Novel tricyclic inhibitors of 1κB kinase

Article abstract of DOI:10.1021/jm8015816

The design and synthesis of a novel series of oxazole-, thiazole-, and imidazole-based inhibitors of IκB kinase (IKK) are reported. Biological activity was improved compared to the pyrazolopurine lead, and the expedient synthesis of the new tricyclic systems allowed for efficient exploration of structure-activity relationships. This, combined with an iterative rat cassette dosing strategy, was used to identify compounds with improved pharmacokinetic (PK) profiles to advance for in vivo evaluation.

Full text of DOI:10.1021/jm8015816

1994
J. Med. Chem. 2009, 52, 1994–2005
Novel Tricyclic Inhibitors of IKB Kinase
James Kempson,*,† Steven H. Spergel,† Junqing Guo,† Claude Quesnelle,† Patrice Gill,† Dominique Belanger,†
Alaric J. Dyckman,† Tianle Li,† Scott H. Watterson,† Charles M. Langevine,† Jagabandhu Das,† Robert V. Moquin,†
Joseph A. Furch,† Anne Marinier,† Marco Dodier,† Alain Martel,† David Nirschl,¶ Katy Van Kirk,¶ James R. Burke,‡
Mark A. Pattoli,‡ Kathleen Gillooly,‡ Kim W. McIntyre,‡ Laishun Chen,§ Zheng Yang,§ Punit H. Marathe,§
David Wang-Iverson,§ John H. Dodd,† Murray McKinnon,‡ Joel C. Barrish,† and William J. Pitts†
Departments of DiscoVery Chemistry, DiscoVery Biology, and Metabolism and Pharmacokinetics and Synthesis and Analysis Technology Team,
Bristol-Myers Squibb Research and DeVelopment, P.O. Box 4000, Princeton, New Jersey, 08543-4000
ReceiVed December 15, 2008
The design and synthesis of a novel series of oxazole-, thiazole-, and imidazole-based inhibitors of IκB
kinase (IKK) are reported. Biological activity was improved compared to the pyrazolopurine lead, and the
expedient synthesis of the new tricyclic systems allowed for efficient exploration of structure-activity
relationships. This, combined with an iterative rat cassette dosing strategy, was used to identify compounds
with improved pharmacokinetic (PK) profiles to advance for in vivo evaluation.
Introduction
The nuclear transcription factor NF-κBa has a crucial role in
the pathogenesis of several human disorders, particularly those
with an inflammatory component.1 In unstimulated cells, NF-
κB is sequestered in the cytoplasm as an inactive complex with
IκB inhibitory protein.2 In the case of IκBR, the most extensively
studied member of this protein family, the stimulation of
proinflammatory receptors such as the tumor necrosis factor
(TNF) receptor, activates the so-called “canonical” pathway of
NF-κB activation in which a multisubunit IKK complex
catalyzes the phosphorylation of IκBR at Ser32 and Ser36.3 This
phosphorylation event is essential for signaling the subsequent
ubiquitination and proteosomal degradation of IκBR, thus
leaving the NF-κB free to translocate to the nucleus and activate
proinflammatory gene transcription.
The IKK complex comprises two catalytic subunits, IKK1
and IKK2 (also termed IKKR and IKKꢀ, respectively).
Although both of these subunits can catalyze the phospho-
rylation of IκB protein, evidence has shown that the IKK2
Figure 1. Previous IKK inhibitors discovered at Bristol-Myers
Squibb.
subunit plays a major role in NF-κB activation arising from
proinflammatory stimuli such as TNF-R, IL1-ꢀ, IL-6, IL-17,
and IL-23.4 Since IKK2 is vital for translating these proin-
flammatory stimuli into the activation of NF-κB, an inhibitor
of IKK2 could be effective in the treatment of autoimmune
and inflammatory disorders such as rheumatoid arthritis,
inflammatory bowel disease, lupus, and multiple sclerosis.
Interestingly, the activity of glucocorticoids, which are among
the most effective anti-inflammatory agents available, is
mediated primarily through the inhibition of NF-κB mediated
transcription. This suggests that an IKK2 inhibitor would
provide an opportunity to develop novel therapeutics that has
the potential for glucocorticoid-like activity but without their
trans-activation related toxicities.5
The discovery of small molecule IKK inhibitors as orally
active therapeutic agents for the treatment of inflammatory
diseases has been the goal of numerous research groups, and
several classes of IKK inhibitors have been reported.6 We
recently reported the identification of the imidazoquinoxaline
based inhibitor of IKK2, 1 (Figure 1).6b Despite promising in
vivo efficacy studies in both acute and chronic preclinical models
of inflammation, the compound has relatively weak in vitro
potency which prompted a search for alternative inhibitors of
IKK2 with greater potency. Discovery of the thiophene6e and
pyrazolopurine6f chemotypes followed by structure-activity
relationship (SAR) modification led to the identification of 2
and 3 as potent and selective inhibitors of IKK2. Both of these
chemotypes, however, were metabolically unstable resulting in
poor PK profiles, and their lengthy chemical syntheses drasti-
* To whom correspondence should be addressed. Phone: (609) 252-4539.
Fax: (609) 252-7410. E-mail: james.kempson@bms.com.
†
Department of Discovery Chemistry.
Synthesis and Analysis Technology Team.
Department of Discovery Biology.
Department of Metabolism and Pharmacokinetics.
¶
‡
§
a Abbreviations: IKK, IκB kinase; TNF, tumor necrosis factor; IL,
interleukin; NF-κB, nuclear factor κ light-chain enhancer of activated
B cells; ser, serine; SAR, structure-activity relationship; PK, pharma-
cokinetic; GST, glutathione s-transferase; LPS, lipopolysaccharide;
PBMC, peripheral blood mononuclear cell; AUC, area under curve; Vss,
volume of distribution; CYP, cytochrome P450.
10.1021/jm8015816 CCC: $40.75
2009 American Chemical Society
Published on Web 03/06/2009

NoVel Tricyclic Inhibitors of IκB Kinase
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7 1995
Scheme 2a
Figure 2. Metabolism driven design of new IKK inhibitors.
a Conditions: (a) benzoyl chloride, CH2Cl2, room temp, 100%; (b)
Na2CO3, DMF, microwave, 220 °C, 60%; (c) MeNH2 ·HCl, (i-Pr)2NEt,
n-BuOH, 140 °C, 40%.
Scheme 3a
Figure 3. Disconnection of newly proposed IKK targets to common
intermediate.
Scheme 1a
a Conditions: (a) Lawesson’s reagent, toluene, 110 °C, then K2CO3,
DMA, 160 °C, 75%; (b) MeNH2 ·HCl, (i-Pr)2NEt, n-BuOH, 140 °C, 33%.
Scheme 4a
a Conditions: (a) fuming HNO3, conc H2SO4, reflux, 85%; (b) POCl3,
PCl5, reflux; (c) 2.0 M MeNH2 in THF, 0 °C, 65% for two steps; (d) SnCl2,
conc HCl, 100 °C, 50-60%; (e) CH(OEt)3, CH3CN, 100 °C, 95%.
cally slowed SAR generation, with no opportunity for chemo-
type diversification.
a Conditions: (a) (i) SOCl2, toluene, 110 °C; (ii) MeNH2 ·HCl, (i-Pr)2NEt,
CH3CN, 80 °C, (83% for two steps); (b) KOtBu, DMF, microwave, 200
°C, 67%; (c) MeNH2 ·HCl, (i-Pr)2NEt, n-BuOH, 185 °C, 18%.
Design of New Tricyclic Based Inhibitors of IKK
Metabolite identification of the pyrazolopurine and thiophene-
based tricyclic chemotypes revealed core oxidation as a major
metabolic pathway. New isosteric tricyclic chemotypes were
designed to block the current site of metabolism, leading to
compounds of the type shown in Figure 2.
Disconnection from these newly proposed tricycle targets led
to the novel Cs-symmetrical pentasubstituted pyridine intermedi-
ate 4 (see Figure 3). It was anticipated that this common
intermediate could be used to access the desired chemotypes
as well as facilitate a more rapid SAR analysis than was
previously possible. This approach was particularly attractive,
since there was no IKK2 protein structure to guide our efforts,
and as such, an efficient chemotype construction/SAR modifica-
tion strategy was of paramount importance.
group with concomitant installation of the chlorine groups at
the 2- and 6-positions of the pyridine ring was accomplished
in a single step by treatment with tin(II) chloride.8 Desymme-
trization was then accomplished by the reaction of 4 with 1
equivalent of triethyl orthoformate to give 9.
Schemes 2-4 outline the synthesis of the oxazole, thiazole,
and imidazole tricyclic cores, 12, 14, and 17, respectively.
In the oxazole case, common intermediate 9 was treated with
1 equiv of benzoyl chloride. The resulting amide, 10, was
isolated as the hydrochloride salt and then cyclized in the
presence of sodium carbonate in DMF at 220 °C under
microwave irradiation to give 11. Displacement of the
2-chloropyridine in 11 was then achieved with methylamine
and led to the first oxazole-based compound 12. The structure
of 12 was confirmed by X-ray crystallographic analysis of
the HCl salt. Amide 10 was also treated with Lawesson’s
reagent (see Scheme 3), which after base-induced cyclization
led to the thiazole 13. In a manner similar to the oxazole
case, final displacement with methylamine then provided the
first thiazole-based tricycle 14, whose structure was also
confirmed by X-ray crystallographic analysis. Scheme 4
depicts the synthesis of a third tricyclic core, which was
Chemistry
The synthesis of the Cs-symmetrical pyridine, 4, and its
conversion to the common intermediate, 9, is outlined in Scheme
1. Nitration of commercially available 4-hydroxypyridine, 5,
was carried out according to literature methods to afford 6.7
Treatment of 6 with PCl5 in phosphorus oxychloride gave the
unstable intermediate 7. Attempts to isolate this compound
resulted in significant hydrolysis back to 6, hence 7 was treated
directly with methylamine to give 8. The reduction of each nitro

