Novel tricyclic glycal-based TRIB1 inducers that reprogram LDL metabolism in hepatic cells

Article abstract of DOI:10.1039/C8MD00297E

Increased expression of the Tribbles pseudokinase 1 gene (TRIB1) is associated with lower plasma levels of LDL cholesterol and triglycerides, higher levels of HDL cholesterol and decreased risk of coronary artery disease and myocardial infarction. We identified a class of tricyclic glycal core-based compounds that upregulate TRIB1 expression in human HepG2 cells and phenocopy the effects of genetic TRIB1 overexpression as they inhibit expression of triglyceride synthesis genes and ApoB secretion in cells. In addition to predicted effects related to downregulation of VLDL assembly and secretion these compounds also have unexpected effects as they upregulate expression of LDLR and stimulate LDL uptake. This activity profile is unique and favorably differs from profiles produced by statins or other lipoprotein targeting therapies. BRD8518, the initial lead compound from the tricyclic glycal class, exhibited stereochemically dependent activity and the potency far exceeding previously described benzofuran BRD0418. Gene expression profiling of cells treated with BRD8518 demonstrated the anticipated changes in lipid metabolic genes and revealed a broad stimulation of early response genes. Consistently, we found that BRD8518 activity is MEK1/2 dependent and the treatment of HepG2 cells with BRD8518 stimulates ERK1/2 phosphorylation. In agreement with down-regulation of genes controlling triglyceride synthesis and assembly of lipoprotein particles, the mass spectrometry analysis of cell extracts showed reduced rate of incorporation of stable isotope labeled glycerol into triglycerides in BRD8518 treated cells. Furthermore, we describe medicinal chemistry efforts that led to identification of BRD8518 analogs with enhanced potency and pharmacokinetic properties suitable for in vivo studies.

Full text of DOI:10.1039/C8MD00297E

View Article Online
View Journal
MedChemComm
Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: M. M. Nagiec, J.
Duvall, A. Skepner, E. Howe, J. Bastien, E. Comer, J. Marie, S. Johnston, J. Negri, M. Eichhorn, J.
Vantourout, C. Clish, K. Musunuru, M. Foley, J. Perez and M. Palmer, Med. Chem. Commun., 2018, DOI:
10.1039/C8MD00297E.
This is an Accepted Manuscript, which has been through the
Volume 7 Number 1 January 2016 Pages 1–204
Royal Society of Chemistry peer review process and has been
accepted for publication.
MedChemComm
Accepted Manuscripts are published online shortly after
acceptance, before technical editing, formatting and proof reading.
Broadening the field of opportunity for medicinal chemists
www.rsc.org/medchemcomm
Using this free service, authors can make their results available
to the community, in citable form, before we publish the edited
article. We will replace this Accepted Manuscript with the edited
and formatted Advance Article as soon as it is available.
You can find more information about Accepted Manuscripts in the
author guidelines.
Please note that technical editing may introduce minor changes
to the text and/or graphics, which may alter content. The journal’s
Themed issue: Antibiotic Resistance
ISSN 2040-2503
standard Terms & Conditions and the ethical guidelines, outlined
in our author and reviewer resource centre, still apply. In no
event shall the Royal Society of Chemistry be held responsible
for any errors or omissions in this Accepted Manuscript or any
consequences arising from the use of any information it contains.
rsc.li/medchemcomm

Please do not adjust margins
MedChemComm
Page 1 of 12
View Article Online
DOI: 10.1039/C8MD00297E
MedChemComm
ARTICLE
Novel Tricyclic Glycal-based TRIB1 Inducers that Reprogram LDL
Metabolism in Hepatic Cells
a
a
a
a
a
Marek M. Nagiec,* Jeremy R. Duvall , Adam P. Skepner , Eleanor A. Howe , Jessica Bastien , Eamon
Received 00th January 20xx,
Accepted 00th January 20xx
a
a
a
a
a
Comer , Jean-Charles Marie , Stephen E. Johnston , Joseph Negri , Michelle Eichhorn , Julien
a
b
c
a
a
a
Vantourout , Clary Clish , Kiran Musunuru , Michael Foley , Jose R. Perez , and Michelle A.J. Palmer
DOI: 10.1039/x0xx00000x
www.rsc.org/
Increased expression of the Tribbles pseudkinase 1 gene (TRIB1) is associated with lower plasma levels of LDL cholesterol
and triglycerides, higher levels of HDL cholesterol and decreased risk of coronary artery disease and myocardial infarction.
We identified a class of tricyclic glycal core-based compounds that upregulate TRIB1 expression in human HepG2 cells and
phenocopy the effects of genetic TRIB1 overexpression as they inhibit expression of triglyceride synthesis genes and ApoB
secretion in cells. In addition to predicted effects related to downregulation of VLDL assembly and secretion these
compounds also have unexpected effects as they upregulate expression of LDLR and stimulate LDL uptake. This activity
profile is unique and favorably differs from profiles produced by statins or other lipoprotein targeting therapies. BRD8518,
the initial lead compound from the tricylclic glycal class, exhibited stereochemically dependent activity and the potency far
exceeding previously described benzofuran BRD0418. Gene expression profiling of cells treated with BRD8518
demonstrated the anticipated changes in lipid metabolic genes and revealed a broad stimulation of early response genes.
Consistently, we found that BRD8518 activity is MEK1/2 dependent and the treatment of HepG2 cells with BRD8518
stimulates ERK1/2 phosphorylation. In agreement with down-regulation of genes controlling triglyceride synthesis and
assembly of lipoprotein particles, the mass spectrometry analysis of cell extracts showed reduced rate of incorporation of
stable isotope labeled glycerol into triglycerides in BRD8518 treated cells. Furthermore, we describe medicinal chemistry
efforts that led to identification of BRD8518 analogs with enhanced potency and pharmacokinetic properties suitable for in
vivo studies.
an adaptor protein in multiple pathways, including the MAPK cascade.
Over-expression of TRIB1 in the liver results in suppression of hepatic
secretion of very-low-density lipoprotein (VLDL) particles, the precursor
of LDL particles, improved plasma lipoprotein profile, and reduction in
Introduction
expression of genes encoding proteins involved in de novo lipogenesis
(7). Beyond this, TRIB1 has recently been implicated in macrophage
differentiation and diabetes (8). Using a cell-based screen of ~9K
compounds from a Diversity Oriented Synthesis (DOS) screening
collection (9, 10) , we identified two classes of lead compounds,
benzofurans and tricyclic glycals, which induce TRIB1 mRNA. In HepG2
cells, both classes of compounds exhibit time- and dose-dependent
effects on TRIB1 expression. In parallel, these compounds induce LDLR
mRNA and protein, increase LDL clearance and reduce PCSK9 mRNA
expression and PCSK9 levels in the media. The structure activity
relationship for induction of TRIB1 mirrors that for induction of LDLR
and suppression of PCSK9. Whereas the benzofuran series was
described previously (11), here we present a more detailed
characterization of the second, more potent series of tricyclic glycals
exemplified by BRD8518, which was undertaken in an effort to better
define its mechanism of action. In addition we present derivatives of
BRD8518 with further enhanced potency and an improved
pharmacokinetic profile, which were obtained through a targeted
medicinal chemistry effort.
Plasma lipoprotein and lipid profiles are heritable risk factors
associated with coronary artery disease. Genome-wide association
studies (GWAS) have identified ~100 loci that associate with blood lipid
concentrations (1). Amongst these, a single nucleotide polymorphism
(SNP) located at chromosomal locus 8q24 is significantly associated
with the levels of triglycerides, low-density lipoprotein cholesterol
(LDL-C) and high-density lipoprotein cholesterol (HDL-C). The minor G
allele at this SNP was associated with lower triglycerides (TG), lower
LDL, and higher HDL, a unique pattern for any lipid modifying SNP. The
SNP is located in the 3’-downstream region of the gene encoding
Tribbles pseudkinase 1 (TRIB1). More recent reports indicate that the
minor SNP allele, which is associated with reduced risk of CAD and MI,
increases the level of TRIB1 mRNA (2). The association of TRIB1 with
CAD has been further validated by the larger GWAS study conducted
by the CARDIoGRAMplusC4D Consortium (3) and replicated in
populations of various ethnic backgrounds (4-6). TRIB1 is a serine
threonine kinase-like protein, which lacks a catalytic domain. The
precise molecular function of TRIB1 is unknown but it is proposed to
act as
a.Therapeutics Platform, Broad Institute of MIT and Harvard, Cambridge,
MA02142. E-mail: marek@broadinstitute.org
Results and discussion
b.Metabolite Profiling Platform, Broad Institute of MIT and Harvard, Cambridge,
MA02142.
c.
