Optimization and SAR research at the piperazine and phenyl rings of JNJ4796 as new anti-influenza A virus agents, part 1

Article abstract of DOI:10.1016/j.ejmech.2021.113591

JNJ4796, a small molecule fuse inhibitor targeting the conserved stem region of hemagglutinin, effectively neutralized a broad spectrum of group 1 influenza A virus (IAV), and protected mice against lethal and sublethal influenza challenge after oral admi

Full text of DOI:10.1016/j.ejmech.2021.113591

European Journal of Medicinal Chemistry 222 (2021) 113591
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Optimization and SAR research at the piperazine and phenyl rings of
JNJ4796 as new anti-influenza A virus agents, part 1
,
,
Aoyu Wang a b, Yuhuan Li a, Kai Lv a *, Rongmei Gao a, Apeng Wang a, Haiyan Yan a,
Xiaoyu Qin a, Shijie Xu a, Chao Ma a, Jiandong Jiang a, Zengquan Wei c, Kai Zhang b
,
,
**
Mingliang Liu a
,
***
a CAMS Key Laboratory of Antiviral Drug Research, Beijing Key Laboratory of Antimicrobial Agents, NHC Key Laboratory of Biotechnology of Antibiotics,
Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100050, China
b Department of Pharmaceutical Chemistry, School of Pharmacy, Hebei Medical University, Shijiazhuang, 050017, China
c Chemical Medicine Department, R&D Center, Tasly Pharmaceutical Group Co. Ltd., Tianjin, 300410, China
a r t i c l e i n f o
a b s t r a c t
Article history:
JNJ4796, a small molecule fuse inhibitor targeting the conserved stem region of hemagglutinin, effec-
tively neutralized a broad spectrum of group 1 influenza A virus (IAV), and protected mice against lethal
and sublethal influenza challenge after oral administration. In this study, we reported the modification
and structure-activity relationship (SAR) of C (piperazine ring) and E (phenyl ring) rings of JNJ4796.
Compound (R)-2c was identified to show excellent in vitro activity against IAV H1N1 and Oseltamivir-
Received 20 March 2021
Received in revised form
24 May 2021
Accepted 26 May 2021
resistant IAV H1N1 stains (IC50: 0.03e0.06
profiles and low inhibition rate of hERG (13.2%, at 10
(R)-2c will begin soon.
mM), low cytotoxicity (CC50 > 200 mM), accepted oral PK
Keywords:
Influenza A virus
JNJ4796
Hemagglutinin inhibitors
Structure-activity relationship
m
M). Evaluation for the in vivo anti-IAV efficacy of
© 2021 Elsevier Masson SAS. All rights reserved.
1. Introduction
Oseltamivir, Zanamivir, and Peramivir (Fig. 1) [6,7], as well as the
most recently developed polymerase acidic (PA) protein inhibitor
Baloxavir (Fig. 1) [8], are major therapeutic options for new influ-
enza outbreaks. Unfortunately, NAIs- and Baloxavir-resistant
strains also have been reported [11e14]. Thus, there is a pressing
and urgent unmet need for new broad-spectrum influenza antivi-
rals to suppress seasonal and pandemic influenza.
IAV has two surface glycoproteins, hemagglutinin (HA) and
neuraminidase (NA). Based on the antigenicity of HA and NA, IAV
has been classified into 18 hemagglutinin subtypes (H1 to H18) and
11 neuraminidase subtypes (N1 to N11) [15]. HA subtypes are
further divided phylogenetically into two groups (Group 1: H1, H2,
H5, H6, H8, H9, H11, H12, H13, H16, H17 and H18; Group 2: H3, H4,
H7, H10, H14 and H15) [16]. The conserved stem region of HA has
been recently recognized as a potential anti-IAV target to develop
broad spectrum inhibitors [17,18]. Through high-throughput
screening and optimization, scientists from Janssen Pharma and
Scripps Institute collaboratively discovered JNJ4796 (Fig. 2), a small
molecule targeting the conserved stem region of HA, effectively
neutralized a broad spectrum of group 1 IAV (H1, H2, H5, H6, H11,
H13 and H16) in vitro. In addition, twice daily oral administration of
JNJ4796 (5 mg/kg) in the H1N1 infection mouse model gave rise to
100% survival and only moderate weight loss effects [18].
Influenza A virus (IAV), an enveloped negative-sense RNA virus
belongs to the orthomyxoviridae family, can cause substantial
morbidity and mortality through seasonal epidemics [1,2]. The
World Health Organization (WHO) estimated that approximately
5e10% of adults and 20e30% of children infected annually, 3e5
million cases of severe diseases and 250,000e500,000 deaths occur
from influenza each year worldwide [3]. Seasonal influenza vacci-
nations remain the principal prophylactic for controlling influenza
infections, but the efficacy is limited by the rapid antigenic drift and
shift in influenza viruses [4,5]. To date, three classes of anti-
influenza drugs are available on the market. The influenza M2 ion
channel blockers (Amantadine and Rimantadine) are not recom-
mended anymore since all of the circulating influenza strains are
resistant to them [9,10]. The neuraminidase inhibitors (NAIs)
* Corresponding author.
** Corresponding author.
*** Corresponding author.
E-mail addresses: Lvkailk@hotmail.com (K. Lv), zhk810728@163.com (K. Zhang),
lmllyx@126.com (M. Liu).
https://doi.org/10.1016/j.ejmech.2021.113591
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

