Design and synthesis of pinane oxime derivatives as novel anti-influenza agents

Article abstract of DOI:10.1016/j.bioorg.2020.104106

Parasitic characteristics, mutations and resistance of influenza A virus make it difficult for current influenza antiviral drugs to maintain long-term effectiveness. Currently, to design non-adamantane compounds targeting the S31N mutant of M2 proton channel is a promising direction for the development of novel anti-influenza drugs. In our previous research, a pinanamine-based antiviral M090 was discovered to target hemagglutinin instead of M2, with its structure being highly similar to reported M2-S31N inhibitors. Herein, a series of pinane oxime derivatives were designed from scratch and evaluated for anti-influenza activity and their cytotoxicity in vitro. Utilizing a combination of structure-activity relationship analysis, electrophysiological assay and molecular docking, the most potent compound 11h, as a M2-S31N blocker, exhibited excellent activity with EC₅₀ value at the low micromolar level against both H3N2 and H1N1. No significant toxicity of 11h was observed. In addition, compound 11h was located tightly in the pore of the drug-binding site with the thiophene moiety facing down toward the C-terminus, and did not adopt a similar position and orientation as the reference inhibitor.

Full text of DOI:10.1016/j.bioorg.2020.104106

Journal Pre-proofs
Design and synthesis of pinane oxime derivatives as novel anti-influenza
agents
Jianghong Dong, Mengjie Xiao, Qinge Ma, Guicheng Zhang, Weijie Zhao,
Mengjie Kong, Yue Zhang, Luyun Qiu, Wenhui Hu
PII:
S0045-2068(20)31403-6
DOI:
https://doi.org/10.1016/j.bioorg.2020.104106
YBIOO 104106
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
3 May 2020
26 June 2020
14 July 2020
Please cite this article as: J. Dong, M. Xiao, Q. Ma, G. Zhang, W. Zhao, M. Kong, Y. Zhang, L. Qiu, W. Hu,
Design and synthesis of pinane oxime derivatives as novel anti-influenza agents, Bioorganic Chemistry (2020),
doi: https://doi.org/10.1016/j.bioorg.2020.104106
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover
page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version
will undergo additional copyediting, typesetting and review before it is published in its final form, but we are
providing this version to give early visibility of the article. Please note that, during the production process, errors
may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
©
2020 Elsevier Inc. All rights reserved.

Design and synthesis of pinane oxime derivatives as novel anti-
influenza agents
a,
c
d
e
a
Jianghong Dong *, Mengjie Xiao , Qinge Ma , Guicheng Zhang , Weijie Zhao ,
a
a
a
b,
Mengjie Kong , Yue Zhang , Luyun Qiu , Wenhui Hu *
a
College of Chemistry and Pharmaceutical Engineering, Huanghuai University,
Zhumadian 463000, China
b
State Key Laboratory of Respiratory Disease, Key Laboratory of Molecular Target &
Clinical Pharmacology, School of Pharmaceutical Sciences, Guangzhou Medical
University, Guangzhou 511436, China
c
School of Life Sciences, Chinese University of Hong Kong, Shatin, N.T, Hong Kong,
SAR 999077, China
d
Key Laboratory of Modern Preparation of TCM of Ministry of Education & Research
Center of Natural Resources of Chinese Medicinal Materials and Ethnic Medicine,
Jiangxi University of Traditional Chinese Medicine, Nanchang 330004, China
e
Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Science, 190
Kaiyuan Avenue, Guangzhou 510530, China
Address correspondence to these authors:
Jianghong Dong, Ph.D.
College of Chemistry and Pharmaceutical Engineering, Huanghuai University,
Zhumadian 463000, China
E-mail: hndxdjh555@163.com
Wenhui Hu, Ph.D.
State Key Laboratory of Respiratory Disease, Key Laboratory of Molecular Target &
Clinical Pharmacology, Guangzhou Medical University, Guangzhou 511436, China
E-mail:huwenhui@gzhmu.edu.cn

Abstract
Parasitic characteristics, mutations and resistance of influenza A virus make it
difficult for current influenza antiviral drugs to maintain long-term effectiveness.
Currently, to design non-adamantane compounds targeting the S31N mutant of M2
proton channel is a promising direction for the development of novel anti-influenza
drugs. In our previous research, a pinanamine-based antiviral M090 was discovered to
target hemagglutinin instead of M2, with its structure being highly similar to reported
M2-S31N inhibitors. Herein, a series of pinane oxime derivatives were designed from
scratch and evaluated for anti-influenza activity and their cytotoxicity in vitro. Utilizing
a combination of structure-activity relationship analysis, electrophysiological assay and
molecular docking, the most potent compound 11h, as a M2-S31N blocker, exhibited
excellent activity with EC value at the low micromolar level against both H3N2 and
5
0
H1N1. No significant toxicity of 11h was observed. In addition, compound 11h was
located tightly in the pore of the drug-binding site with the thiophene moiety facing
down toward the C-terminus, and did not adopt a similar position and orientation as the
reference inhibitor.
Keywords: Influenza A virus, Amantadine-resistant, S31N mutant, Pinane oxime
derivative, antiviral drug
ABBREVIATIONS
M2, matrix-2 protein; S31N, Serine 31 arganine; WT, wild type; EC , half maximal
5
0
effective concentration; TC , 50% cytotoxic concentration; NOE, Nuclear Overhauser
5
0
effect; CPE, cytopathic effect; NMR, nuclear magnetic resonance; MTT, (3-(4,5-
Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide; SAR, structure-activity
relationship; DMF, N,N-dimethylformamide; RMSD, root mean square deviation;
TosMIC, tosylmethyl isocyanide; PDC, pyridinium dichromate; TBTU, 2-(1H-
benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate; DMSO, dimethyl
sulfoxide; SOCl , thionyl chloride; TLC, thin layer chromatography; CDCl ,
2
3
chloroform-d; AMD, amantadine; IFD, induced fit docking; PDB, Protein Data Bank;
MDCK, Madin-Darby canine kidney; PBS, phosphate buffered saline; DMEM,
Dulbecco’s modified Eagle medium.

1
. Introduction
Influenza viruses are highly pathogenic microorganisms that cause acute respiratory
diseases and are responsible for the recurrent seasonal epidemics and occasional
pandemics, which seriously endanger human health [1, 2]. Currently, the best way for
prophylaxis of influenza is to get vaccinated each year. However, due to the production
and manufacturing limitations of pharmaceutical companies, the initial supply of the
vaccines might not be enough to meet the demand for them [3]. Various types and
strains of pathogenic viruses are constantly adapting and developing resistance to
medications and treatments. Therefore, influenza vaccines should be updated annually
based on rigorous international surveillance and scientists' estimations [4]. These
prevention factors are highly effective at minimizing influenza pandemics. It is also
worth noting that once patients are infected with influenza viruses, they will be
promptly treated with antiviral medications to shorten the duration of illness symptoms
and reduce complications [5].
Influenza antiviral drugs, approved by the U.S. Food and Drug Administration
(FDA), include three classes: virion ion-channel (M2 protein) inhibitors (amantadine
and rimantadine), neuraminidase inhibitors (oseltamivir, zanamivir and peramivir) and
cap-dependent endonuclease inhibitor (baloxavir marboxil) [6]. Indeed, the parasitic
nature of influenza virus and its susceptibility to spontaneous mutations make it
difficult for chemical drugs to have a long-lasting effect. For example, drug resistance
of amantadine, oseltamivir and other first-line drugs emerged in varying degrees [7].
Antiviral resistance to baloxavir is currently very low, but this has already happened
[8]. In this regard, there is an urgent need for development of new anti-influenza drugs.
Classical M2 inhibitors (amantadine and rimantadine, Fig. 1 and Fig. 2 )) are rarely
used because the M2-S31N mutant in more than 95% of clinically isolated influenza A
viruses makes those drugs difficult to bind [9]. Thus, it appears that it is logical and
valid to design non-adamantane-based M2-S31N inhibitors to address the problem of
adamantane resistance. For one thing, the crystal structure of the S31N mutant has
recently been reported, and it is possible to study the structural function and drug-

resistant mechanisms of the mutant channels with the development of molecular
modeling techniques, solid (or liquid) nuclear magnetic technology and lipidic cubic
phase crystallization techniques [10-12]. For another, researchers around the world
have spared no effort to develop new small molecular inhibitors of M2-S31N mutant
[13]. Instead, they have summarized a phamacophore structure of adamantyl-1-
+
NH CH −aryl and a series of amantadine derivatives have been reported [14-21].
2
2
Among them, compound WJ332 (Fig. 1) is the first M2-S31N inhibitor as amantadine
derivative and compound 1 (Fig. 1) exhibits favorable absorption, distribution,
metabolism, excretion, and toxicity (ADMET) properties and antiviral activity in vitro
[20]. In addition, M2-S31N inhibitors show a higher in vitro genetic barrier to drug
resistance than amantadine in cell culture, and drug resistance only emerges under high
drug selection pressure after several passages [18, 22]. These research jobs have laid
down a theoretical and realistic foundation for the development of new M2 inhibitors.
(1R,2R,3R,5S)-3-pinanamine(Fig. 2) is a monoterpene amine that blocked the
replication of wild-type influenza A virus as an M2 ion channel inhibitor identified by
our research group [23]. And a series of pinanamine-based antivirals were designed and
synthesized by attaching a bulky and hydrophobic functionality to pinanamine through
a systematic structure−activity relationship (SAR) study [24-27]. Interestingly, an
imidazole-based pinanamine derivative 2 (Fig. 1) exhibited dual inhibitory activity
against the wild-type and drug-resistant mutant of the influenza A virus [25]. The
methyl group was changed to a medium-size n-propyl group to afford the compound 3
(Fig. 1), of which M2-WT ion channel inhibition activity remained but M2-S31N ion-
channel inhibition activity disappeared [26]. However, 3-cyclopropylthiophene-based
pinanamine 4 (Fig. 1, M090) was discovered to inhibit viral replication by targeting the
viral hemagglutinin instead of M2-S31N mutant [27]. In addition, the same
phenomenon occurred in aminoadamantanes and azapropellanes with excellent anti-
influenza A virus activity through M2-S31N independent mechanisms [28, 29]. It
means that subtle change in the scaffold structure of antiviral chemotherapeutic agent
could lead to variety in target protein and mechanism of action.

