Optimisation of estrogen receptor subtype-selectivity of a 4-Aryl-4H-chromene scaffold previously identified by virtual screening

Article abstract of DOI:10.1016/j.bmc.2019.115261

4-Aryl-4H-Chromene derivatives have been previously shown to exhibit anti-proliferative, apoptotic and anti-angiogenic activity in a variety of tumor models in vitro and in vivo generally via activation of caspases through inhibition of tubulin polymerisation. We have previously identified by Virtual Screening (VS) a 4-aryl-4H-chromene scaffold, of which two examples were shown to bind Estrogen Receptor α and β with low nanomolar affinity and <20-fold selectivity for α over β and low micromolar anti-proliferative activity in the MCF-7 cell line. Thus, using the 4-aryl-4H-chromene scaffold as a starting point, a series of compounds with a range of basic arylethers at C-4 and modifications at the C3-ester substituent of the benzopyran ring were synthesised, producing some potent ER antagonists in the MCF-7 cell line which were highly selective for ERα (compound 35; 350-fold selectivity) or ERβ (compound 42; 170-fold selectivity).

Full text of DOI:10.1016/j.bmc.2019.115261

Journal Pre-proofs
Optimisation of Estrogen Receptor Subtype-Selectivity of a 4-Aryl-4H-Chro-
mene Scaffold Previously Identified by Virtual Screening
Miriam Carr, Andrew J.S. Knox, Daniel K. Nevin, Niamh O'Boyle, Shu
Wang, Billy Egan, Thomas McCabe, Brendan Twamley, Daniela M. Zisterer,
David G. Lloyd, Mary J. Meegan
PII:
S0968-0896(19)31245-3
https://doi.org/10.1016/j.bmc.2019.115261
BMC 115261
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
22 July 2019
3 December 2019
9 December 2019
Please cite this article as: M. Carr, A.J.S. Knox, D.K. Nevin, N. O'Boyle, S. Wang, B. Egan, T. McCabe, B.
Twamley, D.M. Zisterer, D.G. Lloyd, M.J. Meegan, Optimisation of Estrogen Receptor Subtype-Selectivity of a
4
-Aryl-4H-Chromene Scaffold Previously Identified by Virtual Screening, Bioorganic & Medicinal Chemistry
(
2019), doi: https://doi.org/10.1016/j.bmc.2019.115261
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Optimisation of Estrogen Receptor Subtype-Selectivity of a 4-Aryl-4H-Chromene
Scaffold Previously Identified by Virtual Screening.
Miriam Carr1,2#, Andrew J.S. Knox1,3#*, Daniel K. Nevin , Niamh O’Boyle , Shu Wang ,
1
2
2
2
4
4
1
Billy Egan , Thomas McCabe , Brendan Twamley , Daniela M. Zisterer , David G.
1
2
Lloyd and Mary J. Meegan
1School of Biochemistry and Immunology, Trinity Biomedical Sciences Institute, 152 - 160
Pearse Street Trinity College Dublin, Dublin 2, Ireland
2School of Pharmacy and Pharmaceutical Sciences, Trinity Biomedical Sciences Institute,
1
52 - 160 Pearse Street Trinity College Dublin, Dublin 2, Ireland
3School of Biological and Health Sciences, Technology University Dublin, Dublin City
Campus, Kevin St., Dublin 8, D08 NF82, Ireland
4School of Chemistry, Trinity College Dublin, Dublin 2, Ireland
1

Abstract
4
-Aryl-4H-Chromene derivatives have been previously shown to exhibit anti-proliferative,
apoptotic and anti-angiogenic activity in a variety of tumor models in vitro and in vivo
generally via activation of caspases through inhibition of tubulin polymerisation. We have
previously identified by Virtual Screening (VS) a 4-aryl-4H-chromene scaffold, of which two
examples were shown to bind Estrogen Receptor α and β with low nanomolar affinity and
<
20-fold selectivity for α over β and low micromolar anti-proliferative activity in the MCF-7
cell line. Thus, using the 4-aryl-4H-chromene scaffold as a starting point, a series of
compounds with a range of basic arylethers at C-4 and modifications at the C3-ester
substituent of the benzopyran ring were synthesised, producing some potent ER antagonists
in the MCF-7 cell line which were highly selective for ERα (compound 35; 350-fold
selectivity) or ERβ (compound 42; 170-fold selectivity).
Keywords
Estrogen receptor alpha, Estrogen receptor beta, Isoform selectivity, Subtype Selectivity, 4-
Aryl-4H-Chromene, Benzopyran, Anticancer, Anti-proliferative, Breast cancer, Cytotoxic,
Knovenagel condensation, Molecular Modeling
2

Abbreviations
Introduction
1
.
Estrogen Receptors (ER) are hormone regulated receptors that are known to produce their
long-term effects on cell structure and function via intracellular signalling and subsequent
modulation of gene expression. In addition to genomic signalling, a rapid non-genomic
signalling pathway has also been identified which is mediated by cell membrane-associated
estrogen receptors. In fact a G-Protein Coupled Estrogen Receptor (GPR30/GPER) has been
identified that binds estrogen, initiating a plasma membrane-initiated signaling cascade 1–4.
Focusing on ‘classical’ ER, two subtypes exist and are expressed in a wide-range of tissues
and cells throughout the body; ER alpha (ERα) is predominantly found in the female bone,
uterus, ovary (thecal cells), mammaries, adipose tissue, cardiovascular system and brain. ER
beta (ERβ) is found also in the adipose tissue, cardiovascular system and brain but also in the
lung, bladder, colon and granulosa cells of the ovary 5–14. The concept of Selective Estrogen
Receptor Modulation (SERM) 15 was first demonstrated with the clinical drug Tamoxifen 16,17
which was introduced in the 1970s as a treatment for advanced breast cancer in
postmenopausal women. Tamoxifen was shown to have anti-estrogenic function in the
breast, however partial estrogenic effects in other target tissues such as bone and the
cardiovascular system where it is beneficial but conversely harmful in the endometrium 18–21
.
However a strong association between its use and the development of endometrial carcinoma
emerged fuelling research for the discovery of alternative compounds with mixed
agonistic/antagonistic profiles (Figure 1).
Figure 1.
Figure 1: Structures of selected SERM structural classes including triarylethylenes tamoxifen
and endoxifen, benzothiophenes raloxifene and arzoxifene, indoles pipendoxifene and
bazedoxifene, benzopyran acolbifene and the tetrahydronaphthalene lasofoxifene.
3

It has been since hypothesised that the optimal route to the development of an ‘ideal’ SERM
can only occur when delineation of the full array of the biological roles orchestrated by ERα
or ERβ in different tissues is revealed, and this is best achieved through the design of
subtype-selective ER ligands. To date, a plethora of both ERα and ERβ selective agonists and
antagonists have been described (compounds 1-12, Figure 2)22–57. The ER agonist 16a-LE2
5
8
(
1) has been reported to inhibit cardiac hypertrophy . The synthesis and characterisation of a
series of pyrazole-based compounds resulted in a >400-fold selective agonist for ERα, 1,3,5-
4
4
tris(4-hydroxyphenyl)-4-propyl-1H-pyrazole, (2) (PPT) . Mortenson et al subsequently
synthesised a library of heterocycle-based (furans, thiophenes, and pyrroles) ER agonists, the
most ERα selective (65-fold) of which was compound 3 - 2,4,5-tris(4-hydroxyphenyl)-3-
3
9
methylfuran . Several SERMs have also been reported with ERα selectivity, for example;
5
9
45
benzothiophenes (4), tetrahydroisoquinolines (5) and pyrazoles , with the most selective
ERα antagonist to date being a pyrazole core incorporating a basic side-chain termed MPP
6
0
(
6)(~220-fold) . To facilitate the study of ERβ specific mechanisms of action, the first
reported full agonist discovered some time ago by Katzenellenbogen and co-workers,
diarylpropionitrile(7) (DPN), exhibited 70-fold preferential binding for ERβ over ERα and
3
7
7
8-fold selectivity in transcriptional assays . ERB-041 (Prinaberel)(8), WAY-200070 and
WAY-202196(9) were all developed by Wyeth and reported in 2004-561–63, followed by a
series of ERβ selective benzopyrans developed by Lilly in 200641,64,65. The wide range of
activities of these compounds both in vitro and in vivo has made it particularly difficult to
assign clinical relevance to them and has also been additionally hampered by the existence of
different isoforms of ERβ. ERβ1 appears to be the isoform displaying the strongest activity,
however, some variant forms may interact with ERβ1 and also with ERα presenting a
6
6
challenge in understanding the full complexity of the biology of ERβ . To date, very few
examples of ERβ antagonists have been reported (Figure 2). Two 30-fold ERβ selective
antagonists have been described; one based on a triazine scaffold (10) was discovered by
4

