Discovery of highly potent DPP-4 inhibitors by hybrid compound design based on linagliptin and alogliptin

Article abstract of DOI:10.1016/j.ejmech.2014.06.044

Highly potent DPP-4 inhibitors have been identified by hybrid compound design based on linagliptin and alogliptin. The most promising compound 2h (IC₅₀ = 0.31 nM) exhibited 8.5-fold and 2.5-fold more potent activity than that of alogliptin (IC₅₀ = 2.63 nM) and linagliptin (IC ₅₀ = 0.77 nM), respectively. Compound 2h had a good inhibition selectivity for DPP-4 over DPP-8/9 and thus was selected for further biological evaluation, including oral glucose tolerance, plasma DPP-4 inhibitory activity, pharmacokinetic profile, acute toxicity and hERG inhibition. The assay results showed that 2h displayed significant in vivo glucose-lowering effect and low risk of toxicity. Further studies are expected to confirm 2h as a potential drug candidate for the treatment of type 2 diabetes.

Full text of DOI:10.1016/j.ejmech.2014.06.044

Accepted Manuscript
Discovery of Highly Potent DPP-4 Inhibitors by Hybrid Compound Design Based on
Linagliptin and Alogliptin
Zengwei Lai, Chunhong Li, Jun Liu, Lingyi Kong, Xiaoan Wen, Hongbin Sun
PII:
S0223-5234(14)00573-X
10.1016/j.ejmech.2014.06.044
EJMECH 7087
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 24 February 2014
Revised Date: 12 June 2014
Accepted Date: 23 June 2014
Please cite this article as: Z. Lai, C. Li, J. Liu, L. Kong, X. Wen, H. Sun, Discovery of Highly Potent
DPP-4 Inhibitors by Hybrid Compound Design Based on Linagliptin and Alogliptin, European Journal of
Medicinal Chemistry (2014), doi: 10.1016/j.ejmech.2014.06.044.
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ACCEPTED MANUSCRIPT
Discovery of Highly Potent DPP-4 Inhibitors by Hybrid Compound
Design Based on Linagliptin and Alogliptin
Zengwei Lai, Chunhong Li, Jun Liu, Lingyi Kong, Xiaoan Wen, Hongbin Sun
Graphical Abstract
A highly potent DPP-4 inhibitor 2h (IC = 0.31 nM) has been identified by hybrid compound
5
0
design on the basis of SAR analysis and binding modes of linagliptin and alogliptin.
O
N
80
NH2
1a
2h
N
N
N
N
7
6
0
0
N
N
O
Linagliptin (1a, IC50 = 0.77 nM)
50
O
4
3
0
0
N
N
N
N
NH
2
O
N
O
CN
20
10
0
N
N
2h (IC50 = 0.31 nM)
NH2
O
N
Contr. 0.1 mg/kg 0.3 mg/kg 1.0 mg/kg
Alogliptin (2a, IC50 = 2.63 nM)

ACCEPTED MANUSCRIPT
Discovery of Highly Potent DPP-4 Inhibitors by Hybrid Compound Design
Based on Linagliptin and Alogliptin
a,1
a,1
b, 1
a
a,
a,
Zengwei Lai, Chunhong Li, Jun Liu, Lingyi Kong, Xiaoan Wen, * Hongbin Sun *
a
State Key Laboratory of Natural Medicines and Center of Drug Discovery, China Pharmaceutical University, 24 Tongjia Xiang,
Nanjing 210009, P. R. China
b
Jiangsu Center for Drug Screening, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, P. R. China
1
Authors contributed equally to this work
*
Corresponding authors
Tel/fax: +86-25-83271198. E-mail: wxagj@126.com; hbsun2000@yahoo.com.
Abastract
Highly potent DPP-4 inhibitors have been identified by hybrid compound design based on linagliptin and
alogliptin. The most promising compound 2h (IC50 = 0.31 nM) exhibited 8.5-fold and 2.5-fold more potent
activity than that of alogliptin (IC50 = 2.63 nM) and linagliptin (IC50 = 0.77 nM), respectively. Compound 2h
had a good inhibition selectivity for DPP-4 over DPP-8/9 and thus was selected for further biological
evaluation, including oral glucose tolerance, plasma DPP-4 inhibitory activity, pharmacokinetic profile, acute
toxicity and hERG inhibition. The assay results showed that 2h displayed significant in vivo glucose-lowering
effect and low risk of toxicity. Further studies are expected to confirm 2h as a potential drug candidate for the
treatment of type 2 diabetes.
Keywords
DPP-4 inhibitors; hybrid compound design; linagliptin; alogliptin; diabetes.
1
. Introduction
Human glucagon-like peptide-1 (GLP-1), an incretin resulted from selective cleavage of
the proglucagon molecule and mainly secreted by ileal L-cells, is a potent antihyperglycemic hormone. It
exerts glucose lowering effect via multi-functions: stimulating insulin secretion and increasing insulin
sensitivity, decreasing glucagon release, inhibiting acid secretion and gastric emptying, etc [1-3]. Most
importantly, the ability of GLP-1 to enhance insulin secretion is glucose-dependent [4]. Nevertheless, the
active forms GLP-1-(7-37) NH and GLP-1-(7-36) NH , once existing in the circulation, quickly lose activity
2
2
due to rapid degradation by the enzyme dipeptidyl peptidase-4 (DPP-4) [5, 6]. Inhibition of DPP-4 has been
proved to prolong and enhance the activity of GLP-1, and thus validated as an effective approach to treatment
of type 2 diabetes [7, 8]. To date, seven DPP-4 inhibitors (sitagliptin, vildagliptin, saxagliptin, linagliptin,
anagliptin, teneligliptin and alogliptin) have been approved for the treatment of diabetes. Yet, the enthusiasm
1

ACCEPTED MANUSCRIPT
in developing new DPP-4 inhibitors remains strong since current drugs still have some undesirable side effects
[
9, 10].
Among the marketed DPP-4 inhibitors, linagliptin and alogliptin (Figure 1) have the same structure
fragment (R)-3-aminopiperidine, which has been identified to provide key hydrogen bonding interactions with
Glu205 and Glu206 of DPP-4 [11, 12]. However, a systematic study on the structure-activity relationship of
this fragment still remains to be explored, particularly, it would be interesting to replace this fragment with
other amino groups. In this regard, we designed and synthesized a series of novel analogues 1b~1j and 2b~2d
with diverse amino groups to replace the (3R)-aminopiperidine moiety (Figure 2). Moreover, we reasoned that
hybrid compound design based on the privileged fragments of linagliptin and alogliptin might result in more
potent DPP-4 inhibititors. Thus, after careful comparison of the binding modes of linagliptin and alogliptin, we
used the quinazolinylmethyl and 2-butynyl groups of linagliptin to take the places of the N -methyl and N -
3
1
orthocyanobenzyl groups of alogliptin, respectively, while (3R)-aminopiperidine group was remained or
displaced by other amines (2e~2k, Figure 3). Upon screening of compounds 2e~2k, a couple of compounds
were found to be highly potent DPP-4 inhibititors, and encouraged by this finding, more derivatives along this
line (2l~2v, Figure 3) were synthesized and evaluated. During this study, very recently Xie et al also reported
their discovery of highly potent DPP-4 inhibitors by this hybrid strategy [13]. Herein, we present our study
results.
2
. Result and discussion
2
.1. Chemistry
Compounds 1a-1j were prepared according to the procedure depicted in Scheme 1 [11]. 8-Bromo-3-methyl-
H-purine-2,6(3H,7H)-dione was selectively alkylated at N-7 by 1-bromo-2-butyne, 3-bromo-1-propyne or 3-
1
bromo-1-propene to give the corresponding N-alkylated purinediones 3a-3c, respectively, which further
reacted with 2-chloromethyl-4-methylquinazoline to afford the purinediones 4a-4c. Amination of 4a-4c gave
the target compounds 1a-1j.
The synthetic route to compounds 2a-2v is outlined in Scheme 2 [12]. In brief, 6-chlorouracil was
selectively alkylated at N-1 by alkyl halides under mild conditions (DIPEA and r.t.) to give the corresponding
alkylated uracils 5a-5d, which were further alkylated at N-3 by alkyl halides under strong conditions
o
(
NaH/LiBr/80 C) to afford the dialkylated uracils 6a-6o. Amination of 6a-6o in the presence of
4
Å molecule
sieve furnished the target compounds 2a-2v.
.2. Biological evaluation and prelimilary SAR analysis of 1a-1j and 2a-2d
The DPP-4 inhibitory activity was screened for analogues 1b-1i and 2b-2d at 10 ꢀM and 100 nM, with 1a
linagliptin) and 2a (alogliptin) as the positive controls. The compounds with good inhibition rates at 100 nM
2
(
were further selected to determine their IC50 values. As shown in Tables 1~2, all the analogues without (3R)-
aminopiperidine substituent exhibited poor DPP-4 inhibitory activity, and the compounds with (S)-
configuration were generally less active than those with (R)-configuration (e.g. 1h vs 1i; 2c vs 2d), implying
that (3R)-aminopiperidine was a predominant fragment for the potency.
2
.3. Comparison of the binding modes of linagliptin and alogliptin
The crystallographic structures of DPP-4 complexed with linagliptin and alogliptin (PDB codes: 2RGU and
G0B) were aligned using PyMol [14]. It was revealed that alogliptin structurally overlaped well with the right
3
2

ACCEPTED MANUSCRIPT
part of linagliptin (Figure 4). The uracil ring of alogliptin and the purinedione skeleton of linagliptin lay nearly
in a plane, both forming hydrogen bonding interactions with Ser630 and π-π stacks with Tyr547. The 2-
cyanobenzyl group of alogliptin and the 2-butynyl group of linagliptin inserted into the S1 pocket with their
distant carbons almost completely overlapping.
Interestingly, although both two primary amino groups formed hydrogen bonding interactions with
Glu205/206, it was found that the primary amino group of alogliptin took an axial conformation, but not
equatorial like in linagliptin. With computational study, Zhang et al explained that the alogliptin molecule
energy with an axial NH was lower than that with an equatorial NH , because the axial NH could make
2
2
2
intromolecular hydrogen bonding interactions with the cyanobenzyl CN [12]. This speculation seems discord
with a very recent crystallography study [15]. In this crystallography study, both axial NH and equatorial NH2
2
were observed in the crystals of alogliptin, indicating that the energy gap between the axial and equatorial NH2
2
is small. Why only axial NH was observed in alogliptin binding to DPP-4? One reason might be that the
alogliptin molecule with equatorial NH was induced to take an axial conformation for fitting the
2
Glu205/Glu206 residues of DPP-4. Another possibility is that it could not interact with Glu205/Glu206 at all.
2
Taken together, it might be not easy for the alogliptin molecule with equatorial NH to interact with DPP-4,
therefore causing alogliptin less potent than linagliptin. Moreover, it was clearly disclosed that the quinazoline
skeleton of linagliptin paralell faced Trp629, indicating that linagliptin had π-π stacks not only with Tyr547 but
also with Trp629. This is probably one main factor leading to that linagliptin is more potent than alogliptin.
2
.4. Discovery of highly potent DPP-4 inhibitors 2h and 2i
Based on the preliminary SAR study and the binding mode analysis, hybrid compounds 2e-2k were
designed, synthesized and evaluated for their DPP-4 inhibitory activity. As shown in Table 3, compound 2h
and its (S)-entiomer 2i exhibited highly potent DPP-4 inhibitory activity, with IC50 of 0.31 nM and 0.35 nM,
respectively, while the others except 2j (IC50 = 12.7 nM) showed poor or no activity. It seems that a proper
combination of R
3 4 2
and R substituents had profound effect on potency (e.g 2e vs 2h; 2f vs 2j; 2h vs 2k). For R
substitution, 3-aminopiperidine was found to be critical for the potency.
To predict the possible interactions of 2h and 2i binding to DPP-4, molecular docking of 2h and 2i into the
inhibitor binding site of DPP-4 was performed based on the DPP-4 co-crystal structure (PDB code: 2RGU) by
the genetic algorithm of GOLD 3.0.1. As expected, apart from keeping hydrogen bonding interactions with
Glu205 and Glu206, and π-π stacks with Try547, 2h and 2i probably have additional π-π stacks with Trp629
(
Figure 5), thus leading to be 7.5-fold and 8.5-fold more potent than alogliptin, respectively.
2
.5. Further compound design and SAR analysis
Based on the discovery of 2h and 2i, compounds 2l-2v were designed and synthesized, mainly to probe the
effect of diverse substituents of R
l-2v displayed DPP-4 inhibitory activity to some degree. Among them, 2l, 2s and 2u showed quite potent
activity with IC50 < 4 nM. Moreover, there was a very clear tendency that the R substituent with electron
3
aromatic groups on th epotency. As shown in Table 4, most of compounds
2
3
4
withdrawn ability benefited the DPP-4 inhibitory activity (2l vs 2m/2n, 2o vs 2p~2s). As for the R substituents,
the allyl group could be an alternative substituent for 2-butynyl to keep th epotency (2l vs 2u), while the 2-
propynyl group obviously reduced the activity (2l vs 2t).
2
.6. Selectivity for DPP-4 over DPP-8 and DPP-9
3

