A novel series of IKKβ inhibitors part I: Initial SAR studies of a HTS hit

Article abstract of DOI:10.1016/j.bmcl.2010.10.126

A novel series of (E)-1-((2-(1-methyl-1H-imidazol-5-yl) quinolin-4-yl) methylene) thiosemicarbazides was discovered as potent inhibitors of IKKβ. In this Letter we document our early efforts at optimization of the quinoline core, the imidazole and the semithiocarbazone moiety. Most potency gains came from substitution around the 6- and 7-positions of the quinoline ring. Replacement of the semithiocarbazone with a semicarbazone decreased potency but led to some measurable exposure.

Full text of DOI:10.1016/j.bmcl.2010.10.126

Bioorganic & Medicinal Chemistry Letters 21 (2011) 417–422
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
A novel series of IKKb inhibitors part I: Initial SAR studies of a HTS hit
a,
⇑
b
d
e
e
e
Timothy D. Cushing , Vijay Baichwal , Karen Berry , Roland Billedeau , Viola Bordunov , Chris Broka ,
c
a
a
f
a
e
Mario Cardozo , Peng Cheng , David Clark , Stacie Dalrymple , Michael DeGraffenreid , Adrian Gill ,
Xiaolin Hao , Ronald C. Hawley , Xiao He , Juan C. Jaen , Sharada S. Labadie , Marc Labelle , Csaba Lehel ,
a
e
a
a
e
a
b
a
e
d
b
a
e
Pu-Ping Lu , Joel McIntosh , Shichang Miao , Camran Parast , Youngsook Shin , Eric B. Sjogren ,
a
e
d
e
c
Marie-Louise Smith , Francisco X. Talamas , George Tonn , Keith M. Walker , Nigel P. C. Walker ,
b
e
d
g
Holger Wesche , Chris Whitehead , Matt Wright , Michelle F. Browner
Medicinal Chemistry, Amgen Inc., 1120 Veterans Blvd., South San Francisco, CA 94080, USA
Inflammation Biology, Amgen Inc., 1120 Veterans Blvd., South San Francisco, CA 94080, USA
Molecular Structure, Amgen Inc., 1120 Veterans Blvd., South San Francisco, CA 94080, USA
Pharmacokinetics and Drug Metabolism, Amgen Inc., 1120 Veterans Blvd., South San Francisco, CA 94080, USA
Chemistry, Roche Palo Alto, Hillview Ave., Palo Alto, CA 94304, USA
Biology, Roche Palo Alto, Hillview Ave., Palo Alto, CA 94304, USA
Discovery Technologies, Roche Palo Alto, Hillview Ave., Palo Alto, CA 94304, USA
a
b
c
d
e
f
g
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of (E)-1-((2-(1-methyl-1H-imidazol-5-yl) quinolin-4-yl) methylene) thiosemicarbazides
was discovered as potent inhibitors of IKKb. In this Letter we document our early efforts at optimization
of the quinoline core, the imidazole and the semithiocarbazone moiety. Most potency gains came from
substitution around the 6- and 7-positions of the quinoline ring. Replacement of the semithiocarbazone
with a semicarbazone decreased potency but led to some measurable exposure.
