Estrogen receptor dependent inhibitors of NF-κB transcriptional activation-1 synthesis and biological evaluation of substituted 2-cyanopropanoic acid derivatives: Pathway selective inhibitors of NF-κB, a potential treatment for rheumatoid arthritis

Article abstract of DOI:10.1021/jm701013k

Pathway selective ligands of the estrogen receptor inhibit transcriptional activation of proinflammatory genes mediated by NF-κB. Substituted 2-cyanopropanoic acid derivatives were developed leading to the discovery of WAY-204688, an orally active, pathway selective, estrogen receptor dependent anti-inflammatory agent. This propanamide was shown to be orally active in preclinical models of inflammatory diseases, such as rheumatoid arthritis, without the proliferative effect associated with traditional estrogens.

Full text of DOI:10.1021/jm701013k

© Copyright 2007 by the American Chemical Society
Volume 50, Number 22
November 1, 2007
Letters
dimeric transcription factors comprising members of the Rel
family. NF-κB was first characterized as a lymphoid specific
protein;1 however, it has been shown to be present, in a latent
form, in the cytoplasm of virtually all cell types. Five mam-
malian NF-κB/Rel proteins have been cloned and characterized.2
It is the dimeric form of these proteins that regulates many
cellular functions such the immune response, response to
bacterial and viral infections, cancer, chronic inflammatory
disorders such as rheumatoid arthritis (RA), ulcerative colitis,
and Crohn’s disease as well as neurodegenerative disorders. NF-
κB has been shown to be overexpressed in the inflamed
synovium3 where it amplifies the recruitment of inflammatory
cells that play a role in the production of proinflammatory
mediators such as IL-6 in RA synovial fibroblasts.4 This central
role of NF-κB in the inflammatory response suggested that
inhibition of NF-κB driven gene transcription might be effective
in the treatment of inflammatory diseases such as RA. For NF-
κB to activate a gene, it must be released from its latent form
by removal of the inhibitory protein IκB; this involves a series
of phosphorylation and ubiquitation reactions that activate
proteosomal degradation of IκB. Once NF-κB translocates into
the nucleus, binding of coactivators facilitates gene transcription.
Several groups have targeted the IκB and ubiquitation pathways
to suppress inappropriate activation of NF-κB for the treatment
of inflammatory diseases5 and cancer.6
Estrogen Receptor Dependent Inhibitors of
NF-KB Transcriptional Activation-1 Synthesis
and Biological Evaluation of Substituted
2-Cyanopropanoic Acid Derivatives: Pathway
Selective Inhibitors of NF-KB, a Potential
Treatment for Rheumatoid Arthritis
Thomas J. Caggiano,*,§ Antony Brazzale,§ Douglas M. Ho,#
Christina M. Kraml,§ Eugene Trybulski,‡
Christopher C. Chadwick,† Sue Chippari,†
Lisa Borges-Marcucci,| Amy Eckert,† James C. Keith,
Thomas Kenney,† and Douglas C. Harnish|
Chemical and Screening Sciences, Wyeth Research, CN 8000,
Princeton, New Jersey 08540-8000, Chemical and Screening
Sciences, Women’s Health Research Institute, and CardioVascular/
Metabolic Disease Research, Wyeth Research, CollegeVille,
PennsylVania, CardioVascular/Metabolic Disease Research, Wyeth
Research, Cambridge, Massachusetts, and Department of Chemistry,
Princeton UniVersity, Princeton, New Jersey 08544
ReceiVed August 14, 2007
Abstract: Pathway selective ligands of the estrogen receptor inhibit
transcriptional activation of proinflammatory genes mediated by NF-
κB. Substituted 2-cyanopropanoic acid derivatives were developed
leading to the discovery of WAY-204688, an orally active, pathway
selective, estrogen receptor dependent anti-inflammatory agent. This
propanamide was shown to be orally active in preclinical models of
inflammatory diseases, such as rheumatoid arthritis, without the
proliferative effect associated with traditional estrogens.
We were interested in exploiting the central role of NF-κB
in inflammatory diseases such as RA, however, using a different
approach. The estrogen receptor is involved in the inhibition
of NF-κB transcriptional activity as demonstrated for IL-6 gene
expression.7 This data and the fact that 17â-estradiol has been
shown to be effective in models of RA8 and that RA went into
remission during pregancy9 suggested the possibility of using
an estrogen receptor-ligand complex to control the up-
regulation of NF-κB in chronic inflammatory states such as RA.
