Quinolone-1-(2: H)-ones as hedgehog signalling pathway inhibitors

Article abstract of DOI:10.1039/c6ob00606j

A series of quinolone-2-(1H)-ones derived from the Ugi-Knoevenagel three- and four-component reaction were prepared exhibiting low micromolar cytotoxicity against a panel of eight human cancer cell lines known to possess the Hedgehog Signalling Pathway (H

Full text of DOI:10.1039/c6ob00606j

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Biomol. Chem., 2016, DOI: 10.1039/C6OB00606J.
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Quinolone-1-(2H)-ones as Hedgehog Signalling Pathway Inhibitors
Trieu N. Trinh,a Eileen A. McLaughlin,b,† Mohammed Abdel-Hamid,a,c Christopher P. Gordon,d Ilana
R. Bernstein,b Victoria Pye, b Peter Cossar,a Jennette A. Sakoff,e and Adam McCluskeya*
Received
January 20xx,
00th
A series of quinolone-2-(1H)-ones derived from a Ugi-Knoevenagel three- and four- component reaction were prepared
exhibiting low micromolar cytotoxicity against a panel of eight human cancer cell lines known to possess the Hedgehog
Signalling Pathway (HSP) components, as well as the seminoma TCAM-2 cell line. A focused SAR study was conducted and
revealed core characteristics of the quinolone-2-(1H)-ones required for cytotoxicity. These requirements included a C3-
tethered indole moiety, an indole C5-methyl moiety, an aliphatic tail or an ester, as well as an additional aromatic moiety.
Further investigation in the SAG-activated Shh-LIGHT 2 cell line with the most active analogues: 2-(3-cyano-2-oxo-4-
phenylquinolin-1(2H)-yl)-2-(1-methyl-1H-indol-3-yl)-N-(pentan-2-yl)acetamide (5), 2-(3-cyano-2-oxo-4-phenylquinolin-1(2H)-yl)-2-
(5-methyl-1H-indol-3-yl)-N-(pentan-2-yl)acetamide (23) and ethyl (2-(3-cyano-2-oxo-4-phenylquinolin-1(2H)-yl)-2-(5-methyl-1H-
indol-3-yl)acetyl)glycinate (24) demonstrated a down regulation of the HSP via a reduction in Gli expression, and in the
mRNA levels of Ptch1 and Gli2. Analogues 5, 23 and 24 returned in cell inhibition values of 11.6, 2.9 and 3.1 µM,
respectively, making this new HSP-inhibitor pharmacophore amongst the most potent non-Smo targeted inhibitors thus
far reported.
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
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Introduction
The Hedgehog (Hh) signalling pathway (HSP) plays a pivotal
role in embryogenesis by controlling the spatial and temporal
regulation of cell proliferation, differentiation, and tissue
patterning.1, 2 Aberrant Hh signalling in humans can initiate the
development of a diverse range of human cancers, including
basal cell carcinoma,3 medulloblastoma,4-6 cancers of the
Figure 1. Chemical structures of the Smo inhibitors Vismodegib (1, GDC-0449,
Erivedge®) and Sonidegib (2, LDE225, Odomzo®) approved by FDA for the treatment of
early and advanced basal cell carcinomas.18, 19
pancreas,7 prostate,8 lung,9, 10 colon,11 stomach,12 breast,13, 14
15
ovary
and perhaps most problematically the formation of
The activation and suppression of the HSP involves an
intricate interplay between proteins, both within the HSP and
with associated signalling networks including the TGF-β, p53,
WIP1, PI3K/AKT and RAS/MEK pathways. Briefly, the canonical
HSP functions in a hierarchical manner, in which a Hedgehog
ligand (Sonic, Desert or Indian hedgehog protein) binds to the
membrane receptor Patched1 (Ptch1), resulting in the
activation of the Smo protein and subsequent release of active
cancer stem cells.16, 17 Consequently, suppressing the HSP is an
attractive and recently validated chemotherapeutic target with
two inhibitors targeting the Smoothened (Smo) protein,
Vismodegib
(1, (2,
GDC-0449, Erivedge®) and Sonidegib
LDE225, Odomzo®) (Figure 1), approved by FDA for the
treatment of early and advanced basal cell carcinomas.18, 19
Glioma-Associated Oncogene Homolog transcriptional factors
2, 20
These Gli transcription factors
(Gli1-3) into the nucleus.1,
facilitate the transcription of Hh target genes, including the
components of the HSP Gli1, Gli2, Ptch1, and Ptch2.21
Alternatively, the HSP can be activated directly at the Smo
level via a synthetic Smo agonist (
3
, SAG) (Figure 2).22
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family of quinolone-2-(1H)-ones recently reported from our
laboratories (exemplified by
; Figure 3).48
5
To assess the potential of quinolone-1-(2H)-one scaffold as
HSP inhibitors, we first evaluated their cytotoxicity in a double-
filter screening against a panel of eight human cancer cell lines
possessing components of the HSP (Table 1; entries 1-8), and
one seminoma cancer cell line (TCAM-2) (Table 1; entry 9).
O
Cl
N
O
NC
O
Figure 2. The canonical HSP is initiated by the binding of the Hedgehog ligand (Sonic,
NC
NH
HN
NH
Cl
N
Desert or Indian) to the membrane receptor Ptch1, resulting in the activation of Smo
O
protein and release of active Gli transcriptional factors (Gli1-3) into the nucleus,
culminating in the transcription of Hh-target genes.20 Alternatively, the HSP can be
activated directly at the Smo level by using SAG (3).22
4
5
HPI-4
Figure 3. N-(sec-butyl)-2-(3-cyano-2-oxo-4-phenylquinolin-1-(2H)-yl)-2-(1-methyl-1H-
indol-3-yl)acetamide (5) from our laboratory with the bolded structure sections
reflecting the structural similarities with the Gli inhibitor HPI-4 (4).
The hierarchical character of the HSP, affords several
opportunities to suppress the pathway including the inhibition
24
inhibition of the
of Hedgehog-ligand-Ptch1 interactions,23,
22, 25-35
Smo protein
or further downstream such as the
inhibition of the Gli transcription factors.36-46 At present the
most clinically advanced HSP inhibitor compounds target Smo.
These clinical studies have identified limitations to this
approach including the development of acquired resistance
resulting from Smo mutations and compensatory amplification
of Gli2 transcription factors by the aforementioned interacting
pathways.20 Targeting the HSP further downstream of Smo at
the Gli transcription factor level, and/or indirectly at
interacting signalling pathways may constitute a more robust
strategy for treating HSP related cancers.20, 43
The TCAM-2 cell line, in addition to expressing the HSP
49
(ESI†), possesses the acꢀve PI3K signalling pathway and the
aberrantly up-regulated mitogen-activated protein kinase
signalling pathway (RAS/RAF/MEK/ERK) due to a mutation at
the BRAF gene (V600E).50-52 Together these signalling pathways
create a complex loop facilitating the non-canonical activation
of Gli activity downstream of Smo.47, 49, 53 Thus the TCAM-2 cell
line provides
a valuable filter to identify potential Gli
transcription factor inhibitors, with this (we believe) to be the
first such use of this system. Active compounds from our
double-filter cytotoxicity screening approach would be further
evaluated in SAG-activated Sonic Hedgehog-(Shh) LIGHT 2 cell
line model for their potential to suppress the HSP using Dual
Luciferase Reporter (DLR), Reverse Transcription PCR (RT-PCR)
and Quantitative PCR (qPCR) assays.
