Discovery of N-{4-[5-(4-Fluorophenyl)-3-methyl-2-methylsulfanyl-3H-imidazol-4-yl]-pyridin-2-yl}-acetamide (CBS-3595), a Dual p38α MAPK/PDE-4 Inhibitor with Activity against TNFα-Related Diseases

Article abstract of DOI:10.1021/acs.jmedchem.6b01647

The anti-inflammatory potential of p38 mitogen-activated protein kinase (MAPK) inhibitors was coincidentally expanded to a dual inhibition of p38α MAPK and phosphodiesterase 4 (PDE4), and the potential benefits arising from the blockage of both inflammation-related enzymes were thoroughly investigated. The most promising compound, CBS-3595 (1), was successively evaluated in in vitro experiments as well as in ex vivo and in vivo preclinical studies after administration of 1 to rodents, dogs, and monkeys. The resulting data clearly indicated a potent suppression of tumor necrosis factor alpha release. For reconfirming the findings of the animal studies when administering 1 to healthy human volunteers, a phase I clinical trial was conducted. Apart from further information regarding the pharmacokinetic and pharmacodynamic characteristics of 1, it was demonstrated that dual inhibition of p38α MAPK and PDE4 is able to synergistically attenuate the excessive anti-inflammatory response.

Full text of DOI:10.1021/acs.jmedchem.6b01647

Subscriber access provided by CORNELL UNIVERSITY LIBRARY
Drug Annotation
Discovery of N-{4-[5-(4-Fluoro-phenyl)-3-methyl-2-methylsulfanyl-3H-
imidazol-4-yl]-pyridin-2-yl}-acetamide (CBS-3595), a Dual p38#
MAPK/PDE-4 Inhibitor with Activity against TNF# -related Diseases
Wolfgang Albrecht, Anke Unger, Silke M Bauer, and Stefan A. Laufer
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 14 Jun 2017
Downloaded from http://pubs.acs.org on June 14, 2017
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Discovery of Nꢀ{4ꢀ[5ꢀ(4ꢀFluoroꢀphenyl)ꢀ3ꢀmethylꢀ2ꢀmethylsulfanylꢀ3Hꢀimidazolꢀ4ꢀyl]ꢀ
pyridinꢀ2ꢀyl}ꢀacetamide (CBSꢀ3595), a Dual p38α MAPK/PDEꢀ4 Inhibitor with Activity
against TNFα ꢀrelated Diseases
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Wolfgang Albrecht , Anke Unger , Silke M. Bauer and Stefan A. Laufer
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Dr. Wolfgang Albrecht, cꢀaꢀiꢀr biosciences GmbH, AlfredꢀMendler Weg 25/1, Dꢀ89075 Ulm,
Germany
2
Eberhard Karls University Tuebingen, Department Pharmacy & Biochemistry, Pharm./Med.
Chemistry, Auf der Morgenstelle 8, Dꢀ72076 Tuebingen, Germany
KEYWORDS: COPD, Rheumatoid Arthritis, Dual inhibition, JNK, p38, PDEꢀ4, Psoriasis,
TNFα
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ABSTRACT: The antiꢀinflammatory potential of p38 mitogenꢀactivated protein kinase (MAPK)
inhibitors was coincidentally expanded to a dual inhibition of p38α MAPK and
phosphodiesterase 4 (PDE4) and the potential benefits arising from the blockage of both
inflammationꢀrelated enzymes were thoroughly investigated. The most promising compound,
CBSꢀ3595 (1), was successively evaluated in in vitro experiments as well as in ex vivo and in
vivo preclinical studies after administration of 1 to rodents, dogs and monkeys. The resulting data
clearly indicated a potent suppression of tumor necrosis factor alpha (TNFα) release. In order to
reconfirm the findings of the animal studies when administering 1 to healthy human volunteers, a
phase I clinical trial was conducted. Apart from further information about the pharmacokinetic
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(PK) and pharmacodynamic (PD) characteristics of 1, it was demonstrated that dual inhibition of
p38α MAPK and PDE4 is able to synergistically attenuate the excessive antiꢀinflammatory
response.
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Introduction. The role of TNFα in inflammatory, infectious and malignant conditions has been
studied for more than two decades. This 17 kDa soluble protein, predominantly produced by
macrophages and T lymphocytes, is found in inflammatory and infectious conditions at elevated
levels in serum and tissue. However, in a healthy state, TNFα is a modulator of an appropriate
host response to bacterial, viral and parasitic infections. TNFα blocking agents, the soꢀcalled
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“biologicals” Infliximab (IFX), Adalimumab, Golimumab, Certolizumab and Etanercept are used
in chronic inflammatory diseases. These substances do not interfere with TNFα biosynthesis or
signaling cascade, but mask the freely circulating protein, thereby preventing it from initiating the
signaling pathway which is triggerd by binding to TNFα receptors. Apart from rheumatoid
arthritis (RA), Crohn’s disease (CD), psoriasis and ankylosing spondylitis represent further
indications which are driven by inappropriate or excessive TNFα levels.
Affecting approximately 1% of the adult population, RA is the consequence of complex series of
pathologic events involving cytokines like ILꢀ1, ILꢀ6 and TNFα, leading to characteristic
1
, 2
symptoms like synovial inflammation, cartilage damage, and bone erosion.
The conventional therapy for this inflammatory and degenerative autoimmune disease applies a
combination of disease modifying drugs (DMARDs) such as methotrexate (MTX),
glucocorticoids and biologicals. Frequently, these therapeutic concepts ameliorate the
inflammation processes only in an inadequate manner and are linked to adverse side effects such
as serious infections and lymphoma. Without disregarding the significant improvement for
treatment of these severe and debilitating diseases by biological TNFαꢀinhibitors, there remain
limitations, such as (a) a still large percentage (approx. 30%) of patients, who do not respond to
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the treatment with these drugs, (b) high costs and (c) the route of administration.
