Peripheral Selective Oxadiazolylphenyl Alanine Derivatives as Tryptophan Hydroxylase 1 Inhibitors for Obesity and Fatty Liver Disease

Article abstract of DOI:10.1021/acs.jmedchem.0c01560

Tryptophan hydroxylase 1 (TPH1) has been recently suggested as a promising therapeutic target for treating obesity and fatty liver disease. A new series of 1,2,4-oxadiazolylphenyl alanine derivatives were identified as TPH1 inhibitors. Among them, compound 23a was the most active in vitro, with an IC50 (half-maximal inhibitory concentration) value of 42 nM, showed good liver microsomal stability, and showed no significant inhibition of CYP and hERG. Compound 23a inhibited TPH1 in the peripheral tissue with limited BBB penetration. In high-fat diet-fed mice, 23a reduced body weight gain, body fat, and hepatic lipid accumulation. Also, 23a improved glucose intolerance and energy expenditure. Taken together, compound 23a shows promise as a therapeutic agent for the treatment of obesity and fatty liver diseases.

Full text of DOI:10.1021/acs.jmedchem.0c01560

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Article
Peripheral Selective Oxadiazolylphenyl Alanine Derivatives as
Tryptophan Hydroxylase 1 Inhibitors for Obesity and Fatty Liver
Disease
Eun Jung Bae,# Won Gun Choi,# Haushabhau S. Pagire, Suvarna H. Pagire, Saravanan Parameswaran,
Jun-Ho Choi, Jihyeon Yoon, Won-il Choi, Ji Hun Lee, Jin Sook Song, Myung Ae Bae, Mijin Kim,
Jae-Han Jeon, In-Kyu Lee, Hail Kim,* and Jin Hee Ahn*
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ABSTRACT: Tryptophan hydroxylase 1 (TPH1) has been recently suggested as a promising therapeutic target for treating obesity
and fatty liver disease. A new series of 1,2,4-oxadiazolylphenyl alanine derivatives were identified as TPH1 inhibitors. Among them,
compound 23a was the most active in vitro, with an IC50 (half-maximal inhibitory concentration) value of 42 nM, showed good liver
microsomal stability, and showed no significant inhibition of CYP and hERG. Compound 23a inhibited TPH1 in the peripheral
tissue with limited BBB penetration. In high-fat diet-fed mice, 23a reduced body weight gain, body fat, and hepatic lipid
accumulation. Also, 23a improved glucose intolerance and energy expenditure. Taken together, compound 23a shows promise as a
therapeutic agent for the treatment of obesity and fatty liver diseases.
adipose tissue (BAT).17,19−21 These results indicate that
peripheral serotonin can contribute to weight gain and fat
INTRODUCTION
■
Serotonin (5-hydroxytryptamine, 5-HT) is a monoamine
found in the central and peripheral systems.1−3 Serotonin is
accumulation and that TPH1 inhibitors could serve as a new
treatment for obesity and fatty liver disease.17,20,22
synthesized from L-tryptophan, sequentially, by the enzymes
Serotonergic systems are divided into the central and
tryptophan hydroxylase (TPH) and aromatic amino acid
peripheral systems by the blood−brain barrier (BBB).23,24 As
decarboxylase (AAAD).4,5 In serotonin biosynthesis, TPH is a
serotonin cannot cross the BBB,24 two serotonergic systems
rate-determining enzyme that catalyzes the hydroxylation of L-
tryptophan6,7 (Figure 1). Thus, serotonin production is
are functionally separated. In this regard, we envisioned new
controlled by the activity of TPH.8
In the central nervous system, serotonin acts as a
peripheral TPH1 inhibitors that do not affect the CNS, for
treating obesity and fatty liver disease.
Several TPH1 inhibitors have been reported previously. p-
neurotransmitter associated with mood, anxiety, cognition,
and appetite and maintains homeostasis.6,9,10 The lack of
Chlorophenylalanine (pCPA) is one of the classic TPH1
inhibitors.25,26 However, its binding activity to TPH1 in vitro is
serotonin in the central nervous system (CNS) causes
neuropsychiatric diseases.11,12 In peripheral tissue, serotonin
Received: September 7, 2020
Published: January 8, 2021
plays a critical role in the regulation of metabolism and is
emerging as a potential target for treating both obesity and
fatty liver disease.13−16 Recently, our laboratory17,18 and others
found that high-fat diet (HFD)-fed TPH1 knockout (KO)
mice showed reduced weight gain and lipogenesis in white
adipose tissue (WAT), and increased thermogenesis in brown
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Figure 1. Biosynthesis of serotonin (5-hydroxytryptamine 5-HT) from L-tryptophan.
Figure 2. Drug design and reported TPH1 inhibitors in clinical trials (and market).
very weak (∼50 μM of IC50 (half-maximal inhibitory
concentration)), and pCPA is associated with central side
effects including depression, because it passes into the
CNS.27−30 Lexicon Pharmaceuticals, Inc. reported a new
class of phenyl alanine derivatives as a TPH1 inhibitor, using
high-throughput screening.31 Among them, telotristat ethyl,
which is a prodrug of telotristat, was recently launched in the
market as a TPH1 inhibitor for the treatment of carcinoid
syndrome.32−34 Karos Pharmaceuticals, Inc. reported a series
of spirocyclic proline and acyl guanidine compound-based
TPH1 inhibitors that were further modified from Lexicon’s
compound.35,36 Phase I and II clinical trials are in progress for
rodatristat ethyl for the treatment of pulmonary arterial
hypertension (PAH).37,38 In addition, the Novartis Research
Foundation discovered a novel allosteric site binding TPH1
inhibitor, using a high-throughput proximity assay.39 TPH1
inhibitors in clinical trials, such as telotristat, LX-1031, and
rodatristat, share a chiral arylsubstituted trifluoroethoxyl group.
These prompted us to design new TPH1 inhibitors, which
have a less chiral center for ease of synthesis and more rigidity
(Figure 2). Based on our previous TPH1 KO results for
obesity and fatty liver17 and reported effects of TPH1
inhibitors, we tried to develop our own new TPH1 inhibitor
acting in the peripheral tissue for treating obesity and fatty liver
disease. Here, we report the design, synthesis, and biological
evaluation of a series of oxadiazolylphenyl alanine derivatives
as TPH1 inhibitors.
RESULTS AND DISCUSSION
■
The synthetic route of 1,3-imidazole derivatives is outlined in
Scheme 1. Commercially available L-tyrosine 1 was converted
into ester 2, followed by N-Boc protection with di-tert-butyl
dicarbonate to give compound 3. Compound 3 reacted with
trifluromethanesulfonic anhydride to give triflate 4.
Triflate 4 was treated with molybdenum hexacarbonyl in the
presence of 1,1′-bis(diphenylphosphino)ferrocene in a micro-
wave to give carboxylic acid 5. The coupling of compound 5
with 2-bromo-1-(4-methoxyphenyl)ethan-1-one resulted in
compound 6. Cyclization of compound 6 with ammonium
acetate provided intermediate 7, followed by ester saponifica-
tion and acidic deprotection to give the desired amino acid
hydrochloride 8.
