Novel Nonsecosteroidal Vitamin D Receptor Modulator Combined with Gemcitabine Enhances Pancreatic Cancer Therapy through Remodeling of the Tumor Microenvironment

Article abstract of DOI:10.1021/acs.jmedchem.0c01197

In a pancreatic tumor microenvironment, activated pancreatic stellate cells (PSCs) produce extracellular matrix (ECM) to form a barrier to drug penetration. Moreover, the interaction between cancer cells and activated PSCs promotes the tumor growth. Vitam

Full text of DOI:10.1021/acs.jmedchem.0c01197

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Article
Novel Nonsecosteroidal Vitamin D Receptor Modulator Combined
with Gemcitabine Enhances Pancreatic Cancer Therapy through
Remodeling of the Tumor Microenvironment
‡
‡
Zisheng Kang, Cong Wang, Yu Tong, Yanyi Li, Yi Gao, Siyuan Hou, Meixi Hao, Xiaolin Han,
Bin Wang, Qianqian Wang, and Can Zhang*
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ABSTRACT: In a pancreatic tumor microenvironment, activated
pancreatic stellate cells (PSCs) produce extracellular matrix (ECM)
to form a barrier to drug penetration. Moreover, the interaction
between cancer cells and activated PSCs promotes the tumor growth.
Vitamin D receptor (VDR), as a key regulator to promote the
recovery of PSCs to the resting state, is an attractive therapeutic
target for pancreatic cancer. Herein, we reported the design and
synthesis of 57 nonsecosteroidal VDR modulators based on the
skeleton of phenyl-pyrrolyl pentane. Among them, compounds C4,
I5, and I8 exhibited excellent VDR affinity and effective inhibition of
the activation of PSCs, as well as potent suppression of the
interaction between cancer cells and PSCs in vitro. In vivo, compound
I5 combined with gemcitabine achieved efficacious antitumor activity
without causing hypercalcemia. In conclusion, the compounds
designed in our study can remodel the tumor microenvironment and are expected to be candidates for the treatment of pancreatic
cancer.
INTRODUCTION
other factors to promote pancreatic cancer cell survival,
■
15,16
proliferation, and migration.
Therefore, a pharmacologic
Due to difficulties in its early diagnosis, pancreatic cancer is
1
,2
strategy to regulate the activation of PSCs would remodel the
pancreatic tumor microenvironment through hindering ECM
deposition and tumor−stroma crosstalk, resulting in enhanced
clinical chemotherapy effect of pancreatic cancer.
one of the most lethal cancers. Unlike many other cancers,
pancreatic cancer is characterized as a denser desmoplastic
reaction, generating more than 50 percent occupation of the
3
stroma in tumor tissue. Extracellular matrix (ECM), which is
Vitamin D receptor (VDR), as a ligand-dependent gene
transcription factor, can interact with other coactivators and
play a vital role in a variety of physiological processes like
the main component of stroma and a barrier for chemo-
therapeutic drugs to penetrate into tumor tissue, has been
implicated in the failure of many chemotherapeutic regi-
4
,5
17
mens.
calcium homeostasis, cell proliferation, and differentiation.
Pancreatic stellate cells (PSCs), located in periacinal or
periductal regions of the exocrine pancreas, are important
stromal cells. PSCs can be activated and acquire proliferative
VDR agonist calcipotriol could inhibit the PSC activation and
remodel the microenvironment of pancreatic cancer, while the
side effects of hypercalcemia and shorter half-life greatly limit
6
18−20
capacity in response to injury, inflammatory stimuli, and
its clinical application.
A series of threats to human health
7
cancer. Activated PSCs synthesize abundant ECM proteins,
including abdominal pain, kidney stones, and an abnormal
leading to a physical barrier formation to resist chemotherapy
21
heart rhythm is attributed to hypercalcemia. Previous studies
8
,9
drugs. Furthermore, the reciprocal relationship between
PSCs and cancer cells has attracted increasing interest. For
instance, pancreatic cancer cells secrete many kinds of
fibrogenic and mitogenic factors that promote activation of
found that the hypercalcemia effect of calcipotriol was caused
Received: September 10, 2020
Published: December 31, 2020
10
PSCs, such as fibroblast growth factor (FGF), platelet-
11
derived growth factor (PDGF), periostin, and hepatocyte
1
2,13
growth factor (HGF).
Reciprocally, the cytokines
produced by activated PSCs are very different from those of
1
4
resting cells, such as insulin-like growth factor 1 (IGF-1) and
©
2020 American Chemical Society
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Figure 1. Design concept of the novel nonsecosteroidal compounds.
Table 1. Chemical Structures of Synthetized Novel Nonsecosteroidal VDR Agonists
a
b
Relative VDR binding affinity (%) = (mP DMSO - mP Testing Compound) / (mP DMSO - mP Calcipotriol) × 100%. Calcipotriol is assigned as 100%. -, no
activity.
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by the secosteroid skeleton structure, which compose almost
all of the reported VDR ligands.
Figure 1, first, for investigating the different side chain length
and electronic property effects on VDR affinity and antitumor
activity, the carbon chain length of the phenyl side chain was
adjusted, and the side chain substituents were changed.
Meanwhile, different fragments containing heteroatoms with
different sizes and electronic properties were introduced into
the pyrrolyl side chain to obtain compounds C4−C19 and
H1−H10. For the sake of further investigation of the pyrrole
ring substitution position effect on VDR affinity and antitumor
activity, compounds D1−D19 and I1−I11 were synthesized.
Overall, 57 new compounds bearing a phenyl-pyrrolyl pentane
scaffold were designed and synthesized.
22−24
In order to discover new VDR agonists without hyper-
calcemia, our group have designed and characterized a series of
nonsecosteroidal VDR agonists that bear a phenyl-pyrrolyl
2
5−32
pentane scaffold.
Among them, compounds 11b and 11d
(
Figure 1) showed satisfactory binding affinity and transcrip-
2
6
tional activation. However, the structure of these compounds
can further be optimized for better activity and application in
more refractory indications. In the present study, 57 new
compounds were designed and synthesized to further improve
the efficacy and potency profile of nonsecosteroidal VDR
agonists, and a variety of biological tests against pancreatic
cancer were carried out. Among them, compounds C4, I5, and
I8 exhibited excellent VDR affinity and could effectively inhibit
the activation of PSCs. In vitro studies indicated that the ECM
deposition in PSC spheres was decreased, and the interaction
between cancer cells and PSCs was inhibited after the
treatment. The combination of compound I5 with gemcitabine
exerts strong antitumor effect in vivo without causing
hypercalcemia.
The intermediates 2a−2l were generated by the Grignard
reaction (Scheme 1).
a
Scheme 1. Synthesis of Intermediate 2a−2l
RESULTS AND DISCUSSION
■
a
Reagents and conditions: (a) RMgBr, Et O, 0 °C, 6 h.
Novel Nonsecosteroidal Compounds Were Designed
and Synthesized through Structure-Based Optimiza-
tion. VDR agonists can be divided into steroidal VDR agonists
and nonsecosteroidal VDR agonists according to whether they
have the core skeleton of cyclopentane polyhephenanthrene.
Steroidal VDR agonists with open-loop cyclopentane poly-
hydrophenanthrene core skeleton are collectively referred to as
steroid VDR agonists. At present, several steroid VDR agonists
have been put on the market and some are in the clinical
research stage. Although the physiological activity of the
improved steroids has been separated from the ability of
increasing blood calcium to a certain extent, it still cannot meet
the needs of clinical treatment due to the side effects of
hypercalcemia. Therefore, many pharmaceutical chemists
began to turn to the research and development of non-
secosteroidal VDR agonists, hoping to retain the VDR agonist
activity and not combine with DBP, so as to completely avoid
the side effects of hypercalcemia. LG190178 is one of the first
synthesized nonsecosteroidal VDR agonist without inducing
hypercalcemia, which mimics various physiological activities of
2
The synthetic route of compounds C2−C6 and C8−C19 is
shown in Scheme 2. In the presence of boron fluoride ethyl
ether (BF ·Et O) at 0 °C, intermediate 3 reacting with 1H-
3
2
pyrrole-2-carboxylate produced intermediate 4. Intermediate 5
was produced following the treatment with iodoethane (EtI) in
DMF. Then, reduction of intermediate 5 offered compound 6.
Subsequently, hydrolyzed by NaOH, compound 7 was
obtained. Following intermediate 7 reaction with different
amines afford 8a or 8b. Then, in the presence of the
corresponding bromine-containing fragment (2a−2l), 8a or 8b
was dissolved in DMF to give target compounds C2−C6 and
C8−C19.
The synthetic pathway of compounds D1−D19 is shown in
Scheme 3. In the presence of BF ·Et O at 30 °C, intermediate
3
2
9 was obtained by reacting with ethyl pyrrole-2-carboxylate.
Following treatment with EtI in DMF, intermediate 9 was to
afford intermediate 10. Then, intermediate 10 was reduced to
obtain intermediate 11. After hydrolysis by KOH, 11 was to
give 12. Then, intermediates 13a or 13b was produced by the
reaction of intermediate 12 with different amines. Target
compounds D1−D19 were prepared by coupling intermedi-
ates 13a or 13b with 2a−2l.
The synthetic pathway of target compounds H1−H10 is
summarized in Scheme 4. In the presence of 4-bromo-2-
methylbutan-2-ol, intermediate 7 was dissolved in DMF to give
14. By reaction of 14 with different amines, target compounds
H1−H10 were produced.
