Discovery of indolyl acrylamide derivatives as human diacylglycerol acyltransferase-2 selective inhibitors

Article abstract of DOI:10.1039/c2ob27114a

A series of indolyl acrylamide derivatives was synthesized as potential diacylglycerol acyltransferase (DGAT) inhibitors. Furfurylamine containing indolyl acrylamide derivative 5h exhibited the most potent DGAT inhibitory activity using microsomes prepare

Full text of DOI:10.1039/c2ob27114a

Organic &
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Discovery of indolyl acrylamide derivatives as human
diacylglycerol acyltransferase-2 selective inhibitors†
Cite this: Org. Biomol. Chem., 2013, 11,
849
Kyeong Lee,‡a Minkyoung Kim,‡b Boah Lee,b Jail Goo,b Jiyoung Kim,b Ravi Naik,a
Jee Hee Seo,c Mun Ock Kim,c Youngjoo Byun,d Gyu-Yong Song,e Hyun Sun Lee*c
and Yongseok Choi*b
A series of indolyl acrylamide derivatives was synthesized as potential diacylglycerol acyltransferase
(DGAT) inhibitors. Furfurylamine containing indolyl acrylamide derivative 5h exhibited the most potent
DGAT inhibitory activity using microsomes prepared from rat liver. Further evaluation against human
DGAT-1 and DGAT-2 identified indolyl acrylamide analogues as selective inhibitors against human
DGAT-2. In addition, the most potent compound 5h inhibited triglyceride synthesis dose-dependently in
HepG2 cell lines.
Received 30th October 2012,
Accepted 28th November 2012
DOI: 10.1039/c2ob27114a
www.rsc.org/obc
Introduction
In mammals, diacylglycerol acyltransferase (DGAT) occurs in
two isoforms, DGAT-1 and DGAT-2, from distinct gene
families. Although both isoforms are widely expressed and
present at high levels in white adipose tissue, DGAT-1 is most
highly expressed in the small intestine, whereas DGAT-2 is pri-
marily expressed in the liver.1 DGAT-1 and DGAT-2 are two of
the enzymes that are responsible for the main component of
triglyceride (TG) synthesis in most organisms, and they have
been studied in many eukaryotic organisms.2
It has been reported that diet-induced obesity caused by a Fig. 1 Synthetic selective hDGAT-1 and hDGAT-2 inhibitors.
high fat diet could effectively be inhibited in DGAT-1 knock-
down mice, and an abnormal glucose metabolism was
expression pattern suggests a fundamentally different role of
improved by an increase of the susceptibility of insulin and
leptin, the dietary hormones.3 Accordingly, DGAT-1 has
emerged as an attractive target for the control of obesity and
other related disorders. Andrew et al. reported that the
improvements in hepatic steatosis produced by DGAT-2 anti-
sense oligonucleotide treatment come at the expense of exacer-
bations in hepatic injury and fibrosis. This differential
each DGAT enzyme in TG metabolism.4
DGAT-1 and DGAT-2 selective inhibitors have recently been
reported, such as T863, A922500, JNJ-DGAT2-A, and
JNJ-DGAT2-B as shown in Fig. 1.5–10 A functional study by Con-
nelly et al. demonstrated different hepatic functions of two
enzymes, in which exogenous fatty acids (FAs) are esterified to
glycerol by DGAT-1, while endogenous FAs are incorporated
into TG by DGAT-2.10 Therefore, selective inhibitors of DGAT-1
or 2 may play an important role for further study to under-
stand the molecular mechanism of these enzymes.
In our effort to identify novel small-molecule DGAT inhibi-
tors, we have carried out a screening of our in-house com-
pound library composed of various heterocyclic compounds
using microsomes from rat liver, which led to the identifi-
cation of indolyl acrylamide derivatives. Herein, we describe
the synthesis and biological evaluation of novel indolyl acryl-
amide derivatives as potential DGAT inhibitors, resulting in
the discovery of human DGAT-2 selective inhibitors.
aCollege of Pharmacy, Dongguk University-Seoul, Seoul 100-715, Korea
bCollege of Life Sciences and Biotechnology, Korea University, Seoul 136-701, Korea
cChemical Biology Research Center, Korea Research Institute of Bioscience and
Biotechnology (KRIBB), Ochang, Cheongwon, Chungbuk 363-883, Korea.
E-mail: leehs@kribb.re.kr
dCollege of Pharmacy, Korea University, Chungnam 339-700, Korea
eCollege of Pharmacy, Chungnam National University, Daejeon 305-764, Korea
†Electronic supplementary information (ESI)
10.1039/c2ob27114a
available. See DOI:
‡Both authors have equally contributed to this work.
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prepared from rat liver (Table 1). Kurarinone,11 a known DGAT
inhibitor, was used as a reference standard for comparison
and showed moderate activity with an IC50 value of 10.9 μM.
Our strategy for structural modification of indolyl acryl-
amide derivatives was focused on derivatization of an amide
group and alteration of the N-protecting group on the indole
ring. We first examined the exploration of the amide side
chain by preparing alkyl amide and cyclic amide analogues
5a–d with isopropylamine, methoxy ethylamine, pyrrolidine,
and piperidine amide linkers. Compounds 5a–c displayed
good inhibitory activity (IC50 = 8.8, 12.3, and 13.2 μM, respecti-
vely), while compound 5d was moderately active with an IC50
value of 28 μM. We then investigated the effect of amide ana-
logues 5e–i linked through aromatic groups. Interestingly, this
derivatization resulted in a potent DGAT inhibitor 5h, which
Scheme 1 Reagents and conditions: (a) methyl 4-aminobenzoate or methyl
3-aminobenzoate or methyl 2-aminobenzoate, EDCI, HOAT, DIPEA, DMF or DCC;
(b) LiOH·H2O, THF, H2O; (c) R1–NH2, HATU, DIPEA, DMF; (d) (Boc)2O, K2CO3, THF.
demonstrated appreciably high inhibitory activity (IC50
=
2.5 μM) in comparison with the reference compound kurarin-
one (IC50 = 10.9 μM). Although pyridinylmethanamine deriva-
tive 5e and benzylamine derivative 5g exhibited considerable
activity as effective as kurarinone, phenylamine derivative 5f
was found to be inactive up to 50 μM.
In view of the potent inhibitory activity of furfurylamine
derivative 5h, we prepared amide analogues with thiophenyl-
methanamine, substituted furfurylamines, and tetrahydrofur-
anylmethanamine derivatives represented by analogues 5i–m.
Compounds 5j and 5k with methyl substitution on a furan
ring displayed good inhibitory activities (IC50 = 9.4 and
9.6 μM, respectively), while 5l and 5m with a tetrahydrofuran
ring were found to be moderate inhibitors. However, 5i with a
thiophene ring did not show inhibitory activity up to 50 μM.
From these results, it is evident that the replacement of the
furanyl group of 5h with the thiophene ring, substituted
furanyl rings, and saturated furanyl rings caused significant
loss of inhibitory activity and that the furan ring is optimal for
DGAT inhibition. Further, in order to evaluate the significance
of the furanyl amide at the meta-position of the phenylamide
of 5h, we prepared corresponding ortho and para regioisomers
5n and 5o. Although the ortho-isomer 5n is moderately active,
the para-isomer 5o displayed significantly poor inhibitory
activity, suggesting that the furanyl amide at the meta-position
is essential for effective inhibition of DGAT. To determine the
conformational preference, we then investigated the effect of a
single bond in place of a double bond while retaining all other
functionalities as shown in 5h. This modification provided
indolyl propylamide analogue 5p, which did not show any
activity suggesting that the double bond is important for DGAT
inhibitory activity.
