Design, synthesis and biological evaluation of nonsecosteroidal vitamin D3receptor ligands as anti-tumor agents

Article abstract of DOI:10.1016/j.bmcl.2017.01.084

1α,25-dihydroxyvitamin D₃(1,25-(OH)₂D₃, also known as calcitriol), the active form of vitamin D₃, is being increasingly recognized for cancer therapy. Our previous work showed that phenyl-pyrrolyl pentane analogs, which mimicked anti-proliferative activities against several cancer cell lines of the natural secosteroidal ligand 1,25-(OH)₂D₃. Here, in order to optimize the structural features and discover more potent derivative, a series of nonsecosteroidal vitamin D₃receptor (VDR) ligands bearing acetylene bond linker was designed, synthesized and evaluated. Most of them showed moderate to good binding affinities and agonistic activities. Especially, compound 19f displayed the most anti-proliferative activities against MCF-7 and PC-3 cells with the IC₅₀values of 1.80 and 5.35 μM, respectively, which was comparable to positive control 1,25-(OH)₂D₃. Moreover, compound 19f exhibited reduced toxicity against human normal liver cell line (L02) compared with the parental compound 7. Besides, the preliminary structure–activity relationships (SARs) were also analyzed.

Full text of DOI:10.1016/j.bmcl.2017.01.084

Accepted Manuscript
Design, synthesis and biological evaluation of nonsecosteroidal vitamin D3 re-
ceptor ligands as anti-tumor agents
Bin Wang, Meixi Hao, Can Zhang
PII:
S0960-894X(17)30107-5
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.01.084
BMCL 24660
Reference:
Bioorganic & Medicinal Chemistry Letters
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Accepted Date:
19 December 2016
17 January 2017
27 January 2017
Please cite this article as: Wang, B., Hao, M., Zhang, C., Design, synthesis and biological evaluation of
nonsecosteroidal vitamin D3 receptor ligands as anti-tumor agents, Bioorganic & Medicinal Chemistry Letters
(2017), doi: http://dx.doi.org/10.1016/j.bmcl.2017.01.084
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Design, synthesis and biological evaluation of nonsecosteroidal vitamin D3
receptor ligands as anti-tumor agents
Bin Wang, Meixi Hao and Can Zhang*
State Key Laboratory of Natural Medicines and Jiangsu Key Laboratory of Drug Discovery for Metabolic
Diseases, Center of New Drug Discovery, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing, 210009,
China.
*
Corresponding author. Tel/Fax: 86-25-83271171; E-mail: zhangcan@cpu.edu.cn
1

ABSTRACT
1α,25-dihydroxyvitamin D3 (1,25-(OH)2D3, also known as calcitriol), the active
form of vitamin D3, is being increasingly recognized for cancer therapy. Our previous
work showed that phenyl-pyrrolyl pentane analogs, which mimicked anti-proliferative
activities against several cancer cell lines of the natural secosteroidal ligand
1,25-(OH)2D3. Here, in order to optimize the structural features and discover more
potent derivative, a series of nonsecosteroidal vitamin D3 receptor (VDR) ligands
bearing acetylene bond linker was designed, synthesized and evaluated. Most of them
showed moderate to good binding affinities and agonistic activities. Especially,
compound 19f displayed the most anti-proliferative activities against MCF-7 and
PC-3 cells with the IC50 values of 1.80 and 5.35 μM, respectively, which was
comparable to positive control 1,25-(OH)2D3. Moreover, compound 19f exhibited
reduced toxicity against human normal liver cell line (L02) compared with the
parental compound 7. Besides, the preliminary structure–activity relationships (SARs)
were also analyzed.
Keywords: Vitamin D3; VDR ligand; Nonsecosteroidal; Phenyl-pyrrolyl pentane
skeleton; Binding affinity; Agonistic activity; Anti-proliferative activity.
The active form of vitamin D3, the hormone 1α,25-dihydroxyvitamin D3
(1,25-(OH)2D3, also known as calcitriol) participates in numerous biological
processes1-3. In addition to its classical role in mineral homeostasis and bone
mineralization4, this hormone regulates numerous cellular pathways that could have a
role in determining cancer risk and prognosis5-9. Because of this “nonclassical” actions,
1,25(OH)2D3 has attracted considerable interest as a potential drug for the treatment of
cancer disease. The effects of 1,25(OH)2D3 are mediated through the vitamin D3
receptor (VDR) which is a ligand-dependent transcription factor belonging to the
superfamily of nuclear hormone receptors10-14
.
So far, more than 3000 secosteroid analogs have been synthesized15, and many of
them exhibit efficient VDR activities and some analogs have been successfully used
2