ACCEPTED MANUSCRIPT
Inhibition selectivity for DPP-4 over DPP-8 and DPP-9 [16] was tested for 8 compounds with DPP-4 IC50
<
1
2 nM. The DPP-8/9 inhibition rates of 8 compounds at 100 ꢀM and 25 ꢀM are listed in Table 5, with 1a
(linagliptin) and 2a (alogliptin) as the controls. As shown in Table 5, except that 1h and 2t at 100 ꢀM
exhibited DPP-8 inhibtion rates >33% and >27%, the other 6 compounds were proved to be highly selective
for DPP-4 over DPP-8 and DPP-9.
2
.7. Further in vitro and in vivo evaluation of 2h
The effect of 2h on glucose tolerance was tested in db/db diabetic mice, with 1a (linagliptin) as the positive
control (Figures 6, 7). As shown in Figure 7, by single oral administration of 2h 45 min prior to glucose load,
the plasma glucose excursion was remarkably reduced in a dose-dependent manner from 0.1 mg/kg to 1.0
mg/kg. Compared with 1a (Figure 6), the improvement effect of 2h on glucose tolerance was observed. For
example, the plasma glucose level 30 min after glucose load was suppressed to below 24 mM by only 0.1
mg/kg dose of 2h (Figure 7), while it was almost not affected by the same dose of 1a (Figure 6). In addition,
the inhibition rates of plasma glucose AUC0→120 min by 0.1 mg/kg and 0.3 mg/kg doses of 2h were much higher
than that by the same doses of 1a (Figure 8). However, at a 1.0 mg/kg dose, the inhibition rate of AUC0→120 min
by 2h was equal to that by 1a. Moreover, the improvement of glucose tolerance by 2h was proved to correlate
with decreased DPP-4 activity (Figure 9).
Preliminary pharmacokinetic profile of 2h was studied in SD rats. The selected parameters including half
life (t ), time to the maximum plasma concentration (Tmax), maximum plasma concentration (Cmax), area
1
/2
under the plasma concentration time curve (AUC0-∞), mean residence time (MRT) and oral bioavailability (F),
are listed in Table 6. The results showed that 2h was orally available.
The safety of 2h was evaluated by acute toxicity study and hERG channel inhibitory activity test.
Compound 2h was proved to have wide safety margin (LD50 = 1.63 g/kg) and low risk of hERG channel
inhibition (IC50 > 100 ꢀM).
3
. Conclusion
Several highly potent selective DPP-4 inhibitors have been discovered based on structure activity
relationship study and hybrid compound design, with the marketed drugs linagliptin and alogliptin as lead
compounds. Among them, 2h (IC50 = 0.31 nM) and 2i (IC50 = 0.35 nM) showed the best DPP-4 inhibitory
activity, with more potent activity than both linagliptin (IC50 = 0.77 nM) and alogliptin (IC50 = 2.63 nM).
Preliminary efficacy, pharmacokinetics and safety studies identified 2h as a potential drug candidate for the
treatment of type 2 diabetes.
4
. Experiments
4
4
.1. Chemistry
.1.1. General
1
13
All commercially available solvents and reagents were used without further purification. H NMR and
NMR spectra were recorded at 300/500 and 75/125 MHz respectively on an ACF * 300Q Bruker or ACF*
00Q Bruker spectrometer with Me Si as the internal reference. Low-resolution and high-resolution mass
C
5
4
spectra (LRMS and HRMS) were given with electron impact mode. The mass analyzer type used for the
HRMS measurements was TOF. Reactions were monitored by TLC on silica gel 60 F254 plates (Qingdao
4

ACCEPTED MANUSCRIPT
Ocean Chemical Company, China). Column chromatography was carried out on silica gel (200−300 mesh,
Qingdao Ocean Chemical Company, China). Optical rotation datas were recorded on Jasco p-1020 Polarimeter.
The optical purity of the key compounds were determined by analytical HPLC (Equipment: Agilent 1100
system with a VWD G1314A UV detector; Column: Chiralpak IC 4.6 mm × 250 mm).
4
.1.2. 8-Bromo-3-methyl-7-(prop-2-ynyl)-1H-purine-2,6(3H,7H)-dione (3b)
To a mixture of 8-bromo-3-methyl-1H-purine-2,6(3H,7H)-dione (244 mg, 1 mmol) and DIPEA (277 ꢀl, 1.3
mmol) in DMF (4 mL) was added 3-bromo-1-butyne (93 ꢀl, 1.2 mmol). The reaction mixture was stirred
overnight at r. t. The precipitate was collected by filtration, washed with water and EtOH, and dried to give 3b
o
-1
as a white solid (212 mg, 75%). mp: 275-276 C. IR (KBr, cm ): 3447, 3263, 3154, 3017, 2833, 2129, 1685,
1
1
598, 1531, 1436, 1366, 1288, 1204, 878, 748, 566. H NMR (300 MHz, DMSO-d ): δ 11.33 (s, 1H), 5.10 (d,
6
1
3
J = 2.1 Hz, 2H), 3.52 (s, 1H), 3.32 (s, 3H). C NMR (75 MHz, DMSO-d
6
): δ 153.8, 150.4, 149.2, 127.8,
+
1
08.1, 76.8, 76.6, 36.1, 28.5. HRMS (ESI) calcd for C H N O Br [M+H] 282.9831, found 282.9834.
9
8
4
2
4
.1.3. 8-Bromo-7-(but-2-ynyl)-3-methyl-1H-purine-2,6(3H,7H)-dione (3a)
Following a similar procedure for the preparation of 3b, 3a was prepared starting from 8-bromo-3-methyl-
o
-1
1
3
1
1
2
H-purine-2,6(3H,7H)-dione and 1-bromo-2-butyne. White solids (5 g, 85%). mp: 256-260 C. IR (KBr, cm ):
1
480, 3160, 3028, 2836, 1723, 1685, 1531, 1362, 1202, 863, 746, 553. H NMR (300 MHz, DMSO-d ): δ
6
13
1.32 (s, 1H), 5.06 (s, 2H), 3.32 (s, 3H), 1.80 (s, 3H). C NMR (75 MHz, DMSO-d ): δ 153.8, 150.5, 149.2,
6
+
27.7, 108.1, 81.8, 72.3, 36.5, 28.5, 3.0. HRMS (ESI) calcd for C10
H
10
N
4
O
2
Br [M+H] 296.9987, found
96.9989.
4
.1.4. 7-Allyl-8-bromo-3-methyl-1H-purine-2,6(3H,7H)-dione (3c)
Following a similar procedure for the preparation of 3b, 3c was prepared starting from 8-bromo-3-methyl-
o
1
1
H-purine-2,6(3H,7H)-dione and 3-bromo-1-butene. White solids (420 mg, 86%). mp: 238~240 C. H NMR
(
300 MHz, CDCl ): δ 9.04 (br s, 1H), 5.95 (ddt, J = 22.3, 10.7, 5.6 Hz, 1H), 5.29 (d, J = 10.2 Hz, 1H), 5.22 (d,
3
13
J = 17.1 Hz, 1H), 4.94 (d, J = 5.3 Hz, 2H), 3.52 (s, 3H). C NMR (75 MHz, CDCl
3
): δ 153.6, 152.4, 150.7,
+
1
30.6, 127.7, 119.4, 114.4, 49.2, 29.1. MS (ESI) m/z 285.0 [M+1] .
4
.1.5.
8-Bromo-3-methyl-1-[(4-methylquinazolin-2-yl)methyl]-7-(prop-2-ynyl)-1H-purine-2,6(3H,7H)-dione
(4b)
To a suspension of 3b (849 mg, 3 mmol) and Na CO (382 mg, 3.6 mmol) in DMF (36 mL) was added 2-
2
3
o
chloromethyl-4-methylquinazoline (636 mg, 3.3 mmol). The reaction mixture was stirred at 80 C for 12 h.
After cooling to r. t., the reaction mixture was diluted with DCM, washed with water and brine, dried over
anhydrous Na SO and concentrated in vacuo. The resulting residue was purified by flash chromatography
2
4
o
-1
(
petroleum ether/ethyl acetate, 1:1) to give 4b as a white solid (1.05 g, 80%). mp: 228-232 C. IR (KBr, cm ):
1
3
455, 3264, 3196, 1709, 1670, 1614, 1567, 1534, 1437, 1399, 1362, 1205, 1189, 949, 769. H NMR (500
MHz, CDCl ): δ 8.02 (d, J = 8.3 Hz, 1H), 7.86 (d, J = 8.4 Hz, 1H), 7.77 (t, J = 7.5 Hz, 1H), 7.53 (t, J = 7.5 Hz,
3
13
1
H), 5.56 (s, 2H), 5.19 (d, J = 2.1 Hz, 2H), 3.60 (s, 3H), 2.89 (s, 3H), 2.42 (s, 1H). C NMR (125 MHz,
CDCl ): δ 168.7, 160.4, 154.2, 151.3, 149.9, 148.6, 133.3, 128.8, 127.6, 126.8, 124.8, 123.2, 108.7, 75.6, 74.3,
3
+
4
6.4, 36.5, 29.9, 21.7. HRMS (ESI) calcd for C H N O Br [M+H] 439.0518, found 439.0522.
19
16
6
2
4
.1.6. 8-Bromo-7-(but-2-ynyl)-3-methyl-1-[(4-methylquinazolin-2-yl)methyl]-1H-purine-2,6(3H,7H)-dione (4a)
5

ACCEPTED MANUSCRIPT
Following a similar procedure for the preparation of 4b, 4a was prepared starting from 3a and 2-
o
-1
chloromethyl-4-methylquinazoline. White solids (1.1 g, 85%). mp: 229-231 C. IR (KBr, cm ): 3447, 3162,
1
1
8
3
1
704, 1672, 1566, 1540, 1400, 1367, 763. H NMR (500 MHz, CDCl ): δ 8.03 (d, J = 8.2 Hz, 1H), 7.88 (d, J =
3
.4 Hz, 1H), 7.78 (t, J = 7.2 Hz, 1H), 7.54 (t, J = 7.5 Hz, 1H), 5.58 (s, 2H), 5.14 (d, J = 2.3 Hz, 2H), 3.60 (s,
13
3
H), 2.90 (s, 3H), 1.80 (t, J = 2.1 Hz, 3H). C NMR (125 MHz, CDCl ): δ 168.6, 160.5, 154.2, 151.4, 149.9,
48.5, 133.3, 128.9, 127.5, 126.8, 124.8, 123.1, 108.6, 82.3, 71.3, 46.4, 37.1, 29.8, 21.7, 3.5. HRMS (ESI)
+
calcd for C20
H
18
N
6
O
2
Br [M+H] 453.0675, found 453.0678.
4
.1.7. 7-Allyl-8-bromo-3-methyl-1-[(4-methylquinazolin-2-yl)methyl]-1H-purine-2,6(3H,7H)-dione (4c)
Following a similar procedure for the preparation of 4b, 4c was prepared starting from 3c and 2-
o
1
chloromethyl-4-methylquinazoline. White solids (450 mg, 72%). mp: 210-214 C. H NMR (300 MHz,
CDCl ): δ 8.02 (d, J = 8.3 Hz, 1H), 7.86 (d, J = 8.3 Hz, 1H), 7.77 (t, J = 7.6 Hz, 1H), 7.54 (t, J = 7.2 Hz, 1H),
.96 (ddt, J = 17.2, 10.7, 5.5 Hz, 1H), 5.56 (s, 2H), 5.27 (d, J = 10.3 Hz, 1H), 5.20 (d, J = 17.2 Hz, 1H), 4.98
3
5
+
(
d, J = 5.5 Hz, 2H), 3.63 (d, J = 16.5 Hz, 3H), 2.88 (s, 3H). MS (ESI) m/z 441.1 [M+1] .
4
.1.8. (R)-8-(3-Aminopiperidin-1-yl)-3-methyl-1-[(4-methylquinazolin-2-yl)methyl]-7-(prop-2-ynyl)-1H-
purine-2,6(3H,7H)-dione (1i)
A mixture of 4b (88 mg, 0.2 mmol), (R)-3-(N-Boc-amino)piperidine (44 mg, 0.22 mmol) and K CO ( 55
2
3
mg, 0.4 mmol) in DMF (6 mL) was stirred at 75 °C for 6 h. After cooling to r. t., the mixture was poured into
water (12 mL) and extracted with DCM (3×10 mL). The combined organic layer was washed with saturated
brine, dried over anhydrous Na SO , and concentrated. The crude product was purified by flash
2
4
chromatography (petroleum ether/ethyl acetate, 1:1) to give the Boc precusor of 1i as a colorless syrup (80 mg,
2%), which was dissolved in DCM (2 mL), and TFA (390 ꢀL) was added. The solution was stirred at room
temperature for 3 h and then poured into ice-cold water (4 mL). The organic phase was separated, and the
aqueous phase was basified with K CO and extracted with DCM (2×10 mL). The organic layers were
combined and washed with saturated brine, dried over anhydrous Na SO , and concentrated. The crude product
7
2
3
2
4
was purified by flash chromatography (DCM/MeOH/TEA, 100:0.5:0.5) to give pure 1i as a white solid (51 mg,
o
20
D
-1
8
1
7
2
1
1
2
5%). mp: 164-167 C. [α]
+17.641 (c 0.034, MeOH). IR (KBr, cm ): 3441, 3135, 1701, 1655, 1570, 1517,
1
400, 1283, 762. H NMR (300 MHz, CDCl ): δ 7.97 (d, J = 8.3 Hz, 1H), 7.83 (d, J = 8.4 Hz, 1H), 7.72 (t, J =
3
.6 Hz, 1H), 7.48 (t, J = 7.5 Hz, 1H), 5.53 (s, 2H), 4.91 (d, J = 2.1 Hz, 2H), 3.76 – 3.42 (m, 5H), 3.08 (m, 2H),
.94 – 2.87 (m, 1H), 2.84 (s, 3H), 2.37 (t, J = 2.2 Hz, 1H), 2.26 (s, 2H), 2.06 – 1.93 (m, 1H), 1.91-1.79 (m, 1H),
13
.73 (ddd, J = 20.0, 11.9, 6.9 Hz, 1H), 1.43-1.26 (m, 1H). C NMR (75 MHz, CDCl ): δ 168.4, 161.0, 156.3,
3
54.4, 151.7, 149.8, 148.1, 133.1, 128.8, 126.6, 124.7, 123.0, 76.6, 73.4, 58.2, 50.4, 47.2, 46.2, 45.8, 35.2, 33.4,
+
9.6, 23.2, 21.6, 9.9. HRMS (ESI) calcd for C H N O [M+H] 459.2257, found 459.2261.
24
27
8
2
4
.1.9.
(S)-8-(3-Aminopiperidin-1-yl)-3-methyl-1-[(4-methylquinazolin-2-yl)methyl]-7-(prop-2-ynyl)-1H-
purine-2,6(3H,7H)-dione (1h)
Following a similar procedure for the preparation of 1i, 1h was prepared starting from 4b and (S)-3-(N-Boc-
2
D
0
1
amino)piperidine. White solids (42 mg, 84%). [α]
-18.182 (c 0.011, MeOH). H NMR (300 MHz, CDCl ): δ
3
7
.99 (d, J = 8.3 Hz, 1H), 7.85 (d, J = 8.4 Hz, 1H), 7.78-7.69 (m, 1H), 7.50 (t, J = 7.6 Hz, 1H), 5.52 (d, J = 11.6
Hz, 2H), 4.94 (d, J = 1.9 Hz, 2H), 3.75-3.61 (m, 1H), 3.62-3.45 (m, 4H), 3.24-3.06 (m, 2H), 3.02-2.80 (m, 4H),
.38 (t, J = 2.3 Hz, 1H), 2.24 (br s, 3H), 1.93 (ddd, J = 13.6, 10.9, 6.0 Hz, 2H), 1.74 (ddd, J = 19.7, 11.7, 8.0
Hz, 1H), 1.49 – 1.36 (m, 1H).
2
6