Scheme 2 Reagents and conditions: (a) R–Br or R–Cl, K2CO3, DMF for 6–8;
t
acetic anhydride, K2CO3, DMAP, DMF for 9; R–Cl, BuOK, DMF for 10; R–Cl, NaH,
DMF for 11.
Chemistry
A series of indolyl acrylamide derivatives (5a–p and 6–11) were
prepared according to procedures depicted in Schemes 1 and
2. As shown in Scheme 1, the reaction of (E)-3-(1H-indol-3yl)-
acrylic acid (1) with appropriate amino benzoates under stan-
dard coupling conditions in dry DMF furnished methyl esters 2.
Lithium hydroxide mediated alkaline hydrolysis of these esters 2
provided corresponding acids 3. The resulting acids 3 were
further coupled with appropriate amines to produce respective
amide derivatives of 4. Further reaction of compounds 4 with
appropriate anhydrides, alkyl halides, and sulfonyl chlorides
under standard coupling conditions afforded N-Boc-protected
indolyl analogues 5a–p (Scheme 1).
As shown in Scheme 2, compounds 6–11, in which the
N-Boc group was replaced with different protecting groups,
were prepared from furfurylamine derivative 4h.
Having established primary structure–activity relationships
of amide groups and conformational preference, we were inter-
ested in exploring the effects of altering the N-Boc group
attached to the indole nitrogen. As shown in Table 1, 4h
without the N-Boc group abolished the DGAT inhibitory
activity of 5h. Accordingly, the derivatization produced N-pro-
tected indolyl analogues 6–11. In comparison with 5h with
Results and discussion
All the newly synthesized compounds 5–11 were evaluated for the N-Boc group, 6–11 with different protecting groups also
their ability to inhibit DGAT activity using microsomes displayed considerable inhibitory activities (Table 2). These
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Table 1 DGAT inhibitory activity of benzamide derivatives 4h and 5a–p
No.
R1
X
R2
H
R3
R4
H
IC50 a (μM)
5a
Boc
HCvCH
8.8
5b
5c
Boc
Boc
HCvCH
HCvCH
H
H
H
H
12.3
13.2
5d
5e
Boc
Boc
HCvCH
HCvCH
H
H
H
H
28
12.1
5f
Boc
Boc
HCvCH
HCvCH
H
H
H
H
>50
10
5g
5h
5i
Boc
Boc
Boc
HCvCH
HCvCH
HCvCH
H
H
H
H
H
H
2.5
>50
5j
9.4
5k
5l
Boc
Boc
HCvCH
HCvCH
H
H
H
H
9.6
26
5m
Boc
HCvCH
H
H
H
22.4
5n
Boc
Boc
Boc
H
HCvCH
HCvCH
H2C–CH2
HCvCH
H
H
23
50
5o
H
H
H
5p
H
H
>50
>50
10.9
4h
Kurarinone
a Rat microsomal DGAT inhibition.
results suggested that N protection for the indole nitrogen is hDGAT-2 (IC50 = 6.9, 6.8, and 6.9 μM, respectively) without
important for DGAT inhibitory activity. inhibitory activities against hDGAT-1 up to 100 μM, while com-
Since indolyl acrylamides have displayed interesting DGAT pound 5h was more potent than 5a in a microsomal fraction
inhibitory activities in the rat microsomal DGAT inhibition prepared from rat liver. 5b was found to lose inhibitory activi-
test, we chose several compounds for a human recombinant ties against both hDGAT-1 and hDGAT-2.
DGAT-1 and DGAT-2 inhibition test. As shown in Table 3, the
TG is synthesized primarily through DGAT and the inhi-
selected compounds were found to exhibit selective inhibitory bition of TG synthesis may reduce the incidence of obesity and
activities against hDGAT-2 in comparison with hDGAT-1. its related diseases. Therefore, 5h possessing potent DGAT
Among them, 5a, 5c, and 5h showed the most potency against inhibitory activity in the rat liver microsomal fraction and
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Table 2 Effect of N-protected indolyl analogues 5h and 6–11
Conclusions
In summary, a series of indolyl acrylamide derivatives was
found to exhibit DGAT inhibitory activities using microsomes
prepared from rat liver. Further evaluation of the selected com-
pounds led to identification of furanylamine derivative 5h as a
selective hDGAT-2 inhibitor. In addition, 5h exhibited a dose-
dependent inhibitory effect on TG synthesis in HepG2 cells,
suggesting its effect on TG formation via selective inhibition of
hDGAT-2. These results merit further investigation on the
mechanism of hDGAT-2 inhibitors and development of precli-
nical candidates for the treatment of hepatic steatosis,12
hepatic injury and fibrosis.
DGAT inhibition
(%) at 25 μM
DGAT inhibition
(%) at 25 μM
No. R1
No. R1
5h
6
Boc
78.7
54.1
9
39.8
10
11
49.9
7
8
>50
50.9
53.2
—
—
—
Experimental
General
All reagent grade chemicals and solvents were used without
further purification. All reactions were carried out under a
nitrogen atmosphere in flame-dried glassware. Melting points
were determined on an Electrothermal capillary melting point
apparatus and were uncorrected. NMR spectra were deter-
mined on a Varian (400 or 500 MHz) spectrometer. Chemical
shifts were measured in parts per million (ppm) downfield
from tetramethylsilane (internal standard) with coupling con-
stants in hertz (Hz). Multiplicity was indicated by abbrevi-
ations like singlet (s), doublet (d), doublet of doublet (dd),
triplet (t), quartet (q), multiplet (m), and broad (brs). Mass
spectra were recorded on a Finnigan ESI mass spectrometer.
HRMS and EI-MS were recorded on a JMS-700 (Jeol, Japan).
Products from all reactions were purified to a minimum purity
of 96% as determined by HPLC, either by flash column chrom-
atography using silica gel (230–400 mesh Kieselgel 60) or by
preparative thin layer chromatography using glass-backed
silica gel plates (1 mm thickness) unless otherwise indicated.
Thin-layer chromatography on 0.25 mm silica plates (E. Merck,
silica gel 60 F254) was used to monitor reactions. The chroma-
tograms were visualized using ultraviolet illumination,
exposure to iodine vapour, and dipping in PMA or Hanessian’s
solution. The purity of the products was checked by reversed
phase high pressure liquid chromatography (RP-HPLC), which
was performed either on a Dionex Corp. HPLC system or on a
Waters Corp. HPLC system equipped with a UV detector set at
254 nm. The mobile phases used were A: H2O containing
0.05% TFA, and B: CH3CN. The HPLC employed a YMC Hydro-
sphere C18 (HS-302) column (5 μ particle size, 12 nM pore
Table 3 Inhibitory activity of the selected compounds against hDGAT-1 and
hDGAT-2
No.
hDGAT-1 IC50 a (μM)
hDGAT-2 IC50a (μM)
5a
5b
5c
5e
5g
5h
5j
>100
>100
89.1
>100
>100
>100
>100
>100
6.9
>100
6.8
31.9
15.7
6.9
7.4
21.5
5k
a Human recombinant DGAT1 and DGAT2 inhibition.
