Identification of novel uracil derivatives incorporating benzoic acid moieties as highly potent Dipeptidyl Peptidase-IV inhibitors

Accepted Manuscript Identification of novel uracil derivatives incorporating benzoic acid moieties as highly potent Dipeptidyl Peptidase-IV inhibitors Junli Huang, Xiaoyan Deng, Siru Zhou, Na Wang, Yujun Qin, Liuwei Meng, Guobao Li, Yuhua Xiong, Yating Fan, Ling Guo, Danni Lan, Junhao Xing, Weizhe Jiang, Qing Li PII: DOI: Reference:

S0968-0896(18)31772-3 https://doi.org/10.1016/j.bmc.2019.01.001 BMC 14693

To appear in:

Bioorganic & Medicinal Chemistry

Received Date: Revised Date: Accepted Date:

10 November 2018 31 December 2018 3 January 2019

Please cite this article as: Huang, J., Deng, X., Zhou, S., Wang, N., Qin, Y., Meng, L., Li, G., Xiong, Y., Fan, Y., Guo, L., Lan, D., Xing, J., Jiang, W., Li, Q., Identification of novel uracil derivatives incorporating benzoic acid moieties as highly potent Dipeptidyl Peptidase-IV inhibitors, Bioorganic & Medicinal Chemistry (2019), doi: https:// doi.org/10.1016/j.bmc.2019.01.001

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Identification of novel uracil derivatives incorporating benzoic acid moieties as highly potent Dipeptidyl Peptidase-IV inhibitors Junli Huanga#, Xiaoyan Denga#, Siru Zhoua, Na Wanga, Yujun Qin a, Liuwei Menga, Guobao Lia, Yuhua Xionga, Yating Fana, Ling Guoa, Danni Lana, Junhao Xingb, Weizhe Jianga, Qing Li a 

a Pharmaceutical b

College, Guangxi Medical University, Nanning 530021, China;

Department of Organic Chemistry and State Key Laboratory of Natural Medicines, China

Pharmaceutical University, Nanjing 210009, China; #

Those authors contributed equally to this works

Abstract Dipeptidyl peptidase-IV (DPP-4) is a validated therapeutic target for type 2 diabetes. Aiming to interact with both residues Try629 and Lys554 in S2’ site, a series of novel uracil derivatives1a-l and 2a-i incorporating benzoic acid moieties at the N3 position were designed and evaluated for their DPP-4 inhibitory activity. Structure-activity relationships (SAR) study led to the identification of the optimal compound 2b as a potent and selective DPP-4 inhibitor (IC50 = 1.7 nM). Docking study revealed the additional salt bridge formed between the carboxylic acid and primary amine of Lys554 has a key role in the enhancement of the activity. Furthermore, compound 2b exhibited no cytotoxicity in human hepatocyte LO2 cells up to 50 μM. Subsequent in vivo evaluations revealed that the ester of 2b robustly improves the glucose tolerance in normal mice. The overall results have shown that compound 2b has the potential to a safe and efficacious treatment for T2DM. Keywords T2DM; DPP-4 inhibitor; uracil derivatives; benzoic acid

Qing Li, Pharmaceutical College, Guangxi Medical University, Nanning 530021, Guangxi, PR China. Email: [email protected](Q. L.).Weizhe Jiang, Pharmaceutical College, Guangxi Medical University, Nanning 530021, Guangxi, PR China. Email: [email protected](W. Z.). 1

1. Introduction Type 2 diabetes mellitus (T2DM) is a progressive chronic metabolic disease manifested by impaired β-cell function and insulin resistance1, 2. Currently, many oral antidiabetic agents, including metformin, sulfonylureas, thiazolidinediones, and insulin etc, are available to control high blood glucose, but these therapies do not address the reduction in β-cell mass and function2-4. As there is a progressive deterioration in β-cell function in patients with T2DM, increasing polypharmacy is needed to control their blood glucose, eventually, insulin is only treatment in most T2DM patients2,

3, 5, 6.

In addition, these agents are associated with

undesirable side effects, such as hypoglycemia, gastric symptoms, and body weight gain7. Thus it is necessary to search for a novel hypoglycemic drug with better pharmacological profiles in terms of both safety and impaired β-cell function7.

The Dipeptidyl Peptidase-IV (DPP-4) is a serine protease enzyme responsible for degradation of incretins, such as glucagon-like peptide-1 (GLP-1), which is a hormone responsible for the glucose-dependent stimulation of insulin by pancreatic β-cells8. Inhibition of DPP-4 increases the endogenous concentration of GLP-1 and reduces hyperglycemia8. To date, four DPP-4 inhibitors, including sitagliptin9, saxagliptin10, linagliptin11, and alogliptin12 are approved by US FDA, and vildagliptin13 is approved in Europe, and a variety of DPP-4 inhibitors have been in clinical trials and reported in the literature14 (Fig. 1). DPP-4 inhibitors not only demonstrate glycaemic control with low risk for hypoglycemia and weight gain15, but also have a number of protective effects on the β-cells, including a reduction in apoptosis and enhancement of βcell proliferation4, 16-18. However, there is still an unmet medical need for more therapeutic options as an increased risk for hospitalization for heart failure was associated with saxagliptin use19, and severe and persistent joint pain with use of sitagliptin was frequently reported15. Discovery of novel DPP-4 inhibitors with different chemical structure may provide a unique safety and efficacy profile differentiating from existing inhibitors20, which will ultimately benefit patients with T2DM. We have previously reported the identification of triazole-based DPP-4 inhibitors through modification at S2’ site21, 22. The triazole ring forms π-π stacking at S2’ site, the introduction of the carboxylic acid led to significant improvement of inhibitory activity through the formation of hydrogen bonds with Tyr752 at S2’ site or salt bridges with 2

Arg125 at S2 pocket21, 22. In order to increase the chemical space at S2’ site, we herein reported our research efforts toward the identification of benzoic acid-based DPP-4 inhibitor. These efforts ultimately led to the discovery of (R)-2-((4-(3-aminopiperidin-1-yl)-3-(but-2-yn-1-yl)2,6-dioxo-3,6-dihydropyrimidin-1(2H)-yl)methyl)-6-fluorobenzoic acid (compound 2b) with the IC50 of 1.7 nM (Fig. 1), which is more potent than the market drug alogliptin. Additionally, compound 2b exhibited no cytotoxicity in human hepatocyte LO2 cells. The in vivo study demonstrated that compound 2b have a robustly hypoglycemic effect in normal mice.

F

O

O

H 2N

F

N

N N

F

N

N

N H

HO

O

CF3

Sitagliptin

N N

O

O

N N N

N

N

Linaglitpin

NH2

O

CN

OH

F

N

O

NH2

N

NC

Saxagliptin

Vildagliptin

O N

N

HO

CN

NH2

Alogliptin

O N

O

N N

NH2

Compound 2b IC50 = 1.7 nM

Figure 1. The structures of marketed DPP-4 inhibitors and compound 2b.

2. Results and Discussion 2.1 Design strategy As supported by X-ray crystallography, the binding modes of DPP-4 inhibitor in DPP-4 enzyme are known8, 23, 24. Most of those inhibitors occupy S1 and S2 pockets of DPP-4 with variedly optimal groups. Alogliptin and linagliptin also bind at the S1’ site besides S1 and S2 pockets. The binding modes of these inhibitors in S1, S2 pockets and S1’ sites have been extensively studied14. Linagliptin has a distinctive feature that the characteristic methylquinazoline group binds at S2’ site, which leads to linagliptin as one of the most potent DPP-4 inhibitors (IC50 = 1 nM)24. S2’ site is a site in which only a few inhibitors bind, and thus leave this site a promising area to be explored25. The crystallographic structure (PDB ID: 2RGU) indicate that the S2’ site is a larger cavity surrounded by residues of Trp629, Trp627, Tyr752, His740, Gly741, and Lys554, in which Trp629 is a residue forming π-π stacking with the characteristic methylquinazoline group of linagliptin24. Furthermore, 3

Lys554 in this site is an attractive anchor that many potent DPP-4 inhibitors interacted with to enhance inhibitory activity, as exemplified by quinoline-based DPP-4 inhibitor developed by Takeda forms a hydrogen-bonding interaction with Lys554 for enhancing DPP-4 inhibition26, and 3H-imidazo[4,5-c]quinolin-4(5H)-ones based DPP-4 inhibitor discovered by Sumitomo Dainippon Pharma makes a salt bridge interaction with Lys554 to significantly improve the inhibitory activity27. Herein, we envisioned our new designed molecules target both residues Trp629 and Lys554. Our design is supported by the evidence that the distance between N3 of methylquinazoline of linagliptin and the primary amine of Lys554 is 5.5 Å (Fig. 2), which is an appropriate distance for adding a carbonyl or a carboxyl group to form a hydrogen bond or salt bridges with Lys554. Therefore, obenzoic acids or amides were incorporated to N3-position of uracil, with the speculation that the benzene ring forms π-π stacking with Trp629, and acid or amide forms salt bridges or hydrogen bond with Lys554. Thus compounds 1a-l and 2a-i were designed with a variety of amides or acids at o-positon of the phenyl ring (Fig. 2).

Figure 2 Structural superposition of crystal structures of DPP-4 binding alogliptin (PDB ID: 3G0B, magenta) and linagliptin (PDB ID: 2RGU, yellow), and the design strategy of compounds 1a-l and 2a-i.

2.2 Chemistry The synthetic routes adopted to obtain target compounds 1a-l are depicted in Scheme 1. The commercially available starting material 6-chlorouracil was selectively alkylated with 1-bromo-but2-yne to afford product 3, which was further converted to intermediate 4a-b by alkylation with methyl 2-(bromomethyl)benzoate or methyl 2-(2-(chloromethyl)phenyl)acetate. Treatment of 4a-b 4

with (R)-3-N-Boc-aminopiperidine afforded the desired compounds 5a-b, which were hydrolyzed with LiOH to give benzoic acid 6a-b. Compounds 5a-b were directly deprotected to give target compounds 1a-b. Condensation of the obtained 6a with a variety of amino-acid ester (7a-c) or amines (7d-f) by using classical amidation reaction, followed by basic hydrolysis and/or deprotection furnished the desired esters 1e-g, acids 1h-i, and amides 1j-l.

