Novel nonsecosteroidal VDR agonists with phenyl-pyrrolyl pentane skeleton

Article abstract of DOI:10.1016/j.ejmech.2013.09.015

In order to find the vitamin D receptor (VDR) ligand whose VDR agonistic activity is separated from the calcemic activity sufficiently, novel nonsecosteroidal analogs with phenyl-pyrrolyl pentane skeleton were synthesized and evaluated for the VDR binding

Full text of DOI:10.1016/j.ejmech.2013.09.015

European Journal of Medicinal Chemistry 69 (2013) 768e778
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Novel nonsecosteroidal VDR agonists with phenyl-pyrrolyl pentane
skeleton
*
Wei Shen, Jingwei Xue, Zekai Zhao, Can Zhang
State Key Laboratory of Natural Medicines, Center of Drug Discovery, China Pharmaceutical University, Nanjing 210009, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
In order to find the vitamin D receptor (VDR) ligand whose VDR agonistic activity is separated from the
calcemic activity sufficiently, novel nonsecosteroidal analogs with phenylepyrrolyl pentane skeleton
were synthesized and evaluated for the VDR binding affinity, antiproliferative activity in vitro and serum
calcium raising ability in vivo (tacalcitol used as control). Among them, several compounds showed
varying degrees of VDR agonistic and growth inhibition activities of the tested cell lines. The most
Received 11 May 2013
Received in revised form
31 August 2013
Accepted 3 September 2013
Available online 18 September 2013
effective compound 2g (EC50: 1.06 nM) exhibited stronger VDR agonistic activity than tacalcitol (EC50
7.05 nM), inhibited the proliferations of HaCaT and MCF-7 cells with IC50 of 2.06 M and 0.307
(tacalcitol: 2.07 M and 0.057 M) and showed no significant effect on serum calcium.
Ó 2013 Elsevier Masson SAS. All rights reserved.
:
m
mM
Keywords:
VDR
Phenyl-pyrrolyl pentane skeleton
VDR agonistic activity
Calcemic activity
Docking study
m
m
1. Introduction
But their pharmacological activities were separated from the cal-
cemic activity insufficiently, so for the consideration of long-term
therapy (especially oral) of diseases like cancers and severe psori-
asis, the risk of hypercalcemia still existed [10]. Therefore, there is
an unmet clinical need for exploring VDR agonists whose calcemic
activity was effectively reduced.
In 1999, a series of nonsecosteroidal ligands of VDR (such as
LG190155, Fig. 2) which mimic various activities of 1,25(OH)2D3
in vitro were identified [11]. Although their VDR agonistic activities
were weaker than 1,25(OH)2D3, they showed no calcemic potential
in vivo. These nonsecosteroidal VDR agonists provided opportu-
nities for developing novel, oral medications to treat cancer, leu-
kemia and psoriasis. In addition, the synthetic accessibility of these
bis-phenyl derivatives made them attractive targets for drug
discovery.
In order to find potent VDR agonists which rival 1,25(OH)2D3 in
biological activities but exhibit no calcemic activity like LG190155,
we designed a series of phenyl-pyrrolyl pentane derivatives, using
LG190155 as the leading compound. In this work, we found the
potential of constructing the phenyl-pyrrolyl pentane skeleton, and
introducing side chains which have abilities to form the hydrogen
bond for the VDR agonists. First, we changed one benzene ring of
the leading compound into the pyrrole ring to create a novel scaf-
fold. Then we retained the 2-oxo-3,3-dimethylbutoxy chain at the
4-position of the benzene ring and introduced different amino acid
esters to the 2-position of the pyrrole ring. At last, we adjusted the
polarities of two side chains by reduction or hydrolysis reaction.
1a,25-Dihydroxyvitamin D3 (1,25(OH)2D3, Fig. 1) is the biolog-
ically active form of vitamin D in human, which plays an important
role in calcium homeostasis [1,2], cell differentiation and prolifer-
ation as well as immunomodulation [3,4]. The physiological effects
of 1,25(OH)2D3 are exerted by binding to vitamin D receptor (VDR)
which belongs to the nuclear receptor superfamily [5]. Once
binding with the ligand, VDR becomes active and forms a complex
with co-factors, which consequently binds to vitamin D responsive
element (VDRE) in the promoter moiety of the target genes leading
to transactivation [6].
The secosteroidal VDR ligands (Fig. 1) have showed their ther-
apeutic abilities in several human diseases such as psoriasis, oste-
oporosis and secondary hyperparathyroidism [7,8]. They have also
exhibited efficacy in various cells and animal models of autoim-
mune diseases like arthritis, multiple sclerosis and inflammatory
bowel disease as well as cancers of prostate, colon and breast [9].
However, the widely use of these classic VDR ligands as drugs is
limited by their associated toxicity, namely hypercalcemia. Among
these ligands, some decreased calcemic activity by structure
modification, such as tacalcitol and calcipotriol (Fig. 1), which have
been approved for topical treatments of mild to moderate psoriasis.
* Corresponding author. Tel./fax: þ86 25 83271171.
E-mail addresses: zhangcan@cpu.edu.cn, canzhang65@yahoo.com.cn (C. Zhang).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.09.015

W. Shen et al. / European Journal of Medicinal Chemistry 69 (2013) 768e778
769
Fig. 1. The structures of secosteroidal VDR ligands.
Among the designed analogs, Compounds 2d, 2e, 2g and 2h
demonstrated promising VDR binding affinities and antiprolifera-
tion activities against keratinocyte and tumor cells. Especially
compound 2g showed better VDR agonistic activity and the closely
antiproliferation activities, compared with tacalcitol. Meanwhile,
the calcium raising ability of 2g was almost lost. Docking study was
also carried out to understand the structureeactivity relationships
of these novel VDR agonists.
1-postion of pyrrole ring of 5 afforded 6. After the deprotection of 6,
in the presence of K2CO3, the phenolic hydroxyl group was
substituted by 1-chloropinacolone in the boiling acetone, and 8 was
formed. Compound 8 was hydrolyzed by potassium hydroxide, and
the exposed carboxyl group was acylated with different amino acid
methyl ester hydrochlorides under the condition of EDCI/HOBT to
form compounds 1ae1h. In methanol, simultaneous reduction of
both carbonyl group and methyl ester group of 1ae1h was given by
sodium borohydride, and compounds 2ae2h could be formed.
Hydrolyzed by lithium hydroxide, the methyl ester group of 1ae1h
produced compounds 3ae3h. At last, reduced by sodium borohy-
dride towards carbonyl group of 3ae3h, compounds 4ae4h were
synthesized in methanol (Fig. 2).
2. Results and discussion
2.1. Chemistry
The synthetic route was shown in Scheme 1. Compound 1,
esterificated by methanol, produced 2. Then, protection of phenolic
hydroxyl group of 2 with benzyl bromide created 3, which, was
given a further nucleophilic attack from ethylmagnesium bromide
and 4 was generated. Coupling with ethylpyrrole-2-carboxylate,
compound 4 produced the key intermediate 5 in the presence of
boron fluoride ethyl ether. With different alkyl iodides, alkylating at
2.2. Biological activities
2.2.1. In vitro HL-60 differentiation-inducing activity (VDR binding
affinity)
It was examined that the HL-60 differentiation-inducing activity
and the VDR binding affinity were relative, so the VDR binding
Fig. 2. The designed VDR agonists.

770
W. Shen et al. / European Journal of Medicinal Chemistry 69 (2013) 768e778
Table 1
Structures and VDR binding affinities of the designed compounds.
Compd R1 R2
VDR binding affinitya
b
EC50
(mM)
1a
1b
1c
1d
1e
Me
Me
Me
Me
Et
Et
Et
Et
Me
Me
Me
Me
Et
eCH2COOCH3
0.023
0.79
dc
eCH2CH2COOCH3
(S)-CH(CH3)COOCH3
(S)-CH(CH2COOCH3)COOCH3
eCH2COOCH3
eCH2CH2COOCH3
(S)-CH(CH3)COOCH3
(S)-CH(CH2COOCH3)COOCH3
eCH2CH2OH
dc
1.83
dc
1f
1g
1h
2a
2b
2c
2d
2e
2f
dc
dc
dc
eCH2CH2CH2OH
0.15
0.57
0.20
5.64
dc
(S)-CH(CH3)CH2OH
(S)-CH(CH2CH2OH)CH2OH
eCH2CH2OH
Et
eCH2CH2CH2OH
2g
2h
3a
3b
3c
3d
3e
3f
3g
3h
4a
4b
4c
Et
Et
(S)-CH(CH3)CH2OH
(S)-CH(CH2CH2OH)CH2OH
eCH2COOH
1.06E-03
0.11
dc
Me
Me
Me
Me
Et
Et
Et
Et
Me
Me
Me
Me
Et
eCH2CH2COOH
dc
(S)-CH(CH3)COOH
(S)-CH(CH2COOH)COOH
eCH2COOH
2.63
0.41
dc
eCH2CH2COOH
dc
(S)-CH(CH3)COOH
(S)-CH(CH2COOH)COOH
eCH2COOH
dc
dc
4.95
1.6
eCH2CH2COOH
(S)-CH(CH3)COOH
(S)-CH(CH2COOH)COOH
eCH2COOH
dc
4d
4e
dc
dc
4f
Et
Et
Et
eCH2CH2COOH
(S)-CH(CH3)COOH
(S)-CH(CH2COOH)COOH
33.22
4.07E-03
dc
4g
4h
Tacalcitol
Scheme 1. Reagents and reaction conditions: (a) conc. H2SO4, CH3OH, 70 ꢀC, over-
night; (b) BnBr, acetone, K2CO3, KI, reflux, 6 h; (c) C2H5MgBr, Et2O, 25 ꢀC, 2 h; (d)
Ethylpyrrole-2-carboxylate, BF3$Et2O, 25 ꢀC, 1 h; (e) alkyl iodide, NaH, DMF, 0 ꢀCe
25 ꢀC, 2 h; (f) Pd/C, HCOONH4, CH3OH, 25 ꢀC, overnight; (g) 1-chloropinacolone,
acetone, K2CO3, KI, reflux, 12 h; (h) KOH, H2O, C2H5OH, 70 ꢀC, overnight; (i) Amino acid
7.05E-03
a
VDR binding affinity was measured as HL-60 differentiation-inducing activity.
Data shown are from triplicate experiments.
Inactive at >50 mM.
b
c
methyl ester hydrochloride, EDCI, HOBt, Et3N, DMF,
0
ꢀCe25 ꢀC, overnight; (j)
LiOH$2H2O, THF, H2O, 25 ꢀC, overnight; (k) NaBH4, CH3OH, 0 ꢀCe25 ꢀC, 2 he6 h.
showed in compounds of 1ae1h and 3ae3h could be explained by
the weak hydrogen-bonding interactions between the compounds
and amino acid residues of VDR when there were no conjunct hy-
droxyl groups or carboxyl groups in two side chains.
affinity was measured in terms of the activity of differentiation of
human promyelocytic leukemia cell line (HL-60) into macrophages,
as had usually been done [12,13]. Tacalcitol was used as control for
its strong VDR agonistic activity as 1,25(OH)2D3 but weaker in-
duction of hypercalcemia [14]. All the designed compounds were
tested for the effect on the differentiation of HL-60 cells, as shown
in Table 1. Among them, six compounds of 2ae2h embodied
2.2.2. In vitro cell cytotoxicity assay
Cell cytotoxicity assay of the compounds exhibiting VDR binding
affinity was evaluated against HaCaT and MCF-7 cells, using tacal-
citol as the positive control. As shown in Table 2, compound 2g
which displayed the strongest VDR agonistic activity exhibited
good antiproliferative activities against HaCaT and MCF-7 cells,
varying degrees of VDR agonistic activities (EC50: 5.64
mMe
1.06 nM), with two side chains containing two or three hydroxyl
groups. In particular, compound 2g revealed the best VDR agonistic
activity with EC50 at 1.06 nM, while the EC50 of tacalcitol was
7.05 nM. Four compounds of 4ae4h revealed the VDR agonistic
with IC50 at 2.06 and 0.32
revealed the moderate VDR binding affinity though, showed the
best antiproliferative effects, with each IC50 at 0.19 M and 7.83 nM
mM respectively. Compound 2d only
activities at the EC50 from 32.22
mM to 4.07 nM, and among them,
m
compound 4g showed the best differentiation-inducing activity
with EC50 at 4.07 nM. There were also three compounds belonged
to 1ae1h and two compounds belonged to 3ae3h showed mod-
erate VDR agonistic activities whose structures had no hydroxyl
against HaCaT and MCF-7 cells, and this result indicated that it may
work though the multiple molecular mechanisms.
