Synthesis and biological evaluation of 3-(2-aminoethyl) uracil derivatives as gonadotropin-releasing hormone (GnRH) receptor antagonists

Article abstract of DOI:10.1016/j.ejmech.2017.12.095

We investigated a series of uracil analogues by introducing various substituents on the phenyl ring of the N-3 aminoethyl side chain and evaluated their antagonistic activity against human gonadotropin-releasing hormone (GnRH) receptors. Analogues with su

Full text of DOI:10.1016/j.ejmech.2017.12.095

Accepted Manuscript
Synthesis and biological evaluation of 3-(2-aminoethyl) uracil derivatives as
gonadotropin-releasing hormone (GnRH) receptor antagonists
Seon-Mi Kim, Minhee Lee, So Young Lee, Soo-Min Lee, Eun Jeong Kim, Jae Sun
Kim, Jihyae Ann, Jiyoun Lee, Jeewoo Lee
PII:
S0223-5234(17)31127-3
10.1016/j.ejmech.2017.12.095
EJMECH 10073
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 1 November 2017
Revised Date: 29 December 2017
Accepted Date: 30 December 2017
Please cite this article as: S.-M. Kim, M. Lee, S.Y. Lee, S.-M. Lee, E.J. Kim, J.S. Kim, J. Ann, J. Lee, J.
Lee, Synthesis and biological evaluation of 3-(2-aminoethyl) uracil derivatives as gonadotropin-releasing
hormone (GnRH) receptor antagonists, European Journal of Medicinal Chemistry (2018), doi: 10.1016/
j.ejmech.2017.12.095.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
Graphical Abstract
F
O
O
N
N
CF3
N
N
NFAT inhibition IC50
9.9 nM
HO2C
NH
O
max. LH inhibition (%, h) 82%, 8 h
F
LH inhibition (24 h)
69%
CF3
12c

Synthesis and biologAicCaClEePvTaElDuMatAioNnUSoCfR3IP-(T2-aminoethyl) uracil
derivatives as gonadotropin-releasing hormone (GnRH)
receptor antagonists
Seon-Mi Kim a, Minhee Lee a, So Young Lee b, Soo-Min Lee b, Eun Jeong Kim b, Jae Sun Kim c,
Jihyae Ann e, Jiyoun Lee d, Jeewoo Lee e,*
a R&D Center, TiumBio Company Ltd., Seongnam-si, Gyeonggi-do, 13493, Korea
b
Life Science R&D Center, SK Chemicals Company Ltd., Seongnam-si, Gyeonggi-do, 13494,
Korea
c J2H Biotech Company, Ansan-si, Gyeonggi-do, 15426, Korea
d Department of Global Medical Science, Sungshin University, Seoul 01133, Korea
e Laboratory of Medicinal Chemistry, College of Pharmacy, Seoul National University, Seoul 08826,
Korea
* To whom correspondence should be addressed.
Phone: 82-2-880-7846
Fax: 82-2-888-0649
E-mail: jeewoo@snu.ac.kr

ACCEPTED MANUSCRIPT
Keywords:
Gonadotropin-releasing hormone receptor, GnRH antagonist, luteinizing hormone, endometriosis
Abstract
We investigated a series of uracil analogues by introducing various substituents on the phenyl ring
of the N-3 aminoethyl side chain and evaluated their antagonistic activity against human
gonadotropin-releasing hormone (GnRH) receptors. Analogues with substituents at the ortho or
meta position demonstrated potent in vitro antagonistic activity. Specifically, the introduction of a
2-OMe group enhanced nuclear factor of activated T-cells (NFAT) inhibition up to 6-fold compared
to the unsubstituted analogue. We identified compound 12c as a highly potent GnRH antagonist
with moderate CYP inhibition. Compound 12c showed potent and prolonged LH suppression after a
single dose was orally administered in castrated monkeys compared to a known antagonist, Elagolix.
We believe that our SAR study offers useful insights to design GnRH antagonists as a potential
treatment option for endometriosis.

ACCEPTED MANUSCRIPT
1. Introduction
Endometriosis is a progressive disease mainly affecting premenopausal women, with a
prevalence rate of 5 - 10% [1]. The disease is histologically characterized by the presence of
endometrial tissue outside of the uterine cavity where the tissue grows in response to the cyclic
rhythm of ovarian sex hormones [2]. Depending on the affected site, patients suffer from pelvic pain,
dysmenorrhea, dyspareunia and infertility [3]. Surgery is a preferred treatment option that can
improve pregnancy rate and relieve the endometriosis-associated pain. However, the high
recurrence of endometriosis leads to reoperation in 50% of the patients within 5 years [4], requiring
continuous management of the patients until they reach menopause. Suppression of ovulation
and/or estrogens by the administration of combined oral contraceptive pills (OCPs) can prevent
recurring endometriosis and maintain the endometrial tissue as thin and compact [1]. However,
OCPs are associated with side effects such as breakthrough bleeding, weight gain, fluid retention,
depression and venous thromboembolism. Other agents such as progestational agents, which are
similar to combined OCPs in mechanistic ways, also exhibit similar adverse effects such as venous
thromboembolism. Human gonadotropin-releasing hormone (GnRH) receptor agonists are another
therapeutic option to maintain a hypoestrogenic state by suppressing the hypothalamic-pituitary-
ovarian axis. However, with an excessively low estrogen level, they cause serious side effects such
as reduction of bone mineral density, which limits the period of their use or necessitates an
additional “add-back therapy” with low dose estrogen [1].
According to the estrogen threshold hypothesis proposed by Barbieri, tissues vary in their
sensitivity to estrogen. For example, an extremely low estrogen level induces bone loss; however,
only a slight elevation of estrogen can restore bone turnover without stimulating the growth of
endometriotic lesions. Such a difference suggests an optimal therapeutic window of 30-45 pg/ml
estradiol for endometriosis which induces atrophy of endometriotic cells without any impact on
bone loss [5]. Given the hierarchy of organ response to estradiol, GnRH antagonists are considered
to be a better alternative due to their partial suppression of estradiol. The hypothesis has been

ACCEPTED MANUSCRIPT
verified by recent clinical trials for an oral GnRH antagonist [6]. In phase III trials, small-molecule
GnRH antagonists appear to have advantages over GnRH agonists in terms of convenience in the
dosing regimen and control of estrogen level.
Elagolix (1a) is an orally available GnRH antagonist currently in phase III trials for the
treatment of endometriosis and uterine fibroids (Figure 1). Elagolix demonstrated improved
potency and CYP3A4 inhibition compared to its parent compound, NBI-42902 (1b) [7, 8]. While
elagolix is the most advanced compound developed to date, given its modest oral bioavailability
and rapid metabolism [9], expanding the compound library with improved pharmacokinetic
parameters will aid in finding better clinical candidates. We previously developed a series of uracil
derivatives containing the N-substituted piperazines at the 5-position and identified SKI2496 (1c) as
a promising candidate with an improved oral bioavailability [10]. As a continued effort to diversify
GnRH antagonists based on a uracil scaffold, we decided to shift our focus on the phenyl ring at the
N-3 position of the uracil core. While previously reported SAR studies indicated that the 3-
aminoethyl group is particularly important for metabolic stability [11, 12], relatively few analogues
have been investigated regarding the SAR of the phenyl ring, probably due to synthetic difficulties.
In this study, we synthesized a series of uracil derivatives with various nonaromatic or substituted
aromatic rings at the 3-position of the uracil core that also contained the 5-piperazinyl substituents.
We evaluated the biological activity of the compound library by measuring the binding affinity and
NFAT promoter inhibition for GnRH receptors in vitro. In addition, we determined the
pharmacokinetic and pharmacodynamic profiles of the selected compound in animal models to
assess in vivo antagonistic effects including luteinizing hormone (LH) suppression and species
sensitivity.

ACCEPTED MANUSCRIPT
O
O
F
F
O
O
N
O
O
N
N
CF3
4
5
N
F
N
3 N
N
6
NH
NH2
NH
O
O
2
N1
O
F
F
CO2H
CO2H
F3C
Elagolix (1a)
F
F3C
1b
NBI-42902 (
)
1c
)
SKI2496 (
Figure 1. Examples of small-molecule GnRH antagonists
2. Results and discussion
2.1 Chemistry
Syntheses of 3-(2-aminoethyl) uracil derivatives began with compound 2, which was
prepared according to previously described procedures [13]. After N-PMB protection of compound
2, the Suzuki coupling reaction of the 5-bromine with 2-fluoro-3-methoxyphenylboronic acid was
performed, followed by deprotection of the N-PMB group using aluminum chloride to yield
compound 3. To diversify the N-3 position of the uracil core, we synthesized the N-Boc-protected
amino alcohol fragment 4 by reducing commercially available unnatural amino acids and protecting
the α-amino group. The amino alcohol fragment 4 was then introduced into the intermediate 3 by
performing the Mitsunobu reaction. Subsequent deprotection of the N-Boc group yielded the
corresponding amine 6 (Scheme 1).