Given our ongoing interest in the development of small
20, 47
molecule HSP inhibitors
our attention was drawn to the
4
).43 Considered as a non-selective
previously reported HIP-4 (
inhibitor of the Gli family of transcription factors, HIP-4
contained a number of structural features present within a
Table 1. Human cancer cell lines known to possess the HSP.
Entry
Cell Line
HT29
SW480
MCF-7
A2780
H460
Cell Type
Colorectal carcinoma
Colorectal carcinoma
Breast adenocarcinoma
Ovarian carcinoma
Lung carcinoma
HSP components expressed
Ihh, Shh, Ptch1, Smo, Gli1,2,3, Hhip at mRNA levels
Shh, Ptch, Smo, Sufu, Gli2,3, Hhip at mRNA levels
Ihh, Shh, Dhh, Ptch1, Smo, Gli1,2 at mRNA levels
Shh, Dhh, Ptch, Smo, Gli1 at mRNA and protein levels
Smo, Ptch1, Gli1 at mRNA levels
Ptch1, Gli1,2 at mRNA levels
Shh, Smo, Gli2 at protein levels
Shh, Ptch1,2, Smo, Gli1,2 at mRNA levels
Ptch1, Smo, SuFu, Gli2, and Gli3 at mRNA levels
Expression of PI3 pathway
Ref
54
55
14
15
56, 57
58, 59
60
61,62
ESI†
49
1
2
3
4
5
6
7
8
9
DU145
BE2-C
Prostate carcinoma
Neuroblastoma
MIA-Paca-2 Pancreatic carcinoma
TCAM-2 Seminoma
Mutation at BRAF gene, overexpression of RAS/RAF/MEK/ERK
pathway
51,50, 52
stirred at room temperature for 48 h, followed by
chromatographic separation of the desired product 10
(Scheme 1).48 Using this approach eleven exemplars were
generated, of which five ( 12 14 16, Table 2) were obtained
as a mixture of diastereomers (see experimental).
Results and discussion
(
)
A targeted library of quinolone-1-(2H)-ones retaining the
5,
,
-
highlighted pharmacophore of
4 (Figure 3) was prepared using
our previously reported sequential Ugi-Knoevenagel
protocol.48 In a typical synthesis, a methanolic solution of 2-
aminobenzophenone
(
6),
4-chlorobenzaldehyde
(7),
cyanoacetic acid (8) and ethyl isocyanate (
9) in methanol was
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approaches. This theory level was used to calculate the 1H
NMR shifts of each conformer and to predict the more
abundant diasteroisomer obtained synthetically. Data relating
1
16 showed distinguishable H NMR peaks for
to analogues 14
-
each pair of diastereomersfor the two methyl and the
methylene moieties of the 2-pentyl substituent. Comparison of
the R,S/S,R and R,S/S,R pairs as well as their computed 1H NMR
chemical shifts (ESI, Table S1) showed favourable DFT energies
for the R,S/S,R pair compared to R,R/S,S (difference of 1.6 - 4.3
kcal/mol) in all instances. This was consistent with the
observed 1HNMR shifts for the major product and the
calculated chemical shifts for R,S/S,R pair of enantiomers. This
11 component library was screened against our panel of eight
human cancer cell lines and the data presented in Table 2.
Scheme 1. Synthesis of quinolone-2-(1H)-ones. Reagents and Conditions: (i) MeOH, rt;
(ii) spontaneous.48
Attempts to separate individual diastereomers proved
unsuccessful. However using Willoughby et al’s computational
approach we identified the relative configuration of the major
isomers in each instance.63 With analogues 14
-16 the
geometry was optimised and free energy calculated using
Density Functional Theory and B3LYP (6-31+G(d,p) basis set)
Table 2. Evaluation of the cytotoxicity of the quinolin-2-(1H)-ones analogues (5, 10-19) against a panel of eight hedgehog signalling pathway expressing cancer cell lines. Values are
the percentage of growth inhibition at 25 µM drug concentration
Compound
5
R1
R2
R3
HT29a
SW480a
MCF-7b
A2780c
H460d
Du145e
BE2-Cf
MIAg
79 ± 2
99 ± 5
92 ± 2
96 ± 2
84 ± 4
94 ± 4
92 ± 2
92 ± 2
10
11
12
13
14
15
16
49 ± 2
42 ± 3
34 ± 6
11 ± 7
11 ± 6
46 ± 1
85 ± 0
43 ± 3
57 ± 1
46 ± 1
2 ± 5
62 ± 3
60 ± 5
65 ± 2
20 ± 5
11 ± 4
29 ± 3
90 ± 2
51 ± 2
38 ± 5
39 ± 6
27 ± 3
29 ± 3
31 ± 1
96 ± 1
43 ± 7
32 ± 4
26 ± 7
4 ± 4
22 ± 2
28 ± 4
18 ± 2
<0
42 ± 0
43 ± 6
41 ± 2
8 ± 3
<0
41 ± 2
45 ± 10
39 ± 15
16 ± 10
14 ± 11
36 ± 2
7 ± 2
5 ± 5
<0
47 ± 5
77 ± 3
28 ± 7
>100
<0
42 ± 3
>100
63 ± 4
81 ± 0
17
18 ± 3
38 ± 3
3 ± 9
17 ± 3
35 ± 1
14 ± 12
<0
<0
19 ± 3
30 ± 3
18
19
26 ± 7
6 ± 3
45 ± 5
9 ± 9
42 ± 3
19 ± 6
26 ± 17
9 ± 5
21 ± 5
4 ± 5
16 ± 1
2 ± 7
6 ± 2
14 ± 5
a
b
c
d
e
f
HT29 and SW480 (colon carcinoma); MCF-7 (breast carcinoma); A2780 (ovarian carcinoma); H460 (lung carcinoma); Du145 (prostate carcinoma); BE2-C
(neuroblastoma); g MIA (pancreatic carcinoma).
Analysis of the data presented in Table
2
showed significant decrease in inhibition. All other analogues displayed
M drug modest (30-75%) to negligible growth inhibition (<30%) (Table
concentration evaluated. The C3-tethered indole group ( ) was 2). The two most promising analogues ( and 16) proceeded to
analogues
5 and 16 as the most promising at the 25 µ
5
5
shown to be crucial for activity, while its replacement by either full dose response evaluation (Table 3).
a 4-methoxyphenyl (12) or phenyl moiety (14) resulted in a
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Table 3. Evaluation of the cytotoxicity, GI50 values (µM), of compounds 5 and 16 against a panel of nine human HSP expressing cancer cell lines. GI50 is the concentration
of drug that reduces cell growth by 50%.