GSK 32 and another by researchers at Organon 67 belonging to the class of 10-aryl substituted
benzo[b]fluorenes (11). The (R,R)-cis-tetrahydrochrysene (12) (THC) was subsequently
demonstrated by the Katzenellenbogen lab to behave as a full agonist in ERα and full
3
6
antagonist in ERβ . Surprisingly, as THC lacks a basic side-chain, the term ‘passive
antagonism’ was proposed for its mechanism of action supported by crystal structures of
6
8
THC bound to both ERα and ERβ ligand binding domains (LBDs) .
We have described in earlier work the identification by in silico screening of a novel 4-aryl-
6
9
4
H-chromene scaffold which was shown to potently bind both ER isoforms . The two 4-
aryl-4H-chromene analogs (13 and 14) displayed modest ERα selectivity (20-fold) and also
antiproliferative activity in an ER positive breast cancer cell line, MCF-7, (Figure 3).
Importantly, it was noted that neither analog incorporated a typical basic arylether and this
formed the premise of the current study. In the present work, a series of 4-aryl-4H-chromene
derivatives were designed and synthesised some of whch incorporated the typical basic
arylethers usually required in ER antagonists (Figure 2). In addition, we have now carried out
modifications at the C3 ester position of the benzopyran ring to probe potential differences in
ER binding pockets thereby maximising isoform selectivity. The most active compound in
the series exhibited 350-fold selectivity for ERα and interestingly one of the series showed
1
70-fold selectivity for ERβ. The 4-aryl-4H-chromene scaffold has been reported in
numerous studies to possess anti-proliferative, apoptotic and anti-angiogenic activity in a
variety of tumor models in vitro and in vivo, generally via activation of caspases through
7
0
inhibition of tubulin polymerisation . In addition, the inhibition of insulin-regulated
7
1
aminopeptidase by related 4-aryl-4H-chromenes has been recently reported . The 4-aryl-
H-chromene Crolibulin (15), Figure 3, (a microtubule destabilizing agent that disrupts
4
vascular endothelial cells, and in turn, blood flow to the tumor) is currently in Phase I/II
clinical trials assessing its toxicity levels when co-administered with cisplatin and
7
2
progression-free survival (PFS) in adults with anaplastic thyroid cancer (ATC) . We
5

envisage that further biochemical analysis of our 4-aryl-4H-chromene compound series could
potentially lead to the developement of novel clinically relevant ER modulating compounds
featuring the 4-aryl-4H-chromene scaffold structure.
Figure 2. Subtype selective ER ligands
Figure 3. 4-Aryl-4H-chromenes 13, 14 and Crolibulin (15)
2
2
.
Results
.1
Chemistry
Many approaches to the preparation of 4-aryl-4H-chromenes have been described71,73–76. In
the present study, the synthetic route to the 4-aryl-4H-chromenes (16-25) initially identified
for the study is outlined in Scheme 1. These compounds contain methyl ester, ethyl ester or
nitrile substituents at C-3 of the chromene ring and also benzyl, ethyl, methyl ether or phenol
substituents on the C-4 aryl ring. The appropriate arylaldehyde is reacted with malononitrile
or the cyanoacrylate ester and resorcinol in the presence of base (piperidine or triethylamine)
in a one-pot synthesis. The reaction proceeds through the formation of an intermediate
cyanoacrylate ester or benzylidene malonitrile by Knoevenagel condensation from the
cyanoacetate ester, malonitrile and the benzaldehyde. This intermediate cyanoacrylate ester
can be observed as a solid forming within ten minutes of heating the mixture. Further reaction
of the resorcinol phenolic OH with the nitrile and subsequent electrophilic ring closure
afforded the desired final chromene products, 16-25. The cyanoacrylate intermediate product
was isolated in the case of 26-28 and subsequently reacted with resorcinol to afford the
desired products. The phenolic methyl ester 19 was also obtained from the benzyl ether 21 by
hydrogenation (H /Pd/C) (Scheme 1).
2
The series of 4-aryl-4H-chromenes containing a basic ether substituent at C-4 (compounds
6

2
4-36) were obtained as outlined in Scheme 2. These compounds also contain methyl, ethyl,
n-butyl or tert-butyl ester or nitrile substituents at C-3 of the chromene. The benzaldehydes
9-31 were obtained by alkylation of 4-hydroxybenzaldehyde with 1-(2-
chloroethyl)pyrrolidine, 1-(2-chloroethyl)piperidine or 1-(2-chloroethyl)morpholine
2
respectively as previously reported77 to afford the pure aldehydes in 65-97% yield.
Subsequent reaction of the aldehydes 29-31 with malononitrile or the appropriate
cyanoacrylate ester in the presence of piperidine or triethylamine afforded desired products
3
2-44 in moderate yield. The cyanoacrylate intermediate 45 was also isolated on reaction of
the aldehyde 30 with ethyl cyaoacrylate and subsequently reacted with resorcinol to afford
the desired product 38.
While attempting the synthesis of the methyl esters 16, 19, 25, 32, 37, 42 using ethanol as
solvent, it was noted that the final product isolated in each case was in fact the corresponding
ethyl ester 17, 20, 21, 33, 38, 43 respectively. It appeared that in the presence of ethanol, a
facile transesterification was occurring. Several different solvents were then employed in an
attempt to obtain the methyl ester products, e.g. methanol, ethyl acetate, acetone,
dichloromethane and tetrahydrofuran. Dichloromethane was found to be successful for the
synthesis of all other methyl ester compounds (16, 19, 25, 32, 37 and 42), however yields
were low. Details of the reaction conditions for the synthesis of compounds 16-25 and 32-44
are presented in Table 1. Attempts to improve the yield using potassium carbonate or
cetyltrimethylammonium chloride in aqueous conditions were not successful75,76. The most
efficient method of preparation for the methyl ester products was by replacing the initially
used triethylamine with piperidine as the base of choice and by performing the reactions
under microwave conditions.
The stability of representative examples of the target compounds 38 and 40 was determined
in phosphate buffer and the half life was determined to be greater than 24 h for each
7

compound at pH values 7.4 and 9. The compounds 38 and 40 degraded at lower pH = 4, with
7% and 32% remaining after 30 min respectively. The compounds showed very high
2
stability in human blood plasma, and the half-life was determined to be greater than 24 h.
The molecular structure of a representative example of the 4-aryl-4H-chromenes series,
compound 22 was determined by single crystal X-ray crystallography (S enantiomer shown).
The ORTEP diagram is displayed in Figure 4 (thermal ellipsoids at 50% probability). It can
be seen that the benzopyran ring is planar, and the aryl ring at C-4 positioned at a dihedral
angle of 78.403(77) ° with respect to the benzopyran ring (Figure 4).
Figure 4. ORTEP representation of the X-Ray crystal structure of 22, ellipsoids at 50%
probability
Scheme 1. Synthesis of 4-aryl-4H-chromenes 16-25
Scheme 2. Synthesis of 4-aryl-4H-chromenes 32-44
2
.2
Biological results and discussion
All compounds were initially evaluated for antiproliferative effects in MCF-7 cells using the
MTT assay (72 h) and the results are displayed in Table 1. The most potent compound was
identified as compound 41 (which contains piperidine aryl ether at C-4 and a tert-butyl ester
at C-3 of the chromene ring) which showed comparative results to that of tamoxifen in the
antiproliferative assay with IC value of 2.65 M (IC value determined for control drug
5
0
50
tamoxifen = 4.12 M). Compounds 16, 21, 35 and 38 also demonstrated low microlmolar
antiproliferative effects with IC50 values of 10.82, 4.50, 7.73 and 8.75 M respectively.
Selected compounds were concurrently tested to assess the extent of their cytotoxicity using a
LDH assay. Compounds displayed negligable cytotoxicity in LDH assay with 0% cell death
observed for compounds 32, 33, 37, 38, 43 and 44.
8

Selected compounds from the series 16-23 and 32-44 were then screened for their binding
affinity to ERα and ERβ in a fluorescence polarisation assay, with tamoxifen as a positive
control. The results are displayed in Table 1. For compounds 16 and 17 tested, the methyl and
ethyl ester compounds showed similar affinity to each other for ERα and ERβ. The cyano
compound 18 did not show any binding affinity for ERα at concentrations up to 10 µM.
Compound 19 with the phenolic substituent at C-4 showed a slightly greater affinity for ERα
than the benzyloxy ether 21, however it showed markedly less affinity for ERβ. The ethyl
ether compounds 22 and the 3,4,dimethoxyether 23 having little antiproliferative activity,
were not further evaluated.
On addition of the basic ether substituent to the core phenol 20, whether pyrolidine,
piperidine or morpholine, greater ERα binding ability was observed. For the pyrolidine
containing compounds, the methyl (32), n-butyl (35) and tert-butyl (36) esters all displayed
similar affinity to ERα with IC binding of less than 100 nM each (e.g. 20 nM, 20 nM and 10
5
0
nM respectively). The ethyl ester (33) demonstrated slightly less affinity with binding of
.41 M. The C-3 cyano compound 34 as observed with the unsubstituted compounds had
decreased affiniy for ER C   , and little affinity for ERβ (IC > 10 M). All
1
5
0
50
compounds showed selectivity for ERα over ERβ with significant difference noted in binding
affinity, this was consistent across all compounds tested. For the compounds with the
piperidine containing side chain, the methyl (37), ethyl (38) and tert-butyl (41) esters all
demonstrated good affinity to ERα with ethyl ester compound 38 showing greatest activity
with IC values of 20 nM and 60 nM for ERα and ER respectively. The tert-butyl ester (41)
5
0
which was the most active in the antiproliferative assay, also demonstrated good affinity for
ERα (IC = 0.090 M) and also sub-micromolar binding to ERβ (IC = 370 M). Once
5
0
50
again the cyano compound 39 was the least effective in the series in the ERα and ER
binding assays, with IC50 values of 1.47 and 10.42 M respectively. Of the compounds
9

examined containing the morpholine side-chain (42-44), the ethyl ester 43 showed the
greatest ER binding activity (IC = 220 nM), while the methyl ester 42 was the most
5
0
effective as a ligand for ER (IC = 0.63 M).
5
0
Table 1. ERα and ERβ binding effects and antiproliferative activity in MCF-7 cells of
selected 4-aryl-4H-chromenes.
Compound
R
1
R
2
ER
IC50(M)
ER
IC50(M)