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Even before the first biological TNFαꢀinhibitor entered the market, researchers at SmithKline
Beechem identified the mechanism of action of antiꢀinflammatory pyridinylimidazoles, which
inhibited the cytokine release (TNFα and IL1β), when cells were preꢀincubated with these
molecules prior to LPSꢀstimulation. These compounds, initially denominated as cytokineꢀ
suppressive antiꢀinflammatory drugs (CSAIDs), were found to bind to a single 38 kDa protein
kinase implicating that the expression of proꢀinflammatory cytokines can be modulated by
inhibition of this unique enzyme, the p38 MAP kinase (p38 MAPK). The concept of development
of an orally available antiꢀcytokine small molecule which could be used for treatment of various
inflammatory autoꢀimmune diseases was soon adopted by essentially all big pharmaceutical
companies and many biotech enterprises.
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Many different small molecule p38α MAPK inhibitors have been discovered; the latest
generation exhibiting high potency and a selectivity, which appeared good enough to avoid the
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modulation of critical offꢀtargets at therapeutically effective doses.
So far, more than 20 candidates entered clinical development. Based on the excellent efficacy of
p38a MAPK inhibitors in various models of experimental arthritis and further to the successful
development of biological antiꢀTNFαꢀtherapies, most candidates were initially evaluated in RA
patients. In standard 12ꢀweek clinical trials, however, despite an initial and substantial reduction
of the inflammation biomarker Cꢀreactive protein (CRP), at the end of the treatment period, the
clinical efficacy was not superior to placebo and the CRPꢀlevels had returned to baseline values.
The mechanism behind this intermittent but not sustained antiꢀinflammatory treatment remains
unknown but an activation of redundant shunt or escape signalling pathways was suspected, e.g.
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via JNKs.
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As of today, in clinical phase 2 studies, p38α MAPK inhibitors have been investigated in other
indications such as Crohn’s disease, neuropathic pain, coronary heart disease, atherosclerosis,
depression, mild symptoms of Alzheimer’s disease as well as in asthma and chronic obstructive
pulmonary disease (COPD).
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In patients with asthma and COPD, proinflammatory cytokines, such as TNFα, ILꢀ1β, and ILꢀ6,
are found in increased amounts in the sputum and BAL fluid. TNFα responses are initiated by
binding of soluble TNFα to either TNF receptor 1 or 2, which leads to an activation of additional
pathways and signaling proteins, including transforming growth factorꢀbeta (TGFβ)ꢀactivated
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kinase (TAK)1 and the MAPKs cꢀJun Nꢀterminal kinases (JNKs) and p38. TNFα is expressed in
various cells in asthmatic airways, particularly mast cells, and may play a key role in amplifying
asthmatic inflammation through the activation of NFꢀκB. In preliminary, small clinical studies
with asthma patients, antiꢀTNFα therapy with either etanercept or infliximab (IFX) led to an
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improvement of lung function and reduced exacerbations in patients.
However, in larger
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clinical trials, the efficacy was not confirmed.
In patients with COPD, IFX failed to provide
any benefit, as assessed by effects on symptoms, lung function, and exercise performance, when
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administered at doses that are effective in individuals with rheumatoid arthritis.
Similarly, clinical studies with singleꢀtargeted therapy against ILꢀ4 (dupilumab), ILꢀ13
tralokinumab, lebrikizumab), ILꢀ17 (brodalumab) or the chemokine receptor 2 (MKꢀ7123; (R)ꢀ2ꢀ
hydroxyꢀN,Nꢀdimethylꢀ3ꢀ((2ꢀ((1ꢀ(5ꢀmethylfuranꢀ2ꢀyl)propyl)amino)ꢀ3,4ꢀdioxocyclobutꢀ1ꢀenꢀ1ꢀ
1
5
yl)amino)benzamide) failed. In 2016, two monoclonal antibodies against ILꢀ5 (mepolizumab
and reslizumab) have been approved in US and Europe as addꢀon therapy for treatment of asthma
patients with a specific eosinophilic phenotype .
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The rationale to explore the efficacy of p38α MAPK inhibitors in COPD is based on the efficacy
in experimental in vivo and in vitro models and on the analysis of biopsy samples which showed
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a correlation between activation of p38α MAPK and disease severity.
Furthermore, in a small
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scale exploratory clinical study, the intermittent shortꢀterm dosing (75 mg on days 1 and 6) with
BCT197 (3ꢀ(5ꢀaminoꢀ4ꢀ(3ꢀcyanobenzoyl)ꢀ1Hꢀpyrazolꢀ1ꢀyl)ꢀNꢀcyclopropylꢀ4ꢀmethylbenzamide)(
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showed a marked improvement in lung function (FEV1) in COPD patients As described in
This compound, now denominated acumapimod is currently being developed as firstꢀline acute
therapy for acute exacerbations of COPD.
On the other side, Watz et al. reported that treatment of COPD patients for 12 week with
losmapimod, another selective and highly potent p38α MAPK inhibitor, did not cause an
improvement in exercise tolerance or lung function, despite being wellꢀtolerated in this COPD
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population. However, Pascoes et al. performed a subgroup analysis and demonstrated a
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significant efficacy in a subpopulation (patients with low eosinophil count).
