Synthesis, biological evaluation and 3D-QSAR studies of 3-keto salicylic acid chalcones and related amides as novel HIV-1 integrase inhibitors

Article abstract of DOI:10.1016/j.bmc.2011.01.047

HIV-1 integrase is one of the three most important enzymes required for viral replication and is therefore an attractive target for anti retroviral therapy. We herein report the design and synthesis of 3-keto salicylic acid chalcone derivatives as novel HIV-1 integrase inhibitors. The most active compound, 5-bromo-2-hydroxy-3-[3-(2,3,6-trichlorophenyl)acryloyl]benzoic acid (25) was selectively active against integrase strand transfer, with an IC ₅₀ of 3.7 μM. While most of the compounds exhibited strand transfer selectivity, a few were nonselective, such as 5-bromo-3-[3-(4- bromophenyl)acryloyl]-2-hydroxybenzoic acid (15), which was active against both 3′-processing and strand transfer with IC₅₀ values of 11 ± 4 and 5 ± 2 μM, respectively. The compounds also inhibited HIV replication with potencies comparable with their integrase inhibitory potencies. Thus, 5-bromo-2-hydroxy-3-[3-(2,3,6-trichlorophenyl)acryloyl]benzoic acid (25) and 5-bromo-3-[3-(4-bromophenyl)acryloyl]-2-hydroxybenzoic acid (15) inhibited HIV-1 replication with EC₅₀ values of 7.3 and 8.7 μM, respectively. A PHASE pharmacophore hypothesis was developed and validated by 3D-QSAR, which gave a predictive r² of 0.57 for an external test set of ten compounds. Phamacophore derived molecular alignments were used for CoMFA and CoMSIA 3D-QSAR modeling. CoMSIA afforded the best model with q² and r² values of 0.54 and 0.94, respectively. This model predicted all the ten compounds of the test set within 0.56 log units of the actual pIC₅₀ values; and can be used to guide the rational design of more potent novel 3-keto salicylic acid integrase inhibitors.

Full text of DOI:10.1016/j.bmc.2011.01.047

Bioorganic & Medicinal Chemistry 19 (2011) 2030–2045
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, biological evaluation and 3D-QSAR studies of 3-keto salicylic acid
chalcones and related amides as novel HIV-1 integrase inhibitors
Horrick Sharma a, Shivaputra Patil a, Tino W. Sanchez b, Nouri Neamati b, Raymond F. Schinazi c,
John K. Buolamwini a,
⇑
a Department of Pharmaceutical Sciences, College of Pharmacy, University of Tennessee Health Science Center, Memphis, 847 Monroe Avenue, Suite 327, TN 38163, USA
b Department of Pharmacology and Pharmaceutical Sciences, School of Pharmacy, University of Southern California, 1985 Zonal Avenue, Los Angeles, CA 90089, USA
c Emory University School of Medicine/VA Medical Center, Medical Research 151H, 1670 Clairmont Road, Decatur, GA 30033, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
HIV-1 integrase is one of the three most important enzymes required for viral replication and is therefore
an attractive target for anti retroviral therapy. We herein report the design and synthesis of 3-keto sal-
icylic acid chalcone derivatives as novel HIV-1 integrase inhibitors. The most active compound, 5-
bromo-2-hydroxy-3-[3-(2,3,6-trichlorophenyl)acryloyl]benzoic acid (25) was selectively active against
Received 24 November 2010
Revised 16 January 2011
Accepted 24 January 2011
Available online 1 March 2011
integrase strand transfer, with an IC50 of 3.7 lM. While most of the compounds exhibited strand transfer
selectivity, a few were nonselective, such as 5-bromo-3-[3-(4-bromophenyl)acryloyl]-2-hydroxybenzoic
Keywords:
HIV-1
acid (15), which was active against both 30-processing and strand transfer with IC50 values of 11 4 and
5
2 lM, respectively. The compounds also inhibited HIV replication with potencies comparable with
HIV integrase
Integrase inhibitors
3-Keto salicylic acids
Antiviral activity
Strand transfer inhibitors
Chalcones
Synthesis
3D-QSAR
CoMFA
CoMSIA
their integrase inhibitory potencies. Thus, 5-bromo-2-hydroxy-3-[3-(2,3,6-trichlorophenyl)acryloyl]ben-
zoic acid (25) and 5-bromo-3-[3-(4-bromophenyl)acryloyl]-2-hydroxybenzoic acid (15) inhibited HIV-1
replication with EC50 values of 7.3 and 8.7 lM, respectively. A PHASE pharmacophore hypothesis was
developed and validated by 3D-QSAR, which gave a predictive r2 of 0.57 for an external test set of ten
compounds. Phamacophore derived molecular alignments were used for CoMFA and CoMSIA 3D-QSAR
modeling. CoMSIA afforded the best model with q2 and r2 values of 0.54 and 0.94, respectively. This
model predicted all the ten compounds of the test set within 0.56 log units of the actual pIC50 values;
and can be used to guide the rational design of more potent novel 3-keto salicylic acid integrase
inhibitors
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
as anti-HIV agents. Though, HAART has been effective in reducing
morbidity and mortality, it does not eliminate the virus from
Acquired immunodeficiency syndrome (AIDS) has been a devas-
tating pandemic with around 33 million people infected with it
worldwide. The human immunodeficiency virus (HIV) is impli-
cated in the etiology of AIDS. The pol gene of HIV-1 encodes three
enzymes that are required for viral replication viz., reverse trans-
criptase (RT), integrase (IN) and protease (PR).1 Currently FDA ap-
proved antiretroviral drugs target mainly RT2 and PR3 and are
widely used in combination as highly active antiretroviral therapy
(HAART). Entry inhibitors, for example, Maraviroc4 and fusion
inhibitors such as Enfuvirtide5 have also recently been approved
patients.6 Furthermore, the emergence of multidrug resistant viral
strains along with severe side effect7,8 associated with existing
drugs and the life-long chronic nature of the infection, demand
the development of novel drugs that target other crucial steps of
viral replication and survival.
The integration of viral cDNA into host chromosomes is an
essential step in the viral replication process9 and is responsible
for stable infection. Moreover, IN has no known structural analogs
in human cells.10–13 Inhibition of this enzyme therefore provides
an attractive strategy for antiretroviral drug development.
The function of IN is to integrate viral cDNA into the host gen-
ome. This process occurs in two steps: 30-end processing (cleavage;
30-P) and joining reaction (strand transfer, ST). In the processing
reaction which occurs in the cytoplasm of an infected cell, a site
specific endonucleolytic activity removes the highly conserved 30
terminal CA dinucleotide from the 30-end of the of viral DNA to
generate a reactive nucleophilic 30-hydroxyl group.14,15 In the
strand transfer reaction, the processed 30-hydroxyl end of the viral
Abbreviations: HIV, human immunodeficiency virus; ST, strand transfer; FDA,
food and drug administration; 3D-QSAR, three dimensional quantitative structure
activity relationship; CoMFA, comparitive molecular field analysis; CoMSIA, com-
parative molecular similarity analysis; VdW, Van der Waals; NMR, nuclear
magnetic resonance; HPLC, high performance liquid chromatography.
⇑
Corresponding author. Tel.: +1 901 448 7533; fax: +1 901 448 6828.
E-mail address: jbuolamwini@uthsc.edu (J.K. Buolamwini).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.01.047

H. Sharma et al. / Bioorg. Med. Chem. 19 (2011) 2030–2045
2031
DNA does a nucleophilic attack at the phosphodiester bond of the
host DNA to effect integration.
concentrated sulfuric acid. The acetylated intermediate 5 was then
subjected to Fries rearrangement reaction using anhydrous alumi-
num chloride and heating at 160 °C to transfer the acetyl group
from the 2-position oxygen to the 3-position to yield 3-acetyl sal-
icylic acid intermediate 6.29 The next step involved the Claisen–
Schmidt condensation reaction of intermediate 6 with various aryl
aldehydes at room temperature using NaOH to obtain the target
b-Diketo acid derivatives as represented by S-136016 was the
first integrase inhibitor class to have entered clinical trials. Naph-
thyridine carboxamides such as L-870,81017 have also advanced
to clinical trials as have novel mono keto quinolone carboxylic
acids18,19 such as GS-9137 which is currently undergoing Phase
III clinical trials. In spite of over two decades of extensive research
leading to promising IN inhibitors,20–25 hitherto only one IN inhib-
itor, raltegravir,26 a pyrimidinone carboxamide, has been approved
for clinical use. Moreover, emergence of resistance against
raltegravir due to viral mutations mostly usually T66I, N155H,
Q148H/R and E92Q),27 demands exploration of novel scaffolds for
the treatment of HIV infection.
chalcones (10–57).30 To determine the importance of the
a,b-
unsaturated bond (chalcone scaffold) to IN inhibitory activity, the
synthesis of compound 60 was undertaken by reduction of com-
pound 36 by Pd/C-catalyzed hydrogenation (Scheme 2). The amide
61 was synthesized according to reaction Scheme 3 such that, com-
mercially available 5-bromo salicylic acid (2) was first formylated
with chloroform at the 2-position (ortho) by Reimer–Tiemann
reaction to yield the aldehyde intermediate 7. However, the low
yield of the above reaction, prompted to employ an alternative
synthetic route. Thus, 3-formyl salicylaldehyde 1 was brominated
by NBS in acetonitrile to afford intermediate 7 in higher yields.
In the next step, compound 7 was condensed with 4-fluorobenzyl
amine to afford intermediate 8 which was subsequently subjected
to oxidation with KMnO4 to yield 61. For SAR purposes, compounds
62 and 63 were synthesized by coupling with p-fluorobenzyl
amine following step c of reaction Scheme 3, from the commer-
cially available starting materials 3a and 3b.
We have previously reported novel DKA IN inhibitors with aryl
moiety modifications that were derived from a phenanthridinone
scaffold.28 In continuing our efforts, we report herein the synthesis
and biological evaluation of chalcone derivatives having 3-keto
salicylic acid moieties as a new class of IN inhibitors. We have
undertaken substantial analog synthesis and applied the PHASE
pharmacophore analysis program in conjunction with three
dimensional structure–activity relationship (3D-QSAR) studies to
gain insights into their binding at the IN active site. CoMFA and
CoMSIA 3D-QSAR models were derived to study the influence of
stereoelectronic, hydrophobic and hydrogen bonding interactions
on activity; and to establish predictive tools for design of novel
inhibitors.
2.2. HIV-1 integrase inhibition structure–activity relationship
(SAR)
2. Results and discussion
2.1. Chemistry
After synthesis, the target compounds were tested for integrase
inhibitory activity to assess their potency and study the SAR. The
intergrase 30-cleavage and strand transfer (ST) inhibitory activity
data are presented in Table 1 along with compound structures. In
general, the compounds behaved as typical diketo acid IN inhibi-
tors, being selective against the strand transfer (ST) step compared
to the 30-end processing (30-P).
In the present study, a novel series of IN inhibitors was discov-
ered based on modification of the diketo acid pharmacophoric
group as represented by structure III (Fig. 1). The 2-position OH
and 3-position keto groups together mimic the keto-enol tautomer
of the b-diketo group. Attachment of the carboxylic acid at the
1-position gave the 3-keto salicylic acid moiety of III, intended to
interact with IN similar to the b-diketo acids. A styryl group was
attached to the 3-position keto group to create chalcones (IV)
(Fig. 1), the type of which have not yet been investigated as HIV
IN inhibitors to our knowledge. The chalcone moiety was also
reduced to give an ethylene linker as shown in compound 60.
Furthermore, replacement of the enone group of the chalcones
with an amide group was also investigated.
2.2.1. Substitution on aromatic ring A
Compound 9, 2-hydroxy-3-(3-phenyl-propyl)-benzoic acid, in
which salicylic acid is connected to an unsubstituted phenyl ring
B by a propylene linker exhibited only weak inhibitory activity
with IC50 of >100
ification to a 3-keto salicylic acid along with substitution with Br at
R1 and an ,b-unsaturated linker (chalcone) in compound 10 (used
as reference) demonstrated significant increase in both activity and
selectivity (IC50 25 M for ST vs IC50 75
M for 30-end processing).
lM for ST and 91 l
M for 30-end processing. Mod-
a
l
l
The synthesis of target chalcones was performed according to
reaction Scheme 1. The 5-substituted salicylic acid (X = COOH)
starting material 4 was first acetylated with acetic anhydride and
With the presence of a bromine substituent at the R1 on aromatic
ring A being shown to be important for activity, chloroine and fluo-
rine substituents were also investigated. Compounds with a Br
substituent were the most active followed by F which did a little
better than the corresponding Cl substituted analogs. To determine
the effect of hydrophobic interaction, a methyl group was substi-
tuted for the halogens at R1 (see compound 57). Compound 57
was found to be 13 times less active than the corresponding
Br-substituted compound 25. Since Br and a methyl group are
similar in steric terms, this implies that it is not the hydrophobic
effect of the halogen, but possibly the electronegativity and/or
polarizability that might be dominant. The 3-keto salicylic acid
pharmacophore was designed to form coordinate bonds with
Mg2+. To test this hypothesis, the carboxylic acid was replaced with
NO2 functionality (58 and 59) and this modification diminished
HIV inhibitory activity. Replacement of the carboxyl group with
another polar group but with weaker coordination ability, CN
(compound 56) also resulted in reduced inhibitory activity. This
probably indicates that the formal negative charge of the free ion-
izable acidic moiety, that would chelate better with the metal ion,
O
OH
O
O
OH
O
OH O
O
2
OH
3
1
OH
OH
II
R1
III
I
O
OH
O
2
OH
3
1
Ar
R1
IV
Figure 1. Design of 3-keto salicylic acid pharmacophore of synthesized chalcones.

