Design of HIV-1 integrase inhibitors targeting the catalytic domain as well as its interaction with LEDGF/p75: A scaffold hopping approach using salicylate and catechol groups

Article abstract of DOI:10.1016/j.bmc.2011.06.058

HIV-1 integrase (IN) is a validated therapeutic target for antiviral drug design. However, the emergence of viral strains resistant to clinically studied IN inhibitors demands the discovery of novel inhibitors that are structurally as well mechanistically different. Herein, we describe the design and discovery of novel IN inhibitors targeting the catalytic domain as well as its interaction with LEDGF/p75, which is essential for the HIV-1 integration as an IN cofactor. By merging the pharmacophores of salicylate and catechol, the 2,3-dihydroxybenzamide (5a) was identified as a new scaffold to inhibit the strand transfer reaction efficiently. Further structural modifications on the 2,3-dihydroxybenzamide scaffold revealed that the heteroaromatic functionality attached on the carboxamide portion and the piperidin-1-ylsulfonyl substituted at the phenyl ring are beneficial for the activity, resulting in a low micromolar IN inhibitor (5p, IC₅₀ = 5 μM) with more than 40-fold selectivity for the strand transfer over the 3′-processing reaction. More significantly, this active scaffold remarkably inhibited the interaction between IN and LEDGF/p75 cofactor. The prototype example, N-(cyclohexylmethyl)-2,3- dihydroxy-5-(piperidin-1-ylsulfonyl) benzamide (5u) inhibited the IN-LEDGF/p75 interaction with an IC₅₀ value of 8 μM. Using molecular modeling, the mechanism of action was hypothesized to involve the chelation of the divalent metal ions inside the IN active site. Furthermore, the inhibitor of IN-LEDGF/p75 interaction was properly bound to the LEDGF/p75 binding site on IN. This work provides a new and efficient approach to evolve novel HIV-1 IN inhibitors from rational integration and optimization of previously reported inhibitors.

Full text of DOI:10.1016/j.bmc.2011.06.058

Bioorganic & Medicinal Chemistry 19 (2011) 4935–4952
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design of HIV-1 integrase inhibitors targeting the catalytic domain as well
as its interaction with LEDGF/p75: A scaffold hopping approach using
salicylate and catechol groups
Xing Fan a, , Feng-Hua Zhang a, , Rasha I. Al-Safi b, Li-Fan Zeng a, Yumna Shabaik b, Bikash Debnath b,
a,
Tino W. Sanchez b, Srinivas Odde b, Nouri Neamati b, , Ya-Qiu Long
⇑
⇑
a State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 555 Zuchongzhi Road,
Shanghai 201203, China
b Department of Pharmacology and Pharmaceutical Sciences, School of Pharmacy, University of Southern California, 1985 Zonal Avenue, Los Angeles, CA 90089, 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 (IN) is a validated therapeutic target for antiviral drug design. However, the emergence of
viral strains resistant to clinically studied IN inhibitors demands the discovery of novel inhibitors that are
structurally as well mechanistically different. Herein, we describe the design and discovery of novel IN
inhibitors targeting the catalytic domain as well as its interaction with LEDGF/p75, which is essential
for the HIV-1 integration as an IN cofactor. By merging the pharmacophores of salicylate and catechol,
the 2,3-dihydroxybenzamide (5a) was identified as a new scaffold to inhibit the strand transfer reaction
efficiently. Further structural modifications on the 2,3-dihydroxybenzamide scaffold revealed that the
heteroaromatic functionality attached on the carboxamide portion and the piperidin-1-ylsulfonyl substi-
tuted at the phenyl ring are beneficial for the activity, resulting in a low micromolar IN inhibitor (5p,
Received 28 May 2011
Revised 18 June 2011
Accepted 20 June 2011
Available online 26 June 2011
Keywords:
HIV-1 integrase inhibitors
2,3-Dihydroxybenzamide
Strand transfer
30-Process
IC50 = 5 l
M) with more than 40-fold selectivity for the strand transfer over the 30-processing reaction.
More significantly, this active scaffold remarkably inhibited the interaction between IN and LEDGF/p75
LEDGF/p75
Chelation
cofactor. The prototype example, N-(cyclohexylmethyl)-2,3-dihydroxy-5-(piperidin-1-ylsulfonyl) benz-
amide (5u) inhibited the IN-LEDGF/p75 interaction with an IC50 value of 8 lM. Using molecular modeling,
the mechanism of action was hypothesized to involve the chelation of the divalent metal ions inside the
IN active site. Furthermore, the inhibitor of IN-LEDGF/p75 interaction was properly bound to the LEDGF/
p75 binding site on IN. This work provides a new and efficient approach to evolve novel HIV-1 IN inhib-
itors from rational integration and optimization of previously reported inhibitors.
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
either function.2 It is generally accepted that Mg2+ is a more rea-
sonable cofactor for integration because of its 1,000,000-fold abun-
HIV-1 IN is a virally encoded enzyme that is critical for viral
replication. This protein catalyzes the insertion of the reverse-
transcribed proviral cDNA into the host cell genome through a
multistep process that includes two catalytic reactions: 30-endonu-
cleolytic processing of the proviral DNA ends (termed 30-process-
ing) and the integration of the 30-processed viral DNA into the
cellular DNA (referred to as strand transfer).1 Divalent metals, such
as Mg2+ or Mn2+, are required for not only the 30-processing and
strand transfer steps, but also for the assembly of IN onto specific
viral donor DNA to form a complex that is competent to carry out
dance over Mn2+ in cells.2,3 Thus the chelation of the critical metal
cofactors can cause the functional impairment of IN, providing an
alternative opportunity for the design and development of highly
efficient IN inhibitors.4 In fact, all of the small molecule HIV-1 IN
inhibitors that exhibit potent antiviral activity by the inhibition
of viral DNA integration, commonly contain a structural motif that
coordinates the two divalent magnesium ions in the enzyme’s ac-
tive site.4 Typically, the most developed and promising b-diketo-
acid, naphthyridine carboxamide, pyrimidinone and quinolone
carboxylic acid classes of IN inhibitors belong to this category.5
Among these chelation inhibitors, the pyrimidinone MK-0518 has
been approved by the FDA and currently as the first and only
HIV-1 IN inhibitor for the treatment of HIV infection.6
⇑
Corresponding authors. Tel./fax: +86 21 50806876 (Y.-Q.L.); tel.: +1 323 442
2341; fax: +1 323 442 1390 (N.N.).
Although great strides have been achieved in the design and
discovery of IN inhibitors as antiviral agents,7,8 the emergence of
viral strains resistant to clinically studied IN inhibitors and the
E-mail addresses: neamati@usc.edu (N. Neamati), yqlong@mail.shcnc.ac.cn
(Y.-Q. Long).
These two authors equally contributed to this work.
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.06.058

4936
X. Fan et al. / Bioorg. Med. Chem. 19 (2011) 4935–4952
dynamic nature of the HIV-1 genome demand a continued effort
toward the discovery of novel inhibitors to keep a therapeutic
advantage over the virus. One approach to discover structurally no-
vel classes of IN inhibitors is to revive previously identified IN
inhibitor chemical classes, which displayed potent IN inhibition,
but were developmentally halted due to unwanted pharmacoki-
netic, pharmacodynamic, or toxicological properties. The salicyl-
hydrazide and polyhydroxyl aromatics (as exemplified by the
structures in Fig. 1) were previously reported as potent IN inhibi-
tors,9,10 but the inherently high cytotoxicity in these compounds
limited their therapeutic application as antiretroviral agents. Re-
cent structural modifications on the salicylhydrazide family re-
sulted in a significant decrease in the cytotoxicity while the IN
inhibitory activity was retained,11,12 thereby confirming the feasi-
bility of reviving old drugs by structural optimization.
In this study, we were interested in designing new IN inhibitors
by merging the pharmacophores of the salicylic acid and catechol
to generate novel chemical scaffolds. Actually, the adjacent carbox-
ylic and hydroxyl groups on salicylic acid could serve as the metal
binding pharmacophore. On the other hand, the polyhydroxylated
aromatic inhibitors are usually active against both 30-processing
and strand transfer reactions13 that might imply a different mech-
anism targeting both steps. Since HIV-1 resistant strains exhibited
cross-resistance to different strand transfer specific chemical clas-
ses in preclinical and clinical development studies,5 the optimal
integration of salicylic acid and catechol pharmacophores is ex-
pected to produce novel inhibitors by chelating a divalent metal
on the IN active site. These inhibitors are likely to be effective
against viral strains that display resistance to strand transfer spe-
cific inhibitors.
terns on the phenyl ring to determine an optimal scaffold (as
shown in Fig. 2). The catechol moiety is susceptible to oxidation
into a quinone species that have a propensity to cross-link with
cellular proteins, thereby leading to cytotoxicity,14 an outcome
that we tried to mitigate by incorporating alkyoxy groups such
as benzyloxy, 40-fluorobenzyloxy and naphthalenylmethoxy into
3-, 4-, 5-, and 6-postion of the 2-hydroxybenzoic acid, respectively
(Fig. 2, compounds 3a–j). Furthermore, the aryl group might serve
as the pharmacophore to interact with the hydrophobic binding
surface of the IN, as revealed by the aryl diketoacid IN inhibitors.
On the other hand, we tried to apply hydroxyamic acid (Fig. 2,
4a–d) or 2,3-dihydroxybenzoyl amide (Fig. 2, 5a–x, 6a–d) as an
isostere of the 3-substituted-2-hydroxybenzoic acid for the design
of metal-chelating IN inhibitors. This idea was also inspired by the
recent discovery of the dihydroxybenzoylnaphthylhydrazone
(DHBNH, Fig. 1) as a novel HIV-1 reverse transcriptase inhibitor
that exerts its inhibitory effect through a metal-chelating mecha-
nism.15,16 We propose that the neighboring carbonyl and two free
hydroxyl groups on the 2,3-dihydroxybenzoyl amide might suffi-
ciently bind to the two metal cofactors in the IN active site, and
the substituent on the amide portion could provide the interac-
tions with the hydrophobic pocket of the enzyme. Herein, we re-
port the synthesis, evaluation and SAR study of these salicylic
acid-based IN inhibitors and establish their binding mode. We fur-
ther tested their ability to inhibit the interaction between IN and
LEDGF/p75 with the idea that some of these compounds may act
as allosteric inhibitors of IN.17
2. Chemistry
Consequently, by combining the privileged fragments of salicyl
and catechol-containing IN inhibitors, we designed four classes of
2-hydroxybenzoic acid derivatives with various substitution pat-
The 3/4/5/6-substituted salicylic acid derivatives were synthe-
sized readily from the corresponding precursors. As outlined in
Scheme 1, the direct O-alkylation of the 3/4/5-hydroxy-2-hydroxy-
OH
O
N
N
O
OH
H
N
OH
OH
O
OH
H
O
N
N
S
N
N
H
H
O
OH
O
2
Caffeic acid phenethyl ester(CAPE)
1
OH
O
HO
O
H3C
O
O
OH
OH
OH
N
O
O
OH
N
OH
OH
O
N
HO
H
OCH3
OH
O
2,3-dihydro-6,7-dihydroxy-2-
phenylmethyl-1H-isoindol-1-one
L-Chicoric acid (L-CA)
DHBNH
Figure 1. Representative examples of salicyl and catechol-containing IN inhibitors.
CONHOH
OH
COOH
OH
4a-d
OR
COOH
OH
OH
3/4/5/6-substituted-2,3-
OH
dihydroxybenzyl hydroxamic acid
OR
CONHR2
OH
3/4/5/6-substituted-2-
hydroxybenzoic acid
3a-j
5a-x, 6a-d
2,3-dihydroxybenzoyl amide
OH
R1
Figure 2. Scaffold hopping approach for design of new IN inhibitors by combining the privileged fragments of salicylate and catechol.

X. Fan et al. / Bioorg. Med. Chem. 19 (2011) 4935–4952
4937
O
OH
O
OH
O
OH
ii
HOHN
i
HO
HO
RO
HO
OH
RO
4a-c
3a-g
O
O
OH
O
OH
O
OH
iv
iii
ii
O
O
HO
RO
HOHN
RO
HO
RO
7h-j
3h-j
4d
Scheme 1. Preparation of the 3/4/5/6-substituted salicylic acid derivatives. Reagents and conditions: (i) NaH, DMF, bromide, 7 h, rt; (ii) ClCO2iBu, NMM, NH2OHHCl, THF, 0 °C,
2 h; (iii) K2CO3, NaI, bromide, acetonitrile, DMF, 14 h, rt; (iv) THF, 1 N NaOH, 80 °C, 24 h.
benzoic acids by the corresponding bromide in the NaH/DMF solu-
tion afforded the desired products (3a–g). The intramolecular
hydrogen bonding between the 1-carboxy group and 2-hydroxy
group secured the selective alkylation on the 3/4/5-hydroxy site.
Similarly, the 6-subtituted analogs (3h–j) were prepared from
methyl 2,6-dihydroxybenzoate by reacting with different bromide
in the presence of NaI and K2CO3 followed by the hydrolysis in 1 N
NaOH/THF solution. However, because the 1-carboxy group can
form intramolecular hydrogen bonds with neighboring 2- and 6-
hydroxyl groups which lowered the reactivity, the methyl 2,6-
dihydroxybenzoate was employed as the starting material for the
synthesis of 6-alkoxy analogs (3h–j). The resulting 3/4/5/6-substi-
tuted salicylic acids were conveniently converted into the corre-
sponding hydroxamic acids (4a–d) by reacting with
hydroxylamine in the presence of activating agent and base.
For the 2,3-dihydroxybenzamide series (5a, 5c–m), the synthe-
sis was generally achieved by the condensation of 2,3-dihydroxy-
benzoic acid with the corresponding amine, as depicted in
Scheme 2. Additionally, the preparation of 1-benzyloxy derivative
(5b) needed one more alkylation prior to the condensation.
In this series, further structural modifications were conducted
on the phenyl ring by incorporating an acidic functionality into
the benzylamino portion or the benzoyl moiety. These analogs re-
quired a separate preparation of the coupling components. For
example, the substituted benzylamine was first synthesized as
the building block for the N-(2,4-substituted-phenyl methyl) 2,3-
dihydroxybenzamide derivatives (5n–o). As indicated in Scheme 3,
the ring-opening of isochroman-3-one by methyl amine produced
the 2-(2-(hydroxymethyl)phenyl)-N-methylacetamide 9, which
was treated with methanesulfonyl chloride to yield the chloride
10 instead of the expected methanesulfonate. The conversion of
the chloride into the amine 12n was achieved by the nucleophilic
attack of azide followed by reduction. The resulting 2-(2-(amino-
methyl)phenyl)-N-methylacetamide was coupled with 2,3-dihy-
droxybenzoic acid afforded the desired product 5n. Another
methylcarbamoyl-substituted benzylamine 12o was synthesized
from 5-fluoro-2-methylbenzoic acid via the sequential bromina-
tion, azido replacement and the reduction. The condensation of
the 2,3-dihydroxybenzoic acid with the resultant 2-(amino-
methyl)-5-fluoro-N-methylbenzamide generated the final product
N-(4-fluoro-2-(methylcarbamoyl)benzyl)-2,3-dihydroxybenza-
mide 5o.
OH
O
O
OH
O
i
HO
HO
OH
OH
NHR
5a, 5c-m
OH
ii
O
OH
i
BnO
BnO
N
H
F
8b
5b
Scheme 2. Synthesis of 2,3-dihydroxybenzamide derivatives. R is as indicated in
Table 2. Reagents and condition: (i) EDCI, DIPEA, HOBt, THF or DCM, various amine,
6–8 h, rt; (ii) BnBr, NaH, DMF.
OH
Cl
O
i
ii
O
O
N3
H2N
O
N
H
N
H
R1
R1
9
10
iii
iv
Br
O
O
CO2H
vi
vii
N
H
N
R2
11n-o
R2
12n-o
H
13
14
n:
F
F
OH
F
O
O
R1
HO
v
H
F
R2 =
R2 =
R1
=
=
N
N
H
H
N
H
o: R1
R2
5n-o
O
Scheme 3. Synthesis of N-(2,4-substitutedphenyl methyl) 2,3-dihydroxybenzamide derivatives. Reagents and conditions: (i) NH2Me, THF; (ii) MsCl, TEA, DCM; (iii) NaN3,
DMF, 80 °C; (iv) Ph3P, THF; (v) 2,3-dihydroxy-benzoic acid, EDCI, HOBt, TEA or NMM, DCM; (vi) NH2Me, EDCI, HOBt, DCM; (vii) NBS, AIBN, CCl4, reflux.

