Synthesis, preliminary biological evaluation and molecular modeling of some new heterocyclic inhibitors of TACE

Article abstract of DOI:10.1016/j.ejmech.2011.09.018

Central heteroaryl ring analogues belonging to a series of potent hydroxamate TACE inhibitors were synthesized. The TACE inhibitory activities of these compounds were evaluated by in vitro WBA and in silico molecular modeling studies using crystal structure of human TACE. Compound 14 showed very good in vitro inhibition, supported by the in silico docking studies.

Full text of DOI:10.1016/j.ejmech.2011.09.018

European Journal of Medicinal Chemistry 46 (2011) 5549e5555
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, preliminary biological evaluation and molecular modeling of some new
heterocyclic inhibitors of TACE
Prabal Senguptaa, Chetan S. Puria, Hemant A. Chokshia, Chetana K. Shetha, Ajay S. Midhaa,
,
a
b
b
Trinadha Rao Chitturia , Rajamannar Thennati , Prashant R. Murumkar , Mange Ram Yadav
*
a Organic Synthesis Division, Sun Pharma Advanced Research Company Ltd., Tandalja, Vadodara 390012, Gujarat, India
b Pharmacy Department, Faculty of Technology and Engineering, Kalabhavan, The M. S. University of Baroda, Vadodara 390001, Gujarat, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Central heteroaryl ring analogues belonging to a series of potent hydroxamate TACE inhibitors were
synthesized. The TACE inhibitory activities of these compounds were evaluated by in vitro WBA and in
silico molecular modeling studies using crystal structure of human TACE. Compound 14 showed very
good in vitro inhibition, supported by the in silico docking studies.
Received 5 March 2011
Received in revised form
16 August 2011
Accepted 13 September 2011
Available online 21 September 2011
Ó 2011 Elsevier Masson SAS. All rights reserved.
Keywords:
TACE inhibitor
Hydroxamates
TNF-
a
Molecular modeling
1. Introduction
(TACE) [13] and inhibition of TACE blocks the release of TNF-a [14].
TACE is a member of the ADAM (a disintegrin and a metalloprotease
domain) subfamily of the metzincin superfamily that also includes
the astacins, serralysins and matrix metalloproteinases (MMPs).
Since broad-spectrum MMP inhibitors have been found to cause
side effects in oncology clinical trials [15], it is desirable to develop
selective small molecule TACE inhibitors for the long-term treat-
Tumor necrosis factor-a (TNF-a) isa pleitropic, pro-inflammatory
cytokine produced by macrophages, monocytes, neutrophils, T-
cells, mast cells, epithelial cells, osteoblasts and dendritic cells [1,2].
Overexpression of TNF-a is responsible for a number of pathological
conditions such as Crohn’s disease, ulcerative colitis [3], diabetes [4],
multiple sclerosis [5] and atherosclerosis [6]. In addition to this,
ment of TNF-a mediated disorders.
there is compelling data tosupport the fact that TNF-
role in the origin and progression of rheumatoid arthritis (RA) and
other immune-mediated disorders [7e11].
a
plays a pivotal
As evident from literature [16], various motifs have been used
for developing small molecule TACE inhibitors which include
succinate based compounds, macrocyclic inhibitors, sulfonamide
The anti-TNF-
a
biological therapeutics etanercept, infliximab
inhibitors, g-lactam inhibitors, b-benzamido inhibitors and
and adalimumab have demonstrated clinical success in inflamma-
tory and autoimmune diseases and have validated the hypothesis of
benzothiadiazepine inhibitors. Majority of the potential new
chemical entities failed in the phase I clinical trials due to their
poor pharmacokinetic profile or lack of efficacy [17]. Apratastat
(TMI-005), developed by Wyeth as a broad spectrum TACE
inhibitor [18], and BMS-566394, a potent and selective orally
bioavailable inhibitor of TACE, developed by Bristol-Myers
Squibb (BMS) [19] had advanced to phase II clinical trials, but
failed to progress further due to either lack of efficacy or asso-
ciated side effects [20,21]. Thus, there is a requirement to
develop orally bioavailable and efficacious small molecule
inhibitors of TACE lacking side effects. Herein, we present
a report towards development of new potent small-molecule
hydroxamate inhibitors of TACE.
TNF-
a
modulation as a drug discovery paradigm [12].
exists in two forms, the membrane-bound proform
TNF-
a
comprising 233 amino acids with a molecular mass of 26 kDa and
the soluble form of 17 kDa comprising 157 nonglycosylated amino
acids. It has been shown recently that the shredding of the bio-
logically active TNF-
mediated by a metalloproteinase called TNF-
a
from its membrane-anchored proform is
converting enzyme
a
* Corresponding author. Tel.: þ91 265 6615500; fax: þ91 265 2354897.
E-mail addresses: ctrao@sunpharma.com, drctrao@yahoo.co.in (T.R. Chitturi).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.09.018

5550
P. Sengupta et al. / European Journal of Medicinal Chemistry 46 (2011) 5549e5555
2. Chemistry
carbocyclic/heterocyclic
b
ꢀamino esters 8aef using EDC [N-(3-
dimethylaminopropyl)-N’-ethylcarbodiimide] and HOBt. The
resulting amido esters 9aef were treated with an excess of
hydroxylamine to give the hydroxamates 10e15.
Researchers from BMS have reported
a series of orally
bioavailable
566394 demonstrated exceptional potency for inhibition of
pTACE as well as in the suppression of LPS induced TNF- in human
b-benzamidohydroxamates, amongst which BMS-
Similarly the central furan and thiophene ring containing
a
b-benzamidohydroxamic acids 16e23 were prepared from the
whole blood [22]. The PCT Application WO 03/24899 which
discloses BMS-566394 generically claims analogues wherein the
central ring system is specifically a phenyl. Although a prior art
cited in this PCT Application includes claims for 5/6-membered
monocyclic heteroaryl central ring, neither their synthesis nor
their biological activity is reported therein [23]. While the
hydroxamic acid part of these molecules attached to the central
phenyl ring is responsible for interaction with the zinc binding site
of TACE, the group in the para position imparts selectivity as well as
cellular potency. It appears that the central phenyl ring remains
dormant and does not contribute to any binding interaction with
the active site of TACE [19,24]. Though considered to be a benign
component of the molecule for the purpose of molecular modifi-
cations of BMS-566394, we intended to replace the central phenyl
ring hitherto with 5/6-membered monocyclic heteroaryl rings with
the expectation to improve the overall pharmacokinetic profile of
the molecule without compromising the binding affinity to the
enzyme.
carbocyclic/heterocyclic
The synthesis of the precursor benzimidazolylmethylheteroaryl
acids for the proposed compounds is outlined in Scheme 2. Methyl
b
ꢀamino esters 8aef and the acids 3cef.