Received 8 September 2010
Revised 22 October 2010
Accepted 25 October 2010
Available online 29 October 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
IKKb
IKKa
Semithiocarbazones
Semicarbazones
The highly homologous IKKb and IKK
a
are members of a small
(TNFR) family members (e.g., BAFF receptor) leads to the activation
of the kinase NIK, which recruits IKK and binds to p100, the
NF- B precursor. NIK phosphorylates IKK , and IKK in turn phos-
phorylates p100 leading to its polyubiquitination, partial degrada-
tion and activation. NF- B then translocates to the nucleus
initiating the transcriptional machinery. IKKb and IKK clearly play
a central role in activation of NF- B with IKKb proving indispens-
able for the activation of the classical NF-
B pathway.3
Aberrant activation of the NF- B pathway has been implicated
in inflammation, autoimmune diseases and cancer. Thus, drugs
1
class of protein kinases known as I
jB Kinases (IKKs). They have a
a
distinct role in the regulation of both adaptive and innate immu-
j
a
a
nity and are critical for most aspects of host defense.2 IKKb and
IKK
a
activity are required for the activation of the transcription
B). NF- B controls much of
j
factor Nuclear Factor kappa B (NF-
j
j
a
the innate immune system and is instrumental in the induction
of genes leading to a pro-inflammatory response. In the classical
j
j
NF-
after activation (cytokine signaling) leads to a larger complex with
the ancillary proteins TRAF2/5, RIP1, TRADD, and IKK . Phosphor-
ylation of two key serines in part of the I B/NF- B complex causes
the protein to be polyubiquitinated, and degraded. This allows the
unbound NF- B dimers to translocate to the nucleus, initiate gene
transcription ultimately leading to pro-inflammatory cytokine pro-
duction. In the alternative NF- B pathway, involved in adaptive
immunity, activation of certain tumor necrosis factor receptor
j
B pathway, IKKb and IKK
a
form a heteromeric dimer, which
j
4
5
c
targeting these kinases are very promising candidates for the treat-
6
j
j
ment of many diseases. While the role of IKKb is limited to the
activation of NF-jB, IKKa is thought to be involved in other pro-
j
cesses, including skin development, and the termination of an
7
inflammatory response, hence selective inhibitors of IKKb are
j
highly desirable.
In this Letter, we describe our preliminary efforts to understand
the SAR, pharmacokinetics and pharmacology of a unique class of
molecules that inhibit IKKb. Compound 1 (Fig. 1) was identified
through high throughput screening (HTS). It is ATP competitive,
⇑
Corresponding author.
E-mail address: tcushing@amgen.com (T.D. Cushing).
0
960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.10.126

4
18
T. D. Cushing et al. / Bioorg. Med. Chem. Lett. 21 (2011) 417–422
Table 1
SAR of the semithiocarbazone
Compds
R
IKKb IC50 (lM)
S
S
S
S
S
HN
N
NH2
NH2
NH
N
1
0.25
Figure 1. Lead compound 1, obtained by high throughput screening.
N
N
reversible, potent, and selective over IKK
a, (IC50 IKKb = 0.25
lM,
2
3
4
5
0.12
1.7
IC50 IKK = 9.19 M). It is also selective (>30 l
a
l
M) over several
other kinases: JAK3, NIK, IRAK, PKA, PKC, c-Src, Abl, and RIP.
Although 1 represents a promising lead with respect to its inhibi-
tion of IKKb and its selectivity profile, it demonstrated poor aque-
HN
N
ous solubility (3.1 lg/mL, PBS buffer).
Our initial SAR efforts focused on the identification of the phar-
macophore, optimization of the potency and improvement of the
physico-chemical properties. Compound 1 could be subdivided
into three main regions; a quinoline core, a semithiocarbazone
and a methylimidazole ring. Analogs were prepared via a variety
HN
N
>30
8
N
N
N
H
of synthetic routes. The main route employed a Pfitzinger conden-
>30
sation between an isatin (A) and an imidazole ketone, such as F.
The resulting carboxylic acid was manipulated by standard meth-
ods to provide an aldehyde (D), which was then condensed with
various nucleophiles to provide a generalized analog E (Scheme 1).
Most of the analogs in Table 1 were prepared by the method of
Scheme 1. To explore the SAR around positions 2 and 4 of the quin-
oline core, Scheme 2 was generally followed. The analogs of Table 2
were prepared by routes 2D, and 2E and Scheme 1. Many of the
analogs in Table 3 were prepared by employing alternative hetero-
cyclic methyl ketones for intermediate F (Scheme 1). Other analogs
in Table 3 were prepared by the chemistry outlined in Scheme 2B
and C. Routes 2A and 2C and variants thereof were used in the syn-
thesis of analogs found in Table 4. The majority of the compounds
6
7
X
X = O: 1.1
X = NH: 17
HN
N
NH2
NH2
O
N
N
8
9
7.8
S
HN
N
NH2
>30
1
in Table 5 followed Scheme 1. Reduction (hydrogenation) of the R -
group prior to the final nucleophilic alkylation provided analogs
such as 69 and 70 (step g, Scheme 1).
One initial goal of our SAR efforts was to understand the role of
the semithiocarbazone, which we believed could prove a liability.
