Full text of DOI:10.1039/d1md00021g

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Amino alcohol acrylonitriles as broad spectrum
and tumour selective cytotoxic agents†
Cite this: DOI: 10.1039/d1md00021g
b
*
Jennifer R. Baker,
Adam McCluskey
a Cecilia C. Russell, a Jayne Gilbert,
and Jennette A. Sakoff
a
b
We have identified specific dichlorophenylacrylonitriles as lead compounds in the development of novel
anticancer compounds, notably, (Z)-N-(4-(2-cyano-2-(3,4-dichlorophenyl)vinyl)phenyl)acetamide (1) and
ANI-7 (2). Herein we specifically probe the SAR associated with the terminal aromatic ring and associated
cytoxicity in a broad range of human cancer cell lines. Synthesis of three focused libraries revealed a poor
tolerance for electron withdrawing and donating moieties (Library A). A clear preference for hydrophobic
substituents on a terminal piperazine moiety (Library B) with good levels of broad spectrum cytotoxicity,
e.g. 13a (GI50 2.5–6.0 μM), as did the introduction of a methylene spacer with 13i (4-CH3PhCH2; GI50 1.5–
4.5 μM). Removal of the aromatic moiety and installation of simple hydrophobic groups (Library C), in
particular an adamantyl moiety, afforded highly active broad spectrum cytotoxic agents with GI50 values
ranging from 1.7 μM (14k; 1-adamantyl) to 5.6 μM (14i; pyrrolidine). Within these libraries we note lung
cancer selectivity, relative to normal cells, of 13h (fluoro substituted acrylonitrile, GI50 1.6 μM, 9.3-fold
selective); the colorectal selectivity of 14h (methylpiperidine analogue, GI50 0.36 μM, 6.9-fold selective) and
the breast cancer selectivity of 13f (nitrile substituted acrylonitrile, GI50 2.3–6.0 μM, up to 20-fold selective).
The latter was confirmed as a novel AhR ligand and a CYP1A1 activating compound, that likely induces cell
death following bioactivation; a phenomenon previously described in breast cancer cell populations.
Received 23rd January 2021,
Accepted 15th February 2021
DOI: 10.1039/d1md00021g
rsc.li/medchem
the AhR include immune suppression, tumorigenesis, the
regulation of genes involved in energy and xenobiotic
Introduction
Despite advances in treatment, globally more than 500 000
women die of breast cancer each year.1 Moreover, there are
no targeted therapies for the more aggressive triple negative
cancers, that lack the estrogen (ER), progesterone (PR) and
HER-2 hormone receptors. Even more disturbingly, resistance
to common broad-spectrum chemotherapeutics is an
increasing issue.2–4 Clearly, there is an urgent need for novel
agents and targets to address this shortfall. Indeed, over the
past few years our team has focused on the development of
novel agents that mediate their effects via the aryl
hydrocarbon receptor (AhR) pathway as a promising approach
for the treatment of breast cancer.5–10 While the AhR pathway
is traditionally known for its role in metabolising
environmental toxins, it also functions in initiating and
maintaining breast cancer growth.11,12 Key oncogenic roles of
metabolism, and subsequent roles in cell proliferation and
the cell cycle.13 The upregulation of AhR in the cytosol of
inflammatory breast cancer cells, with increased levels
correlating with poor prognosis make the AhR an attractive
breast cancer treatment target.14–16
The AhR is a transcription factor found in the cytoplasm
of cells complexed with two Hsp90 (heat shock protein 90)
molecules and associated p23 co-chaperones, the Aryl
Hydrocarbon interacting protein (AIP), and Src kinase.
Subsequent ligand binding initiates translocation to the
nucleus of the cell wherein the AhR heterodimerizes with the
aryl hydrocarbon nuclear translocator (ARNT), and Hsp90
together with Src are released. The heterodimer then binds to
xenobiotic response elements (XRE) (found in the gene
promotor region), upregulating the expression of target genes
including cytochrome P450s CYP1A1, CYP1A2 and CYP1B1.17
In this arena we have developed the dichlorinated
phenylacrylonitriles as potent and selective breast cancer
(in vitro cell lines) agents, that elicit their effects via the AhR
pathway.5,7,17 Within this class of compounds we note (Z)-N-
(4-(2-cyano-2-(3,4-dichlorophenyl)vinyl)phenyl)-acetamide (1)
and ANI-7 (2) (Fig. 1), with the latter showing GI50 values of
0.16–2.0 μM in breast cancer cell lines including those with a
a Chemistry, School of Environmental & Life Sciences, The University of Newcastle,
University Drive, Callaghan, NSW 2308, Australia.
E-mail: Jennifer.R.Baker@newcastle.edu.au
b Experimental Therapeutics Group, Department of Medical Oncology, Calvary
Mater Newcastle Hospital, Edith Street, Waratah, NSW 2298, Australia
† Electronic supplementary information (ESI) available: Including ORCIDs for all
authors, and 1H and 13C NMR for all compounds herein. See DOI: 10.1039/
d1md00021g
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dichlorophenylacrylonitrile epoxide (10), as
a common
intermediate for all subsequent analogues (Scheme 1).28
Library A development saw epoxide (10) ring opening with
a selection of amines, 11a–g, which under flow chemistry
conditions gave compounds 12a–g in moderate yields (18–
43%) (Scheme 1). This library spanned a simple aniline (12a),
a
cyclohexyl (12b), as well as the substituted aniline
derivatives 4-Cl (12c), 4-Br (12d), 4-OCH3 (12e),
4-phenylpiperazine variant (12f), and 4-N(CH3)2 (12g)
moieties (Table 1). Previous work in this area exhibited no
cytotoxicity difference for the pure enantiomers afforded via
the epoxide ring opening,6 so all compounds afforded were
tested as a racemic mixture.
Fig. 1 Chemical structures of selected known AhR ligands, (Z)-N-(4-
(2-cyano-2-(3,4-dichlorophenyl)vinyl)phenyl)acetamide (1), ANI-7 (2),
aminoflavone (3), phortress (4) and the amino alcohol modified
acrylonitrile (5).
The cytotoxicity of Library
A was evaluated in a
sulforhodamine B (SRB) assay across a range of ten human
cancer and one normal cell line, viz: glioblastoma (U87 and
SJ-G2), ovarian (A2780), lung (H460), skin (A431), prostate
(Du145), neuroblastoma (BE2-C), pancreas (MIA), colon
(HT29), breast (MCF-7) and normal breast (MCF10A). The
inclusion of the MCF10A cell line allows for an interpretation
of tumour selectivity it is important to note that this is an
immortal cell line and a poor surrogate for dormant cell
populations, and for predicting human clinical toxicity.
Additional tiered models of toxicity are required to define
triple negative receptor phenotype and up to 270-fold
selectivity over the MCF10A normal breast cell line.7
Clinically, two AhR ligands have progressed to human trials,
aminoflavone (3) and phortress (4) for breast cancer (Phase
II), and 4 for colon cancer (Phase I) (Fig. 1).19,20
Our most recent efforts revealed an unexpected
complication with MTT-based phenotypic screening, with
metabolic inactivation of the MTT reaction.6 During this
study, however, on examination of the amino alcohol
modified acrylonitrile (5) (Fig. 1) an unexpected level of
potency against the colon cancer cell line HT29 was
observed.6 The current study reports the subsequent in vitro
anticancer activity of these amino alcohol acrylonitriles in a
broad spectrum of tumour types, as well as an expanded
range of previously unreported analogues. Compounds
and predict clinical toxicity. Thus,
a strong level of
cytotoxicity in this cell line does not preclude further
development.
Of the Library A analogues, examination of the SRB
cytotoxicity data shown in Table 1 revealed that 12a–e
(excepting 12b and 12g) displayed good to modest levels of
cytotoxicity (GI50 0.75–62 μM). Within this library 12a showed
4-fold selectivity towards the ovarian cell line A2780 (GI50 4.4
μM); while 12e showed preferential activity towards colorectal
HT29 (GI50 3.7 μM, 5.1-fold selectivity) and breast MCF-7
(GI50 5.2 μM, 3.6-fold selectivity) cancer cells relative to
normal MCF10A cells, 12g also showed good colorectal
selectivity (3.7-fold) and the greatest potency (GI50 0.75 μM)
in this class. The presence of electron withdrawing groups
(12c and 12d) was poorly tolerated as was the presence of the
electron donating –OCH3 moiety (12e). The observed GI50
containing
both
the
β-amino
alcohol,21–23
and
phenylacrylonitrile,24–26 moieties have shown great promise
as anticancer agents. Herein we report on the development of
a range of amino alcohol modified acrylonitriles as potent
broad-spectrum cytotoxic agents and some with tumour-
specific activity.
Results and discussion
Our initial intent was to explore the SAR associated with the
phenyl moiety (blue in Fig. 1). Our prior efforts have shown
that coupling via the 4-disposed ether moiety (green, Fig. 1)
afforded the highest level of activity;6 thus, we limited our
efforts to these analogues. Similarly, we have previously
shown the critical nature of the –OH moiety, although no
stereochemical preference was noted. In a typical synthesis,
4-hydroxybenzaldehyde (7) coupling to epi-chlorohydrin (6)
gave the epoxide (8).27 This was achieved by both flow and
batch chemistry approaches in excellent yield and purity.
Subsequent basic ionic liquid (benzyltrimethylammonium
Scheme 1 Reagents and conditions: (i) Vapourtec Easy-Medchem, 5
M 6 (anhy. DMF), 0.1 M 7 (anhy. DMF), 105 °C, 5 bar, 1.0 mL min−1 (tr =
20 min); (ii) 9, PhCH2NMe3(OH), H2O, 50 °C, 5 h; (iii) Vapourtec RS-
400, RNH2 (11a–g) 0.05 M (EtOH), 10 0.01 M (EtOH), 150 °C, 10 bar, 1.0
mL min−1 (tr = 20 min) OR 1–2 eq. amine, EtOH, μw, 120 °C, 20 min.
hydroxide)
mediated
Knoevenagel
coupling
with
3,4-dichlorophenyl acetonitrile (9) gave the desired
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Table 1 Evaluation of the cytotoxicity (GI50 values, μM) of Library A (12a–g) against a panel of ten cancer cell and one normal cell line, in an SRB assay.
