Synthesis of a 2-aryl-3-aroyl indole salt (OXi8007) resembling combretastatin A-4 with application as a vascular disrupting agent

Article abstract of DOI:10.1021/np400374w

The natural products colchicine and combretastatin A-4 are potent inhibitors of tubulin assembly, and they have inspired the design and synthesis of a large number of small-molecule, potential anticancer agents. The indole-based molecular scaffold is prom

Full text of DOI:10.1021/np400374w

Article
pubs.acs.org/jnp
Synthesis of a 2‑Aryl-3-aroyl Indole Salt (OXi8007) Resembling
Combretastatin A‑4 with Application as a Vascular Disrupting Agent
Mallinath B. Hadimani,† Matthew T. MacDonough,† Anjan Ghatak,†,# Tracy E. Strecker,†
Ramona Lopez,‡ Madhavi Sriram,† Benson L. Nguyen,† John J. Hall,† Raymond J. Kessler,†
Anupama R. Shirali,† Li Liu,‡ Charles M. Garner,† George R. Pettit,§ Ernest Hamel,⊥ David J. Chaplin,∥
Ralph P. Mason,‡ Mary Lynn Trawick,† and Kevin G. Pinney*,†
†Department of Chemistry and Biochemistry, Baylor University, One Bear Place #97348, Waco, Texas 76798-7348, United States
‡Department of Radiology, The University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas
75390-9058, United States
§Department of Chemistry and Biochemistry, Arizona State University, Tempe, Arizona 85287-1604, United States
⊥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
∥Oxigene Inc., 701 Gateway Boulevard, Suite 210, South San Francisco, California 94080, United States
S
* Supporting Information
ABSTRACT: The natural products colchicine and combre-
tastatin A-4 are potent inhibitors of tubulin assembly, and they
have inspired the design and synthesis of a large number of
small-molecule, potential anticancer agents. The indole-based
molecular scaffold is prominent among these SAR modifica-
tions, leading to a rapidly increasing number of agents. The
water-soluble phosphate prodrug 33 (OXi8007) of 2-aryl-3-
aroylindole-based phenol 8 (OXi8006) was prepared by
chemical synthesis and found to be strongly cytotoxic against
selected human cancer cell lines (GI50 = 36 nM against DU-145 cells, for example). The free phenol, 8 (OXi8006), was a strong
inhibitor (IC50 = 1.1 μM) of tubulin assembly. The corresponding phosphate prodrug 33 (OXi8007) also demonstrated
pronounced interference with tumor vasculature in a preliminary in vivo study utilizing a SCID mouse model bearing an
orthotopic PC-3 (prostate) tumor as imaged by color Doppler ultrasound. The combination of these results provides evidence
that the indole-based phosphate prodrug 33 (OXi8007) functions as a vascular disrupting agent that may prove useful for the
treatment of cancer.
he vast majority of natural products and synthetic
compounds that bind to the tubulin−microtubule protein
The indole structure is prevalent as a core molecular
component in a variety of inhibitors of tubulin assembly.8 As
an early example, the vinca alkaloid natural products vinblastine
and vincristine, originally isolated from the madagascar
periwinkle plant Catharanthus roseus (L.) G.Don (Apocyna-
T
system and subsequently interfere with the dynamic assembly/
disassembly inherent to the α,β-tubulin−microtubule system do
so by many variations of this overall mechanism of action. The
most thoroughly studied are interactions at the colchicine, vinca
alkaloid, and taxoid sites.1,2 A significant number of
antiproliferative, anticancer agents function through this basic
mechanism of action. The combretastatin family of natural
products consists of a variety of cis-stilbenoid compounds
isolated from the South African bush willow tree, Combretum
caffrum Kuntze (Combretaceae).1 Combretastatin A-4 (CA4)
is among the most potent antimitotic agents from this family of
compounds and binds to the colchicine site on tubulin (Figure
1, compound 2).3−5 As with many natural products, the
challenge of water solubility led to the development of a
disodium phosphate prodrug, 3, combretastatin A-4P (CA4P).6
The discovery of CA4 has led to a diverse library of antitubulin
agents designed to mimic the simple stilbenoid structure.1,7
ceae), both incorporate two key indole ring systems.9,10
A
significant number of compounds that bind to the colchicine
site and inhibit microtubule formation are also indole-based
(see Figure 2 for representative molecules). To the best of our
knowledge, the first examples of such colchicine site interactive,
indole-based compounds were reported by von Angerer et al.11
(Figure 2, compound 6) and separately by Pinney et al.12,13
(Figure 2, compounds 7, 8) in the middle to late 1990s. Our
(Pinney and co-workers) 2-aryl-3-aroylindole analogues12−14
were originally inspired, in part, by the combretastatin
Received: May 9, 2013
© XXXX American Chemical Society and
American Society of Pharmacognosy
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Figure 1. Representative small-molecule inhibitors of tubulin assembly.
Figure 2. Compilation of indole-based inhibitors of tubulin assembly (6, von Angerer, 1998;11 7 and 8, Pinney, 2001;12 9, Mahboobi and Beckers,
2001;19 10, Silvestri, 2004;20 11, Lee and Hsieh, 2004;21 12, Chang, 2007;22 13, Cachet, 2008;23 14 and 15, Chang, 2008;24 16, Romagnoli, 2008;25
17, Silvestri, 2011;26 18, Liou, 201127).
collection of natural products (pioneered by George R.
Pettit)1,15 and the nonsteroidal, selective estrogen receptor
modulator work of Eli Lilly, Inc., featuring benzo[b]thiophene
templates.16 A judicious combination of key structural features
inherent to both of these molecules led to the preparation of
benzo[b]thiophene-based analogues (Figure 1, compounds 4
and 5) that proved to be potent inhibitors of tubulin assembly
preliminary color Doppler ultrasound experiment with a PC-3
tumor in a SCID mouse model is presented, demonstrating a
time-progressive disruption of tumor blood flow, thus aiding in
the establishment of phosphate prodrug 33 (OXi8007) as
among the first indole-based, vascular disrupting agents
(VDAs). A structurally similar 3-aroylindole (Figure 2,
compound 11, also referred to as BPR0L075)21,28,29 was
recently described by Mason et al. as a VDA.30 In addition, N-
methyl-5,6,7-trimethoxyindole VDAs have been recently
reported by Hu et al.31
A previous study by Dalal and Burchill in a TC-32 tumor
model found minimal necrosis and vessel occlusion with indole
salt 33 (OXi8007) and CA4P as compared with OXi4503.32
Compounds delineated as VDAs function by disrupting existing
tumor vasculature, ultimately leading to oxygen and nutrient
deprivation to the tumor. VDAs are a separate class of
anticancer agents that are mechanistically distinct from the
through a binding interaction at the colchicine site.17,18
A
logical course of structure−activity relationship (SAR)
considerations led to the idea of incorporating a nitrogen
atom in the form of an indole-based analogue.
Herein, we report the synthesis of this indole-based
compound, 8 (OXi8006), along with a water-soluble phosphate
salt, 33 (OXi8007), and the corresponding N-methyl
derivatives (30 and 34). Cytotoxicity of these compounds
against selected human cancer cell lines and their ability to
inhibit tubulin polymerization are described. In addition, a
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Scheme 1. Synthesis of Indole Analogues 8 and 30
angiogenesis inhibiting agents.33,34 VDAs cause morphology
changes (rounding up) of the endothelial cells lining the
microvessels feeding tumors, leading to hypoxia and ultimately
to tumor necrosis.35 No VDA has been approved by the FDA
to date, although several are currently progressing in clinical
trials. Our original patent publications inspired Flynn and co-
workers to apply their elegant heteroatom ring formation
synthetic strategy toward the synthesis of indole 8
(OXi8006).36 Their important contributions in this area
ultimately led to their design and synthesis of BNC105, a
benzofuran-based VDA that is currently undergoing clinical
trials.37,38
RESULTS AND DISCUSSION
■
The synthesis of indole analogues 8 and 30 involved substituted
bromoacetophenone 24 as a key intermediate, which was
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Scheme 2. Preparation of Indole-Based Disodium Phosphate Prodrug Salts 33 and 34
prepared from commercially available isovanillin 19 (Scheme
1). After protection of the phenol as its corresponding silyl
ether 20, addition of methyllithium afforded the secondary
alcohol 21, which upon oxidation with pyridinium chlorochro-
mate furnished the expected acetophenone derivative 22.
