Development of the β-lactam type molecular scaffold for selective estrogen receptor α modulator action: synthesis and cytotoxic effects in MCF-7 breast cancer cells

Article abstract of DOI:10.1080/14756366.2016.1210136

The estrogen receptors (ERα and ERβ) which are ligand inducible nuclear receptors are recognized as pharmaceutical targets for diseases such as osteoporosis and breast cancer. There is an increasing interest in the discovery of subtype Selective Estrogen Receptor Modulators (SERMs). A series of novel β-lactam compounds with estrogen receptor modulator properties have been synthesized. The antiproliferative effects of these compounds on human MCF-7 breast tumor cells are reported, together with binding affinity for the ERα and ERβ receptors. The most potent compound 15g demonstrated antiproliferative effects on MCF-7 breast tumor cells (IC₅₀=186 nM) and ERα binding (IC₅₀=4.3 nM) with 75-fold ERα/β receptor binding selectivity. The effect of positioning of the characteristic amine containing substituted aryl ring (on C-4 or N-1 of the β-lactam scaffold) on the antiproliferative activity and ER-binding properties of the β-lactam compounds is rationalized in a molecular modeling study.

Full text of DOI:10.1080/14756366.2016.1210136

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
Development of the β-lactam type molecular
scaffold for selective estrogen receptor α
modulator action: synthesis and cytotoxic effects
in MCF-7 breast cancer cells
Miriam Carr, Andrew J. S. Knox, David G. Lloyd, Daniela M. Zisterer & Mary J.
Meegan
To cite this article: Miriam Carr, Andrew J. S. Knox, David G. Lloyd, Daniela M. Zisterer & Mary
J. Meegan (2016): Development of the β-lactam type molecular scaffold for selective estrogen
receptor α modulator action: synthesis and cytotoxic effects in MCF-7 breast cancer cells,
Journal of Enzyme Inhibition and Medicinal Chemistry, DOI: 10.1080/14756366.2016.1210136
To link to this article: http://dx.doi.org/10.1080/14756366.2016.1210136
Published online: 01 Aug 2016.
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Date: 04 August 2016, At: 10:03

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ISSN: 1475-6366 (print), 1475-6374 (electronic)
J Enzyme Inhib Med Chem, Early Online: 1–14
2016 Informa UK Limited, trading as Taylor & Francis Group. DOI: 10.1080/14756366.2016.1210136
!
RESEARCH ARTICLE
Development of the b-lactam type molecular scaffold for selective
estrogen receptor a modulator action: synthesis and cytotoxic effects
in MCF-7 breast cancer cells
Miriam Carr1, Andrew J. S. Knox1,2, David G. Lloyd2, Daniela M. Zisterer2, and Mary J. Meegan1
1School of Pharmacy and Pharmaceutical Sciences, Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin, Ireland and
2School of Biochemistry and Immunology, Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin, Ireland
Abstract
Keywords
The estrogen receptors (ERa and ERb) which are ligand inducible nuclear receptors are
recognized as pharmaceutical targets for diseases such as osteoporosis and breast cancer.
There is an increasing interest in the discovery of subtype Selective Estrogen Receptor
Modulators (SERMs). A series of novel b-lactam compounds with estrogen receptor modulator
properties have been synthesized. The antiproliferative effects of these compounds on human
MCF-7 breast tumor cells are reported, together with binding affinity for the ERa and ERb
receptors. The most potent compound 15g demonstrated antiproliferative effects on MCF-7
breast tumor cells (IC50 ¼186 nM) and ERa binding (IC50 ¼4.3 nM) with 75-fold ERa/b receptor
binding selectivity. The effect of positioning of the characteristic amine containing substituted
aryl ring (on C-4 or N-1 of the b-lactam scaffold) on the antiproliferative activity and ER-binding
properties of the b-lactam compounds is rationalized in a molecular modeling study.
Antiproliferative activity, azetidin-2-one,
b-lactam, breast cancer, estrogen receptor
History
Received 8 March 2016
Revised 26 June 2016
Accepted 4 July 2016
Published online 28 July 2016
Introduction
potent as antiestrogens than tamoxifen and are most likely
contributors to the base antiproliferative activity observed with
tamoxifen10. The metabolite norendoxifen was shown to have dual
aromatase inhibitory and estrogen receptor modulatory activ-
ities11. Novel tamoxifen analogs that avoid CYPD6 metabolism
have been recently reported12. The SERM raloxifene (4, Figure 1),
a 2,3-disubstitued benzothiophene containing compound, is a
potent antiestrogen that binds to the ER with an affinity higher
than that of tamoxifen or 4-hydroxytamoxifen and equal to that of
estradiol13,14. Raloxifene was approved for the treatment of
osteoporosis in 1997 and has shown modest activity in ER-
positive breast cancer while lacking the increased risk for
endometrial cancer associated with the use of tamoxifen15.
Aromatase inhibitors such as the non-steroidal agent’s letrozole
and anastrozole are reported to be more efficacious than
tamoxifen as a first-line therapy, and are useful for second-line
therapy and against tamoxifen-resistant tumors16,17. The steroid
fulvestrant (5), Figure 1, is also effective as second line therapy
against advanced breast cancer in patients who develop resistance
to tamoxifen18. Due to its pure antiestrogen activity, it is devoid of
endometrial stimulation and therefore the risk of endometrial
cancer; similarly it does not possess the positive side effects on
the skeletal and cardiovascular systems of SERMs.
Breast cancer is by far the most frequent cancer among women
globally, with an estimated 1.67 million new cancer cases
diagnosed in 2012 ranking second overall (25% of all female
cancers), and accounting for 521 817 deaths1,2. Incidence rates
vary from 27 per 100 000 women in Middle Africa and Eastern
Asia to 96 per 100 000 women in Western Europe3. In Europe,
breast cancer is the most frequent cancer in women with 464 000
new diagnoses in 2012, which accounts for 29% of all new female
cancers in Europe. One in three people in Ireland will develop
cancer during their lifetime. Irish statistics note that breast cancer
now accounts for 32% of all cancers in women in Ireland, with
2942 new diagnoses in 20134.
Breast cancers are classified as estrogen receptor (ER) positive
or negative with 70–80% of all primary breast tumors being ER
positive, which is a less aggressive type than ER negative breast
cancer5. The first antiestrogen to show positive clinical results
was tamoxifen (1), (Figure 1), a synthetic non-steroidal antiestro-
gen, which was approved by the Food and Drug Administration in
1977 for the treatment of women with advanced breast cancer and
several years later for adjuvant treatment of primary cancer6,7
.
Tamoxifen is extensively metabolized by the human hepatic
cytochrome P450 enzyme system into several metabolites
including 4-hydroxytamoxifen (2) and 4-hydroxy-N-desmethylta-
moxifen (endoxifen), (3), Figure 18,9. They are ꢀ100-fold more
There are two types of ER assigned as ERa and ERb. A
comparison of the amino acid sequence of ERa and ERb shows
that they share the same functional domain architecture, and the
full length residues are ꢀ50% identical. The high homology
between the DNA binding domain (96%) suggests that ERa and
ERb are expected to bind various estrogen response elements
(EREs) with similar specificity and affinity and therefore interact
Address for correspondence: Mary J. Meegan, School of Pharmacy and
Pharmaceutical Sciences, Trinity Biomedical Sciences Institute, 152–160
Pearse St, Trinity College Dublin, Dublin 2, Ireland. Tel: +353-1-
8962798. E-mail: mmeegan@tcd.ie

2
M. Carr et al.
J Enzyme Inhib Med Chem, Early Online: 1–14
OH
R2
O
N
N
N
O
O
CH3
CH3
O
S
R1
OH
HO
HO
O
HO
N
(1) Tamoxifen R1 = H, R1 = CH3
(7)
(4) Raloxifene
(6) Lasofoxifene
(2) 4-Hydroxytamoxifen R1 = OH, R2 = CH3
(3) Endoxifen R1 =OH, R2 = H
OH
OCH3
OH
N N
N
N
N
H
N
HO
O
HO
(CH3)9SO(CH2)3CF2CF3
(5) Fulvestrant
(9)
(8)
N
OH
OH
O
OH
HO
S
HO
O
O
N
N
O
O
F
O
(10)
(11)
(12)
Figure 1. Structures of clinical SERMs and related ER antagonists.
with, and activate the same genes. However, because there is only
In a study to discover subtype selective ER scaffolds, we have
58% similarity between the ligand-binding domain, various identified a novel estrogen receptor modulator scaffold structure
estrogens may differentially bind to the two ERs19,20. ER subtypes containing the b-lactam ring as a potential scaffold for SERMs,
bind some ligands with different affinity; ligands may also and have reported the antiproliferative effects and ER-binding
demonstrate different agonist or antagonist character mediated by properties of a series of 1,4-diarylsubstituted azetidin-2-ones,
the two receptors21. SERMs such as 4-hydroxytamoxifen (2) and where the required basic amine function was positioned on the
raloxifene (4) act as partial agonists on Era, but they exert benzylic substituent at the C-3 position32,40. We now report the
exclusively antagonistic activity on Erb22.
further investigation of this novel heterocyclic core scaffold
Due to the increased risk of endometrial cancer with the use of structure as ER subtype selective ligands and the subsequent
tamoxifen many different fixed ring structures have been synthesis of a number of structurally varied b-lactam compounds,
developed as potential SERMs to prevent E/Z isomerization of which are substituted at N-1, C-3 and C-4 with the required aryl
the triarylethylene structure23. Some of these are illustrated in rings. We have evaluated the antiproliferative activity of these
Figure 1 and include the tetrahydronaphthalene lasofoxifene (6)24, products in ER-positive MCF-7 human breast tumor cells and also
spiroindene (7)25, pyrazole (8)26, quinoline (9)27, benzopyran in ER-negative MDA-MB-231 cells and have determined their
(10)28, benzoxathiin (11)29 and benzoxepin ring structures (12)30. relative binding affinities for ERa and ERb. The two main
The recently reported benzopyranobenzoxepanes were identified b-lactam structural types now reported contain the important
as potent SERMs for the treatment of postmenopausal basic side-chain substituent positioned on the phenyl ring at the
symptoms31.
C4-position (type I) and at the N1-position (type II) as these were
We are specifically interested in the development of novel the optimal positions for substitution indicated from initial
heterocyclic ring scaffolds as ER antagonists32. Natural and molecular modeling and docking studies. The general features
synthetic azetidinone derivatives occupy a central place among of the b-lactam target scaffold structures selected for synthesis are
medicinally important compounds due to their diverse and illustrated in Figure 2.
interesting biological activities33. Their importance is no longer
exclusive due to the extensive clinical use of the b-lactam Experimental section
antibiotics but also because of their potential as intermediates in
the synthesis of other types of compounds of biological interest.
Chemistry
The antitumour properties of b-lactams have previously been All reagents were commercially available and were used without
reported32,34–36 together with inhibitory activity against serine further purification unless otherwise indicated. Tetrahydrofuran
proteases such as prostate-specific antigen (PSA)37, and other (THF) was distilled immediately prior to use from
serine proteases such as tryptase38 and human leukocyte elastase Na/Benzophenone under a slight positive pressure of nitrogen,
(HLE)39.
toluene was dried by distillation from sodium and stored on

