Synthesis and biological evaluation of 1‐(Diarylmethyl)‐1h‐1,2,4‐triazoles and 1‐(diarylmethyl)‐1h‐imidazoles as a novel class of anti‐mitotic agent for activity in breast cancer

Article abstract of DOI:10.3390/ph14020169

We report the synthesis and biochemical evaluation of compounds that are designed as hybrids of the microtubule targeting benzophenone phenstatin and the aromatase inhibitor letrozole. A preliminary screening in estrogen receptor (ER)‐positive MCF‐7 breast cancer cells identified 5‐((2H‐1,2,3‐triazol‐1‐yl)(3,4,5‐trimethoxyphenyl)methyl)‐2‐methoxyphenol 24 as a potent antiproliferative compound with an IC50 value of 52 nM in MCF‐7 breast cancer cells (ER+/PR+) and 74 nM in triple‐negative MDA‐MB‐231 breast cancer cells. The compounds demonstrated significant G2/M phase cell cycle arrest and induction of apoptosis in the MCF‐7 cell line, inhibited tubulin polymerisation, and were selective for cancer cells when evaluated in non-tumorigenic MCF‐10A breast cells. The immunofluorescence staining of MCF‐7 cells confirmed that the compounds targeted tubulin and induced multinucleation, which is a recognised sign of mitotic catastrophe. Computational docking studies of compounds 19e, 21l, and 24 in the colchicine binding site of tubulin indicated potential binding conformations for the compounds. Compounds 19e and 21l were also shown to selectively inhibit aromatase. These compounds are promising candidates for development as antiproliferative, aromatase inhibitory, and microtubule‐disrupting agents for breast cancer.

Full text of DOI:10.3390/ph14020169

pharmaceuticals
Article
Synthesis and Biological Evaluation of
1
1
-(Diarylmethyl)-1H-1,2,4-triazoles and
-(Diarylmethyl)-1H-imidazoles as a Novel Class of
Anti-Mitotic Agent for Activity in Breast Cancer
1
1
2
1
3
4
Gloria Ana , Patrick M. Kelly , Azizah M. Malebari , Sara Noorani , Seema M. Nathwani , Brendan Twamley ,
3
1
3
5
Darren Fayne , Niamh M. O’Boyle
, Daniela M. Zisterer , Elisangela Flavia Pimentel ,
Denise Coutinho Endringer 5 and Mary J. Meegan *
1,
1
School of Pharmacy and Pharmaceutical Sciences, Trinity College Dublin, Trinity Biomedical Sciences
Institute, 152-160 Pearse Street, Dublin 2, DO2R590 Dublin, Ireland; anag@tcd.ie (G.A.);
kellyp9@tcd.ie (P.M.K.); noorani87@gmail.com (S.N.); NIOBOYLE@tcd.ie (N.M.O.)
Department of Pharmaceutical Chemistry, College of Pharmacy, King Abdulaziz University,
Jeddah 21589, Saudi Arabia; amelibary@kau.edu.sa
2
3
School of Biochemistry and Immunology, Trinity College Dublin, Trinity Biomedical Sciences Institute,
1
52-160 Pearse Street, Dublin 2, DO2R590 Dublin, Ireland; seema.nathwani@outlook.com (S.M.N.);
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀ
ꢁꢂꢃꢄꢅꢆ
FAYNED@tcd.ie (D.F.); dzistrer@tcd.ie (D.M.Z.)
School of Chemistry, Trinity College Dublin, Dublin 2, DO2R590 Dublin, Ireland; TWAMLEYB@tcd.ie
Department of Pharmaceutical Sciences, University Vila Velha, Av. Comissário José Dantas de Melo, n 21,
Boa Vista Vila Velha—Espírito Santo, Vila Velha 29102-920, Brazil; eflapim@hotmail.com (E.F.P.);
endringe@gmail.com (D.C.E.)
4
5
Citation: Ana, G.; Kelly, P.M.;
Malebari, A.M.; Noorani, S.;
◦
Nathwani, S.M.; Twamley, B.; Fayne,
D.; O’Boyle, N.M.; Zisterer, D.M.;
Pimentel, E.F.; et al. Synthesis and
Biological Evaluation of
*
Correspondence: mmeegan@tcd.ie; Tel.: +353-1-896-2798
Abstract: We report the synthesis and biochemical evaluation of compounds that are designed as
hybrids of the microtubule targeting benzophenone phenstatin and the aromatase inhibitor letrozole.
A preliminary screening in estrogen receptor (ER)-positive MCF-7 breast cancer cells identified 5-((2H-
,2,3-triazol-1-yl)(3,4,5-trimethoxyphenyl)methyl)-2-methoxyphenol 24 as a potent antiproliferative
compound with an IC50 value of 52 nM in MCF-7 breast cancer cells (ER+/PR+) and 74 nM in triple-
1
-(Diarylmethyl)-1H-1,2,4-triazoles
and 1-(Diarylmethyl)-1H-imidazoles
as a Novel Class of Anti-Mitotic
Agent for Activity in Breast Cancer.
Pharmaceuticals 2021, 14, 169.
1
negative MDA-MB-231 breast cancer cells. The compounds demonstrated significant G /M phase
https://doi.org/10.3390/ph14020169
2
cell cycle arrest and induction of apoptosis in the MCF-7 cell line, inhibited tubulin polymerisation,
and were selective for cancer cells when evaluated in non-tumorigenic MCF-10A breast cells. The
immunofluorescence staining of MCF-7 cells confirmed that the compounds targeted tubulin and
induced multinucleation, which is a recognised sign of mitotic catastrophe. Computational docking
Academic Editor:
Marialuigia Fantacuzzi
Received: 5 January 2021
Accepted: 18 February 2021
Published: 22 February 2021
studies of compounds 19e, 21l, and 24 in the colchicine binding site of tubulin indicated potential
binding conformations for the compounds. Compounds 19e and 21l were also shown to selectively
inhibit aromatase. These compounds are promising candidates for development as antiproliferative,
aromatase inhibitory, and microtubule-disrupting agents for breast cancer.
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
Keywords: phenstatin; letrozole; tubulin polymerisation inhibitor; aromatase inhibitor; breast cancer;
hybrid molecule; dual-targeting molecule; apoptosis; designed multiple ligand
1
. Introduction
Designing single agents that act against multiple biological targets is of increasing
interest and prominence in medicinal chemistry [ ]. Dual-targeting drugs are designed
with the potential to be more potent and efficient and overcome many of the disadvantages
of single drugs such as low solubility, side effects [ ], and multidrug resistance (MDR).
While the molecular mechanisms of resistance to chemotherapeutics have been identified,
MDR is known to be a key factor in the failure of breast cancer chemotherapy [ ]. Tradition-
ally, drugs have been designed to target a single biological target (protein), aiming for high
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5
6
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selectivity and thus avoiding unwanted effects due to off-target events. The interaction
of a drug with multiple target proteins has been regarded as potentially associated with
adverse side effects. However, for complex diseases such as cancer, it is now recognised
that a single-target drug may not achieve the optimum therapeutic effect. Molecules that
are effective at more than one target protein may overcome incomplete efficacy and demon-
strate an increased safety profile compared to single-targeted ones [
strategies may offer a more favourable outcome of cancer treatment.
2]. Dual-targeting
A possible strategy to improve the outcome for postmenopausal breast cancer patients
is to design compounds with dual aromatase and tubulin targeting activities, which may
offer the potential benefits of improved efficacy and fewer side effects [
of our research is to investigate a new series of 1-(diarylmethyl)-1H-1,2,4-triazoles and 1-
diarylmethyl)-1H-imidazoles as a novel class of antimitotic compounds with an interesting
7,8]. The objective
(
biochemical profile particularly as tubulin-targeting agents and aromatase inhibitors for
the treatment of breast cancer.
Breast cancer is the most commonly diagnosed cancer in women; it is estimated that
approximately one in eight women will develop breast cancer during their lifetime, and it
is the most frequent cause of death for women in the age group 35–55 [
two million new cases in 2018 [10], and the number of cases is predicted to rise due to an
ageing population [11 12]. Mortality has decreased due to improved screening and early
9]. There were over
,
detection together with the use of adjuvant therapy [13]. Approximately 70–80% of breast
cancers are hormone-dependent; their growth is stimulated in response to the hormone
estrogen, with the majority of these estrogen receptor positive (ER+) cancers also expressing
the progesterone receptor (ER+/PR+ cancers). Upregulation of the gene encoding the PR is
directly mediated by ER, and PR modulates ERα action in breast cancer [14].
Aromatase (CYP19A1), a member of the cytochrome P-450 enzyme superfamily, catal-
yses the aromatisation of C-19 androgens to C-18 estrogens in the final step in estrogen
biosynthesis, and it is an attractive target for selective inhibition [15
privation is an effective therapeutic intervention for hormone-dependent breast cancer
HDBC) and has been clinically established by the inhibition of the aromatase enzyme. The
aromatase inhibitors (AIs), e.g., letrozole 18], anastrozole 19], and exemestane [20
Figure 1a), prevent the stimulating effects of estrogen in breast tissue [19], and they are
–17]. Estrogen de-
(
1
[
2
[
]
(
approved in the treatment of a wide spectrum of breast cancers [21]. These AIs have
demonstrated superior efficacy in postmenopausal women and have few associated risks
apart from reduction in bone density [8,21–23], and emerging resistance [24,25].
The selective estrogen receptor modulator (SERM) tamoxifen 3a (Figure 1a) is effective
for the treatment of ER+ breast cancer [13]; however, resistance is a clinical problem [26
together with a small increase in incidences of blood clots and endometrial cancers for
postmenopausal women [27 28]. The potential advantage of the tamoxifen metabolites
]
,
endoxifen (3b) and norendoxifen (3c) in endocrine-refractory metastatic breast cancer is
reported [29]. Breast cancers that are (ER+/PR+) are likely to respond to hormone therapy
such as tamoxifen and anastrozole [23], while the prophylatic use of tamoxifen, raloxifene,
or anastrozole is recommended for postmenopausal women at high risk of developing
breast cancer [30,31]. Approximately 20% of breast cancers overexpress the human epider-
mal growth factor receptor 2 (HER2), which promotes the growth of cancer cells.
Effective treatments for HER2+ breast cancers include the monoclonal antibody
trastuzumab [32], the antibody–drug conjugate ado-trastuzumab emtansine [33], and
the dual tyrosine kinase inhibitor lapatinib which targets both the HER/neu and the epi-
dermal growth factor receptor (EGFR) [34]. Breast cancers are classified as triple negative
(TNBC) when their growth is not supported by estrogen and progesterone nor by the
presence of HER2 receptors. The clinical options for treatment of TNBC are limited due
to poor response to hormonal therapy, resulting in low 5-year survival rates [35]. There is
extensive diversity among breast cancer patients, and each sub-type of breast cancer has
unique characteristics. The identification of sub-type-specific network biomarkers can be
useful in predicting the survivability of breast cancer patients [36].

