Further Developments of the Phenyl-Pyrrolyl Pentane Series of Nonsteroidal Vitamin D Receptor Modulators as Anticancer Agents

Article abstract of DOI:10.1021/acs.jmedchem.8b00106

The vitamin D₃ receptor (VDR), which belongs to the nuclear-receptor superfamily, is a potential molecular target for anticancer-drug discovery. In this study, a series of nonsteroidal vitamin D mimics with phenyl-pyrrolyl pentane skeletons wit

Full text of DOI:10.1021/acs.jmedchem.8b00106

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Further Developments of the Phenyl-Pyrrolyl Pentane Series of Non-
steroidal Vitamin D Receptor Modulators as Anticancer Agents
Meixi Hao, Siyuan Hou, Lingjing Xue, Haoliang Yuan, Lulu
Zhu, Cong Wang, Bin Wang, Chunming Tang, and Can Zhang
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00106 • Publication Date (Web): 08 Mar 2018
Downloaded from http://pubs.acs.org on March 8, 2018
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Further Developments of the Phenyl-Pyrrolyl Pentane Series of
Non-steroidal Vitamin D Receptor Modulators as Anticancer Agents
AUTHORS
＋
Meixi Hao , Siyuan Hou , Lingjing Xue, Haoliang Yuan, Lulu Zhu, Cong Wang, Bin Wang,
＋
*
Chunming Tang, Can Zhang
AFFILIATIONS
State Key Laboratory of Natural Medicines and Jiangsu Key Laboratory of Drug Discovery
for Metabolic Diseases, Center of Drug Discovery, China Pharmaceutical University, 24
Tong Jia Xiang, Nanjing, 210009, China.
*
Correspondence to:
Can Zhang, Ph.D., Center of Drug Discovery, China Pharmaceutical University, Nanjing,
China.
E-mail: zhangcan@cpu.edu.cn
＋
Contributed equally to this work.
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ABSTRACT
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Vitamin D receptor (VDR), belonging to the nuclear receptor superfamily, is a potential
molecular target for anticancer drug discovery. In this study, a series of non-steroidal vitamin
D mimics with a phenyl-pyrrolyl pentane skeleton were synthesized with therapeutic
potential for cancer treatment. Among them, 11b and 11g were identified as the most
effective agents in reducing the viability of four cancer cell lines, particularly breast cancer
cells, with IC50 values in the submicromolar concentration range. In addition, 11b and 11g
possessed VDR binding affinity and displayed significant partial VDR agonistic activity
determined by dual luciferase reporter assay and human leukemia cell line (HL-60 cells)
differentiation assay. Furthermore, 11b and 11g inhibited tumor growth in the orthotopic
breast tumor model due to the inhibition of cell proliferation and induction of cell apoptosis.
More importantly, 11b and 11g, exhibiting favorable pharmacokinetic behavior in vivo, did
not increase serum calcium levels or cause any other apparent side effects. In summary, 11b
and 11g act as novel VDR modulators and may be promising candidates for cancer
chemotherapy.
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Keywords: vitamin D
3
receptor (VDR) modulator, non-steroidal vitamin D analog,
phenyl-pyrrolyl pentane skeleton, antitumor agent, calcitriol
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INTRODUCTION
3 3 2 3
The active form of vitamin D , 1,25-dihydroxyvitamin D (calcitriol, 1,25-(OH) D ),
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regulates multiple signaling pathways through binding to its intracellular receptor, vitamin D3
receptor (VDR), which belongs to the nuclear receptor superfamily of steroid hormone.
Calcitriol and its synthetic analogs play important roles in calcium homeostasis, bone
1
-3
mineralization and immune regulation. However, over the past two decades it has become
clear that calcitriol and its analogs might have therapeutic potential as anticancer agents by
modulating proliferation, apoptosis and differentiation of a variety of cancer cell types
4, 5
through the VDR signaling pathway.
Preclinical research indicates that vitamin D deficiency raises the risk of developing
6, 7
8, 9
10, 11
cancers, such as colon cancer,
breast cancer,
and prostate cancer
while dietary
calcitriol and its analogs result in a significant inhibition of tumor growth and eventual tumor
12,13
burden.
Additionally, Vdr-null mice are more sensitive to carcinogen-induced
1
tumorigenesis. More importantly, clinical studies further support the development of
4
vitamin D modulators as preventative and therapeutic anticancer agents. Garland, et al.
3
discovered that the exposure to sufficient sunlight could induce enough vitamin D and
15
reduce the mortality risk of patients with colon cancer. Lappe, et al. also discovered that
improving calcium and vitamin D nutritional status substantially reduces cancer risk in
1
postmenopausal women. Moreover, seocalcitol, a vitamin D
6
3
analog, displayed antitumour
17
activity in the treatment of the hepatocellular carcinoma (HCC) in the phase II trial study.
Combination therapy with calcitriol and other anticancer agents improved survival of prostate
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cancer patients in the phase II trials.
Therefore, it is essential to develop vitamin D
modulators as preventative and therapeutic anticancer agents.
During the last 30 years, a large number of vitamin D analogs have been synthesized by
introducing different types of chemical modifications to calcitriol, such as maxacalcitol,
calcipotriol, seocalcitol and lexicalcitol, which were used for the treatment of cancer,
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osteoporosis and skin disorders.
