Novel 2-(substituted phenyl)benzimidazole derivatives with potent activity against IgE, cytokines, and CD23 for the treatment of allergy and asthma

Article abstract of DOI:10.1021/jm049288j

The effectiveness of the injectable anti-IgE antibody omalizumab has validated IgE as an important target for allergic diseases, thus spawning the development of small-molecule IgE inhibitors. Herein, a brief SAR is described for novel phenylbenzimidazole

Full text of DOI:10.1021/jm049288j

J. Med. Chem. 2004, 47, 6451-6454
6451
effectiveness requires greater than 95% suppression of
IgE,4 a level that has been unachievable in vivo by
small-molecule IgE inhibitors.
Novel 2-(Substituted
phenyl)benzimidazole Derivatives with
Potent Activity against IgE, Cytokines,
and CD23 for the Treatment of Allergy
and Asthma
Our objective was to address one of the foremost
challenges in the field of asthma pharmacotherapy, to
suppress disease development at its foundation without
imparting significant liability to the patient. Herein, we
describe a group of compounds that is novel in structure
and apparent mechanism. These agents appear to act
via a single target shared by multiple cell types to
suppress their activation to allergic stimuli, including
production of IgE and Th2 cytokines and expression of
CD23 and IL-4 receptor-R. The lead compound in this
series 1i (AVP-13358) is orally active in mouse models
of asthma and currently in phase I clinical trials for the
treatment of asthma and allergic rhinitis.
Mark L. Richards,* Shirley Cruz Lio,† Anjana Sinha,
Kenneth K. Tieu,‡ and Jagadish C. Sircar
Avanir Pharmaceuticals, 11388 Sorrento Valley Road,
San Diego, California 92121
Received August 31, 2004
Abstract: The effectiveness of the injectable anti-IgE antibody
omalizumab has validated IgE as an important target for
allergic diseases, thus spawning the development of small-
molecule IgE inhibitors. Herein, a brief SAR is described for
novel phenylbenzimidazole compounds that potently suppress
IgE responses. In addition to IgE, these agents inhibit other
targets critical for allergic response. The profile of orally active
AVP-13358, the lead compound of this series currently in
clinical trials, is described.
Identification of these compounds started with the
premise that since IgE is central to allergic manifesta-
tions, a compound that interferes with the IgE response
would act on a target that is fundamentally linked to
its development. This allergic cascade was experimen-
tally reconstituted in mice by immunizing BALB/c mice
with an antigen (DNP-KLH) and adjuvant (alum) fol-
lowed 2 weeks later by removing the spleen and
establishing cultures of lymphocytes with specific an-
tigen, in the presence and absence of drug.7 Allergen
sensitization in vivo initiates the cascade of events that
prepares the animal to produce IgE upon subsequent
challenge. The response to allergen challenge (expo-
sure), which is pertinent to the clinical situation, is thus
reconstituted in the spleen cell culture, wherein equal
numbers of B and T cells act in concert to produce IgE.
In this system, B cells likely function to present the
introduced antigen (DNP-KLH) to T cells, which re-
spond by producing IL-4 (and/or IL-13) and expressing
costimulatory molecules such as the T cell receptor and
CD40 ligand, which in turn activate B cells/plasma cells
to produce IgE. Thus, although IgE is the downstream
product of this experimental system, the assay identifies
drug candidates that act on numerous potential targets
within either of two cell types that are involved in the
generation or propagation of allergic responses. To
identify lead structures that would potentially interfere
with this process, an in-house universal informer library
(developed from over 300 000 compounds) was screened
for biological activity using a cell-based ex vivo IgE
response assay. This effort resulted in the identification
of compounds with the 2-phenylbenzimidazole core
structure and modest activity against the IgE response.
This core structure was chosen for follow-up.
About 5% of Americans suffer asthma, and the
incidence is expanding in epidemic proportions. The cost
of hospitalization in the U.S. alone is $1.6 billion, and
the cost of prescription asthma medications is $1.1
billion annually.1 The paucity of novel pharmacological
approaches introduced in the past 30 years for the
treatment of asthma is a striking contrast to the
plethora of new medicines for diseases such as cancer
or arthritis. Moreover, the drugs currently available for
the treatment of asthma are largely palliative; i.e., they
treat the symptoms rather than the underlying disorder.
Drugs such as antihistamines and leukotriene antago-
nists target single effecter molecules and provide some
relief, but the heterogeneous nature of asthma makes
this approach unsuccessful for most patients.2 Corti-
costeroids act in a multifactorial manner, are very
effective at suppressing allergic symptoms, and remain
the mainstay for treating serious asthma.3 However, the
long-term morbidity associated with daily use severely
limits their use, particularly in children and for less
serious allergic manifestations in adults.
An important drug recently approved for the treat-
ment of asthma is the injectable humanized anti-IgE
antibody omalizumab (Xolair).4 The effectiveness of this
antibody validates IgE as an important mediator in
atopic diseases and has led to an influx of small-
molecule IgE inhibitors under development for the
treatment of asthma and allergic rhinitis.5 The latter
compounds work via various mechanisms to suppress
IgE responses such as inhibiting the release of the Th2
cytokine IL-4.6 While their oral bioavailability provides
an advantage over the anti-IgE antibody, previous work
with omalizumab has shown that optimal therapeutic
The synthesis of the 2-(substituted phenyl)benzimi-
dazoles is indicated in Scheme 1.8 Appropriate struc-
tural modifications followed by SAR analyses of this
scaffold yielded compounds with higher potency and
eventually with oral bioactivity (Table 1).8 SAR studies
of bis-substituted phenylbenzimidazole series revealed
that when R1 and R2 are small aliphatic or aromatic
groups, low potency resulted (IC50 > 100 nM).9 The
potency significantly increased when aliphatic cycloalkyl
groups or aromatic groups were inserted in R1 and R2.
Combining aliphatic and aromatic groups yielded com-
* To whom correspondence should be addressed. Phone: (858) 622-
5225. Fax: (858) 658-7447. E-mail: mrichards@avanir.com.
† Current address: Neurocrine Biosciences, Inc., 12790 El Camino
Real, San Diego, CA 92130.
‡ Current address: Penn State College of Medicine, The Milton S.
Hershey Medical Center, 500 University Drive, Student Box 235,
Hershey, PA 17033.
10.1021/jm049288j CCC: $27.50 © 2004 American Chemical Society
Published on Web 11/13/2004

