Bioreductively Activatable Prodrug Conjugates of Combretastatin A-1 and Combretastatin A-4 as Anticancer Agents Targeted toward Tumor-Associated Hypoxia

Article abstract of DOI:10.1021/acs.jnatprod.9b00773

The natural products combretastatin A-1 (CA1) and combretastatin A-4 (CA4) function as potent inhibitors of tubulin polymerization and as selective vascular disrupting agents (VDAs) in tumors. Bioreductively activatable prodrug conjugates (BAPCs) can enha

Full text of DOI:10.1021/acs.jnatprod.9b00773

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Article
Bioreductively Activatable Prodrug Conjugates of Combretastatin
A‑1 and Combretastatin A‑4 as Anticancer Agents Targeted toward
Tumor-Associated Hypoxia
Blake A. Winn,⊥ Laxman Devkota,⊥ Bunnarack Kuch, Matthew T. MacDonough, Tracy E. Strecker,
Yifan Wang, Zhe Shi, Jeni L. Gerberich, Deboprosad Mondal, Alejandro J. Ramirez, Ernest Hamel,
David J. Chaplin, Peter Davis, Ralph P. Mason, Mary Lynn Trawick,* and Kevin G. Pinney*
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ABSTRACT: The natural products combretastatin A-1 (CA1) and combretastatin A-4 (CA4) function as potent inhibitors of
tubulin polymerization and as selective vascular disrupting agents (VDAs) in tumors. Bioreductively activatable prodrug conjugates
(BAPCs) can enhance selectivity by serving as substrates for reductase enzymes specifically in hypoxic regions of tumors. A series of
CA1-BAPCs incorporating nor-methyl, mono-methyl, and gem-dimethyl nitrothiophene triggers were synthesized together with
corresponding CA4-BAPCs, previously reported by Davis (Mol. Cancer Ther. 2006, 5 (11), 2886), for comparison. The CA4-gem-
dimethylnitrothiophene BAPC 45 proved exemplary in comparison to its nor-methyl 43 and mono-methyl 44 congeners. It was
stable in phosphate buffer (pH 7.4, 24 h), was cleaved (25%, 90 min) by NADPH-cytochrome P450 oxidoreductase (POR), was
inactive (desirable prodrug attribute) as an inhibitor of tubulin polymerization (IC50 > 20 μM), and demonstrated hypoxia-selective
activation in the A549 cell line [hypoxia cytotoxicity ratio (HCR) = 41.5]. The related CA1-gem-dimethylnitrothiophene BAPC 41
was also promising (HCR = 12.5) with complete cleavage (90 min) upon treatment with POR. In a preliminary in vivo dynamic
bioluminescence imaging study, BAPC 45 (180 mg/kg, ip) induced a decrease (within 4 h) in light emission in a 4T1 syngeneic
mouse breast tumor model, implying activation and vascular disruption.
he tumor microenvironment exhibits unique attributes,
Tnotably associated with vascular architecture and
associated blood flow dynamics.1−3 Solid tumors, once they
reach approximately 2−5 mm3 in size, must establish their own
vascular network in order to meet their rapidly accelerating
demand for oxygen and nutrients.4−10 This rapid angiogenic
development of tumor-associated vasculature results in
disorganized, fragile, and leaky vessels lacking pericyte support,
thus providing a target for therapeutic intervention.1−3,11−13
Vascular disrupting agents (VDAs) selectively damage
established tumor-associated vasculature, thus denying neces-
sary oxygen and nutrients, resulting in tumor necrosis.2,14−16
VDAs are mechanistically distinct from the established
angiogenesis-inhibiting agents (AIAs) such as bevacizumab
(Avastin).17,18 The natural products combretastatin A-1 (CA1,
Figure 1) and combretastatin A-4 (CA4, Figure 1), isolated
from the bark of the African bush willow tree Combretum
caffrum Kuntze (Combretacae) by Pettit and co-workers, are
potent inhibitors of tubulin polymerization (binding at the
colchicine site) that function biologically as antiproliferative
agents and as VDAs.19−22 These agents cause rapid
morphological changes to the endothelial cells lining tumor-
Received: August 13, 2019
© XXXX American Chemical Society and
American Society of Pharmacognosy
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by release of CA4 in the presence of supersomal P450R) across
a range of oxygen concentrations, the unsubstituted one (nor-
methyl) was only effective under extreme hypoxia (<0.01%
O2).42
Inspired by the premise of targeting tumor hypoxia for the
selective delivery of tubulin-active VDAs, and building on the
encouraging results reported for the CA4-BAPCs, we designed
and synthesized a series of BAPCs that incorporate the natural
product CA1 and evaluated them in preliminary studies to
determine their efficacy as therapeutic agents. A regioselective
protecting group strategy (incorporating tert-butyldimethylsil-
yl, isopropyl, and tosyl groups), which we previously developed
as part of a separate synthetic campaign,43,44 was utilized to
differentiate the catechol functionality (C-2 and C-3 positions)
inherent to CA1. The nitrothiophene triggers previously
described by Davis and co-workers42 were synthesized using
a revised synthetic strategy.41 The synthesized CA1-BAPCs
were evaluated for their ability to inhibit tubulin polymer-
ization and to function as substrates for the reductase enzyme
POR. In addition, differential cytotoxicity studies (normoxia
versus hypoxia) assessed cell-based (A549 lung cancer)
hypoxia-selective activation (evidenced by enhanced cytotox-
icity). Collectively these studies were designed to guide the
potential therapeutic advancement of the most promising
CA1-BAPCs as hypoxia-selective anticancer agents.
Figure 1. Colchicine and combretastatin natural products and
phosphate prodrug salts.
associated blood vessels, resulting in irreversible vascular
damage.1,23,24 The corresponding water-soluble phosphate
prodrugs of CA1 and CA4 [referred to as CA1P (or
OXi4503) and CA4P (fosbretabulin), respectively, Figure 1]
have advanced through preclinical evaluation and clinical
trials.14,21,25−32
In addition to an aberrant vascular network and elevated
interstitial pressure due to immature and leaky vasculature, the
tumor microenvironment is often characterized by regions of
hypoxia and a pH gradient, with cells distant from blood
vessels being in an acidic environment.3,33−37 Hypoxia
represents a characteristic uniquely inherent to many solid
tumors that does not naturally occur in normal tissue,3 thus
offering a specific target based on selective activation of potent
anticancer agents or their selective delivery achieved through
appropriate prodrug strategies.3,38−41 Hypoxia-selective pro-
drugs undergo activation through either one- or two-electron
reductase enzymes.3,34
One type of bioreductively activatable prodrug conjugate
(BAPC) incorporates a bioreductive trigger covalently
attached to an appropriate therapeutic agent, which is designed
to undergo enzyme-mediated cleavage (to release the parent
anticancer agent) under hypoxic conditions (Scheme 1).3,30,34
Davis and co-workers synthesized a series of nor-, mono-, and
gem-dimethyl-nitrothienyl BAPCs (Figure S3, Supporting
Information) that incorporated CA4 and evaluated their
efficacy through the generation of radical anions by pulse
radiolysis and determination (spectrophotometrically) of their
stability and fragmentation.42 They also evaluated these
compounds in the presence of NADPH-cytochrome P450
oxidoreductase (POR) and further assessed their efficacy by
determining their cytotoxicity (normoxia versus hypoxia) in
the A549 human lung cancer cell line.42 It was determined that
the gem-dimethyl-nitrothiophene trigger CA4 prodrug
(Scheme 1) was the most resistant to aerobic metabolism
(in comparison to the nor- and mono-methyl-nitrothiophene
trigger CA4 prodrugs), and the gem-dimethyl CA4-BAPC
remained intact in high oxygen environments.42 While the gem-
and mono-substituted CA4 BAPCs were effective (as evidenced
RESULTS AND DISCUSSION
■
Synthesis. The CA1-BAPCs were synthesized utilizing two
key reactions, a Wittig olefination to generate regioselectively
protected CA1 followed by reaction between the tosyl,
isopropyl, and tert-butyldimethylsilyl protected CA1 analogues
(20, 21, 27, and 28 respectively, Schemes 3 and 4) and the
nitrothienyl triggers (16, 17, and 19, Scheme S1, Supporting
Information) under Mitsunobu conditions.45,46 Synthesis of
the regioselectively protected Z-CA1 analogues (11−13,
Scheme 2) was facilitated by a Wittig olefination reaction
between aldehydes 5−7 (Scheme 2) and triphenyl phospho-
nium salt 10.46 While the Wittig reaction produced a mixture
of Z- and E-stilbene isomers, the Z-isomer was formed
preferentially (Scheme 2).43,44,46,47
Selective demethylation of aldehyde 1 using BCl3 yielded
catechol 2, which was subsequently converted to selectively
protected aldehydes 3−7 (Scheme 2) using a previously
reported synthetic strategy.43,44,46 Phosphonium salt 10 was
prepared upon bromination of benzyl alcohol 8 using PBr3,
followed by a reaction with triphenyl phosphine. A Wittig
reaction between suitably protected aldehydes (5−7) and
Wittig salt 10 yielded a mixture of Z- and E-stilbene isomers
(11−13, favoring the Z-isomer), which were separated by flash
column chromatography.
Scheme 1. Proposed Mechanism for the Biological Reduction and Cleavage of CA4 gem-Dimethyl-Nitrothiophene Trigger
Releasing CA442
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Scheme 2. Synthesis of Regioselectively Protected CA1 Derivatives 11−1332,43
Scheme 3. Regioselective Synthesis of CA1-BAPCs 22−2642,47
Synthesis of the three nitrothiophene triggers (16, 17, 19)
utilized in the Mitsunobu reactions was achieved as previously
described (Scheme S1, Supporting Information).41,42 Depro-
tection of CA1 analogues 11 and 12 using tetra-n-
butylammonium fluoride (TBAF) yielded the corresponding
phenols 20 and 21, respectively, which were subjected to
Mitsunobu conditions that utilized nitrothiophene triggers (16,
17, and 19), phosphine reagents (PPh3 or PBu3), and azo
compounds [diethyl azodicarboxylate (DEAD), diisopropyl
azodicarboxylate (DIAD), or 1,1′-(azodicarbonyl)dipiperdine
(ADDP)] to generate BAPCs 22−26 (Scheme 3).
Attempted deprotection of compounds 22 and 23 with
NaOH (2 M) under either microwave or reflux conditions did
not yield the desired product, but instead cleaved the
nitrothiophene trigger from the starting material to regenerate
compound 20 (Scheme S2, Supporting Information). Sim-
ilarly, compound 24 regenerated compound 21 upon
attempted deprotection using AlCl3 (Scheme S2, Supporting
Information). In an effort to solve this problem, we attempted
to partially cleave the bis-TBS-protected CA1 13 using a
deficiency of TBAF, but this resulted in a mixture of
regioisomers 27 and 28 (Scheme 4), which proved inseparable
by flash column chromatography. This mixture of regioisomers
27 and 28 was further functionalized to incorporate nitro-
thienyl triggers under Mitsunobu conditions to synthesize
regioisomeric TBS/trigger analogues 30−34. The parent
natural product CA1 (29) proved unreactive under analogous
reaction conditions. The protected CA1-BAPC 32 proved
difficult to purify by column chromatography, so the crude
product was taken to the next step. Interestingly, the
conventional tert-butyldimethylsilyl (TBS) deprotection of
compounds 30 and 31 using TBAF yielded ring-cyclized
products 35 and 36 (proposed structures based on analysis of
NMR and HRMS data) without producing any other
discernible side products (Scheme 5). While a plausible
mechanistic explanation for this cyclization has yet to be
established, we place a high degree of confidence on the
structural assignment for cyclic compounds 35 and 36 based
on a combination of 1H NMR, 13C NMR, and mass
spectrometry data (see Supporting Information for further
details). Intrigued by this unusual cyclization (that produced
35 and 36), we investigated whether exposure of phenolic
compounds 37 and 38 to strong base would also facilitate a
similar cyclization reaction, and this indeed proved to be the
case (Scheme 5).
Modification (Scheme 6) of the deprotection conditions
[HCl (2 M)/AcOH, instead of TBAF] afforded the desired
CA1-BAPCs 37−40 (regiochemistry was determined through
1D NOE NMR).
Purification of the TBS-protected gem-dimethyl CA1 BAPC
32 by column chromatography did not result in the requisite
level of purity necessary for meaningful biological evaluation.
Thus, the crude mixture (containing 32, 27, and 28)
underwent deprotection (Scheme 6) prior to purification by
column chromatography. Regioisomeric assignment was
confirmed by 1D NOE NMR. While the overall yield for
this deprotection was quite low, the remaining material
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Scheme 4. Synthesis of TBS-Protected CA1-BAPCs 30−34
Scheme 5. Generation of Cyclized Analogues 35 and 36
consisted only of starting material (crude mixture of 32, 27,
reported by Davis et al., but with several useful modifications
(Scheme 7).42 CA4-BAPC 43 was synthesized through a
Mitsunobu reaction heated to 50 °C with nitrothiophene 16.42
CA4 and nitrothiophene 17 were reacted with DIAD and
and 28).
The known CA4-BAPCs were synthesized (as comparative
compounds) under conditions similar to those previously
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Scheme 6. Synthesis of CA1 BAPCs 37−41
Scheme 7. Synthesis of CA4-BAPCs42
triphenylphosphine to generate BAPC 44.42 The gem-dimethyl
CA4 BAPC 45 was synthesized from CA4, ADDP, nitro-
thiophene 19, and tributylphosphine.42 In order to improve
the yield for the gem-dimethyl CA4 BAPC 45, subsequent
Mitsunobu reactions were performed in toluene.42 While the
overall yield was improved, the new method required a more
extensive purification procedure to remove the remaining CA4
and gem-dimethyl thiophene trigger, both of which had nearly
identical chromatographic retention times to the desired CA4-
BAPC 45. Accordingly, the reaction mixture was subjected to
chemical modification to facilitate chromatographic separation
during purification. The phenolic moiety of CA4 was
converted to its corresponding silyl ether (TBS), and the
unreacted gem-dimethyl trigger was subsequently acetylated,
allowing both of these compounds to be successfully separated
chromatographically from the desired CA4 gem-dimethyl-
nitrothiophene BAPC 45.
Biological Evaluation. Inhibition of Tubulin Polymer-
ization and Colchicine Binding. The BAPCs and their
parent anticancer agents (CA4 and CA1) were evaluated for
their ability to inhibit tubulin polymerization and colchicine
binding (Table 1). The parent anticancer agents [CA4, CA1,
tosyl-protected CA1 (20), and isopropyl-protected CA1 (21)]
utilized in this study were potent inhibitors of tubulin
polymerization (IC50 = 0.64, 1.9, 0.84, and 0.82 μM,
respectively) and strongly inhibited colchicine binding. The
TBS-protected CA1 analogue 26 was only moderately active as
an inhibitor of tubulin polymerization (IC50 = 9.5 μM). Ideally,
the BAPCs prepared from these parent anticancer agents
would be protected from binding to tubulin until cleaved (in
vivo) to generate their corresponding anticancer agents.
Considering the collective group of 15 BAPCs synthesized
for this study, seven BAPCs (22, 23, 25, 36, 43, 44, 45) were
inactive (IC50 > 20 μM) as inhibitors of tubulin polymer-
ization, while four BAPCs (24, 26, 39, 40) were moderately
active inhibitors (IC50 > 3 μM but <20 μM) and four BAPCs
(35, 37, 38, 41) proved to be potent inhibitors (IC50 < 3 μM).