GI50 is the concentration at which 50% of the cell growth is inhibited relative to an untreated control. All values are n = 3. Entries in italic mark
substantial tumour selectivity when compared with normal breast cells (MCF10A) (relative to an untreated control)
R
U87a
A2780b
H460c
A431d
Du145e
BE2-Cf
SJ-G2g
MIAh
HT29i
MCF-7j
13 2.2
MCF10Ak
18 1.9
26 3.2
4.4 0.3 14 0.8 16 0.3
22 2.7 13 0.7 18 0.7 17 1.3
10 2.5
(4-fold)
12a
12b
2.8 0.07 2.9 0.5 2.2 0.1 2.2 0.3
2.4 0.1 2.8 0.1 2.7 0.0 2.7 0.07 2.0 0.2
2.7 0.5 2.8 0.2
16 0.3
16 1.5
29 4.2
5.4 1.3
10 1.8
11 1.2 15 0.7
15 0.7 14 0.3 15 0.0 14 0.0
7.0 2.0
6.0 1.4
11 1.8
5.3 1.5
17 1.2
13 1.2
19 1.5
12c
12d
12e
10 2.4 6.8 2.2 11 3.6
13 0.9 11 3.6 11 2.8 11 1.4
17 6.4
16 6.2 62 16.3 28 9.0 41 7.0 32 7.5 27 1.9
3.7 0.3
(5.1-fold)
5.2 2.1
(3.6-fold)
6.3 0.6 4.0 0.9 6.3 1.0
3.5 0.1 16 0.6 9.0 1.0 5.7 0.8
3.3 0.4
4.1 1.0 4.8 0.3
12f
3.2 0.2
2.5 0.1 2.8 0.2 1.6 0.4
2.7 0.2 2.5 0.4 2.7 0.1 2.3 0.18 0.75 0.20 1.9 0.2 2.8 0.1
(3.7-fold)
12g
a
c
j
Glioblastoma. b Ovarian. Lung. d Skin. e Prostate. f Neuroblastoma. g Glioblastoma. h Pancreas. i Colorectal. Breast. k Breast (normal).
values for these analogues was 4–10-fold lower than that
observed with cyclohexyl (12b), phenylpiperazine (12f) and
dimethylamino (12g). This suggested that the presence of a
terminal hydrophobic moiety was favoured. Of the Library A
analogues, 12b and 12g were the most active displaying low-
micromolar activity against all cell lines tested. The
phenylpiperazine 12f also demonstrated good activity against
most cancer lines tested. This, coupled with the ready access
to substituted piperazine analogues, positioned the
hydrophobic moieties 12b and 12g, and the phenylpiperazine
12f, as the most appropriate development candidates from
this library.
Thus, oxirane (10) ring opening with an expanded
range of amines, in particular 4-phenylpiperazines,
(Table 2) gave Library B, 13a–i using the above microwave
protocol (Scheme 1) in good to excellent yields (60–96%)
with no purification required; a particularly advantageous
(13e), 4-nitrile (13f) and 4-acetyl (13g), and the 2-fluoro
(13h). An additional modification in this library was 13i, a
benzyl analogue of 13a.
On analysis of the SRB toxicity data measured for Library
B, a number of trends were apparent. Within Library B
(13a–i; Table 2), the 4-chloro (13b), 4-bromo (13c),
4-trifluoromethyl (13d), 4-nitro (13e), 4-nitrile (13f) and
4-acetyl (13g) piperazine analogues were essentially
ineffective in most cell lines tested with GI50 values up to or
greater than 50 μM. This strongly suggests a low tolerance for
an electron withdrawing substituent on the terminal
aromatic moiety. Analogues bearing hydrophobic moieties
were the most potent broad-spectrum compounds, as evident
with –CH3 (13a and 13i) which displayed low-micromolar
activity across all cell lines tested, including MCF10A cells.
The U87, MIA and SJ-G2 cell lines were particularly resistant
to this compound library. Tumour selectivity was noted for
13c again in the ovarian cell line (A2780, 3.5-fold selectivity),
13h in the lung cancer cell line (H460, 9.3-fold selectivity),
and 13f in the breast cancer cell line (MCF-7, 8.1-fold
selectivity).
factor for library development. Library
B spanned the
hydrophobic electron donating 4-methyl (13a), the electron
withdrawing halides, 4-chloro (13b), 4-bromo (13c) and
4-trifluoromethyl (13d), heteroatom-substituted 4-nitro
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Table 2 Evaluation of the cytotoxicity (GI50 values, μM) values of Library B (13a–i) against a panel of ten cancer cell and one normal cell line, in an SRB
assay. GI50 is the concentration at which 50% of the cell growth is inhibited, relative to an untreated control. All values are n = 3. Entries in italic mark
substantial tumour selectivity when compared with normal breast cells (MCF10A)
R
U87a
A2780b
H460c
A431d
Du145e BE2-Cf SJ-G2g
MIAh
HT29i
MCF-7j
MCF10Ak
6.0 3.5 3.0 0.52
2.5 0.20
3.1
2.7
2.7
2.9
3.0
2.6
2.3 0.52
3.5
0.09
0.43
0.24
0.07
0.12
0.60
0.32
13a
13b
13c
13d
13e
13f
>50
>50
>50
>50
>50
>50
15 2.5
28 11
>50
27 6.6 32 9.5 >50
24 3.0 17 2.0 9.0
0.61
15 2.4
18 2.3
49 15
>50
14 2.7
(3.5-fold)
>50
>50
>50
31 4.9 40 5.0 21 4.3 57 22 16 2.3 24 2.1
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
50
0.00
>50
>50
>50
>50
>50
>50
15 91
40 10
>50
20 5.0 17 7.0 31 12 >50
50
0.00
5.8 2.8
(8.1-fold)
47 3.3
7.5 2.3
>50
30 2.0 12 3.9 >50
>50
7.7
1.8
11 0.58
13g
13h
20 1.2 17 2.8
1.6 0.41
(9.3-fold)
14
0.88
18 1.2 21 3.8 18
24 1.8 5.4
0.83
11 0.33
1.7 0.12
15 1.5
0.33
3.6
1.5 0.17
2.7 0.12
2.4
0.15
4.5
2.0
2.4
2.6
1.4
0.26
2.4
0.15
0.97
0.59
0.13
0.22
0.26
13i
a
c
j
Glioblastoma. b Ovarian. Lung. d Skin. e Prostate. f Neuroblastoma. g Glioblastoma. h Pancreas. i Colorectal. Breast. k Breast (normal).
Shifting the hydrophobic moiety to a methylene spacer
with benzyl (13i) afforded a significant potency enhancement
with activities ranging from 1.4 μM (HT29) to 4.5 μM
(Du145). These data, in addition to that of 12b and 12g,
support the introduction of additional hydrophobic moieties
with an apparent preference for the moiety to be positioned
close to the amino alcohol; accordingly this was examined
with the development of Library C.
These modifications afforded analogues 14a–l in moderate
to good yields (39–87%) as described (Scheme 1). This library
comprising phenylpiperidine (14a), 4-acetylpiperazine (14b),
4-methylpiperazine (14c), morpholine (14d), piperidine (14e),
methylpiperidine (14f–h), pyrrolidine (14i), azepane (14j) and
adamantyl (14k and l), exhibited improved aqueous solubility
over the previous analogues,5 leading to a general trend of
lower yields.
The introduction of additional hydrophobic moieties with
Library C analogues (14a–l) saw good broad spectrum activity
across the entire Library; with the exception of the oxygen-
bearing N-acetyl (14b) and morpholine (14d) moieties
(Table 3). This data supports the observations noted with the
Library B analogues, that the removal of polar moieties from
the tail of these compounds is of benefit for cytotoxic activity.
Simple methylpiperazine (14c), methylpiperidines (14f–h) and
piperidine (14e) all displayed low-micromolar potencies.
Notwithstanding this, 14b showed a 3-fold selective response
in MCF-7 cells (GI50 5.5 μM), while 14e and 14h showed 3.8
and 6.9-fold selectivity in HT29 cells, respectively (GI50 0.66
μM and 0.36 μM). Further simplification and ring size
modification saw a slight potency drop, with the cyclopentyl
(14i) and azepane (14j) showing reduced activity against the
U87, SJ-G2 and MIA cell lines. Increasing the hydrophilic
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Table 3 Evaluation of the cytotoxicity (GI50 values, μM) values of Library C analogues 14a–l against a panel of ten cancer cell and one normal cell line,
in an SRB assay. GI50 is the concentration at which 50% of the cell growth is inhibited, relative to an untreated control. All values are n = 3. Entries in
italic mark substantial tumour selectivity when compared with normal breast cells (MCF10A)
R
U87a
A2780b
H460c
A431d
Du145e
BE2-Cf
SJ-G2g
MIAh
HT29i
MCF-7j
MCF10Ak
2.9 0.03 2.7 0.13 2.7 0.03 2.6 0.12 2.7 0.12 2.6 0.10 2.5 0.12 2.4 0.07 1.9 0.058 2.5 0.20 2.5 0.22
14a
21 2.2 6.9 2.1
14 1.9
10 1.7
15 1.0
11 0.88 13 1.4
16 1.5 8.6 2.2
5.3 2.6
(3-fold)
16 0.33
14b
14c
14d
14e
14f
2.7 0.34 2.5 0.24 2.6 0.20 1.8 0.12 2.4 0.19 2.4 0.20 2.2 0.07 2.6 0.09 1.2 0.12 2.3 0.38 2.4 0.21
17 1.0 8.9 0.92 8.7 0.20 3.0 0.03 13 1.2 3.0 0.19 9.3 0.87 13 1.0 3.1 0.13 5.3 0.91 5.6 1.2
3.0 0.15 2.7 0.20 3.0 0.06 2.1 0.10 2.9 0.32 2.5 0.12 2.5 0.03 2.6 0.17 0.66 0.24 2.5 0.10 2.5 0.12
(3.8-fold)
2.9 0.20 2.7 0.24 2.9 0.32 2.6 0.22 4.6 0.23 2.6 0.20 3.0 0.07 3.4 0.28 1.7 0.23 2.3 0.30 2.7 0.10
2.8 0.25 2.8 0.12 2.9 0.35 2.5 0.21 4.0 0.62 2.6 0.20 2.8 0.22 3.3 0.42 2.4 1.3
2.3 0.18 2.5 0.07
14g
2.9 0.13 2.7 0.23 2.7 0.25 1.6 0.28 3.0 0.39 2.4 0.12 2.8 0.13 2.7 0.10 0.36 0.07 2.1 0.03 2.5 0.06
(6.9-fold)
14h
14i
5.6 1.5 2.7 0.25 3.0 0.45 2.6 0.30 4.2 1.1 2.8 0.15 3.7 0.73 5.6 1.8 1.5 0.57 2.8 0.49 2.9 0.07
3.8 0.59 3.0 0.21 3.5 0.35 2.9 0.06 4.6 1.1 2.6 0.25 5.3 0.80 5.5 1.0 1.7 0.38 2.8 0.23 3.1 0.67
14j
2.5 0.10 1.7 0.73 1.8 0.70 2.1 0.72 2.5 0.15 2.2 0.38 2.0 0.55 1.9 0.68 0.89 0.41 2.0 0.10 2.5 0.22
2.7 0.15 2.5 0.27 2.7 0.00 2.8 0.07 2.6 0.24 2.6 0.20 2.6 0.12 2.4 0.13 1.4 0.07 2.1 0.07 2.7 0.22
14k
14l
a
c
j
Glioblastoma. b Ovarian. Lung. d Skin. e Prostate. f Neuroblastoma. g Glioblastoma. h Pancreas. i Colorectal. Breast. k Breast (normal).
bulk saw an additional policy enhancement, with both the
1-adamantyl (14l) and 2-adamantyl (14k) demonstrating
excellent broad-spectrum cytotoxic activity, with no
significant cell-line specificity noted.
