Synthesis of mixed (E, Z)-, (E)-, and (Z)-norendoxifen with dual aromatase inhibitory and estrogen receptor modulatory activities

Article abstract of DOI:10.1021/jm400364h

The first synthesis of the tamoxifen metabolite norendoxifen is reported. This included syntheses of (E)-norendoxifen, (Z)-norendoxifen, and (E,Z)-norendoxifen isomers. (Z)-Norendoxifen displayed affinity for aromatase (K_i 442 nM), estrogen receptor-α (EC₅₀ 17 nM), and estrogen receptor-β (EC₅₀ 27.5 nM), while the corresponding values for (E)-norendoxifen were aromatase (K_i 48 nM), estrogen receptor-α (EC₅₀ 58.7 nM), and estrogen receptor-β (EC₅₀ 78.5 nM). Docking and energy minimization studies were performed with (E)-norendoxifen on aromatase, and the results provide a foundation for structure-based drug design. The oral pharmacokinetic parameters for (E,Z)-norendoxifen were determined in mice, and (Z)-norendoxifen was found to result in significantly higher plasma concentrations and exposures (AUC values) than (E)-norendoxifen. The affinities of both isomers for aromatase and the estrogen receptors, as well as the pharmacokinetic results, support the further development of norendoxifen and its analogues for breast cancer treatment.

Full text of DOI:10.1021/jm400364h

Article
pubs.acs.org/jmc
Synthesis of Mixed (E,Z)‑, (E)‑, and (Z)‑Norendoxifen with Dual
Aromatase Inhibitory and Estrogen Receptor Modulatory Activities
Wei Lv,† Jinzhong Liu,‡ Deshun Lu,‡ David A. Flockhart,‡ and Mark Cushman*,†
†Department of Medicinal Chemistry and Molecular Pharmacology, College of Pharmacy and The Purdue University Center for
Cancer Research, Purdue University, West Lafayette, Indiana 47907, United States
‡Division of Clinical Pharmacology, Department of Medicine, Indiana University School of Medicine, Indianapolis, Indiana 46202,
United States
ABSTRACT: The first synthesis of the tamoxifen metabolite
norendoxifen is reported. This included syntheses of (E)-
norendoxifen, (Z)-norendoxifen, and (E,Z)-norendoxifen iso-
mers. (Z)-Norendoxifen displayed affinity for aromatase (Ki
442 nM), estrogen receptor-α (EC50 17 nM), and estrogen
receptor-β (EC50 27.5 nM), while the corresponding values for
(E)-norendoxifen were aromatase (Ki 48 nM), estrogen
receptor-α (EC50 58.7 nM), and estrogen receptor-β (EC50 78.5 nM). Docking and energy minimization studies were
performed with (E)-norendoxifen on aromatase, and the results provide a foundation for structure-based drug design. The oral
pharmacokinetic parameters for (E,Z)-norendoxifen were determined in mice, and (Z)-norendoxifen was found to result in
significantly higher plasma concentrations and exposures (AUC values) than (E)-norendoxifen. The affinities of both isomers for
aromatase and the estrogen receptors, as well as the pharmacokinetic results, support the further development of norendoxifen
and its analogues for breast cancer treatment.
superior to tamoxifen in the treatment of postmenopausal
women with ER-positive breast cancer. Generally, compared to
tamoxifen, AIs are more effective, with increased disease-free
survival, enhanced safety profile, and better tolerability.7−11
However, the use of AIs is also accompanied by significant side
effects, including reduction of the bone density, severe
musculoskeletal pain, and increased frequency of cardiovascular
and thromboembolic events.12−16 Most of these side effects of
AIs have been attributed to global depletion of estrogen. There
has been a need to develop new aromatase inhibitors with novel
mechanisms that may cause fewer side effects.
We recently reported the human metabolism of tamoxifen to
the potent aromatase inhibitor norendoxifen of undetermined
stereochemistry (Figure 1).17 Norendoxifen competitively
inhibits recombinant human aromatase with an IC50 of 90
nM. Norendoxifen also shows good selectivity toward
aromatase among other CYP450 enzymes, including CYP2B6,
2C9, 2C19, 2D6, and 3A. The high potency and selectivity
make norendoxifen an ideal lead compound for developing new
therapeutic agents for breast cancer. Two unique features make
norendoxifen attractive as a lead compound for the develop-
ment of new breast cancer chemotherapeutic agents. (1) The
close structural relationship of norendoxifen to tamoxifen
suggests that it would be possible to make a series of
compounds with dual aromatase inhibitory activity and ER
modulatory activity. The aromatase inhibitory activity could
efficiently block estrogen biosynthesis in the breast to inhibit
INTRODUCTION
■
Breast cancer is the most frequently diagnosed cancer in
women, and it is also the second leading cause of cancer-related
deaths in women in the United States. It is estimated that
226870 new cases of invasive breast cancer and 63300 new
cases of in situ breast cancer occurred among women in the
United States in 2012, along with 39510 breast cancer deaths.1
It has been established that 60% of premenopausal and 75% of
postmenopausal breast cancer patients are estrogen receptor
(ER) positive, with cancers that are dependent on estrogen for
proliferation, making them suitable for hormonal therapy
(antiestrogen treatment).2 The selective estrogen receptor
modulator tamoxifen (Figure 1) has been widely used for more
than 30 years as the first-line hormonal therapy and adjuvant
therapy after surgery for ER-positive breast cancer patients.
