Design, synthesis, and biological evaluation of N-arylpiperazine derivatives as interferon inducers

Article abstract of DOI:10.1016/j.bmcl.2020.127613

Type I Interferon (IFN) signaling plays an important role in the immune defense system against virus infection and in the innate immune response, thus IFNs are widely used as anti-viral agents and treatment for immune disorder or cancer. However, there is a growing demand for novel small-molecule IFN inducer due to tolerance, toxicity, or short duration of action following direct administration of IFNs. In this study, we assessed arylpiperazine (ARP) as a new core skeleton of IFN inducer. To investigate structure–activity relationship, we designed and synthesized a series of ARP analogues and evaluated the ability to stimulate IFN response in THP-1 human monocyte cells. Compound 5i was identified as a potent type I IFN inducer as it significantly increased cytokine secretion and increased expression of various IFN-stimulating genes which are representative biomarkers of type I IFN pathway. Our results suggested a beneficial therapeutic potential of 5i as an anti-viral agent.

Full text of DOI:10.1016/j.bmcl.2020.127613

Journal Pre-proofs
Design, synthesis, and biological evaluation of N-arylpiperazine derivatives as
interferon inducers
Yeonjeong Chu, B. Raja Sekhara Reddy, V Pratap Reddy Gajulapalli, K.
Sudhakar Babu, Eunha Kim, Sanghee Lee
PII:
S0960-894X(20)30724-1
DOI:
Reference:
https://doi.org/10.1016/j.bmcl.2020.127613
BMCL 127613
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Bioorganic & Medicinal Chemistry Letters
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Revised Date:
Accepted Date:
17 August 2020
9 October 2020
12 October 2020
Please cite this article as: Chu, Y., Raja Sekhara Reddy, B., Pratap Reddy Gajulapalli, V., Sudhakar Babu, K.,
Kim, E., Lee, S., Design, synthesis, and biological evaluation of N-arylpiperazine derivatives as interferon
inducers, Bioorganic & Medicinal Chemistry Letters (2020), doi: https://doi.org/10.1016/j.bmcl.2020.127613
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Design, synthesis, and biological evaluation of N-arylpiperazine derivatives as interferon
inducers
Yeonjeong Chua,b,†, B. Raja Sekhara Reddyc,†, V Pratap Reddy Gajulapallib,†, K. Sudhakar Babuc,*,
Eunha Kimb,*, and Sanghee Leea,d,
*
a Brain Science Institute, Korea Institute of Science and Technology, Seoul, South Korea
b Department of Molecular Science and Technology, Ajou University, Suwon, South Korea
c Department of Chemistry, Sri Krishnadevaraya University, Anantapur, India
d Department of HY-KIST Bio-convergence, Hanyang University, Seoul, South Korea
† These authors equally contributed to this work
E mail: slee19@kist.re.kr, ehkim01@ajou.ac.kr, and drksbabu9@gmail.com
Corresponding authors
Dr. Sanghee Lee
Brain Science Institute, Korea Institute of Science and Technology
Hwarangro 14-gil-5, Seoul 02792, Korea
E-mail : slee19@kist.re.kr
Prof. Eunha Kim
Department of Molecular Science and Technology
206 Worldcup-ro, Woncheon-dong, Yeongtong-gu, Suwon, 16499, Korea
E-mail: ehkim01@ajou.ac.kr
Prof. K. Sudhakar Babu
Department of Chemistry,
Sri Krishnadevaraya University, Anantapur, 515055, India
E-mail : drksbabu9@gmail.com

