Synthesis and biological evaluation of 4-amino derivatives of benzimidazoquinoxaline, benzimidazoquinoline, and benzopyrazoloquinazoline as potent IKK inhibitors

Article abstract of DOI:10.1016/j.bmcl.2006.12.017

We have recently identified BMS-345541 (1) as a highly selective and potent inhibitor of IKK-2 (IC₅₀ = 0.30 μM), which however was considerably less potent against IKK-1 (IC₅₀ = 4.0 μM). In order to further explore the SAR around the

Full text of DOI:10.1016/j.bmcl.2006.12.017

Bioorganic & Medicinal Chemistry Letters 17 (2007) 1233–1237
Synthesis and biological evaluation of 4-amino derivatives
of benzimidazoquinoxaline, benzimidazoquinoline,
and benzopyrazoloquinazoline as potent IKK inhibitors
Francis Beaulieu,a,* Carl Ouellet,a Edward H. Ruediger,a Makonen Belema,b Yuping Qiu,b
Xuejie Yang,b Jacques Banville,a James R. Burke,c Kurt R. Gregor,c John F. MacMaster,c
Alain Martel,a Kim W. McIntyre,c Mark A. Pattoli,c
F. Christopher Zusib and Dolatrai Vyasb
aBristol-Myers Squibb Pharmaceutical Research Institute, 100 boul. de l’Industrie, Candiac, Que., Canada J5R 1J1
bDepartment of Discovery Chemistry, 5 Research Parkway, Wallingford, CT 06492, USA
cDepartment of Immunology, Inflammation and Pulmonary Drug Discovery, PO Box 4000 Princeton, NJ 08543, USA
Received 25 October 2006; revised 5 December 2006; accepted 5 December 2006
Available online 9 December 2006
Abstract—We have recently identified BMS-345541 (1) as a highly selective and potent inhibitor of IKK-2 (IC50 = 0.30 lM), which
however was considerably less potent against IKK-1 (IC50 = 4.0 lM). In order to further explore the SAR around the imidazoqui-
noxaline tricyclic structure of 1, we prepared a series of tetracyclic analogues (7, 13, and 18). The synthesis and biological activities
of these potent IKK inhibitors are described.
ꢀ 2006 Elsevier Ltd. All rights reserved.
The nuclear transcription factor NF-jB plays a key role
in regulating the expression of many pro-inflammatory
genes. Examples of genes modulated by NF-jB include
the cytokines tumor necrosis factor (TNF-a), interleu-
kins IL-1, IL-6, IL-8, intercellular adhesion molecule
(ICAM-1), and vascular cellular adhesion molecule
(VCAM-1).1 NF-jB is normally retained in the cyto-
plasm as an inactive form associated with the IjB inhib-
itory proteins. However, upon cellular stimulation IjB
is phosphorylated by the IjB kinase (IKK) for which
IKKa (IKK-1) and IKKb (IKK-2) are the two most
common isoforms,2 and subsequently phosphorylated
IjB is ubiquitinated and degraded. NF-jB is then
released from the IjB/NF-jB complex into the cell,
where it translocates to the nucleus and activates a num-
ber of genes.3 IKK-2 has been shown to be required for
the pro-inflammatory cytokine-induced activation of
NF-jB in inflammatory cells through the so-called ‘clas-
sical’ NF-jB activation pathway (i.e., degradation of
IjB-alpha), whereas IKK-1 appears to be involved in
the ‘alternative’ NF-jB activation pathway, related to
the development and organization of secondary lym-
phoid organs and B-cell maturation.4 This suggests that
inhibitors of IKK could in principle be used in the treat-
ment of inflammatory and related disorders.5
We have recently identified BMS-345541, 4-(20-aminoeth-
yl)amino-1,8-dimethylimidazo[1,2-a]quinoxaline (1, Fig. 1)
as a highly selective and potent inhibitor of IKK-2
(IC50 = 0.3 lM), but which showed considerably less
potency against IKK-1 (IC50 = 4.0 lM).6 BMS-345541
has also been reported to show dose-dependent efficacy
in terms of reducing disease severity in a murine model
of dextran sulfate sodium-induced colitis7 and in a model
of collagen-induced arthritis.8 Our objective was to pre-
Keywords: NF-jB; IKK inhibitors; TNF-a; Quinoxaline.
*
Corresponding author. Tel.: +1 450 444 4103; fax: +1 450 444
4166; e-mail: francis.beaulieu@bms.com
Figure 1. BMS-345541.
0960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.12.017