Department of Stem Cell and Regenerative Biology, Harvard University,
Cambridge, MA 02138.
E-mail: marek@broadinstitute.org; Tel: +617-714-7377
The initial lead compound BRD8518 was developed following the
screen of the DOS-derived small-molecule library for compounds
This journal is © The Royal Society of Chemistry 20xx
Med. Chem. Commun., 2018, 00, 1-3 | 1
Please do not adjust margins

Please do not adjust margins
MedChemComm
Page 2 of 12
View Article Online
DOI: 10.1039/C8MD00297E
ARTICLE
MedChemComm
Figure 1. A qRT-PCR screen of HepG2 cells identified a series of TRIB1 inducers from tricyclic glycal library. (A) Stereochemical dependency of the
tricyclic glycal compounds revealed by the analysis of the primary screening data. Each block of eight fields represents eight stereoisomers tested in the
primary screen. (B) Dose dependent up-regulation of TRIB1 mRNA relative to GAPDH calibrator control. (C) Chemical structures of TRIB1 inducers.
that upon 20 hour incubation at 12 µM concentration upregulated
expression of TRIB1 in HepG2 cells (Figure 1). From the confirmed
hit compounds that (i) upregulated TRIB1 expression in two
hepatocellular cell lines, (ii) had no apparent cytotoxicity and (iii)
inhibited the secretion of the VLDL marker ApoB from HepG2
cells, a series of tricyclic glycal-based hits (10) stood out and
demonstrated stereochemically-dependent activity. BRD8518,
which was not present in the initial screening collection, was
synthesized by introducing the stereochemically preferred SSR
configuration to the most potent hit of the series, BRD3918. This
modification resulted in about 10-fold increase in potency over
BRD3918 as well as the previously described lead benzofuran
BRD0418 (11) (Figure 1B, C and Figure 2A). Alternatively, the SRR
configuration in BRD9697 resulted in a compound with no observed
activity at the highest concentration tested. From the initial ‘sparse
matrix’ (12) designed tricyclic glycal library (10), SAR was observed in
1
2
the HTS at both R and R appendage sites, visualized in figure 1A (13),
but the trifluormethyl phenyl amide and indane motifs were superior at
the respective positions. BRD8518 was characterized further and
showed improved potency in inhibiting ApoB secretion, indicating
inhibition of VLDL secretion, as predicted based on known effect of
TRIB1 overexpression on inhibition of lipogenesis
(7) (Figure 2B). Early in the hit triage process the TRIB1
upregulating compounds were profiled in the Luminex gene
expression assay to measure differential expression of 1000
selected landmark genes (L1000) (14). The L1000 data analysis
Figure 2. BRD8518 modulates expression of the key genes involved in VLDL production and in LDL clearance in HepG2 cells. Transcript levels were
measured by qRT-PCR in HepG2 cells 6 hours (TRIB1) and 24 hours (PCSK9) after treatment with a dilution series of BRD3918 and BRD8518 (A). The levels
of secreted ApoB and PCSK9 proteins were measured in the media by ELISA 24 hours post treatment (B, C). The level of LDL receptor protein was
measured using ELISA in the cell lysates obtained 24 hours after the treatment in indicated concentrations of compounds (C). The cellular levels of ATP
were not affected by BRD3918 and only slightly affected by highest concentrations of BRD8518 treatment as measured by the Cell-titer-Glo assay (D).
2 | Med. Chem. Commun., 2018, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Please do not adjust margins
Page 3 of 12
MedChemComm
View Article Online
DOI: 10.1039/C8MD00297E
MedChemComm
ARTICLE
13C3-D5-C50:2 TAG
2.5E+07
2.0E+07
1.5E+07
1.0E+07
DMSO
2 uM BRD8518
5.0E+06
0.0E+00
0
100
200
time [min]
13C3-D5-C48:2 TAG
8.0E+06
6.0E+06
AUC4.0E+06
2.0E+06
DMSO
2 uM BRD8518
0.0E+00
0
100
200
time [min]
Figure 4. TRIB1 inducers attenuate the rate of triglyceride synthesis in
HepG2 cells. HepG2 cells were incubated with 2 µM compound
BRD8518 for 24 hours and then incubated with the stable isotope
labeled glycerol (13C3-D5-glycerol) for varying amounts of time. The
incorporation of the 13C3-D5-glycerol into triglycerides was
quantified in the lipid extracts by high resolution mass spectroscopy.
The plots shows incorporation of 13C3-D5-glycerol into two of the
species of triacylglycerol as indicated.
Figure 3. TRIB1 inducers stimulate uptake of LDL in HepG2 cells. (A)
HepG2 cells grown under standard culture conditions (10% FBS)
were treated for 20 hours with compounds BRD-3918 and BRD8518
at indicated concentrations and the uptake of BODIPY-FL
conjugated LDL particles was monitored for 5 hours using high
content microscopy. To control for regulation of LDL uptake by
cholesterol the cells were grown in the media depleted for
cholesterol (0.5% LPDS) or supplemented with cholesterol (0.5%
LPDS plus cholesterol (10mg/ml cholesterol and 1mg/ml 25-OH
cholesterol). (B) Dose dependent effect of BRD8518 on LDL uptake.
development. The OSM treatment was included based on our
observation that OSM induced responses in HepG2 cells that
resembled responses to BRD8518 including the upregulation of
TRIB1 and LDLR expression and the downregulation of PCSK9
(Figure 5). The time course observed for BRD8518 induction of
TRIB1 and LDLR was different than that observed for OSM, which
produced peak of responses at 1 hour followed by decay to
revealed that BRD3918 and other tricyclic glycal hits affected
expression of cholesterol metabolic genes and in subsequent
studies we established that BRD3918 stimulated expression of LDL
receptor protein and inhibited secretion of PCSK9 protein from
HepG2 cells (data not shown). Here we found that BRD8518 was
also more potent than BRD3918 in downregulating secretion of
PCSK9 protein (Figure 2C), upregulating expression of LDL receptor
protein (Figure 2C), and, as a functional consequence, stimulating
LDL uptake in HepG2 cells (Figure 3). The effects of TRIB1
upregulating compounds in the LDL uptake pathway were
unexpected and, apparently, are not driven by upregulation of
TRIB1 expression as HepG2 cells transfected with TRIB1 expressing
plasmids have unchanged levels of PCSK9 and LDLR transcripts
(data not shown).
Since TRIB1 overexpression achieved by cDNA transfection was
reported to inhibit the rate of triglyceride synthesis in HepG2 cells
(7), we measured effects of BRD8518 on the rate of incorporation of
stable isotope-labeled glycerol into triglycerides. In HepG2 cells
13
preincubated with 2 µM BRD8518 for 24 hours, the rate of C3-D5-
glycerol incorporation into individual triglycerides was significantly
suppressed for all eight species of triglycerides that were monitored by
mass spectrometry in lipid extracts (Figure 4). To begin defining the
mechanism of action of BRD8518, we performed a complete gene-
expression profiling experiment in HepG2 cells treated for 6 and 24
hours with 1µM BRD8518 and we identified genes differentially
expressed in response to compound treatment compared to DMSO
vehicle treatment (GEO accession number: GSE57753). In addition, we
profiled cells treated for 1 and 24 hours with 100 ng/ml of oncostatin
M (OSM), a cytokine involved in liver
Figure 5. Kinetics of gene expression modulation over a 28 hour
time course after treatment with 2.5 µM BRD8518 or 100 ng/ml
oncostatin M.
This journal is © The Royal Society of Chemistry 20xx
Med. Chem. Commun., 2018, 00, 1-3 | 3
Please do not adjust margins

Please do not adjust margins
MedChemComm
Page 4 of 12
View Article Online
DOI: 10.1039/C8MD00297E
ARTICLE
MedChemComm
Color key
response to both treatments was also broad and it included
upregulation of 122 genes in response to OSM and of 180 genes in
response to BRD8518. Gene set enrichment analysis pointed to
significant upregulation of classic immediate early response genes
such as JUNB, EGR1, FOS, JUN, DUSP1, ZFP36, ATF3 and genes that
were described to be induced as part of immediate early response
in some cells like LDLR (15) (Figure 6). Transcript levels of SREBF1
and SREBF2, transcription factors controlling expression of
cholesterol pathway genes, remained unchanged by any treatment.
In many respects responses to BRD8518 treatment resembled
responses to OSM, a cytokine that was previously reported to
stimulate LDLR expression and to reduce LDL and TG levels in
hyperlipidemic animals (16). BRD8518 treatment produced
expression changes in a larger number of genes, particularly at the
later 24 hour time point, than OSM treatment suggesting that
BRD8518 is less specific and may perturb additional pathways.