4938
X. Fan et al. / Bioorg. Med. Chem. 19 (2011) 4935–4952
O
O
O
O
O
O
O
O
i
OH
OH
ii
iii
O
OH
p
q
: R=1-Piperidinyl
: R=Me
O
S
Cl
O
O
S
NHR
O
16p-r
r
: R=tBu
15
OH
O
OH
O
O
O
HO
O
O
N
H
N
H
N
H
iv
v
F
F
F
O
S
NHR
O
O
S
NHR
O
O
S O
NHR
5p-r
18p-r
17p-r
OH
O
OH
O
R1O
COOH
vi
O
COOH
v
R1O
R2
N
H
O
S
N
O
O
S O
N
O
S O
N
s-v:
R1=H
w
:
R1=Me
5s-v, 6c
17s-w
16p
Scheme 4. Synthesis of 5-sulfoxamide substituted 2,3-dihydroxybenzamide derivatives. Reagents and conditions: (i) chlorosulfonic acid, 15 h, rt; (ii) alkyl amine, Et3N,
CH2Cl2, reflux, 22 h; (iii) EDCI, DIPEA, HOBt, THF, 4-fluorobenzylamine, 6.5 h, rt.; (iv) 15 equiv BBr3, CH2Cl2, ꢀ20 °C, 10 h or LiCl, DMF, 110 °C, 8 h; (v) 15 equiv BBr3, CH2Cl2,
ꢀ20 °C, 10 h; (vi) EDCI, DIPEA, HOBt, DMF, amine, 6.5 h, rt.
OMe O
NO2
OMe
OMe
MeO
MeO
CO2H
iii
i
ii
N
H
MeO
MeO
CO2H
F
20
HO
19
NO2
OH
O
OMe O
OMe O
NH2
MeO
v
N
iv
N
H
H
N
H
F
F
F
w
5w-x
R= Ms,
NHR
OH
NHR
22w-x
Ac, x
21
vi
O
OMe O
HO
vii
MeO
N
H
N
H
F
F
CN
5y
22y
CN
Scheme 5. Synthesis of 5-substituted 2,3-dihydroxybenzamide derivatives. Reagents and conditions: (i) concd H2SO4, HNO3, 0 °C; (ii) 4-fluorobenzylamine, EDCI, DIPEA,
HOBt, DCM,, 6.5 h, rt; (iii) SnCl2, HCl, THF, rt; (iv) MsCl for 22w (or AcCl for 22x), TEA, DCM, rt; (v) LiCl, DMF, 110 °C, 8 h then 15 equiv BBr3, CH2Cl2, ꢀ20 °C, 10 h (for 5w–x) or
AlBr3, MeCN, 0 °C, 8 h (for 5y); (vi) NaNO2, EtOH, H2O, ꢀ15 °C, then CuCN, H2O, 50 °C,1 h, rt overnight.
For the synthesis of 5-substituted 2,3-dihydroxybenzamide
derivatives, the 5-sulfamoyl substituted 2,3-dihydroxybenzoic acid
was first synthesized as the component for the condensation reac-
tion. As shown in Scheme 4, the 2,3-dimethoxybenzoic acid was
treated with chlorosulfonic acid to provide the sulfonylated com-
pound 15. The sulfonamidation of 15 with piperidine, methyl-
amine, or tert-butyl amine afforded compounds 16p–r. Then the
condensation of 16p–r with 4-fluorobenzyl amine in the presence
of HOBt and EDCI provided compounds 17p–r. The sequential
demethylation of the 2,3-dimethoxyl groups by boron tribromide
afforded the desired products 5p–r. More derivatives of 2,3-dihy-
droxy-5-(piperidin-1-ylsulfonyl)benzamide with variation on the
amide portion (5s–v, 6c) were synthesized in a similar fashion,
from the common intermediate of 16p, which underwent the
demethylation before the coupling with various amines. The
demethylation of 16p by boron tribromide yielded the mixture of
3-demethylated and 2,3-demethylated products, which were sepa-
rated by column chromatography.
The amino or cyano group substituted at 5-position of the
2,3-dihydroxybenzamide was introduced via nitration on the 2,3-
dimethoxybenzoic acid. As shown in Scheme 5, after the condensa-
tion with 4-fluorobenzyl amine, the 5-nitro- 2,3-dimethoxybenzoic
OH
OH
O
OH
HO
N
COOH HO
ii
R
HO
COOH
N
H
i
23
6a-b
N
Scheme 6. Synthesis of 4-alkylamino substituted 2,3-dihydroxybenzamide deriv-
atives. Reagents and conditions: (i) 40% HCHO (aq), dimethylamine hydrochloride,
Et3N, EtOH; (ii) EDCI, DIPEA, HOBt, DMF, amine, 6.5 h, rt.

X. Fan et al. / Bioorg. Med. Chem. 19 (2011) 4935–4952
4939
Table 1
Scaffold screening: inhibition of HIV-1 integrase catalytic activities and the cytotoxicity by salicylic acid derivatives with various substitution pattern
OH
O
R2
R1
Compd
R1
R2
IC50
(lM)
CC50 (lM) H630 cells
30-Processing
Strand transfer
>100
3a
3b
3c
3d
3e
3f
OH
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
NDa
ND
O
3-
3-
3-
4-
4-
5-
5-
6-
6-
6-
F
OH
>100
O
OH
140 46
>100
O
O
OH
F
F
F
OH
>100
O
OH
>100
O
3g
3h
3i
OH
>100
O
OH
>100
O
OH
>100
O
3j
OH
186 15
>100
O
F
F
F
F
4a
4b
NHOH
NHOH
O
3-
4-
5-
100
O
O
O
4c
4d
5a
5b
NHOH
NHOH
>100
>100
100
85
97
7
6
ND
ND
>40
>40
6-
F
F
H
N
3-OH
35 25
111
H
N
3-OBn
>333
a
ND: not determined.
acid 20 was reduced into the corresponding 5-amino derivative 21
by stannous chloride in hydrochloric acid. Then the 5-amino deriv-
atives were further converted into amide (5w–x) or cyano analogs
(5y) by amidation and diazo-reaction followed by nucleophilic
replacement, respectively.
The substitution at 4-position was examined with alkylamino
group. The synthesis of 4-alkyamino substituted 2,3-dihydroxyb-
enzamide derivatives (6a–b) was achieved via a formylation on
the phenyl ring followed by reductive amination, as shown in
Scheme 6.

4940
X. Fan et al. / Bioorg. Med. Chem. 19 (2011) 4935–4952
Table 2
Inhibition of HIV-1 integrase catalytic activities, integrase-LEDGF/p75 interaction, and cytotoxicity of the 2,3-dihydroxybenzamide series with variations on the phenyl ring and
the carboxamide moiety
OH
O
HO
R2
N
H
R1
Compd
R1
R2
IC50
(
lM)
IC50
(lM) LEDGF/p75-IN
CC50 (lM) H630 cells
30-Processing
>100
Strand transfer
5a
5c
H
H
35 25
>100
>40
21
F
>100
26
4
34
9
F
F
5d
5e
H
H
>100
>100
23
24
7
2
56
45
8
>40
>40
9
N
H
5f
H
H
100
100
63 20
15
N
H
5g
>100
58 12
88 19
95 11
>40
N
H
S
5h
5i
H
H
83 21
90 14
12
15
4
5
>40
>40
O
89
8
5j
H
H
S
>100
>100
21
5
>100
>100
15
NH
5k
>100
>100
>10
5l
H
H
>100
>100
>100
>100
>40
21
5m
18
9
O
N
H
5n
5o
H
>100
>100
56 19
>100
>100
>40
>40
H
N
O
H
100
F
F
O2S
5p
5q
200
5
ND
13
N
–SO2NHMe
>100
44
7
>100
>40

X. Fan et al. / Bioorg. Med. Chem. 19 (2011) 4935–4952
4941
Table 2 (continued)
Compd
R1
R2
IC50
(
lM)
IC50
(lM) LEDGF/p75-IN
CC50 (lM) H630 cells
30-Processing
Strand transfer
F
F
5r
5s
–SO2NHtBu
70
8
5
100
>10
33
O
O2S
N
>100
13
>100
>100
O2S
N
5t
89
11
19
31
CF3
O2S
N
5u
5v
5w
53
4
3
8
1
>40
>40
S
O2S
N
>100
>100
44 19
34 10
>50
100
HN
S
F
F
F
O
O
HN
O
5x
5y
55
75
7
14
22
4
8
>100
ND
<10
>10
CN
7
OH
OH
O
O
HO
N
N
H
6a
6b
>100
>100
20
39
4
8
>50
>50
>10
7
F
HO
N
O
N
H
OH
O
O
O
N
H
6c
>100
>100
>100
>100
13
51
4
>25
>25
SO2
N
OH
O
H2N
O
6d
4
N
H
ported to facilitate the binding of two Mg2+ ions by the
azaindole-based IN inhibitors,18 which implied the ineffectiveness
of the alkoxy substituted salicylic acid scaffold as IN inhibitor.
However, the evolved 2,3-dihydroxybenzamide (5a–b) displayed
moderate inhibition against the strand transfer reaction. The N-
(4-fluorobenzyl)-2,3-dihydroxybenzamide 5a exhibited IC50 values
3. Results and discussion
3.1. Identification of the 2,3-dihydroxybenzamide as the active
scaffold of HIV-1 IN inhibitors
As depicted in Table 1, the 3/4/5/6-alkyloxy substituted salicylic
acid derivatives (3a–j) generally displayed weak inhibition against
IN regardless of the substituent structure and position. Even the
incorporation of the chelation-advancing hydroxylamino group
into the 3/4/5/6-alkyloxy-salicylic acid scaffold just slightly im-
proved the binding (4a–d). Although hydroxamic acids were re-
of 100 lM and 35 l
M in inhibiting 30-processing and strand trans-
fer, respectively. The elimination of the phenolic hydroxy at the 3-
position through its conversion to a benzyl ether reduced the
inhibitory potency (5b, IC50 = 111
lM for strand transfer) by 2.5-
fold relative to the 3-hydroxy analog 5a, which might result from

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X. Fan et al. / Bioorg. Med. Chem. 19 (2011) 4935–4952
the reduction of the metal-binding region. In addition, the 2,3-
fer inhibition (5c, IC50 = 26 lM; 5d, IC50 = 23 lM). In particular, the
dihydroxybenzamide derivatives (5a and 5b) were not cytotoxic
thiophenyl, furanyl and (thiophen-2-yl)phenyl substitutions mark-
in H630 cells at the concentration of up to 40
l
M.
edly enhanced the potency of strand transfer inhibition (5h,
Consequently, the 2,3-dihydroxybenzamide was chosen as the
template for further structural modification to increase potency.
The SAR study on the 2,3-dihydroxybenzamide-based IN inhibitors
included structural variation on the left side catechol group and
the right side benzamide moiety. The substitution on the phenyl
ring of the catechol core was investigated, and the structural vari-
ation on the ‘right side’ carboxamide group was explored with het-
erocycle (aliphatic and aromatic) and substituted phenyl ring
separately. The activity data is summarized in Table 2 and rational-
ized by molecular modeling.
IC50 = 12 lM; 5i, IC50 = 15 lM; 5j, IC50 = 21 lM). But the effect of
the indolyl substitution varied according to the linker length and
substituted position (5e–g), in which the best substituent was
(1H-indol-5-yl) methyl group (5e, IC50 = 24 lM). Conversely, the
N-methyl carbamoyl substitution at the 2-position of the 4-fluoro-
phenyl ring (5o) resulted in a loss of IN inhibitory potency. Inter-
estingly, the replacement of the phenylmethyl functionality with
the aliphatic counterpart, cyclohexylmethyl group, improved the
activity (5m, IC50 = 18
cyclohexyl or 4-piperidinyl group was deemed unfavorable at this
carboxamide position (5k and 5l, IC50 >100 M). This result indi-
lM vs 5a, IC50 = 35 lM), while the direct
l
3.2. SAR study with respect to the structural variation on the
carboxamide portion and phenyl ring of the 2,3-
dihydroxybenzamide scaffold
cated the importance of the hydrophobic substituent in the inhibi-
tion of the catalytic activities of IN.
As for the substitution effect of the 2,3-dihydroxyphenyl group
on potency, the introduction of a piperidin-1-ylsulfonyl group at 5-
position of the phenyl ring led to a 7-fold improvement in the
We prepared compounds with modifications on the ‘right side’
of the core structure (Table 2, 5a–o). A range of aryl- or alkyl-
substituted amines were investigated, whereby the heteroaromatic
amine and the amide collectively caused an increase in the 30-pro-
cessing inhibitory activity (5h–i, 5r, 5t–u, 5x–y) compared to the
parent compound 5a. Lipophilic substituents such as naphthalenyl
and 3,4-difluorophenyl groups were beneficial for the strand trans-
strand transfer inhibitory potency (5p, IC50 = 5 lM) relative to
the parent compound 5a. However, a more polar substituent such
as N-methyl or butyl sulfonyl group at the 5-position of 2,3-dihy-
droxyphenyl ring caused a decrease in the inhibitory activity (5q,
IC50 = 44
lM; 5r, IC50 = 8 lM), in accordance with the reduction
of the hydrophobicity. More electron-withdrawing groups were
Figure 3. The proposed binding modes of 5p, 5t, 5u and 6c in the modeled HIV-1 intasome. The close contact residues are shown in surface model. Inhibitors are shown as
pink sticks (colored by atom types) and the two Mg+2 are depicted as green spheres.

