Structural interrogation of benzosuberene-based inhibitors of tubulin polymerization

Bioorganic & Medicinal Chemistry xxx (2015) xxx–xxx

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Bioorganic & Medicinal Chemistry

journal homepage: w ww.elsevier.com/locate/bmc

Structural interrogation of benzosuberene-based inhibitors

of tubulin polymerization

Christine A. Herdman ^a, Laxman Devkota ^a, Chen-Ming Lin ^a, Haichan Niu ^a, Tracy E. Strecker ^a,

Ramona Lopez ^b, Li Liu ^b, Clinton S. George ^a, Rajendra P. Tanpure ^a, Ernest Hamel ^c, David J. Chaplin ^a,d

,

Ralph P. Mason ^b, Mary Lynn Trawick ^a, Kevin G. Pinney ^a,

⇑

^aDepartment of Chemistry and Biochemistry, Baylor University, One Bear Place #97348, Waco, TX 76798-7348, United States

^bDepartment of Radiology, The University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390-9058, United States

^cScreening 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, MD 21702, United States

^dOXiGENE Inc., 701 Gateway Boulevard, Suite 210, South San Francisco, CA 94080, United States

a r t i c l e i n f o

a b s t r a c t

The discovery of 3-methoxy-9-(3⁰,4⁰,5⁰-trimethoxyphenyl)-6,7-dihydro-5H-benzo[7]annulen-4-ol (a ben-

zosuberene-based analogue referred to as KGP18) was originally inspired by the natural products colchi-

cine and combretastatin A-4 (CA4). The relative structural simplicity and ease of synthesis of KGP18,

coupled with its potent biological activity as an inhibitor of tubulin polymerization and its cytotoxicity

(in vitro) against human cancer cell lines, has resulted in studies focused on new analogue design and

synthesis. Our goal was to probe the relationship of structure to function in this class of anticancer agents.

A series of twenty-two new benzosuberene-based analogues of KGP18 was designed and synthesized.

These compounds vary in their methoxylation pattern and separately incorporate triﬂuoromethyl groups

around the pendant aryl ring for the evaluation of the effect of functional group modiﬁcations on the

fused six-membered aromatic ring. In addition, the 8,9-saturated congener of KGP18 has been synthe-

sized to assess the necessity of unsaturation at the carbon atom bearing the pendant aryl ring. Six of

Article history:

Received 14 July 2015

Revised 1 October 2015

Accepted 10 October 2015

Available online xxxx

Keywords:

Inhibitors of tubulin polymerization

Benzosuberene analogues

Vascular disrupting agents

Small-molecule synthesis

the molecules from this benzosuberene-series of compounds were active (IC₅₀< 5

tubulin polymerization while four analogues were comparable (IC₅₀approximately 1

l

M) as inhibitors of

M) in their tubulin

inhibitory activity to CA4 and KGP18. The potency of a bis-triﬂuoromethyl analogue 74 and the unsatu-

rated KGP18 derivative 73 as inhibitors of tubulin assembly along with their moderate cytotoxicity sug-

gested the potential utility of these compounds as vascular disrupting agents (VDAs) to selectively target

microvessels feeding tumors. Accordingly, water-soluble and DMSO-soluble phosphate prodrug salts of

each were synthesized for preliminary in vivo studies to assess their potential efﬁcacy as VDAs.

1. Introduction

and chaotic in character.^2–4The primitive nature of the vascular

network in tumors makes it a promising target for cancer therapy.

As solid tumors grow beyond approximately 1 mm³in size, they

require an ever larger vascular network to supply oxygen and

nutrients to the cells and remove cellular waste products.¹Since

the vasculature feeding tumors tends to grow rapidly to keep up

with tumor expansion, it has a tendency to vary in diameter and

incorporate bulges and blind ends, rendering it somewhat fragile

There are two types of antivascular therapies: angiogenesis

inhibiting agents (AIAs) and vascular disrupting agents (VDAs).^5–7

AIAs inhibit the formation of new vasculature in developing

tumors, while VDAs damage the already existing tumor vascula-

ture.^6,8,9VDAs are further subdivided into biologics and small-

molecule anticancer agents. Inhibitors of tubulin polymerization

represent one class of small-molecule VDAs. These compounds dis-

rupt the tubulin-microtubule protein system and cause structural

changes to the endothelial cells lining the vasculature, in response

to cell signaling events. These morphological changes eventually

lead to irreversible damage to the tumor vasculature, thus starving

⇑

Corresponding author. Tel.: +1 254 710 4117; fax: +1 254 710 4272.

E-mail address: Kevin_Pinney@baylor.edu (K.G. Pinney).

http://dx.doi.org/10.1016/j.bmc.2015.10.012

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2

C. A. Herdman et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx

the tumor of nutrients and oxygen, ultimately leading to tumor

necrosis.^10–19

One class of VDAs interact with the colchicine site on b-tubulin

near the a,b-tubulin heterodimer interface. Two of the most potent

colchicine site binding VDAs are the natural products combretas-

tatin A-4 (CA4)²⁰and combretastatin A-1 (CA1)²¹originally iso-

lated from Combretum caffrum, the South African bushwillow tree

(Fig. 1). The corresponding phosphate prodrug salts combretastatin

A-4P (CA4P)^22,23and combretastatin A-1P (CA1P)¹⁵have improved

water solubility and have been extensively evaluated in both pre-

clinical experiments and clinical studies in humans.^24–29

Suzuki coupling to install the pendant aryl ring.⁴⁴They prepared

and evaluated a series of structurally diverse benzosuberene ana-

logues.⁴⁵Using KGP18 and KGP156 as models, we developed a ser-

ies of analogues to analyze further functional group modiﬁcations

for their effects on cytotoxicity and inhibition of tubulin

polymerization.

2. Results and discussion

2.1. Synthesis

Signiﬁcant structural similarities exist among the natural prod-

ucts colchicine, CA4, and CA1 (Fig. 1), including a trimethoxy phe-

nyl ring, a separate p-methoxy phenyl moiety, and bridging

functionality connecting the two rings at a comparable centroid

to centroid distance. The relative structural simplicity of CA4 has

inspired the synthesis of a vast array of synthetic analogues and

derivatives in which both aryl rings and the ethylene bridge have

been structurally modiﬁed. An early initial molecular design para-

digm led us to utilize clinically relevant non-steroidal, selective

estrogen receptor modulators (SERMs) and related compounds as

molecular templates modiﬁed to mimic colchicine and CA4.^37–39

This led us to the discovery and establishment of benzo[b]thio-

phene,^40–42benzofuran,^42,43dihydronaphthalene,^30–32benzo-

suberene,^30–34and indole-based³⁵analogues as potent inhibitors

of tubulin polymerization (Fig. 1). Two benzosuberene analogues

Twenty-two benzosuberene analogues (Fig. 2) were synthesized

and evaluated for both their ability to inhibit tubulin polymeriza-

tion and for their in vitro cytotoxicity against selected human can-

cer cell lines. Structural modiﬁcations to the R₁and R₂positions of

the fused aryl ring as well as the pendant aryl ring were explored in

order to evaluate their impact on tubulin dynamics and cytotoxic-

ity. The synthesis of each benzosuberene analogue involved a Wit-

tig oleﬁnation reaction followed by hydrogenation to afford

carboxylic acid derivatives 7–12. An intramolecular Friedel–Crafts

annulation facilitated by Eaton’s reagent (7.7 weight percent P₂O₅

in CH₃SO₃H)^46,47yielded benzosuberone analogues 13–18, which

were subsequently treated with requisite aryl-lithium reagents to

generate tertiary alcohols 19–27, which upon dehydration afforded

the ﬁnal benzosuberene analogues 28–36 (Scheme 1).

Concomitant elimination accompanied the addition of 4-meth-

oxyphenyl lithium, which resulted in benzosuberene analogue 37

(Scheme 2).

(referred to as KGP18^30,33and its amino congener KGP156^31,34

)

are especially promising anticancer agents based, in part, on their

pronounced cytotoxicity against human cancer cell lines and their

efﬁcacy as inhibitors of tubulin polymerization. Our previous stud-

ies in this area^30,33,34resulted in two separate synthetic strategies

towards the pendant 9-aryl, fused six-seven ring system present in

the benzosuberene analogues KGP18 and KGP156. These studies

included a variety of functional group modiﬁcations designed to

probe structural diversity as it relates to biological function.

Inspired by our original work with these and related benzo-

suberene analogues, Maderna and co-workers at Pﬁzer developed

a separate synthetic approach utilizing a ring-closing metathesis

(RCM) reaction to form the benzosuberene molecular core and a

Benzosuberone analogues 13 and 14 were also subjected to a

demethylation reaction using the ionic liquid [TMAH][Al₂Cl₇]⁴⁸to

afford phenolic derivatives 38–40. We previously demonstrated

the regioselective demethylation of compound 13.^33,48Protection

of the phenolic moieties as their corresponding tert-

butyldimethylsilyl (TBS) ethers followed by nucleophilic addition

with an appropriately substituted lithiated aryl ring produced ter-

tiary alcohols 44–50, which were subsequently dehydrated to yield

TBS protected benzosuberene analogues 51–57 (Scheme 3).

Removal of the TBS protecting groups upon treatment with TBAF

resulted in benzosuberene analogues 64–70 (Scheme 4).

Figure 1. Representative small-molecule inhibitors of tubulin polymerization: colchicine, combretastatins (CA4, CA1),^20,21dihydronaphthalene analogue (OXi6196),^30–32

benzosuberene analogues (KGP18 and KGP156),^30,33,34indole analogue (OXi8006)³⁵and benzo[b]furan analogue (BNC105).³⁶

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Figure 2. Compilation of synthesized benzosuberene analogues.

In our hands, desired benzosuberene analogues 61, 62, and 71

were not accessible by the methodology involving aryl lithium

addition, instead yielding recovered starting material. It is possible

that competing enolate formation was faster than 1,2-carbonyl

addition in these cases. Alternatively, the synthesis of analogues

61, 62, and 71 was accomplished using a Suzuki coupling to attach

the pendant aryl ring, similar to the methodology of Maderna and

co-workers.⁴⁴Vinyl triﬂates 58, 59, and 60 were reacted with the

corresponding boronic acids to generate target benzosuberene

analogues 61 and 62 and TBS protected compound 63, which, after

deprotection with TBAF, afforded benzosuberene analogue 71

(Scheme 4).

The double bond of benzosuberene 69 (KGP18) was reduced to

afford benzosuberane analogue 72, which was subsequently con-

verted to its corresponding phosphate salt 73. Benzosuberene ana-

logue 65 was also converted to its phosphate prodrug salt 74 under

analogous reaction conditions (Scheme 5).

of molecules of inhibitor bound to tubulin in a cell-based assay

versus a pure protein assay, as well as practical assay limits (in

the low micromolar range) in the pure protein assay (no cells

and no additional microtubule-associated proteins). Inhibitor gen-

erated interference within the dynamic that is inherent to the

tubulin-microtubule protein system in cells may inﬂuence signal

transduction leading to an ampliﬁcation of activity in cell-based

assays.^35,49An initial structure–activity analysis for the 22 com-

pounds for which we obtained biological data (Table 1), is perhaps

best accomplished by focusing on the studies on the presumptive

intracellular target, tubulin. The most extensive data were

obtained for effects on tubulin assembly, in part because we have

rarely observed substantial inhibition of colchicine binding (>50%,

with 5

Ten of the evaluated compounds were active inhibitors of tubu-

lin polymerization (IC₅₀values <20 M). Structural variability was

lM inhibitor) if the assembly IC₅₀is >3 lM.

l

tolerated (in terms of retained tubulin inhibitory activity) by the

incorporation of several groups (H, OCH₃, CH₃) at R₁while the pen-

dant 3,4,5-trimethoxyaryl motif reminiscent of CA4 and colchicine

was maintained. This mirrored our previous observations with

other functional groups [OH (parent KGP18), NH₂(parent

KGP156), Br, Cl] situated at R₁in this same molecular template.

Replacement of the R₂methoxy group with a hydroxyl group,

while either maintaining R₁= OH or modifying R₁to be a hydrogen

atom, led to analogues (70 and 68, respectively) that were also

active inhibitors of tubulin polymerization. Variation of the

methoxylation pattern (2,3,4-trimethoxy) within the pendant aryl

ring also led to active inhibitors of tubulin polymerization when R₁

was H or OH (62 and 71, respectively). Replacement of the

trimethoxyaryl ring with either a 3,5-bis-triﬂuoromethyl aryl ring

or a 4-methoxyaryl ring with maintenance of R₁= OH (compounds

65 and 67, respectively) resulted in benzosuberene analogues that

were still inhibitory against tubulin polymerization. Intriguingly,

the double-bond reduced analogue 72 was the most active

2.2. Biological evaluation

Each of the twenty-two analogues was evaluated biologically

for its ability to inhibit tubulin polymerization (cell free assay),

as well as its cytotoxicity against human cancer cell lines

[SK-OV-3 (ovarian), NCI-H460 (lung), and DU-145 (prostate)]. It

is important to note that these two assays, while providing

complementary structure activity relationship (SAR) information,

represent very different approaches to analyzing the biological

activity of the target benzosuberene analogues. It is common for

biologically active, small-molecule inhibitors of tubulin polymer-

ization to demonstrate IC₅₀values in the low micromolar range

(in this type of cell free assay) while demonstrating sub-micromo-

lar to nanomolar GI₅₀values in terms of cytotoxicity against

human cancer cell lines. This activity differential may be due, in

part, to requisite stoichiometry differences in regard to the number

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C. A. Herdman et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx

Scheme 1. Synthesis of benzosuberene analogues 28–36.

resulted in the loss of inhibitory activity (as observed with com-

pounds 31, 32, and 33). While ten of the analogues inhibited tubu-

lin assembly, only 29, 30, 62, and 72 demonstrated inhibition

values (1.0 lM, 1.6 lM, 1.2 lM, and 0.70 lM, respectively) compa-

rable to those observed with lead compounds KGP18 and CA4.

Molecular docking studies were carried out on several

analogues that were active inhibitors of tubulin polymerization

(compounds 29, 62 and 72), and compared to compound 33 with

an IC₅₀>20 lM (inactive) in this assay. Docking placed the

trimethoxyphenyl ring of all three active analogues in a similar

position to that of the trimethoxyphenyl moiety of N-deacetyl-N-

(2-mercaptoacetyl)-colchicine in the structure co-crystallized with

tubulin. In contrast, modeling placed multiple top conformations

of compound 33 with its trimethoxyphenyl ring outside of this

pocket (see Supplementary data).

Among the twenty-two benzosuberene analogues investigated

in this study, the most cytotoxic agents were compounds 29 and

62 (for example, GI₅₀= 0.0516 lM and 0.0432 lM, respectively,

Scheme 2. Synthesis of benzosuberene analogue 37.

inhibitor of tubulin assembly within the entire series of benzo-

suberene analogues analyzed. Loss of inhibition of tubulin polymer-

ization was observed when the trimethoxy aryl substituent was

replaced with an unsubstituted phenyl ring (28 and 64). While

the R₁methyl analogue 30 functioned as an inhibitor of tubulin poly-

merization, extension of the alkyl chain to ethyl, propyl, and butyl

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C. A. Herdman et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx

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Scheme 3. Synthesis of TBS protected analogues 51–57.

Scheme 4. Synthesis of benzosuberene analogues 61, 62, and 64–71.

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C. A. Herdman et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx

Scheme 5. Synthesis of reduced benzosuberane analogue 72 and phosphate salts 73 and 74.

Table 1

Inhibition of tubulin polymerization, percent inhibition of colchicine binding, and cytotoxicity of the target benzosuberene analogues

Compound

Inhibition of tubulin polymerization IC₅₀

(lM) SD

% inhibition of colchicine binding SD

GI₅₀(l

M) SRB assay^a

SK-OV-3

NCI-H460

DU-145

CA4

CA4P

KGP18

28

1.0^b

84 3 (1

nr

l

M), 98 0.007 (5

lM)

0.00455

0.00119

0.0000543^e

32.7

0.00223^c

0.00194^c

0.0000418^e

37.5

0.00327^c

0.00323^c

0.0000249^e

89.3

>40^b

1.4^d

>20

29

30

31

32

33

34

1.0 0.02

1.6 0.2

>20

37 5 (1

65 0.6 (5 l

nr

l

M), 72 0.8 (5

M)

lM)

0.0516

0.330

0.568

2.96

11.5

31.1

0.0527

0.422

0.763

3.32

16.1

25.5

0.0619

0.644

1.51

6.03

12.2

>20

52.1

35

36

3.1 0.03

>20

30 4 (5

nr

lM), 56 4 (50

lM)

0.277

20.5

0.593

33.4

0.708

48.3

37

>20

nr

40.7

57.7

68.7

61

>20

nr

6.96

10.5

26.2

62

64

65

66

1.2 0.007

>20

3.8 0.3

>20

36 5 (1

nr

8.5 4 (5

nr

l

M), 69 3 (5

l

M)

0.0432

0.557

4.81

0.120

0.652

4.39

0.0562

4.40

4.92

l

M), 37 5 (50

lM)

16.8

25.0

21.8

67

68

70

71

72

73

74

7.4 0.06

2.7 0.1

7.7 0.2

11 0.4

0.70 0.1

>20

nr

18.4

10.6

8.59

1.02

1.53

7.54

0.570

0.753

17.5

27 5 (5

nr

21 0.9 (1

nr

l

M)

0.527

0.346

3.53

0.408

0.357

17.2

0.647

0.691

4.24

0.141

0.145

16.3

lM), 67 0.6 (5

lM)

>20

nr = not reported.

a

Average of n P 3 independent determinations.

Data from Ref. 50.

For additional data, see Ref. 50.

Data from Ref. 33.

b

c

d

e

For additional data, see Ref. 30.

against the SK-OV-3 cell line). Both of these compounds bear tri-

methoxy aryl groups, but, unlike KGP18, they each contain a hydro-

gen atom at the R₁position, rather than a hydroxyl group. The

cytotoxicity, inhibition of colchicine binding, and the inhibition of

tubulin polymerization correlate well for these compounds. Com-

pounds 31 and 64 were not inhibitors of tubulin polymerization

(IC₅₀>20

lM), but they were found to be cytotoxic (GI₅₀<1 lM)

against two of the three cell lines utilized in this study. Although

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7

these compounds are structurally similar to others in this library,

4. Experimental section

4.1. Chemistry

they may have an alternate mechanism of inhibiting cell growth.

We note the strong antitubulin activity of compound 72, in which

the double bond in the seven-membered fused ring was reduced,

although this modiﬁcation appears to be associated with reduced

cytotoxicity. This reduction in cytotoxicity (for compound 72) cor-

relates with a decrease in the percent inhibition of colchicine

4.1.1. General materials and methods

Tetrahydrofuran (THF), dichloromethane, ethanol, methanol,

dimethylformamide (DMF), and acetonitrile were used in their

anhydrous forms. Reactions were performed under nitrogen gas,

unless otherwise speciﬁed. Thin-layer chromatography (TLC)

plates (precoated glass plates with silica gel 60 F254, 0.25 mm

thickness) were used to monitor reactions. Puriﬁcation of interme-

diates and products was carried out with a Biotage Isolera or Tele-

dyne Combiﬂash ﬂash puriﬁcation system using silica gel (200–400

mesh, 60 Å) or RP-18 pre-packed columns or manually in glass col-

umns. Intermediates and products synthesized were characterized

on the basis of their ¹H NMR (500 or 300 MHz), ¹³C NMR (125 or

75 MHz), ¹⁹F (470 MHz) and ³¹P NMR (200 or 120 MHz) spectro-

scopic data using a Varian VNMRS 500 MHz or a Bruker DPX

300 MHz instrument. Spectra were recorded in CDCl₃, D₂O, (CD₃)₂CO,

or CD₃OD. All chemical shifts are expressed in ppm (d), and peak

patterns are reported as broad (br), singlet (s), doublet (d), triplet

(t), quartet (q), pentet (p), sextet (sext), septet (sept), double

doublet (dd), double double doublet (ddd), 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

binding (at 1

three compounds (29, 62, and 72) are comparable in the colchicine

binding assay at 5 M.

lM) relative to compounds 29 and 62, although all

l

Two compounds, 65 and the strong tubulin inhibitor 72, were

selected for conversion to prodrugs (74 and 73, respectively) by

phosphorylation, in order to improve water-solubility and poten-

tially bioavailability. As with CA4 (phosphorylated to CA4P), this

synthetic transformation eliminated (IC₅₀>20 lM) the ability to

inhibit tubulin polymerization (cell-free assay), while the cytotox-

icity was maintained presumably due to phosphatase activity

present in the cell-based assay.

We previously demonstrated that dynamic bioluminescence

imaging (BLI) provides a facile indication of vascular disruption

in luciferase transfected tumors.^7,51,52Analysis is noninvasive,

and each tumor acts as its own control. Speciﬁcally, the substrate

luciferin normally diffuses into the blood stream following subcu-

taneous injection, and, when it reaches a tumor, light emission

occurs. Vascular disruption impairs delivery, and reduced light

emission is observed. Analogue 73 showed no obvious acute toxi-

city over 24 h to breast tumor bearing SCID mice following IP

administration of saline solutions delivering doses up to 40 mg/

kg. Doses of 20 or 30 mg/kg showed a similar modest reduction

of BLI signal 4 h after administration. At 40 mg/kg there was

approximately a 50% reduction of the emitted signal. In each case,

the signal generally returned to its original level within 24 h. By

comparison, saline controls showed a highly reproducible signal,

and CA4P (a well-established VDA in clinical development)

showed greater than 90% reduction at 4 h, which remained

depressed (>75%, after 24 h). These data are presented in Figures

3 and 4. Patterns of light emission are presented in Figure 5. The

signal intensity for saline emphasizes reproducibility of signal

(hence assessment of vasculature) for repeat measurements. As

reported previously, CA4P^7,51,52caused signiﬁcant vascular impair-

ment at 4 h. Compound 73 had little effect at 20 or 30 mg/kg, but

caused about 50% reduction in light emission at 40 mg/kg. While

these initial, preliminary studies at 20, 30, and 40 mg/kg suggested

a potential VDA mechanism for analogue 73, future dose escalation

studies will be necessary to establish a maximum tolerated dose

(MTD) in this mouse model and to conﬁrm the extent to which

analogue 73 is capable of disrupting tumor-associated vasculature.

(k = 190–400 nm),

a

Zorbax

XDB-C18

HPLC

column

(4.6 mm Åꢀ150 mm,

5

l

m), 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 0–40 min; post-time

10 min; ﬂow rate 1.0 mL/min; injection volume 20 lL; monitored

at wavelengths of 210, 230, 254, 280, and 320 nm. Mass spectrom-

etry was carried out under positive or negative ESI (electrospray

ionization) using

instrument.

a Thermo Scientiﬁc LTQ OrbitrapDiscovery

4.1.1.1.