1996 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7
Kempson et al.
Scheme 5a
Scheme 7a
a Conditions: (a) PdCl2(PPh3)2, Et3N, 1:1 CH3CN/MeOH, 100 psi CO,
60 °C, 91%; (b) NaOH, THF, 53%; (c) MeNH2 in EtOH, 150 °C, 36%; (d)
RR′NH, PyBOP, NMP, 52-83%.
Scheme 8a
a Conditions: (a) Ppotassium thioxanthate, MeI, DMF, 55%; (b) MeNH2
in EtOH, n-BuOH, 180 °C, 91%; (c) Oxone, CH2Cl2; (d) 5 N NaOH, 48%
(for two steps); (e) POCl3, pyridine, 180 °C, 90%; (d) RR′NH, K2CO3,
NMP, 140 °C, 30-75%.
Scheme 6a
a Conditions: (a) 4-fluorophenylboronic acid, K2CO3, Pd(PPh3)4, 1:1
DME/EtOH, 100 °C, 60%; (b) RNH2 in EtOH, 150 °C, 22-64%.
Scheme 9a
a Conditions: (a) BzNCS, acetone, 75%; (b) (i) KOtBu, NMP; (ii) HCl,
dioxane, 64%; (c) isoamylnitrile, CuBr2, CH3CN, 82%.
achieved via conversion of amide 10 into the corresponding
chloroimidate with thionyl chloride and subsequent displace-
ment with methylamine to provide 15. Closure with potassium
tert-butoxide gave 16, which after final displacement with
methylamine resulted in 17.
Scheme 5 highlights the initial synthetic approach to generate
increased diversity in the thiazole chemotype. Reaction of 9
with potassium thioxanthate followed by in situ alkylation with
methyl iodide afforded 18 in 55% yield. Displacement with
methylamine under microwave conditions then provided 19. The
structure was confirmed by X-ray crystallographic analysis (see
Supporting Information). Oxidation to the sulfone 20 with oxone
followed by hydrolysis to 21 and subsequent chlorination using
phosphorus oxychloride provided the advanced intermediate 22.
Displacement with a variety of amines provided compounds 23.
Scheme 6 highlights a complementary synthetic approach to
the thiazole tricyclic system, again starting from the common
intermediate, 9. Condensation of 9 with benzoylisothiocyanate
gives 24. This undergoes cyclization and amide cleavage to give
the 2-aminothiazole 25. Sandmeyer reaction in the presence of
isoamyl nitrite and copper bromide then provides 26. This
advanced intermediate proved more efficient for the SAR
investigations that were conducted in this series.
a Conditions: (a) 3-(N-Boc-aminomethyl)phenylboronic acid, K2CO3,
Pd(PPh3)4, 1:1 DME/EtOH, 100 °C, 60%; (b) RNH2 in EtOH, 150 °C,
10-75%; (c) HCl in dioxane, 80%; (d) RCO2H, PyBOP, NMP, 36-85%.
Carbonylation of the bromo intermediate 26 provided the ester
27, which after hydrolysis furnished the carboxylic acid 28
(Scheme 7). Displacement with methylamine then gave 29,
which proved useful to probe structural diversity via amide
couplings employing pyBOP and an amine to give 30.
Scheme 8 highlights a Suzuki coupling with 4-fluorophenyl-
boronic acid to give 31. This compound then formed the basis
for SAR exploration at the 2-position of the pyridine ring.
Chemistry described previously using a variety of amines was
used to generate the desired inhibitors 32.
Suzuki coupling reaction with 3-(N-Boc-aminomethyl)phe-
nylboronic acid (Scheme 9) followed by nucleophilic displace-
ment with methylamine and subsequent acid deprotection of

NoVel Tricyclic Inhibitors of IκB Kinase
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7 1997
Table 2. Rat Pharmacokinetic Data for Compounds 3b and 14
Table 1. Enzyme, Cell Activities, and in Vitro Metabolic Stabilities for
Program “Lead” 3b and New Oxazole, Thiazole, and Imidazole
Tricycles 12, 14, and 17, Respectively
clearance,
a
compd Cmax
,
nM AUC, nM ·h T1/2, h (mL/min)/kg Vss, L/kg
3b
14
3600
3000
2200
2800
0.5
1.2
55
42
2.1
2.8
a Maximum serum concentration after intravenous (2 mg/kg) administra-
tion of the compound to rat in Tween 80: H2O (1:9) medium; n ) 3 animals
microsomal clearance
IKK-1
IKK-2
PBMC
in rat, human, mouse,
compd
IC50, µM
IC50, µM
IC50, µM
nmol/(min·mg)
3b
12
14
17
0.99
0.27
3.5
0.034
0.011
0.006
0.52
0.47
0.39
0.31
0.44
0.05, 0.11, 0.17
0.14, 0.09, 0.23
0.12, 0.09, 0.25
0.08, 0.13, 0.12
Figure 4. SAR strategy from new common intermediate 26.
vitro metabolic stability assay had little predictive value with
respect to the metabolism of the tricycle (thiazole) core. In an
attempt to address this issue, we implemented a cassette
screening strategy in which we simultaneously administered
five compounds at a dose of 2 mg/kg.9 In an effort to exclude
false positives resulting from drug interactions, compounds were
prescreened in cytochrome P450 (CYP) assays to exclude potent
inhibitors (IC50 < 10 µM). The thiazole chemotype was selected
not only because of its potent and selective inhibition of IKK2
but also because of the amount of chemical diversity we could
explore in this chemotype arising from the same intermediate.
A focused library effort would allow efficient modification at
the 2- and 6-positions of the thiazole core (see Figure 4). The
linear synthesis of previous tricyclic systems had thus far limited
the 6-position SAR to aryl and heteroaryl groups. Expansion
of this to include amine and amide functionality was planned
for the first time. It was hoped that this strategy, after iterative
cassette screening, would identify compounds that maintained
the potency of 14 and had favorable PK profiles.
Schemes 5-9 highlight the implementation of the SAR
strategy that began with amine modification at the 6-position
of the thiazole core. Despite encouraging results with the
thiomethyl and sulfone (19 and 20, respectively) analogues,
subsequent substitution studies with a variety of amines revealed
a narrow structural requirement at this position. Replacement
of the thiomethyl substituent with primary amines significantly
attenuated potency. Examination of cyclic compounds 23d, 23e,
and 23f suggested that enzyme potency could be optimized to
some extent but cell activity, as with all analogues in this class,
remained weak (Table 3).
Our attention turned next to the modification of the carboxa-
mide substituent on the thiazole core (Table 4). Both the Me
and Et amide analogues (30a and 30b) are equipotent against
IKK2. Replacement with cyclopentylamide 30c leads to a
substantial loss (10-fold) in potency which is further attenuated
with the tertiary di-Et amide 30d. The thienylamide 30e displays
the same enzyme potency as 30a and 30b, although again for
this series of compounds cellular potency is generally weak.
Table 5 summarizes the 2-aminopyridine SAR which was
conducted with a p-fluorophenyl group at the 6-position. SAR
findings in related series restricted this effort to primary amines,
since removal of the N-H bond donor resulted in a drastic loss
of enzyme potency. All amines studied maintained a modest
amount of activity against IKK2. Although the amine and
alcohol functionality of 32a-f were tolerated by the enzyme,
32e and 32f possessed significant cytotoxicity as measured by
an Αlamar blue toxicity assay.10 This is possibly due to the
4.3
the Boc group gave 35. This then allowed for derivatization at
the benzylamine, providing 36 with a variety of carboxylic acids.
Results and Discussion
Compounds were evaluated for their ability to inhibit IKK1
or IKK2 catalyzed phosphorylation of glutathione s-transferase
(GST)-IκBR fusion protein, and a peripheral blood mono-
nuclear cell assay (PBMC) was used to measure the ability of
compounds to inhibit lipopolysaccharide (LPS)-induced TNFR
production in human primary cells.
By use of the previously outlined syntheses (Schemes 1-4),
the oxazole, thiazole, and imidazole tricycles 12, 14, and 17
were initially prepared to test the concept of blocking metabo-
lism. Both the oxazole 12 and thiazole 14 displayed improved
enzyme activites with IKK2 IC50 values of 0.011 and 0.006 µM,
respectively (Table 1), compared to 0.034 µM for the pyrazol-
opurine 37 (3- to 6-fold increase in potency). Cellular (PBMC)
activities were also improved, although not to the same extent
as the enzyme potencies. Interestingly, the thiazole tricycle also
displayed a marked increase in IKK1/IKK2 selectivity (580-
fold). Although 12, 14, and 17 are isosteric, 17 proved to be
somewhat less potent than the other two tricyclic systems.
Encouraged by the improvements in enzyme and cell activities
for two of the three newly synthesized chemotypes, their
metabolic stability in rat, human, and mouse liver microsomes
was then evaluated in in vitro assays. Disappointingly, both the
oxazole and thiazole chemotypes displayed the same moderate-
to-poor metabolic profiles, with negligible improvement in
human liver microsome stability compared to the pyrazolopurine
3b and even worse profiles in mouse and rat. Clearance rates
for the imidazole 17 were comparable to 3b across all three
species. Although the microsomal stability profiles were less
than ideal, the potent thiazole analogue and the pyrazolopurine
3b were further evaluated in rats dosed with an intravenous (2
mg/kg) administration of compound in order to determine their
clearance rate (Table 2). For the pyrazolopurine, 3b, in vivo
clearance was higher than predicted by incubation with liver
microsomes. Despite the apparent increase in in vitro microso-
mal degradation of 14 compared with 3b, in vivo pharmaco-
kinetic evaluation in rats showed a minor improvement in
clearance (Table 2).
Although the concept of blocking metabolism had resulted
in minor improvements to in vivo clearance in the case of 14,
over the course of our studies it became apparent that the in

