Synthesis of dihydronaphthalene analogues inspired by combretastatin A-4 and their biological evaluation as anticancer agents

Article abstract of DOI:10.1039/C8MD00322J

The natural products colchicine and combretastatin A-4 (CA4) have provided inspiration for the discovery and development of a wide array of derivatives and analogues that inhibit tubulin polymerization through a binding interaction at the colchicine site on β-tubulin. A water-soluble phosphate prodrug salt of CA4 (referred to as CA4P) has demonstrated the ability to selectively damage tumor-associated vasculature and ushered in a new class of developmental anticancer agents known as vascular disrupting agents (VDAs). Through a long-term program of structure activity relationship (SAR) driven inquiry, we discovered that the dihydronaphthalene molecular scaffold provided access to small-molecule inhibitors of tubulin polymerization. In particular, a dihydronaphthalene analogue bearing a pendant trimethoxy aryl ring (referred to as KGP03) and a similar aroyl ring (referred to as KGP413) were potent inhibitors of tubulin polymerization (IC₅₀ = 1.0 and 1.2 μM, respectively) and displayed low nM cytotoxicity against human cancer cell lines. In order to enhance water-solubility for in vivo evaluation, the corresponding phosphate prodrug salts (KGP04 and KGP152, respectively) were synthesized. In a preliminary in vivo study in a SCID-BALB/c mouse model bearing the human breast tumor MDA-MB-231-luc, a 99% reduction in signal was observed with bioluminescence imaging (BLI) 4 h after IP administration of KGP152 (200 mg kg^?1) indicating reduced tumor blood flow. In a separate study, disruption of tumor-associated blood flow in a Fischer rat bearing an A549-luc human lung tumor was observed by color Doppler ultrasound following administration of KGP04 (15 mg kg^?1).

Full text of DOI:10.1039/C8MD00322J

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Chen, V. Mocharla, M. Sriram, T. Strecker, E. Hamel, H. Zhou, R. Lopez, Y. Wang, R. Mason, D. J. Chaplin,
M. Trawick and K. G. Pinney, Med. Chem. Commun., 2018, DOI: 10.1039/C8MD00322J.
Volume 7 Number 1 January 2016 Pages 1–204
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Page 1 of 30
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Synthesis of dihydronaphthalene analogues inspired by
combretastatin A-4 and their biological evaluation as anticancer
agents.
a
a
a
a
a
Casey J. Maguire , Zhi Chen , Vani P. Mocharla , Madhavi Sriram , Tracy E. Strecker , Ernest Hamel
b
c
c
a
c
a,d
,
Heling Zhou , Ramona Lopez , Yifan Wang , Ralph P. Mason , David J. Chaplin , Mary Lynn
Trawick , Kevin G. Pinney
a
a,*
a
Department of Chemistry and Biochemistry, Baylor University, One Bear Place #97348, Waco, TX 76798-7348, United States
of America
Screening Technologies Branch, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National
b
Cancer Institute, Frederick National Laboratory for Cancer Research, National Institutes of Health, Frederick, MD 21702,
United States of America
c
Department of Radiology, The University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX
7
5390-9058, United States of America
Mateon Therapeutics, Inc., 701 Gateway Boulevard, Suite 210, South San Francisco, CA 94080, United States of America
d
*Corresponding author. Tel.: (254) 710ꢀ4117; Eꢀmail address: Kevin_Pinney@baylor.edu
Dedication:
Dedicated to the memory of Dr. Anjan Ghatak, an inspiring and creative synthetic organic, medicinal chemist who made seminal
contributions to the science described in this manuscript.
Animal Care and Use Statement:
All animal procedures were approved by the University of Texas Southwestern Medical Center Institutional Animal Care and
Use Committee under APN101222 and 102169.

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Abstract
The natural products colchicine and combretastatin Aꢀ4 (CA4) have provided inspiration for the
discovery and development of a wide array of derivatives and analogues that inhibit tubulin
polymerization through a binding interaction at the colchicine site on βꢀtubulin. A waterꢀsoluble
phosphate prodrug salt of CA4 (referred to as CA4P) has demonstrated the ability to selectively damage
tumorꢀassociated vasculature and ushered in a new class of developmental anticancer agents known as
vascular disrupting agents (VDAs). Through a longꢀterm program of structure activity relationship (SAR)
driven inquiry, we discovered that the dihydronaphthalene molecular scaffold provided access to smallꢀ
molecule inhibitors of tubulin polymerization. In particular, a dihydronaphthalene analogue bearing a
pendant trimethoxy aryl ring (referred to as KGP03) and a similar aroyl ring (referred to as KGP413)
were potent inhibitors of tubulin polymerization (IC = 1.0 and 1.2 ꢀM, respectively) and displayed low
50
nM cytotoxicity against human cancer cell lines. In order to enhance waterꢀsolubility for in vivo
evaluation, the corresponding phosphate prodrug salts (KGP04 and KGP152, respectively) were
synthesized. In a preliminary in vivo study in a SCIDꢀBALB/c mouse model bearing the human breast
tumor MDAꢀMBꢀ231ꢀluc, a 99% reduction in signal was observed with bioluminescence imaging (BLI) 4
h after IP administration of KGP152 (200 mg/kg) indicating reduced tumor blood flow. In a separate
study, disruption of tumorꢀassociated blood flow in a Fischer rat bearing an A549ꢀluc human lung tumor
was observed by color Doppler ultrasound following administration of KGP04 (15 mg/kg).

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1
. Introduction
The natural products colchicine and combretastatin Aꢀ4 (CA4) provide a rich canopy for SARꢀ
1
2,3
guided molecular interrogation directed towards the discovery of highly potent inhibitors of tubulin
polymerization (Fig. 1). This rich, natural productsꢀbased structural landscape has enabled the exploration
of a wide range of structural diversity, resulting in literally thousands of synthetic analogues and
derivatives with structural and functional group modifications of both aryl rings and the ethylene bridge
4–9
of CA4. Key structural features of the combretastatins include the trimethoxy phenyl ring, the pꢀ
methoxyphenyl moiety, cisꢀconfiguration of the aromatic rings and a distance of approximately 4ꢀ5 Å
5,9,10
between the aryl rings.
Our longꢀstanding research agenda in this area has contributed a variety of
1
1–13
12,14–18
12,15,19
functionalized molecular scaffolds including benzo[b]thiophene
indole,
that bear structural analogy to colchicine and CA4.
Our original molecular design motif recognized salient aspects of structural similarity between nonꢀ
benzofuran,
5,9,20–22
23–26
stilbenoid,
and benzosuberene analogues
1
1
27,28
steroidal, antiꢀestrogen agents (such as nafoxidine and LY117018)
and CA4, which led us to the
discovery of a collection of highly potent inhibitors of tubulin polymerization that function through a
12,13,21,23–26,29,30
direct binding interaction at the colchicine site on the tubulin heterodimer.
In addition to
functioning as antiproliferative (cytotoxic agents), colchicine site inhibitors of tubulin polymerization can
also function as vascular disrupting agents (VDAs). Selective disruption of tumorꢀassociated microvessels
31–38
results in loss of nutrients, oxygen deprivation, and ultimately necrosis.
Therefore, this therapeutic
approach offers a unique and promising strategy for the treatment of cancer, which is mechanistically
3
9
TM 40
distinct from that of the angiogenesis inhibiting agents (AIAs), such as bevacizumab (Avastin ) that
3
1–34
target the angiogenesis process.
The subꢀset of colchicine site inhibitors of tubulin polymerization that
function as VDAs induce rapid morphological changes (flat to round) in the endothelial cells lining
33,36,41–43
microvessels, leading to microvessel occlusion, compromised vessel wall integrity, and necrosis.
VDAs are selective for tumorꢀassociated neovasculature due to the primitive nature of these vessels,
which are typically incomplete or missing basement membranes, deficient in smooth muscle and
16,44–48
pericytes, and therefore more reliant on endothelial cells to maintain the shape of the vessel wall.
Herein, we report the design, synthesis, and preliminary biological evaluation of a 5ꢀhydroxyꢀ6ꢀmethoxyꢀ
ꢀaryldihydronaphthalene analogue (referred to as KGP03, Fig. 1) along with its corresponding 1ꢀ
aroyldihydronaphthalene analogue (referred to as KGP413, Fig. 1). Functionalized dihydronaphthalene
1
5,21,25,49–
analogues of this nature bear structural similarity to CA4 and have been previously reported by us
53
54
and others. Our previous studies with indole analogues (such as OXi8006) along with Pettit’s
55,56
discovery of phenstatin (synthetic benzophenone analogue of CA4, Fig. 1)
confirmed the tolerability
(
and potential benefit) of a carbonyl group juxtaposed between functionalized aryl rings in regard to

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inhibition of tubulin polymerization, cytotoxicity, and in vivo efficacy. These molecules along with CA4
have very limited waterꢀsolubility, and in their initial development of the combretastatins, Pettit and coꢀ
workers astutely developed phosphate prodrug salts of CA4 and CA1 (referred to as CA4P and CA1P,
57–59
respectively) that dramatically increased waterꢀsolubility (Fig. 1).
This strategy has also been applied
to these dihydronaphthalene analogues to generate waterꢀsoluble phosphate prodrug salts (KGP04 and
KGP152, respectively, Fig. 1). Synthetic strategies towards these molecules are reported along with
inhibition of tubulin polymerization (cellꢀfree assay) and cytotoxicity against NCIꢀH460 (nonꢀsmall cell
lung carcinoma), DUꢀ145 (prostate carcinoma), and SKꢀOVꢀ3 (ovarian adenocarcinoma) human cancer
cell lines. The two waterꢀsoluble prodrugs (KGP04 and KGP152) were subjected to preliminary in vivo
(
mouse and rat) evaluation to assess their ability to disrupt tumorꢀassociated vasculature and thus function
as VDAs.

