Discovering novel chemical inhibitors of human cyclophilin A: Virtual screening, synthesis, and bioassay

Article abstract of DOI:10.1016/j.bmc.2005.11.006

Cyclophilin A (CypA) is a member of cyclophilins, a family of the highly homologous peptidyl prolyl cis-trans isomerases (PPIases), which can bind to cyclosporin A (CsA). CypA plays critical roles in various biological processes, including protein folding, assembly, transportation, regulation of neuron growth, and HIV replication. The discovery of CypA inhibitor is now of a great special interest in the treatment of immunological disorders. In this study, a series of novel small molecular CypA inhibitors have been discovered by using structure-based virtual screening in conjunction with chemical synthesis and bioassay. The SPECS_1 database containing 85,000 small molecular compounds was searched by virtual screening against the crystal structure of human CypA. After SPR-based binding affinity assay, 15 compounds were found to show binding affinities to CypA at submicro-molar or micro-molar level (compounds 1-15). Seven compounds were selected as the starting point for the further structure modification in considering binding activity, synthesis difficulty, and structure similarity. We thus synthesized 40 new small molecular compounds (1-6, 15, 16a-q, 17a-d, and 18a-l), and four of which (compounds 16b, 16h, 16k, and 18g) showed high CypA PPIase inhibition activities with IC₅₀s of 2.5-6.2 μM. Pharmacological assay indicated that these four compounds demonstrated somewhat inhibition activities against the proliferation of spleen cells.

Full text of DOI:10.1016/j.bmc.2005.11.006

Bioorganic & Medicinal Chemistry 14 (2006) 2209–2224
Discovering novel chemical inhibitors of human cyclophilin A:
Virtual screening, synthesis, and bioassay
Jian Li,a, Jing Chen,a, Chunshan Gui,a, Li Zhang,b Yu Qin,b Qiang Xu,b Jian Zhang,a
a,c,
Hong Liu,a,* Xu Shena,c, and Hualiang Jiang
*
*
aDrug Discovery and Design Centre, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica,
Graduate School of the Chinese Academy of Sciences, Shanghai Institutes for Biological Sciences,
Chinese Academy of Sciences, Shanghai 201203, China
bState Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University,
22 Han Kou Road, Nanjing 210093, China
cSchool of Pharmacy, East China University of Science and Technology, Shanghai 200237, China
Received 7 September 2005; revised 1 November 2005; accepted 2 November 2005
Available online 22 November 2005
Abstract—Cyclophilin A (CypA) is a member of cyclophilins, a family of the highly homologous peptidyl prolyl cis–trans isomerases
(PPIases), which can bind to cyclosporin A (CsA). CypA plays critical roles in various biological processes, including protein fold-
ing, assembly, transportation, regulation of neuron growth, and HIV replication. The discovery of CypA inhibitor is now of a great
special interest in the treatment of immunological disorders. In this study, a series of novel small molecular CypA inhibitors have
been discovered by using structure-based virtual screening in conjunction with chemical synthesis and bioassay. The SPECS_1 data-
base containing 85,000 small molecular compounds was searched by virtual screening against the crystal structure of human CypA.
After SPR-based binding affinity assay, 15 compounds were found to show binding affinities to CypA at submicro-molar or micro-
molar level (compounds 1–15). Seven compounds were selected as the starting point for the further structure modification in con-
sidering binding activity, synthesis difficulty, and structure similarity. We thus synthesized 40 new small molecular compounds (1–6,
15, 16a–q, 17a–d, and 18a–l), and four of which (compounds 16b, 16h, 16k, and 18g) showed high CypA PPIase inhibition activities
with IC50s of 2.5–6.2 lM. Pharmacological assay indicated that these four compounds demonstrated somewhat inhibition activities
against the proliferation of spleen cells.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
prolyl isomerase (PPIase) activity.4 hCypA performs
an essential function in HIV-1 replication by binding
specifically with the capsid domain (CA) of the Gag
polyprotein.5 hCypA may also render neuroprotective/
neurotrophic effects6 when presented at high levels in
the brain.7 Recently, it was discovered that the nucleo-
capsid (N) protein of SARS coronavirus (SARS-CoV)
can bind to hCyPA, which may be associated with
SARS-CoV infection.8 CypA has attracted considerable
attention not only for its novel catalytic activity, but
also for its therapeutic significance. It is reported that
CypA–CsA binding might inhibit the serine/threonine
phosphatase activity of calcineurin, thereby blocking
the production of cytokines including interleukin-29
and interferon c.10
Cyclophilins were discovered originally for their high
affinity against cyclosporin A (CsA), an immunosup-
pressive drug used to prevent allograft rejection.1 The
general biological function of cyclophilins is to catalyze
the cis–trans isomerization of peptide bonds preceding
proline in protein and peptides.2 Of the 15 known hu-
man cyclophilins, cyclophilin A (hCypA) is one of the
most important members and has been widely studied
for mapping its biological functions.3 In addition to a
binding target of CsA, hCypA is able to enhance the rate
of folding (or unfolding) of proteins via its peptidyl-
Keywords: Cyclophilin A; Inhibitor; Drug design; Virtual screening.
The discovery of CsA more than two decades ago her-
alded a new era in the field of organ transplantation.
Since then, the number of transplanted organs has
grown continuously and the search for novel immuno-
*
Corresponding authors. Tel.: +86 21 50806600x1210; fax: +86 21
50807088; e-mail addresses: hliu@mail.shcnc.ac.cn; xshen@mail.
shcnc.ac.cn; hljiang@mail.shcnc.ac.cn
These authors contributed equally to this work.
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.11.006

2210
J. Li et al. / Bioorg. Med. Chem. 14 (2006) 2209–2224
suppressants has intensified.11 However, inhibitors of
CypA are mainly derived from the natural sources (such
as FK506,12 rapamycin,13 and sanglifehrin A14) and
peptide analogues,15 which are all large molecules, and
little has been reported regarding the small molecule
CypA inhibitors.
pocket. During the docking calculation, Kollman-all-
atom charges22 were assigned to the protein and
Gasteiger–Marsili partial charges23 were assigned to
the small molecules in the SPECS_1 database. Confor-
mational flexibility of the compounds from the database
was considered in the docking search.
Although immunosuppressants like CsA have improved
the rates of transplant success and prolonged patient
survivals, some side effects with this agent have been
sequentially found such as nephrotoxicity,16 hyperten-
sion17, and cardiotoxicity.18 This thereby prompted us
to discover novel small molecule CypA inhibitors in or-
der to decrease the side effects. Structure-based ligand
design has led to the identification of compounds that
are currently in clinical trials or into the market.19 The
wealth of structural and functional information of
CypA15 has offered a solid starting point for the rational
structure-based design of inhibitors. By using docking-
based virtual screening approach in conjunction with
surface plasmon resonance (SPR) determination, 15
novel small molecule CypA binders (hits) (compounds
1–15) have been discovered. According to the binding
potency, structural similarity, and synthetic complexity,
seven hits (compounds 1–6 and 15) were selected as the
starting points for further structural optimization.
Totally, 40 new compounds including 1–6 and 15 (the
preparation methods for these seven compounds have
not been reported) and their 33 analogues (16a–q,
17a–d, and 18a–l) have been synthesized and tested with
biological assay. Finally, four compounds (16b, 16h,
16k, and 18g) were found to show high CypA PPIase
(peptidyl-prolyl isomerase) inhibition activities and one
compound (16h) is active in inhibiting the proliferation
of spleen cells.
The DOCK suite of programs is designed to find possi-
ble orientations of a ligand in a ÔreceptorÕ site.21 The ori-
entation of a ligand is evaluated with a shape scoring
function and/or a function approximating the ligand–re-
ceptor binding energy. The shape scoring function is an
empirical function resembling the van der Waals attrac-
tive energy. The ligand–receptor binding energy is taken
to be approximately the sum of the van der Waals and
electrostatic interaction energies. After the initial orien-
tation and scoring evaluation, a grid-based rigid body
minimization is carried out for the ligand to locate the
nearest local energy minimum within the receptor bind-
ing site. The position and conformation of each docked
molecule were optimized using single anchor search and
torsion minimization method of DOCK4.0.21 Thirty
configurations per ligand building a cycle and 50 maxi-
mum anchor orientations were used in the anchor-first
docking algorithm. All docked configurations were ener-
gy minimized using 100 maximum iterations and 1 min-
imization cycle.
The 5000 molecules with the highest score as obtained
by DOCK search were re-scored by using the Consensus
Score (CScore)24 method encoded in Sybyl6.8.25 The
binding free energies and the binding modes of the top
1301 molecules with CScore P4 to CypA were calculat-
ed and modeled using the FlexX26 program encoded in
Sybyl6.8.25 FlexX is a flexible docking method that uses
an incremental construction algorithm to place ligand
into an active site. By dividing the ligand into small frag-
ments and building incrementally, the conformational
flexibility of the ligand can be considered in reasonable
times. The Kollman-all-atom charge22 and Gasteiger-
Marsili partial charge23 were used for cyclophilin A
and candidate molecules, respectively, in the FlexX
simulations.
2. Materials and methods
2.1. Small molecular database for virtual screening
SPECS database contains the structural information of
280,000 small molecules (http://www.specs.net). SPECS
Company supplies all the compound samples collected
from difference sources. First, SPECS database was
evaluated using our own filter of druglikeness.20 Non-
druglike molecules were removed from the database,
and finally 85,000 potentially druglike molecules were
selected out for docking screening. These molecules were
saved in the SPECS_1 database.
Finally 82 compounds were distinguished and selected
for bioassay on the basis of score by virtual screening
and druglikeness analysis. The virtual screening was
performed on
supercomputer.
a 128-processor SGI Origin 3800
2.3. Chemistry
2.2. Virtual screening by molecular docking
2.3.1. Design of analogues of compounds 1–6, 15. Com-
pounds1–6 and 15 (Fig. 1) bearing the highest binding
affinities with CypA as obtained by the surface plasmon
resonance (SPR) determination (see Section 4) for the 82
candidate compounds selected by virtual screening were
used as lead compounds for designing new CypA inhib-
itors. Based on the structural and enzyme binding activ-
ity features of compounds 1–6 and 15, 33 new analogues
(16a–q, 17a–d, and 18a–l) (Tables 1 and 2) were designed
and synthesized for the first round. Keeping the key
groups of compounds 1–4—2-substituted quinoline ring
The crystal structure of CypA in complex with sang-
lifehrin macrolide (SFM) (PDB entry 1NMK)14 recov-
ered from the Brookhaven Protein Data Bank (http://
www.rcsb.org/pdb) was used as a target for virtual
screening on the SPECS_1 database (http://www.specs.-
net). In the present study, the DOCK4.0 program21 was
employed for the primary screening. Residues of CypA
around SFM at a radius of 6.5 A were isolated for con-
structing the grids of the docking screening. The result-
ing substructure included all residues of the binding
˚

