Amino Alcohol Acrylonitriles as Activators of the Aryl Hydrocarbon Receptor Pathway: An Unexpected MTT Phenotypic Screening Outcome

Accepted Article

Title: Amino Alcohol Acrylonitriles as Activators of the Aryl hydrocarbon

Receptor Pathway, An Unexpected MTT Phenotypic Screening

Outcome

Authors: Jennifer Baker, Cecilia C Russel, Jayne Gilbert, Jennette

Sakoff, and Adam McCluskey

This manuscript has been accepted after peer review and appears as an

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of the final Version of Record (VoR). This work is currently citable by

using the Digital Object Identifier (DOI) given below. The VoR will be

published online in Early View as soon as possible and may be different

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the VoR from the journal website shown below when it is published

to ensure accuracy of information. The authors are responsible for the

content of this Accepted Article.

To be cited as: ChemMedChem 10.1002/cmdc.201900643

Link to VoR: http://dx.doi.org/10.1002/cmdc.201900643

A Journal of

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Amino Alcohol Acrylonitriles as Activators of the Aryl

hydrocarbon Receptor Pathway, An Unexpected MTT Phenotypic

Screening Outcome

Jennifer R. Baker,^[a]Cecilia C. Russell,^[a]Jayne Gilbert,^[b]Jennette A. Sakoff *^[b]and Adam McCluskey

[a]

*

[a]

Ms JR Baker (0000-0002-9560-301X), Dr CC Russell (0000-0001-8401-5856) and Prof A McCluskey (0000-0001-7125-863X)

Chemistry

The University of Newcastle

University Drive, Callaghan, NSW 2308, Australia

E-mail: Adam.McCluskey@newcstle.edu.au

[b]

Dr J Gilbert (0000-0001-5034-386X) and Dr JA Sakoff (0000-000207009-5792)

Experimental Therapeutics Group, Department of Medical Oncology

Calvary Mater Hospital

Edith Street, Waratah NSW 2298, Australia.

Supporting information for this article is given via a link at the end of the document.

Abstract:

Lead

(Z)-N-(4-(2-cyano-2-(3,4-

a promising approach for a novel breast cancer treatment.^[5-7]The

AhR pathway localises in the cytosol, with the AhR complexing

two molecules of heat shock protein 90 (Hsp90), co-chaperone

p23 and the Aryl hydrocarbon interacting protein (AIP). Upon

ligand binding by either an exogenous or endogenous ligand, AhR

translocates into the cell nucleus, forms a heterodimer with the

Aryl hydrocarbon receptor nuclear translocator (ARNT), and

Hsp90 is released. The heterodimer then binds to xenobiotic

response elements (XRE) located in the promotor region of critical

genes, resulting in their transcriptional activation. The AhR

targeted genes include the cytochrome P450 metabolising

enzymes CYP1A1, CYP1A2 and CYP1B1. Depending upon the

ligand, this metabolic activation may produce an inert product or

a DNA damaging molecule that induces cell death (Figure 1).^[8]

dichlorophenyl)vinyl)phenyl)acetamide, 1 showed MCF-7 GI₅₀= 30nM

and 400-fold selective c.f. MCF10A (normal breast tissue). Acetamide

moiety modification (13a-g) to introduce additional hydrophobic

moieties was favoured with MCF-7 breast cancer cell activity

enhanced at 1.3 nM. Other analogues were potent against the HT29

colon cancer cell line at 23 nM. Textbook SAR data was observed in

the MCF-7 cell line via the ortho (17a), meta (17b) and para (13f). The

amino alcohol -OH moiety was pivotal, but no stereochemical

preference noted. But, these data did not fit our homology modelling

expectations. Aberrant MTT ((3-[4,5-dimethylthiazol-2-yl]-2,5-

diphenyl-tetrazolium bromide) screening results and metabolic

interference confirmed by sulforhodamine

B (SRB) screening.

Interfering analogues resulted in 120 and 80-fold CYP1A1 and

CYP1A2 amplification, with no upregulation of SULT1A1. This is

consistent with activation of the AhR pathway. Piperidine per-

deuteration reduced metabolic inactivation. 3-OH / 4-OH piperidine

analogues showed differential MTT and SRB activity supporting MTT

assay metabolic inactivation. Data supports piperidine 3-OH, but not

the 4-OH, as a CYP substrate. This family of β-amino alcohol

substituted 3,4-dichlorophenylacetonitriles show broad activity

modulated via the AhR pathway. By SRB analysis the most potent

analogue

was

23b,

(Z)-3-(4-(3-(4-phenylpiperidin-1-yl)-2-

hydroxypropoxy)phenyl)-2-(3,4-dichlorophenyl)-acrylonitrile.

Introduction

Breast cancer is the most common cancer in women worldwide,

and has the second highest mortality rate. In 2018, it was

estimated that more than 600,000 women across the globe

succumbed to this insidious disease.^[1]Moreover, triple negative

breast cancers that lack estrogen (ER), progesterone (PR) and

HER-2 hormone receptors have no available targeted therapies,

and resistance to standard treatment is common.^[2]Combined

with the sobering fact that metastatic breast cancer is essentially

incurable, it is apparent that new targeted therapies are urgently

required.^[3,4]

Figure 1: The AhR pathway showing ligand binding, nuclear translocation and

cytochrome P450 (CYP) activation.

Upregulation of the AhR in the cytosol of inflammatory breast

cancer cells, as well as its poor prognostication for patients with

this disease, makes the AhR an attractive breast cancer treatment

target.^[9,10]

We have previously reported a range of dichlorinated phenyl

acrylonitriles as potent and selective inhibitors of cell growth in the

MCF-7 breast cancer cell line, mediating their effects via

activation of the AhR pathway.^[11,12]Two of these ligands, (Z)-N-

The Aryl hydrocarbon receptor (AhR) is a member of the basic

helix-loop-helix/Per-ARNT-SIM transcription factor family, and

hijacking of its cytosol-to-nuclear pathway has been identified as

(4-(2-cyano-2-(3,4-dichlorophenyl)vinyl)phenyl)acetamide

(1)

and ANI-7 (2) were found to selectively reduce cell viability in the

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MCF-7 breast cancer cell line, at 0.03 and 0.56 µM, respectively;

representing more than 400-fold selectivity for 1, and almost 70-

fold selectivity for 2, compared with the MCF10A normal breast

cell line. Notably, these compounds also exhibited excellent

decreased cell viability in the triple-negative breast cancer cell line

MDAMB468 (0.042 µM for 1 and 0.23 µM for 2), and good activity

against the therapy-resistant triple-negative lines BT20 (2.5 µM

for 1 and 2 µM for 2) and MDAMB231 (5.1 µM for 1 and 17 µM for

2). Other reported AhR ligands that show similar selectivity in

breast carcinoma models are aminoflavone (3) and Phortress (4).

Aminoflavone (3) and Phortress (4), as the pro-drugs, progressed

to clinical trials; 3 for breast cancer (Phase II), and 4 for colon

cancer (Phase I).^[13,14]

access to b-amino alcohols,^[15]thus we hypothesized that addition

of this moiety may provide additional activation of the AhR

pathway.

b-Amino alcohols are known to be biologically active,^[16-18]and

introduction of sp³character (increased Fsp³character) is also

known to improve drug solubility;^[19]which has been an issue with

some previous analogues.^[11,12]The amino alcohol moiety is

present in many clinically relevant compounds, including

alprenolol (5), utilized for the treatment of angina pectoris, and

febrifugine (6, Figure 3), a potent antimalarial agent. Additionally,

we hoped to explore the effect of adding substituents to the phenyl

ring, in an effort to further enhance the biological potency that was

noted with the previously reported N-acetamide 1 against the

MCF-7 breast cancer cell line. Herein we report our recent efforts

in this area through the synthesis and biological analysis of a

range of dichlorinated phenylacrylonitriles bearing a b-amino

alcohol moiety. Critically we also report on an unexpected

outcome from our MTT-based phenotypic screening.

H

CN

N

CN

Cl

N

Cl

O

H

1

2

OH

NH₂

O

N

OH

H

O

F

O

N

F

H

O

5

6

F

NH₂

N

H

Figure 3: Two biologically active amino-alcohol containing drugs; alprenolol (5)

and febrifugine (6).

NH₂

3

NH₂

F

N

S

Results and Discussion

NH

O

To determine approach feasibility, a focused library was prepared

and evaluated. Thus, the desired b-amino alcohols were

accessed via flow chemistry introduction of epi-chlorohydrin (7) to

4-hydroxybenzaldehyde (8) which gave epoxide (9),^[15]

Knoevenagel condensation with 3,4-dichlorophenyl acetonitrile

(10) in the basic ionic liquid benzytrimethyl ammonium hydroxide

gave the key epoxide (11) (64% 2 steps; Scheme 1). With

intermediate 11 in hand, we next examined the synthesis of a pilot

library of b-amino alcohol acrylonitriles using amines 12a-g,

Library A. This library spanned a simple aniline (13a), a

cyclohexyl derivative (13b), as well as the decorated aniline

derivatives 4-chloro (13c), 4-bromo (13d), 4-methoxy (13e), 4-

phenylpiperazine variant (13f), and dimethylamino (13g) moieties.

This afforded a seven-component library in poor to moderate

yields (18-43%). In all instances, excepting 13f, the increased

solubility and by-product profile required these analogues to be

purified by flash chromatography (see Experimental).

NH₂

4

Figure 2: 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) and Phortress (4). Red highlights indicate the presence of the

pro-moiety in 3 and 4.

The mechanism of action of these cytotoxic AhR ligands requires

the in situ formation of a nitrenium ion moiety, which enables

highly localized DNA damage, and cell death to occur. This

process demands a highly orchestrated interaction between the

AhR ligand, the AhR, CYP1s and SULT1A1 to activate the

‘dormant’ amino moieties in these ligands.⁶Given our recent

efforts with the phenyl acrylonitriles, we rationalized that

enhanced breast cancer specificity and potency might result from

increasing the number of amino moieties in these molecules. In

turn, this allowed us to leverage against our in-house facile

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O

Cl

7

5 M in DMF

OH

Cs₂CO₃

4 Å sieves

i

O

Cl

CN

5 bar

O

+

O

8

10 mL 10 mL

150 ˚C 150 ˚C

9

0.1 M in DMF

10

ii

O

CN

Cl

OH

iii

H

O

N

11

CN

R

10 bar

Cl

10 mL

150 ˚C

10 mL

150 ˚C

R

H₂N

5 equiv.

13a-g

12a-g

Scheme 1: Reagents and conditions: (i) Vapourtec Easy-MedChem, 5 M 7 (anhy. DMF), 0.1 M 8 (anhy. DMF), 105 °C, 5 bar, 1.0 mL.min^-1(t_r= 20 min); (ii) 10,

PhCH₂NMe₃(OH), H₂O, 50 °C, 5h; (iii) RNH₂(12a-g) 0.05M (EtOH), 11 0.01 M (EtOH), 150 °C, 10 bar, 1.0 mL.min^-1(t_r= 20 min).

H

The effectiveness of Library A to selectively reduce the

viability of breast cancer cells (MCF-7) relative to the normal

breast cell line MCF10A or cells derived from another tumour type

(HT29 colon) was evaluated in an MTT assay (see Table 1 for

detail). Analogues 13a-13e exhibited good activity against the

MCF-7 cells (2.3 – 16 µM) (Table 1). Hydrophobic-substituted

analogues 13b and 13g also revealed broad spectrum low micro-

molar inhibition across the three cell lines tested. In particular, 13g

exhibited nanomolar activity against the colon cancer cell line

HT29 (23 nM). Phenylpiperazine 13f, however, showed low

nano-molar potency in the MCF-7 cell line (1.3 nM), and sub- to

low micro-molar growth inhibition (0.05 – 2.2 µM) in the remaining

cell lines. Notably, this analogue demonstrates >260 times higher

potency in the MCF-7 cells than the normal breast cells (MCF10A).

This is in keeping with our earlier findings with the parent 1 and

2.^[11,12]Buoyed by this exciting development, we sought to

explore the extreme potency of this derivative. It was noted that

the addition of both electron-withdrawing (13c and 13d) and

electron-donating (13e) functional groups had little effect on the

cell viability, while the presence of a hydrophobic functionality

(13b, 13f and 13g) was favoured.