1998 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7
Kempson et al.
Table 4. Enzyme and Cell Activities for Thiazole Tricycles Derived
Table 3. Enzyme and Cell Activities for Thiazole Tricycles Derived
from Scheme 5
from Scheme 7
a PBMC ) LPS induced TNF-R production in peripheral blood mono-
nuclear cells. b NT ) not tested.
Table 5. Enzyme Activities and Cytotoxicity Data for Thiazole
Tricycles Derived from Scheme 8
a PBMC ) LPS induced TNF-R production in peripheral blood mono-
nuclear cells. b NT ) not tested.
detergent-like nature of the molecules bearing a polar “head”
group and a lipophilic p-fluorophenyl “tail”.
Efforts then turned to the meta-position of the phenyl ring
which in previous efforts had proved successful, allowing
modulation of both enzyme and cell potencies as well as
physicochemical characteristics. Table 6 summarizes the SAR
findings. Addition of m-benzylamine derivatives maintained
reasonable potency (compounds 37a, 37b). Removal of the
p-fluoro group led to significantly improved enzyme and cellular
activities. Sulfonamide 36b in particular represented the most
potent inhibitor studied. Further benzylamine derivatization led
to heterocyclic amides 36c and 36d which, despite a 3- to 5-fold
drop in enzyme potency, still maintained similar potency in
cellular assays to 36b.
Given the initial SAR findings, we next wanted to investigate
the impact of each of these structural modifications on the in
vivo clearance of the compounds. Consistent with all aminothia-
zoles tested, 23f displayed high in vitro microsomal clearance
which translated into high clearance (83 (mL/min)/kg) and low
exposure in rats (Table 7). Improvements seen with the amide
30d were quickly abolished with the first phenyl analogue 32a
which highlights the lack of correlation between the in vitro
system and in vivo clearance. Amine modifications 32d and
32e led to significant (Cl ) 16 (mL/min)/kg) and slight (52
(mL/min)/kg) improvements, respectively, compared to 32a (67
(mL/min)/kg). Meta-substitution significantly improved clear-
a NT ) not tested.
ance over 32a, while removal of the p-fluoro group led to an
increase in in vivo clearance. Optimization of the amides gave

NoVel Tricyclic Inhibitors of IκB Kinase
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7 1999
Table 6. Enzyme and Cell Activities for Thiazole Tricycles Derived
from Scheme 9
Table 7. In Vitro and in Vivo Clearance Data for Different Structural
Elements in the Thiazole Chemotype
microsomal clearance
in rat, human, mouse, Cmax
a
,
AUC,
clearance, Vss,
compd
nmol/(min·mg)
nM nM·h T1/2, h (mL/min)/kg L/kg
23f
0.25, 0.15, 0.29
300
1100
1600 1600
4700 2700
600
800
1200
500
2300 1900
3900 5000
250
880
0.4
1.2
0.6
0.4
2.6
0.3
0.4
0.3
0.4
0.6
83
23
67
16
52
31
20
41
2.6
0.6
3.2
0.5
10.4
0.5
0.4
0.9
0.2
0.2
30d
32a
32d
32e
37a
37b
36b
36c
36d
NTb
0.095, 0.13, 0.21
0.000, 0.003, 0.065
0.000, 0.000, 0.000
NTb
0.12, 0.030, 0.14
0.13, 0.015, 0.16
0.16, 0.18, 0.26
0.14, 0.16, 0.23
590
570
800
400
8.4
3.0
a Maximum serum concentration after intravenous (2 mg/kg) administra-
tion of the compound to rat in Tween-80/H2O (1:9) medium; n ) 3 animals.
b NT ) not tested.
Figure 5. LPS-induced TNF inhibition by 36d in mice. BALB/c female
mice (Harlan), 6-8 weeks of age, were used. Compound was dissolved
in poly(ethylene glycol) (MW ) 300, PEG300) and administered to
mice (n ) 8/treatment) by oral gavage in a volume of 0.1 mL. Control
mice received PEG300 alone (“Veh”). Four hours later, mice were
injected intraperitoneally with 50 µg/kg lipopolysacchride (LPS, E. coli
O111:B4; Sigma). Blood samples were collected 90 min after LPS
injection. Serum was separated and analyzed for the level of TNF-R
by commercial ELISA assay (BioSource) according to the manufac-
turer’s instruction. Data shown are the mean ( SEM.
Table 8. Kinase Selectivity Profile for Compounds 12, 17, and 36aa
compd IKK2 Btk Cdk2 IGF
Itk
<50 17
<50 19
<50 9.1
IKKꢀ Plk1
Src
12
0.011
0.52
23
14
17
16
23
31
17
14
12
<50
12
<50
<50
<50
<50
17
36a
0.022
a Enzyme inhibition in µM.
is orally active with statistically significant (50%) reduction in
serum TNF compared to vehicle control.
Representative examples of each tricyclic core were evaluated
in an in-house kinase selectivity panel (n ) 30), and in general,
the compounds were found to possess a high degree of
selectivity (Table 8).
a PBMC ) LPS induced TNF-R production in peripheral blood mono-
nuclear cells. b NT ) not tested.
Conclusion
In an attempt to improve the low metabolic stability of the
pyrazolopurine and thiophene tricycles, we developed a new
synthetic route to block the proposed common site of metabo-
lism in these molecules. Novel oxazole, thiazole, and imidazole
tricycles were prepared to test this hypothesis. The oxazole and
thiazole displayed improved potency against IKK2; however,
these parent compounds still suffered from poor PK. The
expedient nature of the synthetic route allowed for significant
diversification via a common intermediate, and this, in combina-
tion with a cassette screening strategy, was successful in
36c and 36d which represented the most improved compounds
with respect to both IKK2 and PBMC potency as well as
metabolic stability.
The in vivo activity of the thiazole tricyclic based IKK
inhibitors was demonstrated by assessment of 36d in an acute
murine model of TNFR production. Mice were dosed orally with
36d at 10 mg/kg, 4 h prior to LPS administration, and TNFR
levels were measured 90 min later. As shown in Figure 5, 36d