J. Li et al. / Bioorg. Med. Chem. 14 (2006) 2209–2224
2211
and sulfanilamide moiety—we used various setric, elec-
tronic, and hydrophobic groups to substitute at position
2 of the quinoline ring in compounds 1–4 (Fig. 1) and
obtained 10 analogues (16a–j) (Table 1). Replacing the
quinoline ring with a pyridine ring, we obtained seven
compounds (16k–q) (Table 1). Keeping the key groups
of compounds 5 and 6—phenyl alkyl acylamide and sul-
fanilamide moiety—we changed the length of the alkyl
linkers, coupled with the sulfisoxazole of compound 1,
and obtained four compounds (17a–d) (Table 1). On
the basis of the structural features of compound 15,
we designed compounds 18a–j (Table 2), maintained
dibenzoic acid or dibenzoic acid ester moiety, and re-
placed 3,5-dimethoxybenzamido group in compound
15 with other electronic and hydrophobic substituted
benzamido groups. Substituting the methylene group
of compound 15 with an ethylene group, we obtained
compounds 18k–l (Table 2).
lic acids 20 were prepared by isatins (19) condensation
with ketones in ethanol. Compound 20 was converted
to the corresponding acyl chlorides (21) by refluxing
with SOCl2.27 Then 21 was substituted by using different
sulfanilamides, giving the target compounds 1–4 and
16a–j (Table 1). The key intermediate 25 required by
synthesizing compound 16j was prepared from com-
pound 2328 by sulfamation and deprotection.
Compounds 16k–q were synthesized through the route
outlined in Scheme 2. 2-Bromo-1-arylethanone (26)
was reacted by refluxing with ammonium formate in
98% HCOOH, which afforded 4-aryloxazole (27).29
Compound 28 was synthesized using the Usui Y. meth-
od30 by compound 27 reacting with maleic acid at
110 ꢁC for 15 min. Then, the target compounds 16k–q
were obtained after steps c–d of Scheme 1 were
employed (Table 1).
2.3.2. Synthetic procedures. Scheme 1 depicts the se-
quence of reactions that led to the preparation of com-
pounds 1–4 and 16a–j using isatins (19) as the starting
material. In general, the substituted 4-quinolinecarboxy-
Compounds 5–6 and 17a–d were synthesized by using
the same process as compounds 1–4, except compound
20 was replaced by compound 29. The synthetic route
is outlined in Scheme 3.
Class I
N
H
N
H
N
O
S
O
S
O
O
O
O
N
N
O
2
4
Br
N
N
H
H
N
1
N
5
8
6
2 (KD=6.34 µM)
1 (KD=1.13 µM)
H
N
N
O
H
N
O
S
S
O
O
O
O
O
N
N
N
N
H
H
O
N
N
3 (KD=9.33 µM)
4 (KD=23.2 µM)
H
N
O
H
O
N
N
N
O
N
S
Cl
S
Cl
O
O
O
N
N
H
N
H
Cl
5 (KD=1.23 µM)
6 (KD=8.99 µM)
H
O
O
N
NH2
O
S
S
O
O
O
H
Br
N
N
H
Cl
N
H
O
N
S
O
O
O
8 (KD=34.3 µM)
7 (KD=23.4 µM)
Figure 1. The structures of 15 binders (or hits) of CypA selected from the candidates by virtual screening and SPR-based binding affinity assay.

2212
J. Li et al. / Bioorg. Med. Chem. 14 (2006) 2209–2224
Class II
Br
OH
O
H
OH
O
N
N
O
O
N
Cl
H
N
O
O
N
N
HN
O
O
H
O
10 (KD=53.4 µM)
9 (KD=5.60 µM)
Cl
O
N
O
N
O
N
O
N
H
O
H
O
O
HO
O
HO
12 (KD=0.83 µM)
11 (KD=12.3 µM)
Class III
O
S
N
O
O
O
N
N
O
N
N
H
H
H
N
S
N
S
O
O
13 (KD=3.45 µM)
14 (KD=5.64 µM)
O
O
HO
HN
O
O
O
HN
O
O
O
OH
15 (KD=0.58 µM)
Figure 1. (continued)
Scheme 4 depicts the sequence of reactions that led to the
preparation of compounds 15 and 18a–i using anthranilic
acid (30) as the starting material. Compound 31a31 was
synthesized by heating anthranilic acid (30) with HCHO
in 36% HCl. Refluxing 31a with saturated HCl in metha-
nol afforded compound 31b. Acylation 31a–b with vari-
ous substituted benzoyl chlorides in pyridine afforded
compounds 15 and 18a–f. Compound 31b reacted with
benzyl bromide in refluxing NMP produced compound
18g, which was hydrolyzed using LiOH at 25 and 80 ꢁC to
afford monoester 18h and dicarboxylic acid 18i, respectively.
2.4. Biological assay
2.4.1. Binding assay. CypA was expressed and purified
according to our previously reported procedure.8 The
binding affinities of the virtual screening candidates to
CypA in vitro were determined by employing the surface
plasmon resonance (SPR) technology. The measurement
was performed using the dual flow cell Biacore 3000
instrument (Biacore AB, Uppsala, Sweden). Immobiliza-
tion of the CypA to the hydrophilic carboxymethylated
dextran matrix of the sensor chip CM5 (Biacore) was car-
ried out by the standard primary amine coupling reaction
wizard. The CypA to be covalently bound to the matrix
was diluted in 10 mM sodium acetate buffer (pH 6.0) to
a final concentration of 0.035 mg/mL. Equilibration of
the baseline was completed by a continuous flow of HBS-
EP running buffer (10 mM Hepes, 150 mM NaCl, 3 mM
EDTA, and 0.005% (v/v) surfactant P20, pH 7.4) through
the chip for 1–2 h. All the Biacore data were collected at
25 ꢁC with HBS-EP as running buffer at a constant flow rate
of 30 lL/min. All the sensorgrams were processed by using
automatic correction for non-specific bulk refractive index
effects. The equilibrium constants (KD values) evaluating
the protein–ligand binding affinities were determined by
the steady state affinity fitting analysis of the Biacore data.
Compounds 18j–l were synthesized through the route
outlined in Scheme 5. Refluxing 5-methyl-2-nitrobenzoic
acid (32) with saturated HCl in methanol afforded com-
pound 33, which was then brominated by NBS in chlo-
roform to afford compound 34.32 Compound 34 was
coupled with compound 33, giving the key intermediate
35 in the presence of n-BuLi at ꢀ78 ꢁC, which was then
reduced by Pd-C. Compound 18j was obtained by N-ac-
ylation of 37 with 3,5-dimethoxybenzoyl chloride in pyr-
idine. Compound 18k was obtained under the same
conditions as compound 18a, and compound 18l was
quantitatively obtained by hydrolysis of 18k using LiOH
at room temperature.

J. Li et al. / Bioorg. Med. Chem. 14 (2006) 2209–2224
Table 1. Chemical structures of compounds 1–6 and 16–17, and their activities
2213
O
R9
O
HN
S NHR3
O
I
a
Compound
R9
R3
KD (lM)
% inhibition at 10 lMb
1
3,4-Dimethyl-5-isoxazolyl
4-Methyl-2-pyrimidinyl
2-Pyrimidinyl
1.13
6.34
9.33
23.2
10.2
52.3
4.11
223
3.20
11.2
10.2
26.5
18.0
67.3
10.0
15.0
40.0
30.0
NA
Br
N
CH
3
2
N
N
CH3
3
CH3
3
O CH
4
5-Methyl-3-oxazolyl
N
N
16a
16b
16c
16d
16e
16f
16g
16h
16i
16j
16k
16l
3,4-Dimethyl-5-isoxazolyl
3,4-Dimethyl-5-isoxazolyl
3,4-Dimethyl-5-isoxazolyl
3,4-Dimethyl-5-isoxazolyl
3,4-Dimethyl-5-isoxazolyl
3,4-Dimethyl-5-isoxazolyl
3,4-Dimethyl-5-isoxazolyl
3,4-Dimethyl-5-isoxazolyl
3,4-Dimethyl-5-isoxazolyl
2-Quinoxalinyl
F
Cl
N
N
Br
Br
N
N
10.6
91.3
1.79
10.1
2.25
3.13
46.0
89.5
N
N
N
N
S
77.4
32.0
36.0
74.7
F
Br
N
Br
N
3,4-Dimethyl-5-isoxazolyl
3,4-Dimethyl-5-isoxazolyl
Br
Br
N
N
NA
(continued on next page)

2214
J. Li et al. / Bioorg. Med. Chem. 14 (2006) 2209–2224
Table 1 (continued)
a
Compound
R9
R3
KD (lM)
% inhibition at 10 lMb
16m
3,4-Dimethyl-5-isoxazolyl
14.5
NA
N
16n
16o
16p
3,4-Dimethyl-5-isoxazolyl
3,4-Dimethyl-5-isoxazolyl
3,4-Dimethyl-5-isoxazolyl
1.86
NA
26.0
NA
N
N
Cl
F
42.4
CH3
CH3
7.61
N
N
16q
4-Methyl-2-pyrimidinyl
340
NA
5
6
2-Quinoxalinyl
2-Quinoxalinyl
1.23
8.99
3.2
7.5
O
Cl
O
Cl
Cl
Cl
Cl
O
17a
17b
17c
17d
2-Quinoxalinyl
37.7
NA
5.6
O
2-Quinoxalinyl
23.4
1.06
0.35
O
2-Quinoxalinyl
NA
NA
3,4-Dimethyl-5-isoxazolyl
O
Cl
a Data obtained from SPR-based binding affinity assay. KD represents the equilibrium constant.
b Data are means of three independent experiments.
Table 2. Chemical structures of compounds 15 and 18 and their activities
R10
R11
R12HN
NHR12
n
III
% inhibition at 10 lMb
a
Compound
n
R10
R11
R12
KD (lM)
15
1
1
1
1
1
1
1
1
1
1
1
2
2
COOH
CO2CH3
COOH
CO2CH3
COOH
CO2CH3
CO2CH3
CO2CH3
COOH
COOH
—
COOH
CO2CH3
COOH
CO2CH3
COOH
CO2CH3
CO2CH3
CO2CH3
CO2CH3
COOH
—
3,5-Dimethoxy-benzoyl
3,5-Dimethoxy-benzoyl
Benzoyl
0.58
NA
NAc
32.3
NAc
NA
140
12.4
NA
NAc
11.2
NAc
NA
10.2
86.0
6.5
18a
18b
18c
18d
18e
18f
18g
18h
18i
18j
18k
18l
Benzoyl
4-Methoxy-benzoyl
4-Methoxy-benzoyl
4-Cl-Benzoyl
Benzyl
Benzyl
Benzyl
414
0.23
184
NA
43.0
15.6
NAc
3,5-Dimethoxy-benzoyl
3,5-Dimethoxy-benzoyl
3,5-Dimethoxy-benzoyl
3.3
CO2CH3
COOH
CO2CH3
COOH
87.5
NAc
a Data obtained from SPR-based binding affinity assay. KD represents the equilibrium constant.
b Data are means of three independent experiments.
c These compounds cannot be assayed for poor solubility in DMSO.