1.8±0.1

2.3±0.2

2.7±0.1

N

13b

13c

13d

H

N

12±1.0

11±0.0

4.7±0.3

13±0.0

11±1.0

31±1.1

29±1.0

15±0.0

Cl

H

N

Br

H

N

O

13e

N

0.052±0.019

0.023±0.011

0.0013±0.0004

0.33±0.33

0.34±0.05

0.79±0.26

N

13f

N

13g

Table 1. GI₅₀values (µM) of Library A (13a-g) against HT29 (colon carcinoma),

MCF-7 (breast carcinoma) and MCF10A (normal breast) cell lines, in an MTT

assay. GI₅₀is the concentration at which 50% of the cell growth is inhibited.

[a] Colon carcinoma; [b] breast carcinoma; [c] breast (normal); Data is

presented as ±SEM (n=3).

OH

Based on these data, we sought to further develop the SAR

data associated with 13f. We considered 13f to comprise three

distinct rapidly modifiable regions, the ether linkage (green), the

alcohol moiety (blue) and the phenylpiperazine functionalities

(red) (Figure 4). We have examined the SAR requirements of the

dichlorophenyl moiety in earlier reports.^[11]

O

CN

Cl

R

HT29^a

MCF-7^b

MCF10A^c

H

13±0.0

16±1.0

27±3.0

N

13a

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glycidyl-3-nitrobenzenesulfonate

(19b)

with

8

gave

enantiomerically pure oxirane-benzaldehydes 20a and 20b

respectively, in excellent yields (86 and 91%). Knoevenagel

condensation with 10 gave the β-amino alcohol intermediates 21a

and 21b. Oxirane ring opening with 1-phenylpiperazine (12f)

under microwave conditions (ethanol, 20 min and 120 °C)

afforded the desired (S)-22a and (R)-22b in good to excellent

yields (59-83% and optical purity; electronic supporting

information) (Scheme 2). No observable difference in bioactivity

with 22a and 22b was evident (electronic supporting information).

OH

N

O

N

CN

Cl

13f

MCF-7 GI₅₀= 1.3 ± 0.4 nM

Figure 4: Chemical structure of Library A lead compound 13f.

The initial modification explored was the para-ether linkage of

13f through the synthesis of the corresponding ortho-17a and

meta-17b analogues. This two-component library, Library B, was

generated from the corresponding hydroxybenzaldehydes 14a

and 14b as per Scheme 1 in good yields (72 and 74%).

Knoevenagel condensation with 10 afforded the epoxides 16a

and 16b. Epoxide-opening with 1-phenylpiperazine (12f) gave

the desired 17a and 17b. MTT evaluation demonstrated that

neither analogue showed significant activity in the cell lines tested,

particularly the breast cancer cell line of interest, MCF-7 (Table 2).

In this cell line, these analogues displayed an almost textbook

activity response to ring substituent position changes, viz,

inhibitory activity para > meta > ortho (13f, 1.3 nM; 17b, 6.7 µM;

17a 23 µM).

Table 2:GI₅₀values (µM) of Library B (17a,b and 18) against HT29 (colon

carcinoma), MCF-7 (breast carcinoma) and MCF10A (normal breast) cell line,

in an MTT assay. GI₅₀is the concentration at which 50% of the cell growth is

inhibited.

CN

Cl

MCF-

7^b

MCF10A

R

HT29^a

c

OH

N

8.2±2.5

23±3.0

24±4.0

O

N

Concurrently we also examined the requirement of the alcohol

moiety; as a function of ready access to starting materials,

through the synthesis of the de-hydroxylated derivative of 13g, 18.

As can be surmised from the cytotoxicity data in Table 2, removal

of the hydroxy moiety resulted in a 10-fold decrease in potency in

the MCF-7 cell line, and a similar loss across the remaining cell

populations. The activity of these compounds was typically 3-fold

more potent against the HT29 cell line than the target MCF-7 cell

line. Only poor selectivity for MCF-7 vs. MCF10A was noted,

unlike the lead compounds, 1 and 2.

17a

OH

N

6.7±1.

0

3.3±0.48

13±1.0

2.5±0.6

O

N

17b

O

N

0.70±0.0

1

2.3±0.

1

18

Given the critical nature of the -OH moiety, we next explored

the stereochemical preference at this position through the

synthesis of both enantiomers of 13f. Coupling of commercially

available (S)-(+)-glycidyl-3-nitrobenzenesulfonate (19a) or (R)-(-)-

O O

[a] Colon carcinoma; [b] breast carcinoma; [c] breast (normal); Data is

presented as ±SEM (n=3).

Cl

OH

O

CN

O₂N

S

R

i

+

O

8

10

20a, R =

20b, R =

19a, R =

19b, R =

O

ii

O

OH

N

CN

R

iii

O

N

Cl

+

CN

N

Cl

HN

12f

22a; (2S)-isomer

22b; (2R)-isomer

21a, R =

21b, R =

O

Scheme 2: Reagents and conditions: (i) 1.5 eq. Cs₂CO₃, DMF, 80 °C, 4 h; (ii) PhCH₂NMe₃(OH), H₂O, 50 °C, 5 h; (iii) 2 eq. amine, EtOH, MW, 120 °C, 20 min.

Library C saw further modification of the phenylpiperazine

moiety to determine the effect of this moiety. Using the above

microwave protocol (Scheme 2), and the 12 analogues 23a-m

were afforded in moderate to good yields (39-87%). This library

of compounds exhibited improved aqueous solubility over the

previous analogues,^[12]leading to a general trend of lower yields.

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Subsequent MTT cytotoxicity screening in our cell line panel

revealed all analogues, except 23b, displayed low nano-molar to

pico-molar activity against the MCF-7 cell line, with micro-molar

to nano-molar in the remaining cell lines (Table 3).

The introduction of additional hydrophobic moieties with

Library C analogues with 23a, 23f – 23m saw apparent excellent

activity and selectivity towards the MCF-7 breast cancer cells,

Increase (23k) or reduction (23j) in the hydrophobic ring size had

little impact on broad-spectrum cytotoxicity across the panel of

cell lines evaluated, and with the exception of the slight loss of

activity in the HT29 colon cancer cell line, neither did the addition

of the adamantyl moiety (23l and 23m). Addition of an oxygen,

either in the saturated ring or proximal to it (23c and 23e) resulted

in a concomitant reduction in potency, but these compounds still

retained low nano-molar potency (MCF-7) in the MTT assay.

with each analogue returning

enhancement relative to 13f. Addition of an extra nitrogen

functionality (23d) showed marginal potency reduction.

a >10-fold MCF-7 potency

a

Table 3: GI₅₀values (µM) of Library C analogues 23a-m against HT29 (colon carcinoma), MCF-7 (breast carcinoma) and MCF10A (normal breast) cell line, in an

MTT assay. GI₅₀is the concentration at which 50% of the cell growth is inhibited.

OH

O

CN

Cl

R

HT29^a

MCF-7^b

MCF10A^c

R

HT29^a

MCF-7^b

MCF10A^c

N

ND*

<0.0001

0.0±0.0

0.016±0.0005

<0.0001

0.20±0.00

N

23h

23a

N

1.5±0.27

2.4±0.28

2.9±0.13

1.0±0.20

0.00062

<0.0001

0.072±0.064

N

23i

23b

N

O

N

0.064±0.021

0.014±0.01

0.00059

0.00010

<0.0001

0.032±0.0064

0.14±0.047

N

23j

23c

N

0.011±0.0005

0.0072±0.0027

0.00088

0.0020

0.30±0.25

0.18±0.10

N

23d

23k

H

N

O

N

0.033±0.018

0.031±0.012

0.0±0.0019

0.0±0.00

0.11±0.022

0.32±0.058

23e

23l

HN

N

ND

<0.0001

0.08±0.05

23m

23f

N

0.0025

0.11±0.050

23g

^aColon carcinoma; ^bbreast carcinoma; ^cbreast (normal); *ND denotes that the GI₅₀was not determined; Data is presented as ±SEM (n=3).

These data, excepting that noted with 23b, was in part

consistent with our prior observations that the ligand-binding

PAS-B domain had shown that the binding pocket is lined with

mostly hydrophobic residues, however these prior studies did

not support the introduction of bulky substituents as the

binding pocket was defined as compact and hydrophobic.^[12,20]

However a number of these analogues, e.g. 23a, 23f-k,

returned undeterminable GI₅₀values, which was puzzling.

Moreover, the magnitude in activity reduction with 23b could

not be explained by our PAS-B homology model.^[12,20]Re-

synthesis and re-examination of the Library C compounds

screened by MTT assay showed significant activity variation.

Subsequent morphological examination of the treated cells

using phase contrast microscopy suggested that the visual

morphology was inconsistent with the MTT determined

cytotoxicity with all analogues excepting 23b. As the MTT

assay relies on mitochondrial dehydrogenase enzymes to

reduce the tetrazolium moiety to a formazan precipitate,^[21]it

was hypothesized that a metabolite generated by the majority

of the amino-alcohol products was interfering with this reaction.

Microscopic examination of the MCF-7 cells after compound

exposure (72 h, 1µM, 13f) revealed viable cells with no

evidence of cell death (Figure 5).

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7 breast cancer cells treated with 13f (1µM); C. As for B, with no added 13f

(untreated control cells).

Re-screening of 13f and Library C in a sulforhodamine B

(SRB) protein assay was conducted (Table 4). The SRB assay

relies on the ability of the sulphur-based dye to bind to protein

components of the cells.^[22]Comparisons have been made

between the SRB and MTT assays, with the former demonstrating

greater sensitivity.^[21,23]The outcome of this secondary assay was

significantly different to that of the MTT assay, with the activity of

all analogues starkly lower than previously noted, as shown in

Figure 4 for 13f. Only 23b retained the activity in the SRB assay

compared with the MTT assay.

The key point of differentiation between 13f and 23b is the N

to C isosteric modification (13f, piperazine; 23b, piperidine), and

logically this is the effector of differential activity in these two

assays. Phenylpiperazines are known to form highly reactive

iminoquinone moieties capable of reacting with glutathione

(Supplementary material),^[24-26]which potentially rationalizes the

differential reactivity of 13f and 23b, with the latter lacking the

critical nitrogen to induce the iminoquinone formation. However,

this does not explain the MTT vs. SRB assay differences

observed with other analogues.

In these instances, we

hypothesize that in vitro CYP1 mediated oxidative activation

results in the formation of an additional active metabolite that

prevents the reduction of MTT,^[16,27,28]and yielding aberrant assay

outcomes. SRB cytotoxicity assay operates via an oxidative

mechanism and is thus not affected by this reduction block.

Figure 4: A. Comparison of the effect of 13f in MTT and SRB assays in MCF-

7 cells after 72 h treatment, data is ±SEM (n=3); B. Microscopy images of MCF-

Table 4: GI₅₀values (µM) of 13f and Library D analogues (23a-m) against HT29 (colon carcinoma), MCF-7 (breast carcinoma) and MCF10A (normal breast) cell

line, in an MTT assay and an SRB assay. GI₅₀is the concentration at which 50% of the cell growth is inhibited.

HT29^a

MCF-7^b

MCF10A^c

Compound

13f

SRB

MTT

SRB

MTT

SRB

MTT

3.3±0.38

0.52±0.019

4.1±0.99

0.0013±0.000

4.8±0.26

0.34±0.05

4

0.4

ND

1.2

<0.0001

2.0

0.0±0.0

2.9±0.13

23a**

23b

23c

23d

23e

23f

1.9±0.06

8.6±2.2

1.5±0.27

0.64±0.21

2.5±0.20

5.3±2.6

2.4±0.28

0.014±0.01

0.00088

0.0020

2.5±0.22

16±0.33

1.0±0.20

0.011±0.0005

0.0072±0.0027

ND

2.3±0.38

5.3±0.91

2.5±0.10

2.3±0.30

2.3±0.18

2.1±0.03

2.8±0.49

2.8±0.23

2.0±0.10

2.1±0.07

2.4±0.21

5.6±1.2

0.30±0.25

0.18±0.10

0.08±0.05

0.11±0.050

0.20±0.00

0.072±0.064

0.032±0.0064

0.14±0.047

0.11±0.022

0.32±0.058

1.2±0.12

3.1±0.13

0.66±0.24

1.7±0.23

2.4±1.3

<0.0001

0.0±0.0019

0.0±0.00

2.5±0.12

2.7±0.10

2.5±0.07

2.5±0.06

2.9±0.07

3.1±0.07

2.5±0.22

2.7±0.22

0.0025

23g

23h

23i

0.016±0.0005

0.00062

0.36±0.07

1.5±0.57

1.7±0.38

0.89±0.41

1.4±0.07

0.00059

23j

0.00010

23k

23l

0.033±0.018

0.031±0.012

23m

[a] Colon carcinoma; [b] breast carcinoma; [c] breast (normal); *ND denotes that the GI₅₀was not determined. **n=1 for SRB assay; All other data, MTT assay

values are n=3; SRB values are n=3.

