Investigation of N-aryl-3-alkylidenepyrrolinones as potential Niemann-Pick type C disease therapeutics

Article abstract of DOI:10.1021/jm900707n

A five-step synthesis of an array of N-aryl-3-alkylidenepyrrolinones, which are potential Niemann-Pick type C (NPC) disease therapeutics, is described. The synthetic route allows for the production of analogues, including photoaffinity and biotinylated de

Full text of DOI:10.1021/jm900707n

6494 J. Med. Chem. 2009, 52, 6494–6498
DOI: 10.1021/jm900707n
Investigation of N-Aryl-3-alkylidenepyrrolinones as Potential Niemann-Pick
Type C Disease Therapeutics
Casey C. Cosner,† John T. Markiewicz,† Pauline Bourbon,† Christopher J. Mariani,† Olaf Wiest,† Madalina Rujoi,‡
Anton I. Rosenbaum,‡ Amy Y. Huang,‡ Frederick R. Maxfield,‡ and Paul Helquist*,†
†Department of Chemistry and Biochemistry, University of Notre Dame, 250 Nieuwland Science Hall, Notre Dame, Indiana 46556, and
‡Department of Biochemistry, Weill Medical College of Cornell University, 1300 York Avenue, New York, New York 10065
Received May 25, 2009
A five-step synthesis of an array of N-aryl-3-alkylidenepyrrolinones, which are potential Niemann-
Pick type C (NPC) disease therapeutics, is described. The synthetic route allows for the production of
analogues, including photoaffinity and biotinylated derivatives. Compound 1a increased esterification
by acyl-coenzyme A:cholesteryl acyltransferase in NPC1 mutant cells. It also decreased LDL uptake
and increased cholesterol efflux in both NPC1-deficient and normal cells.
Introduction
Niemann-Pick type C disease (NPCa)1 is a rare autosomal
recessive lipid storage disorder with most commonly a fatal
neurodegenerative course.2 The disease mainly affects chil-
dren, but juvenile and adult forms of the disease are also
known. Some of the clinical symptoms of this disease include
liver abnormalities, learning difficulties, epilepsy, vertical gaze
palsy, seizures, neonatal jaundice, and finally neurodegenera-
tion, which is the most common cause of death.1a On the
cellular level, the most pronounced observation is an abnor-
mal accumulation of lipids such as cholesterol, glycosphingo-
lipids, and some phospholipids in late endosome/lysosome
(LE/LY)-like storage organelles (LSOs).3
Figure 1. Pyrrolinones of general structure 1.
the Maxfield laboratory was able to screen an initial library of
14956 compounds and found that several of these compounds
lowered the filipin fluorescence intensity in the lysosomal
storage compartments of CT60 and CT43 NPC1 mutant
Chinese hamster ovary (CHO) cells. This finding indicated
that the free cholesterol content of these organelles was
NPC disease is caused by mutations in one of two genes,
npc1 (95% of all cases) and npc2.4 The npc1 gene encodes the
1278-residue integral membrane NPC1 protein,5 which con-
tains 13 putative transmembrane domains. The npc2 gene
encodes the soluble protein NPC2 found in endosomes and
possesses a cholesterol binding site.6 It has recently been
reported that while both NPC1 and NPC2 proteins bind
cholesterol, NPC2 accelerates the transfer of cholesterol from
NPC1 to liposomes, indicating a potential necessary function
of the two proteins.7 There is currently no cure for NPC
disease. While attempts have been made to develop effective
treatment options,1d,8 there remains a significant need to
discover and develop useful therapeutics to treat this disease.
In 2006, the Maxfield laboratory reported an automated
filipin-based high-throughput screening procedure that mea-
sures the levels of filipin-bound cholesterol in the LSOs and
also overall in the cell, based on images of cells stained with
filipin, a fluorescent antibiotic.9 By using this new technique,
reduced.