Tamoxifen blocks ER-stimulated tumor growth in the breast,
providing a 47% reduction of recurrence and 26% reduction of
mortality as documented in a 5-year treatment clinical trial.3
Tamoxifen also acts as an ER agonist in bones, uterus, and
other tissues, which is beneficial for preventing bone
demineralization in postmenopausal women.4 Although the
benefits are prominent, the use of tamoxifen suffers from
intrinsic and acquired drug resistance, and it has only been
proven to be effective for a maximum of 10 years.5,6 In
postmenopausal women, an alternative strategy to block the
effects of estrogen in promoting breast cancer is to use
aromatase inhibitors (AIs) to reduce estrogen biosynthesis.
Currently, three AIs (letrozole, anastrozole, and exemestane,
Figure 1) have been approved for breast cancer treatment.
Several clinical trials have demonstrated that the use of AIs is
Received: March 11, 2013
© XXXX American Chemical Society
A
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a
Scheme 1. Synthesis of Mixed (E,Z)-Norendoxifen 4
a
Reagents and conditions: (a) propiophenone, Zn, TiCl4, THF, 88%;
(b) ICH2CONH2, acetone, K2CO3, 45%; (c) LAH, AlCl3, THF, 82%.
The synthesis of mixed (E,Z)-norendoxifen revealed that the
E- and Z- isomers had strikingly different solubilities in
methanol. Trituration of the solid (E,Z)-norendoxifen with
methanol preferentially dissolved the Z isomer, allowing the
pure (E)-norendoxifen (E:Z > 100:1) to be filtered off in nearly
40% yield (Scheme 2). The stereochemistry of (E)-norendox-
Figure 1. The structures of the selective estrogen receptor modulator
tamoxifen, the aromatase inhibitors letrozole, anastrozole, and
exemestane, and the tamoxifen metabolite norendoxifen.
Scheme 2. Synthesis of (E)-Norendoxifen (E-4)
the tumor growth, while the estrogen receptor modulatory
activity could possibly ameliorate the side effects in bones and
other tissues caused by estrogen depletion. In fact, the
combination of the aromatase inhibitor anastrozole with the
selective estrogen receptor modulator tamoxifen has been
reported to result in fewer fractures than when anastrozole was
used alone.18 (2) Because norendoxifen is a metabolite of the
widely used drug tamoxifen, many patients have already been
exposed to it.
This report documents the first synthesis of (E,Z)-
norendoxifen, as well as the syntheses of (E)-norendoxifen
(E:Z > 100:1) and (Z)-norendoxifen (E:Z 1:10). The
aromatase inhibitory activity and ER binding affinities for
mixed (E,Z)-, as well as (E)- and (Z)-norendoxifen, were also
evaluated. The possible binding mode of (E)-norendoxifen with
aromatase was explored by molecular modeling methods. The
results of these studies will facilitate future structure-based drug
design efforts.
ifen was confirmed by NOE NMR spectroscopy. Irradiation of
the Ha aromatic protons (Scheme 2) produced a clear NOE
with the Hb benzylic methylene protons.
RESULTS AND DISCUSSION
■
Synthesis of Mixed (E,Z)-, (E)-, and (Z)-Norendoxifen.
The syntheses of 4-hydroxytamoxifen (with a dimethylaminoe-
thoxy side chain) and endoxifen (with a methylaminoethoxy
side chain) have previously been reported.19−21 However, the
synthesis of norendoxifen (with an aminoethoxy side chain) has
not been reported in the chemical literature. This report
describes the first synthesis of norendoxifen via a short and
efficient synthetic pathway (Scheme 1). Starting from the 4,4′-
dihydroxybenzophenone 1, the McMurry coupling reaction
with propiophenone afforded the diphenol 2 in high yield
(88%).19 The diphenol 2 was then monoalkylated with 2-
iodoacetamide in the presence of potassium carbonate to
provide the amide 3. In the last step, the reduction of the amide
proceeded well with lithium aluminum hydride to afford
norendoxifen 4 in high yield (82%) as a 1:1 mixture of E- and
Z- isomers.
To prepare (Z)-norendoxifen, Gauthier et al.’s procedure22
was used to stereospecifically synthesize the monopivalate 6
(Scheme 3). The McMurry coupling reaction of 522 with
propiophenone provided the crude product of 6 as a mixture
with E:Z ratio 10:1. Trituration of the crude product with
methanol gave 6 as the pure E isomer (E:Z > 100) in 69% yield.
In the subsequent alkylation reaction, the isomerization of 6
occurred. By minimizing the amount of reaction solvent and
use of 2-iodoacetamide in excess, the crude product 7 was
obtained with E:Z ratio 13:1. Further purification by trituration
with methanol improved E:Z to 25:1 with 67% yield. In the last
step, the reduction of the amide and removal of the pivaloyl
group were accomplished in one pot. Unfortunately, partial
isomerization occurred, and (Z)-norendoxifen (Z-4) was
obtained with an E:Z ratio of 1:10. Further trituration of the
product with many different solvents (including methanol,
B
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a
Scheme 3. Synthesis of (Z)-Norendoxifen (Z-4)
Table 1. The Aromatase Inhibitory Activity and Estrogen
Receptor Binding Affinities of Norendoxifen and Structurally
a,b,c,d
Related Compounds
aromatase (IC50,
ER-α (EC50,
ER-β (EC50,
nM, or percent aromatase nM, or percent nM or percent
compd
inhibition)
(Ki, nM)
competition)
competition)
(E,Z)-4
E-4
102.2 32.7
76.8 33.3
1029 318
24880 1360
77 9.5
48 0.4
26.9 4.8
58.7 1.0
17.0 1.9
35.2 16.8
78.5 57.3
27.5 14.3
306.9 + 106.4
Z-4
442
ND
8
2
80%
competition
3
6
7
9257 195
8% inhibition
0% inhibition
ND
ND
ND
57%
71%
competition
competition
68%
competition
68%
competition
56%
competition
45%
competition
a
b
ND = not determined. The values are mean values of at least three
c
experiments. Percent aromatase inhibition was determined at the
concentration of 50 μM for each compound. Percent estrogen
d
receptor competition was determined at the concentration of 100 μM
for each compound.