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F. Beaulieu et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1233–1237
pare tetracyclic analogues related to compound 1 and we
report herein the synthesis and biological activities of
4-amino-substituted benzimidazoquinoxaline, benzopy-
razoloquinazoline, and benzimidazoquinoline inhibitors
of IjB kinase.9
Based on a reported procedure,10 the synthesis of the ben-
zimidazoquinaxoline series is described in Scheme 1.
Commercially available diaminonaphthalene 2 was
reacted with diethyl oxalate to give annulated quinoxal-
inedione 3, which under treatment with phosphorus oxy-
chloride afforded dichloride 4. Compound 4 was then
reacted with propargylamine to give amino-chloro deriv-
ative 5, which was cyclized under acidic conditions
to provide 1-methyl-4-chlorobenzimidazoquinaxoline 6.
Compound 6 then served as a key intermediate to gener-
ate 4-amino-substituted analogues 7a–d.11
Scheme 2. Synthesis of the benzopyrazoloquinazoline series. Reagents
and conditions: (a) concd HCl, H2O, NaNO2, 0 ꢁC, KI, rt, 0.1 h then
95 ꢁC, 1 h; (b) concd H2SO4, MeOH, reflux, 13 h, 69% over two steps; (c)
4-methyl-1-(4-toluenesulfonyl)-5-trimethylstannylpyrazole, Pd2(dba)3,
Ph3As, CuI, DMF, 90 ꢁC, 12 h, 55%; (d) 1 N NaOH, THF, MeOH,
80 ꢁC, 4.2 h, 40%; (e) (PhO)2PON3, Et3N, benzene, 50 ꢁC, 2 h, then
1,2-dichlorobenzene, 150 ꢁC, 4 h, 67%; (f) POCl3, PhNEt2, reflux, 46 h,
77%; (g) RNH2, THF, 60–75 ꢁC, 2–5 h, 98%.
The benzopyrazoloquinazoline series 13a–b was pre-
pared as shown in Scheme 2. 3-Amino-2-naphthoic acid
(8) was converted to 3-iodo-2-naphthoic acid via diazo-
tization followed by treatment with potassium iodide.
Esterification of the resulting acid then gave the ester 9
which was cross-coupled with 4-methyl-1-(4-toluene-
sulfonyl)-5-trimethylstannylpyrazole12 under Stille-type
conditions to afford the coupled product 10. Upon
hydrolysis, the resulting pyrazole acid intermediate
was heated with diphenylphosphoryl azide, producing
an intermediate isocyanate which spontaneously cyc-
lized under the reaction conditions to give compound
11. Exposure of compound 11 to phosphorus oxychlo-
ride then gave the chloride intermediate 12, which was
finally reacted with primary amines to provide benzopy-
razoloquinazoline analogues 13a–b.
The preparation of the benzimidazoquinolines is out-
lined in Scheme 3. Previously prepared methyl 3-iodo-
2-naphthoate (9) was converted to the corresponding
boronate derivative using bis(pinacolato)diboron and
palladium catalysis. This boronate intermediate was
subsequently cross-coupled with 5-bromo-1-methyl-
Scheme 3. Synthesis of the benzimidazoquinoline series. Reagents and
conditions: (a) bis(pinacolato)diboron, PdCl2(dppf), KOAc, DMSO,
85 ꢁC, 18 h, 58%; (b) 5-bromo-1-methyl-1H-imidazole, Pd(PPh3)4,
Na2CO3, toluene, EtOH, H2O, reflux, 20 h, 72%; (c) 1 N NaOH, THF,
MeOH, rt, 20 h, 74%; (d) (PhO)2PON3, Et3N, t-BuOH, 80 ꢁC, 30 min,
51%; (e) TFA, CH2Cl2, rt, 18 h, 70%; (f) CDI, 1,2-dichlorobenzene,
180 ꢁC, 5 h, 42%; (g) POCl3, PhNEt2, reflux, 4 h, 90%; (h) RNH2,
THF, 60–80 ꢁC, 18 h, 5%.
1H-imidazole under Suzuki conditions, and the resulting
ester was hydrolyzed to give the acid 14. A Curtius reac-
tion was carried out by reacting the acid 14 with diphe-
nylphosphoryl azide in tert-butanol, and this was
followed by acid treatment to afford amine 15. Heating
15 in 1,2-dichlorobenzene with carbonyldiimidazole
provided cyclized product 16, which was subsequently
treated with phosphorus oxychloride to give chloride
17. This intermediate was subsequently reacted with pri-
mary amines to give benzimidazoquinolines 18a-b.
Scheme 1. Synthesis of the benzimidazoquinoxaline series. Reagents
and conditions: (a) diethyl oxalate, reflux, 14 h, 82%; (b) POCl3, reflux,
5 h, 85%; (c) propargylamine, Et3N, 1,4-dioxane, reflux, 4.5 h, 73%; (d)
concd H2SO4, 80 ꢁC, 1 h, 30%; (e) RNH2, THF, 80 ꢁC, 18 h, 26–94%.

