Design, Synthesis, and Biological Evaluation of New Peripheral 5HT2A Antagonists for Nonalcoholic Fatty Liver Disease

Article abstract of DOI:10.1021/acs.jmedchem.0c00002

Nonalcoholic fatty liver disease (NAFLD) is increasingly prevalent worldwide, causing serious liver complications, including nonalcoholic steatohepatitis. Recent findings suggest that peripheral serotonin (5-hydroxytryptamine, 5HT) regulates energy homeostasis, including hepatic lipid metabolism. More specifically, liver-specific 5HT_2A knockout mice exhibit alleviated hepatic lipid accumulation and hepatic steatosis. Here, structural modifications of pimavanserin (CNS drug), a 5HT_2A antagonist approved for Parkinson's disease, led us to synthesize new peripherally acting 5HT_2A antagonists. Among the synthesized compounds, compound 14a showed good in vitro activity, good liver microsomal stability, 5HT subtype selectivity, and no significant inhibition of CYP and hERG. The in vitro and in vivo blood-brain barrier permeability study proved that 14a acts peripherally. Compound 14a decreased the liver weight and hepatic lipid accumulation in high-fat-diet-induced obesity mice. Our study suggests new therapeutic possibilities for peripheral 5HT_2A antagonists in NAFLD.

Full text of DOI:10.1021/acs.jmedchem.0c00002

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Design, Synthesis, and Biological Evaluation of New Peripheral
5HT2A Antagonists for Nonalcoholic Fatty Liver Disease
Minhee Kim,‡ Inseon Hwang,‡ Haushabhau S. Pagire, Suvarna H. Pagire, Wonsuk Choi, Won Gun Choi,
Jihyeon Yoon, Won Mi Lee, Jin Sook Song, Eun Kyung Yoo, Seung Mi Lee, Mi-jin Kim, Myung Ae Bae,
Dooseop Kim, Heejong Lee, Eun-Young Lee, Jae-Han Jeon, In-Kyu Lee, Hail Kim,* and Jin Hee Ahn*
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ABSTRACT: Nonalcoholic fatty liver disease (NAFLD) is increasingly
prevalent worldwide, causing serious liver complications, including non-
alcoholic steatohepatitis. Recent findings suggest that peripheral serotonin (5-
hydroxytryptamine, 5HT) regulates energy homeostasis, including hepatic
lipid metabolism. More specifically, liver-specific 5HT2A knockout mice exhibit
alleviated hepatic lipid accumulation and hepatic steatosis. Here, structural
modifications of pimavanserin (CNS drug), a 5HT2A antagonist approved for
Parkinson’s disease, led us to synthesize new peripherally acting 5HT2A
antagonists. Among the synthesized compounds, compound 14a showed
good in vitro activity, good liver microsomal stability, 5HT subtype selectivity,
and no significant inhibition of CYP and hERG. The in vitro and in vivo
blood−brain barrier permeability study proved that 14a acts peripherally.
Compound 14a decreased the liver weight and hepatic lipid accumulation in
high-fat-diet-induced obesity mice. Our study suggests new therapeutic
possibilities for peripheral 5HT2A antagonists in NAFLD.
metabolism.21−24 Among the subtypes of 5HT receptors,
liver-specific 5HT2A knockout mice showed a reduction in liver
size, weight, and lipid accumulation, as indicated by
histological data, NAS (NAFLD activity score), and hepatic
triglyceride (TG) concentrations, without affecting the
systemic energy homeostasis.23
These biological results prompted us to identify new
peripheral 5HT2A antagonists. Many 5HT2A antagonists are
currently being used for CNS disorders, including schizo-
phrenia. We envisioned new peripheral 5HT2A antagonists by
introducing non-BBB-permeable moieties on CNS drugs. We
chose pimavanserin (Nuplazid), 1-(4-fluorobenzyl)-3-(4-iso-
butoxybenzyl)-1-)1-methylpiperidin-4-yl)urea (1), a 5HT2A
antagonist approved in 2016 in the United States for
Parkinson’s disease,25 for structural modification (Figure 1
and Table 1).
INTRODUCTION
■
Nonalcoholic fatty liver disease (NAFLD), which involves
excessive lipid accumulation in the liver, has become a serious
disease worldwide.1,2 It is closely associated with obesity and
chronic liver disease and is considered a leading cause of
progressive liver affections such as nonalcoholic steatohepatitis
(NASH), cirrhosis, and hepatocellular carcinoma.3,4 Serotonin
(5-hydroxytryptamine, 5HT) is a monoamine mostly found in
the gastrointestinal tract, platelets, and the pineal gland, and in
smaller amounts in the brain.5−8 In the central nervous system
(CNS), 5HT acts as a neurotransmitter that modulates mood,
behavior, sleep cycles, and appetite.9−14 Central and peripheral
serotonin systems are regarded as separate systems because
5HT cannot cross the blood−brain barrier (BBB).9,15,16
The physiology of peripheral 5HT has been studied since
the 1960s, and recent findings suggest that peripheral 5HT can
regulate energy homeostasis.16−18 Slc6a4 (serotonin trans-
porter, SERT) knockout mice manifested an obese phenotype
with a metabolic syndrome,17 and TPH1/TPH2 (tryptophan
hydroxylase 1 and 2) double knockout (DKO) mice displayed
reduced body weight.18 In addition, inhibition of 5HT
synthesis elicited decreased lipogenesis in the epididymal
white adipose tissue (WAT), induction of browning in the
inguinal WAT, and activation of adaptive thermogenesis in
brown adipose tissue (BAT).16,19−21 Other studies indicate
that peripheral 5HT can also regulate hepatic lipid
Diverse derivatives are designed and synthesized with the
aim of obtaining peripherally acting compounds. In the present
Received: January 4, 2020
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coupling reaction between compound 6 and compound 9,
which was synthesized through the reductive amination of
commercially available 4-fluorobenzaldehyde (7) and 1-Boc-4-
piperidineamine (8) to give compound 10. Compound 10
underwent acidic deprotection leading to compound 11, N-
desmethylpimavanserin (Scheme 1). Compounds 12a−c were
synthesized through the reaction of compound 11 and acetyl
chloride, methanesulfonyl chloride, and isopropyl thiocyanate,
respectively, in the presence of a base. The condensation of
compound 11 and succinic anhydride afforded compound 13
(Scheme 2). The amide coupling reaction of compound 11
and 1-benzyl N-carbobenzoxy-L-glutamate, 1-benzyl N-benzy-
loxycarbonyl-L-aspartate, and 5-benzyl N-benzyloxycarbonyl-L-
glutamate afforded coupled products followed by hydro-
genation resulting in compounds 14a-c, respectively. Com-
pound 14a was treated with di-tert-butyl dicarbonate under
alkaline conditions to produce compound 15 (Scheme 3).