a
Reagents and conditions: (a) NaH, t-BuCOCl, THF, 36%; (b)
propiophenone, Zn, TiCl4, THF, 69%; (c) ICH2CONH2, acetone,
K2CO3, 67%; (d) LAH, AlCl3, THF, 70%.
(90 nM) vs aromatase,17 while in the present case the IC50
values of (E,Z)-norendoxifen (102 nM) and (E)-norendoxifen
(77 nM) are different. During the course of the present studies,
an appreciation was gained for how readily isomerization can
occur and precautions were implemented to prevent it.
ethanol, hexanol, ethyl acetate, propanol, THF, and 2-
propanol) did not improve the E:Z ratio.
The stabilities of (E)- and (Z)-norendoxifen in different
solvents were also tested. Interestingly, if chloroform (or
dichloromethane) was used as the single solvent, both (E)- and
(Z)-norendoxifen underwent a fast isomerization to the mixed
(E,Z)-norendoxifen. However, if methanol or methanol−
chloroform mixture (v/v 1:1, good solubility for E-norendox-
ifen) was used as solvent, the isomerization rate was
significantly decreased and the sample could be stored for at
least a few weeks without substantial isomerization. This
observation is similar to the isomerization of (E,Z)-4-
hydroxytamoxifen reported by Yu et al.19 The detailed
mechanism of isomerization is still not fully understood.23,24
Biological Activity Evaluation. All of the compounds
were tested for inhibition of aromatase activity and estrogen
receptor binding affinities (including both ER-α and ER-β,
Table 1). The IC50 value against aromatase for the mixed (E,Z)-
norendoxifen (4) is 102 nM, which is quite close to the value
(IC50 90 nM) previously reported.17 As expected, the mixed
(E,Z)-norendoxifen also showed good binding affinities toward
both ER-α and ER-β, with EC50 values in the nanomolar range
(26.9 and 35.2 nM, respectively). The (E)-norendoxifen (E-4)
is about 10-fold more potent than the (Z)-norendoxifen (Z-4)
when tested against aromatase (IC50 77 vs 1029 nM). Because
(Z)-norendoxifen (E:Z ratio 1:10) still contains a certain
amount of (E)-norendoxifen (9%), the actual aromatase
inhibitory activity for (Z)-norendoxifen is even weaker. For
ER binding affinity, (Z)-norendoxifen is about 3-fold more
potent than (E)-norendoxifen when tested against ER-α and
ER-β, which is similar to the observation with 4-hydroxytamox-
ifen.25 The aromatase inhibitory activity of diphenol 2 (IC50 25
μM) is significantly weaker than the mixed (E,Z)-norendoxifen.
Interestingly, the amide 3 (IC50 9.3 μM) is also 90-fold less
potent than the mixed (E,Z)-norendoxifen, indicating the
importance of the aminoethoxy side chain for aromatase
inhibition.
Molecular Modeling. A molecular docking study was
performed to investigate the norendoxifen−aromatase inter-
action and provide a basis for further structure-based drug
design. (E)-Norendoxifen (E-4) was docked into the aromatase
androgen binding site (PDB 3s79) using GOLD 3.0 software,
and the resulting structure was fully energy minimized using the
Amber 10 molecular dynamics package. The hypothetical
binding mode of (E)-norendoxifen in the active site of
aromatase is shown in Figure 2. The phenolic hydroxyl group
forms a hydrogen bond with the backbone carbonyl group of
Leu372. The ether oxygen hydrogen bonds to the side chain
hydroxyl group of Ser478. According to the testing results, the
terminal amino group is crucial for aromatase inhibitory activity
because the amide 3 is much less active than norendoxifen.
Here, the amino group displayed a dual interaction with the
carbonyl group (hydrogen bond) and carboxyl group (salt
bridge) of Asp309. The dual interaction also explains the
previous testing results that replacing the amino group with a
methylamino group (endoxifen, IC50 6 μM) or dimethylamino
group (4-hydroxytamoxifen, IC50 530 μM) significantly reduced
the aromatase inhibitory activity.17 Besides the polar
interactions, the unsubstituted phenyl ring and the ethyl
group point toward the heme and are surrounded by
hydrophobic residues Ile133 and Val370. Because this model
is consistent with the aromatase inhibitory testing results for
our compounds and the previously reported tamoxifen
metabolites, it can provide a rational basis for further
structure-based drug design.
