Dihydrodibenzothiepine: Promising hydrophobic pharmacophore in the influenza cap-dependent endonuclease inhibitor

Article abstract of DOI:10.1016/j.bmcl.2020.127547

This work describes a set of discovery research studies of an influenza cap-dependent endonuclease (CEN) inhibitor with a carbamoyl pyridone bicycle (CAB) scaffold. Using influenza CEN inhibitory activity, antiviral activity and pharmacokinetic (PK) parameters as indices, structure activity relationships (SAR) studies were performed at the N-1 and N-3 positions on the CAB scaffold, which is a unique template to bind two metals. The hydrophobic substituent at the N-1 position is extremely important for CEN inhibitory activity and antiviral activity, and dihydrodibenzothiepine is the most promising pharmacophore. The compound (S)-13i showed potent virus titer reduction over oseltamivir phosphate in an in vivo mouse model. The CAB compound described herein served as the lead compound of baloxavir marboxil with a tricyclic scaffold, which was approved in Japan and the USA in 2018.

Full text of DOI:10.1016/j.bmcl.2020.127547

Bioorganic&MedicinalChemistryLetters30(2020)127547
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Dihydrodibenzothiepine: Promising hydrophobic pharmacophore in the
influenza cap-dependent endonuclease inhibitor
Yoshiyuki Taoda⁎, Masayoshi Miyagawa, Toshiyuki Akiyama, Kenji Tomita, Yasushi Hasegawa,
Ryu Yoshida, Takeshi Noshi, Takao Shishido, Makoto Kawai
Shionogi Pharmaceutical Research Center, Shionogi & Company, Limited, 1-1, Futabacho, 3-chome, Toyonaka 561-0825, Japan
A R T I C L E I N F O
A B S T R A C T
Keywords:
This work describes a set of discovery research studies of an influenza cap-dependent endonuclease (CEN) in-
hibitor with a carbamoyl pyridone bicycle (CAB) scaffold. Using influenza CEN inhibitory activity, antiviral
activity and pharmacokinetic (PK) parameters as indices, structure activity relationships (SAR) studies were
performed at the N-1 and N-3 positions on the CAB scaffold, which is a unique template to bind two metals. The
hydrophobic substituent at the N-1 position is extremely important for CEN inhibitory activity and antiviral
activity, and dihydrodibenzothiepine is the most promising pharmacophore. The compound (S)-13i showed
potent virus titer reduction over oseltamivir phosphate in an in vivo mouse model. The CAB compound described
herein served as the lead compound of baloxavir marboxil with a tricyclic scaffold, which was approved in Japan
and the USA in 2018.
Influenza
Cap-dependent endonuclease
Carbamoyl pyridone
Chelator
Hydrophobic pharmacophore
Dihydrodibenzothiepine
Influenza is a viral respiratory infection caused by influenza virus,
with high fever, general malaise, cough, and sore throat appearing as
symptoms. Seasonal influenza is repeatedly epidemic around the world,
with 3–5 million people becoming infected every year.1 In 2009, the
strain A(H1N1)pdm09 became prevalent and spread rapidly because it
was significantly different in antigenicity from the seasonal influenza
circulating until then and humans had not developed immunity to it.
Types/subtypes including another subtype of new pandemic influenza
might affect the severity profile and lead to a large number of deaths.2,3
Moreover, a pandemic might occur again in the future.
bicycle (CAB) scaffold found by structure activity relationships (SAR)
studies of our compound library, inhibited influenza CEN activity and
thereby interfered with influenza virus replication.10 4a is largely
composed of two parts. The bicyclic carbamoyl pyridone moiety co-
ordinates with the metal cofactor present in the active site of CEN. The
lipophilic benzhydryl group at the N-1 position of the CAB core is
considered to play an important role in binding to the enzyme by hy-
drophobic interaction. In this paper, we report on optimization of the
lipophilic side chain at the N-1 position and the substituent at the N-3
position of the CAB scaffold. As a result, we discovered that the com-
pound having dihydrodibenzothiepine at the N-1 position exerted re-
markably potent CEN inhibitory activity, antiviral activity and reduc-
tion of virus titer in an in vivo mouse model.
At present, neuraminidase inhibitors such as oseltamivir, zanamivir,
peramivir, and laninamivir are used to treat influenza.4 However,
problems are arising such as the emergence of viruses resistant to these
therapeutic agents and new viruses against which they are ineffective.5
Thus, there is a growing need for new medication which has a different
mechanism for its antiviral effect. We started research on an influenza
cap-dependent endonuclease (CEN) inhibitor in 2006 and discovered
baloxavir marboxil, which was approved in Japan and the US in 2018.
CEN has two metal ions (Mg2+ or Mn2+) in its active site and is an
enzyme nesessary for virus replication.6 It has been reported that
compounds 1–3, the scaffold of which can chelate to these two metal
ions, inhibit the CEN activity (Fig. 1).7–9 However, they had insufficient
antiviral activity and were not developed as clinical candidates. We
previously reported that compound 4a, with a carbamoyl pyridone
The synthetic route is shown in Scheme 1. Amination of the com-
mercially available pyrone 5 using ammonia gave pyridone 6. After
condensation of carboxylic acid with the corresponding amine, N-
amination using O-(2,4-dinitrophenyl)hydroxylamine was performed to
give intermediate 8. Bicyclic pyridone derivatives 9 were synthesized
by cyclization with paraformaldehyde. Subsequently, compound 4 and
10–14 were obtained by N-alkylation with alkylhalide and debenzyla-
tion with TFA, or addition of alcohol accompanied by debenzylation
under acidic condition.
The dihydrodibenzothiepines having a substituent on the benzene
ring were synthesized according to the literature.11 First, ethyl
⁎ Corresponding author.
E-mail address: yoshiyuki.taoda@shionogi.co.jp (Y. Taoda).
https://doi.org/10.1016/j.bmcl.2020.127547
Received 8 July 2020; Received in revised form 27 August 2020; Accepted 6 September 2020
Availableonline11September2020
0960-894X/©2020ElsevierLtd.Allrightsreserved.