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H. Sharma et al. / Bioorg. Med. Chem. 19 (2011) 2030–2045
O
CH3
X
O
OH
OH
OH
R1
O
O
X
X
X
c
H3C
b
a
Ar
R1
R1
R1
4
10-57
6
5
Scheme 1. Reagents and conditions: (a) Ac2O, H2SO4; (b) AlCl3, 160 °C; (c) ArCHO, NaOH, EtOH.
We next turned our attention to examining the following. (1)
The effect of a bulky aryl B groups: benzyloxy and chloro substituted
phenoxy in ortho, meta and para-positions of phenyl ring B en-
hanced activity for both 30-P and ST steps; whereas an ortho furan
substituent (compound 50) increased the inhibitory potency
against ST but decreased potency against 30-P; the increase in
activity was most pronounced for the o-benzyloxy substituent
(compound 42), with the 30-P and ST IC50 values lowered to 21
Cl
Cl
OH
F
Cl
O
OH
F
O
COOH
COOH
a
Cl
36
60
Scheme 2. Reagents and conditions: (a) H2, Pd/C (10 mol %), methanol.
and 9
lM, respectively, compared to corresponding IC50 values of
75 and 25
lM for the parent phenyl compound 10. (2) The effects
of electron donating or withdrawing groups: introduction of electron
donating groups such as methoxy, methyl and cyclopentoxy (com-
pounds 19, 39 and 46) decreased integrase inhibitory activity; and,
increasing the number of electron donating groups resulted in
further decrease in activity (compare compounds 19 and 20).
Introduction of strong electron withdrawing groups such as trifluo-
romethyl and nitro also diminished activity as exemplified by
compounds 52 and 53, respectively; (3) Boisosteric replacement
of phenyl ring B with polycyclic and heteroaromatic rings: replace-
ment of phenyl with thiophene or benzothiophene (compounds
45 and 49, respectively) displayed improved activity. Naphthalene
derivatives on the other hand decreased activity (compounds 47
and 48). It thus appears that heteroaromatic B rings may improve
integrase inhibitory activity.
is required for potent activity, supporting the hypothesis. Of course
a chelation study will have to be done to confirm this.
2.2.2. Substitution on aromatic ring B
Various groups were substituted on aromatic ring B to further
explore the SAR. The presence of halogens on phenyl ring B pro-
duced noticeable improvements in potency based on the type,
number and position on the aromatic ring. Fluorine substitution
in the ortho- and meta-positions (compounds 12 and 55) dimin-
ished activity, as did iodine substitution at the para-position (com-
pound 16). However, substitution with Cl enhanced activity
specifically for ST in all the three positions (compounds 11, 13
and 14). Interestingly, compounds 54 and 15 with m-Br and p-Br
substituent, respectively, were potent against both catalytic steps
2.2.3. Reduction of the
chalcones or replacement with an amide moiety
To explore the importance of the ,b-unsaturated bond of the
a,b-unsaturated double bond of the
(compound 15 showed IC50 of 5
11
M against 30-processing). The 2,3,6-trichloro-substituted com-
M against
lM against ST activity and
l
a
pound (25) was the most active, with IC50 of about ꢀ4
l
chalcones on inhibitory activity, we reduced it to a single bond.
Our first attempt at this was with compound 25, but reduction also
led to a loss of the Br substituent. However, reduction (Scheme 2)
of analog 36, having a fluorine substituent, which is not as good a
the ST reaction. This suggests a binding site pocket with high affin-
ity for halogenated phenyl rings. This might be the site that binds
the fluorophenyl group common in potent IN inhibitors like the
clinically used drug raltegravir.
OH
HOOC
a
R1
2
OH
R1
O
OH
O
OH
CHO
CHO
HOOC
c
N
H
d
X
N
H
61 R1= Br, X = COOH
F
F
R1
62 R1 = H, X = CH3
63 R1 = Br, X = H
R1
8
7
b
c
OH
CHO
OH
HOOC
X
HOOC
3a. R1 = H, X = CH3
3b. R1 = Br, X = H
1
R1
Scheme 3. Reagents and conditions: (a) CHCl3, NaOH, H2O; (b) NBS, CH3CN, 2 h (c) 4-F benzylamine, EDCI, HOBT, Et3N; (d) KMnO4, H2O/pyridine, 90 °C.

H. Sharma et al. / Bioorg. Med. Chem. 19 (2011) 2030–2045
2033
Table 1
Structure and HIV-1 integrase inhibitory activities of chalcones
Table 2
Structure and HIV-1 integrase inhibitory activities of synthesized compounds
O
OH
O
OH
X
X
R2
Ar
2
3
1
A
R1
Ar
B
Compd
Ar
R2
R1
X
Activity (IC50
,
l
M)
30-Pa
STb
R1
8
60
61
62
4-F-Ph
2,5-di-Cl-Ph
4-F-Ph
4-F-Ph
4-F-Ph
N/A
NH
CH2
NH
NH
NH
N/A
Br
F
Br
H
Br
N/A
CHO
>100
>100
>100
>100
>100
8
57
15
>100
>100
0.6 0.1
1
5
3
Compd
Ar
Ph
2-Cl-Ph
2-F-Ph
3-Cl-Ph
4-Cl-Ph
4-Br-Ph
4-I-Ph
X
R1
Activity IC50 (lM)
COOH
COOH
CH3
H
N/A
30-Pa
STb
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
S1360c
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
CN
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Cl
Cl
Cl
Cl
Cl
Cl
Cl
F
75 14
52 13
74 25
45 10
81 28
25
14
32
9
12
5
35
7
19 11
34
51 16
18
16
14
13
<3.7
53 21
>100
5
4
3
63
S1360c
11
2
a
b
c
3-P = 30-processing.
ST = strand transfer.
S1360 = standard.
3
1
11
69
4
2
4
2,4-di-Cl-Ph
2,3-di-Cl-Ph
2,3,di-MeO-Ph
2,3,4-tri-MeO-Ph
2,5-di-Cl-Ph
2,6-di-Cl-Ph
3,4-di-Cl-Ph
2,3,5-tri-Cl-Ph
2,3,6-tri-Cl-Ph
4-Cl-Ph
2,4-di-Cl-Ph
2,3-di-Cl-Ph
2,5-di-Cl-Ph
2,3,5-tri-Cl-Ph
2,3,6-tri-Cl-Ph
3,4-di-Cl-Ph
4-Cl-Ph
2,4-di-Cl-Ph
2,3-di-Cl-Ph
2,5-di-Cl-Ph
2,3,5-tri-Cl-Ph
2,3,6-tri-Cl-Ph
3,5-di-Me-Ph
2-Cl-3,4-di-MeO-Ph
6-F-3,4-di-MeO-Ph
2-BzO-Ph
3-(4-Cl-PhO)-Ph
4-(4-Cl-PhO)-Ph
3-Thienyl
3-(Cyclopentyloxy)-Ph
3-(2-OEt-naphthyl)
5-MeO-naphthyl
5-Benzothiophene
2-Furan-2-yl-Ph
3-Cl-4,5-Methylenedioxy-Ph
3-OCF3-Ph
3- NO2-Ph
3-Br-Ph
3-F-Ph
2,3,6-tri-Cl-Ph
2,3,6-tri-Cl-Ph
2,3,5-tri-Cl-Ph
2,3,6-tri-Cl-Ph
N/A
28 12
58 13
62 14
85 20
47 14
52 12
27 11
20 12
23 12
85 13
>100
6
amide moiety (compound 61, see Table 2) was well tolerated with
retention of comparative inhibitory activity but an increase in ST
selectivity. Interestingly, the aldehyde intermediate compound 8,
which was obtained during the synthesis of the amide analog com-
pound 61, was found to be more active than the carboxylic acid
product 61, indicating that an aldehyde group can substitute for
the carboxylic acid group in these compounds, opening an oppor-
tunity for exploring other bioisosteric substitutions.
4
8
4
5
100
75
92 14
25
55 40
77 20
87 13
36 13 17
52 13 24
55 18 30
17
30
41
3
5
9
3
9
9
6
4
6
2.3. Inhibition of HIV replication in cell culture
To evaluate the potential of these new IN inhibitors as antiviral
agents, five potent analogs were tested for the ability to inhibit
HIV-1 replication in primary human peripheral blood mononuclear
cells (PBMCs) infected with the virus. The toxicity of the com-
pounds was also tested against PBMC and human CEM lympho-
blastic leukemia cells. All five compounds tested inhibited HIV
replication (Table 3). The most potent compounds, 25 and 15
F
F
F
F
50
29
33 14 15
82 16 39
F
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
CH3
Br
Br
N/A
>100
>100
>100
21
58
76
76
9
showed comparable EC50 values of 7.3 and 8.3
Compound 49 (integrase ST IC50 = 11 M) exhibited an EC50 of
10 M, while analog 17 (integrase ST IC50 = 7.0 M) displayed an
EC50 of 13.9 M. Thus, in general the biological activity correlated
lM, respectively.
25
22
30 12
100
92
18
16
18
55
46
l
l
l
1
l
well with the IN inhibitory potency. Moreover, the two most po-
tent IN inhibitory compounds of the set tested, 15 and 25, were
also found to have the highest activity against HIV replication, sug-
gesting that IN might indeed be their antiviral target. These results
demonstrate moderate antiviral activity, which is not very differ-
ent from early members of the diketo acid IN inhibitors, which
served as important lead compounds such as L-706,908 which
54
54 25
23
>100
79
3
11
16
63
52
32
18
59
44
50
68
21
1
2
>100
49
19
71
>100
>100
>333
8
4
1
6
1
2
2
9
5
3
had an EC50 of 5.7 4.7
The reason that compound 8 (integrase ST IC50 = 8.0
the carboxylic acid is replaced by an aldehyde group showed a low-
er antiviral activity (EC50 = 30.8 M) than expected is not apparent,
l
M for inhibition of HIV replication.31
M), in which
l
COOH
NO2
NO2
l
36
11
4
2
N/A
0.6 0.1
but may have to do with solubility, absorption, binding to serum
proteins in cell culture media and/or cellular distribution factors.
The compounds showed mild cytotoxicity, with selectivity index
up to fourfold for compound 49. It may be noteworthy that com-
pound 49 is the only compound among the antiviral test set that
had a 5-benzothiophene for ring B; this may offer clues for improv-
ing selectivity index. These results demonstrate reasonable antivi-
ral activity when compared to the antiviral activity of reported
diketo acid inhibitors like L-706,908 which has an EC50 of
a
b
c
3-P = 30-processing.
ST = strand transfer.
S1360 = standard.
leaving group as Br, was successful in achieving our objective. The
reduction product, compound 60, was less active than unsaturated
analog, showing that the reduction of the double bond in the chal-
cone template is detrimental to activity. Bioisosteric replacement
5.7 4.7
spective with regard to AZT’s activity, in the same assay, AZT had
an EC50 of 0.001
M.31
l
M31 for inhibition of HIV replication. To put data in per-
of the
a,b-unsaturated carbonyl system in the chalcone with an
l

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H. Sharma et al. / Bioorg. Med. Chem. 19 (2011) 2030–2045
O
OH
O
N
N
O
N
H
N
O
F
OH
O
O
O
O
S
O
L-706,908
L-870,810
O
OH
F
N
N
NH
S-1360
QSAR. Random external test sets were generated automatically to
determine the predictive ability of the models. The hypothesis that
afforded the best PLS statistics (Table 4) and the highest predicted
ability consisted of six features viz., two H-bond acceptors, two
hydrophobic groups, a negative group and one aromatic ring
(Fig. 2a). Two H-bond acceptor features mapped onto the oxygen
of the hydroxyl and the 3-keto moiety and negative feature
mapped onto the carboxylate group of the salicylic acid pharmaco-
phore. The two hydrophobic features mapped to the halogen sub-
stituent of ring A and the para-substituted halogen in aryl ring B.
The aromatic pharmacophoric feature mapped onto aromatic ring
A. Residuals of the training and the test set are listed in Tables 5
and 6, respectively. The prediction curve for the test set is shown
in Figure 2b. As can be seen, the hypothesis gave a good predictive
model with a predictive r2 of 0.57 for an external test set of ten
3. Molecular modeling
3.1. Generation of a pharmacophore model using the PHASE
program
Knowledge of the structure of target protein inhibitor interac-
tions is limited as no crystal structure of human integrase–
ligand–DNA complex is available. Therefore, to explore the bioactive
conformation of these novel IN inhibitors, the PHASE32 (v3.0) phar-
macophore mapping program implemented in Maestro modeling
program package (v8.5; Shrodinger Inc.) was used. The 3D structures
of molecules were constructed in and imported from the SYBYL
modeling program (v8.3, Tripos).
Conformers of each molecule were generated using the
OPLS_2005 force field in the macromodel conformational analysis
program of PHASE. A set of pharmacophoric sites based on features
defined in PHASE were assigned to the molecules. These included
H-bond acceptor (A), H-bond donor (D), hydrophobic group (H),
negatively charged group (N) and aromatic ring (R). The three most
potent compounds of the series, compound 15, 17 and 25 were
selected as actives for use in identifying a common pharmacophore
hypothesis using a tree-based partitioning technique of PHASE. The
resulting pharmacophores were then scored and ranked.
Table 4
PLS Statistics of PHASE 3D-QSAR model
q2
0.57
0.74
0.17
r2
SD
F
28.3
PLS Factors
Pearson R
4
0.75
The top twenty pharmacophore hypotheses were analyzed and
validated by partial least square (PLS) regression-based PHASE 3D-
Table 3
Anti-HIV activity of selected novel potent derivatives
Compd
Anti-HIV-1 activity in PBMCsa
Slope
R
Cytotoxicity (IC50
PBMCs
, lM)
b
b
EC50
(l
M)
EC90
(
lM)
CEM
15
17
25
49
8
AZT
L-706,908
8.7
13.9
7.3
10.1
30.8
21.0
26.0
20.3
24.3
52.7
0.01
N/A
2.5 0.61
3.5
2.2 0.96
2.5 0.89
4.1
0.97
1.0
0.91
0.94
1.0
24.4
22.1
22.7
39.1
44.0
>100
N/A
27.5
23.8
19.0
20.7
40.2
56.1
N/A
0.0025
1.6 0.30
N/A
0.96
N/A
5.7 4.7c
a
b
c
Human peripheral blood mononuclear cells (PBMCs).
EC50 and EC90 are the effective concentrations inhibiting 50% and 90% HIV replication, respectively.
In the human leukemic T-cell line MOLT-4; in that assay the EC50 of AZT was 0.001 M.
l