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Figure 1. Cont.

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Figure 1.
Raloxifene), kinase inhibitors Lapatinib, Alpelisib, Tucatinib, steroid sulfatase inhibitor Irosustat and mutant p53 inhibitor
PRIMA-1, ( ) Combretastatins 4a–f, Isocombretastatins , phenstatins 7a–e, colchicine binding site inhibitors 8a 9a–c
and target structures 10.
(a) Aromatase inhibitors (letrozole, anastrozole, Exemestane), SERMs (Tamoxifen, Endoxifen, Norendoxifen,
b
5,
6
,b,
FDA-approved drugs for breast cancer in 2019 include the antibody–drug conju-
gate Fam-trastuzumab deruxtecan [37] (HER2-directed antibody and topoisomerase in-
hibitor) for the treatment of unresectable or metastatic HER2-positive breast cancer [38],
the phosphoinositide-3-kinase (PI3Kα) inhibitor alpelisib [39] for the treatment of HER2-
negative, PIK3CA-mutated, advanced or metastatic breast cancer [40] and in 2020, tuca-
tinib, an orally bioavailable, small molecule tyrosine kinase inhibitor for patients with
HER2-positive metastatic breast cancer [41]. The microtubule-stabilising drugs pacli-
taxel, docetaxel, and the epothilone ixabepilone were approved for use in patients with
metastatic breast cancer (MBC), alongside the microtubule destabilising vinca alkaloid
eribulin [42
topoisomerase inhibitor) conjugate sacituzumab govitecan (Trodelvy) for previously
treated metastatic TNBC [44], while ladiratuzumab vedotin (a LIV-1-targeted antibody
,43]. The FDA recently granted accelerated approval to the antibody–drug
(

Pharmaceuticals 2021, 14, 169
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linked to the microtubule-disrupting agent monomethyl auristatin E (MMAE)) is in clinical
trials for locally advanced or metastatic triple-negative breast cancer [45]. The steroid
sulfatase inhibitor (STS) e.g., STX64 (Irosustat) has entered clinical trials for ER+ locally ad-
vanced or metastatic breast cancer [46], while inhibitors of mutant p53, e.g., PRIMA-1 and
MET
PRIMA-1
, overexpressed in TNBC have been demonstrated to be effective in vitro [47].
Combretastatins CA-4 4a, (phosphate prodrug 4b), CA-1 4c (phosphate prodrug 4d),
e, and serine prodrug ombrabulin 4f (Figure 1b) have demonstrated impressive antiprolif-
4
erative potency with microtubule destabilising and anti-vascular effects in many cancers,
including breast cancer [48 50]. While many structurally related colchicine binding site
–
inhibitors have been reported [51–53], problems associated with the poor water solubility
and isomerisation causing an extensive loss of potency have hampered the progression
of combretastatins in clinical trials [54
of a series of CA-4 analogues with structures based on the conformationally constrained
-azetidinone ring, demonstrating potent activity in breast cancer cells [56]. Triazole [57 58
imidazole [59 60], and pyridine-containing analogs [61] of CA-4 are also reported with
antiproliferative activity in human cancer cell lines. Isocombretastatin A4 62] and 1,1-
diheterocyclic ethylenes derived from quinaldine and carbazole e.g., 63] and related
,55]. We have previously reported the synthesis
2
,
]
,
5
[
6
[
conjugates [64] display potent antiproliferative effects in cancer cells and induce G /M
2
cell cycle arrest [65]. The related benzophenone phenstatin 7a (Figure 1b) [66], together
with its sodium phosphate prodrug (7b) and metabolites 7c–e [67], show potent activity
in cancer cells and microtubule destabilising activity. The imidazole and indole hetero-
cycles are widely recognised as nuclei of great interest in the design of molecules with
anti-tumour activity [68,69]. Related fused-ring heterocyclic structures such as imidazo[2,1-
b][1,3,4]thiadiazoles with potent antiproliferative activity have also been reported [70]. A
variety of compounds structurally related to phenstatin, which contain the heterocycles
indole and imidazole, have been synthesised and subsequently evaluated for antimitotic
effects and vascular-disrupting effects in cancer cells [71
aroylindole (OXi8006) 8b are cytotoxic against selected human cancer cell lines and strongly
inhibit tubulin assembly [73 74]. Examples of novel imidazole and indole containing com-
pounds e.g., 9a 75], 9b 76], and 9c (BZML) [77] have been developed as potent tubulin
,72]. The azaindole 8a and 2-aryl-3-
,
[
[
polymerisation-targeting antiproliferative agents. The imidazole derivative BZML 9c is
a novel colchicine binding site inhibitor, which also overcomes multidrug resistance by
inhibiting P-gp function and inducing mitotic catastrophe [77]. Compounds such as 9a
(VERU-111), 9b, and 9c (BZML) containing both imidazole and indole nuclei exhibit potent
activity against a panel of cancer cell lines, are not substrates of P-glycoprotein, and inhibit
tumour growth in paclitaxel-resistant cell lines. 9c inhibits tumour growth and metastasis
in vivo [75–77].
A number of approaches to the design of dual targeting breast cancer agents have
been reported e.g., ER/tubulin [78], tubulin/HSP90 [79], tubulin/HSP27 [80], ER/AI e.g.,
norendoxifen [81,82], and endoxifen [83], sulfatase/AI [84], tubulin/sulfatase [85,86] and
tubulin/angiogenesis (vascular endothelial growth factor receptor-2 (VEGFR2) [87]. We
now report the synthesis and biological evaluation of a series of 1-(diarylmethyl)-1H-1,2,4-
triazoles, 1-(diarylmethyl)-2H-1,2,3-triazoles, and 1-(diarylmethyl)-1H-imidazoles, which
are designed as hybrid scaffolds derived from the benzophenone structure of the tubulin
targeting phenstatin 7a, together with the 1,2,4-triazole of the aromatase inhibitor letrozole
2 [88]. These compounds are designed to provide a selective anti-tumour effect by targeting
tubulin polymerisation and also would be effective by inhibiting estrogen production.
Although aromatase inhibitors such as letrozole are widely used in the treatment of breast
cancer, dual tubulin–aromatase inhibitors have not been reported to date. 1-(Diarylmethyl)-
1H-1,2,4-triazole and 1-(diarylmethyl)-1H-1,2,4-imidazole derivatives have been previously
investigated as dual aromatase-steroid sulfatase inhibitors [89]. The target structures 10
are shown in Figure 1b. In addition, a number of related compounds containing the cyclic
amines pyrrolidine, piperidine, and piperazine are investigated. We wished to develop

Pharmaceuticals 2021, 14, 169
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this strategic approach with the aim of targeting dual tubulin–aromatase inhibition and
have investigated a series of dual-targeting inhibitors.
2. Results and Discussion
2.1. Chemistry
A series of benzophenone-like compounds related in structure to phenstatin 7a were
first prepared (11a–11m). The carbonyl group of the benzophenone was subsequently
reduced to afford a benzhydryl alcohol; the heterocycles 1,2,4-triazole, 1,2,3-triazole, im-
idazole, piperidine, pyrrolidine, and piperazine were introduced in order to afford the
N-benzhydryl-heterocyclic products (Schemes 1–8). These compounds were investigated
as potential dual-active hybrids: the benzophenone scaffold was designed to interact with
tubulin, while the heterocyclic ring was incorporated to target the aromatase enzyme.
◦
◦
Scheme 1. Synthesis of compounds 13a–13o (Series 1). Reagents and conditions: (
a
) NaBH4, MeOH, 0 C, (38–100%);
o
(
b
) 1,2,4-triazole, p-TSA, toluene, 4 h, 120 C, microwave open vessel, (3–98%); (
c
) K CO , MeOH, H O, 20 C, 72 h, (16%);
2
3
2
◦
◦
(
d) H , Pd(OH) , ethyl acetate, 20 C, (67%); (e) TBAF, THF, 0 C, (90%) [TBDMS, tert-butyldimethylsilyl; Bn, CH C H ].
2
2
2
6
5
Scheme 2. Synthesis of letrozole-phenstatin hybrid compounds 16a–16i (Series 2a). Reagents and conditions: (
a
) n-BuLi,
◦
◦
THF,
−
78 C, 1.5 h, (21–89%); (
b
) 1,2,4-triazole, p-TSA, toluene, 4 h, 120 C, microwave open vessel, (34–93%); (
c) Pd(OH)2,
◦
H , ethyl acetate, 20 C, (49%). [Bn, CH C H ].
2
2
6
5

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Scheme 3. Synthesis of letrozole-phenstatin hybrid compounds 19a–19e (Series 2a) and 19f-19i (Series 2b). Reagents and
◦
conditions: (
(
a
): n-BuLi, dry THF,
−
78 C,1.5 h, (16–88%); (
b) 1,2,4-triazole, p-TSA, microwave open vessel, 4h, (64–95%);
c) Pd(OH) , H , (66–82%). [Bn: CH C H ].
2 2 2 6 5
Scheme 4. Synthesis of hybrid imidazole-phenstatin compounds 20a–20l (Series 3). Reagents and conditions: (
a) CDI,
◦
CH CN, reflux, 3 h, (10–64%); (b) K CO , MeOH, H O, 20 C, 72, (50%). [Bn: CH C H ].
3
2
3
2
2
6
5

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Scheme 5. Synthesis of hybrid imidazole-phenstatin compounds 21a–21l, (Series 4). Reagents and conditions: (
a
) CDI,
◦
CH CN, reflux, 3 h, (30–100%). (b) H , Pd(OH) , ethyl acetate, 20 C, (93%). [Bn: CH C H ].
3
2
2
2
6
5
Scheme 6. Synthesis of phenstatin 7a and phenstatin hybrids 21b
,
21c
,
24, (Series 4). Reagents and conditions: (
a) Eaton’s
◦
reagent (P O , CH SO H), 60 C, 3 h, [23a (60%), 23b (57%), 23c (17%]); (
b
) sodium acetate, methanol, refux, 2 h, (89%);
2
5
3
3
o
◦
(
c
) NaBH4, MeOH, 0 C, [15c (50%), 15d (89%),15i (96%)]; (
d) 1,2,3-triazole, p-TSA, toluene, 4 h, 120 C, microwave open
vessel, (77%); (e) CDI, CH CN, reflux, 3 h, [21b (39%), 21c (67%)].
3
The target compounds are arranged as follows:
Series 1: 1-(Diarylmethyl)-1H-1,2,4-triazoles 13a–13o
Series 2a: 1-(Aryl-(3,4,5-trimethoxyphenyl)methyl)-1H-1,2,4-triazoles 16a–i, 19a–e,
-(aryl-(3,4,5-trimethoxyphenyl)methyl)-1H-1,2,3-triazole 24
Series 2b: 4-(Aryl-(1H-1,2,4-triazol-1-yl)methyl)benzonitriles 19f–i
Series 3: 1-(Diarylmethyl)-1H-imidazoles 20a–l
Series 4: 1-(Aryl-(3,4,5-trimethoxyphenyl)methyl)-1H-imidazoles 21a–l,
Series 5: 1-(Diarylmethyl)pyrrolidines 25a–g, 1-(diarylmethyl)piperidines 26a–c and
-(diarylmethyl)piperazines 27a–i, 28
1
1