It is unknown whether this interferant occurs pre- or post-

CYP1 activation in the cell lines evaluated. In an effort to elicit

this information, mono-hydroxylated piperidine analogues of 13f,

viz 25a and 25b were synthesized as per Scheme 3, as potential

CYP1 metabolism products and their activity evaluated in both

assays. In a similar vein, we also prepared the per-deuterated

MTT to examine the possibility that MTT itself was the reacting,

and thus interfering species in these assays.

Combining the hydroxylated analogues 25a and 25b with

MTT in a ‘faux cell-free assay’ as described previously,²⁹and

analysis of the plate contents at 8 different concentrations after

incubation for 4 hours showed no change in the concentration of

MTT, indicating that no reaction was occurring with the MTT

reagent. Further examination of 25a,b under standard MTT and

SRB assay conditions revealed that the 4-OH 25b showed no

piperidine analogue (26), noting that

a CYP1 mediated

transformation would be retarded by deuteration. In addition,

each of the suspect analogues were stirred in the presence of

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MTT vs. SRB assay disparity, while the effect was still evident for

3-OH 25a (Table 5). This strongly suggest that the 4-moiety

blocks the subsequent deactivation of the MTT assay, while the

3-OH moiety enables further reaction to deactivate MTT. This

may be a function of the 3-OH 25a remaining a CYP substrate

and undergoing further metabolic activation of a MTT reactive

species (Scheme 4). This is also consistent with 25a being the

major metabolite responsible for the deactivation of the MTT

assay with this type of compound.

(Table 5) exhibited a stark contrast to the previously observed

results with the non-deuterated analogue 23f. As shown in the

results below, while 23f demonstrated <0.0001 µM activity in the

MCF-7 cell line, 26 exhibited much lower activity at

0.0031±0.0029 µM.

This supports the hypothesis that a

metabolite of these compounds is forming, as the deuteration

would slow the rate of this formation. Gratifyingly, 26 exhibited an

identical cell viability profile to 23f, indicating that the substitution

of hydrogen for deuterium had no effect on the activity of the

analogue.

O

OH

CN

OH

+

O

N

Cl

HN

CN

Cl

11

3-OH; 24a

4-OH; 24b

23f

CYP

O

OH

O

N

CN

OH

Cl

3-OH; 25a

4-OH; 25b

25a

Scheme 4. Possible mechanism of activation of 26a vs. 26b explain the

differential MTT vs. SRB assay data obtained.

Scheme 3: Reagents and conditions: 1.5 eq. amine, EtOH, MW, 120 °C, 20 min.

Undeterred by the non-identification of the specific interfering

compound, analysis of the per-deuterated piperidine analogue 26

Table 5: GI₅₀values (µM) of 25a, 25b and 26 against HT29 (colon carcinoma), MCF-7 (breast carcinoma) and MCF10A (normal breast) cell line, in an MTT assay

and an SRB assay. Compound 23f included as a reference compound. GI₅₀is the concentration at which 50% of the cell growth is inhibited. MTT assay values

are n=3; SRB values are n=3.

Compound

HT29^a

MCF-7^b

MCF10A^c

SRB

MTT

SRB

MTT

SRB

MTT

23f

25a

25b

26

0.66±0.24

0.84±0.53

1.0±0.62

ND*

2.5±0.10

2.5±0.57

3.1±1.5

2.2±0.46

<0.0001

2.5±0.12

2.2±0.46

3.1±0.033

3.5±1.2

0.08±0.05

1.2±0.033

1.9±0.32

0.013±0.010

0.20±0.06

0.0016±0.0014

1.9±0.94

0.29±0.0058

0.0034±0.0018

0.0031±0.0029

0.082±0.069

[a] Colon; [b] breast; ^[c]breast (normal); *ND denotes that the GI₅₀was not determined,Data presented as ±SEM viz, MTT assay (n=3) and SRB (n=3).

To further investigate the mechanism of action of these

compounds and to determine whether they acted via the AhR

pathway, gene expression studies were conducted with 13f. It

has been described that metabolism of aminoflavone (3) occurs

via first a hydroxylation of the primary amines by a CYP, then a

subsequent sulfonylation by sulfotransferase SULT1A1; before

forming the final cytotoxic metabolite, a nitrenium ion.^[30]As we

have reported previously, the expression of enzymes such as

CYP1A1 and CYP1A2 is increased following AhR ligation with our

breast selective compounds. In the same study, it was also noted

that basal expression of CYP1A1 and CYP1A2 did not predict for

the activity of our compounds whilst the expression of SULT1A1

did; highlighting the inducible nature of CYP1s and the need for

SULT1A1 activity.^[31]In order to determine whether our current

cohort of compounds were indeed acting via the AhR pathway,

the expression of CYP1A1 and CYP1A2 following treatment for 4

h was evaluated, with the results portrayed as fold change

compared to untreated cells (Figure 5).

SULT1A1. As previously observed, this expression profile is fully

consistent with this compound acting via the AhR pathway.^[31]

Conclusions

Rapid access to a series of diverse libraries of β-amino alcohol

substituted 3,4-dichlorophenylacetonitriles was possible through

a combination of flow and microwave chemistry approaches. This

enabled the introduction of Fsp³enhancement and additional

amine moieties, with the latter previously shown to be beneficial

for AhR pathway activation. Phenotypic (MTT) evaluation of

these compound libraries across a panel of two cancer and one

normal cell line revealed a marked breast cancer selectivity for a

number of the analogues produced. A particular focus was placed

on the introduction of phenyl substituents based on the excellent

activity and selectivity of the parent (1) (MCF-7 GI₅₀= 30nM and

400-fold selective compared to MCF10A (normal breast tissue)).

Within the first library (13a-g), additional hydrophobic moieties

were favoured, with 13f the most MCF-7 potent (1.3 nM). Other

analogues in this library also showed excellent activities against

As shown in Figure 5, treatment of the breast cancer cell line

MCF-7 with 5 µM 13f for 4 hours resulted in 120- and over 80-fold

increased expression of CYP1A1 and CYP1A2 respectively;

whilst no change was noted for the expression of AhR and

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the colon cancer cell line HT29 (13g, GI₅₀= 23 nM). Within this

series, textbook SAR data was observed with activity increasing

against the MCF-7 cell line via the ortho (17a), meta (17b) and

para (13f) analogues respectively. The pivotal nature of the

amino alcohol -OH moiety was demonstrated with 13g and 18;

and the lack of stereochemical requirement at this position noted

with 22a and 22b. Intriguingly, increasing the hydrophobic nature

of the terminal phenyl substituent afforded almost homeopathic

levels of potency. Examination of this phenomenon revealed a

by-product from cellular metabolism resulted in interference with

the MTT assay, confirmed on alternative phenotypic screening via

an SRB assay.

Our investigations confirmed that these analogues

significantly upregulate both CYP1A1 and CYP1A2 with 13f

resulting in a 120- and 80-fold amplification of CYP1 expression,

respectively. No equivalent upregulation of SULT1A1 was noted.

This is consistent with these compounds acting via the AhR

pathway. In addition, the rate of metabolic inactivation was

slowed with the introduction of per-deuterated piperidine 26.

Further we note that the 3-OH 25a and 4-OH 25b showed

differential activity in the MTT and SRB assay suggesting that it is

a metabolic product from at least two oxidation steps that

interferes with the MTT assay. In this instance we postulate that

the 3-OH 25a or a further metabolite thereof, adversely modulates

the MTT-assay signal leading to false positives in this phenotypic

assay. Notwithstanding this, we clearly demonstrate that these

analogues are capable of AhR pathway upregulation, a feature

recently postulated as a favourable outcome in the treatment of a

range of cancers.^[32]

Critically in this study the application of the SRB assay

revealed that no true SAR data could be extracted, and this adds

a significant note of caution as the MTT assay with this series of

compounds clearly shown

a

bias towards significant

overestimation of compound potency in the MCF-7 cell line.

In addition to identifying these analogues as potential novel

breast cancer targeting compounds, this work also identified 23a,

23f and 23l with sub-micromolar potency in killing the HT29 colon

cancer cell line.

Figure 5: A. Gene expression analysis in MCF-7 cells in response to 5µM 13f for 4h, demonstrating the upregulation of CYP1A1 and CYP1A2 by 120 and 82-fold

over untreated cells respectively; B. As for A with AhR and SULT1A1 showing no change in expression level was observed for AhR and SULT1A1 .

without the test agent was added to each well. After 72 h drug exposure

growth inhibitory effects were evaluated using either the MTT (3-[4,5-

dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) or sulforhodamine

B (SRB) assay with absorbances read at 540 nm. Percentage growth

Experimental Section

Biology

inhibition was determined at a fixed drug concentration of 25 µM. A value

of 100% is indicative of complete cell growth inhibition. Those analogues

Cell culture and stock solutions

showing appreciable percentage growth inhibition underwent further dose

Stock solutions were prepared as follows and stored at -20 °C: drugs were

response analysis allowing for the calculation of a GI₅₀value. An eight-

stored as 40 mM solutions in DMSO. All cell lines were cultured in a

point dose response curve was produced, using MS Excel software. Each

humidified atmosphere 5% CO₂at 37 ºC. The cancer cell lines were

data point is the mean ±S.E.M. calculated from four to five replicates,

maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Trace

which were performed on separate occasions and separate cell line

Biosciences, Australia) supplemented with 10% foetal bovine serum, 10

passages. From these dose-response curves, the GI₅₀value was

mM sodium bicarbonate, penicillin (100 IU/mL), streptomycin (100 µg/mL),

calculated, representing the drug concentration at which cell growth is 50%

and glutamine (4 mM). The non-cancer MCF10A cell line was cultured in

inhibited based on the difference between the optical density values on

DMEM:F12 (1:1) cell culture media, 5% heat inactivated horse serum,

supplemented with penicillin (50 IU/mL), streptomycin (50 µg/mL), 20 mM

day 0 and those at the end of drug exposure.^[33,34]

Hepes, L-glutamine (2 mM), epidermal growth factor (20 ng/mL),

hydrocortisone (500 ng/mL), cholera toxin (100 ng/mL), and insulin (10

µg/mL).

SRB: 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

1hr at 4 °C the plates were washed 5x 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 4x in 1% acetic

acid to remove unbound stain and allowed to air dry. The protein was then

solubilized in 150 µL of 10mM unbuffered TRIS.

In vitro growth inhibition assay

Cells in logarithmic growth were transferred to 96-well plates. Cell viability

was determined by plating cells in duplicate in 100 µL medium at a density

of 2500-4000 cells/well in 96 well plates, viz MCF10A 2500 cells/well,

HT29 3000 cells/well and MCF7 4000 cells/well. On day 0, (24 h after

plating) when the cells were in logarithmic growth 100 µL medium with or

Morphological Assessment: Live cells were examined for morphological

alterations after 72 h exposure with and without 1µM 13f using phase

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contrast microscopy (Olympus CKX41 inverted microscope 100x

magnification).

General Procedure 2: (Z)-2-(3,4-dichlorophenyl)-3-(4-(oxiran-2-

ylmethoxy)phenyl)acrylonitrile (11, 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.

Gene Expression Analysis: For each cell population 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, CYP1A2, AhR and SULT1A1 on

a Rotor-Gene 3000 Thermo -Cycler Instrument using β2-microglobulin as

a housekeeping gene (Qiagen). The primer sequences were purchased

from Qiagen as follows: AhR (QT02422938), CYP1A1 (QT00012341),

(Z)-2-(3,4-Dichlorophenyl)-3-(4-(oxiran-2-ylmethoxy)phenyl)-acrylonitrile

(11)

4-Hydroxybenzaldehyde (8) (305 mg, 2.5 mmol) in 25 mL ACN was

passed through a series of omnifit columns, the first containing molecular

sieves, the second contained cesium carbonate and sand (50%:50% w/w)

at a flow rate of 0.5 mL/min. The reactant stream was then met by a

secondary stream containing epichlorohydrin (17 mL, with 20 mL DMF and

63 mL ACN, flow rate 0.5 mL/min), and together the reactant streams were

passed through a Vapourtec static mixer reactor (20mL) heated at 105 °C

and held at 5 bar back pressure.