The synthetic pathway of compounds I1−I11 is shown in
Scheme 5. In the presence of 1-bromopropan-2-ol, inter-
mediate 12 was dissolved in DMF to give 15. Target
compounds I1−I11 were produced by reaction of intermediate
15 with different amines.
VDR Binding Affinity. To identify whether the synthesized
phenyl-pyrrolyl pentane analogues act as VDR agonists, the
VDR binding affinity of compounds was evaluated by VDR
competitor assay. The relative binding affinity is listed in Table
1. As the results have shown, the volume of substituents was
positively related to the activity, i.e., propyl > ethyl > methyl,
33
1
1
,25(OH) D in vitro and in vivo. However, compared with
2 3
,25(OH) D , the binding affinity and transcriptional activity
2
3
of LG190178 were modest. Therefore, to find more potential
nonsecosteroidal VDR agonists, 225 nonsteroidal 1,25-
(
OH) D analogues using LG190178 as the leading compound
2
3
were designed, synthetized, and screened in our previous
2
5−32
studies.
At present, there are not many studies on the non-
secosteroidal VDR agonists. One of the main disadvantages
of the nonsecosteroidal VDR agonists is that the activation
activity is relatively poor, and the ability to stimulate VDR is
much weaker than that of steroids. Therefore, our research
group has been committed to the study of nonsecosteroidal
VDR agonists with higher activity. After continuous research,
the structure is also constantly optimized. Among these
compounds, 11b and 11d (Figure 1) were identified as the
potent agonists of VDR. To further improve the efficacy and
potency profile of nonsecosteroidal VDR agonists, several
series of chemically modified compounds based on 11b and
11d were generated (Figure 1 and Table 1). As shown in
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a
Scheme 2. Synthesis of Compounds C2−C6 and C8−C19
a
Reagents and conditions: (a) ethyl 1H-pyrrole-2-carboxylate, BF ·Et O, −20-0 °C, 3 h; (b) EtI, NaH, DMF, 0−25 °C, 5 h; (c) Pd/C, H , MeOH,
3
2
2
2
h.
5 °C, 12 h; (d) NaOH, EtOH/H O (10:1), 90 °C, 24 h; (e) EDCI, HOBt, TEA, RNH , DCM, rt, overnight; (f) 2a−2l NaH, DMF, 0−70 °C, 12
2
2
a
Scheme 3. Synthesis of Target Compounds D1−D19
a
Reagents and conditions: (a) ethyl 1H-pyrrole-2-carboxylate, BF ·Et O, 30 °C, 3 h; (b) EtI, NaH, DMF, 0−25 °C, 5 h; (c) Pd/C, H , MeOH, 25
3
2
2
°C, 12 h; (d) NaOH, EtOH/H O (10:1), 90 °C, 24 h; (e) EDCI, HOBt, TEA, RNH , DCM, rt, overnight; (f) 2a−2l, NaH, DMF, 0−70 °C, 12 h.
2
2
a
Scheme 4. Synthesis of Compounds H1−H10
a
Reagents and conditions: (a) 4-bromo-2-methylbutan-2-ol, NaH, DMF, 0−70 °C, 12 h; (b) EDCI, HOBt, TEA, RNH , DCM, rt, overnight.
2
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a
Scheme 5. Synthesis of Compounds I1−I11
a
Reagents and conditions: (a) 1-bromopropan-2-ol, K CO , acetone, reflux, 12 h; (b) EDCI, HOBt, TEA, RNH , DCM, rt, overnight.
2
3
2
1
2
while the activity was partially decreased when R or R is H,
indicating that the side substituents might not be enough to
provide a steric effect, which was adverse to the activity.
However, when the side substituents were phenyl, the activity
was still excellent (C19). When the end substituent is
disubstituted methyl or ethyl, the activity order of the carbon
chain length was 3 > 6 > 5 > 4. When the end substituent was
di-n-propyl, the activity order of carbon chain length was 4 > 5
important indicator of PSC activation. In order to evaluate the
effect of target compounds on the suppression of PSC
activation, LTC-14 cells were treated with different com-
pounds in the presence of 0.5 ng/mL TGF-β1 for 24 h. Acta2
and Col1a1 are mRNA of α-SMA and collagen I, respectively.
The expression levels of Col1a1 and Acta2 in LTC-14 cells
were detected by Q-PCR. Meanwhile, the expression of
collagen I protein was determined by western blot. As shown
in Figure 2, most of the compounds showed the ability to
suppress these gene expressions in varying degrees. Consider-
ing the results of activity, the effect of the side chain, the end
>
3 > 6.
In the second round of design, the substitution position of
the pyrrole ring in the core framework from C5 to C4 was
changed to generate compounds D1−D19. The results showed
that the volume effect of end substituents on the activity was
basically the same as that of compounds C2−C19, but the
effect of the carbon chain length on the activity was slightly
different. When the end was substituted by dimethyl or diethyl,
the effect of “n” on the activity was 4 > 6 > 5 > 3. However, in
this round of design, the most active molecule appeared in the
compound D16 with the length of carbon chain of 1 and the
end substituted by H and methyl. The two optimized
molecules C4 and D16 from the first and second rounds
were taken as the starting point to carry out the third round of
structural transformation, and the right hydrophilic end
segment without modifying side chain structure was optimized.
In the optimization of C4, we found the significantly decreased
activity of shortening one carbon atom of the right carbon
chain, i.e., when “m” was 2 (H2). No matter the simplification
of the end N atom substituent to methyl (H1), cyclization
1
2
hydroxyl side substituents R and R , on the activity was propyl
> ethyl > methyl, indicating that larger substituents had better
1
2
activity. In addition, when R was phenyl and R was H, the
compounds had better activity. The length of the side chain
carbon atom also had an obvious influence on the activity.
When the core framework pyrrole ring was substituted at the
C5 position, the influence of the carbon chain length on the
activity was: 3 > 6 > 5 > 4. When the core framework pyrrole
ring was substituted at the C4 position, the influence of the
length of the carbon chain on the activity was: 4 > 6 > 5 > 3.
The selected compounds C4 and D16 had better ability to
inhibit the activation of PSCs. When optimizing the right
hydrophilic segment without modification of the left side chain
structure, it was found that the structure optimization starting
from C4 did not get better active molecules, while the
optimization starting from D16 produced many better active
compounds, which suggested that the activity of C4
substitution with the core skeleton pyrrole was higher than
that of C5 substitution.
(
H3), or the introduction of the second heteroatom (H4−
H5), could not restore the VDR affinity, and the above effects
also appeared when “m” was shortened to 1, indicating that the
optimal length of the carbon atom on the right side of the core
skeleton was 3 concerning the extraction of pyrrole C5. The
activity would be reduced with the length reduction of the
carbon atom and no more active molecule could be found for
the optimization of C4. The optimization starting from D16
showed that compounds I5 and I8 had better activity. It was
worth mentioning that the right carbon chain length of these
two molecules was 2, which was the preferred length for the
pyrrole C4 substituted skeleton.
Compounds Inhibited the Oncogenic Factor Expres-
sion of PSCs. In the microenvironment of pancreatic tumor,
the interaction between cancer cells and activated PSCs
36
promotes tumor growth and metastasis. Cancer cells can
promote PSCs to secrete many cytokines, such as periosteal
osteoblast specific factor 2 (POSTN), PDGF, FGF and HGF,
which in turn enhance the cancer cell proliferation, migration,
3
7
and invasion . In pancreatic cancer, PSCs play the role of
″accomplice″, and thus inhibiting the interaction of cancer
cells and PSCs may reshape the pancreatic tumor micro-
environment and improve the chemotherapy effect.
Phenyl-pyrrolyl Pentane Analogues Inhibited the
Activation of PSCs. The immortalized rat PSC cell line,
After comprehensive consideration of the affinity and ability
to inhibit activation of PSCs, compounds C4, C15, D11, H2,
H3, H5, I3, I5, and I8 were selected for the follow-up activity
study, calcipotriol as the positive control. LTC-14 cells were
treated with the conditioned media (CM) of rat pancreatic
cancer cell DSL-6A/C1 in the presence of different
compounds (0.5 μM) for 24 h. The expression of POSTN
and PDGF-A protein in LTC-14 cells was determined by
western blot, and the mRNA expression of Pdgfa, Pdgfb, Fgf,
Hgf, and Periodin was measured by Q-PCR. As shown in Figure
3A−C, compounds C4, D11, H5, I5, and I8 can significantly
inhibit the expression of PDGF-A and POSTN protein in
34
LTC-14 cells were selected for the experiment. To avoid the
influence in activation of PSCs due to cytotoxicity, the toxicity
of all compounds to LTC-14 cells were tested by the MTT
assay. as shown in Table S1, the compounds showed no
significant toxicity to LTC-14 cells, and the IC50 values of all
compounds to LTC-14 cells were greater than 10 μM.
Therefore, 0.5 μM was selected for the subsequent activity
screening test.
Collagen I and α-smooth muscle actin (α-SMA) are over
3
5
expressed when PSCs are activated.
Therefore, the
expression level of α-SMA and collagen I can be used as an
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Figure 2. The expression levels of Col1a1, Acta2, and collagen I in LTC-14 cells after different compound treatment. (A and B) The expression of
Col1a1 and Acta2 were determined by Q-PCR. Data are shown as mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001 compared to TGF-β1 alone.