Abstract
Type I Interferon (IFN) signaling plays an important role in the immune defense system against virus
infection and in the innate immune response, thus IFNs are widely used as anti-viral agents and
treatment for immune disorder or cancer. However, there is a growing demand for novel small-molecule
IFN inducer due to tolerance, toxicity, or short duration of action following direct administration of IFNs.
In this study, we assessed arylpiperazine (ARP) as a new core skeleton of IFN inducer. To investigate
structure-activity relationship, we designed and synthesized a series of ARP analogues and evaluated
the ability to stimulate IFN response in THP-1 human monocyte cells. Compound 5i was identified as
a potent type I IFN inducer as it significantly increased cytokine secretion and increased expression of
various IFN-stimulating genes which are representative biomarkers of type I IFN pathway. Our results
suggested a beneficial therapeutic potential of 5i as an anti-viral agent.
Keyword: interferon inducer, type I Interferon, arylpiperazine, anti-viral agent, innate immunity
Highlight
.
.
We designed and synthesized a series of arylpiperazine derivatives as interferon inducers
We tested different linker system and carbonyl moiety resulted in increase of potency
.
.
5i exhibited the activity of interferon inducer with EC50 of 13.1 μM and no cytotoxicity
5i initiated immune responses by mediating type I IFN signaling

Interferons (IFNs) are essential signaling peptides which act as the first line of surveillant in defense
system and modulate innate and adaptive immune activation[1,2]. In response to virus infection, IFNs
are released by host cells, after which they bind to their specific receptors, thereby stimulating
JAK/STAT signaling pathway and initiating transcription of various interferon-stimulated genes (ISGs)
such as CXCL10, IRF7, IFIT3, and OAS etc [3–7]. Activation of IFN signaling pathway regulates not
only anti-viral response but also induces cellular immunity by stimulating macrophage or monocyte [8].
Based on the class of binding receptor and signaling cascade, IFNs are categorized into three major
group: type I, type II and type III. Type I IFNs including IFNα and IFNβ are considered as therapeutic
targets for treatment of hepatitis B and C infections and multiple sclerosis [9,10]. In addition, IFNs are
used in cancer treatments of hematological malignancy such as leukemia and lymphomas [11,12].
Despite their therapeutic potential, there are several limitations of IFNs treatments including toleration,
dose-related toxicity and side-effects such as dizziness, headache, muscle pain, and depression [13].
Moreover, IFNs are typically administrated by intramuscular injection for therapeutic use. PEGylated
IFNs with enhancing stability are generally used in clinical treatment to overcome the short duration of
effect. However, PEGylated IFNs are not able to be used on patients with hyperbilirubinemia [14,15].
For these reasons, continuous efforts have been made to develop small-molecule modulators so as to
stimulate IFNs. For example, tilorone is the first synthetic small-molecule drugs used as orally active
IFN inducer which showed efficient anti-viral activity against broad spectrum of viruses including ebola
virus and middle east respiratory syndrome-related coronavirus (MERS-Cov), especially [16].

Me
N
O
N
Me
(a)
HN
O
O
NO2
N
F
NH
OMe
N
Cl
N
N
Cl
Cl
N
N
Cl
N
CF3
N
N
OH
Anti-malarial agent
Selective 5-HT1A antagonist
DRD3 antagonist
Cariprazine
O
N
N
Cl
Cl
NH
N
N
O
N
N
O
OMe
OMe
Anti-bacterial agent
Anti-cancer agent
Aripiprazol
(b)
S
O
O
R
R
Cl
N
N
Cl
N
N
Cl
N
R
H
H
Cl
N
Cl
N
Cl
N
Figure 1. (a) Various bioactive small molecules embedded in an arylpiperazine (ARP) and a di-chloro ARP core
structure. (b) Design strategy of ARP derivatives for type I IFN inducers with a different linker moiety.
Therefore, we aimed to develop new small-molecule regulators to stimulate type I IFN effect and
identified that particular arylpiperazine (ARP) derivatives exhibited the desired biological activity using
phenotypic screening. Remarkably, the ARP core structure was reported to exert anti-malarial, anti-
microbial, anti-cancer activity or selective 5-HT1A antagonistic effect (Figure 1a) [17–20]. In addition,
2,3-di-chloro substituent in ARP pharmacophore plays important role in particular drugs such as
cariprazine, aripiprazol or bioactive DRD3 antagonist (Figure 1a) [21–23]. Based on these important
characteristics of the ARP skeleton, we designed and synthesized a series of 2,3-dichloro ARP
derivatives by further N-modification on piperazine part with different linker moiety such as thiourea,
urea, and carbonyl functional group in order to assess structure-activity relationship (SAR; Figure 1b),
then we evaluated their biological activities associated with the immune response.