6-methylnicotinate
1 was photochemically chlorinated with
N-chlorosuccinimide (NCS) to give ester 2. Alkylation of 2 with
2-substituted benzimidazoles in presence of cesium carbonate in
N,N-dimethylformamide provided the benzimidazole substituted
esters which were saponified with sodium hydroxide in
tetrahydrofuran-methanol to give the corresponding acids 3aeb.
The related furan and thiophene containing benzimidazolylme-
thylheteroaryl acids 3cef were analogously prepared.
The b-keto esters 5aef were synthesized from the ketones 4aef
using lithium hexamethyldisilazane (LHMDS), methyl cyanoformate
and HMPA in THFat ꢀ78 ꢁC. Condensation of
b-ketoesters 5aef with
(R)-a-methylbenzylamine in presence of ytterbium triflate afforded
the corresponding enamine derivatives 6aef. Following a literature
procedure [25], the enamines 6aef were reduced with sodium tri-
acetoxyborohydride in acetonitrile and acetic acid at 0 ꢁC to obtain
7aef, which upon hydrogenolysis using palladium hydroxide cata-
lyst provided the desired carbocyclic/heterocyclic
8aef (Scheme 3).
Hence we prepared compounds wherein the central phenyl ring
was replaced with monocyclic heteroaryl rings, viz. pyridine,
bꢀamino esters
thiophene and furan. These
synthesized in an analogous manner as previously described [24],
involving amidation of substituted benzimidazolylmethyl hetero-
b
ꢀbenzamidohydroxamates were
aryl acids with carbocyclic/heterocyclic
lowed by conversion of the ester to the corresponding hydroxamic
acids.
b
ꢀamino acid esters, fol-
3. Biological activity
The compounds prepared were tested in vitro by measuring
cellular suppression of LPS-induced TNF- using human whole
Scheme 1 outlines the general synthesis of the central pyridyl
a
ring containing b-benzamidohydroxamic acids 10e15. The benzi-
midazolylmethylpyridyl acids 3aeb were coupled with the
blood assay (WBA) [26], and the inhibitory activities of these were
compared with BMS-566394 (Table 1).
N
N
Y
Y
N
N
O
O
O
NH2.HCl
i
ii
O
N
N
O
HN
O
HN
(n)
z
HO
O
N
H
(n)
(n)
z
z
8a-8f
9a-9f
10, Y = CH , Z = O, n = 1
3
11, Y = CF , Z = O, n = 1
3
12, Y = CH , Z = CH , n = 1
3
2
13, Y = CF , Z = CH , n = 1
3
2
Y
14, Y = CH , Z = CH , n = 0
3
2
X
N
O
N
15, Y = CF , Z = CH , n = 0
3
2
HN
O
HO
N
H
(n)
z
16, X = S, Y = CH , Z = O, n = 1
3
17, X = S, Y = CF Z= O, n = 1
3,
18, X = S, Y = CH , Z = CH , n = 0
3
2
19, X = S, Y = CF , Z = CH , n = 0
3
2
20, X = O, Y = CH , Z =CH , n = 0
3
2
21, X = O, Y = CF , Z = CH , n = 0
3
2
22, X = S, Y = CH , Z = CH , n = 1
3
2
23, X = O, Y = CH , Z = CH , n = 1
3
2
Scheme 1. Reagents and conditions: i) 3ae3b, EDC, HOBt, DMF; ii) NH2OH, NaOCH3, CH3OH.

P. Sengupta et al. / European Journal of Medicinal Chemistry 46 (2011) 5549e5555
5551
N
N
Y
O
O
i
O
O
Cl
OH
N
N
2
N
N
Y
ii
O
3a, Y = CH
1
N
H
3
3b, Y = CF
Y = CH , CF
3
3
3
N
iii
Y
O
N
O
O
i
X
X
X
O
O
OH
3c, X = O, Y = CH
Cl
2c, X = O
2d, X = S
1c, X = O
1d, X = S
3
3d, X = O, Y = CF
3
3
3e, X = S, Y = CH
3f, X = S, Y = CF
3
Scheme 2. Reagents and conditions: i) N-chlorosuccinimide, CCl4; ii) Cs2CO3, DMF; iii) NaOH, THF-CH3OH.
4. Molecular modeling studies
systems, irrespective of changes that have been made in other parts
of the molecule.
The synthesized compounds were energy minimized and
docked in the active site of human TACE obtained from the Protein
Data Bank (USA). The docking score (G score) for the potent
compounds along with the experimentally determined IC50 values
(from WBA assay) of the potent compounds is summarized in
Table 2.
An alteration in the presumably dormant central ring system of
the molecule leading to significant changes in the inhibitory
activity was quite unexpected. Hence we undertook molecular
modeling studies on the potent compounds 10e15 selected from
Table 1 with human TACE enzyme so as to gather insight into their
binding affinity.
Since compound 14 shows the highest binding score (G
score ꢀ13.80) and is the most active compound in the series, its
binding interactions within the binding site of the enzyme were
extensively studied (Fig. 1).
5. Results and discussion
Tetrahydropyran containing b-benzamidohydroxamates 10 and
The docking study for compound 14 suggested that the P10
group (i.e. 2-methylbenzimidazol-1-ylmethyl moiety) is oriented
towards the larger S10 pocket of TACE and interacts with the active
site residues (Ile438, Thr347 and Gly349). Oxygen atom of
hydroxamic acid moiety of compound 14 binds with the catalytic
zinc in the active site. Carbonyl group of hydroxamate interacts
with His415 whereas the NH group interacts with Gly349, Leu350
and Glu406 active site residues (nitrogen of the hydroxamate
11 having central pyridyl ring proved to be better inhibitors than
the corresponding cyclohexane analogues 12 and 13. Similarly,
substituting methyl group at 2-position of benzimidazole ring with
trifluoromethyl group afforded higher inhibitory activity. The
inhibitory potency increased substantially with cyclopentyl ring
analogues 14 and 15, either with methyl or trifluoromethyl groups
at 2-position of the benzimidazole moiety.