PDE4 Inhibitors in chronic inflammatory diseases
Theophylline has been used in the treatment of asthma (and COPD) since the 1930s, but its use
declined with the development of newer and more efficacious drugs. Investigations to elucidate
the mechanism of action of theophylline led to the identification of the protein kinase A (PKA)
pathway, which is an integral part of the signaling cascade of Gꢀproteinꢀcoupled receptors. The
PKA pathway depends on cellular cyclic adenosine phosphate (cAMP) levels that are controlled
by the cAMP forming adenyl cyclases and the cAMP degrading phosphodiesterases, such as
PDE4. As second messenger, cAMP holds key roles in a variety of physiological responses,
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including regulation of proꢀinflammatory cytokine levels. Low cAMP levels are a result of highly
active phosphodiesterases and lead to a low activity of PKA. As a result, the suppression of
TNFα expression via PKA is repealed. Being mainly expressed in inflammatory cells, smooth
muscle cells, endothelial cells and keratinocytes, inhibition of PDE4 is a reasonable strategy for
impeding the pathophysiological events in chronic inflammatory lung diseases by blocking TNFα
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biosynthesis right at the scene of action.
To date, only two PDE4 inhibitors have reached marketing authorization and only one of them,
roflumilast (Daxas EU, Daliresp US), is being marketed for treatment of chronic inflammatory
diseases lung, albeit just for a particular subgroup of COPD affected patients. The other PDE4
inhibitor, apremilast (Otezla EU, US), is used for treatment of psoriatic arthritis and severe
plaque psoriasis. Moreover, results from in vitro studies and experimental murine arthritis models
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suggest apremilast’s potential effectiveness in treatment of RA, as reported by McCann et al.
However, in a phase II RA clinical study, application of apremilast on top of a stable MTX
treatment had no superior effect even though administered at dose levels, which were efficacious
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in patients with psoriasis and psoriatic arthritis. An increase of the dose of PDE4 inhibitors is
limited by a low therapeutic window, as PDE4 inhibition is the associated with nausea and
emesis as major side effects.
Rationale for dual inhibitors of p38α MAPK and PDE4.
The standard therapy for asthma and COPD is dominated by the use of inhaled fixedꢀdose
combinations of a longꢀacting β2 agonist (LABA) and a corticosteroid (ICS). Further
combinations are short and longꢀacting β2 agonist or combinations with β2 agonists and
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muscarinic receptor antagonists. Although asthma is usually well controlled with ICS,
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approximately 20% of asthma patients are particularly difficult to manage, and 3– 5% of patients
have severe disease that is not controlled even by maximal inhaled treatment with an LABA/ICS
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combination . COPD has a high morbidity and no current treatment, including corticosteroids,
reduce disease progression or mortality and have relatively little effect in preventing
exacerbations, reflecting their lack of antiꢀinflammatory effects in this disease. There is an
enormous unmet need for the development of new treatments for COPD that target the
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underlying inflammatory process and aberrant repair mechanisms.
The combination of a bronchodilator with an antiꢀinflammatory agent as a treatment of COPD
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has been extensively discussed. The combination of roflumilast with salmeterol, formoterol, or
dexamethasone on lipopolysaccharide (LPS)ꢀinduced peripheral blood mononuclear cells
(PBMC) cytokine production demonstrated additive effects for the PDE4/LABA or
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PDE4/glucocorticosteroid combination.
Because the development of fixedꢀdose
combinations is sometimes challenging, bifunctional drugs (dual inhibitors) represent a potential
alternative for treatment of diseases with a complex and multiꢀfactorial pathophysiology such as
asthma and COPD. Tannheimer et al. and Baker et al. described and characterized (R)ꢀ6ꢀ[[3ꢀ[[4ꢀ
[
5ꢀ[[2ꢀhydroxyꢀ2ꢀ(8ꢀhydroxyꢀ2ꢀoxoꢀ1,2ꢀdihydroquinolinꢀ5ꢀyl) ethyl]amino]pentꢀ1ꢀ
ynyl]phenyl]carbamoyl]phenyl]sulfonyl]ꢀ4ꢀ[(3ꢀmethoxyphenyl) amino]ꢀ8ꢀmethylquinolineꢀ3ꢀ
carboxamide (GSꢀ5759) as a dual LABA/PDE4ꢀinhibitor which has been suggested to offer a
molecular mechanism for additive or synergistic antiꢀinflammatory effects through elevation of a
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common second messenger.
Despite promising in vitro data, this compound did not progress
to clinical development. As the candidate was designed for inhalation, the localized antiꢀ
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inflammatory effect was probably not sufficient to improve the symptoms associated with asthma
or COPD.
The dual inhibition of p38α MAPK and PDE4 represents a new and so far not explored approach
which may be useful for treatment of pulmonary diseases but also in other inflammatory diseases.
This rationale is supported by results generated with CGH2466 (6) (4ꢀ(3,4ꢀdichlorophenyl)ꢀ5ꢀ
pyridinꢀ4ꢀylꢀ1,3ꢀthiazolꢀ2ꢀamine), a dual p38α MAPK / PDE4 inhibitor described by Trifilieff et.
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al., which additionally exhibits antagonistic effect on adenosine A1, A2b and A3 receptors. This
compound suppressed the production of cytokines and oxygen radicals by human peripheral
blood leucocytes in vitro more potently than the standard p38 MAP kinase inhibitor SB203580,
the PDE4 inhibitor cilomilast and the broad spectrum adenosine receptor antagonist CGS15943
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(
9ꢀChloroꢀ2ꢀ(2ꢀfuranyl)ꢀ[1,2,4]triazolo[1,5ꢀc]quinazolinꢀ5ꢀamine). When given either orally or
locally into the lungs, 6 inhibited the ovalbuminꢀ or lipopolysaccharideꢀinduced airway
inflammation in mice more potently than the single receptor antagonists or enzyme inhibitors
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used alone.
This Drug Annotations article reports the discovery of 1, a dual p38α MAPK / PDE4 inhibitor,
and the background of the medicinal chemistry strategy as well as the synthetic routes and the
resulting structureꢀactivity relationships (SARs). Moreover, the promising synergistic efficacy of
this dual p38α MAPK / PDE4 inhibitor is emphasized by exhaustive preclinical results.