F. Beaulieu et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1233–1237
1235
The analogues described above (7, 13, and 18) were eval-
uated in a primary screen assay measuring the (IKK-2
and IKK-1) enzyme-catalyzed phosphorylation of
GST-IjBa as substrate.13 The secondary assay mea-
sured the inhibition of lipopolysaccharide(LPS)-induced
TNF-a secretion in THP-1 cells.14 The IC50 values for
all tetracyclic analogues are compared with that of
BMS-345541 in Table 1. All tetracyclic analogues tested
showed more potent activity than the tricyclic com-
pound 1, against both IKK-2 and IKK-1.15 The first ser-
ies of tetracyclic analogues (7), which are related to
BMS-345541 by the addition of a fused benzene ring,
resulted in an order of magnitude increase in potency
in the IKK-2 and THP-1 cell assays. Compound 7a
showed good IKK-2 potency, with a 13-fold selectivity
versus IKK-1, comparable to the ratio seen with BMS-
345541. Benzimidazoquinoxalines bearing a solubilizing
sidechain (i.e., 7b–d) showed no particular advantage in
IKK-2 activity over the 4-NHMe analogue 7a, however,
the hydroxyethylamine analogue 7c gave an improved
48-fold selectivity for IKK-2 versus IKK-1. When the
imidazoquinoxaline core was modified to a pyrazoloqui-
nazoline (13) or imidazoquinoline (18) scaffold, the
IKK-2 in vitro activities in general remained compara-
ble to the tetracyclic structures 7, although a significant
loss in cellular potency was observed for compounds 13a
and 18b.16
Since the tetracyclic compounds 7a and 7b were more
potent IKK-2 inhibitors than the corresponding tricyclic
analogue BMS-345541, we examined their in vivo bio-
logical activities in mice. As shown in Figures 2 and 3,
we measured the effect of compounds 7a and 7b on ser-
um TNF-a concentrations induced by intraperitoneal
injection of LPS-treated mice.17 As shown in Figure 2,
compound 7a produced the same effect at 10 mg/kg as
did BMS-345541 at 30 mg/kg, which corresponds to
approximately a 50% reduction of TNF-a levels versus
vehicle control animals. Figure 3 shows that a dose of
100 mg/kg resulted in a nearly complete inhibition of
serum TNF-a for BMS-345541 and 7b, with a good
dose-proportional response being observed.
Table 1. IKK-2, IKK-1, and THP-1 cell inhibitory potencies of tetracyclic analogues 7, 13, and 18
Compound
R
IKK-2 IC50, lMa
IKK-1 IC50, lMa
THP-1 cell IC50, lMa
1
0.30
4.0
4.0
0.34
1.0
1.4
7a
7b
7c
–Me
–CH2CH2NHMeÆHCl
–CH2CH2OH
0.018
0.023
0.018
0.23
0.39
0.87
7d
13a
13b
18a
18b
0.046
0.011
0.035
ndb
1.7
0.60
8.3
CH2CH2N
–Me
0.67
ndb
0.99
1.6
–CH2CH2NHMe
–Me
–CH2CH2NH2
1.5
0.95
5.9
0.058
a Single experiment.
b Not determined.
16000
14000
12000
10000
8000
6000
4000
2000
0
Serum TNFα Levels
(pg/mL)
Figure 2. The effect of BMS-345541 and compound 7a on serum TNF-a concentrations induced by intraperitoneal injection of LPS.