CYP1A2

(QT00000917),

CYP1B1

(QT00209496),

SULT1A1

(QT01665489), AND β2M (QT00088935). HotStar Taq activation at 95°C

for 5 minutes, 40 cycles of denaturation (95°C for 5 seconds), and

annealing/extension (60ºC for 10 seconds). The comparative Ct value

method was used for data analysis. Gene expression was examined in

MCF-7 cells following treatment with 5 µM 13f for 4 h. Results show the

fold change in gene expression relative to untreated control cells.

The product solution was then evaporated under reduced pressure, then

diluted with ethyl acetate (50 mL), extracted with water (3 x 50mL), and

then brine (1 x 50 mL), dried over magnesium sulfate and the solvent

removed

under

reduced

pressure

to

afford

4-(oxiran-2-

Chemistry

ylmethoxy)benzaldehyde (9) as a light brown oil (364 mg, 82%).

General Methods

Alternatively, 4-hydroxybenzaldehyde (8, 2.019 g, 16.4 mmol, 1 eq.) and

cesium carbonate (8.500 g, 49.3 mmol, 1.5 eq.) were combined with 15

mL DMF and 20 mL epichlorohydrin (7) in a round-bottomed flask. The

reaction was heated to 75 °C and left to stir. After 3 h, the reaction was

cooled to ambient temperature, diluted with EtOAc (50 mL), washed with

water (3 x 30 mL, brine (2 x 30 mL), dried over MgSO₄and concentrated

under reduced pressure to afford 4-(oxiran-2-ylmethoxy)benzaldehyde (9)

as a brown oil (2.604 g, 90%). 4-(Oxiran-2-ylmethoxy)benzaldehyde

(9,12.177 g, 68 mmol, 1 eq.), was then added to a vigorously stirred

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

solution of water (40 mL) and heated at 50 °C.

Dichlorophenylacetonitrile (10,12.109 g, 65 mmol, 1.05 eq.) was then

slowly added forming suspension. After 5–10 min of stirring,

3,4-

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-

coated aluminum plates with

chromatography was performed under ‘flash’ conditions on Merck silica gel

60 (230-400 mesh).

a

benzytrimethyl ammonium hydroxide (40 wt.% aq. solution) (25 mL) was

added dropwise. After complete addition, the reaction was left to stir at

50 °C for 5 h. After this period, the solution was filtered hot, washed with

warm water, and dried under suction to yield a solid. The resulting yellow

solid was recrystallised from ethanol to afford the desired product as a

yellow solid (15.805 g, 70%), m.p.: 121-124 ˚C; ¹H NMR (400 MHz,

acetone-d₆) d 8.02 (d, J = 8.8 Hz, 2H), 7.97 (s, 1H), 7.94 (d, J = 1.4 Hz,

1H), 7.73 – 7.68 (m, 2H), 7.15 (d, J = 8.8 Hz, 2H), 4.47 (dd, J = 11.4, 2.6

Hz, 1H), 4.00 (dd, J = 11.4, 6.4 Hz, 1H), 3.36 (td, J = 6.6, 2.6 Hz, 1H), 2.87

(dd, J = 4.8, 4.4 Hz, 1H), 2.75 (dd, J = 5.2, 2.4 Hz, 1H); ¹³C NMR (101

MHz, acetone-d₆) d 162.0, 144.6, 136.4, 133.5, 132.8, 132.5 (2C), 132.0,

128.2, 127.5, 126.5, 118.5, 116.0 (2C), 106.6, 70.4, 50.4, 44.4; IR u_max/cm^-

¹: 3063 (C=C), 2210 (CN), 1255 (arom. C-O), 811 (C-Cl); LRMS: (ESI^-)

m/z: 368 (C₁₈H₁₂Cl₂NNaO₂) [M+Na-H]; HRMS: Material decomposed.

a thickness of 0.2 mm. Column

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. Optical rotation was performed on a

Jasco P-2000 Polarimeter in a 10 x 100 mm cell, and the [α]_Dvalues are

(Z)-2-(3,4-Dichlorophenyl)-3-(4-(2-hydroxy-3-(phenylamino)-

propoxy)phenyl)acrylonitrile (13a)

Prepared according to general procedure

1

with (Z)-2-(3,4-

dichlorophenyl)-3-(4-(oxiran-2-ylmethoxy)phenyl)acrylonitrile (11, 346 mg,

1 mmol, 1 eq) and aniline (12a, 456 µL, 5 mmol, 5 eq.). Column

chromatography (0-100% EtOAc in hexanes) afforded the desired

compound as a pale brown solid (78 mg, 18%), m.p.: 130-133 ˚C. ¹H NMR

(400 MHz, acetone-d₆) d 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, 1H); ¹³C NMR (101 MHz,

acetone-d₆) d 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; IR u_max/cm^-1: 3497 (NH), 3390 (br, OH), 3050

(C=C), 2210 (CN), 1598 (NH), 1253 (C-O), 815 cm^-1(C-Cl). LRMS: (ESI⁺)

m/z: 439 (C₂₄H₂₁Cl₂N₂O₂) [M+H]; HRMS: Exact mass calculated for

C₂₄H₂₁Cl₂N₂O₂[M+H], 439.0975. Found 439.0976.

given in 10⁻¹deg cm²g⁻¹

.

General Procedure 1: Using a Vapourtec RS-400 equipped with fraction

collection kit and auto-sampler, a 2.0 mL sample loop was charged with a

0.4 M solution of epoxide in toluene. An additional 2 mL sample loop was

charged with a 2.8 M amine solution in ethanol (as stated). The solutions

were flowed together and the resulting stream was then passed through

two PFA coil reactors in series at 150 °C, 10 bar back pressure and 0.5

mL min^-1(residence time 40 min). The resulting reaction mixture was

collected, concentrated in vacuo and purified as described below.

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(Z)-3-(4-(3-(Cyclohexylamino)-2-hydroxypropoxy)phenyl)-2-(3,4-

dichlorophenyl)-acrylonitrile (13b)

J = 12.6, 6.0 Hz, 1H); ¹³C NMR (101 MHz, acetone-d₆) d 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;

IR u_max/cm^-1: 3260 (br, OH), 3050 (C=C), 2829 (O-CH₃), 2210 (CN), 1596

(NH), 1272 (C-O), 820 (C-Cl); LRMS: (ESI+) m/z: 469 (C₂₅H₂₃Cl₂N₂O₃)

[M+H]; (ESI^-) m/z: 513 (C₂₆H₂₄Cl₂N₂O₅) [M+FA-H]; HRMS: Exact mass

calculated for C₂₅H₂₃Cl₂N₂O₃[M+H], 469.1081. Found 469.1083.

Prepared according to general procedure

1

with (Z)-2-(3,4-

dichlorophenyl)-3-(4-(oxiran-2-ylmethoxy)phenyl)acrylonitrile (11, 346 mg,

1 mmol, 1 eq) and cyclohexylamine (12b, 458 µL, 5 mmol, 5 eq.). Column

chromatography (0-100% EtOAc in hexanes) afforded the desired

compound as a pale yellow solid (119 mg, 27%), m.p.: 112-114 ˚C. ¹H

NMR (600 MHz, acetone-d₆) d 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) d 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), 34.1 (d, J = 21.1 Hz), 26.9, 25.6;

IR u_max/cm^-1: 3305 (NH), 3119 (br, OH), 2922 (C=C), 2853 (C-O-C), 2209

(CN), 1597 (NH), 1256 (C-O), 823 cm^-1(C-Cl).; LRMS: (ESI⁺) m/z: 445

(C₂₄H₂₇Cl₂N₂O₂) [M+H]; HRMS: Exact mass calculated for C₂₄H₂₇Cl₂N₂O₂

[M+H], 445.1444. Found 445.1447.

(Z)-2-(3,4-Dichlorophenyl)-3-(4-(2-hydroxy-3-(4-phenyl-piperazin-1-

yl)propoxy)-phenyl)acrylonitrile (13f)

Prepared according to general procedure

1

with (Z)-2-(3,4-

dichlorophenyl)-3-(4-(oxiran-2-ylmethoxy)phenyl)acrylonitrile (11, 346 mg,

1 mmol, 1 eq.) and 1-phenylpiperazine (12f, 788 µL, 5 mmol, 5 eq.). The

resulting mixture was filtered under vacuum and washed with cold EtOH

(2 x 10 mL) to afford the desired compound as a pale yellow solid (106 mg,

21%); Prepared according to general procedure 2 (with 346 mg, 1 mmol

epoxide) and 1-phenylpiperazine (17, 316 µL, 2 mmol, 2 eq.) to afford the

desired compound as a pale yellow solid (430 mg, 88%), m.p.: 175-178 °C.

¹H NMR (600 MHz, DMSO-d₆) d 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 = 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) d 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; IR u_max/cm^-1: 3392 (br, OH), 2825 (N-CH₂), 2210 (CN), 1245

(C-O), 818 (C-Cl); LRMS: (ESI⁺) m/z: 508 (C₂₈H₂₈Cl₂N₃O₂) [M+H]; HRMS:

Exact mass calculated for C₂₈H₂₈Cl₂N₃O₂[M+H], 508.1553. Found

508.1557.

(Z)-2-(3,4-Dichlorophenyl)-3-(4-(2-hydroxy-3-((4-chlorophenyl)-

amino)propoxy)-phenyl)acrylonitrile (13c)

Prepared according to general procedure

1

with (Z)-2-(3,4-

dichlorophenyl)-3-(4-(oxiran-2-ylmethoxy)phenyl)acrylonitrile (11, 346 mg,

1 mmol, 1 eq.) and 4-chloroaniline (12c, 446 µL, 5 mmol, 5 eq.). Column

chromatography (0-100% EtOAc in hexanes) afforded the desired

compound as a pale yellow solid (198 mg, 42%), m.p.: 110-113 °C. ¹H

NMR (400 MHz, acetone-d₆) d 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) d 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; IR

(Z)-2-(3,4-Dichlorophenyl)-3-(4-(3-(dimethylamino)propoxy)-

phenyl)acrylonitrile (13g)

Prepared according to general procedure

2

from (Z)-2-(3,4-

dichlorophenyl)-3-(4-(oxiran-2-ylmethoxy)phenyl)acrylonitrile (11, 346 mg,

1 mmol, 1 eq.) and dimethylamine (12g, 68 mg, 1.5 mmol, 1.5 eq.; 3 mL

of a 2.0 M solution in THF) to afford the desired compound as a pale yellow

solid (97 mg, 25%), m.p.: 99-101 ˚C. ¹H NMR (400 MHz, acetone-d₆) d

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) d 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); IR u_max/cm^-1: 3350 (OH), 2939 (C=H),

2208 (CN), 1692 (C-O), 812 (C-Cl); LRMS: (ESI⁺) m/z: 391

(C₂₀H₂₁Cl₂N₂O₂) [M+H]; HRMS: Exact mass calculated for C₂₀H₂₁Cl₂N₂O₂

[M+H], 391.0975. Found 391.0977.

u

_max/cm^-1: 3385 (br, OH), 2926 (C=C), 2210 (CN), 1586 (NH), 1257 (C-O),

828 (C-Cl); LRMS: (ESI⁺) m/z: 473 (C₂₄H₂₀Cl₃N₂O₂) [M+H]; HRMS: Exact

mass calculated for C₂₄H₂₀Cl₃N₂O₂[M+H], 473.0585. Found 473.0575.

(Z)-2-(3,4-Dichlorophenyl)-3-(4-(2-hydroxy-3-((4-

bromophenyl)amino)propoxy)-phenyl)acrylonitrile (13d)

Prepared according to general procedure

1

with (Z)-2-(3,4-

dichlorophenyl)-3-(4-(oxiran-2-ylmethoxy)phenyl)acrylonitrile (11, 346 mg,

1 mmol, 1 eq.) and 4-bromoaniline (12d, 573 µL, 5 mmol, 5 eq.). Column

chromatography (0-100% EtOAc in hexanes) afforded the desired

compound as a yellow solid (225 mg, 43%), m.p.: 121-124 ˚C. ¹H NMR

(400 MHz, acetone-d₆) d 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) d 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; IR u_max/cm^-1: 3380 (br, OH), 3264 (C=C), 2878 (O-CH₃), 2210

(CN), 1594 (NH), 1246 (C-O), 812 (C-Cl); LRMS: (ESI⁺) m/z: 517

(C₂₄H₂₀BrCl₂N₂O₂) [M+H]; HRMS: Exact mass calculated for

C₂₄H₂₀⁷⁹BrCl₂N₂O₂[M+H], 517.0080. Found 517.0086. Exact mass

calculated for C₂₄H₂₀⁸¹BrCl₂N₂O₂[M+H], 519.0060. Found 519.0062.

