Synthesis, biological evaluation and X-ray analysis of bicalutamide sulfoxide analogues for the potential treatment of prostate cancer

Article abstract of DOI:10.1016/j.bmcl.2021.127817

The androgen receptor (AR) is a pivotal target for the treatment of prostate cancer (PC) even when the disease progresses toward androgen-independent or castration-resistant forms. In this study, a series of sulfoxide derivatives were prepared and their antiproliferative activity evaluated in vitro against four different human prostate cancer cell lines (22Rv1, DU-145, LNCaP and VCap). Bicalutamide and enzalutamide were used as positive controls. Compound 28 displayed significant enhancement in anticancer activity across the four PC cell lines with IC₅₀ = 9.09 – 31.11 μM compared to the positive controls: bicalutamide (IC₅₀ = 45.20 –51.61 μM) and enzalutamide (IC₅₀ = 11.47 – 53.04 μM). Sulfoxide derivatives of bicalutamide were prepared efficiently from the corresponding sulfides using only one equivalent of mCPBA, limiting the reaction time to 15–30 min and maintaining the temperature at 0 °C. Interestingly, three pairs of sulfoxide diastereomers were separated and NMR comparison of their diastereotopic methylene (CH₂) group is presented. X-ray diffraction crystal structure analysis provided relative configuration assignment at the chiral sulfur and carbon centres. Molecular modelling study of the four diastereoisomers of compound 28 is described.