(1).^31,33

5-(2⁰,3⁰-Dimethoxyphenyl)pent-4-enoic

To dissolved 3-(carboxypropyl)triphenyl phospho-

acid

nium bromide (13.04 g, 30.39 mmol) in THF (500 mL) was added

potassium tert-butoxide (7.43 g, 66.2 mmol), and the reaction mix-

ture was stirred at room temperature for 1 h. 2,3-Dimethoxyben-

zaldehyde (5.02 g, 30.1 mmol) dissolved in THF (100 mL) was

added, and the mixture was stirred at room temperature for

12 h. The THF was removed under reduced pressure, and the

resulting material was quenched with 2 M HCl (75 mL) and

extracted with EtOAc (3 ꢁ 100 mL). The combined organic layers

were evaporated under reduced pressure, and the crude reaction

product was puriﬁed by ﬂash chromatography using a pre-packed

100 g silica column [solvent A: EtOAc; solvent B: hexanes; gradi-

ent: 10% A/90% B (1 CV), 10% A/90% B ? 50% A/50% B (10 CV),

50% A/50% B (2 CV); ﬂow rate: 40 mL/min; monitored at 254 and

280 nm] to afford compound 1 (5.39 g, 21.5 mmol, 72%) as a yellow

oil. NMR characterization was conducted after the next step.

3. Conclusion

In summary, the results of these experiments have expanded

our SAR knowledge regarding effects that modiﬁcations of the ben-

zosuberene skeleton play in relationship to cytotoxicity and anti-

tubulin activity. The most promising analogues evaluated in this

study demonstrated inhibition of tubulin assembly comparable

to CA4 and KGP18, but these compounds had reduced inhibitory

effects on colchicine binding and on cell growth. Preliminary

in vivo BLI evaluation of the VDA capability of benzosuberene ana-

logue 73 against an MDA-MB-231-luc xenograft (in a SCID mouse

model) showed efﬁcacy, but, at the doses examined, the effect of

73 was less pronounced than that obtained with an established

VDA (in this case, CA4P) currently in clinical development. Future

studies with compound 73 and related benzosuberene analogues

involving dose escalation and other tumor models to assess vascu-

lar damage appear warranted.

4.1.1.2. 5-(2⁰,3⁰-Dimethoxyphenyl)pentanoic acid (7).^31,33,53To

dissolved carboxylic acid

1 (5.39 g, 21.5 mmol) in methanol

(100 mL) was added 10% palladium on carbon (0.43 g) and hydro-

gen gas. The reaction mixture was stirred at room temperature for

12 h and ﬁltered through Celite^Ò, and the Celite^Òwas washed with

EtOAc (3 ꢁ 50 mL). The combined organic phase (MeOH and

EtOAc) was evaporated under reduced pressure. The resulting

organic material was puriﬁed by ﬂash chromatography using a

pre-packed

100 g

silica

column

[solvent

A:

EtOAc;

solvent B: hexanes; gradient: 7% A/93% B (1 CV), 7% A/93% B ?

40% A/60% B (10 CV), 40% A/60% B (2 CV); ﬂow rate: 40 mL/min;

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C. A. Herdman et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx

Figure 3. BLI assessment of vascular disruption caused in MDA-MB-231-luc orthotopic human tumor xenografts by analogue 73. Dynamic BLI was performed at baseline

(bottom row), 4 h after VDA administration (middle), and after 24 h (top), and images are shown for representative mice 17 min after administering fresh luciferin substrate

on each occasion to each animal. Images show bioluminescent signal intensity overlaid on photographs of the mice. Analogue 73 was administered at 20, 30 or 40 mg/kg ip in

saline, and additional mice received saline control or CA4P (120 mg/kg) for comparison. Analogue 73 caused a reduced signal at all doses at 4 h with substantial recovery by

24 h.

monitored at 254 and 280 nm] to afford carboxylic acid 7 (4.45 g,

18.7 mmol, 82%) as a colorless oil. ¹H NMR (500 MHz, CDCl₃) d

11.67 (1H, s), 6.99 (1H, t, J = 8 Hz), 6.78 (2H, m), 3.86 (3H, s), 3.84

(3H, s), 2.68 (2H, t, J = 8 Hz), 2.41 (2H, t, J = 7.5 Hz), 1.70 (4H, m).

¹³C NMR (125 MHz, CDCl₃) d 180.2, 152.7, 147.1, 135.8, 123.8,

121.9, 110.2, 60.6, 55.6, 34.0, 30.8, 29.4, 24.5.

12 h. It was then poured over ice and neutralized with sodium

bicarbonate. The reaction mixture was extracted with EtOAc

(3 ꢁ 50 mL), and the combined organic phase was dried over

sodium sulfate and evaporated under reduced pressure. The reac-

tion product was puriﬁed by ﬂash chromatography using a pre-

packed 100 g silica column [solvent A: EtOAc; solvent B: hexanes;

gradient: 7% A/93% B (1 CV), 7% A/93% B ? 40% A/60% B (10 CV),

40% A/60% B (2 CV); ﬂow rate: 40 mL/min; monitored at 254 and

280 nm] to afford benzosuberone 13 (2.43 g, 11.0 mmol, 74%) as

a light yellow solid. ¹H NMR (500 MHz, CDCl₃) d 7.33 (1H, d,

J = 8.5 Hz), 6.67 (1H, d, J = 9 Hz), 3.72 (3H, s), 3.62 (3H, s), 2.83

4.1.1.3. 1,2-Dimethoxy-6,7,8,9-tetrahydro-5H-benzo[7]annulen-

5-one (13).^31,33,54

To carboxylic acid 7 (3.55 g, 14.9 mmol)

was added Eaton’s reagent (29 mL, 3 g per mmol of compound

7), and the reaction mixture was stirred at room temperature for

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C. A. Herdman et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx

9

4.1.1.4. [TMAH][Al₂Cl₇].⁴⁸

To dry dichloromethane (150 mL)

was added AlCl₃(19.84 g, 149.1 mmol), and the mixture was stir-

red and cooled to 0 °C. Trimethylamine hydrochloride (7.11 g,

74.5 mmol) was added, and the reaction mixture was allowed to

stir for 2 h at room temperature. The resulting liquid was stored

at room temperature under nitrogen.

4.1.1.5.

1-Hydroxy-2-dimethoxy-6,7,8,9-tetrahydro-5H-benzo

To benzosuberone 13 (1.01 g,

[7]annulen-5-one (38).^31,33,55

4.54 mmol) in a 20 mL microwave vial was added [TMAH][Al₂Cl₇]

(18.3 mL, 9.08 mmol), and the mixture was subjected to micro-

wave irradiation for 1 h at 80 °C on high absorbance. The solution

was then poured into water (50 mL) and extracted with EtOAc

(3 ꢁ 25 mL). The combined organic phase was dried over sodium

sulfate and evaporated under reduced pressure. The crude reaction

product was puriﬁed by ﬂash chromatography using a pre-packed

50 g silica column [solvent A: EtOAc; solvent B: hexanes; gradient:

7% A/93% B (1 CV), 7% A/93% B ? 60% A/40% B (10 CV), 60% A/40% B

(2 CV); ﬂow rate: 50 mL/min; monitored at 254 and 280 nm] to

afford benzosuberone 38 (0.61 g, 3.0 mmol, 65%) as a yellow oil.

¹H NMR (500 MHz, CDCl₃) d 7.24 (1H, d, J = 8.5 Hz), 6.67 (1H, d,

J = 9 Hz), 6.26 (1H, s), 3.78 (3H, s), 2.93 (2H, t, J = 5.5 Hz), 2.61

(2H, t, J = 6.5 Hz), 1.71 (4H, m). ¹³C NMR (125 MHz, CDCl₃) d

205.0, 149.5, 142.5, 133.0, 127.8, 120.7, 107.9, 55.9, 40.6, 24.4,

23.0, 21.2.

Figure 4. Vascular disruption caused by analogue 73. Relative signal intensity is

plotted for the mice shown in Figure 3.

(2H, t, J = 6 Hz), 2.50 (2H, t, J = 6 Hz), 1.66 (2H, p, J = 6.5 Hz), 1.59

(2H, p, J = 6.5 Hz). ¹³C NMR (125 MHz, CDCl₃) d 204.2, 155.9,

145.8, 135.5, 132.6, 125.2, 109.6, 60.8, 55.6, 40.4, 24.7, 23.0, 20.7.

Analogue 73 (40 mg/kg)

Analogue 73 (30 mg/kg)

CA4P (120 mg/kg)

Control (NaCl)

Time (mins)

Figure 5. Dynamic bioluminescence with respect to vascular disruption. Graphs show evolution of light emission from individual MDA-MB-231-luc tumors following

administration of luciferin substrate subcutaneously in the fore-back region of each mouse at baseline (blue) and 4 h after administration of agent (red). Analogue 73 had a

modest effect at 30 mg/kg and a greater effect at 40 mg/kg. By comparison control saline showed a high degree of reproducibility and CA4P showed >90% reduction in light

emission.

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C. A. Herdman et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx

4.1.1.6.

1-((tert-Butyldimethylsilyl)oxy)-2-dimethoxy-6,7,8,9-

(125 MHz, CDCl₃) d 153.0, 151.7, 148.5, 142.4, 140.3, 135.8,

132.5, 131.1, 128.2, 124.9, 120.7, 108.0, 106.6, 105.2, 60.6, 60.4,

55.9, 54.6, 33.8, 26.2, 25.5, 24.2, 19.0, ꢂ3.9.

tetrahydro-5H-benzo[7]annulen-5-one (41).^31,33Benzosuberone

38 (2.16 g, 10.5 mmol) was dissolved in dimethylformamide

(50 mL). TBSCl (3.16 g, 21.0 mmol) and DIPEA (5.50 mL, 31.6 mmol)

were added, and the solution was stirred for 12 h at room tempera-

ture. The reaction mixture was washed with water and extracted

with EtOAc (5 ꢁ 30 mL). The combined organic phase was dried over

sodium sulfate and evaporated under reduced pressure. The crude

reaction product was puriﬁed by ﬂash chromatography using a

pre-packed 100 g silica column [solvent A: EtOAc; solvent B: hex-

anes; gradient: 2% A/98% B (1 CV), 2% A/98% B ? 20% A/80% B

(10 CV), 20% A/80% B (2 CV); ﬂow rate: 50 mL/min; monitored at

254 and 280 nm] to afford TBS protected analogue 41 (2.37 g,

7.38 mmol, 71%) as a white solid. ¹H NMR (500 MHz, CDCl₃) d 7.23

(1H, d, J = 8.5 Hz), 6.62 (1H, d, J = 9 Hz), 3.67 (3H, s), 2.88 (2H, t,

J = 5.5 Hz), 2.53 (2H, t, J = 5.5 Hz), 1.64 (4H, m), 0.89 (9H, s), 0.06

(6H, s). ¹³C NMR (125 MHz, CDCl₃) d 204.3, 152.9, 141.6, 132.9,

132.7, 122.2, 108.6, 54.6, 40.5, 26.0, 24.6, 23.8, 21.1, 18.8, ꢂ4.0.

4.1.1.9. 3-Methoxy-9-(2⁰,3,⁰4⁰-trimethoxyphenyl)-6,7-dihydro-

5H-benzo[7]annulen-4-ol

(71).

TBS-protected

benzo-

suberene 63 (0.068 g, 0.146 mmol) was dissolved in THF (5 mL),

and tetrabutylammonium ﬂuoride (0.18 mL, 0.18 mmol) was

added. The reaction was stirred at room temperature for 12 h,

washed with water, extracted with EtOAc (3 ꢁ 25 mL), dried over

sodium sulfate, and evaporated under reduced pressure. The crude

reaction product was puriﬁed by ﬂash chromatography using a

pre-packed 10 g silica column [solvent A: EtOAc; solvent B: hex-

anes; gradient: 7% A/93% B (1 CV), 7% A/93% B ? 60% A/40% B

(10 CV), 60% A/40% B (2 CV); ﬂow rate: 12 mL/min; monitored at

254 and 280 nm] to afford benzosuberene analogue 71 (0.053 g,

0.15 mmol, 96%) as a white solid. ¹H NMR (500 MHz, CDCl₃) d

6.91 (1H, d, J = 8.5 Hz), 6.63 (1H, d, J = 8.5 Hz), 6.61 (1H, d,

J = 8.5 Hz), 6.37 (1H, d, J = 8.5 Hz), 6.10 (1H, t, J = 7 Hz), 5.71 (1H,

s), 3.87 (3H, s), 3.85 (3H, s), 3.82 (3H, s), 3.42 (3H, s), 2.87 (2H, t,

J = 7 Hz), 2.15 (2H, p, J = 7 Hz), 1.96 (2H, q, J = 7 Hz). ¹³C NMR

(125 MHz, CDCl₃) d 153.0, 151.8, 144.7, 142.39, 142.36, 140.0,

136.2, 131.0, 128.6, 126.8, 125.0, 119.0, 107.3, 106.6, 60.7, 60.5,

55.94, 55.90, 33.6, 25.6, 23.4. HRMS: Obsd 379.1516 [M+Na⁺],

Calcd for C₂₁H₂₄O₅Na: 379.1516. HPLC (Method B): 16.18 min.

4.1.1.7.

dro-5H-benzo[7]annulen-9-yl

(60). To an oven-dried ﬂask, diisopropylamine (0.18 mL,

4-((tert-Butyldimethylsilyl)oxy)-3-methoxy-6,7-dihy-

triﬂuoromethanesulfonate

1.3 mmol) dissolved in THF (50 mL) was added and cooled to

ꢂ78 °C, and n-BuLi (0.51 mL, 1.3 mmol) was added. The reaction

mixture was stirred for 15 min. TBS protected 41 (0.37 g,

1.2 mmol) dissolved in THF (10 mL) was added dropwise, and the

reaction mixture was stirred for 2 h at ꢂ78 °C. N-Phenyl-bis(triﬂu-

oromethanesulfonimide) (0.45 g, 1.3 mmol) dissolved in THF

(10 mL) was then added dropwise, and the reaction mixture was

stirred for 12 h while warming from ꢂ78 °C to room temperature.

After 12 h, the THF was evaporated under reduced pressure, and

the resulting solid was washed with water (50 mL) and extracted

with EtOAc (3 ꢁ 50 mL). The organic layer was dried over sodium

sulfate and evaporated under reduced pressure. The crude reaction

product was puriﬁed by ﬂash chromatography using a pre-packed

25 g silica column [solvent A: EtOAc; solvent B: hexanes; gradient:

2% A/98% B (1 CV), 2% A/98% B ? 20% A/80% B (10 CV), 20% A/80% B

(2 CV); ﬂow rate: 25 mL/min; monitored at 254 and 280 nm] to

afford triﬂate 60 (0.17 g, 0.38 mmol, 33%) as a yellow oil. ¹H

NMR (500 MHz, CDCl₃) d 7.10 (1H, d, J = 8.5 Hz), 6.79 (1H, d,

J = 8.5 Hz), 6.09 (1H, t, J = 6.5 Hz), 3.82 (3H, s), 2.88 (2H, t,

J = 6 Hz), 2.15 (2H, q, J = 6.5 Hz), 2.03 (2H, p, J = 6.5 Hz), 1.03 (9H,

s), 0.20 (6H, s). ¹³C NMR (125 MHz, CDCl₃) d 150.8, 146.5, 132.9,

130.9, 129.9, 126.0, 120.9, 119.7, 108.8, 54.7. 30.5, 26.0, 25.0,

24.5, 18.9, 4.0.

4.1.1.10. 3,4-Dimethoxy-6,7-dihydro-5H-benzo[7]annulen-9-yl

triﬂuoromethanesulfonate (58).

Diisopropylamine (0.84 mL,

6.0 mmol) was dissolved in THF (20 mL) and cooled to ꢂ78 °C.

n-BuLi (2.4 mL, 6.0 mmol) was added dropwise, and the solution

was stirred for 15 min. Benzosuberone 13 (1.2 g, 5.4 mmol)

dissolved in THF (10 mL) was added dropwise, and the reaction

was stirred for 2 h at ꢂ78 °C. N-Phenyl-bis(triﬂuoromethanesulfon-

imide) (2.14 g, 5.99 mmol) dissolved in THF (10 mL) was then added

dropwise, and the reaction mixture was stirred for 12 h while

warming from ꢂ78 °C to room temperature. After 12 h, the THF

was evaporated under reduced pressure, and the resulting solid

was washed with water (50 mL) and extracted with EtOAc

(3 ꢁ 50 mL). The organic layer was dried over sodium sulfate and

evaporated under reduced pressure. The crude reaction product

was puriﬁed by ﬂash chromatography using a pre-packed 50 g silica

column [solvent A: EtOAc; solvent B: hexanes; gradient: 5% A/95% B

(1 CV), 5% A/95% B ? 40% A/60% B (10 CV), 40% A/60% B (2 CV); ﬂow

rate: 50 mL/min; monitored at 254 and 280 nm] to afford triﬂate 58

(1.01 g, 2.87 mmol, 50%) as a yellow oil. NMR characterization was

performed after the next step.

4.1.1.8.

6,7-dihydro-5H-benzo[7]annulen-4-yl)oxy)dimethylsilane

(63). Triﬂate 60 (0.17 g, 0.38 mmol) was dissolved in THF

(25 mL) and 2,3,4-trimethoxyphenyl boronic acid (0.09 g,

0.41 mmol), barium hydroxide octahydrate (0.18 g, 0.57 mmol),

tert-Butyl((3-methoxy-9-(2⁰,3⁰,4⁰-trimethoxyphenyl)-

4.1.1.11. 3,4-Dimethoxy-9-(2⁰,3⁰,4⁰-trimethoxyphenyl)-6,7-dihy-

dro-5H-benzo[7]annulene

(61).

Triﬂate

58

(0.46 g,

1.3 mmol) was dissolved in THF (25 mL) and 2,3,4-trimethoxyphe-

nyl boronic acid (0.31 g, 1.4 mmol), barium hydroxide octahydrate

(0.62 g, 2.0 mmol), and tetrakis(triphenylphosphine)palladium(0)

(0.05 g, 0.04 mmol) were added to the solution, which was reﬂuxed

at 80 °C for 2 h. The solution was then ﬁltered through Celite^Ò, and

the Celite^Òwas washed with dichloromethane. The organic solu-

tion (dichloromethane and THF) was evaporated under reduced

pressure. The crude reaction product was puriﬁed using ﬂash chro-

matography using a pre-packed 100 g silica column [solvent A:

EtOAc; solvent B: hexanes; gradient: 7% A/93% B (1 CV), 7%

A/93% B ? 45% A/55% B (10 CV), 45% A/55% B (2 CV); ﬂow rate:

50 mL/min; monitored at 254 and 280 nm] to afford benzo-

suberene 61 (0.07 g, 0.19 mmol, 15%) as a yellow solid. ¹H NMR

(500 MHz, CDCl₃) d 6.91 (1H, d, J = 8.5 Hz), 6.64 (2H, overlapping

d, J = 8.5 Hz), 6.58 (1H, d, J = 8.5 Hz), 6.11 (1H, t, J = 7 Hz), 3.86

(3H, s), 3.84 (3H, s), 3.82 (3H, s), 3.81 (3H, s), 3.38 (3H, s), 2.86

and

tetrakis(triphenylphosphine)palladium(0)

(0.013 g,

0.011 mmol) were added to the solution and reﬂuxed at 80 °C for

2 h. The solution was then ﬁltered through Celite^Ò, and the Celite^Ò

was washed with dichloromethane. The organic solution (dichlor-

omethane and THF) was evaporated under reduced pressure. The

crude reaction product was puriﬁed using ﬂash chromatography

using a pre-packed 10 g silica column [solvent A: EtOAc; solvent

B: hexanes; gradient: 2% A/98% B (1 CV), 2% A/98% B ? 20%

A/80% B (10 CV), 20% A/80% B (2 CV); ﬂow rate: 12 mL/min; mon-

itored at 254 and 280 nm] to afford benzosuberene 63 (0.07 g,

0.15 mmol, 41%) as a yellow oil. ¹H NMR (500 MHz, CDCl₃) d 6.91

(1H, d, J = 8.5 Hz), 6.64 (1H, d, J = 8.5 Hz), 6.60 (1H, d, J = 8 Hz),

6.44 (1H, d, J = 8 Hz), 6.10 (1H, t, J = 7 Hz), 3.87 (3H, s), 3.83 (3H,

s), 3.76 (3H, s), 3.38 (3H, s), 2.89 (2H, t, J = 6.5 Hz), 2.12 (2H, p,

J = 7 Hz), 1.95 (2H, q, J = 7 Hz), 1.05 (9H, s), 0.22 (6H, s). ¹³C NMR

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C. A. Herdman et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx

11

(2H, t, J = 7 Hz), 2.15 (2H, p, J = 6 Hz), 1.95 (2H, q, J = 7 Hz). ¹³C NMR

(125 MHz, CDCl₃) d 153.1, 151.7, 151.0, 146.2, 142.4, 140.1, 135.9,

134.9, 130.8, 128.4, 124.9, 123.5, 108.9, 106.7, 61.2, 60.6, 60.3, 55.9,

55.6, 34.4, 25.4, 23.9. HRMS: Obsd 393.1740 [M+Na⁺], Calcd for

4.1.1.15.

6,7,8,9-tetrahydro-5H-benzo[7]annulen-1-yl)oxy)(tert-butyl)

dimethylsilane (45). 1-Bromo-3,5-bis(triﬂuoromethyl)ben-

((5-(3⁰,5⁰-Bis(triﬂuoromethyl)phenyl)-2-methoxy-

zene (0.36 g, 1.2 mmol) was dissolved in THF (13 mL), and the

reaction ﬂask was cooled to ꢂ78 °C. n-BuLi (0.55 mL, 2.5 M) was

added to the reaction mixture, which was stirred for 1 h. Ketone

41 (0.29 g, 0.91 mmol) was dissolved in THF (5 mL) and slowly

added to the reaction mixture over a period of 15 min. The reaction

mixture was stirred for 20 h while warming from ꢂ78 °C to room

temperature. The reaction mixture was diluted with H₂O (25 mL)

and extracted with EtOAc (2 ꢁ 25 mL), and the organic extract

was dried over Na₂SO₄, ﬁltered, concentrated under reduced pres-

sure, and puriﬁed by ﬂash chromatography using a pre-packed

25 g silica column [solvent A: EtOAc; solvent B: hexanes; gradient:

7% A/93% B (1 CV), 7% A/93% B ? 60% A/40% B (10 CV), 60% A/40% B

(2 CV); ﬂow rate: 25 mL/min; monitored at 254 and 280 nm] to

afford alcohol 45 (0.40 g, 0.75 mmol, 82%) as a yellow solid. ¹H

NMR (CDCl₃, 500 MHz) d 7.77 (1H, s), 7.74 (2H, s), 6.95 (1H, d,

J = 8.7 Hz), 6.70 (1H, d, J = 8.7 Hz), 3.80 (3H, s), 3.32 (1H, ddd,

J = 14.8, 7.6, 2.0 Hz), 2.57 (1H, ddd, J = 14.2, 7.6, 3.0 Hz), 2.41 (1H,

s), 2.34–2.21 (1H, m), 2.16 (1H, ddd, J = 13.8, 10.3, 3.1 Hz), 1.97–

1.90 (1H, m), 1.75–1.68 (1H, m), 1.67–1.57 (1H, m), 1.56–1.48

(1H, m), 0.99 (9H, s), 0.18 (3H, s), 0.17 (3H, s). ¹³C NMR (CDCl₃,

C₂₂H₂₆O₅Na: 393.1672. HPLC (Method B): 18.25 min.