Commercially available L-tyrosine (16) underwent esterifica-
tion to give compound 17. Compound 17 was treated with di-
tert-butyl dicarbonate to afford compound 18. The con-
densation of compound 18 and trifluoromethanesulfonic
anhydride yielded compound 19. Compound 19 was
converted into carboxylic acid to give compound 20.
Compound 11 was coupled with compound 20 affording
compound 21 and after hydrolysis and acidic deprotection
compound 22 was obtained (Scheme 4).
Figure 1. Chemical structure of compound 1 (pimavanserin).
Table 1. Brain/Plasma Ratio in Male ICR Mice (10 mg/kg,
ip, Mean SD, n = 3) for Compound 1
concentration (ng/mL or ng/g)
time (h)
0.5
plasma
brain tissue
C
brain/Cplasma
612.0 49.0
3014 590.6
4.98 1.25
study, we report the synthesis and biological evaluation of new
peripherally acting 5HT2A antagonists.
RESULTS AND DISCUSSION
■
A new series of 5HT2A antagonists were synthesized according
to synthesis Schemes 1−6. The commercially available 4-
hydroxybenzylamine (2) was treated with di-tert-butyl
dicarbonate resulting in compound 3. Compound 3 reacted
with 1-bromo-2-methylpropane under alkaline conditions to
afford compound 4. Compound 5 was synthesized by the
acidic deprotection of compound 4. Compound 5 treated with
1,1′-carbonyldiimidazole (CDI) afforded compound 6. Com-
pound 10 was obtained under alkaline conditions through a
The reaction between 4-hydroxybenzylamine (2) and
phthalic anhydride resulted in compound 23. Compound 23
reacted with epichlorohydrin in the presence of a base to afford
a
Scheme 1. Synthesis of N-Desmethylpimavanserin (Compound 11)
a
Reagents and conditions: (a) di-tert-butyl dicarbonate, NaHCO3, MeOH, H2O, rt, 95%; (b) 1-bromo-2-methylpropane, K2CO3, DMF, 80 °C,
32%; (c) 4 M HCl in 1,4-dioxane, EtOAc, rt, 92%; (d) 1,1’-carbonyldiimidazole, DMF, rt, 68%; (e) NaBH4, MeOH, rt, 65%; (f) K2CO3, DMF, rt,
74%; and (g) 4 M HCl in 1,4-dioxane, EtOAc, rt, 88%.
B
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Scheme 2. Synthesis Acetyl, Methanesulfonyl, Isopropylthiocyanate, and Succinyl Derivatives of Pimavanserin (Compounds
a
12a-c and 13)
a
Reagents and conditions: (a) acetyl chloride or methanesulfonyl chloride, DIPEA, DCM, 0 °C to rt, 61−79%; (b) Isopropyl isothiocyanate,
DIPEA, THF, 40 °C, 97%; and (c) succinic anhydride, THF, reflux, 61%.
a
Scheme 3. Synthesis of Amino Acid Derivatives of Pimavanserin (Compounds 14a−c and 15)
a
Reagents and conditions: (a) 1-benzyl N-carbobenzoxy-L-glutamate or 1-benzyl N-benzyloxycarbonyl-L-aspartate or 5-benzyl N-
benzyloxycarbonyl-L-gluatamte, EDCI, HOBt, DIPEA, DMF, rt, 44−73%; (b) H2, Pd/C, MeOH, rt, 63−75%; and (c) di-tert-butyl dicarbonate,
NaOH, 1,4-dioxane, H2O, rt, 84%.
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a
Scheme 4. Synthesis of the Tyrosine Derivative of Pimavanserin (Compound 22)
a
Reagents and conditions: (a) SOCl2, EtOH, reflux; (b) Boc2O, TEA, MeOH, DCM, rt, 80%; (c) (CF3SO2)2O, pyridine, DCM, rt, 95%; (d)
Pd(OAc)2, dppf, V(CO)6, DMAP, DIPEA, H2O, 1,4-dioxane, microwave, 110 °C, 78%; (e) EDCI, HOBt, DIPEA, DMF, rt, 74%; (f) NaOH, THF,
H2O, rt, 86%; and (g) 4 M HCl in 1,4-dioxane, EtOAc, rt, 83%.
a
Scheme 5. Synthesis of Amino Alcohol Derivatives of Pimavanserin (Compounds 30 and 31)
a
Reagents and conditions: (a) phthalic anhydride, DMF, 140 °C, 32%; (b) epichlorohydrin, K2CO3, ACN, reflux, 50%; (c) 1-Boc-piperazine, IPA,
reflux, 51%; (d) N2H4.H2O, EtOH, reflux, 81%; (e) 1,1′-carbonyldiimidazole, DMF, rt; (f) NaBH4, MeOH, rt, 90%; (g) DMF, rt, 62%; and (h) 4
M HCl in 1,4-dioxane, EtOAc, rt, 91%.