Compounds 2d, 2e, 2g and 2h demonstrated favorable anti-
proliferative activities against both two cell lines. It was found that
all the side chains of these four compounds have been substituted
by hydroxyl groups. In this regard, it could be proved that the
antiproliferative activity of the designed compounds is positively
correlative with VDR binding affinity, and the hydroxyl groups in
the side chains might be the pharmacophores of the phenyl-
pyrrolyl pentane derivatives.
group or carboxyl group in the side chains. (EC50: 2.62 mMe23 nM).
According to the results, 75% compounds of 2ae2h had the
acceptability to an excellent VDR agonistic activities. It may due to
the hydroxyl groups on the two side chains are able to mimic the
function of hydroxyl groups in 1,25(OH)2D3 [6] and form hydrogen
bonds with amino acid residues of VDR. Although compound 4g
showed stronger agonistic activity than tacalcitol did, half com-
pounds of 4ae4h showed no significant agonistic activity, which
suggested introducing carboxyl group into the side chain could
reduce the VDR binding affinities of the compounds. It is presumed
that the reduction of the binding affinity could be attributed to the
increased polarity of these compounds. The least agonistic activity
2.2.3. In vivo calcemic activity assay
Calcemic activity assay in vivo was measured to testify the effect
of the designed nonsecosteroidal analogs on inducing hypercalce-
mic. The two representative compounds, 2g and 2d, were chosen
for the test. The results were shown in Fig. 3.

W. Shen et al. / European Journal of Medicinal Chemistry 69 (2013) 768e778
771
Table 2
group, which makes the near-end of the side chains closer to some
amino acid residues of VDR LBD to form extra hydrogen bonds.
When the structure of the VDR LBD-2g complex overlapped with
the VDR LBD-1,25(OH)2D3 complex, it could be found that, both 2g
and 1,25(OH)2D3 were embed in the same position of the binding
pocket, as shown in Fig. 5. It was reported that the 25-OH group of
1,25(OH)2D3 could interact with His301 and His 393, and the 1-OH
group could interact with Ser233 and Arg274 [15]. In comparison,
the 20-OH group of 2g shared the same amino acid residue His 393
with 25-OH of 1,25(OH)2D3, and the 4-OH group of 2g played the
same role as 1-OH of 1,25(OH)2D3. No interactions with Tyr139 and
Ser274 was observed in 2g, while the 3-OH of 1,25(OH)2D3 formed
hydrogen bonds with them was reported [15]. All of these demon-
strated thatcompound2gworkedsimilarlyastheendogenousligand.
Cellular antiproliferative activities of the selected compounds.
a
a
Compd Cell inhibition IC50
(m
M)
Compd
Cell inhibition IC50 (mM)
HaCaTc
MCF-7
HaCaTc
MCF-7
1a
1b
1e
2b
2c
2d
2e
2g
db
db
2h
3c
3d
4a
4b
4f
1.25
db
db
0.30
db
28.89
db
db
db
db
0.16
db
16.35
db
9.81
db
15.72
0.19
2.08
2.06
db
db
7.83 ꢁ 10ꢂ3
db
1.81
0.32
4g
db
0.18
0.057
Tacalcitol 2.07
a
Data shown are from triplicate experiments.
Inactive at >50
HaCaT cell is an immortal human keratinocyte line commonly used as a model
b
c
mM.
system for vitamin D3 metabolism in human skin.
3. Conclusions
In summary, a series of novel phenyl-pyrrolyl pentane derivatives
weresynthesized and firstreported. TheirVDR bindingaffinities were
measured as the HL-60 differentiation-inducing activities in vitro.
According to the results, the compounds containing groups which
have abilities to form the hydrogen bond in the side chains exhibited
good even excellent activities. Introducing hydroxyl groups to both
side chains seemed to be the best choice to achieve the strongest
activity. The resultsof cellcytotoxicityassay invitro againstHaCaTand
MCF-7 cells demonstrated the conclusion indirectly as well. Com-
pound 2g revealed a remarkable VDR agonistic activity which even
better than tacalcitol, with the EC50 value at 1.06 nM. Meanwhile, it
had little effect on serum calcium. Simulated by the docking study, a
summary of structureeactivity relationships could be reached.
Herein, these findings are suggesting the potential of constructing the
hydrophobic phenyl-pyrrolyl pantene skeleton, and introducing side
chains, about five atoms long, which are substituted by hydroxyl
groups at the far-end for the vitamin D receptor agonists with
extremely low calcemic mobilization.
A significant increase in serum calcium (12.14 mg/dL compared
with 8.76 mg/dL in control mice; P < 0.05) was noticed when mice
were treated with 5 mg/kg tacalcitol each day. Compounds 2g and
2d had no effect on serum calcium at serial concentrations (8.09
and 8.91 mg/dL at 0.5 mg/kg/day, 8.30 and 9.29 mg/dL at 10 mg/kg/
day, 9.08 and 8.94 mg/dL at 30 mg/kg/day). Meanwhile the two
compounds showed
a dramatic decrease in serum calcium,
compared with tacalcitol (P < 0.01 for 2g and P < 0.05 for 2d).
2.3. Molecular docking study
Using Schrödinger Glide version 7.3 and MOE 2009, the docking
study was carried out to understand the strong VDR agonistic ac-
tivity of compound 2g.
The two compounds 2c and 2g shared the same side chains were
docked into the VDR ligand binding domain (VDR LBD, PDB ID:
2ZFX) and the resulting structures of the complexes were showed
and compared in Fig. 4. The most suitable conformations of the
ligands were selected based on the calculated docking scores by
means of the bonding strength.
4. Experiment
It was found that the 20-OH group and the oxygen atom beside
the benzene ring of compound 2g were able to form hydrogen
bonds with the His 393 of VDR LBD. The 4-OH of compound 2g
interacted with Arg270 and Ser233 while 2-NH also formed a
hydrogen bond with Ser233 of VDR LBD. Compound 2c formed
hydrogen-bonding interactions at 20-OH with His 393 and 4-OH, as
well as at Arg270 and Ser233. It is probable that the additional
interactions with those amino acid residues of VDR LBD would
make a stronger binding affinity. In other words, 2g had a better
VDR agonistic activity than 2c did. These additional hydrogen-
bonding interactions might be accredited to that a greater steric
hindrance of ethyl group bonded to the 1-position of pyrrole ring is
forcing the molecular conformation to be more curve than methyl
4.1. Regents and equipments
All commercially available starting materials, reagents and sol-
vents were used without further purification unless otherwise
stated. Melting points were determined and uncorrected employ-
ing an RY-1 apparatus from Tianjin Analytical Instrument Factory
(China). High-resolution mass spectra (HRMS) were recorded on
QSTAR XL Hybrid MS/MS mass spectrometer. 1H NMR and 13C NMR
were recorded employing Bruker AV-300 or AV-500 instruments
using CDCl3 or DMSO-d6. Chemical shifts were given as
d (ppm)
units relative to the internal standard tetramethylsilane (TMS).
Column chromatography separations were progressed on silica gel
(200e300 mesh).
Fig. 3. In vivo calcemic effects of tacalcitol, compounds 2g and 2d.

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W. Shen et al. / European Journal of Medicinal Chemistry 69 (2013) 768e778
Fig. 4. Binding models of compound 2g and 2c docked into VDR ligand binding domain (VDR LBD).
Fig. 5. Structure of the VDR LBD-2g complex overlapped with the VDR LBD-1,25(OH)2D3 complex.
4.2. Methyl-4-hydroxy-3-methylbenzoate (2)
4.4. 3-(4-Benzyloxy-3-methylphenyl)pentan-3-ol (4)
To a solution of compound 1 (0.32 mol) in methanol (300 mL)
was added conc. H2SO4 (45 mL). The reaction mixture was refluxed
for 6 h and then cooled. The solution was adjusted to approx. pH 6
with 2 M NaOH and then poured into cold H2O. The precipitate was
filtered off and washed with cold water. The product was collected
as a pink solid and dried (50.4 g, 92% yield). 1H NMR (300 MHz,
To a solution of compound 3 (3.9 mmol) in ether (10 mL) was
added ethyl Grignard reagent (15.6 mmol) dropwise at 0 ꢀC. The
reaction mixture was stirring at 30 ꢀC for 2 h and then cooled. To
the mixture was added an aqueous saturated solution of NH4Cl and
the two phases were separated. Aqueous phase was extracted with
ethyl acetate. The combined organic phases were washed with
brine, dried over anhydrous Na2SO4 and evaporated to give com-
CDCl3)
d: 7.01 (1H, s), 6.98 (1H, d, J ¼ 8.5 Hz), 6.50 (1H, d, J ¼ 8.5 Hz),
5.40 (1H, bs), 3.88 (3H, s), 2.26 (3H, s).
pound 4 as white oil. 1H NMR (300 MHz, CDCl3)
d: 7.49e7.28 (5H,
m), 7.06 (1H, s), 7.03 (1H, d, J ¼ 8.3 Hz), 6.79 (1H, d, J ¼ 8.3 Hz), 5.04
(2H, s), 2.27 (3H, s), 1.77 (4H, q, J ¼ 7.5 Hz), 0.97 (6H, t, J ¼ 7.5 Hz).
4.3. Methyl-4-benzyloxy-3-methylbenzoate (3)
Benzyl bromide (112 mmol), K2CO3 (306 mmol) and KI
(30.6 mmol) were added to a solution of compound 2 (102 mmol) in
acetone. The mixture was refluxed for 6 h and then cooled. The
precipitate was filtered off and the solution was evaporated. The
residue was precipitated in petroleum ether to give compound 3 as
pink crystal (25.3, 96% yield). Mp: 56e58 ꢀC; 1H NMR (300 MHz,
4.5. Ethyl-5-(3-(4-benzyloxy-3-methylphenyl)pentan-3-yl)-1H-
pyrrole-2-carboxylate (5)
BF3$Et2O (1.3 mL) was added dropwise to a solution of com-
pound
4
(4.6 mmol) and ethyl-1H-pyrrole-2-carboxylate
(5.1 mmol) in dichloromethane (20 mL) at 0 ꢀC. The mixture was
stirred for 1 h at 25 ꢀC. Then the solution was added H2O and
organic phase was separated. The organic phases were washed
CDCl3)
d: 7.46e7.31 (5H, m), 7.10 (1H, s), 7.04 (1H, d, J ¼ 8.3 Hz), 6.69
(1H, d, J ¼ 8.3 Hz), 5.04 (2H, s), 3.88 (3H, s), 2.24 (3H, s).