A. Wang, Y. Li, K. Lv et al.
European Journal of Medicinal Chemistry 222 (2021) 113591
Fig. 1. Structures of anti-IAV drugs.
Fig. 2. Design of new target compounds.
Encouraged by the promising anti-IAV activity and novel
mechanism of JNJ4796, we initiated an optimization campaign with
the goal of identifying new HA inhibitor through systematically
exploring each ring system (A-E rings) of JNJ4796. In this work, we
studied the structure-activity relationship (SAR) of C (piperazine
ring) and E rings (phenyl ring) of JNJ4796, optimization of the other
three rings will be reported in due course (Fig. 2).
2. Results and discussion
2.1. Chemistry
General synthetic routes for key intermediates G7 and target
compounds 1e2 were shown in Schemes 1 and 2, respectively.
Coupling of Boc-protected cyclic amines (piperazine and dia-
zaheterocycles) with substituted benzoic acids (R1-PhCOOH), fol-
lowed by Iridium-catalyzed reductive Strecker reaction of the
resulted amides G2 gave a-amino phenylacetonitriles G3. [3 þ 2]
cycloaddition of the nitriles and trimethylsilyl azide in the presence
of tributyltin oxide furnished tetrazoles G4, which upon nucleo-
philic substitution with CH3I provided a mixture of positional iso-
In our previous studies [19e21], replacement of the piperazine
ring of PBTZ169 (a known anti-TB candidate) with symmetrical
spiro- or bridged diazaheterocycles was proved to be a successful
optimization strategy. Therefore, we started with our modifications
on the C ring of JNJ4796 by displacement of the piperazine with
three diazaheterocycles. After identifying optimal C ring, we
focused on optimization of the E ring of JNJ4796 since phenyl was
prone to be metabolized by CYP450 enzyme [22e24]. Considering
the fact that the phenyl group of JNJ4796 is wrapped in a small
mers (G5: G6
z 1 : 1). The two positional isomers were
conveniently separated by silica gel chromatography (Rf: G5 < G6,
EtOAc: Hexane ¼ 1 : 2), but NOE and 1H NMR failed to distinguish
the two isomers. Fortunately, the single crystal of isomer G5-2e
(Fig. 3) was successfully cultured, and the definite position of the
methyl group on the tetrazole ring was thus confirmed. The key
intermediates G7 were easily prepared by removal of the Boc-group
of G6 with TFA (Scheme 1).
binding pocket and forms CH-
p
bonds with His18 from HA1 [18],
only small size substituents could be accommodated into this
pocket. Thus, some small substituents (such as F, Cl, Br, et al.) were
introduced to the phenyl moiety (E ring) to improve metabolic
stability.
In this study, a series of JNJ4796 derivatives containing dia-
zaheterocycles and substituted phenyls as C and E rings, respec-
tively, were designed and synthesized. Our primary objective was
to find alternative anti-IAV candidates with potent activity and
drug-like properties. A preliminary SAR study was also explored to
facilitate further development of these compounds.
As shown in Scheme 2, nucleophilic addition of 2-amino-4-
nitrophenol G8 with methyl 4-formylpicolinate, followed by
dehydrogenation in the presence of DDQ gave benzimidazole G9.
The key intermediate G12 was obtained from G9 by catalytic hy-
drogenation, acetylation and hydrolysis in sequence. Finally,
condensation of acid G12 with above amines G7 yielded the target
compounds 1a-d and 2a-j.
2