ACCEPTED MANUSCRIPT
4
.1.10.
(R)-8-(3-Aminopiperidin-1-yl)-7-(but-2-ynyl)-3-methyl-1-[(4-methylquinazolin-2-yl)methyl]-1H-
purine-2,6(3H,7H)-dione (1a)
Following a similar procedure for the preparation of 1i, 1a was prepared starting from 4a and (R)-3-(N-Boc-
o
20
D
1
amino)piperidine. Light yellow solids (2.8 g, 80%). mp: 132-135 C. [α]
-15.972 (c 0.144, MeOH). H NMR
(
300 MHz, CDCl ): δ 8.01 (d, J = 8.1 Hz, 1H), 7.88 (d, J = 8.3 Hz, 1H), 7.82-7.69 (m, 1H), 7.52 ( m, 1H), 5.71
3
(
d, J = 6.1 Hz, 1H), 5.57 (s, 2H), 4.91 ( q, 2H), 3.88 (s, 1H), 3.56 (s, 4H), 3.34 (s, 3H), 2.88 (s, 3H), 1.80 (m,
13
7
1
H), 1.45 (s, 9H). C NMR (75 MHz, CDCl ): δ 168.4, 161.1, 155.8, 155.1, 154.4, 151.8, 149.9, 147.6, 133.1,
3
28.9, 126.6, 124.7, 123.1, 104.5, 81.3, 72.8, 54.5, 50.8, 46.2, 46.0, 35.5, 29.6, 29.4, 28.3, 22.0, 21.7, 3.6.
+
HRMS (ESI) calcd for C26
H
29
N
8
O
2
[M+H] 485.2413, found 485.2415. [11]
4
.1.11.
(R)-8-(7-Amino-5-azaspiro[2.4]heptan-5-yl)-7-(but-2-ynyl)-3-methyl-1-[(4-methylquinazolin-2-
yl)methyl]-1H-purine-2,6(3H,7H)-dione (1b)
Following a similar procedure for the preparation of 1i, 1b was prepared starting from 4a and (R)-N-Boc-5-
o
20
D
azaspiro[2.4]heptan-7-amine. White solids (25 mg, 78%). mp: 130-132 C. [α]
+10.000 (c 0.030, MeOH). IR
-1
1
(
KBr, cm ): 3448, 3179, 1693, 1655, 1619, 1571, 1524, 1400, 1237, 1137, 948, 762. H NMR (300 MHz,
CDCl
3
): δ 8.01 (d, J = 8.1 Hz, 1H), 7.87 (d, J = 8.3 Hz, 1H), 7.80-7.71 (m, 1H), 7.52 (m, 1H), 5.56 (s, 2H),
.07 (d, J = 2.2 Hz, 2H), 4.09 (dd, J = 10.1, 5.4 Hz, 1H), 4.00 (d, J = 9.7 Hz, 1H), 3.68 (dd, J = 10.1, 3.4 Hz,
H), 3.58 (s, 1H), 3.54 (s, 3H), 3.17 (dd, J = 5.1, 3.6 Hz, 1H), 2.88 (s, 3H), 1.79 (t, J = 2.1 Hz, 3H), 0.88-0.77
5
1
1
3
(m, 1H), 0.76-0.60 (m, 3H). C NMR (75 MHz, CDCl
3
): δ 168.2, 161.3, 154.5, 153.9, 151.9, 149.9, 149.1,
1
33.0, 128.8, 126.4, 124.6, 123.0, 103.4, 81.4, 73.6, 58.0, 55.9, 55.3, 46.0, 35.2, 29.4, 27.4, 21.6, 10.9, 5.3, 3.6.
+
HRMS (ESI) calcd for C H N O [M+H] 485.2413, found 485.2415.
26
29
8
2
4
.1.12.
(S)-8-(7-Amino-5-azaspiro[2.4]heptan-5-yl)-7-(but-2-ynyl)-3-methyl-1-[(4-methylquinazolin-2-
yl)methyl]-1H-purine-2,6(3H,7H)-dione (1c)
Following a similar procedure for the preparation of 1i, 1c was prepared starting from 4a and (S)-N-Boc-5-
2
D
0
1
azaspiro[2.4]heptan-7-amine. White solids (30 mg, 79%). [α]
-10.638 (c 0.094, MeOH). H NMR (500 MHz,
CDCl ): δ 8.01 (d, J = 8.2 Hz, 1H), 7.87 (d, J = 8.4 Hz, 1H), 7.76 (t, J = 7.6 Hz, 1H), 7.51 (t, J = 7.5 Hz, 1H),
3
5
1
0
1
.56 (s, 2H), 5.07 (s, 2H), 4.11 (dd, J = 10.2, 5.3 Hz, 1H), 4.02 (d, J = 9.6 Hz, 1H), 3.72 (dd, J = 10.2, 2.9 Hz,
H), 3.61-3.52 (m, 4H), 3.20 (d, J = 3.4 Hz, 1H), 2.88 (s, 3H), 1.89 (s, 3H), 1.79 (s, 3H), 0.92-0.84 (m, 1H),
1
3
.76-0.62 (m, 3H). C NMR (126 MHz, CDCl ): δ 168.3, 161.4, 154.4, 154.1, 151.8, 150.0, 149.0, 133.0,
3
28.9, 126.5, 124.7, 123.1, 103.3, 81.5, 73.7, 57.8, 56.1, 55.4, 46.1, 35.3, 29.5, 27.3, 21.7, 11.5, 5.5, 3.6.
4
1
.1.13. 8-(7-Amino-5-azaspiro[2.4]heptan-5-yl)-7-(but-2-ynyl)-3-methyl-1-[(4-methylquinazolin-2-yl)methyl]-
H-purine-2,6(3H,7H)-dione (1d)
Following a similar procedure for the preparation of 1i, 1d was prepared starting from 4a and N-Boc-5-
1
azaspiro[2.4]heptan-7-amine. White solids (32 mg, 78%). H NMR (300 MHz, CDCl ): δ 8.01 (d, J = 8.1 Hz,
3
1
H), 7.87 (d, J = 8.3 Hz, 1H), 7.80-7.71 (m, 1H), 7.52 (dd, J = 11.1, 4.0 Hz, 1H), 5.56 (s, 2H), 5.07 (d, J = 2.2
Hz, 2H), 4.09 (dd, J = 10.1, 5.4 Hz, 1H), 4.00 (d, J = 9.7 Hz, 1H), 3.68 (dd, J = 10.1, 3.4 Hz, 1H), 3.58 (s, 1H),
.54 (s, 3H), 3.17 (dd, J = 5.1, 3.6 Hz, 1H), 2.88 (s, 3H), 1.79 (t, J = 2.1 Hz, 3H), 0.88-0.77 (m, 1H), 0.76-0.60
3
1
3
(m, 3H). C NMR (75 MHz, CDCl ): δ 168.3, 161.3, 154.5, 153.9, 151.9, 149.9, 133.0, 128.8, 126.4, 124.6,
3
1
23.0, 103.4, 81.4, 73.6, 57.9, 55.9, 55.3, 46.0, 35.2, 29.5, 27.4, 21.6, 10.9, 5.3, 3.6.
4
1
.1.14. (S)-8-[2-(Aminomethyl)pyrrolidin-1-yl]-7-(but-2-ynyl)-3-methyl-1-[(4-methylquinazolin-2-yl)methyl]-
H-purine-2,6(3H,7H)-dione (1e)
7

ACCEPTED MANUSCRIPT
Following a similar procedure for the preparation of 1i, 1e was prepared starting from 4a and (S)-2-(N-Boc-
2
D
0
1
aminomethyl)pyrrolidine. White solids (30 mg, 52%). [α]
+6.250 (c 0.016, MeOH). H NMR (300 MHz,
CDCl ): δ 8.01 (d, J = 7.7 Hz, 1H), 7.89 (d, J = 8.2 Hz, 1H), 7.81-7.71 (m, 1H), 7.58-7.47 (m, 1H), 5.56 (d, J =
3
1
7
3
1
.1 Hz, 2H), 5.30 (s, 1H), 4.77 (dd, J = 18.0, 2.3 Hz, 1H), 4.42 (dd, J = 10.5, 5.9 Hz, 1H), 3.84 (dt, J = 14.2,
.1 Hz, 1H), 3.73-3.48 (m, 4H), 3.06-2.81 (m, 5H), 2.22-2.04 (m, 2H), 2.04-1.85 (m, 2H), 1.78 (t, J = 2.2 Hz,
1
3
H). C NMR (75 MHz, CDCl ): δ 168.4, 161.3, 155.5, 154.1,151.9, 149.9, 149.1, 133.1, 128.9, 126.6, 124.7,
3
23.1, 103.4, 81.0, 73.3, 62.7, 51.1, 46.2, 44.3, 35.4, 29.6, 28.1, 25.3, 21.7, 3.5. HRMS (ESI) calcd for
+
C H N O [M+H] 473.2413, found 473.2417.
2
5
29
8
2
4
.1.15.
(R)-8-(3-Aminopyrrolidin-1-yl)-7-(but-2-ynyl)-3-methyl-1-[(4-methylquinazolin-2-yl)methyl]-1H-
purine-2,6(3H,7H)-dione (1f)
A mixture of 4a (181 mg, 0.4 mmol), (R)-3-aminopyrrolidine dihydrochloride (70 mg, 0.44 mmol) and
K CO (110 mg, 0.8 mmol) in DMF (4 mL) was stirred at 75 °C for 6 h. After cooling to r. t., the mixture was
2
3
poured into water (4 mL) and extracted with DCM (3×10 mL). The organic layers were combinded and
washed with saturated brine, dried over anhydrous Na SO , and concentrated. The crude product was purified
2
4
by flash chromatography (DCM/MeOH/TEA, 100:1:0.5) to give pure 1f as a light yellow solid (128 mg, 70%).
o
20
D
-1
mp: 236-240 C. [α]
-2.410 (c 0.083, MeOH). IR (KBr, cm ): 3441, 3168, 2360, 2341, 1697, 1655, 1621,
1
1
1
2
1
1
565, 1524, 1400, 1235, 945, 762. H NMR (300 MHz, CDCl
3
): δ 7.99 (d, J = 8.2 Hz, 1H), 7.85 (d, J = 8.4 Hz,
H), 7.74 (t, J = 7.6 Hz, 1H), 7.50 (t, J = 7.5 Hz, 1H), 5.55 (s, 2H), 5.05 (d, J = 2.2 Hz, 2H), 4.05-3.84 (m,
H), 3.84-3.65 (m, 2H), 3.65-3.40 (m, 4H), 2.88 (d, J = 10.9 Hz, 3H), 2.19 (dt, J = 12.8, 7.1 Hz, 1H), 1.96 –
13
3
.66 (m, 5H). C NMR (75 MHz, CDCl ): δ 168.3, 161.3, 154.6, 153.9, 151.9, 149.9, 149.2, 133.0, 128.8,
26.5, 124.7, 123.0, 103.2, 81.3, 73.7, 57.8, 51.1, 47.9, 46.1, 35.2, 34.6, 29.5, 21.7, 3.6. HRMS (ESI) calcd for
+
C H N O [M+H] 459.2257, found 459.2260.
2
4
27
8
2
4
.1.16.
(S)-8-(3-Aminopyrrolidin-1-yl)-7-(but-2-ynyl)-3-methyl-1-[(4-methylquinazolin-2-yl)methyl]-1H-
purine-2,6(3H,7H)-dione (1g)
Following a similar procedure for the preparation of 1f, 1g was prepared starting from 4c and (S)-3-
2
D
0
1
aminopiperidine. Light yellow solid (120 mg, 65%). [α]
+4.000 (c 0.025, MeOH). H NMR (300 MHz,
CDCl ): δ 7.98 (d, J = 8.2 Hz, 1H), 7.84 (d, J = 8.4 Hz, 1H), 7.73 (t, J = 7.5 Hz, 1H), 7.48 (t, J = 7.4 Hz, 1H),
3
5
2
1
2
.54 (s, 2H), 5.04 (s, 2H), 3.89 (dd, J = 10.0, 5.7 Hz, 2H), 3.81 – 3.58 (m, 2H), 3.59-3.36 (m, 4H), 2.85 (s, 3H),
1
3
3
.17 (dt, J = 12.6, 6.2 Hz, 1H), 1.87-1.62 (m, 4H). C NMR (75 MHz, CDCl ): δ 168.3, 161.3, 154.6, 153.9,
51.9, 149.9, 149.2, 133.0, 128.8, 126.5, 124.7, 123.0, 103.2, 81.3, 73.7, 57.8, 51.1, 47.9, 46.1, 35.2, 34.6,
+
9.5, 21.7, 3.6. MS (ESI) m/z 481.2 [M+Na] .
4
2
.1.17.
(R)-7-Allyl-8-(3-aminopiperidin-1-yl)-3-methyl-1-[(4-methylquinazolin-2-yl)methyl]-1H-purine-
,6(3H,7H)-dione (1j)
Following a similar procedure for the preparation of 1f, 1j was prepared starting from 4c and (R)-3-
o
20
D
1
aminopiperidine. Yellow solids (50 mg, 78%). mp: 176-180 C. [α]
-17.391 (c 0.115, MeOH). H NMR (300
MHz, MeOD): δ 7.99 (d, J = 8.3 Hz, 1H), 7.84 (d, J = 8.4 Hz, 1H), 7.74 (t, J = 7.6 Hz, 1H), 7.50 (t, J = 7.6 Hz,
1
3
2
H), 6.06 (ddd, J = 22.0, 10.2, 5.0 Hz, 1H), 5.54 (s, 2H), 5.31-5.03 (m, 2H), 4.73 (d, J = 4.8 Hz, 2H), 3.56 (s,
H), 3.53-3.48 (m, 1H), 3.42-3.37 (m, 1H), 3.09-2.94 (m, 2H), 2.86 (s, 3H), 2.83-2.76 (m, 1H), 1.99-1.95 (m,
13
H), 1.91-1.78 (m, 3H), 1.38-1.24(m, 1H). C NMR (75 MHz, CDCl
3
): δ 168.4, 161.1, 156.5, 149.9, 133.3,
8