Y. Taoda, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127547
Fig. 1. Reported influenza CEN inhibitors and our design of new inhibitors.
Scheme 1. Synthesis of CAB compounds. Reagents and conditions: (a) 28% NH3 aq., r.t.; (b) (1) WSC-HCl, HOBt, DMF, r.t., (2) amine, r.t.; (c) O-(2,4-dinitrophenyl)
hydroxylamine, K2CO3, DMF, r.t.; (d) paraformaldehyde, AcOH, toluene, 100 °C; (e) (1) R1-X, Cs2CO3, DMF, r.t., (2) TFA, r.t.; (f) R1-OH, H2SO4, AcOH, r.t.; (g) EtI,
K2CO3, DMF, r.t.; (h) NBS, CCl4, reflux; (i) benzenethiol, K2CO3, acetone, reflux; (j) 2 N NaOH aq., EtOH, r.t.; (k) PPA, 120 °C; (l) NaBH4, MeOH, 0 °C ~ r.t.
esterification of 2-methylbenzoic acid 15 having a substituent and
bromination of methyl group afforded 17. After reaction with a ben-
zenethiol, the benzoic acid 19 was obtained by hydrolysis of the ethyl
ester. The dihydrodibenzothiepine ring was constructed by treatment
with polyphosphoric acid (PPA) and reduction of the ketone gave al-
cohol 21.
configuration, respectively (Scheme 2). The relative and absolute ste-
reochemistry of (R)-23 was confirmed by single crystal X-ray and that
of other compounds was determined based on this result (Fig. 2).
First, we studied the effect of substituents on the benzhydryl group
at the N-1 position (Table 1). In the case (4b-4f) of single substitution of
Me or Cl, no great improvement was observed in either CEN inhibitory
activity or antiviral activity. Second, when two substituents were
symmetrically introduced, some improvement in antiviral activity was
observed in 4h having two Cl atoms, but in other cases, the activities
decreased (4g, 4j, 4k). We thought that this was due to the reduction of
cell membrane permeability by the introduction of a polar substituent
In the case of compounds having a 1,1,1-trifluoroisopropyl group at
the N-3 position, acetylation of the hydroxyl group of the CAB scaffold
made it possible to separate the two diastereomers by usual silica gel
column chromatography. By hydrolyzing the acetyl group, we suc-
ceeded in obtaining derivatives with a dihydrodibenzothiepine of R or S
2

