Inhibition of Tpl2 kinase and TNF-α production with 1,7-naphthyridine-3-carbonitriles: Synthesis and structure-activity relationships

Article abstract of DOI:10.1016/j.bmcl.2005.08.029

The synthesis and structure-activity studies of a series of 6-substituted-4-anilino-[1,7]-naphthyridine-3-carbonitriles as inhibitors of Tpl2 kinase are described. The early exploratory work described here may lead to the discovery of compounds with signi

Full text of DOI:10.1016/j.bmcl.2005.08.029

Bioorganic & Medicinal Chemistry Letters 15 (2005) 5288–5292
Inhibition of Tpl2 kinase and TNF-a production with
1,7-naphthyridine-3-carbonitriles: Synthesis and
structure–activity relationships
Lori Krim Gavrin,a Neal Green,a,* Yonghan Hu,a Kristin Janz,a Neelu Kaila,a
Huan-Qiu Li,a Steve Y. Tam,a Jennifer R. Thomason,a Ariamala Gopalsamy,b
Greg Ciszewski,b John W. Cuozzo,c J. Perry Hall,c Sang Hsu,c
Jean-Baptiste Telliezc and Lih-Ling Linc
aChemical and Screening Sciences, Wyeth Research, 200 Cambridge Park Drive, Cambridge, MA 02140, USA
bChemical and Screening Sciences, Wyeth Research, 401 North Middletown Road, Pearl River, NY 10965, USA
cInflammation Signaling, Wyeth Research, 200 Cambridge Park Drive, Cambridge, MA 02140, USA
Received 3 August 2005; revised 11 August 2005; accepted 11 August 2005
Available online 13 September 2005
Abstract—The synthesis and structure–activity studies of a series of 6-substituted-4-anilino-[1,7]-naphthyridine-3-carbonitriles as
inhibitors of Tpl2 kinase are described. The early exploratory work described here may lead to the discovery of compounds with
significant therapeutic potential for treating rheumatoid arthritis and other inflammatory diseases.
ꢀ 2005 Elsevier Ltd. All rights reserved.
Tumor necrosis factor-a (TNF-a), a pro-inflammatory
cytokine, is involved in inflammation in a number of dis-
ease states, most notably in the autoimmune disease
rheumatoid arthritis (RA). RA occurs in nearly 1% of
the population with an annual incidence of 0.04%.1
The debilitating aspects of RA are associated with the
erosion of cartilage and bone leading to joint pain,
swelling, stiffness, and restricted mobility. The protein
therapeutic ENBRELꢁ/etanercept, a soluble form of
human recombinant TNF-a receptor (sTNFR-a), is cur-
rently available to patients with RA and has been a
major advance in treating the disease; however, an oral-
ly available small molecule that inhibits TNF-a synthe-
sis and/or signaling would have widespread therapeutic
potential.2
kines.4 Tpl2 is also required for TNF-a signaling (i.e.,
the cellular response to ligation of the TNF-a receptor),
and thus an inhibitor of Tpl2 would have the double
benefit of blocking both TNF-a production and signal-
ing.5 Furthermore, the unique features of Tpl2 presum-
ably increase the potential for discovering a selective
Tpl2 inhibitor. Tpl2 is not inhibited by staurosporine,
a non-specific kinase inhibitor, and it is the only human
kinase that has a proline instead of a conserved glycine
in the glycine-rich ATP binding loop.6
Screening of our in-house kinase library provided us
with several classes of reversible and ATP-competitive
hits (Fig. 1), one of which, a 6-substituted-4-anilino-
[1,7]-naphthyridine-3-carbonitrile 1, originated in
Tpl2 (Cot/MAP3K8) is a serine/threonine kinase in the
MAP3K family that is upstream of MEK in the ERK
pathway.3 Recent studies using Tpl2 knock-out mice
indicate an important role for Tpl2 in the LPS-induced
production of TNF-a and other pro-inflammatory cyto-
X
Ar
HN
R
N
HN
N
H
N
CN
CN
N
X
O
N
N
N
1
2a-y
Keywords: Tpl2 kinase; 1,7-Naphthyridine-3-carbonitriles.