Compound
HT29a
SW480a
11 ± 1
MCF-7b
4.6 ± 1.1
7.9 ± 1
A2780c
3.9 ± 0.3
7.5 ± 0.6
H460d
5.2 ± 0.1
11 ± 1
Du145e
13 ± 0
18 ± 1
BE2-Cf
MIAg
6.0 ± 0.1
13 ± 1
TCAM-2h
11.6 ± 0.6
>100
5
5.3 ± 0.3
8.7 ± 0.5
3.6 ± 0.1
7.3 ± 0.3
16
17 ± 1
a
b
HT29 and SW480 (colon carcinoma); MCF-7 (breast carcinoma); c A2780 (ovarian carcinoma); d H460 (lung carcinoma); e Du145 (prostate carcinoma); f BE2-C
(neuroblastoma);g MIA (pancreatic carcinoma).
The data in Table 3 shows
5 and 16 to be potent broad focused library, assembled via our Ugi-Knoevenagel approach
spectrum cytotoxic agents with GI50 values of 3.6-11 and 7.3- (Scheme 1). Given the differential activity noted with the
18 µM for respectively. However examination of these two TCAM-2 cell line, these new indole based analogues were
analogues in TCAM-2 cells revealed 16 to be inactive (GI50 >100 screened directly in this cell line only and the data presented in
µM), while the indole-based
5 displayed excellent growth Table 4. This represents the first such use of TCAM-2 cells in
inhibition (GI50 = 11.6 ± 0.6 µM). These data and those the development of HSP inhibitors.
presented in Table 2 support retention of the indole moiety as
a key pharmacophore in this study. To further investigate this
hypothesis we developed a second indole moiety based
Table 4. Synthesis results and the evaluation of the cytotoxicity of the second focused library against the TCAM-2 cell line. Values are the percentage of growth inhibition at 10 µM
drug concentration and GI50 were determined where the growth inhibition > 50% (ESI†) Reagents and conditions: (i) MeOH, rt, 24 h
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Yield
(%)
TCAM-2
TCAM-2
Compound
5
R1
R2
R3
% Inhibition at 10 µM GI50 (μM)
38
52
11.6 ± 0.6
13
11
36
21
41
<0
-
-
-
20
21
22
46
34
72
66
2.9 ± 0
23
24
3.1 ± 0.4
46
26
50
28
45
44
-
-
-
25
26
27
33
<0
-
-
28
47
26
41
44
29
30
activated with 100nM SAG. Treatments were performed in triplicate. *P < .05,
** P < .001 compared with SAG control
Analysis of the DLR assay data indicated moderate
suppression (55, 54 and 31%) of Gli expression at the protein
level by 5, 23 and 24 respectively relative to the DMSO and
Thus, the mRNA level of HSP components in SAG-activated
Shh-LIGHT2 cell line was probed using a combination of
Reverse Transcription PCR (RT-PCR) and Quantitative PCR
(qPCR) assays. Of the individual HSP components identified at
the mRNA level by RT-PCR, only Ptch1 and Gli2 exhibited
significant up-regulation under SAG-sꢀmulaꢀon (ESI†) and
thus became our targets. Unlike previous reports, we found no
evidence for Gli1 expression under the conditions evaluated
herein.65, 66 The outcomes of our qPCR analysis of Ptch1 and
SAG-treated controls (Figure 4). This inhibition over Gli protein
expression does not always result from the suppression of the
HSP due to the complex crosstalk of interacting signalling
pathways sharing Gli2 as the same effector.20
1.2
1
0.8
0.6
0.4
0.2
0
*
**
*
*
Gli2 post treatment at 10 µM of
Figure 5.
5,
23 and 24 are shown in
DMSO SAG
5
23
24
2
Figure 4: Effect of compounds
5
,
23, and 24 at 25 µM and Sonidegib (
2
) at
100nM concentration on the suppression Gli expression in Shh-LIGHT2 cells
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pharmacophore required to elicit the cytotoxicity profile has
been established. Apparent crucial structural features include
an indole moiety at R2 which is tethered to the remainder of
the scaffold through the C3 position. Moreover, the presence
of bulky aliphatic groups within R3 of the scaffold appears to
be required to endow cytotoxicity against the TCAM-2 cell
line. These valuable data undoubtedly will enable us to exploit
the current pharmacophore to develop next generation
analogues with superior properties to combat the hedgehog
signalling related cancers. The results of these efforts will be
reported in due course.
1.4
1.2
1
1.2
1
0.8
0.6
0.4
0.2
0
0.8
0.6
0.4
0.2
0
*
*
*
*
*
DMSO SAG
5
23
24
DMSO SAG
5
23
24
Figure 5. Effect of compounds 5, 23, and 24 at 10 µM concentration on mRNA levels of
Ptch1 (A) and Gli2 (B) in Shh-LIGHT2 cells activated with 100nM SAG. Treatments were
performed in triplicate. * P < .05 compared to SAG controls.
Experimental section
Biology
Cell culture and stock solutions
As illustrated in Figure 5 both Ptch1 and Gli2 mRNA levels
were significantly suppressed by compounds 5, 23, and 24 at
Stock solutions were prepared as follows and stored at -
20ºC: Related compounds were stored as 40 mM solutions in
DMSO. All cell lines were cultured at 37ºC in an automated
CO2 (5%) incubator (HERA cell 150, Thermo Scientific).
HT29, SW480 (colon carcinomas), MCF-7 (breast
carcinoma), A2780 (ovarian carcinoma), H460 (lung
carcinoma), A431 (skin carcinoma), DU145 (prostate
carcinoma), BEC-2 (neuroblastoma), SJ-G2 (glioblastoma) and
MIA (pancreatic carcinoma) cell lines were maintained in
Dulbecco’s modified Eagle’s medium (Trace Biosciences,
Australia) supplemented with 10% foetal bovine serum, 10
mM sodium bicarbonate, penicillin (100 IU/mL), streptomycin
(100 mg/mL), and glutamine (4 mM).
10µM treatments. To clarify the data presented in Figure 5 the
percent inhibition of Ptch1 and Gli2 mRNA levels were
calculated (Table 5), in which the inhibition of mRNA levels of
Gli2 appeared to be larger than 100%. This may arise as a
result of the compounds not only suppressing the elevated
mRNA levels of Gli2 induced by SAG, but also inhibition of Gli2
in inactivated Shh LIGHT2 cells. Together, these results
indicate that compounds 5, 23, and 24 exhibited suppressive
activity over the HSP in Shh LIGHT2 through the inhibition of
Ptch1 at mRNA level and Gli2 at both mRNA and protein levels.
Table 5. Evaluation of compounds 5, 23, and 24 (10 µM) on Ptch1 and Gli2 mRNA
levels in SAG-activated Shh-LIGHT 2 cells. Values are the approximate percentage
reduction relative to the DMSO and SAG-treated controls.
TCAM-2 cell line (testis carcinoma) was maintained in
Hyclone RPMI 1640 medium (GE Healthcare Life Sciences)
supplemented with 10% foetal bovine serum (Gibco®),
penicillin (100 IU/mL) (Gibco®), streptomycin (100 mg/mL)
(Gibco®) and glutamine (4 mM) (Gibco®).
Percent change in Ptch1 and Gli2 mRNA levels (%)
Compound
Ptch1
Gli2
112
5
57
67
55
23
24
117
112
Shh LIGHT2 cell line (derived from NIH-3T3 fibroblast cell
line) was maintained in Gibco® Dulbecco’s modified Eagle’s
medium (Thermo Fisher Scientific) supplemented with 10%
foetal bovine serum (FBS), glutamine (4mM), Zeocin®
(0.15mg/mL, Invitrogen), Genetecin® (0.4mg/mL, Thermo
Fisher Scientific).
