Identification of potent agonists of photoreceptor-specific nuclear receptor (NR2E3) and preparation of a radioligand

Article abstract of DOI:10.1016/j.bmcl.2006.07.056

Agonists of NR2E3 (PNR, RNR) have been identified and optimized to EC₅₀ < 200 nM. A tritiated analogue of one agonist was prepared to aid in the development of a binding assay.

Full text of DOI:10.1016/j.bmcl.2006.07.056

Bioorganic & Medicinal Chemistry Letters 16 (2006) 5001–5004
Identification of potent agonists of photoreceptor-specific nuclear
receptor (NR2E3) and preparation of a radioligand
Scott E. Wolkenberg,a,* Zhijian Zhao,a Marianna Kapitskaya,b Andrea L. Webber,b
Konstantin Petrukhin,b Yui Sing Tang,c Dennis C. Dean,c
George D. Hartmana and Craig W. Lindsleya
aDepartment of Medicinal Chemistry, Technology Enabled Synthesis Group, Merck & Co., Inc., PO Box 4,
West Point, PA 19486, USA
bDepartment of Ophthalmics Research, Merck & Co., Inc., PO Box 4, West Point, PA 19486, USA
cDepartment of Drug Metabolism, Merck & Co., Inc., PO Box 2000, Rahway, NJ 07065, USA
Received 31 May 2006; revised 13 July 2006; accepted 17 July 2006
Available online 1 August 2006
Abstract—Agonists of NR2E3 (PNR, RNR) have been identified and optimized to EC50 < 200 nM. A tritiated analogue of one
agonist was prepared to aid in the development of a binding assay.
ꢀ 2006 Elsevier Ltd. All rights reserved.
Age-related macular degeneration (AMD) is a leading
cause of blindness in the Western world which affects
10 million patients in the United States.1 Loss of vision
and eventual blindness has been related to the degener-
ation of photoreceptor cells in the macula, the center
portion of the retina. The death of photoreceptor cells
is associated with dysfunction of the retinal pigmented
epithelium, which serves many critical roles in mainte-
nance of rods and cones including cycling of visual
pigments derived from retinol (vitamin A).2
cones, and progressive reduction in their function.4
Based on these associations and other evidence,
NR2E3 is an attractive target for intervention in AMD.
High throughput screening of the Merck sample collec-
tion generated a series of leads which exhibited NR2E3
agonism.6 Agonism was assessed using a GAL4DBD–
NR2E3LBD chimera in a recently described transactiva-
tion-based b-lactamase assay.6 Because no endogenous
ligand for NR2E3 is known, this assay was developed
in parallel with a functionally analogous thyroid hor-
mone nuclear receptor GAL4DBD–TRLBD chimera
assay. Since TR is well characterized and its native
ligand is known, this pairing confirmed a functioning
NR2E3 assay and enabled an initial determination of
agonist selectivity. Structures of positive hits, which rep-
resent the first known agonists of NR2E3, are presented
in Figure 1.
Photoreceptor-specific nuclear receptor (PNR3a, also
known as RNR3b and NR2E3) is a recently discovered3
orphan nuclear receptor which, as its name implies, is
expressed exclusively in rod photoreceptor cells of the
retina.3a,4 In addition to its localization in the retina,
several lines of evidence implicate NR2E3 in the pro-
gression of photoreceptor degeneration. For instance,
NR2E3 mutations in humans have been correlated with
various retinal diseases5 and rd7 mice, which contain a
sporadic genomic deletion within the gene coding for
NR2E3, exhibit abnormal development of rods and
It is noteworthy that 13-cis-retinoic acid (1), a natural
retinal pigment, exhibits limited properties of an
NR2E3 agonist in cell-based assays and does so only
at very high, non-physiological concentrations. There-
fore, optimization efforts focused on the more potent
compound 2. An important complement to the
NR2E3 b-lactamase transactivation assay, the NR2E3
NCOR release assay,7 was based on NR2E3’s role as a
transcriptional repressor in its unligated state. This
Keywords: NR2E3; PNR; RNR; Age-related macular degeneration;
AMD; Nuclear receptor; Ophthalmics.
*
Corresponding author. Tel.: +1 215 652 0821; fax: +1 215 652
6345; e-mail: scott_wolkenberg@merck.com
0960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.07.056