*
Figure 1. Optimization of 6-substituted-4-anilino-[1,7]-naphthyridine-
3-carbonitriles.
Corresponding author. Tel.: +1 617 665 5604; fax: +1 617 665
5685; e-mail: ngreen@wyeth.com
0960-894X/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.08.029

L. K. Gavrin et al. / Bioorg. Med. Chem. Lett. 15 (2005) 5288–5292
5289
Table 1. Inhibition of Tpl2 kinase by 4-(arylamino)-6-(2-morpholino-
ethylamino)-3-cyano-[1,7]-naphthyridines (2a–m)
Cl
F
F
CO2Me
NHBoc
a
b
N
N
N
NO2
F
NHBoc
Ar
HN
H
3
c
4
N
CN
N
O
Cl
N
O
N
CN
F
CN
N
d
2a - 2m
N
N
NHBoc
6
7
Compounds
Ar
IC50 (nM)a
Ar
Ar
HN
R
N
HN
2a
2b
2c
2d
2e
2f
3-Chloro-4-fluorophenyl
4-Benzylphenyl
1,200
2
F
CN
CN
f
e
X
4-(Phenylthio)phenyl
4-Phenoxyphenyl
4-(Phenylsulfonyl)phenyl
6
380
N
N
N
N
2a-y
8a-m
>40,000
4,600
4-(Phenylmethanone)phenyl
3-Chloro-4-fluorobenzylamine
4-Trifluoromethylphenyl
4-Chlorophenyl
Scheme 1. Reagents and conditions: (a) i—KF, DMSO, 70 ꢂC, 18 h;
ii—SnCl2–H2O, EtOAc; iii—Boc2O, t-BuOH, 40 ꢂC, 3 h, 53%, 3 steps.
(b) i—n-BuLi, TMEDA, ether, À78 ꢂC then CO2; ii—TMSCH2N2,
CHCl3/MeOH, 51%, 2 steps. (c) CH3CN, n-BuLi, THF À78 ꢂC, 76%.
(d) i—DMF–DMA, rt, 1 h; (ii) oxalyl chloride, DMF, CH2Cl2, 59%,
2 steps. (e) ArNH2, EtOH, reflux 6–12 h, 80–94% or ArNH2, DME,
110 ꢂC microwave, 15–45 min, 95%. (f) RXNH, pyridine, 80 ꢂC, 3
days, 30–50% or RXNH, THF or neat, 120–180 ꢂC microwave, 0.5–
2 h, 12–60%.
2g
2h
2i
>40,000
>40,000
>40,000
10,000
10,000
>40,000
4,200
2j
4-Chloro-2-fluorophenyl
3,4-Dichlorophenyl
2k
2l
3-Bromo-2-methylphenyl
3,5-Dichlorophenyl
2m
a Average of at least two experiments (see Ref. 9).
another kinase program targeting a receptor tyrosine
kinase. In this manuscript, we wish to describe the
early-stage lead exploration of a series of 6-substi-
tuted-4-anilino-[1,7]-naphthyridine-3-carbonitriles 2a–y.
The synthesis, structure–activity relationships, and in
vitro TNF-a inhibition studies of Tpl2 inhibitors are
described herein.
2n–y)]. In the 6-(2-morpholinoethylamino) series (Table
1), large non-rigid substituents in the para position of
the 4-anilino ring, such as benzyl, thiophenyl, and phen-
oxy, greatly improved Tpl2 inhibition with IC50s of 2–
380 nM (2b, 2c, and 2d). In contrast, the addition of a
more polar sulfonyl or carbonyl group between the
two aromatic rings had reduced inhibition (2f,
4600 nM) or loss of inhibition (2e, >40,000 nM). De-
spite the tolerance of Tpl2 for large hydrophobic groups
on the 4-anilino headpiece, a derivative with a methylene
group inserted between the 4-anilino nitrogen atom and
the aromatic ring (3-chloro-4-fluorobenzyl derivative 2g,
Scheme 1 illustrates the synthesis of 6-substituted-4-ani-
lino-[1,7]-naphthyridine-3-carbonitriles. The synthesis of
the key intermediate 4-chloro-6-fluoro-[1,7]-naphthyri-
dine-3-carbonitrile 7 has been previously described.7
Compound 7 was condensed with a variety of anilines
(ÔheadpiecesÕ) either in refluxing ethanol or via micro-
wave irradiation using a slight excess of the aniline to
give the penultimate intermediates 8a–m.