Conclusions
We have successfully identified a new scaffold of HSP
inhibitors derived from the Ugi-Knoevenagel products. At
inhibitor concentration of 10 µM, these quinolone-2-(1H)-one
analogues can effectively inhibit the mRNA levels of Ptch1 and
Gli2 in Sonic Hedgehog LIGHT2 cell line stimulated with 100nM
SAG. Of note, selected compounds demonstrated good
cytotoxicity (GI50 from 2.9 to 18.0 µM) against a panel of eight
human cancer cell lines, as well as the mutant seminoma
TCAM-2 cell line, all of which are known to possess the HSP’s
components (Table 3). Whilst the exact mechanism remains to
be determined, our data is consistent with inhibition
downstream of Smo due to the fact that it is valid in the
presence of SAG, a potent Smo activator. Moreover, the
analogues reported herein suppress Gli2 mRNA level in non-
activated Shh LIGHT 2 cells also supports a downstream of
Smo inhibition. Inhibition of Smo does not display this
phenotype. Furthermore, a preliminary quinolone-2-(1H)-one
In vitro growth inhibition assay
Protocol 1 (HT29, SW480, MCF-7, A2780, H460, DU145,
BEC-2 and MIA cell lines)
Cells in logarithmic growth were transferred to 96-well
plates. Cytotoxicity was determined by plating cells in
duplicate in 100 µL medium at a density of 2500-4000
cells/well. On day 0, (24 h after plating) when the cells were in
logarithmic growth, 100 µL medium with or without the test
agent was added to each well. After 72 h drug exposure
growth inhibitory effects were evaluated using the MTT (3-
[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide)
assay and absorbance read at 540 nm. Percentage growth
inhibition was determined at a fixed drug concentration of 25
µM. A value of 100% is indicative of total cell growth
inhibition. Those analogues showing appreciable percentage
growth inhibition underwent further dose response analysis
allowing for the calculation of a GI50 value. This value is the
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drug concentration at which cell growth is 50% inhibited carried out for 45 amplification cycles. SYBR® Green
based on the difference between the optical density values on fluorescence was measured after the extension step at the
day 0 and those at the end of drug exposure.
end of each amplification cycle and quantified using
LightCycler Analysis Software (Roche). For each sample, a
replicate omitting the reverse transcription step was
Protocol 2 (TCAM-2 cell line)
Cells in logarithmic growth were transferred to 96-well undertaken as a negative control. qPCR data was normalized
plates in triplicates at 2500 cells/well in 200µL media and to the house-keeping control Cyclophilin. Experiments were
cultured in the automated CO2 (5%) incubator. When the cells replicated at least 3 times prior to statistical assessment. Each
reach to about 80% confluency, old media were removed and PCR was performed on at least 3 separate cell isolations, of
replaced with 100 µL fresh media containing testing agents (at which a representative PCR or an average is shown (ESI†).
10 µM), as well as DMSO and 1% Triton X as controls. Cells
were further incubated for another 72 h and were evaluated
using the MTT assay with the absorbance at 550 nm. The
growth inhibition was calculated based on the differences in
the optical densities between those treated by various agents
(10 µM) and controls by DMSO and 1% Triton X treatments.
Only those agents which expressed a growth inhibition greater
than 60% were further subjected to full dose response
evaluation (GI50 values).
Table 6. Primer sequences used in qPCR assay.
Human gene
Forward
Reverse Sequence Annealing
Sequence (5'-3')
ATCTCTTGCCACC
ATTCCAT
(5'-3')
Temp (ºC)
60
Gli2
GGACAGAATGAG
GCTCGTAA
SMO
SuFu
Ptch1
CTGCCACTTCTAC
GACTTCT
GGCCTGACATAGC 56
ACATAGT
GACCCCTTGGACT
ATGTTAG
CTGATGTAGTGCC
AGTGCTC
55
Dual Luciferase Reporter assay
CCCTCACGTCCAT
CAGCAAT
AACACCACTACTA
CCGCTGC
58
Shh-LIGHT2 cells in logarithmic growth were transferred to
96-well plate (3000 cells/well) and cultured to confluency. The
Shh-LIGHT2 cells were then grown in DMEM containing 0.5%
FBS, 4 mM glutamine, 0.15 mg/mL Zeocin®, 0.4 mg/mL
Genetecin®, and combinations of 100 nM SAG (Smo agonist),
with different testing compounds (5, 23, and 24) at 25 µM
each. The SAG- free DMSO treated (25 µM), and SAG-included
Sonidegib (100nM) treated cells were used as controls.
Treatments were done in triplicates. After the cells were
cultured for another 45 h in the automated CO2 (5%)
incubator, the resulting firefly and Renilla luciferase activities
were measured using a Dual Luciferase Reporter kit (Promega)
and a BMG Labtech Pherastar microplate reader (Thermo
Fisher Scientific).
Mouse gene
Gli2
TCCAGTCAATGGT
TCTGTCC
TGGCTCAGCATCG
TCACTTC
60
Gli3
GGCCGTTACCATT
ATGATCC
CTGAGGCTGCAGT 60
GGGATTA
Shh
TGCTTTGTAACCG
CCACTTT
CGCTGCTAGGTGC
ACTTTTA
61
60
55
SMO
SuFu
Ptch1
GAACTCCAATCGC
TACCCTG
ATCTGCTCGGCAA
ACAATCT
GACCCCTTGGACT
ATGTTAG
CTGATGTAGTGCC
AGTGCTC
CATAGCTGCCCAG
TTCAAGT
GGTCGTAAAGTAG 55
GTGCTGG
Statistical analysis
RNA Extraction
Total RNA was isolated from cultured cells using two
rounds of a modified acid guanidinium thiocyanate-phenol-
chloroform protocol:67 washed cells resuspended in lysis
buffer (4 M guanidinium thiocyanate, 25 mM sodium citrate,
0.5% sarkosyl, 0.72% β-mercaptoethanol). RNA was isolated
Statistical analysis was performed using F-test and t-test in Excel
2013. * P < .05, ** P < .001, *** P < .0001.
Chemistry
All reagents were purchased from Sigma-Aldrich, Matrix
Scientific or Lancaster Synthesis and were used without
purification. All solvents were re-distilled from glass prior to
use.
by
phenol/chloroform
extraction
and
isopropanol
precipitated.
Reverse Transcription PCR (RT-PCR) and Quantitative PCR (qPCR)
1H and 13C NMR spectra were recorded on a Brüker
Advance™ AMX 400 MHz spectrometer at 400.13 and 100.62
MHz, respectively. Chemical shifts (δ) are reported in parts per
million (ppm) measured to relative the internal standards.
Coupling constants (J) are expressed in hertz (Hz). Mass
spectra were recorded on a Shimadzu LCMS 2010 EV using a
mobile phase of 1 : 1 acetonitrile–H2O with 0.1% formic acid.