However, most of these classic VDR modulators with a
secosteroidal structure can lead to hypercalcemia or hypercalciuria which impedes their
application for long-term cancer therapy and the translation of basic and clinical research to
therapeutic agents. It was reported that calcitriol was given at a high dose (45 ꢀg) once a
week in combination with docetaxel for prostate cancer treatment was terminated in the phase
III trial. Part of the reason was the side effects of hypercalcaemia along with myocardial
injury, nervous system damage and renal stone resulting in lower survival in the treatment
2
group compared with the placebo group. Moreover, another clinical trial revealed that
2
calcitriol in daily oral doses of more than 2-2.5 ꢀg led to hypercalcaemia in men with
23
recurrent prostate cancer, which limited the ability to raise the administered dose. Hence,
there is an urgent clinical need of designing a new generation of VDR modulators without the
risk of increasing serum calcium for cancer chemotherapy.
As early as 1999, Boehm et al. reported the first generation of non-steroidal vitamin D
modulators which exhibited no hypercalcemic potential in vivo. Among them, LG190119
(Figure 1A) effectively inhibited LNCaP xenograft tumor growth without increased serum
24
calcium levels or any other apparent side effects. Based on this, various groups had reported
25, 26
their non-steroidal derivatives with different structures.
Non-steroidal vitamin D mimics
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based on a hydrophobic core of p-carborane have been developed by Fujii, S. et al.
Furthermore, other non-steroidal derivatives such as bis and tris-aromatic compounds,
2
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carboxylic acid and hydroxamic acid derivatives, hydroxamic acid derivatives have been
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reported afterwards.
Recently, a series of non-steroidal VDR derivatives with phenyl-pyrrolyl pentane skeleton
were designed and synthesized by our group. Among them, sw22 (Figure 1B) significantly
inhibited the proliferation of human breast adenocarcinoma cells (MCF7 cells, IC50 = 320
31
nM). Based on the study, the pyrrolyl side chains were modified to improve the
anti-proliferative activity, and the results showed that designed compounds exhibited
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promising anti-proliferative activity in vitro.
Figure 1. The designed VDR modulators. (A) The structure of calcitriol, lexicalcitol, MC1301 and
LG190119. (B) The design of non-steroidal VDR modulators with phenyl-pyrrolyl pentane skeleton.
In this study, to further improve the anti-proliferative effect against cancer cells and
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pharmacokinetic properties in vivo, twenty-six novel vitamin D analogs with phenyl-pyrrolyl
pentane skeleton have been designed, synthesized and evaluated their bioactivity in vitro and
in vivo. First, we retained the phenyl-pyrrolyl pentane skeleton, which could form strong
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hydrophobic interactions with the hydrophobic pocket of VDR. Next, we introduced the
diethylcarbinol as the terminal hydrophobic group in the phenyl side chain to improve
biological activities, as similar modifications have been reported in both secosteroidal and
27, 33, 34
non-steroidal vitamin D analogs.
Previous report showed that lexicalcitol (Figure 1A)
possessing the diethylcarbinol moiety in the side chain is at least 100-fold more effective in
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the biological activity than calcitriol.
Moreover, the compound MC1301 with
diethylcarbinol moiety also presented improved differentiation-inducing activity (Figure 1A)
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compared to calcitriol. Therefore, we tried to improve the biological activities by
introducing the diethylcarbinol into the benzene ring as the side chain. Then, hydrophilic
groups or hydrophobic groups were added in the pyrrolyl side chain to explore the effects of
different substitutions on the pyrrole ring. Finally, we investigated whether both the biological
activities and the metabolism were enhanced on the basis of the above design and
considerations (Figure 1B). Here, we describe the sythesis and biological activities of novel
non-steroidal VDR modulaters with phenyl-pyrrolyl pentane skeleton and demonstrate their
promising therapeutic potential for the treatment of breast cancer and other cancers without
hypercalcemia intervenes.
RESULTS
Synthetic Procedures of Target Compounds. The synthetic route of non-steroidal
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vitamin D analogs was summarized in Scheme 1. Among these compounds, the key
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intermediate 8 was readily prepared as previously reported by our group.
The
intermediate 8 underwent potassium hydroxide (KOH) hydrolysis to give intermediate 9
containing the carboxyl group. The hydroxyl of 9 was directly reacted with
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1-bromo-3-ethyl-pentan-3-ol to obtain key intermediate 10. The target compounds 11a-11i,
12a-12b, 13a-13d and 14a-14c were prepared in a single step by being coupled with
intermediate 10 through different amines, alcohols, amino acids methyl ester hydrochloride,
aryl amines or esters. Compounds 15a-15d were synthesized through hydrolysis reaction.
The amino acid or ester compounds were reduced to obtain compounds 16a-16d by sodium
borohydride in methanol.
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Scheme 1. Synthesis of phenyl-pyrrolyl pentane series of non-steroidal VDR derivatives.
2 5 2
Reagents and reaction conditions: (a) KOH, C H OH, 70 °C, overnight; (b) EtMgBr, Et O, 30 °C, 2
hours; (c) NaH, DMF, r.t, 2.5 hours; (d) Amines/Amino acids methyl ester hydrochloride,
-nitrobenzenesulfonyl chloride, DMAP, CH CN, 70 °C, overnight; (e) Aryl amines or
4
3
esters/Alcohols, EDCI, DMAP, CHCl
3
, 70 °C, overnight; (f) Amino acid methyl ester
N, CHCl , 25 °C, overnight; (g) LiOH·H O, THF, H O,
hydrochloride/Amines, EDCI, HOBT, Et
3
3
2
2
4 3
25 °C, overnight; (h) NaBH , CH OH, 25 °C, 6 hours.