Treatment with LDA followed by trimethylsilyl chloride
resulted in silyl enol ether 23, which was converted to its
corresponding bromide 24. The 2-aryl-substituted indole
derivative 25 was prepared following the Bischler−Mohlau
method, by condensation of the substituted bromoacetophe-
none 24 with 3-methoxyaniline in the high boiling solvent N,N-
dimethylaniline.39 Subsequent reaction of indole 25 with 3,4,5-
trimethoxybenzoyl chloride yielded the 2-aryl-3-trimethoxyben-
zoyl-substituted indole derivative 27 through a Friedel−Crafts
benzoylation reaction. Desilylation of compound 27 with
tetrabutylammonium fluoride afforded the desired parent
indole-based phenolic ligand 8 (OXi8006). Preparation of the
corresponding N-methyl indole derivative 30 followed a similar
synthetic pathway (Scheme 1) except that the TBS protecting
group on intermediate 25 was replaced with an isopropyl group
(intermediate 26b) prior to the benzoylation reaction. In
general, the benzoylation reaction proceeded with higher yields
with the isopropyl protecting group rather than with the TBS
group. However, the isopropyl group was not used throughout
the synthetic sequence since other reactions tended to have
higher yields with the TBS group in place, and product
purification following deprotection of the isopropyl group was
more involved, hence the preference for the TBS group.
Reaction of either phenol (8 or 30) with in situ-generated
dibenzyl chlorophosphite resulted in the expected indole-based
dibenzyl phosphate ester derivative (31 or 32). Catalytic
hydrogenolysis of the benzylic carbon−oxygen bonds with
hydrogen gas and palladium on activated carbon (10%),
followed by an acid−base reaction between the resulting
phosphoric acid and sodium methoxide in methanol, resulted in
the desired disodium phosphate indole prodrug 33 (OXi8007)
or the N-methyl derivative 34.6 Initial debenzylation attempts
involved reaction of the dibenzyl ester with in situ-generated
iodotrimethylsilane, from chlorotrimethylsilane and sodium
iodide. The hydrogenolysis reaction offered the advantages of
ease of workup and isolation of the product and better yields
compared with the iodotrimethylsilane reaction.6
The two indole-free phenolic analogues 8 (OXi8006) and 30
and their corresponding water-soluble prodrug salts 33
(OXi8007) and 34 were evaluated for their ability to inhibit
tubulin polymerization (in a cell-free, pure protein assay).
Indole 8 was found to be a potent inhibitor of tubulin assembly
(IC50 = 1.1 μM) comparable with CA4 (Table 1). While CA4P
Table 1. Inhibition of Tubulin Polymerization and
Colchicine Binding
inhibition of colchicine binding (%)
inhibition of
tubulin
polymerization
compound
CA4
IC50 (μM)
1 μM
5 μM
50 μM
a
0.96 0.07
1.1 0.04
4.2 0.1
>20
91
1
40 0.2
nd
99 0.8
75 0.2
nd
8 (OXi8006)
nd
nd
34
29
33 (OXi8007)
26
nd
nd
4
30
34
nd
2
2
>20
nd
a
nd = not determined in this study.
(containing the bulky phosphate prodrug moiety) is inactive
(IC50 >40 μM) as an inhibitor of tubulin assembly,40 the
indole-based phosphate prodrug 33 (OXi8007) was found to
be strongly inhibitory (IC50 = 4.2 μM). The addition of the N-
methyl group resulted in a loss of the ability to inhibit tubulin
polymerization (IC50 >20 μM) for both the free phenol 30 and
its corresponding phosphate prodrug salt 34. In a binding assay
against tritium-labeled colchicine, indole 8 (OXi8006)
demonstrated modest inhibition at a concentration of 1 μM
(40%), which increased at 5 μM (75%). CA4, as a control,
strongly inhibited colchicine binding even at 1 μM (91%). The
phosphate prodrug salt, 33 (OXi8007), was only minimally
inhibitory (26%) at a concentration of 5 μM.
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Both of the N-methyl analogues 30 and 34 showed only
modest inhibition (34% and 29%, respectively) at a high
concentration (50 μM). Two possible conclusions can be
inferred from these data. First, it seems likely that the
nonmethylated, free phenol 8 (OXi8006) interacts with the
colchicine binding site in an orientation that places the free
phenolic moiety outside of the binding pocket. This seems
plausible since the corresponding prodrug 33 (OXi8007),
containing the bulky phosphate salt moiety, also demonstrated
strong inhibition of tubulin assembly, which is typically not the
case for small-molecule phosphate prodrugs of active tubulin
binding agents (such as CA4P). Second, incorporation of a
methyl group on the indole-nitrogen atom severely limited the
ability of these analogues to interact with tubulin.
As a preliminary test of vascular disrupting activity in vivo,
color Doppler ultrasound was applied to a PC-3 human tumor
xenograft growing in a SCID mouse. Images were acquired over
a period of 80 min starting immediately after administration of
indole prodrug 33 (OXi8007). The tumor was clearly visible
(Figure 3) with extensive bidirectional blood flow displayed in
red and blue. Blood flow was also observed in vessels outside
the tumor. Blood flow decreased progressively and by 80 min
had essentially ceased in the core of the tumor. The
extratumoral vessels remained undisrupted throughout. The
differential activity in the tumor and animal tissue provides
preliminary evidence of selective vascular disruption in vivo.
Ultrasound has emerged as a valuable imaging technique for
VDAs42 and has recently been validated as a corollary imaging
strategy to bioluminescence imaging33 for the assessment of
VDAs.43
In summary, an indole-based, water-soluble phosphate
prodrug salt, 33 (OXi8007), was prepared by chemical
synthesis and found to be strongly cytotoxic against a selection
of human cancer cell lines. Its parent phenolic analogue 8
(OXi8006) was a potent inhibitor of tubulin assembly. A
preliminary Doppler ultrasound experiment demonstrated that
indole prodrug 33 (OXi8007) has VDA characteristics that are
potentially suitable for the treatment of cancer. Additional
studies are under way with indole prodrug 33 (OXi8007) and
related compounds to further understand the biological
mechanism of action and to investigate structure−activity
relationships within the 2-aryl-3-aroylindole molecular frame-
work.