5002
S. E. Wolkenberg et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5001–5004
Table 1. NR2E3 agonist activity of selected analogues
a
EC50 (nM)
Compound
R0
CO2H
NR2E3
b-lactamase
NR2E3
NCOR
1, NR2E3 β-lactamase EC50 25−50 μM
O
6a
6b
>25,000
7200
4100
Cl
O
O
N
H
F
N
N
Cl
O
>25,000
>25,000
N
H
H
N
N
2, NR2E3 β-lactamase EC50 1.3 μM
Cl
6c
4200
Figure 1. NR2E3 Screening leads.
HO
Cl
assay quantitates the release of the co-repressor NCOR
fragment from the NR2E3 ligand binding domain upon
binding of an agonist.
a Values are means of two experiments (na, not active).
Subsequent analogues retained the 2-phenylbenzimidaz-
ole core and focused on examining the two amide
substituents. In this case (Scheme 2), 4-nitro-1,2-diami-
nobenzene was condensed with polymeric 4-aminobenz-
aldehyde in refluxing pyridine,9 then converted to the
2-chlorobenzamide. Nitro reduction (SnCl2) and amide
formation generated the desired analogues; data are pre-
sented in Table 2.
Initial investigation of lead 2 was carried out with
libraries aimed at incorporating a diversity of benzimid-
azole 2-phenyl replacements, and preparation of these
analogues is described in Scheme 1. Commercially avail-
able 2-nitro-1,4-diaminobenzene (3) was selectively acyl-
ated at the 4-amino position with 2-chlorobenzoyl
chloride, and the resulting amide 4 was reduced under
Zinin conditions to give 5.8 The diamine 5 was con-
densed with aromatic aldehydes (acetic acid, microwave
irradiation, 170 ꢁC, 20 min) to provide analogues 6, a
selection of which is shown in Table 1.
Small ring amides provided the most potent compounds,
with cyclopropyl amide 11a conferring the greatest
NR2E3 activity. Ring expansion (11c and 11d) or open-
ing (11b) significantly decreased activity, and alternative
small alkyl groups (e.g., pivaloyl 11e) exhibited dimin-
ished activity. The corresponding acetamide was devoid
of any activity.
Potency in the b-lactamase assay suffered as a result of
these changes; however, in the NCOR release assay sev-
eral analogues retained some agonist activity. Reconcil-
iation of these results is possible upon consideration that
an agonist signal in the NCOR release assay is registered
as decreased expression of the luciferase reporter; thus
reduction in luciferase activity due to cell death caused
by a toxic test compound could be misinterpreted as
an agonist signal.7 It was possible to confirm toxicity
by testing the effect of compounds on cell survival in a
control cell line; in the case of 6a–6c this secondary
screen confirmed toxicity and, importantly, established
the criteria that test compounds were viable only if they
exhibited activity in both the b-lactamase transactiva-
tion and the luciferase NCOR release assays, and were
non-toxic in control cell lines.
The cyclopropyl amide present in 11a proved to be
uniquely capable of providing NR2E3 agonism at
EC50 < 200 nM. Table
3
indicates how sensitive
NR2E3 activity is to substitution on the cyclopropane,
with even small changes like mono- and difluorination
resulting in a significant decrease in activity. Substitu-
tion with a phenyl group (11j) abolished NR2E3 activity
and introduced cellular toxicity to the compounds.
O2N
NH2
NH2
O2N
N
a
NH2
N
H
7
8
Cl
H
O
N
H2N
NO2
NH2
N
R
a
R
N
b
d
Cl
b
O
NH2
N
H
H
3
4, R = NO2
5, R = NH2
9, R = NO2
10, R = NH2
c
Cl
H
N
O
c
N
H
N
R'
R'
N
Cl
O
N
H
N
H
O
N
H
6
11
Scheme 1. Reagents and conditions: (a) 2-chlorobenzoyl chloride,
CH2Cl2, rt, 99%; (b) Na2S, NaHCO3, MeOH/H2O, 60 ꢁC, 96%; (c)
R0CHO, 10% HOAc/trifluorotoluene, microwave, 170 ꢁC, 20 min, 10–
64%.
Scheme 2. Reagents and conditions: (a) 4-aminobenzaldehyde poly-
mer, pyridine, 115 ꢁC, 68%; (b) 2-chlorobenzoyl chloride, CH2Cl2, rt,
86%; (c) SnCl2, EtOH/EtOAc, reflux; (d) R’COCl, CH2Cl2, rt, 10–50%.