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2
2
. Results and discussion
.1. Synthesis
Our initial synthetic route towards KGP03 (Scheme 1) provided the target compound in a 9%
21,25,49,54
overall yield from 6ꢀmethoxytetralin.
Considering an alternative strategy to the synthesis of
KGP03 in order to improve efficiency and overall yield, 6ꢀmethoxyꢀ1ꢀtetralone was selected as a starting
material (Scheme 2). This precursor is more advanced, commercially available, and approximately half
the cost of the tetralin used in the original synthesis. Regioselective bromination of the tetralone followed
by lithium halogen exchange provided a means to direct the hydroxylation to the regiochemically desired
position in a significantly higher yield (69% for 2 steps) compared to the orthoꢀdirected CꢀH
25,60,61
deprotonation of tetralin used in the original route (23% yield for 1 step).
The phenolic tetralone 4
was subsequently converted to its corresponding silyl ether 5 upon treatment with TBSCl. Installation of
the trimethoxy aryl ring and elimination of the resulting tertiary alcohol to generate KGP03 was
accomplished in a oneꢀpot reaction. The appropriate aryllithium intermediate (prepared in situ from the
corresponding aryl bromide) was allowed to react with tetralone 5, and the resulting tertiary alcohol (in
62
situ) was treated with triethylamine and mesyl chloride to furnish the KGP03ꢀsilyl ether derivative 7.
Desilylation with TBAF afforded KGP03 in a 5ꢀstep synthesis with a 36% overall yield.

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Our initial route to the carbonyl hinged analogue, KGP413, centered on a modified Shapiro
coupling reaction (Scheme 3). Ketone 5 was converted to hydrazine 10, which provided the in situ
o
1
2
3
. TMEDA, sec-BuLi, 0 C
o
. B(OMe) , 0 C to r.t.
3
o
HO
. H O , HOAc, 0 C to r.t., 12 h
2
2
+
H CO
3
H CO
3
H CO
3
OH
1a (23%)
1b (63%)
Ac O, Et N,
DDQ, Dioxane:H O (95:5)
O
2
3
2
CH Cl , r.t., 12 h
r.t., 12 h
2
2
H CO
3
H CO
3
H CO
3
95% yield
65% yield
OH
OAc
OAc
1a
2
3
OCH3
OCH3
H CO
3
NaHCO , MeOH,
H O r.t., 12 h
2
O
TBSCl, imidazole, DMAP,
O
Li
3
o
o
CH Cl , 0 C to r.t., 12 h
Et O, -78 C to r.t., 12 h
2
2
2
H CO
3
H CO
3
98% yield
92% yield
69% yield
OH
OTBS
4
5
OCH3
OCH3
OCH3
OCH3
H CO
3
OCH3
H CO
3
H CO
3
H CO
3
TBAF, CH Cl ,
2
2
OH
o
HOAc, H O, reflux, 12 h
0 C, 10 min
2
H CO
3
H3CO
H3CO
quantitative yield
97% yield
OTBS
OTBS
OH
6
7
8
KGP03
Scheme 1. Synthesis of KGP03 from 6-Methoxytetralin Featuring Early Installation of Phenolic Moiety

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generated vinyl lithium adduct that was subsequently treated with 3,4,5ꢀtrimethoxybenzaldehyde to afford
secondary alcohol 11. DessꢀMartin oxidation provided silylꢀether derivative 12, which was desilylated to
25,49
afford KGP413.
Our improved synthesis of KGP413 (Scheme 4), proceeded through a silyl ether
tetralone intermediate that was highlighted in the KGP03 synthesis (Scheme 2). The ketone was
63
converted to vinyl bromide 14 using phosphorus tribromide. The corresponding vinyllithium
intermediate was prepared in situ followed by the addition of the trimethoxy aryl Weinreb amide to afford
silylꢀether derivative 12. Desilylation using concentrated HCl afforded KGP413 in a 6ꢀstep synthesis
with a 23% overall yield. This was a significant improvement from the original 9ꢀstep synthesis that
resulted in a 1% overall yield.
The reaction of either phenol (8 or 13) with in situꢀgenerated dibenzyl chlorophosphite resulted in
the expected dihydronaphthalene dibenzyl phosphate ester derivative (15 or 17, Scheme 5).
Debenzylation was accomplished using in situꢀgenerated iodomethylsilane, from chloromethylsilane and
sodium iodide. The in situꢀgenerated TMSꢀphosphate ester intermediate was quenched using sodium
22,57
methoxide in methanol to afford the disodium phosphate salt prodrug (16 or 18).

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2
.2. Biological evaluation
The two phenolic dihydronaphthalene analogues (KGP03 and KGP413 along with their corresponding
waterꢀsoluble phosphate prodrug salts KGP04 and KGP152) were evaluated for their ability to inhibit the
polymerization of purified tubulin. Both KGP03 and KGP413 were potent inhibitors (IC = 0.46 and
50
0
.85 ꢀM, respectively) akin to CA4 (IC = 1.2 ꢀM) while their corresponding phosphate prodrug salts
50
were inactive (IC > 20 ꢀM), as anticipated, since this cell free assay is devoid of phosphatase enzymes
5
0
required for cleavage and regeneration of the parent phenolic analogues. Phosphate prodrug salts of this
type are typically substrates for alkaline phosphatase enzymes, readily available in vivo. We confirmed
that KGP04 underwent conversion (essentially quantitative) to its corresponding phenol (KGP03) in the
presence of alkaline phosphatase (see Supplemental data file for details). All four dihydronaphthalene
compounds demonstrated low nM cytotoxicity (range of 2ꢀ52 nM) against the three cancer cell lines
utilized in this study, and thus were comparable to the activity of CA4 and CA4P. In order to assess the
ability of these compounds to interfere with tumorꢀassociated vasculature and thus function as VDAs, the
two waterꢀsoluble prodrugs were evaluated against in vivo models (mouse and rat) of human cancer
(
breast and lung, respectively).

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Table 1. Inhibition of tubulin polymerization, percent inhibition of colchicine binding, and cytotoxicity of
the target dihydronaphthalene analogues
Inhibition of
%
inhibition of
colchicine
GI50 (ꢁM) SRB assay
DUꢀ145
tubulin
Compound
polymerization
IC (µM) ± SD
binding ± SD
NCIꢀH460
SKꢀOVꢀ3
5
0
9
0% ± 2 (1 ꢁM),
KGP03
KGP04
0.46 ± 0.007
> 20
0.00451 ± 0.00104
0.00336 ± 0.000587
0.00327 ± 0.00139
0.00730 ± 0.00869
0.0126 ± 0.0125
0.00219 ± 0.000262
9
8% ± 0.3 (5 ꢁM)
a
nd
0.00373 ± 0.000851
0.0216 ± 0.0227
0.0524 ± 0.0137
0.00190 ± 0.000427
0.00622 ± 0.00296
0.0129 ± 0.0139
8
9
1% ± 2 (1 ꢁM),
8% ± 2 (5 ꢁM)
KGP413
KGP152
1.2 ± 0.007
> 20
a
nd
b
84% ± 1.1 (1 µM),
CA4
1.2 ± 0.05
> 20
0.00500 ± 0.000359 0.00602 ± 0.000661
0.00506 ± 0.00145
0.00188 ± 0.000976
9
8% ± 0.1 (5 µM)
c
a
CA4P
nd
0.00282 ± 0.000497
0.00336 ± 0.00105
a: nd = not determined; b: data from reference 21; c: data from reference 24
Preliminary In Vivo Evaluation of Water-Soluble Prodrugs KGP04 and KGP413 as VDAs
The dynamic and longitudinal effect of VDAs can be assessed with various nonꢀinvasive imaging
38,64
modalities.
Bioluminescence imaging (BLI) is a widely used optical imaging technique for preclinical
research. The light emission of BLI is based on the expression of the luciferase enzyme and the presence
of the substrate luciferin. The effect of VDAs can be observed through reduced delivery of substrate
luciferin and consequent diminished light emission, as has been demonstrated with several
17,38,41,42,65
VDAs.
We performed a preliminary dose escalation study on three BALB/c SCID mice with
orthotopic MDAꢀMBꢀ231ꢀluc tumors with respective doses of 100, 150, and 200 mg/kg KGP152
administered IP. Vascular shutdown at 4 hrs following KGP152 was quite uniform with all three tumors
showing at least 85% decrease in light emission. Figure 2 shows the respective decrease of BLI signal 4
and 24 hrs after administration of KGP152 (200 mg/kg). While dynamic BLI is very effective at
revealing vascular disruption, it does require transfected tumor cells to express luciferase. As an
1
4,38,66,67
alternative, ultrasound can reveal blood flow
been previously applied to demonstrate vascular shutdown following the administration of VDAs.
Figure 3 shows an example of the use of colorꢀDoppler ultrasound to evaluate the reduction of blood flow
h after administration of KGP04 to a nude rat with a subcutaneous A549 human lung tumor growing in
the leg.
and colorꢀDoppler based on the Doppler effect has
14
2