H. Sharma et al. / Bioorg. Med. Chem. 19 (2011) 2030–2045
2035
Figure 2. The best pharmacophore hypothesis (AAHHNR) mapped onto (a) compound 17 (b) alignment of actives and (c) inactives from PHASE pharmacophore mapping.
Pharmacophore features are: red spheres and vectors for H-bond acceptor (A), brown rings for aromatic groups (R), dark red spheres refer to a negative feature (N) and green
spheres indicate hydrophobic groups (H). Atoms in the ligands are represented as O, red; Br, wine red; Cl, dark green; H, white; C, gray. (d) PHASE 3D-QSAR predictions of the
test set.
compounds. It was also able to distinguish active and inactive
compounds, with the latter group not aligning well to the features
(Fig. 2c and d).
27, 28, 55 and 57. The reasons for compound 55 being an outlier
are not apparent. With compound 16 (iodo substituted) and com-
pound 57 (R1 = CH3), their outlier status may stem from structural
uniqueness, whereas the outlier status of compounds 27 and 28
(least active analogs, R1 = Cl) may drive from different binding
modes. The CoMFA and CoMSIA predictions for the training and
the test sets are shown in Figure 4a and b, respectively. The CoMFA
and CoMSIA residual values for the training set are given in Table 8
and the corresponding values for the test set in Table 9. In concor-
dance with the higher q2 value, the CoMSIA model performed
better than CoMFA model, and predicted all test set compounds
within 0.56 log unit of the actual pIC50 values.
3.2. 3D-QSAR analysis
3.2.1. CoMFA and CoMSIA 3D-QSAR models
Statistical analysis by PLS33,34 was done using CoMFA and CoM-
SIA descriptors as independent variables and biological activity in
the form of pIC50 values as dependent variable. The molecular
alignment used for CoMFA and CoMSIA 3D-QSAR modeling was
derived from PHASE pharmacophore mapping (Shrodinger, Inc.).
PHASE derived pharmacophore hypotheses have been successfully
used to derive ‘bioactive conformations’ for 3D-QSAR modeling in
previous work from our laboratory35 and elsewhere (Telvekar
et al.).36 Thus, ‘bioactive conformations’ of all the molecules from
PHASE conformational analysis were superimposed using the
MATCH alignment tool in SYBYL (Fig. 3). Fifty three (53) synthe-
sized target compounds were used for the 3D-QSAR analyses. The
Leave-One-Out (LOO) method of cross-validation was used for ini-
tial assessment of the predictive abilities of the models with the
training sets. The optimal number of components used in the final
QSAR models was that which gave the smallest standard error of
prediction. Reliability of the models was tested by prediction of
10 compounds selected as an external test set using factor analysis.
The PLS analysis results from both the CoMFA and CoMSIA models
are summarised in Table 7. Cross-validated q2 values of 0.43 and
0.54 were obtained for CoMFA and CoMSIA, respectively. Results
of rigorous statistical testing using group cross-validation (20
groups), bootstrapping and randomization of activity data demon-
strated the robustness of the QSAR models. Both CoMSIA and CoM-
FA identified six outliers, which were excluded on the basis of their
extreme residual values (Table 8). Out of these, five outliers were
common to both CoMFA and CoMSIA. These were compounds 16,
3.2.2. CoMFA and CoMSIA contour maps
The CoMFA and CoMSIA contour maps derived from the PLS
coefficients are displayed in Figure 5. Steric CoMFA maps
(Fig. 5a) show green contours around the ortho and para-positions
on aryl ring B indicating that bulky groups are favored at these
positions. This is validated by the fact that a benzyl group substitu-
tion at the ortho-position resulted in higher activity (compound
42). Compounds 44 and 49, which have a bulky chloro phenoxy
and thiophene groups around the green contour at the para-posi-
tion also displayed better inhibitory activities. A large yellow con-
tour surrounds a small green contour at the meta-position on aryl
ring B suggesting that only a limited bulk is favorable for activity.
This is apparent by the loss of inhibitory activity observed with
compound 46, the bulky cyclopentyl ring of which occupies the
sterically disfavored yellow contour region. There is also a green
contour evident at the R1-position on the aromatic ring A. This
may be the reason why among the three halogens at that position,
bromine, which is the bulkiest, afforded the highest activity. At the
aryl ring B, CoMFA electrostatic map reveals a red contour at the 30-
position suggesting that electronegative groups could increase
activity at this position. This is most exemplified by the 30-Cl

2036
H. Sharma et al. / Bioorg. Med. Chem. 19 (2011) 2030–2045
Table 5
Residuals of training set by PHASE 3D-QSAR
Compd Actual ST
pIC50
Predicted ST
pIC50
Residuals
10
11
12
14
15
16
17
18
19
20
21
22
24
25
26
27
28
32
33
34
35
36
37
39
40
41
42
43
44
45
46
47
48
49
53
54
55
56
57
58
59
61
63
4.60
4.85
4.49
4.92
5.30
4.45
5.15
4.72
4.46
4.29
4.74
4.79
4.88
5.40
4.27
4.00
4.03
4.39
4.76
4.62
4.52
4.54
4.82
4.23
4.12
4.12
5.04
4.74
4.79
4.74
4.26
4.33
4.26
4.96
4.49
4.74
4.23
4.40
4.30
4.16
4.68
4.82
4.00
4.85
4.59
4.53
4.84
4.94
4.85
5.02
4.66
4.59
4.29
4.87
4.83
4.82
5.40
4.49
4.21
4.48
4.44
4.63
4.33
4.44
4.63
4.59
4.42
3.94
4.15
4.99
4.79
4.79
4.85
4.32
4.16
4.28
4.86
4.46
4.60
4.32
4.24
4.37
4.10
4.80
4.83
4.26
ꢁ0.25
0.26
ꢁ0.04
0.08
0.36
ꢁ0.4
0.13
0.06
ꢁ0.13
0.00
ꢁ0.13
ꢁ0.04
0.06
0.00
ꢁ0.22
Figure 3. Superimposition of the conformation of molecules obtained from PHASE
pharmacophore analysis. Atoms are represented as O, red; Br, dark green; Cl, green;
F, bright green; H, cyan; C, white; S, yellow; N, blue.
ꢁ0.21
ꢁ0.45
ꢁ0.05
0.13
0.29
0.08
Table 7
ꢁ0.09
PLS Statistics of CoMFA and CoMSIA 3D-QSAR
0.23
PLS statistics
CoMFA
CoMSIA
ꢁ0.19
0.18
q2
0.43
0.94
0.09
78.32
6
0.54
0.94
0.09
78.69
6
r2
ꢁ0.03
0.05
s
F
ꢁ0.05
0.00
ꢁ0.11
ꢁ0.06
0.17
PLS components
Field contribution
Steric
Electrostatic
Hydrophobic
0.474
0.526
0.100
0.428
0.283
0.146
0.044
0.96 0.01
0.39
ꢁ0.02
0.10
Donor
Acceptor
0.03
0.14
Bootstrapping r2 (20 runs)
Randomization q2 (20 runs)
Cross-validation q2 (20 runs)
0.96 0.01
0.33
0.44
ꢁ0.09
0.16
0.50
ꢁ0.07
0.06
ꢁ0.12
ꢁ0.01
ꢁ0.26
these compounds. The red contours around the salicylic acid moi-
ety in aryl ring A suggest that a negative charge is important for
activity in this region, possibly for chelation with the active site
Mg2+ ions. This is in agreement with the proposed mechanism of
action of these compounds as HIV-1 integrase inhibitors. There is
a red contour around the 40-position and both blue and red con-
tours at the 50-position (R1). This suggests that polarizability effect
might be important at R1 and could explain the increased activity
conferred by bromine substitution over other halogens and the
similar-sized hydrophobic methyl group at this position.
The CoMSIA steric contour map (Fig. 5b) also shows a large
green contour around aryl ring B. Like in the CoMFA, the electro-
static contours of CoMSIA also show red contours at the 30 and
the 40-positions on ring B, and at the 50-position (R1) on ring A.
There is a blue contour located at the position of the hydrogen
atom of the hydroxyl group of the salicylic acid moiety. The hydro-
phobic, H-bond donor and acceptor contours are shown in Figure
5c. There is a white contour located near the R1 substituent posi-
tion which suggests hydrophophilic groups increase activity at this
region. This might be the reason why compound 57 with a hydro-
phobic methyl group at this position was less active than the cor-
responding halogen compounds. The improved activity observed
with bromine at R1-position may be the result because of a combi-
nation of polarizability effects. White contours are also evident at
the 20 and the 50-positions on the B ring, indicating that hydrophilic
groups are favored for activity. This might be the explanation, or at
least in part, for the decrease in potency of compounds 19 and 39,
which have hydrophobic methoxy and methyl groups at these re-
Table 6
Residuals of test set by PHASE 3D-QSAR
Compd
Actual ST pIC50
Predicted ST pIC50
Residual
13
23
29
30
31
38
50
51
52
62
5.04
4.85
4.60
4.77
4.52
4.41
4.80
4.20
4.28
4.00
4.86
4.78
4.71
4.63
4.25
4.28
4.91
4.51
4.46
4.35
0.18
0.07
ꢁ0.11
0.14
0.27
0.13
ꢁ0.11
ꢁ0.31
ꢁ0.18
ꢁ0.35
substitution in compound 13 which had an IC50 value of 9 lM.
However, for compound 55, a decrease in activity was observed.
Red contours are also present at the 50- and the 60-positions. The
dichloro-substituted compounds 21 and 22 having Cl at the 50-
and 60-positions showed improved activities. There are also distant
blue contours around aryl ring B, which predicts that electroposi-
tive groups should increase activity at these positions. Electroneg-
ative oxygen atom of the methoxy substituents in compounds 20,
40 and 41 points towards the blue contours and thus may explain
the loss of activity observed by the presence of methoxy groups in