(C) The expression level of collagen I protein on LTC-14 cells was determined by western blot, protein expression levels were quantified by
densitometry. Data are shown as mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001 compared to TGF-β1 alone.
LTC-14 cells after CM treatment. In addition, the mRNA
expression of Pdgfa, Pdgfb, Fgf, Hgf, and Periodin in LTC-14
cells, which is related to PSC activation, was inhibited in
varying degrees after CM treatment (Figure 3D−H). There-
fore, these preferred compounds can inhibit the oncogenic
factor expression of PSCs that are promoted by pancreatic
cancer cells.
Compounds Inhibited Tumor Cell Proliferation that
Were Induced by PSCs and Decreased the ECM
Deposition in PSC Spheres. PSCs secrete many factors to
3
8
increase the cancer cell proliferation . In order to detect the
inhibition ability of selected compounds on cancer cell
proliferation that were induced by PSCs, cancer cells were
treated with culture medium supernatant of PSCs (PSC-CM)
in the presence of the compound (PSC-CM + compound) for
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Figure 3. Compounds inhibit the oncogenic factors expression of PSCs. (A) The expression of PDGF-A and POSTN was determined by western
blot. (B, C) The expression of PDGF-A and POSTN proteins was quantified by densitometry and normalized to β-actin. (D−H) The expression of
Pdgfa, Pdgfb, Fgf2, Hgf, and Periodin in LTC-14 cells after CM treatment was measured by Q-PCR. Three repeats were performed. Data are shown
as mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001 versus CM alone.
Figure 4. Compounds inhibit the promotion effect of PSCs on cancer cells. (A−C) Cancer cells were treated with a PSC culture medium (PSC-
CM) in the presence of the compound (0.5 μM) for 24 h. Otherwise, the culture supernatant of compound-pretreated PSCs for 24 h was used to
culture tumor cells (compound treated PSC-CM). The cancer cell proliferation was determined by MTT assay. Data are shown as mean ± SD. **P
<
0.01; ***P < 0.001 versus PSC-CM. (D, E) LTC-14 cells were used for 3D culture in vitro to form PSC spheres. After the PSC spheres were
treated with compounds, doxorubicin (DOX) with red fluorescence was added. The content of DOX in PSC spheres was detected by a laser
confocal microscope. Three repeats were performed. Data are shown as mean ± SD. *P < 0.05; **P < 0.01 versus control.
2
4 h. Otherwise, the culture supernatant of compound
(compound treated PSC-CM). The cancer cell proliferation
was determined by MTT assay. In order to prevent the residual
pretreated PSCs for 24 h was used to culture tumor cells
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Figure 5. C4, I5, and I8 are VDR agonists. (A) Chemical structure of selected compounds C4, I5, and I8. (B) VDR binding affinity of compounds
C4, I5, and I8. The affinity was determined by fluorescence polarization competition assay (n = 3). (C) Compound transcriptional activities were
determined in HEK293 cells. TK-Spp×3-LUC reporter plasmid, pCMX-Renilla, pENTER-CMV-hRXRα, and pCMX−VDR were cotransfected
into HEK293 cells. Test compounds and calcipotriol were added after 8 h. After 24 h, luciferase activity was performed using the Dual-Luciferase
Assay System. (D) Predicted binding model of C4, I5, and I8. Using the discovery studio, VDR-LBD (PDB code: 2ZFX) was applied for the
molecular docking analysis.
compounds in PSC-CM from directly inhibiting proliferation
of cancer cells, the cytotoxicity of the compounds to DSL-6A/
C1 were tested. All the selected compounds had no obvious
toxicity to DSL-6A/C1 at the concentration of 0.5 μM (Figure
S1). As shown in Figure 4A−C, the proliferation of DSL-6A/
C1 cells increased significantly after PSC-CM treatment
compared with the control. When the selected compounds
were added, the proliferation of cancer cells had no significant
change compared with PSC-CM alone. Meanwhile, it is
noteworthy that the cancer cell proliferation was significantly
decreased after the compound-treated-PSC-CM treatment,
which indicated that the compound could cause PSCs to lose
inhibitory effect on cancer cell proliferation.
Previous studies have shown that the activated PSCs can
secrete a large amount of collagen, resulting in excessive
deposition of ECM in the tumor microenvironment and
providing a physical barrier for cancer cells. In order to
investigate whether compounds can reduce ECM deposition
after inhibiting PSC activation, LTC-14 cells were used for 3D
culture in vitro to form PSC spheres. After the PSC spheres
were treated with different compounds, doxorubicin (DOX)
with red fluorescence was added as the model drug. The
content of DOX in PSC spheres was detected by a laser
confocal microscope, which reflected whether the compounds
could increase the permeation rate of drug via ECM barrier. As
shown in Figure 4D,E, red fluorescence was only observed on
the surface of PSC spheres in the control group after
administration of DOX, while DOX could enter PSC spheres
in varying degrees after administration of calcipotriol, C4, I5,
and I8, indicating that the dense of ECM secreted by PSCs was
inhibited and the permeation rate of drug via physical barrier
was increased by the compounds acting as permeation
enhancers.
Compounds C4, I5, and I8 Showed VDR Agonist
Activities. The structures of compounds C4, I5, and I8 were
shown in Figure 5A. To evaluate the VDR binding affinities of
the compounds, various concentrations of compounds were
tested for the VDR binding ability. Compounds C4 (IC50
=
92.51 nM), I5 (IC50 = 57.19 nM), and I8 (IC50 = 81.59 nM)
showed excellent VDR binding affinity (Figure 5B). Affinity is
the degree of interaction between ligand and VDR protein.
The premise of activating VDR is that the ligand must have a
certain affinity with VDR protein. Therefore, affinity can be
used to characterize the matching degree of ligand and protein
binding, which can be used as a standard to judge whether the
design of the nonsteroidal structure is reasonable and whether
it can simulate the binding of natural steroid ligand and protein
receptor. Meanwhile, there are many other mechanisms
involved in the interaction of VDR, including the ability to
interact with many other cofactors. The ability to activate VDR
can be evaluated in many aspects, including the binding
activity, transcriptional activity, and related biological activity.
To further evaluate the agonistic properties of target
compounds, transactivation assay was performed in HEK293
cells. Compounds C4, I5, and I8 were tested, and calcipotriol
was applied as positive control. pGL4.27-SPP×3-Luc reporter
plasmid acts as the VDRE activated reporter, and pRL-TK
normalizes the expression of luciferase as the internal
reference. pENTER-CMV-hVDR was applied to increase the
VDR expression, and pENTER-CMV-hRXRα expressing
RXRα acts as the VDR heterodimer partner. As shown in
Figure 5C, compounds C4, I5, and I8 showed a concentration-
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Figure 6. C4, I5, and I8 plus gemcitabine shows anti-pancreatic cancer efficacy in vivo. (A) Schematic illustrating the treatment of tumor-bearing
mice. DSL-6A/C1-luci and LTC-14-GFP cells were co-inoculated into the nude mice pancreas to construct the orthotopic xenotransplantation
model of pancreatic cancer. Fourteen days after tumor grafting, mice were randomly divided into groups: vehicle, gemcitabine, or gemcitabine and
compound combination. Gemcitabine was administered once every 3 days and compounds were administered daily (n = 5 per group). After 12
days administration, mice were photographed by in vivo imaging and tumors were harvested. (B) Tumor growth in mice was examined by an IVIS
optical imaging system. Representative animal images are shown. (C) Structures of tumor tissue were determined by H&E staining (×100),
collagen deposition was examined by Masson’s trichrome stain (×100). (D) The expression of α-SMA (red) in the PSCs (green) of tumor sections
was tested by immunofluorescence. Scale bars: 10 μm.
dependent transcriptional activity, indicating that the com-
pounds were potent agonists.
the orthotopic xenotransplantation model of pancreatic cancer.
Fourteen days after tumor grafting, mice were randomly
divided into six groups: vehicle, gemcitabine (Gem), Gem +
calcipotriol, Gem + C4, Gem + I5, and Gem + I8. Once every
3 days, gemcitabine (100 mg/kg) was administered by
intraperitoneal injection. Meanwhile, compounds (C4, I5,
and I8) were administered at 500 μg/kg and calcipotriol was
administered at 60 μg/kg. After 12 days of administration,
mice were photographed by in vivo imaging and tumors were
harvested (Figure 6A). As shown in Figure 6B and Figures S2
and S3, the tumor did not decrease significantly after
gemcitabine administration alone. The tumor volume was
significantly reduced after treatment with gemcitabine
combined with the compounds. In particular, compound I5
combined with gemcitabine showed the best antitumor
activity.
H&E-staining of tumor sections further confirmed that the
density of pancreatic tumor tissue decreased with the
combination of I5 and gemcitabine. According to Masson
staining results, the collagen deposition in the group of I5
combined with gemcitabine was significantly inhibited, and the
inhibition activity was better than that of the positive control
(Figure 6C). The results in Figure 6D and Figure S4 show that
after treatment with gemcitabine + calcipotriol, gemcitabine +
C4, gemcitabine + I5, and gemcitabine + I8, the α-SMA
protein expression of LTC-14 cells decreased in varying
degrees, indicating that the activation of PSCs in the tumor
microenvironment was inhibited, and compound I5 combined
with gemcitabine showed the best efficacy. In addition, the
combination treatment of compounds and gemcitabine can
To further elucidate the interaction mode between these
compounds and VDR at atomic level, C4, I5, and I8 were
selected for molecular docking with VDR ligand binding
domain (rat VDR-LBD). The results of molecular docking
showed that the compound I8 pyrrole ring interacted with the
indole ring of TRP 282 of VDR protein (PDB: 2ZFX). The
hydroxyl group interacted with the imidazole ring of HIS 301
and HIS 393 at the same time, and the hydroxyl group of SER
2
33 at the other end formed a hydrogen bond force and
formed a water molecule-mediated hydrogen bond force with
the ARG 270 guanidine group. Compound I5 was obtained by
replacing the propylene glycol side chain of I8 with
methylpiperazine, which could also form π−π interaction
with TRP 282, but methylpiperazine could not interact with
HIS 301 and HIS 393. Moreover, due to the steric hindrance
of methylpiperazine, the hydroxyl side chain only formed
hydrogen bonding force with the hydroxyl group of SER 233.