ACCEPTED MANUSCRIPT
1
33.1, 128.8, 126.6, 124.8, 123.1, 117.0, 104.8, 58.5, 50.8, 47.7, 47.3, 46.3, 33.4, 29.7, 23.3, 21.7. HRMS (ESI)
+
calcd for C24
H
29
N
8
O
2
[M+H] 461.2408, found 461.2409.
4
.1.18. 1-(But-2-ynyl)-6-chloropyrimidine-2,4(1H,3H)-dione (5b)
To a suspension of 6-chlorouracil (17.58 g, 120 mmol) in DMF (60 mL) were added DIPEA (27.2 mL, 156
mmol) and 1-bromo-2-butyne (12 mL, 132 mmol). The reaction mixture was stirred overnight at r. t. Water
was added. The precipitate was collected by filtration, washed with water and EtOH, and dried to give 5b as a
o
-1
light yellow solid (21 g, 88%). mp: 216-217 C. IR (KBr, cm ): 3176, 3072, 3046, 1707, 1595, 1440, 1405,
1
1
4
342, 1293, 1231, 1166, 1133, 1015, 951, 857, 830. H NMR (300 MHz, CDCl
3
): δ 9.36 (s, 1H), 5.90 (s, 1H),
13
.75 (s, 2H), 1.81 (s, 3H). C NMR (75 MHz, CDCl ): δ 160.8, 149.6, 147.2, 103.1, 81.6, 71.9, 36.0, 3.4.
3
+
HRMS (ESI) calcd for C H N O Cl [M+H] 199.0274, found 199.0276.
8
8
2
2
4
.1.19. 6-Chloro-1-(2-cyanobenzyl)pyrimidine-2,4(1H,3H)-dione (5a)
Following a similar procedure for the preparation of 5b, 5a was prepared starting from 6-chlorouracil and 2-
1
cyanobenzyl bromide. White solids (1.5 g, 58%). H NMR (500 MHz, DMSO): δ 11.78 (s, 1H), 7.88 (dd, J =
7
2
1
.7, 0.8 Hz, 1H), 7.74-7.68 (m, 1H), 7.51 (t, J = 7.6 Hz, 1H), 7.38 (d, J = 7.8 Hz, 1H), 6.06 (s, 1H), 5.33 (s,
1
3
6
H). C NMR (125 MHz, DMSO-d ): δ 161.0, 150.4, 146.3, 139.7, 133.8, 133.3, 128.3, 126.5, 116.8, 109.5,
02.8, 46.8.
4
.1.20. 6-Chloro-1-(prop-2-ynyl)pyrimidine-2,4(1H,3H)-dione (5c)
Following a similar procedure for the preparation of 5b, 5c was prepared starting from 6-chlorouracil and 1-
o
-1
bromo-2-propyne. Yellow solids (512 mg, 70%). mp: 184 C. IR (KBr, cm ): 3297, 3028, 1706, 1592, 1444,
1
1
406, 1381, 1331, 1137, 1019, 953, 830, 681. H NMR (500 MHz, CDCl ): δ 9.15 (brs, 1H), 5.96 (s, 1H), 4.83
3
13
(
d, J = 2.4 Hz, 2H), 2.38 (s, 1H). C NMR (125 MHz, CDCl ): δ 160.5, 149.4, 146.9, 103.3, 77.2, 73.6, 35.4.
3
+
HRMS (ESI) calcd for C
7
H
6
N
2
O
2
Cl [M+H] 185.0118, found 185.0120.
4
.1.21. 1-Allyl-6-chloropyrimidine-2,4(1H,3H)-dione (5d)
Following a similar procedure for the preparation of 5b, 5d was prepared starting from 6-chlorouracil and 1-
o
-1
bromo-2-propene. White solids (1.4 g, 75%). mp: 122-125 C. IR (KBr, cm ): 3011, 2848, 2803, 1669, 1588,
1
1
1
456, 1413, 1373, 1332, 1158, 1093, 1019, 996, 933, 813, 754, 660. H NMR (300 MHz, CDCl ): δ 10.24 (brs,
3
13
H), 5.99-5.78 (m, 2H), 5.35-5.28 (m, 1H), 5.25 (d, J = 10.6 Hz, 1H), 4.66 (d, J = 5.5 Hz, 2H). C NMR (75
+
MHz, CDCl
3
): δ 161.6, 150.1, 147.8, 130.7, 118.8, 102.8, 47.9. HRMS (ESI) calcd for C
7 8 2 2
H N O Cl [M+H]
1
87.0274, found 187.0273.
4
.1.22. 1-(But-2-ynyl)-6-chloro-3-[(4-methylquinazolin-2-yl)methyl]pyrimidine-2,4(1H,3H)-dione (6c)
To a suspension of 5b (14.6 g, 73.5 mmol) and LiBr (5.6 g, 58.8 mmol) in DMF (150 mL) was added NaH
(
60%, 3.82 g, 95.5 mmol) in portions under nitrogen at 0 °C. The mixture was stirred for 0.5 h. 2-
Chloromethyl-4-methylquinazoline (15.6 g, 81 mmol) was added. The mixture was stirred overnight at 80 °C.
The mixture was evaporated and azeotroped with water in vacuo to remove most of the DMF. The crude
product was suspended in the mixture of hot EtOAc (100 mL) and isopropyl ether (200 mL). The suspension
was stirred for 30 min and allowed to stand at -20 °C for 1 h. The formed precipitate was collected by
filtration, washed with water, EtOH and isopropyl ether, and dried to give 6c as a yellow brown solid (21 g,
-1
1
8
8%). mp: 153 °C. IR (KBr, cm ): 3434, 3106, 1716, 1661, 1608, 1572, 1435, 1397, 1202, 821, 768; H NMR
(
300 MHz, CDCl ): δ 8.08 (d, J = 8.3 Hz, 1H), 7.94 (d, J = 8.2 Hz, 1H), 7.85 (ddd, J = 8.4, 6.9, 1.3 Hz, 1H),
3
9

ACCEPTED MANUSCRIPT
7
.60 (ddd, J = 8.1, 6.9, 1.2 Hz, 1H), 6.12 (s, 1H), 5.54 (s, 2H), 4.89 (dd, J = 4.5, 2.2 Hz, 2H), 2.95 (s, 3H), 1.89
13
(t, J = 2.3 Hz, 3H). C NMR (75 MHz, CDCl
3
): δ 168.6, 160.6, 159.6, 150.8, 149.8, 145.4, 133.4, 128.8,
+
1
3
26.9, 124.8, 123.1, 102.6, 81.1, 72.2, 46.7, 36.8, 21.7, 3.5. HRMS (ESI) calcd for C H N O Cl [M+H]
18 16 4 2
55.0962, found 355.0965.
4
.1.23. 6-Chloro-1-(2-cyanobenzyl)-3-methyl-pyrimidine-2,4(1H,3H)-dione (6a)
Following a similar procedure for the preparation of 6c, 6a was prepared starting from 5a and methyl iodide.
-1
White solids (386 mg, 70%). IR (KBr, cm ): 3463, 3096, 2227, 1708, 1654, 1604, 1441, 1398, 1203, 1093,
1
1
030, 981, 942, 838, 766, 521. H NMR (300 MHz, CDCl
3
): δ 7.56 (d, J = 7.7 Hz, 1H), 7.47 (dd, J = 11.1, 4.4
Hz, 1H), 7.30 (t, J = 7.8 Hz, 1H), 7.12 (d, J = 7.9 Hz, 1H), 5.86 (s, 1H), 5.35 (s, 2H).
4
.1.24. 6-Chloro-1-(2-cyanobenzyl)-3-[(4-methylquinazolin-2-yl)methyl]-pyrimidine-2,4(1H,3H)-dione (6b)
Following a similar procedure for the preparation of 6c, 6b was prepared starting from 5a and 2-
o
-1
chloromethyl-4-methylquinazoline. White solids (500 mg, 50%). mp: 204-206 C. IR (KBr, cm ): 2224, 1713,
1
1
2
608, 1569, 1436, 1426, 1209, 814,764. H NMR (300 MHz, CDCl ): δ 8.02 (d, J = 8.3 Hz, 1H), 7.92-7.76 (m,
3
H), 7.66 (d, J = 7.6 Hz, 1H), 7.56 (q, J = 8.2 Hz, 2H), 7.39 (t, J = 7.9 Hz, 2H), 6.10 (s, 1H), 5.55 (s, 2H), 5.51
13
(
s, 2H), 2.89 (s, 3H). C NMR (75 MHz, CDCl
3
): δ 168.7, 160.3, 159.4, 151.4, 149.7, 145.4, 139.2, 133.5,
1
33.3, 133.0, 128.4, 128.2, 127.0, 126.3, 125.0, 123.1, 116.6, 110.9, 103.0, 47.5, 46.5, 21.7. HRMS (ESI)
+
calcd for C H N O Cl [M+H] 418.1071, found 418.1074.
22
17
5
2
4
.1.25 1-(But-2-ynyl)-6-chloro-3-methylpyrimidine-2,4(1H,3H)-dione (6d)
Following a similar procedure for the preparation of 6c, 6d was prepared starting from 5b and methyl iodide.
o
-1
Yellow solids (250 mg, 98%). mp: 133 C. IR (KBr,cm ): 3078, 2304, 2231, 1716, 1653, 1603, 1446, 1427,
1
1
1
3
332, 1202, 1172, 980, 948, 860, 751, H NMR (300 MHz, CDCl ): δ 6.04 (s, 1H), 4.87 (q, 2H), 3.42 (s, 3H),
3
13
.90 (t, J = 2.3 Hz, 3H). C NMR (75 MHz, CDCl
3
): δ 160.7, 150.6, 145.0, 102.4, 81.3, 77.4, 77.0, 76.6, 72.0,
+
6.8, 28.4, 3.6. HRMS (ESI) calcd for C H N O Cl [M+H] 213.0431, found 213.0433.
9
10
2
2
4
.1.26. 1-(But-2-ynyl)-6-chloro-3-(quinoxalin-2-ylmethyl)pyrimidine-2,4(1H,3H)-dione (6e)
Following a similar procedure for the preparation of 6c, 6e was prepared starting from 5b and 2-
-1
chloromethylquinoxaline. Red oil (1.4 g, 82%). IR (KBr, cm ): 3450, 1715, 1668, 1609, 1494, 1431, 1342,
1
1
203, 762. H NMR (500 MHz, CDCl ): δ 8.84 (s, 1H), 8.06 (dd, J = 6.3, 3.5 Hz, 1H), 8.00 (dd, J = 6.3, 3.5
3
13
Hz, 1H), 7.73-7.66 (m, 2H), 6.04 (s, 1H), 5.47 (s, 2H), 4.79 (d, J = 2.4 Hz, 2H), 1.81 (s, 3H). C NMR (125
MHz, CDCl ): δ 160.2, 150.4, 150.3, 145.6, 143.9, 141.7, 141.7, 129.8, 129.4, 129.1, 129.0, 102.3, 81.3, 71.8,
3
+
4
4.6, 36.8, 3.4. HRMS (ESI) calcd for C H N O Cl [M+H] 341.0805, found 341.0808.
17
14
4
2
4
.1.27. 1-(But-2-ynyl)-6-chloro-3-(naphthalen-2-ylmethyl)pyrimidine-2,4(1H,3H)-dione (6f)
Following a similar procedure for the preparation of 6c, 6f was prepared starting from 5b and 2-
-1
bromomethylnaphthalene. Colorless syrup (1.3 g, 77%). IR (KBr, cm ): 3451, 2921, 1710, 1665, 1607, 1432,
1
1
2
331, 1275, 749. H NMR (500 MHz, CDCl ): δ 7.97 (s, 1H), 7.83-7.78 (m, 1H), 7.75 (dd, J = 11.5, 5.3 Hz,
3
H), 7.64 (dd, J = 8.5, 1.0 Hz, 1H), 7.45-7.36 (m, 2H), 5.90 (s, 1H), 5.21 (d, J = 5.1 Hz, 2H), 4.60 (d, J = 2.1
13
Hz, 2H), 1.78 (s, 3H). C NMR (126 MHz, CDCl ): δ 159.8, 149.8, 144.6, 133.4, 132.7, 132.4, 127.8, 127.6,
3
1
27.4, 127.0, 126.6, 125.5, 125.5, 101.9, 80.7, 71.9, 44.5, 36.2, 3.0. HRMS (ESI) calcd for C H N O Cl
19 16 2 2
+
[
M+H] 339.0900, found 339.0902.
10

ACCEPTED MANUSCRIPT
4
.1.28. 1-(But-2-ynyl)-6-chloro-3-(naphthalen-1-ylmethyl)pyrimidine-2,4(1H,3H)-dione (6g)
Following a similar procedure for the preparation of 6c, 6g was prepared starting from 5b and 1-
-1
bromomethylnaphthalene. White solids (1.4 g, 83%). mp: > 300 °C. IR (KBr, cm ): 3461, 1717, 1686, 1608,
1
1
436, 1074, 783, 472. H NMR (500 MHz, CDCl
3
): δ 8.29 (d, J = 8.6 Hz, 1H), 7.89 (d, J = 8.2 Hz, 1H), 7.81
(
dd, J = 6.7, 2.5 Hz, 1H), 7.63-7.57 (m, 1H), 7.56-7.51 (m, 1H), 7.46-7.40 (m, 2H), 6.06 (s, 1H), 5.65 (s, 2H),
1
3
4
1
3
.80 (d, J = 2.3 Hz, 2H), 1.86 (s, 3H). C NMR (126 MHz, CDCl ): δ 160.6, 150.6, 145.5, 133.8, 131.3, 131.2,
28.7, 128.2, 126.3, 125.6, 125.4, 125.2, 123.4, 102.6, 81.4, 72.0, 42.6, 36.9, 3.6. HRMS (ESI) calcd for
+
C H N O Cl [M+H] 339.0900, found 339.0902.
1
9
16
2
2
4
.1.29. 3-Benzyl-1-(but-2-ynyl)-6-chloropyrimidine-2,4(1H,3H)-dione (6h)
Following a similar procedure for the preparation of 6c, 6h was prepared starting from 5b and benzyl
o
-1
chloride. White solids (1 g, 69%). mp: 82-85 C. IR (KBr, cm ): 3103, 2235, 1706, 1661, 1602, 1423, 1326,
1
1
1
1
192, 990, 828, 552,449; H NMR (500 MHz, CDCl ): δ 7.47-7.41 (m, 2H), 7.29-7.24 (m, 2H), 7.24-7.19 (m,
3
13
3
H), 5.92 (s, 1H), 5.05 (s, 2H), 4.74-4.67 (m, 2H), 1.78 (s, 3H). C NMR (126 MHz, CDCl ): δ 160.1, 150.2,
44.9, 136.0, 128.9, 128.1, 128.1, 128.1, 127.5, 102.3, 81.0, 71.9, 44.6, 36.5, 3.3. HRMS (ESI) calcd for
+
C H N O Cl [M+H] 289.0744, found 289.0746.
1
5
14
2
2
4
.1.30. 1-(But-2-ynyl)-6-chloro-3-(2-cyanobenzyl)pyrimidine-2,4(1H,3H)-dione (6i)
Following a similar procedure for the preparation of 6c, 6i was prepared starting from 5b and 2-cyanobenzyl
-1
bromide. White solids (1.1 g, 71%). mp: 162-164 °C. IR (KBr, cm ): 3085, 2225, 1712, 1666, 1600, 1429,
1
1
7
3
1
413, 1355, 1320, 846,765. H NMR (500 MHz, CDCl ): δ 7.65 (d, J = 7.8 Hz, 1H), 7.52 (t, J = 7.7 Hz, 1H),
3
.35 (t, J = 7.6 Hz, 1H), 7.29 (d, J = 7.9 Hz, 1H), 6.01 (s, 1H), 5.33 (s, 2H), 4.78 (d, J = 2.2 Hz, 2H), 1.81 (s,
13
H). C NMR (125 MHz, CDCl ): δ 160.2, 150.2, 145.9, 139.6, 132.9, 132.8, 127.8, 127.4, 117.1, 112.0,
3
+
02.3, 81.5, 71.8, 43.1, 36.9, 3.5. HRMS (ESI) calcd for C H N O Cl [M+H] 314.0696, found 314.0697.
1
6
13
3
2
4
.1.31. 1-(But-2-ynyl)-6-chloro-3-(3-chlorobenzyl)pyrimidine-2,4(1H,3H)-dione (6j)
Following a similar procedure for the preparation of 6c, 6j was prepared starting from 5b and 3-chlrobenzyl
-1
bromide. White solids (1.2 g, 75%). mp: 118 °C; IR (KBr, cm ): 3099, 1700, 1648, 1608, 1429, 1343, 1201,
1
1
4
1
181, 1000, 965, 845, 759; H NMR (300 MHz, CDCl
3
): δ 7.43 (s, 1H), 7.33 (t, J = 4.3 Hz, 1H), 7.21 (d, J =
13
.3 Hz, 2H), 5.95 (s, 1H), 5.03 (s, 2H), 4.74 (s, 2H), 1.80 (s, 3H). C NMR (75 MHz, CDCl ): δ 160.0, 150.2,
3
45.2, 138.0, 134.0, 129.4, 128.8, 127.8, 127.1, 102.3, 81.3, 71.8, 44.1, 36.7, 3.4. HRMS (ESI) calcd for
+
C
15
H
13
N
2
O
2
Cl
2
[M+H] 323.0354, found 323.0357.
4
.1.32. 1-(But-2-ynyl)-6-chloro-3-(3-fluorobenzyl)pyrimidine-2,4(1H,3H)-dione (6k)
Following a similar procedure for the preparation of 6c, 6k was prepared starting from 5b and 3-
-1
fluorobenzyl chloride. White solids (870 mg, 57%). mp: 98-100 °C. IR (KBr, cm ): 3117, 1707, 1663, 1605,
1
1
1
518, 1436, 1346, 1197, 752. H NMR (300 MHz, CDCl
3
): δ 7.25 (d, J = 9.9 Hz, 2H), 7.16 (d, J = 9.8 Hz,
13
H), 6.95 (t, J = 7.0 Hz, 1H), 5.96 (s, 1H), 5.07 (s, 2H), 4.75 (s, 2H), 1.81 (s, 3H). C NMR (75 MHz, CDCl ):
3
δ 164.3, 160.2, 150.4, 145.4, 138.4, 129.9, 129.8, 124.7, 124.6, 116.0, 115.7, 114.8, 114.5, 102.5, 81.4, 71.9,
+
4
4.9, 44.4, 36.8, 29.6, 3.5. HRMS (ESI) calcd for C15
H
13
N
2
O
2
ClF [M+H] 307.0641, found 307.0644.
4
.1.33. 1-(But-2-ynyl)-6-chloro-3-(4-nitrobenzyl)pyrimidine-2,4(1H,3H)-dione (6l)
11

