Discovery of a novel series of indolyl hydrazide derivatives as diacylglycerol acyltransferase-1 inhibitors

Article abstract of DOI:10.1002/bkcs.10123

A novel series of hydrazide derivatives were synthesized as potential diacylglycerol acyltransferase (DGAT) inhibitors. Among them, compounds 8u and 8v exhibited selective and potent DGAT-1 inhibitory activities. In addition, compound 8u dose-dependently inhibited triglyceride synthesis in HepG2 cell lines. Furthermore, treatment with compound 8u for an oral lipid tolerance test showed a significant decrease in plasma triglyceride levels compared with vehicle-treated control animals, indicating delayed absorption of triglyceride after an acute lipid challenge.

Full text of DOI:10.1002/bkcs.10123

Article
DOI: 10.1002/bkcs.10123
BULLETIN OF THE
M. Kim et al.
KOREAN CHEMICAL SOCIETY
Discovery of a Novel Series of Indolyl Hydrazide Derivatives
as Diacylglycerol Acyltransferase-1 Inhibitors
Minkyoung Kim,†,†† Jinsun Kwon,†,†† Mun Ock Kim,‡,†† Sarbjit Singh,† Sang Kyum Kim,§
Kyeong Lee,¶ Kiho Lee,k Hyun Sun Lee,‡ and Yongseok Choi†,
*
†College of Life Sciences and Biotechnology, Korea University, Seoul 136-701, Korea.
*E-mail: ychoi@korea.ac.kr
‡Chemical Biology Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB),
Chungbuk 363-883, Korea
§College of Pharmacy, Chungnam National University, Daejeon 305-764, Korea
¶College of Pharmacy, Dongguk University-Seoul, Goyang 410-820, Korea
kCollege of Pharmacy, Korea University, Sejong 339-700, Korea
Received November 18, 2014, Accepted November 25, 2014, Published online January 23, 2015
A novel series of hydrazide derivatives were synthesized as potential diacylglycerol acyltransferase (DGAT)
inhibitors. Among them, compounds 8u and 8v exhibited selective and potent DGAT-1 inhibitory activities. In
addition, compound 8u dose-dependently inhibited triglyceride synthesis in HepG2 cell lines. Furthermore,
treatment with compound 8u for an oral lipid tolerance test showed a significant decrease in plasma triglyceride
levels compared with vehicle-treated control animals, indicating delayed absorption of triglyceride after an
acute lipid challenge.
Keywords: Diacylglycerol O-acyltransferase inhibitor, Triglyceride, Obesity, Hydrazide,
Diacylglycerol O-acyltransferase
Introduction
fed a high-fat diet, and exhibited increased insulin.6 DGAT-
1 inhibition is expected to reduce the formation of triacylgly-
cerol (TAG) and chylomicrons in enterocytes and thus lower
postprandial TAG levels in the blood.8 Consequently, selec-
tiveinhibitionofDGAT-1has becomean attractive target with
growing potential for the treatment of type II diabetes and
obesity.
In the past few years, DGAT has emerged as an
attractive target for the control of obesity and other related dis-
orders.9 Currently, preclinical trials are under way for the
compound Pradigastat (Novartis, Switzerland),10 compound
PF-04620110 (Pfizer, USA),11 and compound ABT-046
(Abbott, USA) 12 (Figure 1).
In earlier reports, we showed that some benzimidazole deri-
vatives have a tendency to inhibit TG formation in HepG2
cells by acting as potent DGAT inhibitors.3 In our research
to find small organic molecules that are effective DGAT inhi-
bitors, we have discovered a novel class of indolyl hydrazide
derivatives that show potent DGAT inhibitory activities and
also with the potential to control TG formation in whole cells.