Table 4 Inhibitory effect of 5h against TG synthesis in HepG2 cells
[
14C]TG synthesis, % of controla
Compound 5h
3 μM
Substrate
Control (DMSO)
1 μM
10 μM
[
14C] glycerol 100 9.4
83.8 5.4 60.2 2.5 33.2 3.5
a HepG2 cells (1 × 106 cells/1 ml) were incubated in the absence
(control) or presence of compound 5h (1, 3, 10 μM) with [14C]glycerol
for 18 h at 37 °C. Intracellular [14C] lipids were extracted and analyzed
as indicated in “Experimental”. Values are expressed as percentages of
control and are means SD (n = 3).
size), 4.6 mm dia. × 150 mm with a flow rate of 1.0 ml min−1
.
Compound purity was assessed in two different systems, using
one of the following methods, Method A: gradient 20% B to
100% B in 30 min (Waters Corp. HPLC system); Method B: gra-
dient 25% B to 100% B in 30 min (Dionex Corp. HPLC system).
against hDGAT-2 was further evaluated for its ability to inhibit
TG synthesis in HepG2 cells using [14C] glycerol as substrates.
As shown in Table 4, 5h significantly reduced the formation of
TG to 66.7% at the tested concentration of 10 μM. Notably, the
exposure of 5h up to 10 μM did not show any toxic effect on
HepG2 cells (data not shown). This result indicates that 5h
may reduce the formation of TG by inhibiting hDGAT-2 activity
in HepG2 cells.
Chemistry
The synthesis of 5–11 is depicted in Schemes 1 and 2. As
shown in Scheme 1, the reaction of (E)-3-(1H-indol-3yl)acrylic
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acid or 3-(1H-indol-3-yl)propanoic acid (1) with appropriate overnight. Solvent was evaporated under reduced pressure.
amino benzoates under standard coupling conditions in dry Then the mixture was diluted with water and ethyl acetate. The
DMF furnished corresponding methyl esters 2. Lithium combined organic extracts were washed with sat. brine solu-
hydroxide mediated alkaline hydrolysis of these esters 2 pro- tion, and dried with anhydrous MgSO4 to dryness. The result-
vided corresponding acids 3. The resulting acids 3 were further ing brown oil was purified by silica gel column
coupled with appropriate amines to produce respective amide chromatography using DCM and MeOH (30 : 1) to afford
derivatives of 4. Further reaction of compound 4 with appropri- 4 (90%). Similar experimental procedures were used for o-,
ate anhydrides, alkyl halides, and sulfonyl chlorides under p- isomers, and saturated indoles.
standard coupling conditions afforded corresponding N-pro-
Boc protection
tected indolyl analogues 5–11 (Schemes 1 and 2). The spectral
1
data, including H NMR, 13C NMR, and MS spectra of all the The mixture of 4 (147 mg, 0.38 mmole) and potassium carbon-
newly synthesized intermediates and final compounds, were ate (158 mg, 1.14 mmol) in THF was stirred for 20 minutes at
consistent with the assigned structures. Products from all the room temperature and then the reaction mixture was cooled to
reactions were purified to a minimum purity of >96%, as deter-
mined by HPLC.
0 °C. A solution of di-tert-butyl dicarbonate (498 mg,
2.28 mmol) in THF was added dropwise to the above contents
and the reaction mixture was stirred at 0 °C for 20 minutes.
Then the ice bath was removed and the reaction mixture was
Synthesis of amine
trans-3-Indoleacrylic acid (1) (1000 mg, 5.35 mmol) in dry DMF stirred at room temperature for another 20 minutes. Then the
was added to HATU (2440 mg, 6.42 mmol) and DIPEA reaction mixture was refluxed at 50 °C overnight. The reaction
(1.12 ml, 6.42 mmol) in DMF. The reaction mixture was stirred mixture was cooled to room temperature and solvents were
for 20 minutes at room temperature. Then, dry 3-aminoben- evaporated in vacuo. Then the mixture was diluted with water
zoic acid methyl ester (969 mg, 6.42 mmol) was added to the and ethyl acetate (3 × 20 ml). The combined organic extracts
reaction mixture in a dropwise manner. After that, the reaction were washed with sat. brine solution, and dried with anhy-
mixture was refluxed for 4.5 h at 50 °C. As the TLC indicated drous MgSO4 to dryness. The resulting brown oil was purified
the completion of the reaction solvent was evaporated under by silica gel column chromatography using hexane and ethyl
reduced pressure. Then, the mixture was diluted with water acetate (1 : 1) as eluents to afford 5a–p (90%). Similar experi-
and ethyl acetate (3 × 20 ml). The combined organic extracts mental procedures were used for o- and p-isomers and satu-
were washed with sat. brine solution and dried with anhydrous rated indole.
MgSO4 to dryness. The resulting brown oil was purified by
(E)-tert-Butyl-3-(3-(3-(isopropylcarbamoyl)phenylamino)-
silica gel column chromatography using hexane and ethyl 3-oxoprop-1-enyl)-1H-indole-1-carboxylate (5a). Mp 178 °C;
acetate as an eluent to afford 2 (700 mg, 40%) as a pale yellow 1H-NMR (CD3OD, 500 MHz) δ 8.17 (d, J = 7.8 Hz, 1H, aro-
solid. Similar experimental procedures were used for o-, p- matic), 8.09 (s, 1H, aromatic), 7.92 (m, 2H, aromatic), 7.79
isomers, and saturated indoles.
(m, 2H, aromatic, indole–CHvCH–), 7.51 (d, J = 7.8 Hz, 1H,
aromatic), 7.36 (m, 3H, aromatic), 6.91 (d, J = 15.7 Hz, 1H,
indole–CHvCH–), 4.21 (d, J = 6.8 Hz, 1H, N–CH–(CH3)2), 1.68
General procedure for the hydrolysis
A solution of LiOH (183 mg, 4.37 mmol) in H2O was added to (s, 9H, Boc), 1.27 (d, J = 6.6 Hz, 6H, –N–CH–(CH3)2); 13C-NMR
a mixture of methyl 3-((E)-3(1H-indol-3-yl)acrylamido)benzoate (CD3OD, 125 MHz) δ 169.5, 167.4, 150.6, 140.4, 137.8, 137.2,
(2) (700 mg, 2.19 mmol) in a mixture of 20 ml of THF : H2O 135.1, 130.2, 129.3, 126.4, 124.8, 124.0, 123.9, 121.5, 121.4,
(2 : 1). After the completion of the addition the reaction turned 120.2, 118.4, 116.6, 86.0, 43.4, 28.5, 22.7; HRMS [M + H]+ calcd
to an orange color from a lemon one. The mixture was stirred [C26H29N3O4 + H] 448.2236, found 448.2130; HPLC purity:
at room temperature overnight. The solvents were evaporated 99.4% area.
under reduced pressure. The contents were diluted with water,
(E)-tert-Butyl-3-(3-(3-(2-methoxyethylcarbamoyl)phenylamino)-
acidified to pH 5 using 2 N HCl, and extracted with ethyl 3-oxoprop-1-enyl)-1H-indole-1-carboxylate (5b). Mp 114 °C;
acetate. The combined organic extracts were washed with sat. 1H-NMR (CDCl3, 500 MHz) δ 8.21 (d, J = 8.3 Hz, 1H, aromatic),
brine solution and dried with anhydrous MgSO4 to dryness. 7.91 (m, 5H, aromatic, indole–CHvCH–), 7.50 (d, J = 7.8 Hz,
The resultant syrup was purified by silica gel column 1H, aromatic), 7.39 (m, 2H, aromatic), 7.32 (m, 1H, aromatic),
chromatography using DCM and MeOH (30 : 1 → 20 : 1) to 6.91 (d, J = 15.7 Hz, 1H, indole–CHvCH–), 3.65 (t, J = 5.3 Hz,
afford 3 (603 mg, 90%) as a yellow oil. A similar procedure was 2H, –NH–(CH2)2–O), 3.55 (t, J = 5.0 Hz, 2H, –NH–(CH2)2–O),
used for saturated indole.