S
I
O
N
N
N
HN
HN
S
S
HN
HN
NH2
HN
HN
HN
OH
Amantadine
WJ332
1
2
3
4(M090)
Fig. 1. Structure of Amantadine, adamantane-based M2-S31N inhibitors and several reported
pinanamine-based antivirals.
To address the problem of adamantane resistance and the discrepancy of pinanamine-
based antivirals, the structural characteristics of rimantadine were re-examined from
scratch, and a series of novel pinane oxime derivatives (Fig. 2) were designed in the
hope of specifically targeting the M2-S31N ion channel. The new synthesized
compounds were assessed for their in vitro antiviral activity against the pandemic
influenza virus A/HK/68 (H3N2, wild type, WT) and A/WSN/33 (H1N1, S31N mutant).
Meanwhile, cytotoxicity of the compounds was evaluated in uninfected MDCK cells.
And structure-activity relationship (SAR) was also discussed. Moreover, these efforts
resulted in a promising lead compound 11h endowed with the most potency against
amantadine-sensitive, as well as amantadine-resistant influenza A viruses (EC = 3.29
5
0
μM and 2.45 μM, respectively), with a TC value over 173.14 μM. Accordingly, the
5
0
compound was subjected to patch clamp assay and molecular docking to shed light on
whether the observed antiviral activity was by blocking M2 ion channel and reasonable
theoretical binding patterns. This study could enrich the diverse library of pinane-based
compounds and provide a molecular probe for further chemical biology research.

Scaffold
Polar group
NH2
Headgroup
OH
Rimantadine
NH2
R
hybridization
S
I
+
N
Ar
H
3
-Pinanamine
Fig. 2. Design strategy of the target compounds
2
. Results and discussion
2
.1. Chemistry
Synthetic routes are shown in Schemes 1. Using the (-)-isopinocampheol as raw
material, (1R,2R,3R,5S)-2,6,6-trimethylbicyclo[3.1.1]heptane-3-carboxylic acid 6 was
synthesized by oxidation of compound 5, cyanide reaction and nitrile hydrolysis, with
a 36.4% yield over three steps [30]. The carboxylic acid 6 was condensed with N,O-
dimethylhydroxylamine hydrochloride to give Weinreb amide 7 , which was treated
with the lithium reagent of various substituted thiophene or other aromatic compounds
to afford the corresponding Weinreb ketones 8a-j [31]; In another way, carboxylic acid
6
1
was treated with thionyl chloride in the presence of DMF to afford the acyl chloride
0, and the compound 10 underwent smooth Friedel-Crafts acylation with different
substituted thiophenes catalyzed by SnCl to obtain the corresponding ketones 10a-d
4
[32]. The ketones 8a-j and 10a-d were transformed into the corresponding pinane
oxime derivatives 11b-o by heating with hydroxylamine hydrochloride in the presence
of pyridine [33]. Among them, the geometry of pinane oxime derivative 11h was
1
selectively determined as anti-confirmation using H NMR NOE spectrum. It could be
seen that the 2D NOESY spectrum of compound 11h showed strong NOE correlations
between protons from the hydroxyl of oxime and the 2-methyl of pinane backbone,
other than 3’-H of the thiophene moiety (Figure S1 in Supporting Information).

OH
COOH
a, b, c
Ref.12
5
6
O
O
O
e
N
Ar
d
7
HO
N
COOH
8a-j
h
Ar
O
f
COCl
g
S
6
R1
11b-o
R2
9
10a-d
Scheme 1. Reaction Condition: (a) PDC, CH Cl , r.t., overnight; (b) TosMIC, t-BuOK, DMSO,
2
2
6
0 ℃, 48 h; (c) H SO , CH COOH, reflux, overnight; (d) N,O-dimethylhydroxylamine
2 4 3
hydrochloride, TBTU, triethylamine, CH CN, r.t., 1 h; (e) Me/ArLi, ether, 0 ℃, 30 min; -78 ℃, 3
3
h; (f) SOCl , cat. DMF, reflux, 2 h; (g) substituted thiophene, SnCl , dry CH Cl , 0 ℃, 30 min; (h)
2
4
2
2
pyridine, NH OH·HCl, 80 ℃, 24 h.
2
O
O
N
NH2 . HCl
S
S
a
S
b
8a
12
11a
Scheme 2. Reaction Condition: (a) pyridine, methoxyammonium chloride, 80 ℃, 24 h; (b)
LiAlH , diethyl ether, 0-40 ℃, 4h; HCl/ethyl acetate, r.t..
4
Treatment of ketone 8a with methoxyamine hydrochloride gave O-methyl oxime 12,
which was readily reduced by Lithium aluminum hydride to a mixture of two isomeric
amines 11a (Schemes 2) [34]. Compound 11d was reacted with (S)-N-Boc-2-
(bromomethyl)pyrrolidine under basic conditions using sodium hydride in DMF to
yield the compounds 13a and 13b, which were treated with hydrogen chloride/ethyl
acetate solution to give the target compounds 14a and 14b, respectively, as
hydrochlorides (Schemes 3) [35].

HO
R
O
R
O
N
N
S
N
a
b
S
S
. HCl
11d
Boc
N
HN
13a
14a
R=
R= 



1
3b R=
14b R=
N
N
Scheme 3. Reaction Condition: (a) 60 % NaH, RBr, DMF, 0 ℃; (b) HCl/ethyl acetate, r.t.。
2
.2. Viral inhibition assay
All the novel compounds were subjected to antiviral evaluation against influenza A
virus including amantadine-sensitive A/Hong Kong/8/68 strain (H3N2, M2-WT) and
amantadine-resistant A/WS/33 strain (H1N1, M2-S31N) in virus-infected Madin-
Darby canine kidney (MDCK) epithelial cell according to a previous report [26]. In
addition, cytotoxicity of the compounds was performed in uninfected MDCK cells by
3
-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
Amantadine (AMD) was used as the reference drug for comparison. The antiviral data
half maximal effective concentration, EC ) obtained by microscopic scoring of the
bromide
(MTT)
assay.
(
5
0
virus-induced CPE and cytotoxic results (50% cytotoxic concentration, TC ) are
5
0
presented in Table 1.
2
.3. Structure-activity relationship
Table 1
Structures and in vitro inhibition efficiency of test compounds against influenza A viruses
R
Antiviral EC50 a(μM)
Cytotoxicity
Ar
(
TC50 b, μM)
Compd.
1a
R
Ar
A/HK/68
A/WSN/33
NA c
S
1
NH
2
0.27±0.07
193.23

S
1
1b
=NHOH
=NHOH
=NHOH
12.4±1.2
NA
11.8±0.8
NA
>200.46
>276.4
93.43
1
1c
Me
S
1
1d
10.8±1.3
14.81±2.5
2
2.2±3.0
NA
63.09
1
1e
=NHOH
S
2
4
9.1±3.2
.84±0.6
11.9±1.12
30.2±3.6
>191.04
>154.21
S
S
1
1f
=NHOH
=NHOH
1
1g
3
.29±0.10
2.45±0.06
>173.14
>154.35
1
1h
=NHOH
=NHOH
S
Cl
2
5.4±1.5
19.29±0.47
1
1i
S
Br
7
9
.11±0.72
.44±0.91
5.97±0.81
>191.37
>162.51
S
S
1
1j
=NHOH
=NHOH
Cl
Cl
10.15±1.26
1
1k
Cl
NA
18.65±3.1
32±2.61
NA
>170.4
>200.46
>187.89
>196.1
10.15
S
N
1
1l
=NHOH
=NHOH
=NHOH
O
8
.65±0.42
S
1
1m
_ d
_
1
1n
O
NA
1
1o
4a
=NHOH
F
HN
_
NA
S
S
1
N O
N
O
_
NA
9.67
1
4b
N
AMD
3.7±0.6
199.9±21
337.19
Data are presented as the mean ± S.D. of the results of two or three independent tests. a EC50
b
represents half maximal effective concentration. TC represents 50% cytotoxic concentration, as
5
0
c
d
determined by the MTT cell viability test. NA, no activity. The symbol “–” indicates “not tested.”

It has been previously found that 1-((1R,2R,5S)-2,6,6-trimethylbicyclo[3.1.1]heptan
-3-yl)ethan-1-amine, which was similar to rimantadine, exhibited anti-influenza
activity against the M2-WT virus A/HK/68 (EC = 1.93 μM) [30]. As a privileged
5
0
pharmacophore, the thiophene group played a significant role in the activity against
amantadine-resistant influenza virus[16, 20, 27]. Replacement of the methyl group of
this compound with the thiophen-2-yl moiety afforded compound 11a, and clearly
increased the antiviral activity against WT virus, but this analogue exhibited no activity
against amantadine-resistant strain A/WSN/33. Surprisingly, the corresponding oxime
derivative 11b displayed dual inhibitory activity against the M2-MT virus A/HK/68
and the amantadine-resistant strain A/WSN/33, with its EC values of 12.4 μM and
5
0
1
1.8 μM respectively. On the other hand, viral inhibitory potency of the methyl-
substituted analog 11c disappeared. In order to clarify the effectiveness of various
substituted pinane-based thiophene oxime derivatives on the anti-influenza activity,
compounds 11d–l were synthesized by introducing simple methyl, methoxy or halogen
groups. Compounds 11d containing Ar = 4-methyl-thiophen-2-yl and 11f containing
Ar = 5-methyl-thiophen-2-yl maintained antiviral effect against the amantadine-
resistant H1N1 virus while 3-methyl derivative 11e showed no inhibition potency. The
information suggested that modifying 4,5-position of thiophene was beneficial to
antiviral activity. However, compound 11g with 4,5-dimethyl group displayed inferior
activity against H1N1 and superior activity against H3N2 compared with compound
1
1b. Fortunately, among 2,5-disubstituted thiophene derivatives 11g-i and 11k, the
most potent analogue 11h exhibited almost 4-fold increased inhibitory activity against
both wild-type and drug-resistant mutant of the influenza A virus compared with 11b
(
EC = 3.29 μM and 2.45 μM, respectively). As reported halogenation was a valuable
50
approach for improving ligand bioactivity [36]. Comparison of activity of compounds
1h-k indicated that it was preferable for 5-position of the thiophene group to be
1
substituted by electron-withdrawing chlorine atom. Introducing an electron-donating
methoxy group to 5-position of the thiophene ring resulted in compound 11l, which
clearly abolished its anti-influenza activity. Replacing the thiophene moiety of 11b with
the thiazol-2-yl (11m), 4-methoxyphenyl (11n) or 4-fluorophenyl (11o) groups also led

