Design, synthesis and identification of a new class of triarylmethyl amine compounds as inhibitors of apolipoprotein e production

Article abstract of DOI:10.1016/j.bmcl.2012.08.009

We have identified a new class of triarylmethyl amine compounds that can inhibit apolipoprotein E (apoE) production. ApoE is a cholesterol- and lipid-carrier protein implicated in aging, atherosclerosis, Alzheimer's Disease (AD), and other neurological and lipid-related disorders. Attenuation of apoE production is generally considered to be of therapeutic value. A majority of the apoE in the brain is produced by astrocytes. Here, we describe the design, synthesis, and biological screening of a small library of compounds that led to the identification of four triarylmethyl amines as potent inhibitors of apoE production in CCF-STTG1 astrocytoma cells.

Full text of DOI:10.1016/j.bmcl.2012.08.009

Bioorganic & Medicinal Chemistry Letters 22 (2012) 6252–6255
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis and identification of a new class of triarylmethyl amine
compounds as inhibitors of apolipoprotein E production
Mandeep Singh a, Jason T. Schott b, Martin A. Leon a, Robert T. Granata a, Harkiran K. Dhah a,
Jason A. Welles a, Michelle A. Boyce a, Hafeez S. Oseni-Olalemi a, Charles E. Mordaunt b,
a,
Anthony J. Vargas b, Nilay V. Patel b,c, , Santanu Maitra
⇑
⇑
a Department of Chemistry, California State University Fresno, Fresno, CA 93740, USA
b Department of Biological Science, California State University Fullerton, Fullerton, CA 92831, USA
c Center for Applied Biotechnology Studies, California State University Fullerton, Fullerton, CA 92831, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
We have identified a new class of triarylmethyl amine compounds that can inhibit apolipoprotein E
(apoE) production. ApoE is a cholesterol- and lipid-carrier protein implicated in aging, atherosclerosis,
Alzheimer’s Disease (AD), and other neurological and lipid-related disorders. Attenuation of apoE produc-
tion is generally considered to be of therapeutic value. A majority of the apoE in the brain is produced by
astrocytes. Here, we describe the design, synthesis, and biological screening of a small library of com-
pounds that led to the identification of four triarylmethyl amines as potent inhibitors of apoE production
in CCF-STTG1 astrocytoma cells.
Received 9 April 2012
Revised 23 July 2012
Accepted 1 August 2012
Available online 10 August 2012
Keywords:
Triarylmethyl amines
Apolipoprotein E
Ó 2012 Elsevier Ltd. All rights reserved.
Inhibitors
Structure-activity-relationship (SAR) studies
Apolipoprotein E (apoE) is a cholesterol- and lipid-carrier that
has been implicated in aging, atherosclerosis and several neurolog-
ical diseases including Alzheimer Disease (AD).1 ApoE genotype is
the biggest risk factor for AD and may account for 60–90% of the
genetic variance (VG) associated with AD.2 There are three common
isoforms (alleles) of apoE in humans—apoE2, apoE3, and apoE4—
which contribute to the pleiotropic effects observed in human cog-
nition and neurodegenerative diseases.3 In contrast, there is only
one common apoE allele in other primates.4 Increase in levels of
murine apoE in a mouse model of AD by Bexarotene, an FDA-ap-
proved cancer drug, has been considered to be therapeutic because
the treatment attenuated Ab plaque burden.5 However, the human
apoE4 is hypofunctional and is considered to be the ‘bad’ apoE,
while the human apoE3 is the most common isoform and is consid-
ered to be the ‘normal’ apoE.1,3,6 Moreover, high levels of plasma
and brain apoE are risk-factors for AD independent of the apoE
genotype.7 Thus, there is a strong interest in the biomedical com-
munity to find therapies that can reduce apoE levels. These thera-
pies are of value to all individuals, and especially to subjects that
have apoE4 allele(s).
screening was performed on a small library of compounds (Chart
1; scaffolds A–C) which was followed up using traditional struc-
ture-activity-relationship (SAR) approaches on scaffold A (Chart
2). The three scaffolds, A–C were chosen to provide with simple,
readily available and chemically modifiable molecular backbones
that offer the versatility for further functionalization to enable fu-
ture SAR studies should any of these series yields any hit. Herein,
we report on the design, synthesis and biological study leading
to the identification of the triarylmethyl amine compounds that
showed the greatest reduction in the amount of apoE present in
the conditioned medium from the CCF-STTG1 cells.
