Synthesis and biological evaluation of disubstituted pyrimidines as selective 5-HT2C agonists

Article abstract of DOI:10.3390/molecules24183234

Here, we describe the synthesis of disubstituted pyrimidine derivatives and their biological evaluation as selective 5-HT_2C agonists. To improve selectivity for 5-HT_2C over other subtypes, we synthesized two series of disubstituted pyrimidines with fluorophenylalkoxy groups at either the 5-position or 4-position and varying cyclic amines at the 2-position. The in vitro cell-based assay and binding assay identified compounds 10a and 10f as potent 5-HT_2C agonists. Further studies on selectivity to 5-HT subtypes and drug-like properties indicated that 2,4-disubstituted pyrimidine 10a showed a highly agonistic effect on the 5-HT_2C receptor, with excellent selectivity, as well as exceptional drug-like properties, including high plasma and microsomal stability, along with low CYP inhibition. Thus, pyrimidine 10a could be considered a viable lead compound as a 5-HT_2C selective agonist.

Full text of DOI:10.3390/molecules24183234

molecules
Article
Synthesis and Biological Evaluation of Disubstituted
Pyrimidines as Selective 5-HT2C Agonists
Juhyeon Kim 1,2,† , Yoon Jung Kim 3,†, Ashwini M. Londhe 4,5, Ae Nim Pae 4,5, Hyunah Choo 1,4
,
Hak Joong Kim 2 and Sun-Joon Min 3,6,
*
1
Center for Neuro-Medicine, Korea Institute of Science and Technology (KIST), Seoul 02792, Korea
Department of Chemistry, Korea University, Seoul 02841, Korea
Department of Applied Chemistry, Hanyang University, Ansan, Gyeonggi-do 15588, Korea
Division of Bio-Medical Science & Technology, KIST School, Korea University of Science and Technology,
Seoul 02792, Korea
Convergence Research Center for Diagnosis, Treatment and Care System of Dementia, Korea Institute of
Science and Technology, Seoul 02792, Korea
Department of Chemical & Molecular Engineering, Hanyang University, Ansan, Gyeonggi-do 15588, Korea
Correspondence: sjmin@hanyang.ac.kr; Tel.: +82-31-400-5502
2
3
4
5
6
*
†
These authors contributed equally to this work.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 1 August 2019; Accepted: 3 September 2019; Published: 5 September 2019
Abstract: Here, we describe the synthesis of disubstituted pyrimidine derivatives and their biological
evaluation as selective 5-HT2C agonists. To improve selectivity for 5-HT2C over other subtypes,
we synthesized two series of disubstituted pyrimidines with fluorophenylalkoxy groups at either
the 5-position or 4-position and varying cyclic amines at the 2-position. The in vitro cell-based assay
and binding assay identified compounds 10a and 10f as potent 5-HT2C agonists. Further studies on
selectivity to 5-HT subtypes and drug-like properties indicated that 2,4-disubstituted pyrimidine
10a showed a highly agonistic effect on the 5-HT2C receptor, with excellent selectivity, as well as
exceptional drug-like properties, including high plasma and microsomal stability, along with low
CYP inhibition. Thus, pyrimidine 10a could be considered a viable lead compound as a 5-HT2C
selective agonist.
Keywords: disubstituted pyrimidine; 5-HT2C receptor; cell-based assay; binding affinity; selectivity
1. Introduction
Serotonin (5-hydroxytryptamine, 5-HT), a monoamine neurotransmitter, plays a critical role in
the regulation of various neurological functions, including mood, sleep, cognition, anxiety, sexual
behavior, and appetite [1,2]. There are 14 variants of serotonin receptors (5-HT receptors), which mostly
belong to G-protein-coupled receptors (GPCRs), except for the 5-HT3 receptor, a ligand-gated ion
channel. Activation of the 5-HT receptors induces both inhibitory and excitatory signal transduction
by modulating the release of many neurotransmitters, such as glutamate, dopamine, epinephrine,
and acetylcholine. Therefore, they have been considered crucial therapeutic targets for a variety of
neurological diseases, including depression, psychiatric disorders, sexual dysfunction, obesity, and
urinary incontinence [3–5].
The 5-HT2 receptors comprise three subtypes: 5-HT2A, 5-HT2B, and 5-HT2C. Recent studies
revealed that activation of 5-HT2C receptors in the central nerve system (CNS) is a potential drug
target for effective treatment of schizophrenia, as well as obesity [6–10]. One of the crucial problems
faced during the development of 5-HT2C agonists is selectivity over the structurally related 5-HT2A
and 5-HT2B receptors. 5-HT2A agonism in humans is implicated in acute adverse effects, such as
Molecules 2019, 24, 3234; doi:10.3390/molecules24183234
www.mdpi.com/journal/molecules

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hallucination and cardiovascular effects. Stimulation of the 5-HT2B receptor is related to other side
effects, including chronic cardiac valvulopathy and pulmonary hypertension.
Potent and selective 5-HT2C agonists have been developed, as shown in Figure 1. Lorcaserin
1
was approved by the FDA (Food and Drug Administration) in 2012 for treating obesity [11], and
vabicaserin was clinically evaluated as a potential antipsychotic [12]. Numerous 5-HT2C agonists that
have undergone preclinical or early clinical studies have been reported [13 19]. In addition, several
) have been developed as
2
–
5-HT2C-selective positron emission tomography (PET) radioligands (
chemical tools for in vivo imaging of the 5-HT2C receptor [20,21].
5 and 6
Figure 1. The representative 5-HT2C selective agonists and PET radioligands.
Despite the discovery of potential 5-HT2C agonists, the selectivity of these compounds over other
5-HT2 subtypes is still moderate; therefore, the search for highly selective 5-HT2C agonists remains
challenging. In this regard, our group has previously reported optically active pyrimidine derivative
8
as a potent and selective 5-HT2C agonist (Figure 2) [22]. The structure activity relationship (SAR)
between related pyrimidine derivatives and 5-HT2 subtypes revealed that a subtle change in the
fluorophenylalkoxy moiety attached to pyrimidine was essential to control the activation of selectivity
for 5-HT2C over 5-HT2A and 5-HT2B. Although compound was discovered as a lead compound with
8
8
excellent potency and good selectivity for the 5-HT2 subtypes, further structural modification of these
pyrimidine derivatives is required to improve selectivity over other 5-HT subtypes. Furthermore,
selective 5-HT2C agonists could be applied in the development of chemical probes such as PET
radiotracers [23–26].
Figure 2. Design of new pyrimidine derivatives as 5-HT2C agonists.

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We first planned to alter the substitution pattern of pyrimidines for the structural adjustment of
compound toward the 5-HT2C binding pocket. Recent studies demonstrated that pyridazine-fused
azepine containing a remote phenyl group (Figure 1) showed high potency and selectivity to 5-HT2C
8
4
over 5-HT2B with a good stability profile [19]. Accordingly, we hypothesized that the linear form of
2,5-disubstituted pyrimidine 9, possessing a flexible fluorophenylalkoxy group at the 5-position, might
be suitable for exploring the relationship between structural change and selectivity (Figure 2). Based
on the molecular docking study of several 5-HT2C agonists published in literature [27
assumed that the terminal-free amine of the piperazine moiety could be a key functional group binding
to a conserved residue, D134, in 5-HT2C protein. Taken together with the fact that compound derived
,28], we also
8
from (R)-isomer of secondary benzyl alcohol showed better binding affinity and selectivity to 5-HT2C
than its diastereomer [22], we designed an alternative series of pyrimidine derivatives 10 possessing a
variety of cyclic amines to examine their agonistic effect on 5-HT receptor activation. Herein, we report
the synthesis of disubstituted pyrimidine derivatives with different substitution patterns and their
biological evaluation as selective 5-HT2C receptor agonists.
2. Results and Discussion
2.1. Synthesis of 2,5-Disubstituted Pyrimidine Derivatives
Initially, we synthesized 2,5-disubstituted pyrimidines with flexible phenylalkoxy groups at
the 5-position, as shown in Scheme 1. Starting from 2,5-dibromopyrimidine 13, three kinds of
2-aminopyrimidines 11a–c were prepared through SNAr type amination. Pyrimidine derivatives 11a,
11b, and 11c were reacted with 3- or 4-fluoro-phenols, benzyl alcohols, and phenethyl alcohols in the
presence of copper catalyst and 1,10-phenanthroline ligands 14 or 15 [29–31] to afford 2,5-disubstituted
pyrimidines possessing an N-Boc-protecting group at the terminal amine. It is noted that the use
of 15 as a ligand in this transformation led to formation of the desired products in higher yields.
Finally, the 2-amino-5-alkoxypyrimidines 9a–r were obtained after removal of the Boc group under
acidic conditions.
Scheme 1. Synthesis of 2,5-disubstituted pyrimidine derivatives 9a–r. Reagents and conditions:
(a b) 3- or 4-fluorophenol/benzyl
) N-Boc-protected amines, K2CO3, CH3CN, 80 ◦C, 88–97%; (
alcohol/phenethyl alcohol, CuI or CuO2, 14 or 15, Cs2CO3, toluene, 110 ◦C, 42–85%; (
c) TFA, CH2Cl2,
0 ◦C, 52–67%.

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2.2. Synthesis of 2,4-Disubstituted Pyrimidine Derivatives
The synthesis of a series of 2,4-disubstituted pyrimidines 10, possessing different cyclic amines, is
described in Scheme 2. According to our protocol [22], we efficiently synthesized optically active 1-(3-
or 4-fluorophenyl)ethan-1-ol (R)-16a and (R)-16b using an enzymatic kinetic resolution method. As
per the procedure previously reported in literature [32], nucleophilic aromatic substitution (SNAr) of
2,4-dichloropyrimidine with alcohols (R)-16a and (R)-16b selectively produced 4-alkoxypyrimidines 17a
and 17b, which were subjected to an additional SNAr reaction to afford 2-alkoxy-4-aminopyrimidines
as free or N-Boc-protected amine forms. The latter were further treated with a strong acid, such
as HCl or TFA, to give the desired disubstituted pyrimidines. Besides pyrimidine analogues 10
,
we synthesized compounds 20a and 20b possessing a direct connection between the fluorophenyl
group and pyrimidine via ether linkage using a similar experimental process. In this case, these
compounds 20a/b might allow us to examine the optimal size of the fluorophenylalkoxy group through
a comparison of their in vitro activity with that of 8.
Scheme 2. Synthesis of 2,4-disubstituted pyrimidine derivatives 10a–j and 20ab. Reagents and
conditions: (
78%; (
) 2,4-dichloro-5-fluoropyrimidine, t-BuONa, toluene, 0 ◦C, 51–93%; (
(0.5 M), 100 ◦C, 12 h, 61–98%; (
a
) (i) Vinyl acetate (0.4 eq), CAL-B, pyridine, hexane, rt, 66–67%; (ii) 1 M NaOH, MeOH, rt,
b
c) amines, Et3N, toluene
d
) (i) N-Boc-protected amines, Et3N, DMSO (0.5 M), 100 ◦C, 12 h, 44–94%;
(ii) HCl (4.0 M in dioxane), CH3CN, 0 ◦C or TFA, CH2Cl2, 0 ◦C.
2.3. In Vitro Evaluation of Pyrimidine Derivatives
In vitro agonistic activities of pyrimidines against 5-HT2C were evaluated using a
fluorescence-based receptor functional assay, and the results are summarized in Table 1. Among the
first series of pyrimidines possessing different sizes of phenylalkoxy groups at the 5-position, we found
that the cellular agonistic effect of only compound 9b was higher than 50% at a concentration of 10
µM. Regarding the effect of cyclic amines at the 2-position of pyrimidine, only compounds 9g–l with
methyl piperazine showed low to moderate activities, with up to 48% activation. Based on this result,
we concluded that the substitution pattern of cyclic amines and the fluorophenylalkoxy group on
the pyrimidine ring is important for 5-HT2C agonism. Thus, we decided not to proceed with further

Molecules 2019, 24, 3234
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biological evaluation of this first series of compounds because an activation value higher than 50% is
considered to represent significant agonistic effects of the compounds.
Table 1. In vitro agonistic activity of 2,5-disubstituted pyrimidine 9a–r against 5-HT2C
.
%activation
(10 µM)
Entry
Compds
n
R1
R2
1
2
3
4
5
6
9a
9b
9c
9d
9e
9f
0
1
2
0
1
2
3-F
3-F
3-F
4-F
4-F
4-F
7
61
15
5
18
4
7
8
9
10
11
12
9g
9h
9i
9j
9k
9l
0
1
2
0
1
2
3-F
3-F
3-F
4-F
4-F
4-F
35
48
40
26
48
32
13
14
15
16
17
18
19
9m
9n
9o
9p
9q
9r
0
1
2
0
1
2
3-F
3-F
3-F
4-F
4-F
4-F
16
−1
3
14
16
−1
94a
Lorcaserin (1)
a
%activation value at 1 µM, EC50 = 14 nM.
Next, the in vitro biological activities of 2,4-disubstituted pyrimidines 10a–j and 20a–b against the
5-HT2C receptor were evaluated, as shown in Table 2. At this point, the ligand binding assay, as well as
the cell-based functional assay, were performed to identify more potent and selective 5-HT2C agonists.
Among the tested compounds, four pyrimidine derivatives showed greater than 50% activation against
5-HT2C in the cell-based assay. Studies on the binding affinity of this series to 5-HT2C indicated that
compounds 10a and 10f, with 1,4-diazepane at the 2-position of pyrimidine, exhibited excellent Ki
values of 7.9 nM and 19.0 nM, respectively. Although the result of the cell-based assay is important to
explore the cellular function of the 5-HT2C receptor, binding affinity plays a key role in determining
whether the compound is suitable for the development of a 5-HT2C-specific chemical probe. Thus,
we selected compounds 10a and 10f for further examination of the 5-HT subtype selectivity and
drug-like properties.