The challenge was to develop a ligand that provided the
advantages of 17â-estradiol (E2) without the classic steroid side
effects.10 To this end, human aortic endothelial cell lines
(HAECT-1) were created; one cell line was cotransfected with
a human estrogen receptor (ER-R or ER-â) and an NF-κB-
luciferase construct, and a separate cell line was transfected with
just the NF-κB-luciferase construct. This allowed the demon-
stration of ER dependency of the NF-κB translational inhibition
Chronic inflammation is perpetuated and amplified through
a complex series of pathways fed by cytokines, chemokines,
adhesion molecules, enzymes, etc. in the inflamed tissue. This
response is regulated by a relatively small number of inducible
genes in response to transcriptions factors such as STATs, AP-
1, NF-AT, and NF-κB. NF-κB is the name given to a family of
* To whom correspondence should be addressed. Phone: 732-274-4514.
Fax: 732-274-4505. E-mail: caggiat@wyeth.com.
§ Chemical and Screening Sciences, Wyeth Research, NJ.
# Princeton University.
‡ Chemical and Screening Sciences, Wyeth Research, PA.
† Cardiovascular/Metabolic Disease Research, Wyeth Research, PA.
| Women’s Health Research Institute, Wyeth Research, PA.
Cardiovascular/Metabolic Disease Research, Wyeth Research, MA.
10.1021/jm701013k CCC: $37.00 © 2007 American Chemical Society
Published on Web 09/29/2007

5246 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 22
Letters
Table 1. Carboxylate SAR of NFκB and CK Expression in
Table 2. Aryl Substitution SAR of NF-κB and CK Expression in
Ad5-wt-ER-Transfected HAECT-1 Cellsa
Ad5-wt-ER-Transfected HAECT-1 Cellsa
NF-κB-luc
CK
NF-κB-luc
CK
compd
R
IC50 ( SE (nM) % E2 IC50 ( SE (nM) % E2
IC50 ( SE
EC50
% E2 (nM) % E2
compd
R
Ar1
(nM)
1
2
3
Oet
OH
NH2
550 ( 122
IA
96
55
IA
IA
IA
4
5
6
7
8
Me 1-naph
Me 2-NO2Ph
H
206 ( 91
268 ( 41
139 ( 25
105 ( 22
389 ( 2.5
IA
550 ( 133
792 ( 19
19% @ 1 µM
3% @ 1 µM
110 ( 0.5
96
112
90
110
76
IA
IA
233
98
IA
1900 ( 37.2
2-OMePh
32
33
a All compounds were ER-dependent. % E2 ) efficacy (relative inhibition
of test compound at 10 µM vs E2 at 0.1 nM). IA ) inactive.
Me 2-OMePh
H
H
H
H
H
H
H
H
H
2-naph
3-OMePh
4-MePh
Ph
2-F-Ph
4-quin
2-CF3Ph
9
IA
Scheme 1. Synthesis of Propanoatesa
10
11
12
13
14
15
16
17
64
96
32
4
IA
IA
NT
NT
240
IA
106
24
2,4-OMe2Ph IA
2-SMePh 118 ( 31
327 ( 17
102
65
197
IA
24
-
Me 2-SMePh
a All compounds were ER-dependent. % E2 ) efficacy (relative inhibition
of test compound at 10 µM vs E2 at 0.1 nM). IA ) inactive.
Table 3. Ester and R Substitution SAR of NF-κB and CK Expression
in Ad5-wt-ER-Transfected HAECT-1 Cellsa
a Reagents and conditions: (a) piperazine, toluene, reflux; (b) (i) aryl
Grignard in THF, room temp, (ii) HCl; (c) (i) KHMDS in THF, (ii) MeI in
THF.
(our selectivity filter). In a typical assay, cells were treated for
16-18 h with IL-1â and the test compound; transcription was
directly proportional to the amount of luciferase transcribed.
Endogenous creatine kinase (CK) activity in the cells was used
as a measure of classical estrogen activity in vitro.