J. Li et al. / Bioorg. Med. Chem. 14 (2006) 2209–2224
2215
H
O
OH
O
Cl
O
N
O
O
R1
R1
R1
R1
O
b
c
R2
S
O
NHR3
O
a
N
N
R2
N
R2
N
R2
H
19
20
21
1-4 and 16 a-j
H
H
O
O
S
O
N
O
N
N
O
N
N
O
S Cl
O
H2N
N
N
S
d
e
O
HN
+
N
N
H
H2N
22
23
24
25
Scheme 1. Reagents and conditions: (a) (i) KOH, C2H5OH, H2O, reflux, (ii) HCl, H2O; (b) SOCl2, reflux; (c) R3NHSO2C6H6NH2-p, pyridine, 25 ꢁC;
(d) pyridine, 25 ꢁC; (e) (i) NaOH, H2O, reflux, (ii) HCl.
H
O
OH
O
Cl
O
N
O
R
4
Br
O
S
a
b
c
d
R
NHR
4
N
3
O
N
N
N
R
R
4
R
4
4
O
26
27
28
16k-q
Scheme 2. Reagents and conditions: (a) HCOOH (98%), HCOONH4, reflux; (b) maleic acid, 110 ꢁC; (c) SOCl2, reflux; (d) R3NHSO2C6H6NH2-p,
pyridine, 25 ꢁC.
O
NHR
O
3
O
O
S
a
b
O
R
R
5
5
R
5
OH
Cl
n
n
N
n
H
29
5-6 and 17a-d
Scheme 3. Reagents and conditions: (a) SOCl2, reflux; (b) R3NHSO2C6H6NH2-p, pyridine, 25 ꢁC.
O
O
O
O
NH
O
2
R O
6
OR
6
R O
OR
6
a
c
6
OH
HN
NH
H N
NH
2
2
O
O
R
7
R
7
31a: R =H
6
b
30
15and 18a-f
31b: R =CH
6
3
O
O
HO
O
HN
NH
O
O
O
O
e
18h
d
31b
HN
NH
O
O
f
HO
HN
OH
NH
18g
18i
Scheme 4. Reagents and conditions: (a) 3% HCHO, HCl, H2O; (b) CH3OH, HCl; (c) substituted benzoyl chlorides, pyridine, 25 ꢁC; (d) benzyl
bromide, NMP, Na2CO3, H2O; (e) LiOH, THF/CH3OH/H2O = 3:1:1, 25 ꢁC; (f) LiOH, THF/CH3OH/H2O = 3:1:1, 80 ꢁC.

2216
J. Li et al. / Bioorg. Med. Chem. 14 (2006) 2209–2224
OH
O
O
NO2
O
O
O
a
b
33
c
O
O
Br
O
O2N
O2N
O2N
O
O2N
35
32
33
34
OR8
O
O
NH2
O
d
e
HN
O
O
O
N
H
O
O
H2N
OR8
O
O
18k: R8=CH3
18l: R8=H
36
f
O
O
O
O
N
N
H
e
H
H2N
NH2
O
O
37
18j
Scheme 5. Reagents and conditions: (a) CH3OH, HCl; (b) NBS, CHCl3, BzooBz; (c) n-BuLi, THF, ꢀ78 ꢁC; (d) Pd-C, H2, THF; (e) 3,5-
dimethoxybenzoyl chloride, pyridine, 25 ꢁC; (f) LiOH, THF/CH3OH/H2O = 3:1:1, 25 ꢁC.
2.4.2. Enzymatic activity assay. CypA PPIase activity was
measured at 4 ꢁC by using the standard chymotrypsin-
coupled assay.33 The assay buffer (50 mM Hepes,
100 mM NaCl, pH 8.0) and CypA (500 nM stock solu-
tion) were pre-cooled to 4 ꢁC, to which then was added
15 lL of 3 mg/mL chymotrypsin in 1 mM HCl. The reac-
tion was initiated by adding 12 lL of 3.8 mM peptide sub-
strate (Suc-Ala-Ala-cis-Pro-Phe–pNA) in LiCl/THF
solution with rapid inversion. After a delay from the onset
of mixing (usually 6 s), the absorbance of p-nitroaniline
was followed at 390 nM until the reaction was complete
(1 min). The final concentration of LiCl in the assay was
9.6 mM; THF was present at a concentration of 2%
(v/v). Absorbance readings were collected every 0.1 s by
a U-2010 spectrophotometer. The progress curves were
analyzed by non-linear least-squares fit.
The spleen was aseptically taken from mice, crushed gent-
ly, and separated into single cells by squeezing in 5 mL
D-HanksÕ solution (Gibco-BRL). The cells obtained were
passed through eight layers of gauze and centrifuged at
1000 rev/min for 5 min at 4 ꢁC. Pellets were added into
10 mL of sterile 0.17 M Tris-(hydroxymethyl) amino-
methane containing 0.75% NH4Cl (pH 7.5) followed by
centrifugation to remove erythrocytes. After washing
twice with RPMI 1640 containing 100 U mLꢀ1 penicillin,
100 U mLꢀ1 streptomycin, and 10% fetal calf serum
(FCS) (RPMI 1640 medium), they were re-suspended in
the RPMI 1640 medium and used for cell culture.
Spleen cells were cultured in 96-well flat-bottomed
microplates (Falcon) at a density of 5 · 105 cells per well
in RPMI 1640 medium (0.2 mL) and stimulated with
5 lg mLꢀ1 Con A for 72 h at 37 ꢁC in 5% CO2-air in
the presence or absence (control group) of various con-
centrations of compounds. Then the cell growth was
evaluated with modified MTT assay.34 Briefly, 20 lL
of 5 mg mLꢀ1 MTT in RPMI 1640 was added for a fur-
ther 4 h incubation. After removing the supernatant,
200 lL of DMSO was added to dissolve the formazan
crystals. The plate was shaken for 10 min and then read
on an ELISA reader (Sunrise Remote/Touch Screen;
TECAN, Austria) at 540 nm. All assays were run in trip-
licate and the effect of compounds on the proliferation
of mouse spleen cells induced by Con A was calculated
by Eq. 1
The inhibition assays of compounds were performed in
the same manner as mentioned above. A 0.6 lL aliquot
of the compounds in DMSO was added to the CypA
solution in the assay buffer. After being pre-incubated
for 1 h at 4 ꢁC, the assay was started by the addition
of chymotrypsin and the substrate. To calculate the
half-maximal inhibitory concentration (IC50), the per-
cent of remaining PPIase activity is plotted against the
common logarithm of the compound concentration,
and the data were fitted using the sigmoidal fitting mod-
el by the Origin7.0 software.
2.4.3. T cell viability assay. Female ICR strains of mice,
6–8 weeks old and 20 2 g, were purchased from the
Experimental Animal House of China Pharmaceutical
University (Nanjing, China) and were maintained in
plastic cages with free access to pellet food and water
(12 h light/dark cycle, 21 2 ꢁC). This study complied
with the current ethical regulations on animal research
in Nanjing University and the mice used were treated
humanely.
Inhibitory rate ð%Þ ¼ ðcontrol ðOD540
Þ
ꢀ compounds ðOD540ÞÞ=control ðOD540Þ.
ð1Þ
Data were expressed as means SD (significance of dif-
ferences between groups). Statistical analysis was evalu-
ated by one-way analysis of variance, followed by
StudentÕs two-tailed t test for the evaluation of the differ-

J. Li et al. / Bioorg. Med. Chem. 14 (2006) 2209–2224
2217
ence between two groups and DunnettÕs t test between
control group and multiple dose groups. One-way anal-
ysis of variance revealed a significant effect at P < 0.05.
3.2. Analogue design and synthesis
Structurally, the 15 hits can be divided into three classes:
class I includes eight compounds containing sulfanil-
amide moiety (compounds 1–8); class II compounds
are acylhydrazone derivatives (compounds 9–12); class
III is comprised by compounds 13–15, which have no
distinct structural character (Fig. 1). Considering the
structural similarity and the binding affinity with CypA,
we took compounds 1–6 in class I and compound 15 in
class III as leaders for structural optimization. Totally,
40 compounds (1–6, 15, 16a–q, 17a–d, and 18a–l) were
designed and synthesized, and their chemical structures
are shown in Tables 1 and 2. These compounds were syn-
thesized through the routes outlined in Schemes 1–5, and
the details for synthetic procedures and structural char-
acterizations are described in the Experimental section.
3. Results and discussion
3.1. Identification of binders (hits) by virtual screening
Targeting the crystal structure of CypA (PDB entry
1NMK),14 we searched the SPECS_1 database by using
DOCK4.0.21 The small molecules were ranked accord-
ing to their scores calculated by using the scoring func-
tion of DOCK. The top 5000 candidate molecules were
obtained with the best scores by a shape complementar-
ity scoring function in DOCK.21 These compounds were
re-estimated using Cscore.24 A total of 1301 compounds
with Cscore = 4 or 5 (the best score of Cscore is 5) were
subsequently docked and ranked using the FlexX26 pro-
gram. Finally, 82 compounds were selected according to
the score of FlexX for biological assay. Because enzy-
matic assay is time-consuming, therefore, surface plas-
mon resonance (SPR) measurements were used for the
primary screening, determining the binding affinity of
these 82 candidate molecules to CypA. Immobilization
of CypA resulted in a resonance signal at about 5370
resonance units (RUs). Among the 82 compounds, the
biosensor RUs of 15 compounds were concentration-de-
pendent. The collected data indicated that these 15 com-
pounds can bind to CypA in vitro and the binding
affinities to cyclophilin A are in the submicro- or mi-
cro-molar range (KD = 53.4–0.58lM). These com-
pounds could be designated as binders (or hits) of
CypA, their chemical structures and binding affinities
shown in Figure 1.
3.3. Biological activities
3.3.1. The PPIase activity of CypA. For the primary as-
say, the percent inhibitions of the compounds at 10 lM
were measured. The results are listed in Tables 1 and 2.
Four compounds, i.e., 16b, 16h, 16k, and 18g, can
remarkably inhibit the PPIase activity of CyPA (percent
inhibition at 10 lM > 50%), indicating that these four
compounds are CypA inhibitors. Therefore, we deter-
mined their IC50 values, which are, respectively, 4.88,
2.84, 2.48, and 6.23 lM (Fig. 2). In addition, the binding
affinities of the 33 analogues were determined by using
the SPR technology, the results also listed in Tables 1
and 2. The PPIase activities of the compounds do not
consistently correlate with the SPR binding affinities.
The reason may be that the protein was immobilized
to a sensor chip in the SPR assay, which affects the con-
formational flexibility of the protein. Accordingly, good
binders identified by the SPR assay may not be good
inhibitors. To find CypA inhibitors rather than binders,
the effects of compounds on the PPIase activity of CypA
have to be determined.
100
80
60
3.3.2. Inhibition activity against the proliferation of spleen
cells. To test the immunosuppressive effects of 16a, 16h,
16k, and 18g, anti-proliferation activities on spleen cells
in vitro of the four inhibitors have been evaluated. The
result is listed in Table 3, indicating that compounds
16a, 16h, and 16k show partial influence on the viability
of the na¨ıve spleen cells. It is remarkable that the effec-
tive concentration of compound 16h is ꢁ10 lM in inhi-
bition of the proliferation of mouse spleen cells induced
by Con A, which is slightly lower than that of CsA. This
indicates that compound 16h is a good lead for design-
ing more potent immunosuppressive agents.
Compound16b IC50 = 4.88 µM
Compound16h IC50 = 2.84 µM
Compound16k IC50 = 2.48 µM
Compound18g IC50 = 6.23 µM
40
20
0
-20
0.1
1
10
100
Concentration of Compound (µM)
Figure 2. Concentration dependence of PPIase activity by 16b, 16h,
16k, and 18g, the concentration of CyPA was kept constant at
25 nM, while the concentration of compounds ranged from 0.1 to
200 lM.
Table 3. Effect of compounds 16b, 16h, 16k, 18l, and CsA on the proliferation of mouse spleen cells induced by Con A in vitro
Dose (lM)
Inhibitory rate (%)a
CsA
16b
16h
16k
18g
100
10
1
84.31 4.88b
82.57 5.20b
56.52 1.60
47.35 4.98b
29.78 5.00
14.13 9.97
46.74 5.39b
34.26 5.60b
20.76 8.41
49.05 4.69b
22.71 7.06
15.33 7.81
28.83 3.39b
7.46 4.13
1.20 6.41
a Each value indicates the mean SD of three experiments using three mice with triplicate sets in each assay.
b One-way analysis of variance revealed a significant effect at P < 0.05.

