carboxyl

and ester at different position, compound 7b without carboxyl was

also synthesized for comparison. Compound 7b displayed potent

0

DPP-4 activity with IC50 of 18.2 nM, however, 9e-g with 2 , 3 and

0

4

-carboxyl showed reduction of DPP-4 inhibition compared to 7b.

Interestingly, their esters 7f-g retained the inhibitory activities, in

0

which the 7f with 3 -ester had the IC50 of 15.0 nM, and 4 -ester 7g

0

had the IC50 of 20.2 nM, but 7e with 2 -ester did not preserve the

potency, suggesting carboxyl and ester play a complicated role in

DPP-4 binding, and conjugation of benzoic acid and its ester to

cyanobenzyl-containing compounds was not beneﬁcial to DPP-4

potency. Together, 3-substituted benzoic acid and ester coupled

with 2-butynyl were the best compounds with regard to their

similarly potent activities, thus 3a and 3b were selected for further

SAR exploration.

Further optimization was focused on conjugating various amino

acids (compounds 12a-d) and their esters (compounds 11a-d) to

4

Q. Li, X. Deng, N. Jiang et al.

European Journal of Medicinal Chemistry 225 (2021) 113765

Table 2

In vitro DPP-4 inhibitory activities of compounds 11a-d and 12a-d.

Compounds

R

9

%Inhibition at 200 nM^a

%Inhibition at 40 nM^a

IC50 (nM)^a^,^b

9.1

11a

92.4 ± 2.1

81.9 ± 3.8

1

1b

83.4 ± 6.2

80.6 ± 2.8

33.3 ± 1.6

43.3 ± 5.3

NT

11c

47.6

1

1d

2a

2b

87.5 ± 0.7

96.1 ± 2.4

92.7 ± 2.8

61.5 ± 3.2

83.7 ± 1.1

72.2 ± 1.3

23.7

6.9

34.6

1

2c

83.7 ± 6.1

64.1 ± 4.6

19.1

2d

93.1 ± 1.8

96.7 ± 1.8

74.3 ± 1.4

85.1 ± 0.1

9.1

3.1

alogliptin

a

Measured in two independent experiments.

NT: not tested.

b

value > 1

m

M. With the obtained SAR of ﬂuorine, we then intro-

ﬂuoro 19a (IC50 ¼ 7.0 nM) and 2-bromo 19d (IC50 ¼ 4.5 nM) also

showed potent DPP-4 activities, being different from their acids 18a

and 18d.

duced a bromine on 2, 4, 5 and 6-position. The bromine at 4 and 6-

position displayed slightly more potent DPP-4 inhibition than

ﬂuoro-based compounds at 4 and 6-position, in which 18e with 4-

bromo showed the most potent DPP-4 activity in this series, with

the IC50 of 0.8 nM, being 4.5-fold more potent than alogliptin.

Notably, 5-bromo also exhibited low nanomolar potency (18f,

Together, both 3-substituted acids and esters were identiﬁed as

potent DPP-4 inhibitors, especially, 3-substituted acids and esters

with 4-halogen displayed IC50 values down to the low single-digit

nanomolar range, being much better than alogliptin. Further-

more, the activities of esters were comparable to their acid coun-

terparts. In addition, methyl esters 19a-h showed cLogP values with

the range of 1.72e2.88, which was an optimal range for oral drug.

After above described and our previous SAR exploration [19,20,24],

a clear SAR summary of carboxylic acid (ester, amide)-based DPP-4

inhibitors was emerged as shown in Fig. 2.

IC50 ¼ 4.9 nM). However, 2-bromo-acid 18d (IC50 > 1

mM) also lost

DPP-4 activity as that of 2-ﬂuoro 18a. Similar potency trend was

observed in bromine-modiﬁed compounds compared to ﬂuoro-

derived compounds, with the potency order of 4-Br > 6-Br > 2-

Br, indicating 4-position is an optimal position for the introduc-

tion of halogen. Thus we subsequently introduced a chlorine atom

at 4-position, the resulting compound 18h showed potent activity

with IC50 value of 1.0 nM. Its activity is approximately 3-fold more

potent than that of alogliptin.

2.3. Molecular simulation study

Meanwhile, some halogens were introduced to benzene ring of

ester 3b. As shown in Table 3, all of esters 19a-h demonstrated

highly potent DPP-4 activities with IC50 values < 7 nM, and dis-

played similar potency trend to the corresponding acids with the

order of 4 > 6 > 2. 4-Fluoro-substituted 19b, 4-bromo-substituted

To understand molecular basis of their inhibitory activity, mo-

lecular docking study was performed for the acids 18b, 18e, 18h and

ester 19b based on the co-crystal structure of linagliptin (PDB ID:

2RGU). The most appropriate binding modes are displayed in Fig. 3.

All compounds preserve two binding interactions as that of 2-

substituted benzoic acid 1a, the 2-butynyl group inserts into the

S1 pocket to form hydrophobic interaction, and the primary amine

of (3R)-aminopiperidine makes salt bridge interactions with

Glu205 and Glu206. However, signiﬁcant differences were

19e and 4-chloro-substituted 19h are low single-digit nanomolar

DPP-4 inhibitors with IC50 values of 1.6 nM, 1.3 nM and 1.1 nM,

respectively. Most importantly, their activities are comparable to

the acid counterparts, being more active than alogliptin. Notably, 2-

5

Q. Li, X. Deng, N. Jiang et al.

European Journal of Medicinal Chemistry 225 (2021) 113765

Table 3

In vitro DPP-4, DPP-8 and DPP-9 inhibitory activities and cLogP values of compounds 18a-h and 19a-h.

Compounds

R

8

DPP-4 IC50 (nM)^a

DPP-8 IC50

(

m

M)^a^,^c

DPP-9 IC50

(m

M)^a^,^c

cLogP^b

1

8a

8b

8c

8d

8e

8f

8g

8h

9a

9b

9c

9d

9e

9f

2-F

4-F

6-F

2-Br

4-Br

5-Br

6-Br

4-Cl

2-F

>1000

1.0

2.8

>1000

0.8

4.9

1.3

1.0

7.0

1.6

4.3

4.5

1.3

2.6

1.1

3.6

NT

ꢁ0.60

ꢁ0.24

ꢁ0.60

ꢁ0.17

0.48

ꢁ0.17

0.33

1.72

2.16

1.72

2.44

2.88

2.44

2.73

e

>100

NT

>100

NT

>100

1.86

>100

0.12

4-F

6-F

2-Br

4-Br

5-Br

6-Br

4-Cl

e

9g

9h

alogliptin

vildagliptin

e

a

Measured at least in two independent experiments.

clogP were calculated by ChemBioDraw software Ultra 14.0.

NT: not tested.

b

c

Fig. 2. SAR summary of benzoic acids and its derivatives-based DPP-4 inhibitors.

observed in S

1

’ and S

2

’ subsite compared to 1a, the carboxyl of 3-

Lys554 and Trp629, respectively, being similar to that of 3-

substituted benzoic acid. This is the primary reason for the

similar potency between the 3-substituted benzoic acids and their

esters.

substituted benzoic acid impacts the orientation and molecular

interactions of benzoic acid moiety and uracil scaffold. The carboxyl

of 1a makes a salt bridge with the primary amine of Lys554, and the

uracil of compound 1a interacts with Tyr547 via

However, the 3-substituted benzoic acids of 18b, 18e and 18h orient

toward the amino nitrogens of Lys554 and Trp629 in S ’ subsite,

p-p stacking.

2.4. DPP-8 and DPP-9 selectivity study

2

forming a salt bridge interaction and a hydrogen bond with Lys554

and Trp629, respectively, while the benzene rings of benzoic acid

Minimizing binding to off-target proteins, especially some ho-

mologous enzymes, has been identiﬁed as particularly promising

strategies for improving a drug candidate safety index. Studies have

shown that inhibition of DPP-4 homologous enzymes, such as DPP-

8 and DPP-9, causes severe toxicities in animal models [28]. Thus

we evaluated the binding selectivity of compounds 18a-h and 19a-

h against DPP-8 and DPP-9. As presented in Table 3. All compounds

18a-h and 19a-h showed excellent selectivity against DPP-8 and

are positioned for a

p-p stacking with Tyr547. This lead to the loss

of stacking interaction between the uracil ring and Tyr547.

p-p

These interactions, together with the hydrogen bonds are key for

retaining the inhibition in the active site. The ester 19b adopts an

essentially same binding mode to acids 18b, 18e and 18h, the two

oxygen atoms of ester engage in two key hydrogen bonds with

6

Q. Li, X. Deng, N. Jiang et al.

European Journal of Medicinal Chemistry 225 (2021) 113765

Fig. 3. The docking binding modes of compounds 18b, 18e, 18h and 19b in DPP-4 active site. Hydrogen bonds are depicted as dashed yellow lines. Residues and compounds in active

site are shown as sticks. (A) the overlay structures of 1a (lightpink) with compound 18b (green), (B) compound 18e (cyan), (C) compound 18h (orange), (D) compound 19b

(

lightblue).

DPP-9 with the IC50 values > 100

vildagliptin displayed moderate DPP-4 inhibitory activities with the

IC50 of 1.86 M and 0.12 M, respectively.

m

M. However, reference drugs

cell using the MTT assay. Doxorubicin (DOX) was used as control. As

shown in Fig. 4, all of compounds 18b, 18e, 18h, 19b and 19h

m

exhibited approximately >95 % of viability up to 100

low cytotoxicity of these compounds. In contrast, compound 19e

showed moderate cytotoxicity with 69 % of viability at 100 M,

while the positive control DOX exhibited higher toxicity with only

mM, indicating

m

2.5. Cytotoxicity study

25 % of cell viability at 20 mM.

Compounds 18b,18e,18h,19b,19e and 19h were selected for cell

viability test. Cell viability was evaluated on normal hepatic LO2

2.6. Metabolic stability assay in rat plasma and liver microsomes

The metabolic stability inﬂuences pharmacokinetic character-

istics of therapeutic compounds, we therefore determined the

metabolic stability of selected compounds in plasma and liver mi-

crosomes. Compounds 18b, 18e, 18h, 19b, 19e and 19h were incu-

bated with plasma of SD rat for 120 min. As shown in Table 4, the

incubation of esters 19b, 19e and 19h resulted in 19%e27 % of

conversion to corresponding acids 18b, 18e and 18h, and remaining

ratio of esters 19b, 19e and 19h were 81 %, 82 % and 64 %, respec-

tively. Almost all of carboxylic acid 18b, 18e and 18h were retained

after incubation (remaining ratio ¼ ~100 %), indicating the acids

were metabolically stable in rat plasma. To evaluate the microsomal

stability, these compounds were incubated with liver microsomes

of ICR mice. As presented in Table 4, esters 19b, 19e and 19h were

converted into carboxylic acids after incubation, while acids 18b,

18e and 18h were hardly metabolized (remaining ratio > 97 %). The

total ratio of ester and metabolite acid was more than 93 %,

demonstrating that acid is main metabolite. The remaining ratio of

esters 19b, 19e and 19h were 56 %, 38 % and 34 %, respectively,

Fig. 4. Cytotoxicity of compounds 18b, 18e, 18h, 19b, 19e and 19h against normal

hepatic LO2 cell. Values are mean ± SD (n ¼ 5). **P ꢂ 0.01 compared to vehicle-treated

group by using a one-way ANOVA with Dunnett's multiple comparisons test.

7

Q. Li, X. Deng, N. Jiang et al.

European Journal of Medicinal Chemistry 225 (2021) 113765

Table 4

Metabolic stability of compounds in the rat plasma and ICR liver microsomes.

Compounds^a

Analytes

Remaining ratio (%)

Plasma (SD rats)

Liver Microsome (ICR mice, 60 min)

(120 min)

1

8b

8e

8h

9b

18b

18e

18h

19b

100.64 ± 3.57

101.68 ± 2.14

102.66 ± 2.56

81.75 ± 3.52

22.48 ± 2.10

82.67 ± 1.21

19.19 ± 3.51

64.07 ± 2.64

27.82 ± 2.78

98.92 ± 2.58

97.84 ± 4.13

105.89 ± 1.29

56.03 ± 1.69

37.46 ± 4.28

38.07 ± 3.49

58.19 ± 1.69

34.44 ± 5.10

70.74 ± 3.72

1

8b

19e

8e

19h

8h

1

9e

1

9h

1

a

Measured in two independent experiments.

suggesting that the methyl ester 19b had the best metabolic sta-

bility. Considering both ester and acid are highly potent DPP-4 in-

hibitor, ester might exert in vivo effects through both prototype

ester and metabolite acids.

2.8. In vivo DPP-4 inhibition evaluation

We next evaluated plasma DPP-4 inhibition of compounds 19b,

19e and 19h at a dose of 10 mg/kg via oral administration in male

SpragueeDawley rats. As shown in Fig. 5, oral administration of

compounds 19b, 19e and 19h exhibited maximum inhibition (90 %)

as early at 30 min postdose, and sustainedly inhibited > 70 % of

DPP-4 through 12 h postdose, but the DPP-4 inhibition of com-

pounds 19e and 19h was substantially reduced after 12 h postdose,

which might attribute to their short half-life. In contrast, persistent

inhibition (>70 %) of DPP-4 was observed in compound 19b

through 24 h postdose. The DPP-4 inhibition of compound 19b

lasted longer than that of alogliptin, which inhibited 45 % DPP-4 at

2.7. In vivo pharmacokinetic evaluation

In vivo pharmacokinetic proﬁle of compounds 18b, 18e, 18h, 19b,

1

9e and 19h was evaluated in SD rats. Compounds 18b, 18e, 18h and

9b were administrated by i.v. injection at dose of 3 mg/kg, while

9b,19e and 19h were administrated by gavage at dose of 10 mg/kg.

As presented in Table 5, acids 18b, 18e and 18h showed similar

pharmacokinetic properties. All of them exhibited high plasma

exposure, but they suffered from a high plasma clearance, leading

to short half-life (T1/2 ¼ 0.59e0.96 h). As expected, esters 19b, 19e

and 19h showed improved pharmacokinetic proﬁles including low

clearance and high oral bioavailability compared to acids 18b, 18e

and 18h, in which compound 19b had excellent oral bioavailability

24 h postdose. This indicated that compound 19b is a long-acting

DPP-4 inhibitor.

2.9. Acute hypoglycemic effects of 19b in normal mice and db/db

mice

(

F% calculated from 19b and 18b is 98 %). In addition, ester 19b had

Due to its potent DPP-4 activity, excellent selectivity and phar-

macokinetic properties, as well as long-lasting DPP-4 inhibition,

the compound 19b was then selected for in vivo hypoglycemic

activity evaluation. Compound 19b and alogliptin were given orally

at the dose of 10 mg/kg before glucose challenge (3 g/kg) in normal

ICR mice. As shown in Fig. 6A and 6B, oral administration of com-

pound 19b led to signiﬁcantly reduced glucose excursion by

approximately 14.8 %. The improvement of glucose tolerance was

also observed for alogliptin with reduced excursion of 12.4 %.

The in vivo hypoglycemic potency of compound 19b was further

investigated at different doses in db/db mice. 19b were adminis-

the longest half-life (T1/2 ¼ 6.29 h), and compounds 19e and 19h

displayed moderate half-life (T1/2 ¼ 2.52 h and 2.43 h). This was

consistent with the results that compound 19b showed the best

metabolic stability. In addition, esters 19b, 19e and 19h were con-

verted to their corresponding acids 18b, 18e and 18h, respectively.

The time-concentration curve of compound 19b after oral admin-

istration is shown in Fig. 5, compound 19b sustained release of

compound 18b. Due to its long half-life, the concentration of

compound 19b in plasma was 7.5 ng/mL at 24 h postdose. Together,

esters 19b, 19e and 19h demonstrated suitable pharmacokinetic

proﬁles for oral administration, thus we evaluated their DPP-4 in-

hibition in vivo.

ꢁ1

tered by gavage at the doses of 10 mg kg , 3 mg kg and 1 mg

ꢁ1

kg . As shown in Fig. 6C and 6D, compound 19b robustly improved

Table 5

Pharmacokinetic parameters of 18b, 18e, 18h, 19b, 19e and 19h.

Parameters^a

analyte

T

1/2 (h)

T

max (h)

C

(m

g/L)

AUC0-inf

(

mg/L*h)

Cl(L/h/kg)

F%

max

1

8b (iv)

8e (iv)

8h (iv)

9e(po)

18b

18e

18h

19e

0.96 ± 0.24

0.59 ± 0.25

0.91 ± 0.47

2.52 ± 0.47

2.24 ± 0.40

2.43 ± 0.41

2.39 ± 0.59

1.73 ± 0.45

1.81 ± 0.18

6.29 ± 1.20

4.62 ± 1.12

e

26105.01 ± 1072.87

24581.07 ± 6709.55

25697.60 ± 9270.77

1410.94 ± 750.06

1274.61 ± 572.63

3192.06 ± 1067.50

3175.51 ± 154.45

933.67 ± 130.50

732.06 ± 66.82

5356.87 ± 460.36

5684.67 ± 3921.09

8272.25 ± 3026.46

13757.26 ± 5970.26

5597.56 ± 2298.43

26786.92 ± 21659.47

8369.48 ± 3180.12

1735.83 ± 477.37

878.14 ± 202.61

5.62 ± 0.48

6.92 ± 2.48

3.89 ± 1.14

0.85 ± 0.44

2.09 ± 1.19

0.55 ± 0.14

12.88 ± 4.89

0.18 ± 0.04

3.53 ± 0.73

0.23 ± 0.17

6.28 ± 0.18

e

0.25

0.83

0.38

0.75

e

1

8e

19h

8h

19b

8b

19b

8b

e

19h(po)

19b (iv)

19b (po)

e

1

e

1

e

0.50

695.65 ± 147.29

586.22 ± 131.98

5880.28 ± 1051.76

1703.32 ± 583.60

101 %

58 %

1

a

Compounds were dosed to equal number of male Sprague-Dawley rats (n ¼ 3) in po (10 mg/kg) and iv (3 mg/kg) administration.

