Rapid generation of novel benzoic acid–based xanthine derivatives as highly potent, selective and long acting DPP-4 inhibitors: Scaffold-hopping and prodrug study

Article abstract of DOI:10.1016/j.ejmech.2019.07.045

A series of novel xanthine derivatives 2a-l incorporating benzoic acid moieties were rapidly generated by using strategy of scaffold-hopping from our previously reported scaffold uracil to xanthine, a scaffold of approved drug linagliptin. After systematic structure-activity relationship (SAR) study around benzoic acid moieties, 5 novel DPP-4 inhibitors with low picomolar potency range (IC₅₀ < 1 nM) and excellent selectivity against various DPP-4 homologues were identified, in which the best one, compound 2f, with the IC₅₀ value of 0.1 nM for DPP-4, showed 22-fold improvement in inhibitory activity compared to lead compound uracil 1, its activity was 45-fold more potent than alogliptin. 2e, 2f, 2i and 2k were selected for pharmacokinetic evaluation, and 2f and 2i showed the better pharmacokinetic profiles after iv administration, but poor oral bioavailability. To improve the oral pharmacokinetic profile, prodrug design approach was performed around 2f and 2i. Esters of 2f and 2i were synthesized and evaluated for stability, toxicity and pharmacokinetics. Compound 3e, the methyl ester of compound 2f, was identified to demonstrate good stability, low toxicity and improved oral bioavailability, with 3-fold higher blood concentration compared to 2f in rats. The following in vivo evaluations revealed 3e provided a sustained pharmacodynamics effect for 48h, and robustly improved glucose tolerance in normal ICR and db/db mice in dose-dependent manner. Chronic treatments investigations demonstrated that 3e achieved more beneficial effects on fasting blood glucose levels and glucose tolerance than alogliptin in type 2 diabetic db/db mice. The overall results have shown that compound 3e has the potential to efficacious, safety and long-acting treatment for T2DM.

Full text of DOI:10.1016/j.ejmech.2019.07.045

Accepted Manuscript
Rapid generation of novel benzoic acid–based xanthine derivatives as highly potent,
selective and long acting DPP-4 inhibitors: Scaffold-hopping and prodrug study
Qing Li, Liuwei Meng, Siru Zhou, Xiaoyan Deng, Na Wang, Yi Ji, Yichun Peng,
Junhao Xing, Gongmei Yao
PII:
S0223-5234(19)30666-X
DOI:
https://doi.org/10.1016/j.ejmech.2019.07.045
EJMECH 11542
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 7 June 2019
Revised Date: 13 July 2019
Accepted Date: 14 July 2019
Please cite this article as: Q. Li, L. Meng, S. Zhou, X. Deng, N. Wang, Y. Ji, Y. Peng, J. Xing, G. Yao,
Rapid generation of novel benzoic acid–based xanthine derivatives as highly potent, selective and long
acting DPP-4 inhibitors: Scaffold-hopping and prodrug study, European Journal of Medicinal Chemistry
(2019), doi: https://doi.org/10.1016/j.ejmech.2019.07.045.
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ACCEPTED MANUSCRIPT
Graphical Abstract
Rapid generation of novel benzoic acid–based xanthine derivatives as highly potent,
selective and long acting DPP-4 inhibitors: scaffold-hopping and prodrug study
Qing Li , Liuwei Meng, Siru Zhou Xiaoyan Deng, Na Wang, Yi Ji, Junli Huang, Yichun Peng,
Junhao Xing, Gongmei, Yao
Qing Li, Pharmaceutical College, Guangxi Medical University, Nanning 530021, Guangxi, PR
China. Email: qingligxmu@163.com(Q. L.).
1

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Rapid generation of novel benzoic acid–based xanthine derivatives as highly potent, selective and
long acting DPP-4 inhibitors: scaffold-hopping and prodrug study
a
a
Qing Li
#, Liuwei Menga, #, Siru Zhoua#, Xiaoyan Denga, Na Wanga, Yi Jia, Yichun Peng , Junhao Xingb,
Gongmei, Yaoa
a Pharmaceutical College, Guangxi Medical University, Nanning 530021, China;
b 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
A series of novel xanthine derivatives 2a-l incorporating benzoic acid moieties were rapidly generated by using
strategy of scaffold-hopping from our previously reported scaffold uracil to xanthine, a scaffold of approved
drug linagliptin. After systematic structure-activity relationship (SAR) study around benzoic acid moieties, 5
novel DPP-4 inhibitors with low picomolar potency range (IC50 < 1 nM) and excellent selectivity against
various DPP-4 homologues were identified, in which the best one, compound 2f, with the IC50 value of 0.1 nM
for DPP-4, showed 22-fold improvement in inhibitory activity compared to lead compound uracil 1, its activity
was 45-fold more potent than alogliptin. 2e, 2f, 2i and 2k were selected for pharmacokinetic evaluation, and 2f
and 2i showed the better pharmacokinetic profiles after iv admistration, but poor oral bioavailability. To
improve the oral pharmacokinetic profile, prodrug design approach was performed around 2f and 2i. Esters of
2f and 2i were synthesized and evaluated for stability, toxicity and pharmacokinetics. Compound 3e, the methyl
ester of compound 2f, was identified to demonstrate good stability, low toxicity and improved oral
bioavailability, with 3-fold higher blood concentration compared to 2f in rats. The following in vivo evaluations
revealed 3e provided a sustained pharmacodynamics effect for 48h, and robustly improved glucose tolerance in
normal ICR and db/db mice in dose-dependent manner. Chronic treatments investigations demonstrated that 3e
achieved more beneficial effects on fasting blood glucose levels and glucose tolerance than alogliptin in type 2
Qing Li, Pharmaceutical College, Guangxi Medical University, Nanning 530021, Guangxi, PR China. Email:
qingligxmu@163.com(Q. L.).
1

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diabetic db/db mice. The overall results have shown that compound 3e has the potential to efficacious, safety
and long-acting treatment for T2DM.
Keywords DPP-4 inhibitor; xanthine derivatives; benzoic acid; scaffold-hopping; prodrug
1. Introduction
The escalating epidemic of diabetes mellitus poses a major public health menace[1]. Currently, 451
million of adults have diabetes globally, it is estimated that the number will rise to 693 million by 2045[1].
China is the country with the largest numbers of people with diabetes in 2017[2]. As the most common
type of diabetes, Type 2 Diabetes mellitus (T2DM) accounts for around 90% of all cases of diabetes.
T2DM are associated with increased rates of macro- and micro-vascular complications, which contribute
to increased premature mortality and lower quality of life[2-4]. The current treatments for T2DM are
based on reducing the plasma glucose to control the risk of developing long-term complications[3-5].
Many class of hypoglycemic agents, including metformin, sulfonylureas, thiazolidinediones, DPP-4
inhibitors, SGLT2 inhibitors, GLP-1 receptor agonists and insulin, are available in clinic, but the control
of diabetes remains unsatisfactory[1, 2]. A surveys from 2003-2006 indicated that 58.2% of people
achieved their target goals for glycemic control (HbA1c <7%) in the United States[6]. The 3B study
showed that only 47.7% of outpatients with diabetes met their goals for HbA1c control (HbA1c <7%) in
China[7]. Unsatisfactory glycemic control is also related to increased risks for cancer, cognitive disability
and depression[8, 9]. Therefore, there remain vast unmet needs for the effective, durable, and safe
treatment in diabetes management.
Glucagon-like peptide-1 (GLP-1) is an incretin hormone released from the gastrointestinal tract after
a meal, which enhance glucose-dependent insulin secretion from the pancreas, aiding the overall
regulation of glucose homeostasis [10]. In addition, GLP-1 has a number of protective effects on the
β-cells, including a reduction in apoptosis and enhancement of β-cell proliferation and neogenesis [11,
12],. However, the active GLP-1 is rapidly degraded by a serine protease enzyme, dipeptidyl peptidase-IV
(DPP-4). Inhibition of DPP-4 augments the endogenous concentration of GLP-1 and improves
hyperglycemia in T2DM[10]. Recently, several DPP-4 inhibitors have been approved in many countries,
including sitagliptin[13], vildagliptin[14], saxagliptin[15], linagliptin[16], and alogliptin[17] (Figure 1).
DPP-4 inhibitors, as monotherapy or add-on therapy, improve glucose control in patients with T2DM,
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without inducing hypoglycemia and weight gain[18]. However, some safety concerns were raised,
especially a potential increase in the risk of acute pancreatitis pancreatic cancer[18], and an increased risk
of hospitalization for heart failure in saxagliptin treated T2DM patients [19]. In addition, the variability
exists in response to the interventions of DPP-4 inhibitors as the heterogeneity in diabetes
phenotype[20-22]. In the light of precision medicine in diabetes[23], novel DPP-4 inhibitors may provide
differential pharmacological response to subgroups of diabetic individuals[22], and may demonstrate
comparatively greater effectiveness for a specific segment of the population. Introduction of new
treatments will ultimately improve the prevention and treatment strategies of T2DM. Hence, it is requisite
to develop novel DPP-4 inhibitor.
Our previous studies have identified several series of novel DPP-4 inhibitors by exploring building
blocks, including triazole rings[24, 25], benzoic acids[26], targeting the S2’ subsite of DPP-4 active site. In
our recent study, we reported a series of the uracil analogues incorporated o-benzoic acids at N3-position to
interact with both Trp629 and Lys554 in S2’ subsite[26]. The compound
1 (Figure 1) bearing
6-fluorobenzoic acid was identified as one of the most potent DPP-4 inhibitor[26]. Aiming to enrich and
identify alternative DPP-4 inhibitors with better pharmacological profiles, we herein simply replaced the
scaffold uracil of compound
1 with xanthine of linagliptin to generate novel benzoic acid–based xanthine
derivatives, by using scaffold-hopping strategy. An additional structure-activity relationship (SAR) study
led to discovery 2f as a high potent (IC50 = 0.1 nM) and selective DPP-4 inhibitor. Subsequent prodrug
strategy was successfully performed to improve both oral bioavailability and plasma duration. Eventually,
the methyl ester of 2f (3e) was proven to demonstrate good plasma exposure, prolonged duration,
sustained in vivo DPP-4 inhibition and excellent in vivo hypoglycemic activities in both normal ICR and
db/db mice (Figure 1).
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Figure 1. The structures of marketed DPP-4 inhibitors and compound 1, 2f and 3e.
