Inhibitory Kappa B Kinase α (IKKα) Inhibitors That Recapitulate Their Selectivity in Cells against Isoform-Related Biomarkers

Article abstract of DOI:10.1021/acs.jmedchem.7b00484

IKKβ plays a central role in the canonical NF-kB pathway, which has been extensively characterized. The role of IKKα in the noncanonical NF-kB pathway, and indeed in the canonical pathway as a complex with IKKβ, is less well understood. One major reason for this is the absence of chemical tools designed as selective inhibitors for IKKα over IKKβ. Herein, we report for the first time a series of novel, potent, and selective inhibitors of IKKα. We demonstrate effective target engagement and selectivity with IKKα in U2OS cells through inhibition of IKKα-driven p100 phosphorylation in the noncanonical NF-kB pathway without affecting IKKβ-dependent IKappa-Bα loss in the canonical pathway. These compounds represent the first chemical tools that can be used to further characterize the role of IKKα in cellular signaling, to dissect this from IKKβ and to validate it in its own right as a target in inflammatory diseases.

Full text of DOI:10.1021/acs.jmedchem.7b00484

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Article
Inhibitory Kappa B kinse # (IKK#) inhibitors that recapitulate
their selectivity in cells against isoform-related biomarkers
Nahoum G. Anthony, Jessica Baiget, Giacomo Berretta, Marie Boyd, David Breen, Joanne Edwards,
Carly Gamble, Alexander I. Gray, Alan L. Harvey, Sophia Hatziieremia, Ka Ho Ho, Judith K Huggan,
Stuart Lang, Sabin Llona-Minguez, Jia Lin Luo, Kathryn McIntosh, Andrew Paul, Robin J Plevin, Murray N.
Robertson, Rebecca Scott, Colin J. Suckling, Oliver Brook Sutcliffe, Louise C. Young, and Simon Paul Mackay
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b00484 • Publication Date (Web): 24 Jul 2017
Downloaded from http://pubs.acs.org on July 24, 2017
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Young, Louise; University of Strathclyde
Mackay, Simon; University of Strathclyde, Strathclyde Institute of
Pharmacy and Biomedical Science
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Inhibitory Kappa B kinase α (IKKα) inhibitors that recapitulate their selectivity in cells against
isoformꢀrelated biomarkers
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Nahoum G. Anthony , Jessica Baiget , Giacomo Berretta , Marie Boyd , David Breen , Joanne
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Edwards , Carly Gamble , Alexander I. Gray , Alan L. Harvey , Sophia Hatziieremia , Ka Ho Ho ,
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Judith K. Huggan , Stuart Lang , Sabin LlonaꢀMinguez , Jia Lin Luo , Kathryn McIntosh , Andrew
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Paul , Robin J. Plevin , Murray N. Robertson , Rebecca Scott , Colin J. Suckling , Oliver B. Sutcliffe ,
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Louise C. Young and Simon P. Mackay *
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Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, 161 Cathedral Street, Glasgow G4
0NR, Scotland, United Kingdom
¥
WestCHEM Department of Pure and Applied Chemistry, University of Strathclyde, 295 Cathedral Street, Glasgow G1
1XL, Scotland, United Kingdom
§
Wolfson Wohl Cancer Research Centre, Institute of Cancer Sciences, University of Glasgow, Garscube Estate,
Switchback Road, Glasgow, G61 1QH, Scotland, United Kingdom
Abstract
IKKβ plays a central role in the canonical NFꢀkB pathway, which has been extensively characterised.
The role of IKKα in the nonꢀcanonical NFꢀkB pathway, and indeed in the canonical pathway as a
complex with IKKβ, is less well understood. One major reason for this is the absence of chemical
tools designed as selective inhibitors for IKKα over IKKβ. Herein, we report for the first time a series
of novel, potent and selective inhibitors of IKKα. We demonstrate effective target engagement and
selectivity with IKKα in U2OS cells through inhibition of IKKαꢀdriven p100 phosphorylation in the
nonꢀcanonical NFꢀkB pathway without affecting IKKβꢀdependent IKappaꢀBα loss in the canonical
pathway.
These compounds represent the first chemical tools that can be used to further characterise the role of
IKKα in cellular signalling, to dissect this from IKKβ and validate it in its own right as a target in
inflammatory diseases.
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Introduction
Nuclear FactorꢀκB (NFꢀκB) transcription factors are central coordinators of the innate and adaptive
1, 2
immune response and play key roles in cancer development and progression. NFꢀκBs also have a
major role in controlling the ability of both preꢀneoplastic and malignant cells to resist apoptosis and
1, 2
support tumour angiogenesis and invasiveness. The signalling pathways that mediate the activation
of the different NFꢀκB complexes are therefore attractive targets for new chemotherapeutic
interventions.
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The NFꢀκB pathways, which are regulated by the inhibitory κB kinases (IKKs), are elevated when
homeostasis is disrupted. This is represented by an increase in constitutive IKKα/β activity leading to
enhanced NFꢀκB expression and subsequent nuclear localisation. The IKKs are upstream regulators of
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, 2
the NFꢀκBs, which exist as either homoꢀ or heterodimers bound to inhibitory kappa Bs (IκB’s). The
activation of these IKK complexes dictates the phosphorylation, targeted ubiquitination, and
proteolytic removal of IκBs in the canonical pathway and the phosphorylation and processing of high
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, 2
molecular weight NFꢀκB proteins (p100) in the nonꢀcanonical pathway. This in turn allows NFꢀκB
complexes to translocate to the nucleus and bind specific promoter regions of their targeted genes.
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, 4
Studies
have indicated that IKKα and IKKβ play key but divergent roles in the regulation of global
NFꢀκB signalling and many aspects of cellular transcription. IKKβ controls the canonical pathway via
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ꢀ7
activation of p65 RelAꢀp50 heterodimers and its inhibition leads to a reduction in proꢀinflammatory
gene expression in several cell types. This is relevant to cancer because several proꢀinflammatory
species associated with tumour development and progression are encoded by genes regulated through
3, 4, 6
4, 6
the IKKβꢀNFꢀκB axis.
IKKα has been shown to have a minor role in the canonical pathway,
but is pivotal in the nonꢀcanonical pathway, catalysing the phosphorylation and proteolytic processing
of p100 NFꢀκB2 which in turn liberates distinct NFꢀκB p52/RelB dimers and initiates transcription of
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, 8, 9
a specific subset of genes. IKKα and IKKβ have specific cellular functions
and the selective
inhibition of one isoform over the other may provide a useful and novel therapeutic strategy in cancer
and inflammatory diseases.