The phenolic indole 8 (OXi8006) and its corresponding
prodrug salt 33 (OXi8007) demonstrated consistently strong
cytotoxicity (GI50 values ranging from 3.5 to 38 nM) against
the NCI-H460 (lung), DU-145 (prostate), and SK-OV-3
(ovarian) human cancer cell lines (Table 2). Cytotoxicity was
Table 2. Cytotoxicity against Human Cancer Cell Lines NCI-
H460, DU-145, and SK-OV-3
a
GI50 (μM) SRB assay
compound
CA4
NCI-H460
DU-145
SK-OV-3
0.00042
b
b
0.0028
0.00054
c
c
d
CA4P
0.066
0.016
nd
8 (OXi8006)
0.0379
0.0311
4.14
0.0356
0.0297
2.68
0.00345
0.0223
2.05
33 (OXi8007)
30
34
3.56
4.67
8.64
EXPERIMENTAL SECTION
■
a
b
Average of n ≥ 3 independent determinations. For additional data,
General Experimental Methods. Dichloromethane, acetonitrile,
dimethylformamide (DMF), methanol, ethanol, and tetrahydrofuran
(THF) were used in their anhydrous forms, as obtained from the
chemical suppliers. Reactions were performed under an inert
atmosphere using nitrogen gas, unless specified. Thin-layer chroma-
c
d
see ref 40. See ref 41. nd = not determined in this study.
greatly diminished (IC50 approximately 3000 nM) with the
closely related N-methyl phenol 30 and prodrug 34. These data
track with the inhibition of tubulin assembly data. Thus, it is
instructive to briefly address the question (and subsequent
confusion) that occasionally arises in regard to why compounds
that are in the nM concentration range in terms of GI50 values
for cytotoxicity against selected human cancer cell lines rarely
show lower than micromolar activity in terms of their measured
IC50 value in the inhibition of tubulin assembly assay. This is
commonly noted for many compounds including CA4, for
example, as well as indole 8 (OXi8006).
Although the reason is not known with certainty, several
factors may contribute. For example, the stoichiometry that
exists between the concentration of compound and the
concentration of tubulin needed to achieve an IC50 value in
the micromolar range in the pure protein (cell-free) assay may
be drastically different from the requisite stoichiometry that
exists when inhibiting tubulin in a cell-based assay, leading to a
measured GI50 value for cytotoxicity in the nanomolar range. It
should be emphasized that the pure protein assay incorporates
no cellular components other than tubulin. Importantly, tubulin
disassembly (in cells) may release factors that are involved in
intramolecular signal transduction, leading to a major
amplification. In addition, the amount of tubulin needed in
the pure protein assay in order to accurately measure inhibition
of microtubule formation (by monitoring absorbance at 350
nm spectrophotometrically) is large enough that the practical
lower limit for this assay is around 0.5 to 1 μM (IC50 value) for
even the most active inhibitors of tubulin polymerization.
tography (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 flash
purification system using silica gel (200−400 mesh, 60 Å) or RP-18
prepacked columns or manually in glass columns.
Intermediates and products synthesized were characterized on the
1
basis of their H NMR (500 or 300 MHz), 13C NMR (125 or 75
MHz), and 31P NMR (200 or 120 MHz) spectroscopic data using a
Varian VNMRS 500 MHz or a Bruker DPX 300 MHz instrument.
Spectra were recorded in CDCl3, D2O, or CD3OD. All chemical shifts
are expressed in ppm (δ), coupling constants (J) are presented in Hz,
and peak patterns are reported as broad (br), singlet (s), doublet (d),
triplet (t), quartet (q), septet (sept), double doublet (dd), and
multiplet (m).
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 (4.6 mm × 150 mm, 5
μm), and a Zorbax reliance cartridge guard-column; method A: solvent
A, acetonitrile, solvent B, 0.1% TFA in H2O; or method B: solvent A,
acetonitrile, solvent B, H2O; gradient, 10% A/90% B to 100% A/0% B
over 0 to 40 min; post-time 10 min; flow rate 1.0 mL/min; injection
volume 20 μL; monitored at wavelengths of 210, 254, 230, 280, and
360 nm. Mass spectrometry was carried out under positive ESI
(electrospray ionization) using a Thermo Scientific LTQ Orbitrap
Discovery instrument.
3-(tert-Butyldimethylsilyloxy)-4-methoxybenzaldehyde (20) (ref
44). To a solution of 3-hydroxy-4-methoxybenzaldehyde 19 (25.00 g,
164.4 mmol) dissolved in CH2Cl2 (250 mL) at 0 °C was added
triethylamine (Et3N) (25.2 mL, 180.9 mmol) followed by N,N-
dimethylaminopyridine (DMAP) (2.01 g, 16.4 mmol). The reaction
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Figure 3. Dynamics of vascular disruption caused by 33 (OXi8007) in human prostate tumor PC-3 xenograft visualized by Doppler ultrasound.
Sequential transaxial images were acquired over a period of 80 min following injection of indole prodrug 33 (OXi8007) (350 mg/kg ip) in a SCID
mouse. (A) Immediately postinjection, (B) 20 min, (C) 40 min, (D) 60 min, (E) 80 min. Each image was acquired in color Doppler mode with
extensive vasculature initially observed both within the tumor (outlined in yellow) and in surrounding tissue. Within 20 min, the tumor vascular
perfusion had decreased with further progressive decline, so that by 80 min no vascular flow was observed within the tumor. Flow was, however, still
apparent in surrounding normal tissues (white arrow). White scale bar: 2 mm. Heat scale bar representing flow in the range 64.2 mm/s.
mixture was stirred for 10 min, and tert-butyldimethylsilyl chloride
(27.26 g, 180.9 mmol) was then added gradually. The solution was
allowed to warm to room temperature over 12 h. Upon completion of
the reaction, the reaction mixture was quenched with water (150 mL)
and extracted with CH2Cl2. The extracted layers were combined, dried
over Na2SO4, and concentrated under reduced pressure. The TBS
benzaldehyde product 20 [47.09 g, 176.9 mmol, Rf = 0.50 (hexanes−
EtOAc, 70:30)] was isolated quantitatively as a yellow oil and was
allowed to reach room temperature over 12 h. Upon completion of the
reaction, the reaction mixture was slowly quenched with water (200
mL) and extracted with EtOAc. The organic extract was dried over
Na2SO4 and concentrated under reduced pressure, resulting in
secondary alcohol 21 [48.97 g, 173.4 mmol, 98%, Rf = 0.40
(hexanes−EtOAc, 70:30)] as a yellow oil, which was taken to the
next step without further purification. 1H NMR (CDCl3, 500 MHz) δ
6.88 (2H, m, ArH), 6.83 (1H, d, J = 8.1 Hz, ArH), 4.81 (1H, q, J = 6.3
Hz, CH), 3.79 (3H, s, OCH3), 1.82 (1H, s, OH), 1.45 (3H, d, J = 6.3
Hz, CH3), 0.99 (9H, s, (CH3)3), 0.15 (6H, s, Si(CH3)2); 13C NMR
(CDCl3, 125 MHz) δ 149.7, 144.5, 138.9, 118.4, 118.0, 111.7, 69.1,
55.1, 25.5, 24.9, 18.2, −4.8.
3-(tert-Butyldimethylsilyloxy)-4-methoxyacetophenone (22) (refs
17, 44). To a solution of crude alcohol 21 (48.97 g, 173.4 mmol) and
Celite (35 g) in CH2Cl2 (500 mL) at 0 °C was added pyridinium
chlorochromate (41.12 g, 190.7 mmol) in small increments (1−3 g),
allowing 10 min of stirring between each addition. The reaction
mixture was allowed to warm to room temperature over 12 h. Upon
1
taken to the next step without further purification. H NMR (CDCl3,
500 MHz) δ 9.80 (1H, s, CHO), 7.45 (1H, dd, J = 8.5 Hz, J = 2.0 Hz,
ArH), 7.35 (1H, d, J = 2.0 Hz, ArH), 6.93 (1H, d, J = 8.5 Hz, ArH),
3.87 (3H, s, OCH3), 0.99 (9H, s, C(CH3)3), 0.16 (6H, s, Si(CH3)2);
13C NMR (CDCl3, 125 MHz) δ 190.2, 156.2, 145.2, 130.0, 126.0,
119.4, 110.9, 55.1, 25.3, 18.0, −5.0.