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Scheme 2. The structure of phenyl-pyrrolyl pentane series of non-steroidal VDR derivatives.
Anti-proliferative Effects on Cancer Cell Lines in Vitro. The non-steroidal vitamin D
analogs with the phenyl-pyrrolyl pentane skeleton were synthesized and screened for
anti-proliferative activity against a panel of four different cancer cell lines [human breast
36
37
cancer cell line (MCF7 cells) , human prostate cancer cell line (PC3 cells) , human
3
colorectal adenocarcinoma cell line (Caco2 cells) and human liver cancer cell line (HepG2
8
39
cells) ] which expressed VDR as previously reported. We confirmed the VDR protein
expression in different cell types by western blot and observed the relatively high expression
of VDR protein in MCF7 cells (Figure S1). As shown in Table 1, 11b exhibited the best
anti-proliferative activity with IC50 value of 57 nM, 40 nM, 17 nM and 150 nM for MCF7,
PC3, Caco2 and HepG2 cells, respectively, being more potent than both the prototype
compound sw22 and the positive compound calcitriol. In addition, 11a, 11c, 11d, 11f, 11g,
15b and 16b also showed better anti-proliferative activity against the four cancer cell lines
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than sw22 and calcitriol. It was obvious that R substitutions at the end of pyrrole ring side
chain of the compounds (Scheme 2) may be crucial for their anticancer activity. In common,
compounds with a hydrophilic group such as tertiary amine, morpholine ring, and carboxyl at
the end of pyrrole ring side chain (11a, 11b, 11d, 11g and 15b) showed more potent
anti-proliferative activity than those bearing hydrophobic groups such as trifluoromethyl,
benzene, alkynyl and ester group (12a-12b, 13a-13d and 14a-14b).
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Table 1. Cellular anti-proliferative activities of the phenyl-pyrrolyl pentane derivatives
a
IC50(µM)
Compound
MCF7
3.35± 1.26*
0.057± 0.018*
0.43± 0.27*
0.55± 0.16*
>50
PC3
Caco2
0.49± 0.04*
0.017± 0.001*
1.14± 0.25*
1.63± 0.59*
>50
HepG2
0.11± 0.03*
0.15± 0.05*
0.24± 0.03*
0.23± 0.12*
>50
L02
2.42± 1.35
0.37± 0.17*
0.99± 0.21
2.29± 1.28
>50
1
1a
11b
1c
1d
11e
1f
1g
11h
1i
2a
12b
3a
3b
13c
3d
4a
14b
4c
5a
15b
5c
5d
16a
6b
2.15± 0.93*
0.040± 0.006*
0.87± 0.26*
3.35± 0.95*
17.81± 4.18
2.42± 0.83*
1.57± 0.61*
>50
1
1
1
0.67± 0.34*
0.26± 0.08*
>50
1.74± 0.43*
2.72± 0.71*
>50
1.59± 0.21*
1.76± 0.46*
46.28± 14.28
>50
2.94± 0.93
2.86± 1.12
37.80± 10.28
1.19± 0.36
>50
1
1
>50
12.77± 3.86*
14.81± 4.18*
>50
>50
1
>50
>50
>50
>50
>50
>50
>50
1
>50
>50
>50
>50
>50
1
>50
>50
>50
>50
>50
>50
23.77± 7.29
>50
>50
>50
>50
1
>50
>50
>50
>50
1
>50
>50
>50
>50
>50
3.21± 1.57*
0.39± 0.14*
11.27± 2.49
0.079± 0.028*
10.21± 3.21
19.69± 3.37
29.14± 5.76
2.87± 1.04*
7.14± 1.63*
5.84± 1.42*
13.79± 2.91*
2.25± 0.82*
11.17± 1.28*
11.69± 3.19*
28.39± 4.25
3.09± 0.43*
30.26± 10.37
3.02± 0.82*
19.97± 5.15
2.74± 1.16*
25.19± 9.36
46.47± 12.57
>50
25.31± 9.36*
>50
1.99± 0.26
4.28± 1.84
9.99± 2.55
2.15± 0.81
7.59± 1.62
4.04± 0.74
>50
1
1
2.62± 0.91*
5.71± 2.47*
10.71± 1.64*
1.74± 0.22*
>50
1
1
1
0.074± 0.005*
0.51± 0.06*
2.05± 0.86
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16c
6d
31.41± 7.92
12.62± 4.63
2.803± 0.52
5.59± 1.72
8.76± 1.72*
7.87± 1.45*
17.36± 2.65
17.25± 3.83
40.24± 11.68
>50
24.81± 10.83*
43.07± 13.59*
47.55± 3.27*
>50
3.04± 1.51
4.84± 1.22
17.38± 1.75
0.67± 0.05
1
sw22
>50
calcitriol
4.46± 0.82
a
Data were presented mean ± SD, n = 3, *P < 0.05, compared with calcitriol.
1
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Additionally, to evaluate the selective cytotoxicity of designed compounds against normal
cells as a surrogate experiment determining their systemic toxicity in vivo, the human normal
hepatic cell line (L02 cells) was selected. The results revealed that 11a, 11b, 11d, 11g and
1
5b were less toxic (IC50 = 0.67 µM) against L02 cells, compared with their lead compound
sw22 and with calcitriol. Together, these results suggested that 11a, 11b, 11d, 11g and 15b
may possess selective anti-proliferative potential against cancer cells.
