Synthesis and antiviral activity of substituted bisaryl amide compounds as novel influenza virus inhibitors

Article abstract of DOI:10.1016/j.ejmech.2012.07.008

The influenza virus is a persistent cause of mortality and morbidity on an annual basis and thus presents itself as an important target for pharmaceutical investigation. In this work, substituted bisaryl amide compounds were found to be a new class of potential anti-influenza agents, and a series of substituted bisaryl amide compounds were synthesised and evaluated for their anti-influenza virus activities. The analysis of the results produced a preliminary structure-activity relationship study (SAR). Compounds 1a, 1g, 1h, 1j, 1l and 1n exhibited clear antiviral activities against the influenza A (A/Guangdong Luohu/219/2006, H1N1) virus with 50% inhibitory concentrations (IC₅₀) for virus growth ranging from 12.5 to 59.0 μM. Specifically, compound 1j also possessed antiviral activity against both oseltamivir-resistant influenza (A/Jinnan/15/2009) virus and influenza B (B/Jifang/13/97) virus with IC ₅₀ values of 9.2 μM and 21.4 μM, respectively. Compound 1j is thus worth further investigation as an anti-influenza virus candidate.

Full text of DOI:10.1016/j.ejmech.2012.07.008

European Journal of Medicinal Chemistry 55 (2012) 117e124
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and antiviral activity of substituted bisaryl amide compounds as novel
influenza virus inhibitors
1
1
***
Lan-hu Hao , Yan-ping Li , Wei-ying He, Hui-qiang Wang, Guang-zhi Shan, Jian-dong Jiang
,
**
*
Yu-huan Li , Zhuo-rong Li
Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China
a r t i c l e i n f o
a b s t r a c t
Article history:
The influenza virus is a persistent cause of mortality and morbidity on an annual basis and thus presents
itself as an important target for pharmaceutical investigation. In this work, substituted bisaryl amide
compounds were found to be a new class of potential anti-influenza agents, and a series of substituted
bisaryl amide compounds were synthesised and evaluated for their anti-influenza virus activities. The
analysis of the results produced a preliminary structureeactivity relationship study (SAR). Compounds
1a, 1g, 1h, 1j, 1l and 1n exhibited clear antiviral activities against the influenza A (A/Guangdong Luohu/
219/2006, H1N1) virus with 50% inhibitory concentrations (IC50) for virus growth ranging from 12.5 to
Received 6 April 2012
Received in revised form
4 July 2012
Accepted 4 July 2012
Available online 14 July 2012
Keywords:
Influenza virus
Inhibitor
Bisaryl amide
Synthesis
59.0
influenza (A/Jinnan/15/2009) virus and influenza B (B/Jifang/13/97) virus with IC50 values of 9.2
and 21.4 M, respectively. Compound 1j is thus worth further investigation as an anti-influenza virus
candidate.
mM. Specifically, compound 1j also possessed antiviral activity against both oseltamivir-resistant
m
M
m
Ó 2012 Published by Elsevier Masson SAS.
1. Introduction
In our previous study, a novel class of bisaryl amide compounds
were confirmed as APOBEC3G (human apolipoprotein B mRNA-
editing enzyme catalytic polypeptide-like 3G, hA3G) stabilizers
[12,13]. The hA3G protein was initially identified as a host cellular
restriction factor of HIV-1 by Sheehy et al. in 2002 [14], and it was
also found to be active against other reverse transcription viruses,
such as SIV, MLV and HBV [13,15]. Although there has been no
report revealing the relationship between hA3G and the influenza
virus until now, we tested some hA3G-stabilizing compounds that
were available in our laboratory for whether they exhibited any
anti-influenza virus activity. Surprisingly, small molecule LH-905
(4-methoxy-N-phenyl-3-propionamidobenzamide, Fig. 1) was
found to be active against the influenza virus in vitro. However, LH-
Influenza is a type of acute respiratory disease that is caused by
the influenza virus. The typical clinical symptoms include acute
fever, body aches, significant fatigue and mild respiratory symp-
toms. The influenza virus mutates easily and often causes flu
pandemics [1]. For example, the influenza pandemic that occurred
in 2009 in Mexico swept around the globe and caused severe
mortality and morbidity [2,3].
Despite the extensive efforts that are made to control the
infection of the influenza virus, only two classes of drugs are
currently available for clinical use: antagonists of the viral M2
protein, such as amantadine and its derivative rimantadine, and
viral neuraminidase inhibitors, such as zanamivir and oseltamivir
[4e6]. However, both of these classes are limited in their efficacy
due to drug resistance and side effects [7e10]. Recently, Hama et al.
found that oseltamivir could acerbate the illness and occasionally
even lead to death [11]. Therefore, it is necessary to develop novel,
effective antiviral agents against the influenza virus.
905 exhibited only modest antiviral activity with an IC50 of 43.0
mM.
To discover more active candidates for use as anti-influenza virus
agents, we synthesised a series of substituted bisaryl amide
analogues of LH-905 in this study. The preliminary SAR was also
discussed here.
2. Results and discussion
* Corresponding author. Tel.: þ86 10 63027185; fax: þ86 10 63017302.
** Corresponding author. Tel.: þ86 10 63010984; fax: þ86 10 63017302.
*** Corresponding author.
2.1. Chemistry
The analogues mainly differed in their chemical structures at the
following positions of LH-905: 1) replacement of substituents and
ring structure of benzene ring B; 2) the amide linker between the
E-mail addresses: jiang.jdong@163.com (J.-d. Jiang), yuhuanlibj@126.com
(Y.-h. Li), l-z-r@263.net (Z.-r. Li).
1
These authors made equal contributions to this work.
0223-5234/$ e see front matter Ó 2012 Published by Elsevier Masson SAS.
http://dx.doi.org/10.1016/j.ejmech.2012.07.008

118
L.-h. Hao et al. / European Journal of Medicinal Chemistry 55 (2012) 117e124
chloride (9-M2). These chlorides were reacted with various aryl-
O
amines to yield the desired products 1neq and 17 (Scheme 2).
Compound 21 was synthesised using the route shown in
Scheme 3. First, starting material 18 was reacted with propionyl
chloride, using triethylamine (TEA) as a base, to yield intermediate
19, and the nitro group in intermediate 19 was subsequently
reduced to an amino group to yield intermediate 20. Then, inter-
mediate 20 was reacted with benzoyl chloride to afford final
product 21.
HN
A
H3CO
H
N
B
O
Fig. 1. The chemical structure of LH-905.
two benzene rings; and 3) substituents on benzene ring A, espe-
cially the amide side-chain at the C3-position.
2.2. Evaluation of anti-influenza viral activity of substituted bisaryl
amide compounds
LH-905 was synthesised by condensing 3-propionamide-4-
methoxybenzoic acid (1-M1) with aniline using diisopropylcarbo-
diimide (DIC) as a coupling reagent and N-hydroxybenzotriazole
(HOBt) as an activating reagent [16], as shown in Scheme 1. The
intermediates 1-M1e1-M4, 2-M and 3-M were obtained from the
acylation of the amino groups or hydroxy group of starting mate-
rials 1, 2 and 3, respectively, with different chlorides. Next, these
intermediates were condensed with diverse primary amines to
yield products 1ae1k, 10 and 11 (Scheme 1a). The intermediates 1-
M5 and 4-Me7-M were produced by the direct condensation of
starting materials 1 and 4e7 with aniline, respectively, and their
amino groups at the C3-position were then acylated with cyclo-
The antiviral activities of substituted bisaryl amide compounds
against influenza A/Guangdong Luohu/219/2006 (H1N1) were
initially evaluated by cytopathic effects (CPE) using MDCK cells
with Tamiflu and Ribavirin (RBV) as positive drugs. Among the 25
novel synthesized compounds summarized in Table 1, twenty
compounds showed antiviral activity against influenza virus A.
Although all active compounds showed less activity than positive
control drugs Tamiflu (IC50 ¼ 2.0
m
M) and RBV (IC50 ¼ 8.8
mM),
compounds 1a, 1g, 1h, 1j, 1l and 1n exhibited considerable or even
higher antiviral activities when compared with LH-905
(IC50 ¼ 43.0
in range of 12.5e59.0
(IC50 ¼ 12.5 M) which have distinct substituents from other
compounds at C3-position of ring A showed the highest activity.
mM). The IC50 for virus growth of these compounds was
pentane carboxylic acid or
a-fluoropropionic acid to yield the
m
M. Compounds 1h (IC50 ¼ 17.3 M) and 1j
m
desired products 1l, 1m and 13e16 (Scheme 1b). Following the first
step of Scheme 1b, compound 12 was directly synthesised from 4-
methoxybenzoic acid (8) as the starting material.
m
The substituent was a-chloropropionamide and benzamide for 1h
The coupling reagent failed to yield the desired substituted
bisaryl compounds when substituted benzenesulphonic acid was
used as a starting material or when the weakly nucleophilic aryl-
amine was used with benzoic acid for the condensation. The
conjunction of the two benzene rings in these cases was achieved
through sulphonyl chloride or carbonyl chloride intermediates.