Table 2. Inhibition of Tpl2 kinase by 4-(3-chloro-4-fluorophenylami-
no)-6-amino-3-cyano-[1,7]-naphthyridines (2a and 2n–y)
F
Displacement of the C-6-fluoro atom in 8a–m with var-
ious amino-containing groups (ÔtailpiecesÕ) via heating of
the mixture in pyridine for 3–5 days gave the target com-
pounds 2a–y in moderate yields.7 Microwave irradiation
dramatically reduced the reaction times for the conver-
sions and in some cases the final two steps were carried
out in one pot without purification.8 The yields for these
microwave transformations were variable (on average
35–60%) and dependent on the amine nucleophile with
more nucleophilic amines giving higher yields and fewer
by-products.
Cl
R
N
HN
N
CN
X
2a, 2n-2y
N
Compounds R (amine)
X
IC50 (nM)a
2a
2n
2o
2p
2q
2r
2-(Morpholino)CH2CH–
3-(Morpholino)CH2CH2CH2–
H
H
H
H
H
H
H
H
H
H
1,200
3,400
21,000
50
4-Me-(piperazin-l-yl)CH2CH2CH2–
(Pyridin-3-yl)CH2–
Benzyl
73
Naphthyridine derivatives 2a–y were first studied for
inhibition of isolated Tpl2 enzyme, via quantification
of MEK phosphorylation in an ELISA format.9 For
the purpose of independently comparing the effects of
the 4-aminoaryl and 6-amino groups on activity, the
data are separated into Table 1 [4-(arylamino)-6-(2-mor-
pholinoethylamino)-naphthyridine carbonitriles (2a–m)]
2-Morpholino-l-phenylethyl–
(1S)-1-phenylethylamino
(1R)-1-phenylethylamino
(1R)-1-phenylpropylamino
(1S)-1-phenylpropylamino
Benzyl
1,800
27,000
12
2s
2t
2u
2v
2w
2x
2y
414
>40,000
Me >40,000
Cyclopentyl
3-(Pyrrolidinyl)propyl–
H
H
>40,000
3,565
and Table
(alkylamino)-naphthyridine-3- carbonitriles (2a and
2
[4-(3-chloro-4-fluorophenylamino)-6-
a Average of at least two experiments.

5290
L. K. Gavrin et al. / Bioorg. Med. Chem. Lett. 15 (2005) 5288–5292
>40,000 nM) gave dramatically reduced inhibition. In
the absence of a large hydrophobic group at the para po-
sition of the headpiece, a 3-halo group appears to be
necessary for good inhibitory activity, as exemplified
by the two inactive analogs 2h and 2i. Other analogs
containing meta-substituted aniline headpieces bearing
such groups as alkyl, carbonyl, phenoxy, sulfonyl, and
amino were also inactive (not shown).
meric pairs 2s/2t and 2u/2v). In these two cases, the R-
isomer was 100- to >2000 times more potent than the
corresponding S-isomer. The importance of a secondary
rather than a tertiary amino group at C-6 was seen;
when the 6-amino group of 2q was N-methylated to give
2w, activity was lost.
Selectivity studies were carried out with Tpl2 inhibitors
against a panel of kinases, including kinases known to
be involved in TNF-a production (p38 and MK2), and
other serine-threonine and tyrosine kinases (Table 3).
Analog 2a showed moderate selectivity over most ki-
nases; however, in a cell-based assay, 2a showed strong
inhibition of EGFR at 5 lM.10 Analogs with the same
tailpiece as 2a, but with larger hydrophobic headpieces,
showed improved selectivity against MEK (2b, 2c, and
2d) and had reduced inhibition of cellular EGFR,
although 2b and 2c showed poor selectivity against
Src. Of the five compounds tested in this panel, 2l
showed the best combination of potency and selectivity.
