Design, synthesis, inhibitory activity, and SAR studies of hydrophobic p-aminosalicylic acid derivatives as neuraminidase inhibitors.

Abstract

A series of hydrophobic p-aminosalicylic acid derivatives containing a lipophilic side chain at C-2 and an amino or guanidine at C-5 were synthesized and evaluated for their ability to inhibit neuraminidase (NA) of influenza A virus (H3N2). All compounds were synthesized in good yields starting from commercially available p-aminosalicylic acid (PAS) using a suitable synthetic strategy. These compounds showed potent inhibitory activity against influenza A NA. Within this series, six compounds, 11, 12, 13e, 16e, 17c, and 18e, have the good potency (IC(50)=0.032-0.049 microM), which are compared to Oseltamivir (IC(50)=0.021 microM) and could be used as lead compounds in the future.

Full text

Page 1

Design, synthesis, inhibitory activity, and SAR studies
of hydrophobic p-aminosalicylic acid derivatives as
neuraminidase inhibitors
Jie Zhang,aQiang Wang,aHao Fang,aWenfang Xu,a,*Ailin Liuband Guanhua Dub
aDepartment of Medicinal Chemistry, School of Pharmacy, Shandong University, Ji’nan, Shandong 250012, PR China
bInstitute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, PR China
Received 12 December 2007; revised 21 January 2008; accepted 23 January 2008
Available online 30 January 2008
Abstract—A series of hydrophobic p-aminosalicylic acid derivatives containing a lipophilic side chain at C-2 and an amino or gua-
nidine at C-5 were synthesized and evaluated for their ability to inhibit neuraminidase (NA) of influenza A virus (H3N2). All com-
pounds were synthesized in good yields starting from commercially available p-aminosalicylic acid (PAS) using a suitable synthetic
strategy. These compounds showed potent inhibitory activity against influenza A NA. Within this series, six compounds, 11, 12, 13e,
16e, 17c, and 18e, have the good potency (IC50= 0.032–0.049 lM), which are compared to Oseltamivir (IC50= 0.021 lM) and could
be used as lead compounds in the future.
? 2008 Elsevier Ltd. All rights reserved.
1. Introduction
Influenza is worldwide one of the deadliest infectious
diseases that can affect millions of people every year.1
Vaccines against influenza virus are ineffective due to
the rapid emergence of mutant viral antigens. The M2
protein ion channel blockers are only effective on type
A influenza with undesirable side effects and rapidly gen-
erated resistant mutants.2Because effective and safe
anti-influenza therapeutics are lacking, developing effec-
tive anti-influenza agents become a high-priority and
attractive area in drug discovery.
In recent years, virology studies of influenza virus illus-
trated the replication mechanism of the virus and some
molecular targets have been identified for drug interven-
tion such as hemagglutinin (HA), neuraminidase (siali-
dase, NA), M2 protein, and endonuclease.3Among
those potential targets, NA appears to be an attractive
target for drug development. As a glycoprotein in viral
surface, NA is essential for viral replication due to its
ability to catalyze removal of terminal SA linked to gly-
coproteins and glycolipids. Scientific research showed
that NA is not only crucial in the release of virion prog-
eny away from infected cells,4but also important in the
movement of the virus through mucus of respiratory
tract and reducing the propensity of the virus particles
to aggregate. Despite the homology identity of NA in
different strains is only about 30%, the catalytic site of
NA in all influenza A and B virus is completely con-
served.5Mutations of these conserved residues generally
result in enzyme inactivation, suggesting that the virus
may not easily escape NA-targeted drug therapy.
Now, two NA inhibitors, Zanamivir (1) and Oseltamivir
(2), have been confirmed as effective and safe for the
treatment of influenza and approved by FDA.6
O
NHCOCH3
1
N
H
NH
NH2
HO
OH
OH
COOH
NHCOCH3
2
NH2
O
COOH
It is reported that NA exists as tetramer consisting of
four spherical subunits in the influenza virus, and a
hydrophobic region is located in the central.7According
to the X-ray crystal structure of the NA and the inhibi-
tor, Wang et al.8proposed an ‘airplane’ model of the
0968-0896/$ - see front matter ? 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.01.036
Keywords: Influenza; Neuraminidase inhibitor; p-Aminosalicylic acid
derivatives; SAR study.
*Corresponding
author. Tel./fax:
xuwenf@sdu.edu.cn
+8653188382264; e-mail:
Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 3839–3847