The docking pose displayed in Figure 2 is revised relative to a
previously proposed structure.17 The new pose resulted from
the use of the recently released aromatase crystal structure
(PDB ID 3s79), which has slightly higher resolution than the
structure used before (PDB ID 3s79 3eqm). A more significant
difference is that in the present case, all crystal water molecules
were removed before docking, thus making space for the
aminoethoxy side chain of the ligand. According to MM-PBSA
It was previously reported that commercial samples of (E,Z)-
norendoxifen and (E)-norendoxifen had the same IC50 values
C
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Figure 2. The hypothetical binding mode of (E)-norendoxifen (E-4, green) in the active site of human aromatase. The stereoview is programmed for
wall-eyed (relaxed) viewing.
calculations, the docking pose displayed in Figure 2 has a lower
calculated binding energy (ΔG = −46.11 kcal/mol) than the
previous structure (ΔG = −38.80 kcal/mol). As stated above,
this pose also has the advantage of offering an explanation for
the observed decrease in aromatase inhibitory activity when the
primary amino group is changed to a methylamino group or a
dimethylamino group.17
Pharmacokinetics. To explore the pharmacokinetic profile
and oral bioavailability for (E)- and (Z)-norendoxifen, (E,Z)-
norendoxifen (4) was administered to NOD/SCID female
mice. Plasma concentrations of (E)- and (Z)-norendoxifen
were determined after a single oral dose of 100 mg/kg (Figure
3), and the pharmacokinetic parameters for both (E)- and (Z)-
The area under the curve (AUC) for (Z)-norendoxifen is also
much higher (6-fold) than that for (E)-norendoxifen. However,
both (E)- and (Z)-norendoxifen have similar half-lives (21.4 h
vs 22.7 h), which would allow them to be tested as once daily
oral agents.
The factors that could possibly contribute to the lower
exposure of (E)-norendoxifen vs (Z)-norendoxifen include
lower oral absorption, higher excretion rate, and a higher rate of
metabolism. The possibility of lower biological exposure of (E)-
norendoxifen resulting from its in vivo isomerization to (Z)-
norendoxifen is small. The parent drug, tamoxifen, is the pure Z
form, and patients receiving (Z)-tamoxifen have mainly (Z)-
tamoxifen detected in plasma samples.26 In our own work we
have analyzed more than 500 tamoxifen samples from clinical
trials in various settings, and we have not seen the (E)-
tamoxifen isomer in any of these studies, even though in some
cases the concentration of (Z)-tamoxifen was very high.
CONCLUSION
■
Mixed (E,Z)-, (E)-, and (Z)-norendoxifen have been prepared
via a short and convenient synthetic approach. The mixed
(E,Z)-norendoxifen exhibits potent aromatase inhibitory
activity (IC50 102 nM) while also displaying good binding
affinity to both ER-α (EC50 27 nM) and ER-β (EC50 35 nM).
(E)-Norendoxifen is the active component for aromatase
inhibition because it is at least 10 times more active than
(Z)-norendoxifen. In contrast, the (Z)-norendoxifen has
slightly better binding affinity toward ER-α and ER-β than
the (E)-norendoxifen. The possible binding mode of (E)-
norendoxifen with aromatase was investigated by molecular
modeling techniques, providing a result that can serve as the
basis for further structure-based drug design. It is possible that
(E)-norendoxifen or a closely related analogue could be an
effective aromatase inhibitor with fewer side effects than
aromatase inhibitors currently in use because of estrogenic
effects in noncancerous cells in muscular and skeletal tissues.
Figure 3. Time-dependent plasma concentration of (E)- and (Z)-
norendoxifen following a single oral dose of the mixed (E,Z)-
norendoxifen (100 mg/kg).
norendoxifen were calculated (Table 2). The results reveal that
(Z)-norendoxifen has a more rapid absorption than (E)-
norendoxifen (Tmax 1 h vs 2 h), and its peak plasma
concentration is 3-fold higher than that of (E)-norendoxifen.
a
Table 2. Pharmacokinetic Parameters for (E)- and (Z)-Norendoxifen in Mice
compd
Tmax (h)
Cmax (μg/mL)
Clast (μg/mL)
T1/2 (h)
AUClast (h·μg/L)
Vz_F_obs (L)
Cl_F_obs (L/h)
(E)-norendoxifen
(Z)-norendoxifen
2
1
0.7
2.1
0.1
0.9
21.4
22.7
5.8
2.8
0.5
0.091
0.016
35.1
a
The data are mean concentrations in mouse plasma (n = 3) following a single oral dose of the mixed (E,Z)-norendoxifen (100 mg/kg).
D
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CDCl3 and methanol-d4) δ 7.10−6.99 (m, 13 H), 6.84−6.82 (m, 2 H,
E isomer), 6.75−6.71 (m, 3.4 H, Z isomer), 6.63−6.61 (m, 2 H, E
isomer), 6.49−6.47 (m, 1.6 H, Z isomer), 6.40−6.39 (m, 2 H, E
isomer), 3.99 (t, J = 5.1 Hz, 2 H, E isomer), 3.83 (t, J = 5.1 Hz, 1.6 H,
Z isomer), 3.02 (t, J = 5.1 Hz, 2 H, E isomer), 2.92 (t, J = 5.1 Hz, 1.6
H, Z isomer), 2.45−2.39 (m, 3.6 H), 0.87−0.84 (m, 5.5 H). 13C NMR
(125 MHz, CDCl3 and methanol-d4) δ 157.1, 156.2, 155.3, 154.4,
142.6, 142.5, 140.7, 140.5, 137.8, 136.7, 136.3, 135.0, 134.6, 131.8,
130.5, 130.4, 129.5, 127.5, 125.6, 114.6, 113.9, 113.8, 113.0, 68.5, 68.2,
40.5, 40.4, 28.7, 13.2. ESIMS m/z 360 (MH+). HRESIMS m/z calcd
for C24H26NO2 (MH+) 360.1964, found 360.1959. Anal. Calcd for
C24H25NO2: C, 80.19; H, 7.01; N, 3.90. Found: C, 79.98; H, 7.15; N,
3.98.