ACCEPTED MANUSCRIPT
Scheme 1. Synthesis of 3-(2-2-aminoethyl) uracil derivatives
O
N
F
O
N
F
O
N
F
R1
Br
N
O
HN
a
HN
O
c
BocHN
F
F
O
O
O
R1
F
F
F
OH
NHBoc
4
2
3
5
b
O
O
N
R1
R1
OH
N
O
d
NH2
H2N
F
O
F
F
6
o
Reagents and coniditions: (a) i) p-methoxybenzyl chloride, K2CO3, DMF, 60 C, 3 h, 56%, ii) 2-
fluoro-3-methoxyphenylboronic acid, Pd(PPh3)4, saturated aq. Ba(OH)2, benzene:EtOH:DME =
45:5:50, 85 oC, 2 days, 55%, iii) AlCl3, anisole, r.t, 3 h, 93%; (b) i) LiAlH4, THF, 70~80 oC, 3~4 h,
37~58%, ii) (Boc)2O, diisopropylethylamine, CH2Cl2, r.t., overnight, 22~86%; (c) PBu3, DEAD,
DMF, 80 oC, 10~42%; (d) TFA, CH2Cl2, r.t., 3h, 52~81%
To synthesize the 5-piperazinyl uracil derivatives, substitution of N-benzylpiperazine at the
5-position of compound 7, followed by removal of the benzyl group yielded compound 8.
Alkylation of 8 with the proper halide afforded two intermediates 9 and 10, which were subjected to
N-alkylation with various mesylates 4a and subsequent Boc deprotection to give the corresponding
amines 11. The amine compounds were further alkylated with ethyl 4-bromobutyrate, followed by
hydrolysis with aqueous sodium hydroxide to yield the acids 12 (Scheme 2).

ACCEPTED MANUSCRIPT
Scheme 2. Synthesis of 5-(4-piperazinyl) uracil derivatives
R2
X
O
N
O
O
NH
N
Br
N
HN
HN
b
a
HN
O
N
O
N
O
N
F
+
F
OR3
NHBoc
F
CF3
CF3
CF3
9
10
4a
R2 = 3-NO2Ph
R2 = 5-CF3furyl
R3 = Ms
R3 = H
7
8
c
R2
R2
O
N
O
N
N
X
X
N
N
N
N
d
e
HO2C
NH
H2N
O
N
O
F
F
CF3
CF3
11
12
Reagents and conditions: (a) i) benzylpiperazine, MeCN, 120 oC, 2 h, microwave, 48%, ii) Pd/C, H2,
MeOH/CH2Cl2, r.t., 3 h, 87% (b) R2CH2Br, DIPEA, CH2Cl2, 2 h, r.t, 77~89%; (c) MsCl, TEA,
CH2Cl2, 100%; (d) i) K2CO3, DMF, 70 oC, overnight, 65%, ii) TFA, CH2Cl2, r.t., 3 h, 65~85%; (e) i)
Br(CH2)3CO2Et, DIPEA, NaI, MeCN, 95 oC, overnight, 33%, ii) 1N-NaOH, EtOH, 50 oC, overnight,
79%
2.2 Structure-Activity Relationship
To evaluate the biological activity of the synthesized uracil analogues, we determined their
in vitro binding affinities for the human GnRH receptors using the radiolabeled peptide, [125I]
D-
Trp6-LHRH. We first wanted to determine whether the modification of the phenyl ring at the N-3
position of the uracil core affected the binding affinity. As described in Table 1, all of the
heterocyclic analogues (6a-6g) showed reduced binding affinities, regardless of aromaticity, except
for the 2-furyl analogue (6f). As demonstrated by two enantiomers of the 2-furyl derivatives (6f, 6g),
stereochemistry is also an important factor at this specific position. Analogues containing a phenyl
ring (6h-6o) varied in binding affinity depending on the substituents. For example, compounds with
at least one electron-withdrawing group (6h-6j, and 6m) demonstrated comparable activities to that

ACCEPTED MANUSCRIPT
of compound 1a, except 2-trifluoromethyl and 3-fluoromethoxy analogues (6n, 6o). Compound 6i,
which had 2-OMe and 5-F substituents, demonstrated the most potent binding affinity in this series
(IC50 = 0.29 nM). Given that 6i was tested as a racemic mixture, we believe that the specific
stereoisomer would be more potent. On the other hand, benzyl analogues (6p-6v) generally
demonstrated weaker binding affinities, with IC50 values ranging from 9 to 62.3 nM.
Table 1. SAR of 3-(2-aminoethyl) uracil compounds
O
F
O
R1
N
*
H2N
F
O
N
F
hGnRH-R binding
Compound
R1
Stereochemistry
IC50 (nM)
0.59
1a
6a
R
R
> 1000
> 100
49.5
O
6b
6c
6d
RS
RS
>1000
RS
S
69.1
2.3
6e
6f
R
73.3
6g
RS
RS
RS
1.21
0.29
0.74
6h
6i
6j
RS
RS
20.4
5.4
6k
6l

ACCEPTED MANUSCRIPT
RS
RS
RS
0.38
6m
6n
6o
19.1
7.5
RS
16
6p
RS
RS
RS
RS
46
62.3
11.3
9
6q
6r
6s
6t
RS
RS
6.2
6u
6v
17.2
Because several analogues in this series demonstrated potent binding affinity that was
comparable to 1a, we next wanted to incorporate these modifications with the 5-piperazinyluracil
moiety that showed superior potency and bioavailability in our previous study [10]. We selected 4-
(3-nitrobenzyl)-piperazinyl (11a-11l) and 4-((5-(trifluoromethyl)-furan-2-yl)methyl)piperazinyl)
groups (11m-11q, and 12a-12c) for further derivatization of the 5-position. We additionally
replaced the 1-(2,6-difluoro)benzyl group at the N-1 position with a 1-(2-fluoro-6-
trifluoromethyl)benzyl group, which was present in compound 1a and led to a more potent
antagonistic effect than that observed for 1b [14]. For these derivatives (compounds 11a-11q and
12a-12c), we assessed the antagonistic activity by performing a reporter gene assay that measures
the inhibition of NFAT (nuclear factor of activated T-cells) activation. As described in Table 2,
among the analogues with 4-(3-nitrobenzyl)-piperazinyl group (11a-11l), compounds 11i (X = 2-
methoxy, 5-F) and 11k (X = 3-methyl, 5-F) showed 5- to 7-fold higher activities than 11a (X = H).

ACCEPTED MANUSCRIPT
It is interesting to note that compounds with these particular substituents in the previous series (6i
and 6j) also demonstrated potent in vitro binding affinities. While most of the compounds were
tested as a racemic mixture, it appears that the (R)-stereoisomers (11i and 11k) are more potent than
the (S)-stereoisomer (11j and 11l). The four substituents showing the most potent NFAT inhibition,
such as 2-OMe, 2-OH, (2-OMe, 5-F) and (3-Me, 5-F) were additionally incorporated into the 5-(4-
((5-(trifluoromethyl)-furan-2-yl)methyl)piperazinyl)uracil core as (R)-stereoisomers (compounds
11m-11q), and these derivatives demonstrated potent NFAT inhibition, having IC50 values in the
low nanomolar range (1.54 – 15.2 nM). Specifically, compounds 11n, 11o, and 11p exhibited equal
or higher activities than the unsubstituted analogue (11m, IC50 = 8.96 nM) while 11n (X = 2-OMe)
was the most potent (IC50 = 1.54 nM).
On the other hand, when these potent compounds were tested for CYP inhibition, they also
strongly inhibited the CYP3A4 enzyme. Given that elagolix (1a) was modified from NBI-42902
(1b) to improve the CYP inhibition, this result is not surprising. Therefore, we added a butyric acid
group at the amine group of the N-3 side chain, which is the same functional group as that in
elagolix. The strategy, when applied to our compounds, was also effective, significantly reducing
the CYP3A4 inhibition. While compounds 12a (X = 2-OMe) and 12b (X = 2-OMe, 5-F)
significantly lost their antagonistic activities, compound 12c (X = 3-Me, 5-F) maintained its activity
and demonstrated relatively weak CYP3A4 inhibition (30% at 10 µM), which is comparable to the
unsubstituted analogue (21% at 10 µM) in our previous study [10]. Therefore, we decided to focus
on 12c for further in vivo studies.