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Over the past 10 years, many inhibitors of IKKβ have been reported,
primarily towards
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developing clinical agents to treat inflammatory conditions such as asthma. However, recent studies
suggest there may be significant toxicity and side effects associated with IKKβ inhibition, including
the development of inflammatory skin disease and sensitisation of colonic epithelium to a range of
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insults. In addition, IKKβ knockout mice display severe liver dysfunction. Intestinal and liver
toxicity have also been an issue in several clinical trials of IKKβ inhibitors which may further limit
their clinical applications. Some IKKα inhibitors have been described in the patent literature, but with
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little detail regarding activity and specificity. Asamitsu et al. reported that the natural product,
noraristeromycin (NAM), inhibits IκBα phosphorylation and degradation upon TNFα stimulation and
prevents p65 phosphorylation through selective IKKα inhibition. However, the pharmacodynamic
readouts used were reporters for both IKKβ and IKKα activity in cells, and do not focus on specific
biomarkers of the IKKαꢀcontrolled nonꢀcanonical pathway, such as p100 phosphorylation and
subsequent processing to p52.
18ꢀ20
Given the growing evidence that IKKα has an important role in a number of cancers,
selective
IKKα inhibitors are required in order to fully understand and validate its role in cancer development
19, 21
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25ꢀ27
and progression, particularly in prostate,
breast
and pancreatic
cancers. Herein we
describe the design, synthesis and evaluation of a series of 4ꢀsubstituted 2ꢀaminoꢀ5ꢀcyanopyrrolo[2,3ꢀ
d]pyrimidines as part of our programme to develop isoform selective IKKα inhibitors. We present a
comparison of the kinase domains of IKKα and IKKβ based on molecular dynamics simulations to
explore differences in conformational flexibility that would enable small molecule inhibitors to
discriminate between the two isoforms. Finally, we report the first example of IKKαꢀselective
compounds that recapitulate activity in cells against isoformꢀrelated pharmacodynamic readouts.
Results and Discussion
Strategy for discriminating between IKKα and IKKβ
To date, no group has been able to successfully crystallise IKKα and report a high resolution structure
of sufficient detail to guide structureꢀbased inhibitor design. In order to explore differences between
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the two IKK isoforms, we built a homology model of the IKKα kinase domain based on the crystal
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structure of IKKβ (chain B, residues 1ꢀ309, PDB entry 4KIK). keeping the inhibitor (KSA700 in the
pdb file) and water molecules found within 6 Å of the proteinꢀinhibitor complex (Figure 1). Both
IKK kinase domains were solvated and then subjected to extended molecular dynamics, with an
average structure generated for the last 21 ns (IKKα) or 26 ns (IKKβ), and subsequently minimised.
When superimposing the preꢀsimulated structures of both IKK isoforms, it was striking to see that
regions making up the ATPꢀbinding pocket were essentially identical (Figure 2; Left). However,
analysis of descriptors of motion extracted from their MD trajectories such as residual fluctuation
revealed dynamic differences between the two isoforms that could potentially be exploited in an
inhibitor design programme.
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Residual fluctuations obtained from the MD trajectories highlighted areas of the IKKs that acted
differently during the simulations (Figure 2; Right). Overall, the two isoforms behaved very similarly
but IKKα appeared more flexible in several key areas around the ATP binding site, particularly at the
Gꢀloop (residues 22ꢀ27) above the site entrance and the loop located just adjacent to the hinge
(residues 155ꢀ159 in IKKα (VGGKI) and residues 156ꢀ160 in IKKβ (GEQRL)). Two residues could
account for the differences observed with the Gꢀloop: Pro52 and Gln48 in IKKβ (Thr52 and Leu48 in
IKKα) induce a tension at the tip of the first αꢀhelix through proline’s intrinsic structure and the
engagement of the glutamine side chain in a reciprocal Hꢀbond dimer arrangement with the side chain
amide of Asn28 (Figure 3). This asparagine is located at the end of the Gꢀloop and its interaction with
Gln48 restrained the Gꢀloop movement by more than 1 Å in IKKβ when compared with IKKα. A
different dynamic behaviour was observed with IKKα, as the equivalent residues do not impose
restraints on the Gꢀloop movements: Thr52 has no rigid turn restriction like Pro52 in IKKβ and the
side chain of Leu48 seeks a hydrophobic environment and will not engage in Hꢀbond formation with
Asn28, leaving the side chain of this latter residue free to make interactions in the ATP binding site of
IKKα (in contrast to being sequestered by Gln48 as in IKKβ) (Figure 3). The other significant
difference was related to the VGGKI loop (residues 155ꢀ159) in IKKα (residues GEQRL: 156ꢀ160 in
IKKβ). In this case, one residue is responsible for the change observed in residual fluctuation: Lys158
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in IKKα is replaced by Arg159 in IKKβ. The slightly longer arginine side chain and its bifurcate Hꢀ
bonding capacity forms a reciprocal Hꢀbond dimer with the side chain carboxylate of Asp128 in IKKβ
that was maintained throughout the simulation, whilst the equivalent lysine in IKKα never engaged in
a strong interaction with the equivalent Asp127 in IKKα (Figure 4). This interaction in IKKβ is
responsible for tethering the 156ꢀ160 loop to αꢀhelix 3 (Asp128 is located in the middle of this helix),
thus reducing its flexibility compared with IKKα.
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With respect to putative inhibitor binding, the implications for isoform selectivity of these two sets of
differences in the ATP site are twoꢀfold: firstly, it should be possible to design small molecules that
target the free Asn28 side chain amide presented at the back of the IKKα pocket that is otherwise
engaged in IKKβ (Figure 3); and secondly, because the ATP binding pocket has greater flexibility in
IKKα, it has the potential to accommodate larger substituents, particularly below the Gꢀloop.
Figure 1. Sequence alignment for the kinase domains of IKKα and IKKβ (4KIK_chainB) showing 61 % of
identical residues (coloured in turquoise), a further 14 % similar residues (polar for polar, hydrophobic for
hydrophobic; in light blue) and 25 % nonꢀsimilar residues (white).
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Figure 2 Left: Minimized average structure of IKKβ highlighting residues that are identical to (turquoise),
similar to (light blue) or different from (white) IKKα. The ATP analogue marks the ATP binding site and is
surrounded turquoise residues. Right: Residual fluctuations of IKKα (black line) and IKKβ (blue line) arising
during the MD simulations. Several areas were found to be more flexible in IKKα (red underline).