2. Results and Discussion
2.1. Design Strategy
The active site of DPP-4 has multiple binding subsites labeled as S1 and S2 pockets, S2 extensive pockets,
and S1’and S2’ subsites[27, 28]. The analysis of X-ray cocrystal structures of DPP-4 and its inhibitor reveals that
all of the inhibitors bind to the S1 and S2 subsites through hydrophobic and salt bridge interaction[27]. The two
interactions play a key role in retaining DPP-4 inhibitory activity. In addition to the S1 and S2 subsites,
sitagliptin, anagliptin, teneligliptin, and anagliptin also occupy the S2 extensive pocket, while alogliptin and
linagliptin interact with the S1’ subsites[27, 28]. Only linagliptin extends to the S2’ subsites with the
characteristic methylquinazoline group, forming π-π stacking with Trp629. This feature achieves the highest
activity value of linagliptin[28, 29]. Thus exploration of S2’ subsite is a promising strategy to design novel
DPP-4 inhibitors with higher bioactivity[27]. We have designed o-benzoic acids based DPP-4 inhibitors to
interact with both Trp629 and Lys554 in S2’ subsite, leading to identification of compound 1 as potent DPP-4
inhibitor with the IC50 value of 2.19 nM [26]. Docking studies revealed that the benzene ring forms π-π stacking
with Trp629, and the carboxylic acid forms salt bridges with Lys554 in S2’ subsites (Figure 2). Compound 1
takes uracil as core scaffold, a scaffold drived from alogliptin, while linagliptin uses xanthine as scaffold. The
structural superposition of crystal structures of DPP-4 binding alogliptin (PDB ID: 3G0B) and linagliptin (PDB
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ID: 2RGU) indicated that both uracil of alogliptin and compound 1, and xanthine of linagliptin bind in S1’
subsite in a similar fashion (Figure 2 and Figure S-1), in which the aromatic ring forms π-π stacking with
residue Tyr547, and their carbonyls interact with Ser630 through hydrogen bond. Furthermore, the overlay
structures of the docking binding modes of compound 1 against the tricyclic guanines (Figure S-2), a
bioisosteric replacement of the xanthine scaffold [30], revealed that o-carboxylic acid of compound 1 and the
o-cyano group of tricyclic guanines interact with Lys554 in a similar way. These evidences inspired us to
envision a simple replacement of the scaffold uracil of compound 1 with xanthine of linagliptin, by using
scaffold-hopping strategy, might be a practical strategy to discover novel alternative DPP-4 inhibitors (Figure
2), thus xanthine-based compound 2a-l were designed by introducing various o-carboxylic acids into
N1-position of xanthine.
Figure 2. A. Structural superposition of crystal structures of DPP-4 binding linagliptin (PDB ID: 2RGU,
magenta) and docked compound 1 (green); B. the design strategy of compounds 2a-l.
2.2. Chemistry
The synthetic routes adopted to obtain target compounds 2a-l and ester 3a-j are depicted in Scheme 1. The
commercially available 8-bromo-3-methyl-xanthine 4 was selectively alkylated with 1-bromo-but-2-yne to
afford product 5, then compound
5
was converted to intermediate 6a-l by alkylation with
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2-bromomethyl-benzoates 11a-l, which were obtained from bromination of starting materials 10a-l. Treatment
of 6a-l with (R)-3-N-Boc-aminopiperidine afforded the desired compounds 7a-l, which were hydrolyzed with
LiOH to give benzoic acid 8a-l. Boc groups of 7a-l and 8a-l were removed to furnish the desired acids 2a-l,
esters 3a-e and 3h. 8f and 8h was alkylated with various alkyl halides to give 9a-d. Removal of the tert-butoxy
carbonyl group of 9a-d yielded the esters 3f-g and 3i-j. The structures of target compounds 2a-l and 3a-j are
listed in Table 1.
O
O
O
O
O
N
O
N
O
N
O
N
H
N
N
N
N
N
N
N
N
HN
HN
a
b
c
N
N
R1
Br
Br
Br
R1
N
O
O
O
O
NH
Boc
4
5
6a-l
7a-l
e
O
OH
O
OH
O
O
O
N
O
O
N
N
N
N
N
N
N
N
N
d
e
N
R1
N
N
R1
R1
O
N
O
N
O
NH
NH2
NH2
Boc
8a-l
f
2a-l
3a-e, 3h
R2
O
R2
O
O
O
O
O
O
O
O
O
1'
N
6'
R1
N
e
g
N
N
2'
3'
N
Br
R1
N
R1
R1
N
O
N
5'
N
O
N
4'
NH
NH2
Boc
9a-d
10a-l
11a-l
3f, 3g, 3i, 3j
5a, 6a, 7a, 8a, ,10a, 11a, 2a,3a R1 = H
5g, 6g, 7g, 8g, 9g, 10g, 11g, 2g R1 = 3'-Br
5h, 6h, 7h, 8h, 10h, 11h, 3e, 2h R1= 4'-Br
5i, 6i, 7i, 8i, 10i, 11i, 2i, 3h, 9c, 9d, 3i, 3j R1=5'-Br
5j, 6j, 7j, 8j, 10j, 11j, 2j R1=3'-MeO
3f, 3i R2
=
5b, 6b, 7b, 8b,10b, 11b, 2b, 3b R1 = 6'-F
5c, 6c, 7c, 8c, 10c, 11c, 2c R1 = 5'-F
5d, 6d, 7d, 8d, 10d, 11d, 2d, 3c R1 = 3'-Cl
5e, 6e, 7e, 8e, 10e, 11e, 2e, 3d R1 = 4'-Cl
O
O
3g, 3j R2
=
O
5k, 6k, 7k, 8k, 10k, 11k, 2k R1=4'-MeO
5f, 6f, 7f, 8f, 10f, 11f, 2f, 3e, 9a, 9b,3f, 3g R1 = 5'-Cl 5l, 6l, 7l, 8l, 10l, 11l, 2l R1=4'-CN
Scheme 1. Synthesis of target compounds 2a-l and 3a-j. Reagents and conditions: (a) 1-bromo-but-2-yne,
DIPEA, DMF, r.t., 12 h; (b) 11a-l, K2CO3, DMF, 50 °C, 10 h; (c) (R)-3-N-Boc-aminopiperidine, K2CO3, DMF,
65 °C, 10 h; (d) LiOH, H2O/MeOH, r.t. 24h; (e) HCl gas, EtOAc/ether, 0 °C; (f) alkyl halides, K2CO3, DMF, r.t.
6h; (g) NBS, (PhCO2)2, ClCH2CH2Cl, 80 °C, 12 h.
2.3. In vitro DPP-4 inhibition and SAR study
All the target compounds were evaluated for the inhibition of human recombinant DPP-4, while lead
compound 1 and the approved drug alogliptin were chosen as the reference drugs. Compound 1, with a fluorine
at the 6’-position, was identified as the most potent DPP-4 inhibitor (IC50 = 2.19 nM) in the uracil series. As the
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initial idea was to use the xanthine as a bioisostere for uracil in the optimization of the compound series, the
corresponding xanthine 2b of uracil 1 was firstly synthesized and tested. However, 2b (IC50 = 10.21 nM)
suffered from an approximate 5-fold loss of DPP-4 inhibitory activity compared to lead compound 1. On the
contrary, compound 2a (IC50 = 1.98 nM), bearing hydrogen at 6’-position, exhibited an improvement of DPP-4
inhibition compared to the corresponding uracil series[26], whose activity is comparable with that of the most
potent compound 1 in uracil series[26], implying that the xanthine series displayed a different SAR with that of
uracil series[26]. Inspired by the results, a variety of substituent groups were introduced to the benzene ring to
explore the SAR. We were pleased to find that the 5’-fluorine substituted compound 2c displayed 4-fold
increase in DPP-4 activity (IC50 = 2.35 nM) compared with 6’-fluorine substituted compound 2b (IC50 = 10.21
nM). The introduction of the chlorine at the 3’-position (2d, IC50 = 3.90 nM) retained the potent DPP-4 activity.
Notably, the substitution of the chlorine at the 4’-position (2e) and 5’-position (2f) resulted in compounds with
low picomolar potency. Compounds 2e and 2f exhibited the IC50 of 0.18 nM and 0.10 nM, respectively, whose
activities are 12-fold and 22-fold more potent than that of lead compound uracil 1. The activity of compound 2f
was 45-fold much more potent than reference alogliptin (IC50 = 4.45 nM), and was more potent than reported
the activity of linagliptin (IC50 = 1 nM)[29]. Furthermore, compared to compound 2a, the substitutions of
bromine groups at 3’, 4’ and 5’-positions markedly boosted the DPP-4 activity, the bromine at 3’ position
resulted in low nanomolar potency (2g, IC50 = 1.05 nM). 4’ and 5’-bromine groups led to compound 2h and 2i
with picomolar potency, exhibiting the IC50 of 0.72 nM and 0.22 nM, respectively. The installation of methoxy
group on 3’-position eroded potency (2j, IC50 = 5.99 nM) compared to compound 2a, but moving methoxy
group to 4’-position led to a 14-fold enhancement in potency (2k, IC50 = 0.44 nM) relative to 2j. Replacing the
4′-methoxy with 4′-cyano group caused a minor loss of potency (2k, IC50 = 2.43 nM). The comparison of the
activities among these compounds turned out that the order of potency was Cl > Br > F at 5’-position, and Cl >
MeO > Br > CN at 4’-position, indicating that the chlorine is the optimal group at both 4’-position and
5’-position. It is worth to note that all the designed compound with xanthine scaffold had potency less than 10
nM, most of them are more potent than lead compound 1, indicating the xanthine are more favorable than the
uracil for the DPP-4 binding in this series. Together, using scaffold-hopping strategy and SAR study, some
novel xanthine-based DPP-4 inhibitors have been discovered with significant improvement of DPP-4 activity,
the potency of 2e, 2f, 2h, 2i and 2k on inhibition of DPP-4 is within picomolar potency range, and is much
better than the drug alogliptin.
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Considering the low calculated cLogP values of the compound 2a-k, which might lead to poor oral
absorption, some selected compounds were esterified to serve as prodrug to improve the oral absorption. As
shown in Table 1, the esterification improved the cLogP values with the range of 2.15-3.84, which is an optimal
range for oral drug. However, most of the esters, as expected, showed less potent DPP-4 activity than their
corresponding acids, supporting our design that the carboxyl group play a vital role in the DPP-4 affinity[26].
The methyl ester of compound 2a (3a, IC50 = 2.91 nM) showed slight loss of DPP-4 inhibition compared to its
acid 2a, but the methyl ester of compound 2b (3b, IC50 = 5.06 nM) displayed a 2-fold improvement in
inhibition of DPP-4. The introduction of methyl ester to compounds 2d and 2e resulted in a pronounced loss of
potency (3c, IC50 = 50.91 nM, 3d, IC50 = 5.61 nM). The methyl and ethyl esters of compound 2f and 2h (3e,
IC50 = 20.48 nM, 3f, IC50 = 74.43 nM, 3h, IC50 = 22.09 nM, 3i, IC50 = 85.48 nM) also showed decreased
potency compared to compound 2f and 2h. The comparison of the activity of methyl esters and the ethyl esters
indicated that the methyl esters were statistically better than the ethyl ester compounds in DPP-4 activity,
demonstrating that increasing the bulk seems to be detrimental to the activity.