in the treatment of bone, mineral, and skin disorders (Fig. 1) 16-17. However, adverse
effects, particularly hypercalcemia, limit the clinical application of secosteroid
analogs in the management of cancer disease15. Recently, much attention has been
directed towards nonsecosteroidal VDR ligands, which mimic various activities in
vitro and in vivo of the natural ligand 1,25(OH)2D3 without direct structural
relationship to 1,25(OH)2D3, such as VDR binding and inhibition of proliferation of
cancer cells18-19. Moreover, they have simpler structures compared with secosteroid
analogs20-27. Above all, some nonsecosteroidal VDR ligands avoid hypercalcemia
effect, such as LG190178 and its analogs. Therefore, the development of VDR ligands
with nonsecosteroidal skeleton is required to create novel therapy for the VDR-related
cancer
disease.
Figure 1. Chemical structures of secosteroidal and nonsecosteroidal VDR ligands
We previously reported novel phenyl-pyrrolyl pentane skeleton as a
nonsecosteroidal VDR ligand skeleton which had anti-proliferative effects against
3

cancer cells and avoided hypercalcemia effect in vivo28-29. While the VDR binding
affinities of all the synthesized compounds suffered a remarkable decrease as
compared with 1,25(OH)2D3, compound 7 had a moderate VDR binding affinity in
vitro, which deserved further study. Here, we wish to report compound 7 as a lead for
further optimization to explore the structure-activity relationships (SARs) and to
discover more potent derivative with high VDR binding affinity and anti-proliferative
effect against cancer cells. As shown in Figure 2, first, we attempted to introduce
acetylene bond linker to minimize the flexibility and to fix the direction of the side
chain, and then designed 3-pentanolyl as the terminal hydrophobic group of the side
chain based on previous SARs. Additionally, to explore the effects of substitutions of
polarity and steric hindrance on the pyrrole ring, compounds 17c-i and 18a-d were
designed. Second, phenyl-pentane group on C-4 position of pyrrole ring replaced C-5
position to investigate the influence of the substitution positions of the terminal
hydrophilic group of the pyrrole ring to give compounds 19a, 19f-j and 20a-e.
Figure 2. Design of novel nonsecosteroidal vitamin D receptor ligands with phenyl-pyrrolyl
pentane skeleton.
The synthetic pathway of target compounds 17c-i and 18a-d is outlined in Scheme
1. Key intermediate 8 was readily prepared using our previously reported approach25,
4

and then it reacted with ethyl pyrrole-2-carboxylate in the presence of lewis acid
o
BF3·Et2O at 0 C to give intermediate 9a, following the treatment with iodoethane in
DMF to afford intermediate 10a. The intermediate 11a was obtained by reduction
reaction of intermediate 10a in the presence of HCOONH4, which was acylated with
trifluoromethanesulfonic anhydride to give 12a in moderate yield. Intermediate 12a
subjected to sonogashira coupling reaction with trimethylsilylacetylene in the
presence of palladium catalyst afforded 13a and subsequent removal of trimethylsilyl
group using tetrabutylammonium fluoride (TBAF) gave acetylene 14a. Hydrolysis of
intermediate 14a by KOH produced the key intermediate 15a, which was alkylated
with n-butyl lithium and 3-pentanone at -78 oC to give key intermediate alcohol 16a.
Interestingly, followed by moving to room temperature for 2 h, alcohol 16a
transformed to compound 17i, which were obtained by one step from carboxylic acid
group to ketone group. By reaction of intermediate 16a with the corresponding amines
or alcohols, target compounds 17c-h and intermediates 17a-b were obtained. Finally,
target compounds 18a-c were obtained by hydrolysis reactions of intermediates 17a-c
in the presence of KOH. On the other hand, target compound 18d was obtained by
reduction reaction of compound 17c.
The synthetic pathway of target compounds 19a, 19f-j and 20a-e is outlined in
Scheme 2. Intermediate 9b, which was the regioselectivity isomer of intermediate 9a,
was produced by reacting with ethyl pyrrole-2-carboxylate in the presence of lewis
o
o
acid BF3·Et2O at 20 C instead of at 0 C. By the same manner as described for the
preparation of intermediates 10a-16a, intermediates 10b-16b were obtained. As
described before, compound 19j were obtained by one step from carboxylic acid
group to ketone group. By reaction of intermediate 16b with the corresponding
amines or alcohols, target compounds 19a, 19f-j and intermediates 19b-e were
obtained. Finally, target compounds 20a-d were obtained by hydrolysis reactions of
intermediates 19a-d in the presence of KOH. On the other hand, target compound 20e
was obtained by reduction reaction of compound 19e.
5