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Figure 2. BLI monitoring of tumor response to KGP152. KGP152 (200 mg/kg) was administered IP to a
3
BALB/C SCID mouse with a large MDAꢀMBꢀ231ꢀluc orthotopic breast tumor (2 cm ). (A) Images show
signal intensity heat maps overlaid on gray scale photographs of mouse at successive time points.
Following KGP152 administration, the BLI signal was dramatically lower at 4 hr (99% signal reduction)
and recovered slightly at 24 hr (81% signal reduction from baseline). (B) Corresponding dynamic time
courses of total flux obtained following luciferin administration at the three time points.
Figure 3. Color Doppler ultrasound images show reduction of tumor blood perfusion. Images show
baseline and 2 hr after the administration of KGP04 (15 mg/kg, IP) to a nude rat with a subcutaneous
3
A549 human tumor xenograft (0.5 cm ). Red and blue indicate magnitude of flow into and out of plane.

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The layer of skin is apparent at the top of the images, together with structural heterogeneity in the tumor.
Vascular shutdown is indicated by yellow arrow.
3
. Conclusion
In summary, we utilized the dihydronaphthalene molecular scaffold to design and synthesize two
promising inhibitors of tubulin polymerization (KGP03 and KGP413) and their corresponding waterꢀ
soluble phosphate prodrug salts. Initial synthetic routes were further optimized towards these lead
compounds. The analogues were evaluated for inhibition of tubulin polymerization and for their ability to
compete for colchicine binding, which provided robust data [similar to CA4 (positive control compound)]
for both phenolic dihydronaphthalene compounds (KGP03 and KGP413). Potent cytotoxicity (low nM
GI values) was observed for the four compounds (KGP03/KGP04 and KGP413/KGP152) against the
50
three human cancer cell lines (NCIꢀH460, DUꢀ145, and SKꢀOVꢀ3) used in this study. Preliminary in vivo
BLI evaluation of dihydronaphthalene prodrug KGP152 (200 mg/kg) against an MDAꢀMBꢀ231ꢀluc
orthotopic breast tumor (SCID mouse model) showed a dramatic decrease in signal after 4 h. The signal
recovered only slightly after 24 h. Preliminary in vivo assessment (nude rat bearing the A549 tumor) of
KGP04 (15 mg/kg) demonstrated evidence of vascular shutdown (imaged by color Doppler ultrasound).
Thus, these dihydronaphthalene analogues appear to be promising candidates for further development as
antiproliferative agents and VDAs.
4
4
4
. Experimental section
.1. Chemistry
.1.1. General materials and methods
Dichloromethane, methanol, diethylether, and tetrahydrofuran (THF) were used in their
anhydrous forms, as obtained from the chemical suppliers. Reactions were performed under an inert
nitrogen atmosphere. Thinꢀlayer 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 flash purification system using silica gel (200ꢀ400 mesh, 60 Å) or
manually in glass columns. Intermediates and products synthesized were characterized on the basis of
1
13
their H NMR (600, 500 MHz), C NMR (151 or 126 MHz) spectroscopic data using a Bruker Avance
III 600 MHz or a Varian VNMRs 500 MHz instrument. Spectra were recorded in CDCl , CD OD, or
3
3
C D O. All chemical shifts are expressed in ppm (δ), coupling constants (J) are presented in Hz, and peak
3
6
patterns are reported as singlet (s), doublet (d), triplet (t), quartet (q), septet (sept), double doublet (dd),
and multiplet (m).

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o
Purity of the final compounds was further analyzed at 25 C using an Agilent 1200 HPLC system
with a diodeꢀarray detector (λ = 190ꢀ400 nm), a Zorbax XDBꢀC18 HPLC column (4.6 mm x 150 mm, 5
µm), and a Zorbax reliance cartridge guardꢀcolumn; method A: solvent A, acetonitrile, solvent B, 0.1%
TFA in H O; or method B: solvent A, acetonitrile, solvent B, H O; gradient, 10% A/90% B to 100%
2
2
A/0% B over 0 to 40 min; postꢀtime 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.
Experimental procedures for intermediates in Scheme 1.
61,68
4
.1.1.1. 2-Methoxy-5,6,7,8-tetrahydronaphthalen-1-ol (1).
To a wellꢀstirred solution of 6ꢀmethoxyꢀ
1
,2,3,4ꢀtetrahydronapthalene (14.15 g, 86.75 mmol) in secꢀBuLi (100 mL, 110 mmol) at 0 °C, freshly
distilled TMEDA (13.6 mL) was added dropwise. The reaction mixture was then stirred at room
temperature for 1 h. The reaction mixture was cooled to 0 °C and B(OMe) (12.5 mL, 110 mmol) was
3
added dropwise. Then the reaction mixture was stirred for 1 h at room temperature. The reaction mixture
was cooled to 0 °C, and glacial HOAc (7 mL) was added dropwise, followed by the dropwise addition of
3
5 wt.% H O (15 mL). Finally, the reaction mixture was stirred at room temperature for 12 h. Saturated
2 2
NH Cl (100 mL) was added, and product was extracted with Et O (3 x 400 mL). The combined organic
4
2
phases were washed with brine and dried over Na SO , filtered, and concentrated in vacuo. Purification
2
4
by flash column chromatography (silica gel, EtOAc/hexanes, gradient 2:98 to 5:95) yielded 2ꢀmethoxyꢀ
1
5
,6,7,8ꢀtetrahydronaphthalenꢀ1ꢀol 1 (3.50 g, 19.6 mmol, 23% yield) as an offꢀwhite solid. H NMR
(
(
CDCl , 300 MHz): δ 6.68 (1H, d, J = 8.3 Hz,), 6.23 (1H, d, J = 8.8 Hz,), 5.65 (1H, s), 3.84 (3H, s), 2.71
3
4H, t, J = 6.0 Hz), 1.71 (4H, m).
25,49,68
4
.1.1.2. 2-Methoxy-5,6,7,8-tetrahydronaphthalen-1-yl acetate (2).
To a wellꢀstirred solution of 2ꢀ
methoxyꢀ5,6,7,8ꢀtetrahydronaphthalenꢀ1ꢀol 1 (1.25 g, 7.01 mmol) dissolved in anhydrous CH Cl (25 mL)
2
2
was added Et N (1.5 mL, 10.8 mmol), Ac O (1.0 mL, 11 mmol), and DMAP (0.10 g, 0.9 mmol). The
3
2
reaction mixture was stirred for 12 hours at room temperature. The solvents were removed in vacuo, and
the crude product was subjected to flash chromatography (silica gel, 3:97 EtOAc/hexanes) to afford 2ꢀ
1
methoxyꢀ5,6,7,8ꢀtetrahydronaphthalenꢀ1ꢀyl acetate 2 (1.50 g, 6.81 mmol, 95% yield) as a white solid. H
NMR (CDCl , 300 MHz): δ 6.91 (1H, d, J = 8.4 Hz), 6.75 (1H, d, J = 8.4 Hz), 3.78 (3H, s), 2.71 (2H, m),
3
2
.54 (2H, m), 2.32 (3H, s), 1.74 (4H, m).
25,49,68
4
.1.1.3. 2-Methoxy-5-oxo-5,6,7,8-tetrahydronaphthalen-1-yl acetate (3).
A solution of DDQ
(
10.30 g, 22.70 mmol) in dioxane (40 mL) was added dropwise to a well stirred solution of 2ꢀmethoxyꢀ
,6,7,8ꢀtetrahydronaphthalenꢀ1ꢀyl acetate 2 (5.01 g, 23 mmol) in H O/dioxane (5/95) at rt. The reaction
5
2