Structure–activity relationship data previously reported
by Wyeth for 4-anilinonaphthyridine-3-carbonitriles,10
and by other research groups for 4-anilinoquinazo-
lines,11 indicated that substituents at the C-6 and/or C-
7 positions have less influence on isolated kinase activity
than the headpiece unit. Thus, the C-6 and/or C-7 posi-
tions have been used to introduce groups that improve
the solubility and permeability of inhibitors. In the pres-
ent case, it was found that for the [1,7]-naphthyridine-3-
carbonitrile series, a large range of inhibitory activity
was dependent on the length, the hybridization, and
the stereochemistry of the tailpiece moiety (Table 2).
For example, 2a with a 2-(morpholino)ethylamino
group was nearly three times as potent as the 3-methy-
lene containing analogs 2n and 2y, and more than 17
times as potent as the 4-piperazinyl analog 2o (IC50 of
21 lM).
With information on the selectivity profiles of these
[1,7]-naphthyridine-3-carbonitriles, they were then eval-
uated in both cellular13 and blood environments (Table
4).14 Included in Table 4 are the physicochemical
properties that may influence the activity of these
derivatives in these assays, specifically solubility, per-
meability, and protein binding.15 TNF-a inhibition
was diminished in human blood compared to human
monocytes for all the compounds tested. The 2-(mor-
pholino)ethylamino tailpiece analog 2a showed the
lowest potency in human monocytes, and yet, appar-
ently due to lower protein binding, high solubility,
and higher permeability it was the most active in
blood. Analogs 2p and 2b were the most potent in
monocytes. Low solubility and low permeability, cou-
pled with higher protein binding, were presumably
why these two analogs were weaker than 2a in blood.
A significant improvement in inhibitory activity was
achieved with the installation of CH2-aryl and CH2-het-
eroaryl groups at the 6-amino position. The 3-pyridyl-
methylamino analog 2p and the benzyl analog 2q were
potent inhibitors of Tpl2 (50 and 73 nM, respectively).
Alkyl and cycloalkyl analogs, represented by derivative
2x, were not active. Racemic 2-morpholino-1-phenyleth-
ylamino derivative 2r12 was less potent than 2a with an
IC50 of only 1.8 lM. The stereochemical configuration
of the tailpiece affects the inhibition of Tpl2 (enantio-
Table 3. In vitro selectivity profiles of selected 6-amino-4-anilino-3-cyano-[1,7]-naphthyridinesa
Compounds
Tpl2
MEK
p38
58
Src
40
MK2
PKC
EGFRb
2a
2p
2b
2d
2c
1.200
0.050
0.002
0.380
0.006
15
>40
>400
110
30
>400
>400
200
100%–5 lM
50%–5 lM
50%–5 lM
50%–5 lM
10%–5 lM
180
>400
>400
31
>400
0.63
4
90
3
>400
>400
>400
0.80
0.96
>400
a Average of at least two experiments (lM).
b A431 cell-based assay from an average of three experiments.
Table 4. In vitro inhibition of TNF-a production with selected inhibitorsa
Compounds
Human monocytesa,b
HWBb
Solubility (lg/mL)
Permeability (·106 m/s)
Plasma protein bindingc
2a
2p
2b
2c
2d
4.5
0.7
0.8
2.7
1.4
6.8
8.5
64
2
3.78
0.1
85
98
13.6
20.1
14.9
BLDd
BLD
0.01
BLD
99
1
1
>99
>99
a Lipopolysaccharide-induced TNF-a from primary human monocytes and human whole-blood (IC50 lM).
b Average of at least two experiments.
c Percent bound to human serum albumin.
d Below limit of detection.

L. K. Gavrin et al. / Bioorg. Med. Chem. Lett. 15 (2005) 5288–5292
5291
7. Wissner, A.; Hamann, P. R.; Nilakantan, R.; Greenber-
ger, L. M.; Ye, F.; Rapuano, T. A.; Loganzo, F. Bioorg.
