Dichlorophenylacrylonitriles as AhR Ligands That Display Selective Breast Cancer Cytotoxicity in vitro

Article abstract of DOI:10.1002/cmdc.201800256

Knoevenagel condensation of 3,4-dichloro- and 2,6-dichlorophenylacetonitriles gave a library of dichlorophenylacrylonitriles. Our leads (Z)-2-(3,4-dichlorophenyl)-3-(1H-pyrrol-2-yl)acrylonitrile (5) and (Z)-2-(3,4-dichlorophenyl)-3-(4-nitrophenyl)acryloni

Full text of DOI:10.1002/cmdc.201800256

Accepted Article
Title: Dichlorophenylacrylonitriles as AhR Ligands displaying selective
breast cancer cytotoxicity in vitro
Authors: Jennifer R Baker, Jayne Gilbert, Stefan Paula, Xiao Zhu,
Jennette A Sakoff, and Adam McCluskey
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Dichlorophenylacrylonitriles as AhR Ligands displaying selective breast
cancer cytotoxicity in vitro
Jennifer R Baker,[a] Jayne Gilbert,[b] Stefan Paula,[c] Xiao Zhu, [d] Jennette A Sakoff [b] and Adam
McCluskey *[a]
[a]
Ms JR Baker (0000-0002-9560-301X), Prof A McCluskey (0000-0001-7125-863X)
Chemistry, School of Environmental & Life Sciences
The University of Newcastle
University Drive, Callaghan NSW 2308 Australia
E-mail: Adam.McCluskey@newcastle.edu.au
[b]
Dr J Gilbert (0000-0001-5034-386X), Dr JA Sakoff (0000-000207009-5792)
Experimental Therapeutics Group, Department of Medical Oncology
Calvary Mater Hospital
Edith Street, Waratah NSW 2298, Australia.
[c]
[d]
Prof S Paula (0000-0003-4100-1467),
Department of Chemistry
Purdue University, 560 Oval Drive, West Lafayette Indiana USA.
Dr X Zhu (0000-0003-4946-2081)
Research Computing, Information Technology at Purdue (ITaP)
Purdue University, 155 S. Grant St, West Lafayette Indiana USA.
Supporting information for this article is given via a link at the end of the document.
Abstract: Knoevenagel condensation of 3,4-dichloro- and 2,6-
dichloro- phenylacetonitriles gave library of
Globally cancer results in 9 million deaths annually. In women,
breast cancer is the most common cancer both in the developed
and less developed world. While survival prognosis is improving,
this is offset by a continued rise in disease incidence. Breast
cancer treatment is stage and receptor status dependent. Early-
stage breast cancer treatments typically include surgery and
radiotherapy. More advanced and aggressive tumours require
chemotherapy, hormonal, and targeted therapies. Hormone
sensitive tumours can be treated with Tamoxifen and anastrozole,
but this frequently results in drug resistance, and poor response
in these instances. These treatments often have poor tumour
selectivity [1]. Herceptin selectively targets the human epidermal
growth factor receptor (HER2); however, 70% of HER2-positive
patients fail to respond to treatment, with resistance developing
rapidly. Herceptin also induces significant cardiac dysfunction in
a
dichlorophenylacrylonitriles. Our leads (Z)-2-(3,4-dichlorophenyl)-3-
(1H-pyrrol-2-yl)acrylonitrile (5) and (Z)-2-(3,4-dichlorophenyl)-3-(4-
nitrophenyl)acrylonitrile (6) displayed 0.56±0.03 and 0.127±0.04 µM
growth inhibition (GI50) and 260-fold selectivity for the MCF-7 breast
cancer cell line. A 2,6-dichlorophenyl moiety saw a 10-fold potency
loss; additional nitrogen moieties (-NO2) enhanced activity (Z)-2-(2,6-
dichloro-3-nitrophenyl)-3-(2-nitrophenyl)-acrylonitrile (26) and (Z)-2-
(2,6-dichloro-3-nitrophenyl)-3-(3-nitrophenyl)acrylonitrile (27), with
the
corresponding
-NH2
analogues
(Z)-2-(3-amino-2,6-
dichlorophenyl)-3-(2-aminophenyl)acrylonitrile (29) and (Z)-2-(3-
amino-2,6-dichlorophenyl)-3-(3-aminophenyl)-acrylonitrile (30) more
potent. Despite this, both 29 (2.8±0.03 µM) and 30 (2.8±0.03 µM)
were 10-fold less cytotoxic than 6. A bromine moiety effected a 3-fold
enhancement in solubility with (Z)-3-(5-bromo-1H-pyrrol-2-yl)-2-(3,4-
dichlorophenyl)acrylonitrile 18 relative to 5 at 211 µg mL-1. Modelling
led synthesis saw the introduction of 4-aminophenyl substituents (Z)-
3-(4-aminophenyl)-2-(3,4-dichlorophenyl)acrylonitrile (35) and (Z)-N-
2–7% of patients [2]
.
Triple negative breast cancer, i.e. breast cancers lacking the
estrogen (ER), progesterone, and HER2 receptors, are best
described as highly challenging with poor prognosis. Triple-
negative breast cancers are vastly heterogeneous, hindering
targeted therapy development [3]. Regardless of hormonal status,
1 in 3 breast cancer sufferers will experience disease recurrence
or metastasis. The 5-year survival for these advanced breast
cancer patients is only 25%. Metastatic breast cancer remains
incurable [4]. New breast cancer drug targets are required.
The arylhydrocarbon receptor (AhR) belongs to the family of
basic-helix-loop-helix transcription factors and is known to
mediate the effects of polycyclic and polyhalogenated aromatic
hydrocarbon ligands, including environmental toxins such as
benzo[a]pyrene (1) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (2) [5-
9]. Multiple other ligands, including bilirubin, prostaglandins,
tryptophan, and even plant derived ligands, such as resveratrol
and flavones also exist [10,11]. On-going analysis of AhR function
is revealing the increased complexity of this pathway with
evidence that the AhR has the ability to modulate gene
(4-(2-cyano-2-(3,4-dichlorophenyl)vinyl)phenyl)acetamide
(38),
0.030±0.014 and 0.034±0.01 µM potent, respectively. Other
analogues, e.g. 35 and 36, were sub-micromolar potent against our
cell line panel (HT29, colon; U87 and SJ-G2, glioblastoma; A2780,
ovarian; H460, lung; A431, skin; Du145, prostate; BE2-C
neuroblastoma; MIA, pancreas and SMA murine glioblastoma) except
38 against U87. A more extensive evaluation of 38 ((Z)-N-(4-(2-cyano-
2-(3,4-dichlorophenyl)vinyl)phenyl)acetamide), in a panel of drug
resistant breast carcinoma cell lines showed 10-206 nM potency
against MDAMB468, T47D, ZR-75-1, SKBR3 and BT474. MOE
docking scores showed a good correlation between predicted binding
efficiencies and observed MCF-7 cytotoxicity. This supports the use
of this model in developing breast cancer specific drugs.
Introduction
1
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transcription and other cellular events without ligand activation
dichlorophenyl (in red); the centre acrylonitrile (in green), and the
pyrrole / phenyl (in blue) moieties (Figure 1). Previous studies
have found that modification of the central acrylonitrile region, via
either a reduction of the double bond or replacement of the nitrile
moiety with an acid or the introduction of an amide moiety resulted
in loss of activity [22,23]. Rapid access to their common core is via
a Knoevenagel condensation: with 5, of 3,4-dichlorophenyl
acetonitrile 7 and 2-pyrrolecarboxaldehyde 8; and with 6, of 3,4-
[10,12]
.
The AhR complexes with heatshock protein 90, prostaglandin
E synthase 3, and a single molecule of the immunophilin-like
protein hepatitis B virus X–associated protein 2. This complex is
typically localised in the cytosol and serves to maintain the AhR
in a ligand-ready conformation by preventing AhR degradation
and binding of the AhR nuclear translocator (ARNT) [5,6,9]. Ligand
binding to the AhR then facilitates the formation of an AhR-ARNT
heterodimer complex, which translocates to the cell nucleus,
binds to xenobiotic response elements (XREs), and promotes the
transcription of metabolic enzymes such as cytochrome p450
(P450), CYP-1A1, -1A2, and -1B1. The unliganded AhR is then
dichlorophenyl acetonitrile 7 and 4-nitrobenzaldehyde 9 [21]
.
Of the compounds reported thus far, the 3,4-dichlorophenyl
moiety was present in the most cytotoxic analogues [19,21]. Despite
these prior efforts, we had not explored the corresponding 2,6-
dichlorophenyl analogues. Using our standard Knoevenagel
exported back to the cytosol [5,6]
.
condensation
conditions
the
coupling
of
2,6-
The AhR pathway has been linked with breast cancer
initiation and progression [13-16], and has been exploited in the
development of a number of small molecules that target
dichlorophenylacetonitrile 10 and 2-pyrrolecarboxaldehyde 8
failed, as did the equivalent coupling of 10 and 4-
nitrobenzaldehyde 9. However, the corresponding reaction of 10
with the 2-, and 3-nitrobenzaldehyde (11 and 12) afforded the
desired analogues 13 and 14, respectively (Scheme 1).
breast cancer such as aminoflavone (
[18] (Figure 1).
3
)
[17] and phortress (
4)
Cl
O
Cl
Cl
Cl
O
1
2
NH2
O
O
NH2
F
F
N
S
Cl
Cl
CN
Cl
CN
NH
F
i
O
Cl
Cl
CN
O
NH2
NO2
NO2
F
NH2
O2N
3
4
Cl
10
11; 2-NO2
12; 3-NO2
13; 2-NO2
14; 3-NO2
15; 2-NO2
16; 3-NO2
NO2
CN
CN
Cl
Cl
Cl
Cl
N
H
Scheme 1. Synthesis of nitro substituted analogues of 6. Reagents and Conditions. i)
PhCH2NMe3(OH), H2O, 50ºC, 5h. Boxed section shows the equivalent 3,4-
dichlorophenylacrylonitrile analogues 15 and 16.