ACCEPTED MANUSCRIPT
Table 2. SAR of 5-(4-piperazinyl) uracil derivatives
R2
O
N
X
N
N
*
NH
R3
O
N
F
CF3
CYP3A4
inh. %
(1µM
Stereo-
chemist
ry
hGnRHR
luciferase
IC50 (nM)
Compoun
d
R2
R3
X
/10µM)
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
-H
-H
2-Me
2-OMe
2-OH
2-F
R
RS
R
165
1066
45
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
11a
11b
11c
11d
11e
11f
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
R
89
RS
RS
R
142
160
1682
144
32
3-Me
4-F
11g
11h
11i
3-F
RS
R
2-OMe,5-F
2-OMe,5-F
3-Me,5-F
3-Me,5-F
H
S
26%*
22
11j
R
11k
11l
S
35%*
8.96
O
CF3
R
11m

ACCEPTED MANUSCRIPT
O
O
O
O
O
O
O
CF3
CF3
CF3
CF3
CF3
CF3
CF3
-H
2-OMe
2-OMe,5-F
3-Me,5-F
2-OH
R
R
R
R
R
R
R
1.54
3.03
8.40
15.2
65.2
38.5
9.9
88 / ND
89 / ND
90 / ND
ND
11n
-H
11o
-H
11p
-H
11q
-(CH2)3CO2H
-(CH2)3CO2H
-(CH2)3CO2H
2-OMe
ND
12a
2-OMe,5-F
3-Me,5-F
ND / 28
ND / 30
12b
12c
* assayed at 0.4 µM
2.3. Interspecies selectivity
Some known small-molecule GnRH antagonists, including compounds 1a and 1b, were
reported to be highly specific to the human GnRH receptors [15, 16], which limits the use of certain
animal species for the in vivo evaluation of efficacy. We performed a competitive binding assay and
NFAT promoter inhibition assay to examine the species sensitivity of compound 12c (Table 3).
Compound 12c was more specific to the human GnRH receptors compared to those of monkeys and
rats. The binding affinity for the human GnRH receptors was 12-fold greater than that for the
monkey receptors, while the NFAT promotor inhibitory activity for the human GnRH receptors was
34-fold higher than that for the rat receptors.
Table 3. Specificities for human, monkey, and rat GnRH receptors
Compounds
IC50 (nM)
Competitive binding
([125I]-DTrp6-LHRH)
Inhibition of
NFAT reporter activity
human
0.58
monkey
3.5
human
rat
1.6
590
1a (Elagolix)
0.46
0.33
3.8
4.2
6.3
4.9
279
170
1c
12c

ACCEPTED MANUSCRIPT
2.4 In vivo activity in castrated monkeys
To evaluate the functional antagonism of compound 12c in vivo, we measured LH
(luteinizing hormone) suppression in castrated monkeys after the administration of compound 12c
[17]. A single dose of compound 12c (30 mg/kg, po) was administered to castrated male
cynomolgus monkeys, and the blood samples were collected at the designated time points to
analyze circulating LH concentrations using a radioimmunoassay (RIA). As described in Figure 2,
maximal LH suppression (82% of basal level) was achieved at 8 h after the administration of 12c,
which was maintained for 24 h. Considering that compound 1a and 1b demonstrated maximal
suppression of 75% and 50% at 8 h and completely recovered the basal level after 32 h and 20 h
respectively [18], compound 12c appeared to exert more potent and prolonged antagonistic activity.
In addition, we measured the plasma levels of compound 12c as described in Table 4. Compound
12c showed a relatively high plasma level with a Cmax of 6.0 µg/mL and an AUC of 37.1 µg h mL−1,
which was also observed with the 5-piperazinyl uracil analogue in our previous study [10],
explaining the prolonged antagonistic activity of 12c.
Figure 2. Suppression of plasma LH concentrations in castrated male cynomolgus monkeys after
oral administration of compound 12c at 30 mg/kg. Values shown are the mean ±SEM of bioactive
LH levels expressed as a percentage of pretreatment LH levels for each of three individual animals.

ACCEPTED MANUSCRIPT
Table 4. Pharmacokinetic parameters of compound 12c in castrated monkeys after oral
administration (30 mg/kg)
Compound
Cmax (µM)
6.0±2.9
AUCinf (µM*hr)
37.1±20.7
Tmax (hr)
2.3±1.5
12c
3. Conclusion
In this study, we synthesized a series of uracil analogues as orally available GnRH
antagonists. By introducing various substituents in the phenyl ring of the N-3 side chain, we aimed
to explore potency and pharmacokinetic properties of these newly synthesized analogues. In
addition, we incorporated these results into the 5-piperazinyluracil core, which exhibited potent
activity and a favorable pharmacokinetic profile in our previous study. When the aromatic
substituents were introduced at the ortho or meta position, the in vitro antagonistic activity was
improved. In particular, the introduction of a 2-OMe group enhanced NFAT inhibition up to 6-fold
compared to the unsubstituted analogue. However, those analogues appeared to inhibit the CYP3A4
enzyme strongly, which can be overcome by adding a butyric acid group at the side chain amine
group of the N-3 position. We identified compound 12c, which is a highly potent GnRH antagonist
with moderate CYP inhibition. The antagonistic activity of 12c was specific to human GnRH
receptors, showing 12-fold and 34-fold higher activities than those detected towards monkey and rat
receptors respectively. When tested in castrated monkeys, compound 12c demonstrated more
profound and prolonged suppression in LH levels in castrated monkeys than Elagolix, while it
reached the maximum effect in shorter time (8 h) than the previously reported compound 1c (12 h)
[10]. We believe that our study offers useful insights to design orally available GnRH antagonists
with favorable pharmacokinetic properties.
4. Experimental
4.1. Chemistry

ACCEPTED MANUSCRIPT
1H NMR and 13C NMR spectra were recored on a Varian Unity 300, Jeol Oxford 600 at 300 and 150
MHz. Chemical shifts were given in ppm using tetramethylsilane as the reference standard, and
coupling constants (J) are given in hertz (Hz). Mass spectra were recorded on a Thermo LCQ
DECA XP instrument. Chromatographic separations were carried out on silica gel (Kieselgel 60,
230−400 mesh, Merck) or basic silica gel (Chromatorex NHDM1020, 100- 200 mesh, Fuji Silysia
Chemical Ltd.) using the indicated eluents. Yields were not optimized. All final compounds were
assessed for purity by high performance liquid chromatography (HPLC) on Agilent 1120 Compact
LC (G4288A) system via the following conditions. Column: Agilent TC-C18 column (4.6 mm ×
250 mm, 5 µm). Mobile phase A: 0.78% NH4OAc in water (v/v). Mobile phase B: MeCN. Gradient:
65.0% water/ 35.0% MeCN linear to 10% water/ 90% MeCN in 30 min. Wavelength: 272 nM. Flow:
1.0 mL/min. According to the HPLC analyses, all final compounds showed a purity of ≥95%.
4.1.1. Procedure for 5-phenyluracil preparation
4.1.1.1. 1-(2,6-difluorobenzyl)-5-(2-fluoro-3-methoxyphenyl)-6-methylpyrimidine-2,4(1H,3H)
-
dione (3). To a suspension of 2 (10 g, 30.2 mmol) and К2СО3 (8.35 g, 60.4 mmol) in DMF (125 mL)
was added p-methoxybenzyl chloride (4.39 mL, 31.7 mmol). After the mixture was stirred at 60 °С
for 3 h, it was cooled to room temperature. The reaction mixture was diluted with EtOAc (90 mL)
and washed with saturated NH4Cl (270 mL) and brine (100 mL), respectively. The organic layer
was dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by column
chromatography on silica gel using hexane/EtOAc/CH2Cl2 (1:4.5:1) as eluent to give red solid,
followd by recrystallization using EtOAc and hexane to yield 5-bromo-1-(2,6-difluorobenzyl)-3-(4-
methoxybenzyl)-6-methylpyrimidine-2,4(1H,3H)-dione (7.65 g, 56%) as white solid. The mixture
of the N-protected bromide (2.8 g. 6.21 mmol), 2-fluoro-3-methoxyphenylboronic acid (1.27 g, 7.45
mmol) and saturated barium hydroxide (1.08 g, 0.93 mmol) in benzene : EtOH : dimethoxyethane
(45:5:50) (90 mL) was bubbled under nitrogen for 30 min and then treated with Pd(PPh3)4, followed
by stirring at 85 °С for 2 days. The reaction mixture was cooled to room temperature, diluted with

ACCEPTED MANUSCRIPT
EtOAc (90 mL) and then washed with water (90 mL) and brine (90 mL), respectively. The organic
layer was dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by
column chromatography on amine silica gel using hexane/EtOAc (4:1) as eluent to obtain 1-(2,6-
difluorobenzyl)-5-(2-fluoro-3-methoxyphenyl)-3-(4-methoxybenzyl)-6-methylpyrimidine-
2,4(1H,3H)-dione (1.7 g, 55%) as white solid. The uracil (3.4 g, 6.85 mmol) in anisole (50 mL) was
treated with aluminum chloride (4.66 g, 34.2 mmol) under ice bath, followed by warming to room
temperature and stirring for 3h. The resulting red mixture was added dropwise to saturated NaHCO3
(70 mL) to give white suspension. EtOAc (100 mL) was added and the organic layer was taken up.
The inorganic layer was extracted with CH2Cl2/ MeOH (10:1) (100 mL) three times. The combined
organic layer was dissolved with CH2Cl2/ MeOH (10:1) (700 mL) and dried over Na2SO4, filtered,
and concentrated in vacuo. The resulting solid was triturated with EtOAc (20 mL) for 30min, then
filtered. The filtered residue was dried in vauco at 40 °С overnight to obtain compound 3 (2.41 g,
o
1
93%) as white solid. mp 72 C, H NMR (300 MHz, DMSO-d6) δ 11.57 (s, 1H), 7.51 – 7.39 (m,
1H), 7.24 – 7.10 (m, 4H), 6.80 – 6.73 (m, 1H), 5.25 (s, 2H), 3.89 (s, 3H), 2.12 (s, 3H).
4.1.2. General Procedure for N-Boc protected aminoalcohol preparation (4).
The suspension of amino acids (1 mmol) in THF (0.5 mL) was treated with LiAlH4 (2 mmol)
slowly under ice bath. The mixture was heated to 70~80 °С and stirred for 3hrs. After it was cooled
under ice bath, 4 drops of water was added, followed by treatment of potassium carbonate. The
resulting suspension was filtered, then the filtrate solution was concentrated in vacuo. The residue
was purified by column chromatography on silica gel using CH2Cl2/MeOH (20:1~10:1) as eluent to
gain the corresponding amino alcohols. The suspension of amino alcohols (1 mmol ) in CH2Cl2 (4
mL) were treated with DIPEA (3 mmol) and (Boc)2O (3 mmol), followed by stirring at room
temperature overnight. The reaction mixture was diluted with CH2Cl2 (4 mL) and washed with
satuated NH4Cl (4 mL) followed by concentration of the organic layer. The residue was purified by
column chromatography on silica gel using hexane/EtOAc/CH2Cl2 (2:1:0.5) as eluent or triturated