H. Sharma et al. / Bioorg. Med. Chem. 19 (2011) 2030–2045
2037
Table 8
Residuals of training set of CoMFA and CoMSIA models
Table 9
Residuals of the test set predictions given by CoMFA and
CoMSIA models
Compd
Actual ST
pIC50
Residuals
Compd
Actual ST pIC50
Residuals
CoMFA
CoMSIA
CoMFA
CoMSIA
10
11
12
13
14
15
16a
17
18
19
20
21
22
23
24
25
26
27a
28a
29
30
31
33
34
35
36
38
39
40
43
44c
46
47
48
49
51
52b
55a
56
57a
58
62
63
4.60
4.85
4.49
5.04
4.92
5.30
4.45
5.15
4.72
4.46
4.29
4.74
4.79
4.85
4.88
5.40
4.27
4.00
4.03
4.60
4.77
4.52
4.76
4.62
4.52
4.54
4.41
4.23
4.12
4.74
4.79
4.26
4.33
4.26
4.96
4.20
4.28
4.23
4.35
4.30
4.16
4.00
4.00
ꢁ0.13
0.12
ꢁ0.12
0.12
32
37
41
42
45
50
53
54
59
61
4.39
4.82
4.12
5.04
4.74
4.79
4.49
4.74
4.67
4.82
ꢁ0.41
0.35
ꢁ0.28
0.30
ꢁ0.32
0.35
0.37
0.09
0.22
0.05
0.56
0.40
ꢁ0.18
ꢁ0.19
0.05
0.03
ꢁ1.00
ꢁ0.15
0.16
ꢁ0.08
0.11
0.25
0.14
0.09
ꢁ0.62
ꢁ0.96
0.21
0.19
ꢁ0.10
0.10
0.06
ꢁ0.07
ꢁ0.04
ꢁ0.003
0.04
ꢁ0.03
ꢁ0.03
0.11
0.19
0.11
0.76
ꢁ0.05
0.01
ꢁ0.03
ꢁ0.13
0.07
ꢁ0.05
ꢁ0.02
ꢁ0.62
ꢁ0.79
0.02
tionality perfectly fits the magenta contour within its vicinity indi-
cating that they may act as H-bond acceptors. A cyan contour
appears close to, and merges into the magenta contour, suggesting
that the OH group is a H-bond donor at this position. It may be in-
volved in H-bonding interactions with the acceptor carboxylate
groups of the acidic catalytic triad residues of intergase active site.
The general agreement between the maps and the observed biolog-
ical activity trends suggests that these models could aid in the
design of more active compounds.
ꢁ0.05
0.09
ꢁ0.89
ꢁ0.74
0.005
0.11
0.06
0.04
ꢁ0.13
ꢁ0.12
0.04
ꢁ0.04
ꢁ0.05
ꢁ0.02
ꢁ0.01
0.56
0.01
0.03
ꢁ0.009
ꢁ0.02
ꢁ0.03
0.06
ꢁ0.001
ꢁ0.05
ꢁ0.02
ꢁ0.07
ꢁ0.07
ꢁ0.03
0.07
0.002
0.10
0.01
ꢁ0.84
ꢁ0.41
ꢁ0.05
ꢁ1.03
ꢁ0.01
0.07
4. Conclusion
In summary, we have successfully synthesized and evaluated
the HIV-1 integrase inhibitory activities of a series of chalcone
derivatives and related amides harboring a 3-keto salicylic acid
moiety as a diketo acid isostere. Consistent with the DKA class of
IN inhibitors, these series of compounds were also generally selec-
tive against the IN strand transfer catalytic step. Introduction of a
0.00
0.09
ꢁ0.03
0.04
ꢁ0.07
ꢁ0.09
ꢁ0.31
ꢁ0.01
ꢁ1.03
0.11
Br substituent at the R1-position and an a,b-unsaturated carbonyl
linker connecting aryl rings A and B resulted in significant
enhancement of inhibitory activity. The most active compound
has 2,3,6-trichloro substitution on aryl ring B. In the absence of a
crystal structure of the complete human integrase, inhibitor and
DNA complex, bioactive conformational hypothesis was obtained
and validated by PHASE pharmacophore mapping and 3D-QSAR
analysis. Statistically significant CoMFA and CoMSIA 3D-QSAR
models were obtained using an alignment derived from the
pharmacophore analysis, and may be useful for the design and
prediction of the activities of novel related compounds as HIV-1
integrase inhibitors. Last but not the least, most of the potent com-
pounds also inhibited HIV replication in cell culture with moderate
antiviral activity, which supports further development of the
series.
0.04
ꢁ0.05
ꢁ0.047
a
b
c
Represents common outliers.
Outlier only for CoMFA.
Outlier only for CoMSIA.
gions. Another white contour appears at the 3-keto oxygen again
signifying the need for hydrophilic groups at this position. There
is a yellow contour close to the 40-position on ring B, suggesting
that hydrophobic character is favored for activity. The 30-keto func-
Figure 4. Predictions of ST activities of the training (blue diamonds) and test set (red triangles) compounds by (a) CoMFA and (b) CoMSIA

2038
H. Sharma et al. / Bioorg. Med. Chem. 19 (2011) 2030–2045
Figure 5. (a) CoMFA and (b) CoMSIA steric and electrostatic contour plots. The green contours indicate regions where bulky groups increase activity, whereas yellow contours
indicate regions where bulky groups decrease activity. Blue contours indicate regions where electropositive groups increase activity, whereas red contours depict regions
where electronegative groups increase activity. (c) CoMSIA hydrophobic, H-bond donor and H-bond acceptor contour plots; white contours indicate regions where
hydrophilic groups increase activity, whereas yellow contours indicate regions where hydrophobic groups increase activity, cyan indicates region where H-donors increase
activity and magenta contour indicate region where H-bond acceptor groups increase activity.
Na2SO4. The solvent was evaporated under reduced pressure to
obtain a crude solid which was recrystalized from ethyl acetate/
hexane to afford white crystals (5a–5c).
5. Experimental section
All reagents and solvents were purchased from the Aldrich
Chemical Company and used without further purification. Progress
of reactions was monitored by TLC on silica gel GHLF-250 micron
plates (Analtech, Inc.). Fisher scientific Da visil grade 1740 (170–
400 mesh) silica gel was used for flash column chromatography.
1H NMR spectra were recorded on Brucker AR, 300 or 500 MHz
spectrometer: chemical shifts are expressed in d values (ppm)
and coupling constants (J) in Hertz. Mass spectral data were
determined on a Brucker-HP Esquire-LC spectrometer (ESI-MS).
Melting points of final products were determined using a Fisher-
Johns melting point apparatus and are reported uncorrected. HPLC
analysis of final compounds was carried out using a reverse phase
5.1.1. 2-Acetoxy-5-bromobenzoic acid (5a)
Yield 88%; mp 155–157 °C; 1H NMR (300 MHz, DMSO-d6): d
13.44 (br s, 1H, COOH), 8.0 (d, 1H, J = 2.4 Hz, ArH), 7.85 (dd, 1H,
J1 = 2.7 Hz, J2 = 8.7 Hz, ArH), 7.20 (d, 1H, J = 8.7 Hz, ArH), 2.2 (s,
3H, OCOCH3); MS (ESI): m/z 216.6 [MꢁCOCH3]ꢁ.
5.1.2. 2-Acetoxy-5-fluorobenzoic acid (5b)
Yield 85%; mp 133–134 °C; 1H NMR (300 MHz, DMSO-d6): d
13.42 (br s, 1H, COOH), 7.67 (dd, 1H, J1 = 3.3 Hz, J2 = 9.0 Hz, ArH),
7.51 (ddd, 1H, J1 = 8.7 Hz, J2 = 8.1 Hz, J3 = 3.3 Hz, ArH), 7.26 (dd,
1H, J1 = 4.8 Hz, J2 = 8.7 Hz, ArH), 2.24 (s, 3H, OCOCH3); MS (ESI):
m/z 154.7 [MꢁCOCH3]ꢁ.
SUPELCOSIL 5
lm C-18 column of dimensions 25 ꢂ 4.6 cm. Area%
purity was detected at 254 nm. An initial isocratic method com-
prising 40% water (solvent A) and 60% methanol (solvent B) for
20 min at a flow-rate of 1.2 mL/min was optimized to a linear
gradient elution with water (A) and methanol (B) at a flow-rate
of 1.7 mL/min. Gradient elution of the mobile phase was either
(a) 80% A to 35% A from 0 to 5 min, 35% A, 65% B for 5–12 min,
and 35% A to 80% A from 12 to 15 min or (b) 60% A to 20% A from
0 to 4 min, 20% A and 80% B for 4–12 min and 20% A to 60% A from
12 to 15 min. The purity of all, but two, compounds was found to
be P95%.
5.1.3. Acetoxy-5-chlorobenzoic acid (5c)
Yield 85%; mp 150–152 °C; 1H NMR (300 MHz, DMSO-d6): d
7.88 (d, 1H, J = 2.7 Hz, ArH), 7.72 (dd, 1H, J1 = 2.7 Hz, J2 = 9.0 Hz,
ArH), 7.27 (d, 1H, J = 8.4 Hz, ArH), 2.24 (s, 3H, OCOCH3); MS (ESI):
m/z 170.7 [MꢁCOCH3]ꢁ.
5.1.4. Acetic acid-4-bromo-2-cyanophenyl ester (5d)
Yield 76%; mp 60–61 °C; 1H NMR (300 MHz, DMSO-d6): d 8.26
(d, 1H, J = 2.7 Hz, ArH), 8.0 (dd, 1H, J1 = 2.4 Hz, J2 = 8.7 Hz, ArH),
7.44 (d, 1H, J = 8.7 Hz, ArH), 2.29 (s, 3H, OCOCH3); MS (ESI): m/z
197.5 [MꢁCOCH3]ꢁ.
5.1. General procedure for the synthesis of acetylated
intermediates (5a–5e)
5.1.5. 2-Acetoxy-5-methylbenzoic acid (5e)
5-Halogenated salicylic acids (46 mmol) were stirred with
acetic anhydride (20 mL) and 80 lL of concd H2SO4 was then
added. After a few (20) min the reaction mixture solidified and
was poured into cold water. The solid so obtained was filtered
and extracted with ethyl acetate, washed with brine and dried over
Yeild 75%; mp 158–160 °C; 1H NMR (300 MHz, DMSO-d6): d
8.01 (d, 1H, J = 1.5 Hz, ArH), 7.50 (dd, 1H, J1 = 1.8 Hz, J2 = 8.4 Hz,
ArH), 7.12 (d, 1H, J = 8.1 Hz, ArH), 2.43 (s, 3H, OCOCH3), 2.27 (s,
3H, CH3); MS (ESI): m/z 150.7 [MꢁCOCH3]ꢁ.