3.38 (s, 3H, –OCH3), 1.70 (s, 9H, Boc); 13C-NMR (CDCl3,
125 MHz) δ 167.4, 164.8, 149.1, 138.9, 136.2, 135.2, 134.3,
129.3, 128.4, 127.9, 125.2, 123.5, 122.9, 122.2, 120.1, 119.9,
R–NH2 diamine coupling (4)
A solution of benzoic acid (3) (270 mg, 0.88 mmol) in dry DMF 118.5, 116.8, 115.6, 84.6, 71.0, 58.8, 39.8, 28.1; HRMS [M + H]+
was added to a mixture of HATU (500 mg, 1.31 mmol) with calcd [C26H29N3O5 + H] 464.2185, found 464.2085; HPLC
DIPEA (0.23 ml, 1.31 mmol) in DMF under N2 and the mixture purity: 91.5% area.
was stirred for 20 minutes. Then R1–NH2 (1.31 mmol) was
(E)-tert-Butyl-3-(3-oxo-3-(3-(pyrrolidine-1-carbonyl) phenyl-
added and the mixture was stirred at room temperature amino)prop-1-enyl)-1H-indole-1-carboxylate (5c). Mp 108 °C;
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1H-NMR (CDCl3, 500 MHz) δ 8.89 (brs, 1H, –NH), 8.20 (d, J = (s, 1H, aromatic), 7.67 (d, J = 15.7 Hz, 1H, indole–CHvCH–),
8.3 Hz, 1H, aromatic), 7.88 (m, 1H, aromatic), 7.83 (m, 2H, aro- 7.50 (d, J = 7.6 Hz, 1H, aromatic), 7.28 (m, 6H, aromatic), 7.19
matic, indole–CHvCH–), 7.74 (s, 1H, aromatic), 7.38 (t, J = 7.8 (m, 2H, aromatic), 6.79 (d, J = 15.9 Hz, 1H, indole–CHvCH–),
Hz, 1H, aromatic), 7.29 (m, 3H, aromatic), 7.19 (d, J = 7.6 Hz, 4.53 (s, 2H, –NH–CH2), 1.60 (s, 9H, Boc); 13C-NMR (CD3OD,
1H, aromatic), 6.83 (d, J = 15.7 Hz, 1H, indole–CHvCH–), 3.66 125 MHz) δ 170.0, 167.2, 150.4, 140.5, 140.2, 137.6, 136.5,
(t, J = 6.8 Hz, 2H, pyrrolidine), 3.46 (t, J = 6.5 Hz, 2H, pyrroli- 134.9, 130.2, 130.0, 129.6, 129.4, 128.6, 128.3, 126.2, 124.6,
dine), 1.93 (m, 2H, pyrrolidine), 1.87 (m, 2H, pyrrolidine), 1.69 124.1, 123.8, 121.4, 121.3, 120.1, 118.2, 116.5, 85.8, 44.7, 28.5;
(s, 9H, Boc); 13C-NMR (CDCl3, 125 MHz) δ 169.7, 164.9, 149.2, HRMS [M + H]+ calcd [C30H29N3O4 + H] 496.2236, found
138.8, 137.4, 133.6, 128.9, 128.2, 128.1, 125.1, 123.3, 122.1, 496.9650; HPLC purity: 95.3% area.
120.3, 117.1, 115.6, 84.5, 49.7, 46.3, 29.7, 28.1, 26.3, 24.4;
HRMS [M + H]+ calcd [C27H29N3O4 + H] 460.2236, found 3-oxoprop-1-enyl)-1H-indole-1-carboxylate (5h). Mp 122 °C;
460.2129; HPLC purity: 95.2% area.
1H-NMR (CD3OD, 500 MHz) δ 8.12 (m, 2H, aromatic), 7.86
(E)-tert-Butyl-3-(3-(3-(furan-2-ylmethylcarbamoyl)phenylamino)-
(E)-tert-Butyl-3-(3-oxo-3-(3-(piperidine-1-carbonyl)phenyl- (m, 2H, aromatic, indole–CHvCH–), 7.77 (m, 1H, aromatic),
amino)prop-1-enyl)-1H-indole-1-carboxylate (5d). Mp 142 °C; 7.73 (s, 1H, aromatic), 7.51 (d, J = 7.6 Hz, 1H, aromatic), 7.42
1H-NMR (CDCl3, 500 MHz) δ 8.21 (m, 2H, aromatic, –NH), 7.90 (s, 1H, aromatic), 7.33 (m, 3H, aromatic, furan), 6.86 (d, J =
(m, 2H, aromatic), 7.87 (s, 1H, aromatic), 7.75 (d, J = 7.8 Hz, 15.9 Hz, 1H, indole–CHvCH–), 6.32 (m, 2H, furan), 4.55
1H, indole–CHvCH–), 7.56 (s, 1H, aromatic), 7.40 (m, 1H, aro- (s, 2H, –NH–CH2), 1.66 (s, 9H, Boc); 13C-NMR (CD3OD
matic), 7.33 (m, 2H, aromatic), 7.09 (d, J = 7.8 Hz, 1H, aro- 125 MHz) δ 169.9, 167.4, 153.3, 150.5, 143.4, 140.5, 137.8,
matic), 7.02 (d, J = 7.8 Hz, 1H, aromatic), 6.73 (d, J = 15.7 Hz, 136.5, 135.0, 130.2, 129.2, 126.3, 124.7, 124.2, 123.9, 121.5,
1H, indole–CHvCH–), 3.75 (m, 2H, piperidine), 3.39 (m, 2H, 121.3, 120.2, 118.3, 116.6, 111.5, 108.4, 85.9, 37.8, 28.5; HRMS
piperidine), 1.67 (m, 11H, Boc, piperidine), 1.59 (m, 4H, piper- [M + H]+ calcd [C28H27N3O5 + H] 486.2029, found 486.202;
idine); 13C-NMR (CDCl3, 125 MHz) δ 170.5, 165.0, 149.1, 139.0, HPLC purity: 100.0% area.
136.3, 133.3, 129.0, 128.3, 128.1, 125.0, 123.3, 121.3, 120.8,
(E)-tert-Butyl-3-(3-oxo-3-(3-(thiophen-2-ylmethylcarbamoyl)-
120.4, 118.4, 117.1, 115.5, 84.4, 48.8, 43.2, 31.5, 26.4, 24.4, phenylamino)prop-1-enyl)-1H-indole-1-carboxylate (5i). Mp
1
22.6; HRMS [M + H]+ calcd [C28H31N3O4 + H] 474.2393, found 118 °C; H-NMR (CD3OD, 500 MHz) δ 8.18 (d, J = 8.3 Hz, 1H,
474.2378; HPLC purity: 99.5% area.