W. Shen et al. / European Journal of Medicinal Chemistry 69 (2013) 768e778
773
with brine and dried over anhydrous Na2SO4 and evaporated. The
4.8. General procedure for the synthesis of ethyl-5-(3-(4-(3,3-
dimethyl-2-oxobutoxy)-3-methylphenyl)pentan-3-yl)-1-
alkylpyrrole-2-carbocxylate (8a and 8b)
residue was purified by column chromatography with petroleum
ether/ethyl acetate (12/1, v/v) to give compound 5 as yellow solid
(1.35 g, 73% yield). Mp: 86e89 ꢀC; 1H NMR (300 MHz, CDCl3)
d:
7.49e7.28 (5H, m), 7.03 (1H, s), 7.01 (1H, d, J ¼ 8.5 Hz), 6.79 (1H, d,
J ¼ 8.5 Hz), 6.70 (1H, d, J ¼ 2.0 Hz), 6.50 (1H, d, J ¼ 2.0 Hz), 5.04 (2H,
s), 4.30 (2H, q, J ¼ 7.1 Hz), 2.24 (3H, s), 1.97 (4H, q, J ¼ 7.3 Hz), 1.32
(3H, t, J ¼ 7.1 Hz), 0.67 (6H, t, J ¼ 7.3 Hz).
4.8.1. Ethyl-5-(3-(4-(3,3-dimethyl-2-oxobutoxy)-3-methylphenyl)
pentan-3-yl)-1-methylpyrrole-2-carbocxylate (8a)
1-Chloropinacolone (29 mmol), K2CO3 (57 mmol) and KI
(3 mmol) were added to a solution of the compound 7a (19 mmol)
in acetone. The mixture was refluxed for 12 h and then cooled. The
precipitate was filtered off and the solution was concentrated in
vacuo. The residue was purified by column chromatography with
petroleum ether/ethyl acetate (30/1, v/v) to give compound 8a as
4.6. General procedure for the synthesis of ethyl-5-(3-(4-benzyloxy-
3-methylphenyl) pentan-3-yl)-1-alkylpyrrole-2-carboxylate
derivatives (6a and 6b)
yellow oil (6.9 g, 83% yield). 1H NMR (300 MHz, CDCl3)
d: 7.01 (1H,
4.6.1. Ethyl-5-(3-(4-benzyloxy-3-methylphenyl)pentan-3-yl)-1-
methylpyrrole-2-carboxylate (6a)
s), 6.97 (1H, d, J ¼ 8.5 Hz), 6.68 (1H, d, J ¼ 1.9 Hz), 6.50 (1H, d,
J ¼ 8.5 Hz), 6.49 (1H, d, J ¼ 1.9 Hz), 4.84 (2H, s), 4.30 (2H, q,
J ¼ 7.1 Hz), 3.85 (3H, s), 2.26 (3H, s), 1.93 (4H, q, J ¼ 7.3 Hz), 1.32 (3H,
t, J ¼ 7.1 Hz), 1.25 (9H, s), 0.65 (6H, t, J ¼ 7.3 Hz).
To a solution of compound 5 (0.6 mmol) in DMF (5 mL), NaH
(1.2 mmol) was added portionwise at 0 ꢀC. After stirring for 0.5 h,
methyl iodide (0.8 mmol) was added. The reaction mixture was
stirred at 25 ꢀC for 2 h and then H2O (20 mL) was added dropwise
followed by ethyl acetate (10 mL). The organic phase was separated
and the aqueous phase was extracted with ethyl acetate. The
combined organic phases were washed with H2O, brine and then
dried over anhydrous Na2SO4 and evaporated. The residue was
purified by column chromatography with petroleum ether/ethyl
acetate (20/1, v/v) to give compound 6a as yellow oil (205 mg, 82.4%
4.8.2. Ethyl-5-(3-(4-(3,3-dimethyl-2-oxobutoxy)-3-methylphenyl)
pentan-3-yl)-1-ethylpyrrole-2-carbocxylate (8b)
Yellow oil, 5.2 g, 86% yield; 1H NMR (300 MHz, CDCl3)
d: 7.02
(1H, s), 6.97 (1H, d, J ¼ 8.5 Hz), 6.68 (1H, d, J ¼ 2.0 Hz), 6.56 (1H, d,
J ¼ 2.0 Hz), 6.51 (1H, d, J ¼ 8.5 Hz), 4.84 (2H, s), 4.31e4.10 (4H, m),
2.26 (3H, s), 1.93 (4H, q, J ¼ 7.6 Hz), 1.46e1.30 (6H, m), 1.28 (9H, s),
0.66 (6H, t, J ¼ 7.6 Hz).
yield). 1H NMR (300 MHz, CDCl3)
d: 7.49e7.29 (5H, m), 7.03 (1H, s),
7.02 (1H, d, J ¼ 8.4 Hz), 6.79 (1H, d, J ¼ 8.4 Hz), 6.60 (1H, d,
J ¼ 1.9 Hz), 6.49 (1H, d, J ¼ 1.9 Hz), 5.04 (2H, s), 4.25 (2H, q,
J ¼ 7.1 Hz), 3.86 (3H, s), 2.25 (3H, s), 1.94 (4H, q, J ¼ 7.3 Hz), 1.30 (3H,
t, J ¼ 7.1 Hz), 0.67 (6H, t, J ¼ 7.3 Hz).
4.9. General procedure for the synthesis of 5-(3-(4-(3,3-dimethyl-2-
oxobutoxy)-3-methylphenyl) pentan-3-yl)-1-alkylpyrrole-2-
carboxylic acid (9a and 9b)
4.9.1. 5-(3-(4-(3,3-Dimethyl-2-oxobutoxy)-3-methylphenyl)
4.6.2. Ethyl-5-(3-(4-benzyloxy-3-methylphenyl)pentan-3-yl)-1-
pentan-3-yl)-1-methylpyrrole-2-carbocxylic acid (9a)
ethylpyrrole-2-carboxylate (6b)
Compound 8a (4.2 mmol) was dissolved in ethanol (20 mL),
treated with H2O (10 mL) and KOH (20.1 mmol) and the reaction
mixture was stirred at 80 ꢀC for 5 h. The solution was diluted with
H2O (20 mL), the pH value was adjusted to about 3e4 using 1 M HCl.
Then it was extracted with ethyl acetate. The aqueous phase was
extracted with ethyl acetate. The combined organic phases were
washed with brine and then dried over anhydrous Na2SO4 and
evaporated. The residue was purified by column chromatography
with petroleum ether/ethyl acetate (10/1, v/v) to give compound 9a
Yellow oil, 480 mg, 66.9% yield; 1H NMR (300 MHz, CDCl3)
d:
7.50e7.28 (5H, m), 7.04 (1H, s), 7.02 (1H, d, J ¼ 8.6 Hz), 6.77 (1H, d,
J ¼ 8.6 Hz), 6.70 (1H, d, J ¼ 2.0 Hz), 6.57 (1H, d, J ¼ 2.0 Hz), 5.04 (2H,
s), 4.32e4.20 (4H, m), 2.25 (3H, s), 1.95 (4H, q, J ¼ 7.0 Hz), 1.35e1.26
(6H, m), 0.67 (6H, t, J ¼ 7.0 Hz).
4.7. General procedure for the synthesis of ethyl-5-(3-(4-hydroxy-
3-methylphenylpentan-3-yl)-1-alkylpyrrole-2-carboxylate
derivatives (7a and 7b)
as yellow oil (1.5 g, 89% yield). 1H NMR (300 MHz, CDCl3)
d: 6.93 (1H,
s), 6.91 (1H, d, J ¼ 8.2 Hz), 6.78 (1H, d, J ¼ 1.9 Hz), 6.45 (1H, d,
J ¼ 1.9 Hz), 6.43 (1H, d, J ¼ 8.2 Hz), 4.76 (2H, s), 3.78 (3H, s), 2.18 (3H,
s), 1.86 (4H, q, J ¼ 7.3 Hz), 1.24 (9H, s), 0.60 (6H, t, J ¼ 7.3 Hz).
4.7.1. Ethyl-5-(3-(4-hydroxy-3-methylphenyl)pentan-3-yl)-1-
methylpyrrole-2-carboxylate (7a)
To a solution of compound 6a (10.2 mmol) in methanol (20 mL),
Pd/C (0.43 g) and ammonium formate (102 mmol) was added. The
reaction mixture was stirred at 25 ꢀC overnight. The precipitate was
filtered off, H2O (40 mL) and ethyl acetate (10 mL) was added to the
solution. The organic phase was separated and the aqueous phase
was extracted with ethyl acetate. The combined organic phases
were washed with brine, then dried over anhydrous Na2SO4 and
evaporated to give compound 7a as white solid (3.3 g, 98% yield).
4.9.2. 5-(3-(4-(3,3-Dimethyl-2-oxobutoxy)-3-methylphenyl)
pentan-3-yl)-1-ethylpyrrole-2-carbocxylic acid (9b)
Yellow oil, 2.4 g, 86% yield; 1H NMR (300 MHz, CDCl3)
d: 7.01
(1H, s), 6.97 (1H, d, J ¼ 8.5 Hz), 6.85 (1H, d, J ¼ 2.0 Hz), 6.60 (1H, d,
J ¼ 2.0 Hz), 6.51 (1H, d, J ¼ 8.5 Hz), 4.84 (2H, s), 4.26 (2H, q,
J ¼ 7.1 Hz), 2.26 (3H, s), 1.93 (4H, q, J ¼ 7.3 Hz), 1.41 (6H, t, J ¼ 7.1 Hz),
1.28 (9H, s), 0.66 (6H, t, J ¼ 7.3 Hz).
Mp: 78e81 ꢀC; 1H NMR (300 MHz, CDCl3)
d: 6.98 (1H, s), 6.96 (1H,
d, J ¼ 8.3 Hz), 6.70 (1H, d, J ¼ 2.0 Hz), 6.69 (1H, d, J ¼ 8.3 Hz), 6.49
(1H, d, J ¼ 2.0 Hz), 4.25 (2H, q, J ¼ 7.1 Hz), 3.85 (3H, s), 2.21 (3H, s),
1.95 (4H, q, J ¼ 7.4 Hz), 1.31 (3H, t, J ¼ 7.1 Hz), 0.67 (6H, t, J ¼ 7.4 Hz).
4.10. General procedure for the synthesis of phenyl-pyrrolyl
pentane derivatives (1ae1h, 2ae2h, 3ae3h, 4ae4h)
4.10.1. Methyl-2-(5-(3-(4-(3,3-dimethyl-2-oxobutoxy)-3-
methylphenyl)pentan-3-yl)-1-methylpyrrole-2-carboxamido)
acetate (1a)
To a solution of compound 9a (0.75 mmol) in DMF (10 mL) was
added Et3N (3.3 mmol), followed by hydrochloride salt of glycine
methyl ester (0.83 mmol), EDCI (0.91 mmol) and HOBT (0.91 mmol).
The reaction mixture was stirred at 25 ꢀC overnight and then poured
into H2O. The solution was extracted with ethyl acetate, and aqueous
4.7.2. Ethyl-5-(3-(4-hydroxy-3-methylphenyl)pentan-3-yl)-1-
ethypyrrole-2-carboxylate (7b)
White solid, 4.2 g, 97% yield; Mp: 72e74 ꢀC; 1H NMR (300 MHz,
CDCl3)
d
: 6.92 (1H, s), 6.88 (1H, d, J ¼ 8.6 Hz), 6.63 (1H, d, J ¼ 2.1 Hz),
6.60 (1H, d, J ¼ 8.6 Hz), 6.50 (1H, d, J ¼ 2.1 Hz), 4.25e4.06 (4H, m),
2.28 (3H, s), 1.87 (4H, q, J ¼ 7.4 Hz), 1.30e1.25 (6H, m), 0.60 (6H, t,
J ¼ 7.4 Hz).