Full text of DOI:10.1016/j.bmcl.2021.127817

Bioorg. Med. Chem. Lett. 36 (2021) 127817
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis, biological evaluation and X-ray analysis of bicalutamide
sulfoxide analogues for the potential treatment of prostate cancer
,
Sahar B. Kandila *, Benson M. Kariukib, Christopher McGuigana, Andrew D. Westwella
a School of Pharmacy & Pharmaceutical Sciences, Cardiff University, Cardiff, CF10 3NB, Wales, United Kingdom
b School of Chemistry, Cardiff University, Park Place, Cardiff, CF10 3AT, Wales, United Kingdom
A R T I C L E I N F O
A B S T R A C T
Keywords:
The androgen receptor (AR) is a pivotal target for the treatment of prostate cancer (PC) even when the disease
progresses toward androgen-independent or castration-resistant forms. In this study, a series of sulfoxide de-
rivatives were prepared and their antiproliferative activity evaluated in vitro against four different human
prostate cancer cell lines (22Rv1, DU-145, LNCaP and VCap). Bicalutamide and enzalutamide were used as
positive controls. Compound 28 displayed significant enhancement in anticancer activity across the four PC cell
lines with IC50 = 9.09 – 31.11 µM compared to the positive controls: bicalutamide (IC50 = 45.20 –51.61 µM) and
enzalutamide (IC50 = 11.47 – 53.04 µM). Sulfoxide derivatives of bicalutamide were prepared efficiently from
the corresponding sulfides using only one equivalent of mCPBA, limiting the reaction time to 15–30 min and
maintaining the temperature at 0 ◦C. Interestingly, three pairs of sulfoxide diastereomers were separated and
NMR comparison of their diastereotopic methylene (CH2) group is presented. X-ray diffraction crystal structure
analysis provided relative configuration assignment at the chiral sulfur and carbon centres. Molecular modelling
study of the four diastereoisomers of compound 28 is described.
Androgen receptor (AR)
Prostate cancer (PC)
Sulfoxide
Oxidation
Diastereotopic
Diastereoisomer
Diarylpropionamide
Bicalutamide
The androgen receptor (AR) plays substantial anabolic and repro-
ductive roles in men and women. Additionally, AR signaling plays a
crucial function in tumourigenesis and metastasis of different cancer
types, including prostate, bladder, kidney, lung, breast and liver 1–3. AR
is a member of the nuclear receptor family and consists of three main
functional domains: a variable N-terminal domain, a highly conserved
DNA-binding domain (DBD) and a conserved ligand binding domain
(LBD). 4 Binding of testosterone and dihydrotestosterone (DHT) to the
LBD induces AR conformational changes followed by translocation into
the nucleus to interact with DNA and modulate prostate specific antigen
(PSA) levels.5 AR antagonists (anti-androgens) inhibit these processes
More recently, darolutamide (ODM-201) has been approved and clini-
cally used in patients with non-metastatic CRPC.9 New AR antagonists
are continuously needed to improve the efficacy of the clinically used
compounds.
In this paper, we present the design and synthesis of a series of new
sulfoxide bicalutamide analogues, building on our previous work 10–14
to offer new therapeutic possibilities for combating resistance
commonly observed in the clinical use of AR antagonists.
Small chemical changes in the structure of nonsteroidal AR ligands
can play a major role in determining the pharmacological outcome.15,16
We previously published extensive SAR studies on bicalutamide chem-
10–14
and are used for the treatment of advanced prostate cancer (PC).6,7
A
ical structure modification.
Here we are covering additional
variety of non-steroidal anti-androgens (NSAA) are approved for the
treatment of PC. The first generation NSAAs include flutamide,
hydroxyflutamide and bicalutamide, Fig. 1. However, these anti-
androgens eventually fail to inhibit the AR upon long term treatment
switching from being AR antagonists to AR agonists with the develop-
ment of castration resistant prostate cancer (CRPC), an aggressive form
of the disease with poor prognosis. Similarly, resistance to the more
recent second-generation anti-androgens (enzalutamide, apalutamide)
is developing in PC patients via the upregulation of AR expression.8
modifications for further evaluation of the impact on the anti-
proliferative activity in prostate cancer models. Fig. 2 shows the gen-
eral three main areas of modification; ring A, ring B and linker area C.
Herein, a series of sulfoxide bicalutamide derivatives were prepared and
their anti-proliferative activity was evaluated in vitro against four
different human prostate cancer cell lines (22Rv1, DU-145, LNCaP and
VCap).
Phenylacrylamides (4 and 5) were prepared by reacting the corre-
sponding aniline (1 or 2) with methacryloyl chloride (3) in
* Corresponding author.
E-mail address: kandils1@cf.ac.uk (S.B. Kandil).
https://doi.org/10.1016/j.bmcl.2021.127817
Received 20 November 2020; Received in revised form 9 January 2021; Accepted 19 January 2021
Available online 26 January 2021
0960-894X/© 2021 Elsevier Ltd. All rights reserved.