X. Fan et al. / Bioorg. Med. Chem. 19 (2011) 4935–4952
4943
examined with respect to the effect on the potency. The methyl-
sulfonamide, acetylamide or cyano group at 5-postion produced
moderately potent inhibitors against the strand transfer (5w,
aryl or aliphatic cyclic substituent on the right side carboxamide
portion might be responsible for the interaction with the hydro-
phobic surface of the enzyme. These observations can be reason-
ably rationalized from the docking studies by molecular modeling.
IC50 = 34
tural modification with the alkylamino group substituted at 4-po-
sition displayed slight influence on the potency (6a, IC50 = 20 M;
6b, IC50 = 39 M). Comparatively, the replacement of the 3-hydro-
lM; 5x, IC50 = 14 lM; 5y, IC50 = 22 lM). Further struc-
l
3.3. The 2,3-dihydroxybenzamide derivatives inhibit the
interaction of HIV-1 IN and LEDGF/p75 cofactor
l
xyl group with a methoxy or an amino group suffered a severe loss
of potency (6c, 6d), suggesting that the 3-hydroxy group was in-
volved in the two metal-binding interaction.
With the privileged structures disclosed above, we designed
new analogs with simultaneous substitution on both sides (5s–
v). Contrary to our expectation however, the combination of the
favorable substitution on both sides did not result in a synergistic
effect on the inhibitory activity . The resulting active compounds
exhibited low micromolar activity to inhibit the strand transfer
Lens epithelium-derived growth factor (LEDGF/p75) is a cellular
cofactor of IN that promotes viral integration by tethering the pre-
integration complex to the chromatin.19,20 Instead of targeting the
catalytic activity of IN, the disruption of the integrase-LEDGF/p75
interaction, and the consequent inhibition of HIV replication, rep-
resents a new frontier for the design and development of novel
anti-HIV agents for AIDS therapy.21 However, only a few small mol-
ecule inhibitors of the IN-LEDGF/p75 interaction have been re-
ported to date.17,22–25
Considering that the specific protein–protein interaction be-
tween IN catalytic core domain (CCD) and the LEDGF/p75 IN-binding
domain (IBD) was characterized by IBD residues Ile365, Asp366,
Phe406 and Val408,26 we were interested in testing these salicylate
and catechol-merged compounds in the inhibition of IN-LEDGF/
p75 interaction, since our chemical frame contained both the
aromatic moiety and the carboxylic functionality. Interestingly,
reaction (5s, IC50 = 13
IC50 = 44 M).
lM; 5t, IC50 = 11 lM; 5u, IC50 = 19 lM; 5v,
l
These salicylate and catechol-merged IN inhibitors were
hypothesized to function by the chelation of the divalent metal
ions in the active site of IN. According to the SAR study, the 2,3-
dihydroxybenzamide core might serve as the metal-binding motif,
and the substituents on the phenyl ring possibly afforded an addi-
tional interaction with the key residues in the binding pocket. The
Figure 4. A close-up view of the proposed binding modes of 5p, 5t, 5u and 6c in the modeled HIV-1 IN active site. The close interacting residues are depicted as cyan sticks.
5p, 5t, 5u and 6c are shown in pink sticks (colored by atom types) while green sphere represent the Mg2+ ions.

4944
X. Fan et al. / Bioorg. Med. Chem. 19 (2011) 4935–4952
most of our 2,3-dihydroxybenzamide derivatives were able to inhi-
bit the IN-LEDGF/p75 interaction at micromolar concentrations in
the AlphaScreen assay. As shown in Table 2, the active IN strand
transfer inhibitors displayed consistent potency in inhibiting the
interaction of IN-LEDGF/p75, among which the highest potency
occupies the hydrophobic pocket formed by Pro142, Tyr143 and
Asn117. Compounds 5t and 5u exhibited a binding mode that is
similar to 5p, while the inactive compound 6c, has the disadvan-
tage of possessing a single hydroxyl group to form metal chelating
interaction. In addition, its piperidine and furanyl moieties are not
properly positioned into hydrophobic pockets. As a result, 6c forms
weak interactions with the active site residues thereby potentially
contributing to its weak strand transfer activity.
Active LEDGF/p75 inhibitor, 5u, was also docked into the
LEDGF/p75 binding site of the HIV IN crystal structure 3LPU. Fig-
ure 5 shows the predictive binding mode of 5u into the LEDGF/
p75 binding site of HIV IN. The inhibitor develops strong interac-
tions with IN residues in the LEDGF/p75 binding site. One hydroxyl
group from central phenyl ring forms hydrogen bond with the
backbone carbonyl oxygen of Thr125. Cyclohexyl moiety formu-
lates hydrophobic interaction with Tyr99 and Gln95, while the
piperidine moiety creates hydrophobic interactions with Trp132
and Leu102.
was exhibited by 5u (IC50 = 8 lM) bearing the privileged structures
on both sides. The preliminary SAR study indicated that the substi-
tuent attached on the carboxamide portion played an important
role in the disruption of IN-LEDGF/p75 interaction. In this position,
the lipophilic structure as well as the heteroaromatics was favored
(Table 2, 5c–i). However, the catechol structure was not essential
for inhibiting IN-LEDGF/p75 interaction, exemplified by 6c and
6d which were almost inactive against the strand transfer but
moderately active in preventing IN-LEDGF/p75 interaction (6c,
IC50 = 13 lM; 6d, IC50 = 51 lM). Furthermore, the piperidin-1-yls-
ulfonyl substituent at 5-position of 2,3-dihydroxyphenyl ring
might potentiate the interaction with the LEDGF/p75-binding site
of IN when properly oriented (5u, IC50 = 8
lM; 6c, IC50 = 13 lM),
which was depicted in the following molecular modeling.
Our study provides new important chemical frames to disrupt
the protein–protein interaction between IN and its cellular cofactor
LEDGF/p75, which could advance the design and discovery of anti-
HIV agents possessing a novel mechanism of action.
4. Conclusions
In this study, through the optimal combination of the active
structures of salicylic acid and catechol, we successfully identified
a novel class of salicylic acid-based IN inhibitors targeting both the
catalytic domain of IN and the IN-LEDGF/p75 interaction. The hit
compound, 2,3-dihydroxybenzamide, showed low micromolar IN
inhibitory activity without exerting significant cytotoxicity. These
inhibitors are predicted to function by chelating the metal cofactor
in the active site of IN. Moreover, the substituents on the phenyl
ring and the carboxamide portion are responsible for the binding
with the key residues in the drug-binding pocket. Molecular mod-
eling revealed the binding mode of the active and inactive com-
pounds, confirming the metal-chelation mechanism. Our study
also supports a potential for allosteric inhibition by some of these
compounds due to their similar potency in inhibiting the interac-
tion between IN and LEDGF/p75.
3.4. Molecular modeling studies
We selected 5p, 5t and 5u as active and 6c as inactive represen-
tative structures to dock against an HIV-1 IN model which has been
built from a crystal structure of prototype foamy virus (PFV) inta-
some.27 Compounds were then docked into the binding site using
Standard Precision method of Glide from Schrodinger, Inc.28,29
The binding modes of compounds 5p, 5t, 5u and 6c in the IN active
site are shown in Figures 3 and 4. The docking modes in PFV IN of
all active compounds are similar to that of raltegravir. Both hydro-
xyl groups of 5p form metal chelating interactions with the two
Mg2+ ions along with the amino acid residues Asp64, Asp116 and
Glu152. The fluorophenyl moiety of 5p packed into the tight bind-
ing site formed by viral DNA bases DA17, DC16 and amino acid res-
idues Pro145 and Gln146. The phenyl ring forms a stacking
interaction with DC16. Thus, the compound displaces reactive
DNA 30OH group from the active site and causes the deactivation
of the intasome. On the other hand, the piperidine moiety of 5p
5. Experimental section
5.1. General synthetic methods
The 1H NMR spectra were recorded on a Varian 300-MHz spec-
trometer. The data are reported in parts per million relative to TMS
and referenced to the solvent. Elemental analyzes were obtained
using a Vario EL spectrometer. Melting points (uncorrected) were
determined on a Buchi-510 capillary apparatus. The MS spectra
were obtained on an APEXIII 7.0 T FTMS mass spectrometer. The
flash column chromatography was performed on silica gel H (10–
40 lm). Anhydrous solvents were obtained by standard procedure.
For the target compounds the purity was determined by analytical
HPLC using a Dionex P680 equipped with UV detector. HPLC condi-
tions: Global chromatography column (250 ꢁ 4.6 mm); solvent
gradient, A, 0.05% TFA in water; B, 0.05% TFA in 90% acetonitrile
in water (solvent system 1); or A, 0.05% TFA in water; C, methanol
(solvent system 2), with gradient indicated below; flow rate,
1.0 mL/min; UV detector, 254 nm.
5.2. Methods
Method A: 10% B for 2 min, 10% B to 90% B in 13 min, 90% B for
5 min, 90% B to 10% B in 5 min.
Method B: 10% B for 2 min, 10% B to 90% B in 8 min, 90% B for
10 min, 90% B to 10% B in 5 min.
Method C: 10% B for 2 min, 10% B to 90% B in 13 min, 90% B for
5 min, 90% B to 10% B in 3 min.
Figure 5. The proposed binding mode of 5u in to the LEDGF binding site of HIV IN.