Starting materials 1 and 9 were first reacted with propionyl chlo-
ride and isobutyryl chloride, respectively, to yield intermediates 1-
M1, 1-M6 and 9-M1, which were then converted into the corre-
sponding benzoyl chlorides (1-M7 and 1-M8) and benzosulphonyl
and 1j, respectively.
Nine compounds with selectivity indices (SI ¼ TC50/IC50) > 10
against influenza virus A (A/Guangdong Luohu/219/2006 strain)
were further evaluated for their potential antiviral activity against
influenza B and oseltamivir resistant influenza A virus strain.
Tamiflu and RBV were used as control drugs, and the CPE method
was used in these assays. As summarized in Table 2, compounds 1g,
1j and 1n exhibited antiviral activity against influenza B (B/Jifang/
13/97) virus. Compound 1j (IC50 ¼ 21.4
mM) possessed of higher
activity comparing with positive control RBV against influenza B
a
R2
R2
Z
Z
Z
i
ii
R1
R1
R1
OH
OH
Y
R3
O
O
O
1-M1: Z=NH, R2= COC2H5
1-M2: Z=NH, R2=COCH(Cl)CH3
1-M3: Z=NH, R2=COCH3
1-M4: Z=NH, R2= COC6H5
2-M: Z=NH, R2= COC2H5
3-M: Z=O, R2=COC2H5
1: Z=NH2, R1= 4-OCH3
2: Z=NH2, R1= 4-Cl
LH-905: Z=NH, R1= 4-OCH3, R2= COC2H5, Y= NH, R3= phenyl
1a: Z=NH, R1= 4-OCH3, R2= COC2H5, Y= NH, R3= 4-methylphenyl
1b: Z=NH, R1= 4-OCH3, R2= COC2H5, Y= NH, R3= 4-bromophenyl
1c: Z=NH, R1= 4-OCH3, R2= COC2H5, Y= NH, R3= 3-chlorophenyl
1d: Z=NH, R1= 4-OCH3, R2= COC2H5, Y= NH, R3= 3-methylthiophenyl
1e: Z=NH, R1= 4-OCH3, R2= COC2H5, Y= NH, R3= cyclohexyl
1f: Z=NH, R1= 4-OCH3, R2= COC2H5, Y= NH, R3= cyclobutyl
1g: Z=NH, R1= 4-OCH3, R2= COC2H5, Y= NH, R3= thiazol-2-yl
1h: Z=NH, R1= 4-OCH3, R2= COCH(Cl)CH3, Y= NH, R3= phenyl
1i: Z=NH, R1= 4-OCH3, R2= COCH3, Y= NH, R3= pheny
1j: Z=NH, R1= 4-OCH3, R2= COC6H5, Y= NH, R3= phenyl
1k: Z=NH, R1= 4-OCH3, R2= COC2H5, Y= O, R3= phenyl
10: Z=NH, R1= 4-Cl, R2= COC2H5, Y= NH, R3= phenyl
11: Z=O, R1= 4-OCH3, R2= COC2H5, Y= NH, R3= phenyl
3: Z=OH, R1= 4-OCH3
b
R2
Z
Z
Z
ii
iii
R1
R1
R1
H
N
OH
Y
R3
R3
O
O
O
1: Z=NH2, R1= 4-OCH3
4: Z=NH2, R1= 2-OCH3
5: Z=NH2, R1= 5-OCH3
6: Z=NH2, R1= 6-OCH3
7: Z=NH2, R1= H
1l: Z=NH, R1= 4-OCH3, R2= cyclopentanecarboxyl, Y= NH, R3= phenyl
1m: Z=NH, R1= 4-OCH3, R2= COCH(F)CH3, Y= NH, R3= phenyl
13: Z=NH, R1= 2-OCH3, R2= COCH(F)CH3, Y= NH, R3= phenyl
14: Z=NH, R1= 5-OCH3, R2= COCH(F)CH3, Y= NH, R3= phenyl
15: Z=NH, R1= 6-OCH3, R2= COCH(F)CH3, Y= NH, R3= phenyl
16: Z=NH, R1= H, R2= COCH(F)CH3, Y= NH, R3= phenyl
1-M5: R3= phenyl
4-M: R3= phenyl
5-M: R3= phenyl
6-M: R3= phenyl
7-M: R3= phenyl
12: R3=phenyl
8: Z=H, R1= 4-OCH3
Scheme 1. The synthetic route to compounds 1aem and 10e16. Reagents and conditions: (i) acyl chloride, TEA, CH2Cl2; (ii) arylamine or cycloalkylamine, DIC, HOBt, CH2Cl2; (iii)
cyclopentane carboxylic acid or -fluoropropionic acid, DIC, HOBt, CH2Cl2
a

L.-h. Hao et al. / European Journal of Medicinal Chemistry 55 (2012) 117e124
R1
119
R1
R1
HN
NH2
HN
HN
O
O
O
O
ii
iii
i
OH
OH
Cl
Y
X
X
X
X
R6
1: X=CO
1-M1: X=CO, R1=COC2H5
1-M7: X=CO, R1=COC2H5
1n: X=CO, R1= COC2H5, Y= NH, R6= 4-nitrophenyl
1o: X= CO, R1= COC2H5, Y= NH, R6= pyridin-2-yl
1p: X= CO, R1= COC2H5, Y= N(CH3), R6= phenyl
1q: X= CO, R1=COCH(CH3)2,Y= N(CH3), R6= phenyl
1-M6: X=CO, R1=COCH(CH3)2
1-M8: X=CO, R1=COCH(CH3)2
9-M2: X=SO2, R1=COC2H5
9: X=SO2
9-M1: X=SO2, R1=COC2H5
17: X=SO2, R1=COC2H5, Y=NH, R6=phenyl
Scheme 2. The synthetic route to compounds 1neq and 17. Reagents and conditions: (i) acyl chloride, TEA, CH2Cl2; (ii) SOCl2; (iii) arylamine, TEA, CH2Cl2.
virus. Moreover, compounds 1g and 1j also displayed antiviral
activity against influenza A oseltamivir resistant strain. Especially,
the most effective compound 1j exhibited several folds higher
activity than positive control drug RBV, the IC50 and SI values of 1j
for oseltamivir resistant influenza (A/Jinnan/15/2009 strain) virus
IC50 ¼ 48.4
mM) led to decreasing of antiviral activity. Noted that
sulfonamide linker between two aryl rings (17) only induced
a weakly decrease of antiviral activity comparing with carbonyl
amide linker (LH-905). The interchange of CO and NH of amide
linker (21, IC50 ¼ 74.5
mM) weakened the anti-influenza viral
were 9.2
mM and 21.0, respectively.
activity compared with LH-905. So the NH at C1-position of ring B
and aromatic ring B was also beneficial for anti-influenza virus
activity.
2.3. Structureeantiviral activity relationships
Although we still are uncertainty about the exact action mech-
anism, some classical drug targets of influenza virus, for example
viral haemagglutinin, neuraminidase and M2 protein, have been
ruled out for target discovery in our studies. The results indicated
that these compounds have no inhibitory effect on viral haemag-
glutinin, influenza neuraminidase and M2 protein at the tested
The replacement of propionamido side-chain at C3-position in
ring A with acetamido (1i),
a-fluoropropionamido (1m) or pro-
pionyloxy group (11) led to several folds decreased antiviral
activity compared with LH-905. However, the most effective
antiviral activity of corresponding compounds was exhibited
concentrations (over 58
mM). The best compound 1j definitely
when propionamido (LH-905, IC50 ¼ 43.0
with
-chloropropionamido (1h, IC50 ¼ 17.3
group (1j, IC50 ¼ 12.5 M). The removal of this propionamido group
m
M)) group was replaced
showed activity against oseltamivir resistant influenza virus strain
even if it exhibited weaker activity comparing with control drugs
Tamiflu and RBV against influenza A virus, and the activity of 1j
a
m
M) or benzamido
m
at the C3-position in ring A (12) resulted in loss of antiviral activity.
Therefore, the results indicated that substitution at the C3-position
of ring A is required for activity. And from the stronger activity of
compounds 1h and 1j than other compounds, we estimated that
more lipophilic substituents at the C3-position of ring A led to
higher biological activity. Replacement of methoxy group at C4-
position of ring A with chloro atom (10) led to the significantly
decrease of inhibitory activity against influenza virus comparing
with LH-905. The removal of methoxyl from C4-position (16,
(IC50 ¼ 9.2
m
M) against oseltamivir resistant virus strain was much
higher than that of RBV (IC50 ¼ 35.3
mM). Hence, we would like to
conclude that the action mechanism of these substituted bisaryl
amides is different from that of market available drugs.
3. Conclusion
Substituted bisaryl amide compounds were found to be a new
class of potential anti-influenza agents, and a total of 25 novel
bisaryl amide compounds were synthesised and screened for their
antiviral activity against influenza A virus in this paper. Moreover,
the data were analysed to produce a preliminary SAR. Ten
compounds exhibited obvious antiviral activity against tested
influenza A virus strain. The nine compounds with SI > 10 were
further tested for antiviral activity against influenza B and resistant
influenza A virus. Compounds 1g and 1j exhibited additional anti-
viral activity against both influenza B and oseltamivir resistant
influenza A virus strain. Worthy of note, compound 1j with ben-
zamide substituted at C3-position of ring A exhibited the highest
antiviral activity against three influenza virus strains among all test
compounds, which makes compound 1j worthy to be exploited as
a novel candidate against influenza viruses. Without doubt, the
further exploration is needed to produce more potent compounds
and to clear the detailed action mechanism of this novel class of
biaryl amide compounds against influenza virus. These relative
studies are already in progress in our research team.