(E)-tert-Butyl-3-(3-oxo-3-(3-(pyridin-2-ylmethylcarbamoyl)- (m, 2H, aromatic, indole–CHvCH–), 7.53 (d, J = 7.8 Hz, 2H,
phenylamino)prop-1-enyl)-1H-indole-1-carboxylate (5e). Mp aromatic, thiophen), 7.37 (m, 3H, aromatic), 7.28 (d, J =
aromatic), 8.13 (s, 1H, –NH), 7.93 (m, 2H, aromatic, –NH), 7.81
1
130 °C; H-NMR (CD3OD, 500 MHz) δ 8.49 (d, J = 4.6 Hz, 1H, 5.1 Hz, 1H, aromatic), 7.05 (d, J = 3.2 Hz, 1H, thiophen), 6.94
aromatic), 8.18 (m, 2H, aromatic), 7.92 (m, 2H, aromatic), 7.85 (m, 1H, thiophen), 6.90 (s, 1H, aromatic), 4.74 (s, 2H, –NH–
(d, J = 8.8 Hz, 1H, aromatic), 7.80 (m, 2H, aromatic), 7.60 CH2), 1.68 (s, 9H, Boc); 13C-NMR (CD3OD, 125 MHz) δ 169.9,
(d, J = 7.6 Hz, 1H, indole–CHvCH–), 7.43 (m, 2H, aromatic), 167.4, 152.9, 150.6, 149.1, 143.1, 140.6, 137.8, 137.1, 136.6,
7.33 (m, 3H, aromatic), 6.91 (d, J = 15.7 Hz, 1H, indole– 135.1, 130.3, 129.3, 127.8, 126.9, 126.4, 126.0, 123.9, 121.4,
CHvCH–), 4.70 (s, 2H, –NH–CH2), 1.68 (s, 9H, Boc); 13C-NMR 118.4, 116.6, 86.0, 52.7, 39.6, 28.5, 21.1; HRMS [M + H]+ calcd
(CD3OD, 125 MHz) δ 170.2, 167.4, 159.5, 150.6, 149.9, 140.7, [C28H27N3O4S + H] 502.1801, found: 502.9262; HPLC purity:
139.0, 137.8, 136.4, 135.1, 130.3, 130.2, 129.3, 126.4, 124.8, 100.0% area.
123.9, 123.0, 121.4, 120.3, 118.4, 116.6, 86.0, 46.2, 28.5; HRMS
(E)-tert-Butyl-3-(3-(3-(5-methylfuran-2-ylmethylcarbamoyl)-
[M + H]+ calcd [C29H28N4O4 + H] 497.2189, found 497.9719; phenylamino)-3-oxoprop-1-enyl)-1H-indole-1-carboxylate (5j). Mp
1
HPLC purity: 96.4% area.
120 °C; H-NMR (CDCl3, 500 MHz) δ 8.93 (brs, 1H, –NH), 8.09
(E)-tert-Butyl-3-(3-oxo-3-(3-(phenylcarbamoyl)phenylamino)- (m, 2H, aromatic), 7.94 (d, J = 7.8 Hz, 1H, aromatic), 7.81
1
prop-1-enyl)-1H-indole-1-carboxylate (5f). Mp 144 °C; H-NMR (d, J = 15.7 Hz, 1H, indole–CHvCH–), 7.73 (d, J = 8.1 Hz, 1H,
(CD3OD, 500 MHz) δ 8.15 (s, 1H, aromatic), 8.06 (d, J = 8.3 Hz, aromatic), 7.68 (s, 1H, –NH), 7.44 (d, J = 7.6 Hz, 1H, aromatic),
1H, aromatic), 7.79 (d, J = 7.6 Hz, 1H, aromatic), 7.72 (m, 3H, 7.27 (m, 2H, aromatic), 7.13 (t, J = 7.5 Hz, 1H, aromatic), 6.79
aromatic, indole–CHvCH–), 7.66 (d, J = 7.6 Hz, 3H, aromatic, (m, 2H, aromatic, indole–CHvCH–), 6.07 (s, 1H, furan), 5.82
–NH), 7.54 (d, J = 7.8 Hz, 1H, aromatic), 7.29 (m, 4H, aromatic), (s, 1H, furan), 4.51 (d, J = 5.1 Hz, 2H, –NH–CH2), 2.19 (s, 3H,
7.21 (m, 1H, aromatic), 7.08 (m, 1H, aromatic), 6.79 (d, J = –furyl–CH3), 1.63 (s, 9H, Boc); 13C-NMR (CDCl3, 125 MHz)
15.7 Hz, 1H, indole–CHvCH–), 1.59 (s, 9H, Boc); 13C-NMR δ 167.3, 165.2, 152.0, 149.0, 139.1, 136.1, 134.8, 134.0, 129.2,
(CD3OD, 125 MHz) δ 168.6, 167.3, 150.4, 140.5, 139.9, 137.6, 128.4, 127.9, 125.0, 123.3, 122.3, 120.2, 118.6, 116.8, 115.4,
137.1, 135.0, 130.2, 130.1, 129.9, 129.1, 126.2, 125.7, 124.6, 108.6, 106.3, 84.5, 37.2, 28.1, 13.4; HRMS [M + H]+ calcd
124.3, 124.1, 122.4, 121.4, 121.2, 120.3, 118.2, 116.5, 85.8, 28.5; [C29H29N3O5 + H] 500.2185, found 500.2160; HPLC purity:
HRMS [M + H]+ calcd [C29H27N3O4 + H] 482.2080, found 97.8% area.
482.9251; HPLC purity: 100.0% area.
(E)-tert-Butyl-3-(3-(3-(3-methylfuran-2-ylmethylcarbamoyl)-
(E)-tert-Butyl-3-(3-(3-(benzylcarbamoyl)phenylamino)- phenylamino)-3-oxoprop-1-enyl)-1H-indole-1-carboxylate (5k). Mp
1
3-oxoprop-1-enyl)-1H-indole-1-carboxylate (5g). Mp 120 °C; 114 °C; H-NMR (CDCl3, 500 MHz) δ 8.75 (brs, 1H, –NH), 8.11
1H-NMR (CD3OD, 500 MHz) δ 8.13 (s, 1H, aromatic), 8.07 (d, J = 8.1 Hz, 1H, aromatic), 8.05 (s, 1H, –NH), 7.91
(d, J = 8.3 Hz, 1H, aromatic), 7.77 (m, 2H, aromatic), 7.71 (d, J = 8.1 Hz, 1H, aromatic), 7.81 (d, J = 15.4 Hz, 1H,
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indole–CHvCH–), 7.72 (m, 2H, aromatic, furan), 7.44 (d, J = 28.1; HRMS [M + H]+ calcd [C28H27N3O5 + H] 486.2029, found
7.8 Hz, 1H, aromatic), 7.28 (m, 2H, aromatic), 7.21 (s, 1H, aro- 486.1999; HPLC purity: 92.2% area.
matic), 7.14 (t, J = 7.5 Hz, 1H, aromatic), 6.75 (m, 2H, aromatic,
(E)-tert-Butyl-3-(3-(4-(furan-2-ylmethylcarbamoyl)phenyla-
indole–CHvCH–), 6.14 (s, 1H, furan), 4.53 (d, J = 5.1 Hz, 2H, mino)-3-oxoprop-1-enyl)-1H-indole-1-carboxylate (5o). Mp
–NH–CH2), 1.98 (s, 3H, –furyl–CH3), 1.63 (s, 9H, Boc); 13C-NMR 186 °C; 1H-NMR (CDCl3, 500 MHz) δ 8.19 (d, J = 8.1 Hz, 1H,
(CDCl3, 125 MHz) δ 167.3, 165.1, 149.1, 146.1, 141.3, 139.0, aromatic), 7.90 (d, J = 15.4 Hz, 1H, indole–CHvCH–), 7.83
136.1, 134.9, 134.1, 129.2, 128.4, 127.9, 125.0, 123.4, 122.4, (m, 2H, aromatic), 7.77 (m, 2H, aromatic), 7.70 (m, 2H, aro-
120.1, 117.3, 116.8, 115.4, 113.1, 84.5, 35.2, 28.1, 9.64; HRMS matic), 7.37 (m, 1H, aromatic), 7.29 (t, J = 7.5 Hz, 1H, furan),
[M + H]+ calcd [C29H29N3O5 + H] 500.2185, found 500.2130; 6.69 (d, J = 15.7 Hz, 1H, indole–CHvCH–), 6.46 (m, 1H,
HPLC purity: 98.7% area.
