Synthesis and biological evaluation of tricyclic anilinopyrimidines as IKKβ inhibitors

Article abstract of DOI:10.1016/j.bmcl.2010.04.022

A series of tricyclic anilinopyrimidines were synthesized and evaluated as IKKβ inhibitors. Several analogues, including tricyclic phenyl (10, 18a, 18c, 18d, and 18j) and thienyl (26 and 28) derivatives were shown to have good in vitro enzyme potency and excellent cellular activity. Pharmaceutical profiling of a select group of tricyclic compounds compared to the non-tricyclic analogues suggested that in some cases, the improved cellular activity may be due to increased clog P and permeability.

Full text of DOI:10.1016/j.bmcl.2010.04.022

Bioorganic & Medicinal Chemistry Letters 20 (2010) 3821–3825
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of tricyclic anilinopyrimidines as
IKKb inhibitors
b
b
b
b
Aimee L. Crombie a, , Fuk-Wah Sum , Dennis W. Powell , Darrin W. Hopper , Nancy Torres ,
*
Dan M. Berger b, Yixian Zhang c, Maria Gavriil c, Tammy M. Sadler c, Kim Arndt c
a Chemical Sciences, Pfizer Global Research and Development, 500 Arcola Road, Collegeville, PA 19335, USA
b Chemical Sciences, PGRD, 401 North Middletown Road, Pearl River, NY 10965, USA
c Oncology Research, PGRD, 401 North Middletown Road, Pearl River, NY 10965, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of tricyclic anilinopyrimidines were synthesized and evaluated as IKKb inhibitors. Several ana-
logues, including tricyclic phenyl (10, 18a, 18c, 18d, and 18j) and thienyl (26 and 28) derivatives were
shown to have good in vitro enzyme potency and excellent cellular activity. Pharmaceutical profiling
of a select group of tricyclic compounds compared to the non-tricyclic analogues suggested that in some
cases, the improved cellular activity may be due to increased clog P and permeability.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 11 March 2010
Revised 6 April 2010
Accepted 8 April 2010
Available online 18 April 2010
Keywords:
NF-jB
IKK inhibitor
IKKb
IKK-2
The nuclear factor-
plays an important role in many biological processes, including
j
B (NF-
j
B) family of transcription factors
(Fig. 1).27 These analogues, including the lead compound 1, had
an excellent in vitro IKKb inhibitory profile. However, further eval-
uations of the scaffold revealed less impressive activity in cellular
assays28,29 potentially due to poor physical properties. Research ef-
forts were then directed toward improving both the cellular activity
and aqueous solubility of these analogues in an effort to produce
potent IKKb inhibitors with good pharmacokinetic profiles.
Initial SAR studies revealed that substitution of the sulfonamide
of 1 with water solubilizing groups led to compounds with similar
in vitro IKKb inhibitory activity, but improved aqueous solubility
and pharmacokinetic properties.30 Additionally, we found that
substitution of the anilinopyrimidine in the 4-position with alter-
native heteroaryl or aryl moieties produced compounds with sim-
ilar biological profiles. In an effort to further develop our
understanding of the SAR of this series, it was decided to examine
inflammatory immune responses, oncogenesis, tumor metastasis,
and chemo resistance.1–6 NF-
j
B is normally retained in the
cytoplasm as an inactive form associated with the IkB inhibitory
proteins.7 However, upon cellular stimulation, I
is phosphory-
lated by the I B kinase (IKK) and subsequently undergoes ubiqui-
tin-dependent degradation. This liberates NF- B from the I B/NF-
B complex allowing its entry into the nucleus where it regulates
the transcription of an array of genes responsible for cell survival
and growth.8–10 The I
B kinase (IKK) complex is composed of at
least two catalytic subunits IKK , IKKb, and a noncatalytic regula-
tory protein, IKK , also known as IKK-1, IKK-2, and NEMO, respec-
tively.11–13 Although both catalytic subunits are capable of
phosphorylating I , IKKb has been identified as essential for
jBa
j
j
j
j
j
a
c
jBa
the activation of the complex in response to inflammatory stim-
uli.14,15 The potential for utilizing IKKb inhibitors for the treatment
of cancer and immunological disorders has been widely recognized
by the pharmaceutical industry as demonstrated by the abundance
of recent publications16–23 and patents.24,25
Our investigations to identify an IKKb inhibitor began with a
high throughput screening of compounds through an IKKb Lance
assay.26 The studies led to the discovery of a series of compounds
containing an anilinopyrimidine core, such as that contained in 1
* Corresponding author. Tel.: +1 610 354 8840.