2-(Oxiran-2-ylmethoxy)benzaldehyde (15a)

2-Hydroxybenzaldehyde (14a, 300 µL, 2.86 mmol) in 50 mL ACN was

passed through a series of omnifit columns, the first containing molecular

sieves, the second contained cesium carbonate and sand (50%:50% w/w)

at a flow rate of 0.5 mL/min. The reactant stream was then met by a

secondary stream containing epichlorohydrin (17 mL, with 20 mL DMF and

63 mL ACN, flow rate 0.5 mL/min), and together the reactant streams were

passed through a Vapourtec static mixer reactor (20mL) heated at 105 °C

and held at 5 bar back pressure.

The product solution was then evaporated under reduced pressure, then

diluted with ethyl acetate (50 mL), extracted with water (3 x 50mL), and

then brine (1 x 50 mL), dried over magnesium sulfate and the solvent

(Z)-2-(3,4-Dichlorophenyl)-3-(4-(2-hydroxy-3-((4-

methoxyphenyl)amino)propoxy)-phenyl)acrylonitrile (13e)

Prepared according to general procedure

1

with (Z)-2-(3,4-

removed

under

reduced

pressure

to

afford

2-(oxiran-2-

dichlorophenyl)-3-(4-(oxiran-2-ylmethoxy)phenyl)acrylonitrile (11, 346 mg,

1 mmol, 1 eq.) and 4-methoxyaniline (12e, 575 µL, 5 mmol, 5 eq.). Column

chromatography (0-100% EtOAc in hexanes) afforded the desired

compound as a pale yellow solid (153 mg, 33%), m.p.: 130-133 °C. ¹H

NMR (400 MHz, acetone-d₆) d 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,

ylmethoxy)benzaldehyde (15a) as a light brown oil (379 mg, 74%).

Alternatively, 2-hydroxybenzaldehyde (14a, 436 µL, 4.10 mmol, 1 eq.) and

caesium carbonate (1.940 g, 6.14 mmol, 1.5 eq.) was combined with 15

mL DMF and 6 mL epichlorohydrin (7) in a round-bottomed flask. The

reaction was heated to 75 °C and left to stir. After 3 h, the reaction was

cooled to ambient temperature, diluted with EtOAc (50 mL), washed with

water (3 x 30 mL, brine (2 x 30 mL)), dried over MgSO₄and concentrated

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under reduced pressure to afford the desired product as a yellow oil (15a;

634 mg, 86%). LRMS: (ESI⁺) m/z: 179 (C₁₀H₁₁O₃) [M+H]. The material

was carried directly through without further characterization.

(Z)-2-(3,4-Dichlorophenyl)-3-(3-(oxiran-2-ylmethoxy)phenyl)-acrylonitrile

(16b)

3-(Oxiran-2-ylmethoxy)benzaldehyde (15b, 429 mg, 2.41 mmol, 1.05 eq.)

was then combined with 10 mL water in a round-bottomed flask and heated

(Z)-2-(3,4-Dichlorophenyl)-3-(2-(oxiran-2-ylmethoxy)phenyl)-acrylonitrile

(16a)

to 50 °C.

The solution was stirred for 10 min, then 3,4-

dichlorophenylacetonitrile (10, 426 mg, 2.29 mmol, 1 eq.) was added to

the stirring solution. Benzyltrimethyl ammonium hydroxide (40 wt.% aq.

solution) (7 mL) was added dropwise and the reaction left to stir at 50 °C

for 5 h. The reaction mixture was then filtered under vacuum and the

resulting solid was recrystallised from ethanol to afford the desired product

as a crystalline golden solid (495 mg, 59%); m.p.: 126-128 °C. ¹H NMR

(400 MHz, DMSO-d₆) d 8.16 (s, 1H), 8.05 (d, J = 2.1 Hz, 1H), 7.80 (d, J =

8.5 Hz, 1H), 7.72 (dd, J = 8.5, 2.1 Hz, 1H), 7.56 (d, J = 7.3 Hz, 2H), 7.48

(t, J = 8.0 Hz, 1H), 7.16 – 7.14 (m, 1H), 4.39 (dd, J = 11.3, 2.6 Hz, 1H),

3.90 (dd, J = 11.3, 6.5 Hz, 1H), 3.38 (dd, J = 6.5, 3.9 Hz, 1H), 2.87 (t, J =

4.6 Hz, 1H), 2.73 (dd, J = 4.9, 2.6 Hz, 1H); ¹³C NMR (101 MHz, DMSO-d₆)

d 158.4, 144.6, 134.6, 134.4, 132.1, 131.8, 131.3, 130.2, 127.4, 126.2,

122.0, 117.4, 117.3, 115.2, 108.2, 69.1, 49.6, 43.8; IR u_max/cm^-1: 3076

(C=C), 2218 (CN), 1272 (arom. C-O), 830 (C-Cl); LRMS: (ESI⁺) m/z: 346

(C₁₈H₁₄Cl₂NO₂) [M+H]; HRMS: Material decomposed.

2-(Oxiran-2-ylmethoxy)benzaldehyde (15a, 693 mg, 3.9 mmol, 1.05 eq.)

was combined with 10 mL water in a round-bottomed flask and heated to

50 °C.

The solution was stirred for 10 min, then 3,4-

dichlorophenylacetonitrile (10, 689 mg, 3.7 mmol, 1 eq.) was added to the

stirring solution. Benzytrimethyl ammonium hydroxide (40 wt.% aq.

solution) (7 mL) was added dropwise and the reaction left to stir at 50 °C

for 5 h. The reaction mixture was then filtered under vacuum and the

resulting solid was recrystallised from ethanol to afford the desired product

as a pale brown solid (380 mg, 30%). m.p.: 101-104 °C. ¹H NMR (400

MHz, DMSO-d₆) d 8.14 (s, 1H), 7.96 (d, J = 2.1 Hz, 1H), 7.91 (d, J = 7.3

Hz, 1H), 7.78 (d, J = 8.5 Hz, 1H), 7.70 (dd, J = 8.5, 2.1 Hz, 1H), 7.51 (dd,

J = 11.5, 4.2 Hz, 1H), 7.19 (d, J = 8.4 Hz, 1H), 7.13 (t, J = 7.5 Hz, 1H), 4.47

(dd, J = 11.6, 2.3 Hz, 1H), 3.99 (dd, J = 11.6, 6.4 Hz, 1H), 2.86 (t, J = 4.6

Hz, 1H), 2.74 (dd, J = 5.0, 2.6 Hz, 1H); ¹³C NMR (101 MHz, DMSO-d₆) d

156.8, 140.4, 134.5, 132.7, 132.1, 131.8, 131.4, 128.5, 127.5, 126.1, 122.6,

121.0, 117.2, 113.0, 109.4, 69.7, 49.6. 43.8; IR u_max/cm^-1: 3042 (C=C),

2220 (CN), 1252 (arom. C-O), 821 (C-Cl); LRMS: (ESI⁺) m/z: 346

(C₁₈H₁₄Cl₂NO₂) [M+H]; HRMS: Material decomposed.

(Z)-2-(3,4-Dichlorophenyl)-3-(3-(2-hydroxy-3-(4-phenyl-piperazin-1-

yl)propoxy)phenyl)-acrylonitrile (17b)

Prepared according to general procedure

2

with (Z)-2-(3,4-

dichlorophenyl)-3-(3-(oxiran-2-ylmethoxy)phenyl)acrylonitrile (16b, 205

mg, 0.59 mmol, 1 eq.) and 1-phenylpiperazine (12f, 187 µL, 1.18 mmol, 2

eq.) to afford the desired compound as a creamy-coloured solid (137 mg,

63%), m.p.: 132-134 °C. ¹H NMR (400 MHz, DMSO-d₆) d 8.17 (s, 1H),

8.05 (s, 1H), 7.78 (d, J = 8.2 Hz, 1H), 7.71 (d, J = 8.1 Hz, 1H), 7.54 (d, J =

12.3 Hz, 2H), 7.48 (d, J = 7.7 Hz, 1H), 7.16 (dd, J = 16.9, 7.5 Hz, 3H), 6.90

(d, J = 7.7 Hz, 2H), 6.77 (d, J = 6.2 Hz, 1H), 4.99 (s, 1H), 4.08 (d, J = 8.3

Hz, 2H), 3.97 (s, 1H), 3.12 (s, 4H), 2.62 (s, 5H); ¹³C NMR (101 MHz,

DMSO-d₆) d 158.9, 144.8, 134.6, 134.4, 132.1, 131.8, 131.3, 130.3, 130.2,

128.9, 127.4, 126.2, 121.8, 118.8, 117.8, 117.4, 115.4, 114.0, 108.1, 71.2,

69.2, 53.5, 49.6, 48.2; IR u_max/cm^-1: 3190 (O-H), 2978 (C=C), 2216 (CN),

1274 (arom. C-O), 819 (C-Cl); LRMS: (ESI⁺) m/z: 508 (C₂₈H₂₈Cl₂N₃O₂)

[M+H]; HRMS: Exact mass calculated for C₂₈H₂₈Cl₂N₃O₂[M+H], 508.1553.

Found 508.1544.

(Z)-2-(3,4-Dichlorophenyl)-3-(2-(2-hydroxy-3-(4-phenyl-piperazin-1-

yl)propoxy)phenyl)-acrylonitrile (17a)

Prepared according to general procedure

2

with (Z)-2-(3,4-

dichlorophenyl)-3-(2-(oxiran-2-ylmethoxy)phenyl)acrylonitrile (16a, 150

mg, 0.43 mmol, 1 eq.) and 1-phenylpiperazine (12f, 137 µL, 0.87 mmol, 2

eq.) to afford the desired compound as a yellow solid (178 mg, 82%), m.p.:

128-130 °C. ¹H NMR (400 MHz, acetone-d₆) d 8.27 (s, 1H), 8.11 (dd, J =

7.8, 1.2 Hz, 1H), 7.92 (t, J = 1.2 Hz, 1H), 7.72 (d, J = 1.2 Hz, 2H), 7.53 –

7.49 (m, 1H), 7.23 – 7.19 (m, 3H), 7.12 (t, J = 7.6 Hz, 1H), 6.93 (d, J = 7.9

Hz, 2H), 6.77 (t, J = 7.3 Hz, 1H), 4.25 (d, J = 8.9 Hz, 1H), 4.22 – 4.14 (m,

2H), 3.17 (t, J = 5.0 Hz, 4H), 2.72 – 2.61 (m, 6H); ¹³C NMR (101 MHz,

acetone-d₆) d 168.5, 162.2, 150.2, 146.0, 143.33, 143.29, 142.9, 141.8,

139.4 (2C), 138.8, 138.2, 136.4, 133.5, 131.4, 129.6, 127.7, 126.2 (2C),

123.5, 119.5, 82.6, 77.3, 71.6, 64.3 (2C), 59.5 (2C); IR u_max/cm^-1: 3433

(OH), 2814 (N-CH₂), 2214 (CN), 1228 (C-O), 830 (C-Cl); LRMS: (ESI⁺)

m/z: 508 (C₂₈H₂₈Cl₂N₃O₂) [M+H]; HRMS: Exact mass calculated for

C₂₈H₂₈Cl₂N₃O₂[M+H], 508.1553. Found 508.1557.

(Z)-2-(3,4-Dichlorophenyl)-3-(4-(3-(dimethylamino)propoxy)-

phenyl)acrylonitrile (18)

Prepared

according

to

general

procedure

mmol,

2

from

3,4-

dichlorophenylacetonitrile (10, 346 mg,

1

1 eq.) and 4-(3-

3-(Oxiran-2-ylmethoxy)benzaldehyde (15b)

(dimethylamino)propoxy)benzaldehyde (514 mg, 2 mmol, 2 eq.) to afford

the desired compound as a pale brown solid (76 mg, 20%), m.p.: 89-92 ˚C.

¹H NMR (400 MHz, acetone-d₆) d 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), 7.10 (d, J = 8.9 Hz, 2H), 4.16

(t, J = 6.4 Hz, 2H), 2.42 (t, J = 7.0 Hz, 2H), 2.18 (s, 6H), 1.93 (dd, J = 13.4,

6.7 Hz, 2H); ¹³C NMR (101 MHz, acetone-d₆) d 162.6, 144.8, 136.5, 133.9,

132.5 (2C), 132.0, 130.0, 128.2, 127.0, 126.5, 118.5, 115.8 (2C), 106.1,

67.2, 56.7, 45.7 (2C), 28.1; IR u_max/cm^-1: 2953 (C=H), 2216 (CN), 1248 (C-

O), 727 (C-Cl); LRMS: (ESI⁺) m/z: 375 (C₂₀H₂₀Cl₂N₂O) [M+H]; HRMS:

Exact mass calculated for C₂₀H₂₁Cl₂N₂O [M+H], 375.1025. Found

375.1013.