The synthesis of the triarylmethyl amine series started with the
formation of a Grignard reagent from the corresponding aryl bro-
mide, followed by the subsequent reaction with benzophenone to
generate the tertiary alcohol (Scheme 1).8 The alcohol was in turn
converted into the chloride using acetyl chloride, which was substi-
tuted with an amine nucleophile to yield the final triarylmethyl
amine compound.9 Most of the molecules in the scaffold A series
(1–4 and 10–42, Chart 2) were purified by standard procedures,
viz., crystallization, column chromatography, and preparative thin
layer chromatography (prep-TLC). However, the triarylmethyl
amines with para-methoxyphenyl moieties (compounds 21–32 in
Chart 3) proved to be less stable than the other two aromatic groups
used for this scaffold during purification processes. We attribute
this instability to the ease of triarylmethyl carbocation formation
due to the presence of a strong electron-donating methoxy group
We have found a class of small molecules that inhibit apoE pro-
duction in the human CCF-STTG1 astrocytoma cell line. The initial
⇑
Corresponding authors. Tel.: +1 559 278 2961; fax: +1 559 278 4402.
E-mail addresses: npatel@fullerton.edu (N.V. Patel), smaitra@csufresno.edu
(S. Maitra).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.08.009

M. Singh et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6252–6255
6253
Chart 1. Scaffolds A–C, tested for their effects on apoE production.
Chart 2. Triarylmethyl amine- (1–4), benzoin- (5 and 6) and benzophenone-based
(7–9) compounds.
Chart 3. Triarylmethyl amine compounds with 3 aryl variants and 15 amines.
Technologies). Sample apoE concentrations were adjusted for total
cell number and then normalized to DMSO treated wells. Results
for each set of compounds tested in parallel (n = 3) were analyzed
by a one-tailed t-test.
The triarylmethyl amine molecules designated as Scaffold A
inhibited apoE secretion with the greatest potency.
We have used apoE ELISA to monitor apoE production after a
24 h-treatment with a 10 lM concentration of each of the com-
pounds in Scaffolds A–C (Fig. 1). Figure 1 depicts the generally
Scheme 1. Reagents and conditions: (a) Mg, anhyd. ether, reflux, 2 h; (b)
benzophenone, reflux, 24 h; (c) acetyl chloride, benzene, reflux, 40 min; (d)
R1R2NH, triethylamine, methylene chloride, rt, 15 h. Overall yield for steps a-d:
39–85%.6
on one aryl group. Successful purification of these final compounds
was effected by pre-soaking the silica gel with eluent (ethyl ace-
tate:hexane mixture) containing small amounts of triethylamine
while performing flash columns, preparatory TLCs and radial
chromatography.
The human CCF-STTG1 astrocytoma cells (ATCC, CRL-1718)
were maintained in RPMI medium (Mediatech) supplemented with
10 mM HEPES, 1 mM sodium pyruvate, 4.5 g/L glucose, 1% penicil-
lin/streptomycin, and 10% bovine growth serum (HyClone) at 37 °C
with 5% CO2. All compounds were dissolved in DMSO at a stock
concentration of 10 mM. For ELISA assays, 1.6 Â 104 cells / 96-well
were grown for 1 day in RPMI, followed by a 24 h equilibration in
serum-free Opti-MEM (Life Technologies). Treatments were carried
out in quadruplicate with 100
lL of fresh Opti-MEM containing
10 M of compound or vehicle (0.1% DMSO) for 24 h. Conditioned
l
medium from these samples were then diluted twofold with incu-
bation buffer (PBS + 0.05% Tween-20 + 0.1% BSA) and analyzed
with a human apoE HRP ELISA kit (Mabtech) according to the man-
ufacturer’s protocol. A standard curve was used to derive sample
apoE concentrations. The cells in the 96-well plates were used to
determine the total cell number using CyQUANT reagent (Life
Figure 1. Effect of compounds, 1–9 from scaffolds A–C on apoE production in
human CCF-STTG1 astrocytoma cells. The cells were treated with 10 lM compound
or 0.1% DMSO (vehicle) in serum-free Opti-MEM for 24-h and the medium
supernatant was used to quantify apoE production using a human apoE ELISA.
