Discovery of bisamide-heterocycles as inhibitors of scavenger receptor BI (SR-BI)-mediated lipid uptake

Article abstract of DOI:10.1016/j.bmcl.2015.03.074

Abstract A new series of potent inhibitors of cellular lipid uptake from HDL particles mediated by scavenger receptor, class B, type I (SR-BI) was identified. The series was identified via a high-throughput screen of the National Institutes of Health Mole

Full text of DOI:10.1016/j.bmcl.2015.03.074

Bioorganic & Medicinal Chemistry Letters 25 (2015) 2594–2598
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of bisamide-heterocycles as inhibitors of scavenger
receptor BI (SR-BI)-mediated lipid uptake
a
a
c
a
Chris Dockendorff a,b, , Patrick W. Faloon , Andrew Germain , Miao Yu , Willmen Youngsaye ,
⇑
Partha P. Nag a, Melissa Bennion a, Marsha Penman c, Thomas J. F. Nieland c, Sivaraman Dandapani a,
José R. Perez a, Benito Munoz a, Michelle A. Palmer a, Stuart L. Schreiber a,d, Monty Krieger c,
⇑
a Center for the Science of Therapeutics, Broad Institute, 7 Cambridge Center, Cambridge, MA 02142, USA
b Department of Chemistry, Marquette University, PO Box 1881, Milwaukee, WI 53201-1881, USA
c Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA
d Howard Hughes Medical Institute, Broad Institute, 7 Cambridge Center, Cambridge, MA 02142, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A new series of potent inhibitors of cellular lipid uptake from HDL particles mediated by scavenger recep-
tor, class B, type I (SR-BI) was identified. The series was identified via a high-throughput screen of the
National Institutes of Health Molecular Libraries Small Molecule Repository (NIH MLSMR) that measured
the transfer of the fluorescent lipid DiI from HDL particles to CHO cells overexpressing SR-BI. The series is
characterized by a linear peptidomimetic scaffold with two adjacent amide groups, as well as an aryl-
substituted heterocycle. Analogs of the initial hit were rapidly prepared via Ugi 4-component reaction,
and select enantiopure compounds were prepared via a stepwise sequence. Structure–activity relation-
ship (SAR) studies suggest an oxygenated arene is preferred at the western end of the molecule, as well
as highly lipophilic substituents on the central and eastern nitrogens. Compound 5e, with (R)-stereo-
chemistry at the central carbon, was designated as probe ML279. Mechanistic studies indicate that
ML279 stabilizes the interaction of HDL particles with SR-BI, and its effect is reversible. It shows good
potency (IC50 = 17 nM), is non-toxic, plasma stable, and has improved solubility over our alternative
probe ML278.
Received 6 February 2015
Revised 24 March 2015
Accepted 26 March 2015
Available online 11 April 2015
Keywords:
ML279
SR-BI inhibitor
HDL receptor
Cholesterol transport
HCV
Ó 2015 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
The trafficking of lipids (e.g., cholesterol and its esters) between
tissues is critical for lipid homeostasis as well as steroidogenesis.