3-(tert-Butyldimethylsilyloxy)-1-(1′-hydroxyethyl)-4-methoxyben-
zene (21) (refs 12, 44). Crude TBS benzaldehyde 20 (47.09 g, 176.9
mmol) dissolved in tetrahydrofuran (THF, 500 mL) at 0 °C was
treated with CH3Li (144 mL, 230 mmol) dropwise. The solution was
F
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completion of the reaction, the reaction mixture was filtered through a
50:50 plug of silica gel−Celite, and the plug was rinsed well with
CH2Cl2. The filtrate was concentrated under reduced pressure,
providing the desired acetophenone derivative 22 [37.94 g, 135.3
mmol, 78%, Rf = 0.50 (hexanes−EtOAc, 70:30)] as a pale yellow solid.
1H NMR (CDCl3, 500 MHz) δ 7.57 (1H, dd, J = 8.5 Hz, 2.0 Hz,
ArH), 7.46 (1H, d, J = 2.0 Hz, ArH), 6.86 (1H, d, J = 8.5 Hz, ArH),
3.86 (3H, s, OCH3), 2.52 (3H, s, CH3), 1.00 (9H, s, C(CH3)3), 0.16
(6H, s, Si(CH3)2); 13C NMR (CDCl3, 125 MHz) δ 196.7, 155.3,
144.8, 130.5, 123.5, 120.4, 110.7, 55.4, 26.2, 25.6, 18.4, −4.7.
112.4, 109.9, 98.6, 94.5, 55.6, 55.4, 25.7, 18.5, −4.6; HPLC method B,
tR 21.09 min; (+) HRESIMS m/z found 384.1989 [M + H]+ (calcd for
C22H30NO3Si, 384.1989).
2-(3′-Hydroxy-4′-methoxyphenyl)-6-methoxyindole (26a) (ref
46). To a solution of TBS-protected phenol 25 (0.10 g, 0.26 mmol)
in THF (10 mL) at 0 °C was added tetrabutylammonium fluoride
(TBAF, 0.4 mL, 0.4 mmol, 1 M in THF) dropwise. The reaction
mixture was stirred for 30 min and then allowed to reach room
temperature. The reaction mixture was quenched with water (10 mL)
and extracted with EtOAc (3 × 10 mL). The combined organic layers
were dried over Na2SO4 and concentrated under reduced pressure.
Purification by flash column chromatography using a prepacked 25 g
silica column [solvent A: EtOAc; solvent B: hexanes; gradient: 7% A/
93% B (4 CV), 7% A/93% B → 60% A/40% B (10 CV), 60% A/40%
B (5.2 CV); flow rate: 25 mL/min; monitored at 254 and 280 nm]
afforded the desired free phenol indole 26a [0.72 g, 0.27 mmol, Rf =
0.22 (hexanes−EtOAc, 70:30)] as a yellow powder. 1H NMR (CDCl3,
500 MHz) δ 8.12 (1H, br s, NH), 7.47 (1H, d, J = 8.6 Hz, ArH), 7.20
(1H, d, J = 2.1 Hz, ArH), 7.11 (1H, dd, J = 8.3 Hz, 2.1 Hz, ArH), 6.90
(1H, m, ArH), 6.78 (1H, dd, J = 8.6 Hz, 2.2 Hz, ArH), 6.64 (1H, d, J =
1.6 Hz, ArH), 5.67 (1H, br s, OH), 3.93 (3H, s, OCH3), 3.86 (3H, s,
OCH3); 13C NMR (CDCl3, 125 MHz) δ 156.6, 146.20, 146.15, 137.6,
136.9, 126.5, 123.8, 121.2, 116.8, 111.4, 111.2, 110.1, 99.1, 94.6, 56.2,
55.9; HPLC method B, tR 12.33 min; (+) HRESIMS m/z found
270.1128 [M + H]+ (calcd for C16H16NO3, 270.1125).
1-(3-tert-Butyldimethylsilyloxy)-4-(methoxyphenyl)-1-trimethylsi-
lylethene (23) (ref 17). To a solution of diisopropylamine (16.6 mL,
117 mmol) in THF (300 mL) at 0 °C was added n-butyllithium (47.04
mL, 117.4 mmol) dropwise. The LDA solution was allowed to stir for
15 min, and then a solution of TBS−acetophenone 22 (21.95 g, 78.27
mmol) in THF (100 mL) was added dropwise. The solution was
stirred for 10 min, and trimethylsilyl chloride (14.9 mL, 117.4 mmol)
was also added dropwise. The reaction mixture was allowed to reach
room temperature over 12 h. Upon completion of the reaction, the
solution was diluted with NaHCO3 (10%, 200 mL). The reaction
mixture was extracted with diethyl ether. Next, the extract was dried
over Na2SO4, and the organic phase was concentrated under reduced
pressure to provide crude TMS−enol ether 23 [30.45 g, 86.12 mmol]
as a dark yellow oil, which was taken to the next step without
purification. 1H NMR (CDCl3, 500 MHz) δ 7.18 (1H, dd, J = 8.5 Hz,
2.5 Hz, ArH), 7.12 (1H, d, J = 2.5 Hz, ArH), 6.80 (1H, d, J = 8.5 Hz,
ArH), 4.78 (1H, d, J = 1.5 Hz, CH2), 4.34 (1H, d, J = 1.5 Hz, CH2),
3.81 (3H, s, OCH3), 1.03 (9H, s, C(CH3)3), 0.27 (9H, s, Si(CH3)3),
0.18 (6H, s, Si(CH3)2); 13C NMR (CDCl3, 125 MHz) δ 155.3, 151.1,
144.4, 130.6, 118.8, 118.1, 111.2, 89.5, 55.4, 25.7, 18.4, 0.03, −4.7.
3′-(tert-Butyldimethylsilyloxy)-4′-methoxy-2-bromoacetophe-
none (24) (ref 17). To a solution of crude 23 (30.45 g, 86.12 mmol)
and anhydrous K2CO3 (0.51 g, 3.7 mmol) in CH2Cl2 (250 mL) at 0
°C was added bromine (2.66 mL, 51.7 mmol) dropwise. The solution
was allowed to stir for 30 min, diluted with sodium thiosulfate (10%),
and extracted with CH2Cl2. The extract was dried over Na2SO4 and
concentrated under reduced pressure. Purification by flash chromatog-
raphy using a prepacked 25 g silica column [solvent A: EtOAc; solvent
B: hexanes; gradient: 2% A/98% B (4 CV), 2% A/98% B → 20% A/
80% B (10 CV), 20% A/80% B (1.2 CV); flow rate: 25 mL/min;
monitored at 254 and 280 nm] afforded bromoacetophenone analogue
24 [18.10 g, 50.38 mmol, 58%, Rf = 0.37 (hexanes−EtOAc, 90:10)] as
2-(3′-Isopropoxy-4′-methoxyphenyl)-6-methoxyindole (26b) (ref
45). To a well-stirred solution of free phenol 26a (0.07 g, 0.27 mmol)
and K2CO3 (0.12 g, 0.89 mmol) in DMF (10 mL) at 100 °C was
added 2-bromopropane (0.05 mL, 0.54 mmol) dropwise. The reaction
mixture was stirred for 12 h at 100 °C. The mixture was cooled to
room temperature and extracted with EtOAc (3 × 10 mL). The
combined organic extract was dried over Na2SO4 and concentrated
under reduced pressure to afford the desired isopropyl-protected
indole 26b [0.05 g, 0.27 mmol, 60%, Rf = 0.50 (hexanes−EtOAc,
60:40)] as a tan solid, which was carried to the next step without
1
further purification. H NMR (CDCl3, 500 MHz) δ 8.43 (1H, br s,
NH), 7.47 (1H, d, J = 8.6 Hz, ArH), 7.21 (1H, d, J = 2.1 Hz, ArH),
7.16 (1H, dd, J = 8.3 Hz, 2.1 Hz, ArH), 6.90 (1H, d, J = 8.4 Hz, ArH),
6.86 (1H, d, J = 2.1 Hz, ArH), 6.79 (1H, dd, J = 8.6 Hz, 2.3 Hz, ArH),
6.67 (1H, m, ArH), 4.61 (1H, sept, J = 6.0 Hz, CH), 3.87 (3H, s,
OCH3), 3.83 (3H, s, OCH3), 1.39 (6H, d, J = 6.1 Hz, (CH3)2); 13C
NMR (CDCl3, 125 MHz) δ 156.5, 150.2, 147.7, 137.6, 137.2, 125.9,
123.8, 121.0, 117.9, 113.4, 112.5, 110.0, 98.7, 94.7, 71.9, 56.1, 55.8,
22.3; (+) HRESIMS m/z found 312.1596 [M + H]+ (calcd for
C19H22NO3, 312.1594).