DOI: 10.1080/14756366.2016.1210136
Development of ꢂ-lactam type molecular scaffold
3
O
R1
O
N
N
A
C
A
C
O
N
4
3
O
N
N
A
O
B
B
C
B
R2
R1
Type II β-lactam
R2
Type I β-lactam
Tamoxifen (1)
Figure 2. Structure of tamoxifen (1) and Type I and Type II b-lactam ER antagonists.
˚
activated molecular sieves (4 A) and dichloromethane was dried and 4-methoxyaniline (0.1 mol). Colorless crystals, m.p.
1
by distillation from calcium hydride prior to use. Uncorrected 148 ꢁC, (88%). IR ꢁmax (KBr) cmꢃ 1: 1605.9 cmꢃ 1 (C¼N). H
melting points were measured on a Gallenkamp apparatus. NMR (CDCl3): ꢀ 3.84 (s, 3H, OCH3), 5.14 (s, 2H, OCH2), 6.93 (d,
Infrared (IR) spectra were recorded as thin film on NaCl plates, 2H, J ¼ 9.0 Hz, Ar-H), 7.08 (d, 2H, J ¼ 8.5 Hz, Ar-H), 7.24 (d,
or as potassium bromide discs on a Perkin Elmer FT-IR Spectrum 2H, J ¼ 8.5 Hz, Ar-H), 7.41–7.46 (m, 5H, Ar-H), 7.84 (d, 2H,
100 spectrometer (Perkin Elmer, Waltham, MA). 1H, 13C and 19
F
J ¼ 8.5 Hz, Ar-H), 8.42 (s, 1H, CH¼N). 13C NMR (CDCl3): ꢀ
nuclear magnetic resonance (NMR) spectra were recorded at 57.89, 69.63, 113.91, 114.61, 121.64, 127.06, 127.69, 128.21,
27 ꢁC on a Bruker Avance DPX 400 spectrometer (Bruker, 129.13, 129.86, 136.01, 144.70, 157.52, 157.60, 160.71. HRMS:
1
Billerica, MA) (400.13 MHz, H; 100.61 MHz, 13C; 376.47 MHz, Found 318.1490 (M++H); C21H20NO2 requires 318.1494.
19F) at 20 ꢁC in either CDCl3 (internal standard tetramethylsilane
(TMS)) or CD3OD by Dr. John O’Brien and Dr. Manuel Ruether as above from 4-benzyloxyaniline (0.1 mol) and 4-hydroxyben-
4-[(4-Benzyloxyphenylimino)methyl]phenol (13e). Preparation
in the School of Chemistry, Trinity College Dublin. For CDCl3, zaldehdye (0.1 mol). Yellow crystals, m.p. 214 ꢁC, (96%). IR ꢁmax
1H-NMR spectra were assigned relative to the TMS peak at 0.00 ꢀ (KBr) cmꢃ 1: 1607.9 cmꢃ 1 (C¼N). H NMR (DMSO-d6): ꢀ 5.07
1
and 13C-NMR spectra were assigned relative to the middle CDCl3 (s, 2H, O–CH2), 6.86 (d, 2H, J ¼ 8.5 Hz, Ar-H)), 7.00 (d, 2H,
1
triplet at 77.00 ppm. For CD3OD, H and 13C-NMR spectra were J ¼ 8.6 Hz, Ar-H), 7.19 (d, 2H, J ¼ 8.5 Hz, Ar-H), 7.30–7.44 (m,
assigned relative to the center peaks of the CD3OD multiplets at 5H, Ar-H), 7.71 (d, 2H, J ¼ 8.5 Hz, Ar-H), 8.43 (s, 1H, CH¼N).
3.30 d and 49.00 ppm, respectively. 19F-NMR spectra were not HRMS: Found 304.1336 (M++H); C20H18NO2 requires 304.1338.
calibrated. Electrospray ionization mass spectrometry (ESI-MS)
4-[(4-Benzyloxybenzylidene)amino]phenol (13 h). Preparation
was performed in the positive ion mode on a liquid chromatog- was as above from 4-benzyloxybenzaldehyde (0.1 mol) and 4-
raphy time-of-flight (TOF) mass spectrometer (Micromass LCT, aminophenol (0.1 mol). Pale green crystals, Mp. 208 ꢁC, (96%).
Waters Ltd., Manchester, UK) equipped with electrospray IR ꢁmax (KBr) cmꢃ 1: 1606.4 cmꢃ 1 (C¼N), 3437.8 cmꢃ 1 (OH).
ionization (ES) interface operated in the positive ion mode at 1H NMR (CD3OD): ꢀ 5.14 (s, 2H, OCH2), 6.86 (d, 2H, J ¼ 8.6 Hz,
the High Resolution Mass Spectrometry Laboratory by Dr. Martin Ar-H), 7.06 (d, 2H, J ¼ 9.0 Hz, Ar-H), 7.14 (m, 2H, Ar–H), 7.34–
Feeney in the School of Chemistry, Trinity College. Mass 7.46 (m, 5H, Ar-H), 7.84 (d, 2H, J ¼ 9.0 Hz, Ar-H), 8.40 (s, 1H,
measurement accuracies of5 5 ppm were obtained. Low reso- CH¼N). 13C NMR (CD3OD): ꢀ 69.25, 114.34, 114.91, 121.36,
lution mass spectra (LRMS) were acquired on a Hewlett-Packard 126.78, 127.16, 127.70, 129.58, 136.11, 144.25, 153.94, 155.53,
5973 MSD GC-MS system (Hewlett-Packard, Palo Alto, CA) in 158.11. HRMS: Found 304.1328 (M++H); C20H18NO2 requires
electron impact (EI) mode. Rf values are quoted for thin layer 304.1338.
chromatography (TLC) on silica gel Merck F-254 plates, unless
General procedure for alkylation of phenols: preparation of
otherwise stated. Flash column chromatography was carried out 13f–g, 13i and 15a. The appropriate phenol (10 mmol) was
on Merck Kieselgel 60 (particle size 0.040–0.063 mm), Aldrich dissolved in dry acetone (100 mL). Anhydrous potassium carbon-
aluminum oxide, (activated, neutral, Brockmann I, 50 mesh) or ate (0.16 mol, 22 g) was then added and the mixture was
Aldrich aluminum oxide, (activated, acidic, Brockmann I, 50 stirred gently for 10 min under
a N2 atmosphere. 1-(2-
mesh). All products isolated were homogenous on TLC. Chloroethyl)pyrrolidine hydrochloride (40 mmol, 5.78 g) was
Analytical high-performance liquid chromatography (HPLC) to then added and the reaction was refluxed until reaction was
determine the purity of the final compounds was performed using complete when monitored by TLC. On completion, the solution
a Waters 2487 Dual Wavelength Absorbance detector, a Waters was filtered and the solvent was removed under reduced pressure.
1525 binary HPLC pump, a Waters In-Line Degasser AF and a
4-Fluorophenyl-[4-(2-pyrrolidin-1-ylethoxy)benzylidene]amine
Waters 717plus Autosampler (Waters Corporation, Milford, MA). (13f). Preparation was as above from 13d (5mmol, 1.076 g). Brown
The column used was a Varian Pursuit XRs C18 reverse phase oil, (37%). IR ꢁmax (film) cmꢃ 1: 1624.5cmꢃ 1 (CH¼N). 1H NMR
150 ꢂ 4.6 mm chromatography column (Agilent, Santa Clara, (CDCl3): ꢀ 1.80–1.84 (m, 4H, –CH2–CH2–), 2.63–2.66 (m, 4H,
CA). Samples were detected using a wavelength of 254 nm. All CH2–N–CH2), 2.95 (t, 2H, J ¼ 5.8 Hz, N–CH2), 4.19 (t, 2H,
samples were analyzed using acetonitrile (70%): water (30%) over J ¼ 6.0 Hz, OCH2), 7.01 (d, 2H, J ¼ 9.0Hz, Ar-H), 7.04 (d, 2H,
10 min and a flow rate of 1 mL/min. Schiff bases 13a and 13d J ¼ 8.5 Hz, Ar-H), 7.15 (m, 2H, Ar-H), 7.83 (d, 2H, J ¼8.5 Hz, Ar-
were prepared as previously reported41.
General preparation of Schiff bases 13c, 13e, 13 h. A solution 54.49, 66.82, 114.34, 115.21, 115.44, 121.71, 128.59, 129.99,
of the appropriately substituted aryl aldehyde (0.1 mol) and the 147.89, 159.11, 161.14, 161.73. HRMS: Found 313.1709 (M++H);
appropriately substituted aryl amine (0.1 mol) in ethanol (50 mL) C19H23N2OF requires 313.1716.
H), 8.36 (s, 1H, –CH¼N). 13C NMR (CDCl3): ꢀ 23.08, 54.32,
was heated to reflux for 3 h. The reaction mixture was reduced to
4-Benzyloxyphenyl-[4-(2-pyrrolidin-1-ylethoxy)benzylidene]
25 mL under vacuum, the Schiff base product crystallized from amine (13 g). Preparation was as above from 13e (0.02 mol,
the solution. The crude product was then recrystallized from 6.067 g). Orange oil (60%). IR ꢁmax (film) cmꢃ 1: 1622.3 cmꢃ 1
ethanol to afford the purified product.
(4-Benzyloxybenzylidene)-(4-methoxyphenyl)amine
Preparation as above from 4-benzyloxybenzaldehyde (0.1 mol) (t, 2H, J ¼ 5.8 Hz, CH2–N), 5.01 (s, 2H, O–CH2), 6.94 (d, 4H,
(CH¼N). 1H NMR (CDCl3): ꢀ 1.75 (s, br, 4H, –CH2–CH2–), 2.59
(13c). (s, br, 4H, –CH2–N–CH2–), 2.86 (t, 2H, J ¼ 5.8 Hz, CH2–N), 4.12