NF-κB-luc
CK
compd
R
R1
IC50 ( SE (nM) % E2 EC50 (nM) % E2
6
7
18
Et
Et
H
139 ( 25
105 ( 22
119 ( 42
81
110
94
IA
98
38
The target profile was a compound with IC50 < 100 nM (vs
NF-κB or IL-6) with an efficacy similar to that of ethinyl
estradiol (EE, =100%) and lacking CK activity in cells
transfected with the hER in vitro. Our primary in vivo assay
assessed classic estrogen-like activity (i.e., undesireable side
effects) by measured changes in the uterine wet weight in
sexually immature female mice after 5 days of oral dosing. As
a measure of oral activity, the target compound was assessed
for down-regulation of four proinflammatory genes (MHC,
invariant chain (MHI), VCAM-1, RANTES, and TNF-R)
induced by feeding C57BL/6 mice a high-fat diet for 5 weeks.
The results were measured against the effect of EE. Further in
vivo characterization would be performed using preclinical
models of inflammatory diseases such as collagen induced
arthritis (CIA), adjuvant induced arthritis (AIA), and inflam-
matory bowel disease models (HLA-B27 transgenic rat model).
High-throughput screening identified 2-cyano-3,3-dinaphtha-
len-1-ylpropionic acid ethyl ester (1)11 as an inhibitor of NF-
κB transcription. Compound 1 showed IC50 ) 550 nM at 96%
efficacy (vs NF-κB), IC50 ) 185 nM at 91% efficacy (vs IL-6),
and no CK activity. The activity was ER-dependent. The related
carboxylic acid (2) and primary amide (3) were less active
(Table 1). The synthesis of this series, based on a Knoevenagel
condensation and Michael addition sequence, was efficient and
allowed rapid development of an SAR (Scheme 1).
Me
28
24
tBu Me
a All compounds were ER-dependent. % E2 ) efficacy (relative inhibition
of test compound at 10 µM vs E2 at 0.1 nM). IA ) inactive.
was isolated. Subsequent Michael addition of an aryl Grignard
gave the bisaryl propionate in high yield. Replacement of one
naphthalene with substituted aryl or heteroaryl rings improved
the activity; however, this modification also gave rise to mixtures
of diastereoisomers that were configurationally labile (note:
initial studies were performed on the mixture of diastereoiso-
mers). The introduction of a methyl group at C-2 improved
activity in some instances (e.g., 4 f 5) and removed the
configurational lability (Table 2).
Interestingly, the alkylation of many of the diaryl cyano-
acetates was highly disastereoselective, providing only a single
diastereomer (e.g., RR/SS in the case of 7). While replacement
of ethyl ester 7 with tert-butyl ester 18 resulted in an improve-
ment in the stability in mouse plasma (32% hydrolysis in 4 h
at 37 °C vs O% in 24 h at 37 °C), the in vitro profile was
essentially unchanged (Table 3).
HPLC resolution of ester 18 showed the activity to reside in
a single enantiomer. X-ray crystallographic analysis of an
ephedrine carboxylate salt derived from 18 showed the absolute
configuration of the eutomer of 18 to be SS (IC50 ) 21 ( 12
nM at 81% efficacy vs NF-κB with no CK activity). An efficient
resolution of the carboxylic acid core of 18 (SS-2-cyano-3-(2-
Condensation of a cyanoacetate ester with an arylaldehyde
in the presence of a base (e.g., piperidine) provided an excellent
yield of the cinnamate ester; in many cases a single olefin isomer

Letters
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 22 5247
Table 4. Effect of Amide Substituent on NF-κB and CK Expression in
Ad5-wt-ER-Transfected HAECT-1 Cellsa
NF-κB-luc
IC50 ( SE
(nM)
RR/SS 125 ( 38
CK
EC50
% E2 (nM) % E2
Figure 1. Mean clinical joint scores by day in animals receiving oral
daily treatment with T/MC vehicle or 21 (0.3 mg/kg) in the Lewis rat
AIA model.
compd
R2
config
19
20
piperidine
1-(3-Cl-2-CH3Ph)- SS
piperazine
92
99
259
84
37
41
23 ( 7.4
21
4-(3-CF3Ph)-
piperidinetht
SS
122 ( 30
94
IA
a All compounds were ER-dependenrt (activity seen only when hER is
coexpressed with NF-κB-luciferase in HAECT-1 cells). IA ) inactive.