Table 1. In vitro DPP-4, DPP-8 and DPP-9 inhibitory activities of compounds 2a-l and 3a-j.
DPP-4
IC50 (nM) b
2.19
DPP-8
IC50 (µM) b
>100
DPP-9
IC50 (µM) b
>100
Compdsa
R1
R2
cLogPc
-
-
-
-
-
-
-
-
-
-
-
-0.54
-0.34
-0.47
-0.11
0.46
0.46
0.46
0.61
0.61
0.61
1
H
1.98
>100
>100
2a
2b
2c
2d
2e
2f
6’-F
5’-F
3’-Cl
4’-Cl
5’-Cl
3’-Br
4’-Br
5’-Br
10.21
2.35
>100
>100
>100
>100
3.90
>100
>100
0.18
>100
>100
0.10
>100
>100
1.05
>100
>100
2g
2h
2i
0.72
>100
>100
0.22
>100
>100
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3’-MeO
4’-MeO
4’-CN
H
-
5.99
0.44
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
-0.21
-0.21
-0.68
2.45
2.15
3.16
3.16
3.16
3.69
2j
2k
2l
-
-
2.43
OMe
OMe
OMe
OMe
OMe
OEt
2.91
3a
3b
3c
3d
3e
3f
6’-F
5.06
3’-Cl
4’-Cl
5’-Cl
5’-Cl
50.90
5.61
20.48
74.43
5’-Cl
NA
NA
NA
2.46
3g
5’-Br
5’-Br
OMe
OEt
22.09
85.48
>100
>100
>100
>100
3.31
3.84
3h
3i
5’-Br
NA
NA
NA
2.61
3j
-
-
-
-
4.46
-
>100
1.52
>100
0.05
alogliptin
vildagliptin
a not available.
b Measured at least in two independent experiments.
c clogP were calculated by ChemBioDraw software Ultra 14.0.
2.4. DPP-8 and DPP-9 selectivity study
To examine the selectivity against other members of serine protease family (DPP-8 and DPP-9), whose
inhibition might produce severe toxicities [31], all the designed compounds were tested for the inhibition of
DPP-8 and DPP-9. As illustrated in Table 1, all compounds inhibited the DPP-8 and DPP-9 with the IC50 > 100
µM, implying that the compounds do not have inhibition for DPP-8 or DPP-9, while the reference vildagliptin
inhibited both DPP-8 and DPP-9, with IC50 of 1.52 µM and 0.05 µM, respectively. The results indicated that the
designed compounds have excellent selectivity against DPP-8 and DPP-9.
2.5. Molecular docking study
To verify the interaction of the designed compounds with DPP-4 protein, molecular docking study was
performed by using molecular docking program GLIDE 5.9. The DPP-4 crystal structure (PDB ID: 2RGU) was
selected as receptor, and most potent compounds 2f was chosen as ligand. The docking binding modes are
shown in Figure 3. The compound 2f share the key binding interactions with compound 1 and linagliptin.
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2-butynyl groups occupy the hydrophobic S1 pocket. The amino groups of (3R)-aminopiperidine are in close
proximity to the side chains of Glu205 and Glu206 for salt bridges interaction in S2 pocket. The carboxyl
groups of o-benzoic acid make salt bridges interaction with Lys554 in S2 subsite with a distance between the
7-carboxyl group and the residue of Lys554 of 2.6 Å, which have a vital role in the enhancement of DPP-4
affinity. Some differences in the binding of the core scaffold and o-benzoic acid moieties were observed. In
addition to salt bridges interaction with Lys554, the carboxyl of o-benzoic acid moiety of 2f forms hydrogen
bonding with amine of Typ629 in S2’ subsite. The cores of xanthine of 2f stacks with benzene ring of Tyr547
with a closer distance than the uracil of 1 does in S1 subsite, leading to more efficient π-stacking interaction.
These two results may contribute to increasing in potency of xanthine-based compounds, interpreting decreased
potency caused by esterification of the carboxylic acid.
Figure 3. The docking binding modes of compounds 2f and its structural superposition in DPP-4 active site.
Hydrogen bonds are depicted as dashed black lines. DPP-4 is represented in white cartoon. Compounds are
shown in stick. Fig. 4A, the overlay structure of 2f (cyan) with compound 1 (green). Fig. 4B, the superposition
of 2f (cyan) and linagliptin (maganta).
2.6. Pharmacokinetic study of compounds 2e, 2f, 2i and 2k
On the basis of their highly potent and selective DPP4 inhibitory activity, 2e, 2f, 2i and 2k were carried on
for pharmacokinetic studies in SD rats after i.v. administration at the dose of 5 mg/kg. Key parameters are
summarized in Table 2. Compounds 2e and 2k had high clearance values (1.03 and 1.30 L/h/kg, respectively)
and poor plasma exposure of (4859.81 µg/L*h and 3926.21 µg/L*h), leading to a short half-life of 0.82 and 0.26
h, while compounds 2f and 2i had larger plasma exposure with AUC0-inf of 9624.06 and 23543.02 µg/L*h,
bigger maximum plasma concentration Cmax (30554.51 µg/L, 12151.13µg/L) and lower plasma clearance (0.53
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and 0.20 L/h/kg), resulting in a longer terminal half-life of 5.24 h and 5.75 h. However, compounds 2f and 2i
suffered from poor oral bioavailability (< 7%).
Table 2. Pharmacokinetic Parameters of 2e, 2f, 2i and 2k after i.v. administration at the dose of 5 mg/kg
Parameters a
T1/2 (h)
Cmax(µg/L)
AUC0-inf (µg/L*h)
4859.81 ±578.39
9624.06±1813.15
Cl (L/h/kg)
1.03±0.11
0.53 ±0.11
0.22±0.05
1.30±0.21
0.82±0.31
5.24±1.22
5.75±1.07
0.26 ±0.04
14636.64 ±1417.06
30554.51 ±5207.74
2e
2f
2i
65494.80±13073.47 23543.02±4997.73
12151.13 ±1733.24 3926.21 ±717.44
2k
a Compounds were dosed to equal number of male Sprague-Dawley rats in iv (5 mg/kg) administration (n = 3).
2.7. Stability studies of prodrug 3e-j in PBS buffer and rat plasma
Prodrug strategies are often used to improve both oral bioavailability and the plasma duration[32].
Compounds 2f and 2i, with better PK properties, were selected for the design of prodrug. Methyl (3e, 3h), ethyl
(3f, 3i) and dioxolenone (3g, 3j) esters of 2f and 2i were constructed for the stability test using a concentration
of 1 µM of in PBS buffer at pH 7.4 and rat plasma. As shown in Table 3, compounds 2f and 2i showed good
stability in PBS buffer and rat plasma. Methyl (3e, 3h), ethyl (3f, 3i) esters are stable at 37 °C in PBS buffer in
the time span of 2 h, and they (3e, 3h, 3f, 3i) were observed to exhibit good stability in the rat plasma as the
percentage remaining more than 54.21% after 120 min incubation. However, the dioxolenone esters 3g and 3j
suffered from poor stability in both PBS buffer and rat plasma, half of 3g and 3j are rapidly degraded even after
5 min incubation with plasma (Data not shown), and they were almost completely degraded in PBS buffer and
rat plasma after 120 min incubation. Among all the compounds, main metabolites were the corresponding acids
2f and 2i.
Table 3. The stablility of 2f, 2i and 3e-j in PBS buffer and rat plasma.
% compound remaining
Compoundsa
PBS
plasma
101.45±2.23
99.21±2.52
101.25±1.25
99.45±2.14
12.01±0.31
102.10±0.69
98.26±1.24
65.24±5.24
70.24±3.45
1.25±0.24
2f
2i
3e
3f
3g
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99.36±3.45
98.42±4.12
8.32±1.25
54.21±1.65
3h
3i
62.18±3.24
2.11±1.23
3j
a, values are the means±SD of at least two independent determinations
2.8. Effects of compounds 2f, 2i, 3e, 3f, 3h and 3i on cell viability
Compounds 2f, 2i, 3e, 3f, 3h and 3i with good stability were evaluated for cell viability test on normal
hepatic LO2 cell using the MTT assay. Doxorubicin (DOX) was used as reference. As shown in Figure 4,
compounds 3e and 3f displayed approximately 90% and 80% of viability of at 20 µM, indicating those
compounds displayed low cytotoxicity against normal hepatic LO2 cell line. In contrast, 3h and 3i exhibited
higher toxicity (% viability at 20 µM were 70% and 50%) in the cell line, as well as the positive control (%
viability at 10 µM was 35%). In addition, compounds 2f and 2i displayed low cytotoxicity with > 90% viability
up to 50 µM.
Figure 4. Cytotoxicity of compounds 2f, 2i, 3e, 3f, 3h and 3i against normal hepatic LO2 cell. Values are mean
± SD (n = 5). * P≤0.05 and ** P≤0.01 compared to vehicle-treated group by using a one-way ANOVA with
Dunnett's multiple comparisons test.
2.9. Pharmacokinetic evaluation of compounds 3e and 3f
On the basis of the toxicity data, compounds 3e and 3f were further selected for in vivo PK study after po
administration at the dose of 10 mg/kg. The plasma concentration of their active metabolite 2f was determined.
The time-dependent concentrations of 2f, 3e and 3f are shown in Figure 5, and the key parameters are shown in
Table 3. Notably, these compounds 3e and 3f are freely soluble in water, indicating favorable drugability
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property. The directly dosed compound 2f displayed short half-life (1.77 h) and low bioavailability (6.76%). As
expected, active metabolite 2f was released from 3e and 3f after oral administration. 3e and 3f had 3-fold and
2-fold improved plasma exposure compared to that of the directly dosed parent 2f, resulting in improved
bioavailability (19.46% and 15.60% for 3e and 3f, respectively). The plasma clearance of 3e and 3f was reduced
relative to 2f, leading to longer terminal half-life with the T1/2 of 10.08 h and 8.02 h for 3e and 3f, which are
much longer than alogliptin (5 h) in rats[33]. Remarkably, the plasma concentration of 3e and its released 2f
was approximate 30 ng/mL after 36 h of oral administration, while direct dosed 2f did not remain in the plasma
after 12 h as indicated by a concentration below the limit of quantification (LOQ: 1.0 ng/mL), suggesting 3e can
maintain a high plasma concentration for a long time. The PK profiles indicated that administration of prodrugs
led to releasing 2f with prolonged duration. 3e with good bioavailability and plasma duration was selected for
further evaluation.