774
W. Shen et al. / European Journal of Medicinal Chemistry 69 (2013) 768e778
phase was extracted with ethyl acetate. The combined organic phases
were washed with H2O, brine, then dried over anhydrous Na2SO4 and
evaporated. The residue was purified by column chromatography
with petroleum ether/ethyl acetate (4/1, v/v) to give compounþd 1a as
white solid (0.29 g, 82% yield). Mp: 121e122 ꢀC; HRMS, ESI , m/z:
Calcd for C27H39N2O5 (M þ H)þ, 471.2853; found, 471.2863; 1H NMR
80.5% yield. HRMS, ESIþ, m/z: Calcd for C28H41N2O5 (M þ H)þ,
485.3010; found, 485.3016; 1H NMR (300 MHz, CDCl3)
d: 7.01 (1H,
s), 6.97 (1H, d, J ¼ 8.5 Hz), 6.57 (1H, d, J ¼ 1.4 Hz), 6.51 (1H, d,
J ¼ 8.5 Hz), 6.28 (1H, d, J ¼ 1.4 Hz), 4.84 (2H, s), 4.32 (2H, q,
J ¼ 7.1 Hz), 4.10 (2H, d, J ¼ 5.3 Hz), 3.76 (3H, s), 2.26 (3H, s), 1.92 (4H,
q, J ¼ 7.3 Hz), 1.36 (3H, t, J ¼ 7.1 Hz), 1.26 (9H, s), 0.64 (6H, t,
(300 MHz, CDCl3)
d
: 1H NMR (CDCl3, 300 Hz)
d: 7.01 (1H, s), 6.98 (1H,
J ¼ 7.3 Hz); 13C NMR (300 MHz, CDCl3)
d: 210.02, 170.79, 161.83,
d, J ¼ 8.5 Hz), 6.50(1H, d,J ¼ 8.5 Hz), 6.45(1H, d, J ¼ 1.8Hz), 6.18 (1H, d,
J ¼ 1.8 Hz), 4.833 (2H, s), 3.88 (3H, s), 3.69 (3H, s), 3.59 (2H, t,
J ¼ 6.0 Hz), 2.60 (2H, t, J ¼ 6.0 Hz), 2.26 (3H, s), 1.91 (4H, q, J ¼ 7.3 Hz),
154.08, 140.62, 131.29, 130.43, 125.91, 125.78, 125.01, 123.33, 112.14,
110.14, 69.57, 52.25, 44.92, 43.72, 40.97, 34.21, 30.29, 26.33, 17.15,
16.64, 8.50.
1.26 (9H, s), 0.65 (6H, t, J ¼ 7.3 Hz); 13C NMR (500 MHz, CDCl3)
d:
209.99, 170.75, 161.99, 154.09, 140.61, 131.23, 130.39, 126.69, 125.95,
125.71, 124.14, 111.89, 110.16, 69.57, 52.23, 44.82, 43.18, 40.94, 36.52,
30.20, 26.31, 16.59, 8.46.
4.10.6. Methyl-3-(5-(3-(4-(3,3-dimethyl-2-oxobutoxy)-3-
methylphenyl)pentan-3-yl)-1-ethylpyrrole-2-carboxamido)
propanoate (1f)
The same method as 1a and the starting materials were 9b and
4.10.2. Methyl-3-(5-(3-(4-(3,3-dimethyl-2-oxobutoxy)-3-
methylphenyl)pentan-3-yl)-1-methylpyrrole-2-carboxamido)
propanoate (1b)
hydrochloride salt of b-alanine methyl ester. Colorless oil, 0.38 g,
87% yield. HRMS, ESIþ, m/z: Calcd for C29H43N2O5 (M þ H)þ,
499.3166; found, 499.3175; 1H NMR (CDCl3, 300 Hz)
d: 7.01 (1H, s),
The same method as 1a and the starting materials were 9a and
6.97 (1H, d, J ¼ 8.5 Hz), 6.54 (1H, d, J ¼ 1.9 Hz), 6.51 (1H, d,
J ¼ 8.5 Hz), 6.17 (1H, d, J ¼ 1.9 Hz), 4.84 (2H, s), 4.31 (2H, q,
J ¼ 7.1 Hz), 3.68 (3H, s), 3.60 (2H, t, J ¼ 6.1 Hz), 2.60 (2H, t, J ¼ 6.1 Hz),
2.26 (3H, s), 1.90 (4H, q, J ¼ 7.3 Hz), 1.36 (3H, t, J ¼ 7.1 Hz), 1.26 (9H,
hydrochloride salt of b-alanine methyl ester. White solid, 0.25 g,
82% yield. Mp: 97e100 ꢀC; HRMS, ESIþ, m/z: Calcd for C28H41N2O5
(M þ H)þ, 485.3010; found, 485.3016; 1H NMR (CDCl3, 300 Hz)
d:
7.01 (1H, s), 6.98 (1H, d, J ¼ 8.5 Hz), 6.50 (1H, d, J ¼ 8.5 Hz), 6.45 (1H,
d, J ¼ 1.8 Hz), 6.18 (1H, d, J ¼ 1.8 Hz), 4.833 (2H, s), 3.88 (3H, s), 3.69
(3H, s), 3.59 (2H, t, J ¼ 6.0 Hz), 2.60 (2H, t, J ¼ 6.0 Hz), 2.26 (3H, s),
1.91 (4H, q, J ¼ 7.3 Hz), 1.26 (9H, s), 0.65 (6H, t, J ¼ 7.3 Hz); 13C NMR
s), 0.64 (6H, t, J ¼ 7.3 Hz); 13C NMR (300 MHz, CDCl3)
d: 210.00,
173.07, 161.91, 154.05, 140.65, 131.08, 130.43, 125.87, 125.75, 124.65,
123.94, 111.37, 110.11, 69.56, 51.70, 44.89, 43.66, 34.58, 34.11, 30.28,
26.32, 17.17, 16.62, 8.50.
(500 MHz, CDCl3) d: 209.94, 173.06, 162.09, 154.11, 140.65, 131.05,
130.42, 126.36, 125.95, 125.70, 124.77, 111.15, 110.16, 69.61, 51.70,
44.82, 43.21, 36.52, 34.59, 34.12, 30.23, 26.33, 16.62, 8.49.
4.10.7. (S)-Methyl-2-(5-(3-(4-(3,3-dimethyl-2-oxobutoxy)-3-
methylphenyl)pentan-3-yl)-1-ethylpyrrole-2-carboxamido)
propanoate (1g)
4.10.3. (S)-Methyl-2-(5-(3-(4-(3,3-dimethyl-2-oxobutoxy)-3-
methylphenyl)pentan-3-yl)-1-methylpyrrole-2-carboxamido)
propanoate (1c)
The same method as 1a and the starting materials were 9b and
hydrochloride salt of
L
-alanine methþyl ester. White solid, 0.51 g, 80%
yield. Mp: 92e93 ꢀC; HRMS, ESI , m/z: Calcd for C29H43N2O5
The same method as 1a and the starting materials were 9a and
(M þ H)þ, 499.3166; found, 499.3175; 1H NMR (300 MHz, CDCl3)
d:
hydrochloride salt of
L
-alanine methyl ester. White solid, 0.34 g,
7.02 (1H, s), 6.97 (1H, d, J ¼ 8.7 Hz), 6.56 (1H, d, J ¼ 1.5 Hz), 6.51 (1H,
d, J ¼ 8.7 Hz), 6.26 (1H, d, J ¼ 1.5 Hz), 4.85 (2H, s), 4.66 (1H, m), 4.29
(2H, q, J ¼ 6.9 Hz), 3.75 (1H, s), 2.27 (3H, s), 1.90 (4H, q, J ¼ 7.2 Hz),
1.45 (3H, d, J ¼ 7.2 Hz),1.35 (3H, t, J ¼ 6.9 Hz),1.26 (9H, s), 0.64 (6H, t,
89.7% yield. Mp: 92e93 ꢀC; HRMS, ESIþ, m/z: Calcd for C28H41N2O5
(M þ H)þ, 485.3010; found, 485.3019; 1H NMR (500 MHz, CDCl3)
d:
7.02 (1H, s), 6.98 (1H, d, J ¼ 8.6 Hz), 6.51 (1H, d, J ¼ 8.6 Hz), 6.48 (1H,
d, J ¼ 1.8 Hz), 6.28 (1H, d, J ¼ 1.8 Hz), 4.84 (2H, s), 4.66 (1H, m), 3.87
(3H, s), 3.75 (3H, s), 2.26 (3H, s), 1.92 (4H, q, J ¼ 6.5 Hz), 1.44 (3H, d,
J ¼ 7.2 Hz), 1.25 (9H, s), 0.65 (6H, t, J ¼ 6.5 Hz); 13C NMR (500 MHz,
J ¼ 7.2 Hz); 13C NMR (300 MHz, CDCl3)
d: 210.06, 173.88, 161.27,
153.95, 140.60, 131.15, 130.36, 125.80, 125.71, 124.90, 123.38, 111.82,
109.98, 69.42, 52.33, 47.71, 44.78, 43.68, 35.20, 30.10, 26.27, 18.43,
17.14, 16.63, 8.44.
CDCl3)
d: 209.93, 173.85, 161.46, 154.09, 140.65, 131.15, 130.39,
126.64, 125.94, 125.72, 124.31, 111.60, 110.15, 69.57, 52.32, 47.73,
44.81, 43.18, 36.53, 30.17, 26.31, 18.53, 16.60, 8.46.
4.10.8. (S)-Dimethyl-2-(5-(3-(4-(3,3-dimethyl-2-oxobutoxy)-3-
methylphenyl)pentan-3-yl)-1-ethylpyrrole-2-carboxamido)
succinate (1h)
4.10.4. (S)-Dimethyl-2-(5-(3-(4-(3,3-dimethyl-2-oxobutoxy)-3-
methylphenyl)pentan-3-yl)-1-methylpyrrole-2-carboxamido)
succinate (1d)
The same method as 1a and the starting materials were 9b and
hydrochloride salt of L-aspartic acid dimethyl ester. Colorless oil,
The same method as 1a and the starting materials were 9a and
0.52 g, 83% yield. HRMS, ESIþ, m/z: Calcd for C31H45N2O7 (M þ H)þ,
hydrochloridesaltof
L
-asparticacid dimethyl ester. White solid, 0.60 g,
557.3221; found, 557.3223; 1H NMR (300 MHz, CDCl3)
d: 7.02 (1H,
84% yield. Mp: 57e58 ꢀC; HRMS, ESIþ, m/z: Calcd for C30H43N2O7
s), 6.98 (1H, d, J ¼ 8.5 Hz), 6.66 (1H, d, J ¼ 8.5 Hz), 6.54 (1H, d,
J ¼ 1.8 Hz), 6.31 (1H, d, J ¼ 1.8 Hz), 4.95 (1H, m), 4.85 (2H, s), 4.28
(2H, q, J ¼ 7.1 Hz), 3.77 (3H, s), 3.68 (3H, s), 3.01 (2H, m), 2.26 (3H, s),
1.92 (4H, q, J ¼ 6.9 Hz),1.35 (3H, t, J ¼ 7.1 Hz),1.26 (9H, s), 0.65 (6H, t,
(M þ H)þ, 543.3065; found, 543.3078; 1H NMR (300 MHz, CDCl3)
d:
7.01 (1H, s), 6.98 (1H, d, J ¼ 8.7 Hz), 6.68 (1H, d, J ¼ 8.7 Hz), 6.47 (1H, d,
J¼ 1.2Hz), 6.32 (1H, d,J ¼ 1.2Hz), 4.84(2H, s), 4.12(1H, m), 3.86(3H, s),
3.77 (3H, s), 3.69 (3H, s), 3.07 (1H, m), 2.91 (1H, m), 2.32 (3H, s), 1.92
(4H, q, J ¼ 7.0 Hz), 1.26 (9H, s), 0.65 (6H, t, J ¼ 7.0 Hz); 13C NMR
J ¼ 6.9 Hz); 13C NMR (300 MHz, CDCl3)
d: 209.97, 171.56, 161.37,
154.10, 140.57, 131.35, 130.41, 125.93, 125.76, 125.29, 123.22, 112.12,
110.22, 69.64, 52.71, 51.96, 48.31, 44.88, 43.76, 37.10, 36.43, 30.27,
26.33, 17.12, 16.63, 8.50.