5
6
Figure 1. Selected Aryl hydrocarbon receptor ligands: benzo[a]pyrene (1),
2,3,7,8-tetrachlorodibenzo-p-dioxin (2), aminoflavone (3), phortress (4), ANI-7
(5) and (Z)-2-(3,4-dichlorophenyl)-3-(4-nitrophenyl)acrylonitrile (6).
To enable a direct potency comparison, the 2- and 3-nitro
derivatives of 6, analogues 15 and 16 respectively, were also
prepared (Scheme 1, boxed insert).
Screening of these
We have explored (Z)-2-(3,4-dichlorophenyl)-3-(1H-pyrrol-2-
yl)acrylonitrile (ANI-7 (5)) and the related (Z)-2-(3,4-
dichlorophenyl)-3-(4-nitrophenyl)acrylonitrile (6) (Figure 1) and
identified both as potent and selective inhibitors of cell growth in
MCF-7 breast cancer cells [19], while having minimal to no effect
on the growth of normal non-tumour derived breast cells or cells
derived from other tumour types, including colon, ovarian, lung,
skin, neuronal, glial, and pancreatic [20]. Herein we report on the
development of more potent and breast cancer selective
analogues of 5 and 6.
analogues was conducted against our panel of 11 cancer cell
lines (HT29, colon; U87, SJ-G2 and SMA, glioblastoma; MCF-7,
breast; A2780, ovarian; H460, lung; A431, skin, Du145, prostate;
BE2-C, neuroblastoma; MIA, pancreas) and one normal cell line
(MCF-10A), with the outcomes against the breast cancer cell line,
MCF-7, show in Table 1 (data for other cell lines is presented in
Table 8). The 2,6-dichloro variants, with MCF-7 GI50 values of
26±2.7 and 25±4.2 µM for analogues 13 and 14 respectively,
exhibited a 10-fold lower potency than the 3,4-dichloro analogues,
with 15 and 16 displaying 2.9±0.2 and 5.3±0.7 µM potency
respectively, supporting the importance of a 3,4-disposed phenyl
substituent. These findings were consistent with the docked pose
of ANI-7 (5) in the AhR, based on our in-house developed
homology model of the AhR PAS-B domain (Figure 2).
Results and Discussion
Examination of the core scaffold of 5 and 6 suggested three
key areas suitable for synthetic modification; the 3,4-
Table 1. Growth inhibition of the MCF-7 breast carcinoma cell line by dichlorophenylacrylonitriles 5, 6 and 13-16.
Compound
Compound
CN
MCF-7 GI50 (µM)
MCF-7 GI50 (µM)
CN
Cl
Cl
Cl
N
H
NO2
0.56±0.03
25±4.2
Cl
5
14
CN
NO2
CN
Cl
Cl
Cl
0.127±0.43
2.9±0.2
NO2
Cl
15
6
2
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Cl
CN
CN
Cl
Cl
NO2
26±2.7
5.3±0.7
NO2
Cl
16
13
In silico analysis of ANI-7 (5) and 6 in the AhR PAS-B domain
hydrophobic interactions with non-polar structural elements
present in 5 and 6 (Figure 2C and D). In addition, an attractive p-
p interaction was predicted between the side chain of H17 and the
phenyl ring of 5. Moreover, the tight fit between ligand and
receptor suggested the presence of substantial van-der-Waals
interactions in the binding pocket. In the case of 5, the observed
orientation suggests that the 2,6-dichlorophenyl moiety results in
a shift in conjugated system planarity, consistent with the
observed 10-fold reduction in potency. Interestingly, no hydrogen
bonds between ligand and receptor were predicted. Docking of 4-
nitrophenyl 6 into the AhR homology model yielded very similar
results to those noted for 5. Control experiments with AutoDock
Vina generated comparable binding poses, with slight shifts in the
overall position of the ligand observed. However, given that some
of the critical binding interactions (hydrophobic forces, van-der-
Waals interactions) do not display a pronounced level of
directionality (compared to hydrogen bonds, for example), the
variations are expected and certainly do not lessen the confidence
in the accuracy of our results.
An AhR PAS-B domain homology model was developed,
using the MOE platform (see experimental for detail). Analysis of
the model’s geometry revealed no steric clashes or outliers in
backbone bond lengths and angles and a Ramachandran
diagram showed no entries in the “forbidden” regions for
backbone torsion angles. The MOE site finder function was
employed to identify the ligand binding area in the homology
model, which coincided with the location of co-crystallized ligands
in related proteins with PAS-B domains [3F1O, 3H7W, 3H82],
when superimposed onto the homology model (Figure 2A-B; for
sequence alignment, see supporting materials). Inspection of the
ligand binding site of the AhR homology model revealed a
predominately hydrophobic pocket buried in the interior of the
receptor, consistent with the nature of the known AhR ligands
(Figure 1) [9,17,18]. The site is lined by a number of hydrophobic
residues including F21, L34, F50, M66, L79, A93, I105 and V107,
creating
a
non-polar environment capable of generating
A
B
C
D
E
F
G
Figure 2. A. Agreement between the binding site as predicted by MOE’s site finder (red and white spheres) and the position of ligands observed in X-ray crystal
structures. B. As for A with the protein background removed for clarity. C. Docked structure of 6 bound in the PAS-B domain homology model, illustrating the central
and buried nature of the ligand binding site. D. Overlaid images of 6 bound in the PAS-B domain, before and after molecular dynamic simulations of 100 ns. E. A
2D generated ligand interaction diagram of the generated AhR PAS-B domain, shown with ANI-7 (5) docked. F. As for E with lead 6 docked. Hydrophobic residues
are shown in green; hydrophilic residues are shown in purple; the green dashed line depicts p-p interactions. G. Water molecules (space-filling) present in the
binding site as predicted by MD simulations. Polar residues are shown in green, non-polar residues in magenta.
The stability of the docking-predicted poses was evaluated by
subsequent MD simulations. Figure 2D highlights the stability of
the docked pose of 6 with minimal deviations between the initial
and final positions after 100 ns of simulation time, suggesting that
3
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CN
the docked pose was a realistic depiction of the binding event.
Interestingly, the simulations also showed two to three water
molecules forming bridged hydrogen bonds between the ligand’s
nitrile group and the receptor (with the backbone of L315, as well
as the side chains of T289 and H291) (Figure 2G). The presence
of these water-mediated hydrogen bonds is consistent with the
previous observation that removal of the nitrile group results in a
Br
Cl
Cl
N
H
0.89±0.19
1.04±0.18
18
Br
CN
Cl
Cl
N
H
19
Br
loss of activity [24]
.
CN
Br
Cl
Cl
N
H
Enhancing ANI-7 solubility
0.33±0.04
0.48±0.04
Concurrent with efforts to further enhance the potency of
these analogues (see below) we also explored the introduction of
simple bromine moieties in an effort to enhance the solubility of
lead 5 whilst maintaining analogue potency and selectivity
20
Br
Br
CN
Br
Cl
Cl
N
H
towards the MCF-7 cell line.
To effect these proposed
21
modifications, the flow photochemical bromination of 5 with NBS
Effect of additional nitrogen containing substituents on MCF-7
cytotoxicity
[27]
17 [25,26], was investigated
.
A rapid UV-filter screen was
conducted (Table 2). Filter Type 1 afforded both mono-
brominated products 18 and 19 in a 1:1 ratio; filter Type 2 afforded
primarily 19, and filter Type 5 and 6 favoured the formation of 18
(for details of filter UV-vis outputs, see supporting materials).
Each of the brominated species generated was readily separable
by flash chromatography (see experimental). The di- and tri-
bromo derivatives 20 and 21 were afforded in a similar manner,
with increased equivalents of NBS 17. The four brominated
products were then screened for cytotoxicity (Table 3).
Our modelling analysis suggested limited opportunity to
modify this scaffold further (Figure 2). However prior mechanistic
evaluation of aminoflavone (3) and phortress (4) postulated that
the breast cancer activity was a consequence of in situ nitrenium
ion formation from the pendant amino moieties [28,29]. ANI-7 (5)
possesses a pyrrole NH which could give rise to such an
intermediate, but the 4-nitrophenyl 6, does not. Despite this it is
known that CYPs can effect -NO2 to -NH2 transformations as well
as hydroxylations, and this would be consistent with the in situ
formation of a nitrenium ion effecting the observed cytotoxicity [30]
.
Br
CN
CN
CN
Br
Cl
Cl
Thus the presence of additional nitrogen substituents in
analogues 5 and 6 may result in enhanced MCF-7 potency. To
explore this possibility we introduced a -NO2 moiety (H2SO4,
HNO3, RT, 4 h) to 2,6-dichlorophenyl acetonitrile 10 affording 22,
which was coupled with nitrobenzaldehydes 23 and 24 under
standard Knoevenagel conditions, and then the resultant 25 and
26 were subjected to reducing conditions (Fe(s) 12 equiv., 2:2:1
AcOH:EtOH:H2O, sonication) to yield the amino analogues 27
and 28 (Scheme 3). No product was isolable on treatment of 3,4-
dichlorophenyl acetonitrile under nitration conditions.
Cl
Cl
N
H
N
H
Cl
Cl
N
H
5
hυ
0.05 M in ACN
1 mL.min-1
18
19
Br
Br
Br
CN
10 mL
0ºC
Br
CN
O
Br
Br
N
Cl
Cl
Cl
Cl
N
H
N
H
O
0.055 M in ACN
1 mL.min-1
17
20
21
Scheme 2. Flow bromination of 5. Reagents and Conditions: 0.05M 5 in
CH3CN, 0.055 M 17 in CH3CN, hu, 1.0 mL.min-1, 0ºC.
Table 2. Flow-bromination in the Vapourtec easy-Photochem afforded
brominated adducts of ANI-7 (5); the reaction conditions - 0.05M 5 in CH3CN,
0.055 M 17 in CH3CN, hu, 1.0 mL.min-1, 0ºC. Variations in the UV filter were
examined to afford 18-20.
