Discovery of highly potent agents against influenza A virus

Article abstract of DOI:10.1016/j.ejmech.2010.10.010

We previously reported several new M2 inhibitors as active as amantadine against influenza A virus and validated by three types of in vitro assays. Herein, we further modified one of the most potent hits in a viral inhibition assay and conducted structure

Full text of DOI:10.1016/j.ejmech.2010.10.010

European Journal of Medicinal Chemistry 46 (2011) 52e57
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Discovery of highly potent agents against influenza A virus
,b,
Xin Zhao a, Chufang Li a, Shaogao Zeng a, Wenhui Hu a
*
a Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, 190 Kai Yuan Avenue, Guangzhou Science Park, Guangzhou 510530, People’s Republic of China
b State Key Laboratory of Respiratory Disease, Guangzhou, Guangdong 510120, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
We previously reported several new M2 inhibitors as active as amantadine against influenza A virus and
validated by three types of in vitro assays. Herein, we further modified one of the most potent hits in
a viral inhibition assay and conducted structureeactivity relationship studies on this scaffold. As a result,
compound 8e was identified to be the most potent inhibitor against wild-type influenza A virus, being
nearly 240-fold more active than amantadine.
Received 10 August 2010
Received in revised form
8 October 2010
Accepted 9 October 2010
Available online 16 October 2010
Ó 2010 Elsevier Masson SAS. All rights reserved.
Keywords:
Hit-to-lead optimization
Influenza A virus
SAR study
1. Introduction
amine library [13]. The hits were as active as amantadine against
wild-type influenza A virus as determined by three kinds of assays,
Influenza is always
a
major worldwide health threat to
including cell-based, viral inhibition and patch clamp assays.
Among them, compound 5 was the most potent inhibitor and was
three times more active than amantadine for viral inhibition
humankind. In the face of the recent outbreak of highly pathogenic
avian influenza (H5N1) in Southeast Asia and H1N1 influenza
(swine flu) around the world [1,2], there is a great need for anti-
influenza therapeutics. However, very few effective drugs are
available to combat the influenza virus. Amantadine (1) and
rimantadine (2) (Fig. 1), which target the influenza A virus M2
(matrix-2 protein) ion channel, have long been available for both
the prophylaxis and therapy of influenza A viral infections, but their
use has been limited because of the rapid emergence of drug
resistance and, particularly for amantadine, the occurrence of
central nervous system (CNS) side effects [3]. Over the past decade,
nearly all reported M2 inhibitors have been amantadine deriva-
tives, such as compound 3 (Fig. 1) [4e9]. Compound 4 is one of the
very few examples of nonadamantane-based M2 inhibitors that
have been reported [10e12]. Therefore, there is an increasingly
urgent need to discover new types of M2 inhibitors for the devel-
opment of new anti-influenza drugs.
(IC50 ¼ 1.363
mM vs. 5.960 mM).
Encouraged by these results, we decided to modify the hit to
further increase its potency. By keeping the scaffold constant and
modifying the amino functionality, 14 analogs were made and
evaluated for viral inhibition, as assessed by A/WS/33 (H1N1,
amantadine resistant) and A/Hong Kong/8/68 (H3N2, amantadine
sensitive) viruses [14,15]. Most of the compounds in this study
exhibited antiviral inhibition as good as amantadine, and
compound 8e was identified to be the most potent; it was nearly
240-fold more potent than amantadine.
2. Chemistry
The synthesis of (1R,2R,3R,5S)-(ꢀ)-isopinocampheylamine
amido derivatives 7ae7g is shown in Scheme 1. Initially, commer-
cially available compound 5 was N-acylated with methyl chlor-
oformate or acetyl chloride to give 6a and 6b, which were then
reduced with LiAlH4. Following reduction, salification with HCl/
CH3OH gave 7a and 7b [16]. On the other hand, compounds 7ceg
were obtained through a one-pot synthesis in which compound 5
was first coupled with different aldehydes or ketones. This synthesis
was then followed by hydrogenation with NaBH(OAc)3 in methanol
[17] and finally treated with HCl/CH3OH to provide salts 7ceg.
Recently, we reported the identification of several new hits as
M2 inhibitors through the focused screening of a small primary
Abbreviations: M2, matrix-2 protein; SAR, structureeactivity relationship.
* Corresponding author. Guangzhou Institute of Biomedicine and Health, Chinese
Academy of Sciences, 190 Kai Yuan Avenue, Guangzhou Science Park, Guangzhou
510530, People’s Republic of China. Tel.: þ86 20 32015211; fax: þ86 20 32015299.