The breast cancer selectivity of 13f and 14b is suggestive
of targeting and hi-jacking the AhR pathway as previously
noted.7 Thus, we further explored the cytotoxicity of these
compounds in the triple receptor negative breast cancer cell
line MDA-MB-468 and the ER-positive breast cancer cell line,
T47D (Table 4). The selectivity and potency of these
compounds was maintained in the additional breast cancer
cell lines examined. The T47D cells showed greater potency
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Table 4 Evaluation of the cytotoxicity (GI50 values, μM) values of selected analogues 14b and 13f in a broader panel of breast cancer cell lines and one
normal cell line, in an SRB assay. GI50 is the concentration at which 50% of the cell growth is inhibited, relative to an untreated control. All values are n
= 3. Entries in italic mark substantial tumour selectivity when compared with normal breast cells (MCF10A)
MCF-7
MDA-MB-468
T47D
MCF10A
ER+ve breast
carcinoma
5.3 2.6
Triple negative breast
carcinoma
5.9 0.7
ER+ve breast
carcinoma
6.8 1.5
Normal
breast
16 0.33
(3-fold)
(2.7-fold)
(2.3-fold)
14b
5.8 2.8
(8.1-fold)
6.0 1.4
(7.8-fold)
2.3 0.9
(20-fold)
47 3.3
13f
for 13f and therefore a greater selectivity response (GI50 2.3
μM, 20-fold selective) compared with the normal breast cells
(MCF10A).
In order to determine the mode-of-action of these
compounds we exploited the use of an engineered AhR
reporter assay in HT29 cells (Fig. 2a). This data showed that
13f increased the AhR reporter activity 1.84-fold (5 μM) within
48 h, supporting its ability to interact with the AhR and
induce transcription. In addition, 13f (5 μM) increased the
expression of CYP1A1 by 7.6-fold in the MDA-MB-468 breast
predominately lined with hydrophobic residues (Fig. 3).5,29
Previous AhR ligands are typically small, rigid and non-
polar.16,18,28 The larger size of 13f allows the ligand to extend
further into the pocket than is typically observed, this
facilitates additional interactions with polar residues. An
additional π–π stacking interaction between His17 and the
terminal aromatic ring is predicted (Fig. 3). The piperazine
moiety of 13f is predicted to align with the non-polar Phe13,
Leu34 and Phe50 residues. The terminal 4-cyanophenyl,
however, accesses a more exposed area of the pocket,
allowing polar interactions. We previously noted increased
potency for hydrophobic residues, consistent with the data
presented herein.5
cancer cells within
8 h of exposure (Fig. 2b). These
observations are consistent with those noted with other
halogenated aryl hydrocarbons, which activate the AhR and
induce the biological transformation of the ligand into a
DNA targeting molecule.7
These data are consistent with our prior observations that
the ligand-binding PAS-B domain binding pocket of AhR is
Collectively, we have developed a number of acrylonitrile
compounds that present with broad spectrum anticancer
activity, while others present with tumour-specific activity. Of
the tumour selective compounds, we show that 13f represents
Fig. 2 Analysis of (a) AhR activation (fold increase) in response to 13f (25 μM) using a transcription reporter assay (HT29-Luca™ AhR cells) after 24
and 48 h. FICZ (a known AhR ligand 5 μM) and CH223191 (a known AhR inhibitor, 5 μM) are included as positive and negative controls,
respectively; (b) CYP1A1 activation (fold increase) in MDA-MB-468 cells after 8 h exposure to 13f (5 μM) using a qPCR method.
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Fig. 3 Cyanophenylpiperazine analogue 13f bound in the AhR PAS-B ligand-binding domain shows the hydrophobic residues that line the binding
pocket are particularly prevalent where the piperazine group sits. The cyanophenyl, however, allows the ligand to reach previously inaccessible
polar residues. In all images, hydrophobic is green and hydrophilic is pink.
a novel and previously unexplored pharmacophore for the
treatment of breast cancer, and a novel AhR ligand structural
motif.
ovarian, breast and lung cancer cell lines, respectively. While
our efforts have focused on the development of breast cancer
selective molecules the discovery of a potentially selective
lung cancer targeting molecule is quite rare and exciting, and
warrants further investigation.
Library C saw the introduction of an expanded array of
hydrophobic moieties including substituted piperazines and
adamantyl moieties. As before, analogues with polar moieties
Conclusions
A combination of flow chemistry and traditional approaches
enabled rapid access to three focused compound libraries
(Library A–C). This allowed interrogation of the SAR
associated with the dichlorophenylacrylonitriles, cell growth
inhibition and interaction with the AhR. Examination of the
SRB data for Library A highlighted a distinct preference for
displayed lower levels of cytotoxicity, e.g. 14b and 14d (GI50
=
6.9–21 and 3.0–17 μM, respectively). The hydrophobic Library
C analogues displayed good broad-spectrum cytotoxicity, with
increasing hydrophobic bulk preferred, as demonstrated by
both adamantyl analogues (14k and 14l, GI50 values 1.7–2.8
μM). The breast cancer selectivity of 13f precipitated further
biological analysis in our AhR models of transcription,
CYP1A1 expression and PAS-B modelling. These data are
consistent with 13f mediating its breast cancer selectivity
through the AhR pathway.
hydrophobic
moieties.
Cyclohexylamine
(12b)
and
dimethylamino (12g) displayed the most promising broad-
spectrum cytotoxicity in this subset. Ovarian cancer selectivity
was noted for 12a, preferential activity in colon and breast
cancer for 12e and colon selectivity for 12g. These data led to
the development of Library
B with variations to the
hydrophobic terminal moiety via a family of substituted
piperazines. With Library B, again hydrophobic groups were
favoured with the polar 13d (4-CF3) and 13e (4-NO2) inactive
across all cell lines examined (GI50 > 50 μM). Piperazines with
pendant hydrophobic moieties displayed good levels of broad
spectrum cytotoxicity, e.g. 13a (GI50 = 2.5–6.0 μM), as did the
introduction of a methylene spacer with 13i (GI50 1.5–4.5 μM).
Cancer cell line selectivity was noted for 13c, 13f and 13h in
Combined, the data presented herein is consistent with
modulation of the AhR represents a novel approach to the
breast cancer selective agents. The observed cell line
specificity of analogues possessing terminal hydrophobic
moieties is fully consistent with our modelling studies with
all classes of dichlorophenylacrylonitriles reported to
date,5,6,30 with the further observation that an appended
polar functionality allows previously unexplored interactions.
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Experimental
AhR reporter assay. The activity of the AhR signalling
pathway was measured using the stably transfected AhR
cell line, HT29-Lucia™ (Invivogen, USA). This cell line
stably expresses the secreted Lucia luciferase reporter gene
under the control of a minimal promoter coupled with
the human CYP1A1 gene entire regulatory sequence,
containing six DREs. The Lucia luciferase reporter protein
is readily measurable in the cell culture supernatant using
QUANTI-Luc™. For the determination of AhR activity, 6 ×
106 cells in 180 μL was plated into each well of a 96 well
micro-titre plate in the absence of antibiotics and allowed
Biology
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 °C. The cancer cell
lines were maintained in Dulbecco's modified Eagle's
medium
(DMEM)
(Trace
Biosciences,
Australia)
supplemented with 10% foetal bovine serum (FBS), 10 mM
sodium bicarbonate, penicillin (100 IU mL−1), streptomycin
(100 μg mL−1), 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−1), streptomycin (50
μg mL−1), 20 mM Hepes, L-glutamine (2 mM), epidermal
growth factor (20 ng mL−1), hydrocortisone (500 ng mL−1),
cholera toxin (100 ng mL−1), and insulin (10 μg mL−1). The
AhR reporter cell line HT29-Lucia™ was maintained in
DMEM supplemented with 10% heat inactivated FBS,
L-glutamine (2 mM), penicillin (100 IU mL−1), streptomycin
(100 μg mL−1), 100 μg ml−1 Normocin™, and 100 μg mL−1 of
the selective antibiotic Zeocin™.
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 per well in 96 well plates.
On day 0, (24 h after plating) when the cells were in
logarithmic growth 100 μL medium with or without the
to culture for 24
compounds in a volume of 20 μL. At the indicated time,
20 μL of cell supernatant was transferred to white
h prior to the addition of test
a
luminometer plate and 50 μL of QUANTI-Luc was added
immediately prior to reading the luminescence using a
GloMax Explorer Luminescence Plate Reader. The promoter
activity was replicated twice and values are expressed as
fold-change relative to DMSO treated cells.