S. E. Wolkenberg et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5001–5004
Table 2. NR2E3 agonist activity of selected analogues Table 4. NR2E3 agonist activity of selected analogues
5003
a
EC50 (nM)
a
EC50 (nM)
Compound
Compound
Structure
O
NR2E3
b-lactamase
NR2E3
NCOR
NR2E3 NR2E3
R'
b-lactamase
NCOR
610
O
N
O
O
O
6d
6e
270
11a
11b
11c
11d
11e
141
35
147
H
F
O
N
316
1328
3160
6270
270
260
170
330
H
986
O
6f
O
N
H
O
1320
2610
HN
Cl
O
6g
10,000
4100
a Values are means of two experiments (na, not active).
Cl
O
N
N
6h
4800
2200
Table 3. NR2E3 agonist activity of selected analogues
H
a Values are means of two experiments (na, not active).
a
EC50 (nM)
Compound
O
NR2E3
b-lactamase
NR2E3
NCOR
R'
H
tion of 11a, the most potent NR2E3 agonist known to
date, presented the opportunity to develop a binding as-
say, which would confirm direct binding of test com-
pounds to the ligand-binding domain of NR2E3 and
simplify the evaluation of new compounds by eliminat-
ing the complications of membrane permeability and
cellular toxicity. Toward this end, tritiated 11a was pre-
pared from a diiodinated precursor as outlined in
Scheme 3. Intermediate 8 was acylated with 2-chloro-
5-iodobenzoyl chloride (CH2Cl2, rt, 67%) before being
reduced (SnCl2) to the corresponding aniline. Incorpo-
ration of a second iodine substituent was achieved by
treating 13 with benzyltrimethylammonium dichloroio-
O
11f
11g
11h
11i
11j
720
2100
260
3000
H
F
F
O
F
O
2500
2900
O
17,000
>25,000
10,000
370
NC
O
Cl
R'
O
R
N
a
8
a Values are means of two experiments (na, not active).
N
N
H
H
I
12, R = NO2, R' = H
13, R = NH2, R' = H
14, R = NH2, R' = I
b
c
Having optimized the benzimidazole 5-amide, attention
was directed toward improving potency by varying the
right-hand anilide. These analogues were prepared by
the route shown in Scheme 1 and NR2E3 data are given
in Table 4. Benzamides were the most potent agonists,
and while o-substitution with Cl increased activity 2·
(11a), other o-substituents (e.g., F (6e) and Me (6f)) were
equipotent with the parent benzamide 6d. Relocation of
the amide to the m-anilide position (6g) decreased
potency by 100-fold, and incorporation of a 2-chloronic-
otinamide (6h) also decreased activity.
Cl
O
I
H
N
d
N
N
O
O
N
H
H
H
I
15
Cl
T
O
N
H
N
e
N
N
H
T
[3H]-11a
To this point, optimization of NR2E3 agonists was
based on their activity in two cell-based assays, and we
could not exclude the possibility that active compounds
exert their effects on NR2E3 indirectly. The identifica-
Scheme 3. Reagents and conditions: (a) 2-chloro-5-iodobenzoyl chlo-
ride, CH2Cl2, rt, 67%; (b) SnCl2, EtOH/EtOAc, reflux, 79%; (c)
BTMACl2I, CaCO3; (d) cyclopropanecarbonyl chloride, CH2Cl2, rt;
(e) tritium gas, 10% Pd/CaCO3, DMF.