3-Hydroxybenzaldehyde (14b, 389 mg, 3.1 mmol) in 25 mL ACN was

passed through a series of omnifit columns, the first containing molecular

sieves, the second contained caesium carbonate and sand (50%:50%

w/w) at a flow rate of 0.5 mL/min. The reactant stream was then met by a

secondary stream containing epichlorohydrin (17 mL, with 20 mL DMF and

63 mL ACN, flow rate 0.5 mL/min), and together the reactant streams were

passed through a Vapourtec static mixer reactor (20mL) heated at 105 °C

and held at 5 bar back pressure.

The product solution was then evaporated under reduced pressure, then

diluted with ethyl acetate (50 mL), extracted with water (3 x 50mL), and

then brine (1 x 50 mL), dried over magnesium sulfate and the solvent

(S)-4-(Oxiran-2-ylmethoxy)benzaldehyde (20a)

removed

under

reduced

pressure

to

afford

3-(oxiran-2-

(S)-(+)-Glycidyl-3-nitrobenzenesulfonate (19a, 493 mg, 1.9 mmol, 1.1 eq.),

4-hydroxybenzaldehyde (8, 214 mg, 1.75 mmol, 1 eq.) and cesium

carbonate (847 mg, 2.6 mmol, 1.5 eq.) were combined in a round-

bottomed flask with 20 mL DMF. The solution was heated to 80 °C and

stirred for 4 h. The solution was cooled to RT, extracted with ethyl acetate

(50 mL) and washed with water (5 x 30 mL) and brine (30 mL). The organic

extracts were dried over MgSO₄and concentrated under reduced pressure

to afford the desired product as a brown oil (286 mg, 86%).¹H NMR (400

MHz, CDCl₃) d 9.89 (s, 1H), 7.84 (d, J = 8.7 Hz, 2H), 7.03 (d, J = 8.7 Hz,

2H), 4.34 (dd, J = 11.1, 2.9 Hz, 1H), 4.01 (dd, J = 11.1, 5.8 Hz, 1H), 3.38

(ddd, J = 6.7, 4.8, 2.0 Hz, 1H), 2.94 (t, J = 4.5 Hz, 1H), 2.78 (dd, J = 4.7,

2.6 Hz, 1H); ¹³C NMR (101 MHz, CDCl₃) d 190.9, 136.5, 132.1, 130.5,

115.1, 69.1, 50.0, 44.7; IR u_max/cm^-1: 2834 (C-C), 1686 (C=O), 1250 (C-

ylmethoxy)benzaldehyde (15b) as a light brown oil (403 mg, 72%).

Alternatively, 3-hydroxybenzaldehyde (14b, 500 mg, 4.10 mmol, 1 eq.)

and caesium carbonate (1.940 g, 6.14 mmol, 1.5 eq.) were combined with

15 mL DMF and 6 mL epichlorohydrin (7) in a round-bottomed flask. The

reaction was heated to 75 °C and left to stir. After 3 h, the reaction was

cooled to ambient temperature, diluted with EtOAc (50 mL), washed with

water (3 x 30 mL, brine (2 x 30 mL)), dried over MgSO₄and concentrated

under reduced pressure to afford 3-(oxiran-2-ylmethoxy)benzaldehyde

(15b) as a pale yellow oil (660 mg, 90%). LRMS: (ESI⁺) m/z: 179

(C₁₀H₁₁O₃) [M+H]. The material was carried directly through without

further characterization.

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O); LRMS: (ESI⁺) m/z: 179 (C₁₀H₁₁O₃) [M+H]; Material was not completely

dried, contained acetone.

solution) (10 mL) was added dropwise and the reaction left to stir at 50 °C

for 5 h. The reaction mixture was then filtered under vacuum and the

resulting solid was recrystallised from ethanol to afford the desired product

as a yellow solid (1.720 g, 62%), m.p.: 111-113 °C. ¹H NMR (400 MHz,

acetone-d₆) d 8.04 (d, J = 2.5 Hz, 1H), 8.02 (d, J = 8.9 Hz, 2H), 7.97 (d, J

= 0.8 Hz, 1H), 7.71 (d, J = 1.9 Hz, 2H), 7.15 (d, J = 8.9 Hz, 2H), 4.47 (dd,

J = 11.4, 2.6 Hz, 1H), 3.99 (dd, J = 11.4, 6.5 Hz, 1H), 3.37 (ddd, J = 6.6,

2.6, 1.7 Hz, 1H), 2.87 (dd, J = 5.0, 4.3 Hz, 1H), 2.75 (dd, J = 5.1, 2.6 Hz,

1H); ¹³C NMR (101 MHz, acetone-d₆) d 162.0, 144.6, 136.4, 133.5, 132.8,

132.5 (2C), 132.0, 128.2, 127.5, 126.5, 118.5, 115.9 (2C), 106.5, 70.4,

50.4, 44.4; IR u_max/cm^-1: 3390 (OH), 3094 (C=C), 2213 (CN), 1257 (arom.

C-O), 825 (C-Cl); LRMS: (ESI⁺) m/z: 346 (C₁₈H₁₄Cl₂NO₂) [M+H], HRMS:

Material decomposed.

(S,Z)-2-(3,4-Dichlorophenyl)-3-(4-(oxiran-2-ylmethoxy)phenyl)-

acrylonitrile (21a)

(S)-4-(Oxiran-2-ylmethoxy)benzaldehyde (20a, 220 mg, 1.23 mmol, 1.05

eq.) was combined with 10 mL water in a round-bottomed flask and heated

to 50 °C.

The solution was stirred for 10 min, then 3,4-

dichlorophenylacetonitrile (10, 219 mg, 1.18 mmol, 1 eq.) was added to

the stirring solution. Benzytrimethyl ammonium hydroxide (40 wt.% aq.

solution) (7 mL) was added dropwise and the reaction left to stir at 50 °C

for 5 h. The reaction mixture was then filtered under vacuum and the

resulting solid was recrystallised from ethanol to afford the desired product

as a yellow solid (216 mg, 53%), m.p.: 112-115 °C. ¹H NMR (400 MHz,

acetone-d₆) d 8.02 (d, J = 8.9 Hz, 2H), 7.98 (s, 1H), 7.96 – 7.93 (m, 1H),

7.75 – 7.67 (m, 2H), 7.15 (d, J = 8.9 Hz, 2H), 4.47 (dd, J = 11.4, 2.6 Hz,

1H), 4.00 (dd, J = 11.4, 6.4 Hz, 1H), 3.36 (ddd, J = 6.7, 5.2, 2.6 Hz, 1H),

2.87 (dd, J = 5.0, 4.3 Hz, 1H), 2.75 (dd, J = 5.1, 2.6 Hz, 1H); ¹³C NMR (101

MHz, acetone-d₆) d 162.0, 144.7, 136.5, 133.6, 132.8, 132.5 (2C), 132.0,

128.2, 127.5, 126.6, 118.5, 116.0 (2C), 106.6, 70.4, 50.4, 44.4 IR u_max/cm^-

¹: 3093 (C=C), 2213 (CN), 1257 (arom. C-O), 824 (C-Cl); LRMS: (ESI⁺)

m/z: 346 (C₁₈H₁₄Cl₂NO₂) [M+H], HRMS: Material decomposed.

(R,Z)-2-(3,4-Dichlorophenyl)-3-(4-(2-hydroxy-3-(4-phenylpiperazin-1-

yl)propoxy)phenyl)-acrylonitrile (22b)

Prepared according to general procedure

2

from (S,Z)-2-(3,4-

dichlorophenyl)-3-(4-(oxiran-2-ylmethoxy)phenyl)acrylonitrile (21b, 213

mg, 0.62 mmol, 1 eq.) and 1-phenylpiperazine (12f, 194 µL, 1.22 mmol, 2

eq.) to afford the desired compound as a pale yellow solid (312 mg, 82%),

m.p.: 163-165 °C. ¹H NMR (400 MHz, DMSO-d₆) d 8.10 (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), 7.20 (t, J = 7.9 Hz, 2H), 7.14 (d, J = 8.8 Hz, 2H), 6.92

(d, J = 8.2 Hz, 2H), 6.76 (t, J = 7.2 Hz, 1H), 4.98 (d, J = 4.2 Hz, 1H), 4.13

– 4.11 (m, 1H), 4.02 – 3.99 (m, 2H), 3.12 (t, J = 4.7 Hz, 4H), 2.65 – 2.57

(m, 4H), 2.44 (dd, J = 12.6, 5.9 Hz, 1H); ¹³C NMR (101 MHz, DMSO-d₆) d

161.2, 151.1, 144.3, 134.9, 132.0, 131.5 (2C), 131.2, 131.1, 128.9 (2C),

127.0, 125.80, 125.77, 118.7, 118.0, 115.3 (2C), 115.1 (2C), 104.4, 71.4,

66.5, 60.9, 53.5 (2C), 48.3 (2C); IR u_max/cm^-1: 2825 (N-CH₂), 2210 (CN),

1245 (C-O), 818 (C-Cl); LRMS: (ESI⁺) m/z: 508 (C₂₈H₂₈Cl₂N₃O₂) [M+H];

CHIRAL HPLC: Column: Phemonex Lux Cellulose 2 (250 x 4.6 mm);

Conditions: 80% hexane + 0.1% DEA, 20% IPA + 0.1% DEA. Runtime: 30

min; Peak retention time: 17.213 mins; Area (%): 100.0%. Optical rotation:

[α]_D-0.225 (c 0.472 in DMSO). HRMS: Exact mass calculated for

C₂₈H₂₈Cl₂N₃O₂[M+H], 508.1553. Found 508.1558.

(S,Z)-2-(3,4-Dichlorophenyl)-3-(4-(2-hydroxy-3-(4-phenylpiperazin-1-

yl)propoxy)phenyl)-acrylonitrile (22a)

Prepared according to general procedure

2

with (S,Z)-2-(3,4-

dichlorophenyl)-3-(4-(oxiran-2-ylmethoxy)phenyl)acrylonitrile (21a, 134

mg, 0.39 mmol, 1 eq.) and 1-phenylpiperazine (12f, 122 µL, 0.77 mmol, 2

eq.) to afford the desired compound as a pale yellow solid (116 mg, 59%),

m.p.: 157-159 °C. ¹H NMR (400 MHz, DMSO-d₆) d 8.10 (s, 1H), 8.01 (d,

J = 1.5 Hz, 1H), 7.96 (d, J = 8.7 Hz, 2H), 7.76 (d, J = 8.4 Hz, 1H), 7.69 (dd,

J = 8.4, 1.5 Hz, 1H), 7.20 (t, J = 7.7 Hz, 2H), 7.15 (d, J = 8.7 Hz, 2H), 6.92

(d, J = 8.1 Hz, 2H), 6.76 (t, J = 7.1 Hz, 1H), 4.99 (s, 1H), 4.13 – 4.11 (m,

1H), 4.02 – 3.98 (m, 2H), 3.12 (m, 4H), 2.65 – 2.57 (m, 4H), 2.44 (dd, J =

12.8, 5.8 Hz, 1H); ¹³C NMR (101 MHz, DMSO-d₆) d 161.2, 151.1, 144.4,

135.0, 132.1, 131.5 (2C), 131.2, 131.1, 128.9 (2C), 127.0, 125.82, 125.80,

118.8, 117.9, 115.3 (2C), 115.2 (2C), 104.4, 71.4, 66.5, 60.9, 53.5 (2C),

48.3 (2C); IR u_max/cm^-1: 3392 (br, OH), 2825 (N-CH₂), 2210 (CN), 1245 (C-

O), 818 (C-Cl); LRMS: (ESI⁺) m/z: 508 (C₂₈H₂₈Cl₂N₃O₂) [M+H]; CHIRAL

HPLC: Column: Phemonex Lux Cellulose 2 (250 x 4.6 mm); Conditions:

80% hexane + 0.1% DEA, 20% IPA + 0.1% DEA. Runtime: 30 min; Peak

retention time: 15.658 mins; Area (%): 100.0%. Optical rotation: [α]_D0.235

(c 0.514 in DMSO). HRMS: Exact mass calculated for C₂₈H₂₈Cl₂N₃O₂

[M+H], 508.1553. Found 508.1539.