Med. Chem. Lett. 2004, 14, 1411.
8. A typical preparation of target compounds 2b–t is
illustrated by the following procedure for 2h: in a 100 mL
round-bottomed flask fitted with a condenser, 4-chloro-
Derivatives 2d and 2c were the least potent in mono-
cytes and both demonstrated high protein binding,
and poor solubility and permeability. The poor physi-
cochemical properties of 2d and 2c most likely preclud-
ed TNF-a inhibition in blood.
6-fluoro-[1,7]-naphthyridine-3-carbonitrile
(0.250 g,
We have explored Tpl2 inhibition and functional TNF-a
inhibition with a class of 6-alkylamino-4-anilino-[1,7]-
naphthyridine-3-carbonitriles. In this study, it was
found that a large range of inhibitory activity depended
on the length, the hybridization, and the stereochemistry
of the tailpiece moiety, while headpiece modifications
resulted in potent Tpl2 inhibitors with less selectivity
over other kinases than the 3-chloro-4-fluorophenyl
headpiece analogs. This initial SAR study was used to
exploit continued optimization toward the discovery of
Tpl2 inhibitors for the treatment of TNF-a-driven dis-
eases such as rheumatoid arthritis. Further studies and
modifications in physicochemical properties and
improvements in in vitro and in vivo selectivity profiles
for this series will be reported in due course.
1.20 mmol) and 4-chloroaniline (0.168 g, 1.32 mmol) were
taken up in 20 mL of 2-ethoxyethanol and heated at reflux
for 1 h, until t.l.c. analysis (20% EtOAc in hexanes) showed
complete disappearance of the 4-chloronaphthyridine.
After cooling to room temperature, the reaction mixture
was partitioned between 40 mL of each EtOAc and 5%
Na2CO3. The aqueous layer was extracted twice more with
EtOAc, and the combined organic layers were washed
three times with brine, dried over anhydrous MgSO4,
filtered, and evaporated to give 4-(4-chlorophenylamino)-
6-fluoro-[1,7]-naphthyridine-3-carbonitrile as a crystalline
yellow solid, which was of sufficient purity to be used
directly in the next step: 1H NMR (400 MHz, DMSO-d6) d
7.41 (d, J = 8.6 Hz, 2H) 7.47–7.58 (m, 2H) 8.18 (s, 1H) 8.69
(s, 1H) 9.07 (s, 1H) 10.11 (s, 1H). The product of the first
step (0.179 g, 0.600 mmol assuming 100% yield of step 1)
was taken up in a microwave vial in 3.4 mL THF, with 4-
(2-aminoethyl)morpholine (1.6 mL, 1.6 g, 12 mmol). The
sealed vial was heated in a microwave reactor at 150 ꢂC for
1 h, until t.l.c. analysis showed complete disappearance of
the starting material. Then THF was removed under
reduced pressure, and the crude product was purified by
flash chromatography over silica gel (7% MeOH in
CH2Cl2) and lyophilized to give a fluffy, bright yellow
Acknowledgments
The authors wish to thank Drs. Tarek Mansour, David
Simmons, Glenn Larsen, James Clark, and Dennis Pow-
ell for their support. We also gratefully acknowledge the
members of the Wyeth Discovery Analytical Chemistry
group for analytical and spectroscopic determinations,
Dr. Alex Gontcharov of Wyeth Discovery Synthetic
Chemistry for the large-scale preparation of 8a, Dr.
Qin Wang for protein binding results, and Satenig Guler
for assistance in preparing the manuscript.
1
solid (105 mg, 43% yield of 2 steps): H NMR (400 MHz,
DMSO-d6) d 2.43 (br s, 4H) 2.56 (t, J = 6.2 Hz, 2H) 3.37 (q,
J = 6.6 Hz, 2 H) 3.49–3.64 (m, 4H) 6.65 (t, J = 5.8 Hz, 1H)
7.01 (s, 1H) 7.31 (d, J = 8.6 Hz, 2 H) 7.46 (d, J = 8.8 Hz,
2H) 8.28 (s, 1H) 8.85 (s, 1H) 9.63 (s, 1H); HRMS (ESI+)
calcd for C21H22ClN6O (MH+) 409.1538, found 409.1542.