Furthermore, the outcome of a phase I clinical study to elucidate the antiꢀinflammatory efficacy
as well as its toxicological profile and PK / PD data are reported.
Discovery of pyridinylimidazoles as cytokineꢀsuppressive antiꢀinflammatory drugs
(CSAIDs).
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Chemistry. The developed and optimized synthetic routes are extremely flexible and allow
regioselective synthesis, as evident from the overview in Figure 1Error! Reference source not
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6ꢀ38,38ꢀ52
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found.. For more details, see synthesis references
and patents WO 2002066458 , WO
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004018458 , WO 2006089798 , WO 2008023066 . The synthetic route of 1 is described in
detail in the Supporting Information.
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Figure 1: Synthetic strategy to achieve either triꢀ or tetraꢀ substitution of the 2ꢀthioimidazole
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scaffold in a regioselective manner. This overview was modified from and and . Reagents
and conditions: (a) NaNO , AcOH, room temperature; (b) H , Pd/C, 1 atm, HCl in iPrOH, room
2
2
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temperature; (c) DMF, KSCN, reflux; (d) MeI, EtOH / THF, reflux, see also ; (e) DMF, methylꢀ
5
8
/
ethylꢀ/benzylthiocyanate, reflux; see also ; (f) 50% HBF (aq), NaNO , 0 °C to 45 °C; (g)
4 2
NaNO , 70% HF in pyridine (Olah’s reagent), ꢀ15 to ꢀ10 °C to room temperature; (h) NaNO ,
2
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AcOH, room temperature; (i) (H CNR ) , EtOH, reflux; (j) 2,2,4,4ꢀtetramethylcyclobutaneꢀ1,3ꢀ
2
3
3
dithione, DCM, room temperature; (k) HalꢀR , K CO , MeOH, room temperature; (l) Br , AcOH,
2
3
2
2
room temperature; (m) excess H NꢀR , DCM / MeOH, ꢀ5 to 0 °C, then HCl in EtOH, Et O /
2
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3
acetone; (n) KSCN, DMF, reflux; (o) R ꢀSCN, DMF, reflux; (p) HalꢀR , K CO , MeOH, room
2
3
temperature; (q) NBS, CCl , 0 °C to room temperature; (r) Pd(PPh ) or Pd(PPh ) Cl / PPh ,
4
3 4
3 2
2
3
Na CO or KꢀOAc, toluene or DMF, 80 to 120 °C. Parentheses: n=1: 4ꢀpyridinylꢀ or n=0:
2
3
hydrogen.
Identification of CBSꢀ3408 (3) and CBSꢀ3435 (4) as tool compounds.
The first CSAID prototypes, “6ꢀ(4ꢀfluorophenyl)ꢀ5ꢀpyridinꢀ4ꢀylꢀ2,3ꢀdihydroimidazo[2,1ꢀ
b][1,3]thiazole (SKFꢀ86002)” and “4ꢀ[4ꢀ(4ꢀfluorophenyl)ꢀ2ꢀ(4ꢀmethylsulfinylphenyl)ꢀ1Hꢀ
imidazolꢀ5ꢀyl]pyridine (SB203580)” were extremely valuable as tools and chemical probes and
emerged as frequently used reference compounds, thereby facilitating the experimental
5
9
elucidation of various biological functions. However, clinical studies with SB203580 and other
first generation p38α MAPK inhibitors revealed a dose limiting hepatotoxicity, presumably
caused by interactions with the cytochrome P450 system. Second generation p38α MAPK
inhibitors with improved selectivity profile within the p38 MAPK family showed a better safety
profile but, in the end, lacked efficacy. Blockage of the p38 MAPK pathways leads to a positive
feedback mechanism for JNK and other signaling pathways. The intention to inhibit these
bypasses gave rise to the idea that multiꢀtarget drugs inhibiting both key drivers of inflammatory
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processes may exhibit additive or even synergistic effects in vivo. Consequently, SB203580’s
offꢀtarget activity was exploited for the development of dual inhibitors of p38α MAPK and JNKs.
Further investigation demonstrated this dual approach to be a promising strategy for treatment of,
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e.g., Huntington’s disease.
Derived from the SKFꢀ86002 and SB203580 scaffold, the dual p38α MAPK/JNK3 inhibitors still
contained the pyridinylꢀimidazoles scaffold as the core structure. However, the thiazoleꢀring of
SKFꢀ86002 was cleaved in a manner that kept the sulfur atom at position 2 of the imidazole ring,
as illustrated in Figure 2.
In this way, a compound library (“CBS library”) comprising more than 2500 triꢀ und
tetrasubstituted imidazoles was synthesized over the years. Error! Reference source not found.
provides an overview of the structures that are mentioned in the present work. Extensive
structureꢀactivity relationships (SARs) of the CBS library and p38α MAPK and JNK3 were
determined in the course of various in vitro screenings using the isolated enzymes for
determination of IC values and kinome selectivity.
5
0
Figure 2. Template development of SB203580 and triꢀ / tetrasubstituted 2ꢀthioimidazoles derived
from the early dihydrothiazolineꢀderived lead compound SKFꢀ86002.
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Optimizing the substituents in positions R – R led to IC values as low as the singleꢀdigit
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nanomolar range for both kinases. Apart from optimized interactions, the decoration of the
pyridinylꢀimidazoles pharmacophore with appropriate substituents the modulation of selectivity
and potency towards p38α MAPKs and JNKs, as well as substantial reduction of the cytochrome
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P450 interactions and consequently a reduced liver toxicity potential.