4.1.1.12. 1-((tert-Butyldimethylsilyl)oxy)-2-methoxy-5-phenyl-

6,7,8,9-tetrahydro-5H-benzo[7]annulen-5-ol (44). To an

oven dried ﬂask, THF (50 mL) and phenyl bromide (0.69 mL,

6.5 mmol) were added, and the solution was cooled to ꢂ78 °C. n-

BuLi (2.74 mL, 6.86 mmol) was slowly added to the reaction mix-

ture, which was then stirred at ꢂ78 °C for 1 h. TBS-protected 41

(1.55 g, 4.83 mmol) in THF (25 mL) was then added dropwise to

the ﬂask, and the reaction mixture was stirred while warming from

ꢂ78 °C to room temperature over 12 h. The reaction mixture was

washed with water and extracted with EtOAc (3 ꢁ 50 mL). The

combined organic phase was dried over sodium sulfate and evap-

orated under reduced pressure. The crude reaction product was

puriﬁed by ﬂash chromatography using a pre-packed 50 g silica

column [solvent A: EtOAc; solvent B: hexanes; gradient: 2%

A/98% B (1 CV), 2% A/98% B ? 20% A/80% B (10 CV), 20% A/80% B

(2 CV); ﬂow rate: 50 mL/min; monitored at 254 and 280 nm] to

afford tertiary alcohol 44 (0.80 g, 2.01 mmol, 42%) as a clear oil.

NMR characterization was performed after the next step.

125 MHz)

d 149.9, 149.7, 142.5, 137.1, 132.7, 131.6 (q,

J = 33.2 Hz), 127.3 (q, J = 3 Hz), 123.5 (q, J = 272.8 Hz), 121.2 (hept,

J = 3.8 Hz), 120.3, 108.6, 79.8, 54.8, 41.6, 26.7, 26.2, 25.3, 25.3, 19.1,

ꢂ3.94, ꢂ3.95.

4.1.1.13. tert-Butyl((3-methoxy-9-phenyl-6,7-dihydro-5H-benzo

[7]annulen-4-yl)oxy)dimethylsilane

(51).

Acetic

acid

(10 mL) was added to alcohol 44 (0.80 g, 2.0 mmol), and the reac-

tion mixture was stirred for 12 h at room temperature. The mixture

was washed with water and extracted with EtOAc (3 ꢁ 30 mL). The

combined organic phase was dried over sodium sulfate. The

organic phase was evaporated under reduced pressure, and the

crude reaction product was puriﬁed by ﬂash chromatography

using a pre-packed 10 g silica column [solvent A: EtOAc; solvent

B: hexanes; gradient: 2% A/98% B (1 CV), 2% A/98% B ? 20%

A/80% B (10 CV), 20% A/80% B (2 CV); ﬂow rate: 12 mL/min; mon-

itored at 254 and 280 nm] to afford benzosuberene 51 (0.38 g,

1.0 mmol, 49%) as a yellow solid. ¹H NMR (500 MHz, CDCl₃) d

7.28 (5H, m), 6.70 (1H, d, J = 8 Hz), 6.60 (1H, d, J = 8.5 Hz), 6.37

(1H, t, J = 7.5 Hz), 3.81 (3H, s), 2.79 (2H, t, J = 7 Hz), 2.13 (2H, p,

J = 7 Hz), 1.98 (2H, q, J = 7.5 Hz), 1.07 (9H, s), 0.26 (6H, s). ¹³C

NMR (125 MHz, CDCl₃) d 148.6, 143.0, 142.8, 141.6, 134.1, 133.3,

128.0, 127.3, 126.8, 122.1, 108.8, 108.4, 54.7, 33.8, 26.2, 25.6,

24.2, 19.0, ꢂ3.80.

4.1.1.16. ((9-(3⁰,5⁰-Bis(triﬂuoromethyl)phenyl)-3-methoxy-6,7-

dihydro-5H-benzo[7]annulen-4-yl)oxy)(tert-butyl)dimethylsi-

lane (52).

2 M HCl (8 mL, 16 mmol) was added to a well-stirred

solution of alcohol 45 (0.68 g, 1.3 mmol) in EtOH (5 mL), and the

reaction mixture was stirred for 12 h at ambient temperature. The

reaction mixture was then extracted with EtOAc (4 ꢁ 15 mL), and

the organic extract was dried over Na₂SO₄, ﬁltered, concentrated

under reduced pressure, and puriﬁed by ﬂash chromatography using

a pre-packed 25 g silica column [solvent A: EtOAc; solvent B: hex-

anes; gradient: 2% A/98% B (1 CV), 2% A/98% B ? 15% A/85% B

(10 CV), 15% A/85% B (2 CV); ﬂow rate: 25 mL/min; monitored at

254 and 280 nm] to afford benzosuberene 52 (0.43 g, 0.84 mmol,

66%) as a white solid. ¹H NMR (CDCl₃, 500 MHz) d 7.77 (1H, s),

7.74 (2H, s), 6.73 (1H, d, J = 8.5 Hz), 6.50 (1H, t, J = 7.1 Hz), 6.49

(1H, d, J = 8.5 Hz), 3.84 (3H, s), 2.80 (2H, t, J = 6.9 Hz), 2.16 (2H, p,

J = 7.0 Hz), 2.04 (2H, q, J = 7.1 Hz), 1.07 (9H, s), 0.27 (6H, s). ¹³C

NMR (CDCl₃, 125 MHz) d 149.3, 145.0, 142.1, 141.1, 133.5, 132.5,

131.5 (q, J = 33.3 Hz), 130.8, 128.0 (q, J = 3.4 Hz), 123.7 (q,

J = 272.7 Hz), 121.9, 120.6 (hept, J = 3.7 Hz), 109.0, 54.8, 33.8, 26.3,

26.0, 24.4, 19.2, ꢂ3.6.

4.1.1.14. 3-Methoxy-9-phenyl-6,7-dihydro-5H-benzo[7]annu-

len-4-ol (64).

TBS-protected benzosuberene 51 (0.38 g,

1.0 mmol) was dissolved in THF (25 mL), TBAF (1.20 mL,

1.20 mmol) was added, and the reaction mixture was stirred at

room temperature for 12 h. The solution was washed with water

and extracted with EtOAc (3 ꢁ 30 mL). The combined organic

phase was dried over sodium sulfate and evaporated under

reduced pressure. The crude reaction product was puriﬁed by ﬂash

chromatography using a pre-packed 10 g silica column [solvent A:

EtOAc; solvent B: hexanes; gradient: 3% A/97% B (1 CV), 3% A/97%

B ? 30% A/70% B (10 CV), 30% A/70% B (2 CV); ﬂow rate: 12 mL/

min; monitored at 254 and 280 nm] to afford benzosuberene ana-

logue 64 (0.12 g, 0.45 mmol, 44%) as a pale yellow oil. ¹H NMR

(500 MHz, CDCl₃) d 7.28 (5H, m), 6.71 (1H, d, J = 8.5 Hz), 6.54

(1H, d, J = 8.5 Hz), 6.38 (1H, t, J = 7.5 Hz), 5.77 (1H, s), 3.91 (3H,

s), 2.79 (2H, t, J = 7 Hz), 2.16 (2H, p, J = 7 Hz), 1.99 (2H, q,

J = 7 Hz). ¹³C NMR (125 MHz, CDCl₃) d 145.0, 142.8, 142.7, 142.4,

134.6, 128.02, 128.00, 127.8, 127.6, 126.9, 120.6, 107.7, 55.9,

33.5, 25.7, 23.5. HRMS: Obsd 267.1385 [M+H⁺], Calcd for

4.1.1.17.

dihydro-5H-benzo[7]annulen-4-ol (65).

9-(3⁰,5⁰-Bis(triﬂuoromethyl)phenyl)-3-methoxy-6,7-

To solution of

a

TBS-protected analogue 52 (0.43 g, 0.84 mmol) in THF (5 mL) was

added TBAF (1.0 mL, 1 M in THF), and the reaction mixture was

stirred for 18 h at ambient temperature and then concentrated

under reduced pressure. The reaction mixture was washed with

water (5 mL) and extracted with EtOAc (3 ꢁ 10 mL). The organic

extract was dried over Na₂SO₄, ﬁltered, concentrated under

reduced pressure, and puriﬁed by ﬂash chromatography using a

pre-packed 10 g silica column [solvent A: EtOAc; solvent B: hex-

anes; gradient: 6% A/94% B (1 CV), 6% A/94% B ? 50% A/50% B

(10 CV), 50% A/50% B (2 CV); ﬂow rate: 12 mL/min; monitored at

254 and 280 nm] to afford phenolic benzosuberene analogue 65

(0.26 g, 0.66 mmol, 78%) as

a

white solid. ¹H NMR (CDCl₃,

500 MHz) d 7.77 (1H, s), 7.74 (2H, s), 6.74 (1H, d, J = 8.3 Hz), 6.51

(1H, t, J = 7.3 Hz), 6.45 (1H, d, J = 8.4 Hz), 5.83 (1H, s), 3.93 (3H,

C₁₈H₁₉O₂: 267.1380. HPLC (Method B): 17.89 min.

Please cite this article in press as: Herdman, C. A.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.10.012

12

C. A. Herdman et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx

s), 2.79 (2H, t, J = 6.9 Hz), 2.19 (2H, p, J = 7.1 Hz), 2.05 (2H, q,

J = 7.2 Hz). ¹³C NMR (CDCl₃, 125 MHz) d 145.8, 144.9, 142.9,

140.8, 133.0, 131.5 (q, J = 33.4 Hz), 131.2, 128.03, 127.99, 123.6

(q, J = 272.8 Hz), 120.6 (hept, J = 3.9 Hz), 120.4, 108.2, 56.1, 33.4,

26.1, 23.7. ¹⁹F NMR (CDCl₃, 470 MHz) d ꢂ62.80. HRMS: Obsd

401.0963 [M–H]^ꢂ, Calcd for C₂₀H₁₅F₆O₂: 401.0976. HPLC (Method

B): 20.39 min.

brine, dried over Na₂SO₄, ﬁltered, evaporated under reduced pres-

sure, and puriﬁed by ﬂash chromatography using a pre-packed

25 g silica column [solvent A: EtOAc, solvent B: hexanes; gradient:

5% A/95% B (1 CV), 5% A/95% B ? 50% A/50% B (10 CV), 50% A/50% B

(2 CV); ﬂow rate: 25 mL/min; monitored at 254 and 280 nm] to

afford a clear oil that solidiﬁed as a colorless solid of TBS-protected

benzosuberene analogue 54 (0.43 g, 1.1 mmol, 85%). ¹H NMR

(CDCl₃, 500 MHz) d 7.20 (2H, d, J = 8.7 Hz), 6.83 (2H, d, J = 8.8 Hz),

6.68 (1H, d, J = 8.4 Hz), 6.58 (1H, d, J = 8.4 Hz), 6.26 (1H, t,

J = 7.3 Hz), 3.81 (3H, s), 3.79 (3H, s), 2.75 (2H, t, J = 6.9 Hz), 2.09

(2H, p, J = 7.1 Hz), 1.93 (2H, q, J = 7.2 Hz), 1.04 (9H, s), 0.23 (6H,

s). ¹³C NMR (CDCl₃, 126 MHz) d 158.7, 148.5, 142.4, 141.5, 135.5,

134.3, 133.3, 129.0, 125.7, 122.0, 113.4, 108.3, 55.3, 54.7, 33.94,

26.2, 25.5, 24.2, 19.0, ꢂ3.9.

4.1.1.18.

dihydro-5H-benzo[7]annulen-4-yl

(74). To well-stirred solution of phenol 65 (0.10 g,

9-(3⁰,5⁰-Bis(triﬂuoromethyl)phenyl)-3-methoxy-6,7-

disodium phosphate

a

0.25 mmol) in CH₂Cl₂(10 mL), POCl₃(153.3 mg, 1.00 mmol) and

pyridine (70.8 mg, 0.9 mmol) were added to the reaction ﬂask.

After the reaction mixture was stirred for 15 h at ambient temper-

ature, the solvent was evaporated under reduced pressure. Satu-

rated aqueous Na₂CO₃(20 mL) was added to the ﬂask, and the

reaction mixture was stirred for another 2 h. The reaction mixture

was concentrated to dryness with a stream of N₂gas and puriﬁed

by ﬂash chromatography using a prepacked C-18 30 g reversed

phase column [solvent A: acetonitrile; solvent B: water; gradient:

30% A/70% B (1 CV), 30% A/70% B ? 100% A/0% B (10 CV), 100%

A/0% B (2 CV); ﬂow rate: 25 mL/min; monitored at 254 and

280 nm] to afford phosphate salt 74 (0.043 g, 0.082 mmol, 33%)

as a white solid. ¹H NMR (D₂O, 500 MHz) d 7.96 (1H, s), 7.87 (2H,

s), 6.91 (1H, d, J = 8.6 Hz), 6.72 (1H, d, J = 8.4 Hz), 6.62 (1H, t,

J = 7.4 Hz), 3.84 (3H, s), 2.76 (2H, t, J = 7.0 Hz), 2.17 (2H, p,

J = 7.1 Hz), 1.97 (2H, q, J = 7.2 Hz). ¹³C NMR (CD₃OD, 125 MHz) d

153.1, 146.6, 142.0, 141.9, 137.2, 133.3, 132.6 (q, J = 33.0 Hz),

132.4, 129.0 (q, J = 2.5 Hz), 124.9 (q, J = 271.3 Hz), 125.2 (hept,

J = 3.8 Hz), 121.2, 111.1, 56.4, 34.6, 26.9, 25.9. ¹⁹F NMR (D₂O,

470 MHz) d ꢂ62.8. ³¹P NMR (D₂O, 200 MHz) d ꢂ3.5. HRMS: Obsd

527.0429 [M+H]⁺, Calcd for C₁₉H₁₆F₆O₂Na₂O_5-P: 527.0429. HPLC

(Method A): 13.79 min.

4.1.1.21.

benzo[7]annulen-4-ol (67).

3-Methoxy-9-(4⁰-methoxyphenyl)-6,7-dihydro-5H-

The TBS-protected analogue 54

(0.33 g, 0.8 mmol) was dissolved in THF (5 mL). To the solution,

TBAF–3H₂O (0.96 mmol) was added, and the mixture was stirred

for 3 h at room temperature. The reaction was quenched with

water (15 mL), followed by the evaporation of organic solvent

under reduced pressure. The resultant aqueous phase was then

extracted with EtOAc (3 ꢁ 20 mL). The combined organic extracts

were washed with brine solution, dried over Na₂SO₄, ﬁltered, evap-

orated under reduced pressure, and puriﬁed by ﬂash chromatogra-

phy using a pre-packed 40 g silica column [solvent A, EtOAc,

solvent B, hexanes; gradient 0% A/100% B ?100% A/0% B over

9.0 min; ﬂow rate: 50 mL/min; monitored at 254 and 280 nm] to

afford the benzosuberene analogue 67 (0.21 g, 0.71 mmol, 89%)

as a white solid. ¹H NMR (CDCl₃, 500 MHz) d 7.21 (2H, d,

J = 8.7 Hz), 6.83 (2H, d, J = 8.7 Hz), 6.70 (1H, d, J = 8.4 Hz), 6.54

(1H, d, J = 8.4 Hz), 6.28 (1H, t, J = 7.4 Hz), 5.72 (1H, s), 3.90 (3H,

s), 3.81 (3H, s), 2.75 (2H, t, J = 7.0 Hz), 2.13 (2H, p, J = 7.0 Hz),

1.95 (2H, q, J = 7.2 Hz). ¹³C NMR (CDCl₃, 126 MHz) d 158.8, 144.9,

142.4, 142.2, 135.3, 134.8, 129.0, 127.8, 126.1, 120.6, 113.4,

107.6, 55.9, 55.3, 33.7, 25.6, 23.5. HRMS: Obsd 297.1492 [M+H⁺],

Calcd for C₁₉H₂₁O₃: 297.1485. HPLC (Method B): 17.42 min.

4.1.1.19. 1-((tert-Butyldimethylsilyl)oxy)-5-(4-hydroxyphenyl)-

2-methoxy-6,7,8,9-tetrahydro-5H-benzo[7]annulen-5-ol

(47).

To a solution of 4-methoxyphenyl bromide (0.52 g,

2.8 mmol) in THF (40 mL) at ꢂ78 °C was added n-BuLi (0.34 mL,

2.5 M in hexanes), and the reaction mixture was stirred for

30 min. Benzosuberone 41 (0.61 g, 1.9 mmol) in THF (20 mL) was

added dropwise over a period of 15 min. The reaction mixture

was allowed to reach room temperature over 12 h. Upon comple-

tion, water was added, and the mixture was extracted with EtOAc

(3 ꢁ 20 mL). The organic extract was washed with brine, dried over

Na₂SO₄, ﬁltered, concentrated under reduced pressure, and puri-

ﬁed by ﬂash chromatography using a pre-packed 50 g silica col-

umn [solvent A: EtOAc, solvent B: hexanes; gradient: 7% A/93% B

(1 CV), 7% A/93% B ? 60% A/40% B (10 CV), 60% A/40% B (2 CV);

ﬂow rate: 40 mL/min; monitored at 254 and 280 nm] to afford

alcohol 47 (0.32 g, 0.75 mmol, 39%) as a clear oil. ¹H NMR (CDCl₃,

500 MHz) d 7.26 (1H, d, J = 8.6 Hz), 7.15 (2H, d, J = 8.8 Hz), 6.81

(2H, d, J = 8.8 Hz), 6.73 (1H, d, J = 8.7 Hz), 3.81 (3H, s), 3.79 (3H,

s), 3.30 (1H, dd, J = 14.6, 7.5 Hz), 2.67–2.59 (1H, m), 2.14–2.04

(2H, m), 1.77–1.65 (2H, m), 1.35–1.25 (2H, m), 0.99 (9H, s), 0.20

(3H, s), 0.14 (3H, s). ¹³C NMR (CDCl₃, 125 MHz) d 158.7, 149.2,

141.8, 139.0, 137.6, 132.7, 128.3, 119.1, 113.6, 107.8, 79.4, 55.2,

54.6, 41.2, 27.1, 26.7, 26.1, 25.5, ꢂ3.86, ꢂ4.22.

4.1.1.22. 1-((tert-Butyldimethylsilyl)oxy)-2-methoxy-5-(3⁰,4⁰,5⁰-

trimethoxyphenyl)-6,7,8,9-tetrahydro-5H-benzo[7]annulen-5-ol

(49).^31,33

To an oven dried ﬂask, THF (50 mL) and 3,4,5-tri-

methoxyphenyl bromide (2.46 g, 9.97 mmol) were added, and the

solution was cooled to ꢂ78 °C. n-BuLi (4.2 mL, 10 mmol) was

slowly added to the reaction mixture, which was then stirred at

ꢂ78 °C for 1 h. Benzosuberone 41 (2.37 g, 7.38 mmol) was then

added dropwise to the ﬂask, and the reaction mixture was stirred

while warming from ꢂ78 °C to room temperature over 12 h. The

reaction mixture was washed with water and extracted with EtOAc

(3 ꢁ 50 mL). The combined organic phase was dried over sodium

sulfate and evaporated under reduced pressure. The crude reaction

product was puriﬁed by ﬂash chromatography using a pre-packed

100 g silica column [solvent A: EtOAc; solvent B: hexanes; gradi-

ent: 7% A/93% B (1 CV), 7% A/93% B ? 60% A/40% B (10 CV), 60%

A/80% B (2 CV); ﬂow rate: 40 mL/min; monitored at 254 and

280 nm] to afford compound 49 (1.80 g, 3.70 mmol, 50%) as a clear

oil. NMR characterization was performed after the next step.

4.1.1.23. tert-Butyl((3-methoxy-9-(3⁰,4⁰,5⁰-trimethoxyphenyl)-

6,7-dihydro-5H-benzo[7]annulen-4-yl)oxy)dimethylsilane

4.1.1.20. tert-Butyl((3-methoxy-9-(4⁰-methoxyphenyl)-6,7-dihy-

dro-5H-benzo[7]annulen-4-yl)oxy)dimethylsilane

(56).

Acetic acid (20 mL) was added to tertiary alcohol 49

(54).

Tertiary alcohol 47 (0.53 g, 1.2 mmol) was dissolved in

(1.80 g, 3.70 mmol), and the reaction mixture was stirred for 12 h

at room temperature. The mixture was washed with water and

extracted with EtOAc (3 ꢁ 30 mL). The combined organic phase

was dried over sodium sulfate and evaporated under reduced pres-

sure. The crude reaction product was puriﬁed by ﬂash chromatog-

raphy using a pre-packed 50 g silica column [solvent A: EtOAc;

acetic acid (5 mL) and reﬂuxed for 5 h. No apparent change in

TLC was observed, so water (15 mL) was added to the reaction mix-

ture, which was reﬂuxed for 2 h. The solvents were evaporated,

and the resulting product was extracted with EtOAc (3 ꢁ 15 mL).

The combined organic extracts were washed with satd NaHCO₃,

Please cite this article in press as: Herdman, C. A.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.10.012

C. A. Herdman et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx

13

solvent B: hexanes; gradient: 7% A/93% B (1 CV), 7% A/93% B ? 60%

A/40% B (10 CV), 60% A/40% B (2 CV); ﬂow rate: 40 mL/min; mon-

itored at 254 and 280 nm] to afford TBS-protected benzosuberene

56 (1.43 g, 8.38 mmol, 83%) as a clear oil. ¹H NMR (500 MHz,

CDCl₃) d 6.67 (1H, d, J = 8.5 Hz), 6.61 (1H, d, J = 8.5 Hz), 6.50 (2H,

s), 6.32 (1H, t, J = 7.5 Hz), 3.85 (3H, s), 3.78 (3H, s), 3.77 (6H, s),

2.78 (2H, t, J = 7 Hz), 2.11 (2H, p, J = 7 Hz), 1.95 (2H, q, J = 7.5 Hz),

1.06 (9H s), 0.25 (6H, s). ¹³C NMR (125 MHz, CDCl₃) d 152.8,

148.6, 143.1, 141.5, 138.6, 137.3, 133.8, 133.2, 126.7, 122.4,

108.4, 105.3, 60.7, 56.0, 54.5, 34.0, 26.2, 25.6, 24.2, 19.0, ꢂ3.8.

6.07 (1H, d, J = 8.5 Hz), 4.04 (1H, d, J = 9.5 Hz), 3.61 (6H, s) 3.60 (3H,

s), 3.56 (3H, s), 3.26 (1H, m), 2.55 (1H, t, J = 12 Hz), 1.90 (1H, m),

1.65 (4H, m), 1.16 (1H, q, J = 10.5 Hz). ¹³C NMR (125 MHz, D₂O) d

152.2, 150.3, 142.8, 140.5, 140.4, 139.0, 137.3, 134.6, 121.8,

108.6, 105.9, 60.8, 55.4, 48.6, 33.7, 30.0, 33.7, 29.7, 26.6. ³¹P NMR

(200 MHz, D₂O, 85% phosphoric acid reference) d 0.97. HRMS: Obsd

483.1190 [M+H⁺], Calcd for C₂₁H₂₆O₅Na₂P⁺: 483.1155. HPLC

(Method A): 14.23 min.