ACCEPTED MANUSCRIPT
with the eluent in cases of poor solubility.
4.1.3. General Procedure for Alkylation and Deprotection (6).
The suspension of compound 3 (1.1 mmol) and compound 4 (1 mmol) in DMF (9 mL) were treated
with PBu3 (1.5 mmol) and DEAD (1.5 mmol), then stirred at 80 °С for 3 h. The reaction mixture
was cooled to room temperature, diluted with CH2Cl2 (9 mL) and then washed with water (9 mL),
followed by concentration of the organic layer. The residue was purified by two rounds of column
chromatographies on silica gel and subsequently amine silica gel using hexane/EtOAc/CH2Cl2
(3:1:0.7~7:1:1) as eluent to gain compound 5. TFA (10 mL) was added slowly to the solution of
compound 5 (1 mmol) in CH2Cl2 (100 mL) and then stirred for 3h at room temperature. The
reaction mixture was quenched with saturated NaHCO3 under ice bath, which was extracted with
CH2Cl2 three times. The combined organic layer was concentrated, followed by column
chromatographies on silica gel and subsequently amine silica gel using CH2Cl2/MeOH (30:1~10:1)
as eluent.
4.1.3.1.
3-((2R)-2-amino-2-(tetrahydrofuran-3-yl)ethyl)-1-(2,6-difluorobenzyl)-5-(2-fluoro-3-
1
methoxyphenyl)-6-methylpyrimidine-2,4(1H,3H)-dione (6a). Yield 20%, colorless oil, H NMR
(300 MHz, CDCl3) δ 7.36-7.20 (m, 1H), 7.13-7.05 (m, 1H), 7.03-6.87 (m, 3H), 6.82-6.76 (m, 1H),
5.45-5.20 (m, 2H), 4.13-3.55 (m, 9H), 3.20-2.99 (m, 1H), 2.31-2.11 (m, 4H), 2.09-1.95 (m, 1H),
1.85-1.70 (m, 1H). MS (ESI) m/z 490 (MH+). Anal. HPLC 95% (Rt = 10.59 min).
4.1.3.2. (R)-3-(2-amino-2-(tetrahydro-2H-pyran-4-yl)ethyl)-1-(2,6-difluorobenzyl)-5-(2-fluoro -3-
1
methoxyphenyl)-6-methylpyrimidine-2,4(1H,3H)-dione (6b). Yield 8%, white foam, H NMR (300
MHz, CDCl3) δ 7.34-7.23 (m, 1H), 7.10 (td, J = 8.0, 1.4 Hz, 1H), 7.01-6.86 (m, 3H), 6.83-6.76 (m,
1H), 5.41-5.23 (m, 2H), 4.07-3.94 (m, 4H), 3.89 (s, 3H), 3.42-3.28 (m, 2H), 3.04-2.93 (m, 1H), 2.15
(s, 3H), 1.72 (d, J = 11.7 Hz, 2H), 1.62-1.36 (m, 4H). MS (ESI) m/z 504 (MH+). Anal. HPLC 97%

ACCEPTED MANUSCRIPT
(Rt = 10.14 min).
4.1.3.3. 3-(2-amino-2-(tetrahydro-2H-thiopyran-4-yl)ethyl)-1-(2,6-difluorobenzyl)-5-(2-fluoro -3-
1
methoxyphenyl)-6-methylpyrimidine-2,4(1H,3H)-dione (6c). Yield 15%, white foam, H NMR (300
MHz, CDCl3) δ 7.34-7.23 (m, 1H), 7.14-7.06 (m, 1H), 7.01-6.87 (m, 3H), 6.82-6.76 (m, 1H), 5.40-
5.22 (m, 2H), 4.14-3.93 (m, 2H), 3.89 (s, 3H), 3.00 (dt, J = 8.6, 4.4 Hz, 1H), 2.76-2.55 (m, 4H),
2.15 (m, 4H), 2.05-2.93 (m, 1H), 1.73-1.30 (m, 3H). MS (ESI) m/z 520 (MH+). Anal. HPLC 95%
(Rt = 10.96 min).
4.1.3.4. 3-(2-amino-2-(1-methylpiperidin-4-yl)ethyl)-5-(2-fluoro-3-methoxyphenyl)-1-(2-fluoro-6-
(trifluoromethyl)benzyl)-6-methylpyrimidine-2,4(1H,3H)-dione compound (6d). Yield 34%. white
1
foam, H NMR (300 MHz, CDCl3) δ 7.34-7.22 (m, 1H), 7.14-7.06 (m, 1H), 7.01-6.86 (m, 3H),
6.88-6.75 (m, 1H), 5.40-5.22 (m, 2H), ), 4.09-3.94 (m, 2H), 3.89 (s, 3H), 3.07-2.88 (m, 4H), 2.29 (s,
3H), 2.15 (s, 3H), 2.04-1.19 (m, 5H). MS (ESI) m/z 517 (MH+). Anal. HPLC 97% (Rt = 14.45 min).
4.1.3.5. 3-(2-amino-2-(pyridin-3-yl)ethyl)-1-(2,6-difluorobenzyl)-5-(2-fluoro-3-methoxy phenyl)-6-
1
methylpyrimidine-2,4(1H,3H)-dione (6e). Yield 10%, colorless oil, H NMR (300 MHz, CDCl3) δ
8.61 (dd, J = 4.9, 2.3 Hz, 1H), 8.49 (dt, J = 4.9, 1.8 Hz, 1H), 7.78 (dq, J = 7.3, 2.3 Hz, 1H), 7.35-
7.20 (m, 2H), 7.15-7.05 (m, 1H), 7.02-6.87 (m, 3H), 6.81 (ddd, J = 7.7, 6.0, 1.6 Hz, 0.5H), 6.72
(ddd, J = 7.8, 6.1, 1.6 Hz, 0.5H), 5.40-5.19 (m, 2H), 4.50-4.42 (m, 1H), 4.26 (ddd, J = 13.0, 8.8, 6.8
Hz, 1H), 4.15 (ddd, J = 12.9, 7.3, 5.4 Hz, 1H), 3.89 (s, 3H), 2.15 (s, 3H). MS (ESI) m/z 497 (MH+).
Anal. HPLC 99% (Rt = 14.52 min).
4.1.3.6. (S)-3-(2-amino-2-(furan-2-yl)ethyl)-1-(2,6-difluorobenzyl)-5-(2-fluoro-3-methoxy phenyl)-
6-methylpyrimidine-2,4(1H,3H)-dione (6f). Yield 41%, colorless oil, 1H NMR (300 MHz, CDCl3) δ
7.34-7.21 (m, 2H), 7.13-7.05 (m, 1H), 7.01-6.86 (m, 3H), 6.85-6.75 (m, 1H), 6.28-6.24 (m, 1H),

