Synthesis and evaluation of hexahydropyrimidines and diamines as novel hepatitis C virus inhibitors

Article abstract of DOI:10.1016/j.ejmech.2013.09.055

In order to identify novel anti-hepatitis C virus (HCV) agents we devised cell-based strategies and screened phenotypically small molecule chemical libraries with infectious HCV particles, and identified a hit compound (1) containing a hexahydropyrimidine (HHP) core. During our cell-based SAR study, we observed a conversion of HHP 1 into a linear diamine (6), which is the active component in inhibiting HCV and exhibited comparable antiviral activity to the cyclic HHP 1. In addition, we engaged into the biological characterization of HHP and demonstrated that HHP does not interfere with HCV RNA replication, but with entry and release of viral particles. Here we report the results of the preliminary SAR and mechanism of action studies with HHP.

Full text of DOI:10.1016/j.ejmech.2013.09.055

European Journal of Medicinal Chemistry 70 (2013) 315e325
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Preliminary communication
Synthesis and evaluation of hexahydropyrimidines and diamines as
novel hepatitis C virus inhibitors
, ,
,
Jong Yeon Hwang a 3, Hee-Young Kim b 3, Suyeon Jo a, Eunjung Park a, Jihyun Choi a,
*
Sunju Kong a, Dong-Sik Park a, Ja Myung Heo c, Jong Seok Lee a 1, Yoonae Ko a, Inhee Choi a,
,
Jonathan Cechetto c, Jaeseung Kim a, Jinhwa Lee d, Zaesung No a 2, Marc Peter Windisch b
,
,
**
a Medicinal Chemistry Group, Institut Pasteur Korea, Seongnam-si, Gyeonggi-do, Republic of Korea
b Applied Molecular Virology, Institut Pasteur Korea, Seongnam-si, Gyeonggi-do, Republic of Korea
c Screening Technology Platforms Group, Institut Pasteur Korea, Seongnam-si, Gyeonggi-do, Republic of Korea
d Late Discovery Program, Institut Pasteur Korea (IP-K), Seongnam-si, Gyeonggi-do, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
In order to identify novel anti-hepatitis C virus (HCV) agents we devised cell-based strategies and
screened phenotypically small molecule chemical libraries with infectious HCV particles, and identified a
hit compound (1) containing a hexahydropyrimidine (HHP) core. During our cell-based SAR study, we
observed a conversion of HHP 1 into a linear diamine (6), which is the active component in inhibiting
HCV and exhibited comparable antiviral activity to the cyclic HHP 1. In addition, we engaged into the
biological characterization of HHP and demonstrated that HHP does not interfere with HCV RNA repli-
cation, but with entry and release of viral particles. Here we report the results of the preliminary SAR and
mechanism of action studies with HHP.
Received 15 August 2013
Received in revised form
25 September 2013
Accepted 28 September 2013
Available online 6 October 2013
Keywords:
Hepatitis C virus
Phenotypic screening
Hexahydropyrimidine
Diamines
Ó 2013 Elsevier Masson SAS. All rights reserved.
Entry
Release
1. Introduction
severe side effects and still require a combinatorial treatment
regimen with pegylated interferon-alpha (PEG-IFN-a) and ribavirin
Approximately 200 million people worldwide are chronically
infected with the hepatitis C virus (HCV) [1]. This pathogen is the
major cause of acute hepatitis and chronic liver disease including
cirrhosis and liver cancer. The HCV genome encodes a single pol-
yprotein of w3,000 amino acids which is co- and posttranslation-
ally cleaved into structural (C, E1, E2, and p7) and non-structural
proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) [2]. Recently, two
direct acting antivirals (DAAs), telaprevir and boceprevir inhibiting
the viral NS3/4A protease, have been approved by the FDA, but
unfortunately, both therapies are expensive and accompanied by
which is prone to side effects, too [3e6]. To date, a number of anti-
HCV agents mainly targeting the viral non-structural proteins are in
clinical development, e.g. inhibiting NS5A or NS5B and, are await-
ing FDA approval [1,7]. Nevertheless, the unpredictability of drug
development and clear unmet medical needs encouraged us to
screen for new, safer and potent drugs against HCV, ideally with a
pan-genomic activity and a high genetic barrier to resistance. By
using the recently developed infectious cell culture system for HCV
(HCVcc), we devised strategies for a phenotypic high throughput
screening (HTS) assay, which enables targeting the entire viral life
cycle to identify novel HCV inhibitors [8].
* Corresponding author. Current address: Korea Research Institute of Chemical
Technology, Daejeon 305-600, Republic of Korea.
** Corresponding author.
2. Result and discussion
E-mail addresses: jongyeonhwang@gmail.com (J.Y. Hwang), mpwindisch@
gmail.com (M.P. Windisch).
2.1. High throughput screening and hit selection
1
Current address: Korea Institute of Ocean Science and Technology, Ansan 426-
The screening campaign was conducted at 6 mM final compound
concentration in duplicates with quadruplicate measurements.
Selected active hits were subjected to 10-point dose response curve
744, Republic of Korea.
2
Current address: Gyeonggi Bio-Center, Suwon 443-270, Republic of Korea.
These authors contributed equally to this study.
3
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.09.055

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J.Y. Hwang et al. / European Journal of Medicinal Chemistry 70 (2013) 315e325
(DRC) analysis in the primary assay; briefly, serially diluted com-
pounds were incubated with naïve Huh-7 target cells in 384-well
microplates, and subsequently inoculated with HCVcc expressing
an HCV NS5A-GFP fusion protein at a multiplicity of infection (MOI)
of 1. At 72 h post-infection cells were analyzed by automated
confocal microscopy (OperaÒ, PerkinElmer) and the half maximal
effective concentration (EC50) to inhibit the viral life cycle was
determined. Additionally, to rule out that the observed antiviral
effects were due to compound-induced toxicity the cytotoxic con-
centration (CC50) was determined by fully automated quantifica-
tion of cell nuclei, which served as a marker for cell proliferation.
The main focus of our small molecule screening campaign using
cell culture derived HCV (HCVcc) was to identify compounds with a
novel mechanism of action. Therefore, all identified active hits were
counter-screened in the HCV replicon system to filter out inhibitors
interfering with HCV RNA replication. This strategy reduces
competition on already advanced DAAs interfering with viral
replication. Following this rational, we identified amongst others a
scaffold (1, Fig. 1) containing a hexahydropyrimidine (HHP) struc-
ture being inactive in the replicon system. This result suggested
that HHP 1 interferes with early and/or late steps in the HCV life
cycle rather than interfering with viral RNA replication. This drug
mechanism accommodated our strategy and encouraged us to
initiate a preliminary SAR study to move towards the development
of novel HCV interventions. Additionally, to elucidate mode of ac-
tion (MoA) of HHPs we conducted experiments with HCV pseu-
doparticles (HCVpp) [9] and analyzed viral secretion to monitor
early and late steps in the HCV life cycle, respectively. Here we
report the synthesis and cell based SAR study of HHPs and linear
diamine analogs in the infectious HCV cell culture system and
describe the MoA of HHPs.
formation reaction with 4-methoxychlorobenzene gave the corre-
sponding products 24 and 25, respectively.