Table 5. Effect of 17R-Ethinyl 17â-Estradiol (EE) (0.01 (mg/kg)/day)
and Propanamides on NF-κB Mediated Gene Expression in C57BL/6
Mice on a High-Fat Diet
% inhib (% EE)
uterine wet wt,
compd
RANTES
VCAM
MHC
TNF-R
fold increase
20
21
EE
35 (45)
50 (65)
58
24 (47)
48 (94)
58
20 (41)
42 (86)
64
58 (82)
59 (83)
60
1.1
1
4
Figure 2. Mean stool scores by day in HLA-B27 transgenic rats
receiving oral daily treatment with T/MC vehicle or 21 (0.15 mg/kg)
in T/MC vehicle.
methoxyphenyl)-2-methyl-3-naphthalen-1-ylpropionic acid) as
its quinidine salt was developed to facilitate further SAR.12
To improve the pharmaceutical properties of the series, we
looked at several propanamides13 (Table 4). These were prepared
by activation of the carboxylic acid as the acid chloride followed
by condensation with the amine. This amidation not only
improved the pharmaceutical properties of the series but also
significantly improved the in vitro and in vivo profiles.
Compounds 2014 and 21 (WAY-204688, SIM 688)15 were
evaluated in vivo for the ability to inhibit four proinflammatory
genes (MHC, invariant chain (MHI), VCAM-1, RANTES, and
TNF-R). The effect of the test compound on induction of the
gene products and on uterine wet weight was compared to that
of 17R-ethinyl 17â-estradiol (EE at 10 (µg/kg)/day) in the same
paradigm. The results for 20 and 21 at a dose of 5 (mg/kg)/day
are shown in Table 5 (compound dosed po daily for 5 weeks;
for significant inhibition, p < 0.05).
Further characterization of 21 was carried out in several
preclinical models of inflammatory disease. In the Lewis rat
adjuvant-induced arthritis model (AIA),16 21 was active at a
dose of 0.3 (mg/kg)/day, po (Figure 1). The histology scoring
from this experiment is shown in Table 6. 21 demonstrated
approximately 50% improvement in synovial scoring.
In the HLA-B27 transgenic rat model of inflammatory bowel
disease,17 21 reversed the clinical and histologic scores at a dose
of 0.15 (mg/kg)/day (Figure 2). When administered to sexually
immature Sprague Dawley rats, at a dose of 2 (mg/kg)/day, 21
did not stimulate uterine wet weights and did not antagonize
EE’s (0.06 µg/day) 4-fold increase in uterine wet of weights.
Interestingly, it has been shown that NF-κB transcription can
be regulated by an inhibitor that interacts with the hERR and
hERâ receptors.18,19 The interaction of 21 with ERR and ERâ
was examined in vitro. Compound 21 displaces [3H]E2 from
the ERR ligand binding domain protein (LBD) with IC50 ) 2.43
µM and from the ERâ LBD with IC50 ) 1.5 µM. In vivo, 21
inhibits NF-κB induced transcriptional activation in an ERR
selective fashion and shows less activity in the presence of ERâ.
The difference between the binding results and the transcrip-
tional activation results may simply reflect the differences
between the conformation of the E2 bound LBDs vs the
unoccupied ERR receptors or that transcriptional activation
requires 21 to make unique contacts with ERR that E2 does
not make.
In summary, HTS identified 2-cyano-3,3-dinaphthalen-1-
ylpropionic acid ethyl ester 1 as an estrogen receptor dependent
inhibitor of NF-κB transcriptional activity. SAR studies resulted
in the improvement of the series profile. HPLC resolution and
X-ray crystallography showed the absolute stereochemistry of
the eutomer to be SS. Further elaboration of the structure resulted
in the synthesis of 21, an ERR ligand that is active in several
preclinical models of inflammation. 21 is an ERR selective,
orally active inhibitor of NF-κB transcriptional activity, which
may have use in the treatment of inflammatory diseases such
as rheumatoid arthritis.
Acknowledgment. The authors thank Amedeo Failli, Boyd
Harrison, Robert Steffan, and Rick Winneker for helpful
discussions. The authors thank Heather Harris for measuring
rat uterine wet weights, Discovery Analytical Chemistry for
Table 6. Histological Scoring of Synovitis in the Tarsal Joints from Animals with Adjuvant-Induced Arthritis Treated for 10 Days with Once Daily
Oral 21 (0.3 (mg/kg)/day) Beginning on Day 8 after CFA Injection (Mean ( SD)
group
synovial structure (0-3)
fibroplasia (0-3)
inflammatory cells (0-3)
pannus (0-2)
total synovitis score (0-11)
T/MC vehicle
21
2.92 ( 0.20
1.92 ( 0.74a
2.67 ( 0.26
1.08 ( 0.86a
2.92 ( 0.20a,b
2.00 ( 0.00
0.33 ( 0.52a
10.5 ( 0.55
4.17 ( 2.30a
0.83 ( 0.52a
a Total score is the summation of four individuals. b Signal is less than vehicle signal. For significant inhibition, p < 0.05.