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a
Scheme 6. Synthesis of Amino Alcohol Derivatives of Pimavanserin (Compounds 35a-b)
a
Reagents and conditions: (a) epichlorohydrin, K2CO3, ACN, reflux, 37%; (b) 3-(1-piperazinyl)-1,2-benzisothiazole or 6-fluoro-3-(piperidin-4-
yl)benzo[d]isoxazole hydrochloride, TEA, IPA, reflux, 42−81%; (c) 4M HCl in 1,4-dioxane, EtOAc, rt, 94−97%; (d) 1,1′-carbonyldiimidazole,
DMF, rt; and (e) DMF, rt, 11−23%.
Table 2. SAR Data of Pimavanserin Derivatives
compound 24. The ring opening reaction of compound 24
with 1-Boc-piperazine yielded compound 25. Compound 26
was obtained by the phthalimide deprotection of compound
25, and next it was treated with CDI yielding compound 27.
Under alkaline conditions, compound 27 was coupled with
compound 29 (synthesized through the reductive amination of
commercially available 4-fluorobenzaldehyde (7) and 1-
methyl-4-piperidineamine (28)) to give compound 30.
Compound 30 underwent acidic deprotection to give
compound 31 (Scheme 5). Compound 3 reacted with
epichlorohydrin in the presence of a base to afford compound
32. Compound 32 was coupled with 3-(1-piperazinyl)-1,2-
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Table 3. SAR Data of Amino Acid Derivatives
benzisothiazole and 6-fluoro-3-(piperidin-4-yl)benzo[d]-
isoxazole hydrochloride to yield compounds 33a-b. Com-
pounds 33a-b underwent acidic deprotection followed by a
reaction with CDI to afford compounds 34a-b. Compounds
35a-b were synthesized by the reaction of compounds 34a-b
and compound 29 (Scheme 6).
The synthesized 5HT2A antagonists were evaluated for their
5HT2A inhibition activities, and the results are summarized in
Tables 2−4. In a biological assay at 1 μM concentration, N-
desmethylpimavanserin (11) showed superior in vitro activity
compared to pimavanserin; therefore, the nitrogen of
desmethylpimavanserin (11) was further modified. N-acetyl
(12a), N-methylsulfonyl (12b), and isopropyl thiourea
derivative (12c) were evaluated and showed lower activity
than pimavanserin. Furthermore, the succinyl derivate of N-
desmethylpimavanserin was synthesized and evaluated. Suc-
cinyl derivative 13 showed submicromolar in vitro potency
with an IC50 (half maximal inhibitory concentration) value of
317.3 nM. Therefore, carboxylic acid derivatives such as
glutamate, aspartate, and tyrosine derivatives were synthesized
and evaluated (Table 3). Compound 14a, a glutamate
derivative, improved the potency of pimavanserin with an
IC50 value of 8.35 nM. Furthermore, tert-butoxy carbonyl
substitution at the free amine 15 decreased the potency. The
aspartate derivative 14b showed lower potency than 14a. The
reversed glutamate derivative 14c exhibited a poor inhibition
percentage. Next, the tyrosine derivative, 22 was synthesized
and was shown to manifest a good in vitro activity; however, it
was inferior to that shown by 14a.
Amino alcohol derivatives obtained from modifications at
the isobutoxy position of pimavanserin were synthesized and
screened for their ability to inhibit 5HT2A (Table 4). The 1-
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Table 4. SAR Data of Amino Alcohol Derivatives
Table 5. PAMPA Results of Compounds 14a and 35b
Boc-piperazine derivative 30 exhibited a superior in vitro
activity with an IC50 value of 1.94 nM. The acidic deprotection
of 30 caused a loss of potency in 31. Next, 3-(1-piperazinyl)-
1,2-benzisothiazole and 6-fluoro-3-(piperidin-4-yl)benzo[d]-
isoxazole derivatives were synthesized and evaluated. Both
compounds showed good in vitro potency and 35b performed
better than 35a, with an IC50 value of 0.39 nM.
From the in vitro data, we chose the compounds 14a and
35b, with an amino acid and a neutral heterocycle moiety,
respectively, for in vitro and in vivo BBB permeability
evaluation. The parallel artificial membrane permeation assay
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(PAMPA) was used for BBB permeability prediction, and
results are shown in Table 5.
Table 7. Liver Microsomal Stability, Cytotoxicity, and
Selectivity Results of Compound 14a
The peripheral residence of compound 14a was predicted by
the low permeability value (10−6 cm/s) considering that values
under 2.0 predict low permeation. For consistency with the
PAMPA results, compound 14a was evaluated for its BBB
penetration by iv injection in rats (Table 6). The brain-to-
assay
results
liver microsomal phase I
rat: 84% remained after 30 min incubation
human: 89% remained after 30 min
incubation
a
stability
cytotoxicity
VERO IC50: 54.7 μM
HFL-1 IC50: 55.2 μM
L929 IC50: 64.0 μM
NIH 3T3 IC50: 18.2 μM
CHO-K1 IC50: 51.0 μM
18.7% inhibition at 10 μM
1A: −2.8% inhibition
1B: 6% inhibition
Table 6. Brain/Plasma Ratio in Male ICR Mice (5 mg/kg,
IV, Mean SD, n = 3) for Compound 14a and 35b
concentration (ng/mL)
time
hERG
b
selectivity
compound
(h)
plasma
brain tissue
Cbrain/Cplasma
14a
35b
0.5
0.5
415.7 178.0
455.3 19.0
15.1 5.22
740.9 275.5
0.038 0.011
1.63 0.61
2A: 93% inhibition
2B: 7% inhibition
2C: 63.8% inhibition
3: −5% inhibition
4E: −12.1% inhibition
6: −18.5% inhibition
7: 6.3% inhibition
plasma concentration ratio of compound 14a was 0.038 after
0.5 h, indicating a limited BBB penetration (Figure 2).
a
Buspirone was used as a positive control − rat: 15% and human: 4%
b
remained after 30 min of incubation. Test concentration of all assays
was 1 μM.