ACCEPTED MANUSCRIPT
Following a similar procedure for the preparation of 6c, 6l was prepared starting from 5b and 4-nitrobenzyl
-1
bromide. White solids (1.0 g, 60%). mp: 156-160 °C. IR (KBr, cm ): 3117, 2299, 2228, 1707, 1663, 1605,
1
1
522, 1435, 1345, 1316, 1197, 994, 827, 752, 595. H NMR (300 MHz, CDCl ): δ 8.31-8.10 (m, 2H), 7.62 (d,
3
13
J = 8.6 Hz, 2H), 6.00 (s, 1H), 5.17 (s, 2H), 4.94 – 4.72 (m, 2H), 1.82 (d, J = 2.3 Hz, 3H). C NMR (75 MHz,
CDCl
3
): δ 160.2, 150.3, 145.8, 143.2, 129.8, 123.6, 102.4, 81.6, 71.7, 44.2, 37.0, 3.5. HRMS (ESI) calcd for
+
C H N O Cl [M+H] 334.0597, found 334.0595.
1
5
13
3
4
4
.1.34. 6-Chloro-3-[(4-methylquinazolin-2-yl)methyl]-1-(prop-2-ynyl)pyrimidine-2,4(1H,3H)-dione (6m)
Following a similar procedure for the preparation of 6c, 6m was prepared satrting from 5c and 2-
-1
chloromethyl-4-methylquinazoline. White solids (300 mg, 22%). mp: 230 °C. IR (KBr, cm ): 3280, 1720,
1
1
7
709, 1660, 1611, 1573, 1502, 1439, 1393, 815, 767; H NMR (300 MHz, CDCl ): δ 8.03 (d, J = 8.3 Hz, 1H),
3
.89 (d, J = 8.2 Hz, 1H), 7.80 (t, J = 7.6 Hz, 1H), 7.55 (t, J = 7.1 Hz, 1H), 6.08 (s, 1H), 5.47 (s, 2H), 4.89 (d, J
+
=
2.4 Hz, 2H), 2.90 (s, 3H), 2.36 (t, J = 2.3 Hz, 1H). HRMS (ESI) calcd for C H N O Cl [M+H] 341.0805,
17
14
4
2
found 341.0807.
4
.1.35. 1-Allyl-6-chloro-3-[(4-methylquinazolin-2-yl)methyl]pyrimidine-2,4(1H,3H)-dione (6n)
Following a similar procedure for the preparation of 6c, 6n was prepared starting from 5d and 2-
-1
chloromethyl-4-methylquinazoline. Light yellow solids (1.1 g, 65%). mp: 127-130 °C. IR (KBr, cm ): 3075,
1
2
7
5
1
2
962, 1714, 1656, 1604, 1569, 1502, 1442, 1341, 1203, 1097, 988, 927, 763. H NMR (300 MHz, CDCl ): δ
3
.99 (d, J = 8.3 Hz, 1H), 7.84 (d, J = 7.9 Hz, 1H), 7.77 (m, 1H), 7.58-7.46 (m, 1H), 6.03 (s, 1H), 5.90 (m, 1H),
1
3
.48 (s, 2H), 5.29 (dd, J = 14.9, 10.4 Hz, 2H), 4.84-4.64 (m, 2H), 2.86 (s, 3H). C NMR (75 MHz, CDCl ): δ
3
68.7, 160.7, 159.7, 151.0, 149.8, 145.9, 133.4, 131.0, 128.7, 126.9, 124.9, 123.1, 117.7, 102.4, 48.4, 46.5,
+
1.7. HRMS (ESI) calcd for C17
H
16
N
4
O
2
Cl [M+H] 343.0962, found 343.0963.
4
.1.36. 6-Chloro-1,3-di(prop-2-ynyl)pyrimidine-2,4(1H,3H)-dione (6o)
Following a similar procedure for the preparation of 6c, 6o was prepared starting from 5c and 1-bromo-2-
-1
propyne. Light yellow solids (680 mg, 80%). mp: 160 °C. IR (KBr, cm ): 3304, 3256, 3099, 1712, 1672, 1609,
1
1
4
1
2
427, 1332, 1207, 1184, 1138, 1112, 1009, 969, 936, 850, 760. H NMR (500 MHz, CDCl ): δ 6.01 (s, 1H),
3
1
3
.86 (d, J = 2.4 Hz, 2H), 4.69 (d, J = 2.4 Hz, 2H), 2.38 (s, 1H), 2.20 (s, 1H). C NMR (125 MHz, CDCl ): δ
3
+
59.1, 149.6, 145.3, 102.7, 77.3, 76.3, 73.5, 71.1, 36.2, 30.9. HRMS (ESI) calcd for C10
8 2 2
H N O Cl [M+H]
23.0274, found 223.0275
4
2
.1.37.
(R)-6-(3-Aminopiperidin-1-yl)-1-(but-2-ynyl)-3-[(4-methylquinazolin-2-yl)methyl]pyrimidine-
,4(1H,3H)-dione (2h)
A mixture of 6c (8 g, 22.5 mmol), (R)-3-aminopiperidine dihydrochloride (4.67 g, 27 mmol), NaHCO3
9.45g, 112.5 mmol) and activated 4 Å MS (2.6 g) in isopropanol (150 mL) was stirred at 100 °C for 2 h. The
(
reaction mixture was filtered through Celite and concentrated in vacuo. The residue was diluted with DCM,
washed with water and saturated brine, dried over anhydrous Na SO , and concentrated. The crude product
2
4
was purified by flash chromatography (DCM/MeOH/TEA, 100:0.5:0.5) to give pure 2h as a light yellow solid
o
20
D
-1
(
8 g, 85%). mp: 78-81 C. [α]
-1.923 (c 0.102, MeOH). IR (KBr, cm ): 3402, 2924, 1704, 1651, 1440, 1228,
1
7
1
3
1
63, 565. H NMR (300 MHz, CDCl ): δ 8.00 (d, J = 8.2 Hz, 1H), 7.87 (d, J = 8.3 Hz, 1H), 7.76 (t, J = 7.5 Hz,
3
H), 7.51 (t, J = 7.5 Hz, 1H), 5.47 (s, 2H), 5.32 (s, 1H), 4.58 (d, J = 1.9 Hz, 2H), 3.39 (d, J = 10.9 Hz, 1H),
.27 (d, J = 11.7 Hz, 1H), 3.13-2.98 (m, 1H), 2.98-2.82 (m, 3H), 2.76 (dd, J = 15.4, 6.2 Hz, 1H), 2.65-2.47 (m,
13
H), 2.05-1.86 (m, 5H), 1.80 (s, 3H), 1.76-1.62 (m, 1H), 1.41-1.20 (m, 1H). C NMR (75 MHz, CDCl ): δ
3
12

ACCEPTED MANUSCRIPT
1
4
68.3, 162.9, 160.3, 159.4, 152.4, 149.7, 133.1, 128.6, 126.6, 124.7, 122.9, 89.0, 79.7, 73.7, 59.1, 51.3, 47.4,
+
6.2, 35.3, 32.9, 23.0, 21.5, 3.4. HRMS (ESI) calcd for C23
H
27
N
6
O
2
[M+H] 419.2195, found 419.2199.
4
.1.38. (R)-6-(3-Aminopiperidin-1-yl)-1-(2-cyanobenzyl)-3-methylpyrimidine-2,4(1H,3H)-dione (2a)
Following a similar procedure for the preparation of 2h, 2a was prepared starting from 6a and (R)-3-
2
0
1
aminopiperidine dihydrochloride. White solid (2.93 g, 80%). [α] +6.000 (c 0.100, MeOH). H NMR (500
D
3
MHz, CDCl ): δ 7.68 (d, J = 7.6 Hz, 1H), 7.56 (t, J = 7.7 Hz, 1H), 7.38 (t, J = 7.6 Hz, 1H), 7.15 (d, J = 7.9 Hz,
1
1
1
1
H), 5.38 (s, 1H), 5.35 – 5.25 (m, 2H), 3.31 (s, 3H), 3.05 (d, J = 11.2 Hz, 1H), 3.00-2.86 (m, 2H), 2.61 (t, J =
0.5 Hz, 1H), 2.41 (t, J = 9.6 Hz, 1H), 1.94 (dd, J = 9.8, 5.3 Hz, 1H), 1.83-1.72 (m, 1H), 1.60 (m, 4H), 1.35-
1
3
.12 (m, 2H). C NMR (125 MHz, CDCl ): δ 163.0, 159.7, 152.6, 140.7, 133.1, 133.0, 127.8, 126.6, 117.0,
3
10.7, 90.4, 63.3, 59.3, 52.9, 51.8, 47.2, 46.2, 33.2, 29.6, 27.8, 23.1. [12]
4
.1.39. (R)-6-(7-Amino-5-azaspiro[2.4]heptan-5-yl)-1-(2-cyanobenzyl)-3-methylpyrimidine-2,4(1H,3H)-dione
(2b)
Following a similar procedure for the preparation of 2h, 2b was prepared starting from 6a and (R)-5-
2
D
0
azaspiro[2.4]heptan-7-amine dihydrochloride. Colorless syrup (60 mg, 81%). [α]
+40.000 (c 0.035, MeOH).
-1
1
IR (KBr, cm ): 3416, 2925, 2223, 1695, 1643, 1450, 1316, 1157, 1024, 803, 768. H NMR (500 MHz,
CDCl ): δ 7.68 (d, J = 7.6 Hz, 1H), 7.58 (t, J = 7.6 Hz, 1H), 7.39 (t, J = 7.6 Hz, 1H), 7.24 (d, J = 7.9 Hz, 1H),
3
5
3
1
1
1
.31 (q, J = 17.1 Hz, 2H), 5.25 (s, 1H), 3.51 (dd, J = 9.6, 5.5 Hz, 1H), 3.40 (d, J = 9.3 Hz, 1H), 3.31 (s, 3H),
.09 (t, J = 4.6 Hz, 1H), 2.96 (dd, J = 9.6, 3.9 Hz, 1H), 2.91 (d, J = 9.3 Hz, 1H), 1.53 (brs, 2H), 0.79-0.71 (m,
13
H), 0.67-0.59 (m, 1H), 0.56 (dt, J = 10.3, 5.3 Hz, 1H), 0.52-0.43 (m, 1H). C NMR (126 MHz, CDCl ): δ
3
62.8, 156.8, 153.1, 140.6, 133.1, 132.8, 127.7, 126.4, 116.8, 110.2, 84.5, 59.7, 57.1, 55.4, 48.3, 27.7, 27.0,
+
0.8, 5.4. HRMS (ESI) calcd for C19
H
22
N
5
O
2
[M+H] 352.1774, found 352.1777.
4
.1.40. (R)-6-(3-Aminopyrrolidin-1-yl)-1-(2-cyanobenzyl)-3-methylpyrimidine-2,4(1H,3H)-dione (2c)
Following a similar procedure for the preparation of 2h, 2c was prepared satrting from 6a and (R)-3-
20
-1
aminopyrrolidine dihydrochloride. White solids (114 mg, 84%). [α]
D
+10.204 (c 0.049, MeOH). IR (KBr, cm ):
1
3
367, 2223, 1696, 1642, 1446, 1275, 1260, 769, 759, 742. H NMR (300 MHz, CDCl ): δ 7.62 (d, J = 7.7 Hz,
3
1
H), 7.53 (t, J = 7.7 Hz, 1H), 7.33 (t, J = 7.6 Hz, 1H), 7.20 (d, J = 7.9 Hz, 1H), 5.23 (s, 2H), 5.18 (s, 1H), 3.66-
.47 (m, 1H), 3.40-3.17 (m, 5H), 3.07 (m, 1H), 2.78 (dd, J = 9.7, 4.6 Hz, 1H), 2.05 (td, J = 13.0, 6.6 Hz, 1H),
3
13
1
.70-1.56 (m, 2H). C NMR (75 MHz, CDCl ): δ 162.8, 156.8, 153.2, 140.7, 133.2, 132.9, 127.8, 126.5,
3
+
1
17.0, 110.2, 84.5, 59.5, 50.7, 49.7, 48.5, 34.2, 27.8. HRMS (ESI) calcd for C H N O [M+H] 326.1617,
17
20
5
2
found 326.1620.
4
.1.41. (S)-6-(3-Aminopyrrolidin-1-yl)-1-(2-cyanobenzyl)-3-methylpyrimidine-2,4(1H,3H)-dione (2d)
Following a similar procedure for the preparation of 2h, 2d was prepared starting from 6a and (S)-3-
20
1
aminopyrrolidine dihydrochloride. White solids (91 mg, 82%). [α] -10.000 (c 0.097, MeOH). H NMR (500
D
3
MHz, CDCl ): δ 7.60 (d, J = 7.6 Hz, 1H), 7.51 (t, J = 7.6 Hz, 1H), 7.31 (t, J = 7.6 Hz, 1H), 7.18 (d, J = 7.9 Hz,
1
3
H), 5.21 (s, 2H), 5.17 (s, 1H), 3.58 (dd, J = 10.6, 5.3 Hz, 1H), 3.49 (q, J = 7.2 Hz, 1H), 3.32 – 3.16 (m, 5H),
.05 (m, 1H), 2.85 (dd, J = 9.7, 4.6 Hz, 1H), 2.06-2.01 (dt, J = 13.1, 6.5 Hz, 1H), 1.72-1.69 (m, 1H).
4
2
.1.42.
(R)-6-(3-Aminopiperidin-1-yl)-1-(2-cyanobenzyl)-3-[(4-methylquinazolin-2-yl)methyl]pyrimidine-
,4(1H,3H)-dione (2e)
13