4
M. Carr et al.
J Enzyme Inhib Med Chem, Early Online: 1–14
J ¼ 9.1 Hz, Ar-H), 7.15 (d, 2H, J ¼ 9.4 Hz, Ar-H), 7.36 (m, 3H, (6 mmol, 0.79 mL). Brown oil, (78%). This compound was used
Ar-H), 7.39 (d, 2H, J ¼ 8.5 Hz, Ar-H), 7.80 (d, 2H, J ¼ 8.5 Hz, Ar- without further purification in the next experiment. IR ꢁmax (film)
1
H), 8.35 (s, 1H, –CH¼N). 13C NMR (CDCl3): ꢀ 23.03, 54.28, cmꢃ 1: 1750.1 cmꢃ 1 (C¼O, b-lactam). H NMR (CDCl3): ꢀ 0.28
54.66, 66.79, 69.63, 114.59, 114.61, 121.60, 127.06, 128.21, (s, 6H, Si–(CH3)2), 1.03 (s, 9H, Si–C–(CH3)3), 3.70 (s, 3H, O–
129.24, 129.80, 136.03, 144.82, 156.78, 157.40. HRMS: Found CH3), 4.21 (d, 1H, J ¼ 2.0 Hz, H-3), 4.97 (d, 2H, J ¼ 2.0 Hz, H-4),
401.2216 (M++H); C26H29N2O2 requires 401.2229.
4-Benzyloxybenzylidene-[4-(2-pyrrolidin-1-ylethoxy)phenyl]
6.97–7.42 (m, 13H, Ar-H).
4-[4-Benzyloxyphenyl)-1-(4-methoxyphenyl)-3-phenylazetidin-
amine (13i). Preparation was as above from 13h (5 mmol, 2-one (14b). Preparation was as above from 13c (0.02 mol,
1.517 g). Orange solid, (60%), m.p. 118 ꢁC. IR ꢁmax (KBr) 6.347 g) and phenylacetyl chloride (0.02 mol, 2.63 mL). Brown
cmꢃ 1: 1621.6 cmꢃ 1 (CH¼N). 1H NMR (CDCl3): ꢀ 1.81–1.84 (m, solid, (78%). This compound was used without further purifica-
4H, –CH2–CH2–), 2.64–2.67 (m, 4H, CH2–N–CH2), 2.94 (t, 2H, tion in the next experiment. IR ꢁmax (KBr) cmꢃ 1: 1735.2 cmꢃ 1
J ¼ 5.8 Hz, CH2–N), 4.15 (t, 2H, J ¼ 5.8 Hz, CH2–N), 5.13 (s, 2H, (C¼O, b-lactam). 1H NMR (CDCl3): ꢀ 3.69 (s, 3H, O-CH3), 4.19
O–CH2), 6.95 (d, 2H, J ¼ 9.0 Hz, Ar-H), 7.04 (d, 2H, J ¼ 9.0 Hz, (d, 1H, J ¼ 2.5 Hz, H-3), 4.82 (d, 1H, J ¼ 2.5 Hz, H-4), 6.74–6.77
Ar-H), 7.20 (d, 2H, J ¼ 9.04 Hz, Ar-H), 7.34–7.46 (m, 5H, Ar-H), (m, 2H, Ar-H), 6.93–6.99 (m, 2H, Ar-H), 7.15–7.28 (m, 9H, Ar-
7.81 (d, 2H, J ¼ 6.52 Hz, Ar-H), 8.40 (s, 1H, CH¼N). 13C NMR H), 7.30–7.36 (m, 5H, Ar-H). 13C NMR (CDCl3): ꢀ 55.02, 63.10,
(CDCl3): ꢀ 23.03, 54.28, 54.66, 66.79, 69.63, 114.59, 114.61, 64.76, 69.67, 113.90, 113.93, 114.62, 115.10, 118.15, 126.62,
121.60, 127.06, 128.21, 129.24, 129.80, 136.03, 144.82, 156.78, 127.69, 127.06, 127.69, 127.93, 129.25, 129.90, 130.60, 134.50,
157.40. HRMS: Found 401.2216 (M++H); C26H29N2O2 requires 136.25, 155.67, 157.57, 164.82.
401.2229. 1-(4-Fluorophenyl)-3-phenyl)-4-[4-(2-pyrrolidin-1-ylethoxy)-
1-(4-Methoxyphenyl)-3-phenyl-4-[4-(2-pyrrolidin-1-ylethoxy)- phenyl]azetidin-2-one (15b). Preparation was as above from 13f
phenyl]azetidin-2-one (15a).
(2 mmol, 0.624 g). Orange oil, (23%). IR ꢁmax (film) cmꢃ 1
:
1
Preparation was as above from 14c (0.4 mmol, 0.138 g). 1751.2 cmꢃ 1 (C¼O, b-lactam). H NMR (CDCl3): ꢀ 1.82 (s, br,
Yellow oil, (30%), IR ꢁmax (film) cmꢃ 1: 1747.4 cmꢃ 1 (C¼O, 4H, –CH2–CH2–), 2.66 (s, br, 4H, –CH2–N–CH2–), 2.91 (t, 2H,
1
b-lactam). H NMR (CDCl3): ꢀ 1.82–1.84 (m, 4H, –CH2–CH2– J ¼ 5.8 Hz, CH2–N), 4.14 (t, 2H, J ¼ 5.8 Hz, CH2–C), 4.26 (d, 1H,
), 2.67 (s, br, 4H, CH2–N–CH2), 2.91 (t, 2H, J ¼ 6.0 Hz, N– J ¼ 2.0 Hz, H-3), 4.87 (d, 1H, J ¼ 2.0 Hz, H-4), 6.93 (2xd
CH2–C), 3.76 (s, 3H, OCH3), 4.12 (t, 2H, J ¼ 5.8 Hz, OCH2), overlapping, 4H, J ¼ 7.3 Hz, Ar-H), 7.26–7.39 (m, 9H, Ar-H).
4.24 (d, 1H, J ¼ 2.0 Hz, H-3), 4.86 (d, 1H, J ¼ 2.0 Hz, H-4), 13C NMR (CDCl3): ꢀ 23.01, 54.23, 54.49, 63.20, 64.91, 65.54,
6.81 (d, 2H, J ¼ 9.0 Hz, Ar-H), 6.98 (d, 2H, J ¼ 8.5 Hz, Ar-H), 114.89, 115.31, 118.16, 126.80, 126.97, 127.46, 128.60, 133.27,
7.28–7.4 (m, 9H, Ar-H). 13C NMR (CDCl3): ꢀ 23.01, 53.75, 133.30, 134.20, 157.42, 158.76, 165.08. HRMS: Found 431.2131
54.26, 54.99, 63.10, 64.74, 66.57, 113.85, 114.79, 118.09, (M++H); C27H28FN2O2 requires 431.2135.
126.81, 127.02, 127.35, 128.54, 129.02, 130.61, 134.50, 155.61,
1-(4-Fluorophenyl)-3-(4-methoxyphenyl)-4-[4-(2-pyrrolidin-1-
164.75. HRMS: Found 443.2333 (M++H); C28H31N2O3 ylethoxy) phenyl]azetidin-2-one (15c). Preparation was as above
requires 443.2335.
[4-(tert-Butyldimethylsilanyloxy)benzylidene]-(4-methoxyphenyl)
from 13f (2 mmol, 0.624 g). Orange oil, (21%). IR ꢁmax (film)
cmꢃ 1: 1747.8 cmꢃ 1 (C¼O, b-lactam). 1H NMR (CDCl3): ꢀ
amine (13b). To a suspension of the phenol 13a (0.02mol, 4.54g) 1.84 (s, br, 4H, –CH2–CH2–), 2.77 (m, 4H, –CH2–N–CH2–),
and dimethyl-tert-butylchlorosilane (0.024 mol) in dry dichloro- 2.99 (t, 2H, J ¼ 5.5 Hz, CH2–N), 3.79 (s, 3H, O–CH3), 4.16 (t,
methane (60 mL) was added 1,8-diazobicyclo[5.4.0] undec-7-ene 2H, J ¼ 5.5 Hz, CH2–C), 4.20 (d, 1H, J¼ 2.5 Hz, H-3), 4.82 (d,
(DBU) (0.032 mol). The resulting mixture was stirred at room 1H, J ¼ 2.5 Hz, H-4), 6.89–6.97 (m, 6H, Ar-H), 7.20–7.31 (m,
temperature until complete as monitored on TLC. The solution was 6H, Ar-H). 13C NMR (CDCl3): ꢀ 22.90, 53.99, 54.16, 54.88,
then diluted with dichloromethane (80 mL) and washed with water 63.53, 64.37, 65.90, 113.99, 114.85, 115.52, 118.16, 126.19,
(60 mL), 0.1M HCl (60 mL) and finally with saturated aqueous 128.14, 133.31, 158.46, 158.82, 159.81, 165.52 (C¼O, C2).
NaHCO3 (60 mL). The organic layer was dried using anhydrous HRMS: Found 461.2218 (M++H); C28H30FN2O3 requires
sodium sulfate and the solvent removed by evaporation under 461.2218.
reduced pressure to afford the product as orange crystals, (58%), m.p.
1-(4-Benzyloxyphenyl)-3-phenyl-4-[4-(2-pyrrolidin-1-ylethox-
210 ꢁC. This product was used in the following reactions without y)phenyl] azetidin-2-one (15d). Preparation was as above from
further purification. IR ꢁmax (KBr) cmꢃ 1: 1605.3 cmꢃ 1 (CH¼N). 13 g (3 mmol, 1.202 g). Orange oil, (61%), IR ꢁmax (film) cmꢃ 1
:
1H NMR (CDCl3): ꢀ 0.28 (s, 6H, Si-(CH3)2), 1.04 (s, 9H, Si-C- 1744.9 cmꢃ 1 (C¼O, b-lactam). H NMR (CDCl3): ꢀ 1.86 (s, br,
(CH3)3), 3.77 (s, 3H, O-CH3), 6.94 (d, 4H, J¼ 8.9 Hz, Ar-H), 7.22 4H, –CH2–CH2–), 2.77 (s, br, 4H, –CH2–N–CH2–), 2.99–3.04 (m,
(d, 2H, J¼ 8.9 Hz, Ar-H), 7.83 (d, 2H, J¼ 8.9 Hz, Ar-H), 8.38 (s, 1H, 2H, CH2–N), 4.19–4.21 (m, 2H, CH2–C), 4.18 (d, 1H, J ¼ 2.5 Hz,
–CH¼N). 13C NMR (CDCl3): ꢀ – 4.80, 17.82, 25.38, 54.79, 113.89, H-3), 4.84 (d, 1H, J ¼ 2.5 Hz, H-4), 5.06 (s, 2H, CH2), 6.86 (d,
1
119.95, 121.72, 129.72, 144.66, 157.24, 157.59, 157.99.
2H, J ¼ 9.0 Hz, Ar-H), 6.96–7.01 (m, 4H, Ar-H), 7.18 (d, 2H,
General preparation of 3-substituted azetidin-2-ones 14a, 14b, J ¼ 8.5 Hz, Ar-H), 7.27–7.44 (m, 10H, Ar-H). 13C NMR (CDCl3):
15b-e, 15 h–i. A solution consisting of the appropriate acetyl ꢀ 22.89, 53.71, 54.12, 63.07, 64.69, 66.34, 69.79, 114.60, 115.08,
chloride (7.5 mmol, 0.99 mL) in dry dichloromethane (50 mL) 118.10, 126.85, 127.02, 128.55, 132.10, 134.45, 136.21, 158.64,
was added dropwise to a stirring solution containing the 165.78. HRMS: Found 519.2648 (M++H); C34H35N2O3 requires
appropriate imine (5 mmol) and triethylamine (15 mmol, 519.2648.
2.091 mL) in dry dichloromethane (50 mL) at reflux. The solution
1-(4-Benzyloxyphenyl)-3-(4-methoxyphenyl)-4-[4-(2-pyrroli-
was heated at reflux for 10 h and then cooled, washed with din-1-ylethoxy) phenyl] azetidin-2-one (15e). Preparation was as
saturated sodium bicarbonate solution (50 mL), dilute HCl (10%, above from 13 g (3 mmol, 1.202 g). Brown oil, (33%). IR ꢁmax
50 mL) and brine (50 mL). The organic layer was dried and the (film) cmꢃ 1: 1744.2 cmꢃ 1 (C¼O, b-lactam). 1H NMR (CDCl3): ꢀ
solvent was evaporated in vacuo. The product was obtained by 1.80 (s, br, 4H, –CH2–CH2–), 2.65 (s, br, 4H, –CH2–N–CH2–),
column chromatography as required over silica gel (eluent: 2.89 (t, 2H, J ¼ 5.8 Hz, CH2–N), 3.76 (s, 3H, O–CH3), 4.11 (t, 2H,
dicholoromethane).
J ¼ 5.8 Hz, CH2–C), 4.16 (d, 1H, J ¼ 2.0 Hz, H-3), 4.78 (d, 1H,
J ¼ 2.0 Hz, H-4), 4.96 (s, 2H, CH2), 6.84–6.92 (m, 6H, Ar-H),
4-[4-tert-Butyldimethylsilanyloxy)phenyl]-1-(4-methoxyphe-
nyl)-3-phenylazetidin-2-one (14a). Preparation was as above 7.23 (d, 2H, J ¼ 8.5 Hz, Ar-H), 7.26–7.38 (m, 9H, Ar-H). 13C
from 13b (6 mmol, 2.046 g) and phenylacetyl chloride NMR (CDCl3): ꢀ 22.97, 54.17, 54.41, 54.88, 63.47, 64.23, 66.29,

