Sulfonyl-containing phenyl-pyrrolyl pentane analogues: Novel non-secosteroidal vitamin D receptor modulators with favorable physicochemical properties, pharmacokinetic properties and anti-tumor activity

Article abstract of DOI:10.1016/j.ejmech.2018.08.085

Modulating the vitamin D receptor (VDR) is an effective way to treat for cancer. We previously reported a potent non-secosteroidal VDR modulator (sw-22) with modest anti-tumor activity, which could be due to its undesirable physicochemical and pharmacokinetic properties. In this study, we investigated the structure-activity and structure-property relationships around the 2′-hydroxyl group of sw-22 to improve the physicochemical properties, pharmacokinetic properties and anti-tumor activity. Compounds 19a and 27b, the potent non-secosteroidal VDR modulators, were identified as the most effective molecules in inhibiting the proliferation of three cancer cell lines, particularly breast cancer cells, with a low IC₅₀ via the distribution of cell cycle and induction of apoptosis by stimulating the expression of p21, p27 and Bax. Further investigation revealed that 19a and 27b possessed favorable rat microsomal metabolic stability (2.22 and 2.3 times, respectively, more stable than sw-22), solubility (43.9 and 50.2 times, respectively, more soluble than sw-22) and in vivo pharmacokinetic properties. In addition, 19a and 27b showed excellent in vivo anti-tumor activity without cause hypercalcemia, which is the main side effect of marketed VDR modulators. In summary, the favorable physicochemical properties, pharmacokinetic properties and anti-tumor activity of 19a and 27b highlight their potential therapeutic applications in cancer treatment.

Full text of DOI:10.1016/j.ejmech.2018.08.085

Accepted Manuscript
Sulfonyl-containing phenyl-pyrrolyl pentane analogues: Novel non-secosteroidal
vitamin D receptor modulators with favorable physicochemical properties,
pharmacokinetic properties and anti-tumor activity
Zi-Sheng Kang, Cong Wang, Xiao-Lin Han, Bin Wang, Hao-Liang Yuan, Si-Yuan Hou,
Mei-Xi Hao, Jun-Jie Du, Yan-Yi Li, An-Wei Zhou, Can Zhang
PII:
S0223-5234(18)30757-8
10.1016/j.ejmech.2018.08.085
EJMECH 10696
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 14 July 2018
Revised Date: 27 August 2018
Accepted Date: 28 August 2018
Please cite this article as: Z.-S. Kang, C. Wang, X.-L. Han, B. Wang, H.-L. Yuan, S.-Y. Hou, M.-X.
Hao, J.-J. Du, Y.-Y. Li, A.-W. Zhou, C. Zhang, Sulfonyl-containing phenyl-pyrrolyl pentane analogues:
Novel non-secosteroidal vitamin D receptor modulators with favorable physicochemical properties,
pharmacokinetic properties and anti-tumor activity, European Journal of Medicinal Chemistry (2018), doi:
10.1016/j.ejmech.2018.08.085.
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ACCEPTED MANUSCRIPT
Sulfonyl-Containing Phenyl-Pyrrolyl Pentane Analogues: Novel Non-secosteroidal
Vitamin D Receptor Modulators with Favorable Physicochemical Properties,
Pharmacokinetic properties and Anti-tumor Activity
1
1
Zi-sheng Kang, Cong Wang, Xiao-lin Han, Bin Wang, Hao-liang Yuan, Si-yuan
Hou, Mei-xi Hao, Jun-jie Du, Yan-yi Li, An-wei Zhou and Can Zhang*
1
The first two authors contributed equal
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.
Abstract
Modulating the vitamin D receptor (VDR) is an effective way to treat for cancer.
We previously reported a potent non-secosteroidal VDR modulator (sw-22) with
modest anti-tumor activity, which could be due to its undesirable physicochemical
and pharmacokinetic properties. In this study, we investigated the structure-activity
and structure-property relationships around the 2`-hydroxyl group of sw-22 to
improve the physicochemical properties, pharmacokinetic properties and anti-tumor
activity. Compounds 19a and 27b, the potent non-secosteroidal VDR modulators,
were identified as the most effective molecules in inhibiting the proliferation of three
cancer cell lines, particularly breast cancer cells, with a low IC via the distribution
5
0
of cell cycle and induction of apoptosis by stimulating the expression of p21, p27 and
Bax. Further investigation revealed that 19a and 27b possessed favorable rat
microsomal metabolic stability (2.22 and 2.3 times, respectively, more stable than sw-
2
2), solubility (43.9 and 50.2 times, respectively, more soluble than sw-22) and in
vivo pharmacokinetic properties. In addition, 19a and 27b showed excellent in vivo
anti-tumor activity without cause hypercalcemia, which is the main side effect of
marketed VDR modulators. In summary, the favorable physicochemical properties,
pharmacokinetic properties and anti-tumor activity of 19a and 27b highlight their
potential therapeutic applications in cancer treatment.

ACCEPTED MANUSCRIPT
Keywords: vitamin D receptor (VDR), non-secosteroidal, physicochemical properties,
pharmacokinetic properties, anti-tumor activity.
1
. Introduction
Vitamin D receptor (VDR), a member of the steroid–thyroid–retinoid receptor
superfamily of ligand-activated transcription factors, which involve regulating
1
-3
calcium homeostasis and bone metabolism.
Recently, accumulating evidence
suggests that VDR was involved in antineoplastic actions in various malignancies,
4
-8
such as breast, pancreatic and prostate cancer. After being combined with its
modulator, VDR dimerizes with the retinoid X receptor (RXR) and binds to the
vitamin D response elements (VDREs) to alter the rate of target gene transcription.
With recruitment of co-modulators, the activated VDR can decrease tumor growth
9
-10
mainly by suppressing proliferation or promoting apoptosis of cancer cells.
In
order to enable VDR play a more effective antineoplastic activity, large numbers of
1
1-14
VDR modulators had been applied to anti-tumor researches.
The VDR modulators can be classified into “secosteroid” and “non-secosteroidal”
based on their structure specificity. Up to now, more than 3000 secosteroidal VDR
modulators (SVDRMs) have been developed, such as calcipotriol (1), tacalcitol (2)
1
5
and natural VDR modulator 1,25(OH) D (3). However, secosteroidal-based
2
3
1
6
chemical synthesis has proven to be difficult and costly. What's more, in clinical
cancer treatment, the required doses of these SVDRMs induced serious hypercalcemia,
the main side effect of VDR modulators which could induce abdominal pain, kidney
1
7
stones and cardiac arrest. In addition, clinical studies showed that 20 to 30 percent
of cancer patients suffer from hypercalcemia at the same time, which strongly limit
1
8-21
the clinical application of SVDRMs in cancer treatment.
In order to potentiate beneficial properties of SVDRMs and avoide their
disadvantages, non-secosteroidal VDR modulators (NVDRMs) were developed, such
2
2
as LG190178 (4) and CB-16 (5). NVDRMs are easy to synthesis, and possess
distinct activities compared with SVDRMs including VDR binding affinity and
,
VDR-dependent transcriptional activation. More importantly, NVDRMs can carry out
2
3-
their physiological functions without hypercalcemia side effect even at high doses.
2
4

