Optimized synthesis and antiproliferative activity of desTHPdactylolides

Article abstract of DOI:10.1016/j.bmc.2018.05.026

Dactylolide and certain analogues are attractive targets for study due to their structural resemblance to zampanolide, a very promising anticancer lead compound and a unique covalent-binding microtubule stabilizing agent. The primary goal of this project is identification and synthesis of simplified analogues of dactylolide that would be easier to prepare and could be investigated for antiproliferative activity in comparison with zampanolide. Extension of Almann's concept of a simplified zampanolide analogue to dactylolide in the form of desTHPdactylolide was attractive not only for reasons of synthetic simplification but also for the prospect that analogues of dactylolide could be prepared in both (17S) and (17R) configurations. Since Altmann's overall yield for the six-step procedure leading to the C9–C18 fragment of desTHPdactylolide was only 8.7%, a study focused on optimized synthesis and antiproliferative evaluation of each enantiomer of desTHPdactylolide was initiated using Altmann's route as a framework. To this end, two optimized approaches to this fragment C9–C18 were successfully developed by us using allyl iodide or allyl tosylate as the starting material for a critical Williamson ether synthesis. Both (17S) and (17R) desTHPdactylolides were readily synthesized in our laboratory using optimized methods in yields of 37–43%. Antiproliferative activity of the pair of enantiomeric desTHPdactylolides, together with their analogues, was evaluated in three docetaxel-sensitive and two docetaxel-resistant prostate cancer cell models using a WST-1 cell proliferation assay. Surprisingly, (17R) desTHPdactylolide was identified as the eutomer in the prostate cancer cell models. It was found that (17S) and (17R) desTHPdactylolide exhibit equivalent antiproliferative potency towards both docetaxel-sensitive (PC-3 and DU145) and docetaxel-resistant prostate cancer cell lines (PC-3/DTX and DU145/DTX).

Full text of DOI:10.1016/j.bmc.2018.05.026

Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Optimized synthesis and antiproliferative activity of desTHPdactylolides
Guanglin Chena, Rubing Wanga, Bao Vuea, Manee Patanapongpibula, Qiang Zhangb,c
,
Shilong Zhengb,c, Guangdi Wangb,c, James D. Whited, Qiao-Hong Chena,⁎
a
Department of Chemistry, California State University, Fresno, 2555 E. San Ramon Ave. M/S SB70, Fresno, CA 93740, USA
Department of Chemistry, Xavier University of Louisiana, 1 Drexel Drive, New Orleans, LA 70125, USA
RCMI Cancer Research Center, Xavier University of Louisiana, 1 Drexel Drive, New Orleans, LA 70125, USA
b
c
d
Department of Chemistry, Oregon State University, 153 Gilbert Hall, Corvallis, OR 97331, USA
A R T I C L E I N F O
A B S T R A C T
Keywords:
Dactylolide
Zampanolide
Prostate cancer cell line
Antiproliferative activity
Dactylolide and certain analogues are attractive targets for study due to their structural resemblance to zam-
panolide, a very promising anticancer lead compound and a unique covalent-binding microtubule stabilizing
agent. The primary goal of this project is identification and synthesis of simplified analogues of dactylolide that
would be easier to prepare and could be investigated for antiproliferative activity in comparison with zampa-
nolide. Extension of Almann’s concept of a simplified zampanolide analogue to dactylolide in the form of
desTHPdactylolide was attractive not only for reasons of synthetic simplification but also for the prospect that
analogues of dactylolide could be prepared in both (17S) and (17R) configurations. Since Altmann’s overall yield
for the six-step procedure leading to the C9–C18 fragment of desTHPdactylolide was only 8.7%, a study focused
on optimized synthesis and antiproliferative evaluation of each enantiomer of desTHPdactylolide was initiated
using Altmann’s route as a framework. To this end, two optimized approaches to this fragment C9–C18 were
successfully developed by us using allyl iodide or allyl tosylate as the starting material for a critical Williamson
ether synthesis. Both (17S) and (17R) desTHPdactylolides were readily synthesized in our laboratory using
optimized methods in yields of 37–43%. Antiproliferative activity of the pair of enantiomeric
desTHPdactylolides, together with their analogues, was evaluated in three docetaxel-sensitive and two doc-
etaxel-resistant prostate cancer cell models using
a WST-1 cell proliferation assay. Surprisingly, (17R)
desTHPdactylolide was identified as the eutomer in the prostate cancer cell models. It was found that (17S) and
(17R) desTHPdactylolide exhibit equivalent antiproliferative potency towards both docetaxel-sensitive (PC-3
and DU145) and docetaxel-resistant prostate cancer cell lines (PC-3/DTX and DU145/DTX).
1. Introduction
aldehyde and/or ketone functions in dactylolide to enolize. Never-
theless, the structural resemblance of dactylolide to zampanolide, a
Dactylolide was isolated in 2001 from the marine sponge
Dactylospongia sp. by Cutignano and was assigned structure 1 (Fig. 1)
except for configuration at C11, C15, and C19.1 On the supposition that
the twenty-membered lactone of dactylolide is identical in configura-
tion with the same unit in zampanolide (2, Fig. 1) of established relative
and absolute configuration2 and that dactylolide is a likely biogenetic
precursor of 2, we have provisionally assigned the (11S, 15S, 19S)
configuration to natural dactylolide.3 Unfortunately, total syntheses of
dactylolide have failed to remove this uncertainty since natural dacty-
lolide is reported to possess [α]D + 30.01 whereas specific rotations for
synthetic enantiomers of dactylolide range from +134 to +235 and
from −128 to −258.4–11 These discrepancies have been attributed to
(a) the tendency for the aldehyde residue of dactylolide to form a hy-
drate and/or an acetal with methanol, and (b) a propensity for the
unique microtubule stabilizing agent that covalently binds to tubulin12
and which has superior cytotoxic potency in both drug-sensitive and
multi-drug resistant cancer cell models,12,13 makes 1 and certain ana-
logues attractive targets for study. Since published synthetic routes to
dactylolide appear unlikely to produce sufficient material for the
comprehensive in vivo studies we envision, a primary goal of this pro-
ject is identification and synthesis of simplified analogues of 1 that
would be easier to prepare and could be investigated for anti-
proliferative activity in comparison with zampanolide.