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mixture was stirred for 12 h, and the solid that precipitated was filtered and washed with EtOAc. The
filtrates were evaporated under reduced pressure. To this was added saturated NaHCO (100 mL), and the
3
solution was extracted with Et O (3 x 200 mL). The combined organic phases were washed with brine,
2
dried over Na SO , filtered and concentrated in vacuo. The crude product was subjected to flash column
2
4
chromatography (silica gel, 30:70 EtOAc/hexanes) to afford 2ꢀmethoxyꢀ5ꢀoxoꢀ5,6,7,8ꢀ
1
tetrahydronaphthalenꢀ1ꢀyl acetate 3 (3.45 g, 14.7 mmol, 65% yield). H NMR (CDCl , 300 MHz): δ 7.99
3
(
1H, d, J = 8.7 Hz), 6.92 (1H, d, J = 8.7 Hz), 3.88 (3H, s), 2.79 (2H, t, J =6.0 Hz), 2.60 (2H, m), 2.36 (3H,
s), 2.10 (2H, p, J = 6.2 Hz).
25,49,68
4
.1.1.4a. 5-Hydroxy-6-methoxy-3,4-dihydronaphthalen-1(2H)-one (4).
Anhydrous K CO (4.72
2 3
g, 34.1 mmol) was added to a wellꢀstirred solution of 2ꢀmethoxyꢀ5ꢀoxoꢀ5,6,7,8ꢀtetrahydronaphthalenꢀ1ꢀyl
acetate 3 (4.0 g, 17.1 mmol) in MeOH (100 mL) and H O (5 mL). The reaction mixture was stirred for 12
2
h at room temperature. The solvents were removed under reduced pressure followed by the addition of
saturated NaHCO (50 mL). The solution was extracted with CH Cl (3 x 100 mL), and the combined
3
2
2
organic phases were washed with brine, dried over Na SO and filtered. The organic phases were
2
4
evaporated in vacuo to afford 5ꢀhydroxyꢀ6ꢀmethoxyꢀ3,4ꢀdihydronaphthalenꢀ1(2H)ꢀone 4 (3.24 g, 16.9
1
mmol, 98% yield) as red crystals. H NMR (CDCl , 300 MHz): δ 7.67 (1H, d, J = 8.6 Hz), 6.83 (1H, d, J
3
=
8.6 Hz), 5.72 (1H, s), 3.96 (3H, s), 2.93 (2H, t, J =6.0 Hz), 2.60 (2H, m), 2.10 (2H, p, J = 6.1 Hz).
25,49,68
4
.1.1.5a. 5-((Tert-butyldimethylsilyl)oxy)-6-methoxy-3,4-dihydronaphthalen-1(2H)-one (5).
Tetralone 4 (3.24 g, 16.9 mmol) was dissolved in anhydrous CH Cl (75 mL) at room temperature. Et N
2
2
3
(
3.53 mL, 25.3 mmol) and DMAP (0.21 g, 1.69 mmol) were added, and the reaction mixture was stirred
for 10 min. TBSCl (3.05 g, 20.2 mmol) was added in portions, and the reaction mixture was stirred for 12
h. The reaction was ended by the addition of H O (50 mL). The organic layers were separated, and the
2
aqueous phases were extracted with CH Cl (2 x 100 mL). The combined organic phases were washed
2
2
with brine, dried over Na SO , filtered and evaporated to dryness under reduced pressure. Separation of
2
4
the crude product by flash column chromatography (silica gel, 10:90 EtOAc/hexanes) afforded TBSꢀ
1
protected tetralone 5 (4.38 g, 14.3 mmol, 92% yield) as a colorless oil. H NMR (CDCl , 300 MHz): δ
3
7
.72 (1H, d, J = 8.6 Hz), 6.82 (1H, d, J = 8.6 Hz), 3.85 (3H, s), 2.90 (2H, t, J = 6.0 Hz), 2.57 (2H, m),
.07 (2H, p, J = 6.1 Hz), 1.01 (9H, s), 0.18 (6H, s).
2
4
.1.1.6.
5-((Tert-butyldimethylsilyl)oxy)-6-methoxy-1-(3,4,5-trimethoxyphenyl)-1,2,3,4-
49,68
tetrahydronaphthalen-1-ol (6).
To a well stirred solution of 3,4,5ꢀtrimethoxybromobenzene (7.57 g,
o
3
0.6 mmol) in anhydrous Et O (300 mL) at ꢀ78 C, was added nꢀBuLi (20.8 mL, 24.5 mmol) dropwise.
2
o
The reaction was stirred until the temperature reached ꢀ30 C. The TBSꢀprotected tetralone 5 (6.26 g, 20.4

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mmol) dissolved in Et O (50 mL) was added dropwise to the reaction mixture, and the reaction mixture
2
was stirred until it reached room temperature. H O (100 mL) was added, and the organic layer was
2
separated. The aqueous phase was extracted with Et O (2 x 250 mL), and the combined organic phases
2
were washed with brine. Removal of solvent under reduced pressure afforded crude product, which was
subjected to flash column chromatography (silica gel, 20:80 EtOAc/hexanes) to afford tertiary alcohol 6
1
(
6.86 g, 14.5 mmol, 69% yield) as a colorless oil. H NMR (CDCl , 300 MHz): δ 6.67 (2H, m), 6.55 (2H,
3
s), 3.89 (3H, s), 3.78 (9H, s), 2.89 (2H, m), 2.06 (2H, m), 1.60 (2H, m), 1.02 (9H, s), 0.23 (3H, s), 0.20
3H, s).
(
4
.1.1.7a.
Tert-butyl((2-methoxy-5-(3,4,5-trimethoxyphenyl)-7,8-dihydronaphthalen-1-
4
9,68
yl)oxy)dimethylsilane (7).
A mixture of tertiary alcohol 6 (6.86 g, 14.1 mmol), glacial acetic acid
120 mL) and H O (400 mL) was refluxed for 12 h. The reaction was cooled and extracted with CH Cl
(
(
2
2
2
2 x 300 mL). The combined organic phases were washed with brine, dried over Na SO and filtered.
2
4
Evaporation of the solvents under reduced pressure followed by drying under high vacuum afforded tertꢀ
butyl((2ꢀmethoxyꢀ5ꢀ(3,4,5ꢀtrimethoxyphenyl)ꢀ7,8ꢀdihydronaphthalenꢀ1ꢀyl)oxy)dimethylsilane 7 as a
colorless oil (6.43 g, 14.1 mmol, quantitative yield). The crude product was carried on to the next step
without further characterization.
49,68
4
.1.1.8a. 2-Methoxy-5-(3,4,5-trimethoxyphenyl)-7,8-dihydronaphthalen-1-ol (8).
The crude
product 7 (6.43 g, 14.1 mmol) was dissolved in CH Cl (150 mL) and cooled to 0 °C. To this was added
2
2
TBAF (22 mL, 22 mmol) dropwise. The reaction was stirred for 10 min and ended by the addition of H O
2
(
100 mL). The organic layers were separated, and the aqueous phases were extracted with CH Cl (2 x
2 2
3
00 mL). The combined organic phases were washed with brine and dried over Na SO . Filtration
2 4
followed by evaporation of solvents afforded the crude product, which was separated by flash column
chromatography (silica gel, 40:60 EtOAc/ hexanes) to afford 8 (4.50 g, 13.1 mmol, 97% yield) as an offꢀ
1
white solid. H NMR (CDCl , 300 MHz): δ 6.62 (1H, d, J = 8.4 Hz), 6.58 (1H, d, J = 8.4 Hz), 6.56 (2H,
3
s), 5.97 (1H, t, J = 4.6 Hz), 5.74 (1H, s), 3.89 (3H, s), 3.88 (3H, s), 3.84 (6H, s), 2.88 (2H, t, J = 8.2 Hz),
1
3
2
.37 (2H, td, J = 8.2, 4.6 Hz). C NMR (CDCl , 75 MHz): δ 152.9, 145.8, 141.9, 139.0, 136.9, 128.9,
3
1
25.4, 122.3, 117.4, 107.2, 105.9, 60.9, 55.9, 22.8, 20.2. HPLC retention time – 14.00 min.
Experimental procedures for intermediates in Scheme 2.
6
0
4
.1.1.9. 5-Bromo-6-methoxy-3,4-dihydronaphthalen-1(2H)-one (9). 6ꢀMethoxyꢀ1ꢀtetralone (1.06 g,
6
.02 mmol) was stirred in 60 mL of H O. Nꢀbromosuccinimide (1.07 g, 6.01 mmol) was added, and the
2
reaction mixture was heated to 60 °C. H SO (0.67 mL) was then added to the reaction mixture, which
2
4
was heated for 5 h. The reaction mixture was extracted with EtOAc, and the organic layers were dried

