Novel acyl coenzyme A: Diacylglycerol acyltransferase 1 inhibitors - Synthesis and biological activities of N-(substituted heteroaryl)-4-(substituted phenyl)-4-oxobutanamides

Article abstract of DOI:10.1248/cpb.58.673

In a program to discover new small molecule diacylglycerol acyltransferase (DGAT)-1 inhibitors, screening of our in-house chemical library was carried out using recombinant human DGAT-1 enzyme. From this library, the lead compound 1a was identified as a n

Full text of DOI:10.1248/cpb.58.673

May 2010
Regular Article
Chem. Pharm. Bull. 58(5) 673—679 (2010)
673
Novel Acyl Coenzyme A: Diacylglycerol Acyltransferase 1 Inhibitors—
Synthesis and Biological Activities of N-(Substituted heteroaryl)-4-
(substituted phenyl)-4-oxobutanamides
Yoshihisa NAKADA,*,a Masaki OGINO,a Kouhei ASANO,a Kazuko AOKI,a Hiroshi MIKI,b
d
Toshihiro YAMAMOTO,c Koki KATO,d Minori MASAGO,d Norikazu TAMURA,e and Mitsuyuki SHIMADA
a Medicinal Chemistry Research Laboratories, Takeda Pharmaceutical Co., Ltd.; b Discovery Research Center, Takeda
c
d
Pharmaceutical Co., Ltd.; Pharmacology Research Laboratories, Takeda Pharmaceutical Co., Ltd.; Strategic Research
Planning Department, Takeda Pharmaceutical Co., Ltd.; and e Research Administration Department, Takeda
Pharmaceutical Co., Ltd.; 2–17–85 Jusohonmachi, Yodogawa-ku, Osaka 532–8686, Japan.
Received December 25, 2009; accepted February 19, 2010; published online February 22, 2010
In a program to discover new small molecule diacylglycerol acyltransferase (DGAT)-1 inhibitors, screening
of our in-house chemical library was carried out using recombinant human DGAT-1 enzyme. From this library,
the lead compound 1a was identified as a new class of DGAT-1 inhibitor. A series of novel N-(substituted het-
eroaryl)-4-(substituted phenyl)-4-oxobutanamides 2 was designed from 1a, synthesized and evaluated for in-
hibitory activity against DGAT-1 enzyme. Among these compounds, N-(5-benzyl-4-phenyl-1,3-thiazol-2-yl)-4-
(4,5-diethoxy-2-methylphenyl)-4-oxobutanamide 9 was found to exhibit potent inhibitory activity and good en-
zyme selectivities. Following administration in KKAy mice with 3 mg/kg high fat diet admixture for four weeks, 9
reduced body weight gain and white adipose tissue weight without affecting total food intake. These results sug-
gested that the small molecule DGAT-1 inhibitor might have potential in the treatment of obesity and metabolic
syndrome.
Key words acyl coenzyme A: diacylglycerol acyltransferase-1 inhibitor; obesity; KKAy
Obesity is a major health problem and a risk factor for hy- obesity effects previously demonstrated in DGAT-1 deficient
pertension, diabetes, and cardiovascular disease. The current mice. Both of compounds (I, II) have a similar linear struc-
treatments of obesity attempt to restore energy balance pri- ture with a terminal carboxylic acid group. In this report, we
marily by decreasing energy input, either by suppressing ap- present a new class of compounds without a carboxylic acid
petite or interfering with lipid absorption in the small intes- group and with potent DGAT-1 inhibitory activity and bio-
tine. Due to both of the rapid increase in the prevalence of logical properties.
obesity worldwide and the lack of effective medical thera-
Research Strategy Screening of our in-house chemical
pies, efforts to discover novel pharmacological therapies for library was carried out to discover new small molecule
obesity have been intensified.1) One of potential therapeutic DGAT-1 inhibitors. Thus, an amide derivative 1a (IC50ꢀ5.2
strategies involves inhibiting triacylglycerol synthesis. Al- mM) was identified as a new class of DGAT-1 inhibitor (Fig.
though triacylglycerol is essential for normal physiology, ex- 1). Initially we examined a lead library consisting of 1a de-
cess triacylglycerol (TG) accumulation results in obesity and rivatives with modified aromatic amine moieties, synthesized
is associated with insulin resistance. Inhibition of TG synthe- by high throughput synthesis technology. In this library, com-
sis may ameliorate obesity and its related medical conse- pound 1j (IC50ꢀ150 nM) exhibited moderate DGAT-1 in-
quences. One of the key enzymes in TG synthesis is acyl hibitory activity. We designed compounds of type 2 and initi-
coenzyme A (CoA): diacylglycerol acyltransferase (DGAT), ated chemical modification, focusing on the aromatic amine
which catalyzes the final step of the TG synthesis pathway in moieties (NHAr), the phenyl ring substituents (R) and the
mammalian cells by using diacylglycerol and fatty acyl CoA linker, with the goal of improving DGAT-1 inhibitory activity
as substrates. TGs synthesized by DGAT enzymes are either (Fig. 1). In this paper we describe the synthesis, structure–
stored in cytosolic lipid droplets or secreted as components activity relationships (SAR) and biological properties of this
of lipoproteins in organs such as the liver and small intestine. novel series of N-heteroaryl carboxamide derivatives.