IC50 ¼ 143.5
m
M) of ring A showed a slightly increased activity than
M). The antiviral activities of
similar compound 1m (IC50 ¼ 210.8
m
compounds 1m, 13 and 15 indicated that altering the position of
methoxy group from C4-position to other position in ring A may
decrease inhibitory activity against influenza (A/Luohu/219/2006,
H1N1) virus. Thus, the substituent at the C4-position of ring A
seems be important to antiviral activity. However, we can’t
conclude whether the methoxyl was the best substituent at C4-
position of ring
compounds containing various substituted B benzene rings indi-
cated no specific rules between structure and antiviral activity. The
two more effective compounds (IC50 is 23.7 mM and 26.3 mM for 1a
A or not, based on current results. The
and 1n, respectively) possessed of opposite chemical structure
characteristics of substituent in ring B. Replacement of benzene
ring B with saturated cycle (1e, 1f) caused decreased antiviral
activity of corresponding compounds. The amide linker between
ring A and B was methylated (1p, IC50 ¼ 100.8
mM) or replaced
with ester (1k, IC50 ¼ 222.9
m
M) or sulfonamide linker (17,
O
O
O
NH2
HN
HN
HN
O
O
O
iii
O
ii
i
O
NO2
NO2
NH2
N
H
18
20
21
19
Scheme 3. The synthetic route to compound 21. Reagents and conditions: (i) propionyl chloride, TEA, CH2Cl2; (ii) H2, Pd/C; (iii) benzoyl chloride, TEA, CH2Cl2.

120
L.-h. Hao et al. / European Journal of Medicinal Chemistry 55 (2012) 117e124
Table 1
chromatography (HPLC method for all compounds: detector
wavelength: 260 nm, column temperature: 25 ꢀC, column: ODS
Anti-influenza (A/Guangdong Luohu/219/2006 strain) virus activities and cytotox-
icities of the synthesised substituted bisaryl amide compounds.a
(5
m
m, 4.6 ꢁ 250 mm), mobile phase: methanol:water ¼ 75:25, flow
Compd.
TC50
(
m
M)
IC50
(
m
M)
SI
>15.6
rate ¼ 1 mL/min).
LH-905
1a
1b
1c
1d
1e
1f
1g
1h
1i
1j
1k
1l
1m
1n
1o
1p
1q
10
11
12
13
14
15
16
17
21
Tamiflu
RBV
>670.4
162.3
90.4
602.3
324.4
43.0
23.7
>17.6
>66.9
>63.0
73.0
59.0
35.0
17.3
112.7
12.5
6.8
4.2. Synthesis
_
_
_
4.2.1. General procedure for the synthesis of 1-M1e1-M4, 1-M6, 2-
M, 3-M and 9-M1
510.3
6.9
12.3
18.7
11.6
6.2
15.4
2.1
12.1
3.0
11.7
3.0
4.4
4.2
3.5
>724.2
>655.0
200.3
>703.5
192.5
463.6
458.7
>632.3
307.1
>668.6
444.2
491.9
530.3
518.5
>880.0
>632.3
490.7
271.6
578.5
4.2.1.1. 4-Methoxy-3-propionamidobenzoic acid (1-M1). A total of
2.5 mL (30 mmol) of propionyl chloride was added dropwise to
a solution of 5.0 g (30 mmol) of 1 and 4.3 mL (30 mmol) of TEA in
dichloromethane at 0 ꢀC. The mixture was then stirred at room
temperature until the starting material was completely dis-
appeared. The solvent was evaporated in vacuo to produce a sticky
residue that was extracted with dichloromethane and 0.5 N HCl
(25 mL). The organic phase was concentrated, and the residue was
purified by column chromatography (silica gel) eluted with petro-
leum ether and ethyl acetate (v:v ¼ 2:1) to yield the desired
compound as a white solid (yield: 76%). Mp: 372e373 ꢀC. 1H NMR
222.9
37.9
210.8
26.3
222.9
100.8
116.7
152.7
172.9
>293.4
>210.8
210.8
>70.2
143.5
48.4
3.0
(400 MHz, DMSO-d6)
d (ppm): 12.59 (1H, br s, COOH), 9.12 (1H, s,
_
_
NH), 8.56 (1H, s, PhH), 7.68 (1H, d, J ¼ 8.8 Hz, PhH), 7.11 (1H, d,
J ¼ 8.8 Hz, PhH), 3.89 (3H, s, OCH3), 2.42 (2H, q, J ¼ 7.6 Hz, CH2), 1.07
(3H, t, J ¼ 7.6 Hz, CH3). ESI-MS (m/z): 224 (M þ H)þ.
2.3
_
4.0
12.4
5.2
1535.1
541.7
>598.6
387.3
3070.2
4766.8
4.2.1.2. 3-(2-Chloropropanamido)-4-methoxybenzoic acid (1-M2).
Compound 1-M2 was synthesised using a method similar to that of
74.5
2.0
8.8
1-M1 and was isolated as
a
white solid (yield: 66%). Mp:
(ppm): 12.59 (1H, br s,
414e416 ꢀC. 1H NMR (400 MHz, DMSO-d6)
d
a
All data were average values from three independent assays.
COOH), 9.12 (1H, s, NH), 8.56 (1H, s, PhH), 7.68 (1H, d, J ¼ 8.8 Hz,
PhH), 7.11 (1H, d, J ¼ 8.8 Hz, PhH), 4.99 (1H, q, J ¼ 6.4 Hz, CH), 3.90
(3H, s, OCH3), 1.62 (3H, d, J ¼ 6.4 Hz, CH3). ESI-MS (m/z): 258
(M þ H)þ.
4. Experimental protocol
4.1. Chemistry
4.2.1.3. 3-Acetamido-4-methoxybenzoic acid (1-M3). Compound 1-
M3 was synthesised using a method similar to that of 1-M1 and
was isolated as a white solid (yield: 75%). Mp: 331e332 ꢀC. 1H NMR
1H NMR spectra were recorded in DMSO-d6 or CDCl3 on a Varian
Inova 400 or 500 MHz spectrometer. Chemical shifts were reported
in parts per million relative to tetramethylsilane as the internal
standard. The melting points were determined with an X6 micro-
scope melting point apparatus and were uncorrected. Electrospray
ionisation (ESI) mass spectra and high-resolution mass spectra
(HRMS) were obtained on an MDS SCIEX Q-Trap mass spectrometer.
The area normalization purities of the tested compounds were
>95% as determined using analytical high-performance liquid
(400 MHz, DMSO-d6)
d (ppm): 12.59 (1H, br s, COOH), 9.12 (1H, s,
NH), 8.56 (1H, s, PhH), 7.68 (1H, d, J ¼ 8.8 Hz, PhH), 7.11 (1H, d,
J ¼ 8.8 Hz, PhH), 3.90 (3H, s, OCH3), 1.96 (3H, s, CH3CO). ESI-MS (m/
z): 210 (M þ H)þ.
4.2.1.4. 3-Benzamido-4-methoxybenzoic acid (1-M4). Compound 1-
M4 was synthesised using a method similar to that of 1-M1 and was
isolated as a white solid (yield: 69%). Mp: 391e393 ꢀC. 1H NMR
(400 MHz, DMSO-d6)
d (ppm): 12.59 (1H, br s, COOH), 10.07 (1H, s,
Table 2
NH), 9.16 (1H, s, PhH), 7.81 (1H, d, J ¼ 6.0 Hz, PhH), 7.78 (2H, d,
J ¼ 7.6 Hz, PhH), 7.33 (2H, m, PhH), 7.20 (1H, d, J ¼ 8.8 Hz, PhH), 7.07
(1H, m, PhH), 3.90 (3H, s, OCH3). ESI-MS (m/z): 272 (M þ H)þ.
Anti-influenza (B/Jifang/13/97 and A/Jinnan/15/2009) virus activities and cytotox-
icities of selected synthesised bisaryl amide compounds.a
Compd. Influenza A (strain A/luohu/
Influenza B
(strain B/jifang/
13/97)
Resistant
219/2006)
influenza A
(strain A/Jinnan/
15/2009)
4.2.1.5. 3-Isobutyramido-4-methoxybenzoic acid (1-M6). Compound
1-M5 was synthesised using a method similar to that of 1-M1 and
was isolated as a white solid (yield: 72%). Mp: 379e381 ꢀC. 1H NMR
TC50
(
mM) IC50
(m
M) SI
IC50
>223.5
6.84 >23.7
(
m
M) SI
IC50 (mM) SI
(400 MHz, DMSO-d6)
d (ppm): 12.59 (1H, br s, COOH), 9.12 (1H, s,
LH-905 >670.4
1a
1f
1g
1h
1j
1l
1n
17
43.0
23.7
59.0
35.0
17.3
12.5
37.9
26.3
16.2
2.0
15.5
_
_
>223.5
>23.7
>241.4
93.8
>22.3
9.2
>65.7
>21.6
>66.7
>2436.6
_
_
_
NH), 8.56 (1H, d, J ¼ 8.8 Hz, PhH), 7.68 (1H, m, PhH), 7.11 (1H, d,
J ¼ 8.8 Hz, PhH), 3.90 (3H, s, OCH3), 2.71 (1H, m, CH), 1.04 (6H, d,
J ¼ 6.4 Hz, CH3). ESI-MS (m/z): 238 (M þ H)þ.