HN

O

a

NH

HN

Cl

O

O

O

O O

b

N

N

O

n

c

O

Cl

H N

N

Boc

5a n = 0 5b n = 1

4a n = 0 4b n = 1

3

N

N

N

O

Cl

O

O

O

n

d R1

O HO

N

R2

O

O

n

e

N

N

O

N

H N

O

n

f

N

N N

O

Boc

6a n = 0 6b n = 1

R1 O

n N

O

N

O

N

N

N

R4

N N

NH2

OMe

O

7a, 1e N

7d, 1j

7b, 1f N H

7e, 1k

O

7c, 1g

NH2

N

1h-i n = 0

O

OMe

N H

N

N

O

1e-g n = 0 1j-l n = 0

OMe

O

n

N

O

O N H

N

O

O

NH2

NH2

N

e, d R3

n

N

1a n = 0 1b n = 1

R2

1c n = 0 1d n = 1

7a-f 1e-g 1j-l

N O

Boc

d

O

N R1 = R2

H N

O

n

7a-f n = 0 d

HO

O

O

N R3 = R4

1h-i

N H

OH O

1h

OH

N

1i

N H

7f, 1l

Scheme 1. Synthesis of target compounds1a-l. Reagents and conditions: (a) 1-bromo-but-2-yne, DIPEA, DMF, r.t., 12 h; (b) 4a-b: methyl 2-(bromomethyl)benzoate (4a) or methyl 2-(2(chloromethyl)phenyl)acetate (4b), K2CO3, DMF, r.t. 12 h; (c) (R)-3-N-Boc-aminopiperidine, K2CO3, DMF, 60 °C, 10 h; (d) HCl gas, EA/ether, 0 °C; (e) LiOH, H2O/MeOH, r.t.; (f) NHR1R2, EDCI, HOBt, TEA, r.t., 12 h.

The target compounds 2a-i were prepared as shown in Scheme 2. The commercially available substituted methyl 2-methylbenzoates 8a-g were used as starting materials, which were brominated to give compounds 9a-g. Treating 9a-g with 3 gave compounds 10a-g. Compounds 10a-g, 5

followed the similar procedures for the preparation of compounds 1a-d, underwent substitution reaction with (R)-3-N-Boc-aminopiperidine, deprotection of Boc group and/or hydrolysis reaction to afford target compounds 2a-i. The structures of target compounds 1a-l and 2a-i obtained are listed in Table 1 and Table 2. O 6'

O

R5

5'

O

1'

4'

2' 3'

O

O

a

Br

R5

O

O

O

b O

8a-g

O

c

N

R5

N

N

R5

O

O

Cl

N

10a-g

9a-g

N

H N

Boc

11a-g d

O

OH

e

N

R5

O

O

O

OH

d

N N

H N

N

R5 O

Boc

12a-g

O

O N N

NH2

2a-g

O

O N

R5 O

N N

NH2

2h 2i

8e, 9e, 10e, 11e, 12e, 2e R5 = 4'-Br 8a, 9a, 10a, 11a, 12a, 2a R5 = 5'-F 8b, 9b, 10b, 11b, 12a, 2b R5 = 6'-F 8f, 9f, 10f, 11f, 12f, 2f R5 = 4'-MeO 8g, 9g, 10g, 11g, 12g, 2g R5 = 4'-CN 8c, 9c, 10c, 11c, 12c, 2c R5 = 5'-Cl 8d, 9d, 10d, 11d, 12d, 2d R5 = 5'-Br 2h R5 = 6'-F 2i R5 = 4'-Br

Scheme 2. Synthesis of target compounds 2a-i. Reagents and conditions: (a) NBS, (PhCO2)2, ClCH2CH2Cl, 80 °C, 12 h; (b) compound 3, K2CO3, DMF, r.t. 12 h; (c) (R)-3-N-Bocaminopiperidine, K2CO3, DMF, 60 °C, 10 h; (d) HCl gas, EA/ether, 0 °C; (e) LiOH, H2O/MeOH, r.t.. 2.3 In vitro DPP-4, DPP-8, and DPP-9 inhibition and SAR study The in vitro DPP-4 inhibitory activity of the compounds was measured using human recombinant DPP-4. To evaluate our compounds DPP-4 inhibitory activity, some approved drugs sitagliptin and alogliptin were used as references. To interact with both Trp629 and Lys554, we first designed and synthesized compound 1c with o-benzoic acid at the N3 position of uracil. Gratifyingly, compound 1c displayed potent DPP-4 inhibitory activity with an IC50 value of 7.5 nM, being almost comparable with that of sitagliptin (IC50 = 6.9 nM). However, compound 1a, an ester of compound 1c, lost ~10fold of potency (IC50 = 74.6 nM ) compared to compound 1c, which revealed the carboxylic acid group has a key interaction with the active site of DPP-4. To explore the tolerability of the functionalities at o-position of the benzene ring, we introduced various carboxylic acids into oposition of the benzene ring (compounds 1d, 1h, 1i). The activities of their corresponding esters (compounds 1b, 1e, 1f, 1g) were tested for comparison. However, acetic acid-substituted compound 6

1d, with two carbon lengths, exhibited a decrease in potency (IC50 = 89.8 nM). Compound 1b, the ester of compound 1d, showed a slight improvement in potency over its acid compound 1d, but their activities were less potent than that of carboxylic acid 1c. This result may due to their extended carboxylic acid groups lost the key interaction. The introduction of amino acids, such as flexible glycine (compounds 1h) and rigid proline (compounds 1i), did not improve the inhibitory activities. Moreover, there is essentially no difference of inhibitory activities between the acids and their corresponding esters (compounds 1e, 1f, 1h, 1i), which revealed their carboxylic acids may be incapable of having the key interaction to enhance the activity. In addition, replacement of the acid group with amides (compounds 1j, 1k, 1l) led to substantially lose the activity. All the results demonstrated that the carboxylic acid at o-position of the benzene ring is the optimal substituent, and lengthening the carbon chain or covering acid group were detrimental to activity.

Table 1. In vitro DPP-4 inhibitory activities of compounds 1a-l

R6

O N

N N

O

Compounds

R6 O

1a

O

1b

O

O

1c

OH

OH

1d 1e

O

O

O

H N

O

O

NH2

%Inhibition at 200 nM a

%Inhibition at 40 nM a

IC50 (nM) a, b

74.3±2.3

39.1±1.7

74.6

71.5±6.0

44.1±10.8

56.6

93.3±0.4

76.9.1±1.5

7.50

73.0±5.2

21.8±5.4

89.8

64.0±0.8

39.2±2.9

NT

42.0±3.2

18.6±1.0

NT

O O

1f

O

O

1g

O

1h

HO

O

N

H N

O

45.5±4.7

17.1±4.5

NT

H N

O

73.7±0.8

34.3±2.0

NT

7

O

1i

HO

1j

O

62.9±2.5

33.7±2.7

NT

60.2±2.5

23.3±5.0

NT

62.1±9.5

29.3±3.6

NT

42.6±4.5

18.8±4.3

NT

alogliptin

96.7±1.8

85.1±0.1

2.7

sitagliptin

82.7±4.22

73.5±1.8

6.9

N

N

1k

O

H N

1l

O

H N

a Measured b

O

in two independent experiments.

NT: not tested.

Based on these results above, we, therefore, kept o-benzoic acid moiety and focused on the introduction of a variety of substituent group to the benzene ring for further modification. As shown in Table 2, all of the compounds 2a-g were potent in vitro DPP-4 inhibitors with a single-digit nanomolar potency (IC50 < 7.6 nM), indicating good tolerability for a wide variety of substituents on the benzene ring. As a fluorine atom can exert a significant impact on the properties of a molecule that are of beneficial interest in drug design28, a fluorine group was first introduced to the benzene ring. Compound 2a, a 5’-fluoro substituted compound, retained the inhibitory activity (IC50 = 7.6 nM). Furthermore, the substitution of a fluorine group at the 6’-position led to the identification of compound 2b as one of the most potent DPP-4 inhibitor (IC50 = 1.7 nM), which was 4-fold more potent than compound 1c, and more potent than alogliptin. Subsequently, we focused on the introduction of the chlorine and bromine to benzene ring, the resulting compounds 2c (5’-Cl, IC50 = 3.9 nM), 2d (5’-Br, IC50 = 4.6 nM), 2e (4’-Br, IC50 = 2.5 nM) also displayed potent DPP-4 inhibitory activity, in which the activity of compound 2e was comparable with that of alogliptin. The comparison among compounds 2a, 2c, and 2d turned out that the order of potency was Cl > Br >F, indicating that the chlorine is the optimal group at 5’-position. Moreover, electron-donating methoxyl group attached to 4’-position (1f, IC50 = 4.0 nM) also improve the DPP-4 activity compared with compound 1c. Pissarnitski et al reported the cyano group can form extended hydrogen-bonding network in the S2’ site29, thus cyano group was introduced to 4’-position to give compound 2g with an IC50 value of 3.5 nM. The results suggested that both electron-donating and 8

electron-withdrawing groups tolerated the inhibitory activity at the 4’-position. Table 2. In vitro DPP-4, DPP-8 and DPP-9 inhibitory activities of compounds 2a-i O

OH

1' R5

2'

6' 5' 4'

O

O N

N

3' O

O

1' N

NH2

R5

2'

6' 5' 4'

N

3' O

2a-g

Compounds

O N N

NH2

2h-i

%Inhibition

%Inhibition

DPP-4

DPP-8

DPP-9

at 200 nM a

at 40 nM a

IC50 (nM) a

IC50 (μM) a

IC50 (μM) a

cLogP b

R5

1c

H

93.3±0.4

76.9.1±1.5

7.5

>100

>100

-0.78

2a

5’-F

92.8±0.8

76.4±1.0

7.6

>100

>100

-0.54

2b

6’-F

97.3±1.3

88.4±1.5

1.7

>100

>100

-0.54

2c

5’-Cl

95.8±1.1

82.0±0.3

3.9

>100

>100

0.03

2d

5’-Br

96.1±1.8

79.6±2.6

4.6

>100

>100

0.18

2e

4’-Br

93.6±1.7

82.2±3.0

2.5

>100

>100

0.18

2f

4’-MeO

91.1±1.2

75.2±4.6

4.0

>100

>100

-0.64

2g

4’-CN

96.3±2.8

85.0±1.5

3.5

>100

>100

-1.12

2h

6’-F

69.1±2.0

36.6±2.7

93.2

>100

>100

2.15

2i

4’-Br

20.5±4.5

3.2±3.2

395.0

>100

>100

2.88

alogliptin

96.7±1.8

85.1±0.1

2.7

>100

>100

vildagliptin

NT

NT

NT

2.3

0.14

a Measured b

at least in two independent experiments.

clogP were calculated by ChemBioDraw software Ultra 14.0.