Scheme 1. Synthesis of target compounds 17c-i and 18a-d. Reagents and conditions: (a) Ethyl
1H-pyrrole-2-carboxylate, BF3·Et2O, 0 oC, 1 h, 53%; (b) C2H5I, NaH, DMF, 0-25 oC, 2 h, 82%; (c)
Pd/C, HCOONH4, CH3OH/EtOAc (10:1), 25 oC, 1 h, 98%; (d) Tf2O, TEA, toluene, 0 oC, 2 h, 67%;
o
(e) TMS acetylene, PdCl2(dppf)2, TEA, DMF, 100 C, overnight, 63%; (f) TBAF, THF, rt, 1 h,
o
o
95%; (g) 2 mol/L KOH, EtOH, 80 C, 6 h, 95%; (h) 3-pentanone, n-BuLi, THF, -78-0 C, 2 h,
75%; (i) EDCI, HOBt, TEA, RNH2, DCM, rt, overnight, 35-96%; (j) EDCI, DMAP, ROH, DCM,
rt, overnight, 26-64%; (k) n-BuLi, THF, 25 oC, 2 h, 79%; (l) 2 mol/L KOH, EtOH, rt, 1 h, 83-94%;
(m) LiAlH4, EtOAc, rt, 1 h, 86%.
6

Scheme 2. Synthesis of target compounds 19a, 19f-j and 20a-e. Reagents and conditions: (a) Ethyl
1H-pyrrole-2-carboxylate, BF3·Et2O, 25 oC, 1 h, 44%; (b) C2H5I, NaH, DMF, 0-25 oC, 2 h, 85%;
o
(c) Pd/C, HCOONH4, CH3OH/EtOAc (10:1), 25 C, 1 h, 97%; (d) Tf2O, TEA, toluene, 0 oC, 2 h,
64%; (e) TMS acetylene, PdCl2(dppf)2, TEA, DMF, 100 oC, overnight, 53%; (f) TBAF, THF, rt, 1
o
h, 95%; (g) 2 mol/L KOH, EtOH, 80 C, 6 h, 96%; (h) 3-pentanone, n-BuLi, THF, -78-0 oC, 2 h,
54%; (i) EDCI, HOBt, TEA, RNH2, DCM, rt, overnight, 35-96%; (j) EDCI, DMAP, ROH, DCM,
rt, overnight, 64%; (k) n-BuLi, THF, 25 oC, 2 h, 77%; (l) 2 mol/L KOH, EtOH, rt, 1 h, 83-94%; (m)
LiAlH4, EtOAc, rt, 1 h, 89%.
The receptor binding affinity assay of synthesized compounds was initially
performed at a concentration of 1 μM and the binding affinity was displayed by a
relative value based on 1,25(OH)2D3 being assigned as 100%. As shown in Table 1.
the results displayed that although seven compounds (17d, 18a, 18d, and 19f-i)
demonstrated more effective binding affinities than the parental compound 7 (25.5%),
with binding values at the range of 29.7-62.2%, none compound displayed equivalent
7