furan), 6.31 (m, 2H, aromatic, furan), 4.64 (d, J = 4.9 Hz, 2H,
(S,E)-tert-Butyl-3-(3-oxo-3-(3-((tetrahydrofuran-2-yl)methylcarb- –NH–CH2), 1.68 (s, 9H, Boc); 13C-NMR (CDCl3, 125 MHz)
amoyl)phenylamino)prop-1-enyl)-1H-indole-1-carboxylate δ 166.6, 164.6, 151.2, 149.1, 142.3, 141.4, 136.2, 134.9, 129.5,
(5l). Mp 128 °C; 1H-NMR (CDCl3, 500 MHz) δ 8.67 (brs, 1H, 128.7, 128.1, 125.2, 123.5, 120.1, 119.4, 116.8, 115.6,
–NH), 8.16 (d, J = 7.8 Hz, 1H, aromatic), 8.00 (m, 2H, aromatic, 110.5, 107.7, 84.7, 37.1, 29.7, 28.2; HRMS [M + H]+ calcd
–NH), 7.86 (d, J = 15.7 Hz, 1H, indole–CHvCH–), 7.80 (m, 2H, [C28H29N3O5 + H] 488.2185, found 486.2020; HPLC purity:
aromatic), 7.45 (d, J = 7.6 Hz, 1H, aromatic), 7.34 (t, J = 7.8 Hz, 97.8% area.
2H, aromatic), 7.23 (t, J = 7.6 Hz, 1H, aromatic), 6.84 (t, J =
tert-Butyl 3-(3-(3-(furan-2-ylmethylcarbamoyl)phenylamino)-
5.6 Hz, 1H, aromatic), 6.79 (d, J = 15.7 Hz, 1H, indole– 3-oxopropyl)-1H-indole-1-carboxylate (5p). Mp 116 °C;
CHvCH–), 4.03 (m, 1H, tetrahydrofuran), 3.86 (q, J = 7.1 Hz, 1H-NMR (CDCl3, 500 MHz) δ 8.09 (d, J = 6.1 Hz, 1H, aromatic),
1H, tetrahydrofuran), 3.74 (m, 3H, –tetrahydrofuran, –NH– 7.82 (d, J = 9.5 Hz, 1H, aromatic), 7.68 (d, J = 7.8 Hz, 1H, aro-
CH2), 3.30 (m, 1H, –NH–CH2), 1.93 (m, 1H, tetrahydrofuran), matic), 7.52 (d, J = 7.8 Hz, 1H, aromatic), 7.45 (d, J = 7.3 Hz,
1.82 (m, 2H, tetrahydrofuran), 1.67 (s, 9H, Boc); 13C-NMR 1H, aromatic), 7.39 (s, 1H, aromatic), 7.29 (m, 2H, furan), 7.20
(CDCl3, 125 MHz) δ 167.6, 165.0, 149.1, 139.0, 136.2, 135.1, (m, 1H, aromatic), 6.63 (t, J = 5.3 Hz, 1H, furan), 6.30 (s, 1H,
134.1, 129.2, 128.4, 127.9, 125.1, 123.4, 122.2, 120.2, 116.7, –NH–CH2), 6.23 (d, J = 2.2 Hz, 1H, –NH–CH2), 4.57 (d, J =
115.5, 84.6, 77.7, 68.1, 43.9, 28.7, 28.1, 25.8; HRMS [M + H]+ 5.6 Hz, 2H, aromatic), 3.10 (t, J = 7.5 Hz, 2H, indole–CH2–
calcd [C28H31N3O5 + H] 490.2342, found 490.9582; HPLC CH2–), 2.73 (t, J = 7.5 Hz, 2H, indole–CH2–CH2–), 1.63 (s, 9H,
purity: 96.3% area.
Boc); 13C-NMR (CDCl3, 125 MHz) δ 170.9, 167.0, 151.0, 149.7,
(R,E)-tert-Butyl-3-(3-oxo-3-(3-((tetrahydrofuran-2-yl)methyl- 142.3, 138.3, 134.8, 130.2, 129.2, 124.5, 123.1, 122.7, 122.5,
carbamoyl)phenylamino)prop-1-enyl)-1H-indole-1-carboxylate 119.4, 118.8, 118.4, 115.3, 110.5, 107.7, 83.6, 37.0, 28.2, 27.8,
1
(5m). Mp 134 °C; H-NMR (CDCl3, 500 MHz) δ 8.76 (brs, 1H, 20.7; HRMS [M + H]+ calcd [C28H29N3O5 + H] 488.2185, found
–NH), 8.16 (d, J = 8.1 Hz, 1H, aromatic), 8.03 (d, J = 7.6 Hz, 1H, 488.2153; HPLC purity: 96.9% area.
aromatic), 7.98 (brs, 1H, –NH), 7.86 (d, J = 15.7 Hz, 1H, indole–
(E)-3-(3-(1-Benzyl-1H-indol-3-yl)acrylamido)-N-(furan-
CHvCH–), 7.79 (m, 2H, aromatic), 7.45 (d, J = 7.3 Hz, 1H, aro- 2-ylmethyl)-benzamide (6). A suspension of 4h (63.0 mg,
matic), 7.33 (t, J = 7.9 Hz, 2H, aromatic), 7.22 (t, J = 7.5 Hz, 1H, 0.16 mmol) and anhydrous potassium carbonate (34.0 mg,
aromatic), 6.85 (t, J = 5.5 Hz, 1H, aromatic), 6.81 (d, J = 0.24 mmol) in DMF (3.0 ml) was treated with benzyl bromide
15.7 Hz, 1H, indole–CHvCH–), 4.02 (m, 1H, tetrahydrofuran), (0.03 ml, 0.24 mmol) dropwise at room temperature and
3.85 (m, 1H, tetrahydrofuran), 3.74 (m, 2H, –tetrahydrofuran, the resulting mixture was stirred overnight. The reaction
–NH–CH2), 3.30 (m, 1H, –NH–CH2), 1.93 (m, 1H, tetrahydro- mixture was diluted with EtOAc and subsequently washed with
furan), 1.86 (m, 2H, tetrahydrofuran), 1.67 (s, 9H, Boc), 1.53 brine and water, and dried over anhydrous MgSO4. The solvent
(m, 1H, tetrahydrofuran); 13C-NMR (CDCl3, 125 MHz) δ 167.6, was evaporated under reduced pressure to afford a crude solid
165.0, 149.1, 139.0, 136.2, 135.1, 134.0, 129.3, 128.4, 127.9, which was purified by column chromatography using DCM
125.1, 123.4, 122.1, 120.2, 116.9, 115.5, 84.5, 77.7, 68.1, 43.9, and MeOH (30 : 1) to afford 6 as a pale yellow solid (56.0 mg,
28.7, 28.1, 25.8; HRMS [M + H]+ calcd [C28H31N3O5 + H] 73.0% yield). Mp 138 °C; 1H-NMR (DMSO, 400 MHz) δ 10.22
490.2342, found 490.9582; HPLC purity: 95.1% area.