A. Wang, Y. Li, K. Lv et al.
European Journal of Medicinal Chemistry 222 (2021) 113591
Scheme 1. Synthesis of intermediates G7. Reagents and conditions: a): R1-PhCOOH, EDCI, DMAP, DCM, rt; b): IrCl(CO)[P(C6H5)3]2, TMDS, TMSCN, anhydrous Toluene; c): Bis(-
tributyltin) oxide, azidotrimethylsilane, anhydrous toluene, 110 ꢀC; d): MeI, K2CO3, MeCN; e): TFA, DCM, 0 ꢀC-rt.
Scheme 2. Synthesis of target compounds 1a-d and 2a-j. Reagents and conditions: a): (i) Methyl 4-formylpicolinate, MeOH, 60 ꢀC; (ii) DDQ, DCM; b) H2 1 atm, Pd/C, MeOH; c): c):
acetic anhydride, DCM; d): LiOH, H2O, THF; e): G7, EDCI, DMAP, DMF.
Table 1
Structures, anti-IAV H1N1 activity and cytotoxicity (Series 1).
Compd.
Q
IC50
(
mM)
CC50 (mM)
1a
>50
>50
Fig. 3. Crystal structure of isomer G5-2e.
1b
1c
1d
18.50
>50
>50
>50
2.2. In vitro anti-IAV activity and cytotoxicity
7.41
0.35
32.05
The SAR of C ring was first investigated. As shown in Table 1,
replacement of the piperazine ring of ( )-JNJ4796 with three
symmetrical spiro- or bridged diazaheterocycles led to significant
decrease of potency (1a-d vs ( )-JNJ4796). The anti-IAV activity of
(
)-JNJ4796
>50
compounds 1a and 1c were even diminished (IC50: >50
surprisingly, compound 1d with reversed 2.7-diazaspiro [3.5]non-
ane ring was active against H1N1 strain (IC50: 7.41 M), although it
mM). Yet
showed slightly higher cytotoxicity against MDCK cell (CC50
:
32.05 M) than compound 1c (CC50: >50 M). These results
m
m
m
3