Values are means of three independent experiments SEM and are normalized to
the vehicle control.

6254
M. Singh et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6252–6255
Figure 2. Effect of 33 compounds, 10–42 from scaffold A on apoE production in human CCF-STTG1 astrocytoma cells.
consistent inhibitory effects with varying strengths from com-
pounds 1 through 9 (Chart 2). The three scaffolds A–C, although
fundamentally different, share some common features, viz., vary-
ing number of phenyl rings on minimalist scaffolds. The notewor-
thy difference between the three structures is the lack of an amine
group in scaffolds B and C. The first four compounds have triaryl-
methyl amine scaffolds with all three phenyl groups. Four easily
available amines, viz., dimethylamine, diethylamine, dibutylamine
and pyrrolidine were used in compounds 1–4, respectively. O-ben-
zylbenzoin (5) was easily available from its precursor benzoin (6)
through benzylation, and both the compounds (5 and 6) were se-
lected for screening.10,11 Compound 7, a benzyloxy benzophenone,
and two fragments, hydroxyl benzophenone (8) and benzophenone
(9), were also screened in the biological assay.12 Quantification of
apoE in the conditioned medium of cells treated with these nine
compounds identified the triarylmethyl amines (scaffold A) as
the best apoE inhibitors over the benzoins (scaffold B) and benz-
ophenones (scaffold C) (Fig. 1). Lack of apoE inhibitory activity ob-
served in scaffolds B and C could be attributed to scaffold structure
itself or, absent design elements may be crucial for the desired
activity, viz., the amine moiety and the number of aryl groups. Tri-
arylmethyl amine 4 containing a pyrrolidine group with a hydro-
gen-bond acceptor tertiary amine moiety exhibited maximum
apoE inhibition in this early study.
The promising results from the pilot apoE screen (Fig. 1)
prompted us to undertake further SAR studies to design, synthesize
and screen more triarylmethyl amines (Chart 3). Attention was
paid to include hydrogen bonding donor and acceptor amines in
the final compounds, vary the size of the third aromatic ring and
also include acyclic and cyclic amines of different sizes and shapes.
Thus, each compound in this particular series has two phenyl rings,
a third aryl ring, and a unique amine. Para-methoxyphenyl and
naphthyl aromatic groups were selected for the new set of mole-
cules in order to test the effect of varying size without altering
polarity in this area of the molecule. Altogether, fifteen different
primary and secondary amines provided with added diversity for
the molecules. The amines included aliphatic acyclic amines such
as dimethyl amine, diethyl amine, and dibutyl amine. Aliphatic,
cyclic amines were also included in the study with the selection
of pyrrolidine, piperidine and morpholine. Cyclohexyl amine, ani-
line, isobutyl amine, and sec-butyl amine represent the secondary
and 41 in Chart 3) in their final form. Although 45 molecules (15
amines with 3 aryl variations) were attempted for synthesis and
subsequent biological screening, not every candidate was screened
due to occasional challenges in isolation, purification and in certain
cases with final acceptable purities (e.g., compounds comprising a
naphthyl aryl group and one of the following amines: cyclo-
hexylmethyl amine, dibenzylamine, and piperazine).