Synthesized compounds exhibited VDR-modulating activities.
In Vitro HL-60 Differentiation-inducing Activity. Calcitriol was known to induce HL-60
40
cells to differentiate into macrophages or monocytes by activating VDR signaling. Recent
studies showed that the measurement of the differentiation-inducing activity of HL-60 cells
treated with different vitamin D analogs was used for determining their VDR agonistic
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1, 42
activity.
First, we investigated whether our synthesized compounds were efficient
inducers of HL-60 cell differentiation (Table 2).
Table 2. The HL-60 differentiation-inducing activity of synthesized compounds
a
a
Compound
1a
1b
11c
EC50(nM)
Compound
13d
EC50(nM)
1
5.000 ± 1.3*
11.36 ± 2.7
82.00 ± 7.8
1630 ± 486.2
>10000
1
14a
14b
520.0 ± 28.4
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11d
1e
1f
1g
2.300 ± 1.1*
14c
15a
21.00 ± 8.2
860.0 ± 124.7
89.00 ± 15.3
26.00 ± 5.9
66.00 ± 17.9
450.0 ± 26.3
52.74± 11.9
2494± 563.2
96.00 ± 7.3
8.500 ± 1.2
9.000 ± 1.2
1
1080 ± 114.9
156.0 ± 13.7
238.0 ± 6.2
>10000
1
15b
1
15c
11h
11i
15d
>10000
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12a
12b
13a
2653± 83.5
6033± 491.3
>10000
16b
16c
16d
13b
3c
>10000
sw22
Calcitriol
1
5900 ± 1629.2
a
VDR agonistic activity was measured as HL-60 differentiation inducing affinity. Data were presented as
mean ± SD, n = 3, *p < 0.05, compared with calcitriol.
Consistent with their potent effects on cancer cell viability, the compounds (11a-11d, 11f,
11g, 15b and 16b) showed excellent differentiation-inducing activity with EC50 values at the
nanomolar range (EC50 = 2.3-238 nM) when the pyrrolyl side chain contained hydrophilic
group (tertiary amine, primary amine, morpholine ring, carboxyl and hydroxyl), suggesting
that they may possess VDR agonistic activity. In contrast, compounds 11e, 11h, 11i with no
hydrophilic group in the side chain induced HL-60 differentiation with EC50 value at the
micromole range, indicating that the hydrophilic group in the side chain of pyrrole ring may
be important for VDR agonistic activity.
Transactivation and Transcription Activity. VDR transactivation activity of the three
compounds (11b, 11g and 15b) was evaluated by the luciferase reporter assay in human
kidney cell lines (HEK293 cells) to further prove their VDR modulating activity. The three
compounds showed concentration-dependent transactivation activity and significant partial
agonistic activity, compared with the DMSO control. Among them, 15b appeared to be the
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most potent compound while compounds 11b and 11g had the similar activity with the natural
hormone calcitriol (Figure 2). Furthermore, 11b and 11g activated the expression of the
endogenous CYP24a1 gene strongly in MCF7 cells (Figure S2), suggesting that 11b and 11g
may be novel VDR modulators for further investigation.
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Figure 2. Transcriptional activity of compounds (11b, 11g and 15b) compared with calcitriol in
HEK293 cells. HEK293 cells were cotransfected with pGL4.27-SPP×3-Luci reporter plasmid,
pCMV-VDR and pCMV-hRXRα. The cells were treated with several concentrations of 11b, 11g, 15b
and calcitriol. After 24 hours, the cells were harvested to test the luciferase activity.
VDR-binding Affinity. Due to the potency of these vitamin D analogs with phenyl-pyrrolyl
pentane skeleton (11b, 11d, 11g and 15b) on cancer cell viability (Table 1), HL-60 cells
differentiation (Table 2) and VDR signaling activation (Figure 2), we further examined
whether these analogs could bind directly to VDR protein in vitro by the ligand-binding assay.
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Compounds 11e and 13d with little differentiation-inducing activity and anti-proliferative
activity for cancer cells showed lower potency (IC50 >500 nM) than sw22 and calcitriol under
equilibrium conditions in vitro, whereas the more potent analogs (11b, 11d and 11g) in the
cell-based assay (HL-60 differentiation in Table 2 and VDR transactivation in Figure 2) did
demonstrate binding to VDR protein; with 11b and 11d being the most potent (Figure 3). We
hypothesized that the lower VDR binding affinity of phenyl-pyrrolyl pentane derivatives
might result from less hydrogen bonds, the most important bond for ligands and VDR protein
interaction, formed with the ligand binding domain of VDR (VDR-LBD) than the natural
hormone calcitriol.
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Figure 3. The VDR ligand binding ability for compounds measured by fluorescence polarization
assay. Fluorescence polarization competition assay was performed under equilibrium conditions in
vitro and the competence of the Fluormone VDR Red ligand from the VDR-LBD by different
compounds are shown (n = 3).
Molecular Docking Study. Docking study was performed to investigate VDR-binding
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characteristics of phenyl-pyrrolyl pentane derivatives. As the vitamin D analogs (11b, 11d
and 11g) possessed a flexible side chain in the benzene ring, we first investigated the
feasibility of our docking model for predicting the structure complex between such flexible
analogs and VDR-LBD. LG190178 is a representative non-steroidal vitamin D analog
possessing a similar flexible 2-hydroxy-3,3-dimethylbutoxy phenyl side chain (Figure S3A
and S3B), which embedded in the ligand-binding pocket in the same position as calcitriol in
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6, 43, 44
the VDR-LBD-calcitriol complex.