Of the initial set of 14956 compounds, several pyrrolinones
of general structure 1 (Figure 1) exhibited significantly above
average activity and consistent dose response. In addition to
this activity, some of these compounds showed only mild
toxicity toward normal cells in contrast to many of the other
hit compounds. The dual observation of cholesterol reduction
and mild toxicity of this pyrrolinone family made them an
attractive target for further investigation as potential thera-
peutic leads for NPC disease. As such, we sought to develop a
general synthesis that would allow the efficient production of a
series of analogues for assay of activity in NPC1-deficient cells.
The original source of 1 for screening was a commercially
obtained library for which the details of synthesis were not
available and from which only very limited quantities of 1 of
questionable purity could be obtained. Therefore, the initial
goal of the work reported in this paper was to develop an
efficient, flexible, high-throughput pathway to the desired
pyrrolinones with a variety of substitution patterns with both
good quantity and purity. In addition to providing a series of
analogues, a synthesis ofpurecompoundswouldminimizethe
risk of false positives in further assays. A survey of the
literature revealed that there have been previous reports of
the synthesis of the core 3-alkylidene-4-pyrrolin-2-one scaf-
fold.10 Many of these routes utilize 3-alkylidene-furan-2-ones
as starting materials.10a,10b11 We found that these protocols
were not amenable to our needs. Given the deficiencies in
*To whom correspondence should be addressed. Phone: (574)-631-
7822. Fax: (574)-631-6652. E-mail: phelquis@nd.edu.
a Abbreviations: ACAT, acyl-coenzyme A:cholesteryl acyltransfer-
ase; CHO cells, Chinese hamster ovary cells; DiI, 1,10-dioctadecyl-
3,3,30,30-tetramethylindocarbocyanine perchlorate; DMAP, 4-dimethy-
lamino pyridine; EDC, N-(3-dimethylaminopropyl)-N0-ethylcarbodii-
mide hydrochloride; GC, gas chromatography; LDL, low-density
lipoprotein; LE/LY, late endosomes/lysosomes; LSO, LE/LY-like or-
ganelles; NPC, Niemann-Pick type C disease; TMSN3, trimethylsilyl
azide.
r
pubs.acs.org/jmc
Published on Web 09/22/2009
2009 American Chemical Society

Brief Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20 6495
Scheme 1a
a Reagents and conditions: (a) 3-benzoylpropionic acid, EDC, DMAP, CH2Cl2, 0°C, 80%; (b) AcCl, DMAP, reflux, 90%; (c) NaBH4, MeOH, 0 °C,
85%; (d) 6a, NaOAc, Ac2O, 100 °C, 75%; (e) LiI, pyridine, reflux, 95%.
Scheme 2a
a Reagents and conditions: (a) (i) NaNO2, HCl; (ii) 2-furaldehyde,
CuCl2, acetone.
previous syntheses, we set out to establish our own synthetic
pathway toward the production of 1.
Figure 2. Library of pyrrolinone analogues produced by the gen-
eral scheme.
Chemistry
In addition to the study of analogues, another strategy to
develop improved therapeutic agents is to identify their bio-
logicaltargets. Withthisinformation in hand, the mechanisms
of action can be probed and a combination of experimental
and computational methods can be employed to design the
improved compounds. Wethereforesetouttoobtain a deriva-
tive of 1a for photoaffinity labeling studies to identify its
biological target.15 Of the many classes of photoaffinity
labeling agents available, the azides are particularly appealing
for the case of 1a in that the bromide substituent should
be suitable for nucleophilic exchange to produce the azide
derivative 10.