Primary screening of test compounds included the inhibition of p38α MAPKꢀcatalyzed
phosphorylation of the transcription factor ATFꢀ2 and the suppression of TNFα/ILꢀ1βꢀrelease in
LPSꢀstimulated human whole blood. In each enzyme assay or whole blood testing sequence,
SB203580 was included as reference and the potency was expressed as the ratio of IC (SB) vs.
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IC (test). For the top 100 triꢀsubstitued candidates, the values varied between 2.0 and 28.5
5
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(
enzyme assay) and between <0.5 and 77 (whole blood assay). For the top 40 tetraꢀsubstitued
imidazoles, IC ꢀratios between 0.5 and 42 (enzyme assay) as well as between <0.5 and 4.5
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(whole blood assay) were determined. Most promising candidates were subsequently subjected to
a preliminary kinase selectivity screening with a fixed panel of 18 protein kinases and a
cytochrome Pꢀ450 highꢀthroughput fluorimetric assay (CYP 1A2, 2C9, 2C19, 3A4, 2D6). Based
on this panel, the triꢀsubstituted imidazoles are highly potent p38α/βꢀinhibitors with significant
potency on JNKꢀisoforms. In contrast, tetraꢀsubstitued derivatives are less potent but the
selectivity against other protein kinases was higher. Furthermore, the triꢀsubstituted candidates
showed a slightly higher affinity to cytochrome Pꢀ450 isoforms. As final selection criterion, the
doseꢀdependent efficacy of the most promising some 30 hits was investigated in experimental,
LPSꢀ or LPS/galactosamine (GalN)ꢀinduced endotoxaemia in either rats or mice (n=6 or 8 per
group). In this model, the effect of test compound on the plasma concentration of TNFα was used
as readꢀout. One hour after oral administration of test compounds or vehicle, endotoxemia was
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induced in rats with 0.1 mg/kg LPS i.v. or 1.5 µg/kg LPS / 500 mg/kg GalN i.p. After 90 min,
blood samples were collected and used for determination of TNFα plasma concentrations.
Finally, the compounds 3 and 4 were selected as tool compounds. The characteristic data are
summarized in Table 1.
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Assay
Compound 3
Compound 4
Potency
∗1
p38 MAPKα
2.35
5.8
whole blood assay
ILꢀ1β
1.5
2.1
3.2
5.4
TNFα
2
Kinase selectivity* (>70%
inhibition @ 10µM)
JNK3, p38β
JNKꢀ1, ꢀ2, ꢀ3, p38β
3
CYPꢀinhibition @ 10 µM* [%]
18; 46; 4; ꢀ7; 45
72; 60; 90; 16.5; 74.5
4
In vivo endotoxaemia*
Rat: 97% @ 10 mg/kg
Mouse: 60% @ 10 mg/kg
Table 1: Summary of pharmacological profiling data of selected tool compounds
and 4.
1: IC (SB203580)/IC (test)
3
*
*
5
0
50
2: Kinase profiler (Upstate): CaMKII, cꢀRaf, GSK3β, JNKꢀ1, ꢀ2, ꢀ3, Lck, MAPK1, MAPK2,
MEKꢀ1, PKA, PKB, PKC, RockꢀII, p38β,γ,δ
3: CYP1A2, CYP2C9, CYP2C19, CYP3A4, CYP2D6
*
*4: 90 min after administration of 0.1 mg/kg iv to rats or 1.5 µg/kg LPS+500 mg/kg GalN i.p. to
mice, TNFα plasma concentrations were used as readꢀout.
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From pharmacological tool compounds to the clinical candidate.
Prior to the investigation of the efficacy of both tool compounds in collagenꢀinduced arthritis
(CIA) in mice, a singleꢀdose pharmacokinetic study was performed. To allow for repeated blood
samplings from the same animal, the rat was selected as animal species. The results are
summarized in Table 2. Maximum plasma concentrations of 3 and 4 were 2.07 and 1.61 µM,
respectively, which correspond to the 12.5ꢀ and 24ꢀfold the IC for inhibition of TNFαꢀrelease in
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LPSꢀstimulated human whole blood. Based on the rapid systemic elimination, for the CIAꢀstudy,
a twice daily dosing regimen was suggested. Furthermore, both compounds exhibited genderꢀ
specific pharmacokinetics with approx. twoꢀfold higher Cmax and AUC values determined in
females.
Compound
Tmax
Cmax
AUC0ꢀt
T½
[h]
3.0
2.8
[
h]
[ng/ml]
738
[µM]
2.07
1.61
[ng*h/ml]
[µM*h]
13.83
9.86
3
4
1.0
2.0
4,928
3,713
608
Table 2: Pharmacokinetic parameters (mean of males and females) of 3 and 4 after single oral
gavage) administration of 30 mg/kg to male and female Wistar rats.
(
In experimental CIA in male DBA/1 mice, treatment of animals with compounds 3 and 4 (50
mg/kg b.i.d.) commenced on day 22, i.e. one day after the second (boost) injection of type II
collagen. The development of arthritic score, which represents a quantification of both the
number of inflamed joints and the severity of inflammation, is illustrated in Figure 3. Treatment
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of animals with compounds 3 and 4 was associated with a substantial reduction of the disease
severity, which was quantified by scoring of the number of inflamed joints and the severity of
inflammation.
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Figure 3: Effect of 3 and 4 on collagenꢀinduced arthritis in male DBA/1 mice. (50 mg/kg
b.i.d.), treatment started on day 22.
In the CIAꢀstudy, except for a body weight loss, essentially identical to that observed for control
animals, no signs of toxicity were recorded. In order to estimate the maximum tolerated dose in
Wistar rats, a twoꢀstep approach was followed. In a first setting, single doses of 100 (n=1) 500
(n=1) and 2,500 mg/kg (n=3) were administered and the clinical signs observed for 48 hours.