The TACE inhibitory activity decreased remarkably for
compounds 16e23 where the central six-membered pyridyl ring
was replaced with five-membered thiophenyl and furyl ring
interacts with carbonyl group of Glu406 and Gly349 and
a-
hydrogen of Leu350). Oxygen of the carbonyl group adjacent to
H3C
O
z
O
O
ii
O
NH
i
O
O
(n)
(n)
z
(n)
z
6a-6f
5a-5f
4a-4f
Z = O, CH , n = 0, 1
2
H3C
O
NH2.HCl
iv
O
NH
z
iii
O
O
(n)
z
(n)
8a-8f
7a-7f
Scheme 3. Reagents and conditions: i) Methyl cyanoformate, LHMDS; ii) (R)-(a)-methylbenzylamine, Ytterbium (III) trifluoromethanesulfonate, benzene; iii) NaBH(OAc)3,
acetonitrile-acetic acid; iv) H2/Pd(OH)2-C, HCl, methanol.

5552
P. Sengupta et al. / European Journal of Medicinal Chemistry 46 (2011) 5549e5555
Table 1
Table 2
In vitro activity of the compounds for TNF-
a
inhibition.
In vitro TNF-
a
inhibition and docking scores for active compounds.
IC50 M)*
Compound
% inhibition of TNF-
a
*
Compound
(
m
Glide score
(G-score)
1
m
M
0.3 mM
BMS-566394
0.130
0.080
0.160
0.126
0.280
0.070
0.074
ꢀ11.03
ꢀ12.54
ꢀ12.14
ꢀ12.41
ꢀ11.49
ꢀ13.80
ꢀ11.23
BMS-566394
77.78
80.09
83.07
78.10
64.23
84.90
81.20
58.50
3.98
66.26
72.45
66.40
66.48
40.61
80.53
77.98
24.90
0.00
10
11
12
13
14
15
10
11
12
13
14
15
16
17
18
19
20
21
22
23
*LPS induced TNF-
a inhibition in WBA.
0.00
55.80
3.26
3.50
0.00
0.00
29.20
9.57
7.06
0.00
ꢂ Between central pyridine ring and carbon of the carbonyl group
present on Glu406 (2.754 Å)
ꢂ Between the eNH of CONHOH and the oxygen of the carbonyl
group of Gly349 (2.496 Å) and
6.40
13.20
ꢂ Between oxygen of the carbonyl group attached to the pyridine
ring and the NH group of Gly349 (2.182 Å).
*LPS induced TNF-
a inhibition in WBA.
Briefly, the docking data suggests that the hydroxamic acid
moiety of compound 14 binds with the zinc ion in the active site.
Nitrogen present on the central pyridine ring interacts with the a-
the central pyridine ring of compound 14 interacts with the
hydrogen of the NH group present on Gly349 and also with
His415.
hydrogen of Val402 (2.54 Å) through hydrogen bond, which affords
an extra stabilization to the compound 14. Such stabilizing inter-
action however does not exist for compounds having central phenyl
ring or five-membered heterocyclic rings (furan and thiophene). In
fact, docking studies reveal that compounds having central five-
membered rings (compounds 16e23) adopt a pose in which the
zinc binding group (hydroxamate) moves away from the catalytic
zinc. This could probably be the reason for poor TACE inhibition of
the compounds having five-membered central rings.
The nitrogen atom of the central pyridine ring (compound 14)
makes hydrogen bonding with the Val402 of active site residue
(bond distance 2.54 Å). The
central pyridine ring and
a
a
-hydrogen of Val402 interacts with the
-nitrogen of Val402 interacts with
methyl group present on benzimidazole ring. Val434 interacts with
the methylene group present between pyridine ring and benz-
imidazole ring. There is also a favorable hydrogen bonding between
hydrogen of central pyridine and carbon of carbonyl group present
on Ile438.
The most interesting outcome of the docking study is that
central pyridine ring containing compound 14 (highest ranked
compound in the present series) makes crucial hydrogen
6. Conclusions
bonding with the a-hydrogen of Val402. Apart from this, three
other vital interactions have been observed as mentioned
below:
In summary, a new series of central heteroaryl containing
hydroxamates as inhibitors of TACE have been synthesized. It was
envisaged to make changes in the unexplored central phenyl ring of
Fig. 1. Docking of compound 14 (shown in violet color) in the active site of TACE. Interactions are shown in green lines, zinc is shown in red color (for sake of clarity only important
amino acids are given). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

P. Sengupta et al. / European Journal of Medicinal Chemistry 46 (2011) 5549e5555
5553
BMS-566394 primarily with monocyclic five and six-membered
7.3. General procedure for the synthesis of amines 8aef
aromatic heterocyclic rings viz. furan, thiophene and pyridine.
The tetrahydropyranyl ring of the lead compound BMS-566394 was
also replaced with five and six-membered carbocyclic rings. The
synthesized compounds were evaluated by in vitro WBA for their
TACE inhibitory activity. Some of the compounds (10, 14 and 15)
showed promising TACE inhibitory activity.
A tetrahydrofuran solution of LHMDS (5.2 mmol) was added
dropwise to a solution of the cyclic ketone (5.0 mmol) in tetrahy-
drofuran (20 ml) at ꢀ78 ꢁC and the resulting solution was stirred
at ꢀ20 ꢁC for 1 h. The mixture was again cooled to ꢀ78 ꢁC and
methyl cyanoformate (6.0 mmol) was added dropwise. After 10
min, the reaction was quenched with aqueous ammonium chloride
and extracted with ether. The organic layer was washed with brine,
dried (sodium sulfate) and concentrated. Column chromatography
on silica gel (ether-hexane, 1:4 to 3:2) yielded the ester as an oil.
The ester obtained from previous step (3.4 mmol) was dissolved
In order to assess the role played by central heteroaryl ring of
the synthesized compounds for binding to the active site, molecular
modeling studies were carried out using crystal structure of human
TACE obtained from protein data bank. Compound 14 having the
highest activity was found to have an extra binding to the active site
in the form of a hydrogen bond between the pyridine nitrogen and
Val402. Compounds 16e23 possessing central five-membered
heterocyclic rings were shown to have a “different pose” which
disallowed interaction between the zinc-binding hydroxamate
group and the catalytic zinc ion site in these modeling studies. This
study throws new light on the role played by the central aromatic
ring in benzamidohydroxamate series of compounds belonging to
BMS-566394 which was hitherto unexplored. The in silico crystal
structure of these new heterocyclic inhibitors bound to TACE
enhances our understanding of the binding interactions with the
TACE active site which can facilitate the structure-based design of
inhibitors with improved potency for the treatment of disorders
in benzene (20 ml) and treated with (R)-a-methylbenzylamine
(2.3 mmol) and ytterbium (III) trifluoromethanesulfonate
(0.34 mmol). The mixture was heated to reflux under DeaneStark
conditions for 2 h, concentrated and purified by column chroma-
tography on silica gel (ethyl acetate-hexane, 1:4) to yield the
desired enamine as a white solid.