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over Na SO and filtered. The solvent was then removed in vacuo, and the resulting solid was dissolved in
2
4
methanol and recrystallized. The crystals were isolated by filtration and washed with cold methanol to
1
afford the product 9 (1.22 g, 4.78 mmol, 81% yield) as a white solid. H NMR (CDCl , 600 MHz): δ 8.09
3
(
1H, d, J = 8.7 Hz), 6.91 (1H, d, J = 8.7 Hz), 4.00 (3H, s), 3.06 (2H, t, J = 6.2 Hz), 2.66 (2H, m), 2.15
1
3
(
2H, p, J = 6.3 Hz). C NMR (CDCl , 151 MHz): δ 196.8, 159.8, 145.5, 128.4, 127.6, 113.0, 109.6, 56.5,
3
+
+
3
8.0, 30.5, 22.5. HRMS: Obsd 255.0016 [M + H ], Calcd for C H BrO : 255.0015.
11 12 2
6
1
4
.1.1.4b. 5-Hydroxy-6-methoxy-3,4-dihydronaphthalen-1(2H)-one (4). To a wellꢀstirred solution of
5
ꢀbromoꢀ6ꢀmethoxyꢀ3,4ꢀdihydronaphthalenꢀ1(2H)ꢀone 9 (0.50 g, 1.96 mmol) in THF at ꢀ78 °C, nꢀBuLi
(
4.90 mL, 7.84 mmol) was added dropwise. The reaction mixture was then stirred at ꢀ78 °C for 1 h and
then allowed to warm to room temperature. B(OMe) (0.45 mL, 3.92 mmol) was added dropwise, and the
3
reaction mixture was stirred for 1 h at room temperature. Glacial acetic acid (0.22 mL) was added
dropwise, followed by the addition of 35 wt.% H O (0.48 mL) added dropwise. The reaction mixture was
2
2
then stirred at room temperature for 12 h. Saturated NH Cl (20 mL) was added to the solution, and the
4
mixture was extracted with EtOAc (3 x 50 mL). The combined organic phases were washed with brine
and dried over Na SO , filtered, and concentrated in vacuo. Purification by flash column chromatography
2
4
1
(
(
(
silica gel, EtOAc/ hexanes) afforded the phenol 4 (0.32 g, 1.66 mmol, 85% yield) as a tan solid. H NMR
CDCl , 600 MHz): δ 7.70 (1H, d, J = 8.6 Hz), 6.86 (1H, d, J = 8.6 Hz), 5.76 (1H, s), 3.98 (3H, s), 2.95
3
13
2H, t, J = 6.2 Hz), 2.65 (2H, m), 2.13 (2H, p, J = 6.4 Hz). C NMR (CDCl , 151 MHz): δ 197.9, 149.9,
3
+
1
41.9, 130.4, 126.8, 120.0, 108.3, 56.1, 38.8, 22.9, 22.7. HRMS: Obsd 215.0679 [M + Na ], Calcd for
+
C H NaO : 215.0679.
11
12
3
49
4
.1.1.5b. 5-((Tert-butyldimethylsilyl)oxy)-6-methoxy-3,4-dihydronaphthalen-1(2H)-one (5).
5ꢀ
hydroxyꢀ6ꢀmethoxyꢀ3,4ꢀdihydronaphthalenꢀ1(2H)ꢀone 4 (1.05 g, 5.46 mmol) was stirred in 10 mL of
CH Cl at 0 °C. Triethylamine (0.84 mL, 6.03 mmol) and DMAP (0.27 g, 2.18 mmol) were then added.
2
2
The reaction mixture was stirred for an additional 10 min before adding TBSCl (0.90 g, 5.97 mmol). The
reaction was quenched with 100 mL of H O after 1 h. The reaction mixture was extracted with CH Cl (3
2
2
2
x 100 mL), and the organic layer was dried over Na SO . The organic layer was then filtered, and the
2
4
solvent was removed in vacuo. The crude mixture was purified by flash chromatography (silica gel,
1
EtOAc/ hexanes) to afford silyl ether 5 (1.58 g, 5.16 mmol, 95% yield) as an orange oil. H NMR (CDCl ,
3
6
00 MHz): δ 7.54 (1H, d, J = 8.6 Hz), 6.64 (1H, d, J = 8.7 Hz), 3.67 (3H, s), 2.72 (2H, t, J = 6.1 Hz), 2.42
13
(
2H, m), 1.89 (2H, p, J = 6.4 Hz), 0.83 (9H, s), 0.00 (6H, s). C NMR (CDCl , 151 MHz): δ 198.0, 154.0,
3
1
41.3, 136.1, 126.8, 121.5, 109.2, 54.9, 38.7, 26.1, 24.4, 22.9, 18.9, ꢀ3.8. HRMS: Obsd 307.1727 [M +
+
+
H ], Calcd for C H O Si : 307.1724.
1
7
27
3

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4
.1.1.7b.
Tert-butyl((2-methoxy-5-(3,4,5-trimethoxyphenyl)-7,8-dihydronaphthalen-1-
25,62
yl)oxy)dimethylsilane (7).
To a solution of 3,4,5ꢀtrimethoxybromobenzene (1.62 g, 6.55 mmol) in
anhydrous THF (20 mL), nꢀBuLi (2.62 mL, 6.55 mmol) was added dropwise at ꢀ78 °C. The reaction
mixture was stirred for 30 min at ꢀ78 °C. A solution of 5ꢀ((tertꢀbutyldimethylsilyl)oxy)ꢀ6ꢀmethoxyꢀ3,4ꢀ
dihydronaphthalenꢀ1(2H)ꢀone 5 (1.00 g, 3.27 mmol) in THF (10 mL) was added dropwise, and the
reaction mixture was then allowed warm to 0 °C over 3 h. The reaction was then cooled to ꢀ78 °C, and
triethylamine (3.68 mL, 26.16 mmol) and MsCl (1.01 mL, 13.08 mmol) were added dropwise to the
solution. The reaction mixture was allowed to warm to room temperature over a period of 8 h. The
reaction mixture was quenched with H O (100 mL), and the reaction mixture was then extracted with
2
EtOAc (3 x 100 mL). The combined organic phases were washed with brine, dried over Na SO , filtered,
2
4
and the solvent was removed in vacuo. The resulting crude product was then subjected to flash column
chromatography (silica gel, EtOAc/ hexanes) to afford tertꢀbutyl((2ꢀmethoxyꢀ5ꢀ(3,4,5ꢀtrimethoxyphenyl)ꢀ
1
7
,8ꢀdihydronaphthalenꢀ1ꢀyl)oxy)dimethylsilane 7 (0.82 g, 1.80 mmol, 55% yield). HNMR (CDCl , 600
3
MHz): δ 6.44 (1H, d, J = 8.4 Hz), 6.40 (1H, d, J = 8.5 Hz), 6.37 (2H, s), 5.75 (1H, t, J = 4.6 Hz), 3.69
(
(
3H, s), 3.65 (6H, s), 3.58 (3H, s), 2.69ꢀ2.64 (2H, m), 2.13 (2H, td, J = 7.9, 4.7 Hz), 0.83 (9H, s), 0.00
13
6H, s). C NMR (CDCl , 151 MHz): δ 152.9, 149.7, 141.4, 139.8, 137.0, 137.0, 128.7, 128.1, 124.9,
3
1
18.9, 108.1, 106.0, 60.9, 56.1, 26.1, 23.1 21.7, 18.9, ꢀ3.9.
49
4
.1.1.8b. 2-Methoxy-5-(3,4,5-trimethoxyphenyl)-7,8-dihydronaphthalen-1-ol (8).
Tertꢀbutyl((2ꢀ
methoxyꢀ5ꢀ(3,4,5ꢀtrimethoxyphenyl)ꢀ7,8ꢀdihydronaphthalenꢀ1ꢀyl)oxy)dimethylsilane 7 (0.82 g, 1.80
mmol) was dissolved in THF and cooled to 0 °C. TBAF (0.71 g, 2.70 mmol) was added to the solution.
The reaction mixture was stirred 30 min and then quenched with 50 mL of H O. The aqueous layer was
2
extracted with EtOAc (3 x 100 mL), and the organic layer was dried over Na SO . The organic layer was
2
4
then filtered, and the solvent was removed in vacuo. Purification by flash chromatography (silica gel,
EtOAc/ hexanes) afforded 2ꢀmethoxyꢀ5ꢀ(3,4,5ꢀtrimethoxyphenyl)ꢀ7,8ꢀdihydronaphthalenꢀ1ꢀol 8 (0.62 g,
1
1
6
2
1
3
.80 mmol, quantitative yield) as a white solid. H NMR (CDCl , 600 MHz): δ 6.65 (1H, d, J = 8.4 Hz),
3
.61 (1H, d, J = 8.4 Hz), 6.58 (2H, s), 5.99 (1H, t, J = 4.7 Hz), 5.74 (1H, s), 3.91 (6H, s), 3.86 (6H, s),
13
.93 (2H, t, J = 8.4 Hz), 2.40 (2H, td, J = 8.0, 4.7 Hz). C NMR (CDCl , 151 MHz): δ 152.9, 145.8,
3
42.0, 139.5, 136.9, 129.0, 125.4, 122.3, 117.4, 107.2, 105.8, 61.0, 56.1, 56.0, 22.8, 20.2. HRMS: Obsd
+
+
43.1541 [M + H ], Calcd for C H O : 343.1540. HPLC retention time 7.47 min.
2
0
23
5
Experimental procedures for intermediates in Scheme 3.
4
.1.1.10.
N'-(5-((tert-butyldimethylsilyl)oxy)-6-methoxy-3,4-dihydronaphthalen-1(2H)-ylidene)-4-
49
methylbenzenesulfonohydrazide (10). To a wellꢀstirred solution of tetralone 5 (6.20 g, 13.1 mmol) in

