Imidazo[4,5-d]thiazolo[5,4-b]pyridine based inhibitors of IKK2: Synthesis, SAR, PK/PD and activity in a preclinical model of rheumatoid arthritis

Article abstract of DOI:10.1016/j.bmcl.2010.10.133

The synthesis, structure-activity relationships (SAR) and biological evaluation of thiazole based tricyclic inhibitors of IKK2 are described. Compound 9 was determined to be orally efficacious in a murine model of rheumatoid arthritis.

Full text of DOI:10.1016/j.bmcl.2010.10.133

Bioorganic & Medicinal Chemistry Letters 21 (2011) 383–386
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Imidazo[4,5-d]thiazolo[5,4-b]pyridine based inhibitors of IKK2: Synthesis, SAR,
PK/PD and activity in a preclinical model of rheumatoid arthritis
⇑
Alaric J. Dyckman , Charles M. Langevine, Claude Quesnelle, James Kempson, Junqing Guo, Patrice Gill,
Steven H. Spergel, Scott H. Watterson, Tianle Li, David S. Nirschl, Kathleen M. Gillooly, Mark A. Pattoli,
Kim W. McIntyre, Laishun Chen, Murray McKinnon, John H. Dodd, Joel C. Barrish, James R. Burke,
William J. Pitts
Bristol-Myers Squibb, Research and Development, Princeton, NJ 08543-4000, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis, structure–activity relationships (SAR) and biological evaluation of thiazole based tricyclic
inhibitors of IKK2 are described. Compound 9 was determined to be orally efficacious in a murine model
of rheumatoid arthritis.
Received 14 September 2010
Revised 26 October 2010
Accepted 27 October 2010
Available online 31 October 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
IKK
IKK2
IKKb
NF-jB
The NF-jB family of nuclear transcription factors are ubiqui-
tously expressed proteins that regulate the transcription of a vari-
N
N
N
N
N
N
N
ety of genes relating to immune and inflammatory disorders,
F
cancers and diabetes among others.1 NF-
j
B is held in an inactive
state in the cytoplasm of unstimulated cells by a family of inhibitor
proteins known as I Bs, which mask the nuclear localization sig-
nals of NF- B. In response to various stimuli, I B is phosphorylated
by IkB-kinase (IKK), leading to its ubiquitination and subsequent
NH2
N
N
H
N
H
2
1 (BMS-345541)
NH
j
O
IKK2 IC50 279 nM
IKK1 IC50 9700 nM
PBMC IC50 730 nM
IKK2 IC50 200 nM
IKK1 IC50 7000 nM
PBMC IC50 1000 nM
j
j
proteosomal degradation which allows NF-jB to translocate to
the nucleus and activate gene transcription.2a IKK is a high molec-
ular weight (700–900 kD) trimeric complex consisting of IKK1
N
N
N
X
3 X = NR'
4 X = O
5 X = S
(IKK
IKK2 subunit is responsible for the phosphorylation of I
canonical signaling pathway leading to NF-
B activation.2b
Efforts to modulate NF- B activity through small molecule inhi-
a
), IKK2 (IKKb) and NEMO (NF-
j
B essential modulator). The
N
N
H
j
B in the
R
j
Figure 1. Tricyclic inhibitors of IKK2.
j
bition of IKK2 have been reported from several research groups.3
We have previously disclosed several tricyclic based inhibitors of
IKK2 (Fig. 1) including imidazoquinoxaline 1 (BMS-345541),4a
pyrazolopurine (2),4b and more recently tricyclic imidazole (3),
oxazole (4) and thiazole (5) based structures.4c Herein we disclose
SAR studies within the imidazothiazolopyridine (5) series in addi-
tion to results from in vivo evaluation in models of acute and
chronic inflammation.