Y. Taoda, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127547
Scheme 2. Separation of stereoisomers of CAB compounds. Reagents and conditions: (a) Ac2O, Et3N, DMAP, CH2Cl2, r.t.; (b) 2 N NaOH aq., THF-MeOH, r.t.
demonstrated in the previous paper, the region around the N-3 position
is exposed to the solvent and does not have much effect on the potency
of the compounds.10 Therefore, we aimed to use the N-3 subtituent to
adjust the physicochemical properties of the molecule. Since compound
12a had a large rat iv clearance value (32.7 mL/min/kg), we in-
vestigated whether it would be improved by modification of the iso-
propyl group at the N-3 position. Conversion to a cyclopropyl group or
substitution with a polar group (OMe, NMe2) and deuterium instead
worsened clearance and did not significantly alter antiviral activity
(12b-e). However, in the case of substitution with a F atom, the
clearance of (S)-12g with (R)-1,1,1-trifluoro-2-propyl group was im-
proved (15.8 mL/min/kg). This can be attributed to improved stabili-
sation in rat microsomes and a decreased fu value in plasma. In addi-
tion, (S)-12g was the most potent of the four isomers (12g, 12h) and
demonstrated more than 3 times higher antiviral activity than 12a,
indicating a good balanced profile in both activity and PK profile.
Finally, (S)-12g was used as a lead compound to optimize the
substituent on the dihydrodibenzothiepine at the N-1 position
(Table 3). Since introduction of the Cl atom on the benzhydryl group
(4h and 4i) in Table 1 improved the antiviral activity, we decided to
thoroughly investigate the effect of a halogen atom (F or Cl) on the
dihydrodibenzothiepine ring. The F atom led to about 4-fold more CEN
inhibitory activity than (S)-12g when introduced at the positions 7 or 8
((S)-13e, (S)-13f), but there was no significant change at other posi-
tions. In addition, when the F atom was introduced at both positions 7
and 8, further improvement of CEN inhibitory activity was observed
((S)-13i). (S)-14f and (S)-14h having a Cl atom at position 8 or 10
showed CEN inhibitory activity below 10 nM. The enhanced enzyme
inhibitory activity of these compounds suggests the presence of small
lipophilic pockets in the vicinity of the dihydrodibenzothiepine ring
that can accommodate F and Cl atoms. Although many compounds
exhibit antiviral activity of EC50 values of low nM, there is a dis-
crepancy between the values of IC50 and EC50 in some cases. The
reason for this is unknown, but one possibility is that the cellular assay
system reached the limit of detection. As described above, (S)-13i and
Fig. 2. Single crystal X-ray structure of (R)-23: Thermal ellipsoids are drawn at
the 30% probability level.
or an increase in molecular size. To improve membrane permeability,
we designed dihydrodibenzoannulene 10, dihydrodibenzooxepin 11
and dihydrodibenzothiepine 12 derivatives by linking two benzene
rings of a benzhydryl group to constrain the free rotation. Although no
significant improvement in activity was observed in 11, both the CEN
inhibitory activity and the antiviral activity were improved about
fourfold in 10. Moreover, despite its racemic form, 12a demonstrated
almost equal activity to that of 10. The N-1 substituent of compound 11
is less lipophilic than those of 10 and 12. Therefore, the lower activities
of 11 seem to be due to the reduction of affinity for the enzyme and/or
cell membrane permeability. Based on this result, we selected dihy-
drodibenzothiepine as a substituent at the N-1 position for further op-
timization.
Table 2 shows the effect of the substituent at the N-3 position of the
CAB scaffold with dihydrodibenzothiepine at the N-1 position. As we
3