2000 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7
Kempson et al.
and the residue suspended in ethyl acetate (200 mL) which was
then filtered and the filtrate evaporated in vacuo to produce the
crude product. The crude product was recrystallized from methanol
identifying compounds with improved PK. When dosed orally
at 10 mg/kg, 36d inhibited LPS-induced TNF-R production in
mice. Efforts continue to optimize this and other series and will
be reported in the future.
1
(100 mL) to give 8 as a tan solid (7.2 g, 71% for two steps). H
NMR (CDCl3) δ 9.06 (s, 1H), 8.84 (s, 1H), 2.98 (s, 3H). MS (ESI)
m/z 199 (M + H)+. Purity: 98% (method A, tR ) 1.58 min).
2,6-Dichloro-N′-methylpyridine-3,4,5-triamine (4). A solution
of 8 (60.0 g, 0.30 mol) in concentrated hydrochloric acid (300 mL)
was heated to 90 °C. Tin(II) chloride (85.0 g, 0.45 mol) was added
portionwise over 1 h with vigorous effervescence noted for the first
equivalent of tin chloride added. The reaction mixture was heated
for a further hour before the additon of more tin chloride (28.0 g,
0.15mol) and continued heating for 2 more hours. The reaction
mixture was cooled to 0 °C and cautiously basified with concen-
trated ammonium hydroxide (200 mL). The precipitated solid was
filtered off and the filtrate extracted with ethyl acetate (5 × 200
mL). The combined organics were dried (MgSO4) and evaporated
Experimental Section
Proton magnetic resonance (1H) spectra were recorded on either
a Bruker Avance 400 or a JEOL Eclipse 500 spectrometer and are
reported in ppm relative to the reference solvent of the sample in
which they were run. HPLC and LCMS analyses were conducted
using a Shimadzu SCL-10A liquid chromatograph and a SPD
UV-vis detector at 220 or 254 nm with the MS detection performed
with either a Micromass Platform LC spectrometer or a Waters
Micromass ZQ spectrometer. Preparative reverse-phase HPLC
purifications were performed using the following conditions:
Ballistic YMC S5 ODS 20 mm × 100 mm column with a binary
solvent system, where solvent A is 10% methanol, 90% water, 0.1%
trifluoroacetic acid and solvent B is 90% methanol, 10% water,
and 0.1% trifluoroacetic acid; flow rate of 20 mL/min; linear
gradient time of 10 min; start %B ) 20; final %B ) 100. Fractions
containing the product were concentrated in vacuo to remove the
methanol and were neutralized with aqueous sodium bicarbonate.
The products were collected by vacuum filtration or were extracted
with organic solvents and concentrated under reduced pressure. All
flash column chromatography was performed on EM Science silica
gel 60 (particle size of 40-60 µm). All reagents were purchased
from commercial sources and used without further purification
unless otherwise noted. All reactions were performed under an inert
atmosphere. Microwave reactions were performed using a Personal
Chemistry SmithSynthesizer workstation with a power range of 300
W provided from a magnetron at 2.45 GHz. The purity of all tested
compounds was determined by HPLC (see HPLC Methods below)
and were g95% pure.
1
in vacuo to produce 4 as a brown solid (28.0 g, 46%). H NMR
(DMSO-d6) δ 4.94 (br s, 5H), 2.60 (s, 3H). 13C NMR (DMSO-d6)
δ 133.9, 130.7, 121.6, 31.9. MS (ESI) m/z 207 (M + H)+. Purity:
97% (method A, tR ) 0.71 min), 98% (method B, tR ) 1.20 min).
7-Amino-4,6-dichloro-1-methyl-1H-imidazo[4,5-c]pyridine (9).
Triethyl orthoformate (25.0 mL, 0.15mol) was added in one portion
to a suspension of 4 (28 g, 0.14mol) in dry acetonitrile (400 mL). The
reaction mixture was heated to reflux for 4 h and then cooled to room
temperature. The reaction mixture was evaporated in vacuo to produce
9 as a brown powder. 1H NMR (DMSO-d6) δ 8.20 (1H, s), 5.49 (2H,
br s), 4.07 (3H, s). MS (ESI) m/z 217 (M + H)+. Purity: 98% (method
A, tR ) 0.78 min).
N-(4,6-Dichloro-1-methyl-1H-imidazole[4,5-c]pyridin-7-yl)-
benzamide (10). Benzoyl chloride (1.2 g, 10.1 mmol) in dry
acetonitrile (100 mL) was added in one portion to a suspension of
the amine 9 (2.0 g, 9.2 mmol), and the resulting mixture was heated
to 70 °C for 6 h. The reaction mixture was concentrated in vacuo
to produce the crude product which was precipitated with methanol
HPLC Methods. HPLC analyses were performed using the
following conditions.
1
and filtered to give 7 (1.3 g, 44%). H NMR (DMSO-d6) δ: 8.30
(1H, s), 8.20 (2H, app d, J ) 1.0 Hz), 7.87 (2H, app t, J ) 6.0
Hz), 7.70 (2H, app t, J ) 8.0 Hz), 5.55 (1H, s), 4.15 (3H, s). MS
(ESI) m/z 321 (M + H)+. Purity: 98% (method A, tR ) 1.20 min).
7-Chloro-4-methyl-2-phenyl-4H-imidazo[4,5-d]oxazolo[5,4-b]-
pyridine (11). Sodium carbonate (0.35 g, 3.3mmol) was added in
one portion to a solution of 10 (0.96 g, 3.0 mmol) in N,N-
dimethylformamide (30 mL), and the reaction mixture was heated
to 160 °C for 24 h. The reaction mixture was cooled to room
temperature and evaporated in vacuo. The residue was partitioned
between dichloromethane (50 mL) and water (50 mL). The
separated organic layer was dried (MgSO4) and evaporated in vacuo
Method A. A linear gradient program using 10% MeOH-90%
H2O-0.1% TFA (solvent A) and 90% MeOH-10% H2O-0.1%
TFA (solvent B); t ) 0 min, 0% B, t ) 2 min, 100% B; was
employed on a YMC ODS-A S7 C28 3.0 mm × 50 mm column.
Flow rate was 5 mL/min, and UV detection was set to 254 nm.
The LC column was maintained at ambient temperature.
Method B. A linear gradient program using 10% MeOH-90%
H2O-0.2% H3PO4 (solvent A) and 90% MeOH-10% H2O-0.2%
H3PO4 (solvent B); t ) 0 min, 0% B, t ) 4 min, 100% B; was
employed on a YMC ODS-A S7 C28 3.0 mm × 50 mm column.
Flow rate was 4 mL/min, and UV detection was set to 254 nm.
The LC column was maintained at ambient temperature.
1
to produce the oxazole 11 (0.42 g, 49%). H NMR (DMSO-d6) δ
8.34 (1H, s), 8.05 (2H, dd, J ) 6.0 Hz, 1.0 Hz), 7.50-7.45 (3H,
m), 4.02 (3H, s). MS (ESI) m/z 285 (M + H)+. Purity: 98% (method
A, tR ) 1.74 min), 100% (method B, tR ) 3.43 min).
Method C. A linear gradient program using 5% acetoni-
trile-95% H2O-0.05% TFA (solvent A) and 95% acetonitrile-5%
H2O-0.05% TFA (solvent B); t ) 0 min, 10% B, t ) 25 min,
100% B; was employed on a SunFire C18 3.5 µm, 4.6 mm × 150
mm column. Flow rate was 1 mL/min, and UV detection was set
to 254 nm. The LC column was maintained at ambient temperature.
Method D. A linear gradient program using 5% acetoni-
trile-95% H2O-0.05% TFA (solvent A) and 95% acetonitrile-5%
H2O-0.05% TFA (solvent B); t ) 0 min, 10% B, t ) 25 min,
100% B; was employed on a XBridge phenyl 3.5 µm, 4.6 mm ×
150 mm column. Flow rate was 1 mL/min, and UV detection was
set to 254 nm. The LC column was maintained at ambient
temperature.
(3,5-Dinitropyridin-4-yl)methylamine (8). 6 (10.0 g, 0.051 mol)
was added portionwise to a mixture of phosphorus oxychloride (25
mL) and PCl5 (17.0 g, 0.082 mol). The reaction mixture was heated
to reflux under a nitrogen atmosphere for 12 h. The reaction mixture
was allowed to cool to room temperature and the phosphorus
oxychloride removed in vacuo. The residue was suspended in dry
THF (50 mL) and cooled to 0 °C. Methylamine (32 mL, 2.0 M in
THF, 0.064 mol) was added dropwise over 20 min under a nitrogen
atmosphere, and the resulting solution was allowed to warm to room
temperature over 1 h. The reaction mixture was evaporated in vacuo
4-Methyl-7-methylamino-2-phenyl-4H-imidazo[4,5-d]oxazo-
lo[5,4-b]pyridine (12). Methylamine hydrochloride (104 mg, 1.54
mmol) and diisopropylethylamine (0.54 mL, 3.08mmol) were each
added in one portion to a suspension of 11 (200 mg, 0.7 mmol) in
n-butanol (1 mL). The reaction mixture was heated in the
microwave at 180 °C for 4 h. The reaction mixture was evaporated
in vacuo and the residue suspended between ethyl acetate (5 mL)
and water (5 mL). The separated organic layer was dried (MgSO4)
and evaporated in vacuo to leave the crude product which was
purified by preparative HPLC (reverse phase) to produce 12 (38
1
mg, 19%). H NMR (DMSO-d6) δ 8.20 (1H, s), 8.07-7.98 (2H,
m), 7.49-7.38 (3H, m), 4.83 (3H, s), 4.08 (3H, s). HRMS for
C15H14O1N5 (M + H)+, calcd: 280.1193. Found: 280.1191. Purity:
100% (method C, tR ) 12.57 min), 100% (method D, tR ) 10.97
min).
7-Chloro-4-methyl-2-phenyl-4H-imidazo[4,5-d]thiazolo[5,4-b]-
pyridine (13). A mixture of 10 (43 mg, 0.13 mmol) and Lawesson’s
reagent (80 mg, 0.2 mmol) in toluene (2 mL) was heated to 110
°C for 5 h. After the mixture was cooled to room temperature, 1,8-
diazabicyclo[5.4.0]undec-7-ene (0.06 mL, 0.4 mmol) was added