E-mail address: aimeecrombie@hotmail.com (A.L. Crombie).
Figure 1. Lead compound from exploratory investigations.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.04.022

3822
A. L. Crombie et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3821–3825
Scheme 1. Reagents and conditions: (a) DMF-DMA, 110–125 °C, 24 h; (b) 3.0 equiv 4, NMP, 105–120 °C, 24 h to 3 d.
Table 1
IKKb inhibition of non-tricyclic and corresponding tricyclic analogues
a
b
c
Compound
Core
R
IKKb IC50
(lM)
IKKb Reporter IC50
(lM)
Cell growth (LoVo) IC50 (lM)
6
7
8
9
10
I
H
H
H
OMe
OMe
20.16d
1.83
>10,000
0.04h
0.55
nde
3.28
1.05
4.18g
2.55
0.75
II
III
I
1.43
4.06f
0.64i
0.42
II
a
b
c
d
e
f
[ATP] = 2 lM; N = 1, unless otherwise noted; see Ref. 26.
N = 1, unless otherwise noted; see Ref. 28.
N = 1, unless otherwise noted; see Ref. 29.
N = 2, (SD 1.68
lM).
nd = not determined.
N = 2, (SD 1.84
N = 2, (SD 0.16
l
l
M).
M).
g
h
i
N = 7, (SD 0.016
N = 2, (SD 0.16
l
M).
lM).
substitution of the pyrimidine in more detail. One of these investi-
gations was focused on developing a set of anilinopyrimidines with
bridged heteroaryl or aryl substitution (e.g., tricyclic compound 5)
to probe the effects of restricted rotation. These tricyclic com-
pounds were produced using a route similar to that employed for
the synthesis of non-bridged analogues of this series (Scheme 1).
Reaction of ketone 2 with DMF-DMA led to enamine 3, which cy-
clized under thermal conditions with guanidine 4 to form the de-
sired anilinopyrimidine derivatives.
The IKKb activity of the tricyclic phenyl compounds compared to
their corresponding non-tricyclic analogues is outlined in Table 1. It
was found that 6–6–6 tricyclic systems (Table 1, core II) served as
suitable cores, however, 6–7–6 tricyclic systems (Table 1, core III)
were not tolerated. While these tricyclic compounds had moderate
enzyme activity, a significant improvement of cellular activity in
both the reporter and cell growth assays31 was observed.
Additional SAR investigations revealed an alternative approach
for improving cellular activity. Substitution of the phenyl ring para
to the pyrimidine with acylamino groups had led to compounds
with excellent enzyme potency and cellular activity. Compounds
such as 11, 12, and 13 had approximately 10-fold increase in cell
growth potency31 over other anilinopyrimidine analogues
(Fig. 2). This led us to investigate IKKb inhibitory activity of tricy-
clic derivatives of these acylamino compounds.
We focused on developing a small library of acylamino-phenyl
substituted tricyclic analogues (18). Utilizing a route similar to
that described previously, reaction of the amino-substituted tetra-
lone 14 with DMF-DMA formed dienamino compound 15, which
was condensed with substituted guanidines 4 to form the tricyclic
anilinopyrimidine unit 16. Addition of acyl chlorides led to the
formation of the 4-acylamino-phenyl tricyclic analogues 18
(Scheme 2).
Figure 2. Acylamino-phenyl substituted anilinopyrimidines.

A. L. Crombie et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3821–3825
3823
Scheme 2. Reagents and conditions: (a) DMF-DMA, 120 °C, 24 h; (b) 1.3 equiv 4, NMP, 120 °C, 24 h, then 2 N aq NaOH, 100 °C; (c) 1.2 equiv 17, 2.5 equiv diethyl aniline, NMP,
rt, 24 h.
Table 2
IKKb inhibition of acylamino-phenyl substituted tricyclic anilinopyrimidines
a
b
c
Compound
R
IKKb IC50
(lM)
IKKb Reporter IC50
(lM)
Cell growth (LoVo) IC50 (lM)
11
18a
12
18b
13
18c
18d
18e
18f
18g
18h
18i
18j
18k
—
0.01
0.82
0.05
2.38
0.02e
0.51
0.31
1.83
1.94
1.59
2.20
1.11
0.28
2.23
2.46d
1.36
4.47
1.79
6.91f
0.83
0.97
1.60
6.40
8.25
1.57
1.46
ndh
0.79
0.37
0.18
0.19
0.25g
0.29
0.49
0.73
0.38
0.56
0.83
0.66
0.62
1.06
Neopentyl
—
Benzyl
—
Thiophen-2-ylmethyl
Thiophen-3-ylmethyl
4-Methoxybenzyl
isobutyl
2-Methylprop-1-enyl
Cyclopentyl
Isopropyl
3-Methoxy-3-oxo-propionyl
2-Furyl
ndh
a
b
c
[ATP] = 2
l
M; N = 1, unless otherwise noted; see Ref. 26.