Therefore, we determined whether the in silico
docking structure of the VDR-LBD-LG190178 could reproduce the X-ray crystal structure of
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3
the VDR-LBD-LG190178 complex (PDB ID: 2ZFX). The docking results showed that in
silico docking structure of the VDR-LBD-LG190178 complex superimposed on the X-ray
crystal structure of the VDR-LBD-LG190178 (Figure S3C), with the root-mean-squared
deviation (r.m.s.d.) equals 0.5031, suggesting that the docking model was repeatable and
reliable for predicting the structure complex between the VDR-LBD and flexible
non-steroidal vitamin D analogs.
Then the binding models of 11g and 15b with VDR-LBD (PDB ID: 2ZFX) were predicted
and the most suitable conformations of the ligands were selected based on the calculated
docking scores by means of the bonding strength (Figure 4). The results showed that 11g
could form hydrogen bonds with VDR-LBD, including the hydroxyl group at the terminal of
phenyl side chain with His301 and His393, and the O at the morpholine ring of pyrrolyl side
chain with Asp144 and Tyr232. Besides, the hydroxyl group at the terminal of phenyl side
chain of 15b formed the same hydrogen bond with His393 and His301, and the terminal
carboxyl group of pyrrolyl side chain formed a weak hydrogen bond with Arg270. Moreover,
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1g and 15b also had similar intense van der Waals interactions between VDR-LBD and the
45
phenyl-pyrrolyl pentane skeleton to those of calcitriol.
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Figure 4. The predicted binding model of 11g (A) and 15b (B). VDR-LBD of PDB reference 2ZFX
is applied for molecular docking using Glide 5.5 in Schrödinger 2009. The most suitable
conformations of the ligands were selected based on the calculated docking scores by means of the
bonding strength. (A) Hydroxyl group at the end of phenyl ring side chain of compound 11g was able
to form hydrogen bond with the His393 and His301 of VDR-LBD. On the other side of the structure,
the O of morpholine ring bound with Asp144 and Try232. (B) Hydroxyl group beside phenyl of
compound 15b was able to form hydrogen bond with the His393 and His301, and the terminal
carboxyl group formed hydrogen bond with Arg270.
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It was reported that calcitriol could form hydrogen bonds with VDR-LBD, including the
5-OH group with His301 and His393, the 1-OH group with Ser233 and Arg270, and the
2
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3-OH group with Tyr139 and Ser274. In comparison, the hydrogen bonds formed by the
hydroxyl group at the terminal of phenyl side chain of 11g and 15b interacting with His393
and His301 were the same as those of calcitriol. It is interesting that the pyrrolyl side chain of
1
1g formed hydrogen bonds with Asp144 and Tyr232 but not Ser233 or Arg270 on the other
side, resulting from the structure of spatial configuration reversal of the morpholine ring.
However, the 3-OH group of calcitriol also bound with Tyr139 and Ser274 to form additional
hydrogen bonds compared to 11g and 15b, suggesting lower binding affinity of 11g and 15b
4
4
than calcitriol with VDR-LBD.
11b, 11d and 11g Inhibited MCF7 Cell Growth by Inducing Cell Cycle Arrest and
Apoptosis. As compounds 11b, 11d, 11g and 15b showed the most potent VDR binding,
transactivation and anti-proliferative activities for cancer cells, they were selected for further
evaluation of biological activities.
Previous study showed that vitamin D analogs such as calcitriol could prevent tumor
1, 4, 39, 46
progression by inhibiting cell proliferation and inducing apoptosis.
To explore the
underlying mechanism for the reduced cell viability of MCF7 cells, we investigated the effect
of 11b, 11d, 11g and 15b on the cell cycle by flow cytometry. Treatment with 11b, 11d and
1
1g on MCF7 cells effectively arrested cells in the G0/G1 phase of the cell cycle. The
population of cells in G0/G1 phase dramatically increased as compared to that of calcitriol
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(
Figure 5A and 5B). The effect of cell cycle arrest was also observed in HepG2 cells after
treatment with compounds 11b, 11g and 15b (Table S1). Then we investigated whether 11b,
1d, 11g and 15b might induce apoptosis of MCF7 cells. MCF7 cells were treated with each
compound at the concerntration of 1 M for 24 h. The percentage of apoptotic cells in MCF7
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ꢀ
was measured by staining Annexin V-FITC and propidium iodide (PI). After that, we
analyzed the DNA content of the cells by flow cytometry as described in the section of
“
Experimental Section”. We discovered that 11b, 11d, 11g and 15b could promote the
apoptosis of MCF7 cells compared with the DMSO control group. Moreover, the proportion
of early apoptotic cells (Annexin V-FITC positive and propidium iodide negative)
signifcantly increased in the groups after 11d, 11g and 15b treatment compared with the
calcitriol treatment group (Figure 5C and 5D).
Previous studies reported that MART-10, a new analog of calcitriol, could up-regulate the
protein level of p21 and p27, arrest cancer cells at the G
Moreover, other studies also determined that VDR modulators could cause cell apoptosis and
the accumulation of cells in G /G phase which was associated with the up-regulation of p21
and p27 in the protein level in cells.
arrest the tumor cells in G /G phase through the up-regulation of p21 and p27. After MCF7
cells were treated with 11b, 11g at the concentration of 1 M for 24 hours, the expression
0 1
/G phase and promote cell apoptosis.