(Z)-3-(4-(3-(4-Cyclohexylpiperazin-1-yl)-2-hydroxypropoxy)-phenyl)-2-

(3,4-dichlorophenyl)-acrylonitrile (23a)

Prepared according to general procedure

2

from (Z)-2-(3,4-

dichlorophenyl)-3-(4-(oxiran-2-ylmethoxy)phenyl)acrylonitrile (11, 346 mg,

1 mmol, 1 eq.) and 1-cyclohexylpiperazine (12h, 340 mg, 2 mmol, 2 eq.)

to afford the desired compound as a yellow solid (277 mg, 54%), m.p.: 137-

139 °C. ¹H NMR (400 MHz, DMSO-d₆) d 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), 7.69 (d, J = 2.1 Hz,

1H), 7.13 (d, J = 8.8 Hz, 2H), 4.88 (d, J = 4.1 Hz, 1H), 4.10 – 4.06 (m, 1H),

3.95 (d, J = 6.2 Hz, 2H), 2.46 – 2.31 (m, 10H), 2.16 (s, 1H), 1.74 – 1.70 (m,

4H), 1.55 (d, J = 11.7 Hz, 1H), 1.23 – 1.04 (m, 5H); ¹³C NMR (101 MHz,

DMSO-d₆) d 161.2, 144.3, 135.0, 132.0 (2 overlapping signals), 131.5 (2C),

131.2, 131.1, 127.0, 125.8, 117.9 (2C), 115.1, 104.3, 71.5, 66.4, 62.5, 61.1,

54.1 (2C), 48.5 (2C), 28.4, 25.9 (2C), 25.3 (2C); IR u_max/cm^-1: 3498 (OH),

2812 (N-CH₂), 2212 (CN), 1269 (C-O), 813 (C-Cl); LRMS: (ESI⁺) m/z: 514

(C₂₈H₃₄Cl₂N₃O₂) [M+H]; HRMS: Exact mass calculated for C₂₈H₃₄Cl₂N₃O₂

[M+H], 514.2023. Found 514.2027.

(R)-4-(Oxiran-2-ylmethoxy)benzaldehyde (20b)

(R)-(-)-Glycidyl-3-nitrobenzenesulfonate (19b, 508 mg, 1.9 mmol, 1.1 eq.),

4-hydroxybenzaldehyde (8, 215 mg, 1.75 mmol, 1 eq.) and caesium

carbonate (847 mg, 2.6 mmol, 1.5 eq.) were combined in a round-

bottomed flask with 20 mL DMF. The solution was heated to 80 °C and

stirred for 4 h. The solution was cooled to ambient temperature, extracted

with ethyl acetate (50 mL) and washed with water (5 x 30 mL) and brine

(30 mL). The organic extracts were dried over MgSO₄and concentrated

under reduced pressure to afford the desired product as a brown oil (286

mg, 86%); ¹H NMR (400 MHz, CDCl₃) d 9.89 (s, 1H), 7.83 (dd, J = 8.8, 2.6

Hz, 2H)), 7.03 (dd, J = 8.8, 2.6 Hz, 2H), 4.34 (dd, J = 11.1, 2.9 Hz, 1H),

4.02 (dd, J = 11.1, 5.9 Hz, 1H), 3.38 (ddd, J = 5.8, 2.9, 1.3 Hz, 1H), 2.99 –

2.86 (m, 1H), 2.78 (dd, J = 4.8, 2.6 Hz, 1H); ¹³C NMR (101 MHz, CDCl₃) d

190.9, 163.5, 132.1 (2C), 130.5, 115.0 (2C), 69.1, 50.0, 44.7; IR u_max/cm^-

¹: 2825 (C-C), 1686 (C=O), 1250 (C-O); LRMS: (ESI⁺) m/z: 179 (C₁₀H₁₁O₃)

[M+H].

(Z)-3-(4-(3-(4-Phenylpiperidin-1-yl)-2-hydroxypropoxy)phenyl)-2-(3,4-

dichlorophenyl)-acrylonitrile (23b)

Prepared according to general procedure

2

from (Z)-2-(3,4-

dichlorophenyl)-3-(4-(oxiran-2-ylmethoxy)phenyl)acrylonitrile (11, 346 mg,

1 mmol, 1 eq.) and 4-phenylpiperidine (12i, 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) d 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) d 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 u_max/cm^-1: 3422 (OH),

(R,Z)-2-(3,4-Dichlorophenyl)-3-(4-(oxiran-2-ylmethoxy)phenyl)-

acrylonitrile (21b)

(R)-4-(oxiran-2-ylmethoxy)benzaldehyde (20b, 1.603 g, 8.77 mmol, 1.1

eq.) was combined with 15 mL water in a round-bottomed flask and heated

to 50 °C.

The solution was stirred for 10 min, then 3,4-

dichlorophenylacetonitrile (10, 1.483 g, 7.97 mmol, 1 eq.) was added to

the stirring solution. Benzyltrimethyl ammonium hydroxide (40 wt.% aq.

12

10.1002/cmdc.201900643

ChemMedChem

FULL PAPER

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.

(ESI⁺) m/z: 431 (C₂₃H₂₅Cl₂N₂O₂) [M+H]; HRMS: Exact mass calculated for

C₂₃H₂₅Cl₂N₂O₂[M+H], 431.1288. Found 431.1275.

(Z)-2-(3,4-Dichlorophenyl)-3-(4-(2-hydroxy-3-(4-methylpiperidin-1-

(Z)-3-(4-(3-(4-Acetylpiperazin-1-yl)-2-hydroxypropoxy)phenyl)-2-(3,4-

yl)propoxy)phenyl)-acrylonitrile (23g)

dichlorophenyl)-acrylonitrile (23c)

Prepared according to general procedure

2

from (Z)-2-(3,4-

Prepared according to general procedure

2

from (Z)-2-(3,4-

dichlorophenyl)-3-(4-(oxiran-2-ylmethoxy)phenyl)acrylonitrile (11, 346 mg,

1 mmol, 1 eq.) and 4-methylpiperidine (12n, 178 µL, 1.5 mmol, 1.5 eq.) to

afford the desired compound as a pale yellow solid (216 mg, 57%), m.p.:

79-81 °C. ¹H NMR (400 MHz, DMSO-d₆) d 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) d 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; IR u_max/cm^-1: 3300 (OH), 2981 (C=C), 2212 (CN), 1180 (C-O),

815 (C-Cl); LRMS: (ESI⁺) m/z: 445 (C₂₄H₂₇Cl₂N₂O₂) [M+H]; HRMS: Exact

mass calculated for C₂₄H₂₇Cl₂N₂O₂[M+H], 445.1444. Found 445.1461.

dichlorophenyl)-3-(4-(oxiran-2-ylmethoxy)phenyl)acrylonitrile (11, 346 mg,

1 mmol, 1 eq.) and N-acetylpiperazine (12j, 256 mg, 2 mmol, 2 eq.) to

afford the desired compound as a pale yellow solid (346 mg, 73%), m.p.:

88-91 °C. ¹H NMR (400 MHz, DMSO-d₆) d 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) d 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; IR u_max/cm^-

¹: 3367 (br, OH), 2819 (N-CH₂), 2212 (CN), 1182 (C-O), 815 (C-Cl); LRMS

(ESI⁺) m/z: 474 (C₂₄H₂₆Cl₂N₃O₃) [M+H]; HRMS: Exact mass calculated for

C₂₄H₂₆Cl₂N₃O₃[M+H], 474.1352. Found 474.1332.

(Z)-2-(3,4-Dichlorophenyl)-3-(4-(2-hydroxy-3-(4-methylpiperazin-1-

(Z)-2-(3,4-Dichlorophenyl)-3-(4-(2-hydroxy-3-(3-methylpiperidin-1-

yl)propoxy)phenyl)-acrylonitrile (23d)

yl)propoxy)phenyl)-acrylonitrile (23h)

Prepared according to general procedure

2

from (Z)-2-(3,4-

Prepared according to general procedure

2

from (Z)-2-(3,4-

dichlorophenyl)-3-(4-(oxiran-2-ylmethoxy)phenyl)acrylonitrile (11, 346 mg,

1 mmol, 1 eq.) and N-methylpiperazine (12k, 222 µL, 2 mmol, 2 eq.) to

afford the desired compound as a yellow solid (221 mg, 50%), m.p.: 111-

114 °C. ¹H NMR (400 MHz, DMSO-d₆) d 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) d 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; IR u_max/cm^-1: 3070 (br, OH),

2933 (N-CH₂), 2210 (CN), 1183 (C-O), 870 (C-Cl); LRMS (ESI⁺) m/z: 446

(C₂₃H₂₆Cl₂N₃O₂) [M+H]; Exact mass calculated for C₂₃H₂₆Cl₂N₃O₂[M+H],

446.1397. Found 446.1383.

dichlorophenyl)-3-(4-(oxiran-2-ylmethoxy)phenyl)acrylonitrile (11, 346 mg,

1 mmol, 1 eq.) and 3-methylpiperidine (12o, 176 uL, 1.5 mmol, 1.5 eq.) to

afford the desired compound as a pale yellow solid (279 mg, 65%), m.p.:

103-106 °C. ¹H NMR (400 MHz, DMSO-d₆) d 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) d 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; IR u_max/cm^-1: 3270 (OH), 2926 (C=C), 2215 (CN), 1180 (C-O),

814 (C-Cl); LRMS: (ESI⁺) m/z: 445 (C₂₄H₂₇Cl₂N₂O₂) [M+H]; HRMS: Exact

mass calculated for C₂₄H₂₇Cl₂N₂O₂[M+H], 445.1444. Found 445.1448.

(Z)-2-(3,4-Dichlorophenyl)-3-(4-(2-hydroxy-3-

morpholinopropoxy)phenyl)acrylonitrile (23e)

(Z)-2-(3,4-Dichlorophenyl)-3-(4-(2-hydroxy-3-(2-methyl-piperidin-1-

yl)propoxy)phenyl)-acrylonitrile (23i)

Prepared according to general procedure

2

from (Z)-2-(3,4-

dichlorophenyl)-3-(4-(oxiran-2-ylmethoxy)phenyl)acrylonitrile (11, 346 mg,

1 mmol, 1 eq.) and morpholine (12l, 172 µL, 2 mmol, 2 eq.) to afford the

desired compound as a yellow solid (192 mg, 44%), m.p.: 103-105 °C. ¹H

NMR (400 MHz, acetone-d₆) d 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) d 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); IR u_max/cm^-1:

3360 (br, OH), 2861 (N-CH₂), 2214 (CN), 1270 (C-O), 814 (C-Cl); LRMS:

(ESI⁺) m/z: 433 (C₂₂H₂₃Cl₂N₂O₃) [M+H]; HRMS: Exact mass calculated for

C₂₂H₂₃Cl₂N₂O₃[M+H], 433.1080. Found 433.1067.

Prepared according to general procedure

2

from (Z)-2-(3,4-

dichlorophenyl)-3-(4-(oxiran-2-ylmethoxy)phenyl)acrylonitrile (11, 346 mg,

1 mmol, 1 eq.) and 2-methylpiperidine (12p, 177 µL, 1.5 mmol, 1.5 eq.) to

afford the desired compound as a pale yellow solid (165 mg, 39%), m.p.:

94-96 °C. ¹H NMR (400 MHz, DMSO-d₆) d 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) d 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); IR u_max/cm^-1: 3380 (OH), 2926 (C=C), 2221 (CN), 1180 (C-O), 815 (C-

Cl). LRMS: (ESI⁺) m/z: 445 (C₂₄H₂₇Cl₂N₂O₂) [M+H]; HRMS: Exact mass

calculated for C₂₄H₂₇Cl₂N₂O₂[M+H], 445.1444. Found 445.1447.

(Z)-2-(3,4-Dichlorophenyl)-3-(4-(2-hydroxy-3-(piperidin-1-

yl)propoxy)phenyl)acrylonitrile (23f)

Prepared according to general procedure

2

from (Z)-2-(3,4-

dichlorophenyl)-3-(4-(oxiran-2-ylmethoxy)phenyl)acrylonitrile (11, 346 mg,

1 mmol, 1 eq.) and piperidine (12m, 198 µL, 2 mmol, 2 eq.) to afford the

desired compound as a yellow solid (169 mg, 30%), m.p.: 111-115 °C. ¹H

NMR (400 MHz, acetone-d₆) d 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) d 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; IR u_max/cm^-

¹: 3300 (b, OH), 2853 (N-CH₂), 2214 (CN), 1264 (C-O), 816 (C-Cl); LRMS:

(Z)-2-(3,4-Dichlorophenyl)-3-(4-(2-hydroxy-3-(pyrrolidin-1-

yl)propoxy)phenyl)acrylonitrile (23j)

Prepared according to general procedure

2

from (Z)-2-(3,4-

dichlorophenyl)-3-(4-(oxiran-2-ylmethoxy)phenyl)acrylonitrile (11, 346 mg,

1 mmol, 1 eq.) and pyrrolidine (12q, 123 µL, 1.5 mmol, 1.5 eq.) to afford

the desired compound as a yellow solid (169 mg, 42%), m.p.: 90-93 °C.