9. Tpl2/Cot activity was directly assayed using GST-MEK1
as a substrate. The phosphorylation on serine residues 217
and 221 of GST-MEK1 was detected by an ELISA.
Briefly, 0.4 nM Tpl2 was incubated with 35 nM GST-
MEK1 in a kinase reaction buffer containing 20 mM
Mops, pH 7.2, 50 lM ATP, 20 mM MgCl2, 1 mM DTT,
25 mM b-glycerophosphate, 5 mM EGTA, and 1 mM
sodium orthovanadate for 1 h at 30ꢂC. Compounds
solubilized in 100% DMSO were pre-diluted in assay
buffer so that the final concentration of DMSO in the
reaction was 1%. The kinase reaction was carried out in
100 ll volume on 96-well plates. The kinase reaction was
then stopped with the addition of 100 mM EDTA. The
entire reaction mix was then transferred to the detection
plate, a 96-well Immunosorb plate that had been pre-
coated with anti-GST antibody (Amersham). After 1 h
incubation at room temperature, the detection plate was
washed four times with TBST (TBS + 0.05% Tween 20)
and then incubated for another hour at room temperature
with anti- phospho-MEK1 antibody (Cell Signaling)
1:1000 in 10 mM Mops 7.5, 150 mM NaCl, 0.05% Tween
20, 0.1% gelatin, 0.02% NaN3, and 1% BSA. The detection
plate was washed again and incubated for 30 min with
DELFIA Europium (Eu) labeled goat anti-rabbit IgG
(Perkin-Elmer), 1:4000 in the same buffer used for the
primary incubation. After a final wash, Eu detection
solution was added to each well and the Eu signal was
measured in a Wallac Victor Multilabel Counter. Data
were imported into Excel and IC50 calculations were
performed using the Xlfit (IDBS) software package.
References and notes
1. Feldmann, M.; Mani, R. Annu. Rev. Immunol. 2001, 19,
163.
2. Jarvis, B.; Faulds, D. Drugs 1999, 57, 945; Maini, R.; St.
Clair, E. W.; Breedveld, F.; Furst, D.; Kalden, J.;
Weisman, M.; Smolen, J.; Emery, P.; Harriman, G.;
Feldmann, M.; Lipsky, P. Lancet 1999, 354, 1932.
3. (a) Sasai, H.; Higashi, T.; Nakamori, S.; Miyoshi, J.;
Suzuki, F.; Nomura, T.; Kakunaga, T. Br. J. Cancer 1993,
67, 262; (b) Miyoshi, J.; Higashi, T.; Mukai, H.; Ohuchi,
T.; Kakunaga, T. Mol. Cell. Biol. 1991, 11, 4008; (c)
Schlesinger, T. K.; Franger, G. R.; Yujiri, T.; Johnson, G.
L. Front. Biosci. 1998, 3, D1181.
4. (a) Dumitru, C. D.; Ceci, J. D.; Tsatsanis, C.; Konoyian-
nis, D.; Stamatakis, K.; Lin, J. H.; Patriotis, C.; Jenkins,
N. A.; Copeland, N. G.; Kollias, G.; Tsichlis, P. N. Cell
2000, 103, 1071; (b) Eliopoulos, A. G.; Dumitru, C. D.;
Wang, C. C.; Cho, J.; Tsichlis, P. N. EMBO J. 2002, 21,
4831.
5. (a) Lin, X.; Cunningham, E. T., Jr.; Mu, Y.; Gelziunas,
R.; Greene, W. C. Immunity 1999, 10, 271; (b) Chiarello,
M.; Marinissen, M. J.; Gutkind, J. S. Mol. Cell. Biol. 2000,
20, 1747; (c) Salmeron, A.; Ahmad, T. B.; Carlile, G. W.;
Pappin, D.; Narsimhan, R. P.; Ley, S. C. EMBO J. 1996,
15, 817.