1,3-Thidazole derivatives were prepared as described in
Scheme 2. Triflate 4 was coupled with 4,4,4′,4′,5,5,5′,5′-
octamethyl-2,2′-bi(1,3,2-dioxaborolane) in the presence of a
palladium catalyst to yield boronate ester 9. The Suzuki
reaction of compound 9 with 2-bromothiazole gave
intermediate 10, which was further coupled with 1-bromo-4-
methoxybenzene or 4-bromo-1,2-dimethoxybenzene in the
presence of a palladium catalyst to afford 11a or 11b,
respectively. Protected 1,3-thiazole 11 was hydrolyzed with a
2 M sodium hydroxide solution, followed by acidic
deprotection to give the amino acid hydrochloride 12.
1,2,4-Oxadiazole derivatives were synthesized as shown in
Scheme 3. Commercially available (S)-2-amino-3-(4-
cyanophenyl)propanoic acid 13 was converted into protected
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a
Scheme 1. Synthesis of a 1,3-Imidazole Compound
a
Reagents and conditions: (a) SOCl2, ethanol (EtOH), reflux, and 63%; (b) di-tert-butyl dicarbonate, Et3N, methanol (MeOH), 0 °C to rt, and
96%; (c) trifluromethanesulfonic anhydride, pyridine, CH2Cl2, −15 °C to rt, and 83%; (d) Mo(CO)6, N,N-diisopropylethylamine (DIPEA), 4-
dimethylaminopyridine (4-DMAP), Pd(OAc)2, 1,1′-bis(diphenylphosphino)ferrocene, 1,4-dioxane, H2O, microwave, 110 °C, and 55%; (e) 2-
bromo-1-(4-methoxyphenyl)ethan-1-one, Et3N, acetonitrile, rt, and 78%; (f) ammonium acetate, xylene, 160 °C, and 15%; (g) NaOH, THF, H2O,
and rt; and (h) 4 M HCl in 1,4-dioxane, ethyl acetate (EtOAc), rt, and 68%.
compound 14. Compound 14 was reacted with hydroxylamine
hydrochloride under reflux to give intermediate 15. Amidox-
ime 15 was coupled with substituted benzoic acid, followed by
cyclization under reflux conditions to give compound 16.
Protected 1,2,4-oxadiazole 16 was treated with a 2 M sodium
hydroxide solution, followed by acidic deprotection to give
amino acid hydrochloride 17.
Methyl 3-bromo-4-methoxybenzoate 18 was subjected to
Suzuki reaction with substituted phenyl boronic acid in the
presence of a palladium catalyst, followed by hydrolysis to give
carboxylic acid 19. Acetyl protection of compound 19 using
acetyl chloride gave compound 20. The amide coupling
reaction of compound 22a with ammonium hydroxide,
followed by deprotection, resulted in compound 24.
Deacetylation of compound 21a under basic conditions,
followed by acidic deprotection, afforded compound 26.
The synthesis of 1,3,4-oxadiazole derivatives is depicted in
Scheme 4. 3-[Bis(dimethylamino)methyliumyl]-3H-benzotria-
zol-1-oxide hexafluorophosphate (HBTU) coupling of carbox-
ylic acid 5 with substituted benzohydrazide produced
compound 27. Cyclization of compound 27 with p-
toluenesulfonyl chloride gave compound 28. Protected 1,3,4-
oxadiazole 28 was hydrolyzed, followed by acidic deprotection
to give amino acid hydrochloride 29.
The preparation of 1,2,4-thiadiazole derivatives is outlined in
Scheme 5. Commercially available 3-bromo-5-chloro-1,2,4-
thiadiazole 31 was coupled with substituted 4-methoxyphe-
nylboronic acid derivatives 30 in the presence of a palladium
catalyst to produce compound 32. Suzuki reaction of the
bromide of compound 32 with boronate ester 9 provided
compound 33. Compound 33 was subjected to hydrolysis and
acidic deprotection to afford 1,2,4-thiadiazole compound 34.
To identify a new peripherally acting TPH1 inhibitor, we
screened an in-house library with the amino acid moiety, which
generally showed BBB impermeability. Compound 8, contain-
ing a 1,3-imidazole core, was identified as a new and
moderately potent TPH1 inhibitor (80% inhibition at 10 μM
and 21% inhibition at 1 μM). We optimized the imidazole core
and synthesized diverse five-membered heterocyclic deriva-
tives, which are summarized in Table 1. LP533401 was used as
a reference.30 1,3-Thiazole derivatives 12a and 12b showed
decreased activities compared to compound 8, with 10−15%
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a
Scheme 2. Synthesis of a 1,3-Thiazole Compound
a
Reagents and conditions: (a) 4,4,4′,4′,5,5,5′,5′-octamethyl-2,2′-bi(1,3,2-dioxaborolane), potassium acetate (KOAc), PdCl2(dppf).CH2Cl2,
DMSO, 100 °C, and 84%; (b) 2-bromothiazole, K2CO3, 5 mol % Pd(PPh3)4, 1,4-dioxane, H2O, 100 °C, and 79%; (c) 1-bromo-4-methoxybenzene
or 4-bromo-1,2-dimethoxybenzene, KOAc, Pd(OAc)2, N-methyl-2-pyrrolidone (NMP), 120 °C, and 25−57%; (d) NaOH, THF, H2O, rt; and (e)
4 M HCl in 1,4-dioxane, EtOAc, rt, and 23−69%.
inhibitions at 1 μM. 1,2,4-Oxadiazole derivatives (17a, 17f)
showed greatly increased in vitro inhibitory activity with
compound 17f, causing a 68% inhibition of TPH1 at 1 μM.
However, once the 1,2,4-oxadiazole core was converted into a
1,3,4-oxadiazole core (29a,b), which is an isomer of 1,2,4-
oxadiazole, the TPH1-inhibitory activity was lost (Table 1).
Furthermore, the 1,2,4-thiadiazole core (34a,b) had a lower
potency than the 1,2,4-oxadiazole core (17a, 17f). Therefore,
the 1,2,4-oxadiazole core 17a was selected for further
optimization.
As shown in Table 2, compound 17a with a 4-methoxy
group had good in vitro activity, with an IC50 value of 483 nM.
However, the replacement of the 4-methoxy group with a
simple phenyl group in 17b resulted in the loss of inhibition.
The replacement of the 4-methoxy group in 17a with the 4-
fluoro group in 17c resulted in moderate activity, with an IC50
of 1570 nM. The replacement of the 4-methoxy moiety with 4-
hydroxy in 17d, or 3-methoxy in 17e, resulted in no inhibitory
activity at 1 μM. Compound 17f, which has a 3,4-dimethoxy
substituent, showed good in vitro activity, with an IC50 of 490
nM. This suggests that the 4-methoxy substituent is essential
for in vitro activity and prompted us to modify the molecule at
the 3-position. The 4-ethoxy substituent 17g and the 4-
methoxybenzyl derivative 17h had reduced inhibitory activity.