The enamine obtained (1.3 mmol) was taken in acetonitrile-
acetic acid (1:1, 10 ml) and treated with sodium triacetoxybor-
ohydride (1.3 mmol) and stirred for 2 h at 0 ꢁC. Following
concentration in vacuo, the residue was dissolved in ether, washed
with saturated sodium bicarbonate until the aqueous phase was
basic, dried (magnesium sulfate) and concentrated to yield an oil.
The intermediate (7 mmol) in methanol (10 ml) was treated
with 10% palladium hydroxide on carbon (0.6 g) and aqueous 1N
hydrochloric acid (10 ml) and hydrogenated for 72 h at r.t. The
catalyst was removed by filtration; concentration of filtrate
provided the desired amine as a hydrochloric acid salt.
mediated by TNF-a.
7. Experimental
7.1. Chemistry
7.4. General procedure for the synthesis of hydroxamic acids 10e23
Melting points were determined in open cavities and are
uncorrected. 1H and 13C NMR spectra were recorded on a Bruker
AMX 400 spectrometer operating at 400 MHz for 1H and 100 MHz
for 13C in DMSO-d6. The mass spectrum was obtained on a JEOL SX-
102 mass spectrometer. Thin layer chromatography was performed
on silica gel G (Merck). Column chromatography was performed
with silica gel (100e200 mesh).
The amine (8aef) (0.7 mmol) and the acid (3aef) (0.7 mmol) in
N,N-dimethylformamide (2 ml) was treated with EDC (1.12 mmol)
and 1-hydroxybenzotriazole (1.12 mmol) and stirred for 12 h at r.t.
The reaction was quenched with saturated aqueous sodium bicar-
bonate and extracted with ethyl acetate, washed with water, dried
and concentrated to furnish the desired ester.
Hydroxylamine hydrochloride (75 mmol) was suspended in dry
methanol (10 ml) and a solution of potassium hydroxide (77 mmol)
in methanol (10 ml) was added. The reaction mixture was stirred at
r.t. for 30 min. Ester derivative (9aef, 0.7 mmol) was added to the
above reaction mixture, stirred at r.t for 3 h and quenched with ice-
water (15 ml). The mixture was cooled to 0 ꢁC, conc. hydrochloric
acid was added dropwise until pH ¼ 6 and extracted with chloro-
form. The organic extract was dried over anhydrous sodium sulfate
and concentrated to afford an oil which upon trituration with ether
afforded the desired products.
7.2. General procedure for the synthesis of acids 3aef
A solution of ester (33 mmol), N-chlorosuccinimide (33 mmol)
and benzoyl peroxide (0.82 mmol) in carbon tetrachloride
(50 ml) was refluxed for 12 h and then filtered. The filtrate was
washed with saturated solution of sodium metabisulfite, dried
with sodium sulfate and concentrated under vacuum to afford
the crude product. Flash chromatography (hexane/ethyl acetate,
8:2) provided the desired compound as colorless crystalline
solid.
A solution of this chloromethyl ester (1.8 mmol) in N,N-dime-
thylformamide (4 ml) was treated with the 2-substituted benz-
imidazoles (1.8 mmol) and cesium carbonate (2.8 mmol). The
mixture was stirred for 3 h at r.t. and partitioned with water and
ethyl acetate (20 ml). The aqueous layer was further extracted with
ethyl acetate (20 ml) and the combined organic layers washed with
brine (20 ml), dried (sodium sulfate), filtered and concentrated.
Flash chromatography with ethyl acetate provided the desired
product as colored oil.
The compound obtained from the previous reaction was taken
in tetrahydrofuran-methanol (1:1, 10 ml) and to this was added
sodium hydroxide (10N, 0.4 ml) and stirred for 1 h at r.t. The
reaction was concentrated under reduced pressure and the
residue was acidified (pH 5e6) with dil. hydrochloric acid.
The white solid was filtered, washed with water and dried on
vacuum.
7.4.1. N-[(3R, 4R)-3-(hydroxycarbamoyl)tetrahydro-2H-pyran-4-
yl]-6-[(2-methyl-1H-benzimidazol-1-yl)methyl]nicotinamide (10)
Rf:0.3(CH2Cl2-methanol, 9:1); 1H NMR (
d, DMSO-d6):10.51 (s,1H),
8.90 (s,1H), 8.76 (s,1H), 8.48 (d,1H, J ¼ 7.0 Hz), 8.15 (d,1H, J ¼ 7.5 Hz),
7.61e7.57 (m,1H), 7.51e7.47 (m,1H), 7.39 (d,1H, J ¼ 8.0 Hz), 7.20e7.16
(m, 2H), 5.67 (s, 2H), 4.33 (m, 1H), 4.07e3.98 (m, 1H), 3.83e3.72 (m,
1H),3.68e3.55(m, 3H), 2.21e2.10(m,1H),1.72e1.64(m,1H);13CNMR
(d, DMSO-d6): 170.23, 166.44, 159.37, 153.89, 149.53, 142.56, 137.31,
136.07, 130.64, 123.24, 123.02, 122.08, 118.78, 110.91, 67.37, 66.05,
48.97, 47.64, 42.64, 29.71, 13.67; ESI-MS (m/z): 410.1 (M þ H)þ.
7.4.2. N-[(3R, 4R)-3-(hydroxycarbamoyl)tetrahydro-2H-pyran-4-
yl]-6-[(2-trifluoromethyl-1H-benzimidazol-1-yl)methyl]
nicotinamide (11)
Rf: 0.3 (CH2Cl2-methanol, 9:1); 1H NMR (
d, DMSO-d6): 10.50 (s,
1H), 8.81 (s, 1H), 8.75 (s, 1H), 8.49 (d, 1H, J ¼ 7.3 Hz), 8.18 (d, 1H,

5554
P. Sengupta et al. / European Journal of Medicinal Chemistry 46 (2011) 5549e5555
J ¼ 6.8 Hz), 7.92 (d, 1H, J ¼ 7.7 Hz), 7.75 (d, 1H, J ¼ 7.8 Hz), 7.50e7.42
(m, 3H), 5.93 (s, 2H), 4.33 (m, 1H), 4.04e3.99 (m, 1H), 3.83e3.81 (m,
1H), 3.64e3.61 (m, 1H), 3.56e3.55 (m, 1H), 2.72e2.68 (m, 1H),
134.72, 128.40, 126.81, 121.73, 121.53, 118.34, 109.99, 66.69, 64.85,
41.67, 28.81,13.55; ESI-MS (m/z): 415.1 (M þ H)þ.