Altmann and co-workers have reported that deletion of the tetra-
hydropyran moiety from zampanolide and its replacement by a direct
ether linkage within the macrolactone produces
a
structure
(desTHPzampanolide, 3) that retains submicromolar cytotoxicity.8 This
simplification of the zampanolide core not only removes two of three
⁎
Corresponding author.
E-mail address: qchen@csufresno.edu (Q.-H. Chen).
https://doi.org/10.1016/j.bmc.2018.05.026
Received 16 April 2018; Received in revised form 12 May 2018; Accepted 17 May 2018
0968-0896/©2018ElsevierLtd.Allrightsreserved.
Pleasecitethisarticleas:Chen,G.,Bioorganic&MedicinalChemistry(2018),https://doi.org/10.1016/j.bmc.2018.05.026

G. Chen et al.
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Fig. 1. Structures of dactylolide, zampanolide and simplified analogues.
stereocenters from the macrolactone portion but substantially reduces
the total number of synthetic steps from that required for zampanolide
itself. Extension of Altmann’s concept to dactylolide (1) in the form of
desTHPdactylolide (4, Fig. 1) was attractive not only for reasons of
synthetic simplification but also for the prospect that analogues of 1
could be prepared in both (17S) and (17R) configurations. Therefore, a
study focused on optimized synthesis and antiproliferative evaluation
of each enantiomer of desTHPdactylolide (4) was initiated using Alt-
mann’s route to 4 as a framework.
represents a substantial improvement over Altmann’s synthesis, a dis-
advantage of the route is the high volatility of diiodide 12. In order to
circumvent the problem of handling this material on a large scale, a
second modification of the synthesis of 15 was investigated which first
converted iodo alcohol 9 to iodo tosylate 16 (Scheme 4). Williamson
ether synthesis with this substrate and alcohol 14 in the presence of
sodium hydride gave 15 in an optimized yield of 75% after chroma-
tographic purification. Diiodo ether byproduct 17 formed in this reac-
tion by displacement of tosylate 16 with iodo alcohol 9 was minimized
by adding a mixture of 9 and sodium hydride (1.5–1.8 eq) in ether to a
solution of tosyl chloride (1.1–1.6 eq) in ether and then adding alcohol
14 (2 eq). This protocol did not diminish formation of a second by-
product, tosylate 18, from tosylation of alcohol 14, but this is readily
removed upon chromatographic separation.
2. Results and discussion
2.1. Optimized syntheses of fragment C9–C18
A key step in Altmann’s synthesis of desTHPdactylolide (4) is
Yamaguchi esterification of acid 5 (fragment C1–C8) with alcohol 6
(fragment C9–C18) to yield an acyclic precursor 7 for macrocyclization
(Scheme 1). Ring closure of 7 was accomplished by intramolecular
Horner-Wadsworth-Emmons condensation. Construction of the 18-
membered macrolactone 4 by this route required a total of 22 steps
from commercially available material.8 Although the C1–C8 fragment 5
can be synthesized in multi-gram quantities from 2-butyn-1-ol with the
yield of each step exceeding 70%, Altmann’s overall yield for the six-
step procedure leading to the C9–C18 fragment 6 was only 8.7%. This
places a limitation on the availability of material needed for in vivo
testing. Our goal was to establish an efficient route to 6 from a readily
available starting material that significantly improved the overall yield
of this intermediate. Since the low yield of 6 by Altmann’s route is
mainly attributable to an inefficient synthesis of C9-C15 fragment 8, we
focused initially on modifications to the pathway shown in Scheme 2
which employed a Williamson ether synthesis of iodo alcohol 9 with
allyl bromide followed by hydroboration-oxidation of 10 and final
protection of primary alcohol 11 to give 8.
2.2. Preparation of (17S) and (17R) desTHPdactylolides
With an efficient synthesis of iodo ether 15 in hand, its lithiation
with tert-butyllithium followed by reaction with PMB-protected (R)-
glycidol14,15 in toluene at −90 °C in the presence of boron trifluoride
etherate proceeded smoothly to give 19 in 61% yield. The overall yield
of 19 from 9 by this three-step sequence was improved from the lit-
erature value of 8.7% to 37–43%. Subsequent transformations towards
(17S) desTHPdactylolide (4) via macrolides 20 and 21 were patterned
on Altmann’s synthesis8 which used a TBDPS-masked C9 ether rather
than our TBS-protected version 19 (Scheme 5). Our route resulted in an
overall yield of (17S) 4 from 9 of 30%. A parallel series from 15 using
PMB-protected (S)-glycidol led to (17R) 22 and then to (17R)
desTHPdactylolide 23 in 32% overall yield via the six-step sequence
through macrolactone intermediates 24 and 25. Note that spectral data
for known compounds (final and intermediates) are consistent with
data reported by Altmann.8
An attractive strategy for improving access to 8 appeared to be re-
versal of reactant roles in a Williamson ether approach, and to this end
iodo alcohol 9 was converted to diiodide 12 (Scheme 3). Treatment of
the latter with mono-protected 1,3-propanediols 13 and 14 afforded
iodo ethers 8 and 15 in 58–60% yield. Although this sequence
2.3. The antiproliferative activity of the dactylolide analogues
An IC50 value of 0.75
0.07 µM has been reported by Altmann and
co-workers8 for (−)-dactylolide towards the PC-3 prostate cancer cell
line, which is about 260-fold less potent than (−)-zampanolide (cells
Scheme 1. Retrosynthetic analysis of desTHPdactylolide (4).