Antiproliferative
activity
d
d
MCF-7 cells
a
IC50(M)
1
1
1
1
6
7
8
9
H
H
-CO
2
CH
3
3.30
2.36
>10
0.79
1.59
0.61
0.48
0.26
10.82
101.17
182.05
156.80
-CO
2
CH
2
CH
3
H
-CN
8.39
0.84
OH
-CO
2
CH
3
10.02
12.67
2
2
2
2
3
0
1
2
3
2
OH
-CO
-CO
-CO
-CO
2
2
2
2
CH
CH
CH
CH
2
2
2
2
CH
CH
CH
CH
3
3
3
3
nd
40.32
nd
nd
>100
nd
-
>100
4.5
OCH
OCH
3,4-(OCH
2
C
6
H
5
-
2
CH
3
-
-
>100
>100
19.60
3
)
2
nd
nd
-CO
2
CH
3
0.02
0.35
14.31
3
3
CO
2
CH
-CN
CH
2
CH
3
1.41
3.10
2.21
27.29
3
3
4
5
0.89
0.02
>10
11.23
>50
-CO
2
CH
2
2
CH
2
CH
3
6.55
353.34
7.73
3
3
6
7
-CO
2
C(CH
CH
3
)
3
0.01
0.05
0.67
0.22
49.49
4.77
10.03
16.2
-CO
2
3
1
0

3
3
4
4
4
4
4
8
9
0
1
2
3
4
-CO
2
CH
2
CH
CH
3
2
0.02
1.47
0.06
10.42
2.35
0.37
0.63
2.44
>50
2.56
7.10
8.75
23.21
31.82
2.65
-CN
-CO
2
CH
2
CH
2
CH
3
0.03
87.29
3.93
-CO
2
C(CH
3
)
3
0.09
-CO
2
CH
3
109.10
0.22
0.005
11.02
3.58
103.22
18.79
35.31
4.12
2 2 3
-CO CH CH
-CN
-
13.97
0.070
Tamoxifenb, c
-
0.170
2.43
aIC50 values are half maximal inhibitory concentrations required to block the growth stimulation of MCF-7 cells.
b
Values represent the mean for at least two experiments performed in triplicate. The IC50 value obtained for
Tamoxifen is 4.12 μM is in good agreement with the reported IC50 value for tamoxifen using the MTT assay in
7
8 c
human MCF-7 cells . The ER binding values obtained are in agreement with the reported ER IC50 binding data
d
for tamoxifen (ER 60.9 nM, ER 188 nM, Panvera/Invitrogen); Values are an average of at least nine
replicate experiments, for ER with typical standard errors below 15%, and six replicate experiments for ER,
with typical standard errors below 15%
2
.3
Computational
To rationalise the subtype selectivity observed for compounds 35 (350-fold ERα selective)
and 42 (170-fold ERβ selective) we carefully selected the most appropriate X-ray structure to
utilise for subsequent docking experiments, through visual analysis of the binding modes
made by all co-crystallised SERMs/SERDs with their respective ER isoforms. Lasofoxifene
co-crystallised with ERα (PDBID 2OUZ) was considered to be the most suitable LBP to dock
compound 35, however, the electron withdrawing oxygen of the morpholino-ring of 42 has
the potential to H-bond unlike the other pyrrolidine/piperidine side-chains in the chromene
series. Taking this into account, we reasoned that PDBID 1R5K whose co-crystallised ligand
GW5638, uses a bridging water molecule that is perfectly positioned for substitution by the
morpholino-ring oxygen of 42 to enable H-bonding with the backbone amide N-H of Leu536
1
1

(H12) would be most appropriate for docking in this case. As no suitable ERβ isoforms of
these structures existed, we constructed refined models of each as described in the
experimental section. Briefly, the iTASSER server was used for protein structure prediction
using 2OUZ and 1R5K as templates to guide the prediction and the co-ordinates of each were
firstly morphed 79 to the co-ordinates of 2OUZ and 1R5K respectively followed by
8
0
refinement using 3DRefine . Reconstruction of any missing residues in 2OUZ and 1R5K
was also undertaken using the iTASSER server.
Conformers of all R and S enantiomers of compound 35 and 42 were docked and in the LBP
8
1
our refined models of ERα and β using FRED , and scored using Chemgauss4.
Marvin pKa prediction 82 for compound 35 revealed the tertiary amine of the piperidine side-
chain of compound 35 is protonated at pH7.4. Interestingly, Marvin predicted that only 15%
of compound 42 would have the tertiary amine of its morpholine ring protonated at pH7.4
with the remaining >80% predominantly neutral.
Only in the case of compound 35, was there a clear binding preference to both ER subtypes
for the R enantiomer (see supplementary information) with 1.6 and 1.9-fold differences
observed in scoring for top scoring docked poses, indicating that enantiomeric separation of
the R enantiomer might afford additional binding afffinity and selectivity. Figure 5 highlights
the key interactions made by compound 35 in both isoforms, and as is commonly observed
with most Selective Estrogen Receptor Modulators (SERMs), it strongly interacts with
Asp351 (303) via a salt bridge and is stabilised within the binding pocket through a series of
hydrophobic interactions (e.g. with Phe404 (356), Met343 (295)). Methionine residues are
relatively unique in the protein core whereby they can form hydrophobic interactions but also
engage polar oxygen (e.g. carbonyl) as recently reported by Pal et al 83. In the case of
compound 35, direct contact of the nucleophilic oxygen of the carbonyl group of 35 with
divalent sulfur of methionine (Met343 and Met421) through a hypervalent nonbonded S---X
1
2

8
4
interaction is apparent contributing approximately 2.5 kcal/mol for this interaction . This
interaction is not present in ERβ as the mutation of Met421  Ile373 allows 35 to slightly
rotate in the LBP and H-bonding with Phe346, Leu339 and Arg346 occurs rather than H-
8
5
bonding to Glu353 and Arg394 observed in ERα . This mutation has been reported by
Nilsson et al 11 as being critical in achieving subtype-selective ER based therapeutics. A
similar trend is also seen for the bulky tert-butyl moiety of 36 whereby alpha selectivity is
observed for the same reasons but to a lesser degree than 35 (~50-fold). Interestingly,
switching from the pyrrolidinyl (36) to piperidinyl basic side-chain (41) led to a marked
reduction in ERα selectivity (>12-fold) (Table 1).
Figure 5. Top ranked poses of Compound 35 in ERα (green) and ERβ (grey) indicating key
hydrogen bonding interactions.
Figure 6. Side-view of top ranked poses of Compound 35 in ERα (green) and ERβ (grey)
indicating shift in orientation due to Leu384Met336 mutation.
Compound 42 (Figure 7) is stabilised in the binding site of both ERα and ERβ via hydrogen
bonding interactions with Glu353 (305) and Arg394 (346) and also through additional H-
bonding contacts to the morpholino oxygen of 42 (Leu536; 2.12Å (Val487; 2.32 Å)) and the
hydroxy group of Thr347 (299) which is well positioned to H-bond with the morpholino
tertiary amine, irrespective of its protonation state. Interestingly, Figure 7 highlights the
additional polar contact made between Asp303 and Tyr488 in ERβ as a result of rotation of
the carboxylate group of Asp303 which potentially would reduce the conformational entropy
of the ligand bound state of 42, translating to an increase in binding affinity.
Figure 7. Top ranked poses of Compound 42 (R Enantiomer) in ERα (green) and ERβ (grey)
indicating key interactions.
1
3

4
.
Conclusion
We have previously demonstrated the utility of the 4-aryl-4H-chromene scaffold as a potent
modulator of ER activity. Although two 4-aryl-4H-chromene analogues displayed anti-
6
9
proliferative activity in the ER positive MCF-7 cell line, ERα/β selectivity was modest .
Following from these findings, the rationale that 4-aryl-4H-chromene analogues
incorporating a typical basic side-chain could improve ERα/β selectivity was explored.
Alongside this approach, modifications at the 3-position of the benzopyran ring were also
investigated with a view to maximising ER isoform selectivity via probing of binding pocket
topology. Several of these compounds possessed potent ER binding activity indicative of
potential ER antagonistic effects, the most active compound (35) displayed 350-fold
selectivity for ERα, whilst another (42) showed 170-fold selectivity for ERβ. Our
computational study suggests that compound 35 achieves its selectivity in a completely
different manner to compound 42. In the case of compound 35, selectivity is achieved
through exploitation of the differing LBP size of each isoform which forms as a result of the
mutational differences (Met421  Ile373) and results in a hypervalent nonbonded S---X
interaction with Met343 and Met421 in ERα only. Increasing the size of the carboxylate alkyl
chain increases the binding affinity and selectivity for ERα. Compound 42 appears to exhibit
differences in interaction potential to H12 of ERβ via Asp303 (H3) H-bonding to Tyr488
(H12) which may stabilize the ligand bound state and reduce its conformational entropy in
order to achieve this selectivity.
1
4