8

Q. Li, X. Deng, N. Jiang et al.

European Journal of Medicinal Chemistry 225 (2021) 113765

Fig. 5. (A) Time-concentration curve of compound 18b (i.v. 3 mg/kg) and 19b (p.o. 10 mg/kg) after administration. (B) The in vivo % DPP-4 inhibition of compounds 19b, 19e and 19h

10 mg/kg) after p.o. administration.

(

Fig. 6. Effects of compound 19b on blood glucose levels during an OGTT in male ICR (n ¼ 8) and db/db mice (n ¼ 6). (A) blood glucose changes in ICR mice; (B) AUC0e120 min of blood

glucose in ICR mice; (C) blood glucose changes in db/db mice, (D) AUC0e120 min of blood glucose in db/db mice; Data are presented as mean ± SEM. (*) P < 0.05, (**) P < 0.01 vs

vehicle-treated group by using a one-way ANOVA with Dunnett's multiple comparisons test.

the glucose tolerance in a dose-dependent manner, with the

reduced the AUC0e120 min values of 41.6%, 33.2 % and 28.2 % at the

dose of 10 mg/kg, 3 mg/kg and 1 mg/kg, respectively. The hypo-

glycemic activity of the compound 19b is slightly more potent than

that of referenced drug alogliptin at the dose of 10 mg/kg, which

decreased 40.1 % of AUC0e120mim value.

administration of 19b and alogliptin at the dose of 10 mg/kg

signiﬁcantly decreased fasting blood glucose (FBG) levels, and 19b

reduced more FBG levels than alogliptin did at this dose, but the

FBG levels were not signiﬁcantly decreased at the dose of 1 mg/kg.

In addition, non-fasting body weight was gradually increased after

the treatment of 19b (Fig. 7B) as that of alogliptin, indicating that

19b (at both 1 and 10 mg/kg) is well tolerated in db/db mice.

2

.10. Chronic treatment of 19b in type 2 diabetic db/db mice

Moreover, signiﬁcantly improvement in the glucose tolerance was

observed after 4 weeks of treatment of 19b (10 mg/kg) (Fig. 7C), the

decreased the AUC0e120 min values of 19b at the dose of 10 mg/kg

and 1 mg/kg were 21.5% and 6.8 % respectively, while alogliptin

(Fig. 7D) reduced the AUC0e120 min values of 16.7% at the dose of

10 mg/kg, suggesting 19b produced more potent effects than that of

The potential long-term therapeutic effects of 19b were evalu-

ated in type 2 diabetic db/db mice. Based on pharmacokinetic

proﬁle and DPP-4 inhibition, 19b and alogliptin were orally

administrated once daily for 4 weeks. As shown in Fig. 7A,

9

Q. Li, X. Deng, N. Jiang et al.

European Journal of Medicinal Chemistry 225 (2021) 113765

Fig. 7. Chronic treatment effects of 19b in diabetic db/db mice with 1 and 10 mg/kg for 4 weeks (n ¼ 8). (A) FBG changes; (B) body weight changes; (C) blood glucose changes in

OGTT; (D) AUC0e120 min of blood glucose between 0 and 120 min; OGTT was conducted after 4 weeks of treatment of 19b after an oral glucose load (1.5 g/kg). Data are presented as

the mean ± SEM. (*) P < 0.05, (**) P < 0.01 vs vehicle-treated group by using a one-way ANOVA with Dunnett's multiple comparisons test.

alogliptin.

candidate for the treatment of T2DM.

4. Experimental

3

. Conclusion

Here, we identiﬁed a novel series of carboxylic acid and ester

4.1. Chemical materials and methods

derivatives as potent and orally bioavailable DPP-4 inhibitors.

Starting with 3-substituted benzoic acid 3a and its methyl ester 3b,

we performed extensive SAR studies via exploration of the optimal

position of carboxyl, comparison of the 2 -cyanobenzyl and 2-

butynyl groups, conjugation of various amino acid at carboxyl

handle and introduction of the various halogen groups to benzene

ring. A clear SAR summary of carboxylic acid (ester)-based DPP-4

inhibitors was presented, and revealed carboxylic acids (esters)

are an important structural elements required to exert highly

potent activity. Finally, both acids and esters with 4-halogen groups

including ﬂuorine, bromine and chlorine were identiﬁed as

attractive candidates with IC50 values down to the low single-digit

nanomolar range, being more potent than alogliptin. More impor-

tantly, the esters were equipotent as acids. Molecular docking

revealed that both esters and acids exhibited comparable binding

modes with key hydrogen bonds interaction with Lys554 and

Trp629. Further evaluation demonstrated best compound 19b

showed excellent selectivity, low cytotoxicity and good metabolic

stability, and also displayed excellent oral pharmacokinetic proﬁles

and sustained in vivo DPP-4 inhibition. In addition, acute and

chronic treatment with ester 19b robustly improved the glucose

tolerance in normal and db/db mice, and ameliorated FBG levels in

db/db mice. These excellent results qualiﬁed ester 19b as a

All starting reagents and materials were commercially available

and used without further puriﬁcation. Reactions and column

chromatography were monitored by TLC on silica gel 60 F254

plates. Puriﬁcation was performed on column chromatography

with silica gel (200e300 mesh). The purity of the target compounds

was determined on HPLC (Agilent 1260 Series apparatus) using UV

detector (wavelength: 254 nm) with mobile phase composed of

1

13

methanol and H

2

O (0.1 % HCOOH). H NMR and C NMR spectra

3

) were obtained on a Bruker spectrometer (500,

(DMSO‑d , CDCl

6

600 MHz) or Varian spectrometer (400 MHz) in the indicated sol-

vents (TMS as internal standard) at ambient temperature; the

chemical shifts are expressed in

d values (ppm) and the coupling

constants (J) in Hz. MS spectra were recorded on ThermoFisher TSQ

Series Mass spectrometer system (ESI). Melting points were

measured on capillary tube and were uncorrected. Compounds 4a,

5c, 6c, 8c were prepared according to reported methods [20,24,29].

4.1.1. General procedure for the synthesis of 2-((6-chloro-2,4-dioxo-

3,4-dihydropyrimidin-1(2H)-yl)methyl)benzonitrile (4b)

According to published procedure [20], a mixture of 6-

chlorouracil (41 mmol), 2-(bromomethyl)benzonitrile (41 mmol)

and DIPEA (6.3 g, 49 mmol) was stirred at room temperature in

10

Q. Li, X. Deng, N. Jiang et al.

European Journal of Medicinal Chemistry 225 (2021) 113765

DMF (25 mL) for 12 h. Water (100 mL) was added, the precipitate

2 4

washed with saturated brine, dried over anhydrous Na SO . The

was collected by ﬁltration, washed with water, EtOH and ethyl

acetate, and dried to give product 4b. White solid (3.2 g, 53.5 %). H

crude product was puriﬁed by ﬂash chromatography (ethyl acetate/

petroleum ether 10:1e3:1) to give 6a-b and 6d-g.

1

NMR (400 MHz, DMSO‑d

6

)

d

11.79 (s, 1H), 7.87 (dd, J ¼ 7.7, 1.3 Hz,

1

H), 7.69 (td, J ¼ 7.7, 1.4 Hz, 1H), 7.50 (td, J ¼ 7.7, 1.1 Hz, 1H), 7.37 (d,

4.1.3.1. tert-butyl

(R)-(1-(1-benzyl-3-(but-2-yn-1-yl)-2,6-dioxo-

(6a).

Light yellow solid (6.4 g, 86.9 %). H NMR (600 MHz, DMSO‑d

J ¼ 7.9 Hz, 1H), 6.05 (d, J ¼ 1.8 Hz, 1H), 5.30 (s, 2H); MS (ESI) m/z:

1,2,3,6-tetrahydropyrimidin-4-yl)piperidin-3-yl)carbamate

þ

1

2

62.13 [MþH] .

6

)

d

7.34e7.18 (m, 5H), 7.00 (d, J ¼ 7.8 Hz, 1H), 5.23 (s, 1H), 4.94 (s, 2H),

4

5

.1.2. General procedure for the synthesis of compounds 5a-b and

d-g

4.61e4.32 (m, 2H), 3.60e3.52 (m, 1H), 3.28e3.22 (m, 1H), 3.18e3.16

(m, 1H), 2.76e2.72 (m, 1H), 2.58e2.51 (m, 1H), 1.81e1.76 (m, 2H),

1.75 (s, 3H), 1.60e1.58 (m, 2H), 1.38 (s, 9H). MS (ESI) m/z: 453.37

[MþH] .

2 3

To a suspension of 4a-b (1 equiv.) and K CO (2 equiv.) in DMF

þ

(

20 mL) was added substituted benzyl bromide (1 equiv.). The

mixture was stirred at room temperature for 12 h. Water was

added, the precipitate was ﬁltrated, dried, and recrystallized with

ethyl acetate to give compounds 5a-b and 5d-g.

4.1.3.2. tert-butyl

1,2,3,6-tetrahydropyrimidin-4-yl)piperidin-3-yl)carbamate

White solid (0.31 g, 92 %). H NMR (400 MHz, CDCl

(R)-(1-(1-benzyl-3-(2-cyanobenzyl)-2,6-dioxo-

(6b).

1

3

)

d

7.66 (d,

4

.1.2.1. 3-benzyl-1-(but-2-yn-1-yl)-6-chloropyrimidine-2,4(1H,3H)-

J ¼ 7.7 Hz, 1H), 7.50 (t, J ¼ 7.7 Hz, 1H), 7.39e7.34 (m, 3H), 7.31e7.20

(m, 3H), 7.10 (d, J ¼ 8.0 Hz, 1H), 5.36 (s, 1H), 5.32e5.18 (m, 2H), 5.07

(d, J ¼ 3.1 Hz, 2H), 4.54e4.40 (m, 1H), 3.78e3.62 (m, 1H), 3.29e3.10

(m, 1H), 2.98e2.85 (m, 1H), 2.80e2.65 (m, 1H), 2.62e2.42 (m, 1H),

1.94e1.84 (m, 1H), 1.83e1.74 (m, 1H), 1.73e1.62 (m, 1H), 1.39 (s, 9H).

1

3

dione (5a). Yellow solid (5.6 g, 77.0 %). H NMR (400 MHz, CDCl )

d

7.50e7.44 (m, 2H), 7.34e7.24 (m, 3H), 5.96 (s, 1H), 5.09 (s, 2H),

4

.75 (q, J ¼ 2.4 Hz, 2H), 1.81 (t, J ¼ 2.4 Hz, 3H); MS (ESI) m/z: 289.15

þ

[MþH] .

þ

MS (ESI) m/z: 516.36 [Mþ H] .

4

1

.1.2.2. 2-((3-benzyl-6-chloro-2,4-dioxo-3,4-dihydropyrimidin-

(2H)-yl)methyl)benzonitrile (5b). White solid (0.17 g, 42.2 %). ¹

7.70 (d, J ¼ 7.7 Hz, 1H), 7.57 (t, J ¼ 7.8 Hz,

H), 7.47e7.44 (m, 2H), 7.41 (t, J ¼ 7.7 Hz, 1H), 7.37e7.25 (m, 3H),

H

4.1.3.3. methyl

(R)-4-((3-(but-2-yn-1-yl)-4-(3-((tert-butox-

NMR (400 MHz, CDCl

1

3

)

d

ycarbonyl)amino)piperidin-1-yl)-2,6-dioxo-3,6-dihydropyrimidin-

1

1(2H)-yl)methyl)benzoate (6d). White solid (0.45 g, 90.1 %). H NMR

7

.15 (d, J ¼ 7.9 Hz, 1H), 6.02 (s, 1H), 5.50 (s, 2H), 5.14 (s, 2H); MS (ESI)

(400 MHz, CDCl

3

)

d

7.95 (d, J ¼ 8.2 Hz, 2H), 7.51 (d, J ¼ 8.2 Hz, 2H),

þ

m/z: 352.21 [MþH] .

5.25 (s, 1H), 5.18e5.06 (m, 2H), 4.86e4.68 (m, 1H), 4.61 (d,

J ¼ 16.9 Hz, 1H), 4.53e4.41 (m, 1H), 3.88 (s, 3H), 3.86e3.77 (m, 1H),

3.36e3.22 (m, 1H), 3.18e3.00 (m, 1H), 2.93e2.76 (m, 1H), 2.72e2.57

(m, 1H), 1.95e1.83 (m, 2H), 1.80 (s, 3H), 1.75e1.70 (m, 1H), 1.43 (s,

4

.1.2.3. methyl 4-((3-(but-2-yn-1-yl)-4-chloro-2,6-dioxo-3,6-

dihydropyrimidin-1(2H)-yl)methyl)benzoate (5d). White solid

7.97 (d, J ¼ 8.3 Hz, 2H),

1

þ

(

0.24 g, 51.1 %). H NMR (400 MHz, CDCl

3

)

d

9H). MS (ESI) m/z: 511.37 [Mþ H] .

7.50 (d, J ¼ 8.3 Hz, 2H), 5.98 (s, 1H), 5.13 (s, 2H), 4.76 (q, J ¼ 2.3 Hz,

2

H), 3.89 (s, 3H), 1.81 (t, J ¼ 2.4 Hz, 3H); MS (ESI) m/z: 347.19

4.1.3.4. methyl (R)-2-((4-(3-((tert-butoxycarbonyl)amino)piperidin-

1-yl)-3-(2-cyanobenzyl)-2,6-dioxo-3,6-dihydropyrimidin-1(2H)-yl)

þ

[MþH] .

1

methyl)benzoate (6e). White solid (0.27 g, 87.2 %). H NMR

4

.1.2.4. methyl 2-((4-chloro-3-(2-cyanobenzyl)-2,6-dioxo-3,6-

dihydropyrimidin-1(2H)-yl)methyl)benzoate (5e). White solid

7.96 (d, J ¼ 7.8 Hz, 1H),

.70 (d, J ¼ 7.7 Hz, 1H), 7.59 (t, J ¼ 7.8 Hz, 1H), 7.48e7.39 (m, 2H),

(400 MHz, CDCl

3

)

d

7.92 (d, J ¼ 7.8 Hz, 1H), 7.65 (d, J ¼ 7.8 Hz, 1H),

7.52 (t, J ¼ 7.7 Hz, 1H), 7.38e7.30 (m, 2H), 7.29e7.22 (m, 1H), 7.19 (d,

J ¼ 7.9 Hz, 1H), 6.95 (d, J ¼ 7.8 Hz, 1H), 5.50 (s, 2H), 5.40 (s, 1H),

5.31e5.20 (m, 2H), 4.58e4.43 (m, 1H), 3.89 (s, 3H), 3.81e3.67 (m,

1H), 3.35e3.17 (m, 1H), 3.02e2.91 (m, 1H), 2.86e2.70 (m, 1H),

2.66e2.48 (m, 1H), 1.99e1.87 (m, 1H), 1.87e1.77 (m, 1H), 1.77e1.66

1

(

3

0.32 g, 68.1 %). H NMR (400 MHz, CDCl ) d

7

.32 (t, J ¼ 7.6 Hz, 1H), 7.23e7.18 (m, 1H), 7.13 (d, J ¼ 7.9 Hz, 1H), 6.06

(

[

s, 1H), 5.61 (s, 2H), 5.52 (s, 2H), 3.93 (s, 3H); MS (ESI) m/z: 410.18

MþH] .

þ

(m, 1H), 1.41 (s, 9H). MS (ESI) m/z: 574.32 [Mþ H] .

4

.1.2.5. methyl 3-((4-chloro-3-(2-cyanobenzyl)-2,6-dioxo-3,6-

dihydropyrimidin-1(2H)-yl)methyl)benzoate (5f). White solid

8.10e8.08 (m, 1H), 7.96

4.1.3.5. methyl (R)-3-((4-(3-((tert-butoxycarbonyl)amino)piperidin-

1-yl)-3-(2-cyanobenzyl)-2,6-dioxo-3,6-dihydropyrimidin-1(2H)-yl)

1

(

0.2 g, 63.8 %). H NMR (400 MHz, CDCl

t, J ¼ 7.8 Hz, 1H), 7.72e7.67 (m, 1H), 7.66e7.62 (m, 1H), 7.58 (t,

3

)

d

methyl)benzoate (6f). White solid (0.16 g, 82.2 %).

(400 MHz, CDCl

H NMR

3

)

d

8.04 (s, 1H), 7.91 (d, J ¼ 7.8 Hz, 1H), 7.65 (d,

J ¼ 7.8 Hz, 1H), 7.46e7.36 (m, 2H), 7.19e7.15 (m, 1H), 6.03 (s, 1H),

J ¼ 7.7 Hz, 1H), 7.56 (d, J ¼ 7.7 Hz, 1H), 7.50 (t, J ¼ 7.7 Hz, 1H),

þ

5

.51 (s, 2H), 5.18 (s, 2H), 3.90 (s, 3H); MS (ESI) m/z: 432.20 [MþNa] .

7.39e7.29 (m, 2H), 7.12 (d, J ¼ 7.9 Hz,1H), 5.37 (s,1H), 5.30e5.20 (m,

2

H), 5.10 (d, J ¼ 2.0 Hz, 2H), 4.52e4.38 (m, 1H), 3.89 (s, 3H),

4

.1.2.6. methyl 4-((4-chloro-3-(2-cyanobenzyl)-2,6-dioxo-3,6-

3.77e3.64 (m, 1H), 3.30e3.15 (m,1H), 2.98e2.86 (m, 1H), 2.81e2.66

(m, 1H), 2.62e2.45 (m, 1H), 1.95e1.83 (m, 1H), 1.83e1.74 (m, 1H),

dihydropyrimidin-1(2H)-yl)methyl)benzoate(5g). White

solid

7.98 (d, J ¼ 8.4 Hz, 2H),

.70 (d, J ¼ 7.7 Hz, 1H), 7.57 (t, J ¼ 7.8 Hz, 1H), 7.50 (d, J ¼ 8.4 Hz, 2H),

1

þ

(

7

0.26 g, 55.3 %). H NMR (400 MHz, CDCl

3

)

d

1.74e1.62 (m, 1H), 1.39 (s, 9H). MS (ESI) m/z: 574.38 [Mþ H] .