2

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Scheme 2. Synthesis of fragment C9–C15 (8) reported by Zurwerra et. al8.
Scheme 3. Optimized synthesis of C9–C15 fragments 8 and 15.
Scheme 4. Optimized synthesis of fragment C9–C15 15.
were exposed to the compound for 72 h). An IC50 value of
3.05 0.18 µM for 21 and an IC50 value of 4.02 0.10 µM for (17S)
desTHPdactylolide (4), respectively, have been reported towards PC-3
cells in the literature,8 indicating that 21 and 4 are about 4- to 5-fold
less potent than (−)-dactylolide (3).
In this work, the antiproliferative activity of three pairs of en-
antiomeric macrolides, 20 and 24, 21 and 25, and (17S)
desTHPdactylolide (4) and (17R) desTHPdactylolide (23), has been
evaluated in three docetaxel-sensitive prostate cancer cell models (PC-
3, DU145, and LNCaP) and two docetaxel-resistant prostate cancer cell
lines (PC-3/DTX and DU145/DTX) using the WST-1 cell proliferation
assay with docetaxel as a positive control. As illustrated in Table 1, two
(17S) macrolides (21 and 4) show almost equivalent antiproliferative
potency towards both androgen-sensitive prostate cancer cells (LNCaP)
and androgen-insensitive prostate cancer cell lines (PC-3 and DU145),
while two (17R) macrolides (24 and 23) are slightly more potent
against androgen-sensitive LNCaP cells. The IC50 values for 21 and 4
against PC-3 prostate cancer cells are slightly higher than those re-
ported in the literature, a variation which may be caused by the dif-
ferent assay methods and conditions. Interestingly, two (17R) dactylo-
lide analogues (24 and 23) are eutomers because they are generally
more potent than their enantiomers towards the five prostate cancer
cell lines. The eudismic ratios16,17 for compound (17R) 24 over com-
pound (17S) 21 are 1.2, 2.9, and 2.9 against PC-3, DU145, and LNCaP
cell lines. Similarly, the eudismic ratios for (17R) 23 over (17S) 4 are
2.3, 3.5, 2.1. 1.9, and 4.2 towards PC-3, PC-3/DTX, DU145, DU145/
DTX, and LNCaP cell lines. It is worth noting that the antiproliferative
activity of (+)-zampanolide and (+)-dactylolide on prostate cancer
cell models has not yet been reported, even though the C20-epimer of
(−)-zampanolide is reported to be one order of magnitude less potent
than (−)-zampanolide.8 Our data imply that inverting the C19 chiral
center might be a way to develop improved dactylolide analogues.
However, it is not clear whether this activity as well as the activity of
dactylolide is biologically related to that of zampanolide.
3

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Scheme 5. Preparation of (17S) desTHPdactylolide (4) and (17R) desTHPdactylolide (23).
Table 1
Anti-proliferative activity of dactylolide analogues against both docetaxel-sensitive and docetaxel-resistant prostate cancer cells.
Compounds
IC50: (µM)a
R/Sd
IC50: (µM)a
R/Sc
IC50: (µM)a
PC-3b
0.0019
7.66
16.08
11.91
6.31
PC-3/DTXc
DU145e
0.0012
9.21
17.76
11.58
3.16
DU145/DTXf
LNCaPg
0.0002
8.27
19.33
13.46
2.85
Docetaxel
0.0006
0.69
0.44
3.68
0.22
2.34
0.25
1232.0
> 2.6
0.9
1.3
0.6
0.0003
0.13
1.35
0.21
0.25
8.58
0.39
7150.0
> 2.2
0.8
1.3
1.0
0.0001
0.66
2.36
20
21
4
> 20.00
14.40
15.42
> 20.00
14.07
15.33
1.01
1.23
0.45
1.24
0.9
24
25
23
3.77
0.84
3.20
0.15
0.71
> 30.00
3.22 0.31
> 30.00
5.27 0.65
> 30.00
–
0.8
> 30.00
5.41 0.11
> 30.00
–
1.5
4.41
0.44
8.03
0.13
a
IC50 is the drug concentration effective in inhibiting 50% of the cell viability measured by WST-1 cell proliferation assay after 3 days of exposure. The data were
presented as the mean
standard deviation of the mean.
b
Human androgen-insensitive prostate cancer cell line derived from bone metastasis of prostate tumor.
Docetaxel-resistant PC-3 prostate cancer cell line.
The relative resistance of the two cell lines obtained by dividing the IC50 value of the resistance cell line by that of the parental cell line.
Human androgen-insensitive prostate cancer cell line derived from brain metastasis of prostate tumor.
Docetaxel-resistant DU145 prostate cancer cell line.
c
d
e
f
g
Human androgen-sensitive prostate cancer cell line.
Additionally, four dactylolide mimics (21, 4, 24, and 23) were
and 25, has been evaluated in three docetaxel-sensitive and two doc-
found to exhibit equivalent antiproliferative potency towards both
docetaxel-sensitive (PC-3 and DU145) and docetaxel-resistant prostate
cancer cell lines (PC-3/DTX and DU145/DTX) (Table 1). The relative
resistance of PC-3/DTX over PC-3 ranges from 0.6 to 1.3 while the
relative resistance of DU145/DTX over DU145 falls in the range of 0.8
to 1.48. In contrast, the docetaxel-resistant PC-3 cells are 1232 fold
more resistant to docetaxel compared to the parent PC-3 cell line while
the docetaxel-resistant DU145 cells are 7150 fold more resistant to
docetaxel compared to the parent DU145 cell line.
etaxel-resistant prostate cancer cell models using the WST-1 cell pro-
liferation assay, with docetaxel as a positive control. Two (17S) dac-
tylolide analogues (22 and 4) exhibit almost equivalent potency
towards both androgen-sensitive and androgen-insensitive prostate
cancer cells while two (17R)-dactylolide analogues (24 and 23) are
more potent against androgen-sensitive LNCaP cells. For the first time,
we investigated the effect of configuration at C-17 of
desTHPdactylolides (C-19 of dactylolides) on prostate cancer cell pro-
liferation and identified (17R) desTHPdactylolide (23) and the struc-
turally related compound 24 as the eutomers. Our data imply that in-
verting the C19 chiral center may be a way to develop improved
dactylolide analogues.