Triglycerides (TGs), whether derived from secretion of very
low density lipoproteins (VLDLs) or chylomicrons, are major
contributors tothemetabolic syndrome.1 TGsareusuallypres-
ent in cytosolic lipid droplets in cells or in the plasma as core
components of lipoproteins. However, excessive accumula-
tion of TGs in human adipose and nonadipose tissues results
in pathological conditions such as obesity, type 2 diabetes,
coronary heart disease, hypertriglyceridemia, and nonalco-
holic fatty liver disease.2
TGs are produced in the body through two pathways: (1) the
major glycerol phosphate pathway and (2) the minor monoa-
cylglycerol pathway. Diacylglycerol O-acyltransferase
(DGAT), which catalyzes the acyl residue transfer from
acyl-CoA to diacylglycerol (DAG), is an exclusive key
enzyme for the final step common to both pathways.3 In mam-
mals, DGAT occurs in two isoforms, DGAT-1 and DGAT-2,
from distinct gene families.4 DGAT-1 is widely expressed in
skeletal muscle, skin, intestine, and testis, with lower levels of
expression in liver and adipose tissue. DGAT-2 is widely
expressed in hepatocytes and adipocytes. This widely differ-
ent distribution in tissues and their two different protein
familiessuggestthattheseenzymeshavespecializedfunctions
even though they catalyze the same reaction.5–7 DGAT-1
knockouts were viable and resistant to weight gain when
Experimental
A series of indole-based hydrazide derivatives 8a–y were pre-
pared, as depicted in Scheme 1. Synthesis of the appropriate
(E)-3-(indol-3-yl)acrylic acid derivatives 4a–i involves the
esterificationofthecarboxylicacidgroupofcompound1, then
N-protection of corresponding compound 2 by different pro-
tecting groups, and, finally, base hydrolysis of corresponding
†† Theses authors contributed equally to this work.
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Discovery of a Novel Series of Indolyl Hydrazide Derivatives
KOREAN CHEMICAL SOCIETY
Figure 1. Selected structure of DGAT-1 inhibitors.
compounds 3a–i to afford compounds 4a–i. The isopropyl-
hydrazide derivatives 7a–n were synthesized in quantitative
yields by performing reductive amination of hydrazide deriva-
tives 5a–n with acetone, using sodium cyanoborohydride as a
reducing agent. Then the appropriate indole-based hydrazides
8a–y were synthesized with good to high yields by amide cou-
pling of isopropyl-hydrazide derivatives 7a–n with different
(E)-3-(indol-3-yl)acrylic acid derivatives 4a–i using 1-[bis
(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridi-
nium 3-oxid hexafluorophosphate (HATU) and N,N-
diisopropylethylamine (DIPEA) in dimethylformamide
(DMF). Compounds 9a–d were synthesized by the O-
alkylation of hydroxyl function of 8b and 8c using methyl
iodide or 1-bromo-2-methoxyethane (Scheme 1).
The synthesis for compounds 14a–d is also shown in
Scheme 2. First, the aldehyde moiety of different 1H-
indole-3-carbaldehyde derivatives 10a–d was converted to
theester moietyusingethyl(triphenylphosphoranylidene)ace-
tate followed by hydrolysis of corresponding esters to give
compounds 12a–d in moderate to good yields. Then N-Boc
protection of compounds 12a–d was performed, followed
by amide coupling of compounds 13a–d with compound 7h
using standard amide coupling conditions.
Compounds 16a–d were synthesized through the coupling
reaction of compound 1 with phenyl hydrazide derivative 7a
followed by N-acylation of compound 15 (Scheme 3).
Meanwhile, compound 19 was also synthesized based on the
same strategy used for the synthesis of compounds 16a–d start-
ing with indolepropionic acid. Compounds 20a and b were
synthesized by coupling compound 4a with benzohydrazide
7o and benzenesulfonohydrazide 7p, as shown in Scheme 4.
General. All reagent-grade chemicals and solvents were used
without further purification. All reactions were carried out
under a nitrogen atmosphere. Melting points were determined
on an electrothermal capillary melting point apparatus. NMR
spectra were determined ona Varian (500 MHz) spectrometer.
Chemical shifts were measured in parts per million (ppm)
downfield from tetramethylsilane (internal standard), with
coupling constants reported in hertz (Hz). Multiplicity is indi-
cated by abbreviations such as singlet (s), doublet (d), doublet
of doublet (dd), triplet (t), multiplet (m), andbroad (brs). High-
resolution mass spectrometry (HRMS) was carried out on
an Agilent 6530 Accurate-Mass Q-TOF LC/MS (Agilent
Technologies, Korea, Seoul). The purity of the products was
checked by reversed-phase high-pressure liquid chromatogra-
phy (RP-HPLC), which was performed a Waters Corporation
HPLC system equipped with a YMC hydrosphere C18 (HS-
302) column and the UV detector set at 254 nm. The mobile
phase used was a gradient of MeOH and H2O with a flow rate
of 1.0 mL/min.