Enzyme-Mediated Cleavage of BAPCs and Stability in
Phosphate Buffer. In preliminary studies, the CA4-BAPCs
were treated with POR supersomes (Table 2). Complete
cleavage was observed for compound 45 (24 h, anaerobic
conditions). Therefore, a shorter assay time period (90 min)
was selected for compound comparison, and under these
conditions compounds 43 and 44 underwent minimal cleavage
(2.7% and 4.1%, respectively), while 45 was more extensively
cleaved (25.4%). These results are in accordance with the
previously reported results from Davis and co-workers that
utilized supersomal POR with compounds 43, 44, and 45 and
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Figure 2. Compilation of parent anticancer agents and their corresponding BAPCs utilized in this study: (A) CA1-BAPCs; (B) CA4-BAPCs; (C)
parent CA1 and CA4 agents.
demonstrated that 45 was cleaved more readily (to release
CA4) than 43 and 44.42 This trend in cleavage (from gem-
dimethyl to mono-methyl to nor-methyl) was also observed
when the CA1-BAPCs were exposed to POR. The gem-
dimethyl CA1-BAPC (41) and the isopropyl-protected gem-
dimethyl BAPC (27) were cleaved more extensively in
comparison to their corresponding mono-methyl and nor-
methyl BAPCs. The mono-methyl and nor-methyl CA1-BAPCs
(37, 38, 39, and 40) were cleaved by POR to different extents,
depending on the position of the nitrothiophene side chain
(bioreductive trigger) and the hydroxy group. It should be
noted that under these assay conditions these four BAPCs (37,
38, 39, and 40) underwent cyclization to generate their
corresponding cyclized analogue 35 or 36. While the
mechanism of this cyclization is unknown, it is noteworthy
that under these assay conditions cyclization that incorporates
the bioreductive trigger was more favorable than the desired
cleavage of the prodrug trigger. BAPC 41 was the only gem-
dimethyl BAPC that was fully cleaved (100%) by POR (90
min) in this study, and thus its stability was further evaluated
in the pH 7.4 buffer. BAPC 41 showed no apparent
spontaneous hydrolysis for the first 150 min, but it was mostly
hydrolyzed if incubated in the buffer for 24−48 h. Therefore,
the cleavage (100%) of BAPC 41 by POR (90 min) was not
due to spontaneous hydrolysis in buffer.
Hypoxia Cytotoxicity Ratio Determined in A549 Lung
Cancer Cell Line. The initial cytotoxicity data for the CA1
and CA4 BAPCs showed promise for differential activity
between oxic and hypoxic environments (Table 3), with
several BAPCs demonstrating a positive hypoxia cytotoxicity
ratio (HCR). A number of prodrugs stood out, notably
compounds 24, 41, and 45. The most active prodrugs in the
series were the gem-dimethyl BAPCs of CA1 (41) and CA4
(45), with HCRs of 12.5 and 41.5, respectively, consistent with
previous studies by Davis and co-workers that demonstrated
that the gem-dimethyl CA4-BAPC had greater resistance to
cleavage in oxic environments, releasing the parent anticancer
agent (CA4) selectively under hypoxic conditions.42 The
hypoxia-activated compounds tirapazamine and RB6145 were
included as positive controls. The lower HCR for tirapazamine
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Table 1. Inhibition of Tubulin Polymerization and
Colchicine Binding
Table 3. In Vitro Potency and Hypoxia Cytotoxicity Ratio
(HCR) of the CA4 and CA1 BAPCs in the A549 Human
Cancer Cell Line
inhibition of colchicine
b
a
,
b
a,b
binding % inhibition
GI50 [oxic] (μM)
GI50 [anoxic] (μM)
SD
compound
SEM
SEM
HCR
inhibition of tubulin
RB6145
tirapazamine
29 (CA1)
42 (CA4)
20
21
22
23
24
25
26
35
36
37
38
39
40
>89
63 5.7
1.2 0.48
0.0047 0.00021
0.065 0.0030
0.063 0.0087
0.44 0.022
4.6 0.14
24 6.7
6.8 0.39
0.82 0.12
0.0061 0.00048
0.19 0.015
0.046 0.0047
0.52 0.060
3.3 0.36
0.30 0.047
1.2 0.20
0.49 0.033
0.033 0.0012
0.72 0.17
0.28 0.082
0.26 0.077
0.69 0.031
0.051 0.0041
0.48 0.054
0.032 0.0073
0.038 0.0059
>3.7
9.2
a
compound
polymerization IC50 (μM) SD
1 μM
5 μM
c
d
c
29 (CA1)
1.9
ND
99.6 0.7
97 0.7
n/a
n/a
n/a
n/a
c
42 (CA4)
20
21
22
23
24
25
26
35
36
37
38
39
40
41
43
44
0.64 0.01
0.84 0.1
0.82 0.04
>20
84
50
72
2
5
4
c
84
1
c
94 0.7
0.85
1.4
6.3
2.4
3.1
>20
12
1
1.9 0.66
2.9 0.22
1.5 0.39
>20
9.5 0.9
1.7 0.2
>20
1.7 0.01
0.84 0.1
4.3 0.4
6.2 0.3
1.3 0.08
>20
25
3
0.046 0.0037
0.55 0.025
0.17 0.040
0.18 0.061
0.32 0.014
0.31 0.035
6.0 2.4
0.11 0.031
0.15 0.027
2.2 0.64
1.4
0.76
0.61
0.69
0.46
6.1
12.5
3.4
4.0
53
34
3
3
92 0.5
90 0.7
58
72
43
26
15
33
4
3
4
1
5
3
41
43
44
45
>20
>20
45
0.053 0.0088
41.5
a
b
a
b
Average of n ≥ 2 independent determinations. Only compounds
showing significant activity as inhibitors of tubulin polymerization
(defined as having IC50 values below 5 μM) were evaluated for
inhibitory effects on colchicine binding. Data from refs 44, 26, and
48. ND = not determined.
Average of n ≥ 3 independent determinations. Incubation involved
4 h (oxic or anoxic) followed by 48 h oxic exposure. n/a (not
applicable).
c
c
d
conditions, tirapazamine gave an HCR of 9.2 in contrast to
HCRs > 62 in assays in which it was washed out after 4 h.
Preliminary In Vivo Assessment with Biolumines-
cence Imaging (BLI). In line with ARRIVE guidelines, animal
investigations were conducted in accordance with State and
Federal guidelines and approved by the Institutional Animal
Care and Use Committee of UT Southwestern under protocol
APN#2017-102169. A preliminary in vivo study was performed
using orthotopic 4T1-luc breast tumors growing in the left
frontal mammary fat pad of syngeneic BALB/c mice treated
with CA4-BAPC 45 [single dose (180 mg/kg at a
concentration of 30 mg/mL), ip] to gauge initial tolerability
and efficacy of this agent. 4T1 is a murine mammary tumor
that arose spontaneously in an aging BALB/C mouse and is
considered to replicate many of the characteristics of human
breast cancer.49 It is widely used in studies of chemotherapy,
and several reports have used luciferase transfected clones to
facilitate imaging of therapeutic response and metastasis.50−52
We are only aware of one previous report of evaluation of a
VDA in 4T1, specifically OXi4503.53
Since BAPC 45 was insoluble in buffered saline or water, it
was necessary to develop a suitable vehicle to solubilize this
agent for in vivo use. While BAPC 45 proved soluble in
DMSO, there are limits (in terms of volume tolerability)
associated with the use of neat DMSO in mice. A solubilization
study identified 10% DMSO/55% sesame oil/35% PEG 400
(hereafter referred to as DSP) as a suitable vehicle (see
Supporting Information for further details). An initial
evaluation of tolerability of vehicle alone (without added
BAPC) led to the conclusion that 150 μL of DSP
(administered ip) approached the maximum usable volume.
The solubility constraints (of BAPC 45 in this vehicle) limited
Table 2. Stability of BAPCs and Their Cleavage by NADPH-
Cytochrome P450 Oxidoreductase
percent BAPC
hydrolysis/cleavage
(nonenzymatic) in
phosphate buffer
percent BAPC cleavage by POR
(90 min)
compound
(pH 7.4, 48 h)
a
22
23
24
25
26
35
36
37
38
39
40
41
43
44
45
0.25
0.84
1.59
0.69
4.03
0
NC
13.5
1.1
3.8
7.6
NC
NC
0
b
0
14.2 (24% cyclization of 37 to 35)
5.6 (48% cyclization of 38 to 36)
17.9 (46% cyclization of 39 to 35)
25.5 (35% cyclization of 40 to 36)
c
0.5
b
0
c
0
100
0.35
ND
0.69
100
2.7
4.1
25.4
a
b
NC = no cleavage observed. Significant cyclization to 35.
c
Significant cyclization to 36.
(Table 3) compared to literature values was a result of
modification of the assay conditions. The drug removal step
after anoxic (or oxic) exposure was omitted in order to detect
the antimitotic activity of the parent compounds CA1 and CA4
released from their corresponding BAPCs (see Table S7 in the
Supporting Information for additional data). Under these
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the maximum single dose for injection. BLI was performed on
a group of five mice at baseline. BLI was repeated again 4 h
postadministration of BAPC 45 (180 mg/kg, ip) in three mice,
while two mice served as controls, with one mouse treated with
vehicle alone and another mouse treated with CA4P54 (120
mg/kg, ip), a benchmark VDA.
Bioluminescence images are shown for the group of five
mice at various time points (baseline to 48 h in Figure 3). At
Figure 4. Dynamic light emission time courses with respect to
vascular disruption. Following administration of luciferin, BLI was
performed over a period of about 20 min for the group of mice shown
in Figure 3, and variation of signal intensity is shown at baseline, 4 h,
and 24 h. The mice represented by the red line received CA4P (M1);
those represented by the green line received vehicle (M4), and those
represented by the blue lines each received BAPC 45 (triangles, M2;
squares, M3; open diamonds, M0). At baseline, all tumors showed
similar light emission kinetics (upper left panel). Four hours later, two
of three tumors receiving BAPC 45 and the CA4P-treated tumor
showed substantially reduced signals, while one BAPC 45 treated
tumor and the control tumor were relatively unchanged (upper right
panel). Data for 24 h are shown in the lower left panel. The results
over 48 h are summarized for the tumors in the lower right panel: red
bars (CA4P); green bars (vehicle), and blue bars (mean of three
tumors receiving BAPC 45).
attribute this to the stress of anesthesia accompanying the high
tumor burden. Tumors from four of the mice were harvested at
72 h or following additional imaging at 96 h and were stained
with hematoxylin and eosin (H&E). Whole mount sections
(Figure 5) showed substantial necrosis in all tumors including
the vehicle control (46% necrosis), as also reported by others
for this tumor type.50,53 The tumor showing a strong BLI
signal response to BAPC 45 showed more necrosis than the
Figure 3. Bioluminescence images of 4T1-luc tumor-bearing BALB/c
mice at various times following VDA administration. Baseline shows
mice at 20 min following administration of 120 mg/kg luciferin
subcutaneously in the foreback region of five BALB/c mice bearing
orthotopic syngeneic 4T1-luc tumors growing in a frontal upper
mammary fat pad. Immediately following baseline BLI, mice M2, M3,
and M0 were injected ip with 180 mg/kg BAPC 45 dissolved in DPS.
M1 received 120 mg/kg CA4P ip, and M4 received DPS (vehicle
alone). Four, 24, and 48 h later BLI was repeated following
administration of fresh luciferin on each occasion. Light emission time
courses are presented in Figure 4.
baseline each tumor showed an integrated light intensity of
about 5 × 109 photons/s. Four hours following administration
of BAPC 45 (180 mg/kg, ip), two of three mice showed a
dramatic decrease in light emission (>80%) following
administration of fresh luciferin (Figures 3 and 4). At 24 h
these two tumors remained >75% depressed, but showed
substantial recovery by 48 h. By comparison, CA4P caused a
>99% drop in signal within 4 h, which remained reduced by
>90% up to 72 h. The control mouse receiving vehicle alone
showed relative stability up to 72 h. One mouse died during
the night prior to BLI at 72 h, and two mice died under
anesthesia during BLI at 72 h following treatment. We
Figure 5. Histology of 4T1-luc tumors. H&E staining revealed
substantial necrosis in all tumors, including the vehicle control.
However, necrosis is particularly evident following CA4P (upper
right) and in M2, which showed a strong response to BAPC 45.
Expanded views of histology are presented in Figure S9 (Supporting
Information).
H
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DIPEA (2.50 mL, 14.3 mmol) in anhydrous DMF (10 mL) was
added p-TSCl (1.29g, 6.73 mmol) in portions while stirring at room
temperature. After stirring for 5 h, the reaction mixture was quenched
with H2O (20 mL) and extracted with EtOAc (3 × 25 mL). The
combined organic phase was washed with brine, dried over MgSO4,
filtered, and evaporated under reduced pressure. Flash chromatog-
raphy of the residue using a prepacked 50 g silica column [eluents;
solvent A, EtOAc, solvent B, hexanes; gradient, 40% A/60% B over
1.19 min (1 CV), 40% A/60% B →100% A/0% B over 16.3 min (10
CV), 100% A/0% B over 3.18 min (2 CV); flow rate 40.0 mL/min;
monitored at 254 and 280 nm] afforded aldehyde 3 (1.33 g, 4.3
unresponsive one (55% vs 47% respectively). Likewise, the
tumor on the mouse receiving CA4P was highly necrotic
(70%). At higher magnification, extensive hemorrhage was
apparent in several tumors together with congested blood
vessels (see Figure S9, Supporting Information). These BLI
and histology results are potentially indicative of in vivo
cleavage by POR and subsequent vascular disruption by the
released parent anticancer agent CA4. The differential
response is not unexpected since 4T1 tumors show highly
variable levels of hypoxia (see Figure S10, Supporting
Information).
1
mmol, 61% yield) as a white solid: H NMR (600 MHz, CDCl3) δ
9.85 (1H, s, CHO), 7.87 (2H, d, J = 8.3 Hz, ArH), 7.50 (1H, d, J =
8.6 Hz, ArH), 7.36 (2H, d, J = 8.0 Hz, ArH), 6.90 (1H, d, J = 8.6 Hz,
ArH), 5.91 (1H, s, OH), 3.97 (3H, s, OCH3), 2.47 (3H, s, CH3); 13
C
EXPERIMENTAL SECTION
■
NMR (151 MHz, CDCl3) δ 187.0, 153.2, 146.2, 139.2, 138.2, 132.0,
130.0, 128.7, 124.1, 120.6, 109.2, 56.7, 21.8.
General Experimental Procedures. Acetic acid (AcOH), acetic
anhydride, acetonitrile, CH2Cl2, dimethylformamide (DMF), ethanol,
methanol, hexanes, nitric acid, sulfuric acid, ethyl acetate (EtOAc),
and tetrahydrofuran (THF) were used in their anhydrous forms or as
obtained from the chemical suppliers. Reactions were performed
under N2 gas. Thin-layer chromatography (TLC) plates (precoated
glass plates with silica gel 60 F254, 0.25 mm thickness) were used to
monitor reactions. Purification of intermediates and products was
carried out with a Biotage Isolera or Teledyne Combiflash flash
purification system using silica gel (200−400 mesh, 60 Å) or RP-18
prepacked columns or was performed manually in glass columns.