6. Luciano, B.; Hsu, S.; Channavajhala, P. B.; Lin, L.-L.;
Cuozzo, J. W. J. Biol. Chem. 2004, 279, 52117.
10. (a) Wissner, A.; Berger, D. M.; Boschelli, D. H.; Floyd, B.
M.; Greenberger, L. M.; Gruber, B. C.; Johnson, B. D.;

5292
L. K. Gavrin et al. / Bioorg. Med. Chem. Lett. 15 (2005) 5288–5292
Mamuya, N.; Nilakantan, R.; Reich, F. M.; Shen, R.;
A.; Green, T. P.; Hennequin, L. F.; Curwen, J.; Fennell,
M.; Allen, J.; Lambert-van der Brempt, C.; Costello, G.
J. Med. Chem. 2004, 47, 871.
Tsou, H.; Upeslacis, E.; Wang, Y. F.; Wu, B.; Zhang, N.
J. Med. Chem. 2000, 43, 3244; (b) Wang, Y. D.; Miller, K.;
Boschelli, D. H.; Ye, F.; Wu, B.; Floyd, B. M.; Powell, D.
W.; Wissner, A.; Weber, J. M.; Boschelli, F. Bioorg. Med.
Chem. Lett. 2000, 10, 2477; (c) Boschelli, D. H.; Wang, Y.
D.; Ye, F.; Wu, B.; Zhang, N.; Dutia, M.; Powell, D. W.;
Wissner, A.; Arndt, K.; Weber, J. M.; Boschelli, F. J.
Med. Chem. 2001, 44, 822; (d) Boschelli, D. H.; Ye, F.;
Wang, Y. D.; Dutia, M.; Johnson, S. L.; Wu, B.; Miller,
K.; Powell, D. W.; Yaczko, D.; Young, M.; Tischler, M.;
Arndt, K.; Discafani, C.; Etienne, C.; Gibbons, J.; Grod,
J.; Lucas, J.; Weber, J. M.; Boschelli, F. J. Med. Chem.
2001, 44, 3965; (e) Zhang, N.; Wu, B.; Wissner, A.; Powell,
D. W.; Rabindran, S. K.; Kohler, C.; Boschelli, F. Bioorg.
Med. Chem. Lett. 2002, 12, 423; (f) Berger, D.; Dutia, M.;
Powell, D.; Wu, B.; Wissner, A.; Demorin, F.; Weber, J.;
Boschelli, F. Bioorg. Med. Chem. Lett. 2002, 12, 2761; (g)
Wissner, A.; Floyd, B. M.; Rabindran, S. K.; Nilakantan,
R.; Greenberger, L. M.; Shen, R.; Wang, Y. F.; Tsou, H.
Bioorg. Med. Chem. Lett. 2002, 12, 2893; (h) Berger, D.;
Dutia, M.; Powell, D.; Wissner, A.; Demorin, F.; Raifeld,
Y.; Weber, J.; Boschelli, F. Bioorg. Med. Chem. Lett. 2002,
12, 2989; (i) Berger, D.; Dutia, M.; Powell, D.; Wu, B.;
Wissner, A.; Boschelli, D. H.; Floyd, B. M.; Zhang, N.;
Torres, N.; Levin, J.; Du, X.; Wojciechowicz, D.; Disca-
fani, C.; Kohler, C.; Kim, S. C.; Feldberg, L. R.; Collins,
K.; Mallon, R. Bioorg. Med. Chem. Lett. 2003, 13, 3031;
(j) Wissner, A.; Overbeek, E.; Reich, M. F.; Floyd, B.;
Johnson, B. D.; Mamuya, N.; Rosfjord, E. C.; Discafani,
C.; Davis, R.; Shi, X.; Rabindran, S. K.; Gruber, B. C.;
Ye, F.; Hallett, W. A.; Nilakantan, R.; Shen, R.; Wang, Y.
F.; Greenberger, L. M.; Tsou, H. J. Med. Chem. 2003, 46,
49.