Figure 3. Superimposition of IKKα (white) and IKKβ (blue) highlighting the differences near/in the ATP
binding site (marked by the staurosporine analogue as a stick model) between the two isoforms. The expanded
area shows equivalent residues in IKKα (Asn28, Leu48 and Thr52) and IKKβ (Asn28, Gln48 and Pro52)
engaged in different interactions/structural effects resulting in Asn28 being available to interact with putative
ligands in the binding pocket of IKKα but not IKKβ
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Figure 4. Left: Loop conformation located below the active site in IKKα (white) and IKKβ (blue) and its
relationship with αꢀhelix 3 residue Asp127 (IKKα)/Asp128 (IKKβ). In IKKβ, Arg159 makes a reciprocal
hydrogen bond dimer interaction with Asp128, whereas in IKKα Lys158 has no close interactions with Asp127.
Right: Side chain amine nitrogen (Lys158 (IKKα)/Arg159 (IKKβ)) to side chain acid oxygen (Asp127
(IKKα)/Asp128 (IKKβ)) distance throughout the equilibrated phase of the simulation.
Initial hit identification
In order to identify hits that could be developed to exploit the differences between the two isoforms,
we screened our inꢀhouse compound library compiled of fragments designed to target the common
hingeꢀbinding motif found in protein kinases. Using a DELFIA kinase assay kit with minor
29
modifications to measure IKKα and IKKβ inhibitory activity, we identified 2ꢀaminoꢀ4ꢀchloroꢀ5ꢀ
cyanopyrrolo[2,3ꢀd]pyrimidine 4 as our primary hit (Table 1). Based on the assumption that the 2ꢀ
aminopyrrolo[2,3ꢀd]pyrimidine core of compound 4 was binding at the hinge of IKKα, we varied
substitution at the 4ꢀposition widely. Differences in bulk and polar functionality were incorporated to
exploit the greater flexibility in the IKKα ATP binding site and the position of the Asn28 side chain at
the back of the pocket.
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Chemistry
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The general route used to prepare N ꢀsubstituted 2ꢀaminoꢀ5ꢀcyanopyrrolo[2,3ꢀd]pyrimidines for
30
assessment began with the preparation of 1 using a published procedure. 1 was then protected as
either its diacetyl derivative 2 or pivaloyl derivative 3, which were subsequently converted to the aryl
chlorides 4 and 5 (Scheme 1) and used to prepare an initial series of compounds (4ꢀ31) (Table 1) by
nucleophilic aromatic substitution with a range of primary and secondary amines. The second series
of aryl cross coupled compounds exploring the 4ꢀphenyl 2ꢀaminoꢀ5ꢀcyanopyrrolo[2,3ꢀd]pyrimidines
was prepared by SuzukiꢀMiyaura couplings (43ꢀ62) with 5. In general, the use of the Nꢀpivalamide 5
gave higher yields of the final compounds compared to the unprotected form 4.
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Scheme 1. Reagents and conditions: (a) Ac
DMA, (d) Amine, Et N, 1,4ꢀdioxane 200 °C (ꢁW) 20 mins, (e) Amine, Et
EtOH, 80 °C, 20 h, (f) boronic acid /ester, Pd(dppf)Cl , KOAc, H O/dioxane, 110 C 16 h.
2
O, DMF, 150 °C, 4 h, ; (b) PivCl, pyridine, 85 °C, (c) POCl
3
,
3
3
N, nꢀBuOH, reflux, 16 h then KOH,
°
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4
In order to explore polar functionality further in the N ꢀsubstituted 2ꢀaminoꢀ5ꢀcyanopyrrolo[2,3ꢀ
d]pyrimidines, a series of amino alcohol derivatives (23ꢀ31) was prepared as analogues of 16.
Compounds 23ꢀ29 were prepared from commercially available amines using the procedure detailed in
Scheme 1. To incorporate 4ꢀsubstituents with more than one hydroxyl group in the cyclopentane ring
17
31
as in the putative IKKα selective inhibitor NAM, we prepared the diol 30 and triol 31. The
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synthesis of 30 began with bromination of cyclopentene 32 to produce the unstable halide 33, which
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was immediately treated with N,Nꢀdibenzylamine. The resulting allylic amine 34 underwent synꢀ
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dihydroxylation to afford the diol 35 with excellent diastereoselectivity, which was then protected as
the dibenzoate 36. The amine was subsequently deprotected by hydrogenation to yield 37, which was
coupled with 5. Global deprotection gave the desired final product 30 (Scheme 2).
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Table 1: Biochemical inhibitory data for the N ꢀsubstituted 2ꢀaminoꢀ5ꢀcyanopyrrolo[2,3ꢀd]pyrimidines. Ki
values are expressed in ꢁM units and are the result of three independent experiments.
Compound No.
R
IKKα (K
i
± SEM μM)
IKKβ (K
i
± SEM μM)
>30
4
6
4.0 ± 0.9
>30
>30
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8
13.0 ± 0.51
>30
>30
16.0 ± 1.1
1.8 ± 0.31
9
>30
>30
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1
0
1
>30
13 ± 1.1
>30
>30
1
2
>30
1
1
3
4
10 ± 2.9
5.3 ± 1.6
>30
>30
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Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
15
>30
>30
>30
>30
1
1
6
7
1.6 ± 0.48
5.3 ± 0.9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
8
5.3 ± 0.94
5.9 ± 0.34
1
2
9
0
>30
>30
20.0 ± 1.3
>30
2
2
1
2
>30
>30
>30
>30
23
12.1 ± 6.3
>30
>30
>30
2
4
2
2
5
6
6.0 ± 1.6
>30
2.5 ± 0.14
3.1 ± 0.6
>30
>30
27
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1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
28
2.0 ± 1.5
>30
2
9
>30
>30
>30
3
3
0
1
1.0 ± 0.14
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
0
.8 ± 0.71
3.6 ± 0.87
Scheme 2. Reagents and conditions: (a) NBS, (PhCO
OsO , NMO, acetone / H O, rt, 4 h, 71%, 96:4 dr; (d) BzCl, pyridine, 0 °C to rt, 24 h, 84%; (e) H
Pd(OH) , rt, 16 h, 98% (f) 5, Et N, nꢀBuOH, reflux, 16 h then KOH, EtOH 80 °C, 20 h, 42%.