The key players in this transport system include lipoprotein parti-
cles (e.g., LDL and HDL) that carry the water-insoluble lipids
through the bloodstream, the enzymes that modify the lipid cargos
and/or help to transfer them from one particle to another (includ-
ing LCAT and CETP), and the receptors for the lipoprotein particles
that serve to transfer the lipids into and out of cells (i.e., uptake and
efflux). The cellular receptor for high-density lipoprotein (HDL)
particles, scavenger receptor class B, type I (SR-BI),1,2 has been
studied in detail in recent years,3–5 however a complete picture
of its mode of action is still incomplete, despite related structural
data.6 SR-BI has important effects on cardiovascular physiology,7
as well as pathogen entry (e.g., Hepatitis C virus),8–10 immune
response,11–13 and female fertility.7
We herein report our discovery of a second class of SR-BI inhi-
bitors that shows distinct advantages over those previously discov-
ered in our labs (e.g., BLT-1 and BLT-3).14 Concurrent with this
work was our discovery of the indoline–thiazole ML278,15,16 fol-
lowed soon after by the discovery of the benzo-fused lactams rep-
resented by ML312,17 described in the companion paper in this
journal.18 Several other inhibitors of SR-BI have been reported,
including HDL376,19,20 ITX-5061,21–24 R-138329, and R-154716
(Fig. 1).25 Recently, researchers at iTherX reported additional HCV
entry inhibitors, including ITX-4520, which is postulated to be an
inhibitor of SR-BI.26,27 Our discovery of the bisamide inhibitors
described herein was undertaken as part of the NIH Molecular
Libraries Program (MLP).28
To elucidate more details about the mechanism of lipid uptake
and efflux via SR-BI, and to potentially identify less toxic and more
potent small molecule probes, we undertook a high-throughput
screening (HTS) campaign measuring the uptake of the fluorescent
lipid surrogate 1,10-dioctadecyl-3,3,30,30-tetramethylindocarbocya-
nine perchlorate (DiI) from HDL particles into CHO cells overex-
pressing mouse SR-BI (ldlA[mSR-BI]).14 3046 compounds (0.96%)
⇑
Corresponding authors. Tel.: +1 414 288 1617 (C.D.), +1 617 253 6793 (M.K.)
E-mail addresses: christopher.dockendorff@marquette.edu (C. Dockendorff),
krieger@MIT.edu (M. Krieger).
http://dx.doi.org/10.1016/j.bmcl.2015.03.074
0960-894X/Ó 2015 The Authors. Published by Elsevier Ltd.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

C. Dockendorff et al. / Bioorg. Med. Chem. Lett. 25 (2015) 2594–2598
2595
Cl
H
N
S
N
S
NH2
Ph
Ph
N
H
N
H
N
O
BLT-1
HDL376
BLT-3
O
O
N
O
O
N
MeO HN
O
O
N
X
HN
N
H
S
O
O
O
Cl
X = NO2 : R-138329
X = NHAc : R-154716
ITX-5061
Figure 1. Known inhibitors of SR-BI.
on plates with Z0 <0.3 or that were active in 10% or more of the
HTS assays listed in PubChem. A counterscreen was also performed
that rejected hit compounds that quenched the fluorescence of DiI-
HDL in a dose-dependent manner.
Table 1
Amide analogs
MeO
MeO
O
Of the numerous scaffolds verified to inhibit DiI uptake with
N
N
N
N
R1
IC50 of <1
zole series characterized by the commercially-available compound
1a,29 with IC50 of 0.055
M in the primary assay (Table 1). We
lM, we focused our efforts in part on a bisamide–tetra-
O
R2
N
l
Compd R1
IC50
(
lM)
Compd R1
IC50 (lM)
a
a
deduced that such compounds could be made rapidly via Ugi 4-
component coupling reactions (Eq. 1),30–32 and thus would be
amenable to rapid structure–activity relationship (SAR) studies.
1a also showed no measurable cytotoxicity in the cell line used
for our assays (ldlA[mSR-BI]) after incubation for 24 h.
Additionally, 1a lacked non-selective behavior according to data
published in PubChem.33 Finally, in contrast to our hit compounds
in the indoline–thiazole series,16,17 members of the bisamide–te-
1a
0.055 0.014 1b
0.20
0.42
1.6
N
H
N
H
1c
1e
0.81
17
1d
1f
N
H
N
H
N
H
N
Me
trazole series showed improved aqueous solubility (114
lM for
1a in PBS with 1% DMSO).
N
R3
1g
7.1
R3
N
O
Ugi 4-CR
NH2
R
OH
a
Ar1
N
H
Ar1
ð1Þ
Average of at least two measurements in DiI uptake assay, standard error of
C
N R
O
O
R2
mean when n >2.