2.3. Structureeactivity relationship (SAR) study
All synthesized HHPs were evaluated for their ability to inhibit
the HCV life cycle as described earlier and the inhibitory activities of
HHPs are summarized in Table 1. First, we explored R2 effect on the
HHPs (1 and 2, Fig. 1). Replacement of the 4-pyridyl group with a 4-
Me2NePh group significantly reduced anti-HCV potency (2a,
EC50 ¼ 25
(2b, EC50 ¼ 6.5
showed no inhibitory activity (EC50 > 50
substituent 2d was almost equipotent (EC50 ¼ 3.7
HHP 1, whereas no inhibitory potency was observed in unsub-
m
M), while 3-CF3ePh substitution retained the potency
M). Another heteroaryl compound 2c, furanyl,
M). Interestingly, methyl
M) compared to
m
m
m
stituted compound (2e, EC50 > 50 mM). Furthermore, ketone 2f was
inactive. In case of R1 ¼ 4-MeO and 4-Me2N, most of the com-
pounds (2gep) showed similar activity, ranging between 0.83 and
3.2 mM EC50 values. Thus no strong electron effect of R2 by sub-
stituents was observed in this series. Next, we examined the effect
of the R1 group. 4-Methoxy- (2g) and 4-Me2N-substituted (2n)
compounds showed three- and five-fold improved potency
(EC50 ¼ 1.5 and 0.83
m
M, respectively) compared to HHP 1, but also
demonstrated increased cytotoxicity of CC50 ¼ 23 and 14
mM,
respectively. Likewise, compounds 2i and 2m with a 4-MeO group
in R1 exhibited better potency (EC50 ¼ 3.2 and 1.3
m
M, respectively)
M,
in comparison to compounds 2b and 2a (EC50 ¼ 6.5 and 25
m
respectively). Asymmetric HHPs (2qes) were also evaluated and
showed moderate anti-HCV activities (EC50 ¼ 4.7, 2.7, 11 M,
m
respectively). Regarding ring size, five-membered imidazolidine
analogs (2tev) were evaluated and demonstrated a loss of antiviral
activities regardless of the substitution patterns at R2 (Table 1).
Next, we measured the metabolic stability of HHP (1) in the liver
microsomal matrix. Interestingly, only molecular weight of diamine
(6), a ring-cleaved form of HHP, was predominantly detected in the
mass spectrum (data not shown), suggesting that HHP was con-
verted to diamine 6 (Fig. 2). It was recently reported that an HHP,
named aminal, is reversibly in an equilibrium with diamine 6 in an
aqueous environment [11]. Therefore, we examined the chemical
stability of HHP in cell culture medium. HHP 1 was incubated with
cells in culture medium at 37 ꢀC for two days, followed by analysis
of compound 1 in the cell culture supernatant by LC/MS. This
revealed that only linear diamine 6 was detected by mass spectrum
but not even trace amounts of HHP 1. We assumed that the linear
diamine 6 is most likely to be the active component inhibiting HCV
infection and therefore several additional linear diamine com-
pounds were synthesized and characterized. Diamines with
different lengths between the two nitrogen atoms and substituted
diamines were synthesized as shown in Schemes 1 and 2 and their
activities in the HCVcc system are summarized in Table 2.
2.2. Chemistry
To explore the preliminary SAR study of HHPs, we evaluated the
effects of R1 and R2 groups. The general scheme for the synthesis of
HHPs has been previously described and is briefly outlined in
Scheme 1 [9,10]. Diamines 5 were prepared by a condensation of
corresponding aldehydes and primary diamines, followed by
reduction with NaBH4. The diamines 5 were condensed with al-
dehydes to obtain the corresponding HHPs 2.
Compound 2f bearing a ketone moiety in the HHP core was
synthesized from commercially available trimethylene urea 31 by
treatment of benzyl bromide (Scheme 2). Acylation of compound 6
with acetyl chloride provided compound 9 with an acetamido
moiety. Dibenzamide 8 and diphenylacetamide 10 were obtained
from propane-1,3-diamine 26 by treatment of the corresponding
benzoyl chloride and 2-phenylpropionic acid, respectively. Com-
pound 17 having an aniline moiety was prepared from dibromo-
butane 27 and aniline. Compounds 24 and 25 bearing a piperidine
moiety were synthesized from intermediate 30, which was syn-
thesized from commercially available compound 28 by N-alkyl-
ation, followed by deprotection of the N-Boc group. Reductive
alkylation with p-anisaldehyde and CuI-catalyzed CeN bond
The anti-HCV efficacy of diamine 6 is comparable to HHP 1 with
EC50 values of 4.6 mM and 4.3 mM, respectively. This result strongly
suggests that the linear diamine is the active component inhibiting
HCV infection. Surprisingly, imine compound 7, the precursor of
compound 6, was inactive. In addition, no inhibition was observed
Scheme 1. General synthetic procedure of diamine 5 and HHPs 2. Reagents and conditions: (a) diamines, EtOH, room temperature, 2 h; (b) NaBH4, EtOH, room temperature,
overnight; (c) aldehydes, H2O, reflux, overnight.

J.Y. Hwang et al. / European Journal of Medicinal Chemistry 70 (2013) 315e325
317
Scheme 2. Reagents and conditions: (a) Benzyl bromide, NaH, 1,4-dioxane, reflux, 12 h; (b) acetyl chloride, K2CO3, DMF, 120 ꢀC, 24 h; (c) benzoyl chloride, TEA, DCM, rt; (d) 2-
phenylpropionic acid, EDC, HOBT, DCM, rt; (e) aniline, K2CO3, DMF, 120 ꢀC, 24 h; (f) (i) p-anisaldehyde, MeOH, 50 ꢀC, 12 h, (ii) NaBH4, rt, 12 h; (g) 4 N HCl in dioxane, rt, 3 h;
(h) p-anisaldehyde, NaBH4, MeOH; (i) 4-methoxychlorobenzene, CuI, K2CO3, L-proline, DMSO, microwave, 130 ꢀC, 20 min.
with amide compounds (8e10). This result suggests that a sp3-
hybridized nitrogen is required for anti-HCV potency. Based on
this observation, we further explored the effect of various linker
lengths between the two nitrogen atoms. Increasing the linker size
to greater than three-carbon units improved antiviral potency as
shown by compounds 12e15 (0.98 for four-carbon, 0.61 for five-
carbon, and 0.65
shorter linker (two-carbon) completely lost its antiviral activity
(EC50 > 50 M). This result corroborated our observation that five-
mM for six-carbon), while compound 11 with a
m
membered imidazolidine analogs (2tev), having only two carbon
units between two nitrogen atoms, were inactive. Insertion of an
oxygen atom in the linker slightly reduced the activity (16,
EC50 ¼ 1.7
mM). Among longer linkers, four-carbon linker (12)
provided improved profiling in both increased antiviral potency
Fig. 1. Hit compound 1 (HHP) and generic structure of HHPs (2).

318
J.Y. Hwang et al. / European Journal of Medicinal Chemistry 70 (2013) 315e325
Fig. 2. Conversion of HHP (1) to linear diamine in cell culture conditions.
(EC50 ¼ 0.98
m
M) and reduced cytotoxicity (CC50 ¼ 42
m
M), thereby
compounds (18 and 19) increased cytotoxic effects in parallel. A 4-
methoxy or 4-dimethylamino substituent in the HHP structure (2g
and 2n) improved the anti-HCV potency by three- and five-fold,
respectively. This trend was also observed in linear diamine com-
pounds in which substitution with 4-methoxy (20 and 22) and 4-
dimethylamino groups (21 and 23) on the benzyl moiety showed
resulting in a SI value of 43. Replacement of benzyl amine to aniline
resulted in a total loss of potency (17, EC50 > 50
mM), while sub-
stitution to phenethylamine improved potency (18, EC50 ¼ 1.1
mM).
An alpha-methyl substituted phenethylamine compound (19) also
gave improved antiviral potency, but both phenethylamine
Table 1
Activity profiling of HHPs.
No.
R1
R2
EC50
(m
M)
CC50
(
m
M)
SI (CC50/EC50)
1
H
4.3
>50
>12
2a
2b
H
H
4-Me2NePh
3-CF3ePh
25
6.5
>50
>50
>2.0
>7.7
2c
H
>50
>50
N.A.
2d
2e
2f
H
H
H
Me
H
3.7
>50
>50
>50
>50
>50
>14
N.A.
N.A.