EXPERIMENTAL SECTION
■
General. Melting points were determined using capillary tubes with
a Mel-Temp apparatus and are uncorrected. The nuclear magnetic
resonance (1H and 13C NMR) spectra were recorded using a Bruker
ARX300 spectrometer (300 MHz) with a QNP probe or a Bruker
DRX-2 spectrometer (500 MHz) with a BBO probe. High-resolution
mass spectra were recorded on a double-focusing sector mass
spectrometer with magnetic and electrostatic mass analyzers. The
purities of biologically important compounds are ≥95% as determined
by elemental analyses, with the observed percentages differing less that
0.40% from the calculated values. Cytochrome P450 (CYP) inhibitor
screening kit for aromatase (CYP19) was purchased from BD
Biosciences (San Jose, CA). Estrogen receptor α and β competitor
assay kits were purchased from Invitrogen (Carlsbad, CA).
(E)-4-(1-(4-(2-Aminoethoxy)phenyl)-2-phenylbut-1-enyl)phenol
(E-4). Trituration of norendoxifen (4, 35 mg, E:Z = 5:4) with
methanol (2 mL) gave pure (E)-norendoxifen (E-4, E:Z > 100:1) as a
4,4′-(2-Phenylbut-1-ene-1,1-diyl)diphenol (2).19 Zinc powder
(10.11 g, 154 mmol) was suspended in dry THF (100 mL), and the
mixture was cooled to 0 °C. TiCl4 (7.5 mL, 70 mmol) was added
dropwise under argon. When the addition was complete, the mixture
was warmed to room temperature and heated to reflux for 2 h. After
cooling down, a solution of 4,4′-dihydroxybenzophenone (2.63 g, 12.3
mmol) and propiophenone (5.15 g, 38.4 mmol) in dry THF (100 mL)
was added at 0 °C and the mixture was heated at reflux in the dark for
2.5 h. After being cooled to room temperature, the zinc dust was
filtered off and THF was evaporated. The residue was dissolved with
saturated ammonium chloride aqueous solution (150 mL) and
extracted with ethyl acetate (120 mL × 6). The organic layers were
combined and dried over Na2SO4, concentrated in vacuo, and further
purified by silica gel column chromatography (2:1 hexanes−ethyl
acetate) to provide the product 2 as yellow solid (3.4 g, 88%): mp
1
white solid (13.2 mg, 38%): mp 206−208 °C. H NMR (300 MHz,
CDCl3 and methanol-d4) δ 7.13−7.03 (m, 7 H), 6.88−6.85 (m, 2 H),
6.65−6.61 (m, 2 H), 6.42−6.39 (m, 2 H), 4.00 (t, J = 5.1 Hz, 2 H),
3.02 (t, J = 5.1 Hz, 2 H), 2.43 (q, J = 7.4 Hz, 2 H), 0.88 (t, J = 7.4 Hz,
3 H). 13C NMR (125 MHz, CDCl3 and methanol-d4) δ 157.2, 154.4,
142.5, 140.5, 137.8, 136.6, 134.6, 131.8, 130.4, 129.5, 127.5, 125.6,
113.9, 113.7, 69.0, 40.7, 28.7, 13.2. ESIMS m/z 360 (MH+). HRESIMS
m/z calcd for C24H26NO2 (MH+) 360.1964, found 360.1964. Anal.
Calcd for C24H25NO2·0.15CHCl3: C, 76.86; H, 6.72; N, 3.71. Found:
C, 76.75; H, 6.99; N, 3.49.
(E)-4-(1-(4-Hydroxyphenyl)-2-phenylbut-1-enyl)phenyl Pivalate
(6).22 Zinc powder (446 mg, 6.82 mmol) was suspended in dry
THF (8 mL), and the mixture was cooled to 0 °C. TiCl4 (0.4 mL, 3.7
mmol) was added dropwise under argon. When the addition was
complete, the mixture was warmed to room temperature and heated to
reflux for 2 h. After cooling down, a solution of 4-(4-hydroxybenzoyl)-
phenyl pivalate (5, 257 mg, 0.861 mmol) and propiophenone (374
mg, 2.79 mmol) in dry THF (10 mL) was added, and the mixture was
heated at reflux in the dark for 2.5 h. After being cooled to room
temperature, the zinc dust was filtered off and THF was carefully
evaporated. The residue was dissolved in saturated ammonium
chloride aqueous solution (30 mL) and extracted with ethyl acetate
(40 mL × 4). The organic layers were combined, dried over Na2SO4,
concentrated in vacuo, and further purified by silica gel column
chromatography (4:1 hexanes−ethyl acetate) to afford the product 6
as white solid (323 mg, 94%). The NMR spectrum showed an E:Z
ratio of 10:1. Trituration with methanol (2 mL) provided the pure E
isomer (E:Z > 100:1) as white solid (238 mg, 69%): mp 162−164 °C
200−202 °C (lit.19 200.6 °C). H NMR (300 MHz, acetone-d6) δ
1
7.16−7.04 (m, 7 H), 6.84−6.81 (m, 2 H), 6.70−6.67 (m, 2 H), 6.48−
6.45 (m, 2 H), 2.47 (q, J = 7.5 Hz, 2 H), 0.88 (t, J = 7.5 Hz, 3 H).
Anal. Calcd for C22H20O2: C, 83.51; H, 6.37. Found: C, 83.21; H, 6.31.