4.1.1.27. 1,2-Dimethoxy-5-(4⁰-methoxyphenyl)-6,7,8,9-tetrahy-

dro-5H-benzo[7]annulen-5-ol (27).

To a solution of 4-meth-

4.1.1.24. 3-Methoxy-9-(3⁰,4⁰,5⁰-trimethoxyphenyl)-6,7-dihydro-

oxyphenyl bromide (0.719 g, 3.84 mmol) in THF (50 mL) at ꢂ78 °C

was added n-BuLi (2.6 mL, 2.5 M in hexanes), and the reaction mix-

ture was stirred for 30 min. Benzosuberone 13 (0.58 g, 2.6 mmol)

in THF (15 mL) was added dropwise over 15 min. The reaction mix-

ture was warmed to room temperature over 12 h. Upon comple-

tion, water was added, and the mixture was extracted with

EtOAc (3 ꢁ 20 mL). The organic layer was washed with brine, dried

over Na₂SO₄, ﬁltered, concentrated under reduced pressure, and

puriﬁed by ﬂash chromatography using a pre-packed 80 g silica

column [solvent A, EtOAc, solvent B, hexanes; gradient 0%

A/100% B ? 100% A/0% B over 20.3 min; ﬂow rate: 50 mL/min;

monitored at 254 and 280 nm] to afford alcohol 27 (0.45 g,

1.4 mmol, 54%) as a white solid. ¹H NMR (CDCl₃, 500 MHz) d 7.39

(1H, d, J = 8.7 Hz), 7.15 (2H, d, J = 8.8 Hz), 6.83 (2H, d, J = 8.8 Hz),

6.79 (1H, d, J = 8.7 Hz), 3.89 (3H, s), 3.79 (3H, s), 3.75 (3H, s),

3.25–3.18 (1H, m), 2.67–2.59 (1H, m), 2.15–2.08 (2H, m), 1.93–

1.86 (1H, m), 1.80–1.69 (2H, m), 1.43–1.35 (1H, m). ¹³C NMR

(CDCl₃, 126 MHz) d 158.8, 151.7, 146.3, 139.1, 137.5, 135.4,

128.2, 122.31, 113.7, 108.8, 79.4, 61.0, 55.6, 55.2, 41.2, 27.3, 26.6,

25.2.

5H-benzo[7]annulen-4-ol

(69).^{30,31,33,44,56}

Benzosuberene

56 (1.43 g, 3.04 mmol) was dissolved in THF (10 mL), TBAF

(3.65 mL, 3.6 mmol) was added, and the reaction mixture was stir-

red at room temperature for 12 h. The solution was washed with

water and extracted with EtOAc (3 ꢁ 30 mL). The combined

organic phase was dried over sodium sulfate and evaporated under

reduced pressure affording benzosuberene 69 (0.66 g, 1.9 mmol,

61%) as an orange solid. ¹H NMR (500 MHz, CDCl₃) d 6.72 (1H, d,

J = 8.5 Hz), 6.58 (1H, d, J = 8 Hz), 6.51 (2H, s), 6.35 (1H, t, J = 7 Hz),

5.74 (1H, s), 3.93 (3H, s), 3.87 (3H, s), 3.81 (6H, s), 2.77 (2H, t,

J = 7 Hz), 2.15 (2H, p, J = 7 Hz), 1.97 (2H, q, J = 7.5 Hz). ¹³C NMR

(125 MHz, CDCl₃) d 152.8, 145.0, 142.8, 142.3, 138.5, 137.3,

134.2, 127.7, 127.2, 120.8, 107.6, 105.3, 60.9, 56.1, 55.9, 33.6,

25.7, 23.5.

4.1.1.25.

tetrahydro-5H-benzo[7]annulen-1-ol

2-Methoxy-5-(3⁰,4⁰,5⁰-trimethoxyphenyl)-6,7,8,9-

(72). To benzo-

suberene 69 (0.66 g, 1.9 mmol) was added methanol (50 mL) and

10% Pd/C (0.42 g). Hydrogen gas was added, and the reaction mix-

ture was stirred at room temperature for 12 h. The reaction mix-

ture was ﬁltered through Celite^Ò, and the Celite^Òwas washed

with EtOAc (3 ꢁ 30 mL). The organic phase (MeOH and EtOAc)

was evaporated under reduced pressure. The crude reaction pro-

duct was puriﬁed by ﬂash chromatography using a pre-packed

25 g silica column [solvent A: EtOAc; solvent B: hexanes; gradient:

7% A/93% B (1 CV), 7% A/93% B ? 60% A/40% B (10 CV), 60% A/40% B

(2 CV); ﬂow rate: 12 mL/min; monitored at 254 and 280 nm] to

afford benzosuberane analogue 72 (0.16 g, 0.45 mmol, 24%) as a

white solid. ¹H NMR (500 MHz, CDCl₃) d 6.54 (1H, d, J = 8.5 Hz),

6.47 (2H, s), 6.17 (1H, d, J = 8 Hz), 5.84 (1H, s), 4.17 (1H, d,

J = 9.5 Hz), 3.89 (3H, s), 3.84 (9H, s), 3.28 (1H, q, J = 7.5 Hz), 2.73

(1H, t, J = 12 Hz), 2.17 (1H, m), 2.02 (2H, m), 1.86 (2H, m), 1.46

(1H, q, J = 12 Hz). ¹³C NMR (125 MHz, CDCl₃) d 153.1, 144.7,

142.6, 141.2, 139.6, 136.2, 128.4, 118.6, 108.1, 105.7, 60.9, 56.1,

55.9, 49.5, 34.7, 30.6, 27.2, 25.4. HRMS: Obsd 381.1764 [M+Na⁺],

Calcd for C₂₁H₂₆O₅Na⁺: 381.1672. HPLC (Method B): 15.30 min.

4.1.1.28. 3,4-Dimethoxy-9-(4⁰-methoxyphenyl)-6,7-dihydro-5H-

benzo[7]annulene (36).

Tertiary alcohol analogue 27

(0.401 g, 1.22 mmol) was dissolved in acetic acid (10 mL), and

the mixture was stirred for 12 h at ambient temperature. Water

(30 mL) was added, and the reaction mixture was stirred for 2 h.

The reaction mixture was extracted with EtOAc (3 ꢁ 20 mL). The

organic solvent was evaporated under reduced pressure and the

residue extracted with EtOAc (3 ꢁ 20 mL). The combined organic

extracts were washed with brine, dried over Na₂SO₄, ﬁltered, con-

centrated under reduced pressure, and puriﬁed by ﬂash chro-

matography using a pre-packed 25 g silica column [solvent A:

EtOAc, solvent B: hexanes; gradient: 5% A/95% B (1 CV), 5% A/95%

B ? 50% A/50% B (10 CV), 50% A/50% B (2 CV); ﬂow rate: 25 mL/

min; monitored at 254 and 280 nm] to afford benzosuberene ana-

logue 36 (0.31 g, 1.0 mmol, 82%) as a clear oil. ¹H NMR (CD₃OD,

500 MHz) d 7.17 (2H, d, J = 8.8 Hz), 6.87 (1H, d, J = 8.5 Hz), 6.86

(2H, d, J = 8.8 Hz), 6.71 (1H, d, J = 8.5 Hz), 6.30 (1H, t, J = 7.4 Hz),

3.89 (3H, s), 3.85 (3H, s), 3.81 (3H, s), 2.74 (2H, t, J = 7.0 Hz), 2.15

(2H, p, J = 7.1 Hz), 1.94 (2H, q, J = 7.2 Hz). ¹³C NMR (CDCl₃,

125 MHz) d 158.8, 151.3, 146.1, 142.2, 135.9, 135.2, 134.3, 129.0,

125.9, 125.0, 113.5, 109.2, 61.2, 55.6, 55.3, 34.6, 25.5, 24.0. HRMS:

Obsd 281.1597 [M+H⁺], Calcd for C₁₉H₂₃O₂: 281.1536. HPLC

(Method B): 19.08 min.

4.1.1.26.

tetrahydro-5H-benzo[7]annulen-1-yl

(73). To benzosuberane analogue 72 dissolved in CH₂Cl₂

2-Methoxy-5-(3⁰,4⁰,5⁰-trimethoxyphenyl)-6,7,8,9-

disodium phosphate

(25 mL) was added POCl₃(0.11 mL, 1.1 mmol) and pyridine

(0.1 mL, 1 mmol), and the reaction mixture was stirred at room

temperature for 12 h. 2 M NaOH (1.69 mL, 3.38 mmol) was added,

and the reaction was stirred for 5 min and extracted with CH₂Cl₂,

and the organic layer was evaporated under reduced pressure.

NaOH (2 mL, 2 M) was added to the resulting oil, and the solution

was reﬂuxed at 60 °C for 15 min. Water was removed under

reduced pressure. The crude reaction product was puriﬁed by ﬂash

chromatography using a pre-packed 12 g C-18 column [solvent A:

water; solvent B: acetonitrile; gradient: 0% A/100% B (1 CV), 0%

A/100% B ? 100% A/0% B (10 CV), 0% A/100% B (2 CV); ﬂow rate:

12 mL/min; monitored at 254 and 280 nm] to afford the benzo-

suberane phosphate salt 73 (0.024 g, 0.050 mmol, 17%) as a white

solid. ¹H NMR (500 MHz, D₂O) d 6.43 (2H, s), 6.36 (1H, d, J = 8.5 Hz),

4.1.1.29. 1,2-Dimethoxy-5-phenyl-6,7,8,9-tetrahydro-5H-benzo

[7]annulen-5-ol (19).

To an oven dried ﬂask, THF (50 mL)

and phenylbromide (0.25 mL, 2.4 mmol) were added, and the solu-

tion was cooled to ꢂ78 °C. n-BuLi (1.0 mL, 2.5 mmol) was slowly

added to the reaction mixture, which was then stirred at ꢂ78 °C

for 1 h. Benzosuberone 13 (0.39 g, 1.8 mmol) was then added drop-

wise to the ﬂask, and the reaction mixture was stirred while warm-

ing from –78 °C to room temperature over 12 h. The reaction

mixture was washed with water and extracted with EtOAc

(3 ꢁ 50 mL). The combined organic phase was dried over sodium

Please cite this article in press as: Herdman, C. A.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.10.012

14

C. A. Herdman et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx

sulfate and evaporated under reduced pressure. The crude reaction

product was puriﬁed by ﬂash chromatography using a pre-packed

25 g silica column [solvent A: EtOAc; solvent B: hexanes; gradient:

7% A/93% B (1 CV), 7% A/93% B ? 60% A/40% B (10 CV), 60% A/40% B

(2 CV); ﬂow rate: 25 mL/min; monitored at 254 and 280 nm] to

afford tertiary alcohol 19 (0.29 g, 0.97 mmol, 55%) as a pale yellow

oil. NMR characterization was performed after the next step.

+Na⁺], Calcd for C₂₂H₂₆O₅Na: 393.1672. HPLC (Method B):

17.52 min.

4.1.1.33.

6,7,8,9-tetrahydro-5H-benzo[7]annulen-5-ol (25).

5-(3⁰,5⁰-Bis(triﬂuoromethyl)phenyl)-1,2-dimethoxy-

To

a

solution of 1-bromo-3,5-bis(triﬂuoromethyl)benzene (0.36 g,

1.2 mmol) in THF (13 mL) at ꢂ78 °C was added n-BuLi (0.55 mL,

2.5 M), and the reaction mixture was stirred for 1 h. Benzo-

suberone 13 (0.20 g, 0.91 mmol) in THF (5 mL) was added drop-

wise over 15 min. The reaction mixture was stirred for 20 h and

was warmed to room temperature. The reaction mixture was

diluted with H₂O (25 mL) and extracted with EtOAc (2 ꢁ 25 mL).

The organic extract was dried over Na₂SO₄, ﬁltered, concentrated

under reduced pressure, and puriﬁed by ﬂash chromatography

using a pre-packed 25 g silica column [solvent A: EtOAc; solvent

B: hexanes; gradient: 7% A/93% B (1 CV), 7% A/93% B ? 60%

A/40% B (15 CV), 60% A/40% B (2 CV); ﬂow rate: 25 mL/min; mon-

itored at 254 and 280 nm] to afford alcohol 25 (0.45 g, 1.0 mmol,

84%) as a yellow solid. ¹H NMR (CDCl₃, 500 MHz) d 7.70 (1H, s),

7.67 (2H, s), 6.92 (1H, d, J = 8.7 Hz), 6.63 (1H, d, J = 8.8 Hz), 3.76

(3H, s), 3.67 (3H, s), 3.16 (1H, ddd, J = 14.6, 7.8, 1.9 Hz), 2.50 (1H,

s), 2.46 (1H, ddd, J = 14.3, 8.2, 2.8 Hz), 2.37–2.24 (1H, m), 2.06

(1H, ddd, J = 14.2, 9.6, 3.0 Hz), 1.91–1.85 (1H, m), 1.73–1.61 (1H,

m), 1.59–1.50 (2H, m). ¹³C NMR (CDCl₃, 125 MHz) d 152.4, 149.8,

146.6, 137.2, 135.6, 131.6 (q, J = 33.1 Hz), 127.1 (q, J = 3.8 Hz),

123.5 (q, J = 272.7 Hz), 123.7, 121.3 (hept, J = 4.0 Hz), 109.5, 79.8,

61.1, 55.7, 41.6, 27.0, 25.2, 24.8.

4.1.1.30. 3,4-Dimethoxy-9-phenyl-6,7-dihydro-5H-benzo[7]an-

nulene (28).

Acetic acid (15 mL) was added to tertiary alcohol

19 (0.29 g, 0.97 mmol), and the reaction mixture was stirred for

12 h at room temperature. The mixture was washed with water

(50 mL) and extracted with EtOAc (3 ꢁ 30 mL). The combined

organic phase was dried over sodium sulfate and evaporated under

reduced pressure. The crude reaction product was puriﬁed by ﬂash

chromatography using a pre-packed 10 g silica column [solvent A:

EtOAc; solvent B: hexanes; gradient: 5% A/95% B (1 CV), 5% A/95%

B ? 50% A/60% B (10 CV), 40% A/60% B (2 CV); ﬂow rate: 12 mL/

min; monitored at 254 and 280 nm] to afford benzosuberene ana-

logue 28 (0.10 g, 0.36 mmol, 37%) as a yellow oil. ¹H NMR

(500 MHz, CDCl₃) d 7.30 (5H, m), 6.78 (2H, s), 6.40 (1H, t,

J = 7.5 Hz), 3.91 (3H, s), 3.90 (3H, s), 2.81 (2H, t, J = 7 Hz), 2.19

(2H, p, J = 7 Hz), 2.01 (2H, q, J = 7.5 Hz). ¹³C NMR (125 MHz, CDCl₃)

d 151.5, 146.1, 142.9, 142.6, 135.9, 134.2, 128.1, 128.0, 127.5,

127.0, 125.1, 109.4, 61.2, 55.6, 34.5, 25.6, 24.1.HRMS: Obsd

303.1363 [M+Na⁺], Calcd for C₁₉H₂₀O₂Na: 303.1356. HPLC (Method

B): 19.93 min.

4.1.1.31. 1,2-Dimethoxy-5-(3⁰,4⁰,5⁰-trimethoxyphenyl)-6,7,8,9-

4.1.1.34.

6,7-dihydro-5H-benzo[7]annulene (34).

9-(3⁰,5⁰-Bis(triﬂuoromethyl)phenyl)-3,4-dimethoxy-

To a solution of ter-

tetrahydro-5H-benzo[7]annulen-5-ol (26).³¹

To a solution

of 3,4,5-trimethoxyphenyl bromide (0.67 g, 2.7 mmol) in THF

(100 mL) at ꢂ78 °C was added n-BuLi (1.1 mL, 2.5 M in hexanes),

and the reaction mixture was stirred for 30 min. Benzosuberone

13 (0.60 g, 2.7 mmol) in THF (15 mL) was added dropwise over

15 min. The reaction mixture was warmed to room temperature

over 12 h. Water was added, and the mixture was extracted with

EtOAc (100 mL). The organic extract was washed with brine, dried

over Na₂SO₄, ﬁltered, concentrated under reduced pressure, and

puriﬁed by ﬂash chromatography using a pre-packed 25 g silica

column [solvent A: EtOAc, solvent B: hexanes; gradient: 7%

A/93% B (1 CV), 7% A/93% B ? 56% A/44% B (9.2 CV), 56% A/44% B

(1 CV); ﬂow rate: 25 mL/min; monitored at 254 and 280 nm] to

afford alcohol 26 (0.39 g, 0.99 mmol, 34%) as a light yellow oil.

¹H NMR (CDCl_3,500 MHz) d 7.26 (1H, d, J = 8.7 Hz), 6.75 (1H, d,

J = 8.8 Hz), 6.49 (2H, s), 3.87 (3H, s), 3.83 (3H, s), 3.74 (3H, s),

3.73 (6H, s), 3.26–3.20 (1H, m), 2.56 (1H, ddd, J = 14.1, 6.9,

3.0 Hz), 2.38–2.30 (1H, m), 2.21–2.21 (1H, m), 2.11 (1H, ddd,

J = 14.0, 10.7, 3.1 Hz), 1.93 (1H, ddd, J = 15.3, 7.4, 3.8 Hz), 1.81–

1.71 (2H, m), 1.50–1.42 (1H, m). ¹³C NMR (CDCl₃, 126 MHz) d

153.0, 151.8, 146.2, 141.6, 138.6, 137.2, 135.5, 123.0, 108.8,

104.2, 79.9, 61.0, 60.8, 56.1, 55.5, 41.3, 27.1, 26.3, 25.1.

tiary alcohol 25 (0.45 g, 1.0 mmol) in EtOH (10 mL) was added 2 M

HCl (10 mL, 20 mmol), and the reaction mixture was stirred over-

night. The reaction mixture was then extracted using EtOAc

(4 ꢁ 15 mL). The organic extract was dried over Na₂SO₄, ﬁltered,

concentrated under reduced pressure and puriﬁed by ﬂash chro-

matography using a pre-packed 25 g silica column [solvent A:

EtOAc; solvent B: hexanes; gradient: 5% A/95% B (1 CV), 5%

A/95% B ? 40% A/60% B (10 CV), 40% A/60% B (2 CV); ﬂow rate:

25 mL/min; monitored at 254 and 280 nm] to afford benzo-

suberene 34 (0.22 g, 0.54 mmol, 54%) as a white solid. ¹H NMR

(CDCl₃, 500 MHz) d 7.75 (1H, s), 7.71 (2H, s), 6.77 (1H, d,

J = 8.5 Hz), 6.63 (1H, d, J = 8.5 Hz), 6.49 (1H, t, J = 7.3 Hz), 3.90

(3H, s), 3.88 (3H, s), 2.76 (2H, t, J = 6.9 Hz), 2.18 (2H, p,

J = 7.1 Hz), 2.03 (2H, q, J = 7.2 Hz). ¹³C NMR (CDCl₃, 125 MHz) d

152.2, 146.6, 144.7, 140.8, 136.1, 132.4, 131.6 (q, J = 33 Hz),

131.1, 128.0 (q, J = 3.8 Hz), 124.8, 123.6 (q, J = 272.7 Hz), 120.7

(hept, J = 4.0 Hz), 109.9, 61.4, 55.8, 34.3, 26.0, 24.3. ¹⁹F NMR (CDCl₃,

470 MHz) d ꢂ62.81. HRMS: Obsd 401.0965 [M–CH₃]^ꢂ, Calcd for

C₂₀H₁₅F₆O₂: 401.0976. HPLC (Method B): 21.57 min.

4.1.1.35.

1,2-Dihydroxy-6,7,8,9-tetrahydro-5H-benzo[7]annu-

Ketone 13 (0.88 g, 4.0 mmol) was added

len-5-one (40).^31,57

4.1.1.32. 3,4-Dimethoxy-9-(3⁰,4⁰,5⁰-trimethoxyphenyl)-6,7-dihy-

dro-5H-benzo[7]annulene (35).³¹

Tertiary alcohol analogue

to the ionic liquid [TMAH][Al₂Cl₇](20.0 mL, 0.497 M). The reaction

mixture was subjected to microwave irradiation at 80 °C and 1 atm

for 1 h. H₂O (20 mL) was added to the mixture, and the resulting

brown liquid was extracted with dichloromethane (3 ꢁ 20 mL).

The organic extracts were dried over Na₂SO₄, ﬁltered, evaporated

under reduced pressure, and puriﬁed by ﬂash chromatography

using a pre-packed 25 g silica column [solvent A: EtOAc; solvent

B: hexanes; gradient: 5% A/95% B (1 CV), 5% A/95% B ? 60%

A/40% B (13 CV), 60% A/40% B (2 CV); ﬂow rate: 25 mL/min; mon-

itored at 254 and 280 nm] to afford benzosuberone 40 (0.50 g,

2.6 mmol, 65%) as a brown solid. ¹H NMR ((CD₃)₂CO, 500 MHz) d

7.14 (1H, d, J = 8.3 Hz), 6.78 (1H, d, J = 8.8 Hz), 3.07–2.99 (2H, m),

2.67–2.59 (2H, m), 1.84–1.77 (2H, m), 1.77–1.71 (2H, m). ¹³C

26 (0.5 g, 1 mmol) was dissolved in acetic acid (10 mL) and

reﬂuxed for 2 h. Water (30 mL) was added, and the reaction was

reﬂuxed for 2 h. The white precipitate thus obtained was ﬁltered

and washed with hexanes. On drying, it afforded benzosuberene

analogue 35 (0.444 g, 1.2 mmol, 93%) as a colorless solid, which

was not further puriﬁed. ¹H NMR (CDCl₃, 500 MHz) d 6.79 (1H, d,

J = 8.5 Hz), 6.77 (1H, d, J = 8.5 Hz), 6.51 (2H, s), 6.35 (1H, t,

J = 7.3 Hz), 3.90 (3H, s), 3.89 (3H, s), 3.88 (3H, s), 3.82 (6H, s),

2.77 (2H, t, J = 7.0 Hz), 2.17 (2H, p, J = 7.1 Hz), 1.98 (2H, q,

J = 7.2 Hz). ¹³C NMR (CDCl₃, 126 MHz) d 152.9, 151.5, 146.1,

142.9, 138.4, 137.4 135.9, 133.8, 127.1, 125.3, 109.3, 105.3, 61.3,

60.9, 56.17, 55.6, 34.6, 25.6, 24.2. HRMS: Obsd 393.1682 [M

Please cite this article in press as: Herdman, C. A.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.10.012

C. A. Herdman et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx

15

NMR ((CD₃)₂CO, 126 MHz) d 205.4, 148.1, 142.0, 132.3, 128.9,

120.6, 112.3, 40.3, 24.4, 22.8, 21.0.