2-(3′-tert-Butyldimethylsiloxy-4′-methoxyphenyl)-3-(3″,4″,5″-tri-
methoxybenzoyl)-6-methoxyindole (27) (ref 13). To a solution of
compound 25 (3.12 g, 8.14 mmol) in o-dichlorobenzene (30 mL) was
added 3,4,5-trimethoxybenzoylchloride (2.82 g, 12.2 mmol). The
reaction mixture was heated to reflux at 170 °C for 12 h. The o-
dichlorobenzene was removed by simple distillation, and the resulting
dark-colored crude oil was subjected to flash chromatography using a
prepacked 100 g silica column [solvent A: EtOAc; solvent B: hexanes;
gradient: 10% A/90% B (4 CV), 10% A/90% B → 80% A/20% B (10
CV), 80% A/20% B (2.8 CV); flow rate: 40 mL/min; monitored at
254 and 280 nm], resulting in TBS-indole analogue 27 [1.60 g, 2.77
mmol, 34%, Rf = 0.38 (hexanes−EtOAc, 60:40)] as a yellow powder.
1H NMR (CDCl3, 500 MHz) δ 8.42 (1H, br s, NH), 7.93 (1H, d, J =
9.5 Hz, ArH), 6.99 (2H, s, ArH) 6.94 (1H, dd, J = 8.0 Hz, 2.0 Hz,
ArH), 6.91 (1H, dd, J = 9.0 Hz, 2.0 Hz, ArH), 6.91 (1H, d, J = 2.0 Hz,
ArH), 6.77 (1H, d, J = 2.0 Hz, ArH), 6.70 (1H, d, J = 8.5 Hz, ArH),
3.87 (3H, s, OCH3), 3.79 (3H, s, OCH3), 3.74 (3H, s, OCH3), 3.69
(6H, s, OCH3), 0.94 (9H, s, C(CH3)3), 0.04 (6H, s, Si(CH3)2); 13C
NMR (CDCl3, 125 MHz) δ 191.9, 157.4, 152.6, 151.6, 145.2, 142.1,
141.3, 136.5, 134.6, 125.2, 123.4, 122.6, 122.3, 121.9, 112.9, 111.8,
111.7, 107.4, 94.6, 60.9, 56.1, 55.9, 55.5, 25.8, 18.5, −4.7.
2-(3′-Isopropoxy-4′-methoxyphenyl)-3-(3″,4″,5″-trimethoxyben-
zoyl)-6-methoxyindole (28) (ref 36). To a well-stirred solution of
compound 26b (1.07 g, 3.45 mmol) in o-dichlorobenzene (20 mL)
was added 3,4,5-trimethoxybenzoyl chloride (1.39 g, 6.04 mmol). The
1
a tan-red solid. H NMR (CDCl3, 500 MHz) δ 7.61 (1H, dd, J = 8.5
Hz, 2.5 Hz, ArH), 7.48 (1H, d, J = 2.5 Hz, ArH), 6.88 (1H, d, J = 8.5
Hz, ArH), 4.37 (2H, s, CH2), 3.88 (3H, s, OCH3), 1.00 (9H, s,
C(CH3)3), 0.17 (6H, s, Si(CH3)2); 13C NMR (CDCl3, 125 MHz) δ
189.8, 156.1, 145.1, 127.1, 124.2, 121.0, 111.0, 55.5, 30.7, 25.6, 18.4,
−4.6; HPLC method B, tR 9.10 min; (+) HRESIMS m/z found
381.0496 [M + Na]+ (calcd for C15H23BrNaO3Si, 381.0492).
2-(3′-tert-Butyldimethylsilyloxy-4′-methoxyphenyl)-6-methoxyin-
dole (25) (ref 45). To a solution of m-anisidine (2.05 mL, 18.4 mmol)
dissolved in N,N-dimethylaniline (20 mL) at 170 °C was added
dropwise bromoacetophenone 24 (2.0 g, 5.6 mmol) in EtOAc (5 mL).
The reaction mixture was stirred at 170 °C for 12 h. Upon completion
of the reaction, the reaction mixture was cooled to room temperature
and extracted with EtOAc (3 × 50 mL). The combined organic extract
was dried over Na2SO4 and concentrated under reduced pressure.
Purification by flash chromatography using a prepacked 25 g silica gel
column [solvent A: EtOAc; solvent B: hexanes; gradient: 12% A/88%
B (4 CV), 12% A/88% B → 100% A/0% B (10 CV), 100% A/0% B
(2.6 CV); flow rate: 25 mL/min; monitored at 254 and 280 nm]
resulted in the desired 2-phenylindole derivative 25 [1.49 g, 3.88
mmol, 69%, Rf = 0.48 (hexanes−EtOAc, 50:50)] as light tan crystals.