(brs, 1H, –NH), 9.00 (t, J = 8.0 Hz, 1H, –NH), 8.17 (s, 1H, aro-
(E)-tert-Butyl-3-(3-(2-(furan-2-ylmethylcarbamoyl)phenyl- matic), 8.09 (s, 1H, aromatic), 8.02 (m, 2H, aromatic), 7.83
amino)-3-oxoprop-1-enyl)-1H-indole-1-carboxylate (5n). Mp (d, J = 16.0 Hz, 1H, aromatic), 7.61 (m, 3H, aromatic), 7.48
110 °C; 1H-NMR (CDCl3, 500 MHz) δ 11.31 (brs, 1H, –NH), 8.71 (m, 1H, aromatic), 7.36 (m, 7H, aromatic), 6.90 (d, J = 16.0 Hz,
(d, J = 8.3, 1H, aromatic), 8.22 (d, J = 7.8 Hz, 1H, aromatic), 1H, indole–CHvCH–), 6.40 (m, 2H, aromatic), 5.53 (s, 2H,
7.89 (m, 3H, aromatic, –NH), 7.51 (d, J = 7.8 Hz, 1H, aromatic), –N–CH2), 4.52 (d, J = 4.00 Hz, 2H, –NH–CH2); 13C-NMR
7.44 (t, J = 7.7 Hz, 1H, aromatic), 7.38 (m, 2H, aromatic), 7.03 (DMSO, 100 MHz) δ 166.6, 165.4, 152.9, 142.4, 140.3, 137.9,
(t, J = 7.5 Hz, 1H, aromatic), 6.96 (t, J = 5.1 Hz, 1H, furan), 6.69 137.7, 135.4, 134.7, 134.6, 129.2, 129.1, 128.0, 127.6, 126.0,
(d, J = 15.9 Hz, 1H, indole–CHvCH–), 6.35 (m, 2H, furan), 123.1, 121.9, 121.4, 121.3, 120.7, 118.7, 116.8, 112.2, 111.7,
4.66 (d, J = 5.4 Hz, 2H, –NH–CH2), 1.71(s, 9H, Boc). 13C-NMR 110.9, 107.3, 49.8, 36.6; HRMS [M + H]+ calcd [C30H25N3O3 + H]
(CDCl3, 125 MHz) δ 168.9, 164.8, 150.6, 149.1, 142.5, 139.9, 476.2199, found 476.2211; HPLC purity: 99.9% area.
136.1, 133.5, 132.6, 128.1, 127.8, 126.7, 125.1, 123.5, 122.6,
(E)-3-(3-(1-(2-(Dimethylamino)ethyl)-1H-indol-3-yl)acryl-
121.7, 121.2, 120.2, 119.9, 116.9, 115.5, 110.5, 107.9, 84.5, 36.9, amido)-N-(furan-2-ylmethyl)benzamide (7). A suspension of 4h
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(52.0 mg, 0.13 mmol) and anhydrous potassium carbonate anhydrous MgSO4. The solvent was evaporated under reduced
(56.0 mg, 0.41 mmol) in DMF (3.0 ml) was treated with pressure to afford a crude solid which was purified by column
2-(dimethylamino)ethyl chloride (39.0 mg, 0.27 mmol) drop- chromatography using DCM and MeOH (30 : 1) to afford 9 as a
wise at room temperature and the resulting mixture was yellow solid (62.0 mg, 81.0% yield). Mp 124 °C; 1H-NMR
stirred at 50 °C overnight. The reaction mixture was diluted (CD3OD, 400 MHz) δ 8.36 (m, 1H, aromatic), 8.03 (s, 1H,
with EtOAc and subsequently washed with brine and water, aromatic), 7.97 (s, 1H, aromatic), 7.86 (m, 1H, aromatic), 7.64
and dried over anhydrous MgSO4. The solvent was evaporated (m, 2H, aromatic), 7.45 (d, J = 8.0 Hz, 1H, aromatic), 7.33
under reduced pressure to afford a crude solid which was puri- (m, 4H, aromatic), 6.86 (d, J = 16.0 Hz, 1H, indole–CHvCH–),
fied by column chromatography using hexane, ethyl acetate 6.26 (m, 1H, aromatic), 6.21 (d, J = 4.0 Hz, 1H, aromatic), 4.47
and MeOH (6 : 3 : 1) to afford 7 as a yellow solid (47.0 mg, (s, 2H, –NH–CH2), 2.58 (s, 3H, –(CH3); 13C-NMR (CD3OD,
77.0% yield). Mp 122 °C; 1H-NMR (CD3OD, 400 MHz) δ 8.03 100 MHz) δ 169.6, 168.4, 165.8, 151.8, 141.9, 139.0, 136.7,
(s, 1H, aromatic), 7.86 (d, J = 8.0 Hz, 1H, aromatic), 7.78 (d, J = 135.0, 133.5, 129.5, 128.7, 127.7, 125.3, 124.0, 122.7, 122.4,
16.0 Hz, 1H, aromatic), 7.72 (d, J = 8.0 Hz, 1H, aromatic), 7.58 120.3, 119.8, 118.7, 118.0, 116.4, 110.0, 106.8, 36.3, 22.5;
(s, 1H, aromatic), 7.42 (m, 2H, aromatic), 7.33 (m, 2H, aro- HRMS [M + H]+ calcd [C25H21N3O4 + H] 428.1610, found
matic), 7.16 (m, 2H, aromatic), 6.67 (d, J = 16.0 Hz, 1H, indole– 428.1610; HPLC purity: 99.2% area.
CHvCH–), 6.26 (m, 1H, aromatic), 6.20 (d, J = 4.0 Hz, 1H, aro-
(E)-N-(Furan-2-ylmethyl)-3-(3-(1-(thiophen-2-ylsulfonyl)-1H-
matic), 4.63 (s, 2H, –NH–CH2), 4.24 (t, J = 4.0 Hz, 2H, –N–CH2), indol-3-yl)acrylamido)benzamide (10). A suspension of 4h
2.71 (t, J = 4.0 Hz, 2H, –N–CH2), 2.23 (s, 6H, (CH3)2); 13C-NMR (120 mg, 0.31 mmol) and tert-BuOK (107 mg, 0.95 mmol) in
(CD3OD, 100 MHz) δ 168.6, 167.1, 151.8, 141.9, 139.3, 137.6, DMF (4.0 ml) was treated with 2-thiophensulfonyl chloride
135.7, 134.9, 133.1, 128.7, 126.0, 122.7, 122.6, 122.1, 120.9, (85.0 mg, 0.47 mmol) dropwise at room temperature and the
120.1, 118.7, 114.7, 112.3, 110.0, 109.9, 106.8, 57.9, 44.2, 43.7, resulting mixture was stirred at 50 °C overnight. The reaction
36.3; HRMS [M + H]+ calcd [C27H28N4O3 + H] 457.2226, found mixture was diluted with EtOAc and subsequently washed with
457.2236; HPLC purity: 99.6% area.