binding affinity compared to 1,25(OH)2D3. Additionally, five compounds (17e, 18c,
20a, 20c, and 20e) showed moderate binding affinities with the percentage of binding
ranging from 13.6% to 20% and other compounds had no obvious binding affinities.
Our SARs analysis started with C-5 position of pyrrole ring bearing phenyl-
pentane group and showed that compounds with the terminal hydrophilic groups in
the A ring section, such as carboxylic acid (18a), hydroxyl (18d), and amino groups
(17d) showed significant binding affinities. However, by one-atom extension of the
hydrophilic groups of compounds 17d and 18a, 17e and 18c were synthesized and the
binding affinities of them were dramatically decreased. Additionally, introducing a
large carboxylic acid obtained compound 18b, which displayed poor binding affinity,
suggesting sterically hindered substitutes may not be accepted in VDR ligand binding
domains. In order to verify the hypothesis, benzene and aromatic heterocyclic groups
were introduced to synthesize compounds 17f and 17h. As expected, the low binding
affinities of these compounds testify our hypothesis. Besides, when carboxylic acid
group was protected by methyl group, the binding affinity of the resulting compound
17c were dramatically decreased compared with compound 18c, suggesting
carboxylic acid group plays an important role in the VDR binding affinity.
Furthermore, a similar binding affinities were observed when replacing ester group
with hydrophobic groups, such as propargyl and n-butyl groups.
In order to explore the influence of substitution positions on the binding affinities,
compounds 19a, 19f-j and 20a-e were synthesized by removing the substitution on the
C-5 position of pyrrole group to C-4 position. As shown in Table 1, similar SARs
were observed. Although compound 19g showed the most potent affinity among them,
no better result was observed compared to compound 17d. On the contrary, some
compounds bearing same groups showed decreased binding affinity, such as
compounds 20a, 20b, and 19f as compared with 18a, 18c, and 17d. The results
suggest that the position of the substitute is important by affecting the conformation of
the terminal hydrophilic group which is essential for binding, as in the case of the
conformationally restricted A ring of secosteroid. To further explore the difference on
the binding affinities between C-5 position of pyrrole group and C-4 position, we
8

performed the docking analyses of compounds 18a and 20a, which had the same
hydrophilic substitute, but displayed remarkable difference on binding affinities,
based on crystallographic structure of 1,25(OH)2D3 in complex with VDR (PDB code:
1DB1). As shown in figure 3C, docking analyses demonstrated that the side chain of
both compounds present similar conformations. However, substitutuion of pyrrole
group from C-5 position to C-4 position resulted in conformational change in the A
ring section (figure 3A and 3B). In the docking, the introducing carboxylic acid of
compound 18a was beautifully adjusted in VDR and formed two hydrogen bonds with
Ser237 and Arg274. Unfortunately, the change of substitution positions resulted in a
longer distance between the introduced carboxylic acid of compound 20a and Ser237,
so only one hydrogen bond with Arg274 was formed, which maybe the important
reason of different binding affinities.
B
A
C
Figure 3. (A) Docking structure of the complex VDR-compound 18a. (B) Docking structure of the
complex VDR-compound 20a. (C) Superposition of compounds 18a and 20a. Compound 18a is
shown in stick representation with carbon and oxygen atoms in green and red, respectively.
Compound 20a is shown in stick representation with carbon and oxygen atoms in wheat and red,
respectively. The hydrogen bonds formed are shown as yellow dashed lines.
9

Table 1. Structures and binding abilities of target compounds.
Relative VDR
Compd
X
N
Y
C
R1
R2
H
R
binding ability(%)a
C2H5
—
17c
N
N
C
C
C2H5
C2H5
H
H
62.2±3.5
17d
17e
18.8±1.8
N
N
C
C
C2H5
C2H5
H
H
4.0±0.3
17f
—
17g
N
C
C2H5
H
—
—
17h
N
N
C
C
C2H5
C2H5
H
H
17i
55.4±3.1
5.9±0.3
18a
N
N
C
C
C2H5
C2H5
H
H
18b
18c
13.6±1.5
10

N
C
C
N
C2H5
H
H
59.7±2.7
18d
19a
—
C2H5
C
N
H
C2H5
50.3±3.1
19f
C
C
N
N
H
H
C2H5
C2H5
55.3±4.1
31.6±1.7
19g
19h
C
C
N
N
H
H
C2H5
C2H5
29.7±2.1
19i
19j
—
C
C
N
N
H
H
C2H5
C2H5
14.6±1.1
4.3±0.1
20a
20b
C
N
H
C2H5
20.0±2.4
20c
C
C
N
N
H
H
C2H5
C2H5
5.9±0.3
20d
20e
19.8±1.2
—
—
—
—
—
—
—
—
—
—
25.5±1.4
100.0±2.3
7
1,25(OH)2D3
aThe values represent the mean ± SD of three independent experiments. 1,25(OH)2D3 is assigned
as 100%.
Subsequently, we further evaluated the VDR binding affinities (IC50) of
11