ACCEPTED MANUSCRIPT
Following a similar procedure for the preparation of 2h, 2e was prepared starting from 6b and (R)-3-
o
20
D
aminopyrrolidine dihydrochloride. White solids (90 mg, 88%). mp: 128-132 C. [α]
-3.571 (c 0.112, MeOH).
-1
1
IR (KBr, cm ): 2944, 1706, 1653, 1569, 1436, 1225, 761. H NMR (500 MHz, CDCl ): δ 7.99 (d, J = 8.2 Hz,
3
1
H), 7.85 (d, J = 8.3 Hz, 1H), 7.83-7.76 (m, 1H), 7.61 (d, J = 7.6 Hz, 1H), 7.52 (q, J = 7.2 Hz, 2H), 7.39 (d, J
7.9 Hz, 1H), 7.32 (s, 1H), 5.49 (s, 1H), 5.46 (s, 2H), 5.30 (d, J = 20.5 Hz, 2H), 3.88 (brs, 2H), 3.25 (d, J =
.7 Hz, 1H), 3.05 (d, J = 9.6 Hz, 1H), 2.96 (d, J = 11.7 Hz, 1H), 2.85 (s, 3H), 2.63 (d, J = 10.5 Hz, 2H), 2.02
=
9
13
(
d, J = 9.0 Hz, 1H), 1.82-1.69 (m, 1H), 1.66-1.53 (m, 1H), 1.35 (m, 1H). C NMR (126 MHz, CDCl
3
): δ 168.4,
1
1
4
62.8, 160.0, 159.6, 152.5, 149.5, 140.6, 133.2, 133.0, 132.7, 128.2, 127.5, 126.6, 126.4, 124.8, 122.8, 116.9,
+
10.2, 90.7, 57.4, 51.6, 47.1, 45.8, 31.6, 29.4, 22.9, 21.5. HRMS (ESI) calcd for C H N O [M+H]
2
7
28
7
2
82.2304, found 482.2308.
4
.1.43. (R)-6-(7-Amino-5-azaspiro[2.4]heptan-5-yl)-1-(2-cyanobenzyl)-3-[(4-methylquinazolin-2-
yl)methyl]pyrimidine-2,4(1H,3H)-dione (2f)
Following a similar procedure for the preparation of 2h, 2f was prepared starting from 6b and (R)-5-
o
20
D
azaspiro[2.4]heptan-7-amine dihydrochloride. Light yellow solids (50 mg, 88%). mp: 65-70 C. [α]
+45.556
-1
1
(
c 0.090, MeOH). IR (KBr, cm ): 3428, 2944, 2873, 2223, 1697, 1650, 1571, 1449, 1437, 1312, 1295, 763. H
NMR (300 MHz, CDCl ): δ 7.97 (d, J = 8.1 Hz, 1H), 7.84 (d, J = 8.2 Hz, 1H), 7.75 (t, J = 7.5 Hz, 1H), 7.58 (d,
3
J = 7.5 Hz, 1H), 7.49 (d, J = 4.6 Hz, 2H), 7.41 (d, J = 7.7 Hz, 1H), 7.34-7.24 (m, 1H), 5.44 (s, 2H), 5.40-5.20
(
m, 3H), 3.60-3.45 (m, 1H), 3.39 (d, J = 9.0 Hz, 1H), 3.04 (s, 1H), 2.93 (dd, J = 16.8, 7.2 Hz, 2H), 2.83 (s,
13
3
1
1
H), 0.69 (d, J = 3.3 Hz, 1H), 0.63-0.47 (m, 2H), 0.44 (d, J = 4.3 Hz, 1H). C NMR (75 MHz, CDCl ): δ
3
68.3, 162.5, 160.3, 156.8, 153.2, 149.6, 140.6, 133.2, 133.0, 132.6, 128.4, 127.5, 126.6, 126.3, 124.8, 122.9,
16.9, 109.9, 84.8, 59.7, 57.0, 55.3, 47.7, 45.9, 45.9, 21.6, 10.5, 5.3. HRMS (ESI) calcd for C H N O
2
28
28
7
+
[
M+H] 494.2304, found 494.2308.
4
2
.1.44.
(R)-6-(3-Aminopyrrolidin-1-yl)-1-(2-cyanobenzyl)-3-[(4-methylquinazolin-2-yl)methyl]pyrimidine-
,4(1H,3H)-dione (2g)
Following a similar procedure for the preparation of 2h, 2g was prepared starting from 6b and (R)-3-
o
20
D
aminopyrrolidine dihydrochloride. White solids (82 mg, 88%). mp: 113-120 C. [α]
+1.562 (c 0.131, MeOH).
-1
1
IR (KBr, cm ): 3446, 3255, 2925, 1710, 1653, 1436, 1227, 763. H NMR (500 MHz, CDCl ): δ 8.03 (d, J =
3
8
2
1
.2 Hz, 1H), 7.90 (d, J = 8.4 Hz, 1H), 7.87-7.78 (m, 1H), 7.63 (d, J = 7.6 Hz, 1H), 7.54 (dd, J = 10.3, 3.8 Hz,
H), 7.47 (d, J = 7.8 Hz, 1H), 7.34 (t, J = 7.5 Hz, 1H), 5.50 (s, 2H), 5.38 (s, 2H), 5.34 (s, 1H), 3.72-3.60 (m,
H), 3.35 (ddd, J = 15.3, 9.5, 6.4 Hz, 2H), 3.29-3.14 (m, 1H), 2.90 (s, 5H), 2.13 (td, J = 12.9, 6.5 Hz, 1H), 1.71
13
(
dt, J = 18.8, 6.3 Hz, 1H). C NMR (125 MHz, CDCl ): δ 168.4, 162.6, 160.5, 156.9, 153.3, 149.8, 140.8,
3
1
3
33.2, 133.1, 132.7, 128.5, 127.6, 126.7, 126.6, 124.9, 123.1, 117.0, 110.1, 85.1, 59.6, 50.8, 49.8, 47.8, 46.1,
+
4.4, 21.7. HRMS (ESI) calcd for C H N O [M+H] 468.2148, found 468.2150.
2
6
26
7
2
4
2
.1.45.
(S)-6-(3-Aminopiperidin-1-yl)-1-(but-2-ynyl)-3-[(4-methylquinazolin-2-yl)methyl]pyrimidine-
,4(1H,3H)-dione (2i)
Following a similar procedure for the preparation of 2h, 2i was prepared starting from 6c and (S)-3-
2
D
0
1
aminopyrrolidine dihydrochloride. Light yellow oil (144 mg, 83%). [α] +1.923 (c 0.104, MeOH). H NMR
(
500 MHz, CDCl
3
) δ 7.99 (d, J = 8.2 Hz, 1H), 7.86 (d, J = 8.4 Hz, 1H), 7.76 (t, J = 7.6 Hz, 1H), 7.51 (t, J = 7.5
Hz, 1H), 5.46 (s, 2H), 5.32 (s, 1H), 4.58 (s, 2H), 3.29 (d, J = 9.3 Hz, 1H), 3.11 (s, 1H), 2.86 (s, 3H), 2.76 (s,
14

ACCEPTED MANUSCRIPT
1
1
H), 2.64 (s, 1H), 2.02 (d, J = 9.2 Hz, 1H), 1.95-1.84 (m, 1H), 1.79 (s, 3H), 1.72-1.66 (m, 1H), 1.40-1.33 (m,
H).
4
.1.46.
(R)-6-(7-Amino-5-azaspiro[2.4]heptan-5-yl)-1-(but-2-yn-1-yl)-3-((4-methylquinazolin-2-
yl)methyl)pyrimidine-2,4(1H,3H)-dione (2j)
Following a similar procedure for the preparation of 2h, 2j was prepared starting from 6c and (R)-5-
-1
azaspiro[2.4]heptan-7-amine dihydrochloride. Colorless syrup (100 mg, 78%). IR (KBr, cm ): 3390, 2922,
1
1
1
1
2
1
3
699, 1652, 1646, 1572, 1444, 1398, 1294, 1201, 784; H NMR (300 MHz, CDCl ): δ 8.02 (d, J = 8.3 Hz,
3
H), 7.90 (d, J = 8.4 Hz, 1H), 7.77 (t, J = 7.6 Hz, 1H), 7.53 (t, J = 7.5 Hz, 1H), 5.59-5.41 (m, 2H), 5.21 (s,
H), 4.63 (q, 2H), 3.92-3.72 (m, 2H), 3.36 (dd, J = 10.1, 3.2 Hz, 1H), 3.22 (m, 2H), 2.89 (s, 3H), 2.66 (brs,
13
H), 1.80 (s, 3H), 0.90 (dd, J = 8.9, 4.9 Hz, 1H), 0.79-0.60 (m, 3H). C NMR (75 MHz, CDCl ): δ 168.4,
3
62.8, 160.7, 156.4, 152.9, 149.9, 133.1, 128.8, 126.6, 124.8, 123.1, 83.4, 80.0, 73.9, 59.1, 57.1, 55.8, 46.3,
+
7.4, 26.9, 21.7, 11.6, 5.6, 3.6. HRMS (ESI) calcd for C H N O [M+H] 431.2195, found 419.2199.
2
4
27
6
2
4
.1.47. (R)-6-(3-Aminopiperidin-1-yl)-1-(but-2-ynyl)-3-methylpyrimidine-2,4(1H,3H)-dione (2k)
Following a similar procedure for the preparation of 2h, 2k was prepared starting from 6d and (R)-3-
2
D
0
-
aminopyrrolidine dihydrochloride. Colorless syrup (70 mg, 98%). [α] -4.167 (c 0.144, MeOH). IR (KBr, cm
1
1
)
: 3369, 2937, 1700, 1620, 1608, 1436, 1376, 1220, 1098, 798. H NMR (500 MHz, CDCl ): δ 5.17 (s, 1H),
3
4
1
.48 (d, J = 1.8 Hz, 2H), 3.24 (s, 4H), 3.15 (d, J = 10.2 Hz, 1H), 3.07-2.92 (m, 1H), 2.64 (s, 1H), 2.45 (s, 1H),
13
.98-1.87 (m, 1H), 1.86-1.78 (m, 1H), 1.76 (s, 5H), 1.70-1.56 (m, 1H), 1.25 (d, J = 10.0 Hz, 1H). C NMR
(
125 MHz, CDCl ): δ 163.0, 159.1, 152.2, 88.9, 79.9, 73.7, 59.1, 51.4, 47.4, 35.2, 32.9, 29.5, 27.6, 23.0, 3.4.
3
+
HRMS (ESI) calcd for C H N O [M+H] 277.1665, found 277.1667.
14
21
4
2
4
.1.48. (R)-6-(3-Aminopiperidin-1-yl)-1-(but-2-ynyl)-3-(quinoxalin-2-ylmethyl)pyrimidine-2,4(1H,3H)-dione
(2l)
Following a similar procedure for the preparation of 2h, 2l was prepared starting from 6e and (R)-3-
o
20
D
aminopyrrolidine dihydrochloride. Brown solids (1.3 g, 92%). mp: 68-72 C. [α]
-0.800 (c 0.125, MeOH). IR
-1
1
(
KBr, cm ): 3359, 2942, 2855, 1705, 1654, 1606, 1437, 1371, 1226, 1111, 764. H NMR (500 MHz, CDCl ):
3
δ 8.82 (s, 1H), 8.13-7.99 (m, 2H), 7.76-7.59 (m, 2H), 5.47 (s, 2H), 5.32 (s, 1H), 4.56 (d, J = 2.3 Hz, 2H), 3.39
d, J = 13.7 Hz, 1H), 3.27 (d, J = 11.9 Hz, 1H), 3.13-3.02 (m, 1H), 2.77 (s, 1H), 2.59 (s, 1H), 2.26 (brs, 2H),
.06-1.95 (m, 1H), 1.95-1.84 (m, 1H), 1.79 (dt, J = 12.4, 2.3 Hz, 3H), 1.76-1.63 (m, 1H), 1.33 (dt, J = 16.9, 7.2
(
2
1
3
Hz, 1H). C NMR (125 MHz, CDCl ): δ 162.7, 159.6, 152.3, 151.5, 144.1, 141.9, 141.7, 129.8, 129.3, 129.3,
3
+
1
4
29.0, 89.0, 80.3, 73.5, 58.9, 51.5, 47.5, 44.4, 35.7, 32.8, 23.1, 3.6. HRMS (ESI) calcd for C22
25 6 2
H N O [M+H]
05.2039, found 405.2043.
4
.1.49. (R)-6-(3-Aminopiperidin-1-yl)-1-(but-2-ynyl)-3-(naphthalen-2-ylmethyl)pyrimidine-2,4(1H,3H)-dione
(2m)
Following a similar procedure for the preparation of 2h, 2m was prepared starting from 6f and (R)-3-
o
20
D
aminopyrrolidine dihydrochloride. White solids (1 g, 87%). mp: 58-62 C. [α]
+0.840 (c 0.119, MeOH). IR
-1
1
(
KBr, cm ): 3359, 2921, 2852, 1700, 1653, 1600, 1436, 1370, 1224, 1093, 922, 781, 564. H NMR (300 MHz,
CDCl
3
): δ 7.87 (s, 1H), 7.67 (d, J = 8.4 Hz, 3H), 7.55 (d, J = 8.4 Hz, 1H), 7.42 -7.21 (m, 2H), 5.15 (d, J = 11.0
Hz, 3H), 4.38 (s, 2H), 3.09 (dd, J = 24.1, 9.7 Hz, 2H), 2.80 (s, 1H), 2.50 (t, J = 9.5 Hz, 1H), 2.40-2.20 (m, 1H),
13
2
3
.03-1.58 (m, 3H), 1.48 (d, J = 10.2 Hz, 1H), 1.11 (m, 3H). C NMR (75 MHz, CDCl ): δ 162.8, 159.3, 152.2,
15