A small series of changes to 1 were made to explore the SAR of this
unusual functional group (Table 1). Importantly, replacement of
the sulfur with oxygen (6) was tolerated, while NH (7) was not,
and the terminal amino group proved to be very sensitive to
change. While a single methyl substituent was tolerated (3) a
There were several assays used to develop the SAR in this pro-
gram. Our primary biochemical assay was a chemiluminescent
9
ELISA assay against IKKb and IKKa.
Our main cellular assays were an NF-j
B activation assay10
(
(
gel-shift assay induced with TNF) and a human whole blood
HWB) assay induced by lipopolysaccharide (LPS).1
1
Scheme 1. Reagents and conditions: (a) KOH, EtOH, H
2
O 60%; (b) MeOH, H
2
SO
4
, 80%; (c) Te, NaBH
4 2
; (d) Dess–Martin periodinane; (e) AcOH, EtOH, H O, semi(thio)carbazide,
semicarbazide or nucleophile; (f) PdCl (PPh , CuI, DMF–Et
2
3
)
2
3
N, methyl propargyl ether, or Pd(PPh
3
)
4
, Et N, DMF, acrylonitrile; (g) 10%Pd/C, H , MeOH.
3
2

T. D. Cushing et al. / Bioorg. Med. Chem. Lett. 21 (2011) 417–422
419
Table 2
Table 3
SAR of the quinoline 4-position
SAR of the quinoline 2-position
R
Compound
IKKb IC50 (lM)
N
N
N
O
Compds
R
IKKb IC50 (lM)
HN
O
1
0
>30
2
H N
1
N
0.25(X = S)
N
1
1
1
2
7
CN
O
N
24
>30(X = O)
S
S
8.5
NH2
25
1.2(X = S)
1.6(X = O)
26
N
N
O
S
O
13
>30
>30
>30
>30
27
28
>30(X = S)
>30X = O)
NH2
O
N
NH
1
1
1
4
5
6
29
>30(X = S)
NH2
N
N
S
N
30
>30(X = S)
>30X = O)
31
O
CONH2
32
>30(X = S)
>30X = O)
O
33
N
N
CONH2
O
N
3
3
4
5
>30(X = S)
NH
N
>30(X = S)
>30X = O)
1
7
>30
36
H N
2
3
7
>30(X = S)
1
1
8
9
>30
>30
NH2
38
7.5(X = S) a
6.2(X = O)
39
N
HN
N
HN
40
>30(X = S)
>30X = O)
41
CONH2
N
42
NH
4.5(X = O)
1.75(X = S)
2
2
0
1
>30
>30
N
CONH2
43
N
N
N
N
NH
O
N
H
S
44
45
>30(X = S)
N
N
N
2
2
2
3
>30
>30
N
N
>30(X = S)
>30(X = S)
CONH2
CONH2
S
46
N
S
4
4
7
8
N
>30(X = S)
>30X = O)
dimethylamine (4) was not tolerated. Interestingly, methylation as
in 2 improved potency twofold but an additional methyl group at
a
IC50 calculated from measurement at 10 lM.
2
the terminal NH as 5 was not tolerated. Thus a key element in this
SAR was the terminal amine, which proved critical for binding
potency.
Additional SAR studies were undertaken to modify this poten-
tially problematic moiety (Table 2). Many analogs (10, 15, 16, 17,
The potency may be enhanced by the pK
a
of the 3-aza-heterocy-
cles. For example, the pK of thiazole is 2.52 versus 0.8 for oxa-
a
12
zole, the increased potency of 25 and 26 relative to 27 and 28
could be due to the differences in basicity between these analogs.
Methyl substituted analogs that sterically hindered the 3-aza
nitrogen exhibited a potency decline, indicative of a precise bind-
ing orientation or a highly constrained binding pocket. Compare
2
2, and 23) were made in an attempt to mimic the length and ori-
entation of the semithiocarbazone, but none proved to be very ac-
tive. Compounds such as 11 and 12, which retained some of the
orientation of the semithiocarbazone moiety, were more potent.
Most efforts to replace or modify the N-methyl imidazole moi-
ety were unsuccessful. Five-membered rings such as thiazoles (25,
4
3
3 to 29 or 44 and 25 to 46. The weak potency in 32, 33, 35, and
6, which all contain 3-aza nitrogen, imply that steric demands
2
6), the N-linked imidazole 43, pyrazole 42, and the des-methyl
take precedence over a putative H-bond contact (Table 3).