NoVel Tricyclic Inhibitors of IκB Kinase
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7 2001
and the reaction mixture was allowed to stir for 60 h. The volatiles
were removed in vacuo, and the residue was dissolved in N,N-
dimethylacetamide (2 mL). After adding potassium carbonate (50
mg, 0.36 mmol), the reaction mixture was heated to 160 °C for
1 h. The reaction mixture was cooled to room temperature and
partitioned between ethyl acetate (20 mL) and water (20 mL). After
washing with water (2 × 20 mL) and brine (20 mL), the organic
layer was dried (MgSO4). Filtration and concentration afforded a
crude residue that was purified by preparative thin layer chroma-
tography (20 cm × 20 cm, 1 mm thick silica gel plate) using ethyl
acetate as the eluent. Extraction of the desired band with ethyl
acetate, filtration, and concentration afforded 21 mg (75%) of 13
as an off-white solid. 1H NMR (DMSO-d6) δ 8.52 (1H, s),
8.15-8.12 (2H, m), 7.65-7.58 (3H, m), 4.30 (3H, s). MS (ESI)
m/z 301 (M + H)+. Purity: 93% (method A, tR ) 1.89 min), 92%
(method B, tR ) 3.73 min).
4-Methyl-7-methylamino-2-phenyl-4H-imidazo[4,5-d]thiazo-
lo[5,4-b]pyridine (14). A mixture of 13 (19 mg, 0.063 mmol),
methylamine hydrochloride (22 mg, 0.32 mmol), and diisopropy-
lethylamine (0.092 mL, 0.5 mmol) in 0.5 mL of n-butanol was
heated to 180 °C for 4.5 h in a sealed tube, using a microwave
apparatus. After cooling to room temperature, the reaction mixture
was partitioned between ethyl acetate (20 mL) and saturated sodium
bicarbonate solution (20 mL). The aqueous layer was extracted with
ethyl acetate (20 mL), and the combined organic layers were dried
(MgSO4). Filtration and concentration afforded a residue that was
triturated with ethyl ether and dried to afford 6 mg (33%) of 14 as
a yellow powder. 1H NMR (DMSO-d6) δ 8.20 (1 H, s), 8.01 (1 H,
d, J ) 6.80 Hz), 7.42-7.60 (3 H, m), 4.22 (3 H, s), 3.00 (3 H, s).
HRMS for C15H14N5S1 (M + H)+, calcd: 296.0964. Found:
296.0956. Purity: 100% (method C, tR ) 11.12 min), 95% (method
D, tR ) 10.73 min).
N-(4,6-Dichloro-1-methyl-1H-imidazo[4,5-c]pyridin-7-yl)-N′-
methylbenzamidine (15). 10 (2.65 g, 8.3mmol) was suspended in
toluene (100 mL) and thionyl chloride (1.97 g, 16.6mmol) added
in one portion. The resulting mixture was refluxed for 24 h before
cooling to room temperature and evaporating in vacuo. The resulting
tan solid was washed with diethyl ether (100 mL) and dried under
vacuum to afford 2.8 g of a solid that was immediately used in the
next reaction.
The tan solid (1.0 g, 2.9mmol) was added in one portion to a
mixture of methylamine hydrochloride (398 mg, 5.9 mmol) and
N,N-diisopropylethylamine (760 mg, 5.9mmol) in acetonitrile (100
mL). The resulting mixture was refluxed for 48 h before cooling
and evaporating in vacuo. The residue was then partitioned between
dichloromethane (50 mL) and saturated sodium bicarbonate solution
(75 mL). The organic layer was then dried (MgSO4) and evaporated
in vacuo. The residue was triturated with diethyl ether (100 mL)
to afford 0.8 g (83%) of 15 as a white powder. 1H NMR (DMSO-
d6) δ 8.30 (1 H, s), 7.70-7.92 (2 H, m), 7.12-7.38 (3 H, m), 4.02
(3 H, s), 2.94 (3 H, d, J ) 3.27 Hz). HRMS for C15H14N5Cl2 (M
+ H)+, calcd: 334.062 08. Found: 334.061 54. Purity: 95% (method
B, tR ) 1.25 min).
butanol (3 mL). The resulting mixture was heated in the microwave
at 185 °C for 4 h. The reaction mixture was then evaporated in
vacuo and purified by preparative HPLC to afford 124 mg (18%)
1
of 17 as a yellow solid. H NMR (DMSO-d6) δ 8.15 (1 H, s),
7.83-7.93 (1 H, m), 7.54-7.68 (4 H, m), 4.12 (3 H, s), 3.92 (3 H,
s), 3.03 (3 H, s). HRMS for C16H17N6 (M + H)+, calcd: 293.150 92.
Found: 293.150 39. Purity: 97% (method C, tR ) 6.62 min), 95%
(method D, tR ) 8.14 min).
7-Chloro-4-methyl-2-(methylthio)-4H-imidazo[4,5-d]thiazo-
lo[5,4-b]pyridine (18). 9 (2.17 g, 10 mmol) and potassium
thioxanthate (3.05 g, 20mmol) were added to 20 mL of DMF and
heated for 2.5 h at 145 °C. The reaction mixture was cooled to
room temperature and then placed in an ice bath and cooled to ∼0
°C. Methyl iodide (1.24 mL, 20 mmol) was added and the reaction
mixture stirred for 1 h in the ice bath. Volatile liquids were removed
at ∼45 °C under high vacuum. The residue was partitioned between
chloroform (150 mL) and saturated sodium bicarbonate (100 mL).
The aqueous layer was washed with additional chloroform (2 ×
75 mL). The combined organic layers were dried over magnesium
sulfate, filtered, and evaporated to provide a cream-colored solid.
The crude product was triturated with hot ethyl acetate (∼75 mL)
which was allowed to cool to room temperature. The product was
collected by filtration, and 1.49 g (55%) of 18 was isolated as a
cream-colored solid. 1H NMR (DMSO-d6) δ 8.48 (1 H, s), 4.20 (3
H, s), 2.86 (3 H, s). HRMS for C9H8N4Cl1S2 (M + H)+, calcd:
270.9873. Found: 270.9872. Purity: 88% (method A, tR ) 1.58
min), 92% (method B, tR ) 3.06 min).
4-Methyl-7-methylamino-2-(methylthio)-4H-imidazo[4,5-d]thi-
azolo[5,4-b]pyridine (19). 18 (540 mg, 2 mmol) and 1.5 mL of
methylamine in ethyl alcohol (12 mmol) was added to n-butanol
(5 mL) and heated to 180 °C in a sealed tube for 4 h and cooled to
room temperature. HPLC indicated that the reaction had not
proceeded to completion. Additional methylamine in ethyl alcohol
(12 mmol) was added and the reaction vessel sealed and heated
for an additional 4 h. The reaction mixture was cooled to room
temperature and the volatile solvent removed under vacuum. The
residue was triturated with water and the residue dried under high
1
vacuum to provide 428 mg (91%) of 19 as a white powder. H
NMR (DMSO-d6) δ 8.09 (1 H, s), 7.13 (1 H, d, J ) 4.78 Hz), 4.11
(3 H, s), 2.94 (3 H, d, J ) 4.78 Hz), 2.75 (3 H, s). HRMS for
C10H12N5S2 (M + H)+, calcd: 266.0529. Found: 266.0526. Purity:
99% (method C, tR ) 7.50 min), 100% (method D, tR ) 7.64 min).
2-Methanesulfonyl-4-methyl-7-methylamino-4H-imidazo[4,5-d]-
thiazolo[5,4-b]pyridine (20). To a stirred mixture of 19 (5.22 g,
19.6 mmol) in MeOH (80 mL) was added a suspension of Oxone
(35 g) in H2O (80 mL). After 3 h, the mixture was partially
concentrated, diluted with H2O, neutralized with NaHCO3 and then
the solid was collected by filtration to provide 3.22 g (55%) of 20.
1H NMR (DMSO-d6) δ 8.23 (1 H, s), 7.83 (1 H, d, J ) 4.78 Hz),
4.15 (3 H, s), 3.49 (3 H, s), 3.00 (3 H, d, J ) 4.53 Hz). HRMS for
C10H12O2N5S2 (M + H)+, calcd: 298.0427. Found: 298.0423. Purity:
100% (method C, tR ) 8.15 min), 95% (method D, tR ) 7.63 min).
2-Hydroxy-4-methyl-7-methylamino-4H-imidazo[4,5-d]thia-
zolo[5,4-b]pyridine (21). To 20 (3.22 g) as a suspension in H2O
(25 mL) was added 5 N NaOH, and the resulting mixture was heated
to 120 °C for 3 h. The mixture was then allowed to cool to room
temperature before acidifying with 20 mL AcOH and the resulting
solid was collected by filtration to afford 2.22 g (48%) of 21 as a
tan solid. MS (ESI) m/z 236 (M + H)+. Purity: 95% (method A, tR
) 1.58 min).
2-Chloro-4-methyl-7-methylamino-4H-imidazo[4,5-d]thiazo-
lo[5,4-b]pyridine (22). A mixture of 21 (1.00 g, 4.25 mmol) and
pyridine (0.35 mL) in POCl3 (20 mL) was heated to 140 °C with
stirring for 48 h. The mixture was cooled to room temperature and
concentrated. The residue was partitioned between 1% H2SO4 and
CHCl3, and the aqueous phase was separated and extracted with
THF (2×). The combined organic phases were dried (MgSO4),
filtered, and concentrated to dryness. The residue was triturated
with CHCl3/EtOAc 1:10 to provide 975 mg (90%) 22 as a beige
solid. MS (ESI) m/z 254 (M + H)+. Purity: 98% (method A, tR )
1.76 min).
7-Chloro-1,4-dimethyl-2-phenyl-4H-imidazo[4,5-d]imidazo[5,4-b]-
pyridine (16). Potassium tert-butoxide (200 mg, 1.8mmol) was
added in one portion to a solution of 15 (300 mg, 0.9mmol) in
DMF (2.0 mL), and the resulting solution was heated in the
microwave at 200 °C for 1 h. The solvent was then removed in
vacuo and the residue taken up in 95:5 dichloromethane/methanol
(50 mL). This organic layer was then washed with water (2 × 50
mL), and the separated organic layer was then dried (MgSO4) and
evaporated in vacuo. The residue was then triturated with diethyl
1
ether (50 mL) to afford 179 mg (67%) of 16 as a tan solid. H
NMR (DMSO-d6) δ 8.36 (1 H, s), 7.93 (1 H, dd, J ) 7.55, 2.01
Hz), 7.51-7.71 (4 H, m), 4.21 (3 H, s), 3.97 (3 H, s). HRMS for
C15H13N5Cl1 (M + H)+, calcd: 298.085 40. Found: 298.084 85.
Purity: 95% (method B, tR ) 2.84 min).
1,4-Dimethyl-7-methylamino-2-phenyl-4H-imidazo[4,5-d]imi-
dazo[5,4-b]pyridine (17). 16 (700 mg, 2.4mmol) was added in one
portion to a mixture of methylamine hydrochloride (700 mg, 10.3
mmol) and N,N-diisopropylethylamine (1.4 g, 10.8mmol) in n-