Some studies have indicated that inhibition of the other members of serine protease family (DPP-8 and DPP-9) may cause severe toxicities 30, thus it is very important to the development of DPP-4 inhibitor without DPP-8 and DPP-9 inhibitory activity. We evaluated the inhibitory activities of DPP-8 and DPP-9. As shown in Table 2, all selected compounds showed no inhibition for DPP-8 or DPP-9, with the IC50 > 100 μM, while vildagliptin inhibited both DPP-8 and DPP-9, with IC50 of 2.3 μM and 0.14 μM, respectively. The results indicated that the designed compounds have excellent selectivity against DPP-8 and DPP-9.

9

Optimal lipophilicity is of paramount importance for good oral bioavailability and pharmacokinetics31. Studies suggested that the mean cLogP values ranging from 2.3 to 2.5 were the best for oral drugs32. Thus, we calculated clogP values for selected compounds by ChemBioDraw software Ultra 14.0. Although compounds 2a-2g were potent in vitro DPP-4 inhibitors, they had low cLogP values (ranging from -1.12 to 0.18), indicating these compounds may suffer from poor oral adsorption. Thus the methyl ester was introduced to compounds 2b and 2e to construct compounds 2h and 2i, which may convert into the corresponding acid 2b and 2e in vivo. The compounds 2h and 2i dramatically lost potency of inhibiting DPP-4 in vitro (Table 2), but they demonstrated a substantial improvement in cLogP values (2.15-2.88), which may allow improvement of oral adsorption. Then we selected compounds 2b, 2e, 2h, and 2i for further evaluation.

2.4 Molecular docking study To investigate the binding modes of designed compounds, We used the molecular docking program GLIDE 5.9 to dock selected compounds into a DPP-4 crystal structure (PDB ID: 2RGU). The docking results are shown in Figure 3. The overlay of compound 1c against linagliptin shows that the uracil ring, 2-butynyl group and (3R)-aminopiperidine group interact with DPP-4 in a similar way with linagliptin. Furthermore, the phenyl of o-benzoic acid moiety forms π-π stacking with Trp629, and carboxylic acid makes salt bridges with a primary amine of Lys554 with a distance of 3.8 Å. The addition of the salt bridges may contribute to increasing in potency, which therefore provides theoretical evidence supporting our speculation that interacting with both Trp629 and Lys554 could enhance the inhibitory activity, and interprets decreased potency when covering the carboxylic acid by esters or amides.

To interpret the SAR of the designed compounds, the most potent compounds 2b and 2e were docked into a DPP-4 crystal structure (PDB ID: 2RGU). As shown in Figure 3B and 3C, compound 2b displays similar binding modes with compound 1c and linagliptin. But some subtle changes in the binding of o-benzoic acid moiety were observed in the overlayed structure of compound 1c, 2b, and 2e. The benzene ring of o-benzoic acid moiety rotates about its axis to make about 0.5 Å movement. The movement led to the benzene ring is parallel to the indole of Trp629, which may 10

result in forming π–π interaction more efficiently with the side chain of Trp629 in the S2’ site. Recently, Pissarnitski et al reported the crystal structure of the 2-cyano benzyl substituted tricyclic guanines DPP-4 inhibitor29, the o-cyano group interacts with Lys554 through hydrogen bonds29. The structural superposition of compound 2b and the tricyclic guanines showed that o-carboxylic acid and the o-cyano group interact with Lys554 in a similar fashion, and both benzene rings were parallel to the indole of Trp629 (Fig. 3D). These results further supported the feasibility of our design that DPP-4 inhibitors could target both residues Trp629 and Lys554.

Figure 3. The docking binding modes of compounds and their structural superposition in DPP-4 active site. Hydrogen bonds are depicted as dashed black lines. DPP-4 is represented in surface. Compounds are shown in stick. Fig. 3A, the overlayed structure of 1c (cyan) with alogliptin (magenta) and linagliptin (yellow). Fig. 3B, the superposition of 2b (green) and linagliptin (yellow). Fig. 3C, the superposition of 1c (cyan) 2b (green), and 2e (wheat). Fig. 3D, the overlayed structure of 2b (green) against tricyclic guanines29 (blue)

2.5 Effects of compounds 2b, 2e, 2h, and 2i on cell viability The toxicity of the compounds 2b, 2e, 2h, and 2i was evaluated on normal hepatic LO2 cell using the MTT assay. Anticancer drug Doxorubicin (DOX) was served as controls. As present in Figure 11

4, the % viability of compounds 2b, 2e, and 2h at 100 μM and 50 μM were approximately 70% and 90%, which indicated those compounds displayed low cytotoxicity against normal hepatic LO2 cell line. In contrast, compounds 2i was found to exhibit higher toxicity (% viability at 100 μM was 14%) in the cell line, as well as the positive control (% viability at 20 μM was 11%). On the basis of the toxicity data, compounds 2b and 2h were selected for in vivo hypoglycemic studies.

Figure 4. Cytotoxicity of compounds 2b, 2e, 2h, and 2i against normal hepatic LO2 cell.

2.6 Hypoglycemic effects of 2b and 2h in normal mice Based on in vitro potency and toxicity data, compounds 2b and 2h were selected for acute efficacy evaluation by the oral glucose tolerance test (OGTT) in Kunming (KM) mice. Compounds 2b, 2h and reference drug alogliptin at the dose of 10 mg kg−1 were orally administered to KM mice. The time-dependent changes in plasma glucose levels and the corresponding area under the curve (AUC0–120 min) are shown in Figure 5. Alogliptin produced a significant decrease in glucose level with the decreased (AUC)0–120 min of 30.0% at a dose of 10 mg/kg. Compound 2b reduced the AUC0–120 min value to 20.5%, and compound 2h reduced the value to 27.3%. The results indicated that the hypoglycemic activity of the compound 2h approximated that of alogliptin, but was more potent than that of compound 2b. The reason may be: the low cLogP value of compound 2h resulted in low membrane permeability, and compound 2h was converted into 2b in vivo. To confirm that the ester 2h can be converted into acid 2b, we tested the plasma stability of compound 2h. Compound 2h was incubated with rat plasma at 37 °C for 0 and 30 min, the concentration of ester 2h and acid 2b were analyzed by LC/MS/MS system. The results indicated that part of compound 2h molecules were converted into compound 2b after incubation for 30 min (Figure S1-3). 12

In order to investigate the in vivo hypoglycemic potency of compound 2h at different doses, 2h at the doses of 3 mg kg−1 and 1 mg kg−1 were orally administered to KM mice. As shown in Figure 5, compound 2h exerts a dose-dependently hypoglycemic effect, with the reduced the AUC0–120 min value of 18.8% and 13.3% at the dose of 3 mg/kg and 1mg/kg, respectively. The hypoglycemic activity of the compound 2h was comparable with that of referenced drug alogliptin, which decreased 17.5% of (AUC)0–120 min value at a dose of 3 mg/kg.

Figure 5. Effects of compounds 2b and 2h on blood glucose levels during an OGTT in male Kunming mice (A) Time-dependent changes of plasma glucose levels of compounds 2b and 2h at the dose of 10 mg/kg. (B) AUC0–120 min of blood glucose levels of compounds 2b and 2h at the dose of 10 mg/kg. (C) Time-dependent changes of plasma glucose levels of compound 2h at the dose of 3 mg/kg and 1 mg/kg. (D) AUC0–120 min of blood glucose levels of compound 2h at the dose of 3 mg/kg and 1 mg/kg. Values are mean ± SEM (n = 8). * P≤0.05 compared to vehicle-treated group by using a one-way ANOVA with Dunnett's multiple comparisons test. 13

3. Conclusion In summary, the introduction of o-benzoic acids to N3-position of uracil, with the aim to target both residues Trp629 and Lys554, resulted in identification of 1c as a novel DPP-4 inhibitor with potent activity. Further optimization on the benzene ring of 1c obtained a series of novel o-benzoic acidsubstituted uracil derivatives, which were potent DPP-4 inhibitors with IC50 values in 1.7–7.6 nM and showed good selectivity over other related enzymes including DPP-8, and DPP-9. In particular, the 6-fluorine substituted o-benzoic acid 2b displayed the most potent DPP-4 inhibition with an IC50 value of 1.7 nM, which was more potent than alogliptin. Docking study revealed the additional salt bridge interaction formed between the carboxylic acid and primary amine of Lys554 is key to enhance the activity. In addition , compounds 2b and 2h showed hardly any toxicity toward the LO2 cell line. Further in vivo evaluation indicated that the ester of 2b (2h) can reduce blood glucose excursion in both normal mice in a dose-dependent manner. The overall results showed that compound 2b promises to be a safe and efficacious DPP-4 inhibitors for the treatment of T2DM 4. Experimental section All reagents were purchased from commercial sources and used without further purification unless otherwise indicated. Reactions were monitored by TLC on silica gel 60 F254 plates. Column chromatography was performed on silica gel (200-300 mesh). Melting points were measured on capillary tube and were uncorrected. 1H NMR and