to significant decline in activity or inactivity against amantadine-resistant influenza
virus.
To extend the SAR information, the oxime ether derivatives 14a and 14b were also
designed by adding a hydrophilic secondary amine or tertiary amine to compound 11d.
Unfortunately, the two designed compounds totally lost their effectiveness probably as
a result of their higher cytotoxicity.
SAR analysis of the newly synthesized compounds demonstrated that the potent
inhibitors could be obtained if the amine group of 3-pinanamine was substituted by the
oxime group, just as other polar groups such as the hydroxyl, guanidine, amidine, or
aminooxyl groups [37]. In addition, the thiophene ring was the optimal heterocycle
attached to pinane to achieve the best antiviral inhibitory potency and 5-position of the
thiophene moiety favored electron-withdrawing substitutions, such as chlorination.
Significantly, compound 11h bearing the 4-methyl-5-cholo-thiophene substituent
proved to be promising lead compound with low cytotoxicity, inhibiting both drug-
sensitive and -resistant influenza A viruses. The SAR of the synthesized compounds
was summarized in Fig. 3.
An oxime moiety as the polar group
can be tolerated inhibiting the A/M2-S31N
R
The thiophene ring was
the optimal heterocycle
Ar
Different substituted thiophene groups influenced the EC50 values
and 5-position of thiophene favored the electron-withdrawing substitutions, such as Cl
Fig. 3. A preliminary summary of SAR
.4. Whole cell patch clamp assay
2
To verify whether the antiviral efficacy of the novel pinane oxime derivatives was
through M2 ion channel blockage or not, patch clamp electrophysiology in mammalian
cells was conducted. Compounds 11a, 11b and 11h were selected and tested in the

whole cell patch clamp assay using 293T-Rex cells expressing either the M2-WT or the
M2-S31N mutant of the M2 protein, as previously reported [23, 38].The percentage
inhibition of currents through the M2-WT and M2-S31N ion channels at pH 5.5 was
recorded in Fig. 4 after 2 min of co-applied with 100 μM of compounds. All
measurements were repeated three times. The results were consistent with those of the
cell-based phenotypic method.
Fig. 4. Inhibition of the M2 WT and S31N ion channel conductivity by various compounds as
determined by patch clamp assays.
2
.5. Molecular docking study
To figure out the binding modes of the pinane oxime derivatives with the active site
of the M2-S31N channel, Schrödinger's induced fit docking (IFD) protocol [39] was
performed by using the solution NMR structure of M2-S31N (PDB ID code: 2LY0 [15])
as the template. Based on the in vitro inhibition results, compounds 11a, 11b and 11h
were selected as ligand examples. Utilizing molecular docking computations, the
docking conformation of the reference compound WJ332 approximately agreed with
the molecular dynamics simulations performed by Duque (RMSD:1.29) [15]. Two
enantiomers of compound 11a might not enter the drug-binding site cavity of M2-S31N
ion channel and the direction of the molecular skeletons were almost perpendicular to
the reference compound (Figure S2 in Supporting Information). As presented in Fig.
5
A and 5B, binding modes of WJ332, compound11b and 11h in the pore of the M2

channel drug-binding site were visualized by using UCSF Chimera [40]. Hydrophobic
skeletons of the designed ligands 11b and 11h were located in the drug-binding sites
formed by pore facing residues including Val27, Asn31 and Ala30, while the aromatic
group pointed downward the C-terminus. The binding modes and orientations of the
two compounds differed from those of the reference compound. In conclusion, the
distinct docking results accounted for the dissimilarity between compound 11a and
pinane oxime derivatives in inhibitory activity.
A
B
Fig. 5. A) Overlay of the reference compound WJ332 (pink) and 11b (sky blue) bound to M2-
S31N. B) Overlay of WJ332 (pink) and 11h (brown) bound to M2-S31N. One helix was removed
for clarity.
From Fig. 6A, the backbone of derivative 11b could occupy the hydrophobic region,
spanning from residue Val27 to Gly30. And the thiophene moiety of 11b formed
hydrophobic interactions with the Ala30 side chains and the backbone of Gly34. In
addition, the hydroxyl of oxime might form a C=O⋯H hydrogen bond with the carbonyl
group of Ala30, with its distance being 1.939Å. These results indicated that the oxime
group and the thiophene moiety had great impact on the anti-influenza activity.
Compared with compound 11b, the most potent compound 11h could bind more
tightly to M2-S31N protein and enter the cavity created by residues Ala30, Asn31, Ile33,
and Gly34, which was consistent with the results of SAR studies. Moreover, the 4-
methyl-5-cholo-thiphene group proved to be crucial to enhance the binding affinity. It

A
B
Fig. 6A-B. Docking models of compounds 11b (sky blue) and 11h (brown) into the drug-binding
sites of M2-S31N, respectively.
seemed that the chlorine atom of compound 11h formed a C−Cl···π halogen bond[41]
with His37 side chains, which rendered the ligand to move towards the C-terminus of
ion channel. Accordingly, the hydroxyl of the oxime group had a hydrogen-bonding
interaction with Asn31 (OH⋯O=C, distance: 2.115 Å) to enhance the affinity (Fig. 6B).
To sum up, our preliminary computational analysis explained the plausible theoretical
binding modes of pinane oxime derivatives, which were distinct from the reference
compound. And the most potent M2-S31N antagonist 11h could be used as a hit
compound for further structural optimization.
In addition, compound 11b and 11h were modeled into M2 wild-type protein to
clarify their binding modes, as determined by solid-state NMR (PDB code: 2KQT) [42].
Similar to amantadine, the hydrophobic backbones of the two derivatives (Fig. 7A-B)
mainly bound to the region of the channel formed mainly by Ala30 and Ser31 residues
and that the polar group pointed toward the C-termini of the pore. The thiophene moiety
in compound 11b interacted with Ile33 and Gly34 via the hydrophobic interaction,
while one hydrogen bond was formed between the ligand 11h and the residue Ser31
(OH⋯O=C), with its 4-methyl-5-cholo-thiophene group facing towards the C-terminal

end. Therefore, the observed docking results provided rational explanation for the
antiviral activity and the M2-WT channel inhibitory activity.
B
A
Fig. 7A-B Representation of compounds 11b (sky blue, Fig. 7A) and 11h (brown, Fig. 7B)
interacting with residues in the binding site of M2-WT ion channel (PDB code: 2KQT). Amantadine
was displayed as in the wire draw mode, with heteroatoms colored by element.
3
. Conclusion
In conclusion, our study focused on the synthesis and antiviral activity studies of
novel pinane oxime derivatives as M2-S31N inhibitors for anti-influenza agents to
address the drug resistance. Inspired by structural characteristics of rimantadine, a
series of non-adamantane-based pinane oxime derivatives were designed, synthesized
1
13
and characterized by H NMR, C NMR, HRMS and elemental analysis. All new
compounds were evaluated for their in vitro inhibitory activity against influenza A virus
and their cytotoxicity in MDCK Cells. In light of comprehensive structure-activity
relationship, the oxime group as a polar motif instead of the amine group, attached to
the pinane scaffold, could maintain the antiviral activity. And the 4-methyl-5-cholo-
thiophene moiety was the high-priority heterocycle for this series of compounds. In
particular, compound 11h was the most potent inhibitor against wild-type and

adamantane-resistant viruses at low micromolar concentrations through blocking M2
ion channel. Moreover, the molecular docking revealed that compound 11h tightly
bound to the pore of the drug-binding site by forming the hydrophobic interactions,
halogen bonding and hydrogen bonding, with the orientation being different from the
reference compound. Its antiviral efficacy in vivo and pharmacokinetic parameters in
animal models are in progress.
4
4
. Experimental procedure
.1. Chemistry
Commercially available reagents were used without further purification unless
otherwise noted. Reactions were monitored by TLC using Qing Dao Hai Yang GF254
silica gel plates (5×10 cm); zones were detected visually under ultraviolet irradiation
(254 nm). Compounds were purified by silica gel column chromatography which
1
13
performed on silica gel (200–300 mesh) from Qing Dao Hai Yang. H NMR and C
NMR spectra were recorded on a Bruker Avance 400 spectrometer. CDCl and DMSO-
3
d6 were used as solvents. Chemical shifts (δ) are given in relative to tetramethylsilane
(
δ 0.00 ppm) in CDCl . Coupling constants (J) were reported in hertz unit (Hz). High
3
resolution mass spectra were performed on an Agilent 6210 LC/MSD TOF Liquid
Chromatograph-ESI-Mass Spectrometer. Low-resolution mass spectra were measured
on an Agilent MSD-1200 ESI-MS system. Melting points were determined using a
WRS-1C melting point apparatus (Shanghai INESA Physico optiacal instrument Co.
1
13
Ltd.) and were uncorrected. All final products were characterized by H NMR, C
1
NMR and HRMS, selected intermediates were confirmed by H NMR, possibly also
MS. Elemental analysis was performed on an Elemental Analyzer – Elementar Vario
MICRO cube.