CYP1A1 activation. Gene expression was examined in
MDA-MB-468 cells following treatment with 5 μM 13f for 8
h. Total RNA was extracted using the RNeasy Mini Kit
(Qiagen) according to the manufacturer's instructions. One
μg of RNA was reverse transcribed using the QuanitTect
Reverse Transcription Kit (Qiagen) according to the
manufacturer's instructions. Rotor-Gene SYBR Green PCR Kit
(Qiagen) was used to perform qPCR for CYP1A1 on a Rotor-
Gene
3000
Thermo-Cycler
Instrument
using
β2-
microglobulin as a housekeeping gene (Qiagen). The primer
sequences for CYP1A1 (QT00012341) and β2M (QT00088935)
were purchased from Qiagen. HotStar Taq activation (95 °C
for 5 minutes) was followed by 40 cycles of denaturation (95
°C for 5 seconds), and annealing/extension (60 °C for 10
seconds). The comparative Ct value method was used for
data analysis.
test agent was added to each well. After 72
h drug
exposure growth inhibitory effects were evaluated using the
sulforhodamine B (SRB) 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
Docking of compounds into the homology model of the
AhR PAS-B domain. The homology model for the AhR PAS-B
(ligand binding) domain was prepared as previously
described.5,30 The structures of all ligands to be docked were
constructed in MOE and their conformations energy-
minimized using Molecular Mechanics in conjunction with
the MMFF94x force field.
Docking was performed using MOE's default settings,
using the triangle matcher method in combination with the
London dG scoring function for the initial placement of the
ligand, followed by a refinement of the 30 top poses with
rigid receptor setting and the GBVI/WSA scoring function.
Analysis and visualization of the docking output, such as
inhibition underwent further dose response analysis
allowing for the calculation of a GI50 value. An eight-point
dose response curve was produced, using MS Excel
software. Each data point is the mean S.E.M. calculated
from three to four replicates, which were performed on
separate occasions and separate cell line passages. From
these dose–response curves, the GI50 value was calculated,
representing the drug concentration at which cell growth is
50% inhibited based on the difference between the optical
density values on day 0 and those at the end of drug
exposure.31,32
SRB assay. Briefly, cells from each 96 well plate were fixed
by protein precipitation after the addition of 50 μL of 50%
TCA. After an incubation of 1 h at 4 °C the plates were
washed 5 times in tap water and allowed to air dry. The cells
were then stained with 50 μL of 0.4% SRB in 1% acetic acid
for 15 minutes at room temperature. The plates were washed
4 times in 1% acetic acid to remove unbound stain and
allowed to air dry. The protein was then solubilized in 150 μL
of 10 mM unbuffered TRIS.
identification
of
hydrogen
bonds,
steric
clashes,
hydrophobic interactions, or π–π interactions were
performed in MOE.
Chemistry
General experimental. All reactions were performed using
standard laboratory equipment and glassware. Solvents and
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reagents were purchased from Sigma Aldrich, Alfa Aesar or
AK Scientific and used as received. 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 HPLC-grade
10% acetonitrile/H2O (with 0.1% formic acid) as carrier
solvents on an Agilent Technologies 1260 Infinity UPLC
1H); 13C NMR (101 MHz, acetone-d6) δ 162.4, 149.9, 144.7,
136.5, 133.5, 132.8, 132.5 (2C), 132.0, 129.8 (2C), 128.2, 127.2,
126.5, 118.5, 117.4, 116.0 (2C), 113.5 (2C), 106.3, 71.7, 69.1,
47.3.
(Z)-3-(4-(3-(Cyclohexylamino)-2-hydroxypropoxy)phenyl)-2-
(3,4-dichlorophenyl)acrylonitrile (12b). Prepared as previously
1
described,6 m.p.: 112–114 °C. H NMR (600 MHz, acetone-d6)
δ 8.01 (d, J = 8.8 Hz, 2H), 7.97 (s, 1H), 7.94 (d, J = 2.0 Hz, 1H),
7.73–7.68 (m, 2H), 7.14–7.10 (m, 2H), 4.16 (dd, J = 9.7, 4.5 Hz,
1H), 4.09 (dd, J = 9.7, 6.0 Hz, 1H), 4.03 (dd, J = 12.0, 5.4 Hz,
1H), 2.92 (dd, J = 11.9, 4.6 Hz, 1H), 2.77 (dd, J = 11.9, 7.1 Hz,
1H), 2.48 (ddd, J = 10.1, 7.0, 3.8 Hz, 1H), 1.91–1.90 (m, 2H),
1.73–1.70 (m, 2H), 1.60–1.57 (m, 1H), 1.28 (tt, J = 15.7, 3.3 Hz,
2H), 1.21–1.16 (m, 1H), 1.15–1.07 (m, 2H); 13C NMR (151
MHz, acetone-d6) δ 162.5, 144.7, 136.5, 133.5, 132.7, 132.5
(2C), 132.0, 128.2, 127.1, 126.5, 118.5, 116.0 (2C), 106.2, 72.0
(2C), 69.4, 57.5, 50.0 (2C), 34.2, 34.1, 26.9, 25.6.
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-coated aluminium
plates with a thickness of 0.2 mm. Column chromatography
was performed under ‘flash’ conditions on Merck silica gel
60 (230–400 mesh).
Nuclear magnetic resonance (NMR) spectroscopy was
performed on a Brüker Avance III 400 MHz spectrometer,
where proton NMR (1H NMR) spectra and carbon NMR (13-
CNMR) 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 (δ) 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), doublet of doublet of
doublets (ddd), triplet (t), triplet of triplets (tt), quartet (q),
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.
(Z)-2-(3,4-Dichlorophenyl)-3-(4-(2-hydroxy-3-((4-chlorophenyl)
amino)propoxy)phenyl)acrylonitrile
(12c).
Prepared
as
previously described,6 m.p.: 110–113 °C. H NMR (400 MHz,
acetone-d6) δ 8.01 (d, J = 8.8 Hz, 2H), 7.97 (s, 1H), 7.94 (d, J =
1.6 Hz, 1H), 7.71–7.70 (m, 2H), 7.14 (d, J = 8.8 Hz, 2H), 7.09
(d, J = 8.8 Hz, 2H), 6.72 (d, J = 8.8 Hz, 2H), 5.19 (t, J = 5.6 Hz,
NH), 4.49 (d, J = 4.4 Hz, 1H, OH), 4.23–4.17 (m, 3H), 3.45
(ddd, J = 13.1, 6.6, 4.6 Hz, 1H), 3.31–3.25 (m, 1H); 13C NMR
(101 MHz, acetone-d6) δ 162.3, 148.8, 144.7, 136.5, 133.5,
132.8, 132.5 (2C), 132.0, 129.6 (2C), 128.2, 127.2, 126.5, 121.2,
118.5, 116.0 (2C), 114.7 (2C), 106.4, 71.5, 69.0, 47.4.
1
(Z)-2-(3,4-Dichlorophenyl)-3-(4-(2-hydroxy-3-((4-bromophenyl)
amino)propoxy)phenyl)acrylonitrile
(12d).
Prepared
as
previously described,6 m.p.: 121–124 °C. H NMR (400 MHz,
acetone-d6) δ 8.00 (d, J = 7.6 Hz, 2H), 7.95 (d, J = 2.8 Hz, 1H),
7.92 (s, 1H), 7.69–7.68 (m, 2H), 7.21 (d, J = 8.8 Hz, 2H), 7.13–
7.11 (m, 2H), 6.67 (d, J = 8.8 Hz, 2H), 5.21 (bs, NH), 4.49 (d, J
= 4.5 Hz, 1H), 4.25–4.15 (m, 3H), 3.47–3.41 (m, 1H), 3.31–3.24
(m, 1H); 13C NMR (101 MHz, acetone-d6) δ 162.3, 149.1,
144.6, 136.4, 133.5, 132.7, 132.44, 132.40, 131.9, 128.1, 127.2,
126.5, 118.4, 115.9, 115.2, 108.1, 106.3, 71.5, 68.9, 47.2.
1
(Z)-2-(3,4-Dichlorophenyl)-3-(4-(2-hydroxy-3-((4-methoxyphenyl)
amino)propoxy)phenyl)acrylonitrile (12e). Prepared as previously
General method. (Z)-2-(3,4-dichlorophenyl)-3-(4-(oxiran-2-
ylmethoxy)phenyl)acrylonitrile (10, 1 eq.) was combined with
the required amine (either 1.5 or 2 eq., as stated) and 20 mL
ethanol. The solution was irradiated at 120 °C for 20 min.
Upon chilling, the desired product was isolated via vacuum
filtration and washed with cold ethanol (10 mL). A number of
the previously reported analogues were prepared via the flow
protocol (Scheme 1).
1
described,6 m.p.: 130–133 °C. H NMR (400 MHz, acetone-d6) δ
8.01 (d, J = 8.8 Hz, 2H), 7.97 (s, 1H), 7.94 (d, J = 1.5 Hz, 1H),
7.70–7.70 (m, 2H), 7.13 (d, J = 8.8 Hz, 2H), 6.76–6.73 (m, 2H),
6.68–6.66 (m, 2H), 4.59 (bs, NH), 4.41 (d, J = 4.5 Hz, OH), 4.25–
4.14 (m, 3H), 3.68 (s, 3H), 3.39 (dd, J = 12.6, 3.6 Hz, 1H), 3.22
(dd, J = 12.6, 6.0 Hz, 1H); 13C NMR (101 MHz, acetone-d6) δ
162.4, 152.8, 144.7, 144.1, 136.5, 133.5, 132.7, 132.5 (2C), 132.0,
128.2, 127.2, 126.5, 118.5, 116.0 (2C), 115.5 (2C), 114.8 (2C),
106.3, 71.8, 69.2, 55.8, 48.3.