DOI: 10.1080/14756366.2016.1210136
Development of ꢂ-lactam type molecular scaffold
5
69.77, 113.97, 114.79, 114.90, 118.12, 126.52, 126.82, 127.02, 135.13, 155.66, 157.30, 164.34. HRMS: Found 368.1260
127.56, 128.15, 128.20, 129.11, 130.85, 136.40, 154.80, 158.53, (M++Na); C22H19NO3Na requires 368.1263.
158.76, 165.28. HRMS: Found 549.2777 (M++H); C35H37N2O4
requires 549.2753.
4-(4-Benzyloxyphenyl)-3-phenyl-1-[4-(2-pyrrolidin-1-ylethox-
1-(4-Hydroxyphenyl)-3-phenyl)-4-[4-(2-pyrrolidin-1-ylethox-
y)phenyl]azetidin-2-one (15f). Preparation was as above from 15d
(1 mmol, 0.518 g). Yellow oil, (50%). IR ꢁmax (film) cmꢃ 1
:
1
y)phenyl] azetidin-2-one (15 h). Preparation was as above from 13i 1738.82 cmꢃ 1 (C¼O, b-lactam), 3434.80 cmꢃ 1 (OH). H NMR
(5 mmol, 2.088 g). Orange oil, (14%). IR ꢁmax (film) cmꢃ 1
:
(CDCl3): ꢀ 1.89 (s, br, 4H, –CH2–CH2–), 2.83 (s, br, 4H, –CH2–
1746.6 cmꢃ 1 (C¼O, b-lactam). 1H NMR (CDCl3): ꢀ 1.81 (m, 4H, N–CH2–), 3.14-3.16 (m, 2H, CH2–N), 4.20 (d, 1H, J ¼ 2.2 Hz, H-
–CH2–CH2–), 2.66 (m, 4H, –CH2–N–CH2–), 2.89 (t, 2H, 3), 4.23–4.29 (m, 2H, CH2–C), 4.83 (d, 1H, J ¼ 2.2 Hz, H-4), 6.71
J ¼ 5.8 Hz, CH2–N), 4.05 (t, 2H, J ¼ 5.8 Hz, CH2–C), 4.22 (d, (d, 2H, J ¼ 9.0 Hz, Ar-H), 6.87 (d, 2H J ¼ 8.5 Hz Ar-H), 7.01 (d,
1H, J ¼ 2.5 Hz, H-3), 4.85 (d, 1H, J ¼ 2.5 Hz, H-4), 6.80 (m, 2H, 2H, J ¼ 8.5 Hz Ar-H), 7.19 (d, 2H, J ¼ 9.0 Hz, Ar-H), 7.26–7.39
Ar-H), 7.00 (d, 2H, J ¼ 9.0 Hz, Ar-H), 7.41–7.48 (m, 14H, Ar-H). (m, 5H, Ar-H). 13C NMR (CDCl3): ꢀ 22.83, 54.13, 54.22, 63.01,
13C NMR (CDCl3): ꢀ 22.91, 54.02, 63.07, 64.75, 65.01, 69.66, 64.59, 66.09, 114.40, 115.63, 118.35, 126.91, 127.03, 128.57,
114.60, 115.08, 118.10, 126.85, 128.55, 132.10, 134.45, 136.21, 129.51, 129.62, 131.58, 134.42, 153.18, 157.99, 164.75. HRMS:
158.64, 164.79. HRMS: Found 519.2641 (M++H); C34H35N2O3 Found 429.2184 (M++H); C27H29N2O3 requires 429.2178.
requires 519.2648.
4-(4-Benzyloxyphenyl)-3-(4-methoxyphenyl)-1-[4-(2-pyrroli-
1-(4-Hydroxyphenyl)-3-(4-methoxyphenyl)-4-[4-(2-pyrrolidin-
1-ylethoxy) phenyl]azetidin-2-one (15 g). Preparation was as
din-1-ylethoxy)phenyl]azetidin-2-one (15i). Preparation was as above from 15e (0.7 mmol, 0.384 g). Yellow oil, (78%). IR ꢁmax
above from 13i (3 mmol, 1.253 g). Brown oil, (10%). IR ꢁmax (film) cmꢃ 1: 1737.6 cmꢃ 1 (C¼O, b-lactam), 3387.0 cmꢃ 1 (OH).
(film) cmꢃ 1: 1741.9 cmꢃ 1 (C¼O, b-lactam). 1H NMR (CDCl3): ꢀ 1H NMR (CDCl3): ꢀ 1.85 (s, br, 4H, –CH2–CH2–), 2.84 (s, br, 4H,
1.82 (s, br, 4H, –CH2–CH2–), 2.69 (s, br, 4H, –CH2–N–CH2–), –CH2–N–CH2–), 3.04 (t, 2H, J ¼ 5.3 Hz, CH2–N), 3.77 (s, 3H, O–
2.92 (t, 2H, J ¼ 5.4 Hz, CH2–N), 3.80 (s, 3H, OCH3), 4.05 (t, 2H, CH3), 4.11 (d, 1H, J ¼ 2.0 Hz, H-3), 4.12 (t, 2H, J ¼ 5.5 Hz, CH2–
J ¼ 5.8 Hz, CH2–C), 4.17 (d, 1H, J ¼ 2.0 Hz, H-3), 4.79 (d, 1H, C), 4.75 (d, 1H, J ¼ 2.0 Hz, H-4), 4.78 (bs, 1H, OH), 6.69 (d, 2H,
J ¼ 2.0 Hz, H-4), 5.05 (s, 2H, O-CH2), 6.81 (d, 2H, J ¼ 9.2 Hz, Ar- J ¼ 8.6 Hz, Ar-H), 6.82 (2 ꢂ d, overlapping, 4H, J ¼ 8.8 Hz, Ar-
H), 6.91 (d, 2H, J ¼ 8.8 Hz, Ar-H), 6.99 (d, 2H, J ¼ 8.4 Hz, Ar-H), H), 7.14-7.20 (m, 6H, Ar-H). 13C NMR (CDCl3): ꢀ 22.75, 54.03,
7.23 (d, 2H, J ¼ 8.0 Hz, Ar-H), 7.28 (d, 2H, J ¼ 8.0 Hz, Ar-H), 54.12, 54.88, 63.41, 63.96, 65.11, 113.95, 114.69, 115.55, 118.36,
7.39 (d, 2H, J ¼ 7.6 Hz, Ar-H), 7.40–7.44 (m, 5H, Ar-H). 13C 126.42, 126.86, 128.18, 129.38, 153.34, 158.01, 158.72, 165.35.
NMR (CDCl3): ꢀ 23.36, 54.51, 54.77, 55.31, 63.85, 64.66, 70.06, HRMS: Found 459.2276 (M++H); C28H31N2O4 requires
114.36, 115.01, 115.46, 118.49, 126.96, 127.22, 127.48, 128.08, 459.2284.
128.60, 129.72, 130.24, 131.22, 136.65, 155.08, 159.00, 159.17,
165.59. HRMS: Found 549.2764 (M++H); C35H37N2O4 requires ylethoxy)-phenyl] azetidin-2-one (15j). Preparation was as above
549.2753. from 15 h (0.11 mmol, 0.057 g). Yellow oil, (37%). IR ꢁmax (film)
4-(4-Hydroxyphenyl)-3-phenyl)-1-[4-(2-pyrrolidin-1-
4-(4-Hydroxyphenyl)-1-(4-methoxyphenyl)-3-phenylazetidin-2- cmꢃ 1: 1745.1 cmꢃ 1 (C¼O, b-lactam), 3437.5 cmꢃ 1 (OH). 1H
one (14c). To a suspension of the protected phenol 14a (10 mmol) NMR (CDCl3): ꢀ 1.88 (s, br, 4H, –CH2–CH2–), 2.95 (s, br, 4H,
in THF (50 ml) was added tetrabutylammonium fluoride (1M, 1.5 –CH2–N–CH2–), 3.11 (bs, 2H, CH2–N), 4.07 (bs, 2H, O–CH2),
equivalents). The solution was stirred in an ice bath for 15 min to 4.21 (d, 1H, J ¼ 1.8 Hz, H-3), 4.80 (d, 1H, J ¼ 1.8 Hz, H-4), 5.06
avoid decomposition of the b-lactam ring. The reaction mixture (bs, 1H, OH), 6.65 (d, 2H, J ¼ 8.8 Hz, Ar-H), 6.77 (d, 2H,
was then diluted with ethyl acetate (100 mL) and quenched with J ¼ 8.8 Hz, Ar-H), 7.15 (d, 2H, J ¼ 8.0 Hz, Ar-H), 7.25 (d, 2H,
10% HCl (100 mL). The organic layer was separated and the J ¼ 8.8 Hz, (Ar-H), 7.30–7.36 (m, 5H, Ar-H). HRMS: Found
aqueous layer was extracted with ethyl acetate (2 ꢂ 50 mL). The 429.2177 (M++H); C27H29N2O4 requires 429.2178.
organic layers were combined and then washed with water
4-(4-Hydroxyphenyl)-3–(4-methoxyphenyl)-1-[4-(2-pyrrolidin-
(100 mL) and brine (100 mL) and dried with anhydrous sodium 1-ylethoxy) phenyl]azetidin-2-one (15k). Preparation was as above
sulfate. The solvent was removed by evaporation under reduced from 15i (0.153 mmol, 0.084 g). Orange oil, (15%). IR ꢁmax (film)
pressure, to afford the product as a yellow oil (50%). IR ꢁmax cmꢃ 1: 1740.0 cmꢃ 1 (C¼O, b-lactam), 3233.2 cmꢃ 1 (OH). 1H
(film) cmꢃ 1
:
1735.6 cmꢃ 1 (C¼O, b-lactam). 1H NMR NMR (CDCl3): ꢀ 1.91 (s, br, 4H, –CH2–CH2–), 2.93 (s, br, 4H,
((CD3)2CO): ꢀ 3.65 (s, 3H, O–CH3), 4.21 (d, 1H, J ¼ 2.5 Hz, H- –CH2–N–CH2–), 3.12 (s, br, 2H, CH2–N), 3.81 (s, 3H, O–CH3),
3), 4.97 (d, 1H, J ¼ 2.0 Hz, H-4), 6.77–6.83 (m, 4H, Ar-H), 7.24- 4.12 (t, 2H, J ¼ 5.23 Hz, CH2–C), 4.18 (d, 1H, J ¼ 2.2 Hz, H-3),
7.32 (m, 9H, Ar-H), 9.78 (s, 1H, OH). 13C NMR ((CD3)2CO): ꢀ 4.78 (d, 1H, J ¼ 2.24 Hz, H-4), 5.13 (bs, 1H, OH), 6.74 (d, 2H,
54.34, 62.41, 64.58, 113.72, 115.45, 118.02, 127.09, 127.29, J ¼ 9.0 Hz, Ar-H), 6.81 (d, 2H, J ¼ 8.0 Hz Ar-H), 6.91 (d, 2H,
128.16, 128.38, 130.83, 131.50, 135.13, 155.66, 157.30, 164.34. J ¼ 8.5 Hz, Ar-H). 7.18 (d, 2H, J ¼ 8.0 Hz, Ar-H), 7.23–7.24 (m.
HRMS: Found 368.1260 (M++Na); C22H19NO3Na requires 4H Ar-H). HRMS: Found 459.2294 (M++H); C28H31N2O4
368.1263.
4-(4-Hydroxyphenyl)-1-(4-methoxyphenyl)-3-phenylazetidin-2-
requires 459.2284.
one (14c). The benzyloxy protected compound 14b (2 mmol)
was dissolved in ethanol: ethyl acetate, (50 mL, 1:1) and
hydrogenated over 10% palladium on carbon (1.2 g) for 2 h. The
reaction was carefully monitored by TLC. The catalyst was
filtered, the solvent was removed under vacuum and residue
was purified by column chromatography on silica gel (eluent:
dichloromethane, methanol) to afford the product as a yellow
MTT assay procedure
All assays were performed in triplicate for the determination of
mean values reported. Compounds were assayed as the free bases
isolated from reaction. The human breast tumor cell line MCF-7
was cultured in Eagles Minimum Essential (MEM) medium in a
95% air/5% CO2 atmosphere supplemented with 10% fetal bovine
serum, 2 mM L-glutamine and 100 mg/mL penicillin/streptomycin
The medium was further supplemented with 1% non-essential
amino acids. MDA-MB-231 cells are human breast adenocarcin-
oma cells, and representative of ER-negative breast cancer. They
were grown as monolayer cultures at 37 ꢁC, under a humidified
atmosphere of air supplemented with 5% CO2. The cells were
oil, (70%). IR ꢁmax (film) cmꢃ 1
:
1735.6 cmꢃ 1 (C¼O,
b-lactam). 1H NMR ((CD3)2CO): ꢀ 3.65 (s, 3H, O–CH3),
4.21 (d, 1H, J ¼ 2.3 Hz, H-3), 4.97 (d, 1H, J ¼ 2.3 Hz, H-4),
6.77–6.83 (m, 4H, Ar-H), 7.24-7.73 (m, 9H, Ar-H), 9.78 (s, 1H,
OH). 13C NMR ((CD3)2CO): ꢀ 54.34, 62.41, 64.58, 113.72,
115.45, 118.02, 127.09, 127.29, 128.16, 128.38, 130.83, 131.50,