Therefore, we fixed the 4-methoxy substituent 17a and further
modified the molecule at the 3-position.
out in AutoDock4.2.41 The interactions between the inhibitor
17a and TPH1 revealed that the binding mode of 17a is similar
to that of LP-533401, according to the PDB (Protein Data
Bank) structure 3HF8,40 and is shown in Figure 3. The amino
acid head from the inhibitor 17a interacts with Arg257 and
Thr265 of TPH1, while the phenyl group attached to the
amino acid head of 17a has a π−π interaction with His272.
The core, 1,2,4-oxadiazole ring of inhibitor 17a interacts with
Glu317 of TPH1 via a π−anion interaction. The methox-
yphenyl group has a π−π T-shaped interaction with Tyr312 of
TPH1. Docking analysis also revealed an unoccupied sub−
pocket in residues 228 to 252 (forming a secondary structure
of the β strand−α helix−β strand), while the distance between
the 3-methoxy position on 17a and the backbone oxygen of
Gly234 of TPH1 was 8.17 Å, providing additional space for the
optimization of 17a. Based on the docking study and the
activity of 17a and 17f, we further introduced substituents at
the 3-methoxy position of 17a.
Additional 4-methoxyphenyl-substituted 1,2,4-oxadiazole
derivatives were synthesized and evaluated (Table 3). The
introduction of a meta-substituted phenyl group (17i) at the
compound 17a resulted in a significant increase in the in vitro
inhibitory activity (17i, 97% inhibition at 1 μM). We next
introduced an electron-donating group on the meta-phenyl
ring such as methoxy group 17j, which resulted in extremely
effective TPH1 inhibition (∼100% inhibition at 1 μM), with
an IC50 of 65 nM. The 4-hydroxy biphenyl analogue 23a
showed better in vitro activity than 17j and telotristat (64
nM), with an IC50 of 42 nM.
To optimize compound 17a further, a molecular docking
study of 17a with TPH1 was conducted with the crystal
structure of TPH1 (Protein Data Bank access code 3HF8),40
and the compound’s atomic coordinates were generated from
its chemical structure. The docking calculations were carried
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a
Scheme 3. Synthesis of a 1,2,4-Oxadiazole Compound
a
Reagents and conditions: (a) di-tert-butyl dicarbonate, NaHCO3, 1,4-dioxane, H2O, 0 °C to rt, and 72%; (b) iodoethane, K2CO3, DMF, 0 °C to
rt, and 94%; (c) hydroxylamine hydrochloride, Et3N, EtOH, reflux, and 90%; (d) substituted benzoic acid, 1-ethyl-3-(3-dimethylaminopropyl)-
carbodiimide, 1-hydroxybenzotriazole (HOBt), DIPEA, CH2Cl2, and rt; (e) 1,4-dioxane, reflux, 38−97%; (f) NaOH, THF, H2O, and rt; (g) 4 M
HCl in 1,4-dioxane, EtOAc, rt, and 26−94%; (h) substituted phenyl boronic acid, K2CO3, 5 mol % Pd(PPh3)4, 1,4-dioxane, H2O, and 100 °C; (i)
NaOH, THF, H2O, rt, and 73−83%; (j) acetyl chloride, pyridine, CH2Cl2, 0 °C to rt, and 55−74%; (k) ammonium hydroxide, 1-ethyl-3-(3-
diemthylaminopropyl)carbodiimide (EDCI), HOBt, THF, rt, and 35%; and (l) NaHCO3, EtOH, rt, and 83%.
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a
Scheme 4. Synthesis of a 1,3,4-Oxadiazole Compound
a
Reagents and conditions: (a) 4-methoxybenzohydrazide or 3,4-dimethoxybenzohydrazide, HBTU, DIPEA, DMF, rt, and 92−97%; (b) DIPEA, p-
toluenesulfonyl chloride, acetonitrile, rt, and 77−81%; (c) NaOH, THF, H2O, and rt; and (d) 4 M HCl in 1,4-dioxane, EtOAc, rt, and 46−83%.
a
Scheme 5. Synthesis of a 1,2,4-Thiadiazole Compound
a
Reagents and conditions: (a) tripotassium phosphate, PdCl2(dppf).CH2Cl2, DMF, microwave, 80 °C, and 62−77%; (b) K2CO3, 5 mol %
Pd(PPh3)4, 1,4-dioxane, H2O, 100 °C, and 46−76%; (c) NaOH, THF, H2O, and rt; and (j) 4 M HCl in 1,4-dioxane, EtOAc, rt, and 54−66%.
Next, we replaced the amino acid 23a with amino amide 24,
its ethyl ester hydrochloride 26, but this change resulted in the
loss or decrease of TPH1 potency (Table 4).
We synthesized individual regioisomers of 23a and evaluated
their activity as shown in Table 5. Meta-hydroxy-substituted
compounds (23b) and ortho-hydroxy-substituted compounds
(23c) had weaker in vitro TPH1 inhibition activity as
compared to that of para-substituted compounds (23a). In
addition, compound 23d had less in vitro potency than
compound 23a.
Based on the in vitro data, we chose compound 23a as a
prototype for further evaluation of its liver microsomal
stability, and CYP and hERG inhibition (Table 6). Compound
23a had good liver microsomal stability (92% of the parent
remained after a 30 min incubation in human liver micro-
somes) and no significant CYP or hERG inhibition.
tissue and plasma at 1 h after intraperitoneal (IP)
administration (10 mg/kg). The concentration of 23a in
plasma was 3108.00 1077.63 ng/mL, while it was below the
quantification limit (31.3 ng/g) in the brain after 1 h. It
indicates limited BBB penetration of compound 23a.
It has been reported that genetic deletion or inhibition of
TPH1 has an anti-obesity effect through the activation of
thermogenesis in BAT and iWAT.17−19 Thus, we evaluated the
in vivo efficacy of compound 23a for obesity and fatty liver
disease. First, we checked 5-HT levels in serum and brain and
whether 23a has blood−brain barrier (BBB) permeability and
affects 5-HT synthesis in the brain. C57BL6J mice were treated
with 23a or pCPA by a daily intraperitoneal injection for one
week. Although pCPA treatment decreased 5-HT levels in
both serum and brain, 23a treatment decreased 5-HT levels
only in serum but not in brain 5-HT (Figure 4A), indicating
that 23a cannot cross BBB and thereby effectively inhibits 5-
HT synthesis only in peripheral tissues. To further verify the
Further, the in vivo BBB permeability of 23a was evaluated
(Table 7). The concentration of 23a was examined in brain
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Table 1. Structure−Activity Relationship of Five-Membered Heteroarylphenyl Alanine Derivatives
anti-obesity effect of 23a in vivo, C57BL6J mice were treated
with 23a by a daily intraperitoneal injection for eight weeks
with a high-fat diet (HFD) feeding. After 23a treatment for 8
weeks, serum 5-HT levels were robustly reduced (Figure 4B).
Body weight gain upon HFD was attenuated in mice treated
with 23a compared to control mice (Figure 4C). The glucose
tolerance test revealed that glucose tolerance was improved in
HFD-fed mice treated with 23a in a dose-dependent manner
(Figure 4D). Furthermore, fat mass was decreased and lean
body mass was increased by 23a treatment (Figure 4E).
