New zampanolide mimics: Design, synthesis, and antiproliferative evaluation

Article abstract of DOI:10.3390/molecules25020362

Zampanolide is a promising microtubule-stabilizing agent (MSA) with a unique chemical structure. It is superior to the current clinically used MSAs due to the covalent nature of its binding to β-tubulin and high cytotoxic potency toward multidrug-resistan

Full text of DOI:10.3390/molecules25020362

molecules
Article
New Zampanolide Mimics: Design, Synthesis,
and Antiproliferative Evaluation
Guanglin Chen 1, Ziran Jiang 1, Qiang Zhang 2, Guangdi Wang 2 and Qiao-Hong Chen 1,
*
1
Department of Chemistry, California State University, Fresno, CA 93740, USA; chen.bmc@gmail.com (G.C.);
ziranjiang@mail.fresnostate.edu (Z.J.)
Department of Chemistry and RCMI Cancer Research Center, Xavier University of Louisiana,
New Orleans, LA 70125, USA; qzhang1@xula.edu (Q.Z.); gwang@xula.edu (G.W.)
Correspondence: qchen@csufresno.edu; Tel.: +1-559-278-2394
2
*
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 26 December 2019; Accepted: 14 January 2020; Published: 15 January 2020
Abstract: Zampanolide is a promising microtubule-stabilizing agent (MSA) with a unique chemical
structure. It is superior to the current clinically used MSAs due to the covalent nature of its binding
to β-tubulin and high cytotoxic potency toward multidrug-resistant cancer cells. However, its further
development as a viable drug candidate is hindered by its limited availability. More importantly,
conversion of its chemically fragile side chain into a stabilized bioisostere is envisioned to enable
zampanolide to possess more drug-like properties. As part of our ongoing project aiming to develop its
mimics with a stable side chain using straightforward synthetic approaches, 2-fluorobenzyl alcohol was
designed as a bioisosteric surrogate for the side chain based on its binding conformation as confirmed
by the X-ray structure of tubulin complexed with zampanolide. Two new zampanolide mimics
with the newly designed side chain have been successfully synthesized through a 25-step chemical
transformation for each. Yamaguchi esterification and intramolecular Horner–Wadsworth–Emmons
condensation were used as key reactions to construct the lactone core. The chiral centers at C17
and C18 were introduced by the Sharpless asymmetric dihydroxylation. Our WST-1 cell proliferation
assay data in both docetaxel-resistant and docetaxel-naive prostate cancer cell lines revealed that
compound 6 is the optimal mimic and the newly designed side chain can serve as a bioisostere for
the chemically fragile N-acetyl hemiaminal side chain in zampanolide.
Keywords: natural product; anticancer agent; synthesis; zampanolide
1. Introduction
Prostate cancer is still one of the big health concerns, as evidenced by its high incidence
and mortality rate in American men. In 2019, 174,650 new prostate cancer cases that account for
nearly 20% of all cancer cases and 31,620 prostate cancer deaths are projected to occur in the United
States [
initiated by the binding of androgen to the AR [
the main treatment for prostate cancer over 70 years. However, the median duration of ADT response
is merely 18–24 months due to inevitable progression to castration-resistant prostate cancer (CRPC) [ ].
1
]. Prostate cancer is driven by the androgen receptor (AR)-regulated gene expression that is
2
]. Androgen deprivation therapy (ADT) has thus been
3
Consequently, approximately 30,000 prostate cancer deaths in the United State each year are caused
by CRPC as well as resistance to the current treatments. The critical driving force for the continued
progression of lethal CRPC is the reactivation of AR transcriptional activity [
and second-line chemotherapeutics for CRPC are two microtubule-stabilizing cytotoxic agents (MSA),
docetaxel and cabazitaxel [ ]. Promotion of mitotic arrest is the initially well-established mechanism
underlying the action of MSAs as anticancer agents [ ]. The MSAs were recently revealed to impair
AR transcriptional activity by suppressing nuclear importation of AR as downstream of microtubule
4,5]. The current first-line
6–8
9
Molecules 2020, 25, 362; doi:10.3390/molecules25020362
www.mdpi.com/journal/molecules

Molecules 2020, 25, 362
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stabilization [10]. This alternative mechanism is independent of MSA-induced mitotic arrest and can
explain in part the efficacy of MSA in AR-driven CRPC.
The marine macrolide (
and promising MSA, which distinguishes it from the current clinically used MSAs due to (1) the covalent
nature of its binding to the taxane pocket in -tubulin as confirmed by a high-resolution X-ray structure
of zampanolide in complex with -tubulin, and (2) the very high cytotoxic potency as evidenced by
−
)-zampanolide (1, Figure 1) [11] has been demonstrated as a unique
β
α,β
low nanomolar IC50 values in suppressing multidrug-resistant cancer cell proliferation [12].
Figure 1. Zampanolide, desTHP-(−)-zampanolide, desTHPzampanolide mimics 3 and 4.
Although ( )-zampanolide has shown great potential in becoming a more effective
−
chemotherapeutic for CRPC, no in vivo assessment of its antitumor efficacy, pharmacokinetics,
and toxicity in animal models has so far been reported yet mainly because of its limited supply.
Unfortunately, the supply issue of (
sponge sources or by current reported total syntheses. The best yield for isolation of (
from marine sponge sources is merely 0.001% [12 13], and the highest yield for its syntheses is only
0.9% [14 18]. More importantly, the chemically fragile N-acyl hemiaminal side chain of zampanolide can
−
)-zampanolide cannot be addressed either by isolation from marine
−
)-zampanolide
,
–
compromise its pharmacokinetic profiles as well as the feasibility as a viable drug candidate. The easy
cleavage of the N-acyl hemiaminal side chain has been confirmed by a thermolysis reaction carried
out by Professor Smith and his coworkers [16], and the instability of zampanolide in CDCl3 has been
reported by Higa and coworkers when they first identified its structure [13]. The N-acyl hemiaminal side
chain of zampanolide is, however, very vital to retain its excellent cytotoxic potency, and the derivative
without the side chain has 10- to 1000-fold less potency in certain cell models [12
study has confirmed that a suitably designed side chain (e.g., the side chain in desTHPzampanoide
mimics and ) can function as a bioisostere for the chemically unstable N-acyl hemiaminal side chain
in desTHPzampanolide ( ) [21]. However, the bioisosteric side chains that we reported previously
do not contain a hydroxyl substituent, corresponding to the C20 alcohol of zampanolide that is
hydrogen-bonded to the carbonyl oxygen of Thr276 in -tubulin. As part of our ongoing project to
,19,20]. Our previous
3
4
2
β
develop simplified and stabilized zampanolide mimics, this paper presents the design, total synthesis,
and antiproliferative evaluation of zampanolide mimics in which the N-acyl hemiaminal side chain is
substituted by more stable 2-fluorobenzyl alcohol appendages.
2. Results and Discussion
2.1. Design of New Zampanolide Mimics
The first group of side chains in the zampanolide mimics (e.g., 3–4) previously published by
us [21] do not contain a hydroxyl group in order to render a more simplified synthesis. However,
the binding conformation confirmed by the high-resolution crystal structure of zampanolide tubulin
complex [22] indicates the hydroxyl group at the C20 position in zampanolide is hydrogen-bonded
to the carbonyl oxygen of Thr276 in β-tubulin. With this point in mind, a new side chain (S-2) was
designed as a bioisosteric substitution for the N-acyl hemiaminal side chain, an unstable moiety in
zampanolide but important for its potent cytotoxic properties. The stabilized side chain (S-2) was

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designed to replace the chemically fragile N-acylhemiaminal moiety (blue circle in Figure 2) with
a benzyl alcohol. The 20OH group is retained in the target side chain so that it can be hydrogen-bonded
β
to the main-chain carbonyl oxygen of Thr276 in -tubulin. The 10O atom of zampanolide’s side chain is
replaced with fluorine atom in the designed side chain so that it can be hydrogen-bonded to the NH
group of Thr276 in
-tubulin. The E,Z-hexadienoyl amide in zampanolide side chain is replaced with
a planar conjugate phenyl ring to preserve the hydrophobic contacts between the proposed side chain
and residues of -tubulin. The Almann’s desTHP-dactylolide and its enantiomer [23] were further
adopted as the core structure of the new zampanolide mimics ( and ) in the current study because of
the acceptable in vitro potency of desTHP-( )-zampanolide ( ) and the practical synthesis of the core.
β
β
5
6
−
2
Consequently, we set 5 and 6 as our new target mimics of zampanolide in this study.
Figure 2. Design of a new stabilized side chain for zampanolide mimics.
2.2. Synthesis
Our retrosynthetic analysis of the zampanolide mimics
and intramolecular Horner–Wadsworth–Emmons condensation as key reactions, is illustrated in
Scheme 1. The Yamaguchi esterification was employed to connect Fragment C1–C8 (carboxylic acid
with Fragment C9–C18 (alcohol or ) via the ester bond between C1 and C17. The intramolecular
5 and 6, using Yamaguchi esterification
9)
7
8
Horner–Wadsworth–Emmons condensation was used to close the macrolactone ring at C8–C9.
These two key reactions were successfully applied to our syntheses of desTHPdactylolide and the mimics
of desTHPzampanolides [21,23]. The known Fragment C1–C8 (9) was readily synthesized via 10 steps
from commercially available 2-butyn-1-ol according to the reported procedure [24].
Scheme 1. Retrosynthetic analysis of zampanolide mimics 5 and 6.
The preparation of Fragment C9–C18 (
The chiral centers at C17 and C18 in the Fragment C9–C18 (
asymmetric dihydroxylation using the commercially available AD-mix formulations (AD-mix-
and AD-mix- , respectively) with the ligand and the osmium salt as the critical trace component (0.6%
7
and
8) with the desired side chain is shown in Scheme 2.
7
and ) were introduced by the Sharpless
8
α
β