2.10e2.09 (m, 1H), 1.68e1.67 (m, 1H); 13C NMR (
d
, DMSO-d6):
7.4.8. N-[(3R, 4R)-3-(hydroxycarbamoyl)tetrahydro-2H-pyran-4-
yl]-5-[(2-trifluoromethyl-1H-benzimidazol-1-yl)methyl]thiophene-
2-carboxamide (17)
171.39, 167.69, 159.15, 150.02, 142.16, 140.18, 138.03, 131.44, 127.39,
125.69, 122.16, 122.09, 122.05, 113.08, 68.23, 67.14, 50.60, 48.61,
43.43, 30.11; ESI-MS (m/z): 464.43 (M þ H)þ.
Rf: 0.3 (CH2Cl2-methanol, 9:1); 1H NMR (
d, DMSO-d6): 10.47 (s,
1H), 8.75 (s, 1H), 8.29 (d, 1H, J ¼ 7.0 Hz), 7.93e7.89 (m, 1H),
7.74e7.73 (m, 1H), 7.58e7.54 (m, 1H), 7.49e7.45 (m, 1H), 7.19e7.18
(m, 1H), 5.95 (s, 2H), 4.19 (m, 1H), 4.01e3.97 (m, 1H), 3.87e3.84 (m,
1H), 3.53e3.49 (m, 1H), 2.66e2.61 (m, 1H), 2.25e2.22 (m, 1H),
7.4.3. N-[(1S, 2R)-3-(hydroxycarbamoyl)cyclohexyl]-6-[(2-methyl-
1H-benzimidazol-1-yl)methyl]nicotinamide (12)
Rf: 0.3 (CH2Cl2-methanol, 9:1); 1H NMR (
d, DMSO-d6): 10.51 (s,
1H), 8.90 (s, 1H), 8.76 (s, 1H), 8.48 (d, 1H, J ¼ 7.0 Hz), 8.15 (d, 1H,
J ¼ 7.5 Hz), 7.61e7.57 (m, 1H), 7.51e7.47 (m, 1H), 7.39 (d, 1H,
J ¼ 8.0 Hz), 7.20e7.16 (m, 2H), 5.67 (s, 2H), 4.33 (m, 1H), 4.07e3.98
(m, 1H), 3.83e3.72 (m, 1H), 3.68e3.55 (m, 3H), 2.21e2.10 (m, 1H),
1.61e1.59 (m, 1H); 13C NMR (
d, DMSO-d6): 168.25, 160.16, 142.66,
140.48, 139.76, 139.07, 135.06, 128.30, 127.54, 125.75, 124.00, 121.04,
112.12, 66.69, 64.86, 43.36, 28.80; ESI-MS (m/z): 469.0 (M þ H)þ.
1.72e1.64 (m, 1H); 13C NMR (
d
, DMSO-d6): 170.06, 164.38, 158.26,
7.4.9. N-[(1S, 2R)-3-(hydroxycarbamoyl)cyclopentyl]-5-[(2-
methyl-1H-benzimidazol-1-yl)methyl]thiophene-2-carboxamide
(18)
152.29, 148.59, 142.37, 136.31, 135.31, 129.72, 121.51, 121.28, 120.96,
118.20, 109.90, 48.22, 47.87, 41.23, 28.97, 24.91, 22.90, 21.71, 13.70;
ESI-MS (m/z): 408.15 (M þ H)þ.
Rf: 0.1 (CH2Cl2-methanol, 9:1); 1H NMR (
d, DMSO-d6): 10.46 (s,
1H), 8.69 (s,1H), 8.15 (d,1H, J ¼ 6.9 Hz), 7.63e7.58 (m, 3H), 7.26e7.19
(m, 2H), 7.14e7.13 (m, 1H), 5.71 (s, 2H), 4.31 (d, 1H, J ¼ 7.0 Hz),
2.71e2.67 (m,1H), 2.62 (s, 3H),1.90e1.84 (m, 5H),1.54e1.52 (m,1H);
7.4.4. N-[(1S, 2R)-3-(hydroxycarbamoyl)cyclohexyl]-6-[(2-
trifluoromethyl-1H-benzimidazol-1-yl)methyl]nicotinamide (13)
Rf: 0.3 (CH2Cl2-methanol, 9:1); 1H NMR (
d, DMSO-d6): 10.51 (s,
13C NMR (
d, DMSO-d6): 169.71, 160.20, 151.41, 144.36, 142.31, 139.48,
1H), 8.90 (s, 1H), 8.76 (s, 1H), 8.48 (d, 1H, J ¼ 7.0 Hz), 8.15 (d, 1H,
J ¼ 7.5 Hz), 7.61e7.57 (m, 1H), 7.51e7.47 (m, 1H), 7.39 (d, 1H,
J ¼ 8.0 Hz), 7.20e7.16 (m, 2H), 5.67 (s, 2H), 4.33 (m, 1H), 4.07e3.98
(m, 1H), 3.83e3.72 (m, 1H), 368e3.55 (m, 3H), 2.21e2.10 (m, 1H),
134.73, 128.03, 126.75, 123.73, 121.52, 118.33, 110.00, 52.74, 43.54,
41.66, 31.55, 28.04, 22.36, 13.55; ESI-MS (m/z): 399.10 (M þ H)þ.
7.4.10. N-[(1S, 2R)-3-(hydroxycarbamoyl)cyclopentyl]-5-[(2-
trifluoromethyl-1H-benzimidazol-1-yl)methyl]thiophene-2-
carboxamide (19)
1.72e1.64 (m, 1H); 13C NMR (
d, DMSO-d6): 170.04, 164.30, 156.99,
148.43,140.45,140.16,139.86,136.35,135.95,129.88,125.43,123.64,
120.81, 120.66, 120.12, 117.97, 112.06, 64.91, 48.82, 48.19, 41.27,
29.00, 24.82, 22.93, 21.55, 15.16; ESI-MS (m/z): 462.09 (M þ H)þ.
