Indolinyl-thiazole based inhibitors of scavenger receptor-BI (SR-BI)-mediated lipid transport

Article abstract of DOI:10.1021/ml500154q

A potent class of indolinyl-thiazole based inhibitors of cellular lipid uptake mediated by scavenger receptor, class B, type I (SR-BI) was identified via a high-throughput screen of the National Institutes of Health Molecular Libraries Small Molecule Repo

Full text of DOI:10.1021/ml500154q

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Letter
Indolinyl-thiazole based inhibitors of Scavenger
Receptor-BI (SR-BI)-mediated lipid transport
Christopher Dockendorff, Patrick W. Faloon, Miao Yu, Willmen Youngsaye, Marsha
Penman, Thomas J.F. Nieland, Partha Pratim Nag, Timothy A. Lewis, Jun Pu,
Melissa Bennion, Joseph Negri, Conor Paterson, Garrett Lam, Sivaraman Dandapani,
Jose Perez, Benito Munoz, Michelle A Palmer, Stuart Schreiber, and Monty Krieger
ACS Med. Chem. Lett., Just Accepted Manuscript • Publication Date (Web): 02 Feb 2015
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Indolinyl-thiazole based inhibitors of Scavenger Receptor-BI
(SR-BI)-mediated lipid transport
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Chris Dockendorff,*,†,§ Patrick W. Faloon,† Miao Yu,‡ Willmen Youngsaye,† Marsha Penman,‡ Thomas J.
F. Nieland,‡,† Partha P. Nag,† Timothy A. Lewis,† Jun Pu,† Melissa Bennion,† Joseph Negri,† Conor Pater-
son,† Garrett Lam,† Sivaraman Dandapani,† José R. Perez,† Benito Munoz,† Michelle A. Palmer,† Stuart L.
Schreiber†,∞ and Monty Krieger*,‡
† Center for the Science of Therapeutics, Broad Institute, 7 Cambridge Center, Cambridge, MA, 02142, USA
§ Department of Chemistry, Marquette University, P.O. Box 1881, Milwaukee, WI, 53201-1881, USA
‡ Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA, 02139, USA
∞ Howard Hughes Medical Institute, Broad Institute, 7 Cambridge Center, Cambridge, MA, 02142, USA
KEYWORDS: ML278; SR-BI inhibitor; HDL receptor; reverse cholesterol transport; indoline; thiazole; HTS; MLP; HCV
Supporting Information Placeholder
ABSTRACT: A potent class of indolinyl-thiazole based inhibitors of cellular lipid uptake mediated by Scavenger Receptor, class
B, type I (SR-BI) was identified via a high-throughput screen of the National Institutes of Health Molecular Libraries Small Mole-
cule Repository (NIH MLSMR) in an assay measuring the uptake of the fluorescent lipid DiI from HDL particles. This class of
compounds is represented by ML278 (17-11), a potent (average IC50 = 6 nM) and reversible inhibitor of lipid uptake via SR-BI.
ML278 is a plasma-stable, non-cytotoxic probe that exhibits moderate metabolic stability, thus displaying improved properties for
in vitro and in vivo studies. Strikingly, ML278 and previously described inhibitors of lipid transport share the property of increas-
ing the binding of HDL to SR-BI, rather than blocking it, suggesting there may be similarities in their mechanisms of action.