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NA active site as illustrated in Figure 1 to summarize
the basic structural requirements of a potent NA inhib-
itor. Each monomeric subunit has an active site cavity
lined with ten conserved residues and four water mole-
cules. The active site of NA has four main binding sites.
The positively charged site 1 consists of Arg118, Arg292,
and Arg371 and interacts with the carboxylate. The neg-
atively charged site 2 consists of Glu119, Glu227, and
Asp151 and interacts with the amino or guanidine.
The small hydrophobic pocket consists of Ile222 and
Trp178 (site 3) accommodates the acetyl group, and site
4, consisting of Glu276 and Glu277, binds to the hydro-
phobic side chain.
According to the studies on NA active site and SAR of
published NA inhibitors, inhibition of NA is mainly
determined by the relative positions of substituents of
the central ring. And all the reported molecules have
shown the importance of all four substituents (carboxyl-
ate, glycerol or hydrophobic side chain, acetamido, ami-
no or guanidine) for the activity on NA A and B. For
example, Sudbeck et al. reported a series of tri-substi-
tuted benzoic acid analogs which contain a benzene ring
to replace the pyranose ring in Zanamivir (2).9In the
inhibition assay, one compound (BANA 113, 3) ap-
peared to be the strongest inhibitor with an IC50of
10 lM. The crystal structure of BANA113 complexed
with NA shows that the guanidine group extends to
reach a small pocket between residues Glu119 and
Glu227. And the carboxylate group forms an electro-
static interaction with three arginine triad in the active
site. Opposite the Arg pocket, the methyl group of the
N-acetylamino fits into a hydrophobic pocket lined with
residues Trp178 and Ile222.
During our previous work, the 4-hydroxy-L L-proline has
been used to prepare a series of pyrrolidine derivatives
as NA inhibitors.10In our ongoing work, we wanted
to use benzene ring to replace pyrrolidine ring and stud-
ied the substituted benzoic acid derivatives. We first
screened all the benzoic acid derivatives in our com-
pound library including not only the target compounds
and intermediates we synthesized before, but also some
commercially available compounds. The pharmacologi-
cal result showed that p-aminosalicylic acid (PAS), one
antibacterial agent, exhibited modest activity against
influenza virus A (H3N2) NA (IC50= 0.27 lM) and
could be used as lead compound in future.
Considering the SAR of lead compound (BANA 113, 3),
we designed and synthesized several novel aromatic
inhibitors (4) of NA from commercially available PAS.
In order to improve the affinity of lead compounds,
we optimized the structure of PAS with the following
chemical modification: (i) C-1 carboxylic acid was kept
or converted to other derivatives such as methyl ester
or hydroxymate; (ii) C-2 hydroxy group in aromatic ring
was changed to various phenolic ether in order to in-
crease the hydrophobic interaction with site 4; (iii)
hydrogen at C-3 position was kept or replaced with ami-
no group; (iv) amino group at C-4 position was acety-
lated; and (v) hydrogen at C-5 position was converted
to free nitro group or amino group or guanido group.
COOH
NHAc
3
N
H
NH2
NH
COOR1
R3
R4
R1= H or CH3
R2 = lipophilic side chain
R3 = H or NH2
R4 = NH2 or NHC(=NH)NH2
converted to NH2 or NHC(=NH)NH2
Keep or converted
to COOCH3
lipophilic side chain
R2O
NHCOCH3
4
2. Chemistry
The synthesis of p-aminosalicylic acid derivatives pos-
sessing NA inhibitory activities is described in Schemes
1 and 2. Benzoic acids 12 and 13e–19e were synthesized
from commercially available PAS (5). The methyl ester 6
was prepared to avoid side-reactions of the carboxylate
group.11Then 6 underwent selective acetylation on the
4-amino group using acetic anhydride to provide amide
7.12Selective alkylation of compound 7 with various
alkylogen in the presence of K2CO3or NaH to give
intermediate 8 or 13a–19a, respectively.13,14The com-
pound 8 or 13a–19a, on nitration with fuming HNO3
and glacial acetic acid at 0 ?C gave nitro derivatives 9
or 13b–19b.15Reduction of the nitro groups of 9 and
13b–19b proceeded without problem using transfer
hydrogenation, following the procedure described by
Singh et al.16Whereas the synthesis of11 and 13d–19d
was accomplished in 60% yield by reaction of 10 and
13c–19c with cyanamide and HCl.17Hydrolysis of the
methyl esters 11 and 13d–19d with NaOH/H2O yielded