Table 4. Pharmacokinetic Parameters of 2f, 3e and 3f after po administration at the dose of 10 mg/kg
Parameters a
analyte
T1/2 (h)
Tmax(h)
0.17
1
Cmax(µg/L)
2335.96±619.57
1182.03±615.42
693.32±159.86
507.74±113.76
401.06±13.36
AUC0-inf (µg/L*h)
1301.87±377.63
6107.30±1193.97
3746.05 ±160.13
4536.25±1475.16
3001.17±924.70
Cl(L/h/kg)
0.82±0.23
0.16 ±0.03
0.27±0.01
0.23±0.08
0.35±0.11
F%
6.76%
NA
1.77 ±0.76
10.08±1.39
11.81±0.12
8.02±2.89
6.97±2.47
2f
2f
3e
2f
3f
2f
3e
1
19.46%
NA
0.5
3f
0.5
15.60%
a Compounds were dosed to equal number of male Sprague-Dawley rats in po (10 mg/kg) administration (n = 3).
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Figure 5. Time-dependent concentrations of 2f, 3e and 3f in rat plasma after oral administration (10 mg/kg).
2.10. Effect on in vivo DPP-4 activity of 3e
To further assess in vivo efficacy, plasma DPP-4 inhibition study of 3e was evaluated at single oral
administration (10 mg/kg) in SD rats over a time period of 0–48 h, while the 2f, 3f and alogliptin were chosen
as references at same dose. As shown in Figure 6, compound 3e provides a sustained pharmacodynamics effect,
with 85% and 60% inhibition of plasma DPP-4 activity at 12 and 24 h, respectively. Remarkably, plasma DPP-4
inhibition of 3e was sustained to 48 h. The strong and long-lasting inhibitory effect of 3e on DPP-4 observed in
vivo can be attributed to two aspects: DPP-4 inhibition of 3e itself and prolonged duration of 2f released from
3e. Alogliptin also showed potent DPP-4 inhibition, giving >80% inhibition of DPP-4 activity before 12 h, but
their inhibitions were decreased after 12 h, indicating that the effect of 3e was superior to that of alogliptin at
the same dose. Compound 3f had >80% inhibition of DPP-4 activity within 12 h, but was observed rapidly
decreasing in DPP-4 inhibition after 12h, which may due to the higher plasma clearance of 3f. Meanwhile,
owing to low oral bioavailability and high plasma clearance of 2f, oral administration 2f resulted in > 80%
DPP-4 inhibition only in 2 hours, demonstrating our prodrug strategy successfully prolonged the in vivo
pharmacodynamics.
Figure 6. The in vivo DPP-4 inhibition of compounds 3e, 3f and 2f (10 mg/kg).
2.11. Acute hypoglycemic effects of 3e in ICR mice and db/db mice
Based on PK property and in vivo DPP-4 inhibition, compounds 3e were selected for acute efficacy
evaluation by the oral glucose tolerance test (OGTT) in ICR mice. Compounds 3e and references 2f and
alogliptin at the dose of 10 mg kg−1 were orally administered to ICR mice. The time-dependent changes in
plasma glucose levels and the area under the curve (AUC0–120min) are shown in Figure 7A and 7B, compound 3e
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exerts a significant decrease in glucose level with the decreased AUC0–120min of 17.43%, while alogliptin
reduces the AUC0-120min to 10.35% at a dose of 10 mg/kg, indicating that the hypoglycemic activity of the
compound 3e are more potent than that of alogliptin. Interestingly, the compound 2f also produces potent
hypoglycemic activity with the reduced AUC0–120min of 14.30%, which is better than alogliptin. indicating that
compound 2f is a potent antidiabetic agent due to its super-potent in vitro DPP-4 inhibitory activity, even though
its poor oral bioavailability.
In order to further investigate the in vivo hypoglycemic potency of compound 3e at different doses in db/db
mice, 3e at the doses of 10 mg kg−1, 3 mg kg−1 and 1 mg kg−1 were orally administered to db/db mice. As shown
in Figure 7C and 7D, compound 3e robustly improves the glucose tolerance in a dose-dependent manner, with
the reduced the AUC0–120 min values of 54.7 %, 28.8% and 26.4% at the dose of 10 mg/kg, 3 mg/kg and 1mg/kg,
respectively. The hypoglycemic activity of the compound 3e is more potent than that of referenced drug
alogliptin at the dose of 10 mg/kg, which decreased 45.0% of AUC0–120mim value.
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Figure 7. Effects of compounds 3e and 2f on blood glucose levels during an OGTT in male ICR and db/db mice
(A) Time-dependent changes of plasma glucose levels of compounds 3e and 2f at the dose of 10 mg/kg. (B)
AUC0–120 min of blood glucose levels of compounds 3e and 2f at the dose of 10 mg/kg. Data are presented as
mean ± SEM (n = 8). (C) Time-dependent changes of plasma glucose levels of compounds 3e. (D) AUC0–120 min
of blood glucose levels of compounds 3e. Data are presented as mean ± SEM (n = 6). (*) P < 0.05, (**) P < 0.01
vs vehicle-treated group by using a one-way ANOVA with Dunnett's multiple comparisons test.
2.12. Chronic treatment of 3e in type 2 diabetic db/db mice.
The long-term treatment effects were tested to further evaluate the potential therapeutic utility of 3e in
db/db mice. Based on the PK and hypoglycemic duration tests results, 3e and alogliptin were orally
administrated once daily for 3 weeks. As shown in Figure 8a, administration of 3e with the dose of 3 and 10
mg/kg significantly decreased fasting blood glucose (FBG) levels, which was better than alogliptin. Moreover,
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chronic treatment of 3e (10 mg/kg) in db/db mice also significantly improved the glucose tolerance of the
diabetic mice (Figure 8c), 3e reduced the AUC0–120 min values of 21.6 %, 6.0% at the dose of 10 mg/kg, 3 mg/kg
respectively, glucose AUC of 3e was lower than that of alogliptin (Figure 8d) with the AUC0–120 min values of
13.2 % at the dose of 10 mg/kg. In addition, treatment of 3e gradually increased nonfasting body weight (Figure
8b) as that of alogliptin, which are similar to the reports of several other studies of DPP-4 inhibitors,
demonstrating that 3e (both 3 and 10 mg/kg) is well tolerated in db/db mice.
Figure 8. Chronic treatment effects of 3e in diabetic db/db mice with 3 and 10 mg/kg for 3 weeks (n = 6). (a),
FBG changes, (b) body weight changes; (c) blood glucose changes in OGTT, (d) AUC0–120 min of blood glucose
between 0 and 120 min; OGTT was conducted after 3 weeks of treatment of 3e 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.
3. Conclusion
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To summarize, in order to identify alternative DPP-4 inhibitors with better pharmacological profiles,
novel benzoic acid–based xanthine derivatives were rapidly generated by using scaffold-hopping strategy
from uracil of compound
1
to xanthine of linagliptin. This strategy and additional SAR study led us to
2f 2h 2i and 2k) with picomolar potency
successfully discover some novel potent DPP-4 inhibitors (2e
,
,
,
and excellent selectivity against DPP-8 and DPP-9, their DPP-4 activities are much better than that of drug
alogliptin and their corresponding lead uracil derivatives. Molecular simulation revealed an additional
hydrogen bonding between the carboxyl of o-benzoic acid moiety with amine of Typ629 in S2’ subsite
may contribute to increasing in potency observed in xanthine-based compounds. The subsequent PK
studies in SD rats revealed that compounds 2f and 2i have poor oral bioavailability. To address absorption
limitation, ester prodrugs were prepared and screened for stability, toxicity and oral PK. The prodrug
evaluation resulted in the identification of a methyl ester 3e that offered an improved exposure of 2f and
prolonged duration, demonstrating a successful prodrug approach to the oral delivery of 2f. To investigate
the potential therapeutic effects of 3e, the in vivo DPP-4 inhibition was performed and revealed that 3e
had better DPP-4 inhibition duration than that of alogliptin. The acute administration of 3e achieved better
glucose-lowering activities than that of alogliptin in both normal and db/db mice. Chronic treatment of 3e
in type 2 diabetic db/db mice significantly decreased FBG levels and improved glucose tolerance. These
beneficial preclinical results support that 3e have the potential to be a novel DPP-4 inhibitor with
enhanced and longer-lasting in vivo therapeutic efficacy on T2DM.
4. Experimental section
4.1. Chemistry
All starting materials and reagents were obtained from commercial sources and used without further
purification. Reaction progresses were monitored by TLC using silica gel 60 F254 plates and UV light. Column
chromatography was performed on silica gel (200-300 mesh). Evaporations of solvents were carried out under
1
reduced pressure using regular rotary evaporators. Melting points were measured on capillary tube. H NMR
and 13C NMR spectra were recorded with a Bruker spectrometer in the indicated solvents (DMSO-d6, CDCl3,
TMS as internal standard): the values of the chemical shifts are expressed in δ values (ppm) and the coupling
constants (J) in Hz. Mass spectra were run on ThermoFisher TSQ Series Mass spectrometer system (ESI,
TQU04153). %Purity of the target compounds were determined on HPLC (Agilent 1260 Series apparatus) using
UV detector (wavelength: 254 nm) with mobile phase composed of methanol and H2O (0.1% HCOOH).
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4.1.1. 8-bromo-7-(but-2-yn-1-yl)-3-methyl-3,7-dihydro-1H-purine-2,6-dione (5)
To a solution of 8-bromo-3-methyl-xanthine (5 g, 20.4 mmol) in DMF (50 mL) was added successively
DIPEA (2.8 g, 21.4 mmol) and 1-bromo-2-butyne (2.9 g, 21.4 mmol). After the reaction mixture was stirred at
r.t. for 12 h, water (150 mL) was added, the precipitate was separated by filtration, washed with water, and dried
to give compound 5 as white solid (5.2 g, yield: 85.8%); mp: >300 °C; 1H NMR (300 MHz, DMSO-d6): δ
11.31(s, 1H), 5.06 (s, 2H), 3.31 (s, 3H), 1.80 (s, 3H); MS (ESI) m/z: 296.97 [M+H]+.
4.1.2. methyl 3-(bromomethyl)-4-fluorobenzoate (11a)
A mixture of methyl 3-bromo-2-methylbenzoate (500 mg, 3.3 mmol), NBS (770.4 mg, 4.3 mmol), and
di-benzoyl peroxide (BPO, 80.7 mg, 0.3 mmol) in 1,2-dichloroethane (10 mL) was heated at 80 °C for 12 hours.