Compound characterisation
(Z)-2-(3,4-Dichlorophenyl)-3-(4-(2-hydroxy-3-(phenylamino)
propoxy)phenyl)acrylonitrile (12a). Prepared as previously
(Z)-2-(3,4-Dichlorophenyl)-3-(4-(2-hydroxy-3-(4-phenylpiperazin-1-
yl)propoxy)phenyl)acrylonitrile (12f). Prepared as previously
1
described,6 m.p.: 130–133 °C. H NMR (400 MHz, acetone-d6)
1
δ 8.02 (d, J = 8.8 Hz, 2H), 7.98 (s, 1H), 7.94 (d, J = 1.6 Hz, 1H),
7.713–7.706 (m, 2H), 7.14 (d, J = 8.8 Hz, 2H), 7.10 (dd, J = 8.5,
7.4 Hz, 2H), 6.70 (d, J = 7.7 Hz, 2H), 6.60 (t, J = 7.6 Hz, 1H),
4.96 (s, 1H), 4.43 (d, J = 4.6 Hz, 1H), 4.27–4.21 (m, 2H), 4.21–
4.15 (m, 1H), 3.50–3.40 (m, 1H), 3.28 (dd, J = 12.9, 5.7 Hz,
described,6 m.p.: 175–178 °C. H NMR (600 MHz, DMSO-d6) δ
8.10 (s, 1H), 8.01 (d, J = 2.2 Hz, 1H), 7.97 (d, J = 8.9 Hz, 2H),
7.77 (d, J = 8.5 Hz, 1H), 7.69 (dd, J = 8.5, 2.3 Hz, 1H), 7.20 (dd, J
= 8.5, 7.4 Hz, 2H), 7.15 (d, J = 8.9 Hz, 2H), 6.92 (d, J = 8.0 Hz,
2H), 6.76 (t, J = 7.2 Hz, 1H), 4.98 (d, J = 4.7 Hz, 1H), 4.12 (dd, J =
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9.3, 2.9 Hz, 1H), 4.03–4.00 (m, 2H), 3.12 (t, J = 4.9 Hz, 4H), 2.63
(dt, J = 10.0, 4.8 Hz, 2H), 2.63–2.58 (m, 2H), 2.54 (s, 1H), 2.44
(dd, J = 12.7, 6.1 Hz, 1H); 13C NMR (151 MHz, DMSO-d6) δ
161.2, 151.1, 144.3, 134.9, 132.0, 131.5 (2C), 131.2, 131.1, 128.89
(2C), 128.86, 127.0, 125.80, 125.78, 118.7, 117.9, 115.31 (2C),
115.27, 115.1 (2C), 104.4, 71.4, 66.5, 60.9, 53.5, 48.3.
according to the general procedure from (Z)-2-(3,4-
dichlorophenyl)-3-(4-(oxiran-2-ylmethoxy)phenyl)acrylonitrile (10,
200 mg, 0.58 mmol, 1 eq.) and 1-(4-bromophenyl)piperazine
(11j; 279 mg, 1.16 mmol, 2 eq.) to afford the desired compound
1
as a cream coloured solid (326 mg, 96%), m.p.: 162–165 °C. H
NMR (400 MHz, DMSO-d6) δ 8.10 (s, 1H), 8.00 (s, 1H), 7.96 (d, J
= 8.6 Hz, 2H), 7.76 (d, J = 8.6 Hz, 1H), 7.68 (d, J = 6.8 Hz, 1H),
7.33 (d, J = 8.8 Hz, 2H), 7.14 (d, J = 8.6 Hz, 2H), 6.88 (d, J = 8.8
Hz, 2H), 4.99 (s, 1H), 4.11 (d, J = 6.5 Hz, 1H), 4.01–3.99 (m, 2H),
3.12 (s, 4H), 2.63–2.56 (m, 4H), 2.46–2.41 (m, 1H); 13C NMR (101
MHz, DMSO-d6) δ 161.2, 150.2, 144.3, 134.9, 132.0, 131.5 (2C),
131.4 (2C), 131.2, 131.1, 127.0, 125.80, 125.77, 117.9, 117.2 (2C),
115.1 (2C), 109.9, 104.4, 71.4, 66.5, 60.9, 53.3 (2C), 47.9 (2C); IR
νmax/cm−1: 3300 (OH), 2830 cm (N–CH2), 2216 (CN), 1259 cm
(C–O), 814 cm (C–Cl); LRMS: (ESI+) m/z: 588 (C28H27BrCl2N3O2)
[M+H]; HRMS: exact mass calculated for (C28H27BrCl2N3O2) [M
+ H], 588.0638. Found 588.0617.
(Z)-2-(3,4-Dichlorophenyl)-3-(4-(3-(dimethylamino)-2-
hydroxypropoxy)phenyl)acrylonitrile
(12g).
Prepared
as
previously described,6 m.p.: 99–101 °C. 1H NMR (400 MHz,
acetone-d6) δ 8.01 (d, J = 8.4 Hz, 2H), 7.97–7.94 (m, 2H), 7.70
(s, 2H), 7.13 (d, J = 8.4 Hz, 2H), 4.17 (dd, J = 11.3, 5.6 Hz, 1H),
4.07 (d, J = 6.0 Hz, 2H), 2.48 (s, 2H), 2.28 (s, 6H); 13C NMR
(101 MHz, acetone-d6) δ 162.6, 144.7, 136.5, 133.5, 132.7,
132.5 (2C), 132.0, 128.2, 127.1, 126.5, 118.5, 115.9 (2C), 106.2,
72.2, 67.7, 63.0, 46.2 (2C).
(Z)-2-(3,4-Dichlorophenyl)-3-(4-(2-hydroxy-3-(4-(p-tolyl)piperazin-
1-yl)propoxy)phenyl)acrylonitrile (13a). Prepared according to the
general procedure from (Z)-2-(3,4-dichlorophenyl)-3-(4-(oxiran-2-
ylmethoxy)phenyl)acrylonitrile (10, 346 mg, 1 mmol, 1 eq.) and
1-(p-tolyl)piperazine (11h; 264 mg, 1.5 mmol, 1.5 eq.) to afford
the desired compound as a bright yellow solid (448 mg, 89%),
(Z)-2-(3,4-Dichlorophenyl)-3-(4-(2-hydroxy-3-(4-(4-(trifluoromethyl)
phenyl)piperazin-1-yl)propoxy)-phenyl)acrylonitrile (13d). Prepared
according to the general procedure from (Z)-2-(3,4-
dichlorophenyl)-3-(4-(oxiran-2-ylmethoxy)phenyl)acrylonitrile (10,
346 mg, 1 mmol, 1 eq.) and 1-(4-trifluoromethylphenyl)piperazine
(11k; 461 mg, 2 mmol, 2 eq.) to afford the desired compound as
1
m.p.: 147–150 °C. H NMR (400 MHz, DMSO-d6) δ 8.10 (s, 1H),
8.00 (d, J = 1.9 Hz, 1H), 7.96 (d, J = 8.7 Hz, 2H), 7.76 (d, J = 8.5
Hz, 1H), 7.68 (dd, J = 8.5, 1.9 Hz, 1H), 7.14 (d, J = 8.7 Hz, 2H),
7.01 (d, J = 8.2 Hz, 2H), 6.81 (d, J = 8.4 Hz, 2H), 4.97 (d, J = 4.2
Hz, 1H), 4.12 (d, J = 6.7 Hz, 1H), 4.00 (d, J = 8.1 Hz, 2H), 3.06–
3.05 (m, 4H), 2.64–2.56 (m, 4H), 2.43 (dd, J = 12.7, 5.6 Hz, 1H),
2.19 (s, 3H); 13C NMR (101 MHz, DMSO-d6) δ 161.2, 149.0,
144.3, 135.0, 132.0, 131.5 (2C), 131.2, 131.1, 129.4 (2C), 127.5,
127.0, 125.81, 125.78, 117.9, 115.6 (2C), 115.2 (C2), 104.4, 71.4,
66.5, 60.9, 53.5 (2C), 48.8 (2C), 20.0; IR νmax/cm−1: 3333 (OH),
2816 (N–CH2), 2208 (CN), 1182 (C–O), 817 (C–Cl); LRMS (ESI+)
m/z: 522 (C29H30Cl2N3O2) [M + H]; HRMS: exact mass calculated
for C29H30Cl2N3O2 [M + H], 522.1710. Found 522.1713.
1
a pale yellow solid (486 mg, 84%), m.p.: 159–161 °C. H NMR
(400 MHz, DMSO-d6) δ 8.09 (s, 1H), 8.00 (d, J = 1.8 Hz, 1H), 7.96
(d, J = 8.7 Hz, 2H), 7.75 (d, J = 8.5 Hz, 1H), 7.68 (dd, J = 8.5, 1.8
Hz, 1H), 7.48 (d, J = 8.6 Hz, 2H), 7.14 (d, J = 8.7 Hz, 2H), 7.04 (d, J
= 8.6 Hz, 2H), 5.00 (d, J = 3.3 Hz, 1H), 4.12–4.11 (m, 1H), 4.02–
3.99 (m, 2H), 3.27 (s, 4H), 2.64–2.55 (m, 4H), 2.47–2.42 (m, 1H);
13C NMR (101 MHz, DMSO-d6) δ 161.2, 153.3, 144.3, 134.9, 132.1,
131.5, 131.2, 131.1, 127.0, 126.1 (q, J = 3.4 Hz, 2C), 125.83, 125.78,
125.0 (q, J = 272.7 Hz), 117.9, 117.8 (q, J = 32.2 Hz), 115.2 (2C),
114.1 (2C), 104.4, 71.4, 66.6, 60.9, 53.2 (2C), 47.1 (2C); 19F NMR
(376 MHz, DMSO-d6, CF3CO2H) δ −56.1; IR νmax/cm−1: 3300 (OH),
2208 (CN), 1224 (C–F), 1179 (C–F), 1103 (C–O), 819 (C–Cl); LRMS
(ESI+) m/z: 576 (C29H27Cl2F3N3O2) [M + H]; HRMS: exact mass
calculated for (C29H27Cl2F3N3O2) [M + H], 576.4442. Found
576.4311.
(Z)-3-(4-(3-(4-(4-Chlorophenyl)piperazin-1-yl)-2-hydroxypropoxy)
phenyl)-2-(3,4-dichlorophenyl)-acrylonitrile
(13b).
Prepared
according to the general procedure from (Z)-2-(3,4-
dichlorophenyl)-3-(4-(oxiran-2-ylmethoxy)phenyl)acrylonitrile (10,
200 mg, 0.58 mmol, 1 eq.) and 1-(4-chlorophenyl)piperazine
(11i; 227 mg, 1.16 mmol, 2 eq.) to afford the desired compound
(Z)-3-(4-(3-(4-(4-Nitrophenyl)piperazin-1-yl)-2-hydroxypropoxy)
phenyl)-2-(3,4-dichlorophenyl)acrylonitrile
(13e).