the target compounds 12 and 13e–19e.18
3. Results and discussion
All the target compounds were evaluated for in vitro
neuraminidase inhibitory activity. Preliminary result
showed that 33 compounds displayed inhibitory activi-
ties with IC50value from 0.032 to 9.26 lM (Table 1).
Compound 12 with two guanidine groups at C-3 and
C-5 and ethyl as hydrophobic side chain showed the best
inhibitory activity (IC50= 0.032 lM). The other five
compounds containing guanidine (11, 13e, 14e, 16e,
and 18e) exhibited good activities (0.036–0.049 lM).
Generally, the compound with guanidine at C-5 and car-
boxyl group at C-1 showed better activities.
Figure 1. ‘Airplane’ model of NA active site (Ref. 8).
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In summary, our studies have discovered a new series of
p-aminosalicylic acid derivatives that have potent NA
inhibitory activity. The binding of compound 12 in the
active site of NA is shown in Figure 2, and we found
that the carboxylate makes tight salt-bridge interactions
with an arginine triad consisting of Arg118, Arg292, and
Arg371, and the lipophilic side chain binds to the hydro-
phobic pocket (site 4) formed by Glu277 and Glu276,
whereas the guanidine group binds to the negatively
charged site 2 created by Glu227, Glu119, and
Asp151. The carbonyl of the N-acetyl group hydrogen
bonds to Arg152 and the methyl group occupies a
hydrophobic pocket created by Trp178 and Ile222.
Meanwhile, we found that another guanidine interacts
electrostatically with Asn294, Asn347, and Cly348.
Compared to other research, we reported a more conve-
nient and economical method of the synthesis of
p-aminosalicylic acid NA inhibitors. Compared to the
other research, p-aminosalicylic acid we used appeared
to be an ideal starting material because of its low cost
and commercial abundance.
4. SAR studies
4.1. Dataset and molecular modeling
We used Sybyl 7.0 program to carry out the SAR studies
of these p-aminosalicylic acid derivatives. The CoMFA
studies were carried out with the QSAR model of Sybyl.
The test set consisted of compounds 7, 13a, 15b, 17c,
and 18e, considering the last three compounds (19a,
19b, 19c) all had a long side chain R2for CoMFA, so
the other 25 compounds composed of the training set.
The IC50values were converted into pIC50according
to the formula: pIC50= ?lgIC50.
Based on the docking results, the template molecule 12
was taken and the rest of the molecules were aligned
to it using the benzoic acid as scaffold by DATABASE
ALIGNMENT method in the Sybyl.
The steric and electrostatic CoMFA fields were calcu-
lated at each lattice intersection of a regularly spaced
grid of 2.0 A˚in all three dimensions within defined re-
gion. An sp3carbon atom with +1.00 charge was used
as a probe atom. The steric and electrostatic fields were
truncated at +30.00 kal mol?1, and the electrostatic
fields were ignored at the lattice points with maximal
steric interactions.
PLS (partial least square) method was used to linearly
correlate the CoMFA fields to the inhibitory activity
values. The cross-validation analysis was performed
using the leave one out (LOO) method in which one
compound is removed from the dataset and its activity
is predicted using the model derived from the rest of
the dataset. The cross-validated q2(0.526) that resulted
in optimum number of components (n = 7) and lowest
standard error of prediction were considered for further
NH2
5
OH
COOH
NH2
6
OH
COOCH3
NHCOCH3
7
OH
COOCH3
NHCOCH3
8
OCH2CH3
COOCH3
NHCOCH3
9
COOH
OCH2CH3
COOCH3
O2N
NHCOCH3
10
OCH2CH3
COOCH3
H2N
NHCOCH3
11
OCH2CH3
NH
COOCH3
N
H
HN
H2N
NO2
NH2
a
b
cd
ef
N
H
NH2
NHCOCH3
12
OCH2CH3
NH
N
H
HN
H2N
N
H
NH2
g
Scheme 1. Reagents and conditions: (a) MeOH, concd H2SO4, n; (b) Ac2O, acetone; (c) BrCH2CH3, K2CO3, acetone, n; (d) fuming HNO3; (e) 10%
Pd/CaCO3, H2NNH2, EtOH; (f) H2NCN, concd HCl, EtOAC, n; (g) 1—NaOH; 2—HAc.
NH2
5
OH
COOH
NH2
6
OH
COOCH3
NHCOCH3
7
OH
COOCH3
NHCOCH3
13a-19a
O(CH2)nCH3
COOCH3
NHCOCH3
13b-19b
O(CH2)nCH3
COOCH3
O2N
NHCOCH3
13c-19c
O(CH2)nCH3
COOCH3
H2N
NHCOCH3
13d-19d
O(CH2)nCH3
COOCH3
N
H
HN
H2N
a
b
cd
e
f
NHCOCH3
13e-19e
O(CH2)nCH3
COOH
N
H
HN
H2N
g
Scheme 2. Reagents and conditions: (a) MeOH, concd H2SO4, n; (b) Ac2O, acetone; (c) Br(CH2)nCH3, NaH, DMF, n; (d) fuming HNO3; (e) 10%
Pd/CaCO3, H2NNH2, EtOH; (f) H2NCN, concd HCl, EtOAC, n; (g) 1—NaOH; 2—HAc.
J. Zhang et al. / Bioorg. Med. Chem. 16 (2008) 3839–3847
3841