2002 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7
Kempson et al.
4-Methyl-7-methylamino-2-methylamino-4H-imidazo[4,5-d]thi-
azolo[5,4-b]pyridine (23a). To a solution of 22 (10 mg, 0.039
mmol) in NMP (0.5 mL) was added a solution of methylamine
(1 mL of a 0.5 M solution in NMP) and K2CO3 (10 mg) and then
heated to 140 °C overnight. The mixture was cooled to room
temperature, and AcOH (0.50 mL) was added, filtered, and purified
directly by preparative HPLC to afford 3.3 mg (28%) of 23a as a
film. 1H NMR (DMSO-d6) δ 7.96 (1 H, s), 7.55 (1 H, d, J ) 4.78
Hz), 6.49 (1 H, d, J ) 4.78 Hz), 4.06 (3 H, s), 3.33 (6 H, s). HRMS
for C10H13N6S1 (M + H)+, calcd: 249.0917. Found: 249.0914.
oil bath (120 °C) and stirred for 1.5 h before cooling to room
temperature and pouring into an ice/1 N ammonium chloride
solution (150 mL). The resulting solid was collected by filtration
and dried in a vacuum oven at 60 °C overnight to afford 2.55 g
(93%) of 5-chloro-8-methyl-8H-imidazo[4,5-d]thiazolo[5,4-b]py-
1
ridine-2-ylbenzamide as an off-white solid. H NMR (DMSO-d6)
δ 13.17 (1H, s), 8.49 (1H, s), 8.17 (2H, d, J ) 7.3 Hz), 7.70-7.65
(1H, m), 7.60 (2H, t, J ) 7.60 Hz), 4.26 (3H, s). MS (ESI) m/z
344, 346 (M + H)+. Purity: 98% (method A, tR ) 2.02 min).
The off-white solid was added portionwise to a hot (85 °C)
solution of hydrochloric acid (50 mL), anhydrous ethanol (25 mL),
and dioxane (25 mL). The resulting heterogeneous mixture was
heated to 100 °C for 16 h, during which time the solution had
become homogeneous. The reaction mixture was allowed to cool
to room temperature, and then the organics were evaporated under
reduced pressure. The remaining precipitate was cooled in an ice
bath and carefully neutralized with sodium hydroxide (25% w/v
aqueous solution) while keeping the internal temperature below 15
°C. The solid was collected by vacuum filtration and washed with
water. The air-dried solid was then triturated in hot acetonitrile and
then allowed to cool to room temperature before filtering and
allowing to air-dry to afford 1.41 g (67%) of 25 as a light-tan solid.
1H NMR (DMSO-d6) δ 8.32 (1 H, s), 7.93 (2 H, s), 4.05 (3 H, s).
MS (ESI) m/z 240, 242 (M + H)+. Purity: 98% (method A, tR )
0.69 min), 95% (method B, tR ) 1.08 min).
7-Chloro-4-methyl-2-bromo-4H-imidazo[4,5-d]thiazolo[5,4-b]-
pyridine (26). Isoamyl nitrite (5.58 mL, 41.7mmol) was added
dropwise over 5 min to a suspension of copper(II) bromide (4.66
g, 20.8mmol) and 25 (5.0 g, 20.8mmol) in anhydrous acteonitrile
(150 mL). The reaction mixture was stirred at 65 °C for 16 h before
cooling to room temperature and filtering. The solid was slurried
in ammonium chloride (10% w/v aqueous solution, 125 mL) and
concentrated ammonium hydroxide solution (125 mL) with soni-
cation for 30 min. The suspension was then filtered and dried to
afford 4.62 g (73%) 26 as a light-pink solid. 1H NMR (DMSO-d6)
δ 8.61 (1 H, s), 4.25 (3 H, s). HRMS for C8H5N4Br1Cl1S1 (M +
H)+, calcd: 302.9101. Found: 302.9101. Purity: 97% (method A,
tR ) 1.56 min), 97% (method D, tR ) 3.02 min). Anal. Calcd for
C8H5N4Br1Cl1S1: C, 31.65; H, 1.32; N, 18.45; S, 10.56; Br, 26.32;
Cl, 11.67. Found: C, 32.20; H, 1.38; N, 18.57; S, 9.82; Br, 22.33;
Cl, 12.60; Cu, <0.01%.
Purity: 95% (method C, tR ) 4.12 min), 100% (method D, tR
)
4.63 min).
4-Methyl-7-methylamino-2-benzylamino-4H-imidazo[4,5-d]thi-
azolo[5,4-b]pyridine (23b). 23b was prepared as a pale-yellow solid
1
following the procedure described for 23a. H NMR (DMSO-d6)
δ 8.10 (1 H, s), 7.34-7.40 (4 H, m), 7.13-7.17 (1 H, m), 4.63 (2
H, s), 4.15 (3 H, s), 3.00 (3 H, d, J ) 4.53 Hz). HRMS for
C16H16N6S1 (M + H)+, calcd: 324.1157. Found: 327.1148. Purity:
100% (method C, tR ) 8.15 min), 95% (method D, tR ) 7.63 min).
4-Methyl-7-methylamino-2-hydroxyethylamino-4H-imidazo[4,5-d]-
thiazolo[5,4-b]pyridine (23c). 23c was prepared as a pale-yellow
solid following the procedure described for 23a. 1H NMR (DMSO-
d6) δ 7.95 (1 H, s), 7.49-7.74 (1 H, m), 6.38-6.55 (1 H, m), 4.78
(1 H, t, J ) 5.29 Hz), 4.07-4.16 (2 H, m), 4.04 (3 H, s), 3.59 (2
H, q, J ) 5.71 Hz), 3.16 (3 H, s). HRMS for C11H15O1N6S1 (M +
H)+, calcd: 279.1023. Found: 279.1023. Purity: 95% (method C,
tR ) 3.28 min), 100% (method D, tR ) 3.60 min).
4-Methyl-7-methylamino-2-[1,2,3,6-tetrahydropyridine]-4H-
imidazo[4,5-d]thiazolo[5,4-b]pyridine (23d). 23d was prepared as
a pale-yellow solid following the procedure described for 23a. 1H
NMR (DMSO-d6) δ 7.99 (1 H, s), 6.55-6.72 (1 H, m), 5.70-6.00
(2 H, m), 4.04-4.20 (5 H, m), 3.98 (2 H, d, J ) 2.27 Hz), 3.64 (2
H, t, J ) 5.67 Hz), 3.20 (3 H, s). HRMS for C14H17N6S1 (M +
H)+, calcd: 301.1230. Found: 301.1230. Purity: 100% (method C,
tR ) 7.79 min), 100% (method D, tR ) 9.05 min).
4-Methyl-7-methylamino-2-piperidine-4H-imidazo[4,5-d]thia-
zolo[5,4-b]pyridine (23e). 23e was prepared as a pale-yellow solid
1
following the procedure described for 23a. H NMR (DMSO-d6)
δ 8.05 (1 H, s), 4.15 (3 H, s), 3.56-3.60 (4 H, m), 3.10 (3 H, s),
1.66-1.77 (6 H, m). HRMS for C14H18N4S1 (M + H)+, calcd:
302.1314. Found: 302.1310. Purity: 100% (method C, tR ) 8.15
min), 95% (method D, tR ) 7.63 min).
Methyl 7-Chloro-4-methyl-4H-imidazo[4,5-d]thiazolo[5,4-b]-
pyridine-2-carboxylate (27). A mixture of 26 (4.00 g, 13.18 mmol),
PdCl2(PPh3)2 (277 mg, 0.395 mmol), Et3N (2.75 mL, 19.7 mmol)
in CH3CN (60 mL) and MeOH (60 mL) was stirred under an
atmosphere of CO (100 psi) in a bomb at 60 °C. After 24 h, the
mixture was cooled to room temperature and depressurized. The
solution was concentrated to dryness and triturated with hot ethyl
acetate. When the mixture was cooled, the solid was collected by
4-Methyl-7-methylamino-2-(azepan-1-yl)-4H-imidazo[4,5-d]thi-
azolo[5,4-b]pyridine (23f). 23f was prepared as a pale-yellow solid
1
following the procedure described for 23a. H NMR (DMSO-d6)
δ 8.23 (1 H, s), 7.83 (1 H, d, J ) 4.78 Hz), 4.15 (3 H, s), 3.49 (6
H, s), 3.33 (6 H, s), 3.00 (3 H, d, J ) 4.53 Hz). HRMS for
C15H21N6S1 (M + H)+, calcd: 317.1543. Found: 317.1542. Purity:
97% (method C, tR ) 9.03 min), 97% (method D, tR ) 10.50 min).
1-Benzoyl-3-(4,6-dichloro-1-methyl-1H-imidazo[4,5-c]pyridine-
7-yl)thiourea (24). 9 (6.94 g, 31.0 mmol) was suspended in acetone
(120 mL). Benzoyl isothiocyanate (5.8 g, 35 mmol) was added
dropwise over 10 min, and the reaction mixture was allowed to
stir at room temperature overnight before heating to reflux for 1 h.
The reaction mixture was then cooled to room temperature and the
solvent removed under reduced pressure. Ethyl alcohol (50 mL)
was added to the solid and the mixture heated to reflux for 1 h,
then cooled to room temperature and allowed to stand for several
hours. The product was collected by filtration and dried under
vacuum to afford 11.32 g (93%) of 24 as a white powder. 1H NMR
(DMSO-d6) δ 12.18 (1H, s), 12.10 (1H, s), 8.48 (1H, s), 8.10-8.06
(2H, m), 7.70-7.65 (1H, m), 7.59-7.55 (2H, m), 4.26 (3H, s).
MS (ESI) m/z 380, 382 (M + H)+. Purity: 98% (method A, tR )
2.10 min).
1
filtration to afford 3.38 g (91%) of 27 as a yellow solid. H NMR
(DMSO-d6) δ 8.72 (1 H, s), 4.25 (3 H, s), 3.94 (3H, s). MS (ESI)
m/z 283 (M + H)+. Purity: 95% (method A, tR ) 1.60 min)
7-Chloro-4-methyl-4H-imidazo[4,5-d]thiazolo[5,4-b]pyridine-
2-carboxylic Acid (28). To a stirring solution of 27 (3.38 g, 12.0
mmol) in THF (700 mL) was slowly added NaOH (1 N, 40 mL).
After 40 min, the mixture was concentrated to dryness, 25 mL of
cold H2O was added, the mixture was acidified with 40 mL of 1 N
HCl, and the resulting solid was collected by filtration. After the
sample was dried under high vacuum, 1.72 g (53%) of 28 was
obtained as a colorless solid. 1H NMR (DMSO-d6) δ 8.62 (1 H, s),
4.19 (3 H, s). MS (ESI) m/z 269 (M + H)+. Purity: 100% (method
A, tR ) 1.45 min).
7-Methylamino-4-methyl-4H-imidazo[4,5-d]thiazolo[5,4-b]py-
ridine-2-carboxylic Acid (29). Starting with 28 (1.71 g) and
dividing into 150 mg lots, to each lot was added MeNH2 (33 wt %
in EtOH, 1.8 mL) and the suspension heated for 20 min at 150 °C
in the microwave reactor. Once cooled to room temperature, the
solid was collected by filtration. The combined crude solids were
dissolved in a solution of 50 mmol of NH4OAc in H2O (50 mL),
purified on a C18 column eluted with 50 mmol of NH4OAc in H2O
solution and gradient with MeOH. Fractions containing product
7-Chloro-4-methyl-2-amino-4H-imidazo[4,5-d]thiazolo[5,4-b]-
pyridine (25). Potassium tert-butoxide (1 M solution in THF, 24
mL, 24 mmol) was evaporated to dryness under reduced pressure
and the residue dissolved in NMP 15 mL). 24 (3.04 g, 8.0mmol)
was dissolved in NMP (15 mL) and added dropwise to the solution
of potassium butoxide, during which time the mixture had become
warm to the touch. The reaction mixture was placed into a preheated