2218
J. Li et al. / Bioorg. Med. Chem. 14 (2006) 2209–2224
Figure 3. Three-dimensional structural modes of inhibitors 16b (a), 16h (b), 16k (c), and 18g (d) to CypA derived from the docking simulations.
These four images were generated using the Pymol program (http://www.pymol.org/). The CypA surface was colored by electrostatic potential.
3.4. Binding models
pyridine moiety (16k–q) or alkyl groups (5, 6 and
17a–d) act as linkers interacting with the saddle part
between sites A and B. The interacting models for class
III compounds to CypA are mostly similar to those for
class I compounds, except that compounds 15, 18a,
18d–f, and 18j–l extend from site B due to their longer
linkers and/or substituents.
To gain structural information for further structural
optimization, the 3D binding models of the designed
compounds to CypA were generated based on the
docking simulation, Figure 3 shows the interaction
models of four potent inhibitors (16a, 16h, 16k, and
18g) to CypA. From Figure 3, one can see that there
are two binding sites in the surface binding groove of
CypA: site A is a small binding pocket formed by
Lys82, Met100, Ala101, Asn102, Ala103, Thr107,
Asn108, Gly109, Ser110, Gln111, and Phe112; near site
A, there is a larger pocket, which is called site B,
formed by Phe60, Met61, Gln63, Ala101, Asn102,
Ala103, Phe113, Leu122, and His126; there is a ÔsaddleÕ
between the two sites. The binding models (Figs. 3a–c)
indicate that class I compounds (Table 1) interact with
CypA in a similar way: the common sulfanilamide moi-
ety occupies the polar binding site (site B), forming
three to four hydrogen-bonds (H-bonds) with residues
Gln63, Gly75, Asn102, Thr107, Ser110, and Gln111
(Fig. 4); the 2-substituents of quinoline rings (1–4
and 16a–j) and pyridine rings (16k–q) or aryl rings
(5, 6 and 17a–d) fill in the hydrophobic pocket (site
A); and the quinoline moiety (1–4 and 16a–j), the
In summary, we have discovered four potent CypA
inhibitors (16a, 16h, 16k, and 18g) by using a struc-
ture-based virtual screening approach in conjunction
with chemical synthesis and bioassay. One of them
(compound 16h) is active with very close potency to
CsA in inhibiting the proliferation of spleen cells, dem-
onstrating that this compound may be a good lead for
discovering new immunosuppressive agents. Discover-
ing small molecule CypA inhibitors is an urgent need,
so the new chemical structures produced in this study
are of significance.
4. Experimental
The reagents (chemicals) were purchased from Lancas-
ter, Acros, and Shanghai Chemical Reagent Company,

J. Li et al. / Bioorg. Med. Chem. 14 (2006) 2209–2224
2219
Figure 4. Two-dimensional representation for the interacting mode of compounds 16b (a), 16h (b), 16k (c), and 18g (d) with CypA. It is drawn using
the LIGPLOT program.35 A distance between donor and acceptor of less than 3.4 A is considered as a hydrogen-bond, and a 4.1 A distance between
two hydrophobic atoms is considered a hydrophobic interaction.
˚
˚
and used without further purification. Analytical thin-
layer chromatography (TLC) was HSGF 254 (150–
200 lm thickness, Yantai Huiyou Company, China).
Yields were not optimized. Melting points were mea-
sured in capillary tube with a SGW X-4 melting point
apparatus without correction. Nuclear magnetic reso-
nance (NMR) spectra were given on a Brucker AMX-
400 NMR (IS as TMS). Chemical shifts were reported
in parts per million (ppm, d) downfield from tetrameth-
ylsilane. Proton coupling patterns were described as sin-
glet (s), doublet (d), triplet (t), quartet (q), multiplet (m),
and broad (br). Low- and high-resolution mass spectra
(LRMS and HRMS) were given with electric, electro-
spray, and matrix-assisted laser desorption ionization
(EI, ESI, and MALDI) produced by Finnigan MAT-
95, LCQ-DECA spectrometer, and IonSpec 4.7 T.
matography on silica gel, eluted with a mixture of
EtOAc/petroleum ether (1:2, v/v), to afford 24 (1.08 g,
31.6%) as a pale yellow solid; 1H NMR (DMSO): d
2.03 (s, 3H), 7.61 (t, 1H), 7.78 (m, 4H), 7.90 (d, 1H),
8.02 (d, 2H), 8.62 (s, 1H); EI-MS m/z 342 (M+), 277
(100%).
A mixture of compound 24 (1 g, 3 mmol) and NaOH
(1.5 g, 37.5 mmol) in H2O (15 mL) was refluxed for 2 h
to generate a yellow solution, which was acidified to
pH 5.5 at 70–80 ꢁC. After cooling to room temperature,
the precipitate was collected, washed with H2O, and
dried, to afford compound 25 (0.8 g, 91.2%) as a yellow
solid: mp 258–260 ꢁC; EI-MS m/z 300 (M+), 236 (100%).
4.2. General procedures for preparations of 2027 are described
as those for 2-(3-fluorophenyl)-quiniline-4-carboxylic
acid (20a)
4.1. 4-Amino-N-quinoxalin-2-yl benzenesulfonamide (25)
A mixture of 4-acetylamino-benzenesulfonyl chloride 22
(2.34 g, 10 mmol) and compound 1528 (1.45 g, 10 mmol)
in pyridine (20 mL) was stirred overnight at 25 ꢁC,
poured into H2O (50 mL), and extracted with EtOAc.
The combined organic layer was washed, dried, filtered,
and condensed. The residue was purified by flash chro-
As illustrated in Scheme 1, isatin 19 (34.0 mmol), 3-flu-
oroacetophenone (40.8 mmol), and 85% KOH pellets
(102 mmol) were dissolved in EtOH (40 mL), and the
reaction mixture was stirred at 80 ꢁC for 24 h. The mix-
ture was condensed, diluted with water, and acidified
with concd HCl, and the precipitate was collected,