1H NMR (CDCl3, 500 MHz) δ 8.11 (1H, br s, NH), 7.47 (1H, d, J =
8.5 Hz, ArH), 7.16 (1H, dd, J = 8.5 Hz, 2.0 Hz, ArH), 7.13 (1H, d, J =
2.5 Hz, ArH), 6.90 (1H, d, J = 8.5 Hz, ArH), 6.89 (1H, d, J = 2.5 Hz,
ArH), 6.79 (1H, dd, J = 8.5 Hz, 2.5 Hz, ArH), 6.64 (1H, dd, J = 2.0
Hz, 1.0 Hz, ArH), 3.86 (3H, s, OCH3), 3.84 (3H, s, OCH3), 1.04 (9H,
s, C(CH3)3), 0.21 (6H, s, Si(CH3)2); 13C NMR (CDCl3, 125 MHz) δ
156.3, 150.5, 145.4, 137.4, 136.9, 125.8, 123.7, 120.9, 118.2, 117.8,
G
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reaction mixture was heated to reflux at 160 °C for 16 h. The excess o-
dichlorobenzene was removed by simple distillation, and the resulting
dark-colored solid was subjected to glass column flash chromatography
(silica gel, hexanes−EtOAc, 50:50), yielding the desired isopropyl-
indole analogue 28 [0.96 g, 1.90 mmol, 55%, Rf = 0.29 (hexanes−
EtOAc, 50:50)] as a yellow solid. 1H NMR (CDCl3, 300 MHz) δ 8.79
(1H, br s, NH), 7.94 (1H, d, J = 9.4 Hz, ArH), 6.99 (1H, dd, J = 8.2
Hz, J = 2.1 Hz, ArH), 6.98 (2H, s, ArH), 6.92 (1H, dd, J = 6.1 Hz, 2.3
Hz, ArH), 6.90 (1H, d, J = 2.3 Hz, ArH), 6.74 (1H, d, J = 2.1 Hz,
ArH), 6.73 (1H, d, J = 8.4 Hz, ArH), 4.16 (1H, m, CH), 3.85 (3H, s,
OCH3), 3.79 (3H, s, OCH3), 3.78 (3H, s, OCH3), 3.66 (6H, s,
OCH3), 1.21 (3H, d, J = 5.6 Hz, CH3), 1.19 (3H, d, J = 5.6 Hz, CH3);
13C NMR (CDCl3, 75 MHz) δ 191.8, 157.3, 152.5, 150.9, 147.1, 142.3,
AlCl3 was added, and the reaction mixture was stirred for an additional
1.5 h. Upon completion of the reaction, the reaction mixture was
quenched with water (15 mL) and extracted with CH2Cl2 (2 × 10
mL). The combined organic extract was dried over Na2SO4 and
concentrated under reduced pressure. Purification by flash column
chromatography (silica gel, hexanes−EtOAc, 60:40) afforded the free
phenolic N-methyl indole analogue 30 [0.286 g, 0.599 mmol, 73%, Rf
= 0.17 (hexanes−EtOAc, 50:50)] as a colorless solid. 1H NMR
(CDCl3, 500 MHz) δ 8.02 (1H, d, J = 8.7 Hz, ArH), 6.96 (1H, dd, J =
8.7 Hz, 2.2 Hz, ArH), 6.86 (1H, d, J = 2.1 Hz, ArH), 6.79 (1H, d, J =
8.0 Hz, ArH), 6.78 (2H, s, ArH), 6.65 (2H, m, ArH), 5.77 (1H, s,
OH), 3.92 (3H, s, CH3), 3.82 (3H, s, OCH3), 3.77 (3H, s, OCH3),
3.74 (6H, s, OCH3), 3.65 (3H, s, OCH3); 13C NMR (CDCl3, 75
MHz) δ 192.2, 157.3, 152.3, 147.0, 145.4, 145.2, 140.2, 138.2, 135.8,
124.0, 123.6, 122.7, 121.9, 117.0, 114.4, 111.7, 110.2, 106.6, 93.7, 60.8,
56.1, 56.0, 55.9, 31.5; HPLC method B, tR 12.38 min; (+) HRESIMS
m/z found 478.1857 [M + H]+ (calcd for C27H28NO7, 478.1860).
2-(3′-Dibenzyl phosphate-4′-methoxyphenyl)-3-(3″,4″,5″-trime-
thoxybenzoyl)-6-methoxyindole (31) (ref 45). To a solution of
compound 8 (1.90 g, 4.09 mmol) in acetonitrile (70 mL) at −25 °C
was added CCl4 (3.50 mL, 35.98 mmol). The solution was stirred for
10 min, and ethyldiisopropylamine (1.50 mL, 8.63 mmol) and DMAP
(0.05 g, 0.41 mmol) were added. After 5 min of stirring, dibenzyl
phosphite (1.36 mL, 6.17 mmol) was added, and the reaction mixture
was stirred for 2 h while allowing the solution to reach room
temperature. Upon completion of the reaction, the reaction was
terminated by adding a solution of KH2PO4 (15 mL, 0.5 M) and
extracted with EtOAc (3 × 50 mL). The combined organic extract was
dried over Na2SO4 and concentrated under reduced pressure.
Purification by flash column chromatography using a prepacked 25 g
silica column [solvent A: EtOAc; solvent B: hexanes; gradient: 12% A/
88% B (4 CV), 12% A/88% B → 100% A/0% B (10 CV), 100% A/0%
B (5.2 CV); flow rate: 25 mL/min; monitored at 254 and 280 nm]
afforded the desired phosphate ester 31 [2.71 g 3.75 mmol, 91%, Rf =
0.57 (hexanes−EtOAc, 50:50)] as a yellow powder. 1H NMR (CDCl3,
500 MHz) δ 9.20 (1H, br s, NH), 7.78 (1H, d, J = 8.5 Hz, ArH), 7.35
(10H, m, ArH), 7.25 (1H, m, ArH), 6.96 (1H, dd, J = 9.0 Hz, 2.5 Hz,
ArH), 6.93 (2H, s, ArH), 6.91 (1H, d, J = 2.0 Hz, ArH), 6.86 (1H, dd,
J = 9.0 Hz, 2.5 Hz, ArH), 6.51 (1H, d, J = 8.5 Hz, ArH) 5.17 (4H, d, J
= 8.0 Hz, CH2), 3.82 (3H, s, OCH3), 3.79 (3H, s, OCH3), 3.65 (6H, s,
OCH3), 3.54 (3H, s, OCH3); 13C NMR (CDCl3, 125 MHz) δ 192.1,
157.3, 152.7, 150.73, 150.69, 141.3, 141.2, 136.7, 135.6 (d, J = 7.3 Hz),
135.0, 128.84, 128.78, 128.1, 127.8, 124.6, 123.1, 122.3, 121.4, 112.8,
112.0, 111.7, 107.4, 94.9, 70.3 (d, J = 6.0 Hz), 60.9, 56.2, 55.8, 55.7;
31P NMR (CDCl3, 200 MHz) δ −6.1; HPLC method B, tR 16.40 min;
141.2, 136.4, 134.5, 124.8, 123.2, 122.4, 121.0, 117.3, 112.6, 111.6,
111.5, 107.2, 94.5, 71.7, 64.4, 60.8, 55.9, 55.6, 25.3, 21.9.
2-(3′-Isopropoxy-4′-methoxyphenyl)-3-(3″,4″,5″-trimethoxyben-
zoyl)-N-methyl-6-methoxyindole (29) (ref 45). To a solution of
compound 28 (0.52 g, 1.03 mmol) in CH2Cl2 (20 mL) at 0 °C was
added sodium hydride (0.04 g, 1.75 mmol) slowly and carefully. The
solution was stirred for 5 min, and methyl iodide (0.13 mL, 2.16
mmol) was added dropwise. The reaction mixture was allowed to
warm to room temperature over 19 h. Upon completion of the
reaction, the solution was diluted with water (15 mL) and extracted
with CH2Cl2 (2 × 15 mL). The combined organic phases were dried
over Na2SO4 and concentrated under reduced pressure. Purification by
column flash column chromatography (silica gel, hexanes−EtOAc,
70:30) afforded methyl indole analogue 29 [0.43 g, 0.82 mmol, 80%,
Rf = 0.18 (hexanes−EtOAc, 60:40)] as a white solid. 1H NMR
(CDCl3, 300 MHz) δ 8.00 (1H, d, J = 8.7 Hz, ArH), 6.97 (1H, dd, J =
8.7 Hz, 2.3 Hz, ArH), 6.88 (1H, dd, J = 7.9 Hz, 1.9 Hz, ArH), 6.87
(1H, d, J = 2.4 Hz, ArH), 6.84 (2H, s, ArH), 6.81 (1H, d, J = 8.3 Hz,
ArH), 6.66 (1H, d, J = 1.9 Hz, ArH), 4.28 (1H, m, CH), 3.93 (3H, s,
CH3), 3.82 (3H, s, OCH3), 3.78 (3H, s, OCH3), 3.73 (6H, s, OCH3),
3.66 (3H, s, OCH3), 1.27 (6H, d, J = 6.1 Hz, (CH3)2); 13C NMR
(CDCl3, 75 MHz) δ 191.8, 157.2, 152.2, 150.6, 146.7, 145.2, 140.5,
138.1, 135.3, 123.5, 123.3, 122.5, 121.8, 118.4, 114.0, 111.5, 111.2,
106.7, 93.6, 71.4, 60.7, 55.90, 55.86, 55.7, 31.4, 21.9; HPLC method B,
tR 15.14 min; (+) HRESIMS m/z found 542.2150 [M + Na]+ (calcd
for C30H33NNaO7, 542.2149).