Based on the SAR generated around the 2- and 4-quinoline
substituent it appeared that both the semithiocarbazone and the
N-methylimidazole were playingcrucialrolesin theenergetics ofen-
zyme binding. In the absence of enzyme–inhibitor crystallographic
analogs 38 and 39 were tolerated when compared to 1 and 6.
The common feature in these analogs was the unhindered 3-aza
nitrogen. Compounds with nearly identical architecture but lack-
ing this 3-aza-nitrogen (24, 40, and 41) were poorly tolerated.

4
20
T. D. Cushing et al. / Bioorg. Med. Chem. Lett. 21 (2011) 417–422
1
2
Scheme 2. Reagents and conditions: (a) n-BuLi, THF, diethyl oxalate; (b) KOH, R -COMe EtOH, H
2
O; (c) MeI, K
, dioxane; (i) Pd(PPh
, reflux, then DIPEA DCM, NHR ; (l) R -stannane, Pd (dba) , triphenylarsine, CuI, DMF.
2
CO
3
, DMF; (d) POBr
3 3
, (POCl ) toluene; (e) R -stannane,
3
Pd
2
(dba)
3
, triphenylarsine, CuI, DMF; (f) MeMgCl, ZnBr
2
, THF; (g) Pd(PPh
)
3 4
, R -stannane, CuI, DMF; (h) SeO
2
3 4
)
, CuI, 1-methy-5-tributylstannylimidazole,
4
5
6
7
DMF 60 °C 3 h; (j) R R NH
2
, 120 °C sealed tube; (k) SOCl
2
2
3
data we speculate that these moieties were making key contacts to
the hinge region of the protein. A model of these inhibitors bound
to IKKb was constructed that had the NH of the semithiocarbazone
2
Table 4
SAR around the quinoline core structure
acting as a H-bond donor to the hinge carbonyl of Gln-100 of IKKb in
theATPbinding domainandthe3-Nofthemethylimidazoleactingas
an H-bond acceptor to the –NH of the adjacent Cys-99. All subse-
quent SAR development was undertaken with this model in mind.
Attempts were made to optimize the core quinoline moiety (Ta-
ble 4). A pyridine ring (49) was not tolerated but a naphthalene
core (50) had some modest activity. Various other nitrogen con-
taining cores were prepared, with substitutions at the 3-, 6-, 7-,
and 8-positions. The 8-aza (52) and the 3-aza analog 54 had weak
activity. Molecular modeling indicated that the lone pair on the 3-
aza analog 54 could have a repulsive interaction with the adjacent
nitrogen lone pair of the semithiocarbazone (Fig. 2).
Compds
R
IKKb IC50 (lM)
49
50
51
52
53
54
>30
N
It was intriguing to find that 6-aza (51) and 7-aza (53) both had
a level of potency similar to 1. This observation led us to explore
further substitutions at the 6- and 7-positions. We found that the
1.8
0.42
24
6
- and 7-methyl analogs, 55 and 57 were more potent than 3-
methyl (56) and 8-methyl (58) (Table 5). Generally, small lipophilic
and electron-withdrawing groups (EWG) at the quinoline 7-posi-
tion were found to improve activity. For example, 7-Me (57), 7-
N
N
N
3
CF (64), (65), 7-Br (72), 7-CN (73), and 7-Et (78) displayed
improved IKKb potency compared to 1 or 6, and 72 had sub-micro-
molar cellular activity. However, the activity dropped off with
bulkier groups substituted at the 7-position such as t-butyl (66),
n-propyl (74), and phenyl (75).
At the 6-position small groups seemed to have little effect, as
can be seen by 55 and 63 compared to 1, and EWG containing ana-
logs like 62 and 63 were either neutral or detrimental for potency.