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Table 2. In vitro activity of 2,4-disubsitutued pyrimidine against 5-HT2C.
Entry
Compds
10a
R1
R2
%activationa %binding
Ki (nM)
1
81.0
78.2
29.5
81.3
47.6
73.6
7.9
2
3
10b
10c
ND
295.0
4
5
10d
10e
16.4
7.7
88.6
61.4
119.0
1255.0
6
7
8
10f
10g
10h
52.4
ND
10.1
93.9
78.1
50.8
19.0
232.0
466.0
9
10i
10j
20a
ND
0.1
85.9
26.2
64.8
120.0
ND
10
11
56.0
660.0
12
13
20b
8
34.0
54.8
89.0
1107.0
5.9
N/Ab
a
Serotonin was used as a reference compound (EC50 = 2.6 nM). b Not available.
The evaluation of compounds 10a and 10f for binding affinity to the 5-HT receptor subtypes is
illustrated in Table 3. We calculated the selectivity index (SI) of binding affinities between 5-HT2C and
other subtypes by dividing the given subtype Ki value with the 5-HT2C Ki value. The results indicate
that compound 10a displayed higher selectivity for most 5-HT subtypes compared to compound 10f
except for 5-HT1B and 5-HT1E, to which the binding affinities of both compounds were negligible.
Most importantly, compound 10a showed good selectivity for 5-HT2C over 5-HT2A and 5-HT2B, which
are associated with the primary side effects of non-specific 5-HT2C agonists.
,

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Table 3. Evaluation of binding affinity of 10a and 10f to 5-HT receptor subtypes a.
5-HT Subtypes (%Binding at 10 µM/Ki (nM))
1A
1B
1D
1E
2A
2B
2C
3
5A
6
7
74.1
56.1
71.4
43.1
75.5
/1284.0
98.0
/83.0
81.3
/7.9
89.0
/323.0
22.5
/ND
50.7
/348.0 /1016.0
61.3
10a
/578.0 /3237.0 /679.0
/NDb
SI
73.2
67.6
409.7
19.2
85.9
81.3
/937.0
-
162.5
52.9
/2863.0 /118.0
10.5
95.0
1.0
98.9
/19.0
40.9
88.0
/415.0
23.4
15.2
/ND
44.1
56.5
/467.0
128.6
55.4
/616.0
31.3
10f
/1066.0 /NDb
/NDb
SI
8c
56.1
89.1
/271.0
-
49.3
83.4
-
150.7
85.3
/511.0
6.2
98.1
/38.0
1.0
89.0
/5.9
21.8
83.4
/543.0
10.3
18.5
24.6
56.7
/30.0
32.4
63.4
/528.0
4.9
48.0
/NDb
/1052.0 /NDb
/NDb
SI
45.9
-
178.3
-
86.6
6.4
1.0
92.0
-
5.1
89.5
a 5-HT receptor binding was determined by competitive binding assay using radioligands and reference compounds
b
c
in Table S1. Not determined due to low %binding. Compound
selectivity index.
8 was used as a reference compound. SI,
Considering the 5-HT2C cellular agonistic effect, binding affinity, and subtype selectivity of 10a
we further investigated its drug-like properties, such as plasma stability, microsomal stability, and
CYP inhibition, as shown in Table 4. For plasma stability, 10 M of compound 10a was incubated in
human plasma; the remaining amount of 10a was measured after incubation for 30 min and 2 h. For
microsomal stability, human liver microsomes were treated with 10a at a concentration of 1 M; the
,
µ
µ
% remaining amount of 10a was analyzed at 30 min after treatment. CYP inhibition values against
five human CYP450 isozymes were recorded as % remaining activity values of the given isozymes.
We found that compound 10a was stable in both the plasma and liver, indicating that compound 10a
would be less affected by hepatic metabolism or plasma decomposition throughout systemic circulation.
Furthermore, the inhibitory activity of 10a against all the tested CYP isozymes was significantly low
(%remaining activity of isozymes >60%), suggesting that compound 10a might not exert toxic effects
due to drug–drug interaction (DDI). Overall, compound 10a exhibited considerably excellent plasma
and microsomal stability with low CYP inhibitory activity.
Table 4. The results of plasma stability, microsomal stability, and CYP inhibition.
HLM
%Remaining @
1 µM after 0.5 h
96.6
Plasma Stability
%Remaining @
10 µM after 0.5 and 2 h
99.9/98.0
CYP Isozymes (%Remaining @ 10 µM)
Compd
10a
1A2
2D6
2C9
3A4
2C19
86.5
63.6
>100 86.2
86.3
2.4. Molecular Docking Study
In order to investigate the good selectivity of 10a to 5-HT2C over 5-HT2B, the molecular docking
study was performed using Discovery Studio software. Thus, the interaction of 10a and with those
8
receptors were evaluated using the crystal structures of 5-HT2C (PDB ID = 6BQH) and 5-HT2B (PDB ID
= 4IB4), which were recently reported in the literatures [28,33].
In the case of 5-HT2C receptor docking study, the binding orientation of both ligands 10a
(-CDOCKER score 36.31) and
over each other (Figure 3A). Both of these ligands have shown hydrogen bonds with crucial aspartic
acids Asp134 (Figure 3B,C). NH groups of piperazine ( ) and 1,4-diazepane ring (10a) are involved in
8 (-CDOCKER score 36.96) are similar but not precisely overlapping
8
hydrogen bonds and π-cationic interactions with Trp130 and Asp134. Additionally, 8 has shown two
more hydrogen bonds with Tyr358 and Leu209. The hydrogen of the NH group of the piperazine ring
has shown a hydrogen bond with Tyr358 and nitrogen of the pyrimidine ring has shown one hydrogen
bond with Leu209. In total, we recognized four hydrogen bond interactions between 5-HT2C and
Fluorophenyl rings of were involved in stacking interactions with Phe328 residue. In the case of
10a, in addition to hydrogen bonds, strong stacking contacts were observed with Phe327, phe328,
8.
8
π–π
π–π

Molecules 2019, 24, 3234
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and Trp324. Several hydrophobic contacts in both ligands were identified, mediated by Val135 and
Leu209 in 10a and Val135, Val208, and Leu209 in 8.
Figure 3. Interactions of 10a and
shown in green and magenta color stick format. 5-HT2C (wheat color) and 5-HT2B (green color) protein
structures are shown in cartoon format. Hydrogen bond, -cationic, stacking, and hydrophobic
interactions are shown in green, orange, dark pink, and light pink dash line format. ( ) Overlap
of 10a and inside 5-HT2C protein. ( ) Interactions between ligand 10a and the 5-HT2C receptor.
) Interactions between ligand and the 5-HT2C receptor. ( ) Overlap of 10a and inside 5-HT2B
protein. ( ) Interactions between ligand 10a and the 5-HT2B receptor. ( ) Interactions between ligand
and the 5-HT2B receptor.
8 inside the 5-HT2C and 5-HT2B receptors. Ligands 10a and 8 are
π
π–π
A
8
B
(
C
8
D
8
E
F
8
In the case of 5-HT2B receptor docking study, overlap of ligand 10a (-CDOCKER score 37.40) and
8
(-CDOCKER score 39.53) inside 5-HT2B binding pocket (Figure 3D) shows that ligand bind at the
same place but differ in pyrimidine, fluorophenyl ring, and diazepane ring/piperidine orientation.
Compound has shown three hydrogen bonds with binding site residues. The hydrogen (NH group)
of the piperazine ring showed two hydrogen bonds with Asp135 (Figure 3F). The fluorine group on
the phenyl ring in has shown an additional hydrogen bond with Thr114. Beside these hydrogen
8
8
bonding interactions, several hydrophobic residues, Val136, Val208, Leu209, and Leu362, hold the
molecule by several hydrophobic interactions. Compound 10a has only one hydrogen bond with
Asp135 (Figure 3E). Compared to
binding pocket (Val2018, Leu209, and Leu362).
Although both 10a and bind in a similar way inside the 5-HT2B binding pocket, a minor difference
in aromatic ring orientation affects the hydrophobic interactions of 10a. The difference in fluorophenyl
ring orientation is responsible for the additional hydrogen bond between Thr114 and . These extra
hydrogen bonds and hydrophobic contacts inside the 5-HT2B receptors make the potency of compound
superior to that of compound 10a in the 5-HT2B binding assay. Compared to the 5-HT2B receptor, 10a
8, 10a has fewer hydrophobic contacts with the 5-HT2B receptor
8
8
8
has stronger interactions (hydrogen bond and hydrophobic) with the 5-HT2C receptor. Overall, the
decrease in hydrophobic contacts inside the 5-HT2B receptor makes our molecule 10a more selective
towards the 5-HT2C receptor.

Molecules 2019, 24, 3234
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3. Materials and Methods
3.1. General Methods
All reactions were conducted under oven-dried glassware, under an atmosphere of nitrogen. All
commercially available reagents were purchased and used without further purification. Solvents and
gases were dried according to standard procedures. Organic solvents were evaporated with reduced
pressure using a rotary evaporator. Reactions were followed by analytical thin layer chromatography
(TLC) analysis using glass plates precoated with silica gel (0.25 mm). TLC plates were visualized by
exposure to UV light (UV), and then were visualized with a KMnO4 or p-anisaldehyde stain followed
by brief heating on a hot plate. Flash column chromatography was performed using silica gel 60
1
(230–400 mesh, Merck) with the indicated solvents. H-NMR spectra were measured with 400 MHz
and 13C-NMR spectra were measured with 100 MHz using CDCl3 and CD3OD. 1H NMR spectra are
represented as follows: Chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet,
m = multiplet), integration, and coupling constant (J) in Hertz (Hz). 1H NMR chemical shifts are
reported relative to CDCl3 (7.26 ppm). 13C NMR was recorded relative to the central line of CDCl3
(77.0 ppm). High resolution mass spectra (LR-MS) were obtained using positive electrospray ionization
and mass/charge (m/z) ratios are reported as values in atomic mass units.
3.2. Synthesis of 2,5-Disubstituted Pyrimidines
3.2.1. General Procedure for Preparing Compounds 11a–c
In a dry sealed tube under argon were placed, to a solution of 1-Boc-piperazine (5.04 mmol),
potassium carbonate (13.1 mmol) in acetonitrile (12.6 mL) was added 2,5-dibromo pyrimidine 13
◦
(5.04 mmol). The mixture was allowed to stir at 80 C for 12 h. After completion of the reaction
(monitored by TLC), the mixture was then cooled at room temperature, then it was quenched with
saturated aqueous NH4Cl (10 mL) and extracted with EtOAc. The organic layers were dried over
anhydrous MgSO4 and concentrated in vacuo. The resulting residue was purified by flash column
chromatography on silica gel (EtOAc:n-hexane = 1:8) to afford piperazine 11a.
tert-Butyl 4-(5-bromopyrimidin-2-yl)piperazine-1-carboxylate (11a): Yield: 88%; 1H-NMR (400 MHz,
CDCl3)
δ 8.30 (s, J = 2.0 Hz, 2H), 3.78—3.75 (t, J = 5.2 Hz, 4H), 3.49–3.47 (t, J = 5.2 Hz, 4H), 1.48 (s, 9H).
13C-NMR (100 MHz, CD3OD) δ 161.2, 159.2, 156.46, 107.15, 81.5, 44.8, 28.6.
tert-Butyl (R)-4-(5-bromopyrimidin-2-yl)-3-methylpiperazine-1-carboxylate (11b): Yield: 92%; 1H-NMR
(400 MHz, CDCl3)
δ 8.28 (s, J = 2.0 Hz, 1H), 4.81–4.70 (m, 1H), 4.39–4.36 (m, 1H), 4.15–3.90 (m, 2H),
3.19–3.08 (m, 1H), 3.08–2.90 (m, 2H), 1.47 (s, 9H), 1.16 (d, J = 6.7 Hz, 3H). 13C-NMR (100 MHz, CD3OD)
δ 160.9, 159.2, 156.9, 107.0, 81.4, 45.1, 44.0, 39.4, 14.5.
1
tert-Butyl 4-(5-bromopyrimidin-2-yl)-1,4-diazepane-1-carboxylate (11c): Yield: 97%; H-NMR (400
MHz, CDCl3)
3.38–3.35 (t, J = 5.9 Hz, 1H), 3.28–3.25 (t, J = 6.1 Hz, 1H), 1.96–1.90 (m, 2H), 1.43 (d, J = 8.4 Hz, 9H).
13C-NMR (100 MHz, CDCl3)
159.5, 158.1, 155.3, 105.8, 79.6, 49.0, 48.5, 47.4, 46.9, 46.3, 46.1, 28.5, 25.4.
δ 8.28 (s, 2H), 3.86–3.80 (m, 2H), 3.73–3.70 (t, J = 6.1 Hz, 2H), 3.55–3.52 (t, J = 5.4 Hz, 2H),
δ
3.2.2. General Procedure for Preparing Compounds 9a–r
In a dry sealed tube under argon were placed 11b (0.56 mmol), 3-fluorobenzyl alcohol (1.68 mmol),
cesium carbonate (0.84 mmol), copper iodide (0.056 mmol) (or copper(I) oxide), 1,10-phenanthroline
(0.11 mmol) (or 3,4,7,8-tetramethyl-1,10-phenanthroline) in toluene (1.1 mL) and the mixture was
heated at 130 ◦C for 20 h. After completion of the reaction (monitored by TLC), the mixture was then
cooled at room temperature and it was quenched with saturated aqueous NH4Cl (3
×
50 mL), extracted
with EtOAc. The organic layers were dried over anhydrous MgSO4 and concentrated in vacuo. The
resulting residue was purified by flash column chromatography on silica gel (EtOAc: Acetone: Hexane
= 1:1:15) to afford intermediate pre-9h.