4.1.1.39. 9-(3⁰,4⁰,5⁰-Trimethoxyphenyl)-6,7-dihydro-5H-benzo[7]

annulene-3,4-diol (70). The di-TBS-protected analogue 57

(0.32 g, 0.56 mmol) was dissolved in THF (5 mL). To the solution,

TBAF–3H₂O (1.4 mmol) was added and stirred for 3 h at room tem-

perature. The reaction was quenched with water (15 mL), and the

organic solvent was evaporated under reduced pressure. The

resulting aqueous phase was then extracted with EtOAc

(3 ꢁ 20 mL). The combined organic extract was washed with brine,

dried over Na₂SO₄, ﬁltered, evaporated under reduced pressure and

puriﬁed by ﬂash chromatography using a pre-packed 25 g silica gel

column [solvent A, EtOAc, solvent B, hexanes; gradient 7% A/93% B

(1 CV), 7% A/93% B ? 60% A/40% B (10 CV), 60% A/40% B (5.2 CV);

ﬂow rate: 25 mL/min; monitored at 254 and 280 nm] to afford cat-

echol analogue 70 (0.17 g, 0.49 mmol, 88%) as a white solid. ¹H

NMR (CDCl₃, 500 MHz) d 6.68 (1H, d, J = 8.2 Hz), 6.52 (1H, d,

J = 8.2 Hz), 6.49 (2H, s), 6.33 (1H, t, J = 7.4 Hz), 5.29 (1H, s), 5.28

(1H, s), 3.86 (3H, s), 3.79 (6H, s), 2.71 (2H, t, J = 7.0 Hz), 2.15 (2H,

p, J = 7.1 Hz), 1.96 (2H, q, J = 7.2 Hz). ¹³C NMR (CDCl₃, 126 MHz) d

152.8, 142.9, 141.8, 140.8, 138.3, 137.3, 134.1, 128.5, 127.0,

121.7, 112.3, 105.3, 60.9, 56.1, 33.8, 25.6, 23.8. HRMS: Obsd

365.1444 [M+H⁺], Calcd for C₁₂H₂₃O₅: 365.1359. HPLC (Method

B): 16.18 min.

4.1.1.36. 1,2-Bis((tert-butyldimethylsilyl)oxy)-6,7,8,9-tetrahy-

dro-5H-benzo[7]annulen-5-one (43).³¹

To a solution of cat-

echol 40 (0.68 g, 3.5 mmol) and DIPEA (2.7 mL, 16 mmol) in DMF

(5 mL) at 0 °C was added TBSCl (1.60 g, 10.6 mmol) in portions.

The reaction mixture was stirred for 18 h, diluted with H₂O

(5 mL), and extracted with Et₂O (2 ꢁ 20 mL). The organic extract

was dried over Na₂SO₄, ﬁltered, concentrated under reduced pres-

sure, and puriﬁed by ﬂash chromatography using a pre-packed

25 g silica column [solvent A: EtOAc; solvent B: hexanes; gradient:

0% A/100% B (1 CV), 0% A/100% B ? 30% A/70% B (10 CV), 30%

A/70% B (2 CV); ﬂow rate: 25 mL/min; monitored at 254 and

280 nm] to afford ketone 43 (1.51 g, 3.59 mmol, 99%) as a white

solid. ¹H NMR (CDCl₃, 500 MHz) d 7.29 (1H, d, J = 8.5 Hz), 6.75

(1H, d, J = 8.5 Hz), 2.97–2.94 (2H, m), 2.70 (2H, t, J = 6.2 Hz),

1.89–1.70 (4H, m), 1.02 (9H, s), 0.96 (9H, s), 0.24 (6H, s), 0.15

(6H, s). ¹³C NMR (CDCl₃, 125 MHz) d 205.3, 151.2, 143.9, 135.5,

134.0, 122.6, 118.6, 41.0, 26.5, 26.5, 25.3, 25.1, 21.9, 19.2, 18.9,

ꢂ3.15, ꢂ3.19.

4.1.1.37.

methoxyphenyl)-6,7,8,9-tetrahydro-5H-benzo[7]annulen-5-ol

(50).³¹

To solution of 3,4,5-trimethoxyphenyl bromide

1,2-Bis((tert-butyldimethylsilyl)oxy)-5-(3⁰,4⁰,5⁰-tri-

4.1.1.40.

butyldimethylsilyl)oxy)-6,7,8,9-tetrahydro-5H-benzo[7]annu-

len-5-ol (46). 1-Bromo-3,5-bis(triﬂuoromethyl)benzene

5-(3⁰,5⁰-Bis(triﬂuoromethyl)phenyl)-1,2-bis((tert-

a

(0.458 g, 1.85 mmol) in THF (20 mL) at ꢂ78 °C was added n-BuLi

(0.96 mL, 2.5 M in hexanes), and the reaction mixture was stirred

for 1 h. Benzosuberone 43 (0.639 g, 1.51 mmol) in THF (20 mL)

was added dropwise over 15 min. The reaction mixture was stirred

while warming to room temperature over 12 h. Water was added,

and the mixture was extracted with EtOAc (4 ꢁ 20 mL). The

organic extract was washed with brine, dried over Na₂SO₄, ﬁltered,

concentrated under reduced pressure, and puriﬁed by ﬂash chro-

matography using a pre-packed 25 g silica column [solvent A:

EtOAc, solvent B: hexanes; gradient: 10% A/90% B (1 CV), 10%

A/90% B ? 42% A/58% B (6 CV), 42% A/58% B?70% A/30% B

(1 CV), 70% A/30% B ? 100% A/0% B, 100% A/0% B (1.1 CV); ﬂow

rate: 40 mL/min; monitored at 254 and 280 nm] to afford alcohol

50 (0.480 g, 0.81 mmol, 54%) as a clear oil. ¹H NMR (CDCl₃,

500 MHz) d 7.10 (1H, d, J = 8.7 Hz), 6.72 (1H, d, J = 8.6 Hz), 6.46

(2H, s), 3.83 (3H, s), 3.74 (6H, s), 3.23–3.15 (1H, m), 2.57 (1H,

ddd, J = 14.0, 6.2, 3.0 Hz), 2.23–2.07 (3H, m), 1.94–1.84 (1H, m),

1.81–1.66 (2H, m), 1.46–1.33 (1H, m), 1.00 (9H, s), 0.95 (9H, s),

0.24 (3H, s), 0.23 (3H, s), 0.15 (3H, s), 0.10 (3H, s). ¹³C NMR (CDCl₃,

126 MHz) d 152.9, 146.4, 143.7, 141.8, 139.1, 137.1, 133.9, 120.0,

117.5, 104.1, 80.0, 60.8, 56.0, 40.9, 26.8, 26.3, 26.2, 26.1, 25.9,

18.9, 18.6, ꢂ3.4, ꢂ3.6.

(0.36 g, 1.2 mmol) was dissolved in THF (13 mL) at ꢂ78 °C and

n-BuLi (0.55 mL, 2.5 M) was then added. The reaction mixture was

stirred for 1 h, and then ketone 43 (0.38 g, 0.91 mmol) in THF

(5 mL) was added dropwise over 15 min. The reaction mixture

was stirred for 20 h, warming from ꢂ78 °C to room temperature,

and then diluted with H₂O (25 mL) and extracted with EtOAc

(3 ꢁ 25 mL). The organic extract was dried over Na₂SO₄, ﬁltered,

concentrated under reduced pressure, and puriﬁed by ﬂash chro-

matography using a pre-packed 25 g silica column [solvent A:

EtOAc; solvent B: hexanes; gradient: 7% A/93% B (1 CV), 7%

A/93% B ? 100% A/0% B (10 CV), 100% A/0% B (2 CV); ﬂow rate:

25 mL/min; monitored at 254 and 280 nm] to afford alcohol 46

(0.43 g, 0.68 mmol, 74%) as a yellow solid. ¹H NMR (CDCl₃,

500 MHz) d 7.77 (1H, s), 7.72 (2H, s), 6.92 (1H, d, J = 8.6 Hz), 6.73

(1H, d, J = 8.7 Hz), 3.25 (1H, ddd, J = 14.7, 7.6, 1.9 Hz), 2.57 (1H,

ddd, J = 14.2, 7.3, 3.1 Hz), 2.31 (1H, s), 2.22–2.14 (2H, m), 1.96–

190 (1H, m), 1.74–170 (1H, m), 1.64–1.58 (1H, m), 1.54–148 (1H,

m), 1.01 (9H, s), 0.97 (9H, s), 0.26 (3H, s), 0.23 (3H, s), 0.19 (3H,

s), 0.09 (3H, s). ¹³C NMR (CDCl₃, 125 MHz) d 149.7, 147.2, 144.3,

137.6, 134.0, 131.6 (q, J = 33.1 Hz), 127.2 (q, J = 3.2 Hz), 123.5 (q,

J = 273.0 Hz), 121.3 (hept, J = 4.0 Hz), 120.6, 118.3, 79.8, 41.2,

26.8, 26.39, 26.35, 25.9, 25.2, 19.0, 18.7, ꢂ3.1, ꢂ3.2, ꢂ3.5, ꢂ3.9.

4.1.1.38. ((9-(3⁰,4⁰,5⁰-Trimethoxyphenyl)-6,7-dihydro-5H-benzo

[7]annulene-3,4-diyl)bis(oxy))bis(tert-butyldimethylsilane)

4.1.1.41. ((9-(3⁰,5⁰-Bis(triﬂuoromethyl)phenyl)-6,7-dihydro-5H-

benzo[7]annulene-3,4-diyl)bis(oxy))bis(tert-butyldimethylsi-

(57).³¹

Tertiary alcohol 50 (0.44 g, 0.75 mmol) was dissolved

in acetic acid (5 mL) and stirred for 12 h at room temperature.

The reaction was quenched with water (10 mL) and extracted with

Et₂O (3 ꢁ 10 mL). The combined organic extract was washed with

satd NaHCO₃, brine, dried over Na₂SO₄, ﬁltered, and evaporated

under reduced pressure to afford the clear oil of TBS-protected

benzosuberene analogue 57 (0.38 g, 0.66 mmol, 89%), which was

used without further puriﬁcation. ¹H NMR (CDCl₃, 500 MHz) d

6.69 (1H, d, J = 8.4 Hz), 6.52 (1H, d, J = 8.4 Hz), 6.45 (2H, s), 6.33

(1H, t, J = 7.3 Hz), 3.85 (3H, s), 3.78 (6H, s), 2.70 (2H, t, J = 6.9 Hz),

2.10 (2H, q, J = 7.0 Hz), 1.95 (2H, q, J = 7.0 Hz), 1.04 (9H, s), 0.95

(9H, s), 0.24 (6H, s), 0.20 (6H, s). ¹³C NMR (CDCl₃, 125 MHz) d

152.7, 145.9, 143.4, 143.0, 138.6, 137.1, 134.4, 134.2, 126.8,

122.7, 117.9, 105.0, 60.9, 56.0, 33.9, 26.27, 26.25, 25.7, 24.5, 18.9,

18.7, ꢂ3.3, ꢂ3.4.

lane) (53).

Tertiary alcohol 46 (0.43 g, 0.67 mmol) was dis-

solved in EtOH (5 mL) and 2 M HCl (10 mL, 20 mmol) was then

added. The reaction mixture was stirred overnight and then

extracted using EtOAc (4 ꢁ 15 mL). The organic extract was dried

over Na₂SO₄, ﬁltered, concentrated under reduced pressure and

puriﬁed by ﬂash chromatography using a pre-packed 25 g silica

column [solvent A: EtOAc; solvent B: hexanes; gradient: 2%

A/98% B (1 CV), 2% A/98% B ? 15% A/85% B (10 CV), 15% A/85% B

(2 CV); ﬂow rate: 25 mL/min; monitored at 254 and 280 nm] to

afford benzosuberene 53 (0.22 g, 0.35 mmol, 53%) as a colorless

oil. ¹H NMR (CDCl₃, 500 MHz) d 7.75 (1H, s), 7.69 (2H, s), 6.72

(1H, d, J = 8.4 Hz), 6.49 (1H, t, J = 7.2 Hz), 6.40 (1H, d, J = 8.4 Hz),

2.73 (2H, t, J = 6.9 Hz), 2.15 (2H, p, J = 7.0 Hz), 2.03 (2H, q,

J = 7.1 Hz), 1.07 (9H, s), 0.98 (9H, s), 0.26 (6H, s), 0.23 (6H, s). ¹³C

Please cite this article in press as: Herdman, C. A.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.10.012

16

C. A. Herdman et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx

NMR (CDCl₃, 125 MHz) d 146.8, 145.0, 144.0, 141.1, 134.7, 133.0,

131.5 (q, J = 33.0 Hz), 130.8, 127.9 (q, J = 3.1 Hz), 123.6 (q,

J = 272.7 Hz), 122.3, 120.6 (hept, J = 3.9 Hz), 118.5, 33.8, 26.43,

26.39, 26.1, 24.8, 19.1, 18.9, ꢂ3.1, ꢂ3.2.

ate. The aqueous layer was extracted with EtOAc (3 ꢁ 50 mL).

The combined organic phase was dried over sodium sulfate, evap-

orated under reduced pressure, and puriﬁed by ﬂash chromatogra-

phy using a pre-packed 100 g silica column [solvent A: EtOAc;

solvent B: hexanes; gradient: 7% A/93% B (1 CV), 7% A/93% B ?

30% A/70% B (10 CV), 30% A/70% B (2 CV); ﬂow rate: 50 mL/min;

monitored at 254 and 280 nm] to afford benzosuberone 14

(2.80 g, 14.7 mmol, 70%) as a white solid. ¹H NMR (500 MHz,

CDCl₃) d 7.59 (1H, d, J = 8.5 Hz), 6.61 (1H, dd, J = 8.5, 2.5 Hz), 6.51

(1H, d, J = 2.5 Hz), 3.63 (3H, s), 2.70 (2H, t, J = 6 Hz), 2.51 (2H, t,

J = 6 Hz), 1.67 (2H, p, J = 7.5 Hz), 1.59 (2H, p, J = 5.5 Hz). ¹³C NMR

(125 MHz, CDCl₃) d 203.5, 162.5, 144.1, 131.3, 131.0, 114.7,

111.6, 55.1, 40.5, 32.6, 24.9, 20.5.

4.1.1.42.

benzo[7]annulene-3,4-diol (66).

9-(3⁰,5⁰-Bis(triﬂuoromethyl)phenyl)-6,7-dihydro-5H-

TBS-protected analogue 53

(0.43 g, 0.84 mmol) was dissolved in THF (5 mL), and TBAF

(1.00 mL, 1 M in THF) was added to the reaction ﬂask. The reaction

mixture was stirred for 18 h at ambient temperature, concentrated

under reduced pressure, and H₂O (5 mL) was then added. The reac-

tion mixture was extracted with EtOAc (3 ꢁ 10 mL). The organic

extract was dried over Na₂SO₄, ﬁltered, concentrated under

reduced pressure and puriﬁed by ﬂash chromatography using a

pre-packed 25 g silica column [solvent A: EtOAc; solvent B: hex-

anes; gradient: 7% A/93% B (1 CV), 7% A/93% B ? 60% A/40% B

(10 CV), 60% A/40% B (2 CV); ﬂow rate: 25 mL/min; monitored at

254 and 280 nm] to afford catechol 66 (0.077 g, 0.20 mmol, 57%)

as a white solid. ¹H NMR (CDCl₃, 500 MHz) d 7.75 (1H, s), 7.71

(2H, s), 6.71 (1H, d, J = 8.2 Hz), 6.50 (1H, t, J = 7.3 Hz), 6.38 (1H, d,

J = 8.2 Hz), 5.36 (1H, s), 5.25 (1H, s), 2.73 (2H, t, J = 6.9 Hz), 2.19

(2H, p, J = 7.0 Hz), 2.04 (2H, q, J = 7.2 Hz). ¹³C NMR (CDCl₃,

4.1.1.46. 2-Hydroxy-6,7,8,9-tetrahydro-5H-benzo[7]annulen-5-

one (39).^31,55

To benzosuberone 14 (0.89 g, 4.7 mmol) in a

20 mL microwave vial was added [TMAH][Al₂Cl₇] (22 mL,

0.53 M), and the reaction mixture was subjected to microwave

irradiation for 1 h at 80 °C. The reaction mixture was poured into

water (50 mL) and extracted with EtOAc (3 ꢁ 25 mL). The com-

bined organic phase was dried over sodium sulfate and evaporated

under reduced pressure. The crude reaction product was puriﬁed

by ﬂash chromatography using a pre-packed 100 g silica column

[solvent A: EtOAc; solvent B: hexanes; gradient: 7%/93% B (1 CV),

7% A/93% B ? 60% A/40% B (10 CV), 60% A/40% B (2 CV); ﬂow rate:

50 mL/min; monitored at 254 and 280 nm] to afford alcohol 39

(0.66 g, 3.8 mmol, 80%) as a white solid. ¹H NMR (500 MHz, CDCl₃)

d 7.73 (1H, d, J = 8.5 Hz), 6.75 (1H, dd, J = 8.5, 2.5 Hz), 6.67 (1H, d,

J = 2 Hz), 3.84 (1H, s), 2.87 (2H, t, J = 6 Hz), 2.71 (2H, t, J = 6 Hz),

1.82 (4H, m). ¹³C NMR (125 MHz, CDCl₃) d 204.9, 159.9, 144.8,

131.6, 131.3, 116.3, 113.7, 40.7, 32.7, 25.0, 20.7.

125 MHz)

d 144.7, 142.4, 141.43, 140.9, 132.9, 131.6 (q,

J = 32.9 Hz), 131.1, 128.8, 128.0 (q, J = 2.9 Hz), 123.6 (q,

J = 272.7 Hz), 121.2, 120.7 (hept, J = 3.9 Hz), 112.9, 33.7, 26.0,

23.9. ¹⁹F NMR (CDCl₃, 470 MHz) d ꢂ62.83. HRMS: Obsd 387.0805

[MꢂH]^ꢂ, Calcd for C₁₉H₁₃F₆O₂: 387.0820. HPLC (Method B):

18.11 min.

4.1.1.43. 5-(3⁰-Methoxyphenyl)pent-4-enoic acid (2).^31,33

To

dissolved 3-(carboxypropyl)triphenyl phosphonium bromide

(15.92 g, 37.09 mmol) in THF (500 mL) was added potassium tert-

butoxide (8.20 g, 73.4 mmol), and the reaction mixture was stirred

at room temperature for 1 h. 3-Methoxybenzaldehyde (4.5 mL,

37 mmol) was added, and the reaction mixture was stirred at room

temperature for 12 h. The THF was evaporated, and the resulting

material was quenched with 2 M HCl (75 mL) and extracted with

EtOAc (3 ꢁ 50 mL). The combined organic phase was dried over

sodium sulfate, ﬁltered, and evaporated under reduced pressure.

The crude reaction product was puriﬁed by ﬂash chromatography

using a pre-packed 100 g silica column [solvent A: EtOAc; solvent

B: hexanes; gradient: 12% A/88% B (1 CV), 12% A/88% B ? 70%

A/30% B (10 CV), 70% A/30% B (2 CV); ﬂow rate: 50 mL/min; moni-

tored at 254 and 280 nm] to afford carboxylic acid 2 (5.63 g,

27.3 mmol, 74%) as a yellow solid. NMR characterization was per-

formed after the next step.

4.1.1.47. 2-((tert-Butyldimethylsilyl)oxy)-6,7,8,9-tetrahydro-5H-

benzo[7]annulen-5-one (42).³¹

Phenol 39 (0.42 g, 2.4 mmol)

was dissolved in dimethylformamide (50 mL). TBSCl (0.72 g,

4.8 mmol) and DIPEA (1.24 mL, 7.14 mmol) were added, and the

solution was stirred for 12 h at room temperature. The reaction

mixture was washed with water (50 mL) and extracted with EtOAc

(5 ꢁ 30 mL). The combined organic phase was dried over sodium

sulfate and evaporated under reduced pressure. The crude reaction

product was puriﬁed by ﬂash chromatography using a pre-packed

50 g silica column [solvent A: EtOAc; solvent B: hexanes; gradient:

7% A/93% B (1 CV), 7% A/93% B ? 60% A/40% B (10 CV), 60% A/40% B

(2 CV); ﬂow rate: 50 mL/min; monitored at 254 and 280 nm] to

afford TBS-protected 42 (0.53 g, 1.82 mmol, 77%) as a clear oil. ¹H

NMR (500 MHz, CDCl₃) d 7.62 (1H, d, J = 8.5 Hz), 6.64 (1H, dd,

J = 8.5, 2 Hz), 6.56 (1H, d, J = 2 Hz), 2.77 (2H, t, J = 6 Hz), 2.59 (2H,

t, J = 6 Hz), 1.75 (2H, p, J = 6.5 Hz), 1.68 (2H, p, J = 6 Hz), 0.90 (9H,

s), 0.14 (6H, s). ¹³C NMR (125 MHz, CDCl₃) d 203.7, 159.0, 143.9,

132.0, 130.9, 120.8, 117.8, 40.5, 32.5, 25.5, 25.0, 20.6, 18.0, ꢂ4.5.

4.1.1.44. 5-(3⁰-Methoxyphenyl)pentanoic acid (8).^31,33,53

dissolved carboxylic acid (5.63 g, 27.3 mmol) in MeOH

To

2

(100 mL) was added 10% palladium on carbon (0.44 g) and hydro-

gen gas. The reaction was stirred at room temperature for 12 h. The

reaction mixture was then ﬁltered through Celite^Ò, and the Celite^Ò

was washed with EtOAc (3 ꢁ 50 mL). The organic phase (MeOH

and EtOAc) was evaporated under reduced pressure to afford car-

boxylic acid 8 (4.27 g, 20.5 mmol, 75%) as a yellow oil. ¹H NMR

(500 MHz, CDCl₃) d 11.2 (1H, s), 7.03 (1H, d, J = 8 Hz), 6.62 (2H, d,

J = 5 Hz), 6.59 (1H, d, J = 8.5 Hz), 3.59 (3H, s), 2.44 (2H, t,

J = 7.5 Hz), 2.21 (2H, d, J = 7.5 Hz), 1.52 (4H, m). ¹³C NMR

(125 MHz, CDCl₃) d 180.2, 159.7, 143.7, 129.4, 120.9, 114.3,

111.1, 55.0, 35.6, 34.1, 30.7, 24.5.

4.1.1.48. 2-((tert-Butyldimethylsilyl)oxy)-5-(3⁰,4⁰,5⁰-trimethoxy-

phenyl)-6,7,8,9-tetrahydro-5H-benzo[7]annulen-5-ol

(48).³¹

To an oven dried ﬂask, THF (50 mL) and 3,4,5-tri-

methoxyphenyl bromide (1.01 g, 4.04 mmol) were added, and the

solution was cooled to ꢂ78 °C. n-BuLi (1.71 mL, 4.27 mmol) was

slowly added to the reaction mixture, which was then stirred at

ꢂ78 °C for 1 h. Benzosuberone 42 (0.53 g, 3.0 mmol) in THF

(25 mL) was then added dropwise to the ﬂask, and the reaction

mixture was stirred while warming from ꢂ78 °C to room temper-

ature over 12 h. The reaction mixture was washed with water and

extracted with EtOAc (3 ꢁ 50 mL). The combined organic phase

was dried over sodium sulfate and evaporated under reduced pres-

sure. The crude reaction product was puriﬁed by ﬂash chromatog-

raphy using a pre-packed 50 g silica column [solvent A: EtOAc;

solvent B: hexanes; gradient: 5% A/95% B (1 CV), 5% A/95% B ?

4.1.1.45. 2-Methoxy-6,7,8,9-tetrahydro-5H-benzo[7]annulen-5-

one (14).