Reduction of Hydrazides. Various commercially available
hydrazides (5a–n) were poured into a solution of acetone
and hexane and stirred for 5 min. The mixture was refluxed
for 12 h at 65ꢀC, and was then cooled in an ice bath to get solid
precipitates, which were filtered to afford white solids; they
were then washed several times with hexane. Then the corre-
sponding hydrazides (6a–n) were shifted in methanol and stir-
red for 20 min. Sodium cyanoborohydride and acetic acid
were added to this mixture and stirred for 18 h at room temper-
ature. After completion of reaction, a saturated sodium bicar-
bonate solution was added to mixture till the pH reached ~7.
The mixture was extracted with ethyl acetate and water and
dried over by MgSO4. After evaporation under vacuum, the
mixture was purified using column chromatography using
n-hexane/EtOAc/MeOH = 6:3:1 as the eluting system. All
the hydrazide derivatives obtained were colored solids with
yields ranging from 85 to 100%.
Procedure of Amide Coupling. To a solution of the 4a (1 g,
3.48 mmol), N0-isopropylbenzohydrazine (7a) (0.93 g, 5.22
mmol), and HATU (1.98 g, 5.22 mmol) in DMF (15.0 mL)
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Discovery of a Novel Series of Indolyl Hydrazide Derivatives
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Scheme1. Reactionandconditions:(a)MeI,K2CO3,acetone,60 ꢀC,72 h,(90–98%);(b)(Boc)2O, K2CO3,THF,60 ꢀCfor3a,(80–95%);R1–X(X
= Br orCl or I), NaH, DMF, 80 ꢀC, 12 h for 3b–h, (35–80%); cyclopropylboronicacid, DMAP, Cu(CO2CH3)2, NaHMDS, toluene, 95 ꢀC, 12 h for
3i, (45–50%); (c) NaOH, THF/MeOH/H2O = 2:1:1, 50 ꢀC, 12 h, (70–98%); (d) acetone, hexane, 65 ꢀC, 12 h, (85–90%); (e) NaBH3CN, AcOH,
MeOH, roomtemperature,12 h, (85–95%);(f )HATU, DIPEA, DMF, roomtemperature,12 h,(34–70%);(g)MeI, K2CO3, acetone, 60 ꢀC,72 hfor
the synthesis of 9a and 9b, (36–50%); 1-bromo-2-methoxyethane, K2CO3, acetone 60 ꢀC, 2 h for the synthesis of 9c and 9d, (30–55%).
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Discovery of a Novel Series of Indolyl Hydrazide Derivatives
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Scheme 2. Reaction and conditions: (a) ethyl(triphenylphosphoranylidene)acetate, benzene, 80 ꢀC, 12 h, (81–89%); (b) NaOH, THF/MeOH/
H2O = 2:1:1, 50 ꢀC, 12 h, (95–98%); (c) (Boc)2O, DMAP, TEA, CH3CN, THE, H2O, room temperature, 4 h, (82–97%); (d) HATU, DIPEA,
DMF, 80 ꢀC, 12 h, (45–48%).
Scheme 3. Reaction and conditions: (a) HATU, DIPEA, DMF, room temperature, 12 h; (b) Ac2O, DMAP, pyridine, DCM, room temperature,
12 h for 16a; R2–Cl, DIPEA, DMAP, DCM, room temperature, 12 h for 16b and 16c; Piv-Cl, K2CO3, DMAP, DMF, room temperature, 12 h
for 16d.
was added DIPEA (0.90 mL, 5.22 mmol). The reaction mix-
ture was then stirred at room temperature overnight, and then
partitionedbetweenethylacetateandwater. Theorganicphase
was washed with brine, dried over MgSO4, and concentrated.