H. Sharma et al. / Bioorg. Med. Chem. 19 (2011) 2030–2045
2039
5.2. General procedure for the synthesis of 3-acetyl-5-halo
salicylic acids (6a–6e)
J1 = 1.8 Hz, J2 = 8.4 Hz, ArH), 7.46 (ddd, 2H, J1 = 1.8 Hz, J2 = 7.2 Hz,
J3 = 7.5 Hz, ArH); MS (ESI): m/z 378.6, 380.6 [MꢁH]ꢁ HPLC: tR
8.05 min, purity 98.82%.
The acetylated intermediates (38.61 mmol) and AlCl3
(120 mmol) were mixed in a three-necked flask and heated to
160 °C under mechanical stirring. After 3 h, the reaction mixture
was cooled to room temperature and poured into ice containing
20 mL concentrated HCl. The slurry was extracted with ethyl ace-
tate, acidified with 1 M HCl, washed with brine and dried over
Na2SO4. The solvent was evaporated to give the crude product,
which was washed with dichloromethane for removal of impuri-
ties, filtered and dried to give 3-acetyl salicylic acids as pale brown
powders.
5.3.3. 5-Bromo-3-[3-(2-fluorophenyl)-acryloyl]-2-hydroxy-
benzoic acid (12)
Yellow solid, yield 47%; mp 185–187 °C; 1H NMR (300 MHz,
DMSO-d6): d 8.05 (d, 1H, J = 2.1 Hz, ArH), 8.0 (s, 1H, H-b), 7.93 (t,
1H, J = 7.2 Hz, ArH), 7.74 (s, 1H, H-a), 7.73 (d, 1H, J = 2.7 Hz, ArH),
7.51 (d, 1H, J = 6.9 Hz, ArH), 7.31 (dd, 1H, J1 = 7.2 Hz, J2 = 8.7 Hz,
ArH), 7.29 (t, 1H, J = 7.2 Hz, ArH); MS (ESI): m/z 362.7, 364.7
[MꢁH]ꢁ HPLC: tR 7.24 min, purity 99.39%.
5.3.4. 5-Bromo-3-[3-(3-chlorophenyl)-acryloyl]-2-hydroxy-
benzoic acid (13)
5.2.1. 2-Acetoxy-3-acetyl-5-bromobenzoic acid (6a)
Yield 34%; mp 182–185 °C; 1H NMR (300 MHz, DMSO-d6): d
8.06 (d, 1H, J = 2.7 Hz, ArH), 7.97 (d, 1H, J = 2.7 Hz, ArH), 2.60 (s,
3H, COCH3); MS (ESI): m/z 258.7 [MꢁH]ꢁ.
Yellow solid, yield 56%; mp 234–236 °C; 1H NMR (300 MHz,
DMSO-d6): d 8.06 (d, 1H, J = 2.7 Hz, ArH), 8.01 (d, 1H, J = 2.7 Hz,
ArH), 7.92 (s, 1H, ArH), 7.76 (d, 1H, J = 15.9 Hz, H-b), 7.75 (t, 1H,
J = 7.8 Hz ArH), 7.63 (d, 1H, J = 15.9 Hz, H-a), 7.50 (dd, 2H,
5.2.2. 2-Acetoxy-3-acetyl-5-fluorobenzoic acid (6b)
J1 = 1.5 Hz, J2 = 8.7 Hz, ArH); MS (ESI): m/z 378.6, 380.6 [MꢁH]ꢁ.
HPLC: tR 7.67 min, purity 99.30%.
Yield 31%; mp 135–136 °C; 1H NMR (300 MHz, DMSO-d6): d
7.78 (dd, 1H, J1 = 3.3 Hz, J2 = 8.1 Hz, ArH), 7.72 (dd, 1H, J1 = 3.3 Hz,
J2 = 8.7 Hz, ArH), 2.62 (s, 3H, COCH3); MS (ESI): m/z 196.7 [MꢁH]ꢁ.
5.3.5. 5-Bromo-3-[3-(4-chlorophenyl)-acryloyl]-2-hydroxy-
benzoic acid (14)
5.2.3. 2-Acetoxy-3-acetyl-5-chlorobenzoic acid (6c)
Yield 32%; mp 160–163 °C; 1H NMR (300 MHz, DMSO-d6): d
7.95 (d, 1H, J = 3.0 Hz, ArH), 7.85 (d, 1H J = 2.7 Hz, ArH), 2.61 (s,
3H, COCH3); MS (ESI): m/z 212.7 [MꢁH]ꢁ.
Yellow solid, yield 62%; mp 238–241 °C; 1H NMR (300 MHz,
DMSO-d6): d 8.04 (d, 1H, J = 15.3 Hz, H-b), 7.91 (d, 1H, J = 2.4 Hz,
ArH), 7.74 (d, 2H, J = 8.4 Hz, ArH), 7.71 (d, 1H, J = 3.0 Hz, ArH),
7.58 (d, 1H, J = 15.3 Hz, H-a), 7.50 (d, 2H, J = 8.4 Hz, ArH); MS
(ESI): m/z 380.6 [MꢁH]ꢁ. HPLC: tR 8.1 min, purity 99.10%.
5.2.4. 3-Acetyl-5-bromo-2-hydroxybenzonitrile (6d)
Yield 26%; mp 75–77 °C; 1H NMR (300 MHz, DMSO-d6): d 13.17
(s, 1H, OH), 8.41 (d, 1H, J = 2.4 Hz, ArH), 8.32 (d, 1H J = 2.4 Hz, ArH),
2.68 (s, 3H, COCH3); MS (ESI): m/z 239.6 [MꢁH]ꢁ.
5.3.6. 5-Bromo-3-[3-(4-bromophenyl)-acryloyl]-2-hydroxy-
benzoic acid (15)
Yellow solid, yield 34%; mp 236–238 °C; 1H NMR (300 MHz,
DMSO-d6): d 8.06 (d, 1H, J = 2.7 Hz, ArH), 8.02 (d, 1H, J = 2.7 Hz,
ArH), 7.74 (d, 2H, J = 8.7 Hz, ArH), 7.69 (s, 1H, H-b), 7.67 (d, 2H,
5.2.5. 3-Acetyl-2-hydroxy-5-methylbenzoic acid (6e)
Yield 28%; mp >180 °C (dec); 1H NMR (300 MHz, DMSO-d6): d
7.80 (s, 1H, ArH), 7.64 (s, 1H, ArH), 2.59 (s, 3H, COCH3), 2.24 (s,
3H, CH3); MS (ESI): m/z 192.7 [MꢁH]ꢁ.
J = 8.7 Hz, ArH), 7.65 (s, 1H, H-
HPLC: tR 6.05 min, purity: 99.02%.
a
); MS (ESI): m/z 424.6 [MꢁH]ꢁ.
5.3. General procedure for the synthesis of chalcones (10–57)
5.3.7. 5-Bromo-2-hydroxy-3-[3-(4-iodophenyl)-acryloyl]benzoic
acid (16)
Equimolar amounts of aromatic aldehydes and intermediates
6a–6e were dissolved in 10 mL of ethanol, and 5 mL of 25% aq
NaOH was added. More solvent was added to help stirring in cases
of excessive precipitation. The reaction mixture was stirred at rt for
1–5 days or until completion of reaction (6 h for compound 56).
The mixture was then poured into ice and acidified with 3 N HCl
to pH 4. The yellow precipitate formed was filtered and washed
with water. The crude solid was purified by column chromatogra-
phy (10–25% ethyl acetate/hexane) and/or recrystalized with etha-
nol or THF/water to yield pure chalcones.
Yellow solid, yield 48%; mp 237–239 °C; 1H NMR (300 MHz,
DMSO-d6): d 8.06 (d, 1H, J = 2.7 Hz, ArH), 8.01 (d, 1H, J = 2.7 Hz,
ArH), 7.83 (d, 1H, J = 8.4 Hz, ArH), 7.73 (d, 1H, J = 15.9 Hz, H-b),
7.69 (d, 1H, J = 8.4 Hz, ArH), 7.60 (d, 1H, J = 15.3 Hz, H-a), 7.59 (d,
2H J = 8.4 Hz, ArH); MS (ESI): m/z 472.6 [MꢁH]ꢁ. HPLC: tR
6.46 min, purity 94.70%.
5.3.8. 5-Bromo-3-[3-(2,4-dichlorophenyl)-acryloyl]-2-hydroxy-
benzoic acid (17)
Yellow solid, yield 54%; mp 234–235 °C; 1H NMR (300 MHz,
DMSO-d6): d 8.08 (d, 1H, J = 2.7 Hz, ArH), 8.05 (d, 1H, J = 8.4 Hz,
ArH), 8.05 (d, 1H, J = 2.7 Hz, ArH), 7.87 (d, 1H, J = 15.9 Hz, H-b),
5.3.1. 5-Bromo-2-hydroxy-3-(3-phenyl-acryloyl)benzoic acid
(10)
7.76 (d, 1H, J = 15.9 Hz, H-a), 7.76 (d, 1H, J = 2.1 Hz, ArH), 7.52
Yellow solid, yield 58%; mp 210–212 °C; 1H NMR (300 MHz,
MeOH-d4): d 8.08 (d, 1H, J = 2.4 Hz, ArH), 8.04 (d, 1H, J = 2.7 Hz,
(dd, 1H, J1 = 1.5 Hz, J2 = 8.4 Hz, ArH); MS (ESI): m/z 414.8 [MꢁH]ꢁ.
HPLC: tR 10.91 min, purity 98.23%.
ArH), 7.81 (dd, 2H, J1 = 2.1 Hz, J2 = 7.5 Hz, ArH), 7.68 (s, 2H, H-
a
and H-b), 7.42–7.44 (m, 3H, ArH); MS (ESI): m/z 346.6 [MꢁH]ꢁ;
HPLC: tR 7.71 min, purity 99.10%.
5.3.9. 5-Bromo-3-[3-(2,3-dichlorophenyl)-acryloyl]-2-hydroxy-
benzoic acid (18)
Yellow solid, yield 50%; mp 226–228 °C; 1H NMR (300 MHz,
DMSO-d6): d 8.08 (d, 1H, J = 2.7 Hz, ArH), 8.03 (d, 1H, J = 2.4 Hz,
ArH), 7.98 (d, 1H, J = 8.1 Hz, ArH), 7.91 (s, 1H, H-b), 7.77 (d, 1H,
5.3.2. 5-Bromo-3-[3-(2-chlorophenyl)-acryloyl]-2-hydroxy-
benzoic acid (11)
Yellow solid, yield 60%; mp 211–214 °C; 1H NMR (300 MHz,
DMSO-d6): d 8.08 (d, 1H, J = 2.7 Hz, ArH), 8.03 (d, 1H, J = 2.7 Hz,
ArH), 8.01 (dd, 1H, J1 = 1.5 Hz, J2 = 7.5 Hz, ArH), 7.94 (d, 1H,
J = 15.6 Hz, H-a), 7.74 (d, 1H, J = 7.8 Hz, ArH), 7.46 (t, 1H, J = 7.8 Hz,
ArH); MS (ESI): m/z 414.8 [MꢁH]ꢁ. HPLC: tR 6.46 min, purity:
99.08%.
J = 15.9 Hz, H-b), 7.78 (d, 1H, J = 15.9 Hz, H-a), 7.59 (dd, 1H,

2040
H. Sharma et al. / Bioorg. Med. Chem. 19 (2011) 2030–2045
5.3.10. 5-Bromo-3-[3-(2,3-dimethoxyphenyl)-acryloyl]-2-
hydroxybenzoic acid (19)
5.3.18. 5-Chloro-3-[3-(2,4-dichlorophenyl)-acryloyl]-2-hydroxy-
benzoic acid (27)
Yellow solid, yield 51%; mp 195–197 °C; 1H NMR (300 MHz,
DMSO-d6): d 8.02 (s, 1H, ArH), 7.92 (s, 1H, ArH), 7.86 (d, 1H,
Yellow solid, yield 48%; mp 248–250 °C; 1H NMR (300 MHz,
MeOH-d4): d 8.05 (d, 1H, J = 15.9 Hz, H-b), 7.93 (d, 1H, J = 8.7 Hz,
J = 16.2 Hz, H-b), 7.75 (d, 1H, J = 15.9 Hz, H-
a), 7.40 (dd, 1H,
ArH), 7.84 (d, 1H, J = 2.4 Hz, ArH), 7.80 (d, 1H, J = 15.3 Hz, H-a),
J1 = 3.0 Hz, J2 = 5.7 Hz, ArH), 7.13–7.15 (m, 2H, ArH), 3.83 (s, 3H,
OCH3), 3.77 (s, 3H, OCH3); MS (ESI): m/z 406.8 [MꢁH]ꢁ. HPLC: tR
7.03 min, purity: 99.7%.
7.73 (d, 1H, J = 2.1 Hz, ArH), 7.65 (d, 1H, J = 2.7 Hz, ArH), 7.52 (dd,
1H, J1 = 2.1 Hz, J2 = 8.4 Hz, ArH); MS (ESI): m/z 370.8 [MꢁH]ꢁ.
HPLC: tR 6.76 min, purity 99.14%.
5.3.11. 5-Bromo-2-hydroxy-3-[3-(2,3,4-trimethoxyphenyl)-
acryloyl]-benzoic acid (20)
5.3.19. 5-Chloro-3-[3-(2,3-dichlorophenyl)-acryloyl]-2-hydroxy-
benzoic acid (28)
Yellow solid, yield 49%; mp 194–195 °C; 1H NMR (300 MHz,
DMSO-d6): d 8.05 (d, 1H, J = 1.8 Hz, ArH), 8.02 (d, 1H, J = 1.8 Hz
ArH), 7.80 (d, 1H, J = 15.9 Hz, H-b), 7.63 (d, 1H, J = 8.7 Hz, ArH),
Yellow solid, yield 41%; mp 250–252 °C; 1H NMR (300 MHz,
DMSO-d6): d 8.10 (d, 1H, J = 15.3 Hz, H-b), 7.85–7.89 (m, 3H, H-
a
and ArH), 7.70 (d, 1H, J = 7.2 Hz, ArH), 7.62 (s, 1H, ArH), 7.45 (t,
1H, J = 7.5 Hz, ArH); MS (ESI): m/z 370.8 [MꢁH]ꢁ. HPLC: tR
6.06 min, purity 96.61%.
7.10 (d, 1H, J = 15.9 Hz, H-a), 6.92 (d, 1H, J = 8.7 Hz, ArH), 3.87 (s,
1H, OCH3), 3.84 (s, 1H, OCH3), 3.77 (s, 1H, OCH3); MS (ESI): m/z
436.8 [MꢁH]ꢁ. HPLC: tR 6.88 min, purity: 99.36%.
5.3.20. 5-Chloro-3-[3-(2,5-dichlorophenyl)-acryloyl]-2-hydroxy-
benzoic acid (29)
5.3.12. 5-Bromo-3-[3-(2,5-dichlorophenyl)-acryloyl]-2-hydroxy-
benzoic acid (21)
Yellow solid, yield 42%; mp 247–249 °C; 1H NMR (300 MHz,
DMSO-d6): d 8.20 (d, 1H, J = 1.2 Hz, ArH), 8.04 (s, 2H, H-b and
Yellow solid, yield 51%; mp 232–234 °C; 1H NMR (300 MHz,
DMSO-d6): d 8.16 (d, 1H, J = 2.4 Hz, ArH), 8.11 (d, 1H, J = 2.7 Hz,
ArH), 7.90 (s, 2H, H-a and ArH), 7.67 (d, 1H, J = 8.4 Hz, ArH), 7.61
ArH), 8.0 (d, 1H, J = 2.7 Hz, ArH), 7.87 (s, 2H, H-
a and H-b), 7.65
(dd, 1H, J1 = 1.5 Hz, J2 = 8.4 Hz, ArH); MS (ESI): m/z 370.7 [MꢁH]ꢁ.
HPLC: tR 8.39 min, purity 99.0%.
(d, 1H, J = 8.4 Hz, ArH), 7.57 (dd, 1H, J1 = 2.4 Hz, J2 = 8.4 Hz, ArH);
MS (ESI): m/z 414.7 [MꢁH]ꢁ. HPLC: tR 8.03 min, purity 97.30%.
5.3.21. 5-Chloro-2-hydroxy-3-[3-(2,3,5-trichlorophenyl)-acryl-
oyl]benzoic acid (30)
5.3.13. 5-Bromo-3-[3-(2,6-dichlorophenyl)-acryloyl]-2-hydroxy-
benzoic acid (22)
Yellow solid, yield 37%; mp 235–237 °C; 1H NMR (300 MHz,
DMSO-d6): d 8.05 (d, 1H, J = 15.9 Hz, H-b), 7.91 (d, 2H, J = 1.2 Hz,
Yellow solid, yield 54%; mp 235–237 °C; 1H NMR (300 MHz,
DMSO-d6): d 8.08 (d, 1H, J = 2.7 Hz, ArH), 7.96 (d, 1H, J = 2.7 Hz,
ArH), 7.82 (d, 1H, J = 3.0 Hz, ArH), 7.75 (d, 1H, J = 15.6 Hz, H-a),
ArH), 7.79 (d, 1H, J = 16.5 Hz, H-b), 7.67 (d, 1H, J = 16.5 Hz, H-a),
7.60 (d, 1H, J = 2.7 Hz, ArH); MS (ESI): m/z 404.6 [MꢁH]ꢁ. HPLC:
tR 7.18 min, purity 98.81%.
7.60 (d, 2H, J = 8.1 Hz, ArH), 7.41–7.46 (t, 1H, J = 8.7 Hz, ArH); MS
(ESI): m/z 414.6 [MꢁH]ꢁ. HPLC: tR 7.96 min, purity 96.60%.
5.3.22. 5-Chloro-2-hydroxy-3-[3-(2,3,6-trichlorophenyl)-acryl-
oyl]benzoic acid (31)
5.3.14. 5-Bromo-3-[3-(3,4-dichlorophenyl)-acryloyl]-2-hydroxy-
benzoic acid (23)
Yellow solid, yield 49%; mp 229–231 °C; 1H NMR (300 MHz,
DMSO-d6): d 7.99 (d, 1H, J = 16.2 Hz, H-b), 7.86 (d, 1H, J = 2.1 Hz,
ArH), 7.71 (d, 1H, J = 2.7 Hz, ArH), 7.69 (d, 1H, J = 7.8 Hz, ArH),
Yellow solid, yield 48%; mp 218–221 °C; 1H NMR (300 MHz,
DMSO-d6): d 8.07 (d, 1H, J = 15.9 Hz, H-b), 8.0 (s, 1H, ArH), 7.93
(d, 1H, J = 2.7 Hz, ArH), 7.72 (d, 1H, J = 2.7 Hz, ArH), 7.70 (d, 2H,
7.65 (d, 1H, J = 8.4 Hz, ArH), 7.60 (d, 1H, J = 16.2 Hz, H-a); MS
J = 7.5 Hz), 7.56 (d, 1H, J = 15.9 Hz, H-a); MS (ESI): m/z 414.6
(ESI): m/z 404.6 [MꢁH]ꢁ. HPLC: tR 6.58 min, purity 95.66%.
[MꢁH]ꢁ. HPLC: tR 7.25 min, purity 97.0%.
5.3.23. 5-Chloro-3-[3-(3,4-dichlorophenyl)-acryloyl]-2-hydroxy-
benzoic acid (32)
5.3.15. 5-Bromo-2-hydroxy-3-[3-(2,3,5-trichlorophenyl)-acryl-
oyl]benzoic acid (24)
Yellow solid, yield 54%; mp >240 °C (dec); 1H NMR (300 MHz,
DMSO-d6): d 8.05 (d, 1H, J = 15.9 Hz, H-b), 7.99 (s, 1H, ArH), 7.80
(d, 1H, J = 3.0 Hz, ArH), 7.71 (d, 2H, J = 6.0 Hz, ArH), 7.58 (d, 1H,
Yellow solid, yield 54%; mp 212–214 °C; 1H NMR (300 MHz,
DMSO-d6): d 8.10 (d, 1H, J = 2.4 Hz, ArH), 8.08 (d, 1H, J = 2.7 Hz,
ArH), 8.05 (d, 1H, J = 2.7 Hz, ArH), 7.96 (d, 1H, J = 2.4 Hz, ArH),
7.84 (br s, 2H, H-
a
and H-b); MS (ESI): m/z 448.6 [MꢁH]ꢁ. HPLC:
J = 3.0 Hz, ArH), 7.56 (d, 1H, J = 15.9 Hz, H-a); MS (ESI): m/z 370.8
[MꢁH]ꢁ. HPLC: tR 6.78 min, purity 99.60%.
tR 7.41 min, purity 98.21%.
5.3.24. 3-[3-(4-Chlorophenyl)-acryloyl]-5-fluoro-2-hydroxy-
benzoic acid (33)
5.3.16. 5-Bromo-2-hydroxy-3-[3-(2,3,6-trichlorophenyl)-acryl-
oyl]benzoic acid (25)
Yellow solid, yield 56%; mp 198–200 °C; 1H NMR (300 MHz,
DMSO-d6): d 7.91 (d, 1H, J = 15.9 Hz, H-b), 7.60 (d, 1H, J = 15.9 Hz,
Yellow solid, yield 57%; mp 220–223 °C; 1H NMR (300 MHz,
DMSO-d6): d 8.07 (d, 1H, J = 2.7 Hz, ArH), 7.97 (d, 1H, J = 2.4 Hz,
ArH), 7.73 (d, 1H, J = 8.7 Hz, ArH), 7.71 (d, 1H, J = 16.2 Hz, H-b),
H-a), 7.68 (dd, 1H, J1 = 3.3 Hz, J2 = 8.4 Hz, ArH), 7.76 (d, 2H,
7.61–7.64 (m, 2H, H-
a
and ArH); MS (ESI): m/z 448.6 [MꢁH]ꢁ.
J = 8.4 Hz, ArH), 7.49 (d, 2H, J = 8.7 Hz, ArH), 7.54 (dd, 1H,
J1 = 3.3 Hz, J2 = 9.3 Hz, ArH); MS (ESI): m/z 318.8 [MꢁH]ꢁ. HPLC:
tR 7.11 min, purity 99.11%.
HPLC: tR 7.13 min, purity 99.20%.
5.3.17. 5-Chloro-3-[3-(4-chlorophenyl)-acryloyl]-2-hydroxy-
benzoic acid (26)
5.3.25. 3-[3-(2,4-Dichlorophenyl)-acryloyl]-5-fluoro-2-hydroxy-
benzoic acid (34)
Yellow solid, yield 43%; mp 213–215 °C; 1H NMR (300 MHz,
DMSO-d6): d 7.95 (d, 1H, J = 2.1 Hz, ArH), 7.93 (d, 1H, J = 2.7 Hz,
ArH), 7.84 (dd, 2H, J1 = 1.5 Hz, J2 = 6.9 Hz, ArH), 7.66 (s, 2H, H-b
Yellow solid, yield 41%; mp 260–262 °C; 1H NMR (300 MHz,
DMSO-d6): d 8.12 (d, 1H, J = 15.6 Hz, H-b), 7.93 (d, 1H, J = 8.4 Hz,
ArH), 7.81 (d, 1H, J = 15.9 Hz, H-
7.65 (dd, 1H, J1 = 3.3 Hz, J2
a
), 7.74 (d, 1H, J = 2.1 Hz, ArH),
8.7 Hz, ArH), 7.52 (dd, 1H,
and H-a), 7.51 (dd, 2H, J1 = 1.5 Hz, J2 = 6.9 Hz, ArH); MS (ESI): m/z
336.9 [MꢁH]ꢁ. HPLC: tR 6.13 min, purity 99.55%.
=

