Synthesis and SAR Study of Carbamoyl Pyridone Bicycle Derivatives as Potent Inhibitors of Influenza Cap-dependent Endonuclease

Article abstract of DOI:10.1021/acs.jmedchem.9b00861

The medicinal chemistry and structure-activity relationships (SAR) for a novel series of carbamoyl pyridone bicycle (CAB) compounds as influenza Cap-dependent endonuclease (CEN) inhibitors are disclosed. Substituent effects were evaluated at the C (N)-1, N-3, and C-7 positions of the CAB ring system using a docking study. Submicromolar EC₅₀ values were achieved in the cellular assay with C-7-unsubstituted CAB which possessed a benzhydryl group on either the C-1 or the N-1 position. An N-3 substituent was found to be critical for the plasma protein binding effect in vitro, and the CAB-N analogue 2v exhibited reasonable total clearance (CL_tot). More importantly, compound 2v displayed significant efficacy in a mouse model infected with influenza viruses.

Full text of DOI:10.1021/acs.jmedchem.9b00861

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Article
Synthesis and SAR Study of Carbamoyl Pyridone Bicycle Derivatives
as Potent Inhibitors of Influenza Cap-dependent Endonuclease
Masayoshi Miyagawa, Toshiyuki Akiyama, Yoshiyuki Taoda, Kenji Takaya, Chika
Takahashi-Kageyama, Kenji Tomita, Kazuya Yasuo, Kazunari Hattori, Shinya Shano,
Ryu Yoshida, Takao Shishido, Tomokazu Yoshinaga, Akihiko Sato, and Makoto Kawai
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00861 • Publication Date (Web): 06 Aug 2019
Downloaded from pubs.acs.org on August 8, 2019
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Synthesis and SAR Study of Carbamoyl
Pyridone Bicycle Derivatives as Potent
Inhibitors of Influenza Cap-dependent
Endonuclease
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Masayoshi Miyagawa*†, Toshiyuki Akiyama†, Yoshiyuki Taoda†, Kenji Takaya†, Chika
Takahashi-Kageyama†, Kenji Tomita†, Kazuya Yasuo†, Kazunari Hattori†, Shinya
Shano†, Ryu Yoshida†, Takao Shishido†, Tomokazu Yoshinaga†, Akihiko Sato†,
Makoto Kawai†
AUTHOR ADDRESS
†Shionogi Pharmaceutical Research Center, Shionogi & Co., Ltd., 1-1, Futabacho, 3-
chome, Toyonaka, 561-0825, Japan
KEYWORDS
Anti-influenza drug, Cap-dependent endonuclease, Carbamoyl Pyridone, Chelator,
Virtual modeling
Abstract
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The medicinal chemistry and structure activity relationships (SAR) for a novel series of
CArbamoyl pyridone Bicycle (CAB) compounds as influenza Cap-dependent
EndoNuclease (CEN) inhibitors are disclosed. Substituent effects were evaluated at the
C (N)-1, N-3, and C-7 positions of the CAB ring system using docking study.
Submicromolar EC50 values were achieved in the cellular assay with C-7-unsubstituted
CAB which possessed a benzhydryl group on either the C-1 or N-1 position. An N-3
substituent was found to be critical for the plasma protein binding effect in vitro, and the
CAB-N analogue (2v) exhibited reasonable total clearance (CLtot). More importantly,
the compound 2v displayed significant efficacy in a mouse model infected with influenza
viruses.
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Text
1. Introduction
Influenza is an acute respiratory infectious disease that affects 5-10% of the world
population every winter, resulting in 3-5 million severe cases with 250 000-50 000
1
deaths. In the past century, four influenza pandemics have occurred, each causing the
2
death of millions. While vaccination is a reasonable preventive, its efficacy is heavily
dependent on accurate prediction of the predominant infectious strains for each season.3
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Major antiviral drugs, such as zanamivir, oseltamivir and peramivir, target viral
neuraminidase and, can be useful for treatment but must be administered within 48 hours
of infection.4, 5 These therapeutics also can cause undesirable side effects, including
unusual neurologic or psychiatric events such as delirium, hallucinations, confusion, and
abnormal behavior, primarily in children.5, 6 Moreover, problems such as the appearance
of resistant strains, as well as worldwide epidemics caused by new strains of influenza
viruses possessing high pathogenicity and mortality,7-9 have raised the need for the
development of novel anti-influenza drugs that function via different mechanisms.10
The influenza virus is a lipid-enveloped virus with a negative-sense single-strand RNA
with the viral genome having eight genomic segments; PB2, PB1, PA, HA, NP, NA, M
and NS. PB2, PB1 and PA compose the polymerase complex and are responsible for both
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transcription and replication of the viral genome. The viral replication starts with “cap-
snatching” and occurs in three steps . First, the PB2 subunit binds the 5’-mRNA cap of a
host cell, and in the second step the PA subunit cleavages 10-13 nucleotides downstream
to yield 5’-capped RNA fragments. These fragment serve as primers for viral mRNA
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elongation catalyzed by the PB1 subunit in the third step. Those three subunits are
essential for proliferation of the influenza virus, in particular, Cap-dependent
EndoNuclease (CEN) in the N-terminal domain of the PA subunit is highly conserved
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across various influenza types and subtypes.13, 14 Therefore CEN inhibitors are expected
to have broad efficacy against multiple influenza viruses. However, there are no currently
approved drugs.
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Biochemical and crystallographic studies have revealed CEN to contain a dinuclear
2
+
2+
14
metal in the active site, employing two Mn or Mg ions.
In previous researches, several potent inhibitors which bind the CEN active site, have
been reported (Figure 1). Almost all of them(1a~1g) belonged to 1 metal, 2 metal or
water-mediated 2 metal chelator which has a hydrophobic region behind the chelate side
to enhance CEN inhibitory potency.15-22 However their antiviral activities are very weak
compared to their enzyme inhibitory potencies. This may be due to low cell membrane
permeability caused by the high polarity of their chelate motifs. Recently, the non-chelate
2
3
compound 1h was reported, to have low dissociation of enzyme and virus inhibition
activity, but its in vitro activity was not sufficient for clinical applications.
Our research effort focused on a new type of scaffold, a 2-metals chelator, namely,
CArbamoyl pyridone Bicycle (CAB) shown in Figure 2. First, we identified the hit
compound 2a, which binds two metals, as a CEN inhibitor from our compound library.
As described below, the evolution from 2a to the promising lead CAB compounds was
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accompanied by a marked increase in enzyme inhibitory and antiviralactivity. In addition,
one CAB congener which had acceptable in vivo clearance in rat, showed significant
efficacy in a mouse model infected with influenza viruses.
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OH
OH
HO
O
O
N
O
OH
H
N
HO
N
O
O
N
OH
HO
HO
HN
O
F
1a
1b
1c
N
H
Cl
OMe
1d
CEN IC50 = 0.5 M
Antivirus EC50 = not reported
CEN IC50 = 0.43 M
CEN IC50 = 0.8 M
high cytotoxicity
CEN IC50 = 0.94 M
Antivirus EC50 = 16 M
2.21 M (in house)
Antivirus EC50 = 0.35 M
38.3 M (in house)
HO
O
O
OH
O
OH
O
NH
NH
O
H
N
N
HO
N
N
N
N
O
N
S
N
HN
N
F
NH
N
O
1e
1f
1g
1h
N
Cl
N
N
H
N
N
CEN IC50 = 0.011 M
Antivirus EC50 = 11.4 M
CEN IC50 = 0.014 M
Antivirus EC50 = 2.1 M
CEN IC50 = 0.005 M
Antivirus EC50 = 0.201 M
CEN Kd = 1.1 M
Antivirus EC50 = 0.8 M
Figure 1. Reported potent CEN inhibitors. The results of compound 1a tested in our
conditions were shown.
OH
O
OH
O
3
O
O
O
O