compounds 17d, 18a, 18d, and 19f-g with excellent inhibition at 1 μM. As shown in
Table 2, introducing hydroxyl and carboxylic acid groups as the terminal hydrophilic
groups showed decreased affinities compared with the parental compound 7. However,
compounds 17d, 19f, and 19g, all of which were introduced amino groups instead
displayed more potent than lead compound 7, which deserved further investigations.
Table 2. The binding affinities of selected compounds.
Compd
17d
VDR binding (IC50, nM)a
73.21±3.68
Compd
VDR binding (IC50, nM)a
51.31±3.54
19g
7
85.66±2.45
76.76±5.12
18a
123.43±2.32
1,25(OH)2D3
1.13±0.11
18d
56.48±2.17
19f
aIC50: the concentration that causes 50% of cell proliferation inhibition. Data are expressed as
mean ± SD from three independent experiments.
It is proved that vitamin D3-agonistic activity is associated with HL-60 cell
differentiation induction30-31. Therefore, the vitamin D3-agonistic ability can be
estimated as the potential to differentiate human promyelocytic leukemia cell line
(HL-60) into macrophages. All synthesized compounds were tested for HL-60 cell
differentiation using calcitriol as positive control, as shown in Table 3. Most
compounds displayed good agonistic activities with EC50 values in the nanomole
range. Among them, compounds 19f and 20e showed higher agonistic activities than
positive control 1,25(OH)2D3. As similar as the SARs of binding affinities,
compounds with the terminal hydrophilic groups in the A ring section, such as
carboxylic acid (18a) and anime groups (17d) also showed significant agonistic
activities. By introducing a large carboxylic acid substitute, compound 18b was
synthesized and showed a slight decreased agonistic activity compared to compound
18a, although a dramatically decreased binding affinity was observed. This
discrepancy between agonistic activity and binding affinity could be explained by the
interactions between the VDR ligand complex and other cofactors. For the
transcriptional activation of VDR, it is required that the AF-2 transactivation motif of
12

VDR interacts with several types of cofactor such as VDR interacting
proteins(DRIPs)32-35. Furthermore, introducing large sterically hindered substitutes,
such as 17f and 17h displayed remarkable decreased agonistic activities. Additionally,
when removing the substitution on the C-5 position of pyrrole group to C-4 position,
most compounds with the same hydrophilic groups showed decreased agonistic
activities.
However,
compound
19f
(EC50
=
6.98
nM)
bearing
N,N-diethyl-1,2-ethanediamine, which had less binding affinity than 17d, showed the
best agonistic activity. Unfortunately, no improvement in agonistic activities of all the
synthesized compounds was observed compared with the parental compound 7 (EC50
= 6.54 nM). We estimate that acetylene bond introduced has weaker cell permeability
than ether bond and results in the reduced agonistic activity.
To evaluate the anti-proliferative activities of synthesized compounds, human
breast cancer cell line (MCF-7)36-37 and human prostate cancer cell line (PC-3)38,
which over express VDR were selected as cell models to test the anti-proliferative
effects by the standard MTT assay, with 1,25(OH)2D3 as positive control. As shown
in Table 3, the results displayed that most of the synthesized compounds showed
moderate to good activities with IC50 values in the micromole range and in some cases
better than that of 1,25(OH)2D3, while compounds 17h-i, and 17k were proved to be
poor activities against both cell lines. Interestingly, compounds 17d, 18a, 18d, 19f,
19i and 20e showed higher anti-proliferative activities than 1,25(OH)2D3 against
MCF-7 cells. In addition, thirteen compounds exhibited remarkable anti-proliferative
activities with the IC50 values ranging from 2.2 to 15.1 μM, which were comparable to
that of 1,25(OH)2D3 (IC50 = 17.2 μM) on PC-3 cells. Notably, compounds 17d, 18a,
19f, 19i and 20e displayed more effective anti-proliferative activities against both cell
lines compared with 1,25(OH)2D3, although only compounds 19f and 20e showed
better agonistic activities than 1,25(OH)2D3, which suggests that they may work
though the multiple molecular mechanisms. This primary screening results revealed
that phenyl-pyrrolyl pentane derivatives with acetylene bond linker exhibited strong
anti-proliferative activities. As similar as the SARs of the binding affinity and
agonistic activity, compounds bearing hydrophilic groups, such as 17d, 18a, 19f, and
13