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by wt) and ferricyanide and carbonate as the bulk ingredient (99.4% by wt.) [25]. Compound (E)-ethyl
3-(2-fluorophenyl)acrylate was synthesized by the Wittig reaction of the commercially available
2-fluorobenzaldehyde (10) with ethyl 2-(triphenyl- phosphoranylidene)acetate (11) according to
the procedure described in the literature [26]. (E)-3-(2-Fluorophenyl)prop-2-en-1-ol (13) was synthesized
by reducing (E)-ethyl 3-(2-fluorophenyl)acrylate (12) with diisobutylaluminium hydride (DIBAL)
based on the procedure reported in the literature [27]. The Sharpless asymmetric dihydroxylation of
the allylic alcohol (13) with AD-mix-α or AD-mix-β was performed following the standard experimental
procedure reported in the literature and sulfonamide was used to accelerate the hydrolysis of osmate
ester [25]. Epoxy alcohols (18 with R,R configuration, 19 with S,S configuration) have been synthesized
by sequential selective mesylation of primary alcohol (14 or 15) and intermolecular Williamson
ether synthesis [28]. The hydroxy group in epoxy alcohols (18 with R,R configuration, 19 with S,S
configuration) was then protected as the corresponding methoxymethyl (MOM) ethers (20 with
R,R configuration, 21 with S,S configuration). Iodo ether 22 was prepared using the optimized
protocol previously developed in our laboratory [23]. Its lithiation with tert-butyllithium followed by
nucleophilic reaction with epoxy MOM ether (20 with R,R configuration, 21 with S,S configuration)
in toluene at 7 with R,R
90 ◦C mediated by boron trifluoride etherate furnished Fragment C9–C18 (
−
configuration, 8 with S,S configuration) in 22–23% overall yield from 22.
Scheme 2. Synthesis of Fragment C9–C18 (7 and 8).
As illustrated in Scheme 3, the union of Fragment C1–C8 (alcohol 9) and Fragment C9–C18
(carboxylic acid,
7 with R,R configuration, 8 with S,S configuration) has been accomplished
by the Yamaguchi esterification to yield bis-TBS ethers (23 with R,R configuration, 24 with S,S
configuration). Removal of the protecting TBS groups at C7 and C9 with HF-pyridine complex

Molecules 2020, 25, 362
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followed by double oxidation of the corresponding diol (25 with R,R configuration and 26 with S,S
configuration) with Dess–Martin periodinane afforded the keto aldehydes (27 with R,R configuration,
28 with S,S configuration). Without further chromatographic purification, the crude keto aldehydes
were directly subjected to the synthesis of zampanolide mimics (29 with R,R configuration, 30 with S,S
configuration) through the intramolecular HWE condensation catalyzed by Ba(OH)2. Since extractive
work-up resulted in significant hydrolysis of the ester [21], the reaction was quenched by filtration of
the reaction mixture through a pad of silica gel and the crude product was subjected to preparative
TLC purification. The MOM group in 29 was initially planned to be removed using methanolic HCl,
but the methanol participated in a Michael addition to the enone, which resulted in the byproduct 31
(Scheme 4). The removal of the MOM with HCl was, therefore, carried out using THF rather than
methanol as solvent, leading to the desired zampanolide mimics (5 with R,R configuration at C17
and C18, 6 with S,S configuration at C17 and C18).
Scheme 3. Synthesis of zampanolide mimics 5 and 6.
Scheme 4. Deprotection of MOM ether 29 with methanolic HCl.
2.3. Antiproliferative Activity toward Prostate Cancer Cell Lines
The in vitro antiproliferative activity of two pairs of enantiomeric macrolides,
5
and 6, 29 and 30,
has been assessed against a panel of prostate cancer cell lines, including three docetaxel-sensitive
prostate cancer cell models (PC-3 DU145, and LNCaP) and two docetaxel-resistant prostate cancer

Molecules 2020, 25, 362
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cell lines (PC-3/DTX and DU145/DTX). WST-1 cell proliferation assay was used for the in vitro
evaluation according to the procedure described in the Experimental Section. Docetaxel was used
as a positive control while DMSO as a negative control. The half-maximal inhibitory concentrations
(IC50 values) were measured by WST-1 cell proliferation assay after 3 days of exposure, calculated from
the dose–response curves, and presented as the mean
The IC50 values for these four new zampanolide mimics are in the range of 1.00–9.36
has a set of smallest IC50 values (1.00–3.91 M) among the four test macrolides. As summarized in
Figure 3, the IC50 values of new mimic are very close to those for mimic (0.86–2.97 M) and mimic 32
(1.92–3.16 M). The mimic has been evidenced to have similar potency as desTHPzampanolide with
the N-acetyl hemiaminal side chain of zampanolide against prostate cancer cells [21]. The side chain in
mimic was thus identified as a new bioisostere for the chemically unstable N-acetyl hemiaminal side
±
standard deviation of the mean in Table 1.
µM. Mimic
6
µ
6
3
µ
µ
3
2
6
chain of zampanolide. The relative resistance for Mimic
over DU145 is 0.59 and 0.44, respectively.
6 of PC-3/DTX over PC-3 and DU145/DTX
Table 1. Antiproliferative potency of zampanolide mimics against docetaxel-sensitive and docetaxel-resistant
prostate cancer cells.
IC50: (µM) a
R/S a
IC50: (µM) a
R/S a
IC50: (µM)
Compounds
PC-3
PC-3/DTX
DU145
DU145/DTX
LNCaP
Docetaxel
0.0019 ± 0.0006
0.88 ± 0.19
0.35 ± 0.05
9.36 ± 0.15
8.72 ± 2.21
5.80 ± 0.21
3.91 ± 0.73
2.34 ± 0.25
2.97 ± 0.09
0.30 ± 0.05
3.97 ± 0.24
3.63 ± 0.11
3.71 ± 0.72
2.31 ± 0.32
1232
1.3
0.9
0.42
0.42
0.64
0.59
0.0012 ± 0.0003
0.86 ± 0.25
0.29 ± 0.03
3.98 ± 0.57
3.87 ± 0.23
2.28 ± 0.51
2.27 ± 0.39
8.58 ± 0.39
2.78 ± 0.16
0.46 ± 0.10
4.42 ± 0.55
5.03 ± 1.11
4.30 ± 0.79
1.00 ± 0.91
7150
3.2
1.6
1.1
1.3
0.0002 ± 0.00005
0.76 ± 0.29
0.35 ± 0.02
5.29 ± 0.49
6.51 ± 0.60
3.15 ± 0.19
1.92 ± 0.29
3 b
4 b
29
5
30
6
1.9
0.44
a
The relative resistance of the two cell lines obtained by dividing the IC50 value of the resistance cell line by that of
b
the parental cell line. These data were reported in our previous paper [21].
Figure 3. Comparison of antiproliferative potency among different zampanolide mimics.
3. Materials and Methods
3.1. General Procedures
Optical rotations were measured on a RUDOLPH Research Analytical Autopol III Automatic
Polarimeter (RUDOLPH Research Analytical, Hackettstown, NJ, USA). IR spectra were recorded
on a Nicolet Nexus 470 FTIR spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).
High-Resolution MS were obtained on an Orbitrap mass spectrometer (Thermo Fisher Scientific,
Waltham, MA, USA) with electrospray ionization (ESI). NMR spectra (see Supplementary Materials) were
obtainedonaBrukerFourier300spectrometer(Billerica, MA, USA)inCDCl3. Thechemicalshiftsaregiven
in ppm referenced to the respective solvent peak, and coupling constants are reported in Hz. Anhydrous
THF and dichloromethane were purified by PureSolv MD 7 Solvent Purification System from Innovative
Technologies (MB-SPS-800) (Herndon, VA, USA). All other reagents and solvents were purchased from
commercial sources (Fisher Scientific, Portland, OR, USA) and were used without further purification.
Silica gel column chromatography was performed using silica gel (32–63 µM). Preparative thin-layer