Additionally, strongly electron donating groups (EDG) like 61 were
1.2
4.6
N
N
N

T. D. Cushing et al. / Bioorg. Med. Chem. Lett. 21 (2011) 417–422
421
Table 5
SAR of the quinoline 6- and 7-positions
Compds
X
R
IKKb IC50
(l
M)
IKKa
IC50
(lM)
NF-j
B IC50
(lM)
HWB IC50 (lM)
6
O
S
O
S
S
S
O
O
O
S
S
O
S
O
S
S
O
S
S
S
S
S
S
6,7-H
6-Me
3-Me
7-Me
8-Me
8-Ph
6-Ph
6-OMe
6-CN
6-F
1.1
0.23
3.0
25.9
18.2
—
1.9
—
—
—
—
—
5.9
—
—
>30
—
—
>30
>30
>30
1.0
1.0
1.8
>30
—
3.0
0.5
10
30
0.4
1
2.5
>30
10
8
1.0
1.0
26
—
—
—
—
—
—
—
—
—
—
14
—
—
5
5
5
5
5
6
6
6
6
6
6
6
6
6
6
7
7
7
7
7
7
7
7
7
7
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0.12
>30
>30
>30
2.95
3.07
0.26
0.09
0.7
—
7-CF
7-CF
3
17.2
25.2
>30
—
—
1.6
—
>30
2.3
10.6
0.88
20
3
7-t-Bu
7-t-Bu
6-(Pyridin-3-(yl))
2.8
12.6
0.32
0.010
0.10
0.9
0.06
0.06
0.24
2.0
0.13
0.6
0.13
0.6
—
6-CH
6-CH
2
CH
CH
2
CN
CN
OH
2.5
9.4
—
—
—
—
—
—
30
—
2
2
6-CCCH
7-Br
7-CN
7-Propyl
7-Ph
7-Cyclopropyl
7-Cyclopropyl
7-Et
2
—
—
22.5
>30
O
S
O
7-CH@CH
2
31
Table 7
PK parameters
Compds
Rata
Dogb
Cl (L/h/kg)
V
diss(L)
%F
Cl (L/h/kg)
%F
2
6
5
9
1.9
1.6
1.7
1.3
0.8
1.2
0.9
1.1
54
4
2
—
—
1.5 ± 0.8
—
—
75 ± 30
—
—
6
7
2
a
Males 1 mg/kg iv, 5 mg/kg po.
Female beagles 1 mg/kg iv, 5 mg/kg po.
b
Figure 2. Putative repulsive interactions.
7
0. Compound 69 was also potent in cells (0.5 lM in the NF-jB
gel-shift assay) although the corresponding semicarbazone (70)
was 10-fold less active toward IKKb and had limited cellular activ-
ity. In all cases, there was a 4–10-fold biochemical potency differ-
ence between the semithiocarbazones and the semicarbazones. For
example, semithiocarbazones 64, 66, and 76 are more potent than
the corresponding semicarbazones 65, 67, and 77, respectively (Ta-
ble 5).
Because of their poor solubility, we were unable to obtain reli-
able PK; many of the semithiocarbazones could not be formulated
for iv/po administration. However, the semicarbazones proved
somewhat more soluble and we were able to measure the expo-
sure in rat and dog. These values were compared with in vitro
parameters such as permeability and microsomal clearance (Tables
Table 6
Physicochemical and PKDM parameters
Permeabilityc
Compds Aq Soln Solubility 10% FBS
Intrinsic
a
b
clearanced
(
l
g/
in PBS buffer
mL)
(l
g/
PAPP
À6
mL)
(10 cm/s)
(mL/min/kg)
2
6
5
9
0.26
8.3
4.8
16.3
>29
71.2
15.1
10.1
13.9
9
5
—
—
6
7
0.15
a
b
c
pH 6.5.
Turbidity assay.
CaCO-2 cells.
d
Rat microsomes.
6
and 7).
Although 2 and 6 had reasonable intrinsic clearance as mea-
sured by rat microsomes, the in vivo clearance was considerably
higher approaching 50% liver blood flow. In general, the oral
exposure was low (2–4%), with a corresponding low CaCo-2 cell
permeability (PAPP: 10–15 Â 10 cm/s) for 6, 65, and 79. The
also disfavored. Larger groups were more tolerated, including pyri-
din-3-yl (68) and propynol (71), but a real improvement in potency
was found with the flexible cyanoethyl containing analogs 69 and
À6

4
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T. D. Cushing et al. / Bioorg. Med. Chem. Lett. 21 (2011) 417–422
N-methyl semithiocarbazone (2) had a PAPP value nearly fivefold
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Initial SAR was developed around the HTS lead 1. Few changes
were tolerated except substitution at the quinoline 6- and 7-posi-
tions. There were a few examples of submicromolar activity in
cells, notably 69, which had low micromolar potency in a HWB as-
say. In general, semithiocarbazones were found to have increased
potency relative to the semicarbazones but suffered from poor
PK characteristics compared to the semicarbazones. Additional ef-
forts on the further refinement of these analogs are described in
part II, which follows this manuscript.