6452 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 26
Letters
Scheme 1. Synthesis of 2-(Substituted phenyl)benzimidazole Derivativesa
a Reagents: (i) C6H5NO2 at 155-160 °C for 18 h, 79%; (ii) CDI/DMF at 80 °C for 3 h, then amine/DMAP at 100 °C for 18 h or EDC/
DMF at room temp for 3 h, then amine and DMAP at room temp for 18 h, 65%; (iii) MeOH/DMF (9:1)/10% Pd/C, 80%; (iv) dry THF, dry
Py, acid chloride, room temp for 18 h, 45%.
small halogens such as fluorine.9 Potency was optimized
when large hydrophobic residues were placed on the
Table 1. Biological Activity of Phenylbenzimidazole
Compounds
amide linkage; preferred substitutions at R1 and R2 are
bulky fused cycloalkyl groups (1f: R1 ) R2 ) adaman-
tyl; IC50 ) 1 nM).
The oral bioavailability of an asthma/allergy drug is
of paramount importance for application of that com-
pound in the clinical setting. However, the highly
lipophilic character that defines compound potency in
this series is diametrically opposed to achieving good
bioavailability after oral administration. Addition of
polar groups to improve solubility and gastrointestinal
absorption results in an increased IC50 for IgE inhibi-
tion. In the absence of -OH conjugation sites (1h, 1j),
the addition of a pyridine group on the benzimidazole
side of the molecule (1i) substantially improved oral
bioavailability without a significant loss of activity.
Moreover, the pyridinephenylbenzimidazole core struc-
ture is suitable for salt formation. The best counterion
was found to be methansulfonic acid, which improved
the apparent oral bioavailability approximately 3-fold
more than that achieved by the free base (data not
shown).
To address the question of which cell types respond
to compound, individual cell populations were isolated
from the spleens of naive mice and tested for bioactivity.
IgE responses were elicited from B-lymphocytes that
were isolated from spleens of naive BALB/c mice and
cultured 5 days with interleukin-4 (IL-4) and anti-CD40
antibody (in vitro IgE assay). The latter stimuli are
normally provided by T cells and are critical for B cell
production of IgE. As shown in Figure 1 and Table 1,
the activities of these compounds against IgE in both
the ex vivo and in vitro assays roughly follow in parallel,
demonstrating that the compounds are acting directly
on the B cell. The higher potency in the ex vivo assay
suggests that these compounds may also have an effect
on the T cell. This question was addressed directly by
isolating T lymphocytes from mouse spleen and testing
for cytokine responses to various stimuli. The observa-
tion that IL-4, IL-5, and IL-13 production/release were
potently suppressed by 1i demonstrated a direct action
on the T cell.10 The benzimidazole compounds also
target other markers important for development of
allergic response, including suppression of the B cell IgE
receptor (CD23) on human monocytes11 and of CD23 and
IL-4 receptor-R on murine B cells.12 Finally, the revers-
a Includes the combined levels of parent and active metabolite.
Relative oral bioavailability was determined from the levels of
active drug/metabolite in mouse serum samples taken 1 and 4 h
following oral gavage of 40 mg/kg doses of drug.
pounds (e.g., 1d and 1e) with intermediate potency,
although dichlorophenyl substitution at R2 together
with an adamantyl at R1 yielded the most potent
compound of the series (1g). In general, modifications
of the 2-phenylbenzimidazole/bis-amide backbone ab-
lated the bioactivity, including replacement of amide
linkages or alternative amide substitution sites on the
benzimidazole and phenyl ring structures.9 The core
2-phenylbenzimidazole/bis-amide structure also was
found to have little tolerance for substitution except for