The effects of the 33 triarylmethyl amines on apoE production
are shown in Figure 2. The expansion of the series yielded much
greater apoE inhibitory activity than the initial screen. Both the
new aryl groups fared better than the original phenyl series in
terms of apoE inhibition, with the naphthalene moiety resulting
in more pronounced inhibition of apoE production. Among all the
triarylmethyl amine compounds comprising the naphthyl group
(Group C, Chart 3), compounds 36, 38, 40 and 41 inhibited apoE
production by 46%, 49%, 50% and 66%, respectively. Compound 18
from the phenyl series was the only compound that exhibited
respectable apoE inhibition (51%). The corresponding amine func-
tionalities in these four compounds are pyrrolidine (36), morpho-
line (38), isobutyl amine (40), sec-butyl amine (41), and
cyclohexylmethyl amine (18). The presence of hydrogen bond do-
nor vs acceptor groups at the amine functionality, or the acyclic vs
cyclic nature of the amine did not cause any noticeable change in
the inhibitory activity. Six of them (33–38) have hydrogen bond
donor groups and the rest (39–42) have hydrogen bond acceptor
groups and there is an equal mix of acyclic and cyclic structures
around the amine group. The overall size of most of the amine
groups might have played a role in determining the inhibition effi-
cacy since all the four amines in compounds, 36, 38, 40, and 41
comprise approximately 5–6 atoms. The cyclohexylmethyl amine
in compound 18 is slightly larger than these four amines and the
plans are in order to synthesize and screen the naphthyl version
of the triarylmethyl amine molecule with cyclohexylmethyl amine.
The SAR studies have led to the identification of four com-
pounds in the triarylmethyl amines series as preliminary hits for
inhibiting the production of apoE protein. Results have pointed at
the usefulness of the bicyclic naphthalene ring. Additionally, five
specific amine functionalities, viz., pyrrolidine, morpholine, isobu-
tylamine, sec-butylamine, and cyclohexylmethyl amine proved to
be essential. These results also provide additional insights into
structural features necessary for development of more potent
and drug-like scaffolds and compounds to inhibit apoE production.
amines. A racemic version of
a-methylbenzyl amine moiety was
the only chiral example of amine in this study which falls under
the general secondary amine category. The initial nature of the se-
lected amine dictated the hydrogen bonding capability of the final
compounds. Some of these compounds, including 1 (Chart 2), 12,
25 and 38 (Chart 3), have hydrogen-bond acceptor regions as ter-
tiary amines, whereas others are secondary amines (20, 27, 40
Acknowledgments
S.M. thanks Dr. Saeed Attar, the Chair of the Chemistry Depart-
ment, California State University Fresno and Dr. Uday Maitra, the
Chair of the Organic Chemistry Department, Indian Institute of

M. Singh et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6252–6255
6255
was acidic to Litmus paper. The mixture was diluted with ether (30 mL) and the
aqueous layer was removed. The ether layer was washed with water (30 mL),
followed by brine (30 mL) and dried with anhydrous Na2SO4. After filtration,
the solvent was removed under reduced pressure to provide a colorless solid
(triarylmethyl alcohol). The crude triarylmethyl alcohol derivative (6.30 g,
20.3 mmol) was refluxed with acetyl chloride (2.2 mL, 30.45 mmol) in benzene
(10 mL) for 5 min. An additional amount of acetyl chloride (2.5 mL) was added
over the course of 10 min via an addition funnel, and the resulting solution was
allowed to reflux for another 30 min. The solution was quickly cooled by
holding the flask under a stream of running tap water while vigorously shaking
the mixture. The mixture was concentrated under reduced pressure to give the
triarylmethyl chloride derivative. The crude triarylmethyl chloride derivative
and morpholine (5.0 mL, 57.9 mmol) in anhydrous CH2Cl2 (25 mL) were stirred
at 25 °C for 18 h. The organic layer was washed with water (30 mL) followed by
saturated NaHCO3 solution (30 mL) and brine (30 mL), dried with anhydrous
Na2SO4. The solution was filtered and the solvent was evaporated under
reduced pressure to yield crude triarylmethyl amine, 38 as a dirty white solid.