The inverse correlation between human plasma HDL cho-
lesterol (HDL-C) levels and risk for adverse events from ath-
erosclerotic coronary artery disease (CAD)1,2 has generated
considerable interest in developing novel therapies that in-
crease HDL-C levels by several different mechanisms,3 most
prominently by CETP (cholesteryl ester transfer protein) inhi-
bition.4 Despite massive investments in the clinical study of
these inhibitors, the strategy of decreasing CAD risk by artifi-
cially boosting HDL-C levels has yet to be validated, and our
understanding of lipid trafficking and regulation remains in-
complete. New pharmacologic tools that selectively modulate
HDL-C via different targets would be valuable for in vitro
mechanistic studies and in vivo functional analyses. Here we
attempted to identify compounds that can modulate the actions
of Scavenger Receptor, class B type I (SR-BI),5 a mammalian
high density lipoprotein (HDL) receptor that binds HDL parti-
cles on the cell surface and mediates transport of unesterified
cholesterol (UC) or cholesteryl esters (CE). Unlike LDL-
receptor mediated uptake of lipids, this process is independent
of endocytosis.5,6 In vivo, the structure and composition of
plasma HDL, and the metabolic fates of its cholesterol, are
controlled by SR-BI.7 In fact, SR-BI has important influences
on the gastrointestinal, endocrine and reproductive systems, as
well as development, inflammation/host defense, Hepatitis C
Virus (HCV) infection, and cardiovascular physiology.7–13
Thus, SR-BI is an interesting drug target, particularly for
compounds that may modulate cholesterol levels or inhibit
HCV infection. However, despite several informative, mecha-
nistic studies14–16 the precise details of HDL recognition by
SR-BI and consequent lipid uptake and efflux remain unclear;
new chemical probes may help further our understanding of
these processes.17
We and others have previously discovered small molecule
inhibitors of SR-BI (some examples in Figure 1), which have
subsequently proven to be valuable tools in the study of the
activities of SR-BI in vitro and in vivo, including lipid
transport and lipoprotein metabolism (BLTs,18 HDL376,19,20
R-13832921), and HCV infection (ITX-506122,23). Unfortunate-
ly, the reported disadvantages of these tool compounds limit
their utility, including toxicity (BLT-1), weak potency (BLT-
3, BLT-4), specificity (mitogen-activated protein kinase activi-
ty; ITX-5061), and potential safety issues for humans
(HDL376). ITX-5061 also recently showed disappointing re-
sults in a Phase 1b clinical trial.24 It would be valuable to iden-
tify additional potent SR-BI chemical modulators that lack
detrimental side effects25 and that might selectively modulate
lipid uptake or efflux.
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Cl
H
N
Table 1. Amide analogs
S
N
S
1
2
NH2
Ph
Ph
N
H
N
N
O
H
O
R
S
N
3
4
5
6
7
8
BLT-1
HDL376
BLT-3
HN
O
O
N
O
O
N
N
MeO HN
HN
O
O
N
O
X
N
H
S
O
O
Cmp
R
IC50
(µM)a
Cmp
5-14
5-15
5-16
5-17
5-18
R
IC50
(µM)a
Cl
X = NO2 : R-138329
X = NHAc : R-154716
ITX-5061
O
9
0.047
±0.009
5-1
0.14
0.97
2.2
O
O
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11
12
13
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N
FIGURE 1. Some reported inhibitors of SR-BI
Scheme 1. Synthesis of amide analogs
5-2
5-3
9.8
5.4
O
O
S
O
O
Cl
Cl
Cl
Cl
N
H
O
N
AlCl3
5-4
5-5
2.1
1.4
( -Pr) NEt
DCM
95%
i
2
1
2
N
F
DCE, 50°C
85%
3
O
O
O
N
0.90
0.77
R
S
O
Cl
S
S
HN
O
H2
N
N
H2
N
NH
2
N
R
Cl
O
5-6
5-7
14.1
1.6
5-19
5-20
5-21
5-22
5-23
1.5
N
N
N
EtOH, reflux
84%
(i-Pr)2NEt
DCE, 60°C
5
4
CN
O
O
0.17
N
O
MeO
Under the auspices of the NIH’s Molecular Libraries Probe
Production Centers Network (MLPCN), we used a high-
throughput screening approach to identify potent SR-BI modu-
lators with low toxicities and favorable physicochemical prop-
erties that may be useful as in vitro and in vivo probes of SR-
BI mechanism and function, and that might also be promising
lead compounds for drug discovery. The screen involved
measuring the effects of cellular uptake of the fluorescent lipid
1,1’-dioctadecyl-3,3,3’,3’-tetramethylindocarbocyanine per-
chlorate (DiI) from HDL particles into CHO cells overexpress-
ing mouse SR-BI (ldlA[mSR-BI]).26 An initial probe report is
available,27 and assay data may be found in the PubChem da-
tabase (http://pubchem.ncbi.nlm.nih.gov, AID 488952).