Rf: 0.1 (CH2Cl2-methanol, 9:1); 1H NMR (
d, DMSO-d6): 10.45 (s,
1H), 8.68 (s, 1H), 8.19 (d, 1H, J ¼ 7.3 Hz), 7.93e7.90 (m, 2H),
7.65e7.64 (m, 1H), 7.58e7.54 (m, 1H), 7.49e7.45 (m, 1H), 7.17e7.16
(m, 1H), 5.94 (s, 2H), 4.34e4.27 (m, 1H), 2.72e2.67 (m, 1H),
7.4.5. N-[(1S, 2R)-2-(hydroxycarbamoyl)cyclopentyl]-6-[(2-
methyl-1H-benzimidazol-1-yl)methyl]nicotinamide (14)
1.92e1.79 (m, 5H), 1.56e1.52 (m, 1H); 13C NMR (
d, DMSO-d6):
Rf: 0.3 (CH2Cl2-methanol, 9:1); 1H NMR (
d, DMSO-d6): 10.48 (s,
169.69, 160.10, 142.35, 140.47, 140.00, 135.03, 127.90, 127.46, 125.72,
123.97, 121.01, 112.10, 52.75, 43.56, 43.32, 31.52, 28.02, 22.35; ESI-
MS (m/z): 453.04 (M þ H)þ.
1H), 8.85 (s, 1H), 8.69 (s, 1H), 8.34 (d, 1H, J ¼ 7.7 Hz), 8.11 (dd, 1H,
J1 ¼ 8.1 Hz, J2 ¼ 2.0 Hz), 7.59e7.57 (m, 1H), 7.50e7.48 (m, 1H), 7.36
(d, 1H, J ¼ 8.1 Hz), 7.19e7.17 (m, 2H), 5.65 (s, 2H), 4.45e4.40 (m, 1H),
2.74 (dd, 1H, J1 ¼ 15.3 Hz, J2 ¼ 7.7 Hz), 2.62 (s, 3H), 2.00e1.93 (m,
7.4.11. N-[(1S, 2R)-3-(hydroxycarbamoyl)cyclopentyl]-5-[(2-
1H), 1.86e1.78 (m, 4H), 1.56e1.55 (m, 1H); 13C NMR (
d, DMSO-d6):
methyl-1H-benzimidazol-1-yl)methyl]furan-2-carboxamide (20)
169.50, 164.28, 158.28, 152.29, 148.46, 142.37, 136.16, 135.31, 129.59,
121.53, 121.29, 120.95, 118.21, 109.91, 52.60, 47.87, 43.90, 31.57,
27.74, 22.56, 13.71; ESI-MS (m/z): 394.13 (M þ H)þ.
Rf: 0.5 (CH2Cl2-methanol, 9:1); 1H NMR (
d, DMSO-d6): 10.64 (s,
1H), 8.91 (s, 1H), 7.95 (d, 1H, J ¼ 6.9 Hz), 7.70 (d, 1H, J ¼ 7.7 Hz), 7.57
(d, 1H, J ¼ 7.7 Hz), 7.28e7.18 (m, 2H), 7.07 (d, 1H, J ¼ 3.3 Hz), 6.66 (d,
1H, J ¼ 3.2 Hz), 5.56 (s, 2H), 4.31e4.24 (m, 1H), 2.74 (s, 3H),
7.4.6. N-[(1S, 2R)-2-(hydroxycarbamoyl)cyclopentyl]-6-[(2-
2.72e2.68 (m, 1H), 2.00e1.70 (m, 6H); 13C NMR (
d, DMSO-d6):
trifluoromethyl-1H-benzimidazol-1-yl)methyl]nicotinamide (15)
170.02, 156.74, 151.93, 151.82, 147.54, 142.16, 134.82, 121.78, 121.54,
118.23, 114.10,110.63, 110.13, 52.23, 43.14, 32.52, 28.33, 22.28,13.55;
ESI-MS (m/z): 383.13 (M þ H)þ.
Rf: 0.3 (CH2Cl2-methanol, 9:1); 1H NMR (
d, DMSO-d6): 10.47 (s,
1H), 8.76 (s, 1H), 8.68 (s, 1H), 8.35 (d, 1H, J ¼ 7.8 Hz), 8.14 (dd, 1H,
J1 ¼ 8.0 Hz, J2 ¼ 1.8 Hz), 7.91 (d, 1H, J ¼ 7.7 Hz), 7.75 (d, 1H,
J ¼ 7.9 Hz), 7.50e7.42 (m, 3H), 5.92 (s, 2H), 4.47e4.40 (m, 1H), 2.73
(dd, 1H, J1 ¼ 15.2 Hz, J2 ¼ 7.7 Hz), 1.98e1.93 (m, 1H), 1.89e1.78 (m,
7.4.12. N-[(1S, 2R)-3-(hydroxycarbamoyl)cyclopentyl]-5-[(2-
trifluoromethyl-1H-benzimidazol-1-yl)methyl]furan-2-
carboxamide (21)
4H), 1.55e1.51 (m, 1H); 13C NMR (
d, DMSO-d6): 169.45, 164.21,
156.99, 148.28, 140.44, 140.15, 139.85, 136.71, 135.93, 129.80, 125.44,
123.64, 120.81, 120.64, 120.11, 117.96, 112.35, 52.60, 48.81, 43.94,
31.55, 27.69, 22.53; ESI-MS (m/z): 448.07 (M þ H)þ.
Rf:0.3(CH2Cl2-methanol, 9:1); 1HNMR(
d, DMSO-d6):10.61(s,1H),
8.84(s,1H), 8.04 (d,1H, J ¼ 8.2 Hz), 7.90 (d, 2H, J ¼ 7.6 Hz), 7.60 (m,1H),
7.47 (m,1H), 7.09 (d,1H, J ¼ 2.8 Hz), 6.66 (d,1H, J ¼ 2.5 Hz), 5.79(s, 2H),
4.31e4.28 (m, 1H), 2.72e2.67 (m, 1H), 1.92e1.76 (m, 5H), 1.60e1.50
7.4.7. N-[(3R, 4R)-3-(hydroxycarbamoyl)tetrahydro-2H-pyran-4-
yl]-5-[(2-methyl-1H-benzimidazol-1-yl)methyl]thiophene-2-
carboxamide (16)
(m,1H); 13C NMR (
d, DMSO-d6): 169.88,156.63,150.40,147.73,140.32,
135.22, 125.83, 123.92, 120.87, 114.10, 112.32, 110.23, 52.26, 43.19,
41.13, 32.42, 28.16, 22.18; ESI-MS (m/z): 437.07 (M þ H)þ.