Letters
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 26 6453
Figure 2. In vivo efficacy model was performed similarly to
that in previous reports.15 Female BALB/c mice were injected
with 10 µg of ovalbumin (OVA) in 4 mg of alum intraperito-
neally. Two weeks hence, the mice were exposed to OVA by
nebulization (2% in PBS) for 20 min on 2 consecutive days and
treated with 1i or vehicle orally twice daily for 3 days. After 2
additional weeks, the mice were re-administered OVA by
nebulization on 4 consecutive days while receiving drug or
vehicle as above. Control mice were nebulized with PBS only.
One day after the last OVA nebulization and drug dose, mice
were placed in a chamber and tested for airway responsiveness
to increasing concentrations of methacholine by a Buxco whole-
body plethysmograph. The graph compares the data from mice
that received 5 mg/kg AVP-13358 orally twice daily; the ED50
of 1i in this assay system was previously determined to be
2.5 mg/kg when administered as a suspension in a 0.2 M citric
acid formulation (pH 4) by mouth twice daily.
Figure 1. IgE Response. Cultures of spleen cells from antigen-
sensitized mice were established ex vivo in the presence and
absence of antigen and 1i, and antigen-specific IgE response
was measured as described.7 In vitro assays were performed
on B cells that were isolated from naive BALB/c mice and
cultured for 5 days with IL-4 (10 ng/mL) and antimouse CD40
antibody (100 ng/mL). The level of IgE in the supernatants of
both assays was quantified by ELISA and by comparing with
a standard curve using a DNP-specific mouse monoclonal
antibody.
ibility of the biological activity of the phenylbenzimi-
dazoles suggests that their activity is not a result of
nonspecific toxicity; i.e., lymphocytes respond normally
to stimulus following exposure (up to 24 h) and subse-
quent removal of the drug 1i.8 Thus, these compounds
have a direct action on several individual lymphoid cell
types to inhibit their activation by a variety of polyclonal
stimuli.
tion of IgE production in cultures of B-lymphocytes and
in mixed cultures of mouse spleen cells is complemented
by activity against other targets within the allergy
cascade, thus vastly improving their opportunity for
efficacy in asthma. The breadth of in vitro activities is
reflected in the efficacy of 1i in a murine model of
asthma. The structure-activity relationships thus offer
a focus for the discovery and dissection of a potentially
important target for IgE response regulation, which can
be exploited therapeutically in allergic diseases.
The similar potency of these compounds in suppress-
ing multiple targets in diverse cell types suggests that
they act on a specific target that is shared by these cells.
Investigations into the mechanism of action of the
phenylbenzimidazoles have revealed a novel effect on
the processing of proteins in the Golgi.13 This activity
is consistent with the observed effects on IgE, cytokines,
and CD23, all of which putatively require Golgi process-
ing for cellular release or membrane expression.14
Moreover, the mechanism of 1i was clearly shown to be
distinct from the action of other agents known to
perturb the Golgi, such as brefeldin A and monensin.
The in vivo efficacy of 1i was tested in a murine model
of airway hyper-responsiveness.15 As shown in Figure
2, mice that had been sensitized to an allergen (oval-
bumin, OVA) develop significantly greater airway re-
sistance in response to methacholine than mice that
have not been exposed to OVA (PBS group). Mice that
were exposed to OVA and treated with 1i responded to
methacholine in a similar manner as the PBS mice,
indicating that the compound is protecting against the
allergic response elicited by OVA sensitization and
challenge. It is noted that a majority of the in vivo
activity of this compound is contributed by an active
metabolite 1k that has been identified and synthesized;8
1k has about twice the potency of the parent 1i and is
present at about 2-fold higher serum concentrations.
In conclusion, new 2-(substituted phenyl)benzimida-
zoles with potent anti-IgE activity and Th-2 cytokine
inhibitory activity have been identified. These agents
are unique in terms of their chemical structure, high
potency, and putative mechanism of action. The inhibi-
Acknowledgment. The authors thank Homayon
Banie for excellent technical contributions.
Supporting Information Available: Experimental pro-
cedures and spectral data. This material is available free of
charge via the Internet at http://pubs.acs.org.
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JM049288J