E-mail address: hu_wenhui@gibh.ac.cn (W. Hu).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.10.010

X. Zhao et al. / European Journal of Medicinal Chemistry 46 (2011) 52e57
53
Scheme 2. Synthesis of compounds 8aeg. (a) Substituted arylaldehyde, benzene,
piperidine, reflux, yield 64e90%.
Fig. 1. Reported M2 inhibitors.
might need a strategy other than the classic optimization process to
improve the activity. Because the Schiff base, like compound 8a,
was so easy to make from the condensation of compound 5 with
aldehydes, a series of compounds were rapidly synthesized and
screened (Table 2). Compound 8a shows a significantly improved
activity compared with the compounds in Table 1; it was found to
be three times more potent than amantadine.
The preparation of imines 8aeg is shown in Scheme 2. The
starting material 5 was condensed with various aromatic aldehydes
[18] to easily provide compounds 8aeg with a yield of 64e90%.
3. Results and discussion
We further optimized this series by placing diverse substituents
on the phenyl ring: 4-fluorophenyl substituted compound 8b was
3.1. Viral inhibition assay
the exact analog of 7g, but the data (16.3 mM vs. >200 mM) clearly
All the synthesized compounds were evaluated for their inhi-
bition of influenza A virus. The inhibitory activity was measured by
the survival of cells infected with the influenza A virus because
a potent M2 inhibitor can rescue the cell by inhibiting the repli-
cation of the virus. The results showed that the inhibition ability of
the compounds in Table 1 were decreased or even lost completely.
While all the compounds except for 8d in Table 2 were more active
than amantadine, 8e was found to be the most potent one at an
demonstrated that the C]N bond was much more favorable for
inhibition than a CeH bond. In addition, neither 4-methylphenyl
(8c) nor 4-N,N-dimethylphenyl (8d) increased the activity. As we
expected, 4-hydroxyphenyl exhibited highly potent inhibition
(0.088 mM), which was more than 200 times that of adamantan-
amine. The switch of hydroxyl group on the phenyl ring, such as
2-hydroxyphenyl (8f) and 3-hydroxyphenyl (8g), did not retain the
previously observed excellent inhibition, but these compounds
were still more active than amantadine.
inhibition level 240-fold higher than amantadine (IC50 ¼ 0.088
mM)
(for inhibition data, see Fig. 2).
All the secondary amines prepared here exerted decreased
activity, especially the aromatic analogs, which completely lost
their inhibition ability. However, all the imine compounds listed
here, except 8d, were more active than amantadine.
3.2. 3.2. Structureeactivity relationship (SAR) studies
The mechanism of M2 inhibitors is to block proton flow in the
M2 ion channel and thus inhibit viral replication [19,20]. In our
initial investigations, the amine functionality was recognized as the
pharmacophore and the alkyl ring was the scaffold. Therefore, we
decided to modify the primary amine of compound 5 while keeping
the bicyclic ring constant. In the beginning, the primary amine was
changed to a secondary amine (Table 1) by the addition of a diverse
set of alkyl groups, such as methyl (7a), ethyl (7b), isopropyl (7c),
cyclopropylmethyl (7d) and cyclohexylmethyl (7e). All of them
displayed activity lower than adamantanamine. The attachment of
aromatic rings, such as 4-hydroxylbenzyl and 4-fluorobenzyl
(7f and 7g), made this trend worse, as the compounds completely
lost their viral inhibition.
Table 1
In vitroa anti-influenza A virus activity of compounds 7aee
H
N
R
Compound
R
IC50
23.54
>200
>200
(
m
M)
7a
7b
7c
eCH3
eCH2CH3
eCH(CH3)2
The previous structureeactivity relationships of adamantan-
amine [4] showed that there was no significant difference between
the N-mono and N-dialkyl derivatives. This reminded us that we
C
H2
7d
7e
7f
28.48
43.68
OH
F
C
>200
H2
C
7g
>200
H2
Scheme 1. Synthesis of compounds 7aeg. (a) Acyl chloride, Et3N, rt, yield 90%; (b) (i)
LiAlH4, THF, rt, yield 60e66%; (ii) HCl, CH3OH, rt, yield 100%; (c) (i) aldehyde or ketone,
NaBH(OAc)3, CH3OH, rt, yield 30e78%; (ii) HCl, CH3OH, rt, yield 100%.