3. Conclusions
The yield reported in the literature for synthesis of fragment C9-C18
of desTHPdactylolides was significantly improved in this work from
8.7% to 36.6–43.0%, which makes the gram-scale synthesis of
desTHPdactylolide (4) far more efficient. To this end, two optimized
methods have been successfully developed by us using allyl iodide or
allyl tosylate as the electrophile for the critical Williamson ether
synthesis of the C9-C18 unit. With these two methods, both (17S)-
desTHPdactylolide (4) and (17R)-desTHPdactylolide (23) have been
synthesized. The antiproliferative activity of this pair of enantiomers,
together with that of the structurally related compounds 20, 21, 24,
4. Experimental section
4.1. General methods
Optical rotations were measured on
a RUDOLPH Research
Analytical Autopol III Automatic Polarimeter. IR spectra were recorded
on a Nicolet Nexus 470 FTIR spectrophotometer. HRMS were obtained
on an Orbitrap mass spectrometer with electrospray ionization (ESI).
NMR spectra were obtained on a Bruker Fourier 300 spectrometer in
4

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Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
CDCl3. The chemical shifts are given in ppm referenced to the solvent
peak, and coupling constants are reported in Hz. Anhydrous THF and
dichloromethane were purified by a PureSolv MD 7 Solvent Purification
System from Innovative Technologies (MB-SPS-800). All other reagents
and solvents were purchased from commercial sources and were used
without further purification. Silica gel column chromatography was
performed using silica gel (32–63 µM). Preparative thin-layer chroma-
tography (PTLC) separations were carried out on thin layer chromato-
graphy plates precoated with silica gel GF254 (EMD Millipore
Corporation).
concentrated in vacuo. The crude tosylate was used directly for the
next step reaction.
4.2.2.2. Preparation of fragment C9-C15 (15). To a suspension of
sodium hydride (52 mg, 1.3 mmol) in anhydrous THF (4 mL) at 4 °C
under argon was added a solution of alcohol 14 (247 mg, 1.3 mmol) in
anhydrous THF (3 mL), and the reaction mixture was stirred at 4 °C for
2 h prior to addition of a solution of crude tosylate (16, ∼1 mmol)
prepared above. The reaction was allowed to proceed in a cold room
(4 °C) overnight before being quenched with a saturated solution of
ammonium chloride (30 mL). The subsequent mixture was extracted
with dichloromethane (20 mL × 3), and the combined organic extracts
were dried over anhydrous sodium sulfate and concentrated in vacuo to
remove the volatile solvent. The crude product was subjected to PTLC
purification using hexane/ethyl acetate (30:1, v/v) to yield the desired
ether 15 in 27% yield, as well as by products 17 and 18.
4.2. Preparation of fragment C9-C15 (8 & 15)
4.2.1. Method 1
4.2.1.1. Preparation of (E)-1,3-diiodobut-2-ene (12). To a solution of
triphenylphosphine (1.38 g, 5.3 mmol) and imidazole (394 mg,
5.8 mmol) in dichloromethane (10 mL) were sequentially added a
solution of iodine (1.21 g, 4.75 mmol) in dichloromethane (10 mL)
4.2.3. Method 3
and
a
solution of (E)-3-iodobut-2-en-1-ol purchased from Fisher
4.2.3.1. Preparation of tosylate 16. To a suspension of sodium hydride
(360 mg, in 60% mineral oil, 9.0 mmol) in diethyl ether (25 mL) at
room temperature under argon was added a solution of (E)-3-iodobut-2-
en-1-ol (9, 990 mg, 5 mmol) in diethyl ether (10 mL), and the resulting
mixture was stirred for 30 min until no gas evolution occurred. The
reaction mixture was transferred by cannula to a reaction flask charged
with a solution of p-toluenesulfonyl chloride (1.52 g, 8.0 mmoL) in
diethyl ether (15 mL) at 4 °C. The reaction was allowed to proceed with
stirring at room temperature for 1 h before being quenched by adding
aqueous saturated ammonium chloride (150 mL). The subsequent
mixture was stirred for 30 min and then extracted with diethyl ether
(60 mL × 3), and the combined ether extracts were rinsed with brine
(15 mL × 3), dried over anhydrous sodium sulfate, and concentrated in
vacuo. The crude tosylate was used directly for the next step.
Scientific (9, 525 mg, 2.65 mmol) in dichloromethane (5 mL) at 4 °C
(ice bath), and the reaction was allowed to proceed at room
temperature with stirring for 1 h. The reaction was quenched by
adding saturated sodium thiosulfate (50 mL), and the resulting
mixture was extracted with dichloromethane (3 × 30 mL). The
combined organic extracts were dried over anhydrous sodium sulfate
and concentrated in vacuo at less than 20 °C. The crude product was
subjected to PTLC purification eluting with hexane/ethyl acetate (30:1,
v/v) to furnish the title compound as a red oil in 91% yield. 1H NMR
(300 MHz, CDCl3) δ 6.48 (tq, J = 9.0, 1.5 Hz, 1H, vinyl H), 3.76 (d,
J = 9.0 Hz, 2H, allylic CH2), 2.40 (d, J = 1.2 Hz, 3H, allylic CH3).