Consistently, the size of adipose tissues including eWAT,
iWAT, and BAT was reduced in 23a-treated mice (Figure 4F−
H). In the histological analysis using H&E staining, a crownlike
structure was observed less in eWAT of 23a-treated mice,
indicating reduced adipose tissue inflammation (Figure 4I). In
addition, adipocyte size in eWAT and iWAT was reduced in
23a-treated mice (Figure 4I). In BAT, 23a treatment
decreased lipid droplet size and increased multilocular
adipocytes after HFD feeding for eight weeks (Figure 4H,I).
We further measured the metabolic rate using indirect
calorimetry to check whether the anti-obesity effect of 23a is
attributed to the activation of brown fat. VO2, VCO2, and heat
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Table 2. In Vitro Inhibitory Activity of 1,2,4-Oxadiazole Derivatives 17a−h Against TPH1
production were increased in 23a-treated mice (Figure 4J).
However, RER of 23a-treated mice was comparable to that of
control mice, which suggested that 23a treatment increased
both glucose and lipid usage. To test thermogenic activity by
23a treatment, we checked Uncoupling protein 1 (Ucp1)
expression in both BAT and iWAT. Levels of Ucp1 mRNA in
BAT and iWAT were increased by 23a treatment with HFD
feeding, and immunohistochemical staining of UCP1 in BAT
and iWAT showed the robust induction of UCP1 by 23a
treatment with HFD feeding in BAT but not in iWAT (Figure
4K−N). These data demonstrate that inhibition of 5-HT
synthesis by 23a treatment increases energy expenditure in
BAT and thereby prevents obesity.
The inhibition of 5-HT synthesis is also known to reduce
hepatic steatosis by reducing the hepatic lipogenesis through
HTR2A in the liver.42,43 Therefore, we confirmed the efficacy
of 23a for treating hepatic steatosis. Histological analysis
revealed the robust reduction of hepatic lipid accumulation by
23a treatment and, in accordance, hepatic triacylglycerol (TG)
levels were reduced (Figure 4O,P). In addition, plasma TG
and cholesterol levels were reduced in 23a-treated HFD-fed
mice (Figure 4Q,R). Taken together, these data indicate that
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Figure 3. The predicted binding mode of compound 17a at the inhibitor binding pocket of TPH1 has an orientation similar to that of LP-533401,
according to the PDB structure, 3HF8.40 The three-dimensional structure of human TPH1 is shown with a cartoon representation, whereas
compound 17a is shown as sticks. The unoccupied subpocket of the inhibitor binding pocket of TPH1 identified by docking analysis is shown with
surface representation. The key interactions are depicted as dotted lines according to the interaction type.
Table 3. In Vitro Inhibitory Activity of Compounds 17a, 17i, 17j, and 23a against TPH1
23a can protect against diet-induced obesity and hepatic
steatosis in vivo and thus it can be potentially used for anti-
obesity and anti-fatty liver treatment.
The binding mode of 23a with TPH1 was predicted using
available structures of TPH1 (Figure 5). The amino acid head
from the inhibitor 23a interacts with Arg257, Thr265, and
Ser336 via hydrogen bonds, as observed with LP-533401.40
The phenyl group attached to the amino acid head of 23a has a
π−π stacked interaction with Phe318 of TPH1. The 1,2,4-
oxadiazole ring and the methoxyphenyl group of the inhibitor
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Table 4. In Vitro Inhibitory Activities of Compounds 24 and 26 against TPH1
(multiplet). Fast atom bombardment-high-resolution mass spectrom-
etry (FAB-HRMS) data were obtained by a JMS 700 (JEOL, Japan).
The purity of all tested compounds was ≥ 95%, as estimated by high-
performance liquid chromatography (HPLC) analysis. Samples were
analyzed on a Waters Agilent HPLC system equipped with a PDA
detector and a Waters SB-C18 column (1.8 μm, 2.1 × 50 mm2). The
mobile phase was used with buffer A (ultrapure H2O containing 0.1%
TFA) and buffer B (chromatographic-grade CH3CN). The flow rate
was 0.5 mL/min.
23a interact with Glu317 of TPH1 via a π−anion interaction.
The hydroxyphenyl ring, which was introduced adjacent to the
methoxyphenyl group in 23a, has a hydrogen bond with
Gly234 along with a π−π interaction with Phe241 of TPH1,
which is positioned in the subpocket, thereby enhancing the
TPH1 inhibition effect of 23a.
CONCLUSIONS
■
In this study, a series of 1,2,4-oxadiazolylphenyl alanine
derivatives were identified and evaluated for their ability to
inhibit TPH1 inhibitors. Among them, 1,2,4-oxadiazole
derivatives with the 4-methoxyphenyl substituent were found
to be potent TPH1 inhibitors. The binding mode of
compound 17a with TPH1 was predicted for further
optimization. Compound 23a was the most active in vitro,
with an IC50 value of 42 nM, and showed good liver
microsomal stability and no significant inhibitions of CYPs
and hERG. Compound 23a inhibited TPH1 in the peripheral
tissue with limited BBB penetration. In high-fat diet-fed mice,
23a reduced body weight gain and body fats, and improved
glucose tolerance and energy expenditure. In addition, 23a
reduced hepatic lipid accumulation. Taken together, this newly
developed peripheral TPH1 inhibitor can specifically inhibit
the peripheral actions of 5-HT, and shows promise as a
therapeutic agent to ameliorate obesity and fatty liver disease.
(S)-2-amino-3-(4-(5-(4′-hydroxy-6-methoxy-[1,1′-biphenyl]-
3-yl)-1,2,4-oxadiazol-3-yl)phenyl)propanoic Acid Hydrochlor-
ide (23a). Step 1. To a solution of (S)-amino-3-(4-cyanophenyl)-
propanoic acid 13 (3.0 g, 15.77 mmol) in 1,4-dioxane (20 mL) at
0°C, NaHCO3 (3.98 g, 47.32 mmol) in water (10 mL) was added. A
solution of di-tert-butyl dicarbonate (5.16 g, 23.66 mmol) in 1,4-
dioxane was added slowly to the reaction mixture at 0 °C over 1 h.
The reaction mixture was allowed to reach room temperature and
stirred for 18 h. After the completion of the reaction, the solvent was
removed in vacuo. The remaining residue was dissolved in a solution
of 5% KHSO4 and the aqueous phase was extracted with ethyl acetate.
The combined organic phase was dried over anhydrous sodium sulfate
and the product was collected by filtration as a beige solid (3.31 g,
1
72%). H NMR (400 MHz, DMSO-d6) δ 12.72 (s, 1H), 7.75 (d, J =
8.2 Hz, 2H), 7.45 (d, J = 8.2 Hz, 2H), 6.81 (d, J = 7.9 Hz, 1H), 4.11−
4.17 (m, 1H), 3.12 (dd, J = 13.7, 4.3 Hz, 1H), 2.89 (dd, J = 13.6, 10.8
Hz, 1H), 1.30 (s, 9H).