Two DGAT enzymes, DGAT-1 and DGAT-2, have been iden-
Chemistry The target compounds 1a—j, l, m and 3—10
tified and both DGAT enzymes are ubiquitously expressed, were synthesized as illustrated in Chart 1. The nucleophilic
but higher level of expression are found in tissues that are amine fragments 12a—e, g—j, l,10) m11) were condensed
active in TG synthesis, such as white adipose tissue, small with appropriate carboxylic acid fragments 11i—viii using
intestine and liver.2,3) The phenotype of DGAT-1 deficient 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochlo-
mice has already been described.4—6) These animals are ride (EDCI) to yield the corresponding amides (Method A).
viable, resistant to diet-induced obesity (DIO), and have The glycine derivative 10 was prepared from 3,4-diethoxy-
increased sensitivity to insulin and leptin. These findings benzoic acid 13 and the glycine unit by the usual manner.
suggest and encourage our exploration of novel DGAT-1 However, the condensation reaction of carboxylic acids 11i—
inhibitors, since pharmacological inhibition of DGAT-1 may iii, v—viii with less nucleophilic amines frequently gave
be a feasible therapeutic strategy for human obesity and type amides in low yield due to self-condensation. Self-condensa-
2 diabetes. Recent studies7—9) have demonstrated that small tion was apparently occurring through the labile benzylic
molecule DGAT-1 inhibitors (I, II) showed the similar anti- carbonyl group, thus we modified the reaction conditions to
© 2010 Pharmaceutical Society of Japan
∗ To whom correspondence should be addressed. e-mail: Nakada_Yoshihisa@takeda.co.jp

674
Vol. 58, No. 5
Fig. 1. Structure of Known Compounds (I, II), Lead Compounds 1a, j and the General Design 2
Chart 1. Synthesis of the 4-Phenylbutanamide Derivatives and the Glycine Derivative: Method A
Chart 2. Synthesis of the 4-Phenylbutanamide Derivatives: Method B
avoid formation of the by-products as shown in Chart 2 Results and Discussion
(Method B).
The compounds synthesized in this study were evaluated
g-Ketoester 16 was prepared from 1,2-diethoxybenzene 15 for their inhibitory activity against human DGAT-1 enzyme
and ethyl succinyl chloride by Friedel–Crafts acylation.12) (Tables 1—3), measured as % inhibition at 10 mM and/or as
The key intermediate 17 was obtained by treatment with IC50 values.
orthoester in methanol under acidic condition and subse-
We initially examined the 1a-derived lead library focusing
quent treatment potassium hydroxide in methanol. Using the on the amino moiety as illustrated in Table 1. The 3-biphenyl-
Yamaguchi condensation13) and subsequent acidic work-up, amine derivative 1b and 4-benzylaniline derivative 1c main-
target compounds 1k, o, p were prepared from 17 and the tained inhibitory activity; however the unsubstituted aniline
corresponding heterocyclic amine 12k,14) o,15) p.16) On the derivative 1d showed decreased potency. Aralkyl amine de-
other hand, amide 1n17) was obtained by treatment of 17 with rivatives 1e—g showed moderate inhibitory activity. As
EDCI and subsequent removal of acetal.
shown in the marginal difference between 1e and 1f on
DGAT-1 inhibitory activity, it is a fair assumption that the
methyl group at R1 position can be replaced with hydrogen in

May 2010
675
Table 1. Focus Library of Amide Derivatives
Table 3. SAR of the Phenyl and Linker Moieties
Enzyme
% of inhibition
Compd.
R1
R2
IC50
(nM)
Enzyme
IC50 (nM)
Compd.
Carbamoyl moieties
at 10 mM
1a
1b
1c
1d
1e
1f
Me
Me
Me
Me
Me
H
69
77
84
15
72
95
5200
3700
3300
NTa)
1100
510
11
3
3
4
86
3700
10
5
140
3
1g
1h
H
92
0
770
6
6
Me
NTa)
7
8
290
220
1i
H
H
74
90
NTa)
1j
150
a) Not tested.
9
8.5
Table 2. SAR around the Aromatic Ring
rest of the SAR studies. In comparison to compound 1f,
compound 1g did not show significant change in potency.
Similar tendency was observed in other aralkyl amine deriva-
tives (data not shown). Thiazole derivative 1h did not show
enzymatic activity at a concentration of 10 mM. However,
insertion of a phenyl ring in thiazole moiety of 1h improved
inhibitory activity, and this effect is also shown in the case of
1a—c. Furthermore, compound 1j exhibited an even greater
improvement in inhibitory activity by insertion of an addi-
tional phenyl ring (IC50ꢀ150 nM).
Based on the discovery of the moderate DGAT-1 inhibitor
1j, we continued further modification of di-substituted aro-
matic amine derivatives described in the general structure 2,
and the results are shown in Table 2. Thiazole derivative 1k,
which is a regioisomer of 1j, showed no improvement in po-
tency. On the other hand, 5-benzyl derivative 1l (IC50ꢀ3 nM)
exhibited 50-fold greater potency than 1j. Thiophene deriva-
tive 1m (IC50ꢀ14 nM), containing the same ring substituents
as thiazole compound 1l, showed good inhibitory activity;
however the potency of 1m was found to be 4-fold weaker
than 1l. This result suggested that the nitrogen atom in the
thiazole ring might interact with the target protein. In com-
mon with 1i and 1j, the six-membered aromatic amine deriv-
ative 1n, with an additional phenyl substituent on the right-
hand phenyl of 1b, exhibited a 28-fold enhancement in po-
Enzyme
IC50 (nM)
Compd.
Ar
1j
150
1k
1l
310
3
1m
1n
14
130
1o
1p
830
22

676
Vol. 58, No. 5
tency as compared to 1b. We then examined the introduction
of a nitrogen atom into 1n. The 2,6-diphenyl-4-pyridyl deriv-
ative 1o showed 6-fold decreased inhibitory activity as com-
pared to 1n. In contrast, 4,6-diphenyl-2-pyridyl derivative 1p
(IC50ꢀ22 nM) showed a 6-fold enhanced potency, as in the
case of 1l and 1m. These results suggested that high potency
could be obtained by the incorporation of a heterocycle nitro-
gen atom next to the amide group (i.e. ortho or 2-position).