Induction of early response genes suggested that upregulation of
TRIB1 and LDLR expression may depend on stimulation of MAP
kinase signaling pathways. To test this notion we evaluated effects
of two specific kinase inhibitors on ability of OSM and BRD8518 to
stimulate LDLR expression. CP-69550 (a JAK inhibitor, which blocks
receptor proximal signaling) and U01260 (a MEK inhibitor, which
6
7
8
9
10 11
Value
DMSO OSM
1 hour
DMSO BRD8518
6 hour
DMSO BRD8518 OSM
24 hour
Figure 6. Genes differentially expressed in response to treatment of
HepG2 cells with BRD8518 and OSM. Heat map of normalized gene
expression values obtained from Affymetrix microarray hybridization
experiments using RNA isolated from HepG2 cells treated with 1 µM
BRD8518 and 100 ng/ml Oncostatin M. Values below mean expression
level are represented by green and values above the mean are
represented by red color.
Erk
blocks MAPK signaling cascade) were used to pre-treat HepG2
background levels within 6 hours. Maximal induction of TRIB1 and LDLR
was achieved at 6 hours with BRD8518 treatment and was stable up to
24 hrs later suggesting that both agents may trigger response of the
same regulatory network through interaction with different triggering
targets. Evaluation of Affymetrix gene expression profiling data
confirmed upregulation of TRIB1 and LDLR expression at earlier time
points (6 hour for BRD8518 and 1 hour for OSM) and downregulation
of PCSK9 expression 24 hours post treatment. In addition, the 24 hour
treatment with both BRD8518 and OSM resulted in broad
cells for 1 hour prior to treatment with BRD8518 or OSM. The
activity of OSM was both JAK- and MEK-dependent, whereas
activity of BRD8518 was inhibited by the MEK inhibitor, but not by
the JAK inhibitor (Figure 7A). Upregulation of TRIB1 expression
showed similar sensitivity to U0126 (data not shown). Subsequently
we examined the ability of BRD8518 to directly stimulate ERK1/2
phosphorylation. In HepG2 cells treated with 2 µM BRD8518, the
ratio of phospho-ERK1/2 to total ERK1/2 detected by Western blot
was increased 2-fold comparing to cells treated with the vehicle
control as soon as 30 minutes after the treatment and remained
elevated at 6 hours post treatment (Figure 7B). These results
indicate that BRD8518 affects target(s) operating downstream of
JAK and upstream from ERK in the MAPK signaling pathway leading
to MEK1/2 dependent upregulation of TRIB1 and LDLR expression.
Other notable treatments that have been reported to stimulate ERK
phosphorylation and LDL uptake in hepatoma cells include: HGF
(17), FGF21 (18), PMA (19) and AICAR (20).
downregulation of lipid metabolic genes including genes involved in
triacylglycerol biosynthesis (DGAT1, MOGAT2), lipoprotein assembly
(MTTP, APOC3) and cholesterol biosynthesis (HMGCS1, HMGCR,
ACAT2, MVK, LSS) (Figure 6). Early
A
6
6
DMSO
5
DMSO
5
U0126
4
4
3
CP-690550
3
2
2
1
1
Since upregulation of LDLR stimulated by BRD8518 occurs via
ERK1/2-dependent signaling and since BRD8518 downregulates
other genes involved in cholesterol metabolism its mechanism of
action appears to be independent of canonical SREBP2 regulation.
To examine this notion further we tested the effects of BRD8518 on
HepG2 cells subjected to cholesterol depletion. Incubation of
HepG2 cells in cholesterol depleted media stimulated expression of
LDLR and PCSK9 genes, as expected due to coordinated regulation
of those genes by SREBP2, and had no effect on TRIB1 expression.
BRD8518 treatment of HepG2 cells in cholesterol depleted media
led to an additional upregulation of LDLR expression and to a
significant downregulation of PCSK9 expression indicating that the
effects of BRD8518 are additive with the effects of cholesterol
starvation (Figure 8A). In addition, we determined that effects of
BRD8518 are additive with effects of statins, which stimulate the
state of cholesterol depletion pharmacologically through inhibition
of HMGCoA reductase. Stimulation of LDLR by treatment of HepG2
cells with simvastatin was potentiated by BRD8518 co-treatment,
whereas stimulation of PCSK9 expression by atorvastatin was
0
0
DMSO
OSM
BRD3918 BRD8518
DMSO
OSM
BRD3918 BRD8518
B
30 min
6 hours
DMSO EGF BRD8518 DMSO
2.7 2.0
EGF BRD8518
1.72.0
Fold change:
1
1
P-ERK1/2
ERK1/2
Figure 7. Induction of LDLR by BRD8518 depends on MAPK signaling.
HepG2 cells were pretreated for 1 hour with 1 µM JAK inhibitor CP-
690550 (left panel) or 5 µM MEK inhibitor U-0126 (right panel) and
the treated with 100 ng/ml oncostatin M (OSM) for 1 hour and 10 µM
BRD-3918 or 2.5 µM BRD8518 for 6 hours and examined for LDLR
expression by qRT-PCR. (B) Western blot analysis of cell lysates from
HepG2 cells treated for 20 hours with 100 ng/ml EGF or 2 µM
BRD8518.
This journal is © The Royal Society of Chemistry 20xx
4 | Med. Chem. Commun., 2018, 00, 1-3
Please do not adjust margins

Please do not adjust margins
MedChemComm
Page 5 of 12
View Article Online
DOI: 10.1039/C8MD00297E
MedChemComm
ARTICLE
Figure 8. Effects of TRIB1 inducers on LDLR and PCSK9 expression are additive with effects of cholesterol depletion and with effects of the treatment with statins.
(A) HepG2 cells pre-grown for 24 hours in the lipoprotein depleted media in the presence or absence of cholesterol were treated with 2.5 µM BRD8518 or with
DMSO and the levels of transcripts were measured by qRT-PCR 6 hours (for LDLR) and 24 hour (for PCSK9) after compound treatment.
(B) HepG2 cells pre-grown for 24 hours in the presence of indicated concentrations of statin were treated for 6 hours (left panel) or for 24 hours
(right panel) with 10 µM BRD3918, 2 µM BRD8518 and the levels of LDLR (left panel) and PCSK9 (right panel) transcripts were quantified by qRT-PCR.
opposed by co-treatment with BRD8518 (Figure 8B).
studies. The liver cell biomarkers TRIB1, LDLR and PCSK9, were used
to drive chemistry optimization of potency. Changes to solubility,
mouse and human microsomal stability, plasma stability, plasma
protein binding and ATP levels in HepG2 cells were also monitored.
1
The potency and ADME profile of BRD8518 indicated it was not suitable
for in vivo testing and medicinal chemistry work was undertaken to
improve its potency and physical chemical properties with the goal of
identifying a compound for in vivo proof of concept
Efforts were primarily focused on the two appendage sites, R and
2
1
R . Modification at R generally resulted in a loss of activity,
including relatively minor changes such as positional changes of the
2
CF moiety on the aromatic ring. Changes at R were better
3
tolerated and hit to lead optimization produced a number of
promising molecules with improved stability and increased potency
against the liver cell biomarkers (Figure 9, Table 1). The best
molecules (3 and 5), as assessed by maintaining or improving
potency while also improving solubility and stability, were chosen
for in vivo pharmacokinetic assessment. Plasma PK parameters for 1
(BRD8518), 3 and 5 are listed in Table 2. At a single dose of 10mg/kg
delivered by IP injection 3 gave the best liver exposure based on
calculation of free fraction (fu values for 1, 3 and 5 were 0.01, 0.01
and 0.02, respectively) and the potency measured in HepG2 cells. In
vivo dial-up PK with 3 was carried out at four concentrations at
single dose (10, 15, 30 and 45 mg/kg, IP). At the 15, 30 and 45
mg/kg dose, acceptable level of the required concentration of 3 was
observed at 8-12 hrs post dose, indicating that BID dosing would be
required for full target coverage. At all doses including the highest
dose of 45 mg/kg, no adverse effects were observed over 24 hrs.
Figure 9. Improved potency of cpd 3 in transcript modulation assays
in comparison to the screening hit BRD3918 and the initial lead
BRD8518. Transcript levels were measured by qRT-PCR in HepG2
cells treated for 6 hours for TRIB1 (solid symbols) and for 24 hours
for PCSK9 (open symbols) with a dilution series of indicated
compounds.