4.2.1.2. Preparation of fragment C9-C15 (8 and 15). To a suspension of
sodium hydride (404 mg, 60% in mineral oil, 10.1 mmol) in anhydrous
THF (68 mL) at 4 °C was added a solution of alcohol 13 or 14
(10.1 mmol) in THF (5 mL), and the reaction mixture was stirred at
4 °C for 2 h prior to addition of a solution of (E)-1,3-diiodobut-2-ene
(12, 2.39 g, 7.76 mmol) in THF (5 mL). The subsequent reaction
mixture was stirred in a cold room (4 °C) overnight before the
addition of saturated ammonium chloride (200 mL). The mixture was
extracted with dichloromethane (150 mL × 3), the combined organic
extracts were dried over anhydrous sodium sulfate, and volatile solvent
was removed under vacuum. The crude product was subjected to
column chromatography using hexane/ethyl acetate (30:1, v/v) as
eluent to yield the desired ether 8 or 15, respectively. Ether 8 was
obtained as a pale yellow oil in 76% yield. The 1H and 13C NMR data of
ether 8 are consistent with those reported in the literature.8 Ether 15
was obtained as a pale yellow oil in 73% yield. 1H NMR (300 MHz,
CDCl3) δ 6.31 (t, J = 6.9 Hz, 1H, H-14), 3.89 (d, J = 6.8 Hz, 2H, H2-13),
3.67 (t, J = 6.0 Hz, 2H, H2-11), 3.48 (t, J = 6.3 Hz, 2H, H2-9), 2.42 (s,
3H, 15-CH3), 1.75 (quin, J = 6.3 Hz, 2H, H2-10), 0.88 (s, 9H, TBS), 0.03
(s, 6H, TBS). 13C NMR (75 MHz, CDCl3) δ 138.0, 98.4, 67.7, 67.1, 59.9,
33.0, 28.3, 26.1, 18.5, −5.2.
4.2.3.2. Preparation of fragment C9-C15 (15). To a suspension of
sodium hydride (400 mg, 10 mmol) in anhydrous THF (420 mL) at
4 °C under argon was added a solution of alcohol 14 (1.9 g, 10 mmol) in
anhydrous THF (15 mL), and the reaction mixture was stirred at 4 °C for
2 h prior to addition of a solution of crude tosylate (16, ∼1 mmol)
prepared above in anhydrous THF (15 mL). The reaction was allowed to
proceed in a cold room (4 °C) overnight before being quenched with
saturated solution of ammonium chloride (150 mL). The subsequent
mixture was extracted with dichloromethane (60 mL × 3), and the
combined organic extracts were dried over anhydrous sodium sulfate
and concentrated in vacuum to remove the volatile solvent. The crude
product was subjected to column chromatographic purification over
silica gel using hexane/ethyl acetate (50:1, v/v) to yield the desired
ether 15 in 75% yield.
4.3. Synthesis of fragment C9-C18 (compound 19)
To a solution of vinyliodide 15 (942 mg, 2.546 mmol; co-evaporated
twice with pentance) in toluene (16 mL) at −78 °C was added tert-BuLi
(2.68 mL, 1.9 M, 5.09 mmol), and the mixture was stirred at −78 °C for
45 min prior to being cooled down to −90 °C. PMB-protected (S)-gly-
cidol8 (1.33 g, 6.87 mmol; coevaporated twice with pentance) in to-
luene (10 mL) was added dropwise to maintain an interior temperature
below −78 °C. The reaction solution was re-cooled to −90 °C before
BF3%OEt2 (759 mg, 5.35 mmol) was added dropwise. The resulting so-
lution was then stirred at −78 °C overnight prior to being quenched
with EtOAc (40 mL) and saturated aqueous NaHCO3 (100 mL) was
added. The organic layer was separated and the aqueous layer was
extracted with ethyl acetate (100 mL × 3). The combined organic
layers were dried over anhydrous sodium sulfate and concentrated in
vacuo to remove ethyl acetate. The crude mass was subjected to column
chromatographic purification over silica gel, using hexane/ethyl
acetate 4:1 as eluent, to yield secondary alcohol 19 in 61% yield as a
4.2.2. Method 2
4.2.2.1. Preparation of tosylate 16. To a suspension of sodium hydride
(60 mg, in 60% mineral oil, 1.5 mmol) in diethyl ether (3 mL) at room
temperature under argon was added a solution of alcohol 9 (198 mg,
1 mmol) in diethyl ether (5 mL), and the resulting mixture was stirred
for 30 min until no gas evolution occurred. The reaction mixture was
then cooled down to 0 °C and a solution of p-toluenesulfonyl chloride
(210 mg, 1.1 mmoL) in diethyl ether (5 mL) was added. The reaction
was allowed to proceed with stirring at room temperature overnight
before being quenched by adding aqueous saturated ammonium
chloride (30 mL). The subsequent mixture was extracted with diethyl
ether (20 mL × 3), and the combined ether extracts were rinsed with
brine (5 mL × 3), dried over anhydrous sodium sulfate, and
5

G. Chen et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
colorless oil. 1H NMR (300 MHz, CDCl3) δ 7.26 (d, J = 8.7 Hz, 2H,
phenyl-H), 6.88 (d, J = 8.7 Hz, 2H, Phenyl-H), 5.43 (t, J = 5.7 Hz, 1H,
H-14), 4.48 (s, 2H, benzylic CH2), 3.98 (d, J = 6.6 Hz, 2H, H2-18),
3.99–3.91 (m, 1H, H-17), 3.81 (s, 3H, OCH3), 3.69 (t, J = 6.0 Hz, 2H,
H2-11), 3.49 (t, J = 6.6 Hz, 2H, H2-9), 3.49–3.44 (overlapped, 1H, H-
13), 3.32 (dd, J = 9.3, 7.2 Hz, 1H, H-13), 2.19 (d, J = 6.6 Hz, 2H, H2-
16), 1.78 (quin, J = 6.3 Hz, 2H, H2-10), 1.70 (s, 3H, 15-CH3), 0.89 (s,
9H, TBS), 0.05 (s, 6H, TBS).
were dried over anhydrous sodium sulfate and concentrated in vacuum.