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Recently, we successfully designed and characterized a novel series of NVDRM
2
5-27
bearing phenyl-pyrrolyl pentane scaffold.
Among them, compound sw-22 (6), the
most potent NVDRM, showed EC value for inducing the HL-60 leukaemia cells
5
0
differentiation in 1.06 nM, which is better than positive control tacalcitol (2).
2
5
Furthermore, in in vivo study, sw-22 showed no significant change on serum calcium
.
However, sw-22 showed modest anti-tumor activity in in vitro and in vivo studies,
which may be due to its undesirable metabolic stability (t½ = 25.76 min, rat liver
microsomes), solubility (1.7 µg/mL, pH 7.4) and in vivo pharmacokinetics property
(t½ = 2.89 h, intravenous; t½ = 3.47 h, intraperitoneal). Compound sw-22 contain a 2-
hydroxy-3,3-dimethylbutoxy side chain at phenyl part, the 2`-hydroxyl group on this
side chain could form hydrogen bonds with amino acid residues (His 393) of the VDR
ligand binding domain (LBD), which is important to VDR transcriptional activity.
However, the hydroxyl group on the side chain of VDR modulators is easy to be
28
oxidated, which could lead to its inactive and therefore causing undesirable
metabolic stability and pharmacokinetic properties of sw-22. Accordingly, we
hypothesized that suitable chemical modification around the 2`-hydroxyl group could
improve the physicochemical and pharmacokinetic properties of sw-22, and therefore,
lead to the analogues possess better anti-tumor activity.

ACCEPTED MANUSCRIPT
Figure 1. Chemical structures of VDR modulators.
In this work, our first effort was aimed at identifying a suitable group to replace
the 2`-OH group on the side chain based on sw-22. Sulfonyl group commonly used as
2
9
a group to improve the metabolic stability and solubility of molecules and
furthermore, the oxygen atoms on the sulfonyl group could act as hydrogen bond
acceptors, which could form hydrogen bonds with amino acid residues and mimic the
function of 2`-OH group on the side chain. Consequently, we replaced the 2`-OH
group to sulfonyl group while replacing the substituents in the terminal of side chain,
and targeting improved physicochemical properties (i.e., metabolic stability and
solubility) as well as anti-tumor activity. Then, hydrophilic groups were added to the
pyrrol part, which could form hydrogen bonds with amino acid residues of VDR LBD.
Next, to explore the effects of substitutions at terminal of side chain and oxidation
state of sulfur group, compounds 19a-b and 20a were designed. Additionally, in order
to investigate the influence of the substitution positions of the pyrrole ring, phenyl-
pentane group on C-4 position of pyrrole ring instead of C-5 position was designed
and obtained compounds 26a-b, 27a-b (Figure 2). Furthermore, the in vitro and in

ACCEPTED MANUSCRIPT
vivo biological activities of these non-secosteroidal VDR modulators are evaluated in
this study.
Figure 2. Design concept of novel non-secosteroidal VDR modulators.
2
2
. Results and Discussion
.1 Chemistry.
The synthetic pathway of target compounds 15a-o, 16a-l and 17a-k is outlined
in Scheme 1. Key intermediate 7 was readily prepared using our previously reported
approach. Compound 7 coupling with 1H-pyrrole-2-carboxylate in the presence of
o
lewis acid BF ·Et O at 0 C produced the intermediate 8, following the treatment with
3
2
iodoethane in DMF to afford intermediate 9. Subsequently, reduction of compound 9
offered compound 10, then compound 10 was dissolved in DMF in the presence of
chloromethyl phenyl sulfide to give compound 11. The oxidation reaction of
compound 11 obtained the intermediate 12, which was hydrolyzed by NaOH to give
1
3. By reaction of intermediate 13 with the corresponding amines, intermediates 14a-
o were produced. Finally, reduction or hydrolysis of appropriate esters group in 14a-l,
the target compounds 15a-l and 16a-k were obtained, respectively.
a
Scheme 1. Synthesis of target compounds 14a-o, 15a-l and 16a-k

ACCEPTED MANUSCRIPT
a
o
Reagents and conditions: (a) ethyl 1H-pyrrole-2-carboxylate, BF ·Et O, -20-0 C, 3 h;
3
2
o
o
(
b) C H I, NaH, DMF, 0-25 C, 5 h; (c) Pd/C, H , MeOH, 25 C, 12 h; (d)
2
5
2
o
chloromethyl phenyl sulfide, NaH, DMF, 0-70 C, 12 h; (e) potassium
o
peroxymonosulfate, EtOH/H O (10:1), 25 C, 24 h; (f) NaOH, EtOH/H O (10:1), 90
2
2
o
C, 24 h; (g) EDCI, HOBt, TEA, RNH , DCM, rt, overnight; (i) NaBH , MeOH, 0-25
2
4
o
o
C, 8 h; (j) KOH, MeOH/H O (10:1), 70 C, 24 h;
2
The synthetic pathway of target compounds 19a-b and 20a is outlined in Scheme
. Hydrolysis of compound 10 by KOH produced the intermediate 17 in the presence
2
of carboxyl group. Substitution of the carboxyl group with N,N-
diethylethylenediamine provide the compound 18. Compounds 19a-b were prepared
in a single step by the treatment of compound 18 with different halogen substituted
alkyl sulfide. By oxidation of compound 19a, the target compound 20a was obtained.
a
Scheme 2. Synthesis of target compounds 19a-b and 20a

ACCEPTED MANUSCRIPT
a
o
Reagents and conditions: (a) NaOH, EtOH/H O (10:1), 90 C, 24 h; (b) EDCI, HOBt,
2
TEA, N,N-diethylethane-1,2-diamine, DCM, rt, overnight; (c) chloromethyl sulfide,
o
o
NaH, DMF, 0-70 C, 12 h; (d) potassium peroxymonosulfate, EtOH/H O (10:1), 25 C,
2
2
4 h;
The synthetic pathway of target compounds 26a-b and 27a-b is outlined in
Scheme 3. Intermediate 21, which was the regioselectivity isomer of intermediate 8,
was produced by reacting with ethyl pyrrole-2-carboxylate in the presence of lewis
o
acid BF ·Et O at 20 C. Compound 21 treated with iodoethane in DMF to afford
3
2
intermediate 22. The reduction reaction of compound 22 obtained the intermediate 23,
which were hydrolyzed by KOH to give 24. By reaction of intermediate 24 with N,N-
diethylethylenediamine, intermediates 25 was produced. Compounds 26a-b were
prepared in a single step by the treatment of compound 25 with different halogen
substituted alkyl sulfide. By oxidation of compounds 26a-b, the target compounds
2
7a-b were obtained, respectively.
a
Scheme 3. Synthesis of target compounds 26a-b and 27a-b

