Synthesis and physicochemical characterization of novel phenotypic probes targeting the nuclear factor-kappa B signaling pathway

Article abstract of DOI:10.3762/bjoc.9.103

Activation of nuclear factor-kappa B (NF-κB) and related upstream signal transduction pathways have long been associated with the pathogenesis of a variety of inflammatory diseases and has recently been implicated in the onset of cancer. This report provi

Full text of DOI:10.3762/bjoc.9.103

Synthesis and physicochemical characterization of
novel phenotypic probes targeting the nuclear
factor-kappa B signaling pathway
Paul M. Hershberger1, Satyamaheshwar Peddibhotla1,
E. Hampton Sessions1, Daniela B. Divlianska1, Ricardo G. Correa2,
Anthony B. Pinkerton2, John C. Reed2 and Gregory P. Roth*1
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2013, 9, 900–907.
1Conrad Prebys Center for Chemical Genomics, Sanford-Burnham
Medical Research Institute at Lake Nona, 6400 Sanger Road
Orlando, FL 32827, USA and 2Sanford-Burnham Medical Research
Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA
doi:10.3762/bjoc.9.103
Received: 16 January 2013
Accepted: 22 April 2013
Published: 08 May 2013
Email:
Gregory P. Roth* - groth@sanfordburnham.org
This article is part of the Thematic Series "Synthetic probes for the study
of biological function".
* Corresponding author
Guest Editor: J. Aubé
Keywords:
ML029; ML130; ML146; ML236; ML237
© 2013 Hershberger et al; licensee Beilstein-Institut.
License and terms: see end of document.
Abstract
Activation of nuclear factor-kappa B (NF-κB) and related upstream signal transduction pathways have long been associated with
the pathogenesis of a variety of inflammatory diseases and has recently been implicated in the onset of cancer. This report provides
a synthetic and compound-based property summary of five pathway-related small-molecule chemical probes identified and opti-
mized within the National Institutes of Health-Molecular Libraries Probe Center Network (NIH-MLPCN) initiative. The chemical
probes discussed herein represent first-in-class, non-kinase-based modulators of the NF-κB signaling pathway, which were identi-
fied and optimized through either cellular phenotypic or specific protein-target-based screening strategies. Accordingly, the
resulting new chemical probes may allow for better fundamental understanding of this highly complex biochemical signaling
network and could advance future therapeutic translation toward the clinical setting.
Introduction
The Molecular Libraries Network was established in 2005 as an chemical screening (HTS), efforts in medicinal chemistry and
academic pilot-scale (MLSCN) screening effort for exploring exploratory pharmacology were added in order to leverage and
potential new therapeutic targets stemming from the human advance the small-molecule compounds discovered within the
genome project. After initial successes in high-throughput HTS process. The program then advanced a broader probe-
900

Beilstein J. Org. Chem. 2013, 9, 900–907.
production initiative (MLPCN) in 2008. This consortium pathways. Within this project, new probes were sought that
resulted in the harnessing of chemical biology resources from were not active via the currently known receptor-driven path-
four comprehensive centers and five specialty centers, which ways and IκB kinases, but attenuated NF-κB transcriptional
focused on either medicinal chemistry or a specific technical activity as measured by a luciferase-based reporter gene assay
screening capability as an overall academic, collaborative scien- with validation using a panel of known receptor and kinase-
tific network [1]. In general terms, the overall vision and NIH based counter screens [7].
mission encompasses the discovery of unique chemical tools
that will be useful to interested scientific investigators to assist Because activation of NF-κB is known to be initiated through
them with advancing basic in vitro and in vivo studies for protein kinase C (PKC), we hypothesized that selectivity could
testing new hypotheses in disease modulation.