Molecules 2020, 25, 362
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chromatography (PTLC) separations were carried out on thin-layer chromatography plates loaded with
silica gel 60 GF254 (EMD Millipore Corporation, Berlington, MA, USA). Starting materials 22 and were
synthesized using the procedure previously described by us [23]. (E)-ethyl 3-(2-fluorophenyl)acrylate
12) was prepared from commercially available 2-fluorobenzaldehyde (10, CAS 446-52-6) according to
9
(
the reported procedure [26]. (E)-3-(2-Fluorophenyl)prop-2-en-1-ol (13) was synthesized by reducing
(E)-ethyl 3-(2-fluorophenyl)acrylate (12) with DIBAL based on the procedure reported in the literature [27].
Its structure was confirmed by 1H NMR and high resolution mass spectrometry (HRMS) data (calculated
for C9H10FO (M + H): 153.0716; Found: 153.0710).
3.2. Synthesis of (1R,2R)-1-(2-Fluorophenyl)propane-1,2,3-triol [(17R,18R) triol 14]
AD-mix-α (5.9 g) was dissolved in a mixture of tert-butyl alcohol (21 mL) and water (21 mL).
The solution was stirred vigorously at room temperature until two clear phases were observed. At this
point, the lower aqueous phase emerged as bright yellow. Methanesulfonamide (399 mg, 4.2 mmol,
◦
1 equiv) was added and the reaction mixture was cooled down to 0 C. A solution of 13 (640 mg,
4.2 mmol, 1 equiv) in 1 mL of tert-butyl alcohol was then added. The resulting mixture was stirred
vigorously at 0 ◦C overnight and the reaction progress was monitored with TLC. The reaction was
quenched by adding solid sodium sulfate (6.3 g) at 0 ◦C. The mixture was warmed to room temperature
and stirred for 30–60 min. The tert-butyl alcohol was removed under vacuum, and saturated sodium
bicarbonate (30 mL) and water (300 mL) were added to the residue. The mixture was stirred for
additional 30 min and then extracted with ethyl acetate (150 mL
rinsed with brine, dried over anhydrous sodium sulfate, and concentrated. The crude product was
purified through column chromatography eluting with ethyl acetate. The product after purification,
×
3). The combined extracts were
with quantitative yield, still contained a trace amount of methanesulfonamide, which is good enough
1
to be used for the next-step reaction. H NMR (300 MHz, CDCl3)
δ
. 7.38 (t, J = 7.2 Hz, 1H, aromatic H),
7.21 (dd, J = 13.5, 5.7 Hz, 1H, aromatic H), 7.06 (t, J = 7.5 Hz, 1H, aromatic H), 6.95 (t, J = 8.4 Hz, 1H,
aromatic H), 4.88 (d, J = 6.3 Hz, 1H, H-18), 4.23 (br.s, 3H, 3 OH), 3.77 (q, J = 5.1 Hz, 1H, H-17), 3.45 (d, J
= 4.8 Hz, 2H, H2-16). 13C NMR (75 MHz, CDCl3)
159.9 (d, JCF = 243.8 Hz), 129.6, 128.5, 127.9 (d, JCF
×
δ
= 12.8 Hz), 124.6, 115.4 (d, JCF = 21.8 Hz), 75.3, 68.8, 63.5. IR (film) νmax: 3384, 2988, 1617, 1490, 1455,
1320, 1222, 1054 cm−1. HRMS (ESI): m/z calculated for C9H12FO3 [M + H]+: 187.0770. Found: 187.0766.
3.3. Synthesis of (2R,3R)-3-(2-Fluorophenyl)-2,3-dihydroxypropyl methanesulfonate [(17R,18R) mesylate 16
]
To a solution of triol 14 (1.37 g, 7.39 mmol) in pyridine (15 mL, 0.5 M), methane sulfonyl chloride
(0.57 mL, 7.39 mmol) was added dropwise at 0 ◦C under argon. The reaction mixture was stirred
at room temperature for 20 h and the reaction progress was monitored by TLC (Hexane/EtOAc, 1:1,
v/v). The reaction was quenched by diluting with ethyl acetate (800 mL), and the resulting solution was
rinsed with brine (50 mL
×
3) and dried over anhydrous sodium sulfate. After removing the organic
solvent, the residue was subjected to column chromatography, using 40% ethyl acetate in hexane
as eluent, to give the desired product. Colorless oil; 81% yield. H NMR (300 MHz, CDCl3)
1
δ 7.47 (dt, J
= 7.5, 1.8 Hz, 1H, aromatic H), 7.34–7.27 (m, 1H, aromatic H), 7.17 (dt, J = 7.5, 0.9 Hz, 1H, aromatic H),
7.04 (ddd, J = 10.5, 8.1, 0.9 Hz, 1H, aromatic H), 5.00 (d, J = 5.7 Hz, 1H, H-18), 4.27–4.16 (m, 2H, H2-16),
4.06–4.01 (m, 1H, H-17), 3.18 (br.s, 2H, 2
×
OH), 3.03 (s, 3H, SO2CH3). 13C NMR (75 MHz, CDCl3)
δ
159.9 (d, JCF = 244.3 Hz), 130.1 (d, JCF = 8.3 Hz), 128.4 (d, JCF = 3.8 Hz), 127.0 (d, JCF = 12.8 Hz),
124.8 (d, JCF = 3.8 Hz), 115.7 (d, JCF = 21.8 Hz), 72.9, 70.4, 67.9 (d, JCF = 2.3 Hz), 37.6. IR (film) νmax
:
3502, 3029, 2940, 1617, 1587, 1490, 1456, 1346, 1171 cm−1. HRMS (ESI): m/z calculated for C10H14FO5S
[M + H]+: 265.0546. Found: 265.0547.
3.4. Synthesis of (R)-(2-Fluorophenyl)((R)-oxiran-2-yl)methanol [(17R,18R) epoxide 18]
To a suspension of sodium hydride (275 mg, 60%, 6.87 mmol, 1.5 equiv) in anhydrous THF (36 mL),
a solution of 16 (1.21 g, 4.58 mmol, 1 equiv) in anhydrous THF was added at about
−
30 ◦C under
argon. The reaction mixture was stirred at 0 ◦C overnight and the reaction progress was monitored

Molecules 2020, 25, 362
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with TLC (hexane:EtOAc, 3:1). The mixture was filtered through a silica gel pad eluting with ethyl
acetate. After concentration to remove the solvent, the residue was subjected to PTLC purification
1
eluting with toluene:EtOAc (3:1, v/v) to furnish the desired epoxide. Colorless oil; 57% yield. H NMR
(300 MHz, CDCl3) δ 7.57 (dd, J = 7.8, 1.8 Hz, 1H, aromatic H), 7.35–7.28 (m, 1H, aromatic H), 7.20 (dt,
J = 7.5, 1.2 Hz, 1H, aromatic H), 7.06 (ddd, J = 10.5, 8.1, 1.2 Hz, 1H, aromatic H), 4.84 (t, J = 6.6 Hz,
1H, H-18), 3.26–3.22 (m, 1H, H-17), 2.92–2.85 (overlapped, 2H, H2-16), 2.47 (d, J = 5.7 Hz, 1H, OH).
13C NMR (75 MHz, CDCl3)
δ 159.9 (d, JCF = 244.5 Hz), 129.6 (d, JCF = 8.3 Hz), 127.9 (d, JCF = 4.5 Hz),
127.3 (d, JCF = 13.5 Hz), 124.5 (d, JCF = 3.8 Hz), 115.4 (d, JCF = 21.8 Hz), 68.7 (d, JCF = 2.3 Hz), 55.6, 45.5
(d, J = 2.3 Hz). IR (film) νmax: 3420, 3067, 3002, 2928, 1617, 1586 1488, 1456, 1224 cm−1. HRMS (ESI): m/z
calculated for C9H10FO2 [M + H]+: 169.0665. Found: 169.0657.
3.5. Synthesis of (R)-2-((R)-(2-Fluorophenyl)(methoxymethoxy)methyl)oxirane [(17R,18R)MOM ether 20]
To a solution of alcohol◦18 (716 mg, 4.3 mmol) in DMF (4.2 mL), sodium hydride (204 mg, 60%,
◦
5.1 mmol) was added at 4 C, and the mixture was stirred at 4 C for 30 min. Methoxymethyl
chloride (0.39 mL, 5.1 mmol) followed by tetrabutylammonium iodide (157 mg, 0.43 mmol) was
added to the reaction mixture. The reaction was proceeded with stirring at room temperature for 20 h.
The reaction mixture was diluted with ethyl acetate (300 mL) and diethyl ether (300 mL) and then rinsed
with brine (50 mL
×
3). The organic layer was dried over anhydrous sodium sulfate and concentrated
to give a crude mass, which was purified by column chromatography eluting with hexane/ethyl acetate
(3:1, v/v) to furnish the MOM ether 20 as a pale-yellow oil in 67% yield. H NMR (300 MHz, CDCl3)
1
δ
7.51 (dt, J = 7.2, 1.8 Hz, 1H, aromatic H), 7.33–7.25 (m, 1H, aromatic H), 7.17 (dt, J = 7.5, 1.2 Hz, 1H,
aromatic H), 7.04 (ddd, J = 9.9, 8.1, 1.2 Hz, 1H, aromatic H), 4.75 (d, J = 6.9 Hz, 1H, OCH2OCH3), 4.67
(d, J = 6.9 Hz, 1H, OCH2OCH3), 4.62 (d, J = 6.6 Hz, 1H, H-18), 3.36 (s, 3H, OCH3), 3.26–3.22 (m, 1H,
H-17), 2.73–2.72 (overlapped, 2H, H2-16). 13C NMR (75 MHz, CDCl3)
δ 160.3 (d, J = 244.5 Hz), 129.8
(d, J = 8.3 Hz), 128.6 (d, J = 4.5 Hz), 125.2 (d, J = 14.3 Hz), 124.4 (d, J = 3.0 Hz), 115.5 (d, J = 21.8 Hz),
94.6, 73.0, 55.6, 54.4, 44.2 (d, J = 2.3 Hz). IR (film) νmax: 2891, 1615, 1587, 1488, 1455, 1022 cm−1. HRMS
(ESI): m/z calculated for C11H14FO3 [M + H]+: 213.0927. Found: 213.0921.
3.6. Synthesis of (5R,6R,E)-5-(2-Fluorophenyl)-8,16,16,17,17-pentamethyl-2,4,11,15-tetraoxa-16-
silaoctadec-8-en-6-ol [(17R,18R) Fragment C9–C18 (7)]
To a soluti◦on of vinyliodide 22 (164 mg, 0.44 mmol; co-evaporated twice with pentane) in toluene
(3 mL) at
stirred at
−
78 C, tert-butyllithium (0.46 mL, 1.9 M, 0.89 mmol) was added, and the mixture was
−
78 ◦C for 45 min prior to being cooled down to 90 ◦C. Epoxide 20 (188 mg, 0.89 mmol;
−
co-evaporated twice with pentane) in toluene (1.5 mL) was added dropwise to make sure that interior
temperature lower than
(0.11 mL, 0.89 mmoL) was added dropwise. The resulting solution was then stirred at
prior to being quenched with ethyl acetate (15 mL) and saturated aqueous sodium bicarbonate (35 mL)
was added. The organic layer was separated and the aqueous layer was extracted with ethyl acetate
◦
◦
−
78 C. The reaction solution was re-cooled to
−
90 C before BF3
•
OEt2
−
78 ◦C overnight
(60 mL
×
3). The combined organic layers were dried over anhydrous sodium sulfate and concentrated
in Vacuum to remove ethyl acetate. The crude mass was subjected to PTLC purification over silica
gel using hexane/ethyl acetate (4:1, v/v) as eluent to yield secondary alcohol
7
as a pale-yellow oil in
1
23% yield. H NMR (300 MHz, CDCl3)
δ
. 7.41 (dt, J = 7.2, 1.5 Hz, 1H, aromatic H), 7.32–7.24 (m, 1H,
aromatic H), 7.15 (dt, J = 7.5, 0.9 Hz, 1H, aromatic H), 7.53 (ddd, J = 9.9, 8.1, 0.9 Hz, 1H, aromatic H),
5.41 (t, J = 6.6 Hz, 1H, H-14), 4.81 (d, J = 6.3 Hz, 1H, H-17), 4.62 (d, J = 6.9 Hz, 1H, OCH2OCH3), 4.58
(d, J = 6.6 Hz, 1H, OCH2OCH3), 3.94 (d, J = 6.6 Hz, 2H, H2-13), 3.67 (t, J = 6.3 Hz, 2H, H2-11), 3.46
(t, J = 6.3 Hz, 2H, H2-9), 3.36 (s, 3H), OCH3), 1.75 (quin, J = 6.3 Hz, 2H, H2-10), 1.64 (s, 3H, 15-CH3),
0.87 (s, 9H, TBS), 0.03 (s, 6H, TBS). 13C NMR (75 MHz, CDCl3)
δ 160.9 (d, JCF = 244.8 Hz), 136.4, 129.8,
129.1, 128.7, 126.1 (d, JCF = 13.5 Hz), 124.5 (d, JCF = 3.8 Hz), 115.6 (d, JCF = 21.8 Hz), 95.3, 75.6, 72.5 (d,
JCF = 12.1 Hz), 67.3, 67.0, 60.2, 56.2, 43.0, 33.2, 26.1, 26.07, 18.5, 16.6, 16.5. IR (film) νmax: 3420, 2929,