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anhydrous EtOH (120 mL) was added pꢀtoluenesulfonylhydrazide (3.77 g, 20.3 mmol). Solution was
achieved within 5 min, at which point pꢀTSA monohydrate (0.17 g, 1.01 mmol) was added, and the
reaction mixture was stirred at room temperature for 12 h. Hydrazone 10 precipitated as a white solid,
which was then filtered, washed with iceꢀcold EtOH, and dried under reduced pressure to afford 10 (5.20
1
g, 11.0 mmol, 54% yield) as a white solid. H NMR (CDCl , 500 MHz): δ 7.93 (2H, d, J = 8.3 Hz), 7.86
3
(
1H, s), 7.61 (1H, d, J = 8.7 Hz), 7.31 (2H, d, J = 8.0 Hz), 6.73 (1H, d, J = 8.8 Hz), 3.79 (3H, s), 2.71ꢀ
2
.65 (2H, m), 2.42 (2H, t, J = 6.6 Hz), 2.40 (3H, s), 1.80 (2H, p, J = 6.6 Hz), 0.97 (9H, s), 0.13 (6H, s).
4
.1.1.11. (5-((Tert-butyldimethylsilyl)oxy)-6-methoxy-3,4-dihydronaphthalen-1-yl)(3,4,5-
49
trimethoxyphenyl)methanol (11). nꢀBuLi (13.5 mL, 33.8 mmol) was added to freshly distilled
o
TMEDA (30 mL), and the mixture was cooled to ꢀ50 C. At this point, hydrazine 10 (4.00 g, 8.44 mmol)
o
was added, and the reaction mixture was stirred until the temperature reached 25 C. To the reaction
mixture 3,4,5ꢀtrimethoxybenzaldhyde (6.62 g, 33.8 mmol) was added, and the reaction was stirred for 1 h.
H O (25 mL) was added, and the product was extracted with EtOAc (2 x 100 mL). The combined organic
2
phases were washed with 10% aqueous CuSO solution (100 mL) and brine, dried over Na SO , filtered,
4
2
3
and evaporated under reduced pressure. Purification of the crude product by flash column
chromatography (silica gel, 16:84 EtOAc/hexanes) yielded alcohol 11 (2.22 g, 4.56 mmol, 54% yield) as
a paleꢀyellow oil. The resulting secondary alcohol was carried on to the next step without
characterization.
4
.1.1.12a.
(5-((Tert-butyldimethylsilyl)oxy)-6-methoxy-3,4-dihydronaphthalen-1-yl)(3,4,5-
49
trimethoxyphenyl)methanone (12). To a wellꢀstirred solution of DessꢀMartin periodinane (1.30 g, 2.24
mmol) in dry CH Cl (20 mL) at room temperature was added a solution of alcohol 11 (1.00 g, 2.04
2
2
mmol) in anhydrous CH Cl (20 mL) followed by 10% aqueous Na S O (0.05 mL). After stirring for 5
2
2
2
2
4
min, a solution of 10% aqueous Na S O and saturated NaHCO (30 mL, 1:1 ratio) was added, and the
2
2
3
3
reaction mixture was stirred for 5 min. The product was extracted into CH Cl (3 x 50 mL). The
2
2
combined organic phases were filtered, and the solvent was removed under reduced pressure. The crude
product was purified by flash column chromatography (silica gel, 10:90 EtOAc/hexanes) to afford ketone
1
1
2 (0.68 g, 1.41 mmol, 69% yield) as a paleꢀyellow oil. H NMR (CDCl , 500 MHz): δ 7.14 (2H, s), 6.80
3
(
1H, d, J = 8.4 Hz), 6.63 (1H, d, J = 8.5 Hz), 6.32 (1H, t, J = 4.7 Hz), 3.91 (3H, s), 3.84 (6H, s), 3.77 (3H,
s), 2.88 (2H, t, J = 8.0 Hz), 2.43 (2H, td, J = 7.9, 4.8 Hz), 1.01 (9H, s), 0.17 (6H, s).
4
.1.1.13a. (5-Hydroxy-6-methoxy-3,4-dihydronaphthalen-1-yl)(3,4,5-trimethoxyphenyl)methanone
4
9
(
13). Ketone 12 (0.68 g, 1.41 mmol) was dissolved in CH Cl (25 mL), and the reaction mixture was
2 2
cooled to 0 °C. To this was added TBAF (2.82 mL, 2.82 mmol) dropwise. The reaction mixture was

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stirred for 10 min and terminated by the addition of H O (25 mL). The organic layers were separated, and
2
the aqueous phases were extracted with CH Cl (2 x 25 mL). The combined organic phases were washed
2
2
with brine and dried over Na SO . Filtration followed by evaporation of solvents afforded the crude
2
4
product, which was separated by flash column chromatography (silica gel, 40:60 EtOAc/ hexanes) to
1
afford 13 (0.29 g, 0.78 mmol, 55% yield) as an offꢀwhite solid. H NMR (CDCl , 500 MHz): δ 7.16 (2H,
3
s), 6.77 (1H, d, J = 8.4 Hz), 6.65 (1H, d, J = 8.4 Hz), 6.33 (1H, t, J = 4.7 Hz), 5.74 (1H, s), 3.92 (3H, s),
13
3
.88 (3H, s), 3.85 (6H, s), 2.91 (2H, t, J = 8.0 Hz), 2.47 (2H, td, J = 8.0, 4.7 Hz). C NMR (CDCl , 151
3
MHz): δ 196.3, 152.8, 146.4, 142.4, 142.2, 138.3, 133.0, 133.0, 125.9, 121.4, 117.5, 107.8, 107.5, 60.9,
+
+
5
6.2, 55.9, 22.5, 19.8. HRMS: Obsd 371.1489 [M + H ], Calcd for C H O 371.1489.
21 23 6
Experimental procedures for intermediates in Scheme 4.
6
3
4
.1.1.14. ((5-Bromo-2-methoxy-7,8-dihydronaphthalen-1-yl)oxy)(tert-butyl)dimethylsilane (14). To
a solution of 5ꢀ((tertꢀbutyldimethylsilyl)oxy)ꢀ6ꢀmethoxyꢀ3,4ꢀdihydronaphthalenꢀ1(2H)ꢀone 5 (0.66 g,
2
.17 mmol) in toluene (50 mL) phosphorus tribromide (3.25 mL, 1 M in dichloromethane) was added.
o
The reaction mixture was heated to 80 C for 3 h. Water was added, and the mixture was extracted with
EtOAc (3 x 50 mL). The organic layer was dried over Na SO , filtered, concentrated under reduced
2
4
pressure, and purified by flash chromatography (silica gel, EtOAc/ hexanes) to afford vinyl bromide 14
1
(
0.51 g, 1.4 mmol, 63% yield) as an orange oil. H NMR (CDCl , 500 MHz): δ 7.16 (1H, d, J = 8.5 Hz),
3
6
4
1
.69 (1H, d, J = 8.5 Hz), 6.29 (1H, t, J = 4.8 Hz), 3.80 (3H, s), 2.86ꢀ2.82 (2H, m), 2.29 (2H, td, J = 8.0,
1
3
.9 Hz), 1.00 (9H, s), 0.16 (6H, s). C NMR (CDCl , 151 MHz): δ 150.6, 141.1, 128.7, 127.7, 126.7,
3
21.1, 120.1, 108.2, 54.9, 26.1, 26.0, 25.0, 21.5, ꢀ4.0.
4
.1.1.12b.
(5-((Tert-butyldimethylsilyl)oxy)-6-methoxy-3,4-dihydronaphthalen-1-yl)(3,4,5-
trimethoxyphenyl)methanone (12). To a solution of vinyl bromide 14 (0.51 g, 1.4 mmol) in THF (50
o
mL) at ꢀ78 C, nꢀBuLi (0.56 mL, 2.5 M in hexanes) was added. The reaction mixture was stirred for 15
min, then N,3,4,5ꢀtetramethoxyꢀNꢀmethylbenzamide dissolved in THF was added. The reaction was
allowed to warm to room temperature over a period of 8 h. Upon completion, water was added, and the
mixture was extracted with EtOAc (3 x 50 mL). The organic layer was dried over Na SO , filtered,
2
4
concentrated under reduced pressure, and purified by flash chromatography (silica gel, EtOAc/hexanes) to
1
afford dihydronaphthalene analog 12 (0.32 g, 0.66 mmol, 47% yield) as a clear oil. H NMR (CDCl , 500
3
MHz): δ 7.14 (2H, s), 6.80 (1H, d, J = 8.4 Hz), 6.63 (1H, d, J = 8.5 Hz), 6.32 (1H, t, J = 4.7 Hz), 3.91
(
3H, s), 3.84 (6H, s), 3.77 (3H, s), 2.88 (2H, t, J = 8.0 Hz), 2.43 (2H, td, J = 7.9, 4.8 Hz), 1.01 (9H, s),
1
3
0
.17 (6H, s). C NMR (CDCl , 126 MHz): δ 196.4, 152.8, 150.2, 142.3, 141.5, 138.6, 133.1, 133.0,
3
1
27.3, 125.8, 119.2, 108.7, 107.4, 60.9, 56.2, 54.8, 26.0, 22.8, 21.3, 18.8, ꢀ4.0.