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To a solution of methyl piperazine carboxylate pre-9h (0.136 mmol) in dichloromethane (1.3 mL)
◦
was added trifluoroacetic acid (0.21 mL). The mixture was allowed to stir at 0 C for 3 h. After
completion of the reaction (monitored by TLC), then neutralized by sodium hydrogen carbonate until
the mixture to be pH 8 and extracted with EtOAc. The organic layers were dried over anhydrous
MgSO4 and concentrated in vacuo. The resulting residue was purified by flash column chromatography
on silica gel (MeOH:DCM = 1:15) to afford pyrimidine 9h, which was isolated as HCl salt form after
treating with 4 M HCl in diethyl ether.
tert-Butyl 4-(5-((3-fluorophenoxy)pyrimidin-2-yl)piperazine-1-carboxylate (pre-9a): Yield: 21%;
1H-NMR (400 MHz, CDCl3)
δ
8.17 (s, 2H), 7.27–7.21 (m, 1H), 6.77–6.73 (m, 1H), 6.71–6.62 (m,
2H), 3.80 (t, J = 5.1 Hz, 4H), 3.52 (t, J = 5.0 Hz, 4H), 1.49 (s, 9H).
4-(5-((3-fluorophenoxy)pyrimidin-2-yl)piperazin-1-ium
hydrochloride (9a): Yield: 21%; 1H-NMR
(400MHz, CDCl3) 8.17 (s, 2H), 7.24 (dd, J = 15.4, 7.5 Hz, 1H), 6.75 (m, 1H), 6.70 (d, J = 8.4
Hz, 1H), 6.64 (d, J = 10.2 Hz, 1H), 3.89 (t, J = 4.5 Hz, 4H), 3.07 (t, J = 4.7 Hz, 4H). 13C-NMR (100 MHz,
·
δ
CD3OD) δ
162.4 (d, 1J = 259 Hz), 159.7, 152.3, 152.0, 144.5, 132.2 (d, 3J = 9 Hz), 113.4 (d, 4J = 3 Hz), 110.9
(d, 2J = 21 Hz), 105.4 (d, 2J = 25 Hz), 44.4, 42.5; LRMS-EI (m/z): [M–Cl]+ calcd for C14H15FN4O: 274.30,
found: 274.30
tert-Butyl 4-(5-((3-fluorobenzyl)oxy)pyrimidin-2-yl)piperazine-1-carboxylate (pre-9b): Yield: 79%;
1H-NMR (400 MHz, CDCl3)
δ
8.11 (s, 2H), 7.37–7.32 (m, 1H), 7.17–7.11 (m, 2H), 7.05–6.99 (m, 1H), 5.01
(s, 2H), 3.71 (t, J = 5.2 Hz, 4H), 3.49 (t, J = 5. 2 Hz, 4H), 1.48 (s, 9H).
4-(5-((3-fluorobenzyl)oxy)pyrimidin-2-yl)piperazin-1-ium hydrochloride (9b): Yield: 58%; H-NMR
(400 MHz, CD3OD) 8.23 (s, 1H), 7.42–7.37 (m, 1H), 7.25–7.17 (m, 2H), 7.06 (td, J = 8.5, 2.4 Hz, 1H),
7.07–7.02 (m, 1H), 5.12 (s, 2H), 3.95 (t, J = 5.3 Hz, 4H), 3.23 (t, J = 5.3 Hz, 4H). 13C-NMR (100 MHz,
1
·
δ
CD3OD)
δ
164.3 (d, 1J = 243 Hz), 158.2, 147.7, 147.4, 140.8 (d, 3J = 7 Hz), 131.5 (d, 3J = 8 Hz), 124.4 (d, 4J
= 3 Hz), 115.8 (d, 2J = 21 Hz), 115.3 (d, 2J = 22 Hz), 71.9, 44.3, 42.6; LRMS-EI (m/z): [M–Cl]+ calcd for
C15H17FN4O: 288.33, found: 288.33
tert-Butyl 4-(5-((3-fluorophenethoxy)pyrimidin-2-yl)piperazine-1-carboxylate (pre-9c): Yield: 46%;
1H-NMR (400 MHz, CDCl3)
δ
8.05 (s, 1H), 7.24–7.20 (m, 2H), 7.02–6.98 (m, 2H), 4.12 (t, J = 6.8 Hz, 2H),
3.69 (t, J = 5.2 Hz, 2H), 3.48 (t, J = 5.2 Hz, 2H), 3.03 (t, J = 6.8 Hz, 2H), 1.48 (s, 9H).
4-(5-((3-fluorophenethoxy)pyrimidin-2-yl)piperazin-1-ium hydrochloride (9c): Two-step yield: 46%;
1H-NMR (400 MHz, CD3OD)
8.17 (s, 2H), 7.34–7.29 (m, 1H), 7.13 (d, J = 8 Hz, 1H), 7.07 (d, J = 10.1
Hz, 1H), 6.96 (td, J = 8.7, 2.3 Hz, 1H), 4.25 (t, J = 6.5 Hz, 2H), 3.97 (t, J = 5.2 Hz, 4H), 3.27 (t, J = 5.2 Hz,
·
δ
4H), 3.09 (t, J = 6.5 Hz, 2H). 13C-NMR (100 MHz, CD3OD) 164.3 (d, 1J = 242 Hz), 158.1, 148.2, 146.8,
δ
142.5 (d, 3J = 7 Hz), 131.1 (d, 3J = 8 Hz), 125.9 (d, 4J = 3 Hz), 116.7 (d, 2J = 21 Hz), 114.3 (d, 2J = 21 Hz),
71.28, 44.3, 42.7, 36.4, 36.4; LRMS-EI (m/z): [M–Cl]+ calcd for C16H19FN4O: 302.35, found: 302.35
tert-Butyl 4-(5-((4-fluorophenoxy)pyrimidin-2-yl)piperazine-1-carboxylate (pre-9d): Yield: 21%;
1H-NMR (400 MHz, CDCl3)
δ 8.17 (s, 2H), 7.03–6.99 (m, 2H), 6.92–6.89 (m, 2H), 3.80 (t, J = 5.2
Hz, 4H), 3.52 (t, J = 5.2 Hz, 4H), 1.49 (s, 9H).
4-(5-((4-fluorophenoxy)pyrimidin-2-yl)piperazin-1-ium
hydrochloride (9d): Yield: 60%; 1H-NMR (400
·
MHz, CDCl3)
δ 8.15 (s, 2H), 7.02-6.98 (m, 2H), 6.91–6.88 (m, 2H), 3.77 (t, J = 5.2 Hz, 4H), 3.51 (t, J = 5.1 Hz,
4H). 13C-NMR (100 MHz, CD3OD)
δ
158.9 (d, 1J = 241 Hz), 157.9, 153.8, 150.1, 144.4, 118.6 (d, 3J = 8 Hz),
116.6 (d, 2J = 23 Hz), 43.4, 41.5; LRMS-EI (m/z): [M–Cl]+ calcd for C14H15FN4O: 274.30, found: 274.30.
tert-Butyl 4-(5-((4-fluorobenzyl)oxy)pyrimidin-2-yl)piperazine-1-carboxylate (pre-9e): Yield: 76%;
1H-NMR (400 MHz, CDCl3)
δ 8.10 (s, 1H), 7.37 (dd, J = 8.7, 5.3 Hz, 2H), 7.09–7.05 (m, 2H), 4.98 (s, 2H),
3.71 (t, J = 5.2 Hz, 4H), 3.49 (t, J = 5.2 Hz, 4H), 1.48 (s, 9H).
4-(5-((4-fluorobenzyl)oxy)pyrimidin-2-yl)piperazin-1-ium hydrochloride (9e): Two-step yield: 67%;
·
1H-NMR (400 MHz, CD3OD)
δ 8.22 (s, 2H), 7.47–7.43 (m, 2H), 7.13–7.08 (m, 2H), 5.08 (s, J = 2.5 Hz,
2H), 3.95 (t, J = 5.3 Hz, 4H), 3.24 (t, J = 5.2 Hz, 4H). 13C-NMR (100 MHz, CD3OD) 164.0 (d, 1J = 243
δ
Hz), 158.4, 147.4, 134.0 (d, 4J = 3 Hz), 132.3, 131.0 (d, 3J = 8 Hz), 116.3 (d, 2J = 22 Hz), 72.1, 44.7, 43.3;
LRMS-EI (m/z): [M–Cl]+ calcd for C15H17FN4O: 288.33, found: 288.33.