S.B. Kandil et al.
Bioorganic & Medicinal Chemistry Letters 36 (2021) 127817
Fig. 1. Chemical structures of the non-steroidal anti-androgens (NSAA); flutamide, hydroxyflutamide, nilutamide, bicalutamide, enzalutamide, apalutamide and
darolutamide.
workers demonstrated that the nature of the linker (C), Fig. 2 plays a
pivotal role in controlling the ultimate antagonistic/agonistic effect of
these kinds of molecules.26–28
Previous literature states that the sulfoxide derivatives are usually
obtained by the oxidation of the sulfide precursors using sodium meta-
17,21,29
periodate (NaIO4) in aqueous methanol for 48 h.
It is also re-
ported that the oxidation reaction of the sulfide bicalutamide derivatives
with m-chloroperbenzoic acid (mCPBA) would give sulfones as the sole
product. 17,21,29 However, we efficiently managed to prepare the bica-
lutamide sulfoxide analogues (25–34) from the corresponding sulfide
precursors (15–24) using only one equivalent of mCPBA (rather than 2
equivalents in case of preparing the sulfone), limiting the reaction time
to 15–30 min and maintaining the temperature at 0 ◦C while monitoring
the progress of reaction using TLC, as outlined in Scheme 1. Introduction
of fluorinated groups into the chemical structure provides a combination
of electronegativity, size and lipophilicity impacts and can affect phys-
icochemical properties which in turn influences the biological
Fig. 2. Chemical structure of bicalutamide (X = SO2) and the areas of chemical
structure modifications, ring A, ring B and the linker area C.
dimethylacetamide (DMA).17
Compounds 4 and 5 were epoxidised using hydrogen peroxide and
trifluoroacetic anhydride (TFAA) in dichloromethane to give 6 and 7.
18,19 Subsequently the epoxides were reacted with thiols (8–14) to afford
the corresponding sulfide derivatives (15–24), Scheme 1.
activity30–33
.
Previous studies of the non-steroidal propionamides showed that the
heteroatom (X) linked to the B-ring (Fig. 2) is the main point of meta-
bolic lability and that there was no significant in vivo activity of some
sulfide analogues because of the rapid hepatic metabolism into the
sulfoxide and sulfone analogues.20-22
Interestingly, during the synthesis and purification of sulfoxide an-
alogues (32–34)34 using column chromatography, we obtained two sets
of diastereoisomers, a fast-moving (32a, 33a, 34a) and a slow-moving
(32b, 33b, 34b) products. The comparison of the NMR data of the
separated sets of diastereoisomers of compounds (32–34) revealed
Bicalutamide and its analogues are clinically used in a single enan-
tiomeric form at the asymmetric carbon atom (S-configuration if X = O,
NH and R-configuration if X = S, SO2) and this chirality has an
important effect on the anti-androgenic activity.23-25 Upon oxidation of
the sulfide bicalutamide analogues to the corresponding sulfoxide (SO),
another chiral centre at the sulfur atom is created.21 Miller and co-
1
consistent trends in the H and 13C NMR chemical shifts. The clearest
trend is observed in the chemical shift separation of the two protons of
the diastereotopic methylene (CH2) group next to the chiral sulfoxide
moiety (Fig. 3 and Table 1). The coupling constant (J-value) between the
two protons and the carbon chemical shift also show a consistent trend
2

S.B. Kandil et al.
Bioorganic & Medicinal Chemistry Letters 36 (2021) 127817
Scheme 1. Reagents and conditions, i) DMA, rt, 3 h, ii) H2O2, TFAA, DCM, rt, 24 h; iii) NaH, THF, RT, 24 h, iv) mCPBA (1 equiv), DCM, 0◦ C, 15–30 min.
Fig. 3. 1H NMR spectra of the three pairs of sulfoxide compounds (32–34) showing the chemical shift separation between the diastereotopic methylene (CH2)
protons. The top panel represents the fast-moving products (32a-33a-34a) while the bottom panel shows the slow-moving products (32b-33b-34b).
3