(E,Z)-2-(4-(1-(4-Hydroxyphenyl)-2-phenylbut-1-enyl)phenoxy)-
acetamide (3). A suspension of 2 (3.4 g, 10.7 mmol) and K2CO3
(4.22 g, 30.5 mmol) in acetone (60 mL) was heated to reflux for 10
min. A solution of 2-iodoacetamide (2.4 g, 13 mmol) in acetone (20
mL) was added in small portions over 3 h, and the mixture was stirred
for 1 h. After cooling down, acetone was evaporated and the residue
was dissolved in saturated ammonium chloride aqueous solution (120
mL) and extracted with ethyl acetate (120 mL × 5). The organic layers
were combined, dried over Na2SO4, concentrated in vacuo, and further
purified by silica gel column chromatography (1:2 hexanes−ethyl
acetate) to provide the product 3 as white solid (1.8 g, 45%) as a 5:4
1
(lit.22 165−167 °C). H NMR (300 MHz, methanol-d4) δ 7.25−7.22
mixture of E and Z isomers: mp 193−196 °C. H NMR (500 MHz,
1
methanol-d4 and CDCl3) δ 7.14−6.98 (m, 13 H), 6.91−6.89 (m, 2 H,
isomer 1), 6.76−6.73 (m, 3.2 H, isomer 2), 6.63−6.61 (m, 2 H, isomer
1), 6.55−6.53 (m, 1.6 H, isomer 2), 6.40−6.38 (m, 2 H, isomer 1),
4.46 (s, 2 H, isomer 1), 4.30 (s, 1.6 H, isomer 2), 2.45−2.40 (m, 3.8
H), 0.88−0.85 (m, 5.7 H). 13C NMR (125 MHz, methanol-d4 and
CDCl3) δ 172.5, 156.0, 155.5, 155.2, 154.6, 142.4, 141.0, 140.7, 137.7,
137.5, 137.2, 134.8, 134.4, 131.8, 131.7, 130.5, 130.3, 129.4, 127.5,
125.6, 125.5, 114.6, 114.0, 113.8, 113.2, 66.6, 66.4, 28.7, 28.6, 12.9.
Negative ion ESIMS m/z 372 (M − H+)−. HRESIMS m/z calcd for
C24H24NO3 (MH+) 374.1756, found 374.1749. Anal. Calcd for
C24H23NO3: C, 77.19; H, 6.21; N, 3.75. Found: C, 77.17; H, 6.27;
N, 3.72.
(m, 2 H), 7.16−7.08 (m, 5 H), 7.05−7.02 (m, 2 H), 6.68−6.64 (m, 2
H), 6.42−6.39 (m, 2 H), 2.46 (q, J = 7.4 Hz, 2 H), 1.36 (s, 9 H), 0.91
(t, J = 7.4 Hz, 3 H). Anal. Calcd for C27H28O3·0.8MeOH: C, 78.35; H,
7.38. Found: C, 78.26; H, 6.99.
(E)-4-(1-(4-(2-Amino-2-oxoethoxy)phenyl)-2-phenylbut-1-enyl)-
phenyl Pivalate (7). A mixture of 6 (226 mg, 0.565 mmol), 2-
iodoacetamide (434 mg, 2.35 mmol), and K2CO3 (440 mg, 3.18
mmol) was dissolved in acetone (8 mL). The suspension was heated
to reflux and stirred for 2 h. After cooling down, acetone was carefully
evaporated and the residue was dissolved in saturated ammonium
chloride aqueous solution (20 mL) and extracted with ethyl acetate
(20 mL × 3). The organic layers were combined, dried over Na2SO4,
concentrated in vacuo, and further purified by silica gel column
chromatography (1:1 hexanes−ethyl acetate) to provide the product 7
as a white solid (203 mg, 78.5%). The NMR spectrum shows an E:Z
ratio of 13:1. Trituration with methanol (3 mL) provided the pure E
isomer (E:Z > 25:1) as a white solid (174 mg, 67%): mp 167−169 °C.
1H NMR (300 MHz, CDCl3) δ 7.26−7.20 (m, 2 H), 7.18−7.04 (m, 7
H), 6.82−6.79 (m, 2 H), 6.57−6.54 (m, 2 H), 4.35 (s, 2 H), 2.47 (q, J
= 7.4 Hz, 2 H), 1.37 (s, 9 H), 0.92 (t, J = 7.4 Hz, 3 H). 13C NMR (75
MHz, CDCl3) δ 177.1, 171.2, 155.0, 149.7, 142.3, 142.0, 140.8, 137.0,
136.8, 132.2, 130.4, 129.6, 127.9, 126.2, 121.2, 113.5, 66.9, 39.1, 29.0,
27.1, 13.5. ESIMS m/z 480 (MNa+). HRESIMS m/z calcd for
C29H31NO4Na (MNa+) 480.2151, found 480.2148. Anal. Calcd for
(E,Z)-4-(1-(4-(2-Aminoethoxy)phenyl)-2-phenylbut-1-enyl)phenol
(4). A suspension of AlCl3 (492 mg, 3.69 mmol) and LiAlH4 (510 mg,
12.7 mmol) in dry THF (10 mL) was stirred under argon and cooled
to 0 °C. A solution of 3 (312 mg, 0.835 mmol) in dry THF (10 mL)
was added. The mixture was warmed to room temperature and stirred
under argon overnight. The reaction was quenched with H2O (3 mL),
and THF was evaporated. The residue was dissolved in saturated
ammonium chloride aqueous solution (20 mL) and extracted with
ethyl acetate (25 mL × 4). The organic layers were combined, dried
over Na2SO4, concentrated in vacuo, and further purified by silica gel
column chromatography (1:9 methanol−dichloromethane) to provide
the product 4 as a white solid (245 mg, 82%) consisting of a 5:4
1
mixture of E and Z isomers: mp 204−209 °C. H NMR (500 MHz,
E
dx.doi.org/10.1021/jm400364h | J. Med. Chem. XXXX, XXX, XXX−XXX

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Article
C29H31NO4: C, 76.12; H, 6.83; N, 3.06. Found: C, 75.88; H, 6.88; N,
3.06.