13C

NMR spectra (DMSO-d6, CDCl3) were

recorded with a Bruker spectrometer in the indicated solvents (TMS as internal standard): the values of the chemical shifts are expressed in δ values (ppm) and the coupling constants (J) in Hz. MS spectra were determined on ThermoFisher TSQ Series Mass spectrometer system (ESI, TQU04153). %Purity of the target compounds were determined by HPLC analysis (UV detector, wavelength: 254 nm, mobile phase composed of mathanol (0.1% HCOOH) and H2O. 1-(but-2-ynyl)-6-chloropyrimidine-2,4(1H,3H)-dione (3) To a mixture of 6-chlorouracil (10 g, 69.0 mmol) and DIPEA (9.7 g, 75.0 mmol) in DMF (30 mL) was added 1-bromo-2-butyne (9.9 g, 75.0 mmol). The reaction mixture was stirred at r.t. for 12 h. Water (150 mL) was added, the precipitate was collected by filtration, washed with water and EtOH, and dried to give compound 3 as a light yellow solid (11.0 g, 80%). mp: 216-217 °C; 1H NMR (300 MHz, DMSO-d6): δ 11.71 (s, 1H), 5.99 (s, 1H), 4.65 (s, 2H), 1.80 (s, 3H); MS (ESI) m/z: 199.02 14

[M+H]+. methyl

2-((3-(but-2-yn-1-yl)-4-chloro-2,6-dioxo-3,6-dihydropyrimidin-1(2H)-yl)methyl)

benzoate (4a) To a suspension of 3 (8 g, 40.3 mmol) and K2CO3 (16.7 g, 120.8 mmol) in DMF (50 mL) was added methyl 2-(bromomethyl)benzoate (9.7 g, 42.3 mmol). The reaction mixture was stirred at r.t. for 10 h. The reaction mixture was poured into water. The precipitate was collected by filtration, washed with water, and dried to give compound 4 as a brown solid (10.5 g, 75%). mp: 140-141 °C; 1H NMR (400 MHz, CDCl3) δ 8.10 (s, 1H), 7.95 (d, J = 7.8 Hz, 1H), 7.65 (d, J = 7.7 Hz, 1H), 7.38 (t, J = 7.7 Hz, 1H), 5.97 (s, 1H), 5.13 (s, 2H), 4.84 – 4.67 (m, 2H), 3.90 (s, 3H), 1.81 (s, 3H); MS (ESI) m/z: 347.26 [M+H]+. methyl (R)-2-((3-(but-2-yn-1-yl)-4-(3-((tert-butoxycarbonyl)amino)piperidin-1-yl)-2,6-dioxo3,6-dihydropyrimidin-1(2H)-yl)methyl)benzoate (5a) A mixture of compound 4 (4.0 g, 11.5 mmol), (R)-3-(N-Boc-amino)piperidine (2.8 g, 13.8 mmol) and K2CO3 (4.8 g, 34.6 mmol) in DMF (20 mL) was stirred at 65 °C for 10 h. After cooling to r.t., water was added, and extracted with dichloromethane (3 × 30 mL). The combined organic layer was washed with saturated brine, dried over anhydrous Na2SO4, and concentrated to give the crude product, which was purified by flash chromatography (dichloromethane/MeOH 100:1~30:1) to give 5a as a light yellow solid (3.6 g, 61%). mp: 100-101 °C. 1H NMR (400 MHz, CDCl3) δ 7.94 (d, J = 7.8 Hz, 1H), 7.40 (t, J = 7.8 Hz, 1H), 7.28 (t, J = 7.8 Hz, 1H), 7.08 (d, J = 7.8 Hz, 1H), 5.54 (s, 2H), 5.29 (s, 1H), 4.66 (d, J = 16.9 Hz, 1H), 4.48 (d, J = 16.9 Hz, 1H), 3.92 (s, 3H), 3.90 – 3.76 (m, 1H), 3.42 – 3.23 (m, 1H), 3.16 – 3.17 (m, 1H), 2.95 – 2.78 (m, 1H), 2.71 – 2.64 (m, 1H), 1.97 – 1.84 (m, 2H), 1.80 (s, 3H), 1.78 – 1.70 (m, 1H), 1.66 – 1.62 (m, 1H), 1.45 (s, 9 H); MS (ESI) m/z: 511.28 [M+H]+. (R)-2-((3-(but-2-yn-1-yl)-4-(3-((tert-butoxycarbonyl)amino)piperidin-1-yl)-2,6-dioxo-3,6dihydropyrimidin-1(2H)-yl)methyl)benzoic acid (6a) To the solution of compound 5a (5 g, 9.4 mmol) in methanol (30 mL) was added LiOH (1M, 20 mL). The resulting mixture was stirred at r.t. for 24 h. After evaporation of solvent, the residue was dissolved in water (80 mL) and acidified with HCl to adjust the pH value to 3. The precipitate was collected by filtration give compound 6a (3.5 g, 71%) as a light yellow solid. mp: 122-126 °C; 1H NMR (400 MHz, DMSO-d6) δ 13.07 (s, 1H), 7.87 (d, J = 7.6 Hz, 1H), 7.43 (t, J = 7.6 Hz, 1H), 7.31 15

(t, J = 7.6 Hz, 1H), 6.97 (d, J = 7.6 Hz, 1H), 6.89 (d, J = 8.0 Hz, 1H), 5.29 (s, 2H), 5.23 (s, 1H), 4.57 – 4.33 (m, 2H), 3.58 – 3.51 (m, 1H), 3.29 – 3.26 (m, 2H), 3.22 – 3.17 (m, 1H), 2.77 – 2.69 (m, 1H), 2.57 – 2.51 (m, 1H), 2.81 – 1.77 (m, 2H), 1.74 (s, 3H), 1.63 – 1.54 (m, 1H), 1.37 (s, 9H); MS (ESI) m/z: 495.40 [M-H]-. methyl (R)-2-((4-(3-aminopiperidin-1-yl)-3-(but-2-yn-1-yl)-2,6-dioxo-3,6-dihydropyrimidin1(2H)-yl)methyl)benzoate (1a) Compound 5a (300 mg, 0.6 mmol) was dissolved in EtOAc (10 mL) and ether (30 mL). Freshly prepared HCl gas was bubbled into the solution at °C. After the completed consumption of starting materials, the mixture was concentrated under reduced presure. The residue was dissolved in water (20 mL), and neutralized with NaHCO3, and extracted with dichloromethane (30 mL). The organic layer was washed with brine, dried over anhydrous Na2SO4, concentrated to give compound 1a as a light yellow solid (238 mg, 98%); HPLC purity: 95.8%; mp: 63-65 °C; 1H NMR (500MHz, DMSOd6): δ 7.90 (dd, J = 7.8, 1.2 Hz, 1H), 7.53 (td, J = 7.8, 1.2 Hz, 1H), 7.39 (t, J = 7.8 Hz, 1H), 7.00 (d, J = 7.8 Hz, 1H), 5.29 (s, 2H), 5.22 (s, 1H), 4.62 – 4.43 (m, 2H), 3.89 (s, 3H), 3.27 – 3.16 (m, 3H), 2.87 – 2.82 (m, 1H), 2.75– 2.71 (m, 1H), 2.49 – 2.44 (m, 1H), 1.91 – 1.83 (m, 1H), 1.79 (t, J = 2.2 Hz, 3H), 1.78 – 1.76 (m, 1H), 1.65 – 1.54 (m, 1H);

13C

NMR (101 MHz, DMSO-d6) δ 167.39,

162.31, 160.02, 152.32, 138.74, 132.97, 130.72, 128.78, 127.28, 125.97, 87.83, 80.06, 75.00, 59.19, 52.60, 51.19, 47.68, 42.63, 36.15, 33.28, 23.38, 3.52; MS (ESI) m/z: 433.59 [M+Na]+. methyl

(R)-2-(2-((4-(3-aminopiperidin-1-yl)-3-(but-2-yn-1-yl)-2,6-dioxo-3,6-dihydro

pyrimidin-1(2H)-yl)methyl)phenyl)acetate hydrochloride (1b) Following a similar procedure for the preparation of 1a. 1b was prepared starting from compound 3 and methyl 2-(2-(chloromethyl)phenyl)acetate. White solid (50 mg, yield 45%); HPLC purity: 99.5%; mp: 134-136 °C; 1H NMR (400 MHz, DMSO-d6) δ 8.43 (s, 3H), 7.18 – 7.17 (m, 3H), 7.05 – 6.95 (m, 1H), 5.24 (s, 1H), 4.90 (s, 2H), 4.62 (d, J = 17.3 Hz, 1H), 4.43 (d, J = 17.5 Hz, 1H), 3.87 (s, 2H), 3.59 (s, 3H), 3.39 – 3.28 (m, 2H), 3.21 – 3.03 (m, 1H), 2.99 – 2.83 (m, 2H), 2.01 – 1.83 (m, 2H), 1.75 (s, 3H), 1.70 – 1.54 (m, 2H); 13C NMR (101 MHz, DMSO-d6) δ 171.85, 162.28, 159.45, 152.12, 136.41, 132.90, 131.11, 127.68, 127.40, 126.96, 88.77, 80.18, 74.97, 52.58, 52.21, 51.80, 46.46, 41.35, 38.22, 36.10, 27.43, 21.73, 3.60; MS (ESI) m/z: 425.35 [M+H]+.