1
21.11
a
Using influenza A/Hong Kong/8/68 (H3N2) strain.

54
X. Zhao et al. / European Journal of Medicinal Chemistry 46 (2011) 52e57
Table 2
3.3. Plaque reduction assay
In vitroa anti-influenza A virus activity of compounds 8aeg
The inhibitory effect of compound 8e was further assessed in
a plaque reduction assay using influenza A viruses. The photos
in Fig. 3A show that plaque formation by wild-type influenza
virus (A/Hong Kong/8/68) was inhibited by amantadine 1 and
N
R
compound 8e at concentrations ranging from 0.01 to 10 mM. From
the results shown in Fig. 3B, compound 8e was an obviously potent
inhibitor.
Compound
R
IC50 (mM)
8a
8b
8c
8d
8e
8f
8g
1
Ph
Phe4-F
Phe4-CH3
Phe4-N(CH3)2
Phe4-OH
7.78
16.30
13.91
38.36
0.088
20.28
5.99
4. Conclusion
We have identified a highly potent anti-influenza A agent
through a general modification of hit 5 followed by a viral inhibition
assay. From the study, compound 8e was identified as the most
active inhibitor against wild-type influenza A virus. The IC50 value
Phe2-OH
Phe3-OH
21.11
a
Using influenza A/Hong Kong/8/68 (H3N2) strain.
for this compound was found to be 0.088 mM, which was nearly
240-fold more potent than amantadine in the same assay. Although
there was no inhibition of mutant virus observed by any of the
compounds listed here, compound 8e might be a novel chemical
probe for mechanism studies and further investigation of the M2
ion channel.
5. Experimental procedure
5.1. Chemistry
All commercially available compounds and solvents were
reagent grade and were used without further treatment unless
otherwise noted. Reactions were monitored by TLC using Qing Dao
Fig. 2. Inhibition efficiency of amantadine and 8e on A/Hong Kong/8/68.
Fig. 3. Reduction of plaque formation by A/Hong Kong/8/68 (H3N2, amantadine sensitive) in MDCK cells upon treatment with 1 and 8e. MDCK cells were infected with virus at
approximate 45 plaque forming units/well. (A) The effect of 1 and 8e on viral plaque formation. (B) Quantification of viral plaque formation following treatment with serial dilutions
of the compound.

X. Zhao et al. / European Journal of Medicinal Chemistry 46 (2011) 52e57
55
Hai Yang GF254 silica gel plates (5 ꢁ10 cm); zones were detected
visually under ultraviolet irradiation (254 nm) and by spraying
with an ethanol solution of 2,4-DNP or Ninhydrin or by being
fumed with iodine steam. Silica gel column chromatography was
performed on silica gel (200e300 mesh) from Qing Dao Hai Yang.
NMR spectra were recorded on a Bruker NMR AVANCE 400
(400 MHz) or a Bruker NMR AVANCE 500 (500 MHz), and nuclear
magnetism reagents were used as internal standards. Chemical
d ppm: 20.68, 23.32, 23.61, 27.97, 35.30, 37.37, 38.37, 41.59, 46.49,
47.78, 47.84, 169.27; ESI-MS: calculated for C12H21NO (M þ Hþ):
196.30, found: 196.1.
5.1.5. (1R,2R,3R,5S)-N,2,6,6-Tetramethylbicyclo[3.1.1]heptan-3-
amine hydrochloride (7a)
A solution of 6a (1 g, 4.73 mmol) in anhydrous THF (5 mL) was
added dropwise to
a stirred suspension of LiAlH4 (0.54 g,
shifts (
d
) were recorded in parts per million and coupling constants
14.2 mmol) in anhydrous THF (20 mL) at 0 ꢂC. The resulting solu-
tion was stirred for 10 h at reflux. The solution was then cooled
to 0 ꢂC and filtered following the portionwise addition of
Na2SO4$10H2O (5.0 g). The collected solids were washed with
CH2Cl2 (2 ꢁ 20 mL). The combined organics were evaporated to
dryness and treated with saturated HCl/CH3OH (20 mL) then
evaporated to dryness, and the insoluble material was washed with
diethyl ether (3 ꢁ 20 mL) to afford the corresponding hydrochlo-
(J) in hertz (Hz). MS data were measured on an Agilent MSD-1200
ESI-MS system.