H. Sharma et al. / Bioorg. Med. Chem. 19 (2011) 2030–2045
2041
J1 = 2.1 Hz, J2 = 8.4 Hz, ArH), 7.43 (dd, 1H, J1 = 3.3 Hz, J2 = 9.3 Hz,
ArH); MS (ESI): m/z 352.8 [MꢁH]ꢁ. HPLC: tR 6.51 min, purity
99.5%.
5.3.33. 3-[3-(2-Benzyloxyphenyl)-acryloyl]-5-bromo-2-hydroxy-
benzoic acid (42)
Yellow solid, yield 26%; mp >165 °C (dec); 1H NMR (300 MHz,
MeOH-d4): d 7.92–7.95 (m, 2H, H-b and ArH), 7.72 (d, 1H,
J = 7.5 Hz, ArH), 7.65 (d, 1H, J = 2.7 Hz, ArH), 7.47 (d, 2H,
5.3.26. 3-[3-(2,3-Dichlorophenyl)-acryloyl]-5-fluoro-2-hydroxy-
benzoic acid (35)
J = 7.2 Hz, ArH), 7.30–7.42 (m, 5H, H-a, and ArH), 7.20 (d, 1H,
Yellow solid, yield 47%; mp 222–224 °C; 1H NMR (500 MHz,
J = 8.1 Hz, ArH), 7.0 (t, 1H, J = 7.5 Hz, ArH), 5.22 (s, 2H, CH2),; MS
(ESI): m/z 452.8 [MꢁH]ꢁ. HPLC: tR 7.83 min, purity 95.65%.
DMSO-d6): d 7.94 (d, 1H, J = 7.8 Hz, ArH), 7.90 (s, 2H, H-
a and H-
b), 7.77 (dd, 1H, J1 = 3.0 Hz, J2 = 8.5 Hz, ArH), 7.73 (d, 1H,
J = 8.0 Hz, ArH), 7.46 (t, 1H, J = 8.0 Hz, ArH), 7.66 (dd, 1H,
J1 = 2.5 Hz, J2 = 9.0 Hz, ArH); MS (ESI): m/z 352.8 [MꢁH]ꢁ. HPLC:
tR 5.68 min, purity 97.20%.
5.3.34. 5-Bromo-3-{3-[3-(4-chlorophenoxy)-phenyl]acryloyl}-2-
hydroxybenzoic acid (43)
Yellow solid, yield 42%; mp 162–165 °C; 1H NMR (300 MHz,
DMSO-d6): d 7.94 (s, 2H, ArH), 7.74 (d, 1H, J = 6.9 Hz, ArH), 7.70
(d, 1H, J = 15.9 Hz, H-b), 7.40–7.46 (m, 6H, H-
a and ArH), 7.20
5.3.27. 3-[3-(2,5-Dichlorophenyl)-acryloyl]-5-fluoro-2-hydroxy-
benzoic acid (36)
(d, 1H, J = 7.8 Hz, ArH), 7.0 (t, 1H, J = 6.9 Hz, ArH); (ESI): m/z
472.6 [MꢁH]ꢁ. HPLC: tR 5.86 min, purity 96.8%.
Yellow solid, yield 59%; mp 238–240 °C; 1H NMR (300 MHz,
DMSO-d6): d 8.07 (d, 1H, J = 15.9 Hz, H-b), 7.93 (d, 1H, J = 2.4 Hz,
ArH), 7.76 (d, 1H, J = 15.9 Hz, H-
a
), 7.66 (dd, 1H, J1 = 3.6 Hz, J2
=
5.3.35. 5-Bromo-3-{3-[4-(4-chlorophenoxy)phenyl]acryloyl}-2-
hydroxybenzoic acid (44)
8.4 Hz, ArH), 7.60 (d, 1H J = 8.7 Hz, ArH), 7.52 (dd, 1H, J1 = 2.4 Hz,
J2 = 8.7 Hz, ArH), 7.42 (dd, 1H, J1 = 3.6 Hz, J2 = 9.3 Hz, ArH); MS
(ESI): m/z 352.8 [MꢁH]ꢁ. HPLC: tR 6.02 min, purity 98.43%.
Yellow solid, yield 35%; mp 219–220 °C; 1H NMR (300 MHz,
DMSO-d6): d 8.06 (d, 1H, J = 2.1 Hz, ArH), 8.02 (d, 1H, J = 2.1 Hz,
ArH), 7.84 (d, 2H, J = 8.4 Hz, ArH), 7.67 (d, 1H, J = 15.9 Hz, H-b),
7.59 (d, 1H, J = 15.9 Hz, H-a), 7.48 (d, 2H, J = 8.7 Hz, ArH), 7.13 (d,
5.3.28. 5-Fluoro-2-hydroxy-3-[3-(2,3,5-trichlorophenyl)-acryloyl]-
benzoic acid (37)
2H, J = 8.7 Hz, ArH), 7.07 (d, 2H, J = 8.7 Hz, ArH); MS (ESI): m/z
472.6 [MꢁH]ꢁ. HPLC: tR 7.23 min, purity 99.44%.
Yellow solid, yield 53%; mp 231–233 °C; 1HNMR(300 MHz, DMSO-
d6): d 8.02 (d, 1H, J = 2.4 Hz, ArH), 7.95 (d, 1H, J = 15.9 Hz, H-b), 7.92 (d,
1H, J = 2.4 Hz, ArH), 7.79 (d, 1H, J = 15.6 Hz, H-
a
), 7.74 (dd, 1H,
5.3.36. 5-Bromo-2-hydroxy-3-(3-thiophen-3-yl-acryloyl-
)benzoic acid (45)
J1 = 3.3 Hz, J2 = 8.4 Hz, ArH), 7.64 (dd, 1H, J1 = 3.3 Hz, J2 = 9 Hz, ArH);
MS (ESI): m/z 388.6 [MꢁH]ꢁ. HPLC: tR 8.11 min, purity 97.86%.
Yellow solid, yield 54%; mp 207–210 °C; 1H NMR (300 MHz,
DMSO-d6): d 7.97 (s, 2H, ArH), 7.75 (d, 1H, J = 15.9 Hz, H-b), 7.65
(s, 1H, ArH), 7.61 (s, 1H, ArH), 7.58 (d, 1H, J = 15.6 Hz, H-a), 7.48
5.3.29. 5-Fluoro-2-hydroxy-3-[3-(2,3,6-trichlorophenyl)acry-
loyl]benzoic acid (38)
(d, 1H, J = 4.2 Hz, ArH); MS (ESI): m/z 352.6 [MꢁH]ꢁ. HPLC: tR
Yellow solid, yield 46%; mp 235–237 °C; 1HNMR(300 MHz, DMSO-
d6): d 8.11 (d, 1H, J = 16.2 Hz, H-b), 7.70 (d, 1H, J = 8.7 Hz, ArH), 7.62 (d,
6.91 min, purity 99.71%.
1H, J = 8.7 Hz, ArH), 7.58 (d, 1H, J = 16.2 Hz, H-
a
), 7.66 (dd, 1H,
5.3.37. 5-Bromo-3-[3-(3-cyclopentyloxy-4-methoxyphenyl)-
acryloyl]-2-hydroxybenzoic acid (46)
J1 = 3.6 Hz, J2 = 8.4 Hz, ArH), 7.44 (dd, 1H, J1 = 3.6 Hz, J2 = 9.3 Hz,
ArH); MS (ESI): m/z 388.6 [MꢁH]ꢁ. HPLC: tR 7.57 min, purity 97.57%.
Yellow solid, yield 31%; mp 233–235 °C; 1H NMR (300 MHz,
MeOH-d4): d 7.91 (d, 1H, J = 2.7 Hz, ArH), 7.82 (d, 1H, J = 15.6 Hz,
H-b), 7.67 (d, 1H, J = 3.0 Hz, ArH), 7.59 (d, 1H, J = 15.9 Hz, H-a),
5.3.30. 5-Bromo-3-[3-(3,5-dimethylphenyl)-acryloyl]-2-
hydroxy-
7.26 (d, 1H, J = 8.7 Hz, ArH), 7.25 (s, 1H, ArH), 6.99 (d, 1H,
J = 8.1 Hz, ArH), 4.86 (p, 1H, J = 5.7 Hz, cyclopentyl), 3.79 (s, 3H,
CH3) 1.87–1.92 (m, 2H, cyclopentyl), 1.66–1.72 (m, 4H, cyclopen-
tyl), 1.57–1.60 (m, 2H, cyclopentyl); MS (ESI): m/z 460.8 [MꢁH]ꢁ.
HPLC: tR 7.19 min, purity 97.51%.
benzoic acid (39)
Yellow solid, yield 51%; mp 223–225 °C; 1H NMR (300 MHz,
DMSO-d6): d 8.06 (d, 1H, J = 2.7 Hz, ArH), 7.99 (d, 1H, J = 1.8 Hz,
ArH), 7.61 (d, 1H, J = 16.5 Hz, H-b), 7.58 (d, 1H, J = 16.5 Hz, H-a),
7.40 (s, 2H, Hz, ArH), 7.10 (s, 1H, ArH), 2.32 (s, 6H, CH3); MS
(ESI): m/z 374.9 [MꢁH]ꢁ. HPLC: tR 6.59 min, purity 99.0%.
5.3.38. 5-Bromo-3-[3-(2-ethoxynaphthalen-1-yl)acryloyl]-2-
hydroxybenzoic acid (47)
Yellow solid, yield 23%; mp 216–218 °C; 1H NMR (300 MHz,
MeOH-d4): d 8.48 (d, 1H, J = 15.9 Hz, H-b), 8.30 (d, 1H, J = 8.4 Hz,
5.3.31. 5-Bromo-3-[3-(2-chloro-3,4-dimethoxyphenyl)-acryl-
oyl]-2-hydroxybenzoic acid (40)
Yellow solid, yield 21%; mp 217–220 °C; 1H NMR (300 MHz,
DMSO-d6): d 8.05 (d, 1H, J = 2.7 Hz, ArH), 8.00 (d, 1H, J = 2.7 Hz,
ArH), 7.90 (d, 1H, J = 15.6 Hz, H-b), 7.81 (d, 1H, J = 8.7 Hz, ArH),
ArH), 8.19 (d, 1H, J = 2.7 Hz, ArH), 8.17 (d, 1H, J = 15.9 Hz, H-a),
7.92 (d, 1H, J = 9.0 Hz, ArH), 7.88 (d, 1H, J = 2.7 Hz, ArH), 7.84 (d,
1H, J = 8.4 Hz, ArH), 7.55 (t, 1H, J = 7.2 Hz, ArH), 7.37–7.44 (m,
2H, ArH), 4.33 (q, 2H, J = 6.9 Hz, CH2), 1.55 (t, 3H, J = 6.9 Hz, CH3);
MS (ESI): m/z 440.9 [MꢁH]ꢁ. HPLC: tR 8.53 min, purity 95.0%.
7.68 (d, 1H, J = 15.6 Hz, H-a), 7.16 (d, 1H, J = 8.7 Hz, ArH), 3.90 (s,
3H, OCH3), 3.77 (s, 3H, OCH3); MS (ESI): m/z 440.8 [MꢁH]ꢁ. HPLC:
tR 6.46 min, purity 99.0%.
5.3.39. 5-Bromo-2-hydroxy-3-[3-(6-methoxynaphthalen-2-yl)-
acryloyl]benzoic acid (48)
5.3.32. 5-Bromo-3-[3-(2-fluoro-4,5-dimethoxyphenyl)-acryl-
oyl]-2-hydroxybenzoic acid (41)
Yellow solid, yield 15%; mp 239–241 °C; 1H NMR (300 Hz,
MeOH-d4): d 8.17 (d, 1H, J = 2.4 Hz, ArH), 8.01 (s, 1H, H-b), 7.86
(d, 1H, J = 7.9 Hz, ArH), 7.84 (d, 2H, J = 8.1 Hz, ArH), 7.83 (d, 1H,
Yellow solid, yield 32%; mp 193–195 °C; 1H NMR (300 Hz,
DMSO-d6): d 8.03 (d, 1H, J = 2.7 Hz, ArH), 7.93 (d, 1H, J = 2.7 Hz,
J = 2.4 Hz, ArH), 7.80 (s, 2H, H-a, and ArH), 7.26 (d, 1H, J = 2.4 Hz,
ArH), 7.66 (s, 2H, H-b and H-a), 7.38 (d, 1H, J = 7.2 Hz, ArH),
ArH), 7.16 (dd, 1H, J1 = 8.7 Hz, J2 = 2.4 Hz, ArH), 3.93 (s, 3H,
OCH3); MS (ESI): m/z 426.8 [MꢁH]ꢁ. HPLC: tR 6.07 min, purity
98.93%.
7.00 (d, 1H, J = 12.3 Hz, ArH), 3.84 (s, 3H, OCH3), 3.82 (s, 3H,
OCH3); MS (ESI): m/z 424.8 [MꢁH]ꢁ. HPLC: tR 7.19 min, purity
97.51%.