2220
J. Li et al. / Bioorg. Med. Chem. 14 (2006) 2209–2224
washed, and dried to afford 20a; yield 80%; mp 232–
EtOAc/petroleum ether (1:4, v/v), yield 31.3%: mp 291–
293 ꢁC; H NMR (DMSO): d 2.29 (s, 3H), 2.44 (s, 3H),
6.91 (d, 1H), 7.21 (m, 3H), 7.62 (d, 1H), 7.71 (t, 1H),
7.87 (t, 1H), 7.89 (s, 1H), 7.95 (d, 2H), 8.02 (d, 2H),
8.18 (q, 2H), 8.32 (d, 1H); EI-MS m/z 509 (M+), 336
(100%); HRMS (EI) m/z calcd C28H23N5O3S (M+)
509.1518, found 509.1521.
1
1
234 ꢁC; H NMR (DMSO): d 7.33–7.42 (m, 3H), 7.55
(d, 1H), 7.75 (m, 1H), 7.87 (m, 1H), 8.02 (s, 1H), 8.13
(d, 1H), 8.71 (d, 1H); EI-MS m/z 266 (M+ꢀ1), 222
(100%).
4.3. General procedures for preparations of 28 are
described as those for 2-phenylpyridine-4-carbonic acid
(28m)
4.4.3. 4-(2-(2,4-Dimethylphenyl)-quinoline-4-carboxami-
do)-N-pyrimidin-2-yl benzenesulfonamide (3). This com-
pound was purified by flash chromatography with
EtOAc/petroleum ether (1:2, v/v), yield 40.0%: mp
A
mixture of a-bromoacetophenone 26m (10 g,
50 mmol), HCOONH4 (11 g, 174 mmol), and 98%
HCOOH (53 mL) was refluxed under stirring for 2.5 h.
The reaction mixture was poured into H2O, alkalinized
with concentrated NaOH, and extracted with Et2O.
The combined organic layer was washed, dried, filtered,
and condensed. The crude product obtained was puri-
fied by flash chromatography on silica gel, eluted with
a mixture of EtOAc/petroleum ether (1:10, v/v), to af-
ford 27m (1.2 g, 16.7%) as a yellow oil; 1H NMR
(CDCl3): d 7.33 (d, 1H), 7.42 (d, 2H), 7.75 (d, 2H),
7.94 (m, 2H).
1
240–241 ꢁC; H NMR (DMSO): d 2.36 (s, 3H), 2.42 (s,
3H), 7.05 (t, 1H), 7.21 (m, 2H), 7.57 (d, 1H), 7.71 (t,
1H), 7.87 (m, 2H), 7.96 (d, 2H), 8.02 (d, 2H), 8.17 (q,
2H), 8.52 (d, 2H); EI-MS m/z 509 (M+), 350 (100%);
HRMS (EI) m/z calcd C28H23N5O3S (M+) 509.1515,
found 509.1521.
4.4.4. 4-(2-(2-Methoxyphenyl)-quinoline-4-carboxamido)-
N-(5-methyl-oxazol-2-yl) benzenesulfonamide (4). This
compound was purified by flash chromatography with
EtOAc/petroleum ether (1:2, v/v), yield 46.9%: mp
1
A mixture of 4-phenyloxazole 27m (1 g, 6.9 mmol) and
maleic acid (0.83 g, 7.1 mmol) was heated at 110 ꢁC until
melting for 2 h. The solid obtained after cooling was trit-
urated with Et2O, collected by suction filtration. The fil-
ter cake was recrystallized with 95% EtOH to yield 28m
(0.2 g, 14.6%) as a yellow solid: mp 263–264 ꢁC; 1H
NMR (DMCO): d 7.56 (m, 3H), 7.85 (d, 1H), 8.20 (d,
2H), 8.38 (s, 1H), 8.88 (d, 1H). EI-MS m/z 199 (M+)
(100%), 155.
266–268 ꢁC; H NMR (DMSO): d 2.31 (s, 3H), 3.82 (s,
3H), 6.15 (s, 1H), 7.17 (t, 1H), 7.22 (d, 1H), 7.56 (t,
1H), 7.67 (t, 1H), 7.92 (m, 4H), 8.02 (d, 2H), 8.10 (s,
1H), 8.18 (t, 2H); EI-MS m/z 514 (M+), 234 (100%);
HRMS (EI) m/z calcd C27H22N4O5S (M+) 514.1314,
found 514.1311.
4.4.5. 4-(4-Chlorophenylacetamido)-N-quinoxalin-2-yl
benzenesulfonamide (5). This compound was purified by
flash chromatography with EtOAc/petroleum ether (1:2,
1
4.4. General procedures for preparations of 4-substituted
carboxamido sulfanilamides 1–6, 16a–q, and 17a–d are
described as those for 1
v/v), yield 33.2%: mp 263–265 ꢁC; H NMR (DMSO): d
3.65 (s, 2H), 7.35 (m, 4H), 7.62 (t, 1H), 7.78 (m, 4H),
7.92 (d, 1H), 8.05 (d, 2H), 8.62 (br, 1H); EI-MS m/z
452 (M+), 387 (100%); HRMS (EI) m/z calcd
C22H17ClN4O3S (M+) 452.0711, found 452.0710.
4.4.1. 4-(2-(3-Bromophenyl)-quinoline-4-carboxamido)-N-
(3,4-dimethyl-isoxazol-5-yl)benzene sulfonamide (1). A sus-
pension of 3-bromophenyl-quinoline-4-carboxylic acid
(0.33 g, 1 mmol) in redistilled SOCl2 (6 mL) was refluxed
gently for 3 h. A clear solution resulted. The SOCl2 was
then evaporated under reduced pressure. The yellow solid
thus obtained was used in the next step without further
purification.
4.4.6. 4-(2,4-Dichlorobenzamido)-N-quinoxalin-2-yl benzene-
sulfonamide (6). THis compound was purified by flash chro-
matography with EtOAc/petroleum ether (1:2, v/v), yield
1
43.8%: mp 217–219 ꢁC; H NMR (DMSO): d 7.51 (dd,
1H), 7.68 (m, 3H), 7.78 (t, 1H), 7.89 (d, 1H), 7.99 (m,
3H), 8.21 (d, 2H), 8.72 (s, 1H); EI-MS m/z 472 (M+), 173
(100%); HRMS (EI) m/z calcd C21H14Cl2 N4O3S (M+)
472.0163, found 472.0164.
A mixture of yellow solid thus obtained, sulfisoxazole
(0.27 g, 1 mmol), and pyridine (5 mL) was stirred over-
night at 25 ꢁC, poured into H2O (30 mL), and extracted
with EtOAc. The combined organic layer was washed,
dried, filtered, and condensed. The residue was purified
by flash chromatography on silica gel, eluted with a mix-
ture of EtOAc/petroleum ether (1:4, v/v), to afford 1
4.4.7. 4-(2-(3-Fluorophenyl)-quinoline-4-carboxamido)-N-
(3,4-dimethyl-isoxazol-5-yl) benzenesulfonamide (16a).
This compound was purified by flash chromatography
with EtOAc/petroleum ether (1:2, v/v), yield 26.7%: mp
1
257–258 ꢁC; H NMR (DMSO): d 1.85 (s, 3H), 2.12 (s,
1
(120 mg, 20.8%) as a white solid: mp 254–256 ꢁC; H
3H), 7.40 (t, 1H), 7.67 (q, 1H), 7.73 (t, 1H), 7.84 (d, 2H),
7.92 (t, 1H), 8.05 (d, 2H), 8.25 (m, 4H), 8.47 (s, 1H); EI-
MS m/z 516 (M+), 222 (100%); HRMS (EI) m/z calcd
C27H21FN4O4S (M+) 516.1261, found 516.1268.
NMR (DMSO): d 1.71 (s, 3H), 2.10 (s, 3H), 7.57 (t, 1H),
7.75 (m, 2H), 7.84 (d, 2H), 7.90 (m, 1H), 8.03 (d, 2H),
8.22 (m, 2H), 8.40 (d, 1H), 8.48 (s, 1H), 8.58 (t, 1H); EI-
MS m/z 578 (M++1), 203 (100%); HRMS (EI) m/z calcd
C27H21BrN4O4S (M+) 576.0473, found 576.0467.
4.4.8. 4-(2-(3-Chlorophenyl)-quinoline-4-carboxamido)-N-
(3,4-dimethyl-isoxazol-5-yl) benzenesulfonamide (16b).
This compound was purified by flash chromatography
with EtOAc/petroleum ether (1:2, v/v), yield 30.5%: mp
4.4.2. 4-(2-(2-Methylphenyl)-quinoline-4-carboxamido)-N-
(4-methyl-pyrimidin-2-yl) benzenesulfonamide (2). This
compound was purified by flash chromatography with
1
237–238 ꢁC; H NMR (DMSO): d 1.70 (s, 3H), 2.10 (s,

J. Li et al. / Bioorg. Med. Chem. 14 (2006) 2209–2224
2221
3H), 7.62 (m, 2H), 7.72 (t, 1H), 7.86 (d, 2H), 7.89 (t, 1H),
8.04 (d, 2H), 8.22 (t, 2H), 8.35 (d, 1H), 8.45 (s, 1H), 8.48
(s, 1H); EI-MS m/z 532 (M+), 266 (100%); HRMS (EI) m/
z calcd C27H21ClN4O4S (M+) 532.0969, found 532.0972.
(100%); HRMS (EI) m/z calcd C31H24N4O4S (M+)
548.1509, found 548.1518.
4.4.15. 4-(2-(3-Bromophenyl)-6-fluoro-quinoline-4-carbox-
amido)-N-(3,4-dimethyl-isoxazol-5-yl) benzenesulfonamide
(16i). This compound was purified by flash chromatogra-
phy with EtOAc/petroleum ether (1:2, v/v), yield 34.9%:
4.4.9. 4-(2-(4-Bromophenyl)-quinoline-4-carboxamido)-N-
(3,4-dimethyl-isoxazol-5-yl) benzenesulfonamide (16c).
This compound was purified by flash chromatography
with EtOAc/petroleum ether (1:2, v/v), yield 35.2%: mp
1
mp 267–269 ꢁC; H NMR (DMSO): d 1.70 (s, 3H), 2.10
(s, 3H), 7.56 (t, 1H), 7.74 (d, 1H), 7.87 (m, 3H), 7.96 (d,
1H), 8.04 (d, 2H), 8.29 (q, 1H), 8.38 (d, 1H), 8.56 (m,
2H); EI-MS m/z 596 (M+), 221 (100%); HRMS (EI) m/z
calcd C27H20FBrN4O4S (M+) 594.0376, found 594.0373.
1
248–250 ꢁC; H NMR (DMSO): d 1.70 (s, 3H), 2.10 (s,
3H), 7.67 (t, 1H), 7.79 (d, 2H), 7.82 (d, 2H), 7.88 (t,
1H), 8.02 (d, 2H), 8.18 (d, 2H), 8.33 (d, 2H), 8.42 (s,
1H); EI-MS m/z 578 (M+), 203 (100%); HRMS (EI) m/
z calcd C27H21BrN4O4S (M+) 576.0431, found 576.0467.
4.4.16. 4-(2-(3-Bromophenyl)-quinoline-4-carboxamido)-
N-quinoxalin-2-yl benzenesulfonamide (16j). This com-
pound was purified by flash chromatography with
EtOAc/petroleum ether (1:2, v/v), yield 38.2%: mp
285–286 ꢁC; 1H NMR (DMSO): d 7.53 (t, 1H), 7.67
(m, 2H), 7.76 (m, 2H), 7.86 (m, 2H), 7.95 (d, 1H), 8.00
(d, 2H), 8.14 (d, 2H), 8.18 (d, 2H), 8.35 (d, 1H), 8.40
(s, 1H), 8.53 (t, 1H), 8.72 (br, 1H); EI-MS m/z 611
(M+), 203 (100%); HRMS (EI) m/z calcd
C30H20BrN5O3S (M+) 609.0452, found 609.0470.
4.4.10. 4-(2-(2-Bromophenyl)-quinoline-4-carboxamido)-
N-(3,4-dimethyl-isoxazol-5-yl) benzenesulfonamide (16d).
This compound was purified by flash chromatography
with EtOAc/petroleum ether (1:2, v/v), yield 40.3%: mp
1
243–244 ꢁC; H NMR (DMSO): d 1.74 (s, 3H), 2.15 (s,
3H), 7.47 (t, 1H), 7.57 (t, 1H), 7.75 (m, 2H), 7.81 (t, 3H),
7.90 (t, 1H), 8.00 (m, 3H), 8.17 (d, 1H), 8.22 (d, 1H); EI-
MS m/z 578 (M+), 203 (100%); HRMS (EI) m/z calcd
C27H21BrN4O4S (M+) 576.0464, found 576.0467.
4.4.17. 4-(2-(3-Bromophenyl)-pyridine-4-carboxamido)-N-
(3,4-dimethyl-isoxazol-5-yl) benzenesulfonamide (16k).
This compound was purified by flash chromatography
with EtOAc/petroleum ether (1:2, v/v), yield 38.7%: mp
4.4.11. 4-(2-Pyridin-4-yl-quinoline-4-carboxamido)-N-(3,4-
dimethyl-isoxazol-5-yl) benzenesulfonamide (16e). This
compound was purified by flash chromatography with
EtOAc/petroleum ether (1:2, v/v), yield 37.7%: mp 238–
1
240–242 ꢁC; H NMR (DMSO): d 1.70 (s, 3H), 2.10 (s,
1
239 ꢁC; H NMR (DMSO): d 2.04 (s, 3H), 2.35 (s, 3H),
3H), 7.54 (t, 1H), 7.71 (d, 1H), 7.82 (d, 2H), 7.86 (d, 1H),
8.03 (d, 2H), 8.21 (d, 1H), 8.38 (t, 1H), 8.46 (s, 1H), 8.90
(d, 1H); EI-MS m/z 528 (M+), 260 (100%); HRMS (EI)
m/z calcd C23H19BrN4O4S (M+) 526.0292, found 526.0311.
7.97 (m, 1H), 8.14 (m, 3H), 8.32 (d, 2H), 8.47 (d, 1H),
8.52 (dd, 2H), 8.59 (d, 1H), 8.77 (s, 1H), 9.01 (dd, 2H);
EI-MS m/z 499 (M+), 205 (100%); HRMS (EI) m/z calcd
C26H21N5O4S (M+) 499.1316, found 499.1314.
4.4.18. 4-(2-(4-Bromophenyl)-pyridine-4-carboxamido)-N-
(3,4-dimethyl-isoxazol-5-yl) benzenesulfonamide (16l). This
compound was purified by flash chromatography with
EtOAc/petroleum ether (1:2, v/v), yield 35.2%: mp 248–
4.4.12. 4-(2-Thiophen-2-yl-quinoline-4-carboxamido)-N-(3,4-
dimethyl-isoxazol-5-yl) benzenesulfonamide (16f). This com-
pound was purifiedby flash chromatography with EtOAc/pe-
troleum ether (1:2, v/v), yield 24.3%: mp 234–235 ꢁC; H
1
1
249 ꢁC; H NMR (DMSO): d 1.67 (s, 3H), 2.08 (s, 3H),
NMR (DMSO): d 1.70 (s, 3H), 2.10 (s, 3H), 7.25 (t, 1H),
7.64 (t, 1H), 7.76 (d, 1H), 7.82 (m, 3H), 8.11 (m, 5H), 8.34
(s, 1H); EI-MS m/z 504 (M+), 210 (100%); HRMS (EI) m/z
calcd C25H20N4O4S2 (M+) 504.0916, found 504.0926.
7.76 (d, 2H), 7.84 (m, 3H), 8.02 (d, 2H), 8.16 (d, 2H),
8.41 (s, 1H), 8.89 (d, 1H); EI-MS m/z 526 (M+), 126
(100%); HRMS (EI) m/z calcd C23H19BrN4O4S (M+)
526.0308, found 526.0310.
4.4.13. 4-(2-Cyclohexanyl-quinoline-4-carboxamido)-N-
(3,4-dimethyl-isoxazol-5-yl) benzenesulfonamide (16g).
This compound was purified by flash chromatography
with EtOAc/petroleum ether (1:2, v/v), yield 21.6%: mp
4.4.19. 4-(2-Phenyl-pyridine-4-carboxamido)-N-(3,4-dimeth-
yl-isoxazol-5-yl) benzenesulfonamide (16m). This compound
was purified by flash chromatography with EtOAc/petro-
leum ether (1:2, v/v), yield 48.2%: mp 219–220 ꢁC; H
NMR (DMSO): d 1.66 (s, 3H), 2.08 (s, 3H), 7.56 (m,
3H), 7.80 (m, 3H), 8.02 (d, 2H), 8.15 (d, 2H), 8.17 (s,
1H), 8.87 (d, 1H); EI-MS m/z 448 (M+), 57 (100%);
HRMS (EI) m/z calcd C23H20N4O4S (M+) 448.1214,
found 448.1206.
1
1
222–223 ꢁC; H NMR (DMSO): d 1.35 (m, 1H), 1.48
(m, 2H), 1.75 (m, 6H), 1.87 (m, 2H), 1.99 (m, 2H), 2.10
(s, 3H), 2.96 (m, 1H), 7.61 (t, 1H), 7.66 (s, 1H), 7.80 (m,
3H), 7.99 (d, 2H), 8.03 (d, 1H), 8.07 (d, 1H); EI-MS m/z
504 (M+), 210 (100%); HRMS (EI) m/z calcd
C27H28N4O4S (M+) 504.1822, found 504.1831.
4.4.20. 4-(2-(4-Chlorophenyl)-pyridine-4-carboxamido)-N-
(3,4-dimethyl-isoxazol-5-yl) benzenesulfonamide (16n).
This compound was purified by flash chromatography
with EtOAc/petroleum ether (1:2, v/v), yield 38.1%: mp
4.4.14. 4-(2-Naphthalen-2-yl-quinoline-4-carboxamido)-N-
(3,4-dimethyl-isoxazol-5-yl) benzenesulfonamide (16h).
This compound was purified by flash chromatography
with EtOAc/petroleum ether (1:3, v/v), yield 37.1%: mp
264–266 ꢁC; H NMR (DMSO): d 1.71 (s, 3H), 2.05 (s,
3H), 7.60 (m, 2H), 7.70 (t, 1H), 7.88 (m, 3H), 8.00 (m,
1H), 8.05 (d, 2H), 8.11 (d, 2H), 8.19 (d, 1H), 8.24 (d, 1H),
8.59 (m, 2H), 8.95 (s, 1H); EI-MS m/z 548 (M+), 254
1
244–245 ꢁC; H NMR (DMSO): d 1.66 (s, 3H), 2.08 (s,
1
3H), 7.62 (d, 2H), 7.82 (m, 3H), 8.02 (d, 2H), 8.22 (d,
2H), 8.41 (s, 1H), 8.87 (d, 1H); EI-MS m/z 482 (M+),
216 (100%); HRMS (EI) m/z calcd C23H19ClN4O4S
(M+) 482.0829, found 482.0816.