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Scheme 7. Synthesis of pyrrolidine 25a–g and piperidine derivatives 26a–c, (Series 5). Reagents and conditions:
o
(
a
) SOCl ,CH Cl , 12 h, 20 C; (
b) pyrrolidine, acetonitrile,12 h, reflux (23–93%); (
c) piperidine, acetonitrile,12 h, re-
2
2
2
flux (84–91%).
Scheme 8. Synthesis of piperazine-phenstatin compounds 27a–g, 28, (Series 5). Reagents and conditions: (
a
) SOCl2,
o
CH Cl ,12 h, 20 C; (
b
) N-phenylpiperazine, N-benzylpiperazine, N-Boc-piperazine or p-methoxyphenylpiperazine,
2
2
o
acetonitrile, reflux, 12 h (6–80%) (
c) TFA, CH Cl , 30 min, 20 C (42%); (d) H , Pd(OH) (45%); (e): piperazine, ACN,
2 2 2 2
acetonitrile, reflux, 12 h (12%). [Boc:tert-Butoxycarbony; Bn: CH C H ].
2
6
5

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2
.1.1. 1-(Diarylmethyl)-1H-1,2,4-triazoles (Series 1 and 2)
The general reaction scheme for the preparation of the 1-(diarylmethyl)-1H-1,2,4-
triazoles 13a–o (Series 1) heterocyclic derivatives of benzophenones is shown in Scheme 1
These initial compounds carry a single substituent at the para position on one or both
of the aryl rings (Cl, F, Br, OH, OCH , CH etc.). The benzophenones (11a–m) were
.
3
3
reduced with sodium borohydride to afford the secondary alcohols 12a–m, in good yields,
Scheme 1. Coupling of 1,2,4-triazole with the secondary alcohols 12a–12m to afford the
benzhydryl-1H-1,2,4-triazoles 13a–l was achieved using p-TSA as a catalyst in an open-
vessel microwave reactor. Amine 13m was obtained on treatment of amide 13i with
potassium carbonate, while the phenol 13n was obtained by hydrogenolysis of the benzyl
ether 13j with palladium hydroxide. Deprotection of the silyl ether 13k with TBAF afforded
1
the diphenol 13o. In the H-NMR spectrum of compound 13o, the singlets (8.41 and
7
6
.97 ppm) were identified as the triazole H5 and H3 respectively, while the singlet at
.75 ppm corresponds to the methine proton. In the 13C-NMR spectrum of 13o, the signal
at 65.12 ppm was assigned to the tertiary carbon, while the signals at 151.52 and 143.93 ppm
are assigned to C3 and C5 of the triazole (see Supplementary Information).
Since the potent tubulin-inhibiting activity of the 3,4,5-trimethoxyaryl function is very
well documented in colchicine-binding site inhibitors [90], the 1,2,4-triazole heterocycle
was next reacted with several phenstatin-type 3,4,5-trimethoxyaryl substituted benzhydryl
alcohols in order to maximise the potential tubulin activity in the scaffold structures with
aromatase-inhibiting action (Series 2). It was decided to retain in most compounds the
3
,4,5-trimethoxyaryl group substitution (ring A) and introduce alternative substituents on
the second ring (ring B). A modified synthetic procedure allowing access to the desired
benzhydryl alcohol intermediates 15a–h and 18a–f is shown in Schemes 2 and 3 (step a) [91].
Scheme 2 shows the alcohols (15a–h) obtained by treatment of the appropriate aryl bro-
mides 14a–h with n-butyllithium followed by reaction with 3,4,5-trimethoxybenzaldehyde
(
(
A ring) to afford the alcohols 15a–h in yields of 21–89%. For the preparation of compounds
18a–d) (Scheme 3), the A ring was derived from 3,4,5-trimethoxybromobenzene followed
by reaction with the appropriate aldehyde 17a–d. The nitrile-containing compounds 18e
,f
were similarly obtained from the aldehydes 17e and 4-bromobenzonitrile (Scheme 3).
,
f
The benzhydryl compounds were obtained in good yield after purification via flash col-
umn chromatography and the presence of the hydroxyl group was confirmed from IR
−
1
(
ν 3200–3600 cm ).
Then, the secondary alcohols 15a–h and 18a–f were reacted with 1,2,4-triazole to
afford the hybrid phenstatin/letrozole compounds 16a–h and 19a–d
,f
,g
as racemates,
except for 19b, (Schemes 2 and 3, step b). The phenolic compounds 16i
,
19e 19h, and 19i
,
were obtained by hydrogenolysis over palladium hydroxide of the benzyl ethers 16b
,
19a
,
1
19f, and 19g respectively. From the H-NMR spectrum of compound 16c, the singlet at
6
.62 ppm was assigned the tertiary aliphatic proton. The singlets at 7.91 and 8.01 ppm were
13
assigned to the triazole H-3 and H-5. In the C-NMR spectrum, the tertiary CH signal was
identified at 67.4 ppm, while the triazole ring C3 and C5 signals were identified at 143.5
and 152.3 ppm, respectively.
X-ray crystal structures of the triazole compounds 16e, 16f, and 19c (recrystallised
from dichloromethane/n-hexane) are displayed in Figure 2, while the crystal data and
structure refinement are displayed in Table 1. The length of the C-N bond between the
methine carbon and the triazole N-1 for compounds 16e
, 16f, and 19c was measured at
1.470, 1.471, and 1.479 Å, respectively. The N1-N2 bond length was 1.366 Å (16e), 1.363 Å
(
(
16f), and 1.365 Å (19c). The N1-C5 bond length of the triazole ring was observed as 1.334 Å
16e), 1.342 Å (16f), and 1.343 Å (19c). The angle between the methine carbon and the two
◦
◦
◦
aromatic rings (Ar-C1-Ar) was measured as 112.51 , 115.08 , and 113.53 respectively for
compounds 16e
◦
,
16f, and 19c. The corresponding value for the letrozole structure is 114.0 ,
while the C-N bond between the methine carbon and the triazole N-1 was 1.46 Å [92].

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Figure 2. X-ray crystallography structures of 16e
,
16f, and 19c with heteroatoms labelled and
displacement shown at 50% probability.
2
.1.2. 1-(Diarylmethyl)-1H-imidazoles (Series 3 and 4)
A series of related imidazole-containing compounds were also prepared 20a–l (Series
) and 21a–k (Series 4). The secondary alcohols 12a–h , and were coupled to imida-
3
,
j,
k
m
zole using CDI (carbonyldiimidazole) [93] to afford products 20a–k, Series 3, (Scheme 4,
step a). The associated carbamate derivatives were not isolated in our reactions [94]. The
hydrolysis of 20i afforded the amine 20l in 50% yield (Scheme 4, step b). Structures were op-
timised with variations in electron-releasing and electron-withdrawing substituents on the
aryl rings. A further series of compounds containing the ring A type 3,4,5-trimethoxyaryl
substituents was prepared by reacting alcohols 15a,c–h, and 18a–d with CDI to afford imi-
dazole products 21a–k, Series 4, (Scheme 5, step a). The benzyl ether 21h was treated with
1
Pd(OH) to afford the phenol 21l as a racemate in 93% yield (Scheme 5, step b). In the H
2
NMR spectrum of compound 21i, the imidazole H4 was observed as a singlet at 6.88 ppm,
while the H2 and H5 were observed at 7.44, and 7.12 ppm, respectively. The singlet at 6.38
ppm was assigned to the tertiary aliphatic CH. From the 13C-NMR spectrum, the aliphatic
tertiary CH was identified at 65.2, while the signals at 138.0, 129.4, and 119.4 ppm were
assigned to the imidazole C2, C4, and C5, respectively.

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Table 1. Crystal data and structure refinement details for compounds 16e, 16f, 19c, 21i, and 26a.
Compound
16e
16f
19c
21i
26a
CCDC no.
Empirical formula
M (g/mol)
T (K)
Crystal System
SG
2015431
2015432
2015433
2015434
2015435
C H N O
C H N O
C H N O
C H N O
C H FN
1
8
19
3
3
19 21
3
3
20 23
3
4
22 26
2
6
18 20
325.36
100(2)
monoclinic
339.39
100(2)
monoclinic
369.41
100(2)
monoclinic
414.45
100(2)
monoclinic
269.35
100(2)
monoclinic
P2 /n
1
P2 /c
1
P2 /c
1
P2 /c
1
P2 /c
1
a (Å)
b (Å)
8.1796(5)
14.1725(10)
14.4742(10)
90
98.7882(11)
90
9.2487(4)
9.6620(4)
20.0006(9)
90
97.8110(10)
90
12.8748(6)
9.8550(5)
14.8091(7)
90
93.1792(8)
90
14.1937(6)
13.1553(5)
12.4613(5)
90
113.2250(10)
90
5.9228(2)
14.0705(5)
17.7920(6)
90
98.275(2)
90
1467.29(9)
4
1.219
c (Å)
◦
α ( )
◦
β ( )
◦
γ ( )
3
V (Å )
1658.23(19)
4
1770.69(13)
4
1876.10(16)
4
2138.25(15)
4
Z
3
Dcalc (g/cm )
1.303
0.09
688
1.273
0.088
720
1.308
0.092
784
1.287
0.094
880
−
1
µ (mm
F(000)
)
0.628
576
MoKα
λ = 0.71073)
MoKα
(λ = 0.71073)
MoKα
(λ = 0.71073)
MoKα
(λ = 0.71073)
CuKα
(λ = 1.54178)
Radiation
(
Reflections
collected
6
1043
298
Rint = 0.0532,
Rsigma = 0.0237]
298/0/217
115661
95764
85391
20214
4
5438
[Rint = 0.0264,
Rsigma = 0.0097]
5504
[Rint = 0.0364,
Rsigma = 0.0143]
6261
[Rint = 0.0385,
Rsigma = 0.0188]
2684
[Rint = 0.0634,
Rsigma = 0.0356]
Independent
reflections
[
Data/restraints/
parameters
4
5438/0/230
1.043
5504/0/248
1.046
6261/0/277
1.005
2684/1/191
Goodness-of-fit on
1
.018
1.063
2
F (S)
Final R indexes
R = 0.0407,
R = 0.0374,
R = 0.0381,
R = 0.0405,
R1 = 0.0479,
wR2 = 0.1325
R1 = 0.0547,
wR2 = 0.1369
1
1
1
1
[
I ≥ 2σ (I)] *
Final R indexes
all data]
wR = 0.0904
wR = 0.1018
wR = 0.0938
wR = 0.0967
2
2
2
2
R = 0.0613,
R = 0.0445,
R = 0.0506,
R = 0.0580,
1
1
1
1
[
wR = 0.1005
wR = 0.1083
wR = 0.1025
wR = 0.1079
2
2
2
2
Largest diff.
peak/hole/e Å-3
0
.31/−0.21
0.42/−0.39
0.42/−0.21
0.38/−0.24
0.19/−0.21
R1 = Σ||Fo| − |Fc||/ Σ|Fo|, wR2 = [Σ w(Fo2 − Fc ) /Σw(Fo ) ]
2 2
2 2 1/2.
*
Single crystal X-ray analysis was obtained for compound 21i (recrystallised from
dichloromethane/n-hexane), and the crystal structure is shown in Figure 3. The crystal data
and structure refinement for compound 21i are displayed in Table 1. The angle between the
◦
methine carbon and the aryl rings (114.16 ) and also the bond length between the methine
carbon and the N-1 imidazole nitrogen (1.471 Å) were similar to the corresponding values
obtained for the triazole compounds 16e
, 16f, and 19c (Table 1). The bond angles between
◦
◦
the aryl rings and the imidazole ring were determined as 111.36 and 111.80 , also similar
◦
◦
to the corresponding values of 109.99 and 112.6 reported for letrozole [92].
An alternative approach for the preparation of phenstatin and related azole compounds
using a Friedel–Crafts acylation with Eaton’s reagent was also investigated (Scheme 6) [67].
3
,4,5-Trimethoxybenzoic acid was reacted with anisole (22a), 1,2-dimethoxybenzene (22b),
or compound 22c (prepared by the protection of 2-methoxyphenol with chloroacetyl chlo-
ride) using Eaton’s reagent (readily prepared from phosphorus pentoxide and methane-
sulfonic acid) to afford respectively benzophenones 23a
Then, these benzophenones were reduced to the benzhydryl alcohols 15c
,
23b, and 23c (Scheme 6, Step a).
15d, and 15i
,
,
respectively with sodium borohydride (Scheme 6, step a), with the concomitant removal
of the chloroacetyl protecting group of 23c. Although requiring an additional step, this
method was followed after the reaction of the aryl bromide with the aldehyde to afford
the alcohol as shown in Schemes 2 and 3 was not successful or did not afford a sufficient