Subsequently, 300 mg/kg (n=5) were administered twice daily for 7 days. The second dose was
given 8 h after the first dose. During the 7 days study, 3 was tolerated well while one animal
receiving 4 was found dead on day 6, and two further animals had to be sacrificed due to bad
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conditions. After 7 days, treatmentꢀrelated effects on body weight gain, number and composition
of blood cells, liver enzymes and macroscopic appearance of liver and spleen was recorded. In
addition, a hERGꢀassay (manual patchꢀclamp technique) was performed and both compounds
were found to inhibit the hERGꢀmediated potassium current in a concentrationꢀdependent manner
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with IC ꢀvalues of 28.6 µM (3) and 2.76 µM (4).
50
Based on these data, it was concluded that the tool compounds were useful to demonstrate that
inhibition of p38α MAPK and TNFαꢀrelease in LPSꢀstimulated whole blood translated into a
relevant efficacy in acute (endotoxaemia) and chronic (CIA) inflammation in vivo. However, the
observed genderꢀspecific pharmacokinetics, the signs of toxicity after treatment of Wistar rats for
7
days and the results of the hERGꢀassay did not allow to consider either compound as a clinical
candidate. Irrespective of this conclusion, the genderꢀspecific pharmacokinetics of 3, which was
also observed for further tetraꢀsubstituted imidazoles, was investigated. In vitro metabolism
studies with liver microsomes from rats and humans demonstrated a fast oxidation of the
methylsulfanyl group to the corresponding sulfoxide. Except for traces of the sulfone, no
additional metabolites were detected.
Thus, based on in vitro metabolism studies, the sulfoxide represents a metabolically stable
compound and represented another candidate for pharmacological testing. The compound CBSꢀ
3595 (1), bearing a chiral sulfoxide function, was synthesized as racemate. The potency against
p38α MAPK and in the whole blood assay, however, was lower compared to 3 and would
therefore not immediately suggest further evaluation. Nevertheless, a rat pharmacokinetic study
was performed followed by an in vivo study to investigate the efficacy in experimental
endotoxaemia. In contrast to 3, the pharmacokinetics of 1 were essentially identical for males and
females. Surprisingly, the in vivo efficacy was substantially higher, which could not be explained
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by in vitro data. Further screening on 39 GPCRs (adenosine, serotonin, dopamine, tachykinine,
adrenergic and muscarinic receptors) and phosphodiesterases (PDE), the inhibition of PDE4 with
an IC of 400 nM and a minor interaction with the adenosine A1 receptor (58%ꢀinhibition at 10
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µM) were the only additional interaction of 1. Futher to this finding, some 90 triꢀ and
tetrasubstituted 2ꢀ(methylsulfanyl) and 2ꢀ(methylsulfinyl)ꢀ1Hꢀimidazoles, were tested on PDE4
inhibition potency. The 2ꢀ(methylsulfinyl)ꢀderivatives were used as either racemates or pure
enantiomers. Experimental data ( %ꢀinhibition at 1 µM and 10 µM) demonstrated that sulfoxides
are more potent than corresponding 2ꢀ(methylsulfanyl)ꢀderivatives and that one enantiomer is 2ꢀ
4ꢀfold more potent than the opposite isomer. Single crystal analysis of the enantiomers of 1
demonstrated (R)ꢀconfiguration for the more potent enantiomer. For all other investigated
enantiomeric pairs, no single crystal analysis was performed but correlation of the order of
elution in chiral chromatography with PDE4ꢀinhibition data suggest that (R)ꢀsulfoxides are more
potent. Further SARꢀanalysis demonstrated that tetraꢀsubstituted imidazoles are more potent
PDE4ꢀinhibitors compared to the triꢀsubstitued analogues. However, no substantial differences
were observed for Nꢀmethylꢀ, Nꢀethyl and Nꢀ2ꢀmethoxyethylꢀsubstitution as well as for
modification of X (2ꢀpyridylꢀsubstitutions). With the most potent compounds, at 1 µM, PDE4
activity was inhibited by 86%, compared with 73% for 1 and 94% for the selective PDE4ꢀ
inhibitor roflumilast.
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CBS-3728 (2)
CBS-3595 (1)
CBS-3408 (3)
CBS-3435 (4)
CBS-3846 (7)
CBS-3733 (5)
CBS-3831 (8)
CBS-3835 (9)
Figure 4: Relevant structures of the CBS library. CBSꢀ3831 (8 = TAKꢀ715) was frequently used
as a second reference compound.
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Results and Discussion. Profiling of 1, a dual p38α MAPK / PDE4 inhibitor in nonclinical
studies. Additional information for all conducted experiments is provided in the Supporting
Information.
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The common synthetic route of 1 yielded the compound as racemate. By separation of the
sulfoxide enantiomers, it was demonstrated that the (R)ꢀenantiomer (IC =129 nM) is
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substantially more potent than the (S)ꢀenantiomer (11% inhibition @ 1 µM). In contrast, both
enantiomers are equally active against p38α MAPK. Single enantiomers are accessible via an
enantioselective sulfoxidation in a good yield of 77% and an excellent optical purity of 98% ee,
3
8
as described in more detail by Bühler et al. Nevertheless, for preclinical development, the
racemate was used.
The dual inhibitory properties of 1 towards p38α MAPK and PDE4 were characterized in a
variety of screening approaches, including in vitro characterization of the inhibitory potency on
the isolated enzymes and kinome selectivity. In vivo, CBSꢀ3728 (2), a systemically predominant
and pharmacologically active metabolite was identified (PDE4 IC =2µM).
5
0
For both, parent compound and metabolite, a selectivity assay with different human PDE4 splice
variants, expressed in Sf9ꢀcells, was performed. In general, the potency of 1 and 2 to inhibit the
PDE4 splice variants of the different subtypes is in the lower micromolar range. For both
compounds, the rank order of potency for inhibition of the splice variants are
D3~D2≥B1≥A4>C1. Halfꢀmaximum inhibition values (IC ) are given in Table 3 and illustrated
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in Figure 5.