Preparation of the target compounds was accomplished follow-
ing several routes that permitted the SAR evaluation at the meta
position of the C2 aryl group as well as the substitution at C5
(Scheme 1). The previously described advanced intermediate 6
was key to facilitating this evaluation.4c Suzuki–Miyaura reaction
of 6 with boronic acid 16 proceeded selectively at the C2-bromo
position to provide chloro-tricyclic compound 7. Displacement of
the chlorine of 7 with a variety of amines was effected under
microwave heating. Alternatively, 6 could be coupled with boronic
ester 17 and then reacted with ethylamine to afford, after Boc
deprotection, intermediate 13. Acylation of 13 with a variety of
⇑
Corresponding author. Tel.: +1 609 252 3593; fax: +1 609 252 7410.
E-mail address: alaric.dyckman@bms.com (A.J. Dyckman).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.10.133

384
A. J. Dyckman et al. / Bioorg. Med. Chem. Lett. 21 (2011) 383–386
N
N
N
N
16
N
S
8
9
10
b or c or d
R = Me
R = Et
N
N
N
S
N
S
2
Br
5
Cl
N
Cl
R
R = PMB
a
N
N
N
H
e
Me
11 R = H
Me
N
6
7
NHAc
Et
NHAc
N
N
N
N
17
a, c
f
g
N
N
N
S
N
S
6
Et
Et
S
N
N
H
N
N
H
N
N
H
Me
Me
O
Me
12
13
14
NH2
Br
NH
R
NHBoc
NHAc
O
B(OH)2
B
h, i
j, k
O
Me
Me
Me
15
16
17
NH2
NHBoc
Scheme 1. Reagents and conditions: (a) Pd(PPh3)4, K2CO3 (aq), DME, 120 °C; (b) MeNH2, EtOH, microwave 150 °C; (c) EtNH2, EtOH, microwave 150 °C; (d) PMBNH2, THF, DMF,
microwave 200 °C; (e) TFA, CH2Cl2; (f) HCl (4 N in 1,4-dioxane); (g) R-COOH, EDCI, HOBt, DMF, 60 °C; (h) Ac2O, pyr.; (i) MeLi, tBuLi, (iPrO)3B, THF, À78 °C; (j) Boc2O, THF; (k)
PdCl2(dppf), dppf, bis(pinacolato)diboron, KOAc, 1,4-dioxane, 80 °C.
Table 1
C2-Aryl SAR
tolerated. When combined with alkyl branching at the benzylic
carbon (21), a significant improvement in potency was observed.
Resolution of the enantiomers demonstrated a preference for the
N
(S) isomer (8) relative to the (R) isomer (22), providing an increase
in potency against IKK2 of approximately fivefold. The (S)-1-ace-
tamidoethyl template was therefore selected for subsequent eval-
uation of the C5 SAR.
The SAR of the C5 position within a series of compounds bearing
the preferred meta-(S)-1-acetamidoethyl substitution on the phe-
nyl group is shown in Table 2. Optimal in vitro activity was
achieved with amino (11) or small alkylamino (8, 9) groups. Fluo-
rination or amination of the alkylamino group (23, 24) resulted in
N
N
S
R
Me
N
N
H
Compd
R
IKK2a IC50
,
IKK1a IC50
,
hPBMCa,b
IC50
lM
lM
, lM
0.310 0.03
(2)
18
19
0.006
0.060
3.50
NTc
AcHN
0.530
Table 2
C5-SAR with (S) 1-acetamidoethyl template
20
0.022
1.90
0.350
NHAc
Me
N
N
0.009 0.005
(10)
(R) 0.028 0.02
0.170 0.03
(43)
0.560
N
21
22
( )
0.430
NT
S
N
R
(3)
Me
NHAc
8
(S) 0.006 0.004 0.230 0.0 0.080
(17) (2)
NHAc
IKK2 IC50
Compd
R
,
l
M
IKK1 IC50
,
hPBMC
IC50, lM
l
M
a
Where values are derived from more than a single experiment, the standard
deviation is noted followed by the number of test occasions in parenthesis.