High resolution mass spectra (HRMS) were determined using
nanoflow reversed phased Liquid Chromatography (Dionex
Ultimate 3000 RSLCnano, Thermo Fischer Scientific) coupled
directly to a High Resolution mode equipped, Q-Exactive Plus
Reverse transcription was performed with 2 μg of isolated
RNA, 500 ng oligo(dT)15 primer, 40 U of RNasin, 0.5 mM
dNTPs, and 20 U of M-MLV-Reverse Transcriptase (Promega).
Total RNA was DNase treated prior to reverse transcription to
remove genomic DNA. Reverse transcription reactions were
verified by β-actin RT-PCR using cDNA amplified with GoTaq
Flexi (Promega). qPCR was performed using SYBR Green
GoTaq qPCR master mix (Promega) according to
manufacturer's instructions on LightCycler 96 SW 1.0 (Roche).
Primer sequences have been supplied (Table 6). Reactions
were performed on cDNA equivalent to 50 ng of total RNA and
This journal is © The Royal Society of Chemistry 20xx
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DOI: 10.1039/C6OB00606J
ARTICLE
Journal Name
Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo chromatography (1:4 hexanes–EtOAc) to afford
Fischer Scientific). This system was fitted with 5µm C18 as an off white solid (mp 243-245°C).
4 (70 mg, 13%)
nanoViper trap column (100um x 2cm, Acclaim PepMap100,
IR (cm-1): 3246 (NH), 3083 (CH), 2972 (CH), 2229 (CN), 1637
Thermo) for desalting and pre-concentration, and separation (CO); The 1H NMR displays a mixture of isomers, with the ratio
1
was then performed at 300nl/min over an EASY-Spray PepMap 1.35 : 1.0 calculated at 0.74 and 0.60 ppm, respectively. H is
column (3um C18, 75um x 15cm) utilising a gradient of 2-99% reported as a whole without splitting due to the complex
overlapping. All peaks detected in 13C are reported. 1H NMR
Buffer B (80% Acetonitrile, 0.1% Formic Acid) over 25 minutes.
Analytical HPLC traces were obtained using a Shimadzu (400 MHz, DMSO-d6) δ 7.91 (d, J = 8.2 Hz, 1H), 7.83 – 7.69 (m,
system possessing a SIL-20A auto-sampler, dual LC-20AP 2H), 7.67 – 7.51 (m, 2H), 7.47 – 7.35 (m, 3H), 7.29 (dd, J = 9.8,
pumps, CBM-20A bus module, CTO-20A column heater, and a 5.4 Hz, 1H), 7.13 (t, J = 7.6 Hz, 1H), 7.01 (t, J = 7.4 Hz, 1H),
SPD-20A UV/vis detector. This system was fitted with an 3.98-3.86 (m, 1H), 3.75 (s, 3H), 2.75 (d, J = 3.2 Hz, 3H), 1.54 –
Alltima™ C18 5 µm 150 mm × 4.6 mm column with solvent A: 1.15 (m, 4H), 0.93-0.87 (m, 3H), 0.77-0.56 (m, 2H); 13C NMR
0.06% Trifluoroacetic acid (TFA) in water and solvent B: 0.06% (101 MHz, DMSO-d6) δ 167.4, 166.8, 159.2, 159.2, 158.3,
TFA in CH3CN–H2O (90 : 10). In each case HPLC traces were 158.3, 139.1, 136.6, 136.5, 133.3, 133.2, 130.9, 130.81, 127.7,
acquired at a flow rate of 2.0 mL min-1, gradient 10–100 (%B), 127.6, 127.6, 123.4, 121.9, 120.1, 120.1, 119. 8, 118.9, 118.1,
over 15.0 min, with detection at 220 nm and 254 nm.
118.1, 116.2, 110.4, 107.7, 106.2, 106.1, 106.1, 60.2, 53.8,
Melting points were recorded on a Büchi Melting Point M- 53.7, 52.9, 45.3, 45.2, 38.3, 38.0, 33.0 (Cx2), 27.4, 26.8, 21.2,
565. IR spectra were recorded on a PerkinElmer Spectrum 21.1, 20.8, 19.6, 19.1, 18.8, 14.6, 14.3, 14.2, 11.2, 10.8; LRMS
Two™ FTIR Spectrometer with the UATR accessories. Thin (ESI-) m/z 440, 520 [M+DMSO+2H]+ 100%. HRMS (ES+) for
layer chromatography (TLC) was performed on Merck 60 F254 C27H28N4O2Na; calculated 463.2110, found 463.2104; RP-HPLC
pre-coated aluminium plates with a thickness of 0.2 mm. Alltima™ C18 5 µm 150 mm x 4.6 mm, 10–100% B in 15 min, Rt
Column chromatography was performed under ‘flash’ min = 7.07, 93 %.
conditions on Merck silica gel 60 (230–400 mesh).
2-(3-Cyano-2-oxo-4-phenylquinolin-1(2H)-yl)-2-(1H-indol-3-yl)-N-
(pentan-2-yl)acetamide (21)
Experimental data
Synthesized utilizing the general procedure described
Compounds
5 and 10-19 were prepared as described in ref
above, from 2-aminobenzophenone (0.252 g, 1.28 mmol),
indole-3-carboxaldehyde (0.186g, 1.28 mmol), cyanoacetic
acid (0.109 g, 1.28 mmol) and 2-pentylisocyanide (0.158 mL,
48. The relative configuration for the obtained products was
assigned computationally as follow: Each of the initial
geometry of each analogue (14-16) was built using the
1.28 mmol) in MeOH (5.0 mL) to afford
off white solid (mp 182–183 ºC).
6 (0.07 g, 11%) as an
molecular builder of Molecular Operating Environment (MOE).
Each molecule was relaxed using the semi-empirical AM1
method in MOE with a root mean square (rms) gradient of
0.01. Each analogue was subjected to conformational analysis
using Stochastic Conformational Search method. The most
stable conformation for each analogue was retained and
saved as a mol2 file format. Each conformer was subjected to
geometry optimization at DFT level of theory using B3LYP
function with the 6-31+G(d,p) basis set. At this stage the
DFT energy was calculated. The optimized structures were
used for the calculation of NMR chemical shifts (relative to
TMS) using the GIAO (gauge-independent (or including)
atomic orbitals) method and the B3LYP functional with the
6-311+G(2d,p) basis set. The calculated DFT energy and
1HNMR chemical shifts for selected peaks were used for
assigning the configuration for the major and minor
isomeric products (ESI, Table S1).
IR (cm-1): 3420 (NH), 2229 (CN), 1678 (CONH), 1646 (CON);
The 1H NMR displays a mixture of isomers, with the ratio 5.5 :
1.0 calculated at 3.96 and 3.72 ppm, respectively. 1H is
reported as a whole without splitting due to the complex
1
overlapping. All peaks detected in 13C are reported H NMR
(400 MHz, DMSO-d6) δ 11.26 (s, 1H), 7.85 (s, 2H), 7.73 – 7.32
(m, 10H), 7.29 – 6.87 (m, 4H), 3.96 (s, 1H), 1.84 – 0.09 (m,
11H); 13C NMR (101 MHz, DMSO) δ 167.4, 166.8, 160.1, 159.3,
140.1, 136.2, 136.1, 134.1, 133.3, 130.4, 129.3, 129.2, 129.1,
127.4, 127.3, 127.0, 126.9, 123.5, 122.0, 119.9, 119.8, 118.8,
118.6, 116.0, 112.2, 108.5, 106.0, 54.3, 54.2, 53.0, 45.4, 45.3,
38.4, 38.2, 27.4, 26.9, 21.1, 20.9, 19.6, 19.2, 14.4, 14.2, 11.3,
10.8; LRMS (ESI+) m/z 488, 489 [M+H]+, 40%. HRMS (ES+) for
C31H28N4O2; calculated 489.2285, found 489.2284; RP-HPLC
Phenomenex Onyx™ Monolithic C18 5 µm 100 mm x 4 mm,
10–100% B in 15 min, Rt min = 12.24, 100 %.