When the side chain of methylpiperazine was replaced by the
diethylamino group to obtain C4, due to the further increase of
steric hindrance, the torsion of the pyrrole ring resulted in the
destruction of the formation of π−π interaction, resulting in
the formation of only the end hydroxyl group with SER 233
and ARG 270.
C4, I5, and I8 plus Gemcitabine Showed Anti-
Pancreatic Cancer Effect. Based on in vitro results, we
selected the compounds C4, I5, and I8 for the pharmacody-
namic test in vivo. DSL-6A/C1-luci and LTC-14-GFP cells
were co-inoculated into the nude mice pancreas to construct
6
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prolong the survival time of tumor-bearing mice in a certain
degree (Figure S5). More importantly, the compounds do not
cause hypercalcemia in mice (Figure S6). These results
indicated that the compounds designed in our study can
provide new effective candidate strategies for the treatment of
pancreatic cancer.
Ethyl-5-(3-(4-(Benzyloxy)-3-methylphenyl)pentan-3-yl)-1H-pyr-
role-2-carboxylate (4). BF ·Et O (5 mL) was added dropwise to a
3
2
solution of compound 3 (2.0 g, 7.03 mmol) and ethyl 1H-pyrrole-2-
carboxylate (1.17 g, 8.44 mmol) in dichloromethane (20 mL) at −20
C. The mixture was stirred for 3 h at 0 °C, water (10 mL) and
°
EtOAc (30 mL) was carefully added to the mixture at 0 °C, and the
organic phase was separated. The organic phase was washed with
brine and dried over anhydrous Na SO , filtered, and concentrated.
2
4
The residue was purified by column chromatography with petroleum
ether/ethyl acetate (100/1, v/v) to give compound 4 as colorless oil
CONCLUSIONS
■
In summary, 57 new compounds were designed and
synthesized in our study on the basis of previous works.
Among them, compounds C4, I5, and I8 exhibited excellent
VDR affinity and effective inhibition of the activated PSCs. In
vitro studies indicated that after the compound treatment, the
ECM deposition in PSC spheres was decreased and the
interaction between pancreatic cancer cells and PSCs was
inhibited. Meanwhile, in the antitumor study in vivo,
compound I5 combined with gemcitabine had a strong
antitumor effect without causing hypercalcemia in mice. In
light of the presented findings, we concluded that these novel
nonsecosteroidal VDR modulators may have potent applica-
tion in combined treatment of pancreatic cancer.
(
1.73 g, 61% yield).
Ethyl-5-(3-(4-(Benzyloxy)-3-methylphenyl)pentan-3-yl)-1-ethyl-
1H-pyrrole-2-carboxylate (5). To a solution of compound 4 (0.87 g,
2
.15 mmol) in DMF (15 mL) was added NaH (0.10 mg, 4.29 mmol)
at 0 °C and stirred for 1 h. Ethyl bromide was added to the mixture at
°C. The reaction mixture was stirred for 5 h at room temperature,
0
water (10 mL) and EtOAc (30 mL) was added and the organic phase
was separated. The organic phase were washed with brine and dried
over anhydrous Na SO , filtered, and concentrated. The residue was
2
4
purified by column chromatography with petroleum ether/ethyl
acetate (80/1, v/v) to give compound 5 as oil (0.97 g, 85% yield).
Ethyl-1-ethyl-5-(3-(4-hydroxy-3-methylphenyl)pentan-3-yl)-1H-
pyrrole-2-carboxylate (6). To a solution of 5 (2.00 g, 4.61 mmol) in
methanol (30 mL), Pd/C (0.2 g) was added. The reaction mixture
was stirred under a H atmosphere at for 12 h. The Pd/C was filtered
2
off, and then the residue was purified by column chromatography with
petroleum ether/ethyl acetate (20/1, v/v) to give compound 6 as oil
(1.33 g, 84% yield).
1-Ethyl-5-(3-(4-hydroxy-3-methylphenyl)pentan-3-yl)-1H-pyr-
role-2-carboxylic acid (7). To a stirred solution of 6 (0.30 g, 0.87
EXPERIMENTAL SECTION
■
General Materials and Methods. All material and reagents were
purchased from commercial sources and, unless otherwise stated,
were used without further purification. High-resolution mass spectra
mmol) in a mixture of EtOH/H O 10:1 (40 mL) was added NaOH
(
HRMS) were recorded on a QSTAR XL Hybrid MS/MS mass
2
1
13
(
0.35 g, 8.73 mmol) at 90 °C. After 24 h, the solution was evaporated,
and water (20 mL) and EtOAc (80 mL) was added. The organic
phase was washed with brine and dried over anhydrous Na SO ,
spectrometer. H NMR and C NMR were recorded employing
Bruker AV-300 or AV-500 instruments using CDCl . Chemical shifts
3
were given as δ (ppm) units relative to the internal standard
tetramethylsilane (TMS). Column chromatography separations were
progressed on a silica gel (200−300 mesh). Purity of all final
compounds was ≥95% as determined by HPLC.
Synthesis of the New Compounds. General Procedure 1:
Synthesis of Compounds 2a−2l. To a stirred solution of 1 (55
mmol) in ether (100 mL) was added RMgBr (138 mmol) dropwise at
2
4
filtered, and concentrated. The residue was purified by column
chromatography with dichloromethane/methanol (50/1, v/v) to give
compound 7 as oil (0.21 g, 76% yield).
N-(2-(Diethylamino)ethyl)-1-ethyl-5-(3-(4-hydroxy-3-
methylphenyl)pentan-3-yl)-1H-pyrrole-2-carboxamide (8a). To a
solution of compound 7 (100.93 mg, 0.32 mmol) in dichloromethane
(50 mL), 3-(ethyliminomethylideneamino)-N,N-dimethylpropan-1-
amine (hydrochloride) (74.38 mg, 0.48 mmol), 1-hydroxybenzo-
triazole (64.74 mg, 0.48 mmol), and triethylamine (48.49 mg, 0.48
mmol) were added at 0 °C. After stirring for 0.5 h, N,N-diethylethane-
1,2-diamine (55.78 mg, 0.48 mmol) was added. The reaction mixture
0
°C. The mixture was stirred at 30 °C for 6 h, water (50 mL) and
EtOAc (200 mL) was carefully added to the mixture at 0 °C, and the
sorganic phase was separated. The organic phase was washed with
brine and dried over anhydrous Na SO , filtered, and concentrated.
2
4
The residue was purified by silica gel column chromatography eluting
with an appropriate mixture as indicated in each case.
was stirred at room temperature for 12 h and then H
added. The organic phase was washed with brine and dried over
anhydrous Na SO , filtered, and concentrated. The residue was
O (100 mL) was
2
4
-Bromo-2-methylbutan-2-ol (2a). Petroleum ether/ethyl acetate
1
(
100/1). Colorless oil. 42% yield.
-Bromo-2-methylpentan-2-ol (2b). Petroleum ether/ethyl ac-
etate (100/1). Colorless oil. 38% yield.
-Bromo-2-methylhexan-2-ol (2c). Petroleum ether/ethyl acetate
80/1). Colorless oil. 53% yield.
-Bromo-2-methylheptan-2-ol (2d). Petroleum ether/ethyl ac-
etate (70/1). Colorless oil. 62% yield.
-Bromo-3-ethylpentan-3-ol (2e). Petroleum ether/ethyl acetate
30/1). Colorless oil. 35% yield.
-Bromo-3-ethylhexan-3-ol (2f). Petroleum ether/ethyl acetate
40/1). Colorless oil. 40% yield.
-Bromo-3-ethylheptan-3-ol (2g). Petroleum ether/ethyl acetate
50/1). Colorless oil. 32% yield.
-Bromo-3-ethyloctan-3-ol (2h). Petroleum ether/ethyl acetate
100/1). Colorless oil. 20% yield.
-(2-Bromoethyl)heptan-4-ol (2i). Petroleum ether/ethyl acetate
100/1). Colorless oil. 22% yield.
-Bromo-4-propylheptan-4-ol (2j). Petroleum ether/ethyl acetate
50/1). Colorless oil. 52% yield.
-Bromo-4-propyloctan-4-ol (2k). Petroleum ether/ethyl acetate
50/1). Colorless oil. 41% yield.
-Bromo-4-propylnonan-4-ol (2l). Petroleum ether/ethyl acetate
70/1). Colorless oil. 37% yield.
2
4
5
purified by column chromatography with dichloromethane/methanol
(30/1, v/v) to give compound 8a as oil (0.11 g, 83% yield).