Molecules 2020, 25, 362
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1715, 1647, 1615, 1488, 1029 cm−1. HRMS (ESI): m/z calculated for C24H42FO5Si [M + H]+: 457.2786.
Found: 457.2780.
3.7. Synthesis of
(2E,4Z)-(5R,6R,E)-5-(2-Fluorophenyl)-8,16,16,17,17-pentamethyl-2,4,11,15-tetraoxa-16-silaoctadec-8-en-6-yl
7-((tert-butyldimethylsilyl)oxy)-8-(diethoxyphosphoryl)-5- methylocta-2,4-dienoate [(17R,18R) ester 23]
To a solution of Fragment C1–C8 (
toluene (0.5 mL) at room temperature, trimethylamine (28
chloride (18 L, 0.12 mmol) was sequentially added, and the reaction mixture was stirred at room
temperature for 1.5 h. A solution of alcohol (35 mg, 0.08 mmol) and DMAP (9.3 mg, 0.08 mmol) in
9
, 37 mg, 0.09 mmol; co-evaporated with pentane twice) in
µL, 0.20 mmol) and 2,4,6-trichlorobenzoyl
µ
7
toluene (0.5 mL) was then added to the reaction mixture. The reaction was allowed to proceed with
stirring at room temperature for 19 h prior to being quenched with water (5 mL) and saturated aqueous
sodium bicarbonate (5 mL). The mixture was extracted with ethyl acetate (5 mL
organic extracts were dried over anhydrous sodium sulfate and concentrated in vacuum. PTLC
purification of the crude product over silica gel eluting with hexane/ethyl acetate (60:40, v/v) yielded
×
3), the combined
1
the ester 23 as a mixture of diastereoisomers in a 1:1 ratio as a colorless oil in 77% yield. H NMR
(300 MHz, CDCl3)
δ 7.50 (dd, J = 15.0, 11.7 Hz, H-3), 7.42 (t, J = 7.5 Hz, 1H, aromatic H), 7.25–7.20
(m, 1H, aromatic H), 7.10 (t, J = 7.5 Hz, 1H, aromatic H), 7.00 (t, J = 8.4 Hz, 1H, aromatic H), 6.03 (d, J =
11.4 Hz, H-4), 5.75 (d, J = 15.3 Hz, 1H, H-2), 5.43–5.37 (m, 1H, H-17), 5.33 (t, J = 6.6 Hz, 1H, H-14), 5.08
(d, J = 5.4 Hz, 1H, H-18), 4.58 (d, J = 6.6 Hz, 1H, -OCH2OCH3), 4.51 (d, J = 6.6 Hz, 1H, OCH2OCH3),
4.18–4.04 (overlapped, 5H, 2 x OCH2CH3, H-7), 3.93–3.79 (m, 2H, H2-13), 3.64 (t, J = 6.3 Hz, 2H, H2-11),
3.40 (t, J = 6.3 Hz, 2H, H2-9), 3.32 (s, 3H, -OCH2OCH3), 2.58–2.48 (m, 2H, H2-16), 2.33–2.24 (m, 2H,
H2-8), 2.00–1.92 (m, 2H, H2-6), 1.87 (s, 3H, 5-CH3), 1.72 (quin, J = 6.3 Hz, 2H, H2-10), 1.64 (1.63) (s, 3H,
15-CH3), 1.31 (t, J = 7.2 Hz, 6H, 2
×
OCH2CH3), 0.86 (s, 9H, TBS), 0.81 (0.80) (s, 9H, TBS), 0.04 (0.03)
(s, 3H, TBS), 0.01 (s, 6H, TBS), 0.04(
−
−
0.05) (s, 3H, TBS). 13C NMR (75 MHz, CDCl3)
δ
166.8 (166.7),
160.8 (d, J = 244.5 Hz), 146.4 (d, J = 9.8 Hz), 141.6, 135.2 (d, J = 3.8 Hz), 129.7, 129.0, 126.8, 125.7 (d, J =
13.5 Hz), 125.1, 124.3, 119.3, 115.6 (115.3), 95.0, 72.9, 72.3, 67.3, 66.8, 61.7(61.6), 60.5, 60.1, 56.1, 41.8 (41.2),
36.1, 34.3, 33.1, 27.0, 26.11, 26.05, 25.90, 25.87, 25.2, 25.0, 18.5, 18.0, 16.6 (16.5),
−
4.6, 5.2. IR (film) νmax:
−
2929, 2856, 1716, 1489, 1251, 1020 cm−1. HRMS (ESI): m/z calculated for C43H77FO10PSi2 [M + H]+:
859.4777. Found: 859.4756.
3.8. Synthesis of (2E,4Z)-(1R,2R,E)-1-(2-Fluorophenyl)-6-(3-hydroxypropoxy)-1-(methoxymethoxy)
-4-methylhex-4-en-2-yl 8-(diethoxyphosphoryl)-7-hydroxy-5-methylocta-2,4-dienoate [(17R,18R) diol 25]
◦
To a solution of di-TBS ether 23 (90 mg, 0.105 mmol) in THF (4.2 mL) at 0 C in a plastic
bottle, hydrogen fluoride-pyridine complex (70%, 1.0 mL) was added dropwise. The solution was
◦
stirred at 0 C for 5 min and then at room temperature for 16 h. Saturated sodium bicarbonate
(30 mL) was added to quench the reaction and the suspension was extracted with ethyl acetate
(30 mL
×
3). The combined organic extracts were dried over anhydrous sodium sulfate and the dried
extract was concentrated under vacuum. The crude product was purified by PTLC, eluting with
dichloromethane/methanol (93:7. v/v), and the pure diol was washed out from the PTLC silica gel with
1
acetone as a pale-yellow oil in 92% yield. H NMR (300 MHz, CDCl3)
δ
7.50 (7.49) (dd, J = 15.3, 11.7 Hz,
1H, H-3), 7.43 (t, J = 9.0 Hz, 1H, aromatic H), 7.30–7.23 (m, 1H, aromatic H), 7.13 (7.12) (t, J = 7.5 Hz, 1H,
aromatic H), 7.031 (7.028) (dd, J = 10.2, 9.0 Hz, 1H, aromatic H), 6.11 (d, J = 11.4 Hz, 1H, H-4), 5.80 (d, J
= 15.3 Hz, 1H, H-2), 5.47–5.40 (m, 1H, H-17), 5.32 (t, J = 5.7 Hz, 1H, H-14), 5.09 (d, J = 5.7 Hz, 1H, H-18),
4.58 (d, J = 6.9 Hz, 1H, -OCH2OCH3), 4.51 (d, J = 6.9 Hz, 1H, OCH2OCH3), 4.18–4.06 (overlapped, 5H,
2
×
OCH2CH3, H-7), 3.90 (t, J = 5.7 Hz, 2H, H2-13), 3.69 (3.68) (t, J = 5.7 Hz, 2H, H2-11), 3.48 (3.47) (t, J
= 5.7 Hz, 2H, H2-9), 3.33 (3.32) (s, 3H, -OCH2OCH3), 3.03 (s, 2H, 2 OH), 2.66–2.53 (m, 1H, H-16),
2.46–2.19 (m, 3H, H2-8 & H-16), 1.92 (S, 3H, 5-CH3), 1.96–1.88 (m, 2H, H2-6), 1.75 (quin, J = 5.7 Hz,
2H, H2-10), 1.65 (s, 3H, 15-CH3), 1.33 (t, J = 7.2 Hz, 6H, 2
OCH2CH3). 13C NMR (75 MHz, CDCl3)
167.0 (166.8), 160.9 (d, J = 247.5 Hz), 146.0, 140.8 (d, J = 17.3 Hz), 135.7 (d, J = 9.0 Hz), 129.8, 129.0,
×
×
δ