(300 MHz, CDCl3) d: 209.99, 171.57, 161.52, 154.09, 140.58, 131.32,
130.36,126.96,125.96,125.67,123.98,111.88,110.21, 69.61, 52.73, 51.97,
48.25, 44.75, 43.19, 36.60, 36.41, 30.11, 26.32, 16.62, 8.46.
4.10.9. 5-(3-(4-(2-Hydroxy-3,3-dimethylbutoxy)-3-methylphenyl)
pentan-3-yl)-N-(2-hydroxyethyl)-1-methylpyrrole-2-carboxamide
(2a)
To a solution of 1a (0.23 mmol) in methanol (10 mL), NaBH4
(2.3 mmol) was added portionwise at 0 ꢀC. The reaction mixture
was stirred at 25 ꢀC for 0.5 h and then added H2O (10 mL). The
4.10.5. Methyl-2-(5-(3-(4-(3,3-dimethyl-2-oxobutoxy)-3-
methylphenyl)pentan-3-yl)-1-ethylpyrrole-2-carboxamido)acetate
(1e)
The same method as 1a and the starting materials were 9b and
hydrochloride salt of glycine methyl ester. Colorless oil, 0.49 g,

W. Shen et al. / European Journal of Medicinal Chemistry 69 (2013) 768e778
775
solution was extracted with ethyl acetate, and aqueous phase was
extracted with ethyl acetate. The combined organic phases were
washed with brine and then dried over anhydrous Na2SO4 and
evaporated. The residue was purified by column chromatography
with petroleum ether/ethyl acetate (1/1, v/v) to give compound 2a
as white solid (0.10 mg, 96% yield). Mp: 98e100 ꢀC; HRMS, ESIþ, m/
z: Calcd for C26H41N2O4 (M þ H)þ, 445.3061; found, 445.3069; 1H
J ¼ 8.3 Hz), 6.58 (1H, d, J ¼ 1.8 Hz), 6.20 (1H, d, J ¼ 1.8 Hz), 4.31 (2H,
q, J ¼ 7.1 Hz), 4.08 (1H, m), 3.86 (1H, m), 3.74 (1H, m), 3.70 (2H, m),
3.47 (2H, m), 2.04 (3H, s), 1.92 (4H, q, J ¼ 7.2 Hz), 1.36 (3H, t,
J ¼ 7.1 Hz), 1.01 (9H, s), 0.64 (6H, t, J ¼ 7.2 Hz); 13C NMR (300 MHz,
CDCl3)
d: 163.21, 154.42, 140.35, 131.33, 130.30, 125.89, 125.44,
124.84, 123.67, 111.82, 110.13, 69.25, 62.85, 44.90, 43.72, 42.38,
33.56, 30.20, 26.03, 17.17, 16.61, 8.48.
NMR (500 MHz, CDCl3)
d
: 7.02 (1H, d, J ¼ 8.5 Hz), 7.00 (1H, s), 6.70
(1H, d, J ¼ 8.5 Hz), 6.49 (1H, d, J ¼ 1.9 Hz), 6.21 (1H, d, J ¼ 1.9 Hz),
4.09 (2H, m), 3.87 (3H, s), 3.72 (2H, m), 3.70 (1H, m), 3.46 (2H, m),
2.19 (3H, s), 1.91 (4H, q, J ¼ 7.5 Hz), 1.01 (9H, s), 0.65 (6H, t,
4.10.14. 5-(3-(4-(2-Hydroxy-3,3-dimethylbutoxy)-3-methylphenyl)
pentan-3-yl)-N-(3-hydroxypropyl)-1-ethylpyrrole-2-carboxamide
(2f)
J ¼ 7.5 Hz); 13C NMR (500 MHz, CDCl3)
d
: 163.35, 154.46, 140.35,
The same method as 2a and the starting material was 1f. White
131.28, 130.30, 126.28, 125.83, 125.52, 124.49, 111.59, 110.18, 69.30,
62.79, 44.82, 42.35, 36.58, 33.58, 30.14, 26.03, 16.59, 8.47.
solid, 0.24 g, 93% yield. Mp: 104e106 ꢀC; HRMS, ESIþ, m/z: Calcd for
C
28H45N2O4 (M þ H)þ, 473.3374; found, 473.3379; 1H NMR (CDCl3,
300 Hz)
d
: 7.01 (1H, s), 7.02 (1H, d, J ¼ 8.2 Hz), 6.71 (1H, d,
4.10.10. 5-(3-(4-(2-Hydroxy-3,3-dimethylbutoxy)-3-methylphenyl)
pentan-3-yl)-N-(3-hydroxypropyl)-1-methylpyrrole-2-
carboxamide (2b)
J ¼ 8.2 Hz), 6.58 (1H, d, J ¼ 1.5 Hz), 6.17 (1H, d, J ¼ 1.5 Hz), 4.32 (2H,
q, J ¼ 6.9 Hz), 4.11 (1H, m), 3.86 (1H, m), 3.70 (1H, m), 3.63 (2H, t,
J ¼ 5.6 Hz), 3.47 (2H, t, J ¼ 5.7 Hz), 2.20 (3H, s), 1.94 (4H, q,
J ¼ 7.1 Hz), 1.68 (2H, m), 1.36 (3H, t, J ¼ 6.9 Hz), 1.00 (9H, s), 0.65 (6H,
The same method as 2a and the starting material was 1b. White
solid, 0.19 g, 98% yield. Mp: 80e81 ꢀC; HRMS, ESIþ, m/z: Calcd for
t, J ¼ 7.1 Hz); 13C NMR (300 MHz, CDCl3)
d: 163.26, 154.44, 140.33,
C
27H43N2O4 (M þ H)þ, 459.3217; found, 459.3223; 1H NMR (CDCl3,
131.29, 130.34, 125.93, 125.45, 124.83, 123.70, 111.58, 110.14, 69.27,
58.97, 44.94, 43.77, 35.56, 33.58, 32.72, 30.29, 26.05, 17.21, 16.63,
8.52.
300 Hz)
d
: 7.00 (1H, s), 7.01 (1H, d, J ¼ 8.3 Hz), 6.70 (1H, d,
J ¼ 8.3 Hz), 6.50 (1H, d, J ¼ 1.4 Hz), 6.19 (1H, d, J ¼ 1.4 Hz), 4.11 (2H,
m), 3.88 (3H, s), 3.70 (1H, m), 3.63 (2H, m), 3.48 (2H, m), 2.19 (3H, s),
1.93 (4H, q, J ¼ 7.2 Hz), 1.70 (2H, m), 1.00 (9H, s), 0.65 (6H, t,
4.10.15. 5-(3-(4-(2-Hydroxy-3,3-dimethylbutoxy)-3-methylphenyl)
pentan-3-yl)-N-((S)-1-hydroxypropan-2-yl)-1-ethylpyrrole-2-
carboxamide (2g)
J ¼ 7.2 Hz); 13C NMR (300 MHz, CDCl3)
d: 163.32, 154.41, 140.28,
131.20, 130.27, 126.43, 125.80, 125.45, 124.52, 111.25, 110.09, 69.23,
59.00, 44.77, 36.56, 35.59, 33.55, 32.59, 30.14, 26.01, 16.58, 8.45.
The same method as 2a and the starting material was 1g. White
solid, 0.28 g, 94% yield. Mp: 89e90 ꢀC; HRMS, ESIþ, m/z: Calcd for
4.10.11. (S)-5-(3-(4-(2-Hydroxy-3,3-dimethylbutoxy)-3-
methylphenyl)pentan-3-yl)-N-(1-hydroxypropan-2-yl)-1-
methylpyrrole-2-carboxamide (2c)
C
28H45N2O4 (M þ H)þ, 473.3374; found, 499.3388; 1H NMR
(300 MHz, CDCl3)
d
: 7.01 (1H, s), 7.02 (1H, d, J ¼ 8.1 Hz), 6.72 (1H, d,
J ¼ 8.1 Hz), 6.58 (1H, d, J ¼ 1.5 Hz), 6.18 (1H, d, J ¼ 1.5 Hz), 4.31 (2H,
q, J ¼ 7.1 Hz), 4.11 (2H, m), 3.85 (1H, m), 3.70 (2H, m), 3.56 (1H, m),
2.20 (3H, s),1.92 (4H, q, J ¼ 7.2 Hz),1.36 (3H, t, J ¼ 7.1 Hz),1.20 (3H, d,
J ¼ 6.8 Hz), 1.01 (9H, s), 0.65 (6H, t, J ¼ 7.2 Hz); 13C NMR (300 MHz,
The same method as 2a and the starting materiþal was 1c. White
solid, 0.30 g, 95% yield. Mp: 88e89 ꢀC; HRMS, ESI , m/z: Calcd for
C
27H43N2O4 (M þ H)þ, 459.3217; found, 459.3223; 1H NMR
(300 MHz, CDCl3)
d
: 7.02 (1H, s), 7.02 (1H, d, J ¼ 9.7 Hz), 6.71 (1H, d,
CDCl3) d: 162.79, 154.35, 140.33, 131.24, 130.28, 125.86, 125.40,
J ¼ 9.7 Hz), 6.51 (1H, d, J ¼ 1.5 Hz), 6.20 (1H, d, J ¼ 1.5 Hz), 4.10 (2H,
m), 3.86 (2H, m), 3.83 (1H, s), 3.69 (1H, m), 3.54 (1H, m), 2.19 (1H, s),
1.92 (4H, q, J ¼ 7.2 Hz), 1.20 (3H, d, J ¼ 6.9 Hz), 1.01 (9H, s), 0.65 (6H,
124.94, 123.69, 111.54, 109.95, 69.10, 67.77, 47.77, 44.82, 43.76,
33.52, 30.10, 26.01, 17.20, 17.12, 16.65, 8.46.
t, J ¼ 7.2 Hz); 13C NMR (300 MHz, CDCl3)
d: 162.92, 154.37, 140.34,
4.10.16. (S)-N-(1,4-Dihydroxybutan-2-yl)-5-(3-(4-(2-hydroxy-3,3-
dimethylbutoxy)-3-methylphenyl)pentan-3-yl)-1-ethylpyrrole-2-
carboxamide (2h)
131.20, 130.28, 126.62, 125.77, 125.46, 124.47, 111.29, 109.94, 69.10,
67.71, 47.74, 44.71, 36.68, 33.53, 29.98, 26.02, 17.13, 16.64, 8.44.