2
)
2
, CCl
4
, 90 °C, 1h; (b) NH(Bn)
2
, CCl
4
, rt, 12 h, 70%; (c)
4
2
2
(1 atm),
2
3
3
4,
To prepare the triol 31, the allyl acetate 38 was reacted with sodium diꢀtertꢀbutyl iminodicarbonate
35
to produce 39, which was oxidised to the synꢀdiol 40 followed by benzoate protection of the
hydroxyl functionalities to give 41, and removal of the BOC groups under acidic conditions to yield
the amine 42. This was then coupled with 5 followed by basic hydrolysis to remove the protecting
groups to give the desired final product 31 (Scheme 3).
An obvious progression from this series was to replace the bulky saturated ring with a phenyl ring
bearing a selection of polar oꢀ, mꢀ or pꢀsubstituents. This series was prepared by reacting 5 with a
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Journal of Medicinal Chemistry
variety of boronic acids or esters under SuzukiꢀMiyaura coupling conditions to afford the products 43ꢀ
62 (Scheme 1; Table 2).
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Scheme 3. Reagents and conditions: (a) Pd(PPh
OsO , NMO, acetone / H O, rt, 24 h, 89%; (c) BzCl, pyridine, 0 °C to rt, 17 h, 74%; (d) 4N HCl in 1,4ꢀdioxane,
°C to r.t, 16 h, 76%; (e) 7, Et N, nBuOH, reflux, 16 h then KOH, EtOH 80 °C, 20 h, 33%.
3 4 3 2
) , PPh , NaH, NH(BOC) , THF / DMF, 50 °C 24 h, 42%; (b)
4
2
0
3
Table 2: Biochemical inhibitory data for the 4ꢀphenyl 2ꢀaminoꢀ5ꢀcyanopyrrolo[2,3ꢀd]pyrimidine series. K
i
values are expressed in ꢁM units and are the result of three independent experiments.
Compound No.
R
H-
IKKα (K
i
± SEM μM)
IKKβ (K
i
± SEM μM)
>30
4
4
4
4
4
4
4
5
5
5
3
4
5
6
7
8
9
0
1
2
1.7 ± 0.95
1.6 ± 0.35
10 ± 1.0
4-HO-
4-F-
20 ± 1.2
>30
4-H
2
NCO-
16 ± 1.1
>30
4-HOCH2-
0.65 ± 0.06
0.08 ± 0.07
2.4 ± 0.24
1.7 ± 1.2
16 ± 5.8
2.3 ± 1.1
1.0 ± 0.28
0.3 ± 0.6
0.4 ± 0.48
1.4 ± 0.9
0.1 ± 0.6
4-H
2
NSO
2
-
4-CH
3
SO
2
-
4-CH
4-ToluylNHSO
4- CH SO NH-
3 2
NHSO -
2
-
3
2
1.4 ± 0.2
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1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
53
3-HO-
2.4 ± 0.2
7.7 ± 1.3
>30
3.8 ± 1.7
>30
5
5
5
5
5
5
6
6
6
4
5
6
7
8
9
0
1
2
3-F-
3-H NCO-
>30
2
3-HOCH
2
-
9.8 ± 0.54
5.3 ± 0.95
15 ± 1.11
2.0 ± 0.5
>30
4.0 ± 1.7
5.9 ± 3.4
>30
3- H NSO -
2
2
2-HO-
2-F-
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
>30
2-H
2-HOCH2-
2-H NSO
2
NCO-
>30
>30
>30
2
2
-
>30
>30
In order to assess the importance of the 2ꢀamino and 5ꢀcyano groups for activity and selectivity, we
purchased (63) or prepared compounds (64ꢀ71) without either or both substituents (Scheme 4, Table
3
).
3
Scheme 4. Reagents and conditions: (a) Alkylamine, Et N, 1,4ꢀdioxane 200 °C (ꢁW) 20 mins, (b) boronic acid
°
/
ester, Pd(dppf)Cl
2
, KOAc, H
2
O / dioxane, 110 C, 16 h.
Table 3: Biochemical inhibitory data for the 4ꢀsubstituted pyrrolo[2,3ꢀd]pyrimidines with and without the 2ꢀ
i
amino and/or 5ꢀcyano groups. K values are expressed in ꢁM units and are the result of three independent
experiments.
Compound
R
1
R
2
R
3
IKKα (K
i
± SEM μM)
IKKβ (K
i
± SEM μM)
No.
4
4.0 ± 0.9
>30
>30
>30
6
3
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Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
48
0.08 ± 0.07
1.0 ± 0.28
0.61 ± 0.23
2.5 ± 1.1
>30
6
6
6
4
5
6
2.9 ± 0.71
1.5 ± 0.36
2.1 ± 0.5
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
67
>30
>30
>30
>30
>30
>30
>30
>30
6
8
69
7
0
71
>30
>30
Structureꢀactivity relationship studies
4
In the N ꢀsubstituted series (Table 1) several compounds had similar K
i
values against IKKα
compared to the initial hit 4. For example, compounds with aliphatic substituents retained potency and
selectivity against IKKα and had the potential to be improved through further derivatisation.
Inhibitory activity for the secondary amines (7, 8, and 9) suggests the presence of a restricted
lipophilic pocket in IKKα, which could be responsible for a fiveꢀfold increase in potency from the
methylamine analogue 7 to the cyclohexylamine derivative 9. However, the inactivity of the
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compound with the bulkier cyclohexylmethylamino group (10) suggested that this lipophilic pocket
was limited in size. In contrast, the more planar phenyl and benzyl analogues (11 and 14) were found
to be less active than 9, and the insertion of polar substituents in the para position of the aniline group
produced no significant improvement in activity (12 and 13). Conversely, the insertion of polar groups
in more flexible side chains was tolerated (16), which suggested the presence of a polar region
adjacent to the lipophilic pocket.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Although compounds with a piperidyl side chain (17 and 18) were found to be active, other tertiary
amines at position 4 were not tolerated (19ꢀ22), which could be due to additional heteroatoms not
being accepted in a lipophilic area of the binding directly proximal to position 4 of the pyrrolo[2,3ꢀ
d]pyrimidine scaffold. By contrast, the activity of 16 suggested that a flexible ethyl chain is able to
direct the heteroatom away from this lipophilic region, and possibly engage an adjacent polar region
more effectively.
Removal of the hydrogen bond donor (HBD) from the 4ꢀamino substituent by replacing the secondary
cyclohexylamino group in 9 with the tertiary Nꢀmethylcyclohexylamine (10) abrogated activity.