Molecules 2020, 25, 362
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126.8, 125.6 (d, J = 12.8 Hz), 124.8 (d, J = 11.3 Hz), 124.4, 119.7 (d, J = 11.3 Hz), 115.7 (115.4), 94.9, 73.0,
72.4, 68.8, 67.4, 65.3, 62.2, 61.8, 56.1, 41.1, 34.6 (34.5), 32.7, 32.3, 25.3, 25.0, 16.6 (16.5). IR (film) νmax
:
3366, 2929, 1711, 1633, 1488, 1222, 1019 cm−1. HRMS (ESI): m/z calculated for C31H49FO10P [M + H]+:
631.3048. Found: 631.3041.
3.9. Synthesis of (R,3E,9E,11Z,15E)-6-((R)-(2-Fluorophenyl)(methoxymethoxy)methyl)-4,12-
dimethyl-1,7-dioxacyclooctadeca-3,9,11,15-tetraene-8,14-dione [(17R,18R) macrolactone 29]
To a solution of diol 25 (65 mg, 0.10 mmol) in dichloromethane (6.2 mL) at room temperature,
Dess–Martin periodinane (DMP, 131 mg, 0.30 mmol) was added. The reaction mixture was stirred for
30 min before a second portion of DMP (131 mg, 0.30 mmol) was added. The reaction was allowed to
proceed with stirring at room temperature for an additional 1 h. The reaction mixture was poured into
a stirred mixture of saturated sodium bicarbonate (30 mL) and saturated sodium thiosulfate (30 mL),
and the resulting suspension was stirred for 30 min before being extracted with dichloromethane
(30 mL
×
3). The combined organic extracts were dried over anhydrous sodium sulfate and the dried
extract was concentrated under vacuum. The crude product was used for the next reaction without
further purification. To a solution of the crude ketoaldehyde (0.10 mmol) obtained above in THF
(103 mL) at 0 ◦C was added water (2.2 mL) followed by activated barium hydroxide (15 mg, 0.08 mmol).
The reaction mixture was stirred at 0 ◦C for 30 min and at room temperature for 2.5 h, then was filtered
through a pad of sodium sulfate and a pad of silica gel that was rinsed with ethyl acetate. After
evaporation of the solvent, the crude product was subjected to PTLC purification, using hexane/ethyl
acetate (73:37) as eluent, to yield the pure macrolactone 29. Colorless oil, 46% yield for two steps. [
α]:
−
15.6 (c = 0.23, MeOH). 1H NMR (300 MHz, CDCl3)
δ
7.60 (dd, J = 15.0, 11.4 Hz, 1H, H-3), 7.46 (dt, J
= 7.5, 1.8 Hz, 1H, aromatic H), 7.33–7.25 (m, 1H, aromatic H), 7.16 (dt, J = 7.5, 1.2 Hz, 1H, aromatic
H), 7.05 (ddd, J = 9.9, 8.4, 1.2 Hz, 1H, aromatic H), 6.82 (dt, J = 16.2, 6.6 Hz, 1H, H-9), 6.12 (d, J =
11.4 Hz, 1H, H-4), 6.01 (d, J = 16.2 Hz, 1H, H-8), 5.91 (d, J = 15.0 Hz, 1H, H-2), 5.51 (ddd, J = 11.4,
6.3, 1.2 Hz, 1H, H-17), 5.27 (dd, J = 7.8, 4.8 Hz, 1H, H-14), 5.12 (d, J = 6.3 Hz, 1H, H-18), 4.60 (d, J =
6.9 Hz, 1H, O-CH2OCH3), 4.53 (d, J = 6.9 Hz, 1H, OCH2OCH3), 3.99 (dd, J = 12.0, 8.1 Hz, 1H, H-16),
3.88–3.81 (m, 2H, H2-13), 3.47–3.28 (m, 2H, H2-11), 3.34 (s, 3H, OCH2OCH3), 3.14 (d, J = 12.6 Hz, 1H,
H-16), 2.43–2.36 (m, 2H, H2-10), 2.33 (d, J = 12.0 Hz, 1H, H-6), 2.05 (d, J = 12.1 Hz, 1H, H-6), 1.83 (s,
3H, 5-CH3), 1.63 (S, 3H, 15-CH3). 13C NMR (75 MHz, CDCl3)
δ 197.2, 166.6, 160.9 (d, J = 244.5 Hz),
146.8, 142.5, 139.7, 138.9, 134.3, 130.3, 128.9 125.9 (d, J = 7.5 Hz), 125.6 (d, J = 13.5 Hz), 125.2 (d, J =
5.6 Hz), 124.5, 120.9 (d, J = 5.3 Hz), 115.6 (d, J = 21.8 Hz), 94.9, 72.9, 72.6, 67.9, 67.8, 56.0, 46.0, 41.4, 33.1,
24.09, 16.7. IR (film) νmax: 2931, 1713, 1633, 1489, 1358, 1226, 1030 cm−1. HRMS (ESI): m/z calculated for
C27H34FO6 [M + H]+: 473.2339. Found: 473.2333.
3.10. Synthesis of (R,3E,9E,11Z,15E)-6-((R)-(2-Fluorophenyl)(hydroxy)methyl)-4,12-dimethyl-
1,7-dioxacyclooctadeca-3,9,11,15-tetraene-8,14-dione [(17R,18R) macrolactone 5]
To a solution of 29 (30 mg, 0.064 mmol) in a mixture of tetrahydrofuran/water (0.92 mL, 1:1,
v/v), concentrated hydrochloric acid (130
µL) was added. The two more portions of concentrated
hydrochloric acid (130 2) were added sequentially after each one-hour stirring at room temperature.
µL
×
The reaction mixture was stirred at room temperature overnight before the reaction was quenched by
adding saturated ammonium chloride (60 mL). The subsequent mixture was extracted with ethyl acetate
(40 mL
×
3). The combined extracts were dried over anhydrous sodium sulfate and the solvents were
evaporated in vacuum. The crude product was purified over preparative thin layer chromatography
eluting with toluene/ethyl acetate (3:1, v/v) to furnish the desired product (11 mg). Colorless syrup,
1
41% yield. H NMR (300 MHz, CDCl3)
δ
7.62 (dd, J = 15.0, 11.7 Hz, 1H, H-3), 7.49 (dt, J = 7.5, 1.8 Hz,
1H, aromatic H), 7.31–7.27 (m, 1H, aromatic H), 7.17 (dt, J = 7.5, 1.2 Hz, 1H), 7.05 (ddd, J = 10.2, 8.1,
0.9 Hz, 1H), 6.83 (dt, J = 15.9, 6.9 Hz, 1H, H-9), 6.12 (d, J = 11.4 Hz, 1H, H-4), 6.02 (d, J = 16.2 Hz, 1H,
H-8), 5.91 (d, J = 15.0 Hz, 1H, H-2), 5.48–5.38 (m, 1H, H-17), 5.27 (t, J = 6.0 Hz, 1H, H-14), 5.10 (d, J
= 6.3 Hz, 1H, H-18), 3.99 (dd, J = 12.0, 8.1 Hz, 1H, H-16), 3.86 (br.s, 1H, H-13), 3.82 (br.s, 1H, H-13),