N
N
7
1
HO
N
N

X

Hit compound
X = CH; CAB-C
X = N; CAB-N
2a
Figure 2. Screening hit compound 2a, and the chemical structure of the CAB scaffold,
and each substituent positions.
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2
. Chemistry
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Scheme 1 shows the synthetic routes to the Carbamoyl Pyridone Bicycle (CAB) moiety.
2
4
Cyclization of 3 with cinnamoyl chloride and LHMDS yielded pyrone 4. Ruthenium-
catalyzed oxidative cleavage of olefin provided aldehyde 5. This compound 5 was
oxidized with sodium sulfite to produce the corresponding carboxylic acid 6.
Condensation of 6 with 2-methoxyethylamine afforded compound 7. Insertion of various
amino-alcohol yielded pyridone derivatives 8. Cyclization of 8 under Mitsunobu
conditions gave CAB-C moiety 11. Insertion of N-Boc hydrazine to compound 7 yielded
N-amino pyridone 9. Compound 9 was deprotected with TFA followed by cyclization to
provide CAB-N moiety 10. Alkylation of 10 with various alkyl bromides gave 11(d, j).
Compound 11k was deprotected with TFA to produce 2k. Hydrolysis of ethyl ester and
deprotection of benzyl ether furnished compound 2(b-j). 1-Benzhydryl-7-carboxylic acid
1
2i was converted to its N-methyl amide 13 and Weinreb amide 14 derivatives by
condensation with the corresponding amines. Compound 13 was deprotected with TFA
to produce 2l. Treatment of 14 with MeMgBr in THF afforded 7-acetyl compound 15.
Baeyer-Villiger oxidation of 15 with m-CPBA and hydrolysis of acetyl group gave 7-
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hydroxy pyridone 17. Compound 17 was alkylated with MeI in the presence of NaH to
afford 7-methoxy-pyridone 18. Compound 18 was treated with TFA to yield 2n. On the
other hand, Curtius rearrangement of 12i by stepwise method provided 7-
methoxycarbonylaminopyridone 19. Hydrolysis of 19 and deprotection of benzyl group
gave 2m. Decarboxylation of 12i at high temperature (245 °C) in sealed tube under
microwave condition produced 7-nonsubstituted pyridone 21. Deprotection of the benzyl
group gave compound 2q. In the same way, compound 2t was synthesized from
compound 12j via compound 24. Compound 21 was treated with NBS to convert 7-
bromo-pyridone 22. Treatment of 22 with CuCl in DMSO afforded 7-chloro derivative
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p in which benzyl ether was deprotected under the reaction conditions. Palladium-
catalyzed methylation of compound 22 at the C-7 position was performed to give
compound 23. Deprotection of benzyl ether furnished compound 2o.
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O
N
O
OBn
OBn
OBn
O
O
O
O
CHO
i
ii
iii
O
EtO
EtO
O
EtO
O
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OBn
OBn
O
OBn
O
CO2H
O
O
O
O
N
N
iv
H
vii
H
EtO
O
EtO
O
7
EtO
N
NH
Boc
O
6
O
O
9
v
viii
OBn
N
O
OBn
N
O
OBn
O
O
O
vi
O
O
O
O
O
ix
N
N
N
H
EtO