However the activity of piperidyl analogue 17 suggests that a HBD at the 4ꢀposition is not an absolute
requirement for inhibition of IKKα. The inactivity of compound 10 can be explained by rotation of the
Nꢀmethyl (and hence the cyclohexyl group) substituent orthogonal to the pyrrolo[2,3ꢀd]pyrimidine
moiety to reduce steric clash with the 5ꢀcyano substituent and generate a conformation that is no
longer compatible with the binding site.
Based on the activity of the 4ꢀhydroxyethylamino derivative 16, we prepared a second series of amino
alcohol derivatives (Table 1). Extending the alkyl chain of 16 from ethyl to propyl was detrimental to
activity (23), whereas the cyclic analogues produced more interesting results. The (R)ꢀpyrrolidinꢀ3ꢀol
enantiomer 25 was active and selective against IKKα whereas the (S)ꢀenantiomer 24 was inactive,
suggesting the directionality of the hydroxyl group had a major influence on activity and selectivity.
On the other hand, the (R)ꢀ and (S)ꢀpyrrolidinꢀ2ꢀyl methanol enantiomers 26 and 27 shared the same
activity and selectivity against IKKα implying that free rotation around the methyl alcohol is
sufficient to allow equivalent interactions.
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The transꢀaminocyclohexanol derivative 28 was also active and selective against IKKα, but the
cyclohexylamino ethyl alcohol 29, designed as a hybrid of compounds 8 and 16, proved to be inactive.
Like compound 10, this is probably due to the aliphatic ring and the ethyl chain being twisted 90°
away from the nitrile axis to adopt a more energetically favoured conformation that is incompatible
with the binding site. The diol 30 was active and selective, but did not offer significant improvement.
The triol 31, prepared as an analogue of NAM, proved to be as active as 30 but less selective. Overall,
no significant improvement in binding or selectivity was gained by the introduction of more than one
hydroxyl group in this series of secondary cyclic amine derivatives.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
The direct attachment of an aromatic ring to position 4 of the pyrrolo[2,3ꢀd]pyrimidine scaffold
exemplified by compound 43 retained the potency and selectivity of the original hit 4 (Table 2). In
general, compounds with para substituents in the phenyl ring had better activity against IKKα and
produced our first nanomolar IKKα inhibitors in 47 and 48. Although activity against IKKβ was
evident for these two compounds, their higher potency for IKKα ensured significant isoform
selectivity was maintained. Compounds with meta substituents were low micromolar inhibitors of
IKKα (for example 53, 56 and 57) but were equipotent against IKKβ, apart from 55, which was
inactive against both isoforms. As with compounds 10 and 28, the inactivity of the orthoꢀsubstituted
derivatives in this series can be attributed to enforced conformational rotation of the phenyl ring to a
more orthogonal relationship with the pyrrolo[2,3ꢀd]pyrimidine scaffold, which introduces steric
clashes in the binding site. This is less pronounced with an oꢀfluoro substituent and consequently, 59
had similar potency to the unsubstituted 4ꢀphenyl derivative 43.
Compound 48 (proprietary code SU909), with a paraꢀprimary sulfonamide represents the most potent
IKKα inhibitor reported to date that has significant selectivity over IKKβ, although a limited kinase
profile using 40 kinases representative of the kinome identified offꢀtarget effects (>80% inhibitory
activity) with other kinases (Aurora A; CaMK1; CHK2; CK1; GSK3; MEK1 and PKC: Figure 5).
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1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 5: % Residual activity of 40 kinases challenged with compound 48 at 10 ꢁM.
To explore the role of the primary sulfonamide, the 4ꢀmethyl sulfone analogue 49 and the secondary
sulfonamides 50 and 51 were prepared. Notably, 49 and 50 had reduced potency against IKKα, but
both were more active against IKKβ, reversing the isoform selectivity for the first time in this series.
Similarly, when the orientation of the sulfonamide with respect to the phenyl ring (52) was reversed, a
reduction in potency against IKKα occurred but an improvement against IKKβ was again evident,
inverting isoform selectivity.
The absence of a 5ꢀcyano group in 48 (compound 64) reduced activity against IKKα more than 30ꢀ
fold (Table 3), although there was a slight improvement against IKKβ. Compound 65, which lacked
both the 5ꢀcyano and the 2ꢀamino substituent had lower activity against both isoforms. Compound 66,
without the 2ꢀamino group but with the 5ꢀcyano, attenuated IKKβ activity but did not reꢀestablish the
potency against IKKα seen for 48. The importance of the 5ꢀcyano group for activity against IKKα was
4
further demonstrated by preparing analogues of selected active N ꢀsubstituted exemplars from Table
1: removal of the 5ꢀcyano substituent produced inactive compounds against both isoforms (67ꢀ69),
and its replacement with other electronꢀwithdrawing substituents such as a trifluoromethyl (70) or a
carboxamide (71) moiety also attenuated all IKKα inhibitory activity (Scheme 4, Table 3).
Furthermore, compound 63, which represents the original hit compound (4) without the 5ꢀcyano
group, was also inactive against IKKα (Table 3).
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Overall, our SAR data suggest that the 5ꢀcyano substituent is essential for IKKα activity in this series
of compounds, but must be combined with a 2ꢀamino group to promote selectivity. To enhance
potency for IKKα, a 4ꢀphenyl group bearing a specific polar paraꢀsubstituent appears to be crucial.
Docking studies
1
2
3
4
5
6
7
8
9
In order to explain the general SAR profile observed with our series and to relate these to the
differences between the two IKK isoforms that our MD studies had revealed, we performed docking
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
3
6
studies using GOLD. A limited flexibility was enabled in the side chains of specific residues that
had shown significant fluctuation in the MD trajectories, namely Asn28, Val29 and Lys44. In the coꢀ
2
8
crystal structure of IKKβ that had formed the basis of our simulations, the staurosporine analogue
interacts though Hꢀbonds with the backbone groups of GK+1 (Glu97) and GK+3 (Cys99) in the
conserved hinge region. Initially, no attempt was made to direct the ligands to interact specifically
with the hinge region in our docking simulations; the only requirement was to occupy the ATP
binding site. This first docking study was performed to find a common binding mode that could
explain the IKKα inhibitory activity and selectivity reported for exemplars from our two series. To
explore this, compounds bearing different hydrophobic substituents were selected for their similar
potency and selectivity (e.g. 4, 9 and 43). The poses where the 2ꢀaminopyrrolo[2,3ꢀd]pyrimidine
scaffold interacted with the hinge region by Hꢀbonds were studied in detail and compared to similar
37
hingeꢀbinding protein kinase inhibitors. For example, aminopurines are known to adopt two
38
different binding modes in interactions with the hinge region of CDK2 via three hydrogen bonds.