Due to the discovery of the potent DGAT-1 inhibitor 1l,
our attention focused on the optimization of the phenyl ring
substituents and the linker of 1l (Table 3). Benzodioxine
derivative 3 (IC50ꢀ86 nM), which is a derivative of 1l
cyclized at the ethoxy subsituents, demonstrated reduced
potency. Benzodioxine derivative 4, without a benzylic car-
bonyl group, showed drastically decreased potency compared
to 3. Derivative 10, containing an additional amide group,
exhibited moderate activity. Both mono-ethoxyphenyl deriva-
tives 5 (IC50ꢀ3 nM) and 6 (IC50ꢀ6 nM) maintained potent in-
hibitory activity. These results suggested that the benzylic
carbonyl group significantly increased the inhibitory activity
and that straight-chain substituents at the 3- or 4-positions of
the phenyl ring are more suitable than a fused ring. Removal
of an oxygen atom from the ethoxy group of 6 results in a
48-fold decrease in activity (7, IC50ꢀ290 nM). Unsubstituted
compound 8 showed similarly decreased potency. These re-
sults suggested that the alkoxy substituent is needed to
achieve potent inhibitory activity. The 2-methyl derivative 9
(IC50ꢀ8.5 nM) was found to be an equipotent inhibitor to 1l.
From these SAR studies, three moieties were identified
as key elements of a pharmacophore with potent DGAT-1
inhibitory activity: the alkoxyphenyl group, the carbonyl
group at the benzylic position and the heterocycle nitrogen
atom alpha to the amide group. This suggested that there
might be at least three key interactions involved in binding to
DGAT-1.
Fig. 2. Reduction of in Vitro TG Synthesis by Using Plasma after Oral
Administration of Compound 9
Data expressed as meanꢂS.D. for
10 mg/kg/4 ml in Labrasol solution. ∗ pꢃ0.05, ∗ pꢃ0.01 vs. initial value by paired t-test.
3 3 and
KKAy mice. Compound dosed at
Table 4. Enzyme and Cellular Inhibitory Potenciesa) of Compound 9
hDGAT1
hDGAT2
ꢄ10000
hACAT1
mDGAT1
C2C12 cell
8.9
8.5
8400
16
a) Compound 9 potencies measured as IC50 values (nM).
Table 5. Quantity, Purity and Mass Spectroscopic Data of the Compounds
in Hit Lead Library
Compd.
Amount (mg) Yield (%)
Purity (%)
LC/MS (m/z)
1a
1b
1c
1d
1e
1f
1g
1h
1i
38
40
37
31
35
15
16
10
6
44
46
42
44
91
16
19
14
7
92
89
91
99
85
93
94
100
99
97
432
432
446
356
474
460
432
363
425
501
Before examining the in vivo efficacy of these compounds,
we evaluated their activity in a cell-based assay and their se-
lectivities against DGAT-1 related enzymes. We also tested
their pharmacodynamic properties and efficacy in an ex vivo
assay, in which the compound was orally administered to
1j
7
7
mice and plasma sample was taken after 2 h to assay reduc- obtained in this study revealed the importance of the alkoxy
tion of TG synthesis in vitro [using recombinant DGAT-1]. substituent on the terminal phenyl ring, the presence of a
Among the various compounds tested, compound 9 showed benzylic carbonyl group and a heterocycle nitrogen atom
good potency in cell-based assay and good enzyme selectivi- located next to the amide group. Compound 9 has a unique
ties (Table 4). Furthermore, compound 9 significantly inhib- structure, and in contrast to many known DGAT-1 inhibitors,
ited TG synthesis in our ex vivo assay (Fig. 2).
does not possess carboxylic acid or amino functionalities.
The anti-obesity efficacy of potent DGAT-1 inhibitor 9 was Compound 9 exhibited significant anti-obesity effects in
examined in spontaneously diabetic and obese KKAy mice. KKAy mice. Further in vivo characterization of this com-
Assessed by a four-week study in KKAy mice (3 mg/kg/d as pound will be disclosed in due course.
0.003% food admixture), 9 reduced body weight gain (ꢁ74
%) and white adipose tissue weight in visceral and subcuta-
neous tissue (visc: ꢁ16%, subc: ꢁ18%) without affecting
total food intake normalized with body weight.18) These
results suggested that the small molecule DGAT-1 inhibitor
might have potential in the treatment of obesity and meta-
bolic syndrome.
Experimental
Chemistry All melting points were determined on a Yanagimoto mi-
cromelting point apparatus or Stanford Research Systems OptiMelt auto-
mated melting point system and are uncorrected. Proton nuclear magnetic
resonance (1H-NMR) spectra were recorded on a Varian Mercury-300
(300 MHz), Bruker DPX-300 (300 MHz) or Bruker-400 (400 MHz) with
tetramethylsilane as internal standard. The following abbreviations are used:
sꢀsinglet, dꢀdoublet, tꢀtriplet, qꢀquartet, mꢀmultiplet and brꢀbroad.
Coupling constants (J values) are given in hertz (Hz). LC/MS (ESI-MS)
analyses were carried out using a Waters Open-Lynx system. Elemental
analyses were carried out by Takeda Analytical Laboratories Ltd. and were
within ꢂ0.4% of the theoretical values for the elements indicated unless
otherwise noted.
Conclusion
Modification of the N-hetero-aryl amide derivative 1a led
to potent DGAT-1 inhibitor 9, which showed approximately
600-fold more potent in vitro activity than 1a. The SAR data

May 2010
677
oxin-6-yl)butanamide (4) Yield 90% as amorphous powder. 1H-NMR
(CDCl3) d: 1.64—1.74 (2H, m), 1.75—1.82 (2H, m), 2.29 (2H, t, Jꢀ
7.3 Hz), 4.17—4.24 (6H, m), 6.53 (1H, dd, Jꢀ8.1, 2.0 Hz), 6.58 (1H, d, Jꢀ
1.7 Hz), 6.70—6.74 (1H, m), 7.20—7.25 (3H, m), 7.28—7.39 (3H, m),
7.39—7.45 (2H, m), 7.57 (2H, d, Jꢀ7.1 Hz), 11.26 (1H, br s.). LC/MS m/z:
471 (MHꢆ). Anal. Calcd for C28H26N2O3S·0.3H2O: C, 70.65; H, 5.63; N,
5.89. Found: C, 70.74; H, 5.72; N, 5.93.
N-(5-Benzyl-4-phenyl-1,3-thiazol-2-yl)-4-(3-ethoxyphenyl)-4-oxobu-
tanamide (5) Yield 15%. mp 179 °C. 1H-NMR (DMSO-d6) d: 1.34 (3H, t,
Jꢀ6.9 Hz), 2.78 (2H, t, Jꢀ6.1 Hz), 3.33—3.38 (2H, m), 4.09 (2H, q, Jꢀ
7.0 Hz), 4.22 (2H, s), 7.18—7.25 (4H, m), 7.28—7.34 (2H, m), 7.36—7.48
(5H, m), 7.54—7.65 (3H, m), 12.24 (1H, s). LC/MS m/z: 471 (MHꢆ). Anal.