Me
Et
0.006 0.004
(17)
0.230 0.10
(2)
b
N
H
hPBMC = inhibition of LPS-induced TNF-
mononuclear cells.
a release from human peripheral blood
8
0.080
c
NT = not tested.
0.019 0.01
(34)
0.220 0.08
(7)
N
H
9
0.420
NT
0.013 0.002
(3)
0.290 0.10
(3)
11
23a
NH2
carboxylic acids provided the final compounds. The requisite chiral
boronic acid/ester fragments (16/17) were prepared in two steps
from the commercially available homochiral aryl bromide 15.
Final compounds were initially evaluated for inhibition of hu-
man IKK2 enzyme activity followed by inhibition of lipopolysac-
N
H
CF3
NH2
0.057 0.05 (2) NT
1.36
4.89
24a
25b
0.049
0.130
NT
NT
N
H
charide (LPS) induced TNF
a production in human PBMCs as a
O
measure of cellular potency.5,6 Counter screening of select com-
pounds against human IKK1 provided early selectivity determina-
tion. Based on SAR from our earlier chemotypes4b–d we directed
our efforts toward benzylamine substitution on the C-2 aryl group
(Table 1).
NT
N
H
NMe2
26a
27c
0.100
NT
NT
0.99
>10
NMe2
Me
O
>0.500
Acetamidomethyl substitution at the para-position (19) led to a
10-fold loss in enzymatic activity against IKK2 along with a reduc-
tion in cellular potency relative to the unsubstituted 18. The iso-
meric analog 20 indicated that meta substitution was better
a
b
c
Prepared in analogy to 8 by substituting with the appropriate amine.
Prepared from 11 by reaction with N,N-dimethylcarbamoyl chloride, NaH, DMF.
Prepared in analogy to 8 by heating 7 with MeOH, NaOH.

A. J. Dyckman et al. / Bioorg. Med. Chem. Lett. 21 (2011) 383–386
385
decreased potency, as did incorporation of the amine into a urea
(25). An important role for the NH of the C5 substituent was sug-
gested by the reduced potency for N,N-dimethylamino and meth-
has prevented generation of a binding model. Thus, the exact role
of the C5 NH is not understood.
While the methylamino group at C5 afforded slightly improved
in vitro potency over the ethylamino group, the latter generally
conferred improved microsomal stability and reduced in vivo
clearance (data not shown). Further optimization efforts around
the meta-benzylamine side chain were therefore investigated using
ethylamino substitution at C5. Extensive variations were explored
for the acyl group of the benzylamine, with select examples pre-
sented in Table 3. In addition to in vitro potency, the pharmacoki-
netics (PK) in rats was evaluated for many compounds utilizing a
cassette dosing strategy.4c Consistent with earlier SAR, the (S)
enantiomer within this series continued to provide improved
in vitro activity (9 vs 28) and was also found to be superior to
the dimethyl analog (29). There was a high degree of tolerance
for the nature of the acyl substituent in vitro, with alkyl, function-
alized alkyl, aryl, heteroaryl and heterocyclic based acyl groups
providing IKK2 inhibitors with reasonable enzyme and cellular
activity. Differentiation was more pronounced on the basis of
in vivo properties, with acyl group modifications leading to
significantly improved rat PK properties in several examples. In
comparison to acetamide 9, the 1-methylcyclopropanecarboxa-
mide (30), 5-methylisoxazole-3-carboxamide (34) and meth-
ylsulfonylbenzamide (35) analogs displayed reduced clearance
(Cl = 7.5, 2.8, 5.1 mL/min/kg, respectively) with increased half life
values (t1/2 = 2.0, 2.8. 1.5 h, respectively). While 1-methylpiperi-
dinecarboxamide 36 proved to be rapidly cleared (Cl = 130 mL/
min/kg), the des-methyl analog (37) was significantly more stable
in vivo (Cl = 3.0 mL/min/kg, t1/2 = 2.9 h).