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4
.1.1.13b. (5-Hydroxy-6-methoxy-3,4-dihydronaphthalen-1-yl)(3,4,5-trimethoxyphenyl)methanone
(
13). To a solution of silyl ether 12 (0.32 g, 0.66 mmol) in THF concentrated HCl (5 mL) was added. The
reaction was stirred at room temperature for 3 h. Upon completion, saturated aqueous NaHCO was
3
added, and the mixture was extracted with EtOAc (3 x 50 mL). The organic layer was dried over Na SO ,
2
4
filtered, concentrated under reduced pressure, and purified by flash chromatography (silica gel, EtOAc/
hexanes) to afford 13 (0.24 g, 0.66 mmol, quantitative yield) as a white solid. To prevent the
dihydronaphthalene ring from aromatizing the reaction mixture was not heated above room temperature
1
during the deprotection reaction or during the purification process. H NMR (CDCl , 500 MHz): δ 7.16
3
(
2H, s), 6.77 (1H, d, J = 8.4 Hz), 6.65 (1H, d, J = 8.4 Hz), 6.33 (1H, t, J = 4.7 Hz), 5.74 (1H, s), 3.92 (3H,
13
s), 3.88 (3H, s), 3.85 (6H, s), 2.91 (2H, t, J = 8.0 Hz), 2.47 (2H, td, J = 8.0, 4.7 Hz). C NMR (CDCl ,
3
1
51 MHz): δ 196.3, 152.8, 146.4, 142.4, 142.2, 138.3, 133.0, 133.0, 125.9, 121.4, 117.5, 107.8, 107.5,
+
+
6
0.9, 56.2, 55.9, 22.5, 19.8. HRMS: Obsd 371.1489 [M + H ], Calcd for C H O 371.1489. HPLC
21 23 6
retention time – 5.18 min.
Experimental procedures for intermediates in Scheme 5.
4
.1.1.15. Dibenzyl (2-methoxy-5-(3,4,5-trimethoxyphenyl)-7,8-dihydronaphthalen-1-yl) phosphate
47,49
o
(
15).
To a wellꢀstirred solution of 13 (1.40 g, 4.09 mmol) in CH CN (25 mL) at ꢀ25 C, CCl (25 mL)
3 4
was added, and the reaction mixture was stirred for approximately 15 min. DIPEA (1.51 mL, 8.61 mmol)
and DMAP (0.05 g, 0.41 mmol) were added, followed by the addition of dibenzyl phosphonate (1.36 mL,
6
.14 mmol), and the reaction mixture was stirred for approximately 2 h. The reaction was quenched with
a 0.5 M KH PO solution (30 mL). The solution was extracted with EtOAc (3 x 100 mL). The combined
2
4
organic phases were washed with brine, dried over Na SO , filtered and concentrated in vacuo.
2
4
Purification by flash column chromatography (silica gel, 40:60 EtOAc/hexanes) afforded dibenzylꢀ
KGP03ꢀphosphate 15 (2.16 g, 3.58 mmol, 88% yield) as a yellow oil. The resulting dibenzyl ester was
carried on to the next step without characterization.
4
.1.1.16. Disodium 2-methoxy-5-(3,4,5-trimethoxyphenyl)-7,8-dihydronaphthalen-1-yl phosphate
47,49
(
16).
To a solution of dibenzylꢀKGP03ꢀphosphate 15 (2.16 g, 3.58 mmol) in acetonitrile (60 mL),
sodium iodide (1.07 g, 7.16 mmol) was added. Before dropwise addition of chlorotrimethylsilane (0.91
mL, 7.16 mmol), the mixture was stirred for 2 min, and approximately 30 min later the reaction was
terminated with 1% aqueous sodium thiosulfate (1 mL). Removal of the acetonitrile in vacuo afforded a
crude mixture, which was dissolved in waterꢀdichloromethane and washed with water (4 x 60 mL).
Concentration (facilitated by toluene azeotrope) of the aqueous layer resulted in isolation of the crude
phosphoric acid intermediate, which was subjected to drying under vacuum for approximately 1 h, and

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then dissolved in dry methanol (30 mL). Next, sodium methoxide (0.39 g, 7.16 mmol) was added. The
mixture was stirred 6 h and additional methanol was added to effect dissolution. After filtration of the
solution, concentration of the methanol in vacuo led to an offꢀwhite solid, which was reprecipitated from
1
waterꢀethanol to yield 16 (1.10 g, 2.36 mmol, 66% yield). H NMR (D O, 300 MHz): δ 6.67 (1H, d, J =
2
8
.4 Hz), 6.65 (2H, s), 6.64 (1H, d, J =8.4 Hz), 5.98 (1H, t, J = 4.6 Hz), 3.75 (6H, s), 3.74 (6H, s), 2.86
13
(
2H, t, J = 8.2 Hz), 2.23 (2H, td, J =8.2, 4.6 Hz). C NMR (D O, 75 MHz): δ 152.2, 151.6, 140.0, 140.0,
2
1
38.5, 137.8, 135.8, 131.5, 131.5, 128.2, 128.1, 126.7, 120.7, 109.1, 106.3, 61.0, 56.0, 55.5, 22.5, 21.8.
HPLC retention time 3.07 min.
4
.1.1.17. Dibenzyl (2-methoxy-5-(3,4,5-trimethoxybenzoyl)-7,8-dihydronaphthalen-1-yl) phosphate
47,49
o
(
17).
To a wellꢀstirred solution of 13 (0.74 g, 2.00 mmol) in CH CN (15 mL) at ꢀ25 C CCl (15 mL)
3 4
was added, and the reaction mixture was stirred for approximately 15 min. DIPEA (0.51 mL, 2.91 mmol)
and DMAP (0.02 g, 0.16 mmol) were added, followed by the addition of dibenzyl phosphonate (0.53 mL,
2
.40 mmol), and the reaction mixture was stirred for approximately 2 h. The reaction was quenched with
a 0.5 M KH PO solution (30 mL). The solution was extracted with EtOAc (3 x 100 mL). The combined
2
4
organic phases were washed with brine, dried over Na SO , filtered and concentrated in vacuo.
2
4
Purification by flash column chromatography (silica gel, 40:60 EtOAc/hexanes) afforded dibenzylꢀ
KGP413ꢀphosphate 17 (0.67 g, 1.06 mmol, 84% yield) as a yellow oil. The resulting dibenzyl ester was
carried on to the next step without characterization.
4
.1.1.18. Disodium 2-methoxy-5-(3,4,5-trimethoxybenzoyl)-7,8-dihydronaphthalen-1-yl phosphate
47,49
(
18).
To a solution of dibenzylꢀKGP413ꢀphosphate 17 (0.67 g, 1.06 mmol) in acetonitrile (20 mL),
sodium iodide (0.32 g, 2.12 mmol) was added. Before dropwise addition of chlorotrimethylsilane (0.27
mL, 2.12 mmol), the mixture was stirred for 2 min, and approximately 30 min later the reaction was
terminated with 1% aqueous sodium thiosulfate (1 mL). Removal of the acetonitrile in vacuo afforded a
crude mixture, which was dissolved in waterꢀdichloromethane and washed with water (4 x 20 mL).
Concentration (facilitated by toluene azeotrope) of the aqueous layer resulted in isolation of the crude
phosphoric acid intermediate, which was subjected to drying under vacuum for approximately 1 h, and
then dissolved in dry methanol (10 mL). Next, sodium methoxide (0.12 g, 2.12 mmol) was added. The
mixture was stirred 6 h, and additional methanol was added to effect dissolution. After filtration of the
solution, concentration of the methanol in vacuo led to an offꢀwhite solid, which was reprecipitated from
1
waterꢀethanol to yield 18 (0.22 g, 0.45 mmol, 42% yield). H NMR (D O, 300 MHz): δ 7.11 (2H, s), 6.77
2
(
1H, d, J = 8.4 Hz), 6.69 (1H, d, J = 8.4 Hz), 6.44 (1H, t, J = 4.7), 3.75 (6H, s), 3.75 (3H, s), 3.70 (3H, s),
1
3
2
1
.86 (2H, t, J = 8.0 Hz), 2.34 (2H, td, J = 8.0, 4.7 Hz). C NMR (D O, 75 MHz): δ 200.1, 152.3, 152.2,
2
52.1, 141.5, 140.1, 139.0, 137.3, 133.5, 130.8, 130.8, 124.9, 120.9, 109.5, 108.5, 107.9, 61.0, 56.1, 55.4,