Calcd for C28H26N2O3S: C, 71.46; H, 5.57; N, 5.95. Found: C, 71.42; H,
5.54; N, 5.93.
Reactions were carried out at room temperature unless otherwise noted
and monitored by thin-layer chromatography (TLC) on silica gel 60 F254
precoated TLC plates (E. Merck) or by HPLC using an octadecyl silica
(ODS) column (A-303, 4.6 mm i.d.ꢅ250 mm, YMC Co., Ltd.). Standard
workup procedures were as follows. The reaction mixture was partitioned
between the indicated solvent and water. Organic extracts were combined
and washed in the indicated order using the following aqueous solutions:
water, 5% aqueous sodium hydrogen carbonate solution (aqueous NaHCO3),
saturated sodium chloride (NaCl) solution (brine), 1 N aqueous sodium hy-
droxide solution (1 N NaOH) and 1 N hydrochloric acid (1 N HCl). Extracts
were dried over anhydrous magnesium sulfate (MgSO4), filtered, and evapo-
rated in vacuo. Chromatographic separations were carried out on Silica gel
60 (0.063—0.200 mm, E. Merck) on Purif-Pack (SI 60 mm or NH 60 mm,
Fuji Silysia, Ltd.) or ODS (CPO-273L, prepacked column, 22—300 mm,
Kusano Kagaku Kikai Co.) using the indicated eluents. Yields were not
maximized.
N-(5-Benzyl-4-phenyl-1,3-thiazol-2-yl)-4-(4-ethoxyphenyl)-4-oxobu-
1
tanamide (6) Yield 6%. mp 193—195 °C. H-NMR (DMSO-d6) d: 1.35
Amide derivatives 1a—j were prepared by using high throughput synthe-
sis technology.
(3H, t, Jꢀ7.0 Hz), 2.76 (2H, t, Jꢀ6.4 Hz), 3.27—3.34 (2H, m), 4.12 (2H, q,
Jꢀ7.0 Hz), 4.22 (2H, s), 7.03 (2H, d, Jꢀ8.7 Hz), 7.18—7.25 (2H, m), 7.34
(3H, ddd, Jꢀ16.2, 7.3, 7.1 Hz), 7.45 (2H, t, Jꢀ7.4 Hz), 7.62 (2H, d, Jꢀ
7.2 Hz), 7.94 (2H, d, Jꢀ8.9 Hz), 12.22 (1H, s). LC/MS m/z: 471 (MHꢆ).
Anal. Calcd for C28H26N2O3S: C, 71.46; H, 5.57; N, 5.95. Found: C, 71.42;
H, 5.63; N, 5.95.
General Method of Hit Lead Library FlexChem reaction block with
48 well was placed in a rack where the following reagents were added: 1)
dichloromethane (DCM) (0.30 ml), 2) a solution of EDCI/HOBt coupling
reagent (1.0 ml, 0.20 M/0.20 M in DCM, 0.20 mmol/0.20 mmol, 2.0 eq), 3)
carboxylic acids 11i and 11ii (1.0 ml, 0.18 M in DCM, 0.18 mmol) and 4)
amines 12a—j (0.50 ml, 0.20 M in DCM, 0.10 mmol). The FlexChem block
was transported to the incubator module (FlexChem Oven) and heated to
35 ° and stirred overnight. To each well, DCM and water were added, and
the two phases were mixed. The mixture was allowed to settle, and the aque-
ous phase was removed. Further washings with a portion of aqueous hy-
drochloric acid and brine were carried out in a similar way, respectively. The
organic phase was concentrated in a GeneVac Atlas vacuum centrifuge. The
residue was dissolved in DMSO and purified using a Gilson automated
preparative HPLC instrument to give the products (Table 5).
Method A. N-(5-Benzyl-4-phenyl-1,3-thiazol-2-yl)-4-(2,3-dihydro-1,4-
benzodioxin-6-yl)-4-oxobutanamide (3) To a mixture of 4-(2,3-dihydro-
1,4-benzodioxin-6-yl)-4-oxobutanoic acid 11iii (354 mg, 1.50 mmol), 5-ben-
zyl-4-phenyl-1,3-thiazol-2-amine 12l (266 mg, 1.00 mmol) and 1-hydroxy-
benzotriazole monohydrate (HOBt) (230 mg, 1.50 mmol) in N,N-dimethyl-
formamide (DMF) (20 ml) was added EDCI (345 mg, 1.80 mmol), and the
mixture was stirred for 2 d. The mixture was diluted with ethyl acetate and
aqueous NaHCO3. The precipitate was collected by filtration, and the solid
N-(5-Benzyl-4-phenyl-1,3-thiazol-2-yl)-4-oxo-4-(4-propylphenyl)bu-
tanamide (7) Yield 38%. mp 198—199 °C. 1H-NMR (CDCl3) d: 0.94
(3H, t, Jꢀ7.5 Hz), 1.65 (2H, m), 2.42 (2H, t, Jꢀ6.6 Hz), 2.64 (2H, t, Jꢀ
7.5 Hz), 3.18 (2H, t, Jꢀ6.6 Hz), 4.20 (2H, s), 7.19—7.30 (8H, m), 7.38 (2H,
t, Jꢀ7.2 Hz), 7.59 (2H, d, Jꢀ7.2 Hz), 7.85 (2H, d, Jꢀ7.8 Hz), 10.68 (1H,
br s). Anal. Calcd for C29H28N2O2S·0.1H2O: C, 74.04; H, 6.04; N, 5.95.