5004
S. E. Wolkenberg et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5001–5004
date10 (4 equiv BTMACl2I, MeOH, CH2Cl2, and
CaCO3), and iodination occurred exclusively in the
benzimidazole 4-position. Tritiation was carried out
using tritium gas in the presence of catalytic 10% Pd/
CaCO3, and after HPLC purification provided [3H]-
11a with a specific activity of 46 Ci/mmol.
D. B. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 5551; (b)
Haider, N. B.; Naggert, J. K.; Nishina, P. M. Hum.
Mol. Genet. 2001, 10, 1619; (c) Bumsted O’Brien, K.
M.; Cheng, H.; Jiang, Y.; Schulte, D.; Swaroop, A.;
Hendrickson, A. E. Invest. Ophthalmol. Vis. Sci. 2004,
45, 2807; (d) Chen, J.; Rattner, A.; Nathans, J. J
Neurosci. 2005, 25, 118.
5. (a) Haider, N. B.; Jacobson, S. G.; Cideciyan, A. V.;
Swiderski, R.; Streb, L. M.; Searby, C.; Beck, G.; Hockey,
R.; Hanna, D. B.; Gorman, S.; Duhl, D.; Carmi, R.;
Bennett, J.; Weleber, R. G.; Fishman, G. A.; Wright, A.
F.; Stone, E. M.; Sheffield, V. C. Nat. Genet. 2000, 24, 127;
(b) Milam, A. H.; Rose, L.; Cideciyan, A. V.; Barakat, M.
R.; Tang, S.-X.; Gupta, N.; Aleman, T. S.; Wright, A. F.;
Stone, E. M.; Sheffield, V. C.; Jacobson, S. G. Proc. Natl.
Acad. Sci. U.S.A. 2002, 99, 473; (c) Sharon, D.; Sandberg,
M. A.; Caruso, R. C.; Berson, E. L.; Dryja, T. P. Arch.
Ophthalmol. 2003, 121, 1316; (d) Michaelides, M.; Holder,
G. E.; Webster, A. R.; Hunt, D. M.; Bird, A. C.; Fitzke, F.
W.; Mollon, J. D.; Moore, A. T. Br. J. Ophthalmol. 2006,
89, 332.
In summary, a new class of potential NR2E3 agonists
has been identified which is based on a 2-phenylbenzim-
idazole core. Compound 11a exhibits agonism of
NR2E3 in two cell-based assays and a tritiated analogue
with high specific activity has been prepared. Develop-
ment of a radioligand binding assay is in progress and
will be reported in due course.
Acknowledgments
We are grateful to Joan Murphy and Charles W. Ross,
III, for HRMS measurements and Sandor L. Varga for
NMR structure determinations.
6. Kapitskaya, M.; Cunningham, M. E.; Lacson, R.;
Kornienko, O.; Bednar, B.; Petrukhin, K. Assay and Drug
Dev. Technol. 2006, 4, 253.
7. Luciferase reporter gene assays were performed with
extracts from CHO cells which were transiently co-
transfected with pG5Luc luciferase reporter plasmid
containing GAL4 response elements in its promoter region
in combination with pVP16_NR2E3LBD plasmid express-
ing VP16 activator linked to NR2E3LBD, and
pGALDBD_NCOR plasmid expressing GAL4 fusion of
co-repressor NCOR. In the absence of ligand, VP16-
NR2E3LBD interacts with GALDBD_NCOR which
results in constitutative activation of luciferase reporter.
Addition of NR2E3 agonists leads to co-repressor release
and reduction in luciferase activity.
8. Porter, H. K. Org. React. 1973, 20, 455.
9. Bamfield, P.; Harvey, E. D.; Hughes, N.; Stead, C. V. G.B.
Patent 2,125,791.
10. De Los Angeles, J. E.; Nikulin, V. I.; Shams, G.; Konkar,
A. A.; Mehta, R.; Feller, D. R.; Miller, D. D. J. Med.
Chem. 1996, 39, 3701.
References and notes
1. Zarbin, M. A. Arch. Ophthalmol. 2004, 122, 598.
2. (a) Bok, D. J. Cell Sci. Suppl. 1993, 17, 189; (b) Blomhoff,
R.; Green, M. H.; Norum, K. R. Annu. Rev. Nutr. 1992,
12, 37.
3. (a) Kobayashi, M.; Takezawa, S.; Hara, K.; Yu, R. T.;
Umesono, Y.; Agata, K.; Taniwaki, M.; Yasuda, K.;
Umesono, K. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 4814;
(b) Chen, F.; Figueroa, D. J.; Marmorstein, A. D.; Zhang,
Q.; Petrukhin, K.; Caskey, C. T.; Austin, C. P. Proc. Natl.
Acad. Sci. U.S.A. 1999, 96, 15149.
4. (a) Akhmedov, N. B.; Piriev, N. I.; Chang, B.;
Rapoport, A. L.; Hawes, N. L.; Nishina, P. M.;
Nusinowitz, S.; Heckenlively, J. R.; Roderick, T. H.;
Kozak, C. A.; Danciger, M.; Davisson, M. T.; Farber,