A. Wang, Y. Li, K. Lv et al.
European Journal of Medicinal Chemistry 222 (2021) 113591
Table 2
which is consistent with the analysis result of binding pattern as we
mentioned above.
Structures, anti-IAV H1N1 activity and cytotoxicity (Series 2).
In order to determine influence of the chiral center to anti-IAV
activity, we conducted chiral separation of the most potent com-
pound 2c. The absolute configurations of two enantiomers were
confirmed by ECD spectrum (Fig. S1.), and optical rotations were
measured. The two enantiomers were evaluated for their anti-IAV
activity against IAV H1N1 and Oseltamivir-resistant IAV H1N1
stains along with JNJ4796, Ribavirin and Oseltamivir for compari-
son. As shown in Table 3, The two enantiomers displayed potent
Compd.
R
IC50
(
mM)
CC50 (mM)
anti-IAV activity against both of IAV H1N1 (IC50: 0.03e5.51
Oseltamivir resistant-IAV H1N1strains (IC50: 0.06e6.65
enantiomer (R)-2c (IC50: 0.03e0.06 M) was found to be much
more active than (S)-2c (IC50: 5.51e6.65 M), indicating the anti-
IAV activity of 2c is the same enantioselective as ( )-JNJ4796
(Table 2). Moreover, it is worth to note that (R)-2c showed lower
m
M) and
2a
2b
2c
2d
2e
2f
2g
2h
2i
2-F
3-F
4-F
4-Cl
4-Br
4-MeO
4-OCF3
4-Me
4-CF3
4-NO2
H
7.41
2.47
0.27
1.71
1.43
11.98
3.18
4.28
12.83
7.41
0.35
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
mM). The
m
m
cytotoxicity (CC50 > 200
mM) and higher selective index than
JNJ4796.
2j
(
)-JNJ4796
2.3. Further appraisal of compound (R)-2c
Subsequently, enantiomer (R)-2c was evaluated for its in vivo PK
profiles in Balb/c mice after a single oral administration of 25 mg/
kg. As shown in Table 3, (R)-2c displayed better drug exposures
(Cmax: 866 ng/mL, AUC0-inf: 1913 h ng/mL), and longer half-life (T1/
2: 5.84 h) than JNJ4796, indicating that introduction of a fluorine
atom to phenyl group (C ring) led to an improvement in metabolic
stability as we originally speculated.
In most cases, compounds with high affinity for hERG ion
channel can induce QT interval prolongation frequently associated
with potentially lethal arrhythmias [25]. Therefore, compounds
(R)-2c and JNJ4796 were further examined for their hERG inhibi-
revealed that employing the piperazine as C ring is essential for
excellent activity.
The effect of substituents on phenyl ring (E ring) was then
explored. Compounds 2a-c with a fluorine atom at different posi-
tions on the phenyl ring of ( )-JNJ4796 were first synthesized. As
shown in Table 2, introduction of a fluorine atom at para-position,
as in compound 2c (IC50: 0.27
(2c vs ( )-JNJ4796), but the presence of the fluorine atom at ortho-
and meta-positions (IC50: 7.41 and 2.47 M, respectively) was found
mM), resulted in an increased potency
m
to be unfavorable (2a, b vs ( )-JNJ4796), suggesting that the posi-
tion of the substituent greatly influences activity, and the fluorine
atom at para-is preferred over ortho- and meta-positions.
tion rates (IRs) at 10 mM. To our delight, the IR of (R)-2c (13.2%) was
significantly lower than that of JNJ4796 (Table 4).
Based on above SAR, derivatives with halogens (Cl, Br), electron-
donating groups (CH3, OCH3) or electron-withdrawing ones (CF3,
OCF3, NO2) at para-position on the phenyl ring were further
designed and synthesized. Unfortunately, all of them (2d-j) showed
2.4. Molecule docking study of compound (R)-2c
decreased potency (IC50: 1.43e12.83 mM). These results indicated
that both the size and nature of substituent impacted the activity,
We predicted the binding mode of compound (R)-2c in the HA1-
HA2 interface (PDB code: 6CF7) through molecular docking with
Table 3
Anti-IAV activity and cytotoxicity of (S)-2c and (R)-2c.
a
Compd.
CC50
(m
M)
IAV H1N1
IC50
M)a
Oseltamivir-resistant IAV H1N1b
(
m
SI
IC50
(
m
M)a
SI
(S)-2c
(R)-2c
(S)-JNJ4796
(R)-JNJ4796 (JNJ4796)
Ribavirin
>200
>200
>200
143.65 48.80
>200
5.51 1.74
0.03 0.05
1.90 0.18
0.04 0.04
4.28 0.00
3.44 0.84
>36
6.65 1.31
0.06 0.02
8.50 1.89
0.05 0.02
4.28 0.00
179.56 35.41
>30
>3333
>23
2873
>46
>1
>6666
>105
3591
>46
Oseltamivir
>200
>58
a
The cytotoxicity and anti-virus activity were tested for 3 times.
Oseltamivir-resistant IAV H1N1 (clinical isolated IAV/Tianjin Jinnan/15/2009) was kindly provided by Professor Yuelong Shu at the Institute for Viral Disease Control and
b
Prevention, China Centers for Disease Control and Prevention, Beijing, China.
Table 4
Further appraisal of (R)-2c and JNJ4796.
Compd.
Pharmacokinetic profilesa
hERG IRb (n ¼ 3)
T1/2 (h)
Tmax (h)
Cmax (ng$mLꢁ1
)
AUC0-inf (h$ng$mLꢁ1
)
(R)-2c
JNJ4796
5.84 0.63
3.69 0.60
0.33
0.25
0
0
866 205
572 166
1913 554
1214 561
13.2 2.87%
53.8 4.04%
a
Pharmacokinetic properties in Balb/c mice after a single oral administration of 25 mg/kg, n ¼ 3.
b
hERG Kþ channel inhibition rate at 10
m
M, n ¼ 3. Abbreviations: t1/2, elimination half-life; Tmax, the time at which the Cmax is observed; Cmax, the maximum serum
concentration; AUC0-inf, area under the concentration-time curve up to infinite time; hERG, human Ether-a-gogo related gene.
4