4
.1.1 Synthesis of (1R,2R,3R,5S)-2,6,6-trimethylbicyclo[3.1.1]heptane-3-carboxylic
acid 6
A dry 500 mL single-necked rounded-bottomed flask, equipped for magnetic stirring,
was charged with (-)-isopinocampheol (20.0 g, 129.66 mmol) and 200 mL
dichloromethane. Pyridinium dichromate (181.5 g, 1.4 mol) was added in portions at 0
℃
to the reaction mixture over a period of 15 min. And then the reaction mixture was
stirred vigorously at room temperature until the starting material was completely
consumed. The mixture was diluted with 200 mL ether and the unsolved solid was
removed by filtration. The organic layer was washed with brine (200 mL), dried over
anhydrous Na SO , filtered and concentrated to give the crude product. Purification by
2
4
silica gel chromatography ((petroleum ether/ ethyl acetate, 50:1) yielded the desired
compound (1R,2R,5S)-2,6,6-trimethylbicyclo[3.1.1]heptan-3-one (16.4 g, 83%) as a
colorless liquid.
To an ice-cooled solution of tosylmethyl isocyanide (11.7 g, 60.00 mmol) in 40 mL
dry dimethyl sulfoxide was added in portions 15.7 g of potassium tert-butylate (140.00
mmol). After stirring for 5 min, methanol (1.45 mL, 28.36 mmol) and the obtained
ketone (3.0 g, 20.00 mmol) were added successively. The mixture was stirred for 48 h
at 60 ℃. The reaction mixture was cooled to room temperature, diluted with 150 mL
water, acidified with 2 M HCl to pH 6 and extracted with ether. The combined organic
layers were washed once with saturated NaCl solution, dried and concentrated to give
a brown oil. The crude product was used in the next step without further purification.
The obtained oil was dissolved in 20.0 mL AcOH, 5.0 mL H SO and 10.0 mL H O,
2
4
2
and the mixture solution was refluxed overnight. The solution was cooled to room
temperature and diluted with 100 mL H O. The aqueous layer was extracted with
2
dichloromethane (40 mL×3), the combined organic layers were dried over anhydrous
MgSO and concentrated in vacuo. The product was purified by flash column
4
chromatography ((petroleum ether/ ethyl acetate, 40:1) to give the product (1.6g, 43.9%,
1
over two steps) as a white powder. H NMR (400 MHz, CDCl ): δ 2.44–2.36 (m, 2H,
3
H-3, 4β), 2.16–2.07 (m, 2H, H-2, 7β), 2.06-2.00 (m, 1H, H-2), 1.95 (q, J = 5.6 Hz, 1H,

H-5), 1.68 (t, J = 5.6 Hz, 1H, H-1), 1.29 (q, J = 10.0 Hz, 2H, H-7α, 4β), 1.21 (s, 3H,
CH -9), 0.99 (d, J = 5.7 Hz, 3H, CH -10), 0.88 (s, 3H, CH -8).
3
3
3
4
.1.2 Method A: General Procedures for Synthesizing Corresponding Weinreb
Ketone 8a-j
Preparation of N-Methoxy-N-methylamide. To a 100 mL round-bottomed flask was
charged with carboxyl acid 6 (1.4 g, 7.68 mmol), N,O-dimethylhydroxylamine
hydrochloride (824.2 mg, 8.45 mmol) and acetonitrile 20 mL. 2-(1H-benzotriazole-1-
yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) (1.87 g, 8.45 mmol) was
added in portions to the reaction solution. The reaction solution was stirred at room
temperature for 30min. A saturated NaCl solution was added and the product was
extracted with ethyl acetate (3×50 mL). The combined organic extracts are washed with
2
M HCl, H O, 5% NaHCO , and then brine successively, dried over MgSO , filtered,
2 3 4
and concentrated in vacuo to afford the amide 7 as a yellow liquid (1.3 g, 75 %), which
1
was purified by silica gel chromatography (petroleum ether/ethyl acetate, 15:1). H
NMR (400 MHz, CDCl ): δ 3.64 (s, 3H, OCH ), 3.20 (s, 3H, NCH ), 3.11 (s, 1H, H-3),
3
3
3
1
1
.91–1.80 (m, 2H, H-4β, 7β), 1.71–1.58 (m, 3H, H-2, 5, 1), 1.44-1.33 (m, 1H, H-7α),
.32–1.22 (m, 1H, H-4α), 0.94 (s, 3H, CH -9), 0.90 (s, 3H, CH -10), 0.88 (s, 3H, CH -8).
3
3
3
+
+
ESI-MS: calculated for C H NO (M+H ): 226.2, found: 226.2.
1
3
24
2
Synthesis of thiophen-2-yl((1R,2R,3R,5S)-2,6,6-trimethylbicyclo[3.1.1]heptan-3-yl)
methanone (8a). A 50 mL single-necked round-bottomed flask was charged with
thiophene (0.25 mL, 3.19 mmol), and redistilled diethyl ether (5 mL), evacuated and
refilled with nitrogen three times. A solution of n-BuLi (2.5 M in hexane, 2.98 mmol,
1
.2 mL) was added dropwise to the solution at 0 ℃. The reaction mixture solution was
stirred for 30 min and then cooled to -78 ℃. A solution of the Weinreb amide 7 (480
mg, 2.13 mmol) in dry diethyl ether (8 mL) was added over a period of 30 min. The
reaction mixture was stirred for additional 2.5 h and quenched by 15 mL saturated
ammonium chloride solution. The resulting solution was extracted with diethyl ether
(3×15 mL). The combined organic layers were washed with brine (40 mL), dried over
anhydrous Na SO and evaporated in vacuo. The residue was purified by silica gel
2
4
chromatography (petroleum ether/ ethyl acetate, 20:1) to give the Weinreb ketone 8a

1
(
404.0 mg, 76.3%) as a light blue liquid. H NMR (400 MHz, CDCl ): δ 7.63 (d, J =
3
3
.6 Hz, 1H, H-5’), 7.59 (d, J = 5.2 Hz, 1H, H-3’), 7.10 (t, J = 4.4 Hz, 1H, H-4’), 3.60
(ddd, J = 11.2, 5.6, 2.0 Hz, 1H, H-3), 1.97–1.89 (m, 1H, H-4β), 1.82 (dd, J = 12.4, 5.2
Hz, 1H, H-7β), 1.77–1.67 (m, 2H, H-2, 5), 1.67–1.53 (m, 2H, H-1), 1.46–1.40 (m, 1H,
H-7α), 1.29 (t, J = 11.2 Hz, 1H, H-4α), 1.03 (s, 3H, CH -9), 0.92 (s, 3H, CH -10), 0.90
3
3
1
3
(
s, 3H, CH -8); C NMR (100 MHz, CDCl ) δ 195.9 (C=O), 146.8 (C-2’), 133.1 (C-
3
3
5
2
’), 131.6 (C-3’), 127.8 (C-4’), 53.1 (C-3), 51.0(C-1), 50.7 (C-5), 45.4 (C-6), 32.1 (C-
), 29.5 (C-7), 27.7 (C-4), 19.2 (C-9), 18.9 (C-8), 16.2 (C-10). ESI-MS: calculated for
+
+
C H OS (M+H ): 249.1, found: 249.1。
1
5
21
The following compounds were synthesized using method A: 1-((1R,2R,3R,5S)-
,6,6-trimethylbicyclo[3.1.1]heptan-3-yl)ethan-1-one (8b), (4-methylthiophen-2-yl)
(1R,2R,3R,5S)-2,6,6-trimethylbicyclo[3.1.1]heptan-3-yl)methanone (8c), (3-methyl-
thiophen-2-yl)((1R,2R,3R,5S)-2,6,6-trimethylbicyclo[3.1.1]heptan-3-yl)methanone
8d), (5-chloro-4-methylthiophen-2-yl)((1R,2R,3R,5S)-2,6,6-trimethylbicyclo[3.1.1]-
2
(
(
heptan-3-yl)methanone (8e), (5-chlorothiophen-2-yl)((1R,2R,3R,5S)-2,6,6-trimethyl-
bicyclo[3.1.1]heptan-3-yl)methanone (8f), (4,5-dichlorothiophen-2-yl)((1R,2R,5S)-2,6,
6
6
5
3
-trimethylbicyclo[3.1.1]heptan-3-yl)methanone (8g), thiazol-2-yl((1R,2R,3R,5S)-2,6,
-trimethylbicyclo[3.1.1]heptan-3-yl)methanone (8h), (4-methoxyphenyl) ((1R,2R,3R,
S)-2,6,6-trimethylbicyclo[3.1.1]heptan-3-yl)methanone (8i), (4-fluorophenyl)((1R,2R,
R,5S)-2,6,6-trimethylbicyclo[3.1.1]heptan-3-yl)methanone(8j).
4
.1.3 Method A: General Procedures for Synthesizing Corresponding Ketone 10a-d
by Friede-Crafts acylation
Using the synthetic procedure of (5-bromo-4-methylthiophen-2-yl)((1R,2R,3R,5S)-
2
,6,6-trimethylbicyclo[3.1.1]heptan-3-yl)methanone (10c) as an example.
In a 100 mL round-bottomed flask equipped with a dropping funnel and a reflux
condenser connected at the top to a gas absorption trap, a solution of the acid 6 (1.1 g,
mmol), a catalytic amount of DMF and 15 mL anhydrous CH Cl was prepared. The
6
2
2
resultant mixture was cooled to 0 ℃, and then thionyl chloride (1.43 g, 12.00 mmol)