6
M. Carr et al.
J Enzyme Inhib Med Chem, Early Online: 1–14
maintained in Dulbecco’s Modified Eagle’s Medium (DMEM), untagged human ER obtained from recombinant baculovirus-
supplemented with 10% (v/v) Fetal Bovine Serum (FBS), 2 mM infected insect cells) and the fluorescent estrogen ligand were
L-glutamine and 100 mg/mL penicillin/streptomycin. This medium removed from the ꢃ80 ꢁC freezer and thawed on ice for 1 h prior
contained phenol red, which is suspected to have estrogen-effects. to use. The fluorescent estrogen ligand (2 nM) was added to the
However, as this assay was only concerned with anti-proliferative ER (40 nM for ERa and 30 nM for ERb) and screening buffer
effects of the compounds, and these were compared to control (100 mM potassium phosphate (pH 7.4), 100 mg/ml BGG, 0.02%
cells grown in the same media, it was not deemed necessary to NaN3 was added to make up to a final volume that was dependent
remove the phenol red prior to the assay being conducted. on the number of tubes used (number of tubes (e.g. 50) ꢂ volume
Similarly, the serum was not stripped with charcoal so estrogen of complex in each tube (50 mL) ¼ total volume (e.g. 2500 mL).
levels were not reduced in this assay.
Test compound (1 mL, concentration range 100 nM–1 mM) was
Cells were trypsinized and seeded at a density of 0.5 ꢂ 104 added to 49 mL screening buffer in each borosilicate tube (6 mm
cells/well into a 96-well plate and incubated for 24 h. After this diameter). To this, 50 mL of the fluorescent estrogen/ER complex
time, they were treated with 2 ml volumes of test compound which was added to make up a final volume of 100 mL.
had been pre-prepared as stock solutions in ethanol to furnish the
final concentration range of study, 1 nM–100 mM, and re- Estrogenic activity: alkaline phosphatase assay
incubated for a further 72 h. Control wells contained the
Following the procedure of Littlefield et al.43, human Ishikawa
equivalent volume of the vehicle ethanol (1% v/v). This vehicle
cells were maintained in Eagle’s Minimum Essential Medium
had no adverse effect on the cells. The culture medium was then
(MEM containing 10% v/v fetal bovine serum (FBS) and
removed and the cells were washed with 100 mL phosphate buffer
supplemented with 100 U/mL penicillin and 10 mg/mL strepto-
saline (PBS) and 50 mL of 1 mg/ml MTT solution was added.
mycin, 2 mM glutamine and 1 mM sodium pyruvate. Twenty four
Cells were incubated for 2 h in darkness at 37 ꢁC. At this point,
hours before the start of the experiment, near confluent cells were
solubilization was begun through the addition of 200 mL DMSO
changed to an estrogen-free basal medium (EFBM), A 1:1
and the cells maintained at room temperature in darkness for
mixture of phenol-free Ham’s F-12 and Dulbecco’s Modified
20 min to ensure thorough color diffusion before reading the
Eagles Medium, together with the supplements listed above, and
absorbance at 595 nm. The absorbance value of control cells
5% calf serum, stripped of endogenous estrogens with dextran-
(vehicle treated) was set to 100% cell viability and from this
coated charcoal. On the day of the experiment, cells were
graphs of absorbance versus cell density per well were prepared to
harvested with 0.25% trypsin and plated in 96-well flat bottomed
assess cell viability and from these, graphs of percentage cell
microtitre plates in EFBM at a density of 2.5 ꢂ 104 cells/well.
viability versus concentration of subject compound were drawn.
Test compounds were dissolved in ethanol at 10ꢃ 3M, diluted with
Effect of compounds on MCF-7 cells treated with estradiol: MCF-
EFBM (final concentration of ethanol 0.1%) and filter sterilized.
7 cells were trypsinised and seeded at a density of 0.5 ꢂ 104
After addition of the test compounds, (plated in 50 mL, added
cells/well into a 96-well plate as described above. The cells were
estradiol in 50 mL and blank medium to give a final volume
then treated with 1 ml of test compound and 1 ml of estradiol with
150 mL) the cells were incubated at 37 ꢁC in a humidified
(starting concentration is 1 in 200 dilution), which had been pre-
atmosphere containing 95%air/5% CO2 for 72 h. All experimental
prepared as stock solutions in ethanol to furnish the final
values were obtained in triplicate. The microtitre plates were then
concentration range of study, 1 nM–100 mM, and re-incubated
inverted and the growth medium removed. The plates were then
for a further 72 h. Control wells contained the equivalent volume
rinsed by gentle immersion and swirling in 2 L of PBS (0.15M
of the vehicle ethanol (1% v/v). The antiproliferative assay is then
NaCl, 10 mM sodium phosphate, pH 7.4). The plates were
completed in the same manner as described above.
removed from the container, the residual saline in the plate was
not removed, and the wash was repeated. The buffered saline was
Cytotoxicity studies
then shaken out, and the plate blotted on paper towel. The covers
Human MCF-7 breast cancer cells or human MDA-MB-231 were replaced and the plates were placed at ꢃ80 ꢁC for at least
breast cancer cells were plated at a density of 0.5 ꢂ 104 cells per 15 min, and then thawed at room temperature for 5–10 min. The
well into a 96-well plate (200 mL per well) and incubated at 37 ꢁC plates were then placed on ice and 50 mL ice cold solution
in air supplemented with 5% CO2 for 24 h. Cells were treated with containing 50 mM p-nitrophenyl phosphate, 0.24 mM MgCl2 and
2 mL volumes of test compound which had been pre-prepared as 1M diethanolamine (pH 9.8) was added. The plates were warmed
stock solutions in ethanol to furnish the final concentration range to room temperature (time zero), and the yellow color from the
of study, 1 nM–100 mM, and re-incubated for a further 72 h. production of p-nitrophenol was allowed to develop. The plates
Control wells contained the equivalent volume of the vehicle were monitored at 405 nm until maximum stimulation of the cells
ethanol (1% v/v). Following incubation, 20 mL of lysis solution showed an absorbance of ꢀ1.2.
was added to one row of wells to act as a 100% lysis control. After
30 min, 50 mL aliquots of medium were removed from all wells
and placed in a clean 96-well plate. Cytotoxicity was determined
Computational procedures: protein preparation
PDB entry 3ERT44 (4-hydroxytamoxifen co-crystallised with
ERa) was downloaded from the Protein Data Bank (PDB) was
utilized for all ERa dockings and also as a template for creation of
a homology model of ERb. The model was constructed in an
automated fashion using EsyPred3D.45 Hydrogens were added to
both structures using MOE46 (Chemical Computing Group,
Montreal, QC, Canada) and their positions minimized with the
AMBER99 force field47.
using the LDH assay kit42 obtained from Promega (Madison, WI),
following the manufacturer’s instructions for use. A 50 mL per
well LDH substrate mixture was added and the plate left in
darkness at room temperature for equilibrium. Stop solution
(50 mL) was added to all the wells and absorbance read at 490 nM.
Data were presented following calculation of percentage cell lysis
versus concentration of subject compound.
Estrogen receptor binding assay
Docking/scoring
ERa and ERb fluorescence polarization based competitor assay
kits were obtained from Panvera at Invitrogen Life Technologies,
Carlsbad, CA. The recombinant ER (insect expressed, full length,
50 conformations of both compounds 15g and 15k were built
using OMEGAv2.2.148 and docked using FREDv2.2.349