A. Wang, Y. Li, K. Lv et al.
European Journal of Medicinal Chemistry 222 (2021) 113591
Fig. 4. Binding mode of compound (R)-2c in complex with HA: (A) comparison of the predicted binding model of compound (R)-2c and JNJ4796 in the HA X-ray structure (PDB
code: 6CF7), surface of HA1 is in blue color, surface of HA2 is in cyan color, carbons of JNJ4796 are in green color, carbons of (R)-2c are in red color; (B) The detailed interactions
between JNJ4796 and HA1-HA2 interface; (C) The predicted interactions between compound (R)-2c and HA1-HA2 interface.
CDOCK module of Discovery Studio 3.5. The docked model of the
compound (R)-2c was compared with JNJ4796 which is an intrinsic
ligand from the X-ray structure (PDB code: 6CF7). As the chemical
structure of compound (R)-2c was similar to JNJ4796, the binding
model of compound (R)-2c also showed a closed interaction
pattern with JNJ4796 (Fig. 4A). The ligand-receptor interaction
analyzation showed that the binding site of (R)-2c and JNJ4796
comprised similar residues (Thr49, Val 52, Ile 48, Thr318, Ile 45, His
38, Trp 21, Gly 20 and His18) (Fig. 4B and C). Notably, the fluo-
rophenyl moiety of (R)-2c could adjusted into the small binding
pocket, and the fluorine atom interacted with His18 and Val 18,
which may enhance the binding affinity. In contrast, compounds
2d-j with Cl, Br or Me et al. substituents may experience steric
clashes and led to reduced activity.
3. Conclusion
In summary, two series of JNJ4796 analogues were synthesized
and evaluated for their anti-IAV activity. Replacement of the
piperazine ring with spiro- or bridged diazaheterocycles resulted in
a dramatically decreased potency, suggesting that the piperazine
ring is an irreplaceable portion and essential for ligand-target in-
teractions. Introduction of substituents to the phenyl ring of
JNJ4796 was also detrimental, and the only exception is 4-
flurophenyl analogue 2c (IC50: 0.27
m
M) with better potency
(IC50: 0.27 M) than ( )-JNJ4796 (IC50: 0.35
m
mM).
The racemic 2c was then resolved via Chiral preparation-HPLC.
The absolute configurations of enantiomers were confirmed by
ECD spectrum, and optical rotations were measured. Both of them
5

A. Wang, Y. Li, K. Lv et al.
European Journal of Medicinal Chemistry 222 (2021) 113591
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and Oseltamivir-resistant IAV H1N1 stains. Enantiomer (R)-2c
(IC50: 0.03e0.06
than (S)-2c (IC50: 5.51e6.65
JNJ4796 (IC50: 0.04e0.05 M). More importantly, (R)-2c displayed
lower cytotoxicity (CC50 > 200 M), better oral PK profiles and
significantly lower hERG IR (13.2%, at 10 M) than JNJ4796. Studies
mM) showed significantly higher anti-IAV potency
mM), and comparable activity to
m
m
m
to determine the in vivo anti-IAV efficacy of (R)-2c will begin soon.
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All animal experiments were carried out in accordance with the
guidelines of the Chinese Association for Laboratory Animal Sci-
ences and approved by the institutional ethical committee (IEC) of
Peking Union Medical College.
Funding sources
This work is supported by CAMS Initiative for Innovative Med-
icine (CAMS-2018-I2M-3e004, CAMS-2020-I2M-2e010), the Na-
tional Mega-project for Innovative Drugs (2018ZX09711001-007-
002), Foundation of the Hebei Education Department Science
Research Project (ZD2020164), Biomedicine Special Project of
Hebei Province of China (20372607D).
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.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2021.113591.
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