Prepared
1
as a cream coloured solid (250 mg, 82%), m.p.: 159–162 °C. H
according to the general procedure from (Z)-2-(3,4-
dichlorophenyl)-3-(4-(oxiran-2-ylmethoxy)phenyl)acrylonitrile
(10, 346 mg, 1 mmol, 1 eq.) and 1-(4-nitrophenyl)piperazine
(11l; 308 mg, 1.5 mmol, 1.5 eq.) to afford the desired
compound as a bright yellow solid (437 mg, 79%), m.p.: 90–
NMR (400 MHz, DMSO-d6) δ 8.10 (s, 1H), 8.01 (d, J = 2.0 Hz,
1H), 7.96 (d, J = 8.8 Hz, 2H), 7.77 (d, J = 8.5 Hz, 1H), 7.69 (dd, J
= 8.5, 2.1 Hz, 1H), 7.21 (d, J = 8.9 Hz, 2H), 7.14 (d, J = 8.8 Hz,
2H), 6.93 (d, J = 9.0 Hz, 2H), 4.99 (d, J = 2.6 Hz, 1H), 4.12–4.11
(m, 1H), 4.05–4.00 (m, 2H), 3.12 (t, J = 4.3 Hz, 4H), 2.65–2.57
(m, 4H), 2.44 (dd, J = 13.1, 4.4 Hz, 1H); 13C NMR (101 MHz,
DMSO-d6) δ 161.2, 149.9, 144.3, 135.0, 132.1, 131.6 (2C), 131.24,
131.15, 128.6 (2C), 127.0, 125.83, 125.81, 122.3, 117.9, 116.8
(2C), 115.2 (2C), 104.4, 71.4, 66.5, 60.9, 53.3 (2C), 48.1 (2C); IR
νmax/cm−1: 3355 (br, OH), 2830 (N–CH2), 2216 (CN), 1260 (C–O),
815 (C–Cl); LRMS: (ESI+) m/z: 542 (C28H27Cl3N3O2) [M + H];
HRMS: exact mass calculated for (C28H27Cl3N3O2) [M+H],
542.1163. Found 542.1147.
1
92 °C. H NMR (600 MHz, DMSO-d6) δ 8.10 (s, 1H), 8.04 (d, J
= 9.2 Hz, 2H), 8.00 (s, 1H), 7.96 (d, J = 8.5 Hz, 2H), 7.76 (d, J =
8.3 Hz, 1H), 7.68 (d, J = 8.3 Hz, 1H), 7.14 (d, J = 8.5 Hz, 2H),
7.02 (d, J = 9.2 Hz, 2H), 5.02 (d, J = 4.1 Hz, 1H), 4.12 (d, J =
6.8 Hz, 1H), 4.01 (dd, J = 16.0, 6.8 Hz, 2H), 3.45 (s, 4H), 2.63–
2.54 (m, 5H), 2.45 (dd, J = 12.6, 6.0 Hz, 1H); 13C NMR (101
MHz, DMSO-d6) δ 161.2, 154.7, 144.3, 136.8, 134.9, 132.0,
131.5 (2C), 131.2, 131.1, 127.0, 125.8, 125.7 (2C), 117.9, 115.1
(2C), 112.6 (2C), 104.4, 71.3, 66.6, 60.7, 53.0 (2C), 46.4 (2C);
IR νmax/cm−1: 3337 (OH), 2835 (N–CH2), 2212 (CN), 1589
(NO2), 1332 (NO2), 1244 (C–O), 827 (C–Cl); LRMS: (ESI+) m/z:
(Z)-3-(4-(3-(4-(4-Bromophenyl)piperazin-1-yl)-2-hydroxypropoxy)
phenyl)-2-(3,4-dichlorophenyl)acrylonitrile
(13c).
Prepared
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553 (C28H27Cl2N4O4) [M + H]; HRMS: exact mass calculated
for (C28H27Cl2N4O4) [M + H], 553.1404. Found 553.1408.
(Z)-2-(3,4-Dichlorophenyl)-3-(4-(2-hydroxy-3-(4-(4-cyanophenyl)
116.7 (d, J = 21.2 Hz), 116.0 (2C), 106.3, 72.2, 67.5, 61.8, 54.6
(2C), 51.36 (d, J = 3.3 Hz, 2C); 19F NMR (376 MHz, acetone-d6,
CF3CO2H) δ −124.4; IR νmax/cm−1: 3200 (br, OH), 2825 (N–CH2),
2214 (CN), 1262 (C–O), 818 (C–Cl); LRMS: (ESI+) m/z: 526 (C28-
H27Cl2FN3O2) [M + H]; HRMS: exact mass calculated for C28H27-
Cl2FN3O2 [M + H], 526.1459. Found 526.1465.
piperazin-1-yl)propoxy)phenyl)-acrylonitrile
(13f).
Prepared
according to the general procedure from (Z)-2-(3,4-
dichlorophenyl)-3-(4-(oxiran-2-ylmethoxy)phenyl)acrylonitrile (10,
346 mg, 1 mmol, 1 eq.) and 1-(4-cyanophenyl)piperazine (11m;
375 mg, 2 mmol, 2 eq.) to afford the desired compound as a
pale yellow solid (443 mg, 83%), m.p.: 185–187 °C. 1H NMR
(400 MHz, acetone-d6) δ 8.04–8.00 (m, 2H), 7.98 (s, 1H), 7.94 (d,
J = 1.3 Hz, 1H), 7.73–7.68 (m, 2H), 7.56–7.52 (m, 2H), 7.16–7.13
(m, 2H), 7.07–7.03 (m, 2H), 4.23 (dd, J = 9.1, 3.5 Hz, 1H), 4.21–
4.17 (m, 1H), 4.13 (dd, J = 9.1, 5.4 Hz, 1H), 3.41 (t, J = 5.1 Hz,
4H), 2.78–2.57 (m, 6H); 13C NMR (101 MHz, DMSO-d6) δ 161.2,
153.2, 144.3, 135.0, 133.3 (2C), 132.0, 131.5 (2C), 131.2, 131.1,
127.0, 125.83, 125.79, 120.1, 117.9, 115.2 (2C), 114.0 (2C), 104.0,
98.2, 71.3, 66.6, 60.8, 53.1 (2C), 46.4 (2C); IR νmax/cm−1: 3300
(OH), 2219 (CN), 2217 (CN), 1180 (C–O), 819 (C–Cl); LRMS:
(ESI+) m/z: 533 (C28H27Cl2N4O4) [M + H]; HRMS: exact mass
calculated for C28H27Cl2N4O4 [M+H], 533.1404. Found 533.1409.
(Z)-3-(4-(3-(4-(4-Acetylphenyl)piperazin-1-yl)-2-hydroxypropoxy)
(Z)-3-(4-(3-(4-(4-Methylbenzyl)piperazin-1-yl)-2-hydroxypropoxy)
phenyl)-2-(3,4-dichlorophenyl)-acrylonitrile
(13i).
Prepared
according to the general procedure from (Z)-2-(3,4-
dichlorophenyl)-3-(4-(oxiran-2-ylmethoxy)phenyl)acrylonitrile (10,
346 mg, 1 mmol, 1 eq.) and 1-(4-methylbenzyl)piperazine (11p;
381 mg, 2 mmol, 2 eq.) to afford the desired compound as a
yellow solid (325 mg, 60%), m.p.: 124–126 °C. 1H NMR (400
MHz, DMSO-d6) δ 8.09 (s, 1H), 8.00 (s, 1H), 7.95 (d, J = 8.4 Hz,
2H), 7.76 (d, J = 8.4 Hz, 1H), 7.69 (d, J = 8.1 Hz, 1H), 7.16–7.11
(m, 6H), 4.90 (s, 1H), 4.08 (d, J = 5.8 Hz, 1H), 3.95 (d, J = 5.6 Hz,
2H), 3.38 (s, 2H), 2.47–2.35 (m, 10H), 2.27 (s, 3H); 13C NMR (101
MHz, DMSO-d6) δ 161.2, 144.4, 135.9, 135.1, 135.0, 132.1 (2
overlapping signals), 131.6 (2C), 131.24, 131.16, 128.9 (2C),
128.7 (2C), 127.0, 125.8, 117.9, 115.2 (2C), 104.4, 71.4, 66.4, 61.9,
60.9, 53.6 (2C), 52.7 (2C), 20.7; IR νmax/cm−1: 3498 cm−1 (OH),
2915 cm−1 (C–CH3), 2810 cm−1 (N–CH2), 2207 cm−1 (CN), 1265
cm−1 (C–O), 811 cm−1 (C–Cl); LRMS: (ESI+) m/z: 536 (C30H32Cl2-
N3O2) [M+H]; HRMS: exact mass calculated for C30H32Cl2N3O2
[M + H], 536.1866. Found 536.1872.
(Z)-2-(3,4-Dichlorophenyl)-3-(4-(2-hydroxy-3-(4-phenylpiperidin-1-
yl)propoxy)phenyl)acrylonitrile (14a). Prepared according to the
general procedure from (Z)-2-(3,4-dichlorophenyl)-3-(4-(oxiran-2-
ylmethoxy)phenyl)acrylonitrile (10, 346 mg, 1 mmol, 1 eq.) and
4-phenylpiperidine (11q, 340 mg, 2 mmol, 2 eq.) to afford the
desired compound as a pale yellow solid (448 mg, 87%), m.p.:
170–172 °C. 1H NMR (400 MHz, DMSO-d6) δ 8.10 (s, 1H), 8.01 (d,
J = 2.2 Hz, 1H), 7.97 (d, J = 8.8 Hz, 2H), 7.76 (d, J = 8.5 Hz, 1H),
7.68 (dd, J = 8.5, 2.2 Hz, 1H), 7.30–7.22 (m, 4H), 7.18–7.14 (m,
3H), 4.91 (d, J = 4.1 Hz, 1H), 4.13–4.11 (m, 1H), 4.00–3.98 (m,
2H), 3.00 (dd, J = 26.9, 10.9 Hz, 2H), 2.49–2.35 (m, 3H), 2.12 (dt, J
= 11.3, 8.4 Hz, 2H), 1.70–1.60 (m, 4H); 13C NMR (101 MHz,
DMSO-d6) δ 161.2, 146.3, 144.3, 135.0, 132.0 (2 overlapping
signals), 131.5 (2C), 131.2, 131.1, 128.3 (2C), 127.0, 126.7 (2C),
126.0, 125.8, 117.9, 115.2 (2C), 104.3, 71.5, 66.6, 61.3, 54.6 (d, J =
20.8 Hz, 2C), 41.8, 33.2 (d, J = 3.0 Hz, 2C); IR νmax/cm−1: 3422
(OH), 2808 (N–CH2), 2207 (CN), 1263 (C–O), 810 (C–Cl); LRMS:
(ESI+) m/z: 507 (C29H29Cl2N2O2) [M + H]; HRMS: exact mass
calculated for C29H29Cl2N2O2 [M + H], 507.1601. Found 507.1620.