O
R2
We explored the SAR of the dipeptide scaffold by varying each
of the components of the Ugi reaction. Reactions were typically
performed using a 1:1:1:1 ratio of aldehyde, amine, carboxylic
were classified as inhibitors of DiI-HDL uptake out of 319,533 com-
pounds tested, with inhibition at 12.5 lM of P70% relative to 1 lM
BLT-1 as positive control. Hit compounds were omitted that were
Table 2
Tetrazole replacements
O
N
Ar1
O
N
H
a
a
Compd
Ar1
IC50
(l
M)
Compd
Ar1
IC50
37
(lM)
MeO
MeO
N
1a
0.055 0.014
2a
N
N
N
MeO
MeO
MeO
MeO
N
N
S
2b
2d
0.13
2c
6.3
O
MeO
MeO
0.023
N
O
a
Average of at least two measurements in DiI uptake assay, standard error of mean when n >2.

2596
C. Dockendorff et al. / Bioorg. Med. Chem. Lett. 25 (2015) 2594–2598
Table 3
Arene SAR
O
N
N
Ar2
N
N
N
H
O
N
Ar2
MeO
IC50
(lM)
Compd
Ar2
IC50 (lM)
a
a
Compd
1a
0.055 0.014
0.18
3a
0.37
0.38
0.62
MeO
MeO
F
3b
3d
3c
3e
OMe
Cl
F
>25
3f
3h
3j
3.8
3g
3i
0.49
0.13
Cl
Cl
F
0.17
0.18
O
3k
0.027 0.008
O
Cl
F
O
F
3l
0.23
O
a
Average of at least two measurements in DiI uptake assay, standard error of mean when n >2.
Table 4
Our results exploring tetrazole replacements are summarized in
Table 2. Replacement of the aryl tetrazole with a simple phenyl
group (2a) abrogated activity. Several heterocyclic replacements
for the tetrazole were prepared from commercially available ary-
lacetic acids: oxazole 2b showed approximately two-fold
SAR of central amide N-substituent
O
MeO
MeO
R4
N
O
N
O
Ar2
N
N
N
H
decreased activity (IC50 = 0.13
activity (IC50 = 6.3 M), and isoxazole 2d demonstrated excellent
potency (IC50 = 0.023 M). One possible explanation for these
lM), thiazole 2c possessed poor
O
N
Ar2=
A
B
l
l
R4
IC50
(
lM)
Compd
R4
Me
IC50
19
(lM)
a
a
Compd
results is that a hydrogen-bond acceptor at the 4-position (num-
bering of tetrazole ring system) is required for optimal activity.
Despite the excellent potency with isoxazole 2d, we elected to con-
tinue our studies with the tetrazole analogs, for which we had a
larger collection of building blocks.
1a
4a
Ar2 = A
Ar2 = A
0.055 0.014
7.5
4c
4b
Ar2 = A
0.11
>25
Next, the western end of the scaffold was examined in detail by
varying the aromatic substituent (Table 3). Removal of the 4-meth-
Ar2 = A
Me
4e
4d
Ar2 = A
N
oxy group of 1a led to
a significant drop in potency (3b,
Ar2 = A
1.6
IC50 = 0.18 M). Several 2- and 3-substituted arenes were exam-
l
ined with both electron-withdrawing and donating groups; these
were uniformly inferior to the 3,4-dimethoxyarene of 1a.
Several 4-substituents were tolerated to some extent, including
O
4f
Ar2 = B
0.84
chlorine (3h, IC50 = 0.17
best results were obtained with the 3,4-methylenedioxy group
(3k, IC50 = 0.027 M), which is more potent than 1a. We also exam-
ined the difluoromethylene analog of 3k (3l, IC50 = 0.23 M). Its
decreased activity relative to 1a, along with the decreased activity
of the 4-methyl analog (3g, IC50 = 0.49 M), suggests that an
lM) and fluorine (3i, IC50 = 0.13 lM). The
a
Average of at least two measurements in DiI uptake assay, standard error of
l
mean when n >2.
l
l
acid, and isonitrile in methanol, and stirring for 18 h. Variation of
the isonitrile component allowed us to explore the eastern amide
of the scaffold (Table 1). The SAR in this area was very sensitive
to modifications, as slight changes in structure from N-cyclopentyl
diminished the activity substantially, such as with N-cyclohexyl 1b
appropriate 4-aryl substituent may act as a hydrogen bond accep-
tor with the target.