be possible by the fact that PKC activation occurs downstream
from cell membrane antigen and growth-factor receptors yet is
Within this report, the outcomes of two Sanford-Burnham still upstream of IKKγ, potentially by inhibition of a new target
projects for chemical probe discovery are discussed, high- protein or novel protein–protein interaction. Using cell-based
lighting five examples of thematic small-molecule chemical HTS reporter gene assays, a series of chemical probes were
probes discovered through our center’s efforts. Within the realm identified that selectively inhibit this unique PKC-induced
of immunology and inflammation research, many cellular path- NF-κB pathway without modulating other NF-κB activation
ways leading to the activation of NF-κB family of transcription pathways such as those including the cytosolic proteins
factors have been identified and several excellent reviews are CARMA1, Bcl-10, MALT1, TRAF6 and Ubc13 [8].
available [2-5]. In general, these pathways have been shown to
participate in host defense, immunity, and inflammation and The first probe identified within this series was the benzimida-
even have implications in cancer. Thus, dysregulation of NF-κB zole ML029 (4), which exhibited an IC50 of 0.07 μM in the
activity contributes to numerous autoimmune and inflamma- HEK 293 cell assay with corresponding well-defined
tory disease states. With this, the availability of new chemical structure–activity relationships (SAR) through analogue syn-
pathway probes will further enhance understanding of this com- thesis [9]. This probe was discovered after two separate
plex network.
screening campaigns totaling ~110,000 compounds. At concen-
trations up to 8 μM, 4 failed to suppress PKCβ and PKCθ (the
The first project encompassed the synthesis and characteriza- PKC family members implicated in TCR/BCR signaling) and
tion of three phenotypic probes designed to be selective modu- IKKβ, while known PKC and IKK inhibitors and the broad-
lators of the NF-κB signaling pathway in either human embry- spectrum kinase inhibitor staurosporine afforded potent inhibi-
onic kidney (HEK) 293, HEK 293T or pre-B leukemia 697 tion [9-11]. Further selectivity profiling revealed that 4 inhib-
cells. The second project focused on two probes that are target- ited (>50% at 10 uM) only 3 out of the 353 kinases surveyed by
oriented within the NF-κB pathway and are specific modulators using a KINOMEscan™ (DiscoveRx) platform. None of these 3
of the intracellular signaling protein, nucleotide-binding (TLK1 (70% inhibition), Raf (57%), and JAK2 (53%)) are rele-
oligomerization domain-1 (NOD1).
vant to NF-κB pathway regulation.
Within each project area first-in-class chemical probes were To prepare 4, a synthetic route (Scheme 1) was optimized in a
identified and characterized as new research-tool compounds. manner that allowed for the preparation of related analogues
The synthetic routes for each probe, including their general [9]. The intermediate 1 was prepared through condensation of
physicochemical and pharmacological properties, are summa- 4,5-dimethylbenzene-1,2-diamine with potassium ethyl xanthate
rized in this report.
in ethanol under reflux. Bromination of 1 led to the key inter-
mediate 2-bromo-5,6-dimethylbenzimidazole (2), which reacted
smoothly with 3-aminopropanol to give the amine 3 in high
Results and Discussion
Phenotypic screening for noncanonical NF-κB pathway yield and purity after extraction. The solvent-free microwave
selective inhibitors of IL-8 production in HEK 293, HEK process employed was superior to traditional oil-bath thermal
293T (ML029) and 697 pre-B cells (ML236 and ML237): heating, which tended to generate crude material requiring
Most pathways for NF-κB activation converge on the IκB extensive purification. Ultimately, probe 4 was prepared
kinases (IKKs), and more than nine signaling routes have been (Scheme 1, Method A) in moderate yield by reaction of 3 with
identified [6]. While IKKs therefore represent attractive targets α-bromo-3,5-di-tert-butyl-4-hydroxyacetophenone in n-butanol
for drug discovery programs, the selectivity envisioned for an under reflux. Importantly, formation of the tricyclic conden-
acceptable therapeutic index has remained elusive as inhibitors sation side product 5 was completely eliminated by performing
of IKKs indiscriminately suppress all known NF-κB activation the reaction with sodium bicarbonate at 23 °C in methanol
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Beilstein J. Org. Chem. 2013, 9, 900–907.