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31
+
2
2.6, 21.0. P NMR (D O, 122 MHz): δ 0.92, 0.79. HRMS: Obsd 495.0791 [M + H ], Calcd for
2
+
C H Na O P 495.0791. HPLC retention time 8.05 min.
21
22
2
9
4
4
.2. Biological evaluation
.2.1. Cell lines and Sulforhodamine B (SRB) assay.
Inhibition of growth of human cancer cells was assessed using the sulforhodamine B assay (SRB), as
69–71
previously described.
Cancer cell lines (DUꢀ145, SKꢀOVꢀ3, and NCIꢀH460) (obtained from ATCC)
were plated at 7000ꢀ8000 cells/well into 96ꢀwell plates using DMEM supplemented with 5% fetal bovine
serum / 1% gentamicin sulfate and incubated for 24 h at 37 °C in a humidified incubator in a 5% CO2
atmosphere. Compounds to be tested were dissolved in DMSO to generate a 10 mg/mL stock solution.
Serial dilutions were made and added to the plates. Doxorubicin and paclitaxel were used as positive
controls. After a 48 h treatment, the cells were fixed with trichloroacetic acid (10% final concentration),
washed, dried, stained with SRB dye, washed to remove excess dye, and dried. SRB dye was solubilized,
and absorbances were measured at wavelength 540 nm and normalized to values at wavelength 630 nm
using an automated Biotek plate reader. A growth inhibition of 50% (GI50 or the drug concentration
causing 50% reduction in the net protein increase) was calculated from the absorbance data.
72
4
.2.2. Colchicine binding assay.
3
Inhibition of [ H]colchicine binding to tubulin was measured using 100 µL reaction mixtures containing
3
1
.0 µM tubulin, 5.0 µM [ H]colchicine (from PerkinꢀElmer), 5% (v/v) dimethyl sulfoxide, potential
inhibitors at 1.0 or 5.0 µM, as indicated, and components that stabilize the colchicine binding activity of
tubulin (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 glucoseꢀ1ꢀphosphate, 1.0 mM MgCl , and 1.0 mM GTP).
2
Incubation was for 10 min at 37 °C, when the binding reaction in control reaction mixtures is 40ꢀ60%
complete. Reactions were stopped with 2.0 mL of iceꢀcold water, and the reaction mixtures were placed
on ice. Each sample was poured onto a stack of two DEAEꢀcellulose filters (from Whatman), followed by
6
mL of iceꢀcold water. The samples were aspirated under reduced vacuum. The filters were washed three
times with 2 mL water and placed into vials containing 5 mL of Biosafe II scintillation cocktail. Samples
were counted 18 h later in a Beckman scintillation counter. Samples with inhibitors were compared to
samples with no inhibitor, and percent inhibition was determined. All samples were corrected for the
amount of radiolabel bound to the filters in the absence of tubulin.
43
4
.2.3. Inhibition of tubulin polymerization.

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Tubulin polymerization experiments were performed in 0.25 mL reaction mixtures (final volume) that
contained 1 mg/mL (10 µM) purified bovine brain tubulin, 0.8 M monosodium glutamate (pH 6.6), 4%
(
v/v) dimethyl sulfoxide, 0.4 mM GTP, and different compound concentrations. All reaction components
except GTP were preincubated for 15 min at 30 °C in 0.24 mL. The mixtures were cooled to 0 °C, and 10
µL of 10 mM GTP was added. Reaction mixtures were transferred to cuvettes held at 0 °C in Beckman
DUꢀ7400 and DUꢀ7500 spectrophotometers equipped with electronic temperature controllers. The
temperature was jumped to 30 °C, taking about 30 s, and polymerization was followed at 350 nm for 20
min. The IC was defined as the compound concentration that inhibited extent of polymerization by 50%
5
0
after 20 min.
4
.2.4. In vivo color Doppler ultrasound imaging with KGP04.
6
®
A549 human lung cancer cells (3x10 , in 200 ꢁL serum free medium with 50% Matrigel ; cell line
provided by Professor John Minna (UT Southwestern)) were implanted subcutaneously in the right hind
thigh of anesthetized nude rats (8ꢀ10 week old female; Tꢀcellꢀdeficient, athymic homozygous nude rat;
73
Charles River, NCI at Frederick, Frederick, MD). The tumor was allowed to grow until it reached about
mm diameter. At this time the rat was anesthetized with isoflurane and colorꢀDoppler ultrasound was
performed using a Vevo 2100 [Fuji (VisualSonics), Toronto, Canada] before and 2 h after administration
9
14
of KGP04 IP (15 mg/kg dissolved in 500 µl saline) in situ. All animal procedures were approved by the
University of Texas Southwestern Medical Center Institutional Animal Care and Use Committee under
APN101222 and 102169.
17
4
.2.5. In vivo bioluminescence imaging (BLI) with KGP152.
6
®
MDAꢀMBꢀ231ꢀluc cells (1x10 in 100 ꢁL of PBS with 50% Matrigel ; original cell line from ATCC,
with transfected cell line recently provided by Dr. Edward Graves, Stanford University) were injected
directly into the left upper mammary fat pad of three anesthetized BALB/c SCID mice. Tumors were
®
allowed to grow over about 3 months and then BLI was performed using an IVIS Spectrum system
(
PerkinꢀElmer (Xenogen), Alameda, CA). Dꢀluciferin (128 mg/kg in PBS in a total volume of 80 µL;
Gold Biotechnology Inc., St. Louis, MO) was administered subcutaneously (SC) in the foreback neck
region. Immediately after luciferin injection, a series of BLI images was acquired over a period of 30 min
17
using auto exposure time. Following baseline BLI, mice were injected intraperitoneally (IP) respectively
with 100, 150, and 200 mg/kg KGP152 in saline. Dynamic BLI was repeated 4 and 24 h postꢀtreatment
with administration of fresh luciferin on each occasion. All animal procedures were approved by the
University of Texas Southwestern Medical Center Institutional Animal Care and Use Committee under
APN101222 and 102169.

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Supplementary data:
Electronic supplementary information (ESI) available: Supplementary data including ( H NMR,
1
13
C
31
1
13
NMR, P NMR, HRMS, HPLC) for targets compounds and intermediates ( H NMR, C NMR, only),
phosphatase enzyme cleavage data, and Xꢀray crystallography (CCDC 1018601 and 1842517) for
KGP03 and KGP413 associated with this article can be found in the online supplementary data file.
Conflicts of Interest:
A significant portion of the work presented in this manuscript was funded through Mateon Therapeutics,
Inc. (formerly OXiGENE Inc.), and this relationship is properly indicated in the acknowledgement
section. Dr. David Chaplin is a former employee and current member of the Board of Directors of Mateon
Therapeutics, Inc. He is included as a coꢀauthor on this manuscript for his valuable contributions to the
overall project. In addition, one of the authors (KGP) is a former paid consultant with Mateon
Therapeutics, Inc. and current shareholder. We are pleased with this productive and useful longꢀterm
scientific collaboration and funding relationship with Mateon Therapeutics, Inc., and it is important to
note that there is absolutely no actual conflict of interest associated with the science presented in this
manuscript.
Acknowledgements:
The research was supported in part by the National Cancer Institute of the National Institutes of Health
(
Grant No. 5R01CA140674 to K.G.P, M.L.T, and R.P.M), the Cancer Prevention and Research Institute
of Texas (CPRIT, Grant No. RP140399 to K.G.P., M.L.T., and R.P.M., Grant No. RP170696 to K.G.P.,
M.L.T., and MIRA RP120670ꢀP3 to RPM), and Mateon Therapeutics, Inc. (grant to K.G.P. and M.L.T.)
for their financial support of this project. The content is solely the responsibility of the authors and does
not necessarily reflect the official views of the National Institutes of Health. The authors also thank Dr.
Craig Moehnke and Dr. Michelle Nemec (Director) for the use of the shared Molecular Biosciences
Center at Baylor University, Dr. Alejandro Ramirez (Mass Spectrometry Core Facility, Baylor
University), Dr. Christine A. Herdman (help with spectral characterization), and Dr. Kevin Klausmeyer
(
Xꢀray analysis). Imaging was facilitated with the assistance of the Southwestern Small Animal Imaging
Resource (SWꢀSAIRP), which is supported in part by NCI U24 CA126608, the Harold C. Simmons
Cancer Center through an NCI Cancer Center Support Grant, 1P30CA142543, and the Department of
Radiology. The IVIS Spectrum was acquired with the assistance of NIH Shared Instrumentation Grant
S10RR024757. We are grateful to Professor J. Hill (Cardiology, UTSW) for providing access to the Vevo
2
100, which was acquired under the NIH Shared Instrumentation Grant S10 RR031859.

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