162.3
>724.2
>655.0
200.3
192.5
458.7
12.2
>241.4
126.1
>22.3
21.4
>65.7
64.8
>66.7
ND
45.5
_
5.2
_
9.0
_
4.7
_
18.7
11.7
15.4
12.1
11.6
12.4
6.9
_
20.9
4.2.1.6. 4-Chloro-3-propionamidobenzoic acid (2-M). Compound 2-
M was synthesised using a method similar to that of 1-M1 and was
isolated as a white solid (yield: 70%). Mp: 387e389 ꢀC. 1H NMR
_
_
_
307.1
>200
(400 MHz, DMSO-d6)
d (ppm): 12.59 (1H, br s, COOH), 9.12 (1H, s,
Tamiflu 3070.2
RBV 4766.8
1535.1
541.7
ND
103.9
e
8.8
35.3 135.0
NH), 8.56 (1H, d, J ¼ 8.8 Hz, PhH), 7.68 (1H, m, PhH), 7.11 (1H, d,
J ¼ 8.8 Hz, PhH), 2.42 (2H, q, J ¼ 7.6 Hz, CH2), 1.07 (3H, t, J ¼ 7.6 Hz,
CH3). ESI-MS (m/z): 228 (M þ H)þ.
ND: not determined.
a
All data were average values from three independent assays.

L.-h. Hao et al. / European Journal of Medicinal Chemistry 55 (2012) 117e124
121
4.2.1.7. 4-Methoxy-3-(propionyloxy)benzoic acid (3-M). Compound
3-M was synthesised using a method similar to that of 1-M1 and
was isolated as a white solid (yield: 72%). Mp: 241e243 ꢀC. 1H NMR
(2H, m, PhH), 7.36 (1H, t, J ¼ 6.4 Hz, PhH), 7.12e7.17 (2H, m, PhH),
3.91 (3H, s, OCH3), 2.4 (2H, q, J ¼ 6.0 Hz, CH2), 1.07 (3H, t, J ¼ 6.0 Hz,
CH3). HRMS-ESI (m/z): calculated for C17H18ClN2O3 (M þ H)þ:
333.10200; measured: 333.10059.
(400 MHz, DMSO-d6)
d (ppm): 12.74 (1H, br s, COOH), 8.56 (1H, s,
PhH), 7.68 (2H, m, PhH), 3.83 (3H, s, OCH3), 2.42 (2H, q, J ¼ 7.6 Hz,
CH2), 1.07 (3H, t, J ¼ 7.6 Hz, CH3). ESI-MS (m/z): 225 (M þ H)þ.
4.2.2.5. 4-Methoxy-N-(3-(methylthio)phenyl)-3-propionamidobenz-
amide (1d). Compound1dwas synthesisedusinga methodsimilarto
that of LH-905 and was isolated (eluent: petroleum ether:ethyl
acetate ¼ 4:1) as a white solid (yield: 45%, HPLC purity: 97.55%). Mp:
4.2.1.8. 4-Methoxy-3-propionamidobenzenesulphonic acid (9-M1).
Compound 9-M1 was synthesised using a method similar to that of
1-M1 and was isolated as
a
white solid (yield: 47%). Mp:
(ppm): 12.21 (1H, br s,
159e161 ꢀC.1H NMR (400 MHz, DMSO-d6)
d (ppm): 8.90 (1H, s, PhH),
379e381 ꢀC. 1H NMR (400 MHz, DMSO-d6)
d
7.93 (1H, s, NH), 7.83 (1H, s, NH), 7.78 (1H, dd, J1 ¼ 8.8 Hz, J2 ¼ 2.4 Hz,
PhH), 7.66 (1H, s, PhH), 7.37 (1H, d, J ¼ 8.4 Hz, PhH), 7.25 (1H, t,
J ¼ 8.4 Hz, PhH), 7.03 (1H, d, J ¼ 7.6 Hz, PhH), 7.00 (1H, d, J ¼ 8.8 Hz,
PhH), 3.96 (3H, s, OCH3), 2.55(3H, s, SCH3), 2.46(2H, q, J ¼ 7.6Hz, CH2),
1.28 (3H, t, J ¼ 7.6 Hz, CH3). HRMS-ESI (m/z): calculated for
C18H21N2O3S (M þ H)þ: 345.12674; measured: 345.12674.
COOH), 8.93 (1H,s, NH), 8.02 (1H, s, PhH), 7.23 (2H, m, PhH), 3.90
(3H, s, OCH3), 2.42 (2H, q, J ¼ 7.6 Hz, CH2), 1.07 (3H, t, J ¼ 7.6 Hz,
CH3). ESI-MS (m/z): 260 (M þ H)þ.
4.2.2. General procedure for the synthesis of LH-905, 1ae1k, 10
and 11
4.2.2.1. 4-Methoxy-N-phenyl-3-propionamidobenzamide (LH-905).
A total of 0.17 mL (0.18 mmol) of DIC and 0.24 g (0.18 mmol) of HOBt
were added to a solution of 0.20 g (0.12 mmol) of 1-M1 in
dichloromethane. The reaction mixture was stirred for 0.5 h at
room temperature, and 0.13 mL (0.14 mmol) of aniline was then
added to the mixture. After 3 h, 0.5 N NaOH was added to the
solution. The lower layer was removed, and 0.5 N HCl was added
until the aqueous solution was neutral. The dichloromethane layer
was washed three times with 10 mL of water, and then MgSO4 was
added. After 2 h, the mixture was filtered, and the solvent was
removed. The residue was purified by column chromatography
(silica gel) eluted with petroleum ether and ethyl acetate (v:
v ¼ 3:1) to yield the desired compound as a white solid (yield: 72%,
HPLC purity: 99.16%). Mp: 178e179 ꢀC. 1H NMR (400 MHz, DMSO-
4.2.2.6. N-cyclohexyl-4-methoxy-3-propionamidobenzamide
(1e). Compound 1e was synthesised using a method similar to that
of LH-905 and was isolated (eluent: petroleum ether:ethyl
acetate ¼ 3:1) as a white solid (yield: 67%, HPLC purity: 98.36%).
Mp: 192e194 ꢀC. 1H NMR (400 MHz, DMSO-d6)
d (ppm): 9.08 (1H, s,
NH), 8.31 (1H, s, NH), 8.00 (1H, d, J ¼ 8.0 Hz, PhH), 7.59 (1H, m, PhH),
7.05 (1H, d, J ¼ 8.4 Hz, PhH), 3.85 (3H, s, OCH3), 3.73 (1H, m, CH),
2.38 (2H, q, J ¼ 7.6 Hz, CH2),1.72 (4H, m, CH2),1.26 (4H, m, CH2),1.07
(3H, t, J ¼ 7.6 Hz, CH3), 0.99 (m, 2H, CH2). HRMS-ESI (m/z): calcu-
lated for C17H25N2O3 (M þ H)þ: 305.18593; measured: 305.18597.
4.2.2.7. N-Cyclobutyl-4-methoxy-3-propionamidobenzamide
(1f). Compound 1f was synthesised using a method similar to that
of LH-905 and was isolated (eluent: petroleum ether:ethyl
acetate ¼ 3:1) as a white solid (yield: 63%, HPLC purity: 99.42%).
d6)
d (ppm): 10.07 (1H, s, NH), 9.16 (1H, s, NH), 8.50 (1H, s, PhH),
7.81 (1H, d, J ¼ 6.0 Hz, PhH), 7.78 (2H, d, J ¼ 7.6 Hz, PhH), 7.33 (2H,
m, PhH), 7.20 (1H, d, J ¼ 8.8 Hz, PhH), 7.07 (1H, m, PhH), 3.90 (3H, s,
OCH3), 2.43 (2H, q, J ¼ 7.6 Hz, CH2), 1.16 (3H, t, J ¼ 7.6 Hz, CH3).
HRMS-ESI (m/z): calculated for C17H18N2O3 (M)þ: 298.12702;
measured: 298.13119.
Mp: 183e185 ꢀC. 1H NMR (400 MHz, DMSO-d6)
d (ppm): 9.08 (1H, s,
NH), 8.42 (1H, d, J ¼ 7.6 Hz, PhH), 8.33 (1H, s, NH), 7.60 (1H, d,
J ¼ 8.4 Hz, PhH), 7.06 (1H, d, J ¼ 7.6 Hz, PhH), 4.37 (1H, m), 3.85 (3H,
s, OCH3), 2.36 (2H, q, J ¼ 7.6 Hz, CH2), 2.17 (2H, m), 2.04 (2H, m), 1.63
(2H, m), 1.06 (3H, t, J ¼ 7.6 Hz, CH3). HRMS-ESI (m/z): calculated for
C15H19N2O3 (M ꢂ H)ꢂ: 275.13983; measured: 275.13902.