The synthesis of 10 (Scheme 3) commenced with an at-
tempted copper-catalyzed aromatic substitution known to
produce azides from the corresponding bromides.16 We found
that the azide was presumably produced but was quickly
converted to the amine 9 before consumption of the starting
material was completed. This reaction was driven fully to the
amine by the use of a stoichiometric equivalent of cuprous
iodide. Amine 9 was converted to azide 10 using Moses’ mild
diazo transfer reaction.17
Alternatively, we also sought to obtain a derivative for
biological target identification by using a biotin label18 in the
form of 11. The synthesis of this labeled compound began by
coupling known aminoazide 1219 to 1a using EDC to produce
13 (Scheme 4). The completion of the synthesis of the labeled
compound was based on using the biotin propargylamide
15.20 This compound was prepared from the readily available
biotin-NHS ester 14 and propargyl amine.21 With both units
in hand, a copper-catalyzed cyclization22 between the azide
13 and terminal alkyne 15 resulted in the final biotinylated
Our general route is illustrated by the synthesis of 1a
(Scheme 1). Coupling methyl p-aminobenzoate 2 to benzoyl-
propionic acid using 1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide hydrochloride (EDC) and catalytic 4-dimethyl-
aminopyridine (DMAP) afforded ketoamide 3. Ketoamide
3 was cyclized to acetoxypyrrole 4 after refluxing in acetyl
chloride.12 The acetyl group was cleaved using sodium boro-
hydride in methanol, providing an inconsequential mixture
of 5a and 5b in approximately a 95:5 ratio. The use of strong
hydrolyzing agents such as hydroxide in this step led to
decomposition of the substrates and ring-opened products.
We next prepared 6a (Scheme 2) by diazotization of
4-bromoaniline (8a), followed by a copper-promoted reac-
tion with furfural.13 The penultimate aldol step involved
submitting the mixture of 5a and 5b with 6a to Perkin
condensation conditions. The Z-olefin 7 was the only
observed product as determined by NOESY NMR studies.
The final deprotection step using lithium iodide in reflux-
ing pyridine afforded the free acid 1a.14 Using this path-
way, we have produced up to 5 g of 1a in highly pure form
compared to the mg quantities of less pure material
obtained from the commercial library.
To demonstrate the flexibility of our pathway and to obtain
a series of analogues for NPC assays, we applied our route to
the syntheses of 1b-g (Figure 2). To obtain these analogues,
the individual components were modified. The methyl ester
2 was replaced with a benzyl ester, the starting benzoylpro-
pionic acid was derivatized to include a chlorine, and the
5-arylfurfural 6a was replaced by aldehydes 6b and 6c as well
as by p-nitrobenzaldehyde.

6496 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20
Cosner et al.
analogue 17. The use ofboththe biotinylated analogue11and
the azide 10 will be the subjects of a separate investigation of
cellular targets.
delivery to LE/LY are all normal in NPC-deficient cells, the
rate of cholesterol efflux from the LE/LY is severely re-
duced.25 As a consequence, there is an obstruction in choles-
terol trafficking and hence a significant decrease in the
homeostatic responses in NPC-deficient cells.
Biological Activity
We investigated the effect of 1a on cholesterol efflux and on
the uptake of LDL-derived cholesterol in NPC1-deficient
cells. We also evaluated the overall levels of cholesteryl ester
in the cell and the esterification promoted by ACAT in the
presence of this compound. Experimental results, discussed
below, are summarized in Figure 3 and Table 1.
The effects of pyrrolinones 1a-g on cholesterol levels in the
LSOs in CT60 and CT43 NPC1 mutant CHO cells had been
previously assayed using a filipin-based microscopy method.9
Of these compounds, only 1a (labeled as 1a13 in the previous
publication)9 exhibited significant reduction of cholesterol
accumulated in the lysosomal storage organelles. Using a
filipin-based image analysis assay that quantifies cholesterol
accumulation in the LSOs (see details in ref 9 for the assay
called LSO compartment ratio assay), it had been shown
that 1a caused an 80% decrease in fluorescence in CT43
cells and 30% in CT60 cells, at a concentration of 10 μM.9
The analogues 1b-1g showed no corresponding activity.
Therefore, 1a was selected for more thorough study.