PTLC purification of the crude product eluting with hexane/acetyl
acetate (1:1, v/v) furnished the desired dactylolide analogue 4 as a pale
yellow syrup in 70% yield. [α]D20: −37.4 (c = 0.065, MeOH). 1H NMR
(300 MHz, CDCl3) δ 9.65 (s, 1H, H-20), 7.66 (dd, J = 15.3, 11.7 Hz, 1H,
H-3), 6.87 (dt, J = 15.9, 6.9 Hz, 1H, H-9), 6.17 (d, J = 11.7 Hz, 1H, H-
4), 6.11 (d, J = 16.2 Hz, 1H, H-8), 5.93 (d, J = 15.0 Hz, 1H, H-2), 5.39
(t, J = 7.2 Hz, 1H, H-14), 5.31 (dd, J = 10.8, 2.7 Hz, 1H, H-17), 4.01
(dd, J = 11.7, 7.5 Hz, 1H, H-13), 3.91 (dd, J = 11.7, 4.5 Hz, 1H, H-13),
3.57 (d, J = 13.2 Hz, 1H, H-6), 3.53–3.46 (m, 3H, H2-11 & H-6), 2.60
(d, J = 13.8 Hz, 1H, H-16), 2.46–2.38 (m, 3H, H2-10 & H-16), 1.89 (s,
3H, 5-CH3), 1.71 (s, 3H, 15-CH3). 13C NMR (75 MHz, CDCl3) δ 199.0,
196.6, 166.3, 146.8, 143.8, 140.6, 133.1, 130.6, 126.00, 125.99, 120.1,
75.9, 68.0, 67.6, 45.8, 39.2, 32.9, 24.5, 16.5. IR (film) νmax: 2923,
2855, 1711, 1666, 1631, 1436 cm−1. HRMS (ESI): m/z calculated for
4.4. Synthesis of (17S) macrolide 20
The (17S) macrolide 20 was prepared from compound 19 and 5
20
through a four-step procedure as described in the literature.8 [α]D
:
−38.7 (c = 0.46, MeOH). 1H NMR (300 MHz, CDCl3) δ 7.60 (dd,
J = 15.0, 11.4 Hz, 1H, H-3), 7.25 (d, J = 8.7 Hz, 2H, phenyl-H), 6.87
(d, J = 8.7 Hz, 2H, phenyl-H), 6.89–6.78 (overlapped, 1H, H-9), 6.12
(d, J = 11.4 Hz, 1H, H-4), 6.04 (d, J = 15.9 Hz, 1H, H-8), 5.90 (d,
J = 15.0 Hz, 1H, H-2), 5.42–5.34 (m, 1H, H-17), 5.28 (t, J = 5.7 Hz,
1H, H-14), 4.53 (d, J = 11.7 Hz, 1H, benzylic H), 4.47 (d, J = 11.7 Hz,
1H, benzylic H), 4.01 (dd, J = 12.0, 8.1 Hz, 1H, H-18), 3.88 (dd,
J = 12.0, 4.8 Hz, 1H, H-18), 3.80 (s, 3H, OCH3), 3.76 (d, J = 12.0 Hz,
1H, H-6), 3.60–3.36 (m, 4H, H2-13 & H2-11), 3.26 (d, J = 12.6 Hz, 1H,
H-6), 2.44–2.23 (m, 4H, H2-10 & H2-16), 1.83 (s, 3H, 5-CH3), 1.69 (s,
3H, 15-CH3). 13C NMR (75 MHz, CDCl3) δ 197.1, 166.7, 159.5, 146.8,
142.4, 139.5, 134.7, 130.3, 130.2, 129.5, 126.0, 124.9, 121.4, 114.0,
73.1, 71.6, 69.8, 67.9, 67.8, 55.4, 45.9, 42.1, 33.0, 24.1, 16.8. IR (film)
νmax: 2914, 2857, 1708, 1668, 1635, 1513 cm−1. HRMS (ESI): m/z
calculated for C27H35O6 [M+H]+: 455.2434. Found: 455.2431.
C
19H25O5 [M+H]+: 333.1702. Found: 333.1696.
4.7. Synthesis of fragment C9–C18 (compound 22)
A similar procedure to that used to prepare 19 from iodide 15 was
employed to prepare 22. 1H NMR (300 MHz, CDCl3) δ 7.25 (d,
J = 8.4 Hz, 2H, phenyl-H), 6.87 (d, J = 8.7 Hz, 2H, phenyl-H), 5.42 (t,
J = 6.0 Hz, 1H, H-14), 4.47 (s, 2H, benzylic CH2), 3.96 (d, J = 6.6 Hz,
2H, H2-18), 3.97–3.89 (m, 1H, H-17), 3.79 (s, 3H, OCH3), 3.68 (t,
J = 6.3 Hz, 2H, H2-11), 3.48 (t, J = 6.3 Hz, 2H, H2-9), 3.48–3.43
(overlapped, 1H, H-13), 3.32 (dd, J = 9.3, 7.2 Hz, 1H, H-13), 2.18 (d,
J = 6.6 Hz, 2H, H2-16), 1.77 (quin, J = 6.3 Hz, 2H, H2-10), 1.69 (s, 3H,
15-CH3), 0.89 (s, 9H, TBS), 0.04 (s, 6H, TBS). 13C NMR (75 MHz, CDCl3)
δ 159.4, 136.2, 130.2, 129.5, 124.5, 114.0, 79.1, 74.0, 73.2, 68.4, 67.3,
60.1, 55.4, 43.7, 33.1, 26.1, 18.5, 16.7, −5.2.
4.5. Synthesis of (17S) macrolide 21
To
a
solution of PMB ether 20 (35 mg, 0.077 mmol) in di-
4.8. Synthesis of (17R) macrolide 24
chloromethane (2 mL) was sequentially added water (0.4 mL) and 2,3-
dichloro-5,6-dicyano-p-benzoquinone (DDQ, 35 mg, 0.154 mmol), and
the reaction mixture was vigorously stirred at room temperature for 1 h.