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tert-Butyl 4-(5-((4-fluorophenethoxy)pyrimidin-2-yl)piperazine-1-carboxylate (pre-9f): Yield: 54%;
1H-NMR (400 MHz, CDCl3)
8.05 (s, 1H), 7.24–7.20 (m, 2H), 7.02–6.98 (m, 2H), 4.12 (t, J = 6.8 Hz, 2H),
3.70 (t, J = 5.2 Hz, 2H), 3.48 (t, J = 5.2 Hz, 2H), 3.03 (t, J = 6.8 Hz, 2H), 1.48 (s, 9H).
4-(5-((4-fluorophenethoxy)pyrimidin-2-yl)piperazin-1-ium hydrochloride (9f): Two-step yield: 53%;
1H-NMR (400 MHz, CD3OD)
8.16 (s, 2H), 7.32–7.28 (m, 2H), 7.04–6.99 (m, 2H), 4.21 (t, J = 6.6 Hz, 2H),
δ
·
δ
3.95 (t, J = 5.1 Hz, 4H), 3.27 (t, J = 5.1 Hz, 4H), 3.05 (t, J = 6.6 Hz, 2H). 13C-NMR (100 MHz, CD3OD)
δ
163.1 (d, 1J = 241 Hz), 158.6, 147.7, 146.8, 135.51, 131.7 (d, 3J = 8 Hz), 116.0 (d, 2J = 21 Hz), 71.7, 45.3,
44.5, 35.9; LRMS-EI (m/z): [M–Cl]+ calcd for C16H19FN4O: 302.35, found: 302.35.
tert-Butyl (R)-4-(5-((3-fluorophenoxy)pyrimidin-2-yl)-3-methylpiperazine-1-carboxylate (pre-9g): Yield:
17%; 1H-NMR (400 MHz, CDCl3)
δ 8.18 (s, 2H), 7.27–7.22 (m, 1H), 6.78–6.70 (m, 2H), 6.65–6.63 (m, 1H),
4.90–4.84 (m, 1H), 4.43–4.40 (m, 1H), 4.18–3.93 (m, 2H), 3.24–3.18 (m, 1H), 3.13–2.94 (m, 2H), 1.49 (s,
9H), 1.20 (d, J = 6.8 Hz, 3H).
(R)-4-(5-((3-fluorophenoxy)pyrimidin-2-yl)-3-methylpiperazine-1-ium
1H-NMR (400 MHz, CDCl3)
8.18 (s, 2H), 7.27–7.21 (m, 1H), 6.77–6.65 (m, 2H), 6.63–6.62 (m, 1H),
4.78–4.75 (m, 1H), 4.41–4.38 (m, 1H), 3.16–3.08 (m, 2H), 3.04–3.00 (m, 1H), 2.92–2.89 (m, 1H), 2.83–2.76
·
hydrochloride (9g): Yield: 21%;
δ
(m, 1H), 1.28 (d, J = 6.8 Hz, 3H). 13C-NMR (100 MHz, CD3OD)
δ 159.3, 152.1, 146.9, 144.3, 132.2 (d,
3J = 9 Hz), 131.8, 113.4, 110.8 (d, 2J = 21 Hz), 105.4 (d, 2J = 25 Hz), 45.8, 44.8, 36.9, 35.9, 13.7; LRMS-EI
(m/z): [M–Cl]+ calcd for C15H17FN4O: 288.33, found: 288.33.
tert-Butyl (R)-4-(5-((3-fluorobenzyl)oxy)pyrimidin-2-yl)-3-methylpiperazine-1-carboxylate (pre-9h):
Yield: 43%; 1H-NMR (400 MHz, CDCl3)
δ 8.11 (s, 2H), 7.37–7.32 (m, 1H), 7.15–7.11 (m, 2H), 7.04–6.99
(m, 1H), 5.00 (s, 2H), 4.75 (s, 1H), 4.31 (d, J = 12.5 Hz, 1H), 4.10 (d, J = 12.5 Hz, 1H, broad), 3.17–3.10 (m,
1H), 3.10–2.91 (brs, 1H), 1.48 (s, 9H), 1.14 (d, J = 6.7 Hz, 3H).
(R)-4-(5-((3-fluorobenzyl)oxy)pyrimidin-2-yl)-3-methylpiperazine-1-ium
1H-NMR (400 MHz, CDCl3)
8.12 (s, 2H), 7.37–7.33 (m, 1H), 7.17–7.11 (m, 2H), 7.05–7.00 (td, J = 8.4,
2.3 Hz, 1H), 5.01 (s, 1H), 4.85–4.82 (m, 1H), 4.45–4.40 (m, 1H), 3.23–3.17 (m, 2H), 3.10–3.01 (m, 2H),
·
hydrochloride (9h): Yield: 70%;
δ
2.89–2.82(m, 1H), 1.30 (d, J = 6.9 Hz, 3H). 13C-NMR (100 MHz, CD3OD)
δ
165.6 (d, 1J = 244 Hz), 157.8,
147.4, 140.8, 131.4 (d, 3J = 9 Hz), 124.3, 115.8 (d, 2J = 20 Hz), 115.3 (d, 2J = 22 Hz), 71.9, 45.8, 44.8, 37.2,
13.4; LRMS-EI (m/z): [M–Cl]+ calcd for C16H19FN4O: 302.35, found: 302.35
tert-Butyl (R)-4-(5-((3-fluorophenethoxy)pyrimidin-2-yl)-3-methylpiperazine-1-carboxylate (pre-9i):
Yield: 24%; 1H-NMR (400 MHz, CDCl3)
δ 8.06 (s, 2H), 7.30–7.26 (m, 1H), 7.04–7.02 (m, 1H), 6.98–6.91
(m, 2H), 4.98 (s, 2H), 4.80–4.70 (m, 1H), 4.31–4.28 (m, 1H), 4.14 (t, J = 6.7 Hz, 2H), 3.97–3.90 (m, 2H),
3.17–3.10 (m, 2H), 3.06 (t, J = 6.7 Hz, 2H), 3.00–2.80 (m. 2H), 1.48 (s, 9H), 1.14 (d, J = 6.7 Hz, 3H).
(R)-4-(5-((3-fluorophenethoxy)pyrimidin-2-yl)-3-methylpiperazin-1-ium
·
hydrochloride (9i): Two-step
yield: 37%; 1H-NMR (400 MHz, CDCl3)
δ
8.06 (s, 2H), 7.28 (dd, J = 13.9, 7.9 Hz, 1H), 7.03 (d, J = 7.6
Hz, 1H), 6.98–6.91 (m, 2H), 4.86 (s, 2H), 4.45 (d, J = 12.4 Hz, 1H), 4.15 (t, J = 6.7 Hz, 2H), 3.28–3.14 (m,
2H), 3.11–3.08 (m, 1H), 3.06 (t, J = 6.7 Hz, 2H), 2.87 (td, J = 12.6, 4.1 Hz, 2H), 1.26 (d, J = 6.9 Hz, 2H).
13C-NMR (100 MHz, CD3OD)
δ
163.10 (d, 1J = 245 Hz), 157.6, 147.9, 146.1, 142.5 (d, 3J = 7 Hz), 131.1 (d,
3J = 8 Hz), 125.9 (d, 4J = 3 Hz), 116.7 (d, 2J = 21 Hz), 114.2 (d, 2J = 21 Hz), 71.3, 45.6, 44.6, 36.7, 36.4, 30.9,
13.3; LRMS-EI (m/z): [M–Cl]+ calcd for C17H21FN4O: 316.38, found: 316.38.
tert-Butyl (R)-4-(5-((4-fluorophenoxy)pyrimidin-2-yl)-3-methylpiperazine-1-carboxylate (pre-9j): Yield:
27%; 1H-NMR (400 MHz, CDCl3)
δ 8.17 (s, 2H), 7.02–6.98 (m, 2H), 6.91–6.89 (m, 2H), 4.90–4.83 (m,
1H), 4.43–4.39 (m, 1H), 4.18–3.92 (m, 3H), 3.25–3.18 (m, 1H), 3.15–2.93 (m, 2H), 1.49 (s, 9H), 1.20 (d,
J = 6.7 Hz, 3H).
(R)-4-(5-((4-fluorophenoxy)pyrimidin-2-yl)-3-methylpiperazine-1-ium
1H-NMR (400 MHz, CDCl3)
8.15 (s, 2H), 7.01–6.96 (m, 2H), 6.91–6.87 (m, 2H), 4.79–4.76 (m, 1H),
4.42–4.38 (m, 1H), 3.16–3.09 (m, 2H), 3.05–3.01 (m, 1H), 2.94–2.91 (m, 1H), 2.84–2.77 (m, 1H), 2.77–2.70
·hydrochloride (9j): Yield: 39%;
δ
(m, 1H), 1.28 (d, J = 6.8 Hz, 3H). 13C-NMR (100 MHz, CD3OD) 160.1 (d, 1J = 239 Hz), 159.0, 155.5,
δ
155.5 (d, 4J = 2 Hz), 145.5, 119.8 (d, 3J = 8 Hz), 117.4 (d, 2J = 24 Hz), 45.9, 44.9, 37.2, 30.9, 30.7, 13.5;
LRMS-EI (m/z): [M–Cl]+ calcd for C15H17FN4O: 288.33, found: 288.33.

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tert-Butyl (R)-4-(5-((4-fluorobenzyl)oxy)pyrimidin-2-yl)-3-methylpiperazine-1-carboxylate (pre-9k):
1
Yield: 41%; H-NMR (400 MHz, CDCl3):
δ 8.10 (s, 2H), 7.37 (dd, J = 8.5, 5.4 Hz, 2H), 7.09–7.05
(m, 2H), 4.97 (s, 2H), 4.80–4.74 (m, 1H), 4.32–4.28 (m, 1H), 4.15–3.89 (m, 3H), 3.17–3.10 (m, 1H), 3.10–2.90
(m, 2H), 1.48 (s, 9H), 1.14 (d, J = 6.7 Hz, 3H).
(R)-4-(5-((4-fluorobenzyl)oxy)pyrimidin-2-yl)-3-methylpiperazin-1-ium
1H-NMR (400 MHz, CDCl3)
8.11 (s, 2H), 7.39–7.35 (m, 2H), 7.09–7.05 (m, 2H), 4.98 (s, 2H), 4.94–4.89
(m, 1H), 4.64–4.61 (m, 1H), 3.48–3.39 (m, 2H), 3.28–3.25 (m, 1H), 3.20–3.12 (m, 1H), 3.01–2.94 (m, 1H),
·hydrochloride (9k): Yield: 73%;
δ
1.42 (d, J = 7.0 Hz, 3H). 13C-NMR (100 MHz, CD3OD)
δ
162.8 (d, 1J = 245 Hz), 156.4, 146.4, 146.1, 131.9,
129.7 (d, 3J = 8 Hz), 115.8 (d, 2J = 21 Hz), 71.5, 47.2, 44.3, 43.3, 35.7, 14.0; LRMS-EI (m/z): [M Cl]+ calcd
–
for C16H19FN4O: 302.35, found: 302.35.
tert-Butyl (R)-4-(5-((4-fluorophenethoxy)pyrimidin-2-yl)-3-methylpiperazine-1-carboxylate (pre-9l):
Yield: 39%; 1H-NMR (400MHz, CDCl3)
8.05 (s, 2H), 7.23–7.20 (m, 2H), 7.02–6.98 (m, 2H), 4.80–4.70
δ
(m, 1H), 4.30–4.27 (m, 1H), 4.12 (t, J = 6.9 Hz, 2H), 3.98–3.87 (m, 2H), 3.17–3.09 (m, 1H), 3.03 (t, J = 6.8
Hz, 2H), 3.01–2.89 (m, 2H), 1.48 (s, 9H), 1.14 (d, J = 6.7 Hz, 3H).
(R)-4-(5-((4-fluorophenethoxy)pyrimidin-2-yl)-3-methylpiperazin-1-ium·hydrochloride (9l): Yield: 85%;
1H-NMR (400 MHz, CDCl3):
δ 8.06 (s, 2H), 7.22 (dd, J = 8.4, 5.5 Hz, 2H), 7.00 (t, J = 8.7 Hz, 2H),
4.87–4.85 (m, 1H), 4.46–4.42 (m, 1H), 4.12 (t, J = 6.8 Hz, 2H), 3.27–3.16 (m, 2H), 3.09–3.05 (m, 2H), 3.03 (t,
J = 6.7 Hz, 2H), 2.90–2.84 (m, 1H), 1.37 (d, J = 7.0 Hz, 3H). 13C-NMR (100 MHz, CD3OD): 163.08 (d, 1J
δ
= 241 Hz), 157.7, 147.8, 146.9, 135.5 (d, 4J = 3 Hz), 131.7 (d, 3J = 8 Hz), 116.0 (d, 2J = 21 Hz), 71.7, 45.8,
44.8, 37.2, 35.9, 30.9, 30.7, 13.3; LRMS-EI (m/z): [M–Cl]+ calcd for C17H21FN4: 316.38, found: 316.38.
tert-Butyl 4-(5-((3-fluorophenoxy)pyrimidin-2-yl)-1,4-diazepane-1-carboxylate (pre-9m): Yield: 19%;
1H-NMR (400 MHz, CDCl3)
δ 8.17 (s, 2H), 7.28–7.22 (m, 1H), 6.78–6.70 (m, 2H), 6.71 (m, 1H), 6.65–6.60
(m, 1H), 3.90–3.88 (m, 2H), 3.80–3.78 (m, 2H), 3.59–3.58 (m, 2H), 3.40–3.39 (m, 1H), 3.33–3.30 (m, 1H),
1.99–1.96 (m, 2H), 1.44 (s, 9H).
4-(5-((3-fluorophenoxy)pyrimidin-2-yl)-1,4-diazepan-1-ium
(400 MHz, CDCl3) 8.17 (s, 2H), 7.26–7.22 (m, 1H), 6.77–6.73 (m, 1H), 6.70–6.67 (m, 1H), 6.65–6.62 (m,
1H), 4.08–4.01 (s, 2H), 3.90 (t, J = 6.3 Hz, 2H), 3.36–3.27 (m, 2H), 3.16–3.15 (m, 2H), 2.16–2.14 (m, 2H).
·
hydrochloride (9m): Yield: 40%; 1H-NMR
δ
13C-NMR (100 MHz, CD3OD) 165.0 (d, 1J = 244 Hz), 159.6, 152.3, 143.9, 132.1 (d, 3J = 10 Hz), 131.6
δ
(d, 3J = 8 Hz), 113.3, 110.7 (d, 2J = 21 Hz), 105.2 (d, 2J = 25 Hz), 46.8, 46.5, 44.7, 26.8; LRMS-EI (m/z):
[M–Cl]+ calcd for C15H17FN4O: 288.33, found: 288.33.
tert-Butyl 4-(5-((3-fluorobenzyl)oxy)pyrimidin-2-yl)-1,4-diazepane-1-carboxylate (pre-9n): Yield: 79%;
1H-NMR (400 MHz, CDCl3):
δ 8.09 (s, 2H), 7.37–7.32 (m, 1H), 7.17–7.11 (m, 2H), 7.04–6.99 (m, 1H), 4.99
(s, 2H), 3.84–3.78 (m, 2H), 3.71–3.68 (m, 2H), 3.53 (s, 2H), 3.35 (t, J = 5.8 Hz, 1H), 3.25 (t, J = 5.9 Hz, 1H),
1.96–1.90 (m, 2H), 1.42 (s, 9H, a mixture of rotamers).
4-(5-((3-fluorobenzyl)oxy)pyrimidin-2-yl)-1,4-diazepan-1-ium
1H-NMR (400 MHz, CDCl3)
8.10 (s, 2H), 7.38–7.32 (m, 1H), 7.17–7.11 (m, 2H), 7.05–7.01 (m, 1H), 5.01
(s, 2H), 3.92–3.90 (m, 2H), 3.81–3.77 (m, 2H), 3.76–3.71 (m, 2H), 3.58 (t, J = 6.0 Hz, 1H), 3.52 (t, J = 6.0
·
hydrochloride (9n): Two-step yield: 53%;
δ
Hz, 1H), 2.07–1.97 (m, 2H). 13C-NMR (100 MHz, CD3OD) 164.4 (d, 1J = 243 Hz), 157.9, 147.8, 147.1,
δ
140.9 (d, 3J = 7 Hz), 131.4 (d, 3J = 8 Hz), 124.3 (d, 4J = 3 Hz), 115.8 (d, 2J = 21 Hz), 115.3 (d, 2J = 22 Hz),
72.1, 72.1, 47.0, 46.6, 44.6, 26.9; LRMS-EI (m/z): [M–Cl]+ calcd for C16H19FN4O: 302.35, found: 302.35.
tert-Butyl 4-(5-((4-fluorophenoxy)pyrimidin-2-yl)-1,4-diazepane-1-carboxylate (pre-9p): Yield: 31%;
1H-NMR (400 MHz, CDCl3)
δ 8.14 (s, 2H), 7.01–6.96 (m, 2H), 6.87–6.75 (m, 2H), 3.89–3.84 (m, 2H),
3.76–3.73 (m, 2H), 3.58–5.55 (m, 2H), 3.39 (t, J = 6.0 Hz, 1H), 3.31 (d, J = 6.0 Hz, 1H), 3.31 (t, J = 6.0 Hz,
1H), 1.98–1.92 (m, 2H), 1.44 (s, 9H).
4-(5-((4-fluorophenoxy)pyrimidin-2-yl)-1,4-diazepan-1-ium
·
hydrochloride (9p): Yield: 34%; 1H-NMR
(400 MHz, CDCl3) 8.14 (s, 2H), 7.00–6.96 (m, 2H), 6.90–6.87 (m, 2H), 4.10–4.01 (m, 2H), 3.98–3.88 (m,
δ
1H), 3.38–3.30 (m, 2H), 3.27–3.20 (m, 2H), 2.20–2.15 (m, 2H). 13C-NMR (100 MHz, CD3OD)
δ 163.9 (d,
1J = 244 Hz), 157.9, 147.9, 147.1, 134.1, 131.0 (d, 3J = 8 Hz), 116.3 (d, 2J = 21 Hz), 72.4, 47.1, 46.6, 46.5,
44.5, 26.8; LRMS-EI (m/z): [M–Cl]+ calcd for C15H17FN4O: 288.33, found: 288.33.