ACCEPTED MANUSCRIPT
a
o
Reagents and conditions: (a) ethyl 1H-pyrrole-2-carboxylate, BF ·Et O, 30 C, 3 h;
3
2
o
o
(
b) C H I, NaH, DMF, 0-25 C, 5 h; (c) Pd/C, H , MeOH, 25 C, 12 h; (d) NaOH,
2 5 2
o
EtOH/H O (10:1), 90 C, 24 h; (e) EDCI, HOBt, TEA, N,N-diethylethane-1,2-diamine,
2
o
DCM, rt, overnight; (f) chloromethyl sulfide, NaH, DMF, 0-70 C, 12 h; (g)
o
potassium peroxymonosulfate, EtOH/H O (10:1), 25 C, 24 h;
2
2
.2. In Vitro Antiproliferative Activity Screening and SARs Analysis.
The structures of synthesized compounds are shown in Table 1. And the effect
of these synthesized compounds on cell proliferation was investigated in a panel of
human cancer cell lines from diverse tissue origins by MTT assay, the IC values are
5
0
listed in Table 2. Replacing the 2`-OH group with sulfonyl group (15a) led to a
modest improvement in potency, maintained VDR binding affinity (Table 3) and
more importantly, the better physicochemical properties (Table 4). Given these
results, we decided to proceed with the sulfonyl group for the next round of analogues
which were aimed at modifying the hydrophilic groups in the pyrrol part. Introduction
of different amino acid esters decreased cytotoxicity. Next, we focused on exploring
SAR at the α-position of amide bond by replacing the methyl group with various
steric bulk groups as well as varying the chiral in this portion of the molecule. The
steric bulk of the α-position modulated the potency, and the methyl group was the
optimal group for the antiproliferative effect (15a-g). And introducing another chain
contain hydroxyl group at this position is unfavorite (15i-l). In addition, oxidative
product 16a-k was devoid of activity, similar detrimental effect was observed in

ACCEPTED MANUSCRIPT
compounds with methyl ester group at terminal of this side chain, which suggest that
carboxyl acid and methyl ester groups may not preferred. Our biggest potency
improvement occurred when we introduced the N,N-diethylpropan-1-amine chain in
the pyrrol part (14o), which had an IC value of 6.27 µM, 4.68 µM and 7.13 µM for
5
0
BXPC-3, MCF-7 and PC-3 cells, respectively. Given these results, we decided to
proceed with the N, N-diethylpropan-1-amine chain for the next round of analogues.
Changes in substituents at the terminal of side chain at phenyl part affected activity,
replaced of the phenyl group with the methyl group result in decreased acitivity (20a),
and change oxidation state of sulfur group can not improve activity. Exploration of
the SAR around the substitution positions of the pyrrole ring indicated that
substitution at the C-4 position was greatly favored compared to the C-5 positions.
To confirm that compounds exhibit anti-tumor activity through VDR, we used
RNA interference (RNAi) in MCF-7 cells. As shown in Figure 3, BrdU was
employed, we found that silence the VDR abolished representative compounds
induced anti-tumor activity. These data suggest that representative compounds exert
their anti-tumor activity through VDR.
Table 1. The structures of all target compounds.
1
2
3
4
Compd.
4a
R
M
X
N
Y
C
R
R
R
1
C
C
C
C
C
6
6
6
6
6
H
H
H
H
H
5
5
5
5
5
C
C
C
C
C
2
2
2
2
2
H
H
H
H
H
5
5
5
5
5
H
H
H
H
H
1
4b
N
N
N
N
C
C
C
C
1
4c
1
4h
1
4i

ACCEPTED MANUSCRIPT
1
4m
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
H
5
H
5
H
5
H
5
H
5
H
5
H
5
H
5
H
5
H
5
H
5
H
5
H
5
H
5
H
5
H
5
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
H
5
H
5
H
5
H
5
H
5
H
5
H
5
H
5
H
5
H
5
H
5
H
5
H
5
H
5
H
5
H
5
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
1
4n
1
4o
1
5a
OH
N
H
1
5b
1
5c
OH
N
H
1
5d
1
5e
1
5f
1
5g
1
5h
1
5i
1
5j
1
5k
1
5l
1
6a

ACCEPTED MANUSCRIPT
1
6b
C
C
C
C
C
C
C
C
C
C
6
6
6
6
6
6
6
6
6
6
H
H
H
H
H
H
H
H
H
H
5
5
5
5
5
5
5
5
5
5
N
N
N
N
N
N
N
N
N
N
N
N
N
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
N
N
N
C
C
C
C
C
C
C
C
C
C
C
C
C
2
2
2
2
2
2
2
2
2
2
2
2
2
H
H
H
H
H
H
H
H
H
H
H
H
H
5
5
5
5
5
5
5
5
5
5
5
5
5
H
H
H
H
H
H
H
H
H
H
H
H
H
1
6c
1
6d
OH
N
H
O
1
6e
1
6f
1
6g
1
6h
1
6i
1
6j
1
6k
1
9a
CH
3
1
9b
C H
6 5
2
2
0a
6a
CH
3
CH
H
H
H
C
C
C
H
3
2
2
2
5
5
5
2
6b
C
6
H
5
H
H
2
7a
CH
3