(S)-2-((tert-butoxycarbonyl)amino)-3-(4-cyanophenyl)propanoic
acid (3.31 g, 11.40 mmol) and potassium carbonate (3.15 g, 22.80
mmol) were dissolved in DMF (10 mL), and the reaction mixture was
cooled to 0 °C. Iodoethane (1.1 mL, 13.68 mmol) was slowly added
to this mixture, and the reaction mixture was allowed to reach room
temperature and stirred for 16 h. After the completion of the reaction,
the mixture was diluted with ethyl acetate and water. The combined
organic layer was washed with brine, dried over anhydrous sodium
sulfate, and concentrated under the reduced pressure. The reaction
mixture was purified by silica gel column chromatography to give
ethyl (S)-2-((tert-butoxycarbonyl)amino)-3-(4-cyanophenyl)-
EXPERIMENTAL SECTION
■
General. All solvents and chemicals were used as purchased
without further purification. All reported yields are isolated yields after
column chromatography or crystallization. H NMR spectra and 13C
1
spectra were recorded on a JEOL JNM-ECS400 spectrometer at 400
1
MHz for H NMR and 100 MHz for 13C NMR, respectively. The
chemical shift (δ) is reported in ppm relative to tetramethylsilane
(TMS) as an internal standard, and CDCl3 and DMSO-d were used as
solvents. Multiplicity of peaks is reported 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), dq
(doublet of quartets), ddd (doublet of doublets of doublets), and m
1
propanoate 14 as a white solid (3.42 g, 94%). H NMR (400 MHz,
chloroform-d) δ 7.59 (d, J = 8.2 Hz, 2H), 7.27 (d, J = 8.2 Hz, 2H),
5.05 (d, J = 7.6 Hz, 1H), 4.58 (dd, J = 13.6, 6.4 Hz, 1H), 4.17 (q, J =
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Table 5. In Vitro Inhibitory Activities of Compounds 23a−d against TPH1
Table 6. Liver Microsomal Stability, and CYP and hERG
Inhibition of Compound 23a
Table 7. Brain/Plasma Concentration in Male ICR Mice
(IP, 10 mg/kg, Mean SD, n = 3) for Compound 23a
assay
results
concentration (ng/mL or ng/g)
liver microsomal stability
85% of the parent remained after a 30 min
incubation
92% of the parent remained after a 30 min
incubation
Time (h)
1
plasma
brain tissue
a
(rat)
a
3108.00 1077.63
BQL
liver microsomal stability
(human)
a
Below the quantification limit (31.3 ng/g).
CYP inhibition
1A2: 3.2% inhibition at 10 μM
2C9: <1% inhibition at 10 μM
2C19: 6.7% inhibition at 20 μM
2D6: <1% inhibition at 10 μM
3A4: <1% inhibition at 10 μM
24.3% inhibition at 10 μM
After the reaction mixture was cooled to room temperature, the
volume was reduced by evaporation and water was added to the
resulting mixture, followed by extraction with ethyl acetate. The
combined organic phase was dried over anhydrous sodium sulfate,
filtered, and evaporated in vacuo. The crude product was purified by
silica gel column chromatography to give ethyl (S)-2-((tert-
butoxycarbonyl)amino)-3-(4-(N-hydroxycarbamimidoyl)phenyl)-
hERG inhibition
a
Buspirone was used as a positive control (0.1% (rat) and 4%
(human) remained after a 30 min incubation).
1
propanoate 15 (2.49 g, 90%) as a white solid. H NMR (400 MHz,
DMSO-d6) δ 9.58 (s, 1H), 7.58 (d, J = 8.4 Hz, 2H), 7.32 (d, J = 8.0
Hz, 1H), 7.22 (d, J = 8.4 Hz, 2H), 5.77 (s, 2H), 4.10−4.16 (m, 1H),
4.07 (q, J = 7.1 Hz, 2H) 2.98 (dd, J = 13.7, 5.3 Hz, 1H), 2.86 (dd, J =
13.5, 10.1 Hz, 1H), 1.33 (s, 9H), 1.12 (t, J = 7.1 Hz, 3H).
Step 3. A mixture of methyl 3-bromo-4-methoxybenzoate 18 (3.9
g, 15.91 mmol), (4-hydroxyphenyl)boronic acid (2.41 g, 17.51
mmol), tetrakis(triphenylphosphine)palladium(0) (919.49 mg, 0.80
7.1Hz, 2H), 3.14 (ddd, J = 53.7, 13.7, 6.1 Hz, 2H), 1.41 (s, 9H), 1.24
(t, J = 7.2 Hz, 3H).
Step 2. A mixture of ethyl (S)-2-((tert-butoxycarbonyl)amino)-3-
(4-cyanophenyl)propanoate 14 (2.5 g, 7.85 mmol), hydroxylamine
hydrochloride (1.64 g, 23.56 mmol), and triethylamine (5.48 mL,
39.26 mmol) in ethanol (20 mL) was stirred under reflux for 2 h.
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Figure 4. TPH1 inhibitor 23a protects against diet-induced obesity in vivo in a mouse model by increasing energy expenditure via brown and beige
adipose tissue activation. (A) Serum and brain 5-HT levels. Twelve week old mice treated with vehicle or pCPA (300 mg/kg) or 23a (10 mg/kg)
by a daily intraperitoneal injection with standard chow diet for one week (vehicle, n = 4/group; pCPA, n = 3/group; 23a, n = 3/group). (B−R)
Twelve week old mice were treated with vehicle or 23a (1, 5, or 10 mg/kg) by a daily intraperitoneal injection with a high-fat diet for eight weeks.
(B) Serum 5-HT levels of vehicle or 10 mg/kg 23a-treated mice (n = 5/group). (C) Body weight curve during compound treatment with HFD (n
= 6/group). (D) Intraperitoneal glucose tolerance test (IPGTT) after 16 hours of fasting (n = 6/group). (E) Percent fat mass and lean mass (n =
6/group). (F−H) Gross appearance of eWAT (F), iWAT (G), and BAT (H) with 10 mg/kg 23a treatment. One block = 1 cm×1 cm. (I)
Representative eWAT, iWAT, and BAT histology by H&E staining in vehicle- and 23a-treated mice. Scale bars, 100 μm. (J) Metabolic parameters:
oxygen (O2) consumption, VO2; carbon dioxide (CO2) production, VCO2; and heat production in vehicle or 23a-treated mice (n = 5/group). (K
and L) Relative mRNA expression of Ucp1 (K) and immunohistochemical staining for UCP1 (L) in BAT of vehicle- and 23a-treated mice (n = 5/
group). Scale bars, 100 μm. (M and N) Relative mRNA expression of Ucp1 (M) and immunohistochemical staining for UCP1 (N) in iWAT of
vehicle- and 23a-treated mice (n = 5/group). Scale bars, 100 μm. (O) Representative liver histology by H&E staining in vehicle- and 23a-treated
mice. Scale bars, 100 μm. (P) Hepatic TG levels (n = 6/group). (Q, R) Plasma TG (Q) and cholesterol (R) levels (n = 5/group). Data are
$
#
$$
<
$$$
expressed as means SEM. , , *P < 0.05,
,
##, **P 0.01, and
,
###, ***P < 0.001. Student’s t-test.
mmol), and a 2 M potassium carbonate solution (6.60 g, 47.74 mmol)
in 1,4-dioxane (20 mL) was stirred for 2 h at 100 °C. After the
reaction mixture was cooled to room temperature, the mixture was
extracted with ethyl acetate and water. The organic phase was washed
with brine. Then, the combined organic fraction was dried over
anhydrous sodium sulfate and filtered. The solvent was removed
under reduced pressure to afford a residue that was purified by silica
gel column chromatography to give methyl 4′-hydroxy-6-methoxy-
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Figure 5. Compound 23a binds at the inhibitor binding pocket of TPH1 with key interactions similar to those of compounds 17a and LP-533401,
according to the PDB structure, 3HF8.40 The amino acid head and the 1,2,4-oxadiazole core of compound 23a stabilize its binding with TPH1,
while the introduced hydroxyphenyl ring enhances the inhibition effect of compound 23a. The structure of human TPH1 is shown with a cartoon
representation, whereas compound 23a is shown as sticks. The key interactions are depicted as dotted lines according to the interaction type.