2-(3′-Hydroxy-4′-methoxyphenyl)-3-(3″,4″,5″-trimethoxybenzo-
yl)-6-methoxyindole (8) (OXi8006) (ref 12). To a well-stirred solution
of compound 27 (4.45 g, 7.70 mmol) in THF (15 mL) at 0 °C was
added TBAF (11.55 mL, 11.55 mmol, 1 M in THF) dropwise. The
reaction mixture was stirred for 30 min while warming to room
temperature. The reaction mixture was quenched with water (10 mL)
and extracted with EtOAc (3 × 10 mL). The combined organic extract
was dried over Na2SO4 and concentrated under reduced pressure.
Purification by flash column 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 → 100% A/0% B (10 CV), 100% A/0%
B (5 CV); flow rate: 40 mL/min; monitored at 254 and 280 nm]
afforded the desired phenolic indole 8 (OXi8006) [2.49 g, 5.37 mmol,
(+) HRESIMS m/z found 746.2126 [M + Na]+ (calcd for
C40H38NNaO10P, 746.2126).
2-(3′-Dibenzyl phosphate-4′-methoxyphenyl)-3-(3″,4″,5″-trime-
thoxybenzoyl)-N-methyl-6-methoxyindole (32) (ref 45). To a well-
stirred solution of compound 30 (0.312 g, 0.653 mmol) dissolved in
acetonitrile (15 mL) at −25 °C was added CCl4 (0.56 mL, 5.75
mmol). The reaction mixture was stirred for 10 min, and ethyl
diisopropylamine (0.24 mL, 1.37 mmol) and DMAP (0.01 g, 0.06
mmol) were added. After 5 min, dibenzyl phosphite (0.22 mL, 0.99
mmol) was added, and the reaction mixture was allowed to reach room
temperature over 1.5 h. A solution of KH2PO4 (20 mL, 0.5 M) was
added, and the reaction mixture was extracted with EtOAc (3 × 50
mL). The combined organic extract was dried over Na2SO4 and
concentrated under reduced pressure. Purification by flash column
chromatography (silica gel, hexanes−EtOAc, 50:50) yielded the
desired phosphate ester 32 [0.35 g, 0.47 mmol, 73%, Rf = 0.52
1
70%, Rf = 0.28 (hexanes−EtOAc, 50:50)] as a yellow powder. H
NMR (CDCl3, 500 MHz) δ 8.30 (1H, br s, NH), 7.93 (1H, d, J = 9.5
Hz, ArH), 6.96 (2H, s, ArH), 6.95 (1H, d, J = 2.0 Hz, ArH), 6.93 (1H,
dd, J = 9.5 Hz, 2.5 Hz, ArH), 6.92 (1H, d, J = 2.5 Hz, ArH), 6.78 (1H,
dd, J = 8.0 Hz, 2.0 Hz, ArH), 6.65 (1H, d, J = 8.5 Hz, ArH), 5.55 (1H,
s, OH), 3.89 (3H, s, OCH3), 3.84 (3H, s, OCH3), 3.80 (3H, s, OCH3),
3.71 (6H, s, OCH3); 13C NMR (CDCl3, 125 MHz) δ 192.7, 157.1,
152.5, 147.0, 145.3, 143.3, 141.0, 136.6, 135.0, 125.1, 123.0, 122.1,
121.5, 115.1, 112.6, 111.6, 110.3, 107.4, 94.8, 60.8, 56.0, 55.8, 55.6;
HPLC method B, tR 11.43 min; (+) HRESIMS m/z found 464.1706
[M + H]+ (calcd for C26H26NO7, 464.1704).
1
(hexanes−EtOAc, 30:70)] as a colorless solid. H NMR (CDCl3, 300
MHz) δ 7.88 (1H, d, J = 8.7 Hz, ArH), 7.31 (10H, m, ArH), 7.17 (1H,
m, ArH), 7.03 (1H, m, ArH), 6.93 (1H, dd, J = 8.8 Hz, 2.2 Hz, ArH),
6.84 (1H, d, J = 2.2 Hz, ArH), 6.83 (2H, s, ArH), 6.78 (1H, d, J = 8.5
Hz, ArH), 5.14 (4H, d, J = 8.1 Hz, CH2), 3.91 (3H, s, CH3), 3.79 (3H,
s, OCH3), 3.76 (3H, s, OCH3), 3.70 (6H, s, OCH3), 3.54 (3H, s,
OCH3); 13C NMR (CDCl3, 125 MHz) δ 191.7, 157.4, 152.5, 150.99,
150.95, 144.1, 140.7, 138.2, 135.7 (d, J = 6.8 Hz), 135.6, 129.0, 128.74,
128.70, 128.1, 123.84, 123.82, 122.8, 121.7, 114.5, 112.2, 111.7, 106.9,
2-(3′-Hydroxy-4′-methoxyphenyl)-3-(3″,4″,5″-trimethoxybenzo-
yl)-N-methyl-6-methoxyindole (30) (ref 45). To a solution of
isopropyl ether 29 (0.426 g, 0.819 mmol) in CH2Cl2 (15 mL) at 0
°C was added AlCl3 (0.33 g, 2.46 mmol). The reaction mixture was
allowed to warm to room temperature, and stirring at this temperature
continued for 2 h. Examination by TLC indicated the presence of
starting material, so an additional 3 molar equiv (0.33 g, 2.46 mmol) of
H
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93.7, 70.1 (d, J = 5.8 Hz), 60.9, 56.2, 56.1, 55.9, 31.4; 31P NMR
(CDCl3, 120 MHz) δ 5.95; HPLC method B, tR 16.84 min; (+)
HRESIMS m/z found 760.2280 [M
Colchicine Binding Assay. Colchicine binding was measured as
previously described.49,50 Solutions containing 1 μM tubulin, 5.0 μM
[3H]colchicine, and inhibitor at 50, 5, and/or 1 μM (as specified) were
used.
+
Na]+ (calcd for
C41H40NNaO10P, 760.2282).
Cell Lines and Sulforhodamine B (SRB) Assay. Cancer cell lines
were obtained from ATCC (DU-145 (prostate), SK-OV-3 (ovarian),
and NCI-H460 (lung)) and maintained as recommended. Media
included gentamicin and amphotericin B. The National Cancer
Institute’s standard SRB assay assessed cancer cell line growth
inhibition, as previously described, with the GI50 values being the
drug concentrations calculated to cause a 50% reduction in net protein
increase relative to untreated cells.51−53 Results reported are averages
of at least three separate experiments, each of which was carried out in
triplicate.
Color Doppler Ultrasound Imaging with Indole 33
(OXi8007). PC-3-luc cells were grown in culture and harvested, as
described previously.43 Cells (2 × 106) were implanted orthotopically
in the prostate of a SCID mouse (NIH, Frederick, MD), and the
tumor was allowed to grow to approximately 5−7 mm in diameter. At
this stage NAIR lotion (Church and Dwight, Princeton, NJ, USA) was
applied to ensure complete local hair removal. The mouse was
anesthetized (2% isoflurane in oxygen), and gel was applied to ensure
effective ultrasound coupling to the solid-state transducers: MS400 (24
MHz). Data were acquired using a Vevo 2100 (Visual Sonics, Inc.,
Toronto, Ontario, Canada) in both B and color Doppler modes.
Compound 33 (OXi8007) (350 mg/kg in saline) was injected ip, and
ultrasound images were acquired over the next 80 min.
2-(3′-Disodium phosphate-4′-methoxyphenyl)-3-(3″,4″,5″-trime-
thoxybenzoyl)-6-methoxyindole (33) (OXi8007) (ref 12). To a
solution of dibenzyl ester 31 (2.71 g, 3.74 mmol) in methanol (70
mL) was added 10% palladium−carbon (1.2 g). The flask was
evacuated under vacuum, and H2 gas was introduced via a balloon.