The molecular target of BRD8518 is currently unknown, however our
data indicate that its perturbation produces a broad response
This journal is © The Royal Society of Chemistry 20xx
Med. Chem. Commun., 2018, 00, 1-3 | 5
Please do not adjust margins

Please do not adjust margins
MedChemComm
Page 6 of 12
View Article Online
DOI: 10.1039/C8MD00297E
ARTICLE
MedChemComm
Table 1. Activity of R2 substituted tricyclic glycals in RT-qPCR assays for TRIB1, LDLR and PSCK9 modulation in Hap1 cells and in in vitro pharmacokinetics
assessment of solubility and mouse liver microsomal stability.
a
b
c
d
6 hour treatment. 24 h treatment. in 1% DMSO in PBS. % remaining after 1 hour
incubation. EC50 values represent average of two or more replicates.
erk
cholesterol depletion experiments cells were grown in the DMEM
that involves activation of MAPK signaling followed by
upregulation of number of early response genes including TRIB1
and LDLR and leads to remarkable changes in the lipoprotein
metabolism, resulting in a decrease in VLDL production and an
upregulation of LDL uptake in cells of hepatic origin. The profile of
hepatic cells responses evoked by BRD8518 and its analogues is
similar to previously described responses to known hypolipidemic
agents, a natural product berberine and the endogenous cytokine
oncostatin M (OSM). Cpd 3 is the most potent molecule in the
class of TRIB1 inducers and its discovery will enable in vivo proof of
concept studies and will enhance target identification efforts and
uncovering o the molecular nature of this novel hepatic regulatory
mechanism with potential therapeutic utility.
media containing the indicated concentration of the lipoprotein
deficient serum (LPDS, Sigma). Oncostatin M was obtained from
R&D Systems. Berberine (Sigma) and the compounds from the
screening collection were prepared as 10 mM solutions in DMSO,
diluted appropriately in DMSO and delivered to cell culture at
indicated concentrations keeping the concentration of vehicle
constant at 0.25%.
Compound screening and generation of cDNA. HepG2 cells were
plated in 40 µL of growth media in white 384 well plates
Table 2. Pharmacokinetic parameters of 1 (BRD8518), 3 and 5 in plasma
and liver following a single intraperitoneal administration in male C57BL/6
mice at 10 mg/kg dose.
Experimental
Materials and methods
Cell culture and chemicals. Growth media for HepG2 cells (ATCC)
contained DMEM High glucose with sodium pyruvate and glutamine
(Invitrogen), 10% FBS (Hyclone), Penicillin (100 units/mL),
Streptomycin (100 µg/mL) and glutamine (2 mM) (Invitrogen).
HepG2 cells were cultured at 37°C, 5% CO2. For
6 | Med. Chem. Commun., 2018, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Please do not adjust margins
MedChemComm
Page 7 of 12
View Article Online
DOI: 10.1039/C8MD00297E
MedChemComm
ARTICLE
(Corning) and incubated overnight. The next day 100 nanoliters of (Hybridization, Washing and Staining Kit, Affymetrix, Santa Clara,
compound was transferred to the plates using CyBio Well pinning CA). The hybridized samples were washed and stained using an
tool and incubated overnight. After 18-22 hr of incubation RNA was Affymetrix fluidics station 450. After staining, microarrays were
harvested and cDNA was synthesized using the Ambion Cells-to-CT immediately scanned using an Affymetrix GeneArray Scanner 3000
reagents (Life Technologies). Specifically, the cells were washed 2x 7G Plus. All procedures were performed at Boston University
with PBS and then lysed with 10 µL of Cells-to-CT Lysis with DNase Microarray Resource Facility exactly as described in Affymetrix
reagent and incubated at room temperature for 10 minutes. After GeneChip Expression Analysis Technical Manual (Affymetrix, Santa
the incubation 1 µL of Stop solution was added. Two µL of the RNA Clara, CA, current version available at www.affymetrix.com). Data
lysis was added to 8 µL of Cells-to-CT cDNA reagent in a 384 well were normalized using the Robust Multi-Array Average method as
plate (Axygen) and incubated as follows: 37 °C for 1 hr, 95 °C for 5 implemented in the R package oligo (22). Differential expression
min then 4 °C until frozen at -80 °C.
analysis was performed using the R package limma (23).
Gene expression analysis by qRT-PCR. Gene expression was ELISA assays. HepG2 cells were plated in black clear bottom (Costar)
measured by quantitative PCR in a 5 µL reaction in a 384 well plate
(Roche) as follows: 2.5 µL Probes Master Mix (Roche)
96-well plates at 40,000 cells/well in 100 µL of growth media. On
Day 2 media was changed and cells were treated with compounds
for 24 hr. After 24 hr medium was removed and used for the PCSK9
(R&D Systems) and apoB (MAb Tech) ELISAs. For the apoB and
PCSK9 ELISAs 50 µL and 10 µL of medium were used, respectfully.
For the LDLR ELISA (R&D Systems), cells were washed once with PBS
(Invitrogen), lysed with 100 µL PBS/0.1% Igepal (Sigma) and frozen
at -80 °C until analyzed. Ten µL of lysate was used for the analysis.
Table 3. RT-qPCR primer / probes.
Gene
TRIB1
GAPDH
B2M
Assay ID/Sequence
Hs00921832_m1
4326317E
Source
Applied Biosystems
Applied Biosystems
Applied Biosystems
IDT
4310886E
PCSK9
LDLR
Hs.PT.49a.3004786
Hs.PT.49a.20193252
Cell viability assay. HepG2 cells were plated in white 384 well plates
(Corning) at 2000 cells/well in 40 µl of growth medium and
incubated overnight at 37°C with 5% CO2. The next day cells were
treated with 100 nL of compound for 24 hr. Cellular ATP levels were
measured using CellTiter-Glo (Promega).
IDT
0.125 µL of human target gene primer/probe, 0.125 µL of human
calibrator gene primer/probe (GAPDH or B2M) and 1.25 µL of PCR
grade water. The identities and sources of the primer/probe sets
used are listed in Table 3. Thermocycling was performed in a Roche
Light Cycler 480 II instrument using
Stable isotope labeling and analysis of triglycerides in HepG2 cells.
The cells were plated at 40,000 cells/well in 96-well plate in growth
media, incubated overnight and then treated for 24 hours with
compounds as indicated. For stable isotope labeling of TG the cells
were washed with PBS, incubated for at least 15 minutes with
DMEM supplemented with 25 mM HEPES (pH 7.5) containing 0.1%
FAF-BSA and then after media replacement incubated at 37°C with
DMEM containing 25 mM HEPES (pH7.5), 0.1 % FAF-BSA and 20 µM
the following conditions: 95°C for 10 minutes, 95°C for 10s, 60°C for
30s for 55 cycles then 40°C for 30s. Relative gene expression was
- CT
calculated using the equation 2
(21).
Transcriptional profiling. (GEO accession number: GSE57753)
HepG2 cells were plated in a black 96-well tissue culture plate
(Corning 3904) in 100μL of DMEM/10% FBS/1x Pen-Strep-
Glutamine media at the density of 10,000 cells per well. The cells
were incubated overnight in a tissue culture incubator at 37°C and
5% CO2. The cells (6-wells each treatment) were treated with 1 µM
BRD8518 for 6 hr at 37°C. The cells were also treated with ML8518
(1 µM) or Oncostatin M (100 ng/mL) for 1 hr and 24 hr at 37°C.
DMSO controls were set up for each time point. Compounds were
added to the cells to a final DMSO concentration of 0.25%.
Oncostatin M was dissolved in PBS and DMSO was added to the
wells to bring the final concentration to 0.25%. RNA was prepared
using RNeasy Minipreps (Qiagen) following the manufacture’s
protocol. Biotin labeling was performed using the Ambion WT
Expression Kit (Life Technologies, Grand Island, NY) according to the
manufacturer's protocol, followed by the GeneChip WT Terminal
Labeling and Controls Kit (Affymetrix, Santa Clara, CA). The labeled,
fragmented DNA was hybridized to the Affymetrix GeneChip
Human Gene ST 2.0 Array for 18 hours in a GeneChip Hybridization
oven 640 at 45°C with rotation (60 rpm). The hybridized samples
were washed and stained using Affymetrix fluidics station 450 with
streptavidin-R-phycoerythrin (SAPE) and the signal was amplified
using a biotinylated goat anti-streptavidin antibody followed by
another SAPE staining
13
C3-D5-glycerol for indicated periods of time. The lipids were
extracted after the media removal by incubating the cells with 150
µl isopropanol at room temperature for 15 min with shaking. The
cell extracts (120 µl) were cleared by centrifugation and
concentrated 5-fold through drying and resuspension in
isopropanol. Liquid chromatography-tandem mass spectrometry
(LC-MS) analyses of cell extracts were conducted using an Open
Accela 1250 U-HPLC coupled to a Q Exactive hybrid quadrupole
orbitrap mass spectrometer (Thermo Fisher Scientific; Waltham,
MA). Extracts (10 µL) were injected directly onto a 150 x 3.0 mm
Prosphere HP C4 column (Grace, Columbia, MD). The column was
eluted isocratically at a flow rate of 350 µL/min with 80% mobile
phase
A (95:5:0.1 vol/vol/vol 10 mM ammonium acetate/methanol/acetic
acid) for 2 minutes followed by a linear gradient to 80% mobile
phase B (99.9:0.1 vol/vol methanol/acetic acid) over 1 minute, a
linear gradient to 100% mobile phase B over 12 minutes, and then
10 minutes at 100% mobile-phase B. MS analyses were conducted
using electrospray ionization in the positive ion mode using full scan
analysis at 70,000 resolution and 3 Hz data acquisition rate.