Cl
Cl
Cl
CN
O
ii
i
O2N
O2N
CN
CN
NO2
O2N
Cl
Cl
Cl
10
22
23; 2-NO2
24; 3-NO2
25; 4-NO2
26; 2-NO2
27; 3-NO2
28; 4-NO2
Filter-Type
Ratio
1
0
2
3
6
3
0
18
50
37
63
46
62
66
19
46
57
32
43
32
32
20a
4
5
2
4
1
2
3
4
5
6
iii
Cl
CN
H2N
2
2
NH2
Cl
29; 2-NH2
30; 3-NH2
31; 4-NH2
a product ratio determined by UPLC-MS analysis
Scheme 3. Reagents and Conditions: i) HNO3 / H2SO4, 0ºC to rt, 2h; ii) PhCH2NMe3(OH),
H2O, 50ºC, 5h; iii) Fe(s) 12 equiv., 2:2:1 AcOH:EtOH:H2O, sonication, 30ºC, 2h.
The brominated analogues 18-21 displayed modest to good
levels of MCF-7 cytotoxicity with GI50 values of 0.33±0.04 (20) to
1.04±0.18 µM (19). Analysis of the solubility of 18-21 and 5 in 1:1
H2O:DMSO highlighted a 3-fold increase in solubility of 18 vs. 5
with observed solubilities of 211 µg.mL-1 (18) and 78.7 µg.mL-1 (5).
However concurrent with this was a modest loss in potency with
18 to a MCF-7 GI50 of 0.89±0.19 µM (Table 3).
Evaluation of these analogues, 26-30, against the MCF-7 cell
line, with the data presented in Table 4, revealed an increase in
potency relative to the lead -NO2 and -NH2 analogues.
Table 4. Growth inhibition of the MCF-7 breast carcinoma cell line by
dichlorophenylacrylonitriles 26-30.
Table 3. Growth inhibition of the MCF-7 breast carcinoma cell line by
dichlorophenylacrylonitriles 18-21.
Compound
µ
MCF-7 GI50 ( M)
Compound
MCF-7 GI50 ( M)
µ
4
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Cl
CN
Cl
20, 21, and 32, offered little advance on the lead analogues
beyond a critically important improvement in solubility, we
expanded our examination of the 3,4-dichlorophenylacrylonitrile
O2N
3.5±0.25
12±1.3
NO2
26
pharmacophore.
To this end an additional series of 4-
Cl
CN
O2N
aminophenyl substituted analogues, 35-39, were synthesised as
per Scheme 1 and screened for activity against the MCF-7 cell
line and this data is presented in Table 6.
NO2
Cl
27
NO2
Cl
CN
Table 6. Growth inhibition of the MCF-7 breast carcinoma cell line by
dichlorophenylacrylonitriles 35-39.
O2
N
7.4±1.2
2.8±0.03
4.5±0.5
Cl
28
Compound
MCF-7 GI50 (µM)
NH2
Cl
CN
CN
H2
N
Cl
Cl
0.030±0.014
NH2
Cl
29
CN
35
H
N
Cl
CN
H2N
NH2
Cl
Cl
0.166±0.013
0.28±0.04
Cl
30
36
N
In all instances, the amino substituted analogues were
equipotent or marginally more cytotoxic than the corresponding
nitro analogue, c.f. 29 and 26, MCF-7 GI50 of 2.8±0.03 vs.
3.5±0.25 µM; and 30 and 27, MCF-7 GI50 of 4.5±0.5 vs. 12±1.3
µM. While supporting the nitrenium ion hypothesis, it is equally
CN
Cl
Cl
37
H
N
CN
possible that the modest potency enhancements were
a
Cl
O
consequence of enhanced cellular uptake. Regardless, no
analogue was more potent than leads 5 and 6. We were unable
to isolate the 4-amino 31 in sufficient quantities and purity to
examine its potency against the MCF-7 cell line. The introduction
of a bromine moiety, to enhance solubility, with 32 was equipotent
with 5, but 2.5-fold less active than 6. Of note was the significant
potency reduction of the amino 33. Introduction of additional polar
substituents such as those present in 34 was similarly detrimental
to MCF-7 cytotoxicity (Table 5).
0.034±0.01
2.1±0.15
Cl
38
N
CN
Cl
Cl
N
H
39
Of analogues 35-39 excellent, and enhanced, activities
against the MCF-7 cell line was observed with 4-NH2 35 (GI50
Table 5. Growth inhibition of the MCF-7 breast carcinoma cell line by
dichlorophenylacrylonitriles 32-34.
=
0.030±0.014 µM) and 4-NHC(O)CH3 38 (GI50 = 0.034±0.01 µM).
Methylation of the 4-NH2 moiety yielded NCH3 36 and N(CH3)2 37
which were both more, or as, active than lead 5 with GI50 values
of 0.166±0.13 and 0.28±0.04 µM respectively, but not more active
than 6. The introduction of an imidazole moiety to encapsulate
the amino moiety was poorly tolerated, giving 39 with a MCF-7
GI50 value of 2.1 µM, ca 60-fold less active than both 35 and 38.
A more detailed examination of the effect of 38, which
displayed the highest level of MCF-7 selectivity (see Table 8) on
a broader panel of breast cancer cell lines (with varying estrogen,
progesterone and HER2 receptor status) revealed nanomolar
potencies against the MDAMB468, T47D, ZR-75-1, SKBR3 and
BT474, ranging from 10 to 206 nM. Against the BT20 and the
MDAMB231, the activity was more modest at 2.5±0.3 and 5.1±0.8
µM. However, these additional breast cancer cell lines are known
as drug resistant cells, thus this activity is highly promising.
Compound
MCF-7 GI50 (µM)
Br
CN
Cl
Cl
NO2
0.32±0.03
32
Br
CN
Cl
72±9% @ 25 µM
51±3% @ 25 µM
NH2
Cl
33
O
OH
CN
Cl
NO2
Cl
34
Given that our efforts thus far, while producing a number of
sub-micromolar potency inhibitors of MCF-7 cell growth, e.g. 18,
Table 7. Growth inhibition (GI50 values) of the MDAMB468, T47D, ZR-75-1, SKBR3, BT474, BT20 and MDAMB231 drug resistant breast carcinoma cell line by
dichlorophenylacrylonitrile 38.
Compound
MCF-7 [a]
MDAMB468[b]
T47D[a]
ZR-75-1[a]
SKBR3[c]
BT474[a]
BT20[b]
MDAMB231[b]
MCF10A[c]
GI50 (µM)
38
0.034±0.01
0.042±0.01
0.027±0.01
0.021±0.01
0.010±0.00
0.206±0.16
2.5±0.3
5.1±0.8
14.1±0.7
[a] Estrogen receptor (ER) positive; [b] Triple negative for ER, progesterone (PR) and human epidermal growth factor (HER2) receptors; [c] ER negative and
HER2 positive;[d]normal breast cell line.
Cytotoxicity across a panel of cell lines
5
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Our focus has been the examination of the breast cancer cell
line, MCF-7, based on our initial report of >250-fold selectivity of
the leads 5 and 6 relative to our cell line panel. The data for the
growth inhibition of the cell lines examined is presented in Table
8. Of these analogues it is noteworthy that 38 shows high levels
of specificity for the MCF-7 cell line with >400-fold selectivity over
the normal breast cell line MCF10A (Table 8).
6
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Table 8. Growth inhibition against a panel of carcinoma cell lines by dichlorophenylacrylonitriles 5, 6, 13-21, and 26-42.
Compound
HT29
U87
MCF-7
A2780
H460
A431
Du145
µ
GI50 ( M)
BE2-C
SJ-G2
MIA
SMA
MCF10A
a
5
6
15±1
3.1±1.
8
-
0.56±0.03
0.127±0.0
4
16±0
12±1
5.7±0.7
69±2
3.2±0.1
41±8
25±1
20±1
14±1
46±7
27±4
-
-
35.5±0.5
36.5±3.5
-
7.1 ± 0.1
18±3
8.9±0.7
24±1.
6
40±9.
7
7.7±0.
4
3.0±0.
4
40.0±4.
13
14
15
16
49± 1.0
>50
26±2.7
25±4.2
2.9±0.2
5.3±0.7
27±4.4
33±8.7
8.9±1.8
9.4±2.1
32±4.0
35±13
28±3.0
29±5.2
4.0±0.1
1.5±0.1
27±6.5
34±14
45±2.9
42±8.3
38±1.5
45±5.3
9.2±1.2
8.8±1.5
>50
28±1.7
36±9.1
0
29.5±8.
5
>50
10.8±0.
2
13.2±2.
7
14.1±3.
2
7.6±1.8
5.6± 0.9
4.4±0.6
2.4±1.0
-
-
5.8±0.8
10.6±1.8
10.3±5.
3
28±2.0
6.7±3.2
18
19
12±1
11±1
2.2±0.
1
2.3±0.
4
33±1
17±1
0.89±0.19
1.04±0.18
13±1
13±0
11±1
15±0
3.1±0.1
1.4± 0.2
19±2
16±0
16±1
16±0
16±1
15±0
24±1
16±0
-
-
22±2
15±0
20
21
26
3.7±0.2
0.33±0.04
0.48±0.04
3.5±0.25
2.2 ±0.0
0.61±0.10
2.9±0.048
2.4±0.2
2.4 ± 0.2
3.1±0.1
2.0±1.1
2.6±0.20
2.8±0.1
2.7±0.1
3.5±0.1
0.49±0.09
2.2±0.058
-
3.3±0.1
3.2±0.2
2.7±0.10
0.77±0.
12
2.1±0.1
5
0.77±0.
04
2.5±0.2
1
0.69±0.
11
2.3±0.2
6
2.3±0.1
3.2±0.2
-
0.77±
0.11
3.4±0.3
0
3.8±0.3
1
2.6±
0.27
13±1.
1
2.9±0.
16
3.4±0.6
2
27
17±0.50
12±1.3
9.4±1.6
11±2.3
10±1.1
6.6±0.85
12±1.0
4.1±0.39
9.7±1.5
2.4±0.