6
N-(3-(Diethylamino)propyl)-1-ethyl-5-(3-(4-hydroxy-3-
methylphenyl)pentan-3-yl)-1H-pyrrole-2-carboxamide (8b). To a
solution of compound 7 (100.93 mg, 0.32 mmol) in dichloromethane
(50 mL), 3-(ethyliminomethylideneamino)-N,N-dimethylpropan-1-
amine (hydrochloride) (74.38 mg, 0.48 mmol), 1-hydroxybenzo-
triazole (64.74 mg, 0.48 mmol), and triethylamine (48.49 mg, 0.48
mmol) were added at 0 °C. After stirring for 0.5 h, N,N-
dimethylpropan-1,3-diamine (62.46 mg, 0.48 mmol) was added.
The reaction mixture was stirred at room temperature for 12 h, and
(
7
1
(
(
(
(
(
(
(
(
6
7
then H
O (100 mL) was added. The organic phase was washed with
SO , filtered, and concentrated.
2
8
brine and dried over anhydrous Na
2
4
The residue was purified by column chromatography with dichloro-
methane/methanol (30/1, v/v) to give compound 8b as oil (0.11 g,
83% yield).
General Procedure 2: Synthesis of Compounds C2 and C3. To a
solution of compound 8a (200 mg, 0.48 mmol) in DMF (50 mL),
NaH (18.28 mg, 0.72 mmol) was added portionwise at 0 °C. After
stirring for 0.5 h, 2f or 2g (0.72 mmol) was added. The reaction
4
1
8
9
mixture was stirred at 70 °C for 12 h and then H O (100 mL) was
2
added dropwise followed by EtOAc (80 mL). The organic phase was
6
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separated and washed with brine and dried over anhydrous Na SO ,
by silica gel chromatography with petroleum ether/ethyl acetate (50/
1, v/v). Yield: 40%.
Ethyl-4-(3-(4-(benzyloxy)-3-methylphenyl)pentan-3-yl)-1-ethyl-
1H-pyrrole-2-carboxylate (10). By the same manner as described for
the preparation of 5, intermediate 10 was prepared from intermediate
9 and purified by silica gel chromatography with petroleum ether/
ethyl acetate (50/1, v/v). Yield: 76%.
Ethyl-1-ethyl-4-(3-(4-hydroxy-3-methylphenyl)pentan-3-yl)-1H-
pyrrole-2-carboxylate (11). By the same manner as described for the
preparation of 6, intermediate 11 was prepared from intermediate 10
and purified by silica gel chromatography with petroleum ether/ethyl
acetate (20/1, v/v). Yield: 73%.
2
4
filtered, and concentrated. The residue was purified by silica gel
column chromatography eluting with appropriate mixture as indicated
in each case.
N-(2-(Diethylamino)ethyl)-1-ethyl-5-(3-(4-((4-ethyl-4-
hydroxyhexyl)oxy)-3-methylphenyl)pentan-3-yl)-1H-pyrrole-2-car-
boxamide (C2). Dichloromethane/methanol (100/1). Oil. 46% yield.
N-(2-(Diethylamino)ethyl)-1-ethyl-5-(3-(4-((5-ethyl-5-
hydroxyheptyl)oxy)-3-methylphenyl)pentan-3-yl)-1H-pyrrole-2-
carboxamide (C3). Dichloromethane/methanol (50/1). Oil. 52%
yield.
General Procedure 3; Synthesis of Compounds C4−C6 and C8−
C19. To a solution of compound 8b (205 mg, 0.48 mmol) in DMF
1
-Ethyl-4-(3-(4-hydroxy-3-methylphenyl)pentan-3-yl)-1H-pyr-
(
50 mL), NaH (18.28 mg, 0.72 mmol) was added portionwise at 0
role-2-carboxylic acid (12). By the same manner as described for the
preparation of 7, intermediate 12 was prepared from intermediate 11
and purified by silica gel chromatography with dichloromethane/
methanol (70/1, v/v). Yield: 68%.
°
C. After stirring for 0.5 h, one of 2a−2l (0.72 mmol) was added. The
reaction mixture was stirred at 70 °C for 12 h, and then H O (100
2
mL) was added dropwise followed by EtOAc (80 mL). The organic
phase was separated, washed with brine, dried over anhydrous
N-(2-(Diethylamino)ethyl)-1-ethyl-4-(3-(4-hydroxy-3-
methylphenyl)pentan-3-yl)-1H-pyrrole-2-carboxamide (13a). By
the same manner as described for the preparation of 8a, the
intermediate 13a was prepared from the intermediate 12 and purified
by silica gel chromatography with dichloromethane/methanol (30/1,
v/v). Yield: 84%.
N-(3-(Diethylamino)propyl)-1-ethyl-4-(3-(4-hydroxy-3-
methylphenyl)pentan-3-yl)-1H-pyrrole-2-carboxamide (13b). By
the same manner as described for the preparation of 8b, the
intermediate 13b was prepared from the intermediate 12 and purified
by silica gel chromatography with dichloromethane/methanol (60/1,
v/v). Yield: 90%.
Na SO , filtered, and concentrated. The residue was purified by silica
2
4
gel column chromatography eluting with the appropriate mixture as
indicated in each case.
N-(3-(Diethylamino)propyl)-1-ethyl-5-(3-(4-(3-hydroxy-3-meth-
ylbutoxy)-3-methylphenyl)pentan-3-yl)-1H-pyrrole-2-carboxamide
(
C4). Dichloromethane/methanol (70/1). Oil. 67% yield.
N-(3-(Diethylamino)propyl)-1-ethyl-5-(3-(4-((5-hydroxy-5-
methylhexyl)oxy)-3-methylphenyl)pentan-3-yl)-1H-pyrrole-2-car-
boxamide (C5). Dichloromethane/methanol (60/1). Oil. 44% yield.
N-(3-(Diethylamino)propyl)-1-ethyl-5-(3-(4-((6-hydroxy-6-
methylheptyl)oxy)-3-methylphenyl)pentan-3-yl)-1H-pyrrole-2-car-
boxamide (C6). Dichloromethane/methanol (70/1). Oil. 48% yield.
N-(3-(Diethylamino)propyl)-1-ethyl-5-(3-(4-((5-ethyl-5-
hydroxyheptyl)oxy)-3-methylphenyl)pentan-3-yl)-1H-pyrrole-2-
carboxamide (C8). Dichloromethane/methanol (70/1). Oil. 71%
yield.
General Procedure 4: Synthesis of Compounds D1−D3. To a
solution of compound 13a (200 mg, 0.48 mmol) in DMF (50 mL),
NaH (18.28 mg, 0.72 mmol) was added portionwise at 0 °C. After
stirring for 0.5 h, 2f or 2g (0.72 mmol) was added. The reaction
N-(3-(Diethylamino)propyl)-1-ethyl-5-(3-(4-((6-ethyl-6-
hydroxyoctyl)oxy)-3-methylphenyl)pentan-3-yl)-1H-pyrrole-2-car-
boxamide (C9). Dichloromethane/methanol (100/1). Oil. 56% yield.
N-(3-(Diethylamino)propyl)-1-ethyl-5-(3-(4-((3-hydroxy-3-
propylhexyl)oxy)-3-methylphenyl)pentan-3-yl)-1H-pyrrole-2-car-
boxamide (C10). Dichloromethane/methanol (100/1). Oil. 41%
yield.
N-(3-(Diethylamino)propyl)-1-ethyl-5-(3-(4-((5-hydroxy-5-
propyloctyl)oxy)-3-methylphenyl)pentan-3-yl)-1H-pyrrole-2-car-
boxamide (C11). Dichloromethane/methanol (70/1). Oil. 40% yield.
N-(3-(Diethylamino)propyl)-1-ethyl-5-(3-(4-((6-hydroxy-6-
propylnonyl)oxy)-3-methylphenyl)pentan-3-yl)-1H-pyrrole-2-car-
boxamide (C12). Dichloromethane/methanol (70/1). Oil. 39% yield.
N-(3-(Diethylamino)propyl)-1-ethyl-5-(3-(4-((4-hydroxy-4-
methylpentyl)oxy)-3-methylphenyl)pentan-3-yl)-1H-pyrrole-2-car-
boxamide (C13). Dichloromethane/methanol (100/1). Oil. 53%
yield.
N-(3-(Diethylamino)propyl)-1-ethyl-5-(3-(4-((4-ethyl-4-
hydroxyhexyl)oxy)-3-methylphenyl)pentan-3-yl)-1H-pyrrole-2-car-
boxamide (C14). Dichloromethane/methanol (70/1). Oil. 42% yield.
N-(3-(Diethylamino)propyl)-1-ethyl-5-(3-(4-((4-hydroxy-4-
propylheptyl)oxy)-3-methylphenyl)pentan-3-yl)-1H-pyrrole-2-car-
boxamide (C15). Dichloromethane/methanol (100/1). Oil. 48%
yield.
mixture was stirred at 70 °C for 12 h, and then H O (100 mL) was
2
added dropwise followed by EtOAc (80 mL). The organic phase was
separated and washed with brine and dried over anhydrous Na SO ,
2
4
filtered, and concentrated. The residue was purified by silica gel
column chromatography eluting with appropriate mixture as indicated
in each case.
N-(2-(Diethylamino)ethyl)-1-ethyl-4-(3-(4-((3-ethyl-3-
hydroxypentyl)oxy)-3-methylphenyl)pentan-3-yl)-1H-pyrrole-2-
carboxamide (D1). Dichloromethane/methanol (50/1). Oil. 45%
yield.