However in similar poses for our compounds in IKKα, the analogous triplet of hydrogen bonds was
poorly supported by the IKKα hinge region in terms of bond distances and bond angles. Not
surprisingly, similar poses were found when the same compounds were docked with IKKβ, which is
due to the high isoform homology and subtle differences in topography in this hinge region of the
ATP binding site. These poses could not account for the selectivity reported in our assays and
suggested that this was not the binding mode responsible for conveying such discriminatory activity
between the isoforms. However, a binding mode where the 2ꢀaminopyrrolo[2,3ꢀd]pyrimidine scaffold
interacted with the back pocket of the IKKα active site explained the potency and selectivity displayed
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by our compounds more effectively. Figures 6AꢀC illustrate how compounds 4, 9 and 43 were
predicted to interact with IKKα, specifically targeting the carboxylate group of Glu61, the ammonium
group of Lys44 and, most significantly, the side chain carbonyl group of Asn28 via four Hꢀbonds. In
IKKβ, because Asn28 is involved in a dimeric Hꢀbond interaction with Gln48 revealed by our
simulations (Figure 3), which also changes the position of Glu61, the triple Hꢀbond interaction
observed in IKKα is lost. We therefore propose that the aminopyrrolo[2,3ꢀd]pyrimidine scaffold is
anchored to the back of the ATP site specifically targeting Asn28 in IKKα, which accounts for the
observed selectivity. In this orientation, the importance of the 5ꢀcyano group when binding with IKKα
can also be explained. Examination of the trajectory from an MD simulation of 47 revealed a water
molecule present for the majority of the run that formed a 3ꢀcentred hydrogen bond bridge between
the nitrile, the side chain carboxylate of Asp102, and the backbone NH of the same residue (Figure
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
6D). Overall, binding to IKKα appears to be facilitated through four centres on the 2ꢀaminoꢀ5ꢀ
cyanopyrrolo[2,3ꢀd]pyrimidine scaffold with four residues in the ATP binding site: Glu61, Lys44,
Asn28 and Asp102. A similar binding orientation with IKKβ is less likely because the two of the
equivalent sites are less accessible (Asn28 and Glu61) and this would account for the lack of activity
in this isoform.
Although the poses described for compounds 4, 9 and 43 showed no interaction with the hinge region,
the equivalent poses for the nanomolar inhibitors 47 and 48 had significant Hꢀbond interactions with
the GK+3 residue via their pꢀhydroxymethyl and pꢀsulfonamide substituents (Figures 6D and 7). The
equivalent binding pose in IKKβ did not feature the hydrogen bonds HB1 and HB2 due to local
structural differences observed between the kinase isoforms, and could explain the lower affinity of 48
for IKKβ. Interestingly, the presence of the methyl group in the secondary sulfonamide 50
compromised its ability to form an Hꢀbond with the HBA carbonyl group of GK+3 because of a steric
clash with the hinge region, thus reducing affinity for IKKα. The sulfonyl group could still interact
with the NH of GK+3, which explains why 50 and the sulfone 49 both have similar potency against
IKKα.
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The reason for both 49 and 50 also having greater inhibitory activity against IKKβ that reverses
isoform selectivity is because the polar side chain of Asn28 in IKKβ rotates out of the back pocket to
Hꢀbond with Gln48 (Figure 3). When 49 and 50 were docked in the IKKβ structure, they commonly
adopted a flipped pose, where the 2ꢀaminopyrrolo[2,3ꢀd]pyrimidine engaged with the GK+1 and +3
residues at the hinge through Hꢀbonding, whilst the sulfonyl oxygen and the 5ꢀcyano group Hꢀbonded
with K44 in the back pocket (Figure 8). Binding to IKKβ by 49 and 50 in this orientation is
facilitated by moving the polar Asn28 from the immediate vicinity creating a cleft that can more easily
accommodate the hydrophobic methyl moiety of 49 and 50. A similar reversal of selectivity was seen
with 64, which lacks the 5ꢀcyano substituent of 48. The drop in potency against IKKα can be
attributed to the absence of a waterꢀbridging Hꢀbond interaction with Asp102, but also to the removal
of the conformationallyꢀrestraining effect of the 5ꢀcyano group on the adjacent 4ꢀphenyl ring which
would normally optimise alignment of the pꢀsulfonamide Hꢀbonding interaction with GK+3 in the
hinge. The enhanced affinity for IKKβ by 64 can again be explained by the adoption of a flipped pose
when binding to this isoform, which is enabled by a more flexible pꢀsulfonamide that can Hꢀbond
more effectively with Lys44. Just as importantly, this flexibility allows the HBD of the sulfonamide
group to access the less accessible Glu41 at the back of IKKβ pocket.
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In summary, in line with our SAR data and the hypothesis derived from our MD simulation studies,
we suggest that nanomolar potency against IKKα with selectivity over IKKβ can be accomplished by
having molecules that can bind to the hinge region of the ATP binding site and to the Asn28 residue at
the back of the pocket that is available in IKKα but not in IKKβ (Figure 8). Furthermore, inhibitory
potency against IKKα can be enhanced when compounds can form Hꢀbonding interactions with the
three residues in the back pocket (Glu41, Lys44, Asn28) and the GK+3 and Asp102 residues in the
hinge.
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Figure 6. Docked poses of 4 , 9, 43 and 47 (AꢀD respectively) binding to the back of the ATP pocket in IKKα,
where the 2ꢀ aminopyrrolo[2,3ꢀd]pyrimidine motif forms Hꢀbonds (shown in green) with the side chain
carboxylate of Glu61, the side chain ammonium group of Lys44 and the side chain carbonyl group of Asn28.
No equivalent poses were identified when these compounds were docked with IKKβ. Figure 6D shows that an
MD simulation of 47 revealed an additional interaction with a molecule of water that formed an Hꢀbond
bridging interaction between the 5ꢀcyano group, the side chain of Asp102 and the backbone NH of the same
residue.
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Journal of Medicinal Chemistry
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Figure 7. Left: docked pose of 48. As before, the 2ꢀ aminopyrrolo[2,3ꢀd]pyrimidine moiety engages with the
back of the ATP pocket in IKKα and the sulfonamide group generates additional Hꢀbonds with the main chain
carbonyl and NH of GK+3 that produce nanomolar activity. Right: Schematic representation of the proposed
binding mode in the IKKαꢀATP binding site showing key interactions for compound 48.