NoVel Tricyclic Inhibitors of IκB Kinase
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7 2003
were combined and concentrated. The resulting solid was dissolved
in H2O and then lyophilized to provide 605 mg (36%) of 29 as a
colorless solid. 1H NMR (DMSO-d6) δ 8.62 (1 H, s), 4.19 (3 H, s),
3.00 (3 H, s). MS (ESI) m/z 264 (M + H)+. Purity: 95% (method
A, tR ) 1.45 min).
1.20 (3 H, t, J ) 7.18 Hz). HRMS for C16H15N5F1S1 (M + H)+,
calcd: 328.102 67. Found: 328.102 12. Purity: 100% (method C,
tR ) 13.52 min), 95% (method D, tR ) 12.41 min).
8-Methyl-2-(4-fluorophenyl)-8H-imidazo[4,5-d]thiazolo[5,4-b]-
pyridine-5-amine (32c). 32c was prepared as a pale-yellow solid
1
4-Methyl-7-methylamino-2-methylamido-4H-imidazo[4,5-d]thi-
azolo[5,4-b]pyridine (30a). To a stirring solution of 29 (32.4 mg,
0.123 mmol) in NMP (0.50 mL) was added MeNH2 (33 wt % in
EtOH, 0.20 mL) followed by PyBop (64.1 mg, 0.123 mmol). After
30 min, to the mixture was added AcOH (1 mL), DMF (1 mL).
This solution was applied directly onto preparative HPLC to provide
following the procedure described for 32a. H NMR (DMSO-d6)
δ 8.43 (1 H, s), 8.01-8.21 (2 H, m), 7.41 (2 H, t, J ) 8.81 Hz),
4.25 (3 H, s). HRMS for C14H11N5F1S1 (M + H)+, calcd: 300.0714.
Found: 300.0711. Purity: 97.9% (method C, tR ) 10.47 min), 94.0%
(method D, tR ) 10.41 min).
Acetamide,N-[2-[[2-(4-Fluorophenyl)-8-methyl-8H-imidazo[4,5-d]-
thiazolo[5,4-b]pyridine-5-yl]amino]ethyl]-(32d). 32d was prepared
as a pale-yellow solid following the procedure described for 32a.
1H NMR (DMSO-d6) δ 8.16 (1 H, s), 7.96-8.10 (2 H, m),
7.26-7.47 (2 H, m), 4.19 (3 H, s), 3.48-3.65 (2 H, m), 3.23-3.41
(2 H, m), 1.70 (3 H, s). HRMS for C17H18O4N5F1S1 (M + H)+,
calcd: 407.105 81. Found: 407.105 59. Purity: 98.2% (method C,
tR ) 11.23 min), 91.4% (method D, tR ) 10.85 min).
Ethanol, N-[2-[[2-(4-Fluorophenyl)-8-methyl-8H-imidazo[4,5-d]-
thiazolo[5,4-b]pyridine-5-yl]amino]ethyl]-(32e). 32e was prepared
as a pale-yellow solid following the procedure described for 32a.
1H NMR (DMSO-d6) δ 8.22 (1 H, s), 8.00-8.15 (2 H, m),
7.52-7.64 (1 H, m), 7.24-7.47 (2 H, m), 5.24 (1 H, t, J ) 4.66
Hz), 4.20 (3 H, s), 3.81 (2 H, d, J ) 6.04 Hz), 3.65 (2 H, q, J )
4.95 Hz), 3.24 (2 H, t, J ) 5.92 Hz), 3.02-3.12 (2 H, m). HRMS
for C18H20O1N6F1S1 (M + H)+, calcd: 387.139 79. Found: 387.139 19.
Purity: 98.0% (method C, tR ) 8.76 min), 95.0% (method D, tR )
10.05 min).
1
14 mg (36%) of 30a as a pale-yellow solid. H NMR (DMSO-d6)
δ 8.23 (1 H, s), 3.49 (3 H, s), 3.13 (3 H, s), 3.00 (3 H, d, J ) 4.53
Hz). HRMS for C11H12O1N6S1 (M + H)+, calcd: 276.0793. Found:
276.0785. Purity: 95% (method C, tR ) 8.04 min), 95% (method
D, tR ) 7.21 min).
4-Methyl-7-methylamino-2-ethylamido-4H-imidazo[4,5-d]thi-
azolo[5,4-b]pyridine (30b). 30b was prepared as a pale-yellow solid
1
following the procedure described for 30a. H NMR (DMSO-d6)
δ 8.21 (1 H, s), 3.92 (3 H, s), 3.40 (2 H, q, J ) 4.25 Hz), 2.97 (3
H, s), 1.20 (3 H, t, J ) 4.25 Hz). HRMS for C12H14O1N6S1 (M +
H)+, calcd: 290.0590. Found: 290.0582. Purity: 100% (method C,
tR ) 8.25 min), 95% (method D, tR ) 7.54 min).
4-Methyl-7-methylamino-2-cyclopentylamido-4H-imidazo[4,5-d]-
thiazolo[5,4-b]pyridine (30c). 30c was prepared as a pale-yellow
solid following the procedure described for 30a. 1H NMR (DMSO-
d6) δ 8.44 (1 H, d, J ) 8.06 Hz), 8.16 (1 H, s), 7.52 (1 H, d, J )
4.78 Hz), 4.24 (3 H, s), 2.98 (3 H, d, J ) 4.78 Hz), 2.62-2.70 (1
H, m), 2.27-2.36 (2 H, m), 1.48-1.68 (3 H, m), 1.22 (3 H, s).
HRMS for C15H19O1N6S1 (M + H)+, calcd: 331.133 56. Found:
331.133 73. Purity: 100% (method C, tR ) 9.86 min), 95% (method
D, tR ) 9.38 min).
8H-Imidazo[4,5-d]thiazolo[5,4-b]pyridine-5-amine,2-(4-fluo-
rophenyl)-8-methyl-N-[2-(1-piperidinyl)ethyl]-(32f). 32f was pre-
pared as a pale-yellow solid following the procedure described for
1
32a. H NMR (DMSO-d6) δ 8.15 (1 H, s), 8.00-8.11 (2 H, m),
4-Methyl-7-methylamino-2-diethylamido-4H-imidazo[4,5-d]thi-
azolo[5,4-b]pyridine (30d). 30d was prepared as a pale-yellow solid
7.37 (2 H, t, J ) 8.81 Hz), 6.96-7.01 (1 H, m), 4.21 (3 H, s), 3.59
(2 H, q, J ) 6.55 Hz), 2.55 (2 H, t, J ) 6.67 Hz), 2.35-2.44 (4 H,
m), 1.44-1.58 (3 H, m), 1.32-1.43 (3 H, m). HRMS for
C21H24N6F1S1 (M + H)+, calcd: 411.1762. Found: 411.1760. Purity:
100.0% (method C, tR ) 10.69 min), 94.7% (method D, tR ) 12.57
min).
1
following the procedure described for 30a. H NMR (DMSO-d6)
δ 8.12 (1 H, s), 4.12 (3 H, s), 3.06-3.19 (4 H, m), 2.99 (3 H, d,
J ) 4.28 Hz), 1.26-1.40 (3 H, m), 1.17 (3 H, t, J ) 7.05 Hz).
HRMS for C14H19O1N6S1 (M + H)+, calcd: 319.133 56. Found:
319.133 48. Purity: 95% (method C, tR ) 8.19 min), 95% (method
D, tR ) 8.11 min).
N-[[3-[8-Methyl-5-chloro-8H-imidazo[4,5-d]thiazolo[5,4-b]py-
ridine-2-yl]phenyl]methyl]-tert-butyl Carbamate (33). To a
stirred solution of 26 (500 mg, 1.65 mmol), 3-(N-Boc-aminometh-
yl)phenylboronic acid (497 mg, 1.98 mmol), and Pd(PPh3)4 (83 mg,
0.083 mmol) in DME (10 mL) and EtOH (10 mL) was added
K2CO3 (1.65 mL, 2 M in H2O, 3.3 mmol), and then the mixture
was heated to reflux. After 2 h, the mixture was cooled to room
temperature and the solvent evaporated under reduced pressure. The
residue was partitioned between water (20 mL) and ethyl acetate
(20 mL). The organic layer was dried (MgSO4) and evaporated
under reduced pressure before purifying by column chromatography
using ethyl acetate as eluent to afford 350 mg (49%) of 33 as a
4-Methyl-7-methylamino-2-thienylamido-4H-imidazo[4,5-d]thi-
azolo[5,4-b]pyridine (30e). 30e was prepared as a pale-yellow solid
1
following the procedure described for 30a. H NMR (DMSO-d6)
δ 8.19 (1 H, s), 7.54-7.50 (1 H, m), 7.22-7.20 (1 H, m), 7.03-7.01
(1 H, m), 4.63 (2 H, s), 4.15 (3 H, s), 2.95 (3 H, s). HRMS for
C15H15O1N6S2 (M + H)+, calcd: 358.0671. Found: 356.0668. Purity:
95% (method C, tR ) 8.15 min), 95% (method D, tR ) 7.63 min).
8-Methyl-2-(4-fluorophenyl)-5-chloro-8H-imidazo[4,5-d]thia-
zolo[5,4-b]pyridine (31). To a stirred solution of 26 (156.2 mg,
0.50 mmol), 4-fluorophenylboronic acid (170 mg, 1.21 mmol), and
Pd(PPh3)4 (34 mg, 0.0294 mmol) in DME (5 mL) and EtOH (2
mL) was added K2CO3 (1 mL, 2 M in H2O), and then the mixture
was heated to reflux. After 1 h, the mixture was cooled to room
temperature and the solid collected by filtration and washed with
EtOH to afford 61 mg (39%) of 31 as a yellow solid. MS (ESI)
m/z 319 (M + H)+. Purity: 99% (method A, tR ) 1.45 min).
N-Methyl-2-(4-fluorophenyl)-8-methyl-8H-imidazo[4,5-d]thia-
zolo[5,4-b]pyridine-5-amine (32a). A solution of 31 (26.5 mg,
0.0831 mmol) and MeNH2 (33 wt % in EtOH, 0.60 mL) was heated
for 60 min at 150 °C in the microwave reactor. The resulting solid
was collected by filtration and washed with EtOH to afford 13.6
1
yellow solid. H NMR (DMSO-d6) δ 8.53 (1 H, s), 7.90-8.19 (2
H, m), 7.50-7.66 (2 H, m), 7.40-7.49 (1 H, m), 4.32 (3 H, s),
4.21-4.28 (2 H, m), 1.43 (9 H, s). MS (ESI) m/z 430, 432 (M +
H)+. Purity: 87% (method B, tR ) 3.40 min).
N-[[3-[8-Methyl-5-methylamino-8H-imidazo[4,5-d]thiazolo[5,4-b]-
pyridine-2-yl]phenyl]methyl]-tert-butyl Carbamate (34). A solu-
tion of 33 (250 mg, 0.58 mmol) and MeNH2 (8 M in EtOH, 4.0
mL, 32mmol) was heated for 10 min at 150 °C in the microwave.
The resulting solid was collected by filtration and washed with
EtOH to afford 209 mg (85%) of 34 as a pale-yellow solid. MS
(ESI) m/z 425 (M + H)+. Purity: 90% (method B, tR ) 2.91 min).
2-[3-(Aminomethyl)phenyl]-N,8-dimethyl-8H-imidazo[4,5-d]-
thiazolo[5,4-b]pyridine-5-amine (35). Hydrochloric acid (4 M in
dioxane, 6 mL, 24 mmol) was added in one portion to 34. The
resulting heterogeneous mixture was stirred at room temperature
for 20 min before removing the solvent under reduced pressure.
The residue was dissolved in water (5 mL) and neutralized with
concentrated ammonium hydroxide solution before extracting
with chloroform (3 × 15 mL). The combined organics were washed
with water (10 mL), then brine (10 mL) and dried (MgSO4) before
evaporating under reduced pressure to afford 105 mg (76%) of 35
1
mg (52%) of 32a as a pale-yellow solid. H NMR (DMSO-d6) δ
8.14 (1 H, s), 7.99-8.12 (2 H, m), 7.39-7.49 (2 H, m), 4.20 (3 H,
s), 2.98 (3 H, d, J ) 4.53 Hz). HRMS for C15H13N5F1S1 (M +
H)+, calcd: 314.087 02. Found: 314.086 49. Purity: 100% (method
C, tR ) 11.88 min), 95% (method D, tR ) 11.31 min).
N-Ethyl-2-(4-fluorophenyl)-N-methyl-8H-imidazo[4,5-d]thia-
zolo[5,4-b]pyridine-5-amine (32b). 32b was prepared as a pale-
1
yellow solid following the procedure described for 32a. H NMR
(DMSO-d6) δ 8.16 (1 H, s), 8.02-8.09 (2 H, m), 7.33-7.41 (2 H,
m), 7.27 (1 H, t, J ) 5.79 Hz), 4.21 (3 H, s), 3.48-3.59 (2 H, m),