Found: C, 73.85; H, 5.99; N, 6.01.
N-(5-Benzyl-4-phenyl-1,3-thiazol-2-yl)-4-phenyl-4-oxobutanamide (8)
Yield 76%. mp 234—236 °C. 1H-NMR (DMSO-d6) d: 2.79 (2H, t, Jꢀ
6.4 Hz), 3.37 (2H, t, Jꢀ6.4 Hz), 4.22 (2H, s), 7.17—7.25 (3H, m), 7.27—
7.40 (3H, m), 7.42—7.48 (2H, m), 7.51—7.57 (2H, m), 7.60—7.68 (3H, m),
7.96—8.01 (2H, m), 12.27 (1H, s). LC/MS m/z: 427 (MHꢆ). Anal. Calcd for
C26H22N2O2S: C, 73.21; H, 5.20; N, 6.57. Found: C, 73.11; H, 5.28; N, 6.60.
N-(5-Benzyl-4-phenyl-1,3-thiazol-2-yl)-4-(4,5-diethoxy-2-methyl-
phenyl)-4-oxobutanamide (9) Yield 38%. mp 134—137 °C. 1H-NMR
(DMSO-d6) d: 1.34 (3H, t, Jꢀ7.0 Hz), 1.32 (3H, t, Jꢀ7.0 Hz), 2.36 (3H, s),
2.73 (2H, t, Jꢀ6.4 Hz), 3.26 (2H, t, Jꢀ6.2 Hz), 4.02—4.12 (4H, m), 4.23
(2H, s), 6.86 (1H, s), 7.18—7.26 (3H, m), 7.28—7.40 (3H, m), 7.41 (1H, s),
7.43—7.48 (2H, m), 7.60—7.65 (2H, m), 12.23 (1H, s). LC/MS m/z: 529
(MHꢆ). Anal. Calcd for C31H32N2O4S: C, 70.43; H, 6.10; N, 5.30. Found: C,
69.93; H, 6.17; N, 5.55.
was recrystallized from tetrahydrofurane (THF)–methanol to give
3
1
(287 mg, 59%) as a white solid. mp 245—246 °C. H-NMR (DMSO-d6) d:
2.74 (2H, t, Jꢀ6.2 Hz), 3.27 (2H, t, Jꢀ6.2 Hz), 4.22 (2H, s), 4.26—4.30
(2H, m), 4.31—4.35 (2H, m), 6.98 (1H, d, Jꢀ8.3 Hz), 7.18—7.25 (3 H, m),
7.27—7.40 (3H, m), 7.42—7.48 (3H, m), 7.51 (1H, dd, Jꢀ8.6, 2.0 Hz),
7.60—7.65 (2H, m), 12.24 (1H, s). LC/MS m/z: 485 (MHꢆ). Anal. Calcd for
C28H24N2O4S: C, 69.40; H, 4.99; N, 5.78. Found: C, 69.14; H, 4.99; N, 5.69.
Following compounds 1j, l, m and 4—9 were prepared from carboxylic
acids 11i—viii and amines 12j, l, m in a manner similar to that described for
3. The carboxylic acid 11i was obtained by a manner as described in Murata
et al., Euro. J. Med. Chem., 12, 17—20 (1977). The carboxylic acids 11ii—
viii were commercially available.
N-[(3,4-Diethoxyphenyl)carbonyl]glycine (14) To a solution of 3,4-
diethoxybenzoic acid 13 (5.0 g, 0.0238 mol) in THF (100 ml) was added cat.
DMF (3 drops) and oxalyl chloride (2.5 ml, 0.0286 mol) at room temperature
(rt). The mixture was stirred for 1 h and then concentrated in vacuo. The
residue was dissolved with chloroform (50 ml) and added methyl glycinate
hydrochloride (3.0 g, 0.0239 mol). To the mixture was added triethylamine
(6.7 ml, 0.0476 mol) at 0 °C and stirred at same temperature. The mixture
was diluted with water and extracted with chloroform. The extracts were
washed with diluted hydrochloric acid, aqueous NaHCO3 and water, dried
over anhydrous magnesium sulfate and concentrated in vacuo. The residue
was dissolved in methanol (100 ml), added 2 N NaOH (50 ml, 0.100 mol) and
the mixture was stirred overnight. The reaction mixture was concentrated
in vacuo. The residue was acidified by 1 N HCl and extracted with ethyl ac-
etate. The extracts were washed with water, dried over anhydrous magne-
sium sulfate and concentrated in vacuo to give 14 (4.5 g, 71%) as a white
N-(4,5-Diphenyl-1,3-thiazol-2-yl)-4-(3,4-diethoxyphenyl)-4-oxobu-
tanamide (1j) Compound 1j was synthesized by method A. Yield 40%.
mp 217—219 °C. 1H-NMR (DMSO-d6) d: 1.28—1.41 (6H, m), 2.82 (2H, t,
Jꢀ6.0 Hz), 3.31—3.43 (2H, m), 4.03—4.17 (4H, m), 7.07 (1H, d,
Jꢀ8.3 Hz), 7.25—7.40 (8H, m), 7.40—7.49 (3H, m), 7.66 (1H, d,
Jꢀ8.3 Hz), 12.44 (1H, br s). LC/MS m/z: 501 (MHꢆ). Anal. Calcd for
C29H28N2O4S: C, 69.58; H, 5.64; N, 5.60. Found: C, 69.42; H, 5.61; N, 5.48.
N-(5-Benzyl-4-phenyl-1,3-thiazol-2-yl)-4-(3,4-diethoxyphenyl)-4-
1
solid. mp 167—168 °C. H-NMR (CDCl3) d: 1.46 (6H, m), 4.15 (6H, m),
6.87 (1H, d, Jꢀ8.4 Hz), 7.01 (1H, br s), 7.37 (1H, m), 7.44 (1H, m). Anal.