N = 1, unless otherwise noted; see Ref. 28.
N = 1, unless otherwise noted; see Ref. 29.
N = 2, (SD 0.49
N = 2, (SD 0.006
N = 3, (SD 1.69
N = 4, (SD 0.24
d
e
f
lM).
lM).
lM).
g
h
lM).
nd = not determined.
Scheme 3. Reagents and conditions: (a) DMF-DMA, 120–125 °C, 24 h to 6 d; (b) 3 equiv 4, NMP, 140 °C, 24 h; (c) 3.0 equiv. guanidine carbonate, NMP, 115 °C, 3 d; (d)
1.2 equiv 23, 15 mol % Pd2dba3, 30 mol % BINAP, 3.5 equiv NaOt-Bu, dioxane, NMP, microwave, 115 °C, 1–2 h.

3824
A. L. Crombie et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3821–3825
Table 3
IKKb inhibition of non-tricyclic and corresponding tricyclic thiophene analogues
a
b
c
Compound
Core
R
IKKb IC50
(
lM)
IKKb Reporter IC50
(
lM)
Cell growth (LoVo) IC50 (lM)
25
26
27
28
29
30
31
32
IV
V
IV
V
IV
V
IV
V
H
H
0.18
0.79
0.23
0.77
0.17
0.78
0.13
1.22
1.70
0.80
0.77
0.51
1.06
0.62
3.39
1.96
2.62
0.33
2.81
0.68
3.38
1.10
4.11
0.80
2-Dimethylaminoethyl
2-Dimethylaminoethyl
3-Dimethylaminopropyl
3-Dimethylaminopropyl
Ethanol-2yl
Ethanol-2yl
a
b
c
[ATP] = 2
N = 1; see Ref. 28.
N = 1; see Ref. 29.
l
M; N = 1; see Ref. 26.
A diverse set of targets containing aryl- and heteroaryl-substi-
tains activity while improving aqueous solubility and pharmaco-
kinetic properties.30 We hoped to produce tricyclic analogues
that would have improved cellular activity and pharmaceutical
profile.
The scheme below outlines the synthesis of both non-tricyclic
and tricyclic thiophene analogues. The cyclocondensation route
described in Schemes 1 and 2 was used to produce thiophene
analogues without substitution of the sulfonamide (21). To incor-
porate water solubilizing groups, it was necessary to synthesize
2-amino-substituted pyrimidine 22, which underwent micro-
wave-assisted Suzuki couplings with a variety of aryl bromides
that contained substituted sulfonamides to form the desired ana-
logues 24 (Scheme 3).
tuted acylamino groups were synthesized and evaluated for IKKb
activity (Table 2). Several compounds exhibited potent cellular
activity in the reporter assay and modest enzyme activity. How-
ever, some analogues demonstrated potency in both assays. In par-
ticular, 18a, 18c, and 18d and 18j had excellent cellular potency
and good IKKb enzyme activity.
In addition to investigating phenyl tricyclic derivatives, we also
explored tricyclic thiophenes. In this case, we examined both the
effect of bridging the aniline pyrimidine substitution and the
incorporation of water solubilizing groups substituted at the sul-
fonamide. As determined from previous SAR studies, substitution
of the sulfonamide with amino- or hydroxyl-alkyl side chains re-
Table 4
IKKb inhibition and pharmaceutical profiling of non-tricyclic and corresponding tricyclic analogues
M) clog P Permeabilityd (Pe  10À6 cm/
Solubility pH: 7.4e
mL)
(lg/
a
b
c
Compound Core IKKb IC50
(l
M) IKKb Reporter IC50
M)
Cell growth (Lovo) IC50
(l
(l
s)
6
7
9
I
II
I
II
I
II
I
20.16
1.83
0.04
0.55
0.01
0.82
0.05
2.38
0.02
0.51
0.18
0.79
0.23
0.77
ndf
3.28
1.05
2.55
0.75
0.79
0.37
0.18
0.19
0.25
0.29
2.62
0.33
2.81
0.68
2.79
3.24
2.83
3.28
3.88
4.34
3.57
4.02
3.21
3.67
2.48
2.93
3.49
3.94
2.26
2.92
<0.01
1.80
0.60
2.30
<0.01
3.37
0.30
0.51
0.50
0.38
2.40
1.54
1.0
7.0
2.3
<1.0
<1.0
9.0
1.0
9.0
<1.0
6.0
2.0
1.43
0.64
0.42
2.46
1.36
4.47
1.79
6.91
0.83
1.70
0.80
0.77
0.51
10
11
18a
12
18b
13
18c
25
26
27
28
II
I
II
IV
V
IV
V
<1.0
>100
36.0
a
b
c
d
e
f
[ATP] = 2 lM; N = 1, unless noted in previous tables; see Ref. 26.