S
R1
Cl
N
N
ii)
H
Cl
N
NCS
R1
3
ARP-thiourea derivatives
O
R2
Cl
N
N
Cl
NH
Cl
H
Cl
N
Cl
N
Cl
iii)
i)
NH2
4
NH2
ARP-urea derivatives
R2
2
1
Core structure of
ARP derivatives
O
Cl
N
R3
iv)
Cl
N
COOH
R3
5
ARP-carbonyl derivatives
Scheme 1. Synthesis of Arylpiperazine derivatives.a
aReaction conditions and reagents: (i) bis(2-chloroethyl)ethylamine, PTSA, tetrabutylammonium bromide, xylene,
135 °C, 48 h, reflux, NH4OH, (yield 88 %); (ii) triethylamine, DCM, 15 min, room temperature, (yield 85-95%).; (iii)
Triphosgene, triethylamine, DCM, room temperature, 1 h, (yield 85-95%); (iv) HATU, DIPEA, dry DMF, room
temperature, 12 h, (yield 85-90%).
The series of ARP derivatives (3, 4, and 5) was completed in a two-step approach, as shown in Scheme
1. In the first step, 2,3-dichloroaniline (1) reacted with bis(2-chloroethyl)ethylamine to obtain 1-(2,3-
dichlorophenyl)
piperazine
hydrochloride.
Subsequently,
1-(2,3-dichlorophenyl)piperazine
hydrochloride in the presence of NH4OH was converted to a free amine (-NH2) compound, 1-(2,3-
dichlorophenyl)piperazine (2). The crude product 2 was modified with diverse substituents based on
thiourea (3), urea (4), and a carbonyl linker (5). For instance, derivatives 3a to 3f were produced from
the reaction between 2 and various thiocyanates with triethylamine (TEA) in dry DCM. The intermediate
2 reacted with aromatic amines in the presence of triphosgene (1.5 equiv.) to produce compounds 4a
to 4h (see Supplementary material). The amide coupling reaction between 2 and acid derivatives (1
equiv.) in presence of HATU and DIPEA (3 equiv.) in DMF produced compounds 5a to 5k (see
Supplementary material). Most of the compounds occurred at moderate yield (85%–90%) and were
characterized by low-resolution mass spectroscopy, 1H NMR, and 13C NMR. Purity of the compounds
was analyzed using high-performance liquid chromatography (HPLC) before performing biological

assays.
All the synthesized compounds were evaluated using ISRE reporter assay on THP-1 human monocyte
cells for monitoring immune response. In this system, ISG54 minimal promoter in conjunction with five
ISRE elicits transcription of secreted luciferase reporter gene upon stimulation of the type I IFN pathway,
and IFN-related immune activation was measured based on luminescence [24].

Table 1. ISRE reporter assay of ARP derivatives with thiourea or urea linker.
X
R1
Cl
N
N
H
Cl
N
Compound
3a
X
S
R1
RUa
Cell viability (%)b
84 ± 15
1.2 ± 0.4
NO2
3b
3c
3d
3e
3f
S
S
1.1 ± 0.3
1.3 ± 0.3
1.2 ± 0.2
0.4 ± 0.2
1.5 ± 0.2
1.0 ± 0.3
2.1 ± 1.7
3.9 ± 1.4
4.2 ± 1.3
3.3 ± 2.0
3.3 ± 1.1
3.0 ± 1.3
2.7 ± 1.0
97 ± 2
81 ± 7
F
Cl
Br
S
99 ± 4
S
46 ± 54
85 ± 9
S
Br
NO2
4a
4b
4c
4d
4e
4f
O
O
O
O
O
O
O
O
90 ± 10
95 ± 11
61 ± 16
96 ± 11
98 ± 16
76 ± 17
70 ± 26
87 ± 15
F
CF3
OCF3
OCH3
CF3
4g
4h
OCF3
OCH3
a Relative unit of ISRE reporter signal. Normalized response using DMSO as a control. b THP-1 cell viability. RU
and cell viability were measured by treatment with 40 μM of each compound and are shown as mean ± standard
deviation.