The reaction was quenched by adding aqueous saturated sodium bi-
carbonate aqueous solution (10 mL), and the mixture was extracted
with dichloromethane (10 mL × 4). The combined extracts were dried
over anhydrous sodium sulfate and concentrated in vacuo. The crude
product was subjected to PTLC purification using hexanes/ethyl acetate
(1:1, v/v) as eluent to give alcohol 21 as a pale yellow syrup in 73%
yield. [α]D20: −86.0 (c = 0.23, MeOH); −79.7 (c = 0.20, CHCl3). 1H
NMR (300 MHz, CDCl3) δ 7.62 (dd, J = 15.0, 11.7 Hz, 1H, H-3), 6.85
(dt, J = 16.2, 6.6 Hz, 1H, H-9), 6.13 (d, J = 11.1 Hz, 1H, H-4), 6.06 (d,
J = 15.9 Hz, 1H, H-8), 5.91 (d, J = 15.0 Hz, 1H, H-2), 5.35–5.25 (m,
2H, H-17 & H-14), 4.02 (dd, J = 12.0, 7.8 Hz, 1H, H-18), 3.89 (dd,
Lactone 24 was prepared from 22 and 5 through a four-step reaction
sequence as described in the literature.8 [α]D20: +38.6 (c = 0.68,
CHCl3). 1H NMR (300 MHz, CDCl3) δ 7.61 (dd, J = 15.0, 11.4 Hz, 1H,
H-3), 7.26 (d, J = 8.1 Hz, 2H, phenyl-H), 6.88 (d, J = 8.7 Hz, 2H,
phenyl-H), 6.91–6.80 (overlapped, 1H, H-9), 6.13 (d, J = 11.4 Hz, 1H,
H-4), 6.05 (d, J = 15.9 Hz, 1H, H-8), 5.91 (d, J = 15.0 Hz, 1H, H-2),
5.43–5.35 (m, 1H, H-17), 5.29 (t, J = 5.7 Hz, 1H, H-14), 4.54 (d,
J = 11.7 Hz, 1H, benzylic H), 4.48 (d, J = 11.7 Hz, 1H, benzylic H),
4.02 (dd, J = 12.0, 8.1 Hz, 1H, H-18), 3.89 (dd, J = 12.0, 4.5 Hz, 1H,
H-18), 3.81 (s, 3H, OCH3), 3.77 (d, J = 12.9 Hz, 1H, H-6), 3.61–3.36
(m, 4H, H2-13 & H2-11), 3.27 (d, J = 12.9 Hz, 1H, H-6), 2.45–2.24 (m,
4H, H2-10 & H2-16), 1.84 (s, 3H, 5-CH3), 1.70 (s, 3H, 15-CH3). 13C NMR
(75 MHz, CDCl3) δ 197.1, 166.7, 159.4, 146.9, 142.4, 139.5, 134.6,
130.3, 130.2, 129.5, 126.0, 124.9, 121.4, 114.0, 73.0, 71.6, 69.7, 67.9,
67.8, 55.4, 45.9, 42.1, 33.0, 24.1, 16.8. IR (film) νmax: 2923, 2854,
1708, 1668, 1633, 1513, 1462 cm−1. HRMS (ESI): m/z calculated for
J = 12.6, 4.5 Hz, 1H, H-18), 3.79–3.67 (m, 3H, H2-13
& H-6),
3.53–3.37 (m, 2H, H2-11), 3.29 (d, J = 12.6 Hz, 1H, H-6), 2.43–2.36
(m, 2H, H2-10), 2.35 (d, J = 13.8 Hz, 1H, H-16), 2.22 (d, J = 13.5 Hz,
1H, H-16), 1.85 (s, 3H, 5-CH3), 1.72 (s, 3H, 15-CH3). 13C NMR (75 MHz,
CDCl3) δ 197.0, 167.1, 146.8, 142.8, 139.8, 134.4, 130.3, 126.0, 125.0,
121.1, 72.2, 68.0, 67.8, 65.5, 46.0, 41.5, 33.0, 24.2, 16.8. IR (film)
νmax: 3435, 2921, 2863, 1693, 1667, 1631, 1436 cm−1. HRMS (ESI): m/
z calculated for C19H27O5 [M+H]+: 335.1859. Found: 335.1848.
C
27H35O6 [M+H]+: 455.2434. Found: 455.2430.
4.9. Synthesis of (17R) macrolide 25
Lactone 25 (31 mg, 80%, pale yellow oil) was synthesized from
lactone 24 using a procedure similar to that employed for conversion of
20 to 21. [α]D20: +70.4 (c = 0.19, CHCl3). 1H NMR (300 MHz, CDCl3)
δ 7.61 (dd, J = 15.0, 11.4 Hz, 1H, H-3), 6.84 (dt, J = 16.2, 6.9 Hz, 1H,
H-9), 6.12 (d, J = 11.4 Hz, 1H, H-4), 6.04 (dt, J = 16.2, 1.2 Hz, 1H, H-
8), 5.90 (d, J = 15.0 Hz, 1H, H-2), 5.31–5.25 (m, 2H, H-14 & H-17),
4.01 (dd, J = 12.0, 7.8 Hz, 1H, H-18), 3.88 (dd, J = 12.0, 4.8 Hz, 1H,
H-18), 3.77–3.72 (m, 3H, H2-13 & H-6), 3.52–3.38 (m, 2H, H2-11), 3.28
(d, J = 12.6 Hz, 1H, H-6), 2.45–2.37 (m, 2H, H2-10), 2.32 (d,
J = 13.5 Hz, 1H, H-16), 2.21 (d, J = 13.5 Hz, 1H, H-16), 1.83 (s, 3H, 5-
CH3), 1.70 (s, 3H, 15-CH3). 13C NMR (75 MHz, CDCl3) δ 197.1, 167.1,
146.9, 142.8, 139.8, 134.4, 130.3, 125.9, 125.0, 121.1, 72.2, 68.0,
67.8, 65.4, 46.0, 41.5, 33.0, 24.2, 16.8. IR (film) νmax: 3446, 2920,
4.6. Synthesis of (17S) desTHPdactylolide (4)
To
a solution of alcohol 21 (15 mg, 0.045 mmol) in di-
chloromethane (0.5 mL) at room temperature was added Dess-Martin
periodinane (29 mg, 0.0675 mmol), and the reaction mixture was
stirred for 10 min before a further quantity of Dess-Martin periodinane
(29 mg, 0.0675 mmol) was added. The reaction was allowed to proceed
at room temperature for an additional 30 min prior to being quenched
with saturated sodium bicarbonate (5 mL) and saturated sodium thio-
sulfate (5 mL). The resulting mixture was stirred for 15 min, and then
extracted with dichloromethane (5 mL × 3). The combined extracts
6

G. Chen et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
2851, 1705, 1632, 1437 cm−1
.