14
3.1±0.
3
3.1±0.
3
0.27±
0.04
68±4
%
2.5±0.3
2
8.2±1
.5
28
29
30
32
33 b
34
35
36
12±0.29
7.4±1.2
2.8±0.03
4.5±0.5
0.32±0.03
72±9%
4.6±0.86
3.4±0.2
3.0±0.2
2.6±0.1
68±4%
11±2.4
4.9 ± 0.4
8.1±2.0
5.1±1.6
40±5%
6.2±2.2
2.7±0.0
2.9±0.1
7.7±1.3
6.8±1.5
13.8±0.8
0.1±0.03
71±11%
40±5%
11±1.4
4.6±0.5
6.6±1.7
21±5
3.1±0.31
8.0±1.1
8.3±1.6
3.5±0.3
4.0±0.2
0.89±0.20
47±16%
12±0%
13.0±0.
2.0±0.3
-
-
-
-
-
-
-
0
15.7±0.
3
2.9±0.1
5.5±0.1
0.45±0.
12
0.41±0.
05
1.5±0.1
33±14%
<10%
8.0±0.4
44±11%
55±8%
42±9%
<10%
55±7%
17±6
%
51±3%
26±4%
16±5%
50±4%
49±4%
15±3%
0.47±
0.05
0.23±
0.01
0.241
±0.07
1
0.030±0.0
14
0.166±0.0
13
0.89±0.
12
0.043±
0.012
0.26±0.
02
0.041±
0.010
0.14±0.
02
0.021±
0.003
0.90±0.
07
0.083±
0.022
1.2 ±0.2
0.25±0.04
0.41±0.05
0.71±0.07
0.039±0.009
1.6±0.1
0.082±0
.020
0.040±
0.011
0.101±
0.023
0.172±0.0
23
0.93±0.
25
0.58±0.
23
0.30±0.
12
0.20±0.
05
0.94±0.
33
37
38
0.28±0.04
0.58±0.23
9.1±0.6
0.82±0.26
1.5± 0.2
0.46±0.12
10.8±2.1
-
-
0.62±0.21
14.1± 0.7
10.
0.034±0.0
1
11.9±2.
5
0.85±0.
59
12.9±4.
4
24±5.3
3.8± 0.4
±1.9
0.95±
0.16
>100
51±4
%
39
2.9±0.2
2.1±0.15
28±14
3.1±0.0
93±3
2.6±0.1
>100
2.5±0.1
88±6
2.1±0.2
>100
2.4±0.1
>100
2.0±0.1
95
3.3±0.1
>100
n.d
2.8±0.1
-
40 19
41 19, c
-
-
-
-
84±1%
63±1%
52±24%
39±4%
42±6%
38±1%
50±6%
46±6%
36±0%
a ‘–‘ = not determined; b percentage growth inhibition at 25 µM compound concentration; c percentage growth inhibition at 100 µM compound concentration
Within these analogues we also observe sub-micromolar
potent growth inhibition with 26 (HT29, colon), 32 (HT29; A431,
skin; Du145, prostate; BE2-C, neuroblastoma), 37 all cell lines
except U87 (glioblastoma; 1.2±0.2 µM), 38 all cell lines and 41
(HT29).
Table 9. MOE docking score for a representative selection of compounds
compared to the biologically derived MCF-7 GI50 (µM) inhibitory values. Lower
values for the MOE scoring function and lower values for MCF-7 GI50 are
indicative of better binding and anti-cancer cell activity, respectively.
MCF-7 GI50
Scoring function-
MOE
Compound
µ
( M)
AhR homology model and MOE binding score
H
Having developed novel analogues with high levels of potency
(nanomolar potent against the MCF-7 breast cancer cell line), we
sought to correlate the measured cytotoxicity with their predicted
binding scores using MOE within our AhR homology model.
Detecting such a correlation would support the notion that binding
to the AhR is the critical step in a sequence of several steps that
also involve interactions with other enzymes in the nucleus. The
data generated via the in silico docking, scoring and ranking of
the calculated binding values of selected analogues, spanning 3-
log values of cytotoxicity, with 5, 6, 18, 38, 40 and 41 (activity
range 0.034 to 28 µM) is presented in Table 9.
N
CN
Cl
Cl
O
-7.36
0.034 ± 0.01
38
NO2
CN
Cl
-7.12
-6.80
0.127 ± 0.043
0.38 ± 0.03
Cl
6
Table 1
CN
Cl
Cl
N
H
7
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5
A number of the analogues reported herein were sub-
micromolar potent, the most notable being 35 and 36 which were
active against all the cell lines evaluated (except 38 against U87).
CN
Br
Cl
N
H
-6.52
-6.47
-6.62
0.89 ± 0.19
Cl
18
Acknowledgements
OH
CN
Cl
OH
28 ± 14
JRB is the recipient of an Australia government HDR scholarship.
Funding from the Australian Cancer Research Foundation and
the Ramaciotti Foundation is gratefully acknowledged. SP is the
recipient of a Fulbright Senior Fellowship.
Cl
40
CN
Cl
N
H
84 ± 1%a
Cl
41
Experimental
a percentage growth inhibition at 100 µM compound concentration.
Biology
As can be seen from the data presented in Table 9, there is a
positive correlation between the levels of observed cytotoxicity
and the MOE docking score (see supporting material). There is a
clear correlation seen for all the docked compounds. Our earlier
hypothesis saw the NO2 moiety of 6 reduced by a CYP to the
amino moiety, and scission of the acetyl moiety would afford the
same common outcome, the in situ generation of 35. The
difference in the observed activity may result from a combination
of cellular uptake rates and the efficiency of the in situ enzymes
at effecting the required transformation. The correlation is strong
for all analysed compounds, supporting AhR as the compound
target and increasing the validation of the binding pose and
binding site location.
Cell culture and stock solutions
Stock solutions were prepared as follows and stored at -20 °C: drugs were
stored as 40 mM solutions in DMSO. All cell lines were cultured in a
humidified atmosphere 5% CO2 at 37 ºC. The cancer cell lines were
maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Trace
Biosciences, Australia) supplemented with 10% foetal bovine serum, 10
mM sodium bicarbonate, penicillin (100 IU/mL), streptomycin (100 µg/mL),
and glutamine (4 mM). The non-cancer MCF10A cell line was cultured in
DMEM:F12 (1:1) cell culture media, 5% heat inactivated horse serum,
supplemented with penicillin (50 IU/mL), streptomycin (50 µg/mL), 20mM
Hepes, L-glutamine (2mM), epidermal growth factor (20ng/mL),
hydrocortisone (500ng/mL), cholera toxin (100ng/mL), and insulin (10
µg/mL).
In vitro growth inhibition assay
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 complete 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 drug concentration at which cell growth is 50% inhibited
based on the difference between the optical density values on day 0 and
Conclusions
Synthetic modification of leads 5 and 6 saw enhanced solubility
through the introduction of a bromine moiety with a modest
potency reduction. The brominated 18, was 3-fold more soluble
that parent 5 at 211 µg mL-1, while retaining sub-micromolar
potency against the MCF-7 cell line. The introduction of additional
nitrogen containing substituents afforded modest levels of activity,
with the -NH2 analogues uniformly more active than their -NO2
counterparts.
In silico evaluation, in an AhR-PAS-B domain homology
model, of these dichlorophenylacrylonitriles supported tight
binding to a largely hydrophobic binding pocket. However, this
model also supported limited scope for additional potency
enhancements resulting in synthesis of additional 3,4-
dichlorophenylacrylonitiles with 4-aminophenyl substituents.
Evaluation of the MOE docking scores and observed activity
against MCF-7 cells showed a strong and positive correlation,
supporting the use of this model in developing additional MCF-7
targeted analogues.
All SAR data presented herein was consistent with the 4-
position most favourable. These additional analogues enhanced
potency 10-fold returning MCF-7 GI50 values of 0.030±0.014 and
0.034±0.01 µM for 35 and 38 respectively. Analysis of the AhR
PAS-B docking scores via the MOE system highlighted excellent
correlation between predicted binding efficiencies and observed
MCF-7 cytotoxicity. A more extensive evaluation of analogue 38
in a panel of drug resistant breast carcinoma cell lines showed
10-206 nM potency against MDAMB468, T47D, ZR-75-1, SKBR3
and BT474.
those at the end of drug exposure [31,32]
.
In silico approaches
Development of a homology model for the AhR PAS-B domain
Homology modelling was performed using the program MOE (Molecular
Operating Environment (MOE), 2013.08; Chemical Computing Group ULC,
1010 Sherbooke St. West, Suite #910, Montreal, QC, Canada, H3A 2R7,
2018). The sequence of the human AhR was obtained from the NCBI
website (https://www.ncbi.nlm.nih.gov/, accession code NP_001612.1)
imported into MOE for a homology search, which returned the three crystal
structures 4F3L [33], 3RTY [34], and 2KDK as the closest matches. The
sequences of these three template candidates were aligned with that of
the AhR (supporting material). For building the model, only 4F3L (a
transcriptional activator complex with a basic helix-loop-helix PAS-domain)
was used as a template (24.8% identity and 48.8% similarity) and the
model generation was executed at default settings (Amber force field).
Subsequent analysis of the model’s geometry revealed no steric clashes
or outliers in backbone bond lengths and angles. Likewise,
a
Ramachandran diagram showed no entries in the “forbidden” regions for
backbone torsion angles.
Docking of compounds into the homology model
8
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The structures of all ligands to be docked were modelled in MOE and
their conformations were energy-minimized using Molecular Mechanics in
combination with the MMFF94x force field.
coated aluminium plates with
chromatography was performed under ‘flash’ conditions on Merck silica gel
60 (230-400 mesh).
a
thickness of 0.2 mm. Column
MOE’s site finder functionality was employed to identify the ligand
binding area in the homology model, which coincided with the location of
co-crystallized ligands in related proteins with PAS-D domains, e.g. 3F1O
[35], 3H7W [36], and 3H82 [36], when superimposed onto the homology model.