.42 (t, J ¼ 7.7 Hz, 1H), 7.14 (d, J ¼ 7.9 Hz, 1H), 6.04 (s, 1H), 5.50 (s,

4.1.3.6. methyl (R)-4-((4-(3-((tert-butoxycarbonyl)amino)piperidin-

1-yl)-3-(2-cyanobenzyl)-2,6-dioxo-3,6-dihydropyrimidin-1(2H)-yl)

þ

2

H), 5.17 (s, 2H), 3.90 (s, 1H); MS (ESI) m/z: 410.16 [MþH] .

1

methyl)benzoate (6g). White solid (0.22 g, 88.1 %). H NMR

4

6

.1.3. General procedure for the synthesis of compounds 6a-b and

d-g

To a solution of compound 5a-g (1 equiv.) and (R)-3-(N-Boc-

(400 MHz, CDCl

3

)

d

7.91 (d, J ¼ 8.3 Hz, 2H), 7.66 (d, J ¼ 7.7 Hz, 1H),

7.50 (t, J ¼ 7.7 Hz, 1H), 7.39 (d, J ¼ 8.3 Hz, 2H), 7.35 (t, J ¼ 7.7 Hz, 1H),

7.12 (d, J ¼ 7.9 Hz, 1H), 5.37 (s, 1H), 5.31e5.19 (m, 2H), 5.09 (d,

J ¼ 1.9 Hz, 2H), 4.55e4.41 (m, 1H), 3.89 (s, 3H), 3.78e3.66 (m, 1H),

3.32e3.17 (m, 1H), 3.01e2.88 (m, 1H), 2.82e2.67 (m, 1H), 2.61e2.47

(m, 1H), 1.96e1.86 (m, 1H), 1.84e1.75 (m, 1H), 1.75e1.64 (m, 1H),

2 3

amino)piperidine (1.25 equiv.) in DMF was added K CO (2 equiv.).

ꢀ

The reaction was heated to 60 C and stirred for 12 h. Water was

added to the cooled mixture, and extracted three times with

dichloromethane (3 ꢃ 30 mL). The combined organic layer was

þ

1.39 (s, 9H). MS (ESI) m/z: 574.39 [Mþ H] .

11

Q. Li, X. Deng, N. Jiang et al.

European Journal of Medicinal Chemistry 225 (2021) 113765

4

7

.1.4. General procedure for the synthesis of compounds 7a-b and

d-g

Freshly prepared HCl gas was bubbled into the solution of the

d

8.20 (s, 3H), 7.84e7.78 (m, 2H), 7.77 (s, 1H), 7.67e7.59 (m,1H), 7.46

(td, J ¼ 7.7, 1.1 Hz, 1H), 7.42e7.34 (m, 2H), 7.23 (d, J ¼ 7.9 Hz, 1H),

5.46 (s, 1H), 5.25 (d, J ¼ 15.6 Hz, 1H), 5.11 (d, J ¼ 15.5 Hz, 1H),

4.99e4.86 (m, 2H), 3.92e3.81 (m, 3H), 3.43e3.34 (m, 2H), 2.98 (s,

1H), 2.93e2.73 (m, 2H), 2.02e1.89 (m, 1H), 1.85 (d, J ¼ 12.5 Hz, 1H),

compounds 6a-b and 6d-g (1 equiv.) in EtOAc (20 mL) and ether

ꢀ

(

20 mL) at 0 C until the completed consumption of starting ma-

13

terials, the precipitate was ﬁltrated, and dried in vaccuo to give

compounds 7a-b and 7d-g.

6

1.56 (s, 2H). C NMR (126 MHz, DMSO‑d ) d 166.49, 162.31, 160.20,

152.00, 141.71, 138.25, 133.89, 133.44, 132.75, 130.10, 129.20, 128.54,

1

28.40, 127.96, 117.88, 110.80, 89.79, 52.90, 52.66, 51.66, 46.77,

þ

4

.1.4.1. (R)-6-(3-aminopiperidin-1-yl)-3-benzyl-1-(but-2-yn-1-yl)

46.44, 43.59, 27.41. MS (ESI) m/z: 474.51[Mþ1] .

pyrimidine-2,4(1H,3H)-dione hydrochloride (7a). White solid

ꢀ

1

(

360 mg, yield 83.8 %); HPLC purity: 96.4 %; mp: 204.6e206.5 C; H

4.1.4.6. methyl (R)-4-((4-(3-aminopiperidin-1-yl)-3-(2-

cyanobenzyl)-2,6-dioxo-3,6-dihydropyrimidin-1(2H)-yl)methyl)ben-

zoate hydrochloride (7g). White solid (176 mg, yield 65.0 %). HPLC

NMR (500 MHz, DMSO‑d 8.45 (s, 3H), 7.40e7.08 (m, 5H), 5.27 (s,

6

) d

1

3

2

H), 4.95 (s, 2H), 4.65 (d, J ¼ 17.4 Hz, 1H), 4.52e4.40 (m, 1H),

ꢀ

1

.48e3.40 (m, 1H), 3.37e3.34 (m, 1H), 3.09 (s, 1H), 2.98 (s, 1H),

.94e2.86 (m, 1H), 2.02e1.93 (m, 1H), 1.93e1.85 (m, 1H), 1.78 (t,

purity: 95.3%; mp:169.0e173.0 C H NMR (500 MHz, DMSO‑d )

6

d

8.19 (d, J ¼ 5.0 Hz, 3H), 7.86e7.79 (m, 3H), 7.68 (td, J ¼ 7.7, 1.4 Hz,

13

J ¼ 2.3 Hz, 3H), 1.70e1.60 (m, 2H). C NMR (126 MHz, DMSO‑d

6

)

1H), 7.53e7.43 (m, 1H), 7.26 (d, J ¼ 7.9 Hz, 1H), 7.21 (d, J ¼ 8.0 Hz,

2H), 5.47 (s, 1H), 5.26 (d, J ¼ 15.6 Hz, 1H), 5.12 (d, J ¼ 15.5 Hz, 1H),

4.95 (s, 2H), 3.84 (s, 3H), 3.43e3.36 (m, 2H), 3.00 (s, 1H), 2.88 (s,

d

162.23, 159.44, 152.07, 137.72, 128.77, 128.05, 127.58, 88.87, 80.20,

7

5.03, 52.61, 51.82, 46.48, 43.94, 36.04, 27.45, 21.80, 3.63; MS (ESI)

þ

13

m/z: 353.03[Mþ1] .

2H), 1.97 (m, J ¼ 20.7 Hz, 1H), 1.85 (s, 1H), 1.57 (s, 2H). C NMR

6

(126 MHz, DMSO‑d ) d 166.43, 162.28, 160.21, 152.02, 143.01, 141.73,

4

.1.4.2. (R)-2-((6-(3-aminopiperidin-1-yl)-3-benzyl-2,4-dioxo-3,4-

133.94, 133.45, 129.58, 128.80, 128.44, 128.07, 127.64, 117.89, 110.90,

dihydropyrimidin-1(2H)-yl)methyl) benzonitrile hydrochloride hy-

89.69, 52.92, 52.58, 51.63, 46.85, 46.44, 43.70, 27.47. MS (ESI) m/z:

474.44[Mþ1] .

þ

drate (7b). White solid (154 mg, yield 59.0 %). HPLC purity: 97.7 %;

ꢀ

1

mp: 128.4e134.6 C; H NMR (500 MHz, DMSO‑d

6

) d 8.55e8.28 (m,

3

H), 7.83 (dd, J ¼ 7.8, 1.3 Hz, 1H), 7.66 (td, J ¼ 7.7, 1.4 Hz, 1H), 7.47 (td,

4.1.5. General procedure for the synthesis of compounds 8d-g

To the solution of compound 6d-g (1 equiv.) in methanol was

added LiOH (1 M, 2 equiv.). The solution was stirred at room tem-

perature until complete consumption of 6d-g. The methanol was

removed under reduced pressure, and residue was dissolved in

water, and acidiﬁed with HCl (1 M) to give products 8d-g.

J ¼ 7.7,1.0 Hz,1H), 7.28e7.16 (m, 4H), 7.12e7.00 (m, 2H), 5.43 (s, 1H),

5

3

.27 (d, J ¼ 15.5 Hz, 1H), 5.11 (d, J ¼ 15.4 Hz, 1H), 4.92e4.82 (m, 2H),

.45e3.36 (m, 3H), 3.09e2.85 (m, 2H), 2.02e1.93 (s, 1H), 1.90e1.79

1

3

(

s, 1H), 1.70e1.45 (m, 2H); C NMR (126 MHz, DMSO‑d

6

) d 162.31,

160.08, 152.03, 141.77, 137.47, 133.92, 133.43, 128.65, 128.45, 128.09,

1

3

27.54, 127.44, 117.91, 110.95, 89.76, 52.91, 51.67, 46.74, 46.45, 43.77,

4.53, 27.45; MS (ESI) m/z: 416.43[Mþ1] .

þ

4.1.5.1. (R)-4-((3-(but-2-yn-1-yl)-4-(3-((tert-butoxycarbonyl)amino)

piperidin-1-yl)-2,6-dioxo-3,6-dihydropyrimidin-1(2H)-yl)methyl)

1

4

2

.1.4.3. methyl (R)-4-((4-(3-aminopiperidin-1-yl)-3-(but-2-yn-1-yl)-

benzoic acid (8d). Light yellow solid (0.12 g, 52.0 %). H NMR

,6-dioxo-3,6-dihydropyrimidin-1(2H)-yl)methyl)benzoate

hydro-

(400 MHz, DMSO‑d

6

)

d

7.85 (d, J ¼ 8.3 Hz, 1H), 7.33 (d, J ¼ 8.2 Hz,

chloride (7d). White solid (138 mg, yield 75.5 %). HPLC purity:

9

d

1H), 6.96 (d, J ¼ 7.7 Hz, 1H), 5.22 (s, 1H), 4.98 (s, 2H), 4.63e4.33 (m,

2H), 3.60e3.49 (m, 1H), 3.28e3.20 (m, 1H), 3.19e3.11 (m, 1H),

2.79e2.65 (m, 1H), 2.55e2.51 (m, 1H), 1.81e1.76 (m, 1H), 1.73 (s,

ꢀ

1

5.5 %; mp: 155.0e157.2 C; H NMR (500 MHz, Chloroform-d)

8.65 (s, 3H), 7.96 (d, J ¼ 7.8 Hz, 2H), 7.49 (d, J ¼ 7.9 Hz, 2H), 5.36 (s,

H), 5.11 (s, 2H), 4.58 (d, J ¼ 16.6 Hz, 1H), 4.46 (d, J ¼ 17.0 Hz, 1H),

.90 (s, 3H), 3.67e3.45 (m, 2H), 3.45e3.34 (m, 1H), 3.23e3.06 (m,

H), 3.01e2.83 (m, 1H), 2.57e2.34 (m, 1H), 2.29e2.12 (m, 1H),

.02e1.88 (m, 1H), 1.82 (s, 3H), 1.74e1.62 (m, 1H); ¹C NMR

þ

1

3

1

2

3H), 1.64e1.50 (m, 2H), 1.36 (s, 9H). MS (ESI) m/z: 497.41 [MþH] .

4.1.5.2. (R)-2-((4-(3-((tert-butoxycarbonyl)amino)piperidin-1-yl)-3-

(2-cyanobenzyl)-2,6-dioxo-3,6-dihydropyrimidin-1(2H)-yl)methyl)

benzoic acid (8e). White solid (0.11 g, 47.6 %). H NMR (400 MHz,

3

1

(

126 MHz, DMSO‑d

6

)

d

166.46, 162.19, 159.57, 152.06, 143.22, 129.72,

1

28.90, 128.07, 88.75, 80.25, 74.96, 52.58, 51.82, 46.46, 43.87, 36.16,

DMSO‑d

6

)

d

7.85 (d, J ¼ 7.6 Hz, 1H), 7.78 (d, J ¼ 7.8 Hz, 1H), 7.66 (t,

þ

27.45, 21.78, 3.63. MS (ESI) m/z: 411.29 [MþH] .

J ¼ 7.8 Hz, 1H), 7.43 (t, J ¼ 7.6 Hz, 1H), 7.34 (t, J ¼ 7.5 Hz, 1H),

7

.32e7.26 (m, 2H), 6.90 (d, J ¼ 7.5 Hz, 1H), 6.75 (d, J ¼ 7.8 Hz, 1H),

4

.1.4.4. methyl (R)-2-((4-(3-aminopiperidin-1-yl)-3-(2-

5.39 (s, 1H), 5.22 (s, 2H), 5.15 (s, 2H), 3.52e3.40 (m, 1H), 3.23e3.13

(m, 1H), 3.11e3.00 (m, 1H), 2.80e2.63 (m, 1H), 2.57e2.51 (m, 1H),

1.82e1.69 (m, 2H), 1.59e1.45 (m, 2H), 1.34 (s, 9H). MS (ESI) m/z:

cyanobenzyl)-2,6-dioxo-3,6-dihydropyrimidin-1(2H)-yl)methyl)ben-

zoate hydrochloride (7e). White solid (198 mg, yield 73.6 %). HPLC

purity: 95.0%; mp:172.0e175.0 C; H NMR (500 MHz, DMSO‑d

d

ꢀ

1

þ

6

)

560.43 [MþH] .

8.22 (s, 3H), 7.89e7.84 (m, 1H), 7.82 (d, J ¼ 7.2 Hz, 1H), 7.69 (t,

J ¼ 7.7 Hz,1H), 7.47 (t, J ¼ 7.6 Hz,1H), 7.39 (t, J ¼ 6.9 Hz,1H), 7.33 (dd,

J ¼ 15.8, 7.7 Hz, 2H), 6.75 (d, J ¼ 7.6 Hz, 1H), 5.48 (s, 1H), 5.27 (d,

J ¼ 15.4 Hz,1H), 5.21 (d, J ¼ 2.8 Hz, 2H), 5.12 (d, J ¼ 15.5 Hz,1H), 3.85

4.1.5.3. (R)-3-((4-(3-((tert-butoxycarbonyl)amino)piperidin-1-yl)-3-

(2-cyanobenzyl)-2,6-dioxo-3,6-dihydropyrimidin-1(2H)-yl)methyl)

benzoic acid (8f). White solid (0.23 g, 42.7 %). H NMR (400 MHz,

1

(

s, 3H), 3.43e3.37 (m, 2H), 3.03 (s, 1H), 2.90 (s, 2H), 1.98 (d,

DMSO‑d

6

)

d

7.83e7.72 (m, 3H), 7.60 (t, J ¼ 7.7 Hz, 1H), 7.42 (t,

13

J ¼ 12.0 Hz, 1H), 1.86 (s, 1H), 1.59 (s, 2H). C NMR (126 MHz,

DMSO‑d 167.29, 162.41, 160.31, 152.01, 141.77, 138.33, 133.93,

33.41, 132.86, 130.82, 128.53, 128.46, 128.19, 127.32, 125.52, 117.91,

J ¼ 7.7 Hz, 1H), 7.38e7.32 (m, 2H), 7.19 (d, J ¼ 7.9 Hz, 1H), 6.86 (d,

J ¼ 7.6 Hz, 1H), 5.37 (s, 1H), 5.14 (s, 2H), 4.92 (s, 2H), 3.47e3.38 (m,

1H), 3.17e3.07 (m, 1H), 3.05e2.95 (m, 1H), 2.74e2.62 (m, 1H),

2.54e2.50 (m, 1H), 1.80e1.66 (m, 2H), 1.56e1.42 (m, 2H), 1.32 (s,

6

) d

1

110.95, 89.62, 52.95, 52.64, 51.63, 46.88, 46.45, 42.77, 27.48, 14.56;

þ

MS (ESI) m/z: 474.44[Mþ1] .

9H). MS (ESI) m/z: 560.41 [MþH] .

4

.1.4.5. methyl (R)-3-((4-(3-aminopiperidin-1-yl)-3-(2-

4.1.5.4. (R)-4-((4-(3-((tert-butoxycarbonyl)amino)piperidin-1-yl)-3-

(2-cyanobenzyl)-2,6-dioxo-3,6-dihydropyrimidin-1(2H)-yl)methyl)

benzoic acid (8g). White solid (0.14 g, 48.70 %). H NMR (400 MHz,

cyanobenzyl)-2,6-dioxo-3,6-dihydropyrimidin-1(2H)-yl)methyl)ben-

zoate hydrochloride(7f). White solid (126 mg, yield 71.4 %). HPLC

purity: 95.5%; mp:158.0e160.0 C; H NMR (500 MHz, DMSO‑d

1

ꢀ

1

6

)

DMSO‑d

6

)

d

12.85 (s, 1H), 7.83e7.75 (m, 3H), 7.64 (t, J ¼ 7.7 Hz, 1H),

12

Q. Li, X. Deng, N. Jiang et al.

European Journal of Medicinal Chemistry 225 (2021) 113765

7

6

.43 (t, J ¼ 7.6 Hz, 1H), 7.23 (d, J ¼ 8.0 Hz, 1H), 7.19 (d, J ¼ 8.1 Hz, 2H),

4.1.7. General procedure for preparation of compound 10a-d

To a mixture of compound 9 (1 equiv.), amino acid ester hy-

drochloride (1.1 equiv.), EDCI (2.1 equiv.) and HOBt (2.1 equiv.) in

dry acetonitrile was added DIPEA (3 equiv.). The mixture was stir-

red at r.t. until the starting material was absent. Water (10 mL) was

added. The organic layer was washed successively with 0.5 M of

.88 (d, J ¼ 7.5 Hz, 1H), 5.37 (s, 1H), 5.14 (s, 2H), 4.93 (s, 2H),

3

.51e3.38 (m, 1H), 3.22e3.10 (m, 1H), 3.08e2.96 (m, 1H), 2.76e2.61

(

m, 1H), 2.58e2.51 (m, 1H), 1.84e1.65 (m, 2H), 1.61e1.43 (m, 2H),

þ

1.33 (s, 9H). MS (ESI) m/z: 560.49 [MþH] .

NaOH, 0.5 M of HCl and brine, dried over anhydrous Na

concentrated to give crude product. The crude product was puriﬁed

by ﬂash chromatography (dichloromethane/methanol,

2

SO4,

4

.1.6. General procedure for the synthesis of compounds 9d-g

Following a similar procedure for the preparation of 7a-g,

compounds 9d-g was prepared from 8d-g.