The same method as 2a and the starting material was 1h. White
solid, 0.17 g, 91% yield. Mp: 93e94 ꢀC; HRMS, ESIþ, m/z: Calcd for
C29H47N2O5 (M þ H)þ, 503.3479; found, 503.3483; 1H NMR
4.10.12. (S)-N-(1,4-Dihydroxybutan-2-yl)-5-(3-(4-(2-hydroxy-3,3-
dimethylbutoxy)-3-methylphenyl)pentan-3-yl)-1-methylpyrrole-2-
carboxamide (2d)
(300 MHz, CDCl3)
d: 7.02 (1H, d, J ¼ 8.9 Hz), 7.00 (1H, s), 6.71 (1H, d,
The same method as 2a and the starting material was 1d. White
J ¼ 8.9 Hz), 6.58 (1H, d, J ¼ 2.0 Hz), 6.26 (1H, d, J ¼ 2.0 Hz), 4.30 (2H,
m), 4.07 (2H, q, J ¼ 7.0 Hz), 3.85 (1H, m), 3.71 (2H, m), 3.67 (1H, m),
3.62 (2H, m), 2.19 (3H, s), 1.92 (4H, q, J ¼ 7.2 Hz), 1.35 (3H, t,
J ¼ 7.0 Hz), 1.01 (9H, s), 0.65 (6H, t, J ¼ 7.2 Hz); 13C NMR (300 MHz,
solid, 0.31 g, 90% yield. Mp: 80e82 ꢀC; HRMS, ESIþ, m/z: Calcd for
C
28H45N2O5 (M þ H)þ, 489.3323; found, 489.3316; 1H NMR
(300 MHz, CDCl3)
d
: 6.94 (1H, d, J ¼ 8.6 Hz), 6.93 (1H, s), 6.63 (1H, d,
J ¼ 8.6 Hz), 6.44 (1H, d, J ¼ 1.3 Hz), 6.20 (1H, d, J ¼ 1.3 Hz), 4.11 (1H,
m), 4.02 (1H, m), 3.78 (3H, s), 3.75 (1H, m), 3.62 (2H, m), 3.59 (2H,
m), 3.56 (1H, m), 2.11 (3H, s), 1.85 (4H, q, J ¼ 7.1 Hz), 1.55 (2H, m),
CDCl3) d: 163.10, 154.43, 140.32, 131.40, 130.29, 125.92, 125.46,
125.18, 123.45, 111.93, 110.16, 69.26, 65.39, 58.78, 48.26, 44.90,
43.83, 34.54, 33.59, 30.21, 26.04, 17.19, 16.62, 8.49.
0.93 (9H, s), 0.57 (6H, t, J ¼ 7.1 Hz); 13C NMR (300 MHz, CDCl3)
d:
164.25, 153.27, 139.33, 130.39, 129.29, 125.85, 124.86, 124.52,
123.29, 110.71, 109.18, 68.29, 64.32, 57.78, 47.34, 43.80, 35.73, 33.51,
32.62, 29.10, 25.06, 15.63, 7.49.
4.10.17. 2-(5-(3-(4-(3,3-Dimethyl-2-oxobutoxy)-3-methylphenyl)
pentan-3-yl)-1-methylpyrrole-2-carboxamido)acetic acid (3a)
To a solution of 1a (0.33 mmol) in the THF (5 mL) and H2O
(1 mL), LiOH$2H2O (1.65 mmol) was added. The reaction mixture
was stirred at 25 ꢀC overnight and then H2O (10 mL) was added
followed by the pH value was adjusted to about 3e4. Then it was
extracted with ethyl acetate, and the organic phase was separated
and the aqueous phase was extracted with ethyl acetate. The
combined organic phases were washed with brine, then dried over
anhydrous Na2SO4 and evaporated. The residue was purified by
column chromatography with dichloromethane/methanol (40/1, v/
4.10.13. 5-(3-(4-(2-Hydroxy-3,3-dimethylbutoxy)-3-methylphenyl)
pentan-3-yl)-N-(2-hydroxyethyl)-1-ethylpyrrole-2-carboxamide
(2e)
The same method as 2a and the starting materiþal was 1e. White
solid, 0.22 g, 97% yield. Mp: 81e82 ꢀC; HRMS, ESI , m/z: Calcd for
C
27H43N2O4 (M þ H)þ, 459.3217; found, 459.3217; 1H NMR
(300 MHz, CDCl3)
d
: 7.03 (1H, d, J ¼ 8.3 Hz), 7.00 (1H, s), 6.71 (1H, d,

776
W. Shen et al. / European Journal of Medicinal Chemistry 69 (2013) 768e778
v) to give compound 3a as faint yellow solid (0.13 g, 83.9% yield).
Mp: 104e105 ꢀC; HRMS, ESIþ, m/z: Calcd for C26H37N2O5 (M þ H)þ,
4.10.22. 3-(5-(3-(4-(3,3-Dimethyl-2-oxobutoxy)-3-methylphenyl)
pentan-3-yl)-1-ethylpyrrole-2-carboxamido)propanoic acid (3f)
The same method as 3a and the starting material was 1þf. Faint
yellow solid, 0.24 g, 89% yield. Mp: 90e91 ꢀC; HRMS, ESI , m/z:
Calcd for C28H41N2O5 (M þ H)þ, 485.3010; found, 485.3010; 1H
457.2697; found, 457.2704; 1H NMR (500 MHz, CDCl3)
d: 7.00 (1H,
s), 6.95 (1H, d, J ¼ 8.5 Hz), 6.50 (1H, d, J ¼ 1.9 Hz), 6.49 (1H, d,
J ¼ 8.5 Hz), 6.32 (1H, d, J ¼ 1.9 Hz), 4.83 (2H, s), 4.07 (2H, d,
J ¼ 5.3 Hz), 3.85 (3H, s), 2.24 (3H, s), 1.90 (4H, q, J ¼ 7.3 Hz), 1.24 (9H,
NMR (CDCl3, 300 Hz)
d
: 6.93 (1H, s), 6.89 (1H, d, J ¼ 8.5 Hz), 6.49
s), 0.64 (6H, t, J ¼ 7.3 Hz); 13C NMR (500 MHz, CDCl3)
d
: 210.37,
(1H, d, J ¼ 1.9 Hz), 6.40 (1H, d, J ¼ 8.5 Hz), 6.11 (1H, d, J ¼ 1.9 Hz),
4.77 (2H, s), 4.23 (2H, q, J ¼ 7.1 Hz), 3.60 (2H, t, J ¼ 5.6 Hz), 2.53 (2H,
t, J ¼ 5.6 Hz), 2.16 (3H, s), 1.82 (4H, q, J ¼ 7.1 Hz), 1.28 (3H, t,
J ¼ 7.1 Hz), 1.17 (9H, s), 0.56 (6H, t, J ¼ 7.1 Hz); 13C NMR (300 MHz,
172.99, 162.69, 154.07, 140.59, 131.46, 130.44, 127.15, 125.98, 125.73,
123.71, 112.58, 110.17, 69.53, 44.85, 43.20, 41.38, 36.63, 30.19, 26.33,
16.59, 8.47.
CDCl3)
d: 210.45, 176.21, 162.27, 154.00, 140.65, 131.19, 130.47,
4.10.18. 3-(5-(3-(4-(3,3-Dimethyl-2-oxobutoxy)-3-methylphenyl)
pentan-3-yl)-1-methylpyrrole-2-carboxamido)propanoic acid (3b)
The same method as 3a and the starting material was 1b. Faint
yellow solid, 0.22 g, 81% yield. Mp: 124e126 ꢀC; HRMS, ESIþ, m/z:
Calcd for C27H39N2O5 (M þ H)þ, 471.2853; found, 471.2859; 1H
125.86, 125.77, 124.86, 123.67, 111.89, 110.07, 69.46, 44.92, 43.70,
34.64, 34.37, 34.27, 30.27, 26.31, 17.14, 16.59, 8.50.
4.10.23. (S)-2-(5-(3-(4-(3,3-dimethyl-2-oxobutoxy)-3-
methylphenyl)pentan-3-yl)-1-methylpyrrole-2-carboxamido)
propanoic acid (3g)
The same method as 3a and the starting material was 1g. Faint
yellow solid, 0.19 g, 83% yield. Mp: 114e117 ꢀC; HRMS, ESIþ, m/z:
Calcd for C28H41N2O5 (M þ H)þ, 485.3010; found, 485.3020; 1H
NMR (CDCl3, 300 Hz)
d
: 6.98 (1H, s), 6.93 (1H, d, J ¼ 8.5 Hz), 6.45
(1H, d, J ¼ 8.5 Hz), 6.21 (1H, d, J ¼ 1.4 Hz), 6.04 (1H, d, J ¼ 1.4 Hz),
4.81 (2H, s), 3.78 (3H, s), 3.45 (2H, t, J ¼ 5.9 Hz), 2.41 (2H, t,
J ¼ 5.9 Hz), 2.20 (3H, s), 1.87 (4H, q, J ¼ 7.2 Hz), 1.23 (9H, s), 0.60
(6H, t, J ¼ 7.2 Hz); 13C NMR (500 MHz, CDCl3)
d
: 210.37, 176.05,
NMR (300 MHz, CDCl3)
d
: 6.93 (1H, s), 6.90 (1H, d, J ¼ 8.4 Hz), 6.54
162.48, 154.03, 140.75, 131.02, 130.43, 126.37, 125.89, 125.73,
124.70, 111.59, 110.17, 69.51, 44.79, 43.17, 36.53, 35.44, 35.14, 30.14,
26.33, 16.58, 8.49.
(1H, d, J ¼ 1.9 Hz), 6.43 (1H, d, J ¼ 8.4 Hz), 6.20 (1H, d, J ¼ 1.9 Hz),
4.78 (2H, s), 4.53 (1H, m), 4.23 (2H, q, J ¼ 7.1 Hz), 2.19 (3H, s), 1.84
(4H, q, J ¼ 7.0 Hz), 1.43 (3H,d, J ¼ 7.1 Hz), 1.29 (3H,t, J ¼ 7.1 Hz), 1.19
(9H, s), 0.57 (6H, t, J ¼ 7.0 Hz); 13C NMR (300 MHz, CDCl3)
d: 210.25,
4.10.19. (S)-2-(5-(3-(4-(3,3-Dimethyl-2-oxobutoxy)-3-
methylphenyl)pentan-3-yl)-1-methylpyrrole-2-carboxamido)
propanoic acid (3c)
179.78, 162.04, 153.89, 140.67, 131.01, 130.35, 126.61, 125.76, 124.66,
123.64, 111.97, 110.16, 69.49, 50.52, 44.75, 43.51, 43.11, 30.23, 26.26,
17.92, 17.05, 16.61, 8.47.