0.06
±0.02
5-8
5-9
0.58
0.26
0.29
S
Cl
OMe
NTb
N
OMe
0.09
±0.06
5-10
N
OCF3
MeO
0.03
±0.01
5-11
5-12
5-13
NTb
0.53
0.26
5-24
5-25
5-26
N
OMe
N
0.22
O
O
OMe
Of the 319,533 compounds tested in duplicate at 12.5 µM,
3,046 compounds (0.96%) reduced cellular fluorescence with
inhibition of ≥ 70% of that of 1 µM BLT-1, the positive con-
trol and one of the most potent inhibitors readily available. Hit
compounds that were on plates with Z’ < 0.3 or that were ac-
tive in 10% or more of the HTS assays listed in PubChem
were eliminated as either unreliable or too nonspecific. From
the primary screen, a variety of scaffolds was verified to inhib-
it DiI uptake with IC50s of < 1 µM. 127 out of 186 of the se-
lected hits that showed dose-dependent inhibition of DiI up-
take were rejected for further study because secondary screen-
ing established that they quench the intrinsic fluorescence of
DiI in DiI-HDL. We describe here our structure–activity anal-
ysis of one of the most potent scaffolds, represented by the
commercially available indolinyl-thiazole 5-1, with an IC50 of
0.047 µM (Table 1). This scaffold was chosen for further
analysis in part for its modularity and synthetic tractability,
and for its lack of measurable toxicity after incubation with
CHO cells for 24 h. Furthermore, 5-1 appeared to lack non-
selective behavior based on data in PubChem.28
0.12
±0.09
O
F
OMe
a Average of at least two measurements in DiI uptake assay, ± standard
error of mean when n > 2. b Insoluble in DMSO. NT = not tested.
We explored structure–activity relationships (SAR) of the
scaffold by first varying the N-substituent of the aminothia-
zole. The indolinyl-aminothiazole core of 5-1 was prepared
via a simple 3-step sequence (Scheme 1). Indoline 1 was acyl-
ated with propionyl chloride, and the resulting amide 2 was
subjected to Friedel-Crafts acylation with chloroacetyl chlo-
ride. The chloroketone 3 was condensed with thiourea to pro-
vide the desired aminothiazole 4. The poor solubility and nu-
cleophilicity of 4 required heating with the acid chloride cou-
pling partners for optimal preparation of a series of amide
derivatives of 5 (Table 1). Caution should be taken with the
interpretation of the SAR analysis in this series, as the com-
pounds nearly all have measured solubility in PBS of <1 µM.
Despite these issues, the top compounds in this report showed
reproducible inhibition and were very potent, providing low
nanomolar IC50 values.
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Table 2. Functional group modifications and central ring
SAR
Table 3. Modifications to indolinyl amide
4
5
6
7
8
9
Cmp
Structure
IC50
(µM)a
S
N
H2N
Cmp
5-1
Ar
R
IC50
(µM)a
4
16.5
0.12
N
0.047
±0.009
O
2-furyl
O
MeO
MeO
O
S
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
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N
N
3,5-
(MeO)Ph
0.03
±0.01
Me
5-24
17-1
17-2
17-3
17-4
17-5
17-6
17-7
17-8
17-9
17-10
17-11
6
O
N
O
3,5-
(MeO)Ph
0.11
MeO
MeO
H
S
N
HN
3,5-
(MeO)Ph
0.08
±0.06
7
8
0.37
18.9
O
N
O
O
2-furyl
2-furyl
2-furyl
2-furyl
0.24
0.39
0.44
4.1
O
O
N
HN
O
O
O
N
O
S
O
Me
O
O
HN
N
O
S
O
HN
Ph
9
10.1
14.3
N
O
O
O
N
HN
3,5-
(MeO)Ph
0.046
±0.13
N
10
O
OMe
N
Boc
O
3,5-
(MeO)Ph
0.004
±0.003
S
N
O
O
HN
0.3
±0.3
11
F3CO
N
3,5-
(MeO)Ph
0.002
O
N
H
O
3,5-
(MeO)Ph
0.013
±0.007
NMe2
O
a Average of at least two measurements in DiI uptake assay, ± standard
error of mean when n > 2. b Insoluble in DMSO. NT = not tested.