ACCEPTED MANUSCRIPT
2
7b
C
6
H
5
C
N
H
C H
2 5
Table 2. In vitro antiproliferative activity of synthesized compounds against various
human cancer cell lines
a
IC (µM)
5
0
Compd.
4a
4b
4c
4h
4i
4m
4n
BXPC-3
17.92±0.73
19.98±0.18
NA
MCF-7
PC-3
b
1
43.67±0.63
NA
NA
1
NA
1
NA
NA
1
NA
NA
40.37±2.82
6.49±0.15
NA
1
NA
NA
1
NA
NA
1
6.43±0.19
6.27±0.04
15.30±6.16
19.94±0.05
10.01±0.30
18.28±0.89
45.39±1.67
7.72±0.85
13.15±0.76
26.58±1.70
15.90±0.75
NA
6.47±0.04
4.68±0.20
10.78±2.27
15.47±1.40
10.63±1.44
20.65±0.88
28.84±1.27
25.47±0.60
18.82±1.22
41.95±0.89
28.52±2.07
27.42±0.34
31.37±0.41
29.57±0.91
35.47±0.12
33.08±0.87
6.89±0.36
7.13±0.39
9.52±0.66
17.74±1.57
7.79±0.28
9.85±0.69
3.56±0.19
7.30±0.19
10.61±0.58
11.32±0.69
12.14±0.95
14.07±0.47
6.81±0.76
13.43±0.96
16.79±0.78
30.05±1.17
1
4o
5a
1
1
5b
5c
5d
5e
5f
5g
5h
1
1
1
1
1
1
1
1
5i
5j
1
5k
NA
1
5l
16.44±0.74
20.79±1.03
NA
1
6a
1
6b

ACCEPTED MANUSCRIPT
1
6c
6d
6e
6f
6g
6h
20.07±0.88
NA
NA
20.07±0.26
26.30±0.20
3.65±0.30
3.07±0.79
14.36±0.56
NA
1
41.19±0.32
24.46±1.91
44.71±0.29
NA
1
16.22±0.20
13.90±0.66
NA
1
1
1
NA
NA
1
6i
NA
NA
NA
1
6j
NA
NA
NA
1
6k
9a
9b
NA
NA
NA
1
5.13±0.35
5.67±0.40
33.67±1.24
4.11±0.34
5.37±0.35
30.39±0.71
4.69±0.61
15.78±1.58
21.07±0.29
5.16±0.10
4.90±0.85
14.72±0.45
4.64±0.34
4.70±0.86
16.29±1.13
2.03±0.20
18.23±0.34
11.43±0.32
4.01±0.80
2.23±0.14
46.68±0.12
9.54±0.38
2.62±0.21
36.57±0.02
6.08±0.89
8.50±0.02
9.53±0.35
1
2
2
0a
6a
2
6b
7a
7b
2
2
sw-22
c
1
,25(OH) D3
2
a
b
c
Results are expressed as the mean IC of three experiments.
5
0
Not active (IC > 50 µM).
5
0
1
,25(OH) D was used as the positive control.
2 3

ACCEPTED MANUSCRIPT
Figure 3. Representative compounds inhibited the proliferation of MCF-7 cells
through VDR. Negative control (siNC) or VDR-specific (siVDR) siRNA-transfected
MCF-7 cells were treated with 19a, 27b, sw-22 and 1,25(OH)
2
D3 16 hours. BrdU
incorporation was determined via a commercial available ELISA kit (Abcam). BrdU
ELISA optic density (OD) at 450 nm was recorded. Dose-activity curves and IC50
value are shown.(A, B, C and D)
2
.3. In Vitro VDR Binding Affinity.
In vitro receptor binding affinity of compounds for VDR was evaluated with
VDR competitor assay and 1,25(OH) D was used as the positive control. The target
2
3
compounds were performed at a concentration of 1 µM and we defined VDR binding
affinity of the 1,25(OH) D as 100%, so the relative VDR binding affinity of testing
2
3
compounds (%) = (mP DMSO - mP Testing Compound) / (mP DMSO - mP 1,25(OH)2D3) ×100%,
and the relative binding affinity are listed in Table 3. Replacing the 2`-OH group with
sulfonyl group (15a) was generally well tolerated, with only slight changes in binding

ACCEPTED MANUSCRIPT
affinity being observed. By modifying the hydrophilic groups in the pyrrol part,
change the chiral of α-position carbon atom to give (15c) resulted in the complete loss
of VDR binding affinity. Similar detrimental effect was observed after its replacement
the methyl group with large steric bulk groups (15d, 15e and 15f) as well as with
hydroxyl contained alkyl groups (15i, 15k and 15l). As similar as SARs of the
antiproliferative activity (section 2.2), compounds with the carboxyl acid and methyl
ester groups at terminal of side chain show weak binding affinity. In contrast, in vitro
VDR binding affinity recovered with the introduction of nitrogen atoms contained
side chain (14n, 14o). In particular, high activity was obtained with the N, N-
diethylpropan-1-amine (14o) derivatives, showing 1.45-fold better VDR binding
affinity than sw-22. Reduction of sulfonyl (19b) as well as replacement the phenyl
adjacent sulfonyl with methyl group (20a) resulted in weakly active analogues.
Interestingly, reduce the sulfonyl and replace the phenyl with methyl group
simultaneously (19a), resulted in maintained binding affinity, which we postulate to
be due to sulfur atom formed interaction with amino acid residues of the VDR LBD.
Substitution at the C-4 position of pyrrole ring lead to an activity maintained
compound 27b.
Table 3. The relative VDR binding affinity of all target compounds at 1 µM.
Relative VDR
Relative VDR
Compd.
Compd.
a
a
binding ability(%)
14
binding ability(%)
43
1
4a
4b
4c
4h
4i
4m
4n
16a
16b
16c
16d
16e
16f
1
31
28
42
73
78
51
5
1
1
1
-
b
1
-
88
28
69
25
41
59
76
72
1
65
80
54
83
-
16g
16h
16i
1
4o
5a
1
1
5b
5c
5d
5e
16j
1
16k
19a
19b
1
14
-
1

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1
5f
5g
5h
-
20a
26a
53
62
1
42
71
23
67
42
-
1
26b
28
1
5i
27a
60
1
5j
27b
82
1
5k
5l
sw-22
55
1
1,25(OH) D
100
2
3
a
Relative VDR binding affinity of testing compounds (%) = (mP DMSO - mP Testing
Compound) / (mP DMSO - mP 1,25(OH)2D3) ×100%.
b
-
means no activity.
Subsequently, we further evaluated the VDR binding affinities (IC ) of
5
0
compounds 19a and 27b with excellent relative VDR binding affinity at 1 µM, sw-22
and 1,25(OH) D were used as control. As shown in Figure 4, compounds 19a (IC =
2
3
50
8
9.1 nM) and 27b (IC50 = 125.4 nM) shown maintained VDR binding affinity
compare with the lead compound sw-22 (IC = 115.3 nM). However, none of these
5
0
compounds showed similar VDR binding affinity with the natural hormone
,25(OH) D (IC = 1.42 nM). We hypothesized that the lower VDR binding affinity
1
2
3
50
of phenyl-pyrrolyl pentane derivatives might result from less hydrogen bonds
interaction with the ligand binding domain of VDR (VDR-LBD) than 1,25(OH) D3.
2