.1.6.1. (R)-4-((4-(3-aminopiperidin-1-yl)-3-(but-2-yn-1-yl)-2,6-

100:1e30:1) to afford amide 10a-d.

4

4.1.7.1. methyl

(R)-(3-((3-(but-2-yn-1-yl)-4-(3-((tert-butox-

dioxo-3,6-dihydropyrimidin-1(2H)-yl)methyl)benzoic acid hydro-

chloride (9d). White solid (201 mg, yield 73.6 %). HPLC purity:

9

ycarbonyl)amino)piperidin-1-yl)-2,6-dioxo-3,6-dihydropyrimidin-

ꢀ

1

1(2H)-yl)methyl)benzoyl)glycinate (10a). Yellow solid (278 mg,

8.3 %; mp: 204.6e208.8 C; H NMR (600 MHz, DMSO‑d ) d 12.92

6

1

yield 51.7 %). H NMR (400 MHz, DMSO‑d

7

(

6

)

d

8.94 (t, J ¼ 5.9 Hz, 1H),

(

s, 1H), 8.40 (s, 3H), 7.89 (d, J ¼ 8.3 Hz, 2H), 7.36 (d, J ¼ 8.3 Hz, 2H),

.78e7.63 (m, 2H), 7.50e7.33 (m, 2H), 6.96 (d, J ¼ 7.8 Hz, 1H), 5.22

5

1

2

.29 (s, 1H), 5.01 (s, 2H), 4.65 (d, J ¼ 17.3 Hz, 1H), 4.47 (d, J ¼ 17.4 Hz,

s, 1H), 4.97 (s, 2H), 4.54e4.34 (m, 2H), 3.97 (d, J ¼ 5.8 Hz, 2H), 3.63

H), 3.36e3.33 (m, 2H), 3.16e3.06 (m, 1H), 3.06e2.95 (m, 1H),

s, 3H), 3.58e3.48 (m, 1H), 3.27e3.20 (m, 1H), 3.19e3.11 (m, 1H),

.94e2.85 (m, 1H), 2.03e1.87 (m, 2H), 1.79 (t, J ¼ 2.4 Hz, 3H),

_.₇₀_e₁_.₅₈₍_m_,₂_H₎_;1 C NMR (151 MHz, DMSO‑d

3

2.77e2.69 (m, 1H), 2.56e2.50 (m, 1H), 1.81e1.74 (m, 2H), 1.73 (s,

1

6

)

d

167.52, 162.20,

þ

3

H), 1.64e1.47 (m, 2H), 1.36 (s, 9H). MS (ESI) m/z: 568.42[Mþ1] .

159.55, 152.06, 142.72, 130.07, 129.86, 127.91, 88.76, 80.25, 74.98,

5

3

2.57, 51.83, 46.45, 43.86, 36.14, 27.45, 21.75, 3.62; MS (ESI) m/z:

97.31 [MþH] .

þ

4.1.7.2. methyl

ycarbonyl)amino)piperidin-1-yl)-2,6-dioxo-3,6-dihydropyrimidin-

(2H)-yl)methyl)benzoyl)-L-prolinate (10b). Yellow solid (152 mg,

(3-((3-(but-2-yn-1-yl)-4-((R)-3-((tert-butox-

1

4

.1.6.2. (R)-2-((4-(3-aminopiperidin-1-yl)-3-(2-cyanobenzyl)-2,6-

1

yield 49.1 %). H NMR (400 MHz, DMSO‑d

(

6

)

d

7.45e7.13 (m, 4H), 6.95

d, J ¼ 7.8 Hz, 1H), 5.22 (s, 1H), 4.96 (s, 2H), 4.53e4.28 (m, 3H), 3.64

s, 3H), 3.58e3.41 (m, 3H), 3.27e3.19 (m, 1H), 3.19e3.09 (m, 1H),

.75e2.68 (m, 1H), 2.54e2.50 (m, 1H), 2.32e2.18 (m, 2H), 1.92e1.74

dioxo-3,6-dihydropyrimidin-1(2H)-yl)methyl)benzoic acid hydro-

chloride (9e). White solid (231 mg, yield 70.1 %). HPLC purity:

ꢀ

1

9

(

7

6

5.3 %; mp: 236.0e238.0 C; H NMR (500 MHz, DMSO‑d

s, 1H), 8.23 (s, 3H), 7.88 (dd, J ¼ 7.6, 1.5 Hz, 1H), 7.84e7.80 (m, 1H),

.70 (td, J ¼ 7.8,1.2 Hz,1H), 7.47 (t, J ¼ 7.4 Hz,1H), 7.39e7.28 (m, 3H),

.68 (d, J ¼ 7.5 Hz, 1H), 5.48 (s, 1H), 5.34e5.17 (m, 3H), 5.12 (d,

J ¼ 15.5 Hz, 1H), 3.39 (m, J ¼ 7.0 Hz, 2H), 3.04 (s, 1H), 2.92 (s, 2H),

.98 (m, J ¼ 10.4 Hz,1H),1.89 (m, J ¼ 26.0 Hz,1H), 1.70e1.47 (m, 2H).

6

) d 13.06

2

(

6

m, 4H), 1.73 (s, 3H), 1.65e1.48 (m, 2H), 1.36 (s, 9H). MS (ESI) m/z:

30.51[MþNa] .

þ

4.1.7.3. methyl

(3-((3-(but-2-yn-1-yl)-4-((R)-3-((tert-butox-

1

ycarbonyl)amino)piperidin-1-yl)-2,6-dioxo-3,6-dihydropyrimidin-

(2H)-yl)methyl)benzoyl)-D-prolinate (10c). Yellow solid (168 mg,

1

3

C NMR (126 MHz, DMSO‑d

41.81, 138.42, 133.93, 133.42, 132.48, 131.09, 129.40, 128.46, 128.18,

27.11, 125.11, 117.91, 110.92, 89.63, 52.94, 51.64, 46.87, 46.46, 43.04,

6

) d 168.57, 162.45, 160.28, 152.04,

1

6

yield 54.9 %). H NMR (400 MHz, DMSO‑d ) d 7.42e7.16 (m, 4H),

6

3

1

.95 (d, J ¼ 7.8 Hz, 1H), 5.22 (s, 1H), 4.96 (s, 2H), 4.54e4.29 (m, 3H),

þ

15.65, 14.56; MS (ESI) m/z: 460.44[Mþ1] .

.64 (s, 3H), 3.59e3.41 (m, 3H), 3.28e3.19 (m, 1H), 3.19e3.09 (m,

H), 2.76e2.68 (m, 1H), 2.55e2.50 (m, 1H), 2.32e2.18 (m, 2H),

4

.1.6.3. (R)-3-((4-(3-aminopiperidin-1-yl)-3-(2-cyanobenzyl)-2,6-

dioxo-3,6-dihydropyrimidin-1(2H)-yl)methyl)benzoic acid hydro-

chloride (9f). White solid (136 mg, yield 68.6 %). HPLC purity:

1

.93e1.75 (m, 4H), 1.73 (s, 3H), 1.63e1.50 (m, 2H), 1.36 (s, 9H). MS

þ

(ESI) m/z: 630.50[MþNa] .

ꢀ

1

97.7 %; mp: 211.8e215.9 C; H NMR (500 MHz, DMSO‑d ) d 12.95

6

4

.1.7.4. methyl

ycarbonyl)amino)piperidin-1-yl)-2,6-dioxo-3,6-dihydropyrimidin-

(2H)-yl)methyl)benzoyl)-L-alaninate (10d). Yellow solid (213 mg,

(3-((3-(but-2-yn-1-yl)-4-((R)-3-((tert-butox-

(

s, 1H), 8.18 (s, 3H), 7.86e7.77 (m, 2H), 7.74 (s, 1H), 7.64 (t, J ¼ 7.7 Hz,

1

5

4

H), 7.46 (t, J ¼ 7.5 Hz,1H), 7.41e7.32 (m, 2H), 7.23 (d, J ¼ 7.9 Hz,1H),

.47 (s, 1H), 5.26 (d, J ¼ 15.5 Hz, 1H), 5.12 (d, J ¼ 15.6 Hz, 1H),

.99e4.88 (m, 2H), 3.43e3.36 (m, 2H), 2.98 (s, 1H), 2.87 (s, 2H),

1

yield 46.3 %). H NMR (400 MHz, DMSO‑d

6

)

d

8.79 (d, J ¼ 6.9 Hz,1H),

7.81e7.70 (m, 2H), 7.42e7.36 (m, 2H), 6.96 (d, J ¼ 7.7 Hz,1H), 5.22 (s,

1

3

1

.98e1.89 (m, 1H), 1.84 (s, 1H), 1.64e1.48 (m, 2H). ¹³C NMR

H), 4.97 (s, 2H), 4.54e4.37 (m, 3H), 3.62 (s, 3H), 3.57e3.47 (m, 1H),

.27e3.19 (m, 1H), 3.19e3.09 (m, 1H), 2.76e2.69 (m, 1H), 2.55e2.50

(

126 MHz, DMSO‑d

6

) d 167.54, 162.31, 160.18, 152.02, 141.71, 138.01,

133.93, 133.47, 132.26, 131.27, 128.99, 128.57, 128.52, 128.40, 127.86,

(

9

m, 1H), 1.83e1.75 (m, 2H), 1.74 (s, 3H), 1.63e1.50 (m, 2H), 1.36 (s,

H). MS (ESI) m/z: 604.47[MþNa] .

117.88, 110.79, 89.82, 52.92, 51.67, 46.73, 46.45, 43.64, 15.64, 14.56;

þ

MS (ESI) m/z: 460.37[Mþ1] .

4.1.8. General procedure for preparation of compounds 11a-d and

4

.1.6.4. (R)-4-((4-(3-aminopiperidin-1-yl)-3-(2-cyanobenzyl)-2,6-

12a-d

Compounds 11a-d were prepared from compounds 10a-d by

dioxo-3,6-dihydropyrimidin-1(2H)-yl)methyl)benzoic acid hydro-

chloride (9g). White solid (159 mg, yield 64.7 %). HPLC purity:

using similar procedure for the preparation of 7a-d. Compounds

10a-d was hydrolyzed with LiOH, and deprotected with HCl by

using a similar procedure for the preparation of 9d-g to give

compounds 12a-d.

ꢀ

1

9

4.7 %; mp:198.6e203.8 C; H NMR (500 MHz, DMSO‑d ) d 12.85

6

(

s, 1H), 8.20 (s, 3H), 7.85e7.82 (m, 1H), 7.80 (d, J ¼ 8.3 Hz, 2H), 7.68

(

7

td, J ¼ 7.8, 1.1 Hz, 1H), 7.48 (t, J ¼ 7.6 Hz, 1H), 7.27 (d, J ¼ 7.9 Hz, 1H),

.18 (d, J ¼ 8.2 Hz, 2H), 5.46 (s, 1H), 5.26 (d, J ¼ 15.4 Hz, 1H),

5

3

1

.16e5.05 (m, 1H), 4.98e4.89 (m, 2H), 3.42e3.35 (m, 2H),

.07e2.95 (m, 1H), 2.88 (s, 2H), 1.99e1.89 (m, 1H), 1.85 (s, 1H),

4.1.8.1. methyl (R)-(3-((4-(3-aminopiperidin-1-yl)-3-(but-2-yn-1-

yl)-2,6-dioxo-3,6-dihydropyrimidin-1(2H)-yl)methyl)benzoyl)glyci-

nate hydrochloride (11a). White solid (0.55 g, yield 65.2%). HPLC

13

.66e1.48 (m, 2H). C NMR (126 MHz, DMSO‑d

6

) d 167.48, 162.28,

ꢀ

1

160.19, 152.02, 142.50, 141.74, 133.93, 133.44, 129.97, 129.79, 129.72,

purity: 96.3 %; mp: 190.0e192.0 C; H NMR (500 MHz, DMSO‑d

9.01 (t, J ¼ 5.9 Hz, 1H), 8.21 (s, 3H), 7.80e7.69 (m, 2H), 7.49e7.38

(m, 2H), 5.31 (s, 1H), 5.06e4.95 (m, 2H), 4.69e4.58 (m, 1H),

6

)

1

4

28.45, 128.10, 127.94, 127.45, 117.90, 110.93, 89.67, 52.90, 51.65,

6.84, 46.44, 43.69, 27.47, 25.60; MS (ESI) m/z: 460.23[Mþ1] .

d

þ

13

Q. Li, X. Deng, N. Jiang et al.

European Journal of Medicinal Chemistry 225 (2021) 113765

ꢀ

1

4

.52e4.42 (m, 1H), 4.00 (d, J ¼ 5.8 Hz, 2H), 3.93e3.87 (m, 1H), 3.66

95.5 %; mp: 219.2e221.7 C; H NMR (500 MHz, DMSO‑d ) d 12.54

6

(

s, 3H), 3.34e3.27 (m, 1H), 3.16e2.98 (m, 2H), 2.93e2.85 (m, 1H),

(s, 1H), 8.20 (s, 3H), 7.44e7.43 (m, 1H), 7.41e7.38 (m, 2H), 7.34e7.24

(m, 1H), 5.29 (s, 1H), 5.03e4.99 (m, 2H), 4.65e4.62 (m, 1H),

4.48e4.45 (m, 1H), 4.40e4.30 (m, 1H), 3.58e3.35 (m, 2H),

3.31e3.29 (m, 2H), 3.10e2.98 (m, 2H), 2.94e2.85 (m,1H), 2.30e2.24

(m, 1H), 1.99e1.81 (m, 5H), 1.78 (s, 3H), 1.66e1.60 (m, 2H); C NMR

(126 MHz, DMSO‑d 173.70, 168.37, 162.22, 159.51, 152.04, 137.87,

36.71, 129.97, 128.76, 127.04, 126.42, 88.85, 80.26, 74.95, 52.58,

13

2

.02e1.86 (m, 2H), 1.78 (t, J ¼ 2.4 Hz, 3H), 1.69e1.57 (m, 2H);

170.83, 166.92, 162.22, 159.51, 152.07,

38.11, 134.13, 131.21, 128.84, 127.13, 126.39, 88.84, 80.27, 74.97,

C

6

NMR (126 MHz, DMSO‑d ) d

1

5

13

2.58, 52.20, 51.84, 43.92, 41.71, 36.10, 27.42, 21.72, 15.64, 3.61; MS

þ

(

ESI) m/z: 468.35 [MþH] .

6

) d

1

4

.1.8.2. methyl(3-((4-((R)-3-aminopiperidin-1-yl)-3-(but-2-yn-1-

yl)-2,6-dioxo-3,6-dihydropyrimidin-1(2H)-yl)methyl)benzoyl)- -pro-

linate hydrochloride (11b). White solid (131 mg, yield 65.4 %). HPLC

51.82, 49.98, 46.46, 43.83, 36.08, 29.41, 27.42, 25.45, 21.74, 15.64,

3.61; MS (ESI) m/z: 494.28 [MþH] .

þ

L

ꢀ

1

purity: 95.2%; mp:181.0e183.0 C; H NMR (500 MHz, DMSO‑d

d

6

)

4

.1.8.7. (3-((4-((R)-3-aminopiperidin-1-yl)-3-(but-2-yn-1-yl)-2,6-

dioxo-3,6-dihydropyrimidin-1(2H)-yl)methyl)benzoyl)- -proline hy-

drochloride (12c). White solid (231 mg, yield 74.2 %); HPLC purity:

8.25 (s, 3H), 7.45 (s,1H), 7.44e7.39 (m, 2H), 7.36e7.20 (m,1H), 5.30

D

(

2

s, 1H), 4.99 (s, 2H), 4.64 (d, J ¼ 17.4 Hz, 1H), 4.53e4.33 (m, 2H), 3.67

s, 3H), 3.60e3.46 (m, 2H), 3.30 (d, J ¼ 11.6 Hz, 1H), 3.17e2.94 (m,

ꢀ

1

9

5.3 %; mp: 222.0e223.6 C; H NMR (500 MHz, DMSO‑d

6

) d 12.55

H), 2.93e2.82 (m, 1H), 2.33e2.22 (m, 1H), 2.02e1.83 (m, 5H), 1.78

(s, 1H), 8.23 (s, 3H), 7.44 (s, 1H), 7.43e7.38 (m, 2H), 7.35e7.22 (m,

13

(

1

s, 3H), 1.71e1.57 (m, 2H); C NMR (126 MHz, DMSO‑d

68.46, 162.22, 159.51, 152.04, 137.93, 136.41, 130.11, 128.80, 127.09,

26.43, 88.85, 80.25, 74.95, 52.59, 52.32, 51.82, 49.99, 46.45, 43.82,

6

) d 172.80,

1

4

3

H), 5.30 (s, 1H), 4.99 (s, 2H), 4.69e4.59 (m, 1H), 4.51e4.43 (m, 1H),

.40e4.28 (m, 1H), 3.59e3.42 (m, 2H), 3.41e3.38 (m, 2H),

.33e3.25 (m, 1H), 3.13e2.97 (m, 1H), 2.91e2.83 (m, 1H), 2.31e2.22

3

6.08, 34.60, 29.33, 27.42, 25.47, 21.75, 3.60. MS (ESI) m/z: 508.31

13

(m, 1H), 2.02e1.81 (m, 5H), 1.78 (s, 3H), 1.70e1.54 (m, 2H); C NMR

þ

[MþH] .

(126 MHz, DMSO‑d

6

) d 173.70, 168.37, 162.22, 159.51, 152.04, 137.87,

1

36.72, 129.97, 128.76, 127.02, 126.42, 88.85, 80.25, 74.95, 52.59,

4

.1.8.3. methyl(3-((4-((R)-3-aminopiperidin-1-yl)-3-(but-2-yn-1-

yl)-2,6-dioxo-3,6-dihydropyrimidin-1(2H)-yl)methyl)benzoyl)- -pro-

linate hydrochloride (11c). White solid (96.4 mg, yield 54.0 %). HPLC

51.82, 49.98, 46.45, 43.83, 36.08, 29.41, 27.42, 25.45, 21.74, 15.64,

D

þ

3.61; MS (ESI) m/z: 494.36 [MþH] .