5.1.1. General synthetic procedure for amines (method A; Scheme 1)
To a solution of (1R,2R,3R,5S)-(ꢀ)-isopinocampheylamine (1 g,
6.5 mmol) in CH3OH (20 mL), aldehyde or ketone (1.5 equiv,
9.8 mmol) was added. The reaction mixture was stirred at rt for 1 h,
followed by addition of sodium triacetoxyborohydride (5.5 g,
26 mmol). The reaction mixture was then stirred for 10 h and
quenched by the addition of H2O and extracted with ethyl acetate
(3 ꢁ 20 mL). The combined organic layers were washed two times
with brine, dried (Na2SO4) and evaporated to dryness. The residue
was purified by silica gel column chromatography and treated with
saturated HCl/CH3OH (20 mL), then evaporated to dryness. The
insoluble material was washed with diethyl ether (3 ꢁ 20 mL) to
afford the secondary amines as the corresponding hydrochloride
salts.
ride as a white powder. Yield: 60%; 1HNMR (400 MHz, D2O)
d ppm:
0.82 (d, 1H, J ¼ 10.4 Hz), 0.87 (s, 3H), 1.10 (d, 3H, J ¼ 7.2 Hz), 1.15
(s, 3H), 1.68e1.73 (m, 1H), 1.81e1.84 (m, 1H), 1.95e2.03 (m, 2H),
2.35e2.46 (m, 2H), 2.64 (s, 3H), 3.38e3.40 (m, 1H); 13CNMR
(125 MHz, D2O)
d ppm: 20.09, 22.62, 26.66, 30.77, 30.99, 32.77,
37.91, 40.37, 40.72, 46.93, 57.84; ESI-MS: calculated for C11H21
N
(M þ Hþ): 168.29, found: 168.2.
5.1.6. (1R,2R,3R,5S)-N-Ethyl-2,6,6-trimethylbicyclo[3.1.1]heptan-
3-amine hydrochloride (7b)
Synthesis of 7b was accomplished using the same procedure
described for 7a. Compound 7b was obtained as a white powder.
5.1.2. General synthetic procedure for Schiff bases (method B;
Scheme 2)
Yield: 66%; 1HNMR (400 MHz, DMSO-d6)
d ppm: 0.92 (s, 3H), 1.14
To a solution of (1R,2R,3R,5S)-(ꢀ)-isopinocampheylamine (1 g,
6.5 mmol) in benzene (20 mL), the aromatic aldehydes (4.35 mmol)
and one drop of piperidine were added. The solution was heated at
reflux with a DeaneStark trap condenser until no further water
appeared. Then, the reaction solution was concentrated and puri-
fied by flash silica gel column chromatography to obtain the desired
Schiff bases.
(d, 3H, J ¼ 7.2 Hz), 1.20 (s, 3H), 1.25 (t, 3H, J ¼ 6.8 Hz), 1.31 (d, 1H,
J ¼ 10.0 Hz), 1.77e1.85 (m, 2H), 1.94 (br s, 1H), 2.08 (t, 1H,
J ¼ 6.0 Hz), 2.25e2.34 (m, 2H), 2.97 (br s, 2H), 3.35 (br s, 1H), 8.68
(br s, 1H), 8.96 (br s, 1H); 13CNMR (125 MHz, D2O)
d ppm: 10.66,
20.10, 22.61, 26.62, 31.40, 32.65, 37.91, 40.37, 40.72, 40.96, 46.96,
56.32; ESI-MS: calculated for C12H23N (M þ Hþ): 182.32, found:
182.2.
5.1.3. Ethyl ((1R,2R,3R,5S)-2,6,6-trimethylbicyclo[3.1.1]heptan-3-yl)
carbamate (6a)
5.1.7. (1R,2R,3R,5S)-N-Isopropyl-2,6,6-trimethylbicyclo[3.1.1]
heptan-3-amine (7c)
A solution of methyl chloroformate (0.92 g, 9.8 mmol) in CH2Cl2
Method A; yellow powder; yield: 71%; 1HNMR (400 MHz, CDCl3)
was added dropwise to
a
solution of (1R,2R,3R,5S)-(ꢀ)-iso-
d
ppm: 0.95 (s, 3H), 1.23 (s, 3H), 1.24 (d, 3H, J ¼ 7.2 Hz), 1.27 (s, 1H),
pinocampheylamine (1 g, 6.5 mmol) and Et3N (1.03 mL) in CH2Cl2
(20 mL) in an ice bath. The reaction mixture was stirred for 1 h,
quenched by the addition of H2O and extracted with ethyl acetate
(3 ꢁ 20 mL). The combined organic layers were washed two times
with brine, dried (Na2SO4) and evaporated to dryness. The residue
was purified by silica gel column chromatography. The product was
a white powder and was directly used in the next step. Yield: 90%;
1.44 (d, 3H, J ¼ 6.4 Hz), 1.58 (d, 3H, J ¼ 6.4 Hz), 1.69 (d, 1H,
J ¼ 10.4 Hz), 1.81 (t, 1H, J ¼ 5.6 Hz), 2.00 (t, 1H, J ¼ 2.8 Hz), 2.16e2.22
(m, 1H), 2.29e2.34 (m, 2H), 2.42e2.45 (m, 1H), 3.36e3.41 (m, 1H);
13CNMR (125 MHz, CDCl3)
d ppm: 18.51, 19.91, 21.16, 23.71, 27.48,
31.52, 32.52, 38.99, 40.51, 41.00, 47.67, 48.23, 54.10; ESI-MS:
calculated for C12H25N (M þ Hþ): 196.34, found: 196.1.