Figure 8. Left: docked pose of 50 in IKKβ. Unlike IKKα, the aminopyrrolo[2,3ꢀd]pyrimidine moiety has
flipped and Hꢀbonds to GK+1 and GK+3. The rotation of Asn28 out of the pocket to Hꢀbond with Glu48
allows the sulfonyl group to Hꢀbond to Lys44 and accommodates the lipophilic methyl group in the cleft
opened up by this rotation. Right: Schematic representation of the proposed binding mode in the IKKβꢀATP
binding site showing key interactions for compound 50.
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Page 24 of 67
Demonstrating IKKα activity and selectivity is recapitulated in cells.
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The objective of the cellꢀbased assessment was to characterise whether the two most active
compounds, 47 and 48 against IKKα recapitulated their activity in a cellular environment. IKKα but
not IKKβ plays a role in the regulation of the nonꢀcanonical NFꢀκB cascade which initially involves
phosphorylation of p100, followed by p100 processing to p52 to regulate a number of genes that
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contribute to and promote cellular growth. More recent studies have shown that overꢀactivation of
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the nonꢀcanonical NFꢀκB pathway in a variety of cellular settings e.g. prostate,
pancreas
is
inherently associated with cell survival and proliferation. The U2OS osteosarcoma cell line is
representative of these proliferative characteristics and is dependent on constitutively activated IKKα
4
0
signalling; it was therefore selected as the system to demonstrate perturbation of IKKα activity in
the cellular environment.
Preꢀtreatment of U2OS cells with increasing concentration of 48 and 47 resulted in concentrationꢀ
dependent inhibition of FCSꢀstimulated phosphorylation of p100 (Figures 9a and 10a). The IC50
values for 48 and 47 were calculated as 8.8 ꢁM and 13.9 ꢁM respectively. Two IKKβꢀdependent
readouts were selected to assess selectivity over IKKβ . Firstly, TNFα stimulates IκBα degradation
through activation of IKKβ and if IKKβ is inhibited, then a band corresponding to the IκBα protein
should be evident by Western analysis. Secondly, IKKβ phosphorylates p65 on Ser536, which can
also be assessed by Western blot analysis. Only at the highest concentration (100 ꢁM) of 48 was IκBα
degradation and p65 (Ser536) phosphorylation inhibited (Figure 9b). Even at this high concentration
these effects were not apparent following preꢀtreatment with 47 (Figure 10b).
To provide further evidence for cellular activity, mouse embryonic fibroblasts lacking the IKKβ
kinase subꢀunit were utilised to investigate the potency of both compounds against agonistꢀstimulated
IKKαꢀregulated NFꢀκB transcriptional activation. Cells were infected with an adenovirus encoding an
NFꢀκBꢀluciferase promoter gene. NFꢀκB transcriptional activity driven by IKKα was stimulated
4
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specifically using ILꢀ1β, and the ability of the compounds to inhibit this response was assessed. At
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maximal concentrations of 50ꢀ100 ꢁM 48 abolished transcriptional activity compared to 47 which
inhibited activity by approximately 81% (% stim: Ilꢀ1β+DMSO = 100%, 48+Ilꢀ1β = ꢀ40.9 ± 4.6%,
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47+ILꢀ1β = 18.79 ± 7.2%, both **P<0.01). Both 47 and 48 demonstrated concentrationꢀdependent
inhibitory effects on FCSꢀinduced phosphorylation of p100 by IKKα. As IKKα plays a lesser role in
42
the canonical NFꢀκB cascade, particularly the rapid and transient stimulated degradation of IκBα,
selective IKKα inhibitors should not affect TNFꢀα induced IκBα degradation and p65
phosphorylation. Compound 47 did not affect either agonist stimulated IκBα degradation or p65
phosphorylation, whilst 48 only had a small effect on both markers at the highest concentration tested
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(100 ꢁM). Taken together, these data demonstrate that 47 and 48 are the first examples of IKKαꢀ
selective compounds that inhibit agonistꢀstimulated NFꢀκB signalling of the nonꢀcanonical pathway at
much lower concentrations than the canonical pathway.
c
Figure 9: The effect of compound 48 on (a) FCSꢀstimulated nonꢀcanonical and (b) TNFꢀαꢀinduced canonical
NFꢀkB activation in U2OS cells. Cells were preꢀtreated with 48 1 h prior to stimulation with FCS (10%) for 4 h
or TNFꢀα (10 ng/ml) for 30 min. Whole cell lysate were prepared, separated by SDSꢀPAGE and assessed for
(
a) inhibition of p100 phosphorylation (Ser866/870) and (b) IkBꢀα and pꢀp65 (Ser536) status. (c) Blots were
quantified and the IC50 value for inhibition of p100 phosphorylation was determined (48 IC50 = 5.8 ꢁM). The
results are representative of 3 independent experiments.
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Figure 10: The effect of compound 47 on (a) FCSꢀstimulated nonꢀcanonical and (b) TNFꢀαꢀinduced canonical
NFꢀkB activation in U2OS cells. Cells were preꢀtreated with 47 1h prior to stimulation with FCS (10%) for 4 h
or TNFꢀα (10ng/ml) for 30 min. Whole cell lysate were prepared, separated by SDSꢀPAGE and assessed for (a)
inhibition of p100 phosphorylation (Ser866/870) and (b) IkBꢀα and pꢀp65 (Ser536) status. (c) Blots were
quantified and the IC50 value for inhibition of pꢀp100 was determined (47 IC50 = 19.1ꢁM). The results are
representative of 3 independent experiments.
ꢀ
/ꢀ
ꢀ/ꢀ
Table 4: Ilꢀ1βꢀstimulated NFꢀκB transcriptional activation in IKKα MEFs. IKKβ MEFs infected with
Adv.NFꢀkB.luc were grown to near confluency and rendered quiescent by serum deprivation for 24h. Cells
were then preꢀtreated for 1 hour with increasing concentrations of compounds 48 or 47 prior to stimulation with
ILꢀ1β (10ng/ml) for a further 6 h. Cells were assayed for NFꢀκBꢀlinked luciferase reporter activity, as outlined
in the methods section. Values (RLUs) were collated and converted to percentage stimulation (of positive
control = ILꢀ1β + DMSO). Each value represents the mean ± s.e.m from at least 4 independent experiments and
data was quantified using one way ANOVA, with DUNETTS postꢀtest.