Docking was performed using MOE’s default settings, using the
triangle matcher method in conjunction with the London dG scoring
function for the initial placement of the ligand and the rigid receptor setting
with the GBVI/WSA scoring function for the refinement of the 30 top poses.
Analysis and visualization of the docking output, such as identification of
hydrogen bonds, steric clashes, hydrophobic interactions, or p-p
interactions were performed in MOE.
In addition to MOE, docking runs were also conducted with AutoDock
Vina as a control. The centre of the docking box (16 x 16 x 16 Å) was
placed at the location of the central nitrogen atom in the N-[2-nitro-4-
(trifluoromethyl)phenyl]morpholin-4-amine ligand of the crystal structure
3F1O after superimposition onto the homology model. Docking runs were
executed at default settings with the highest level of exhaustiveness
selected.
Nuclear magnetic resonance (NMR) spectroscopy was performed on
a Brüker Avance III 400 MHz spectrometer, where proton NMR (1H NMR)
spectra and carbon NMR (13C NMR) spectra were acquired at 400 and 100
MHz respectively, or a Brüker Avance III 600 MHz spectrometer, where
proton NMR (1H NMR) spectra and carbon NMR (13C NMR) spectra were
acquired at 600 and 150 MHz respectively. All spectra were recorded in
deuterated dimethyl sulfoxide (DMSO-d6), deuterated acetone (acetone-
d6) or deuterated chloroform (CDCl3) obtained from Cambridge Isotope
Laboratories Inc. Chemical shifts (d) were measured in parts per million
(ppm) and referenced against the internal reference peaks. Coupling
constants (J) were measured in Hertz (Hz). NMR assignments were
determined through the interpretation of one- and two-dimensional spectra.
Multiplicities are denoted as singlet (s), broad singlet (bs), doublet (d),
doublet of doublets (dd), triplet (t), quartet (q), triplet of doublets (td),
doublet of triplets (dt) and multiplet (m). Peaks are listed in decreasing
chemical shift in the following format: chemical shift (integration (1H),
multiplicity (1H), coupling constant (1H). The Biotage® initiator+ was used
to perform microwave reactions.
Molecular Dynamics Simulations
(Z)-2-(2,6-dichlorophenyl)-3-(2-nitrophenyl)acrylonitrile (13)
The stability of docking-predicted ligand poses were evaluated in
Molecular Dynamics (MD) simulations using the program NAMD (version
2.10) [37]. The simulations were conducted using periodic boundary
conditions in all directions. The cut-off distance for both Coulombic and
van-der-Waals (vdW) interactions was set to 12 Å and the force-based
switching function was used for the vdW interactions with a switching
range of 10-12 Å. Long-range Coulombic interactions were evaluated by
the Particle Mesh Ewald method [38]. The temperature was maintained at
2-Nitrobenzaldehyde (1.05 eq., 151 mg, 1.0 mmol), was added to a
vigorously stirred solution of water (10 mL) and heated to 50 °C. 2,6-
dichlorophenyl acetonitrile (176 mg, 0.95 mmol) was then slowly added
forming
a
suspension. After 5-10 minutes of stirring, 40%
PhCH2NMe3(OH) (7 mL) was added dropwise. After complete addition, the
reaction vessel stirred at 50 °C for 5 hours. After this period, the solution
was filtered hot, washed with warm water and dried under suction to yield
a solid. The crude solid was then purified via column chromatography (0-
100% EtOAc in hexanes) to afford 13 as a white solid (112 mg, 35%), m.p.
144-146 °C. 1H NMR (400 MHz, acetone-d6) d 8.34 (dd, J = 8.2, 0.8 Hz,
1H), 8.07 – 8.00 (m, 2H), 7.90 (s, 1H), 7.89 – 7.84 (m, 1H), 7.65 (d, J = 1.6
Hz, 1H), 7.63 (s, 1H), 7.57 (dd, J = 9.2, 6.9 Hz, 1H); 13C NMR (101 MHz,
acetone-d6) δ 149.5, 148.4, 135.9, 135.5, 132.9 (2C), 132.53 (2C), 132.49,
131.9, 130.2, 129.7, 126.1, 116.0, 110.8; IR umax/cm-1: 3049 (C=C), 2213
(CN), 1518 (NO2), 1339 (NO2), 779 (C-Cl); LRMS (ESI+) m/z: 341 [M+Na].
HRMS cal’d for C15H9Cl2N2O2 (M+H): 319.0046; found:
303.15 K, controlled by Langevin dynamics while the pressure was
[39]
maintained at 1 atm using the Nosé-Hoover Langevin piston method
.
The bonds with hydrogen atoms were constrained to their equilibrium
lengths using the RATTLE algorithm [40]. Simulations were performed with
an energy minimization of 10,000 steps, six equilibrium stages, totalling
0.75 ns, followed by a production phase of 100 ns with a time step of 2 fs.
During the equilibration, all heavy atoms of the protein-ligand complex
were harmonically restrained to their initial positions, with an initial force
constant of 25 kcal/mol/Å2 that was gradually reduced at each stage.
(Z)-2-(2,6-dichlorophenyl)-3-(3-nitrophenyl)acrylonitrile (14)
Simulations were visualized with the program VMD [41]
.
Synthesized using the general procedure as for 13, using 3-
nitrobenzaldehyde (1.05 eq., 151 mg, 1.0 mmol) and 2,6-dichlorophenyl
acetonitrile (176 mg, 0.95 mmol). The crude solid was purified via column
chromatography (0-100% EtOAc in hexanes) to afford 14 as a white solid
(145 mg, 45%), m.p. 167-169 °C. 1H NMR (400 MHz, acetone-d6) d 8.85
(s, 1H), 8.43 (t, J = 6.8 Hz, 2H), 7.93 (t, J = 8.0 Hz, 1H), 7.70 (s, 1H), 7.66-
7.64 (m, 2H), 7.59-7.55 (m, 1H); 13C NMR (101 MHz, acetone-d6) d 149.6,
148.9, 135.8, 135.7, 135.4, 133.0 (2C), 132.9, 131.6, 129.8 (2C), 126.5,
124.5, 116.5, 109.2; IR umax/cm-1: 3082 (C=C), 2214 (CN), 1525 (NO2),
1347 (NO2), 779 (C-Cl); LRMS (ESI+) m/z: 341 [M+Na]. HRMS cal’d for
C15H9Cl2N2O2 (M+H): 319.0046; found:
Starting from a docked ligand/receptor pose, the system was solvated,
potassium and chloride ions were added at 150 mM to mimic physiological
conditions while preserving charge neutrality. The system was then
solvated in a cubic box of water. The dimensions of the box were chosen
such that the minimum distance between any atom of the solute and the
box walls was 12 Å. All simulations used the CHARMM36 force field for
[42]
the protein
and the ions and the CHARMM-modified TIP3P model for
water molecules [43-45]. The ligand parameters were generated from the
CHARMM general force field [46]
.
(Z)-2-(3,4-dichlorophenyl)-3-(2-nitrophenyl)acrylonitrile (15)
Chemistry
Synthesized using the general procedure as for 13, using 2-
nitrobenzaldehyde (1.05 eq., 500 mg, 3.3 mmol) and 3,4-dichlorophenyl
acetonitrile (587 mg, 3.15 mmol). The crude solid was recrystallized from
ethanol to afford 15 as a golden crystalline solid (526 mg, 52%), m.p. 172-
174 °C. 1H NMR (600 MHz, acetone-d6) δ 8.43 (s, 1H), 8.32 (d, J = 8.2 Hz,
1H), 8.03 (d, J = 7.6 Hz, 1H), 7.99 (s, 1H), 7.97 (d, J = 7.5 Hz, 1H), 7.83 (t,
J = 7.8 Hz, 1H), 7.78 (s, 2H); 13C NMR (151 MHz, acetone-d6) δ 148.7,
142.9, 135.3, 134.9, 134.1, 133.8, 132.3, 132.2, 131.9, 130.8, 128.9, 127.1,
126.1, 116.7, 114.2; IR umax/cm-1: 3073 (C=C), 2223 (CN), 1521 (NO2),
1341 (NO2), 721 (C-Cl); LRMS (ESI+) m/z: 341 [M+Na]. HRMS cal’d for
C15H9Cl2N2O2 (M+H): 319.0046; found:
General Methods
All reactions were performed using standard laboratory equipment and
glassware. Solvents and reagents were purchased from Sigma Aldrich,
Alfa Aesar or AK Scientific and used as received. Organic solvents were
of bulk quality, and were distilled from glass prior to use. Organic solvent
extracts were dried with magnesium sulfate (MgSO4), and dried under
reduced pressure with either Büchi or Heidolph rotary evaporators. Melting
points were recorded in open capillaries on a Stuart SMP11 Melting Point
Apparatus.
Where available, literature values are provided and
appropriately referenced. Electrospray mass spectra were recorded using
10% DMSO/H2O or HPLC-grade methanol or acetonitrile as carrier
solvents on an Agilent Technologies 1260 Infinity UPLC system with a
6120 Quadrupole LC/MS in electrospray ionization (ESI) positive and
negative modes. TLC was performed on Merck silica gel 60 F254 pre-
(Z)-2-(3,4-dichlorophenyl)-3-(3-nitrophenyl)acrylonitrile (16)
Synthesized using the general procedure as for 13, using 3-
nitrobenzaldehyde (1.05 eq., 500 mg, 3.3 mmol) and 3,4-dichlorophenyl
9
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10.1002/cmdc.201800256
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acetonitrile (587 mg, 3.15 mmol). The crude solid was recrystallized from
ethanol to afford 15 as a pale pink solid (871 mg, 87%), m.p. 217-218 °C.