Molecules 2020, 25, 362
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3.51–3.36 (m, 2H, H2-11), 3.18 (d, J = 12.9 Hz, 1H, H-16), 2.44–2.34 (m, 3H, H2-10, H-6), 2.06 (d, J =
14.4 Hz, 1H, H-6), 1.83 (s, 3H, 5-CH3), 1.62 (s, 3H, 15-CH3). 13C NMR (75 MHz, CDCl3)
197.2, 167.0,
160.1 (d, JCF = 244.5 Hz), 146.9, 143.0, 140.1 (d, JCF = 12.8 Hz), 134.2, 130.2, 129.9, 128.8, 127.6 (d, JCF
12.8 Hz), 126.0, 125.2, 124.6, 120.7, 115.6 (d, JCF = 21.8 Hz), 79.4, 74.4, 70.3, 68.0, 45.9, 41.4, 33.0, 24.1,
δ
=
16.7. [
α
]:
−
15.2 (c = 0.16, MeOH). IR (film) νmax: 3428, 2924, 2854, 1706, 1633, 1558, 1488, 1360, 1221,
1036 cm−1. HRMS (ESI): m/z calculated for C25H30FO5 [M + H]+: 429.2077. Found: 429.2074.
3.11. Synthesis of (1S,2S)-1-(2-Fluorophenyl)propane-1,2,3-triol [(17S,18S) triol 15]
Triol 15 was prepared in 68% yield from 13 catalyzed by AD-mix-
β
using a similar procedure
1
used to synthesize its antipode 14. H NMR (300 MHz, CDCl3)
δ
7.39 (t, J = 7.5 Hz, 1H, aromatic H),
7.22 (dd, J = 13.8, 7.2 Hz, 1H, aromatic H), 7.07 (t, J = 7.5 Hz, 1H, aromatic H), 6.96 (t, J = 9.3 Hz, 1H,
aromatic H), 4.90 (d, J = 6.3 Hz, 1H, H-18), 4.21 (br.s, 3H, 3 OH), 3.78 (dd, J = 10.2, 4.8 Hz, 1H, H-17),
3.48 (d, J = 4.8 Hz, 2H, H2-16). 13C NMR (75 MHz, CDCl3)
160.0 (d, JCF = 244.5 Hz), 129.6, 128.5
×
δ
(d, JCF = 16.5 Hz), 127.8 (d, JCF = 12.8 Hz), 124.6, 115.5 (d, JCF = 21.8 Hz), 75.4, 68.8, 63.4. IR (film) νmax
:
3375, 2930, 1781, 1489, 1456, 1319 cm−1. HRMS (ESI): m/z calculated for C9H12FO3 [M + H]+: 187.0770.
Found: 187.0765.
3.12. Synthesis of (2S,3S)-3-(2-Fluorophenyl)-2,3-dihydroxypropyl methanesulfonate [(17S,18S) mesylate 17
]
Mesylate 17 was prepared from triol 15 as a colorless oil in 72% yield, employing a procedure
1
similar to that used for the mesylation of triol 14. H NMR (300 MHz, CDCl3)
δ 7.48 (dt, J = 7.5, 1.8 Hz,
1H, Aromatic H), 7.32–7.29 (m, 1H, aromatic H), 7.18 (dt, J = 8.4, 0.9 Hz, 1H, aromatic H), 7.05 (ddd, J
= 9.3, 8.1, 0.9 Hz, 1H, aromatic H), 5.01 (d, J = 5.7 Hz, 1H, H-18), 4.28–4.15 (m, 2H, H2-16), 4.08–4.03
(m, 1H, H-17), 3.04 (s, 3H, SO2CH3). 13C NMR (75 MHz, CDCl3)
δ 159.9 (d, JCF = 244.5 Hz, 1H), 130.1,
128.4, 127.0 (d, JCF = 12.8 Hz, 1H), 124.8, 115.7 (d, JCF = 21.8 Hz, 1H), 72.9, 70.3, 67.9, 37.6. IR (film) νmax
:
3482, 3029, 2939, 1616, 1587, 1489, 1456, 1332, 1168 cm−1. HRMS (ESI): m/z calculated for C10H14FO5S
[M + H]+: 265.0546. Found: 265.0542.
3.13. Synthesis of (S)-(2-Fluorophenyl)((S)-oxiran-2-yl)methanol [(17S,18S) epoxide 19]
Epoxide 19 (52%, pale yellow oil) was obtained from mesylate 17 according to the internal
1
Williamson ether synthesis procedure employed for the conversion of mesylate 16 to epoxide 18. H
NMR (300 MHz, CDCl3)
(dt, J = 7.5, 1.2 Hz, 1H, aromatic H), 7.06 (ddd, J = 10.5, 8.1, 1.2 Hz, 1H, aromatic H), 4.83 (d, J = 5.1 Hz,
1H, H-18), 3.26–3.21 (m, 1H, H-17), 2.92–2.84 (m, 2H, H2-16). 13C NMR (75 MHz, CDCl3)
160.0 (d, JCF
δ 7.57 (dt, J = 7.5, 1.8 Hz, 1H, aromatic H), 7.35–7.25 (m, 1H, aromatic H), 7.20
δ
= 244.5 Hz), 129.8, 127.6 (d, JCF = 9.0 Hz), 127.4 (d, JCF = 13.5 Hz), 124.7, 115.6 (d, JCF = 21.0 Hz), 68.6,
55.4, 45.6. IR (film) νmax: 3482, 3029, 2939, 1617, 1587, 1489, 1456, 1331, 1168 cm−1. HRMS (ESI): m/z
calculated for C9H10FO2 [M + H]+: 169.0665. Found: 169.0659.
3.14. Synthesis of (S)-2-((S)-(2-Fluorophenyl)(methoxymethoxy)methyl)oxirane [(17S,18S) MOM ether 21]
MOM ether 21 was prepared from epoxide 19 as a pale-yellow oil in 73% yield employing
1
a procedure similar to that used for conversion of 18. H NMR (300 MHz, CDCl3)
δ
7.44 (dt, J = 7.5,
1.8 Hz, 1H, aromatic H), 7.32–7.27 (m, 1H, aromatic H), 7.17 (dt, J = 7.5, 0.9 Hz, 1H, aromatic H), 7.05
(ddd, J = 10.2, 8.4, 1.2 Hz, 1H, aromatic H), 4.76 (d, J = 6.6 Hz, 1H, -OCH2OCH3), 4.68 (d, J = 6.9 Hz,
1H, H-18), 4.63 (d, J = 6.6 Hz, 1H, -OCH2OCH3), 3.37 (s, 3H, -OCH2OCH3), 3.27 – 3.23 (m, 1H, H-17),
2.75–2.73 (m, 2H, H2-16). 13C NMR (75 MHz, CDCl3)
δ 160 (d, JCF = 245.3 Hz), 129.9 (d, JCF = 8.3 Hz),
128.7 (d, JCF = 3.8 Hz), 125.3 (d, JCF = 14.3 Hz), 124.5 (d, JCF = 3.8 Hz), 115.7 (d, JCF = 21.8 Hz), 94.8,
73.1, 55.8, 54.6, 44.4. IR (film) νmax: 2891, 2825, 1616, 1587, 1488, 1455, 1149, 1022 cm−1. HRMS (ESI):
m/z calculated for C11H14FO3 [M + H]+: 213.0927. Found: 213.0920.