Calcd for C13H17NO5: C, 58.42; H, 6.41; N, 5.24. Found: C, 58.34; H, 6.43;
N, 5.18.
1
oxobutanamide (1l) Yield 45%. mp 213—215 °C. H-NMR (CDCl3) d:
1.46 (3H, t, Jꢀ7.2 Hz), 1.49 (3H, t, Jꢀ7.2 Hz), 2.40 (2H, t, Jꢀ6.6 Hz), 3.15
(2H, t, Jꢀ6.6 Hz), 4.10—4.17 (4H, m), 4.20 (2H, s), 6.86 (1H, d, Jꢀ8.1 Hz),
7.19—7.31 (6H, m), 7.38 (2H, m), 7.48—7.60 (4H, m), 10.72 (1H, br s).
LC/MS m/z: 515 (MHꢆ). Anal. Calcd for C30H30N2O4S: C, 70.01; H, 5.88;
N, 5.44. Found: C, 69.77; H, 5.97; N, 5.54.
N-{2-[(5-Benzyl-4-phenyl-1,3-thiazol-2-yl)amino]-2-oxoethyl}-3,4-di-
ethoxybenzamide (10) To
a mixture of N-[(3,4-diethoxyphenyl)car-
bonyl]glycine 14 (481 mg, 1.80 mmol), 5-benzyl-4-phenyl-1,3-thiazol-
2-amine 12l (266 mg, 1.00 mmol), HOBt (306 mg, 2.00 mmol) and acetoni-
trile (20 ml) was added EDCI (0.351 ml, 2.00 mmol) and the mixture was
stirred at 50 °C for 4 h. The precipitate was collected by filtration to give 10
(281 mg, 54%) as a white solid. mp 221—222 °C. 1H-NMR (CDCl3) d: 1.40
(3H, t, Jꢀ6.9 Hz), 1.46 (3H, t, Jꢀ6.9 Hz), 4.08 (6H, m), 4.21 (2H, s), 6.80
(1H, d, Jꢀ8.4 Hz), 6.90 (1H, br s), 7.21—7.55 (11H, m), 7.56 (1H, m),
10.85 (1H, br s). Anal. Calcd for C29H28N3O4S: C, 67.55; H, 5.67; N, 8.15.
Found: C, 67.49; H, 5.78; N, 8.20.
N-(5-Benzyl-4-phenylthiophen-2-yl)-4-(3,4-diethoxyphenyl)-4-oxobu-
1
tanamide (1m) Yield 43%. mp 178—179 °C. H-NMR (CDCl3) d: 1.47
(6H, m), 2.80 (2H, t, Jꢀ6.6 Hz), 3.41 (2H, d, Jꢀ6.6 Hz), 4.10 (2H, s), 4.16
(4H, m), 6.64 (1H, s), 6.87 (1H, d, Jꢀ8.4 Hz), 7.16—7.39 (10H, m), 7.51
(1H, d, Jꢀ1.8 Hz), 7.59 (1H, dd, Jꢀ8.4, 1.8 Hz), 8.63(1H, br s). Anal. Calcd
for C31H31NO4S: C, 72.49; H, 6.08; N, 2.73. Found: C, 72.31; H, 6.05; N,
2.54.
N-(5-Benzyl-4-phenyl-1,3-thiazol-2-yl)-4-(2,3-dihydro-1,4-benzodi-
Method B. Ethyl 4-(3,4-Diethoxyphenyl)-4-oxobutanoate (16) To a

678
Vol. 58, No. 5
mixture of 1,2-diethoxybenzene (20.0 g, 120 mmol) and ethyl 4-chloro-4-
DGAT-1 (hDGAT-1), hDGAT-2, hACAT-1 and mouse DGAT-1 (mDGAT-1)
oxobutyrate (18.0 ml, 126 mmol) in dichloromethane (200 ml) in an ice bath were expressed in Sf9 insect cells using a baculovirus expression system.
was added aluminum chloride (33.6 g, 252 mmol) portionwise and the mix-
ture stirred for 45 min. The reaction mixture was poured into ice, added
conc. hydrochloric acid (100 ml) and stirred for 30 min. The mixture was
The microsome of the Sf9 cells was obtained as an enzyme source.
The hDGAT1 reaction mixtures contained 100 mM Tris–HCl (pH 7.5),
250 mM sucrose, 150 mM MgCl2, 0.01% bovine serum albumin (BSA fatty
extracted with chloroform, washed with brine, dried over anhydrous magne- acid free), 1% acetone, 5 mg/ml the microsome of Sf9 expressing hDGAT-1,
sium sulfate and concentrated in vacuo. The residue was purified by silica
and 25 mM 1,2-dioleoyl-sn-glycerol at the final concentration. For inhibitor
gel column chromatography and recrystallized from ethyl acetate–hexane to testing, serial dilutions of the compounds were added to the reaction mixture
give 16 (24.2 g, 69%) as a white solid. 1H-NMR (CDCl3) d: 1.27 (3H, t, Jꢀ in the final concentration of 0.1% DMSO. The reaction was initiated by
7.2 Hz), 1.47 (3H, t, Jꢀ7.1 Hz), 1.49 (3H, t, Jꢀ7.2 Hz), 2.74 (2H, t, Jꢀ6.7
adding 25 mM [14C]-oleoyl-CoA at the final concentration. The reactions
Hz), 3.27 (2H, t, Jꢀ6.7 Hz), 4.11—4.21 (6H, m), 6.88 (1H, d, Jꢀ8.5 Hz), were performed in a 96-well plate in a final volume of 100 ml and were ter-
7.53 (1H, d, Jꢀ2.1 Hz), 7.59 (1H, dd, Jꢀ8.4, 2.0 Hz). LC/MS m/z: 317 minated after 15 min at 32 °C by addition of 300 ml of chloroform/methanol
(MNaꢆ).