2042
H. Sharma et al. / Bioorg. Med. Chem. 19 (2011) 2030–2045
5.3.40. 3-(3-Benzo[b]thiophen-5-yl-acryloyl)-5-bromo-2-
hydroxybenzoic acid (49)
7.46 (d, 1H, J = 2.4 Hz, ArH), 7.43 (d, 1H, J = 16.2 Hz, H-a); MS
(ESI): m/z 431.6 [MꢁH]ꢁ. HPLC: tR 6.37 min, purity: 95.69%.
Yellow solid, yield 21%; mp 235–237 °C; 1H NMR (300 MHz,
DMSO-d6): d 8.21 (s, 1H, ArH), 8.1 (d, 1H, J = 15.9 Hz, H-b), 8.0 (s,
1H, ArH), 7.92 (d, 1H, J = 2.7 Hz, ArH), 7.82 (d, 1H, J = 5.4 Hz,
5.3.48. 2-Hydroxy-5-methyl-3-[3-(2,3,6-trichlorophenyl)-acryloyl]-
benzoic acid (57)
ArH), 7.73 (d, 1H, J = 15.9 Hz, H-a), 7.72 (d, 1H, J = 3 Hz, ArH),
Yellow solid, yield 58.5%; mp 205–208 °C; 1H NMR (300 MHz,
DMSO-d6): d 7.87 (s, 1H, ArH), 7.74 (s, 1H, ArH), 7.70 (d, 1H,
7.72 (s, 1H, ArH), 7.53 (d, 1H, J = 5.1 Hz, ArH); MS (ESI): m/z
402.8 [MꢁH]ꢁ. HPLC: tR 5.53 min, purity 98.31%.
J = 8.7 Hz, ArH), 7.59–7.64 (m, 3H, H-a, H-b and ArH), 2.29 (s, 3H,
CH3); MS (ESI): m/z 384.8 [MꢁH]ꢁ. HPLC: tR 6.01 min, purity
5.3.41. 5-Bromo-3-[3-(2-furan-2-yl-phenyl)-acryloyl]-2-
hydroxybenzoic acid (50)
97.84%.
Yellow solid, yield 36%; mp 208–209 °C; 1H NMR (300 MHz,
5.4. General procedure for the synthesis of chalcones (58–59)
DMSO-d6): d 7.92 (m, 3H, H-b and ArH), 7.87 (s, 1H, H-a), 7.81
(d, 1H, J = 2.7 Hz, ArH), 7.70 (s, 1H, ArH), 7.68 (d, 1H, 9.6 Hz,
ArH), 7.48 (t, 2H, J = 7.5 Hz, ArH), 6.66 (s, 1H, ArH), 6.63 (s, 1H,
ArH); MS (ESI): m/z 412.8 [MꢁH]ꢁ. HPLC: tR 5.77 min, purity
95.02%.
A similar procedure to the one mentioned above was followed
with appropriate aromatic aldehydes and commercially available
1-(5-bromo-2-hydroxy-3-nitro-phenyl)-ethanone
material.
starting
5.3.42. 5-Bromo-3-[3-(6-chlorobenzo[1,3]dioxol-5-yl)-acryloyl]-
2-hydroxybenzoic acid (51)
5.4.1. 1-(5-Bromo-2-hydroxy-3-nitrophenyl)-3-(2,3,5-trichloro-
phenyl)propenone (58)
Yellow solid, yield 18%; mp 228–231 °C; 1H NMR (300 MHz,
DMSO-d6): d 8.10 (s, 1H, ArH), 8.07 (s, 1H, ArH), 7.90 (d, 1H,
Yellow solid, yield 65%; mp 175–178 °C; 1H NMR (300 MHz,
DMSO-d6): d 8.13 (d, 1H, J = 15.6 Hz, H-b), 7.76 (d, 1H, J = 15.9 Hz,
J = 15 Hz, H-b), 7.66 (s, 1H, ArH), 7.63 (d, 1H, J = 14.7 Hz, H-a),
H-a), 8.05 (d, 1H, J = 2.7 Hz, ArH), 8.0 (d, 2H, J = 2.4 Hz, ArH), 7.92
7.21 (s, 1H, ArH), 6.16 (s, 2H, CH2); MS (ESI): m/z 424.6 [MꢁH]ꢁ.
HPLC: tR 7.82 min, purity 99.0%.
(d, 1H, J = 2.4 Hz, ArH); MS (ESI): m/z 451.8 [MꢁH]ꢁ. HPLC: tR
5.46 min, purity 97.30%.
5.3.43. 5-Bromo-2-hydroxy-3-[3-(3-trifluoromethoxyphenyl)-
acryloyl]benzoic acid (52)
5.4.2. 1-(5-Bromo-2-hydroxy-3-nitrophenyl)-3-(2,3,6-trichloro-
phenyl)propenone (59)
Yellow solid, yield 44%; mp 217–219 °C; 1H NMR (300 MHz,
DMSO-d6): d 8.07 (d, 1H, J = 2.7 Hz, ArH), 8.03 (d, 1H, J = 2.7 Hz,
Yellow solid, yield 58%; mp 153–154 °C; 1H NMR (500 MHz,
DMSO-d6): d 8.32 (s, 1H, ArH), 8.24 (s, 1H, ArH), 7.77 (d, 1H,
J = 16.5 Hz, H-b), 7.75 (d, 1H, J = 9.0 Hz, ArH), 7.65 (d, 1H,
ArH), 7.80–7.84 (br s, 2H, H-a and H-b), 7.72 (s, 1H, ArH), 7.70 (s,
1H, J = 4.0 Hz, ArH), 7.58 (t, 1H, J = 7.8 Hz, ArH), 7.45 (d, 1H,
J = 7.5 Hz, ArH); MS (ESI): m/z 430.8 [MꢁH]ꢁ. HPLC: tR 6.06 min,
purity 99.30%.
J = 15.0 Hz, H-a), 7.63 (d, 1H, J = 8.0 Hz, ArH); MS (ESI): m/z 451.8
[MꢁH]ꢁ. HPLC: tR 6.53 min, purity 98.0%.
5.5. General procedure for the synthesis of 3-[3-(2,5-dichloro-
phenyl)propionyl]-5-fluoro-2-hydroxybenzoic acid (60)
5.3.44. 5-Bromo-2-hydroxy-3-[3-(3-nitrophenyl)-acryloyl]-
benzoic acid (53)
Yellow solid, yield 62%; mp 200–202 °C; 1H NMR (500 MHz,
DMSO-d6): d 8.62 (s, 1H, ArH), 8.26 (s, 2H, ArH), 8.07 (s, 1H, H-b),
Compound 36 (0.2 g, 0.563 mmol) was dissolved in methanol
(10 mL) and Pd/C (10 mol %, 20 mg) was added. The resulting solu-
tion was stirred under hydrogen at atmospheric pressure for 2 h at
room temperature. After the reaction was complete, the reaction
mixture was filtered through celite, and evaporated to dryness.
The resulting crude solid was recrystalized from ethanol to give a
white solid, yield 43%; mp 163–164 °C; 1H NMR (300 MHz,
DMSO-d6): d 7.79 (d, 1H, J = 8.4 Hz, ArH), 7.74 (d, 1H, J = 9.0 Hz,
ArH), 7.49 (s, 1H, ArH), 7.45 (d, 1H, J = 8.7 Hz, ArH), 7.31 (d, 1H,
J = 8.1 Hz, ArH), 3.39 (t, 2H, J = 6.9 Hz, CH2), 3.02 (t, 2H, J = 6.9 Hz,
CH2); MS (ESI): m/z 356.9 [MꢁH]ꢁ. HPLC: tR 8.34 min, purity
96.36%.
8.02 (s, 1H, H-a), 7.81 (d, 2H, J = 7.0 Hz, ArH), 7.75 (t, 1H,
J = 7.0 Hz, ArH); MS (ESI): m/z 391.8 [MꢁH]ꢁ. HPLC: tR 11.47 min,
purity 95.59%.
5.3.45. 5-Bromo-3-[3-(3-bromophenyl)-acryloyl]-2-hydroxy-
benzoic acid (54)
Yellow solid, yield 31%; mp 235–237 °C; 1H NMR (300 MHz,
DMSO-d6): d 8.0 (d, 1H, J = 15.9 Hz, H-b), 7.92 (d, 1H, J = 2.7 Hz,
ArH), 7.90 (s, 1H, ArH), 7.72 (d, 1H, J = 6.6 Hz, ArH), 7.70 (d, 1H,
J = 2.4 Hz, ArH), 7.59 (d, 1H, J = 8.4 Hz, ArH), 7.55 (d, 1H,
J = 15.9 Hz, H-a), 7.39 (t, 1H, J = 7.8 Hz, ArH); MS (ESI): m/z 424.6
[MꢁH]ꢁ. HPLC: tR 6.14 min, purity 98.81%.
5.6. General procedure for the synthesis of 5-bromo-N-(4-flu-
orobenzyl)-2-hydroxyisophthalamic acid (61)
5.3.46. 5-Bromo-3-[3-(3-fluorophenyl)-acryloyl]-2-hydroxy-
benzoic acid (55)
5.6.1. 5-Bromo-3-formyl-2-hydroxybenzoic acid (7)
Yellow solid, yield 59%; mp 190–193 °C; 1H NMR (500 MHz,
DMSO-d6): d 8.0 (d, 1H, J = 2.0 Hz, ArH), 7.93 (s, 1H, ArH), 7.80 (d,
Method a: NaOH (5.53 g, 0.138 mol) was added to a solution of
5-bromosalicylic acid (5 g, 23.0 mmol) in CHCl3 (45 mL) and water
(1 mL, 55.55 mmol) at room temperature. The reaction mixture
was refluxed for 48 h, diluted with water and acidified with 6 N
HCl to pH 1.0. The resulting solution was extracted with ethyl ace-
tate washed with brine and dried over Na2SO4. The organic layer
was evaporated in vacuo to yield a crude product which was puri-
fied by column chromatography (25–35% methanol/dichlorometh-
ane) to give a white solid, yield 13%.
1H, J = 16.0 Hz, H-b), 7.60–7.77 (m, 3H, H-a and ArH), 7.48 (d,
1H, J = 6.5 Hz, ArH), 7.28 (d, 1H, J = 8.0 Hz, ArH); MS (ESI): m/z
364.7 [MꢁH]ꢁ. HPLC: tR 5.39 min, purity 92.5%.
5.3.47. 5-Bromo-2-hydroxy-3-[3-(2,3,6–trichlorophenyl)-acryl-
oyl]benzonitrile (56)
Orange solid, yield 74%; mp 139–141 °C; 1H NMR (300 MHz,
DMSO-d6): d 8.34 (d, 1H, J = 16.2 Hz, H-b), 7.69 (d, 1H, J = 3.0 Hz,
ArH), 7.66 (d, 1H, J = 9.0 Hz, ArH), 7.58 (d, 1H, J = 8.7 Hz, ArH),
Method b: 3-Formyl salicylic acid hydrate (1 g, 6.0 mmol) was
dissolved in dry acetonitrile (20 mL). N-Bromo succinimide