samples was performed by applying the linear regression equation of
the standard curve to the fluorescence response. The limit of
quantification for the metabolites of MFC was 24.7 pmol with intra-
and interassay coefficients of variation less than 10%.
(Z)-4-(1-(4-(2-Aminoethoxy)phenyl)-2-phenylbut-1-enyl)phenol
(Z-4). A suspension of AlCl3 (135 mg, 1.01 mmol) and LiAlH4 (195
mg, 5.14 mmol) in dry THF (5 mL) was stirred under argon and the
mixture was cooled to 0 °C. A solution of 7 (95.1 mg, 0.208 mmol,
E:Z > 25:1) in dry THF (5 mL) was added. The mixture was warmed
to room temperature and stirred under argon for 3 h. The reaction was
quenched with H2O (0.5 mL), and THF was evaporated. The residue
was dissolved in saturated ammonium chloride aqueous solution (20
mL) and extracted with ethyl acetate (20 mL × 5). The organic layers
were combined, dried over Na2SO4, concentrated in vacuo, and further
purified by silica gel column chromatography (1:9 methanol−
dichloromethane) to provide the product Z-4 as white solid (52 mg,
70%): mp 176−179 °C. The NMR spectrum indicated an E:Z ratio of
Kinetic Analysis of Recombinant Human Aromatase
(CYP19). The rates of metabolite formation in the presence of the
test inhibitors were compared with those in the control, in which the
inhibitor was replaced with vehicle. The extent of enzyme inhibition
was expressed as percentage of remaining enzyme activity compared to
the control. IC50 values were determined as the inhibitor
concentrations which brought about half reduction in enzyme activity
by fitting all the data to a one-site competition equation using
Graphpad Prism 5.0 (GraphPad Software Inc., San Diego, CA). To
characterize the inhibitory mechanism of norendoxifen against
aromatase (CYP19), all inhibitory data by norendoxifen at different
substrate concentrations were plotted as Lineweaver−Burk plots. The
inhibitory constant Ki values were determined by nonlinear least-
squares regression analysis using Graphpad Prism 5.0 (GraphPad
Software Inc., San Diego, CA). Before modeling the data using
nonlinear models, initial information about the inhibitory mechanism
was obtained by visual inspection of Lineweaver−Burk plots. Final
decision on the mechanism of inhibition was made on model-derived
parameters such as R2 (or R Square) and absolute sum of squares.
Binding Affinities for Recombinant Human Estrogen
Receptor α and β (ER-α and ER-β). The binding affinities for
estrogen receptor-α and -β were determined by measuring the change
of polarization value when the fluorescent estrogen ligand, ES2, was
displaced by the tested compounds. Experimental procedures were
consistent with the protocol provided by Invitrogen. The fluorescent
estrogen ligand, ES2, was provided in methanol/water (4:1, v/v) with
the concentration of 1800 nM. Recombinant human estrogen
receptor-α and -β (ER-α and ER-β) were provided in buffer (50
mM bis-tris propane, 400 mM KCl, 2 mM DTT, 1 mM EDTA, and
10% glycerol), with the concentration of 734 nM and 3800 nM,
respectively. All tested samples were dissolved in either methanol or
methanol/dichloromethane (1:1, v/v). The sample solutions (1 μL)
were mixed well with 49 μL of ES2 screening buffer (100 mM
potassium phosphate, 100 μg/mL BGG, and 0.02% NaN3). The ER-
α/ES2 complex was prepared with the fluorescent estrogen ligand ES2,
human recombinant estrogen receptor (ER), and ES2 screening buffer
with the concentration of 9 nM ES2 and 30 nM ER-α. The ER-β/ES2
complex was prepared with the fluorescent estrogen ligand ES2,
human recombinant estrogen receptor-β (ER-β), and ES2 screening
buffer with the concentration of 9 nM ES2 and 20 nM ER-β. Reactions
were initiated by adding 50 μL of ER/ES2 complex to bring the
incubation volume to 100 μL and incubated for 2 h avoiding light. The
polarization value was determined by measuring fluorescent response
using a BioTek (Winooski, VT) Synergy 2 fluorometric plate reader.
Excitation−emission wavelengths for fluorescence polarization were
485−530 nm. The polarization value in the presence of the test
competitors were compared with those in control in which the
competitor was replaced with vehicle. The extent of competition was
expressed as percentage of remaining polarization compared to the
control. EC50 values were determined as the competitor concen-
trations which brought about half reduction in polarization value by
fitting all the data to a one-site competition equation using Graphpad
Prism 5.0 (GraphPad Software Inc., San Diego, CA).