This journal is © The Royal Society of Chemistry 20xx
Med. Chem. Commun., 2018, 00, 1-3 | 7
Please do not adjust margins

Please do not adjust margins
MedChemComm
Page 8 of 12
View Article Online
DOI: 10.1039/C8MD00297E
ARTICLE
MedChemComm
13
with 1% DMSO. Compounds were allowed to equilibrate at room
temperature with a 750 rpm vortex shake for 18 hours. After
equilibration, samples were analyzed by UPLC-MS (Waters, Milford,
MA) with compounds detected by SIR detection on a single
quadrupole mass spectrometer. The DMSO samples were used to
create a two-point calibration curve to which the response in PBS
was fit.
Monoisotopic and C3-D5-TAG LC-MS peaks were integrated using
TraceFinder 2.0 (Thermo Fisher Scientific; Waltham, MA). LDL
uptake. HepG2 cells were plated at 3000 cells/well in a 384 well
plate (Corning) in growth medium. After an overnight incubation 10
µL of 5x compound was added and the plates were incubated
overnight. The cells were then washed with phenol red free DMEM
(Invitrogen) and incubated with 30 µl of BODIPY-FL-LDL (Invitrogen)
diluted to final concentration 5 µg protein/ml in phenol red free
DMEM for 0.1 to 5 hr. LDL uptake was stopped by fixing the cells
Plasma Protein Binding. Plasma protein binding was determined by
equilibrium dialysis using the Rapid Equilibrium Dialysis (RED) device
with 3% formaldehyde. Nuclei were stained with Hoechst 33342 (Pierce Biotechnology, Rockford, IL) for both human and mouse
diluted in PBS for 10 minutes. The cells were then washed with PBS plasma. Each compound was prepared in duplicate at 5 uM in
and imaged using an IXM microscope. Image analysis and
quantification was performed with MetaXpress software (ver.
3.1.0.97, Molecular Devices) using the Multi Wavelength Cell
Scoring application module.
plasma (0.95% acetonitrile, 0.05% DMSO) and added to one side of
the membrane (200 uL) with PBS pH 7.4 added to the other side
(350 uL). Compounds were incubated at 37ºC for 5 hours with a
350-rpm orbital shake. After incubation, samples were analyzed by
UPLC-MS (Waters, Milford, MA) with compounds detected by SIR
detection on a single quadrupole mass spectrometer.
Western blot analysis. HepG2 cells were seeded in 6-well plates at
approximately 800,000 cells /well, incubated in growth media at
37°C for 24 hours, further incubated for 20 hours in serum-free
DMEM and then treated with EGF (100 ng/ml), BRD3918 (10 µM) or Microsomal Stability. Microsomal stability was determined at 37ºC
BRD8518 (2 µM) for the indicated times. At the end of treatment, at 60 minutes in both human and mouse microsomes. Each
compound was prepared in duplicate at 1 uM with 0.3 mg/mL
microsomes in PBS pH 7.4 (1% DMSO). Compounds were incubated
at 37ºC for 60 minutes with a 350-rpm orbital shake with time
points taken at 0 minutes and 60 minutes. Samples were analyzed
by UPLC-MS (Waters, Milford, MA) with compounds detected by SIR
detection on a single quadrupole mass spectrometer.
cells were washed with PBS and lysed in RIPA lysis buffer (50mM
Tris-HCl, 1% IGEPAL, 0.5% Na-deoxycholate, 0.1% SDS, 150 nM
NaCl, 2 mM EDTA, 50 mM NaF) with protease inhibitor cocktail
(Roche). Lysates were incubated on ice for 1 hour with frequent
agitation, centrifuged at 13,000 rpm for 15 minutes at 4°C and then
equal amount of protein from each lysate (~ 50 µg) was denatured,
subjected to electrophoresis using NuPAGE® Novex® 4-12% Bis-Tris
Gels and transferred to PVDF membranes with iBlot Gel Transfer
Device (Invitrogen) according to manufacturer’s instructions. The
ERK1/2 phosphorylation was detected using rabbit polyclonal
antibodies directed against p44/42 MAPK (Erk1/2) and Phospho-
p44/42 MAPK (Erk1/2) (Cell Signaling Technology) and the goat
anti-rabbit IgG (H+L) secondary antibodies conjugated with HRP
(Thermo Scientific). After standard incubations and washing the
membranes were exposed with SuperSignal West Femto Maximum
Sensitivity Substrate (Thermo Scientific), images captured using
Kodak Image Station 4000mm PRO and Carestream Molecular
Imaging Software. Band intensities were calculated using Image J
software.The main text of the article should appear here with
headings as appropriate. Pharmacokinetic studies. Plasma
pharmacokinetics and liver distribution of selected compounds
were performed at Sai Life Sciences (Hinjewadi, India) following
animal welfare guidelines provided by the Committee for the
Purpose of Control and Supervision of Experiments on Animals
(CPCSEA). All study protocols were approved by both Broad
Institute and Sai IACUCs. Compound concentrations were measured
by mass spectrometry in plasma and liver homogenates of male
Synthetic procedures
Synthesis of BRD8518: Step1 - Methyl 2-((2S,4aS,12aR)-5-methyl-6-
oxo-8-(4-(trifluoromethyl)benzamido)-2,3,4,4a,5,6,12,12a-
octahydrobenzo[b]pyrano[3,2-f][1,5]oxazocin-2-yl)acetate: At 0
o
C, 4-trifluoromethylbenzoyl chloride (0.49 mL, 3.3 mmol, 1.1 equiv)
was added dropwise to
a solution of methyl 2-((2S,4aS,12aR)-8-amino-5-methyl-6-oxo-
2,3,4,4a,5,6,12,12a-octahydrobenzo[b]pyrano[3,2-
f][1,5]oxazocin-2-yl)acetate (1.0 g, 3.0 mmol, 1.0 equiv) and
triethylamine (0.63 mL, 4.49 m\mol, 1.5 equiv) in CH Cl (60 mL).
2
2
After complete conversion of the starting material (LCMS, 30 min),
the reaction was quenched with a saturated solution of ammonium
chloride (30 mL) and the crude mixture was partially concentrated.
The aqueous phase was extracted with EtOAc (3x50 mL) and the
combined organic phases were washed with brine, dried over
Na2SO4, filtered and concentrated under reduced pressure. The
crude residue was purified by silica gel chromatography (gradient:
0–80% EtOAc in Hexanes), which yielded 1.2 g (2.36 mmol, 79 %
yield) of methyl 2-((2S,4aS,12aR)-5-methyl-6-oxo-8-(4-
C57BL/6 mice following
a
single intraperitoneal dose (trifluoromethyl)benzamido)-2,3,4,4a,5,6,12,12a-
administration. The samples were collected and analysed 0.08, 0.5,
1, 2, 4 and 8 hr post injection. Pharmacokinetic parameters were
assessed using the Non-Compartmental Analysis module in Phoenix
WinNonlin (Version 6.3).
octahydrobenzo[b]pyrano[3,2-f][1,5]oxazocin-2-yl)acetate, as
a
white solid. 1H NMR (300 MHz, CDCl ) δ 8.66 (s, 1H), 7.98 (d, J = 8.1
3
Hz, 2H), 7.87 (d, J = 9.3 Hz, 1H), 7.73 (d, J = 8.1 Hz, 2H), 7.45 (d, J =
2.7 Hz, 1H), 6.82 (d, J = 9.0 Hz, 1H), 4.07 (dd, J = 12.2, 3.2 Hz, 1H),
4.02 – 3.76 (m, 4H), 3.72 (s, 3H), 3.26 (s, 3H), 2.61 (dd, J = 15.7, 7.3
Hz, 1H), 2.48 (dd, J = 15.7, 5.3 Hz, 1H), 2.21 – 2.08
Solubility. Solubility was determined in phosphate buffered saline
(PBS) pH 7.4 with 1% DMSO. Each compound was prepared in
triplicate at 100 uM in both 100% DMSO and PBS
(m, 2H), 1.98 – 1.80 (m, 1H), 1.80 – 1.69 (m, 1H). LRMS (ESI) calcd
for C
H
F N O [M + H]+ 507.17, found 507.22.