(R)-2-((4-(3-aminopiperidin-1-yl)-3-(but-2-yn-1-yl)-2,6-dioxo-3,6-dihydropyrimidin-1(2H)yl)methyl)benzoic acid hydrochloride (1c) 16

The compound 6a (500 mg, 1.0 mmol) was dissolved in EtOAc (10 mL) and ether (30 mL). Freshly prepared HCl gas was bubbled into the solution at °C. After TLC analysis indicated the completed consumption of starting materials, the precipitate was collected by filtration, dried in vacuo to afford compound 1a as a white solid (242 mg, yield: 63%); HPLC HPLC purity: 97.4%; mp: 210-212 °C; 1H

NMR (500MHz, DMSO-d6): δ 13.06 (s, 1H), 8.28 (s, 3H), 7.92 (dd, J = 7.8, 1.2 Hz, 1H), 7.54 –

7.44 (m, 1H), 7.36 (t, J = 7.5 Hz, 1H), 6.94 (d, J = 7.8 Hz, 1H), 5.32 (s, 3H), 4.64 (d, J = 17.0 Hz, 1H), 4.49 (d, J = 17.0 Hz, 1H), 3.39 (m, 2H), 3.14 (s, 1H), 3.03 (s, 1H), 2.95 – 2.85 (m, 1H), 2.03 – 1.88 (m, 2H), 1.65 (s, 3H), 1.69 – 1.60 (m, 2H). 13C NMR (101 MHz, DMSO-d6) δ 168.64, 162.32, 159.58, 152.15, 138.65, 132.62, 130.97, 129.71, 127.12, 125.53, 88.72, 80.18, 74.97, 52.61, 51.79, 46.49, 42.95, 36.18, 27.49, 21.86, 3.60; MS (ESI) m/z: 395.58 [M-H]-. methyl

(R)-2-(2-((4-(3-aminopiperidin-1-yl)-3-(but-2-yn-1-yl)-2,6-dioxo-3,6-dihydro

pyrimidin-1(2H)-yl)methyl)phenyl)acetate hydrochloride (1d) Following a similar procedure for the preparation of 1c. 1d was prepared starting from compound 3 and methyl 2-(2-(chloromethyl)phenyl)acetate. White solid (80 mg, yield 52%); HPLC purity: 96.4%; mp: 193-195 °C; 1H NMR (400 MHz, DMSO-d6) δ 12.45 (s, 1H), 8.43 (s, 3H), 7.18 – 7.14 (m, 3H), 7.06 – 6.85 (m, 1H), 5.24 (s, 1H), 4.91 (s, 2H), 4.62 (d, J = 17.3 Hz, 1H), 4.43 (d, J = 17.2 Hz, 1H), 3.77 (s, 2H), 3.44 – 3.32 (m, 2H), 3.15 – 3.04 (m, 1H), 3.01 – 2.80 (m, 2H), 2.03 – 1.83 (m, 2H), 1.75 (s, 3H), 1.70 – 1.50 (m, 2H); 13C NMR (101 MHz, DMSO-d6) δ 172.85, 162.30, 159.45, 152.12, 136.36, 133.52, 131.06, 127.46, 127.23, 126.39, 88.78, 80.18, 74.98, 52.59, 51.79, 46.47, 41.33, 38.79, 36.12, 27.45, 21.79, 3.61; MS (ESI) m/z: 411.29 [M+H]+.

methyl (R)-(2-((4-(3-aminopiperidin-1-yl)-3-(but-2-yn-1-yl)-2,6-dioxo-3,6-dihydropyrimidin1(2H)-yl)methyl)benzoyl)glycinate hydrochloride (1e) Compound 6a (500 mg, 1.0 mmol), methyl glycinate hydrochloride (139 mg, 1.1 mmol), EDCI (289 mg, 1.5 mmol), HOBt (204 mg, 1.5 mmol) and DIPEA (0.4 mL, 2.5 mmol) were added to dry dichloromethane (20 mL). The mixture was stirred at r.t. for 12 h. After TLC analysis indicated the completed consumption of starting materials, water (20 mL) was added. The organic layer was washed successively with 0.5 M of NaOH, 0.5 M of HCl and brine, dried over anhydrous Na2SO4, concentrated to give crude product. The crude product was purified by flash chromatography (dichloromethane/methanol, 100:1~ 30:1) to afford amide intermediate. The Boc group of the intermediate was deprotected using a similar procedure for the preparation of compound 1c. 17

Compound 1e, white solid (378 mg, 79%); HPLC purity: 96.4%; mp: 173-176 °C; 1H NMR (300MHz, DMSO-d6): δ 8.91 (t, J = 5.8 Hz, 1H), 8.29 (s, 3H), 7.51 (d, J = 7.5 Hz, 1H), 7.40 (t, J = 7.5 Hz, 1H), 7.33 (t, J = 7.5 Hz, 1H), 6.90 (d, J = 7.5 Hz, 1H), 5.31 (s, 1H), 5.17 (s, 2H), 4.64 (d, J = 17.0 Hz, 1H), 4.52 – 4.45 (m, 1H), 4.10 – 4.00 (m, 2H), 3.68 (s, 3H), 3.43 – 3.36 (m, 2H), 3.14 (s, 1H), 3.02 (s, 1H), 2.92 – 2.90 (m, 1H), 2.04 – 1.87 (m, 2H), 1.79 (s, 3H), 1.70 – 1.60 (m, 2H); 13C

NMR (101 MHz, DMSO-d6) δ 170.69, 169.18, 162.32, 159.54, 152.12, 135.86, 134.76, 130.54,

128.00, 126.93, 125.22, 88.71, 80.18, 74.98, 52.61, 52.20, 51.78, 46.48, 42.19, 41.49, 36.16, 27.48, 21.86, 3.59; MS (ESI) m/z: 490.71 [M+Na]+. methyl (2-((4-((R)-3-aminopiperidin-1-yl)-3-(but-2-yn-1-yl)-2,6-dioxo-3,6-dihydropyrimidin1(2H)-yl)methyl)benzoyl)-L-prolinate hydrochloride (1f) Following a similar procedure for the preparation of 1e. 1f was prepared starting from compound 6a and methyl L-prolinate hydrochloride. White solid (120 mg, yield 56%); HPLC purity: 95.6%; mp: 180-183 °C; 1H NMR (300MHz, DMSO-d6): δ 8.25 (s, 3H), 7.44 – 7.22 (m, 3H), 7.12 – 6.99 (m, 1H), 5.30 (s, 1H), 5.12 – 4.90 (m, 2H), 4.78 – 4.57 (m, 1H), 4.57 – 4.42 (m, 2H), 3.69 (s, 3H), 3.45 – 3.35 (m, 3H), 3.30 – 3.24 (m, 1H), 3.12 (s, 1H), 3.03 (s, 1H), 2.95 – 2.82 (m, 1H), 2.36 – 2.30 (m, 1H), 2.03 – 1.86 (m, 5H), 1.84 – 1.74 (m, 3H), 1.66 – 1.55 (m, 2H); 13C NMR (101 MHz, DMSO-d6) δ 172.69, 168.46, 162.18, 159.55, 152.12, 136.27, 134.09, 129.58, 127.29, 126.09, 88.74, 80.19, 74.97, 58.50, 52.60, 52.34, 51.80, 49.17, 46.45, 41.32, 36.16, 29.58, 27.46, 24.96, 21.75, 3.59. MS (ESI) m/z: 530.76 [M+Na]+. methyl (2-((4-((R)-3-aminopiperidin-1-yl)-3-(but-2-yn-1-yl)-2,6-dioxo-3,6-dihydropyrimidin1(2H)-yl)methyl)benzoyl)-L-alaninate hydrochloride (1g) Following a similar procedure for the preparation of 1e. 1g was prepared starting from compound 6a and methyl L-alaninate hydrochloride. White solid (275 mg, yield 62%); HPLC purity: 95.8%; mp: 172-173 °C; 1H NMR (300MHz, DMSO-d6): δ 8.89 (d, J = 6.9 Hz, 1H), 8.23 (s, 3H), 7.49 (dd, J = 7.5, 1.0 Hz, 1H), 7.39 (dt, J = 7.8, 3.8 Hz, 1H), 7.32 (t, J = 7.5 Hz, 1H), 6.91 (d, J = 7.8 Hz, 1H), 5.32 (s, 1H), 5.22 – 5.10 (m, 2H), 4.64 (d, J = 17.5 Hz, 1H), 4.51 – 4.46 (m, 2H), 3.67 (s, 3H), 3.44 – 3.37 (m, 2H), 3.13 (s, 1H), 3.02 (s, 1H), 2.92 – 2.90 (m, 1H), 2.04 – 1.85 (m, 2H), 1.85 – 1.74 (m, 3H), 1.69 – 1.60 (m, 2H), 1.40 (d, J = 7.3 Hz, 3H).

13C

NMR (101 MHz, DMSO-d6) δ 173.48,

168.73, 162.34, 159.54, 152.16, 135.74, 134.95, 130.43, 128.19, 126.87, 125.29, 88.70, 80.17, 74.97,

18

52.60, 52.32, 51.77, 48.58, 46.47, 42.07, 36.18, 27.48, 21.84, 17.06, 3.59; MS (ESI) m/z: 482.73 [M+H]+. (R)-(2-((4-(3-aminopiperidin-1-yl)-3-(but-2-yn-1-yl)-2,6-dioxo-3,6-dihydropyrimidin-1(2H)yl)methyl)benzoyl)glycine hydrochloride (1h) To the solution of amide intermediate of compound 6a (500 mg, 1.0 mmol) in methanol (8 mL) was added LiOH (1M, 1.2 mL). The resulting mixture was stirred at r.t. for 12 h. After evaporation of solvent, the residue was dissolved in water (80 mL), and acidified with HCl to adjust the pH value to 3. The precipitate was collected by filtration, purified by flash chromatography (dichloromethane/methanol, 50:1~10:1) to give acid intermediate, which was undergone deprotection of the Boc group by using a similar procedure for the preparation of compound 1c to give compound 1h as a white solid (340 mg, 75%);. HPLC purity: 95.2%; mp: 210-211 °C; 1H NMR (500 MHz, DMSO-d6): δ 12.63 (s, 1H), 8.78 (t, J = 6.0 Hz, 1H), 8.22 (s, 3H), 7.52 (dd, J = 7.5, 1.2 Hz, 1H), 7.39 (td, J = 7.5, 1.2 Hz, 1H), 7.33 (t, J = 7.5 Hz, 1H), 6.89 (d, J = 7.5 Hz, 1H), 5.31 (s, 1H), 5.18 (s, 2H), 4.64 (d, J = 17.5 Hz, 1H), 4.49 (d, J = 17.5 Hz, 1H), 3.94 (d, J = 6.0 Hz, 2H), 3.41 – 3.36 (m, 2H), 3.14 (s, 1H), 3.02 (s, 1H), 2.93 – 2.85 (m, 1H), 2.02 – 1.87 (m, 2H), 1.79 (t, J = 2.1 Hz, 3H), 1.73 – 1.58 (m, 2H).