HRMS (ESI): m/z calculated for
reagent (Clontech) was added to each well. After mixing gently for one
minute on an orbital shaker, the cells were incubated for additional 3 h
at 37 °C. To ensure homogeneous distribution of color, it is important to
mix gently on an orbital shaker for one minute. The absorbance of each
well was measured using a microplate-reader (Synergy HT, BioTek) at a
wavelength of 430 nm. The IC50 value is the concentration of each
compound that inhibits cell proliferation by 50% under the experi-
mental conditions and is the average from at least triplicate determi-
nations that were reproducible and statistically significant. The IC50
values were calculated from the dose-response curves based on at least
five dosages for each compound.
C19H27O5 [M+H]+: 335.1859. Found: 335.1851.
4.10. Synthesis of (17R) desTHPdactylolide (23)
(17R) DesTHPdactylolide (23) (14 mg, 75%, pale yellow oil) was
synthesized according to the oxidation procedure employed for the
conversion of 21 to (17S) desTHPdactylolide (4). [α]D20: +38.4
(c = 0.06, MeOH). 1H NMR (300 MHz, CDCl3) δ 9.65 (s, 1H, H-18),
7.65 (dd, J = 15.3, 11.7 Hz, 1H, H-3), 6.85 (dt, J = 15.9, 6.9 Hz, 1H, H-
9), 6.17 (d, J = 11.7 Hz, 1H, H-4), 6.10 (dt, J = 15.9, 1.5 Hz, 1H, H-8),
5.93 (d, J = 15.3 Hz, 1H, H-2), 5.38 (t, J = 5.4 Hz, 1H, H-14), 5.31 (dd,
J = 10.8, 3.0 Hz, 1H, H-17), 4.01 (dd, J = 12.0, 7.8 Hz, 1H, H-13), 3.91
(dd, J = 12.0, 5.1 Hz, 1H, H-13), 3.54 (d, J = 5.4 Hz, 1H, H-6),
3.49–3.44 (m, 3H, H2-11 & H-6), 2.59 (d, J = 13.8 Hz, 1H, H-16),
2.46–2.37 (m, 3H, H2-10 & H-16), 1.88 (s, 3H, 5-CH3), 1.71 (s, 3H, 15-
CH3). 13C NMR (75 MHz, CDCl3) δ 199.1, 196.7, 166.3, 146.9, 143.8,
140.6, 133.1, 130.5, 125.99, 125.96, 120.0, 75.8, 68.0, 67.5, 45.8,
39.2, 32.9, 24.5, 16.5. IR (film) νmax: 2919, 1733, 1706, 1669, 1635,
Acknowledgements
This work was financially supported by the National Institutes of
Health (National Institute of General Medical Sciences) under Award
Number SC2GM121185, and California State University (CSU) Fresno.
HRMS were supported by NIH RCMI program at Xavier University of
Louisiana through Grant 2G12MD007595-064 (G. Wang). We also
thank the Graduate Net Initiative at CSU-Fresno for a Graduate
Research Fellowship (to M.P.) and the Division of Graduate Studies at
CSU-Fresno for a Graduate Student Research and Creative Activities
Support Award (to M.P.).
1558, 1521 cm−1. HRMS (ESI): m/z calculated for C19H25O5 [M+H]+
333.1702. Found: 333.1700.
:
4.11. Cell culture
All cell lines were initially purchased from American Type Culture
Collection (ATCC™). The PC-3 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 prostate cancer cells were routinely
cultured in Eagle’s Minimum Essential Medium (EMEM) supplemented
with 10% FBS and 1% penicillin/streptomycin.
Docetaxel-resistant prostate cancer cell lines were established based
on the procedure illustrated in the literature.18,19 Docetaxel-resistant
DU145 and PC-3 cell lines (DU145/DTX and PC-3/DTX) were devel-
oped over a period of one year by stepwise increased concentrations of
docetaxel. Cells were continuously maintained in docetaxel, with
treatments beginning at the initial IC50 of the respective parent cell
lines. Media containing docetaxel will be changed every 2–3 days. As
cells displayed resistance to treatments of docetaxel the concentration
was subsequently increased.
A. Supplementary data
Supplementary data associated with this article can be found, in the
online version, at http://dx.doi.org/10.1016/j.bmc.2018.05.026.
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4.12. WST-1 cell proliferation assay20
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PC-3, LNCap, or DU145 cells were plated in 96-well plates at a
density of 3200 each well in 200 µL of culture medium. The cells were
then treated with the positive control, or the synthesized compounds at
different doses for 3 days, while equal treatment volumes of DMSO
were used as vehicle control. The cells were cultured in a CO2 incubator
at 37 °C for three days. 10 µL of the premixed WST-1 cell proliferation
7