To carboxylic acid 8 (4.43 g, 21.3 mmol) was added

Eaton’s reagent (43 mL, 3 g per mmol of compound 8), and the

mixture was stirred at room temperature for 12 h. The mixture

was then poured over ice and neutralized with sodium bicarbon-

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C. A. Herdman et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx

17

40% A/60% B (10 CV), 40% A/60% B (2 CV); ﬂow rate: 50 mL/min;

monitored at 254 and 280 nm] to afford tertiary alcohol 48

(0.470 g, 1.02 mmol, 34%) as a clear oil. NMR characterization

was performed after the next step.

The combined organic phase was dried over sodium sulfate and

evaporated under reduced pressure. The crude reaction product

was puriﬁed by ﬂash chromatography using a pre-packed 50 g sil-

ica column [solvent A: EtOAc; solvent B: hexanes; gradient: 7%

A/93% B (1 CV), 7% A/93% B ? 60% A/40% B (10 CV), 60% A/40% B

(2 CV); ﬂow rate: 50 mL/min; monitored at 254 and 280 nm] to

afford benzosuberene 29 (0.362 g, 1.06 mmol, 17%) as a white

solid. ¹H NMR (500 MHz, CDCl₃) d 6.99 (1H, d, J = 8.5 Hz), 6.84

(1H, d, J = 2.5 Hz), 6.74 (1H, dd, J = 8.5, 2.5 Hz), 6.53 (2H, s), 6.37

(1H, t, J = 7.5 Hz), 3.78 (3H, s), 3.81 (3H, s), 3.79 (6H, s) 2.65 (2H,

t, J = 7 Hz), 2.18 (2H, p, J = 7 Hz), 1.98 (2H, q, J = 7 Hz). ¹³C NMR

(125 MHz, CDCl₃) d 158.5, 152.8, 143.7, 142.7, 138.5, 137.3,

132.3, 130.5, 126.8, 113.8, 111.1, 105.1, 60.7, 55.9, 54.9, 35.0,

32.7, 25.4. HRMS: Obsd 363.1574 [M+Na⁺], Calcd for C₂₁H₂₄O₄Na:

363.1567. HPLC (Method B): 18.33 min.

4.1.1.49. tert-Butyldimethyl((9-(3⁰,4⁰,5⁰-trimethoxyphenyl)-6,7-

dihydro-5H-benzo[7]annulen-3-yl)oxy)silane (55).³¹

Acetic

acid (10 mL) was added to tertiary alcohol 48 (0.47 g, 1.0 mmol),

and the reaction mixture was stirred for 12 h at room temperature.

The mixture was washed with water and extracted with EtOAc

(3 ꢁ 30 mL). The combined organic phase was dried over sodium

sulfate and evaporated under reduced pressure to afford protected

benzosuberene 55 (0.39 g, 0.89 mmol, 87%) as a pale yellow oil. ¹H

NMR (500 MHz, CDCl₃) d 6.90 (1H, d, J = 8 Hz), 6.77 (1H, d,

J = 2.5 Hz), 6.67 (1H, dd, J = 8.5, 2.5 Hz), 6.49 (2H, s), 6.34 (1H, t,

J = 7 Hz), 3.86 (3H, s), 3.79 (6H, s), 2.60 (2H, t, J = 7 Hz), 2.15 (2H, p,

J = 7.5 Hz), 1.96 (2H, q, J = 7 Hz), 1.00 (9H, s), 0.23 (6H, s). ¹³C NMR

(125 MHz, CDCl₃) d 154.4, 152.8, 143.7, 142.8, 138.4, 137.3, 133.0,

130.5, 127.0, 120.0, 117.3, 105.2, 60.9, 56.1, 35.0, 32.6, 25.7, 25.5,

18.2, ꢂ4.3.

4.1.1.53. 3-Methoxy-6,7-dihydro-5H-benzo[7]annulen-9-yl tri-

ﬂuoromethanesulfonate (59).

To an oven dried ﬂask, diiso-

propylamine (1.74 mL, 12.4 mmol) dissolved in THF (50 mL) was

added and cooled to ꢂ78 °C. Then n-BuLi (5.0 mL, 12 mmol) was

added, and the reaction was stirred for 15 min. Benzosuberone

14 (2.14 g, 11.3 mmol) dissolved in THF was added dropwise and

stirred for 2 h at ꢂ78 °C. N-Phenyl-bis(triﬂuoromethanesulfon-

imide) (4.42 g, 12.4 mmol) dissolved in THF was then added drop-

wise, and the reaction mixture was stirred for 12 h while warming

from ꢂ78 °C to room temperature. After 12 h, the THF was evapo-

rated under reduced pressure, and the resulting solid was washed

with water and extracted with EtOAc (3 ꢁ 50 mL). The combined

organic layer was dried over sodium sulfate and evaporated under

reduced pressure. The crude reaction product was puriﬁed by ﬂash

chromatography using a pre-packed 25 g silica column [solvent A:

EtOAc; solvent B: hexanes; gradient: 2% A/98% B (1 CV), 2% A/98%

B ? 20% A/80% B (10 CV), 20% A/80% B (2 CV); ﬂow rate: 25 mL/

min; monitored at 254 and 280 nm] to afford triﬂate 59 (2.28 g,

7.07 mmol, 68%) as a yellow solid. ¹H NMR (500 MHz, CDCl₃)

7.51 (1H, d, J = 8.5 Hz), 6.86 (1H, dd, J = 8.5, 2.5 Hz), 6.81 (1H, d,

J = 3 Hz), 6.15 (1H, t, J = 6 Hz), 3.79 (3H, s), 2.77 (2H, t, J = 6.5 Hz),

2.20 (2H, q, J = 7 Hz), 2.04 (2H, q, J = 7 Hz).

4.1.1.50. 9-(3⁰,4⁰,5⁰-Trimethoxyphenyl)-6,7-dihydro-5H-benzo[7]

annulen-3-ol (68).³¹

TBS-protected 55 (0.39 g, 0.89 mmol)

was dissolved in THF (25 mL), TBAF (1.06 mL, 1.06 mmol) was

added, and the reaction mixture was stirred at room temperature

for 12 h. The solution was washed with water and extracted with

EtOAc (3 ꢁ 30 mL). The combined organic phase was dried over

sodium sulfate and evaporated under reduced pressure. The crude

reaction product was puriﬁed by ﬂash chromatography using a

pre-packed 25 g silica column [solvent A: EtOAc; solvent B: hex-

anes; gradient: 7% A/93% B (1 CV), 7% A/93% B ? 60% A/40% B

(10 CV), 60% A/40% B (2 CV); ﬂow rate: 25 mL/min; monitored at

254 and 280 nm] to afford benzosuberene 68 (0.13 g, 0.40 mmol,

45%) as a pale yellow oil. ¹H NMR (500 MHz, CDCl₃) d 6.89 (1H,

d, J = 8 Hz), 6.78 (1H, d, J = 3 Hz), 6.67 (1H, dd, J = 8.5 Hz, 2.5 Hz),

6.50 (2H, s), 6.33 (1H, t, J = 7.5 Hz), 6.21 (1H, s), 3.88 (3H, s), 3.79

(6H, s), 2.59 (2H, t, J = 7 Hz), 2.14 (2H, p, J = 7 Hz), 1.95 (2H, q,

J = 7 Hz). ¹³C NMR (125 MHz, CDCl₃) d 154.9, 152.8, 144.0, 142.7,

138.7, 137.0, 132.1, 130.7, 127.1, 115.4, 112.9, 105.3, 61.0, 56.1,

35.0, 32.6, 25.5. HRMS: Obsd 349.1417 [M+Na⁺], Calcd for

C₂₀H₂₂O₄Na: 349.1410. HPLC (Method B): 14.30 min.

4.1.1.54. 3-Methoxy-9-(2⁰,3⁰,4⁰-trimethoxyphenyl)-6,7-dihydro-

5H-benzo[7]annulene (62).

Triﬂate 59 (2.28 g, 7.07 mmol)

was dissolved in THF, and 2,3,4-trimethoxyphenyl boronic acid

(1.65 g, 7.78 mmol), barium hydroxide octahydrate (3.35 g,

4.1.1.51.

tetrahydro-5H-benzo[7]annulen-5-ol (20).^30,31,56

2-Methoxy-5-(3⁰,4⁰,5⁰-trimethoxyphenyl)-6,7,8,9-

To an oven

10.6 mmol),

and

tetrakis(triphenylphosphine)palladium(0)

dried ﬂask, THF (50 mL) and 3,4,5-trimethoxyphenyl bromide

(2.82 g, 11.4 mmol) were added, and the solution was cooled to

ꢂ78 °C. n-BuLi (4.9 mL,12 mmol) was slowly added to the reaction

mixture, which was then stirred at ꢂ78 °C for 1 h. Benzosuberone

14 (1.60 g, 8.41 mmol) was then added dropwise to the ﬂask, and

the reaction mixture was stirred while warming from ꢂ78 °C to

room temperature over 12 h. The reaction mixture was washed with

water and extracted with EtOAc (3 ꢁ 50 mL). The combined organic

phase was dried over sodium sulfate and evaporated under reduced

pressure. The crude reaction product was puriﬁed by ﬂash chro-

matography using a pre-packed 100 g silica column [solvent A:

EtOAc; solvent B: hexanes; gradient: 10% A/90% B (1 CV), 10%

A/90% B ? 60% A/40% B (10 CV), 60% A/40% B (2 CV); ﬂow rate:

50 mL/min; monitored at 254 and 280 nm] to afford tertiary alcohol

20 (2.29 g, 6.48 mmol, 76%) as a light yellow oil. NMR characteriza-

tion was performed after the next step.

(0.24 g, 0.21 mmol) were added to the solution and reﬂuxed at

80 °C for 2 h. The solution was then ﬁltered through Celite^Ò, and

the Celite^Òwas washed with dichloromethane (3 ꢁ 30 mL). The

combined organic solution (THF and dichloromethane) was evapo-

rated under reduced pressure. The crude reaction mixture was

puriﬁed by ﬂash chromatography using a pre-packed 50 g silica

column [solvent A: EtOAc; solvent B: hexanes; gradient: 7%

A/93% B (1 CV), 7% A/93% B ? 45% A/55% B (10 CV), 45% A/55% B

(2 CV); ﬂow rate: 50 mL/min; monitored at 254 and 280 nm] to

afford benzosuberene 62 (1.05 g, 3.08 mmol, 44%) as a yellow solid.

¹H NMR (500 MHz, CDCl₃) d 7.02 (1H, d, J = 8.5 Hz), 6.88 (1H, d,

J = 2.5 Hz), 6.77 (1H, dd, J = 8.5, 2.5 Hz), 6.58 (2H, s), 6.41 (1H, t,

J = 7.5 Hz), 3.91 (3H, s), 3.83 (3H s), 3.82 (6H, s), 2.69 (2H, t,

J = 7 Hz), 2.21 (2H, p, J = 7 Hz), 2.00 (2H, q, J = 7.5 Hz). ¹³C NMR

(125 MHz, CDCl₃) d 158.5, 153.4, 152.8, 143.6, 142.7, 138.3,

137.4, 132.3, 130.4, 126.7, 123.5, 113.8, 111.1, 105.2, 60.6, 60.1,

55.8, 54.8, 35.0, 32.7, 25.4. HRMS: Obsd 363.1573 [M+Na⁺], Calcd

for C₂₁H₂₄O₄Na: 363.1567. HPLC (Method B): 18.23 min.

4.1.1.52. 3-Methoxy-9-(3⁰,4⁰,5⁰-trimethoxyphenyl)-6,7-dihydro-

5H-benzo[7]annulene (29).^30,31,56

Acetic acid (15 mL) was

added to tertiary alcohol 20 (2.29 g, 6.38 mmol), and the reaction

mixture was stirred for 12 h at room temperature. The mixture

was washed with water and extracted with EtOAc (3 ꢁ 30 mL).

4.1.1.55.

benzo[7]annulene (37).

bromide (0.886 g, 4.73 mmol) in THF (50 mL) at ꢂ78 °C was added

3-Methoxy-9-(4⁰-methoxyphenyl)-6,7-dihydro-5H-

To a solution of 4-methoxyphenyl

Please cite this article in press as: Herdman, C. A.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.10.012

18

C. A. Herdman et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx

n-BuLi (3.4 mL, 2.5 M in hexanes), and the reaction mixture was

stirred for 30 min. Benzosuberone 14 (0.601 g, 3.15 mmol) in THF

(25 mL) was added dropwise over a period of 15 min. The reaction

mixture was stirred while warming to room temperature over

12 h. Water was added, and the mixture was extracted with EtOAc

(100 mL). The organic extract was washed with brine, dried over

Na₂SO₄, ﬁltered, concentrated under reduced pressure, and sub-

jected to ﬂash chromatography using a pre-packed 40 g silica col-

umn [solvent A, EtOAc, solvent B, hexanes; gradient 0% A/100%

B ? 100% A/0% B over 22.9 min; ﬂow rate: 50 mL/min; monitored

at 254 and 280 nm] to afford benzosuberene analogue 37 (0.32 g,

1.1 mmol, 36%) as a white solid. ¹H NMR (CDCl₃, 500 MHz) d 7.20

(2H, d, J = 8.7 Hz), 6.94 (1H, d, J = 8.4 Hz), 6.83 (2H, d, J = 8.5 Hz),

6.82 (1H, d, J = 2.5 Hz), 6.73 (1H, dd, J = 8.5, 2.7 Hz), 6.29 (1H, t,

J = 7.5 Hz) 3.83 (3H, s), 3.81 (3H, s), 2.63 (2H, t, J = 7.0 Hz), 2.16

(2H, p, J = 7.1 Hz), 1.96 (2H, q, J = 7.2 Hz). ¹³C NMR (CDCl₃,

126 MHz) d 158.8, 158.3, 143.8, 142.1, 135.3, 133.0, 130.4, 125.9,

113.9, 113.50, 113.49, 111.1, 55.3, 55.2, 35.2, 32.8, 25.4. HRMS:

Obsd 311.1713 [M+H⁺], Calcd for C₂₀H₂₁O₃: 311.1642. HPLC

(Method B): 17.65 min.

4.1.1.58.

(9).

5-(3⁰-Methoxy-2⁰-methylphenyl)pentanoic

To dissolved carboxylic acid 3 (2.12 g, 9.62 mmol) in

acid

MeOH (100 mL) was added 10% palladium on carbon (0.44 g) and

hydrogen gas. The reaction mixture was stirred at room tempera-

ture for 12 h. The mixture was then ﬁltered through Celite^Ò, and

the Celite^Òwas washed with EtOAc (3 ꢁ 50 mL). The combined

organic phase (MeOH and EtOAc) was evaporated under reduced

pressure. The residue was puriﬁed by ﬂash chromatography using

a pre-packed 100 g silica column [solvent A: EtOAc; solvent B: hex-

anes; gradient: 7% A/93% B (1 CV), 7% A/93% B ? 40% A/60% B

(10 CV), 40% A/60% B (2 CV); ﬂow rate: 40 mL/min; monitored at

254 and 280 nm] to afford carboxylic acid 9 (1.20 g, 5.40 mmol,

59%) as a white solid. ¹H NMR (500 MHz, CDCl₃) d 11.94 (1H, s),

7.23 (1H, t, J = 8 Hz), 6.91 (1H, d, J = 7.5 Hz), 6.84 (1H, d,

J = 8.5 Hz), 3.93 (3H, s), 2.77 (2H, t, J = 8 Hz), 2.51 (2H, t, J = 7 Hz),

2.34 (3H, s), 1.86 (2H, p, J = 7.5 Hz), 1.75 (2H, m). ¹³C NMR

(125 MHz, CDCl₃) d 180.4, 157.9, 141.6, 126.1, 124.5, 121.6,

108.0, 55.5, 34.1, 33.4, 30.0, 24.7, 11.3.

4.1.1.59. 2-Methoxy-1-methyl-6,7,8,9-tetrahydro-5H-benzo[7]

annulen-5-one (15).

To carboxylic acid 9 (1.20 g, 5.40 mmol)

4.1.1.56. 3-Methoxy-2-methylbenzaldehyde.⁵⁸

N⁰,N⁰,N⁰-Tri-

was added Eaton’s reagent (10.8 mL, 3 g per mmol of compound 9),

and the mixture was stirred at room temperature for 12 h. It was

then poured over ice and neutralized with sodium bicarbonate.

The aqueous layer was extracted with EtOAc (3 ꢁ 50 mL). The com-

bined organic phase was dried over sodium sulfate, evaporated

under reduced pressure, and puriﬁed by ﬂash chromatography

using a pre-packed 100 g silica column [solvent A: EtOAc; solvent

B: hexanes; gradient: 5% A/95% B (1 CV), 5% A/95% B ? 40%

A/60% B (10 CV), 40% A/60% B (2 CV); ﬂow rate: 40 mL/min; mon-

itored at 254 and 280 nm] to afford benzosuberone 15 (2.43 g,

11.0 mmol, 74%) as a light yellow solid. ¹H NMR (500 MHz, CDCl₃)

d 7.42 (1H, d, J = 8.5 Hz), 6.64 (1H, d, J = 8.5 Hz), 3.72 (3H, s), 2.76

(2H, t, J = 6 Hz), 2.53 (2H, t, J = 6 Hz), 2.09 (3H, s), 1.69 (2H, p,

J = 7.5 Hz), 1.61 (2H, p, J = 6.5 Hz). ¹³C NMR (125 MHz, CDCl₃) d

205.9, 160.4, 140.4, 132.6, 127.4, 123.7, 107.6, 55.4, 40.4, 27.0,

24.1, 20.6, 11.0.

methylethylene diamine (1.84 mL, 14.3 mmol) was dissolved in

benzene (25 mL) and cooled to 0 °C. n-BuLi (5.5 mL, 13 mmol)

was added dropwise, and the reaction mixture was stirred for

10 min at room temperature. The reaction mixture was again

cooled to 0 °C, and m-anisaldehyde (1.54 mL, 13.3 mmol) was

added. The reaction mixture was stirred for 15 min at room tem-

perature and then cooled to 0 °C. Phenyllithium (22.0 mL,

40.0 mmol) was added dropwise. The reaction mixture was stirred

for 12 h at room temperature. THF (20 mL) was then added, and

the reaction mixture was cooled to ꢂ78 °C. Methyl iodide

(5.0 mL, 80 mmol) was added dropwise, and the reaction mixture

was stirred for 30 min at room temperature. The reaction mixture

was poured into cold 10% HCl (50 mL) and extracted with EtOAc

(3 ꢁ 30 mL). The combined organic phase was dried over sodium

sulfate and evaporated under reduced pressure. The crude reaction

product was puriﬁed using a pre-packed 50 g silica column [sol-

vent A: EtOAc; solvent B: hexanes; gradient: 2% A/98% B (1 CV),

2% A/98% B ? 20% A/80% B (10 CV), 20% A/80% B (2 CV); ﬂow rate:

40 mL/min; monitored at 254 and 280 nm] to afford 3-methoxy-2-

methylbenzaldehyde (2.01 g, 14.3 mmol, 98%) as a yellow oil. ¹H

NMR (500 MHz, CDCl₃) d 9.97 (1H, s), 7.10 (1H, d, J = 8 Hz), 6.98

(1H, t, J = 8 Hz), 6.74 (1H, d, J = 8 Hz), 3.54 (3H, s), 2.23 (3H, s).

¹³C NMR (125 MHz, CDCl₃) d 192.0, 157.8, 134.9, 128.9, 126.3,

122.7, 114.8, 55.3, 10.0.

4.1.1.60.

6,7,8,9-tetrahydro-5H-benzo[7]annulen-5-ol (21).

2-Methoxy-1-methyl-5-(3,⁰4⁰,5-trimethoxyphenyl)-

To an

oven dried ﬂask, THF (50 mL) and 3,4,5-trimethoxyphenyl bromide

(0.85 g, 3.4 mmol) were added, and the solution was cooled to

ꢂ78 °C. n-BuLi (1.5 mL, 3.6 mmol) was slowly added to the reac-

tion mixture, which was then stirred at ꢂ78 °C for 1 h. Benzo-

suberone 15 (0.52 g, 2.6 mmol) was then added dropwise to the

ﬂask, and the reaction mixture was stirred while warming from

ꢂ78 °C to room temperature over 12 h. The reaction mixture was

washed with water and extracted with EtOAc (3 ꢁ 50 mL). The

combined organic phase was dried over sodium sulfate and evap-

orated under reduced pressure. The crude reaction product was

puriﬁed by ﬂash chromatography using a pre-packed 100 g silica

column [solvent A: EtOAc; solvent B: hexanes; gradient: 10%

A/90% B (1 CV), 10% A/90% B ? 60% A/50% B (10 CV), 60% A/50% B

(2 CV); ﬂow rate: 40 mL/min; monitored at 254 and 280 nm] to

afford tertiary alcohol 21 (2.29 g, 6.39 mmol, 76%) as a light yellow

oil. NMR characterization was performed after the next step.

4.1.1.57. 5-(3⁰-Methoxy-2⁰-methylphenyl)pent-4-enoic acid

(3).

To dissolved 3-(carboxypropyl)triphenyl phosphonium

bromide (6.55 g, 15.3 mmol) in THF was added potassium tert-

butoxide (3.30 g, 29.0 mmol), and the reaction mixture was stirred

at room temperature for 1 h. 3-Methoxy-2-methylbenzaldehyde

(2.01 g, 13.4 mmol) dissolved in THF was added to the original

reaction mixture, and the reaction mixture was stirred at room

temperature for 12 h. The THF was evaporated, and the resulting

material was quenched with 2 M HCl (75 mL) and extracted with

EtOAc (3 ꢁ 100 mL). The combined organic phase was evaporated

under reduced pressure and puriﬁed by ﬂash chromatography

using a pre-packed 100 g silica column [solvent A: EtOAc; solvent

B: hexanes; gradient: 12% A/88% B (1 CV), 12% A/88% B ? 75%

A/25% B (10 CV), 75% A/25% B (2 CV); ﬂow rate: 40 mL/min; mon-

itored at 254 and 280 nm] to afford carboxylic acid 3 (2.12 g,

9.62 mmol, 69%) as a yellow oil. NMR characterization was per-

formed after the next step.

4.1.1.61.

6,7-dihydro-5H-benzo[7]annulene (30).