ꢀ

1

purity: 96.3 %; mp: 181.0e182.6 C; H NMR (500 MHz, DMSO‑d

8.28 (s, 3H), 7.45 (s,1H), 7.44e7.39 (m, 2H), 7.36e7.20 (m,1H), 5.30

s, 1H), 5.06e4.90 (m, 2H), 4.64 (d, J ¼ 17.3 Hz, 1H), 4.53e4.39 (m,

H), 3.67 (s, 3H), 3.53e3.46 (m, 2H), 3.34e3.27 (m, 1H), 3.17e2.94

m, 2H), 2.93e2.82 (m, 1H), 2.33e2.21 (m, 1H), 2.01e1.85 (m, 5H),

6

)

4

.1.8.8. (3-((4-((R)-3-aminopiperidin-1-yl)-3-(but-2-yn-1-yl)-2,6-

d

dioxo-3,6-dihydropyrimidin-1(2H)-yl)methyl)benzoyl)-

drochloride (12d). White solid (175 mg, yield 64.7 %); HPLC purity:

L-alanine hy-

(

2

(

ꢀ

1

97.0%; mp: 219.2e221.7 C; H NMR (500 MHz, DMSO‑d ) d 12.57 (s,

6

13

1H), 8.70 (d, J ¼ 7.2 Hz, 1H), 8.22 (s, 3H), 7.83e7.66 (m, 2H), 7.42 (dd,

1

3

.78 (s, 3H), 1.60 (s, 2H); C NMR (126 MHz, DMSO‑d

68.46, 162.22, 159.51, 152.04, 137.93, 136.41, 130.11, 128.80, 127.07,

26.43, 88.85, 80.25, 74.95, 52.59, 52.32, 51.82, 49.99, 46.45, 43.82,

6.08, 34.60, 29.33, 27.43, 25.47, 21.74, 3.60; MS (ESI) m/z: 508.35

6

) d 172.80,

J ¼ 4.7, 2.0 Hz, 2H), 5.31 (s, 1H), 5.00 (s, 2H), 4.70e4.56 (m, 1H),

4.51e4.43 (m, 1H), 4.43e4.35 (m, 1H), 3.33e3.26 (m, 2H), 3.16e2.95

(

m, 2H), 2.92e2.83 (m, 1H), 2.06e1.83 (m, 2H), 1.78 (t, J ¼ 2.3 Hz,

13

þ

3H), 1.70e1.53 (m, 2H), 1.39 (d, J ¼ 7.4 Hz, 3H); C NMR (126 MHz,

DMSO‑d 174.61, 166.49, 162.22, 159.50, 152.06, 137.95, 134.48,

30.91, 128.67, 127.35, 126.55, 88.85, 80.27, 74.97, 52.59, 51.82,

[MþH] .

6

) d

1

4

.1.8.4. methyl (3-((4-((R)-3-aminopiperidin-1-yl)-3-(but-2-yn-1-

8.65, 46.46, 43.90, 36.09, 27.42, 21.73, 17.32, 3.62; MS (ESI) m/z:

yl)-2,6-dioxo-3,6-dihydropyrimidin-1(2H)-yl)methyl)benzoyl)-L-ala-

ninate hydrochloride (11d). White solid (164 mg, yield 68.4%). HPLC

purity: 96.3 %; mp: 218.0e220.0 C; H NMR (500 MHz, DMSO‑d )

6

e

66.70 [M ꢁ H] .

ꢀ

1

d

8.85 (d, J ¼ 6.9 Hz, 1H), 8.26 (s, 3H), 7.88e7.74 (m, 2H), 7.57e7.38

4

.1.9. General procedure for preparation of 14a-h

A mixture of 13a-h (3.0 mmol), NBS (3.0 mmol), and di-benzoyl

(

2

m, 2H), 5.30 (s,1H), 5.00 (s, 2H), 4.64 (d, J ¼ 16.9 Hz,1H), 4.53e4.32

m, 2H), 3.64 (s, 3H), 3.31 (d, J ¼ 11.2 Hz, 2H), 3.15e2.95 (m, 2H),

peroxide (BPO, 0.15 mmol) in 1,2-dichloroethane (5 mL) was heated

at 80 C for 12 h. The reaction was cooled to room temperature, and

the precipitated solid was removed by ﬁltration and washed with

ether (10 mL). The ﬁltrate was concentrated in vacuo, and the

residue was partitioned between NaHCO

.92e2.83 (m, 1H), 2.07e1.88 (m, 2H), 1.78 (s, 3H), 1.70e1.57 (m,

ꢀ

1

3

H), 1.40 (d, J ¼ 7.3 Hz, 3H); C NMR (126 MHz, DMSO‑d

6

) d 173.61,

1

3

66.59, 162.22, 159.50, 152.07, 138.01, 134.20, 131.04, 128.72, 127.39,

26.58, 88.84, 80.27, 74.96, 52.59, 52.35, 51.82, 48.77, 46.46, 43.90,

3

(2 N, 15 mL) and ether

, ﬁltered

þ

6.10, 27.42, 21.74, 17.17, 3.61; MS (ESI) m/z: 482.66 [MþH] .

(15 mL). The organic layer was separated, dried over MgSO

4

and concentrated to give a crude product 14a-h, which was used in

the next step reaction without further puriﬁcation.

4

.1.8.5. (R)-(3-((4-(3-aminopiperidin-1-yl)-3-(but-2-yn-1-yl)-2,6-

dioxo-3,6-dihydropyrimidin-1(2H)-yl)methyl)benzoyl)glycine hydro-

chloride (12a). White solid (1.1 g, 65.2 %). HPLC purity: 90.7 %; mp:

ꢀ

1

4.1.10. General procedure for preparation of 15a-h

2

10.6e211.5 C; H NMR (500 MHz, DMSO‑d ) d 12.57 (s, 1H), 8.88

6

1

5a-h were prepared according to our previously published

procedures [20]. Compounds 14a-h (1 equiv.), compound 4a (0.65

equiv.) and K CO (2 equiv.) were stirred in DMF at r.t. The mixture

(

t, J ¼ 5.9 Hz,1H), 8.22 (s, 3H), 7.82e7.69 (m, 2H), 7.50e7.35 (m, 2H),

5

1

3

.30 (s, 1H), 5.05e4.94 (m, 2H), 4.73e4.58 (m, 1H), 4.52e4.40 (m,

H), 3.94e3.87 (m, 2H), 3.42e3.38 (m, 1H), 3.34e3.27 (m, 1H),

.19e2.94 (m, 2H), 2.93e2.85 (m, 1H), 2.01e1.85 (m, 2H), 1.78 (t,

2

3

was added to ice water, ﬁltrated, washed with ethyl acetate and

dried to give 15a-h.

13

J ¼ 2.3 Hz, 3H), 1.70e1.58 (m, 2H); C NMR (126 MHz, DMSO‑d

171.73, 166.80, 162.22, 159.50, 152.07, 138.06, 134.39, 131.09,

28.79, 127.10, 126.35, 88.85, 80.27, 74.97, 52.58, 51.84, 46.45, 43.92,

6

)

d

1

4

4.1.10.1. methyl 3-((3-(but-2-yn-1-yl)-4-chloro-2,6-dioxo-3,6-

þ

1.70, 36.09, 27.41, 21.71, 3.62; MS (ESI) m/z: 454.25 [MþH] .

dihydropyrimidin-1(2H)-yl)methyl)-2-ﬂuorobenzoate

(15a).

7.84 (dd,

1

White solid (0.37 g, 34.1%). H NMR (400 MHz, CDCl

3

)

d

4

.1.8.6. (3-((4-((R)-3-aminopiperidin-1-yl)-3-(but-2-yn-1-yl)-2,6-

J ¼ 7.8, 6.8 Hz, 1H), 7.45 (dd, J ¼ 7.7, 6.7 Hz, 1H), 7.13 (t, J ¼ 7.8 Hz,

dioxo-3,6-dihydropyrimidin-1(2H)-yl)methyl)benzoyl)- -proline hy-

drochloride (12b). White solid (196 mg, yield 72.6 %). HPLC purity:

L

1H), 5.99 (s, 1H), 5.21 (s, 2H), 4.77 (q, J ¼ 2.3 Hz, 2H), 3.92 (s, 3H),

þ

1.82 (t, J ¼ 2.4 Hz, 3H). MS (ESI) m/z: 365.14 [MþH] .

14

Q. Li, X. Deng, N. Jiang et al.

European Journal of Medicinal Chemistry 225 (2021) 113765

4

.1.10.2. methyl 3-((3-(but-2-yn-1-yl)-4-chloro-2,6-dioxo-3,6-

4.1.11.2. methyl

(R)-3-((3-(but-2-yn-1-yl)-4-(3-((tert-butox-

dihydropyrimidin-1(2H)-yl)methyl)-4-ﬂuorobenzoate

White solid (0.49 g, 45.2%). H NMR (400 MHz, CDCl

(15b).

8.01e7.88

ycarbonyl)amino)piperidin-1-yl)-2,6-dioxo-3,6-dihydropyrimidin-

1

3

)

d

1(2H)-yl)methyl)-4-ﬂuorobenzoate (16b). White solid (168 mg,

1

(

3

m, 2H), 7.10 (dd, J ¼ 9.6, 8.6 Hz, 1H), 6.01 (s, 1H), 5.21 (s, 2H), 4.77

yield 89.2 %). H NMR (400 MHz, CDCl

3

)

d

7.95e7.9 (m, 2H),

q, J ¼ 2.4 Hz, 2H), 3.88 (s, 3H), 1.82 (t, J ¼ 2.4 Hz, 3H). MS (ESI) m/z:

7.14e7.03 (m, 1H), 5.29 (s, 1H), 5.20 (s, 2H), 4.89e4.73 (m, 1H),

4.74e4.57 (m, 1H), 4.48 (d, J ¼ 16.8 Hz, 1H), 3.87 (s, 3H), 3.85e3.80

þ

65.11 [MþH] .

(m, 1H), 3.40e3.24 (m, 1H), 3.19e3.02 (m, 1H), 2.96e2.83 (m, 1H),

2

.77e2.63 (m, 1H), 1.96e1.84 (m, 2H), 1.81 (t, J ¼ 2.3 Hz, 3H),

4

.1.10.3. methyl 5-((3-(but-2-yn-1-yl)-4-chloro-2,6-dioxo-3,6-

þ

1.78e1.67 (m, 1H), 1.45 (s, 9H). MS (ESI) m/z: 529.32 [MþH] .

dihydropyrimidin-1(2H)-yl)methyl)-2-ﬂuorobenzoate

White solid (0.46 g, 42.4%). H NMR (400 MHz, CDCl

(15c).

1

3

)

d

8.03 (dd,

J ¼ 6.9, 2.4 Hz, 1H), 7.67e7.63 (m, 1H), 7.07 (dd, J ¼ 10.5, 8.5 Hz, 1H),

4.1.11.3. methyl

(R)-5-((3-(but-2-yn-1-yl)-4-(3-((tert-butox-

5

.97 (s, 1H), 5.07 (s, 2H), 4.76 (q, J ¼ 2.3 Hz, 2H), 3.91 (s, 3H), 1.81 (t,

ycarbonyl)amino)piperidin-1-yl)-2,6-dioxo-3,6-dihydropyrimidin-

þ

J ¼ 2.4 Hz, 3H). MS (ESI) m/z: 365.03 [MþH] .

1(2H)-yl)methyl)-2-ﬂuorobenzoate (16c). White solid (350 mg, yield

1

8

3.7 %). H NMR (400 MHz, CDCl

3

)

d

8.04 (dd, J ¼ 7.0, 2.4 Hz, 1H),

7

.68e7.64 (m, 1H), 7.05 (dd, J ¼ 10.6, 8.5 Hz, 1H), 5.24 (s, 1H),

.11e5.00 (m, 2H), 4.82e4.68 (m, 1H), 4.66e4.54 (m, 1H), 4.51e4.40

(m, 1H), 3.90 (s, 3H), 3.86e3.76 (m, 1H), 3.36e3.21 (m, 1H),

.14e3.01 (m, 1H), 2.92e2.76 (m, 1H), 2.72e2.57 (m, 1H), 1.94e1.83

4

3

.1.10.4. methyl 2-bromo-3-((3-(but-2-yn-1-yl)-4-chloro-2,6-dioxo-

5

,6-dihydropyrimidin-1(2H)-yl)methyl)benzoate (15d). White solid

1

(

0.33 g, 35.5%). H NMR (400 MHz, CDCl

3

)

d

7.55 (dd, J ¼ 7.7, 1.6 Hz,

3

1

2

4

H), 7.29 (d, J ¼ 7.7 Hz, 1H), 7.09e6.97 (m, 1H), 6.03 (s, 1H), 5.25 (s,

(

m, 2H), 1.80 (t, J ¼ 2.3 Hz, 3H), 1.77e1.67 (m, 1H), 1.43 (s, 9H). MS

H), 4.79 (q, J ¼ 2.4 Hz, 3H), 1.83 (t, J ¼ 2.4 Hz, 3H). MS (ESI) m/z:

þ

(ESI) m/z: 529.32 [MþH] .

25.09 [MþH] .

4

.1.11.4. methyl

(R)-2-bromo-3-((3-(but-2-yn-1-yl)-4-(3-((tert-

4

3

.1.10.5. methyl 4-bromo-3-((3-(but-2-yn-1-yl)-4-chloro-2,6-dioxo-

butoxycarbonyl)amino)piperidin-1-yl)-2, 6-dioxo-3, 6-

dihydropyrimidin-1(2H)-yl)methyl)benzoate (16d). White solid

,6-dihydropyrimidin-1(2H)-yl)methyl)benzoate (15e). White solid

1

(

0.26 g, 28.0 %). H NMR (400 MHz, CDCl

3

)

d

7.79e7.73 (m, 1H), 7.65

1

(390 mg, yield 93.7 %). H NMR (400 MHz, CDCl

3

)

d

7.53 (dd, J ¼ 7.7,

(

4

d, J ¼ 8.3 Hz, 1H), 7.59 (d, J ¼ 2.0 Hz, 1H), 6.04 (s, 1H), 5.22 (s, 2H),

1

.6 Hz, 1H), 7.29e7.23 (m, 1H), 7.05e6.99 (m, 1H), 5.30 (s, 1H), 5.23

(s, 2H), 4.85e4.72 (m, 1H), 4.70e4.60 (m, 1H), 4.54e4.45 (m, 1H),

.79 (q, J ¼ 2.3 Hz, 3H), 1.83 (t, J ¼ 2.4 Hz, 3H). MS (ESI) m/z: 425.01

þ

[MþH] .

3

1

.92 (s, 3H), 3.90e3.80 (m, 1H), 3.40e3.29 (m, 1H), 3.22e3.09 (m,

H), 2.93e2.80 (m, 1H), 2.79e2.62 (m, 1H), 1.99e1.84 (m, 2H),

4

3

.1.10.6. methyl 3-bromo-5-((3-(but-2-yn-1-yl)-4-chloro-2,6-dioxo-

1.83e1.78 (m, 3H), 1.78e1.69 (m, 1H), 1.45 (s, 9H). MS (ESI) m/z:

,6-dihydropyrimidin-1(2H)-yl)methyl)benzoate (15f). White solid

þ

5

89.65 [MþH] .

1

(

0.41 g, 44.1%). H NMR (400 MHz, CDCl

3

)

d

8.08 (t, J ¼ 1.7 Hz, 1H),

8

2

.02 (t, J ¼ 1.6 Hz, 1H), 7.77 (t, J ¼ 1.8 Hz, 1H), 5.98 (s, 1H), 5.09 (s,

4.1.11.5. methyl

(R)-4-bromo-3-((3-(but-2-yn-1-yl)-4-(3-((tert-

H), 4.76 (q, J ¼ 2.4 Hz, 2H), 3.90 (s, 3H), 1.82 (t, J ¼ 2.4 Hz, 3H). MS

þ

butoxycarbonyl)amino)piperidin-1-yl)-2, 6-dioxo-3, 6-

dihydropyrimidin-1(2H)-yl)methyl)benzoate(16e). White solid

7.74 (dd, J ¼ 8.3,

.1 Hz, 1H), 7.62 (d, J ¼ 8.3 Hz, 1H), 7.58 (d, J ¼ 2.0 Hz, 1H), 5.32 (s,

H), 5.21 (s, 2H), 4.89e4.75 (m, 1H), 4.69e4.64 (m, 1H), 4.57e4.42

(

ESI) m/z: 425.07 [MþH] .

1

3

(210 mg, yield 81.8 %). H NMR (400 MHz, CDCl ) d

4

3

.1.10.7. methyl 2-bromo-5-((3-(but-2-yn-1-yl)-4-chloro-2,6-dioxo-

2

1

,6-dihydropyrimidin-1(2H)-yl)methyl)benzoate (15g). White solid

1

(

0.44 g, 47.4%). H NMR (400 MHz, CDCl

3

)

d

7.88 (d, J ¼ 2.2 Hz, 1H),

(m, 1H), 3.92e3.87 (m, 1H), 3.85 (s, 3H), 3.40e3.28 (m, 1H),

7

.58 (d, J ¼ 8.2 Hz, 1H), 7.45 (dd, J ¼ 8.3, 2.3 Hz,1H), 5.97 (s, 1H), 5.05

3

.20e3.06 (m, 1H), 2.99e2.83 (m, 1H), 2.81e2.63 (m, 1H), 1.97e1.84

(

s, 2H), 4.75 (q, J ¼ 2.3 Hz, 2H), 3.91 (s, 3H), 1.81 (t, J ¼ 2.4 Hz, 3H).

(

m, 2H), 1.81 (t, J ¼ 2.3 Hz, 3H), 1.79e1.71 (m, 1H), 1.45 (s, 9H). MS

þ

MS (ESI) m/z: 424.95 [MþH] .

þ

ESI) m/z: 589.65 [MþH] .

4

.1.10.8. methyl 3-((3-(but-2-yn-1-yl)-4-chloro-2,6-dioxo-3,6-

4

.1.11.6. methyl

butoxycarbonyl)amino)piperidin-1-yl)-2, 6-dioxo-3, 6-

dihydropyrimidin-1(2H)-yl)methyl)benzoate (16f). White solid

470 mg, yield 95.3 %). H NMR (400 MHz, CDCl 8.06e8.04 (m,

(R)-3-bromo-5-((3-(but-2-yn-1-yl)-4-(3-((tert-

dihydropyrimidin-1(2H)-yl)methyl)-4-chlorobenzoate

White solid (0.38 g, 40.1%). H NMR (400 MHz, CDCl

(15

7.89e7.80

h).