Intermediates and products synthesized were characterized on the
basis of their 1H NMR (600 or 500 MHz), 13C NMR (150, 125, or 90
MHz), and 31P NMR (240 MHz) spectroscopic data using a Varian
VNMRS 500 MHz, a Bruker DRX 600 MHz, or a Bruker DPX 360
MHz instrument. Spectra were recorded in CDCl3 or (CD3)2CO. All
chemical shifts are expressed in ppm (δ), and peak patterns are
reported as broad (br), singlet (s), doublet (d), triplet (t), quartet
(q), quintet (quint), sextet (sext), septet (sept), double doublet (dd),
double double doublet (ddd), and multiplet (m). The purity of the
final compounds was further analyzed at 25 °C using an Agilent 1200
HPLC system with a diode-array detector (λ = 190−400 nm), a
Zorbax XDB-C18 HPLC column (150 mm, 5 μm), and a Zorbax
reliance cartridge guard-column; solvent A acetonitrile, solvent B
H2O; method A: H2O; gradient, 10% A/90% B to 100% A/0% B over
0 to 40 min; post-time 10 min; method B: H2O; gradient, 50% A/50%
B to 90% A/10% B over 0 to 30 min; post-time 10 min; flow rate 1.0
mL/min; injection volume 20 μL; monitored at wavelengths of 210,
230, 254, 280, and 320 nm. Mass spectrometry was carried out under
either positive or negative ESI (electrospray ionization) or positive or
negative APCI/APPI (atmospheric pressure chemical ionization/
atmospheric pressure photoionization) using a Thermo Scientific
LTQ Orbitrap Discovery instrument.
3-Hydroxy-2-isopropoxy-4-methoxybenzaldehyde (4).43,44 2,3-
Dihydroxy-4-methoxybenzaldehyde (0.400 g, 2.34 mmol), K2CO3
(0.330 g, 2.38 mmol), and 2-bromopropane (0.21 mL, 2.3 mmol)
were dissolved in dry DMF (5 mL) in a 5 mL Biotage microwave vial.
The reaction was run in a Biotage microwave reactor (2 h, 90 °C,
normal absorbance). The reaction was then quenched with water, and
the reaction mixture was extracted with EtOAc. The organic phase
was washed with water and brine, dried with Na2SO4, and evaporated
under reduced pressure. Flash column chromatography of the crude
product using a prepacked 50 g silica column [eluents: solvent A,
EtOAc; solvent B, hexanes; gradient, 10% A/90% B over 1.19 min (1
CV), 10% A/90% B → 54% A/46% B over 13.12 min (10 CV), 54%
A/46% B over 2.38 min (2 CV); flow rate 40.0 mL/min; monitored at
254 and 280 nm] yielded 3-hydroxy-2-isopropoxy-4-methoxybenzal-
dehyde (4) (0.220 g, 1.05 mmol, 44%) as a tan solid: 1H NMR
(CDCl3, 600 MHz) δ 10.24 (1H, s, CHO), 7.41 (1H, d, J = 8.7 Hz,
ArH), 6.71 (1H, d, J = 8.7 Hz, ArH), 5.77 (1H, d, J = 4.8 Hz, OH),
4.67 (1H, sept, J = 6.1 Hz, CH), 3.94 (3H, s, OCH3), 1.34 (6H, d, J =
6.2 Hz, C(CH3)2); 13C NMR (151 MHz, CDCl3) δ 189.68, 152.75,
147.90, 138.62, 124.34, 120.36, 106.22, 77.00, 56.44, 22.43.
2,3-Bis((tert-butyldimethylsilyl)oxy)-4-methoxybenzaldehyde
(5).43,44 To a solution of 2,3-dihydroxy-4-methoxybenzaldehyde (1.00
g, 5.95 mmol), Et3N (2.00 mL, 14.3 mmol), and DMAP (0.025 g,
0.200 mmol) in CH2Cl2 (30 mL) was added dropwise TBSCl (2.10 g,
13.9 mmol) dissolved in DMF. The reaction mixture was stirred for
12 h at room temperature. H2O was used to quench the reaction, and
the residue was extracted with CH2Cl2 (3 × 20 mL). The combined
extracts were washed with brine, dried over Na2SO4, filtered, and
concentrated under reduced pressure. The crude product was purified
by flash chromatography using a prepacked 25 g silica column
[solvent A: EtOAc; solvent B: hexanes; gradient: 5% A/95% B (1
CV), 5% A/95% B → 40% A/60% B (10 CV), 40% A/60% B (2 CV);
flow rate: 75 mL/min; monitored at 254 and 280 nm], affording 2,3-
bis((tert-butyldimethylsilyl)oxy)-4-methoxybenzaldehyde (0.650 g,
2,3-Dihydroxy-4-methoxybenzaldehyde (2).43,44 2,3,4-Trime-
thoxybenzaldehyde (4.00 g, 20.4 mmol) was added to dry CH2Cl2
(80 mL) in an ice bath (0 °C). BCl3 (45 mL, 45 mmol, 1.0 M) was
added dropwise to the reaction mixture, and it was stirred for 18 h.
The reaction was then quenched with NaHCO3 and acidified to pH 2
with concentrated HCl. The reaction mixture was extracted with
EtOAc, and the organic phase was dried with Na2SO4 and evaporated
under reduced pressure. The crude mixture was then filtered through
silica gel in a frit funnel with CH2Cl2, and the solvent was evaporated
under reduced pressure. Flash chromatography of the crude product
using a prepacked 100 g silica column [eluents: solvent A, EtOAc;
solvent B, hexanes; gradient, 10% A/90% B over 1.19 min (1 CV),
10% A/90% B → 69% A/31% B over 13.12 min (10 CV), 69% A/
31% B over 2.38 min (2 CV); flow rate 50.0 mL/min; monitored at
254 and 280 nm] yielded 2,3-dihydroxy-4-methoxybenzaldehyde (2)
1
1.64 mmol, 65%) as a white solid: H NMR (600 MHz, CDCl3) δ
10.22 (1H, s, CHO), 7.48 (1H, d, J = 8.8 Hz, ArH), 6.62 (1H, d, J =
8.8 Hz, ArH), 3.84 (3H, s, OCH3), 1.04 (9H, s, C(CH3)3), 0.99 (9H,
s, C(CH3)3), 0.13 (12H, s, Si (CH3)2); 13C NMR (151 MHz, CDCl3)
δ 189.64, 157.88, 151.32, 137.10, 123.64, 121.69, 105.73, 55.53,
26.51, 26.36, 19.07, 18.89, −3.51.
2-((tert-Butyldimethylsilyl)oxy)-6-formyl-3-methoxyphenyl 4-
Methylbenzenesulfonate (6).43,44 Aldehyde 3 (0.501 g, 1.77
mmol), Et3N (2.00 mL, 14.3 mmol), and DMAP (0.035 g, 0.28
mmol) were dissolved in dry CH2Cl2 (45 mL). TBSCl (0.327 g, 2.17
mmol) was added, and the reaction mixture was stirred for 18 h. The
reaction was quenched with water and extracted with diethyl ether.
The organic phase was washed with water and brine, dried with
Na2SO4, and evaporated under reduced pressure. Flash column
chromatography of the residue using a prepacked 50 g silica column
[eluents: solvent A, EtOAc; solvent B, hexanes; gradient, 12% A/88%
B over 1.19 min (1 CV), 12% A/88% B → 54% A/46% B over 13.12
min (10 CV), 54% A/46% B over 2.38 min (2 CV); flow rate 35.0
mL/min; monitored at 254 and 280 nm] yielded aldehyde 6 (0.610 g,
1
(2.64 g, 15.7 mmol, 77%) as a yellow solid: H NMR (500 MHz,
CDCl3) δ 11.12 (1H, s, OH), 9.76 (1H, s, CHO), 7.15 (1H, d, J = 8.5
Hz, ArH), 6.63 (1H, d, J = 8.5 Hz, ArH), 5.46 (1H, s, OH), 3.99 (3H,
s, OCH3); 13C NMR (125 MHz, CDCl3) δ 195.2, 153.0, 149.0, 133.0,
126.1, 116.1, 103.6, 56.4.
6-Formyl-2-hydroxy-3-methoxyphenyl 4-Methylbenzenesulfo-
nate (3).43,44 To a solution of aldehyde 2 (1.15 g, 6.76 mmol) and
I
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1
1.40 mmol, 79%) as a white solid: H NMR (500 MHz, CDCl3) δ
9.60 (1H, d, J = 0.47 Hz, CHO), 7.71 (2H, d, J = 8.34 Hz, ArH), 7.52
(1H, d, J = 8.70 Hz, ArH), 7.32 (2H, d, J = 8.05 Hz, ArH), 6.87 (1H,
d, J = 8.63 Hz, ArH), 3.87 (3H, s, OCH3), 2.45 (3H, s, CH3), 0.97
(9H, s, C(CH3)3), 0.10 (6H, s, Si (CH3)2); 13C NMR (126 MHz,
CDCl3) δ 186.7, 157.3, 145.9, 143.0, 138.9, 132.1, 129.9, 128.5, 124.0,
121.3, 109.8, 55.6, 25.7, 21.7, 18.6, −4.4.
(2H, d, J = 8.5 Hz, ArH), 7.25 (2H, d, J = 8 Hz, ArH), 6.77 (1H, d, J
= 8.5 Hz, ArH), 6.61 (1H, d, 8.5 Hz, ArH), 6.44 (2H, s, ArH), 6.19
(1H, d, J = 12 Hz, CH), 6.16 (1H, d, J = 12 Hz, CH), 3.82 (3H, s,
OCH3), 3.76 (3H, s, OCH3), 3.67 (6H, s, OCH3), 0.95 (9H, s,
C(CH3)3), 0.04 (6H, s, Si(CH3)2); 13C NMR (125 MHz, CDCl3) δ
152.6, 151.3, 144.8, 140.2, 139.1, 134.5, 132.2, 130.4, 129.5, 128.4,
125.3, 124.7, 122.1, 109.5, 106.1, 60.8, 55.8, 55.4, 25.8, 25.7, 25.6,
21.6, 18.7, −4.5.
3-((tert-Butyldimethylsilyl)oxy)-2-isopropoxy-4-methoxybenzal-
dehyde (7).43,44 Aldehyde 4 (1.39 g, 6.61 mmol), Et3N (1.40 mL,
9.91 mmol), and DMAP (0.050 g, 0.40 mmol) were dissolved in dry
CH2Cl2 (50 mL). TBSCl (1.50 g, 9.95 mmol) was added, and the
reaction mixture was stirred for 18 h. The reaction was quenched with
water and extracted with diethyl ether. The organic phase was washed
with water and brine, dried with Na2SO4, and evaporated under
reduced pressure. Flash column chromatography of the residue using
a prepacked 50 g silica column [eluents: solvent A, EtOAc; solvent B,
hexanes; gradient, 12% A/88% B over 1.19 min (1 CV), 12% A/88%
B → 54% A/46% B over 13.12 min (10 CV), 54% A/46% B over 2.38
min (2 CV); flow rate 35.0 mL/min; monitored at 254 and 280 nm]
(Z)-tert-Butyl(2-iso propoxy- 6- methoxy-3-(3, 4, 5-
trimethoxystyryl)phenoxy)dimethylsilane (12).43,44 Triphenyl(3,4,5-
trimethoxybenzyl)phosphonium bromide (1.94 g, 3.70 mmol) was
dissolved in dry THF (50 mL) and cooled to −15 °C. n-Butyllithium
(2.5 M in hexanes, 1.78 mL, 4.44 mmol, 2.5 M) was added dropwise,
and the reaction mixture was stirred for 25 min. The reaction mixture
was cooled to −78 °C, and a solution of aldehyde 7 in THF (30 mL)
was added dropwise. The reaction mixture was stirred for 5 h. The
reaction was quenched with water, and the THF was evaporated
under reduced pressure. The mixture was extracted with EtOAc, and
the organic phase was washed with water and brine, dried with
Na2SO4, and evaporated under reduced pressure. The crude product
was purified using flash column chromatography to yield the Z-isomer
(resolved from the E-isomer) (0.982 g, 2.01 mmol, 65%) as a reddish-
1
yielded aldehyde 7 (1.53 g, 4.71 mmol, 71%) as a white solid: H
NMR (600 MHz, CDCl3) δ 10.11 (1H, s, CHO), 7.35 (1H, d, J = 8.7
Hz, ArH), 6.56 (1H, d, J = 8.7 Hz, ArH), 4.60−4.45 (1H, m, CH),
3.71 (3H, s, OCH3), 1.10 (6H, d, J = 6.2 Hz, C(CH3)2), 0.86 (9H, d,
J = 2.0 Hz, C(CH3)3), 0.00 (6H, s, Si(CH3)2); 13C NMR (151 MHz,
CDCl3) δ 190.0, 157.4, 152.7, 138.4, 125.2, 121.4, 106.9, 75.5, 55.5,
25.9, 22.3, 18.7, −4.3.
1
white solid: H NMR (600 MHz, CDCl3) δ 6.83 (1H, d, J = 8.6 Hz,
ArH), 6.62 (1H, d, J = 12.1 Hz, ArH), 6.52 (2H, s, ArH), 6.45 (1H, d,
J = 8.6 Hz, CH), 6.41 (1H, d, J = 12.1 Hz, CH), 4.61 (1H, sept, J =
6.1 Hz, CH), 3.82 (3H, s, OCH3), 3.76 (3H, s, OCH3), 3.65 (6H, s,
OCH3), 1.27 (6H, d, J = 6.1 Hz, CH3), 1.02 (9H, s, C(CH3)3), 0.14
(6H, s, Si(CH3)2); 13C NMR (151 MHz, CDCl3) δ 152.7, 151.4,
148.0, 138.6, 136.8, 132.9, 128.5, 126.9, 125.1, 122.4, 106.0, 105.9,
74.2, 60.9, 55.8, 55.2, 25.9, 22.3, 18.7, −4.4.
3,4,5-Trimethoxybenzyl Bromide (9).43,44 A mixture of 3,4,5-
trimethoxybenzyl alcohol (20.1 g, 101.4 mmol) and PBr3 (4.8 mL,
50.7 mmol) in anhydrous CH2Cl2 was stirred for 1 h at 0 °C under
N2. Water (10 mL) was added, and the organic layer was separated
and extracted with CH2Cl2 (2 × 100 mL). The combined organic
layer was washed with brine, dried over Na2SO4, filtered, and
evaporated under reduced pressure. After the recrystallization of the
crude solid from 10% (EtOAc/hexanes), the off-white solid of
bromide 9 (23.6 g, 90.3 mmol, 89% yield) was obtained and needed
(Z)-((3-Methoxy-6-(3,4,5-trimethoxystyryl)-1,2-phenylene)bis-
(oxy))bis(tert-butyldimethylsilane) (13).43,44 n-Butyllithium (11.4
mL, 2.5 M) was added to a solution of phosphonium salt (11.2 g,
21.4 mmol) in THF (350 mL). The resulting solution was stirred for
15 min at −78 °C. Aldehyde 5 (5.66 g, 14.3 mmol) was dissolved in
THF and added dropwise using a dropping funnel. The reaction
mixture was stirred for 5 h. H2O was used to quench the reaction, and
the residue was extracted with Et2O. The combined extracts were
washed with brine, dried over Na2SO4, filtered, and concentrated
under reduced pressure. The crude product was purified by flash
column chromatography using a prepacked 340 g silica column
[solvent A: EtOAc; solvent B: hexanes; gradient: 5% A/95% B (1
CV), 5% A/95% B → 30% A/70% B (10 CV), 30% A/70% B (2 CV);
flow rate: 85 mL/min; monitored at 254 and 280 nm], affording
1
no further purification: H NMR (500 MHz, CDCl3) δ 6.62 (2H, s,
ArH), 4.47 (2H, s, CH2), 3.87 (6H, s, OCH3), 3.85 (3H, s, OCH3);
13C NMR (125 MHz, CDCl3) δ 153.3, 138.2, 133.2, 106.1, 60.9, 56.1,
34.3.