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Figure 4. The VDR ligand binding affinity of representative compounds.
Fluorescence polarization competition assay was performed under equilibrium
conditions in vitro and the competence of the Fluormone VDR Red ligand from the
VDR-LBD by different compounds are shown (n = 3).
2
.4. Transactivation.
3
3
To estimate agonistic properties of representative compounds, a transactivation
assay in HEK293 cells was performed using pGL4.27-SPP×3-Luc reporter plasmid as
the VDRE activated reporter, pRL-TK as the internal reference and to normalize the
expression of luciferase, pENTER-CMV-hVDR was to enhance the expression of
VDR and pENTER-CMV-hRXRα expressing RXRα as the heterodimer partner of
VDR. Representative compounds 19a and 27b were tested, sw-22 and 1,25(OH) D
2
3
were used as positive control. As shown in Figure 5, compounds 19a and 27b showed
concentration-dependent transcriptional activity and acted as potent agonists.
However, none of the compounds activated the reporter gene transcription better than
positive controls 1,25(OH) D .
2
3
Figure 5. Transcriptional activities of compounds were determined. HEK293 cells
were cotransfected with TK-Spp × 3-LUC reporter plasmid, pRL-TK, pENTER-
CMV-hRXRα and pENTER-CMV-hVDR. Eight hours after transfection, test
compounds (19a and 27b) and positive control (sw-22 and 1,25(OH) D ) were added.
2
3
Luciferase activity assay was performed 24 hours later using the Dual-Luciferase
Assay System. Firefly luciferase activity was normalized to the corresponding Renilla

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luciferase activity. Data are shown as means ± SD. *P< 0.05 vs. DMSO. All the
experiments were performed three times.
2
.5. Molecular docking study
Docking studies were carried out to understand the atomic level interaction of
1
9a and 27b with VDR. The binding models of 19a and 27b with VDR-LBD (PDB
ID: 2ZFX) were predicted and the most suitable conformations of the ligands were
selected based on the calculated docking scores by means of the bonding strength. As
shown in Figure 6A, the oxygen atom on the sulfonyl group could form hydrogen
2
5
bond with His393, the same interaction was found in lead compound sw-22, which
support the hypothesis that the sulfonyl group on the side chain could mimic the
function of 2`-OH group. And the heteroatom on amide bond could make interactions
with Ser233 and Ser274. Besides, the sulfur atom at the side chain of 27b formed the
similar hydrogen bond with His393 and His301, and the N atom on amide bond could
form hydrogen bond with Ser233 (Figure 6B).

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Figure 6. The predicted binding model of 19a (A) and 27b (B). VDR-LBD of PDB
reference 2ZFX is applied for molecular docking using Glide 5.5 in Schrödinger 2009.
The most suitable conformations of the ligands were selected based on the calculated
docking scores by means of the bonding strength. (A) Oxygen atom on the sulfonyl
group at the side chain of compound 19a was able to form hydrogen bond with the
His393 of VDR-LBD. On the other side of the structure, the heteroatom on amide
bond could make interactions with Ser233 and Ser274. (B) Sulfur atom at the side
chain of 27b formed the similar hydrogen bond with His393 and His301, and the N
atom on amide bond could form hydrogen bond with Ser233.
2
.6. Cell Cycle Assay and Cell Apoptosis.

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Given the inhibitory effect of compounds 14n, 14o, 15a, 15f, 19a, 19b, 26a, 26b
and 27b on MCF-7 cells, we analyzed changes in cell cycle distribution and apoptosis
in response to these representative compounds. Flow cytometry analysis of MCF-7
cells treated with representative compounds (1 µM) for 24 h revealed that compared
with the control (49.3% of cells in G0/G1 phase), selected compounds induced cell
cycle arrest at G0/G1 phase (Figure 7A). Among them, the percentage of compounds
1
9a and 27b induced cells in G0/G1 phase was 55.3 and 56.0%, respectively (Figure
B). And silence the VDR abolished the cell cycle arrest effect induced by
7
representative compounds, which suggest that 19a and 27b exert their cell cycle arrest
effect through VDR. The results in Figure 8 indicate that 19a and 27b induced
apoptosis levels in MCF-7 cells by VDR.
Figure 7. Effects of representative compounds arrested the cell cycle in Negative
control (siNC) or VDR-specific (siVDR) siRNA-transfected MCF-7 cells. (A) Flow
cytometry analysis of cell cycle populations after treatment with 1 µM compounds for
2
4 h. (B) The fold differences of cell population in the G0/G1 phase of the cell cycle
after treatment of each compound. Data are shown as means ± SD. *P< 0.05 vs.
Control. All the experiments were performed three times.

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Figure 8. Effects of representative compounds induced apoptosis in Negative control
(siNC) or VDR-specific (siVDR) siRNA-transfected MCF-7 cells. (A) Flow
cytometry analysis of apoptosis. Cells were exposed to 1 µM compounds for 24 h.
Cells were collected and stained with Annexin V-fluorescein isothiocyanate (FITC)
and PI. (B) The fold differences of apoptotic quantification after exposure to each
compound for 24 hours. Data are shown as means ± SD. *P< 0.05 vs. Control. All the
experiments were performed three times.
There were many of anticancer mechanisms of VDR modulators, including
cyclin-dependent kinase (CDK) inhibitors p21, p27 stimulation and pro-apoptotic
7
gene Bax stimulation. To determine if representative compounds stimulate the
expression of p21, p27 and Bax, MCF-7 cells were treated with 14n, 14o, 15a, 15f,
1
9a, 19b, 26a, 26b and 27b at the concentrations of 1 µM for 24 h, and the expression
of p21, p27 and Bax were examined. As shown in Figure S1, compounds 19a and
2
2
7b significantly increased the expression of p21, p27 and Bax.
.7. Physicochemical and Pharmacokinetic properties of Representative
Compounds
We evaluated the in vitro metabolic stability (rat microsomal) and water
solubility of target compounds with significant anti-tumor activity and VDR binding