5248 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 22
Letters
(10) Keith, J. C.; Albert, L. M.; Leathurby, Y.; Follettie, M.; Wang, L.;
Borges-Marcucci, L.; Chadwick, C. C.; Steffan, R. J.; Harnish, D.
C. The utility of pathway selective estrogen receptor ligands that
inhibit nuclear factor-κB transcriptional activity in models of
rheumatoid arthritis. Arthritis Res. Ther. 2005, 7, R427-R438.
(11) Latif, N.; Mishriky, N.; El-Bayouki, K. Naphthalenes. Naphthyl-
cyanoalkanoates, -cyano alcohols and -malonamic acids of anticipated
broad spectrum biological activity. Egypt. J. Chem. 1976, 17, 879-
887.
(12) Caggiano, T. J.; Brazzale, A. A.; Moore, W. J.; Kim, C. Y.
Preparation of 2-cyanopropanoic acid amide and ester derivatives as
estrogen receptor binding agents for use as antiinflammatory and
immunomodulatory agents. WO 2004099150, 2004; Chem. Abstr.
2004, 141, 423980.
(13) A discussion of the synthesis and SAR of this series is beyond the
scope of this Letter and will be the subject of a separate report.
(14) (2S)-3-[4-(3-Chloro-2-methylphenyl)piperazin-1-yl]-2-[(S)-(2-meth-
oxyphenyl)(1- naphthyl)methyl]-2-methyl-3-oxopropanenitrile.
(15) (2S,3S)-3-(2-Methoxyphenyl)-2-methyl-3-(1-naphthyl)-2-({4-[3-(tri-
fluoromethyl)phenyl]piperidin-1-yl}carbonyl)propanenitrile.
(16) AIA model: 12 week old ovariectomized female Lewis rats were
injected with Freund’s adjuvant. After 8 days, inflammation is
established and the animals are dosed for 16 days with the test
compound. The hindpaw joint is monitored and scored for swelling
and erythema (1-3 scale; max ) 12). Tissue is collected for histology
scoring (synovial hyperplasia, inflammatory cell infiltration, pannus
formation, and articular cartilage destruction).
(17) Harnish, D. C.; Albert, L. M.; Leathurby, Y.; Eckert, A. M.; Ciarletta,
Kasaian, A.; Keith, J. C. Beneficial effects of estrogen treatment in
the HLA-B27 transgenic rat model of inflammatory bowel disease.
Am. J. Physiol. 2004, 286, G118-G125.
(18) Steffan, R. J.; Matelan, E.; Ashwell, M. A.; Moore, W. J.; Solvibile,
W. R.; Trybulski, E.; Chadwick, C. C.; Chippari, S.; Kenney, T.;
Eckert, A.; Borges-Marcucci, L.; Keith, J. C.; Xu, Z.; Mosyak, L.;
Harnish, D. C. Synthesis and activity of substituted 4-(indazol-3-
yl)phenols as pathway-selective estrogen receptor ligands useful in
the treatment of rheumatoid arthritis. J. Med Chem. 2004, 47, 6435-
6438.
(19) Chadwick, C. C.; Chippari, S.; Matelan, E.; Borges-Marcucci, L.;
Eckert, A. M.; Keith, J. C., Jr.; Albert, L. M.; Leathurby, Y.; Harris,
H. A.; Bhat, R. A.; Ashwell, M.; Trybulski, E.; Winneker, R. C.;
Adelman, S. J.; Steffan, R. J.; Harnish, D. C. Identification of
pathway-selective estrogen receptor ligands that inhibit NF-κB
transcriptional activity. Proc. Nat. Acad. Sci. U.S.A. 2005, 102,
2543-2548.
spectral data, and Mark Ashwell for the evaluation and
prioritization of HTS data.
Supporting Information Available: Experimental procedures
and spectral data. This material is available free of charge via the
Internet at http://pubs.acs.org.
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