2-(3-Cyano-2-oxo-4-methylquinolin-1(2H)-yl)-2-(1-methyl-1H-indol-
2-(3-Cyano-2-oxo-4-phenylquinolin-1(2H)-yl)-2-(1H-indol-5-yl)-N-
3-yl)-N-(pentan-2-yl)acetamide (20)
(pentan-2-yl)acetamide (22)
General procedure: A solution of MeOH (5.0 mL), 2-
aminoacetophenone (0.148 mL, 1.23 mmol) and 1-methyl-1H-
indole-3-carboxaldehyde (0.196 g, 1.23 mmol) was stirred at
room temperature for 0.5 h. To the stirred solution was added
cyanoacetic acid (0.105 g, 1.23 mmol) followed by the addition
of 2-pentylisocyanide (0.152 mL, 1.23 mmol). The reaction
mixture was stirred at room temperature for 24 h and the
crude material was subjected to silica gel column
Synthesized utilizing the general procedure described
above, from 2-aminobenzophenone (0.267 g, 1.35 mmol),
indole-5-carboxaldehyde (0.197g, 1.35 mmol), cyanoacetic
acid (0.115 g, 1.35 mmol) and 2-pentylisocyanide (0.167 mL,
1.35 mmol) in MeOH (5.0 mL) to afford
off white solid (mp 271–272 ºC).
6 (0.238 g, 36%) as an
IR (cm-1): 3403 (NH), 3338 (NH), 2956 (CH), 2235(CN), 1647
(CO); The 1H NMR displays a mixture of isomers, with the ratio
8 | J. Name., 2012, 00, 1-3
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ARTICLE
1
2.45 : 1.0 calculated at 0.74 and 0.64 ppm, respectively. H is 8H), 7.32 – 7.17 (m, 4H), 6.92 (d, J = 8.3 Hz, 1H), 4.14 (q, J = 7.1
reported as a whole without splitting due to the complex Hz, 2H), 4.02-3.84 (m, 2H), 2.34 (s, 3H), 1.22 (t, J = 7.1 Hz, 3H);
overlapping. All peaks detected in 13C are reported 1H NMR 13C NMR (101 MHz, DMSO-d6) δ 170.2, 168.4, 160.3, 159.3,
(400 MHz, DMSO-d6) δ 11.13 (s, 1H), 7.89 (dd, J = 14.3, 8.1 Hz, 139.7, 134.5, 134.1, 133.5, 130.5, 129.4, 129.4, 129.4, 129.1,
1H), 7.71 – 7.52 (m, 7H), 7.52 – 7.44 (m, 1H), 7.41 – 7.29 (m, 129.0, 128.3, 127.5, 127.1, 123.7 (Cx2), 119.9, 118.5, 118.2,
2H), 7.26 – 7.01 (m, 4H), 6.40 (d, J = 1.8 Hz, 1H), 3.91 (dd, J = 115.8, 111.9, 107.3, 105.8, 61.0, 53.8, 41.9, 21.9, 14.6; LRMS
13.4, 7.0 Hz, 1H), 1.59 – 1.19 (m, 3H), 1.16 – 0.99 (m, 2H), 0.99 (ESI+) m/z518, 541 [M+Na-H]+ 60%. HRMS (ES+) for
– 0.84 (m, 3H), 0.81-0.55 (m, 2H);
C31H26N4O4; calculated 519.2027, found 519.2026; RP-HPLC
13C NMR (101 MHz, DMSO-d6) δ 167.5, 167.0, 166.9, 160.1, Alltima™ C18 5 µm 150 mm x 4.6 mm, 10–100% B in 15 min, Rt
160.0, 159.4, 140.6, 135.6, 134.2, 134.2, 133.1, 133.0, 130.4, min = 13.72, >97%.
129.3 (Cx2), 129.2 (Cx2), 129.1, 129.1, 128.0, 126.5, 126.5,
Ethyl-[2-(3-Cyano-2-oxo-4-phenyl-2H-quinolin-1-yl)-2-(1H-
125.8, 125.7, 125.6, 123.5, 121.8, 120.1, 120.1, 120.0, 119.1,
indol-3-yl)-acetamido]-acetate (25
)
119.0, 116.0, 116.0, 111.9, 111.9, 106.3, 106.3, 106.2, 101.8,
101.7, 61.3, 61.1, 61.1, 52.8, 45.3, 45.2, 38.3, 38.2, 27.2, 26.8,
21.2, 20.9, 19.5, 19.0, 14.4, 14.3, 11.1, 10.6; LRMS (ESI-) m/z -
488, 520 [M+CH3OH-H] 95%. HRMS (ES+) for C31H28N4O2;
calculated 489.2285, found 489.2284.
Synthesized utilizing the general procedure described
above, from 2-aminobenzophenone (0.366 g, 1.86 mmol), 1H-
indole carbaldehyde (0.269 g, 1.86 mmol), cyanoacetic acid
(0.157 g, 1.86 mmol), and ethyl isocyanoacetate (0.202 mL,
1.86 mmol) to afford 25 (0.30 g, 46%) as an off white solid (mp
179.3-180.5 °C).
IR (cm-1): 3420 (NH), 2236 (CN), 1737 (COO), 1686 (CONH),
1646 (CON); 1H NMR (400 MHz, DMSO-d6) δ 11.35 (s, 1H),
8.58 (s, 1H), 7.93 – 7.75 (m, 2H), 7.75-7.45 (m, 8H), 7.39 (d, J =
RP-HPLC Alltima™ C18 5 µm 150 mm x 4.6 mm, 10–100% B
in 15 min, Rt min = 7.07, >98 %.
2-(3-Cyano-2-oxo-4-phenylquinolin-1(2H)-yl)-2-(5-methyl-1H-
indole-3-yl)-N-(pentan-2-yl) acetamide (23
)
Synthesized utilizing the general procedure described 8.0 Hz, 1H), 7.21 (d, J = 3.7 Hz, 2H), 7.15-6.91 (m, 2H), 4.25 –
above, from 2-aminobenzophenone (0.378 g, 1.92 mmol), 5- 4.06 (m, 2H), 4.04-3.80 (m, 2H), 1.22 (t, J = 7.0 Hz, 3H); 13C
methyl-1H-indole carbaldehyde (0.305 g, 1.92 mmol), NMR (101 MHz, DMSO-d6) δ 170.2, 168.4, 160.3, 159.3, 139.7,
cyanoacetic acid (0.163 g, 1.92 mmol), and 2-pentylisocyanide 136.1, 134.0, 133.5, 130.5, 129.5, 129.4 (Cx2), 129.3 (Cx2),
(0.237 mL, 1.92 mmol) to afford 23 (0.445 g, 46%) as an off 129.2, 129.0, 127.3, 123.7, 122.1, 120.0 (Cx2), 118.6, 118.5,
white solid (mp 178–180 ºC).