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quantity of product for the next step e.g., for compound 15d, the overall yield increased to
5
1% compared with 30%. Then, compounds 15c and 15d were treated with CDI azole to
afford the imidazole-containing products 21b and 21c (Scheme 6, step e).
Figure 3. X-ray crystallography of compounds 21i and 26a with heteroatoms labelled and displace-
a
ment shown at 50% probability. Disordered molecular structure of 26a showing the major occupied
moiety F1a. b Disordered molecular structure of 26a showing the minor occupied moiety F1B.
The phenol 15i was also reacted with 1,2,3-triazole to afford the product 24 in 77%
yield, Series 4, (Scheme 6, step d). Compound 24 is the only phenstatin derivative substi-
tuted with 1,2,3-triazole synthesised in this project and was investigated for comparison
1
with the 1,3,4-triazole compound series. In the H NMR spectrum of 24, the signal at
6
.99 ppm was assigned to the tertiary CH. Interestingly, the two protons of the 1,2,3-triazole
ring were observed as a singlet with an integration of 2H at 7.83 ppm, while the signal at
34.9 ppm in the 13C-NMR spectrum of 24 was assigned to the C4 and C5 of the triazole
1
ring, indicating that alkylation occurred at N2 of the 1,2,3-triazole [95]. The alkylation
of 1,2,3-triazoles may result in the formation of regioisomers depending on the reaction
conditions e.g., solvent, temperature, and catalyst used [96]. The signal for the tertiary
CH was observed at 71.0 ppm. The benzophenone 23c was also used in the preparation of
phenstatin 7a [67]; the deprotection of 23c by reaction with sodium acetate afforded 7a in
8
9% yield (Scheme 6, step b), which was used as a positive control in the cell viability tests.

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2
.1.3. 1-(Diarylmethyl)Pyrrolidines, 1-(Diarylmethyl)Piperidines, and 1-(Diarylmethyl)
Piperazines (Series 5)
The preparation of a series of benzhydryl derivatives substituted on the tertiary
carbon with the heterocycles pyrrolidine, piperidine, and piperazine was next investigated
(Series 5, Schemes 7 and 8). These products allow a comparison of biochemical activity
with the related imidazole and triazole compounds from Series 1–4. The advantages of
incorporating such heterocyclic rings into drugs are well known; i.e., they can increase the
lipophilicity, polarity, and aqueous solubility of the drug [97]. In particular, piperazine is
rd
ranked 3 among the 25 most common heterocycles contained in FDA-approved drugs [98].
In the present work, the corresponding secondary benzhydryl chloride was prepared from
the secondary alcohols 12b–12g, 15c, and 18a using thionyl chloride (Schemes 7 and 8,
step a) [93]. The intermediate alkyl chlorides were reacted with piperidine to afford
products 26a–c (Scheme 7, step c), while reaction with pyrrolidine yielded derivatives
25a–g (Scheme 7, step b).
An alternative synthesis of 1-(diarylmethyl)piperidines is reported using a copper(I)-
catalysed coupling reaction of aryl boronic acids with N,O-acetals and N,N-aminals [99].
All compounds are racemates apart from compound 25e and were obtained in moderate
1
yields (23–93%). In the H-NMR spectrum of compound 25b, the multiplets at 1.71–
1
.80 and 2.35–2.43 ppm were assigned to the pyrrolidine methylene protons at H-3,4
and H-2,5 respectively, while the tertiary CH was observed as a singlet at 4.11 ppm. In
13
the C-NMR spectrum, the pyrrolidine C-3 and C-2 signals were at 23.5 and 53.6 ppm,
respectively. The signal at 75.7 ppm was assigned to the tertiary carbon. Single crystal
X-ray analysis for compound 26a is shown below in Figure 3 (obtained by crystallisation in
dichloromethane/n-hexane). The crystal data and structure refinement for compound 26a
are displayed in Table 1. In 26a, the disordered fluorine was modelled in two positions with
occupancies of 84% and 16%. The C1-N bond length was observed as 1.473 Å and the central
◦
◦
C -C -C and C -C -N angles were observed as 109.28 and 112.09 , respectively. The
14
1
8
14
1
2
piperidine ring bond lengths were 1.471 Å (N2-C3), 1.474 Å (N2-C7), and 1.514 Å (C3-C4),
which differ from the N1-C bond length of the triazole ring 1.334 Å due to unsaturation.
As a further extension of this research, a related series of piperazine-containing
compounds was prepared by coupling selected secondary alcohols with the appropriate
piperazine derivative (Series 5, Scheme 8). The preparation of diarylmethylamines has
been reported by Le Gall et al. by reaction of the aldehyde and piperidine derivative to a
solution of the organozinc reagent in acetonitrile in a Mannich-type reaction [100
,101]. The
secondary alcohols 12e 15c, and 18a were treated with thionyl chloride (Scheme 8, step
,
a), and the resulting alkyl chloride was used immediately for the next reaction step (step
b) by addition of the appropriate piperazine (N-phenylpiperazine, N-benzylpiperazine, p-
methoxyphenylpiperazine, or N-Boc-piperazine) to afford the products 27a–g in yields up
to 80%. For the preparation of compound 27e, Boc-protected piperazine was used to avoid
1
the possible formation of the dimer. In the H-NMR spectrum of compound 27d, the broad
signal at 2.47 ppm is assigned to piperazine methylene protons; the singlet at 3.50 ppm is
assigned to the benzyl methylene, while the singlet at 4.09 ppm corresponds to the tertiary
C-1 proton. The 13C-NMR spectrum of compound 27d further confirms the proposed
structure. The signals at 51.8 and 53.3 ppm were characteristic of the piperazine ring
protons, the signals at 63.0 ppm and 75.6 ppm are assigned to the benzyl methylene and
tertiary CH, respectively. The deprotection of compound 27e with TFA afforded compound
27h as a yellow oil (42%), (Scheme 8, step c). A palladium-catalysed hydrogenolysis of 27g
afforded the phenolic compound 27i in 45% yield (step d), which is the phenylpiperazine
−
1
derivative of phenstatin. Its formation was confirmed by IR spectroscopy (3475 cm ).
When the secondary alcohol 15c was treated with thionyl chloride followed by an
excess of piperazine (5 equivalents), the product obtained was a piperazine dimer 28
1
(
Scheme 8). In the H-NMR spectrum of the dimer 28, the broad signal (2.40 ppm) is
characteristic of the piperazine methylene protons, while the signal at 4.08 ppm integrating
for 2H was assigned to the two tertiary CH protons. In the 1 C-NMR spectrum, the signal
3