3,4,5-Trimethoxybenzyltriphenylphosphonium Bromide
(10).43,44 A mixture of bromide 9 (11.00 g, 42.1 mmol) and PPh3
(12.1 g, 46.3 mmol) in acetone (100 mL, anhydrous) was stirred in a
flask under N2. After 5 h, the resulting suspension was filtered through
a Buchner funnel, and the solid was washed with acetone (100 mL)
and hexanes (50 mL) to afford an off-white solid. The solid was dried
in vacuo to obtain the phosphonium salt 10 (20.3 g, 38.2 mmol, 92%
1
compound 13 (2.89 g, 5.15 mmol, 51%) as a white solid: H NMR
(500 MHz, CDCl3) δ 6.91 (1H, d, J = 8.6 Hz, ArH), 6.62 (2H, s,
ArH), 6.58 (1H, d, J = 12.2 Hz, CH), 6.37 (1H, d, J = 9.2 Hz, ArH),
6.37 (1H, d, J = 12 Hz, CH), 3.83 (3H, s, OCH3), 3.74 (3H, s,
OCH3), 3.67 (6H, s, OCH3), 1.04 (9H, s, C(CH3)3), 1.00 (9H, s,
C(CH3)3), 0.19 (6H, s, Si(CH3)2), 0.10 (6H, s, Si(CH3)2); 13C NMR
(151 MHz, CDCl3) δ 153.0, 152.0, 146.5, 137.1, 133.1, 128.0, 127.7,
123.5, 122.5, 106.2, 104.5, 61.2, 56.1, 55.3, 26.7, 26.4, 19.1, −2.9,
−3.6.
(Z)-2-Hydroxy-3-methoxy-6-(3,4,5-trimethoxystyryl)phenyl 4-
Methylbenzenesulfonate (20).43,44 To a solution of Z-stilbene 11
(0.754 g, 1.26 mmol) in dry THF (40 mL) at −15 °C was added
dropwise a solution of TBAF·3H2O (3.8 mL, 3.8 mmol) dissolved in
THF (10 mL). The reaction was stirred for 12 h. H2O (40 mL) was
used to quench the reaction, THF was removed by evaporation, and
the residue was extracted with EtOAc (3 × 20 mL). The combined
extracts were washed with brine, dried over Na2SO4, filtered, and
concentrated under reduced pressure. The residue was purified by
flash chromatography using a prepacked 50 g silica column [solvent
A: EtOAc; solvent B: hexanes; gradient: 12% A/88% B (1 CV), 12%
A/88% B → 82% A/18% B (10 CV), 82% A/18% B (2 CV); flow
rate: 35 mL/min; monitored at 254 and 280 nm], affording
compound 20 (0.429 g, 0.882 mmol, 70%) as a dark green solid:
1H NMR (500 MHz, CDCl3) δ 7.91 (2H, d, J = 8.1 Hz, ArH), 7.29
1
yield) as a white solid: H NMR (600 MHz, CDCl3) δ 7.74−7.64
(9H, m, ArH), 7.58−7.50 (6H, m, ArH), 6.43 (2H, d, J = 2.6 Hz,
ArH), 5.29 (2H, d, J = 14.1 Hz, CH2), 3.70 (3H, d, J = 3.4 Hz,
OCH3), 3.43 (6H, d, J = 3.7 Hz, OCH3); 13C NMR (125 MHz,
CDCl3) δ 153.0, 137.6, 134.8, 134.6, 130.0, 122.4, 117.8, 108.8, 60.8,
56.2, 30.8; 31P NMR (243 MHz, CDCl3) δ 23.2.
(Z)-2-((tert-Butyldimethylsilyl)oxy)-3-methoxy-6-(3,4,5-
trimethoxystyryl)phenyl 4-Methylbenzenesulfonate (11).43,44
Triphenyl(3,4,5-trimethoxybenzyl)phosphonium bromide (3.25 g,
6.20 mmol) was dissolved in dry THF (90 mL) in an ice/salt bath
(−10 °C). n-Butyllithium (2.4 mL, 6.0 mmol, 2.5 M) was added
dropwise, and the reaction mixture was stirred for 30 min. The
aldehyde 6 (2.01 g, 4.60 mmol) was dissolved in dry THF (30 mL)
and added dropwise to the reaction mixture, which was stirred for 5 h.
The reaction was quenched with water, and the THF was evaporated
under reduced pressure. The mixture was extracted with EtOAc, and
the organic phase was washed with water and brine, dried with
Na2SO4, and evaporated under reduced pressure. Flash chromatog-
raphy of the residue using a prepacked 100 g silica column [eluents:
solvent A, EtOAc; solvent B, hexanes; gradient, 10% A/90% B over
1.19 min (1 CV), 10% A/90% B → 80% A/20% B over 13.12 min (10
CV), 80% A/20% B over 2.38 min (2 CV); flow rate 35.0 mL/min;
monitored at 254 and 280 nm] yielded Z-isomer 11 (1.11 g, 1.84
(2H, d, J = 8.0 Hz, ArH), 6.71 (1H, d, J = 8.6 Hz, ArH), 6.62 (1H, d,
J = 8.6 Hz, ArH), 6.42 (2H, s, ArH), 6.36 (1H, d, J = 12.0 Hz, CH),
1
mmol, 40%) as a white solid: H NMR (500 MHz, CDCl3) δ 7.82
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6.32 (1H, d, J = 12.0 Hz, CH), 5.89 (1H, s, OH), 3.86 (3H, s,
OCH3), 3.82 (3H, s, OCH3), 3.66 (6H, s, OCH3), 2.42 (3H, s, CH3);
13C NMR (126 MHz, CDCl3) δ 151.9, 146.6, 144.5, 138.5, 136.4,
134.5, 132.7, 131.2, 130.5, 128.7, 127.7, 124.9, 123.4, 119.9, 108.3,
105.4, 75.9, 60.0, 55.6, 55.0, 20.9.
(2H, d, J = 8.3 Hz, ArH), 7.01 (1H, d, J = 8.8 Hz, ArH), 6.99 (2H, m,
ArH), 6.57 (2H, s, ArH), 6.51 (1H, d, J = 12.0 Hz, CH), 6.44 (1H, d,
J = 11.9 Hz, CH), 5.47 (1H, q, J = 6.5 Hz, CH), 3.90 (3H, s, OCH3),
3.73 (3H, s, OCH3), 3.66 (6H, s, OCH3), 2.44 (3H, s, CH3), 1.43
(3H, d, J = 6.5 Hz, CH3); 13C NMR (125 MHz, CDCl3) δ 159.6,
158.2, 158.1, 150.5, 147.2, 144.2, 139.9, 137.2, 136.8, 134.9, 133.5,
131.1, 130.9, 129.5, 129.1, 116.0, 111.7, 80.7, 73.4, 64.8, 60.9, 60.5,
26.5, 26.0, 25.8; HRMS m/z 642.1465 [M + H]+ (calcd for
(Z)-2-Isopropoxy-6-methoxy-3-(3,4,5-trimethoxystyryl)phenol
(21).43,44 To a solution of compound 12 (0.150 g, 0.251 mmol) in
THF (5 mL) at room temperature was added dropwise TBAF·3H2O
(0.0952 g, 0.302 mmol) dissolved in THF. The reaction was stirred
for 0.5 h. H2O (5 mL) was used to quench the reaction, THF was
removed by evaporation, and the residue was extracted with EtOAc (3
× 10 mL). The combined extracts were washed with brine, dried over
Na2SO4, filtered, and concentrated under reduced pressure. The
residue was purified by flash chromatography using a prepacked 10 g
silica column [solvent A: EtOAc; solvent B: hexanes; gradient: 7% A/
93% B (1 CV), 7% A/93% B → 60% A/40% B (13 CV), 60% A/40%
B (2 CV); flow rate: 8 mL/min; monitored at 254 and 280 nm],
affording compound 21 (0.135 g, 0.361 mmol, 90%) as a white solid:
1H NMR (500 MHz, CDCl3) δ 6.75 (1H, d, J = 8.6 Hz, ArH), 6.59
+
C31H32NO10S2 , 642.1462); HPLC (method A) 18.2 min, 99%, 1.0%
parent (Ts-protected CA1 analogue 20), 0% CA1.
(Z)-2-((2-Isopropoxy-6-methoxy-3-(3,4,5-trimethoxystyryl)-
phenoxy)methyl)-5-nitrothiophene (24). To a solution of isopropyl-
protected CA1 21 (0.350 g, 0.843 mmol), nor-methyl trigger 16
(0.162 g, 1.02 mmol), and DEAD (0.220 mL) in CH2Cl2 (10 mL)
was added dropwise PPh3 (0.430 g, 1.64 mmol) dissolved in CH2Cl2.
The reaction mixture was stirred for 24 h at room temperature. H2O
(40 mL) was added to quench the reaction, and the resultant liquid
mixture was extracted with CH2Cl2 (3 × 20 mL). The combined
extracts were washed with brine, dried over Na2SO4, filtered, and
concentrated under reduced pressure. The residue was purified by
flash chromatography using a prepacked 25 g silica column [solvent
A: EtOAc; solvent B: hexanes; gradient: 10% A/90% B (1 CV), 10%
A/90% B → 80% A/20% B (10 CV), 80% A/20% B (2 CV); flow
rate: 17 mL/min; monitored at 254 and 280 nm], affording CA1-
BAPC 24 (0.0600 g, 0.116 mmol, 17%) as a yellow oil: 1H NMR (500
MHz, CDCl3) δ 7.82 (1H, d, J = 4.1 Hz, ArH), 7.02 (1H, d, J = 8.8
Hz, ArH), 7.00 (1H, d, J = 4.2 Hz, ArH), 6.60 (1H, d, J = 12.1 Hz,
CH), 6.53 (1H, d, J = 8.7 Hz, ArH), 6.50 (2H, s, ArH), 6.47 (1H, d, J
= 12.1 Hz, CH), 5.17 (2H, s), 4.60 (1H, quint, J = 6.2 Hz, CH), 3.83
(3H, s, OCH3), 3.82 (3H, s, OCH3), 3.67 (6H, s, OCH3), 1.32 (3H,
s, CH3), 1.31 (3H, s, CH3); 13C NMR (126 MHz, CDCl3) δ 152.8,
152.8, 151.7, 149.9, 149.0, 140.6, 137.1, 132.6, 129.3, 128.2, 125.9,
125.7, 125.3, 125.1, 106.8, 106.0, 76.0, 69.2, 60.9, 55.9, 55.8, 22.6;
HRMS m/z 538.1506 [M + Na]+ (calcd for NaC26H29NO8S+,
538.1506); HPLC (method A) 14.7 min, 93%, 2.1% parent
(isopropyl-protected CA1 analogue 21), <1% (trace) CA1.
(1H, d, J = 12.1 Hz, CH), 6.51 (1H, d, J = 8.7 Hz, ArH), 6.51 (2H, s,
ArH), 6.46 (1H, d, J = 12.1 Hz, CH), 5.60 (1H, s, OH), 4.56 (1H,
sept, J = 6.1 Hz, CH), 3.86 (3H, s, OCH3), 3.82 (3H, s, OCH3), 3.66
(6H, s, OCH3), 1.32 (6H, d, J = 6.2 Hz, CH(CH3)2); 13C NMR (126
MHz, CDCl3) δ 152.7, 146.9, 143.2, 138.9, 137.1, 132.5, 129.4, 125.8,
124.1, 120.5, 106.3, 106.0, 75.7, 60.9, 56.2, 55.8, 22.5; HRMS m/z
+
397.1713 [M + Na]+ (calcd for NaC21H26O6 , 397.1713); HPLC
(method A) 14.7 min, 98%.
(Z)-3-Methoxy-2-((5-nitrothiophen-2-yl)methoxy)-6-(3,4,5-
trimethoxystyryl)phenyl 4-Methylbenzenesulfonate (22). To a
solution of compound 20 (0.700 g, 1.44 mmol), nor-methyl trigger
16 (0.191 g, 1.20 mmol), and DIAD (0.32 mL) in CH2Cl2 (10 mL)
was added dropwise PPh3 (0.610 g, 2.33 mmol) dissolved in CH2Cl2.
The reaction mixture was stirred for 12 h at room temperature. The
reaction mixture was then quenched with H2O and extracted with
CH2Cl2 (3 × 30 mL). The combined extracts were washed with brine,
dried over Na2SO4, filtered, and concentrated under reduced pressure.
The residue was purified by flash chromatography using a prepacked
25 g silica column [solvent A: EtOAc; solvent B: hexanes; gradient:
10% A/90% B (1 CV), 10% A/90% B → 80% A/20% B (10 CV),
80% A/20% B (2 CV); flow rate: 25 mL/min; monitored at 254 and
280 nm], affording tosyl-protected CA1 nor-methyl BAPC 22 (0.125
g, 0.236 mmol, 47%) as a tan-white solid: 1H NMR (600 MHz,
CDCl3) δ 7.84 (2H, d, J = 8.2 Hz, ArH), 7.77 (1H, d, J = 4.1 Hz,
ArH), 7.24 (2H, d, J = 8.1 Hz, ArH), 6.94 (1H, d, J = 8.7 Hz, ArH),
6.90 (1H, d, J = 4.1 Hz, ArH), 6.69 (1H, d, J = 8.8 Hz, ArH), 6.46
(2H, s, ArH), 6.40 (1H, d, J = 11.9 Hz, CH), 6.33 (1H, d, J = 11.9 Hz,
CH), 5.06 (2H, s, CH2), 3.85 (3H, s, OCH3), 3.83 (3H, s, OCH3),
3.68 (6H, s, OCH3), 2.40 (3H, s, CH3); 13C NMR (151 MHz,
CDCl3) δ 152.8, 152.5, 151.8, 147.9, 145.2, 141.8, 140.3, 137.2, 134.3,
132.0, 131.7, 129.6, 128.3, 128.1, 126.1, 125.9, 125.8, 124.0, 110.4,
106.1, 69.0, 60.9, 56.2, 55.9, 21.7; HRMS m/z 650.1120 [M + Na]+
(Z)-2-(1-(2-Isopropoxy-6-methoxy-3-(3,4,5-trimethoxystyryl)-
phenoxy)ethyl)-5-nitrothiophene (25).45 To a solution of isopropyl-
protected CA1 21 (0.267 g, 0.715 mmol), monomethyl trigger 17
(0.136 g, 0.785 mmol), and DIAD (0.190 mL) in CH2Cl2 (10 mL)
was added dropwise PPh3 (0.364 g, 1.39 mmol) dissolved in CH2Cl2.
The reaction mixture was stirred for 12 h at room temperature. H2O
was added to quench the reaction, and the resultant liquid mixture
was extracted with CH2Cl2 (3 × 20 mL). The combined extracts were
washed with brine, dried over Na2SO4, filtered, and concentrated
under reduced pressure. The residue was purified by flash column
chromatography using a prepacked 25 g silica column [solvent A:
EtOAc; solvent B: hexanes; gradient: 10% A/90% B (1 CV), 10% A/
90% B → 80% A/20% B (10 CV), 80% A/20% B (2 CV); flow rate:
75 mL/min; monitored at 254 and 280 nm], yielding CA1-BAPC 25
1
(0.125 g, 0.236 mmol, 47%) as a yellow oil: H NMR (600 MHz,
+
CDCl3) δ 7.78 (1H, d, J = 4.2 Hz, ArH), 6.99 (1H, d, J = 8.7 Hz,
ArH), 6.91 (1H, d, J = 4.1 Hz, ArH), 6.59 (1H, d, J = 12.1 Hz, CH),
6.49 (1H, d, J = 8.6 Hz, ArH), 6.47 (2H, s, ArH), 6.45 (1H, d, J =
12.2 Hz, CH), 5.49 (1H, q, J = 6.4 Hz, CH), 4.61 (1H, sept, J = 6.1
Hz, CH), 3.82 (3H, s, OCH3), 3.75 (3H, s, OCH3), 3.65 (6H, s,
OCH3), 1.66 (3H, d, J = 6.5 Hz, CH3), 1.30 (3H, d, J = 6.1 Hz, CH3),
1.26 (3H, d, J = 6.1 Hz, CH3); 13C NMR (151 MHz, CDCl3) δ 155.2,
153.1, 152.8, 151.0, 150.2, 139.2, 137.0, 132.6, 129.2, 128.1, 125.9,
125.9, 125.3, 123.5, 106.6, 105.9, 75.7, 75.4, 60.9, 55.9, 55.8, 22.6,
22.5, 22.2; HRMS m/z 552.1660 [M + Na]+ (calcd for
NaC27H31NO8S+, 552.1663); HPLC (method B) 20.5 min, 95%,
0.3% parent (isopropyl-protected CA1 analogue 21), <0.5% (trace)
CA1.