Molecules 2020, 25, 362
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3.15. Synthesis of (5S,6S,E)-5-(2-Fluorophenyl)-8,16,16,17,17-pentamethyl-2,4,11,15-tetraoxa-16-
silaoctadec-8-en-6-ol [(17S, 18S) Fragment C9–C18 (8)]
(17S,18S) Fragment C9–C18 (
21 in a similar way to the synthesis of its antipode (17R,18R) Fragment C9–C18 (7). 1H NMR (300
MHz, CDCl3) 7.41 (dt, J = 7.5, 1.8 Hz, 1H, aromatic H), 7.32–7.24 (m, 1H, aromatic H), 7.15 (dt, J = 7.5,
8) was synthesized as a colorless oil in 22% yield from MOM ether
δ
1.2 Hz, 1H, aromatic H), 7.03 (ddd, J = 10.2, 8.4, 1.2 Hz, 1H, aromatic H), 5.41 (t, J = 6.6 Hz, 1H, H-14),
4.81 (d, J = 6.6 Hz, 1H, H-18), 4.63 (d, J = 6.6 Hz, 1H, OCH2OCH3), 4.58 (d, J = 6.6 Hz, 1H, OCH2OCH3),
3.95 (d, J = 6.6 Hz, 2H, H2-13), 3.82 (dt, J = 10.2, 5.7 Hz, 1H, H-17), 3.67 (t, J = 6.0 Hz, 2H, H2-11), 3.46
(t, J = 6.6 Hz, 2H, H2-9), 3.36 (s, 3H, OCH3), 2.23 (dd, J = 12.6, 9.6 Hz, 1H, H-16), 2.05 (dd, J = 12.6,
2.7 Hz, 1H, H-16), 1.76 (quin, J = 6.3 Hz, 2H, H2-10), 1.64 (s, 3H, 15-CH3), 0.87 (s, 9H, TBS), 0.03 (s, 6H,
TBS). 13C NMR (75 MHz, CDCl3)
δ 160.9 (d, JCF = 244.7 Hz), 136.4, 129.2, 128.9, 128.6, 126.1 (d, JCF =
12.8 Hz), 124.5, 115.5, 95.3, 75.6, 72.7, 67.3, 67.0, 60.2, 56.2, 43.0, 33.2, 26.0, 18.5, 16.6, 16.5. IR (film) νmax
:
3461, 2928, 2855, 1616, 1586, 1252, 1090, 1031 cm−1. HRMS (ESI): m/z calculated for C24H42FO5Si [M +
H]+: 457.2786. Found: 457.2782.
3.16. Synthesis of (2E,4Z)-(5S,6S,E)-5-(2-Fluorophenyl)-8,16,16,17,17-pentamethyl-2,4,11,15-
tetraoxa-16-silaoctadec-8-en-6-yl 7-((tert-butyldimethylsilyl)oxy)-8-(diethoxyphosphoryl)-
5-methylocta-2,4-dienoate [(17S,18S) ester 24]
(17S,18S) Ester 24 was obtained as a pale-yellow oil in 91% yield from (17S,18S) Fragment C9–C18
(
8
) and Fragment C1–C8 (
9
), according to the esterification procedure employed for the preparation
1
of ester 23. H NMR (300 MHz, CDCl3)
δ
7.50 (dd, J = 14.4, 12.0 Hz, 1H, H-3), 7.42 (t, J = 7.2 Hz, 1H,
aromatic H), 7.34–7.21 (m, 1H, aromatic H), 7.10 (t, J = 7.5 Hz, 1H, aromatic H), 7.01 (t, J = 9.3 Hz, 1H,
aromatic H), 6.03 (d, J = 11.7 Hz, 1H, H-4), 5.76 (d, J = 15.0 Hz, 1H, H-2), 5.45–5.37 (m, 1H, H-17), 5.33
(t, J = 6.0 Hz, 1H, H-14), 5.08 (d, J = 5.1 Hz, 1H, H-18), 4.58 (d, J = 6.9 Hz, 1H, OCH2OCH3), 4.51 (d, J =
6.6 Hz, 1H, OCH2OCH3), 4.23–4.04 (m, 5H, 2
= 6.6 Hz, 2H, H2-11), 3.41 (t, J = 6.3 Hz, H2-9), 3.32 (s, 3H, OCH2OCH3), 2.58–2.54 (m, 2H, H2-16),
2.33–2.28 (m, 2H, H2-8), 2.03–1.93 (m, 2H, H2-6), 1.87 (s, 3H, 5-CH3), 1.77–1.70 (m, 2H. H2-10), 1.64
×
OCH2CH3, H-7), 3.93–3.80 (m, 2H, H2-13), 3.65 (t, J
(s, 3H, 15-CH3), 1.31 (t, J = 6.9 Hz, 6H, 2
×
OCH2CH3), 0.90 (0.88) (s, 3H, TBS), 0.86 (s, 6H, TBS), 0.81
166.7,
(0.80) (s, 9H, TBS), 0.08 (0.04) (s, 6H, TBS), 0.02 (-0.04) (s, 6H, TBS). 13C NMR (75 MHz, CDCl3)
δ
160.8 (d, JCF = 245.3 Hz), 146.5 (146.4), 146.3 (146.2), 135.7 (135.2), 129.8, 129.0, 126.8, 125.8 (125.6),125.1
(124.7), 124.3 119.2, 115.5 (d, JCF = 27.5 Hz), 95.0, 72.9, 72.3, 68.9, 66.8, 61.8, 61.7, 60.1, 56.1, 41.7 (41.2),
36.0 (34.2), 33.1, 32.3, 29.8, 27.0, 26.0, 25.9, 25.8, 24.9, 18.5, 18.1, 18.0, 16.6, 16.5. IR (film) νmax: 2928,
2856, 1714, 1636, 1250, 1020 cm−1. HRMS (ESI): m/z calculated for C43H77FO10PSi2 [M + H]+: 859.4777.
Found: 859.4774.
3.17. Synthesis of (2E,4Z)-(1S,2S,E)-1-(2-Fluorophenyl)-6-(3-hydroxypropoxy)-1-(methoxymethoxy)-
4-methylhex-4-en-2-yl 8-(diethoxyphosphoryl)-7-hydroxy-5-methylocta-2,4-dienoate [(17S,18S) diol 26]
(17S,18S) Diol 26 was obtained as a colorless syrup in 65% yield according to the TBS deprotection
procedure used for the conversion of (17R,18R) ester 23 to (17R,18R) diol 25
CD3COCD3) 7.63–7.55 (m, 1H, H-3), 7.52 (t, J = 7.8 Hz, 1H, aromatic H), 7.41–7.33 (m, 1H, aromatic
.
1H NMR (300 MHz,
δ
H), 7.23 (t, J = 7.2 Hz, 1H, aromatic H), 7.14 (t, J = 9.0 Hz, 1H, aromatic H), 6.16 (d, J = 11.4 Hz, 1H, H-4),
5.80 (d, J = 15.0 Hz, 1H, H-2), 5.46 (quin, J = 5.1 Hz, 1H, H-17), 5.31 (t, J = 6.6 Hz, 1H, H-14), 5.10 (d, J =
5.7 Hz, 1H, H-18), 4.61 (d, J = 6.6 Hz, 1H, -OCH2OCH3), 4.51 (d, J = 6.6 Hz, 1H, -OCH2OCH3), 4.22–4.04
(overlapped, 5H, 2
3.40 (t, J = 6.3 Hz, 2H, H2-9), 3.30 (s, 3H, -OCH2OCH3), 2.92 (br.s, 2H, 2
2H, H2-16), 2.29 (d, J = 9.3 Hz, 2H, H2-8), 2.07–2.04 (m, 2H, H2-6), 1.95 (s, 3H, 5-CH3), 1.68 (quin, J
= 6.0 Hz, H2-10), 1.65 (s, 3H, 15-CH3), 1.29 (t, J = 7.2 Hz, 6H, 2
–OCH2CH3). 13C NMR (75 MHz,
CDCl3) 166.0, 160.7 (d, JCF = 243.8 Hz), 147.0 (d, JCF = 9.8 Hz), 140.8, 134.2, 129.8, 129.3, 125.8 (d, JCF
×
–OCH2CH3, H-7), 3.88 (d, J = 6.0 Hz, 2H, H2-13), 3.57 (t, J = 4.8 Hz, 2H, H2-11),
×
OH), 2.60–2.57(overlapped,
×
δ
= 9.8 Hz), 125.6, 125.4 (d, JCF = 4.5 Hz), 124.3, 119.1, 115.3 (115.0), 94.7, 72.3, 66.8, 66.7, 65.5, 61.4(61.3),
61.2 (61.1), 59.1, 55.1, 41.2, 40.7, 34.6, 33.02(32.98), 32.0, 24.4, 15.91(15.86), 15.7(15.6). IR (film) νmax: 3381,