As a prototypical example of this series, compound 9 was se-
lected for further profiling and in vivo evaluation in PK, pharmaco-
dynamic, and efficacy models. Discrete PK properties for 9 (iv and
po in mouse/rat and iv in dog) are shown in Table 4. The compound
displayed good bioavailability in rodents and a half life of 0.6–1.9 h
across the three species. The clearance was higher than expected
based on previous rat cassette PK results, however the compound
achieved reasonable Cmax and AUC levels. A major metabolic path-
way for 9 was found to be N-dealkylation. The extent of N-deethy-
lation was examined across species, with the formation of the
major metabolite (11) observed in an AUC ratio (metabolite/par-
ent) ranging from 0.2 in dog to 1.1 in mouse (see Fig. 1).
oxy derivatives (26, IKK2 IC50 = 0.10 lM; 27, IKK2 IC50 >0.50 lM
which was the highest compound concentration evaluated).
Crystal structures have not been reported for IKK2 and this lack
of structural information, coupled with the observation that
compounds in this class may bind to a region outside of the tradi-
tional ATP binding site and allosterically modulate the enzyme,4a
Table 3
m-Phenyl SAR
N
N
N
Et
S
N
N
H
R
Compd
R
IKK2
IC50
hPBMC
IC50, lM
Rat cassette PK
Cl (mL/ min/kg)
t1/2 (h) Vss (L/kg)
,
lM
Cl = 25
t1/2 = 0.4
Vss = 0.8
Me
Me
O
0.019 0.01 0.220 0.08
9
HN
HN
HN
HN
HN
(34)
(7)
Me
Me
O
0.055 0.01
(20)
28
29a
30
31
0.700
NT
Me
Me
Cl = 19
t1/2 = 0.3
Vss = 0.5
Me
O
0.036
0.400
0.440
0.600
Cl = 7.5
t1/2 = 2.0
Vss = 1.1
Me
Me
0.034 0.02
(3)
O
O
Cl = 11
t1/2 = 0.95
Vss = 0.7
OEt
0.016 0.01
(2)
Cl = 19
t1/2 = 0.7
Vss = 0.7
Me
Me
NHAc
NHEt
32
33
0.027
0.039
0.480
0.440
HN
HN
Compounds 9 and 11 displayed similar potency in whole blood
O
O
assays measuring inhibition of TNF
stimulation with LPS (human whole blood IC50 ꢀ2
whole blood IC50 ꢀ1.0 M). General kinase selectivity was assessed
through evaluation in a panel of more than 40 diverse kinases. At
10 M concentration, activity of less than 10% of control was ob-
a
production in response to
Cl = 29
t1/2 = 1.8
Vss = 2.4
lM, mouse
l
l
O
N
Cl = 2.8
t1/2 = 2.8
Vss = 0.7
served for only 4 kinases for 9 (DAPK2, GAK, JNK3, STK16) and 2 ki-
nases for 11 (DAPK2, STK16). The compounds were subsequently
0.078 0.02
(2)
34
35
Me
Me
0.940
0.220
HN
O
MeO2S
Table 4
Pharmacokinetic propertiesa of 9 in mice, rats and dogs
Cl = 5.1
t1/2 = 1.5
Vss = 0.6
0.041
Mouse
Rat
Dog
HN
(iv only)
O
% Fpo
Cmax (nM)
Tmax (h)
57%
1330
0.50
1.9
88%
1650
2.00
0.6
—
—
—
0.7
N
Cl = 130
t1/2 = 1.5
Vss = 17
Me
Me
36
37
0.010
0.020
0.560
0.290
t1/2 (h)
HN
HN
MRT (h)
Cl (mL/min/kg)
Vss (L/kg)
2.9
62
1.5
3000
3370
2.1
63
2.3
4630
2400
O
O
43
3.1
1980
400
NH
*
AUCtot (nM h)
Cl = 3.0
t1/2 = 2.9
Vss = 5.6
*
11 AUCtot (nM h) (major metabolite
produced after dosing with 9)
a
Vehicle: iv, PEG400:water = 80:20; po, PEG400 = 100%. Dose: iv, 2 mg/kg; po,
10 mg/kg.
a
Prepared in analogy to 14 using the corresponding boronic ester.