5
5
.
Experimental section
Chemistry
.1
Uncorrected melting points were measured on a Gallenkamp apparatus. Infra-red (IR)
spectra were recorded on a Perkin Elmer FT-IR Paragon 1000 spectrometer. 1H and 13C
nuclear magnetic resonance (NMR) spectra were recorded at 27 °C on a Brucker DPX 400
1
13
19
spectrometer (400.13 MHz, H; 100.61 MHz, C; 376.47 MHz, F) in either CDCl (internal
3
1
standard tetramethylsilane (TMS)) or CD OD or DMSO-d6. For CDCl , H-NMR spectra
3
3
were assigned relative to the TMS peak at 0.00  and 13C-NMR spectra were assigned
1
13
relative to the middle CDCl triplet at 77.00 ppm. For CD OD, H and C-NMR spectra
3
3
were assigned relative to the center peaks of the CD OD multiplets at 3.30  and 49.00 ppm
3
1
respectively. Coupling constants are reported in Hertz. For H-NMR assignments, chemical
shifts are reported: shift value (number of protons, description of absorption, coupling
constant(s) where applicable). Low resolution mass spectra were run on a Hewlett-Packard
5
973 MSD GC–MS system in an electron impact mode. Electrospray ionisation mass
spectrometry (ESI-MS) was performed in the positive ion mode on a liquid chromatography
time-of-flight mass spectrometer (Micromass LCT, Waters Ltd., Manchester, UK). The
samples were introduced into the ion source by an LC system (Waters Alliance 2795, Waters
Corporation, USA) in acetonitrile : water (60:40 %v/v) at 200 μL/min. The capillary voltage
1
5

of the mass spectrometer was at 3 kV. The sample cone (de-clustering) voltage was set at 40
V. For exact mass determination, the instrument was externally calibrated for the mass range
m/z 100 to m/z 1000. A lock (reference) mass (m/z 556.2771) was used. Mass measurement
accuracies of < 5 ppm were obtained. R values are quoted for thin layer chromatography on
f
silica gel Merck F-254 plates, unless otherwise stated. Flash column chromatography was
carried out on Merck Kieselgel 60 (particle size 0.040-0.063 mm), Aldrich aluminium oxide,
(activated, neutral, Brockmann I, 50 mesh) or Aldrich aluminium oxide, (activated, acidic,
Brockmann I, 50 mesh). Chromatographic separations were also carried out on Biotage SP4
instrument. All products isolated were homogenous on TLC. Microwave experiments were
carried out with in the Biotage initiator and Discover CEM microwave synthesisers.
5
.1.2 General procedures for synthesis of benzopyrans.
Method A: To the appropriate aldehyde (benzaldehyde, 4-(2-pyrrolidin-1-
ylethoxy)benzaldehyde or 4-(2-piperidin-1-ylethoxy)benzaldehyde) (10 mmol, 1 equiv) was
added the cyanoacetate ester (10 mmol, 1 equiv) or malononitrile (10 mmol, 1 equiv) and
resorcinol (10 mmol, 1 equiv). Ethanol (or dichloromethane for methyl ester) (30 mL) was
added and the reaction was heated briefly to reflux. The solution was then cooled and
triethylamine (0.5 mL) was added dropwise, and the reaction solution was then heated at
reflux for 4 h. Solvent was removed in vacuo and the product was recrystallized in ethanol or
methanol (for the methyl esters). Method B: To the appropriate aldehyde (4-
hydroxybenzaldehyde,
4-benzyloxybenzaldehyde,
4-ethoxybenzaldehyde,
3,4-
dimethoxybenzaldehyde,
4-(2-pyrrolidin-1-ylethoxy)benzaldehyde,
4-(2-piperidin-1-
ylethoxy)benzaldehyde or 4-(2-morpholin-1-ylethoxy)benzaldehyde)) (10 mmol, 1 equiv)
was added the cyanoacetate ester (10 mmol, 1 equiv) or malononitrile (10 mmol, 1 equiv) and
resorcinol (10 mmol, 1 equiv). Ethanol (dichloromethane for methyl ester) (30 mL) was
added and the reaction was heated briefly to reflux. The solution was then cooled and
1
6

piperidine (0.5 mL) was added dropwise and the reaction solution was then heated at reflux
for 4 h. The solvent was removed in vacuo and product was recrystallized in ethanol (or
methanol for methyl esters). Method C: To the appropriate aldehyde (4-
benzyloxybenzaldehyde,
4-(2-pyrrolidin-1-ylethoxy)benzaldehyde,
4-(2-piperidin-1-
ylethoxy)benzaldehyde or 4-(2-morpholin-1-yl)ethoxybenzaldehyde, (5 mmol, 1 equiv) was
added the cyanoacetate ester (5 mmol, 1 equiv) or malononitrile (5 mmol, 1 equiv) together
with resorcinol (5 mmol, 1 equiv). Ethanol (10 mL), (dichloromethane for methyl esters) was
added and the reaction heated in the microwave reactor for 5 min. The solution was then
cooled and piperidine (0.25 mL) was added dropwise and the reaction solution was then
heated in the microwave reactor for a further 30 min. The solvent was removed in vacuo and
the product was recrystallized from ethanol (or methanol for the methyl esters).
5
.1.2.1.
2-Amino-7-hydroxy-4-phenyl-4H-chromeme-3-carboxylic acid methyl
ester (16).
Preparation following method C from benzaldehyde (5 mmol, 0.51 mL), resorcinol (5 mmol,
50 mg) and methyl cyanoacetate(5 mmol, 0.50 g). Yield 5.5%, yellow powder, M.p. 231°C.
5
-1
-1 1
IR ʋmax (KBr) cm : 3417.5, 3305.5 (NH ), 1664.7 (C=O) cm . H NMR (400 MHz, CDCl ):
2
3
δ 3.55 (s, 3H, O-CH ), 4.88 (s, 1H, CH), 6.50 (d, 1H, J=2.52 Hz, Ar-H), 6.55 (dd, 1H,
3
J=2.52 Hz, 8.4 Hz, Ar-H), 7.00 (d, 1H, J=8.56 Hz, Ar-H), 7.06-7.09 (m, 1H, Ar-H), 7.20-
1
3
7
1
.23 (m, 4H, Ar-H). C NMR (100 MHz, CDCl ): δ 39.1 (CH,), 49.4 (O-CH ), 78.1 (C),
3 3
01.8 (CH), 111.6 (CH), 117.3 (C), 125.3 (CH), 126.7 (CH), 127.7 (CH), 129.5 (CH), 149.2
(
C), 156.3 (C), 160.8(C), 168.7(C=O), 168.8 (C-NH ). HRMS: Found 320.0898;
2
+
C H NO Na requires 320.0899(M +Na).
1
7
15
4
5
.1.2.2.
2-Amino-7-hydroxy-4-phenyl-4H-chromeme-3-carboxylic acid ethyl ester
(17).
1
7

Preparation following Method A from benzaldehyde (10 mmol, 1.01 mL), resorcinol (1.10 g)
and ethyl cyanoacetate(10 mmol, 1.13g). Yield 17%, white powder, M.p. 243°C. IR ʋmax
-1
-1 1
(
KBr) cm : 3306.4, 3300.0 (NH ), 1662.8 (C=O) cm . H NMR (400 MHz, CDCl ): δ 1.11
2
3
(
t, 3H, J=7.02 Hz, -CH ), 2.06 (m, 2H, NH ), 4.00 (q, 2H, J=7.01 Hz, CH ), 4.89 (s, 1H, CH),
3 2 2
6
.51 (d, 1H, J=2.64 Hz, Ar-H), 6.54 (dd, 1H, J=2.52 Hz, 6.00 Hz, Ar-H), 7.00 (d, 1H, J=8.00
1
3
Hz, Ar-H), 7.08-7.11 (m, 1H, Ar-H), 7.19-7.25 (m, 4H, Ar-H). C NMR (100 MHz, CDCl ):
3
δ 13.33 (CH ), 39.21 (CH), δ 58.18 (CH ), 77.01 (C), 101.84 (CH), 111.59 (CH), 117.12 (C),
3
2
1
1
3
25.24 (CH), 126.87 (CH), 127.55 (CH), 129.58 (CH), 148.48 (C), 149.12 (C), 156.30 (C),
60.59(C-NH2), 168.37(C=O). HRMS: Found 334.1042; C H NO Na requires
18
17
4
+
34.1055(M +Na).
5
.1.2.3.
2-Amino-7-hydroxy-4-phenyl-4H-chromeme-3-carbonitrile
(18).
Preparation following Method A from benzaldehyde (10 mmol, 1.01 mL), resorcinol (1.10 g,
7
3
1
0 mmol) and malononitrile (10 mmol, 0.66 g). Yield 56%, Yellow powder, M.p. 246°C .
-1
IR ʋmax (KBr) cm : 3401.1 (OH), 3336.3, 3218.99 (NH ), 2180.9 (CN), 1638.46,
2
-1 1
1
624.90(C=C) cm . H NMR (400 MHz, CDCl ): δ 1.54 (s(br), 2H, NH ), 2.76 (s(br), 1H,
3 2
OH), 4.68 (s, 1H, CH), 6.51 (d, 1H, J=2.00 Hz, Ar-H), 6.57 (dd, 1H, J=2.34 Hz, 8.76 Hz, Ar-
H), 6.86 (d, 1H, J=8.76 Hz, Ar-H), 7.22-7.25 (m, 3H, Ar-H), 7.31 (d, 2H, J=7.6 Hz, Ar-H).
1
3
C NMR (100 MHz, CDCl ): δ 40.21 (CH), 58.21 (C), 101.96 (CH), 111.99 (CH), 113.79
3
(CN), 118.99 (C), 126.26 (CH), 127.19 (CH), 128.08 (CH), 129.71 (CH), 145.88 (C), 148.89
(
C), 156.83(C-O), 159.59 (C-NH ). HRMS: C H N NaO requires 287.0796, found
2
16 12
2
2
+
2
87.0794(M +Na).
5
.1.2.4.
Ethyl
2-amino-7-hydroxy-4-(4-hydroxyphenyl)-4H-chromene-3-
carboxylate 20.
Preparation following the general method B above from ethylcyanoacetate (10 mmol, 1.13 g),
resorcinol (1.10 g, 10 mmol) and 4-hydroxybenzaldehyde (10 mmol, 1.22 g). The product
1
8