X. Fan et al. / Bioorg. Med. Chem. 19 (2011) 4935–4952
4945
2
Method D: 10% B for 2 min, 10% B to 50% B in 8 min, 50% B to 90%
B in 5 min, 90% B to 10% B in 3 min.
Method E: 10% B for 2 min, 10% B to 20% B in 3 min, 20% B to 90%
B in 5 min, 90% B for 5 min, 90% B to 10% B in 5 min.
Method F: 10% C for 2 min, 10% C to 90% C in 8 min, 90% C for
10 min, 90% C to 10% C in 5 min.
Method G: 10% C for 2 min, 10% to 95% C in 3 min, 95% C for
15 min, 95% to 10% C in 5 min, 10% C for 3 min.
Method H: 10% C for 2 min, 10% C to 20% C in 15 min, 10% C to
90% C in 5 min, 90% C to 10% C in 6 min.
3JCF = 8.2 Hz), 116.8 (d, JCF = 21.6 Hz), 109.0, 107.6, 103.1, 70.9.
EI-MS m/z: 262 (M)+. Purity: system 1, 95.1% (method A,
tR = 16.33 min); system 2, 94.7% (method F, tR = 15.34 min).
5.2.6. 5-(Benzyloxy)-2-hydroxybenzoic acid (3f)
Compound 3f was prepared as a white solid according to the
same procedure described for 3a, (66 mg, 46% yield). Mp: 162–
164 °C; 1H NMR (300 MHz, CDCl3): d 7.45–7.33 (m, 6H), 7.21 (m,
1H), 6.95 (m, 1H), 5.04 (s, 2H). 13C NMR (100 MHz, CD3OD): d
158.2, 152.9, 139.1, 130.0, 129.4, 129.2, 125.9, 119.5, 115.9, 72.3.
EI-MS m/z: 244 (M)+. Purity: system 1, 95.1% (method A,
tR = 16.01 min); system 2, 95.1% (method F, tR = 14.60 min).
5.2.1. 3-(Benzyloxy)-2-hydroxybenzoic acid (3a)
To the solution 60% NaH (100 mg, 2.2 mmol) in DMF (5 mL) was
added 2,3-dihydroxy benzoic acid at room temperature and stirred
for 1 h, then benzyl bromide (0.119 mL, 1 mmol) was added to the
above solution and stirred for 7 h. Then the reaction was quenched
by adding H2O (20 mL) and the reaction mixture was acidified with
1 N HCl to pH 1–3. The solution was extracted with EtOAc (20 mL),
washed by brine (3 ꢁ 20 mL) and dried over anhydrous Na2SO4.
The solvent was removed under vacuum. The residue was purified
by flash chromatography using petroleum ether/ethyl acetate (1:1)
to give compound 3a as a white solid (54 mg, 22% yield). Mp: 151–
153 °C; 1H NMR (300 MHz, CDCl3): d 7.55–7.31 (m, 6H), 7.11 (m,
1H), 6.81 (m, 1H), 5.19(s, 2H). 13C NMR (100 MHz, CD3OD): d
174.3, 149.0, 139.0, 130.0, 129.5, 129.2, 124.1, 121.6, 120.0, 72.8.
EI-MS m/z: 244 (M)+. Purity: system 1, 97.6% (method A,
tR = 15.63 min); system 2, 96.8% (method F, tR = 14.61 min).
5.2.7. 5-(4-Fluorobenzyloxy)-2-hydroxybenzoic acid (3g)
Compound 3g was prepared as a white solid according to the
same procedure described for 3a (49 mg, 40% yield). 1H NMR
(300 MHz, CDCl3): d 10.08 (br s, 1H), 7.41 (m, 3H), 7.20 (m, 1H),
7.08 (m, 2H), 6.96 (m, 1H), 5.00 (s, 2H). 13C NMR (100 MHz,
1
CD3OD): d 173.6, 164.4 (d, JCF = 243.3 Hz), 158.3, 152.8, 135.2,
3
2
131.2 (d, JCF = 8.2 Hz), 126.0, 119.6, 116.7 (d, JCF = 21.5 Hz),
115.9, 114.0, 71.6. EI-MS m/z: 262 (M)+. Purity: system 1, 94.7%
(method A, tR = 16.10 min); system 2, 95.4% (method F,
tR = 15.16 min).
5.2.8. Methyl 2-(benzyloxy)-6-hydroxybenzoate (7h)
To a suspension of methyl 2,6-dihydroxybenzoate (84 mg,
0.5 mmol), K2CO3(83 mg, 0.6 mmol), and NaI (22 mg, 0.15 mmol)
in CH3CN (5 mL) and DMF (5 mL) was added benzyl bromide, the
mixture was stirred for 14 h at room temperature. Evaporated to
remove most of the CH3CN, then the mixture was acidified with
1 N HClꢂH2O (20 mL) and EtOAc (20 mL) were added. The organic
layer was washed with brine (3 ꢁ 20 mL), dried over anhydrous
Na2SO4 and concentrated in vacuo. The residue was chromatogra-
phied on silica gel (PE/EtOAc = 20:1) to provide methyl 2-(benzyl-
oxy)-6-hydroxybenzoate 7h (51 mg, 40% yield) as white solid, with
starting material being recovered (44 mg, 52%). 1H NMR (300 MHz,
CDCl3): d 11.54 (s, 1H), 7.50–7.30 (m, 6H), 6.64 (m, 1H), 6.48 (m,
1H), 5.13 (s, 2H), 3.96 (s, 3H).
5.2.2. 3-(4-Fluorobenzyloxy)-2-hydroxybenzoic acid (3b)
Compound 3b was prepared as a white solid according to the
same procedure described for 3a, (42 mg, 28% yield). 1H NMR
(300 MHz, DMSO-d6): d 7.49 (dd, J = 5.7 Hz, 8.7 Hz 2H), 7.36 (dd,
J = 1.5 Hz, 8.1 Hz, 1H), 7.22 (m, 3H), 6.81 (t, J = 8.1 Hz, 1H), 5.09
(s, 2H). 13C NMR (100 MHz, CD3OD):
d 174.3, 164.4 (d,
3
1JCF = 242.6 Hz), 154.7, 148.9, 135.0, 131.4 (d, JCF = 8.2 Hz), 124.3,
2
121.7, 120.0, 116.6 (d, JCF = 21.5 Hz), 115.0, 72.2. EI-MS m/z: 262
(M)+. Purity: system 1, 99.2% (method A, tR = 15.71 min); system
2, 98.1% (method F, tR = 14.63 min).
5.2.3. 3-((Naphthalen-2-yl)methoxy)-2-hydroxybenzoic acid
(3c)
5.2.9. 2-(Benzyloxy)-6-hydroxybenzoic acid (3h)
The reaction mixture of 2-(benzyloxy)-6-hydroxybenzoate 7h
(39 mg, 0.15 mmol) in 3 mL THF and 3 mL 1 N NaOH was heated
to 80 °C for 24 h, then the solution was acidified with 1 N HCl to
pH 1–2 and extracted with ethyl acetate (2 ꢁ 15 mL). Washed with
brine and dried over Na2SO4, the solution was evaporated under
vacuum to give 2-(benzyloxy)-6-hydroxybenzoic acid 3h (34 mg,
92% yield) as a white solid. Mp: 123–126 °C; 1H NMR (300 MHz,
CDCl3): d 12.10 (s, 1H), 11.38 (br s, 1H), 7.41 (m, 6H), 6.75 (m,
1H), 6.59 (m, 1H), 5.46 (s, 2H). 13C NMR (100 MHz, CD3OD): d
173.5, 164.6, 160.6, 138.0, 136.5, 130.2, 129.8, 129.2, 112.1,
105.3, 73.0. EI-MS m/z: 244 (M)+. Purity: system 1, 97.5% (method
A, tR = 16.57 min); system 2, 95.5% (method F, tR = 14.29 min).
Compound 3c was prepared as a white solid according to the
same procedure described for 3a (58 mg, 26% yield). 1H NMR
(300 MHz, DMSO-d6): d 7.96–7.89 (m, 4H), 7.58 (m, 1H), 7.50 (m,
2H), 7.36 (m, 1H), 7.26 (m, 1H), 6.78 (m, 1H), 5.28 (s, 2H). 13C
NMR (100 MHz, CD3OD): d 174.3, 165.3, 154.6, 136.5, 135.2,
135.1, 129.7, 129.5, 129.2, 128.0, 127.7, 127.6, 127.0, 124.2,
121.8, 120.0, 115.0, 73.0. EI-MS m/z: 294 (M)+. Purity: system 1,
96.2% (method A, tR = 17.22 min); system 2, 97.8% (method F,
tR = 16.44 min).
5.2.4. 4-(Benzyloxy)-2-hydroxybenzoic acid (3d)
Compound 3d was prepared as a white solid according to the
same procedure described for 3a, (62 mg, 39% yield). Mp: 173–
175 °C; 1H NMR (300 MHz, CDCl3): d 7.81 (m, 1H), 7.37 (m, 5H),
6.55 (m, 2H), 5.10 (s, 2H). 13C NMR (100 MHz, CD3OD): d 164.5,
137.4, 132.5, 128.9, 128.4, 128.1, 107.2, 101.8, 70.1. EI-MS m/z:
244 (M)+. Purity: system 1, 95.4% (method A, tR = 16.26 min); sys-
tem 2, 99.3% (method F, tR = 15.43 min).
5.2.10. Methyl 2-(4-fluorobenzyloxy)-6-hydroxybenzoate (7i)
Compound 7i was prepared as a white solid according to the
same procedure described for 7h, (42 mg, 22% yield). 1H NMR
(300 MHz, CDCl3): d 11.49 (s, 1H), 7.45–7.35 (m, 3H), 7.09 (m,
2H), 6.63 (m, 1H), 6.47 (m, 1H), 5.08(s, 2H), 3.93(s, 3H).
5.2.11. 2-(4-Fluorobenzyloxy)-6-hydroxybenzoic acid (3i)
Compound 3i was prepared as a white solid according to the
same procedure described for 3h (55 mg, 90% yield). 1H NMR
(300 MHz, CDCl3): d 12.18 (s, 1H), 11.30 (br s, 1H), 7.45–7.38 (m,
3H), 7.17–7.11 (m, 2H), 6.75 (d, J = 8.4 Hz, 1H), 6.56 (d, J = 8.4 Hz,
1H), 5.22(s, 2H). 13C NMR (100 MHz, CD3OD): d 173.5, 165.8 (d,
5.2.5. 4-(4-Fluorobenzyloxy)-2-hydroxybenzoic acid (3e)
Compound 3e was prepared as a white solid according to the
same procedure described for 3a, (72 mg, 53% yield). 1H NMR
(300 MHz, DMSO-d6): d 7.68 (m, 1H), 7.48 (m, 2H), 7.21 (m, 2H),
6.54 (m, 2H), 5.11 (s, 2H). 13C NMR (100 MHz, CD3OD): d 173.9,
1
3
166.5 (d, JCF = 244.1 Hz), 165.8, 163.3, 134.5, 133.5, 131.3 (d,
1JCF = 243.3 Hz), 164.4, 160.6, 136.3, 134.3, 131.2 (d, JCF = 8.9 Hz),

4946
X. Fan et al. / Bioorg. Med. Chem. 19 (2011) 4935–4952
2
116.8 (d, JCF = 21.6 Hz), 112.0, 105.4, 72.2. EI-MS m/z: 262 (M)+.
Purity: system 1, 99.2% (method A, tR = 16.48 min); system 2,
99.6% (method F, tR = 14.27 min).
most of THF, 20 mL of EtOAc was added to the residue. The solution
was washed by 1 N HCl, satd NH4Cl and dried over Na2SO4. The
concentration provided the residue which was purified by chroma-
tography using petroleum ether/ethyl acetate (1:1) as eluent to
give compound 5a as a white solid (51 mg, 57% yield). 1H NMR
(300 MHz, DMSO-d6): d 12.43 (br s, 1H), 8.33 (m, 1H), 7.40–7.32
(m, 3H), 7.20–7.14 (m, 2H), 6.94 (m, 1H), 6.71 (m, 1H), 4.48 (s,
2H). 13C NMR (100 MHz, CD3OD): d 171.9, 163.9 (d, 1JCF = 242.6 Hz),
5.2.12. Methyl 2-((naphthalen-3-yl)methoxy)-6-
hydroxybenzoate (7j)
Compound 7j was prepared as a white solid according to the
same procedure described for 7h (39 mg, 20% yield). 1H NMR
(300 MHz, CDCl3): d 11.55 (s, 1H), 7.97 (s, 1H), 7.90–7.84 (m,
3H), 7.59–7.49 (m, 3H), 7.34 (m, 1H), 6.64 (d, J = 8.4 Hz, 1H), 6.54
(d, J = 8.4 Hz, 1H), 5.28 (s, 2H), 3.99 (s, 3H).
3
150.8, 147.9, 136.6, 130.9 (d, JCF = 8.2 Hz), 120.2, 119.2, 119.1,
2
117.1, 116.6 (d, JCF = 21.6 Hz), 43.7. EI-MS m/z: 261 (M)+. Purity:
system 1, 96.3% (method A, tR = 14.44 min); system 2, 95.0% (meth-
od F, tR = 13.13 min).
5.2.13. 2-((Naphthalen-2-yl)methoxy)-6-hydroxybenzoic acid
(3j)
5.2.19. 3-(Benzyloxy)-2-hydroxybenzoic acid (8b)
Compound 3j was prepared as a white solid according to the
same procedure described for 3h (47 mg, 30% yield). 1H NMR
(300 MHz, CDCl3): d 12.21 (s, 1H), 7.95–7.87 (m, 4H), 7.55 (m,
3H), 7.41 (m, 1H), 6.75 (d, J = 8.4 Hz, 1H), 6.63 (d, J = 8.4 Hz, 1H),
5.43 (s, 2H). 13C NMR (100 MHz, CD3OD): d 173.6, 164.5, 160.7,
137.5, 135.6, 135.3, 130.0, 129.9, 129.7, 129.5, 129.3, 129.1,
128.3, 127.9, 126.8, 126.6, 112.0, 73.4. EI-MS m/z: 294 (M)+. Purity:
system 1, 98.4% (method A, tR = 18.13 min); system 2, 98.9% (meth-
od F, tR = 15.89 min).
To a stirred solution of 2,3-dihydroxybenzoic acid (154 mg,
1 mmol) in DMF (5 mL) was added NaH (48 mg, 2 mmol) and BnBr
(0.125 mL, 1.05 mmol). The above mixture was stirred for 8 h and
the excess DMF was removed under reduced pressure. The residue
was diluted with DCM (10 mL) and the organic phase was washed
with brine (5 mL), dried with anhydrous Na2SO4, filtered, concen-
trated under vacuum to give the 8b (61 mg, 25% yield) as colorless
oil. 1H NMR (300 MHz, CDCl3): d 7.41–7.20 (m, 6H), 6.79 (br, 1H),
6.50 (br, 1H), 4.99 (s, 2H).
5.2.14. 3-(4-Fluorobenzyloxy)-N,2-dihydroxybenzamide (4a)
To a solution of 3b (1.31 g, 5.0 mmol) in dry THF (25.0 ml) was
added NMM (0.58 ml, 5.25 mmol) at ꢀ15 °C, stirred for 15 min,
5.2.20. 3-(Benzyloxy)-N-(4-fluorobenzyl)-2-hydroxybenzamide
(5b)
Compound 5b was prepared from 8b and (4-fluoro-
phenyl)methanamine according to the same procedure described
for 5a. Purification via column chromatography using PE/EA = 5:1
i
then added ClCO2 Bu (0.69 ml, 5.25 mmol) during 15 min, and the
mixture was stirred for 1 h at ꢀ15 °C. Then hydroxylamine hydro-
chloride (0.52 g, 7.5 mmol) which was dissolved in H2O (10 ml)
was added to above system during 30 min at 0 °C. The reaction
mixture was stirred for 1.5 h, then poured into ice-water, washed
with ether three times. The organic phases were combined and
washed with satd NaHCO3 and brine, dried with anhydrous
Na2SO4, filtered, and concentrated under vacuum to give 4a as a
white solid. 1H NMR (CD3OD, 300 MHz) d: 7.41 – 7.49 (m, 3H),
7.22 – 7.30 (m, 2H), 7.08 (t, J = 8.7 Hz, 2H), 5.09 (s, 2H), 3.97 (s,
1H), 3.95 (s, 1H). EI-MS: m/z 279 (M + 2)+.
afforded 5b (31 mg, 35% yield) as
a
white solid. 1H NMR
(300 MHz, CDCl3): d 11.20 (s, 1H), 7.45–7.28 (m, 6H), 7.18 (dd,
J = 1.5 Hz, 8.1 Hz, 1H), 7.14–7.12 (m, 1H), 7.04–6.99 (m, 3H), 6.74
(t, J = 7.8 Hz, 1H), 5.16 (s, 2H), 4.59 (d, J = 5.7 Hz, 2H). 13C NMR
1
(100 MHz, CDCl3): d 168.7, 162.2 (d, JCF = 245.0 Hz), 150.6, 147.6,
3
136.5, 133.5, 129.5 (d, JCF = 7.7 Hz), 128.6, 128.1, 127.5, 118.8,
2
118.4, 117.2, 115.6 (d, JCF = 21.4 Hz), 71.2, 42.9. EI-MS: m/z 351
(M)+. HRMS (EI): calcd for C21H18FNO3 (M)+ 351.1271, found
351.1278. Purity: system 1, 99.3% (method B, tR = 14.20 min); sys-
tem 2, 96.5% (method F, tR = 15.59 min).
5.2.15. 4-(4-Fluorobenzyloxy)-N,2-dihydroxybenzamide (4b)
Compound 4b was prepared from 3d according to the same pro-
cedure as 4a. Compound 4b was purified as a white solid. 1H NMR
(CDCl3, 300 MHz) d: 7.35 – 7.40 (m, 2H), 7.04 – 7.11 (m, 3H),
6.48 – 6.53 (m, 2H), 5.57 (br, 1H), 5.40 (br, 1H), 5.32 – 5.36 (m,
1H), 5.01 (s, 2H). EI-MS: m/z 277 (M)+.
5.2.21. 2,3-Dihydroxy-N-(naphthalen-1-ylmethyl)benzamide
(5c)
Compound 5c was prepared from 2,3-dihydroxybenzoic acid
and naphthalen-1-ylmethanamine according to the same proce-
dure described for 5a. Compound 5c was purified as a white solid.
(78 mg, 45% yield) 1H NMR (300 MHz, CDCl3): d 8.03 (d, J = 8.4 Hz,
1H), 7.92–7.85 (m, 2H), 7.51–7.43 (m, 5H), 6.79 (d, J = 7.8 Hz, 1H),
6.68 (t, J = 8.1 Hz, 1H), 6.52 (br, 1H), 5.82 (s, 1H), 5.08 (d, J = 4.5 Hz,
2H). 13C NMR (100 MHz, CDCl3): d 169.5, 148.7, 145.5, 133.6, 132.6,
131.1, 128.6, 128.3, 126.4, 126.0, 125.8, 125.2, 123.0, 118.5, 118.4,
116.9, 114.6, 41.3. EI-MS: m/z 293 (M)+. HRMS (EI): calcd for
5.2.16. 5-(4-Fluorobenzyloxy)-N,2-dihydroxybenzamide (4c)
Compound 4c was prepared from 3g according to the same pro-
cedure as 4a. Compound 4c was purified as a white solid. 1H NMR
(CDCl3, 300 MHz) d: 7.42 – 7.47 (m, 3H), 7.32 (d, J = 2.4 Hz, 1H),
7.04 – 7.16 (m, 4H), 6.84 (d, J = 8.7 Hz, 1H), 5.00 (s, 2H). EI-MS:
m/z 277 (M)+.
C
18H15NO3 (M)+ 293.1052, found 293.1058. Purity: system 1,
98.0% (method A, tR = 15.96 min); system 2, 99.1% (method F,
tR = 14.32 min).
5.2.17. 2-(4-Fluorobenzyloxy)-N,6-dihydroxybenzamide (4d)
Compound 4d was prepared from 3i according to the same pro-
cedure as 4a. Compound 4d was purified as a white solid. 1H NMR
(CDCl3, 300 MHz) d: 10.36 (br, 1H), 7.39 – 7.44 (m, 2H), 7.23 – 7.33
(m, 1H), 7.00 – 7.11 (m, 2H), 6.82 – 7.00 (m, 1H), 6.67 (d, J = 8.4 Hz,
1H), 6.47 (d, J = 8.7 Hz, 1H), 5.12 (s, 2H). EI-MS: m/z 277 (M)+.
5.2.22. N-(3,4-Difluorobenzyl)-2,3-dihydroxybenzamide (5d)
Compound 5d was prepared from 2,3-dihydroxybenzoic acid
and (3,4-difluorophenyl)methanamine as a white solid according
to the same procedure described for 5a (52 mg, 42% yield). 1H
NMR (300 MHz, CDCl3): d 7.29–7.13 (m, 4H), 6.94 (dd, J = 1.5 Hz,
7.8 Hz, 1H), 6.73 (t, J = 8.1 Hz, 1H), 5.49 (s, 1H), 4.53 (s, 2H). 13C
5.2.18. N-(4-Fluorobenzyl)-2,3-dihydroxybenzamide (5a)
A solution of 2,3-dihydroxybenzoic acid (154 mg, 1 mmol), EDCI
(380 mg, 2 mmol), DIPEA (2 mmol), and HOBt (290 mg, 2 mmol) in
dry THF (15 mL) was stirred at rt under N2. To this solution was
added 90% (4-fluorophenyl)methanamine (0.152 mL, 1.2 mmol).
The reaction mixture was stirred for 6.5 h at rt. After removal of
1
NMR (100 MHz, CDCl3): d 169.7, 148.5 (d, JCF = 266.4 Hz), 147.9
1
3
(d, JCF = 268.2 Hz), 145.3, 135.3, 123.0, 118.2 (d, JCF = 8.1 Hz),
2
2
117.1, 116.7 (d, JCF = 17.2 Hz), 115.9 (d, JCF = 17.8 Hz), 114.7,
41.6. EI-MS: m/z 279 (M)+, 280 (M+1)+. HRMS (EI): calcd for
C
14H11F2NO3 279.0707 (M)+, found 279.0704. Purity: system 1,