(Z)-3-(4-(3-(4-Acetylpiperazin-1-yl)-2-hydroxypropoxy)phenyl)-
phenyl)-2-(3,4-dichlorophenyl)acrylonitrile
(13g).
Prepared
according to the general procedure from (Z)-2-(3,4-
dichlorophenyl)-3-(4-(oxiran-2-ylmethoxy)phenyl)acrylonitrile (10,
193 mg, 0.56 mmol, 1 eq.) and 4′-piperazineacetophenone
(11n; 227 mg, 1.11 mmol, 2 eq.) to afford the desired
compound as an orange solid (240 mg, 78%), m.p.: 149–153
1
°C. H NMR (400 MHz, acetone-d6) δ 8.02 (dd, J = 8.8, 1.6 Hz,
2H), 7.98 (s, 1H), 7.94 (d, J = 1.4 Hz, 1H), 7.86 (dd, J = 8.8, 1.6
Hz, 1H), 7.75–7.69 (m, 2H), 7.15 (dd, J = 8.8, 1.6 Hz, 2H), 6.99
(dd, J = 8.8, 1.6 Hz, 2H) 4.24 (dd, J = 8.8, 3.6 Hz, 1H), 4.20–
4.16 (m, 1H), 4.13 (dd, J = 8.9, 5.2 Hz, 1H), 3.41 (t, J = 5.1 Hz,
4H), 2.78–2.58 (m, 6H), 2.45 (s, 3H); 13C NMR (101 MHz,
acetone-d6) δ 195.8, 162.5, 155.2, 144.7, 136.5, 133.5, 132.7,
132.5 (2C), 132.0, 130.9 (2C), 128.4, 128.2, 127.2, 126.5, 118.5,
116.0 (2C), 114.2 (2C), 106.3, 72.1, 67.6, 61.7, 54.3 (2C), 48.1
(2C), 26.1; IR νmax/cm−1: 3100 (br, OH), 2981 cm−1 (CH3), 2890
(N–CH2), 2210 (CN) 1660 (CO), 1272 (C–O), 818 (C–Cl);
LRMS: (ESI+) m/z: 550 (C30H30Cl2N3O3) [M + H]; HRMS: exact
mass calculated for C30H30Cl2N3O3 [M + H], 550.1659. Found
550.1671.
(Z)-2-(3,4-Dichlorophenyl)-3-(4-(3-(4-(2-fluorophenyl)piperazin-1-
yl)-2-hydroxypropoxy)phenyl)-acrylonitrile
(13h).
Prepared
according to the general procedure from (Z)-2-(3,4-
dichlorophenyl)-3-(4-(oxiran-2-ylmethoxy)phenyl)acrylonitrile (10,
200 mg, 0.58 mmol, 1 eq.) and 1-(2-fluorophenyl)piperazine
(11o; 208 mg, 1.16 mmol, 2 eq.) to afford the desired compound
as a pale yellow solid (225 mg, 74%), m.p.: 119–121 °C. 1H NMR
(400 MHz, acetone-d6) δ 8.02 (d, J = 8.8 Hz, 2H), 7.98 (s, 1H),
7.94 (d, J = 1.6 Hz, 1H), 7.73–7.67 (m, 2H), 7.15 (d, J = 8.8, Hz,
2H), 7.11–7.01 (m, 3H), 6.98–6.93 (m, 1H) 4.23 (dd, J = 9.1, 3.4
Hz, 1H), 4.21–4.16 (m, 1H), 4.12 (dd, J = 9.1, 5.3 Hz, 1H), 3.12 (t,
J = 4.8 Hz, 4H), 2.82–2.59 (m, 6H); 13C NMR (101 MHz, acetone-
d6) δ 162.6, 156.5 (d, J = 245.4 Hz), 144.7, 141.23 (d, J = 8.5 Hz),
136.5, 133.5, 132.7, 132.5 (2C), 132.0, 128.2, 127.2, 126.5, 125.5
(d, J = 3.0 Hz), 123.1 (d, J = 8.1 Hz), 120.0 (d, J = 3.0 Hz), 118.5,
2-(3,4-dichlorophenyl)-acrylonitrile
(14b).
Prepared
as
previously described,6 m.p.: 88–91 °C. 1H NMR (400 MHz,
DMSO-d6) δ 8.10 (s, 1H), 8.01 (d, J = 2.2 Hz, 1H), 7.96 (d, J =
8.9 Hz, 2H), 7.77 (d, J = 8.5 Hz, 1H), 7.69 (dd, J = 8.5, 2.2 Hz,
1H), 7.14 (d, J = 8.9 Hz, 2H), 4.97 (d, J = 4.3 Hz, 1H), 4.10–
4.07 (m, 1H), 4.00–3.96 (m, 2H), 3.42–3.39 (m, 4H), 2.48–2.37
(m, 10H), 1.97 (s, 3H); 13C NMR (101 MHz, DMSO-d6) δ 168.1,
161.2, 144.4, 135.0, 132.1, 131.5 (2C), 131.3, 131.2, 127.0,
125.84, 125.81, 117.9, 115.2 (2C), 104.4, 71.3, 66.5, 60.8, 53.7*
(2C), 53.2* (2C), 45.8* (2C), 40.9* (2C), 21.2. *Isomerization
confirmed by 2D correlations.
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(Z)-2-(3,4-Dichlorophenyl)-3-(4-(2-hydroxy-3-(4-methylpiperazin-1-
(Z)-2-(3,4-Dichlorophenyl)-3-(4-(2-hydroxy-3-(2-methylpiperidin-1-
yl)propoxy)phenyl)-acrylonitrile (14h). Prepared as previously
described,6 m.p.: 94–96 °C. 1H NMR (400 MHz, DMSO-d6) δ 8.09
(s, 1H), 8.00 (s, 1H), 7.96 (d, J = 7.8 Hz, 2H), 7.76 (d, J = 8.1 Hz,
1H), 7.68 (d, J = 7.4 Hz, 1H), 7.14 (s, 2H), 4.83 (d, J = 37.4 Hz,
1H), 4.10–3.90 (m, 3H), 3.45–3.33 (m, 2H), 2.77 (dd, J = 51.7,
42.7 Hz, 2H), 2.42–2.03 (m, 2H), 1.57–1.45 (m, 3H), 1.23 (s, 2H),
1.00 (s, 2H); 13C NMR (101 MHz, DMSO-d6) δ 161.2 (d, J = 7.0
Hz), 144.3, 135.0, 132.0, 131.5 (2C), 131.2, 131.1, 127.0, 125.8,
125.7 (d, J = 3.6 Hz), 117.9, 115.1 (d, J = 2.9 Hz, 2C), 104.3, 71.2
(d, J = 5.8 Hz), 69.3 (d, J = 18.0 Hz), 56.5 (d, J = 80.5 Hz), 56.0 (d,
J = 18.4 Hz), 52.9 (d, J = 21.0 Hz), 34.10 (d, J = 28.1 Hz, 25.8 (d, J
= 4.4 Hz), 23.1 (d, J = 38.4 Hz).
yl)propoxy)phenyl)acrylonitrile (14c). Prepared as previously
1
described,6 m.p.: 111–114 °C. H NMR (400 MHz, DMSO-d6) δ
8.10 (s, 1H), 8.00 (d, J = 2.1 Hz, 1H), 7.96 (d, J = 8.8 Hz, 2H),
7.76 (d, J = 8.5 Hz, 1H), 7.68 (dd, J = 8.5, 2.1 Hz, 1H), 7.13 (d, J =
8.8 Hz, 2H), 4.90 (d, J = 3.7 Hz, OH), 4.09–4.06 (m, 1H), 3.99–
3.95 (m, 2H), 2.46–2.42 (m, 3H), 2.38–2.23 (m, 4H), 2.13 (s, 3H);
13C NMR (101 MHz, DMSO-d6) δ 161.2, 144.3, 134.9, 132.0,
131.5 (2C), 131.2, 131.1, 127.0, 125.8, 117.9, 115.2, 115.1 (2C),
104.4, 71.4, 66.4, 60.9, 54.8 (2C), 53.4 (2C), 45.8.
(Z)-2-(3,4-Dichlorophenyl)-3-(4-(2-hydroxy-3-morpholinopropoxy)
phenyl)acrylonitrile (14d). Prepared as previously described,6 m.
1
p.: 103–105 °C. H NMR (400 MHz, acetone-d6) δ 8.01 (d, J = 8.9
Hz, 2H), 7.97 (s, 1H), 7.94 (d, J = 1.6 Hz, 1H), 7.71–7.70 (m, 2H),
7.13 (d, J = 8.9 Hz, 2H), 4.20 (dd, J = 9.1, 3.4 Hz, 1H), 4.17–4.13
(m, 1H), 4.09 (dd, J = 9.0, 5.4 Hz, 1H), 3.64 (s, 4H), 2.61–2.51 (m,
6H); 13C NMR (101 MHz, acetone-d6) δ 162.5, 144.7, 136.5,
133.5, 132.7, 132.5 (2C), 132.0, 128.2, 127.1, 126.5, 118.5, 116.0
(2C), 106.3, 72.1, 67.4 (2C), 67.3, 62.3, 55.1 (2C).
(Z)-2-(3,4-Dichlorophenyl)-3-(4-(2-hydroxy-3-(pyrrolidin-1-yl)
propoxy)phenyl)acrylonitrile (14i). Prepared as previously
1
described,6 m.p.: 90–93 °C. H NMR (400 MHz, acetone-d6) δ
8.01 (d, J = 8.8 Hz, 2H), 7.96 (s, 1H), 7.93 (d, J = 1.5 Hz, 1H),
7.71–7.70 (m, 2H), 7.12 (d, J = 8.8 Hz, 2H), 4.19–4.16 (m, 1H),
4.10–4.05 (m, 2H), 3.56 (q, J = 7.0 Hz, 1H), 2.65 (d, J = 5.6 Hz,
1H), 2.61–2.56 (m, 3H), 1.74 (s, 3H), 1.12 (t, J = 7.0 Hz, 1H);
13C NMR (101 MHz, acetone-d6) δ 162.6, 144.7, 136.5, 133.5,
132.7, 132.5 (2C), 132.0, 128.2, 127.1, 126.5, 118.5, 116.0 (2C),
106.2, 72.2, 68.7, 59.5, 55.1 (2C), 24.3 (2C).