ACCEPTED MANUSCRIPT
6.16 (dq, J = 3.3, 0.7 Hz, 1H), 5.38-5.20 (m, 2H), 4.45-4.28 (m, 2H), 4.22 (ddd, J = 12.3, 5.9, 5.1
Hz, 1H), 3.89 (s, 3H), 2.16 (m, 3H). MS (ESI) m/z 486 (MH+). Anal. HPLC 95% (Rt = 11.78 min).
4.1.3.7. (R)-3-(2-amino-2-(furan-2-yl)ethyl)-1-(2,6-difluorobenzyl)-5-(2-fluoro-3-methoxy phenyl)-
6-methylpyrimidine-2,4(1H,3H)-dione (6g). Yield 38%, white foam, 1H NMR (300 MHz, CDCl3) δ
7.34-7.21 (m, 2H), 7.13-7.05 (m, 1H), 7.01-6.86 (m, 3H), 6.85-6.75 (m, 1H), 6.28-6.24 (m, 1H),
6.16 (dq, J = 3.3, 0.7 Hz, 1H), 5.38-5.20 (m, 2H), 4.45-4.28 (m, 2H), 4.22 (ddd, J = 12.3, 5.9, 5.1
Hz, 1H), 3.89 (s, 3H), 2.16 (m, 3H). MS (ESI) m/z 486 (MH+). Anal. HPLC 96% (Rt = 11.55 min).
4.1.3.8.
3-(2-amino-2-(3,5-dichlorophenyl)ethyl)-1-(2,6-difluorobenzyl)-5-(2-fluoro-3-
methoxyphenyl)-6-methylpyrimidine-2,4(1H,3H)-dione (6h). Yield 12%, white foam, 1H NMR (300
MHz, CDCl3) δ 7.34-7.21 (m, 4H), 7.13-7.05 (m, 1H), 7.01-6.86 (m, 3H), 6.85-6.75 (m, 1H), 5.33-
5.26 (m, 2H), 4.45-4.33 (m, 1H), 4.25-4.11 (m, 1H), 4.09-4.01 (m, 1H), 3.89 (s, 3H), 2.16 (s, 3H).
MS (ESI) m/z 564 (MH+). Anal. HPLC 99% (Rt = 16.53 min).
4.1.3.9.
3-(2-amino-2-(5-fluoro-2-methoxyphenyl)ethyl)-1-(2,6-difluorobenzyl)-5-(2-fluoro-3-
1
methoxyphenyl)-6-methylpyrimidine-2,4(1H,3H)-dione (6i). Yield 20%, white foam, H NMR (300
MHz, CDCl3) δ 7.34-7.21 (m, 1H), 7.13-7.05 (m, 1H), 7.01-6.71 (m, 7H), 5.33-5.26 (m, 2H), 4.53-
4.45 (m, 1H), 4.38-4.26 (m, 1H), 4.22-4.20 (m, 1H), 3.88 (s, 3H), 3.80 (s, 3H), 2.12 (s, 3H). MS
(ESI) m/z 544 (MH+). Anal. HPLC 99% (Rt = 12.72 min).
4.1.3.10.
3-(2-amino-2-(3-fluoro-5-methylphenyl)ethyl)-1-(2,6-difluorobenzyl)-5-(2-fluoro-3-
o
1
methoxyphenyl)-6-methylpyrimidine-2,4(1H,3H)-dione (6j). Yield 9%, white solid, mp 70 C, H
NMR (300 MHz, CDCl3) δ 7.34-7.21 (m, 1H), 7.13-7.05 (m, 1H), 7.01-6.71 (m, 7H), 5.33-5.26 (m,
2H), 4.45-4.32 (m, 1H), 4.27-4.17 (m, 1H), 4.09-4.05 (m, 1H), 3.89 (s, 3H), 2.30 (s, 3H), 2.16 (s,
3H). MS (ESI) m/z 528 (MH+). Anal. HPLC 99% (Rt = 14.25 min).

ACCEPTED MANUSCRIPT
4.1.3.11.
3-(2-amino-2-(4-(methylthio)phenyl)ethyl)-1-(2,6-difluorobenzyl)-5-(2-fluoro-3-
1
methoxyphenyl)-6-methylpyrimidine-2,4(1H,3H)-dione (6k). Yield 7%, white foam, H NMR (300
MHz, CDCl3) δ 7.40-7.19 (m, 5H), 7.13-7.05 (m, 1H), 7.01-6.71 (m, 4H), 5.36-5.27 (m, 2H), 4.45-
4.32 (m, 1H), 4.27-4.17 (m, 1H), 4.12-4.06 (m, 1H), 3.89 (s, 3H), 2.46 (s, 3H), 2.15 (s, 3H). MS
(ESI) m/z 542 (MH+). Anal. HPLC 98% (Rt = 13.90 min).
4.1.3.12.
3-(2-amino-2-(2,4-dimethylphenyl)ethyl)-1-(2,6-difluorobenzyl)-5-(2-fluoro-3-
o
1
methoxyphenyl)-6-methylpyrimidine-2,4(1H,3H)-dione (6l). Yield 11%, white solid, mp 68 C, H
NMR (300 MHz, CDCl3) δ 7.40-7.19 (m, 2H), 7.13-7.05 (m, 1H), 7.03-6.80 (m, 6H), 5.36-5.27 (m,
2H), 4.63-4.56 (m, 1H), 4.25-4.17 (m, 1H), 4.02-3.98 (m, 1H), 3.89 (s, 3H), 2.40 (s, 3H), 2.28 (s,
3H), 2.14 (s, 3H). MS (ESI) m/z 524 (MH+). Anal. HPLC 95% (Rt = 14.62 min).
4.1.3.13.
3-(2-amino-2-(2-chlorophenyl)ethyl)-1-(2,6-difluorobenzyl)-5-(2-fluoro-3-methoxy
o
1
phenyl)-6-methylpyrimidine-2,4(1H,3H)-dione (6m). Yield 6%, white solid, mp 72 C, H NMR
(300 MHz, CDCl3) δ 7.54-7.51 (m, 1H), 7.37-7.07 (m, 5H), 6.98-6.75 (m, 4H), 5.36-5.27 (m, 2H),
4.87-4.84 (m, 1H), 4.32-4.22 (m, 2H), 3.89 (s, 3H), 2.14 (s, 3H). MS (ESI) m/z 530 (MH+). Anal.
HPLC 96% (Rt = 13.59 min).
4.1.3.14.
3-(2-amino-2-(2-(trifluoromethyl)phenyl)ethyl)-1-(2,6-difluorobenzyl)-5-(2-fluoro-3-
1
methoxyphenyl)-6-methylpyrimidine-2,4(1H,3H)-dione (6n). Yield 8%, white foam, H NMR (300
MHz, CDCl3) δ 7.82-7.79 (m, 1H), 7.59-7.51 (m, 2H), 7.39-7.24 (m, 2H), 7.17-7.05 (m, 1H), 6.98-
6.75 (m, 4H), 5.36-5.27 (m, 2H), 4.85-4.75 (m, 1H), 4.45-4.22 (m, 2H), 3.89 (s, 3H), 2.13 (m, 3H).
MS (ESI) m/z 564 (MH+). Anal. HPLC 96% (Rt = 14.63 min).
4.1.3.15.
3-(2-amino-2-(3-(trifluoromethoxy)phenyl)ethyl)-1-(2,6-difluorobenzyl)-5-(2-fluoro-3-

ACCEPTED MANUSCRIPT
1
methoxyphenyl)-6-methylpyrimidine-2,4(1H,3H)-dione (6o). Yield 28%, yellow foam, H NMR
(300 MHz, CDCl3) δ 7.49-6.75 (m, 10H), 5.36-5.08 (m, 2H), 4.65-4.30 (m, 2H), 4.08-3.82 (m, 4H),
2.09 (s, 3H). MS (ESI) m/z 580 (MH+). Anal. HPLC 98% (Rt = 15.96 min).
4.1.3.16.
3-(2-amino-3-(2-chloro-6-fluorophenyl)propyl)-1-(2,6-difluorobenzyl)-5-(2-fluoro-3-
methoxyphenyl)-6-methylpyrimidine-2,4(1H,3H)-dione (6p). Yield 17%, white foam, 1H NMR (300
MHz, CDCl3) δ 7.27-6.75 (m, 9H), 5.36-5.25 (m, 2H), 4.21-4.06 (m, 2H), 3.59-3.46 (m, 1H), 2.95-
2.74 (m, 2H), 2.15 (s, 3H). MS (ESI) m/z 562 (MH+). Anal. HPLC 97% (Rt = 15.03 min).
4.1.3.17.
3-(2-amino-3-(3-fluorophenyl)propyl)-1-(2,6-difluorobenzyl)-5-(2-fluoro-3-methoxy
1
phenyl)-6-methylpyrimidine-2,4(1H,3H)-dione (6q). Yield 48%, white foam, H NMR (300 MHz,
CDCl3) δ 7.34-7.19 (m, 2H), 7.12-7.07 (m, 1H), 6.99-6.79 (m, 7H), 5.39-5.25 (m, 2H), 4.04 (d, , J =
3.9Hz, 2H), 3.88 (s, 3H), 3.51-3.46 (m, 1H), 2.88-2.82 (m, 1H), 2.59-2.56 (m, 1H), 2.14 (s, 3H).
MS (ESI) m/z 528 (MH+). Anal. HPLC 98% (Rt = 13.91 min).
4.1.3.18.
3-(2-amino-3-(2-chloro-4-fluorophenyl)propyl)-1-(2,6-difluorobenzyl)-5-(2-fluoro-3-
methoxyphenyl)-6-methylpyrimidine-2,4(1H,3H)-dione (6r). Yield 29%, white foam, 1H NMR (300
MHz, CDCl3) δ 7.34-7.24 (m, 2H), 7.16-7.09 (m, 2H), 7.02-6.90 (m, 4H), 6.85-6.80 (m, 1H), 5.37-
5.25 (m, 2H), 4.09 (d, J = 4.2Hz, 2H), 3.91 (s, 3H), 3.53-3.46 (m, 1H), 3.02-2.91 (m, 1H), 2.75-2.67
(m, 1H), 2.18 (s, 3H). MS (ESI) m/z 562 (MH+). Anal. HPLC 98% (Rt = 15.43 min).
4.1.3.19.
3-(2-amino-3-(2-fluoro-4-(trifluoromethyl)phenyl)propyl)-1-(2,6-difluorobenzyl)-5-(2-
fluoro-3-methoxyphenyl)-6-methylpyrimidine-2,4(1H,3H)-dione (6s). Yield 7%, white solid, mp 60
oC, 1H NMR (300 MHz, CDCl3) δ 7.34-7.24 (m, 2H), 7.19-6.77 (m, 7H), 5.43-5.25 (m, 2H), 4.09 (d,
J = 4.2Hz, 2H), 3.91 (s, 3H), 3.53-3.43 (m, 1H), 2.87-2.82 (m, 1H), 2.75-2.67 (m, 1H), 2.59-2.51 (m,
1H), 2.18 (s, 3H). MS (ESI) m/z 596 (MH+). Anal. HPLC 99% (Rt = 14.53 min).