* ¼ O (ketone)
2g
4-MeO
1.5
23
15
2h
2i
2j
2k
2l
2m
4-MeO
4-MeO
4-MeO
4-MeO
4-MeO
4-MeO
4-CF3ePh
3-CF3ePh
4-CNePh
4-FePh
3-Fe4-CNePh
4-Me2NePh
1.6
3.2
1.5
1.9
1.9
1.3
34
43
20
38
29
28
21
13
13
20
15
22
2n
4-Me2N
0.83
14
17
2o
2p
4-Me2N
4-Me2N
4-FePh
4-CF3ePh
1.3
1.5
15
23
12
15
2q
3-MeO/2-F
4.7
>50
>11
2r
2s
3-MeO/2-F
2-Cl/2-F
4-CF3ePh
3-CF3ePh
2.7
11
23
>50
8.5
>4.5
2t
H
>50
>50
N.A.
2u
2v
H
H
Me
4-Me2NePh
>50
>50
>50
>50
N.A.
N.A.
EC50 and CC50 values were determined by 10-point DRC analysis in duplicates with quadruplicate measurements. N.A. not applicable.

J.Y. Hwang et al. / European Journal of Medicinal Chemistry 70 (2013) 315e325
319
Table 2
Activity profiling of diamines.
No.
Structure
EC50
4.6
(m
M)
CC50
27
(
m
M)
SI (CC50/EC50)
6
5.9
7
>50
>50
N.A.
8
9
>50
>50
>50
>50
N.A.
N.A.
10
11
12
>50
>50
>50
>50
42
N.A.
N.A.
43
0.98
14
15
0.61
0.65
29
17
48
26
16
17
1.7
28
16
>50
>50
N.A.
18
1.1
12
11
19
20
1.2
1.5
18
33
15
22
21
22
0.88
0.38
17
32
19
84
(continued on next page)

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J.Y. Hwang et al. / European Journal of Medicinal Chemistry 70 (2013) 315e325
Table 2 (continued )
No.
Structure
EC50
(
m
M)
CC50
(
m
M)
SI (CC50/EC50)
23
0.32
11
34
24
25
1.1
11
34.5
31
>50
>4.7
Putrescine
Spermidine
>50
>50
>50
N.A.
N.A.
>50
>50
Spermine
>50
N.A.
EC50 and CC50 values were determined by 10-point DRC analysis in duplicates with quadruplicate measurements. N.A. not applicable.
increased potencies of three to five-fold. Finally, compound 22 with
3. Conclusion
four carbon linker and 4-methoxy group exhibited not only
significantly improved antiviral efficacy (EC50 ¼ 0.38
m
M), but also
We identified a HHP compound in our phenotypic HTS
reduced cytotoxicity (CC50 ¼ 32
m
M), resulting in an SI value of 84.
campaign using the infectious HCVcc system. The aim of the pre-
sent research work was to synthesize novel HHP lead compounds
and to elucidate the MoA of HHP. During the cell based SAR study
we characterized HHPs and observed a conversion into a linear
diamine under cell culture conditions, which is the active compo-
nent in inhibiting HCV. This observation led to an adjustment of our
chemistry strategy and focusing on a systematic analysis of linear
diamines. This study revealed that diamines to be potent have to
have two crucial substitution patterns containing: (i) more than
two carbon units between two nitrogen atoms, (ii) a basic diamine
with sp3-hybridized nitrogen.
In parallel to our SAR studies we performed a preliminary MoA
study and observed that HHPs exclusively interfere with viral entry
and release. Interestingly, a compound similar to our HHP was
recently described interfering with entry steps of Marburg virus as
demonstrated by using pseudoparticles [20]. Although the exact
MoA of this HHP series has yet to be determined, HHPs and di-
amines can be useful compounds to further investigate the poorly
understood HCV life cycle or for future drug development
campaigns.
Two compounds 24 and 25 with piperidine moiety in a linear
diamine were examined. Interestingly, compound 24 with EC50
value of 1.1
mM is equipotent with acyclic diamine 20, whereas
compound 25 significantly reduced the activity (EC50 ¼ 11
mM). This
result revealed that the cyclic and rigid form of diamine is com-
parable to linear diamine but diamines bearing at least an aniline
moiety demonstrated poor activity or were inactive (17 and 25).
Since diamine structures appear similar to polyamines, which play
important roles in cells [12e14], we tested polyamines including
putrescine, [15] spermidine, [16] and spermine, [17,18] but none of
these compounds was active in the infectious HCVcc system.
2.4. Characterization of HHPs by virological assays
After screening >220,000 small chemical molecules, selected hit
compounds were re-ordered and re-confirmed in the primary HTS
assay followed by counter-screening with HCV replicon cells. Only
re-confirmed hit compounds inactive in the replicon system suited
our strategy. Among others, HHP 1 did not interfere with HCV RNA
replication (EC50 > 50 mM and CC50 > 50 mM), which encouraged us
to initiate a SAR study. In parallel, we engaged in the character-
ization of HHPs and performed preliminary MoA studies. The
HCVpp system was used to monitor early steps of the viral life cycle,
especially binding and internalization of viral particles. Thereby, we
observed that HHP interfered with E1/E2-mediated viral entry
4. Experimental section
4.1. Chemistry
4.1.1. General
(HHP 1 EC50
4
m
M and CC50 > 50
m
M). Furthermore, in order to
All materials were obtained from commercial suppliers and
used without further purification. All solvents used were dried
using an aluminum oxide column. Thin-layer chromatography was
performed on pre-coated silica gel 60 F254 plates. Purification of
compounds was carried out by normal phase column chromatog-
raphy (MPLC, Silica gel 230e400 mesh). NMR spectra were recor-
ded on a Varian 400 MHz. LC/MS data were obtained using a Waters
2695 LC and Micromass ZQ spectrometer. Identity of all final
analyze late steps of the HCV life cycle, the secretion of virions was
determined by a viral supernatant transfer assay [19]. Under this
experimental conditions HHP prevented the release of infectious
virions into the cell culture supernatant (data not shown). Thus,
these results suggested that HHPs have a novel MoA by interfering
with early and late steps of the HCV life cycle. Continuous effort will
be required to characterize the MoA of HHPs in more detail.

J.Y. Hwang et al. / European Journal of Medicinal Chemistry 70 (2013) 315e325
321
compounds was confirmed by proton NMR and by mass spec-
4.1.7. Synthesis of 1,3-dibenzyl-2-(3-(trifluoromethyl)phenyl)
hexahydropyrimidine (2b)
trometry and carbon NMR data of representative compounds 1, 2n,
6,18, 22, and 23 were reported. Yields refer to purified products and
are not optimized.
This compound was synthesized by the procedure described
above for the synthesis of compound 2n. Yield 54%, white solid; 1H
NMR (400 MHz, CDCl3)
d
7.93 (s, 1H), 7.86 (d, J ¼ 7.6 Hz, 1H), 7.52 (d,
4.1.2. General procedure for the preparation of compound 4:
synthesis of 4,40-((1E,10E)-(propane-1,3-diylbis(azanylylidene))
bis(methanylylidene))bis(N,N-dimethylaniline) (7)
J¼ 7.6Hz,1H), 7.45(t, J¼ 7.6Hz,1H), 7.21(m,10H), 3.68(s,1H), 3.51(d,
J ¼ 13.6 Hz, 2H), 2.99 (m, 2H), 2.94 (d, J ¼ 13.6 Hz, 2H), 2.09 (m, 2H),
1.82 (m,1H),1.46 (m,1H); LC/MS (electrospray) m/z (M þ H)þ 410.23.
A solution of propane-1,3-diamine (1 g, 13.491 mmol) and 4-
(dimethylamino)benzaldehyde (3.82 g, 25.634 mmol) in anhydrous
EtOH (100 mL) was stirred at room temperature for 2 h. After re-
action was completed, the precipitate was filtered and washed with
n-hexane to give the desired product 7 (4.0 g, 91%) as white solid.
4.1.8. Synthesis of 1,3-dibenzyl-2-(5-methylfuran-2-yl)-
hexahydropyrimidine (2c)
This compound was synthesized by the procedure described
above for the synthesis of compound 2n. Yield 15%, brown solid; 1H
1H NMR (400 MHz, CDCl3)
d
8.12 (s, 2H), 7.57 (d, J ¼ 8.4 Hz, 4H), 6.67
NMR (400 MHz, CDCl3) d 7.27e7.29 (m, 2H), 7.22e7.23 (m, 4H),
(d, J ¼ 8.4 Hz, 4H), 3.61 (t, J ¼ 6.8 Hz, 4H), 2.99 (s, 12H), 2.05 (p,
J ¼ 6.8 Hz, J ¼ 13.7 Hz, 2H); LC/MS (electrospray) m/z (M þ H)þ
337.27.