EtO
EtO
N
OH
X
R
N
H
O
R
O
8
b, c, e-i
11b-j
10
x
xi
OH
O
OBn
N
O
OBn O
O
O
O
O
O
O
xvii
N
N
N
EtO
N
HO
N
X
R'
O
BH
O
O
R
BH
2
k
12b-j
19: R' = NHCO Me
20: R' = NH2
2
xviii
xii
x
x
OR''
O
OBn
N
OH
O
O
O
O
O
O
O
R1
N
N
N
x
x
N
N
N
R2
R'
R'
X
R
BH
O
BH
1
3: R1 = H, R2 = Me
4: R1 = OMe, R2 = Me
2b-j, l-o, q, t
xix
xx
12i: R' = COOH, R'' = Bn
1: R' = H, R'' = Bn
1
2
x
b: X = CH(S), R = Bn, R' = COOH
c: X = CH(R), R = Bn, R' = COOH
d: X = N, R = p-F-Bn, R' = COOH
e: X = CH(S), R = Ph, R' = COOH
f: X = CH(S), R = Phenethyl, R' = COOH
g: X = CH(S), R = i-Pr, R' = COOH
h: X = CH(S), R = i-butyl, R' = COOH
i: X = CH(S), R = BH, R' = COOH
j: X = N, R = BH, R' = COOH
l: X = CH(S), R = BH, R' = CONHMe
m: X = CH(S), R = BH, R' = NH2
n: X = CH(S), R = BH, R' = OMe
o: X = CH(S), R = BH, R' = Me
q: X = CH(S), R = BH, R' = H
22: R' = Br, R'' = Bn
2p: R' = Cl, R'' = H
xxi
xiii
x
xxii
23: R' = Me, R'' = Bn
OBn
N
O
OBn
N
O
O
O
O
O
N
N
R'
R'
N
BH
BH
1
1
1
5: R' = Ac
6: R' = OAc
7: R' = OH
12j: R' = COOH
24: R' = H
xiv
xv
xix
xvi
18: R' = OMe
t: X = N, R = BH, R' = H
Scheme 1. Synthesis of Compounds 2b-q and 2t
Reagents and conditions: (i) cinnamoyl chloride, LHMDS, THF, -78 °C; (ii) RuCl3,
NaIO , conc.H SO , MeCN, r.t.; (iii) conc.H SO , amidosulfuric acid, sodium sulfite,
4
2
4
2
4
MeCN, r.t.; (iv) (1) WSC-HCl, HOBt, DMF, rt; (2) 2-methoxyethanamine, r.t.; (v) amino-
alcohol, xylene, 120 °C; (vi) PPh , DEAD, THF, r.t.; (vii) N-Boc hydrazine, AcOH,
3
toluene, reflux; (viii) (1) TFA, DCM, r.t.; (2) formaldehyde, EtOH, microwave, 100 °C;
(
ix) R-Br, Cs CO , DMF, r.t.; (x) TFA, r.t.; (xi) 2N NaOH aq., EtOH, r.t.; (xii) (1) ethyl
2 3
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chloroformate, TEA, DMF, r.t.; (2) amine, DMAP, r.t.; (xiii) MeMgBr, THF, -50 °C;
(xiv) m-CPBA, DCM, r.t.; (xv) EtOH, reflux; (xvi) MeI, NaH, DMF, r.t.; (xvii) (1) ethyl
chloroformate, TEA, DMF, r.t.; (2) NaN , r.t.; (3) MeOH, 50 °C; (xviii) 2N NaOH aq.,
3
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EtOH, 60 °C; (xix) microwave, Ph O, 245 °C; (xx) NBS, DCM, reflux; (xxi) CuCl,
2
DMSO, 120 °C; (xxii) hexamethyldistannane, Pd(PPh ) , toluene, reflux; BH means
3 4
benzhydryl
The synthetic routes to 7-decarboxylated CAB-C and CAB-N analogues are shown in
Scheme 2. The commercially available pyrone 25 was converted to pyridone 26 via
amination with N-Boc-ethylene diamine derivatives. Deprotection of Boc group and
neutralization led 26 to CAB-C ring 27. Hydrolysis of methyl ester followed by
decarboxylation yielded compound 29. Alkylation of 29 with the corresponding alkyl
bromides gave 30r and 30s. Deprotection of the benzyl ether furnished compound 2r and
2
s. On the other hand, amination of the commercially available pyrone 31 with ammonia
gave pyridone 32. After condensation of carboxylic acid with the corresponding amine,
N-amination was performed to give intermediates 34u-w. Cyclization of 34 and followed
by alkylation provided CAB-N ring 35u-w. Deprotection of the benzyl group gave
compounds 2u-w.
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
OBn
N
O
OBn
N
O
OBn
N
O
OBn
O
O
O
O
O
O
O
O
O
NH
NH
O
O
O
i
O
ii
O
iii
HO
NHBoc
O
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
25
26
O
27
28
OBn
N
O
OBn
N
O
OH
O
O
R
O
R
NH
N
N
iv
v
vi
N
r: R = Me
s: R = i-Pr
2
9
30r, 30s
O
2r, 2s
O
OBn
O
OBn
O
OBn
O
OBn O
O
vii
O
viii
R
ix
R
N
H
x
OH
OH
N
H
O
NH HCl
NH
33u-w
N
NH2
3
1
32
34u-w
OBn
O
N
OH
O
N
O
R
O
R
N
N
xi
N
N
u: R = Me
v: R = i-Pr
w: R = 2-methyl-furan
3
5u-w
2u-w
Scheme 2. Synthesis of Compounds 2r, 2s, and 2u-w
Reagents and conditions: (i) amine, toluene, reflux; (ii) (1) 4N HCl EtOAc, r.t.; (2)
NaHCO aq., r.t.; (iii) 2N NaOH aq., THF-MeOH, r.t.; (iv) microwave, Ph O, 240 °C; (v)
3
2
R-Br, Cs CO , r.t.; (vi) TFA, r.t.; (vii) 28% NH aq., r.t.; (viii) (1) WSC-HCl, HOBt,
2
3
3
DMF, r.t., (2) amine, r.t.; (ix) O-(2,4-dinitrophenyl)hydroxylamine, K CO , DMF, r.t.;
2
3
(
x) (1) paraformaldehyde, AcOH, toluene, 100 °C, then concentration, (2)
bromodiphenylmethane, Cs CO , DMF, r.t., (xi) TFA, r.t.
2
3
3
3
. Results and Discussion
.1. In Vitro SAR.
We identified several hit compounds in our library. One of them was compound 2a
which was a CEN inhibitor without a hydrophobic region. From our previous research22,
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introduction of the hydrophobic domain to the chelator moiety led to enhanced inhibitory
activity of CEN. A compound substituted by a benzyl group at the C-1 position, 2b
showed remarkable activity compared to 2a, whereas its enantiomer 2c had very low
activity. In addition, replacement of the nitrogen atom at the 1-position (CAB-N)
maintained activity against CEN. On the other hand, either a shorter or longer linker, 2e
and 2f, was clearly associated with a dramatic loss of potency compared to 2b. In the
same way, conversion to an alkyl group, 2g and 2h, reduced in the potency. A branched
structure closer to the CAB ring was found to be favorable for the enzyme inhibition.
From these findings, we hypothesized that substitution at the benzylic position of
compound 2b and 2d would be effective. Benzhydryl-substituted compounds 2i and 2j,
were found to show, 4-5 fold higher inhibition potency than 2b and 2d, respectively
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(corresponding benzyl analogues).
Table 1. SAR of 1-Substitited CAB Inhibitors
OH
O
O
O
O
N
HO
N
X
R
CEN IC50a (M)
68.6
CPE EC50b (M)
Compound X (Chirality)
R
2
a
CH
H
N.D.
> 50
2b
CH (S)
Bn
0.241
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
2
2
2
c
d
e
f
CH (R)
N
Bn
27.4
0.419
27.8
1.35
> 50
> 25
> 50
N.D.
p-F-Bn
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
CH (S)
CH (S)
Ph
Phenethyl