was added over dropwise. The reaction solution was refluxed for 2 h. Concentration in
vacuo offered quantitatively the crude acyl chloride 9 as a brown liquid to be used
directly in the next step.
SnCl (293 mg, 1.13 mmol) was slowly added to a mixture of acyl chloride 9 (150
4
mg, 0.75 mmol) and 2-bromo-3-methylthiophene (132.8 mg, 0.75 mmol) in anhydrous
CH Cl (10 mL) at 0 °C. The reaction mixture was stirred at 0 °C under nitrogen
2
2
atmosphere for 1 h. TLC (petroleum ether: ethyl acetate = 10:1) indicated that the
reaction was complete. The reaction mixture was quenched with 1M dilute hydrochloric
acid (8 mL). The resulting solution was extracted with dichloromethane (10 mL×3).
The combined organic layers were washed with brine, dried over anhydrous MgSO4,
filtered and concentrated. The crude product was purified by silica gel chromatography
eluted with petroleum ether/ ethyl acetate (20:1) to afford the product 10c (133 mg,
1
5
3
2
1.9%) as a yellow viscous liquid. H NMR (400 MHz, CDCl ): δ 7.39 (s, 1H, H-3’),
3
.08 (q, J = 9.6 Hz, 1H, H-3), 2.66 (q, J = 7.2 Hz, 1H, H-4β), 2.23 (s, 3H, CH -4’),
3
.19–2.10 (m, 2H, H-2, 7β), 1.98 (q, J = 5.6Hz, 1H, H-5), 1.94–1.82 (m, 1H, H-1), 1.73
(
t, J = 5.6 Hz, 1H, H-7α), 1.43 (d, J = 10.0 Hz, 1H, H-4α), 1.21 (s, 3H, , CH -9), 0.94
3
(
s, 3H, CH -8), 0.87 (d, J = 6.4 Hz, 3H, CH -10). ESI-MS: calculated for
3
3
+
+
C H BrOS (M+H ): 341.05, found: 341.1.
1
6
22
The following compounds were synthesized using method B: (5-methylthiophen-2-
yl)((1R,2R,5S)-2,6,6-trimethylbicyclo[3.1.1]heptan-3-yl)methanone (10a), (4,5-dime-
thylthiophen-2-yl)((1R,2R,5S)-2,6,6-trimethylbicyclo[3.1.1]heptan-3-yl)methanone
(10b), (5-methoxythiophen-2-yl)((1R,2R,5S)-2,6,6-trimethylbicyclo[3.1.1]heptan-3-yl)
methanone (10d).
4
3
.1.4 Synthesis of thiophen-2-yl((1R,2R,3R,5S)-2,6,6-trimethylbicyclo[3.1.1]heptan-
-yl)methanamine·HCl (11a)
A 25 mL round bottom flask was charged with compound 8a (372 mg, 1.5 mmol),
methoxyamine hydrochloride (250.6 mg, 3 mmol) and pyridine 10 mL. The reaction
mixture was stirred at 80 ℃ for 24 h. After azeotropic removal of solvent by evaporation

with n-hexane, the residue was partitioned between 50 mL dichloromethane (50 mL)
and brine (50 mL). The separated organic layer was dried over anhydrous Na SO ,
2
4
filtered, and concentrated in vacuo. The crude product was purified by column
chromatography (petroleum ether/ ethyl acetate, 100:1) to give O-methyl oxime
1
derivative 12 (220 mg, 52.8%) as a yellow oil. H NMR (400 MHz, CDCl ): δ 7.57 (d,
3
J = 5.0 Hz, 1H), 7.33 (d, J = 3.5 Hz, 1H), 7.06 (t, J = 4.4 Hz, 1H), 2.97–2.64 (m, 2H,
H-3, 4β), 2.28 (s, 3H, OCH ), 2.24–2.17 (m, 2H, H-2, 7β), 2.15–2.09 (m, 1H, H-5),
3
1
.95 (q, J = 5.6 Hz, 1H, H-1), 1.78 (d, J = 5.6 Hz, 1H, H-7α), 1.73 (t, J = 10.0 Hz, 1H,
H-4α), 1.22 (s, 3H, , CH -9), 0.95 (s, 3H, CH -8), 0.88 (d, J = 6.4 Hz, 3H, CH -10).
3
3
3
+
+
ESI-MS: calculated for C H NOS (M+H ): 292.17, found: 292.2.
1
7
26
To a solution of obtained oxime ester 12 (220 mg, 0.79 mmol) in 15 mL dry ether
was added dropwise a solution of (1.58 mL, 5 equiv.) 2.4 M lithium aluminum hydride
solution in tetrahydrofuran under argon atmosphere at 0 ℃. The resulting suspension
was stirred at 40 ℃ for 4 h. After the reaction mixture was cooled to 0 ℃, moist Na SO
2
4
solid was added slowly, and the resulting precipitate was removed by Celite filtration
and washed by THF several times. The combined filtrates were concentrated to dryness
and the residue was purified by silica gel chromatography using petroleum ether/ethyl
acetate (5:1) as an eluent to afford the free amine (60 mg, 30.4 %) as a light yellow
liquid. Corresponding salt 11a was prepared by addition of saturated hydrogen chloride
1
in ethyl acetate solution. White solid, m.p. 216.3–217.6 ℃. H NMR (400 MHz,
+
DMSO-d ): δ 8.68 (m, 3H, -NH ), 7.58 (d, J = 4.8 Hz, 1H, H-5’), 7.40 (d, J = 2.4 Hz,
6
3
1
H, H-3’), 7.08 (t, J = 4.0 Hz, 1H, H-4’), 4.50-4.46 (m, 1H, N-CH), 2.05–1.96 (m, 2H,
H-3, 4β), 1.94–1.88 (m, 2H, H-7β, 2), 1.83–1.70 (m, 2H, H-1, 5), 1.52 (t, J = 5.6 Hz,
1
6
1
3
H, H-7α), 1.22 (s, 1H, H-4α), 1.16 (s, 3H, CH -9), 0.80 (s, 3H, CH -8), 0.24 (d, J =
3 3
1
3
.4 Hz, 3H, CH -10); C NMR (100 MHz, DMSO-d ): δ 141.0 (C-2’), 127.7(C-5’),
3
6
27.6 (C-3’), 126.8 (C-4’), 55.1(N-CH), 48.7(C-3), 41.1(C-1), 40.1(C-5), 39.3(C-6),
3.6(C-2), 29.2(C-7), 26.9(C-4), 24.1(C-9), 20.7 (C-8), 20.5(C-10). HRMS calcd for
+
+
C H NS (M+H ), 250.1624; found, 250.1619. Anal. Calcd. (%) for C H ClNS: C,
1
5
24
15 24
6
3.02; H, 8.46; N, 4.90; Found: C, 62.94; H, 8.49; N, 5.3.

4
.1.5 General Procedures for Synthesizing Pinane Oxime Derivatives 11b-o.
Using
the
synthetic
procedure
of
(E)-(5-chloro-4-methylthiophen-2-
yl)((1R,2R,3R,5S) -2,6,6-trimethylbicyclo[3.1.1]heptan-3-yl)methanone oxime (11h)
as an example.
A 25 mL round bottom flask was charged with the corresponding ketone 8e (430 mg,
1
.44 mmol), hydroxylamine hydrochloride (300 mg, 4.32 mmol) and pyridine 10 mL.
The reaction mixture was stirred at 80 ℃ for 24 h. After azeotropic removal of solvent
by evaporation with n-hexane, the residue was partitioned between dichloromethane
and water. The separated organic layer was washed with brine, dried over anhydrous
Na SO and evaporated to dryness. The crude product was purified by silica gel
2
4
chromatography to give single ant-isomer 11h (247 mg, 55.01 %) as a white solid. m.p.
1
1
68.4–170.1 ℃. H NMR (400 MHz, DMSO-d6): δ 12.00 (s, 1H, -OH), 7.50 (s, 1H,
H-3’), 2.98 (q, J = 9.6 Hz, 1H, H-3), 2.71 (q, J = 7.2 Hz, 1H, H-4β), 2.26–2.20 (m, 1H,
H-7β), 2.18 (s, 3H, Ar-CH ), 2.13–2.03 (m, 1H, H-2), 1.92 (q, J = 5.6 Hz, 1H, H-5),
3
1
.70–1.67 (m, 2H, H-1, 7α), 1.64–1.55 (m, 1H, H-4α), 1.20 (s, 3H, CH -9), 0.91 (s, 3H,
3
1
3
CH -8), 0.82 (d, J = 6.4 Hz, 3H, CH -10)； C NMR (100 MHz, DMSO-d ): δ 150.8
3
3
6
(C=N), 133.0 (C-4’), 130.5(C-5’), 128.8 (C-2’), 128.6 (C-3’), 47.2 (C-3), 40.9 (C-1),
4
0.2 (C-5), 38.82 (C-6), 32.9 (C-2), 32.6 (C-7), 27.1 (C-4), 23.2 (C-9), 20.5 (C-8), 20.4
+
+
(
C-10), 13.5 (Ar-C). HRMS calcd for C H ClNOS (M+H ): 312.1183, found:
16 23
3
6
4
12.1188. Anal. Calcd. (%) for C H ClNOS: C, 61.62; H, 7.11; N, 4.49; Found: C,
16 22
1.56; H, 7.07; N, 4.44.
.1.5.1. (thiophen-2-yl)((1R,2R,3R,5S)-2,6,6-trimethylbicyclo[3.1.1]heptan-3-yl)meth-
1
anone oxime (11b). Pale yellow solid, yield: 62.8%, m.p. 159.6–161.2 ℃. H NMR
(
400 MHz, CDCl ): δ 9.10 (s, 1H, -OH), 7.52–7.50 (m, 2H, H-3’, 5’), 7.08 (t, J = 4.4
3
Hz, 1H, H-4’), 3.46–3.42 (m, 1H, H-3), 2.10–2.02 (m, 1H, H-4β), 1.95–1.89 (m, 1H,
H-7β), 1.80–1.70 (m, 3H, H-2, 5, 1), 1.40–1.22 (m, 2H, H-4α,7α), 1.04 (s, 3H, Me-9),
1
3
0
.92 (s, 3H, Me-10), 0.70 (s, 3H, Me-8). C NMR (100 MHz, DMSO-d ): δ 156.0
6
(C=N), 142.4 (C-2’), 141.6 (C-5’), 127.6 (C-3’), 121.6 (C-4’), 47.5 (C-3), 46.8 (C-1),
4
1.3 (C-5), 34.3 (C-6), 33.1 (C-2), 31.4 (C-7), 28.9 (C-4), 26.8 (C-9), 19.6 (C-8), 19.5