7.19e7.21 (m, 4H), 6.27 (d, J ¼ 4.8 Hz, 1H), 6.12 (d, J ¼ 5.6 Hz, 1H),
3.82 (s, 1H), 3.57 (d, J ¼ 13.6 Hz, 2H), 3.11 (d, J ¼ 4.8 Hz, 2H), 2.42e
2.43 (m, 2H), 2.32e2.33 (m, 2H), 2.13 (s, 3H), 1.48e1.51 (m, 2H); LC/
MS (electrospray) m/z (M þ H)þ 347.20.
4.1.3. General procedure for the preparation of compound 5:
synthesis of 4-(1,3-bis(4-methoxybenzyl)hexahydropyrimidin-2-yl)
benzonitrile (21)
4.1.9. Synthesis of 1,3-dibenzyl-2-methyl-hexahydropyrimidine
(2d)
To a solution of 7 (2.0 g, 5.944 mmol) in anhydrous EtOH (40 mL)
was added NaBH4 (270 mg, 7.132 mmol) and stirred at room tem-
perature for 2 h. The reaction was quenched by the addition of H2O
and concentrated in vacuo. The reaction residue was diluted with
DCM and washed with water. The organic layer was dried over
anhydrous Na2SO4 and concentrated in vacuo to give the desired
product 21 as white solid (1.51 g, 75%). The product was used for the
next reaction without any purification. 1H NMR (400 MHz, CDCl3)
This compound was synthesized by the procedure described
above for the synthesis of compound 2n. Yield 36%, yellow solid; 1H
NMR (400 MHz, CDCl3) d 7.27e7.29 (m, 2H), 7.22e7.23 (m, 4H), 7.19e
7.21 (m, 4H), 3.82 (d, J ¼ 13.6 Hz, 2H), 3.63 (q, J ¼ 4.8 Hz,1H), 3.58 (d,
J ¼ 6.4 Hz, 2H), 2.42e2.43 (m, 2H), 2.32e2.33 (m, 2H), 1.47e1.49 (m,
2H), 1.28 (d, J ¼ 5.6 Hz, 3H); LC/MS (electrospray) m/z (M þ H)þ
281.20.
d
7.14 (d, J ¼ 8.4 Hz, 4H), 6.69 (d, J ¼ 8.4 Hz, 4H), 3.66 (s, 4H), 2.91 (s,
4.1.10. Synthesis of 1,3-dibenzyl-hexahydropyrimidine (2e)
This compound was synthesized by the procedure described
above for the synthesis of compound 2n. Yield 45%, yellow solid; 1H
12H), 2.66 (t, J ¼ 6.8 Hz, 4H), 1.68 (p, J ¼ 6.8 Hz, J ¼ 13.7 Hz, 2H); LC/
MS (electrospray) m/z (M þ H)þ 341.27.
NMR (400 MHz, CDCl3)
d 7.27e7.29 (m, 2H), 7.22e7.23 (m, 4H),
4.1.4. General procedure for the preparation of compound 2:
synthesis of 4,40-((2-(pyridin-4-yl)dihydropyrimidine-1,3(2H,4H)-
diyl)bis(methylene))bis(N,N-dimethylaniline) (2n)
7.19e7.21 (m, 4H), 3.86 (q, J ¼ 4.8 Hz, 2H), 3.58 (d, J ¼ 6.4 Hz, 2H),
3.47 (s, 2H), 2.34 (t, J ¼ 5.6 Hz, 4H), 1.50e1.52 (m, 2H); LC/MS
(electrospray) m/z (M þ H)þ 267.20.
A solution of 21 (100 mg, 0.293 mmol) and isonicotinaldehyde
(27
m
L, 0.293 mmol) in H2O (2 mL) was refluxed for 2 h. The reaction
4.1.11. Synthesis of 1,3-dibenzyl-tetrahydropyrimidin-2(1H)-one (2f)
To a solution of trimethylene urea (0.30 g, 2.996 mmol) in 1,4-
dioxane (3 mL) were sequentially added NaH (0.18 g,
4.494 mmol) and benzyl bromide (1.02 g, 5.992 mmol) at 0 ꢀC. The
reaction mixture was refluxed for 12 h and cooled to room tem-
perature. The solvent was removed under reduced pressure, the
residue was diluted with ethyl acetate and washed with saturated
NaHCO3. The organic layer was dried over Na2SO4, concentrated in
vacuo, and purified by column chromatography (silica gel, gradient
10e50 percent, ethyl acetate in hexane) to give compound 2f
mixture was concentrated in vacuo and the precipitate was filtered
and washed with ether to give the desired product 2n (40 mg, Yield
31%) as white solid. 1H NMR (400 MHz, CDCl3)
d
8.59 (d, J ¼ 8.0 Hz,
2H), 7.59 (d, J ¼ 8.0 Hz, 2H), 7.02 (d, J ¼ 8.8 Hz, 4H), 6.61 (d,
J ¼ 8.8 Hz, 4H), 3.66 (s, 1H), 3.48 (d, J ¼ 13.2 Hz, 2H), 2.98 (m, 4H),
2.88 (s, 12H), 2.03 (t, J ¼ 11.6 Hz, 2H), 1.69 (m, 1H), 1.48 (m, 1H); LC/
MS (electrospray) m/z (M þ H)þ 430.38.
4.1.5. Synthesis of 1,3-dibenzyl-2-(pyridin-4-yl)
hexahydropyrimidine (1)
(0.39 g, yield 42%) as white solid. 1H NMR (400 MHz, CDCl3)
d 7.37e
This compound was synthesized by the procedure described
7.39 (m, 4H), 7.32e7.33 (m, 4H), 7.29e7.30 (m, 2H), 3.84 (q,
J ¼ 5.6 Hz, 2H), 3.54 (d, J ¼ 8.8 Hz, 2H), 3.28 (t, J ¼ 4.8 Hz, 4H), 1.81e
1.82 (m, 2H); LC/MS (electrospray) m/z (M þ H)þ 281.30.
above for the synthesis of compound 2n. 1H NMR (400 MHz, CDCl3)
d
8.64 (d, J ¼ 8.0 Hz, 2H), 7.64 (d, J ¼ 8.0 Hz, 2H), 7.21 (m, 10H), 3.75
(s, 1H), 3.59 (d, J ¼ 13.2 Hz, 2H), 3.03 (d, J ¼ 13.2 Hz, 2H), 2.97 (m,
2H), 2.13 (t, J ¼ 11.6 Hz, 2H), 1.79 (m, 1H), 1.56 (m, 1H); 13C NMR
4.1.12. Synthesis of 1,3-bis(4-methoxybenzyl)-2-(pyridin-4-yl)
hexahydropyrimidine (2g)
(100 MHz, CDCl3) d 151.5, 150.2, 139.1, 128.7, 128.4, 127.1, 124.9, 86.8,
58.5, 51.0, 23.6; LC/MS (electrospray) m/z (M þ H)þ 344.64.
This compound was synthesized by the procedure described
above for the synthesis of compound 2n. Yield 56%, white solid, 1H
4.1.6. Synthesis of 4-(1,3-dibenzyl-hexahydropyrimidin-2-yl)-N,N-
dimethylbenzenamine (2a)
NMR (400 MHz, CDCl3)
d
8.60 (d, J ¼ 5.6 Hz, 2H), 7.59 (d, J ¼ 5.6 Hz,
2H), 7.08 (d, J ¼ 4.8 Hz, 4H), 6.78 (d, J ¼ 4.8 Hz, 4H), 3.75 (s, 6H), 3.66
(s,1H), 3.49 (d, J ¼ 13.2 Hz, 2H), 2.93 (m, 4H), 2.06 (t, J ¼ 11.6 Hz, 2H),
1.72 (m,1H),1.52 (m,1H); LC/MS (electrospray) m/z (M þ H)þ 404.26.