4.2.2.2. 4-Methoxy-3-propionamido-N-p-tolylbenzamide (1a). Co-
mpound 1a was synthesised using a method similar to that of LH-
905 and was isolated (eluent: petroleum ether:ethyl acetate ¼ 3:1)
as a white solid (yield: 76%, HPLC purity: 99.61%). Mp: 201e203 ꢀC.
4.2.2.8. 4-Methoxy-3-propionamido-N-(thiazol-2-yl)benzamide
(1g). Compound 1g was synthesised using a method similar to that
of LH-905 and was isolated (eluent: petroleum ether:ethyl
acetate ¼ 3:1) as a white solid (yield: 76%, HPLC purity: 99.33%).
1H NMR (500 MHz, DMSO-d6)
d (ppm): 10.00 (1H, s, NH), 9.16 (1H, s,
NH), 8.49 (1H, s, PhH), 7.73 (3H, m, PhH), 7.14 (3H, m, PhH), 3.90
(3H, s, OCH3), 2.42 (2H, q, J ¼ 8.0 Hz, CH2), 2.26 (3H, s, Ph-CH3), 1.07
(3H, t, J ¼ 7.5 Hz, CH3). HRMS-ESI (m/z): calcd for C18H21N2O3
(M þ H)þ: 313.15522; measured: 313.15349.
Mp: 183e185 ꢀC. 1H NMR (400 MHz, DMSO-d6)
d (ppm): 12.45 (1H,
s, NH), 9.18 (1H, s, NH), 8.65 (1H, s, PhH), 7.92 (1H, m, PhH), 7.52
(1H, d, J ¼ 7.6 Hz, PhH), 7.24 (1H, d, J ¼ 7.6 Hz, PhH), 7.16 (1H, d,
J ¼ 8.4 Hz, PhH), 3.91 (3H, s, OCH3), 2.43 (2H, q, J ¼ 7.6 Hz, CH2), 1.07
(3H, t, J ¼ 7.6 Hz, CH3). HRMS-ESI (m/z): calculated for C14H15N3O3S
(M þ H)þ: 306.08988; measured: 306.09069.
4.2.2.3. N-(4-bromophenyl)-4-methoxy-3-propionamidobenzamide
(1b). Compound 1bwassynthesisedusing a method similartothatof
LH-905 and was isolated (eluent: petroleum ether:ethyl
acetate ¼ 3:1) as a white solid (yield: 42%, HPLC purity: 96.77%). Mp:
4.2.2.9. 3-(2-Chloropropanamido)-4-methoxy-N-phenylbenzamide
(1h). Compound 1h was synthesised using a method similar to that
of LH-905 and was isolated (eluent: petroleum ether:ethyl
acetate ¼ 3:1) as a white solid (yield: 69%, HPLC purity: 97.98%). Mp:
172e174 ꢀC. 1H NMR (400 MHz, DMSO-d6)
d (ppm): 10.19 (1H, s, NH),
9.17 (1H, s, NH), 8.50 (1H, s, PhH), 7.71e7.74 (1H, dd, J1 ¼ 8.4 Hz,
J2 ¼ 2.0 Hz, PhH), 7.72 (2H, d, J ¼ 8.88 Hz, PhH), 7.50 (2H, d, J ¼ 8.8 Hz,
PhH), 7.14 (1H, d, J ¼ 8.4 Hz, PhH), 3.90 (3H, s, OCH3), 2.40 (2H, q,
J¼ 7.6Hz,CH2),1.07(3H, t, J¼ 7.6Hz,CH3). HRMS-ESI(m/z):calculated
for C17H18BrN2O3 (M þ H)þ: 377.05003; measured: 377.05008.
161e163 ꢀC.1H NMR (400 MHz, DMSO-d6)
d (ppm): 10.07 (1H, s, NH),
9.16 (1H, s, NH), 8.50 (1H, s, PhH), 7.81 (1H, d, J ¼ 6.0 Hz, PhH), 7.78
(2H, d, J ¼ 7.6 Hz, PhH), 7.33 (2H, m, PhH), 7.20 (1H, d, J ¼ 8.8 Hz,
PhH), 7.07 (1H, m, PhH), 4.99 (1H, q, J ¼ 6.4 Hz, CH), 3.90 (3H, s,
OCH3), 1.62 (3H, d, J ¼ 6.4 Hz, CH3). HRMS-ESI (m/z): calculated for
C17H16N2O3Cl (M ꢂ H)ꢂ: 331.08477; measured: 331.08440.
4.2.2.4. N-(3-chlorophenyl)-4-methoxy-3-propionamidobenzamide
(1c). Compound 1c was synthesised using a method similar to that
of LH-905 and was isolated (eluent: petroleum ether:ethyl
acetate ¼ 3:1) as a white solid (yield: 60%, HPLC purity: 97.38%).
4.2.2.10. 3-Acetamido-4-methoxy-N-phenylbenzamide (1i). Compo-
und 1i was synthesised using a method similar to that of LH-905
and was isolated (eluent: petroleum ether:ethyl acetate ¼ 3:1) as
a white solid (yield: 75%, HPLC purity: 98.25%). Mp: 171e173 ꢀC. 1H
Mp: 133e134 ꢀC. 1H NMR (400 MHz, DMSO-d6)
d (ppm): 10.24 (1H,
s, NH), 9.18 (1H, s, NH), 8.51 (1H, s, PhH), 7.94 (1H, s, PhH), 7.68e7.74

122
L.-h. Hao et al. / European Journal of Medicinal Chemistry 55 (2012) 117e124
NMR (400 MHz, DMSO-d6)
d
(ppm): 10.07 (1H, s, NH), 9.16 (1H, s,
4.2.4. General procedure for the synthesis of 1ne1q and 17
NH), 8.50 (1H, s, PhH), 7.81 (1H, d, J ¼ 6.0 Hz, PhH), 7.78 (2H, d,
J ¼ 7.6 Hz, PhH), 7.33 (2H, m, PhH), 7.20 (1H, d, J ¼ 8.8 Hz, PhH), 7.07
(1H, m, PhH), 3.90 (3H, s, OCH3), 2.09 (3H, s, CH3). HRMS-ESI (m/z):
4.2.4.1. 4-Methoxy-3-propionamido-N-(pyridin-2-yl)benzamide
(1o). A total of 0.50
g (2.2 mmol) of 1-M7 was added to
a dichloromethane solution of 0.21 g (2.2 mmol) pyridin-2-amine
and 0.30 mL (2.2 mmol) of TEA at 0 ꢀC. The mixture was reacted
at room temperature until the starting material was completely
disappeared. The solvent was then evaporated in vacuo to produce
a sticky residue that was then extracted with dichloromethane and
0.5 N HCl (25 mL). The organic phase was concentrated, and the
solid was purified by column chromatography on silica gel (eluent:
calculated for C16H15N2O3 (M
283.10772.
ꢂ
H)ꢂ: 283.10811; measured:
4.2.2.11. 3-Benzamido-4-methoxy-N-phenylbenzamide (1j). Compo-
und 1j was synthesised using a method similar to that of LH-905
and was isolated (eluent: petroleum ether:ethyl acetate ¼ 3:1) as
a white solid (yield: 68%, HPLC purity: 99.72%). Mp: 196e197 ꢀC. 1H
petroleum ether:ethyl acetate
compound as a white solid (yield: 51%, HPLC purity: 99.54%). Mp:
184e186 ꢀC. 1H NMR (400 MHz, DMSO-d6)
(ppm): 12.45 (1H, s,
¼
2:1) to yield the desired
NMR (400 MHz, DMSO-d6)
d (ppm): 10.13 (1H, s, NH), 9.62 (1H, s,
NH), 8.35 (1H, s, PhH), 7.99 (2H, d, J ¼ 6.8 Hz, PhH), 7.87 (1H, d,
J ¼ 6.4 Hz, PhH), 7.77 (2H, d, J ¼ 8.0 Hz, PhH), 7.60 (3H, m, PhH), 7.33
(2H, m, PhH), 7.21 (1H, d, J ¼ 8.8 Hz, PhH), 7.09 (1H,m, PhH), 3.90
(3H, s, OCH3). HRMS-ESI (m/z): calculated for C21H17N2O3 (M ꢂ H)ꢂ:
345.12320; measured: 345.12337.
d
NH), 9.18 (1H, s, NH), 8.65 (1H, s, PhH), 7.92 (1H,m, PhH), 7.53 (1H,
d, J ¼ 7.6 Hz, PhH)), 7.24 (1H, d, J ¼ 7.6 Hz, PhH), 7.16 (1H, d,
J ¼ 8.4 Hz, PhH), 6.91 (1H, m, PhH), 6.53 (1H, d, J ¼ 7.6 Hz, PhH), 3.90
(3H, s, OCH3), 2.41 (2H, q, J ¼ 7.6 Hz, CH2), 1.07 (3H, t, J ¼ 7.6 Hz,
CH3). HRMS-ESI (m/z): calculated for C16H18N3O3 (M þ H)þ:
300.13425; measured: 300.13427.