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at 52.0 ppm was assigned to the piperazine ring carbons; the signal at 75.7 ppm was
assigned to the CH, while the duplication of the aromatic signals confirmed the formation
of the product.
2.2. Stability Studies
HPLC stability studies were performed on representative compounds 21l and 24 to es-
tablish their stability at different pH systems, which mimic in vivo conditions. Compound
1l was chosen among these imidazole compounds for HPLC stability studies at three
2
different pH systems; acidic pH 4, pH 7.4, and basic pH 9 (acid pH found in the stomach,
basic found in the intestine, and pH 7.4 in the plasma). The degradation of compound 21l
was minimal with 80% of 23l remaining at both pH 7.4 and pH 9 and 90% at pH 4 after
2
4 h. The 1,2,3-triazole compound 24 was observed to be most stable at pH 4 with 65%
remaining after 24 h compared to 60% at pH 9 and 50% at pH 7.4.
3. Biochemical Results and Discussion
3.1. In Vitro Antiproliferative Activity in MCF-7 Breast Cancer Cells
The antiproliferative activity of the panel of hybrid compounds 1-(diarylmethyl)-1H-
1
,2,4-triazoles (Series 1 and 2) and 1-(diarylmethyl)-1H-imidazoles (Series 3 and 4) was
initially evaluated in the MCF-7 human breast cancer cell using the standard alamarBlue
assay. In addition, a number of related compounds containing the aliphatic amines pyrroli-
dine, piperidine, and piperazine were investigated (Series 5). The MCF-7 human breast
cancer cell line is estrogen receptor (ER)-positive, progesterone receptor (PR)-positive, and
HER2 negative. Compounds were initially screened at two concentrations (1 and 0.1 µM)
for antiproliferative activity in MCF-7 cells to determine the structure–activity relationship
for these hybrid compounds and to identify the most potent compounds for further inves-
tigation. Compounds that were synthetic intermediates for the final compound were not
screened, as they were not considered as potential actives in the study. The results obtained
from this preliminary screen are displayed in Figures 4–6. Then, those compounds showing
potential activity (cell viability <50%) were selected for further evaluation at different
concentrations and in other cell lines. CA-4 (4a) (24% viable cells at 1
µM) and phenstatin
(
7a) (30% viable cells at 1 M) induced a potent antiproliferative effect and were used as
µ
positive controls. Ethanol (1% v/v) was used as the vehicle control (with 99% cell viability).
The preliminary results obtained for these novel compounds (Series 1–5) are discussed by
structural type.
3.1.1. Series 1: 1-(Diarylmethyl)-1H-1,2,4-triazoles 13b–g, l–o
The first class of compounds tested 1-(diarylmethyl)-1H-1,2,4-triazoles (13b–g, 13l–o,
Figure 4A) were weakly active, with 68–90% viability for the two concentrations tested
M and 0.1 M). These compounds carry a single substituent at the para position on one
or both aryl rings (Cl, F, Br, OH, OCH , CH , etc.) indicating that the triazole ring alone is
(
1
µ
µ
3
3
not sufficient for the induction of antiproliferative activity in MCF-7 cells. The most active
compounds were the diphenolic derivative 13o with 68% viability (1 M) and the amino
compound 13m (72% viability 1 M). It appears that specific substituents are required on
µ
µ
both the A and B rings of the benzophenone for activity, as also observed for phenstatin
and analogues [67].
3
.1.2. Series 2: 1-(Aryl-(3,4,5-Trimethoxyphenyl)Methyl)-1H-1,2,4-Triazoles 16a
,c–i, 19b–e,
1
9h, 19i and 1-(Phenyl(3,4,5-Trimethoxyphenyl)Methyl)-1H-1,2,3-Triazole 24
Since the potent tubulin inhibiting activity of the 3,4,5-trimethoxyaryl function is very
well documented [90], the preliminary screening in MCF-7 cells of the panel of 1,2,4-triazole
containing compounds (16a, c–i 19b–e) synthesised having the 3,4,5-trimethoxyphenyl
motif (A ring) together with various substituents on the B ring was next investigated
Figure 4B, two concentrations of 1 M and 0.1 M). The most potent compound was
identified as 19e having the characteristic 3-hydroxy-4-methoxyaryl B ring as in phenstatin
,
(
µ
µ

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and CA-4 (29% viability at 1
µM), while the ethanol control (1% v/v) resulted in 99%
viability. Two compounds with moderate activity were identified as 16c (4-methoxy group
in the B ring) with 75% cell viability at 1 M and 16g (4-fluoro in B ring) with 77% viable
cells at 1 µM. The remaining 3,4,5-trimethoxyphenylmethyl-1H-1,2,4-triazole compounds
investigated having various substituents on the B ring e.g., 4-F, 4-CN, 4-OH, 4-CH were
µ
3
not as potent as the lead compound with viability >80% at 1
µM, while compounds 19h
and 19i were found to be inactive with half maximal inhibitory concentration (IC ) values
5
0
greater than 100 µM. This result demonstrated that even small changes to the phenstatin
scaffold were unfavourable for antiproliferative activity. From the initial screening results,
it was concluded that the 1,2,4-triazole heterocycle alone was not sufficient to improve
activity in the benzhydryl compounds compared to phenstatin.
Figure 4. Preliminary cell viability data for (
diarylmethyl)-1H-1,2,4-triazoles 16a c–i 19b–e, 1-(diarylmethyl)-2H-1,2,3-triazole 24 in MCF-7 breast cancer cells. Cell
proliferation of MCF-7 cells was determined with an alamarBlue assay (seeding density 2.5 104 cells/mL per well for
6-well plates). Compound concentrations of either 1 or 0.1 M for 72 h were used to treat the cells (in triplicate) with
A) Series 1: 1-(diarylmethyl)-1H-1,2,4-triazoles 13b–g, l–o (B) Series 2: 1-
(
,
,
×
9
µ
control wells containing vehicle ethanol (1% v/v). The mean value + SEM for three independent experiments is shown. The
positive controls used are CA-4 and phenstatin.

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Figure 5. Preliminary cell viability data for (A) Series 3: 1-(diarylmethyl)-1H-imidazoles 20b-h, k, l and (B) Series 4:
1
-(diarylmethyl)-1H-imidazoles 21a–g, i–l in MCF-7 breast cancer cells. Cell proliferation of MCF-7 cells was determined
4
with an alamarBlue assay (seeding density 2.5
either 1 or 0.1
×
10 cells/mL per well for 96-well plates). Compound concentrations of
µM for 72 h were used to treat the cells (in triplicate) with control wells containing vehicle ethanol (1% v/v).
The mean value
±
SEM for three independent experiments is shown. The positive controls used are CA-4 and phenstatin.
The IC50 value for the most potent triazole–phenstatin hybrid compound 19e was
determined in MCF-7 as 0.42
±
0.07 µM at 72 h (Table 2). 19e is a hybrid of phenstatin with
the 3,4,5-trimethoxyaryl motif (ring A) and the 3-hydroxy-4-methoxyaryl B ring, but it is
also related to the aromatase inhibitor letrozole due to the 1,2,4-triazole heterocycle. The
hybrid structure suggests a potential for dual tubulin/aromatase activity, and therefore,
this compound was selected for aromatase inhibition assay.

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Figure 6. Preliminary cell viability data for (
A) Series 5: 1-(diarylmethyl)pyrrolidines and 1-
(
diarylmethyl)piperidines 25a–g, 26a–c and (B) Series 5: 1-(diarylmethyl)piperazines 27c,d,f,i, and h
in MCF-7 breast cancer cells. Cell proliferation of MCF-7 cells was determined with an alamarBlue
4
assay (seeding density 2.5
either 10, 1, or 0.1
vehicle ethanol (1% v/v). The mean value
×
10 cells/mL per well for 96-well plates). Compound concentrations of
M for 72 h were used to treat the cells (in triplicate) with control wells containing
µ
±
SEM for three independent experiments is shown. The
positive controls used are CA-4 and phenstatin.
Table 2. Antiproliferative effects of selected azole compounds 19e, 21l, and 24 in MCF-7, MDA-MB-
2
31 human breast cancer cells, and HL-60 leukaemia cell line.
Antiproliferative
Antiproliferative
Antiproliferative
Activity a
a
a
Activity
Activity
Compound
MCF-7 Cells
IC50 (µM)
MDA-MB-231 Cells
HL-60 Cells
IC50 (µM)
IC50 (µM)
0.261 b
1
2
2
9e
1l
4
0.042 ± 0.070
0.132 ± 0.007
0.052 ± 0.040
0.978 ± 0.130
0.237 ± 0.040
0.074 ± 0.030
0.156 ± 0.052
0.173 ± 0.055
c
c
c
CA-4
0.0039 ± 0.00032
0.0430
0.0019 ± 0.0005
a
IC50 values are half maximal inhibitory concentrations required to block the growth stimulation of MCF-7,
−
6
MDA-MB-231, and HL-60 cells. Values represent the mean (SEM (error values 10 ) for three experiments
performed in triplicate. Antiproliferative activity for compound 19e against HL-60 from NCI. c The IC50 value
obtained for CA-4 in this assay are in agreement with the reported values for CA-4 in MCF-7 and MDA-MB-231
human breast cancer and leukaemia cell lines (see Refs. [62,102,103]).
b

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Compound 24 is the only example synthesised containing the 1,2,3-triazole hete-
rocycle and is also a direct analogue of phenstatin because of the presence of the 3,4,5-
trimethoxyaryl motif (ring A) and the 3-hydroxy-4-methoxyaryl B ring. This structure
showed excellent activity in MCF-7 cells with 27% cell viability at 1
value for the compound was determined as 52 nM (Table 2), which compares with CA-4
IC50 = 3.9 nM) [102 103]. 24 was selected for further studies on different cell lines and for
µM and the IC50
(
,
cell cycle analysis. Phenstatin (7a) was synthesised in our laboratory for use as a positive
control (IC50 = 1.61 ± 2.7 nM) [104].
3.1.3. Series 3: 1-(Diarylmethyl)-1H-Imidazoles 20b–h,k,l
The results obtained from the preliminary screening of the benzhydryl imidazole
derivatives, 20b–k and
l are shown in Figure 5A. These compounds carry a single sub-
stituent at the para position on one or both aryl rings (Cl, F, Br, OH, OCH , CH , etc). This
3
3
library of compounds did not show any significant activity, with cell viability of 67–90%
at concentrations of 1 and 0.1 M, as observed for the Series 1 1,2,4-triazole derivatives
3b and l–o, indicating that the imidazole ring alone is not sufficient for antiproliferative
activity. The most active compounds in this panel were identified as the 4-nitro derivative
0b and the 4-fluoro substituted compound 20d (73% and 67% cell viability respectively
at 1 µM).
µ
1
–g
2
3.1.4. Series 4: 1-(Aryl-(3,4,5-Trimethoxyphenyl)Methyl)-1H-Imidazoles 21a-g, i-l
The results obtained from the preliminary screening of the panel of phenstatin hybrid
compounds carrying imidazole as the heterocyclic ring (21a–g
shown in Figure 5B. From the library of 3,4,5-trimethoxydiphenylmethyl-1H-imidazole
derivatives (21a–g i–l), compound 21l was significantly the most active (31% viable cells
at 1 M), confirming the observation that the phenstatin scaffold is required for optimum
activity. The remaining compounds in the series demonstrated weak activity, with viability
80% at 1 M. The IC50 value of the most potent imidazole containing compound 21l was
determined as 0.132 0.007 M in MCF-7 cells (Table 2), and this compound was selected
for further evaluations in other cancer cell lines and cell cycle analysis in MCF-7 cells.
, i–l) in MCF-7 cells are
,
µ
>
µ
±
µ
3
.1.5. Series 5: 1-(Diarylmethyl)Pyrrolidines 25a–g and 1-(Diarylmethyl)Piperidines 26a–c
,
and 1-(Diarylmethyl)Piperazines 27a–g, 28
The results of preliminary evaluation of the panel of pyrrolidine and piperidine
derivatives 25a–g and 26a–c in MCF-7 cells are shown Figure 6A. These compounds
were not sufficiently active when compared to the positive controls CA-4 and phen-
statin (7a). The most potent examples were identified as the piperidine derivative 26b
showing the lowest percentage of viable cells (78%) at 1 M and containing the 3,4,5-
trimethoxyphenyl (ring A) and 4-methoxyphenyl (Ring B), together with the correspond-
ing pyrrolidine containing compounds 25g and 25d (82% and 80% viability at 1 M). The
3,4,5-trimethoxyphenyl)(methyl)piperazine derivatives (27c ) were screened at three
concentrations (10, 1, 0.1 M) (Figure 6B). Compound 27f was identified as the most ac-
tive, with a percentage of viable cells of 42% at 10 M, 76% at 1 M and 84% at 0.1 M.
,
µ
µ
(
,d,f,h,i
µ
µ
µ
µ
Benzylpiperazine 27i, which is more closely related in structure to phenstatin, displayed
promising antiproliferative activity at 10 µM (48% cell viability).
From the results obtained above, it is interesting to see that inclusion of the triazole
heterocycle on the phenstatin scaffold (as in compounds 21l and 24) results in greater
antiproliferative effects in the MCF-7 cell line than the corresponding imidazole compound
(
19e). By comparison, replacement of the azole with pyrrolidine, piperidine, or piperazine
resulted in decreased antiproliferative activity. The antiproliferative activity of the most
potent azole compounds 19e 21l, and 24 may be correlated to the logP values (see Sup-
,
plementary Information). The imidazole compound 19e has a lower logP (2.41) when
compared to the 1,2,4-triazole compound 21l (logP of 2.91) and the 1,2,3-triazole compound
24
(logP 3.50); the antiproliferative activity of the compounds 19e, 21l, and 24 in MCF-7
cells are determined as IC50 = 0.42, 0.13, and 0.052 M, respectively. In addition, the total
µ