0
1
39, 46-48
Hence, we speculated that our derivatives could
0
1
ꢀ
levels of p21 and p27 were examined. Western blot assay showed that treatment with 11b and
11g increased p21 and p27 expression levels in MCF7 cells (Figure S5). The real-time
quantitative PCR assay revealed that 11b and 11g also increased the mRNA levels of p21 and
p27 (Figure S4). Furthermore, relative expression levels of Bax and Bak mRNA were
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elevated 24 hours after treatment with different compounds in MCF7 cells. The results
revealed that treatment with 11b and 11g dramatically increased the levels of pro-apoptotic
Bax and Bak expression in MCF7 cells (Figure S4). Taken together, the results demonstrated
that 11b, 11d and 11g inhibited the growth of MCF7 cells by inducing cell cycle arrest and
cell apoptosis.
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Figure 5. Cell cycle arrest and apoptosis after treatment with vitamin D modulators in MCF7
cells. (A) Cell cycle phase distribution was determined by flow-cytometry. The MCF7 cells were
treated with each compound at 1 ꢀM for 24 hours. (B) The fold differences of cell population in the
G0/G1 phase of the cell cycle after treatment of each compound (n = 4 for each compound). The
relative difference was normalized to the group of calcitriol treatment. (C) Representative flow
cytometry dot plot of Annexin V-FITC and PI staining for the detection of apoptotic MCF7 cells. The
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MCF7 cells were treated with each compound at 1 ꢀM for 24 hours. (D) The fold differences of
apoptotic quantification after exposure to each compound for 24 hours (n = 3 for each compound).
The relative difference was normalized to the group of calcitriol treatment.
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Cellular Uptake and Metabolic Stability of 11b, 11d and 11g. To provide more basic
information on drug-like properties, cellular uptake and metabolic stability of 11b, 11d and
11g in MCF7 cells were further investigated. MCF7 cells were cultured with 1
ꢀM of 11b,
11d, 11g, sw22, LG190119 and calcitriol in the presence of 10% FBS for 10 and 30 min, and
the concentrations of these compounds in both the culture medium and cells were determined
by HPLC. Cellular uptake of 11g was 37.9% and 38.2% at 10 and 30 min, respectively, while
that of calcitriol was 6.5% and 10.4% at 10 and 30 min, respectively. Thus, compound 11g
was shown to be 4-5 times more effectively incorporated into the MCF7 cells than calcitriol,
and 2-3 times more effectively than sw22 and LG190119. In addition, more effective uptake
of 11b and 11d than calcitriol incorporated into the MCF7 cells was also observed (Figure
6A).
When MCF7 cells were cultured up to 24 hours, 27% of calcitriol was metabolically
consumed, while only 6% of 11g was consumed, indicating that compound 11g was four
times more stable than calcitriol in MCF7 cells (Figure 6B). Overall, the results suggested
that 11b, 11d and 11g possessed comparable uptake activity and metabolic stability to that of
calcitriol.
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Figure 6. Uptake and metabolism stability of 11b, 11d, 11g, calcitriol, LG190119 and sw22 in
MCF7 cells. (A) MCF7 cells were incubated with 1 ꢀM of different compounds for 10 minutes or 30
minutes. The cells and medium were extracted separately by chloroform/methanol (3:1, v/v). The
extracts were analyzed by HPLC. (B) MCF7 cells were incubated with 1 ꢀM of different compounds
for 24 hours (with cells). Similarly, each of the substrates (11b, 11d, 11g, calcitriol, LG190119 and
sw22) was incubated with medium only for 24 hours (without cells). Each experimental mixture was
extracted with chloroform/methanol (3:1, v/v). The extracts were analyzed by HPLC. Data were
shown as the mean ± SEM (n = 3).
In vivo Calcemic Activity Assay. In vivo calcemic activity assay was performed to
evaluate the potential hypercalcemic possibility of designed non-steroidal modulators. The
compounds 11b, 11d, 11g and 15b were chosen for calcemic activity assay in vivo, with
calcitriol, sw22 and LG190119 as the positive controls and vehicle as the negative control.
The results were shown in Figure 7.
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Figure 7. In vivo hypercalcemic effects of calcitriol, LG190119, sw22 and compounds 11b, 11d,
1
1g and 15b. Mice were treated with 11b, 11d, 11g and 15b in different concentration (0.5 mg/kg, 5
mg/kg and 30 mg/kg. i.p.) over a 7 day period. Calcitriol (0.5 µg/kg or 5 µg/kg. i.p.) and LG190119
30 mg/kg. i.p.), sw22 (30 mg/kg. i.p.) were employed as reference drugs. Data were shown as the
(
mean ± SEM (n = 6). Statistical significance (*, p < 0.05; **, p < 0.01; ***, p < 0.001) for each
concentration of serum calcium compared to the vehicle control.
A significant increase in serum calcium was observed after the administration of calcitriol
(5 µg/kg) for 7 days (11.04 mg/dL for calcitriol vs 7.05 mg/dL for saline; p < 0.001), while a
dose of 0.5 µg/kg showed normal serum calcium level compared with the vehicle group.
Furthermore, 11b, 11d, 11g and 15b at the dosage of 0.5 mg/kg/day and 5 mg/kg/day had no
effect on serum calcium. Intraperitoneal administration of 11d and 11g even at the dosage of
30 mg/kg/day resulted in a slight elevation of serum calcium level compared with the vehicle
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group. Therefore, the designed non-steroidal modulators had no hypercalcemic effect in vivo.