Purification by silica gel column chromatography using n-
hexane/EtOAc = 3:1 as eluting solvents gave compound 8a
as a white solid. Compounds 8a–y, 14a–d, 15, 18, 20a, and
20b were also prepared using same procedure.
and potassium carbonate (35.8 mg, 0.26 mmol) in
anhydrous acetonitrile (10.0 mL) was added iodomethane
(0.0673 mL, 1.08 mmol) drop by drop at 0ꢀC. The reaction
mixture was refluxed at 85ꢀC overnight. The remaining of
the solvent was evaporated. Purification by silica gel column
chromatography (n-hexane/EtOAc = 2:1) gave (E)-tert-butyl-
3-(3-(1-isopropyl-2-(3-methoxybenzoyl)hydrazinyl)-3-oxo-
prop-1-enyl)-1H-indole-1-carboxylate (9a) as a yellow solid.
Compounds 9b–d were also prepared using same procedure.
Data for all prepared compounds can be found in Supporting
Information.
O-Alkylation of 8b. To a solution of the (E)-tert-butyl-3-(3-
(2-(3-hydroxybenzoyl)-1-isopropylhydrazinyl)-3-oxoprop-
1-enyl)-1H-indole-1-carboxylate (8b) (100 mg, 0.22 mmol)
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Discovery of a Novel Series of Indolyl Hydrazide Derivatives
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Scheme 4. Reaction and conditions: (a) HATU, DIPEA, DMF, 80 ꢀC 12 h, (60–73%); (b) (Boc)2O, K2CO3, THF, 60 ꢀC, 12 h, (92–98%); (c)
HATU, DIPEA, DMF, 80 ꢀC, 12 h, (60–75%).
(E)-4-fluoro-N0-isopropyl-N0-(3-(1-(3-methyl-but-2-enyl)-
CH2 CH2 CH (CH3)2), 1.19 (6H, m, N–CH (CH3)2),
0.89 (6H, m, N–CH2 CH2 (CH3)2); 13C NMR (CDCl3,
1H-indol-3-yl)acryloyl)benzohydrazide (8u): White solid; m.
1
p. = 196 ꢀC; H NMR (CDCl3, 500 MHz): δ 9.16 (1H, brs,
NH), 7.96 (2H, m, aromatic), 7.83 (1H, d, J = 15.4 Hz,
125 MHz): δ 166.4, 163.6, 161.6, 137.1, 134.5, 132.0,
126.0, 123.1, 122.4, 120.7, 120.3, 115.0, 114.8, 112.4,
109.9, 60.4, 44.7, 38.5, 25.6, 22.2, 19.8, 14.1; HRMS [M +
H]+ calcd. [C26H30FN3O2 + H] 436.2395, found: 436.2392;
HPLC purity = 92.7% area.
indole CH CH ), 7.52 (1H, s, aromatic), 7.24 (1H, d, J =
10.5 Hz, aromatic), 7.11 (4H, m, aromatic), 6.93 (1H, s, aro-
matic), 6.75 (1H, d, J = 15.7 Hz, indole CH CH ), 5.22
(1H, s, N–CH2 CH (CH3)2), 4.89 (1H, m, N–CH
(CH3)2), 4.49 (2H, s, N–CH2 CH (CH3)2), 1.74 (6H, d, J
= 12.5 Hz, N–CH2 CH (CH3)2), 1.18 (6H, s, N–CH
Results and Discussion
(CH3)2); 13C NMR (CDCl3, 125 MHz): δ 166.5, 166.1, 164.1,
137.3, 131.8, 130.1, 126.2, 122.4, 120.9, 120.2, 118.8, 115.9,
115.7, 112.4, 110.2, 44.3, 25.5, 18.0; HRMS [M + H]+ calcd.
[C26H28FN3O2 + H] 434.2239, found: 434.2233; HPLC purity
= 100.0% area.