ACCEPTED MANUSCRIPT
4.1.3.20.
3-(2-amino-3-(3-(trifluoromethyl)phenyl)propyl)-1-(2,6-difluorobenzyl)-5-(2-fluoro-3-
o
1
methoxyphenyl)-6-methylpyrimidine-2,4(1H,3H)-dione (6t). Yield 8%, white solid, mp 62 C, H
NMR (300 MHz, CDCl3) δ 7.34-7.12 (m, 5H), 7.08-6.84 (m, 5H), 5.43-5.25 (m, 2H), 4.21-4.08 (m,
2H), 3.91 (s, 3H), 3.57-3.50 (m, 1H), 3.02-2.82 (m, 2H), 2.18 (s, 3H). MS (ESI) m/z 578 (MH+).
Anal. HPLC 99% (Rt = 15.14 min).
4.1.3.21.
3-(2-amino-3-(3,5-difluorophenyl)propyl)-1-(2,6-difluorobenzyl)-5-(2-fluoro-3-
o
1
methoxyphenyl)-6-methylpyrimidine-2,4(1H,3H)-dione (6u). Yield 5%, white solid, mp 65 C, H
NMR (300 MHz, CDCl3) δ 7.34-7.12 (m, 3H), 7.10-6.78 (m, 6H), 5.43-5.25 (m, 2H), 4.12 (d, J =
4.2Hz, 2H), 3.91 (s, 3H), 3.57-3.44 (m, 1H), 2.98-2.82 (m, 1H), 2.58-2.49 (m, 1H), 2.18 (s, 3H).
MS (ESI) m/z 546 (MH+). Anal. HPLC 99% (Rt = 15.06 min).
4.1.3.22.
3-(2-amino-3-(thiophen-2-yl)propyl)-1-(2,6-difluorobenzyl)-5-(2-fluoro-3-methoxy
1
phenyl)-6-methylpyrimidine-2,4(1H,3H)-dione (6v). Yield 38%, white foam, H NMR (300 MHz,
CDCl3) δ 7.34-7.07 (m, 3H), 6.99-6.78 (m, 6H), 5.39-5.24 (m, 2H), 4.06 (d, J = 6.6 Hz, 2H), 3.88 (s,
3H), 3.54-3.44 (m, 1H), 3.10-3.02 (m, 1H), 2.87-2.75 (m, 1H), 2.15 (s, 3H). MS (ESI) m/z 516
(MH+). Anal. HPLC 99% (Rt = 13.19 min).
4.1.4. Procedure for 5-piperazinyluracil preparation
4.1.4.1. 1-(2-fluoro-6-(trifluoromethyl)benzyl)-6-methyl-5-(piperazin-1-yl)pyrimidine-2,4(1H,3H)-
dione (8). The mixture of compound 7 (1.50 g, 3.94 mmol) and 1-benzylpiperazine (5.5 mL, 31.5
mmol) in acetonitrile (1 mL) was reacted under . microwave irradiation at 120 °C for 1.5 h. The
mixture was concentrated, followed by purification using silica gel chromatography
(hexane/EtOAc/CH2Cl2, 1:2:1) to afford 4-benzyl piperazine adduct as a white solid (1.48 g, 79%).
1H NMR (300 MHz, CDCl3) δ 2.15 (m, 2H), 2.31 (s, 3H), 2.50 (m, 2H), 2.78 (m, 2H), 3.52 (s, 3H),

ACCEPTED MANUSCRIPT
3.58 (m, 2H), 5.36 (s, 2H), 7.19-7.41 (m, 7H), 7.53 (m, 1H). A solution of the above compound
(1.48 g, 3.11 mmol) dissolved in CH2Cl2/MeOH (1:1, 10 mL) was hydrogenated over 10% Pd/C
(280 mg) under atmospheric pressure at room temperature for 5 h. The mixture was filtered through
celite, followed by concentration of the filtrate solution. The residue was purified using column
chromatography using amine silica gel (CH2Cl2/MeOH, 10:1) to yield compound 8 as white solid
o
1
(934 mg, 78%). mp 78 C, H NMR (300 MHz, CDCl3) δ 7.54 (d, J = 7.8 Hz, 1H), 7.41 (m, 1H),
7.24 (m, 1H), 5.38 (s, 2H), 3.43 (m, 2H), 2.96 (m, 2H), 2.81 (m, 2H), 2.52 (m, 2H), 2.33 (s, 3H).
4.1.5. General Procedure for Alkylation (9, 10).
A solution of compound 8 (1 mmol) and N,N-diisopropylethylamine (5 mmol), in 1,2-
dichloroethane (10 mL) was treated with 3-nitrobenzyl bromide (1.5 mmol), then stirred at 50 °C
for 2 h. The mixture was cooled to room temperature and washed with saturated ammonium
chloride solution and concentrated in vacuo. The residue was purified by column chromatography
using silica gel (CH2Cl2/MeOH, 30:1).
4.1.5.1.
1-(2-fluoro-6-(trifluoromethyl)benzyl)-6-methyl-5-(4-(3-nitrobenzyl)piperazin-1-
o
1
yl)pyrimidine-2,4(1H,3H)-dione (9). Yield 77%, yellowish solid, mp 74 C, H NMR (300 MHz,
CDCl3) δ 8.57 (s, 1H), 8.16 (s, 1H), 8.10 (d, J = 8.1 Hz, 1H), 7.77 (d, J = 7.5 Hz, 1H), 7.41 (m, 1H),
7.55-7.46 (m, 2H), 7.23 (m, 1H), 5.37 (s, 2H), 3.66-3.60 (m, 4H), 2.75 (m, 2H), 2.52 (m, 2H), 2.32
(s, 3H), 2.20 (m, 2H).
4.1.5.2.
1-(2-fluoro-6-(trifluoromethyl)benzyl)-6-methyl-5-(4-((5-(trifluoromethyl)furan-2-
o
yl)methyl)piperazin-1-yl)pyrimidine-2,4(1H,3H)-dione (10). Yield 89%, white solid, mp 83 C, 1H
NMR (300 MHz, CDCl3) δ 8.57 (s, 1H), 7.54 (d, J = 7.8 Hz, 1H), 7.41 (m, 1H), 7.24 (m, 1H), 6.72
(d, J = 3.0 Hz, 1H), 6.29 (d, J = 3.0 Hz, 1H), 5.40 (s, 2H), 3.72-3.49 (m, 4H), 2.81 (m, 2H), 2.54 (m,
2H), 2.27 (s, 3H), 2.26 (m, 2H).