was obtained as a dark brown solid which purified by flash column chromatography over
silica gel (eluent: CH Cl – MeOH; 85:15) and then recrystalised from ethanol. Yield: 1.04 g,
2
2
8
6
3
1
2%, M.p. 228-230˚C . IR ʋ (KBr): 3614.49, 3595.36, (OH), 3485.10, 3357.31 (NH2),
max
-1 1
659.71 (C=O), 1629.41 (C=C), 1630.48 (C=C), 1591.36, 1538.14 cm . H NMR (400 MHz,
DMSO-d ) δ 0.98 (t, 3H, J=7.2, CH ), 4.28 - 4.47 (m, 2H, OCH ), 4.68 (s, 1H, CH), 6.13 -
6
3
2
6
.23 (m, 3H, Ar-H), 6.61 - 6.68 (m, 2H, Ar-H), 6.85 - 6.99 (m, 2H, Ar-H), 8.14 (s (br), 1H,
1
3
OH), 9.15 (s (br), 2H, NH ). C NMR (101 MHz, DMSO-d ): δ 14.31 (CH ), 56.03 (CH),
2
6
3
5
1
1
8.49 (CH ), 76.97 (C), 101.96 (CH), 102.44 (CH), 106.17 (CH), 114.75 (C), 127.81 (CH),
2
29.70 (CH), 132.74 (C), 152.56 (C-O), 154.93 (C-OH), 155.24 (C-OH), 158.44 (O-C-N),
+
60.88 (C=O). HRMS: C H NNaO requires 350.1004, found 350.0997(M +Na).
1
8
17
5
5
.1.2.5.
2-Amino-4-(4-benzyloxyphenyl)-7-hydroxy-4H-chromene-3-carboxylic
acid ethyl ester 21.
Preparation following the general method B above from ethyl cyanoacetate (10 mmol, 1.13
g), resorcinol (1.10 g, 10 mmol) and 4-benzyloxybenzaldehyde (10 mmol, 2.12 g). The
product was obtained as yellow powder which was recrystallised from ethanol. Yield: 1.54 g,
3
1
7%. Mp 192-194˚C. IR ʋmax (KBr): 3421.76 (OH), 3304.84, 3248.32 (NH ), 1659.05 (C=O),
2
-1
1
610.67 (C=C), 1598.45 (C=C), 1503.32, 1454.01, 1311.69 cm . H NMR (400 MHz,
DMSO-d ): δ 1.10 (t, 3H, J=7 Hz, CH ), 2.07 (s (br), 2H, NH ), 3.96 (q, 2H, J=7.04 Hz,
6
3
2
OCH ), 4.75 (s, 1H, CH), 5.01 (s, 2H, OCH ), 6.43 (d, 1H, J=6.52 Hz, Ar-H), 6.47 (d, 1H,
2
2
J=8.04 Hz, Ar-H),4 (d, 1H, J=8.52 Hz, Ar-H), 6.94 (d, 2H, J=8.52 Hz, Ar-H), 7.23 (d, 2H,
J=8 Hz, Ar-H), 7.31 (d, 1H, J=7.04 Hz, Ar-H), 7.34 - 7.39 (m, 2H, Ar-H), 7.42 (d, 1H,
13
J=6.76 Hz, Ar-H), OH not observed. C NMR (100 MHz, DMSO-d ): δ 14.78 (CH ), 38.58
6
3
(CH), 58.19 (CH ), 69.58 (CH ), 77.56 (C), 102.46 (CH), 112.53 (CH), 114.80 (C), 117.53
2 2
(CH), 128.08 (CH), 128.35 (CH), 128.85 (CH), 130.24 (CH), 137.68 (C), 141.58 (C), 149.51
1
9

(
C-O), 156.89 (C-OH), 157.09 (C-O), 161.39 (O-C-N), 168.87 (C=O). HRMS: C H NNaO
25 23 5
+
requires 440.1474, found 440.1463 (M +Na).
5
.1.2.6.
2-Amino-4-(4-ethoxyphenyl)-7-hydroxy-4H-chromene-3-carboxylic acid
ethyl ester 22.
Preparation following the general method B above from ethyl cyanoacetate (10 mmol, 1.13
g), resorcinol (1.10 g, 10 mmol) and 4-ethoxybenzaldehyde (10 mmol, 1.50 g). The product
was obtained as yellow crystals following recrystallisation from ethanol, yield 0.78 g, 22%.
M.p. 258-263˚C. IR ʋmax (KBr): 3563.54 (OH), 3431.59, 3419.15 (NH ), 1660.91 (C=O),
2
-1
1
1
634.86 (C=C), 1613.89 (C=C), 1583.43, 1568,42, 1458.83 cm . H NMR (400 MHz,
DMSO-d ): δ 1.05 (t, 3H, J=7.04 Hz, CH ), 1.25 (t, 3H, J=7.02 Hz, CH ), 3.93 - 4.18 (m,
6
3
3
4
6
H, 2xOCH ), 4.72 (s, 1H, CH), 6.42 - 6.50 (m, 2H, Ar-H), 6.73 (d, 2H, J=9.04 Hz, Ar-H),
2
.94 (d, 2H, J=8.04 Hz, Ar-H), 7.01 (d, 1H, J=8.12 Hz, Ar-H), OH not observed, NH not
2
1
3
observed. C NMR (100 MHz, DMSO-d ): δ 14.31 (CH ), 14.68 (CH ), 38.09 (CH), 58.51
6
3
3
(
(
(
CH ), 62.79 (CH ), 76.81 (C), 101.97 (CH), 112.04 (CH), 113.91 (CH), 117.13 (C), 127.84
2 2
CH), 129.76 (CH), 140.71 (C), 149.02 (C-O), 156.53 (C-OH), 160.91 (O-C-N), 168.40
+
C=O). HRMS: C H NO requires 356.1498, found 356.1488(M +H).
2
0
22
5
5
.1.2.7.
Ethyl 2-amino-7-hydroxy-4-(3,4-dimethoxyphenyl)-4H-chromene-3-
carboxylate 23
(E)-Ethyl 2-cyano-3-(3,4-dimethoxyphenyl)acrylate 28 (10 mmol, 2.62 g) and resorcinol
(1.10 g, 10 mmol) were dissolved in ethanol and the reaction mixture was briefly heated to
reflux. Piperidine (0.5 mL) was added dropwise and the reaction solution was heated at reflux
for 4 h. following the general method B above. The solvent was removed in vacuo and
product was recrystallized from ethanol. Yield: 0.07 g, 2%, yellow crystals, M.p. 284-288˚C
71.
629.45 (C=O), 1562.75 cm . H NMR (400 MHz, DMSO-d ): δ 1.09 (t, 3H, J=7.04 Hz,
IR ʋmax (KBr): 3488.34 (OH), 3431.11, 3412.48 (NH ), 1681.40 (C=C), 1642.76 (C=C),
2
-1 1
1
6
2
0

CH ), 3.66 (s, 3H, OCH ), 3.71 (s, 3H, OCH ), 3.95 (q, 2H, J=7.04 Hz, OCH ), 4.73 (s, 1H,
3
3
3
2
CH), 6.42 (s, 1H, Ar-H), 6.46 (dd, 1H, J=2.48, J=7.02 Hz, Ar-H), 6.55 (d, 1H, J=9.04 Hz,
Ar-H), 6.77 (d, 2H, J=9.04 Hz, Ar-H), 6.99 (d, 1H, J=8.52 Hz, Ar-H), 7.55 (s (br), 2H, NH2),
1
3
9
.57 (s (br), 1H, OH). C NMR (100 MHz, DMSO-d ): δ 14.36 (CH ), 38.45 (CH), 55.40
6 3
(
CH ), 58.53 (CH ), 76.72 (C), 101.97 (CH), 110.23 (CH), 111.15 (CH), 111.86 (C), 117.04
3 2
(
CH), 118.64 (CH), 129.73 (CH), 141.44 (C), 146.83 (C-OCH ), 148.19 (C-OCH ), 149.03
3 3
(
C-O), 156.60 (C-O), 160.97 (O-C-N), 168.43 (C=O). HRMS: C H NNaO requires
20 21 6
+
3
94.1267, found 394.1262(M +Na).
5
.1.2.8.
2-Amino-4-(4-benzyloxyphenyl)-7-hydroxy-4H-chromene-3-carbonitrile
(24).
Preparation following the general method B above from malononitrile (0.66 g, 10 mmol) and
resorcinol (1.1 g, 10 mmol) and 4-benxyloxybenzaldehyde (2.12 g, 10 mmol). The product
was obtained as a pink solid, 2.08 g (56%) following recrystallisation from ethanol. M.p.
o
-1 1
2
64-266 C. IR ʋ : 3258.49 (OH), 3032.13 (CH), 2191.16 (CN), 1637.44 (C=C) cm . H
max
NMR (400 MHz, DMSO-d )  9.71 (s, 1H, OH), 7.24 - 7.51 (m, 5H, ArH), 7.11 (d, J = 8.54
6
Hz, 2H, ArH), 6.96 (d, J = 8.54 Hz, 2H, ArH), 6.75 - 6.89 (m, 3H, ArH), 6.41 - 6.60 (m, 2H,
1
3
ArH), 5.05 (s, 2H, CH ), 4.59 (s, 1H, CH). C NMR (101 MHz, DMSO-d )  160.2 (O-C-
2
6
N), 157.2 (C-O), 157.1 (C-O), 148.8 (C-O), 138.8 (C), 137.1 (C), 130.0 (CH), 128.5 (CH),
28.4 (CH), 127.8 (CH), 127.7 (CH), 120.8 (CN), 114.8 (CH), 114.1 (CH), 112.4 (C), 102.2
1
(
CH), 69.3 (CH ), 56.7 (C), 39.3 (CH). HRMS: C H N O requires 369.1239; found
2
23 17
2
3
3
69.1232 [M-H]+.
5
.1.2.9.
2-Amino-4-(4-benzyloxyphenyl)-7-hydroxy-4H-chromeme-3-carboxylic
acid methyl ester (25).
Preparation as above using Method C from 4-benzyloxybenzaldehyde (10 mmol, 2.12 g),
resorcinol (1.10 g, 10 mmol) and methyl cyanoacetate, (10 mmol, 0.99 g). Yield 46%, cream
2
1