X. Fan et al. / Bioorg. Med. Chem. 19 (2011) 4935–4952
4947
97.9% (method A, tR = 14.78 min); system 2, 95.0% (method F,
tR = 13.43 min).
6.75 (t, J = 8.1 Hz, 1H), 6.62 (br s, 1H), 6.36 (br s, 1H), 6.32 (d,
J = 3.0 Hz, 1H), 5.80 (br s, 1H), 4.63 (d, J = 6.0 Hz, 2H). 13C NMR
(100 MHz, CDCl3): d 169.7, 150.5, 148.9, 145.7, 142.4, 118.6,
118.4, 116.4, 114.0, 110.5, 107.9, 36.4. EI-MS: m/z 234.1 [M+H]+.
HRMS (EI): calcd for C12H11NO4 (M)+ 233.0688, found 233.0689.
Purity: system 1, 97.2% (method C, tR = 12.84 min); system 2,
95.9% (method G, tR = 8.73 min).
5.2.23. N-((1H-Indol-5-yl)methyl)-2,3-dihydroxybenzamide (5e)
Compound 5e was prepared from 2,3-dihydroxybenzoic acid
and (1H-indol-5-yl)methanamine according to the same procedure
described for 5a. Compound 5e was purified as a brown solid.
(31 mg, 36% yield) 1H NMR (300 MHz, CDCl3): d 8.23 (br, 1H),
7.63 (s, 1H), 7.41 (d, J = 8.4 Hz, 1H), 7.19 (d, J = 8.4 Hz, 1H), 7.04
(d, J = 7.8 Hz, 1H), 6.85 (d, J = 7.8 Hz, 1H), 6.72 (t, J = 8.1 Hz, 1H),
6.56–6.52 (m, 2H), 5.79 (br, 1H), 4.72 (d, J = 5.1 Hz, 2H). 13C NMR
(100 MHz, CDCl3): d 169.5, 148.8, 145.6, 135.3, 128.2, 127.7,
124.9, 121.6, 119.7, 118.5, 118.3, 116.6, 114.6, 111.4, 101.6, 44.0.
EI-MS: m/z 282 (M)+. HRMS (EI): calcd for C16H14N2O3 282.1004
(M)+, found 282.1006. Purity: system 1, 95.0% (method A,
tR = 13.58 min); system 2, 96.0% (method F, tR = 12.33 min).
5.2.28. 2,3-Dihydroxy-N-(4-(thiophen-2-yl)benzyl)benzamide
(5j)
Compound 5j was prepared from 2,3-dihydroxybenzoic acid
and (4-(thiophen-2-yl)phenyl)methanamine according to the same
procedure described for 5a. Compound 5j (78 mg, 31% yield) was
purified as a pale-yellow solid. 1H NMR (300 MHz, CDCl3): d 7.61
(d, J = 8.4 Hz, 2H), 7.35 (d, J = 8.7 Hz, 2H), 7.30 (t, J = 6.0 Hz, 2H,),
7.04–7.10 (m, 2H), 6.89 (dd, J = 1.8 Hz, 8.1 Hz, 1H), 6.76 (t,
J = 8.1 Hz, 1H), 6.62 (br s, 1H), 5.81 (br s, 1H), 4.65 (d, J = 5.7 Hz,
2H). 13C NMR (100 MHz, CDCl3): d 169.8, 149.2, 145.9, 143.7,
128.4, 128.1, 126.4, 125.0, 123.3, 118.7, 118.2, 115.9, 113.7, 43.4.
ESI-MS: m/z 326.1 [M+H]+. HRMS (ESI): calcd for C18H15NO3Na
(M+Na)+ 348.0675, found 348.0670. Purity: system 1, 99.0% (meth-
od C, tR = 16.59 min); system 2, 99.1% (method G, tR = 9.58 min).
5.2.24. N-(2-(1H-Indol-3-yl)ethyl)-2,3-dihydroxybenzamide (5f)
Compound 5f was prepared from 2,3-dihydroxybenzoic acid
and 2-(1H-indol-3-yl)ethanamine according to the same procedure
described for 5a. Compound 5f was purified as a yellow solid.
(36 mg, 42% yield) 1H NMR (300 MHz, CDCl3): d 8.08 (br, 1H),
7.64 (d, J = 8.1 Hz, 1H), 7.41 (d, J = 8.1 Hz, 1H), 7.25–7.22 (m, 1H),
7.18–7.13 (m, 1H), 7.09–7.08 (m, 1H), 7.01 (dd, J = 2.7 Hz, 6.6 Hz,
1H), 6.67–6.64 (m, 2H), 6.40 (br, 1H), 5.77 (s, 1H), 3.82–3.76 (m,
2H), 3.11 (t, J = 6.3 Hz, 2H). 13C NMR (100 MHz, CDCl3): d 169.7,
148.8, 145.6, 136.5, 136.3, 127.0, 122.3, 121.8, 119.1, 118.4,
118.3, 116.5, 114.5, 112.0, 111.3, 39.8, 24.9. EI-MS: m/z 296 (M)+.
HRMS (EI): calcd for C17H16N2O3 (M)+ 296.1161, found 296.1159.
Purity: system 1, 95.4% (method A, tR = 14.43 min); system 2,
95.1% (method F, tR = 13.09 min).
5.2.29. 2,3-Dihydroxy-N-(piperidin-4-yl)benzamide (5k)
To a stirred solution of tert-butyl 4-oxopiperidine-1-carboxylate
(184 mg, 1.86 mmol) and triethylamine (1.29 mL, 9.29 mmol) in
CH2Cl2 (200 mL, anhydrous) was added the solution of di-tert-butyl
dicarbonate (810 mg, 3.72 mmol) in CH2Cl2 (20 mL, anhydrous)
dropwise. After stirring at rt for 16 h, the solvent was evaporated
and the residue was purified by chromatography using petroleum
ether/ethyl acetate (5:1) as eluent to give compound tert-butyl 4-
oxopiperidine-1-carboxylate as white solid (318 mg, 86% yield).
To the solution of tert-butyl 4-oxopiperidine-1-carboxylate
(360 mg) in NH3/MeOH (15 ml) was added Pd/C (15 mg). The
mixture was stirred under H2 atmosphere at rt overnight. The Pd/C
was filtered and the filtrate was concentrated under vacuum. The
concentration provided the residue which was purified by chroma-
tography using DCM/MeOH (12:1) as eluent to give compound 4-
aminopiperidine-1-carboxylate as white solid (226 mg, 63% yield).
1H NMR (CDCl3, 300 MHz): d 4.07 (m, 2H), 2.81–2.65 (m, 3H), 1.84–
1.79 (m, 2H), 1.48 (s, 9H), 1.38 (m, 2H). EI-MS: m/z 200 (M)+.
A solution of 2,3-dihydroxybenzoic acid (339 mg, 2.2 mmol),
EDCI (422 mg, 2.2 mmol), DIPEA (2.2 mmol), and HOBt (297 mg,
2.2 mmol) in dry CH2Cl2 (11 mL) was stirred at room temperature.
To this solution was added 4-aminopiperidine-1-carboxylate
(220 mg, 1.1 mmol). The reaction mixture was stirred for 6.5 h at
room temperature. After removal of most of CH2Cl2, 20 mL of
EtOAc was added to the residue. The solution was washed by 1 N
HCl, saturated NH4Cl and dried over Na2SO4. The concentration
provided the residue that was purified by chromatography using
petroleum ether/ethyl acetate (5:1) as eluent to give compound
tert-butyl 4-(2,3-dihydroxybenzamido)piperidine-1-carboxylate
as white solid (190 mg, 51% yield). 1H NMR (CDCl3, 300 MHz): d
7.04 (d, J = 8.1 Hz, 1H), 6.85 (d, J = 7.8 Hz, 1H), 6.76 (t, J = 8.1 Hz,
1H), 6.16 (s, 1H), 5.79 (br s, 1H), 4.15–4.10 (m, 4H), 2.90 (t,
J = 9.9 Hz,2H), 2.04–1.98 (m, 3H), 1.49 (s, 9H).
5.2.25. 2,3-Dihydroxy-N-(1H-indol-5-yl)benzamide (5g)
Compound 5g was prepared from 2,3-dihydroxybenzoic acid
and 1H-indol-5-amine according to the same procedure described
for 5a. Compound 5g was purified as a white solid. (40 mg, 40%
yield) 1H NMR (300 MHz, CDCl3): d 8.35 (br, 1H), 8.11 (br, 1H),
7.87 (d, J = 1.8 Hz, 1H), 7.41 (d, J = 8.4 Hz, 1H), 7.31–7.26 (m, 2H),
7.10 (dt, J = 1.2 Hz, 7.8 Hz, 2H), 6.84 (t, J = 7.8 Hz, 1H), 6.57–6.56
(m, 1H), 5.41 (s, 1H). 13C NMR (100 MHz, CDCl3): d 168.4, 148.8,
145.6, 133.8, 128.5, 127.7, 125.3, 118.6, 118.5, 117.3, 117.1,
115.3, 114.3, 111.2, 101.9. EI-MS: m/z 268 (M)+, 269 (M+1)+. HRMS
(EI): calcd for C15H12N2O3 (M)+ 268.0848, found 268.0846. Purity:
system 1, 95.2% (method A, tR = 13.15 min); system 2, 99.5% (method
F, tR = 12.22 min).
5.2.26. 2,3-Dihydroxy-N-(thiophen-2-ylmethyl)benzamide (5h)
Compound 5h was prepared from 2,3-dihydroxybenzoic acid
and thiophen-2-ylmethanamine according to the same procedure
described for 5a. Compound 5h (190 mg, 19% yield) was purified
as a white solid. 1H NMR (300 MHz, CDCl3): d 12.55 (s, 1H), 7.27
(dd, J = 1.5 Hz, 5.1 Hz, 1H), 7.06–7.03 (m, 2H), 6.98 (t, J = 2.7 Hz,
1H), 6.87 (dd, J = 1.5 Hz, 8.4 Hz, 2H), 6.74 (t, J = 8.4 Hz, 1H), 5.91
(br s, 1H), 5.81 (s, 1H), 4.80 (d, J = 5.7 Hz, 2H). 13C NMR
(100 MHz, CDCl3): d 169.5, 148.7, 145.5, 140.4, 126.6, 125.9,
125.0, 118.5, 118.4, 116.9, 114.6, 37.9. EI-MS: m/z 249 (M)+. HRMS
(EI): calcd for C12H11NSO3 249.0456 (M)+, found 249.0460. Purity:
system 1, 96.1% (method C, tR = 13.71 min); system 2, 96.8% (method
G, tR = 8.96 min).
To the solution of 4-(2,3-dihydroxybenzoyl)piperadine-1-car-
boxylate (67 mg, 0.2 mmol) in anhydrous DCM (5 ml) on ice bath
was added TFA(0.578 ml) dropwise. The solution was allowed to
warm to rt for 2 h. The solvent was evaporated and the residue
was purified by chromatography using methylene chloride/ace-
tone (2:1) as eluent to give 5k as white solid (47 mg, 100% yield).
1H NMR (300 MHz, CD3OD): d 7.11 (d, J = 8.4 Hz, 1H), 6.86 (d,
J = 8.1 Hz, 1H), 6.62 (t, J = 7.8 Hz, 1H), 4.08–4.06 (m, 1H), 3.34 (d,
J = 6.3 Hz, 2H), 2.88 (t, J = 12.3 Hz, 2H), 2.00 (d, J = 6.6 Hz, 2H),
1.82 (q, 2H). 13C NMR (100 MHz, CDCl3): d 171.6, 150.7, 147.8,
5.2.27. N-(Furan-2-ylmethyl)-2,3-dihydroxybenzamide (5i)
Compound 5i was prepared from 2,3-dihydroxybenzoic acid
and furan-2-ylmethanamine according to the same procedure de-
scribed for 5a. Compound 5i was purified as an off white solid
(102 mg, 33% yield). 1H NMR (300 MHz, CDCl3): d 7.39 (q, 1H),
7.04 (dd, J = 1.5 Hz, 7.8 Hz, 1H), 6.89 (dd, J = 1.5 Hz, 8.1 Hz, 1H),