Table 8. Pharmacokinetic Parameters of Compound 14a in
Male Rats (iv, 5mpk)
Figure 2. Lead optimization from pimavanserin 1 to compound 14a.
parameters
T1/2 (h)
AUCt (μg·h/mL)
AUC∞ (μg·h/mL)
CL (L/h/kg)
14a
16.9 11.05
1.91 0.186
2.03 0.115
2.46 0.137
11.03 6.62
Furthermore, the unbound fraction of 14a increased peripheral
selectivity (0.02, data not shown). By introducing a glutamic
acid moiety, we could drastically increase the peripheral
selectivity. Generally, compounds with a brain/plasma ratio
greater than 0.3 to 0.5 are considered to have a sufficient CNS
diffusion, and compounds with a value of greater than 1 freely
cross the BBB, whereas compounds with a brain/plasma ratio
smaller than 0.1 may be unable to enter the CNS.26
In contrast, compound 35b seemed to penetrate the BBB at
a brain-to-plasma concentration ratio of 1.63 after 0.5 h.
Compound 14a showed good liver microsomal stability, no
significant cytotoxicity in 5 representative mammalian cell
lines, and low hERG inhibition (Table 7). For further
evaluation, the inhibition percentages of other subtypes of
serotonin receptors were screened and compound 14a showed
selective inhibition of 5HT2A among the subtypes. In addition,
the pharmacokinetic profiles of compound 14a were studied in
rats and are summarized in Table 8. Compound 14 showed
reasonable AUC and a half-life of 17 h.
To determine the in vivo efficacy of 14a for NAFLD, the
mice were subjected to a high-fat diet (HFD) for 10 weeks and
administered 14a by daily intraperitoneal injection. Liver tissue
weight (Figure 3a) and hepatic triglyceride (TG) accumulation
decreased in the mice treated with 14a, 50 and 100 mg/kg
(Figure 3b,c). We also examined the systemic metabolic effects
of 14a considering the 5HT2A expression in other metabolic
organs such as adipose tissues. Compared to the control, the
mice treated with 100 mg/kg 14a gained less body weight after
the HFD and their glucose tolerance was improved (Figure
3d,e). Likewise, body fat decreased while lean body mass
increased in mice treated with 14a (Figure 3f). The mice that
received 14a manifested a fat mass reduction in the inguinal
WAT, epididymal WAT, and brown adipose tissue (Figure 3g).
VSS (L/kg)
In addition, they manifested adipocyte size reduction in the
inguinal WAT (Figure 3h) and less frequent crownlike
structure (CLS) in the epididymal WAT (Figure 3i).
Furthermore, BAT was more active in the mice treated with
14a (Figure 3j). These results demonstrate that 14a as a
selective 5HT2A antagonist has therapeutic potential in
NAFLD and in the metabolic syndrome.
CONCLUSIONS
■
A new series of peripheral 5HT2A antagonists was synthesized
and evaluated for their inhibitory ability. Among them,
compound 14a exhibited a good in vitro activity with an
IC50 value of 8.35 nM. It also showed liver microsomal stability
and no significant inhibition of CYP and hERG. In addition,
compound 14a selectively inhibited 5HT2A among other 5HT
receptor subtypes. From the in vitro and in vivo BBB
permeability study, 14a proved to be a peripherally acting
agent. Compound 14a improved glucose tolerance in HFD-fed
mice. By 5HT2A inhibition, it also decreased the liver weight
and alleviated lipid accumulation and hepatic steatosis. The
treated mice additionally showed decreased fat mass. In
conclusion, these newly synthesized peripherally acting 5HT2A
antagonists have the potential for further development as drugs
in the treatment of fatty liver disease.
EXPERIMENTAL SECTION
General. All solvents and chemicals were used as purchased
without further purification. All the reported yields are isolated yields
■
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Figure 3. In vivo efficacy of 14a for HFD-induced NAFLD mice. C57BL/6J mice (12 weeks old) underwent 10 weeks of HFD feeding and for the
same period intraperitoneal injections of 14a. (a) Whole liver weight of HFD-fed control and 14a-treated mice. (b) Representative liver histology
by hematoxylin and eosin staining from HFD-subjected control and 14a-treated mice. Scale bars, 100 μm. (c) Hepatic triglyceride measurement.