-1
-1
powder, M.p. 216 - 218°C. IR ʋmax (KBr) cm : 3415.4, 3302.9 (NH ), 1660.7 (C=O) cm .
2
1
H NMR (400 MHz, Acetone-d ): δ 3.57 (s, 3H, O-CH ), 4.84 (s, 1H, CH), 5.04 (s, 2H, CH ),
6
3
2
6
.50 (d, 1H, J=2.04 Hz, Ar-H), 6.55 (dd, 1H, J=2.26 Hz, 6.04 Hz, Ar-H), 6.85 (d, 2H, J=8.52
Hz, Ar-H), 7.00 (d, 1H, J=8.56 Hz, Ar-H), 7.14 (d, 2H, J=8.56 Hz, Ar-H), 7.31 (d, 1H,
1
3
J=7.04 Hz, Ar-H), 7.37 (t, 2H, J=7.78 Hz, Ar-H), 7.44 (d, 2H, J=7.88 Hz, Ar-H). C NMR
(
100 MHz, Acetone-d ): δ 38.2 (CH), 49.4 (O-CH ), 68.9 (O-CH ), 77.4 (C), 101.8 (CH),
6 3 2
1
11.6 (CH), 113.9 (CH), 117.6 (C), 127.1 (CH), 127.6 (CH), 127.9 (CH), 129.4 (CH), 137.2
(
C), 140.8 (C), 149.2 (C), 156.2 (C), 160.6 (C-O), 160.7 (C-NH ), 168.7 (C=O). HRMS:
2
+
Found 426.1297; C H NO Na requires 426.1317(M +Na).
2
4
21
5
5
.1.3. 2-Amino-7-hydroxy-4-(4-hydroxyphenyl)-4H-chromeme-3-carboxylic
acid
methyl ester (19).
2
-Amino-4-(4-benzyloxyphenyl)-7-hydroxy-4H-chromene-3-carboxylic acid methyl ester
(25) (1.6 mmol, 0.77 g) was dissolved in ethyl acetate (20 mL) and Pd/C (10%, 0.8 g) was
added. The mixture was stirred under an atmosphere of hydrogen for 12 h until reaction was
complete as monitored by TLC. The catalyst was removed by filtrations and the residue was
purified by chromatography to afford the product as a waxy solid, IR: KBr ʋmax: 3415.4,
-1
1
3
302.9 (NH ), 1660.7 (C=O) cm . H NMR (400 MHz, CDCl ): δ 3.57 (s, 3H, O-CH ), 4.79
2 3 3
(
(
(
(
(
3
s, 1H, CH), 6.49 (d, 1H, J=2.00 Hz, Ar-H), 6.56 (dd, 1H, J=2.02 Hz, 6.00 Hz, Ar-H), 6.69
1
3
d, 2H, J=8.52 Hz, Ar-H), 6.99-7.04 (m, 3H, Ar-H). C NMR (100 MHz, CDCl ): δ 38.2
3
CH), 49.4 (O-CH ), 68.9 (O-CH ), 77.4 (C), 101.8 (CH), 111.6 (CH), 113.9 (CH), 117.6
3
2
C), 127.1 (CH), 127.6 (CH), 127.9 (CH), 129.4 (CH), 137.2 (C), 140.8 (C), 149.2 (C), 156.2
C), 160.6 (C-O), 160.7 (C-NH ), 168.7 (C=O). Found 336.0852; C H NO Na requires
2
17 15
5
+
36.0848(M +Na).
5
.1.4. General procedure for synthesis of cyanoacrylate esters 26-28.
2
2

A solution of methyl or ethyl cyanoacetate (10 mmol) and the appropriate arylaldehyde (10
mmol) in methanol 30 mL was heated briefly to reflux. The solution was cooled and
piperidine (0.5 mL) was added dropwise and the reaction solution was then heated at reflux
for 4 h. The solution was cooled and the solvent was removed in vacuo and product was
recrystallized from ethanol.
5
.1.4.1.
(E)-Methyl 2-cyano-3-(4-hydroxyphenyl)acrylate 26.
A mixture of methyl cyanoacetate (10 mmol, 1.13 g) and 4-hydroxybenzaldehyde (10 mmol,
1
1
.22 g) was melted under vacuum in a Kugelrohr short-path vacuum distillation apparatus at
70 ˚C for 40 min. The product was then dried at 80 ˚C and then recrystallized from ethanol
6
9
as beige coloured crystals. Yield: 1.802 g, 90%, Mp: 208-210 ˚C . IR v (KBr): 3415.50
max
-1
1
(
OH), 2224.80 (CN), 1725.19 (C=O), 1589.33 (C=C) cm . H NMR (400 MHz, DMSO-d ):
6
3
.78 (s, 3H, OCH ), 6.91 (d, J=8.54 Hz, 2H), 7.94 (d, J=9.16, Hz, 2H), 8.18 (s, 1H, CH=C).
3
1
3
C NMR (100 MHz, CDCl ): δ 51.99 (OCH ), 97.62 (C), 115.53 (CN), 115.88 (CH), 123.06
3
3
(C), 133.50 (CH), 153.89 (CH), 162.08 (C-OH), 162.76 (C=O).
5
.1.4.2.
(E)-Ethyl 2-cyano-3-(4-benzyloxyphenyl)acrylate 27.
Following the procedure outlined above, a solution of ethyl cyanoacetate (10 mmol, 1.13 g)
and 4-benzyloxybenzaldehyde (10 mmol, 2.12 g) in benzene (20 mL) was heated briefly to
reflux and then cooled. Piperidine (0.99 mL) was added dropwise and the reaction solution
was then heated at reflux for 4 h. The solution was washed with HCl (10 %) and then
extracted with dichloromethane. The solvent was removed in vacuo and the product was
recrystallized from ethanol as cream coloured crystals. Yield: 2.53 g, 83%, M.p. 99-100 ˚C
87.
IRvmax (KBr): 2221.96 (CN), 1714.07 (C=O), 1688.82 (C=C), 1563.74, 1473.48 cm . H
-1 1
NMR (400 MHz, CDCl ): 1.40 (t, J=7.02 Hz, 3 H, CH ), 4.38 (q, J=7.12 Hz, 2 H, CH ), 5.16
3
3
2
(
s, 2 H, CH ), 7.07 (d, J=9.16 Hz, 2 H, ArH), 7.32 - 7.47 (m, 5 H, ArH), 8.01 (d, J=8.54 Hz,
2
2
H, ArH), 8.18 (s, 1 H, CH=C).
2
3

5
.1.4.3.
(E)-Ethyl 2-cyano-3-(3,4-dimethoxyphenyl)acrylate 28.
Following the procedure outlined above, a solution of ethyl cyanoacetate (10 mmol, 1.13 g)
and 3,4-dimethoxybenzaldehyde (10 mmol, 1.66 g) in ethanol 30 mL was heated briefly to
reflux and then cooled. Piperidine (0.5 mL) was then added dropwise and the reaction
solution heated at reflux for 4 h. The solvent was removed in vacuo and product was
recrystallized from ethanol to afford the product as yellow crystals. Yield: 0.34 g, 13%, M.p.
4
1
1
60 ˚C . IR ʋ (KBr): 2244.68 (CN), 1673.71 (C=C), 1643.64 (C=O), 1563.74, 1473.48
max
-1 1
cm . H NMR (400 MHz, DMSO-d ): δ 0.94 (t, 3H, J=7.14 Hz, CH ), 3.72 (s, 6H, 2xOCH ),
6
3
3
3
7
.86 (q, 2H, J=7.04, CH ), 6.38 (d, 1H, J=2.28, Ar-H), 6.45 – 6.51 (m, 1H, Ar-H), 7.18 -
2
1
3
.27 (m, 1H, Ar-H), 8.46 (d, 1H, J=6.98, CH). C NMR (100 MHz, DMSO-d ): δ 14.33
6
(
CH ), 58.85 (OCH ), 62.03 (CH ), 78.01 (C), 106.63 (CH), 112.53 (CN), 120.75 (CH),
3 3 2
1
28.77 (C), 148.70 (C-OCH ), 149.77 (C-OCH ), 157.73 (CH), 168.72 (C=O). HRMS:
3 3
+
C H NO requires 262.1079, found 262.1086(M +H).
1
4
16
4
5
.1.5.
General procedure for preparation of 29-31.
4
-Hydroxybenzaldehyde (16 mmol (1 equiv)) was dissolved in ethyl acetate (20 mL). To this
solution was added anhydrous potassium carbonate (18 mmol, 2.50 g) (1.125 equiv) and
tetra-n-butylammonium bromide (0.054 g, 0.2 mmol). The mixture was then heated at reflux
for 10 min and the appropriate basic ether was then added (32 mmol, 2 equiv). [The basic
ethers 1-(2-chloroethyl)pyrrolidine, 1-(2-chloroethyl)piperidine and 1-(2-chloroethyl)
morpholine were obtained as their HCl salt and were washed with water (45 mL) and sodium
hydroxide (10.5 g) in toluene (6 mL) to extract the free base for use in the reaction]. The
reaction mixture was heated at reflux until reaction was complete as monitored by thin layer
chromatography. On completion, the solution was filtered and the solvent was removed
under reduced pressure to afford product, which was used in the following reactions without
further purification.
2
4