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4-(5-((3-fluorophenethoxy)pyrimidin-2-yl)-1,4-diazepan-1-ium
·
hydrochloride (9o): Yield: 6.5% (2 steps);
1H-NMR (400 MHz, CDCl3)
δ
8.04 (s, 2H), 7.30–7.28 (m, 2H), 7.04–7.03 (m, 1H), 6.99–6.94 (m, 2H), 4.15
(t, J = 6.6 Hz, 2H), 3.91–3.89 (m, 2H), 3.79–3.77 (m, 2H), 3.75–3.72 (m, 2H), 3.57–3.55(m, 1H), 3,50–3.49
(m, 1H), 3.06 (t, J = 6.5 Hz, 2H), 2.05–1.98 (m, 2H). 13C-NMR (100 MHz, CD3OD)
147.4, 147.3, 147.2,
δ
131.1, 131.0, 125.9, 125.9, 116.8, 116.6, 114.2, 114.0, 71.59, 47.7, 46.66, 46.5, 36.5, 25.9; LRMS-EI (m/z):
[M–Cl]+ calcd for C17H21FN4O: 316.38, found: 316.38.
4-(5-((4-fluorobenzyl)oxy)pyrimidin-2-yl)-1,4-diazepan-1-ium
1H-NMR (400 MHz, CDCl3)
8.11 (s, 2H), 7.37 (dd, J = 8.4, 5.4 Hz, 2H), 7.08 (t, J = 8.6 Hz, 2H), 4.97
(s, 2H), 4.07–4.03 (s, 2H), 3.89–3.86 (m, 2H), 3.35–3.29 (m, 2H), 3.20–3.17 (m, 2H), 2.23–2.22 (m, 2H).
·
hydrochloride (9q): Two-step yield: 7%;
δ
13C-NMR (100 MHz, CD3OD) 165.4 (d, 1J = 243 Hz), 155.0, 147.0, 146.8, 133.5, 131.2 (d, 3J = 8 Hz),
δ
116.4 (d, 2J = 21 Hz), 72.5, 47.2, 46.9, 46.6, 27.0; LRMS-EI (m/z): [M Cl]+ calcd for C16H19FN4O: 302.35,
–
found: 302.35.
4-(5-((4-fluorophenethoxy)pyrimidin-2-yl)-1,4-diazepan-1-ium
1H-NMR (400 MHz, CDCl3)
8.04 (s, 2H), 7.22 (dd, J = 8.4, 5.5 Hz, 2H), 7.01 (t, J = 8.6 Hz, 2H), 4.12 (t,
J = 6.8 Hz, 2H), 3.92–3.89 (m, 2H), 3.81–3.77 (m, 2H), 3.75–3.71 (m, 2H), 3.57 (t, J = 6.0 Hz, 1H), 3.51
·hydrochloride (9r): Two-step yield: 3.5%;
δ
(t, J = 6.0 Hz, 1H), 3.03 (t, J = 6.7 Hz, 2H), 2.07–1.96 (m, 2H). 13C-NMR (100 MHz, CD3OD)
δ 156.4,
146.2, 145.8, 134.2, 130.3 (d, 3J = 8 Hz), 114.6 (d, 2J = 21 Hz), 70.5, 60.1, 45.6, 45.2, 43.2, 34.6, 29.5, 25.5;
LRMS-EI (m/z): [M–Cl]+ calcd for C17H21FN4O: 316.38, found: 316.38.
3.3. Synthesis of 2,4-Disubstituted Pyrimidines 10a–j
3.3.1. General Procedure for Preparing Compound (R)-(+)-16
To a solution of 1-(3 or 4-fluorophenyl)ethanol (6.67 mmol) in n-hexane (22.2 mL) was added
CAL-B (147 mg), vinyl acetate (3.34 mmol), and triethylamine (0.667 mmol). The reaction mixture
was allowed to stir at the room temperature for 1 h. After completion of the reaction (monitored by
TLC), the mixture was filtered and concentrated in vacuo. The resulting residue was purified by flash
column chromatography on silica gel (EtOAc:n-hexane = 1:8) to afford acetate intermediate (315 mg) as
a colorless oil. To a solution acetate (1.73 mmol) in MeOH (3.45 mL) was added 1M NaOH (2.59 mmol).
The reaction mixture was allowed to stir at the room temperature for 1 h. After completion of the
reaction (monitored by TLC), it was quenched with distilled water and extracted with EtOAc. The
organic layers were dried over anhydrous MgSO4 and concentrated in vacuo. The resulting residue
was purified by flash column chromatography on silica gel (EtOAc:n-hexane = 1:8) to afford alcohol
(R)-(+)-16.
(R)-1-(3-Fluorophenyl)ethan-1-ol ((R)-(+)-16a): Yield: 17%; 1H-NMR (400 MHz, CDCl3)
(m, 1H), 7.12–7.07 (m, 2H), 6.97–6.92 (m, 1H), 4.87 (q, J = 6.4 Hz, 1H), 2.18 (s, 1H), 1.47 (d, J = 6.4 Hz,
δ 7.32–7.26
3H). 13C-NMR (100 MHz, CDCl3)
δ
163.0 (d, 1J = 244 Hz), 148.5 (d, 3J = 6 Hz), 130.0 (d, 3J = 8 Hz),
121.0 (d, 4J = 3 Hz), 114.2 (d, 2J = 21 Hz), 112.3 (d, 2J = 21 Hz), 69.8, 25.2; optical rotation for (R)-(+)-16a
[α]D26 + 43.7 ◦ (c 0.7, CHCl3).
:
(R)-1-(4-Fluorophenyl)ethan-1-ol ((R)-(+)-16b): Yield: 32%; 1H-NMR (400 MHz, CDCl3)
δ
7.32–7.26
(m, 2H), 7.03–6.97 (m, 2H), 4.84 (q, J = 6.1 Hz, 1H), 2.34 (s, 1H), 1.44 (d, J = 6.4 Hz, 3H). 13C-NMR (100
MHz, CDCl3) δ
162.1 (d, 1J = 244 Hz), 141.6 (d, 4J = 3 Hz), 127.1 (d, 3J = 8 Hz), 115.2 (d, 2J = 21 Hz),
69.7, 25.3; optical rotation for (R)-(+)-16b: [α]D27 + 51.9 ◦ (c 0.5, CHCl3).
3.3.2. General Procedure for Preparing Compound 17
A solution of sodium tert-butoxide (1.43 mmol) in toluene (7.10 mL) was treated with primary
or secondary alcohol 16 (0.713 mmol) dropwise at 0 ◦C. After 5 min, 2,4-dichloro-5-fluoropyrimidine
(0.713 mmol) was added to the mixture. The reaction mixture was allowed to stir at the room
temperature for 1 h. After completion of the reaction (monitored by TLC), it was quenched with
saturated aqueous NH4Cl, extracted with EtOAc, and washed with brine. The organic layers were