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affinity (Table 4). These data provide information to further guide lead optimization,
with the ultimate goal of testing compounds in animals and identification of a
preclinical candidate. We are targeting solubility of greater than 50 µg/mL (in pH 7.4
buffer), and rat microsomal stability of approximately 60 min half life for compounds
3
0
to be further evaluated in vivo. As expected, the compound containing sulfonyl
group (15a) versus compound without sulfonyl group (sw-22) had better stability (t½
=
43.76 vs. 25.76 min) in rat microsomes. By modifying the hydrophilic groups in the
pyrrol part, introduce N, N-diethylpropan-1-amine side chain result in compound 14o
with desirable metabolic stability (t½ = 62.43 min). And compounds 19a and 27b
showed desirable metabolic stability (t½ = 57.26 and 57.23 min) either. In addition,
sulfonyl contained compound (15a) showed a 21-fold better solubility than the
compound without sulfonyl (sw-22) in solubility assay (35.7 vs. 1.7 µg/mL).
Furthermore, compounds 14n, 14o, 19a, 26a and 27b showed excellent solubility
with 65.0, 117.3, 74.6, 198.2 and 85.3 µg/mL, respectively.
a
Table 4. In vitro metabolic stability and water solubility of selected compounds
Solubility
Rat Microsome
Stability t½ (min)
Compd.
4n
(µg/mL)
1
65.0
34.91
62.43
43.76
46.32
57.26
53.72
48.13
71.44
59.23
25.76
1
1
4o
117.3
35.7
27.8
74.6
25.6
198.2
0.6
5a
1
5f
9a
9b
6a
1
1
2
2
6b
7b
2
85.3
1.7
sw-22
a
See Experimental Section for assay descriptions
Consequently, we profiled the pharmacokinetic properties of our two most potent
analogues, 19a and 27b, which showed desirable metabolic stability and solubility, as
well as excellent anti-tumor activity. As control, our lead compound sw-22 was
selected, and the pharmacokinetic parameters were shown in Table 5. Introduction of

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the sulfonyl group led to a modification of the PK profile and we clearly saw an
improvement in terms of half-lives and bioavailability. Compound 19a showed with
bioavailability of 43.2% and t½ value of 10.75 hours after intraperitoneal
administration. And compound 27b displayed with bioavailability of 40.3% and t½
value of 7.65 hours after intraperitoneal administration. Both of 19a and 27b
possessed better pharmacokinetic properties than lead compound sw-22, whose
bioavailability was 32.5% and t½ value was 3.47 hours after intraperitoneal
administration. These results were in line with the trend we preconceived that
compounds contained the sulfonyl group will possess better pharmacokinetic
properties. In our another work, we improved pharmacokinetic properties with
another approach. We modified the hydroxyl group on the side chain from secondary
alcohol to tertiary alcohol, the steric hinerance around hydroxyl group was enhanced,
which could delay the metabolish of the hydroxyl group on the side chain and lead to
3
1
a longer t½ value.
a
Table 5. Pharmacokinetic Parameters of compounds 19a, 27b and sw-22 in rats
Parameters
sw-22
19a
27b
i.v.
i.p.
i.v.
i.p.
i.v.
i.p.
5
mg/kg
20 mg/kg
6.04±0.23
3.47±0.18
5.5±0.24
3.31±0.13
32.47%
5 mg/kg
11.79±0.34
4.72±0.21
8.08±0.3
0.42±0.01
-
20 mg/kg
20.37±3.54
10.75±0.07
15.67±0.02
0.99±0.17
43.19%
5 mg/kg
7.47±0.43
6.6±0.25
9.72±0.27
0.67±0.04
-
20 mg/kg
12.04±0.19
7.65±0.06
11.87±0.11
1.66±0.02
40.29%
AUC(mg/L*h)
t½(h)
4.65±0.07
2.89±0.12
5.54±0.13
1.08±0.02
-
MRT(h)
CL(L/h)
F
a
SD rats (male, 3 animals per group) weighted 180−220 g were used for the study (n =
3
). Data were presented as Mean ± SD.
2
.8. In Vivo Anticancer Activity Assay
On the basis of their potent antiproliferative effects, desirable physicochemical
and pharmacokinetic properties, compounds 19a and 27b were selected for in vivo
studies. Breast tumor mouse model was generated by orthotopic transplantation of
human MCF-7 cells in athymic nude mice. Our lead compound sw-22 was selected as
positive control. In addition, the natural VDR modulator 1,25(OH) D , which has
2
3
been used in a number of animal models of cancer and clinical trials for cancer

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5
, 7
treatment (prostate cancer and breast cancer) , was selected as positive control either.
As shown in Figure 9A and B, 19a and 27b showed better antitumor efficacy
compared with sw-22 or 1,25(OH) D . In addition, treated with compounds 19a and
2
3
2
7b did not affect mouse body weight (Figure 9C), which indicated that the 19a and
7b have no apparent toxicity to mice during the period of cancer treatment.
2
We further determined the expression of p21, p27 and Bax in vivo by
immunohistochemistry assay (Figure 9D). Compared with control and 1,25(OH) D ,
2
3
there was a significantly increased expression of p21, p27 and Bax in sections of the
MCF-7 orthotopic tumors after they were treated with 19a or 27b. The results
revealed that treatment with 19a or 27b dramatically increased the expression levels
of the proapoptotic p21, p27 and Bax in MCF-7 cells, suggesting that 19a or 27b
inhibited the tumor growth by inducing cell-cycle arrest.
Figure 9. Compounds 19a and 27b exert antitumor efficiency in vivo. (A) Differences
in tumor weight in mice treated with vehicle, 19a, 27b, sw-22 and 1,25(OH) D . *P <
2
3

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0
.05 compared to the placebo group. (B) Differences in tumor size in mice exposed to
vehicle, 19a, 27b, sw-22 and 1,25(OH) D . (C) Changes in body weight of mice
2
3
treated with vehicle, 19a, 27b, sw-22 and 1,25(OH) D . (D) Expression of p21, p27
2
3
and Bax in the tumor sections of MCF-7-bearing mice was examined by
immunohistochemistry (×200).
2
.9. In Vivo Calcemic Activity Assay.
The level of serum calcium was measured to evaluate the safety profile of
compounds 19a and 27b. 1,25(OH) D and sw-22 were selected as the positive
2
3
controls and vehicle as the negative control. As shown in Figure 10, the serum
calcium concentration of 1,25(OH) D group was 0.31 µmol/mL before injection (day
2
3
7
2
), 0.60 µmol/mL ongoing injection (day 17) and 0.61 µmol/mL after injection (day
7), indicating significant risk of hypercalcemia was induced by 1,25(OH) D .
2
3
However, there was no significant change on serum calcium in mice when treated
with compounds 19a and 27b. Therefore, compounds 19a and 27b had no
hypercalcemia side effect in vivo at therapeutic doses.
Figure 10. (A) In vivo hypercalcemic effects of compounds 19a, 27b, sw-22 and
1
,25(OH) D in the MCF-7 tumor-bearing mice The venous blood were withdrawn
2 3 .
before injection (day 7), ongoing injection ( day 17) and after injection (day 27). The
total calcium ion concentration was determined by calcium assay kit. Data are shown
as means ± SD. *P< 0.05 vs. Control.