The same method as 3a and the starting material was 1c. Faint
yellow solid, 0.20 g, 84% yield. Mp: 148e150 ꢀC; HRMS, ESIþ, m/z:
Calcd for C27H39N2O5 (M þ H)þ, 471.2853; found, 471.2860; 1H NMR
4.10.24. (S)-2-(5-(3-(4-(3,3-Dimethyl-2-oxobutoxy)-3-
methylphenyl)pentan-3-yl)-1-ethylpyrrole-2-carboxamido)succinic
acid (3h)
The same method as 3a and the starting material was 1h. Faint
yellow solid, 0.15 g, 81% yield. Mp: 252e255 ꢀC; HRMS, ESIþ, m/z:
Calcd for C29H41N2O7 (M þ H)þ, 529.2908; found, 529.2906; 1H
(300 MHz, CDCl3)
d
: 6.93 (1H, s), 6.90 (1H, d, J ¼ 8.9 Hz), 6.42 (1H, d,
J ¼ 2.0 Hz), 6.21 (1H, d, J ¼ 2.0 Hz), 6.20 (1H, d, J ¼ 8.9 Hz), 4.78 (2H,
s), 4.54 (1H, m), 3.79 (3H, s), 2.18 (3H, s), 1.85 (4H, q, J ¼ 7.0 Hz), 1.42
(3H,d, J ¼ 6.8 Hz), 1.18 (9H, s), 0.56 (6H, t, J ¼ 7.0 Hz); 13C NMR
(300 MHz, CDCl3)
d
: 210.34, 176.50, 162.26, 153.97, 140.55, 131.40,
NMR (300 MHz, CDCl3)
d
: 6.96 (1H, s), 6.95 (1H, d, J ¼ 8.4 Hz), 6.47
130.37, 127.16, 125.90, 125.65, 123.68, 112.23, 109.99, 69.39, 48.15,
44.71, 43.16, 36.70, 30.01, 26.29, 17.86, 16.64, 8.43.
(1H, d, J ¼ 8.4 Hz), 6.52 (1H, d, J ¼ 1.8 Hz), 6.30 (1H, d, J ¼ 1.8 Hz),
4.85 (1H, m), 4.78 (2H, s), 4.24 (2H, q, J ¼ 7.1 Hz), 2.95 (2H, m), 2.20
(3H, s), 1.92 (4H, q, J ¼ 6.8 Hz), 1.35 (3H, t, J ¼ 7.1 Hz), 1.26 (9H, s),
4.10.20. (S)-2-(5-(3-(4-(3,3-Dimethyl-2-oxobutoxy)-3-
methylphenyl)pentan-3-yl)-1-methylpyrrole-2-carboxamido)
succinic acid (3d)
0.65 (6H, t, J ¼ 6.8 Hz); 13C NMR (300 MHz, CDCl3)
d: 210.76, 175.37,
174.77, 162.02, 153.97, 140.60, 131.61, 130.46, 126.36, 125.95, 125.80,
122.69,112.96,110.33, 69.55, 48.37, 44.92, 43.85, 43.19, 36.09, 30.25,
25.52, 17.07, 16.56, 8.50.
The same method as 3a and the starting material was 1d. Faint
yellow solid, 0.15 g, 87% yield. Mp: 92e94 ꢀC; HRMS, ESIþ, m/z:
Calcd for C28H39N2O7 (M þ H)þ, 515.2752; found, 515.2740; 1H NMR
4.10.25. 2-(5-(3-(4-(2-Hydroxy-3,3-dimethylbutoxy)-3-
methylphenyl)pentan-3-yl)-1-methylpyrrole-2-carboxamido)acetic
acid (4a)
(300 MHz, CDCl3)
d
: 6.93 (1H, s), 6.81 (1H, d, J ¼ 2.3 Hz), 6.73 (1H, d,
J ¼ 2.3 Hz), 6.42 (1H, d, J ¼ 8.5 Hz), 6.29 (1H, d, J ¼ 8.5 Hz), 4.89 (1H,
m), 4.78 (2H, s), 3.75 (3H, s), 2.98 (1H, m), 2.87 (1H, m), 2.16 (3H, s),
1.84 (4H, q, J ¼ 7.2 Hz), 1.17 (9H, s), 0.56 (6H, t, J ¼ 7.2 Hz); 13C NMR
To a solution of 3a (0.31 mmol) in methanol (10 mL), NaBH4
(6.2 mmol) was added portionwise at 0 ꢀC. The reaction mixture
was stirred at 25 ꢀC for 0.5 h and then added H2O (10 mL). The
solution was extracted with ethyl acetate, then the organic phase
was separated and the aqueous phase was extracted with ethyl
acetate. The combined organic phases were washed with brine and
then dried over anhydrous Na2SO4 and evaporated. The residue was
purified by column chromatography with dichloromethane/meth-
anol (20/1, v/v) to give compound 4aþas faint yellow solid (0.12 g,
85% yield). Mp: 96e98 ꢀC; HRMS, ESI , m/z: Calcd for C26H39N2O5
(300 MHz, CDCl3) d: 210.76, 175.49, 174.89, 162.18, 153.97, 140.59,
131.57, 130.42, 127.54, 125.98, 125.74, 123.45, 112.79, 110.34, 69.59,
48.33, 44.78, 43.19, 36.80, 36.16, 30.16, 26.33, 16.58, 8.48.
4.10.21. 2-(5-(3-(4-(3,3-Dimethyl-2-oxobutoxy)-3-methylphenyl)
pentan-3-yl)-1-ethylpyrrole-2-carboxamido)acetic acid (3e)
The same method as 3a and the starting material was 1e. Faint
yellow solid, 0.26 g, 82% yield. Mp: 104e106 ꢀC; HRMS, ESIþ, m/z:
Calcd for C27H39N2O5 (M þ H)þ, 471.2853; found, 471.2853; 1H NMR
(M þ H)þ, 459.2853; found, 459.2858; 1H NMR (500 MHz, CDCl3)
d:
(300 MHz, CDCl3)
d
: 6.92 (1H, s), 6.88 (1H, d, J ¼ 8.5 Hz), 6.52 (1H, d,
6.93 (1H, s), 6.92 (1H, d, J ¼ 8.4 Hz), 6.63 (1H, d, J ¼ 8.4 Hz), 6.37 (1H,
d, J ¼ 1.8 Hz), 6.25 (1H, d, J ¼ 1.8 Hz), 4.01 (2H, m), 3.92 (2H, m), 3.81
(1H, m), 3.62 (3H, s), 2.10 (3H, s), 1.92 (4H, q, J ¼ 6.8 Hz), 0.96 (9H, s),
J ¼ 1.9 Hz), 6.41 (1H, d, J ¼ 8.5 Hz), 6.23 (1H, d, J ¼ 1.9 Hz), 4.77 (2H,
s), 4.21 (2H, q, J ¼ 7.1 Hz), 4.00 (2H, m), 2.17 (3H, s), 1.83 (4H, q,
J ¼ 7.3 Hz), 1.27 (3H,t, J ¼ 7.1 Hz), 1.17 (9H, s), 0.56 (6H, t, J ¼ 7.3 Hz);
0.57 (6H, t, J ¼ 6.8 Hz); 13C NMR (500 MHz, CDCl3)
d: 177.31, 163.04,
13C NMR (300 MHz, CDCl3)
d: 210.42, 173.13, 162.48, 154.02, 140.59,
154.45, 140.46, 131.14, 130.13, 126.14, 125.69, 125.51, 124.68, 112.04,
110.38, 69.48, 67.67, 44.62, 43.97, 36.42, 33.63, 29.10, 26.06, 16.52,
8.45.
131.47, 130.44, 125.90, 125.78, 125.41, 122.89, 112.81, 110.13, 69.49,
44.91, 43.79, 43.18, 41.37, 30.26, 26.31, 17.09, 16.59, 8.47.

W. Shen et al. / European Journal of Medicinal Chemistry 69 (2013) 768e778
777
4.10.26. 3-(5-(3-(4-(2-Hydroxy-3,3-dimethylbutoxy)-3-
methylphenyl)pentan-3-yl)-1-methylpyrrole-2-carboxamido)
propanoic acid (4b)
The same method as 4a and the starting material was 3b. Faint
yellow solid, 0.16 g, 83% yield. Mp: 158e160 ꢀC; HRMS, ESIþ, m/z:
Calcd for C27H41N2O5 (M þ H)þ, 473.3010; found, 473.3014; 1H NMR
J ¼ 8.4 Hz), 6.40 (1H, d, J ¼ 1.9 Hz) 6.20 (1H, d, J ¼ 1.9 Hz), 4.12 (2H, q,
J ¼ 6.9 Hz), 4.02 (1H, m), 3.86 (1H, m), 3.65 (1H, m), 3.29 (2H, t,
J ¼ 5.5 Hz), 2.22 (2H, t, J ¼ 5.5 Hz), 2.11 (3H, s), 1.85 (4H, q,
J ¼ 6.6 Hz), 1.19 (3H, t, J ¼ 6.9 Hz), 0.96 (9H, s), 0.58 (6H, t,
J ¼ 6.6 Hz); 13C NMR (300 MHz, CDCl3)
d: 179.30, 162.31, 154.48,
140.47, 131.14, 130.21, 125.79, 125.50, 124.23, 111.62, 110.34, 69.51,
44.75, 43.51, 39.70, 37.08, 36.11, 33.66, 30.00, 26.07, 17.11, 16.56,
14.17, 8.47.
(CDCl3, 300 Hz)
d
: 6.92 (1H, s), 6.93 (1H, d, J ¼ 8.1 Hz), 6.80 (1H, d,
J ¼ 1.3 Hz), 6.63 (1H, d, J ¼ 8.1 Hz), 6.25 (1H, d, J ¼ 1.3 Hz), 4.01 (2H,
m), 3.81 (1H, m), 3.61 (3H, s), 3.27 (2H, t, J ¼ 5.6 Hz), 2.21 (2H, t,
J ¼ 5.6 Hz), 2.09 (3H, s),1.83 (4H, q, J ¼ 6.9 Hz), 0.94 (9H, s), 0.57 (6H,
4.10.31. (S)-2-(5-(3-(4-(2-Hydroxy-3,3-dimethylbutoxy)-3-
methylphenyl)pentan-3-yl)-1-ethylpyrrole-2-carboxamido)
propanoic acid (4g)
The same method as 4a and the starting material was 3g. Faint
yellow solid, 0.23 g, 88% yield. Mp: 110e111 ꢀC; HRMS, ESIþ, m/z:
Calcd for C28H43N2O5 (M þ H)þ, 487.3166; found, 487.3179; 1H NMR
t, J ¼ 6.9 Hz); 13C NMR (300 MHz, CDCl3)
d: 179.66, 162.58, 154.47,
140.41, 131.10, 130.11, 125.91, 125.63, 125.52, 124.91, 111.56, 110.31,
69.50, 44.54, 37.53, 36.48, 36.39, 33.66, 29.75, 29.66, 26.06, 16.52,
8.43.
4.10.27. (S)-2-(5-(3-(4-(2-Hydroxy-3,3-dimethylbutoxy)-3-
methylphenyl)pentan-3-yl)-1-methylpyrrole-2-carboxamido)
propanoic acid (4c)
The same method as 4a and the starting material was 3c. Faint
yellow solid, 0.18 g, 87% yield. Mp: 92e93 ꢀC; HRMS, ESIþ, m/z: Calcd
for C27H41N2O5 (M þ H)þ, 473.3010; found, 473.3016; 1H NMR
(300 MHz, CDCl3)
d
: 7.02 (1H, d, J ¼ 8.1 Hz), 7.01 (1H, s), 6.72 (1H, d,
J ¼ 8.1 Hz), 6.61 (1H, d, J ¼ 1.3 Hz), 6.27 (1H, d, J ¼ 1.3 Hz), 4.61 (1H,
m), 4.31 (2H, m), 4.09 (2H, q, J ¼ 7.2 Hz), 3.73 (1H, m), 2.20 (3H, s),
1.92 (4H, q, J ¼ 6.9 Hz),1.49 (3H, d, J ¼ 6.9 Hz),1.36 (3H, t, J ¼ 7.2 Hz),
1.01 (9H, s), 0.65 (6H, t, J ¼ 6.9 Hz); 13C NMR (300 MHz, CDCl3)
d:
180.16, 162.11, 154.43, 140.41, 131.24, 130.23, 125.81, 125.46, 124.44,
123.99, 112.04, 110.34, 69.50, 65.54, 50.83, 44.82, 43.50, 33.65,
30.12, 26.07, 18.13, 17.09, 16.57, 8.51.