The reaction proceeded for 30 min, and the solution was filtered using
Celite with EtOAc. The filtrate was concentrated under reduced
pressure to give the crude phosphoric acid derivative as a greenish-
yellow oil (2.29 g, 4.22 mmol). The oil was dissolved in methanol (20
mL), and a solution of sodium methoxide (2.0 mL, 25% w/v in
methanol) was added. The reaction mixture was stirred at room
temperature for 12 h, and the methanol was removed under reduced
pressure. Purification by flash chromatography using a prepacked 25 g
reversed-phase silica column [solvent A: water; solvent B: acetonitrile;
gradient: 100% A/0% B (3 CV), 100% A/0% B → 20% A/80% B (10
CV), 0% A/100% B (7.3 CV); flow rate: 25 mL/min; monitored at
254 and 280 nm] resulted in the desired disodium phosphate salt 33
1
(OXi8007) [1.49 g, 2.54 mmol, 60%] as a yellow powder. H NMR
(D2O, 500 MHz) δ 8.03 (1H, d, J = 9.0 Hz, ArH), 7.71 (1H, m, ArH),
7.21 (1H, d, J = 2.0 Hz, ArH) 7.03 (1H, dd, J = 9.0 Hz, 2.5 Hz, ArH),
6.93 (2H, s, ArH), 6.67 (1H, d, J = 8.5 Hz, ArH), 6.63 (1H, dd, J = 9.0
Hz, 2.0 Hz, ArH), 3.95 (3H, s, OCH3), 3.77 (3H, s, OCH3), 3.75 (6H,
s, OCH3), 3.72 (3H, s, OCH3); 13C NMR (D2O, 125 MHz) δ 195.1,
156.3, 151.7, 150.5, 150.4, 147.7, 142.8 (d, J = 5.6 Hz), 139.7, 136.6,
135.4, 125.8, 123.8, 122.2, 121.5, 120.4, 111.7, 111.2, 107.8, 94.5, 60.8,
56.1, 55.8, 55.7; 31P NMR (D2O, 200 MHz) δ 0.57; HPLC method A,
tR 8.32 min; (+) HRESIMS m/z found 588.1008 [M + H]+ (calcd for
C26H25NNa2O10P, 588.1006).
ASSOCIATED CONTENT
* Supporting Information
■
S
Characterization data for selected intermediates and the final
compounds are available free of charge via the Internet at
http://pubs.acs.org.
2-(3′-Disodium phosphate-4′-methoxyphenyl)-3-(3″,4″,5″-trime-
thoxybenzoyl)-N-methyl-6-methoxyindole (34) (ref 45). To a
solution of dibenzyl ester 32 (0.32 g, 0.43 mmol) in ethanol (10
mL) was added 5% palladium−carbon (0.10 g). The flask was
evacuated by aspirator vacuum, and H2 gas was introduced into the
solution via balloon. The reaction proceeded for 30 min, and the
solution was filtered through Celite in EtOAc. The filtrate was
concentrated under reduced pressure to afford the crude phosphoric
acid derivative as a yellow foam (0.223 g, 0.400 mmol). The foam
product was dissolved in methanol (15 mL), and a solution of sodium
methoxide (0.18 mL, 0.80 mmol, 4.37 M solution in methanol) was
added. The reaction mixture was stirred at room temperature for 12 h.
Next, the methanol was removed under reduced pressure. Purification
by recrystallization from water−acetone afforded the desired salt 34
(0.168 g, 0.279 mmol, 64%) as a yellow solid. 1H NMR (CD3OD, 500
MHz) δ 7.89 (1H, d, J = 8.8 Hz, ArH), 7.79 (1H, m, ArH), 7.02 (1H,
d, J = 2.0 Hz, ArH), 6.88 (1H, dd, J = 8.8 Hz, 2.2 Hz, ArH), 6.76 (2H,
s, ArH), 6.62 (1H, d, J = 8.3 Hz, ArH), 6.58 (1H, dd, J = 8.4 Hz, 2.1
Hz, ArH), 3.90 (3H, s, CH3), 3.77 (3H, s, OCH3), 3.76 (3H, s,
OCH3), 3.74 (6H, s, OCH3), 3.69 (3H, s, OCH3); 13C NMR
(CD3OD, 75 MHz) δ 194.9, 158.8, 153.7, 152.2, 149.1, 148.9, 145.6
(d, J = 5.5 Hz), 141.5, 139.9, 137.4, 126.5, 124.3, 124.0, 123.2, 114.6,
112.9, 112.6, 108.0, 94.8, 61.1, 56.8, 56.6, 56.2, 32.4; 31P NMR
(CD3OD, 120 MHz) δ 1.84; HPLC method B, tR 8.74 min; (+)
AUTHOR INFORMATION
Corresponding Author
*Tel: 1-254-710-4117. Fax: 1-254-710-4272. E-mail: Kevin_
Pinney@baylor.edu.
■
Notes
The authors declare the following competing financial
interest(s): One of the co-authors (K.G.P.) is a paid consultant
(and stock shareholder) of a corporate sponsor of this research
(Oxigene Inc.), and another co-author (D.J.C.) is the former
Chief Scientific Officer and a current Board member and stock
shareholder with Oxigene Inc.
ACKNOWLEDGMENTS
■
The authors are grateful to the Cancer Prevention and Research
Institute of Texas [CPRIT (RP100406), to K.G.P. and M.L.T.],
Oxigene, Inc. (to K.G.P. and M.L.T), the Welch Foundation
(grant no. AA-1278 to K.G.P.), the Baylor University Research
Committee (URC grant to K.G.P.), and the National Cancer
Institute Award Number 5R01CA140674 (to K.G.P. and
M.L.T. with subcontract to R.P.M.) for their financial support
of this project. The content is solely the responsibility of the
authors and does not necessarily represent the official views of
the National Cancer Institute or the National Institutes of
Health. The authors also thank Dr. A. Ramirez (Mass
Spectrometry Core Facility, Baylor University) and Dr. J.
Karban and Dr. M. Nemec (Director) for use of the shared
Molecular Biosciences Center at Baylor University. Imaging
was facilitated with the assistance of the Southwestern Small
Animal Imaging Resource (SW-SAIRP), which is supported in
part by NCI U24 CA126608, the Harold C. Simmons Cancer
Center through an NCI Cancer Center Support Grant, 1P30
HRESIMS m/z found 602.1180 [M
+
H]+ (calcd for
C27H27NNa2O10P, 602.1162).
Effects on Tubulin Polymerization. Bovine brain tubulin was
purified using methods previously described.47 The effect of
compounds on tubulin assembly in vitro was determined by using a
series of concentrations that were preincubated with 10 μM tubulin
(1.0 mg/mL) in glutamate buffer at 30 °C, followed by cooling to 0
°C. After GTP was added, the samples were mixed and transferred to
cuvettes at 0 °C in a recording spectrophotometer and warmed to 30
°C to initiate polymerization. Tubulin assembly was observed
turbidimetrically at 350 nm.48 Polymer disassembly was confirmed
by cooling to 0 °C. The calculated compound concentration that
inhibited the extent of tubulin assembly by 50% after a 20 min
incubation was defined as the IC50 value.
I
dx.doi.org/10.1021/np400374w | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
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CA142543, and the Department of Radiology. We are grateful
to Professor J. Hill (Cardiology, UTSW) for providing access to
the Vevo 2100, which was acquired thanks to the NIH Shared
Instrumentation Grant S10 RR031859.
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DEDICATION
■
#Dedicated to the memory of Dr. Anjan Ghatak, who passed
away on July 22, 2003.
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