(Z)-2-(2-(2-Isopropoxy-6-methoxy-3-(3,4,5-trimethoxystyryl)-
phenoxy)propan-2-yl)-5-nitrothiophene (26).45 To a solution of
isopropyl-protected CA1 21 (0.150 g, 0.402 mmol), gem-dimethyl
trigger 19 (0.091 g, 0.486 mmol), and ADDP (0.137 g, 0.543 mmol)
in CH2Cl2 (10 mL) was added dropwise PBu3 (0.199 mL). The
reaction was stirred for 24 h at room temperature. H2O was added to
(calcd for NaC30H29NO10S2 , 650.1125); HPLC (method A) 18.5
min, 97%, 1.3% parent (Ts-protected CA1 analogue 20), 0% CA1.
(Z)-3-Methoxy-2-(2-(5-nitrothiophen-2-yl)propoxy)-6-(3,4,5-
trimethoxystyryl)pheny l-4-Methylbenzenesulfonate (23). To a
solution of compound 20 (0.200 g, 0.411 mmol), DIAD (0.100 g,
0.495 mmol), and 1-(5-nitrothiophen-2-yl)ethanol (0.059 g, 0.34
mmol) in CH2Cl2 (25 mL) was added triphenylphosphine (0.216 g,
0.822 mmol), and the reaction mixture was stirred for 24 h. The
reaction mixture was quenched with water and extracted with EtOAc.
The organic phase was dried with Na2SO4 and evaporated under
reduced pressure. Flash chromatography of the residue using a
prepacked 25 g silica column [eluents: solvent A, EtOAc; solvent B,
hexanes; gradient, 12% A/88% B over 1.19 min (1 CV), 12% A/88%
B → 100% A/0% B over 13.12 min (10 CV), 100% A/0% B over 2.38
min (2 CV); flow rate 25.0 mL/min; monitored at 254 and 280 nm]
yielded (Z)-3-methoxy-2-(2-(5-nitrothiophen-2-yl)propoxy)-6-(3,4,5-
trimethoxystyryl)phenyl 4-methylbenzenesulfonate (23) (0.160 g,
1
0.249 mmol, 61%) as a yellow solid: H NMR (500 MHz, CDCl3) δ
7.90 (1H, d, J = 4.3 Hz, ArH), 7.86 (2H, d, J = 8.3 Hz, ArH), 7.44
K
https://dx.doi.org/10.1021/acs.jnatprod.9b00773
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
pubs.acs.org/jnp
Article
quench the reaction, and the resultant liquid mixture was extracted
with CH2Cl2 (3 × 20 mL). The combined extracts were washed with
brine, dried over Na2SO4, filtered, and concentrated under reduced
pressure. The residue was purified by flash chromatography using a
prepacked 25 g silica column [solvent A: EtOAc; solvent B: hexanes;
gradient: 7% A/93% B (1 CV), 7% A/93% B → 60% A/40% B (10
CV), 60% A/40% B (2 CV); flow rate: 75 mL/min; monitored at 254
and 280 nm], yielding CA1-BAPC 26 (0.020 g, 0.037 mmol, 13%) as
an orange oil: 1H NMR (600 MHz, CDCl3) δ 7.80 (1H, d, J = 4.2 Hz,
ArH), 7.01 (1H, d, J = 8.7 Hz, ArH), 6.93 (1H, d, J = 4.3 Hz, ArH),
6.58 (1H, d, J = 12.1 Hz, CH), 6.49 (2H, s, ArH), 6.47 (1H, d, J = 8.6
Hz, ArH), 6.45 (1H, d, J = 12.1 Hz, CH), 4.60 (1H, sept, J = 6.0 Hz,
CH), 3.82 (3H, s, OCH3), 3.67 (3H, s, OCH3), 3.66 (6H, s, OCH3),
1.71 (6H, s, CH3), 1.23 (6H, d, J = 6.1 Hz, CH3); 13C NMR (151
MHz, CDCl3) δ 161.4, 154.6, 152.8, 151.6, 150.4, 137.1, 137.0, 132.7,
129.0, 128.1, 126.4, 126.2, 125.3, 122.1, 106.4, 105.9, 81.7, 75.1, 60.9,
55.8, 55.5, 28.8, 22.4; HRMS m/z 566.1819 [M + Na]+ (calcd for
NaC28H33NO8S+, 566.1819); HPLC (method B) 22.3 min, 91%, 4.9%
parent (isopropyl-protected CA1 analogue 21), 0% CA1.
gradient: 5% A/95% B (1 CV), 5% A/95% B → 40% A/60% B (10
CV), 40% A/60% B (2 CV); flow rate: 75 mL/min; monitored at 254
and 280 nm].
(Z)-tert-Butyl(6-methoxy-2-((5-nitrothiophen-2-yl)methoxy)-3-
(3,4,5-trimethoxystyryl)phenoxy)dimethylsilane (30). This isomer
30 (0.350 g, 0.739 mmol, 35%) was isolated as a brownish-yellow oil:
1H NMR (600 MHz, CDCl3) δ 7.76 (1H, d, J = 4.1 Hz, ArH), 6.91
(1H, d, J = 4.1 Hz, ArH), 6.87 (1H, dd, J = 8.6, 0.8 Hz, ArH), 6.57
(1H, d, J = 8.6 Hz, ArH), 6.50 (1H, d, J = 12.0 Hz, CH), 6.45 (1H, d,
J = 12.2 Hz, CH), 6.44 (2H, s, ArH), 5.12 (2H, s, CH2), 3.82 (3H, s,
OCH3), 3.79 (3H, s, OCH3), 3.65 (6H, s, OCH3), 0.99 (9H, s,
C(CH3)3), 0.13 (6H, s, Si(CH3)2); 13C NMR (126 MHz, CDCl3) δ
152.7, 151.6, 151.4, 148.6, 147.6, 138.4, 137.1, 132.4, 130.4, 128.2,
125.1, 124.9, 124.4, 122.3, 107.5, 105.9, 68.5, 60.9, 55.8, 55.4, 25.8,
18.6, −4.6.
(Z)-tert-Butyl(3-methoxy-2-((5-nitrothiophen-2-yl)methoxy)-6-
(3,4,5-trimethoxystyryl)phenoxy)dimethylsilane (33). Isomer 33
(0.250 g, 0.425 mmol, 25%) was isolated as a brownish-yellow oil:
1H NMR (600 MHz, CDCl3) δ 7.81 (1H, d, J = 4.1 Hz, ArH), 7.00
(1H, d, J = 8.7 Hz, ArH), 6.96 (1H, d, J = 4.1 Hz, ArH), 6.56 (1H, d,
J = 12.2 Hz, CH), 6.52 (2H, s, ArH), 6.44 (1H, d, J = 11.6 Hz, CH),
6.42 (1H, d, J = 8.5 Hz, ArH), 5.30 (2H, s, CH2), 3.83 (3H, s,
OCH3), 3.80 (3H, s, OCH3), 3.67 (6H, s, OCH3), 1.01 (9H, s,
C(CH3)3), 0.18 (6H, s, Si(CH3)2); 13C NMR (151 MHz, CDCl3) δ
153.4, 152.8, 152.7, 148.8, 147.8, 138.4, 137.0, 132.6, 129.1, 128.2,
126.3, 125.9, 125.4, 123.3, 105.9, 104.9, 68.8, 60.9, 55.9, 55.8, 26.1,
18.6, −3.9.
Synthesis of Compounds 31 and 34. Mono-TBS CA1 [mixture of
27 and 28 (0.680 g, 1.52 mmol)], diisopropylazodicarboxylate (0.415
g, 2.05 mmol), and 1-(5-nitrothiophen-2-yl)ethan-1-ol (0.317 g, 1.82
mmol) were dissolved in THF (50 mL). Triphenylphosphine (0.793
g, 3.04 mmol) was added, and the reaction mixture was stirred (3 d).
The reaction mixture was concentrated under reduced pressure, and
the residue was purified by flash column chromatography using a
prepacked 50 g silica column [solvent A: EtOAc; solvent B: hexanes;
gradient: 5% A/95% B (1 CV), 5% A/95% B → 40% A/60% B (13
CV), 40% A/60% B (2 CV); flow rate: 80 mL/min; monitored at 254
and 280 nm].
Synthesis of Compounds 27, 28, and 29. Deprotection of the
TBS group of compound 13 using TBAF (0.9 equiv) yielded an
inseparable mixture of compounds 27 and 28. At the same time,
about 15% CA1 (compound 29) was also isolated.
(Z)-2-((tert-Butyldimethylsilyl)oxy)-3-methoxy-6-(3,4,5-
trimethoxystyryl)phenol (27) and (Z)-2-((tert-butyldimethylsilyl)-
oxy)-6-methoxy-3-(3,4,5-trimethoxystyryl)phenol (28).43,44 To a
solution of di-TBS CA1 13 (2.00 g, 3.57 mmol) in THF (150 mL)
at −15 °C was added dropwise TBAF·3H2O (1.01 g, 3.20 mmol)
dissolved in THF (10 mL). The reaction was stirred for 0.5 h. H2O
was used to quench the reaction, THF was removed by evaporation,
and the residue was extracted with EtOAc (3 × 30 mL). The
combined extracts were washed with brine, dried over Na2SO4,
filtered, and concentrated under reduced pressure. The crude product
was purified by flash chromatography using a prepacked 100 g silica
column [solvent A: EtOAc; solvent B: hexanes; gradient: 5% A/95%
B (1 CV), 5% A/95% B → 70% A/30% B (13 CV), 70% A/30% B (2
CV); flow rate: 100 mL/min; monitored at 254 and 280 nm],
affording a mixture of compounds 27 and 28 (0.860 g, 2.59 mmol,
43%) as a white solid: 1H NMR (500 MHz, CDCl3) δ 6.80 (1H, d, J
= 8.7 Hz, ArH), 6.71 (1H, d, J = 8.7 Hz, ArH), 6.58 (2H, d, J = 12.0
Hz, CH), 6.52 (4H, s, ArH), 6.47 (1H, d, J = 12.1 Hz, CH), 6.41
(1H, d, J = 12.2 Hz, CH), 6.36 (1H, d, J = 8.5 Hz, ArH), 6.30 (1H, d,
J = 8.6 Hz, ArH), 5.66 (1H, s, OH), 5.45 (1H, s, OH), 3.81 (6H, s,
OCH3), 3.78 (3H, s, OCH3), 3.74 (3H, s, OCH3), 3.64 (12H, d, J =
2.2 Hz, OCH3), 1.01 (9H, d, J = 5.2 Hz, C(CH3)3), 1.00 (9H, s,
C(CH3)3), 0.22 (6H, s, Si(CH3)2), 0.19 (6H, s, Si(CH3)2); 13C NMR
(126 MHz, CDCl3) δ 152.7, 152.7, 149.3, 146.9, 145.9, 141.2, 137.0,
137.0, 136.8, 132.9, 132.8, 131.6, 129.6, 129.0, 126.8, 124.5, 123.2,
122.0, 120.1, 117.1, 106.1, 106.0, 103.8, 103.0, 60.9, 60.8, 56.1, 55.8,
55.7, 55.2, 26.0, 26.0, 18.6, 18.6, −3.9, −4.4.
(Z)-tert-Butyl(6-methoxy-2-(1-(5-nitrothiophen-2-yl)ethoxy)-3-
(3,4,5-trimethoxystyryl)phenoxy)dimethylsilane (31). Isomer 31
1
(0.375 g, 0.623 mmol, 41%) was isolated as a yellow oil: H NMR
(600 MHz, CDCl3) δ 7.60 (1H, d, J = 4.2 Hz, ArH), 6.76−6.69 (2H,
m, ArH), 6.41 (1H, d, J = 8.2 Hz, ArH), 6.39 (1H, d, J = 11.9 Hz,
CH), 6.32 (2H, s, ArH), 6.29 (1H, d, J = 12.1 Hz, CH), 5.58 (1H, q, J
= 6.4 Hz, CH), 3.69 (3H, s, OCH3), 3.65 (3H, s, OCH3), 3.51 (6H, s,
OCH3), 1.48 (3H, d, J = 6.4 Hz, CH3), 0.85 (9H, s, C(CH3)3), 0.00
(3H, s, Si(CH3)), −0.02 (3H, s, Si(CH3)); 13C NMR (151 MHz,
CDCl3) δ 155.0, 152.7, 151.4, 150.9, 146.1, 138.5, 137.2, 132.4, 129.8,
128.1, 125.6, 124.9, 123.4, 122.5, 107.0, 106.0, 74.4, 60.9, 55.8, 55.3,
25.8, 22.3, 18.6, −4.3, −4.4.
(Z)-3-Methoxy-6-(3,4,5-trimethoxystyryl)benzene-1,2-diol (29).
Combretastatin A-1 29 (0.179 mg, 0.538 mmol, 15%) was isolated
as a white solid: 1H NMR (500 MHz, CDCl3) δ 6.76 (1H, d, J = 8.6
Hz, ArH), 6.59 (1H, d, J = 12.1 Hz, CH), 6.54 (1H, d, J = 11.9 Hz,
CH), 6.52 (2H, s, ArH), 6.39 (1H, d, J = 8.6 Hz, ArH), 5.39 (2H, s,
OH), 3.86 (3H, s, OCH3), 3.83 (3H, s, OCH3), 3.67 (6H, s, OCH3);
13C NMR (126 MHz, CDCl3) δ 152.9, 146.5, 141.7, 137.4, 132.7,
(Z)-tert-Butyl(3-methoxy-2-(1-(5-nitrothiophen-2-yl)ethoxy)-6-
(3,4,5-trimethoxystyryl)phenoxy)dimethylsilane (34). Isomer 34
1
(0.073 g, 0.122 mmol, 12%) was isolated as a yellow oil: H NMR
(600 MHz, CDCl3) δ 7.61 (1H, d, J = 4.2 Hz, ArH), 6.84 (1H, d, J =
8.7 Hz, ArH), 6.71 (1H, d, J = 4.2 Hz, ArH), 6.38 (1H, d, J = 12.0 Hz,
CH), 6.37 (2H, s, ArH), 6.26 (1H, d, J = 12.3 Hz, CH), 6.24 (1H, d, J
= 8.6 Hz, ArH), 5.26 (1H, q, J = 6.5 Hz, CH), 3.67 (3H, s, OCH3),
3.61 (3H, s, OCH3), 3.50 (6H, s, OCH3), 1.47 (3H, d, J = 6.5 Hz,
CH3), 0.84 (9H, s, C(CH3)3), 0.03 (3H, s, Si(CH3)), 0.00 (3H, s,
Si(CH3)); 13C NMR (151 MHz, CDCl3) δ 153.3, 151.1, 150.9, 149.1,
146.1, 135.5, 135.2, 130.7, 126.9, 126.2, 124.6, 123.7, 121.5, 121.5,
104.1, 103.0, 73.1, 59.0, 53.9, 24.2, 24.2, 19.8, 16.7, −5.2, −5.7.