2222
J. Li et al. / Bioorg. Med. Chem. 14 (2006) 2209–2224
1
4.4.21. 4-(2-(4-Fluorophenyl)-pyridine-4-carboxamido)-N-
(3,4-dimethyl-isoxazol-5-yl) benzenesulfonamide (16o).
This compound was purified by flash chromatography
with EtOAc/petroleum ether (1:2, v/v), yield 55.6%: mp
leum ether (1:2, v/v), yield 49.3%: mp 193–194 ꢁC; H
NMR (DMSO): d 1.60 (s, 3H), 2.07 (s, 3H), 3.69 (s,
2H), 7.39 (q, 4H), 7.69 (d, 2H), 7.78 (d, 2H); EI-MS m/
z 419 (M+), 125 (100%); HRMS (EI) m/z calcd
C19H18ClN4O3S (M+) 419.0704, found 419.0706.
1
232–234 ꢁC; H NMR (DMSO): d 1.68 (s, 3H), 2.08 (s,
3H), 7.39 (t, 2H), 7.82 (m, 3H), 8.03 (d, 2H), 8.26 (m,
2H), 8.39 (s, 1H), 8.87 (d, 1H); EI-MS m/z 466 (M+), 200
(100%); HRMS (EI) m/z calcd C23H19FN4O4S (M+)
466.1107, found 466.1111.
4.4.28. 4,40-Methylenebisanthranilic acid (31a).31 To a
stirred mixture of anthranilic acid 30 (6.85, 50 mmol),
H2O (63 mL), and 36% HCl (13 mL) at 50 ꢁC was added
3% aqueous formaldehyde (18 mL). The resulting solu-
tion was heated at 70 ꢁC and stirred for 4 h. Upon neu-
tralization with ammonia, the precipitate was filtered,
washed successively with H2O and hot acetic acid, fol-
lowed by drying to afford 31a (4.8 g, 33.6%) as yellow
solid: mp 258–260 ꢁC.
4.4.22. 4-(2-(2-Methylphenyl)-pyridine-4-carboxamido)-N-
(3,4-dimethyl-isoxazol-5-yl) benzenesulfonamide (16p).
This compound was purified by flash chromatography
with EtOAc/petroleum ether (1:2, v/v), yield 25.1%: mp
1
202–203 ꢁC; H NMR (DMSO): d 1.66 (s, 3H), 2.08 (s,
3H), 2.35 (s, 3H), 7.36 (m, 3H), 7.48 (d, 1H), 7.79 (d,
2H), 7.84 (d, 1H), 8.01 (m, 3H), 8.87 (d, 1H); EI-MS
m/z 462 (M+), 167 (100%); HRMS (EI) m/z calcd
C24H22N4O4S (M+) 462.1345, found 462.1361.
4.4.29. 4,40-Methylenebisanthranilic acid dimethyl ester
(31b). A mixture of 31a (1 g, 3.5 mmol) in methanol
(30 mL) saturated with HCl was refluxed for 5 h, poured
into crushed ice, and pH adjusted to 9 with 2 N NaOH.
The precipitate was collected, dried, and purified by
flash chromatography on silica gel, eluted with a mix-
ture of EtOAc/petroleum ether (1:10, v/v), to afford
31b (0.30 g, 27.5%) as a white solid: mp 144–145 ꢁC;
1H NMR (CDCl3): d 3.80 (s, 2H), 3.88 (s, 6H), 5.60
(br, 2H), 6.62 (d, 2H), 7.08 (dd, 2H), 7.65 (d, 2H).
4.4.23. 4-(2-(2-Methylphenyl)-pyridine-4-carboxamido)-N-
(4-methyl-pyrimidin-2-yl) benzenesulfonamide (16q). This
compound was purified by flash chromatography with
EtOAc/petroleum ether (1:2, v/v), yield 28.9%: mp
242–243 ꢁC; 1H NMR (DMSO): d 2.31 (s, 3H), 2.35
(s, 3H), 6.90 (d, 2H), 7.35 (m, 3H), 7.47 (d, 1H),
7.82 (d, 1H), 8.01 (q, 4H), 8.32 (m, 1H), 8.87 (d,
1H); EI-MS m/z 459 (M+), 286 (100%); HRMS (EI)
m/z calcd C24H21N5O3S (M+) 459.1369, found
459.1365.
4.4.30. 6,60-Bis-(3,5-dimethoxybenzamido)-3,30-methanediyl-
di-benzoic acid (15). A mixture of 31a (0.2 g, 0.7 mmol), 3,5-
dimethoxybenzoyl chloride (0.31 g, 1.54 mmol), and pyridine
(5 mL) was stirred at 0 ꢁC for 5 h. The resulting mixture was
poured into H2O, and the precipitate was collected, dried, and
purified by flash chromatography on silica gel, eluted with a
mixture of CH3OH/CHCl3 (1:15, v/v), to afford 15 (70 mg,
4.4.24. 4-(2,4-Dichlorophenylacetamido)-N-quinoxalin-2-yl
benzenesulfonamide (17a). This compound was purified
by flash chromatography with EtOAc/petroleum ether
(1:2, v/v), yield 35.2%: mp 235–236 ꢁC; 1H NMR
(DMSO): d 3.83 (s, 2H), 7.38 (m, 1H), 7.42 (d, 1H),
7.55 (d, 1H), 7.62 (t, 1H), 7.79 (m, 4H), 7.92 (d, 1H),
8.04 (d, 2H), 8.62 (br, 1H); EI-MS m/z 486 (M+), 236
(100%); HRMS (EI) m/z calcd C22H16Cl2N4O3S (M+)
486.0309, found 486.0321.
1
16.3%) as a white solid: mp > 300 ꢁC; H NMR (DMSO):
d 3.80 (s, 12H), 3.90 (s, 2H), 6.68 (s, 2H), 7.14 (s, 4H), 7.30
(d, 2H), 7.92 (s, 2H), 8.58 (d, 2H); MALDI-MS m/z 637
[M+Na]+; HRMS (MALDI) m/z calcd C33H30N2O10Na
[M+Na]+ 637.17927, found 637.18080.
4.4.31. 6,60-Bis-benzamido-3,30-methanediyl-di-benzoic acid
(18b). In the same manner as described for 15, 18b was pre-
pared from benzoyl chloride, yield 20.0%: mp 288–289 ꢁC;
1H NMR (CDCl3): d 4.24 (s, 2H), 7.58 (m, 4H), 7.61 (m,
2H), 7.67 (s, 4H), 8.09 (s, 2H), 8.32 (m, 4H); EI-MS m/z
494 (M+), 105 (100%). HRMS (EI) m/z calcd C29H22N2O6
(M+) 494.1487, found 494.1479.
4.4.25. 4-Benzamido-N-quinoxalin-2-yl benzenesulfona-
mide (17b). This compound was purified by flash chroma-
tography with EtOAc/petroleum ether (1:2, v/v), yield
45.4%: mp 258–259 ꢁC; H NMR (DMSO): d 7.63 (m,
4H), 7.75 (t, 1H), 7.82 (m, 1H), 7.93 (m, 3H), 8.03 (d,
2H), 8.13 (d, 2H), 8.65 (br, 1H); EI-MS m/z 404 (M+),
105 (100%); HRMS (EI) m/z calcd C21H16N4O3S (M+)
404.0943, found 404.0943.
1
4.4.32. 6,60-Bis-(4-methoxybenzamido)-3,30-methanediyl-
di-benzoic acid (18d). In the same manner as described
for 15, 18d was prepared from 4-methoxybenzoyl chlo-
ride; yield 20.0%: mp 280–282 ꢁC;1H NMR (DMSO):
d 3.86 (s, 6H), 4.07 (s, 2H), 7.05 (d, 2H), 7.13 (d, 2H),
7.30 (d, 1H), 7.62 (d, 1H), 7.79 (d, 1H), 7.99 (m, 4H),
8.13 (d, 2H), 8.62 (d, 1H); EI-MS m/z 554 (M+), 135
(100%). HRMS (EI) m/z calcd C31H26N2O8 (M+)
554.1678, found 554.1690.
4.4.26.
4-(3-Phenyl-propionamido)-N-quinoxalin-2-yl
benzenesulfonamide (17c). This compound was purified
by flash chromatography with EtOAc/petroleum ether
(1:2, v/v), yield 39.3%: mp 213–214 ꢁC; 1H NMR
(DMSO): d 2.65 (t, 2H), 2.90 (t, 2H), 7.16 (t, 1H), 7.26
(m, 4H), 7.62 (t, 1H), 7.79 (m, 4H), 7.93 (d, 1H), 8.03
(d, 2H), 8.63 (br, 1H); EI-MS m/z 432 (M+), 367
(100%); HRMS (EI) m/z calcd C23H20N4O3S (M+)
432.1261, found 432.1256.
4.4.33. 6,60-Bis-(3,5-dimethoxybenzamido)-3,30-methanediyl-
di-benzoic acid dimethyl ester (18a). A mixture of 31b
(50 mg, 0.15 mmol), 3,5-dimethoxybenzoyl chloride (68 mg,
0.34 mmol), and pyridine (5 mL) was stirred at 0 ꢁC for 5 h.
The resulting mixture was poured into H2O, and the precipi-
4.4.27. 4-(4-Chlorophenylacetamido)-N-(3,4-dimethyl-iso-
xazol-5-yl) benzenesulfonamide (17d). This compound
was purified by flash chromatography with EtOAc/petro-