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3
. Conclusions
On the basis of the potent non
-secosteroidal VDR modulator reported by our
group previously, we investigated the structure-activity and structure-property
relationships around the 2`-hydroxyl group to improve the physicochemical properties,
pharmacokinetic properties and anti-tumor activities. In this study, a new series of
sulfonyl-containing phenyl-pyrrolyl pentane analogues were synthesized. Screening
assays of these derivatives identified compounds 19a and 27b as potential anti-tumor
agents. Novel compounds 19a and 27b, feature sulfonyl groups on the side chain, are
particularly attractive for future studies because of the following features: (i)
Inhibition of cell proliferation with a low IC50 via the arrest of cell cycle and
induction of apoptosis by stimulating the expression of p21, p27 and Bax. (ii) The
desirable drug-like properties including metabolic stability, water solubility and
pharmacokinetic profiles that are crucial for compounds to exert activity in vivo. (iii)
The excellent in vivo anti-tumor activity without hypercalcemia side effect. In
summary, the favorable physicochemical properties, pharmacokinetic properties and
anti-tumor activity of 19a and 27b highlight their potential therapeutic applications in
cancer treatment.
4
4
. Experiment section
.1. 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
1
(
HRMS) were recorded on QSTAR XL Hybrid MS/MS mass spectrometer. H NMR
1
3
and C NMR were recorded employing Bruker AV-300 or AV-500 instruments using
CDCl . Chemical shifts were given as δ (ppm) units relative to the internal standard
3

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tetramethylsilane (TMS). Column chromatography separations were progressed on
silica gel (200-300 mesh).
4
.2. General procedure 1 - synthesis of compounds 14a-o
To a solution of compound 13 (0.15 g, 0.32 mmol) in dichloromethane (50 mL),
-(ethyliminomethylideneamino)-N,N-dimethylpropan-1-amine,hydrochloride (74.38
3
mg, 0.48 mmol), 1-Hydroxybenzotriazole (64.74 mg, 0.48 mmol) and triethylamine
o
(
48.49 mg, 0.48 mmol) was added at 0 C. After stirring for 0.5 h, appropriate amine
0.48 mmol) was added. The reaction mixture was stirred at room temperature for 12
(
h and then H O (100 mL) was added. The organic phase were washed with brine and
2
dried over anhydrous Na SO , filtered and concentrated. The residue was purified by
2
4
silica gel column chromatography eluting with appropriate mixture as indicated in
each case.
4
.2.1. methyl (1-ethyl-5-(3-(3-methyl-4-((phenylsulfonyl)methoxy)phenyl)pentan-
-yl)-1H-pyrrole-2-carbonyl)-L-alaninate (14a)
3
1
Petroleum ether/ethyl acetate (25/1). Colorless oil. 87% yield. H NMR (300
MHz, CDCl ) δ (ppm) 7.98 (d, J = 7.2 Hz, 2H), 7.67 (t, J = 7.3 Hz, 1H), 7.55 (t, J =
3
7
.3 Hz, 2H), 6.84 (d, J = 8.5 Hz, 1H), 6.81 (s, 1H), 6.67 (d, J = 8.5 Hz, 1H), 6.62 (d, J
4.0 Hz, 1H), 6.16 (d, J = 4.0 Hz, 1H), 5.03 (s, 2H), 4.66-4.59 (m, 1H), 3.85-3.68 (m,
H), 3.75 (s, 3H), 2.05 (s, 3H), 2.01-1.91 (m, 4H), 1.45 (d, J = 7.1 Hz, 3H), 0.79 (t, J
=
2
13
=
6.9 Hz, 3H), 0.56 (t, J = 7.3 Hz, 6H). C NMR (75 MHz, CDCl ) δ (ppm) 173.40,
3
1
61.14, 152.88, 143.09, 140.39, 136.63, 133.81, 129.55, 128.70, 128.60, 126.35,
24.93, 124.49, 111.53, 111.30, 107.82, 82.54, 51.89, 47.38, 45.96, 40.33, 27.95,
1
+
1
8.11, 15.88, 15.51, 7.65. ESI-HRMS calcd for C H N O S [M+Na] 577.2451,
30
38
2
6
found 577.2355.
4
3
.2.2. methyl (1-ethyl-5-(3-(3-methyl-4-((phenylsulfonyl)methoxy)phenyl)pentan-
-yl)-1H-pyrrole-2-carbonyl)glycinate (14b)
1
Petroleum ether/ethyl acetate (30/1). Colorless oil. 79% yield. H NMR (300
MHz, CDCl ) δ (ppm) 7.98 (d, J = 7.2 Hz, 2H), 7.67 (t, J = 7.3 Hz, 1H), 7.56 (t, J =
3
7
.3 Hz, 2H), 6.86-6.81 (m, 2H), 6.68 (d, J = 8.5 Hz, 1H), 6.63 (d, J = 4.0 Hz, 1H),