All the newly synthesized compounds were evaluated for their
ability to inhibit DGAT activity with human recombinant
DGAT-1 and DGAT-2 (Table 1). The inhibitory effects
of these compounds were compared with T863 (IC50
=
0.068 μM)13 as a positive control. First, structural modifica-
tion of hydrazide derivatives was focused on derivatization
of the phenyl ring replaced with different substituted phenyl
rings. We initially checked the DGAT inhibitory activity of
N-Boc-protected compound 8a (containing phenyl group as
a substituent). This compound showed potent DGAT inhibi-
tory activity with an IC50 value of 9.6 μM for DGAT-1 and
7.5%inhibitionforDGAT-2at10 μM(Table1, entry2). Com-
pound 8a also inhibited the biosynthesis of TG in HepG2 cells
with treatment of 10 μM of 8a (Table 1, entry 2). Thus com-
pound 8a might be considered to be a selective inhibitor of
DGAT-1. Based on these results, derivatives of compound
8a were screened to search for more potent compounds, and
(E)-3-fluoro-N0-(3-(1-isopentyl-1H-indol-3-yl)acry-
loyl)-N0-isopropylbenzohydrazide (8v): Yellow solid; m.p.
= 96 ꢀC; 1H NMR (CDCl3, 500 MHz): δ 9.53 (1H, brs, NH),
7.82 (1H, d, J = 15.4 Hz, indole CH CH ), 7.75 (1H, d, J =
7.6 Hz, aromatic), 7.70 (1H, d, J = 6.6 Hz, aromatic), 7.55
(1H, s, aromatic), 7.38 (1H, d, J = 5.4 Hz, aromatic), 7.24
(1H, d, J = 9.3 Hz, aromatic), 7.15 (3H, m, aromatic), 6.94
(1H, s, aromatic), 6.78 (1H, d, J = 15.7 Hz,
indole CH CH ), 4.91 (1H, m, N–CH (CH3)2), 3.91
(2H, m, N–CH2 CH2 CH (CH3)2), 1.58 (2H, d, J = 6.8
Hz, −N–CH2 CH2 CH (CH3)2), 1.49 (1H, m,
N–
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KOREAN CHEMICAL SOCIETY
Table 1. DGAT inhibitory activity of hydrazide derivatives.
entries 21–23). We speculated that the undesirable result of
these examples was due to the elimination of the Boc group
under physiological conditions, which led to the release of
the corresponding Boc-free compounds. Notably, compound
15 containing no protecting group was found to be inactive in
our preliminary studies (Table 1, entry 28); thus, we replaced
the Boc group with other groups to check the role of the N-
protecting groups. Compound 16a with N-acyl protection
did not show DGAT inhibitory activity (Table 1, entry 24).
Although compounds 16b–d, which was protected by pivalic,
benzoyl, and butyric groups, showed good DGAT-1 inhibi-
tory activities, they also showed poor control on TG formation
at the cellular level (Table 1, entries 25–27). The loss of the
activities in cellular assay would be speculated to result from
the elimination of these labile groups under physiological con-
ditions. Next, we focused on protection of the indole moiety
with some stronger protecting groups in order to minimize
the possibility of elimination in whole cells.
In compound 8o, an N-Boc was replaced with an N-methyl
group, but it did not show any improvement in activity
(Table 2, entry 2). Although compound 8p, which
contains an N-benzyl protecting group, showed a moderate
IC50 value of 13.6 μM, it presented excellent control on TG
formation at the cellular level (Table 2, entry 3). Replacing
the benzyl group with a 3-methyl-2-butene group (compound
8q) improved the IC50 value to 11.8 μM and showed
increased inhibition of TG (Table 2, entry 4). Methylcyclopro-
pyl (compound 8r) and cyclopropyl (compound 8s) as
N-protecting groups resulted in decreased level of the IC50
values with 41.6 and 24.6 μM and decreased inhibition of
TG in 35.6 and 40.4%, respectively (Table 2, entries 5, 6).
Based on these results, compound 8q containing N-(3-
methyl-2-butenyl) as a protecting group emerged as the better
potent compound.