19i, showed better agonistic activities than that of hydrophobic groups, such as 17c,
17i and 19j, which suggested that it is necessary to introduce hydrophilic groups into
the A ring section. Compound 18d bearing hydroxyl group displayed poor
anti-proliferative activity against PC-3 cells, although it showed significant binding
affinity and moderate agonistic activity, which suggests that chemical modification of
compound 18d may induce AF2 conformations and cofactor interactions distinct from
those of natural ligands and can result in cell type-selective modulation of target gene
expression39. Besides, compound 18b also showed a slight decreased anti-proliferative
activity compared to compound 18a alike that of agonistic activities. Furthermore,
compound 17f-i bearing sterically hindered or hydrophobic substitutes also exhibited
poor anti-proliferative activities against both cell lines as similar as the binding
affinities and agonistic activities. Additionally, as similar as the SARs of agonistic
activities, most synthesized compounds exhibited decreased anti-proliferative
activities compared with that of the same A ring section, such as 18a, 18b and 18c to
20a, 20c and 20b, respectively. In this regard, it could be proved that the
anti-proliferative activities of synthesized compounds are positively correlative with
VDR agonistic activities. Moreover, compound 19f also showed the most
anti-proliferative activities against MCF-7 and PC-3 cells with the IC50 values of 1.80
and 5.35 μM, respectively, which was comparable to positive control 1,25-(OH)2D3.
Table 3. The HL-60 differentiation-inducing activities and anti-proliferative activities of
synthesized compounds in vitro.
In vitro anti-proliferative activity
HL-60 differentiation
(IC50, μM)b
Compd
-inducing activity(EC50, nM)a
MCF-7
> 50
PC-3
36.27±0.76
5.85±0.37
8.50±0.63
> 50
> 50
10.12±0.86
68.91±2.29
> 50
17c
17d
17e
17f
3.77±0.26
17.80±0.56
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
17g
17h
17i
9.36±0.86
17.67±1.15
293.44±5.68
5.14±0.22
13.00±0.25
> 50
2.25±0.14
5.00±0.28
5.13±0.35
18a
18b
18c
14

136.86±7.43
287.65±10.59
6.98±0.36
19.29±0.86
142.17±7.13
12.56±0.59
> 50
5.95±0.14
26.46±0.44
1.80±0.19
13.44±0.12
20.17±0.69
1.88±0.17
> 50
17.54±0.57
30.55±0.32
12.60±0.31
18.02±0.26
1.91±0.16
1.37±0.02
11.10±0.26
> 50
18d
19a
19f
19g
19h
19i
19j
20a
20b
20c
20d
20e
7
17.45±0.66
5.35±0.31
10.07±0.47
15.06±0.50
11.40±0.21
48.12±0.22
6.30±0.44
> 50
3.75±0.14
10.28±0.33
6.05±0.40
2.8±0.58
54.33±4.33
> 50
12.68±0.76
68.32±4.13
7.33±0.28
6.54±0.07
8.12±0.03
1,25(OH)2D3
16.20±0.29
aEC50: Vitamin D3-agonistic activity was estimated as HL-60 differentiation inducing ability. Data
represent mean ± SD, n = 3, *P < 0.05.
bIC50: the concentration that causes 50% of cell proliferation inhibition. Data are expressed as
mean ± SD from three independent experiments.
As further evaluation for the selective cytotoxicities of the promising compounds
17d, 19f, and 20e, they were tested over human normal liver cell line (L02) using
MTT assay. As illustrated in Table 4, all compounds displayed moderate
cytotoxicities against L02 cells. Notably, compound 19f (IC50 = 42.14 μM) had
remarkable decreased cytotoxicity compared with the parental compound 7 (IC50
=
21.24 μM) and showed better selectivities for both cancer cells (L02/MCF-7 = 23.4,
L02/PC-3 = 7.9). Finally, an investigation on the binding affinities and
anti-proliferative activities for cancer and normal cells of these derivatives showed
that compound 19f exhibited desirable results.
Table 4. The anti-proliferative activities of selected compounds and 1,25(OH)2D3 over L02 normal
cell line and selectivities for both cancer cells.
In vitro anti-proliferative activities (IC50,
Selectivities
μM)a
Compd
L02
MCF-7
PC-3
L02/MCF-7 L02/PC-3
20.52±0.12
42.14±0.46
3.77±0.26
1.80±0.19
5.85±0.37
5.35±0.31
5.4
3.5
7.9
17d
19f
23.4
15