7
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References and notes
1
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(a) Solt, L. A.; May, M. J. Immunol. Res. 2008, 42, 3l; (b) Hacker, H.; Karin, M. Sci.
STKE 2006, 357; (c) Woronicz, J. D.; Gao, X.; Cao, Z.; Rothe, M.; Goeddel, D. V.
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9.
The IKKb biochemical assay was performed in the following manner: a neutravidin
coated 96-well microtiter plate was treated with to 80 L/well of 1
biotinylated IKB derived substrate peptide (KKERLLDDRHDSGLDSMKDEEYE-
QGK-bio) and buffer (20 mM Tris–HCl, pH 7.5, 10 mM MgCl , 2 mM EGTA,
mM NaF, 0.5 mM benzamide, 1 mM DTT, 0.1% NP-40, 10% ATP).
Compound was added (10 L/well) in DMSO from 1 nM to 30 M followed
l
lM
a
2
2
3
.
.
Bonizzi, G.; Karin, M. Trends Immunol. 2004, 25, 280.
Hu, Y.; Baud, V.; Delhase, M.; Zhang, P.; Deerinck, T.; Ellisman, M.; Jounson, R.;
Karin, M. Science 1999, 284, 316.
1
lM
l
l
by 10 mL buffer and baculovirus produced (insect cells) full-length
recombinant IKKb. The mixture was incubated at rt for 45 min, after which
4
.
(a) Karin, M. Cell Res. 2008, 18, 334; (b) Pande, V.; Ramos, M. J. Curr. Med. Chem.
2
005, 12, 357; (c) Karin, M.; Greten, F. R. Nat. Rev. Immunol. 2005, 5, 749; (d)
the reaction was stopped by washing the plates 3Â with 150
2
lL H O. At this
Karin, M.; Yamamoto, Y.; Wang, Q. M. Nat. Rev. Drug Disc. 2004, 3, 17; (e) Luo, J.-
L.; Kamata, H.; Karin, M. J. Clin. Invest. 2005, 115, 2625.
time, 2% BSA in PBS 100 L/well and primary monoclonal antibody recognizing
l
the phosphorylated epitope at 1:10,000 dilution, premixed with conjugated
anti-mouse secondary antibody horse-radish peroxidase (HRP) 1:10,000
dilution. The solution was incubated on a shaker at rt for 40 min then
5
.
.
Millennium (Takeda) had entered MLN-0415 a small molecule inhibitor of IKKb
in a phase I study in 2006. The structure has not yet been disclosed.
6
(a) Liddle, J.; Bamborough, P.; Barker, M. D.; Campos, S.; Cousins, R. P. C.; Cutler,
G. J.; Hobbs, H.; Holmes, D. S.; Ioannou, C.; Mellor, G. W.; Morse, M. A.; Payne, J.
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washed 3Â 150
l
L H
2
O. SuperSignal HRP (Pierce), 100 mL/well 10Â diluted
was added and incubated 5 min. The chemiluminescent signal was found with
a Labsystems LuminoSkan luminometer. IC50s were determined by curve fitting
with LSW data analysis software (MDL).
1
0. NF-kB gel shift assays were carried out as described (Schindler, U.; Baichwal, V.
R. Mol. Cell Biol. 1994, 14, 5820). In summary, IL8/ECV304 cells were stimulated
with TNF for 1 h in the absence or presence of compounds and NF-kB. DNA
binding activity was determined by gel shift experiments from crude nuclear
extracts.
1. Human whole blood assay: blood samples in a 96-well microtiter plate were
stimulated for 5 h with LPS in the presence or absence of test compounds.
Following the incubation period, levels of TNF in the culture supernatants were
determined with an ELISA kit (R&D systems).
1
2
008087933.; (g) Moffat, D.; Festus, C.; Davies, S. J.; Charlton, M. H.; Hirst, S.
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1
2. Zoltewicz, J. A.; Deady, L. W. Adv. Heterocycl. Chem. 1978, 22, 71.
(
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