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6
2
7
1
4
.16 (d, J = 4.0 Hz, 1H), 5.03 (s, 2H), 4.11 (d, J = 4.8 Hz, 2H), 3.80 (q, J = 7.0 Hz,
H), 3.76 (s, 3H), 2.06 (s, 3H), 2.04-1.89 (m, 4H), 0.81 (t, J = 7.0 Hz, 3H), 0.57 (t, J =
1
3
.3 Hz, 6H). C NMR (75 MHz, CDCl ) δ (ppm) 170.35, 152.88, 143.24, 140.37,
3
36.63, 133.82, 129.55, 128.71, 128.60, 126.36, 124.93, 111.53, 107.92, 82.54, 51.80,
5.97, 40.64, 40.37, 27.95, 15.88, 15.53, 7.66. ESI-HRMS calcd for C H N O S
2
9
36
2
6
+
[
M+Na] 563.2294, found 563.2195.
4
.2.3. methyl (1-ethyl-5-(3-(3-methyl-4-((phenylsulfonyl)methoxy)phenyl)pentan-
-yl)-1H-pyrrole-2-carbonyl)-D-alaninate (14c)
3
1
Petroleum ether/ethyl acetate (30/1). Colorless oil. 83% yield. H NMR (300
MHz, CDCl ) δ (ppm) 7.98 (d, J = 7.2 Hz, 2H), 7.67 (tt, J = 7.3 Hz, J = 1.3 Hz, 1H),
3
7
.55 (t, J = 7.3 Hz, 2H), 6.85-6.81 (m, 2H), 6.67 (d, J = 8.5 Hz, 1H), 6.62 (d, J = 4.0
Hz, 1H), 6.15 (d, J = 4.0 Hz, 1H), 5.03 (s, 2H), 4.69-4.59 (m, 1H), 3.85-3.68 (m, 2H),
.75 (s, 3H), 2.06 (s, 3H), 2.03-1.91 (m, 4H), 1.45 (d, J = 7.2 Hz, 3H), 0.79 (t, J = 6.9
3
13
Hz, 3H), 0.56 (t, J = 7.3 Hz, 6H). C NMR (75 MHz, CDCl ) δ (ppm) 173.83, 161.58,
3
1
1
1
5
53.30, 143.52, 140.82, 137.06, 134.24, 129.98, 129.13, 129.03, 126.78, 125.36,
24.92, 111.97, 111.74, 108.25, 82.97, 52.31, 47.81, 46.39, 40.76, 29.62, 28.39, 18.53,
+
6.30, 15.94, 8.09. ESI-HRMS calcd for C H N O S [M+Na] 577.2451, found
30
38
2
6
77.2363.
4
.2.4.
methyl
3-(1-ethyl-5-(3-(3-methyl-4-
((phenylsulfonyl)methoxy)phenyl)pentan-3-yl)-1H-pyrrole-2-
carboxamido)propanoate (14h)
1
Petroleum ether/ethyl acetate (30/1). Colorless oil. 69% yield. H NMR (300
MHz, CDCl ) δ (ppm) 7.97 (d, J = 7.3 Hz, 2H), 7.67 (tt, J = 7.3 Hz, J = 1.3 Hz, 1H),
3
7
.55 (t, J = 7.3 Hz, 2H), 6.84-6.81 (m, 2H), 6.67 (d, J = 8.7 Hz, 1H), 6.51 (d, J = 4.0
Hz, 1H), 6.13 (d, J = 4.0 Hz, 1H), 5.03 (s, 2H), 3.80 (q, J = 6.9 Hz, 2H), 3.69 (s, 3H),
3
0
1
1
3
5
.59 (d, J = 6.0 Hz, 2H), 2.58 (t, J = 6.0 Hz, 2H), 2.05 (s, 3H), 2.03-1.90 (m, 4H),
1
3
.79 (t, J = 6.9 Hz, 3H), 0.55 (t, J = 7.3 Hz, 6H). C NMR (75 MHz, CDCl ) δ (ppm)
3
73.10, 162.08, 153.29, 143.20, 140.87, 137.06, 134.23, 129.96, 129.12, 129.02,
26.75, 125.37, 111.96, 111.21, 108.12, 82.97, 51.69, 46.37, 40.74, 34.59, 34.03,
+
0.17, 28.37, 16.29, 15.99, 8.08, 0.94. ESI-HRMS calcd for C H N O S [M+Na]
3
0
38
2
6
77.2451, found 577.2356.

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4
.2.5.
dimethyl
(1-ethyl-5-(3-(3-methyl-4-
((phenylsulfonyl)methoxy)phenyl)pentan-3-yl)-1H-pyrrole-2-carbonyl)-L-
aspartate (14i)
1
Petroleum ether/ethyl acetate (20/1). Colorless oil. 72% yield. H NMR (300
MHz, CDCl ) δ (ppm) 7.97 (d, J = 7.3 Hz, 2H), 7.67 (tt, J = 7.3 Hz, J = 1.3 Hz, 1H),
3
7
.55 (tt, J = 7.3 Hz, J = 1.3 Hz, 2H), 6.84-6.81 (m, 3H), 6.64 (d, J = 4.0 Hz, 1H), 6.16
d, J = 4.0 Hz, 1H), 5.03 (s, 2H), 4.96-4.90 (m, 1H), 3.83-3.77 (m, 2H), 3.75 (s, 3H),
.69 (s, 3H), 3.10-3.03 (m, 1H), 2.91-2.84 (m, 1H), 2.06 (s, 3H), 2.04-1.91 (m, 4H),
(
3
0
1
1
4
1
3
.79 (t, J = 6.9 Hz, 3H), 0.56 (t, J = 7.3 Hz, 6H). C NMR (75 MHz, CDCl ) δ (ppm)
3
71.57, 171.54, 161.55, 153.32, 143.80, 140.77, 137.07, 134.24, 129.97, 129.13,
29.02, 126.79, 125.37, 124.65, 112.21, 111.98, 108.38, 82.96, 52.67, 51.94, 48.26,
6.40, 40.81, 36.31, 28.38, 16.30, 15.92, 8.08. ESI-HRMS calcd for C H N O S
3
2
40
2
8
+
[
M+Na] 635.2505, found 635.2414.
4
.2.6.
(phenylsulfonyl)methoxy)phenyl)pentan-3-yl)-1H-pyrrole-2-
carboxamido)ethyl)carbamate (14m)
tert-butyl
(2-(1-ethyl-5-(3-(3-methyl-4-
(
1
Petroleum ether/ethyl acetate (20/1). Colorless oil. 76% yield. H NMR (300
MHz, CDCl ) δ (ppm) 7.97 (d, J = 7.2 Hz, 2H), 7.66 (tt, J = 7.4 Hz, J = 1.3 Hz, 1H),
3
7
.55 (t, J = 7.4 Hz, 2H), 6.85-6.80 (m, 2H),6.67 (d, J = 8.5 Hz, 1H), 6.57 (d, J = 4.0
Hz, 1H), 6.14 (d, J = 4.0 Hz, 1H), 5.03 (s, 2H), 3.82 (q, J = 7.1 Hz, 2H), 3.43 (t, J =
5
0
1
1
1
6
.3 Hz, 2H), 3.30 (t, J = 5.3 Hz, 2H), 2.05 (s, 3H), 2.01-1.90 (m, 4H), 1.41 (s, 9H),
1
3
.80 (t, J = 7.0 Hz, 3H), 0.56 (t, J = 7.1 Hz, 6H). C NMR (75 MHz, CDCl ) δ (ppm)
3
62.35, 152.85, 142.73, 140.49, 136.62, 133.80, 129.55, 128.69, 128.59, 126.31,
24.93, 124.75, 111.51, 111.03, 107.78, 82.54, 45.94, 40.32, 40.15, 39.82, 27.89,
+
5.86, 15.60, 7.65. ESI-HRMS calcd for C H N O S [M+Na] 634.3029, found
33
45
3
6
34.2902.
4
.2.7.
N-(2-aminoethyl)-1-ethyl-5-(3-(3-methyl-4-
((phenylsulfonyl)methoxy)phenyl)pentan-3-yl)-1H-pyrrole-2-carboxamide (14n)
1
Dichloromethane / methanol (15/1). Colorless oil. 41% yield. H NMR (300 MHz,
CDCl ) δ (ppm) 7.96 (d, J = 7.2 Hz, 2H), 7.66 (tt, J = 7.4 Hz, J = 1.3 Hz, 1H), 7.55 (t,
3
J = 7.4 Hz, 2H), 6.83-6.80 (m, 2H), 6.67-6.66 (m, 2H), 6.13 (d, J = 4.0 Hz, 1H), 5.03