[1,1′-biphenyl]-3-carboxylate (2.875 g, 70%) as a white solid. 1H
NMR (400 MHz, chloroform-d) δ 8.01−7.98 (m, 2H), 7.41 (dt, J =
8.8, 2.4 Hz, 2H), 6.98 (dd, J = 5.0, 4.1 Hz, 1H), 6.91 (dt, J = 8.8, 2.4
Hz, 2H), 3.91 (s, 3H), 3.87 (s, 3H).
4.57 (q, J = 6.4 Hz, 1H), 4.17 (q, J = 7.2 Hz, 2H), 3.89 (s, 3H), 3.14
(ddd, J = 31.4, 13.5, 5.7 Hz, 2H), 2.33 (s, 3H), 1.43 (s, 9H), 1.25 (t, J
= 7.1 Hz, 3H).
(S)-3-(4-(N′-((4′-acetoxy-6-methoxy-[1,1′-biphenyl]-3-carbonyl)-
oxy)carbamimidoyl)phenyl)-2-((tert-butoxycarbonyl)amino)-
propanoate (2.45 g, 3.96 mmol) in 1,4-dioxane (10 mL) was heated
to reflux for 18 h. After the reaction mixture was cooled to room
temperature, the residue was extracted with ethyl acetate and water,
and dried over anhydrous sodium sulfate. The reaction mixture was
purified by silica gel column chromatography to give (S)-3-(4-(5-(4′-
acetoxy-6-methoxy-[1,1′-biphenyl]-3-yl)-1,2,4-oxadiazol-3-yl)-
phenyl)-2-((tert-butoxycarbonyl)amino)propanoate 21a (1.94 g,
Methyl 4′-hydroxy-6-methoxy-[1,1′-biphenyl]-3-carboxylate (2.88
g, 11.13 mmol) in THF (20 mL) was treated with a 2 M sodium
hydroxide solution. The reaction mixture was stirred for 16 h at
ambient temperature. The solvent was removed in vacuo and the
resulting solution was acidified with 1 N hydrochloric acid to pH 2.
More water was added (50 mL) and the aqueous solution was
extracted with ethyl acetate. The combined organic layer was washed
with brine, dried over anhydrous sodium sulfate, and concentrated.
The residue was purified by silica gel column chromatography to give
4′-hydroxy-6-methoxy-[1,1′-biphenyl]-3-carboxylic acid 19a as a
1
78%) as a white solid. H NMR (400 MHz, chloroform-d) δ 8.19−
8.16 (m, 2H), 8.09 (d, J = 8.4 Hz, 2H), 7.59 (dt, J = 9.2, 2.4 Hz, 2H),
7.29 (d, J = 8.4 Hz, 2H), 7.18 (dt, J = 9.2, 2.4 Hz, 2H), 7.11 (t, J = 4.6
Hz, 1H), 5.04 (d, J = 8.0 Hz, 1H), 4.61 (dd, J = 13.4, 6.4 Hz, 1H),
4.18 (q, J = 7.2 Hz, 2H), 3.92 (s, 3H), 3.17 (ddd, J = 26.6, 13.4, 6.0
Hz, 2H), 2.34 (s, 3H), 1.43 (s, 9H), 1.23 (d, J = 7.2 Hz, 3H).
Step 6. Ethyl (S)-3-(4-(5-(4′-acetoxy-6-methoxy-[1,1′-biphenyl]-3-
yl)-1,2,4-oxadiazol-3-yl)phenyl)-2-((tert-butoxycarbonyl)amino)-
propanoate 21a (1.94 g, 3.23 mmol) in THF (20 mL) was treated
with 2 M sodium hydroxide solution. The reaction mixture was stirred
for 16 h at ambient temperature. The solvent was removed in vacuo
and the resulting solution was acidified with 1 N hydrochloric acid to
pH 3−4. The aqueous solution was extracted with ethyl acetate. The
combined organic layer was washed with brine, dried over anhydrous
sodium sulfate, and concentrated. The residue was purified by silica
gel column chromatography to give (S)-2-((tert-butoxycarbonyl)-
amino)-3-(4-(5-(4′-hydroxy-6-methoxy-[1,1′-biphenyl]-3-yl)-1,2,4-
oxadiazol-3-yl)phenyl)propanoic acid 22a (1.58 g, 92%) as a white
1
white solid (1.83 g, 67%). H NMR (400 MHz, DMSO-d6) δ 12.71
(s, 1H), 9.52 (s, 1H), 7.88 (dd, J = 8.7, 2.3 Hz, 1H), 7.77 (d, J = 2.4
Hz, 1H), 7.30 (dt, J = 9.3, 2.4 Hz, 2H), 7.16 (d, J = 8.9 Hz, 1H), 6.81
(dt, J = 9.2, 2.6 Hz, 2H), 3.83 (s, 3H).
Step 4. 4′-Hydroxy-6-methoxy-[1,1′-biphenyl]-3-carboxylic acid
19a (1.83 g, 7.49 mmol) and pyridine (4.83 mL, 59.94 mmol) were
dissolved in dichloromethane (20 mL) and the reaction mixture was
cooled to 0 °C, and acetyl chloride (3.73 mL, 52.45 mmol) was added
dropwise to this mixture. The reaction mixture was allowed to reach
room temperature and stirred for 18 h. After the completion of the
reaction, the mixture was diluted with dichloromethane and brine.
The combined organic fraction was dried over anhydrous sodium
sulfate, concentrated, and purified by silica gel column chromatog-
raphy to give 4′-acetoxy-6-methoxy-[1,1′-biphenyl]-3-carboxylic acid
20a as a white solid (1.185 g, 55%). 1H NMR (400 MHz, chloroform-
d) δ 8.10 (dd, J = 8.8, 2.3 Hz, 1H), 8.07 (d, J = 2.3 Hz, 1H), 7.55 (d, J
= 8.4 Hz, 2H), 7.15 (d, J = 8.4 Hz, 2H), 7.03 (d, J = 8.8 Hz, 1H), 3.90
(s, 3H), 2.33 (s, 3H).