4.2.2.12. Phenyl 4-methoxy-3-propionamidobenzoate (1k). Compo-
und 1k was synthesised using a method similar to that of LH-905
and was isolated (eluent: petroleum ether:ethyl acetate ¼ 3:1) as
a white solid (yield: 53%, HPLC purity: 98.02%). Mp: 147e149 ꢀC. 1H
4.2.4.2. 4-Methoxy-N-(4-nitrophenyl)-3-propionamidobenzamide
(1n). Compound 1n was synthesised using a method similar to that
of 1o and was isolated (eluent: petroleum ether:ethyl acetate ¼ 3:1)
as a white solid (yield: 53%, HPLC purity: 98.18%). Mp: 154e156 ꢀC.1H
NMR (400 MHz, DMSO-d6)
d (ppm): 10.07 (1H, s, NH), 9.16 (1H, s,
PhH), 7.81 (1H, d, J ¼ 6.0 Hz, PhH), 7.78 (2H, d, J ¼ 7.6 Hz, PhH), 7.33
(2H,m, PhH), 7.20 (1H, d, J ¼ 8.8 Hz, PhH), 7.07 (1H, m, PhH), 4.00
(3H, s, OCH3), 2.43 (2H, q, J ¼ 7.6 Hz, CH2), 1.07 (3H, t, J ¼ 7.6 Hz,
NMR (500 MHz, DMSO-d6) d (ppm): 9.99 (1H, s, NH), 9.16 (1H, s, NH),
8.49 (1H, s, PhH), 8.25 (2H, m, PhH), 8.05 (2H, m, PhH), 7.78 (1H, m,
PhH), 7.19(1H, d, J ¼ 8.5 Hz, PhH), 3.90 (3H, s, OCH3), 2.42 (2H, q,
J ¼ 8.0 Hz, CH2), 1.07 (3H, t, J ¼ 7.5 Hz, CH3). HRMS-ESI (m/z): calcu-
lated for C17H18N3O5 (M þ H)þ: 344.12403; measured: 344.12410.
CH3). HRMS-ESI (m/z): calculated for C17H18NO4 (M
300.12358; measured: 300.12299.
þ
H)þ:
4.2.2.13. 4-Chloro-N-phenyl-3-propionamidobenzamide (10). Com-
pound 10 was synthesised using a method similar to that of LH-905
and was isolated (eluent: petroleum ether:ethyl acetate ¼ 4:1) as
a white solid (yield: 53%, HPLC purity: 98.21%). Mp: 147e149 ꢀC. 1H
4.2.4.3. 4-Methoxy-N-methyl-N-phenyl-3-propionamidobenzamide
(1p). Compound 1p was synthesised using a method similar to that
of 1o and was isolated (eluent: petroleum ether:ethyl
acetate ¼ 3:1) as a white solid (yield: 54%, HPLC purity: 96.09%).
NMR (400 MHz, DMSO-d6)
d (ppm): 9.87 (1H, s, NH), 9.16 (1H, s,
NH), 8.50 (1H, s, PhH), 7.81 (1H, d, J ¼ 6.0 Hz, PhH), 7.78 (2H, d,
J ¼ 7.6 Hz, PhH), 7.33 (2H, m, PhH), 7.20 (1H, d, J ¼ 8.8 Hz, PhH), 7.07
(1H, m, PhH), 2.43 (2H, q, J ¼ 7.6 Hz, CH2),1.16 (3H, t, J ¼ 7.6 Hz, CH3).
HRMS-ESI (m/z): calculated for C16H16ClN2O2 (M þ H)þ: 303.08950;
measured: 303.08948.
Mp: 143e145 ꢀC. 1H NMR (500 MHz, CDCl3)
d (ppm): 8.93 (1H, s,
NH), 8.07 (1H, s, PhH), 7.27 (2H, m, PhH), 7.15 (3H, m, PhH), 6.84
(2H, m, PhH), 3.90 (3H, s, OCH3), 3.30 (3H, s, NCH3), 2.34 (2H, q,
J ¼ 8.0 Hz, CH2), 1.03 (3H, t, J ¼ 8.0 Hz, CH3). HRMS-ESI (m/z):
calculated for C18H21N2O3 (M
313.15467.
þ
H)þ: 313.15461; measured:
4.2.2.14. 2-Methoxy-5-(phenylcarbamoyl)phenyl propionate (11). Com-
pound 11 was synthesised using a method similar to that of LH-905 and
was isolated (eluent: petroleum ether:ethyl acetate ¼ 4:1) as a white
solid (yield: 56%, HPLC purity: 99.31%). Mp: 173e175 ꢀC. 1H NMR
4.2.4.4. 3-Isobutyramido-4-methoxy-N-methyl-N-phenylbenzamide
(1q). Compound 1q was synthesised using a method similar to that
of 1o and was isolated (eluent: petroleum ether:ethyl
acetate ¼ 3:1) as a white solid (yield: 56%, HPLC purity: 99.15%).
(400 MHz, DMSO-d6)
d
(ppm): 10.10 (1H, s, NH), 7.93 (1H, d, J ¼ 6.4 Hz,
PhH), 7.75 (3H, m, PhH), 7.35 (2H, m, PhH), 7.27 (1H, d, J ¼ 8.4 Hz, PhH),
7.09 (1H, m, PhH), 3.85 (3H, s, OCH3), 2.28 (2H, q, J ¼ 7.6 Hz, CH2), 1.07
(3H, t, J ¼ 7.6 Hz, CH3). HRMS-ESI (m/z): calculated for C17H18NO4
(M þ H)þ: 300.12303; measured: 300.12303.
Mp: 150e151 ꢀC. 1H NMR (400 MHz, CDCl3)
d (ppm): 8.89 (1H, s,
NH), 8.05 (1H, s, PhH), 7.27 (2H, m, PhH), 7.15 (3H, m, PhH), 6.79
(2H, m, PhH), 3.90 (3H, s, OCH3), 3.30 (3H, s, NCH3), 2.71 (1H, m,
CH), 1.04 (6H, d, J ¼ 6.4 Hz, CH3). HRMS-ESI (m/z): calculated for
C19H23N2O3 (M þ H)þ: 327.16943; measured: 327.17032.
4.2.3. General procedure for the synthesis of 1-M7, 1-M8 and 9-M2
4.2.3.1. 4-Methoxy-3-propionamidobenzoyl chloride (1-M7). SOCl2
was added to a flask containing 5.0 g (22 mmol) of 1-M1 until the
white solid was completely dissolved. After the starting material
had completely disappeared, the excess SOCl2 was removed,
producing a brown oil that was used in the next step without
further purification.
4.2.4.5. N-(2-methoxy-5-(N-phenylsulphamoyl)phenyl)propiona-
mide (17). Compound 17 was synthesised using a method similar
to that of 1o and was isolated (eluent: petroleum ether:ethyl
acetate ¼ 3:1) as a white solid (yield: 47%, HPLC purity: 97.97%).
Mp: 195e197 ꢀC. 1H NMR (400 MHz, DMSO-d6)
d (ppm): 10.07 (1H,
s, NH), 8.53 (1H, s, NH), 8.17 (1H, s, PhH), 7.78 (2H, d, J ¼ 7.6 Hz,
PhH), 7.33 (2H, m, PhH), 7.81 (2H, m, PhH), 7.20 (1H, d, J ¼ 8.8 Hz,
PhH), 3.90 (3H, s, OCH3), 2.44 (2H, q, J ¼ 7.6 Hz, CH2), 1.07 (3H, t,
J ¼ 7.6 Hz, CH3). HRMS-ESI (m/z): calculated for C16H18N2O4S (M)þ:
334.09956; measured: 334.09873.
4.2.3.2. 3-Isobutyramido-4-methoxybenzoyl chloride (1-M8). Com-
pound 1-M8 was synthesised using a method similar to that of 1-M7
and was isolated as a brown oil (yield: 47%).
4.2.3.3. 4-Methoxy-3-propionamidobenzene-1-sulphonyl
(9-M2). Compound 9-M2 was synthesised using a method similar
to that of 1-M7 and was isolated as a brown oil (yield: 47%).
chloride
4.2.5. General procedure for the synthesis of 1-M5, 4-Me7-M and 12
4.2.5.1. 3-Amino-4-methoxy-N-phenylbenzamide (1-M5). A total of
0.17 mL (0.18 mmol) of DIC and 0.24 g (0.18 mmol) of HOBt were

L.-h. Hao et al. / European Journal of Medicinal Chemistry 55 (2012) 117e124
123
added to a dichloromethane solution of 0.20 g (0.12 mmol) of 1 at
room temperature, and the reaction mixture was stirred for 0.5 h.
Then, 0.13 mL (0.14 mmol) of aniline was added to the mixture.