1

3

)

d

(

5

3

m, 1H), 7.67 (d, J ¼ 2.0 Hz, 1H), 7.44 (d, J ¼ 8.3 Hz, 1H), 6.04 (s, 1H),

1

(

3

) d

.25 (s, 2H), 4.79 (q, J ¼ 2.3 Hz, 2H), 3.87 (s, 3H), 1.82 (t, J ¼ 2.4 Hz,

2

4

3

H), 7.79 (t, J ¼ 1.8 Hz, 1H), 5.26 (s, 1H), 5.08 (d, J ¼ 3.0 Hz, 2H),

þ

H). MS (ESI) m/z: 381.10 [MþH] .

.86e4.68 (m, 1H), 4.62 (dd, J ¼ 16.9, 2.5 Hz, 1H), 4.53e4.41 (m, 1H),

.89 (s, 3H), 3.87e3.79 (m, 1H), 3.37e3.22 (m, 1H), 3.15e3.01 (m,

4

.1.11. General procedure for preparation of 16a-h and 17a-h

According to our previously published procedures [20], 16a-h

1H), 2.93e2.79 (m, 1H), 2.74e2.58 (m, 1H), 1.97e1.83 (m, 2H),

1.83e1.80 (m, 3H), 1.79e1.68 (m, 1H), 1.44 (s, 9H). MS (ESI) m/z:

589.65 [MþH] .

þ

were prepared from 15a-h, and 17a-h were prepared 16a-h.

4

.1.11.1. methyl

ycarbonyl)amino)piperidin-1-yl)-2,6-dioxo-3,6-dihydropyrimidin-

(2H)-yl)methyl)-2-ﬂuorobenzoate (16a). White solid (260 mg,

(R)-3-((3-(but-2-yn-1-yl)-4-(3-((tert-butox-

4.1.11.7. methyl

(R)-2-bromo-5-((3-(but-2-yn-1-yl)-4-(3-((tert-

butoxycarbonyl)amino)piperidin-1-yl)-2, 6-dioxo-3, 6-

dihydropyrimidin-1(2H)-yl)methyl)benzoate (16g). White solid

(410 mg, yield 92.7 %). H NMR (400 MHz, CDCl

1

yield 86.0 %). H NMR (400 MHz, CDCl

3

)

d

7.78e7.72 (m, 1H),

3

)

d

7.91 (d,

7

4

3

.46e7.38 (m, 1H), 7.10 (t, J ¼ 7.7 Hz, 1H), 5.27 (s, 1H), 5.20 (s, 2H),

J ¼ 2.2 Hz, 1H), 7.56 (d, J ¼ 8.2 Hz, 1H), 7.48 (dd, J ¼ 8.2, 2.2 Hz, 1H),

5.24 (s, 1H), 5.05 (d, J ¼ 3.5 Hz, 2H), 4.80e4.68 (m, 1H), 4.60 (dd,

J ¼ 16.9, 2.5 Hz, 1H), 4.52e4.39 (m, 1H), 3.91 (s, 3H), 3.87e3.78 (m,

1H), 3.34e3.22 (m, 1H), 3.16e3.00 (m, 1H), 2.91e2.77 (m, 1H),

2.72e2.57 (m, 1H), 1.93e1.83 (m, 2H), 1.81 (t, J ¼ 2.3 Hz, 3H),

.87e4.73 (m, 1H), 4.63 (d, J ¼ 16.9, 1H), 4.53e4.40 (m, 1H),

.88e3.79 (m, 1H),3.38e3.24 (m, 1H), 3.19e3.02 (m, 1H), 2.92e2.78

(

3

m, 1H), 2.74e2.58 (m, 1H), 1.96e1.82 (m, 2H), 1.80 (t, J ¼ 2.3 Hz,

H), 1.74 (d, J ¼ 8.6 Hz, 1H), 1.44 (s, 9H). MS (ESI) m/z: 529.53

þ

[MþH] .

1.77e1.67 (m, 1H), 1.44 (s, 9H). MS (ESI) m/z: 589.47 [MþH] .

15

Q. Li, X. Deng, N. Jiang et al.

European Journal of Medicinal Chemistry 225 (2021) 113765

4

.1.11.8. methyl

ycarbonyl)amino)piperidin-1-yl)-2,6-dioxo-3,6-dihydropyrimidin-

(2H)-yl)methyl)-4-chlorobenzoate (16h). White solid (512 mg,

(R)-3-((3-(but-2-yn-1-yl)-4-(3-((tert-butox-

(t, J ¼ 1.5 Hz, 1H), 7.70 (t, J ¼ 1.8 Hz, 1H), 6.96 (d, J ¼ 7.7 Hz, 1H), 5.22

(s, 1H), 4.95 (s, 2H), 4.55e4.37 (m, 2H), 3.60e3.48 (m, 1H),

3.28e3.19 (m, 1H), 3.19e3.10 (m, 1H), 2.77e2.64 (m, 1H), 2.55e2.51

(m, 1H), 1.81e1.75 (m, 2H), 1.74 (t, J ¼ 2.3 Hz, 3H), 1.64e1.48 (m, 2H),

1

yield 91.8 %). H NMR (400 MHz, CDCl

H), 7.66 (d, J ¼ 2.0 Hz, 1H), 7.42 (d, J ¼ 8.3 Hz, 1H), 5.31 (s, 1H), 5.24

s, 2H), 4.86e4.76 (m, 1H), 4.66 (d, J ¼ 17.0 Hz, 1H), 4.49 (d,

J ¼ 16.9 Hz, 1H), 3.92e3.86 (m, 1H), 3.85 (s, 3H), 3.40e3.27 (m, 1H),

.19e3.05 (m, 1H), 2.99e2.83 (m, 1H), 2.79e2.63 (m, 1H), 1.97e1.84

3

)

d

7.83 (dd, J ¼ 8.3, 2.1 Hz,

þ

1

(

1.36 (s, 9H). MS (ESI) m/z: 575.22 [MþH] .

4.1.11.15. (R)-2-bromo-5-((3-(but-2-yn-1-yl)-4-(3-((tert-butox-

ycarbonyl)amino)piperidin-1-yl)-2,6-dioxo-3,6-dihydropyrimidin-

3

(

m, 2H), 1.81 (t, J ¼ 2.3 Hz, 3H), 1.78e1.72 (m, 1H), 1.45 (s, 9H). MS

1(2H)-yl)methyl)benzoic acid (17g). White solid (271 mg, yield

þ

1

ESI) m/z: 545.33 [MþH] .

60.4 %). H NMR (400 MHz, DMSO‑d

6

)

d

7.64e7.58 (m, 2H), 7.29 (dd,

J ¼ 8.3, 2.3 Hz, 1H), 6.95 (d, J ¼ 7.7 Hz, 1H), 5.21 (s, 1H), 4.90 (s, 2H),

4.54e4.36 (m, 2H), 3.59e3.49 (m, 1H), 3.26e3.19 (m,1H), 3.16e3.09

(m, 1H), 2.77e2.64 (m, 1H), 2.54e2.50 (m, 1H), 1.82e1.75 (m, 2H),

1.73 (t, J ¼ 2.3 Hz, 3H), 1.64e1.50 (m, 2H), 1.36 (s, 9H). MS (ESI) m/z:

4

.1.11.9. (R)-3-((3-(but-2-yn-1-yl)-4-(3-((tert-butoxycarbonyl)

amino)piperidin-1-yl)-2,6-dioxo-3,6-dihydropyrimidin-1(2H)-yl)

methyl)-2-ﬂuorobenzoic acid (17a). White solid (2.4 g, yield 86.1%).

1

þ

H NMR (400 MHz, DMSO‑d

6

)

d

7.70 (td, J ¼ 7.4, 1.9 Hz, 1H), 7.27 (td,

575.22 [MþH] .

J ¼ 7.2, 1.8 Hz,1H), 7.18 (t, J ¼ 7.7 Hz,1H), 6.97 (d, J ¼ 7.8 Hz,1H), 5.23

s, 1H), 4.99 (s, 2H), 4.54e4.38 (m, 2H), 3.61e3.49 (m, 1H),

.33e3.18 (m, 1H), 3.23e3.12 (m, 1H), 2.79e2.65 (m, 1H), 2.57e2.50

(

3

4.1.11.16. (R)-3-((3-(but-2-yn-1-yl)-4-(3-((tert-butoxycarbonyl)

amino)piperidin-1-yl)-2,6-dioxo-3,6-dihydropyrimidin-1(2H)-yl)

(

1

m, 1H), 1.83e1.76 (m, 2H), 1.76e1.72 (m, 3H), 1.67e1.50 (m, 2H),

.37 (s, 9H). MS (ESI) m/z: 515.48[MþH] .

methyl)-4-chlorobenzoic acid (17h). White solid (118 mg, yield

þ

1

61.3 %). H NMR (400 MHz, DMSO‑d

6

)

d

7.80 (dd, J ¼ 8.3, 2.1 Hz, 1H),

7

.58 (d, J ¼ 8.3 Hz, 1H), 7.48 (d, J ¼ 2.0 Hz, 1H), 6.99 (d, J ¼ 7.7 Hz,

4

.1.11.10. (R)-3-((3-(but-2-yn-1-yl)-4-(3-((tert-butoxycarbonyl)

1H), 5.29 (s, 1H), 5.02 (s, 2H), 4.56e4.41 (m, 2H), 3.63e3.50 (m, 1H),

3.27e3.24 (m, 1H), 3.21e3.17 (m, 1H), 2.84e2.68 (m, 1H), 2.60e2.52

(m, 1H),1.81e1.75 (m, 2H), 1.73 (t, J ¼ 2.3 Hz, 3H),1.67e1.52 (m, 2H),

amino)piperidin-1-yl)-2,6-dioxo-3,6-dihydropyrimidin-1(2H)-yl)

methyl)-4-ﬂuorobenzoic acid (17b). White solid (110 mg, yield

5

1

(

3

(

3

1

þ

1.2 %). H NMR (400 MHz, DMSO‑d

6

)

d

13.03 (s, 1H), 7.88e7.84 (m,

1.36 (s, 9H). MS (ESI) m/z: 553.23 [MþH] .

H), 7.70 (dd, J ¼ 7.4, 2.2 Hz, 1H), 7.29 (dd, J ¼ 9.9, 8.6 Hz, 1H), 6.97

d, J ¼ 7.7 Hz, 1H), 5.25 (s, 1H), 5.00 (s, 2H), 4.55e4.38 (m, 2H),

.60e3.47 (m, 1H), 3.32e3.24 (m, 1H), 3.21e3.11 (m, 1H), 2.81e2.65

4.1.12. General procedure for preparation of 18a-h and 19a-h

According to our previously published procedures [20], 18a-h

were prepared from 17a-h, and 19a-h were prepared from 16a-h.

m, 1H), 2.53 (d, J ¼ 6.2 Hz,1H),1.84e1.74 (m, 2H),1.73 (d, J ¼ 2.3 Hz,

þ

H), 1.65e1.50 (m, 2H), 1.37 (s, 9H). MS (ESI) m/z: 515.59 [MþH] .

4

.1.12.1. (R)-3-((4-(3-aminopiperidin-1-yl)-3-(but-2-yn-1-yl)-2,6-

4

.1.11.11. (R)-5-((3-(but-2-yn-1-yl)-4-(3-((tert-butoxycarbonyl)

dioxo-3,6-dihydropyrimidin-1(2H)-yl)methyl)-2-ﬂuorobenzoic acid

hydrochloride (18a). White solid (153 mg, yield 77.3 %); HPLC pu-

amino)piperidin-1-yl)-2,6-dioxo-3,6-dihydropyrimidin-1(2H)-yl)

methyl)-2-ﬂuorobenzoic acid (17c). White solid (182 mg, yield

5

ꢀ

1

rity: 98.4 %; mp:188.2e189.6 C; H NMR (500 MHz, DMSO‑d )

6

1

8.2 %). H NMR (400 MHz, DMSO‑d

6

)

d

7.78 (dd, J ¼ 7.1, 2.4 Hz, 1H),

d

13.26 (s, 1H), 8.44 (s, 3H), 7.74 (td, J ¼ 7.3, 1.9 Hz, 1H), 7.32 (td,

7

1

.54e7.50 (m, 1H), 7.22 (dd, J ¼ 10.8, 8.5 Hz, 1H), 6.95 (d, J ¼ 7.7 Hz,

J ¼ 7.3, 1.9 Hz, 1H), 7.22 (t, J ¼ 7.7 Hz, 1H), 5.29 (s, 1H), 5.03 (s, 2H),

4.65 (d, J ¼ 17.3 Hz, 1H), 4.52e4.44 (m, 1H), 3.41e3.33 (m, 2H), 3.12

(s, 1H), 3.01 (s, 1H), 2.95e2.89 (m, 1H), 1.99e1.96 (m, 1H), 1.94e1.88

H), 5.21 (s, 1H), 4.92 (s, 2H), 4.55e4.36 (m, 2H), 3.60e3.47 (m, 1H),

.26e3.18 (m, 1H), 3.16e3.08 (m, 1H), 2.78e2.63 (m, 1H), 2.57e2.49

3

13

(

m, 1H), 1.83e1.75 (m, 2H), 1.73 (s, 3H), 1.66e1.48 (m, 2H), 1.36 (s,

(m, 1H), 1.79 (t, J ¼ 2.3 Hz, 3H), 1.73e1.60 (m, 2H). C NMR

þ

9

H). MS (ESI) m/z: 515.76 [MþH] .

(126 MHz, DMSO‑d

6

) d

165.48 (d, J ¼ 2.4 Hz), 162.10, 160.15, 159.63,

1

58.10, 152.03, 132.96 (d, J ¼ 4.6 Hz), 130.94, 126.05 (d, J ¼ 14.8 Hz),

4

.1.11.12. (R)-2-bromo-3-((3-(but-2-yn-1-yl)-4-(3-((tert-butox-

124.53 (d, J ¼ 4.4 Hz), 120.01 (d, J ¼ 10.6 Hz), 88.68, 80.28, 74.96,

52.57, 51.82, 46.45, 38.04 (d, J ¼ 6.7 Hz), 36.24, 27.47, 21.79, 3.63. MS

ycarbonyl)amino)piperidin-1-yl)-2,6-dioxo-3,6-dihydropyrimidin-

1

6

þ

(2H)-yl)methyl)benzoic acid (17d). White solid (97 mg, yield

(ESI) m/z: 415.47 [MþH] .

1

3.8 %). H NMR (400 MHz, DMSO‑d

6

)

d

7.48 (dd, J ¼ 7.6, 1.6 Hz, 1H),

7

5

.34 (t, J ¼ 7.7 Hz, 1H), 6.99 (d, J ¼ 7.8 Hz, 1H), 6.94 (d, J ¼ 7.8 Hz, 1H),

4.1.12.2. (R)-3-((4-(3-aminopiperidin-1-yl)-3-(but-2-yn-1-yl)-2,6-

dioxo-3,6-dihydropyrimidin-1(2H)-yl)methyl)-4-ﬂuorobenzoic acid

hydrochloride (18b). White solid (203 mg, yield 63.7 %); HPLC pu-

.26 (s, 1H), 4.96 (s, 2H), 4.59e4.39 (m, 2H), 3.62e3.50 (m, 1H),

.29e3.26 (m,1H), 3.22e3.16 (m, 1H), 2.83e2.68 (m, 1H), 2.59e2.51

3

ꢀ

1

(

m, 1H), 1.85e1.76 (m, 2H), 1.75 (s, 3H), 1.67e1.51 (m, 2H), 1.37 (s,

rity: 97.8 %; mp: 199.2e201.2 C; H NMR (400 MHz, DMSO‑d )

6

þ

9

H). MS (ESI) m/z: 575.26 [MþH] .

d

13.07 (s, 1H), 8.21 (s, 3H), 7.90e7.82 (m, 1H), 7.68 (dd, J ¼ 7.4,

2

.2 Hz, 1H), 7.30 (dd, J ¼ 9.9, 8.6 Hz, 1H), 5.31 (s, 1H), 5.03e4.97 (m,

4

.1.11.13. (R)-4-bromo-3-((3-(but-2-yn-1-yl)-4-(3-((tert-butox-

2H), 4.61 (d, J ¼ 17.4 Hz, 1H), 4.49e4.37 (m, 1H), 3.40e3.34 (m, 1H),

3.31e3.25 (m, 1H), 3.14e2.94 (m, 2H), 2.92e2.80 (m, 1H), 1.99e1.83

ycarbonyl)amino)piperidin-1-yl)-2,6-dioxo-3,6-dihydropyrimidin-

1

4

13

(2H)-yl)methyl)benzoic acid (17e). White solid (205 mg, yield

(m, 2H), 1.74 (t, J ¼ 2.0 Hz, 3H), 1.68e1.54 (m, 2H); C NMR

1

6.6 %). H NMR (400 MHz, DMSO‑d

6

)

d

7.76 (d, J ¼ 8.3 Hz, 1H), 7.71

(126 MHz, DMSO‑d

6

)

d

166.70, 163.11 (d, J ¼ 252.6 Hz), 162.14,

(

1

3

dd, J ¼ 8.3, 2.0 Hz, 1H), 7.40 (d, J ¼ 2.0 Hz, 1H), 6.99 (d, J ¼ 7.6 Hz,

159.66, 151.95, 131.13 (d, J ¼ 9.5 Hz), 130.52 (d, J ¼ 5.1 Hz), 127.65 (d,

J ¼ 3.0 Hz), 125.03 (d, J ¼ 15.3 Hz), 116.23 (d, J ¼ 22.5 Hz), 88.70,

80.35, 74.81, 52.60, 51.79, 46.44, 37.82 (d, J ¼ 4.8 Hz), 36.18, 27.40,

H), 5.29 (s, 1H), 4.97 (s, 2H), 4.59e4.38 (m, 2H), 3.60e3.48 (m, 1H),

.29e3.23 (m, 1H), 3.23e3.15 (m, 1H), 2.83e2.68 (m, 1H), 2.62e2.51

þ

(

m, 1H), 1.86e1.74 (m, 2H), 1.72 (s, 3H), 1.67e1.51 (m, 2H), 1.37 (s,

21.70, 3.59. MS (ESI) m/z: 415.32 [MþH] .