1H NMR (600 MHz, acetone-d6) δ 8.85 (s, 1H), 8.44 (d, J = 7.8 Hz, 1H),
8.39 (dd, J = 8.2, 1.9 Hz, 1H), 8.28 (s, 1H), 8.06 (d, J = 2.2 Hz, 1H), 7.89
(t, J = 8.0 Hz, 1H), 7.81 (dd, J = 8.5, 2.2 Hz, 1H), 7.77 (d, J = 8.5 Hz, 1H);
13C NMR (101 MHz, DMSO-d6) δ 148.0, 142.3, 135.0, 134.9, 133.9, 132.4,
132.2, 131.4, 130.7, 127.6, 126.4, 125.2, 124.0, 116.8, 110.6; IR
127.10, 127.05, 125.8, 117.0, 108.0, 106.5, 106.2, 103.4; IR umax/cm-1:
3218 cm-1 (NH), 2215 cm-1 (CN), 1603 cm-1 (NH), 815 cm-1 (C-Cl) 734 cm-1
(C-Br); LRMS (ESI+) m/z: 499 (M+H). HRMS cal’d for C13H6Br3Cl2N2
(M+H): 498.7443; found:
(Z)-2-(2,6-dichloro-3-nitrophenyl)-3-(2-nitrophenyl)acrylonitrile (26)
2,6-dichlorophenyl acetonitrile (1000 mg, 5.4 mmol) was combined with 10
mL conc. H2SO4 at 0 °C. The mixture was maintained at this temperature
for 10 minutes; 90% fuming nitric acid (253 µL, 5.4 mmol) was added
dropwise. The reaction was sealed and left to stir at 0 °C for 1.5 h. The
mixture was poured into an ice slurry, and the desired product was isolated
via vacuum filtration to afford 22 as a cream/white coloured solid (1007 mg,
81%), m.p. 80-82 °C. 1H NMR (400 MHz, CDCl3) δ 7.83 (d, J = 8.8 Hz,
1H), 7.57 (d, J = 8.8 Hz, 1H), 4.12 (s, 2H); 13C NMR (101 MHz, CDCl3) δ
147.9, 139.7, 130.3, 129.1, 128.8, 126.1, 114.4, 20.5; IR umax/cm-1: 3081
(C=C), 2253 (CN), 1530 (NO2), 1350 (NO2), 754 (C-Cl); LRMS (ESI+) m/z:
231 [M+H]. HRMS cal’d for C8H5Cl2N2O2 (M+H): 230.9733; found:
u
max/cm 1: 3086 (C=C), 2200 (CN), 1526 (NO2), 1352 (NO2), 734 (C-Cl);
LRMS (ESI+) m/z: 319 [M+H]. HRMS cal’d for C15H9Cl2N2O2 (M+H):
319.0046; found:
(Z)-3-(5-bromo-1H-pyrrol-2-yl)-2-(3,4-dichlorophenyl)acrylonitrile (18)
The Vapourtec easyPhotochem was fitted with a Type 1 filter and the light
was set to 50%. (Z)-2-(3,4-dichlorophenyl)-3-(1H-pyrrol-2-yl)acrylonitrile
(ANI-7, 5) (197 mg, 0.75 mmol) in acetonitrile (0.05 M, 15 mL) was
combined with N-bromosuccinimide (1.1 eq., 147 mg, 0.83 mmol) in
acetonitrile (0.055 M, 15 mL) and flowed through the photoreactor at a
combined rate of 2 mL.min-1 (residence time, 5 min) at 0 °C. The reaction
stream was collected, concentrated under reduced pressure and purified
via column chromatography (0-100% EtOAc in hexanes) to afford both 18
and 19.
Compound 26 was then synthesized using the general procedure as for
13, using 2-nitrobenzaldehyde (1.05 eq., 318 mg, 2.1 mmol) and 2-(2,6-
dichloro-3-nitrophenyl) acetonitrile (22) (462 mg, 2.0 mmol). The crude
solid was purified via column chromatography (0-100% EtOAc in hexanes)
to afford 26 as a pale yellow solid (108 mg, 15%), m.p. 150-152 °C. 1H
NMR (400 MHz, CDCl3) δ 8.33 (dd, J = 8.2, 1.0 Hz, 1H), 8.02 (d, J = 7.7
Hz, 1H), 7.91 (d, J = 8.8 Hz, 1H), 7.87 (td, J = 7.6, 0.9 Hz, 1H), 7.76 – 7.71
(m, 2H), 7.64 (d, J = 8.8 Hz, 1H); 13C NMR (101 MHz, CDCl3) δ 149.2,
147.7, 147.1, 139.8, 134.8, 134.3, 131.9, 131.2, 129.2, 129.0, 128.9, 126.9,
125.6, 114.6, 109.5; IR umax/cm-1: 3066 (C=C), 2215 (CN), 1522 (NO2),
1521 (NO2), 1339 (NO2), 1338 (NO2), 797 (C-Cl); LRMS (ESI+) m/z: 386
[M+Na-H]. HRMS cal’d for C15H8Cl2N3O4 (M+H): 363.9897; found:
Compound 18 was afforded as a yellow solid (75 mg, 30%), m.p. decomp
>125 °C; 1H NMR (600 MHz, CDCl3) δ 7.91 (s, 1H), 7.63 (d, J = 2.1 Hz,
1H), 7.58 (d, J = 8.3 Hz, 1H), 7.36 (dd, J = 8.3, 2.1 Hz, 1H), 7.01 (s, 1H),
6.33 – 6.31 (m, 1H), 6.19 (dd, J = 3.8, 2.4 Hz, 1H); 13C NMR (151 MHz,
CDCl3) δ 134.4, 134.3, 133.2, 132.9, 131.8, 130.8, 128.04, 127.96, 120.1,
119.9, 113.6, 106.1, 102.8; IR umax/cm-1: 3256 (NH), 2213 (CN), 1585
(C=C), 773 (C-Cl); LRMS (ESI+) m/z: 341 [M+H]. HRMS cal’d for
C13H8BrCl2N2 (M+H): 340.9253; found:
(Z)-3-(4-bromo-1H-pyrrol-2-yl)-2-(3,4-dichlorophenyl)acrylonitrile (19)
(Z)-2-(2,6-dichloro-3-nitrophenyl)-3-(3-nitrophenyl)acrylonitrile (27)
Synthesised according to the procedure outlined above for compound 18.
Compound 19 was afforded as a mustard-yellow solid (129 mg, 50%), m.p.
decomp >135 °C; 1H NMR (600 MHz, CDCl3) δ 9.61 (s, NH), 7.64 (s, 1H),
7.48 (d, J = 8.4 Hz, 1H), 7.39 (dd, J = 8.4, 1.6 Hz, 1H), 7.23 (s, 1H), 6.65
(s, 1H), 6.34 – 6.33 (m, 1H); 13C NMR (151 MHz, CDCl3) δ 133.8, 133.7,
132.5, 131.2, 130.7, 128.8, 126.8, 124.3, 121.4, 119.8, 107.5, 99.8; IR
Synthesized using the general procedure as for 13, using 3-
nitrobenzaldehyde (1.05 eq., 474 mg, 3.1 mmol) and 2-(2,6-dichloro-3-
nitrophenyl) acetonitrile (22) (688 mg, 2.6 mmol). The crude solid was
purified via column chromatography (0-100% EtOAc in hexanes) to afford
27 as a white solid (214 mg, 15%), m.p. 151-152 °C; 1H NMR (400 MHz,
acetone-d6) δ 8.87 – 8.86 (m, 1H), 8.48 – 8.42 (m, 2H), 8.20 (d, J = 8.8 Hz,
1H), 7.98 – 7.94 (m, 2H), 7.87 (s, 1H); 13C NMR (101 MHz, acetone-d6) δ
150.2, 149.7, 149.0, 139.7, 135.8, 135.4, 135.1, 131.8, 130.8, 128.3, 128.0,
126.8, 124.7, 116.0, 108.1 (one quaternary carbon not observed in spectra,
confirmed by 2D correlations (149.0)); IR umax/cm-1: 3085 (C=C), 2219
(CN), 1523 (NO2), 1351 (NO2), 758 (C-Cl); LRMS (ESI+) m/z: 386 [M+Na-
H]. HRMS cal’d for C15H8Cl2N3O4 (M+H): 363.9897; found:
u
max/cm-1: 3263 (NH), 2205 (CN), 1596 (C=C), 767 (C-Cl); LRMS (ESI+)
m/z: 341 [M+H]. HRMS cal’d for C13H8BrCl2N2 (M+H): 340.9253; found:
(Z)-3-(4,5-dibromo-1H-pyrrol-2-yl)-2-(3,4-dichlorophenyl)acrylonitrile
(20)
The Vapourtec easyPhotochem was fitted with a Type 4 filter and the light
was set to 50%. (Z)-2-(3,4-dichlorophenyl)-3-(1H-pyrrol-2-yl)acrylonitrile
(5) (263 mg, 1.0 mmol) in acetonitrile (0.05 M, 20 mL) was combined with
N-bromosuccinimide (2.0 eq., 356 mg, 2.0 mmol) in acetonitrile (0.10 M,
20 mL) and flowed through the photoreactor at a combined rate of 4
mL.min-1 (residence time, 2.5 min) at 0 °C. The reaction stream was
collected, concentrated under reduced pressure and purified via column
chromatography (0-5% MeOH in DCM) to afford compound 20 as a yellow
solid (188 mg, 45%); 1H NMR (600 MHz, acetone-d6) δ 7.81 (d, J = 2.3 Hz,
1H), 7.71 (s, 1H), 7.69 (d, J = 8.5 Hz, 1H), 7.61 (dd, J = 8.5, 2.3 Hz, 1H),
7.33 (s, 1H); 13C NMR (151 MHz, acetone-d6) δ 135.6, 133.7, 132.7, 132.2,
131.9, 130.3, 127.7, 126.0, 118.3, 116.2, 107.2, 103.6, 102.4; IR
umax/cm-1: 3184 (NH), 2224 (CN), 1601 (C=C), 797 (C-Cl); LRMS (ESI+)
m/z: 421 [M+H]; HRMS cal’d for C13H7Br2Cl2N2 (M+H): 420.8338; found:
(Z)-2-(2,6-dichloro-3-nitrophenyl)-3-(4-nitrophenyl)acrylonitrile (28)
Synthesized using the general procedure as for 13, using 4-
nitrobenzaldehyde (1.05 eq., 159 mg, 1.05 mmol) and 2-(2,6-dichloro-3-