Scheme 1: Synthesis of ML029 (4).
(Scheme 1, Method B). Thus, compound 4 was isolated as a T cell (HEK 293T) specific assay. The first oxadiazole-based
lyophilized white powder in 78% yield and >98% purity.
probe ML236 (8) was potent (0.035 μM) in the 697 pre-B cell
line and >400-fold selective over both the NF-κB activation in
Deeper in vitro biological investigation using 4 as a chemical HEK 293T cells and against TNFα-mediated NF-κB activation.
probe showed that it was not active (>30 μM) in more physiolo- In comparison, the second oxazole-based probe 12 is also potent
gically relevant HEK 293T and 697 pre-B cell lines. Therefore, (0.2 μM) in the 697 pre-B cell line and >400-fold selective over
an HTS campaign was initiated to identify compounds active in both the NF-κB activation in HEK 293T cells and against TNFα
these specific cell subtypes by using the established HTS mediated NF-κB activation as measured in our reporter gene
NF-κB reporter gene assay format. This included screening of a assay format.
larger (~330,600 compound) MLSMR collection to ensure iden-
tification of additional chemical scaffolds not previously avail- Probe 8 was synthesized as shown in Scheme 2 [12]. Reaction
able. From this campaign two new cell-active NF-κB inhibitors of 4-tert-butyl-cyanobenzene with hydroxylamine hydrochlo-
were derived that were both selective against the NF-κB ride under microwave heating conditions resulted in the
pathway induced by known PKC activators (phorbol myristic hydroxyamidine 6. This intermediate was condensed with
acetate (PMA)/ionomycin) and were selective toward the 697 succinic anhydride to provide the oxadiazole acid 7. Conver-
pre-B cell line. It is interesting to note that no compounds sion of 7 to the corresponding acid chloride and subsequent
meeting probe selectivity criteria were identified within the amidation with excess methylamine afforded 8 in excellent
Scheme 2: Synthesis of ML236 (8).
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Beilstein J. Org. Chem. 2013, 9, 900–907.
yield after normal phase chromatography. This route was precise signaling mechanism is the subject of ongoing research
employed to make a variety of analogues and was generally in our laboratories.
conducted efficiently without purification of any intermediates.
This enabled several analogues to be prepared in parallel, using Discovering modulators of NF-κB signaling via selective
mass-directed reverse-phase preparative HPLC to recover the nucleotide-binding oligomerization domain-1 (NOD1) inhi-
pure isolates.
bition (ML130 and ML146): The second program targeted
proteins in the mammalian innate immune system that confer
The second related probe, ML237, (12) was synthesized as defense by detection of specific microbial ligands, or pathogen-
shown in Scheme 3 [12]. Commercially available 4-ethoxy- associated molecular pattern microbial sensors known to house
benzaldehyde was condensed with diacetyl monoxime under NOD1 (nucleotide-binding oligomerization domain-1 protein).
acidic conditions. The resulting oxazole N-oxide 9 was Dysregulation of the NOD signaling pathway is also strongly
converted to the chloromethyloxazole 10 via selective chlorina- associated with exacerbation of postinfection systemic disease
tion with phosphorous oxychloride in good yield. Addition of states; however, its upregulation at the gene level can enhance
thioglycolic acid led to the carboxylic acid 11, which was systemic innate immunity [13]. The NOD1 cytosolic bacterial
coupled to cyclopropylamine to give 12 using a standard amida- sensor falls within this class of NF-κB transcription pathway
tion protocol. The compound was purified by normal phase activators [14]. The NOD-like receptor (NLR) family report-
chromatography and was recovered as a white solid with a edly comprises a large number of proteins from both vertebrate
sharply defined melting point. The route was also amenable and invertebrate animal species, with >20 human proteins
toward the preparation of related analogues. Generally, 9 was recognized [15-21]. Some NLR proteins have been shown to
used without purification and the entire route was conducted detect bacterial cell-wall components including lipopolysaccha-
efficiently without chromatographic purification of intermedi- rides and/or peptidoglycan (NOD1 or 2) along with those of
ates.