Y. Taoda, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127547
Table 1
SAR of N-1 position of CAB scaffold.
compound
R1
CEN IC50a (nM)
286
CPE EC50b (nM)
81.6
4a
4b
4c
4d
142
276
210
73.4
219
77.8
4e
4f
101
291
75.9
94.0
4g
4h
394
440
37.0
48.2
4i
4j
230
65.8
589
386
1310
456
4k
10
11
60.1
264
20.1
71.0
12a
61.9
16.9
Reported values were the median of two or more experiments. a CEN inhibitory activity was measured by enzymatic assay as reported previously.9 b
Madin-Darby bovine kidney (MDBK) cells were infected with influenza A virus (A/WSN/33 dilutions at 50 TCID50/well in the 96 well plate), and
incubated with test compounds at 37 °C in a CO2 incubator for 72 hr, and the concentration of test compound resulting in 50% cell protection was
reported as the EC50
.
4

Y. Taoda, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127547
Table 2
SAR of N-3 position of CAB scaffold.
compound
R1
R2
CEN IC50a (nM)
61.9
CPE EC50b (nM)
16.9
Met. Stab. Microsomec (%)
72.6
Rat iv Cld (mL/min/kg)
32.7
Fue (%)
7.68
12a
12b
12c
12d
35.0
57.9
151
13.7
20.7
84.1
73.7
58.6
76.4
46.0
49.1
98.7
7.83
10.6
NC
12e
43.4
11.7
74.4
39.1
6.01
12f
113
27.5
131
84.9
NT
33.5
NT
NC
NT
(R)-12 g
1890
(S)-12 g
(R)-12 h
(S)-12 h
49.7
285
112
4.69
21.7
18.6
98.1
NT
15.8
NT
1.03
NT
79.8
NT
NT
a
Reported values were the median of two or more experiments. Values of PK parameters are the means of duplicate experiments. CEN inhibitory activity was
measured by enzymatic assay as reported previously.9 b Madin-Darby bovine kidney (MDBK) cells were infected with influenza A virus (A/WSN/33 dilutions at 50
TCID50/well in the 96 well plate), and incubated with test compounds at 37 °C in a CO2 incubator for 72 hr, and the concentration of test compound resulting in 50%
c
cell protection was reported as the EC50
.
Metabolic stability in the presence of rat liver microsomes was represented as the % compound remaining at a con-
centration of 2 μM after 30 min, incubated at 37 °C. d Rat clearance was measured by LC/MS/MS after a single intravenous administration. e Free fraction ratio in the
presence of rat serum. NT means not tested. NC means not calculated.
(S)-14h were discovered as compounds having not only strong CEN
inhibitory activity but antiviral activity. Furthermore, when the PK
profile was examined, (S)-13i showed lower rat iv clearance value than
(S)-14h ((S)-13i: 11.0 mL/min/kg vs. (S)-14h: 19.6 mL/min/kg).
Therefore compound (S)-13i was selected for further evaluation.
In the mouse model inoculated with A/WSN/33 influenza virus
(100 TCID50/mouse), (S)-13i or oseltamivir phosphate was orally ad-
ministered twice a day (BID) at 5 days after infection (Fig. 3). At 24 h
after the first dosage, the amount of virus titer (log TCID50/mL) in the
lung was measured using Madin-Darby canine kidney (MDCK) cells.
When (S)-13i was administered at 0.4 mg/kg BID, the viral load was
reduced by one-tenth compared to the administration of a clinical dose
equivalent to oseltamivir phosphate (5 mg/kg BID). Furthermore, by
increasing the dose of (S)-13i to 10 mg/kg BID, the lung viral load was
reduced to about one-thirtieth of that after administration of oselta-
mivir phosphate (5 mg/kg BID). Thus, (S)-13i exhibited a superior virus
reduction effect to that of oseltamivir phosphate in vivo. This result
suggests that a CEN inhibitor is potentially useful for the treatment of
influenza.
In conclusion, we optimized the N-1 and N-3 positions of the CAB
scaffold of 4a as a lead compound which was reported in the previous
paper. As a result of SAR studies using CEN inhibitory activity and
antiviral activity as an index, we found that the lipophilic substituent at
the N-1 position was extremely important for the activity, with dihy-
drodibenzothiepine being the most promising. Furthermore, by con-
sidering PK parameters, we discovered that (S)-13i having (S)-7,8-di-
fluoro-6,11-dihydrodibenzothiepine at the N-1 position and (R)-1,1,1-
trifluoroisopropyl group at the N-3 position was an outstanding
5