(Z)-tert-Butyl(6-methoxy-2-((2-(5-nitrothiophen-2-yl)propan-2-
yl)oxy)-3-(3,4,5-trimethoxystyryl)phenoxy)dimethylsilane (32).
Mono-TBS CA1 [mixture of 27 and 28, (1.07 g, 2.40 mmol)], gem-
dimethyl trigger 19 (0.540 g, 2.88 mmol), and ADDP (0.832 g, 3.30
mmol) were dissolved in CH2Cl2 (100 mL). Tributylphosphine (1.26
mL, 5.04 mmol) was added dropwise, and the reaction mixture was
stirred (2 d). The reaction mixture was then concentrated under
132.6, 130.5, 124.2, 120.5, 118.0, 106.1, 103.1, 76.9, 61.0, 56.3, 56.0;
+
HRMS m/z 355.1154 [M + Na]+ (calcd for NaC18H20O6 ,
355.1152); HPLC (method A) 11.3 min, 99%.
Synthesis of Compounds 30 and 33. To a mixture of compounds
27 and 28 (1.00 g, 2.24 mmol), nor-methyl trigger 16 (0.428 g, 2.69
mmol), and DIAD (0.867 mL) in CH2Cl2 (50 mL) was added
dropwise PPh3 (1.47 g, 5.60 mmol). The reaction mixture was stirred
(24 h) at room temperature. H2O (40 mL) was added to quench the
reaction, and the resultant liquid mixture was extracted with CH2Cl2
(3 × 20 mL). The combined extracts were washed with brine, dried
over Na2SO4, filtered, and concentrated under reduced pressure. The
crude product was purified by flash column chromatography using a
prepacked 25 g silica column [solvent A: EtOAc; solvent B: hexanes;
L
https://dx.doi.org/10.1021/acs.jnatprod.9b00773
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Article
reduced pressure. Flash chromatography yielded the crude product,
which was taken to the next step for deprotection.
C24H23NO8S+, 486.1217); HPLC (method A) 14.9 min, 96%, 0%
parent (CA1).
(Z)-4-Methoxy-2-(5-nitrothiophen-2-yl)-7-(3, 4, 5-
trimethoxystyryl)benzo[d][1,3]dioxole (35). To a solution of 30
(0.095 g, 0.162 mmol) in THF (10 mL) at 0 °C was added dropwise
TBAF·3H2O (0.0672 g, 0.213 mmol) dissolved in THF (10 mL). The
reaction mixture was stirred (30 min), and H2O (5 mL) was added.
THF was removed by evaporation, and the residue was extracted with
CH2Cl2 (3 × 20 mL). The combined extracts were washed with brine,
dried over Na2SO4, filtered, and concentrated under reduced pressure.
The crude organic product was purified by flash column
chromatography using a prepacked 10 g silica column [solvent A:
EtOAc; solvent B: hexanes; gradient: 7% A/93% B (1 CV), 7% A/
93% B → 60% A/40% B (13 CV), 60% A/40% B (2 CV); flow rate:
20 mL/min; monitored at 254 and 280 nm], affording compound 35
(Z)-4-Methoxy-2-methyl-2-(5-nitrothiophen-2-yl)-7-(3,4,5-
trimethoxystyryl)benzo[d][1,3]dioxole (36) [Base Cyclization Meth-
od]. Compound 38 (0.0500 g, 0.103 mmol) was dissolved in THF (5
mL) at room temperature. NaOH (1 mL, 2 M) was added dropwise,
and the reaction mixture was then stirred (5 min). THF was removed
by evaporation, and the residue was extracted with CH2Cl2 (3 × 10
mL). The combined extracts were washed with brine, dried over
Na2SO4, filtered, and concentrated under reduced pressure. The
residue was purified by flash chromatography using a prepacked 10 g
silica column [solvent A: EtOAc; solvent B: hexanes; gradient: 10%
A/90% B (1 CV), 10% A/90% B → 80% A/20% B (10 CV), 80% A/
20% B (2 CV); flow rate: 36 mL/min; monitored at 254 and 280
nm], affording compound 36 (0.0470 g, 0.0964 mmol, 93%) as a
1
1
yellow oil: H NMR (500 MHz, acetone) δ 7.76 (1H, d, J = 4.3 Hz,
(0.0510 g, 0.108 mmol, 54%) as a yellow oil: H NMR (600 MHz,
ArH), 7.03 (1H, d, J = 4.2 Hz, ArH), 6.81 (1H, d, J = 8.8 Hz, ArH),
6.56 (1H, d, J = 12.0 Hz, CH), 6.48 (2H, s, ArH), 6.45 (1H, d, J = 8.8
Hz, ArH), 6.42 (1H, d, J = 11.9 Hz, CH), 3.88 (3H, s, OCH3), 3.82
(3H, s, OCH3), 3.67 (6H, s, OCH3), 2.02 (3H, s, CH3); 13C NMR
(126 MHz, acetone) δ 152.8, 151.5, 145.1, 143.1, 137.2, 133.8, 132.7,
131.1, 128.3, 124.0, 123.0, 121.9, 113.8, 113.4, 107.4, 105.7, 60.9,
56.5, 55.9, 26.6.
CDCl3) δ 7.83 (1H, d, J = 4.2 Hz, ArH), 7.15 (1H, d, J = 4.2 Hz,
ArH), 7.07 (1H, s, CH), 6.86 (1H, d, J = 8.8 Hz, ArH), 6.56 (1H, d, J
= 12.0 Hz, CH), 6.50 (2H, s, ArH), 6.48 (1H, d, J = 8.8 Hz, ArH),
6.44 (1H, d, J = 12.0 Hz, CH), 3.90 (3H, s, OCH3), 3.83 (3H, s,
OCH3), 3.69 (6H, s, OCH3); 13C NMR (151 MHz, CDCl3) δ 152.9,
146.0, 145.2, 143.2, 137.3, 133.8, 132.6, 131.2, 128.1, 126.0, 123.4,
121.7, 113.5, 107.8, 105.7, 105.6, 105.2, 60.9, 56.6, 55.9; 13C NMR
DEPT (CDCl3, 151 MHz) δ 131.2, 128.1, 126.0, 123.4, 121.7, 107.8,
105.6, 105.2, 60.9, 56.6, 55.9; HRMS m/z 494.0881 [M + Na]+ (calcd
for NaC23H21NO8S+, 494.0880); HPLC (method A) 17.2 min, 98%,
0% parent (CA1).
(Z)-6-Methoxy-2-((5-nitrothiophen-2-yl)methoxy)-3-(3,4,5-
trimethoxystyryl)phenol (37). AcOH (7 mL) and HCl (5 mL, 2 M)
were added dropwise to a solution of compound 30 (0.115 g, 0.196
mmol) in THF (30 mL). The reaction mixture was stirred for 8 h at
room temperature. H2O (40 mL) was used to quench the reaction,
THF was removed by evaporation, and the residue was extracted with
CH2Cl2 (3 × 20 mL). The combined extracts were washed with brine,
dried over Na2SO4, filtered, and concentrated under reduced pressure.
The crude product was purified by flash chromatography using a
prepacked 10 g silica column [solvent A: EtOAc; solvent B: hexanes;
gradient: 10% A/90% B (1 CV), 10% A/90% B → 80% A/20% B (10
CV), 80% A/20% B (2 CV); flow rate: 20 mL/min; monitored at 254
and 280 nm], affording compound 37 (0.020 g, 0.0422 mmol, 17%)
(Z)-4-Methoxy-2-(5-nitrothiophen-2-yl)-7-(3, 4, 5-
trimethoxystyryl)benzo[d][1,3]dioxole (35) [Base Cyclization Meth-
od]. Compound 37 (0.0380 g, 0.0803 mmol) was dissolved in THF
(5 mL) at room temperature. NaOH (1 mL, 2 M) was added
dropwise, and the reaction mixture was then stirred (5 min). THF was
removed by evaporation, and the residue was extracted with CH2Cl2
(3 × 10 mL). The combined extracts were washed with brine, dried
over Na2SO4, filtered, and concentrated under reduced pressure. The
residue was purified by flash chromatography using a prepacked 10 g
silica column [solvent A: EtOAc; solvent B: hexanes; gradient: 10%
A/90% B (1 CV), 10% A/90% B → 80% A/20% B (10 CV), 80% A/
20% B (2 CV); flow rate: 36 mL/min; monitored at 254 and 280
nm], affording compound 35 (0.0090 g, 0.019 mmol, 23%) as a
1
as a brown oil: H NMR (600 MHz, acetone) δ 8.15 (1H, s, OH),
7.93 (1H, d, J = 4.2 Hz, ArH), 7.21 (1H, d, J = 4.1 Hz, ArH), 6.95
(1H, d, J = 8.7 Hz, ArH), 6.58 (2H, s, ArH), 6.54 (1H, d, J = 12.2 Hz,
CH), 6.49 (1H, d, J = 8.7 Hz, ArH), 6.44 (1H, d, J = 12.2 Hz, CH),
5.29 (2H, s, CH2), 3.86 (3H, s, OCH3), 3.68 (3H, s, OCH3), 3.62
(6H, s, OCH3); 13C NMR (151 MHz, acetone) δ 153.1, 152.3, 149.2,
148.6, 137.4, 134.1, 132.7, 129.0, 128.6, 126.5, 125.1, 124.5, 124.5,
117.9, 106.2, 103.0, 68.5, 59.6, 55.4, 55.2; 13C NMR DEPT (151
MHz, acetone) δ 129.0, 128.6, 126.5, 125.1, 124.5, 106.2, 103.0, 68.5,
59.6, 55.4, 55.2; HRMS m/z 496.1034 [M + Na]+ (calcd for
NaC23H23NO8S+, 496.1037); HPLC (method B) 10.0 min, 95%, 0%
parent (CA1).
1
yellow oil: H NMR (600 MHz, CDCl3) δ 7.83 (1H, d, J = 4.2 Hz,
ArH), 7.15 (1H, d, J = 4.2 Hz, ArH), 7.07 (1H, s, CH), 6.86 (1H, d, J
= 8.8 Hz, ArH), 6.56 (1H, d, J = 12.0 Hz, CH), 6.50 (2H, s, ArH),
6.48 (1H, d, J = 8.8 Hz, ArH), 6.44 (1H, d, J = 12.0 Hz, CH), 3.90
(3H, s, OCH3), 3.83 (3H, s, OCH3), 3.69 (6H, s, OCH3); 13C NMR
(151 MHz, CDCl3) δ 152.9, 146.0, 145.2, 143.2, 137.3, 133.8, 132.6,
131.2, 128.1, 126.0, 123.4, 121.7, 113.5, 107.8, 105.7, 105.6, 105.2,
60.9, 56.6, 55.9.
(Z)-6-Methoxy-2-(1-(5-nitrothiophen-2-yl)ethoxy)-3-(3,4,5-
trimethoxystyryl)phenol (38). Compound 31 (0.200 g, 0.333 mmol)
was dissolved in THF (5 mL). Glacial acetic acid (7 mL) and HCl (2
M, 4 mL) were added dropwise, and the reaction mixture was stirred
(30 min). Glacial acetic acid (4 mL) and HCl (2 M, 2.5 mL) were
added dropwise, and the reaction mixture was stirred (8 h). H2O (30
mL) was used to quench the reaction, and the reaction mixture was
concentrated under reduced pressure. The residue was extracted with
CH2Cl2 (3 × 30 mL), and the combined organic phase was washed
multiple times with brine, dried over Na2SO4, filtered, and
concentrated under reduced pressure. The residue was purified by
flash column chromatography using a prepacked 50 g silica column
[solvent A: EtOAc; solvent B: hexanes; gradient: 10% A/90% B (1
CV), 10% A/90% B → 80% A/20% B (13 CV), 80% A/20% B (2
CV); flow rate: 100 mL/min; monitored at 254 and 280 nm],
affording compound 38 (0.094 g, 0.199 mmol, 60%) as a yellow oil:
1H NMR (600 MHz, CDCl3) δ 7.74 (1H, d, J = 4.2 Hz, ArH), 6.93
(Z)-4-Methoxy-2-methyl-2-(5-nitrothiophen-2-yl)-7-(3,4,5-
trimethoxystyryl)benzo[d][1,3]dioxole (36). Compound 31 (0.105 g,
0.174 mmol) was dissolved in CH2Cl2 (20 mL) at −10 °C. tert-
Butylammonium fluoride trihydrate (0.0620 g, 0.191 mmol) was
dissolved in CH2Cl2 (2 mL) and slowly added dropwise to the
reaction mixture, which was then stirred (18 min). H2O (5 mL) was
used to quench the reaction, and the layers were partitioned. The
residue was extracted with CH2Cl2 (3 × 10 mL), washed with brine,
dried over Na2SO4, and concentrated under reduced pressure. The
crude product was purified by flash column chromatography using a
prepacked 10 g silica column [solvent A: EtOAc; solvent B: hexanes;
gradient: 12% A/88% B (1 CV), 12% A/88% B → 100% A/0% B (13
CV), 100% A/0% B (2 CV); flow rate: 10 mL/min; monitored at 254
and 280 nm], affording compound 36 (0.044 g, 0.0906 mmol, 52%)
1
as a yellow oil: H NMR (500 MHz, acetone) δ 7.76 (1H, d, J = 4.3
Hz, ArH), 7.03 (1H, d, J = 4.2 Hz, ArH), 6.81 (1H, d, J = 8.8 Hz,
ArH), 6.56 (1H, d, J = 12.0 Hz, CH), 6.48 (2H, s, ArH), 6.45 (1H, d,
J = 8.8 Hz, ArH), 6.42 (1H, d, J = 11.9 Hz, CH), 3.88 (3H, s, OCH3),
3.82 (3H, s, OCH3), 3.67 (6H, s, OCH3), 2.02 (3H, s, CH3); 13C
NMR (126 MHz, acetone) δ 152.8, 151.5, 145.1, 143.1, 137.2, 133.8,
132.7, 131.1, 128.3, 124.0, 123.0, 121.9, 113.8, 113.4, 107.4, 105.7,
60.9, 56.5, 55.9, 26.6; HRMS m/z 486.1219 [M + H]+ (calcd for
(1H, d, J = 4.2 Hz, ArH), 6.80 (1H, d, J = 8.5 Hz, ArH), 6.55 (1H, d,
J = 8.6 Hz, ArH), 6.53 (1H, d, J = 12.5 Hz, CH), 6.47 (2H s, ArH),
6.45 (1H, d, J = 12.2 Hz, CH), 5.71 (1H, q, J = 6.4 Hz, CH), 3.87
(3H, s, OCH3), 3.84 (3H, s, OCH3), 3.66 (6H, s, OCH3), 1.71 (3H,
d, J = 6.4 Hz, CH3); 13C NMR (126 MHz, CDCl3) δ 153.6, 152.8,
M
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151.4, 147.5, 137.1, 132.6, 132.2, 130.2, 128.2, 125.4, 123.8, 123.8,
117.5, 105.8, 103.4, 75.2, 60.9, 55.9, 55.8, 29.7, 21.7; HRMS m/z
488.1363 [M + H]+ (calcd for C24H25NO8S+, 488.1374); HPLC
(method B) 10.1 min, 96%, 4% parent (CA1).
13C NMR (CDCl3, 151 MHz) δ 161.5, 152.9, 150.6, 147.1, 140.6,
140.2, 137.3, 132.5, 129.3, 128.4, 127.2, 126.6, 122.2, 120.6, 106.9,
106.0, 81.9, 61.1, 56.4, 56.0, 29.5; HRMS m/z 524.1352 [M + Na]+
(calcd for NaC25H27NO8S+, 524.1350); HPLC (method B) 12.3 min,
98%, 0% parent (CA1).