This compound was synthesized by the procedure described
above for the synthesis of compound 2n. Yield 63%, white solid; 1H
NMR (400 MHz, CDCl3)
d
7.32 (d, J ¼ 4.8 Hz, 4H), 7.22e7.23 (m, 4H),
7.19e7.21 (m, 4H), 6.57 (d, J ¼ 5.6 Hz, 2H), 3.82 (s, 1H), 3.54 (d,
J ¼ 4.8 Hz, 2H), 3.08 (d, J ¼ 7.2 Hz, 2H), 3.04 (s, 6H), 2.42e2.43 (m,
2H), 2.32e2.33 (m, 2H), 1.48e1.52 (m, 2H); LC/MS (electrospray) m/
z (M þ H)þ 386.50.
4.1.13. Synthesis of 1,3-bis(4-methoxybenzyl)-2-(4-(trifluoromethyl)
phenyl)hexahydropyrimidine (2h)
This compound was synthesized by the procedure described
above for the synthesis of compound 2n. Yield 30%, white solid; 1H

322
J.Y. Hwang et al. / European Journal of Medicinal Chemistry 70 (2013) 315e325
NMR (400 MHz, CDCl3)
d
7.76 (d, J ¼ 5.6 Hz, 2H), 7.60 (d, J ¼ 5.6 Hz,
4.1.20. Synthesis of 4,40-((2-(4-fluorophenyl)dihydropyrimidine-
1,3(2H,4H)-diyl)bis(methylene))bis(N,N-dimethylaniline) (2o)
This compound was synthesized by the procedure described
above for the synthesis of compound 2n. Yield 45%, white solid; 1H
2H), 7.07 (d, J ¼ 4.8 Hz, 4H), 6.75 (d, J ¼ 4.8 Hz, 4H), 3.74 (s, 6H), 3.64
(s, 1H), 3.44 (d, J ¼ 13.2 Hz, 2H), 2.97 (m, 2H), 2.83 (d, J ¼ 13.2 Hz,
2H), 2.02 (t, J ¼ 11.6 Hz, 2H), 1.78 (m, 1H), 1.45 (m, 1H); LC/MS
(electrospray) m/z (M þ H)þ 471.26.
NMR (400 MHz, CDCl3)
d 7.60 (m, 2H), 7.02 (m, 6H), 6.61 (d,
J ¼ 4.8 Hz, 4H), 3.51 (s, 1H), 3.46 (d, J ¼ 13.2 Hz, 2H), 2.98 (d,
J ¼ 11.6 Hz, 2H), 2.71 (d, J ¼ 13.2 Hz, 2H), 1.97 (t, J ¼ 11.6 Hz, 2H), 1.75
(m, 1H), 1.42 (m, 1H); LC/MS (electrospray) m/z (M þ H)þ 447.39.
4.1.14. Synthesis of 1,3-bis(4-methoxybenzyl)-2-(3-
(trifluoromethyl)phenyl)hexahydropyrimidine (2i)
This compound was synthesized by the procedure described
above for the synthesis of compound 2n. Yield 12%, white solid; 1H
4.1.21. Synthesis of 4,40-((2-(4-(trifluoromethyl)phenyl)
dihydropyrimidine-1,3(2H,4H)-diyl)bis(methylene))bis(N,N-
dimethylaniline) (2p)
NMR (400 MHz, CDCl3)
d
7.94 (s, 1H), 7.85 (d, J ¼ 7.2 Hz, 1H), 7.55 (d,
J ¼ 8.0 Hz,1H), 7.48 (t, J ¼ 7.6 Hz,1H), 7.08 (d, J ¼ 4.8 Hz, 4H), 6.78 (d,
J ¼ 4.8 Hz, 4H), 3.77 (s, 6H), 3.66 (s,1H), 3.46 (d, J ¼ 13.2 Hz, 2H), 2.99
(d, J ¼ 11.6 Hz, 2H), 2.85 (d, J ¼ 13.2 Hz, 2H), 2.05 (t, J ¼ 11.6 Hz, 2H),
1.82 (m,1H),1.50 (m,1H); LC/MS (electrospray) m/z (M þ H)þ 469.22.
This compound was synthesized by the procedure described
above for the synthesis of compound 2n. Yield 24%, white solid; 1H
NMR (400 MHz, CDCl3)
d
7.77 (d, J ¼ 8.0 Hz, 2H), 7.59 (d, J ¼ 8.0 Hz,
2H), 7.02 (d, J ¼ 8.8 Hz, 4H), 6.61 (d, J ¼ 8.8 Hz, 4H), 3.62 (s, 1H), 3.43
(d, J ¼ 13.2 Hz, 2H), 2.98 (d, J ¼ 11.6 Hz, 2H), 2.87 (s, 12H), 2.80 (d,
J ¼ 13.2 Hz, 2H), 2.00 (t, J ¼ 11.6 Hz, 2H), 1.76 (m, 1H), 1.48 (m, 1H);
LC/MS (electrospray) m/z (M þ H)þ 497.45.
4.1.15. Synthesis of 4-(1,3-bis(4-methoxybenzyl)
hexahydropyrimidin-2-yl)benzonitrile (2j)
This compound was synthesized by the procedure described
above for the synthesis of compound 2n. Yield 33%, white solid; 1H
NMR (400 MHz, CDCl3)
d
7.82 (d, J ¼ 5.6 Hz, 2H), 7.64 (d, J ¼ 5.6 Hz,
4.1.22. Synthesis of 4-(1,3-dibenzylimidazolidin-2-yl)pyridine (2t)
This compound was synthesized by the procedure described
above for the synthesis of compound 2n. Yield 36%; White solid; 1H
2H), 7.05 (d, J ¼ 4.8 Hz, 4H), 6.76 (d, J ¼ 4.8 Hz, 4H), 3.74 (s, 6H), 3.70
(s, 1H), 3.24 (d, J ¼ 13.2 Hz, 2H), 2.95 (m, 2H), 2.87 (d, J ¼ 13.2 Hz,
2H), 2.02 (t, J ¼ 11.6 Hz, 2H), 1.75 (m, 1H), 1.50 (m, 1H); LC/MS
(electrospray) m/z (M þ H)þ 428.27.
NMR (400 MHz, CDCl3)
d
8.57 (d, J ¼ 8.0 Hz, 2H), 7.35 (d, J ¼ 7.2 Hz,
2H), 7.29e7.30 (m, 2H), 7.22e7.23 (m, 4H), 7.20e7.21 (m, 4H), 3.82
(s,1H), 3.55 (d, J ¼ 8.4 Hz, 2H), 3.38 (d, J ¼ 6.4 Hz, 2H), 2.51e2.52 (m,
2H), 2.41e2.42 (m, 2H); LC/MS (electrospray) m/z (M þ H)þ 330.20.
4.1.16. Synthesis of 2-(4-fluorophenyl)-1,3-bis(4-methoxybenzyl)
hexahydropyrimidine (2k)
This compound was synthesized by the procedure described
4.1.23. Synthesis of 1,3-dibenzyl-2-methylimidazolidine (2u)
This compound was synthesized by the procedure described
above for the synthesis of compound 2n. Yield 30%, yellow solid; 1H
above for the synthesis of compound 2n. Yield 14%, white solid; 1H
NMR (400 MHz, CDCl3)
d 7.60 (m, 2H), 7.04 (m, 6H), 6.76 (d,
J ¼ 4.8 Hz, 4H), 3.74 (s, 6H), 3.56 (s, 1H), 3.48 (d, J ¼ 13.2 Hz, 2H),
2.94 (d, J ¼ 11.6 Hz, 2H), 2.76 (d, J ¼ 13.2 Hz, 2H), 1.98 (t, J ¼ 11.6 Hz,
2H), 1.78 (m, 1H), 1.43 (m, 1H); LC/MS (electrospray) m/z (M þ H)þ
419.15.