1HNMR (400 MHz, CDCl3)
d
ppm: 0.82 (d, 1H, J ¼ 10.0 Hz), 1.02
5.1.8. (1R,2R,3R,5S)-N-(Cyclopropylmethyl)-2,6,6-trimethylbicyclo
[3.1.1]heptan-3-amine hydrochloride (7d)
(s, 3H), 1.12 (d, 3H, J ¼ 6.8 Hz), 1.21 (s, 3H), 1.52e1.57 (m, 1H),
1.73e1.81 (m, 3H), 1.92e1.94 (m, 1H), 2.36e2.41 (m, 1H), 2.58 (m,
Method A; white powder; yield: 73%; 1HNMR (400 MHz, D2O)
ppm: 0.24e0.30 (m, 2H), 0.60 (d, 2H, J ¼ 8.4 Hz), 0.86 (s, 3H), 0.88
1H), 3.66 (s, 3H), 3.97 (br s, 1H); 13CNMR (125 MHz, CDCl3)
d
ppm:
d
20.63, 23.28, 27.96, 35.22, 37.45, 38.33, 41.57, 46.36, 47.72, 49.78,
51.86, 156.82; ESI-MS: calculated for C12H21NO2 (M þ Hþ): 212.30,
found: 212.2.
(s, 1H), 0.98e1.02 (m, 1H), 1.09 (d, 3H, J ¼ 7.2 Hz), 1.14 (s, 3H),
1.66e1.72 (m, 1H), 1.80e1.84 (m, 1H), 1.93e1.96 (m, 1H), 2.00e2.04
(m, 1H), 2.33e2.37 (m, 1H), 2.40e2.47 (m, 1H), 2.82e2.88 (m, 1H),
2.93e2.98 (m, 1H), 3.43e3.48 (m, 1H); 13CNMR (125 MHz, D2O)
5.1.4. N-((1R,2R,3R,5S)-2,6,6-Trimethylbicyclo[3.1.1]heptan-3-yl)
acetamide (6b)
d ppm: 0.97, 1.24, 4.44, 17.77, 20.29, 24.31, 29.12, 30.28, 35.60, 38.05,
38.28, 44.61, 48.38, 54.00; ESI-MS: calculated for C14H25
N
Synthesis of 6b was accomplished using the same procedure
described for 6a. Compound 6b was obtained as a white powder.
(M þ Hþ): 208.35, found: 208.2.
Yield: 90%; 1HNMR (400 MHz, CDCl3)
d
ppm: 0.81 (d, 1H,
5.1.9. (1R,2R,3R,5S)-N-Cyclohexyl-2,6,6-trimethylbicyclo[3.1.1]
J ¼ 10.0 Hz), 1.04 (s, 3H), 1.10 (d, 3H, J ¼ 7.2 Hz), 1.21 (s, 3H),
1.47e1.52 (m, 1H), 1.65 (br s, 1H), 1.73 (t, 1H, J ¼ 4.0 Hz), 1.82 (t, 1H,
J ¼ 8.0 Hz), 1.94e1.95 (m, 1H), 1.98 (s, 3H), 2.39e2.42 (m, 1H), 2.60
(m, 1H), 4.25 (m, 1H), 5.37 (br s, 1H); 13CNMR (125 MHz, CDCl3)
heptan-3-amine hydrochloride (7e)
Method A; white powder; yield: 30%; 1HNMR (400 MHz, D2O)
d
ppm: 0.88 (s, 4H), 1.08 (d, 4H, J ¼ 7.2 Hz), 1.15 (s, 3H), 1.21e1.32 (m,
4H), 1.59 (d, 1H, J ¼ 12 Hz), 1.70e1.84 (m, 4H), 1.96e2.00 (m, 3H),

56
X. Zhao et al. / European Journal of Medicinal Chemistry 46 (2011) 52e57
2.11 (br s, 1H), 2.35e2.48 (m, 2H), 3.15 (br s, 1H), 3.52e3.58 (m, 1H);
13CNMR (125 MHz, D2O)
ppm: 19.86, 22.64, 23.95, 24.01, 24.52,
26.68, 28.50, 29.75, 31.49, 32.70, 37.99, 40.49, 40.89, 47.00, 52.99,
54.62; ESI-MS: calculated for C16H29N (M þ Hþ): 236.41, found:
236.2.