(1/2 by vol) mixed solvent. After mixing the solution, 200 ml of phosphate
buffer saline (PBS) was added to facilitate phase separation. After centrifu-
gation (3000 rpm, 3 min), 50 ml of the chloroform layer was spotted on a thin
layer chromatography (TLC) plate. Lipids were separated by TLC with a
solvent system of n-hexane : diethyl ether : ethyl acetate : acetic acid (74 :
Potassium 4-(3,4-Diethoxyphenyl)-4,4-dimethoxybutanoate (17) To a
solution of ethyl 4-(3,4-diethoxyphenyl)-4-oxobutanoate 16 (24.2 g,
82.2 mmol) in methanol (250 ml) were added trimethyl orthoformate
(45.0 ml, 41.1 mmol) and conc. sulfuric acid (0.1 ml), and the reaction mix-
ture was stirred at 50 °C overnight. To the reaction mixture was added 15 : 15 : 1 by vol). The radioactivity of [14C]-triglyceride was counted with
sodium hydrogen carbonate (5.0 g) and the reaction mixture was concen- BAS-2500 imaging system (Fujifilm) to calculate the hDGAT-1 activity or
trated in vacuo. The residue was extracted with ethyl acetate, and washed inhibition rates of the compounds. The hDGAT-2, hACAT-1 and mDGAT-1
with saturated aqueous sodium hydrogen carbonate solution and brine. The assays were performed as described above with the following modifications.
organic layer was dried over anhydrous magnesium sulfate and concentrated
in vacuo to give methyl 4-(3,4-diethoxyphenyl)-4,4-dimethoxybutanoate 20 mM MgCl2, 0.01% BSA, 1% acetone, 40 mg/ml the microsome of Sf9 ex-
(27.0 g, quant.) as an oil. 1H-NMR (CDCl3) d: 1.45 (6H, t, Jꢀ7.0 Hz), pressing hDGAT-2, and 40 mM 1,2-dioleoyl-sn-glycerol, 20 mM [14C]-Oleoyl-
1.99—2.10 (2H, m), 2.13—2.24 (2H, m), 3.15 (6H, s), 3.57 (3H, s), 4.05— CoA. The hDGAT-2 reaction was allowed to proceed for 20 min at 32 °C,
The hDGAT-2 assay contained: 100 mM Tris–HCl (pH 7.5), 250 mM sucrose,
4.16 (4H, m), 6.80—6.90 (1H, m), 6.92—6.99 (2H, m).
terminated, and quantified as described above. The hACAT-1 assay con-
tained: 100 mM phosphate buffer (pH 7.2), 1 mM EDTA, 0.01% BSA, 1%
acetone, 200 mg/ml the microsome of Sf9 expressing hACAT-1, and 100 mM
cholesterol, 40 mM [14C]-oleoyl-CoA. The hACAT-1 reaction was allowed to
proceed for 25 min at 32 °C, terminated, and quantified [14C]-oleoyl-choles-
terol as described above. The mDGAT-1 assay contained: 100 mM Tris–HCl
(pH 7.5), 250 mM sucrose, 150 mM MgCl2, 0.01% BSA, 1% acetone, 5 mg/ml
the microsome of Sf9 expressing mDGAT-1, and 25 mM 1,2-dioleoyl-sn-
glycerol, 25 mM [14C]-oleoyl-CoA. The mDGAT-1 reaction was allowed to
To a solution of methyl 4-(3,4-diethoxyphenyl)-4,4-dimethoxybutanoate
(27.0 g, 82.7 mmol) in methanol (150 ml) was added potassium hydroxide
(4.60 g, 69.7 mmol) and the mixture was stirred at 30 °C for 18 h. The reac-
tion mixture was poured into diethyl ether (300 ml) and stirred for 30 min.
The precipitate was collected by filtration to give 17 (11 g, 37%) as a solid.
1H-NMR (DMSO6) d: 1.31 (3H, t, Jꢀ7.0 Hz), 1.32 (3H, t, Jꢀ6.8 Hz),
1.35—1.43 (2H, m), 1.87—1.97 (2H, m), 2.97—3.05 (6H, m), 3.94—4.05
(4H, m), 6.82—6.92 (3H, m).
N-(2,5-Diphenyl-1,3-thiazol-4-yl)-4-(3,4-diethoxyphenyl)-4-oxobu- proceed for 20 min at 32 °C, terminated, and quantified as described above.
tanamide (1k) To a suspension of 17 (310 mg, 0.885 mmol) in THF (5 ml) Assay of Intracellular DGAT Inhibitory Activity in Mouse Myoblast
were added triethylamine (0.247 ml, 1.76 mmol) and 2,4,6-trichlorobenzoyl C2C12 Cell Line in Vitro C2C12 cells were cultured with Dulbecco’s Modi-
chloride (0.152 ml, 0.973 mmol). The mixture was stirred for 4 h. To the re-
fied Eagle Medium (D-MEM) supplemented with 10% FBS, 50 U/ml of
action mixture was added a solution of 12k (200 mg, 0.793 mmol) in THF
(5 ml) and the reaction mixture was stirred at 50 °C overnight. The reaction ferentiation to myotube and were treated by trypsine-EDTA, following were
penicillin and 50 mg/ml of streptomycin for 2 d as myoblast cells without dif-
mixture was diluted with 1 N HCl and extracted with ethyl acetate. The ex-
suspended in phosphate buffer saline (PBS). The serial dilutions of com-
tracts were washed with saturated aqueous sodium hydrogen carbonate solu- pound were added in the C2C12 cells suspension and incubated at 37 °C for
tion and brine, dried over anhydrous sodium sulfate and concentrated in 20 min. Then, [14C]-oleic acid was added as a substrate of DGAT reaction.
vacuo. The residue was recrystallized from acetonitrile to give 1k (227 mg,
The reactions were performed in a 96-well plate in a final volume of 100 ml
57%) as a white solid. mp 164—165 °C. H-NMR (DMSO-d6) d: 1.30–— at the concentration of 106 C2C12 cells/ml, 5 mM [14C]-oleic acid, 0.001%
1.38 (6H, m), 2.69 (2H, br s), 3.25 (2H, t, Jꢀ5.6 Hz), 4.04—4.15 (4H, m), BSA (fatty acid free) and 0.2% ethanol and 0.1% DMSO. The reactions
1
7.06 (1H, d, Jꢀ8.6 Hz), 7.32—7.39 (1H, m), 7.41—7.49 (3H, m), 7.49— were terminated after 20 min at 37 °C by addition of 300 ml of chloroform/
7.57 (3H, m), 7.57—7.67 (3H, m), 7.90—7.97 (2H, m), 10.24 (1H, br s). methanol (1/2 by vol) mixed solvent. After mixing the solution, 200 ml
LC/MS m/z: 501 (MHꢆ). Anal. Calcd for C29H28N2O4S: C, 69.58; H, 5.64; of DW was added to facilitate phase separation. After centrifugation
N, 5.60. Found: C, 69.49; H, 5.63; N, 5.59.