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PDE4 splice
variant
A4
IC50 [µM]
1
2
0
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4
2.9
1.9
6.4
1.1
0.8
9.9
3.5
4
4
B1
C1
>10
2.4
0.7
4
4
D2
D3
Table 3:
Potency (IC ) of 1and its metabolite 2 to inhibit human PDE4 splice variants.
50
Figure 5
:
Inhibition of selected PDE4 splice variants by
SE from n=3ꢀ4 experiments
1 and 2. Results are shown as mean
±
In a series of in vitro, ex vivo and in vivo experiments, the potency of 1, its main active
metabolite 2 and the reference compound SB203580 on the suppression of TNFα production was
thoroughly demonstrated. These data indicate that 1 is a promising clinical candidate for
treatment of human immune diseases based on the overproduction of TNFα. Additionally, 1’s
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safety, tolerability as well as its PK and ex vivo PD properties were investigated in a phase I firstꢀ
useꢀinꢀhuman study.
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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
. In vitro experiments: investigation of the binding mode and affinity of 1 to p38α MAPK
by Surface Plasmone Resonance (SPR) Spectroscopy. For 1, an ATPꢀcompetitive binding
mode similar to that of SB203580 was suggested when comparing the SPR data.
2. In vitro experiments: inhibition of p38α MAPK and PDEꢀ4, kinase selectivity profile and
the effect on G ProteinꢀCoupled Receptors (GPCRs) of 1 and 2. The inhibitory effect of 1
towards the isolated p38α MAPK kinase was investigated. An IC value of 0.5 µM was
5
0
6
1
determined using the HitHunter p38 MAPK binding assay (DiscoveRx, Fremont, CA, USA).
Investigation of the inhibitory effect of 1 on PDE4 revealed an IC value of 0.2 µM. The activity
5
0
of PDEꢀ3 and PDEꢀ5 was affected by neither 1 nor the metabolite 2 at a concentration of 10
61
µM.
Kinase
Inhibition
Kinase
Inhibition
[
%]
36
51
76
35
94
[%]
62
cꢀRAF
MKK6
JNK2α2
JNK3
p38δ
MKK7β
Lyn
101
31
EphB2
RIPK2
60
CK1δ
33
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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
CK1
97
37
CDK3/cyclinE
CDK7/
55
54
MKK4
cyclinH/MAT1
p70S6K
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
32
Table 4:
Effect of 1 on the activity of selected ATPꢀdependent protein kinases.
The effect of 1 on the activity of a set of 134 protein kinases was investigated at a test
concentration of 10 µM. Kinases that were inhibited by more than 30% were considered as
relevant offꢀtargets and are listed in Table 4. All other 119 kinases, among those the p38 MAPK
isoforms β and γ as well as JNK1α1, were not affected by 1.
Based on these data, the inhibition of the enzymes, JNK2α2, JNK3 and CK1δ may be considered
relevant. The relevance of interactions with other kinases however, is doubtful. CK1 is a yeast
enzyme and MKK7β is the protein kinase which phosphorylates p38α MAPK. In this assay, the
activity of MKK7β was measured indirectly via p38α MAPK activity. Therefore, the observed
complete inhibition is most likely the result of p38α MAPK blockade. Finally, the effect of 1 on
the MKK6ꢀmediated p38α MAPK activation was investigated. The results indicate that 1 did not
inhibit MKK6ꢀmediated p38α MAPK phosphorylation at concentrations up to 10 µM. The effect
of 2 was tested on the following kinases: cꢀRAF, JNK1α1, JNK2α2, JNK2α2, Lck, MAPK1,
MAPK2, ROCKꢀII and the p38 MAPK isoforms α, β and γ. At a final assay concentration of 10
µM no relevant interaction was observed. In an additional study, CK1δ was inhibited by 88% at a
concentration of 3 µM.
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1
2
3
3. In vitro experiments: effect of 1 and 2 on TNFα and LTB4 release in human whole blood.
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
Different concentrations of both test and reference compounds were preꢀincubated with fresh
6
2
human whole blood from healthy donors as described in . TNFα release was induced by
addition of LPS. Quantification of the TNFα levels in the plasma supernatant allowed the
calculation of IC values ± SD of 0.71 ± 2.15 µM (SEM = 0.47; n = 21) for 1, while 2
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
5
0
demonstrated a comparable effect on inhibition of TNFα release.
Above that, the influence of 1 on the formation of leukotriene B4 (LTB4) was investigated and
compared with the references roflumilast and SB203580. A clear doseꢀresponse was observed for
1
1
and roflumilast whilst SB203580 had no effect. Roflumilast was more potent when compared to
which correlates with stronger inhibition of PDEꢀ4.
4. Ex vivo experiments: effect of orally administered 1 on TNFα release from stimulated
whole blood from Cynomolgus Monkeys. As part of the 4ꢀweek toxicity study in Cynomolgus
monkeys, the effect of orally administered 1 on TNFα release in ex vivoꢀstimulated whole blood
was investigated. Animals were treated with either vehicle or three different doses of 1 (6, 12, 20
mg/kg/day). On day 1 and day 27, blood samples were taken and the resulting TNFα
concentrations were determined after ex vivo stimulation of the blood samples with LPS. In
blood samples of all animals treated with 1, substantially reduced TNFα levels were detected and
the degree of TNFα release was significantly suppressed in a doseꢀdependent manner, when
compared to the TNFα levels of control animals that received vehicle. In Figure 6, TNFα levels
after day 1 administration of 1 or vehicle are illustrated.
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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 6: Effect of orally administered 1 on TNFα synthesis in ex vivo LPSꢀstimulated whole
blood from Cynomolgus monkeys.