5.1.15. (1R,2R,3R,5S,E)-N-(4-(Dimethylamino)benzylidene)-2,6,6-
trimethylbicyclo[3.1.1]heptan-3-amine (8d)
d
Method B; white powder; yield: 84%; 1HNMR (400 MHz, CDCl3)
d
ppm: 0.99 (d, 3H, J ¼ 7.2 Hz), 1.08 (s, 3H), 1.26 (s, 3H), 1.28 (s, 1H),
1.84e1.99 (m, 3H), 2.12 (t, 1H, J ¼ 7.6 Hz), 2.27e2.28 (m, 1H),
2.38e2.41 (m, 1H), 3.00 (s, 6H), 3.41e3.44 (m, 1H), 6.71 (d, 2H,
J ¼ 9.2 Hz), 7.61 (d, 2H, J ¼ 8.8 Hz), 8.06 (s, 1H); 13CNMR (125 MHz,
5.1.10. 4-((((1R,2R,3R,5S)-2,6,6-Trimethylbicyclo[3.1.1]heptan-3-yl)
amino)methyl)-phenol hydrochloride (7f)
CDCl3)
d ppm: 19.87, 23.56, 28.11 34.06, 36.20, 38.92, 40.28, 41.83,
Method A; white powder; yield: 62%; 1HNMR (400 MHz, DMSO-
43,68, 47.70, 70.36, 111.71, 124.92, 129.37, 151.82, 157.84; ESI-MS:
d6)
d
ppm: 0.84 (s, 3H), 1.06 (d, 3H, J ¼ 7.2 Hz), 1.19 (s, 3H), 1.38
calculated for C19H28N2 (M þ Hþ): 285.44, found: 285.2.
(d, 1H, J ¼ 9.6 Hz), 1.77 (t, 1H, J ¼ 4.8 Hz), 1.94e1.98 (m, 2H), 2.14
(t, 1H, J ¼ 6 Hz), 2.27e2.32 (m, 2H), 3.23 (br d, 1H), 3.96 (d, 1H,
J ¼ 12.8 Hz), 4.08 (d, 1H, J ¼ 8.8 Hz), 6.80 (d, 2H, J ¼ 8.4 Hz),
7.40 (d, 2H, J ¼ 8.4 Hz), 9.00 (br s, 1H), 9.43 (br s, 1H), 9.75 (br s,
5.1.16. 4-((E)-(((1R,2R,3R,5S)-2,6,6-Trimethylbicyclo[3.1.1]heptan-
3-yl)imino)methyl)phenol (8e)
Method B; yellow powder; yield: 90%; 1HNMR (400 MHz, CDCl3)
1H); 13CNMR (125 MHz, D2O)
d
ppm: 19.88, 22.64, 26.62, 31.38,
d
ppm: 1.00 (d, 3H, J ¼ 7.2 Hz), 1.04 (s, 3H), 1.25 (s, 3H), 1.28 (t, 1H,
32.69, 37.96, 40.46, 40.68, 46.93, 48.65, 55.90, 115.93, 122.59,
131.62, 156.54; ESI-MS: calculated for C17H25NO (M þ Hþ): 260.39,
found: 260.2.
J ¼ 3.6 Hz), 1.83 (t, 1H, J ¼ 4.8 Hz), 1.95e2.00 (m, 2H), 2.16e2.19 (m,
1H), 2.28e2.38 (m, 1H), 3.55e3.61 (m, 1H), 6.61 (d, 2H, J ¼ 8.8 Hz),
7.46 (d, 2H, J ¼ 8.4 Hz), 8.11 (s, 1H); 13CNMR (125 MHz, CDCl3)
d
ppm: 19.77, 23.58, 28.09, 34.01, 35.45, 38.98, 41.66, 42.93, 47.50,
5.1.11. (1R,2R,3R,5S)-N-(4-Fluorobenzyl)-2,6,6-trimethylbicyclo
[3.1.1]heptan-3-amine hydrochloride (7g)
70.47, 116.12, 125.40, 130.48, 160.63, 161.22; ESI-MS: calculated for
C17H23NO (M þ Hþ): 258.37, found: 258.2.