(300 MHz, CDCl3)
d
: 6.93 (1H, s), 6.64 (1H, d, J ¼ 8.2 Hz), 6.44 (1H, d,
J ¼ 1.8 Hz), 6.22 (1H, d, J ¼ 1.8 Hz), 6.16 (1H, d, J ¼ 8.2 Hz), 4.55 (1H, m),
4.04 (1H, m), 3.80 (3H, s), 3.77 (1H, m), 3.65 (1H, m), 2.10 (3H, s), 1.85
(4H, q, J ¼ 6.8 Hz), 1.41 (3H, d, J ¼ 7.0 Hz), 0.94 (9H, s), 0.58 (6H, t,
4.10.32. (S)-2-(5-(3-(4-(2-Hydroxy-3,3-dimethylbutoxy)-3-
methylphenyl)pentan-3-yl)-1-ethylpyrrole-2-carboxamido)succinic
acid (4h)
J ¼ 6.8 Hz); 13C NMR (300 MHz, CDCl3)
d: 176.53, 162.15, 154.36,
140.24, 131.45, 130.27, 127.13, 125.76, 125.50, 123.75, 112.09, 109.98,
69.03, 48.08, 44.72, 36.73, 33.53, 30.04, 26.02, 20.71,17.99,16.63, 8.44.
The same method as 4a and the starting material was 3g. Faint
yellow solid, 0.24 g, 82% yield. Mp: 280e284 ꢀC; HRMS, ESIþ, m/z:
Calcd for C29H43N2O7 (M þ H)þ, 531.3065; found, 531.3064; 1H
4.10.28. (S)-2-(5-(3-(4-(2-Hydroxy-3,3-dimethylbutoxy)-3-
methylphenyl)pentan-3-yl)-1-methylpyrrole-2-carboxamido)
succinic acid (4d)
The same method as 4a and the starting material was 3d. Faint
yellow solid, 0.16 g, 82% yield. Mp: 223e225 ꢀC; HRMS, ESIþ, m/z:
Calcd for C28H41N2O7 (M þ H)þ, 517.2908; found, 517.2903; 1H NMR
NMR (300 MHz, CDCl3)
d
: 6.96 (1H, s), 6.96 (1H, d, J ¼ 8.5 Hz), 6.79
(1H, d, J ¼ 8.5 Hz), 6.71 (1H, d, J ¼ 1.8 Hz), 6.36 (1H, d, J ¼ 1.8 Hz),
4.75 (1H, m), 4.25 (2H, m), 3.76 (2H, q, J ¼ 6.3 Hz), 3.45 (1H, m), 2.50
(2H, m), 2.12 (3H, s), 1.90 (4H, q, J ¼ 6.0 Hz), 1.22 (3H, t, J ¼ 6.3 Hz),
0.92 (9H, s), 0.58 (6H, t, J ¼ 6.0 Hz); 13C NMR (300 MHz, DMSO-d6)
(300 MHz, CDCl3)
d
: 7.00 (1H, d, J ¼ 7.9 Hz), 6.97 (1H, s), 6.79 (1H, d,
d: 177.68, 173.25, 160.20, 154.43, 139.41, 130.20, 129.30, 125.49,
J ¼ 7.9 Hz), 6.67 (1H, d, J ¼ 2.2 Hz), 6.38 (1H, d, J ¼ 2.2 Hz), 4.81 (1H,
m), 4.29 (1H, m), 4.02 (1H, m), 3.77 (3H, s), 3.45 (1H, m), 2.50 (1H,
m), 2.39 (1H, m), 2.12 (3H, s), 1.89 (4H, q, J ¼ 6.9 Hz), 0.92 (9H, s),
124.51, 124.09, 124.03, 111.15, 110.22, 75.91, 69.83, 44.14, 42.75,
40.33, 38.66, 33.92, 29.34, 26.05, 17.26, 16.48, 8.42.
0.59 (6H, t, J ¼ 6.9 Hz); 13C NMR (300 MHz, DMSO-d6)
d
: 179.45,
4.11. Measurement of VDR binding affinity by HL-60 differentiation-
inducting assay
175.24, 160.35, 154.45, 139.44, 130.15, 129.31, 125.59, 125.44, 124.87,
124.58, 110.94, 110.22, 75.92, 69.85, 44.06, 40.06, 38.95, 36.07,
33.93, 29.29, 26.06, 16.49, 8.41.
Differentiation of HL-60 cells was quantified in terms of
morphology using nitroblue tetrazolium (NBT) reduction assay [12].
Target cell line which was grown well was diluted to 1 ꢁ 104 cells/
4.10.29. 2-(5-(3-(4-(2-Hydroxy-3,3-dimethylbutoxy)-3-
methylphenyl)pentan-3-yl)-1-ethylpyrrole-2-carboxamido)acetic
acid (4e)
The same method as 4a and the starting material was 3e. Faint
yellow solid, 0.18 g, 84% yield. Mp: 160e162 ꢀC; HRMS, ESIþ, m/z:
Calcd for C27H41N2O5 (M þ H)þ, 473.3010; found, 473.3017; 1H NMR
mL. Then 200 mL of the obtained cell suspension was added to each
well of 96-well culture plates and the tested compounds at pre-set
concentration in DMSO were added. The HL-60 cells were incu-
bated for 96 h at 37 ꢀC in a humidified atmosphere of 5% CO2/air
without medium change. After incubation, the nitroblue tetrazo-
lium (NBT) and 12-O-tetradecanoylphorbol-13-acetate (TPA) was
added. Then the mixture was incubated at 37 ꢀC for 25 min, and cells
(300 MHz, CDCl3)
d
: 7.02 (1H, d, J ¼ 8.4 Hz), 6.94 (1H, s), 6.53 (1H, d,
J ¼ 8.4 Hz), 6.38 (1H, d, J ¼ 1.3 Hz), 6.33 (1H, d, J ¼ 1.3 Hz), 4.13 (2H,
m), 4.01 (2H, m), 3.82 (2H, q, J ¼ 6.9 Hz), 3.63 (1H, m), 2.11 (3H, s),
1.85 (4H, q, J ¼ 6.3 Hz), 1.16 (3H, t, J ¼ 6.9 Hz), 0.96 (9H, s), 0.57 (6H,
were collected by centrifugation. Each sample was added 150 mL
DMSO and the value of absorbance was measured at 570 nm then
the EC50 value was calculated. The compounds whose efficacies
were larger than 85% were tested of the second screening. And the
final EC50 value was calculated for each tested compound.
t, J ¼ 6.3 Hz); 13C NMR (500 MHz, CDCl3)
d: 177.14, 162.71, 154.47,
140.48, 131.28, 130.19, 125.79, 125.51, 124.36, 123.93, 112.26, 110.42,
69.52, 44.77, 43.51, 33.65, 30.06, 26.08, 17.05, 16.53, 8.49.
4.10.30. 3-(5-(3-(4-(2-Hydroxy-3,3-dimethylbutoxy)-3-
methylphenyl)pentan-3-yl)-1-ethylpyrrole-2-carboxamido)
propanoic acid (4f)
4.12. Measurement of antiproliferation activities by MTT assay
The cytotoxicity was evaluated by MTT assay using HaCaT cells
and MCF-7 cells. First, target cells which were grown well were
The same method as 4a and the starting material was 3f. Faint
yellow solid, 0.17 g, 85% yield. Mp: 163e166 ꢀC; HRMS, ESIþ, m/z:
Calcd for C28H43N2O5 (M þ H)þ, 487.3166; found, 487.3175; 1H NMR
diluted to 1 ꢁ 104 cells/mL. Then 200
mL of the obtained cell sus-
pension was added to each well of 96-well culture plates and the
tested compounds at pre-set concentration in DMSO were added.
(CDCl3, 300 Hz)
d
: 6.94 (1H, s), 6.95 (1H, d, J ¼ 8.4 Hz), 6.65 (1H, d,

778
W. Shen et al. / European Journal of Medicinal Chemistry 69 (2013) 768e778
The target cells were incubated at 37 ꢀC in a humidified atmosphere
of 5% CO2/air without medium change. After 48 h incubation, MTT
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(5 mg/mL) and DMEM culture medium (200
ml) was added to each
well and reacted for 4 h. The medium was replaced by 150
mL DMSO
to solubilize the purple formazan crystals produced. The absor-
bance at 570 nm of each well was measured on an ELISA plate
reader. And the IC50 value was calculated by a dose of Logarithmic
linear regression analysis. The compounds whose efficacies were
larger than 85% were tested of the second screening. And the final
IC50 value was calculated for each tested compound.
4.13. In vivo calcemic activity assay
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ICR mice, aged 10 weeks, of clean grade, weighing 18e22 g, were
provided by Jiangsu University and fed with a vitamin D-replete diet
(0.2% calcium, 1% phosphate, and 2000 units vitamin D/kg) for 7
days. The hypercalcemic effect of the analogs was tested by daily s.c.
injections of serial dilutions of tacalcitol or analogs for 7 consecutive
days. Serum calcium was measured as calcemic parameter using a
commercially available kit (Shanghai ShiFeng Biological) [16].
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R.P. Bissonnette, R.A. Heyman, A.M. Nadzan, M. Reichman, E.A. Allegretto,
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nuclear vitamin D and androgen receptors: dual activities and metabolic
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Acknowledgments
We would like to thank Professor Tao Lu and Dr. Haoliang Yuan
for docking study. This study was supported by the Project Program
of State Key Laboratory of Natural Medicines, China Pharmaceutical
University (SKLNMZZ201206, SKLNMZZCX201306) and 111 Project
from the Ministry of Education of China and the State Adminis-
tration of Foreign Expert Affairs of China (No. 111-2-07).
[13] K. Maruyama, T. Noguchi-Yachide, K. Sugita, Y. Hashimoto, M. Ishikawa, Novel
selective anti-androgens with
a
diphenylpentane skeleton, Bioorg. Med.
Chem. Lett. 20 (2010) 6661e6666.
ꢀ
ꢀ
[14] J. Wietrzyk, M. Pe1czynska, J. Madej, S. Dzimira, H. Kusnierczyk, A. Kutner,
W. Szelejewski, A. Opolski, Toxicity and antineoplastic effect of (24R)-1,24-
dihydroxyvitamin D3 (PRI-2191), Steroids 69 (2004) 629e635.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2013.09.015.
[15] S. Kakuda, K. Okada, H. Eguchi, K. Takenouchi, W. Hakamata, M. Kurihara,
M. Takimoto-Kamimura, Structure of the ligand-binding domain of rat VDR in
complex with the nonsecosteroidal vitamin D3 analogue YR301, Acta Crys-
tallogr. Sect. F Struct. Biol. Cryst Commun. 64 (2008) 970e973.
[16] L. Verlinden, A. Verstuyf, M. Van Camp, S. Marcelis, K. Sabbe, X.Y. Zhao, P. De
Clercq, M. Vandewalle, R. Bouillon, Two novel 14-Epi-analogues of 1,25-
dihydroxyvitamin D3 inhibit the growth of human breast cancer cells in vitro
and in vivo, Cancer Res. 60 (2000) 2673e2679.
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