(d) Bodyweight trends. (e) Intraperitoneal glucose tolerance test (IPGTT) after 16 h of fasting. (f) Body composition, analyzed by NMR. (g)
Inguinal WAT (iWAT), epididymal WAT (eWAT), BAT, and muscle (quadriceps) tissue mass. (h) Representative H&E staining images of (h)
iWAT, (i) eWAT, and (j) BAT. The data are presented as mean SEM (n = 7 per group). *p < 0.05; **p < 0.01; ***p < 0.00; Con, Control; 14a-
50mpk, 14a at 50 mg/kg; 14a-100mpk, 14a at 100 mg/kg.
after column chromatography or crystallization. 1H NMR spectra and
13C spectra were recorded on a JEOL JNM-ECS400 spectrometer at
Step 2. To a solution of compound 3 (3.45 g, 15.452 mmol) in
dimethylformamide (12 mL), 1-bromo-2-methylpropane (2.016 mL,
18.543 mmol) and K2CO3 (3.203 g, 23.984 mmol) were added and
stirred at 80 °C for 16 h. The mixture was cooled to room
temperature and extracted with ethyl acetate and water. The
combined organic layer was washed with brine and dried over
anhydrous Na2SO4, filtered, and concentrated. The residue was then
purified by silica gel chromatography to provide tert-butyl (4-
hydroxybenzyl)carbamate (4) (1.44 g, 33%) as a white solid. 1H
NMR (400 MHz, DMSO-d6) δ 7.29 (t, J = 6.0 Hz, 1H), 7.13 (d, J =
8.5 Hz, 2H), 6.85 (d, J = 8.5 Hz, 2H), 4.03 (d, J = 6.1 Hz, 2H), 3.70
(d, J = 6.4 Hz, 2H), 2.04−1.94 (m, 1H), 1.38 (s, 9H), 0.96 (d, J = 6.7
Hz, 6H).
Step 3. Compound 4 (1.44 g, 5.154 mmol) in ethyl acetate (6 mL)
was treated with 4 M solution of hydrogen chloride in 1,4-dioxane (6
mL). The reaction mixture was stirred for 4 h at ambient temperature.
The mixture was distilled and the product was collected by filtration
and washed with ethyl acetate to yield (4-isobutoxyphenyl)-
methanamine hydrochloride (5) (1.025 g, 92%) as a white solid.
1H NMR (400 MHz, DMSO-d6) δ 8.35 (s, 2H), 7.39 (d, J = 8.2 Hz,
2H), 6.95 (d, J = 8.5 Hz, 2H), 3.92 (s, 2H), 3.74 (d, J = 6.7 Hz, 2H),
2.05−1.95 (m, 1H), 0.97 (d, J = 6.7 Hz, 6H).
Step 4. A mixture of compound 5 (1.025 g, 4.752 mmol) and 1,1′-
carbonyldiimidazole (847.51 mg, 5.227 mmol) in dimethylformamide
(4 mL) was stirred for 16 h at ambient temperature. The mixture was
extracted with ethyl acetate and water. The combined organic layer
was washed with brine and dried over anhydrous Na2SO4, filtered, and
evaporated. The residue was purified by silica gel chromatography to
give N-(4-isobutoxybenzyl)-1H-imidazole-1-carboxamide (6) (1.38 g,
68%) as a pale yellow oil. 1H NMR (400 MHz, chloroform-d) δ 8.50
(t, J = 4.7 Hz, 1H), 8.09 (s, 1H), 7.48 (s, 1H), 7.17 (d, J = 8.5 Hz,
400 MHz for 1H NMR and 100 MHz for 13C NMR, respectively. The
chemical shift (δ) is expressed in ppm relative to tetramethylsilane
(TMS) as an internal standard, and CDCl3, DMSO-d6, and CD3OD
were used as solvents. Multiplicity of peaks is expressed as s (singlet),
d (doublet), t (triplet), q (quartet), dd (doublet of doublets), td
(triplet of doublets), qd (quartet of doublets), dt (doublet of triplets),
and m (multiplet). HRMS data were obtained by a JMS 700 (JEOL,
Japan). Melting points were determined on a Melting Point M-560,
purchased from Buchi. Optical rotations were measured on a P-2000
polarimeter, purchased from Jasco. High-performance liquid chroma-
tography (HPLC) analyses were performed with a Waters Agilent
HPLC system equipped with a PDA detector and an Agilent SB-C18
column (1.8 μm, 2.1 × 50 mm). The mobile phase consisted of buffer
A (ultrapure H2O containing 0.1% trifluoroacetic acid) and buffer B
(chromatographic grade CH3CN) for method A and buffer C
(chromatographic grade MeOH) for method B was applied at a flow
rate of 0.3 mL/min.
1-(4-Fluorobenzyl)-3-(4-isobutoxybenzyl)-1-(piperidin-4-
yl)urea Hydrochloride (11). Step 1. A mixture of 4-hydroxybenzyl-
amine (2) (2 g, 16.239 mmol), di-tert-butylcarbonate (7.085 g, 32.478
mmol), and NaHCO3 (3.411 g, 40.598 mmol) in water (30 mL) and
methanol (30 mL) were stirred for 18 h at ambient temperature. The
reaction mixture was concentrated in vacuo and extracted with ethyl
acetate. The combined organic layer was dried over anhydrous
Na2SO4 and evaporated. The residue was purified by silica gel
chromatography to give tert-butyl (4-isobutoxybenzyl)carbamate (3)
(3.45 g, 95%) as a white solid. 1H NMR (400 MHz, chloroform-d) δ
7.12 (d, J = 7.6 Hz, 2H), 6.79−6.75 (m, 2H), 5.63 (s, 1H), 4.82 (s,
1H), 4.23 (d, J = 5.2 Hz, 2H), 1.46 (s, 9H).
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2H), 6.82−6.80 (m, 3H), 4.42 (d, J = 5.5 Hz, 2H), 3.67 (d, J = 6.4
Hz, 2H), 2.10−2.00 (m, 1H), 1.00 (d, J = 6.7 Hz, 6H).
157.47, 136.91, 133.05, 128.35, 128.27, 128.18, 114.88, 114.67,
114.09, 73.77, 53.70, 52.74, 44.40, 44.05, 43.12, 40.73, 30.53, 29.96,
29.00, 27.74, 26.77, 19.10; mp 147−149 °C; HRMS (FAB) m/z
calculated for C29H40FN4O5 [M + H]+ 543.2983, found 543.2984;
HPLC purity 98.2157% (method A); [α]D: −0.1586 (c 1.0406,
methanol).