2004 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7
Kempson et al.
as a yellow solid. (DMSO-d6) δ 8.16 (1 H, s), 8.03 (1 H, d, J )
7.15 Hz), 7.52-7.61 (2 H, m), 7.33 (1 H, d, J ) 4.95 Hz),
4.21-4.26 (3 H, s), 4.13 (2 H, s), 3.00 (3 H, d, J ) 4.40 Hz). MS
(ESI) m/z 325 (M + H)+. Purity: 100% (method B, tR ) 1.70 min).
N-[[3-[8-Methyl-5-(methylamino)-8H-imidazo[4,5-d]thiazolo[5,4-b]-
pyridine-2-yl]phenyl]methyl]acetamide (36a). O-(7-Azabenzot-
riazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (126
mg, 0.33mmol) was added in one portion to a stirred mixture of
35 (90 mg, 0.28 mmol) and acetic acid (20 mg, 0.33mmol) in DMF
(1 mL). N,N-Diisopropylethylamine (1.45 mL, 0.832mmol) was
then added and the mixture stirred at room temperature for 1 h.
Water was added (2 mL) and the precipitated solid was then purified
by column chromatography using 10% methanol in dichloromethane
mM sodium phosphate, 3 mM NaCl, 0.6 mM potassium phosphate,
1 mM KCl, 1 mM dithiothreitol, 5% (w/v) glycerol, and 470 µg/
mL bovine serum albumin. After 60 min, the kinase reactions were
stopped by the addition of EDTA to 33 mM. IκBR phosphorylation
was quantitated by competition for binding to an antiphospho-IκBR
antibody (SantaCruz Biotechnology no. sc-8404) with fluorescein-
labeled phosphopeptide ([FL]-Asp-Asp-Arg-His-Asp-[p]Ser-Gly-
Leu-Asp-Ser-Met-Lys-NH2) as measured using fluorescence
polarization.
LPS-Induced TNF-r Production in Human Peripheral Blood
Mononuclear Cells. Human PBMCs were isolated from whole
blood collected from healthy donors. Blood was diluted into RPMI
1640 (Life Technologies) containing 2.5 mM EDTA (Life Tech-
nologies) and 10 µg/mL polymyxin (Sigma) and then underlaid
with ficoll (Accurate Scientific Co.) and centrifuged at 600g for
25 min. The interface was collected, and cells were washed twice
and resuspended in RPMI, 10% FBS. Cells are then distributed
(200 mL/well) into 96-well tissue culture treated plates (Falcon) at
1 × 106 cells/mL in RPMI, 10% FBS. Test compounds were added
to appropriate wells and incubated with cells for 30 min. Cells were
then stimulated by the addition of lipopolysaccharide (LPS,
BioWhittaker), with a final concentration of 25 ng/mL, and
incubated for 6 h at 37 °C, 5% CO2. The cell supernatants were
removed and assayed for TNF-R by ELISA (R&D Systems).
Inhibition of TNF-r Release in Mice. BALB\c female mice,
6-8 weeks of age, were obtained from Harlan Laboratories and
maintained ad libitum on water and standard rodent chow (Harlan
Teklad). Mice were acclimated to ambient conditions for at least 1
week prior to use. For oral dosing, the compounds were prepared
in a solution of 100% poly(ethylene glycol) (MW 300), and a dosing
volume of 0.1 mL per mouse was administered on average 4 h
prior to LPS injection (0.1 mL of LPS suspended at 10 µg/mL in
PBS, administered ip). Blood samples were obtained 90 min after
LPS injection. Serum was separated from clotted blood samples
by centrifugation (5 min, 5000g, room temperature) and analyzed
for levels of TNF-R by ELISA assay (R&D Systems) according to
the manufacturer’s directions. Results are shown as the mean (
SEM of n ) 8 mice per treatment group. All procedures involving
animals were reviewed and approved by the Institutional Animal
Care and Use Committee.
1
as eluent to afford 14.5 mg (12%) of 36a as a yellow solid. H
NMR (DMSO-d6) δ 8.47 (1 H, t, J ) 5.79 Hz), 8.16 (1 H, s),
7.86-7.91 (2 H, m), 7.44-7.50 (1 H, m), 7.35 (1 H, d, J ) 7.55
Hz), 4.34 (2 H, d, J ) 5.79 Hz), 4.22 (3 H, s), 2.99 (3 H, s), 1.90
(3 H, s). HRMS for C18H19O1N6S1 (M + H)+, calcd: 367.133 56.
Found: 367.132 95. Purity: 95% (method C, tR ) 6.98 min), 95%
(method D, tR ) 7.10 min).
N-[[3-[8-Methyl-5-(methylamino)-8H-imidazo[4,5-d]thiazolo[5,4-b]-
pyridine-2-yl]phenyl]methyl]methanesulfonamide (36b). 36b was
prepared as a pale-yellow solid following the procedure described
1
for 36a. H NMR (DMSO-d6) δ 8.15 (1 H, s), 7.97 (1 H, s), 7.92
(1 H, d, J ) 7.81 Hz), 7.70 (1 H, t, J ) 6.30 Hz), 7.51 (1 H, t, J
) 7.55 Hz), 7.43-7.47 (1 H, m), 7.33 (1 H, d, J ) 4.78 Hz), 4.26
(2 H, d, J ) 6.30 Hz), 4.23 (3 H, s), 2.99 (3 H, d, J ) 4.53 Hz),
2.91 (3 H, s). HRMS for C17H19O2N6S2 (M + H)+, calcd:
403.100 54. Found: 403.099 91. Purity: 96% (method C, tR ) 8.28
min), 95% (method D, tR ) 8.50 min).
N-[[3-[8-Methyl-5-(methylamino)-8H-imidazo[4,5-d]thiazolo[5,4-b]-
pyridine-2-yl]phenyl]methyl]isoxazole-5-amide (36c). 36c was
prepared as a pale-yellow solid following the procedure described
1
for 36a. H NMR (DMSO-d6) δ 8.42 (1 H, s), 8.29-8.32 (1 H,
m), 7.86 (1 H, s), 7.61-7.68 (2 H, m), 7.36-7.38 (1 H, m), 7.17
(1 H, s), 4.58-4.62 (2 H, m), 4.21 (3 H, s), 3.02 (3 H, s). HRMS
for C20H18O2N7S1 (M + H)+, calcd: 419.1164. Found: 419.1158.
Purity: 100% (method C, tR ) 8.25 min), 95% (method D, tR
8.67 min).
)
N-[[3-[8-Methyl-5-(methylamino)-8H-imidazo[4,5-d]thiazolo[5,4-b]-
pyridine-2-yl]phenyl]methyl]-1,2,3-thiadiazole-5-amide (36d).
36d was prepared as a pale-yellow solid following the procedure
described for 36a. 1H NMR (DMSO-d6) δ 9.71 (1 H, s), 8.14 (1 H,
s), 8.01 (1 H, s), 7.84-7.93 (1 H, m), 7.41-7.53 (2 H, m),
4.57-4.66 (2 H, m), 4.17 (3 H, s), 3.00 (3 H, s). HRMS for
C19H17O1N8S2 (M + H)+, calcd: 437.096 13. Found: 437.095 55.
Metabolic Stability in Liver Microsomes. Incubations with
mouse, human, and rat liver microsomes (BD Gentest, Woburn,
MA) were conducted at 1 mg/mL protein concentration, 3 µM
compound in 65 mM phosphate buffer (pH 7.4), and 1 mM
ꢀ-nicotinamide adenine dinucleotide phosphate (ꢀ-NADPH) at 37
°C for 1 h. Aliquots of incubation mixtures (0.2 mL) were taken at
0 and 15 min, and the reaction was quenched with one volume of
acetonitrile. The rate of metabolism was calculated on the basis of
the fraction of parent disappearance by comparing 15-0 min time
points.
Purity: 98% (method C, tR ) 9.31 min), 95% (method D, tR
9.59 min).
)
N-[[2-Fluoro-5-[8-methyl-5-methylamino-8H-imidazo[4,5-d]-
thiazolo[5,4-b]pyridine-2-yl]phenyl]methyl]acetamide (37a). 37a
was prepared as a pale-yellow solid following the procedure
Supporting Information Available: X-ray crystallaographic
data for compounds 12 and 19. This material is available free of
charge via the Internet at http://pubs.acs.org.
1
described for 36a. H NMR (DMSO-d6) δ 8.40 (1 H, t, J ) 5.69
Hz), 8.20 (1 H, s), 7.80-7.89 (2 H, m), 7.35 (1 H, d, J ) 7.45
Hz), 4.30 (2 H, d, J ) 5.62 Hz), 4.20 (3 H, s), 2.7 (3 H, s), 1.86
(3 H, s). HRMS for C18H18O1N6F1S1 (M + H)+, calcd: 384.1169.
Found: 384.1158. Purity: 95% (method C, tR ) 7.24 min), 95%
(method D, tR ) 7.56 min).
References
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N-[[2-Fluoro-5-[8-methyl-5-methylamino-8H-imidazo[4,5-d]-
thiazolo[5,4-b]pyridine-2-yl]phenyl]methyl]methanesulfona-
mide (37b). 37b was prepared as a pale-yellow solid following
the procedure described for 36a. 1H NMR (DMSO-d6) δ 8.38-8.40
(1 H, m), 7.96-7.99 (1 H, m), 7.90 (1 H, s), 7.41-7.45 (1 H, m),
4.24 (2 H, d, J ) 6.25 Hz), 4.20 (3 H, s), 2.95 (3 H, d, J ) 4.50
Hz), 2.89 (3 H, s). HRMS for C17H18O2F1N6S2 (M + H)+, calcd:
420.0838. Found: 420.0831. Purity: 97% (method C, tR ) 8.54 min),
95% (method D, tR ) 8.67 min).
IKK Enzymatic Activity Assay. Assays measuring the enzyme-
catalyzed phosphorylation of GST-IκBR were performed by adding
enzyme (IKK-2, typically to a final concentration of 3 µg/mL) at
room temperature to solutions of 50 µg/mL GST-IκBR and 20 µM
ATP in 25 mM Tris-HCl, pH 7.5, containing 7.5 mM MgCl2, 34

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