ACCEPTED MANUSCRIPT
4.1.6. General Procedure for Alkylation and Deprotection (11).
To a solution of compound 4 (1 mmol) in CH2Cl2 (3 mL) were added trimethylamine (1.3 mmol)
and methansulfonylchloride (1.1 mmol). After stirring at room temperature for 30 min, the mixture
was diluted with dichloromethane and washed with saturated sodium bicarbonate solution. The
organic layer was dried over sodium sulfate and filtered. After concentration, the filtrate was dried
in vacuo to give compound 4a. The mixture of compound 9 or 10 (1 mmol), the mesylate 4a
(2.5mmol) and K2CO3 (5mmol) in DMF (10 mL) was stirred at 70 °C overnight. The reaction
mixture was cooled to ambient temperature, diluted with ethyl acetate, and washed with saturated
ammonium chloride solution. The organic layer was concentrated, then the residue was purified
using silica gel and amine silica gel chromatography (hexane/EtOAc, 2:1). The alkylated compound
(1 mmol) in CH2Cl2 (40 mL) was treated with TFA (2 mL), stirring at room temperature for 3 h. The
reaction mixture was neutralized with saturated NaHCO3 and extracted with CH2Cl2 twice. The
organic layer was concentrated, then purified using silica gel chromatography (CH2Cl2/MeOH,
15:1~20:1).
4.1.6.1.
(R)-3-(2-Amino-2-phenylethyl)-1-(2-fluoro-6-(trifluoromethyl)-benzyl)-6-methyl-5-(4-(3-
nitrobenzyl)piperazin-1-yl)pyrimidine-2,4-(1H,3H)-dione (11a). Yield 50%, yellowish solid, mp 71
oC, 1H NMR (300 MHz, CDCl3) δ 8.17 (t, J = 1.8 Hz, 1H), 8.11 (dd, J = 8.1, 1.3 Hz, 1H), 7.77 (d, J
= 7.7 Hz, 1H), 7.55 (d, J = 7.8 Hz, 1H), 7.48 (t, J = 7.9 Hz, 1H), 7.44-7.29 (m, 5H), 7.26-7.18 (m,
2H), 5.41 (s, 2H), 4.37 (dd, J = 9.5, 4.9 Hz, 1H), 4.21 (dd, J = 12.9, 9.5 Hz, 1H), 4.06 (dd, J = 12.9,
4.9 Hz, 1H), 3.70-3.51 (m, 4H), 2.75 (d, J = 10.4 Hz, 2H), 2.49 (m, 2H), 2.34 (s, 3H), 2.21 (m, 2H).
MS (ESI) m/z 641 (MH+). Anal. HPLC 99% (Rt = 19.36 min).
4.1.6.2.
3-(2-amino-2-(o-tolyl)ethyl)-1-(2-fluoro-6-(trifluoromethyl)benzyl)-6-methyl-5-(4-(3-
1
nitrobenzyl)piperazin-1-yl)pyrimidine-2,4(1H,3H)-dione (11b). Yield 25%, white foam, H NMR

ACCEPTED MANUSCRIPT
(300 MHz, CDCl3) δ 8.17 (d, J = 1.8 Hz, 1H), 8.11 (dd, J = 7.2, 1.2 Hz, 1H), 7.76 (d, J = 7.8 Hz,
1H), 7.55-7.46 (m, 3H), 7.25-7.12 (m, 4H), 5.42 (s, 2H), 4.63 (dd, J = 9.9, 4.5 Hz, 1H), 4.21 (dd, J =
13.2, 9.9 Hz, 1H), 3.96 (dd, J = 13.2, 4.5Hz, 1H), 3.78-3.60 (m, 4H), 2.75 (d, J = 9.9 Hz, 2H), 2.51
(m, 2H), 2.46 (s, 3H), 2.32 (s, 3H), 2.25-2.18 (m, 2H). MS (ESI) m/z 655 (MH+). Anal. HPLC 99%
(Rt = 20.59 min).
4.1.6.3. (R)-3-(2-amino-2-(2-methoxyphenyl)ethyl)-1-(2-fluoro-6-(trifluoromethyl)benzyl)-6-methyl-
1
5-(4-(3-nitrobenzyl)piperazin-1-yl)pyrimidine-2,4(1H,3H)-dione (11c). Yield 34%, white foam, H
NMR (300 MHz, CDCl3) δ 8.17 (s, 1H), 8.09 (dd, J = 7.2, 1.2 Hz, 1H), 7.75 (d, J = 7.5 Hz, 1H),
7.55 - 7.45 (m, 2H), 7.44-7.29 (m, 1H), 7.25-7.16 (m, 2H), 6.90-6.83 (m, 2H), 5.39 (s, 2H), 4.47-
4.31 (m, 2H), 4.21 (dd, J = 12.6, 5.4 Hz, 1H), 3.86 (s, 3H), 3.70-3.49 (m, 4H), 2.73 (d, J = 10.2 Hz,
2H), 2.52-2.37 (m, 2H), 2.30 (s, 3H), 2.25-2.12 (m, 2H). MS (ESI) m/z 671 (MH+). Anal. HPLC 99%
(Rt = 19.49 min).
4.1.6.4. 3-(2-amino-2-(2-fluorophenyl)ethyl)-1-(2-fluoro-6-(trifluoromethyl)benzyl)-6-methyl-5-(4-
(3-nitrobenzyl)piperazin-1-yl)pyrimidine-2,4(1H,3H)-dione (11e). Yield 28%, white foam, 1H NMR
(300 MHz, CDCl3) δ 8.16 (s, 1H), 8.09 (dd, J = 7.2, 1.2 Hz, 1H), 7.75 (d, J = 7.5 Hz, 1H), 7.55-7.37
(m, 4H), 7.24-7.16 (m, 2H), 7.12-6.96 (m, 2H), 5.39 (s, 2H), 4.59 (dd, J = 8.7, 6.0 Hz, 1H), 4.26 (dd,
J = 12.9, 8.7 Hz, 1H), 4.16 (dd, J = 12.9, 5.7 Hz, 1H), 3.70-3.49 (m, 4H), 2.73 (d, J = 9.3 Hz, 2H),
2.52-2.41 (m, 2H), 2.30 (s, 3H), 2.25-2.12 (m, 2H). MS (ESI) m/z 659 (MH+). Anal. HPLC 99% (Rt
= 19.60 min).
4.1.6.5.
3-(2-amino-2-(m-tolyl)ethyl)-1-(2-fluoro-6-(trifluoromethyl)benzyl)-6-methyl-5-(4-(3-
1
nitrobenzyl)piperazin-1-yl)pyrimidine-2,4(1H,3H)-dione (11f). Yield 37%, white foam, H NMR
(300 MHz, CDCl3) δ 8.17 (d, J = 1.5 Hz, 1H), 8.10 (dd, J = 8.1, 1.3 Hz, 1H), 7.76 (d, J = 7.5 Hz,
1H), 7.55-7.45 (m, 2H), 7.44-7.29 (m, 1H), 7.26-7.18 (m, 4H), 7.07-7.04 (m, 1H), 5.41 (s, 2H), 4.32

ACCEPTED MANUSCRIPT
(dd, J = 9.9, 4.2 Hz, 1H), 4.21 (dd, J = 12.9, 9.9 Hz, 1H), 4.02 (dd, J = 12.6, 4.2Hz, 1H), 3.70-3.52
(m, 4H), 2.75 (d, J = 9.9 Hz, 2H), 2.57-2.45 (m, 2H), 2.33 (s, 3H), 2.30-2.21 (m, 2H). MS (ESI) m/z
655 (MH+). Anal. HPLC 98% (Rt = 21.03 min).
4.1.6.6. (R)-3-(2-amino-2-(4-fluorophenyl)ethyl)-1-(2-fluoro-6-(trifluoromethyl)benzyl)-6-methyl-5-
1
(4-(3-nitrobenzyl)piperazin-1-yl)pyrimidine-2,4(1H,3H)-dione (11g). Yield 53%, white foam, H
NMR (300 MHz, CDCl3) δ 8.17 (s, 1H), 8.10 (dd, J = 8.1, 1.2 Hz, 1H), 7.75 (d, J = 7.5 Hz, 1H),
7.55-7.36 (m, 3H), 7.31-7.18 (m, 3H), 7.13-7.09 (m, 1H), 7.94-6.88 (m, 1H), 5.39 (s, 2H), 4.36 (dd,
J = 8.7, 5.4 Hz, 1H), 4.21-4.03 (m, 2H), 3.68-3.50 (m, 4H), 2.74 (d, J = 9.9 Hz, 2H), 2.53-2.42 (m,
2H), 2.33 (s, 3H), 2.25-2.18 (m, 2H). MS (ESI) m/z 659 (MH+). Anal. HPLC 98% (Rt = 20.03 min).
4.1.6.7. 3-(2-amino-2-(3-fluorophenyl)ethyl)-1-(2-fluoro-6-(trifluoromethyl)benzyl)-6-methyl-5-(4-
(3-nitrobenzyl)piperazin-1-yl)pyrimidine-2,4(1H,3H)-dione (11h). Yield 54%, white foam, 1H NMR
(300 MHz, CDCl3) δ 8.17 (d, J = 1.5 Hz, 1H), 8.10 (dd, J = 8.1, 1.3 Hz, 1H), 7.76 (d, J = 7.5 Hz,
1H), 7.55-7.45 (m, 2H), 7.44-7.29 (m, 1H), 7.26-7.18 (m, 4H), 7.07-7.04 (m, 1H), 5.41 (s, 2H), 4.32
(dd, J = 9.9, 4.2 Hz, 1H), 4.21 (dd, J = 12.9, 9.9 Hz, 1H), 4.02 (dd, J = 12.6, 4.2Hz, 1H), 3.70-3.52
(m, 4H), 2.75 (d, J = 9.9 Hz, 2H), 2.57-2.45 (m, 2H), 2.33 (s, 3H), 2.30-2.21 (m, 2H). MS (ESI) m/z
659 (MH+). Anal. HPLC 98% (Rt = 20.45 min).
4.1.6.8. (R)-3-(2-amino-2-(5-fluoro-2-methoxyphenyl)ethyl)-1-(2-fluoro-6-(trifluoromethyl) benzyl)-
6-methyl-5-(4-(3-nitrobenzyl)piperazin-1-yl)pyrimidine-2,4(1H,3H)-dione (11i). Yield 33%, white
foam, 1H NMR (300 MHz, CDCl3) δ 8.18 (s, 1H), 8.11 (dd, J = 7.8, 1.2 Hz, 1H), 7.76 (d, J = 7.5 Hz,
1H), 7.55-7.46 (m, 2H), 7.45-7.36 (m, 1H), 7.22-7.19 (m, 1H), 6.97 (dd, J = 9.3, 3.0Hz, 1H), 6.89-
6.82 (m, 1H), ), 6.75 (dd, J = 9.0, 4.5Hz, 1H), 5.38 (s, 2H), 4.45 (m, 1H), 4.29-4.25 (m, 2H), 3.85 (s,
3H), 3.60 (s, 2H), 3.72-3.49 (m, 2H), 2.75-2.72 (m, 2H), 2.51-2.40 (m, 2H), 2.31 (s, 3H), 2.30-2.16
(m, 2H). MS (ESI) m/z 689 (MH+). Anal. HPLC 99% (Rt = 20.19 min).