brine and water, and dried over anhydrous MgSO4. The solvent
(E)-tert-Butyl-2-(3-(3-(3-(furan-2-ylmethylcarbamoyl)phenyl- was evaporated under reduced pressure to afford a crude solid
amino)-3-oxoprop-1-enyl)-1H-indol-1-yl)acetate (8). A suspen- which was purified by column chromatography using hexane,
sion of 4h (60.0 mg, 0.16 mmol) and anhydrous potassium ethyl acetate and MeOH (6 : 3 : 1) to afford 10 as a yellow solid
carbonate (33.0 mg, 0.24 mmol) in DMF (3.0 ml) was treated (115 mg, 71.0% yield). Mp 130 °C; 1H-NMR (CD3OD, 400 MHz)
with tert-butyl bromoacetate (47.0 mg, 0.24 mmol) dropwise at δ 8.03 (s, 1H, aromatic), 7.88 (m, 2H, aromatic), 7.85 (d, J = 4.0
room temperature and the resulting mixture was stirred at Hz, 1H, aromatic), 7.73 (m, 4H, aromatic), 7.45 (d, J = 8.0 Hz,
50 °C overnight. The reaction mixture was diluted with EtOAc 1H, aromatic), 7.33 (m, 4H, aromatic), 7.03 (t, J = 8.0 Hz, 1H,
and subsequently washed with brine and water, and dried over aromatic), 6.85 (d, J = 16.0 Hz, 1H, indole–CHvCH–), 6.26
anhydrous MgSO4. The solvent was evaporated under reduced (m, 1H, aromatic), 6.20 (d, J = 4.0 Hz, 1H, aromatic), 4.45
pressure to afford a crude solid which was purified by column (s, 2H, –NH–CH2); 13C-NMR (CD3OD, 100 MHz) δ 168.4, 165.4,
chromatography using hexane, ethyl acetate and MeOH 151.8, 141.9, 138.9, 137.1, 135.6, 135.0, 134.9, 134.1, 132.6,
(6 : 3 : 1) to afford 8 as a yellow solid (52.0 mg, 68% yield). Mp 128.7, 128.5, 128.2, 127.6, 125.3, 124.1, 122.7, 122.4, 121.1,
118 °C; 1H-NMR (CD3OD, 400 MHz) δ 8.22 (s, 1H, aromatic), 120.4, 119.1, 118.7, 113.7, 110.0, 106.8, 36.3; HRMS [M + H]+
7.88 (d, J = 4.0 Hz, 1H, aromatic), 7.79 (d, J = 16.0 Hz, 1H, aro- calcd [C27H21N3O5S2 + H] 532.1001, found 532.1003; HPLC
matic), 7.73 (d, J = 8.0 Hz, 1H, aromatic), 7.52 (s, 1H, aro- purity: 99.3% area.
matic), 7.43 (d, 1H J = 8.0 Hz, aromatic), 7.33 (m, 2H,
(E)-3-(3-(1-(Benzylsulfonyl)-1H-indol-3-yl)acrylamido)-N-(furan-
aromatic), 7.28 (m, 1H, aromatic), 7.16 (m, 2H, aromatic), 6.70 2-ylmethyl)benzamide (11). A suspension of 4h (45.0 mg,
(d, J = 12.0 Hz, 1H, indole–CHvCH–), 6.26 (m, 1H, aromatic), 0.12 mmol) and sodium hydride (6.40 mg, 0.12 mmol) in DMF
6.20 (d, J = 4.0 Hz, 1H, aromatic), 4.85 (s, 2H, –NH–CH2), 4.46 (4.0 ml) was treated with benzyl sulfonyl chloride (33.0 mg,
(s, 2H, –N–CH2), 1.35 (s, 9H, –O–(CH3)3; 13C-NMR (CD3OD, 0.17 mmol) dropwise at 0 °C and the resulting mixture was
100 MHz) δ 168.6, 167.8, 167.0, 151.8, 141.9, 139.3, 138.2, stirred at room temperature overnight. The reaction mixture
135.5, 135.0, 133.9, 128.7, 125.9, 122.7, 122.1, 121.0, 120.0, was diluted with EtOAc and subsequently washed with brine
118.7, 115.1, 112.7, 110.0, 109.8, 106.8, 82.3, 48.2, 36.3, 26.8; and water, and dried over anhydrous MgSO4. The solvent was
HRMS [M + H]+ calcd [C29H29N3O5 + H] 500.2185, found evaporated under reduced pressure to afford a crude solid
500.2178; HPLC purity: 99.8% area.
which was purified by column chromatography using hexane,
(E)-3-(3-(1-Acetyl-1H-indol-3-yl)acrylamido)-N-(furan-2-yl- ethyl acetate and MeOH (6 : 3 : 1) to afford 11 as a yellow solid
1
methyl)benzamide (9). A suspension of 4h (61.0 mg, (46.0 mg, 73% yield). Mp 114 °C; H-NMR (CD3OD, 400 MHz)
0.16 mmol) and anhydrous potassium carbonate (66.0 mg, δ 8.03 (s, 1H, aromatic), 7.87 (d, J = 8.00 Hz, 1H, aromatic),
0.48 mmol) and DMAP (2.40 mg, 0.02 mmol) in DMF (3.0 ml) 7.74 (d, J = 4.0 Hz, 1H, aromatic), 7.68 (d, J = 8.0 Hz, 1H, aro-
was treated with acetic anhydride (0.03 ml, 0.24 mmol) drop- matic), 7.59 (d, J = 16.0 Hz, 1H, aromatic), 7.45 (d, J = 8.0 Hz,
wise at room temperature and the resulting mixture was stirred 1H, aromatic), 7.36 (m, 3H, aromatic), 7.28 (m, 3H, aromatic),
overnight. The reaction mixture was diluted with EtOAc and 7.17 (t, J = 8.0 Hz, 1H, aromatic), 7.05 (t, J = 8.0 Hz, 2H, aro-
subsequently washed with brine and water, and dried over matic), 6.82 (m, 2H, indole–CHvCH–), 6.26 (m, 1H, aromatic),
856 | Org. Biomol. Chem., 2013, 11, 849–858
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6.21 (d, J = 4.0 Hz, 1H, aromatic), 4.74 (s, 2H, –N–CH2), 4.46 NaOH–water (50 : 10 : 40, v/v)]. The radioactivity of the
(s, 2H, –NH–CH2); 13C-NMR (CD3OD, 100 MHz) δ 168.4, 165.6, top phase was determined by
liquid scintillation
a
151.8, 141.9, 139.0, 136.4, 135.0, 132.8, 130.3, 129.5, 128.9, counter (Tri-Carb 2900TR Liquid Scintillation Analyzer, Perkin
128.7, 128.2, 127.5, 127.2, 125.0, 123.6, 122.7, 122.4, 120.3, Elmer).
120.2, 118.8, 116.9, 113.3, 110.0, 106.8, 59.2, 36.3; HRMS
[M + H]+ calcd [C30H25N3O5S + H] 540.1593, found 540.1582;
HPLC purity: 99.1% area.
Acknowledgements
Biological procedures
This work was supported by a grant from KRIBB Research
Cell culture and viability test. Human hepatocyte HepG2 Initiative Program and Basic Science Research Program
cells were obtained from the American Type Culture Collection through the National Research Foundation of Korea (NRF)
(Manassas, VA). Cells were maintained in a DMEM medium funded by the Ministry of Education, Science and Technology
(Invitrogen) supplemented with 10% heat-inactivated FBS and (Grant 2010-0024662 to Y. Choi and 2012053532 to K. Lee).
1% penicillin–streptomycin (Sigma) in 5% CO2 at 37 °C. To
test whether the 5h has cytotoxic effects on HepG2 cells, we
determined it by using an MTT assay. Cells were seeded in 96-
Notes and references
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