+
(
C-10). HRMS calcd for C H NOS+ (M+H ): 264.1417, found: 264.1421. Anal.
15 22
Calcd. (%) for C H NOS: C, 68.40; H, 8.04; N, 5.32; Found: C, 68.51; H, 7.99; N,
1
5
21
5
.22.
4
.1.5.2. 1-((1R,2R,3R,5S)-2,6,6-trimethylbicyclo[3.1.1]heptan-3-yl)ethan-1-one oxime
1
(
11c). White solid, yield: 70.3%, m.p. 140.2–141.9 ℃. H NMR (400 MHz, DMSO-
d6): δ 10.34 (s, 1H, -OH), 2.38 (p, J = 7.2 Hz, 1H, H-3), 2.20 (q, J = 9.6 Hz, 1H, H-4β),
2
1
.07–1.88 (m, 3H, H-7β, 5, 2), 1.79 (s, 3H, N=C-CH ), 1.65–1.56 (m, 2H, H-1, 7α),
3
.42 (d, J = 10.0 Hz, 1H, 4α), 1.18 (s, 3H, CH -9), 0.81 (s, 3H, CH -8), 0.80 (d, J = 6.4
3
3
1
3
Hz, 3H, CH -10); C NMR (100 MHz, DMSO-d ): δ 157.2 (C=N), 47.1 (C-3), 42.4
3
6
(C-1), 40.6 (C-5), 40.0 (C-6), 30.9 (C-2), 29.9 (C-7), 27.1 (C-4), 23.3 (C-9), 20.4 (C-
+
+
8
), 20.1 (C-10), 13.3 (N=C-CH ). HRMS calcd for C H NO (M+H ): 196.1696,
3 12 22
found: 196.1700. Anal. Calcd. (%) for C H NO: C, 73.80; H, 10.84; N, 7.17; Found:
1
2
21
C, 73.86; H, 10.78; N, 7.05.
4
.1.5.3. (4-methylthiophen-2-yl)((1R,2R,3R,5S)-2,6,6-trimethylbicyclo[3.1.1]heptan-
3
-yl)methanone oxime (11d). Light yellow solid, yield: 68.4%, m.p. 177.7–180.1 ℃.
1
H NMR (400 MHz, DMSO-d ): δ 11.64 (s, 1H, -OH), 7.46 (s, 1H, H-5’), 7.28 (s, 1H
6
H-3’), 2.97 (q, J = 9.6 Hz, 1H, H-3), 2.77–2.69 (m, 1H, H-4β), 2.26–2.19 (m, 4H, CH3-
4
’, 7β), 2.09–2.07 (m, 1H, H-2), 1.92 (q, J = 5.6 Hz, 1H, H-5), 1.72–1.55 (m, 3H, H-1,
1
3
7
α, 4α), 1.20 (s, 3H, CH -9), 0.91 (s, 3H, CH -8), 0.82 (d, J = 6.4 Hz, 3H, CH -10); C
3 3 3
NMR (100 MHz, DMSO-d ): δ 150.6 (C=N), 135.5 (C-4’), 132.8 (C-2’), 130.7 (C-5’),
6
1
4
2
5
25.6 (C-3’), 47.3 (C-3), 40.8 (C-1), 32.6 (C-5), 32.5 (C-6,C-2), 26.7 (C-7), 22.9 (C-
+
+
), 20.2(C-8, C-9), 20.0 (C-10), 15.2 (Ar-CH ). HRMS calcd for C H NOS (M+H ):
3
16 24
78.1573, found: 278.1569. Anal. Calcd. (%) for C H NOS: C, 69.27; H, 8.36; N,
1
6
23
.05; Found: C, 69.23; H, 8.32; N, 5.11.
4
.1.5.4. (3-methylthiophen-2-yl)((1R,2R,3R,5S)-2,6,6-trimethylbicyclo[3.1.1]heptan-
3
-yl) methanone oxime (11e). Light yellow solid, yield: 64.9%, m.p. 174.8–176.4 ℃.
1
H NMR (400 MHz, DMSO-d ): δ 10.94 (s, 1H, -OH), 7.50 (d, J = 5.0 Hz, 1H, H-5’),
6
6
.91 (d, J = 5.0 Hz, 1H, H-4’), 2.48–2.44 (m, 1H, H-3), 2.07 (s, 3H, Ar-CH ), 2.04–
3
2
.01 (m, 1H, H-4β), 1.92–1.79 (m, 2H, H-2, 7β), 1.75–1.70 (m, 1H, H-5), 1.64 (t, J =

5
.6 Hz, 1H, H-1), 1.41 (d, J = 10.0 Hz, 1H, H-7α),1.30-1.21 (m, 1H, H-4α) 1.16 (s, 3H,
1
3
CH -9), 0.87 (d, J = 5.6 Hz, 3H, CH -10), 0.72 (s, 3H, CH -8). C NMR (100 MHz,
3
3
3
DMSO-d ): δ 142.7 (C=N), 135.9 (C-3’), 129.9 (C-2’), 128.7 (C-4’), 126.3(C-5’), 47.3
6
(C-3), 44.7 (C-1), 40.5 (C-5), 39.7 (C-6), 32.0 (C-2), 30.0 (C-7), 26.9 (C-4), 23.2 (C-
+
+
9
2
5
4
), 20.1 (C-8), 19.5 (C-10), 14.5 (Ar-CH ). HRMS calcd for C H NOS (M+H ):
3 16 24
78.1573, found: 278.1571. Anal. Calcd. (%) for C H NOS: C, 69.27; H, 8.36; N,
1
6
23
.05; Found: C, 69.30; H, 8.41; N, 5.02.
.1.5.5. (5-methylthiophen-2-yl)((1R,2R,3R,5S)-2,6,6-trimethylbicyclo[3.1.1]heptan-
3
-yl)methanone oxime (11f). Light yellow solid, yield: 71.9%, m.p. 175.2–176.7 ℃.
1
H NMR (400 MHz, CDCl ): δ 8.25 (s, 1H, -OH), 7.37 (s, 1H, H-3’), 6.77 (s, 1H, H-
3
4
’), 2.95–2.87 (m, 1H, H-3), 2.70 (s, 1H, H-4β), 2.52 (s, 3H, Ar-CH ), 2.21 (s, 1H, H-
3
7
β), 2.13 (s, 1H, H-2), 1.96 (s, 1H, H-5), 1.82–1.76 (m, 2H, H-1), 1.58–1.52 (m, 2H,
H-7α, 4α), 1.24 (s, 3H, CH -9), 0.94 (s, 3H, CH -8), 0.90 (d, J = 6.4 Hz, 3H, CH -10);
3
3
3
1
3
C NMR (100 MHz, CDCl ): δ 153.7 (C=N), 144.8 (C-2’), 131.5 (C-5’), 130.1 (C-4’),
3
1
24.7 (C-3’), 48.3 (C-3), 41.8 (C-1), 41.7 (C-5), 40.7 (C-6), 33.2 (C-2), 33.1 (C-7), 27.4
(
C-4), 23.8 (C-9), 20.6 (C-8), 20.4 (C-10), 15.3 (Ar-CH ). HRMS calcd for
3
+
+
C H NOS (M+H ): 278.1573, found: 278.1578. Anal. Calcd. (%) for C H NOS: C,
1
6
24
16 23
6
9.27; H, 8.36; N, 5.05; Found: C, 69.22; H, 8.29; N, 5.10.
4
.1.5.6. (4,5-dimethylthiophen-2-yl)((1R,2R,3R,5S)-2,6,6-trimethylbicyclo[3.1.1]hep-
1
tan-3-yl)methanone oxime (11g). White solid, yield: 63.2%, m.p. 189.1–190.4 ℃. H
NMR (400 MHz, DMSO-d ): δ 11.47 (s, 1H, -OH), 7.34 (s, 1H, H-3’), 2.93 (q, J = 9.6
6
Hz, 1H, H-3), 2.71 (s, 1H, H-4β), 2.29 (s, 3H, CH -5’), 2.23–2.16 (m, 1H, H-7β), 2.11
3
(
s, 3H, CH -4’), 2.07 (s, 1H, H-2), 1.91 (d, J = 5.7 Hz, 1H, H-5), 1.69–1.57 (m, 3H,
3
H-1, 7α, 4α), 1.20 (s, 3H, CH -9), 0.91 (s, 3H CH -8), 0.81 (d, J = 6.4 Hz, 3H, CH -
3
3
3
1
3
1
4
2
0); C NMR(100 MHz, CDCl ): δ 153.6 (C=N), 138.4 (C-5’), 132.9 (C-2’), 132.7 (C-
3
’), 129.0 (C-3’), 48.3 (C-3), 41.8 (C-1), 41.7 (C-5), 40.8 (C-6), 33.3 (C-2), 33.1 (C-7),
7.4 (C-4), 23.8 (C-9), 20.6 (C-8), 20.4 (C-10), 13.8 (Ar-CH -4’), 13.2 (Ar-CH -5’).
3
3
+
+
HRMS calcd for C H NOS (M+H ): 292.1730, found: 292.1736. Anal. Calcd. (%)
1
7
26
for C H NOS: C, 70.06; H, 8.65; N, 4.81; Found: C, 70.00; H, 8.59; N, 4.91.
1
7
25
4
.1.5.7. (5-bromo-4-methylthiophen-2-yl)((1R,2R,3R,5S)-2,6,6-trimethylbicyclo[3.1.1]