(Z)-3-Methoxy-2-((5-nitrothiophen-2-yl)methoxy)-6-(3,4,5-
trimethoxystyryl)phenol (39). AcOH (10 mL) and HCl (10 mL, 2
M) were added dropwise to a solution of compound 33 (0.250 g,
0.425 mmol) in THF (25 mL). The reaction mixture was stirred for 8
h at room temperature. H2O (40 mL) was used to quench the
reaction, THF was removed by evaporation, and the residue was
extracted with CH2Cl2 (3 × 20 mL). The combined extracts were
washed with brine, dried over Na2SO4, filtered, and concentrated
under reduced pressure. The residue was purified by flash
chromatography using a prepacked 10 g silica column [solvent A:
EtOAc; solvent B: hexanes; gradient: 10% A/90% B (1 CV), 10% A/
90% B → 80% A/20% B (10 CV), 80% A/20% B (2 CV); flow rate:
20 mL/min; monitored at 254 and 280 nm], affording compound 33
[(Z)-2-((2-Methoxy-5-(3,4,5-trimethoxystyryl)phenoxy)methyl)-5-
nitrothiophene (43).42 (5-Nitrothiophen-2-yl)methanol (0.100 g,
0.628 mmol), triphenylphosphine (0.336 g, 1.28 mmol), and
combretastatin A-4 (0.396 g, 1.25 mmol) were dissolved in
tetrahydrofuran (2 mL). DEAD (0.218 g, 1.25 mmol) was added,
and the reaction mixture was stirred for 4 h at 50 °C. The reaction
mixture solvent was then removed by evaporation under reduced
pressure. The residue was extracted with EtOAc, and the solution was
washed with water and brine, dried with Na2SO4, and evaporated
under reduced pressure. Flash chromatography of the residue using a
prepacked 100 g silica column [eluents: solvent A, EtOAc; solvent B,
hexanes; gradient, 10% A/90% B over 1.19 min (1 CV), 10% A/90%
B → 67% A/33% B over 13.12 min (10 CV), 67% A/33% B over 2.38
min (2 CV); flow rate 50.0 mL/min; monitored at 254 and 280 nm]
and recrystallization from EtOAc and hexanes yielded [(Z)-2-((2-
methoxy-5-(3,4,5-trimethoxystyryl)phenoxy)methyl)-5-nitrothio-
1
(0.030 g, 0.0634 mmol, 12%) as a brown oil: H NMR (CDCl3, 600
MHz) δ 7.77 (1H, d, J = 4.1 Hz, ArH), 6.97 (1H, d, J = 4.1 Hz, ArH),
6.79 (1H, d, J = 8.4 Hz, ArH), 6.56 (1H, d, J = 8.4 Hz, ArH), 6.55
(1H, d, J = 12.2 Hz, CH), 6.50 (1H, d, J = 12.2 Hz, CH), 6.45 (2H, s,
ArH), 5.59 (1H, s, OH), 5.24 (2H, s, CH2), 3.88 (3H, s, OCH3), 3.82
(3H, s, OCH3), 3.65 (6H, s, OCH3); 13C NMR (CDCl3, 151 MHz) δ
152.8, 151.8, 148.5, 147.0, 142.6, 138.4, 137.2, 132.4, 130.7, 128.2,
125.5, 124.6, 124.3, 120.5, 106.6, 106.0, 68.7, 60.9, 56.4, 55.8; HRMS
m/z 496.1033 [M + Na]+ (calcd for NaC23H23NO8S+, 496.1037);
HPLC (method B): 12.5 min, 96%, 0% parent (CA1).
1
phene (43) (0.286 g, 0.625 mmol, 50%) as a yellow solid: H NMR
(500 MHz, CDCl3) δ 7.80 (1H, d, J = 4 Hz, ArH), 6.97 (1H, dd, J = 8
Hz, J = 1.5 Hz, ArH), 6.89 (1H, d, J = 4 Hz, ArH), 6.87 (1H, d, J = 2
Hz, ArH), 6.84 (1H, d, J = 8.5 Hz, ArH), 6.50 (2H, s, ArH), 6.48
(2H, d, J = 12 Hz, CH), 5.07 (2H, s, CH2), 3.89 (3H, s, OCH3), 3.86
(3H, s, OCH3), 3.72 (6H, s, OCH3); 13C NMR (125 MHz, CDCl3) δ
153.0, 149.1, 148.3, 146.4, 137.1, 132.9, 129.8, 129.2, 129.1, 128.4,
124.8, 124.0, 115.4, 111.7, 105.8, 66.4, 60.9, 56.0, 56.0; HRMS m/z
480.1088 [M + Na]+ (calcd for NaC23H23NO7S+, 480.1087); HPLC
(method A) 17.1 min, 97%, 1.5% parent (CA4).
(Z)-3-Methoxy-2-(1-(5-nitrothiophen-2-yl)ethoxy)-6-(3,4,5-
trimethoxystyryl)phenol (40). Compound 34 (0.100 g, 0.167 mmol)
was dissolved in THF (3 mL). Glacial acetic acid (5.6 mL) and HCl
(2 M, 3.3 mL) were added dropwise, and the reaction mixture was
stirred (8 h). H2O (20 mL) was used to quench the reaction, and the
reaction mixture was concentrated under reduced pressure. The
residue was extracted with CH2Cl2 (3 × 20 mL), and the combined
organic phase was washed multiple times with brine, dried over
Na2SO4, filtered, and concentrated under reduced pressure. The
residue was purified by flash column chromatography using a
prepacked 10 g silica column [solvent A: EtOAc; solvent B: hexanes;
gradient: 10% A/90% B (1 CV), 10% A/90% B → 80% A/20% B (13
CV), 80% A/20% B (2 CV); flow rate: 100 mL/min; monitored at
254 and 280 nm], affording compound 40 (0.026 g, 0.055 mmol,
33%) as a yellow oil: 1H NMR (600 MHz, CDCl3) δ 7.79 (1H, d, J =
4.2 Hz, ArH), 6.96 (1H, d, J = 8.7 Hz, ArH), 6.93 (1H, d, J = 4.2 Hz,
ArH), 6.53 (2H, d, J = 12.4 Hz, CH), 6.49 (2H, s, ArH), 6.37 (1H, d,
J = 8.8 Hz, ArH), 5.56 (1H, q, J = 6.5 Hz, CH), 3.84 (3H, s, OCH3),
3.83 (3H, s, OCH3), 3.67 (6H, s, OCH3), 1.73 (3H, d, J = 6.5 Hz,
CH3); 13C NMR (126 MHz, CDCl3) δ 153.6, 152.8, 151.4, 147.5,
132.6, 132.2, 130.2, 128.2, 125.3, 123.8, 123.7, 117.5, 105.8, 103.4,
103.3, 75.2, 60.9, 55.9, 55.8, 29.7, 21.7; HRMS m/z 488.1373 [M +
H]+ (calcd for C23H23NO8S+), 488.1374; HPLC (method B) 11.1
min, 93%, 1% parent (CA1).
(Z)-6-Methoxy-2-((2-(5-nitrothiophen-2-yl)propan-2-yl)oxy)-3-
(3,4,5-trimethoxystyryl)phenol (41). To a solution of compound 32
[as a mixture of 32, 27, and 28 (2.35 g, 3.82 mmol)] in THF (250
mL) at −15 °C was added dropwise TBAF·3H2O (1.32 g, 4.19
mmol) dissolved in THF (10 mL). The reaction was stirred for 1 h.
H2O (40 mL) was used to quench the reaction, THF was removed by
evaporation, and the residue was extracted with CH2Cl2 (3 × 20 mL).
The combined extracts were washed with brine, dried over Na2SO4,
filtered, and concentrated under reduced pressure. The residue was
purified by flash chromatography using a prepacked 10 g silica column
[solvent A: EtOAc; solvent B: hexanes; gradient: 7% A/93% B (1
CV), 7% A/93% B → 60% A/40% B (13 CV), 60% A/40% B (2 CV);
flow rate: 20 mL/min; monitored at 254 and 280 nm], affording
compound 41 (0.050 g, 0.980 mmol, 2%) as a brownish-yellow solid:
1H NMR (CDCl3, 600 MHz) δ 7.77 (1H, d, J = 4.2 Hz, ArH), 6.93
(Z)-2-(1-(2-Methoxy-5-(3,4,5-trimethoxystyryl)phenoxy)ethyl)-5-
nitrothiophene (44).42 Combretastatin A-4 (0.251 g, 0.79 mmol),
triphenylphosphine (0.105 g, 0.400 mmol), and 1-(5-nitrothiophen-2-
yl)ethanol (0.197 g, 1.14 mmol) were dissolved in dry THF (10 mL).
DEAD (0.155 g, 0.890 mmol) was added dropwise, and the reaction
mixture was stirred for 24 h. The reaction was quenched with water
and partitioned, and the aqueous phase was extracted with EtOAc.
The EtOAc phase was dried with Na2SO4 and evaporated under
reduced pressure. Flash chromatography of the residue using a
prepacked 25 g silica column [eluents: solvent A, EtOAc; solvent B,
hexanes; gradient, 15% A/85% B over 1.19 min (1 CV), 15% A/85%
B → 100% A/0% B over 13.12 min (10 CV), 100% A/0% B over 2.38
min (2 CV); flow rate 25.0 mL/min; monitored at 254 and 280 nm]
yielded (Z)-2-(1-(2-methoxy-5-(3,4,5-trimethoxystyryl)phenoxy)-
ethyl)-5-nitrothiophene (44) (0.090 g, 0.19 mmol, 24%) as a yellow
solid: 1H NMR (500 MHz, CDCl3) δ 7.75 (1H, d, J = 4.5 Hz, ArH),
6.94 (1H, dd, J = 8 Hz, J = 2 Hz, ArH), 6.81 (3H, m, ArH), 6.46 (1H,
d, J = 12.5 Hz, CH), 6.45 (2H, s, ArH), 6.44 (1H, d, J = 12 Hz, CH),
5.25 (1H, q, J = 6 Hz, CH), 3.86 (3H, s, OCH3), 3.84 (3H, s, OCH3),
3.69 (6H, s, OCH3), 1.63 (3H, d, J = 6.5 Hz, CH3); 13C NMR (125
MHz, CDCl3) δ 155.3, 153.0, 149.9, 145.7, 137.1, 132.9, 129.9, 129.2,
129.1, 128.4, 124.4, 123.0, 118.2, 112.0, 105.8, 73.8, 60.9, 55.9, 55.9,
23.1; HRMS m/z 472.1428 [M + H]+ (calcd for C24H25NO8S+,
472.1424); HPLC (method A) 17.6 min, 98%, 0% parent (CA4).
[(Z)-2-(2-(2-Methoxy-5-(3,4,5-trimethoxystyryl)phenoxy)propan-
2-yl)-5-nitrothiophene (45).42 Combretastatin A-4 (1.87 g, 5.91
mmol), 2-(5-nitrothiophen-2-yl)propan-2-ol (1.17 g, 6.25 mmol), and
ADDP (1.46 g, 5.79 mmol) were dissolved in benzene (15 mL).
Tributylphosphine (1.43 mL, 5.91 mmol) was added dropwise, and
the reaction mixture was stirred for 24 h. The reaction was quenched
with water, and the reaction mixture was extracted with EtOAc. The
organic phase was dried with Na2SO4 and evaporated under reduced
pressure. Flash chromatography of the residue using a prepacked 25 g
silica column [eluents: solvent A, EtOAc; solvent B, hexanes; gradient,
15% A/85% B over 1.19 min (1 CV), 15% A/85% B → 100% A/0% B
over 13.12 min (10 CV), 100% A/0% B over 2.38 min (2 CV); flow
rate 25.0 mL/min; monitored at 254 and 280 nm] yielded (Z)-2-(2-
(2-methoxy-5-(3,4,5-trimethoxystyryl)phenoxy)propan-2-yl)-5-nitro-
thiophene (45) (0.670 g, 1.38 mmol, 23%) as a dark red oil: 1H NMR
(1H, d, J = 4.2 Hz, ArH), 6.84 (1H, d, J = 8.6 Hz, ArH), 6.56 (1H, d,
J = 8.6 Hz, ArH), 6.52 (2H, s, ArH), 6.48 (1H, d, J = 12.2 Hz, CH),
6.30 (1H, d, J = 12.2 Hz, CH), 5.47 (1H, s, OH), 3.86 (3H, s,
OCH3), 3.84 (3H, s, OCH3), 3.67 (6H, s, OCH3), 1.79 (6H, s, CH3);
N
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(500 MHz, CDCl3) δ 7.72 (1H, d, J = 4.2 Hz, ArH), 7.01 (1H, dd, J =
2.0 Hz, 8.4 Hz, ArH), 6.83 (1H, d, J = 8.4 Hz, ArH), 6.77 (1H, d, J =
4.2 Hz, ArH), 6.72 (1H, d, J = 2.0 Hz, ArH), 6.43 (4H, m, ArH and
CH), 3.85 (3H, s, OCH3), 3.80 (3H, s, OCH3), 3.71 (6H, s, OCH3),
1.59 (6H, s, CH3); 13C NMR (125 MHz, CDCl3): δ 161.0, 152.9,
152.4, 150.3, 142.8, 136.8, 132.9, 129.5, 129.2, 129.1, 128.3, 125.8,
124.0, 122.2, 111.8, 105.7, 79.9, 60.7, 55.8, 55.6, 28.6; HRMS m/z
508.1399 [M + Na]+ (calcd for NaC25H27NO8S+, 508.1400); HPLC
(method A) 16.2 min, 97%, 0% parent (CA4); X-ray crystallography
single-crystal X-ray diffraction (CCDC 1502383) provided further
structural characterization for compound 45 (see Supporting
Information).
to twice the required concentrations in preconditioned medium, and
100 μL was added per well in duplicate for each experiment. The
highest final concentration of control drugs and test compounds was
50 μg/mL. Plates were incubated for 4 h under anaerobic conditions
and then for 48 h under aerobic conditions prior to sulforhodamine B
determination of cytotoxicity.56−59 The procedure for the normoxic
arm of the assay was identical to that described above except that all
manipulations were carried out under ambient conditions with drug
exposure carried out under 95% air plus 5% CO2. Tirapazamine was
used as a positive control as previously described.60−62
Inhibition of Tubulin Polymerization.63 Tubulin assembly
reaction mixtures (0.25 mL final volume)63 contained 1.0 mg/mL
(10 μM) purified bovine brain tubulin, 0.8 M monosodium glutamate
(pH 6.6 in a 2 M stock solution), 4% (v/v) DMSO, 0.4 mM GTP,
and varying compound concentrations. Initially, all components
except GTP were preincubated for 15 min at 30 °C in 0.24 mL. The
reaction mixtures were placed on ice, and 10 μL of 0.01 M GTP was
added. The reaction mixtures were transferred to cuvettes held at 0 °C
in Beckman DU-7400 and DU-7500 spectrophotometers equipped
with electronic temperature controllers. The temperature was jumped
to 30 °C over about 30 s, and the polymerization reaction was
followed turbidimetrically at 350 nM for 20 min. Each reaction set
included a reaction mixture without compound, and the IC50 was
defined as the concentration of compound that inhibited the extent of
assembly by 50%.