35.43±0.34
21.24±0.32
> 50
1.91±0.16
1.37±0.02
6.05±0.40
2.8±0.58
18.5
15.5
> 4.5
5.9
7.6
20e
7
1,25(OH)2D3
11.10±0.26 16.20±0.29
> 3.1
aIC50: the concentration that causes 50% of cell proliferation inhibition. Data are expressed as
mean ± SD from three independent experiments.
To confirm the detailed interactions of the most promising compound 19f,
molecular docking study was conducted based on crystallographic structure of
1,25(OH)2D3 in complex with VDR (PDB code: 1DB1). Compound 19f was manually
docked into the crystal structure of VDR using software Discovery Studio 3.0. Figure
4 shows the superposition of the conformations of compound 19f and the natural
ligand 1,25(OH)2D3. Docking analyses demonstrated that the side chain and A ring
part of compound 19f present similar conformations to those observed in the presence
of 1,25(OH)2D3. As shown in figure 5, the hydroxyl group in the side chain was able
to form the same hydrogen-bonding interactions with His 305 and His 397 as the
hVDR LBD bound to 1α,25-(OH)2-D3 complex. However, the A ring part of
compound 19f form hydrogen-bonding interaction only with Ser 237 by amide bond,
while 1α,25-(OH)2-D3 binded with Ser 237, Arg 274, Tyr 143, and Ser 278. In
addition, the N,N-diethyl ethyl amine group introduced made longer A ring part than
that of 1α,25-(OH)2-D3. These factors might play important roles in reducing the
binding affinity of compound 19f to VDR.
Figure 4. Superposition of compounds 19f and 1α,25-(OH)2-D3. Compound 19f is depicted in
magenta and 1α,25-(OH)2-D3 is depicted in cyan.
16

B
A
Figure 5. (A) Structure of the complex VDR-1α,25-(OH)2-D3. (B) Docking structure of the
complex VDR-compound 19f. The ligands are shown in stick representation with carbon and
oxygen atoms in cyan and red, respectively. The hydrogen bonds formed are shown as red dashed
lines.
According to the above results (Table 1 and 3), we can draw some conclusions: (1)
introducing the hydrophilic moieties at the R group is important to improve the VDR
binding affinities and anti-proliferative activities, and introduction of the hydrophobic
segments may lead to a remarkable decrease or even loss of binding affinities and
anti-proliferative activities. (2) Introduction of large groups such as benzene ring
cannot be tolerated, nearly leading to a loss of affinities and activities. (3) C-5
position of pyrrole ring bearing phenyl-pentane group is not absolutely required but
may increase the binding affinity.
Figure 6. Schematic diagram of structure-activity relationships.
17

In summary, we synthesized and evaluated a series of novel phenyl-pyrrolyl
pentane derivatives with acetylene bond linker as VDR ligands. Structural
optimization of the parental compound 7 led to the synthesis of 22 derivatives. Seven
analogues (17d, 18a, 18d, and 19f-i) demonstrated more effective binding affinities
than the parental compound 7. Moreover, compound 19f not only showed excellent
agonistic activity to VDR but also displayed more anti-proliferative effect against
MCF-7 and PC-3 cells with the IC50 values of 1.80 and 5.35 μM, respectively.
Besides, compound 19f exhibited reduced toxicity against human normal liver cell
line (L02) compared with the parental compound 7. In conclusion, on the basis of the
abilities of these compounds, introducing acetylene bond linker for phenyl-pyrrolyl
pentane derivatives may be a strategy for the discovery of new drugs for the treatment
of cancer diseases.
Based on the preliminary investigation results, our efforts are now focused on the
modification and understanding the mode of action of these novel molecules. It is
expected that the biological results described and further modification studies will
expedite the development of new chemotherapeutic agents for the clinical intervention
of cancer disease.
Acknowledgments
This work was supported by the National Natural Science Foundation of China
(81273468, 81473153), National Basic Research Program of China (2015CB755500),
Natural Science Foundation of Jiangsu Province of China (BK20140672), and 111
Project from the Ministry of Education of China and the State Administration of
Foreign Expert Affairs of China (No. 111-2-07).
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Graphical abstract
In order to further explore the structure-activity relationships of phenyl-pyrrolyl
pentane skeleton, a series of novel derivatives which had acetylene bond linker was
designed, synthesized and evaluated as anti-cancer agents.
21