4948
X. Fan et al. / Bioorg. Med. Chem. 19 (2011) 4935–4952
120.3, 120.2, 119.4, 117.2, 48.9, 46.4, 44.8, 30.0. ESI-MS: m/z 237.1
[M+H]+. HRMS (ESI): calcd for C12H17N2O3 (M+H)+ 237.1239, found
237.1225. Purity: system 1, 97.0% (method D, tR = 3.38 min); sys-
tem 2, 96.8% (method H, tR = 3.79 min).
evaporated to remove THF. The residue was diluted with DCM
(10 mL) and the organic phase was washed with brine (5 mL), dried
over anhydrous Na2SO4, filtered and concentrated under vacuum.
The residue was chromatographed using DCM/MeOH = 10:1 to af-
ford 12n (94 mg, 39% yield in two steps) as yellow oil.
5.2.30. N-Cyclohexyl-2,3-dihydroxybenzamide (5l)
Compound 5l was prepared from 2,3-dihydroxybenzoic acid
and cyclohexanamine according to the same procedure as 5a. Com-
pound 5l (570 mg, 38% yield) was purified as a white solid. 1H NMR
(300 MHz, CDCl3): d 7.03 (d, J = 7.5 Hz, 1H), 6.86 (d, J = 8.1 Hz, 1H),
6.75 (t, J = 8.1 Hz, 1H), 6.09 (br s, 1H), 5.91 (br s, 1H), 4.02–3.90 (m,
1H), 2.05–2.00 (m, 2H), 1.82–1.74 (m, 2H), 1.71–1.32 (m, 4H),
1.28–1.20 (m, 2H). 13C NMR (100 MHz, CD3OD): d 169.0, 149.2,
146.0, 118.5, 117.9, 115.7, 114.1, 48.6, 33.0, 25.4, 24.8. EI-MS: m/
z 235 (M)+. HRMS (EI): calcd for C13H17NO3 (M)+ 235.1205, found
235.1208. Purity: system 1, 97.8% (method C, tR = 15.27 min); sys-
tem 2, 97.9% (method G, tR = 9.48 min).
5.2.36. 2,3-Dihydroxy-N-(2-(2-(methylamino)-2-
oxoethyl)benzyl)benzamide (5n)
Compound 5n was prepared from 2,3-dihydroxybenzoic acid
and 12q according to the same procedure described for 5a. Com-
pound 5n was purified as a white solid. (28 mg, 39% yield) 1H
NMR (300 MHz, CDCl3): d 7.49–7.46 (m, 1H), 7.32–7.29 (m, 2H),
7.23–7.17 (m, 4H), 4.64 (d, J = 3.6 Hz, 2H), 3.64 (s, 2H), 2.78 (d,
J = 4.5 Hz, 3H). 13C NMR (100 MHz, CDCl3): d 172.4, 169.7, 148.9,
145.4, 136.1, 133.7, 130.6, 130.1, 128.2, 127.7, 118.5, 118.3,
117.2, 114.4, 41.5, 40.0, 26.1. EI-MS: m/z 314 (M)+, 316 (M+2)+.
HRMS (EI): calcd for C17H18N2O4 (M)+ 314.1267, found 314.1265.
Purity: system 1, 98.9% (method A, tR = 11.82 min); system 2,
98.7% (method F, tR = 11.95 min).
5.2.31. N-(Cyclohexylmethyl)-2,3-dihydroxybenzamide (5m)
Compound 5m was prepared from 2,3-dihydroxybenzoic acid
and cyclohexylmethanamine according to the same procedure as
5a. Compound 5m (222 mg, 31% yield) was purified as a white so-
lid. 1H NMR (300 MHz, CDCl3): d 7.04 (d, J = 7.8 Hz, 1H), 6.87 (d,
J = 8.1 Hz, 1H), 6.75 (t, J = 8.1 Hz, 1H), 6.38 (br s, 1H), 5.81 (br s,
1H), 3.29 (t, J = 6.6 Hz, 2H), 1.77 (m, 5H), 1.35–1.12 (m, 4H), 1.01
(m, 2H). EI-MS: m/z 249 (M)+. HRMS (EI): calcd for C14H19NO3
(M)+ 249.1365, found 249.1365. Anal. Calcd (found): C, 67.45
(67.29); H, 7.68 (7.68); N, 5.62 (5.67). Purity: system 1, 99.1%
(method C, tR = 16.40 min); system 2, 96.0% (method G,
tR = 9.88 min).
5.2.37. 5-Fluoro-N,2-dimethylbenzamide (13)
Compound 13 was prepared from 5-fluoro-2-methylbenzoic
acid and methanamine according to the same procedure as 5a.
Compound 13 was purified as a white solid. (173 mg, 82% yield)
1H NMR (300 MHz, CDCl3): d 7.20–7.15 (m, 1H), 7.07–6.98 (m,
2H), 3.00 (d, J = 4.2 Hz, 3H), 2.39 (s, 3H).
5.2.38. 2-(Bromomethyl)-5-fluoro-N-methylbenzamide (14)
To a solution of 13 (581 mg, 3.48 mmol) in CCl4 (10 mL) was
added NBS (743 mg, 4.17 mmol) and AIBN (171 mg, 1.04 mmol).
The above mixture was stirred at reflux for 8 h and evaporated to
remove CCl4 under vacuum. The residue was diluted with DCM
(10 mL) and the organic phase was washed with brine (10 mL),
dried with anhydrous Na2SO4, filtered, concentrated under vac-
uum, the residue was chromatographed using DCM/MeOH = 10:1
to provide 14 (467 mg, 55% yield) as a yellow solid. 1H NMR
(300 MHz, CDCl3): d 7.59–7.41 (m, 3H), 5.75 (s, 2H), 3.32 (s, 3H).
5.2.32. 2-(2-(Hydroxymethyl)phenyl)-N-methylacetamide (9)
To a stirred solution of isochroman-3-one (316 mg, 2.13 mmol)
in THF (10 mL) was added NH2Me (662 mg, 21.35 mmol). The
above mixture was stirred at rt for 8 h and excess THF was re-
moved under reduced pressure. The residue was diluted with
DCM (10 mL) and the organic phase was washed with brine
(5 mL), dried over anhydrous Na2SO4, filtered, concentrated under
vacuum. The residue was chromatographied using PE/EA = 1:1 to
provide 9 (377 mg, 99% yield) as colorless oil. 1H NMR (300 MHz,
CDCl3): d 7.39–7.37 (m, 1H), 7.29–7.24 (m, 3H), 6.37 (br, 1H),
4.66 (s, 2H), 4.38 (br, 1H), 3.62 (s, 2H), 2.74 (d, J = 4.8 Hz, 3H).
5.2.39. 2-(Azidomethyl)-5-fluoro-N-methylbenzamide (11o)
Compound 11o was prepared from 14 according to the same
procedure described for 11n. The crude product 11o was used di-
rectly for next step without further purification.
5.2.33. 2-(2-(Chloromethyl)phenyl)-N-methylacetamide (10)
To a stirred solution of 9 (0.45 mL, 5.9 mmol) in DCM (10 mL)
was added MsCl (0.45 mL, 5.9 mmol) and TEA (1 mL, 7.4 mmol).
The above mixture was stirred at rt for 8 h and the residue was di-
luted with DCM (10 mL) and the organic phase was washed with
brine (20 mL), dried with anhydrous Na2SO4, filtered, concentrated
under vacuum. The residue was chromatographied using PE/
EA = 1:1 to give 10 (267 mg, 28% yield) as colorless oil. 1H NMR
(300 MHz, CDCl3): d 7.42–7.29 (m, 4H), 5.42 (br, 1H), 4.65 (s, 2H),
3.73 (s, 2H), 2.77 (d, J = 5.1 Hz, 3H).
5.2.40. 2-(Aminomethyl)-5-fluoro-N-methylbenzamide (12o)
Compound 12o was prepared from 11o according to the same
procedure described for 12n. Compound 12o was purified as a yel-
low oil. (92 mg, 68% yield) 1H NMR (300 MHz, CD3OD): d 7.60–7.55
(m, 1H), 7.47–7.43 (m, 1H), 7.37–7.30 (m, 1H), 4.12 (s, 2H), 2.93 (s,
3H).
5.2.41. N-(4-Fluoro-2-(methylcarbamoyl)benzyl)-2,3-
dihydroxybenzamide (5o)
Compound 5o was prepared from 2,3-dihydroxybenzoic acid
and 12o as a white solid according to the same procedure de-
scribed for 5a (31 mg, 61% yield) 1H NMR (300 MHz, CDCl3): d
7.57–7.53 (m, 1H), 7.47–7.43 (m, 1H), 7.19–7.01 (m, 3H), 6.91–
6.86 (m, 1H), 6.16 (br, 1H), 5.73 (br, 1H), 4.58 (d, J = 6.3 Hz, 2H),
3.06 (s, 3H). 13C NMR (100 MHz, CDCl3): d 169.7, 169.4, 161.3 (d,
5.2.34. 2-(2-(Azidomethyl)phenyl)-N-methylacetamide (11n)
To a stirred solution of 10 (287 mg, 1.36 mmol) in DMF (5 mL)
was added NaN3 (106 mg, 1.63 mmol), the mixture was stirred at
80 °C for 8 h. The residue was diluted with DCM (10 mL) and the
organic phase was washed with brine (5 mL), dried over anhydrous
Na2SO4, filtered, concentrated under vacuum to give 11n which
was used directly for the next step without further purification.
4
1JCF = 247.0 Hz), 148.7, 145.4, 137.0, 131.9 (d, JCF = 8.2 Hz), 131.7
3
3
(d, JCF = 10.5 Hz), 128.5 (d, JCF = 12.7 Hz), 120.3, 120.1, 118.8,
2
2
117.1 (d, JCF = 21.6 Hz), 114.1 (d, JCF = 26.0 Hz), 40.5, 26.2. EI-
MS: m/z 318 (M)+, 319 (M+1)+. HRMS (EI): calcd for C16H15FN2O4
(M)+ 318.1016, found 318.1020. Purity: system 1, 95.3% (method
A, tR = 14.49 min); system 2, 97.7% (method F, tR = 13.15 min).
5.2.35. 2-(2-(Aminomethyl)phenyl)-N-methylacetamide (12n)
To a stirred solution of 11n in THF (5 mL) was added Ph3P
(713 mg, 2.72 mmol), the mixture was stirred at rt for 8 h and