DOI: 10.1080/14756366.2016.1210136
Development of ꢂ-lactam type molecular scaffold
7
(Openeye Scientific Software, Santa Fe, NM) retaining all default chloride in the presence of triethylamine. To optimize the yield
settings and employing the Chemgauss3 scoring function50 for of the b-lactam product, a number of reaction conditions
prioritizing correct binding orientations. The active site of top were investigated. b-Lactam 14a was initially obtained success-
docked poses of each compound was minimized and refined using fully using DMF as the solvent52. This method resulted in
LigX (Chemical Computing Group)46 to allow side-chain exclusive synthesis of the trans product in a moderate yield (45%),
repositioning.
(one enantiomer only shown for the products 14a–c and 15a–g in
Scheme 1). However, the reaction required over 24-h reflux so an
alternative method was investigated. When toluene was used as
the solvent and the acid chloride was added dropwise to a
refluxing solution of appropriate imine and triethylamine53, an
Results and discussion
Chemistry
The synthesis of the Type I series of b-lactam compounds which improved yield (65%) was obtained with exclusive isolation of the
contain the basic pyrrolidinylethoxy substituent located on the C- trans product. Finally, reaction conditions were optimized using
4 aryl ring is illustrated in Scheme 1. The Staudinger reaction dichloromethane as the solvent and with the dropwise addition of
requiring a cycloaddition reaction of a ketene with an imine is the the acid chloride to a mixture of the imine 13b and triethylamine,
most commonly used method for synthesis of 1,3,4-trisubstituted initially at ꢃ20 ꢁC34. The trans product 14a was again isolated
b-lactams34,51. The initial target compound 15a, containing a exclusively and an improved yield of 78% obtained. This method
methyl ether on the N-1 aryl ring (which is important for was then employed for all subsequent Staudinger reactions. As
interaction with His524 in the ligand binding domain of the indicated, all three methods of reaction above yielded the trans
estrogen receptor), was synthesized using this reaction. The b-lactam product, with the stereochemistry of the b-lactam
required Schiff base 13a was prepared in high yield (88%) by deduced from the coupling constants of the C-3 and C-4 protons
condensation of 4-hydroxybenzaldehyde with 4-methoxyaniline. and were found to be in the region 1–3 Hz. The cis-b-lactams have
To avoid difficulties in the following cycloaddition reaction, the larger coupling constants (5–6 Hz) than the trans-b-lactams which
phenol was protected as a tert-butyldimethylsilyl ether using tert- are usually 1–3 Hz34,52
.
Stereoselectivity in the Staudinger
butyldimethylsilyl chloride and the base DBU to afford the reaction that depends on a number of experimental factors
protected Schiff base 13b. The b-lactam product 14a was obtained including structure of the imine and acid chloride, sequence of
by subsequent Staudinger reaction of imine 13b with phenylacetyl reagent addition, solvent, temperature and organic amine base34.
Scheme 1. Synthesis of Type I b-lactam ER antagonist compounds (one enantiomer shown). Scheme reagents and conditions: (a) Ethanol, reflux;
(b) tert-Butyldimethylsilyl chloride, DBU, DCM; (c) Phenylacetyl chloride, (CH3CH2)3N, DCM; (d) TBAF, THF; (e) H2, 10% Pd/C, ethyl
acetate:ethanol; (f) K2CO3, 1-(2-chloroethyl)pyrrolidine, acetone.

8
M. Carr et al.
J Enzyme Inhib Med Chem, Early Online: 1–14
In general, when the acyl chloride is added dropwise, preferably at interactions with Glu353 and Arg394. Therefore, in the present
low temperature, to a solution of imine and a tertiary amine such work it was critical to include a phenolic substituent group on the
as triethylamine, the cis cycloadduct is the major or exclusive N-1 aryl ring of the Type-1 b-lactam products to provide a 4-
stereoisomer detected. In contrast, when the tertiary base is added hydroxytamoxifen analog containing the b-lactam ring. Synthesis
to a mixture of imine and acetyl chloride, mixtures of cis and of 15f by direct demethylation of 15a proved unsuccessful using
trans cycloadducts are obtained, in which the trans is the major or several different reagents including ethanethiol and boron
exclusive product34,51. However in our synthesis, using all the trifluoride-methyl sulfide; in both cases resulting in degradation
above conditions with reaction temperatures varied from ꢃ20 to of the b-lactam ring. An alternative method of synthesis of 15f
150 ꢁC, the trans isomer was always exclusively formed. We also and 15g was pursued which involved the preparation of a Schiff
observed a similar stereochemical outcome with 4-methoxyphe- base 13g with both the required benzyl protected phenol and the
nylacetyl chloride and 4-benzyloxyphenylacetyl chloride in this basic side-chain are in position. This protected Schiff base was
reaction indicating that the specific acid chloride is an important then treated with the relevant acid chloride to form the b-lactam
variable in determining the stereochemical selectivity. Alonso which could then be deprotected by hydrogenation to yield the
et al reported synthesis of a mixture of cis and trans isomers free phenol as required on the N-1 aryl ring. Schiff base 13e
when tetrahydrofuroyl chloride was the acid component53. was prepared in 96% yield by condensation of 4-hydroxybenzal-
Phthalimidoacetyl chloride and crotonyl chloride are other dehyde with 4-benzyloxyaniline and was then alkylated with
reported acid chlorides, which form exclusively trans b-lactams. 1-(2-chloroethyl)pyrrolidine hydrochloride to afford 13g (60%).
Removal of the silyl protecting group from 14a using Subsequent treatment of 13g with phenylacetyl chloride under the
tetrabutylammonium fluoride (TBAF) resulted in the isolation usual Staudinger conditions resulted in the isolation of the
of the phenolic 14c (50%), which was confirmed in the IR protected b-lactam 15d (61%). The 1H-NMR spectrum of 15d
spectrum that shows a carbonyl absorption at ꢁ 1735.6 cmꢃ 1 and showed characteristic b-lactam doublet signals for H-3 at ꢀ 4.18
a broad absorption corresponding to the hydroxy group at ꢁ and H-4 at ꢀ 4.85, (J3,4 ¼2.52 Hz). The benzyloxy group was
1
1
3300.0 cmꢃ 1. In the H NMR spectrum, coupled doublet signals carefully removed by hydrogenation yielding 15f (50%); the H-
at ꢀ 4.22 and ꢀ 4.98 (J ¼ 2.26 Hz) are assigned to H-3 and H-4 NMR spectrum confirmed the presence of the trans b-lactam ring
respectively, indicating the trans b-lactam stereochemistry.
with H-3 at ꢀ 4.20 and H-4 at ꢀ 4.84, (J3,4 ¼2.24 Hz). Compound
The alternative method used for the preparation of 14c 15g was synthesized in a similar reaction sequence. The protected
involves the use of a benzyl ether as the protecting group in Schiff base 13g was treated with 4-methoxyphenylacetyl chloride
place of the silyl ether (Scheme 1), which can be easily removed resulting in the isolation of 15e (33%). Subsequent removal of the
using catalytic hydrogenolysis. Catalytic hydrogenation has been benzyl protecting group from 15e by careful hydrogenation yields
reported under ambient pressure of hydrogen at 50 ꢁC in methanol the phenolic b-lactam product 15g in 78% yield, with no evidence
with Pd/C as the catalyst to remove the benzyloxy group without of ring hydrogenolysis (Scheme 1).
any effect on the b-lactam ring34,54,55. However, care must be
The synthetic route used for the Type II b-lactams (containing
taken to avoid over-hydrogenation as C-4-N-1 bond cleavage has the basic side chain substituent on N1 aryl ring) is shown in
been reported to proceed by palladium catalyzed hydrogenolysis Scheme 2, which again employs the Staudinger reaction for
when an aryl substituent is located at the C-4 position i.e. a b-lactam formation. The phenolic Schiff base 13h (obtained in
benzylic carbon.56,57 The Schiff base 13c (obtained following the 96% yield on reaction of 4-benzyloxybenzaldehyde with 4-
condensation of 4-methoxyaniline with 4-benzyloxybenzalde- aminophenol) was treated with 1-(2-chloroethyl)pyrrolidine
hyde) was reacted with phenylacetyl chloride under Staudinger hydrochloride yielding the alkylated product 13i (60%).
conditions to afford the benxyloxy protected product 14b, (78%). Reaction of 13i with phenylacetyl chloride and 4-methoxypheny-
The IR spectrum contained the characteristic b-lactam carbonyl lacetyl chloride under the usual Staudinger reaction conditions
absorption band at ꢁ1735 cmꢃ 1 while the 1H-NMR spectrum afforded the trans b-lactams 15h (15%) and 15i (33%) as
confirmed the trans b-lactam isomer with H-3 and H-4 observed confirmed by 1H NMR spectra: 15h (J3,4 ¼2.5 Hz) and 15i
as coupled doublets at ꢀ 4.19 and ꢀ 4.81, respectively, (J3,4 ¼2.0 Hz). Removal of the benzyl protecting group from 15h
(J ¼ 2.26 Hz). Careful removal of the benzyl protecting group and 15i by hydrogenation yielded the required phenolic products
from 14b by hydrogenation yielded 14c (70%). Alkylation of the 15j and 15k respectively, (one enantiomer shown for compounds
phenolic 14c with 1-(2-chloroethyl)pyrrolidine affords the 15h–15k, Scheme 2).
required product 15a (30%).
Fluoro-substituted tamoxifen and cyclofenol derivatives have
Antiproliferative activity in MCF-7 and MDA-MB-231
been investigated as ER-imaging agents for breast cancer58,59 and
breast cancer cells
we have previously reported the potent ER-binding properties of
fluorine-containing benzoxepine type ER antagonists30. We now
The b-lactam compounds prepared above were evaluated in a
series of in vitro assays which determined their antiproliferative
activity in ER positive MCF-7 and ER negative MDA-MB-231
breast cancer cell lines and also their affinity for the estrogen
receptor and estrogenic effects in Ishikawa cells.
wished to examine the effect of the inclusion of the lipophilic
fluorine substituent on the ER activity of the b-lactam ER
antagonist compounds 15b and 15c. The Schiff base 13d,
obtained in 58% by the standard method (Scheme 1), was directly
alkylated with 1-(2-chloroethyl)pyrrolidine hydrochloride to yield
13f which was used without further purification in the subsequent
reactions. Treatment of 13f with phenylacetyl chloride under the
usual Staudinger reaction conditions afforded the trans b-lactam
Compounds 15a–15c, 15f–g and 15j–k were initially screened
for their antiproliferative activity using the ER expressing (ER-
dependent) MCF-7 human breast cancer cell line and the ER-
independent MDA-MB-231 human breast cancer cell line. Table 1
shows the antiproliferative effects of type I and type II b-lactams
in both MCF-7 and MDA-MB-231 cell lines. The majority of the
compounds show anti-proliferative effects at concentrations
similar to that of the positive control tamoxifen60,61. Many of
the compounds (e.g. 15a, 15f, 15g and 15j) have IC50 values in the
range 0.185–7.54 mM in MCF-7 cells, but have significantly
higher IC50 values in MDA-MB-231 cells (11–40 mM), a result
1
product 15b, (Scheme 1). The H-NMR spectrum indicated two
coupled doublets at ꢀ 4.27 and ꢀ 4.87 (J3,4 ¼2.04 Hz) assigned to
H-3 and H-4, respectively. 15c was similarly prepared from 13f
and 4-methoxyphenylacetyl chloride
The requirement for a phenolic substituent in many ER
antagonists such as 4-hydroxytamoxifen4 and raloxifene is
significant for successful binding to the ER as shown by