The reaction mixture 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 (15 mL) and ethers (15 mL). The organic layer was separated, dried over NaSO4, filtered and
concentrated to give a crude product (683.2 mg, 89.6 %), which was used in the next step reaction without
further purification.
4.4.3. Methyl 2-((8-bromo-7-(but-2-yn-1-yl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)methyl)
benzoate (6a)
Compound 11a (385.5 mg, 1.7 mmol) was added to a mixture of compound 5 (500 mg, 1.7 mmol) and
K2CO3 (697.7 mg, 5.1 mmol) in DMF (30 mL). After stirring at 50 °C. for 10 h, the mixture was poured into
water. The precipitate was collected by filtration, washed with water, and dried to give compound 6a as a white
solid (703.5 mg, yield: 93.9%); mp: 214.8 - 215.7 °C; 1H NMR (500 MHz, DMSO-d6) δ 7.92 (dd, J = 7.8, 1.4
Hz, 1H), 7.49 (td, J = 7.6, 1.5 Hz, 1H), 7.39 (td, J = 7.6, 1.2 Hz, 1H), 7.09 (dd, J = 7.9, 1.1 Hz, 1H), 5.39 (s,
2H), 5.11 (q, J = 2.4 Hz, 2H), 3.90 (s, 3H), 3.42 (s, 3H), 1.80 (t, J = 2.4 Hz, 3H); MS (ESI) m/z: 467.27
[M+H]+.
4.4.4.
Methyl
(R)-2-((7-(but-2-yn-1-yl)-8-(3-((tert-butoxycarbonyl)amino)piperidin-1-yl)-3-methyl-2,6-dioxo-2,3,6,7-tetr
ahydro-1H-purin-1-yl)methyl)benzoate (7a)
A flask charged with a stir bar, compound 6a (703.5 mg, 1.6 mmol), (R)-3-(N-Boc-amino)piperidine
(393.6 mg, 2.0 mmol) and K2CO3 (654.6 mg, 4.7 mmol) and DMF (30 mL) was stirred at 65 °C for 10 h, then
the mixture was poured into water. The precipitate was collected by filtration, washed with water, and dried to
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give compound 7a as
a
white solid, which was purified by chromatography on silica gel
(dichloromethane/MeOH 100:1~80:1) to give product 7a as a white solid (763.6 mg, yield: 85.7 %). mp: 107.9 -
109.5 °C; 1H NMR (500 MHz, DMSO-d6) δ 7.91 (dd, J = 7.8, 1.5 Hz, 1H), 7.49 (td, J = 7.6, 1.5 Hz, 1H), 7.37
(td, J = 7.6, 1.2 Hz, 1H), 7.05 – 6.97 (m, 2H), 5.37 (s, 2H), 4.86 (s, 2H), 3.90 (s, 3H), 3.68 – 3.65 (m, 1H), 3.59
– 3.63 (m, 2H), 3.39 (s, 3H), 3.04 – 2.98 (m, 1H), 2.90 – 2.85 (m, 1H), 1.86 – 1.80 (m, 2H), 1.77 (t, J = 2.4 Hz,
3H), 1.72 – 1.61 (m, 1H), 1.45 – 1.42 (m, 1H),1.40 (s, 9H). MS (ESI) m/z: 565.56 [M+H]+.
4.4.5.
(R)-2-((7-(but-2-yn-1-yl)-8-(3-((tert-butoxycarbonyl)amino)piperidin-1-yl)-3-methyl-2,6-dioxo-2,3,6,7-tetr
ahydro-1H-purin-1-yl)methyl)benzoic acid (8a)
To the solution of compound 7a (620 mg, 1.1 mmol) in methanol (20 mL) was added LiOH (1M, 2.2 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 8a (513.2 mg, 84.9 %) as a light yellow solid. mp: 186.23 - 188.74 °C; 1H NMR (500 MHz,
DMSO-d6) δ 12.0 (s, 1H), 7.90 (dd, J = 7.7, 1.5 Hz, 1H), 7.46 (d, J = 7.6, 1.5 Hz, 1H), 7.32 (td, J = 7.5, 1.2 Hz,
1H), 7.02 – 6.95 (m, 2H), 5.42 (s, 2H), 5.04 – 5.00 (m, 1H), 4.96 – 4.90 (m, 1H), 3.72 – 3.68 (m, 1H), 3.66 –
3.60 (m, 1H), 3.40 (s, 3H), 3.38 – 3.35 (m, 1H), 3.26 – 3.22 (m, 1H), 3.22 – 3.18 (m, 1H), 2.04 – 2.00 (m, 1H),
1.95 – 1.90 (m, 1H), 1.78 (t, J = 2.4 Hz, 3H), 1.70 – 1.66 (m, 2H), 1.38 (s, 9H); MS (ESI) m/z: 551.26 [M+H]+.
4.4.6.
(R)-2-((8-(3-aminopiperidin-1-yl)-7-(but-2-yn-1-yl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)
methyl)benzoic acid hydrochloride (2a)
A solution of compound 8a (500 mg, 0.9 mmol) in EtOAc/ether (v/v, 1/3, 20 mL) was bubbled with freshly
prepared HCl gas at 0 °C. After the completed consumption of starting materials, the precipitate was separated
by filtration, dried in vacuum to afford compound 2a as white solid (323 mg, yield 78.9 %); HPLC purity:
95.4 %; mp: 164.5 – 167.2 °C; 1H NMR (500 MHz, DMSO-d6) δ 8.47 (d, J = 5.5 Hz, 3H), 7.92 (dd, J = 7.7, 1.5
Hz, 1H), 7.45 (td, J = 7.6, 1.5 Hz, 1H), 7.34 (td, J = 7.5, 1.2 Hz, 1H), 6.97 (d, J = 7.8 Hz, 1H), 5.40 (s, 2H),
5.11 – 4.97 (m, 1H), 4.97 – 4.86 (m, 1H), 3.73 – 3.70 (m, 1H), 3.68 – 3.58 (m, 1H), 3.41 (s, 3H), 3.39 – 3.36 (m,
1H), 3.25 – 3.21 (m, 1H), 3.20 – 3.16 (m, 1H), 2.05 – 2.01 (m, 1H), 1.96 – 1.91 (m, 1H), 1.79 (t, J = 2.4 Hz,
3H), 1.71 – 1.67 (m, 2H); 13C NMR (126 MHz, DMSO-d6) δ 168.77, 155.87, 153.90, 151.35, 147.92, 139.42,
132.71, 130.96, 129.62, 127.02, 125.74, 104.18, 81.75, 74.17, 51.82, 50.83, 46.53, 43.01, 35.84, 30.06, 27.69,
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22.33, 3.61; MS (ESI) m/z: 451.34 [M+H]+.
4.4.7.
(R)-2-((8-(3-aminopiperidin-1-yl)-7-(but-2-yn-1-yl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)
methyl)-6-fluorobenzoic acid hydrochloride (2b)
Following a similar procedure for the preparation of 2a. 2b was prepared from compound 5 and methyl
2-methyl-6-fluorobenzoate. White solid (89 mg, yield 74.1 %). HPLC purity: 98.7 %; mp: 212.3 - 214.7 °C; 1H
NMR (500 MHz, DMSO-d6) δ 8.49 – 8.48 (m, 3H), 7.41 – 7.36 (m, 1H), 7.18 (t, J = 9.0 Hz, 1H), 6.85 (d, J =
7.8 Hz, 1H), 5.14 (s, 2H), 5.05 – 5.00 (m, 1H), 4.99 – 4.89 (m, 1H), 3.73 – 3.70 (m, 1H), 3.49 – 3.47 (m, 1H),
3.46 – 3.44 (m, 1H), 3.40 (s, 3H), 3.24 – 3.20 (m, 1H), 3.18 – 3.14 (m, 1H), 2.04 – 2.00 (m, 1H), 1.96 – 1.91 (m,
1H), 1.79 (t, J = 2.4 Hz, 3H), 1.71 – 1.67 (m, 2H); 13C NMR (126 MHz, DMSO-d6) δ 166.25, 160.51, 158.53,
155.89, 153.70, 151.25, 147.94, 138.57 (d, J = 2.6 Hz), 132.12 (d, J = 9.0 Hz), 124.18 – 120.49 (m), 114.73 (d,
J = 22.0 Hz), 104.12, 81.79, 74.15, 51.79, 50.80, 46.52, 41.81 (d, J = 2.6 Hz), 35.85, 30.07, 27.68, 22.32, 3.62;
MS (ESI) m/z: 469.30 [M+H]+.
4.4.8.
(R)-2-((8-(3-aminopiperidin-1-yl)-7-(but-2-yn-1-yl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)
methyl)-5-fluorobenzoic acid hydrochloride (2c)
Following a similar procedure for the preparation of 2a. 2c was prepared from compound 5 and methyl
2-methyl-5-fluorobenzoate. White solid (238 mg, yield 76.8 %). HPLC purity: 95.2 %; mp: 181.3-183.5 °C; 1H
NMR (500 MHz, DMSO-d6) δ 8.46 – 8.45 (m, 3H), 7.65 – 7.63 (dd, J = 9.4, 2.9 Hz, 1H), 7.31 – 7.29 (dd, J =
8.4, 2.9 Hz, 1H), 7.06 – 7.03 (dd, J = 8.8, 5.4 Hz, 1H), 5.36 (s, 2H), 5.03 – 4.99 (m, 1H), 4.93 – 4.88 (m, 1H),
3.73 – 3.69 (m, 1H), 3.49 – 3.43 (m, 1H), 3.40 (s, 3H), 3.38 – 3.34 (m, 1H), 3.25 – 3.21 (m, 1H), 3.21 – 3.13 (m,
1H), 2.05 – 2.00 (m, 1H), 1.97 – 1.90 (m, 1H), 1.79 (t, J = 2.4 Hz, 3H), 1.71 – 1.69 (m, 2H); 13C NMR (126
MHz, DMSO-d6) δ 167.64 (d, J = 2.6 Hz), 161.75, 159.81, 155.87, 153.86, 151.33, 147.95, 135.53 (d, J = 3.1
Hz), 131.78, 128.40 (d, J = 7.9 Hz), 119.55, 119.38, 117.32, 117.14, 104.18, 81.77, 74.15, 51.81, 50.82, 46.53,
42.42, 35.83, 30.06, 27.68, 22.32, 3.61; MS (ESI) m/z: 469.32 [M+H]+.
4.4.9.