Step 5. 4-Fluorobenzaldehyde (7) (1.702 mL, 16.115 mmol) and
4-amino-1-bocpiperidine (8) (3.227 g, 16.115 mmol) in methanol
(30 mL) were stirred for 16 h at ambient temperature. NaBH4 (1.837
g, 48.344 mmol) was added to the reaction mixture in portions, and
the resulting mixture was stirred for 16 h. The mixture was evaporated
and extracted with ethyl acetate and water. The combined organic
layer was dried over anhydrous Na2SO4, filtered, and concentrated.
The residue was then purified by silica gel chromatography to give
tert-butyl 4-((4-fluorobenzyl)amino)piperidine-1-carboxylate (9)
In Vitro Activity. The pimavanserin derivatives were evaluated at
a concentration of 1 μM for their antagonist activity on the 5-HT2A
receptor.27,28 5-HT2A stable HEK293 cells were seeded in a 96-well
plate (Costar; 3603) at a density of 8 ∼ 9 × 104 in 100 μL of
minimum essential medium (Welgene; LM007-86) containing 10%
dialyzed fetal bovine serum (Gibco; 30067-334) and 1% penicillin/
streptomycin (Gibco; 15140-122) and kept in a 37 °C incubator for
24 h. The cells were stained with 100 μL of cytosolic calcium dye
using the FLIPR calcium 6 assay solution (Molecular devices,
#R8190) supplemented with 2.5 mM probenecid (Sigma; P8761) and
incubated for 1 h at 37 °C. After 1 h, the designated amount of the
compound was treated in each well and incubated for another 1 h at
37 °C. Then, the cells were loaded in a Flexstation 3 microplate
reader (Molecular Devices; FlexStaion) and 50 μL of 5 μM 5-HT2A
(serotonin hydrochloride, Sigma; H9523) was injected from a source
plate (Nunc 96-well polystyrene conical bottom MicroWell Plate,
Thermo; 249662) and the florescence intensity (Ex/Em-485nm/
525nm) was measured.
Blood−Brain Barrier (BBB) Penetration Study in Mice. The
previously reported methods were used for the BBB penetration of
14a in mice.29 Male ICR mice were intravenously administered 14a
(5 mg/kg) prepared as a solution (DMSO: PEG400: saline = 5:40:55,
v/v/v %). After 0.5 h, the mice were euthanized using CO2 gas, and
the blood was collected from the heart immediately and the plasma
was separated by centrifugation at 3000 rpm for 10 min at 4 °C. The
rest of the blood was washed out from the circulation by performing
cardiac perfusion with physiological saline containing 10 U/mL
heparin. Then, the brain was removed from the skull and added to
three volumes of PBS buffer per weight, homogenized, and stored at
−20 °C until analyzed by LC-MS/MS. The concentrations of 14a in
plasma and brain samples were determined using an API 4000 Q trap
mass spectrometer (Applied Biosystems, Foster City, CA, USA)
coupled with a 1200 series HPLC system (Agilent, Santa Clara, CA,
USA). Isocratic elution was used with 80% acetonitrile and 20% water
with 0.1% formic acid for the separation of analytes. The total run
time was 3 min, and the flow rate was 0.3 mL/min.
Pharmacokinetic Study in Rats. The compound was adminis-
tered by intravenous injection (5 mg/kg) in male SD rats. It was
prepared with the same vehicle as was used in the BBB penetration
study. Blood samples were collected at time points of 0.083, 0.167,
0.5, 1, 2, 4, 8, and 24 h post-injection. After the separation of plasma
by centrifugation, the concentration of 14a in plasma was determined
by the LC−MS/MS system. Pharmacokinetic parameters were
obtained by noncompartmental analysis using the WinNolin program
(ver. 6.3, Pharsight, Mountain View, CA).
In Vivo Efficacy for NAFLD. C57BL/6J mice were purchased
from the Charles River Japan (Yokohama, Japan). The mice were
housed in climate-controlled, specific pathogen-free barrier facilities
under a 12 h light−dark cycle, and chow and water were provided ad
libitum. Mice (12 weeks old) were fed with a HFD (Research Diet
D12492, 60% fat calories) for 10 weeks and administered 14a by daily
intraperitoneal injection for the same period. On the ninth week of
HFD feeding, intraperitoneal glucose tolerance test (2 g/kg of
glucose) was performed and body composition was measured by
NMR analysis (LF50 BCA-analyzer, Bruker) a day before sacrifice. All
animal experiments complied with the relevant ethical regulations.
Experimental protocols for this study were approved by the
institutional animal care and use committee at the Korea Advanced
Institute of Science and Technology.
1
(3.24 g, 65%) as a white solid. H NMR (400 MHz, chloroform-d)
δ 7.32−7.27 (m, 2H), 7.03−6.98 (m, 2H), 4.03 (s, 2H), 3.79 (s, 2H),
2.80 (t, J = 11.6 Hz, 2H), 2.68−2.61 (m, 1H), 1.85 (d, J = 11.3 Hz,
2H), 1.45 (s, 9H), 1.34−1.24 (m, 2H).