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dried over anhydrous MgSO4 and concentrated in vacuo. The resulting residue was purified by flash
column chromatography on silica gel (EtOAc:n-hexane = 1:8) to afford pyrimidine 17.
1
(R)-2-Chloro-5-fluoro-4-(1-(3-fluorophenyl)ethoxy)pyrimidine ((R)-17a): Yield: 72%; H-NMR (400
MHz, CDCl3) δ 8.19 (d, J = 2.2 Hz, 1H), 7.37–7.31 (m, 1H), 7.23 (d, J = 7.7 Hz, 1H), 7.18–7.15 (m, 1H),
7.03–6.99 (m, 1H), 6.30 (q, J = 6.5 Hz, 1H), 1.71 (d, J = 6.6 Hz, 3H). 13C-NMR (100 MHz, CDCl3)
δ 162.9
(d, 1J = 245 Hz), 158.6 (d, 2J = 11 Hz), 153.2 (d, 4J = 5 Hz), 146.0 (d, 1J = 263 Hz), 144.4 (d, 2J = 20 Hz),
142.9 (d, 3J = 7 Hz), 130.3 (d, 3J = 8 Hz), 122.0 (d, 4J = 3 Hz), 115.3 (d, 2J = 21 Hz), 113.3 (d, 2J = 22 Hz),
27
75.6 (d, 4J = 2 Hz), 22.2; optical rotation for (R)-6d: [α]D +178.3 ◦ (c 0.7, CHCl3).
(R)-2-Chloro-5-fluoro-4-(1-(4-fluorophenyl)ethoxy)pyrimidine ((R)-17b): Yield: 68%; 1H-NMR (400
MHz, CDCl3)
δ 8.16 (d, J = 2.2 Hz, 1H), 7.47–7.42 (m, 2H), 7.08–7.02 (m, 2H), 6.30 (q, J = 6.6 Hz, 1H),
1.71 (d, J = 6.6 Hz, 3H). 13C-NMR (100 MHz, CDCl3)
δ
162.6 (d, 1J = 245 Hz), 158.7 (d, 2J = 11 Hz), 153.1
(d, 4J = 4 Hz), 146.0 (d, 1J = 263 Hz), 144.3 (d, 2J = 20 Hz), 136.1 (d, 4J = 4 Hz), 128.4 (d, 3J = 9 Hz), 115.6
(d, 2J = 22 Hz), 75.9, 22.2; optical rotation for (R)-6e: [α]D27 +197.3 ◦ (c 0.8, CHCl3).
2-Chloro-5-fluoro-4-(3-fluorophenoxy)pyrimidine (19a): Yield: 51%; 1H-NMR (400MHz, CDCl3)
δ
8.38 (d, J = 2.0 Hz, 1H), 7.45–7.39 (m, 1H), 7.06–7.01 (m, 2H), 6.99–6.96 (m, 1H). 13C-NMR (100 MHz,
CD3OD) δ
164.5 (d, 1J = 245 Hz), 159.8 (d, 2J = 11 Hz), 154.2 (d, 4J = 4 Hz), 153.7 (d, 3J = 11 Hz), 147.4
(d, 1J = 262 Hz), 147.4 (d, 2J = 20 Hz), 132.0 (d, 3J = 9 Hz), 118.5 (d, 4J = 3 Hz), 114.4 (d, 2J = 21 Hz),
110.5 (d, 2J = 25 Hz).
2-Chloro-5-fluoro-4-(4-fluorophenoxy)pyrimidine (19b): Yield: 52%; 1H-NMR (400MHz, CDCl3)
δ
8.36 (d, J = 2.0 Hz, 1H), 7.19–7.11 (m, 4H). 13C-NMR (100 MHz, CD3OD) 162.0 (d, 1J = 242 Hz),
δ
160.2 (d, 2J = 11 Hz), 154.2 (d, 4J = 5 Hz), 148.7 (d, 4J = 2 Hz), 147.2 (d, 2J = 20 Hz), 146.10, 124.3 (d,
3J = 9 Hz), 117.4 (d, 2J = 24 Hz).
3.3.3. General Procedure for Preparing Compounds pre-10b–e and pre-10g–j
To a solution of pyrimidine 17 (0.154 mmol) in toluene (0.300 mL) was added cyclic amine (0.231
mmol) and triethylamine (0.231 mmol). The reaction mixture was allowed to stir at 90 ◦C for 12 h.
After completion of the reaction (monitored by TLC), it was quenched with saturated aqueous NH4Cl,
extracted with EtOAc, and washed with brine. The organic layers were dried over anhydrous MgSO4
and concentrated in vacuo. The resulting residue was purified by flash column chromatography on
silica gel (EtOAc/n-hexane = 1:3) to afford carboxylate pre-10b–e and pre-10g–j.
tert-Butyl 7-(5-fluoro-4-((R)-1-(3-fluorophenyl)ethoxy)pyrimidin-2-yl)-2,7-diazaspiro[4.4]nonane-2-
carboxylate (pre-10b): Yield: 92%; 1H-NMR (400 MHz, CDCl3, a mixture of rotamers)
δ 7.94 (s, 1H),
7.31–7.26 (m, 1H), 7.17 (d, J = 7.6 Hz, 1H), 7.11 (d, J = 9.6 Hz, 1H) 6.95 (t, J = 8.3 Hz, 1H), 6.15–6.10 (m,
1H), 3.59–3.23 (m, 8H), 1.94–1.83 (m, 4H), 1.66 (d, J = 6.6 Hz, 3H), 1.45 (s, 9H). 13C-NMR (100 MHz,
CDCl3, a mixture of rotamers)
δ
162.9 (d, 1J = 244 Hz), 157.1 (d, 2J = 11 Hz), 156.0, 154.6, 145.0 (d, 3J = 7
Hz), 143.4 (d, 2J = 20 Hz), 140.0 (d, 1J = 245 Hz), 130.0 (d, 3J = 8 Hz), 121.6, 114.6 (d, 2J = 21 Hz), 113.1
(d, 2J = 22 Hz), 79.4, 73.7, 55.9, 55.3, 54.7, 54.6, 48.6, 47.8, 46.0, 45.2, 35.3, 34.9, 34.5, 28.5, 22.7.
tert-Butyl (3aR,6aS)-5-(5-fluoro-4-((R)-1-(3-fluorophenyl)ethoxy)pyrimidin-2-yl) hexahydropyrrolo
[3,4-c]pyrrole-2(1H)-carboxylate (pre-10c): Yield: 82%; 1H-NMR (400 MHz, CDCl3, a mixture of rotamers)
δ 7.94 (d, J = 3.1 Hz, 1H), 7.32–7.28 (m, 1H), 7.17 (d, J = 7.7 Hz, 1H), 7.11 (dd, J = 2.0, 9.7 Hz, 1H), 6.95
(td, J = 2.0, 8.4 Hz, 1H), 6.14 (q, J = 6.6 Hz, 1H), 3.69–3.59 (m, 4H), 3.40–3.21 (m, 4H), 2.94 (bs, 2H), 1.66
(d, J = 6.6 Hz, 3H), 1.47 (s, 9H). 13C-NMR (100 MHz, CDCl3, a mixture of rotamers)
δ
162.9 (d, 1J = 244
Hz), 157.1 (d, 2J = 11 Hz), 156.0 (d, 4J = 3 Hz), 154.5, 145.0 (d, 3J = 10 Hz), 143.4 (d, 2J = 20 Hz), 140.1 (d,
1J = 246 Hz), 130.0 (d, 3J = 8 Hz), 121.6, 114.6 (d, 2J = 21 Hz), 113.0 (d, 2J = 21 Hz), 79.5, 73.6, 50.8, 50.1,
49.8, 42.2, 41.2, 28.5, 22.7.
tert-Butyl
(R)-3-((5-fluoro-4-((R)-1-(3-fluorophenyl)ethoxy)pyrimidin-2-yl)aminopyrrolidine-1-
7.89 (s, 1H),
carboxylate (pre-10d): Yield: 68%; 1H-NMR (400 MHz, CDCl3, a mixture of rotamers)
δ
7.31–7.28 (m, 1H), 7.14 (d, J = 7.7 Hz, 1H), 7.08 (d, J = 9.6 Hz, 1H) 6.94 (t, J = 7.9 Hz, 1H), 6.08 (d, J =
4.4 Hz, 1H), 5.12 (bs, 1H), 4.24–4.16 (m, 1H), 3.71–3.65 (m, 1H), 3.43–3.37 (m, 2H), 3.28–3.12 (m, 1H),
2.01–1.98 (m, 1 H), 1.65 (d, J = 6.6 Hz, 3H), 1.45 (s, 9H). 13C-NMR (100 MHz, CDCl3, a mixture of

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rotamers) δ
162.9 (d, 1J = 245 Hz), 157.6 (d, 2J = 11 Hz), 157.3, 154.6, 144.7 (d, 3J = 6 Hz), 143.7 (d, 2J =
20 Hz), 140.6 (d, 1J = 248 Hz), 130.1 (d, 3J = 8 Hz), 121.3, 114.6 (d, 2J = 22 Hz), 112.8 (d, 2J = 22 Hz), 79.5,
73.9, 51.8, 51.7, 51.4, 51.0, 44.1, 43.7, 31.8, 31.1, 28.5, 22.8.
tert-Butyl
(S)-3-((5-fluoro-4-((R)-1-(3-fluorophenyl)ethoxy)pyrimidin-2-yl)aminopyrrolidine-1-
7.90 (d, J = 2.9
carboxylate (pre-10e): Yield: 52%; 1H-NMR (400 MHz, CDCl3, a mixture of rotamers)
δ
Hz, 1H), 7.33–7.26 (m, 1H), 7.15 (d, J = 7.7 Hz, 1H), 7.10 (d, J = 9.6 Hz, 1H) 6.98–6.94 (m, 1H), 6.11 (q, J
= 6.5 Hz, 1H), 5.12 (bs, 1H), 4.28 (bs, 1H), 3.47–3.42 (m, 3H), 3.21–3.04 (m, 1H), 2.20–2.12 (m, 1H), 1.86
(bs, 1H), 1.65 (d, J = 6.6 Hz, 3H), 1.45 (s, 9H). 13C-NMR (100 MHz, CDCl3, a mixture of rotamers)
δ
162.9 (d, 1J = 245 Hz), 157.6 (d, 2J = 11 Hz), 157.2, 154.6, 143.6 (d, 2J = 20 Hz), 143.3 (d, 1J = 271 Hz),
139.4, 130.1 (d, 3J = 8 Hz), 121.4, 114.7 (d, 2J = 21 Hz), 112.8 (d, 2J = 22 Hz), 79.5, 73.8, 51.9, 51.6, 51.4,
51.0, 44.1, 43.8, 31.9, 31.1, 28.5, 22.8.
tert-Butyl 7-(5-fluoro-4-((R)-1-(4-fluorophenyl)ethoxy)pyrimidin-2-yl)-2,7-diazaspiro[4.4]nonane-2-
carboxylate (pre-10g): Yield: 98%; 1H-NMR (400 MHz, CDCl3, a mixture of rotamers)
δ
7.93 (s, 1H),
7.40–7.37 (m, 2H), 7.03–6.99 (m, 2H), 6.18–6.13 (m, 1H), 3.61–3.22 (m, 8H), 1.95–1.82 (m, 4H), 1.66 (d, J =
6.6 Hz, 3H), 1.45 (s, 9H). 13C-NMR (100 MHz, CDCl3, a mixture of rotamers) 162.2 (d, 1J = 244 Hz),
δ
157.2 (d, 2J = 11 Hz), 156.0, 154.6, 143.3 (d, 2J = 20 Hz), 140.1 (d, 1J = 246 Hz), 138.0, 127.9 (d, 3J = 8 Hz),
115.3 (d, 2J = 21 Hz), 79.4, 73.6, 55.9, 55.4, 54.8, 54.6, 48.6, 47.8, 46.0, 45.2, 44.9, 35.3, 34.9, 34.5, 28.5, 22.8.
tert-Butyl
[3,4-c]pyrrole-2(1H)-carboxylate (pre-10h): Yield: 93%; 1H-NMR (400 MHz, CDCl3, a mixture of
rotamers) 7.92 (d, J = 3.1 Hz, 1H), 7.40–7.36 (m, 2H), 7.00 (t, J = 8.6 Hz, 2H), 6.15 (d, J = 5.7 Hz, 1H),
3.73–3.60 (m, 4H), 3.41–3.21 (m, 4H), 2.91 (bs, 2H), 1.64 (d, J = 6.6 Hz, 3H), 1.44 (s, 9H). 13C-NMR (100
(3aR,6aS)-5-(5-fluoro-4-((R)-1-(4-fluorophenyl)ethoxy)pyrimidin-2-yl)hexahydropyrrolo
δ
MHz, CDCl3, a mixture of rotamers)
δ
162.2 (d, 1J = 245 Hz), 157.2 (d, 2J = 11 Hz), 156.0 (d, 4J = 3 Hz),
154.5, 143.3 (d, 2J = 20 Hz), 140.1 (d, 1J = 245 Hz), 138.0, 127.8 (d, 3J = 8 Hz), 115.3 (d, 2J = 21 Hz), 79.5,
73.6, 50.8, 50.1, 49.8, 42.2, 41.2, 28.5, 22.8.
tert-Butyl
(R)-3-((5-fluoro-4-((R)-1-(4-fluorophenyl)ethoxy)pyrimidin-2-yl)amino)pyrrolidine-1-
7.88 (d, J = 2.0
carboxylate (pre-10i): Yield: 52%; 1H-NMR (400 MHz, CDCl3, a mixture of rotamers)
δ
Hz, 1H), 7.38–7.34 (m, 2H), 7.01 (t, J = 8.5 Hz, 2H), 6.14–6.09 (m, 1H), 5.13 (bs, 1H), 4.26–4.19 (m, 1H),
3.69–3.66 (m, 1H), 3.40 (bs, 2H), 3.29–3.16 (m, 1H), 2.09–2.01 (m, 1H), 1.74–1.73 (m, 1H), 1.64 (d, J = 6.6
Hz, 3H), 1.46 (s, 9H). 13C-NMR (100 MHz, CDCl3, a mixture of rotamers)
δ
162.2 (d, 1J = 245 Hz), 157.7
(d, 2J = 11 Hz), 157.3, 154.6, 143.5 (d, 2J = 21 Hz), 140.6 (d, 1J = 246 Hz), 137.8 (d, 2J = 20 Hz), 127.6 (d, 3J
= 12 Hz), 115.4 (d, 2J = 21 Hz), 79.5, 73.9, 51.8, 51.7, 51.5, 51.0, 44.1, 43.8, 31.8, 31.1, 28.5, 22.8.
tert-Butyl
(S)-3-((5-fluoro-4-((R)-1-(4-fluorophenyl)ethoxy)pyrimidin-2-yl)aminopyrrolidine-1-
7.89 (d, J = 3.0
carboxylate (pre-10j): Yield: 44%; 1H-NMR (400 MHz, CDCl3, a mixture of rotamers)
δ
Hz, 1H), 7.39–7.36 (m, 2H), 7.03 (t, J = 8.6 Hz, 2H), 6.14 (q, J = 6.5 Hz, 1H), 4.98 (bs, 1H), 4.31 (m, 1H),
3.62–3.44 (m, 3H), 3.24–3.06 (m, 1H), 2.28-2.13 (m, 1H), 1.86 (bs, 1H), 1.64 (d, J = 6.6 Hz, 3H), 1.45 (s,
9H). 13C-NMR (100 MHz, CDCl3, a mixture of rotamers)
δ
162.3 (d, 1J = 244 Hz), 157.7 (d, 2J = 11 Hz),
157.2, 154.6, 143.5 (d, 2J = 19 Hz), 140.8 (d, 1J = 247 Hz), 137.7, 127.7, 130.1, 115.4 (d, 2J = 21 Hz), 79.5,
73.8, 52.0, 51.7, 51.5, 51.0, 44.1, 43.8, 31.9, 31.1, 29.8, 28.5, 22.8.
tert-Butyl
(R)-4-(5-fluoro-4-(3-fluorophenoxy)pyrimidin-2-yl)-3-methylpiperazine-1-carboxylate
8.13 (d, J = 2.7 Hz, 1H), 7.39–7.33 (m, 1H), 6.99–6.94
(
pre-20a): Yield: 74%; 1H-NMR (400MHz, CDCl3)
δ
(m, 3H), 4.44 (m, 1H), 4.05–3.80 (m, 3H), 3.06–3.2.99 (m, 2H), 2.99–2.82 (m, 1H), 1.45 (s, 9H), 1.04 (d,
J = 6.6 Hz, 3H).
tert-Butyl
(R)-4-(5-fluoro-4-(4-fluorophenoxy)pyrimidin-2-yl)-3-methylpiperazine-1-carboxylate
8.11 (d, J = 2.7 Hz, 1H), 7.15–7.12 (m, 2H), 7.11–7.06
(
pre-20b): Yield: 61%; 1H-NMR (400MHz, CDCl3)
δ
(m, 2H), 4.50–4.30 (m, 1H), 4.03–3.80 (m, 3H), 3.04–2.97 (m, 2H), 2.97–2.81 (m, 1H), 1.45 (s, 9H), 1.03 (d,
J = 6.6 Hz, 3H).
3.3.4. General Procedure for Preparing Compounds 10a and 10f
To a solution of pyrimidine 17 (0.115 mmol) in toluene (0.250 mL) was added 1,4-diazepane
(0.230 mmol) and triethylamine (0.230 mmol). The reaction mixture was allowed to stir at 90 ◦C for 12 h.