¹H NMR (400 MHz, acetone-d₆) d 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 =

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5.6 Hz, 1H), 2.61 – 2.56 (m, 3H), 1.74 (s, 3H), 1.12 (t, J = 7.0 Hz, 1H); ¹³

C

Prepared according to general procedure

2

from (Z)-2-(3,4-

NMR (101 MHz, acetone-d₆) d 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); IR u_max/cm^-1: 3340 (OH), 2931 (C=C), 2211 (CN),

1183 (C-O), 815 (C-Cl). LRMS: (ESI⁺) m/z: 417 (C₂₂H₂₃Cl₂N₂O₂) [M+H];

HRMS: Exact mass calculated for C₂₂H₂₃Cl₂N₂O₂[M+H], 417.1131. Found

417.1136.

dichlorophenyl)-3-(4-(oxiran-2-ylmethoxy)phenyl)acrylonitrile (11, 346 mg,

1 mmol, 1 eq.), 3-hydroxypiperidine hydrochloride (12u, 262 mg, 2 mmol,

1 eq.) and triethylamine (280 µL, 2 mmol, 2 eq.). Reaction mixture was

evaporated to dryness, loaded onto silica and purified by flash column

chromatography (0-10% MeOH in DCM with 1% NH₄OH) to afford the

desired compound as a yellow crystalline solid (trimethylamine salt) (327

mg, 60%), m.p.: 178-180 °C. ¹H NMR (600 MHz, MeOD) δ 7.96 (s, 2H),

7.86 – 7.82 (m, 2H), 7.63 – 7.61 (m, 2H), 7.09 (s, 2H), 4.59 (s, OH), 4.28

(s, 1H), 4.08 (d, J = 18.3 Hz, 2H), 3.90 – 3.86 (m, 1H), 3.20 (s, 6H)*, 3.10

(s, 1H), 2.95 (s, 3H), 2.69 (s, 2H), 1.94 (s, 1H), 1.86 (s, 1H), 1.67 (s, 1H),

1.49 (s, 1H), 1.32 (d, J = 3.3 Hz, 9H)*; ¹³C NMR (151 MHz, MeOD) δ 162.5,

144.9 (d, J = 1.0 Hz), 136.6, 134.2, 133.5, 132.7 (2C), 132.2, 128.4, 127.94

(d, J = 4.4 Hz), 126.4, 118.9, 116.1, 107.0, 71.9 (2 overlapping signals),

71.8, 67.2, 61.7, 55.1 (2 overlapping signals), 49.6, 47.9*, 9.25*; *denotes

peaks associated with trimethylamine salt; IR u_max/cm^-1: 3308 (OH), 2939

(C=C), 2215 (CN), 1182 (C-O), 816 (C-Cl). LRMS: (ESI⁺) m/z: 447

(C₂₃H₂₅Cl₂N₂O₃) [M+H]; HRMS: Exact mass calculated for C₂₃H₂₅Cl₂N₂O₃

[M+H], 447.1237. Found 447.1263.

(Z)-3-(4-(3-(Azepan-1-yl)-2-hydroxypropoxy)phenyl)-2-(3,4-

dichlorophenyl)acrylonitrile (23k)

Prepared according to general procedure

2

from (Z)-2-(3,4-

dichlorophenyl)-3-(4-(oxiran-2-ylmethoxy)phenyl)acrylonitrile (11, 346 mg,

1 mmol, 1 eq.) and azepane (12r, 169 µL, 1.5 mmol, 1.5 eq.) to afford the

desired compound as a pale yellow solid (177 mg, 42%), m.p.: 76-78 °C.

¹H NMR (400 MHz, DMSO-d₆) d 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) d 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); IR u_max/cm^-1: 3366

(OH), 2926 (C=C), 2215 (CN), 1180 (C-O), 816 (C-Cl). LRMS: (ESI⁺) m/z:

445 (C₂₄H₂₇Cl₂N₂O₂) [M+H]; HRMS: Exact mass calculated for

C₂₄H₂₇Cl₂N₂O₂[M+H], 445.1444. Found 445.1450.

(Z)-2-(3,4-dichlorophenyl)-3-(4-(2-hydroxy-3-(4-hydroxy-piperidin-1-

yl)propoxy)phenyl)-acrylonitrile (25b)

Prepared according to general procedure

2

from (Z)-2-(3,4-

dichlorophenyl)-3-(4-(oxiran-2-ylmethoxy)phenyl)acrylonitrile (11, 346 mg,

1 mmol, 1 eq.) and 4-hydroxypiperidine (12v, 101 mg, 1 mmol, 1 eq.) to

afford the desired compound as a pale yellow solid (254 mg, 60%), m.p.:

101-103 °C. 1H NMR (400 MHz, DMSO) δ 8.10 (s, 1H), 8.00 (d, J = 2.2

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), 4.86 (s, 1H), 4.52 (d, J = 2.8 Hz,

1H), 4.08 (d, J = 6.6 Hz, 1H), 4.01 – 3.87 (m, 2H), 3.47 – 3.39 (m, 1H),

2.83 – 2.64 (m, 2H), 2.37 (ddd, J = 32.4, 12.7, 5.8 Hz, 2H), 2.09 (dd, J =

21.6, 10.6 Hz, 2H), 1.77 – 1.60 (m, 2H), 1.38 (dd, J = 19.0, 9.3 Hz, 2H);

¹³C NMR (151 MHz, Acetone) δ 151.5, 134.6, 125.3, 122.3, 121.8 (2C),

121.5, 121.4, 117.3, 116.3, 116.1, 108.2, 105.4 (2C), 94.6, 61.8, 56.9 (2

overlapping signals), 51.2, 42.1 (d, J = 27.2 Hz, 2C), 24.9 (d, J = 4.1 Hz,

2C); IR u_max/cm^-1: 3314 (OH), 2933 (C=C), 2215 (CN), 1183 (C-O), 815 (C-

Cl). LRMS: (ESI⁺) m/z: 447 (C₂₃H₂₅Cl₂N₂O₃) [M+H]; HRMS: Exact mass

calculated for C₂₃H₂₅Cl₂N₂O₃[M+H], 447.1237. Found 447.1274.

(Z)-3-(4-(3-((1S,3S)-Adamantan-1-ylamino)-2-hydroxy-propoxy)phenyl)-

2-(3,4-dichlorophenyl)acrylonitrile (23l)

Prepared according to general procedure

2

from (Z)-2-(3,4-

dichlorophenyl)-3-(4-(oxiran-2-ylmethoxy)phenyl)acrylonitrile (11, 346 mg,

1 mmol, 1 eq.) and 1-adamantylamine (12s, 303 mg, 2 mmol, 2 eq.) to

afford the desired compound as a pale yellow solid (307 mg, 64%), m.p.:

170-172 °C. ¹H NMR (400 MHz, DMSO-d₆) d 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) d 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); IR u_max/cm^-1: 2903 (CH₂), 2215 (CN), 1142 (C-O), 816 (C-Cl);

LRMS (ESI) m/z (%): LRMS (ESI⁺): 497 (C₂₈H₃₁Cl₂N₂O₂) [M+H]; HRMS:

Exact mass calculated for C₂₈H₃₁Cl₂N₂O₂[M+H], 497.1757. Found

497.1742.

(Z)-2-(3,4-Dichlorophenyl)-3-(4-(2-hydroxy-3-(D-11-piperidin-1-

yl)propoxy)phenyl)-acrylonitrile (26)

Prepared according to general procedure

2

from (Z)-2-(3,4-

dichlorophenyl)-3-(4-(oxiran-2-ylmethoxy)phenyl)acrylonitrile (11) (346 mg,

1 mmol, 1 eq.) and piperidine-d₁₁(12w, 148 µL, 1.5 mmol, 1.5 eq.) to afford

the desired compound as a pale yellow solid (345 mg, 82%), m.p.: 103-

105 °C. ¹H NMR (600 MHz, DMSO-d₆) d 8.10 (s, 1H), 8.01 (d, J = 1.7 Hz,

1H), 7.96 (d, J = 8.7 Hz, 2H), 7.77 (d, J = 8.5 Hz, 1H), 7.69 (dd, J = 8.5,

1.9 Hz, 1H), 7.13 (d, J = 8.7 Hz, 2H), 4.85 (d, J = 3.0 Hz, 1H), 4.08 (t, J =

6.4 Hz, 1H), 3.97 – 3.95 (m, 2H), 2.39 (dd, J = 12.6, 5.8 Hz, 1H), 2.32 (dd,

J = 12.6, 5.8 Hz, 1H); ¹³C NMR (151 MHz, DMSO-d₆) d 161.2, 144.3, 135.0,

132.0, 131.5 (2C), 131.2, 131.1, 127.0, 125.78, 125.75, 117.9, 115.1 (2C),

104.3, 71.5, 66.4, 61.6; IR u_max/cm^-1: 3300 (OH), 2931 (N-CH₂), 2211 (CN),

1182 (C-O), 815 (C-Cl); LRMS (ESI⁺) m/z: 441 (C₂₃H₁₅D₁₀³⁵Cl₂N₂O₂)

[M+H]; HRMS: Exact mass calculated for C₂₃H₁₅D₁₀Cl₂N₂O₂[M+H],

441.1922. Found 441.1925.

(Z)-3-(4-(3-((1R,3S,5r,7r)-Adamantan-2-ylamino)-2-hydroxy-

propoxy)phenyl)-2-(3,4-dichlorophenyl)acrylonitrile (23m)

2-Adamantylamine hydrochloride (12t, 513 mg, 2.66 mmol, 1 eq.) was

combined with sodium hydroxide (126 mg, 3.14 mmol, 1.2 eq.) and water

(30 mL). Stirred at ambient temperature for 5 min. The solution was

diluted with ethyl acetate (20 mL), washed with water (3 x 20 mL) dried

over MgSO₄and concentrated under reduced pressure. The resulting

white powder was then carried directly through to the next step. Prepared

according to general procedure 2 from (Z)-2-(3,4-dichlorophenyl)-3-(4-

(oxiran-2-ylmethoxy)phenyl)-acrylonitrile (11, 500 mg, 1.44 mmol, 1 eq.)

and 2-adamantylamine (12ta, 328 mg, 2.17 mmol, 1.5 eq.) to afford the

desired compound as a pale yellow solid (325 mg, 60%), m.p.: 123-126 °C.

¹H NMR (400 MHz, DMSO-d₆) d 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) d 161.2,

144.3, 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, 68.4, 61.3, 49.6, 37.5, 39.91, 39.89, 31.6, 31.5,

30.79, 30.77, 27.3, 27.2; IR u_max/cm^-1: 2903 (CH₂), 2215 (CN), 1141 (C-O),

818 (C-Cl); LRMS (ESI) m/z (%): LRMS (ESI⁺): 497 (C₂₈H₃₁Cl₂N₂O₂)

[M+H]; HRMS: Exact mass calculated for C₂₈H₃₁Cl₂N₂O₂[M+H], 497.1757.

Found 497.1742.

Acknowledgements

JRB is the recipient of an Australian Government Research

Training Program scholarship. Funding from the Australian

Cancer Research Foundation and the Ramaciotti Foundation is

gratefully acknowledged.

Keywords: dichlorophenylacrylonitriles • aryl hydrocarbon

receptor 2 • breast cancer • MTT assay 4 • SRB assay

Triethylammonium(Z)-1-(3-(4-(2-cyano-2-(3,4-

dichlorophenyl)vinyl)phenoxy)-2-hydroxypropyl)piperidin-3-olate (25a)

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Entry for the Table of Contents

Microscopy examination reveals and unexpected interference in the MTT assay screening of a range of dichlorophenylacrylonitrles

amino alcohols. SRB assays reveal significantly reduced cell viability.

16

Article Doi

Amino Alcohol Acrylonitriles as Activators of the Aryl Hydrocarbon Receptor Pathway: An Unexpected MTT Phenotypic Screening Outcome

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