Molecules 2020, 25, 362
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2927, 1709, 1633, 1488, 1019 cm−1. HRMS (ESI): m/z calculated for C31H49FO10P [M + H]+: 631.3048.
Found: 631.3045.
3.18. Synthesis of (2E,4Z)-(1S,2S,E)-1-(2-Fluorophenyl)-1-(methoxymethoxy)-4-methyl-6-(3-oxopropoxy)
hex-4-en-2-yl 8-(diethoxyphosphoryl)-5-methyl-7-oxoocta-2,4-dienoate [ketoaldehyde (17S,18S) 28]
The crude product was obtained from (17S,18S) diol 26 employing the oxidation procedure used
for the synthesis of its antipode 27. After being confirmed by its 1H NMR data, the crude product
1
was directly used for the next step reaction without further purification. H NMR (300 MHz, CDCl3)
δ
9.72 (t, J = 1.8 Hz, 1H, CHO), 7.41 (dt, J = 7.5, 1.8 Hz, 1H, aromatic H), 7.31 (dd, J = 15.0, 11.7 Hz,
1H, H-3), 7.29–7.22 (m, 1H), 7.11 (dt, J = 7.5, 0.9 Hz, 1H), 7.51 (ddd, J = 10.2, 8.1, 0.9 Hz, 1H), 6.17 (d, J
= 11.1 Hz, 1H, H-4), 5.83 (d, J = 15.0 Hz, 1H, H-2), 5.45–5.38 (m, 1H), 5.31 (t, J = 5.7 Hz, 1H, H-14),
5.07 (d, J = 5.4 Hz, 1H), 4.57 (d, J = 6.9 Hz, 1H, OCH2OCH3), 4.50 (d, J = 6.9 Hz, 1 H, OCH2OCH3),
4.20–4.11 (m, 5H, 2
×
OCH2CH3, H-7), 3.90 (t, J = 7.2 Hz, 2H, H2-13), 3.65 (t, J = 6.0 Hz, 2H, H2-11), 3.31
(s, 3H, OCH3), 3.10 (d, J = 22.8 Hz, 2H, H2-8), 2.39–2.19 (m, 2H, H2-10), 1.87 (s, 3H, 5-CH3), 1.63 (s, 3H,
15-CH3).
3.19. Synthesis of (S,3E,9E,11Z,15E)-6-((S)-(2-Fluorophenyl)(methoxymethoxy)methyl)-4,12-
dimethyl-1,7-dioxacyclooctadeca-3,9,11,15-tetraene-8,14-dione [(17S,18S) macrolide 30]
(17S,18S) Macrolide 30 was synthesized according to the HWE ring closing procedure employed
for the conversion of 27 to 29. Colorless syrup, 68% yield for two steps. [
α]: +8.1 (c = 0.22, MeOH).
1H NMR (300 MHz, CDCl3)
δ
7.59 (dd, J = 15.0, 11.4 Hz, 1H, H-3), 7.46 (dt, J = 7.5, 1.8 Hz, 1H, aromatic
H), 7.36–7.25 (m, 1H, aromatic H), 7.15 (dt, J = 7.5, 1.2 Hz, 1H, aromatic H), 7.04 (ddd, J = 9.9, 8.1,
1.2 Hz, 1H, aromatic H), 6.81 (dt, J = 15.9, 6.9 Hz, 1H, H-9), 6.11 (d, J = 11.4 Hz, 1H, H-4), 6.00 (d, J
= 16.2 Hz, 1H, H-8), 5.90 (d, J = 15.0 Hz, 1H, H-2), 5.50 (dd, J = 11.4, 6.3 Hz, 1H, H-17), 5.26 (dd, J =
7.5, 4.5 Hz, 1H, H-14), 5.11 (d, J = 6.3 Hz, 1H, H-18), 4.59 (d, J = 6.9 Hz, 1H,–OCH2OCH3), 4.52 (d, J =
6.6 Hz, 1H, –OCH2OCH3), 3.98 (dd, J = 12.0, 8.1 Hz, 1H, H-16), 3.88 – 3.81 (overlapped, 2H, H2-13),
3.46 – 3.31 (overlapped, 2H, H2-11), 3.33 (s, 3H, OCH3), 3.14 (d, J = 12.6 Hz, 1H, H-16), 2.42–2.35 (m,
2H, H2-10), 2.33 (d, J = 11.7 Hz, 1H, H-6), 2.04 (d, J = 11.7 Hz, 1H, H-6), 1.82 (s, 3H, 5-CH3), 1.63 (s, 3H,
15-CH3). 13C NMR (75 MHz, CDCl3)
130.2, 129.9, 129.0, 125.9 (d, JCF = 7.5 Hz), 125.5 (d, J = 13.5 Hz), 125.2 (d, JCF = 4.5 Hz), 124.5, 120.9 (d,
δ 197.1, 166.6, 160.9 (d, JCF = 244.5 Hz), 146.8, 142.5, 139.8, 134.3,
J
CF = 3.8 Hz), 115.6 (d, JCF = 21.0 Hz), 94.9, 72.9, 72.6, 67.9, 67.8, 56.0, 45.9, 41.4, 33.1, 24.1, 16.7. IR (film)
νmax: 2925, 2854, 1714, 1669, 1634, 1489, 1359, 1280 cm−1. HRMS (ESI): m/z calculated for C27H34FO6
[M + H]+: 473.2339. Found: 473.2332.
3.20. Synthesis of (S,3E,9E,11Z,15E)-6-((S)-(2-Fluorophenyl)(hydroxy)methyl)-4,12-dimethyl-
1,7-dioxacyclooctadeca-3,9,11,15-tetraene-8,14-dione [(17S,18S) zampanolide mimic 6]
(17S,18S) Zampanolide mimic
used for the conversion of (17R,18R) macrolide 29 to (17S,18S) zampanolide mimic
45% yield. [ 7.61 (dd, J = 15.0, 11.1 Hz,
]: +15.6 (c = 0.15, MeOH). 1H NMR (300 MHz, CD3COCD3)
6
was obtained according to the MOM deprotection procedure
5. Colorless syrup,
α
δ
1H, H-3), 7.34 (dd, J = 14.4, 7.2 Hz, 1H, aromatic H), 7.21 (t, J = 7.5 Hz, 1H, aromatic H), 7.10 (dd, J =
10.5, 8.1 Hz, 1H, aromatic H), 6.83 (dt, J = 16.2, 6.3 Hz, 1H, H-9), 6.20 (d, J = 11.1 Hz, 1H, H-4), 6.06 (d, J
= 15.9 Hz, H-8), 5.88 (d, J = 12.6 Hz, 1H, H-2), 5.44 (dd, J = 11.1, 5.4 Hz, 1H, H-17), 5.27 (t, J = 5.4 Hz,
1H, H-14), 5.17 (t, J = 5.4 Hz, 1H, H-18), 4.81 (d, J = 5.4 Hz, 1H, OH), 4.07–3.97 (m, 1H, H-16), 3.90 (d, J
= 12.9 Hz, 1H, H-13), 3.83 (dd, J = 12.3, 4.5 Hz, 1H, H-13), 3.48– 3.32 (m, 2H, H2-11), 3.17 (d, J = 12.6 Hz,
1H, H-16), 2.42–2.33 (overlapped, 3H, H2-10, H-6), 2.19 (d, J = 13.8 Hz, 1H, H-6), 1.81 (s, 3H, 5-CH3),
1.64 (s, 3H, 15-CH3). 13C NMR (75 MHz, CD3COCD3)
δ 196.5, 166.5, 160.5 (d, JCF = 242.3 Hz), 146.6,
142.7, 139.8, 134.7, 130.6, 129.7, 129.5, 126.3, 126.2, 125.7, 124.8, 121.7, 115.6, 73.8, 69.0, 68.9, 68.2, 45.8,
41.6, 33.2, 23.8, 16.3. IR (film) νmax: 3447, 2916, 2855, 1710, 1669, 1633, 1489, 1456, 1359, 1281, 1148 cm−1
HRMS (ESI): m/z calculated for C25H30FO5 [M + H]+: 429.2077. Found: 429.2073.
.

Molecules 2020, 25, 362
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3.21. Cell Culture
All cell lines were initially purchased from American Type Culture Collection (ATCC). The PC-3,
PC-3/DTX, and LNCaP prostate cancer cell lines were routinely cultured in RPMI-1640 medium
supplemented with 10% FBS and 1% penicillin/streptomycin. Cultures were maintained in a high
humidity environment supplemented with 5% carbon dioxide at a temperature of 37 ◦C. The DU145
and DU145/DTX prostate cancer cells were routinely cultured in Eagle’s Minimum Essential Medium
(EMEM) supplemented with 10% FBS and 1% penicillin/streptomycin.
The procedure illustrated in the literature [29,30] was adapted to establish docetaxel-resistant
prostate cancer cell lines. Specifically, docetaxel-resistant DU145 and PC-3 cell lines (DU145/DTX
and PC-3/DTX) were developed over a period of one year by stepwise increased concentrations of
docetaxel. Cells were repeatedly conserved in an appropriate concentration of docetaxel, starting with
IC50 value of the respective parent cell lines. Docetaxel-containing media will be replaced every 2–3 days.
The concentration of docetaxel was increased when cells exhibited resistance to treatments.
3.22. WST-1 Cell Proliferation Assay
PC-3, PC-3/DTX, DU145, DU145/DTX, or LNCaP cells were placed in 96-well plates at a density of
3200 cells each well in 200 µL of culture medium. The cells were then treated with synthesized mimics,
or positive reference separately at different doses for 3 days, while equal treatment volumes of DMSO
were used as vehicle control. After the cells were cultured in a CO2 incubator at 37 ◦C for three days,
the premixed WST-1 cell proliferation reagent (10 µL, Clontech) was added to each well. The cells
were incubated for additional 3 h at 37 ◦C before mixing gently for one minute on an orbital shaker
to ensure homogeneous distribution of color. A microplate-reader (Synergy HT, BioTek) was used
to measure the absorbance of each well at a wavelength of 430 nm. The half-maximal inhibitory
concentration (IC50 value) is the concentration of each compound that inhibits cell proliferation by 50%
under the experimental conditions. Each of IC50 value is represented as the average from triplicate
determinations that were reproducible and statistically significant. The IC50 values were calculated
based on dose–response curves from at least five dosages for each compound.
3.23. Statistical Analysis
All data are represented as the mean
indicated. Other differences between treated and control groups were analyzed using the Student’s
t-test. A p-value < 0.05 was considered statistically significant.
±
standard deviation (S.D.) for the number of experiments
4. Conclusions
In summary, two new zampanolide mimics have been designed and each of them has been achieved
through a 25-step chemical transformation starting from commercially available 2-butyn-1-ol. Yamaguchi
esterification and intramolecular Horner–Wadsworth–Emmons condensation were employed as crucial
reactions to build up the C-17 and C-1 ester moiety and to close the lactone ring. The chiral centers at C17
and C18 in the Fragment C9–C18 (
using the commercially available AD-mix formulations. Our WST-1 cell proliferation assay data in
both docetaxel-resistant and docetaxel-sensitive prostate cancer cell lines revealed that compound is
7 and 8) were introduced by the Sharpless asymmetric dihydroxylation
6
the optimal mimic and the newly designed side chain can act as a bioisostere for the chemically fragile
N-acetyl hemiminal side chain in zampanolide.
Supplementary Materials: The following are available online at http://www.mdpi.com/1420-3049/25/2/362/s1,
NMR spectra (1H and 13C) of the zampanolide mimics
5 and 6, as well as the macrolides 30 and 31, and intermediates
7–8, 14–21, and 23–26.
Author Contributions: Conceptualization and design, Q.C.; experimentation and data analysis, G.C., Z.J.,
Q.Z., G.W., and Q.-H.C.; funding acquisition, Q.-H.C. and G.W.; all authors finally reviewed and approved
the manuscript. All authors have read and agreed to the published version of the manuscript.

Molecules 2020, 25, 362
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Funding: This research was funded by the National Institutes of Health (National Institute of General Medical
Sciences) under Award Number SC2GM121185 (Q. Chen). HRMS were supported by the National Institutes of
Health RCMI program at Xavier University of Louisiana through Grant 2G12MD007595 (G. Wang).
Acknowledgments: We thank the Department of Chemistry and College of Science and Mathematics at CSU-Fresno
for all administrative support.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study;
in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish
the results.
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Sample Availability: Samples of the compounds 5, 6, 29 and 30 are available from the authors.
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