3-Methoxy-4-methyl-9-(3⁰,4⁰,5⁰-trimethoxyphenyl)-

Acetic acid (15 mL)

was added to tertiary alcohol 21 (0.48 g, 1.3 mmol), and the reac-

tion mixture was stirred for 12 h at room temperature. The mix-

ture was washed with water and extracted with EtOAc

(3 ꢁ 30 mL). The combined organic phase was dried over sodium

sulfate and evaporated under reduced pressure. The crude reaction

Please cite this article in press as: Herdman, C. A.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.10.012

C. A. Herdman et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx

19

product was puriﬁed by ﬂash chromatography using a pre-packed

10 g silica column [solvent A: EtOAc; solvent B: hexanes; gradient:

5% A/95% B (1 CV), 5% A/95% B ? 40% A/60% B (10 CV), 40% A/60% B

(2 CV); ﬂow rate: 12 mL/min; monitored at 254 and 280 nm] to

afford benzosuberene 30 (0.16. g, 0.45 mmol, 36%) as a yellow

solid. ¹H NMR (500 MHz, CDCl₃) d 6.86 (1H, d, J = 8.5 Hz), 6.69

(1H, d, J = 8.5 Hz), 6.53 (2H, s), 6.33 (1H, t, J = 7 Hz), 3.87 (3H, s),

3.82 (3H s,) 3.80 (6H, s), 2.69 (2H, t, J = 6.5 Hz), 2.29 (3H, s), 2.12

(2H, p, J = 7 Hz), 1.91 (2H, q, J = 7 Hz). ¹³C NMR (125 MHz, CDCl₃)

d 156.5, 152.8, 143.5, 141.6, 138.5, 137.3, 132.9, 127.4, 126.4,

123.1, 107.4, 105.3, 60.8, 56.1, 55.4, 34.0, 27.7, 25.5, 11.8. HRMS:

Obsd 377.1731 [M+Na⁺], Calcd for C₂₂H₂₆O₅Na: 377.1723. HPLC

(Method B): 19.56 min.

pressure. The crude reaction product was puriﬁed by ﬂash chro-

matography using a pre-packed 100 g silica column [solvent A:

EtOAc; solvent B: hexanes; gradient: 7% A/93% B (1 CV), 7%

A/93% B ? 40% A/60% B (10 CV), 40% A/60% B (2 CV); ﬂow rate:

40 mL/min; monitored at 254 and 280 nm] to afford carboxylic

acid 10 (0.79 g, 3.3 mmol, 44%) as a clear oil. It is likely in this case

that the carboxylic acid became methylated. ¹H NMR (500 MHz,

CDCl₃) d 7.14 (1H, t, J = 8 Hz), 6.82 (1H, d, J = 7.5 Hz), 6.75 (1H, d,

J = 8.5 Hz), 3.83 (3H, s), 3.70 (3H, s), 2.76 (2H, q, J = 7.5 Hz), 2.70

(2H, t, J = 8 Hz), 2.39 (2H, J = 7.5 Hz), 1.79 (2H, p, J = 7 Hz), 1.68

(2H, p, J = 8 Hz), 1.22, (3H, t, J = 7.5 Hz). ¹³C NMR (125 MHz, CDCl₃)

d 173.7, 157.6, 140.9, 130.6, 126.2, 121.7, 108.1, 55.2, 21.2, 33.9,

32.6, 21.1, 25.1, 19.2, 14.5.

4.1.1.62.

2-Ethyl-3-methoxybenzaldehyde.⁵⁹

N⁰,N⁰N⁰-

4.1.1.65. 1-Ethyl-2-methoxy-6,7,8,9-tetrahydro-5H-benzo[7]an-

Trimethylethlene diamine (1.36 mL, 10.5 mmol) was dissolved in

benzene (25 mL) and cooled to 0 °C. n-BuLi (4.05 mL, 10.1 mmol)

was added dropwise, and the reaction mixture was stirred for

10 min at room temperature. The reaction mixture was again

cooled to 0 °C, m-anisaldehyde (1.34 mL, 9.84 mmol) was added,

and the reaction mixture was stirred for 15 min at room tempera-

ture. The reaction mixture was then cooled to 0 °C, and phenyl-

lithium (16.4 mL, 29.5 mmol) was added dropwise. The reaction

mixture was stirred overnight at room temperature. THF (20 mL)

was then added, and the reaction mixture was cooled to ꢂ78 °C.

Ethyl iodide (3.7 mL, 59 mmol) was added dropwise, and the reac-

tion mixture was stirred for 30 min at room temperature. The reac-

tion mixture was poured into cold 10% HCl (50 mL) and extracted

with EtOAc (3 ꢁ 30 mL). The combined organic phase was dried

over sodium sulfate and evaporated under reduced pressure. The

crude reaction product was puriﬁed using a pre-packed 100 g silica

column [solvent A: EtOAc; solvent B: hexanes; gradient: 5% A/95%

B (1 CV), 5% A/95% B ? 20% A/80% B (10 CV), 20% A/80% B (2 CV);

ﬂow rate: 50 mL/min; monitored at 254 and 280 nm] to afford 2-

ethyl-3-methoxybenzaldehyde (0.91 g, 5.6 mmol, 57%) as a yellow

oil. ¹H NMR (500 MHz, CDCl₃) d 10.24 (1H, s), 7.37 (1H, dd, J = 7.5,

1 Hz), 7.23 (1H, t, J = 8 Hz), 7.01 (1H, dd, J = 8.5, 1 Hz), 3.79 (3H, s),

3.01 (2H, q, J = 7.5 Hz), 1.14 (3H, t, J = 7.5 Hz). ¹³C NMR (125 MHz,

CDCl₃) d 192.1, 157.6, 135.9, 134.4., 126.7, 122.6, 115.5, 55.7,

17.5, 15.3.

nulen-5-one (16).

To carboxylic acid 10 (0.79 g, 3.3 mmol)

was added Eaton’s reagent (6.7 mL, 3 g per mmol of compound

10), and the reaction mixture was stirred at room temperature

for 12 h. The mixture was then poured over ice and neutralized

with sodium bicarbonate. The aqueous layer was extracted with

EtOAc (3 ꢁ 50 mL). The combined organic phase was dried over

sodium sulfate, evaporated under reduced pressure, and puriﬁed

by ﬂash chromatography using a pre-packed 100 g silica column

[solvent A: EtOAc; solvent B: hexanes; gradient: 7% A/93% B

(1 CV), 7% A/93% B ? 60% A/40% B (10 CV), 60% A/40% B (2 CV);

ﬂow rate: 40 mL/min; monitored at 254 and 280 nm] to afford

benzosuberone 16 (0.32 g, 1.6 mmol, 52%) as a yellow solid. ¹H

NMR (500 MHz, CDCl₃) d 7.46 (1H, d, J = 8.5 Hz), 6.70 (1H, d,

J = 8.5 Hz), 3.77 (3H, s), 2.82 (2H, t, J = 6 Hz), 2.65 (2H, q,

J = 7.5 Hz), 2.57 (2H, t, J = 6 Hz), 1.75 (2H, p, J = 7 Hz), 1.66 (2H, p,

J = 6.5 Hz), 1.03 (3H, t, J = 7.5 Hz). ¹³C NMR (125 MHz, CDCl₃) d

206.3, 160.3, 139.5, 132.9, 130.0, 127.7, 108.0, 55.4, 40.4, 25.3,

24.9, 20.4, 18.9, 14.5.

4.1.1.66.

6,7,8,9-tetrahydro-5H-benzo[7]annulen-5-ol (22).

1-Ethyl-2-methoxy-5-(3⁰,4⁰,5⁰-trimethoxyphenyl)-

To an

oven dried ﬂask, THF (50 mL) and 3,4,5-trimethoxyphenyl bromide

(0.52 g, 2.1 mmol) were added, and the solution was cooled to

ꢂ78 °C. n-BuLi (0.88 mL, 2.2 mmol) was slowly added to the reac-

tion mixture, which was then stirred at ꢂ78 °C for 1 h. Benzo-

suberone 16 (0.32 g, 1.54 mmol) was then added dropwise to the

ﬂask, and the reaction mixture was stirred while warming from

ꢂ78 °C to room temperature over 12 h. The reaction mixture was

washed with water and extracted with EtOAc (3 ꢁ 50 mL). The

combined organic phase was dried over sodium sulfate and evap-

orated under reduced pressure. The crude reaction product was

puriﬁed by ﬂash chromatography using a pre-packed 25 g silica

column [solvent A: EtOAc; solvent B: hexanes; gradient: 7%

A/93% B (1 CV), 7% A/93% B ? 60% A/40% B (10 CV), 60% A/40% B

(2 CV); ﬂow rate: 25 mL/min; monitored at 254 and 280 nm] to

afford tertiary alcohol 22 (0.20 g, 0.52 mmol, 33%) as a light yellow

oil. NMR characterization was performed after the next step.

4.1.1.63.

(4).

5-(2⁰-Ethyl-3⁰-methoxyphenyl)pent-4-enoic

To dissolved 3-(carboxypropyl)triphenyl phosphonium

acid

bromide (3.56 g, 8.30 mmol) in THF (500 mL) was added potassium

tert-butoxide (2.03 g, 18.1 mmol), and the reaction mixture was

stirred at room temperature for 1 h. 2-Ethyl-3-methoxybenzalde-

hyde (1.35 g, 8.22 mmol) dissolved in THF (100 mL) was added to

the original reaction mixture and stirred at room temperature for

12 h. The THF was evaporated, and the resulting material was

quenched with 2 M HCl (75 mL) and extracted with EtOAc

(3 ꢁ 100 mL). The combined organic layer was evaporated under

reduced pressure and puriﬁed by ﬂash chromatography using a

pre-packed 100 g silica column [solvent A: EtOAc; solvent B: hex-

anes; gradient: 7% A/93% B (1 CV), 7% A/93% B ? 60% A/40% B

(10 CV), 60% A/40% B (2 CV); ﬂow rate: 50 mL/min; monitored at

254 and 280 nm] to afford carboxylic acid 4 (1.78 g, 7.60 mmol,

92%) as an orange-yellow oil. NMR characterization was performed

after the next step.

4.1.1.67. 4-Ethyl-3-methoxy-9-(3⁰,4⁰,5⁰-trimethoxyphenyl)-6,7-

dihydro-5H-benzo[7]annulene (31).

Acetic acid (15 mL)

was added to tertiary alcohol 22 (0.20 g, 0.52 mmol), and the reac-

tion mixture was stirred for 12 h at room temperature. The reac-

tion mixture was washed with water and extracted with EtOAc

(3 ꢁ 30 mL). The combined organic phase was dried over sodium

sulfate and evaporated under reduced pressure. The crude reaction

product was puriﬁed by ﬂash chromatography using a pre-packed

25 g silica column [solvent A: EtOAc; solvent B: hexanes; gradient:

7% A/93% B (1 CV), 7% A/93% B ? 60% A/50% B (10 CV), 60% A/40% B

(2 CV); ﬂow rate: 25 mL/min; monitored at 254 and 280 nm] to

afford benzosuberene 31 (0.085 g, 0.32 mmol, 45%) as a yellow

solid. ¹H NMR (500 MHz, CDCl₃) d 6.85 (1H, d, J = 8.5 Hz), 6.69

4.1.1.64.

(10).

5-(2⁰-Ethyl-3⁰-methoxyphenyl)pentanoic

To dissolved carboxylic acid 4 (1.78 g, 7.60 mmol) in

acid

MeOH (50 mL) was added 10% palladium on carbon (0.58 g) and

hydrogen gas. The reaction was stirred at room temperature for

12 h. The reaction mixture was then ﬁltered through Celite^Ò, and

the Celite^Òwas washed with EtOAc (3 ꢁ 50 mL). The combined

organic phase (MeOH and EtOAc) was evaporated under reduced

Please cite this article in press as: Herdman, C. A.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.10.012

20

C. A. Herdman et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx

(1H, d, J = 8.5 Hz), 6.52 (2H, s), 6.32 (1H, t, J = 7.5 Hz), 3.86 (3H, s),

3.83 (3H, s), 3.81 (6H, s), 2.78 (2H, q, J = 7.5 Hz), 2.69 (2H, t,

J = 6.5 Hz), 2.14 (2H, p, J = 7 Hz), 1.92 (2H, q, J = 7 Hz), 1.18 (3H, t,

J = 7.5 Hz). ¹³C NMR (125 MHz, CDCl₃) d 156.5, 153.1, 143.8,

141.3, 139.0, 137.6, 133.4, 129.9, 127.9, 126.7, 107.8, 105.6, 61.2,

56.4, 55.7, 35.2, 27.5, 25.7, 20.0, 15.2. HRMS: Obsd 391.1891 [M

+Na⁺], Calcd for C₂₃H₂₈O₄Na: 391.1880. HPLC (Method B):

20.66 min.

monitored at 254 and 280 nm] to afford compound 11 (0.87 g,

3.5 mmol, 38%) as a clear oil. It is likely that the carboxylic acid

was methylated during this reaction. ¹H NMR (500 MHz, CDCl₃) d

7.14 (1H, t, J = 8 Hz), 6.83 (1H, d, J = 7.5 Hz), 6.75 (1H, d, J = 8 Hz),

3.82 (3H, s), 3.71 (3H, s), 2.71 (4H, m), 2.40 (2H, t, J = 7.5 Hz),

1.79 (2H, p, J = 7.5 Hz), 1.66 (4H, m), 1.09 (3H, t, J = 7.5 Hz). ¹³C

NMR (125 MHz, CDCl₃) d 173.4, 157.5, 140.9, 128.8, 125.9, 121.2,

107.6, 54.8, 50.9, 33.5, 32.3, 30.7, 27.8, 24.7, 23.1, 14.2.

4.1.1.68.

3-Methoxy-2-propylbenzaldehyde.⁶⁰

N⁰,N⁰,

4.1.1.71.

annulen-5-one (17).

2-Methoxy-1-propyl-6,7,8,9-tetrahydro-5H-benzo[7]

To carboxylic acid 11 (0.87 g, 3.5 mmol)

N⁰-Trimethylethlene diamine (1.55 mL, 12.0 mmol) was dissolved

in benzene (25 mL) and cooled to 0 °C. n-BuLi (4.6 mL, 11 mmol)

was added dropwise, and the reaction mixture was stirred for

10 min at room temperature. The reaction mixture was again

cooled to 0 °C, and m-anisaldehyde (1.30 mL, 11.2 mmol) was

added. The reaction mixture was stirred for 15 min at room tem-

perature and cooled to 0 °C. Phenyllithium (18.7 mL, 33.7 mmol)

was added dropwise. The reaction mixture was stirred overnight

at room temperature. THF (20 mL) was then added, and the reac-

tion mixture was cooled to ꢂ78 °C. Propyl iodide (6.7 mL,

67.32 mmol) was added dropwise, and the reaction mixture was

stirred for 30 min at room temperature. The reaction mixture

was poured into cold 10% HCl (50 mL) and extracted with EtOAc

(3 ꢁ 30 mL). The combined organic phase was dried over sodium

sulfate and evaporated under reduced pressure. The crude reaction

product was puriﬁed using a pre-packed 100 g silica column [sol-

vent A: EtOAc; solvent B: hexanes; gradient: 2% A/98% B (1 CV),

2% A/98% B ? 20% A/80% B (10 CV), 20% A/80% B (2 CV); ﬂow rate:

50 mL/min; monitored at 254 and 280 nm] to afford 3-methoxy-2-

propylbenzaldehyde (1.82 g, 10.2 mmol, 91%) as a yellow oil. ¹H

NMR (500 MHz, CDCl₃) d 10.19 (1H, s), 7.32 (1H, d, J = 8 Hz), 7.16

(1H, t, J = 7.5 Hz), 6.95 (1H, d, J = 8 Hz), 3.71 (3H, s), 2.94 (2H, t,

J = 7 Hz), 1.48 (2H, sext, J = 7.5 Hz), 0.88 (3H, t, J = 7.5 Hz). ¹³C

NMR (125 MHz, CDCl₃) d 191.9, 157.8, 134.8, 134.3, 126.7, 122.3,

115.3, 55.5, 25.8, 24.2, 14.0.

was added Eaton’s reagent (7.0 mL, 3 g per mmol of compound 11),

and the reaction mixture was stirred at room temperature for 12 h.

The reaction mixture was then poured over ice and neutralized

with sodium bicarbonate. The aqueous layer was extracted with

EtOAc (3 ꢁ 50 mL). The combined organic phase was dried over

sodium sulfate and evaporated under reduced pressure. The crude

reaction product was puriﬁed by ﬂash chromatography using a

pre-packed 50 g silica column [solvent A: EtOAc; solvent B: hex-

anes; gradient: 7% A/93% B (1 CV), 7% A/93% B ? 60% A/40% B

(10 CV), 60% A/40% B (2 CV); ﬂow rate: 50 mL/min; monitored at

254 and 280 nm] to afford benzosuberone 17 (0.56 g, 2.41 mmol,

69%) as a yellow oil. ¹H NMR (500 MHz, CDCl₃) d 7.45 (1H, d,

J = 8.5 Hz), 6.68 (1H, d, J = 8.5 Hz), 3.75 (3H, s), 2.81 (2H, t,

J = 6.5 Hz), 2.59 (4H, m), 1.74 (2H, p, J = 7 Hz), 1.65 (2H, p,

J = 6 Hz), 1.42 (2H, sext, J = 7.5 Hz), 0.91 (3H, t, J = 7.5 Hz). ¹³C

NMR (125 MHz, CDCl₃) d 206.2, 160.4, 139.7, 132.9, 128.5, 127.6,

107.8, 55.2, 40.3, 27.6, 25.3, 24.8, 23.2, 20.3, 14.2.

4.1.1.72.

6,7,8,9-tetrahydro-5H-benzo[7]annulen-5-ol (23).

2-Methoxy-1-propyl-5-(3⁰,4⁰,5⁰-trimethoxyphenyl)-

To an

oven dried ﬂask, THF (50 mL) and 3,4,5-trimethoxyphenyl bromide

(0.80 g, 3.3 mmol) were added, and the solution was cooled to

ꢂ78 °C. n-BuLi (1.4 mL, 3.4 mmol) was slowly added to the reac-

tion mixture, which was then stirred at ꢂ78 °C for 1 h. Benzo-

suberone 17 (0.56 g, 2.4 mmol) was then added dropwise to the

ﬂask, and the reaction mixture was stirred while warming from

ꢂ78 °C to room temperature over 12 h. The reaction mixture was

washed with water and extracted with EtOAc (3 ꢁ 50 mL). The

combined organic phase was dried over sodium sulfate and evap-

orated under reduced pressure. The crude reaction product was

puriﬁed by ﬂash chromatography using a pre-packed 25 g silica

column [solvent A: EtOAc; solvent B: hexanes; gradient: 7%

A/93% B (1 CV), 7% A/93% B ? 60% A/40% B (10 CV), 60% A/40% B

(2 CV); ﬂow rate: 25 mL/min; monitored at 254 and 280 nm] to

afford tertiary alcohol 23 (0.53 g, 1.3 mmol, 52%) as a colorless

oil. NMR characterization was performed after the next step.

4.1.1.69.

(5).

5-(3⁰-Methoxy-2⁰-propylphenyl)pent-4-enoic

To dissolved 3-(carboxypropyl)triphenyl phosphonium

acid

bromide (4.43 g, 10.3 mmol) in THF (500 mL) was added potassium

tert-butoxide (2.52 g, 22.5 mmol), and the reaction mixture was

stirred at room temperature for 1 h. 3-Methoxy-2-propylbenzalde-

hyde (1.82 g, 10.2 mmol) dissolved in THF (100 mL) was added to

the original reaction mixture and stirred at room temperature for

12 h. The THF was evaporated, and the residue was quenched with

2 M HCl (75 mL) and extracted with EtOAc (3 ꢁ 100 mL). The com-

bined organic phase was evaporated under reduced pressure and

puriﬁed by ﬂash chromatography using a pre-packed 100 g silica

column [solvent A: EtOAc; solvent B: hexanes; gradient: 7%

A/93% B (1 CV), 7% A/93% B ? 60% A/40% B (10 CV), 60% A/40% B

(2 CV); ﬂow rate: 50 mL/min; monitored at 254 and 280 nm] to

afford compound 5 (2.25 g, 9.06 mmol, 89%) as a yellow oil. NMR

characterization was performed after the next step.

4.1.1.73. 3-Methoxy-4-propyl-9-(3⁰,4⁰,5⁰-trimethoxyphenyl)-6,7-

dihydro-5H-benzo[7]annulene (32).

Acetic acid (10 mL) was

added to tertiary alcohol 23 (0.53 g, 1.3 mmol), and the reaction

mixture was stirred for 12 h at room temperature. The reaction

mixture was washed with water and extracted with EtOAc

(3 ꢁ 30 mL). The combined organic phase was dried over sodium

sulfate and evaporated under reduced pressure. The crude reaction

mixture was puriﬁed by ﬂash chromatography using a pre-packed

10 g silica column [solvent A: EtOAc; solvent B: hexanes; gradient:

7% A/93% B (1 CV), 7% A/93% B ? 60% A/40% B (10 CV), 60% A/40% B

(2 CV); ﬂow rate: 12 mL/min; monitored at 254 and 280 nm] to

afford benzosuberene 32 (0.13 g, 0.34 mmol, 25%) as a cream-

colored solid. ¹H NMR (500 MHz, CDCl₃) d 6.86 (1H, d, J = 8.5 Hz),

6.70 (1H, d, J = 8.5 Hz), 6.53 (2H, s), 6.33 (1H, t, J = 7.5 Hz), 3.88

(3H, s), 3.84 (3H, s), 3.82 (6H, s), 2.72 (4H, m), 2.15 (2H, p,

J = 7 Hz), 1.93 (2H, q, J = 7 Hz), 1.59 (2H, sext, J = 7 Hz), 1.04 (3H,

4.1.1.70.

(11).

5-(3⁰-Methoxy-2⁰-propylphenyl)pentanoic

To dissolved compound 5 (2.25 g, 9.06 mmol) in MeOH

acid

(50 mL) was added 10% palladium on carbon (0.47 g) and hydrogen

gas. The reaction mixture was stirred at room temperature for 12 h.

The mixture was then ﬁltered through Celite^Ò, and the Celite^Òwas

washed with EtOAc (3 ꢁ 50 mL). The combined organic phase

(MeOH and EtOAc) was evaporated under reduced pressure. The

crude reaction product was puriﬁed by ﬂash chromatography

using a pre-packed 100 g silica column [solvent A: EtOAc; solvent

B: hexanes; gradient: 7% A/93% B (1 CV), 7% A/93% B ? 40%

A/60% B (10 CV), 40% A/60% B (2 CV); ﬂow rate: 40 mL/min;

Please cite this article in press as: Herdman, C. A.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.10.012

C. A. Herdman et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx

21

t, J = 7.5 Hz). ¹³C NMR (125 MHz, CDCl₃) d 156.4, 152.8, 143.5,

141.3, 138.7, 137.3, 133.1, 128.3, 127.6, 126.4, 107.5, 105.3, 60.9,

56.2, 55.4, 35.0, 28.6, 27.3, 25.5, 23.8, 14.5. HRMS: Obsd

405.2043 [M+Na⁺], Calcd for C₂₄H₃₀O₄Na⁺: 405.2036. HPLC

(Method B): 21.53 min.

(2.45 g, 11.8 mmol, 65%). ¹H NMR (CDCl₃, 500 MHz) d 7.55 (1H,

dd, J = 7.9 Hz, 1.1 Hz), 7.24 (1H, t, J = 8.0 Hz), 7.05 (1H, dd,

J = 8.3 Hz, 0.9 Hz), 3.87 (3H, s), 3.01 (2H, m), 1.54 (2H, m), 1.43

(2H, m), 0.95 (3H, t, J = 7.3 Hz). ¹³C NMR (CDCl₃, 125 MHz) d

173.1, 158.0, 134.5, 129.9, 126.2, 122.9, 114.4, 55.8, 32.4, 26.3,

23.1, 13.9.

4.1.1.74.

drooxazole.^61,62

2-(2⁰,3⁰-Dimethoxyphenyl)-4,4-dimethyl-4,5-dihy-

mixture of 2,3-dimethoxybenzoic acid

A

4.1.1.77. (2-Butyl-3-methoxyphenyl)methanol.