[(Z)-2-(2-(2-Methoxy-5-(3,4,5-trimethoxystyryl)phenoxy)propan-
2-yl)-5-nitrothiophene (45)42 (Alternate Purification Route). Com-
bretastatin A-4 (1.61 g, 5.09 mmol), 2-(5-nitrothiophen-2-yl)propan-
2-ol (1.00 g, 5.34 mmol), and ADDP (1.35 g, 5.34 mmol) were
dissolved in toluene (101 mL). Tributylphosphine (1.3 mL, 5.34
mmol) was added dropwise, and the reaction mixture was stirred for
20 h. The reaction mixture was diluted with EtOAc and quenched
with water. The resulting mixture was extracted with EtOAc (30 mL
× 3), and the organic phase was dried with Na2SO4 and evaporated
under reduced pressure. The residue was purified by flash
chromatography using 5−20% EtOAc/hexanes. The column-purified
product contained free alcohol, CA4, and BAPC (product) in the
ratio of 1.0:2.4:3.0. The amount of BAPC was 921 mg, 1.90 mmol
(calculated from NMR) (theoretical yield: 2.469 g). The amount of
free alcohol was 118 mg, 0.63 mmol. The amount of CA4 was 480
mg, 1.52 mmol. To a solution of these three compounds in CH2Cl2
(30.0 mL) was added imidazole (1.03 g, 15.2 mmol), followed by
TBSCl (2.52 g, 16.7 mmol). The solution was stirred at room
temperature for 4 h. The reaction was quenched with water and
extracted with EtOAc (30 mL × 3). The EtOAc phase was dried with
Na2SO4 and evaporated under reduced pressure. The product was
purified by flash chromatography using 5−20% EtOAc/hexanes. This
resulted in removal of the CA4, leaving a mixture of BAPC and the
free alcohol in a ratio of 3:1 by NMR (807 mg, 1.66 mmol BAPC and
103 mg, 0.55 mmol free alcohol). A mixture of BAPC, free alcohol,
DMAP (67 mg, 0.55 mmol), and TEA (0.85 mL, 6.05 mmol) in
CH2Cl2 (11 mL) was treated with acetic anhydride (0.52 mL, 5.5
mmol) and stirred at room temperature for 4.0 h. The reaction was
quenched with water and extracted with EtOAc (30 mL × 3). The
EtOAc phase was dried with Na2SO4 and evaporated under reduced
pressure. The product was purified by flash chromatography using 5−
15% EtOAc/hexanes. (Z)-2-(2-(2-Methoxy-5-(3,4,5-
trimethoxystyryl)phenoxy)propan-2-yl)-5-nitrothiophene (45)
(0.760 g, 1.56 mmol, 31%) was isolated as a dark red oil. By NMR
Colchicine Binding Assay.64 Inhibition of [3H]colchicine binding
to tubulin was determined in reaction mixtures (100 μL) containing
1.0 μM tubulin, 5.0 μM [3H]colchicine (PerkinElmer), 5% (v/v)
DMSO, potential inhibitors at the indicated concentrations, and
components demonstrated to stabilize the colchicine binding activity
of tubulin64 (1.0 M monosodium glutamate [adjusted to pH 6.6 with
HCl in a 2.0 M stock solution], 0.5 mg/mL bovine serum albumin,
0.1 M glucose-1-phosphate, 1.0 mM MgCl2, and 1.0 mM GTP). Only
compounds showing significant activity as inhibitors of tubulin
polymerization (defined as having IC50 values below 5 μM) were
evaluated for inhibitory effects on colchicine binding. Incubation was
for 10 min at 37 °C (at this time point the binding reaction in the
control is about 50% complete). Reactions were stopped by adding
2.0 mL of 0 °C water and chilling the samples to 0 °C. Each sample
was poured onto a stack of two DEAE-cellulose filters, followed
immediately by 6 mL of 0 °C water. The water was aspirated under a
weak vacuum, and the filters were washed three times with 2 mL of
water. Each set of two filters was placed into a scintillation vial
containing 5 mL of Biosafe II scintillation cocktail. Samples were
counted at least 18 h later in a Beckman scintillation counter. Samples
with potential inhibitors were compared to controls with no inhibitor
to determine percent inhibition. All samples were corrected for
colchicine that bound to the filters in the absence of tubulin.
1
the free alcohol was no longer observed in the purified product: H
NMR (500 MHz, CDCl3) δ 7.72 (1H, d, J = 4.2 Hz, ArH), 7.01 (1H,
dd, J = 2.0 Hz, 8.4 Hz, ArH), 6.83 (1H, d, J = 8.4 Hz, ArH), 6.77 (1H,
d, J = 4.2 Hz, ArH), 6.72 (1H, d, J = 2.0 Hz, ArH), 6.43 (4H, m, ArH
and CH), 3.85 (3H, s, OCH3), 3.80 (3H, s, OCH3), 3.71 (6H, s,
OCH3), 1.59 (6H, s, CH3); 13C NMR (125 MHz, CDCl3) δ 161.0,
152.9, 152.4, 150.3, 142.8, 136.8, 132.9, 129.5, 129.2, 129.1, 128.3,
125.8, 124.0, 122.2, 111.8, 105.7, 79.9, 60.7, 55.8, 55.6, 28.6.
Biological Evaluation. Cell Culture. Non-small-cell lung
carcinoma A549 wt (ATCC) cells were grown and maintained in
MEM-alpha medium containing 10% fetal bovine serum (FBS), 17
mM D-glucose (Sigma-Aldrich), and 1% gentamycin sulfate (Teknova,
Hollister, CA, USA). These cells were maintained in a log growth
phase in a humidified 37 °C incubator under 95% air plus 5% CO2.
Differential Cytotoxicity. The bioreductive assay used was
modified from Jaffar et al.55 For the anoxic arm of the assay, A549
cells were introduced as a small sample into an anaerobic chamber
(Coy Chamber) and then resuspended to the appropriate volume
using medium that had been preconditioned for a minimum of 48 h in
the anaerobic chamber. Cells were plated at 6000 cells/well in a 100
μL volume into plastic 96-well plates (Corning Costar) that had been
degassed in the anaerobic chamber for a minimum of 48 h, with a
maximum limit of 10 ppm oxygen. Cells were allowed to attach for 2
h. Serial dilutions were made from 10 mg/mL DMSO stock solutions
NADPH-Cytochrome P450 Oxidoreductase Cleavage Assay.64,65
Rat NADPH-cytochrome P450 oxidoreductase supersomes and
protocatechuate 3,4-dioxygenase (PCD) were purchased from
Corning and Sigma-Aldrich, respectively, and their enzymatic
activities were determined in terms of enzyme units (U) with their
corresponding substrates (Sigma-Aldrich), cytochrome c, and
protocatechuic acid (PCA, 3,4-dihydroxybenzoic acid), respectively.
All bioreductive prodrugs were dissolved in DMSO as 10 mM stock
solutions. Aliquots of the compound stock solution were added to 200
mM pH 7.4 potassium phosphate buffer containing 0.1% Triton X-
100 (to facilitate BAPC solubility) and 400 μM freshly dissolved
protocatechuic acid. The components were fully mixed in a
microvessel capped with a rubber septum stopper and subjected to
three cycles of evacuation and flushing with N2 using a manifold,
followed by sparging with N2 for an additional 20 min. PCD (0.08
units) was added to remove remaining traces of O2 by PCA/PCD.
POR stock (0.006 units) and NADPH (0.8 mM final concentration)
were introduced sequentially. The anaerobic reaction mixture was
incubated for designated times at 37 °C, cooled on ice, and treated
with a 2× volume of acetonitrile. After centrifugation and syringe
filtration, the samples were analyzed by HPLC using a gradient of
water/acetonitrile for elution. Solutions without POR were used as
O
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controls. Standard curves for CA4, CA1, and each BAPC were used
for quantitation.
Authors
Blake A. Winn − Department of Chemistry and Biochemistry,
Baylor University, Waco, Texas 76798-7348, United States
Laxman Devkota − Department of Chemistry and Biochemistry,
Baylor University, Waco, Texas 76798-7348, United States
Bunnarack Kuch − Department of Chemistry and Biochemistry,
Baylor University, Waco, Texas 76798-7348, United States
Matthew T. MacDonough − Department of Chemistry and
Biochemistry, Baylor University, Waco, Texas 76798-7348,
United States
Tracy E. Strecker − Department of Chemistry and Biochemistry,
Baylor University, Waco, Texas 76798-7348, United States
Yifan Wang − Department of Chemistry and Biochemistry,
Baylor University, Waco, Texas 76798-7348, United States
Zhe Shi − Department of Chemistry and Biochemistry, Baylor
University, Waco, Texas 76798-7348, United States
Jeni L. Gerberich − Predictive Imaging Research Laboratory,
Department of Radiology, The University of Texas Southwestern
Medical Center, Dallas, Texas 75390-9058, United States
Deboprosad Mondal − Department of Chemistry and
Biochemistry, Baylor University, Waco, Texas 76798-7348,
United States
Alejandro J. Ramirez − Mass Spectrometry Center, Baylor
University, Waco, Texas 76798-7046, United States
Ernest Hamel − Screening Technologies Branch, Developmental
Therapeutics Program, Division of Cancer Treatment and
Diagnosis, National Cancer Institute, Frederick National
Laboratory for Cancer Research, National Institutes of Health,
Frederick, Maryland 21702, United States
David J. Chaplin − Department of Chemistry and Biochemistry,
Baylor University, Waco, Texas 76798-7348, United States;
Fast Biopharma Ltd., Aston Rowant OX49 5SW, United
Kingdom
In Vivo Tumor Model.66 Murine 4T1-luc breast cancer cells that
stably express firefly luciferase reporter66 were grown in RPMI1640
medium (Hyclone Laboratories, Logan, UT, USA) with 10% FBS
(Sigma) under 5% CO2 at 37 °C. Induction of tumors was carried out
by injecting 50 μL containing 106 cells in PBS mixed with 30%
Matrigel (BD Biosciences, San Jose, CA, USA) into the left upper
ventral mammary fat pads of anesthetized adult female BALB/CJ
mice (18−24.4 g, age 56 days, UTSW breeding colony). Mice were
housed as a group of 5 in a shoe box cage with nestlet and free access
to water and chow in a conventional vivarium with a 12 h light/dark
cycle (6 AM/6 PM). Tumors were allowed to grow to a size of 10−12
mm in diameter, determined by calipers (carbon fiber composite
digital caliper, Fisher Scientific), over 11 days before BLI.
In Vivo Bioluminescence Imaging. BLI was carried out as
described previously.67,68 Briefly, anesthetized, tumor-bearing mice
(O2, 2% isoflurane, Henry Schein Inc., Melville, NY, USA) were
injected subcutaneously in the foreback neck region with 80 μL of a
solution of luciferase substrate, D-luciferin (sodium salt, 120 mg/kg, in
saline, Gold Biotechnology, St. Louis, MO, USA). Mice were
maintained under anesthesia (2% isoflurane in oxygen, 1 dm3/min)
while baseline BLI was performed using a Caliper Xenogen IVIS
Spectrum (PerkinElmer, Alameda, CA, USA). A series of BLI images
was collected using the following settings: exposure time 0.5 to 2 s
depending on signal intensity, f-stop = 2, field of view = D, binning =
4 (medium). Light intensity−time curves obtained from these images
were analyzed using Living Image software, and light emission was
compared based on area under the light emission curve.69 Mice were
injected intraperitoneally with either 120 μL of DPS vehicle, CA4P
(120 mg/kg in saline), or BAPC 45 (180 mg/kg in DPS vehicle)
immediately after baseline BLI was obtained. BLI was repeated, with
new luciferin injections, 4, 24, and 48 h later. Surviving mice were
sacrificed after 96 h by cervical dislocation under isoflurane
anesthesia, and tumors harvested for routine H&E histology.
ASSOCIATED CONTENT
■
Peter Davis − Fast Biopharma Ltd., Aston Rowant OX49 5SW,
United Kingdom
Ralph P. Mason − Predictive Imaging Research Laboratory,
Department of Radiology, The University of Texas Southwestern
Medical Center, Dallas, Texas 75390-9058, United States
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jnatprod.9b00773.
1H NMR and 13C NMR data for compounds 2−7, 10−
13, 15−17, 19−31, 33−41, 43−45; HRMS and HPLC
data for compounds 21−26, 29, 35−41, 43−45; X-ray
crystallography data for compound 45; details regarding
the synthesis of bioreductive triggers 16, 17, 19;
NADPH-cytochrome P450 oxidoreductase cleavage
assay HPLC traces; NOE analysis for compounds 37,
38, 41; details regarding a preliminary PK study;
cytotoxicity data; and additional histology associated
with BAPC 45. (PDF)
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jnatprod.9b00773
Author Contributions
⊥B. A. Winn and L. Devkota contributed equally to the studies
presented herein.
Notes
The authors declare the following competing financial
interest(s): One of the authors (KGP) previously served as a
paid consultant with Mateon Therapeutics, Inc., and another
author (DJC) served as a former Chief Scientific Officer
(CSO) of Mateon Therapeutics, Inc.
X-ray data (CIF)
AUTHOR INFORMATION
■
ACKNOWLEDGMENTS
Corresponding Authors
■
Mary Lynn Trawick − Department of Chemistry and
Biochemistry, Baylor University, Waco, Texas 76798-7348,
United States; Phone: (254) 710-6857;
Email: Mary_Lynn_Trawick@baylor.edu; Fax: (254) 710-
4272
Kevin G. Pinney − Department of Chemistry and Biochemistry,
Baylor University, Waco, Texas 76798-7348, United States;
orcid.org/0000-0003-1262-8631; Phone: (254) 710-
4117; Email: Kevin_Pinney@baylor.edu; Fax: (254) 710-
4272
Dedicated to Professor George R. Pettit on the occasion of his
most recent birthday and in celebration of his lifetime scientific
accomplishments that continue to define and advance the
fields of natural products chemistry, medicinal chemistry, and
synthetic organic chemistry. The authors are grateful to the
Cancer Prevention and Research Institute of Texas (CPRIT,
Grant No. RP140399 to K.G.P., M.L.T., and R.P.M.), the
National Cancer Institute of the National Institutes of Health
(Grant No. 5R01CA140674 to K.G.P., M.L.T., and R.P.M.),
Mateon Therapeutics, Inc. (grant to K.G.P. and M.L.T.), and
P
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the Undergraduate Research and Scholarly Activity Small
Grant Program, the University Research Committee (URC),
and the Vice Provost for Research at Baylor University (grants
to M.L.T.) for their financial support of this project. BLI was
facilitated by the Southwestern Small Animal Imaging
Resource supported in part by the NIH Comprehensive
Cancer Center Grant P30 CA142543 and a Shared
Instrumentation Grant 1S10 RR024757. The authors also
thank Dr. Michelle Nemec (Director) for the use of the shared
Molecular Biosciences Center at Baylor University, the Mass
Spectrometry Core Facility, Baylor University, and Dr. Kevin
Klausmeyer (X-ray analysis, Baylor University). The authors
are grateful to Mr. David Ross IV (Baylor University), Ms.
Taylor Deushane (Baylor University), Mr. Benji Gomez
(Baylor University), and Ms. Evelyn Le (Baylor University)
for their contributions to the synthesis of advanced
intermediates. We appreciate the technical support of Alex
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Histology was performed by Dr. John Shelton in the Histo
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Brain Microscopy Facility (WBMF) in the UT Southwestern
Department of Neurology and Neurotherapeutics for use of
high-resolution scanning equipment used to obtain histological
images of the 4T1-luc tumors. The WBMF is supported by the
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supported in part by the Developmental Therapeutics Program
in the Division of Cancer Treatment and Diagnosis of the
National Cancer Institute, which includes federal funds under
Contract No. HHSN261200800001E. The content of this
publication does not necessarily reflect the views or policies of
the Department of Health and Human Services, nor does
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https://dx.doi.org/10.1021/acs.jnatprod.9b00773
J. Nat. Prod. XXXX, XXX, XXX−XXX