After approximately 3 h, 0.5 N NaOH was added to the solution. The
lower layer was isolated, and 0.5 N HCl was added until the aqueous
solution was neutral. The dichloromethane layer was washed three
times with 10 mL of water, and then MgSO4 was added. After 2 h,
the solution was filtered, and the solvent was removed. The residue
was purified by column chromatography on silica gel (eluent:
4.2.6.2. 3-(2-Fluoropropanamido)-4-methoxy-N-phenylbenzamide
(1m). A total of 0.17 mL (0.18 mmol) of DIC and 0.24 g (0.18 mmol)
of HOBt were added to a dichloromethane solution of 14.0 mL
(0.12 mmol) of 2-fluoropropanoic acid and THF at room tempera-
ture, which was stirred for 0.5 h. Then, 0.35 g (0.14 mmol) of 1-M5
was added to the mixture, and after approximately 3 h, 0.5 N NaOH
was added to the solution. The lower layer was isolated, and 0.5 N
HCl was added until the aqueous solution was neutral. The
dichloromethane layer was washed three times with 10 mL of
water, and MgSO4 was then added. After 2 h, the solution was
filtered, and the solvent was removed. The residue was purified by
column chromatography on silica gel (eluent: petroleum ether:-
ethyl acetate ¼ 4:1) to yield the desired compound as a white solid
(yield: 67%, HPLC purity: 99.40%). Mp: 182e184 ꢀC. 1H NMR
petroleum ether:ethyl acetate
compound as a white solid (yield: 76%). Mp: 197e198 ꢀC. 1H NMR
(400 MHz, DMSO-d6) (ppm): 9.92 (1H, s, NH), 7.74 (2H, d,
¼
3:1) to yield the desired
d
J ¼ 9.0 Hz, PhH), 7.32 (2H, m, PhH), 7.21 (2H, d, J ¼ 8.0 Hz, PhH), 7.06
(1H, m, PhH), 6.88 (1H, d, J ¼ 8.0 Hz, PhH), 4.90 (2H, br s, NH2), 3.83
(3H, s, OCH3). ESI-MS (m/z): 243 (M þ H)þ.
(400 MHz, DMSO-d6)
d (ppm): 10.12 (1H, s, NH), 9.22 (1H, s, NH),
8.49 (1H, s, PhH), 7.81 (1H, d, J ¼ 6.0 Hz, PhH), 7.78 (2H,d, J ¼ 7.6 Hz,
PhH), 7.33 (2H, t, J ¼ 7.6 Hz, PhH), 7.20 (1H, d, J ¼ 8.8 Hz, PhH), 7.07
(1H, m, PhH), 5.34 (1H, qq, JHeH ¼ 6.4 Hz, JHeF ¼ 48.8 Hz, CH), 3.93
(3H, s, OCH3), 1.55 (3H, dd, JHeH ¼ 6.4 Hz, JHeF ¼ 20.8 Hz, CH3).
HRMS-ESI (m/z): calculated for C17H17N2O3F (M)þ: 316.11803;
measured: 316.12177.
4.2.5.2. 3-Amino-2-methoxy-N-phenylbenzamide (4-M). Compound
4-M was synthesised using a method similar to that of 1-M5 and was
isolated as a white solid (yield: 59%). Mp: 205e206 ꢀC. 1H NMR
(400 MHz, DMSO-d6)
d (ppm): 10.33 (1H, s, NH), 8.00 (1H, d,
J ¼ 6.0 Hz, PhH), 7.72 (2H, d, J ¼ 6.4 Hz, PhH), 7.33 (3H, m, PhH), 7.22
(1H, d, J ¼ 6.4 Hz, PhH), 7.07 (1H, t, J ¼ 5.6 Hz, PhH), 5.54 (2H, br s,
NH2), 3.87 (3H, s, OCH3). ESI-MS (m/z): 243 (M þ H)þ.
4.2.6.3. 3-(Cyclopentanecarboxamido)-4-methoxy-N-phenylbenzamide
(1l). Compound 1l was synthesised using a method similar to that of
1m and was isolated (eluent: petroleum ether:ethyl acetate ¼ 4:1) as
a white solid (yield: 63%, HPLC purity: 96.42%). Mp: 192e194 ꢀC. 1H
4.2.5.3. 3-Amino-5-methoxy-N-phenylbenzamide (5-M). Compound
5-M was synthesised using a method similar to that of 1-M5 and was
isolated as a white solid (yield: 54%). Mp: 207e208 ꢀC. 1H NMR
NMR (400 MHz, DMSO-d6) d (ppm): 10.07 (1H, s, NH), 9.16 (1H, s, NH),
(400 MHz, DMSO-d6)
d
(ppm): 10.23 (1H, s, NH), 7.78 (1H, s, PhH),
8.50 (1H, s, PhH), 7.81 (1H, d, J ¼ 6.0 Hz, PhH), 7.78 (2H, d, J ¼ 7.6 Hz,
PhH), 7.33 (2H, m, PhH), 7.20 (1H, d, J ¼ 8.8 Hz, PhH), 7.07 (1H, m,
PhH), 3.90 (3H, s, OCH3), 2.96 (1H, m), 1.83 (2H, m), 1.64 (4H, m), 1.55
(2H, m). HRMS-ESI (m/z): calculated for C20H23N2O3 (M þ H)þ:
339.17056; measured: 339.17032.
7.48 (1H, d, J ¼ 8.0 Hz, PhH), 7.55 (1H, s, PhH), 7.36 (3H, m, PhH), 7.23
(1H,s, PhH), 7.11 (1H, m, PhH), 5.54 (2H, br s, NH2), 3.82 (3H, s, OCH3).
ESI-MS (m/z): 243 (M þ H)þ.
4.2.5.4. 5-Amino-2-methoxy-N-phenylbenzamide (6-M). Compound
6-M was synthesised using a method similar to that of 1-M5 and was
isolated as a white solid (yield: 61%). Mp: 201e202 ꢀC. 1H NMR
4.2.6.4. 3-(2-Fluoropropanamido)-2-methoxy-N-phenylbenzamide
(13). Compound 13 was synthesised using a method similar to that
of 1m and was isolated (eluent: petroleum ether:ethyl
acetate ¼ 4:1) as a white solid (yield: 65%, HPLC purity: 98.76%).
(400 MHz, DMSO-d6)
d
(ppm): 9.75 (1H, s, NH), 7.73 (2H, d, J ¼ 6.4 Hz,
PhH), 7.44 (1H, d, J ¼ 6.8 Hz, PhH), 7.30 (3H, m, PhH), 7.02 (1H, m,
PhH), 6.45 (1H, d, J ¼ 6.8 Hz, PhH), 5.54 (2H, br s, NH2), 3.90 (3H, s,
OCH3). ESI-MS (m/z): 243 (M þ H)þ.
Mp: 177e179 ꢀC. 1H NMR (500 MHz, DMSO-d6)
d (ppm): 10.33 (1H,
s, NH), 9.43 (1H, s, NH), 8.00 (1H, d, J ¼ 7.5 Hz, PhH), 7.72 (2H, d,
J ¼ 9.0 Hz, PhH), 7.33 (3H, m, PhH), 7.22 (1H, d, J ¼ 8.0 Hz, PhH), 7.07
(1H, t, J ¼ 8.0 Hz, PhH), 5.34 (1H, qq, JHeH ¼ 6.5 Hz, JHeF ¼ 49 Hz,
4.2.5.5. 3-Amino-N-phenylbenzamide (7-M). Compound 7-M was
synthesised using a method similar to that of 1-M5 and was isolated
as a white solid (yield: 65%). Mp: 173e175 ꢀC. 1H NMR (400 MHz,
CH), 3.87 (3H, s, OCH3), 1.55 (3H, dd, JHeH
¼
6.5 Hz,
JHeF ¼ 24.5 Hz, CH3). HRMS-ESI (m/z): calculated for C17H18N2O3F
DMSO-d6)
d (ppm): 10.24 (1H, s, NH), 8.18 (1H, s, PhH), 7.88 (1H, d,
(M þ H)þ: 317.12956; measured: 317.12960.
J ¼ 8.0 Hz, PhH), 7.76 (2H, d, J ¼ 8.0 Hz, PhH), 7.67 (1H, d, J ¼ 8.0 Hz,
PhH), 7.49 (1H, m, PhH), 7.36 (2H, m, PhH), 7.11 (1H, m, PhH), 5.54
(2H, br s, NH2). ESI-MS (m/z): 213 (M þ H)þ.
4.2.6.5. 3-(2-Fluoropropanamido)-5-methoxy-N-phenylbenzamide
(14). Compound 14 was synthesised using a method similar to that
of 1m and was isolated (eluent: petroleum ether:ethyl
acetate ¼ 4:1) as a white solid (yield: 65%, HPLC purity: 98.64%).
4.2.5.6. 4-Methoxy-N-phenylbenzamide (12). Compound 12 was
synthesised using a method similar to that of 1-M5 and was isolated
(eluent: petroleum ether:ethyl acetate ¼ 3:1) as a white solid
(yield: 63%, HPLC purity: 98.75%.). Mp: 152e153 ꢀC. 1H NMR
Mp: 186e187 ꢀC. 1H NMR (500 MHz, DMSO-d6)
d (ppm): 10.23 (1H,
s, NH), 10.21 (1H, s, NH), 7.78 (1H, s, PhH), 7.55 (1H, s, PhH), 7.48
(1H, d, J ¼ 8.0 Hz, PhH), 7.36 (2H, q, J ¼ 8.0 Hz, PhH), 7.23 (1H, s,
PhH), 7.11 (2H, m, PhH), 5.13 (1H, qq, JHeH ¼ 6.5 Hz, JHeF ¼ 49 Hz,
(400 MHz, DMSO-d6)
d (ppm): 10.05 (1H, s, NH), 7.95 (2H, d,
J ¼ 7.6 Hz, PhH), 7.75 (2H, d, J ¼ 7.6 Hz, PhH), 7.34 (2H, m, PhH), 7.08
(3H, m, PhH), 3.83 (3H, s, OCH3). HRMS-ESI (m/z): calculated for
C14H14NO2 (M þ H)þ: 228.10169; measured: 228.10191.