hDGAT-
1
IC50
hDGAT-2
(%
inhibition)
(10 μM)
[14C]TG
synthesis (% of
control) (10 μ
M), HepG2
Entry Compound
(μM)
1
2
3
4
5
6
7
8
T86314
8a
8b
8c
8d
8e
8f
8g
8h
8i
8j
8k
8l
8m
8n
9a
9b
0.068
9.6
>100
22.5
6.8
20.4
2.2
8.8
17.1
7.5
<0.3
<0.3
3.9
51.1
60.1
86.7
62.2
93.5
87.1
97.9
84.4
71.2
94.8
74.7
96.7
64.2
66.8
81.3
74.8
85.8
62.0
72.6
57.5
75.0
79.2
85.7
89.0
93.3
91.4
78.2
115.6
2.3
28.7
<0.3
<0.3
28.9
34.1
22.1
<0.3
<0.3
0.6
0.3
<0.3
8.3
<0.3
2.0
3.6
9
20.4
2.1
2.9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
9.8
>100
>100
4.0
>100
>100
36.4
>100
3.2
>100
>100
>100
65.3
9.3
9c
9d
14a
14b
14c
14d
16a
16b
16c
16d
15
2.9
<0.3
<0.3
4.4
11.0
<0.3
<0.3
5.7
5.3
>100
Notes: hDGAT-1 and -2, human recombinant DGAT-1 and -2 in vitro
It has been observed in many cases that replacement of the
hydrogen atom of organic compounds by a fluoro group
imparts substantial effects on their characteristics.14 There-
fore, we made fluoro derivatives of the best molecule found
so far in this study, namely compound 8q. Compounds 8t
and 8u containing 3-fluoro and 4-fluoro phenyl rings, respec-
tively, were synthesized and screened. As expected, both com-
pounds showed better IC50 values of 7.2 and 1.5 μM,
respectively, and were more potent than compound 8q
(IC50, 11.8 μM). Also, compound 8u showed improvement
in control of TG synthesis, with 44.3% inhibition in whole
cells compared with compound 8q (Table 2, entries 7, 8). With
compound 8w, which contains a 4-fluoro-phenyl ring and O-
isopentyl group, a potent IC50 value of 1.0 μM was observed,
but it only showed moderate control on TG formation with
36.5% inhibition (Table 2, entry 10). Changing the position
of the fluoro group from 3 to 4 (compound 8v) resulted in
excellent control on TG formation in whole cells, with
49.2% inhibition (Table 2, entry 9). Compounds 8x and 8y
with O-ethyl and O-n-propyl group, respectively, showed
moderate DGAT-1 inhibitory activity with poor control on
TG (Table 2, entries 11, 12).
enzymatic inhibition.
various results were obtained from compounds 8b–k (Table 1,
entries 3–12).
Although we observed good IC50 values for compounds 8f,
8i, and 8j, those compounds showed poor control on biosyn-
thesis of TG, suggesting that replacing the phenyl group with
2-pyridine and 3-pyridine groups (compounds 8l and 8m)
resulted in complete loss of activity (Table 1, entries 13,
14). Although potent activity was observed with compound
8n containing the 4-pyridine moiety, there was poor control
on TG formation(Table 1, entry 15). Compounds 9a–d, where
OH group of the phenyl ring was alkylated with methyl and
2-methoxyethane groups, also showed no activity (Table 1,
entries 16–19). Compound 14a containing 5-methyl substitu-
entonthe indoleringshowed potent DGAT-1 inhibitory activ-
ity (IC50 = 3.2 μM) with 42.5% inhibition of TG biosynthesis
at the cellular level (Table 1, entry 20). However, compounds
14b–d containing 6-isopropyl, 6-methyl, and 6-fluoro substi-
tuents on the indole ring did not show any DGAT-1 inhibitory
activity and exhibited poor control on TG formation (Table 1,
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Table 2. DGAT inhibitory activity of hydrazide derivatives.
hDGAT-
1
IC50
hDGAT-2
(%
inhibition)
(10 μM)
[14C]TG
synthesis (% of
control) (10 μ
M), HepG2
Entry Compound
(μM)
1
2
3
4
5
6
7
8
T86314
8o
8p
8q
8r
8s
8t
8u
8v
8w
8x
8y
19
0.068
28.3
13.6
11.8
41.6
24.6
7.2
1.5
1.8
1.0
8.1
17.1
<0.3
5.0
51.1
88.6
59.8
58.5
64.4
59.6
58.0
55.7
50.8
63.5
79.4
79.9
60.7
61.7
81.7
1.2
<0.3
<0.3
18.5
28.0
42.3
64.0
20.3
32.4
<0.3
3.7
Figure 2. Oral lipid tolerance test (OLTT) of 8u.
Table 3. Oral lipid tolerance test (OLTT) of 8u.