J. Li et al. / Bioorg. Med. Chem. 14 (2006) 2209–2224
2223
tate was collected, dried, and purified by flash chromatogra-
phy on silica gel, eluted with a mixture of EtOAc/petroleum
ether (1:4, v/v), to afford 18a (23 mg, 22.5%) as a white solid:
mp 194–196 ꢁC; 1H NMR (DMSO): d 3.85 (s, 12H), 3.88 (s,
6H), 4.05 (s, 2H), 6.78 (s, 2H), 7.07 (s, 4H), 7.59 (d, 2H), 7.88
(s, 2H), 8.44(d, 2H);MALDI-MSm/z 665 [M+Na]+. HRMS
(MALDI) m/z calcd C35H34N2O10Na [M+Na]+ 665.21057,
found 665.21140.
tography on silica gel, eluted with a mixture of EtOAc/pe-
troleum ether (1:4, v/v), to afford 18h (20 mg, 41.6%) as a
white solid: mp 173–175 ꢁC; 1H NMR (DMSO): d 3.64 (s,
2H), 3.77 (s, 3H), 4.43 (t, 4H), 6.65 (q, 2H), 7.17 (q, 2H),
7.33 (m, 10H), 7.62 (q, 2H), 7.95 (t, 2H); MALDI-MS
m/z 503 [M+Na]+. HRMS (MALDI) m/z calcd
C30H28N2O4Na [M+Na]+ 503.19413, found 503.19610.
4.4.39. 6,60-Bis-benzylamino-3,30-methanediyl-di-benzoic
acid (18i). In the same manner as described for 18h but
room temperature was changed into refluxing tempera-
ture, 18i was prepared as a white solid, yield 21.2%:
mp 179–181 ꢁC; 1H NMR (DMSO): d 3.64 (s, 2H),
4.41 (s, 4H), 6.61 (d, 2H), 7.14 (d, 2H), 7.33 (m, 10H),
7.60 (s, 2H); MALDI-MS m/z 489 [M+Na]+. HRMS
(MALDI) m/z calcd C29H26N2O4Na [M+Na]+
489.17848, found 489.17930.
4.4.34. 6,60-Bis-benzamido-3,30-methanediyl-di-benzoic acid
dimethyl ester (18c). In the same manner as described for
18a, 18c was prepared from benzoyl chloride, yield 20.3%:
1
mp 214–216 ꢁC; H NMR (DMSO): d 3.88 (s, 6H), 4.06
(s, 2H), 7.65 (m, 8H), 7.90 (s, 2H), 7.97 (d, 4H), 8.50 (d,
2H); EI-MS m/z 522 (M+), 105 (100%); HRMS (EI) m/z
calcd C31H26N2O6 (M+) 522.1781, found 522.1791.
4.4.35. 6,60-Bis-(4-methoxybenzamido)-3,30-methanediyl-
di-benzoic acid dimethyl ester (18e). In the same manner as
described for 18a, 18e was prepared from 4-meth-
4.4.40. 5-Bromomethyl-2-nitro-benzoic acid methyl ester
(34).32 A mixture of 5-methyl-2-nitro-benzoic acid 32
(1 g, 0.55 mmol) in methanol (30 mL) saturated with
HCl was refluxed for 5 h, and most of solvent was evap-
orated, filtered, washed, and dried to afford 33 (0.91 g,
84.5%) as a white solid: mp 76–78 ꢁC.
1
oxybenzoyl chloride, yield 28.0%: mp 234–236 ꢁC; H
NMR (DMSO): d 3.84 (s, 6H), 3.88 (s, 6H), 4.04 (s,
2H), 7.14 (d, 4H), 7.58 (d, 2H), 7.93 (m, 6H), 8.51 (d,
2H); MALDI-MS m/z 605 [M+Na]+. HRMS (MALDI)
m/z calcd C33H30N2O8Na [M+Na]+ 605.18944, found
605.18960.
A mixture of 33 (0.9 g, 4.7 mmol), NBS (1.25 g,
7.0 mmol), benzoyl peroxide (0.05 g), and CH3Cl
(30 mL) was stirred for 24 h. The resulting solution was
washed successively with H2O, aqueous NaCO3, brine,
dried, filtered, and concentrated. The residue was purified
by flash chromatography on silica gel, eluted with a mix-
ture of EtOAc/petroleum ether (1:10, v/v), to afford 34
(0.43 g, 33.8%) as a white solid: mp 58–61 ꢁC.
4.4.36. 6,60-Bis-(4-chlorobenzamido)-3,30-methanediyl-di-
benzoic acid dimethyl ester (18f). In the same manner as
described for 18a, 18f was prepared from 4-chloro-
benzoyl chloride, yield 26.0%: mp 241–242 ꢁC; 1H
NMR (CDCl3): d 3.95 (s, 6H), 3.99 (s, 2H), 7.46 (d,
2H), 7.50 (d, 4H), 7.90 (s, 2H), 7.99 (d, 4H), 8.85 (d,
2H); EI-MS m/z 590 (M+), 139 (100%). HRMS (EI) m/
z calcd C31H24Cl2N2O6 (M+) 590.1030, found 590.1011.
4.4.41. 6,60-Bis-nitro-3,30-ethanediyl-di-benzoic acid
dimethyl ester (35). To a stirred mixture of 33 (0.5 g
2.5 mmol), n-BuLi (2.5 M in hexane) (2 mL, 5 mmol),
and redistilled THF (10 mL) was added dropwise 34
(0.35 g, 1.3 mmol) in redistilled THF (5 mL) at ꢀ78 ꢁC
under N2. The resulting mixture was stirred for 3 h at
ꢀ78 ꢁC and then warmed to room temperature overnight.
The solution was poured into aqueous NH4Cl, extracted
with EtOAc. The combined organic phases were dried, fil-
tered, and concentrated. The residue was purified by flash
chromatography on silica gel, eluted with a mixture of
EtOAc/petroleum ether (1:4, v/v), to afford 35 (0.11 g,
4.4.37. 6,60-Bis-benzylamino-3,30-methanediyl-di-benzoic
acid dimethyl ester (18g). To a stirred mixture of 31b
(100 mg, 0.32 mmol), NaCO3 (80 mg, 0.75 mmol), and
NMP (3 mL) was added benzyl bromide (0.09 mL,
0.75 mmol). The mixture was then heated at 90 ꢁC for
10 h. The reaction mixture after cooling was transferred
to a separatory funnel using a 1:1 mixture of H2O and
EtOAc. The product was extracted with EtOAc. The
combined organic phases were washed, dried, filtered,
and concentrated. The residue was purified by flash
chromatography on silica gel, eluted with a mixture of
EtOAc/petroleum ether (1:20, v/v), to afford 18g
(43 mg, 27.4%) as a white solid: mp 86–88 ꢁC; 1H
NMR (DMSO): d 3.64 (s, 2H), 3.76 (s, 6H), 4.42 (d,
4H), 6.63 (d, 2H), 7.15 (d, 2H), 7.26 (m, 2H), 7.32 (m,
8H), 7.60 (d, 2H), 7.96 (t, 2H); MALDI-MS m/z 517
[M+Na]+. HRMS (MALDI) m/z calcd C31H30N2O4Na
[M+Na]+ 517.20978, found 517.21090.
1
21.8%) as a yellow solid: H NMR (CDCl3): d 3.07 (s,
4H), 3.93 (s, 6H), 7.37 (d, 2H), 7.50 (d, 2H), 7.89 (d,
2H); EI-MS m/z 388 (M+), 194 (100%).
4.4.42. 6,60-Bis-amino-3,30-ethanediyl-di-benzoic acid
dimethyl ester (36). A mixture of 35 (110 mg, 0.28 mmol),
10% palladium on charcoal (20 mg), and THF (20 mL)
was stirred at 25 ꢁC for 8 h in an atmosphere of hydrogen.
The catalyst was filtered and the filtrate was concentrated
to dryness. The residue was washed with a few methanol
to afford 36 (90 mg) as a white solid: mp 216–218 ꢁC.
4.4.38. 6,60-Bis-benzylamino-3,30-methanediyl-di-benzoic
acid monomethyl ester (18h). A mixture of 18g (50 mg,
0.1 mmol) and LiOH (20 mg, 0.8 mmol) in THF/Metha-
nol/H2O (3:1:1, v/v/v, 10 mL) was stirred at room temper-
ature for 12 h. The resulting solution was acidified to pH 2
using 1 N HCl. The product was extracted into the organ-
ic phase using EtOAc (3 · 25 mL), dried, filtered, and
concentrated. The residue was purified by flash chroma-
4.4.43. 6,60-Bis-(3,5-dimethoxybenzamido)-3,30-ethanediyl-
di-benzoic acid dimethyl ester (18k). In the same manner as
described for 18a, 18k was prepared from 36, yield 58.0%:
mp 215–216 ꢁC; 1H NMR (CDCl3): d 2.93 (s, 4H), 3.88 (s,
12H), 3.94 (s, 6H), 6.64 (s, 2H), 7.18 (s, 4H), 7.44 (d, 2H),