Method A; white powder; yield: 67.1%; 1HNMR (400 MHz, D2O)
d
ppm: 0.84 (s, 3H), 0.88 (d, 1H, J ¼ 10.4 Hz), 1.05 (d, 3H, J ¼ 7.2 Hz),
5.1.17. 2-((E)-(((1R,2R,3R,5S)-2,6,6-Trimethylbicyclo[3.1.1]heptan-
3-yl)imino)methyl)phenol (8f)
1.15 (s, 3H), 1.78e1.85 (m, 2H), 1.96e1.99 (m, 2H), 2.00e2.10 (m,
2H), 2.34e2.38 (m, 1H), 2.40e2.54 (m,1H), 3.41e3.47 (m, 1H), 4.15
(q, 2H, J ¼ 13.2 Hz), 7.12e7.17 (m, 2H), 7.42e7.46 (m, 2H); 13CNMR
Method B; yellow oil; yield: 90%; 1HNMR (400 MHz, CDCl3)
d
ppm: 1.06 (d, 3H, J ¼ 7.2 Hz), 1.08 (s, 3H), 1.17 (d, 1H, J ¼ 10.0 Hz),
(125 MHz, D2O)
d
ppm: 19.94, 22.65, 26.61, 31.41, 32.66, 37.97,
1.28 (s, 3H), 1.88e2.05 (m, 3H), 2.14e2.17 (m, 1H), 2.37e2.48 (m,
2H), 3.46e3.51 (m, 1H), 6.86e6.90 (m, 1H), 6.97 (d, 1H, J ¼ 8.0 Hz),
7.24e7.32 (m, 2H), 8.25 (s, 1H), 13.70 (br s, 1H); 13CNMR (125 MHz,
40.45, 40.64, 46.94, 48.49, 56,37, 115.99, 116.16, 126.84, 131.91
(d, JCeF ¼ 8.9); ESI-MS: calculated for C17H24NF (M þ Hþ): 262.38,
found: 262.1.
CDCl3) d ppm: 19.78, 23.40, 27.79, 34.02, 36.34, 38.50, 41.40, 44.34,
47.29, 68.60, 116.80, 118.32, 118.67, 130.93, 131.74, 161.14, 161.61;
5.1.12. (1R,2R,3R,5S,E)-N-Benzylidene-2,6,6-trimethylbicyclo[3.1.1]
heptan-3-amine (8a)
ESI-MS: calculated for C17H23NO (M þ Hþ): 258.37, found: 258.2.
Method B; yellow oil; yield: 64%; 1HNMR (400 MHz, CDCl3)
5.1.18. 3-((E)-(((1R,2R,3R,5S)-2,6,6-Trimethylbicyclo[3.1.1]heptan-
3-yl)imino)methyl)phenol (8g)
d
ppm: 1.08 (d, 3H, J ¼ 7.2 Hz), 1.16 (s, 3H), 1.34 (s, 3H), 1.37 (d, 1H,
J ¼ 9.6 Hz), 1.94e1.97 (m, 1H), 2.02e2.08 (m, 2H), 2.21e2.25 (m,
1H), 2.33e2.36 (m, 1H), 2.47e2.50 (m, 1H), 3.55e3.59 (m, 1H),
7.43e7.46 (m, 3H), 7.82e7.84 (m, 2H), 8.23 (s, 1H); 13CNMR
Method B; yellow powder; yield: 72%; 1HNMR (400 MHz, CDCl3)
d
ppm: 0.99 (d, 3H, J ¼ 7.2 Hz), 1.07 (s, 3H), 1.22 (s, 1H), 1.26 (s, 3H),
1.86 (t, 1H, J ¼ 4 Hz), 1.94e1.98 (m, 2H), 2.15 (t, 1H, J ¼ 6.8 Hz),
2.25e2.38 (m, 2H), 3.51e3.57 (m, 1H), 6.86 (q, 1H, J ¼ 3.2 Hz), 7.19
(q, 2H, J ¼ 3.2 Hz), 7.27 (s, 1H), 8.12 (s, 1H); 13CNMR (125 MHz,
(100 MHz, CDCl3)
d ppm: 19.99, 23.66, 28.17, 33.94, 36.06, 38.97,
41.88, 43.58, 47.78, 70.39, 128.20, 128.53, 130.21, 136.81, 157.60; ESI-
MS: calculated for C17H23N (M þ Hþ): 242.37, found: 242.2.