13C

NMR (101 MHz, DMSO-d6) δ 171.53, 169.00, 162.32,

159.54, 152.13, 142.80, 135.83, 134.95, 130.45, 128.06, 126.89, 125.20, 88.73, 80.19, 74.97, 52.62, 51.78, 46.48, 42.21, 41.52, 36.15, 27.48, 21.81, 3.60; MS (ESI) m/z: 452.70 [M-H]-. (2-((4-((R)-3-aminopiperidin-1-yl)-3-(but-2-yn-1-yl)-2,6-dioxo-3,6-dihydropyrimidin-1(2H)yl)methyl)benzoyl)-L-proline hydrochloride(1i) Following a similar procedure for the preparation of 1i. 1i was prepared starting from compound 6a and methyl L-prolinate hydrochloride. White solid (85 mg, yield 42%); HPLC purity: 96.1%; mp: 215-218 °C; 1H NMR (300MHz, DMSO-d6): δ 12.58 (s, 1H), 8.23 (s, 3H), 7.41 – 7.21 (m, 3H), 7.18 – 6.98 (m, 1H), 5.34 – 5.28 (m, 1H), 5.16 – 4.89 (m, 2H), 4.76 – 4.56 (m, 1H), 4.54 – 4.42 (m, 2H), 3.52 – 3.45 (m, 3H), 3.27 – 3.24 (m, 1H), 3.10 (s, 1H), 3.04 (s, 1H), 2.96 – 2.82 (m, 1H), 2.39 – 2.26 (m, 1H), 2.04 – 1.85 (m, 5H), 1.84 – 1.74 (m, 3H), 1.73 – 1.56 (m, 2H). 13C NMR (101 MHz, DMSO-d6) δ 173.59, 168.35, 162.18, 159.50 (d, J = 7.5 Hz), 152.10, 136.53, 134.02, 129.47, 127.28, 126.14, 88.72, 80.20, 74.96, 58.59, 52.58, 51.80, 49.17, 46.43, 41.35, 36.15, 29.66, 27.43, 24.97, 22.23 (d, J = 94.6 Hz), 3.59. MS (ESI) m/z: 494.70 [M+H]+. (R)-2-((4-(3-aminopiperidin-1-yl)-3-(but-2-yn-1-yl)-2,6-dioxo-3,6-dihydropyrimidin-1(2H)19

yl)methyl)-N,N-dimethylbenzamide hydrochloride (1j) Following a similar procedure for the preparation of 1e. 1j was prepared starting from compound 6a and dimethylamine hydrochloride. White solid (110 mg, yield 74%); HPLC purity: 95. 3%; mp: 168-170 °C; 1H NMR (400 MHz, DMSO-d6) δ 8.24 (s, 3H), 7.28 (p, J = 7.2 Hz, 2H), 7.18 (d, J = 7.1 Hz, 1H), 7.03 (d, J = 7.3 Hz, 1H), 5.25 (s, 1H), 4.87 (s, 2H), 4.61 (d, J = 17.1 Hz, 1H), 4.44 (d, J = 17.7 Hz, 1H), 3.32 – 3.24 (m, 3H), 3.17 – 3.04 (m, 1H), 2.98 (s, 3H), 2.89 – 2.83 (m, 1H), 2.79 (s, 3H), 2.02 – 1.81 (m, 2H), 1.76 (s, 3H), 1.71 – 1.52 (m, 2H). 13C NMR (101 MHz, DMSO-d6) δ 169.69, 162.11, 159.47, 152.04, 136.23, 134.04, 129.07, 127.18, 126.69, 126.39, 88.76, 80.17, 74.99, 52.62, 51.79, 46.44 (d, J = 13.4 Hz), 41.63, 36.09, 34.65, 27.48, 26.31 (d, J = 7.9 Hz), 21.91, 3.57. MS (ESI) m/z: 424.30 [M+H]+. (R)-2-((4-(3-aminopiperidin-1-yl)-3-(but-2-yn-1-yl)-2,6-dioxo-3,6-dihydropyrimidin-1(2H)yl)methyl)-N-ethylbenzamide (1k) Following a similar procedure for the preparation of 1e. 1k was prepared starting from compound 6a and ethylamine hydrochloride. After completion of reaction, the solvent was concentrated in reduced presure. The resulting residues were dissolved in dichloromethane, washed with NaHCO3, dried over anhydrous Na2SO4, concentrated to give compound 1k as a light yellow solid (171 mg, 83%); HPLC purity: 96.1%; mp: 96-100 °C; 1H NMR (400 MHz, DMSO-d6) δ 8.39 (d, J = 5.9 Hz, 1H), 7.40 (d, J = 7.5 Hz, 1H), 7.32 (t, J = 7.6 Hz, 1H), 7.26 (t, J = 7.4 Hz, 1H), 6.87 (d, J = 7.7 Hz, 1H), 5.19 (s, 1H), 5.09 (s, 2H), 4.47 (s, 2H), 3.25 (t, J = 7.2, 2H), 3.25 – 3.12 (m, 3H), 2.88 – 2.83 (m, 1H), 2.74 – 2.69 (m, 1H), 2.45 – 2.42 (m, 1H), 1.86 – 1.79 (m, 1H), 1.75 (s, 3H), 1.61 – 1.52 (m, 2H), 1.11 (t, J = 7.2 Hz, 3H); 13C NMR (101 MHz, DMSO-d6) δ 168.39, 162.39, 159.90, 152.31, 135.94, 135.72, 130.03, 127.83, 126.82, 125.35, 87.93, 80.04, 75.02, 58.63, 51.25, 47.59, 42.09, 36.11, 34.33, 32.80, 23.25, 15.11, 3.52; MS (ESI) m/z: 424.30 [M+H]+. (R)-2-((4-(3-aminopiperidin-1-yl)-3-(but-2-yn-1-yl)-2,6-dioxo-3,6-dihydropyrimidin-1(2H)yl)methyl)-N-(tert-butyl)benzamide (1l) Following a similar procedure for the preparation of 1e. 1l was prepared starting from compound 6a and tert-butylamine. White solid (106 mg, yield 79%); HPLC purity: 97.7%; mp: 190-193 °C; 1H

NMR (400 MHz, DMSO-d6) δ 8.23 (s, 3H), 7.99 (s, 1H), 7.41 – 7.18 (m, 3H), 6.86 (d, J = 7.7

Hz, 1H), 5.28 (s, 1H), 5.08 (s, 2H), 4.61 (d, J = 17.4 Hz, 1H), 4.46 (d, J = 17.4 Hz, 1H), 3.34 – 3.31 (m, 3H), 3.13 – 3.08 (m, 1H), 3.01 – 2.95 (m, 1H), 2.90 – 2.84 (m, 1H), 2.03 – 1.81 (m, 2H), 1.76 20

(s, 3H), 1.72 – 1.54 (m, 2H), 1.37 (s, 9H). 13C NMR (101 MHz, DMSO-d6) δ 168.51, 162.38, 159.53, 152.20, 137.18, 134.93, 129.66, 128.01, 126.80, 125.20, 88.72, 80.15, 74.97, 52.59, 51.77, 51.25, 46.47, 41.94, 36.17, 28.99, 27.45, 21.81, 3.60. MS (ESI) m/z: 452.36 [M+H]+. methyl 2-(bromomethyl)-5-fluorobenzoate (9a) A mixture of methyl 5-fluoro-2-methylbenzoate (0.5 g, 3.0 mmol), NBS (0.18 g, 3.0 mmol), and dibenzoyl peroxide (BPO) (36 mg, 0.15 mmol) in 1,2-dichloroethane (5 ml) was heated at 80 °C for 12 hours until all starting material was consumed. The reaction was cooled to room temperature, and the precipitated solid was removed by filtration and washed with ethers (10 mL). The filtrate was concentrated in vacuo and the residue was partitioned between 2 N NaHCO3 (15mL) and ethers (15mL). The organic layer was separated, dried over MgSO4, filtered and concentrated to give a crude product (0.65 g, 89%), which was used in the next step reaction without further purification. (R)-2-((4-(3-aminopiperidin-1-yl)-3-(but-2-yn-1-yl)-2,6-dioxo-3,6-dihydropyrimidin-1(2H)yl)methyl)-5-fluorobenzoic acid hydrochloride (2a) Following a similar procedure for the preparation of 1c. 2a was prepared starting from compound 3 and 9a. White solid (180 mg, yield 82%); HPLC purity: 97.6%; mp: 217-220 °C; 1H NMR (400 MHz, DMSO-d6) δ 13.39 (s, 1H), 8.41 (s, 3H), 7.61 (d, J = 11.2 Hz, 1H), 7.41 – 7.24 (m, 1H), 7.05 – 6.91 (m, 1H), 5.26 (s, 1H), 5.24 (s, 2H), 4.62 (d, J = 17.2 Hz, 1H), 4.44 (d, J = 17.2 Hz, 1H), 3.40 – 3.25 (m, 2H), 3.19 – 3.06 (m, 1H), 3.01 – 2.96 (m, 1H), 2.94 – 2.79 (m, 1H), 2.02 – 1.83 (m, 2H), 1.76 (s, 3H), 1.70 – 1.53 (m, 2H). 13C NMR (101 MHz, DMSO-d6) δ 167.51, 162.28, 160.82 (d, J = 242 Hz), 159.61, 152.13, 134.77 (d, J = 2.9 Hz), 131.92 (d, J = 6.7 Hz), 128.20 (d, J = 8.5 Hz), 119.43 (d, J = 21.7 Hz), 117.26 (d, J = 22.3 Hz), 88.74, 80.21, 74.94, 52.60, 51.79, 46.47, 42.32, 36.19, 27.49, 21.83, 3.59. MS (ESI) m/z: 415.28 [M+H]+. (R)-2-((4-(3-aminopiperidin-1-yl)-3-(but-2-yn-1-yl)-2,6-dioxo-3,6-dihydropyrimidin-1(2H)yl)methyl)-6-fluorobenzoic acid hydrochloride (2b) Following a similar procedure for the preparation of 1c. 2b was prepared starting from compound 3 and 9b.. White solid (68 mg, yield 52%); HPLC purity: 96.6%; mp: 217-219 °C; 1H NMR (400 MHz, DMSO-d6) δ 13.65 (s, 1H), 8.41 (s, 3H), 7.40 (q, J = 7.5 Hz, 1H), 7.16 (t, J = 9.1 Hz, 1H), 6.80 (d, J = 7.8 Hz, 1H), 5.26 (s, 1H), 5.03 (s, 2H), 4.62 (d, J = 17.2 Hz, 1H), 4.44 (d, J = 17.2 Hz, 1H), 3.37 – 3.30 (m, 2H), 3.20 – 3.05 (m, 1H), 3.04 – 2.94 (m, 1H), 2.92 – 2.88 (m, 1H), 2.04 – 1.83 (m, 2H), 1.76 (s, 3H), 1.71 – 1.55 (m, 2H).