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After completion of the reaction (monitored by TLC), it was quenched with saturated aqueous NH4Cl,
extracted with EtOAc, and washed with brine. The organic layers were dried over anhydrous MgSO4
and concentrated in vacuo. The resulting residue was purified by flash column chromatography on
silica gel (DCM/MeOH = 10:1) to afford 2,4-disubstituted pyrimidine 10a and 10f.
(R)-1-(5-fluoro-4-(1-(3-fluorophenyl)ethoxy)pyrimidin-2-yl)-1,4-diazepane (10a): Yield: 68%; 1H-NMR
(400 MHz, CD3OD)
δ 8.06 (d, J = 3.2 Hz, 1H), 7.23 (d, J = 7.7 Hz, 1H), 7.15 (d, J = 9.8 Hz, 1H), 7.00 (td, J
= 2.1, 8.5 Hz, 1H), 6.11 (q, J = 6.5 Hz, 1H), 3.90–3.77 (m, 4H), 3.26–3.03 (m, 4H), 2.07–1.94 (m, 2H), 1.66
(d, J = 6.6 Hz, 3H). 13C-NMR (100 MHz, CD3OD) 163.0 (d, 1J = 244 Hz), 157.7 (d, 2J = 11 Hz), 155.9,
δ
145.5 (d, 3J = 7 Hz), 142.5 (d, 2J = 21 Hz), 140.3 (d, 1J = 246 Hz), 130.3 (d, 3J = 8 Hz), 121.1 (d, 4J = 3 Hz),
114.1 (d, 2J = 21 Hz), 112.0 (d, 2J = 22 Hz), 74.7, 45.5, 45.3, 45.0, 43.3, 25.3, 22.0. LRMS-EI (m/z): [M+H]+
calcd for C17H21F2N4O: 335.17, found: 335.10.
(R)-1-(5-fluoro-4-(1-(4-fluorophenyl)ethoxy)pyrimidin-2-yl)-1,4-diazepane (10f): Yield: 79%; 1H-NMR
(400 MHz, CD3OD)
δ 8.05 (d, J = 3.2 Hz, 1H), 7.45 (dd, J = 5.4, 8.4 Hz, 1H), 7.11–7.07 (m, 2H), 6.16 (q, J =
6.5 Hz, 1H), 4.10–3.72 (m, 4H), 3.42–3.15 (m, 4H), 2.21–1.98 (m, 2H), 1.66 (d, J = 6.5 Hz, 3H). 13C-NMR
(100 MHz, CD3OD)
δ
162.3 (d, 1J = 243 Hz), 158.0 (d, 2J = 12 Hz), 155.60, 141.7 (d, 2J = 20 Hz), 140.3 (d,
1J = 246 Hz), 138.3 (d, 4J = 3 Hz), 127.4 (d, 3J = 8 Hz), 115.0 (d, 2J = 22 Hz), 75.0, 45.6, 45.4, 45.0, 43.4,
25.3, 22.1. LRMS-EI (m/z): [M+H]+ calcd for C17H21F2N4O: 335.17, found: 335.10.
3.3.5. General Procedure for Preparing Compounds 10b–e and 10g–j
To a solution of carboxylate 17 (0.144 mmol) in CH3CN (1.45 mL) was added 4M HCl in dioxane
(1.44 mmol) at 0 ◦C. The reaction mixture was allowed to stir at the same temperature for 1 h. After
completion of the reaction (monitored by TLC), the mixture was diluted with saturated aqueous
NaHCO3 and extracted with EtOAc. The organic layers were dried over anhydrous MgSO4 and
concentrated in vacuo. The resulting residue was purified by flash column chromatography on silica
gel (DCM/MeOH = 10:1) to afford compound 10.
2-(5-fluoro-4-((R)-1-(3-fluorophenyl)ethoxy)pyrimidin-2-yl)-2,7-diazaspiro[4.4]nonane (10b): Yield: 66%;
1H-NMR (400 MHz, CD3OD, a mixture of rotamers)
δ 8.00 (d, J = 3.4 Hz, 1H), 7.35–7.30 (m, 1H), 7.21
(d, J = 7.7 Hz, 1H), 7.13 (d, J = 9.8 Hz, 1H), 6.97 (td, J = 1.7, 8.4 Hz, 1 H), 6.23–6.16 (m, 1H), 3.59–3.37
(m, 6H), 3.30–3.23 (m, 2H), 2.11–1.98 (m, 4H), 1.64 (d, J = 6.6 Hz, 3H). 13C-NMR (100 MHz, CD3OD, a
mixture of rotamers) δ
162.9 (d, 1J = 243 Hz), 158.3 (d, 2J = 12 Hz), 154.39, 144.7 (d, 3J = 9 Hz), 140.0
(d, 2J = 21 Hz), 139.8 (d, 1J = 246 Hz), 130.1 (d, 3J = 8 Hz), 121.5 (d, 4J = 3 Hz), 114.3 (d, 2J = 21 Hz),
112.5 (d, 2J = 22 Hz), 75.0, 74.9, 55.3, 55.5, 46.0, 44.8, 44.7, 34.3, 34.2, 33.8, 33.7, 21.5, 21.5. LRMS-EI (m/z):
[M+H]+ calcd for C19H23F2N4O: 361.18, found: 361.10.
(3aR,6aS)-2-(5-fluoro-4-((R)-1-(3-fluorophenyl)ethoxy)pyrimidin-2-yl)octahydropyrrolo[3,4-c]pyrrole
1
(
10c): Yield: 92%; H-NMR (400 MHz, CD3OD, a mixture of rotamers)
δ
8.26 (d, J = 4.4 Hz, 1H),
7.44–7.38 (m, 1H), 7.32 (d, J = 7.7 Hz, 1H), 7.24 (dd, J = 1.9, 9.7 Hz, 1H), 7.06 (td, J = 2.3, 8.4 Hz, 1H),
3.91 (bs, 1H), 3.80 (bs, 2H), 3.68–3.58 (m, 3H), 3.37–3.28 (m, 4H), 1.75 (d, J = 6.5 Hz, 3H). 13C-NMR (100
MHz, CD3OD, a mixture of rotamers)
δ
162.9 (d, 1J = 244 Hz), 157.8 (d, 2J = 12 Hz), 152.12, 149.9, 143.9
(d, 3J = 7 Hz), 139.6 (d, 1J = 248 Hz), 135.6 (d, 2J = 26 Hz), 130.3 (d, 3J = 8 Hz), 121.7 (d, 4J = 3 Hz), 114.6
(d, 2J = 21 Hz), 112.6 (d, 2J = 22 Hz), 79.6, 50.9, 50.8, 50.7, 49.6, 49.4, 41.6, 41.5, 21.3. LRMS-EI (m/z):
[M+H]+ calcd for C18H21F2N4O: 347.17, found: 347.05.
5-fluoro-4-((R)-1-(3-fluorophenyl)ethoxy)-N-((R)-pyrrolidin-3-yl)pyrimidin-2-amine (10d): Yield: 71%;
1H-NMR (400 MHz, CD3OD, a mixture of rotamers)
δ 7.97 (d, J = 3.1 Hz, 1H), 7.36–7.29 (m, 1H), 7.18 (d,
J = 7.7 Hz, 1H), 7.10 (d, J = 9.8 Hz, 1H), 6.96 (td, J = 2.3, 8.5 Hz, 1H), 6.17 (q, J = 6.5 Hz, 1H), 4.38–4.33
(m, 1H), 3.49–3.30 (m, 1H), 3.26–3.25 (m, 3H), 2.30–2.14 (m, 1H), 2.09–1.93 (m, 1 H), 1.61 (d, J = 6.6 Hz,
3H). 13C-NMR (100 MHz, CD3OD, a mixture of rotamers)
δ
163.0 (d, 1J = 243 Hz), 158.0 (d, 2J = 12 Hz),
156.9, 144.9 (d, 3J = 7 Hz), 142.2 (d, 2J = 17 Hz), 140.6 (d, 1J = 247 Hz), 130.2 (d, 3J = 8 Hz), 121.2 (d, 4J
= 3 Hz), 114.1 (d, 2J = 21 Hz), 112.1 (d, 2J = 22 Hz), 74.1, 51.0, 50.3, 50.1, 44.1, 29.7, 29.5, 21.74, 21.66.
LRMS-EI (m/z): [M+H]+ calcd for C16H19F2N4O: 321.15, found: 321.05.