2224
J. Li et al. / Bioorg. Med. Chem. 14 (2006) 2209–2224
7.87 (s, 2H), 8.83 (d, 2H); MALDI-MS m/z 679 [M+Na]+.
HRMS (MALDI) m/z calcd C36H36N2O10Na [M+Na]+
679.22622, found 679.22800.
8. (a) Luo, C.; Luo, H. B., et al. Biochem. Biophys. Res.
Commun. 2004, 321, 557–565; (b) Chen, Z. N.; Mi, L.; Xu,
J.; Yu, J., et al. J. Infect. Dis. 2005, 191, 755–760.
9. (a) Friedman, J.; Weissman, I. Cell 1991, 66, 799–806; (b)
Liu, J.; Farmer, J. D.; Lane, W. S.; Friedman, J.;
Weissman, I.; Schreiber, S. L. Cell 1991, 66, 807–815.
10. (a) Barbara, E. B.; Georg, H.; David, F.; Steven, J. B.
Curr. Opin. Immunol. 1993, 5, 763–773; (b) Zou, X. J.;
Matsumura, Y. J.; John, P.; Ryo, S.; Alberto, M.; Jack,
M.; Stanley, C. J. Transpl. Immunol. 1995, 3, 151–161.
11. (a) Dumont, F. J. Exp. Opin. Ther. Patents 2001, 11, 377–
404(b) Anderson, P. S., Kenyon, G. L., Marshall, G. R.,
Eds.; Perspectives in Drug Discovery and Design; ESCOM
Science Publishers: Leiden, The Netherlands, 1994; Vol. 2,
pp 3–248.
12. Siekierka, J. J.; Hung, S. H.; Poe, M.; Lin, C. S.; Sigal, N.
H. Nature 1989, 341, 755–757.
13. Clane, R. Y.; Lim, S.; Samaan, A.; Collier, D. S. T., et al.
Lancet 1989, 334, 227.
14. Rechard, S.; Jorg, K.; Luisa, M. C.; Charles, D. P.;
Francesco, S. J. Am. Chem. Soc. 2003, 125, 3849–3859.
15. Cui, M.; Huang, X.; Luo, X.; James, M. B.; Ji, R.; Chen,
K.; Shen, J.; Jiang, H. J. Med. Chem. 2002, 45, 5249–5259.
16. Su, Q.; Weber, L.; Le Hir, M.; Zenke, G.; Ryffel, B. Ren.
Physiol. Biochem. 1995, 18, 128–139.
4.4.44. 6,60-Bis-(3,5-dimethoxybenzamido)-3,30-ethanediyl-
di-benzoic acid (18l). A mixture of 18k (32 mg, 0.05 mmol)
and LiOH (12 mg, 0.5 mmol) in THF/methanol/H2O
(3:1:1, v/v/v, 20 mL) was stirred at room temperature for
12 h. The resulting solution was acidified to pH 2 using
1 N HCl. The product was extracted, dried, filtered, con-
centrated, and purified by flash chromatography using
EtOAc/petroleum ether (2:1, v/v), to afford 18l (18 mg,
58.1%) as a pale red solid: mp > 300 ꢁC; 1H NMR
(DMSO): d 2.91 (s, 4H), 3.86 (s, 12H), 6.74 (s, 2H), 7.07
(s, 4H), 7.54 (d, 2H), 7.90 (s, 2H), 8.59 (d, 2H); ESI-MS
m/z 627 [M-H]+. HRMS (ESI) m/z calcd C34H31N2O10
[MꢀH]+ 627.1979, found 627.1989.
4.4.45. Bis-(4-(3,5-dimethoxybenzamido)-phenyl)-meth-
ane (18j). In the same manner as described for 18a, 18j
was prepared from 4,40-methanediyl-bis-aniline 37, yield
1
56.0%; mp 185–187 ꢁC; H NMR (CDCl3): d 3.83 (s,
12H), 3.96 (s, 2H), 6.61 (t, 2H), 6.97 (d, 4H), 7.19 (d,
4H), 7.56 (d, 4H); ESI-MS m/z 525 [MꢀH]+. HRMS
(ESI) m/z calcd C31H29N2O6 [MꢀH]+ 525.2026, found
525.2078.
17. Bennet, W. M.; Porter, G. A. Am. J. Med. 1988, 85, 131–
133.
18. Miller, L. W. Am. J. Transplant. 2002, 2, 807–818.
19. (a) Kuntz, I. D.; Meng, E. C.; Shoichet, B. K. Acc. Chem.
Res. 1994, 27, 117–123; (b) Wang, J. L.; Liu, D. X.;
Zhang, Z. J.; Shan, S.; Han, X. B.; Srinivasula, S. M.;
Croce, C. M.; Alnemri, E. S.; Huang, Z. W. Proc. Natl.
Acad. Sci. U.S.A. 2000, 97, 7124–7129; (c) Liu, H.; Li, Y.;
Song, M.; Tan, X.; Cheng, F.; Zheng, S.; Shen, J.; Luo, X.;
Ji, R.; Yue, J.; Hu, G.; Jiang, H.; Chen, K. Chem. Biol.
2003, 10, 1103–1113.
20. Zheng, S.; Luo, X.; Chen, G.; Zhu, W.; Jiang, H. J. Chem.
Inf. Model. 2005, 45, 856–862.
21. (a) Ewing, T.; Kuntz, I. D. J. Comput. Chem. 1997, 18,
1175–1189; (b) Kuntz, I. D. Science 1992, 257, 1078–1082.
22. Weiner, S. J.; Kollman, P. A.; Nguyen, D. T.; Case, D. A.
J. Comput. Chem. 1986, 7, 230–252.
23. Gasteiger, J.; Marsili, M. Tetrahedron 1980, 36, 3219–3228.
24. (a) Muegge, I.; Rarey, M. Small molecule docking and
scoring. In Reviews in Computational Chemistry; Lipko-
witz, K. B., Boyd, D. B., Eds.; Wiley: New York, 2001; pp
1–60; (b) Charifson, P. S.; Corkery, J. J.; Murcko, M. A.;
Walters, W. P. J. Med. Chem. 1999, 42, 5100–5109.
25. Sybyl [molecular modeling package], version 6.8; Tripos
Associates: St. Louis, MO, 2000.
Acknowledgments
We gratefully acknowledge financial support from the
State Key Program of Basic Research of China (Grant
2002CB512802), the National Natural Science Founda-
tion of China (Grants 20372069, 29725203, 20472094,
and 20102007), the Basic Research Project for Talent Re-
search Group from the Shanghai Science and Technology
Commission, the Key Project from the Shanghai Science
and Technology Commission (Grant 02DJ14006), the
Key Project for New Drug Research from CAS, the Qi
Ming Xing Foundation of Shanghai Ministry of Science
and Technology (Grant 03QD14065), and the 863
Hi-Tech Programm (Grants 2002AA233061, 2002AA
104270, 2002AA233011, and 2003AA235030).
References and notes
26. Rarey, M.; Kramer, B.; Lengauer, T.; Klebe, G. J. Mol.
Biol. 1996, 261, 470–489.
1. Handschumacher, R. E.; Harding, M. W.; Rice, J.;
Drugge, R. J.; Speicher, D. W. Science 1984, 226, 544–547.
2. (a) Fisher, G.; Whittman-Liebold, B.; Lang, K.; Kiefhaber,
T.; Schmid, F. X. Nature 1989, 337, 476–478; (b) Takahashi,
N.; Hayano, T.; Suzuki, M. Nature 1989, 337, 473–475.
3. Braaren, D.; Luban, J. EMBO J. 2001, 20, 1300–1309.
4. (a) Galat, A. Curr. Top. Med. Chem. 2003, 3, 1315–1347;
(b) Dornan, J.; Taylor, P.; Walkinshaw, M. D. Curr. Top.
Med. Chem. 2003, 3, 1392–1409.
27. Robert, E. L.; Philip, S. B.; Marion, T. C.; John, F. C.,
et al. J. Am. Chem. Soc. 1946, 68, 1813–1831.
28. Pfister, K.; Sullivan, A. P.; John, W.; Tishler, M. J. Am.
Chem. Soc. 1951, 7, 4955–4957.
29. Bredereck, H.; Gompper, R. Chem. Ber. 1954, 87, 700–706.
30. Usui, Y.; Hara, Y.; Shimamoto, N.; Yurugi, S.; Masada,
T. Heterocycles 1975, 3, 155–161.
31. Reddy, L. M.; Reddy, P. P.; Reddy, P. S. N. Indian J.
Chem. 2002, 41, 2405–2409.
5. Luban, J.; Bossolt, K. L.; Franke, E. K.; Kalpana, G. V.;
Goff, S. P. Cell 1993, 73, 1067–1078.
32. Zrihen, M.; Labia, R.; Wakselman, M. Eur. J. Med.
Chem. Chim. Ther. 1983, 18, 307–314.
6. (a) Capano, M.; Virji, S.; Crompton, M. Biochem. J. 2002,
363, 29–36; (b) Yoshimoto, T.; Siesjo, B. K. Brain Res.
1999, 832, 283–291; (c) Crompton, M. J. Physiol. 2000,
529, 11–21.
33. Kofron, J. L.; Kuzmic, P.; Kishore, V.; Colon-Bonilla, E.;
Rich, D. H. Biochemistry 1991, 30, 6127–6134.
34. Sargent, J. M.; Taylor, C. G. Br. J. Cancer 1989, 60, 206–
210.
7. Dawson, T. M.; Steiner, J. P.; Lyons, W. E.; Fotuhi, M.;
Blue, M.; Snyder, S. H. Neuroscience 1994, 62, 569–580.
35. Wallace, A. C.; Laskowski, R. A.; Thornton, J. M. Protein
Eng. 1995, 8, 127–134.