Flash chromatography using 230–400 mesh silica gel was performed to purify
compound 38. Elution of the column with 30% ethyl acetate in hexanes,
followed by concentration of the appropriate fractions and subsequent
evaporation of solvents yielded 6.88 g (47% overall) of triarylmethyl amine
38 as an off-white amorphous solid. 1H NMR (400 MHz/CDCl3) d 2.88–1.81 (m,
4H), 4.10–3.66 (m, 4H), 7.21–7.15 (m, 2H), 7.35–7.23 (m, 4H), 7.86–7.36 (m,
10H) 7.44–7.41 (m, 2H), 7.61–7.46 (m, 4H) 7.66 (s, 1H), 7.72 (d, J = 8.4 Hz, 1H)
7.79–7.74 (m, 2H), 7.81 (d, J = 7.3 Hz, 1H), 7.96 (s, 1H); 13C NMR (100 MHz/
CDCl3) d 141.6 (C), 132.9, 131.8, 130.2, 129.2, 128.8, 128.1, 127.8, 127.7, 127.2,
Science, Bangalore, India for critical scientific and technical discus-
sions. S.M. also thanks Dr. Brandon White, Assistant Professor at
the Department of Biological Sciences, California State University
at San Jose for assistance with the HRMS.
References and notes
1. Mahley, R. W.; Weisgraber, K. H.; Huang, Y. Proc Natl Acad Sci U.S.A. 2006, 103,
5644. Epub 2006 Mar 27. 2006, 103, 5644.
2. Raber, J.; Huang, Y.; Ashford, J. W. Neurobiol. Aging 2004, 25, 641. 2004, 25, 641..
3. Mahley, R. W.; Rall, S. C., Jr. Annu. Rev. Genomics Hum. Genet. 2000, 1, 507. 2000,
1, 507.
4. Hanlon, C. S.; Rubinsztein, D. C. Atherosclerosis 1995, 112, 85.
5. Cramer, P. E.; Cirrito, J. R.; Wesson, D. W.; Lee, C. Y.; Karlo, J. C.; Zinn, A. E.;
Casali, B. T.; Restivo, J. L.; Goebel, W. D.; James, M. J.; Brunden, K. R.; Wilson, D.
A.; Landreth, G. E. Science 2012, 335, 1503.
6. Hauser, P. S.; Narayanaswami, V.; Ryan, R. O. Prog. Lipid Res. 2011, 50, 62.
7. Dodart, J. C.; Marr, R. A.; Koistinaho, M.; Gregersen, B. M.; Malkani, S.; Verma, I.
M.; Paul, S. M. Proc Natl Acad Sci U.S.A. 2006, 103, 5644. Epub 2006 Mar 27. 2005,
102, 1211.
8. Experimental. Triarylmethyl amine 38: 4-(Naphthalen-2-yl-diphenyl-methyl)-
morpholine: Scratched magnesium turnings (1.056 g, 43.4 mmol) were placed
in a 250 mL round-bottomed, 3-neck flask at room temperature.
A small
portion of the solution of 2-bromonaphthalene (11.84 g, 47.7 mmol) in
anhydrous ether (40 mL) was added through an addition funnel and the
solution was heated to gentle reflux maintaining the temperature at 35 °C. The
rest of the solution was added in small fractions over a period of 1 h, and the
resulting reaction mixture was refluxed for an additional 1 h. Disappearance of
all magnesium indicated the completion of the reaction. The solution
containing benzophenone (7.04 g, 38.6 mmol) in anhydrous ether (20 mL)
was added through an addition funnel over 2–4 min. The resulting reaction was
gently refluxed for 24 h. The cooled mixture was slowly and carefully quenched
(Note: exotherm!) by adding aq HCl (6M) to the reaction mixture until the pH
126.7, 126.3, 125.9, 125.2, 77.6, 67.7, 48.7; IR
m
(cmÀ1): 3690, 2955, 2842, 1488,
1446, 1262, 1112, 1033, 998, 908, 877, 817, 743, 703, 571, 479; Mp: 113–
114 °C; HRMS: (M++Na+) calcd for C27H25NO, 402.1828, found, 402.1818.
9. Verkade, P.; Nijon, H.; Tollenaar, F.; Van Rij, J.; van Leeuwen, M. Recl. Trav. Chim.
Pays-Bas 1952, 71, 1007.
10. Curtin, D. Y.; Proops, W. R. J. Am. Chem. Soc. 1954, 76, 494.
11. Zolotareva, V.; Klimova, L.; Malanina, G.; Sokolova, A.; Grinenko, G. Pharm.
Chem. J. 1990, 24, 241.
12. Olsson, R.; Hyldtoft, L.; Piu, F.; Gustafsson, M.; WO Patent 056500, 2007.