Pharmaceuticals 2021, 14, 169
20 of 54
2
2
polar surface (TPSA) area for these compounds is in the range 74.22–87.86 Å < 140 Å .
However, compounds with higher logP values e.g., the piperazine compounds 27f (5.50)
and 27d (4.67) display poor activity.
3.2. Antiproliferative Activity of Selected Analogues in MDA-MB-231 and HL60 Cell Lines
A number of the more potent compounds synthesised were evaluated in the triple-
negative MDA-MB-231 cell line with 72 h incubation time (see Table 2). For the triazole
compound 19e, an IC50 value of 0.98 M was obtained in MDA-MB-231 cells, although this
is not as potent as observed in the MCF-7 cells (IC50 = 0.42 M, Table 2). The lower IC50
value for the imidazole compound 21l (0.237 M) indicates that the imidazole heterocycle
µ
µ
µ
in 21l contributes to the antiproliferative activity more effectively than the 1,2,4-triazole
ring in compound 19e. The novel 1,2,3-triazole compound 24 was the best of all analogues
tested in MCF-7 cells (IC50 = 0.052
line was also very promising (IC50 = 0.074
the reported activity of phenstatin in MDA-MB-231 cells (IC50 = 1.5
that the 1,2,3-triazole has very potent antiproliferative effects compared to imidazole or
,2,4-triazole present in the related compounds 21l and 19e. Since the antiproliferative
µ
M). The result obtained for 24 in the MDA-MB-231 cell
M), Table 2, and compares very favourably with
M [105]), indicating
µ
µ
1
effects of 1,2,3-triazole–phenstatin hybrid compounds has not previously been investigated,
this heterocycle is especially interesting for further development.
In a further study, the antiproliferative effects of the novel imidazole compound 21l,
1
,3,4-triazole compound 19e, and the 1,2,3-triazole compound 24 (structurally related to
letrozole and phenstatin) in HL-60 leukaemia cells was also investigated. HL-60 leukaemia
cells were used as an in vitro model for acute myeloid leukaemia. Both MCF-7 and HL-
6
0 cell lines are CA-4 sensitive are highly susceptible to the effects of tubulin-targeting
compounds [102]. The IC50 value of 0.156 M obtained for imidazole compound 21l
identifies it as a lead compound for future development. The 1,2,3-triazole compound 24
was also potent in the leukaemia HL-60 cell line with an IC50 value of 0.173 M, while
9e was less potent, IC50 = 261 M. (IC50 value for phenstatin = 0.031 M [106]). This
µ
µ
1
µ
µ
experiment demonstrated the selective effect of interchanging the imidazole, 1,3,4-triazole,
and 1,2,3-triazole heterocycles on cell viability in HL-60 cells.
3.3. NCI Cell Line Screening for 19e, 21l, 25g, 26b, and 27d
Five novel substituted phenstatin compounds from the present work ((19e, (Series 2)
21l, (Series 4), 25g, 26b and 27d (Series 5)) were selected for evaluation in the NCI 60 cell
line screen [107] following initial analysis of the Lipinski (drug-like) properties from the
Tier-1 profiling screen, together with predictions of the relevant absorption, distribution,
metabolism, excretion, and toxicity (ADMET) properties e.g., metabolic stability, permeabil-
ity, blood–brain barrier partition, plasma protein binding, and human intestinal absorption
properties (see Tables S1 and S2 Supplementary Information). The compounds are pre-
dicted to be moderately lipophilic–hydrophilic, revealing their drug-like pharmacokinetic
profiles and are potentially suitable candidates for further investigation.
The results obtained for the triazole compound 19e in the NCI 60 cancer cell line
screening (GI50 values, five doses) [107] are shown in Table 3. (GI50 is defined as the
concentration for 50% of maximal inhibition of cell proliferation). In general, 19e showed
good activity on most of the cell lines with GI50 values in the sub-micromolar range. The
activity was particularly potent in all of the leukaemia, CNS, and prostate cancer cell lines.
The activity in MCF-7 cells (GI50 = 0.347
from our in-house viability assay of 0.424
in the TNBC cell lines HS-578T (GI50 = 0.548
and in the BT-549 invasive ductal carcinoma cell line (GI50 = 0.618
activity was also observed against the ovarian cancer cells, e.g., OVOCAR-3 cell line
0.323 M) and colon cancer, e.g., chemoresistant HT-29 cells (GI50 = 0.330 M). The best
activity for 19e among all of the 60 cell lines tested was the melanoma cell line MDA-MB-
35 in which the GI50 value was 0.181 M. The MID GI50 was calculated as 0.243 M over
µ
M) was in close agreement with the value obtained
M. The compound displayed significant activity
M) and MDA-MB-468 (GI50 = 0.371 M)
M). Potent anti-cancer
µ
µ
µ
µ
(
µ
µ
4
µ
µ

Pharmaceuticals 2021, 14, 169
21 of 54
all 60 cell lines. The MID value obtained for TGI (total growth inhibition) was 53.7
µM,
and for LC , it was 97.7 M, indicating that the lethal concentration of the drug is very
µ
50
high and well above the GI50 value, indicating that 19e has low toxicity. The results of the
NCI COMPARE analysis for compound 19e are shown in Table 4. Based on the GI50 mean
graph and on the TGI mean graph, the compound with the highest rank was vinblastine
sulphate with r values of 0.586 and 0.737, respectively. Correlation values (r) are Pearson
correlation coefficients.
Table 3. Antiproliferative evaluation of compounds 21l and 19e in the NCI 60 cell line in vitro screen a.
Compound 21l c
Compound 19e d
Compound 21l c
Compound 19e d
Cell Line
Cell Line
GI50 (µM) b
GI50 (µM) b
GI50 (µM) b
GI50 (µM) b
Leukemia
CCRF-CEM
HL-60 (TB)
K-562
MOLT-4
RPMI-8226
SR
Melanoma
LOX IMV1
MALME-3M
M14
MDA-MB-435
SK-MEL-2
UACC-62
0.289
0.229
0.225
0.565
0.385
0.182
0.402
0.261
0.427
0.510
0.452
0.376
0.523
Nd
0.180
0.119
0.324
0.668
0.523
Nd
0.374
0.181
0.361
0.550
e
e
Non-Small Cell
Lung Cancer
A549/ATCC
HOP-62
SK-MEL-28
Nd e
Nd e
0.836
0.516
28.6
0.702
0.640
0.882
0.789
0.377
0.247
0.969
0.514
9.69
2.24
1.43
0.977
0.696
0.414
0.395
SK-MEL-5
UACC-257
Ovarian Cancer
IGROV1
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
0.367
1.44
0.440
>100
HOP-92
EKVX
0.862
0.264
3.44
0.648
0.450
1.92
0.959
0.323
3.02
1.99
1.14
1.18
0.402
NCI-H226
NCI-H23
NCI-H332M
NCI-H460
NCI-H552
Colon Cancer
COLO 205
HCT-2998
HCT-116
HCT-15
HT29
KM12
SW-620
CNS Cancer
0.465
0.361
1.72
0.249
0.264
0.312
0.389
0.387
0.332
1.35
0.448
0.438
0.330
0.418
0.431
Renal Cancer
786-0
A498
ACHN
CAKI-1
RXF 393
SN12C
TK-10
UO-31
0.369
0.304
0.693
0.681
0.475
0.324
0.653
0.567
0.321
0.853
>100
0.903
e
Nd
0.910
e
Nd
SF-268
SF295
SF539
0.731
0.307
0.217
0.469
0.325
0.300
0.883
Breast Cancer
MCF-7
MDA-MB-
0.339
0.664
0.347
1.50
SNB-19
0.532
0.578
Nde
0.446
2
31/ATCC
HS 578T
BT-549
SNB-75
0.192
0.391
0.414
0.306
0.548
0.618
AC
Prostate cancer
PC-3
e
e
T-47D
MDA-MB-468
Nd
Nd
0.384
0.407
0.234
0.431
0.530
0.243
0.316
0.371
DU-145
MG-MID (µM) f
a
b
Data obtained from NCI in vitro human tumour cell screen 5 dose assay. GI50 is the molar concentration of the compound causing 50%
inhibition of growth of the tumour cells; NSC 78806; d NSC 788805; Nd: Not determined; MG-MID: the mean of GI50 values over all
c
e
f
cell lines for the tested compound.
The National Cancer Institute (NCI) screening of imidazole compound 21l also demon-
strated very good results showing that the compound not only is active against breast
cancer cells but also against other types of cancer (see Table 2). Compound 21l proved
active against all of the leukaemia cell lines; in particular, very promising activity was
measured in SR cells (GI50 = 0.182
µM) and HL60 (GI50 = 0.229 µM), confirming our in-
house evaluation. The activity against CNS cancer varied in a range between GI = 0.192
5
0