2g
CH (S)
CH (S)
2.99
9.31
N.D.

2
h
N.D.
2
2
i
CH (S)
N

0.0478
0.116
0.293
j
1.47
a
b
Reported values are the means of three or more experiments. Enzyme inhibitory
activity of CEN was measured by a fluorescence recovery assay of the enzyme reaction.
cMDBK cells were incubated with test compounds and influenza A virus (A/WSN/33)
for 72 hr, and the concentration of test compound resulting in 50% cell protection was
reported as the EC . N.D., not determined.
5
0
We attempted to validate the chelating mode of our inhibitors to the active site in CEN.
We hypothesized that chelation by the CAB scaffold could done by the four modes shown
in Figure 3. In order to estimate the chelate binding mode, constrained docking and
minimization studies were performed for a reported CEN crystal structure (PDB code
14
2
W69) with Glide and MacroModel (Schrödinger Inc. U.S.A.). Metal binding oxygen
atoms of 2i were fixed in each optimal geometry to bind the two metals in the active site.
The ligand molecule 2i and the receptor protein were minimized in the flexible mode.
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The coordinating phenolic oxygen atom of inhibitor 2i was deprotonated and the charge
on each metal center was assigned as 2+ in the simulation.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
As a result, docking analysis showed that the CAB derivative 2i could chelate to the
active site metals in the most stabilized mode (c). C-7 carboxylic acid on 2i did not chelate
to metals, but interacted with the side chains of Lys134 and Tyr130 mediated by the
hydrogen bond. More interestingly, the two benzenes on 2i nicely filled two hydrophobic
pockets (Pocket 1 and 2 in Figure 4). In addition, the methoxyethyl moiety binding pocket
neighboring N-3 position (Pocket 3) could tolerate a variety of substitutions, which
suggests that this region of the binding site is not spatially constrained.
(a)
(b)




N
N
N
N
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
E80
D108
E119
E80
D108
E119
(c)
(d)


O
O
O
N
N
N

N
O

O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
E80
D108
E119
E80
D108
E119
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Figure 3. Conceivable chelate modes of the CEN active site with CAB-C analogue.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 4. Docking simulation of 2i (green) binding to the active site of CEN. Two metals
and lipophilic sites are shown in light blue and yellow respectively.
In the light of these findings, we considered the conversion of carboxylic acid at C-7 to
be acceptable for the CEN active pocket. Ethyl ester 2k and N-methyl amide 2l were less
potent in both the CEN and CPE assays but the activities did not disappear. When
sterically restricted and non-chelatable substituents, such as an amino, methoxy, methyl
or chloro group were evaluated, the potency decreased by 2~8 fold. By removing the
carboxylic acid at the C-7 position, we obtained a new type of inhibitor 2q that was 2-
fold less potent than
2i in the CEN assay. This may have been due to the absence of
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the hydrogen bond. However, compound 2q showed the same range of activity in the CPE
assay. Consequently, it is reasonable to assume that non-substitution at the C-7 position
increases membrane permeability to result in enhancing cell activity, because
decarboxylation reduces down the molecular weight and increases the lipophilicity.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
Calculated LogP of 2i and 2q were 1.29 and 2.06, respectively)25
Table 2. SAR of 7-Substitited CAB Inhibitorsa
OH
O
O
R
O
N
N
CEN IC50b (M)
0.0478
0.298
CPE EC50c (M)
0.293
Compound
R
2i
COOH
2
2
k
l
CO Et
2.53
2
CONHMe
NH2
OMe
Me
1.59
7.11
2m
0.358
3.86
2
2
n
o
0.110
1.68
0.281
2.47
2p
Cl
0.114
0.541
2
q
H
0.115
0.134
a
b
Reported values are the mean of three or more experiments. Enzyme inhibitory
activity of CEN was measured by a fluorescence recovery assay of the enzyme reaction.
cMDBK cells were incubated with test compounds and influenza A virus (A/WSN/33)
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
for 72 hr, and the concentration of test compound resulting in 50% cell protection was
reported as the EC .
5
0
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Next, we explored the N-3 position of the CAB scaffold. We thought conversion of the
N-3 position could adjust the physicochemical property of molecules without loss of
potency, because the docking study indicated that this region was not spatially
constrained in the CEN active site. The presence of an N-3 methyl or isopropyl substituent
in CAB-C series (2r, 2s) appeared to lead to the retaining of antiviral potency. The CAB-
N series (2t, 2u, 2v) were similarly found to be equipotent to CAB-C analogues in the
CPE assay except for the 2-methyl-furan-substituted 2w which showed reduced
potency in both assays. In silico docking study using 2v demonstrated the isopropyl group
at N-3 position did not interact any residues in CEN, because it was located in the solvent
exposure resion (Figure S3). Interestingly, PK study with rat showed that more
hydrophobic substituent groups improved rat iv clearance in each series. This was caused
by thet decreasing fu value in plasma in a group with high metabolic stability (2q-2v). On
the other hand, 2w was lower than the clearance value in spite of having the lowest fu
value. It may be due to low stability in the rat microsome. As a result, we obtained the
promising inhibitor 2v which exhibited good antiviral potency with 81.6 nM and
reasonable in vivo clearance with 10.9 mL/min/kg.
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Table 3. SAR of 3-Substitited CAB Inhibitors and Rat PK Parametersb
a
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
OH
O
X
O
R
N
N
Rat
iv Clf
X
CEN
IC50c
CPE
EC50d
Met. Stab.
Microsomee
(%)
fug
Comp
ound
(Chiralit
R
(mL/mi
(%)
y)
(nM)
(nM)
n/kg)
O
2q
115
80.4
239
103
164
286
134
79.7
79.9
78.7
83.4
92.1
86.0
42.2
31.6
25.3
47.4
18.8
10.9
29.7
28.5
18.2
12.9
7.00
3.77