In particular, the effects of 1a on cholesterol metabolism
and transport in CT60 NPC1-defective CHO cells were
investigated. In cells with normal sterol trafficking, cholester-
ol (mostly esterified) is internalized into cells via lipoproteins
and delivered to LE/LY, where hydrolysis of cholesteryl esters
by lysosomal acid lipase takes place.23 Free cholesterol is then
exported from the LE/LY and delivered to the plasma mem-
brane and extracellular acceptors, as well as the endoplasmic
reticulum, where cholesterol is esterified by the acyl-coenzyme
A:cholesterol acyltransferase (ACAT) and deposited as lipid
droplets.24 Cholesterol esters formed by ACAT are then
hydrolyzed by cytoplasmic neutral cholesterol ester hydro-
lases. While low-density lipoprotein (LDL) uptake and its
We measured the effect of compound 1a on cholesterol
effluxtoextracellularacceptorsinthe serum (e.g., highdensity
lipoproteins) in the following cell lines: NPC1-deficient cell
line CT60, its non-NPC1 parental line 25RA, and the non-
NPC1 cell line TRVb1. 25RA cells are CHO cells with a gain
of function mutation in the sterol regulatory element-binding
protein cleavage activating protein (SCAP) gene. TRVb1 cells
are apparently normal CHO cells transfected with a human
transferrin receptor.27 The major cholesterol efflux from the
CHO cells would be mediated by ATP-binding cassette
transporter.28 Changes in the efflux in the presence of 1a were
compared to solvent-treated control cells. We observed
(Figure 3) that compound 1a increased efflux not only in
NPC1-deficient CT60 cells but also in the 25RA and TRVb1
cells, which have normal NPC1.
We studied the direct effect of 1a on LDL endocytosis by
incubating the cells for a short time (35 min) with 1a and DiI-
labeled LDL. We also tested the longer term effect of 1a on LDL
uptake by incubating the cells for 18 h with 1a and DiI-LDL. We
observed that both in NPC1-deficient CT60 cells as well as the
NPC wild type 25RA parental cells 1a causes a significant
inhibition of LDL internalization (DiI-LDL uptake) during
long (18 h) treatments. However, no significant changes in DiI-
LDL uptake during short (35 min) incubations were observed,
indicating that there is not a direct effect on LDL binding.
When we measured the overall cellular content of choles-
teryl esters by gas chromatography (GC), we found no
significant change in the overall cholesteryl ester levels
(Table 1). However, when we tested the effect of 1a on
cholesterol esterification by ACAT by quantifying the incor-
poration of [14C]-oleic acid into cholesteryl-[14C]-oleate, we
Scheme 3a
a Reagents and conditions: (a) Na2CO3, NaN3, CuI, N,N0-dimethyl-
ethylenediamine, DMSO, 85%; (b) tBuONO, TMSN3, DMSO, 74%.
Scheme 4a
a Reagents and conditions: (a) 1a, EDC, DMAP, pyridine, 65%; (b) propargylamine, DMF, 92%; (c) 15, CuSO4, sodium ascorbate, acetone, 73%.

Brief Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20 6497
cellular cholesteryl esters (Table 1) may seem paradoxical
because there is increased esterification by ACAT. However,
it has been reported that net hydrolysis of lipoprotein-derived
cholesteryl esters in lysosomes stalls when levels of unesteri-
fied cholesterol in these organelles becomes high, as would be
the case in NPC1-deficient cells.29 The amounts of cholesteryl
esters in the LSOs of untreated NPC1-deficient cells is not
known, but loss of this cholesteryl ester pool may balance the
increase in the cytoplasmic lipid droplets. We also found that
total unesterified cellular cholesterol as measured by GC was
increased in the cells treated with 1a, and total filipin fluores-
cence from the whole cell area was essentially unchanged.9
Although NPC1-deficient cells have high amounts of choles-
terol in the LSOs, their other membranes may actually be
cholesterol-poor as compared to wild type cells.1 Thus, a
treatment that releases cholesterol from the LSOs would not
be expected to decrease the overall cholesterol level in the cells.
The previously reported increase in cholesterol in response to
1a is not fully understood. It may reflect a transient condition
as the cells deal with release of a large amount of cholesterol
from the LSOs.