ACCEPTED MANUSCRIPT
4.1.6.9. (S)-3-(2-amino-2-(5-fluoro-2-methoxyphenyl)ethyl)-1-(2-fluoro-6-(trifluoromethyl) benzyl)-
6-methyl-5-(4-(3-nitrobenzyl)piperazin-1-yl)pyrimidine-2,4(1H,3H)-dione (11j). Yield 25%, white
foam, 1H NMR (300 MHz, CDCl3) δ 8.18 (s, 1H), 8.11 (dd, J = 7.8, 1.2 Hz, 1H), 7.76 (d, J = 7.5 Hz,
1H), 7.55-7.46 (m, 2H), 7.45-7.36 (m, 1H), 7.22-7.19 (m, 1H), 6.97 (dd, J = 9.3, 3.0 Hz, 1H), 6.89-
6.82 (m, 1H), 6.75 (dd, J = 9.0, 4.5Hz, 1H), 5.38 (s, 2H), 4.45 (m, 1H), 4.29-4.25 (m, 2H), 3.85 (s,
3H), 3.60 (s, 2H), 3.72-3.49 (m, 2H), 2.75-2.72 (m, 2H), 2.51-2.40 (m, 2H), 2.31 (s, 3H), 2.30-2.16
(m, 2H). MS (ESI) m/z 689 (MH+). Anal. HPLC 97% (Rt = 20.14 min).
4.1.6.10. (R)-3-(2-amino-2-(3-fluoro-5-methylphenyl)ethyl)-1-(2-fluoro-6-(trifluoromethyl) benzyl)-
6-methyl-5-(4-(3-nitrobenzyl)piperazin-1-yl)pyrimidine-2,4(1H,3H)-dione (11k). Yield 34%, white
foam, 1H NMR (300 MHz, CDCl3) δ 8.18 (s, 1H), 8.11 (dd, J = 7.8, 1.2 Hz, 1H), 7.77 (d, J = 7.8 Hz,
1H), 7.56-7.46 (m, 2H), 7.45-7.36 (m, 1H), 7.22-7.19 (m, 1H), 7.02 (s, 1H), 6.92 (d, J = 9.9 Hz, 1H),
6.75 (d, J = 9.3 Hz, 1H), 5.50-5.35 (m, 2H), 4.33 (dd, J = 9.3, 4.8 Hz, 1H), 4.18 (dd, J = 12.6, 9.3
Hz, 1H), 3.99 (dd, J = 12.9, 4.8 Hz, 1H), 3.70-3.55 (m, 4H), 2.81-2.69 (m, 2H), 2.57-2.43 (m, 2H),
2.34 (s, 3H), 2.33 (s, 3H), 2.30-2.17 (m, 2H). MS (ESI) m/z 673 (MH+). Anal. HPLC 95% (Rt =
21.78 min).
4.1.6.11. (S)-3-(2-amino-2-(3-fluoro-5-methylphenyl)ethyl)-1-(2-fluoro-6-(trifluoromethyl) benzyl)-
6-methyl-5-(4-(3-nitrobenzyl)piperazin-1-yl)pyrimidine-2,4(1H,3H)-dione (11l). Yield 22%, white
foam, 1H NMR (300 MHz, CDCl3) δ 8.18 (s, 1H), 8.11 (dd, J = 7.8, 1.2 Hz, 1H), 7.77 (d, J = 7.8 Hz,
1H), 7.56-7.46 (m, 2H), 7.45-7.36 (m, 1H), 7.22-7.19 (m, 1H), 7.02 (s, 1H), 6.92 (d, J = 9.9 Hz, 1H),
6.75 (d, J = 9.3 Hz, 1H), 5.50-5.35 (m, 2H), 4.33 (dd, J = 9.3, 4.8 Hz, 1H), 4.18 (dd, J = 12.6, 9.3
Hz, 1H), 3.99 (dd, J = 12.9, 4.8 Hz, 1H), 3.70-3.55 (m, 4H), 2.81-2.69 (m, 2H), 2.57-2.43 (m, 2H),
2.34 (s, 3H), 2.33 (s, 3H), 2.30-2.17 (m, 2H). MS (ESI) m/z 673 (MH+). Anal. HPLC 97% (Rt =
21.74 min).

ACCEPTED MANUSCRIPT
4.1.6.12. (R)-3-(2-amino-2-phenylethyl)-1-(2-fluoro-6-(trifluoromethyl)benzyl)-6-methyl-5-(4-((5-
(trifluoromethyl)furan-2-yl)methyl)piperazin-1-yl)pyrimidine-2,4(1H,3H)-dione (11m). Yield 74%,
1
white foam, H NMR (300 MHz, CDCl3) δ 7.57-7.51 (m, 1H), 7.44-7.35 (m, 3H), 7.35-7.28 (m,
2H), 7.26-7.17 (m, 2H), 6.73 (dq, J = 3.7, 1.2 Hz, 1H), 6.30 (dd, J = 3.4, 0.9 Hz, 1H), 5.40 (s, 2H),
4.36 (dd, J = 9.4, 4.9 Hz, 1H), 4.21 (dd, J = 12.9, 9.5 Hz, 1H), 4.06 (dd, J = 12.9, 4.9 Hz, 1H), 3.70-
3.49 (m, 4H), 2.80 (d, J = 10.4 Hz, 2H), 2.49 (t, J =14.3 Hz, 2H), 2.34-2.16 (m, 5H). MS (ESI) m/z
654 (MH+). Anal. HPLC 99% (Rt = 20.50 min).
4.1.6.13.
(R)-3-(2-amino-2-(2-methoxyphenyl)ethyl)-1-(2-fluoro-6-(trifluoromethyl)benzyl)-6-
methyl-5-(4-((5-(trifluoromethyl)furan-2-yl)methyl)piperazin-1-yl)pyrimidine-2,4(1H,3H)-dione
1
(11n). Yield 32%, colorless oil, H NMR (300 MHz, CDCl3) δ 7.53 (d, J = 7.8 Hz, 1H), 7.41-7.34
(m, 1H), 7.24-7.16 (m, 3H), 6.90-6.82 (m, 2H), 6.72 (d, J = 3.0 Hz, 1H), 6.29 (d, J = 3.0 Hz, 1H),
6.29 (d, J = 3.3 Hz, 1H), 5.38 (s, 2H), 4.46 (dd, J = 8.7, 5.4 Hz, 1H), 4.36 (dd, J = 12.6, 9.0 Hz, 1H),
4.20 (dd, J = 12.6, 5.4 Hz, 1H), 3.86 (s, 3H), 3.60 (s, 2H), 3.72-3.49 (m, 2H), 2.81-2.74 (m, 2H),
2.51-2.37 (m, 2H), 2.27 (s, 3H), 2.26 – 2.19 (m, 2H). MS (ESI) m/z 684 (MH+). Anal. HPLC 98%
(Rt = 20.58 min).
4.1.6.14.
(R)-3-(2-amino-2-(5-fluoro-2-methoxyphenyl)ethyl)-1-(2-fluoro-6-(trifluoromethyl)
benzyl)-6-methyl-5-(4-((5-(trifluoromethyl)furan-2-yl)methyl)piperazin-1-yl)pyrimidine-
2,4(1H,3H)-dione (11o). Yield 39%, colorless oil, 1H NMR (300 MHz, CDCl3) δ 7.53 (d, J = 7.8 Hz,
1H), 7.45-7.35 (m, 1H), 7.25-7.18 (m, 1H), 6.94 (dd, J = 9.3, 3.0Hz, 1H), 6.89-6.81 (m, 1H), 6.76-
6.71 (m, 2H), 6.29 (d, J = 3.3 Hz, 1H), 5.37 (s, 2H), 4.48-4.25 (m, 1H), 4.29-4.23 (m, 2H), 3.85 (s,
3H), 3.60 (s, 2H), 3.72-3.49 (m, 2H), 2.81-2.74 (m, 2H), 2.51-2.37 (m, 2H), 2.28 (s, 3H), 2.26-2.16
(m, 2H). MS (ESI) m/z 702 (MH+). Anal. HPLC 96% (Rt = 20.96 min).