Pharmaceuticals 2021, 14, 169
22 of 54
and 0.731
µM. Particularly good was also the activity against the breast cancer panel with
GI50 values in the range of 0.306–0.664
GI50 = 0.316 µM). Of all the cell lines evaluated in the panel, compound 21l was most
potent against melanoma MDA-MB-435 cells with GI50 = 0.119 M. The MID GI50 value
for the 60 cell line panel was 0.234 M. MID TGI and LC50 values of 40.7 and 100
µM, including the TNBC cell line MDA-MB-468
(
µ
µ
µM
respectively are an indication of the low toxicity of the compound, as the median lethal
dose is very high compared to the GI50 values. From the COMPARE analysis results shown
in Table 3, it was observed that based on the mean GI50 value, the activity of our 21l is
most closely related to paclitaxel (r = 0.587). Based on TGI values, the compound with the
highest ranking was maytansine (r = 0.775); both are tubulin-targeting agents. Correlation
values (r) are Pearson correlation coefficients and LC50 values all >0.1 mM.
Table 4. Standard COMPARE analysis of compounds 19e and 21l a.
Rank
19e
r
21l
r
Based on GI50
mean graph
Based on GI50
mean graph
Vinblastine
sulphate,
hiConc:-7.6
S-Trityl-L-
cysteine
Paclitaxel (Taxol)
hiConc:-6.0
1
2
0.586
0.575
0.587
0.58
S-Trityl-L-
cysteine
Paclitaxel (Taxol)
hiConc:-4.0
Maytansine
3
4
Maytansine
0.547
0.544
0.544
0.53
Rhizoxin
Vinblastine
sulphate,
Paclitaxel (Taxol)
hiConc:-4.6
5
0.509
0.518
hiConc:-5.6
Based on TGI
mean graph
Based on TGI
mean graph
Vinblastine
sulfate
Maytansine
hiConc: -9.0
Vinblastine
sulfate
1
0.737
0.775
2
3
4
Rhizoxin
0.726
0.704
0.696
0.765
0.748
0.703
Paclitaxel (Taxol)
Maytansine
hiConc:-9.0
Rhizoxin
Paclitaxel (Taxol)
Maytansine
hiConc:-4.0
Maytansine
hiConc:-9.0
5
0.689
0.700
a
The target set was the standard agent database and the target set endpoints were selected to be equal to the
seed endpoints. Standard COMPARE analysis was performed. Correlation values (r) are Pearson correlation
coefficients. Vinblastine sulfate and maytansine appear at different concentrations, as it has been tested by the
NCI at multiple concentration ranges (see reference 107).
Compounds 25g
one-dose screen (see Table S4, Supplementary Information). The mean growth percentages
for 25g 26b, and 27d were 73.1%, 34.2%, and 65.5% over the 60 cell line panel at 10 M.
, 26b, and 27d were also selected for evaluation in the NCI 60 cell line
,
µ
Interestingly, the piperidine compound 26b displayed significant potency in the breast
cancer panel, with mean growth of 30.3% over this cell panel, with notable potency in the
triple negative breast cancer cell lines HS587T (16.6% growth) and MDA-MB-468 (9.3%
growth). In the leukaemia panel, the mean growth obtained is 23.5% over this 60 cell panel
and significantly 4.36% growth for the acute myeloid leukaemia HL-60 cell line. Compound
26b also displayed notable potency in the CA-4 resistant colon cancer cell line HT-29 with
7.93% growth recorded.
3.4. Evaluation of Toxicity in MCF-10A Cells
The potent phenstatin derivatives 19e
, 21l, and 24 were selected for toxicity evaluation
in the non-tumorigenic MCF-10A epithelial breast cancer cell line. The human mammary

Pharmaceuticals 2021, 14, 169
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epithelial cell line MCF10A is widely used as an in vitro model for normal breast cell
function and transformation [108]. The viability of the MCF-10A cells was determined after
treatment with compounds 19e and 21l at four different concentrations of 10, 1, 0.5, and
0
.4
compounds 19e and 21l show a cell death of approximately 50%. At 1
compound 19e does not show any loss in cell viability (99% viability), while compound 21l
resulted in 73% cell viability, still above the IC50 values of 0.42 M (19e) and 0.13 M (21l
in MCF-7 cells. When the experiment was repeated with an increased incubation time of
8 h, it was observed that the percentage of viable cells at 10 M concentration decreased
for compounds 19e and 21l to approximately 30% (Figure 7A,B). The percentage of viable
cells at 1 M decreased to 64% for compound 21l, while it did not change significantly
for 19e (>94%). For both compounds, viability at 0.5 M and 0.4 M is close to 100%,
µ
M for 24 h (Figure 7A,B). It was observed that at the highest concentration (10
µM),
µM concentration,
µ
µ
)
4
µ
µ
µ
µ
which means that the compounds are not toxic toward healthy cells at lower concentrations
corresponding to their IC50 values. The third screening for 19e and 21l was performed at
7
2 h, which is the incubation time used through all the screenings in MCF-7 (Figure 7A,B).
It is interesting to note that as the concentration of the drug decreases from 1 to 0.5 and
.4 M, the percentage of viable cells increases significantly, with viable cells percentage
80% at 0.4 M for all compounds tested. This demonstrates that even at concentrations
0
>
µ
µ
that are toxic to the MCF-7 cancer cells, the MCF-10A are not killed by the drug. Therefore,
the compounds selected demonstrate good antiproliferative activity and additionally show
good selectivity and low cytotoxicity to normal cells. Compound 24 was evaluated in
MCF-10A cells at three different concentrations: 10, 1, and 0.1 µM over 72 h (Figure 7C).
The percentage of viable cells at the three different concentrations was 61%, 71%, and 96%,
respectively, with higher percentage of cells alive at the lower concentration. Compound 24
demonstrates good selectivity for cancer cells and low cytotoxicity even if the percentage of
viable cells at 1
µM was slightly lower than the value observed previously for compounds
19e and 21l (>80%) at 72 h. These results are also supported by the low toxicity of the
compounds determined from the NCI evaluation. Tubulin-targeting drugs such as taxanes
and vinca alkaloids are among the most effective anti-cancer therapeutics in the treatment
of castration-resistant prostate cancer and triple-negative breast cancer. However, their use
is limited by toxicities including neutropenia and neurotoxicity; additionally, tumour cells
can develop resistance to these drugs [109]. Our results demonstrate that azoles 19e, 21l,
and 24 were less toxic to normal human breast cells than to breast cancer cells, providing a
potential window of selectivity.
3.5. Effects of Compounds 21l and 24 on Cell Cycle Arrest and Apoptosis
To investigate further the mechanism of action of the novel azole compounds synthe-
sised, the effect of selected potent compounds 21l and 24 was investigated in MCF-7 cells
by flow cytometry and propidium iodide (PI) staining, allowing the percentage of cells in
each phase of the cell cycle to be quantified (Figure 8). For the imidazole compound 21l
,
three time points were analysed (24, 48, and 72 h), and the values obtained for apoptosis
and the G /M phase of the cell cycle were quantified (concentration 1 µM), as shown in
2
Figure 8A. It was observed that the percentage of cells undergoing apoptosis (sub-G1)
increases significantly at all three time points to 15%, 31%, and 37% respectively compared
to the background level of apoptosis with the vehicle ethanol (2%, 4%, and 2%) at the
corresponding time points. It is also interesting to notice how the percentage of cells in the
G /M phase for the treated sample (47%, 43%, and 40%) is statistically higher than the cells
2
in the same phase for the control sample treated with the vehicle (26%, 25%, 25%) at the
corresponding time points. G /M cell cycle arrest is strongly associated with an inhibition
2
of tubulin polymerisation. CA-4 and related tubulin targeting compounds cause G2/M
arrest. Hence, the higher percentage of cells observed in cells treated with 21l may suggest
that the mechanism of action is indeed the inhibition of tubulin polymerisation.

Pharmaceuticals 2021, 14, 169
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Figure 7. Screening of phenstatin derivatives 19e
(
A) and 21l
(
B) in MCF-10A cells at 24, 48, 72 h
and ( 24 in MCF-10A and MCF-7 cells at 72 h. (A,B) Effect of compounds 19e and 21l on the
C
)
viability of non-tumorigenic MCF-10A human mammary epithelial cells. Cells were treated with
the indicated concentrations for 21, 48, or 72 h. Cell viability was expressed as a percentage of
vehicle control (ethanol 1% (v/v)) and was determined by alamarBlue assay (average + SEM of three
independent experiments). (C) Effect of compound 24 on the viability of non-tumorigenic MCF-10A
human mammary epithelial cells and MCF-7 breast cancer cells. Cells were treated with the indicated
concentrations for 72 h. Cell viability was expressed as a percentage of vehicle control (ethanol 1%
(
v/v)) and was determined by alamarBlue assay (average
±
SEM of three independent experiments).
Statistical analysis was performed using two-way ANOVA (***, p < 0.001).

Pharmaceuticals 2021, 14, 169
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Figure 8. Compound (
A
)
19e, (
B)
24, and (
C) phenstatin (7a) in MCF-7 induced G2/M arrest followed
by apoptosis in a time-dependent manner in MCF-7 cells. Cells were treated with either vehicle
control (v) (0.1% ethanol (v/v)] or compound 19e 24, or phenstatin (7a) (1 M) for 24, 48, and 72 h).
,
µ
Then, cells were fixed, stained with PI, and analysed by flow cytometry. Cell cycle analysis was
performed on histograms of gated counts per DNA area (FL2-A). The number of cells with <2 N
(
sub-G1), 2 N (G0G1), and 4 N (G2/M) DNA content was determined with CellQuest software. Values
represent as the mean SEM for three separate experiments. Statistical analysis was performed
±
using two-way ANOVA (**, p < 0.01, ***, p < 0.001).
The 1,2,3-triazole compound 24, which was the most potent compound evaluated in
the viability assay, demonstrated the same effects on the relative percentages of cells in

Pharmaceuticals 2021, 14, 169
26 of 54
apoptosis and the G /M phase, as shown in Figure 8B. Apoptosis increased with time, with
2
a statistically significant difference compared to vehicle control at 72 h. A high percentage
of cells were arrested in the G /M phase (52%, 56%, and 59%) at time points 24, 48, and
2
7
2 h respectively, following treatment with compound 24 with a much lower percentage of
cells in the G /M phase for the sample treated with the vehicle (28%, 23%, and 24%) at the
2
same time points.
Phenstatin 7a was used as a positive control through all the biological experiments.
Cell cycle analysis of MCF-7 cells treated with phenstatin at time points 24, 48, and 72 h and
a concentration of 1
µM showed a very low percentage of cells undergoing apoptosis at 24
and 48 h, as shown in Figure 8C. Apoptosis increased to 18% between 48 and 72 h, while
the percentage of cells in the G /M phase was correspondingly high (65%, 49%, and 51%
2
at 24, 48, and 72 h, respectively). This pattern was also observed in the compounds 21l and
24 tested, but the percentage of cells in apoptosis was always higher than for phenstatin,
possibly suggesting differences in the effects of these compounds on tubulin arising from
the presence of the azole in the modified structures.
The role of apoptosis in the inhibition of MCF-7 and MDA-MB-231 cell growth was
further examined. MCF-7 and MDA-MB-231cells were treated with compound 21l for
4
8 h and stained with Annexin V-fluorescein isothiocyante (FITC)/propidium iodide (PI)
and then analysed by flow cytometry. Dual staining with Annexin-V and PI facilitates dis-
−
−
+
−
crimination between live cells (annexin-V /PI ), early apoptotic cells (annexin- V /PI ),
+
+
−
+
late apoptotic cells (annexin-V /PI ), and necrotic cells (annexin-V /PI ). Compound
1l induced both early and late apoptosis in MCF-7 cells in a concentration-dependent
manner when compared to the untreated control cells (Figure 9A). When MCF- 7 cells were
treated with 21l (0.1, 0.5, and 1 M), the average proportion of Annexin V-stained positive
2
µ
cells (total apoptotic cells) increased from 0.9% in control cells to 14.1%, 17.5%, and 19.3%,
respectively. These results suggested that compound 21l induces the apoptosis of MCF-7
cells in a dose-dependent manner. In MDA-MB-231 cells, the percentage of cells observed
in apoptosis following treatment with 21l was significantly lower with 3.6%, 5.9%, and
6
.9% at 0.1, 0.5, and 1.0
the Annexin V-stained positive cells (total apoptotic) cells were determined as 36.1% and
6% in MCF-7 cells at 0.1 M and 0.5 M, respectively, as shown in Figure 9A. The total
µM respectively, as shown in Figure 9B. In contrast, for phenstatin,
4
µ
µ
apoptotic MDA-MB-231 cells were determined as 16.6% and 17.9% following treatment
with phenstatin (0.1 and 0.5 µM), respectively, as shown in Figure 9B.
3.6. Tubulin Polymerisation
Compound 21l was selected for further analysis using a tubulin polymerisation assay.
Its promising antiproliferative activity (IC50 = 0.237 µM in MCF-7 cells) combined with
structural features related to phenstatin 7a and CA-4 indicate that the mechanism of action
of this compound could be the inhibition of tubulin polymerisation. The assay is based
on the capacity of microtubules to scatter light proportionally to their concentration. The
imidazole compound 21l (red) showed good inhibition of tubulin polymerisation after
6
0 min (Vmax value 2.84
±
0.10 mOD/min at 10 µM), corresponding to a 1.34-fold reduction
of the polymer mass compared to the vehicle, as shown in Figure 10A. Paclitaxel (in green)
was used as a positive control as it stabilises polymerised tubulin, as shown in Figure 10A
Phenstatin 7a is a potent inhibitor of tubulin polymerisation comparable to CA-4 [66],
as shown in Figure 10B. Incubation with either imidazole 21l or phenstatin resulted in a
significant inhibition of tubulin polymerisation and assembly.
.