Y. Taoda, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127547
6.0
5.0
4.0
3.0
2.0
Table 3
Optimization of the substituent on the dihydrodibenzothiepine ring.
(S)-compound X or Y CEN
CPE
Met. Stab.
Microsomec
(%)
Rat iv
Cld
Fue (%)
IC50a
(nM)
EC50b
(nM)
(mL/
min/
kg)
control
oseltamivir
5 mg/kg, bid0.4 mg/kg, bid2 mg/kg, bid 10 mg/kg, bid
p.o. p.o. p.o. p.o.
(S)-13i
(S)-13i
(S)-13i
12g
13a
13b
13c
13d
13e
13f
H
49.7
37.1
39.2
48.2
66.4
12.5
12.3
44.3
31.8
4.69
15.1
8.16
14.3
3.41
3.85
3.54
4.68
3.75
4.28
18.8
70.8
21.2
13.5
3.04
10.5
4.21
3.52
98.1
97.4
103
15.8
9.09
15.4
13.4
10.3
8.37
11.6
21.3
18.0
11.0
10.2
NT
1.03
1-F
1.19
2-F
0.975
0.807
0.574
0.978
0.383
1.29
3-F
93.9
95.6
91.8
95.3
98.9
102
Fig. 3. In vivo efficacy of (S)-13i in the mouse model inoculated with A/WSN/
33 influenza virus. (S)-13i or oseltamivir phosphate was orally administered
twice a day (BID) at 5 days after infection. Next, the amount of virus titer (log
TCID50/mL) in the lung at 24 h after the first dosage was measured.
4-F
7-F
8-F
13g
13h
13i
9-F
10-F
7,8-F,F
1-Cl
2-Cl
3-Cl
4-Cl
7-Cl
8-Cl
9-Cl
10-Cl
1.2
5.57
81.1
85.4
NT
0.411
0.589
NT
14a
14b
14c
14d
14e
14f
76.2
24.9
62.1
23.2
30.0
101
12.2
6.78
7.36
5.70
12.4
19.6
0.303
0.329
0.293
0.117
0.514
1.79
92.3
102
6.99
96.1
94.1
93.3
14g
14h
16.6
9.95
Reported values were the median of two or more experiments. Values of PK
a
parameters are the means of duplicate experiments. CEN inhibitory activity
was measured by enzymatic assay as reported previously.9 b Madin-Darby bo-
vine kidney (MDBK) cells were infected with influenza A virus (A/WSN/33
dilutions at 50 TCID50/well in the 96 well plate), and incubated with test
Fig. 4. Road to baloxavir marboxil from (S)-13i.
compounds at 37 °C in a CO2 incubator for 72 hr, and the concentration of test
c
compound resulting in 50% cell protection was reported as the EC50
.
Meta-
bolic stability in the presence of rat liver microsomes was represented as the %
compound remaining at a concentration of 2 μM after 30 min, incubated at
the PK studies. We are grateful to all biology members for the biological
assays.
d
37 °C. Rat clearance was measured by LC/MS/MS after a single intravenous
administration. e Free fraction ratio in the presence of rat serum. NT means not
tested. NC means not calculated.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bmcl.2020.127547.
compound. In a mouse model inoculated with A/WSN/33 influenza
virus, the virus reduction effect of (S)-13i exceeded oseltamivir phos-
phate. We continued optimizing the compound, which led to the dis-
covery of baloxavir marboxil (Fig. 4). Details of this will be reported in
the near future.
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The authors thank Masayoshi Ogawa for assistance in characterizing
the compounds presented in this manuscript. We thank Kenji Sato for
6