Step 6. A mixture of compound 6 (1.38 g, 5.049 mmol), compound
9 (1.557 g, 5.049 mmol), and K2CO3 (1.047 g, 7.573 mmol) in
dimethylformamide (6 mL) were stirred for 18 h at ambient
temperature. The reaction mixture was extracted with ethyl acetate
and water. The combined organic layer was dried over anhydrous
Na2SO4, filtered, and evaporated. The residue was then purified by
silica gel chromatography to give tert-butyl 4-(1-(4-fluorobenzyl)-3-
(4-isobutoxybenzyl)ureido)piperidine-1-carboxylate (10) (1.930 g,
74%) as a colorless oil. 1H NMR (400 MHz, DMSO-d6) δ 7.23 (dd, J
= 8.6, 5.4 Hz, 2H), 7.17−7.06 (m, 4H), 6.89 (t, J = 5.6 Hz, 1H),
6.91−6.81 (m, 2H), 4.40 (s, 2H), 4.18 (d, J = 5.5 Hz, 2H), 4.14−3.99
(m, 1H), 3.94−3.91(m, 2H), 3.74−3.67 (m, 2H), 2.68 (s, 2H), 2.03−
1.93 (m, 1H), 1.50−1.30 (m, 4H), 1.36 (s, 9H), 0.96 (dd, J = 6.6, 1.7
Hz, 6H).
Step 7. A 4 M hydrogen chloride solution in 1,4-dioxane (6 mL)
was added to the solution of compound 10 (1.930 g, 3.757 mmol) in
ethyl acetate (6 mL). The resulting mixture was stirred for 6 h at
ambient temperature. The mixture was concentrated in vacuo and the
1
product was collected by filtration (1.5 g, 88%) as a white solid. H
NMR (400 MHz, methanol-d4) δ 7.30−7.22 (m, 2H), 7.14−7.01 (m,
4H), 6.84−6.78 (m, 2H), 4.50 (s, 2H), 4.37−4.24 (m, 3H), 3.71 (d, J
= 6.5 Hz, 2H), 3.66 (s, 1H), 3.45−3.37 (m, 2H), 3.04 (td, J = 12.7,
4.4 Hz, 2H), 2.09−2.00 (m, 1H), 1.95−1.87 (m, 4H), 1.02 (d, J = 6.7
Hz, 6H); 13C NMR (100 MHz, DMSO-d6) δ 162.19, 159.78, 157.49,
157.33, 136.61, 132.92, 128.38, 128.30, 128.26, 115.02, 114.81,
114.08, 73.75, 66.39, 50.14, 44.24, 43.11, 42.99, 27.74, 26.63, 19.09;
mp 171−173 °C; HRMS (FAB) m/z calculated for C24H33FN3O2 [M
+ H]+ 414.2557, found 414.2557; HPLC purity 95.9277% (method
A).
(S)-2-Amino-5-(4-(1-(4-fluorobenzyl)-3-(4-isobutoxybenzyl)-
ureido)piperidin-1-yl)-5-oxopentanoic Acid (14a). Step 1. A
mixture of compound 11 (250 mg, 0.557 mmol), 1-benzyl N-
carbobenzoxy-L-glutamate (207 mg, 0.557 mmol), EDCI (267.12 mg,
1.393 mmol), HOBt (113.04 mg, 0.836 mmol), and DIPEA (0.341
mL, 1.951 mmol) in dimethylforamide (2 mL) was stirred for 16 h at
ambient temperature. The reaction mixture was extracted with ethyl
acetate and water. The combined organic layer was dried over
anhydrous Na2SO4, filtered, and evaporated. The residue was then
purified by silica gel column chromatography go give benzyl (S)-2-
(((benzyloxy)carbonyl)amino)-5-(4-(1-(4-fluorobenzyl)-3-(4-
isobutoxybenzyl)ureido)piperidin-1-yl)-5-oxopentanoate (260 mg,
61%) as a colorless oil.
Step 2. To a solution of benzyl (S)-2-(((benzyloxy)carbonyl)-
amino)-5-(4-(1-(4-fluorobenzyl)-3-(4-isobutoxybenzyl)ureido)-
piperidin-1-yl)-5-oxopentanoate in methanol (100 mL), Pd/C (100
mg) was added and stirred for 2 h under a hydrogen atmosphere. The
reaction mixture was filtered through Celite and evaporated. The
residue was filtered with diethyl ether to give compound 14a (138 mg,
75%) as a pale gray solid. 1H NMR (400 MHz, methanol-d4) δ 7.27−
7.18 (m, 2H), 7.12−7.06 (m, 2H), 7.06−6.96 (m, 2H), 6.84−6.76
(m, 2H), 4.58 (d, J = 13.7 Hz, 1H), 4.47 (s, 2H), 4.35−4.27 (d, J =
4.5 Hz, 3H), 4.01−3.92 (m, 1H), 3.71 (d, J = 6.5 Hz, 2H), 3.58 (dt, J
= 7.7, 5.8 Hz, 1H), 3.17−3.06 (m, 1H), 2.69−2.54 (m, 3H), 2.14−
1.96 (m, 3H), 1.77−1.45 (m, 4H), 1.02 (d, J = 6.7 Hz, 6H); 13C
NMR (100 MHz, DMSO-d6) δ 170.31, 170.00, 162.09, 159.69,
Quantification of Hepatic Triglyceride. Liver tissues were
homogenized in 5% NP-40 using FastPrep-24 (MP Biomedicals). To
solubilize fat, the homogenates were heated to 95 °C for 5 min and
cooled at 23 °C and repeated. A triglyceride reagent (Sigma-Aldrich)
or PBS was added and incubated at 37 °C for 30 min to hydrolyze TG
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pubs.acs.org/jmc
Article
into glycerol. For the colorimetric assay of hydrolyzed TG levels,
samples were incubated with a free glycerol reagent (Sigma-Aldrich)
at 37 °C for 5 min. Differences in absorbance at 540 nm between
hydrolyzed or nonhydrolyzed TG were quantified using a glycerol
standard (Sigma-Aldrich). TG contents were normalized by the
protein concentrations of homogenates, which were measured with a
BCA protein assay kit (Thermo Scientific).