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Figure 9. Compound 21l induced cell apoptosis in (
A
) MCF-7 breast cancer cells and (
B
) MDA-MB-
2
31 breast cancer cells. MCF-7 breast cancer cells and MDA-MB-231 breast cancer cells were treated
with 21l or phenstatin (7a) (1 M) or control vehicle (0.1% ethanol (v/v)), and the percentage of
µ
apoptotic cells was determined by staining with Annexin V-FITC and PI. In each panel, the lower
left quadrant shows cells that are negative for both PI and Annexin V-FITC, the upper left shows
only PI cells that are necrotic. The lower right quadrant shows Annexin-positive cells that are in
the early apoptotic stage and the upper right shows both Annexin/PI positive, which are in late
apoptosis/necrosis. Control cells and cells treated with phenstatin 7a and 21l at 48 h are shown,
respectively. Values represent the mean of three independent experiments.

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Figure 10. Effect of compound 21l on tubulin polymerisation in vitro. (
A) Tubulin polymerisation
assay for compound 21l at 10 M. ( ) Paclitaxel (10 µM) and phenstatin (7a) (10 µM) were used as
µ
B
references while ethanol (1% v/v) and DMSO (1% v/v) were used as vehicle controls. Purified bovine
tubulin and guanosine-5’-triphosphate (GTP) were mixed in a 96-well plate. The polymerisation
◦
reaction was initiated by warming the solution from 4 to 37 C. The effect on tubulin assembly
was monitored in a Spectramax 340PC spectrophotometer at 340 nm at 30 s intervals for 60 min at
◦
3
7 C. DMSO. Fold inhibition of tubulin polymerisation was calculated using the Vmax value for each
reaction. The results represent the mean for three separate experiments.
Following the experiment above, the in vitro effects of compounds 19e and 21l were
examined on the microtubule structure of MCF-7 breast cancer cells with confocal mi-
croscopy using anti-tubulin antibodies. Paclitaxel and phenstatin, a known polymeriser
and depolymeriser of tubulin respectively, were used as controls. In Figure 11A, a well-
organised microtubule network (stained green) is clearly seen for the vehicle control,
together with the MCF-7 cell nuclei (stained blue). Hyperpolymerisation of tubulin was
demonstrated in the paclitaxel-treated sample (Figure 11B), whereas the phenstatin-treated
sample Figure 11C shows an extensive depolymerisation of tubulin. Cells treated with the
azoles 19e (Figure 11D) and 21l (Figure 11E) displayed disorganised microtubule networks
with similar effects to phenstatin, together with multinucleation (formation of multiple
micronuclei), which is a recognised sign of mitotic catastrophe [110] previously observed
by us and others upon treatment with tubulin-targeting agents such as CA-4 and related
compounds in non-small cell lung cancer cells and breast cancer MCF-7 cells [111,112].

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Figure 11. Compounds 19e and 21l depolymerise the microtubule network of MCF-7 breast can-
cer cells. Cells were treated with (
) phenstatin (7a) (1 M), ( ) compound 19e (10
were fixed in ice-cold methanol and stained with mouse monoclonal anti-
iocyante (FITC) antibody (clone DM1A) (green), Alexa Fluor 488 dye, and counterstained with DAPI
’-6’-diamidino-2-phenylindole (blue). Images obtained with Leica SP8 confocal microscopy, Leica
A
) vehicle control (0.1% ethanol (v/v)), (
M), or ( ) compound 21l (10
-tubulin–fluorescein isoth-
B
) paclitaxel (1
µM),
(C
µ
D
µ
E
µM) for 16 h. Cells
α
4
application suite X software. Representative confocal micrographs of three separate experiments are
shown. Scale bar indicates 25 µm.
3.7. Effects of Compounds 21l and 24 on Expression Levels of Apoptosis-Associated Proteins
Some of the novel compounds synthesised during the project were selected for further
investigation of their mechanism of action as pro-apoptotic agents based on their effect on
the expression of proteins that can regulate apoptosis or proteins involved in the regulation
of DNA repair. The effects of compounds 21l and 24 on apoptosis were evaluated by
Western blotting. Apoptosis regulating proteins Bcl-2 and Mcl-1 were investigated along
with PARP. PARP (poly ADP-ribose polymerase) is involved in the repair of DNA single-
strand breaks in response to environmental stress [113]; and PARP cleavage is considered
a hallmark of apoptosis. Bcl-2 is an anti-apoptotic protein that prevents apoptosis by
sequestering caspases (apoptosis promoters) or by preventing the release of pro-apoptotic
cytochrome c and AIF (apoptosis inducing factor) from the mitochondria into the cyto-
plasm [114]. The Mcl-1 protein belongs to the Bcl-2 family; it is also an anti-apoptotic
protein localised in the mitochondrial outer membrane that acts at a very early stage in the
cascade, leading to the release of the cytochrome c [115]. Pro- and anti-apoptotic members
of the Bcl-2 family can heterodimerise and titrate each other’s functions. If the expression
levels of Mcl-1 and Bcl-2 are reduced (by drug treatment), apoptosis may be triggered.
From the results obtained, no change in the expression levels of two anti-apoptotic
proteins was observed, indicating that Bcl-2 and Mcl-1 may not play a critical role in the
pro-apoptotic mechanism of action of the compounds (Figure 12). A significant reduction
in the expression of full-length PARP (116 kDa) between the vehicle and the treated MCF-7
cells was observed (Figure 12), suggesting that 21l and 24 cause PARP cleavage. PARP

Pharmaceuticals 2021, 14, 169
30 of 54
enzymes play a crucial role in the DNA repair, and PARP cleavage is affected by caspase
3
activity. PARP enzymes are found in the cell nucleus and are activated by damage of
the DNA single strand; therefore, the inhibition of DNA repair in cancer cells represents
an attractive strategy in cancer therapy [116]. In conclusion, the proposed mechanism of
action of these compounds as pro-apoptotic drugs is supported by the observed increase
in the percentage of cell in subG1 in the cell cycle profile, the flow cytometric analysis of
Annexin V/PI-stained cells, and also by PARP cleavage.
Figure 12. Effects of 21l and 24 on expression of PARP (poly ADP-ribose polymerase) and anti-
apoptotic proteins Bcl-2 and Mcl-1. MCF-7 cells were treated with either vehicle control (ethanol,
0
.1% v/v) or with compounds 21l and 24 (1 µM) for 24 h. After the required time, cells were harvested
and separated by SDS-PAGE to detect the level of the apoptosis-related proteins. The membrane was
probed with anti-PARP or anti-cleaved PARP antibodies. Results are representative of three separate
experiments. To confirm equal protein loading, each membrane was stripped and re-probed with
glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody.
3.8. Aromatase Inhibition
An objective of this research was the design of dual-acting tubulin/aromatase in-
hibitors. The evaluation of the aromatase inhibitory activity of the most potent compounds
prepared was next investigated. Three compounds of the phenstatin hybrid panel 21l
and 19e were selected for evaluation against two cytochrome members of the P450
,
24
family: CYP19 and CYP1A1. CYP19 is the aromatase cytochrome directly responsible for
the synthesis of estradiol by the aromatisation of its steroid precursors testosterone and
androstenedione, while CYP1A1 is involved in the metabolism of estrogen. The specificity
of aromatase inhibition was evaluated by an assay carried out with xenobiotic-metabolising
cytochrome P450 enzymes CYP1A1. The methodology applied in this study requires the
detection of the hydrolysed dibenzylfluorescein (DBF) by the aromatase enzyme [117].
Aromatase and CYP1A1 inhibition were quantified by measuring the fluorescent inten-
sity of fluorescein, the hydrolysis product of dibenzylfluorescein (DBF), by aromatase
as previously described [118
IC50 values of 4.9 M. The test was initially conducted at one concentration (20
Further experiments to determine the IC50 value were performed if the compound caused
greater than 90% inhibition at 20 g/mL. The results are presented in Table 5. Of these,
,2,3-triazole 24 was inactive, as it did not show any inhibition of the enzyme at 20 g/mL,
.05 M (0.01% for CYP19 and 12.81% for CYP1A1), whereas imidazole 21l (0.05 M) and
,2,4-triazole 19e (0.05 M) were active in the first screen against CYP19 (Table 5). The
,119]. Naringenin was used as a positive control, yielding
µ
µg/mL).
µ
1
µ
0
µ
µ
1
µ
inhibition for imidazole 21l, although potent, was not concentration-dependent, and the
IC50 could not be determined. 1,2,4-Triazole 19e inhibited aromatase in a concentration-
dependent manner, and its IC50 was determined as 29
µ
M. Of all the tested compounds (21l
, and 19e), none showed significant inhibition of CYP1A1, yielding IC50 values above
M, which is regarded as inactive [119 120]. From the results obtained, we can suggest
,
24
5
3
µ
,
that the 1,2,4-triazole heterocycle is required for aromatase inhibition in the phenstatin
related compound 19e. Therefore, the 1,2,4-triazole compound 19e could be identified as