2
2
r
s
CH (S)
Me
144
i-Pr
57.5
75.2
81.6
81.6
O
2t

2
2
u
Me
N
v
i-Pr
O

2
w
588
478
50.6
42.6
2.17
a
b
Reported values are the means of three or more experiments. Values are the means of
c
duplicate experiments. Enzyme inhibitory activity of CEN was measured by a
d
fluorescence recovery assay of the enzyme reaction. MDBK cells were incubated with
test compounds and influenza A virus (A/WSN/33) for 72 hr, and the concentration of
e
test compound resulting in 50% cell protection was reported as the EC . Metabolic
50
stability in the presence of rat liver microsomes was represented as the %compound
f
remaining at a concentration of 2 M after 30 min, incubated at 37 °C. Rat clearance was
measured by LC/MS/MS after a single intravenous administration. Free fraction ratio in
g
the presence of rat serum.
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
As the active site of CEN is highly conserved among all influenza virus families, we
confirmed the broad antiviral spectrum using compound 2s and 2v . They showed similar
antiviral behavior in the CPE assay. The atom at the 1-position (C or N) may not have
affected the antiviral activity as described previously. They displayed equivalent antiviral
activities against the H1N1 type, including the reverse genetic strain (rgA/WSN/33),
oseltamivir-resistant strain (rgA/WSN/33–NA/H274Y), and the clinically isolated strain
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(A/PR/8/34). Although their antiviral activities against H3N2 (A/Victoria/3/75,
A/HongKong/8/68) were slightly reduced, those of influenza B exhibited the same range
as that against H1N1.
Table 4. Antiviral potency of compound 2s and 2v against various influenza viruses
Mean CPE EC50a (nM)
Virus
2s
2v
A/PR/8/34
194.4±6.0
154.8±45.9
183.0±11.3
165.1±15.8
80.4±50.0
828.8±94.4
301.5±77.9
124.3±39.8
A / H1N1
rgA/WSN/33 b
rgA/WSN/33 –NA/H274Y b 89.2±67.7
A/Victoria/3/75
487.6±185.5
232.4±17.4
100.1±19.0
A / H3N2
B
A/HongKong/8/68
B/Hong Kong/5/72
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B/Maryland/1/59
177.0±11.6
176.0±6.4
aMDCK cells were incubated with test compounds and influenza viruses for 72 hr, and
the concentration of test compound resulting in 50% cell protection was reported as the
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
b
EC . rg; means reverse genetics.
5
0
3
.2.
In vivo efficacy
Finally, compound 2v was advanced to a mouse influenza B (B/Maryland/1/59) model
Figure 5) where it showed dose-dependent efficacy in an immediate treatment model
after infection at 0.08-10 mg/kg(q.d., 1 day) by intravenous administration (ED =0.89
(
5
0
mg/kg/day). Moreover, we confirmed 100% survival rate at 10 mg/kg. This demonstrated
that CEN is an attractive target for influenza treatment and its efficacy against influenza
26
B is superior to other target of influenza virus (PB2) . However, we confirmed that 2v
was deficient with respect to antiviralpotency because a low dose (0.08, 0.4, and 2 mg/kg)
of 2v was not able to protect all mice infected with influenza virus. Furthermore, thet oral
bioavailability of 2v was not sufficient to confirm its in vivo activity with the same model.
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Figure 5. In vivo activity of 2v in mouse influenza B model when administered
immediately after infection: survival curve of female BALB/C mice (5 mice/group)
inoculated with influenza viruses B/Maryland/1/59 (100 TCID /mouse) by intranasal
5
0
instillation.
4
. Conclusion
In summary, we established the synthesis of two carbamoyl pyridone bicycle analogues
CAB-C, N) focused on the 1-, 3-, and 7-positions, which were identified asCEN
(
inhibitors of the influenza virus starting from our library compounds. Our research on the
substituent effects of 1- and 7-positions of the core templates, led to improved enzyme
inhibition and antiviral activity with corroboration by docking analysis. Additionally,
more hydrophobic substituents at the 3-position, in particular of thtet CAB-N moiety,
presenteda reasonable pharmacokinetic profile in rat coupled with increased protein
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binding. Further testing showed that compound 2s and 2v were potent against all
influenza A and B strains tested, including pandemic H1N1 flu strains. Moreover, 2v was
effective in the mouse model infected with influenza B virus in a dose-dependent manner.
Additional SAR studies in the title series leading to the discovery and development of