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A. J. Dyckman et al. / Bioorg. Med. Chem. Lett. 21 (2011) 383–386
examined in a murine model of acute inflammation for their ability
initiated on day 25, coinciding with the time of disease onset. In
this pseudo-established mode, treatment with 9 provided signifi-
cant inhibition in disease progression throughout the study, with
80–90% reduction in clinical scores (Fig. 3). Exposures obtained
on day 40 indicated that the ratio of parent to metabolite (9:11)
was nearly 1:1 and the combined drug levels remained above the
to inhibit TNF-a production in response to an LPS challenge in vivo
( Fig. 2).4g The compounds were administered orally at 30 and
100 mg/kg 5 h prior to LPS challenge and were found to dose-
dependently inhibit the TNF-a response, with the 100 mg/kg dose
of 9 affording the most robust response (75% inhibition). Drug
exposures were obtained at 6.5 h post-dose and demonstrated that
at the higher doses tested, the delivery of 11 from metabolism of 9
was more efficient than from direct dosing of compound 11. As ex-
pected, the IKK2 activity of 11 (see Table 2) appeared to be helping
drive the pharmacology resulting from administration of 9.
Based on the in vitro profile, results in the murine LPS-TNF
model and pharmacokinetic properties, compound 9 was exam-
ined for efficacy in a rodent model of arthritis (murine collagen-in-
duced arthritis).4g Twice daily administration of 9 at 60 mg/kg was
mouse whole blood IC50 (>1 lM) for at least 6 h post-dose.
In conclusion, SAR investigation of C2 and C5-substituted imi-
dazothiazolopyridines provided potent inhibitors of IKK2 with im-
proved PK properties. Select compounds were shown to be active
in a model of acute inflammation and compound 9 was found to
be highly efficacious in a rodent model of chronic inflammation.
Further studies on the evaluation of this chemotype will be re-
ported in due course.
References and notes
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Schmid, J. A.; Richmond, A. Clin. Cancer Res. 2006, 950; (d) Ammann, J. U.; Haag,
C.; Kasperczyk, H.; Debatin, K. M.; Fulda, S. Int. J. Cancer 2009, 1301; (e) Cai, D.;
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Med. 2004, 183.
2. (a) Ghosh, S.; Karin, M. Cell 2002, 109, S81; (b) Sun, S.-C.; Ley, S. C. Trends
Immunol. 2008, 469.
3. (a) Pitts, W. J.; Kempson, J. Annu. Rep. Med. Chem. 2008, 43, 155; (b) Bamborough,
P.; Callahan, J. F.; Christoper, J. A.; Kerns, J. K.; Liddle, J.; Miller, D. D.; Morse, M.
A.; Rumsey, W. L.; Williamson, R. Curr. Top. Med. Chem. 2009, 623.
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M.; Qiu, Y.; Yang, X.; Banville, J.; Burke, J. R.; Gregor, K. R.; MacMaster, J. F.;
Martel, A.; McIntyre, K. W.; Pattoli, M. A.; Zusi, F. C.; Vyas, D. Bioorg. Med. Chem.
Lett. 2007, 17, 1233; (c) Kempson, J.; Spergel, S. H.; Guo, J.; Quesnelle, C.; Gill, P.;
Belanger, D.; Dyckman, A. J.; Li, T.; Watterson, S. H.; Langevine, C. M.; Das, J.;
Moquin, R. V.; Furch, J. A.; Marinier, A.; Dodier, M.; Martel, A.; Nirschl, D.; Van
Kirk, K.; Burke, J. R.; Pattoli, M. A.; Gillooly, K. M.; McIntyre, K. M.; Chen, L.; Yang,
Z.; Marathe, P. H.; Wang-Iverson, D.; Dodd, J. H.; McKinnon, M.; Barrish, J. C.;
Pitts, W. J. J. Med. Chem. 2009, 52, 1994; (d) Belema, M.; Bunker, A.; Nguyen, V.
