Fluoro analogs of bioactive oxy-sterols: Synthesis of an EBI2 agonist with enhanced metabolic stability

Article abstract of DOI:10.1016/j.bmcl.2016.09.029

Synthesis of several 7-hydroxy oxysterols and their potential roles as signaling molecules in the innate and adaptive immune responses is discussed. Discovery of a new, fluorinated, synthetic analog of the 7α,25-dihydroxycholesterol—the endogenous ligand of GPR 183 (EBI2), a G-protein coupled receptor highly expressed upon Epstein–Barr virus infection is described. Fluoro oxysterol 12 showed good metabolic stability while maintaining excellent EBI2 agonist activity.

Full text of DOI:10.1016/j.bmcl.2016.09.029

Bioorganic & Medicinal Chemistry Letters 26 (2016) 4888–4891
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Fluoro analogs of bioactive oxy-sterols: Synthesis of an EBI2 agonist
with enhanced metabolic stability
⇑
Xiaohu Deng, Siquan Sun, Jiejun Wu, Chester Kuei, Victory Joseph, Changlu Liu, Neelakandha S. Mani
Janssen Research & Development, LLC, 3210 Merryfield Row, San Diego, CA 92121, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 7 July 2016
Revised 8 September 2016
Accepted 10 September 2016
Available online 12 September 2016
Synthesis of several 7-hydroxy oxysterols and their potential roles as signaling molecules in the innate
and adaptive immune responses is discussed. Discovery of a new, fluorinated, synthetic analog of the
7a,25-dihydroxycholesterol—the endogenous ligand of GPR 183 (EBI2), a G-protein coupled receptor
highly expressed upon Epstein–Barr virus infection is described. Fluoro oxysterol 12 showed good meta-
bolic stability while maintaining excellent EBI2 agonist activity.
Ó 2016 Elsevier Ltd. All rights reserved.
Keywords:
Oxysterols
Fluoro oxysterol
Immune response
Epstein–Barr virus
GPR 183
EBI2 agonist
Oxysterols are oxygenated derivatives of cholesterol produced
by the oxidation by the CYP family of enzymes in the cells and falls
in the bile acid synthesis or steroid synthesis pathway.1 Few oxys-
terols such as 7-hydroxy and 7-keto are also known to be produced
in vivo by non-enzymatic radical oxidation mechanism.2 Although
found in very low concentrations in most mammalian tissues,
oxysterols play a crucial role in cholesterol and fatty acid metabo-
lism, regulation of immune response and are also believed to be
mediators in neurodegenerative disorders.3
While oxysterols were most extensively studied for its potent
ability to mediate feedback regulation of cholesterol biosynthesis,
some early investigations showed that the enzymes involved in
its syntheses were highly upregulated in macrophages and den-
dritic cells—an indication of possible roles in immune related func-
tions.3,4 Subsequent research has shown that oxysterols have a
broad range of roles in innate and adaptive immune responses.
For example, 25-hydroxycholesterol (25-HC) is induced in macro-
phages by type 1 interferon (IFN) signaling and has broad ability
to prevent viral entry, replication and budding.3,5 In addition to
the pro-inflammatory effects, many studies show that, 25-HC also
mediate the anti-inflammatory effects downstream of the IFN
pathway.3
receptors (LXRs).6 Recent work from our labs as well as from others
has shown additional receptors linking oxysterols and immunity.7
For example, we found that 7a,25-dihydroxycholesterol as a key
molecule involved in directing the migration of naive B cells, T cells
and dendritic cells by engaging an orphan GPCR, GPR183, a gene
which was initially found to be highly induced by Epstein–Barr
virus infection of B cells. In another study, an oxysterol was found
to be the most potent endogenous ligand of RORct, an orphan
nuclear receptor whose activation is the key step in the down-
stream production of IL-17—a key inflammatory pathway in sev-
eral autoimmune disorders.8 Thus, many new biological functions
of oxysterols, especially their broad range of roles as signaling
molecules in the innate and adaptive immune system continues
to be discovered and hold the promise of finding new targets for
the therapeutic intervention for autoimmune disorders such as
psoriasis, RA, IBD, and MS.9,10
Our interest in oxysterols as signaling molecules and in partic-
ular their role in innate and adaptive immune response mecha-
nisms stems to a large extend from our long standing focus in
understanding the pathogenesis of autoimmune diseases. One of
the major difficulties in the study of oxysterols is their poor
in vivo metabolic stability. As a result, we became interested in
synthetic oxysterol derivatives as tool molecules that are metabol-
ically more stable yet can maintain signaling competency compa-
rable to the endogenous ligands. In this Letter we report the first
Immunomodulation effects of certain oxysterols were previ-
ously shown to depend on activation of oxysterol-binding liver X
example of
a trifluoromethyl substituted derivative of 7a25
⇑
Corresponding author.
E-mail address: nmani@its.jnj.com (N.S. Mani).
dihydroxycholesterol that is almost equipotent to natural product
http://dx.doi.org/10.1016/j.bmcl.2016.09.029
0960-894X/Ó 2016 Elsevier Ltd. All rights reserved.