3,5-
(MeO)Ph
0.006
±0.0003
OMe
O
A number of heterocyclic analogs (Table 1, 5-2–5-14) were
examined to find a replacement for the furan of 5-1, which is a
potential toxicophore.29 None of these compounds provided a
level of inhibition comparable to 5-1. A number of aliphatic
(5-15) and aromatic (5-16–5-26) analogs were prepared, and
analogs with a 3-alkoxybenzene substituent (5-21, 5-23, 5-24,
5-26) provided high levels of inhibition with IC50s in the range
of 30 to 120 nM.
a Average of at least two measurements in DiI uptake assay, ± standard
error of mean when n > 2.
SAR studies were continued by modifying the indoline N-
substituent; a representative synthesis is provided in Scheme
2. Protection of the indoline nitrogen with a phenylsulfonyl
group provided an intermediate (12) that underwent Friedel-
Crafts acylation with chloroacetyl chloride to yield ketone 13
in high yield. The sulfonamide could be hydrolyzed in the
presence of the chloroketone by heating in sulfuric acid. The
resulting indoline 14 was subsequently condensed with thiou-
rea to generate a 2-aminothiazole, which reacted with Boc2O
exclusively at the indoline nitrogen to generate carbamate 15.
The free amine of 15 was acylated with the desired acid chlo-
rides, the Boc group was removed with TFA, and the indoline
nitrogen was acylated to provide compounds 17 (Table 3).
We next modified the central heterocyclic ring, as well as
the adjacent amide functionality (Table 2). The parent amino-
thiazole 4 showed poor activity. N-methylation of 5-24 (6) or
reduction of the amide (7) gave compounds with attenuated
activity. A number of heterocyclic replacements for the central
thiazole were prepared, including oxazole 8, imidazole 9, and
1,2,4-oxadiazole 10. The activities sharply decreased in all
cases. A 5-methyl group was tolerated on the thiazole (11),
which may decrease the potential of thiazole ring oxidation by
CYP enzymes to give toxic metabolites.30 Compound 11 also
demonstrates that a trifluoromethoxy group could be a poten-
tial replacement of the 3-methoxy substituent.
Removal of (17-1) or shortening (17-2) the indolinyl acyl
group of 5-24 did not improve activity; whereas, the addition
of a methyl group to the ethyl chain of compound 5-1 (17-3)
decreased potency. The sulfonamides 17-4 and 17-5 showed
an approximately 10-fold drop in potency relative to 5-24, and
the N-allyl indoline 17-6 showed only weak inhibition. More
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positive results were obtained with compounds 17-7 to 17-11. potential for further development of the indolinyl-thiazole
1
2
3
4
5
6
7
8
Both smaller and bulkier substituents were well tolerated with
compounds possessing the western 3,5-dimethoxybenzene
moiety. The N-Boc compound 17-8 showed excellent potency
in the DiI-uptake assay (4 nM), as did the urea 17-9 (2 nM)
and the methoxyacetamide 17-11 (6 nM).
compound class. None of the compounds showed any signifi-
cant cytotoxicity after incubation with the ldlA[mSR-BI] cells
for 24 h, and in fact compounds 6 (CC50 = 15 µM) and 17-10
(CC50 = 20 µM) were the only ones with measurable cytotox-
icities.31 Solubility is an issue with this series of compounds,
as all of the compounds tested with low nanomolar IC50s have
solubilities of < 1 µM. The methoxyacetamide 17-11 showed
excellent potency (IC50 = 6 nM), measurable solubility (0.57
µM), and excellent stability in human plasma (>99% remain-
ing after 5 h, with 94% plasma protein-bound). 17-11 was
nominated as a probe (ML278) as part of the NIH Molecular
Libraries Probe Production Centers Network (MLPCN) initia-
tive.
Modifications to the indoline ring itself were also examined.
A selection of our results is provided in the Supporting Infor-
mation (Table S1). A range of anilines and oxindoles showed
good to excellent potencies, though none were superior to the
top indoline compounds, and they also suffered from very low
solubilities (<1 µM).