N-(2-(Diethylamino)ethyl)-1-ethyl-4-(3-(4-((4-ethyl-4-
hydroxyhexyl)oxy)-3-methylphenyl)pentan-3-yl)-1H-pyrrole-2-car-
boxamide (D2). Dichloromethane/methanol (70/1). Oil. 46% yield.
N-(2-(Diethylamino)ethyl)-1-ethyl-4-(3-(4-((5-ethyl-5-
hydroxyheptyl)oxy)-3-methylphenyl)pentan-3-yl)-1H-pyrrole-2-
carboxamide (D3). Dichloromethane/methanol (100/1). Oil. 53%
yield.
General Procedure 5: Synthesis of Compounds D4−D19. To a
solution of compound 13b (205 mg, 0.48 mmol) in DMF (50 mL),
NaH (18.28 mg, 0.72 mmol) was added portionwise at 0 °C. After
stirring for 0.5 h, one of 2a−2l (0.72 mmol) was added. The reaction
mixture was stirred at 70 °C for 12 h and then H
added dropwise followed by EtOAc (80 mL). Organic phase was
separated and washed with brine and dried over anhydrous Na SO ,
4
O (100 mL) was
2
N-(3-(Diethylamino)propyl)-1-ethyl-5-(3-(4-(2-hydroxypropoxy)-
-methylphenyl)pentan-3-yl)-1H-pyrrole-2-carboxamide (C16). Di-
2
3
filtered and concentrated. The residue was purified by silica gel
column chromatography eluting with appropriate mixture as indicated
in each case.
chloromethane/methanol (70/1). Oil. 54% yield.
N-(3-(Diethylamino)propyl)-1-ethyl-5-(3-(4-(2-hydroxybutoxy)-
-methylphenyl) pentan-3-yl)-1H-pyrrole-2-carboxamide (C17).
3
N-(3-(Diethylamino)propyl)-1-ethyl-4-(3-(4-(3-hydroxy-3-meth-
ylbutoxy)-3-methylphenyl)pentan-3-yl)-1H-pyrrole-2-carboxamide
(D4). Dichloromethane/methanol (100/1). Oil. 35% yield.
N-(3-(Diethylamino)propyl)-1-ethyl-4-(3-(4-((5-hydroxy-5-
methylhexyl)oxy)-3-methylphenyl)pentan-3-yl)-1H-pyrrole-2-car-
boxamide (D5). Dichloromethane/methanol (100/1). Oil. 45% yield.
N-(3-(Diethylamino)propyl)-1-ethyl-4-(3-(4-((6-hydroxy-6-
methylheptyl)oxy)-3-methylphenyl)pentan-3-yl)-1H-pyrrole-2-car-
boxamide (D6). Dichloromethane/methanol (100/1). Oil. 62% yield.
N-(3-(Diethylamino)propyl)-1-ethyl-4-(3-(4-((3-ethyl-3-
hydroxypentyl)oxy)-3-methylphenyl)pentan-3-yl)-1H-pyrrole-2-
Dichloromethane/methanol (70/1). Oil. 43% yield.
5
-(3-(4-(2-Cyclopropyl-2-hydroxyethoxy)-3-methylphenyl)-
pentan-3-yl)-N-(3-(diethylamino)propyl)-1-ethyl-1H-pyrrole-2-car-
boxamide (C18). Dichloromethane/methanol (70/1). Oil. 41% yield.
N-(3-(Diethylamino)propyl)-1-ethyl-5-(3-(4-(2-hydroxy-2-phenyl-
ethoxy)-3-methylphenyl)pentan-3-yl)-1H-pyrrole-2-carboxamide
(
C19). Dichloromethane/methanol (70/1). Oil. 33% yield.
Ethy-4-(3-(4-(benzyloxy)-3-methylphenyl)pentan-3-yl)-1H-pyr-
role-2-carboxylate (9). By the same manner as described for the
preparation of 4, intermediate 9 was prepared at 30 °C and purified
6
39
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Article
carboxamide (D7). Dichloromethane/methanol (100/1). Oil. 42%
yield.
N-(3-(Diethylamino)propyl)-1-ethyl-4-(3-(4-((5-ethyl-5-
hydroxyheptyl)oxy)-3-methylphenyl)pentan-3-yl)-1H-pyrrole-2-
carboxamide (D8). Dichloromethane/methanol (100/1). Oil. 66%
yield.
N-(3-(Diethylamino)propyl)-1-ethyl-4-(3-(4-((6-ethyl-6-
hydroxyoctyl)oxy)-3-methylphenyl)pentan-3-yl)-1H-pyrrole-2-car-
boxamide (D9). Dichloromethane/methanol (100/1). Oil. 43% yield.
N-(3-(Diethylamino)propyl)-1-ethyl-4-(3-(4-((3-hydroxy-3-
propylhexyl)oxy)-3-methylphenyl)pentan-3-yl)-1H-pyrrole-2-car-
boxamide (D10). Dichloromethane/methanol (100/1). Oil. 49%
yield.
N-(3-(Diethylamino)propyl)-1-ethyl-4-(3-(4-((5-hydroxy-5-
propyloctyl)oxy)-3-methylphenyl)pentan-3-yl)-1H-pyrrole-2-car-
boxamide (D11). Dichloromethane/methanol (100/1). Oil. 34%
yield.
N-(2-(Diethylamino)ethyl)-1-ethyl-5-(3-(4-(3-hydroxy-3-methyl-
butoxy)-3-methylphenyl)pentan-3-yl)-1H-pyrrole-2-carboxamide
(H2). Dichloromethane/methanol (100/1). Oil. 73% yield.
1
-Ethyl-5-(3-(4-(3-hydroxy-3-methylbutoxy)-3-methylphenyl)-
pentan-3-yl)-N-(2-(piperidin-1-yl)ethyl)-1H-pyrrole-2-carboxamide
H3). Dichloromethane/methanol (50/1). Oil. 66% yield.
-Ethyl-5-(3-(4-(3-hydroxy-3-methylbutoxy)-3-methylphenyl)-
pentan-3-yl)-N-(2-morpholinoethyl)-1H-pyrrole-2-carboxamide
H4). Dichloromethane/methanol (100/1). Oil. 71% yield.
(
1
(
1
-Ethyl-5-(3-(4-(3-hydroxy-3-methylbutoxy)-3-methylphenyl)-
pentan-3-yl)-N-(2-(4-methylpiperazin-1-yl)ethyl)-1H-pyrrole-2-car-
boxamide (H5). Dichloromethane/methanol (40/1). Oil. 80% yield.
1
-Ethyl-5-(3-(4-(3-hydroxy-3-methylbutoxy)-3-methylphenyl)-
pentan-3-yl)-N-((tetrahydrofuran-2-yl)methyl)-1H-pyrrole-2-car-
boxamide (H6). Dichloromethane/methanol (50/1). Oil. 61% yield.
1
-Ethyl-5-(3-(4-(3-hydroxy-3-methylbutoxy)-3-methylphenyl)-
pentan-3-yl)-N-((tetrahydro-2H-pyran-4-yl)methyl)-1H-pyrrole-2-
carboxamide (H7). Dichloromethane/methanol (40/1). Oil. 51%
yield.
N-(3-(Diethylamino)propyl)-1-ethyl-4-(3-(4-((6-hydroxy-6-
propylnonyl)oxy)-3-methylphenyl)pentan-3-yl)-1H-pyrrole-2-car-
boxamide (D12). Dichloromethane/methanol (100/1). Oil. 62%
yield.
N-(2,3-Dihydroxypropyl)-1-ethyl-5-(3-(4-(3-hydroxy-3-methylbu-
toxy)-3-methylphenyl)pentan-3-yl)-1H-pyrrole-2-carboxamide
(H8). Dichloromethane/methanol (70/1). Oil. 90% yield.
N-(3-(Diethylamino)propyl)-1-ethyl-4-(3-(4-((4-hydroxy-4-
methylpentyl)oxy)-3-methylphenyl)pentan-3-yl)-1H-pyrrole-2-car-
boxamide (D13). Dichloromethane/methanol (100/1). Oil. 50%
yield.
N-(3-(Diethylamino)propyl)-1-ethyl-4-(3-(4-((4-ethyl-4-
hydroxyhexyl)oxy)-3-methylphenyl)pentan-3-yl)-1H-pyrrole-2-car-
boxamide (D14). Dichloromethane/methanol (100/1). Oil. 61%
yield.
N-(3-(Diethylamino)propyl)-1-ethyl-4-(3-(4-((4-hydroxy-4-
propylheptyl)oxy)-3-methylphenyl)pentan-3-yl)-1H-pyrrole-2-car-
boxamide (D15). Dichloromethane/methanol (100/1). Oil. 53%
yield.
1-Ethyl-5-(3-(4-(3-hydroxy-3-methylbutoxy)-3-methylphenyl)-
pentan-3-yl)-N-(3-morpholinopropyl)-1H-pyrrole-2-carboxamide
(H9). Dichloromethane/methanol (50/1). Oil. 53% yield.
1-Ethyl-5-(3-(4-(3-hydroxy-3-methylbutoxy)-3-methylphenyl)-
pentan-3-yl)-N-(3-(4-methylpiperazin-1-yl)propyl)-1H-pyrrole-2-
carboxamide (H10). Dichloromethane/methanol (40/1). Oil. 88%
yield.