Page 4

analysis. We have evaluated different filter value r and
at least selected r as 2.00 kal mol?1to speed up the anal-
ysis and reduce noise.
4.2. Results and discussion
From the docking results and the actual results, we
can both obtain the conclusions: the order of increas-
ing activity is R4: –N@C(NH2)2> –NH2> –NO2> –H.
The LOO cross-validated q2of the CoMFA model is
0.528, and the noncross-validated r2for the model
established by the study is 0.971. The value of the var-
iance ratio F (n1= 7, n2= 17) is 81.631 and standard
error of the estimate (SEE) is 0.147. The contribution
ofelectrostaticand steric
respectively.
is 68.2%and 31.8%,
From Figure 3(b) we can find that the CoMFA model
can predict compounds 7, 15b, 18e well, but not very
well to 13a and 17c in the test set. The poor predictabil-
ity may be caused by the sample size being more lower
when the actual pIC50< 5.2 (including 1 compounds)
than it >5.2(including 24 compounds). From Table 1
we can see that for R4, the activity of compound with
–NH2is 2- to 3-fold than that of compound with –
NO2while they have the same R2and R3except 17b
and 17c (the activity of 17c is nearly 10-fold than 17b),
and it maybe the reason for the poor predictability of
17c. We also try to use the model to predict the last three
compounds with very long R2side chain. The points of
the three compounds are all in ±0.5log unit in Figure
3(b), which shows that large capacity change of R2has
little influence on the activity. What is more, from the
Table 1. The structure and in vitro inhibitory activities of compounds against NA
COOR1
R3
R4
R2O
NHCOCH3
CompoundR1
R2
R3
R4
IC50(lM) pICpre
50
pICpre
50
Res.
7
8
9
10
11
12
13a
13b
13c
13d
13e
14a
14b
14c
14e
15a
15b
15c
15d
16a
16b
16c
16e
CH3
CH3
CH3
CH3
CH3
H
CH3
CH3
CH3
CH3
H
CH3
CH3
CH3
H
CH3
CH3
CH3
CH3
CH3
CH3
CH3
H
H
CH3CH2
CH3CH2
CH3CH2
CH3CH2
CH3CH2
(CH3)2CH
(CH3)2CH
(CH3)2CH
(CH3)2CH
(CH3)2CH
CH3CH2CH2
CH3CH2CH2
CH3CH2CH2
CH3CH2CH2
CH3CH2(CH3)CH
CH3CH2(CH3)CH
CH3CH2(CH3)CH
CH3CH2(CH3)CH
CH3(CH2)3
CH3(CH2)3
CH3(CH2)3
CH3(CH2)3
H
H
NO2
NH2
N@C(NH2)2
N@C(NH2)2
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
NO2
NH2
N@C(NH2)2
N@C(NH2)2
H
NO2
NH2
N@C(NH2)2
N@C(NH2)2
H
NO2
NH2
N@C(NH2)2
H
NO2
NH2
N@C(NH2)2
H
NO2
NH2
N@C(NH2)2
0.99
0.22
2.15
0.31
0.036
0.032
5.32
0.57
0.23
0.12
0.049
9.26
3.57
1.26
0.72
2.97
1.59
0.95
0.74
0.23
0.14
0.07
0.04
6.00
6.66
5.67
6.51
7.44
7.49
5.27
6.24
6.64
6.92
7.31
5.03
5.45
5.90
6.14
5.53
5.80
6.02
6.13
6.64
6.85
7.15
7.40
6.23
6.70
5.69
6.57
7.49
7.46
5.87
6.26
6.46
7.00
7.18
5.13
5.48
5.61
6.37
5.52
5.87
6.24
6.11
6.52
6.84
7.12
7.57
?0.22
?0.04
?0.02
?0.06
?0.05
0.03
?0.60
?0.01
0.18
?0.08
0.13
?0.10
?0.03
0.29
?0.23
0.00
?0.08
?0.22
0.02
0.12
0.02
0.04
?0.18
17a CH3
HH 0.496.316.130.18
17b CH3
HNO2
0.336.48 6.68
?0.20
17cCH3
H NH2
0.038 7.426.74 0.68
18a
18b
18c
18e
19a
19b
19c
2
CH3
CH3
CH3
H
CH3
CH3
CH3
(CH3)2CH(CH2)2
(CH3)2CH(CH2)2
(CH3)2CH(CH2)2
(CH3)2CH(CH2)2
CH3(CH2)15
CH3(CH2)15
CH3(CH2)15
H
H
H
H
H
H
H
H
NO2
NH2
N@C(NH2)2
H
NO2
NH2
3.81
2.96
0.97
0.041
2.69
1.54
0.59
0.021
5.42
5.53
6.01
7.39
5.57
5.81
6.23
5.53
5.63
5.97
7.25
6.05
6.40
6.72
?0.11
?0.10
0.04
0.14
?0.48
?0.58
?0.49
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Page 5