þ

9

H). MS (ESI) m/z: 575.30 [MþH] .

4

.1.12.3. (R)-5-((4-(3-aminopiperidin-1-yl)-3-(but-2-yn-1-yl)-2,6-

4

.1.11.14. (R)-3-bromo-5-((3-(but-2-yn-1-yl)-4-(3-((tert-butox-

dioxo-3,6-dihydropyrimidin-1(2H)-yl)methyl)-2-ﬂuorobenzoic acid

hydrochloride (18c). White solid (122 mg, yield 67.4 %); HPLC pu-

ycarbonyl)amino)piperidin-1-yl)-2,6-dioxo-3,6-dihydropyrimidin-

ꢀ

1

1(2H)-yl)methyl)benzoic acid (17f). White solid (165 mg, yield

rity: 99.2 %; mp:190.4e191.8 C; H NMR (500 MHz, DMSO‑d

6

)

1

4

2.5 %). H NMR (400 MHz, DMSO‑d

6

)

d

7.90 (t, J ¼ 1.8 Hz, 1H), 7.82

d

13.25 (s, 1H), 8.41 (s, 3H), 7.80 (dd, J ¼ 7.0, 2.4 Hz, 1H), 7.55 (dd,

16

Q. Li, X. Deng, N. Jiang et al.

European Journal of Medicinal Chemistry 225 (2021) 113765

ꢀ

J ¼ 8.5, 4.6, 1H), 7.26 (dd, J ¼ 10.7, 8.5 Hz, 1H), 5.28 (s, 1H), 4.95 (s,

purity: 97.4 %; mp:136.5e138.2 C; ¹H NMR (500 MHz, DMSO‑d

d

6

)

2

3

2

H), 4.64 (d, J ¼ 17.3 Hz, 1H), 4.46 (dq, J ¼ 17.2, 2.3 Hz, 1H),

.40e3.29 (m, 2H), 3.08 (s, 1H), 2.99 (s, 1H), 2.94e2.85 (m, 1H),

.01e1.93 (m, 1H), 1.93e1.86 (m, 1H), 1.78 (t, J ¼ 2.3 Hz, 3H),

13.18 (s, 1H), 8.38 (s, 3H), 7.83 (dd, J ¼ 8.3, 2.1 Hz, 1H), 7.62 (d,

J ¼ 8.3 Hz, 1H), 7.50 (d, J ¼ 2.0 Hz, 1H), 5.36 (s, 1H), 5.05 (s, 2H), 4.67

(d, J ¼ 17.4 Hz,1H), 4.56e4.43 (m,1H), 3.38e3.32 (m, 2H), 3.19e3.00

(m, 2H), 2.99e2.87 (m, 1H), 2.01e1.90 (m, 2H), 1.77 (t, J ¼ 2.3 Hz,

13

1

6

.70e1.57 (m, 2H). C NMR (126 MHz, DMSO‑d ) d 165.34 (d,

13

J ¼ 2.9 Hz), 162.20, 161.77, 159.64 (d, J ¼ 23.9 Hz), 152.02, 134.77 (d,

6

3H), 1.72e1.60 (m, 2H); C NMR (126 MHz, DMSO‑d ) d 166.79,

J ¼ 9.0 Hz), 133.98 (d, J ¼ 3.6 Hz), 131.83, 119.54 (d, J ¼ 10.6 Hz),

162.16, 159.76, 151.98, 136.90, 135.13, 130.28, 130.26, 129.87, 127.89,

88.66, 80.40, 74.75, 52.62, 51.81, 46.44, 41.99, 36.27, 27.42, 21.69,

1

17.39 (d, J ¼ 22.7 Hz), 88.83, 80.28, 74.93, 52.59, 51.80, 46.45, 43.13,

þ

3

6.10, 27.44, 21.84, 3.60. MS (ESI) m/z: 415.24 [MþH] .

3.59; MS (ESI) m/z: 431.40[Mþ1] .

4

.1.12.4. (R)-3-((4-(3-aminopiperidin-1-yl)-3-(but-2-yn-1-yl)-2,6-

4.1.12.9. methyl (R)-3-((4-(3-aminopiperidin-1-yl)-3-(but-2-yn-1-

yl)-2,6-dioxo-3,6-dihydropyrimidin-1(2H)-yl)methyl)-2-

ﬂuorobenzoate hydrochloride (19a). White solid (96 mg, yield

dioxo-3,6-dihydropyrimidin-1(2H)-yl)methyl)-2-bromobenzoic acid

hydrochloride(18d). White solid (173 mg, yield 62.6 %); HPLC pu-

ꢀ

1

ꢀ

1

rity: 95.4 %; mp:199.5e201.3 C; H NMR (500 MHz, DMSO‑d

13.45 (s, 1H), 8.45 (s, 3H), 7.52 (dd, J ¼ 7.6, 1.7 Hz, 1H), 7.39 (t,

J ¼ 7.7 Hz, 1H), 6.97 (dd, J ¼ 7.8, 1.6 Hz, 1H), 5.33 (s, 1H), 4.99 (s, 2H),

6

)

69.5 %); HPLC purity: 98.3 %; mp: 68.8e70.8 C; H NMR (400 MHz,

DMSO‑d 8.48 (s, 3H), 7.83e7.64 (m, 1H), 7.38e7.30 (m, 1H), 7.23

d

6

) d

(t, J ¼ 7.7 Hz, 1H), 5.25 (s, 1H), 4.99 (s, 2H), 4.62 (d, J ¼ 17.3 Hz, 1H),

4

.67 (d, J ¼ 17.3 Hz, 1H), 4.54e4.46 (m, 1H), 3.41e3.37 (m, 2H) 3.16

4.48e4.34 (m, 1H), 3.83 (s, 3H), 3.33e3.28 (m, 2H), 3.18e2.95 (m,

(

1

4

s, 1H), 3.02 (s, 1H), 2.97e2.89 (m, 1H), 2.03e1.96 (m, 1H), 1.96e1.90

2H), 2.92e2.82 (m, 1H), 2.01e1.82 (m, 2H), 1.75 (s, 3H), 1.68e1.54

13

m, 1H), 1.79 (t, J ¼ 2.3 Hz, 3H), 1.71e1.62 (m, 2H). C NMR

126 MHz, DMSO‑d 168.50, 162.13, 159.79, 152.09, 137.18, 136.53,

28.44, 128.19, 128.15, 119.49, 88.65, 80.32, 74.96, 52.59, 51.84,

(m, 2H); C NMR (126 MHz, DMSO‑d

6

)

d

164.46 (d, J ¼ 3.0 Hz),

6

)

d

162.07, 159.69, 158.96 (d, J ¼ 5.0 Hz), 152.03, 133.55 (d, J ¼ 5.0 Hz),

130.80, 126.18 (d, J ¼ 14.8 Hz), 124.78 (d, J ¼ 4.3 Hz), 118.66 (d,

J ¼ 10.4 Hz), 88.52, 80.26, 74.95, 52.90, 52.61, 51.75, 46.54, 37.95 (d,

þ

6.47, 45.06, 36.37, 27.52, 21.84, 3.65; MS (ESI) m/z: 475.42[Mþ1] .

þ

J ¼ 6.4 Hz), 36.30, 27.51, 21.98, 3.64. MS (ESI) m/z: 429.32[Mþ1] .

4

.1.12.5. (R)-3-((4-(3-aminopiperidin-1-yl)-3-(but-2-yn-1-yl)-2,6-

dioxo-3,6-dihydropyrimidin-1(2H)-yl)methyl)-4-bromobenzoic acid

4.1.12.10. methyl (R)-3-((4-(3-aminopiperidin-1-yl)-3-(but-2-yn-1-

yl)-2,6-dioxo-3,6-dihydropyrimidin-1(2H)-yl)methyl)-4-

ﬂuorobenzoate hydrochloride (19b). White solid (163 mg, yield

hydrochloride(18e). White solid (163 mg, yield 69.7 %); HPLC pu-

ꢀ

1

6

rity: 98.7 %; mp: 207.7e209.7 C; H NMR (400 MHz, DMSO‑d )

ꢀ

1

d

13.18 (s, 1H), 8.26 (s, 3H), 7.78 (d, J ¼ 8.3 Hz, 1H), 7.71 (dd, J ¼ 8.3,

64.7 %); HPLC purity: 98.5%; mp: 196.4e198.4 C; H NMR

(400 MHz, DMSO‑d 8.24 (s, 3H), 7.93e7.85 (m, 1H), 7.73 (dd,

2

.1 Hz, 1H), 7.40e7.35 (m, 1H), 5.35 (s, 1H), 5.10e4.85 (m, 2H), 4.64

6

) d

(

3

d, J ¼ 17.4 Hz, 1H), 4.54e4.32 (m, 1H), 3.39e3.34 (m, 1H),

J ¼ 7.2, 2.3 Hz, 1H), 7.38e7.27 (m, 1H), 5.31 (s, 1H), 5.04e4.98 (m,

2H), 4.61 (d, J ¼ 17.3 Hz, 1H), 4.48e4.35 (m, 1H), 3.79 (s, 3H),

3.27e3.32 (m, 2H), 3.04e3.00 (m, 2H), 2.92e2.80 (m,1H),1.96e1.87

.32e3.28 (m,1H), 3.19e2.99 (m, 2H), 2.96e2.84 (m,1H), 2.00e1.85

13

(

m, 2H), 1.74 (s, 3H), 1.67e1.56 (m, 2H); C NMR (126 MHz,

DMSO‑d 165.22, 162.21, 159.64, 152.05, 141.10, 135.77, 132.26,

30.99, 128.10, 122.12, 88.77, 80.28, 74.90, 52.57, 51.78, 46.43, 43.36,

13

6

)

d

(m, 2H), 1.75 (t, J ¼ 2.0 Hz, 3H), 1.63e1.59 (m, 2H); C NMR

1

3

(126 MHz, DMSO‑d

6

)

d

165.66,164.35,162.24 (d, J ¼ 15.5 Hz),159.67,

þ

6.19, 27.45, 21.75, 3.62. MS (ESI) m/z: 475.42[Mþ1] .

151.94, 131.08 (d, J ¼ 9.7 Hz), 130.77 (d, J ¼ 5.4 Hz), 126.51 (d,

J ¼ 3.1 Hz), 125.34 (d, J ¼ 15.4 Hz), 116.50 (d, J ¼ 22.7 Hz), 88.72,

80.31, 74.83, 52.79, 52.60, 51.79, 46.44, 37.81 (d, J ¼ 4.6 Hz), 36.16,

4

.1.12.6. (R)-3-((4-(3-aminopiperidin-1-yl)-3-(but-2-yn-1-yl)-2,6-

þ

dioxo-3,6-dihydropyrimidin-1(2H)-yl)methyl)-5-bromobenzoic acid

hydrochloride (18f). White solid (198 mg, yield 69.3 %); HPLC pu-

rity: 100.0%; mp: 201.9e204.6 C; H NMR (500 MHz, DMSO‑d

d

27.41, 21.74, 3.58. MS (ESI) m/z: 428.97 [MþH] .

ꢀ

1

6

)

4.1.12.11. methyl (R)-5-((4-(3-aminopiperidin-1-yl)-3-(but-2-yn-1-

yl)-2,6-dioxo-3,6-dihydropyrimidin-1(2H)-yl)methyl)-2-

ﬂuorobenzoate hydrochloride (19c). White solid (126 mg, yield

13.36 (s, 1H), 8.41 (s, 3H), 7.93 (s, 1H), 7.84 (s, 1H), 7.73 (s, 1H), 5.29

(

3

2

s, 1H), 4.99 (s, 2H), 4.64 (d, J ¼ 17.3 Hz, 1H), 4.50e4.44 (m, 1H),

ꢀ

1

.38e3.34 (m, 2H), 3.10 (s, 1H), 2.99 (s, 1H), 2.92e2.89 (m, 1H),

.00e1.94 (m, 1H), 1.94e1.88 (m, 1H), 1.78 (t, J ¼ 2.3 Hz, 3H),

61.6 %); HPLC purity: 95.2 %; mp: 99.3e101.2 C; H NMR (400 MHz,

DMSO‑d

6

)

d

8.24 (s, 3H), 7.81 (dd, J ¼ 7.0, 2.4 Hz, 1H), 7.60e7.53 (m,

13

1

8

.68e1.62 (m, 2H). C NMR (126 MHz, DMSO‑d

6

)

d

166.23, 162.22,

1H), 7.29 (dd, J ¼ 10.9, 8.5 Hz, 1H), 5.26 (s, 1H), 4.95e4.90 (m, 2H),

59.63, 152.06, 140.92, 135.29, 133.59, 131.13, 128.10, 121.98, 88.78,

4.60 (d, J ¼ 17.3 Hz, 1H), 4.47e4.36 (m, 1H), 3.82 (s, 3H), 3.38e3.34

0.30, 74.90, 52.59, 51.78, 46.45, 43.38, 36.19, 27.45, 21.76, 3.63. MS

(m, 1H), 3.30e3.21 (m, 1H), 3.10e2.90 (m, 2H), 2.89e2.77 (m, 1H),

þ

13

(

ESI) m/z: 475.42[Mþ1] .

1.97e1.84 (m, 2H), 1.75 (t, J ¼ 2.0 Hz, 3H), 1.67e1.55 (m, 2H);

C

NMR (126 MHz, DMSO‑d

6

)

d

164.34 (d, J ¼ 3.6 Hz), 162.19, 161.60,

4

.1.12.7. (R)-5-((4-(3-aminopiperidin-1-yl)-3-(but-2-yn-1-yl)-2,6-

159.55, 152.01, 135.33 (d, J ¼ 9.3 Hz), 134.22 (d, J ¼ 3.7 Hz), 131.77,

118.30 (d, J ¼ 10.5 Hz), 117.56 (d, J ¼ 22.5 Hz), 88.82, 80.26, 74.93,

52.90, 52.58, 51.80, 46.43, 43.09, 36.10, 27.43, 21.74, 3.60. MS (ESI)

m/z: 429.42 [MþH] .

dioxo-3,6-dihydropyrimidin-1(2H)-yl)methyl)-2-bromobenzoic acid

hydrochloride (18g). White solid (155 mg, yield 73.2 %); HPLC pu-

rity: 98.0 %; mp: 198.4e201.5 C; H NMR (500 MHz, DMSO‑d

d

ꢀ

1

þ

6

)

13.42 (s,1H), 8.40 (s, 3H), 7.66 (d, J ¼ 8.3 Hz,1H), 7.64 (d, J ¼ 2.3 Hz,

1

H), 7.33 (dd, J ¼ 8.3, 2.3 Hz, 1H), 5.28 (s, 1H), 4.94 (s, 2H), 4.63 (d,

4.1.12.12. methyl (R)-3-((4-(3-aminopiperidin-1-yl)-3-(but-2-yn-1-

yl)-2,6-dioxo-3,6-dihydropyrimidin-1(2H)-yl)methyl)-2-

bromobenzoate hydrochloride (19d). White solid (205 mg, yield

J ¼ 17.3 Hz, 1H), 4.50e4.42 (m, 1H), 3.36e3.34 (m, 2H), 3.14 (s, 1H),

3

1

.09 (s, 1H), 2.93e2.87 (m, 1H), 2.01e1.94 (m, 1H), 1.93e1.87 (m,

13

ꢀ

1

H), 1.78 (t, J ¼ 2.3 Hz, 3H), 1.67e1.61 (m, 2H). C NMR (126 MHz,

167.69, 162.19, 159.58, 152.03, 137.58, 134.23, 134.14,

32.31, 130.33, 119.04, 88.78, 80.29, 74.92, 52.58, 51.80, 46.45, 43.27,

69.0 %); HPLC purity: 98.5 %; mp: 161.0e163.0 C; H NMR

(400 MHz, DMSO‑d

DMSO‑d

1

3

6

)

d

6

)

d

8.23 (s, 3H), 7.53 (dd, J ¼ 7.6, 1.6 Hz, 1H), 7.40

(t, J ¼ 7.7 Hz, 1H), 7.00 (dd, J ¼ 7.8, 1.6 Hz, 1H), 5.32 (s, 1H), 4.96 (s,

þ

6.14, 27.44, 21.76, 3.61. MS (ESI) m/z: 475.63[Mþ1] .

2H), 4.62 (d, J ¼ 17.4 Hz, 1H), 4.51e4.38 (m, 1H), 3.85 (s, 3H),

3

.42e3.34 (m, 1H), 3.32e3.28 (m, 1H), 3.18e2.95 (m, 2H),

4

.1.12.8. (R)-3-((4-(3-aminopiperidin-1-yl)-3-(but-2-yn-1-yl)-2,6-

2.92e2.83 (m, 1H), 2.00e1.85 (m, 2H), 1.76 (s, 3H), 1.70e1.55 (m,

13

dioxo-3,6-dihydropyrimidin-1(2H)-yl)methyl)-4-chlorobenzoic acid

6

2H); C NMR (126 MHz, DMSO‑d ) d 167.37, 162.11, 159.79, 152.08,

hydrochloride (18 h). White solid (173 mg, yield 77.3 %); HPLC

137.44, 134.99, 128.92, 128.84, 128.29, 119.76, 88.63, 80.32, 74.95,

17

Q. Li, X. Deng, N. Jiang et al.

European Journal of Medicinal Chemistry 225 (2021) 113765

5

3.18, 52.58, 51.84, 46.46, 45.00, 36.38, 27.52, 21.83, 3.65; MS (ESI)

concentration 10 mM), and then diluted into assay buffer (25 mM

HEPES, pH 7.5, 150 mM NaCl, 0.12 mg/mL BSA) to obtain varying

concentrations. The dilutions were added to 17.3 mU/mL of human

þ

m/z: 489.17 [MþH] .