heptan-3-yl)methanone oxime (11i). White solid, yield: 66.6%, m.p. 165.4–167.1 ℃.
1
H NMR (400 MHz, DMSO-d ): δ 11.98 (s, 1H, -OH), 7.47 (s, 1H, H-3’), 2.97 (q, J =
6
9
.6 Hz, 1H, H-3), 2.71 (p, J = 7.2 Hz, 1H, H-4β), 2.25–2.21 (m, 1H, H-7β), 2.18 (s, 3H,
Ar-CH ), 2.11–2.05 (m, 1H, H-2), 1.91 (q, J = 5.6 Hz, 1H, H-5), 1.70–1.66 (m, 2H,
3
H-1, 7α), 1.64–1.52 (m, 1H, H-4α), 1.20 (s, 3H, CH -9), 0.90 (s, 3H, CH -8), 0.81 (d,
3
3
1
3
J = 6.4 Hz, 3H, CH -10); C NMR (100 MHz, DMSO-d ): δ 150.8 (C=N), 135.7 (C-
3
6
4
’), 131.9 (C-5’), 130.6 (C-2’), 115.2 (C-3’), 55.3 (C-3), 47.2 (C-1), 40.9 (C-5), 38.9
(C-6), 32.9 (C-2), 32.6 (C-7), 27.1 (C-4), 23.2 (C-9), 20.5 (C-8), 20.4 (C-10), 15.1 (Ar-
+
+
C). HRMS calcd for C H BrNOS (M+H ): 356.0678, found: 356.0680. Anal. Calcd.
1
6
23
(
%) for C H BrNOS: C, 53.93; H, 6.22; N, 3.93; Found: C, 53.88; H, 6.20; N, 4.01.
16 22
4
.1.5.8. (5-chlorothiophen-2-yl)((1R,2R,3R,5S)-2,6,6-trimethylbicyclo[3.1.1]heptan-3-
1
yl) methanone oxime (11j). White solid, yield: 56.1%, m.p. 170.8–172.2 ℃. H NMR
(
400 MHz, DMSO-d ): δ 12.09 (s, 1H, -OH), 7.54 (d, J = 4.0 Hz, 1H, H-3’), 7.15 (d, J
6
=
4.0 Hz, 1H, H-4’), 3.00 (q, J = 9.6 Hz, 1H, H-3), 2.70 (p, J = 7.2, 1H, H-4β), 2.22–
2
3
.16 (m, 1H, H-7β), 2.09–2.06 (m, 1H, H-2), 1.94–1.88 (m, 1H, H-5), 1.71–1.57 (m,
H, H-1, 7α, 4α) ), 1.20 (s, 3H, CH -9), 0.90 (s, 3H, CH -8), 0.81 (d, J = 6.4 Hz, 3H,
3
3
1
3
CH -10); C NMR (100 MHz, DMSO-d ): δ 160.0 (C=N), 132.1 (C-2’), 130.2 (C-5’),
3
6
1
3
27.7 (C-4’), 124.6 (C-3’), 46.3 (C-3), 39.0 (C-1), 38.8 (C-5), 37.8 (C-6), 31.9 (C-2),
1.8 (C-7), 26.1(C-4), 22.2(C-9), 19.6(C-8), 19.4(C-10). HRMS calcd for
+
+
C H ClNOS (M+H ): 298.1027, found: 298.1034. Anal. Calcd. (%) for
1
5
21
C H ClNOS: C, 60.49; H, 6.77; N, 4.70; Found: C, 60.43; H, 6.72; N, 4.76.
1
5
20
4
.1.5.9. (4,5-dichlorothiophen-2-yl)((1R,2R,3R,5S)-2,6,6-trimethylbicyclo[3.1.1]hept-
1
an-3-yl)methanone oxime (11k). White solid, yield: 79.7%, m.p. 186.4–187.9 ℃. H
NMR (400 MHz, DMSO-d ): δ 12.42 (s, 1H, -OH), 7.76 (s, 1H, H-3’), 3.02 (q, J = 9.6
6
Hz, 1H, H-3), 2.69 (t, J = 7.2 Hz, 1H, H-4β), 2.24–2.14 (m, 1H, H-7β), 2.07–2.03 (m,
1
1
H, H-2), 1.93–1.88 (m, 1H, H-5), 1.73 (d, J = 10.0 Hz, 1H, H-1), 1.68 (d, J = 5.6 Hz,
H, H-7α), 1.64–1.53 (m, 1H, H-4α), 1.20 (s, 3H, CH -9), 0.90 (s, 3H, CH -8), 0.82 (d,
3
3
1
3
J = 6.4 Hz, 3H, CH -10); C NMR (100 MHz, DMSO-d ): δ 143.9 (C=N), 132.4 (C-
3
6
2
’), 130.0 (C-5’), 127.0 (C-4’), 121.6 (C-3’), 56.1 (C-3), 47.5 (C-1), 40.8 (C-5), 40.6
(C-6), 38.8 (C-2), 32.3 (C-7), 31.2 (C-4), 27.7 (C-9), 23. 8 (C-8), 21.2 (C-10). HRMS

+
calcd for C H Cl NOS (M+H ): 332.0637, found: 332.0642. Anal. Calcd. (%) for
1
5
20
2
C H Cl NOS: C, 54.22; H, 5.76; N, 4.22; Found: C, 54.18; H, 5.78; N, 4.19.
1
5
19
2
4
.1.5.10. (5-methoxythiophen-2-yl)((1R,2R,3R,5S)-2,6,6-trimethylbicyclo[3.1.1]hept-
1
an-3-yl)methanone oxime(11l). Yellow solid, yield: 65.1%, m.p. 154.6–155.9 ℃. H
NMR (400 MHz, DMSO-d ): δ 11.46 (s, 1H, -OH), 7.32 (d, J = 4.4 Hz, 1H, H-3’), 6.32
6
(
d, J = 4.4 Hz, 1H, H-4’), 3.88 (s, 3H, OCH ), 2.94 (t, J = 9.6 Hz, 1H, H-3), 2.73–2.66
3
(m, 1H, H-4β), 2.20–2.14 (m, 1H, H-7β), 2.11–2.02 (m, 1H, H-2), 1.90 (d, J = 6.0 Hz,
1
0
1
H, H-5), 1.69–1.59 (m, 3H, H-1, 7α, 4α), 1.20 (s, 3H, CH -9), 0.92 (s, 3H, CH -8),
3 3
1
3
.81 (d, J = 6.4 Hz, 3H, CH -10); C NMR (100 MHz, DMSO-d ): δ 169.7 (C=N),
3
6
51.3 (C-5’), 127.8 (C-2’), 118.8 (C-3’), 103.0 (C-4’), 60.1 (OCH ), 47.3 (C-3), 40.9
3
(C-1), 38.7 (C-5), 33.1 (C-6), 32.7 (C-2), 27.1 (C-7), 23.2 (C-4), 20.6 (C-8, C-9), 20.4
+
+
(
C-10). HRMS calcd for C H Cl NO S (M+H ): 294.1522, found: 294.1525. Anal.
1
6
24
2
2
Calcd. (%) for C H NO2S: C, 65.49; H, 7.90; N, 4.77; Found: C, 65.41; H, 7.96; N,
1
6
23
4
.75.
4
.1.5.11. thiazol-2-yl((1R,2R,3R,5S)-2,6,6-trimethylbicyclo[3.1.1]heptan-3-yl)methan-
1
one oxime (11m). Pale yellow solid, yield: 70.2%, m.p. 170.5–172.4 ℃. H NMR(400
MHz, DMSO-d ): δ 12.54 (s, 1H, -OH), 8.03 (d, J = 3.2 Hz, 1H, H-5’), 7.96 (d, J = 3.2
6
Hz, 1H, H-4’), 3.54 (q, J = 9.6 Hz, 1H, H-3), 2.74–2.67 (m, 1H, H-4β), 2.35–2.28 (m,
1
1
H, H-7β), 2.14–2.09 (m, 1H, H-2), 1.93 (q, J = 5.0 Hz, 1H, H-5), 1.71 (t, J = 5.6 Hz,
H, H-1), 1.62–1.54 (m, 2H, H-7α, 4α), 1.21 (s, 3H, CH -9), 0.93 (s, 3H, CH -8), 0.81
3
3
1
3
(
(
(
d, J = 6.4 Hz, 3H, CH -10); C NMR (100 MHz, DMSO-d ): δ 155.1 (C=N), 152.1
3
6
C-2’), 142.0 (C-4’), 124.3 (C-5’), 47.3 (C-3), 40.9 (C-1), 40.2 (C-5), 38.7 (C-6), 32.5
C-2), 32.4 (C-7), 27.1 (C-4), 23.4 (C-8), 20.5 (C-9), 20.3(C-10). HRMS calcd for
+
+
C H N OS (M+H ): 265.1369, found: 265.1372. Anal. Calcd. (%) for C H N OS:
1
4
21
2
14 20
2
C, 63.60; H, 7.63; N, 10.60; Found: C, 63.55; H, 7.65; N, 10.58.
4
.1.5.12. (4-methoxyphenyl)((1R,2R,3R,5S)-2,6,6-trimethylbicyclo[3.1.1]heptan-3-yl)
1
methanone oxime (11n). Pale yellow solid, yield: 53.5%, m.p. 194.3–196.1 ℃. H NMR
(
400 MHz, DMSO-d ) δ 10.46 (s, 1H, -OH), 7.25 (d, J = 8.0 Hz, 2H, H-2’, 6’), 6.93 (d,
6
J = 8.0 Hz, 2H, H-3’, 5’), 3.76 (s, 3H, OCH ), 2.57 (q, J = 9.6 Hz, 1H, H-3), 2.48–2.44
3
(m, 1H, H-4β), 2.04–2.00 (m, 1H, H-7β), 1.90–1.72 (m, 2H, H-2, 5), 1.71–1.57 (m, 2H,