Rf: 0.14 (CH2Cl2-methanol, 9:1); 1H NMR (
d, DMSO-d6): 10.46 (s,
1H), 8.76 (s,1H), 8.25 (d,1H, J ¼ 7.0 Hz), 7.73e7.58 (m, 2H), 7.26e7.19
(m, 2H), 7.16e7.15 (m,1H), 5.72 (s, 2H), 4.19e4.18 (m,1H), 4.01e3.97
(m, 1H), 3.87e3.84 (m, 1H), 3.63e3.59 (m, 1H), 3.53e3.49 (m, 1H),
2.66e2.64 (m,1H), 2.62 (s, 3H), 2.25e2.23 (m,1H),1.61e1.58 (m,1H);
7.4.13. N-[(1S, 2R)-3-(hydroxycarbamoyl)cyclohexyl]-5-[(2-
methyl-1H-benzimidazol-1-yl)methyl]thiophene-2-carboxamide
(22)
Rf: 0.1 (CH2Cl2-methanol, 9:1); 1H NMR (
d, DMSO-d6): 10.43 (s,
13C NMR (
d, DMSO-d6): 168.26,160.23,151.41,144.34,142.32,139.20,
1H), 8.65 (s, 1H), 7.98e7.96 (m, 1H), 7.85e7.72 (m, 1H), 7.63e7.58

P. Sengupta et al. / European Journal of Medicinal Chemistry 46 (2011) 5549e5555
5555
(m, 2H), 7.23 (d,1H, J ¼ 7.5 Hz), 7.14 (m,1H), 5.71 (s, 2H), 4.02 (s,1H),
2.62 (s, 3H), 2.38e2.23 (m, 1H), 2.13e2.04 (m, 1H), 1.96e1.88 (m,
2H), 1.68e1.53 (m, 2H), 1.49e1.37 (m, 2H), 1.29e1.14 (m, 1H); 13C
docking. Formal charge of þ2 was assigned to the zinc metal.
Docking was performed using GLIDE software according to re-
ported protocol [26,28].
NMR (d, DMSO-d6): 170.13, 160.19, 151.40, 144.13, 142.32, 139.53,
134.71, 128.21, 126.74, 121.71, 121.50, 118.32, 109.99, 48.66, 41.67,
40.77, 28.59, 25.73, 22.43, 22.22, 13.54; ESI-MS (m/z): 413.09
(M þ H)þ.
Acknowledgments
This study was funded by Sun Pharma Advanced Research
Company Limited (SPARC Ltd). The authors thank Ms. Dipali Desai
of the Systems Biology Division, SPARC Ltd., for assistance in
enzymatic assays and Mr. S. P. Sahoo of Analytical Development
Division, SPARC Ltd., for assistance in analytical services.
7.4.14. N-[(1S, 2R)-3-(hydroxycarbamoyl)cyclohexyl]-5-[(2-
methyl-1H-benzimidazol-1-yl)methyl]furan-2-carboxamide (23)
Rf:0.3 (CH2Cl2-methanol,9:1);1H NMR(
d, DMSO-d6):10.57 (s,1H),
8.83 (s,1H), 8.04 (d,1H, J ¼ 8.2 Hz), 7.70 (d, 2H, J ¼ 7.3 Hz), 7.57 (d,1H,
J ¼ 7.6 Hz), 7.27e7.18 (m, 2H), 7.10 (d, 1H, J ¼ 3.1 Hz), 6.67 (d, 1H,
J ¼ 3.0 Hz), 5.58 (s, 2H), 4.00 (m, 1H), 2.73 (s, 3H), 2.52 (m, 1H),
2.12e2.11 (m,1H),1.87e1.85 (m,1H),1.60e1.57 (m, 3H),1.49e1.38 (m,
Appendix. Supplementary data
3H); 13C NMR (
d, DMSO-d6): 170.21, 156.61, 151.78, 151.73, 147.47,
Supplementary data related to this article can be found online at
doi:10.1016/j.ejmech.2011.09.018.
142.14, 134.80, 121.68, 121.42, 118.17, 114.00, 110.43, 109.96, 47.83,
40.92, 28.76, 25.76, 22.59, 21.73,13.41; ESI-MS (m/z): 397.12 (M þ H)þ.
References
7.5. Biological screening
[1] K.J. Tracey, A. Cerami, Annu. Rev. Med. 45 (1994) 491e503.
[2] A.E. Goldfield, J.M. Strominger, C. Doyle, J. Exp. Med. 174 (1991) 73e81.
[3] R.C. Newton, C.P. Decicco, J. Med. Chem. 42 (1999) 2295e2314.
[4] G. Hotamisligil, P. Arner, J. Caro, R. Atkinson, B. Spiegelman, J. Clin. Invest. 95
(1995) 2409e2415.
[5] K. Selmaj, C. Raine, B. Cannella, C. Brosnon, J. Clin. Invest. 87 (1991) 247e254.
[6] H. Rus, F. Niculescu, R. Vlaicu, Atherosclerosis 89 (1991) 247e254.
[7] M. Feldmann, F.M. Brennan, R.N. Maini, Ann. Rev. Immunol. 14 (1996) 397e440.
[8] D.R. Bertolini, G.E. Nedwin, T.S. Bringman, D.D. Smith, G.R. Mundy, Nature 319
(1986) 516e518.
Blood was drawn from normal donors into tubes containing 143
USP units of heparin/10 ml. The heparinized human blood (225
was plated directly into ml sterile polypropylene tubes.
Compounds were diluted in DMSO/serum-free medium and added
to the blood samples so the final concentrations of the compounds
were 50, 10, 5, 1, 0.5, 0.1 and 0.01 mM. The final concentration of
DMSO did not exceed 0.5%. Compounds were preincubated for
15 min before the addition of 100 ng/ml LPS. Plates were incubated
for 5 h in an atmosphere of 5% CO2 in air. At the end of 5 h, 750 ml of
serum-free medium was added to each tube and the samples were
spun at 1200 rpm for 10 min. The supernatant was collected off the
top and assayed for TNF-
The ability of the compounds to inhibit TNF-
compared to DMSO-treated cultures was given by the IC50 value.
ml)
1
[9] J. Sakalatvala, Nature 319 (1986) 547e549.
[10] K.A. Papadakis, S.R. Targan, Annu. Rev. Med. 51 (2000) 289e298.
[11] D. Tracey, L. Klareskog, E.H. Sasso, J.G. Salfeld, P.P. Tak, Pharmacol. Ther. 117
(2008) 244e279.