DOI: 10.1080/14756366.2016.1210136
Development of ꢂ-lactam type molecular scaffold
9
Scheme 2. Synthesis of Type II b-lactam ER antagonist compounds (one enantiomer shown). Scheme reagents and conditions: (a) Ethanol, reflux;
(b) K2CO3, 1-(2-chloroethyl)pyrrolidine, acetone; (c) Phenylacetyl chloride, (CH3CH2)3N, DCM; (d) H2, 10% Pd/C, ethyl acetate: ethanol.
Table 1. Antiproliferative and cytotoxic effects for compounds 15a–c, 15f–g and 15j–k in MCF-7 and MDA-MB-231 cells; ERa and ERb binding
affinities for compounds 15a–c, 15f–g and 15j–k.
O
R
1
O
N
N
A
C
A
O
N
N
O
B
B
C
R
2
HO
R
Type I: 15a-c, 15f-g
Type II: 15j-k
MCF-7 IC50
(mM)*
Cytotoxicity
10 mMx
MDA-MB-231
IC50 (mM)*
Cytotoxicity
(%) 10 mMx
5.3
ER a IC50
(mM)||
ER b IC50
(mM)||
Compound
a/b ratio
15a
R1¼OCH3
R2¼H
6.22
1
12
26
4
12.77
12.7
1.70
15.49
4100 mM
1.64
9
15b
15c
15f
15g
R1¼F
4.82
0
1.05
495
7
R2¼H
R1¼F
3.49
4.62
25
0
0.23
R2¼OCH3
R1¼OH
R2¼H
0.519
0.186
43.08
19.65
0.060
0.0043
0.66
11
75
R1¼OH
R2¼OCH3
R ¼ H
4.9
3.5
0.32
15j
15k
7.54
11
0
13.4
0
17.54
5.03
20z
2
4.8
0
-yy
0.21
3.04
0.070#
0.040
2.31
11
416
2.42
0.5
R ¼OCH3
–
3.30
450 mM
0.170#
0.020
Tamoxifen
4-Hydroxy-tamoxifen
4.12y
0.107
–
18**
*IC50 values are half maximal inhibitory concentrations required to block the growth stimulation of MCF-7 or MDA-MB-231 cells. Values represent
the mean SEM (error values ꢂ 10 ꢃ 6) for three experiments performed in triplicate.
yThe IC50 value obtained for Tamoxifen using the MTT assay is 4.12 0.038 mM, with cytotoxicity value 13.4% (10 mM) is in good agreement with the
reported IC50 value for tamoxifen on human MCF-7 cells60
.
zThe IC50 value obtained for Tamoxifen in MDA-MB-231 cells (20 mM) is in agreement with reported values for tamoxifen in MDA-MB-231 cells63,64
.
xLactate dehydrogenase assay: following treatment of the cells, the amount of LDH was determined using LDH assay kit from Promega. Data are
presented as % cell lysis at compound concentration of 10 mM42
.
||Values are an average of at least nine replicate experiments, for ERa with typical standard errors below 15%, and six replicate experiments for ERb,
with typical standard errors below 15%.
#The ER binding values obtained are in agreement with the reported ER IC50 binding data for tamoxifen (ERa 60.9 nM ERb188 nM, Panvera/
Invitrogen).
**Work by Seeger et al.61
.
yyNo cytotoxic effect could be demonstrated for 4-hydroxytamoxifen in MDA-MB-321 cells60
.
which is not unexpected. However, compounds 15c and 15k required for interaction with the Asp351 of the estrogen receptor
unusually show moderate potency in MDA-MB-231 cell line LBD14. This indicates that the possible mechanism of action of
(IC50 ¼4.62 mM, 5.03 mM, respectively). The most potent com- the compound is mediated through binding to the estrogen
pound in the series examined in MCF-7 cells is 15g (IC50 receptor. Tamoxifen shows some antiproliferative effects in MDA-
value ¼ 0.185 mM), representative of the Type-I structure, con- MB-231 ER-negative cell lines at much higher concentrations
taining the phenolic substitution in Ring C, which would be (approx. 20 mM) than in MCF-7 cells62,63. The cytotoxic effect of

10 M. Carr et al.
J Enzyme Inhib Med Chem, Early Online: 1–14
(A) 120
100
80
15g + Estradiol
15g
tamoxifen + Estradiol
tamoxifen
110
90
60
70
40
50
15g + Estradiol
15g
20
30
0
10
−11
−10
−9
−8
−7
−6
−5
−4
10
10
10
10
10
10
10
10
−10
log [conc (M)]
−10
−9
−8
−7
−6
10
10
10
Log [conc (molar)]
10
10
(B) 110
90
70
Figure 4. Effects of compound 15g and tamoxifen (control) in Ishikawa
cells (endometrial cancer cells) in the presence and absence of estradiol.
Cells were plated in 96-well flat bottomed microtitre plates in EFBM at a
density of 2.5 ꢂ 104 cells/well. Test compound 15g and tamoxifen
(control) were dissolved in ethanol at 10ꢃ3 M, diluted with EFBM (final
concentration of ethanol 0.1%) and filter sterilized. After addition of the
test compound, (plated in 50 mL, added estradiol (1 nM) in 50 mL, and
blank medium to give a final volume of 150 mL), the cells were incubated
for 72 h. 50 mL ice cold solution containing 50 mM p-nitrophenyl
phosphate, 0.24 mM MgCl2 and 1M diethanolamine (pH 9.8) was
added to the cooled plates, (0ꢁC). The production of p-nitrophenol was
monitored at 405nm until maximum stimulation of the cells showed an
absorbance of ꢀ1.2. Values represent the mean S.E.M (error values) for
three experiments performed in triplicate.
50
30
Tamoxifen +Estradiol
Tamoxifen
10
−10
−11
−10
−9
−8
−7
−6
−5
−4
10
10
10
10
10
10
10
10
log [conc (M)]
Figure 3. Effect of compounds 15g and tamoxifen (1) (control) on the
inhibition of proliferation of MCF-7 breast cancer cells in the absence and
presence of Estradiol. MCF-7 cells were seeded at a density of 2.5 ꢂ 104
cells per well in 96-well plates. The plates were left for 24 h to allow the
cells to adhere to the surface of the wells. A range of concentrations
(0.01 nM–50 mM) of the compound were added in triplicate and the cells
irreversible components to their inhibition of cell proliferation
in vitro, with the former being highly correlated with affinity for
ER. The antiproliferative effect appears to be estradiol reversible
were left for another 72 h. An MTT assay was performed to determine the with the cytotoxic effect being irreversible62. This effect is seen at
level of anti-proliferation. (A, B) are representative of results for 15g, and
tamoxifen (1) (control); the values represent the mean S.E.M (error
the higher ER antagonist concentrations, and is demonstrated for
tamoxifen where estradiol is shown to reverse the antagonist effect
values) for three experiments performed in triplicate. Effect of com-
at lower concentrations of tamoxifen, but not at higher concen-
pounds on MCF-7 cells treated with estradiol – cells were treated with
trations; (IC50 value ¼ 4.22 mM for tamoxifen in the presence of
1 ml of test compound and 1 ml of estradiol, which had been pre-prepared
estradiol (50 nM), Figure 3B). Compounds 15g and 15f show
estradiol–reversible effects at concentrations higher than tamoxi-
fen, indicating that they have less estradiol–irreversible effects
than tamoxifen, and suggesting that the main mechanism of action
of these b-lactam compounds is through affinity for the ER.
as stock solutions in ethanol to furnish the final concentration range of
study, 1nM–100mM, and re-incubated for a further 72 h. Control wells
contained the equivalent volume of the vehicle ethanol (1% v/v). (A)
Compound 15g in the absence and presence of estradiol (50nM); inhibited
proliferation of MCF-7 cells. (B) Tamoxifen (1) (control) in the absence
and presence of estradiol (50nM); inhibited proliferation of MCF-7 cells.
Estrogen receptor binding studies
these b-lactam compounds in MCF-7 cells as determined in the The affinity for the ER of the b-lactam compounds 15a–15c, 15f–
lactose dehydrogenase (LDH) assay (Table 1) is also lower than g and 15j–k is confirmed through estrogen receptor binding
that of tamoxifen64 with the exception of compound 15c, which studies. Table 1 shows the results of the competitive ER binding
resulted in 26% and 25% toxicity in MCF-7 and MDA-MB-231 assay for b-lactam SERM Types I and II with both ERa and ERb.
While the level of affinity to the ER varies depending on the aryl
equal antiproliferative effects in both MCF-7 and MDA-MB-231 substituents present, compounds of structural Type I appear to
cell lines observed for compound 15c.
have better binding ability than compounds of structural type II.
cell lines, respectively. This cytotoxic action may explain the
The antiproliferative effect of 15g (Type I) in MCF-7 cells was Of the Type I compounds, 15g established the most potent binding
significantly reduced with the addition of estradiol (50 nM), with to the ER (IC50 ERa ¼ 4.3 nM, ERb ¼ 322 nM) and significantly
IC50 value increased from 0.186 mM to 9.17 mM, (Figure 3A). A more potent than both tamoxifen (IC50 ERa ¼ 70 nM) and 4-
similar result is also obtained for compound 15f (IC50 values hydroxytamoxifen (IC50 ERa ¼ 40 nM) for ERa. This result also
increased from 0.519 to 27.85 mM). Compound 15k (Type II) correlated with the antiproliferative activity of 15g in the MCF-7
demonstrated a small increase in IC50 value on addition of cell line (IC50 ¼185 nM). The fluoro-substituted b-lactam 15c
estradiol (50 nM) from 3.30 to 8.29 mM). These results support the indicated some improved ERa and ERb interaction when
indication that the antiproliferative effects for compounds 15f and compared to the methoxy substituted product 15a, (Table 1).
15g are a result of interaction of the compounds with the ER and
The IC50 value for ERa binding of the Type II compound 15k
therefore preventing estrogen mediated proliferation. Reversal of (3.04 mM) is much greater than tamoxifen or that of the type I
antiestrogen-mediated cell growth antagonism by estradiol has b-lactam compounds 15f and 15g. However, the moderate
been suggested to indicate the degree to which antagonism is antiproliferative effect in MCF-7 cells is very similar to
mediated through ER65. Tamoxifen and 4-hydroxytamoxifen have 15c with an IC50 of 3.30 mM. The IC50 value for 15k in the
been shown to have an estradiol reversible and estradiol MDA-MB-231 cell line is 5.03 mM indicating that in this case