H-1, 7α), 1.44 (d, J = 10.0 Hz, 1H, H-4α), 1.16 (s, 3H, CH -9), 0.86 (d, J = 6.4 Hz, 3H,
3
1
3
CH -10), 0.72 (s, 3H, CH -8); C NMR (100 MHz, DMSO-d ) δ 159.1 (C=N), 158.0
3
3
6
(
C-4’), 129.6 (C-2’, C-6’), 127.9 (C-1’), 113.6 (C-3’, C-5’), 55.4 (OCH ), 47.3 (C-3),
3
4
2
2.1 (C-1), 40.6 (C-5), 31.9 (C-6), 30.9 (C-2), 27.0 (C-7), 23.4 (C-4), 20.4 (C-8, C-9 ),
+
+
0.1 (C-10). HRMS calcd for C H NO (M+H ): 288.1958, found: 288.1960. Anal.
1
8
26
2
Calcd. (%) for C H NO : C, 75.22; H, 8.77; N, 4.87; Found: C, 75.28; H, 8.82; N,
1
8
25
2
4
.83.
4
.1.5.13. (4-fluorophenyl)(( 1R,2R,3R,5S)-2,6,6-trimethylbicyclo[3.1.1]heptan-3-yl)
1
methanone oxime (11o). White solid, yield: 72.6%, m.p. 190.5–191.1 ℃. H NMR (400
MHz, DMSO-d ): δ 10.63 (s, 1H, -OH), 7.33 (t, J = 7.6 Hz, 2H, H-2’, 6’), 7.22 (t, J =
6
8
2
1
=
.8 Hz, 2H, H-3’, 5’), 2.57 (q, J = 9.6 Hz, 1H, H-3), 2.45 (t, J = 7.2 Hz, 1H, H-4β),
.05–2.00 (m, 1H, H-7β), 1.86 (q, J = 5.6 Hz, 1H, H-2), 1.80–1.73 (m, 1H, H-5), 1.70–
.62 (m, 2H, H-1, 7α), 1.44 (d, J = 10.0 Hz, 1H, H-4α), 1.15 (s, 3H, CH -9), 0.87 (d, J
3
1
3
6.4 Hz, 3H, CH -10), 0.69 (s, 3H, CH -8); C NMR (100 MHz, DMSO-d ): δ 161.9
3
3
6
1
3
(
d, J = 242.8 Hz, C-4’),157.8 (C=N), 132.0 (C-1’), 130.4 (d, J = 8.1 Hz, C-2’, C-
CF CF
6
6
’), 115.2 (d, 2JCF = 21.3 Hz, C-3’, C-5’), 47.2 (C-3), 42.1 (C-1), 40.5 (C-5), 31.8(C-
), 30.6 (C-2), 27.0 (C-7), 23.3 (C-4), 20.4 (C-8, C-9 ), 20.0 (C-10). HRMS calcd for
+
+
C H FNO (M+H ): 276.1758, found: 276.1755. Anal. Calcd. (%) for C H FNO: C,
1
7
23
17 22
7
4.15; H, 8.05; N, 5.09; Found: C, 74.12; H, 8.01; N, 5.13.
4
.1.6 Synthesis of Compound 14a and 14b
A solution of compound 11d (50 mg, 0.18 mmol) in 5 mL anhydrous DMF was
treated with 60% sodium hydride (18 mg, 0.45 mmol) at 0 ℃ and stirred for 1 h. Then
tert-butyl (S)-2-(bromomethyl)pyrrolidine-1-carboxylate (57 mg, 0.22 mmol) was
added in portions. The reaction mixture was stirred at room temperature for 4 h,
quenched by adding saturated NaHCO solution (8 mL), and then extracted with ethyl
3
acetate (3×10 mL). The combined organic layers were washed with brine (15 mL), dried,
filtered, and concentrated under reduced pressure. The crude residue was purified by
column chromatography (petroleum ether/ethyl acetate, 50:1) to give a colorless oil.

The product was dissolved in ethyl acetate (2 mL) and treated with excess saturated
hydrogen chloride in ethyl acetate solution. Upon removal of the solvent, the residue
was triturated with ether, filtered and dried to afford compound 14a as a white solid (55
1
mg, 76.9%). m.p. 210.2–212.0 ℃. H NMR (400 MHz, DMSO-d ): δ 9.62 (s, 1H,
6
pyrrolidine-NH), 9.05 (s, 1H s, pyrrolidine-NH), 7.62 (s, 1H, H-5’), 7.42 (s, 1H, H-3’),
4
.65 – 4.13 (m, 2H, -OCH -), 3.84 (s, 1H, NCH), 3.19 (s, 2H, pyrrolidine-NCH ), 3.01
2 2
(
q, J = 9.6 Hz, 1H, H-3), 2.71–2.67 (m, 1H, H-4β), 2.25 (s, 3H, Ar-CH ), 2.21-2.18 (m,
3
1
1
9
H, H-7β), 2.09 (s, 2H, pyrrolidine-NCH CH ), 2.01–1.82 (m, 3H, H-2, 5, 1), 1.81–
2 2
.71 (m, 1H, H-7α), 1.72–1.59 (m, 3H, pyrrolidine-NCHCH , H-4α), 1.20 (s, 3H, CH -
2
3
1
3
), 0.91 (s, 3H, CH -8), 0.83 (d, J = 6.4 Hz, 3H, CH -10). C NMR (100 MHz, DMSO-
3
3
d ): δ 153.2 (C=N), 136.6 (C-4’), 133.1 (C-2’), 132.3 (C-5’), 127.4 (C-3’), 73.3 (OCH ),
6
2
5
3
2
8.1 (pyrrolidine-C-2’’), 47.3 (C-3), 45.6 (pyrrolidine-C-5’’), 40.9 (C-1), 40.3 (C-5),
3.0 (C-6), 32.7 (C-2), 27.4 (pyrrolidine-C-3’’) , 27.2 (C-7), 23.6 (pyrrolidine-C-4’’),
3.2 (C-4), 20.6 (C-8, C-9), 20.2 (C-10), 15.6 (Ar-C). HRMS calcd for
+
+
C H N OS (M+H ): 361.2308, found: 361.2306. Anal. Calcd. (%) for C H ClN OS:
2
1
33
2
21 33
2
C, 63.53; H, 8.38; N, 7.06; Found: C, 63.48; H, 8.36; N, 7.10.
Compound 14b (48 mg, 71.2%) as a yellow solid was prepared from compound 11d
(50 mg, 0.18 mmol), 2-pyrrolidinoethyl chloride hydrochloride (36.7 mg, 0.22 mmol)
and 60% sodium hydride (18 mg, 0.45 mmol) according to standard procedure similar
1
to preparation of compound 14a. m.p. 223.0–224.8 ℃. H NMR (400 MHz, DMSO-
d ): δ 7.53 (s, 1H, H-5’), 7.35 (s, 1H, H-3’), 4.26 (t, J = 5.6 Hz, 2H, OCH CH ), 2.98
6
2
2
(
q, J = 9.6 Hz, 1H, H-3), 2.83 (s, 2H, OCH CH N), 2.72 (p, J = 7.2 Hz, 1H, H-4β), 2.55
2 2
(
s, 4H, 2×pyrrolidine-N-CH ), 2.24 (s, 3H, Ar-CH ), 2.19 (t, J = 7.2 Hz, 1H, H-7β),
2 3
2
.11–2.05 (m, 1H, H-2), 1.91 (q, J = 5.6 Hz, 1H, H-5), 1.68–1.58 (m, 7H, 2×pyrrolidine-
NCH CH , H-1, 7α, 4α ), 1.20 (s, 3H, CH -9), 0.92 (s, 3H, CH -8), 0.82 (d, J = 6.4 Hz,
2
2
3
3
1
3
3
2
2
4
H, CH -10)； C NMR (100 MHz, CDCl ): δ 151.7 (C=N), 136.1 (C-4’), 133.6 (C-
3 3
’), 131.1 (C-5’), 125.3 (C-3’), 73.2 (OCH ), 54.6 (OCH CH N), 54.5 (pyrrolidine-C-
2
2
2
’’, 5’’), 47.8 (C-3), 41.3 (C-1), 40.4 (C-5), 33.1 (C-6), 32.9 (C-2), 29.6 (C-7), 27.0 (C-
), 23.6 (pyrrolidine-C-3’’, 4’’), 23.4 (C-9), 20.3 (C-8), 19.8 (C-10), 15.4 (Ar-C).
+
+
HRMS calcd for C H N OS (M+H ): 375.2465, found: 375.2470. Anal. Calcd. (%)
2
2
35
2

for C H ClN OS: C, 64.29; H, 8.58; N, 6.82; Found: C, 68.32; H, 8.63; N, 6.78.
2
2
35
2
4
.2 Cytopathic Effect (CPE) Assay Protocol
In CPE assay, MDCK cells were grown to a confluent monolayer in a 96-well
4
culture plate at a concentration of 5×10 /well for 24 h. The medium was removed, and
the cells were rinsed twice. An infectious virus at 100 TCID was inoculated into the
5
0
MDCK cells, which were then incubated for 2 h at 37 °C in 5% CO . The virus
2
supernatant was then removed, followed by the addition of serial two-fold dilutions of
antiviral compounds in DMEM containing 1.5 µg/mL trypsin. After being incubated at
3
4 °C in 5% CO for 48 h, the infected cells displayed 100% CPE under the microscope,
2
and the CPE percentages in the antiviral compound-treated groups were recorded. The
EC values were calculated using a non-linear regression model in GraphPad Prism 6.
5
0
All data were obtained for at least three independent experiments.
4
.3 Cytotoxicity Assay
Cytotoxicity of compounds was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) assay. Madin Darby canine kidney (MDCK) cells
on 96-well plates were washed with sterile phosphate buffered saline (PBS). Then 100
μl of the compound in serum-free MEM at two-fold dilution was added to the cells and
the cells were incubated in CO incubation at 37 °C. Cell controls were also performed.
2
After 48h, 5 mg/mL of fresh MTT was added to each well and the plates were incubated
o
at 37 C for 4 h. Afterwards, the medium was removed and formazan crystal was
dissolved in DMSO (100μl per well). Absorbance of each well at 490 nm was read by
a CLARIOstar multi-mode microplate reader (BMG Labtech, Germany). The cell
viability (%) = OD of compound well / average OD of control wells. The TC of each
5
0
compound was obtained using a non-linear regression model in GraphPad Prism 6.
4
.4 Patch clamp assay
The stable cell lines of transformed 293T-Rex expressed WT and S31N mutant M2
channels of avian H5N1 were created by pCDNA4/TO plasmid prior to
electrophysiological assay according to the literature. M2-293T-Rex cells was induced
with 1 μg/mL tetracycline for 24-48 hours. Patch clamp recordings are performed in