5
.1.5.1.
4-(2-Pyrrolidin-1-ylethoxy)benzaldehyde (29).
The preparation was according to the general procedure above. The product was isolated as
7
7
1
orange oil yield 65%. H NMR (400 MHz, CDCl ): δ 1.59 (s, 4H, (CH ) ), 2.41 (s, 4H,
3
2 2
CH -N-CH ), 2.70 (2H, t, J=5.78 Hz, -CH ), 3.96 (t, J=5.78 Hz, 2H, O-CH ), 6.79 (d, 2H,
2
2
2
2
J=8.56 Hz, Ar-H), 7.58 (d, 2H, J=8.52 Hz, Ar-H), 9.64 (s, 1H, CHO).
5
.1.5.2
4-(2-Piperidin-1-ylethoxy)benzaldehyde (30).
The preparation was according to the general procedure above (32 mmol scale). The product
7
7 1
was isolated as orange coloured oil, (5.42 g, 73%) . H NMR (400 MHz, CDCl ): δ 1.53-
3
1
.55 (m, 6H, 3xCH ), 2.51-2.66 (m, 6H, CH ), 4.29-4.36 (m, 2H, CH ), 7.13 (d, 2H, J=8.0
2 2 2
Hz, Ar-H), 7.85 (d, 2H, J=8.0 Hz, Ar-H), 9.89 (s, 1H, CHO).
5
.1.5.3.
4-(2-Morpholin-1-ylethoxy)benzaldehyde (31).
The preparation was according to the general procedure above and the product was isolated
7
7 1
as an orange coloured oil (97%) . H NMR (400 MHz, CDCl ): δ 2.60 (m, 4H, 2xCH ), 2.68
3
2
(
s, 2H, CH ), 3.56 (m, 4H, 2xCH ), 4.05 (m, 2H, 2xO-CH ), 6.86 (m, 2H, Ar-H), 7.67 (m,
2
2
2
2
H, Ar-H), 9.71 (s, 1H, CHO).
5
3
.1.6.1.
2-Amino-7-hydroxy-4-[4-(2-pyrrolidin-1-ylethoxy)phenyl]-4H-chromeme-
-carboxylic methyl ester (32).
Preparation was as described above from 4-(2-pyrrolidin-1-ylethoxy)benzaldehyde (29) (10
mmol, 2.19 g), resorcinol (1.10 g, 10 mmol) and methyl cyanoacetate (10 mmol, 0.99 g),
-1
using general Method C. Yield 10%, M.p. 167°C, yellow crystals. IR νmax (KBr) cm :
-1
1
3
413.53, 3296.89 (NH ), 1701.9(C=O), 1656.4, 1610.6(C=C) cm . H NMR (400 MHz,
2
CDCl ): δ 1.74-1.77 (m, 4H, C-(CH ) -C), 2.64-2.67 (m, 4H, CH -N-CH ), 2.87-2.90 (t, 2H,
3
2 2
2
2
J=5.88 Hz, CH -N), 3.57 (s, 3H, O-CH ), 4.05-4.08 (t, 2H, J=5.90 Hz, CH -N), 4.83 (s, 1H,
2
3
2
CH), 6.50-6.51 (d, 2H, J=2.52 Hz, Ar-H), 6.54-6.57 (dd, 1H, J=2.36 Hz, 6.04 Hz, Ar-H),
2
5

6
.77-6.79 (d, 2H, J=8.76 Hz, Ar-H), 6.99-7.02 (m, 1H, Ar-H), 7.11-7.13 (d, 2H, J=8.52 Hz,
1
3
Ar-H). C NMR (151 MHz, DMSO-d )  161.5 (Ar-C), 156.9 (Ar-C), 149.4 (Ar-C), 141.0
6
(
Ar-C), 128.0 (Ar-CH), 117.6 (Ar-CH), 114.4 (Ar-CH), 112.4 (Ar-CH), 102.4 (Ar-CH), 77.0
-C-), 66.9 (NCH ), 54.7 (OCH ), 54.3 (NCH ), 40.37 (CH), 38.3 (CH ), 23.4 (CH ). HRMS:
(
2
3
2
2
2
+
Found 411.1927; C H N O requires 411.1920(M +H).
2
3
27
2
5
5
3
.1.6.2.
2-Amino-7-hydroxy-4-[4-(2-pyrrolidin-1-ylethoxy)phenyl]-4H-chromeme-
-carboxylic acid ethyl ester (33).
Preparation was as described above from 4-(2-pyrrolidin-1-ylethoxy)benzaldehyde (29) (10
mmol, 2.19 g), resorcinol (1.10 g, 10 mmol) and ethyl cyanoacetate (10 mmol, 1.13 g) using
Method B. Purification was achieved by flash column chromatography over silica gel (eluent:
dichloromethane:methanol; 9:1) to afford the product as a yellow gel, yield 10%. IR ʋmax
-1 1
(
film) 1671.15 (C=O) cm . H NMR (400 MHz, CDCl ): δ 1.15 (t, 3H, J=7.28 Hz, -CH ),
3
3
1
.76-1.90 (m, 4H, C-(CH ) -C), 2.03-2.07 (m, 4H, CH -N-CH ), 2.93-2.94 (t, 2H, J=5.78 Hz,
2 2 2 2
CH -N), 4.01-4.06 (q, 2H, J=7.02 Hz, CH -N), 4.22-4.24 (t, 2H, J=4.76 Hz, O-CH ), 4.80 (d,
2
2
2
1
8
2
1
H, CH), 5.12 (bs, 1H, OH), 6.47-6.49 (d, 2H, J=3 Hz, Ar-H), 6.85-6.90 (dd, 3H, J=5.04 Hz,
1
3
.78 Hz, Ar-H), 7.14 (d, 2H, J=9.04 Hz, Ar-H). C NMR (100 MHz, CDCl ): δ 13.09 (CH ),
3
3
2.14 (CH ), 38.40 (CH), 53.32 (CH ), 53.84 (CH ), 58.53 (CH ), 62.56 (CH ), 77.09 (C),
2
2
2
2
2
01.73 (Ar-CH), 111.44 (Ar-CH), 113.62 (Ar-CH), 116.96 (ArC), 127.76 (Ar-CH), 129.22
Ar-C), 131.38 (Ar-C), 129.88 (Ar-CH), 138.48 (Ar-C), 148.92 (Ar-C), 155.48 (Ar-C-O),
56.12 (Ar-C-O), 162.50 (Ar-C-N), 169.209 (C=O). HRMS: Found 425.2081; C H N O
5
(
1
2
4
29
2
+
requires 425.2076(M +H).
5
3
.1.6.3.
2-Amino-7-hydroxy-4-[4-(2-pyrrolidin-1-ylethoxy)phenyl]-4H-chromeme-
-carbonitrile (34).
Preparation following General Method B from 4-(2-pyrrolidin-1-ylethoxy)benzaldehyde (29)
1.3 mmol, 0.285 g), resorcinol (143 mg, 1.30 mmol) and malononitrile (1.3 mmol, 85.9 mg).
(
2
6

Yield 20.4%, M.p. 181°C, cream powder. IR ʋmax (KBr) 3439.6, 3360.1 (NH ), 2185.1 (CN)
2
-1 1
cm . H NMR (400 MHz, CDCl ): δ 1.74 (s, 4H, C-(CH ) -C), 2.64 (t, 4H, J=5.10 Hz, CH -
3
2 2
2
N-CH ), 2.80 (t, 2H, J=6.02 Hz, CH -N), 4.01 (t, 2H, J=6.04 Hz, CH -N), 4.55 (s, 1H, CH),
2
2
2
6
1
.10 (bs, 1H, OH), 6.38 (d, 1H, J=2.36 Hz, Ar-H), 6.46 (dd, 1H, J=2.56 Hz, Ar-H), 6.78 (d,
H, J=8.48 Hz, Ar-H), 6.85 (d, 2H, J=4.96 Hz, Ar-H), 7.13-7.15 (d, 2H, J=8.52 Hz). 13C
NMR (100 MHz, CDCl ): δ 23.12 (CH ), 26.45 (CH ), 38.15 (CH), 46.66 (CH ), 53.97
3
2
2
2
(
CH ), 54.35 (CH ), 56.52 (C), 66.63 (CH ), 102.09 (Ar-CH), 106.15 (CH), 112.35 (CH),
2 2 2
1
1
13.94 (C), 114.40 (CH), 120.75 (CH), 128.42 (CH), 129.88 (CH), 138.48 (C), 148.76 (C),
57.13 (C), 157.22 (C), 158.49 (C-O), 160.09 (C-N). HRMS: Found 378.1801; C H N O
2
2
24
3
3
+
requires 378.1818(M +H).
5
3
.1.6.4.
2-Amino-7-hydroxy-4-[4-(2-pyrrolidin-1-ylethoxy)phenyl]-4H-chromeme-
-carboxylic n-butyl ester (35).
Preparation following the general Method A from 4-(2-pyrrolidine-1-yl-ethoxy)-
benzaldehyde (29) (2.5 mmol, 0.547 g), resorcinol (2.5 mmol, 280 mg), and n-butyl
-1
cyanoacetate (2.5 mmol, 353 mg). Yield 14.2%, yellow powder. IR ʋmax (KBr) cm . 3407.44,
-1
1
3
294.68 (NH ), 1673.91 (C=O) cm . H NMR (400 MHz, DMSO-d ): δ 0.78 (t, 1H, J=7.54
2 6
Hz, CH ), 1.06 (t, 3H, J=3.52 Hz, CH ), 1.40 (m, 1H, CH ), 1.66 (bs, 4H, (CH ) ), 2.51 (d,
2
3
2
2 2
6
1
2
H, J=11.04 Hz, (CH ) ), 2.73 (t, 2H, J=5.76 Hz, N-CH ), 3.71 (m, 4H, 2 x O-CH ), 4.73 (s,
2 3 2 2
H, CH), 6.42 (d, 1H, J=2.17 Hz, Ar-H), 6.48 (dd, 1H, J=2.24 Hz, 6.04 Hz, Ar-H), 6.78 (d,
1
3
H, J=8.52 Hz, Ar-H), 6.99 (d, 1H, J=8.28 Hz, Ar-H), 7.03 (m, 2H, Ar-H). C NMR (100
MHz, DMSO-d ): δ 14.32 (CH ), 18.56 (CH ), 23.10 (CH ), 30.50 (CH ), 38.10 (CH), δ
6
3
2
2
2
5
1
3.95 (CH ), 54.30 (CH ), 54.32 (CH ), 58.52 (CH ), 62.20 (CH ), 66.50 (CH ), 76.81 (C),
3 2 2 2 2 2
01.97 (CH), 112.04 (CH), 113.98 (CH), 117.05 (C), 127.74 (CH), 127.86 (CH), 129.75
(CH), 140.83 (C), 148.85 (CH), 149.02 (C), 156.51 (C), 156.61 (C), 160.91 (C), 160.99 (C),
+
1
68.49 (C=O). HRMS: Found 453.2393; C H N O requires 453.2389(M +H).
2
6
33
2
5
2
7