NMR (400 MHz, CDCl3) d 7.28e7.29 (m, 2H), 7.22e7.23 (m, 4H),
7.20e7.21 (m, 4H), 3.83 (d, J ¼ 6.4 Hz, 2H), 3.65 (q, J ¼ 4.0 Hz, 1H),
3.60 (d, J ¼ 6.4 Hz, 2H), 2.52e2.53 (m, 2H), 2.42e2.43 (m, 2H), 1.18
(d, J ¼ 5.6 Hz, 3H); LC/MS (electrospray) m/z (M þ H)þ 267.10.
4.1.17. Synthesis of 4-(1,3-bis(4-methoxybenzyl)
hexahydropyrimidin-2-yl)-2-fluorobenzonitrile (2l)
4.1.24. Synthesis of 4-(1,3-dibenzylimidazolidin-2-yl)-N,N-
dimethylbenzenamine (2v)
This compound was synthesized by the procedure described
This compound was synthesized by the procedure described
above for the synthesis of compound 2n. Yield 56%, yellow solid;
above for the synthesis of compound 2n. Yield 49%, oil; 1H NMR
(400 MHz, CDCl3)
d
7.54 (m, 3H), 7.07 (d, J ¼ 4.8 Hz, 4H), 6.75 (d,
1H NMR (400 MHz, CDCl3)
d
7.29e7.31 (d, J ¼ 7.2 Hz, 4H), 7.22e
J ¼ 4.8 Hz, 4H), 3.75 (s, 6H), 3.72 (s, 1H), 3.46 (d, J ¼ 13.2 Hz, 2H),
2.99 (d, J ¼ 13.2 Hz, 2H), 2.92 (m, 2H), 2.08 (t, J ¼ 11.6 Hz, 2H), 1.68
(m, 1H), 1.56 (m, 1H); LC/MS (electrospray) m/z (M þ H)þ 446.30.
7.23 (m, 4H), 7.20e7.21 (m, 4H), 6.57 (d, J ¼ 5.6 Hz, 2H), 3.83 (s,
1H), 3.54 (d, J ¼ 8.4 Hz, 2H), 3.39 (d, J ¼ 6.4 Hz, 2H), 3.02 (s, 6H),
2.42e2.43 (m, 2H), 2.32e2.33 (m, 2H); LC/MS (electrospray) m/z
(M þ H)þ 372.30.
4.1.18. Synthesis of 4-(1,3-bis(4-methoxybenzyl)
hexahydropyrimidin-2-yl)-N,N-dimethylaniline (2m)
4.1.25. Synthesis of N1,N3-dibenzylpropane-1,3-diamine (6)
This compound was synthesized by the procedure described
above for the synthesis of compound 21. Yield 75%, oil; 1H NMR
This compound was synthesized by the procedure described
above for the synthesis of compound 2n. Yield 56%, white solid; 1H
NMR (400 MHz, CDCl3)
d
7.44 (d, J ¼ 8.0 Hz, 2H), 7.08 (d, J ¼ 8.8 Hz,
(400 MHz, acetone-d6)
d
7.37 (d, J ¼ 7.2 Hz, 4H), 7.31 (t, J ¼ 5.8 Hz,
4H), 6.76 (d, J ¼ 8.8 Hz, 4H), 6.70 (d, J ¼ 8.0 Hz, 2H), 3.74 (s, 6H), 3.49
(d, J ¼ 13.2 Hz, 2H), 3.42 (s, 1H), 2.92 (m, 8H), 2.74 (d, J ¼ 13.2 Hz,
2H), 1.93 (t, J ¼ 11.6 Hz, 2H), 1.76 (m, 1H), 1.40 (m, 1H); LC/MS
(electrospray) m/z (M þ H)þ 446.37.
4H), 7.23 (t, J ¼ 7.2 Hz, 2H), 3.76 (s, 4H), 2.69 (t, J ¼ 6.8 Hz, 4H), 1.70
(p, J12 ¼ 6.8 Hz, J13 ¼ 13.7 Hz, 2H); 13C NMR (100 MHz, acetone-d6)
d
142.1, 128.9,128.7, 127.2, 54.5, 48.6, 30.9; LC/MS (electrospray) m/z
(M þ H)þ 255.29.
4.1.19. Synthesis of 4,40-((2-(pyridin-4-yl)dihydropyrimidine-
1,3(2H,4H)-diyl)bis(methylene))bis(N,N-dimethylaniline) (2n)
This compound was synthesized by the procedure described
above for the synthesis of compound 2n. Yield 31%, white solid; 1H
4.1.26. Synthesis of N,N0-(propane-1,3-diyl)dibenzamide (8)
To a stirred solution of diamine (58 mg, 0.65 mmol) in anhy-
drous dichloromethane (2.2 mL) were added benzoyl chloride
(0.23 mL, 1.95 mmol) and triethylamine (0.28 mL, 1.95 mmol) at
NMR (400 MHz, CDCl3)
d
8.59 (d, J ¼ 8.0 Hz, 2H), 7.59 (d, J ¼ 8.0 Hz,
0
ꢀC. After 10 min, the reaction mixture was warmed to room
2H), 7.02 (d, J ¼ 8.8 Hz, 4H), 6.61 (d, J ¼ 8.8 Hz, 4H), 3.66 (s, 1H), 3.48
(d, J ¼ 13.2 Hz, 2H), 2.98 (m, 4H), 2.88 (s, 12H), 2.03 (t, J ¼ 11.6 Hz,
temperature and stirred for 5 h. White precipitate was filtered,
washed with dichloromethane and purified by flash column chro-
matography (dichloromethane:methanol ¼ 30:1e10:1) to give the
product 8 as white solid (119 mg, yield 62%). 1H NMR (400 MHz,
2H), 1.69 (m, 1H), 1.48 (m, 1H); 13C NMR (100 MHz, CDCl3)
d 150.1,
150.0, 129.7, 126.9, 124.9, 112.6, 86.5, 57.9, 50.6, 40.9, 23.4; LC/MS
(electrospray) m/z (M þ H)þ 430.38.
CDCl3)
d
7.88 (d, J ¼ 8.4 Hz, 4H), 7.49 (m, 6H), 7.19 (NH, 2H),3.56 (t,

J.Y. Hwang et al. / European Journal of Medicinal Chemistry 70 (2013) 315e325
323
J ¼ 6.8 Hz, 4H), 1.82 (p, J ¼ 6.8 Hz, J ¼ 13.7 Hz, 2H); LC/MS (elec-
trospray) m/z (M þ H)þ 283.45.
to room temperature. The reaction mixture was concentrated under
reduced pressure, diluted with ethyl acetate, and washed with sat.
NaHCO3. The organic layer was dried over Na2SO4 and concentrated
in vacuo. The resulting residue was purified by column chroma-
tography (silica gel, gradient 5e12.5 percent, ethyl acetate in hex-
ane) to yield the title compound 17 (0.05 g, yield 23%) as pale yellow
4.1.27. Synthesis of N,N0-(butane-1,4-diyl)bis(N-benzylethanamide)
(9)
To a solution of 12 (0.17 g, 0.633 mmol) in dimethylformamide
(5 mL) were treated potassium carbonate (0.17 g, 1.852 mmol) and
acetyl chloride (0.11 mL, 1.583 mmol) at room temperature. The
reaction mixture was heated for 24 h and cooled to room temper-
ature. The reaction mixture was concentrated under reduced
pressure, diluted with ethyl acetate, and washed with sat. NaHCO3.
The organic layer was dried over Na2SO4 and concentrated in vacuo.
The resulting residue was purified by column chromatography
(silica gel, gradient 10e25%, ethyl acetate in hexane) to yield the
compound 9 (0.17 g, yield 78%) as pale yellow oil; 1H NMR
oil. 1H NMR (400 MHz, CDCl3)
d
7.07 (t, J ¼ 5.6 Hz, 4H), 6.67 (t,
J ¼ 7.2 Hz, 2H), 6.57 (d, J ¼ 8.0 Hz, 4H), 3.32 (t, J ¼ 5.2 Hz, 4H), 1.50 (t,
J ¼ 4.8 Hz, 4H); LC/MS (electrospray) m/z (M þ H)þ 241.14.