1-Ethyl-4-(3-(4-(2-hydroxypropoxy)-3-methylphenyl)pentan-3-
yl)-1H-pyrrole-2-carboxylic acid (15). To a stirred solution of 12
(200 mg, 0.63 mmol) in acetone (50 mL) was added K CO (131.3
2 3
mg, 0.95 mmol) and 1-bromopropan-2-ol (131.1 mg, 0.95 mmol) at 0
°C. The mixture was refluxed for 6 h and then cooled. The precipitate
was filtered off, the solution was evaporated, and water (100 mL) and
EtOAc (80 mL) was added. The organic phase was washed with brine
and dried over anhydrous Na SO , filtered, and concentrated. The
N-(3-(Diethylamino)propyl)-1-ethyl-4-(3-(4-(2-hydroxypropoxy)-
-methylphenyl)pentan-3-yl)-1H-pyrrole-2-carboxamide (D16). Di-
3
chloromethane/methanol (50/1). Oil. 71% yield.
2
4
N-(3-(Diethylamino)propyl)-1-ethyl-4-(3-(4-(2-hydroxybutoxy)-
residue was purified by column chromatography with dichloro-
methane/methanol (100/1, v/v) to give compound 15 as oil (0.25 g,
71% yield).
General Procedure 7: Synthesis of Compounds I1−I11. To a
solution of compound 15 (186.6 mg, 0.50 mmol) in dichloromethane
3
-methylphenyl) pentan-3-yl)-1H-pyrrole-2-carboxamide (D17).
Dichloromethane/methanol (50/1). Oil. 58% yield.
4
-(3-(4-(2-Cyclopropyl-2-hydroxyethoxy)-3-methylphenyl)-
pentan-3-yl)-N-(3-(diethylamino)propyl)-1-ethyl-1H-pyrrole-2-car-
boxamide (D18). Dichloromethane/methanol (50/1). Oil. 43% yield.
N-(3-(Diethylamino)propyl)-1-ethyl-4-(3-(4-(2-hydroxy-2-phenyl-
ethoxy)-3-methylphenyl)pentan-3-yl)-1H-pyrrole-2-carboxamide
(50 mL), 3-(ethyliminomethylideneamino)-N,N-dimethylpropan-1-
amine (hydrochloride) (116.2 mg, 0.75 mmol), 1-hydroxybenzo-
triazole (101.0 mg, 0.75 mmol), and triethylamine (75.6 mg, 0.75
mmol) was added at 0 °C. After stirring for 0.5 h, the corresponding
amine fragment (0.75 mmol) was added. The reaction mixture was
(
D19). Dichloromethane/methanol (50/1). Oil. 51% yield.
1
-Ethyl-5-(3-(4-(3-hydroxy-3-methylbutoxy)-3-methylphenyl)-
pentan-3-yl)-1H-pyrrole-2-carboxylic acid (14). To a solution of
compound 7 (200 mg, 0.63 mmol) in DMF (50 mL) was added NaH
stirred at room temperature for 12 h, and then H O (100 mL) was
2
added. The organic phase was washed with brine and dried over
(
22.84 mg, 0.95 mmol) at 0 °C and stirred for 1 h. 4-Bromo-2-
methylbutan-2-ol (157 mg, 0.95 mmol) was added to the mixture at 0
C. The mixture reacted for 12 h at 70 °C, water (10 mL) and EtOAc
30 mL) was added, and the organic phase was separated. The
anhydrous Na SO , filtered, and concentrated. The residue was
2
4
purified by silica gel column chromatography eluting with appropriate
mixture as indicated in each case.
N-(2-(Dimethylamino)ethyl)-1-ethyl-4-(3-(4-(2-hydroxypropoxy)-
3-methylphenyl)pentan-3-yl)-1H-pyrrole-2-carboxamide (I1). Di-
chloromethane/methanol (40/1). Oil. 70% yield.
N-(2-(Diethylamino)ethyl)-1-ethyl-4-(3-(4-(2-hydroxypropoxy)-3-
methylphenyl) pentan-3-yl)-1H-pyrrole-2-carboxamide (I2). Di-
chloromethane/methanol (40/1). Oil. 60% yield.
1-Ethyl-4-(3-(4-(2-hydroxypropoxy)-3-methylphenyl)pentan-3-
yl)-N-(2-(piperidin-1-yl)ethyl)-1H-pyrrole-2-carboxamide (I3). Di-
chloromethane/methanol (30/1). Oil. 73% yield.
°
(
organic phase was washed with brine and dried over anhydrous
Na SO , filtered, and concentrated. The residue was purified by
2
4
column chromatography with dichloromethane/methanol (100/1, v/
v) to give compound 14 as oil (0.18 g, 71% yield).
General Procedure 6: Synthesis of Compounds H1−H10. To a
solution of compound 14 (200 mg, 0.50 mmol) in dichloromethane
50 mL), 3-(ethyliminomethylideneamino)-N,N-dimethylpropan-1-
amine (hydrochloride) (116.2 mg, 0.75 mmol), 1-hydroxybenzo-
triazole (101.0 mg, 0.75 mmol), and triethylamine (75.6 mg, 0.75
mmol) was added at 0 °C. After stirring for 0.5 h, the corresponding
amine fragment (0.75 mmol) was added. The reaction mixture was
(
1
-Ethyl-4-(3-(4-(2-hydroxypropoxy)-3-methylphenyl)pentan-3-
yl)-N-(2-morpholinoethyl)-1H-pyrrole-2-carboxamide (I4). Di-
chloromethane/methanol (40/1). Oil. 82% yield.
1
-Ethyl-4-(3-(4-(2-hydroxypropoxy)-3-methylphenyl)pentan-3-
stirred at room temperature for 12 h, and then H O (100 mL) was
2
yl)-N-(2-(4-methylpiperazin-1-yl)ethyl)-1H-pyrrole-2-carboxamide
added. The organic phase was washed with brine and dried over
(
I5). Dichloromethane/methanol (30/1). Oil. 85% yield.
anhydrous Na SO , filtered, and concentrated. The residue was
2
4
1-Ethyl-4-(3-(4-(2-hydroxypropoxy)-3-methylphenyl)pentan-3-
yl)-N-((tetrahydrofuran-2-yl)methyl)-1H-pyrrole-2-carboxamide
(I6). Dichloromethane/methanol (50/1). Oil. 84% yield.
purified by silica gel column chromatography eluting with the
appropriate mixture as indicated in each case.
N-(2-(Dimethylamino)ethyl)-1-ethyl-5-(3-(4-(3-hydroxy-3-meth-
ylbutoxy)-3-methylphenyl)pentan-3-yl)-1H-pyrrole-2-carboxamide
(H1). Dichloromethane/methanol (70/1). Oil. 83% yield.
1-Ethyl-4-(3-(4-(2-hydroxypropoxy)-3-methylphenyl)pentan-3-
yl)-N-((tetrahydro-2H-pyran-4-yl)methyl)-1H-pyrrole-2-carboxa-
mide (I7). Dichloromethane/methanol (40/1). Oil. 88% yield.
6
40
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Journal of Medicinal Chemistry
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Article
N-(2,3-Dihydroxypropyl)-1-ethyl-4-(3-(4-(2-hydroxypropoxy)-3-
methylphenyl) pentan-3-yl)-1H-pyrrole-2-carboxamide (I8). Di-
chloromethane/methanol (70/1). Oil. 67% yield.
Antitumor Effect of Compound In Vivo. Female BALB/c nude
mice (18−22 g) were purchased from the Beijing Vital River
Laboratory Animal Technology Co., Ltd. All animals were treated
according to the guidelines for the care and use of laboratory animals
approved by the Animal Science Ethics Committee of China
Pharmaceutical University. Mice were randomly divided into six
groups (five mice in each group). All mice were maintained under
standard conditions with free access to water and laboratory rodent
1
-Ethyl-4-(3-(4-(2-hydroxypropoxy)-3-methylphenyl)pentan-3-
yl)-N-(3-morpholinopropyl)-1H-pyrrole-2-carboxamide (I9). Di-
chloromethane/methanol (40/1). Oil. 79% yield.
1
-Ethyl-4-(3-(4-(2-hydroxypropoxy)-3-methylphenyl)pentan-3-
yl)-N-(3-(4-methylpiperazin-1-yl)propyl)-1H-pyrrole-2-carboxa-
mide (I10). Dichloromethane/methanol (40/1). Oil. 81% yield.
N-(3-(bis(2-Hydroxyethyl)amino)propyl)-1-ethyl-4-(3-(4-(2-hy-
droxypropoxy)-3-methylphenyl)pentan-3-yl)-1H-pyrrole-2-carbox-
amide (I11). Dichloromethane/methanol (30/1). Oil. 66% yield.
VDR Binding Assay. The VDR binding affinity of non-
secosteriodal VDR ligands was measured by a PolarScreen VDR
Competitor Assay following the procedure previously described. All
compounds were tested for their binding affinity at 1 μM in triplicates.
Fluorescence polarization was measured on an Ultra384 microplate
reader (Biotek) using a 535 nm excitation filter (25 nm bandwidth)
and a 590 nm emission filter (20 nm bandwidth).