In vivo antitumor activity assay. To investigate the in vivo antitumor effect of 11b, 11d
and 11g, breast tumor mouse model was established by orthotopic transplantation of human
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MCF7 cells in athymic nude mice. LG190119 and calcitriol as controls were selected as
positive controls because of their antitumor effects in animal models or human clinical trials.
4
4
LG190119, the representative of nonsteroidal compounds, is the only reported compound of
the non-steroidal analogs that displayed antitumor effects in animal models of cancer.
Specifically, previous report showed that LG190119 effectively inhibited LNCaP prostate
2
cancer xenograft tumor growth without causing hypercalcemia. In addition, calcitriol has
4
been used in a number of animal models of cancer and clinical trials for cancer treatment,
1, 4
such as prostate cancer and breast cancer.
The results showed that 11b, 11d and 11g
significantly inhibited the tumor growth compared with calcitriol (0.5 µg/kg, in which
concentration antitumor effects but no significant increase of the serum calcium were
4
observed as shown in Figure 7) and sw22 (Figure 8B and 8C). In addition, these three
9
compounds also have better antitumor efficacy compared with LG190119 (10 mg/kg, in
24
which concentration the inhibition of prostate tumor growth was observed ). However, there
was no significant change of the body weight in the treatment of compounds 11b, 11d and
11g compared with the saline or vehicle group (Figure 8A), indicating that the three
compounds have no apparent toxicity to mice during the period of cancer treatment.
Importantly, the treatment of 11b, 11d and 11g did not increase mouse serum calcium
levels in comparison with the control groups in breast tumor-bearing mice (Figure 8D). We
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further determined the proliferation and apoptosis rate of MCF7 cells by Ki67
immunohistochemistry and terminal deoxynucleotidyl transferase dUTP nick end labeling
assay (TUNEL). There was a significantly low intensity of Ki67 staining in sections of MCF7
orthotopic tumor after treatment with 11b and 11g compared with calcitriol group (Figure 8E
and 8F), which was consistent with tumor volume results (Figure 8B and 8C), suggesting
that 11b and 11g inhibited MCF7 cells proliferation in vivo. In addition, TUNEL staining
showed significantly increasing apoptosis in the breast tumor after treatment with 11b, 11d
and 11g compared with sw22, LG190119 and calcitriol treatment (Figure 8G and 8H).
Collectively, these findings suggested that 11b and 11g could inhibit the growth of breast
cancer through suppressing proliferation and inducing apoptosis of cancer cells.
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Figure 8. Antitumor effects of compounds 11b, 11d and 11g in vivo. (A) The change in the body
weight, (B) tumor volume of MCF7-bearing mice after treatment with vehicle (polyoxyethylated
castor oil (EL)/ethanol/saline=1:1:18), saline, sw22 (10 mg/kg, i.p.), LG190119 (10 mg/kg, i.p.), 11b
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(
10 mg/kg, i.p.), 11d (10 mg/kg, i.p.), 11g (10 mg/kg, i.p.) and calcitriol (0.5 ꢀg/kg, i.p.). (C) The
images of excised tumors in each group. (D) The hypercalcemic effects of calcitriol, LG190119, sw22
and 11b, 11d and 11g in the MCF7 tumor-bearing mice. (E) Immunohistochemistry of Ki-67 in tumor
sections of MCF7-bearing mice. Scale bar, 50 ꢀm. (F) Quantification of percentages of positive Ki-67
staining area in total nuclei area in each field (n=10, 400 fold magnification). The relative difference
was normalized to the group of calcitriol treatment. (G) Immunohistochemistry of TUNEL in tumor
sections of MCF7-bearing mice. Scale bar, 50 ꢀm. (H) Quantification of percentages of positive
TUNEL staining area in total nuclei area in each field (n=10, 400 fold magnification). The relative
difference was normalized to the group of calcitriol treatment. Statistical significance (*, p < 0.05; **,
p < 0.01; ***, p < 0.001) for 11b, 11d and 11g compared to calcitriol.
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Pharmacokinetics Study In Vivo. Given their promising in vivo anti-tumor activities,
preliminary in vivo pharmacokinetic (PK) studies of compounds 11g and 11b were
determined in rats and sw22 was used as the control. The results were summarized in Table 3.
1
1b and 11g displayed better pharmacokinetic properties than sw22, with an oral
bioavailability of 36.8% and a T1/2 value of 11.31 hours after oral administration. Moreover,
the T1/2 value of 11g (T1/2 = 4.75 h) was similar to calcitriol (T1/2 = 4.27 h) after intravenous
50
administration. The results suggested that 11b and 11g could possess therapeutic potential
for cancer treatment.
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a
Table 3. The pharmacokinetic profile of compounds 11b, 11g, sw22 in rats
1
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1b
11g
i.p.
20 mg/kg
sw22
i.p.
Parameter
i.v.
i.p.
p.o.
i.v.
p.o.
i.v.
p.o.