CDCl3) d ppm: 19.78, 23.57, 28.06, 33.92, 35.56, 38.95, 41.68, 43.02,
47.54, 70.58, 114.67, 118.46, 120.85, 129.78, 137.06, 156.67, 159.67;
5.1.13. (1R,2R,3R,5S,E)-N-(4-Fluorobenzylidene)-2,6,6-
trimethylbicyclo[3.1.1]heptan-3-amine (8b)
ESI-MS: calculated for C17H23NO (M þ Hþ): 258.37, found: 258.2.
Method B; yellow oil; yield: 78%; 1HNMR (400 MHz, CDCl3)
5.2. Viral inhibition assay
d
ppm: 1.02 (d, 3H, J ¼ 7.2 Hz), 1.10 (s, 3H), 1.28 (s, 3H), 1.31 (s, 1H),
1.88e2.02 (m, 3H), 2.13e2.16 (m, 1H), 2.27e2.30 (m, 1H), 2.41e2.44
(m, 1H), 3.47e3.52 (m, 1H), 7.07e7.11 (m, 2H), 7.74e7.78 (m, 2H),
MDCK cells were grown to confluence in 96-well microtiter
plates, the medium was removed, and the cells were covered with
8.14 (s, 1H); 13CNMR (125 MHz, CDCl3)
d
ppm: 19.85, 23.55,
50
mL of medium containing various amounts of amantadineeHCl
28.02, 33.85, 35.92, 38.86, 41.70, 43.46, 47.56, 70.20, 115.40
(d, JCeF ¼ 21.6 Hz), 129.87 (d, JCeF ¼ 8.6 Hz), 132.90 (d, JCeF ¼ 2.6 Hz),
156.17, 163.03, 165.02; ESI-MS: calculated for C17H22NF (M þ Hþ):
260.36, found: 260.1.
or synthesized compounds in the presence of 1 mg/mL TPCK and
0.3% BSA. The plates were then incubated at 37 ꢂC for 30 min. Fifty
microliters, equal to approximately 0.01 MOI of A/Hong Kong/8/68
(H3N2) virus was then added to the plates. After incubation in 5%
CO2 at 37 ꢂC for 72 h,10
mL of CCK-8 reagent was added to each well,
5.1.14. (1R,2R,3R,5S,E)-2,6,6-Trimethyl-N-(4-methylbenzylidene)
bicyclo[3.1.1]heptan-3-amine (8c)
and the mixture was incubated for 3 h. The A450 was then
measured with an UVstar-Microplates Synergy HT. Data were
analyzed using GraphPad Prism Demo.
Method B; yellow oil; yield: 64%; 1HNMR (400 MHz, CDCl3)
d
ppm: 1.04 (d, 3H, J ¼ 7.2 Hz), 1.14 (s, 3H), 1.31 (s, 3H), 1.33 (d, 1H,
J ¼ 9.6 Hz), 1.91e2.05 (m, 3H), 2.17e2.20 (m, 1H), 2.30(t, 1H,
J ¼ 10.4 Hz), 2.33 (s, 3H), 2.35e2.47 (m, 1H), 3.49e3.54 (m, 1H), 7.23
(d, 2H, J ¼ 8 Hz), 7.68 (d, 2H, J ¼ 8 Hz), 8.18 (s, 1H); 13CNMR
5.3. Plaque reduction assay
A monolayer of MDCK cells was infected with 0.01 MOI A/Hong
Kong/8/68 virus for 1 h at 37 ꢂC. The inoculums were then
removed, and the cells were washed twice with phosphate-
buffered saline (PBS). The cells were then overlaid with 1% agar
DMEM-containing amantadine or the synthesized compounds in
(125 MHz, CDCl3)
d ppm: 19.91, 21.48, 23.61, 28.11, 33.95,
36.00, 38.93, 41.78, 43.52, 47.64, 70.38, 128.11, 129.22, 134.06,
140.30, 157.65; ESI-MS: calculated for C18H25N (M þ Hþ): 256.40,
found: 256.2.

X. Zhao et al. / European Journal of Medicinal Chemistry 46 (2011) 52e57
57
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