clinical candidate S-033188 will be reported in future publications.
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EXPERIMENTAL SECTION
Compound Purity and Identity. 1H NMR(400 MHz) spectra were measured on a Varian
MERCURY or Bruker in a solution of either CDCl or DMSO-d , using tetramethylsilane
3
6
as the internal standard. Chemical shifts are expressed as δ (ppm) values for protons
relative to the internal standard. The purity of the test compounds was confirmed by
UPLC-MS with UV diode array detection to determine purity and by MS to confirm
molecular weight. A Waters Acquity UPLC system comprising Binary Solvent manager,
Sample Manager, PDA Detector, Waters ZQ or SQD mass spectrometer, and Waters
Acquity evaporative light scattering detector were employed. The Column: ACQUITY
UPLC® BEH C18 (1.7 μm ､ i.d.2.1x 50 mm) (Waters) Flow rate: 0.8 mL/min, UV
detection wavelength: 254 nm. Mobile phase: [A] is 0.1 % formic acid-containing
aqueous solution, and [B] is 0.1 % formic acid-containing acetonitrile solution. Gradient:
Linear gradient of 5 % to 100 % solvent [B] for 3.5 minutes was performed, and 100 %
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solvent [B] was maintained for 0.5 minute. Purity of all tested compounds were ≥95%.
Analytical HPLC was also used in some cases to monitor reactions and establish final
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compound purity using a C-18 column (5.0 μm, 0→100% CH CN /water with 0.1% TFA
3
and UV detection with mass spectrometer detection. Preparative HPLC conditions were
as follows: C-18 column, 5 μm, 21.2 mm × 150 mm; flow rate = 4 mL/min;mobile phase:
1
0→100% CH CN/H O/0.1%HCOOH (10 min run).
3 2
Ethyl-(E)-5-(benzyloxy)-4-oxo-6-styryl-4H-pyran-3-carboxylate (4).
A
1N
lithiumhexamethyldisilazane THF solution (4.29 ml, 4.29 mmol) was cooled to -78° C.,
and a THF solution (4 ml) of compound 3 (500 mg, 1.72 mmol) and cinnamoyl chloride
(343.2 mg, 2.06 mmol) were added dropwise thereto over 3 minutes while the same
temperature was retained. After the reaction solution was stirred at the same temperature
for 25 minutes, 2N hydrochloric acid (10 ml) was added, and the mixture was further
stirred at room temperature for 10 minutes. To the reaction solution was added ethyl
acetate, the organic layer was separated, and the aqueous layer was extracted with ethyl
acetate three times. The combined extracts were dried with sodium sulfate. The solvent
was distilled off and the resulting oil was purified by silica gel column chromatography.
From fraction eluted with n-hexane-ethyl acetate (1:1, v/v), 364.3 mg (yield 56%) of
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compound 4 was obtained as a white solid. H NMR (CDC1 ) δ 1.40 (3H, t, J = 7.2 Hz),
3
4
7
.39 (2H, q, J = 7.2 Hz), 5.27 (2H, s), 6.99 (1H, d, J = 16.2 Hz), 7.23 (1H, d, J = 16.2),
.26-7.48 (10H, m), 8.45 (1H, s).
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Ethyl-5-(benzyloxy)-6-formyl-4-oxo-4H-pyran-3-carboxylate (5). To a MeCN (5
ml) solution of compound 4 and ruthenium chloride (2.76 mg, 0.0133 mmol) was added
dropwise an aqueous solution (8 ml) of sodium periodate (625.8 mg, 2.93 mmol) and 96%
sulfuric acid (287.4 mg, 2.93 mmol) over 10 minutes at room temperature under nitrogen
stream. After the reaction solution was stirred at the same temperature for 5 minutes, ethyl
acetate was added, the organic layer was separated, and the aqueous layer was extracted
with ethyl acetate two times. The combined extracts were dried with sodium sulfate. The
solvent was distilled off, and the resulting oil was purified by silica gel column
chromatography. From fraction eluted with n-hexane-ethyl acetate (1:1, v/v), 303.2 mg
1
(
yield 75%) of compound 5 was obtained as a colorless oil. H NMR (CDC1 ) δ 1.39 (3H,
3
t, J = 6.9 Hz), 4.40 (2H, q, J = 6.9 Hz), 5.54 (2H, s), 7.37 (5H, s), 8.48 (1H, s), 9.85 (1H,
s).
3
-(Benzyloxy)-5-(ethoxycarbonyl)-4-oxo-4H-pyran-2-carboxylic acid (6). To a
MeCN (15 ml) solution of compound 5 (1.00 g, 3.31 mmol) was added an aqueous
solution (10 ml) of 96% sulfuric acid (421.7 mg, 4.30 mmol) and amidosulfuric acid
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(
642.7 mg, 6.62 mmol) at room temperature, the mixture was stirred, and an aqueous
solution (10 ml) of sodium chlorite (388.9 mg, 4.30 mmol) was added dropwise over 5
minutes while the same temperature was retained. After the reaction solution was stirred
at the same temperature for 5 minutes, an aqueous saturated sodium chloride solution was