5
mg/kg
20 mg/kg
20 mg/kg
5 mg/kg
20 mg/kg
5 mg/kg
20 mg/kg
20 mg/kg
AUC(mg/L*h) 25.21±3.40 30.47±5.09 21.47±1.26
10.37±1.91
4.75±0.85
7.55±0.78
0.49±0.089
25.91±2.57 15.27±1.68 13.40±1.07 24.76±2.26 13.49±1.75
T1/2(h)
MRT(h)
CL(L/h)
F(%)
2.97±0.16
3.85±0.49
6.21±1.62
6.82±1.18
11.73±1.6 11.31±2.75 2.44±0.43
17.59±2.42 15.90±3.18 2.82±0.04
3.96±0.53
6.89±2.52
9.30±2.32 10.97±1.53
8.15±0.97 10.76±3.18
0.21±0.032 0.68±0.15 0.93±0.065
30.22% 21.29%
0.78±0.082 1.31±0.14 0.41±0.036 0.81±0.085 1.50±0.21
62.46% 36.80% 46.19% 25.17%
a
SD rats (male, 4 animals per group) weighted 180−220 g were used for the study (n = 4). Data were
presented as Mean ± SD.
DISCUSSION AND CONCLUSIONS
In this study, a series of novel vitaimin D analogs with phenyl-pyrrolyl pentane skeleton were
synthesized and a comprehensive series of experiments were performed to validate their VDR
modulating activities and in vivo anticancer activities. This study was the first to show that
11b and 11g with the phenyl-pyrrolyl pentane skeleton markedly inhibited MCF7 breast
tumor growth in athymic nude mice without causing increased serum calcium level or animal
weight loss, suggesting that they may be suitable for long-term cancer chemotherapy.
31
sw22, previously reported by our group, was selected as the lead compound. First, we
retained the phenyl-pyrrolyl pentane skeleton to keep the VDR binding affinity. Then, we
introduced the diethylcarbinol as the terminal hydrophobic group into the phenyl side chain to
give 16d. The in vitro anti-proliferation results revealed 16d had similar IC50 range against
different cancer cells compared to those of sw22 (Table 1). Based on this, we further
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explored the effects of substitutions groups on the pyrrole ring, 11a-11i, 12a-12b, 13a-13d,
14a-14c, 15a-15d and 16a-16c were designed and synthesized.
Based on the results of the in vitro anti-proliferative assay and HL-60
differentiation-inducing assay, we summarized the following structure-activity relationships
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(SARs) of phenyl-pyrrolyl pentane derivatives. (1) Introducing the 3-ethyl-3-hydroxypentoxy
into the phenyl side chain could retain the anti-proliferative activity against cancer cells. (2)
2
Introducing the hydrophilic moieties at the R group is important to improve the
anti-proliferative and HL-60 differentiation-inducing activities, such as introducing the
hydroxy, amines and carboxyl groups to give compounds 11a-11d, 15a-15d and 16a-16d,
while the introduction of hydrophobic segments may lead to a remarkable decrease or even
loss of anti-proliferative and HL-60 differentiation-inducing activities, such as 11e and 11h.
2
3) The introduction of large groups such as benzene ring into R group cannot be tolerated,
(
nearly leading to a loss of the anti-proliferative and HL-60 differentiation-inducing activities,
such as 13a-13d. (4) Introducing alkenyl and alkynyl to the pyrroyl side chain could result in
a complete disappearance of anti-proliferative and HL-60 differentiation-inducing activities,
such as 11i, 12a-12b.
1
1b and 11g showed significant VDR agonistic activity determined by both dual luciferase
reporter assay (Figure 2) and human leukemia cell line (HL-60 cells) differentiation assay
Table 2), though the new analogs (11b, 11d and 11g) showed lower competence with
(
Fluormone VDR Red ligand (PolarScreen™ Vitamin D Receptor Competitor Assay,
Invitrogen) than calcitriol in the VDR binding assay performed under equilibrium conditions
in vitro. Therefore, the VDR-dependent cell-based assay may be more sensitive than the in
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vitro VDR equilibrium-binding assay in detection of VDR activators. We speculated that
detection of VDR binding of the less potent non-steroidal vitamin D analogs under
equilibrium conditions in the competition binding assay with the Fluormone VDR Red ligand,
may be precluded by the high on-rate and low off-rate of the high-affinity Fluormone VDR
Red ligand. Similar results have been reported in the VDR-binding characteristics of
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non-steroidal vitamin D analogs including LG190119, LG190155 and LG190178.
For the
transcriptional activity of VDR downstream target gene Cyp24a1, Cyp24a1 could be strongly
induced by calcitriol and often serves as a marker of VDR activation in the cell. In the present
study, the activation of Cyp24a1 transcription after treatment of 11b and 11g is lower than
that of calcitriol treatment group (Figure S2). The differences of the activation level of
Cyp24a1 gene between calcitriol and the non-steroidal VDR modulators could be due to their
51-54
distinctions in metabolism and coactivator recruitment.
Both preclinical and clinical studies support the development of vitamin D analogs as
1
preventative and therapeutic anticancer agents, however, clinically approved anticancer
vitamin D drugs remain elusive. Part of the reason for the clinical failure is that drug
concentrations, which are thought to be necessary for antitumor activity on the basis of
preclinical results, are not achievable in the blood/tumor of patients as well as hypercalcemia
4
, 55, 56
intervenes.
One possibility for an effective cancer therapy is attempting to overcome
poor bioavailability and designing novel VDR modulators without the risk of increasing
serum calcium. Another would be the combination of a low-calcemic vitamin D analog with
1, 55
other anticancer agents to achieve a synergistic effect on cancer therapy.
In this study, we
found that 11b and 11g possessing good bioavailability could inhibit breast tumor growth
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