We examined the effects of varying internal positions of the
structure while maintaining the tetrazole along with either a ter-
minal 3,4-dimethoxyphenyl or 3,4-methylenedioxyphenyl group
at the western end of the scaffold. A variety of amines were
screened in the Ugi reaction, leading to different substituents at
R4 (Table 4). The cyclohexyl substituent of 1a again proved
(IC50 = 0.20 lM) and N-isopropyl 1c (0.81 lM). The amide N–H is
required for high activity, as the tertiary amide 1f had low activity.
Compound 1f was prepared via a sequential peptide synthesis, as
selective methylation of 1a proved to be problematic.

C. Dockendorff et al. / Bioorg. Med. Chem. Lett. 25 (2015) 2594–2598
2597
Table 5
cyclohexyl group and the backbone nitrogen decreased activity
by greater than 20-fold (4d, IC50 = 1.6 M). Introducing a nitrogen
atom expected to be protonated at physiological pH abrogated all
SAR analysis of central carbon substituents
l
a
Compd
Structure
IC50 (lM)
activity (4e).
Next, substitution at the alpha carbon atom of the eastern
amide was investigated (Table 5). Introducing a second methyl
group gave a drastic drop in activity (5a), as did the replacement
of the methyl group of 1a with a bulkier isopropyl group (5b).
Compound 5c, lacking the methyl group of the parent compound
MeO
MeO
O
O
O
1a
0.055 0.014
N
N
N
N
N
N
N
H
O
O
O
N
3k, showed slightly improved activity (IC50 = 0.027 lM).
MeO
MeO
Finally, each enantiomer of 3k was prepared in a stepwise fash-
ion from (R)- or (S)-alanine (Scheme 1). The required tetrazole
acetic acid building block was made by cycloaddition between
benzonitrile 6 and sodium azide, then alkylation with methyl bro-
moacetate and hydrolysis of the resulting ester to give 8. (R)-N-Boc
alanine (9) was then coupled with aminocyclopentane using stan-
dard peptide coupling conditions (EDC, HOBt, (i-Pr)2NEt). The
resulting amide 10 was treated with TFA to liberate the amine,
then subjected to reductive alkylation with cyclohexanone and
sodium cyanoborohydride to generate the N-cyclohexylamine 12.
Peptide coupling reactions with this relatively hindered secondary
amine were sluggish, so 12 was reacted with the acid chloride of
tetrazole acetic acid 8 to generate the desired bisamide 5e. The
enantiomer 5d was prepared analogously from (S)-N-Boc-alanine.
5a
5b
3k
5c
5d
5e
19
N
N
N
N
H
N
MeO
MeO
>25
N
N
N
N
H
N
O
O
O
O
O
O
0.027 0.008
0.026 0.012
1.6
N
N
N
N
N
H
O
O
O
O
N
The (R)-isomer 5e (IC50 = 0.017
than its enantiomer 5d.
lM) was 100-fold more potent
O
O
N
N
N
N
N
N
N
H
Additional studies with 5e were performed to study its mode of
action. Its inhibitory action is reversible. In experiments where
cells were pre-treated with ML279 for 2 h, washed extensively
with PBS and then incubated with DiI-HDL, no inhibition was
observed. In addition to measuring the uptake of DiI from HDL in
the presence of 5e, the uptake of 3H-labeled cholesteryl oleate
ester ([3H]CE) from [3H]CE–HDL was measured with an IC50 of
H
N
O
O
N
N
N
N
H
N
0.005 lM (Supporting information Fig. 2), and is significantly more
potent than the clinical compound ITX-5061 in a head-to-head
study. The binding of Alexa-488-labeled HDL particles to
ldlA[mSR-BI] cells via SR-BI was also measured. As with BLT-1
and other inhibitors, including our recently disclosed probe
ML278, 5e enhanced the level of HDL binding to SR-BI, with a mea-
O
O
0.017 0.004
N
N
N
N
H
N
a
Average of at least two measurements in DiI uptake assay, standard error of
mean when n >2.
sured EC50 of 0.27 lM (Supporting information Fig. 3). One possi-
bility is that such compounds act to inhibit lipid transport by
slowing the turnover (release) of the bound HDL particles.