Step 5. A mixture of 4′-acetoxy-6-methoxy-[1,1′-biphenyl]-3-
carboxylic acid 20a (1.185 g, 4.139 mmol), ethyl (S)-2-((tert-
butoxycarbonyl)amino)-3-(4-(N-hydroxycarbamimidoyl)phenyl)-
propanoate 15 (1.75 g, 4.97 mmol), N-ethyl-N′-(3-
dimethylaminopropyl)carbodiimide hydrochloride (1.98 g, 10.35
mmol), 1-hydroxybenzotriazole hydrate (950.78 mg, 6.21 mmol),
and N,N-diisopropylethylamine (2.50 mL, 14.49 mmol) in dichloro-
methane (20 mL) was stirred for 18 h at ambient temperature. The
reaction mixture was diluted with water and extracted with
dichloromethane. The combined organic layer was dried over
anhydrous sodium sulfate and evaporated. The residue was purified
by silica gel chromatography to give ethyl (S)-3-(4-(N′-((4′-acetoxy-
6-methoxy-[1,1′-biphenyl]-3-carbonyl)oxy)carbamimidoyl)phenyl)-
2-((tert-butoxycarbonyl)amino)propanoate (2.45 g, 96%) as a white
solid. 1H NMR (400 MHz, chloroform-d) δ 8.11 (dd, J = 8.7, 2.3 Hz,
1H), 8.02 (d, J = 2.3 Hz, 1H), 7.70 (d, J = 8.2 Hz, 2H), 7.55 (dt, J =
9.2, 2.4 Hz, 2H), 7.21 (d, J = 8.2 Hz, 2H), 7.15 (dt, J = 9.0, 2.3 Hz,
2H), 7.04 (d, J = 8.7 Hz, 1H), 5.15 (s, 2H), 4.99 (d, J = 7.8 Hz, 1H),
1
solid. H NMR (400 MHz, DMSO-d6) δ 9.74 (s, 1H), 8.09 (dd, J =
8.7, 2.3 Hz, 1H), 7.96 (d, J = 2.1 Hz, 1H), 7.94 (d, J = 8.2 Hz, 2H),
7.39 (d, J = 4.9 Hz, 2H), 7.37 (d, J = 6.7 Hz, 2H), 7.32 (d, J = 8.9 Hz,
1H), 6.84 (dt, J = 9.3, 2.4 Hz, 2H), 6.50 (d, J = 3.4 Hz, 1H), 3.98 (td,
J = 8.4, 4.9, 1H), 3.88 (s, 3H), 3.15 (dd, J = 13.4, 4.6 Hz, 1H), 2.97
(dd, J = 13.4, 7.6 Hz, 1H), 1.33 (s, 9H).
Step 7. To a solution of (S)-2-((tert-butoxycarbonyl)amino)-3-(4-
(5-(4′-hydroxy-6-methoxy-[1,1′-biphenyl]-3-yl)-1,2,4-oxadiazol-3-yl)-
phenyl)propanoic acid 22a (600 mg, 1.13 mmol) in ethyl acetate (3
mL) was added a 4 M solution of hydrogen chloride in 1,4-dioxane (3
mL), and the reaction mixture was stirred for 12 h at ambient
temperature. The mixture was concentrated and the residue was
collected by filtration, and washed with ethyl acetate to afford (S)-2-
amino-3-(4-(5-(4′-hydroxy-6-methoxy-[1,1′-biphenyl]-3-yl)-1,2,4-ox-
adiazol-3-yl)phenyl)propanoic acid hydrochloride 23a (510 mg, 97%)
as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 9.62 (s, 1H), 8.44
(s, 3H), 8.12 (dd, J = 8.5, 2.1 Hz, 1H), 8.05 (d, J = 8.2 Hz, 2H), 7.99
(d, J = 2.4 Hz, 1H), 7.50 (d, J = 8.2 Hz, 2H), 7.38 (dt, J = 9.2, 2.3 Hz,
2H), 7.35 (d, J = 8.9 Hz, 1H), 6.86 (dt, J = 9.2, 2.3 Hz, 2H), 4.23 (t, J
= 6.4 Hz, 1H), 3.89 (s, 3H), 3.22 (d, J = 6.4 Hz, 2H); 13C NMR (100
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MHz, DMSO-d6) δ 175.35, 170.25, 168.03, 160.06, 157.20, 138.99,
130.97, 130.51, 130.43, 129.55, 128.48, 127.33, 127.13, 125.22,
115.79, 115.15, 112.52, 56.08, 53.12, 35.63; HRMS (FAB) m/z
calculated for C24H22N3O5 [M + H]+ 432.1559, found 432.1556;
HPLC purity 100%.
In Vitro Screening Method. TPH1 enzyme activity was measured
with a commercially available kit (BPS Bioscience). Compounds were
dissolved in DMSO (Sigma). Then, 10 μL of the compound was
dispensed into a 96-well microplate, and 40 μL of the TPH1 enzyme
solution (320 ng enzyme/reaction) was added. Then, 50 μL of the
TPH1 reaction solution was added, and the microplate was sealed
with aluminum foil. The microplate was immediately cooled to 4 °C,
gently shaken, and incubated for 4 hours. After a defined time, 10 μL
of the TPH1 quench solution was added. Then, the fluorescence was
measured with a Flexstation3 microplate reader at 300 nm for
excitation and 360 nm for emission.
Blood−Brain Barrier (BBB) Penetration Study in Mice. Com-
pound 23a was evaluated for BBB penetration in male ICR mice as
previously reported.44 Male ICR mice were intraperitoneally
administered 23a at 10 mg/kg in a solution of DMSO/PEG400/
saline = 5:40:55, v/v/v % (5 mL/kg). After 1 hour, the mice were
euthanized using a CO2 chamber, and blood from the heart was
separated to plasma by centrifugation (13 000 rpm, 4 °C, 10 min).
The remaining blood was washed out 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 kept at −20 °C until LC-MS/MS
analysis for 23a. The concentrations of 23a were obtained in plasma
and brain samples by an API 4000 Q trap mass spectrometer (Applied
Biosystems, USA) coupled with a 1200 series HPLC system (Agilent,
USA). Isocratic elution consisted of 80% acetonitrile and 20% water
with 0.1% formic acid. The total run time was 3 minutes, and the flow
rate was 0.3mL/min.
Animal Experiments. The experimental protocol for this study was
approved by the Institutional Animal Care and Use Committee at the
Korea Advanced Institute of Science and Technology. C57BL/6J
mice were purchased from the Charles River Japan (Yokohama,
Japan). The mice were housed in a specific pathogen-free barrier
facility under a 12-h light−dark cycle. Food and water were provided
ad libitum. At 12 weeks of age, mice were fed a high-fat diet (HFD,
60% fat calories) with vehicle or 23a treatment by a daily
intraperitoneal injection. Mice were euthanized by the method of
cervical dislocation. The measurement of fluid, lean, and fat mass in
live mice was performed by a body composition analyzer (LF50,
Bruker). After 6 hours of fasting, mice were sacrificed and tissue
samples were obtained.
Aldrich) or PBS was added, and TGs were hydrolyzed into glycerol
and free fatty acids by incubating samples at 37 °C for 30 minutes.