13C

NMR (101 MHz, DMSO-d6) δ 166.08, 162.12,

21

159.62, 159.49 (d, J = 248.9 Hz), 152.04, 137.86 (d, J = 2.6 Hz), 132.06 (d, J = 8.7 Hz), 121.80, 121.64 (m), 114.86 (d, J = 21.8 Hz), 88.68, 80.24, 74.92, 52.60, 51.78, 46.46, 41.73, 36.21, 27.49, 21.84, 3.59. MS (ESI) m/z: 415.21 [M+H]+. (R)-2-((4-(3-aminopiperidin-1-yl)-3-(but-2-yn-1-yl)-2,6-dioxo-3,6-dihydropyrimidin-1(2H)yl)methyl)-5-chlorobenzoic acid hydrochloride (2c) Following a similar procedure for the preparation of 1c. 2c was prepared starting from compound 3 and 9c. White solid (95 mg, yield 48%); HPLC purity: 99.0%; mp: 221-224 °C; 1H NMR (400 MHz, DMSO-d6) δ 13.39 (s, 1H), 8.43 (s, 3H), 7.83 (s, 1H), 7.53 (d, J = 8.4 Hz, 1H), 6.96 (d, J = 8.4 Hz, 1H), 5.26 (s, 1H), 5.24 (s, 2H), 4.61 (d, J = 17.2 Hz, 1H), 4.44 (d, J = 17.2 Hz, 1H), 3.42 – 3.31 (m, 2H), 3.18 – 3.04 (m, 1H), 3.02 – 2.95 (m, 1H), 2.93 – 2.86 (m, 1H), 2.01 – 1.84 (m, 2H), 1.75 (s, 3H), 1.69 – 1.56 (m, 2H). 13C NMR (101 MHz, DMSO-d6) δ 167.38, 162.26, 159.64, 152.11, 137.63, 132.30, 131.80, 131.71, 130.28, 128.02, 88.69, 80.23, 74.92, 52.59, 51.79, 46.47, 42.43, 36.21, 27.48, 21.83, 3.59. MS (ESI) m/z: 431.02 [M+H]+. methyl (R)-2-((4-(3-aminopiperidin-1-yl)-3-(but-2-yn-1-yl)-2,6-dioxo-3,6-dihydropyrimidin1(2H)-yl)methyl)-5-bromobenzoate hydrochloride (2d) Following a similar procedure for the preparation of 1c. 2d was prepared starting from compound 3 and 9d. White solid (68 mg, yield 43%); HPLC purity: 98.9%; mp: 225-227 °C; 1H NMR (400 MHz, DMSO-d6) δ 13.41 (s, 2H), 8.23 (s, 3H), 7.97 (d, J = 2.2 Hz, 1H), 7.71 – 7.62 (m, 1H), 6.90 (d, J = 8.4 Hz, 1H), 5.29 (s, 1H), 5.23 (s, 2H), 4.60 (d, J = 17.5 Hz, 1H), 4.44 (d, J = 17.5 Hz, 1H), 3.39 – 3.34 (m, 2H), 3.14 – 3.09 (m, 1H), 3.03 – 2.96 (m, 1H), 2.93 – 2.81 (m, 1H), 2.01 – 1.83 (m, 2H), 1.83 – 1.73 (m, 3H), 1.73 – 1.55 (m, 2H). 13C NMR (101 MHz, DMSO-d6) δ 167.27, 162.24, 159.64, 152.10, 138.05, 135.20, 133.14, 132.06, 128.28, 119.89, 88.70, 80.22, 74.90, 52.59, 51.79, 46.48, 42.48, 36.21, 27.49, 21.85, 3.59. MS (ESI) m/z: 475.17 [M+H]+. methyl (R)-2-((4-(3-aminopiperidin-1-yl)-3-(but-2-yn-1-yl)-2,6-dioxo-3,6-dihydropyrimidin1(2H)-yl)methyl)-4-bromobenzoate hydrochloride (2e) Following a similar procedure for the preparation of 1c. 2e was prepared starting from compound 3 and 9e.White solid (157 mg, yield 69%); HPLC purity: 96.3%; mp: 214-215 °C; 1H NMR (400 MHz, DMSO-d6) δ 13.22 (s, 1H), 8.39 (s, 3H), 7.78 (d, J = 9.0 Hz, 1H), 7.54 (s, 1H), 7.03 (d, J = 9.3 Hz, 1H), 5.26 (d, J = 7.6 Hz, 3H), 4.59 (s, 1H), 4.46 (s, 1H), 3.47 – 3.33 (m, 7H), 3.18 – 3.04 (m, 1H), 2.99 – 2.85 (m, 2H), 2.05 – 1.81 (m, 2H), 1.75 (d, J = 9.2 Hz, 3H), 1.69 – 1.54 (m, 2H). 22

13C

NMR (101 MHz, DMSO-d6) δ 167.93, 162.29, 159.71, 152.13, 141.21, 133.03, 130.39, 129.14,

128.54, 126.50, 88.72, 80.18, 74.85, 52.61, 51.72, 46.48, 42.67, 36.24, 27.49, 21.83, 3.66. MS (ESI) m/z: 475.17 [M+H]+. methyl (R)-2-((4-(3-aminopiperidin-1-yl)-3-(but-2-yn-1-yl)-2,6-dioxo-3,6-dihydropyrimidin1(2H)-yl)methyl)-4-methoxybenzoate hydrochloride (2f) Following a similar procedure for the preparation of 1c. 2f was prepared starting from compound 3 and 9f.. White solid (117 mg, yield 54%); HPLC purity: 98.5%; mp: 217-219 °C; 1H NMR (400 MHz, DMSO-d6) δ 12.75 (s, 1H), 8.46 (s, 3H), 7.92 (d, J = 8.4 Hz, 1H), 6.90 (d, J = 8.6 Hz, 1H), 6.28 (s, 1H), 5.26 (s, 3H), 4.62 (d, J = 17.7 Hz, 1H), 4.45 (d, J = 17.9 Hz, 1H), 3.70 (s, 3H), 3.56 – 3.38 (m, 2H), 3.23 – 3.04 (m, 2H), 3.04 – 2.83 (m, 2H), 2.07 – 1.84 (m, 2H), 1.74 (s, 3H), 1.69 – 1.55 (m, 2H). 13C

NMR (101 MHz, DMSO-d6) δ 168.08, 162.68, 162.26, 159.60, 152.08, 141.31, 133.92, 121.42,

112.32, 110.67, 88.69, 80.16, 74.91, 55.68, 52.61, 51.72, 46.48, 43.12, 36.17, 27.47, 21.86, 3.54; MS

(ESI) m/z: 427.31 [M+H]+. (R)-2-((4-(3-aminopiperidin-1-yl)-3-(but-2-yn-1-yl)-2,6-dioxo-3,6-dihydropyrimidin-1(2H)yl)methyl)-4-cyanobenzoic acid hydrochloride (2g) Following a similar procedure for the preparation of 1c. 2g was prepared starting from compound 3 and 9g. White solid (47 mg, yield 45%); HPLC purity: 97.7%; mp: 227-228 °C; 1H NMR (400 MHz, DMSO-d6) δ 13.62 (m, 1H), 8.40 (s, 3H), 7.98 (d, J = 7.9 Hz, 1H), 7.81 (d, J = 8.0 Hz, 1H), 7.42 (s, 1H), 5.26 (s, 3H), 4.61 (d, J = 17.3 Hz, 1H), 4.45 (d, J = 17.3 Hz, 1H), 3.40 – 3.36 (m, 2H), 3.20 – 3.11 (m, 1H), 3.00 – 2.96 (m, 1H), 2.92 – 2.87 (m, 1H), 2.05 – 1.84 (m, 2H), 1.77 (s, 3H), 1.69 – 1.59 (m, 3H). 13C NMR (101 MHz, DMSO-d6) δ 167.63, 162.34, 159.76, 152.28, 139.71, 134.57, 131.47, 131.25, 129.73, 118.59, 114.78, 88.77, 80.14, 74.97, 52.61, 51.77, 46.52, 42.63, 36.34, 27.58, 21.92, 3.59; MS (ESI) m/z: 422.13 [M+H]+. methyl (R)-2-((4-(3-aminopiperidin-1-yl)-3-(but-2-yn-1-yl)-2,6-dioxo-3,6-dihydropyrimidin1(2H)-yl)methyl)-6-fluorobenzoate hydrochloride (2h) Following a similar procedure for the preparation of 1a. 2h was prepared starting from compound 3 and 9b.. White solid (117 mg, yield 54%); HPLC purity: 96.4%; mp: 217-219 °C; 1H NMR (400 MHz, DMSO-d6) δ 8.48 (s, 3H), 7.45 (d, J = 7.2 Hz, 1H), 7.20 (t, J = 9.1 Hz, 1H), 6.94 (d, J = 7.6 Hz, 1H), 5.24 (s, 1H), 5.00 (s, 2H), 4.62 (d, J = 17.3 Hz, 1H), 4.43 (d, J = 17.2 Hz, 1H), 3.87 (s, 3H), 3.51 – 3.35 (m, 2H), 3.18 – 3.03 (m, 1H), 3.03 – 2.81 (m, 2H), 2.09 – 1.83 (m, 2H), 1.75 (s, 23