2-butyl-3-

(5.00 g, 27.5 mmol) in SOCl₂(10 mL) was stirred at room tempera-

ture for 24 h. The excess of SOCl₂was evaporated under reduced

pressure, and the residue was diluted with CH₂Cl₂(80 mL). This

solution was added dropwise to a solution of 2-amino-2-methyl-

propan-1-ol (4.89 g, 55 mmol) in CH₂Cl₂(150 mL) at ꢂ10 °C and

stirred for 20 h while warming to room temperature. The resulting

suspension was ﬁltered, and the ﬁltrate was evaporated to give the

crystalline amide. The latter was treated dropwise with SOCl₂

(10 mL) and stirred at room temperature for another 7 h. The mix-

ture was then poured into diethyl ether (400 mL) to form a suspen-

sion, which was then cooled to 0 °C, and aqueous NaOH solution

(20%, 100 mL) was added. The aqueous layer was extracted with

diethyl ether (3 ꢁ 100 mL). The combined organic phase was dried

with Na₂SO₄, ﬁltered, and concentrated under reduced pressure.

The crude reaction product was puriﬁed by ﬂash chromatography

using a prepacked 100 g silica column [solvent A: EtOAc; solvent

B: hexanes; gradient: 7% A/93% B (3 CV), 7% A/93% B ? 60%

A/40% B (10 CV), 60% A/40% B (1 CV); ﬂow rate: 40 mL/min;

monitored at 254 and 280 nm] to afford 2-(2,3-dimethoxyphenyl)-

4,4-dimethyl-4,5-dihydrooxazole (5.22 g, 22.2 mmol, 81%) as a yel-

low solid. ¹H NMR (CDCl₃, 500 MHz) d 7.31–7.00 (3H, m), 4.11 (2H,

s), 3.86 (6H, m), 1.39 (6H, s). ¹³C NMR (CDCl₃, 125 MHz) d 161.3,

153.3, 148.7, 123.8, 123.4, 122.6, 115.0, 79.2, 67.3, 61.4, 56.1, 28.3.

methoxybenzoic acid (2.45 g, 11.8 mmol) was dissolved in THF

(40 mL) and stirred for 10 min. LiAlH₄(7.6 mL, 2.0 M) was then

added dropwise, and the reaction mixture was stirred for 10 h

while warming to room temperature. The reaction was carefully

quenched with a H₂O/THF (1:4) solution, followed by aqueous

NaOH (15%, 20 mL), and a precipitate formed. The unwanted pre-

cipitate was removed by ﬁltration through Celite^Ò. The Celite^Ò

and unwanted precipitate were washed with CH₂Cl₂. The H₂O/

THF/CH₂Cl₂ﬁltrate was extracted with EtOAc (3 ꢁ 30 mL), and

the organic layer was rinsed with brine and dried with Na₂SO₄.

The crude reaction product was puriﬁed by ﬂash chromatography

using a prepacked 100 g silica column [solvent A: EtOAc; solvent

B: hexanes; gradient: 7% A/93% B (3 CV), 7% A/93% B ? 60%

A/40% B (10 CV), 60% A/40% B (1 CV); ﬂow rate: 40 mL/min; mon-

itored at 254 and 280 nm] to afford (2-butyl-3-methoxyphenyl)

methanol as a colorless liquid (1.94 g, 9.97 mmol, 85%). ¹H NMR

(CDCl₃, 500 MHz) d 7.19 (1H, t, J = 8.5 Hz), 7.01 (1H, d, J = 7.6 Hz),

6.83 (1H, d, J = 8.3 Hz), 4.72 (2H, d, J = 5.3 Hz), 3.83 (3H, s), 2.69

(2H, t, J = 7.4 Hz), 1.49 (4H, m), 0.95 (3H, t, J = 7.2 Hz). ¹³C NMR

(CDCl₃, 125 MHz) d 157.7,139.6, 129.8, 126.6, 120.4, 110.0, 63.2,

55.5, 32.4, 25.5, 23.1, 14.0.

4.1.1.78. 2-Butyl-3-methoxybenzaldehyde.

To a well stirred

solution of pyridinium chlorochromate (2.586 g, 12 mmol) in

CH₂Cl₂(20 mL) at room temperature, (2-butyl-3-methoxyphenyl)

methanol (1.936 g, 9.97 mmol) dissolved in CH₂Cl₂(15 mL) was

slowly added via syringe. The reaction mixture was stirred at room

temperature for 12 h. The reaction mixture was ﬁltered through

Celite^Ò, and the Celite^Òwas washed thoroughly with CH₂Cl₂. The

ﬁltrate was concentrated under reduced pressure and further puri-

ﬁed by ﬂash chromatography using a prepacked 100 g silica col-

umn [solvent A: EtOAc; solvent B: hexanes; gradient: 7% A/93% B

(3 CV), 7% A/93% B ? 60% A/40% B (10 CV), 60% A/40% B (1 CV);

ﬂow rate: 40 mL/min; monitored at 254 and 280 nm] to afford 2-

butyl-3-methoxybenzaldehyde as a dark yellow liquid (1.91 g,

9.93 mmol, 99%). ¹H NMR (CDCl₃, 500 MHz) d 10.34 (1H, s), 7.46

(1H, dd, J = 7.8 Hz, 1.1 Hz), 7.30 (1H, t, J = 8.0 Hz), 7.09 (1H, dd,

J = 8.1 Hz, 0.75 Hz), 3.87 (3H, s), 3.06 (2H, m), 1.53 (2H, m), 1.42

(2H, m), 0.95 (3H, t, J = 7.3 Hz). ¹³C NMR (CDCl₃, 125 MHz) d

192.3, 157.8, 135.0, 134.7, 126.7, 122.1, 115.5, 55.8, 33.5, 23.8,

22.8, 13.9.

4.1.1.75. 2-(2⁰-Butyl-3⁰-methoxyphenyl)-4,4-dimethyl-4,5-dihy-

drooxazole.⁶³

A solution of 2-(2,3-dimethoxyphenyl)-4,4-

dimethyl-4,5-dihydrooxazole (5.02 g, 21.4 mmol) in THF (80 mL)

was stirred and cooled to ꢂ40 °C in a cyclohexanone/dry ice bath.

n-BuLi (13 mL, 2.5 M) was added dropwise to the reaction ﬂask.

The reaction mixture was stirred for 5 h while warming to 0 °C.

The reaction was quenched with saturated aqueous NH₄Cl and

extracted with diethyl ether (3 ꢁ 30 mL). The combined organic

phase was dried with Na₂SO₄and concentrated under reduced

pressure. The crude reaction product was puriﬁed by ﬂash chro-

matography using a prepacked 100 g silica column [solvent A:

EtOAc; solvent B: hexanes; gradient: 7% A/93% B (3 CV), 7%

A/93% B ? 60% A/40% B (10 CV), 60% A/40% B (1 CV); ﬂow rate:

40 mL/min; monitored at 254 and 280 nm] to afford 2-(2-butyl-

3-methoxyphenyl)-4,4-dimethyl-4,5-dihydrooxazole as yellow liq-

uid (4.99 g, 19.1 mmol, 89%). ¹H NMR (CDCl₃, 500 MHz) d 7.23 (1H,

dd, J = 7.5 Hz, 1.2 Hz), 7.15 (1H, t, J = 8.0 Hz), 6.91 (1H, dd, J = 8.1,

1.1 Hz), 4.07 (2H, s), 3.82 (3H, s), 1.53–1.47 (2H, m), 1.38 (6H, s),

1.41–1.33 (4H, m), 0.93 (3H, t, J = 7.4 Hz). ¹³C NMR (CDCl₃,

125 MHz) d 163.0, 157.7, 132.0, 129.2, 126.1, 121.9, 112.3, 78.8,

67.7, 55.7, 32.4, 28.4, 26.5, 23.1, 14.0.

4.1.1.79.

(6).

5-(2⁰-Butyl-3⁰-methoxyphenyl)pent-4-enoic

A mixture of 3-(carboxypropyl) triphenylphosphonium

acid

bromide (4.52 g, 10.6 mmol) and potassium tert-butoxide (2.62 g,

23.2 mmol) in THF (100 mL) was stirred for 1 h at room tempera-

ture. 2-Butyl-3-methoxybenzaldehyde (2.03 g, 10.6 mmol) in THF

(20 mL) was added dropwise to the reaction mixture and stirred

for 12 h at room temperature. The reaction was quenched with

2 M HCl (15 mL), then extracted with EtOAc (3 ꢁ 50 mL). The com-

bined organic phase was washed with brine, dried with Na₂SO₄,

and concentrated under reduced pressure. The crude reaction pro-

duct was puriﬁed by ﬂash chromatography using a prepacked

100 g silica column [solvent A: EtOAc; solvent B: hexanes; gradi-

ent: 7% A/93% B (3 CV), 7% A/3% B ? 60% A/40% B (10 CV), 60%

A/40% B (1 CV); ﬂow rate: 40 mL/min; monitored at 254 and

280 nm] to afford carboxylic acid 6 (2.38 g, 9.07 mmol, 86%).

NMR characterization was performed after the next step.

4.1.1.76. 2-Butyl-3-methoxybenzoic acid.^63,64

A solution of

2-(2-butyl-3-methoxyphenyl)-4,4-dimethyl-4,5-dihydrooxazole

(4.69 g, 17.95 mmol) in 4.5 M HCl (100 mL) was reﬂuxed for 21 h.

After cooling to room temperature, the reaction mixture was

extracted with diethyl ether (3 ꢁ 30 mL), and the combined

organic phase was washed with brine, dried with Na₂SO₄, and con-

centrated under reduced pressure. The crude reaction product was

puriﬁed by ﬂash chromatography using a prepacked 100 g silica

column [solvent A: EtOAc; solvent B: hexanes; gradient: 7%

A/93% B (3 CV), 7% A/93% B ? 60% A/40% B (10 CV), 60% A/40% B

(1 CV); ﬂow rate: 40 mL/min; monitored at 254 and 280 nm] to

afford 2-butyl-3-methoxybenzoic acid as

a colorless liquid

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22

C. A. Herdman et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx

4.1.1.80.

(12).

5-(2⁰-Butyl-3⁰-methoxyphenyl)pentanoic

Carboxylic acid 6 (2.38 g, 9.07 mmol) was mixed with

acid

Na₂SO₄and concentrated under reduced pressure. The crude reac-

tion product was puriﬁed by ﬂash chromatography using a pre-

packed 50 g silica column [solvent A: EtOAc; solvent B: hexanes;

gradient: 7% A/93% B (3 CV), 7% A/93% B ? 60% A/40% B (10 CV),

60% A/40% B (1 CV); ﬂow rate: 40 mL/min; monitored at 254 and

280 nm] to afford benzosuberene 33 (0.76 g, 1.8 mmol, quantita-

tive) as a yellowish oil. ¹H NMR (CDCl₃, 500 MHz) d 6.84 (1H, d,

J = 8.5 Hz), 6.69 (1H, d, J = 8.5 Hz), 6.51 (2H, s), 6.32 (1H, t,

J = 7.4 Hz), 3.86 (3H, s), 3.83 (3H, s), 3.81 (6H, s), 2.74 (2H, m),

2.68 (2H, t, J = 6.9 Hz), 2.13 (2H, p, J = 7.0 Hz), 1.91 (2H, q,

J = 7.3 Hz), 1.53 (2H, m), 1.46 (2H, m), 0.98 (3H, t, J = 7.3 Hz). ¹³C

NMR (CDCl₃, 125 MHz) d 156.4, 152.8, 143.5, 141.2, 138.7, 137.3,

133.1, 128.5, 127.5, 126.4, 107.5, 105.3, 60.9, 56.2, 55.4, 34.9,

32.9, 27.3, 26.3, 25.5, 23.2, 14.1. HRMS: Obsd 397.2374 [M+H]⁺,

calcd for C₂₅H₃₃O₄: 397.2373. HPLC (Method B): 22.30 min.

10% Pd/C (0.15 g). Methanol (25 mL) was slowly added, and the

reaction mixture was stirred for 12 h at room temperature under

a H₂atmosphere. The suspension was ﬁltered through Celite^Ò,

and the Celite^Òwas rinsed with EtOAc. The ﬁltrate (MeOH and

EtOAc) was concentrated under reduced pressure, and the crude

reaction product was puriﬁed by ﬂash chromatography using a

prepacked 100 g silica column [solvent A: EtOAc; solvent B: hex-

anes; gradient: 20% A/80% B (3 CV), 20% A/80% B ? 100% A/0% B

(10 CV), 100% A/0% B (1 CV); ﬂow rate: 40 mL/min; monitored at

254 and 280 nm] to afford carboxylic acid 12 (1.48 g, 56.0 mmol,

61%) as a yellow-brown liquid. ¹H NMR (CDCl₃, 500 MHz) d 7.09

(1H, t, J = 7.9 Hz), 6.77 (1H, d, J = 7.6 Hz), 6.72 (1H, d, J = 8.1 Hz),

3.81 (3H, s), 2.63 (4H, m), 2.40 (2H, t, J = 7.3 Hz), 1.75 (2H, m),

1.64 (2H, m), 1.44 (4H, m), 0.96 (3H, t, J = 7.2 Hz). ¹³C NMR (CDCl₃,

125 MHz) d 179.4, 157.7, 141.1, 129.5, 126.1, 121.5, 108.1, 55.4,

34.0, 32.5, 32.3, 30.8, 25.7, 24.8, 23.2, 14.0.

4.2. Biological evaluations

4.2.1. SRB assay^65,66

4.1.1.81. 1-Butyl-2-methoxy-6,7,8,9-tetrahydro-5H-benzo[7]an-

Inhibition of human cancer cell growth was assessed using the

sulforhodamine B assay, as previously described.⁶⁵Cancer cell lines

were plated at 9000 cells/well into 96-well plates using DMEM

supplemented with 5% fetal bovine serum/1% gentamicin sulfate

and incubated for 24 h. Serial dilutions of the compounds were

then added. After 48 h, the cells were ﬁxed with trichloroacetic

acid, washed, dried, stained with sulforhodamine B dye (Acid red

52), solubilized, and read at 540 nm and normalized to 630 nm

with an automated Biotek plate reader. A growth inhibition of

50% (GI₅₀or the drug concentration causing 50% reduction in the

net protein increase) was calculated from the absorbance data.

nulen-5-one (18).

Eaton’s reagent (28 mL) was added to car-

boxylic acid 12 (1.48 g, 5.6 mmol) and sonicated until the 12

dissolved. The reaction mixture was then stirred at room temper-

ature for 12 h. Ice was poured into the reaction ﬂask, and then

the reaction mixture was neutralized with a satd NaHCO₃solution

and extracted with EtOAc (3 ꢁ 50 mL). The combined organic

phase was dried with Na₂SO₄and concentrated under reduced

pressure. The crude reaction product was puriﬁed by ﬂash chro-

matography using a prepacked 100 g silica column [solvent A:

EtOAc; solvent B: hexanes; gradient: 7% A/93% B (3 CV), 7%

A/93% B ? 60% A/40% B (10 CV), 60% A/40% B (1 CV); ﬂow rate:

40 mL/min; monitored at 254 and 280 nm] to afford ketone 18

(1.13 g, 4.59 mmol, 82%) as a yellow liquid. ¹H NMR (CDCl₃,

500 MHz) d 7.53 (1H, d, J = 8.6 Hz), 6.77 (1H, d, J = 8.6 Hz), 3.86

(3H, s), 2.89 (2H, m), 2.67 (4H, m), 1.83 (2H, p, J = 6.5 Hz), 1.75

(2H, m), 1.42 (4H, m), 0.96 (3H, t, J = 7.1 Hz). ¹³C NMR (CDCl₃,

125 MHz) d 206.8, 160.6, 139.8, 133.0, 128.9, 127.7, 108.0, 55.5,

40.5, 32.5, 26.5, 25.5, 25.5, 23.0, 20.0, 20.5, 14.0.

4.2.2. Colchicine binding assay

Inhibition of [³H]colchicine binding to tubulin was determined

using 0.1 mL reaction mixtures. Each reaction mixture contained

1.0

(v/v) dimethyl sulfoxide, potential inhibitors at 1.0, 5.0 or 50

lM tubulin, 5.0 l

M [³H]colchicine (from Perkin–Elmer), 5%

l

M

and components that were previously demonstrated to stabilize

the colchicine binding activity of tubulin⁶⁷(1.0 M monosodium

glutamate [adjusted to pH 6.6 with HCl in a 2.0 M stock solution],

0.5 mg/mL bovine serum albumin, 0.1 M glucose-1-phosphate,

1.0 mM MgCl₂, and 1.0 mM GTP). Incubation was for 10 min at

37 °C, a time point at which the binding reaction in the control is

40–60% complete. Reactions were stopped by adding 2.0 mL of

ice-cold water and placing the samples on ice. Each sample was

poured onto a stack of two DEAE-cellulose ﬁlters, followed imme-

diately by 6 mL of ice-cold water, and the water was aspirated

under reduced vacuum. The ﬁlters were washed with 2 mL

water ꢁ 3 and, following removal of excess water under a strong

vacuum, placed into vials containing 5 mL of Biosafe II scintillation

cocktail. Samples were counted the next day in a Beckman scintil-

lation counter. Samples with potential inhibitors were compared to

controls with no inhibitor to determine percent inhibition. All sam-

ples were corrected for the amount of colchicine that bound to the

ﬁlters in the absence of tubulin.

4.1.1.82.

6,7,8,9-tetrahydro-5H-benzo[7]annulen-5-ol

1-Butyl-2-methoxy-5-(3⁰,4⁰,5⁰-trimethoxyphenyl)-

(24). 5-

Bromo-1,2,3-trimethoxybenzene (1.70 g, 6.9 mmol) was dissolved

in THF (20 mL) and cooled to ꢂ78 °C. n-BuLi (4.06 mL, 2.5 M) was

added dropwise, and the reaction mixture was stirred at ꢂ78 °C.

After 1 h, ketone 18 (1.13 g, 4.6 mmol) in THF (15 mL) was added

dropwise to the reaction ﬂask. The reaction mixture was stirred

for 12 h while warming to room temperature. The reaction was

quenched with water (40 mL) and extracted with EtOAc

(3 ꢁ 30 mL). The combined organic phase was dried with Na₂SO₄

and concentrated under reduced pressure. The crude reaction pro-

duct was puriﬁed by ﬂash chromatography using a prepacked

100 g silica column [solvent A: EtOAc; solvent B: hexanes; gradi-

ent: 7% A/93% B (3 CV), 7% A/93% B ? 60% A/40% B (10 CV), 60%

A/40% B (1 CV); ﬂow rate: 40 mL/min; monitored at 254 and

280 nm] to afford tertiary alcohol 24 (0.76 g, 1.8 mmol, 40%). ¹H

NMR (CDCl₃, 500 MHz) d 7.39 (1H, d, J = 8.7 Hz), 6.72 (1H, d,

J = 8.8 Hz), 6.51 (2H, s), 3.84 (3H, s), 3.82 (3H, s), 3.74 (6H, s),

2.96 (2H, m), 2.67 (4H, m), 1.82 (4H, m), 1.39 (4H, m), 0.92 (3H,

t, J = 6.9 Hz).

4.2.3. Inhibition of tubulin polymerization

Tubulin assembly experiments were performed using 0.25 mL

reaction mixtures (ﬁnal volume).⁶⁸The mixtures contained 1 mg/

mL (10 lM) puriﬁed bovine brain tubulin, 0.8 M monosodium glu-

tamate (pH 6.6, as above), 4% (v/v) dimethyl sulfoxide, 0.4 mM GTP,

and varying compound concentrations. Initially, all components

except GTP were preincubated for 15 min at 30 °C in 0.24 mL. After

4.1.1.83. 4-Butyl-3-methoxy-9-(3⁰,4⁰,5⁰-trimethoxyphenyl)-6,7-

dihydro-5H-benzo[7]annulene (33).

The tertiary alcohol 24

(0.76 g, 1.8 mmol) was dissolved in acetic acid (10 mL), and the

reaction mixture was stirred for 6 h. The reaction was quenched

with water (50 mL) and extracted with EtOAc (3 ꢁ 20 mL). The

combined organic phase was washed with brine, dried with

chilling the mixtures on ice, 10 lL of 10 mM GTP was added. The

reaction mixtures were then transferred to cuvettes held at 0 °C

in Beckman DU-7400 and DU-7500 spectrophotometers equipped

with electronic temperature controllers. The temperature was

Please cite this article in press as: Herdman, C. A.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.10.012

C. A. Herdman et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx

23

jumped to 30 °C over about 30 s, and polymerization was followed

turbidimetrically at 350 nm for 20 min. Each reaction set included

a reaction mixture without compound, and the IC₅₀was deﬁned as

compound concentration that inhibited extent of assembly by 50%

after 20 min at 30 °C.

Small Animal Imaging Resource, and Live Cell Imaging Resource.

The IVIS Spectrum was purchased with support of 1S10RR024757.

Supplementary data

Supplementary data (¹H NMR, ¹³C NMR, ¹⁹F NMR, ³¹P NMR,

HPLC, HRMS for ﬁnal target compounds and intermediates (¹H

NMR, ¹³C NMR, ¹⁹F NMR only), X-ray crystallography for com-

pound 72, an alternative synthetic procedure for compound 30,

and molecular docking for compounds 29, 62, and 72) associated

with this article can be found, in the online version, at http://

dx.doi.org/10.1016/j.bmc.2015.10.012.

4.2.4. In vivo tumor model

Human breast cancer cells, MDA-MB-231 (ATCC), were trans-

fected with a lentivirus containing a ﬁreﬂy luciferase reporter.

Highly expressing stable clones were isolated to create the cell line,

MDA-MB-231-luc, which was kindly provided by Dr. Edward

Graves, Stanford University.⁵¹Induction of tumors was carried

out by injecting 10⁶cells mixed with 30% Matrigel^TM(BD Bio-

sciences, San Jose, CA) into the mammary fat pads of female SCID

mice (UTSW breeding colony). Tumors were allowed to grow to a

size of approximately 5 mm in diameter, determined by calipers,

before selection for BLI or histological analysis. All animal proce-

dures were approved by the University of Texas Southwestern

Medical Center Institutional Animal Care and Use Committee.

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Acknowledgements

The authors are grateful to 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.),

and OXiGENE, Inc. (Grant to K.G.P. and M.L.T.) for their ﬁnancial

support of this project, and to the NSF for funding the Varian

500 MHz NMR spectrometer (Grant No. CHE-0420802). The con-

tent is solely the responsibility of the authors and does not neces-

sarily reﬂect the ofﬁcial views of the National Institutes of Health.

The authors would also thank Dr. James Karban and Dr. Michelle

Nemec (Director) for the use of the shared Molecular Biosciences

Center at Baylor University, Dr. Alejandro Ramirez (Mass Spec-

trometry Core Facility, Baylor University) and Dr. Kevin Klaus-

meyer and Marissa Penney (X-ray analysis). The authors are

grateful to Mr. Tyler Goddard (Baylor University) for his contribu-

tions to the synthesis of certain analogues, and to Jeni Gerberich

(UTSW) and Dr. Li Li (UTSW) for valuable technical assistance.

Imaging was facilitated with the assistance of Resources of the

Harold C. Simmons Cancer Center supported through an National

Institutes of Health National Cancer Institute Cancer Center Sup-

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