12. The amino unit of this tailpiece: Giardina, G. A. M.;
Raveglia, L. F.; Grugni, M.; Sarau, H. M.; Farina, C.;
Medhurst, A. D.; Graziani, D.; Schmidt, D. B.; Rigolio,
R.; Luttmann, M.; Cavagners, S.; Foley, J. J.; Vecchietti,
V.; Hay, D. W. P. J. Med. Chem. 1999, 42, 1053.
13. Inhibition of TNF-a in primary human monocytes:
human blood buffy coat (containing 1 mM EDTA) is
incubated with RosetteSep human monocyte enrichment
antibody cocktail (Stem Cell Technologies #15068). This
mixture is then diluted with an equal volume of PBS/2%
FBS/1 mM EDTA, layered onto an equal volume of
Histopaque-1077 (Sigma #H8889), and centrifuged for
20 min at 1500 rpm. The monocytes are then recovered
from the interface, diluted, and reisolated on a Hist-
opaque-1077 cushion. After this second round of purifi-
cation, the monocytes are washed twice in PBS/2% FBS/
1 mM EDTA, resuspended in RPMI/0.5% FBS to
2 · 106 cells/mL, plated at 800,000 cells per well in 48-
well formats, and incubated at 37 ꢂC/5% CO2. Thirty
minutes prior to LPS stimulation, compounds (in 100%
DMSO) are added. LPS is then added to 10 ng/mL and
the cultures are allowed to incubate for an additional
ꢀ3 h. Media supernatants are then harvested and
analyzed for TNF-a by standard ELISA or electrochem-
iluminescence on
Discovery).
a Sector6000 reader (Meso Scale
14. Inhibition of TNF-a in human blood: blood is drawn from
healthy male volunteers into heparin tubes. At 37 ꢂC, the
blood is diluted 1-to-5 with pre-warmed RPMI/3% FCS,
and then dispensed into 96-well-formatted 200 lL tube
strips and capped. Thirty minutes prior to LPS stimulation
compounds (in 100% DMSO) are added. Final DMSO
concentrations equal 1.0%. LPS is then added to 10 ng/mL
and the cultures are allowed to incubate for an additional
ꢀ3 h. Samples are centrifuged in a strip rotor at 6500 rpm,
and the plasma supernatants are analyzed by standard
ELISA or electrochemiluminescence on a Sector6000
reader (Meso Scale Discovery).
15. For an example see: Cherney, R. J.; Duan, J. J. W.; Voss,
M. E.; Chen, L.; Wang, L.; Meyer, D. T.; Wasserman, Z.
R.; Hardman, K. D.; Liu, R. Q.; Covington, M. B.; Qian,
M.; Mandlekar, S.; Christ, D. D.; Trzaskos, J. M.;
Newton, R. C.; Magolda, R. L.; Wexler, R. R.; Decicco,
C. J. Med. Chem. 2003, 46, 1811.
11. (a) Tsou, H.; Mamuya, N.; Johnson, B. D.; Reich, M. F.;
Gruber, B. C.; Ye, F.; Nilakantan, R.; Shen, R.; Disca-
fani, C.; DeBlanc, R.; Davis, R.; Koehn, F. E.; Green-
berger, L. M.; Wang, Y. F.; Wissner, A. J. Med. Chem.
2001, 44, 2719; (b) Smaill, J. B.; Showalter, H. D. H.;
Zhou, H.; Bridges, A. J.; McNamara, D. J.; Fry, D. W.;
Nelson, J. M.; Sherwood, V.; Vincent, P. W.; Roberts, B.
J.; Elliott, W. L.; Denny, W. A. J. Med. Chem. 2001, 44,
429; (c) Hennequin, L. F.; Stokes, E. S. E.; Thomas, A. P.;
´
Johnstone, C.; Ple, P. A.; Ogilvie, D. J.; Dukes, M.;
Wedge, S. R.; Kendrew, J.; Curwen, J. O. J. Med. Chem.
2002, 45, 1300; (d) Banerjee, R.; Rachid, Z.; McNamee, J.;
´
Jean-Claude, B. J. J. Med. Chem. 2003, 46, 5546; (e) Ple, P.