N.; Beaulieu, F.; Ouellet, C.; Qiu, Y.; Zhang, Y.; Martel, A.; Burke, J. R.; McIntyre, K.
W.; Pattoli, M. A.; Daloisio, C.; Gillooly, K. M.; Clarke, W. J.; Brassil, P. J.; Zusi, F.
C.; Vyas, D. M. Bioorg. Med. Chem. Lett. 2007, 17, 4284; (e) Kempson, J.; Guo, J.;
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A. J.; Pattoli, M.; Burke, J. R.; Yang, X.; Gillooly, K. M.; McIntyre, K. W.; Chen, L.;
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5. IKK enzymatic activity assay. Assays measuring the enzyme-catalyzed
Figure 2. Inhibition of LPS-induced TNF-
a release by 9 and 11 in mouse. BALB/c
female mice (Harlan), 6–8 weeks of age, were used. Compounds were dosed in
PEG400/HCl/Tween80/water (Vehicle) to mice (n = 7–8/treatment) by oral gavage
in a volume of 0.1 mL. Control mice received vehicle alone. Five hours later, mice
were injected intraperitoneally with 50
O111:B4; Sigma). Blood samples were collected 90 min after LPS injection. Serum
was separated and analyzed for the level of TNF- by commercial ELISA assay
lg/kg lipopolysaccharide (LPS; E. coli
a
(BioSource) according to the manufacturer’s instructions. Data shown are
mean standard error.
phosphorylation of GST-I
typically to a final concentration of 3
of 50 g/mL GST-I and 20
M ATP in 25 mM TrisÁHCl, pH 7.5, containing
7.5 mM MgCl2, 34 mM sodium phosphate, 3 mM NaCl, 0.6 mM potassium
phosphate, 1 mM KCl, 1 mM dithiothreitol, 5% (w/v) glycerol, and 470 g/mL
bovine serum albumin. After 60 min, the kinase reactions were stopped by the
addition of EDTA to 33 mM. phosphorylation was quantitated by
competition for binding to an anti-phospho-I antibody (SantaCruz
jB
a
were performed by adding enzyme (IKK-2,
lg/ml) at room temperature to solutions
l
jB
a
l
l
IjBa
jBa
Biotechnology #sc-8404) with fluorescein-labeled phospho-peptide ([FL]-Asp-
Asp-Arg-His-Asp-[p]Ser-Gly-Leu-Asp-Ser-Met-Lys-NH2) as measured using
fluorescence polarization.
6. Experimental procedure for PBMC assay: Heparinized human whole blood was
obtained from healthy volunteers. Peripheral blood mononuclear cells (PBMCs)
were purified from human whole blood by Ficoll-Hypaque density gradient
centrifugation and re-suspended at a concentration of 5 Â 106/mL in assay
medium (RPMI medium containing 10% fetal bovine serum). Fifty microliters of
cell suspension was incubated with 50
assay medium containing 0.2% DMSO) in 96-well tissue culture plates for 5 min
at room temperature. 100 l of LPS (200 ng/mL stock) was then added to the cell
suspension and the plate was incubated for 6 h at 37 °C. Following incubation,
concentration in
ll of test compound (4Â concentration in
l
Figure 3. Efficacy of 9 (blue circles) in the murine model of collagen-induced
arthritis. Mice (n = 14–15/group) were primed with collagen injection on day 1 and
again on day 25. Compound 9 at 60 mg/kg or vehicle control (red squares) was
administered by oral gavage twice daily beginning at the time of the second
collagen injection on day 25. Average gross clinical score for all mice within each
treatment group.
the culture medium was collected and stored at À20 °C. TNF-
a
the medium was quantified using a standard ELISA kit (Pharmingen-San Diego,
CA). Concentrations of TNF- and IC50 values for test compounds (concentration
of compound that inhibited LPS-stimulated TNF-
calculated by linear regression analysis.
a production by 50%) were