(R)-2-((8-(3-aminopiperidin-1-yl)-7-(but-2-yn-1-yl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)
methyl)-3-chlorobenzoic acid hydrochloride (2d)
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ACCEPTED MANUSCRIPT
Using the procedure described for compound 2a, 2d was prepared from compound 5 and methyl
2-methyl-3-chlorobenzoate. White solid (110 mg, yield 57.9 %). HPLC purity: 99.0 %; mp: 134.7-136.8 °C;
1H NMR (500 MHz, DMSO-d6) δ 8.45 – 8.44 (m, 3H), 7.57 (dd, J = 7.7, 1.4 Hz, 1H), 7.51 (dd, J = 8.0, 1.3 Hz,
1H), 7.34 (t, J = 7.8 Hz, 1H), 5.47 (s, 2H), 4.99 – 4.95 (m, 1H), 4.90 – 4.86 (m, 1H), 3.71 – 3.66 (m, 1H), 3.45 –
3.42 (m, 1H), 3.41 – 3.33 (m, 1H), 3.30 (s, 3H), 3.20 – 3.18 (m, 1H), 3.17 – 3.13 (m, 1H), 2.03 – 1.99 (m, 1H),
1.93 – 1.91 (m, 1H), 1.79 (t, J = 2.4 Hz, 3H), 1.71 – 1.65 (m, 2H); 13C NMR (126 MHz, DMSO-d6) δ 169.14,
155.67, 153.80, 151.08, 147.63, 136.77, 134.64, 133.92, 132.47, 128.68, 128.10, 104.15, 81.66, 74.21, 51.85,
50.85, 46.55, 39.36, 35.81, 29.87, 27.67, 22.32, 3.61; MS (ESI) m/z: 485.29 [M+H]+.
4.4.10.
(R)-2-((8-(3-aminopiperidin-1-yl)-7-(but-2-yn-1-yl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)
methyl)-4-chlorobenzoic acid hydrochloride (2e)
Following a similar procedure for the preparation of 2a. 2e was prepared from compound 5 and and methyl
2-methyl-4-chlorobenzoate. White solid (184 mg, yield 63.4 %). HPLC purity: 98.3 %; mp: 214.4-216.3 °C;
1H NMR (500 MHz, DMSO-d6) δ 8.46 – 8.45 (m, 3H), 7.93 (d, J = 8.4 Hz, 1H), 7.43 (dd, J = 8.4, 2.2 Hz, 1H),
7.03 (d, J = 2.1 Hz, 1H), 5.38 (s, 2H), 5.03 – 5.01 (m, 1H), 4.94 – 4.89 (m, 1H), 3.74 – 3.71 (m, 1H), 3.50 –
3.46 (m, 1H), 3.41 (s, 3H), 3.38 – 3.36 (m, 1H), 3.24 – 3.20 (m, 1H), 3.18 – 3.14 (m, 1H), 2.05 – 1.98 (m, 1H),
1.94 – 1.91 (m, 1H), 1.79 (t, J = 2.4 Hz, 3H), 1.73 – 1.67 (m, 2H); 13C NMR (126 MHz, DMSO-d6) δ 167.94,
155.90, 153.87, 151.39, 148.13, 142.09, 137.68, 132.97, 128.59, 127.31, 125.76, 104.20, 81.74, 74.17, 51.83,
50.83, 46.53, 42.92, 35.87, 30.12, 27.70, 22.36, 3.60; MS (ESI) m/z: 485.31 [M+H]+.
4.4.11.
(R)-2-((8-(3-aminopiperidin-1-yl)-7-(but-2-yn-1-yl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)
methyl)-5-chlorobenzoic acid hydrochloride (2f)
Following a similar procedure for the preparation of 2a. 2f was prepared from compound 5 and and methyl
2-methyl-5-chlorobenzoate. White solid (302 mg, yield 75.5 %). HPLC purity: 95.4 %; mp: 204.4-207.3 °C; 1H
NMR (500 MHz, DMSO-d6) δ 8.50 – 8.49 (m, 3H), 7.87 (d, J = 2.4 Hz, 1H), 7.51 (dd, J = 8.5, 2.4 Hz, 1H),
7.03 (d, J = 8.4 Hz, 1H), 5.36 (s, 2H), 5.07 – 4.97 (m, 1H), 4.96 – 4.87 (m, 1H), 3.73 – 3.70 (m, 1H), 3.48 –
3.44 (m, 1H), 3.40 (s, 3H), 3.39 – 3.36 (m, 1H), 3.25 – 3.19 (m, 1H), 3.19 – 3.14 (m, 1H), 2.02 – 2.01 (m, 1H),
1.95 – 1.92 (m, 1H), 1.79 (t, J = 2.4 Hz, 3H), 1.73 – 1.66 (m, 2H); 13C NMR (126 MHz, DMSO-d6) δ 167.51,
22

ACCEPTED MANUSCRIPT
155.88, 153.82, 151.31, 147.97, 138.42, 132.38, 131.68, 131.59, 130.27, 128.23, 104.17, 81.78, 74.14, 51.80,
50.82, 46.53, 42.52, 35.83, 30.07, 27.67, 22.32, 3.61; MS (ESI) m/z: 485.29 [M+H]+.
4.4.12.
(R)-2-((8-(3-aminopiperidin-1-yl)-7-(but-2-yn-1-yl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)
methyl)-3-bromobenzoic acid hydrochloride (2g)
Following a similar procedure for the preparation of 2a. 2g was prepared from compound 5 and methyl
3-bromo-2-methylbenzoate. White solid (168 mg, yield 62.2 %). HPLC purity: 98.6 %; mp: 163.4-165.7 °C; 1H
NMR (500 MHz, DMSO-d6) δ 8.44 – 8.43 (m, 3H), 7.70 (dd, J = 8.0, 1.3 Hz, 1H), 7.55 (dd, J = 7.7, 1.3 Hz,
1H), 7.25 (t, J = 7.8 Hz, 1H), 5.40 (s, 2H), 5.01 – 4.95 (m, 1H), 4.89 – 4.84 (m, 1H), 3.69 – 3.66 (m, 1H) 3.46 –
3.43 (m, 1H), 3.37 – 3.34 (m, 1H), 3.30 (s, 3H), 3.22 – 3.16 (m, 1H), 3.16 – 3.11 (m, 1H), 2.23 – 1.99 (m, 1H),
1.95 – 1.91 (m, 1H), 1.79 (t, J = 2.4 Hz, 3H), 1.71 – 1.66 (m, 2H); 13C NMR (126 MHz, DMSO-d6) δ 169.25,
155.64, 153.89, 147.64, 136.59, 135.68, 135.64, 128.87, 128.47, 123.85, 104.24, 81.67, 74.23, 51.84, 50.87,
46.55, 41.95, 35.81, 29.89, 27.67, 22.31, 3.64; MS (ESI) m/z: 529.22 [M+H]+.
4.4.13.
(R)-2-((8-(3-aminopiperidin-1-yl)-7-(but-2-yn-1-yl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)
methyl)-4-bromobenzoic acid hydrochloride (2h)
Following a similar procedure for the preparation of 2a. 2h was prepared from compound 5 and methyl
4-bromo-2-methylbenzoate. White solid (244 mg, yield 73.2 %). HPLC purity: 96.9 %; mp: 208.3 - 211.5 °C;
1H NMR (500 MHz, DMSO-d6) δ 8.44 – 8.43 (m, 3H), 7.84 (d, J = 8.3 Hz, 1H), 7.57 (dd, J = 8.4, 2.0 Hz, 1H),
7.15 (d, J = 1.9 Hz, 1H), 5.38 (s, 2H), 5.03 – 5.02 (m, 1H), 4.99 – 4.98 (m, 1H), 3.74 – 3.70 (m, 1H), 3.50 –
3.46 (m, 1H), 3.41 (s, 3H), 3.41 – 3.36 (m, 1H), 3.24 – 3.17 (m, 1H), 3.16 – 3.14 (m, 1H), 2.03 – 1.94 (m, 1H),
1.94 – 1.91 (m, 1H), 1.80 (t, J = 2.4 Hz, 3H), 1.74 – 1.67 (m, 2H); 13C NMR (126 MHz, DMSO-d6) δ 168.09,
155.90, 153.86, 151.39, 148.12, 142.06, 133.00, 130.34, 129.05,128.03, 126.69, 104.16, 81.76, 74.17, 51.82,
50.81, 46.53, 42.82, 35.88, 30.12, 27.70, 22.35, 3.63; MS (ESI) m/z: 529.26 [M+H]+.
4.4.14.
(R)-2-((8-(3-aminopiperidin-1-yl)-7-(but-2-yn-1-yl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)
methyl)-5-bromobenzoic acid hydrochloride (2i)
Following a similar procedure for the preparation of 2a. 2i was prepared from compound 5 and methyl
5-bromo-2-methylbenzoate. White solid (186 mg, yield 66.4 %). HPLC purity: 97.6 %; mp: 204.1 - 206.7 °C;
23

ACCEPTED MANUSCRIPT
1H NMR (500 MHz, DMSO-d6) δ 8.46 – 8.45 (m, 3H), 8.00 (d, J = 2.3 Hz, 1H), 7.64 (dd, J = 8.4, 2.3 Hz, 1H),
6.97 (d, J = 8.5 Hz, 1H), 5.34 (s, 2H), 5.04 – 4.98 (m, 1H), 4.94 – 4.87 (m, 1H), 3.72– 3.69 (m, 1H), 3.50 – 3.43
(m, 1H), 3.40 (s, 3H), 3.37– 3.36 (m, 1H), 3.25 – 3.21 (m, 1H), 3.20 – 3.13 (m, 1H), 2.04 – 1.99 (m, 1H), 1.96 –
1.91 (m, 1H), 1.79 (t, J = 2.4 Hz, 3H), 1.74 – 1.63 (m, 2H); 13C NMR (126 MHz, DMSO-d6) δ 167.41, 155.88,
153.81, 151.30, 147.97, 138.86, 135.30, 133.15, 131.92, 128.49, 119.80, 104.17, 81.79, 74.14, 51.80, 50.82,
46.52, 42.57, 35.84, 30.07, 27.67, 22.31, 3.61; MS (ESI) m/z: 529.27 [M+H]+.
4.4.15.