N = 1, unless noted in previous tables; see Ref. 28.
N = 1, unless noted in previous tables; see Ref. 29.
Effective permeability via passive diffusion determined using Parallel Artificial Membrane Permeability Assay (PAMPA).
Aqueous solubility.
nd = not determined.

A. L. Crombie et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3821–3825
3825
D.; Morse, M. A.; Pancholi, K. D.; Rumsey, W.; Solanke, Y. E.; Williamson, R. J.
Med. Chem. 2009, 52, 3098.
20. Kempson, J.; Guo, J.; Das, J.; Moquin, R. V.; Spergel, S. H.; Watterson, S. H.;
Langevine, C. M.; Dyckman, A. J.; Pattoli, M.; Burke, J. R.; Yang, X. X.; Gillooly, K.
M.; McIntyre, K. W.; Chen, L.; Dodd, J. H.; McKinnon, M.; Barrish, J. C.; Pitts, W.
J. Bioorg. Med. Chem. Lett. 2009, 19, 2646.
The IKKb activity of the tricyclic thiophene compounds com-
pared to their corresponding non-tricyclic analogues is shown in
Table 3. Similar to the tricyclic phenyl compounds, the thiophene
derivatives also exhibited improved cellular activity in the reporter
assay, but a decrease in IKKb enzyme inhibition. Additionally,
incorporation of water solubilizing groups had small effects on
the in vitro IKKb activity of these tricyclic compounds.
21. 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.; McIntyre, K. W.; 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.
22. Sugiyama, H.; Yoshida, M.; Mori, K.; Kawamoto, T.; Sogabe, S.; Takagi, T.; Oki,
H.; Tanaka, T.; Kimura, H.; Ikeura, Y. Chem. Pharm. Bull. 2007, 55, 613.
23. Wu, J.-P.; Fleck, R.; Brickwood, J.; Capolino, A.; Catron, K.; Chen, Z.; Cywin, C.;
Emeigh, J.; Foerst, M.; Ginn, J.; Hrapchak, M.; Hickey, E.; Hao, M.-H.; Kashem,
M.; Li, J.; Liu, W.; Morwick, T.; Nelson, R.; Marshall, D.; Martin, L.; Nemoto, P.;
Potocki, I.; Liuzzi, M.; Peet, G. W.; Scouten, E.; Stefany, D.; Turner, M.; Weldon,
S.; Zimmitti, C.; Spero, D.; Kelly, T. A. Bioorg. Med. Chem. Lett. 2009, 19, 5547.
24. Coish, P. D.; Wickens, P. L.; Lowinger, T. B. Expert Opin. Ther. Patents 2006, 16, 1.
25. Bamborough, P.; Callahan, J. F.; Christopher, J. A.; Kerns, J. K.; Liddle, J.; Miller, D.
D.; Morse, M. A.; Rumsey, W. L.; Williamson, R. Curr. Top. Med. Chem. 2009, 9, 623.
26. LANCE reactions are carried out based upon the suggestions of Wallac/Perkin–
Elmer. Purified Flag-IKKb enzyme (2 nM final concentration) is typically used
in the kinase reaction buffer supplemented with 0.0025% Brij solution (Sigma)
The pharmaceutical properties of a select group of tricyclic
compounds and their corresponding non-tricyclic counterparts
were evaluated. In general, the tricyclic phenyl compounds (Ta-
ble 4, core II) had increased cellular permeability, as predicted by
the clog P values. However, the tricyclic thiophene derivatives (Ta-
ble 4, core V) did not have the same permeability profile. Despite
having higher clog P values than their corresponding non-tricyclic
analogues, the thiophene tricyclics did not have improved perme-
ability. Further investigation using Caco-2 analysis confirmed a de-
crease in permeability for these analogues.32 This suggested the
importance of closely monitoring off target activity or modes of
cellular transport when pursuing the compounds. In addition, the
incorporation of a water solubilizing groups greatly increased the
compounds solubility and potentially pharmacokinetic properties,
which would be of importance for the design of future analogues.