X. Deng et al. / Bioorg. Med. Chem. Lett. 26 (2016) 4888–4891
4889
yet with enhanced metabolic stability suitable for in vivo
experiments.
OH
Our initial foray into oxysterols began with our efforts in estab-
lishing the identity of the endogenous ligand of GPR183. GPR183
also known as EBI2 belongs to the family of G-protein-coupled
receptors and is expressed in spleen and other secondary lymphoid
organs and is highly up-regulated upon infection by Epstein–Bar-
virus (EBV). Gas chromatography–mass spectrometry analysis of
most active tissue extracts showed it possibly contained dihydrox-
OH
HO
OH
HO
OH
9
8
Testing the synthesized oxysterols for activation of EBI2 (based
on the stimulation of 35S-GTP-
S incorporation in EBI2-expressing
membranes) showed that 7 ,25-OHC is the most potent ligand (5,
EC50 = 140 pM) while 7b,25-OHC (7, EC50 = 2.1 nM), 7 ,27-OHC (8,
c
ycholesterol derivatives, most likely 7
terol (7 /7b, 25-OHC) and/or /7b, 27-dihydroxycholesterol
(7 /7b,27-OHC). To establish identity of this ligand conclusively,
we synthesized 7 /7b,25-OHC and 7 /7b,27-OHC in stereochemi-
a
/7b,25-dihydroxycholes-
a
a
7
a
a
a
EC50 = 1.3 nM) and 7b,27-OHC (9, EC50 = 51 nM) were less potent in
a
a
activating EBI2.
cally pure forms starting from commercially available 25-hydroxy-
cholesterol and 27-hydroxycholesterol respectively.7,8 Thus, as
outlined in Scheme 1, Manganese(III) catalyzed allylic oxidation
of 3-acetoxy-25-hydroxycholestrol (2) furnished the 7-keto deriva-
7-Hydroxylated sterols especially 7
is metabolically unstable. When testing in vitro in mouse, rat or
human liver microsomal stability assay, the half-life of 7 ,25-dihy-
droxycholesterol was 11, 15 and 30 min respectively. We have
observed that the allylic hydroxyl group at the 7-position is also
chemically unstable—prone to isomerization and degradation
under strongly acidic or oxidizing conditions. After administration
a,25-dihydroxycholesterol
a
tive.11 Reduction of ketone 3 using L-selectride13 furnished the 7
a-
epimer 4 in excellent stereoselectivity (95%). The corresponding
7b-epimer 6 was obtained by Luche reduction14 (CeCl3–NaBH4)
as exclusive product. Hydrolysis of the benzoyl groups by aqueous
in mice via subcutaneous or intraperitoneal injections, 7a,25-dihy-
NaOH followed by chromatographic purification furnished both
a,
droxycholesterol was quickly eliminated from blood with the half-
and b-epimers (5 and 7) in excellent overall yields and isomeric
purity.
life of ꢀ30 min (Fig. 1).
Thus, in our efforts to develop suitable EBI2 agonist molecules
with enhanced metabolic stability, we directed our focus to
synthetic analogs in which this allylic hydroxyl group is stabilized
Both 7
a (8) and 7b (9)-27-dihydroxycholesterols were synthe-
sized following reported methods.13,8
OBz
OH
OBz
CH2Cl2, 0 oC- RT
Mn3O(OAc)9, tBuO2H
Mol Sieves (3A)
EtOAc, RT, 48 h
90%
PhCOCl/Pyridine
79%
BzO
HO
BzO
O
2
1
3
OH
OH
L-Selectride
OBz
OBz
NaOH
THF-MeOH, RT, 8h
3
THF, -78 o
C
76%
81%
HO
HO
OH
OH
BzO
OH
5
4
NaBH4-CeCl3
NaOH
THF-MeOH, RT, 8h
91%
3
THF-MeOH
RT, 1 h
BzO
OH
6
86%
7
Scheme 1. Synthesis of 7a/7b,25-dihydroxycholesterols.
IP, 20mg/kg
SC, 20mg/kg
Mouse PK of 7α, 25-OHC, IP or SC (20 mg/kg)
10000
IP
SC
1000
100
10
0
1
2
3
4
5
Time (hr)
Figure 1. Mouse PK of 7a,25-dihydroxycholesterol.