(R)-2-((8-(3-aminopiperidin-1-yl)-7-(but-2-yn-1-yl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)
methyl)-3-methoxybenzoic acid hydrochloride (2j)
Following a similar procedure for the preparation of 2a. 2j was prepared from compound 5 and methyl
2-methyl-3-methoxybenzoate. White solid (65mg, yield 59.1 %). HPLC purity: 99.1 %; mp: 104.9-106.2 °C; 1H
NMR (500 MHz, DMSO-d6) δ 8.53 – 8.48 (m, 3H), 7.27 (t, J = 7.9 Hz, 1H), 7.22 (dd, J = 7.8, 1.4 Hz, 1H), 7.06
(dd, J = 8.2, 1.4 Hz, 1H), 5.40 (s, 2H), 5.00 (d, J = 2.5 Hz, 1H), 4.93 (d, J = 2.5 Hz, 1H), 3.67 – 3.64 (m, 1H),
3.64 (s, 3H), 3.44 – 3.40 (m, 1H), 3.36 – 3.33 (m, 1H), 3.29 (s, 3H), 3.21 – 3.15 (m, 1H), 3.16 – 3.11 (m, 1H),
2.03 – 1.98 (m, 1H), 1.95 – 1.90 (m, 1H), 1.80 (t, J = 2.4 Hz, 3H), 1.69 (m, 2H); 13C NMR (126 MHz,
DMSO-d6) δ 169.66, 158.42, 155.36, 153.81, 151.03, 147.21, 135.40, 128.17, 125.72, 121.40, 114.38, 104.07,
81.51, 74.38, 56.19, 51.90, 50.89, 46.57, 37.09, 35.58, 29.75, 27.67, 22.36, 3.61; MS (ESI) m/z: 481.32
[M+H]+.
4.4.16.
(R)-2-((8-(3-aminopiperidin-1-yl)-7-(but-2-yn-1-yl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)
methyl)-4-methoxybenzoic acid hydrochloride (2k)
Following a similar procedure for the preparation of 2a. 2k was prepared from compound 5 and methyl
2-methyl-4-methoxybenzoate. White solid (236 mg, yield 73.7 %). HPLC purity: 95.5 %; mp: 142.8-145.2 °C;
1H NMR (500 MHz, DMSO-d6) δ 8.59 – 8.58 (m, 3H), 7.96 (d, J = 8.7 Hz, 1H), 6.92 (dd, J = 8.7, 2.6 Hz, 1H),
6.35 (d, J = 2.5 Hz, 1H), 5.38 (s, 2H), 5.04 – 4.99 (m, 1H), 4.93 – 4.88 (m, 1H), 3.77 – 3.75 (m, 1H), 3.72 (s,
3H), 3.51 – 3.46 (m, 1H), 3.40 (s, 3H), 3.39 – 3.35 (m, 1H), 3.25 – 3.22 (m, 1H), 3.14 – 3.09 (m, 1H), 2.06 –
2.03 (m, 1H), 1.95 – 1.91 (m, 1H), 1.78 (t, J = 2.4 Hz, 3H), 1.74 – 1.66 (m, 2H); 13C NMR (126 MHz,
DMSO-d6) δ 168.20, 162.76, 155.96, 153.85, 151.32, 148.02, 142.15, 133.90, 121.44, 112.52, 110.59, 104.11,
81.70, 74.17, 55.75, 51.79, 50.81, 46.57, 43.20, 35.88, 30.09, 27.69, 22.44, 3.58; MS (ESI) m/z: 481.35
24

ACCEPTED MANUSCRIPT
[M+H]+.
4.4.17.
(R)-2-((8-(3-aminopiperidin-1-yl)-7-(but-2-yn-1-yl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)
methyl)-4-cyanobenzoic acid hydrochloride (2l)
Following a similar procedure for the preparation of 2a. 2l was prepared from compound 5 and methyl
1
2-methyl-4-cyanobenzoate. White solid (195 mg, yield 65.0%). HPLC purity: 97.9 %; mp: 207.5-209.2 °C; H
NMR (500 MHz, DMSO-d6) δ 8.47 – 8.46 (m, 3H), 8.02 (d, J = 8.0 Hz, 1H), 7.83 (dd, J = 8.0, 1.6 Hz, 1H),
7.58 (d, J = 1.5 Hz, 1H), 5.37 (s, 2H), 5.03 – 4.99 (m, 1H), 4.93 – 4.89 (m, 1H), 3.75 – 3.70 (m, 1H), 3.49 –
3.41 (m, 1H), 3.40 (s, 3H), 3.38 – 3.35 (m, 1H), 3.23 – 3.19 (m, 1H), 3.18 – 3.14 (m, 1H), 2.03 (m, 1H), 1.94 –
1.92 (m, 1H), 1.79 (t, J = 2.4 Hz, 3H), 1.73 – 1.70 (m, 2H); 13C NMR (126 MHz, DMSO-d6) δ 167.77, 155.77,
153.95, 151.47, 148.19, 140.57, 134.19, 131.43, 131.0, 129.93, 118.64, 114.98, 104.37, 81.72, 74.20, 51.87,
50.90, 46.53, 42.83, 35.79, 30.12, 27.69, 22.35, 3.60; MS (ESI) m/z: 476.33 [M+H]+.
4.4.18.
methyl
(R)-2-((8-(3-aminopiperidin-1-yl)-7-(but-2-yn-1-yl)-3-methyl-2,6-dioxo-2,3,6,7-
tetrahydro-1H-purin-1-yl)methyl)benzoate hydrochloride (3a)
Using the procedure described for compound 2a, compound 7a was converted to 3a as white solid (258mg,
1
yield 78.3 %); HPLC purity: 95.5 %; mp: 153.5-155.2 °C; H NMR (500 MHz, DMSO-d6) δ 8.47 – 8.46 (m,
3H), 7.91 (dd, J = 7.8, 1.5 Hz, 1H), 7.49 (td, J = 7.6, 1.5 Hz, 1H), 7.38 (dd, J = 7.6, 1.3 Hz, 1H), 7.03 (dd, J =
7.8, 1.2 Hz, 1H), 5.38 (s, 2H), 5.04 – 4.99 (m, 1H), 4.93 – 4.92 (m, 1H), 3.90 (s, 3H), 3.73 – 3.70 (m, 1H), 3.49
– 3.45 (m, 1H), 3.40 (s, 3H), 3.39 – 3.38 (m, 1H), 3.24 – 3.22 (m, 1H), 3.21 – 3.16 (m, 1H), 2.05 – 2.01 (m, 1H),
1.94 – 1.93 (m, 1H), 1.79 (t, J = 2.4 Hz, 3H), 1.72 – 1.67 (m, 2H); 13C NMR (126 MHz, DMSO-d6) δ 167.49,
155.88, 153.85, 151.32, 147.95, 139.35, 133.10, 130.73, 128.69, 127.23, 126.12, 104.17, 81.75, 74.16, 52.67,
51.81, 50.82, 46.53, 42.76, 35.84, 30.06, 27.69, 22.33, 3.61; MS (ESI) m/z: 465.38 [M+H]+.
4.4.19.
methyl
(R)-2-((8-(3-aminopiperidin-1-yl)-7-(but-2-yn-1-yl)-3-methyl-2,6-dioxo-2,3,6,7-
tetrahydro-1H-purin-1-yl)methyl)-6-fluorobenzoate hydrochloride (3b)
Using the procedure described for compound 2a, compound 7b was converted to 3b as white solid (109mg,
yield 85.3%). HPLC purity: 96.5 %; mp: 147.1-150.4 °C; 1H NMR (500 MHz, DMSO-d6) δ 8.43 – 8.42 (m, 3H),
7.45 (td, J = 8.1, 5.8 Hz, 1H), 7.23 (t, J = 9.1 Hz, 1H), 6.98 (d, J = 7.9 Hz, 1H), 5.12 (s, 2H), 5.03 – 4.98 (m,
1H), 4.93 – 4.89 (m, 1H), 3.91 (s, 3H), 3.72 – 3.69 (m, 1H), 3.46 – 3.45 (m, 1H), 3.39 (s, 3H), 3.37 – 3.33 (m,
1H), 3.24 – 3.19 (m, 1H), 3.19 – 3.15 (m, 1H), 2.04 – 2. 01 (m, 1H), 1.96 – 1.91 (m, 1H), 1.80 (t, J = 2.4 Hz,
25

ACCEPTED MANUSCRIPT
3H), 1.72 – 1.65 (m, 2H). 13C NMR (126 MHz, DMSO-d6) δ 165.24, 160.64, 158.65, 155.90, 153.66, 151.20,
147.95, 138.97, 132.88 (d, J = 9.2 Hz), 122.86 (d, J = 2.9 Hz), 120.18 (d, J = 16.4 Hz), 104.08, 81.80, 74.13,
53.17, 51.78, 50.78, 46.51, 41.64, 35.86, 30.05, 27.68, 22.31, 3.61; MS (ESI) m/z: 483.45 [M+H]+.
4.4.20.
methyl
(R)-2-((8-(3-aminopiperidin-1-yl)-7-(but-2-yn-1-yl)-3-methyl-2,6-dioxo-2,3,6,7-
tetrahydro-1H-purin-1-yl)methyl)-3-chlorobenzoate hydrochloride (3c)
Using the procedure described for compound 2a, compound 7d was converted to 3c as white solid (75 mg,
1
yield 64.5 %); HPLC purity: 98.9 %; mp: 136.5-138.4 °C; H NMR (500 MHz, DMSO-d6) δ 8.44 – 8.41 (m,
3H), 7.57 (dd, J = 8.1, 1.3 Hz, 1H), 7.52 (dd, J = 7.8, 1.3 Hz, 1H), 7.36 (t, J = 7.8 Hz, 1H), 5.40 (s, 2H), 5.02 –
4.94 (m, 1H), 4.91 – 4.87 (m, 1H), 3.73 (s, 3H), 3.70 – 3.67 (m, 1H), 3.47 – 3.43 (m, 1H), 3.37 – 3.34 (m, 1H),
3.29 (s, 3H), 3.23 – 3.14 (m, 1H), 3.18 – 3.14 (m, 1H), 2.02 – 1.99 (m, 1H), 1.95 – 1.91 (m, 1H), 1.79 (t, J = 2.4
Hz, 3H), 1.69 – 1.61 (m, 2H); 13C NMR (126 MHz, DMSO-d6) δ 168.11, 155.70, 153.65, 151.01, 147.68,
135.20, 134.40, 134.25, 132.69, 128.84, 128.02, 104.01, 81.72, 74.15, 52.88, 51.80, 50.75, 46.52, 39.31, 35.82,
29.84, 27.67, 22.32, 3.57. MS (ESI) m/z: 499.36 [M+H]+.