9
10
11
12
13
14
15
16
17
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Several of our more promising compounds were profiled in
secondary assays to gain insights into the mode of action and
Scheme 2. Representative synthesis of analogs with alternative indoline N-substituents
O
O
O
Cl
Cl
PhSO2Cl
H2SO4
Cl
Cl
14
13
N
H
N
N
N
NEt3
DCM, 92%
AlCl3
DCE, 88%
H
12
1
SO2Ph
SO2Ph
MeO
MeO
MeO
S
S
O
O
H2N
H2N
NH2
1.
S
Cl
N
MeO
HN
16
EtOH
2. Boc2O
56% (over 3 steps)
15
N
N
Boc
(i-Pr)2NEt
DCM, 67%
N
Boc
1. TFA, Et3SiH
93%
MeO
MeO
O
O
S
N
2.
MeO
HN
Cl
(i-Pr)2NEt, DMAP
DCM, 79%
N
O
17-11
ML278
OMe
0
AC50= 0.035 !M
100
50
0
-50
-100
IC50= 0.00093 !M
Log Concentration [M]
Log Concentration [M]
FIGURE 2. DiI-HDL uptake assay with ML278 (left); Alexa488-HDL binding assay with ML278 (right).
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Funding Sources
Additional mechanistic studies with ML278 were per-
Funding for this work was provided in part by the NIH-MLPCN
program (1 U54 HG005032-1 awarded to S.L.S.) and NIH
grants HL052212 and HL066105 to M.K.
formed to obtain details on its mode of action. First, in ex-
periments where cells were pre-treated with ML278 for 2
hours, washed extensively with PBS and then incubated with
DiI-HDL, sharply reduced levels of inhibition were ob-
served. This demonstrates that the inhibitory action of
ML278 is reversible. In addition to inhibiting the selective
uptake of the synthetic lipid tracer DiI from HDL into
ldlA[m-SR-BI] cells (Table 3 and Figure 2, left frame),
ML278 inhibited uptake of the physiological relevant
[3H]labeled cholesteryl oleate ester ([3H]CE) from [3H]CE-
HDL (calculated IC50 = 7 nM, Supporting Information Fig-
ure S1). Its potency in these assays is far greater than the
clinical compound ITX-5061 (IC50 = 0.94 µM , see compari-
son in Table S2). We also showed that, as was the case for
BLT-1 and other SR-BI inhibitors, ML278 enhanced the
Notes
The authors declare no competing financial interests.
ACKNOWLEDGMENT
We thank Stephen Johnston, Carrie Mosher, Travis Anthoine,
and Mike Lewandowski for analytical chemistry support.
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ABBREVIATIONS
Boc, tert-butoxycarbonyl; CC50, half-maximal cytotoxic concen-
tration; CE, cholesteryl ester; CETP, cholesteryl ester transfer
protein; CHO, Chinese Hamster Ovary; CYP, cytochrome P450;
DCM, dichloromethane; DCE, 1,2-dichloroethane; DMAP, 4-
(N,N-dimethylamino)-pyridine; DiI, 1,1’-dioctadecyl-3,3,3’,3’-
tetramethylindocarbocyanine perchlorate; EC50, half-maximal
effective concentration; EtOH, ethanol; HCV, Hepatitis C virus;
HDL, High Density Lipoprotein; HDL-C; High Density Lipo-
protein Cholesterol; HTS, high-throughput screen; IC50, half-
maximal inhibitory concentration; MAPK, mitogen-activated
protein kinase; MLP, Molecular Libraries Program; MLPCN,
Molecular Libraries Probe Production Centers Network;
MLSMR, Molecular Libraries Small Molecule Repository; NIH,
National Institutes of Health; NO, nitric oxide; NT, not tested;
PPR, Pattern Recognition Receptor; SAR, structure-activity
relationship; SR-BI, Scavenger Receptor Class B, Type I; TFA,
2,2,2-trifluoroacetic acid; Z’, Z’-factor, a measure of assay qual-
ity calculated from the variability of positive and negative con-
trols.32
binding of fluorescent Alexa448-HDL to SR-BI (EC50
=
0.035 µM) (Figure 2, right frame). Thus, ML278 joins a
growing list of small molecules that inhibit lipid transport
mediated by SR-BI yet increase the binding of HDL to SR-
BI.18
The potential utility of ML278 as a probe for in vivo stud-
ies was investigated by measuring its metabolic stability in
the presence of liver microsomes (Table S2). The compound
shows moderate stability, with 75% remaining after 1 h in-
cubation with CD-1 mouse liver microsomes, and 48% re-
maining with human liver microsomes. Additionally, com-
petitive binding studies were performed with a panel of 67
different receptors and secondary targets (Eurofins Panlabs).