nitrophenyl) acetonitrile (22) (231 mg, 1.0 mmol). The crude solid was
purified via column chromatography (0-100% EtOAc in hexanes) to afford
28 as a yellow solid (98 mg, 27%), m.p. 154-156 °C; 1H NMR (400 MHz,
acetone-d6) δ 8.48 (dd, J = 7.0, 1.8 Hz, 2H), 8.26 (d, J = 8.7 Hz, 2H), 8.20
(d, J = 8.8 Hz, 1H), 7.95 (d, J = 8.8 Hz, 1H), 7.86 (s, 1H); 13C NMR (101
MHz, acetone-d6) δ 150.3, 150.2, 149.0, 139.6, 139.4, 135.3, 131.3 (2C),
130.7, 128.3, 128.0, 125.1 (2C), 115.9, 109.1; IR umax/cm-1: 3082 (C=C),
2223 (CN), 1514 (NO2), 1339 (NO2), 747 (C-Cl); LRMS (ESI+) m/z: 364
[M+H]. HRMS cal’d for C15H8Cl2N3O4 (M+H): 363.9897; found:
(Z)-2-(3,4-dichlorophenyl)-3-(3,4,5-tribromo-1H-pyrrol-2-
yl)acrylonitrile (21)
(Z)-2-(3-amino-2,6-dichlorophenyl)-3-(2-aminophenyl)acrylonitrile
(29)
The Vapourtec easyPhotochem was fitted with a Type 4 filter and the light
was set to 50%. (Z)-2-(3,4-dichlorophenyl)-3-(1H-pyrrol-2-yl)acrylonitrile
(5) (250 mg, 0.95 mmol) in acetonitrile (0.05 M, 19.5 mL) was combined
with N-bromosuccinimide (3.0 eq., 507 mg, 2.85 mmol) in acetonitrile (0.15
M, 19.5 mL) and flowed through the photoreactor at a combined rate of 4
mL.min-1 (residence time, 2.5 min) at 0 °C. The reaction stream was
collected and filtered to afford compound 21 as an orange/yellow solid (224
mg, 47%); 1H NMR (600 MHz, DMSO) δ 13.02 (bs, NH), 8.00 (d, J = 2.2
Hz, 1H), 7.75 (d, J = 8.5 Hz, 1H), 7.68 (dd, J = 8.5, 2.2 Hz, 1H), 7.63 (s,
1H); 13C NMR (151 MHz, DMSO) δ 133.9, 132.2, 131.7, 131.4, 130.9,
(Z)-2-(2,6-dichloro-3-nitrophenyl)-3-(2-nitrophenyl)acrylonitrile (26) (160
mg, 0.44 mmol) and iron powder (12 eq., 295 mg, 5.3 mmol) were
combined with a mixture of glacial acetic acid (2 mL), ethanol (2 mL), and
water (1 mL) and exposed to ultrasonic irradiation at 30 °C for 2 h. The
reaction mixture was diluted with ethanol (30 mL) and filtered through a
pad of Celite, before concentration under reduced pressure. The crude
solid was then purified via column chromatography (0-100% EtOAc in
hexanes) to afford 29 as a pale brown solid (32 mg, 23%), m.p. 226-
228 °C; 1H NMR (400 MHz, acetone-d6) δ 7.76 (s, 1H), 7.71 (d, J = 8.0
Hz, 1H), 7.61 (d, J = 8.4 Hz, 1H), 7.54 (t, J = 7.6 Hz, 1H), 7.27 – 7.21 (m,
10
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10.1002/cmdc.201800256
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2H), 6.98 (d, J = 8.8 Hz, 1H), 5.60 (bs, NH2), 5.25 (bs, NH2); 13C NMR
(101 MHz, acetone-d6) δ 156.3, 149.1, 145.4, 138.3, 135.7, 130.2, 129.3,
128.5, 126.6, 124.4, 122.7, 122.3, 122.2, 119.8, 116.9; IR umax/cm-1: 3149
(C=C), 1635 (NH2), 1615 (NH2), 788 (C-Cl); LRMS (ESI+) m/z: 305 [M+H].
HRMS cal’d for C15H12Cl2N3 (M+H): 304.0413; found:
as a crystalline yellow solid (258 mg, 80%), m.p. 71-73 °C; 1H NMR (400
MHz, acetone-d6) δ 9.67 (s, 1H), 7.61 (d, J = 8.6 Hz, 2H), 6.75 (d, J = 8.6
Hz, 2H), 5.69 (bs, NH2); 13C NMR (101 MHz, acetone-d6) δ 190.0, 155.5,
132.7 (2C), 127.3, 114.2 (2C); IR umax/cm-1: 3470 (NH2), 3349 (NH2), 1662
(C=O) 1557 (C=C); LRMS (ESI+) m/z: 122 [M+H] [47]
.
(Z)-2-(3-amino-2,6-dichlorophenyl)-3-(3-aminophenyl)acrylonitrile
(30)
Compound 35 was synthesized using the general procedure as for 13,
using 4-aminobenzaldehyde (1.05 eq., 258 mg, 2.13 mmol) and 3,4-
dichlorophenyl acetonitrile (378 mg, 2.03 mmol). The crude solid was
recrystallized from ethanol to afford 35 as a yellow solid (512 mg, 87%),
m.p. 177-180 °C; 1H NMR (400 MHz, DMSO-d6) δ 7.91 (d, J = 1.9 Hz, 1H),
7.87 (s, 1H), 7.76 (d, J = 8.6 Hz, 2H), 7.70 (d, J = 8.5 Hz, 1H), 7.61 (dd, J
= 8.6, 2.0 Hz, 1H), 6.65 (d, J = 8.6 Hz, 2H), 6.17 (s, NH2); 13C NMR (101
MHz, DMSO-d6) δ 152.5, 144.9, 135.9, 132.0 (2C), 131.9, 131.1, 129.9,
126.3, 125.1, 120.4, 118.9, 113.4 (2C), 98.7; IR umax/cm-1: 3489 (NH2),
3373 (NH2), 2206 (CN), 1619 (NH) 821 (C-Cl); LRMS (ESI-) m/z: 288 [M-
H]. Product contains solvent impurities calculated at 0.43% for acetone.
HRMS cal’d for C15H11Cl2N2 (M+H): 289.0304; found:
Synthesized using the general procedure as for 29, with 27 (134 mg, 0.37
mmol) and iron powder (12 eq., 247 mg, 4.4 mmol). After 2 h, the reaction
mixture was diluted with ethanol (30 mL) and filtered through a pad of
Celite, before concentration under reduced pressure, to afford 30 as a
yellow crystalline solid (109 mg, 97%), m.p. 49-52 °C; 1H NMR (400 MHz,
acetone-d6) δ 7.26 – 7.17 (m, 4H), 7.15 (s, 1H), 6.98 (d, J = 8.8 Hz, 1H),
6.85 (ddd, J = 7.7, 2.2, 1.4 Hz, 1H), 5.34 (bs, NH2), 4.97 (bs, NH2); 13C
NMR (101 MHz, acetone-d6) δ 151.1, 150.0, 145.5, 134.9, 134.0, 130.5,
129.4, 124.6, 123.3, 121.6, 120.5, 118.7, 118.1, 117.4, 114.9; IR
u
max/cm-1: 3221 (C=C), 2209 (CN), 1615 (NH2), 1581 (NH2), 783 (C-Cl);
LRMS (ESI+) m/z: 305 [M+H]. Product contains solvent impurities
calculated at 1.16% for acetone and 1.24% for diethyl ether. HRMS cal’d
for C15H12Cl2N3 (M+H): 304.0413; found:
(Z)-2-(3,4-dichlorophenyl)-3-(4-(methylamino)phenyl)acrylonitrile
(36) [48]
4-Bromobenzaldehyde (377 mg, 2.0 mmol), copper powder (0.05 eq., 17
mg, 0.1 mmol) and 40 % aq. methylamine (5 eq., 1.1 mL, 10 mmol) were
combined in a 2-5 mL vial, sealed and heated at 100 °C in a sand bath for
19 h. The reaction mixture was diluted with ethyl acetate (30 mL), washed
with water (3 x 30 mL), dried over MgSO4 and concentrated under reduced
pressure to afford 4-(methylamino)benzaldehyde as a crystalline yellow
solid (234 mg, 87%), m.p. 42-43 °C; 1H NMR (400 MHz, acetone-d6) δ
8.10 (d, J = 1.4 Hz, 1H), 7.50 (d, J = 8.6 Hz, 2H), 6.60 (d, J = 8.6 Hz, 2H),
5.37 (bs, NH), 3.34 (d, J = 1.5 Hz, 3H); 13C NMR (101 MHz, acetone-d6)
δ 162.3, 152.9, 130.1 (2C), 126.2, 112.2 (2C), 48.0; IR umax/cm-1: 3247
(Z)-3-(4-bromo-3-nitrophenyl)-2-(3,4-dichlorophenyl)acrylonitrile (32)
Synthesized using the general procedure as for 13, using 4-bromo-3-
nitrobenzaldehyde (600 mg, 2.6 mmol) and 3,4-dichlorophenyl acetonitrile
(462 mg, 2.5 mmol). The crude solid was recrystallized from ethanol to
afford 32 as a grey/purple solid (644 mg, 65%), m.p. 189-192 °C; 1H NMR
(400 MHz, acetone-d6) δ 8.50 (d, J = 2.1 Hz, 1H), 8.22 (dd, J = 8.5, 2.1 Hz,
1H), 8.19 (s, 1H), 8.10 (d, J = 8.5 Hz, 1H), 8.03 (d, J = 2.0 Hz, 1H), 7.81 –
7.75 (m, 2H); 13C NMR (101 MHz, acetone-d6) δ 141.5, 136.6, 135.5,
135.1, 134.2, 134.1, 133.9, 133.8, 132.3, 128.8, 127.2, 127.1, 117.2, 116.3,
113.1; IR umax/cm-1: 3096 (C=C), 2222 (CN), 1531 (NO2), 1338 (NO2), 734
(C-Cl); LRMS (ESI+) m/z: 399 [M+H]. HRMS cal’d for C15H8BrCl2N2O2
(M+H): 398.9131; found:
(NH2), 1600 (C=O) 1537 (C=C); LRMS (ESI+) m/z: 136 [M+H] [48]
.