H. Sharma et al. / Bioorg. Med. Chem. 19 (2011) 2030–2045
2043
(1.02 g, 5.73 mmol) was added and the reaction mixture stirred at
5.8. General procedure for the synthesis of 2-hydroxy-3-(3-
phenylpropyl)benzoic acid (9)
rt for 3 h. The white crystals formed were filtered and washed with
water. The filtrate was extracted with ethyl acetate, and washed
with brine. The organic layer was dried over Na2SO4 and evapo-
rated to dryness. The two fractions were combined to give interme-
diate 7 as white crystals, yield 90%; mp 172–174 °C; 1H NMR
(300 MHz, DMSO-d6): d 10.27 (s, 1H, CHO), 8.12 (d, 1H, J = 2.7 Hz,
ArH), 7.93 (d, 1H, J = 2.7 ArH); MS (ESI): m/z 244.7 [MꢁH]ꢁ.
Compound 10 (0.05 g, 0.195 mmol) was dissolved in ethanol
(10 mL) and Pd/C (10 mol %, 5 mg) was added. The resulting solu-
tion was stirred under hydrogen at atmospheric pressure for 10 h
at room temperature. The reaction mixture was then filtered
through celite, and evaporated to dryness. The resulting crude solid
was purified using preparative chromatography to give a white so-
lid, yield 72%; mp 125–127 °C; 1H NMR (300 MHz, DMSO-d6): d
11.66 (s, 1H, OH), 7.67 (dd, 1H, J1 = 1.5 Hz, J2 = 7.8 Hz, ArH), 7.39
(dd, 1H, J1 = 1.5 Hz, J2 = 7.2 Hz, ArH), 7.16–7.30 (m, 5H, ArH), 6.85
(t, 1H, J = 7.8 Hz, ArH), 2.62 (t, 4H, J = 7.2 Hz, CH2), 1.81–1.91 (m,
2H, CH2); MS (ESI): m/z 254.8 [MꢁH]ꢁ. HPLC: tR 7.61 min, purity
98.05%.
5.6.2. 5-Bromo-N-(4-fluorobenzyl)-3-formyl-2-hydroxy-
benzamide (8)
To a solution of intermediate 7 (0.4 g, 1.63 mmol), HOBt
(0.441 g, 3.26 mmol) and Et3N (0.45 mL, 3.26 mmol) in DCM
(20 mL) was added EDCI (0.344 g, 1.8 mmol) at 0 °C. 4-Fluoroben-
zylamine (0.20 mL, 1.8 mmol) was then added and the reaction
mixture stirred overnight at room temperature. The reaction mix-
ture was extracted with ethyl acetate, washed with dilute hydro-
chloric acid and sodium bicarbonate solution. The organic layer
was further washed with brine and dried over sodium sulfate.
The resulting crude was purified by recrystalization with ethyl ace-
tate/hexane to give a yellow solid. Yield 41%; mp 135–137 °C; 1H
NMR (300 MHz, DMSO-d6): d 10.27 (s, 1H, CHO), 9.90 (s, 1H, NH),
8.41 (s, 1H, ArH), 7.87 (s, 1H, ArH), 7.40 (d, 2H, J = 8.1 Hz, ArH),
7.18 (t, 2H, J = 8.1 Hz, ArH), 4.52 (s, 2H, CH2); MS (ESI): m/z 351.9
[MꢁH]ꢁ.
5.9. CoMFA and CoMSIA studies
Three-dimensional structure building and CoMFA and CoMSIA
studies were performed on a Dell computer workstation with the
Red Hat Linux operating system using the SYBYL program, version
8.0 (Tripos Associates Inc.). Partial atomic charges were assigned
with MOPAC. The CoMFA descriptors, steric (Lennard-Jones 6–12
potential) and electrostatic (Coulombic potential) field energies,
were calculated using Tripos force field with a distance-dependent
dielectric and employing the SYBYL default parameters: 2 Å grid
points spacing, an sp3 carbon probe atom with +1 charge and
vdW radius of 1.52 Å, and energy cut-off of 30 kcal/mol. The five
similarity indices in CoMSIA, that is, steric, electrostatic, hydropho-
bic, H-bond donor and H-bond acceptor descriptors, were calcu-
lated37 using a C1+ probe atom with a radius of 1.0 Å placed at
regular grid spacing of 2 Å. A Gaussian type distance dependence
was used between the grid point q and each atom i of the molecule.
The default value of 0.3 was used as the attenuation factor (R).
Column filtering was set to 2.0 kcal/mol. CoMSIA steric indices
are related to the third power of the atomic radii, electrostatic
descriptors were derived from atomic partial charges, hydrophobic
fields were derived from atom-based parameters,38 and H-bond
donor and acceptor indices were obtained by a rule-based method
based on experimental results.39 The CoMFA and CoMSIA
descriptors were used as independent variables, and pIC50
(ꢁlog IC50) values were used as the dependent variable in PLS
regression analyses to derive 3D-QSAR models. The predictive va-
lue of the models was evaluated by leave-one-out (LOO) cross-val-
idation. The cross validated coefficient, q2, was calculated using Eq
1:
5.6.3. 5-Bromo-N-(4-fluorobenzyl)-2-hydroxyisophthalamic
acid (61)
Intermediate 8 (0.2 g, 0.568 mmol) was added to a mixture of
water (2.5 mL) and pyridine (2.5 mL). The reaction mixture was
heated to 90 °C, KMnO4 (0.18 g, 1.13 mmol) was added to the
hot solution was and the heating continued overnight at 90 °C.
After completion, the mixture was filtered through celite and
evaporated to dryness. The resulting residue was purified by
recrystalization from ethyl acetate/hexane. Yield 80%; mp 208–
210 °C; 1H NMR (300 MHz, DMSO-d6): d 10.20 (s, 1H, NH), 7.98
(s, 1H, ArH), 7.85 (s, 1H, ArH), 7.13–7.34 (m, 4H, ArH), 4.50 (s,
2H, CH2); MS (ESI): m/z 367.9 [MꢁH]ꢁ. HPLC: tR 4.21 min, purity
97.7%.
5.7. General procedure for the synthesis of N-(4-fluoro-benzyl)-
2-hydroxy-3-methylbenzamide (62) and 5-bromo-N-(4-fluoro-
benzyl)-2-hydroxybenzamide (63)
A similar procedure to the one described for the synthesis of
intermediate 8 was followed by condensing 4-fluorobenzyl amine
and commercially available 2-hydroxy-3-methylbenzoic acid (3a)
and 5-bromosalicylic acid (3b) to give compounds 62 and 63,
respectively.
P
2
ðYpredicted ꢁ Yactual
Þ
Þ
q2 ¼ 1 ꢁ
ð1Þ
P
2
ðYobserved ꢁ Ymean
where Ypredicted
,
Yactual and Ymean are predicted, actual and
,
mean values of the target property (pIC50), respectively.
5.7.1. N-(4-Fluoro-benzyl)-2-hydroxy-3-methylbenzamide (62)
Yield76%;mp165–166 °C; 1H NMR(300 MHz, DMSO-d6):d13.10
(s, 1H, OH), 9.43 (t, 1H, J = 6.0 Hz, NH), 7.72 (d, 1H, J = 7.2 Hz, ArH),
7.37 (d, 1H, J = 8.7 Hz, ArH), 7.35 (d, 1H, J = 8.4 Hz, ArH), 7.31 (d,
1H, J = 7.2 Hz, ArH), 7.15 (t, 2H, J = 9.0 Hz, ArH), 6.78 (t, 1H,
J = 7.5 Hz, ArH), 4.48 (d, 1H, J = 6.0 Hz, CH2), 2.15 (s, 3H, CH3); MS
(ESI): m/z 257.9 [MꢁH]ꢁ. HPLC: tR 8.63 min, purity 99.11%.
P
2
(Ypredicted ꢁ Yactual
)
is the predictive sum of squares (PRESS).
Conventional correlation coefficient r2 and its standard error,
s, were also computed for the final PLS models. CoMFA and CoMSIA
coefficient maps were generated by interpolation of the pair wise
products between the PLS coefficients and the standard deviations
of the corresponding CoMFA or CoMSIA descriptor values.
5.10. Biological assays
5.7.2. 5-Bromo-N-(4-fluoro-benzyl)-2-hydroxybenzamide (63)
Yield 62%; mp 83–84 °C; 1H NMR (300 MHz, DMSO-d6): d 12.47
(s, 1H, OH), 9.37 (t, 1H, J = 5.7 Hz, NH), 8.07 (d, 1H, J = 2.4 Hz, ArH),
7.55 (dd, 1H, J1 = 2.4 Hz, J2 = 8.7 Hz, ArH), 7.36 (dd, 2H, J1 = 5.7 Hz,
J2 = 8.1 Hz, ArH), 7.15 (t, 2H, J = 9.0 Hz, ArH), 6.89 (d, 1H,
J = 8.7 Hz, ArH), 4.47 (d, 2H, J = 5.7 Hz, CH2); MS (ESI): m/z 323.8
[MꢁH]ꢁ. HPLC: tR 9.7 min, purity 98.66%.
5.10.1. Materials, chemicals, and enzymes
All compounds were dissolved in DMSO and the stock solutions
were stored at ꢁ20 °C.
c[
32P]-ATP was purchased from either
Amersham Biosciences or ICN. The expression systems for wild-
type IN were a generous gift from Dr. Robert Craigie, Laboratory

2044
H. Sharma et al. / Bioorg. Med. Chem. 19 (2011) 2030–2045
of Molecular Biology, NIDDK, NIH, Bethesda, MD. Cell lines were
obtained from American Type Culture Collection (ATCC, Rockville,
MD).
Acknowledgments
Financial support from the National Institute of Allergyand Infec-
tious Diseases (NIAID), NIH Grant No. AI084710 and the Department
of Pharmaceutical Sciences is gratefully acknowledged. RFS is also
supported by NIH CFAR grant 2P30-AI-050409 and the Department
of Veterans Affairs.
5.10.2. Preparation of oligonucleotide substrates
The oligonucleotides 21top, 50-GTGTGGAAAATCTCTAGCAGT-30,
and 21-bot, 50-ACTGCTAGAGATTTTCCACAC-30, were purchased
from the Norris Cancer Center Microsequencing Core Facility (Uni-
versity of Southern California) and purified by UV shadowing on
polyacrylamide gel. To analyze the extent of 30-end processing
and strand transfer using 50-end labeled substrates, 21top was 50-
end labeled using T4 polynucleotide kinase (Epicentre, Madison,
References and notes
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WI) and
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To determine the extent of 30-processing (30-P) and strand
transfer (ST), wild-type IN was preincubated at a final concentra-
tion of 200 nM with the inhibitor in reaction buffer (50 mM NaCl,
1 mM HEPES, pH 7.5, 50 lM EDTA, 50 lM dithiothreitol, 10% glyc-
erol (w/v), 7.5 mM MnCl2, 0.1 mg/mL bovine serum albumin,
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MOPS, pH 7.2) at 30 °C for 30 min. An aliquot (5 lL) was electro-
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%I ¼ 100 ꢃ ½1 ꢁ ðD ꢁ CÞ=ðN ꢁ CÞꢄ
where C, N, and D are the fractions of 21-mer substrate converted to
19-mer (30-P product) or strand transfer products for DNA alone,
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5.11. Anti-HIV-1 activity assay
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ity according to the previously reported procedure.42

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