added, and the mixture was extracted with ethyl acetate three times. The combined
extracts were dried with sodium sulfate. The solvent was distilled off, and the resulting
oil was purified by silica gel column chromatography. The materials were eluted firstly
with chloroform and then with chloroform-MeOH (7:3, v/v). Concentration of the
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objective fraction afforded 748.8 mg (yield 71%) of compound 6 as a colorless oil. H
NMR (CDC1 ) δ 1.40 (3H, t, J = 7.2 Hz), 3.93 (1H, br s), 4.40 (2H, q, J = 7.2 Hz), 5.61
3
(2H, s), 7.38-7.44 (10H, m), 8.52 (1H, s).
Ethyl-5-(benzyloxy)-6-((2-methoxyethyl)carbamoyl)-4-oxo-4H-pyran-3-
carboxylate (7). To a DMF (10 ml) solution of compound 6 (1.00 g, 3.14 mmol) were
added WSC.HC1 (1.20 g, 6.28 mmol) and HOBt (551.6 mg, 4.08 mmol) at room
temperature, and the mixture was stirred at the same temperature for 90 minutes. The
reaction solution was cooled to 0° C, and a DMF (2 ml) solution of 2-methoxyethanamine
(236.0 mg, 3.14 mmol) was added dropwise over 3 minutes. The reaction solution was
stirred at the same temperature for 1 hour, water was added, and the mixture was extracted
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with ethyl acetate three times. The extract was washed with water three times, and dried
with sodium sulfate. The solvent was distilled off and the resulting oil was purified by
silica gel chromatography. The materials were eluted firstly with n-hexane-ethyl acetate
1
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(1:1, v/v) and, then, with n-hexane-ethyl acetate (1:9, v/v). Concentration of the objective
1
fraction afforded 928.5 mg (yield 79%) of compound 7 as a brown oil. H NMR (CDC1 )
3
δ 1.39 (3H, t, J = 7.2 Hz), 3.29 (3H, s), 3.41 (2H, t, J = 5.4 Hz), 3.47-3.53 (2H, m), 4.39
(
2H, q, J = 7.2 Hz), 5.44 (2H, s), 7.36 (3H, m), 7.44-7.47 (2H, m), 8.07 (1H, br s), 8.54
1H, s).
(
Ethyl-(S)-5-(benzyloxy)-1-(1-hydroxy-3-phenylpropan-2-yl)-6-((2-methoxyethyl)
carbamoyl)-4-oxo-1,4-dihydropyridine-3-carboxylate (8b). A xylene (2 ml) solution
of compound 7 (500 mg, 1.33 mmol) and (S)-2-amino-3-phenylpropan-l-ol (604.2 mg,
4
.0 mmol) was heated to 120° C, and stirred for 30 minutes. After the reaction solution
was cooled to room temperature, the solvent was distilled off, and the resulting oil was
purified by silica gel chromatography. The materials were eluted first with chloroform
and then with chloroform-MeOH (9:1, v/v). Concentration of the objective fraction
1
afforded 487 mg (yield 72%) of compound 8b as a colorless oil. H NMR (CDC1 ) 8:1.41
3
(3H, t, J=6.9 Hz), 2.24-2.34 (1H, m), 2.24-3.00 (1H, m), 3.03-3.16 (1H, m), 3.05 (3H, m),
3
.25-3.32 (2H, m), 4.13-4.19 (1H, m), 4.17-4.30 (1H, m), 4.36-4.47 (1H, m), 4.51-4.54
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(
1H, m), 4.55 (1H, d, J = 10.5 Hz), 5.78 (1 H, t, J = 6.9 Hz), 7.17-7.26 (4H, m), 7.28-7.35
5H, m), 7.49 (1H, t, J 5.4 Hz), 6.32 (1H, s).
(
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Compound 8i was prepared by the procedure used for compound 11b.
Ethyl-(S)-5-(benzyloxy)-1-(3-hydroxy-1,1-diphenylpropan-2-yl)-6-((2-
methoxyethyl)carbamoyl)-4-oxo-1,4-dihydropyridine-3-carboxylate (8i). Brown oil,
1
5
2 % yield. H NMR (CDCl ) δ 8.15 (1H, s), 7.58-7.27 (12H, m), 7.20 (1H, d, J = 7.1
3
Hz), 7.09 (2H, t, J = 7.3 Hz), 7.00 (1H, d, J = 7.2 Hz), 5.02-4.92 (2H, m), 4.74 (1H, d, J
11.4 Hz), 4.58 (1H, d, J = 10.4 Hz), 4.46-4.32 (1H, m), 4.12 (2H, q, J = 7.2 Hz), 3.65
=
(1H, t, J = 9.5 Hz), 3.45-3.34 (2H, m), 3.17-2.97 (5H, m), 1.35 (3H, t, J = 7.1 Hz).
Compound 8c, 8e, 8f, 8g, and 8h were not identified because of the parallel synthesis.
Used reagents were same as the synthesis of 8b.
Ethyl-5-(benzyloxy)-1-((tert-butoxycarbonyl)amino)-6-((2-
methoxyethyl)carbamoyl)-4-oxo-1,4-dihydropyridine-3-carboxylate (9). To
a
solution of compound 7 (3.5 g, 9.32 mmol) in toluene (30 ml) was added tert-butyl
hydrazinecarboxylate (2.465 g, 18.65 mmol) and acetic acid (0.213 ml, 3.73 mmol) at r.t.
under N atm.. The mixture was stirred at reflux for 3 hours. After evaporation, the crude
2
product was purified by silica gel chromatography. The material was eluted with
chloroform-MeOH (20:1, v/v). Collected fractions were evaporated to afford compound
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(3.8 g, 7.76 mmol, 83 %) as a white solid. H NMR (CDCl ) δ 8.12 (1H, br s), 7.41-
3
7
.29 (5H, m), 5.20 (2H, s), 4.35 (2H, q, J = 7.2 Hz), 3.39-3.24 (7H, m), 1.46 (9H, s), 1.38