X. Fan et al. / Bioorg. Med. Chem. 19 (2011) 4935–4952
4949
5.2.42. 5-Chlorosulfonyl-2,3-dimethoxy-benzoic acid (15)
2,3-Dimethoxybenzoic acid (10 mmol, 1.82 g) was added in sev-
eral portions to chlorosulfonic acid (10 mL) and stirred at rt. for
15 h. The solution was diluted with CH2Cl2 (50 mL) and poured
onto a mixture of CH2Cl2 and ice on a chilled salt bath. The organic
phase was washed with water (3 ꢁ 20 mL), then with brine
(3 ꢁ 20 mL), dried over Na2SO4 and concentrated to give a white
solid 15 (2.246 g, 80% yield). 1H NMR (300 MHz, CDCl3): d 8.34
(d, J = 2.4 Hz, 1H), 7.67 (d, J = 2.4 Hz, 1H), 4.17 (s, 3H), 4.04 (s, 3H).
under vacuum, the residue was chromatographed using DCM/
MeOH = 50:1 to separate the 16q (303 mg, 89% yield) as a white so-
lid. 1H NMR (300 MHz, CD3OD): d 7.68 (d, J = 2.1 Hz, 1H), 7.43 (d,
J = 2.1 Hz, 1H), 3.92 (s, 3H), 3.91 (s, 3H), 2.52 (s, 3H).
5.2.48. N-(4-Fluorobenzyl)-2,3-dimethoxy-5-(N-methyl-
sulfamoyl)benzamide (17q)
Compound 17q was prepared from 16q and (4-fluoro-
phenyl)methanamine according to the same procedure described
for 5a. Compound 17q was purified as a white solid. (94 mg, 62%
yield) 1H NMR (300 MHz, CDCl3): d 8.21 (s, 1H), 7.52 (s, 1H),
7.35–7.31 (m, 2H), 7.07–7.01 (m, 2H), 4.63 (d, J = 5.1 Hz, 2H),
4.52 (br, 1H), 3.96 (s, 3H), 3.90 (s, 3H), 2.69 (s, 3H).
5.2.43. 2,3-Dimethoxy-5-(piperidine-1-sulfonyl)-benzoic acid
(16p)
Piperidine (0.356 mL, 3.6 mmol) was added to a stirred solution
of 15 (0.84 g, 3.0 mmol) and Et3N (0.5 mL, 3.6 mmol) in CH2Cl2
(20 mL). The solution was heated to reflux for 22 h. The reaction
mixture was washed with dilute HCl (2 ꢁ 30 mL), then brine, dried
over anhydrous Na2SO4 and concentrated in vacuo. Flash chroma-
5.2.49. N-(4-Fluorobenzyl)-2-hydroxy-3-methoxy-5-(N-methyl-
sulfamoyl)benzamide (18q)
To the solution of 17q (402 mg, 1.05 mmol) in DMF (20 mL) was
added LiCl (269 mg, 6.31 mmol). The above mixture was stirred at
110 °C for 8 h and excess DMF was removed under reduced pres-
sure. The residue was diluted with DCM (10 mL) and the organic
phase was washed with brine (10 mL), dried over anhydrous
Na2SO4, filtered, concentrated under vacuum, the residue was
chromatographed using PE/EA = 1:1 to afford 18q (183 mg, 65%
yield) as yellow oil. 1H NMR (300 MHz, CDCl3): d 7.66–7.62 (m,
1H), 7.36–7.32 (m, 3H), 7.04–6.98 (m, 2H), 4.59 (d, J = 5.4 Hz,
2H), 3.94 (s, 3H), 2.60 (s, 3H).
tography (hexane/EtOAc = 2:1) provide 16p as
a
white solid
8.01 (d,
(0.83 g, 84% yield). 1H NMR (300 MHz, CDCl3):
d
J = 1.2 Hz, 1H), 7.46 (d, J = 1.2 Hz, 1H), 4.13 (s, 3H), 3.97 (s, 3H),
3.03 (m, 4H), 1.65 (m, 4H), 1.45 (m, 2H).
5.2.44. N-(4-Fluoro-benzyl)-2,3-dimethoxy-5-(piperidine-1-
sulfonyl)-benzamide (17p)
Compound 17p was prepared as a white solid according to the
same procedure described for 5a (85 mg, 83% yield). 1H NMR
(300 MHz, CDCl3): d 8.51 (m, 1H), 8.07 (s, 1H), 7.38 (s, 1H), 7.31
(m, 2H), 7.02 (m, 2H), 4.61 (d, J = 4.8 Hz, 2H), 3.92 (s, 3H), 3.89 (s,
3H), 3.02 (m, 4H), 1.63 (m, 4H), 1.42 (m, 2H).
5.2.50. N-(4-Fluorobenzyl)-2,3-dihydroxy-5-(N-methylsul-
famoyl)benzamide (5q)
To the solution of 18q (55 mg, 0.15 mmol) in DCM (2 mL) was
added 4 N BBr3 (38uL). The above mixture was stirred at rt for
4 h and treated with MeOH (0.5 mL), concentrated under vacuum,
the residue was chromatographied using PE/EA = 1:1 to give 5q
(12 mg, 24% yield) as a red solid. 1H NMR (300 MHz, CDCl3): d
7.45 (s, 1H), 7.38–7.33 (m, 3H), 7.05 (t, J = 8.4 Hz, 2H), 4.61 (s,
2H), 2.62 (s, 3H). 13C NMR (100 MHz, CDCl3): d 168.4, 162.0 (d,
5.2.45. N-(4-Fluorobenzyl)-2-methoxy-3-hydroxy-5-(piperidin-
1-ylsulfonyl)benzamide (18p)
The solution of 1 M BBr3 (2.61 mmol) in CH2Cl2 was added
dropwise to a stirred solution of 17p (76 mg, 0.174 mmol) in anhy-
drous CH2Cl2 (20 mL) at ꢀ40 °C. The mixture was stirred at ꢀ20 °C
for 10 h. Methanol (2 mL) was added to quench the reaction and
the solution was washed with H2O and brine, dried over Na2SO4,
and concentrated. The residue was purified by chromatography
(hexane/EtOAc = 2:1) to give 18p as a white solid (60 mg, 82%
yield). 1H NMR (300 MHz, CDCl3): d 12.27 (s, 1H), 7.71 (s, 1H),
7.67 (m, 1H), 7.39 (m, 2H), 7.23 (s, 1H), 7.03 (m, 2H), 4.61 (d,
J = 6.0 Hz, 2H), 3.94 (s, 3H), 2.96 (m, 4H), 1.59 (m, 4H), 1.43 (m, 2H).
3
1JCF = 244.8 Hz), 149.0, 133.4, 129.4 (d, JCF = 8.2 Hz), 127.9, 118.5,
2
115.3 (d, JCF = 21.6 Hz), 114.5, 111.9, 42.6, 28.7. EI-MS: m/z 354
(M)+, 356 (M+2)+. HRMS (EI): calcd for C15H15FN2O5S (M)+
354.0686, found 354.0682. Purity: system 1, 95.6% (method A,
tR = 14.73 min); system 2, 98.9% (method F, tR = 12.95 min).
5.2.51. 5-(N-tert-Butylsulfamoyl)-2,3-dimethoxybenzoic acid
(16r)
5.2.46. N-(4-Fluoro-benzyl)-2,3-dihydroxy-5-(piperidine-1-
sulfonyl)-benzamide (5p)
Compound 16r was prepared from 15 and 2-methylpropan-2-
amine according to the same procedure described for 16q. Com-
pound 16r was a white solid (379 mg, 35% yield). 1H NMR
(300 MHz, CD3OD): d 7.75 (d, J = 2.1 Hz, 1H), 7.58 (d, J = 2.1 Hz,
1H), 3.93 (s, 3H), 3.91 (s, 3H), 1.19 (s, 9H).
The solution of 1 M BBr3 (2.13 mmol) in CH2Cl2 was added drop-
wise to a stirred solution of 18p (60 mg, 0.142 mmol) in anhydrous
CH2Cl2 (20 mL) at ꢀ40 °C. The mixture was stirred at ꢀ20 °C for 10 h.
Methanol (2 mL) was added to quench the reaction and the solution
was washed with H2O and brine, dried over Na2SO4, and concen-
trated. The residue was purified by chromatography (hexane/
EtOAc = 2:1) to give 5p as a white solid (25 mg, 42% yield). 1H
NMR (300 MHz, CDCl3): d 13.65 (s, 1H), 7.55 (m, 1H), 7.38 (m, 4H),
7.04 (m, 2H), 6.02 (m, 1H), 4.60 (d, J = 5.7 Hz, 2H), 2.97 (m, 4H),
1.62 (m, 4H), 1.41 (m, 2H). 13C NMR (100 MHz, CD3OD): d 170.5,
164.0 (d, 1JCF = 243.3 Hz), 154.8, 148.7, 136.6, 131.2 (d, 3JCF = 8.2 Hz),
127.6, 120.0, 118.0, 117.1, 116.7 (d, 2JCF = 21.6 Hz), 48.7, 44.0, 26.9,
25.0. EI-MS m/z: 408 (M)+. Purity: system 1, 99.8% (method A,
tR = 16.34 min); system 2, 97.6% (method F, tR = 14.31 min).
5.2.52. 5-(N-tert-Butylsulfamoyl)-N-(4-fluorobenzyl)-2,3-
dimethoxybenzamide (17r)
Compound 17r was prepared from 16r and (4-fluoro-
phenyl)methanamine according to the same procedure described
for 17q. Compound 17r was a white solid (250 mg, 50% yield). 1H
NMR (300 MHz, CDCl3): d 8.25 (d, J = 2.1 Hz, 1H), 8.20–8.17 (m,
1H), 7.55 (d, J = 2.1 Hz, 1H), 7.35–7.30 (m, 2H), 7.04 (t, J = 8.7 Hz,
2H), 4.63 (d, J = 5.4 Hz, 2H), 3.94 (s, 3H), 3.88 (s, 3H), 1.26 (s, 9H).
5.2.53. 5-(N-tert-Butylsulfamoyl)-N-(4-fluorobenzyl)-2-
hydroxy-3-methoxybenzamide (18r)
5.2.47. 2,3-Dimethoxy-5-(N-methylsulfamoyl)benzoic acid (16q)
To the solution of 15 (349 mg, 1.24 mmol) in DCM (5 mL) was
added MeNH2 (0.2 mL, 1.87 mmol) and TEA (0.35 mL, 2.49 mmol).
The above mixture was stirred at rt for 8 h and the residue was di-
luted with DCM (10 mL) and the organic phase was washed with
brine (10 mL), dried over anhydrous Na2SO4, filtered, concentrated
Compound 18r was prepared from 17r according to the same
procedure described for 18q. Compound 18r was purified as a
white solid (95 mg, 88% yield). 1H NMR (300 MHz, CDCl3): d 7.78
(d, J = 1.5 Hz, 1H), 7.36 (d, J = 1.5 Hz, 1H), 7.32 (q, 2H), 7.02 (t,
J = 8.7 Hz, 2H), 4.58 (d, J = 5.7 Hz, 2H), 3.93 (s, 3H), 1.21 (s, 9H).

4950
X. Fan et al. / Bioorg. Med. Chem. 19 (2011) 4935–4952
5.2.54. 5-(N-tert-Butylsulfamoyl)-N-(4-fluorobenzyl)-2,3-
dihydroxybenzamide (5r)
1H), 7.34–7.25 (m, 3H), 4.31 (t, J = 5.4 Hz, 2H), 2.97–2.93 (m, 4H),
1.83–1.79 (m, 4H), 1.73–1.70 (m, 2H).
Compound 5r was prepared from 18r according to the same
procedure described for 5q. Compound 5r was purified as a white
solid (22 mg, 30% yield). 1H NMR (300 MHz, CDCl3): d 7.77 (d,
J = 1.5 Hz, 1H), 7.37–7.30 (m, 3H), 7.05–7.00 (m, 2H), 4.58 (d,
J = 5.7 Hz, 2H), 1.21 (s, 9H). 13C NMR (100 MHz, CDCl3): d 169.4,
5.2.60. 2,3-Dimethoxy-5-nitrobenzoic acid (19)
To a stirred solution of 2,3-dimethoxybenzoic acid (1.96 g,
10 mmol) in concd H2SO4 (10 ml) was added the solution of
HNO3 (0.43 ml) in concentrated H2SO4 (4.5 ml) dropwise at 0 °C.
After stirring at 0 °C for 1 h, the mixture was poured into ice water
and diluted with DCM. The organic layer was washed with brine,
then concentrated under vacuum. The residue was chromato-
graphed using DCM/MeOH = 10:1 to give 19 as a white solid
(634 mg, 25%). 1H NMR (300 MHz, CDCl3): d 8.26 (d, J = 2.7 Hz,
1H), 7.89 (d, J = 2.7 Hz, 1H), 4.01 (s, 3H), 3.99 (s, 3H), 3.95 (s, 3H).
1
163.9 (d, JCF = 242.5 Hz), 153.9, 150.5, 136.2, 135.5, 131.1 (d,
2
3JCF = 7.9 Hz), 120.3, 118.2, 117.9, 116.9 (d, JCF = 19.4 Hz), 44.1,
43.9. EI-MS: m/z 396 (M)+. HRMS (EI): calcd for C18H21FN2O5S
(M)+ 396.1155, found 396.1143. Purity: system 1, 99.4% (method
A, tR = 12.46 min); system 2, 99.1% (method F, tR = 12.67 min).
5.2.55. 2,3-Dihydroxy-5-(piperidin-1-ylsulfonyl)benzoic acid
(17s)
5.2.61. N-(4-Fluorobenzyl)-2,3-dimethoxy-5-nitrobenzamide
(20)
Compound 17s was prepared from 16p and BBr3 according to
the same procedure described for 18p. Compound 17s was purified
as a white solid (195 mg, 68% yield). 1H NMR (300 MHz, CD3OD): d
7.73 (d, J = 2.1 Hz, 1H), 7.28 (d, J = 1.8 Hz, 1H), 2.96 (t, J = 5.7 Hz,
4H), 1.67–1.59 (m, 4H), 1.48–1.41 (m, 2H).
Compound 20 was prepared from 19 and (4-fluoro-
phenyl)methanamine according to the same procedure described
for 5a. Compound 20 was a white solid (45%). 1H NMR (300 MHz,
CDCl3): d 8.66 (d, J = 2.7 Hz, 1H), 8.09 (br, 1H), 7.88 (d, J = 2.7 Hz,
1H), 7.31–7.36 (m, 2H), 7.05 (d, J = 8.7 Hz, 2H), 4.64 (d, J = 5.7 Hz,
2H), 3.99 (s, 3H), 3.94 (s, 3H).
5.2.56. N-(Furan-2-ylmethyl)-2,3-dihydroxy-5-(piperidin-1-
ylsulfonyl)benzamide (5s)
Compound 5s was prepared from 17s and furan-2-ylmethan-
amine according to the same procedure described for 5a. Com-
pound 5s was purified as a white soild (14 mg, 33% yield). 1H
NMR (300 MHz, CD3OD): d 7.42 (d, J = 1.2 Hz, 1H), 7.36 (d,
J = 1.5 Hz, 1H), 6.95 (br s, 1H), 6.39–6.36 (m, 2H), 5.98 (s, 1H),
4.64 (d, J = 5.4 Hz, 2H), 2.98 (t, J = 5.4 Hz, 4H), 1.66–1.62 (m, 4H),
1.47–1.41 (m, 2H). 13C NMR (100 MHz, CD3OD) d: 170.3, 153.2,
148.6, 143.9, 131.4, 127.7, 120.2, 118.0, 117.2, 111.9, 109.0, 48.7,
37.7, 26.9, 25.0. ESI-MS: m/z 381.1 [M+H]+. HRMS (ESI): calcd for
5.2.62. 5-Amino-N-(4-fluorobenzyl)-2,3-dimethoxybenzamide
(21)
To the solution of compound 20 (498 mg, 1.49 mmol) and con-
centrated HCl (0.62 mL) in 10 mL of THF, was added SnCl2
(1414 mg, 7.44 mmol). The reaction mixture was stirred for 8 h
at room temperature, then diluted with DCM (20 mL). Usual
work-up followed by purification on column chromatography
(DCM/MeOH = 10:1) afforded 21 as a yellow solid (272 mg, yield
60%). 1H NMR (300 MHz, CDCl3): d 7.29–7.34 (m, 2H), 6.99–7.04
(m, 3H), 6.39 (d, J = 2.4 Hz, 1H), 4.61 (d, J = 6.0 Hz, 2H), 3.83 (s,
3H), 3.70 (s, 3H).
C
17H10N2O6Na (M+Na)+ 403.0940, found 403.0989. Purity: system
1, 95.5% (method E, tR = 13.00 min); system 2, 98.4% (method G,
tR = 7.76 min).
5.2.63. N-(4-Fluorobenzyl)-2,3-dimethoxy-5-(methylsulfona-
mido)benzamide (22w)
5.2.57. 2,3-Dihydroxy-5-(piperidin-1-ylsulfonyl)-N-(4-
(trifluoromethyl)benzyl) benzamide (5t)
Compound 22w was prepared from 21 and methanesulfonyl
chloride according to the same procedure described for 16p. Com-
pound 22w was a white solid in yield of 80%. 1H NMR (300 MHz,
CDCl3): d 7.86 (d, J = 2.7 Hz, 1H), 7.32–7.37 (m, 3H), 7.00–7.06
(m, 2H), 4.74 (d, J = 5.7 Hz, 2H), 3.91 (s, 3H), 3.82 (s, 3H), 2.92 (s,
3H).
Compound 5t was prepared from 17s and (4-(trifluoro-
methyl)phenyl) methanamine according to the same procedure
described for 5a. Compound 5t was purified as an off white solid
(51 mg, 32% yield). 1H NMR (300 MHz, CD3OD):
d 7.77 (d,
J = 1.8 Hz, 1H), 7.63 (d, J = 4.5 Hz, 2H), 7.54 (d, J = 4.8 Hz, 2H),
7.25 (d, J = 1.8 Hz, 1H), 4.67 (d, J = 6.0 Hz, 2H), 2.97–2.93 (m, 4H),
1.83–1.79 (m, 4H), 1.73–1.70 (m, 2H). 13C NMR (100 MHz, CD3OD):
d 170.7, 154.8, 148.7, 144.9, 129.6, 127.8, 127.0, 126.9, 121.9,
120.1, 119.8, 118.1, 117.1, 114.3, 48.7, 44.3, 26.9, 25.0. EI-MS: m/
z 458 (M)+. HRMS (EI): calcd for C20H21SN2O5F3 (M)+ 458.1123,
found 458.1118. Purity: system 1, 99.6% (method C,
tR = 17.35 min); system 2, 99.8% (method G, tR = 9.61 min).
5.2.64. 5-Acetamido-N-(4-fluorobenzyl)-2,3-dimethoxy-
benzamide (22x)
Compound 22x was prepared from 21 and acetyl chloride
according to the same procedure described for 16p. Compound
22x was a white solid in yield of 88%. 1H NMR (300 MHz, CDCl3):
d 8.07 (d, J = 2.7 Hz, 1H), 7.99 (br, 1H), 7.38 (d, J = 2.7 Hz, 1H),
7.28–7.33 (m, 2H), 7.00–7.06 (m, 2H), 4.63 (d, J = 5.7 Hz, 2H),
4.31 (t, J = 6.6 Hz, 1H), 3.91 (s, 3H), 3.79 (s, 3H), 2.16 (s, 3H).
5.2.58. N-(Cyclohexylmethyl)-2,3-dihydroxy-5-(piperidin-1-
ylsulfonyl)benzamide (5u)
Compound 5u was prepared from 17s and cyclohexylmethan-
amine according to the same procedure described for 5a. Com-
pound 5u was purified as an off white solid (20 mg, 21% yield).
1H NMR (300 MHz, CDCl3): d 8.07 (d, J = 1.8 Hz, 1H), 7.48 (d,
J = 1.8 Hz, 1H), 3.04 (t, J = 5.1 Hz, 4H), 1.84–1.41 (m, 11H), 1.29–
1.14 (m, 4H), 1.07–0.96 (m, 4H).
5.2.65. N-(4-Fluorobenzyl)-2,3-dihydroxy-5-(methylsulfona-
mido)benzamide (5w)
Compound 5w was prepared from 22w and BBr3 according to
the same procedure described for 18p. Compound 5w was a yellow
solid (42%). 1H NMR (300 MHz, CD3OD): d 7.34–7.39 (m, 2H), 7.14
(d, J = 2.7 Hz, 1H), 7.01–7.07 (m, 2H), 6.93 (d, J = 2.7 Hz, 1H), 4.54 (s,
2H), 4.28 (t, J = 6.6 Hz, 1H), 2.90 (s, 3H). MS (EI): m/z 354 (M)+.
5.2.59. N-(Thiophen-2-ylmethyl)-2,3-dihydroxy-5-(piperidin-1-
ylsulfonyl)benzamide (5v)
Compound 5v was prepared from 17s and thiophen-2-ylme-
thanamine according to the same procedure described for 5a. Com-
pound 5v was purified as an off white solid (17 mg, 23% yield). 1H
NMR (300 MHz, CDCl3): d 8.12 (d, J = 1.8 Hz, 1H), 7.54 (d, J = 1.8 Hz,
5.2.66. 5-Acetamido-N-(4-fluorobenzyl)-2,3-dihydroxy-
benzamide (5x)
Compound 5x was prepared from 22x and BBr3 according to the
same procedure described for 18p. Compound 5x was a yellow so-
lid (47%). 1H NMR (300 MHz, CD3OD): d 7.34–7.38 (m, 3H), 7.14 (s,