115.9, 112.2, 107.8, 105.8, 61.1, 53.7 41.9, 14.6; LRMS (ESI+)
IR (cm-1): 3427 (br NH), 2962(CH), 2236 (CN), 1645(CON); m/z 504, 505 [M+H]+, 100%. HRMS (ES+) for C30H24N4O4;
The 1H NMR displays a mixture of isomers, with the ratio 2.1 : calculated 505.1870, found 505.1869; RP-HPLC Phenomenex
1.0 calculated at 0.77 and 0.68 ppm, respectively. 1H is Onyx™ Monolithic C18 5 µm 100 mm x 4 mm, 10–100% B in 15
reported as a whole without splitting due to the complex min, Rt min = 11.09, 100%.
overlapping. All peaks detected in 13C are reported H NMR
1
Ethyl-[2-(3-Cyano-2-oxo-4-phenyl-2H-quinolin-1-yl)-2-(1-
(400 MHz, DMSO-d6) δ 11.13 (d, J = 4.9 Hz, 1H), 7.90 – 7.37 (m,
methylindole-3-yl)-acetamido]-acetate (26
)
10H), 7.29-7.16 (m, 4H), 6.92 (d, J = 8.3 Hz, 1H), 4.03 – 3.87 (m,
1H), 2.34 (s, 3H), 1.57 – 1.20 (m, 3H), 1.20 – 0.86 (m, 5H), 0.82-
0.60 (m, 3H); 13C NMR (101 MHz, DMSO-d6) δ 167.5, 166.9,
160.1, 160.1, 159.3, 140.1, 140.1, 134.6, 134.6, 134.5, 134.1,
133.3, 130.4, 129.4, 129.2, 129.1, 128.2, 128.1, 127.5, 127.5,
126.8, 126.6, 123.5, 119.9, 118.5, 118.4, 118.3, 116.0, 111.9,
107.9, 107.9, 106.0, 105.9, 54.5, 54.4, 52.9, 45.4, 45.2, 38.4,
38.2, 27.3, 26.9, 21.9, 21.1, 20.9, 19.6, 19.2, 14.4, 14.2, 11.2,
10.8; LRMS (ESI-) m/z 502, 521 [M+NH4]+ 40%. HRMS (ES+) for
C32H30N4O2; calculated 503.2442, found 503.2444; RP-HPLC
Alltima™ C18 5 µm 150 mm x 4.6 mm, 10–100% B in 15 min, Rt
min = 10.89, 100%.
Synthesized utilizing the general procedure described
above, from 2-aminobenzophenone (0.281 g, 1.43 mmol), 1-
methyl-indole-3-carboxaldehyde (0.227 g, 1.43 mmol),
cyanoacetic acid (0.121 g, 1.43 mmol) and ethyl
isocyanoacetate (0.156 mL, 1.43 mmol) in MeOH (5.0 mL). The
crude material was subjected to silica gel column
chromatography (1:1 hexanes–EtOAc) to afford 26 (0.192 g,
26%) as an off white solid (mp 209-211°C).
IR (cm-1): 3422 (NH), 2920 (CH), 2229 (CN), 1743 (COO),
1639 (CON); 1H NMR (400 MHz, DMSO-d6) δ 8.53 (bs, 1H), 7.85
(d, J = 8.8 Hz, 1H), 7.75 (s, 1H), 7.70 – 7.48 (m, 7H), 7.43 (d, J =
8.2 Hz, 1H), 7.25 – 7.19 (m, 2H), 7.17 (t, J = 7.2 Hz, 1H), 7.06 (t,
J = 7.2 Hz, 1H),, 4.12 (q, J = 7.1 Hz, 2H), 3.90 (d, J = 6.6 Hz, 2H),
3.79 (s, 3H), 1.20 (t, J = 7.1 Hz, 3H); 13C NMR (101 MHz, DMSO-
Ethyl-[2-(3-Cyano-2-oxo-4-phenyl-2H-quinolin-1-yl)-2-(5-
methyl-1H-indol-3-yl)-acetamido]-acetate (24
)
Synthesized utilizing the general procedure described d6) δ 170.1, 168.3, 160.3, 159.2, 139.6, 136.5, 134.0, 133.7,
above, from 2-aminobenzophenone (0.390 g, 1.98 mmol), 5- 131.4, 130.5, 129.5, 129.4 (Cx2), 129.2, 129.0, 127.7, 123.7,
methyl-indole-3-carboxaldehyde (0.315g, 1.98 mmol), 122.1, 120.1, 120.0, 118.9, 118.2, 115.9, 110.5, 106.8, 105.9,
cyanoacetic acid (0.168 g, 1.98 mmol) and ethyl 105.9, 61.0, 41.9, 33.2, 14.6; LRMS (ESI-) m/z 518, 540 [M+ Na-
isocyanoacetate (0.216 mL, 1.98 mmol) in MeOH (5.0 mL) to H]+, 100%. HRMS (ES+) for C31H26N4O4 ; calculated 519.2027,
afford
9
(0.347 g, 34%) as a greenish solid (mp 199-200 °C).
found 519.2027; RP-HPLC Alltima™ C18 5 µm 150 mm x 4.6
IR (cm-1): 3423 (NH), 3410 (NH), 2232 (CN), 1731 (COO), mm, 10–100% B in 15 min, Rt min = 14.26, >98%.
1
1673 (CON); H NMR (400 MHz, DMSO-d6) δ 11.21 (d, J = 1.8
Hz, 1H), 8.53 (s, 1H), 7.83 (d, J = 8.7 Hz, 1H), 7.72 – 7.48 (m,
This journal is © The Royal Society of Chemistry 20xx
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DOI: 10.1039/C6OB00606J
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Journal Name
Ethyl-3-[2-(3-cyano-2-oxo-4-phenyl-2H-quinolin-1-yl)-2-(1-
8.3 Hz, 1H), 4.03 – 3.87 (m, 1H), 2.34 (s, 3H), 1.57 – 1.20 (m,
3H), 1.20 –0.86 (m, 5H), 0.82-0.60 (m, 3H); 13C NMR (101 MHz,
methyl-1H-indol-3-yl)-acetylamino]-propionate (27
)
Synthesized utilizing the general procedure described DMSO) (Isomeric mixture) δ 167.5, 166.9, 160.1, 160.1, 159.3,
above, from 2-aminobenzophenone (0.186 g, 0.94 mmol), 1- 140.1, 140.1, 134.6, 134.6, 134.5, 134.1, 133.3, 130.4, 129.4,
methyl-indole-3-carboxaldehyde
(0.15g,
0.94
mmol), 129.2, 129.1, 128.2, 128.1, 127.5, 127.5, 126.8, 126.6, 23.5,
cyanoacetic acid (0.08 g, 0.94 mmol) and ethyl 19.9, 118.48, 118.4, 118.3, 116.0, 111.9, 107.9, 107.9, 106.0,
isocyanopropionate (0.12 mL, 0.94 mmol) in MeOH (5.0 mL) to 105.9, 54.5, 54.4, 52.9, 45.4, 45.2, 38.4, 38.2, 27.3, 26.9, 21.9,
afford 27 (0.149 g, 50%) as a white solid (mp 267-268°C).