Figure 3. Relative increase in cholesterol efflux by 1a in normal
(TRVb1, 25RA) and mutant (CT60) CHO cells. Cells were labeled
with 1 μCi/ml [3H]-cholesterol for 24 h and then incubated for 18 h
with 10 μΜ 1a. Efflux was expressed as the radioactivity of the
supernatant relative to the total of the radioactivity in the super-
natant and the cell monolayer. For solvent-treated (control) cells,
the mean values of supernatant/total were: 0.069 ( 0.003 for CT60
cells, 0.066 ( 0.004 for 25RA cells, and 0.214 ( 0.006 for TRVb1
cells. Experimental data of 1a-treated cells are presented as relative
efflux with respect to the control (p < 0.001). Five independent
experiments (n = 25) were run in CT60 cells, and two independent
experiments (n = 10) for 25RA and TRVb1 cells.
Table 1. Summary of the Effects of 10 μM 1a in NPC1-Deficient CT60
Cellsa
Because compound 1a increased not only the esterification
by ACAT but also cholesterol efflux to extracellular accep-
tors, this compound could be considered a valuable candidate
for directly promoting sterol efflux from the LE/LY in NPC1-
deficient and other cells.
average
value ( SE
(fraction of the
control)
assay
endocytosis of DiI-LDL by CT60 cells in 35 min
endocytosis of DiI-LDL by 25RA cells in 35 min
endocytosis of DiI-LDL by CT60 cells in 18 h
endocytosis of DiI-LDL by 25RA cells in 18 h
0.93 ( 0.05
1.16 ( 0.31
0.59 ( 0.06*b
0.67 ( 0.03*b
Conclusion
In summary, we have devised a concise and efficient
route for the production of the goal N-aryl-3-alkylidene-
pyrrolinones comprising five simple steps. When applied
to initial compound 1a, the route provided the desired
product in 43% overall yield. The route has also facili-
tated the synthesis of analogues. Furthermore, using our
primary scaffold, we were able to synthesize photoaffinity
labeled and biotinylated derivatives for use in separate
investigations. Studies of 1a show that it decreases the LDL
uptake and increases ACAT esterification in mutant-NPC1
cells and it increases cholesterol efflux in NPC1 mutant and
normal cells.
ACAT esterification: Incorporation of [14C]-oleic acid 1.49 ( 0.13*b
cholesteryl esters
1.05 ( 0.10
a The effect of 1a on LDL internalization was evaluated by quantifying
the fluorescence intensity of DiI per µg cell protein, normalized tothe mean
value of solvent-treated samples. The impact of 1a on esterification by
ACAT was estimated by quantifying the incorporation of [14C]-oleic acid
into esters per cellular protein content. For solvent-treated cells the mean
value was (14 ( 1) pmol cholesteryl-[14C] ester/μg cell protein.26 The effect
of 1a on overall cholesteryl ester levels in the cell was determined by GC.
The mean value for solvent-treated cells was (0.042 ( 0.004) μg cholesteryl
ester/μg cell protein.26 b * indicates p < 0.02.
observed an increase in ACAT esterification upon treatment
of CT60 cells with 1a for 18 h. For the solvent-treated CT60
cells, the average experimental value corresponding to ACAT
esterification is (14 ( 1) pmol cholesteryl-[14C]-oleate/μg
cellular protein,9 while for 1a-treated CT60 cells, the value is
about 20.9 pmol/μg protein. The restoration by 1a of the
ACAT esterification in NPC-deficient CT60 cells is signi-
ficant, because in the solvent-treated non-NPC mutant
25RA, the experimental value is 37 ( 3 pmol cholesteryl-[14C]-
oleate/μg cellular (n = 27, six independent experiments).
Experimental Section
All experiments were run under inert atmosphere unless other-
wise stated. Thin layer chromatography (TLC) was conducted
using precoated silica gel 60 F254 plates from EMD Chemicals.