Samples were incubated with Free Glycerol Reagent (Sigma-Aldrich)
at 37 °C for 5 minutes for detecting hydrolyzed TG levels using a
colorimetric assay. Differences in absorbance at 540 nm between
hydrolyzed and nonhydrolyzed TGs were calculated using a glycerol
standard (Sigma-Aldrich). The TG content was normalized to the
protein concentration in homogenates, measured using a BCA Protein
Assay Kit (Thermo Scientific).
Glucose Tolerance Tests. For the glucose tolerance test, overnight-
fasted mice were intraperitoneally injected with 2 g/kg D-glucose
(Sigma-Aldrich). Glucose concentrations were measured using a
Gluco DR. TOP glucometer (Allmedicus, Korea).
Quantitative RT-PCR. TRIzol reagent (Ambion, USA) was used for
total RNA extraction from harvested tissues. Complementary DNA
was generated with the High Capacity cDNA Reverse Transcription
kit (Applied Biosystems, Foster City, CA) from 1 μg of total RNA.
Real-time qRT-PCR was performed with the Fast SYBR Green
Master Mix (Applied Biosystems, Foster City, CA) and a ViiA 7 Real-
Time PCR system (Applied Biosystems) according to the
manufacturer’s instructions. Expressional profiles were quantified
based on the relative ddCt method with 36B4 as a reference gene.
Docking Studies of the Discovered Inhibitors with TPH1. The
three-dimensional structure of discovered inhibitors was generated
with ChemDraw and Chem3D, while the three-dimensional structure
of TPH1 was obtained from the Protein Data Bank (Protein Data
Bank access code 3HF8).40 The structures of discovered inhibitors
and TPH1 were preprocessed with Chem3D and Discovery Studio
Visualizer, respectively, while the input structures for docking studies
were generated with AutoDockTools with the standard protocol.41
The docking calculations were performed with AutoDock4.2 of 200
Lamarckian genetic algorithm (LGA) runs, 300 initial population size,
100000000 evaluations along with 0.325 grid spacing, and 90x90x90
grid points focused on the inhibitor binding pocket deduced from LP-
533401 binding. The rest of the parameters were set to default. The
detailed docking methodology was described elsewhere.45 The
synthesized compounds were docked with the 3D structure of
TPH1 as described in the methodology. Based upon the binding of
the synthesized compounds, notably compound 17a, the new
compounds were synthesized. The docking poses and interactions
of the discovered inhibitors with TPH1 were visualized in Discovery
̀
Studio Visualizer. They were created with Dassault Systemes BIOVIA,
̀
Discovery Studio Visualizer, Dassault Systemes: San Diego, 2002.
ASSOCIATED CONTENT
■
sı
* Supporting Information
Metabolic analysis. To measure the metabolic rate, the mice were
housed individually in an eight-chamber, open-circuit Oxymax/
CLAMS (Columbus Instruments Comprehensive Lab Animal
Monitoring System) system. Each mouse was assessed for 72 h in
the fed state to measure metabolic rates. The respiratory exchange
ratio (RER = VCO2/VO2) and heat production (HP = (3.185 + 1.232
x RER) x VO2) were calculated.
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c01560.
1
Synthesis procedures; H NMR, 13C NMR, and HRMS
spectra; and HPLC assessment of purity for all of the
final compounds (PDF).
Histological Analysis. Epididymal, inguinal, brown adipose tissue,
and liver were harvested, fixed in 4% (w/v) paraformaldehyde, and
embedded in paraffin. Five-micron-thick tissue sections were
deparaffinized, rehydrated, and stained with hematoxylin and eosin
or used for immunohistochemistry, as described previously.17
Blood Profiling. Blood samples were obtained by cardiac puncture,
and serum or plasma was isolated. Blood profiling was performed by
GC Pharma (Green Cross Corporation). Plasma TG and cholesterol
were detected with an in vitro enzymatic colorimetric assay (CHOL2
and TRIGL; Roche, Germany) using a Cobas 8000 clinical analyzer
with a c702 module (Roche). Serum 5-HT was detected by LC-MS/
MS (Liquid chromatography-tandem mass spectrometry) using a
5500 QTRAP LC-MS/MS system (AB SCIEX).
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;
orcid.org/0000-0002-6957-6062; Phone: +82-62-715-
4621; Email: jhahn@gist.ac.kr
Quantification of Hepatic TGs. Liver tissues were homogenized in
5% NP-40 using FastPrep-24 (MP Biomedicals). TGs were
solubilized by heating homogenates to 95 °C for 5 minutes and
then cooling to 23 °C two times. Triglyceride reagent (Sigma-
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Authors
Health & Welfare (HI16C1501). We thank Jueun Kim and
Hwajin Kim for their technical support.
Eun Jung Bae − Department of Chemistry, Gwangju Institute
of Science and Technology, Gwangju 61005, 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
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
Saravanan Parameswaran − Department of Chemistry,
Gwangju Institute of Science and Technology, Gwangju
61005, Republic of Korea
Jun-Ho Choi − Department of Chemistry, Gwangju Institute
of Science and Technology, Gwangju 61005, Republic of
Korea; orcid.org/0000-0001-5237-5566
Jihyeon Yoon − Department of Chemistry, Gwangju Institute
of Science and Technology, Gwangju 61005, Republic of
Korea
Won-il Choi − Graduate School of Medical Science and
Engineering, Korea Advanced Institute of Science and
Technology, Daejeon 34141, Republic of Korea
Ji Hun Lee − Bio and Drug Discovery Division, Korea
Research Institute of Chemical Technology, Daejeon 34114,
Republic of Korea
Jin Sook Song − Bio and Drug Discovery Division, Korea
Research Institute of Chemical Technology, Daejeon 34114,
Republic of Korea
Myung Ae Bae − Bio and Drug Discovery Division, Korea
Research Institute of Chemical Technology, Daejeon 34114,
Republic of Korea
ABBREVIATIONS
■
TPH, tryptophan hydroxylase; BBB, blood−brain barrier;
PAH, pulmonary arterial hypertension; WAT, white adipose
tissue; IC50, half-maximal inhibitory concentration; BAT,
brown adipose tissue; KO, knockout; HFD, high-fat diet;
IPGTT, intraperitoneal glucose tolerance test; UCP1,
uncoupling protein 1; MeOH, methanol; EtOH, ethanol;
EtOAc, ethyl acetate; DIPEA, N,N-diisopropylethylamine;
EDCI, 1-ethyl-3-(3-diemthylaminopropyl)carbodiimide;
HOBt, hydroxybenzotriazole; 4-DMAP, 4-dimethylaminopyr-
idine; KOAc, potassium acetate; NMP, N-methyl-2-pyrroli-
done; HBTU, 3-[bis(dimethylamino)methyliumyl]-3H-benzo-
triazol-1-oxide hexafluorophosphate; IP, intraperitoneal; PDB,
Protein Data Bank
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https://pubs.acs.org/10.1021/acs.jmedchem.0c01560
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#E.J.B. and W.G.C. contributed equally to this work.
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by grants from the Ministry of
Science, ICT & Future Planning(MSIP)/National Research
Foundation of Korea (NRF) (2016M3A9B6902868 and
2020M3A9E4038695), the Ministry of Trade, Industry and
Energy (2020-10063396) and the Korea Health Industry
Development Institute (KHIDI), funded by the Ministry of
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