1236
F. Beaulieu et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1233–1237
20000
15000
Serum TNFα Levels
(pg/mL)
10000
5000
0
Figure 3. The effect of BMS-345541 and compound 7b on serum TNF-a concentrations induced by intraperitoneal injection of LPS.
Peters, R. T.; Liao, S.-M.; Maniatis, T. Mol. Cell 2000, 5,
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MacMaster, J. F.; McIntyre, K. W.; Yang, X.; Iotzova, V.
S.; Clarke, W.; Strnad, J.; Qiu, Y.; Zusi, F. C. J. Biol.
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52, 1.
In summary, a series of tetracyclic structures, based on
BMS-345541 as a structural lead, were efficiently synthe-
sized and subsequently evaluated as IKK-2 inhibitors
in vitro and in vivo. Most of the tetracyclic compounds
were more potent than the parent in vitro and two new
benzimidazoquinoxalines showed improved overall
activity when compared to BMS-345541. Future studies
will be directed toward further optimization of these tet-
racyclic scaffolds.
Acknowledgment
´
We thank Dr. Rejean Ruel for fruitful discussions and
suggestions in the preparation of the manuscript.
References and notes
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Qiu, Y.; Zusi, F. C.; Burke, J. R. Arthritis Rheum. 2003,
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9. (a) Beaulieu, F.; Ouellet, C.; Belema, M.; Qiu, Y.; Yang,
X.; Zusi, F. C. U.S. Patent 6,960,585; (b) Burke, J. R.;
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Patent 6,896,956.
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11. Synthesis of 7a: To a solution of benzo[g]-4-chloro-1-
methylimidazo[1,2-a]quinoxaline (0.063 g, 0.236 mmol) in
4 mL THF was added MeNH2 (40% in H2O, 0.16 mL,
1.88 mmol). The reaction tube was sealed, and the mixture
was stirred at 80 ꢁC for 18 h. The cooled mixture was then
taken up in EtOAc, washed with water and brine, dried
(Na2SO4), filtered, and evaporated. The resulting residue
was recrystallized using i-PrOH to give the desired product
´
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1237
as beige needles (0.043 g, 69%); mp 201–202 ꢁC, IR (KBr,
of LPS stimulation. Specific enzyme immunoassay kits
from Pharmingen (OptEIA) were used in quantitating
each cytokine.
1
cmꢀ1) 3324, 1575, 1557, 1411, 1121; H NMR (400 MHz,
DMSO-d6) d 8.62 (s, 1H), 8.10 (s, 1H), 8.08 (d, J ¼ 8:1 Hz,
1H), 7.94 (d, J ¼ 8:1 Hz , 1H), 7.77 (br d, J ¼ 4:5 Hz,
1H), 7.49–7.45 (m, 2H), 7.35 (s, 1H), 3.06 (d, J ¼ 4:6 Hz,
3H), 2.98 (s, 3H); MS (+ESI, M + H+) m/z 263; HPLC
purity: 99.5% (230 nm); HRMS calcd for C16H14N4:
262.1219; found 262.1226.
15. Compound 7b was screened against other kinases (IKK-e,
LCK, EMT) and failed to show inhibition at concentra-
tions as high as 50 lM (data not shown). Enzyme kinetic
analysis of inhibition was not performed to investigate the
binding mechanism of these new inhibitors.
12. This compound was prepared in 43% yield from 4-methyl
pyrazole in two steps (a, p-TsCl, pyridine; b, t-BuLi,
Me3SnCl).
16. Based on inspection of the structures and physicochemical
properties, the reason for the significant loss in cellular
potency for 13a and 18b remains unclear.
13. Enzyme Assays. Assays measuring the enzyme-catalyzed
phosphorylation of GST-IjBa were performed essentially
as described in reference , by adding enzyme (IKK-2 or
17. LPS-induced serum TNF-a in mice. The general proce-
dure of Ghezzi et al. (Cytokine 2000, 12, 1205–1210) was
followed. Briefly, 0.2 mL of an aqueous solution of an
inhibitor was administered by peroral gavage to 6- to 8-
week-old female BALB/c mice (Harlan) 1 h prior to a
challenge with an intraperitoneal dose of 1 lg Escherichia
coli LPS (from strain O111:B4, Sigma) in 500 lL phos-
phate-buffered saline. Blood was collected from mice
90 min after LPS challenge and the levels of TNF-a in the
serum measured by EIA (R&D systems). Inhibition by the
inhibitor was calculated from control animals which
received LPS challenge but no inhibitor (vehicle only).
Mice receiving no LPS challenge gave no detectable serum
TNF-a.
5
IKK-1, typically to a final concentration of 0.5 lg/mL) at
30 ꢁC to solutions of 50 lg/mL GST-IjBa and 20 lM
ATP in 30 mM Tris–HCl, pH 8, containing 7.5 mM
MgCl2, 30 mM sodium phosphate, 3 mM NaCl, 0.6 mM
potassium phosphate, 1 mM KCl, 1 mM dithiothreitol,
5% (w/v) glycerol, and 475 lg/mL bovine serum albumin.
14. Cytokine production in THP-1 cells. THP-1 cells in 96-
well plates (2.5 · 105 cells/well in 180 lL RPMI-1640/10%
FBS) were treated with inhibitors for 1 h prior to
stimulation with 100 ng/mL LPS. Measurements of the
amount of TNF-a in the supernatants were made after 6 h