6
food. LTC-14-GFP (1 × 10 cells per mouse) and DSL-6A/C1 Luci
6
(
1 × 10 cells per mouse) were co-inoculated in situ on the pancreas
of nude mice, and the administration was started 7 days after tumor
grafting. The compound was dissolved in ethanol/polyoxyethylene
castor oil/normal saline (1:1:18) mixed solvent, and the mice were
intraperitoneally injected with normal saline, gemcitabine, gemcita-
bine + calcipotriol (60 μg/kg), gemcitabine + C4 (500 μg/kg),
gemcitabine + I5 (500 μg/kg), and gemcitabine + I8 (500 μg/kg),
once every 3 days. The dosage of gemcitabine was 100 mg/kg in each
group. Tumor images were taken on the Maestro in vivo imaging
system (PerkinElmer) during the 12 day treatment study. Mice were
sacrificed 3 days after the final administration. Mouse pancreas and
serum were obtained for histopathology, collagen assay, biochemical,
and molecular analyses.
Transcription Assay. Luciferase activity assay was performed
using the Dual-Luciferase Reporter Assay System (Promega, Madison,
WI) according to the manufacturer’s instructions. HEK293 cells of
8
1
5%−90% confluence were seeded in 48-well plates. Transfections of
40 ng of TK-Spp × 3-LUC reporter plasmid, 20 ng of pCMX-
Immunofluorescence Analysis. Identification of the α-SMA by
immunofluorescence microscopy was carried out as follows: The
tumor sections were fixed with 4% paraformaldehyde for 10 min,
followed by 0.5% Triton X-100 to perforate cell membranes at room
temperature. After careful washing with PBS three times, the samples
were blocked at 37 °C for 30 min with 5% BSA. The tumor sections
were incubated with primary antibodies to α-SMA overnight at 4 °C.
After careful washing with PBS, tumor sections were incubated with
Renilla, 30 ng of pENTER-CMV-hRXRα, and 100 ng of pCMX-VDR
for each well using a Lipofectamine2000 Reagent (Invitrogen). Eight
hours after transfection, test compounds were added. Luciferase
activity assay was performed 24 h later using the Dual-Luciferase
Assay System. Firefly luciferase activity was normalized to the
corresponding Renilla luciferase activity. All the experiments were
performed three times.
MTT Assay. The effects of the compounds on the proliferation of
the DSL-6A/C1 cells were evaluated by MTT (3-(4,5-dimethylth-
iazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide; Sigma) assay.
Briefly, after the PSC cells (LTC-14) were treated with compounds
for 24 h, the medium supernatant of PSC (PSC-CM) was collected
and added into the pancreatic cancer cells DSL-6A/C1. After
incubation with 10 μL of MTT (5 mg/mL) for 4 h, absorbance of
the soluble MTT product was measured at 570 nm. The
antiproliferation assay was performed in triplicate.
TM
the fluorescent secondary antibody Alexa Fluor 594 donkey anti-
rabbit IgG (H + L) in a 37 °C incubator for 1 h. The clusters were
washed twice with PBS and stained with DAPI for 15 min at room
temperature. After washing twice more, the samples were observed
using a laser confocal scanning microscope (LSM880, Carl Zeiss
Jena).
Statistical Analysis. Statistical analyses were performed using
GraphPad Prism 8.0. All plots show mean ± SD from at least three
independent experiments. Student’s t-test was applied for compar-
isons between two groups, one-way ANOVA with Dunnett’s post hoc
test was used for comparisons of multiple groups, and a log-rank
Anti-PSC Activity Assay. Rat PSC line LTC-14, provided by
Prof. Gisela Sparmann (Department of Gastroenterology, University
of Rostock, Germany) was maintained in Iscove’s Modified
Dulbecco’s medium (IMDM, Gibco) supplement with 10% fetal
(
Mantel−Cox) test was used to analyze the statistical significance of
difference for survival analysis. Statistical significance was set at *P <
0.05, **P < 0.01 and ***P < 0.001.
bovine serum (FBS) and 1% penicillin-streotomycin. Approximate 1
5
×
10 cells, suspended in the medium, were plated into each well of a
2
4-well plate and grown at 37 °C in a humidified atmosphere with 5%
2
CO for 24 h. The following day, tested compounds (0.5 μM) and
ASSOCIATED CONTENT
■
TGF-β1 (5 ng/mL) were added to the culture medium and incubated
for 24 h. RNA extracts were derived from LTC-14 cells for Q-PCR
assay and proteins were purified for western blot.
RNA Extraction and Quantitative Real-Time Polymer-
asechain Reaction (Q-PCR). cDNA was generated from RNA
extracts derived from cultured LTC-14 cells and pancreas tissues
using a reverse transcription kit (Vazyme). β-actin (rat) was used as
an internal control. Q-PCR was performed using the SYBR Green
Master Mix (Yeasen Biotech Co., Ltd.) using a StepOnePlus Real-
Time PCR System (Applied Biosystems).
*
sı Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c01197.
(
Table S1) Cell toxicity of all target compounds against
LTC-14; (Figure S1) cell toxicity of representative
compounds against DSL-6A/C1 cells; (Figure S2)
relative BLI intensities of mice that received different
treatments; (Figure S3) inhibition rate of tumor growth
after tumor-bearing mice received different treatments;
Western Blot. Proteins were purified from LTC-14 cells. Proteins
were separated using 10% SDS-polyacrylamide gel electrophoresis and
were electrophoretically transferred to polyvinylidene fluoride
(
Figure S4) relative fluorescence intensities of α-SMA
after mice received different treatments; (Figure S5)
survival percentages of mice that received different
treatments; (Figure S6) in vivo hypercalcemic effects in
the orthotopic transplantation pancreatic cancer model;
(PVDF) membranes using standard procedures. The following
primary antibodies were employed: rabbit anti-α-SMA, rabbit anti-
collagen I, rabbit anti- PDGF-A, rabbit anti-POSTN, and rabbit anti-
β-actin (Absin, Shanghai, China). Horseradish peroxidase-conjugated
goat anti-rabbit/mouse IgG (Boster, Wuhan, China) was used as the
secondary antibody. Immunoreactive bands were visualized with a
Tanon 5200 Multi Chemiluminescence Imaging System and analyzed
with Image J.
1
NMR spectra information; HPLC traces; and H NMR
1
3
and C NMR spectra (PDF)
Molecular Formula Strings (CSV)
6
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Article
AUTHOR INFORMATION
Author Contributions
Z.K. and C.W. contributed equally to this work.
■
‡
Corresponding Author
Notes
Can Zhang − State Key Laboratory of Natural Medicines,
Jiangsu Key Laboratory of Drug Discovery for Metabolic
Diseases, Center of Advanced Pharmaceuticals and
The authors declare no competing financial interest.
Biomaterials, China Pharmaceutical University, Nanjing
ACKNOWLEDGMENTS
■
2
10009, P.R. China; orcid.org/0000-0003-3529-5438;
We thank the Public Platform of the State Key Laboratory of
Natural Medicines for assistance with the pathological-section
imaging. This work was supported by the National Natural
Science Foundation of China (81930099, 81773664,
Phone: 86-25-83271171; Email: zhangcan@cpu.edu.cn
Authors
Zisheng Kang − State Key Laboratory of Natural Medicines,
Jiangsu Key Laboratory of Drug Discovery for Metabolic
Diseases, Center of Advanced Pharmaceuticals and
8
1703585), National Major Scientific and Technological
Special Project for ″Significant New Drugs Development″
(
(
2019ZX09301163), ″Double First-Class″ University project
Biomaterials, China Pharmaceutical University, Nanjing
CPU2018GY47, CPU2018GF10), 111 Project from the
2
10009, P.R. China
Ministry of Education of China and the State Administration
of Foreign Expert Affairs of China (no. 111-2-07, B17047), and
the Open Project of State Key Laboratory of Natural
Medicines (no. SKLNMZZ202017).
Cong Wang − State Key Laboratory of Natural Medicines,
Jiangsu Key Laboratory of Drug Discovery for Metabolic
Diseases, Center of Advanced Pharmaceuticals and
Biomaterials, China Pharmaceutical University, Nanjing
2
10009, P.R. China
ABBREVIATIONS USED
■
Yu Tong − State Key Laboratory of Natural Medicines,
Jiangsu Key Laboratory of Drug Discovery for Metabolic
Diseases, Center of Advanced Pharmaceuticals and
Biomaterials, China Pharmaceutical University, Nanjing
ECM, extracellular matrix; LBP, ligand binding pocket; VDR,
vitamin D receptor; PSC, pancreatic stellate cell; FGF,
fibroblast growth factor; PDGF, platelet-derived growth factor;
HGF, periostin and hepatocyte growth factor; IGF-1, insulin-
like growth factor 1; TGF, transforming growth factor; α-SMA,
α-smooth muscle actin; POSTN, periosteal osteoblast specific
factor 2; CM, conditioned media; DOX, doxorubicin; RXRα,
retinoid X receptor alpha
2
10009, P.R. China
Yanyi Li − State Key Laboratory of Natural Medicines, Jiangsu
Key Laboratory of Drug Discovery for Metabolic Diseases,
Center of Advanced Pharmaceuticals and Biomaterials,
China Pharmaceutical University, Nanjing 210009, P.R.
China
Yi Gao − State Key Laboratory of Natural Medicines, Jiangsu
Key Laboratory of Drug Discovery for Metabolic Diseases,
Center of Advanced Pharmaceuticals and Biomaterials,
China Pharmaceutical University, Nanjing 210009, P.R.
China
Siyuan Hou − State Key Laboratory of Natural Medicines,
Jiangsu Key Laboratory of Drug Discovery for Metabolic
Diseases, Center of Advanced Pharmaceuticals and
Biomaterials, China Pharmaceutical University, Nanjing
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