4

.1.12.13. methyl(R)-3-((4-(3-aminopiperidin-1-yl)-3-(but-2-yn-1-

recombinant DPP-4 in assay buffer. After 15 min of incubation at

ꢀ

yl)-2,6-dioxo-3,6-dihydropyrimidin-1(2H)-yl)methyl)-4-

37 C, 10

mM of Gly-Pro-AMC was added to initiate the reaction,

bromobenzoate hydrochloride (19e). White solid (136 mg, yield

which is cleaved by DPP-4 to release the ﬂuorescent AMC leaving

group. The ﬂuorescence was measured (380 nm excitation/460 nm

emission) every 1 min for 20 min using BioTek microplate reader.

The inhibitory potency (IC50) was calculated using the software

GraphPad Prism 7. The potency of DPP-8 and DPP-9 were deter-

mined using the same method as the DPP-4 assay with pH of the

assay buffer of 8.0. The test was carried out in duplicate.

ꢀ

1

6

0.8 %); HPLC purity: 98.7 %; mp:156.0e158.0 C; H NMR

(

500 MHz, DMSO‑d

6

) d

8.46 (s, 3H), 7.82 (d, J ¼ 8.3 Hz, 1H), 7.75 (dd,

J ¼ 8.3, 2.1 Hz, 1H), 7.44 (d, J ¼ 2.1 Hz, 1H), 5.37 (s, 1H), 5.02 (s, 2H),

4

3

.68 (d, J ¼ 17.4 Hz, 1H), 4.50 (dq, J ¼ 17.2, 2.3 Hz, 1H), 3.81 (s, 3H),

.40 (s, 1H), 3.37 (m, 1H), 3.14 (m, 1H), 3.05 (m, 1H), 2.95 (m, 1H),

13

1

(

1

.99 (m, 1H), 1.94 (m, 1H), 1.78 (m, 3H), 1.68 (m, 2H). C NMR

126 MHz, DMSO‑d 165.91, 162.16, 159.83, 152.00, 136.95, 133.78,

29.73, 129.66, 127.99, 127.48, 88.70, 80.38, 74.76, 52.94, 52.65,

6

) d

5

1.80, 46.46, 44.49, 36.28, 27.44, 21.78, 3.58; MS (ESI) m/z: 489.17

4.3. Measure of plasma and microsomal stability

þ

[MþH] .

Plasma stability of tested compounds was evaluated as previ-

ously described [19,24]. Rat blood was collected from adult male

4

.1.12.14. methyl (R)-3-((4-(3-aminopiperidin-1-yl)-3-(but-2-yn-1-

yl)-2,6-dioxo-3,6-dihydropyrimidin-1(2H)-yl)methyl)-5-

bromobenzoate hydrochloride (19f). White solid (185 mg, yield

6

SpragueeDawley rats, and plasma was separated from whole blood

ꢀ

by centrifugation (10,000g for 2 min) and stored at ꢁ20 C. The

ꢀ

1

3.8 %); HPLC purity: 98.1 %; mp: 168.8e170.8 C; H NMR

water solution of tested compounds (10 mM) were diluted into rat

(

400 MHz, DMSO‑d 8.22 (s, 3H), 7.93 (s, 1H), 7.85 (s, 1H), 7.74 (s,

6

) d

ꢀ

plasma to a ﬁnal concentration of 1

00

5, 30, 60, 120 min time points, 300

m

M, and incubated at 37 C. A

1

4

1

2

H), 5.28 (s, 1H), 5.03e4.90 (m, 2H), 4.60 (d, J ¼ 17.5 Hz, 1H),

.54e4.33 (m, 1H), 3.83 (s, 3H), 3.39e3.32 (m, 1H), 3.31e3.23 (m,

H), 3.13e3.03 (m, 1H), 3.03e2.92 (m, 1H), 2.92e2.78 (m, 1H),

.01e1.82 (m, 2H), 1.76 (s, 3H), 1.69e1.48 (m, 2H); ¹C NMR

1

0

m

L of plasma was aliquoted from the incubation solution at 0, 5,

L of an ice-cold methanol with

.5 % of formic acid was then added to quench the reaction, fol-

m

3

lowed by centrifugation (14000 RMP, 10 min) to precipitate and

remove proteins. The aliquots of the supernatants were injected

onto LC-MS/MS (Waters, ACQUITY-TQ-XS). Remaining ratio (%) (1-

(

1

5

126 MHz, DMSO‑d

29.72, 129.65, 127.99, 127.47, 88.70, 80.38, 74.76, 52.94, 52.64,

1.81, 46.44, 44.48, 36.26, 27.44, 21.70, 3.58. MS (ESI) m/z: 489.24

6

) d 165.91, 162.15, 159.82, 151.98, 136.94, 133.79,

C

120/C

0

) ꢃ 100, where C

0

is compound concentration at 0 min

þ

[MþH] .

and C120 is compound concentration at 120 min.

Microsomal incubations were performed as previously reported

4

.1.12.15. methyl (R)-5-((4-(3-aminopiperidin-1-yl)-3-(but-2-yn-1-

[

19,24]. In brief, the suspension of ICR mice liver microsomes (1 mg/

mL, Meilunbio, Dalian, China) with test compounds (1 M) in

00 mM Tris buffer (pH 7.4) containing 10 mM MgCl was pre-

incubated at 37 C for 5 min. By the addition of NADPH (Sigma-

eAldrich, St. Louis, MO, USA), the reactions (ﬁnal volumes 600 L)

yl)-2,6-dioxo-3,6-dihydropyrimidin-1(2H)-yl)methyl)-2-

bromobenzoate hydrochloride (19g). White solid (84 mg, yield

m

1

2

ꢀ

1

6

(

0.6 %); HPLC purity: 95.8%; mp: 147.0e149.0 C; H NMR

400 MHz, DMSO‑d

8.24 (s, 3H), 7.68 (d, J ¼ 8.3 Hz, 1H), 7.65 (d,

J ¼ 2.3 Hz, 1H), 7.34 (dd, J ¼ 8.3, 2.3 Hz, 1H), 5.26 (s, 1H), 4.96e4.85

m, 2H), 4.59 (d, J ¼ 17.3 Hz, 1H), 4.48e4.33 (m, 1H), 3.83 (s, 3H),

.39e3.36 (m,1H), 3.30e3.22 (m,1H), 3.14e2.92 (m, 2H), 2.89e2.76

m,1H),1.98e1.81 (m, 2H),1.75 (t, J ¼ 2.1 Hz, 3H),1.67e1.51 (m, 2H);

ꢀ

6

) d

m

were started. The ﬁnal reaction concentration of tested compounds

(

3

ꢀ

was 0.1

m

M. The incubations were carried out for 0e60 min at 37 C,

an aliquot of the reaction mixture were quenched by the addition of

00 L of an ice-cold methanol with 0.5 % of formic acid, followed

(

3

m

1

3

C NMR (126 MHz, DMSO‑d

6

) d 166.54, 162.18, 159.58, 152.02,

by the centrifugation (10 000 g, 10 min). An aliquot of the super-

natants were injected onto LC-MS/MS system to determine com-

pound concentration. Control experiments were carried out under

identical conditions but without microsomes. The values for %

remaining were analyzed. All procedures were run in duplicate.

137.76, 134.38, 132.88, 132.73, 130.67, 119.19, 88.76, 80.27, 74.92,

5

3.11, 52.56, 51.81, 46.44, 43.26, 36.15, 27.45, 21.75, 3.61; MS (ESI)

þ

m/z: 489.17 [MþH] .

4

.1.12.16. methyl (R)-3-((4-(3-aminopiperidin-1-yl)-3-(but-2-yn-1-

yl)-2,6-dioxo-3,6-dihydropyrimidin-1(2H)-yl)methyl)-4-

chlorobenzoate hydrochloride (19 h). White solid (171 mg, yield

ꢀ

1

7

3.2 %); HPLC purity: 95.3%; mp:150.1e152.1 C; H NMR (400 MHz,

4.4. Cell viability assay

DMSO‑d

6

) d

8.26 (s, 3H), 7.82 (dd, J ¼ 8.4, 2.2 Hz, 1H), 7.63 (d,

J ¼ 8.3 Hz, 1H), 7.49 (d, J ¼ 2.1 Hz, 1H), 5.35 (s, 1H), 5.06e5.00 (m,

MTT assay was performed to determine the cell viability of the

tested compounds. In brief, hepatic LO2 cells were cultured in

2

3

H), 4.63 (d, J ¼ 17.4 Hz, 1H), 4.52e4.40 (m, 1H), 3.79 (s, 3H),

.36e3.25 (m, 2H), 3.16e2.96 (m, 2H), 2.95e2.83 (m, 1H), 1.96e1.81

DMEM medium (Gibco, Invitrogen) supplemented with 10 % fetal

13

ꢀ

(

m, 2H), 1.79e1.70 (m, 3H), 1.68e1.58 (m, 2H); C NMR (126 MHz,

DMSO‑d 165.77, 162.16, 159.79, 151.98, 137.41, 135.44, 130.47,

29.66,129.10,128.02, 88.68, 80.36, 74.77, 52.92, 52.63, 51.79, 46.45,

bovine serum (FBS) and antibiotics at 37 C in a 5% CO

2

atmosphere

6

)

d

and 95 % humidity. Hepatic LO2 cells during logarithmic growth

4

1

phase were seeded in 96-well plates at 1 ꢃ 10 cells/well, and were

þ

41.93, 36.25, 27.43, 21.76, 3.58. MS (ESI) m/z: 445.28 [MþH] .

incubated with tested compounds, or vehicle for 48 h. MTT re-

agents were added at 20

were incubated for 4 h in a 37 C incubator containing 5 % CO

m

L per well (5 mg/mL in PBS), and cells

ꢀ

4.2. In vitro DPP-4, DPP-8, and DPP-9 inhibition potency test

2

. After

that, the media was removed without disturbing the formazan

crystals and cells in the wells,150 L of DMSO was added to dissolve

Human recombinant DPP-4, DPP-8 and DPP-9 were obtained

m

from Sigma. The potency of the targeted compounds was assayed as

described previously in a continuous ﬂuorescent (CF) assay by us-

ing Gly-Pro-aminomethylcoumarin (AMC) as substrate [19,24,25].

Brieﬂy, test compounds were dissolved in dimethyl sulfoxide (ﬁnal

the formazan crystals. After 10 min of shaking, absorption at

490 nm was read on a microplate reader (Molecular Devices, USA).

The % cell viability relative to vehicle control for each compound

was calculated.

18

Q. Li, X. Deng, N. Jiang et al.

European Journal of Medicinal Chemistry 225 (2021) 113765

4.5. Animals

postdose of glucose.

Overnight fasted db/db mice (n ¼ 6 in each group) was orally

administered with compound 19b (1, 3 and 10 mg/kg), alogliptin

(10 mg/kg), or vehicle (water). 30 min after the administration,

animals were challenged with oral glucose load (2 g/kg). Blood

samples for glucose measurement were obtained by tail bleed

predose and at serial time points (0, 15, 30, 60 and 120 min) after

the glucose load. Blood glucose was tested using a glucometer

(SanNuo Changsha, China). The blood glucose excursion proﬁle

from t ¼ 0 to t ¼ 120 min was used to integrate an area under the

curve (AUC).

SpragueeDawley rats (9e10 weeks old, 230 ± 20 g) were pur-

chased from the Laboratory Animal Center of Guangxi Medical

University (Nanning, China). Male ICR mice weighing (6e7 weeks,

0 ± 2 g) and Male C57BL/6 db/db mice (6e8 weeks old, 22 ± 2 g)

were obtained from the Model Animal Research Centre of Nanjing

University (Jiangsu, China). Animals were housed in cages in a

temperature and humidity controlled room with 12 h lightedark

cycle, and were maintained with free access to food and water for

2

1

week prior to the experiment. The care and use of the animals

were strictly performed in accordance with the protocols issued by

the Animal Research Committee and the Institutional Animal Care

and Use Committee of Guangxi Medical University.

4.9. Chronic treatment in type 2 diabetic db/db mice

According to their blood glucose (nonfasting and 12 h fasting)

and nonfasting body weight, male db/db mice (10 weeks old) were

divided into 4 treatment groups (n ¼ 8). Lean littermates were used

as the lean control. Animals were orally administered with vehicle

(water), or compound 19b (1 and 10 mg/kg in water), or alogliptin

(10 mg/kg in water) once daily for 4 weeks. Nonfasting body weight

were measured every day. 12 h fasting glucose were measured

every 7 days (at 9 a.m.) using a glucometer (SanNuo Changsha,

China). After 4 weeks of treatment, all mice were fasted 12 h, and

were given a glucose challenge (p.o., 1.5 g/kg). Blood glucose was

measured at 0 min, at 0, 15, 30, 60 and 120 min postglucose chal-

lenge using a one-touch glucometer (SanNuo Changsha, China).

4

.6. Pharmacokinetics assay in SpragueeDawley rats

Male SD rats (n ¼ 3, 230 ± 20 g) were fasted overnight. Tested

compounds in water were administrated by intravenous injection

(

0

3 mg/kg) or orally gavage (10 mg/kg). Serial blood samples

300 L) were collected into tubes containing EDTA at predose,

.083, 0.17, 0.5, 1, 1.5, 2, 4, 6, 8, 12, 24, 36 and 48 h postdose. After

m

centrifugation at 10 000 rpm for 2 min, the plasma samples were

ꢀ

stored at ꢁ20 C until analysis. The plasma samples were mixed

with two volumes of 0.5 % formic acid/acetonitrile (v/v) to precip-

itate proteins. After centrifugation (14,000 rpm, 14 min), the su-

pernatants were analyzed via LC-MS/MS (Waters, ACQUITY-TQ-XS).

Pharmacokinetic parameters were estimated by DAS 2.0 software

4.10. Molecular modeling

(

China).

Docking simulation was performed on Glide 5.9 in Schr o€ dinger

2017 suite. The crystal structure of DPP-4 protein (PDB ID: 2RGU)

was retrieved from RCSB Protein Data Bank. Protein structures were

prepared using Maestro protein preparation wizard applying the

default parameters. Hydrogen atom was added to the proteins and

charges were assigned. All the water molecules and other hetero-

atoms were removed. A docking grid was created by using the

centroid of the bound ligand. Ligands were built using Maestro

build panel and prepared by the LigPrep application using default

parameters. Molecular docking of all molecules into the generated

grid was performed by using the extra precision (XP) docking

mode. Glide docking score was used to determine the best docked

conﬁrmation from the output.

4

.7. Measurement of plasma DPP-4 inhibition

The plasma DPP-4 activity was evaluated as previously

described. Adult male SD rats (n ¼ 3/group) were randomly

assigned to treatment or vehicle groups and fasted overnight. An-

imals were treated orally (gavage) with vehicle (distilled water) and

test compounds (10 mg/kg in vehicle). Blood samples (100 mL) were

collected into a tube containing heparin sodium from the orbit at

the time points 0, 1, 2, 4, 6, 8, 12, 24, 36, 48 h. The plasma was

prepared by centrifugation at 10,000g for 2 min. Aliquots of plasma

ꢀ

samples were stored at ꢁ20 C until measurement. The plasma

DPP-4 activity was measured by a continuous ﬂuorometric assay

using Gly-Pro-AMC as substrates (Sigma). The assay media con-

tained plasma (20

mL), 125

mM Gly-Pro-AMC (20

mL) and buffer

4.11. Statistical analysis

(

100 mM HEPES, pH 7.5, 0.1 mg/mL BSA). Release of AMC was

ꢀ

measured continuously in a 96-well plate in 10 min at 37 C using

an excitation wavelength of 360 nm and an emission wavelength of

The Data are expressed as the means ± SEM. Data and statistical

analyses were performed using GraphPad version 7.00 (GraphPad

Software, San Diego, CA, USA). General effects were analyzed using

a one-way ANOVA with Dunnett's multiple comparisons test.

4

60 nm. The data plasma DPP-4 inhibition was calculated as % DPP-

activity as follows: % activity ¼ (Vts/Vcs) ꢃ 100, where Vts is the

rate of reaction of treated sample and Vcs is the rate of reaction of

vehicle sample.

Declaration of competing interest

4

.8. Acute oral glucose tolerance test (OGTT) in ICR and C57BL/6 db/

The authors declare that they have no known competing

ﬁnancial interests or personal relationships that could have

appeared to inﬂuence the work reported in this paper.

db mice

Male ICR mice (n ¼ 8/group) were randomly assigned to vehicle

and treatment groups, and fasted overnight. Blood glucose con-

centration was determined by a one-touch glucometer (SanNuo,

Changsha, China) from tail tip blood. Animals were treated orally by

gavage with a single dose of vehicle (water), alogliptin (dissolved in

the water; 10 mg/kg) or tested compounds (dissolved in water,

Acknowledgments

This study was supported by grants from the National Natural

Science Foundation of China (Grants 21762009, 31970371), Guangxi

Key R&D Project (Grant No. AB17195009), Guangxi Science and

Technology Development Under the Central Government (Grant

No. ZY20198020), and Training Programs of Innovation and

Entrepreneurship for Undergraduates (Grants 202010598036,

202010598132, 202010598155).

1

0 mg/kg). Blood glucose concentration was measured 60 min after

treatment (t ¼ 0 min) and was immediately followed by orally

administration of glucose (3 g/kg). Blood glucose levels were

determined from tail bleeds taken at 15, 30, 60 and 120 min

19

Q. Li, X. Deng, N. Jiang et al.

European Journal of Medicinal Chemistry 225 (2021) 113765

Appendix A. Supplementary data

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N.R. Desai, O. Mosenzon, D.K. McGuire, K.K. Ray, L.A. Leiter, I. Raz, S.-

T.S. Committee, Investigators, Saxagliptin and cardiovascular outcomes in

patients with type 2 diabetes mellitus, N. Engl. J. Med. 369 (2013) 1317 e 1326.

Supplementary data to this article can be found online at

https://doi.org/10.1016/j.ejmech.2021.113765.

[

17] C.B. Burness, Omarigliptin: ﬁ rst global approval, Drugs 75 (2015) 1947 e 1952 .

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20

Article Doi

Identification and structure–activity relationship exploration of uracil-based benzoic acid and ester derivatives as novel dipeptidyl Peptidase-4 inhibitors for the treatment of type 2 diabetes mellitus

DOI: 10.1016/j.ejmech.2021.113765

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