Infrared spectra were recorded on a Perkin-Elmer Paragon 1000
FT-IR spectrometer. HRMS data were recorded on a JEOL
JMS-AX505HA double sector mass spectrometer using FAB.
NMR spectra were recorded using Varian-INOVA 500 and 300
spectrometersoperatingat500 MHz(1H) and 125 MHz (13C) and
300 MHz (1H) and 75 MHz (13C), respectively. Chemical shifts
are given in ppm relative to residual solvent peaks: 1H (7.27 for
CDCl3 and 2.5 for DMSO-d6) and 13C (77.23 for CDCl3 and
39.51 for DMSO-d6). Flash chromatography was performed
In this paper and in our previous study,9 we described
several effects of 1a on NPC1-deficient cells. There is reduced
filipin labeling of unesterified cholesterol in the LSOs,9 and
there is an increase in efflux of cholesterol to extracellular
acceptors (Figure 3), decreased uptake of LDL (Table 1), and
increased cholesterol esterification by ACAT (Table 1). All of
these changes would be consistent with release of cholesterol
from the LSOs in response to treatment with 1a. The choles-
terol released from the LSOs would be a substrate for ACAT
and would be expected to increase incorporation of radiola-
beled oleate into cholesteryl ester droplets. This released
cholesterol would also be expected to increase cholesterol
efflux and to down-regulate the expression of LDL receptors
through the SREBP pathway.30 The lack of an increase in
ꢀ
˚
using EcoChrome 60 A silica gel. All tested compounds were
>95% pure by HPLC.
(Z)-4-(3-((5-(4-Bromophenyl)furan-2-yl)methylene)-2-oxo-5-
phenyl-2,3-dihydro-1H-pyrrol-1-yl)benzoic Acid (1a). Pyrroli-
none 7 (1.15 g, 1.9 mmol) was added to freshly distilled pyridine
(8 mL) at 23 °C. To this mixture was added anhydrous lithium
iodide (3.56 g, 26.6 mmol). This mixture was heated to reflux
and stirred at this temperature for 14 h. During this time, the
mixture became homogeneous and darkened in color. The
reaction mixture was cooled to 23 °C and poured into ice cold
1 N HCl. The pH of the solution was adjusted to 2, and the

6498 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20
Cosner et al.
precipitated red solid was collected by vacuum filtration. The
solid cake was washed with 1 N HCl (2 ꢀ 20 mL) and DI water
(2 ꢀ 20 mL). The solid was allowed to air-dry, affording 1.12 g
of 1a (95%). 1H NMR matched that of an a commercial
sample; mp = > 200 °C. 1H NMR (500 MHz, DMSO-d6)
δ 7.92 (d, J = 8 Hz, 2H), 7.83 (d, J = 8 Hz, 2H), 7.71 (d, J =
8 Hz), 7.38-7.34 (m, 5H), 7.32-7.29 (m, 2H), 7.23-7.20 (m,
3H), 6.84 (s, 1H). 13C (125 MHz, DMSO-d6) δ 169.2, 167.4,
156.4, 152.7, 146.7, 140.2, 132.9, 131.0, 130.4, 129.9, 129.5,
129.3, 128.9, 128.3, 127.6, 127.2, 126.9, 125.7, 122.6, 118.1,
111.4, 111.4, 104.5. HRMS (FAB) calcd for C28H18BrNO4 (M
þ H)þ 512.0497, found 512.0485. IR (KBr pellet): 3418, 3057,
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Acknowledgment. We thank the Ara Parseghian Medical
Research Foundation and the University of Notre Dame
for financial support of this research. This work was also
supported by NIH grant R37-DK27083 to F.R.M. Also, we
thank Prof. J. David Warren and Dr. Guangtao Zhang of
the Milstein Organic Synthesis Core Facility of Cornell
University for their help with DiI chromatography and
B. Attawut for help in preparing DiI-LDL. M.R. was
supported by a grant from the W. M. Keck Foundation.
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