9
10
11
12
13
14
15
Serum TG (mg/dL)
Doses
Group (n = 7) (mg/kg)
0 h
2 h
4 h
3.6
Control (0.5% 0
CMC)
45.2 9.8 196.6 74.6 110.2 49.5
>100
>100
>100
20a
20b
8u
10
30
10
78.8 23.2 90.8 28.2∗ 43.2 12.9∗
45.8 6.1 86.4 11.1∗ 50.0 11.9∗
45.4 7.4 33.2 5.9∗∗ 36.6 14.1∗
<0.3
Notes: hDGAT-1 and -2, human recombinant DGAT-1 and -2 in vitro
T863
enzymatic inhibition.
Significant figures (t-test): ∗p < 0.05, ∗∗p < 0.001 vs. (vehicle control).
The saturated analog 19 was synthesized to check the role of
the double bond of 8a. It did not show any DGAT-1 inhibitory
activity (Table 2, entry13)andwaslesspotent (IC50 = 9.6 μM)
than compound 8a. This suggests that the rigid conformation
with the double bond plays some role in controlling the activ-
ities of these compounds. Moreover, the isopropyl group on
hydrazide nitrogen has also some role in enhancing the activ-
ities of these compounds because compound 20a (without N-
isopropylgroup) didnotshowanyDGAT-1 inhibitory activity
while compound 8a showed moderate activity (Table 1, entry
14). Replacing the carbonyl group with sulfonyl (compound
20b) was also not useful (Table 1, entry 14). This structure–
activity relationship study clearly showed that compounds
with the indole moiety protected by long-chain alkyl groups
such as isopentyl and 3-methyl-2-butene generally show bet-
ter activities than the same compounds protected by labile or
short-chain groups. Also, compounds with fluoro-substituted
phenyl rings generally show better activities than compounds
without fluoro substituents (Table 2). Thus, from this screen-
ing, compounds8uand8vhave emergedaspotentcompounds
that show high DGAT inhibitory activities and have a ten-
dency to control effectively TG biosynthesis in whole cells.
Next, we chose compound 8u for an oral lipid tolerance test
(OLTT) (an acute lipid challenge model measuring TG change
after corn oil bolus dosing). Mice were treated with a 10 or 30
mg/kg dose, respectively, of compounds 8u and T863. T863
had been previously reported to inhibit TG absorption through
DGAT1 inhibition and was used as a positive control. After 4
h, both compounds showed good control over absorption of
TG (Figure 2 and Table 3). After 4 h, compound 8u (10 mg/
kg) showed a significant decrease in plasma TG levels com-
pared with vehicle-treated control animals, indicating delayed
absorption of TG after an acute lipid challenge; however, this
compound was less potent than T863, which showed a
dynamic drop of serum TG level.
Conclusion
In summary, by using human recombinant DGAT-1 and
DGAT-2, a novel series of hydrazide derivatives were found
to exhibit selective DGAT-1 inhibitory activities. Among all
the compounds screened, compounds 8u and 8v were found
to be potent DGAT-1 inhibitors. In addition, compound 8u
exhibited an excellent dose-dependent inhibitory effect on
TG synthesis in HepG2 cells. Furthermore, treatment with
compound 8u for an OLTT showed a significant decrease in
plasma TG levels compared with vehicle-treated control ani-
mals, indicating delayed absorption of TG after an acute lipid
challenge. Accordingly, this study demonstrates that very sim-
ple andsmall organic molecules can be efficient leads for treat-
ment of metabolic disorders such as obesity.
Acknowledgment. This work was supported by a grant
of KRIBB Research Initiative Program and Basic
Science Research Program through the National Research
Foundation of Korea (NRF) funded by the Ministry of Educa-
tion, Science and Technology (2103R1A1A2013610 and
2014R1A2A2A01005455), Republic of Korea.
SupportingInformation. Additionalsupportinginformation
is available in the online version of this article.
Bull. Korean Chem. Soc. 2015, Vol. 36, 628–635
© 2015 Korean Chemical Society, Seoul & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.bkcs.wiley-vch.de
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Discovery of a Novel Series of Indolyl Hydrazide Derivatives
KOREAN CHEMICAL SOCIETY
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