DOI: 10.1080/14756366.2016.1210136
Development of ꢂ-lactam type molecular scaffold 11
Figure 5. Key anti-estrogenic interactions are
observed in the docking of 15g in ERa.
Hydrogen bonding between the C-ring 3-OH
substituent and Glu353, Arg394, HOH is
depicted. A salt bridge between the basic side
chain and Asp351 is also formed.
Figure 6. Key anti-estrogenic interactions are
also observed in the docking of 15k in ERa.
Hydrogen bonding between the B-ring 3-OH
substituent and Glu353, Arg394, HOH is
depicted. A salt bridge between the basic side
chain and Asp351 is also formed.

12 M. Carr et al.
J Enzyme Inhib Med Chem, Early Online: 1–14
Figure 7. Docked position of 15g and 15k in
both ERa/b superimposed by backbone.
Residues Met336, met295 and Ile373 are
from dockings in ERb. Residues of ERb
depicted in orange for docking of 15k.
Residues Leu384, Met343, Met421 are from
dockings in ERa.
anti-proliferation does not seem to be mediated through the ER. dependent manner, (Figure 4). The estrogenic stimulatory prop-
(Addition of estradiol (50 nM) with compound 15k in MCF-7 erty of this compound was also monitored in Ishikawa cells by
cells produced only a small increase in the IC50 value). This measuring the stimulation of alkaline phosphatase (AP) in these
indicates that the antiproliferative effect is not estradiol reversible cells in the absence of estradiol. 15g showed little ability to inhibit
and therefore compound 15k does not appear to act solely as an the effect of estradiol in Ishikawa cells at concentrations of up to
ER antagonist, but may also have an alternative mode of action. 1 mM, however, at the same time 15g itself shows a higher level of
Although, the main mechanism of action of tamoxifen is primarily estrogen stimulation (21% at 1 mM concentration) when compared
mediated through the ER, studies have shown non-ER mediated to tamoxifen, (10% stimulation at 1 mM concentration). This may
mechanisms are present including the involvement of certain indicate that the stimulatory effect is due to both estradiol and 15g
signaling proteins, proto-oncogenes and transforming growth and is not indicative of the inability of estradiol to be displaced by
factor b in tamoxifen-mediated apoptosis66.
the b-lactam compound. Other known anti-estrogens also show
All of the Type I b-lactam compounds examined also show a estrogen stimulation in Ishikawa cells with reports having shown
preference for ERa binding over ERb. In the case of the most that 4-hydroxytamoxifen stimulates AP activity to a level 47% of
potent example 15 g, the a:b binding selectivity ratio is 75:1 and that of estradiol and the SERM lasofoxifene increases AP activity
for 15b no binding to ERb was observed at concentrations of up to by 18%68.
100 mM. Tamoxifen demonstrates almost equal binding affinity
for ERa and ERb. Type II compounds 15j and 15k also shows Molecular modeling studies of novel b-lactam
competitive binding to ERa in excess of ERb, while compound compounds
15k shows no binding to ERb up to 50 mM (see Supplementary
To rationalize the observed ERa/b affinity of Type I and Type II
Information: Figure S1: Effect of compounds 15a, 15f , 15g, 15k,
compounds, a semi-flexible ligand receptor docking study of
15j on the inhibition of proliferation of MCF-7 breast cancer
compounds 15g and 15k was undertaken. The PDB entry 3ERT
cells; Figure S2: Effect of compounds 15a, 15f, 15g, 15k, 15j on
was used (ERa co-crystallized with 4-hydroxytamoxifen) to
the inhibition of proliferation of MDA-MB-231 breast cancer
examine the ERa binding mode of these compounds44. Figures
cells; Figure S3: Estrogen receptor a binding affinities for
5 and 6 illustrate the docked binding poses of compounds 15g and
compounds 15a, 15f, 15g, 15k, 15j; Figure S4: Estrogen receptor
15k respectively in the binding site of ERa. It is immediately
b binding affinities for compounds 15a, 15f, 15g, 15k, 15j).
evident that both compounds adopt a typical antiestrogenic
orientation in the ligand binding site. Key hydrogen bonding
Antiestrogenic activity in ishikawa cells
interactions between the phenolic group of the ligands and
The estrogen stimulation and antagonistic properties of the most Glu353, Arg394 and bridging water are observed. A salt bridge is
active ER compound 15g was determined in an estrogen bioassay also formed between the basic side chain nitrogens and Asp351 of
˚
˚
which is based on the stimulation of alkaline phosphatase (AP) in Helix-12 (15g – 2.8 A, 15k – 2.6 A). As there is currently no
the Ishikawa human endometrial adenocarcinoma cell line43. antiestrogen co-crystallized in both isoforms of ERa and ERb, it
Enmark et al have reported that Ishikawa cells contain both ERa was deemed necessary to construct a homology model of ERb
and ERb receptors but with ERa being far in excess67. Compound using PDB entry 3ERT as a template45. This process ensured that
15g was examined as an estrogen antagonist by its effect on the bias of ligand induced residue motion would be reduced and the
inhibition of estradiol stimulation in the Ishikawa cells in a dose- process would provide more realistic dockings when used in

DOI: 10.1080/14756366.2016.1210136
Development of ꢂ-lactam type molecular scaffold 13
7. Vogel VG, Costantino JP, Wickerham DL, et al. Update of the
national surgical adjuvant breast and bowel project study of
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conjunction with our semi-flexible docking approach. Figure 7
illustrates the docked positions of both compounds 15g and 15k in
ERa/b superimposed by backbone. It is firstly apparent that the
b-lactam ring of 15g lies in close proximity to the ERa Leu384/
ERb Met336 residue mutation and favorable interaction with
Leu384 could account for some of the ERa selectivity observed
(&75-fold). For the case of 15k (Type II structure), a different
binding pose occurs compared with 15g whereby the b-lactam
ring carbonyl oxygen of 15k is positioned towards ERa residue
Met343 (ERb Met295) making a favorable interaction, whilst
Met295 seems to reposition itself so as not to cause any steric
hindrance. It would appear for the most part that the significant
ERa selectivity observed is mainly a result of the tighter packing
of 15g in the active site of ERa by surrounding residue side
chains.
12. Ahmed NS, Elghazawy NH, ElHady AK, et al. Design and synthesis
of novel tamoxifen analogues that avoid CYP2D6 metabolism. Eur J
Med Chem 2016;112:171–9.
Conclusion
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agonism and antagonism in the oestrogen receptor. Nature 1997;389:
753–8.
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tors as multifunctional medicines. 2. Clinical considerations and
new agents. J Med Chem 2003;46:1081–111.
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in postmenopausal women after five years of tamoxifen therapy for
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There is currently much interest in the discovery of novel
molecular scaffolds with SERM profile properties which could be
suitable for development of new therapies for the treatment of
breast cancer, osteoporosis, and related hormone-dependent
conditions. Both raloxifene and tamoxifen are good preventive
choices for treatment of postmenopausal women with elevated
risk for breast cancer. Because of the known importance of ERa
as a pharmaceutical target and also the potential importance of
ERb, molecules that act as agonists or antagonists selectively a or
b ER subtypes are currently being investigated for their
therapeutic potential. ERa predominates in the breast and in
reproductive tissues such as the uterus, whereas ERb is the
principal subtype in the ovary and certain regions of the brain. We
have synthesized a number of novel b-lactam compounds
designed as potential estrogen receptor ligands, which demon-
18. Robertson JF. Faslodex (ICI 182, 780), a novel estrogen receptor
downregulator-future possibilities in breast cancer.
Biochem Mol Biol 2001;79:209–12.
J Steroid
strate antiproliferative activity against the MCF-7 human breast 19. Pike ACW. Lessons learnt from structural studies of the oestrogen
cancer cell line. The compounds also demonstrate good affinity
receptor. Best Pract Res Clin Endocrinol Metab 2006;20:1–14.
20. Ruff M, Gangloff M, Wurtz JM, Moras D. Estrogen receptor
transcription and transactivation: structure-function relationship in
DNA- and ligand-binding domains of estrogen receptors. Breast
Cancer Res 2000;2:353–9.
21. Sun J, Meyers MJ, Fink BE, et al. Novel ligands that function as
for the estrogen receptor and selectivity for ERa. The most potent
antiproliferative compound 15g having Type I structural scaffold,
demonstrated ERa binding with IC50 ¼4.3 nM and relative
binding affinity ERa/ERb of 75:1. Further biochemical studies
will determine the effects of these novel analogs on ERE
transcription and ERa stability in MCF-7 cells, and will
determine the mechanistic differences between their activity and
that of tamoxifen.
selective estrogens or antiestrogens for estrogen receptor-alpha or
estrogen receptor-beta. Endocrinology 1999;140:800–4.
22. Pike AC, Brzozowski AM, Hubbard RE, et al. Structure of the
ligand-binding domain of oestrogen receptor beta in the presence of
a partial agonist and a full antagonist. Embo J 1999;18:4608–18.
23. Blizzard TA. Selective estrogen receptor modulator medicinal
chemistry at Merck. A review. Curr Top Med Chem 2008;8:
792–812.
Declaration of interest
The authors report no declarations of interest. This work was supported
through funding from the Trinity College IITAC research initiative (HEA
PRTLI, Cycle 3).
24. Gennari L, Merlotti D, Martini G, Nuti R. Lasofoxifene: a third-
generation selective estrogen receptor modulator for the prevention
and treatment of osteoporosis. Expert Opin Investig Drugs 2006;15:
1091–103.
25. Blizzard TA, Morgan II JD, Mosley RT, et al. 2-Phenylspiroindenes:
a novel class of selective estrogen receptor modulators (SERMs).
Bioorg Med Chem Lett 2003;13:479–83.
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Supplementary material available online