4.1.35. Synthesis of N1,N3-diphenethylpropane-1,3-diamine (18)
This compound was synthesized by the procedure described
above for the synthesis of compound 21. Yield 64%, white solid; 1H
NMR (400 MHz, DMSO-d6)
d 8.51 (br s, 2H), 7.34e7.38 (m, 4H),
7.26e7.30 (m, 6H), 3.17e3.21 (m, 4H), 3.02e3.06 (m, 4H), 2.90e2.94
(400 MHz, CDCl3) d 7.34e7.36 (m, 4H), 7.30e7.33 (m, 4H), 7.28e7.29
(m, 4H), 1.94 (q, J ¼ 7.6 Hz, 2H); 13C NMR (100 MHz, DMSO-d6)
(m, 2H), 4.92 (s, 4H), 3.19 (t, J ¼ 4.8 Hz, 4H), 2.42 (s, 6H), 1.52 (t,
d 159.1, 137.6, 129.4, 129.3, 127.6, 48.3, 44.7, 32.3, 23.0; LC/MS
J ¼ 3.6 Hz, 4H); LC/MS (electrospray) m/z(M þ H)þ 353.30.
(electrospray) m/z (M þ H)þ 283.
4.1.28. Synthesis of N,N0-(propane-1,3-diyl)bis(2-
4.1.36. Synthesis of N1,N3-bis(2-phenylpropyl)propane-1,3-diamine
(19)
phenylpropanamide) (10)
Yield 75%, white solid; 1H NMR (400 MHz, CDCl3)
d
7.34 (m,
This compound was synthesized by the procedure described
10H), 5.98 (NH, 2H), 3.55 (p, J12 ¼ 6.8 Hz, J13 ¼ 13.7 Hz, 2H), 3.11 (m,
4H), 1.47 (d, J ¼ 7.2 Hz, 6H), 1.43 (m, 2H); LC/MS (electrospray) m/z
(M þ H)þ 339.50.
above for the synthesis of compound 21. Yield 20%, oil; 1H NMR
(400 MHz, CDCl3) d 7.32 (m, 4H), 7.19 (m, 6H), 2.89 (m, 2H), 2.69 (d,
J ¼ 4.8 Hz, 4H), 2.54 (m, 4H), 1.55 (p, J ¼ 6.8 Hz, J ¼ 13.7 Hz, 4H), 1.22
(d, J ¼ 7.2 Hz, 6H); LC/MS (electrospray) m/z (M þ H)þ 311.48.
4.1.29. Synthesis of N1,N2-dibenzylethane-1,2-diamine (11)
This compound was synthesized by the procedure described
above for the synthesis of compound 21. Yield 80%, yellow oil; 1H
4.1.37. Synthesis of 4-(1,3-bis(4-methoxybenzyl)
hexahydropyrimidin-2-yl)benzonitrile (20)
NMR (400 MHz, CDCl3)
d
7.32 (d, J ¼ 4.4 Hz, 8H), 7.23e7.26 (m, 2H),
This compound was synthesized by the procedure described
3.71 (s, 4H), 2.71 (s, 4H); LC/MS (electrospray) m/z (M þ H)þ 241.30.
above for the synthesis of compound 21. Yield 43%, white solid; 1H
NMR (400 MHz, CDCl3)
4H), 3.76 (s, 6H), 3.69 (s, 4H), 2.66 (t, J ¼ 6.8 Hz, 4H),1.68 (p, J ¼ 6.8 Hz,
d
7.20 (d, J ¼ 8.4 Hz, 4H), 6.82 (d, J ¼ 8.4 Hz,
4.1.30. Synthesis of N1,N4-dibenzylbutane-1,4-diamine (12)
This compound was synthesized by the procedure described
above for the synthesis of compound 21. Yield 93%, yellow oil; 1H
J ¼ 13.7 Hz, 2H); LC/MS (electrospray) m/z (M þ H)þ 315.27.
NMR (400 MHz, CDCl3)
d
7.38e7.36 (m, 4H), 7.31e7.32 (m, 4H),
4.1.38. Synthesis of 4-(1,3-bis(4-methoxybenzyl)
7.29e7.30 (m, 2H), 3.78 (s, 4H), 2.52 (t, J ¼ 4.8 Hz, 4H), 1.42 (t,
hexahydropyrimidin-2-yl)benzonitrile (22)
J ¼ 5.6 Hz, 4H); LC/MS (electrospray) m/z (M þ H)þ 269.30.
This compound was synthesized by the procedure described
above for the synthesis of compound 21. Yield 85%, white solid; 1H
4.1.31. Synthesis of N1,N5-dibenzylpentane-1,5-diamine (14)
This compound was synthesized by the procedure described
above for the synthesis of compound 21. Yield 75%, oil; 1H NMR
NMR (400 MHz, CDCl3)
d
7.21 (d, J ¼ 8.4 Hz, 4H), 6.84 (d, J ¼ 8.4 Hz,
4H), 3.80 (s, 6), 3.71 (s, 4H), 2.62 (t, J ¼ 6.8 Hz, 4H), 1.55 (p,
J12 ¼ 6.8 Hz, J13 ¼ 13.7 Hz, 4H); 13C NMR (100 MHz, CDCl3)
d 158.8,
(400 MHz, CDCl3)
d
7.51 (m, 8H), 7.42 (m, 2H), 3.96 (s, 4H), 2.81 (t,
132.5, 129.5, 114.0, 55.4, 53.5, 49.3, 28.0; LC/MS (electrospray) m/z
J ¼ 6.8 Hz, 4H), 1.71 (p, J ¼ 6.8 Hz, J ¼ 13.7 Hz, 2H), 1.55 (m, 4H);
(M þ H)þ 329.50.
LRMS (electrospray) m/z (M þ H)þ 283.38.
4.1.39. Synthesis of 4-(1,3-bis(4-methoxybenzyl)
hexahydropyrimidin-2-yl)benzonitrile (23)
4.1.32. Synthesis of N1,N6-dibenzylhexane-1,6-diamine (15)
This compound was synthesized by the procedure described
above for the synthesis of compound 21. Yield 71%, oil; 1H NMR
This compound was synthesized by the procedure described
above for the synthesis of compound 21. Yield 80%, white solid; 1H
(400 MHz, CDCl3)
d
7.32 (m, 8H), 7.30 (m, 2H), 3.77 (s, 4H), 2.61 (t,
NMR (400 MHz, CDCl3)
d
7.16 (d, J ¼ 8.4 Hz, 4H), 6.70 (d, J ¼ 8.4 Hz,
J ¼ 6.8 Hz, 4H), 1.51 (p, J12 ¼ 6.8 Hz, J13 ¼ 13.7 Hz, 4H), 1.35 (m, 4H);
4H), 3.68 (s, 4), 2.92 (s,12H), 2.62 (t, J ¼ 6.8 Hz, 4H),1.55 (p, J ¼ 6.8 Hz,
LC/MS (electrospray) m/z (M þ H)þ 297.40.
J¼ 13.7 Hz, 4H);13CNMR (100 MHz, CDCl3)
d 150.1,129.4,127.9,112.9,
53.4, 49.1, 40.9, 28.0; LC/MS (electrospray) m/z (M þ H)þ 355.56.
4.1.33. Synthesis of 2,20-oxybis(N-benzylethan-1-amine) (16)
This compound was synthesized by the procedure described
above for the synthesis of compound 21. Yield 68%, oil; 1H NMR
4.1.40. Synthesis of compound 29
To a stirred solution of 28 (500 mg, 2.333 mmol) in MeOH
(5.0 mL) was added p-anisaldehyde (0.3 mL, 2.566 mmol). The
mixture was stirred at 50 ꢀC for overnight. After reaction was
completed, sodium borohydride (264 mg, 6.999 mmol) was added
at 0 ꢀC. The mixture was stirred at room temperature for overnight.