(Z)-3-(4-(3-(Azepan-1-yl)-2-hydroxypropoxy)phenyl)-2-(3,4-
dichlorophenyl)acrylonitrile (14j). Prepared as previously
described,6 m.p.: 76–78 °C. 1H NMR (400 MHz, DMSO-d6) δ
8.10 (s, 1H), 8.02–8.01 (m, 1H), 7.96 (d, J = 8.9 Hz, 2H), 7.77
(dd, J = 8.5, 2.5 Hz, 1H), 7.69 (dd, J = 8.5, 2.3 Hz, 1H), 7.14 (d,
J = 8.9 Hz, 2H), 4.82 (s, 1H), 4.12 (dd, J = 10.1, 3.3 Hz, 1H),
3.98 (dd, J = 10.1, 6.1 Hz, 1H), 3.87 (s, 1H), 2.65 (m, 3H),
2.57–2.54 (m, 1H), 1.53 (s, 6H); 13C NMR (101 MHz, DMSO-
d6) δ 161.3, 144.4, 135.0, 132.0, 131.5 (2C), 131.2, 131.1,
127.0, 125.8, 125.7, 117.9, 115.1 (2C), 104.3, 71.4, 67.3, 60.3,
55.6 (2C), 28.1 (2C), 26.6 (2C).
(Z)-2-(3,4-Dichlorophenyl)-3-(4-(2-hydroxy-3-(piperidin-1-yl)
propoxy)phenyl)acrylonitrile (14e). Prepared as previously
1
described,6 m.p.: 111–115 °C. H NMR (400 MHz, acetone-d6)
δ 8.01 (d, J = 8.8 Hz, 2H), 7.97 (s, 1H), 7.94 (d, J = 1.6 Hz, 1H),
7.73–7.68 (m, 2H), 7.13 (d, J = 8.8 Hz, 2H), 4.18–4.15 (m, 1H),
4.12–4.05 (m, 2H), 2.53–2.44 (m, 6H), 1.57 (dt, J = 10.7, 5.4
Hz, 4H), 1.46–1.41 (m, 2H); 13C NMR (101 MHz, acetone-d6) δ
162.6, 144.8, 136.5, 133.5, 132.7, 132.5 (2C), 132.0, 128.2,
127.1, 126.5, 118.5, 116.0 (2C), 106.2, 72.3, 67.2, 62.4, 55.8
(2C), 26.89 (2C), 25.0.
(Z)-2-(3,4-Dichlorophenyl)-3-(4-(2-hydroxy-3-(4-methylpiperidin-1-
yl)propoxy)phenyl)acrylonitrile (14f). Prepared as previously
described,6 m.p.: 79–81 °C. 1H NMR (400 MHz, DMSO-d6) δ 8.10
(s, 1H), 8.00 (d, J = 2.1 Hz, 1H), 7.96 (d, J = 8.8 Hz, 2H), 7.76 (d,
J = 8.5 Hz, 1H), 7.68 (dd, J = 8.5, 2.2 Hz, 1H), 7.13 (d, J = 8.8 Hz,
2H), 4.85 (d, J = 3.4 Hz, 1H), 4.08 (d, J = 6.5 Hz, 1H), 3.95 (d, J =
6.6 Hz, 2H), 2.84 (dd, J = 25.6, 10.6 Hz, 2H), 2.37 (ddd, J = 32.9,
12.6, 5.7 Hz, 2H), 1.97 (q, J = 9.4 Hz, 2H), 1.55 (d, J = 12.6 Hz,
2H), 1.38–1.22 (m, 1H), 1.12 (qd, J = 12.0, 3.5 Hz, 2H), 0.87 (d, J
= 6.4 Hz, 3H); 13C NMR (101 MHz, DMSO-d6) δ 161.2, 144.3,
134.9, 132.0, 131.5 (2C), 131.2, 131.1, 127.0, 125.8, 117.9, 115.2,
115.1 (2C), 104.3, 71.5, 66.5, 61.3, 54.2, (d, J = 21.3 Hz, 2C), 34.1
(d, J = 4.6 Hz, 2C), 30.2, 21.9.
(Z)-2-(3,4-Dichlorophenyl)-3-(4-(2-hydroxy-3-(3-methylpiperidin-1-
yl)propoxy)phenyl)acrylonitrile (14g). Prepared as previously
described,6 m.p.: 103–106 °C. 1H NMR (400 MHz, DMSO-d6)
δ 8.10 (s, 1H), 8.01 (t, J = 3.0 Hz, 1H), 7.96 (d, J = 8.9 Hz,
2H), 7.77 (dd, J = 8.5, 2.6 Hz, 1H), 7.69 (dd, J = 8.5, 2.3 Hz,
1H), 7.14 (t, J = 6.2 Hz, 2H), 4.88 (s, 1H), 4.08 (t, J = 6.4
Hz, 1H), 4.02–3.88 (m, 2H), 2.91–2.71 (m, 2H), 2.39 (d, J =
20.5 Hz, 2H), 2.03–1.87 (m, 1H), 1.69–1.53 (m, 3H), 1.45 (dd,
J = 24.5, 12.4 Hz, 1H), 0.82 (dd, J = 6.4, 2.8 Hz, 3H); 13C
NMR (101 MHz, DMSO-d6) δ 161.2, 144.3, 135.0, 132.0, 131.5
(2C), 131.2, 131.1, 127.0, 125.78, 125.76, 117.9, 115.1 (2C),
104.3, 71.5, 66.4 (d, J = 1.7 Hz), 62.3 (d, J = 12.5 Hz), 61.4
(d, J = 3.4 Hz), 54.3, (d, J = 12.9 Hz, 2C), 32.5, 30.7 (d, J =
2.4 Hz), 25.1, 19.6.
(Z)-3-(4-(3-((1S,3S)-Adamantan-1-ylamino)-2-hydroxypropoxy)
phenyl)-2-(3,4-dichlorophenyl)acrylonitrile (14k). Prepared as
1
previously described,6 m.p.: 170–172 °C. H NMR (400 MHz,
DMSO-d6) δ 8.10 (s, 1H), 8.01 (d, J = 2.2 Hz, 1H), 7.96 (d, J =
8.8 Hz, 2H), 7.77 (d, J = 8.5 Hz, 1H), 7.69 (dd, J = 8.5, 2.2 Hz,
1H), 7.13 (d, J = 8.8 Hz, 2H), 4.97 (bs, OH), 4.09 (dd, J = 9.9,
4.3 Hz, 1H), 3.97 (dd, J = 10.0, 6.2 Hz, 1H), 3.83–3.74 (m, 1H),
2.65–2.59 (m, 2H), 2.00 (s, 3H), 1.63–1.56 (m, 12H); 13C NMR
(101 MHz, DMSO-d6) δ 161.2, 144.4, 135.0, 132.0, 131.5 (2C),
131.2, 131.1, 127.0, 125.8, 125.7, 117.9, 115.1 (2C), 104.3,
71.1, 69.1, 43.0, 42.4, 36.3 (6C), 29.0 (3C).
(Z)-3-(4-(3-((1R,3S,5R,7R)-Adamantan-2-ylamino)-2-
hydroxypropoxy)phenyl)-2-(3,4-dichlorophenyl)acrylonitrile (14l).
1
Prepared as previously described,6 m.p.: 123–126 °C. H NMR
(400 MHz, DMSO-d6) δ 8.09 (s, 1H), 8.00 (d, J = 2.0 Hz, 1H),
7.96 (d, J = 8.8 Hz, 2H), 7.76 (d, J = 8.5 Hz, 1H), 7.68 (dd, J =
8.5, 2.0 Hz, 1H), 7.13 (d, J = 8.8 Hz, 2H), 5.02 (d, J = 4.4 Hz,
1H), 4.11 (dd, J = 9.9, 4.3 Hz, 1H), 4.00 (dd, J = 9.9, 6.0 Hz,
1H), 3.90 (dd, J = 8.7, 3.8 Hz, 1H), 2.70–2.57 (m, 3H), 2.01 (d,
J = 12.2 Hz, 2H), 1.79–1.77 (m, 5H), 1.70–1.66 (m, 6H), 1.37
(d, J = 11.8 Hz, 2H); 13C NMR (101 MHz, DMSO-d6) δ 161.2,
144.3, 135.0, 132.0, 131.5 (2C), 131.2, 131.1, 127.0, 125.8,
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125.7, 117.9, 115.1 (2C), 104.3, 71.1, 68.4, 61.3, 49.6, 37.5,
39.91, 39.89, 31.6, 31.5, 30.79, 30.77, 27.3, 27.2.
20 V. Trapani, V. Patel, C. O. Leong, H. P. Ciolino, G. C. Yeh, C.
Hose, J. B. Trepel, M. F. G. Stevens, E. A. Sausville and A. I.
Loaiza-Pérez, Br. J. Cancer, 2003, 88, 599–605.
Conflicts of interest
21 P. Mahendran, A. J. Rajendran, C. Balachandran, A.
Stalin, S. Rangan, L. Kothandapani, K. C. Rao, S. Awale
and B. N. Hiteshkumar, Res. Chem. Intermed., 2018, 44,
535–552.
22 B. Bai, X.-Y. Li, Y. Li and H.-J. Zhu, Bioorg. Med. Chem. Lett.,
2011, 21, 2302–2304.
23 S. El-Meligie, A. T. Taher, A. M. Kamal and A. Youssef, Eur. J.
Med. Chem., 2017, 126, 52–60.
24 S. Maddila, K. Naicker, M. I. K. Momin, S. Rana, S. Gorle, S.
Maddila, K. Yalagala, M. Singh, N. A. Koorbanally and S. B.
Jonnalagadda, Med. Chem. Res., 2016, 25, 283–291.
25 F. Özen, S. Tekin, K. Koran, S. Sandal and A. O. Görgülü,
Appl. Biol. Chem., 2016, 59, 239–248.
26 N. R. Penthala, V. N. Sonar, J. Horn, M. Leggas, J. S. K. B.
Yadlapalli and P. A. Crooks, MedChemComm, 2013, 4,
1073–1078.
27 P. J. Cossar, J. R. Baker, N. Cain and A. McCluskey, R. Soc.
Open Sci., 2018, 5(1–22), 171190.
28 S. Larsson, D. Fraccalvieri, C. D. Andersson, L. Bonati, A.
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There are no conflicts to declare.
Acknowledgements
JRB is the recipient of an Australian government RTP
scholarship. Funding from the Australian Cancer Research
Foundation and the Ramaciotti Foundation is gratefully
acknowledged.
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