4.4.21.
methyl
(R)-2-((8-(3-aminopiperidin-1-yl)-7-(but-2-yn-1-yl)-3-methyl-2,6-dioxo-2,3,6,7
-tetrahydro-1H-purin-1-yl)methyl)-4-chlorobenzoate hydrochloride (3d)
Using the procedure described for compound 2a, compound 7e was converted to 3d as white solid (138 mg,
1
yield 78.2 %). HPLC purity: 97.4 %; mp: 159.6-162.9 °C; H NMR (500 MHz, DMSO-d6) δ 8.55 – 8.42 (m,
3H), 7.92 (d, J = 8.4 Hz, 1H), 7.46 (dd, J = 8.4, 2.2 Hz, 1H), 7.09 (d, J = 2.1 Hz, 1H), 5.35 (s, 2H), 5.06 – 4.97
(m, 1H), 4.94 – 4.89 (m, 1H), 3.90 (s, 3H), 3.80 – 3.65 (m, 1H), 3.54 – 3.44 (m, 1H), 3.40 (s, 3H), 3.38 – 3.36
(m, 1H), 3.25 – 3.20 (m, 1H), 3.18 – 3.13 (m, 1H), 2.05 – 1.95 (m, 1H), 1.94 – 1.91 (m, 1H), 1.79 (t, J = 2.4 Hz,
3H), 1.75 – 1.62 (m, 2H); 13C NMR (126 MHz, DMSO-d6) δ 166.68, 155.89, 153.82, 151.36, 148.15, 141.95,
138.08, 132.72, 127.61, 127.52, 126.14, 104.18, 81.74, 74.16, 52.89, 51.81, 50.82, 46.53, 42.68, 35.87, 30.11,
27.69, 22.37, 3.60; MS (ESI) m/z: 499.41 [M+H]+.
4.4.22.
methyl
(R)-2-((8-(3-aminopiperidin-1-yl)-7-(but-2-yn-1-yl)-3-methyl-2,6-dioxo-
2,3,6,7-tetrahydro-1H-purin-1-yl)methyl)-5-chlorobenzoate (3e)
Using the procedure described for compound 2a, compound 7f was converted to 3e as white solid (200 mg,
yield 88.5 %). HPLC purity: 99.3 %; mp: 110.0-112.7 °C; 1H NMR (500 MHz, DMSO-d6) δ 7.88 (d, J = 2.4 Hz,
1H), 7.56 (dd, J = 8.5, 2.4 Hz, 1H), 7.10 (d, J = 8.5 Hz, 1H), 5.34 (s, 2H), 5.05 – 4.96 (m, 1H), 4.95 – 4.86 (m,
1H), 3.91 (s, 3H), 3.75 – 3.67 (m, 1H), 3.52 – 3.44 (m, 1H), 3.39 (s, 3H), 3.37 – 3.31 (m, 1H), 3.23 – 3.17 (m,
26

ACCEPTED MANUSCRIPT
1H), 3.20 – 3.12 (m, 1H), 2.02 – 1.98 (m, 1H), 1.98 – 1.90 (m, 1H), 1.80 (t, J = 2.4 Hz, 3H), 1.73 – 1.63 (m,
2H); 13C NMR (126 MHz, DMSO-d6) δ 166.29, 155.93, 153.77, 151.28, 148.02, 138.33, 132.76, 131.78, 130.68,
130.10, 128.61, 104.13, 81.78, 74.13, 53.05, 52.07, 50.76, 46.58, 42.25, 35.85, 30.06, 27.94, 22.38, 3.61. MS
(ESI) m/z: 499.41 [M+H]+.
4.4.23.
ethyl
(R)-2-((8-(3-aminopiperidin-1-yl)-7-(but-2-yn-1-yl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)
methyl)-5-chlorobenzoate (3f)
A mixture of compound 8f (300 mg, 0.51mmol), bromoethane (70.0 mg, 0.64 mmol) and K2CO3 (177 mg,
1.28 mmol) was stirred at r.t for 6h. Water was added, and the precipitate was separated by filtration, dried in
vacuum to afford compound 9a, which underwent deprotection of tert-butoxy carbonyl group using the
procedure described for compound 2a to give 3f as white solid (199 mg, yield 86.3 %). HPLC purity: 95.5 %;
mp: 105.3-107.5 °C; 1H NMR (500 MHz, DMSO-d6) δ 7.87 (d, J = 2.4 Hz, 1H), 7.55 (dd, J = 8.5, 2.4 Hz, 1H),
7.09 (d, J = 8.5 Hz, 1H), 5.34 (s, 2H), 5.05 – 4.96 (m, 1H), 4.91 (dd, J = 17.8, 2.5 Hz, 1H), 4.37 (q, J = 7.1 Hz,
2H), 3.74 – 3.68 (m, 1H), 3.51 – 3.45 (m, 1H), 3.40 (s, 3H), 3.36 – 3.31 (m, 1H), 3.21 – 3.17 (m, 1H), 3.17 –
3.13 (m, 1H), 2.04 – 1.98 (m, 1H), 1.95 – 1.89 (m, 1H), 1.80 (t, J = 2.4 Hz, 3H), 1.74 – 1.63 (m, 2H), 1.37 (t, J
= 7.1 Hz, 3H); 13C NMR (126 MHz, DMSO-d6) δ 165.82, 155.94, 153.77, 151.28, 148.03, 138.27, 132.67,
131.77, 130.96, 130.01, 128.57, 104.13, 81.78, 74.13, 61.88, 52.15, 50.75, 46.59, 42.25, 35.85, 30.06, 28.03,
22.39, 14.50, 3.60; MS (ESI) m/z: 513.42 [M+H]+.
4.4.24.
(5-methyl-2-oxo-1,3-dioxol-4-yl)methyl
(R)-2-((8-(3-aminopiperidin-1-yl)-7-(but-2-yn-1-yl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)
methyl)-5-chlorobenzoate (3g)
Using the procedure described for compound 3f, compound 3g was prepared from compound 8f and
4-(chloromethyl)-5-methyl-1,3-dioxol-2-one. White solid (140 mg, yield 66.5 %). HPLC purity: 98.3 %; mp:
219.1-221.4 °C; 1H NMR (500 MHz, DMSO-d6) δ 8.64 (s, 3H), 7.88 (d, J = 2.4 Hz, 1H), 7.58 (dd, 1H), 7.14 (d,
J = 8.5 Hz, 1H), 5.34 (s, 2H), 5.27 (s, 2H), 5.07 – 4.99 (m, 1H), 4.95 – 4.89 (m, 1H), 3.77 – 3.69 (m, 1H), 3.49
– 3.44 (m, 1H), 3.38 (s, 3H), 3.36 (s, 1H), 3.27 – 3.21 (m, 1H), 3.19 – 3.11 (m, 1H), 2.24 (s, 3H), 2.08 – 1.98 (m,
1H), 1.98 – 1.89 (m, 1H), 1.79 (t, J = 2.3 Hz, 3H), 1.76 – 1.66 (m, 2H); 13C NMR (126 MHz, DMSO-d6) δ
165.28, 155.95, 153.78, 152.42, 151.27, 148.03, 141.02, 138.51, 133.62, 133.13, 131.87, 130.18, 128.96,
27

ACCEPTED MANUSCRIPT
104.14, 81.77, 74.11, 55.41, 51.75, 50.81, 46.56, 42.02, 35.84, 30.05, 27.64, 22.40, 9.44, 3.61. MS (ESI) m/z:
597.49 [M+H]+.
4.4.25.
methyl
(R)-2-((8-(3-aminopiperidin-1-yl)-7-(but-2-yn-1-yl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)
methyl)-5-bromobenzoate (3h)
Using the procedure described for compound 2a, compound 7i was converted to 3h as white solid (288 mg,
yield 86.8 %). HPLC purity: 96.4 %; mp: 169.1-171.1 °C; 1H NMR (500 MHz, DMSO-d6) δ 8.01 (d, J = 2.3 Hz,
1H), 7.68 (dd, J = 8.5, 2.3 Hz, 1H), 7.03 (d, J = 8.5 Hz, 1H), 5.31 (s, 2H), 5.08 – 4.96 (m, 1H), 4.96 – 4.83 (m,
1H), 3.91 (s, 3H), 3.75 – 3.67 (m, 1H), 3.52 – 3.45 (m, 1H), 3.39 (s, 3H), 3.33 – 3.24 (m, 1H), 3.20 – 3.10 (m,
2H), 2.09 – 1.97 (m, 1H), 1.96 – 1.89 (m, 1H), 1.79 (t, J = 2.4 Hz, 3H), 1.74 – 1.59 (m, 2H). 13C NMR (126
MHz, DMSO-d6) δ 166.19, 156.01, 153.75, 151.28, 148.07, 138.77, 135.69, 132.95, 130.90, 128.84, 119.94,
104.09, 81.76, 74.13, 53.05, 52.52, 50.72, 46.68, 42.30, 35.86, 30.06, 28.37, 22.51, 3.61. MS (ESI) m/z: 543.40
[M+H]+.
4.4.26.
ethyl
(R)-2-((8-(3-aminopiperidin-1-yl)-7-(but-2-yn-1-yl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)
methyl)-5-bromobenzoate (3i)
Using the procedure described for compound 3f, compound 3i was prepared from compound 8i and
1
bromoethane. White solid (199 mg, yield 76.8 %). HPLC purity: 95.3 %; mp: 158.8-160.7 °C; H NMR (500
MHz, DMSO-d6) δ 8.08 – 7.94 (m, 1H), 7.68 (d, J = 7.8 Hz, 1H), 7.02 (d, J = 8.5 Hz, 1H), 5.32 (s, 2H), 5.04 –
4.96 (m, 1H), 4.95 – 4.88 (m, 1H), 4.42 – 4.33 (m, 2H), 3.71 (d, J = 12.6, 3.5 Hz, 1H), 3.48 (d, J = 13.0 Hz, 1H),
3.39 (s, 3H), 3.37 – 3.32 (m, 1H), 3.23 – 3.13 (m, 2H), 2.04 – 1.97 (m, 1H), 1.95 – 1.89 (m, 1H), 1.79 (s, 3H),
1.71 – 1.63 (m, 2H), 1.37 (t, J = 7.1 Hz, 3H); 13C NMR (126 MHz, DMSO-d6) δ 165.73, 155.92, 153.76,
151.28, 148.02, 138.70, 135.60, 132.85, 131.21, 128.81, 119.95, 104.13, 81.78, 74.13, 61.89, 52.06, 50.76,
46.57, 42.31, 35.85, 30.06, 27.94, 22.37, 14.51, 3.61; MS (ESI) m/z: 557.41 [M+H]+.
4.4.27.
(5-methyl-2-oxo-1,3-dioxol-4-yl)methyl
(R)-2-((8-(3-aminopiperidin-1-yl)-7-(but-2-yn-1-yl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)
methyl)-5-bromobenzoate (3j)
Using the procedure described for compound 3f, compound 3j was prepared from compound 8i and
4-(chloromethyl)-5-methyl-1,3-dioxol-2-one. White solid (123 mg, yield 57.2 %). HPLC purity: 98.9 %; mp:
28