IR (cm-1): 3410 (NH), 2232 (CN), 1725 (COO), 1686 (CON); 488, 243 [M-2H]2+ , 90%. HRMS for C31H28N4O2; calculated
1H NMR (400 MHz, DMSO-d6) δ 8.05 (bs, 1H), 7.83 (d, J = 8.7 489.2285, found 489.2283
RP-HPLC Alltima™ C18 5µµm 150
21.1, 20.9, 19.6, 19.2, 14.4, 14.2, 11.2, 10.8; LRMS (ESI-) m/z
;
Hz, 1H), 7.71 – 7.56 (m, 6H), 7.56 – 7.47 (m, 2H), 7.45-7.38 (m, mm x 4.6 mm, 10–100% B in 15 min, Rt min = 14.59, >95%.
2H), 7.26 – 7.20 (m, 2H), 7.17 (t, J = 7.2 Hz, 1H), 7.06 (t, J = 7.2
N-tert-Butyl-2-(3-cyano-2-oxo-4-phenyl-2H-quinolin-1-yl)-2-(1-
Hz, 1H), 4.03 (q, J = 7.1 Hz, 2H), 3.78 (s, 3H), 3.42 – 3.35 (m,
methyl-1H-indole-3-yl)-acetamide (30
)
2H), 2.57-2.44 (m, 2H), 1.16 (t, J = 7.1 Hz, 3H); 13C NMR (101
MHz, DMSO-d6) δ 171.7, 167.7, 160.1, 159.0, 139.9, 136.5,
134.1, 133.8, 131.3, 130.5, 129.6, 129.4, 129.2, 129.0, 127.8,
123. 7, 122.1, 120.0, 119.9, 119.0, 117.8, 115.9, 110. 5, 107.1,
106.1, 60.4, 54.1, 35.9, 34.0, 33.1, 14.5; LRMS (ESI+) m/z 532,
287 [M+ACN+ 2H]2+ 100%. HRMS (ES+) for C16H11N2O+ (main
fragment); calculated 247.087, found 247.0865; RP-HPLC
Alltima™ C18 5 µm 150 mm x 4.6 mm, 10–100% B in 15 min, Rt
min = 14.46, >95%.
Synthesized utilizing the general procedure described
above, from 2-aminobenzophenone (0.311 g, 1.58 mmol), 1-
methyl-indole-3-carboxaldehyde (0.251g, 1.58 mmol),
cyanoacetic acid (0.134 g, 1.58 mmol) and tert-butyl
isocyanide (0.178 mL, 1.58 mmol) in MeOH (5.0 mL) to afford
29 (0.200 g, 26%) as a white solid (mp 232-234°C).
IR (cm-1): 3357 (NH), 2979 (CH), 2229 (CN), 1650 (CO); H
1
NMR (400 MHz, DMSO) δ 7.89 (d, J = 8.8 Hz, 1H), 7.68 – 7.46
(m, 9H), 7.43 (d, J = 7.8 Hz, 2H), 7.22-7.13 (m, 3H), 7.06 (t, J =
Ethyl-2-(2-(5-chloro- indole (1H)-3-yl)-2-(3-cyano-2-oxo-4- 7.4 Hz, 1H), 3.79 (s, 3H), 1.32 (s, 9H); 13C NMR (101MHz,
phenyl-1(2H)-quinolin-yl)-acetamido)-acetate (28
)
DMSO) δ 166.66, 160.13, 159.17, 140.28, 136.75, 134.09,
Synthesized utilizing the general procedure described 133.34, 130.54, 130.42, 129.31 (Cx3), 129.15 (Cx2), 127.48,
above, from 2-aminobenzophenone (0.478 g, 2.4 mmol), 5- 123.52, 122.17, 119.99, 119.78, 119.04, 118.55, 115.91,
chloro-indole-3-carboxaldehyde
(0.434g,
2.4
mmol), 110.53, 108.04, 105.89, 54.94, 51.57, 33.09, 28.83 (Cx3); LRMS
cyanoacetic acid (0.204 g, 2.4 mmol) and ethyl (ESI+) m/z 488, 243 [M-2H]2+, 100%. HRMS (ES+) for
isocyanoacetate (0.271 mL, 2.4 mmol) in MeOH (5.0 mL) to C31H28N4O2; calculated 489.2285, found 489.2287; RP-HPLC
afford 28 (0.435 g, 33%) as a yellowish precipitate (mp 201– Alltima™ C18 5µm 150 mm x 4.6 mm, 10–100% B in 15 min, Rt
203°C).
IR (cm-1): 3415 (NH), 3406 (NH), 2236(CN), 1736(COO),
min =7.03, 96%
More information on the synthesis and characterization of
1671(CON); 1H NMR (400 MHz, DMSO-d6) (Isomeric mixture) δ the analogues can be found in the ESI.
11.54 (d, J = 1.4 Hz, 1H), 8.53 (s, 1H), 7.82 (dd, J = 11.9, 5.5 Hz,
Acknowledgements
2H), 7.71 – 7.61 (m, 4H), 7.61-7.5 (m, 4H), 7.42 (d, J = 8.6 Hz,
1H), 7.29 – 7.18 (m, 2H), 7.12 (dd, J = 8.6, 1.7 Hz, 1H), 4.14 (q, J
The authors thank the NHMRC (Australia) for project
= 7.0 Hz, 2H), 3.93 (qd, J = 17.2, 5.8 Hz, 2H), 1.22 (t, J = 7.1 Hz, support, Nathan Druery Smith (Analytical and Biomolecular
3H); 13C NMR (101 MHz, DMSO-d6) δ 170.1, 168.2, 160.4, Research Facility at the University of Newcastle) for the HRMS
159.2, 139.6, 134.6, 134.0, 133.7, 130.5, 129.6(Cx2), 129.4, data, James K. Chen for helpful discussions and T.N.T. is the
129.2, 129.0 (Cx2), 128.5, 124.5, 123.8, 122.0, 120.1, 118.3 recipient of a Prime Minister's Australia Asia Postgraduate
(Cx2), 118.2, 115.8, 113.8, 107.8, 106.0, 61.0, 42.0, 14.6; LRMS Endeavour Award.
(ESI+) m/z 538, 292 [M+2Na]2+, 60%. HRMS for C30H23ClN4O4;
Notes and References
calculated 539.1481, found 539.1481; RP-HPLC Alltima™ C18 5
1
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2
3
4
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N-tert-Butyl-2-(3-cyano-2-oxo-4-phenyl-2H-quinolin-1-yl)-2-(5-
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methyl-1H-indol-3-yl)-acetamide (29
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5
6
7
1
IR (cm-1): 3427(NH), 2978 (CH), 2228 (CN), 1650 (CON); H
NMR (400 MHz, DMSO-d6) (Isomeric mixture) δ 11.13 (d, J =
4.9 Hz, 1H), 7.90 – 7.37 (m, 10H), 7.29-7.16 (m, 4H), 6.92 (d, J =
10 | J. Name., 2012, 00, 1-3
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