CH), 3.82 (3H, s, OCH3), 1.55 (3H, dd, JHeH
¼
6.5 Hz,
JHeF ¼ 24.5 Hz, CH3). HRMS-ESI (m/z): calculated for C17H18N2O3F
(M þ H)þ: 317.12955; measured: 317.12960.
4.2.6. General procedure for the synthesis of 1l, 1m, and 13e16
4.2.6.1. 2-Fluoropropanoic acid. A total of 6.6 mL (11.0 mmol) of
a 1.0 g/20 mL NaOH solution was added to a solution of 1.0 mL
(10.0 mmol) of ethyl 2-fluoropropanoate in tetrahydrofuran (THF),
and the mixture was reacted under backstreaming for 2 h. After the
solution reached room temperature, the pH was adjusted to 6.0,
and dichloromethane was added until a total volume of 100 mL was
achieved.
4.2.6.6. 5-(2-Fluoropropanamido)-2-methoxy-N-phenylbenzamide
(15). Compound 15 was synthesised using a method similar to that
of 1m and was isolated (eluent: petroleum ether:ethyl
acetate ¼ 4:1) as a white solid (yield: 65%, HPLC purity: 99.53%).
Mp: 172e174 ꢀC. 1H NMR (500 MHz, DMSO-d6)
d (ppm): 9.75 (1H, s,
NH), 9.49 (1H, s, NH), 7.73 (2H, d, J ¼ 7.0 Hz, PhH), 7.44 (1H, d,
J ¼ 7.5 Hz, PhH), 7.30 (3H, m, PhH), 7.02 (1H, m, PhH), 6.45 (1H, d,
J ¼ 7.5 Hz, PhH), 5.34 (1H, qq, JHeH ¼ 6.5 Hz, JHeF ¼ 49 Hz, CH), 3.84

124
L.-h. Hao et al. / European Journal of Medicinal Chemistry 55 (2012) 117e124
(3H, s, OCH3), 1.55 (3H, dd, JHeH ¼ 6.5 Hz, JHeF ¼ 24.5 Hz, CH3).
HRMS-ESI (m/z): calculated for C17H18N2O3F (M þ H)þ: 317.12957;
measured 317.12960.
strain) and influenza B (B/Jifang/13/97) were obtained from the
Institute of Virology, Chinese Academy of Preventive Medicine.
4.3.1. Cytotoxicity assay
4.2.6.7. 3-(2-Fluoropropanamido)-N-phenylbenzamide (16). Comp-
ound 16 was synthesised using a method similar to that of 1m and
was isolated (eluent: petroleum ether:ethyl acetate ¼ 4:1) as
a white solid (yield: 57%, HPLC purity: 99.09%). Mp: 142e143 ꢀC. 1H
The cytotoxicities of the compounds in the presence of MDCK
cells were monitored by CPE method [17]. MDCK cells (2.5 ꢁ 104/
well) were plated into a 96-well plate. After 24 h, the monolayer
cells were incubated in the presence of various concentrations of
the test compounds. After 48 h of culture at 37 ꢀC and under a 5%
CO2 atmosphere in a carbon dioxide incubator, the cells were
monitored by CPE. Median toxic concentrations (TC50) were
calculated by Reed and Muench [18] analyses.
NMR (500 MHz, DMSO-d6)
d (ppm): 10.27 (1H, s, NH), 10.24 (1H, s,
NH), 8.18 (1H, s, PhH), 7.88 (1H, d, J ¼ 8.0 Hz, PhH), 7.76 (2H, d,
J ¼ 8.0 Hz, PhH), 7.67 (1H, d, J ¼ 8.0 Hz, PhH), 7.49 (1H, m, PhH), 7.36
(2H, m, PhH), 7.11 (1H, m, PhH), 5.34 (1H, qq, JHeH ¼ 6.5 Hz,
JHeF ¼ 49 Hz, CH), 1.55 (3H, dd, JHeH ¼ 6.5 Hz, JHeF ¼ 24.5 Hz,
CH3). HRMS-ESI (m/z): calculated for C16H16N2O2F (M þ H)þ:
287.11901; measured: 287.11903.
4.3.2. Anti-influenza A assays
Confluent MDCK cells grown in 96-well microplates were
infected with 100 median tissue culture infective doses
(100TCID50) of the test strains. After 2 h of adsorption at 37 ꢀC, the
monolayers were washed with phosphate-buffered saline (PBS)
and incubated at 37 ꢀC in the maintenance medium with or
without various concentrations of the test compounds. The viral
CPE was observed when the viral control group reached 4, and the
antiviral activities (IC50) of the bisaryl amide derivatives were
determined with Reed and Muench analyses. The SI value was
4.2.7. General procedure for the synthesis of 21
4.2.7.1. N-(2-methoxy-5-nitrophenyl)propionamide (19). A total of
2.5 mL (30 mmol) of propionyl chloride was added dropwise to
a mixture of 5.0 g of 2-methoxy-5-nitroaniline (18) and 4.3 mL
(30 mmol) of TEA in dichloromethane at 0 ꢀC. The mixture was then
stirred at room temperature until the starting material was
completely disappeared. The solvent was evaporated in vacuo to
produce a sticky residue that was extracted with dichloromethane
and 0.5 N HCl (25 mL). The organic phase was concentrated, and the
solid was purified by column chromatography on silica gel (eluent:
calculated with TC50/IC50
.
Acknowledgements
petroleum ether:ethyl acetate
compound as a white solid (yield: 74%). Mp: 192e193 ꢀC. 1H NMR
(400 MHz, DMSO-d6) (ppm): 10.12 (1H, s, NH), 8.51 (1H, s, PhH),
¼
3:1) to yield the desired
This work was supported by the National Natural Science
Foundation of China (Grant 30873138), the National S&T Major
Special Project on Major New Drug Innovation (Item Number:
2012ZX09102-101-001) and the New Teachers’ Fund for Doctor
Stations, Ministry of Education (20101106120032).
d
7.72 (1H, d, J ¼ 7.6 Hz, PhH), 7.07 (1H, d, J ¼ 7.6 Hz, PhH), 3.90 (3H,s,
OCH3), 2.41 (2H, q, J ¼ 7.6 Hz, CH2), 1.07 (3H, t, J ¼ 7.6 Hz, CH3). ESI-
MS (m/z): 225 (M þ H)þ.
4.2.7.2. N-(5-amino-2-methoxyphenyl)propionamide (20). A total of
0.50 g (2.2 mmol) of 19 was dissolved in 20 mL of methanol, and
10% Pd/C was added. The mixture was reacted under H2 at 50 bar
for 5 h. Then, the solution was filtered, and the methanol was
removed (yield: 57%). Mp: 167e169 ꢀC. 1H NMR (400 MHz, DMSO-
Appendix A. Supplementary data
Supplementary data associated with this article can be found in
online version at http://dx.doi.org/10.1016/j.ejmech.2012.07.008.
d6)
d (ppm): 10.12 (1H, s, NH), 8.51 (1H, s, PhH), 7.72 (1H, d,
J ¼ 7.6 Hz, PhH), 7.07 (1H, d, J ¼ 7.6 Hz, PhH), 5.32 (2H, br s, NH2),
3.90 (3H, s, OCH3), 2.41 (2H, q, J ¼ 7.6 Hz, CH2), 1.07 (3H, t, J ¼ 7.6 Hz,
CH3). ESI-MS (m/z): 195 (M þ H)þ.
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total of 0.35 mL (3.0 mmol) of benzoyl chloride was added dropwise
to a mixture of 0.58 g (3.0 mmol) of 20 and 0.25 mL (3.0 mmol) of
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leum ether:ethyl acetate ¼ 4:1) to yield the desired compound as
a white solid (yield: 73%, HPLC purity: 98.65%). Mp: 183e185 ꢀC. 1H
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d (ppm): 10.12 (1H, s, NH), 9.00 (1H, s,
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NH), 8.30 (1H, s, PhH), 7.95 (2H, d, J ¼ 7.0 Hz, PhH), 7.56 (4H, m,
PhH), 7.00 (1H, d, J ¼ 9.0 Hz, PhH), 3.81 (3H, s, OCH3), 2.40 (2H, q,
J ¼ 7.5 Hz, CH2), 1.08 (3H, t, J ¼ 7.5 Hz, CH3). HRMS-ESI (m/z):
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299.13902.
þ
H)þ: 299.13907; measured:
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4.3. Antiviral assays
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influenza A/Tianjin Jinnan/15/2009 (H1N1, oseltamivir-resistant
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