25 26 3
2
6
8 | Med. Chem. Commun., 2018, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Please do not adjust margins
MedChemComm
Page 9 of 12
View Article Online
DOI: 10.1039/C8MD00297E
MedChemComm
ARTICLE
Hz, 1H), 3.16 (s, 3H), 2.83 (dd, J = 4.1, 4.1 Hz, 1H), 2.78 (dd, J = 4.0,
4.0 Hz, 1H), 2.33 (d, J = 5.9 Hz, 2H), 2.13 – 2.06 (m, 2H), 1.88
Step 2 - 2-((2S,4aS,12aR)-5-methyl-6-oxo-8-(4-
– 1.76 (m, 1H), 1.76 – 1.65 (m, 1H). LRMS (ESI) calcd for
(trifluoromethyl)benzamido)-2,3,4,4a,5,6,12,12a-
octahydrobenzo[b]pyrano[3,2-f][1,5]oxazocin-2-yl)acetic acid:
C
H
F N O [M + H]+ 608.24, found 608.28.
33 33 3
3
5
A THF solution (43 mL) of methyl 2-((2S,4aS,12aR)-5-methyl-6-oxo-
8-(4-(trifluoromethyl)benzamido)-2,3,4,4a,5,6,12,12a-
octahydrobenzo[b]pyrano[3,2-f][1,5]oxazocin-2-yl)acetate (1.1
General Synthetic details for “Reverse amide” capping on the
right-side:
o
General protocol for Curtius reaction: At rt and under argon
atmosphere, DPPA (2.0 equiv) was added dropwise to a solution
g, 2.2 mmol, 1.0 equiv) was cooled in an iced bath (0 C) and
hydrogen peroxide (30 % by weight, 2.7 mL, 26.0 mmol, 12.0 equiv)
was added to the mixture, followed by lithium hydroxide (1.0 M in
water, 13.0 mL, 13.0 mmol, 6.0 equiv). The reaction was stirred
overnight, reaching rt progressively upon which LCMS analysis
showed complete disappearance of the starting
of 2-((2S,4aS,12aR)-5-methyl-6-oxo-8-(4-
(trifluoromethyl)benzamido)-2,3,4,4a,5,6,12,12a-
octahydrobenzo[b]pyrano[3,2-f][1,5]oxazocin-2-yl)acetic acid (1.0
equiv) and triethylamine (3.0 equiv) in dry acetonitrile (0.01 M). The
o
o
reaction mixture was heated at 50 C for two hours, cooled to rt for
material. At 0 C, the reaction mixture was acidified with 1N HCl
15 min and transferred dropwise via Pasteur pipet to an aqueous
solution until pH ~3, diluted with brine (75 mL) and extracted with
EtOAc (3x40 mL). Combined organic layers were dried over Na2SO4,
filtered and concentrated in vacuo to give 2-((2S,4aS,12aR)-5-
methyl-6-oxo-8-(4-(trifluoromethyl)benzamido)-
2,3,4,4a,5,6,12,12a-octahydrobenzo[b]pyrano[3,2-f][1,5]oxazocin-
2-yl)acetic acid
o
solution of 1M NaOH, under vigorous stirring and cooled at 0 C.
After 30 min, Boc O (10 equiv) was quickly added to the solution
2
and the reaction mixture was stirred overnight, gradually warming
to rt. Solvents were partially removed in vacuo and the mixture was
extracted with CH Cl . Combined organic phases were washed with
2
2
as a white solid (1.1 g, 2.2 mmol), which was used in the next step
brine, dried over MgSO , filtered and concentrated and the residue
4
was purified by silica gel chromatography.
without further purification. 1H NMR (300 MHz, CDCl ) δ 9.21 (s,
3
1H), 7.99 (d, J = 8.1 Hz, 2H), 7.91 (d, J = 9.8 Hz, 1H), 7.69 (d, J = 8.1
Hz, 2H), 7.47 (d, J = 2.7 Hz, 1H), 6.77 (d, J = 9.0 Hz, 1H), 4.10 – 3.88
(m, 2H), 3.87 – 3.70 (m, 3H), 3.23 (s, 3H), 2.63 (dd, J = 15.9, 7.2 Hz,
1H), 2.52 (dd, J = 15.9, 5.2 Hz, 1H), 2.19 – 2.06 (m,
o
General protocol for Boc deprotection: At 0 C, trifluoroacetic acid
(TFA, 20.0 equiv) was added dropwise to a solution of the
2H), 1.97 – 1.82 (m, 1H), 1.82 – 1.69 (m, 1H). LRMS (ESI) calcd for
appropriate Boc protected amine (1.0 equiv) in CH Cl (0.05 M).
2
2
C
H
F N O [M + H]+ 493.16, found 493.42.
24 24 3
2
6
After 10 min, the reaction mixture was warmed to rt and stirred
until complete disappearance of the starting material (LCMS). The
crude was then concentrated to dryness, redissolved in EtOAc and
washed with a saturated solution of sodium bicarbonate until pH ~
7. The combined aqueous phases were extracted with EtOAc and
the combined organic layers were washed with brine, dried over
Step 3 - N-((2S,4aS,12aR)-2-(2-((2,3-dihydro-1H-inden-2-yl)amino)-
2-oxoethyl)-5-methyl-6-oxo-2,3,4,4a,5,6,12,12a-
octahydrobenzo[b]pyrano[3,2-f][1,5]oxazocin-8-yl)-4-
o
(trifluoromethyl)benzamide (BRD8518): At 0 C, 2-aminoindane
(13.7 uL, 0.1 mmol, 1.3 equiv) was added to a solution of crude 2-
((2S,4aS,12aR)-5-methyl-6-oxo-8-(4-(trifluoromethyl)benzamido)-
2,3,4,4a,5,6,12,12a-octahydrobenzo[b]pyrano[3,2-f][1,5]oxazocin-
2-yl)acetic acid (40 mg, 0.08 mmol, 1.0 equiv) and DIEA (42.6 uL,
Na SO , filtered and concentrated to give the desired product,
2
4
which was used without further purification.
General protocol for amine capping:
0.24 mmol, 3.0 equiv) in CH Cl (0.16 mL), followed by PyBOP (67.6
2
2
o
mg, 0.13 mmol, 1.6 equiv). The reaction mixture was stirred
overnight, reaching rt progressively. The reaction mixture was
Formation of carbomate: At 0 C, the appropriate chloroformate
(1.1 equiv) was added to a solution of the appropriate amine (1.0
diluted with CH Cl (10 mL) and washed with a saturated solution
2
2
equiv) and triethylamine (5.0 equiv) in CH Cl (0.05 M). After 15
2
2
of ammonium chloride (2x10 mL). The aqueous phase was
min, the iced bath was removed and the reaction mixture was
stirred at rt overnight. Silica gel was then added and the crude was
concentrated to dryness. Purification was done via silica gel
chromatography affording the desired product.
extracted with CH Cl (3x10 mL) and the combined organic layers
2
2
were dried over Na SO , filtered and concentrated to yield a crude
2
4
material which was purified by chromatography on silica gel
(gradient: 10–100% EtOAc in Hexanes). N-((2S,4aS,12aR)-2-(2-((2,3-
dihydro-1H-inden-2-yl)amino)-2-oxoethyl)-5-methyl-6-oxo-
2,3,4,4a,5,6,12,12a-octahydrobenzo[b]pyrano[3,2-f][1,5]oxazocin-
8-yl)-4-(trifluoromethyl)benzamide (BRD8518) was isolated as an
off-white solid (42 mg, 0.07 mmol, 85 % yield over two steps).1H
o
Alternative protocol for the formation of carbomate: At 0 C, the
appropriate chloroformate (1.1 equiv) was added to a solution of
the appropriate amine (1.0 equiv) in a mixture of 1,4-dioxane and
saturated aqueous sodium bicarbonate solution (dioxane:sat.
NMR (300 MHz, CDCl ) δ 8.77 (s, 1H), 7.96 (d, J = 8.1 Hz, 2H), 7.89
3
(dd, J = 9.1, 2.7 Hz, 1H), 7.68 (d, J = 8.2 Hz, 2H), 7.42 (d, J =
NaHCO =2:1, 0.05M concentration). Upon completion of the
3
reaction (LCMS), extraction of the mixture with EtOAc followed by
evaporation of the solvents afforded a crude residue which was
purified via silica gel chromatography.