As a result of the SAR study, all thiourea derivatives revealed negligible or very weak potency,
regardless of alkyl and aryl moiety at R1 (3a to 3f). No significant effects of p-nitro, fluoro, chloro
substituents were observed on the aryl ring, however, the p-bromo substitution revealed undesired
cellular toxicity (3c to 3e). In the aryl part, meta substitution lead to slightly increased activity compared
to para substitution (3e and 3f).
To enhance activity, we introduced a urea moiety as a linker with diverse substitution on the aryl ring,
which increased potency of ARP analogues for immune stimulation. Compared with the thiourea linker,
para substitution on the phenyl ring exhibited stronger potency than meta substitution in most urea
linker cases (4a to 4h). In para-substituted derivatives, no significant activity was observed in the nitro
moiety (4a) which corresponded to thiourea linker, whereas the fluoro and trifluoromethyl moieties
showed increased activity (4b and 4c). However, compound 4c showed undesired cellular toxicity,
thereby a trifluoromethyl substituent was replaced by a trifluoromethoxy moiety which substantially
enhanced potency (4.2-fold change) and generated no toxicity (4d).

Table 2. ISRE reporter assay of ARP derivatives with carbonyl linkers.
O
Cl
N
R1
Cl
N
Compound
R1
RUa
Cell viability (%)
89 ± 16
5a
5b
5c
5d
5e
1.3 ± 0.7
2.5 ± 0.8
2.8 ± 0.3
1.8 ± 0.1
3.7 ± 0.9
11
N
89 ± 8
N
105 ± 20
90 ± 15
CF3
CF3
OCH3
81 ± 16
OCH3
5f
1.9 ± 1.1
83 ± 14
OCH3
NO2
H3C
Cl
5g
5h
1.4 ± 0.6
2.3 ± 1.2
84 ± 16
86 ± 18
Cl
OCH3
OCH3
5i
5.2 ± 1.5
98 ± 22
OCH3
F
F
F
5j
2.8 ± 1.7
2.5 ± 1.4
101 ± 15
96 ± 12
F3C
5k
a Relative unit for ISRE reporter signal. Normalized response by DMSO as a control. b THP-1 cell viability. RU
and cell viability were measured by treatment with 40 μM of each compounds and are shown as mean ± standard
deviation.

Considering the interesting results of thiourea and urea linker, we next incorporated carbonyl moiety as
a linker for ARP derivatives. Somewhat weaker or stronger potency was observed in case of
trifluoromethyl and methoxy substituents, compared to individual functional group in urea linker (5c to
5e). For the further investigation, aliphatic chain or heteroaryl group were introduced at R1 position
which produced no significant difference and only marginally increased activity (5a and 5b).
Furthermore, disubstituted ARP analogues were considered to increase activity (5f to 5h) that resulted
in weak activity which was below that of p-methoxy analogue (5e).
To increase potency of ARP derivatives, we examined the effects on carbon element by incorporating
a benzyl group with a trisubstituted functional group. For this purpose, trimethoxy benzyl (5i) or tri-fluoro
benzyl (5j) were introduced at the phenyl ring. As a result, immune response was considerably improved
due to tri-methoxy substitution on the aryl ring (5.2-fold change), whereas tri-fluoro benzyl substitution
showed no drastic change on ISRE result with a bit increase of activity (2.8-fold change). In addition,
we confirmed no effect on cell viability by benzyl group. For the further modification, we inserted a
phenethyl moiety at R1 position (5k) to assess effects of carbon chain which showed activity similar to
that of dichlorophenyl (5h) or trifluorobenzyl (5j) moiety (2.5, 2.3 and 2.8, respectively) Based on these
SAR results, we identified the compound 5i as an effective type I IFN inducer and conducted further
biological evaluation.
5i
5i
(a)
(b)
150
100
50
6
4
2
0
0
0
5
5
0
1
0
0
0
.
1
10
Concentration (M)
100
0
2
3
4
2
Concentration (M)
Figure 2. (a) Dose-response curve of the ISRE reporter assay. (b) THP-1 cell viability in 5i treatments.
To estimate the potency of compound, we tested dose-dependency of immune responses elicited by
5i, which clearly induced stimulation of ISRE reporter signal. EC50 was calculated as 13.1 μM and Emax
was interpolated as 4.9-fold change (Figure 2a). No cellular toxicity to THP-1 cells was observed at
treatment dosages of up to 40 μM (Figure 2b).