4890
X. Deng et al. / Bioorg. Med. Chem. Lett. 26 (2016) 4888–4891
by introducing substituents. We speculated that by introducing a
suitable alkyl substituent at the 7-position, forming thus a more
stable tertiary allylic hydroxyl group would render the oxysterol
stable to oxidation and metabolic degradation. We thus chose a tri-
fluoromethyl group as it would be more resistant to dehydrative
elimination and subsequent isomerizations. We envisaged the syn-
thesis of the desired trifluoromethyl derivative by direct addition
of trifluoromethyl group to the 7-keto derivative 10, accomplished
usually by in situ activation of the Rupert—Prakash reagent, Me3-
SiCF3, with catalytic fluoride source.15 Addition of small nucle-
ophiles such as MeMgBr to C-7 ketone in the androst-5-en-7-
ones series has been reported to takes place from the b equatorial
face leading to the 7
lated that the addition of trifluoromethyl anion to ketone 10 would
a
alcohol.12 Based on these reports we specu-
be from the b equatorial face to yield the desired 7a-hydroxy
isomer.
For the addition of CF3 group to ketone we followed the proce-
dure developed by Shreeve16 using Me3SiCF3 and catalytic CsF.
OH
CF3
HO
OH
OH
OH
12, 36%
TMSCF3, CsF
NaOH
THF-H2O, RT, 6 h
CF3
OH
Mol Sieves, 3A
AcO
O
AcO
THF, RT, 48 h
OH
11
10
50%
CF3
OH
HO
13, 33%
Scheme 2. Synthesis of 7-trifluoromethyl analogs.
Table 1
The EC50 and Emax values of EBI2 agonists to human, rat and mouse receptors
7
a,25-OHC
13
12
EC50 (nM)
% Emax
EC50 (nM)
% Emax
EC50 (nM)
% Emax
Human EBI2
Rat EBI2
Mouse EBI2
0.032
1.31
0.702
100
100
100
4.1
14.4
14.4
100
88
60
0.102
1.08
1.64
122
116
136
Rat EBI2/GO2
GTPγS Assay
Human EBI2/GO2
GTPγS Assay
120
140
120
100
80
7α,25-DHC
13
12
7α,25-DHC
13
12
100
80
60
40
20
0
60
40
20
0
-13 -12 -11 -10 -9
-8
-7
-6
-5
-13 -12 -11 -10 -9
-8
-7
-6
-5
Log [Compound] (M)
Log [Compound] (M)
(a)
(b)
(b)
Mouse EBI2/GO2
GTPγS Assay
200
180
160
140
120
100
80
7α,25-DHC
13
12
60
40
20
0
-13 -12 -11 -10 -9
-8
-7
-6
-5
Log [Compound] (M)
Figure 2. Agonist potencies of 12, 13, and 7
G-protein. Activation of EBI2 leads to activation of GO2 protein, which then binds GTP. The agonist induced receptor activation and GTP binding to GO2 protein were
measured by GTP
S assay as detailed by Liu et al.6
a,25-OHC for human, rat, and mouse EBI2. EBI2 signals through Gi/Go G-proteins. In our studies, we co-expressed EBI2 with GO2
c