2.8 Hz, 1H), 7.30 – 7.17 (m, 4H), 6.82 (d, J = 9.0 Hz, 1H), 6.17 (d, J =
7.9 Hz, 1H), 4.81 – 4.69 (m, 1H), 3.94 – 3.81 (m, 1H), 3.79 – 3.66 (m,
4H), 3.36 (dd, J = 7.0, 4.3 Hz, 1H), 3.31 (dd, J = 6.7, 4.2
This journal is © The Royal Society of Chemistry 20xx
Med. Chem. Commun., 2018, 00, 1-3 | 9
Please do not adjust margins

Please do not adjust margins
MedChemComm
Page 10 of 12
View Article Online
DOI: 10.1039/C8MD00297E
ARTICLE
MedChemComm
o
benzo-[b]pyrano[3,2-f][1,5]oxazocin-2-yl)methyl)carbamate (16.6
mg, 29 umol, 46 % yield) as a white powder. Alternatively, the title
compound was also prepared from N-((2S,4aS,12aR)-2-
(aminomethyl)-5-methyl-6-oxo-2,3,4,4a,5,6,12,12a-
Formation of amide: At 0 C, the appropriate carboxylic acid (2.0
equiv) was added to a solution of the amine (1.0 equiv) and TEA
(6.0 equiv) in CH Cl /DMF (40:1 ratio, 0.05 M), followed by PyBOP
(2.2 equiv). The reaction mixture was stirred overnight, reaching rt
2
2
octahydrobenzo[b]pyrano[3,2-f][1,5]oxazocin-8-yl)-4-
(trifluoromethyl)benzamide and isobutylchloroformate (1.1 equiv),
in a mixture of 1,4-dioxane and saturated aqueous sodium
progressively. The reaction mixture was diluted with CH Cl and
washed with a saturated solution of sodium bicarbonate. The
2
2
aqueous phase was extracted with CH Cl and the combined
2
2
bicarbonate solution (dioxane:sat. NaHCO =2:1, 0.05M
3
organic layers were dried over Na SO , filtered and concentrated to
2
4
o
concentration), at 0 C. Upon completion of the reaction (LCMS,
yield a crude material which was purified by chromatography on
silica gel.
2h), extraction of the mixture with EtOAc followed by evaporation
of the solvents afforded a crude residue which was purified on silica
using the same conditions as described above. 1H NMR (400 MHz,
Formation of amino oxadiazole: The appropriate oxadiazolone (1.3
equiv) was added to the appropriate amine (1.0 equiv) in DMF
(0.05M), followed by DIPEA (4 equiv) and BOP (1.5 equiv). The
reaction was stirred at room temperature until complete
conversion was observed. The mixture was partially concentrated,
taken up in EtOAc and washed with a saturated solution of sodium
bicarbonate. The layers separated and the aqueous layer extracted
with EtOAc. The combined organic extracts were washed with
CDCl ) δ 9.69 (br s, 1H), 8.04 (br d, J = 8.0 Hz, 1H), 7.99 (d, J = 8.1 Hz,
3
2H), 7.67 (d, J = 7.9 Hz, 2H), 7.47 (s, 1H), 6.74 (d, J = 9.0 Hz, 1H), 5.08
– 4.99 (m, 1H), 3.93 (d, J = 14.7 Hz, 1H), 3.90 – 3.79 (m, 2H), 3.79 –
3.66 (m, 2H), 3.59 (br s, 1H), 3.55
– 3.40 (m, 2H), 3.19 (s, 3H), 3.12 – 3.00 (m, 1H), 2.15 – 2.00 (m, 2H),
1.93 (ddd, J = 13.7, 6.9, 6.9 Hz, 1H), 1.86 – 1.74 (m, 1H), 1.63 (br d, J
= 13.7 Hz, 1H), 0.95 (s, 3H), 0.93 (s, 3H). 13C NMR (100 MHz, CDCl3)
δ 170.8, 165.1, 157.1, 150.9, 138.9, 133.0 (q, JC-F = 32.9 Hz), 132.3,
128.3, 125.4 (q, JC-F = 3.6 Hz), 124.8, 123.9 (q, JC-F = 272.6 Hz),
123.4, 119.8, 119.3, 77.8, 71.4, 64.9,
brine, dried over MgSO , filtered and concentrated to yield a crude
4
material which was purified by chromatography on silica gel.
51.0, 45.6, 32.2, 28.2, 24.5, 23.6, 19.1. LRMS (ESI) calcd for
Synthesis and characterization of
3:
C
H
F N O [M + H]+ 564.23, found 564.13.
28 33 3
3
6
Isobutyl (((2S,4aS,12aR)-5-methyl-6-oxo-8-(4-
Characterization of 4:
(trifluoromethyl)benzamido)-2,3,4,4a,5,6,12,12a-octahydrobenzo-
o
(S)-N-(((2S,4aS,12aR)-5-methyl-6-oxo-8-(4-
(trifluoromethyl)benzamido)-2,3,4,4a,5,6,12,12a-
octahydrobenzo[b]pyrano[3,2-f][1,5]oxazocin-2-
[b]pyrano[3,2-f][1,5]oxazocin-2-yl)methyl)carbamate: At 0 C,
trifluoroacetic acid (TFA, 0.60 mL, 7.81 mmol, 20.0 equiv) was
added dropwise to a solution of
yl)methyl)tetrahydrofuran-2-carboxamide: 1H NMR (400 MHz,
tert-butyl (((2S,4aS,12aR)-5-methyl-6-oxo-8-(4-
(trifluoromethyl)benzamido)-2,3,4,4a,5,6,12,12a-
octahydrobenzo[b]pyrano[3,2-f][1,5]oxazocin-2-
CDCl ) δ 9.69 (s, 1H), 8.06 (d, J = 8.7 Hz, 1H), 8.00 (d, J = 7.8 Hz, 2H),
3
7.68 (d, J = 7.9 Hz, 2H), 7.49 (s, 1H), 7.04 (br s, 1H), 6.73 (d, J = 9.0
Hz, 1H), 4.36 (dd, J = 7.0 Hz, 1H), 4.01 – 3.83 (m, 3H), 3.79
yl)methyl)carbamate (220 mg, 0.39 mmol, 1.0 equiv) in CH Cl (8
2
2
– 3.66 (m, 2H), 3.65 – 3.42 (m, 3H), 3.23 (s, 4H), 2.37 – 2.24 (m,
1H), 2.15 – 1.99 (m, 3H), 1.99 – 1.71 (m, 3H), 1.68 – 1.57 (m, 1H).
mL). After 10 min, the reaction mixture was warmed to rt and
stirred until complete disappearance of the starting material (LCMS,
1h40min). The crude was then concentrated to dryness, redissolved
in EtOAc (20 mL) and washed with a saturated solution of sodium
bicarbonate until pH ~ 7. The combined aqueous phases were
extracted with EtOAc (3x50 mL) and the combined organic layers
LRMS (ESI) calcd for C
H
F N O [M + H]+ 562.22, found 562.44.
28 31 3
3
6
Characterization of 5:
were washed with brine, dried over Na SO , filtered and
N-((2S,4aS,12aR)-2-(((5-cyclobutyl-1,3,4-oxadiazol-2-
yl)amino)methyl)-5-methyl-6-oxo-2,3,4,4a,5,6,12,12a-
octahydrobenzo[b]pyrano[3,2-f][1,5]oxazocin-8-yl)-4-
2
4
concentrated to give N-((2S,4aS,12aR)-2-(aminomethyl)-5-methyl-6-
oxo-2,3,4,4a,5,6,12,12a-octahydrobenzo[b]pyrano[3,2-
f][1,5]oxazocin-8-yl)-4-(trifluoromethyl)benzamide as a white solid
(trifluoromethyl)benzamide: 1H NMR (400 MHz, CDCl ) δ
3
(166 mg, 0.36 mmol, 92% yield) which was used without further
9.01 (s, 1H), 8.01 (d, J = 7.8 Hz, 2H), 7.93 (br d, J = 9.0 Hz, 1H),
7.72 (d, J = 8.0 Hz, 2H), 7.49 (s, 1H), 6.80 (d, J = 9.0 Hz, 1H),
5.97 (br s, 1H), 4.14 – 4.00 (m, 1H), 3.88 – 3.72 (m, 4H), 3.70 –
3.54 (m, 1H), 3.37 – 3.25 (m, 1H), 3.23 (s, 3H), 2.47 – 2.33 (m,
4H), 2.22 – 2.06 (m, 3H), 2.06 – 1.95 (m, 1H), 1.94 – 1.79 (m,
o
purification. At 0 C, isobutylchloroformate (9.3 uL, 71 umol, 1.1
equiv) was added to a solution of crude N-((2S,4aS,12aR)-2-
(aminomethyl)-5-methyl-6-oxo-2,3,4,4a,5,6,12,12a-
octahydrobenzo[b]pyrano[3,2-f][1,5]oxazocin-8-yl)-4-
(trifluoromethyl)benzamide (30 mg, 65 umol, 1.0 equiv) and
1H), 1.78 – 1.67 (m, 1H). LRMS (ESI) calcd for C
H
F N O [M +
29 31 3
5
5
H]+ 586.23, found 586.25.
triethylamine (45.1 uL, 0.32 mmol, 5.0 equiv) in CH Cl (1.2 mL).