In summary, we have reported several tricyclic anilinopyrimi-
dine derivatives that inhibit IKKb. These tricyclic compounds have
increased cellular potency over the corresponding non-tricyclic
analogues. In general, the improved cellular activity may be due
to the compounds’ increased clog P value and permeability profile.
However, this relationship was not observed in a select group of
thiophene analogues, indicating the possibility that biological
activities in addition to IKKb inhibition are contributing to the ob-
served cellular activity.
to help stabilize the enzyme. Biotinylated substrate I
purified (>95% pure) and is used at 500 nM final concentration. ATP is used at a
final concentration of 2 M. The total reaction volumes are 25 L and the
jBa is synthesized and
l
l
inhibitor compounds are preincubated with enzyme before substrate and ATP
are added. Reactions are conducted for 30 min at room temperature in black,
low binding plates (Dynex). 25
reactions and then 100 L of detection mixture [0.25 nM europium labeled
anti-phopho-I (prepared by Wallac) and 0.25 g/mL final concentrations
lL of 20 mM EDTA is added to terminate the
l
jB
a
l
streptavidin-APC, Wallac] is added 30 min before reading the plates in a Wallac
VICTOR plate reader. The energy transfer signal data is used to calculate IC50
values. See: Wisniewski, D.; LoGrasso, P.; Calaycay, J.; Marcy, A. Anal. Biochem.
1999, 274, 220. See also: Sadler, T. M.; Achilleos, M.; Ragunathan, S.; Pitkin, A.;
LaRocque, J.; Morin, J.; Annable, R.; Greenberger, L. M.; Frost, P.; Zhang, Y. Anal.
Biochem. 2004, 326, 106.
27. Sum, F.-W.; Powell, D. W.; Zhang, Y.; Chen, L.; Kincaid, S. L.; Jennings, L. D.; Hu,
Y.; Gilbert, A. M.; Bursavich, M. G. U.S. Patent 2006079543, 2006.
28. NF
expression of a NF
NF B Luciferace assay is as follows: Hela cells are stably transfected with the
pNF B-Luciferace construct. Plate 10,000 Hela + pNF B-Luciferace cells/well in
a total volume (media = DMEM + 10% FBS + 1% pen/strep + 0.1 mg/mL G418) of
100 L in either solid or clear white bottom luminescence plates. Incubate in
37 °C/5% CO2 incubator overnight. Treat the cells with necessary compounds for
1 h (10 l volume; starting concentration will be 2 or 4 g/mL final and then
titrations maybe be done later for IC50 values), followed by 2.5–5 ng/mL final
concentration of TNF- (BioSource) for 6 h inthe incubator. After this incubation,
add 100 L of Steady GloTM Luciferace reagent from Promega and gently mix for
at least 10 min at room temperature. Read on VICTOR5 machine using standard
luminescence protocol. Controls = wells that only have TNF- and wells that
only are treated with DMSO (in place of compound) and PBS (in place of TNF- ).
j
B Luciferace assay test the compound ability to inhibit TNF-
a induced
jB response element-Luciferace reporter gene. Protocol for
Acknowledgments
j
j
j
We thank Drs. Magid Abou-Gharbia and Tarek Mansour, for
their support, the Discovery Analytical Chemistry group for analyt-
ical and spectral determinations, and the Pharmaceutical Profiling
groups for providing profiling data.
l
l
l
a
l
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2 day and then treat the cells with compound. Alternatively, plate the cells on
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l
l
l
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31. Inhibition of cell growth may be to off target activity.
32. Caco-2 Cell-Layer Permeability Assay (Papp  10À6 cm/s): 25 (a–b) 53.08, (b–a)
38.89; 26 (a–b) 33.95, (b–a) 20.68; 27 (a–b) <1.00, (b–a) 13.16; 28 (a–b) <1.00,
(b–a) 3.08.
19. Christopher, J. A.; Bamborough, P.; Alder, C.; Campbell, A.; Cutler, G. J.; Down,
K.; Hamadi, A. M.; Jolly, A. M.; Kerns, J. K.; Lucas, F. S.; Mellor, G. W.; Miller, D.