X. Deng et al. / Bioorg. Med. Chem. Lett. 26 (2016) 4888–4891
4891
Figure 3. Structure with atom numbering of compound 12 and molecular drawing with the refined configuration.18
While very good conversion to the trifluoromethyl alcohol was
Acknowledgment
observed, the reaction was completely non-stereoselective yield-
ing a nearly1:1 mixture of both epimers (Scheme 2). After hydrol-
ysis of the acetate and chromatographic separation of the epimers,
one of the epimers (12) was found crystalline and thus readily
obtained in isomerically pure form by crystallization of an enriched
mixture from ethyl acetate. The other isomer (13) was also
obtained in pure form by subsequent silica gel column chromatog-
raphy of the mother liquor.
We thank Mrs. Heather McAllister for analytical support.
Supplementary data
Experimental procedures, proton and carbon NMR spectra of all
compounds synthesized; X-ray crystallography data for compound
12; materials and methods for all biological experiments. This
material is available free of charge via the Internet. Supplementary
data associated with this article can be found, in the online version,
at http://dx.doi.org/10.1016/j.bmcl.2016.09.029.
Agonist potencies of 7
a,25-OHC and both isoforms (12 and 13)
of 7-trifluoromethyl-7 ,25-OHC for human, rat and mouse EBI2
a
were determined using [35S]-GTP
cS binding assay in COS7 cells
transiently transfected with human, rat or mouse EBI2.17 Com-
pounds were screened in a concentration response with the high-
References and notes
est concentration at 1
to 7 ,25-OHC. As shown in Table 1, the crystalline isomer (12)
was the most potent. Based on the stereochemistry of the endoge-
nous ligand 7 ,25-OHC (5) we tentatively assigned compound 12
-stereochemistry (Fig. 2).
When testing in vitro in mouse, rat and human liver microsomal
stability assay, the half-life of CF3-7 ,25-dihydroxycholesterol was
significantly increased to >180 min, 31 min, and 131 min respec-
tively. The pharmacokinetic property of CF3-7 ,25-dihydroxyc-
holesterol was also significantly improved over ,25-
dihydroxycholesterol. After administration in mice via PO gavage,
CF3-7 ,25-dihydroxycholesterol showed half-life of elimination
of ꢀ10 h.
Our initial assignment of stereochemistry for the active isomer
lM. %Emax was determined by normalizing
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In summary, we have synthesized 7-trifluoromethyl-7
dihydroxycholesterol 12—a new fluorinated, synthetic analog of
,25-dihydroxycholesterol that demonstrated good metabolic
a,25-
7a
stability while maintaining excellent EBI2 agonist activity. We
have also demonstrated that the synthesis of can be accomplished
by nucleophilic trifluoromethylation of the corresponding 7-keto
derivative in very good yields. This compound should prove valu-
able as a tool compound in the study of EBI2 function in vitro
and in vivo. Due to its favorable pharmacokinetic properties, this
compound may be useful to explore whether such stable EBI2 ago-
nists shows any therapeutic benefits in various animal models on
immune, cardiac, neurological, and metabolic disorders.