S.B. Kandil et al.
Bioorganic & Medicinal Chemistry Letters 36 (2021) 127817
guanidine group of Arg 752 of helix 5 (Fig. 5). Another H-bond was
observed between the nitro group (NO2) and the side chain amide (NH2)
group of Gln 711 in three out of four diastereoisomers (Fig. 5B-D).
Table 1
1H and 13C NMR data of the diastereotopic methylene (CH2) group of the
separated diastereoisomers of the sulfoxide bicalutamide derivatives (32–34).
13C - chemical
shift
Diastereoisomer C(R)S(S) shows
π-π stacking between the terminal
ID
Difference in H -
chemical shift
coupling constant (J-value)
between the two protons
phenyl ring and the indole side chain of Trp 741 (Fig. 5A). In addition,
hydrophobic interactions were observed with the surrounding hydro-
phobic pocket formed of residues; Trp 741, Met 745, Leu 712 and Met
787.
(32a)
(32b)
(33a)
(33b)
(34a)
(34b)
0.11
0.73
0.12
0.73
0.20
0.51
14
13
14
13
14
13
57.35
60.14
57.50
59.39
59.62
62.30
In summary, sulfoxide bicalutamide derivatives were prepared effi-
ciently and their antiproliferative activity was evaluated in vitro against
four different human prostate cancer cell lines (22Rv1, DU-145, LNCaP
and VCap). These modifications offer an insight into the SAR of various
propionanilide analogues. Bicalutamide and enzalutamide were used as
positive controls. The results summarised in Tables 2 indicated that two
compounds; 27 and 28 have displayed more potent antiproliferative
activity than the positive controls; bicalutamide (IC50 = 45.20 – 51.61
µM) and enzalutamide (IC50 = 11.47–53.04 µM). Sulfoxide analogues
were prepared via controlling the oxidation reaction time, temperature
and the number of equivalents of the oxidising agent m-CPBA. Three
pairs of diastereomers were separated and a comparison of their dia-
stereotopic methylene (CH2) group NMR data is presented in Table 1
and Fig. 3. Interestingly, X-ray diffraction crystal structure analysis
provided relative configuration assignment at the chiral sulfur and
carbon centres, Fig. 4. An in silico molecular modelling study performed
on the four diastereoisomers of compound 28 is described in Fig. 5,
indicating similar key interactions within the AR-LBD.
(Table 1).
Closer examination of compound 34 using X-ray diffraction crystal
structure analysis35,36 of the two separated sets of diastereoisomers,
namely 34a and 34b obtained in approximately 1 fast moving : 2 slow
moving ratio, established the configuration of 34a (minor product) to be
a mixture of C(R), S(R) and its C(S), S(S) antipode. Meanwhile 34b
(major product) was shown to be a mixture of C(R), S(S) and its C(S), S
(R) antipode, Fig. 4, (CCDC 2040881-2040882).
Miller and co-workers established the absolute configuration of the
chiral sulfoxide group of two bicalutamide sulfoxide analogues (carbon
atom with R configuration) using X-ray analysis, NMR measurements
and quantum chemical calculations. 21,28,37
Evaluation of the antiproliferative activity of the sulfoxide com-
pounds (25–34) in 22Rv1, DU-145, LNCaP and VCap human prostate
cancer cell lines38, showed that compound 28 has the most potent ac-
tivity (IC50 ¼ 9.09 – 31.11 µM) followed by compounds 27, 29 and 30.
Loss of activity was observed with compounds 25, 26, 31–34, Table 2.
A docking study was performed using MOE39 to compare the pre-
dicted binding modes of the four diastereoisomers of sulfoxide com-
pound 28 (IC50 ¼ 9.09 – 31.11 µM). All the diastereoisomers share key
interactions including a H-bond between the nitro group (NO2) and the
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Fig. 4. X-ray crystal structure and absolute configuration of the two sets of diastereoisomers of compound 34, (A) C(R), S(R) and C(S), S(S) antipode, (B) C(R), S(S)
and C(S), S(R) antipode, of 34a and 34b respectively. (CCDC 2040881–2040882).
4

S.B. Kandil et al.
Bioorganic & Medicinal Chemistry Letters 36 (2021) 127817
Fig. 5. The predicted binding modes of compound 28A [C(R), S(S)], 28B [C(S), S(R)], 28C [C(S), S(S)] and 28D [C(R), S(R)] within the hAR-LBD showing H-bond
interactions (blue dashed line) with Arg752, Gln711, Asn 705 and Thr 877.
Table 2
online version. These data include NMR, MS and HPLC data. https://doi.
org/10.1016/j.bmcl.2021.127817.
in vitro antiproliferative activity of the sulfoxide analogues of bicalutamide
(25–34) across four human prostate cancer cell lines (DU-145, 22Rv, LNCaP and
VCap). All data are mean values from experiments carried out on three separate
occasions.
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Appendix A. Supplementary data
Supplementary data associated with this article can be found in the
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Bioorganic & Medicinal Chemistry Letters 36 (2021) 127817
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