Treatment
% stimulation
100
IL-1β + DMSO
Il-1β + 47 (100 μM)
Il-1β + 48 (100 μM)
-40.9 +4.6 **
18.79 + 7.2 **
*
P < 0.05, **P < 0.01 compared to ILꢀ1β+DMSO alone.
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Conclusion
There are many reported inhibitors of IKKβ, primarily because it was considered a viable target in
inflammatory disease. Designing inhibitors of IKKα that are selective over IKKβ has to this date not
been reported, despite the former isoform now being recognised as a potential target in a number of
cancers. One reason for this is the high sequenceꢀhomology in the ATPꢀbinding site of the two
isoforms and the absence of any high resolution crystal structure of IKKα to guide structureꢀbased
inhibitor design.
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By employing molecular dynamics simulations on a homology model of IKKα based on IKKβ, we
identified key dynamic differences at the ATPꢀbinding site and exploited these to design the first
selective inhibitors of IKKα. Our compounds demonstrated target engagement with IKKαꢀrelated
pharmacodynamic markers and nonꢀengagement with IKKβ markers in cells. These compounds
therefore represent the first chemical tools that can be used to further characterise the role of IKKα in
cellular signalling, to dissect this from the very similar IKKβ isoform and validate IKKα in its own
right as a target in cancers, such as prostate, breast and pancreatic cancer.
The discovery of a 2ꢀaminopyrrolo[2,3ꢀd]pyrimidine chemical series also provides valuable
information with respect to SAR for IKKα inhibitory activity. Substituents at position 4 of the
pyrrolo[2,3ꢀd]pyrimidine scaffold allowed for diversification, with IKKα selectivity achievable
through the introduction of groups that specifically target residues in its binding site. However, further
significant improvements in activity through aliphatic 4ꢀamino substituents appear to be unlikely.
Modifying the 4ꢀphenyl substituent offers a more promising route towards generating compounds
with improved activity and selectivity. Using this strategy, we are currently developing more potent
IKKαꢀinhibitors that display functional outputs in a number of cell lines, all of which will be reported
in due course.
Experimental
IKKα homology model
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Building a homology model of IKKα: the kinase domain of IKKβ (chain B, residues 1ꢀ309, PDB entry
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4KIK was used as a template to build the kinase domain of IKKα, keeping the inhibitor (KSA700 in
the pdb file) and waters found within 6 Å of the proteinꢀinhibitor complex. The residue alignment
(Figure Mod1) and homology building were performed using Discovery Studio 3.1 (Accelrys Inc.,
San Diego, USA) after missing residues D174, Q175 and G176 were added in an extended
conformation.
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Both IKK kinase domains were subjected to molecular dynamics using the AMBER12 simulation
software. The inhibitor present (residue name KSA700) was kept and parameterised using
antechamber and charges calculated and fitted using the AM1ꢀBCC scheme. The two systems were
placed in a periodic octahedral box and solvated with TIP3P water with outer edges 6 Å in each
direction from the closest solute atom. The systems were then neutralized and physiological salt
conditions applied (~150 mM) by adding 24 Clꢀ and 29 Na+ ions to the IKKα system and 24 Clꢀ and
30 Na+ ions to the IKKβ system. The AMBER ff12SB was applied to all protein atoms whilst gaff
was used for the ligand. Parameters for the phosphoserine residues were taken from Craft and Legge
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. Before the MD production phase, minimization and equilibration (to reach 310 K) were performed
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in two stages as described . The NPT ensemble was used at 310 K until the systems had stabilized
for at least 20 ns (50 and 100 ns simulation time for IKKβ and IKKα respectively). All MD steps used
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the SHAKE algorithm
with a 2 fs timeꢀstep and a 10 Å cutoff for longꢀrange electrostatic
interactions. An average structure was generated (using ptraj within the AMBER suite) for the last 21
ns (IKKα) or 26 ns (IKKβ) and subsequently minimised in three steps, with the solvent, ions, and
−
1
hydrogen atoms initially minimized while the protein and inhibitor were restrained by 100 kcal mol
−2
Å . The restraint was then removed from the protein side chain atoms, and finally the whole system
−1
−2
was allowed to minimize until a derivative of 0.1 kcal mol Å was achieved. These structures were
then utilized for further docking studies with the GOLD software.
General Experimental
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Solvents (reagent grade or better) were purchased from SigmaꢀAldrich or Fischer Scientific.
Anhydrous solvents where purchased from SigmaꢀAldrich. Deuterated solvents were purchased from
SigmaꢀAldrich. Chemicals (95% purity or above) were purchased from Acros Organics, Alfa Aesar,
Apollo, Fluorochem or SigmaꢀAldrich. Solvents and chemicals were used as received without further
purification or treatment.
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Oxygenꢀ or moistureꢀsensitive reactions were carried out under a nitrogen atmosphere.
Microwave reactions were performed with a Biotage Initiator system. High absorbance was selected
for polar solvents and normal absorbance was selected for nonꢀpolar solvents.
The progress of the reactions was monitored on Merck 60F254 TLC plates. Spots were visualized by
irradiation with ultraviolet light (254/366 nm) or KMnO4, ninhydrin or phosphomolibdic acid (PMA)
TLC stains.
Column chromatography was performed with a Biotage SP4 system; cartridge size and eluent
specified in the corresponding experiments (% is referring to the most polar solvent in the mixture);
using silica gel as the stationary phase (particle size 0.040ꢀ0.063 mm, Merck or Fisher Scientific).
Specific rotations were measured in a Perkin Elmer Polarimeter 341 apparatus at 20 °C and a
wavelength of 589 nm (sodium D line) in DMSO UV spectrophotometric analysis grade.
1H and 13C NMR data were recorded on either a JEOL ECXꢀ400 (400 MHz) or Bruker
Avance3/DPX400 (400 MHz) spectrometers at 400.0 and 100.6 MHz respectively. Chemical shifts (δ)
are expressed in parts per million (ppm) coupling constants (J) are in hertz (Hz). Chemical shifts (δ)
are reported relative to TMS (δ = 0 ppm) and/or referenced to the solvent in which they were
measured. All measurements were carried out at 298 K (except when stated). Abbreviations used in
the description of NMR data are as follows: app, apparent; s, singlet; bs, broad singlet; d, doublet; t,
triplet; q, quartet; p, pentuplet; m, multiplet.
HRꢀMS was conducted using a Thermo Scientific Exactive Orbitrap Mass Analyzer. LRꢀMS was
conducted using a ThermoQuest Finnigan LCQ Duo instrument. GCꢀMS was conducted using a
ThermoQuest Finnigan Polaris Q instrument.
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