ACCEPTED MANUSCRIPT
1
2
34.7, 133.2, 132.7, 127.9, 127.5, 127.1, 125.9, 125.7, 88.9, 80.1, 73.9, 58.9, 51.3, 47.4, 44.3, 35.5, 32.9, 29.6,
+
3.1, 3.6. HRMS (ESI) calcd for C24
H
27
N
4
O
2
[M+H] 403.2134, found 403.2137.
4
.1.50. (R)-6-(3-Aminopiperidin-1-yl)-1-(but-2-ynyl)-3-(naphthalen-1-ylmethyl)pyrimidine-2,4(1H,3H)-dione
(2n)
Following a similar procedure for the preparation of 2h, 2n was prepared starting from 6g and (R)-3-
2
D
0
-
aminopyrrolidine dihydrochloride. Colorless syrup (300 mg, 75%). [α]
-0.962 (c 0.119, MeOH). IR (KBr, cm
1
1
)
: 3428, 2935, 1704, 1653, 1600, 1437, 1375, 1226, 766, 566. H NMR (300 MHz, CDCl ): δ 8.25 (d, J = 8.4
3
Hz, 1H), 7.80 (d, J = 7.8 Hz, 1H), 7.74 – 7.65 (m, 1H), 7.48 (ddd, J = 22.1, 11.3, 4.3 Hz, 2H), 7.39-7.26 (m,
2
2
1
1
H), 5.58 (s, 2H), 5.23 (s, 1H), 4.47 (d, J = 2.2 Hz, 2H), 3.16 (dd, J = 24.2, 11.6 Hz, 2H), 2.99 – 2.78 (m, 1H),
.59 (t, J = 10.2 Hz, 1H), 2.49-2.28 (m, 1H), 1.91-1.79 (m, 1H), 1.79-1.70 (m, 4H), 1.64 (d, J = 10.8 Hz, 3H),
13
3
.62-1.46 (m, 1H), 1.21-1.08 (m, 1H). C NMR (75 MHz, CDCl ): δ 163.0, 159.5, 152.4, 133.7, 132.2, 131.3,
28.6, 127.8, 126.1, 125.6, 125.2, 124.9, 123.6, 89.0, 80.1, 73.8, 59.1, 51.4, 47.4, 41.9, 35.6, 33.0, 23.2, 3.6.
+
HRMS (ESI) calcd for C H N O [M+H] 403.2134, found 403.2137.
24
27
4
2
4
.1.51. (R)-6-(3-Aminopiperidin-1-yl)-3-benzyl-1-(but-2-ynyl)pyrimidine-2,4(1H,3H)-dione (2o)
Following a similar procedure for the preparation of 2h, 2o was prepared starting from 6h and (R)-3-
2
D
0
-
aminopyrrolidine dihydrochloride. Colorless syrup (81 mg, 38%). [α] -0.806 (c 0.124, MeOH). IR (KBr, cm
1
1
)
: 3442, 2940, 1701, 1653, 1602, 1437, 1275, 1225, 763, 749. H NMR (500 MHz, CDCl ): δ 7.54-7.45 (m,
3
2
1
2
H), 7.31-7.25 (m, 2H), 7.25-7.20 (m, 1H), 5.23 (s, 1H), 5.08 (s, 2H), 4.51 (d, J = 2.0 Hz, 2H), 3.30 (d, J =
0.6 Hz, 1H), 3.19 (d, J = 11.2 Hz, 1H), 3.00 (td, J = 8.8, 4.3 Hz, 1H), 2.69 (t, J = 9.9 Hz, 1H), 2.48 (s, 1H),
13
.02-1.91 (m, 1H), 1.89-1.82 (m, 1H), 1.81 (t, J = 2.3 Hz, 3H), 1.71-1.63 (m, 3H), 1.32-1.24 (m, 1H).
C
NMR (125 MHz, CDCl
3
): δ 162.7, 159.2, 152.1, 137.0, 129.0, 128.1, 127.2, 89.0, 80.0, 73.6, 59.1, 51.3, 47.3,
+
4
4.1, 35.3, 33.0, 23.0, 3.4. HRMS (ESI) calcd for C H N O [M+H] 353.1978, found 353.1980.
20
25
4
2
4
.1.52. (R)-6-(3-Aminopiperidin-1-yl)-1-(but-2-ynyl)-3-(2-cyanobenzyl)pyrimidine-2,4(1H,3H)-dione (2p)
Following a similar procedure for the preparation of 2h, 2p was prepared starting from 6i and (R)-3-
20
aminopyrrolidine dihydrochloride. Light yellow solid (400 mg, 90%). [α] +1.316 (c 0.154, MeOH). IR (KBr,
D
-1
1
cm ): 3418, 2946, 2224, 1703, 1653, 1601, 1437, 1227, 763. H NMR (500 MHz, CDCl ): δ 7.39 (d, J = 6.1
3
Hz, 1H), 7.28 (s, 1H), 7.10 (s, 1H), 7.03 (d, J = 6.5 Hz, 1H), 5.07 (s, 2H), 5.04 (s, 1H), 4.33 (s, 2H), 3.14 (d, J
=
=
8.4 Hz, 1H), 3.04 (s, 1H), 2.80 (s, 1H), 2.53 (s, 1H), 2.34 (s, 1H), 1.81 (m, 2H), 1.69-1.38 (m, 5H), 1.14 (d, J
13
39.1 Hz, 2H). C NMR (125 MHz, CDCl ): δ 161.7, 159.1, 151.4, 139.9, 132.2, 132.0, 126.8, 126.3, 116.6,
3
1
24 5 2
10.7, 87.7, 79.5, 73.0, 58.3, 52.1, 46.7, 41.7, 35.1, 32.3, 22.4, 2.8. HRMS (ESI) calcd for C21H N O
+
[
M+H] 378.1930, found 378.1933.
4
.1.53. (R)-6-(3-Aminopiperidin-1-yl)-1-(but-2-ynyl)-3-(3-chlorobenzyl)pyrimidine-2,4(1H,3H)-dione (2q)
Following a similar procedure for the preparation of 2h, 2q was prepared starting from 6j and (R)-3-
20
-1
aminopyrrolidine dihydrochloride. Colorless syrup (1 g, 84%). [α]
D
-1.531 (c 0.196, MeOH). IR (KBr, cm ):
1
3
1
442, 2941, 1701, 1652, 1600, 1433, 1225, 765. H NMR (500 MHz, CDCl ): δ 7.45 (s, 1H), 7.38-7.33 (m,
3
H), 7.25-7.17 (m, 2H), 5.23 (s, 1H), 5.04 (s, 2H), 4.52 (d, J = 2.1 Hz, 2H), 3.31 (d, J = 10.3 Hz, 1H), 3.21 (d,
J = 11.1 Hz, 1H), 3.08-2.94 (m, 1H), 2.70 (t, J = 7.2 Hz, 1H), 2.51 (d, J = 8.9 Hz, 1H), 2.05-1.92 (m, 1H),
13
1
.92-1.78 (m, 4H), 1.77-1.58 (m, 1H), 1.29 (m, 1H). C NMR (125 MHz, CDCl ): δ 162.2, 159.1, 151.7,
3
16

ACCEPTED MANUSCRIPT
1
38.7, 133.5, 129.1, 128.5, 127.1, 126.8, 88.5, 79.8, 73.3, 58.8, 51.0, 47.1, 43.3, 35.2, 32.7, 22.8, 3.2. HRMS
+
(
ESI) calcd for C20
H
24
N
4
O
2
Cl [M+H] 387.1588, found 387.1590.
4
.1.54. (R)-6-(3-Aminopiperidin-1-yl)-1-(but-2-ynyl)-3-(3-fluorobenzyl)pyrimidine-2,4(1H,3H)-dione (2r)
Following a similar procedure for the preparation of 2h, 2r was prepared starting from 6k and (R)-3-
20
-
aminopyrrolidine dihydrochloride. Colorless syrup (810 mg, 77%). [α]
D
+0.508 (c 0.197, MeOH). IR (KBr, cm
): δ 7.00 (s, 2H), 6.93 (d,
J = 9.8 Hz, 1H), 6.67 (d, J = 6.0 Hz, 1H), 5.02 (s, 1H), 4.83 (s, 2H), 4.30 (s, 2H), 3.10 (d, J = 10.8 Hz, 1H),
1
1
)
: 3449, 2923, 2854, 1700, 1653, 1600, 1437, 1226, 765. H NMR (300 MHz, CDCl
3
3
2
1
4
.00 (d, J = 11.5 Hz, 1H), 2.77 (s, 1H), 2.48 (t, J = 10.4 Hz, 1H), 2.39-2.20 (m, 1H), 1.75 (s, 3H), 1.62 (d, J =
13
1.3 Hz, 4H), 1.55-1.34 (m, 1H), 1.06 (d, J = 8.9 Hz, 1H). C NMR (75 MHz, CDCl ): δ 163.5, 162.0, 160.3,
3
58.9, 151.5, 139.0, 138.9, 129.1, 129.0, 123.8, 123.8, 115.0, 114.8, 113.7, 113.4, 88.1, 79.5, 73.2, 58.4, 50.7,
+
6.8, 43.0, 35.0, 32.4, 22.6, 2.9. HRMS (ESI) calcd for C20
H
24
N
4
O
2
F [M+H] 371.1883, found 371.1886.
4
.1.55. (R)-6-(3-Aminopiperidin-1-yl)-1-(but-2-ynyl)-3-(4-nitrobenzyl)pyrimidine-2,4(1H,3H)-dione (2s)
Following a similar procedure for the preparation of 2h, 2s was prepared starting from 6l and (R)-3-
2
0
-1
aminopyrrolidine dihydrochloride. Yellow oil (800 mg, 85%). [α]
D
-0.769 (c 0.130, MeOH). IR (KBr, cm ):
1
3
455, 2940, 2855, 1696, 1649, 1604, 1521, 1434, 1343, 1226, 1108, 763. H NMR (300 MHz, CDCl
3
): δ 7.84
(
dd, J = 8.6, 3.2 Hz, 2H), 7.49-7.27 (m, 2H), 5.00 (d, J = 3.0 Hz, 1H), 4.90 (s, 2H), 4.29 (s, 2H), 3.11 (d, J =
1
1
1.2 Hz, 1H), 3.01 (d, J = 11.3 Hz, 1H), 2.77 (d, J = 3.0 Hz, 1H), 2.49 (t, J = 10.7 Hz, 1H), 2.30 (t, J = 9.8 Hz,
13
H), 1.67 (dd, J = 39.8, 21.5 Hz, 7H), 1.49-1.32 (m, 1H), 1.06 (d, J = 10.4 Hz, 1H). C NMR (75 MHz,
CDCl ): δ 161.8, 159.0, 151.4, 146.4, 143.9, 128.9, 122.7, 87.9, 79.6, 73.0, 58.4, 50.7, 46.8, 42.9, 35.1, 32.4,
3
+
2
2.5, 2.9. HRMS (ESI) calcd for C H N O [M+H] 398.1828, found 398.1832.
2
0
24
5
4
4
2
.1.56.
(R)-6-(3-Aminopiperidin-1-yl)-3-[(4-methylquinazolin-2-yl)methyl]-1-(prop-2-ynyl)pyrimidine-
,4(1H,3H)-dione (2t)
Following a similar procedure for the preparation of 2h, 2t was prepared starting from 6m and (R)-3-
2
D
0
-
aminopyrrolidine dihydrochloride. Colorless syrup (50 mg, 85%). [α]
-0.833 (c 0.121, MeOH). IR (KBr, cm
1
1
)
: 3419, 3244, 2927, 2853, 1700, 1653, 1609, 1569, 1436, 1226, 806, 764. H NMR (500 MHz, CDCl ): δ
3
7
2
2
1
1
3
.96 (d, J = 8.2 Hz, 1H), 7.82 (d, J = 8.4 Hz, 1H), 7.72 (t, J = 7.6 Hz, 1H), 7.47 (t, J = 7.5 Hz, 1H), 5.41 (s,
H), 5.30 (s, 1H), 4.61 (s, 2H), 3.34 (d, J = 10.1 Hz, 1H), 3.20 (s, 1H), 3.05 (d, J = 8.7 Hz, 1H), 2.82 (s, 3H),
.71 (dd, J = 14.1, 7.0 Hz, 1H), 2.55 (s, 1H), 2.33 (s, 2H), 2.31 (d, J = 2.0 Hz, 1H), 1.97 (d, J = 9.0 Hz, 1H),
13
.91-1.79 (m, 1H), 1.67 (dt, J = 14.0, 10.5 Hz, 1H), 1.21 (s, 1H). C NMR (125 MHz, CDCl ): δ 168.4, 162.8,
3
60.2, 159.2, 152.4, 149.7, 133.2, 128.6, 126.7, 124.7, 123.0, 89.5, 78.5, 72.1, 63.1, 58.9, 52.8, 51.4, 46.2,
+
4.8, 32.8, 21.6. HRMS (ESI) calcd for C H N O [M+H] 405.2039, found 405.2041.
22
25
6
2
4
.1.57.
(R)-1-Allyl-6-(3-aminopiperidin-1-yl)-3-((4-methylquinazolin-2-yl)methyl)pyrimidine-2,4(1H,3H)-
dione (2u)
Following a similar procedure for the preparation of 2h, 2u was prepared starting from 6n and (R)-3-
o
20
D
aminopyrrolidine dihydrochloride. Light yellow solid (714 mg, 61%). mp: 82-84 C. [α]
+1.875 (c 0.162,
-1
1
MeOH). IR (KBr, cm ): 3456, 2995, 1769, 1759, 1654, 1383, 1247, 1055, 749. H NMR (300 MHz, CDCl ): δ
3
8
1
.01 (d, J = 8.2 Hz, 1H), 7.86 (d, J = 8.2 Hz, 1H), 7.77 (t, J = 7.6 Hz, 1H), 7.52 (t, J = 7.5 Hz, 1H), 5.96 (m,
H), 5.47 (s, 2H), 5.37 (s, 1H), 5.32-5.11 (m, 2H), 4.51 (d, J = 2.5 Hz, 2H), 3.24 (d, J = 9.7 Hz, 1H), 3.12 (d, J
=
12.0 Hz, 1H), 3.01 (td, J = 9.0, 4.4 Hz, 1H), 2.87 (s, 3H), 2.76-2.61 (m, 1H), 2.47 (t, J = 10.0 Hz, 1H), 1.94
17