1
1
1
1
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0
(3H, t, J = 7.2 Hz).
Ethyl-5-(benzyloxy)-3-(2-methoxyethyl)-4,6-dioxo-2,3,4,6-tetrahydro-1H-
pyrido[2,1-f][1,2,4]triazine-7-carboxylate (10). To a solution of compound 9 (500 mg,
.021 mmol) in DCM(10 mL) was added TFA (2 mL) at room temperature. The mixture
1
was stirred at room temperature for 3 hours. After toluene (10 mL) was added, the mixture
was concentrated under reduced pressure. The resulting residue was diluted with sat
NaHCO , and then extracted with CH Cl to afford the deprotected crude product. The
3
2
2
crude compound was taken to the next step without purification. To a dried sealed tube
were added crude product in EtOH (10 mL) and paraformaldehyde (92 mg, 3.06 mmol)
at room temperature. After the mixture was stirred at 140 °C for 10 minutes, the reaction
mixture was concentrated under reduced pressure. The crude product was subjected to a
silica gel chromatography and eluted with CHCl /MeOH. Collected fractions were
3
1
evaporated to afford compound 10 (352 mg, 85.9 % yield) as a pale yellow solid. H NMR
(
(
(
CDCl ) δ 8.16 (1H, s), 7.37-7.28 (5H, m), 6.18 (1H, t, J = 7.8 Hz), 5.27 (2H, s), 4.46
3
2H, d, J = 7.8 Hz), 4.33 (2H, q, J = 7.2 Hz), 3.57-3.51 (2H, m), 3.51-3.46 (2H, m), 3.31
3H, s), 1.35 (3H, t, J = 7.2 Hz).
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Ethyl-(S)-4-benzyl-9-(benzyloxy)-2-(2-methoxyethyl)-1,8-dioxo-1,3,4,8-
tetrahydro-2H-pyrido[1,2-a]pyrazine-7-carboxylate (11b). To a THF (6 ml) solution
of compound 8b (2.86 g, 5.63 mmol) and triphenylphosphine (2.21 g, 8.45 mmol) was
added dropwise a DEAD 40 wt % toluene solution (3.68 g, 8.45 mmol) at room
temperature over 3 minutes. The reaction solution was stirred at the same temperature for
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0 minutes, the solvent was distilled off, and the resulting oil was purified by silica gel
chromatography. From a fraction eluted with ethyl acetate-MeOH (9:1, v/v), 1.37 g (yield
1
5
0%) of compound 11b was obtained as a colorless oil. H NMR (CDCl ) δ 1.31 (3H, t,
3
J = 7.2 Hz), 3.07 (2H, d, J = 6.9 Hz), 3.33 (3H, s), 3.57-3.80 (4H, m), 3.95 (1H, dd, J =
.0 Hz, 6.6 Hz), 4.01-4.14 (1H, m), 4.16-4.34 (2H, m), 5.24 (1H, d, J = 9.9 Hz), 5.51 (1H,
3
d, J = 9.9 Hz), 7.01-7.03 (2H, m), 7.21-7.37 (5H, m), 7.41-7.58 (1H, m), 7.64-7.69 (2H,
m).
Ethyl-5-(benzyloxy)-1-(4-fluorobenzyl)-3-(2-methoxyethyl)-4,6-dioxo-2,3,4,6-
tetrahydro-1H-pyrido[2,1-f][1,2,4]triazine-7-carboxylate (11d). To a solution of
compound 10 (50 mg, 0.125 mmol) in DMF (0.5 ml) was added 1-(bromomethyl)-4-
fluorobenzene (0.023 ml, 0.187 mmol) and Cs CO (101 mg, 0.311 mmol) at room
2
3
temperature. After the mixture had been stirred at r.t. for 18 hours, the reaction mixture
was diluted with H O, then extracted with CHCl (2 x 20 mL). The organic layers were
2
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combined and washed with H O, and brine. The organic layer was dried over MgSO ,
2
4
filtered and concentrated under reduced pressure. The crude product was purified by silica
1
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gel chromatography. From a fraction eluted with CHCl /MeOH, collected fractions were
3
1
evaporated to afford compound 11d (45.8 mg, 72 % yield) as a white solid. H NMR
(
CDCl ) δ 7.70 (1H, s), 7.64 (2H, d, J = 7.3 Hz), 7.38-7.28 (3H, m), 7.18 (2H, t, J = 6.5
3
Hz), 7.05 (2H, t, J = 8.2 Hz), 5.44 (2H, br s), 4.29 (2H, q, J = 6.9 Hz), 4.10 (2H, s), 3.68-
3
.50 (4H, m), 3.31 (3H, s), 1.31 (3H, t, J = 7.2 Hz).
Compound 11i was prepared by the procedure used for compound 11b.
Ethyl-(S)-4-benzhydryl-9-(benzyloxy)-2-(2-methoxyethyl)-1,8-dioxo-1,3,4,8-
tetrahydro-2H-pyrido[1,2-a]pyrazine-7-carboxylate (11i). Yellow solid, 33% yield.
1
H NMR (DMSO-d ) δ 1.18 (3H, m), 3.11 (3H, s), 3.16 (1H, m), 3.28 (1H, m), 3.76 (1H,
6
m), 3.97-4.13 (3H, m), 4.31(1H, d, J = 11.3 Hz),5.08(2H, s), 5.52 (1H, d, J = 12.0Hz),
7
.18-7.25 (6H, m), 7.25-7.45 (6H, m), 7.55-7.66 (6H, m). MS: m/z 567.7 [M+H]+.
Compound 11j was prepared by the procedure used for compound 11d.
Ethyl-1-benzhydryl-5-(benzyloxy)-3-(2-methoxyethyl)-4,6-dioxo-2,3,4,6-
tetrahydro-1H-pyrido[2,1-f][1,2,4]triazine-7-carboxylate (11j). White solid, 71 %
1
yield. H NMR (CDCl ) δ 7.66 (2H, d, J = 7.3 Hz), 7.59 (1H, s), 7.53 (2H, d, J = 7.2 Hz),
3
7
.44 (2H, t, J = 7.1 Hz), 7.40-7.27 (5H, m), 7.15 (4H, d, J = 13.1 Hz), 5.44 (2H, q, J =
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