bacterial flagellin [22-25]. Importantly, the NOD1 protein
primarily recognizes Gram-negative bacteria, in contrast to the
As a summary of project one, three novel chemical probes were more widely studied sensor NOD2, which participates in immu-
discovered and optimized within this initiative. Tractable and nity against Gram-positive bacteria. Additionally, NOD1 has
efficient synthetic routes have been developed and are reported. been associated with the induction of NF-κB activation,
One HEK 293T and two 697 pre-B cell specific probes are now caspase-1 activation and apoptosis [26-28]. Genetic mutations
available as research tools either through the Sanford-Burnham in NOD are associated with numerous inflammatory conditions,
Center or through commercial sources. These probes were iden- including Crohn’s disease and pancreatitis [29-34]. The identifi-
tified from a phenotypic screen and thus the precise cellular cation of probes relevant to the NOD family of NLR proteins
protein target is yet to be identified. Each probe performs in a will improve understanding of the NOD1-mediated signaling
noncanonical manner and inhibits NF-κB activity outside of the pathway and also may translate to the discovery of new thera-
known, well-characterized pathways [7]. Determination of the peutics for inflammatory diseases.
Scheme 3: Synthesis of ML237 (12).
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Beilstein J. Org. Chem. 2013, 9, 900–907.
Probes ML130 (13) and ML146 (17) were identified as NOD1- biologically relevant MCF-7 cells. While 13 is the more potent
selective molecules from an HTS campaign involving and selective of these probes, 17 also met probe criteria and
~290,000 compounds. Compound 13 (IC50 = 0.52 μM) and represents a second bonafide scaffold for a NOD1-selective
compound 17 (IC50 = 1.54 μM) each inhibited γ-tri-DAP-stimu- probe.
lated (a gamma-tri-diaminopimelic acid derivative and NOD1-
dependent signaling ligand) luciferase production in HEK ML130 (13) was synthesized as shown in Scheme 4 [35].
293T cells, which has endogenous NOD1 levels at submicro- Commercially available 2-aminobenzimidazole was treated with
molar concentration as determined in a NF-κB-linked reporter p-toluenesulfonyl chloride in the presence of pyridine to obtain
assay. Both probes selectively (>40-fold for 13, >8-fold for 17) 13. By performing the reaction at high concentration, it was
inhibited NOD1-dependent activation of the NF-κB pathway generally simple to purify the reaction mixture directly without
without inhibiting MDP-stimulated (muramyl dipeptide and a work up using normal-phase chromatography.
NOD2-dependent signaling ligand) signal transduction in
reporter cell lines containing either low or overexpressed NOD2 ML146 (17) was synthesized as shown in Scheme 5. Commer-
proteins. Probes 13 and 17 were also selective over the non- cially available 6-amino-1-methyluracil was brominated to give
NOD-stimulated NF-κB pathways (TNF-α, doxorubicin and 14, which was reacted without purification with crotylamine
PMA/ionomycin) in these reporter assays. Both of the under microwave heating conditions to provide 15. Reaction of
probes, and their close analogues as discovered through 15 with potassium ethyl xanthogenate, again under microwave
SAR studies, selectively inhibited IL-8 secretion and the bio- heating conditions, led to the bicyclic thiol 16. Without purifi-
logically relevant terminal effect of NOD1 (γ-tri-DAP) cation, 16 was added to 1-bromo-3-phenylpropane in the pres-
dependent NF-κB activation. Additionally, they neither inhib- ence of potassium carbonate to provide 17. Several analogues
ited NOD2-dependent nor TNF-α-dependent IL-8 secretion in were efficiently prepared without chromatographic purification
Scheme 4: Synthesis of ML130 (13).