After reaction was completed, the reaction mixture was quenched
by adding H2O. The residue was diluted with EtOAc and washed
with saturated brine. The organic layer was dried over anhydrous
MgSO4 and concentrated in vacuo. The crude product was purified
by flash column chromatography to give 29 (yield 49%) as white
(400 MHz, CDCl3)
d 7.32 (m, 8H), 7.23 (m, 2H), 3.80 (s, 4H), 3.56 (t,
J ¼ 5.2 Hz, 4H), 2.80 (t, J ¼ 5.2 Hz, 4H), 1.74 (NH, 2H); LC/MS
(electrospray) m/z (M þ H)þ 285.42.
4.1.34. Synthesis of N1,N4-diphenylbutane-1,4-diamine (17)
To a solution of 1,4-dibromobutane 26 (0.20 g, 0.926 mmol) in
dimethylformamide (3 mL) were treated potassium carbonate
(0.26 g, 1.852 mmol) and aniline (0.18 mL, 1.945 mmol) at room
temperature. The reaction mixture was refluxed for 24 h and cooled

324
J.Y. Hwang et al. / European Journal of Medicinal Chemistry 70 (2013) 315e325
solid. 1H NMR (400 MHz, CDCl3)
J ¼ 8.4 Hz, 2H), 4.62 (br s, 1H), 3.80 (s, 3H), 3.45e3.37 (m, 2H), 3.00
(br s, 2H), 2.76e2.68 (m, 2H), 1.98e1.93 (m, 1H), 1.73e1.53 (m, 5H),
1.41 (s, 9H), 1.01e0.97 (m, 1H).
d
7.20 (d, J ¼ 8.8 Hz, 2H), 6.84 (d,
cytotoxicity were determined by GFP expression and nucleus
quantification, respectively.
4.2.2. HCV replicon assay
Huh-7 replicon cells were plated at 2000 cells/well in 40 mL of
4.1.41. Synthesis of compound 30
culture media in 384-well plates (Greiner bio-one,
After overnight incubation, serially diluted compounds in 20
cell culture media were added. At 72 h post compound treatment,
plates with GFP expressing replicon cell lines were fixed with 2%
m
clear black).
L of
To a solution of 29 (184 mg, 0.550 mmol) in dioxane (2 mL) were
added a solution of 4 N HCl in dioxane (1.1 mL, 4.400 mmol) and the
mixture was stirred at room temperature for 3 h. After reaction was
completed, the reaction mixture was diluted with DCM and washed
with saturated 2 N NaOH. The organic layer was dried over anhy-
drous MgSO4 and concentrated to give 30 (yield 71%) as white solid.
m
paraformaldehyde (PFA) and applied for nuclei staining with 10 mg/
mL of Hoechst 33342 (Sigma Aldrich). Cells were visualized by fully
automated confocal microscopy (Opera, PerkinElmer) and analyzed
by in-house software. HCV RNA replication was measured by GFP
expression and cytotoxicity was measured by nucleus counting.
EC50 and CC50 were calculated by non-linear regression analysis
using GraphPad Prism (GraphPad software).
1H NMR (400 MHz, CDCl3)
J ¼ 8.4 Hz, 2H), 3.79 (s, 3H), 3.43 (q, J ¼ 20.0, 12.8 Hz, 2H), 2.85 (d,
J ¼ 9.6 Hz, 1H), 2.76 (d, J ¼ 10.8 Hz, 1H), 2.54 (d, J ¼ 6.0 Hz, 2H),
1.93e1.88 (m, 1H), 1.76e1.53 (m, 5H), 0.92e0.88 (m, 1H).
d
7.21 (d, J ¼ 8.4 Hz, 2H), 6.84 (d,
4.1.42. Synthesis of N-(4-methoxybenzyl)-1-(1-(4-methoxybenzyl)
piperidin-3-yl)methanamine (24)
4.2.3. HCV pseudoparticle production and entry inhibition assay
HCV E1/E2-pseudotyped (or VSV-G pseudotyped as a control)
lentiviral particles bearing the GFP reporter gene were produced as
described previously [9]. Comparable amounts of infectious HCVpp
and VSVpp were used in every experiment. Briefly, Huh-7 cells
were plated in 384-well plates and treated with compounds. After
1 h incubation at 37 ꢀC cells were inoculated with HCVpp or VSVpp
and incubated at 37 ꢀC for 12 h followed by a wash step. At 72 h post
inoculation the transduction efficiency was evaluated by measuring
GFP reporter gene expression by confocal microscopy at 20ꢁ
magnification (ImageXpress ULTRA, Molecular Device) and quan-
tified by in-house image processing software. Relative transduction
values were obtained using DMSO-treated cells.
To a stirred solution of 30 (42 mg, 0.180 mmol) in MeOH (0.5 mL)
was added p-anisaldehyde (0.024 mL, 0.200 mmol). The mixture
was stirred at 50 ꢀC for overnight. After reaction was completed,
sodium borohydride (8.0 mg, 0.200 mmol) was added at 0 ꢀC. The
mixture was stirred at room temperature for overnight. After reac-
tion was completed, the reaction mixture was quenched by adding
H2O, diluted with EtOAc, and washed with saturated brine. The
organic layer was dried over anhydrous MgSO4 and concentrated in
vacuo. The crude product was purified by flash column chroma-
tography to give 3 (yield 52%) as yellow oil; 1H NMR (400 MHz,
CDCl3)
d
7.21 (t, J ¼ 8.0 Hz, 4H), 6.85 (dd, J ¼ 8.4, 1.2 Hz, 4H), 3.79 (s,
6H), 3.68 (s, 2H), 3.44 (q, J ¼ 25.6, 12.8 Hz, 2H), 2.88 (d, J ¼ 10.0 Hz,
1H), 2.77 (d, J ¼ 11.2 Hz, 1H), 2.48 (d, J ¼ 6.4 Hz, 2H), 1.95e1.54 (m,
6H), 0.95e0.88 (m, 1H); LCMS (electrospray) m/z (M þ H)þ 355.
Acknowledgments
We are grateful to Drs. Thierry Christophe and Peter Sommer for
valuable discussions. In memoriam Dr. Junwon Kim. This work was
supported by the National Research foundation of Korea (NRF)
grant funded by the Korea government (MEST) (No. 2012-00011),
Gyeonggi-do and KISTI.
4.1.43. Synthesis of 4-methoxy-N-((1-(4-methoxybenzyl)piperidin-
3-yl)methyl)aniline (25)
To a stirred solution of 30 (20 mg, 0.140 mmol) in DMSO (1.0 mL)
were added 4-methoxychlorobenzene (49 mg, 0.210 mmol), CuI
(2.7 mg, 0.014 mmol), K2CO3 (39 mg, 0.280 mmol) and L-proline
(3.2 mg, 0.028 mmol). The reaction mixture was heated under mi-
crowave at 130 ꢀC for 20 min. After reaction was completed, the re-
action mixture was diluted with EtOAc and washed with saturated
brine. The organic layer was dried over anhydrous MgSO4 and
concentrated invacuo. Thecrudeproductwaspurified by flashcolumn
chromatography to give 25 (yield 28%) as clear oil; 1H NMR (400 MHz,
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2013.09.055.
References
CDCl3)
d 7.26e7.19 (m, 2H), 6.87e6.83 (m, 2H), 6.78e6.74 (m, 2H),
6.55e6.51 (m, 2H), 3.80 (s, 3H), 3.74 (s, 3H), 3.44 (q, J ¼ 12.8 Hz, 2H),
3.01e2.91 (m, 2H), 2.86 (d, J ¼ 9.6 Hz, 1H), 2.74e2.71 (m, 1H), 2.02e
1.97 (m, 1H), 1.92e1.78 (m, 3H), 1.71e1.66 (m, 2H), 1.60e1.54 (m, 1H),
1.09e1.00 (m, 1H); LC/MS (electrospray) m/z (M þ H)þ 341.
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