A concentration of 12 mg/kg of 1 was able to reduce the release of TNFα by more than 50%,
when compared to the TNFα achieved with vehicle only. The dose level of 20 mg/kg completely
inhibited the TNFα release in this experiment.
5. ADME experiments: membrane permeability, plasma protein binding and in vitro
metabolism of 1. The compound’s membrane permeability was investigated in Cacoꢀ2 cell
monolayers at the final test concentration of 10 µM and the obtained results were compared to
those of the reference compounds (atenolol for low, propranolol for high permeability and
talinolol as Pꢀglycoprotein substrate). 1 demonstrated a fairly good permeability (Papp= 18.5 ±
ꢀ6
ꢀ6
6
.2 x10 cm/s compared to 8.7 ± 2.1 and 71.1 ± 6.8 x10 cm/s for atenolol and propranolol,
respectively) and a Pꢀglycoproteinꢀmediated transport could be excluded.
Unbound Fraction [%]
1 µM
10 µM
30 µM
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2
3
4
5
6
7
8
9
Human
90.0 ± 0.0
80.6 ± 0.9
78.1 ± 2.8
75.4 ± 5.2
SpragueꢀDawley
rats
86.3 ± 12.2
63.1 ± 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
Cynomolgus
monkey
66.7 ± 4.1
61.1 ± 1.7
58.3 ± 3.1
Table 5: Plasma protein binding of 1.
The extent of plasma protein binding was determined by equilibrium dialysis in plasma from
humans, SpragueꢀDawley rats and Cynomolgus monkeys for 1, 10 and 30 µM of compound 1.
Low protein binding was confirmed across all species, as shown in Table 5. In comparison, at
5
µM in human plasma, the unbound fraction of 3 was 3.7%.
For investigation of 1’s in vitro metabolism, concentrations of 10, 30 and 100 µM of the
compound were incubated with liver microsomes from either rats, Cynomolgus monkeys or
humans. Two major metabolites were detected: 2, the deacetylated 1 and the oxidized sulfone
metabolite CBSꢀ3733 (5), as shown in Figure 7. Investigation of the biotransformation of 1 in
primary human hepatocytes revealed 2 to be the main metabolite. The sulfone metabolite 5 and
its deacetylated product CBSꢀ3846 (7) were also detected, but at substantially lower
concentrations.
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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
NH2
N
O
N
S
N
O
HN
2
NH2
F
N
N
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
O
S
O
S
N
N
O
N
N
O
HN
1
7
N
F
F
O
S
N
N
O
5
F
Figure 7: Metabolic pathways of the dual p38α MAPK / PDEꢀ4 inhibitor 1.
6
. Efficacy of 1 on TNFα release in LPSꢀinduced endotoxaemia in rats and mice.
First, the effect of orally administered 1 was assessed in Sprague Dawley rats. Five treatment
groups (eight animals/group) received either vehicle (control) or 1, 3, 10 mg/kg of compound 1 or
3
mg/kg of SB203580, respectively. One hour after dosing, 0.1 mg/kg LPS was administered by
intravenous injection. Two hours later (i.e. three hours after dosing), blood samples were
collected and TNFα serum levels were determined. In comparison to the mean TNFαꢀ
concentration determined in vehicleꢀtreated animals, 1, 3, and 10 mg/kg of 1 inhibited the TNFα
release by an average of 43%, 88% and 96%, respectively. Regarding the benchmark of 80% of
TNFα inhibition, a dose of 3 mg/kg of compound 1, reduced TNFα serum levels below this value
in seven out of the eight animals, while 10 mg/kg of 1 were sufficient to diminish the TNFα
concentrations in all animals below this 80% limit. Administration of 3 mg/kg of SB203580
decreased the TNFα serum levels by an average of 37% .
Next, the effect of the time period between administration of 1 and LPSꢀchallenge on TNFα
release and the existence of a genderꢀspecific efficacy was investigated. To this end, female
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2
3
4
5
Lewis rats (n=8/group) received 10 mg/kg one, three and five hours prior to LPS challenge
(
relative time points: ꢀ1h, ꢀ3h, ꢀ5h) . In addition, one group of male Lewis rats was treated with
10 mg/kg at time point ꢀ1 h. As controls, each of one group with male and female animals
received vehicle and one group of females 30 mg/kg of SB203580, all administered at time point
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
ꢀ1 h. In addition, aliquots of the serum samples were used for determination of drug
concentrations.
The extent of TNFα suppression was dependent on the relative time point of dosing. The closer
the time of dosing to LPS challenge, the higher the plasma concentration at 1.5 hour post dose,
and the higher the inhibition of TNFα release. Administration of 10 mg/kg of 1 five hours before
LPS challenge resulted in comparable TNFα plasma levels than those achieved with 30 mg/kg of
SB203580 administered one hour prior to LPS challenge.
The comparison of data obtained with male and female animals indicated the absence of genderꢀ
specific PK or efficacy. However, application of 1 three or one hour before injection of LPS was
more effective than the reference compound SB203580 in blocking TNFα release in male and in
female animals.
Serum levels of 237 ± 20, 526 ± 40 and 871 ± 21 ng/mL of 1 were measured 1.5 hours after LPS
administration in female animals. In the group of male rats that received 10 mg/kg of 1 one hour
before LPS administration, the serum concentration was 689 ± 20 ng/mL and thus in a similar
range when compared to the female animals.
Third, the effect of the p38α MAPK inhibitor properties of 1 was compared to the effects of an
intermediate p38α MAPK inhibitor (8) (4 times as potent as SB203580), a very potent p38α
MAPK inhibitor CBSꢀ3835 (9) (15 times as potent as SB203580) and roflumilast in a headꢀtoꢀ
head setting in eight Lewis rats.
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