Histological Analysis. Mouse liver, inguinal white adipose tissue,
epididymal white adipose tissue, and interscapular brown adipose
tissue were harvested and fixed in 10% neutral buffered formalin
solution (Sigma-Aldrich) and embedded in paraffin. The 5 μm-thick
tissue sections were deparaffinized, rehydrated, and stained with
hematoxylin and eosin (H&E).
Kyungpook National University Hospital, Daegu 41010,
Republic of Korea
Seung Mi Lee − Leading-edge Research Center for Drug
Discovery and Development for Diabetes and Metabolic Disease,
Kyungpook National University Hospital, Daegu 41010,
Republic of Korea
Mi-jin Kim − Leading-edge Research Center for Drug Discovery
and Development for Diabetes and Metabolic Disease,
Kyungpook National University Hospital, Daegu 41010,
Republic of Korea
Myung Ae Bae − Bio & Drug Discovery Division, Korea
Research Institute of Chemical Technology, Daejeon, Republic of
Korea
Dooseop Kim − R&D center, JD Bioscience, Gwangju 61005,
Republic of Korea
Heejong Lee − R&D center, JD Bioscience, Gwangju 61005,
Republic of Korea
Eun-Young Lee − R&D center, JD Bioscience, Gwangju 61005,
Republic of Korea
Jae-Han Jeon − Department of Internal Medicine, School of
Medicine, Kyungpook National University, Kyungpook National
University Hospital, Daegu 41944, Republic of Korea; Leading-
edge Research Center for Drug Discovery and Development for
Diabetes and Metabolic Disease, Kyungpook National
University Hospital, Daegu 41010, Republic of Korea
In-Kyu Lee − Department of Internal Medicine, School of
Medicine, Kyungpook National University, Kyungpook National
University Hospital, Daegu 41944, Republic of Korea; Leading-
edge Research Center for Drug Discovery and Development for
Diabetes and Metabolic Disease, Kyungpook National
University Hospital, Daegu 41010, Republic of Korea
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c00002.
■
sı
Synthesis procedures, 1H NMR, 13C NMR, HRMS
spectra, and HPLC assessment of purity for all the final
compounds, dose response curve, and selectivity data of
compound 14a (PDF)
Molecular formula strings (CSV)
AUTHOR INFORMATION
Corresponding Authors
■
Hail Kim − Graduate School of Medical Science and Engineering,
Korea Advanced Institute of Science and Technology, Daejeon
34141, Republic of Korea; Phone: +82-42-350-4243;
Email: hailkim@kaist.edu; Fax: +82-42-350-4287
Jin Hee Ahn − Department of Chemistry, Gwangju Institute of
Science and Technology, Gwangju 61005, Republic of Korea;
R&D center, JD Bioscience, Gwangju 61005, Republic of
Korea; orcid.org/0000-0002-6957-6062; Phone: +82-62-
715-4621; Email: jhahn@gist.ac.kr
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.0c00002
Author Contributions
‡M.K. and I.H. equally contributed to this work.
Authors
Minhee Kim − Department of Chemistry, Gwangju Institute of
Science and Technology, Gwangju 61005, Republic of Korea;
orcid.org/0000-0001-5384-7342
Notes
The authors declare no competing financial interest.
Inseon Hwang − Graduate School of Medical Science and
Engineering, Korea Advanced Institute of Science and
Technology, Daejeon 34141, Republic of Korea
Haushabhau S. Pagire − Department of Chemistry, Gwangju
Institute of Science and Technology, Gwangju 61005, Republic
of Korea
Suvarna H. Pagire − Department of Chemistry, Gwangju
Institute of Science and Technology, Gwangju 61005, Republic
of Korea
Wonsuk Choi − Graduate School of Medical Science and
Engineering, Korea Advanced Institute of Science and
Technology, Daejeon 34141, Republic of Korea
Won Gun Choi − Graduate School of Medical Science and
Engineering, Korea Advanced Institute of Science and
Technology, Daejeon 34141, Republic of Korea
Jihyeon Yoon − Department of Chemistry, Gwangju Institute of
Science and Technology, Gwangju 61005, Republic of Korea
Won Mi Lee − Department of Chemistry, Gwangju Institute of
Science and Technology, Gwangju 61005, Republic of Korea
Jin Sook Song − Bio & Drug Discovery Division, Korea
Research Institute of Chemical Technology, Daejeon, Republic of
Korea
ACKNOWLEDGMENTS
■
This work was supported by grants from the Korea Health
Industry Development Institute (KHIDI), funded by the
Ministry of Health and Welfare (HI16C1501, HI12C1006),
the Ministry of Science, ICT and Future Planning(MSIP)/
National Research Foundation of Korea(NRF)
(2016M3A9B6902868).
ABBREVIATIONS
■
NAFLD, nonalcoholic fatty liver disease; NASH, nonalcoholic
steatohepatitis; TPH, tryptophan hydroxylase; DKO, double
knockout; WAT, white adipose tissue; BAT, brown adipose
tissue; NAS, NAFLD activity score; TG, hepatic triglyceride;
CDI, 1,1′-carbonyldiimidazole; MeOH, methanol; EtOAc,
ethyl acetate; DIPEA, N,N-diisopropylethylamine; EDCI, 1-
ethyl-3-(3-diemthylaminopropyl)carbodiimide; HOBt, hydrox-
ybenzotriazole; EtOH, ethanol; TEA, triethylamine; dppf, 1,1′-
bis(dipheynlphosphino)ferrocene; ACN, acetonitrile; IPA,
isopropylalcohol; HFD, high-fat diet; CLS, crownlike struc-
ture; IPGTT, intraperitoneal glucose tolerance test
Eun Kyung Yoo − Leading-edge Research Center for Drug
Discovery and Development for Diabetes and Metabolic Disease,
K
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Article
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