(2000 rpm, 2 min), 45 ml of the chloroform layer was spotted on a thin layer
4-(3,4-Diethoxyphenyl)-4-oxo-N-1,1ꢀ:3ꢀ,1ꢁ-terphenyl-5ꢀ-ylbutanamide chromatography (TLC) plate. Lipids were separated by TLC with a solvent
(1n) Compound 1n was prepared from 17 and 12n in a manner similar to system of n-hexane : diethyl ether : ethyl acetate : acetic acid (255 : 30 : 15 :
that described for 3. White solid (yield 57%); mp 164 °C.1H-NMR (CDCl3) 0.6 by vol). The radioactivity of [14C]-triglyceride was counted with BAS-
d: 1.47 (6H, dt, Jꢀ8.1, 7.0 Hz), 2.85 (2H, t, Jꢀ6.3 Hz), 3.45 (2H, t, Jꢀ 2500 imaging system (Fujifilm) to calculate intracellular DGAT inhibitory
6.3 Hz), 4.09—4.20 (4H, m), 6.88 (1H, d, Jꢀ8.5 Hz), 7.33—7.47 (6H, m),
7.51—7.58 (2H, m), 7.60—7.66 (5H, m), 7.77 (2H, d, Jꢀ1.3 Hz), 7.98 (1H,
activity of the compounds.
Measurement of DGAT1 Inhibitory Activity after Oral Administra-
s). LC/MS m/z: 494 (MHꢆ). Anal. Calcd for C32H31NO4: C, 77.87; H, 6.33; tion Compounds were administered orally as a Labrasol solution to female
N, 2.84. Found: C, 77.60; H, 6.42; N, 2.76.
4-(3,4-Diethoxyphenyl)-N-(2,6-diphenylpyridin-4-yl)-4-oxobu- administration, blood was collected via orbital venous plexus and prepared
KKAy mice at 13—15 weeks of age. Half an hour, 1 and 2 h after drug
1
tanamide (1o) Yield (10 mg, 17%) as a white solid. H-NMR (CDCl3) d:
1.49 (6H, q, Jꢀ6.8 Hz), 2.87 (2H, t, Jꢀ5.4 Hz), 3.47 (2H, t, Jꢀ5.4 Hz), 4.16
plasma. Forty microliters of 62.5 mM 1,2-dioleoylglycerol buffered solution
(2.5% acetone solution in 250 mM sucrose, 150 mM MgCl2 and 100 mM Tris
(6H, t, Jꢀ6.8 Hz), 6.90 (1H, d, Jꢀ7.2 Hz), 7.42—7.65 (8H, m), 7.92 (2H, s), buffer; pH 7.5), 10 ml of 10% plasma and 25 ml of 20 mg/ml human DGAT1
8.15 (4H, d, Jꢀ7.2 Hz), 8.18 (1H, br s). LC/MS m/z: 495 (MHꢆ).
microsome fraction in which human DGAT1 was overexpressed in SF9
4-(3,4-Diethoxyphenyl)-N-(4,6-diphenylpyridin-2-yl)-4-oxobu- insect cells by genetic engineering method, were premixed and incubated at
tanamide (1p) Compound 1p was prepared from 17 and 12p in a manner
37 °C for 10 min. And then 25 ml of 100 mM [14C]-oleoyl-CoA (0.23 MBq/
similar to that described for 1k. Yield (300 mg, 51%) as a pale-brown solid. ml) was added and further incubated for 15 min. The reaction was termi-
mp 140—141 °C. 1H-NMR (DMSO-d6) d: 1.30—1.41 (6H, m), 2.85 (2H, t, nated by adding 300 ml of chloroform : MeOH (1 : 2) solution, and then
Jꢀ6.1 Hz), 3.28—3.37 (2H, m), 4.03—4.17 (4H, m), 7.08 (1H, d, Jꢀ8.5 200 ml of phosphate buffered saline was added, extracted and separated to
Hz), 7.43—7.58 (7H, m), 7.67 (1H, dd, Jꢀ8.5, 2.1 Hz), 7.79—7.85 (2H, m),
organic and aqueous layer. Fifty microliters of organic layer was applied on
7.93 (1H, d, Jꢀ1.3 Hz), 8.18—8.24 (2H, m), 8.34 (1H, s), 10.71 (1H, s). TLC plate and separated TG with n-hexane : diethyleter : acetic acid
Anal. Calcd for C31H30N2O4: C, 75.28; H, 6.11; N, 5.66. Found: C, 75.25; H, (85 : 15 : 0.5), and the radioactivity incorporated in TG spot was measured
6.18; N, 5.60.
by BAS2500.
Assay of hDGAT1, hDGAT2, Human Acyl-CoA Cholesterol Acyltrans-
ferase 1 (hACAT1) and mDGAT1 Inhibitory Activity in Vitro Human
Acknowledgment We thank Dr. S. Kitamura, Dr. S. Marui and Dr. K.

May 2010
679
Kubo for encouragement and helpful advice. We thank Mr. Y. Nakano for
technical assistance.
9) Birch A. M., Birtles S., Buckett L. K., Kemmitt P. D., Smith G. J.,
Smith T. J. D., Turnbull A. V., Wang S. J. Y., J. Med. Chem., 52,
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