1
1:10. H NMR (300 MHz, methanol-d4) δ 7.15−7.05 (m, 5.6 H),
7.03−6.98 (m, 2.2 H), 6.95−6.92 (m, 0.20 H, E isomer), 6.76−6.72
(m, 4 H, Z isomer), 6.66−6.63 (m, 0.20 H, E isomer), 6.58−6.54 (m, 2
H, Z isomer), 6.41−6.38 (m, 0.20 H, E isomer), 4.04 (t, J = 5.3 Hz,
0.20 H, E isomer), 3.86 (t, J = 5.3 Hz, 2 H, Z isomer), 3.03 (t, J = 5.3
Hz, 0.20 H, E isomer), 2.92 (t, J = 5.3 Hz, 2 H, Z isomer), 2.48 (q, J =
7.3 Hz, 2.2 H), 0.90 (t, J = 7.3 Hz, 3.4 H). ESIMS m/z 360 (MH+).
HRESIMS m/z calcd for C24H26NO2 (MH+) 360.1964, found
360.1965. Anal. Calcd for C24H25NO2: C, 80.19; H, 7.01; N, 3.90.
Found: C, 79.99; H, 7.08; N, 3.87.
Molecular Modeling. The structure of (E)-norendoxifen was
constructed with Sybyl 7.1 software and energy minimized to a
gradient of 0.01 kcal/mol by the Powell method using Gasteiger−
Huckel charges and the Tripos force field. The crystal structure of
aromatase was obtained from the Protein Data Bank (ID: 3s79), and
the natural ligand (androstenedione) and all crystal water molecules
were removed. The (E)-norendoxifen was docked into the
androstenedione binding pocket in aromatase using the GOLD 3.0
program. The top five docking solutions according to the GOLD
fitness score were selected, and the corresponding ligand−protein
complexes were thoroughly energy minimized by the Amber 10
molecular dynamics package. The Amber parm99 force field was used
for the protein during energy minimization. For the ligand, the force
field parameters were taken from the General Amber Force Field
(GAFF), whereas the atomic partial charges were derived from the
AM1-BCC method implemented by antechamber. The ligand−protein
binding energy for each energy-minimized complex was calculated
using the MM-PBSA method. The complex with the lowest binding
energy was selected out as the best binding pose for (E)-norendoxifen.
Inhibition of Recombinant Human Aromatase (CYP19) by
Microsomal Incubations. The activity of recombinant aromatase
(CYP19) was determined by measuring the conversion rate of
fluorometric substrate 7-methoxy-4-trifluoromethylcoumarin (MFC)
to its fluorescent metabolite 7-hydroxytrifluoromethylcoumarin
(HFC). Experimental procedures were consistent with the published
methodology.27 All the incubations were performed using incubation
times and protein concentrations that were within the linear range for
reaction velocity. The fluorometric substrate, MFC, was dissolved in
acetonitrile with the final concentration of 25 mM. All tested samples
were dissolved in either methanol or methanol/dichloromethane (1:1,
v/v). The sample solutions (2 μL) were mixed well with 98 μL of
NADPH-Cofactor Mix (16.25 μM NADP+, 825.14 μM MgCl2, 825.14
μM glucose-6-phosphate, and 0.4 units/mL glucose-6-phosphate
dehydrogenase) and were prewarmed for 10 min at 37 °C. Enzyme/
substrate mix was prepared with fluorometric substrate, recombinant
human aromatase (CYP19), and 0.1 M potassium phosphate buffer
(pH 7.4). Reactions were initiated by adding 100 μL of enzyme/
substrate mix to bring the incubation volume to 200 μL and incubated
for 30 min. All the reactions were stopped by adding 75 μL of 0.1 M
Tris base dissolved in acetonitrile. The amount of fluorescent product
was determined immediately by measuring fluorescent response using
a BioTek (Winooski, VT) Synergy 2 fluorometric plate reader.
Excitation−emission wavelengths for MFC metabolite were 409−530
nm. The standard curve for MFC metabolite was constructed using the
appropriate fluorescent metabolite standards. Quantification of
Pharmacokinetics. The mixed (E,Z)-norendoxifen (100 mg/kg,
E:Z of 45:55 determined by LC-MS-MS analysis) was given to 10-
week-old NOD/SCID female mice (The Jackson Laboratory, Bar
Harbor, Maine) by oral gavages according to local IUCUC approved
animal protocol. Each mouse was bled for only one time point. Blood
samples were collected by tail vein bleeding (n = 3 for each time
point) at different time points up to 24 h. Plasma levels of both (E)-
and (Z)-norendoxifen were analyzed by LC-MS-MS method and
plotted against time. The relative concentrations of E and Z isoforms
are determined by their AUC in LC-MS-MS analysis. The identity of
each peak was confirmed by purified E and Z isomers.
Pharmacokinetic parameters were further obtained by noncompart-
mental modeling (Winnonlin 6.2).
F
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AUTHOR INFORMATION
■
Corresponding Author
*Phone: 765-494-1465. Fax: 765-494-6790. E-mail: cushman@
purdue.edu.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This research was supported by the Purdue University Center
for Cancer Research and the Indiana University Center Joint
Funding Award 206330. This work was supported in part by an
award from the Floss Endowment, provided to the Department
of Medicinal Chemistry and Molecular Pharmacology, Purdue
University. The authors acknowledge Dr. Richard F. Bergstrom
for advice on pharmacokinetic analysis, and Dr. Huaping Mo
for his help with NMR spectroscopy.
ABBREVIATIONS USED
■
AIs, aromatase inhibitors; AUC, area under the curve; ER,
estrogen receptor; LAH, lithium aluminum hydride; PDB,
Protein Data Bank
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Survivors: Review of Potential Cardiac Problems. Clin. Cancer Res.
2008, 14, 14−24.
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