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1
3
(
m, 5H), 1.75-1.57 (m, 1H). C NMR (75 MHz, CDCl ): δ 168.4, 163.1, 160.4, 160.1, 152.8, 149.8, 133.2,
3
1
32.9, 128.6, 126.6, 124.7, 123.0, 116.2, 89.6, 59.4, 51.6, 47.4, 46.9, 46.1, 33.3, 23.1, 21.6, 7.9. HRMS (ESI)
+
calcd for C H N O [M+H] 407.2195, found 407.2197.
22
27
6
2
4
.1.58. (R)-6-(3-Aminopiperidin-1-yl)-1,3-di(prop-2-yn-1-yl)pyrimidine-2,4(1H,3H)-dione (2v)
Following a similar procedure for the preparation of 2h, 2v was prepared starting from 6o and (R)-3-
2
D
0
-
aminopyrrolidine dihydrochloride. Colorless syrup (85 mg, 98%). [α]
-1.911 (c 0.157, MeOH). IR (KBr, cm
1
1
)
: 3284, 2940, 2855, 1704, 1653, 1605, 1436, 1374, 1342, 1227, 764, 749; H NMR (500 MHz, CDCl ): δ
3
5
1
1
.16 (s, 1H), 4.56 (d, J = 2.2 Hz, 2H), 4.53 (dd, J = 6.7, 2.1 Hz, 2H), 3.22 (d, J = 10.7 Hz, 1H), 3.12 (d, J =
1.1 Hz, 1H), 3.01- 2.89 (m, 1H), 2.64 (t, J = 9.8 Hz, 1H), 2.45 (s, 1H), 2.32 (s, 1H), 2.10 (t, J = 2.2 Hz, 1H),
13
.90 (d, J = 12.8 Hz, 3H), 1.83-1.75 (m, 1H), 1.66-1.55 (m, 1H), 1.23 (dd, J = 19.4, 9.2 Hz, 1H). C NMR
(
125 MHz, CDCl
3
): δ 161.5, 159.4, 151.4, 89.1, 78.3, 78.3, 72.6, 70.6, 59.1, 51.4, 47.4, 35.0, 33.0, 30.2, 23.1.
+
HRMS (ESI) calcd for C H N O [M+H] 287.1508, found 287.1510.
15
19
4
2
4
.2. In vitro assay for inhibition of DPP-4, DPP-8 and DPP-9
The DPP-4 Drug Discovery Kit (Enzo Life Sciences International, Inc.) was used for the assay of inhibition
of DPP-4 activity, which was determined by measuring the rate of hydrolysis of a surrogate substrate, H-Gly-
Pro-7-amino-4-methylcoumarin (H-Gly-Pro-AMC). The DPP-4 inhibitor P32/98A was selected as the
reference inhibitor. The 500 µM substrate solution was diluted with assay buffer (50 mM Tris, pH=7.5) to give
a 5 ꢀM substrate solution. 10 ꢀL of appropriately diluted solutions of the test compounds and 25 ꢀl of assay
buffer were added to 96-well microtiter plates, followed by addition of 15 ꢀL of assay buffer containing 0.26
MU of recombinant human DPP-4. The reaction was initiated by addition of 50 ꢀL of 5 ꢀM substrate solution.
After incubation at room temperature for 10 min, fluorescence was measured using an excitation wavelength
of 380 nm and an emission wavelength of 460 nm by a SpectraMax M5 microplate reader. A fluorescence
standard curve for 7-amino-4-methylcoumarin (AMC) was generated using 0.12-15 ꢀM of AMC buffer
solutions. The inhibitory rate relative to the control without inhibitor was calculated and IC values were
5
0
determined by nonlinear regression. By the similar principle, the assays for inhibition of DPP-8 and DPP-9
activity were performed using DPP-8 and DPP-9 fluorescent activity assay kits (Cat: 80208 and 80209, BPS).
4
.3. Molecular docking
The crystal structures of linagliptin (1a) and alogliptin (2a) were extracted from the X-ray crystallographical
structures of DPP-4 (PDB codes: 2RGU and 3G0B) and used as the templates to construct the 3D structures of
h and 2i in Sybyl 6.9. After hydrogens were added and Gasteiger-Hückel charges were assigned, the
2
structures of 2h and 2i were further optimized with Tripos force field. The protein structure was prepared by
removal of all ligands and waters from the X-ray crystallographical structure of DPP-IV (PDB code: 2RGU)
and protonated. The optimized structures of 2h and 2i were docked into the inhibitor binding pocket of DPP-4
A chain with GOLD 3.0.1. The residues within a radius of 10 Å around the original ligand were selected to
define the active site. Default parameters were set. Maximum 10 solutions were adapted for each compound
and ranked according to Goldscore. Computational interaction modes of the best solutions and atom distances
were pictured with PyMol 1.3.
18

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Acknowledgements
This project was supported by the “111 Project” from the Ministry of Education of China, the State
Administration of Foreign Expert Affairs of China (No. 111-2-07), and program for Changjiang Scholars and
Innovative Research Team in University (IRT1193).
References and notes
[
[
[
[
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2] J.J. Holst, M.B. Toft-Nielsen, C. Orskov, M. Nauck, B. Willms, On the effects of glucagon-like peptide-1
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4] P.E. MacDonald, W. El-Kholy, M.J. Riedel, A.M. Salapatek, P.E. Light, M.B. Wheeler, The multiple
actions of GLP-1 on the process of glucose-stimulated insulin secretion, Diabetes 51 (2002) Suppl 3:
S434-442.
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5] C.F. Deacon, A.H. Johnsen, J.J. Holst, Degradation of glucagon-like peptide-1 by human plasma in vitro
yields an N-terminally truncated peptide that is a major endogenous metabolite in vivo, J. Clin.
Endocrinol Metab. 80 (1995) 952-957.
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6] C.F. Deacon, Circulation and degradation of GIP and GLP-1, Horm. Metab. Res. 36 (2004) 761-765.
7] D.J. Drucker, M.A. Nauck, The incretin system: glucagon-like peptide-1 receptor agonists and dipeptidyl
peptidase-4 inhibitors in type 2 diabetes, Lancet. 368 (2006) 1696-1705.
[
[
8] D.J. Drucker, Dipeptidyl peptidase-4 inhibition and the treatment of type 2 diabetes: Preclinical biology
and mechanisms of action, Diabetes Care 30 (2007) 1335-1343.
9] B. Charbonnel, A. Karasik, J. Liu, M. Wu, G. Meininger, Sitagliptin Study 020 Group, Efficacy and safety
of the dipeptidyl peptidase-4 inhibitor sitagliptin added to ongoing metformin therapy in patients with
type 2 diabetes inadequately controlled with metformin alone, Diabetes Care 29 (2006) 2638-2643.
10] P. Aschner, M.S. Kipnes, J.K. Lunceford, M. Sanchez, C. Mickel, D.E. Williams-Herman, Sitagliptin
Study 021 Group, Effect of the dipeptidyl peptidase-4 inhibitor sitagliptin as monotherapy on glycemic
control in patients with type 2 diabetes, Diabetes Care 29 (2006) 2632-2637.
[
[
11] M. Eckhardt, E. Langkopf, M. Mark, M. Tadayyon, L. Thomas, H. Nar, W. Pfrengle, B. Guth, R. Lotz, P.
Sieger, H. Fuchs, F. Himmelsbach, 8-(3-(R)-aminopiperidin-1-yl)-7-but-2-ynyl-3-methyl-1-(4-methyl-
quinazolin-2-ylmethyl)-3, 7-dihydropurine-2,6-dione (BI 1356), a highly potent, selective, long-acting,
and orally bioavailable DPP-4 inhibitor for the treatment of type 2 diabetes, J. Med. Chem. 50 (2007)
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450-6453.
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[
12] Z. Zhang, M.B. Wallace, J. Feng, J.A. Stafford, R.J. Skene, L. Shi, B. Lee, K. Aertgeerts, A. Jennings, R.
Xu, D.B. Kassel, S.W. Kaldor, M. Navre, D.R. Webb, S.L. Gwaltney, Design and synthesis of
pyrimidinone and pyrimidinedione inhibitors of dipeptidyl peptidase IV, J. Med. Chem. 54 (2011) 510-
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13] H. Xie, L. Zeng, S. Zeng, X. Lu, X. Zhao, G. Zhang, Z. Tu , H. Xu, L. Yang, X. Zhang, S. Wang, W. Hu,
Highly potent dipeptidyl peptidase IV inhibitors derived from Alogliptin through pharmacophore
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14] The PyMOL Molecular Graphics System, Version 1.3 Schrödinger, LLC.
15] T. Gelbrich, V. Kahlenberg, U. J. Griesser, Alogliptin and its benzoate salt, Acta Cryst. C69 (2013) 674-
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16] G.R. Lankas, B. Leiting, R.S. Roy, G.J. Eiermann, M.G. Beconi, T. Biftu, C.C. Chan, S. Edmondson,
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Qian, L. Reigle, A. Woods, J. K. Wu, D. Zaller, X. Zhang, L. Zhu, A.E. Weber, N.A. Thornberry,
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Figure captions
Figure 1. Structures of linagliptin (1a) and alogliptin (2a).
Figure 2. Compounds designed based on 1a and 2a with diverse amino groups.
Figure 3. Hybrid design based on 1a and 2a.
Figure 4. Binding mode comparison of linaliptin and alogliptin in DPP-4.
Figure 5. Predicted docking interactions of 2h/2i binding to DPP-4.
Figure 6. Effect of 1a (linagliptin) on glucose tolerance in db/db mice.
Figure 7. Effect of 2h on glucose tolerance in db/db mice.
Figure 8. Effect of 1a (linagliptin) and 2h on plasma glucose AUC0→120 min.
Figure 9. Effect of 1a (linagliptin) and 2h on plasma DPP-4 activity.
Scheme 1. Synthesis of compounds 1a-1j.
Scheme 2. Synthesis of compounds 2a-2v.
21

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Table 1. DPP-4 inhibitory activity of 1a-1j.
O
R1
N
N
N
R2
N
N
O
N
Inhibition (%)
Cpd
R1
R2
IC50 (nM)
0.77
1
0 ꢀM
100 nM
1
a
94.8
94.7
66.0
94.4
96.8
a
1
b
5.7
NT
a
1
c
-17.4
13.1
NT
a
1
d
NT
a
1
e
91.3
39.2
NT
a
1
f
94.5
91.0
25.8
13.6
NT
a
1
g
NT
1
h
97.1
93.0
NT
92.2
27.6
NT
1.33
a
1
1
i
NT
j
3.77
a
NT means “not tested”.
1

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Table 2. DPP-4 inhibitory activity of 2a-2d.
Inhibition (%)
Cpd
R
2
IC50 (nM)
2.63
1
0 ꢀM
100 nM
86.5
2
2
a
102.2
93.0
a
b
-0.26
NT
a
2
c
97.3
57.9
23.9
NT
a
2
d
-0.96
NT
a
NT means “not tested”.
2

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Table 3. DPP-4 inhibitory activity of 2e-2k.
O
R3
O
R
4
N
N
R2
Inhibition (%)
Cpd
R3
R4
R2
IC50 (nM)
2.63
1
00 nM
2
a
e
-CH
3
86.5
a
2
34.8
-0.3
NT
a
2f
NT
a
2
2
g
3.1
NT
h
95.9
0.31
0.35
12.7
2
i
102.5
71.8
-2.1
2
j
a
2
k
-CH
3
NT
a
NT means “not tested”.
3

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Table 4. DPP-4 inhibitory activity of compounds 2l-2v.
O
R3
O
R4
N
N
N
NH2
Inhibition (%)
Cpd
R3
R4
IC50 (nM)
0.31
1
00 nM
2
h
95.9
2
l
98.6
3.6
a
2
m
21.8
43.2
NT
2
n
164
2
o
p
q
51.3
87.7
66.5
65.9
7.6
2
2
66.4
2
r
s
61.1
89.7
83.6
30.8
2.5
2
2
t
11.4.
2
u
v
92.6
29.7
3.8
a
2
NT
a
NT means “not tested”.
4

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Table 5. Selectivity for DPP-4 over DPP-8 and DPP-9.
DPP-4 IC50
nM)
DPP-8 inhibition (%)
DPP-9 inhibition (%)
Cpd
(
100 µM
25 µM
6.1
100 µM
25 µM
4.2
1
1
2
2
2
2
2
2
2
2
a (linagliptin)
0.77
1.33
2.63
0.31
0.35
3.6
19.6
33.6
-1.4
2.9
9.5
23.1
7.0
8.7
0.1
9.9
5.2
-2.4
8.0
9.0
h
14.6
3.1
5.9
a (alogliptin)
8.6
h
i
4.0
11.1
5.8
16.2
2.8
5.7
l
-3.6
4.1
3.1
p
s
t
7.6
-1.0
9.7
9.5
2.5
1.1
4.9
11.4
3.8
27.2
10.1
11.8
1.5
8.6
u
3.6
Table 6. Selected pharmacokinetics parameters for 2h in rats.
Dose (5 mg/kg)
t1/2 (h)
3.8±2.5
7.5±NA
Tmax (h)
0.1±0.0
5.5±4.3
Cmax (ꢀg/mL)
2.75±0.49
AUC0-∞ (ꢀgꢁh/mL)
2.37±0.48
MRT0-∞ (h)
1.4±0.4
F (%)
iv
/
oral
0.04±0.02
0.36±NA
9.7±NA
15.1
5

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O
N
O
CN
NH2
N
N
N
N
N
N
N
N
NH2
O
O
N
1a, Linagliptin
2a, Alogliptin
Figure 1.
O
N
R1
O
CN
N
N
N
R2
N
N
N
N
O
O
R2
1
b-1j
2
b-2d
Figure 2.
O
N
NH2
N
N
N
N
N
N
Me
or
O
or
1a
O
CN
N
N
N
N
NH2
O
N
Ring
O
CN
N
N
2e-2k
NH2
O
N
2a
O
R3
R
4
N
N
NH2
O
N
2
l-2v
Figure 3.
1

ACCEPTED MANUSCRIPT
A) Structural alignment of linagliptin (pink) and alogliptin (green).
B) Interactions of linagliptin (pink) binding to DPP-4.
C) Interactions of alogliptin (green) binding to DPP-4.
Figure 4.
A) 2h (purple) binding to DPP-4.
B) 2i (green) binding to DPP-4.
Figure 5.
2