5
3
.1.6.5.
2-Amino-7-hydroxy-4-[4-(2-pyrolidine-1-ylethoxy)phenyl]-4H-chromeme-
-carboxylic tert-butyl ester (36).
Preparation following general method
A
above from 4-(2-pyrrolidine-1-yl-
ethoxy)benzaldehyde (29) (2.5 mmol, 0.547 g), resorcinol (2.5 mmol, 280 mg) and tert-butyl
-1
cyanoacetate (2.5 mmol, 353 mg). Yield 63%, white powder. IR ʋmax (KBr) cm : 3407.03,
-
1 1
3
1
291.65 (NH ), 1672.28 (C=O) cm . H NMR (400 MHz, DMSO-d ): δ 1.36 (s, 9H, (CH ) ),
2 6 3 3
.78 (s, 4H, (CH ) ), 2.47 (bs, 4H, (CH ) ), 2.72 (t, 2H, J=6.04 Hz, CH ), 3.97 (t, 2H, J=5.78
2
2
2 2
2
Hz, CH ), 4.65 (s, 1H, CH), 6.39 (d, 1H, J=2.48 Hz, Ar-H), 6.44 (dd, 1H, J=2.26 Hz, 8.52
2
Hz, Ar-H), 6.77 (d, 2H, J=8.52 Hz, Ar-H), 6.93 (d, 1H, J=8.56 Hz, Ar-H), 7.01 (d, 2H,
1
3
J=8.52 Hz, Ar-H). 7.48 (s, 2H, NH ), 9.57 (s, 1H, OH). C NMR (100 MHz, DMSO-d ): δ
2
6
2
7
1
3.11 (CH ), 28.10 (CH ), 38.58 (CH), 53.99 (CH ), 54.34 (CH ), 56.03 (CH ), 66.59 (CH ),
2 3 2 2 2 2
7.86 (C), 78.01 (C), 101.90 (CH), 111.92 (CH), 113.90 (CH), 116.93 (C), 127.88 (CH),
29.82 (CH), 141.19 (C), 148.85 (C), 156.46 (C), 156.52 (C), 160.81 (C), 168.50 (C=O).
+
HRMS: Found 453.2393; C H N O requires 453.2389(M +H).
2
6
33
2
5
5
3
.1.6.6.
2-Amino-7-hydroxy-4-[4-(2-piperidin-1-ylethoxy)phenyl]-4H-chromeme-
-carboxylic methyl ester (37).
Preparation following the general method C above from 4-(2-piperidine-1-ylethoxy)-
benzaldehyde (30) (5 mmol, 1.16 g) and methyl cyanoacetate (5 mmol, 495 mg) and
resorcinol (5 mmol, 560 mg). Yield 25%, M.p. 180-181°C, yellow powder. IR ʋmax (KBr)
-1
-1
1
cm : 3400.00, 3299.51 (NH ), 1660.34 (C=O) cm . H NMR (400 MHz, DMSO-d ): δ
2
6
1
2
6
.35 (d, 2H, J=4.88 Hz, CH ), 1.46 (t, 4H, J=5.38 Hz, (CH ) ), 2.39 (bs, 4H, (CH ) ), 2.51 (t,
2 2 2 2 2
H, J=1.96 Hz, CH ), 3.51 (s, 3H, O-CH ), 3.96 (t, 2H, J=5.86 Hz, CH ), 4.75 (s, 1H, CH),
2
3
2
.44 (d, 1H, J=2.44 Hz, Ar-H), 6.48 (dd, 1H, J=2.20 Hz, 6.36 Hz, Ar-H), 6.78 (d, 2H, J=8.80
1
3
Hz, Ar-H), 6.99 (d, 1H, J=8.32 Hz, Ar-H), 7.03 (d, 2H, J=8.80 Hz, Ar-H). C NMR (100
MHz, DMSO-d ): δ 24.39 (CH ), 26.03 (CH ), 38.47 (CH), 50.86 (CH ), 54.85 (CH ), 57.89
6
2
2
3
2
2
8

(CH ), 65.85 (CH ), 77.07 (C), 102.50 (CH), 112.52 (CH), 114.58 (CH), 117.79 (C), 128.12
2 2
(CH), 130.07 (CH), 141.08 (C), 149.54 (C), 157.08 (C), 161.64 (C), 169.20 (C=O). HRMS:
+
Found 425.2082; C H N O requires 425.2076(M +H).
2
4
29
2
5
5
3
.1.6.7.
2-Amino-7-hydroxy-4-[4-(2-piperidin-1-ylethoxy)phenyl]-4H-chromeme-
-carboxylic ethyl ester (38).
(i) Following the method outlined above for 28, a solution of ethyl cyanoacetate (10 mmol,
1
.13 g) and 4-(2-piperidin-1-ylethoxy)benzaldehyde (30) (10 mmol, 2.33 g) in ethanol (30
mL) was heated briefly to reflux and then cooled. Piperidine (0.5 mL) was then added
dropwise and the reaction solution heated at reflux for 4 h. The solvent was removed in
vacuo to afford the product (E)-ethyl 2-cyano-3-[4-(2-piperidin-1-ylethoxy)phenyl]acrylate
4
5 which was recrystallized from ethanol as orange coloured crystals and used immediately
in the next reaction. Yield: 334 mg, 10.2%, M.p. 88-90 ˚C. IR ʋmax (KBr): 2213(CN),
-1 1
1
702(C=O), 1610(C=C) cm . H NMR (400 MHz, CDCl ): δ 8.06 (s, 1H, CH=C), 7.92 (d, J
3
=
9.16 Hz, 2H, ArH), 6.86 (d, J = 8.54 Hz, 2H, ArH), 4.34 (q, J = 7.12 Hz, 2H, OCH ), 3.45
2
1
3
(
br. s., 4H, 2xCH ), 1.69 (br. s., 6H, 3xCH ), 1.38 (t, J = 7.32 Hz, 3H, OCH CH ). C NMR
2 2 2 3
(
100 MHz, CDCl ): δ 14.26 (CH ), 24.31 (CH ), 25.32 (CH ), 48.10 (CH ), 61.90 (CH ),
3 3 2 2 2 2
9
4.64 (C=CH), 113.24 (CH), 117.38 (CN), 120.08 (C), 134.06 (CH), 154.01 (C), 154.21
(CH=C), 164.14 (C=O). (ii) Preparation following the general method B above from (E)-
ethyl 2-cyano-3-[4-(2-piperidin-1-ylethoxy)phenyl]acrylate 45 (10 mmol, 2.33 g) and
resorcinol (10 mmol, 1.13 g). Yield 20%, M.p. 176-178 °C, yellow powder. IR ʋmax (KBr)
-1
-1 1
cm : 3415.04, 3293.18 (NH ), 1672.17 (C=O) cm . H NMR (400 MHz, DMSO-d ): δ 1.04
2
6
(
m, 9H, CH , CH ), 1.98 (m, 4H, CH ), 2.78 (2H, CH ), 3.94 (t, 4H, J=6.13 Hz, CH ), 4.74
3 2 2 2 2
(s, 1H, CH), 6.44 (d, 1H, J=2.44 Hz, Ar-H), 6.48 (dd, 1H, J=2.44 Hz, 5.88 Hz, Ar-H), 6.69
(d, 2H, J=8.80 Hz, Ar-H), 6.88 (d, 1H, J=8.32 Hz, Ar-H), 7.02 (d, 2H, J=6.84 Hz, Ar-H),
1
3
7
.09 (d, 1H, J=8.80 Hz, Ar-H). C NMR (100 MHz, DMSO-d ): δ 14.32 (CH ), 23.92 (CH ),
6 3 2
2
9