3H), 1.68 – 1.50 (m, 2H).13C NMR (101 MHz, DMSO-d6) δ 165.10, 162.05, 159.62, 159.59 (d, J = 249.6 Hz), 152.01, 138.24, 132.82 (d, J = 9.5 Hz), 122.99, 120.30 (d, J = 16.0 Hz), 115.11 (d, J = 21.8 Hz), 88.64, 80.24, 74.89, 53.15, 52.56, 51.77, 46.46, 41.60, 36.20, 27.45, 21.84, 3.60; MS (ESI) m/z: 429.27 [M+H]+. methyl (R)-2-((4-(3-aminopiperidin-1-yl)-3-(but-2-yn-1-yl)-2,6-dioxo-3,6-dihydropyrimidin1(2H)-yl)methyl)-4-bromobenzoate hydrochloride (2i) Following a similar procedure for the preparation of 1a. 2i was prepared starting from compound 3 and 9e. White solid (143 mg, yield 62%); HPLC purity: 95.8%; mp: 167-169 °C; 1H NMR (400 MHz, DMSO-d6) δ 8.48 (s, 3H), 7.79 (dd, J = 8.3, 2.8 Hz, 1H), 7.58 (d, J = 8.0 Hz, 1H), 7.12 (s, 1H), 5.27 (s, 1H), 5.23 (s, 2H), 4.62 (d, J = 17.5 Hz, 1H), 4.46 (d, J = 17.2 Hz, 1H), 3.85 (s, 3H), 3.43 – 3.36 (m, 2H), 3.20 – 3.06 (m, 2H), 3.02 – 2.82 (m, 2H), 2.07 – 1.84 (m, 2H), 1.76 (s, 3H), 1.72 – 1.51 (m, 2H); 13C NMR (101 MHz, DMSO-d6) δ 166.72, 162.25, 159.74, 152.13, 141.07, 132.77, 130.60, 128.98, 128.15, 126.94, 88.68, 80.18, 74.85, 52.86, 52.59, 51.72, 46.48, 42.48, 36.27, 27.49, 21.87, 3.66; MS (ESI) m/z: 489.10 [M+H]+. In vitro assay for inhibition of DPP-4, DPP-8, and DPP-9 The inhibition of DPP-4, DPP-8 and DPP-9 activity was tested according to the literature33, 34. Human recombinant DPP-4, DPP-8 and DPP-9 enzymes (Sigma), hydrolyzed a substrate Gly-ProAMC (Sigma), which release the fluorescent aminomethylcoumarin (AMC). Liberation of AMC was continuously monitored using an excitation wavelength of 360 nm and an emission wavelength of 460 nm every 60 s for 20 min using a BioTek microplate reader. The reaction contained 17.3μU/μL enzyme, 10 μM Gly-Pro-AMC, different concentrations of the test compounds, and assay buffer (25 mM HEPES, pH 7.5, 150 mM NaCl, 0.12 mg/mL BSA) in a total reaction volume of 100 μL. The test was carried out in triplicate. And the IC50 data were calculated using the software GraphPad Prism 7. The inhibitory effect of selected compounds on DPP-8 and DPP-9 were determined by the continuous fluorometric method, being the same as the DPP-4 assay system. The pH of the assay buffer for DPP-8 and DPP-9 was 8.0. The selective dose response of inhibition was tested in triplicate and data analysis is the same as the DPP-4 assay system. Molecular modeling Docking studies were carried out using Glide 5.9 in Schrödinger 2013 suite. The DPP-4 protein was extracted from RCSB Protein Data Bank (PDB ID: 2RGU). Protein structures were prepared using 24

Maestro protein preparation wizard applying the default parameters. Ligands were built using Maestro build panel and prepared by the LigPrep application using default parameters. A docking grid was constructed by using the centroid of the bound ligand. Molecular docking of all molecules into the generated grid was performed by using the extra precision (XP) docking mode. Cytotoxicity test Cell culture Hepatic LO2 cells were maintained in DMEM medium (Gibco, Invitrogen) supplemented with 10% fetal bovine serum (FBS) and antibiotics. The cell lines were grown at 37 °C in a 5 % CO2 atmosphere. Cell viability assay Cell viability was determined by MTT method. Hepatic LO2 cells during logarithmic growth phase were seeded in 96-well microtiter plates at 1 × 104 cells per well. Cells were incubated in the presence of compounds 2b, 2e, 2h, and 2i for 48 h. MTT dye (20 μL of 5 mg/mL in PBS) was added to each well 4 h prior to experiment termination. The supernatant was discarded without disturbing the formazan crystals and cells in the wells, while the MTT formazan crystals were dissolved in 150 μL of DMSO and the plates agitated on a plate shaker for 10 min. The optical density (OD) was read on a microplate reader (Molecular Devices, USA) with a wavelength of 490 nm. In vivo study Animals

Male KM mice weighing 20±2 g were purchased from the Laboratory Animal Center of Guangxi Medical University (Nanning, China). The mice were housed at a temperature and humidity controlled environment with free access to food and water and a 12 h light–dark cycle. All the animal studies were strictly performed according to the protocols issued by the Animal Research Committee and the Institutional Animal Care and Use Committee of Guangxi Medical University. In vivo oral glucose tolerance test (OGTT) in Kunming mice The male Kunming mice were fasted overnight (12 h), weighed, bled via tail tip, and randomized into groups (n = 8). Mice were administrated orally with a single dose of vehicle (water solution), alogliptin (suspended in the vehicle; 3 mg/kg) or tested compounds (suspended in the vehicle; 3 or 1 mg/kg), subsequently dosed orally with glucose aqueous solution (3 g/kg) after 30 min. Blood 25

samples were collected immediately before drug administration (-30 min), before glucose challenge (0 min), and at 15, 30, 60 and 120 min postdose. The blood glucose was measured by blood glucose test strips (SanNuo Changsha, China). 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. Conflict of interest The authors have no conflicts of interest to declare. Acknowledgments This study was supported by grants from the National Natural Science Foundation of China (Grants 21762009), Guangxi Province Science Foundation for Youths (Grants 2016JJB140166), Guangxi key R&D Program (Grants AB17195009), Major Science and Technology Projects of Guangxi (AA17202050), Joint Cultivation Base of Innovation & Entrepreneurship for Pharmaceutical Postgraduates (Grants 20170703), Guangxi First-class Discipline Project for Pharmaceutical Sciences (No. GXFCDP-PS-2018), and Training Programs of Innovation and Entrepreneurship for Undergraduates (Grants 201710598139, 2018188, 2018379). References and notes 1.

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Figures, Schemes and Tables captions Figure 1. The structures of marketed DPP-4 inhibitors and compound 2b Figure 2. Structural superposition of crystal structures of DPP-4 binding alogliptin (PDB ID: 3G0B, magenta) and linagliptin (PDB ID: 2RGU, yellow), and the design strategy of compounds 1a-l and 2a-i. Figure 3. The docking binding modes of compounds and their structural superposition in DPP-4 active site. Hydrogen bonds are depicted as dashed black lines. DPP-4 is represented in surface. Compounds are shown in stick. Fig. 3A, the overlayed structure of 1c (cyan) with alogliptin (magenta) and linagliptin (yellow). Fig. 3B, the superposition of 2b (green) and linagliptin (yellow). Fig. 3C, the superposition of 1c (cyan) 2b (green), and 2e (wheat). Fig. 3D, the overlayed structure of 2b (green) against tricyclic guanines DPP-4 inhibitor29 (blue). Figure 4. Cytotoxicity of compounds 2b, 2e, 2h, and 2i against normal hepatic LO2 cell. Figure 5. Effects of compounds 2b and 2h on blood glucose levels during an OGTT in male Kunming mice (A) Time-dependent changes of plasma glucose levels of compounds 2b and 2h at the dose of 10 mg/kg. (B) AUC0–120 min of blood glucose levels of compounds 2b and 2h at the dose of 10 mg/kg. (C) Time-dependent changes of plasma glucose levels of compound 2h at the dose of 3 mg/kg and 1 mg/kg. (D) AUC0–120 min of blood glucose levels of compound 2h at the dose of 3 mg/kg and 1 mg/kg. Values are mean ± SEM (n = 8). * P≤0.05 compared to vehicle-treated group by using a one-way ANOVA with Dunnett's multiple comparisons test. Scheme 1. Synthesis of target compounds1a-l. Reagents and conditions: (a) 1-bromo-but-2-yne, DIPEA, DMF, r.t., 12 h; (b) 4a-b: methyl 2-(bromomethyl)benzoate (4a) or methyl 2-(2(chloromethyl)phenyl)acetate (4b), K2CO3, DMF, r.t. 12 h; (c) (R)-3-N-Boc-aminopiperidine, K2CO3, DMF, 60 °C, 10 h; (d) HCl gas, EA/ether, 0 oC; (e) LiOH, H2O/MeOH, r.t.; (f) NHR1R2, EDCI, HOBt, TEA, r.t., 12 h. Scheme 2. Synthesis of target compounds 2a-i. Reagents and conditions: (a) NBS, (PhCO2)2, ClCH2CH2Cl,, 80 °C, 12 h (b) compound 3, K2CO3, DMF, r.t. 12 h; (c) (R)-3-N-Bocaminopiperidine, K2CO3, DMF, 60 °C, 10 h; (d) HCl gas, EA/ether, 0 oC; (e) LiOH, H2O/MeOH, r.t. Table 1. In vitro DPP-4 inhibitory activities of compounds 1a-l Table 2. In vitro DPP-4, DPP-8 and DPP-9 inhibitory activities of compounds 2a-i 28

Graphical Abstract Identification of novel uracil derivatives incorporating benzoic acid moieties as highly potent Dipeptidyl Peptidase-IV inhibitors

Junli Huanga#, Xiaoyan Denga#, Siru Zhoua, Na Wanga, Qin Yujuna, Liuwei Menga, Guobao Lia Yuhua Xionga, Yating Fana, Ling Guoa, Danni Lana, Junhao Xingb, Weizhe Jianga, Qing Li a  a Pharmaceutical b

College, Guangxi Medical University, Nanning 530021, China;

Department of Organic Chemistry and State Key Laboratory of Natural Medicines, China

Pharmaceutical University, Nanjing 210009, China; #

Those authors contributed equally to this works

Qing Li, Pharmaceutical College, Guangxi Medical University, Nanning 530021, Guangxi, PR China. Email: [email protected](Q. L.).Weizhe Jiang, Pharmaceutical College, Guangxi Medical University, Nanning 530021, Guangxi, PR China. Email: [email protected](W. Z.). 29