Compound 36 was synthesized using the general procedure as for 13,
using 4-(methylamino)benzaldehyde (1.05 eq., 226 mg, 1.67 mmol) and
3,4-dichlorophenyl acetonitrile (297 mg, 1.59 mmol). The crude solid was
recrystallized from ethanol to afford 36 as a bright orange solid (465 mg,
92%), m.p. 140-143 °C; 1H NMR (400 MHz, DMSO-d6) δ 7.91 (d, J = 2.0
Hz, 1H), 7.90 (s, 1H), 7.83 (d, J = 8.8 Hz, 2H), 7.70 (d, J = 8.5 Hz, 1H),
7.61 (dd, J = 8.5, 2.1 Hz, 1H), 6.74 (q, J = 4.7 Hz, NH), 6.65 (d, J = 8.8 Hz,
2H), 2.77 (d, J = 5.0 Hz, 3H); 13C NMR (101 MHz, DMSO-d6) δ 152.7,
144.9, 135.9, 131.9, 131.1 (2C), 129.9, 126.3, 125.1, 120.3, 118.9 (2C),
98.6, 29.2; IR umax/cm-1: 3389 (NH), 2200 (CN), 1611 (NH) 807 (C-Cl);
LRMS (ESI+) m/z: 303 [M+H]. HRMS cal’d for C16H13Cl2N2 (M+H):
303.0461; found:
(Z)-3-(2-amino-4-bromophenyl)-2-(3,4-dichlorophenyl)acrylonitrile
(33)
Synthesized using the general procedure as for 13, using 2-amino-4-
bromobenzaldehyde (1.05 eq., 210 mg, 1.05 mmol) and 3,4-
dichlorophenyl acetonitrile (186 mg, 1.0 mmol). The crude solid was
recrystallized from ethanol to afford 33 as a pale yellow crystalline solid
(223 mg, 61%), m.p. 215-217 ºC; n1H NMR (600 MHz, acetone-d6) δ 7.91
(s, 1H), 7.76 (d, J = 2.0 Hz, 1H), 7.74 (d, J = 1.7 Hz, 1H), 7.71 (d, J = 8.2
Hz, 1H), 7.67 (d, J = 8.5 Hz, 1H), 7.55 (dd, J = 8.2, 2.0 Hz, 1H), 7.35 (dd,
J = 8.5, 1.9 Hz, 1H), 5.94 (bs, NH2); 13C NMR (151 MHz, acetone-d6) δ
157.2, 149.9, 139.3, 138.0, 133.3, 132.4, 132.0, 131.9, 130.3, 130.0, 128.6,
126.0, 124.1, 124.0, 123.4; IR umax/cm-1: 3473 (NH2), 3291 (C=C), 1644
(NH2), 778 (C-Cl); LRMS (ESI+) m/z: 369 [M+H]. HRMS cal’d for
C15H10BrCl2N2 (M+H): 368.9389; found:
(Z)-2-(3,4-dichlorophenyl)-3-(4-(dimethylamino)phenyl)acrylonitrile
(37)
Synthesized using the general procedure as for 13, using 4-
(dimethylamino)benzaldehyde (1.05 eq., 250 mg, 1.67 mmol) and 3,4-
dichlorophenyl acetonitrile (297 mg, 1.59 mmol). The crude solid was
recrystallized from ethanol to afford 37 as a bright yellow solid (388 mg,
77%), m.p. 205-208 °C; 1H NMR (400 MHz, DMSO-d6) δ 7.96 (s, 1H), 7.94
(d, J = 2.2 Hz, 1H), 7.90 (d, J = 9.0 Hz, 2H), 7.72 (d, J = 8.5 Hz, 1H), 7.64
(dd, J = 8.5, 2.2 Hz, 1H), 6.83 (d, J = 9.0 Hz, 2H), 3.04 (s, 6H); 13C NMR
(101 MHz, DMSO-d6) δ 152.1, 144.6, 135.8, 131.9, 131.6 (2C), 131.1,
130.0, 126.3, 125.2, 120.4, 118.8, 111.6 (2C), 99.4, 39.6 (2C); IR
(Z)-2-(3,4-dichlorophenyl)-3-(4-hydroxy-3-methoxy-5-
nitrophenyl)acrylonitrile (34)
Synthesized using the general procedure as for 13, using 5-nitro vanillin
(1.05 eq., 207 mg, 1.05 mmol) and 3,4-dichlorophenyl acetonitrile (186 mg,
1.0 mmol). The crude solid was recrystallized from ethanol to afford 34 as
a black/red crystalline solid (193 mg, 53%), m.p. >260 ºC (dec.); 1H NMR
(600 MHz, DMSO-d6) δ 8.07 (s, 1H), 7.88 (d, J = 35.2 Hz, 2H), 7.66 (d, J
= 8.1 Hz, 1H), 7.58 (d, J = 7.5 Hz, 1H), 7.53 (s, 1H), 3.67 (s, 3H); 13C NMR
(151 MHz, DMSO-d6) δ 163.7, 155.3, 144.9, 136.6, 135.6, 131.8, 130.9,
128.9, 128.0, 125.6, 124.6, 119.7, 111.0, 107.2, 95.0, 55.1; IR umax/cm-1:
2970 (C=C), 2204 (CN), 1539 (NO2), 1355 (NO2), 782 (C-Cl) LRMS (ESI-)
m/z: 363 [M-H]; HRMS cal’d for C16H11Cl2N2O4 (M+H): 365.0101; found:
u
max/cm-1: 2910 (C=C), 2210 (CN), 806 (C-Cl); LRMS (ESI+) m/z: 317
[M+H]. HRMS cal’d for C17H15Cl2N2 (M+H): 317.0617; found:
(Z)-N-(4-(2-cyano-2-(3,4-dichlorophenyl)vinyl)phenyl)acetamide (38)
Synthesized using the general procedure as for 13, using 4-
acetamidobenzaldehyde (1.05 eq., 250 mg, 1.67 mmol) and 3,4-
dichlorophenyl acetonitrile (297 mg, 1.59 mmol). The crude solid was
recrystallized from ethanol to afford 38 as a bright yellow solid (169 mg,
51%), m.p. 275-277 °C; 1H NMR (600 MHz, DMSO-d6) δ 10.30 (s, 1H),
8.09 (s, 1H), 8.02 (s, 1H), 7.93 (d, J = 7.9 Hz, 2H), 7.78 – 7.74 (m, 3H),
7.70 (d, J = 7.8 Hz, 1H), 2.09 (s, 3H); 13C NMR (151 MHz, DMSO-d6) δ
168.9, 144.2, 142.0, 134.8, 132.1, 131.3, 131.2, 130.5 (2C), 127.8, 127.1,
125.9 (2C), 118.8, 117.8, 105.3, 24.2; IR umax/cm-1: 3335 (NH), 3033
(Z)-3-(4-aminophenyl)-2-(3,4-dichlorophenyl)acrylonitrile (35) [47]
4-Nitrobenzaldehyde (402 mg, 2.66 mmol) and iron powder (6 eq., 891 mg,
16.0 mmol) were combined with methanol (5 mL) and saturated aq.
ammonium chloride (5 mL) in a 10-20 mL vial, which was sealed and
irradiated at 120 °C for 5 min. The reaction mixture was then diluted with
CH2Cl2 (30 mL), washed with aq. NaHCO3 (3 x 30 mL), dried over MgSO4
and concentrated under reduced pressure to afford 4-amino benzaldehyde
11
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(C=C), 2221 (CN), 1686 (C=O), 815 (C-Cl); LRMS (ESI+) m/z: 331 [M+H].
HRMS cal’d for C17H13Cl2N2O (M+H): 331.0410; found:
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(Z)-3-(1H-benzo[d]imidazol-6-yl)-2-(3,4-dichlorophenyl)acrylonitrile
(39)
Synthesized using the general procedure as for 13, using 1H-
benzo[d]imidazole-5-carbaldehyde (1.05 eq., 153 mg, 1.05 mmol) and 3,4-
dichlorophenyl acetonitrile (186 mg, 1.0 mmol). The crude solid was
recrystallized from ethanol to afford 39 as a pale yellow solid (128 mg,
41%), m.p. 270-273 °C; 1H NMR (600 MHz, DMSO-d6) δ 12.82 (bs, NH),
8.39 (s, 1H), 8.32 – 8.30 (m, 2H), 8.06 (d, J = 1.7 Hz, 1H), 7.86 (d, J = 8.1
Hz, 1H), 7.79 (d, J = 8.4 Hz, 1H), 7.75 – 7.73 (m, 2H); 13C NMR (151 MHz,
DMSO-d6) δ 145.9, 144.58, 144.57, 135.0 (2 overlapping signals), 132.0,
131.23, 131.17, 127.2, 127.1, 125.9 (2 overlapping signals), 123.9, 118.0,
105.0; LRMS (ESI+) m/z: 314 [M+H]. HRMS cal’d for C16H10Cl2N3 (M+H):
314.0257; found:
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Entry for the Table of Contents
Insert graphic for Table of Contents here. ((Please ensure your graphic is in one of following formats))
CN
Cl
N
H
Cl
H
N
CN
Cl
Cl
O
38; MCF-7; GI50 = 34 nM
Breast cancer specificity
of up to 1400-fold
Targeting the aryl hydrocarbon receptor reveals novel breast cancer cytotoxic specific small molecules. Selectivity levels of 250-fold
and 10 nM potency is noted against the SKBR3 cell line.
13
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