X. Fan et al. / Bioorg. Med. Chem. 19 (2011) 4935–4952
4951
1H), 7.01–7.07 (m, 2H), 4.54 (s, 2H), 4.28 (t, J = 6.6 Hz, 1H), 2.07 (s,
3H). MS (EI): m/z 318 (M)+, 319 (M+1)+.
232.0848, found 232.0839. Anal. Calcd (found): C, 62.06 (61.89);
H, 5.21 (4.93); N, 12.06 (12.37). Purity: system 1, 99.7% (method
C, tR = 9.65 min); system 2, 97.0% (method G, tR = 7.77 min).
5.2.67. 4-((Dimethylamino)methyl)-2,3-dihydroxybenzoic acid
(23)
5.3. Homology modeling and molecular docking
To a stirred solution of 2,3-dihydroxybenzoic acid (300 mg,
1.95 mmol), dimethylamine hydrochloride (159 mg, 1.95 mmol),
Given the lack of a full length crystal structure of HIV-1 IN, a
homology model was built based on the X-ray crystal structure
of PFV intasome (PDB: 3OYA)27,30 a retro-lentivirus belonging to
the same viral genus as HIV. For the generation of this model we
used secondary structural alignment on the basis of 3D structures
of PFV IN and HIV-1 IN.31 Prime v2.2 from Schrodinger, Inc. was
used for the modeling.32 As the nucleic acid residues in the binding
sites (DC16 and DA17) are conserved we did not model DNA for
HIV, instead we used PFV DNA and two Mg2+ ions in the binding
site. The modeled protein was minimized using OPLS-2005 force
filed within MacroModel.33 The structure of the modeled protein
was validated by PROCHECK34 as 89.6% residues fall within most
favorable region and another 9.5% in additional allowed region.
The RMSD value of the modeled protein with catalytic core domain
crystal structure (PDB: 1BL3) was 3.18. HIV IN crystal structure
(PDB: 3LPU) co-crystallized with a LEDGF/p75 inhibitor was used
to dock compounds with LEDGF/p75 inhibitory activity. Com-
pounds were docked into the binding site using Standard Precision
method of Glide from Schrodinger, Inc.28,29 with default values for
all parameters. Prior to docking, compounds were minimized and
all possible combinations of stereo isomers were generated by Lig-
Prep from Schrodinger using OPLS-2005 force filed. Glide per-
formed an exhaustive search of the positional, orientational,
conformational space available to the docked ligand using a series
of hierarchical filters and followed by energy optimization. Finally,
the conformations were further refined via a Monte Carlo sampling
that examines nearby torsional minima.29
Et3N (544
lL, 3.9 mmol) in EtOH (20 mL) was added HCHO (aq)
(146 L, 1.95 mmol, 40%) at 0 °C. The above mixture was refluxed
l
overnight. The white precipitate was filtered and washed three
times with anhydrous EtOH. The residue was dried under vacuum
to give 23 (186 mg, 45% yield) as a white solid. 1H NMR (300 MHz,
DMSO-d6): d 7.16 (d, J = 7.8 Hz, 1H), 6.47 (d, J = 7.5 Hz, 1H), 4.14 (s,
1H), 2.72 (s, 6H). EI-MS m/z: 211 (M)+.
5.2.68. 4-((Dimethylamino)methyl)-N-(4-fluorobenzyl)-2,3-
dihydroxybenzamide (6a)
Compound 6a was prepared from 23 and (4-fluoro-
phenyl)methanamine according to the same procedure described
for 5a. Compound 6a was purified as a white solid. (91 mg, 61%
yield) 1H NMR (300 MHz, CD3OD): d 7.36 (dd, J = 5.4 Hz, 8.4 Hz,
2H), 7.27 (d, J = 7.8 Hz, 1H), 7.04 (t, J = 8.7 Hz, 2H), 6.64 (d,
J = 8.4 Hz, 1H), 4.56 (s, 2H), 4.03 (s, 2H), 2.63 (s, 6H).
5.2.69. 4-((Dimethylamino)methyl)-N-(furan-2-ylmethyl)-2,3-
dihydroxybenzamide (6b)
Compound 6b was prepared from 23 and furan-2-ylmethan-
amine according to the same procedure described for 5a. Com-
pound 6b was purified as a white solid. (95 mg, 63% yield) 1H
NMR (300 MHz, CD3OD): d 7.32 (d, J = 1.8 Hz, 1H), 7.14 (d,
J = 7.8 Hz, 1H), 6.47 (d, J = 7.5 Hz, 1H), 6.33 (d, J = 8.7 Hz, 2H),
4.63(s, 2H), 4.09 (s, 2H), 2.65 (s, 6H).
5.2.70. 2-Hydroxy-3-methoxy-5-(piperidin-1-ylsulfonyl)benzoic
acid (17w)
5.4. Biological materials, chemicals and enzymes
Compound 17w was prepared from 16p and BBr3 according to
the same procedure as 18p. Compound 17w was purified as a
white solid (21 mg, 16% yield). 1H NMR (300 MHz, CD3OD): d
7.73 (d, J = 2.1 Hz, 1H), 7.28 (d, J = 1.8 Hz, 1H), 3.98 (s, 3H), 2.96
(t, J = 5.4 Hz, 4H), 1.65–1.63 (m, 4H), 1.46–1.40 (m, 2H).
All compounds were dissolved in DMSO and the 10 mM stock
solutions were stored at ꢀ20 °C. Further dilutions were also per-
formed in DMSO. The expression system used in purifying IN
was a kind gift from Dr. Robert Craigie, Laboratory of Molecular
Biology, NIDDK, NIH, Bethesda, MD. The oligonucleotides used in
the HIV-1 IN catalytic activity assay were synthesized at the USC
5.2.71. N-(Furan-2-ylmethyl)-2-hydroxy-3-methoxy-5-
(piperidin-1-ylsulfonyl)benzamide. (6c)
Compound 6c was prepared from 17w and furan-2-ylmethan-
amine according to the same procedure described for 5a. Com-
pound 6c was purified as a yellow solid (51 mg, 32% yield). 1H
Norris Cancer Center microsequencing core facility. The
ATP was purchased from Perkin–Elmer (Waltham, MA).
c [
32P]-
5.5. Preparation of oligonucleotide substrate
NMR (300 MHz, CDCl3):
d 7.54 (d, J = 1.8 Hz, 1H), 7.24 (d,
J = 2.1 Hz, 1H), 7.09 (br s, 1H), 6.38–6.34 (m, 1H), 6.32 (q, 1H),
6.23 (d, J = 3.6 Hz, 1H), 4.64 (d, J = 6.0 Hz, 2H), 3.95 (s, 3H), 2.98
(t, J = 5.4 Hz, 4H), 1.68–1.62 (m, 4H), 1.47–1.41 (m, 2H). 13C NMR
(100 MHz, CD3OD): d 168.5, 154.8, 150.5, 149.1, 142.1, 125.3,
118.9, 114.3, 112.3, 110.3, 108.0, 56.4, 46.8, 36.3, 25.0, 23.2. EI-
MS: m/z 394 (M)+. HRMS (EI): calcd for C18H22SN2O6 (M)+
394.1199, found 394.1198. Purity: system 1, 99.4% (method E,
tR = 13.60 min); system 2, 98.5% (method G, tR = 9.27 min).
The HIV-1 IN catalytic activity assay uses a 21’mer top strand:
(50-GTGTGGAAAATCTCTAGCAGT-30), and a 21’mer bottom strand:
(50-ACTGCTAGAGATTTTCCACAC-30). The top strand was labeled at
the 50end with
c [
32P]-ATP by T4 polynucleotide kinase (Epicenter,
Madison, WI). The mixture was then incubated at 95 °C for 15 min
to inactivate the kinase and the bottom strand was added in 1.5
molar excess. The strands were allowed to anneal by cooling the
mixture slowly to room temperature. Any unincorporated material
was subsequently removed by centrifuging the mixture through a
Spin-25 mini-column (USA Scientific, Ocala, FL).
5.2.72. 3-Amino-N-(furan-2-ylmethyl)-2-hydroxybenzamide
(6d)
Compound 6d was prepared from 3-amino-2-hydroxybenzoic
acid and furan-2-ylmethanamine according to the same procedure
described for 5a. Compound 6d (45 mg, 30% yield) was purified as
an off white solid. 1H NMR (300 MHz, CDCl3): d 12.38 (s, 1H), 7.37
(s, 1H), 6.81 (dd, J = 1.5 Hz, 7.2 Hz, 1H), 6.74 (dd, J = 1.5 Hz, 7.8 Hz,
1H), 6.66 (d, J = 7.8 Hz, 1H), 6.56 (br s, 1H), 6.36 (t, J = 2.4 Hz, 1H),
6.31 (d, J = 3.0 Hz, 1H), 4.62 (d, J = 5.4 Hz, 2H), 3.93 (br s, 2H). EI-
MS: m/z 232 (M)+. HRMS (EI): calcd for C12H12N2O3 (M)+
5.6. Integrase catalytic activity assay
The extent of 30-processing and strand transfer was analyzed by
preincubating recombinant wild-type HIV-1 IN, at a final concen-
tration of 200 nM, with the inhibitor in reaction buffer (50 mM
NaCl, I mM HEPES, pH 7.5, 50 lM EDTA, 50 lM dithiothreitol,
10% glycerol (w/v), 7.5 mM MnCl2, 0.1 mg/mL bovine serum albu-
min, 10 mM 2-mercaptoethanol, 10% DMSO, and 25 mM MOPS,

4952
X. Fan et al. / Bioorg. Med. Chem. 19 (2011) 4935–4952
32
pH 7.2) at 30 °C for 30 min. Then 20 nM of the P 50end-labeled
linear 21’mer substrate was added, and incubation was continued
for an additional 1 h. Reactions were then quenched by the addi-
and National Science & Technology Major Project ‘Key New Drug
Creation and Manufacturing Program’, China (Numbers:
2009ZX09301-001 and 2009ZX09103-067). The work in N.N. labo-
ratory was supported by funds from an NIH/NIAID (R21AI081610)
grant.
tion of an equal volume (16
formamide, 10 mM EDTA, 0.025% xylene cyanol, and 0.025% bro-
mophenol blue). An aliquot (7 L) was electrophoresed on a dena-
lL) of loading dye (98% deionized
l
turing 20% polyacrylamide gel (0.09 M tris–borate pH 8.3, 2 mM
References and notes
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The work in Y.-Q.L. laboratory was supported by Science and
Technology Commission of Shanghai Municipality (08JC1422200),
National Natural Science Foundation of China (No. 81072527)