Scheme 5: Synthesis of ML146 (17).
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Beilstein J. Org. Chem. 2013, 9, 900–907.
Table 1: Calculated properties of probes.
Property
4
8
12
13
17
Molecular weight [g/mol]
Molecular formula
XLogP3-AA
465.6
287.3
346.4
287.3
370.4
C28H39N3O3
C16H21N3O2
C18H22N2O3S
C14H13N3O2S
C19H22N4O2S
7.2
3
3.0
1
3.1
1
3.2
1
3.5
1
H-Bond donors
H-Bond acceptors
Polar surface area [Å2]
Heavy atom count
5
4
4
4
3
85.2
34
68.0
21
89.7
24
86.4
20
67.2
26
of intermediates, and the final compounds were purified by ranging from 67 to 90 Å2. Based on current hit-to-lead trends
normal-phase chromatography if necessary [12].
within the pharmaceutical industry, all probes discussed
provide qualified starting points for advanced lead-optimiz-
As a summary of the second project, two novel chemical probes ation programs.
that are selective for NOD1 but not NOD2 signaling, were
discovered and optimized within the scope of the program. The probes were evaluated using known in vitro ADME/T
Tractable and efficient synthetic methods have been employed assays to understand their overall pharmacological properties
and are reported. Both NOD1 selective probes are now avail- (Table 2). Probe 4 exhibited good solubility especially as the
able as research tools either through the Sanford-Burnham pH decreased from 6.2 to 5, thereby mitigating the high calcu-
Center or through commercial sources.
lated log P to some degree. Probes 8 and 12 also showed good
solubility. Probe 13 had moderate solubility, and 17 showed the
lowest solubility across the pH range but still approximately
equal to its IC50 value. The PAMPA (parallel artificial
membrane permeability assay) assay is used as an in vitro
model of passive, transcellular permeability. An artificial
membrane immobilized on a filter is placed between a donor
and acceptor compartment. In this test, the probes exhibited
good cell permeability overall. Although metabolic stability was
poor in the presence of murine microsomes, all five probes were
more stable in the presence of human hepatic microsomes. Most
notably, 8 showed no apparent instability when subjected to
Chemical probe physicochemical and
pharmacological properties
Overall, the project probes presented physicochemical prop-
erties consistent with those of well-recognized drug-like mole-
cules (Table 1). Molecular weights ranged from 287 to
465 g/mol. Calculated log P values were between 3.1 and 3.5
with the exception of 4, which had an exceptionally high
lipophilic value of 7.2 due to the presence of two tert-butyl
moieties on the phenyl ring. The probes all contain from one to
five H-bond donors and acceptors, and polar surface areas
Table 2: Summary of in vitro ADME/T properties of probes.
Property
4
8
12
13
17
Aqueous solubility
(μM)
pH 5.0/6.2/7.4
90/1.1/1.5
299/710/441
92/101/103
751/755/746
112/133/160
513/541/59
7.0/5.9/7.0
491/562/382
1.5/1.8/1.7
1269/1516/1344
PAMPA Pe (10−6 cm/s)a
Donor pH: 5.0/6.2/7.4
Acceptor pH: 7.4
Hepatic microsome stabilityb
Human/mouse
(% remaining)
26/0.5
>50
100/4.9
>50
22.7/0
>50
41.8/0.8
>50
8.8/0.86
>50
Hepatic toxicity
LC50 (µM)
aCompound at 50 μM (typical PAMPA Pe permeability classification: low 5 × 10−6, moderate 250 × 10−6, high 1000 × 10−6); bCompound at 1 μM,
60 min.
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Beilstein J. Org. Chem. 2013, 9, 900–907.
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projects. This work was supported under federal grants U54
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