2
2
After 15 min, the iced bath was removed and the reaction mixture
was stirred at rt overnight. Silica gel was then added and the crude
was concentrated to dryness. Purification was done via silica gel
chromatography (gradient: 5–65% EtOAc in Hexanes) affording
isobutyl (((2S,4aS,12aR)-5-methyl-6-oxo-8-(4-
Conclusions
Current report presents a DOS-derived molecule with remarkable
effects on lipid metabolism in hepatic cells. BRD8518, initially
identified as an inducer of expression of the
(trifluoromethyl)benzamido)-2,3,4,4a,5,6,12,12a-octahydro-
10 | Med. Chem. Commun., 2018, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Please do not adjust margins
MedChemComm
Page 11 of 12
View Article Online
DOI: 10.1039/C8MD00297E
MedChemComm
ARTICLE
regulates hepatic lipogenesis and VLDL production in mice. J Clin
Invest. 2010;120(12):4410-4.
cardioprotective gene TRIB1, turned out to broadly reprogram the
lipoprotein metabolism in HepG2 cells from lipogenesis driven VLDL
secretion to lipid scavenging through LDL uptake. This activity
profile is unique as currently used cardiovascular drugs lower the
blood lipid levels either by stimulating LDL clearance through
elevation of hepatic LDL uptake (statins by inducing LDL receptor
expression or PCSK9 blocking antibodies by reducing the LDL
receptor degradation) or by reducing hepatic production of VLDL
particles, the precursor to LDL particles (e.g. MTTP inhibitor
lomitapide or ApoB targeting anti-sense oligonucleotide
mipomersen). BRD8518 appears to regulate both arms of
lipoprotein metabolism and to combine both activities in one
molecule. The limiting ADME properties of BRD8518 were remedied
by medicinal chemistry efforts that led to identification of cpd 3,
which has enhanced potency and improved pharmacokinetic
properties. Development of cpd 3, a new lead in the series of
tricyclic glycal class of TRIB1 inducers, should enable future in vivo
proof of concept studies and will facilitate target identification
efforts.
8. Satoh T, Kidoya H, Naito H, Yamamoto M, Takemura N,
Nakagawa K, et al. Critical role of Trib1 in differentiation of tissue-
resident M2-like macrophages. Nature. 2013;495(7442):524-8.
9. Schreiber SL. Organic chemistry: Molecular diversity by design.
Nature. 2009;457(7226):153-4.
10. Gerard B, Lee MDt, Dandapani S, Duvall JR, Fitzgerald ME,
Kesavan S, et al. Synthesis of stereochemically and skeletally diverse
fused ring systems from functionalized C-glycosides. The Journal of
organic chemistry. 2013;78(11):5160-71.
11. Nagiec MM, Skepner AP, Negri J, Eichhorn M, Kuperwasser N,
Comer E, et al. Modulators of hepatic lipoprotein metabolism
identified in a search for small-molecule inducers of tribbles
pseudokinase 1 expression. PLoS One. 2015;10(3):e0120295.
12. Akella LB, Marcaurelle LA. Application of a sparse matrix design
strategy to the synthesis of dos libraries. ACS Comb Sci.
2011;13(4):357-64.
13. Mulrooney CA, Lahr DL, Quintin MJ, Youngsaye W, Moccia D, Asiedu
JK, et al. An informatic pipeline for managing high-throughput screening
experiments and analyzing data from stereochemically diverse libraries.
J Comput Aided Mol Des. 2013;27(5):455-68.
Conflicts of interest
14. Peck D, Crawford ED, Ross KN, Stegmaier K, Golub TR, Lamb J. A
method for high-throughput gene expression signature analysis.
Genome biology. 2006;7(7):R61.
There are no conflicts to declare.
15. Tullai JW, Schaffer ME, Mullenbrock S, Sholder G, Kasif S,
Cooper GM. Immediate-early and delayed primary response genes
are distinct in function and genomic architecture. J Biol Chem.
2007;282(33):23981-95.
Acknowledgements
This work was sponsored in part by the Broad Institute Gift and
the National Institutes of Health (NIH) Molecular Libraries Probe
Production Centers Network (MLPCN) Program. The authors wish
to thank Amy Deik for conducting LC-MS analyses, Melissa
Bennion, Chaewon Hwang and Katie Loveluck for expert technical
assistance.
16. Cao A, Wu M, Li H, Liu J. Janus kinase activation by cytokine
oncostatin M decreases PCSK9 expression in liver cells. J Lipid Res.
2011;52(3):518-30.
17. Pak YK, Kanuck MP, Berrios D, Briggs MR, Cooper AD, Ellsworth
JL. Activation of LDL receptor gene expression in HepG2 cells by
hepatocyte growth factor. J Lipid Res. 1996;37(5):985-98.
18. Do HT, Tselykh TV, Makela J, Ho TH, Olkkonen VM, Bornhauser
BC, et al. Fibroblast growth factor-21 (FGF21) regulates low-density
lipoprotein receptor (LDLR) levels in cells via the E3-ubiquitin ligase
Mylip/Idol and the Canopy2 (Cnpy2)/Mylip-interacting saposin-like
protein (Msap). J Biol Chem. 2012;287(16):12602-11.
References
1. Teslovich TM, Musunuru K, Smith AV, Edmondson AC, Stylianou
IM, Koseki M, et al. Biological, clinical and population relevance of 19. Yashiro T, Nanmoku M, Shimizu M, Inoue J, Sato R. 5-
95 loci for blood lipids. Nature. 2010;466(7307):707-13.
Aminoimidazole-4-carboxamide ribonucleoside stabilizes low
density lipoprotein receptor mRNA in hepatocytes via ERK-
dependent HuR binding to an AU-rich element. Atherosclerosis.
2013;226(1):95-101.
2. Ishizuka Y, Nakayama K, Ogawa A, Makishima S, Boonvisut S,
Hirao A, et al. TRIB1 downregulates hepatic lipogenesis and
glycogenesis via multiple molecular interactions. Journal of
molecular endocrinology. 2014;52(2):145-58.
20. Vargas NB, Brewer BY, Rogers TB, Wilson GM. Protein kinase C
activation stabilizes LDL receptor mRNA via the JNK pathway in
HepG2 cells. J Lipid Res. 2009;50(3):386-97.
3. Consortium CAD, Deloukas P, Kanoni S, Willenborg C, Farrall M,
Assimes TL, et al. Large-scale association analysis identifies new risk
loci for coronary artery disease. Nat Genet. 2013;45(1):25-33.
21. Schmittgen TD, Livak KJ. Analyzing real-time PCR data by the
comparative C(T) method. Nature protocols. 2008;3(6):1101-8.
4. Musunuru K, Romaine SP, Lettre G, Wilson JG, Volcik KA, Tsai
MY, et al. Multi-ethnic analysis of lipid-associated loci: the NHLBI
CARe project. PLoS One. 2012;7(5):e36473.
22. Carvalho BS, Irizarry RA. A framework for oligonucleotide
microarray preprocessing. Bioinformatics. 2010;26(19):2363-7.
23. K. SG. Limma: linear models for microarray data. Bioinformatics
and computational biology solutions using R and Bioconductor:
Springer New York; 2005. p. 397-420.
5. Walia GK, Gupta V, Aggarwal A, Asghar M, Dudbridge F,
Timpson N, et al. Association of common genetic variants with lipid
traits in the Indian population. PLoS One. 2014;9(7):e101688.
6. Aung LH, Yin RX, Wu DF, Li Q, Yan TT, Wang YM, et al.
Association of the TRIB1 tribbles homolog 1 gene rs17321515 A>G
polymorphism and serum lipid levels in the Mulao and Han
populations. Lipids Health Dis. 2011;10:230.
7. Burkhardt R, Toh SA, Lagor WR, Birkeland A, Levin M, Li X, et al.
Trib1 is a lipid- and myocardial infarction-associated gene that
This journal is © The Royal Society of Chemistry 20xx
Med. Chem. Commun., 2018, 00, 1-3 | 11
Please do not adjust margins

MedChemComm
Page 12 of 12
BRD3918
BRD8518
cpd 3