Finally, we tested to see if 5e was a general inhibitor of receptor-
mediated endocytosis by examining its effects on the endocytosis
of Alexa-594-labeled transferrin by ldlA[mSR-BI] cells. 5e showed
optimal. Conservative changes were tolerated to some degree
(such as 4c, R4 = cyclopentyl, IC50 = 0.11
lM), but clearly a bulky,
lipophilic group is optimal. Inserting a carbon atom between the
O
NH
N
N
Br
1)
O
OMe
CN
NaN3
O
O
O
O
N
OH
N
O
N
N
DMF
86%
K2CO3, acetone
76%
2) NaOH, DCM/MeOH
95%
O
N
7
6
8
O
H2N
O
O
O
TFA
H2N
BocHN
BocHN
OH
N
H
EDCI, HOBt
(i-Pr)2NEt
DCM
N
H
Et3SiH
NaCNBH3
83% (2 steps)
9
10
11
92%
O
OH
N
N
O
O
O
N
O
O
N
N
N
HN
O
N
N
N
H
N
(COCl)2
H
12
O
N
(i-Pr)2NEt, DCM
5e
72%
ML279
Scheme 1. Synthesis of ML279.

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C. Dockendorff et al. / Bioorg. Med. Chem. Lett. 25 (2015) 2594–2598
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no inhibition of this process, at concentrations up to 35 lM. This
result is consistent with the previous studies that indicated that
SR-BI does not mediate lipid uptake via receptor-mediated
endocytosis.34
In summary, potent inhibitors of SR-BI-mediated lipid uptake
were discovered as part of the NIH Molecular Libraries Probe
Production Centers Network (MLPCN) initiative. Profiling of several
top compounds led to the nomination of the bisamide tetrazole 5e
(ML279) as a probe compound. ML279 has superior solubility to
ML278 (28 lM vs 0.57 lM), though it is slightly less potent than
ML278 (IC50 = 17 vs 6 nM in the diI-uptake assay). It is also not
cytotoxic, has no significant chemical liabilities, shows reversible
inhibition, and appears to be selective, as determined by inspection
of PubChem assay results. ML279 is plasma stable, with >99%
remaining after incubation with human or mouse plasma, though
it suffers from a lack of metabolic stability as determined in a
microsomal stability assay (<1% remaining after 1 h with mouse
or human microsomes). Altogether, ML279 represents a promising
lead compound for the blocking of SR-BI.
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Acknowledgments
We thank Stephen Johnston, Carrie Mosher, Travis Anthoine,
and Mike Lewandowski for analytical chemistry support.
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Supplementary data
Supplementary data (general protocol for Ugi reactions, prepa-
ration and characterization of 5e (ML279), compound profiling
protocols, representative dose–response curves of ML279 in DiI-
HDL, [3H]CE uptake, and HDL binding assays, and assay protocols)
associated with this article can be found, in the online version, at
http://dx.doi.org/10.1016/j.bmcl.2015.03.074. These data include
MOL files and InChiKeys of the most important compounds
described in this article.
28. http://www.ncbi.nlm.nih.gov/books/NBK133438/ (accessed February 4, 2015).
29. Purchased from Asinex Ltd.
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33. It was listed as a hit in 4 assays not involving SR-BI out of 614 bioassays listed
on December 10, 2011, and a lack of dose–response data on any of these hits
means that none had apparently been confirmed as actives.
34. Nieland, T. J.; Ehrlich, M.; Krieger, M.; Kirchhausen, T. Biochim. Biophys. Acta
2005, 1734, 44.