docking result we find that R2is to stretch out of the ac-
tive pocket of NA in Figure 2(b), which can explain why
it cannot show distinguished influence to activity.
5. Conclusions
We have described the synthesis and properties of aseries
of p-aminosalicylic acid derivatives as influenza NA
inhibitors. All of the compounds were shown to possess
potent influenza NA inhibitory activity, and the most po-
tentcompoundofthe
(IC50= 0.032 lM), which in addition to good enzyme
inhibitory activity, displays potent anti-viral activity
in vitro. We reported a more convenient and economical
method of the synthesis of p-aminosalicylic acid NA
inhibitors. Compared to other research, p-aminosalicylic
acid we used appeared to be an ideal starting material be-
cause of its low cost and commercial abundance. Estab-
lishing a consistent binding mode was critical to
predictive structure-based drug design and discovering
potent compounds in the nanomolar range that would
seriesiscompound12
potentially be useful for anti-viral therapy. The com-
pounds we have got all showed potent NA inhibitory
activity, and this finding could be used to design further
influenza NA inhibitors. The properly substituted
p-aminosalicylic acids provide an attractive structural
templatefordevelopingpotentinhibitorsofinfluenzaNA.
6. Experimental
6.1. Neuraminidase inhibition assay
All compounds were evaluated for in vitro inhibitory ac-
tions using the method reported by Guanhua Du.20,21
The strain A (Yuefang 72-243 A) influenza virus, which
was donated by Chinese Centers for Disease Control,
was used as source of NA. The NA was obtained by
the method described by Laver.22The assay employed
a spectrofluorometric technique that uses the compound
20-(4-methylumbelliferyl)-a-D D-acetylneuraminic
(MUNANA) as substrate. And cleavage of this sub-
strate by NA produces a fluorescent product, which
acid
Figure 2. FlexX docked result. Compound 12 in the active site of NA (PDB ID:1nnc).19(a) Compound 12 reacting with the amino acids of the active
pocket of NA. (b) Comparing the docking orientation of 12 to the ligand-SA (showed in red color) in the complex.
Figure 3. (a) The most active molecule 12 is shown in the background. Red (R) color represents the negative charge region, blue (B) is the positive
charge region, green (G) is the more bulky region, yellow (Y) is the less bulky region. (b) The predictability of the CoMFA model.
J. Zhang et al. / Bioorg. Med. Chem. 16 (2008) 3839–3847
3843