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5-fluoro-4-((R)-1-(3-fluorophenyl)ethoxy)-N-((S)-pyrrolidin-3-yl)pyrimidin-2-amine (10e): Yield: 42%;
1H-NMR (400 MHz, CD3OD, a mixture of rotamers)
7.93–7.92 (m, 1H), 7.34–7.28 (m, 1H), 7.17 (d, J
δ
= 7.7 Hz, 1H), 7.09 (d, J = 9.9 Hz, 1H), 6.97–6.92 (m, 1H), 6.15 (q, J = 6.5 Hz, 1H), 4.34–4.29 (m, 1H),
3.49–3.38 (m, 2H), 3.35–3.25 (m, 2H), 2.98–2.11 (m, 1H), 2.10–1.89 (m, 1 H), 1.59 (d, J = 6.6 Hz, 3H).
13C-NMR (100 MHz, CD3OD, a mixture of rotamers)
δ
162.9 (d, 1J = 243 Hz), 157.7 (d, 2J = 11 Hz),
157.3 (d, 4J = 3 Hz), 145.0 (d, 3J = 7 Hz), 143.0 (d, 2J = 20 Hz), 140.7 (d, 1J = 246 Hz), 130.1 (d, 3J = 8 Hz),
121.3 (d, 4J = 3 Hz), 114.1 (d, 2J = 21 Hz), 112.1 (d, 2J = 22 Hz), 73.8, 51.0, 50.2, 44.1, 29.7, 29.6, 21.8, 21.7.
LRMS-EI (m/z): [M+H]+ calcd for C16H19F2N4O: 321.15, found: 321.05.
2-(5-fluoro-4-((R)-1-(4-fluorophenyl)ethoxy)pyrimidin-2-yl)-2,7-diazaspiro[4.4]nonane (10g): Yield: 58%;
1H-NMR (400 MHz, CD3OD, a mixture of rotamers)
δ
7.90 (d, J = 3.3 Hz, 1H), 7.44–7.40 (m, 2H), 7.03 (t,
J = 8.8 Hz, 2H), 6.22–6.15 (m, 1H), 3.59–3.35 (m, 6H), 3.30–3.19 (m, 2H), 2.09–1.96 (m, 4H), 1.61 (d, J =
6.6 Hz, 3H). 13C-NMR (100 MHz, CD3OD, a mixture of rotamers)
δ
162.3 (d, 1J = 243 Hz), 157.5 (d, 2J =
11 Hz), 155.9, 142.7 (d, 2J = 20 Hz), 140.0 (d, 1J = 245 Hz), 138.3 (d, 4J = 7 Hz), 127.8 (d, 3J = 8 Hz), 114.8
(d, 2J = 21 Hz), 74.03, 73.97, 55.2, 52.7, 45.7, 44.8, 34.4, 34.3, 33.9, 33.8, 21.7, 21.6. LRMS-EI (m/z): [M+H]+
calcd for C19H23F2N4O: 361.18, found: 361.10.
(3aR,6aS)-2-(5-fluoro-4-((R)-1-(4-fluorophenyl)ethoxy)pyrimidin-2-yl)octahydropyrrolo[3,4-c]pyrrole
(
10h): Yield: 53%; 1H-NMR (400 MHz, CD3OD, a mixture of rotamers)
δ
7.96 (d, J = 2.8 Hz, 1H), 7.27
(bs, 2H), 6.86 (t, J = 8.0 Hz, 1H), 6.11 (bs, 1H), 3.66–3.54 (m, 3H), 3.43–3.36 (m, 3H), 3.07–3.03 (m, 4H),
1.48 (d, J = 3.0 Hz, 3H). 13C-NMR (100 MHz, CD3OD, a mixture of rotamers) 162.8 (d, 1J = 244 Hz),
δ
161.1 (d, 2J = 12 Hz), 150.0, 139.3 (d, 1J = 250 Hz), 136.4 (d, 4J = 3 Hz), 131.6 (d, 2J = 20 Hz), 128.4 (d, 3J
= 8 Hz), 115.2 (d, 2J = 21 Hz), 78.2, 51.4, 51.2, 49.5, 41.7, 41.6, 21.2. LRMS-EI (m/z): [M+H]+ calcd for
C18H21F2N4O: 347.17, found: 347.05.
5-fluoro-4-((R)-1-(4-fluorophenyl)ethoxy)-N-((R)-pyrrolidin-3-yl)pyrimidin-2-amine (10i): Yield: 51%;
1H-NMR (400 MHz, CD3OD, a mixture of rotamers) δ 7.93 (d, J = 3.3 Hz, 1H), 7.49–7.45 (m, 2H), 7.10 (t,
J = 8.7 Hz, 2H), 6.25–6.22 (m, 1H), 4.31–4.30 (m, 1H), 3.56–3.40 (m, 3H), 3.15–3.06 (m, 1H), 2.23–2.16
(m, 1H), 1.94–1.90 (m, 1 H), 1.66 (d, J = 6.6 Hz, 3H). LRMS-EI (m/z): [M+H]+ calcd for C16H19F2N4O:
321.15, found: 321.00.
5-Fluoro-4-((R)-1-(4-fluorophenyl)ethoxy)-N-((S)-pyrrolidin-3-yl)pyrimidin-2-amine (10j): Yield: 27%;
1H-NMR (400 MHz, CD3OD, a mixture of rotamers)
δ 7.92–7.91 (m, 1H), 7.42–7.39 (m, 2H), 7.06–7.01
(m, 2H), 6.18 (td, J = 4.8, 6.5 Hz, 1H), 4.39–4.30 (m, 1H), 3.50–3.39 (m, 2H), 3.36–3.28 (m, 2H), 2.39–2.14
(m, 1H), 2.10–1.90 (m, 1 H), 1.59 (d, J = 6.6 Hz, 3H). 13C-NMR (100 MHz, CD3OD, a mixture of rotamers)
δ
162.3 (d, 1J = 243 Hz), 157.8 (d, 2J = 11 Hz), 157.3 (d, 4J = 3 Hz), 143.0 (d, 2J = 20 Hz), 140.8 (d, 1J = 233
Hz), 138.1, 127.7 (d, 3J = 8 Hz), 114.9 (d, 2J = 22 Hz), 73.9, 51.0, 50.4, 50.3, 44.2, 29.7, 29.6, 21.8, 21.7.
LRMS-EI (m/z): [M+H]+ calcd for C16H19F2N4O: 321.15, found: 321.05.
(R)-4-(5-Fluoro-4-(3-fluorophenoxy)pyrimidin-2-yl)-3-methylpiperazin-1-ium
·
hydrochloride (20a): Yield:
1
58%; H-NMR (400MHz, CDCl3)
δ 8.13 (s, 1H), 7.37–7.31 (m, 1H), 7.00–6.93 (m, 3H), 4.39–4.38 (m,
1H), 4.07–4.03 (m, 1H), 2.98–2.86 (m, 3H), 2.80–2.77 (m, 1H), 2.70–2.62 (m, 1H), 1.12 (d, J = 6.8 Hz,
3H). 13C-NMR (100 MHz, CD3OD) 164.4 (d, 1J = 245 Hz), 158.7 (d, 2J = 11 Hz), 157.2 (d, 3J = 8 Hz),
δ
154.4 (d, 3J = 11 Hz), 146.7 (d, 3J = 20 Hz), 141.4 (d, 1J = 248 Hz), 131.7 (d, 3J = 9 Hz), 118.8 (d, 4J = 3
Hz), 113.7 (d, 2J = 21 Hz), 110.6 (d, 2J = 24 Hz), 45.7, 44.3, 36.6, 13.5. LRMS-EI (m/z): [M-Cl]+ calcd for
C15H17F2N4O: 306.32, found: 306.32.
(R)-4-(5-Fluoro-4-(4-fluorophenoxy)pyrimidin-2-yl)-3-methylpiperazin-1-ium
65%; 1H-NMR (400MHz, CDCl3):
8.08 (d, J = 2.6 Hz, 1H), 7.13 (dd, J = 9.0, 4.5 Hz, 2H), 7.09–7.03 (m,
2H), 4.50–4.30 (s, 1H), 4.03–4.00 (m, 1H), 2.96–2.83 (m, 3H), 2.77–2.74 (m, 1H), 2.68–2.60 (m, 1H), 1.09 (d,
·
hydrochloride (20b): Yield:
δ
J = 6.6 Hz, 3H). 13C-NMR (100 MHz, CD3OD): 161.6 (d, 1J = 241 Hz), 159.2 (d, 2J = 11 Hz), 149.3 (d, 4J
δ
= 22 Hz), 146.4 (d, 2J = 20 Hz), 141.5 (d, 1J = 248 Hz), 124.5 (d, 3J = 8 Hz), 117.1 (d, 2J = 23 Hz), 48.0,
45.6, 44.2, 36.5, 13.5. LRMS-EI (m/z): [M-Cl]+ calcd for C15H17F2N4O: 306.32, found: 306.32

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3.4. Molecular Docking Study
Discovery studio 2019 software were used for modeling study [34]. Crystal structures of 5-HT2B
(PDB ID = 4IB4) and 5-HT2C (PDB ID= 6BQH) proteins were downloaded from protein data bank.
Ligand 10a and
8 were prepared at 7.4 pH. Proteins and ligands were prepared and minimized using
CHARm forcefield. Binding sites were created over co-crystal ligands binding position. Ligands were
docked using CDOCKER scoring function. Twenty binding poses were generated for each ligands.
Obtained docked poses were analyzed based on docking scores and binding site interactions. Images
were rendered using PyMOL software (www.pymol.org).
3.5. In Vitro Assay
3.5.1. Serotonin Receptor Cell-Based Functional Assays
The synthesized compounds were tested in agonist mode with the 5-HT2C receptor assay using
recombinant human HEK-293 cells serviced by either Eurofins Cerep or DGMIF (Daegu-Gyeongbuk
Medical Innovation Foundation). All the experiments were duplicated. The detailed assay protocols
refer to the literature procedure [35].
3.5.2. Serotonin Receptor Binding Affinity Assays
Eleven dilutions (5 x assay concentration) of the test and reference compounds (Table S1) were
prepared in standard binding buffer (50 mM tris(hydroxymethyl)-aminomethane–HCl (Tris-HCl),
10 mM MgCl2, 1 mM ethylenediaminetetraacetate (EDTA), pH 7.4) by serial dilution: 0.05 nM, 0.5 nM,
1.5 nM, 5 nM, 15 nM, 50 nM, 150 nM, 500 nM, 1.5
was diluted to five times the assay concentration in standard binding buffer. Aliquots (50
radioligand were dispensed into the wells of a 96-well plate containing 100 L of standard binding
buffer. Triplicate aliquots (50 L) of the test and reference compound dilutions were then added.
Finally, crude membrane fractions (50 L) of cells (HEK293 or CHO) expressing human recombinant
receptors were dispensed into each well. A total 250 L of the reaction mixtures was incubated at room
µM, 5
µM, and 50
µM. The radioligand (Table S1)
µL) of the
µ
µ
µ
µ
temperature and shielded from light for 1.5 h, then harvested by rapid filtration onto Whatman GF/B
glass fiber filters presoaked with 0.3% polyethyleneimine, by using a 96-well Brandel harvester.
Four rapid washes were performed with chilled standard binding buffer (500 µL) to decrease
nonspecific binding. Filters were placed in 6 mL scintillation tubes and allowed to dry overnight. The
next day, 4 ml of EcoScint scintillation cocktail (National Diagnostics) was added to each tube. The
tubes were capped, labeled, and counted by liquid scintillation counting. The filter mats were dried,
and the scintillant was melted onto the filters, then the radioactivity retained on the filters was counted
in a Microbeta scintillation counter. The IC50 values were obtained by using the Prism 4.0 program
(GraphPad Software, San Diego, CA) and converted into Ki values. Each compound was tested in
triplicate at least.
3.6. Drug-Like Properties
3.6.1. Plasma Stability
Human plasma in each culture tube, treated with test compound (10 µ
M), was incubated 37 ◦C
for 0, 30, and 120 min, respectively. In a determined period of time, an internal standard solution of
chlorpropamide in acetonitrile was added into each culture tube, which was shaken with a vortex
mixer for 5 min and centrifuged for additional 5 min (14,000 rpm, 4 ◦C). The supernatant was then
injected into thr LC-MS/MS system to analyze the plasma stability of compound 10a.
3.6.2. Microsomal Stability
To human liver microsomes (0.5 mg/mL) were added 0.1 M phosphate buffer (pH 7.4) and tested
compounds (1 µ
M). After incubation at 37 ◦C for 5 min, NADPH generation system solution was also

Molecules 2019, 24, 3234
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added and incubated at 37 ◦C for 30 min again. To terminate reaction, acetonitrile including internal
standard (chlorpropamide) was added, and the solution was centrifuged for 5 min (14,000 rpm, 4 ◦C).
The supernatant was then injected into the LC-MS/MS system to analyze the microsomal stability of
compound 10a.
3.6.3. CYP Inhibition
To human liver microsomes (0.25 mg/mL), 0.1 M phosphate buffer (pH 7.4), a cocktail of five probe
substrates (Phenacetin 50
and Midazolam 2.5 M), and tested compounds were added at concentrations of 0
and 10
M. After incubation at 37 ◦C for 5 min, NADPH generation system solution was also added
µM, Diclofenac 10
µM, S-mephenytoin 100
µM, Dextromethorphan 5
µM,
µ
µM (as a control)
µ
and incubated at 37 ◦C for 15 min again. To terminate the reaction, acetonitrile including internal
standard (Terfenadine) was added, and the solution was centrifuged for 5 min (14,000 rpm, 4 ◦C). The
supernatant was then injected into the LC-MS/MS system to simultaneously analyze the metabolites of
the probe substrates and evaluate the % CYP inhibition of the tested compound 10a.
4. Conclusions
Thus, we synthesized two series of disubstituted pyrimidine derivatives as potential 5-HT2C
selective agonists. Initially, a cell-based assay of 2,5-disubstituted pyrimidines revealed that
most compounds did not have good agonistic effects towards the 5-HT2C receptor, although
2-piperazinyl-5-(3-fluorobenzyloxy)pyrimidine 9b showed effective biological activity, with 61%
activation. In vitro evaluation of the second series of pyrimidines, possessing different cyclic amines,
demonstrated that four compounds showed greater than 50% activation against 5-HT2C in the primary
cell-based assay. In the secondary binding affinity assay, compounds 10a and 10f, with 1,4-diazepane
at the 2-position of pyrimidine, were identified as the most potent 5-HT2C ligands, with excellent Ki
values of 7.9 nM and 19.0 nM, respectively. Compound 10a showed high selectivity profiles for other
5-HT receptor subtypes, with a greater than 10-fold difference in potency. Additional in vitro stability
experiments indicated that compound 10a has excellent plasma and microsomal stability, along with
low CYP inhibition. Based on these results, 2,4-disubstituted pyrimidine 10a could be considered a
potential lead compound as a 5-HT2C selective agonist for further application in chemical probes such
as a 5-HT2C PET radiotracer.
Supplementary Materials: The following are available online at http://www.mdpi.com/1420-3049/24/18/3234/s1,
(NMR spectral data of compounds 9a–9r, 10a–10j, and 20a/b; Table S1: A list of 5-HT receptor radioligands and
reference compounds for binding assay).
Author Contributions: S.-J.M. initiated the project and designed the experiments; J.K., and Y.J.K. performed the
experiments; S.-J.M. and J.K. analyzed the data; H.C. and H.J.K. contributed to analyze drug-like properties;
A.M.L. and A.N.P. performed molecular docking studies; S.-J.M., J.K., and Y.J.K. wrote the paper.
Acknowledgments: This work was financially supported by the Korea Health Industry Development Institute
(KHIDI, HI16C1677, HI17C1037), the National Research Foundation of Korea (NRF-2019R1A2C1008186) and the
research fund of Hanyang University (HY-2015-N). Binding affinity data were generously provided by the US
National Institute of Mental Health (NIMH) Psychoactive Drug Screening Program (HHSN-271-2008-00025-C).
Conflicts of Interest: The authors declare no conflict of interest.
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