[12] (a) L.W. Moreland, S.W. Baumgartner, M.H. Schiff, E.A. Tindall,
R.M. Fleischmann, A.L. Weaver, R.E. Ettlinger, S. Cohen, W.J. Koopman,
K. Mohler, M.B. Widmer, C.M. Blosch, N. Engl. J. Med. 337 (1997) 141e147;
(b) P.E. Lipsky, D.M.F.M.E. van der Heijde, W. St. Clair, D.E. Furst,
F.C. Breedveld, J.R. Kalden, J.S. Smolen, M. Weisman, P. Emery, M. Feldman,
G.R. Harrisman, R.N. Maini, N. Engl. J. Med. 343 (2000) 1594e1602;
(c) K.P. Machold, J.S. Smolen, Exp. Opin. Biol. Ther. 3 (2003) 351e360.
[13] A. Cameron, InPharma 1123 (1998) 9e10.
[14] R. Black, C. Rauch, C. Kozlosky, J. Peschon, J. Slack, M. Wolfson, B. Castner,
K. Stocking, P. Reddy, S. Srinivasan, N. Nelson, N. Bolani, K.A. Schooley,
M. Gerhart, R. Devis, J. Fitzner, R. Johnson, R. Paxton, C. March, D. Cerretti,
Nature 385 (1997) 729e733.
[15] M. Rothenberg, A. Nelson, K. Hande, Oncologist 3 (1998) 271e274.
[16] S. Dasgupta, P.R. Murumkar, R. Giridhar, M.R. Yadav, Bioorg. Med. Chem. 17
(2009) 444e459.
[17] H. Kubinyi, Nat. Rev. Drug Discov. 2 (2003) 665e668.
[18] A. Zask, J. Kaplan, X. Du, G. MacEwan, V. Sandanayaka, N. Eudy, J. Levin, G. Jin,
J. Xu, T. Cummons, D. Barone, S. Ayral-Kaloustian, J. Skotnicki, Bioorg. Med.
Chem. Lett. 15 (2005) 1641e1645.
[19] M.M. Thabet, T.W. Huizinga, Curr. Opin. Invest. Drugs 7 (2006) 1014e1019.
[20] G.R. Ott, N. Asakawa, Z. Lu, R. Anand, R.-Q. Liu, M.B. Covington, K. Vaddi,
M. Qian, R.C. Newton, D.D. Christ, J.M. Trzaskos, J.J. Duan, Bioorg. Med. Chem.
Lett. 18 (2008) 1577e1582.
[21] B. Car, Society of Toxicology, 46th Annual Meeting. Charlotte, NC, 2007, March
25e29.
[22] G. Ott, X.-T. Chen, J. Duan, Z. Lu, PCT Int. Appl. 24 (2003) 899 WO 03.
[23] J. Duan, G. Ott, L. Chen, Z. Lu, T.P. Maduskuie, M.E. Voss, C.-B. Xue, PCT Int.
Appl. 70 (2001) 673 WO 01.
[24] J.J. Duan, L. Chen, Z. Lu, C.-B. Xue, R.-Q. Liu, M.B. Covington, M. Qian,
Z.R. Wasserman, K. Vaddi, D.D. Christ, J.M. Trzaskos, R.C. Newton, C.P. Decicco,
Bioorg. Med. Chem. Lett. 18 (2008) 241e246.
a
production bya standard sandwich ELISA.
production by 50%
a
7.6. Molecular modeling studies
All the molecular modeling studies reported herein were per-
formed on a Silicon Graphics Fuel Workstation running on the IRIX
6.5 operating system using SYBYL 6.9 molecular modeling software
from Tripos, Inc., USA [27] and GLIDE from Schröndinger Inc., USA
[28,29]. All compounds used for docking were built from the frag-
ments in the SYBYL database, keeping the hydroxamic acid in its
singly deprotonated form, and the carboxylate of the catalytic Glu
406 was protonated following the finding from the early publica-
tion of Cross et al. [30]. All compounds used for docking were built
from the fragments in the SYBYL database. Each structure was fully
geometry optimized using the standard Tripos force field employ-
ing the Gasteiger Huckel charges with a distance-dependent
dielectric function until a root mean square deviation (RMSD) of
0.01 kcal/mol Å was achieved. Conformational search was carried
out using MULTISEARCH option in SYBYL 6.9. The lowest energy
conformer thus obtained was further minimized using the Tripos
force field and was subsequently used in docking. As number of
human crystal structures of TACE are available in the Protein Data
Bank (USA), we have used crystal structure of human TACE com-
plexed with IK682 (pdb code: 2FV5) since it is a highly tight binding
hydroxamate inhibitor of TACE besides having chemical similarity
with the synthesized compounds. Docking protocol was validated
by docking IK682, which was built, minimized and docked in
a similar manner like the compounds under study. Further it was
superimposed on the co-crystallized structure of IK682 (extracted
one) and the RMSD was found to be in the acceptable range. The
bond orders and formal charges of IK682 were adjusted prior to
[25] C. Cimarelli, G. Palmieri, J. Org. Chem. 61 (1996) 5557e5563.
[26] C.-B. Xue, M.E. Voss, D.J. Nelson, J.J. Duan, R.J. Cherney, I.C. Jacobson, X. He,
J. Roderick, L. Chen, R.L. Corbett, L. Wang, D.T. Meyer, K. Kennedy,
W.F. DeGrado, K.D. Hardman, C.A. Teleha, B.D. Jaffee, R.-Q. Liu, R.A. Copeland,
M.B. Covington, D.D. Christ, J.M. Trzaskos, R.C. Newton, R.L. Magolda,
R.R. Wexler, C.P. Decicco, J. Med. Chem. 44 (2001) 2636e2660.
[27] SYBYL Molecular modeling system, version 6.9. Tripos, Inc., St. Louis, USA, 2003.
[28] R.A. Friesner, J.L. Banks, R.B. Murphy, T.A. Halgren, J.J. Klicic, D.T. Mainz,
M.P. Repasky, E.H. Knoll, M. Shelly, J.K. Perry, D.E. Shaw, P. Francis,
P.S. Shenkin, J. Med. Chem. 47 (2004) 1739e1749.
[29] M. Clark, R.D. Crammer, N. van Opdenbosh, J. Comput. Chem. 10 (1989)
982e1012.
[30] J.B. Cross, J.S. Duca, J.J. Kaminiski, V.S. Madison, J. Amer. Chem. Soc. 124 (2002)
11004e11007.