At a concentration of 10 µM, 11 targets were inhibited by
20% or more, with the highest level of inhibition observed
with the Adenosine A3 receptor (43% inhibition).
In summary, potent inhibitors of SR-BI-mediated lipid
(DiI) uptake were discovered as part of the NIH Molecular
Libraries Program. Profiling of several top compounds led to
the nomination of the indolinyl-thiazole 17-11 (ML278) as a
probe compound. SAR studies demonstrated that the thiazole
of ML278 was required for activity, flanked by a benzamide,
optimally with a 3-methoxy substituent. ML278 shows supe-
rior potency in the uptake of the synthetic lipid tracer DiI , as
well as [3H]CE, compared to the prior art compounds BLT-1
and ITX-5061. ML278 also shows no cytotoxicity, has no
significant chemical liabilities, shows reversible inhibition,
and appears to act selectively at SR-BI. Additionally, it has
excellent plasma stability and moderate metabolic stability.
ML278 is expected be a valuable tool compound for further
in vitro and in vivo studies involving SR-BI.
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ASSOCIATED CONTENT
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Supporting Information. Additional SAR data; preparation
and characterization of 17-11 (ML278); probe comparison table;
representative dose-response curves of ML278 and ITX-5061 in
[3H]CE uptake assay; compound profiling and assay protocols.
This material is available free of charge via the internet at
http://pubs.acs.org.
(6) Brown, M. S.; Goldstein, J. L. A receptor-mediated
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(7) Rigotti, A.; Miettinen, H. E.; Krieger, M. The role of the
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lism of endocrine and other tissues. Endocr Rev. 2003, 24,
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AUTHOR INFORMATION
Corresponding Authors
Email: christopher.dockendorff@marquette.edu. (C.D.)
Email: Krieger@mit.edu (M.K.)
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enger receptor type B, class I: A promising immunotherapy
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scavenger receptor B1 antagonist: resistance selection and
activity in combination with other hepatitis C virus antivi-
rals. J. Infect. Dis. 2012, 205, 656-62.
(12) Catanese, M. T.; Graziani, R.; von Hahn, T.; Moreau,
M.; Huby, T.; Paonessa, G.; Santini, C.; Luzzago, A.; Rice,
C. M.; Cortese, R.; Vitelli, A.; Nicosia, A. High-avidity
monoclonal antibodies against the human scavenger class B
type I receptor efficiently block hepatitis C virus infection in
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(25) Researchers at iTherX recently reported the structure of
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son, A.; Gao, H.; Lee, H.; Chow, S.; Pal Kaur, M.; Nguyen,
N.; Zamboni, R.; McKelvy, J.; Wong-Staal, F.; Macdonald,
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SR-B1 required for normal receptor oligomerization and
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(17) During the preparation of this manuscript, the x-ray
crystal structure of LIMP-2 was determined, a pattern-
recognition recognition receptor in the same CD36 super-
family as SR-BI: Neculai, D.; Schwake, M.; Ravichandran,
M.; Zunke, F.; Collins, R. F.; Peters, J.; Neculai, M.; Plumb,
J.; Loppnau, P.; Pizarro, J.-C.; Seitova, A.; Trimble, W. S.;
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Amide required for optimal activity
Thiazole required
MeO
MeO
O
Indoline preferred, but substitutions
and ring cleavage tolerated
S
HN
N
Arene required
N
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Optimal activity with
H-bond acceptor at 3-position
O
OMe
ML278
Numerous groups are tolerated
diI-HDL uptake assay: IC50 = 6 nM
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