15
10
5
400
300
200
100
0

0
0
20
0
20
Concentration (M)
Concentration (M)
Figure 3. Evaluation of the effect on IFNβ and IP-10 secretion by compound 5i. Cytokine levels were measured
using ELISA.
We further evaluated whether 5i-mediated activation of ISRE reporter signal would be correlated with
activation of innate immunity. Using ELISA assay, we measured extracellular release of IFNβ and IP-
10 which are associated with activation of the type I IFN pathway. The results confirmed that 5i certainly
induced secretion of IFNβ and IP-10 (Figure 3).
IFNB
CXCL10
IRF7
IFIT3
OAS1
5
4
3
2
1
0
5
4
3
2
1
0
5
4
3
2
1
0
10
8
5
4
3
2
1
0
6
4
2
0
0
50
0
50
0
50
0
50
0
50
Concentration (M)
Concentration (M)
Concentration (M)
Concentration (M)
Concentration (M)
Figure 4. Gene expression results of various interferon-stimulated genes. mRNA expression level of IFNB,
CXCL10, IRF7, IFIT3, OAS1 was examined by qPCR and was normalized using GAPDH expression. Graph are
shown as the average value and RQmax.
In response to virus infection, host cells released various cytokines such as IFNs and the cytokine-
mediated anti-viral status is established and maintained by production of various ISGs. To validate
effects of 5i on anti-viral state, we explored the impact of 5i on type I IFN-induced ISG expression.
Corresponding to the cytokine level, mRNA expression of IFNB and CXCL10 was clearly increased by
5i treatment (Figure 4). In addition to IFNB and CXCL10, 5i significantly elicited transcription of various
ISGs such as IRF7, IFIT3, and OAS1 which are considered biomarkers of type I IFN signaling pathway
(Figure 4). In conclusion, all these results indicated the potential ability of 5i as an efficient type I IFN
inducer without undesired toxicity. Although, it is hard to conclude that 5i showed superior potency

compared to tilorone, an efficient small molecule anti-viral agent inducing type I IFN, due to the different
evaluating system. Considering 50 mg/kg required to treat Evola virus by tilorone [25], the fact that 5i
induced significant increased IFNβ secretion at 20 μM suggested structure insight for developing new
anti-viral therapy.
In conclusion, we confirmed ARP motif as a new core skeleton of type I IFN regulation. Moreover, we
synthesized various ARP analogues and investigated their biological activity for anti-viral state and
innate immunity. Based on the SAR analysis, we verified the importance of the carbonyl linker and the
benzyl moiety with a trimethoxy attachment on adjacent aryl ring in the ARP pharmacophore. As a result,
compound 5i was identified as a potent type I IFN inducer. Further investigation about cytokine
secretion and IFN-mediated ISG expression by 5i indicated effective stimulation of type I IFN pathway.
All these results suggest a beneficial therapeutic potential of 5i for anti-viral agent.

Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.
Acknowledgment.
This study was financially supported by Korea Institute of Science and Technology (KIST) Institutional
Program(2E30180)andBio & Medical Technology Development Program(NRF-2019M3E5D4066905)
of the National Research Foundation funded by Korean government (MSIT), a grant from Priority
Research Centers Program (2019R1A6A1A11051471), 2020R1C1C1010044 funded by the National
Research Foundation of Korea (NRF) and Convergence brain research through cross-institute
collaboration (KBRI 20-BR-03-01/ IBS-R001-D1-2020-b02).
Appendix A. Supplemental data
Supplementary data to this article including detailed experimental procedures can be found online
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Declaration of interests
☒ The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may
be considered as potential competing interests: