阅读说明：本技术 具有作为抗癌剂活性的苯并环庚烯类似物和相关化合物 (Benzocycloheptene analogs and related compounds having activity as anticancer agents ) 是由 K·G·平尼 H·牛 D·蒙达尔 于 2019-08-16 设计创作，主要内容包括：一系列苯并环庚烯类似物表现出了有效的微管蛋白聚合抑制、对人癌细胞的细胞毒性以及在肿瘤中的血管破坏作用。(A series of benzocycloheptene analogs exhibit potent tubulin polymerization inhibition, cytotoxicity against human cancer cells, and vascular damaging effects in tumors.)

1. A compound of the formula:

wherein R is CH₃、(CH₂)₃CH₃、O(CH₂)₂O(CH₂)₂OCH₃、O(CH₂)₂OH、COOEt、CH₂OH, CN or CHO, in the presence of oxygen,

n is a number of 0 or 1,

R₁is CH₃、OH、OCH₃Or an OH group, or a mixture of OH,

R₂is Br or H, and

R₃h, OH or NHAc.

2. The compound of claim 1, wherein R is CH₃、(CH₂)₃CH₃、O(CH₂)₂O(CH₂)₂OCH₃、O(CH₂)₂OH、COOEt、CH₂OH, CN or CHO, n is 1.

3. The compound of claim 1, wherein R₁Is CH₃，R₂Is Br, and R₃Is H.

4. The compound of claim 1, wherein R₁Is OH, R₂Is Br, and R₃Is H.

5. The compound of claim 1, wherein R₁Is OCH₃，R₂Is H, and R₃Is OH.

6. A pharmaceutical formulation comprising a therapeutically effective amount of a compound of claim 1.

7. A method for inhibiting tubulin polymerization and disrupting angiogenesis in a tumor in a patient comprising administering the pharmaceutical formulation of claim 6.

8. A compound of the formula:

wherein R is₄Is H, OH or (═ O), and

R₅is PO (ONa)₂。

9. The compound of claim 8, wherein R₄Is (═ O).

10. A pharmaceutical formulation comprising a therapeutically effective amount of a compound of claim 8.

11. A method for inhibiting tubulin polymerization and disrupting angiogenesis in a tumor in a patient comprising administering the pharmaceutical formulation of claim 10.

12. A compound of the formula:

13. a pharmaceutical formulation comprising a therapeutically effective amount of a compound of claim 12.

14. A method for inhibiting tubulin polymerization and disrupting angiogenesis in a tumor in a patient comprising administering the pharmaceutical formulation of claim 13.

Background

Priority of U.S. provisional patent application serial No. 62/719,362 entitled "benzocycloheptene (benzocycloheptene) analogs and related compounds having activity as anticancer agents" filed on 8/17 of 2018, the entire contents of which are incorporated herein by reference.

The present disclosure relates to potent small molecule inhibitors of tubulin polymerization and uses thereof.

Cancer, medically known as malignant tumors, is composed of a variety of diseases involving uncontrolled cell growth. In cancer, cells divide and grow uncontrollably, forming malignant tumors that can invade the body locally to the extremities. In 2007, cancer accounts for about 13% of all human deaths worldwide (790 ten thousand). The incidence is rising with the life extension of more people and the change in lifestyle in developing countries. There is an urgent need to discover and develop new anticancer drugs.

Solid tumors are over 1mm in size³Functional vasculature is required to provide oxygen and nutrients to its cells. Unlike normal vasculature, the tumor-associated vascular network tends to expand irregularly, incorporating fragile, chaotic bulges and blind ends. The primitive character and inherent fragility of tumor-associated vasculature, as well as the important observation that blocking established tumor-associated blood flow leads to tumor regression in mice, makes tumor-associated vasculature a promising therapeutic target for cancer. Two classes of small molecules, vascular-targeted therapies, have been developed: blood vessels that inhibit the formation of new blood vessels in developing tumorsProduction of inhibitors (AIAs); and Vascular Disrupting Agents (VDAs) that irreversibly damage established tumor-associated vasculature, respectively. Two major sub-areas of VDA include biologics and small molecule anticancer drugs. Most small molecule VDAs act as tubulin polymerization inhibitors, destabilizing the tubulin-tubulin system by binding to colchicine sites on β -tubulin near the α, β -tubulin heterodimer interface. In response to inhibition of the microvascular endothelial cell tubulin-tubulin system cytoskeleton triggered by VDA binding to the colchicine site, the microvascular endothelial cells rapidly undergo cytoskeletal destruction, manifested as morphological changes (flat to round). This rapid endothelial cytoskeletal rearrangement causes irreversible damage to tumor-associated vasculature, ultimately leading to tumor necrosis.

Summary of The Invention

The present disclosure relates to a series of benzocycloheptene analogs that act as tubulin polymerization small molecule inhibitors, which act as both antiproliferative agents (cytotoxins) and Vascular Disrupting Agents (VDAs), causing selective and irreversible damage to the tumor-associated vasculature, thereby depriving the tumor of the blood, nutrients, and oxygen necessary for survival.

The natural products colchicine, combretastatin A-4(CA4) and combretastatin A-1(CA1) and the synthetic analogue non-statin (phenstatin) are effective colchicine site inhibitors of tubulin polymerization and can play a promising role in VDA. These molecules provide structural inspiration and guidance for the design, synthesis, and biological evaluation of many second (and later) generation molecules when efforts are made worldwide to identify small molecule colchicine site agents that are useful as cancer therapeutics, with the necessary efficacy and safety in humans. To date, no small molecule therapeutic that interacts with colchicine sites and acts as an antiproliferative or VDA (or exhibits a dual mechanism of action) has received FDA approval. Structural similarities between these natural products include trimethoxyphenyl rings, hydroxylated para-methoxyaryl moieties alone, and bridging functionalities connecting the two rings at comparable centroid-to-centroid distances. Figure 1A shows representative small molecule inhibitors of tubulin polymerization: colchicine, combretastatin (CA4, CA1), non-statins (phenstatin), dihydronaphthalene analogs (KGP03, KGP05), benzocycloheptene analogs (KGP18, KGP156), indole analogs (OXi8006), and benzo [ b ] furan analogs (BNC 105). Molecular recognition of the colchicine site has led to the discovery of promising synthetic analogs and derivatives, including stilbenes (stilbenoids), benzo [ b ] thiophenes, benzofurans, dihydronaphthalenes, benzocycloheptenes, and indole-based molecules, as shown in figure 1A. Numerous other studies have investigated various structural and functional modifications to the a-ring, B-ring and ethylene bridge of combretastatin a-4.

Two benzocycloheptene-based analogs (known as KGP18 and its amino homolog KGP156) were discovered from studies as molecules with high relevance to potential preclinical candidates, due in part to their potent inhibitory effects on tubulin polymerization, significant cytotoxicity on human cancer cells, and promising activity as VDA. Studies have explored various functional group modifications on the fused and pendant aryl rings of tubulin binding to benzocycloheptene and dihydronaphthalene molecular frameworks. FIG. 1B shows selected KGP18 derivatives as inhibitors of tubulin polymerization, with modifications at the C-4 position of the A-ring and the C-6, 7,8 positions of the B-ring. Interestingly, the benzocycloheptene B-cyclodiene analog was identified as one of the most potent cytotoxic agents in a series of 11 members synthesized, and this same molecule (compound 88 herein) was obtained herein as an unexpected product. Notably, a new class of benzodiazepines has been reported as tubulin polymerization inhibitors.

Brief description of the drawings

Figure 1A shows representative small molecule inhibitors of tubulin polymerization.

Fig. 1B shows selected KGP18 derivatives as tubulin polymerization inhibitors.

Fig. 2 shows exemplary benzocycloheptene and dihydronaphthalene analogs according to preferred embodiments described herein.

Figure 3 shows synthesis scheme 1 for the synthesis of representative compounds described herein.

Figure 4 shows synthesis scheme 2 for the synthesis of representative compounds described herein.

FIG. 5 shows synthesis scheme 3 for the synthesis of representative compounds described herein.

FIG. 6 shows synthesis scheme 4 for the synthesis of representative compounds described herein.

FIG. 7 shows synthesis scheme 5 for the synthesis of representative compounds described herein.

FIG. 8 shows synthesis scheme 6 for the synthesis of representative compounds described herein.

Figure 9 shows synthesis scheme 7 for the synthesis of representative compounds described herein.

FIG. 10 shows synthesis scheme 8 for the synthesis of representative compounds described herein.

FIG. 11 shows synthesis scheme 9 for the synthesis of representative compounds described herein.

Fig. 12 shows the results of BLI assessment of vascular response to exemplary Vascular Disrupting Agents (VDAs) in rats.

Figure 13 shows the relative luminescence following VDA administration in rats.

Fig. 14 shows a mechanism of forming a beta-lactam by chlorosulfonyl isocyanate (CSI).

Detailed Description

The present disclosure relates to benzocycloheptene analogs as tubulin polymerization inhibitors. In particular, the present disclosure relates to a series of structurally varied analogs using KGP18 and KGP05 as lead compounds, and further employs our approach to study the effect of functional group modifications on the a-ring (C-4 position) and B-ring (C-6, 7,8,9 positions) and chemical translocation of the pendant aryl ring (C-ring) region on inhibition of tubulin polymerization and cytotoxicity against several human cancer cell lines.

Various functional group modifications have been explored and it has been determined that the C-1 position of these substituted benzocycloheptene analogs is of particular importance for maintaining effective inhibition of tubulin assembly, as exemplified by molecules incorporating hydroxyl, methoxy, and halogen moieties. Due to the strong activity implications of these benzocycloheptene analogs, methods have been described to achieve efficient ring-closing metathesis (RCM) of the benzocycloheptene backbone, and other benzocycloheptene analogs having substituents at different positions have been developed. However, the incorporation of carbon chain homologues and other functional groups at the C-1 position and benzocycloheptene analogues with modified functional groups on the seven-membered ring have not been previously investigated.

The novel benzocycloheptene and dihydronaphthalene analogs depicted in FIG. 2 have been synthesized and investigated for their cytotoxicity and ability to inhibit tubulin polymerization against selected human cancer cell lines.

Preferred embodiments of the benzocycloheptene and dihydronaphthalene analogs described herein include compounds having the following structures:

wherein R is CH₃、(CH₂)₃CH₃、O(CH₂)₂O(CH₂)₂OCH₃、O(CH₂)₂OH、COOEt、CH₂OH, CN or CHO, and wherein n is 0 or 1. In other preferred embodiments, when R is CN, n is 0. In other preferred embodiments, when R is CH₃、(CH₂)₃CH₃、O(CH₂)₂O(CH₂)₂OCH₃、O(CH₂)₂OH、COOEt、CH₂And n is 1 when OH, CN or CHO exists.

Other preferred embodiments of the benzocycloheptene and dihydronaphthalene analogs described herein include compounds having the following structures:

wherein R is₁Is CH₃、OH、OCH₃Or OH, R₂Is Br or H, and R₃H, OH or NHAc. In other preferred embodiments, R₁Is CH₃，R₂Is Br, and R₃Is H. In other preferred embodiments, R₁Is OH, R₂Is Br, and R₃Is H. In other preferred embodiments, R₁Is OCH₃，R₂Is H, and R₃Is OH. In other preferred embodiments, R₁Is OH, R₂Is H, and R₃Is NHAc.

Other preferred embodiments of the benzocycloheptene and dihydronaphthalene analogs described herein include compounds having the following structures:

wherein R is₄H, OH or (═ O).

Other preferred embodiments of the benzocycloheptene and dihydronaphthalene analogs described herein include compounds having the following structures:

wherein R is₅Is PO (ONa)₂。

Other preferred embodiments of the benzocycloheptene and dihydronaphthalene analogs described herein include compounds having one of the following structures:

additional preferred embodiments relate to methods of inhibiting tubulin polymerization, methods of disrupting angiogenesis, and methods of treating cancer comprising administering to an individual having cancer or a tumor a benzocycloheptene analog of the preferred embodiments described herein.

Another aspect of the invention provides a pharmaceutical composition comprising a therapeutically effective amount of a benzocycloheptene analog as defined above and a pharmaceutically acceptable excipient, adjuvant, carrier, buffer or stabilizer. By "therapeutically effective amount" is understood an amount of an exemplary prebiotic (prebiotic) sufficient to exhibit an inhibitory effect on tubulin polymerization, angiogenesis and/or proliferation of tumor or cancer cells. The actual amount, rate and time course of administration will depend on the nature and severity of the condition being treated. Treatment prescriptions are the responsibility of general practitioners and other physicians. Pharmaceutically acceptable excipients, adjuvants, carriers, buffers or stabilizers should be non-toxic and should not interfere with the efficacy of the active ingredient. The exact nature of the carrier or other material will depend on the route of administration, which may be oral or by injection, for example cutaneous, subcutaneous or intravenous injection, or by dry powder inhaler.

Pharmaceutical compositions for oral administration may be in the form of tablets, capsules, powders or liquids. Tablets may contain solid carriers or adjuvants. Liquid pharmaceutical compositions typically comprise a liquid carrier, such as water, petroleum, animal or vegetable oil, mineral oil or synthetic oil. Physiological saline solution, glucose or other sugar solution or glycol such as ethylene glycol, propylene glycol or polyethylene glycol may be included. Capsules may contain a solid carrier, for example, gelatin. For intravenous, cutaneous or subcutaneous injection, the active ingredient should be in the form of a parenterally acceptable aqueous solution free of pyrogens and having suitable pH, isotonicity and stability. Those skilled in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as sodium chloride solution, Ringer's solution, or lactated Ringer's solution. Preservatives, stabilizers, buffers, antioxidants and/or other additives may be included as desired.

In another aspect, there is provided the use of a therapeutically effective amount of a benzocycloheptene analog, as defined above, in the manufacture of a medicament for administration to a subject.

The term "pharmaceutically acceptable salt" as used throughout the specification should be understood to mean any acid or base derived salt formed from hydrochloric acid, sulphuric acid, phosphoric acid, acetic acid, citric acid, oxalic acid, malonic acid, salicylic acid, malic acid, fumaric acid, succinic acid, ascorbic acid, maleic acid, methanesulphonic acid, isoethanedisulphonic acid (isoethonic acid) and the like, as well as potassium carbonate, sodium or potassium hydroxide, ammonia, triethylamine, triethanolamine and the like.

The term "prodrug" refers to a pharmacological agent that is administered in an inactive or significantly inactive form. Upon administration, the prodrug is metabolized in vivo to an active metabolite.

The term "therapeutically effective amount" refers to an amount of a drug that is non-toxic but sufficient to provide the desired therapeutic effect. The amount "effective" will vary from individual to individual, depending on the age and general condition of the individual, the particular concentration and composition administered, and the like. Thus, it is not always possible to specify an exact effective amount. However, one of ordinary skill in the art can use routine experimentation to determine an appropriate effective amount in any individual case. Further, an effective amount is a concentration within a range sufficient to allow immediate application of the formulation for delivery of an amount of drug within a therapeutically effective range.

Certain preferred embodiments of the benzocycloheptene analogs described herein relate to chlorosulfonyl isocyanate-induced cyclic ketone formation. Chlorosulfonyl isocyanate (CSI), which was first discovered by Graf and colleagues in germany in the early fifties of the twentieth century, was liquid at room temperature, strongly reacts with water in humid air, and is incompatible with protic solvents. CSI is probably the most chemically reactive isocyanate. CSI is a universal reagent, in part because the molecule has two electrophilic sites for nucleophile attack, namely the carbonyl carbon and the sulfur of the sulfonyl group, and a cycloaddition reaction can occur at the isocyanate moiety. The first method of synthesizing β -lactams from olefins using CSI was developed by Graf and colleagues in the sixties of the twentieth century.

One of the most common types of CSI reactivity is the addition involving an initial attack on the isocyanate carbon. CSI may be attacked by nucleophiles such as alcohols (thiols/phenols) and amines, providing N-chlorosulfonylcarbamate and urea derivatives. In some cases, primary alcohols can be selectively derivatized by CSI without affecting other stereocenters and other groups in complex molecules. Figure 14 shows the mechanism of β -lactam formation by CSI. For monosubstituted olefins, the process produces primarily the desired β -lactam product. However, for tri-substituted olefins, different types of reactions may occur, including substitution or cyclization of the olefin hydrogen. For these reactions, both uniform and non-uniform 1, 4-dipolar mechanisms have been proposed. Various olefins that produce beta-lactams via [2+2] cycloaddition have been thoroughly investigated. However, there is no report of the formation of cyclic ketones involving adjacent aryl rings.

Example 1 Synthesis

Tetrahydrofuran (THF), carbon tetrachloride, dichloromethane, methanol, Dimethylformamide (DMF) and acetonitrile were used in anhydrous form. The reaction was carried out under nitrogen. Thin Layer Chromatography (TLC) plates (glass plates precoated with silica gel 60F254, 0.25mm thick) were used to monitor the reaction. Purification of intermediates and products was performed using a Biotage Isolera rapid purification system using silica gel (200-400 mesh,) Or RP-18 pre-packed column or manually in a glass column. According to intermediates and products, using a Varian VNMRS500MHz or Bruker DPX 600MHz instrument¹H NMR (500 or 600MHz),¹³It was characterized by C NMR (125 or 150MHz) spectral data. Recorded in CDCl₃、D₂O、(CD₃)₂CO or CD₃Spectrum in OD. All chemical shifts are expressed in ppm (δ) and the peak patterns are reported as broad (br), singlet(s), doublet (d), triplet (t), quartet (q), quintet (p), sext (sext), sept (sept), doublet (dd), doublet-doublet (ddd) and multiplet (m).

Using a column (H.sub.190-400 nm) HPLC with a diode array detector (Zorbax XDB-C18: (H.sub.H.)) 5 μm) and a Zorbax reliable cassette guard column (reliability cartridge guard-column) were further analyzed for purity of the final compound at 25 ℃; the method comprises the following steps: solvent A, acetonitrile, solventAgents B, H₂O; gradient from 10% A/90% B to 100% A/0% B within 0-40 min; equilibration time (post-time)10min (minutes); the flow rate is 1.0 mL/min; the sample volume is 20 mu L; monitoring was at wavelengths of 210, 230, 254, 280 and 320 nm. With the exception of compound 27 (94.3% at 254 nm), the target molecule (with reported biological data) was 95% pure (as determined by HPLC at one or more scanning wavelengths). Mass spectrometry was performed using a Thermo Scientific LTQ Orbitrap Discovery instrument at positive or negative ESI (electrospray ionization).

Figure 3 shows scheme 1, synthesis of compound 9: 1-methyl-2-methoxy-5- (3 ', 4 ', 5 ' -trimethoxyphenyl) -benzocyclohept-5-ene. The tertiary alcohol (2.38g,6.4mmol) was dissolved in acetic acid (15mL) and stirred for 6 h. The reaction was quenched with water (100mL), then extracted with EtOAc, washed with brine, and Na₂SO₄And (5) drying. The organic layer was concentrated and purified by flash chromatography using a pre-packed 100g silica gel column [ solvent a: EtOAc; solvent B: hexane; gradient: 7% A/93% B (3CV), 7% A/93% B → 60% A/40% B (10CV), 60% A/40% B (1 CV); flow rate: 25 mL/min; monitoring at 254nm and 280nm]The title molecule was obtained as a white powder (1.78g,5.0mmol, 78%).¹H NMR(CDCl₃,500MHz)δ6.86(1H,d,J＝10.0Hz),6.70(1H,d,J＝10.0Hz),6.52(2H,s),6.32(1H,t,J＝7.5Hz),2.68(2H,t,J＝6.5Hz),2.29(3H,s),2.12(2H,p,J＝7.0Hz),1.91(2H,q,J＝7.5Hz)。¹³C NMR(CDCl₃125MHz) delta 156.5,152.8,143.5,141.7,138.6,137.3,133.0,127.4,126.5,123.2,107.4,105.3,60.9,56.1,55.5,34.0,27.7,25.5, 11.8. HRMS, found 355.1906[ M + H]⁺Theoretical value C₂₂H₂₇O₅:355.1904。HPLC:19.87min。

Figure 4 shows scheme 2, synthesis of compound 20: 4-butyl-3-methoxy-9- (30,40, 50-trimethoxyphenyl) -6, 7-dihydro-5H-benzo [7 ]]And (4) performing cyclonene. The tertiary alcohol (0.76g,1.8 mmol) was dissolved in acetic acid (10mL) and the reaction mixture was stirred for 6 h. The reaction was quenched with water (50mL) and extracted with EtOAc (3X 20 mL). The combined organic phases were washed with brine, washed with Na₂SO₄Dried and concentrated under reduced pressure. The crude reaction product was purified by flash chromatography using a pre-packed 50g silica gel column[ solvent A: EtOAc; solvent B: hexane; gradient: 7% A/93% B (3CV), 7% A/93% B60% A/40% B (10CV), 60% A/40% B (1 CV); flow rate: 40 mL/min; monitoring at 254nm and 280nm]The expected molecule was obtained as a pale yellow oil (0.76g,1.8mmol, quant.). 1H NMR (cdcl3,500mhz) d 6.84(1H, d, J ═ 8.5Hz),6.69(1H, d, J ═ 8.5Hz),6.51(2H, s),6.32(1H, t, J ═ 7.4Hz),3.86(3H, s),3.83(3H, s),3.81(6H, s),2.74(2H, m),2.68(2H, t, J ═ 6.9Hz),2.13(2H, p, J ═ 7.0Hz),1.91(2H, q, J ═ 7.3Hz),1.53(2H, m),1.46(2H, m),0.98(3H, t, J ═ 7.3 Hz).¹³C NMR(CDCl3,125MHz)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:22.30min。

Figure 5 shows scheme 3, synthesis of compounds 23, 24, 28, 31, 33, 34 and 35. Structural modifications include: 1) functional group (R) modifications on the fused aryl ring, including the installation of alcohol, aldehyde, nitrile, ester groups, and ether linkages, to facilitate extension of the polar alcohol moiety from the fused six-seven ring system; 2) incorporation of R at the olefinic and allylic positions of seven-membered rings incorporating-Br, -OH and-NHAc groups₂And R₃(ii) a 3) Modification of the fused aliphatic Ring Adjacent to the Tertiary alcohol site (R)₄Location); 4) regiochemistry of an alkenylated and trimethoxyphenyl ring pendant on a fused non-aromatic ring. The synthesis of analogs 23, 24, 28, 31, 33, 35 began with the common intermediate ketone 21, which intermediate ketone 21 was readily available using previous methodologies. Treatment of benzocycloheptane 21 with trimethoxyphenyllithium (prepared from the corresponding bromide) yields tertiary alcohol 22, which is subsequently converted to diol 23 after removal of the phenolic TBS protecting group. Separately, tertiary alcohol 22 is converted to its corresponding benzocycloheptene 26, which is treated with a series of oxidizing agents (m-CPBA, NBS, OsO)₄) Treatment to promote epoxidation followed by ring opening and oxidation, bromination and Upjohn dihydroxylation. After removal of the protecting groups, the target compounds 24, 33 and 35 were obtained. Similarly, the reaction of 4-methylbenzcycloheptene 32(9) (previously prepared) with NBS/AIBN affords vinyl bromide 33. The lead compound 27 (named KGP18) which can also be obtained by the method is directly transformedTo their corresponding ether analogs 28 and 31 (scheme 3).

1- ((tert-butyldimethylsilyl) oxy) -2-methoxy-5- (3,4, 5-trimethoxyphenyl) -6,7,8, 9-tetrahydro-5H-benzo [7 ]]Annulene (annulen) -5-ol (22). To an oven dried flask, THF (10mL) and 3,4, 5-trimethoxyphenyl bromide (0.89g, 3.6mmol) were added and the solution was cooled to-78 ℃. N-butyllithium (1.44mL, 3.60mmol) was added dropwise to the reaction mixture, which was then stirred at-78 deg.C for 1 h. TBS protected ketone (21) (0.77g, 2.4mmol) in THF (5mL) was then added slowly to the flask and the reaction mixture was stirred while warming from-78 deg.C to room temperature over 12 h. The reaction mixture was quenched with water and extracted with EtOAc (3X 30 mL). The combined organic phases were dried over sodium sulfate and evaporated under reduced pressure. The crude reaction product was purified by flash chromatography using a pre-packed 50g silica gel column [ solvent a: EtOAc; solvent B: hexane; gradient: 2% A/98% B (1CV), 2% A/98% B → 20% A/80% B (10CV), 20% A/80% B (2 CV); flow rate: 50 mL/min; monitoring at 254nm and 280nm]Tertiary alcohol 22(1.05g,2.15mmol, 89%) was obtained as a clear oil.¹H NMR(500MHz,CDCl₃)δ7.15(1H,d,J＝9Hz),6.69(1H,d,J＝9Hz),6.50(2H,s),3.84(3H,s),3.80(3H,s),3.75(6H,s),3.29(1H,m),2.56(1H,m),2.26(1H,m),2.12(2H,m),1.90(1H,m),1.75(2H,m),0.99(9H,s),0.17(3H,s),0.15(3H,s)。¹³C NMR(125MHz,CDCl₃)δ153.1,149.4,142.0,141.9,138.7,137.3,132.9,119.8,108.0,104.4,80.2,61.0,56.2,54.8,41.4,27.1,26.4,26.2,25.5,19.1,-3.8,-4.0。

2-methoxy-5- (3,4, 5-trimethoxyphenyl) -6,7,8, 9-tetrahydro-5H-benzo [7 ]]Rotalene-1, 5-diol (23). TBS protected tertiary alcohol 22(0.41g, 0.84mmol) was dissolved in THF (6mL) and TBAF (1.01mL, 1M in THF, 1.01mmol) was added and the reaction mixture was stirred at room temperature for 4 h. The solution was washed with water and extracted with EtOAc (3X 20 mL). The combined organic phases were dried over sodium sulfate and evaporated under reduced pressure. The crude reaction product was purified by flash chromatography using a pre-packed 10g silica gel column [ solvent a: EtOAc; solvent B: hexane; gradient: 3% A/97% B (1CV), 3% A/97% B → 30% A/70% B (10CV), 30% A/70% B (2 CV); flow rate: 12 mL/min; at 254nm and 280nmMonitoring]Phenol (0.11g,0.29mmol, 35%) was obtained as a colorless oil.¹H NMR(500MHz,CDCl₃)δ7.04(1H,d,J＝9Hz),6.70(1H,d,J＝9Hz),6.52(2H,s),5.79(1H,s),3.91(3H,s),3.84(3H,s),3.76(6H,s),3.23(1H,m),2.56(1H,m),2.35(1H,m),2.11(1H,m),1.92(1H,m),1,75(2H,m),1.47(1H,m)。¹³C NMR(125MHz,CDCl₃) δ 153.1,145.6,142.7,141.9,139.4,137.3,127.2,118.2,107.3,104.4,80.2,61.0,56.3,56.0,41.5,26.8,26.3, 24.7. HRMS, found 397.1623[ M + Na⁺]Theoretical value C₂₁H₂₆O₆Na:397.1622。HPLC:16.33min。

Tert-butyl ((3-methoxy-9- (3,4, 5-trimethoxyphenyl) -6, 7-dihydro-5H-benzo [7 ]]Rotalen-4-yl) oxy) dimethylsilane (26). TBS protected tertiary alcohol 22(0.64g, 1.3mmol) was dissolved in acetic acid (10mL) and the reaction mixture was stirred at room temperature for 6 h. The unreacted acetic acid was removed under reduced pressure. The resulting reaction mixture was washed with water and extracted with EtOAc (3X 30 mL). The combined organic extracts were washed with brine, dried over sodium sulfate, evaporated under reduced pressure and purified by flash chromatography using a pre-packed 25g silica gel column [ solvent a: EtOAc, solvent B: hexane; gradient: 5% A/95% B (1CV), 5% A/95% B → 50% A/50% B (10CV), 50% A/50% B (2 CV); flow rate: 25 mL/min; monitoring at 254nm and 280nm]This gave a clear oil which solidified to TBS-protected benzocycloheptene analog 26 as a colorless solid (0.41g,0.87mmol, 66%).¹H NMR(CDCl₃,500MHz)δ6.68(1H,d,J＝8.5Hz),6.61(1H,d,J＝8.5Hz),6.48(2H,s),6.32(1H,t,J＝7Hz),3.85(3H,s),3.81(3H,s),3.79(6H,s),2.76(2H,t,J＝7Hz),2.10(2H,m),1.95(2H,m),1.04(9H,s),0.23(6H,s)。¹³CNMR(CDCl₃,125MHz)δ152.9,148.8,143.2,141.6,138.8,137.3,133.9,133.4,127.0,122.5,108.5,105.3,61.0,56.2,54.8,34.1,26.3,25.7,24.4,19.2,-3.7。

1- (tert-butyldimethylsilyl) oxy) -5-hydroxy-2-methoxy-5- (3,4, 5-trimethoxyphenyl) -5,7,8, 9-tetrahydro-6H-benzo [7 ]]Rotalen-6-one (25). At-5 deg.C, to dissolve in CH₂Cl₂To a solution of TBS protected benzocycloheptene 26(0.51g,1.1mmol) (20mL) was added m-CPBA (0.36g,2.1mmol) and the reaction mixture was stirred for 2h, thenThen, the mixture was left at room temperature for 12 hours. The solution was saturated with Na₂S₂O₃And saturated NaHCO₃Washing with CH₂Cl₂(3X 20 mL). The combined organic layers were dried over sodium sulfate and evaporated under reduced pressure. The crude product was purified by flash chromatography using a pre-packed 50g silica gel column [ solvent a: EtOAc; solvent B: hexane; gradient: 5% A/95% B (1CV), 5% A/95% B → 60% A/40% B (10CV), 60% A/40% B (2 CV); flow rate: 100 mL/min; monitoring at 254nm and 280nm]This gave tertiary alcohol 25(0.256g,0.51mmol, 47%) as a yellow oil.¹H NMR(600MHz,CDCl₃)δ7.26(1H,d,J＝9Hz),6.76(1H,d,J＝9Hz),6.42(2H,s),5.01(1H,s),3.83(3H,s),3.81(3H,s),3.75(6H,s),3.10(2H,m),2.82(1H,m),2.70(1H,m),1.98(1H,m),1.76(1H,m)。¹³C NMR(150MHz,CDCl₃)δ211.1,153.4,150.2,142.1,138.0,137.5,131.4,131.1,127.8,109.1,105.2,85.6,61.0,56.3,54.8,39.7,26.2,25.8,24.2,19.1,-3.7,-3.9。

1, 5-dihydroxy-2-methoxy-5- (3,4, 5-trimethoxyphenyl) -5,7,8, 9-tetrahydro-6H-benzo [7 ]]Rotalen-6-one (24). TBS-protected benzocycloheptane (benzosuberane)25(0.17g,0.33mmol) was dissolved in THF (10 mL). TBAF (0.33mL,1M,0.33mmol) was added and the reaction mixture was stirred at 0 ℃ for 1h at room temperature. A solution of brine (30mL) was added and the reaction mixture was extracted with EtOAc (3X 30 mL). The combined organic phases were dried over sodium sulfate, filtered, evaporated under reduced pressure and purified by flash chromatography using a pre-packed 50g silica gel column [ solvent a: EtOAc; solvent B: hexane; gradient: 5% A/95% B (1CV), 5% A/95% B → 60% A/40% B (10CV), 60% A/40% B (2 CV); flow rate: 100 mL/min; monitoring at 254nm and 280nm]To phenol 24 as a white solid (123mg,0.320mmol, 96%).¹H NMR(600MHz,CDCl₃)δ7.17(1H,d,J＝8.4Hz),6.77(1H,d,J＝9Hz),6.43(2H,s),5.88(1H,s),5.00(1H,s),3.90(3H,s),3.82(3H,s),3.74(6H,s),3.08(2H,m),2.84(1H,m),2.68(1H,m),1.99(1H,m),1.83(1H,m)。¹³C NMR(150MHz,CDCl₃) Δ 211.0,153.3,146.4,142.8,138.0,137.2,131.6,125.7,120.2,108.4,105.2,85.5,60.9,56.3,56.0,39.4,25.4,23.3 HRMS, found 411.1414[ M + Na ]⁺]Theoretical value C₂₁H₂₄O₇Na:411.1414。HPLC:15.75min。

3-methoxy-9- (3,4, 5-trimethoxyphenyl) -6, 7-dihydro-5H-benzo [7 ]]Rotalen-4-ol (27). TBS-protected benzocycloheptene 24(0.41g,0.87mmol) was dissolved in THF (10 mL). TBAF (1.13mL,1.13mmol) was added and the reaction mixture was stirred at room temperature for 1 h. The solution was washed with water and extracted with EtOAc (3X 20 mL). The combined organic phases were dried over sodium sulfate and evaporated under reduced pressure. The crude reaction product was purified by flash chromatography using a pre-packed 10g silica gel column [ solvent a: EtOAc; solvent B: hexane; gradient: 3% A/97% B (1CV), 3% A/97% B → 30% A/70% B (10CV), 30% A/70% B (2 CV); flow rate: 12 mL/min; monitoring at 254nm and 280nm]Phenol 27(0.25g,0.70mmol, 81%) was obtained as a white solid.¹H NMR(CDCl₃,500MHz)δ6.71(1H,d,J＝9Hz),6.56(1H,d,J＝9Hz),6.50(2H,s),6.34(1H,t,J＝7.5Hz),5.74(1H,s),3.91(3H,s),3.86(3H,s),3.80(6H,s),2.76(2H,t,J＝7Hz),2.14(2H,m),1.97(2H,m)。¹³C NMR(CDCl₃,125MHz)δ152.9,145.2,142.9,142.4,138.6,134.4,127.9,127.4,121.0,110.1,107.8,105.4,61.1,56.3,56.1,33.7,25.9,23.7。

3-methoxy-4- (2- (2-methoxyethoxy) ethoxy) -9- (3,4, 5-trimethoxyphenyl) -6, 7-dihydro-5H-benzo [7 ]]A rotalene (28). Phenol 27(0.11g,0.31mmol) was dissolved in DMF (6mL) and K was added₂CO₃(0.12g,0.86 mmol). The solution was stirred at room temperature for 20 min. 1-bromo-2- (2-methoxyethoxy) ethane (0.07mL,0.5mmol) was added at 90% purity and the reaction mixture was stirred at room temperature for 15 h. The solution was washed with water and extracted with EtOAc (3X 40 mL). The combined organic phases were dried over sodium sulfate and evaporated under reduced pressure. The crude reaction product was purified by flash chromatography using a pre-packed 10g silica gel column [ solvent a: EtOAc; solvent B: hexane; gradient: 3% A/97% B (1CV), 3% A/97% B → 30% A/70% B (20CV), 30% A/70% B (2 CV); flow rate: 12 mL/min; monitoring at 254nm and 280nm]Ether 28(25mg,0.06mmol, 18%) was obtained as a colorless oil.¹H NMR(500MHz,CDCl₃)δ6.74(2H,m),6.48(2H,s),6.32(1H,t,J＝7.5Hz),4.18(2H,t,J＝5Hz),3.87(2H,t,J＝5.5Hz),3.85(6H,s),3.79(6H,s),3.76(2H,m),3.60(2H,m),3.40(3H,s),2.78(2H,t,J＝6.5Hz),2.13(2H,m),1.93(2H,m)。¹³C NMR(125MHz,CDCl₃) δ 152.9,151.5,145.1,142.9,138.5,137.4,136.1,133.8,127.3,125.3,109.3,105.3,72.8,72.2,70.78,70.77,61.0,59.2,56.2,55.7,34.5,25.7, 24.2. HRMS, found 481.2198[ M + Na⁺]Theoretical value C₂₆H₃₄O₇Na:481.2197。HPLC:21.65min。

((8-bromo-3-methoxy-9- (3,4, 5-trimethoxyphenyl) -6, 7-dihydro-5H-benzo [7 ]]Rotalen-4-yl) oxy) (tert-butyl) dimethylsilane (29). To TBS protected benzocycloheptene (102mg,0.22mmol) in CCl₄To a solution in (30mL) was added NBS (46mg,0.26mmol) and AIBN (3.6mg,0.02 mmol). The solution was heated at reflux for 2h, then water (20mL) was added and CH was used₂Cl₂(3X 30 mL). The combined organic phases were dried over sodium sulfate, filtered and the solvent was removed under reduced pressure. The crude product was obtained as a yellow oil and used directly in the next step without further purification.

1- ((tert-butyldimethylsilyl) oxy) -2-methoxy-5- (3,4, 5-trimethoxyphenyl) -6,7,8, 9-tetrahydro-5H-benzo [7 ]]Rotalene-5, 6-diol (30). OsO was added to a TBS-protected benzocycloheptene 26(1.00g,2.12mmol) solution in acetone/water (35mL/15mL) at room temperature₄(270mg,1.06mmol) and N-methylmorpholine-N-oxide (0.66mL,4.8M,3.2mmol) and the reaction mixture was stirred for 12 h. A saturated solution of sodium bisulfite (20mL) was added and the reaction mixture was extracted with EtOAc (5X 20 mL). The combined organic extracts were washed with brine, dried over sodium sulfate, filtered, evaporated under reduced pressure, and purified by flash chromatography using a pre-loaded 50g silica gel column [ solvent a: EtOAc; solvent B: hexane; gradient: 5% A/95% B (1CV), 5% A/95% B → 60% A/40% B (10CV), 60% A/40% B (2 CV); flow rate: 100 mL/min; monitoring at 254nm and 280nm]Diol 30(0.35mg,0.69mmol, 33%) was obtained as an orange oil.¹H NMR(600MHz,CDCl₃)δ7.30(1H,d,J＝8.4Hz),6.74(1H,d,J＝9Hz),6.47(2H,s),4.51(1H,s,b),3.81(3H,s),3.80(3H,s),3.72(6H,s),3.42(1H,m),3.33(1H,s),2.15(1H,m),1.96(2H,m),1.83(1H,m),1.62(1H,m),1.51(1H,m),0.98(9H,s),0.15(6H,d,J＝3.6Hz)。¹³C NMR(150MHz,CDCl₃)δ153.1,149.8,142.0,138.8,137.7,133.1,132.7,122.3,108.5,105.0,83.1,76.5,60.9,56.2,54.7,32.7,26.2,25.9,21.3,19.0,-3.9,-4.0。

2- ((3-methoxy-9- (3,4, 5-trimethoxyphenyl) -6, 7-dihydro-5H-benzo [7 ]]Rotalen-4-yl) oxy) ethan-1-ol (31). Phenol 27(0.14g,0.39mmol) was dissolved in DMF (3mL) and then ethylene carbonate (70mg,0.79mmol) and tetrabutylammonium bromide (0.13g,0.39mmol) were added simultaneously. The solution was stirred and heated at reflux for 24 h. The reaction mixture was diluted with brine, extracted with EtOAc (3 × 10mL), and the combined organic layers were dried over sodium sulfate, filtered, concentrated under reduced pressure, and purified by flash chromatography using a pre-loaded 25g silica gel column [ solvent a: EtOAc; solvent B: hexane; gradient: 7% A/93% B (1CV), 7% A/93% B → 60% A/40% B (10CV), 60% A/40% B (2 CV); flow rate: 25 mL/min; monitoring at 254nm and 280nm]Alcohol 31(0.11g,0.27mmol, 68%) was obtained.¹HNMR(500MHz,CDCl₃)δ6.76(2H,m),6.47(2H,s),6.33(1H,t,J＝7.5Hz),4.12(2H,m),3.89(2H,m),3.88(3H,s),3.84(3H,s),3.79(6H,s),2.75(2H,t,J＝7Hz),2.15(2H,m),1.95(2H,m)。¹³C NMR(125MHz,CDCl₃) δ 152.9,151.1,145.0,142.8,138.3,137.4,136.1,134.2,127.3,125.7,109.2,105.3,76.1,62.2,61.0,56.2,55.8,34.6,25.6, 24.6. HRMS, found 423.1780[ M + Na⁺]Theoretical value C₂₃H₂₈O₆Na:423.1778。HPLC:13.77min。

8-bromo-3-methoxy-9- (3,4, 5-trimethoxyphenyl) -6, 7-dihydro-5H-benzo [7 ]]Rotalen-4-ol (33). Brominated benzocycloheptene 29(0.12g,0.22mmol, crude) was dissolved in THF (20mL) and TBAF (0.22mL,1M,0.22mmol) was added to the solution at 0 ℃. The reaction mixture was stirred for 1h, washed with brine (20mL), and extracted with EtOAc (3X 30 mL). The combined organic phases were dried over sodium sulfate, filtered and evaporated under reduced pressure. The resulting material was purified by flash chromatography using a pre-packed 50g silica gel column [ solvent a: EtOAc; solvent B: hexane; gradient: 7% A/93% B (1CV), 7% A/93% B → 40% A/60% B (10CV), 40% A/60% B (2 CV); flow rate: 100 mL/min; monitoring at 254nm and 280nm]This gave brominated phenol 333 as a white crystalline solid (97 mg,0.22mmol, 100% in two steps).¹H NMR(600MHz,CDCl₃)δ6.63(1H,d,J＝8.4Hz),6.45(2H,s),6.41(1H,d,J＝8.4Hz),5.74(1H,s),3.88(3H,s),3.87(3H,s),3.80(6H,s),2.88(2H,t,J＝7.2Hz),2.58(2H,t,J＝7.2Hz),2.26(2H,m)。¹³C NMR(150MHz,CDCl₃) δ 152.7,145.5,142.5,140.8,137.9,137.3,135.3,126.4,121.5,121.1,108.0,107.5,61.0,56.3,56.1,38.5,32.5, 23.2. HRMS, found 457.0621[ M + Na⁺]Theoretical value C₂₁H₂₃BrO₅Na:457.0621。HPLC:17.54min。

8-bromo-3-methoxy-4-methyl-9- (3,4, 5-trimethoxyphenyl) -6, 7-dihydro-5H-benzo [7 ]]A rotalene (34). KGP 39132 (9) (68mg,0.19mmol) was dissolved in CCl₄(20mL) and NBS (37mg,0.21mmol) and AIBN (3.1mg,0.02mmol) were carefully added, avoiding shaking or scraping with a metal spatula, as AIBN could explode. The reaction mixture was refluxed and stirred for 2 h. The solution was washed with water and CH₂Cl₂The organic phase was washed with brine, dried over sodium sulfate, filtered, concentrated under reduced pressure, and purified by flash chromatography using a pre-packed 10g silica gel column [ solvent a: EtOAc; solvent B: hexane; gradient: 6% A/94% B (1CV), 6% A/94% B → 70% A/30% B (10CV), 70% A/30% B (2 CV); flow rate: 12 mL/min; monitoring at 254nm and 280nm]This gave brominated benzocycloheptene analog 34(66mg,0.15mmol, 80%) as a white solid.¹H NMR(600MHz,CDCl₃)δ6.70(1H,d,J＝7Hz),6.62(1H,d,J＝7Hz),6.47(2H,s),3.88(3H,s),3.81(9H,s),2.81(2H,m),2.53(2H,m),2.26(3H,s),2.24(2H,m)。¹³C NMR(150MHz,CDCl₃) δ 156.8,152.7,141.4,140.2,138.0,137.2,134.2,127.6,123.5,120.5,107.7,107.4,61.0,56.3,55.6,38.3,33.0,27.5, 11.9. HRMS, found 457.0808[ M + Na⁺]Theoretical value C₂₂H₂₅BrO₄Na:455.0828。HPLC:25.38min。

2-methoxy-5- (3,4, 5-trimethoxyphenyl) -6,7,8, 9-tetrahydro-5H-benzo [7 ]]Rotalene-1, 5, 6-triol (35). TBAF (0.76mL, 1M in THF, 0.76mmol) was added to a TBS-protected solution of phenol 30(0.35g,0.69mmol) in THF (20mL) at 0 ℃. The reaction mixture was stirred for 1h, then washed with brine (30mL) and extracted with EtOAc (3X 30 mL). Sulfur for the organic phase obtainedSodium salt was dried, filtered, evaporated under reduced pressure, and purified by flash chromatography using a pre-packed 50g silica gel column [ solvent a: EtOAc; solvent B: hexane; gradient: 12% A/88% B (1CV), 12% A/88% B → 100% A/0% B (10CV), 100% A/0% B (2 CV); flow rate: 100 mL/min; monitoring at 254nm and 280nm]This gave dihydric phenol 35(138mg,0.35mmol, 51%) as a yellow solid.¹HNMR(600MHz,CDCl₃)δ7.24(1H,d,J＝8.4Hz),6.78(1H,d,J＝8.4Hz),6.51(2H,s),5.82(1H,s),4.56(1H,m),3.93(3H,s),3.83(3H,s),3.75(6H,s),3.37(1H,m),3.21(1H,s),2.24(1H,m),2.05(1H,m),1.96(1H,m),1.69(2H,m)。¹³C NMR(150MHz,CDCl₃) δ 153.3,146.1,142.8,138.6,137.9,133.8,127.0,120.6,107.9,105.2,83.3,76.8,61.0,56.3,56.0,32.7,25.1, 21.2. HRMS, found 413.1571[ M + Na⁺]Theoretical value C₂₁H₂₆O₇Na:413.1571。HPLC:13.71min。

FIG. 6 shows the synthesis of scheme 4, compounds 38, 39 and 40. The lead compound benzocycloheptene 36 (referred to as KGP156) and its corresponding dihydronaphthalene analog 37 (referred to as KGP05), which are readily available from our previous synthetic studies, were subjected to the Sandmeyer free radical-nucleophilic aromatic substitution scheme to yield nitrile analogs 38 and 39 (scheme 4). Benzocycloheptenal analog 40 is obtained after subsequent reduction of the nitrile analog.

3-methoxy-9- (3,4, 5-trimethoxyphenyl) -6, 7-dihydro-5H-benzo [7 ]]Rotacene-4-carbonitrile (38). At 0 ℃ in 2M HCl/CH₃To KGP156(0.10g,0.28mmol) in OH solution (5mL/5mL) was added NaNO₂(77.7mg,1.12mmol) and the mixture was stirred for 1 h. CuCN (50.4mg,0.56mmol) was added and the reaction mixture was heated at 60 ℃ for 2 h. Adding Na₂CO₃And NaCN (50mg each), and the reaction mixture was stirred at room temperature for 12 h. Addition of saturated FeCl₃The solution (50mL) was quenched and the reaction mixture was extracted with EtOAc (3X 30 mL). The combined organic phases were washed with brine and saturated NaHCO₃The solution was washed, then dried over sodium sulfate and evaporated under reduced pressure. The crude reaction was purified by flash chromatography using a pre-packed 50g silica gel column [ solvent a: EtOAc; solvent B: hexane; gradient: 5% A/95% B (1CV), 5% A/95% B → 60% A/40%B (10CV), 60% A/40% B (2 CV); flow rate: 100 mL/min; monitoring at 254nm and 280nm]Nitrile 38(41mg,0.11mmol, 40%) was obtained as a yellow solid.¹H NMR(600MHz,CDCl₃)δ7.20(1H,d,J＝8.4Hz),6.80(1H,d,J＝8.4Hz),6.43(1H,t,J＝7.8Hz),6.42(2H,s),3.94(3H,s),3.86(3H,s),3.80(6H,s),2.90(2H,t,J＝7.2Hz),2.26(2H,m),1.94(2H,m)。¹³C NMR(150MHz,CDCl₃) δ 160.7,153.1,147.5,141.6,137.6,137.5,134.9,133.4,128.7,116.1,108.5,105.0,101.7,61.1,56.3,56.2,34.8,25.7, 25.4. HRMS, found 388.1521[ M + Na⁺]Theoretical value C₂₂H₂₃NO₄Na:388.1519。HPLC:20.75min。

2-methoxy-5- (3,4, 5-trimethoxyphenyl) -7, 8-dihydronaphthalene-1-carbonitrile (39). KGP05(48.6mg,0.14mmol) was dissolved in 2M HCl/CH₃OH (2mL/2 mL). Cooling the solution to 0 deg.C, adding NaNO₂(39.2mg,0.56mmol) and the resulting reaction mixture was stirred at 0 ℃ for 1 h. The reaction mixture was heated at 60 ℃ for 2h, then CuCN (25.5mg,0.28mmol) was added. The reaction mixture was cooled to room temperature and Na was added₂CO₃And NaCN to adjust pH to 10 and provide more nitrile ion to increase yield, followed by stirring for an additional 12 h. Adding FeCl₃The reaction was quenched and then extracted with EtOAc (3X 20 mL). The combined organic phases were washed with brine and saturated NaHCO₃The solution was washed, dried over sodium sulfate and evaporated under reduced pressure. The crude reaction product was purified by flash chromatography using a pre-packed 50g silica gel column [ solvent a: EtOAc; solvent B: hexane; gradient: 12% A/88% B (1CV), 12% A/88% B → 60% A/40% B (10CV), 60% A/40% B (2 CV); flow rate: 100 mL/min; monitoring at 254nm and 280nm]Nitrile 39(17mg,0.046mmol, 32%) was obtained as a white foam.¹H NMR(600MHz,CDCl₃)δ7.20(1H,d,J＝8.4Hz),6.69(1H,d,J＝9Hz),6.50(2H,s),6.03(1H,t,J＝4.8Hz),3.91(3H,s),3.88(3H,s),3.84(6H,s),3.06(2H,t,J＝7.8Hz),2.43(2H,m)。¹³C NMR(150MHz,CDCl₃) δ 160.3,153.3,142.9,138.5,137.6,135.8,130.8,128.8,126.1,115.6,108.3,105.8,101.6,61.1,56.3,56.2,26.8, 22.7. HRMS, found 374.1363[ M + Na⁺]Theoretical value C₂₁H₂₁NO₄Na:374.1363.HPLC:20.92min。

3-methoxy-9- (3,4, 5-trimethoxyphenyl) -6, 7-dihydro-5H-benzo [7 ]]Rotalene-4-carbaldehyde (40). DIBAL-H (0.16mL,1M,0.16mmol) was added to a solution of nitrile 38(48mg,0.13mmol) in toluene (15mL) at 0 deg.C, and the resulting solution was stirred for 12H while warming to room temperature. To the reaction mixture was added 1M HCl (100mL), which was stirred at room temperature for 30min while the solution turned yellow in color. The organic compound was extracted with EtOAc (3X 50 mL). The combined organic phases were dried over sodium sulfate and concentrated under reduced pressure. The crude product was purified by flash chromatography using a pre-packed 50g silica gel column [ solvent a: EtOAc; solvent B: hexane; gradient: 5% A/95% B (1CV), 5% A/95% B → 60% A/40% B (10CV), 60% A/40% B (2 CV); flow rate: 100 mL/min; monitoring at 254nm and 280nm]Aldehyde 40(37.8mg,0.10mmol, 78%) was obtained as a white solid.¹H NMR(600MHz,CDCl₃)δ10.68(1H,s),7.18(1H,d,J＝7.2Hz),6.82(1H,d,J＝8.4Hz),6.47(2H,s),6.43(1H,t,J＝7.8Hz),3.92(3H,s),3.86(3H,s),3.81(6H,s),2.99(2H,m),2.27(2H,m),1.91(2H,m)。¹³C NMR(150MHz,CDCl₃) δ 193.3,161.8,153.2,145.2,141.9,138.2,137.6,135.5,134.7,128.8,123.7,108.7,105.3,61.1,56.3,55.9,31.7,25.7, 22.8. HRMS, found 391.1519[ M + Na⁺]Theoretical value C₂₂H₂₄O₅Na:391.1516。HPLC:22.39min。

FIG. 7 shows the synthesis of scheme 5, compounds 47 and 48. As part of a larger project focused on the use of strongly cytotoxic benzocycloheptenes and dihydronaphthalene analogs as payloads in antibody-drug conjugates (ADCs) and as prodrugs for targeted selective release in the hypoxic region of severe tumors, it is contemplated that the 4-position heteroatom [ oxygen (phenol) or nitrogen (aniline) be substituted with a short carbon chain terminated with a primary alcohol (or amino) moiety]Thereby maintaining its hydrogen bond donor properties and serving as a possibility for future attachment of feasible sites for various linkers. Thus, methylation of the phenolic bromoaldehyde 41 (scheme 5), followed by Wittig olefination, followed by hydrogenation (at Ph)₂S-mediated maintenance of aryl bromo) to provide methyl ester 44. By Eaton's reagent (CH)₃SO₃P in H₂O₅7.7% by weight) to yield benzocycloheptone 45, followed by treatment of compound 45 with trimethoxyphenyllithium, followed by post-reaction treatment (reaction work-up) under acidic conditions to yield benzocycloheptene intermediate 46. The corresponding fluoro and chloro benzocycloheptene analogs have been previously studied. Halogen-lithium exchange followed by reaction with ethyl chloroformate gave ethyl ester 47, which was reduced (LiAlH)₄) To produce benzyl alcohol 48. Notably, although it has been demonstrated that analog 48 can be obtained directly from intermediate 46, the isolated yield under these conditions is very low (< 8%), probably due in part to the low solubility of paraformaldehyde in THF at low temperatures and its low reactivity as a polymer.

2-bromo-3-methoxybenzaldehyde (42). To a solution of 2-bromo-3-hydroxybenzaldehyde 41(2.50g,12.4mmol) in DMF (50mL) was added CH₃I (1.01mL,16.2mmol) and K₂CO₃(1.35g,13.7 mmol). The reaction mixture was stirred at room temperature for 3 h. The solvent was removed under reduced pressure and the residue was washed with water (50mL) and extracted with EtOAc (3X 50 mL). The combined organic phases were concentrated without further purification to give 2-bromo-3-methoxybenzaldehyde 42 as a brown solid (2.67g,12.4mmol, 100%).¹H NMR(600MHz,CDCl₃)δ10.44(1H,s),7.52(1H,d,J＝7.8Hz),7.38(1H,t,J＝7.8Hz),7.13(1H,d,J＝8.4Hz),3.96(3H,s)。¹³C NMR(150MHz,CDCl₃)δ192.4,156.4,134.9,128.5,121.6,117.3,117.1,56.8。

5- (2-bromo-3-methoxyphenyl) -4-pentenoic acid (43). To 3- (carboxypropyl) triphenylphosphonium bromide (5.33g,12.4mmol) dissolved in THF (250mL) was added potassium tert-butoxide (3.08g,27.3mmol) and the reaction mixture was stirred at room temperature for 1 h. 2-bromo-3-methoxybenzaldehyde 42(2.67g,16.2mmol) was added and the reaction mixture was stirred at room temperature for 12 h. THF was removed under reduced pressure and the resulting material was quenched, acidified with 2M HCl (30mL) and extracted with EtOAc (3X 50 mL). The combined organic phases were dried over sodium sulfate, filtered and evaporated under reduced pressure. The crude reaction product was purified by flash chromatography using a pre-packed 100g silica gel column [ solvent a: EtOAc; solvent B: hexane; gradient: 12% A/88% B (1CV), 12% A/88% B → 60% A/40% B (10CV), 60% A/40% B (2 CV); flow rate: 100 mL/min; monitoring at 254nm and 280nm ] gave carboxylic acid 43 as a white solid (1.74g,6.10mmol, 49%). NMR characterization was performed after the next step.

Methyl 5- (2-bromo-3-methoxyphenyl) pentanoate (44). To dissolve in CH₃To carboxylic acid 43(0.69g,2.42mmol) in OH (30mL) was added 10% palladium on carbon (0.26g), Ph₂S (40. mu.L, 0.24mmol) and two hydrogen filled balloons. After stirring for 24h, the mixture was washed withFiltering, and mixingWash with EtOAc (3X 50 mL). The combined organic phases were evaporated under reduced pressure (CH)₃OH and EtOAc). The residue was purified by flash chromatography using a pre-packed 50g silica gel column [ solvent a: EtOAc; solvent B: hexane; gradient: 7% A/93% B (1CV), 7% A/93% B → 40% A/60% B (10CV), 40% A/60% B (2 CV); flow rate: 100 mL/min; monitoring at 254nm and 280nm]Saturated ester 44(0.48g,1.6mmol, 66%) was obtained as a colorless oil.¹H NMR(600MHz,CDCl₃)δ7.54(1H,t,J＝7.8Hz),7.19(1H,d,J＝8.4Hz),7.10(1H,d,J＝7.8Hz),4.24(3H,s),4.02(3H,s),3.14(2H,m),2.72(2H,m),2.07(2H,m),2.02(2H,m)。¹³C NMR(150MHz,CDCl₃)δ174.2,156.1,143.3,127.8,122.5,113.9,109.5,56.4,51.6,36.1,34.0,29.4,24.8。

1-bromo-2-methoxy-6, 7,8, 9-tetrahydro-5H-benzo [7 ]]Rotalen-5-one (45). To ester 44(0.48g,1.6 mmol) was added Eton's reagent (8.5mL) and the mixture was stirred at room temperature for 12 h. The reaction mixture was then poured onto ice and neutralized with sodium carbonate. The aqueous layer was extracted with EtOAc (3X 40 mL). The combined organic phases were dried over sodium sulfate, evaporated under reduced pressure and purified by flash chromatography using a pre-packed 50g silica gel column [ solvent a: EtOAc; solvent B: hexane; gradient: 5% A/95% B (1CV), 5% A/95% B → 40% A/60% B (10CV), 40% A/60% B (2 CV); flow rate: 100 mL/min; monitoring at 254nm and 280nm]To give benzocycloheptanone 45(0.20g,0.74mmol, 47%))。¹H NMR(600MHz,CDCl₃)δ7.63(1H,d,J＝8.4Hz),6.82(1H,d,J＝8.4Hz),3.94(3H,s),3.17(2H,m),2.69(2H,m),1.85(2H,m),1.75(2H,m)。¹³C NMR(150MHz,CDCl₃)δ205.2,159.0,142.1,133.7,129.1,114.1,109.4,56.6,40.5,31.2,23.9,20.7。

4-bromo-3-methoxy-9- (3,4, 5-trimethoxyphenyl) -6, 7-dihydro-5H-benzo [7 ]]A rotalene (46). To an oven dried flask, THF (20mL) and 3,4, 5-trimethoxyphenyl bromide (1.12g, 4.53mmol) were added and the solution was cooled to-78 ℃. To the reaction mixture was slowly added n-butyllithium (1.81mL, 2.5M, 4.52mmol), followed by stirring at-78 ℃ for 45 min. Benzocycloheptane 45(0.61g,2.3mmol) was then added dropwise to the flask and the reaction mixture was stirred while warming from-78 ℃ to room temperature over 12 h. The reaction mixture was washed with water and extracted with EtOAc (3X 40 mL). The combined organic phases were dried over sodium sulfate and evaporated under reduced pressure. The crude reaction product was purified by flash chromatography using a pre-packed 50g silica gel column [ solvent a: EtOAc; solvent B: hexane; gradient: 7% A/93% B (1CV), 7% A/93% B → 20% A/80% B (10CV), 20% A/80% B (2 CV); flow rate: 100 mL/min; monitoring at 254nm and 280nm]This gave brominated benzocycloheptene 46(0.46g,1.1mmol, 49%) as a white solid.¹HNMR(600MHz,CDCl₃)δ6.97(1H,d,J＝8.4Hz),6.75(1H,d,J＝8.4Hz),6.48(2H,s),6.37(1H,t,J＝7.8Hz),3.92(3H,s),3.86(3H,s),3.81(6H,s),2.95(2H,t,J＝7.2Hz),2.17(2H,m),1.92(2H,m)。¹³C NMR(150MHz,CDCl₃)δ154.9,153.1,143.0,142.8,138.0,137.6,134.4,129.1,127.8,113.4,109.1,105.3,61.0,56.4,56.3,33.8,31.9,25.4。

3-methoxy-9- (3,4, 5-trimethoxyphenyl) -6, 7-dihydro-5H-benzo [7 ]]Rotalene-4-carboxylic acid ethyl ester (47). To a solution of benzocycloheptene bromide 46(0.15g,0.36mmol) in THF (20mL) at-78 deg.C was added n-butyllithium (0.34mL,1.6M,0.54mmol) dropwise. The reaction mixture was stirred at-78 deg.C for 30min, then ethyl chloroformate (61.5 μ L,0.64mmol) was added. The reaction mixture was stirred while warming from-78 ℃ to room temperature over 12 h. The reaction mixture was washed with water and extracted with EtOAc (3X 30 mL). The combined organic phases were dried over sodium sulfate and evaporated under reduced pressure.The crude product was purified by flash chromatography using a pre-packed 50g silica gel column [ solvent a: EtOAc; solvent B: hexane; gradient: 7% A/93% B (1CV), 7% A/93% B → 40% A/60% B (10CV), 40% A/60% B (2 CV); flow rate: 100 mL/min; monitoring at 254nm and 280nm]This gave benzocycloheptene ester 47(51.8mg,0.13mmol, 35%) as a colorless oil.¹H NMR(600MHz,CDCl₃)δ7.03(1H,d,J＝8.4Hz),6.76(1H,d,J＝9Hz),6.47(2H,s),6.37(1H,t,J＝7.8Hz),4.43(2H,q,J＝7.2Hz),3.85(3H,s),3.84(3H,s),3.79(6H,s),2.56(2H,t,J＝6.6Hz),2.17(2H,m),1.96(2H,m),1.41(3H,t,J＝7.2Hz)。¹³C NMR(150MHz,CDCl₃) δ 168.8,155.2,153.0,142.3,140.2,138.1,137.5,133.2,131.6,127.7,123.4,108.6,105.2,61.4,61.0,56.3,55.9,34.9,29.6,25.3, 14.4. HRMS, found 435.1778[ M + Na⁺]Theoretical value C₂₄H₂₈O₆Na:435.1778。HPLC:22.42min。

(3-methoxy-9- (3,4, 5-trimethoxyphenyl) -6, 7-dihydro-5H-benzo [7 ]]Rotalen-4-yl) methanol (48). Ester 47(0.12g, 0.28mmol) was dissolved in THF (10mL) and the solution was cooled to 0 deg.C. Mixing LiAlH₄(77 μ L, 4M in ether, 0.31mmol) was added dropwise to the solution and the reaction mixture was stirred for 1h while warming to room temperature. The reaction mixture was washed with water and extracted with EtOAc (3X 20 mL). The combined organic phases were dried over sodium sulfate and evaporated under reduced pressure. The crude reaction product was purified by flash chromatography using a pre-packed 50g silica gel column [ solvent a: EtOAc; solvent B: hexane; gradient: 12% A/88% B (1CV), 12% A/88% B → 60% A/40% B (10CV), 60% A/40% B (2 CV); flow rate: 100 mL/min; monitoring at 254nm and 280nm]To give benzyl alcohol 48 as a white solid (54.3mg,0.15mmol, 52%).¹H NMR(600MHz,CDCl₃)δ6.97(1H,d,J＝9Hz),6.75(1H,d,J＝9Hz),6.50(2H,s),6.35(1H,t,J＝7.8Hz),4.87(2H,s),3.89(3H,s),3.86(3H,s),3.81(6H,s),2.79(2H,t,J＝7.2Hz),2.16(2H,p,J＝7.2Hz),1.92(2H,m)。¹³C NMR(150MHz,CDCl₃) δ 157.1,153.1,143.3,142.4,138.5,137.5,133.8,130.2,127.1,126.0,107.9,105.4,61.1,57.7,56.3,55.7,35.3,27.6, 25.4. HRMS, found 393.1672[ M + Na⁺]Theoretical value C₂₂H₂₆O₅Na:393.1672。HPLC:19.09min。

FIG. 8 shows the synthesis of scheme 6, compounds 57 and 69. Secondary allylic alcohols 57 and their corresponding N-acetyl homologues 69 were prepared (scheme 6) to investigate the structure-activity relationship correlations associated with heteroatom incorporation on conformationally flexible fused seven-membered rings. Meldrum's acid (to 57) and the Wittig-ylide method (to 69) promote appropriate aldehyde chain extension followed by functional group conversion (including installation of the carboxylic acid moiety obtained under saponification conditions) to yield protected alcohol 54 and N-acetamide 65, respectively. Lewis acid mediated cyclization to give the benzocycloheptane molecular core is achieved by treating the requisite acid chloride with tin tetrachloride (to give ketone 55) or eaton's reagent (to give ketone 66). In each case, the side rings were mounted by reaction with 3,4, 5-trimethoxyphenyl lithium. The secondary alcohol moiety (target compound 57) is exposed (by deprotection) prior to the organolithium step, while the phenolic moiety (target compound 69) is exposed after the organolithium reaction (scheme 6). If Lewis acids are used, e.g. AlCl₃Or BCl₃Both compounds 56 and 57 were treated with a first removal (allyl alcohol) followed by demethylation.

S3- (2, 3-dimethoxyphenyl) propionic acid (50). To cinnamic acid 49(5.0g,24mmol) was added methanol (50mL) and 10% Pd/C (0.8 g). Two hydrogen balloons were fitted through a rubber septum and the reaction mixture was stirred at room temperature for 4 h. By usingThe reaction mixture was filtered and washed with EtOAc (3X 50mL)Evaporation of organic solvent (CH) under reduced pressure₃OH and EtOAc) to give carboxylic acid 50 as a white solid (5.0g,24mmol, quantitative). No further purification was required.¹H NMR(600MHz,CDCl₃)δ6.98(1H,t,J＝7.8Hz),6.78(2H,m),3.86(3H,s),3.84(3H,s),2.95(2H,t,J＝7.8Hz),2.66(2H,t,J＝7.8Hz)。¹³C NMR(150MHz,CDCl₃)δ178.9,152.8,147.2,134.1,124.1,121.8,110.9,60.7,55.8,34.8,25.4。

5- (2, 3-dimethoxyphenyl) -3-mevalonate (51). To carboxylic acid 50(5.05g, 24.0mmol) dissolved in dichloromethane (96mL) was added oxalyl chloride (4.12mL, 47.2mmol) and a catalytic amount of DMF (0.15 mL). The reaction mixture was stirred at room temperature for 1h, at which time a further catalytic amount of DMF (0.15mL) was added and the reaction solution was stirred at room temperature for 1 h. The solvent and unreacted oxalyl chloride were removed under reduced pressure to give the acid chloride as a yellow crystalline solid, which was redissolved in dichloromethane (50mL) and cooled to 0 ℃. Meldrum's acid (3.47g, 24.1mmol) and pyridine (4.33mL, 53.8mmol) were added and the reaction mixture was stirred at 0 deg.C for 30min and then at room temperature for 1 h. The mixture was diluted with dichloromethane (50mL) and washed with 2M HCl (20mL) then brine (30 mL). The organic layer was dried over sodium sulfate and concentrated in vacuo. The residue was dissolved in CH₃OH (50mL) and heated at reflux for 3 h. The solvent was removed under reduced pressure. The crude product was purified by flash chromatography using a pre-packed 100g silica gel column [ solvent a: EtOAc; solvent B: hexane; gradient: 7% A/93% B (1CV), 7% A/93% B → 60% A/40% B (10CV), 60% A/40% B (2 CV); flow rate: 40 mL/min; monitoring at 254nm and 280nm]Ester 51(3.57g,13.4mmol, 56%) was obtained as a pale yellow oil.¹H NMR(600MHz,CDCl₃)δ6.96(1H,t,J＝7.8Hz),6.77(1H,d,J＝9Hz),6.74(1H,d,J＝7.8Hz),3.84(3H,s),3.81(3H,s),3.71(3H,s),3.44(2H,s),2.89(2H,m),2.83(2H,m)。¹³C NMR(150MHz,CDCl₃)δ202.1,167.6,152.7,147.0,134.2,124.0,121.8,110.6,60.5,55.6,52.3,49.0,43.6,24.2。

Methyl 5- (2, 3-dimethoxyphenyl) -3-hydroxypentanoate (52). To ketone 51(0.50g,1.9mmol) in CH at 0 deg.C₃To a well stirred solution in OH (8mL) was added an aliquot of sodium borohydride (24mg,0.63 mmol). The reaction mixture was first stirred at 0 ℃ for 1h and then at room temperature for a further 1 h. The solvent was removed under reduced pressure. The residue was washed with water (10mL) and extracted with diethyl ether (3X 10 mL). The combined organic phases were dried over sodium sulfate and concentrated under reduced pressure. The crude product was purified by flash chromatography using a pre-packed 50g silica gel column [ solvent a: EtOAc; solvent B: hexane; gradient: 7% A/93% B (3CV), 7% A/93% B → 60% A/40% B (10CV), 60% A/40% B (1 CV); flow rate: 40 mL/min; at 254nm and 280nmMonitoring]Alcohol 52(0.40g,1.5mmol, 79%) was obtained.¹H NMR(600MHz,CDCl₃)δ6.98(1H,t,7.8Hz),6.78(2H,m),3.97(1H,m),3.85(3H,s),3.82(3H,s),3.69(3H,s),2.76(2H,t,J＝7.8Hz),2.48(2H,m),1.78(2H,m)。¹³C NMR(150MHz,CDCl₃)δ173.3,152.8,147.2,135.4,124.2,122.1,110.5,67.2,60.8,55.8,51.8,41.4,37.6,25.9。

Methyl 3- ((tert-butyldiphenylsilyl) oxy) -5- (2, 3-dimethoxyphenyl) pentanoate (53). To a solution of ethanol 52(0.38g,0.14mmol) and imidazole (0.16g, 2.3mmol) in DMF (2.6mL) at room temperature was added an aliquot of TBDPSCl (0.55mL,2.1 mmol). The reaction mixture was stirred for 14h, diluted with brine (10mL) and Et₂O (3X 10 mL). The combined organic extracts were dried over sodium sulfate, filtered, concentrated under reduced pressure, and purified by flash chromatography using a pre-packed 25g silica gel column [ solvent a: EtOAc; solvent B: hexane; gradient: 7% A/93% B (1CV), 7% A/93% B → 30% A/70% B (10CV), 30% A/70% B (2 CV); flow rate: 75 mL/min; monitoring at 254nm and 280nm]Ester 53(0.35g,0.69mmol, 49%) was obtained as a colorless oil.¹H NMR(600MHz,CDCl₃)δ7.72(3H,m),7.67(1H,m),7.38(6H,m),6.91(1H,t,J＝7.8Hz),6.73(1H,d,J＝9.6Hz),6.55(1H,d,J＝9Hz),4.29(1H,m),3.83(3H,s),3.71(3H,s),3.54(3H,s),2.58(4H,m),1.76(2H,m),1.06(9H,s)。¹³C NMR(150MHz,CDCl₃)δ172.0,152.8,147.1,136.1,136.0,135.9,135.3,134.9,134.2,134.1,129.8,129.7,127.9,127.7,127.6,123.9,121.8,110.2,70.5,60.7,55.8,51.5,41.9,38.2,27.1,26.7,25.4。

3- ((tert-butyldiphenylsilyl) oxy) -5- (2, 3-dimethoxyphenyl) pentanoic acid (54). To ester 53(0.67g,1.3mmol) in CH at 0 deg.C₃To a solution in OH/THF (2.2mL/1.1mL) was added 2.5M NaOH (1.76 mL). The reaction mixture was stirred at 0 ℃ for 1h, then at room temperature for 13h, acidified with 2M HCl (10mL), and Et₂O (3X 10 mL). The combined organic extracts were dried over sodium sulfate, filtered, concentrated under reduced pressure, and purified by flash chromatography using a pre-packed 25g silica gel column [ solvent a: EtOAc; solvent B: hexane; gradient: 7% A/93% B (1CV), 7% A/93% B → 30% A/70% B (10CV), 30% A/70% B (2 CV); flow rate: 75 mL/min; monitoring at 254nm and 280nm]Carboxylic acid 54(0.26g,0.53mmol, 40%) was obtained as a colorless oil.¹H NMR(600MHz,CDCl₃)δ7.67(4H,m),7.41(6H,m),6.89(1H,t,J＝8.4Hz),6.72(1H,d,J＝8.4Hz),6.52(1H,m),4.20(1H,m),3.83(3H,s),3.69(3H,s),2.50(4H,m),1.80(2H,m),1.06(9H,s)。¹³C NMR(150MHz,CDCl₃)δ152.6,146.94,146.93,135.9,135.8,129.9,129.8,129.77,127.7,127.6,123.81,123.80,121.6,110.2,70.2,60.5,55.6,40.8,37.6,26.9,25.3,19.3。

7- ((tert-butyldiphenylsilyl) oxy) -1, 2-dimethoxy-6, 7,8, 9-tetrahydro-5H-benzo [7 ]]Rotalen-5-one (55). To a solution of carboxylic acid 54(5.37g,10.9mmol) in dichloromethane (40mL) was added oxalyl chloride (4.5mL, 52mmol) and 3 drops of DMF as catalyst at room temperature. The resulting reaction mixture was stirred for 2 h. The solvent and unreacted oxalyl chloride were removed under reduced pressure. The residual acid chloride was dissolved in dichloromethane (50 mL). The solution was cooled to-10 ℃ at which time SnCl was added₄(3.63mL in CH₂Cl₂Medium 1M, 3.63mmol) and then stirred at-10 ℃ for 1 h. The reaction was quenched with cold water and extracted with EtOAc (3X 50 mL). The organic extracts were combined, dried over sodium sulfate, filtered, concentrated under reduced pressure, and purified by flash chromatography using a pre-packed 100g silica gel column [ solvent a: EtOAc; solvent B: hexane; gradient: 7% A/93% B (1CV), 7% A/93% B → 40% A/60% B (10CV), 40% A/60% B (2 CV); flow rate: 75 mL/min; monitoring at 254nm and 280nm]This gave cyclized ketone 55 as a colorless oil (2.80g,5.90mmol, 54%).¹H NMR(600MHz,CDCl₃)δ7.63(4H,m),7.56(1H,d,J＝8.4Hz),7.39(6H,m),6.81(1H,d,J＝9Hz),6.29(1H,m),3.90(3H,s),3.78(3H,s),3.17(1H,m),3.03(2H,m),2.88(1H,m),1.98(1H,m),1.84(1H,m),1.03(9H,s)。¹³C NMR(150MHz,CDCl₃)δ199.5,155.8,146.2,137.9,136.0,135.9,134.1,133.9,133.1,129.9,129.8,127.81,127.77,125.7,109.6,68.3,60.9,55.9,50.4,36.2,27.0,21.3,19.3。

7-hydroxy-1, 2-dimethoxy-6, 7,8, 9-tetrahydro-5H-benzo [7 ]]Rotalen-5-one (56). To a solution of ketone 55(0.73g,1.5mmol) in THF (10mL) was added TBAF (3.1mL of 1M in THF, 3.1mmol) and the reaction mixture was taken up inStirring was carried out at 0 ℃ for 30min and at room temperature for 16 h. The reaction was quenched with brine (10mL) and extracted with EtOAc (3X 20 mL). The organic extracts were combined, dried over sodium sulfate, filtered, concentrated under reduced pressure, and purified by flash chromatography using a pre-packed 50g silica gel column [ solvent a: EtOAc; solvent B: hexane; gradient: 12% A/88% B (1CV), 12% A/88% B → 20% A/80% B (10CV), 20% A/80% B (2 CV); flow rate: 75 mL/min; monitoring at 254nm and 280nm]Alcohol 56(0.16g,0.66mmol, 49%) was obtained as a yellow oil.¹H NMR(600MHz,CDCl₃)δ7.60(1H,d,J＝8.4Hz),6.84(1H,d,J＝8.4Hz),4.33(1H,m),3.91(3H,s),3.80(3H,s),3.08(3H,m),2.99(1H,m),1.89(2H,m)。¹³C NMR(150MHz,CDCl₃)δ199.3,156.0,146.3,137.7,132.6,125.8,109.8,67.3,60.9,56.0,50.3,35.8,21.4。

3, 4-dimethoxy-9- (3,4, 5-trimethoxyphenyl) -6, 7-dihydro-5H-benzo [7 ]]Rotalen-7-ol (57). To a solution of 3,4, 5-trimethoxyphenyl bromide (0.49g, 2.0mmol) in THF (20mL) at-78 deg.C was added n-butyllithium (1.85mL, 1.6M in hexanes, 2.98mmol) and the reaction mixture was stirred for 1 h. Benzocycloheptane 56(0.16g,0.66mmol) in THF (5mL) was added slowly. The reaction mixture was stirred at 0 ℃ for 20 h. 2M HCl (20mL) was added and the mixture was extracted with EtOAc (4X 20 mL). The combined organic phases were further washed with brine, dried over sodium sulfate, filtered, and concentrated under reduced pressure and purified by flash chromatography using a pre-packed 25g silica gel column [ solvent a: EtOAc; solvent B: hexane; gradient: 12% A/88% B (1CV), 12% A/88% B → 80% A/20% B (10CV), 80% A/20% B (2 CV); flow rate: 100 mL/min; monitoring at 254nm and 280nm]Alcohol 57(0.10g,0.26mmol, 39%) was obtained as a brown solid.¹H NMR(600MHz,CDCl₃)δ6.75(2H,m),6.51(2H,s),6.28(1H,d,J＝4.8Hz),4.18(1H,m),3.877(3H,s),3.875(3H,s),3.86(3H,s),3.80(6H,s),3.16(1H,m),2.53(1H,m),2.43(1H,m),2.15(1H,m)。¹³C NMR(150MHz,CDCl₃) δ 153.1,152.0,146.1,139.4,137.8,137.4,135.7,132.9,131.5,125.5,109.6,105.5,70.0,61.4,61.1,56.3,55.8,43.2, 22.4. HRMS, found 409.1621[ M + Na⁺]Theoretical value C₂₂H₂₆O₆Na:409.1622。HPLC:16.79min。

2- ((tert-butyldimethylsilyl) oxy) -3-methoxybenzaldehyde (59). To a well stirred solution of 2-hydroxy-3-methoxybenzaldehyde 58(0.50g,3.3mmol) in dichloromethane (30mL) was added TBSCl (0.74g,4.9mmol), DMAP (0.12g,0.99mmol) and Et₃N (0.69mL,4.9 mmol). The reaction mixture was stirred at room temperature for 12h, at which time brine (50mL) was added and the reaction mixture was extracted with dichloromethane (3X 40 mL). The organic extracts were combined, dried over sodium sulfate, filtered, concentrated under reduced pressure, and purified by flash chromatography using a pre-packed 50g silica gel column [ solvent a: EtOAc; solvent B: hexane; gradient: 7% A/93% B (1CV), 7% A/93% B → 40% A/60% B (10CV), 40% A/60% B (2 CV); flow rate: 75 mL/min; monitoring at 254nm and 280nm]Protected aldehyde 59(0.50g,1.86mmol, 57%) was obtained as a pale yellow oil.¹H NMR(600MHz,CDCl₃)δ10.51(1H,s),7.36(1H,d,J＝7.8Hz),7.03(1H,d,J＝7.8Hz),6.94(1H,t,J＝8.4Hz),3.81(3H,s),0.99(9H,s),0.20(6H,s)。¹³C NMR(150MHz,CDCl₃)δ190.4,150.8,149.2,127.9,121.2,119.1,117.0,55.2,26.0,19.0,4.1。

Ethyl 5- (2- ((tert-butyldimethylsilyl) oxy) -3-methoxyphenyl) -3-oxo-4-pentenoate (60). To ethyl 3-oxo-4- (triphenylphosphine) butyrate (3.22g, 8.26mmol) dissolved in THF (20mL) was added protected aldehyde 59(2.2g, 8.3mmol) and the reaction mixture was heated at reflux and stirred for 17 h. The solvent was removed under reduced pressure, and the residue was slurried and purified by flash chromatography using a pre-loaded 100g silica gel column [ solvent a: EtOAc; solvent B: hexane; gradient: 12% A/88% B (1CV), 12% A/88% B → 40% A/60% B (10CV), 40% A/60% B (2 CV); flow rate: 100 mL/min; monitoring at 254nm and 280nm ] gave ester 60 as an off-white solid (2.50g,6.59mmol, 80%). NMR characterization was performed after the next step.

Ethyl 5- (2- ((tert-butyldimethylsilyl) oxy) -3-methoxyphenyl) -3-oxopentanoate (61). To ester 60(2.50g,6.59mmol) dissolved in methanol (60mL) was added 10% palladium on carbon (0.54g) and hydrogen was introduced with a balloon. The reaction mixture was stirred at room temperature for 12h withFiltered and washed with EtOAc (3X 40mL)The combined organic phases were evaporated under reduced pressure (CH)₃OH and EtOAc). The resulting organic material was purified by flash chromatography using a pre-packed 100g silica gel column [ solvent a: EtOAc; solvent B: hexane; gradient: 7% A/93% B (1CV), 7% A/93% B → 40% A/60% B (10CV), 40% A/60% B (2 CV); flow rate: 100 mL/min; monitoring at 254nm and 280nm]Saturated ester 61(1.15g,3.02mmol, 46%) was obtained as a pale yellow oil.¹H NMR(600MHz,CDCl₃)δ6.81(1H,m),6.71(1H,d,J＝7.8Hz),4.16(2H,q,J＝7.2Hz),3.76(3H,s),3.39(2H,s),2.90(2H,m),2.83(2H,m),1.25(3H,t,J＝7.2Hz),0.98(9H,s),0.18(6H,s)。¹³C NMR(150MHz,CDCl₃)δ202.3,167.2,150.0,142.8,131.8,121.9,121.0,109.7,61.4,54.8,49.4,43.2,26.2,24.8,18.9,14.2,-3.7。

(Z) -3-amino-5- (2- ((tert-butyldimethylsilyl) oxy) -3-methoxyphenyl) -2-pentenoic acid ethyl ester (62). To ketoester 61(1.10g,2.89mmol) dissolved in methanol (15mL) was added dry ammonium acetate (1.11g,14.5 mmol). The reaction mixture was stirred at 35 ℃ for 16 h. The methanol was removed in vacuo and the residue was suspended in EtOAc (30mL) and filtered. The filtrate was washed with EtOAc (4X 20 mL). The combined organic layers were dried over sodium sulfate and concentrated under reduced pressure to give amine 62(1.02g,2.69mmol, 93%) as pale yellow crystals. No further purification was performed.¹HNMR(600MHz,CDCl₃)δ6.82(1H,m),6.72(2H,m),4.58(1H,s),4.11(2H,m),3.78(3H,s),2.87(2H,m),2.40(2H,m),1.26(3H,t,J＝7.2Hz),1.00(9H,s),0.19(6H,s)。¹³C NMR(150MHz,CDCl₃)δ170.7,163.6,150.0,142.8,131.8,122.0,121.1,109.8,83.5,58.7,54.8,36.7,29.3,26.3,19.0,14.7,-3.6。

(Z) -3-acetylamino-5- (2- ((tert-butyldimethylsilyl) oxy) -3-methoxyphenyl) -2-pentenoic acid ethyl ester (63). To amine 62(4.02g,10.6mmol) dissolved in THF (50mL) was added pyridine (1.71mL, 21.2mmol) and acetic anhydride (6.00mL, 63.6 mmol)mmol). The reaction mixture was stirred at reflux for 48 h. THF was removed in vacuo, and the residue was dissolved in EtOAc (50mL) and washed with water (50mL), 2M HCl (20mL), saturated NaHCO₃(50mL) and brine (50 mL). The combined organic layers were dried over sodium sulfate and concentrated under reduced pressure. The resulting organic material was purified by flash chromatography using a pre-packed 100g silica gel column [ solvent a: EtOAc; solvent B: hexane; gradient: 7% A/93% B (1CV), 7% A/93% B → 40% A/60% B (10CV), 40% A/60% B (2 CV); flow rate: 100 mL/min; detection at 254nm and 280nm]N-acetamide 63(2.17g,5.15mmol, 49%) was obtained as a yellow oil.¹H NMR(600MHz,CDCl₃)δ6.80(2H,m),6.70(1H,m),4.90(1H,s),4.14(2H,m),3.76(3H,s),3.00(2H,m),2.86(2H,m),2.15(3H,s),1.26(3H,m),0.98(9H,s),0.17(6H,s)。¹³C NMR(150MHz,CDCl₃)δ169.3,168.3,158.2,149.8,142.7,132.0,122.2,120.9,109.5,96.2,59.8,54.7,34.5,28.7,26.2,25.3,18.9,14.3,-3.8。

Ethyl 3-acetylamino-5- (2- ((tert-butyldimethylsilyl) oxy) -3-methoxyphenyl) pentanoate (64). Unsaturated N-acetamide 63(2.17g,5.15mmol) was dissolved in CH₃OH (30 mL). Palladium (10%) carbon (0.53g) and hydrogen balloon were introduced, and the solution was stirred at room temperature for 60h and usedAnd (5) filtering. Will be provided withWash with EtOAc (3X 50 mL). The combined organic phases were evaporated under reduced pressure (CH)₃OH and EtOAc). The resulting organic material was purified by flash chromatography using a pre-packed 100g silica gel column [ solvent a: EtOAc; solvent B: hexane; gradient: 7% A/93% B (1CV), 7% A/93% B → 50% A/50% B (10CV), 50% A/50% B (2 CV); flow rate: 100 mL/min; monitoring at 254nm and 280nm]Saturated N-acetamide 64(0.96g,2.3mmol, 44%) was obtained as a colorless oil.¹H NMR(600MHz,CDCl₃)δ6.83(1H,t,J＝7.8Hz),6.71(2H,m),6.03(1H,d,J＝9Hz),4.29(1H,m),4.11(2H,q,J＝7.2Hz),3.77(3H,s),2.74(1H,m),2.62(1H,m),2.59(1H,m),2.51(1H,m),1.96(3H,s),1.82(2H,m),1.24(3H,t,J＝7.2Hz),1.00(9H,s),0.17(6H,d,J＝10.8Hz)。¹³C NMR(150MHz,CDCl₃)δ172.1,169.6,150.0,142.7,132.7,121.9,121.0,109.4,60.7,54.8,46.3,38.8,34.3,27.6,26.3,23.7,19.0,14.3,-3.6,-3.7。

3-acetylamino-5- (2-hydroxy-3-methoxyphenyl) pentanoic acid (65). To unsaturated ester 64(0.96g,2.3mmol) dissolved in methanol (5mL) was added 1M KOH (7.48 mL). The reaction was stirred from 0 ℃ to room temperature over 3 h. Methanol was removed under vacuum and 2M HCl (5mL) was added to the residue, which was then extracted with EtOAc (3X 20 mL). The combined organic phases were evaporated under reduced pressure. The resulting organic material was purified by flash chromatography using a pre-packed 100g silica gel column [ solvent a: EtOAc; solvent B: hexane; gradient: 12% A/88% B (1CV), 12% A/88% B → 100% A/0% B (35CV), 100% A/0% B (2 CV); flow rate: 100 mL/min; monitoring at 254nm and 280nm]Carboxylic acid 65(0.38g,1.8mmol, 58%) was obtained as a colorless oil.¹H NMR(600MHz,CDCl₃)δ6.74(3H,m),6.34(1H,d,J＝9Hz),4.25(1H,m),3.85(3H,s),2.64(4H,m),1.97(3H,s),1.91(2H,m)。¹³C NMR(150MHz,CDCl₃)δ175.6,171.0,146.6,143.5,127.3,122.5,119.8,108.9,56.2,46.7,38.9,34.1,26.8,23.5。

N- (1-hydroxy-2-methoxy-5-oxo-6, 7,8, 9-tetrahydro-5H-benzo [7 ]]Rotalen-7-yl) acetamide (66). Carboxylic acid 65(0.70g,2.5mmol) was dissolved in eaton reagent (14mL) and the reaction mixture was stirred at room temperature for 12 h. Ice is added to the reaction mixture, which generates a large amount of heat. Saturated sodium carbonate solution was added until a neutral pH was reached. The mixture was extracted with dichloromethane (4X 30 mL). The organic phase was further washed with brine, dried over sodium sulfate, filtered, and concentrated under reduced pressure, and purified by flash chromatography using a pre-packed 50g silica gel column [ solvent a: CH (CH)₃OH; solvent B: CH (CH)₂Cl₂(ii) a Gradient: 1% A/99% B (1CV), 1% A/99% B → 10% A/90% B (10CV), 10% A/90% B (2 CV); flow rate: 100 mL/min; monitoring at 254nm and 280nm]This gave cyclized ketone 66(0.37g,1.4mmol, 57%) as a yellow oil.¹H NMR(600MHz,CDCl₃)δ7.34(1H,d,J＝8.4Hz),6.79(1H,d,J＝9Hz),5.91(1H,s),4.48(1H,m),3.94(3H,s),3.23(1H,m),3.13(1H,m),2.83(2H,m),2.73(1H,m),2.44(1H,m),1.96(3H,s)。¹³C NMR(150MHz,CDCl₃)δ201.1,169.6,149.3,142.9,133.1,129.0,121.1,108.2,56.3,47.0,45.7,32.9,23.6,22.6。

N- (1- (((tert-butyldimethylsilyl) oxy) -2-methoxy-5-oxo-6, 7,8, 9-tetrahydro-5H-benzo [7 ]]Rotalen-7-yl) acetamide (67). To a solution of cyclized ketone 66(0.37g,1.4mmol) in dichloromethane (20mL) was added TBSCl (0.32g,2.1mmol), DMAP (52mg,0.42mmol) and trimethylamine (0.30mL, 2.1mmol) at room temperature and the resulting reaction mixture was stirred for 12 h. The reaction mixture was then washed with brine (30mL) and extracted with dichloromethane (3X 40 mL). The combined organic layers were dried over sodium sulfate and concentrated under reduced pressure. The resulting organic material was purified by flash chromatography using a pre-packed 50g silica gel column [ solvent a: CH (CH)₃OH; solvent B: CH (CH)₂Cl₂(ii) a Gradient: 0% A/100% B (1CV), 0% A/100% B → 5% A/95% B (10CV), 5% A/95% B (2 CV); flow rate: 100 mL/min; monitoring at 254nm and 280nm]Protected ketone 67 was obtained as an off-white solid (0.36g,0.95mmol, 68%).¹H NMR(600MHz,CDCl₃)δ7.41(1H,d,J＝8.4Hz),6.78(1H,d,J＝9Hz),5.61(1H,br),4.46(1H,m),3.84(3H,s),3.26(1H,m),3.16(1H,m),2.80(2H,m),2.71(1H,m),2.45(1H,m),1.96(3H,s),1.00(9H,s),0.17(6H,d,J＝14.4Hz)。¹³C NMR(150MHz,CDCl₃)δ201.1,169.6,153.4,142.4,134.7,129.0,122.6,109.1,55.1,46.9,45.8,40.0,33.2,26.2,23.6,19.1,-3.7,-3.8。

N- (1- ((tert-butyldimethylsilyl) oxy) -2-methoxy-5- (3,4, 5-trimethoxyphenyl) -6,7,8, 9-tetrahydro-5H-benzo [7 ]]Rotalen-7-yl) acetamide (68). To an oven dried flask, THF (20mL) and 3,4, 5-trimethoxyphenyl bromide (0.16g, 0.64mmol) were added and the solution was cooled to-78 ℃. N-butyllithium (1.6M, 0.59mL, 0.94mmol) was slowly added to the reaction mixture, which was then stirred at-78 ℃ for 45 min. Benzocycloheptane 67(80mg,0.21mmol) was then added dropwise to the flask and the reaction mixture was stirred while warming from-78 ℃ to room temperature over 12 h. 2M HCl (20mL) was added and the reaction mixture was stirred for 30min, then extracted with EtOAc (3X 50 mL). For combined organic phasesDried over sodium sulfate and evaporated under reduced pressure. The crude reaction product was purified by flash chromatography using a pre-packed 20g silica gel column [ solvent a: EtOAc; solvent B: hexane; gradient: 12% A/88% B (1CV), 12% A/88% B → 100% A/0% B (10CV), 100% A/0% B (2 CV); flow rate: 100 mL/min; monitoring at 254nm and 280nm]To give benzocycloheptene 68(63.6mg,0.120mmol, 57%) as a crystalline white solid.¹H NMR(600MHz,CDCl₃)δ6.68(1H,d,J＝9Hz),6.58(1H,d,J＝8.4Hz),6.47(2H,s),5.98(1H,d,J＝6Hz),5.54(1H,d,J＝8.4Hz,br),4.39(1H,m),3.85(3H,s),3.80(3H,s),3.79(9H,s),3.20-2.46(4H,m),1.95(3H,s),1.03(9H,s),0.25(3H,s),0.22(3H)。¹³C NMR(150MHz,CDCl₃)δ169.0,152.8,149.1,141.7,141.5,137.9,137.6,132.7,132.3,128.4,122.9,108.9,105.4,60.9,56.2,54.7,47.8,41.0,26.2,23.6,22.9,19.0,-3.6,-3.9。

N- (1-hydroxy-2-methoxy-5- (3,4, 5-trimethoxyphenyl) -6,7,8, 9-tetrahydro-5H-benzo [7 ]]Rotalen-7-yl) acetamide (69). The protected benzocycloheptene N-acetamide 68(63.6mg,0.12mmol) was dissolved in THF (2mL) and cooled to 0 deg.C. TBAF (0.24mL, 0.24mmol) was added and the reaction mixture was stirred at 0 ℃ for 2 h. The solution was washed with water and extracted with EtOAc (3X 20 mL). The combined organic phases were dried over sodium sulfate, filtered and concentrated under reduced pressure and purified by flash chromatography using a pre-packed 25g silica gel column [ solvent a: CH (CH)₃OH; solvent B: CH (CH)₂Cl₂(ii) a Gradient: 5% A/95% B (1CV), 10% A/90% B → 10% A/90% B (10CV), 10% A/90% B (2 CV); flow rate: 100 mL/min; monitoring at 254nm and 280nm]Benzocycloheptene N-acetamide 69(22mg,0.05mmol, 44%) was obtained as a white solid.¹H NMR(600MHz,CDCl₃)δ6.71(1H,d,J＝9Hz),6.54(1H,d,J＝8.4Hz),6.48(2H,s),5.98(1H,d,J＝6Hz),5.76(1H,s),5.56(1H,d,J＝9Hz,br),4.38(1H,m),3.91(3H,s),3.86(3H,s),3.80(6H,s),3.17-2.43(4H,m),1.96(3H,s)。¹³CNMR(150MHz,CDCl₃) δ 169.2,153.0,145.7,142.5,141.4,137.83,137.75,133.3,128.8,126.9,121.5,108.3,105.6,61.1,56.4,56.2,48.1,40.8,23.8, 22.3. HRMS, found 436.1730[ M + Na⁺]Theoretical value C₂₃H₂₉NO₆Na:436.1731。HPLC:9.92min。

Figure 9 shows the synthesis of scheme 7, compounds 76 and 77. The metathesis of trimethoxyphenyl is achieved by: carboxylic acid 74 is first cyclized (eaton reagent) with concomitant elimination, thereby yielding α, β -unsaturated ketone 75. In the case of an α, β -unsaturated ketone on hand, 1, 2-and 1, 4-addition reactions are performed using appropriate aryl lithiums and a gelman reagent (Gilman reagent) to provide a tertiary alcohol analog 76 (with an unsaturated 7-membered ring to maintain rigidity) and a michael adduct, a trimethoxy-side phenyl ring-shifted analog 77, respectively (scheme 7).

Ethyl 5- (2, 3-dimethoxyphenyl) -3-oxopentanoate (72). To a solution of 2, 3-dimethoxybenzaldehyde 70(1.06g,6.38mmol) in THF (50mL) was added ethyl 3-oxo-4- (triphenylphosphine) butyrate (3.00g, 7.65mmol) and potassium tert-butoxide (1.73g,15.3mmol) at room temperature and the reaction mixture was stirred for 12 h. THF was removed under reduced pressure and the resulting material was quenched with 2M HCl (20mL) and extracted with EtOAc (3X 50 mL). The combined organic layers were evaporated under reduced pressure and the crude product (71) was dissolved in CH₃OH (40 mL). To this solution was added 10% palladium on carbon (0.24g) and a balloon filled with hydrogen. The reaction mixture was stirred at room temperature for 12h withFiltered and washed with EtOAc (3X 30mL)The combined organic phases were evaporated under reduced pressure (CH)₃OH and EtOAc). The resulting organic material was purified by flash chromatography using a pre-packed 100g silica gel column [ solvent a: EtOAc; solvent B: hexane; gradient: 7% A/93% B (1CV), 7% A/93% B → 40% A/60% B (10CV), 40% A/60% B (2 CV); flow rate: 100 mL/min; monitoring at 254nm and 280nm]Saturated ester 72(1.45g,5.17mmol, 81%) was obtained as a colorless oil.¹H NMR(600MHz,CDCl₃)δ6.97(1H,t,J＝7.8Hz),6.78(1H,d,J＝9Hz),6.75(1H,d,J＝7.8Hz),4.17,(2H,q,J＝7.2Hz),3.85(3H,s),3.82(3H,s),3.43(2H,s),2.90(2H,m),2.84(2H,m),1.26(3H,t,J＝6.6Hz)。¹³C NMR(150MHz,CDCl₃)δ202.4,167.3,152.9,147.2,134.5,124.1,122.0,110.8,61.5,60.7,55.8,49.5,43.8,24.5,14.2。

Ethyl 5- (2, 3-dimethoxyphenyl) -3-hydroxypentanoate (73). To ketone 72(1.45g,5.17mmol) in CH at 0 deg.C₃NaBH was added to a solution in OH (20mL)₄(110mg,2.91mmol) and the reaction mixture was stirred at 0 ℃ for 1h and then at ambient temperature for 3 h. Removal of CH under reduced pressure₃OH, and the resulting material was washed with water and extracted with EtOAc (3X 30 mL). The combined organic phases were concentrated under reduced pressure to give the crude alcohol product 73(1.20g,4.25mmol, 82%) as a colorless oil.¹H NMR(600MHz,CDCl₃)δ6.98(1H,t,J＝7.8Hz),6.78(2H,m),4.15(2H,m),3.97(1H,m),3.85(3H,s),3.83(3H,s),2.76(2H,m),2.47(2H,m),1.80(1H,m),1.72(1H,m),1.25(3H,t,J＝7.2Hz)。¹³C NMR(150MHz,CDCl₃)δ173.0,152.8,147.2,135.5,124.2,122.1,110.4,67.3,60.8,55.8,41.5,37.6,25.9,14.3。

5- (2, 3-Dimethoxyphenyl) -3-hydroxypentanoic acid (74). At 0 ℃ to ester 73 in CH₃To a solution in OH/THF (8mL/4mL) was added 2.5M aqueous NaOH (6mL) and the reaction mixture was stirred at 0 deg.C for 1h, then warmed to room temperature over 12 h. Removal of CH under reduced pressure₃OH and THF, and the resulting material was washed with water and extracted with EtOAc (3X 30 mL). The combined organic extracts were extracted with Na₂SO₄Drying, filtration, and concentration under pressure gave carboxylic acid 74 as an orange oil (0.88g,3.3mmol, 77%) (without further purification).¹H NMR(600MHz,CDCl₃)δ7.00(1H,t,J＝8.4Hz),6.78(2H,dd,J＝6.6Hz,6.6Hz),3.92(1H,m),3.86(3H,s),3.84(3H,s),2.76(2H,m),2.51(2H,m),1.82(1H,m),1.73(1H,m)。¹³C NMR(150MHz,CDCl₃)δ176.8,152.7,146.9,134.9,124.5,122.2,110.6,66.9,61.0,55.8,41.2,37.6,25.6。

1, 2-dimethoxy-8, 9-dihydro-5H-benzo [7 ]]Rotalen-5-one (75). To carboxylic acid 74(0.88g,3.3mmol) was added Eton's reagent (15.7mL) and the mixture was stirred at room temperature for 14 h. The mixture was then poured onto ice and neutralized with sodium carbonate. The aqueous layer was extracted with EtOAc (3X 40 mL). The combined organic phases were dried over sodium sulfate and evaporated under reduced pressureAnd purified by flash chromatography using a pre-packed 100g silica gel column [ solvent a: EtOAc; solvent B: hexane; gradient: 7% A/93% B (1CV), 7% A/93% B → 30% A/70% B (10CV), 30% A/70% B (2 CV); flow rate: 50 mL/min; monitoring at 254nm and 280nm]Unsaturated cyclic ketone 75(0.360g,1.65mmol, 50%) was obtained as a yellow crystalline solid.¹H NMR(600MHz,CDCl₃)δ7.55(1H,d,J＝9Hz),6.83(1H,d,J＝9Hz),6.71(1H,td,J＝4.8Hz,12Hz),6.23(1H,td,J＝1.8Hz,12Hz),3.91(3H,s),3.78(3H,s),3.16(2H,m),2.55(2H,m)。¹³C NMR(150MHz,CDCl₃)δ193.9,156.2,147.0,145.2,134.4,134.3,132.7,126.7,110.0,61.2,55.9,29.6,25.1。

1, 2-dimethoxy-5- (3,4, 5-trimethoxyphenyl) -8, 9-dihydro-5H-benzo [7 ]]Rotalen-5-ol (76). To an oven dried flask, THF (30mL) and 3,4, 5-trimethoxyphenyl bromide (0.87g, 3.5mmol) were added and the solution was cooled to-78 ℃. N-butyllithium (1.41mL, 2.5M, 3.52mmol) was slowly added to the reaction mixture, which was then stirred at-78 ℃ for 30 min. The unsaturated cyclic ketone 75(0.35g, 1.6mmol) in THF (5mL) was then added dropwise to the flask and the reaction mixture was stirred while warming from-78 deg.C to room temperature over 12 h. The reaction mixture was washed with 2M HCl (10mL) and extracted with EtOAc (3X 30 mL). The combined organic phases were dried over sodium sulfate and evaporated under reduced pressure. The crude reaction product was purified by flash chromatography using a pre-packed 50g silica gel column [ solvent a: EtOAc; solvent B: hexane; gradient: 12% A/88% B (1CV), 12% A/88% B → 60% A/40% B (10CV), 60% A/40% B (5 CV); flow rate: 100 mL/min; monitoring at 254nm and 280nm]Tertiary alcohol 76(0.457g,1.18mmol, 74%) was obtained as a white solid.¹H NMR(600MHz,CDCl₃)δ7.54(1H,d,J＝9Hz),6.82(1H,d,J＝8.4Hz),6.59(2H,s),6.07(1H,d,J＝12Hz),5.77(1H,td,J＝1.2Hz,12Hz),3.90(3H,s),3.82(3H,s),3.78(6H,s),3.75(3H,s),3.05(1H,m),2.48(1H,m),2.43(1H,m),2.15(1H,m)。¹³C NMR(150MHz,CD₃OD) δ 153.9,153.2,147.5,145.1,141.7,138.3,137.2,134.1,130.8,121.6,109.9,106.0,78.6,61.4,61.1,56.5,56.1,30.0, 23.4. HRMS, found 409.1622[ M + Na⁺]Theoretical value C₂₂H₂₆O₆Na:409.1622。HPLC:17.36min。

1, 2-dimethoxy-7- (3,4, 5-trimethoxyphenyl) -6,7,8, 9-tetrahydro-5H-benzo [7 ]]Rotalen-5-one (77). To an oven dried flask, THF (30mL) and 3,4, 5-trimethoxyphenyl bromide (0.47g,1.9mmol) were added and the solution was cooled to-78 ℃. N-butyllithium (0.76mL, 2.5M, 1.9mmol) was slowly added to the reaction mixture, which was stirred at-78 ℃ for 45min, then moved to the-10 ℃ bath. An aliquot of CuI (0.181g, 0.95mmol) was added to the flask and the reaction mixture was stirred at-10 ℃ for 1 h. Unsaturated ketone 75(0.104g, 0.47mmol) in THF (10mL) was then added dropwise to the flask and the reaction mixture was stirred while warming from-78 deg.C to room temperature over 7 h. Adding saturated NH₄Cl solution and ammonium hydroxide (20mL/20mL), then stirred at room temperature for 30min, followed by extraction with EtOAc (3X 50 mL). The combined organic phases were dried over sodium sulfate and evaporated under reduced pressure. The crude reaction was purified by flash chromatography using a pre-packed 50g silica gel column [ solvent a: EtOAc; solvent B: hexane; gradient: 5% A/95% B (1CV), 5% A/95% B → 60% A/40% B (10CV), 60% A/40% B (2 CV); flow rate: 110 mL/min; monitoring at 254nm and 280nm]This gave Michael addition ketone 77(81.7mg,0.21mmol, 44%) as an off-white solid.¹H NMR(600MHz,CDCl₃)δ7.56(1H,d,J＝8.4Hz),6.88(1H,d,J＝9Hz),6.41(2H,s),3.93(3H,s),3.83(6H,s),3.824(3H,s),3.819(3H,s),3.34(1H,td,J＝4.8Hz,15Hz),3.09(1H,m),3.04(1H,m),2.94(2H,m),2.16(1H,m),1.97(1H,m)。¹³C NMR(150MHz,CDCl₃) δ 203.0,156.4,153.4,146.1,141.4,136.6,135.5,132.5,125.7,110.1,104.0,61.3,61.0,56.2,56.0,47.9,39.9,34.6, 23.4. HRMS, found 409.1624[ M + Na⁺]Theoretical value C₂₂H₂₆O₆Na:409.1622.HPLC:18.77min。

FIG. 10 shows the synthesis of scheme 8, compounds 88 and 89. During the course of the study centered on the variability of the protective groups of the phenol moiety of the benzocycloheptene analogue subset, the formation of the diene 87, which is obtained by addition of trimethoxyphenyllithium 1,2 to the ketone 86 followed by a post-reaction treatment, occurred. In this case, the secondary alcohol is present even under mildly acidic or basic conditions(e.g. TBAF deprotection or BCl at lower temperatures)₃Cracking) also showed a tendency to eliminate. Various combinations of 4-position (phenolic moiety on fused aryl ring) and allyl alcohol protecting group strategies were tried, ultimately leading to the unexpected formation of diene 88. It is important to note that the diene (88) was previously obtained. Holding this compound in hand and noting its excellent biological activity (inhibition of tubulin polymerization and cytotoxicity against human cancer cell lines, table 1), there was an incentive to prepare the corresponding water-soluble phosphate prodrug disodium salt 89 to facilitate in vivo studies in a mouse model of prostate cancer to evaluate the efficacy of this compound as a VDA as demonstrated by bioluminescent imaging (BLI).

2-isopropoxy-3-methoxybenzaldehyde (78). To a solution of 2-hydroxy-3-methoxybenzaldehyde 58(5.00g,32.9mmol) in DMF (100mL) was added K₂CO₃(14.97g,98.58mmol) and 2-iodopropane (6.54mL,65.7mmol), and the mixture was stirred at 50 ℃ for 20 h. DMF was removed under reduced pressure and the resulting material was washed with water (100mL) to remove excess salt and extracted with EtOAc (3X 100 mL). The combined organic phases were dried over sodium sulfate and concentrated to give the protected aldehyde 78(6.12g,31.6mmol, 96%) as a colorless oil without further purification.¹H NMR(600MHz,CDCl₃)δ10.44(1H,s),7.41(1H,d,J＝7.8Hz),7.10(2H,m),4.62(1H,m),3.86(3H,s),1.31(6H,d,J＝6Hz)。¹³C NMR(150MHz,CDCl₃)δ191.0,153.4,150.7,131.0,123.7,119.0,118.0,76.3,56.1,22.4。

Ethyl 5- (2-isopropoxy-3-methoxyphenyl) -3-oxo-4-pentenoate (79). To ethyl 3-oxo-4- (triphenylphosphine) butyrate (0.85g, 2.2mmol) dissolved in THF (50mL) was added potassium tert-butoxide (0.50g, 4.4mmol) and aldehyde 78(0.35g,1.8mmol), and the resulting reaction mixture was stirred at room temperature for 12 h. THF was removed under reduced pressure and the resulting material was neutralized with 2M HCl (10mL) and extracted with EtOAc (3X 20 mL). The combined organic layers were evaporated under reduced pressure and the crude reaction product was purified by flash chromatography using a pre-packed 50g silica gel column [ solvent a: EtOAc; b: hexane; gradient: 10% A/90% B (1CV), 10% A/90% B → 40% A/60% B (10CV), 40% A/60% B (2 CV); flow rate: 100 mL/min; monitoring at 254nm and 280nm ] gave the unsaturated ester 79 as a yellow oil (0.40g,1.3mmol, 72%). NMR characterization was performed after the next step.

Ethyl 5- (2-isopropoxy-3-methoxyphenyl) -3-oxopentanoate (80). To dissolve in CH₃To an unsaturated ester 79(0.39g,1.3mmol) in OH (20mL) was added 10% palladium on carbon (0.2g) and a balloon filled with hydrogen. The reaction mixture was stirred at room temperature for 12h and then washed withFiltered and washed with EtOAc (3X 20mL)The combined organic phases were evaporated under reduced pressure (CH)₃OH and EtOAc) to give saturated ester 80 as colorless oil (0.23g,0.73mmol, 57%).¹H NMR(600MHz,CDCl₃)δ6.93(1H,t,J＝7.8Hz),6.75(2H,m),4.51(1H,sept,J＝6Hz),4.17(2H,q,J＝7.2Hz),3.81(3H,s),3.41(2H,s),2.92(2H,t,J＝7.8Hz),2.83(2H,t,J＝8.4Hz),1.25(6H,d,J＝6Hz),1.25(3H,t,7.2Hz)。¹³C NMR(150MHz,CDCl₃)δ202.5,167.2,152.9,144.9,135.0,123.5,121.9,110.7,74.6,61.4,55.7,49.4,43.6,25.0,22.7,14.2。

Ethyl 3-hydroxy-5- (2-isopropoxy-3-methoxyphenyl) pentanoate (81). At 0 deg.C, to dissolve in CH₃To ketone 80(2.32g,7.52mmol) in OH (30mL) was added NaBH₄(96mg,2.5 mmol). The reaction was stirred for 1h, then returned to room temperature for an additional 1 h. Removal of CH under reduced pressure₃OH, the residue was washed with water and extracted with EtOAc (3X 30 mL). The combined organic phases were evaporated under pressure and the crude reaction product was purified by flash chromatography using a pre-packed 50g silica gel column [ solvent a: EtOAc; solvent B: hexane; gradient: 10% A/90% B (1CV), 10% A/90% B → 40% A/60% B (10CV), 40% A/60% B (2 CV); flow rate: 100 mL/min; monitoring at 254nm and 280nm]Alcohol 81(1.80g,5.80mmol, 77%) was obtained as a colorless oil.¹H NMR(600MHz,CDCl₃)δ6.96(1H,t,J＝8.4Hz),6.77(1H,d,J＝6.6Hz),6.75(1H,d,J＝7.8Hz),4.52(1H,sept,J＝6.6Hz),4.13(2H,q,J＝7.2Hz),3.91(1H,m),3.82(3H,s),2.82(1H,m),2.74(1H,m),2.45(1H,m),2.44(1H,m),1.78(1H,m),1.71(1H,m),1.28(3H,t,J＝6.6Hz),1.26(3H,t,J＝6.6Hz),1.25(3H,t,J＝7.2Hz)。¹³C NMR(150MHz,CDCl₃)δ172.8,152.9,144.8,136.0,123.7,122.1,110.3,74.9,67.1,60.7,55.7,41.6,37.5,41.6,37.5,26.3,22.9,22.6,14.3。

Ethyl 3- ((tert-butyldiphenylsilyl) oxy) -5- (2-isopropoxy-3-methoxyphenyl) pentanoate (82). To alcohol 81(0.77g,2.5mmol) dissolved in DMF (10mL) was added tert-butyl (chloro) diphenylsilane (TBDPSCl) (0.96mL,3.7mmol) and imidazole (0.280g,3.98mmol) at room temperature and the solution was stirred at room temperature for 12 h. DMF was removed under reduced pressure and the resulting material was washed with brine (50mL) and extracted with ether (3X 30 mL). The combined organic phases were dried over sodium sulfate and evaporated under reduced pressure. The crude product was purified by flash chromatography using a pre-packed 50g silica gel column [ solvent a: EtOAc; solvent B: hexane; gradient: 10% A/90% B (1CV), 10% A/90% B → 40% A/60% B (10CV), 40% A/60% B (2 CV); flow rate: 100 mL/min; monitoring at 254nm and 280nm]Yield protected alcohol 82 as a colorless oil (0.960g,1.75mmol, 71%).¹H NMR(600MHz,CDCl₃)δ7.70(4H,m),7.39(6H,m),6.87(1H,t,J＝7.8Hz),6.70(1H,d,J＝8.4Hz),6.52(1H,d,J＝7.8Hz),4.41(1H,m),4.29(1H,m),4.00(2H,m),3.80(3H,s),2.55(4H,m),1.76(2H,m),1.19(6H,m),1.18(3H,t,J＝7.2Hz),1.08(9H,s)。¹³C NMR(150MHz,CDCl₃)δ171.6,152.9,144.9,136.5,136.1,135.3,135.0,134.3,129.8,129.7,127.9,127.6,123.3,121.6,110.1,74.4,70.6,60.4,55.8,42.1,37.8,26.7,25.6,22.7,19.5,19.2,14.2。

3- ((tert-butyldiphenylsilyl) oxy) -5- (2-isopropoxy-3-methoxyphenyl) pentanoic acid (83). At 0 deg.C, to dissolve in CH₃To the protected alcohol 82(7.80g,14.2mmol) in OH/THF (60mL/30mL) was added 2.5M NaOH (20mL), and the solution was stirred for 1h, then 13h at room temperature. The organic solvent (CH) was removed under reduced pressure₃OH and THF) and water (30mL) was added to the resulting suspension, followed by extraction with diethyl ether (3 × 50 mL). The combined organic phases were dried over sodium sulfate and evaporated under reduced pressure. The crude product was purified by flash chromatography using a pre-packed 100g silica gel column [ solvent a: EtOAc;solvent B: hexane; gradient: 10% A/90% B (1CV), 10% A/90% B → 60% A/40% B (15CV), 60% A/40% B (2 CV); flow rate: 100 mL/min; monitoring at 254nm and 280nm]Carboxylic acid 83(3.49g,6.70mmol, 46%) was obtained as a colorless oil.¹H NMR(600MHz,CDCl₃)δ7.67(4H,m),7.39(6H,m),6.86(1H,t,J＝7.8Hz),6.70(1H,d,J＝8.4Hz),6.50(1H,d,J＝7.2Hz),4.41(1H,m),4.20(1H,m),3.79(3H,s),2.56(4H,m),1.82(2H,m),1.19(6H,t,J＝6Hz),1.06(9H,s)。¹³C NMR(150MHz,CDCl₃)δ175.5,152.9,144.8,136.0,133.7,133.6,129.9,127.8,123.4,121.6,110.2,74.5,70.5,55.8,41.2,37.5,27.1,25.7,22.7,19.4。

7- ((tert-butyldiphenylsilyl) oxy) -1-isopropoxy-2-methoxy-6, 7,8, 9-tetrahydro-5H-benzo [7 ]]Rotalen-5-one (84). To carboxylic acid 83(3.49g,6.70mmol) dissolved in dichloromethane (30mL) was added oxalyl chloride (2.77mL,31.8mmol) and DMF (0.1mL) as catalyst at room temperature and the solution was stirred at room temperature for 2 h. The solvent and unreacted oxalyl chloride were removed under reduced pressure. The yellow acid chloride was then dissolved in dichloromethane (40mL) and cooled to-10 ℃. To this solution was added 1M SnCl₄Was added to the reaction solution, and the reaction mixture was stirred at-10 ℃ for 40 min. The reaction was quenched by addition of water and then extracted with dichloromethane (3X 40 mL). The combined organic phases were dried over sodium sulfate and concentrated. The crude product was purified by flash chromatography using a pre-packed 100g silica gel column [ solvent a: EtOAc; solvent B: hexane; gradient: 10% A/90% B (1CV), 10% A/90% B → 40% A/60% B (10CV), 40% A/60% B (2 CV); flow rate: 100 mL/min; monitoring at 254nm and 280nm]Ketone 84(2.19g,4.50mmol, 67%) was obtained as a pale yellow gel.¹H NMR(600MHz,CDCl₃)δ7.66(4H,m),7.54(1H,d,J＝9.6Hz),7.38(6H,m),6.79(1H,d,J＝9Hz),4.36(1H,m),4.28(1H,m),3.86(3H,s),3.14(1H,m),3.03(2H,m),2.88(1H,m),1.94(2H,m),1.27(6H,m),1.03(9H,s)。¹³C NMR(150MHz,CDCl₃)δ199.7,156.0,144.1,138.5,136.1,136.0,135.9,134.2,133.9,133.1,129.9,129.8,127.9,127.8,127.7,125.3,109.5,75.2,68.4,55.9,50.4,36.1,27.0,22.7,22.6,22.0,19.3。

7-hydroxy-1-isopropoxy-2-methoxy6,7,8, 9-tetrahydro-5H-benzo [7 ] yl]Rotalen-5-one (85). Ketone 84(2.19g,4.50mmol) was dissolved in THF (20mL), TBAF (9.00mL, 9.00mmol) was added at 0 ℃, and the reaction mixture was stirred for 30min and then at room temperature for 6 h. Brine (30mL) was added and the resulting solution was extracted with EtOAc (3X 30 mL). The combined organic phases were dried over sodium sulfate and evaporated under reduced pressure. The crude product was purified by flash chromatography using a pre-packed 100g silica gel column [ solvent a: EtOAc; solvent B: hexane; gradient: 10% A/90% B (1CV), 10% A/90% B → 40% A/60% B (10CV), 40% A/60% B (2 CV); flow rate: 100 mL/min; monitoring at 254nm and 280nm]Alcohol 85(0.41g,1.7mmol, 37%) was obtained as a yellow oil.¹H NMR(600MHz,CDCl₃)δ7.57(1H,d,J＝8.4Hz),6.8(1H,d,J＝8.4Hz),4.41(1H,m),4.31(1H,m),3.88(3H,s),3.11(2H,m),3.06(1H,m),2.99(1H,m),2.15(1H,m),1.87(1H,m),1.29(6H,m)。¹³C NMR(150MHz,CDCl₃)δ199.6,156.3,144.2,138.4,132.6,125.4,109.7,75.3,67.3,55.9,50.3,35.8,22.7,22.6,22.1。

1-isopropoxy-2-methoxy-7- ((trimethylsilyl) oxy) -6,7,8, 9-tetrahydro-5H-benzo [7 ]]Rotalen-5-one (86). To a solution of alcohol 85(0.35g,1.33mmol) in DMF (20mL) was added imidazole (0.27g, 6.4mmol) and TMSCl (4.26mmol) at room temperature and the reaction mixture was stirred for 12 h. The solvent was removed under reduced pressure and brine (20mL) was added, followed by extraction with EtOAc (3X 30 mL). The organic extract was dried over sodium sulfate, filtered, and concentrated under reduced pressure, and the residue was purified by flash chromatography using a pre-packed 50g silica gel column [ solvent a: EtOAc; solvent B: hexane; gradient: 10% A/90% B (1CV), 10% A/90% B → 40% A/60% B (10CV), 40% A/60% B (2 CV); flow rate: 100 mL/min; monitoring at 254nm and 280nm]This gave TMS-protected ketone 86 as a yellow oil (0.19g,0.56mmol, 43%).¹H NMR(500MHz,CDCl₃)δ7.54(1H,d,J＝10.2Hz),6.79(1H,d,J＝10.8Hz),4.39(1H,m),4.19(1H,m),3.86(3H,s),3.22(1H,m),2.99(2H,m),2.90(1H,m),1.91(2H,m),1.27(6H,d,J＝9Hz),0.11(9H,s)。¹³C NMR(125MHz,CDCl₃)δ199.7,156.1,144.0,138.6,132.6,125.3,109.5,75.1,67.3,55.8,51.0,36.4,22.6,22.5,21.7。

4-Isopropoxy-3-methoxy-9- (3,4, 5-trimethoxyphenyl) -5H-benzo [7 ]]A rotalene (87). To an oven dried flask, THF (20mL) and 3,4, 5-trimethoxyphenyl bromide (0.21g, 0.85mmol) were added and the solution was cooled to-78 ℃. N-butyllithium (0.34mL, 2.5M, 0.85mmol) was slowly added to the reaction mixture, which was then stirred at-78 ℃ for 45 min. Ketone 86(0.19g,0.56mmol) was then added dropwise to the flask and the reaction mixture was stirred (-78 ℃ to 0 ℃) over 4h and further stirred at 0 ℃ for 1 h. 2M HCl (20mL) was added at deg.C, then stirred for 10min and extracted with EtOAc (3X 30 mL). The combined organic phases were dried over sodium sulfate and evaporated under reduced pressure. The crude product was purified by flash chromatography using a pre-packed 50g silica gel column [ solvent a: EtOAc; solvent B: hexane; gradient: 10% A/90% B (1CV), 10% A/90% B → 40% A/60% B (10CV), 40% A/60% B (2 CV); flow rate: 100 mL/min; monitoring at 254nm and 280nm]Diene 87 was obtained as a colorless oil (60mg,0.15mmol, 27%).¹H NMR(600MHz,CDCl₃)δ6.81(1H,d,J＝8.4Hz),6.71(1H,d,J＝9Hz),6.61(2H,s),6.60(1H,d,J＝5.4Hz),6.17(1H,m),5.87(1H,m),4.48(1H,p,J＝6.6Hz),3.91(3H,s),3.86(6H,s),3.85(3H,s),3.26(2H,m,b),1.38(6H,d,J＝6Hz)。¹³C NMR(150MHz,CDCl₃)δ153.8,153.0,145.6,142.0,140.4,137.5,134.5,131.7,128.5,126.9,125.9,124.8,109.1,106.7,75.0,61.0,56.3,55.8,26.7,22.8。

3-methoxy-9- (3,4, 5-trimethoxyphenyl) -5H-benzo [7 ]]Rotalen-4-ol (88). Isopropyl protected phenol 87(60mg,0.15mmol) was dissolved in CH₂Cl₂(10mL), to which BCl was added₃(0.17mL,1M,0.17mmol) and the reaction mixture was stirred at 0 ℃ for 1 h. The solution was washed with water and 2M HCl and extracted with EtOAc (3X 20 mL). The combined organic phases were dried over sodium sulfate and evaporated under reduced pressure. The crude reaction was purified by flash chromatography using a pre-packed 50g silica gel column [ solvent a: EtOAc; solvent B: hexane; gradient: 5% A/95% B (1CV), 5% A/95% B → 60% A/40% B (10CV), 60% A/40% B (2 CV); flow rate: 100 mL/min; monitoring at 254nm and 280nm]Phenol 88(40mg,0.11mmol, 75%) was obtained as a white solid.¹H NMR(600MHz,CDCl₃)δ6.67(1H,d,J＝8.4Hz),6.62(1H,d,J＝9Hz),6.60(2H,s),6.59(1H,d,J＝5.4Hz),6.15(1H,m),5.94(1H,m),3.89(6H,s),3.84(6H,s),3.23(2H,m,b)。¹³C NMR(150MHz,CDCl₃) δ 153.0,147.1,145.5,140.6,140.3,137.5,132.2,128.3,127.0,126.2,126.0,120.1,107.6,106.7,61.1,56.3,56.2, 25.7. HRMS, found 377.1361[ M + Na⁺]Theoretical value C₂₁H₂₂O₅Na:377.1359。HPLC:19.52min。

3-methoxy-9- (3,4, 5-trimethoxyphenyl) -5H-benzo [7 ]]Rotalen-4-yl sodium phosphate (89). To dissolve in CH₂Cl₂To phenol 88(95mg,0.27mmol) (25mL) was added POCl₃(0.11mL,1.1mmol) and pyridine (0.078mL,0.97mmol), and the reaction mixture was stirred from 0 ℃ to room temperature for 8 h. Removal of CH under reduced pressure₂Cl₂The residue was dissolved in H at room temperature₂O/THF (10mL/5mL) and the solution was stirred for 1 h. NaOH solution (0.1M) was added to the reaction mixture to adjust the pH to 10 at 0 ℃ and the solution was stirred at 0 ℃ for 30 min. The water was removed under reduced pressure. The crude product was purified by flash chromatography using a pre-packed 12g C-18 column [ solvent a: acetonitrile; solvent B: water; gradient: 0% A/100% B (1CV), 0% A/100% B → 10% A/90% B (10CV), 10% A/90% B (2 CV); flow rate: 12 mL/min; monitoring at 254nm and 280nm]Phosphate 89(65.8mg,0.14mmol, 51%) was obtained as a yellow solid.¹H NMR(500MHz,D₂O)δ6.65(2H,s),6.60(2H,m),6.53(1H,d,J＝6Hz),6.10(1H,m),5.98(1H,m),3.78(3H,s),3.72(3H,s),3.70(6H,s),3.29(2H,b)。¹³CNMR(125MHz,D₂O)δ153.0,152.1,144.5,140.8,138.4,136.0,134.5,131.1,129.8,126.2,126.1,124.2,109.1,106.7,60.9,55.9,55.7,26.9。³¹P NMR(200MHz,D₂O) δ 0.81. HRMS, found 479.0841[ M + H⁺]Theoretical value C₂₁H₂₂O₈Na₂P⁺:479.0842。HPLC:14.22min。

Figure 11 shows the synthesis of scheme 9, compounds 91 and 93. For this synthesis, Tetrahydrofuran (THF), carbon tetrachloride, dichloromethane, methanol, Dimethylformamide (DMF) and acetonitrile were used in anhydrous form. Unless otherwise stated, the reaction was carried out under nitrogen. Thin Layer Chromatography (TLC) plates (glass plates precoated with silica gel 60F254, thickness 0.25mm) were usedThe reaction was monitored. Purification of intermediates and products a Biotage Isolera rapid purification system was used, using silica gel (200 and 400 mesh,) Or RP-18 pre-packed column or manually in a glass column. Synthesis of intermediates and products using the Varian VNMRS500MHz or Bruker DPX 600MHz instrument¹H NMR (500 or 600MHz),¹³CNMR (125 or 150MHz) spectroscopic data, characterizing synthetic intermediates and products. Recorded in CDCl₃、D₂O、(CD₃)₂CO or CD₃Spectrum in OD. All chemical shifts are expressed in ppm (δ) and peak patterns are reported as broad (br), singlet(s), doublet (d), triplet (t), quartet (q), quintet (p), hexamer (sextet), heptamer (septet), doublet (dd), doubledoublet (ddd), and multiplet (m).

Using a column (H.sub.190-400 nm) HPLC with a diode array detector (Zorbax XDB-C18: (H.sub.H.)) 5 μm) and an Agilent1200HPLC system with Zorbax reliable cassette protection column, further analyzing the purity of the final compound at 25 ℃; the method comprises the following steps: solvent A, acetonitrile, solvent B, H₂O; gradient: from 10% A/90% B to 100% A/0% B in 0-40 min; the balance time is 10 min; the flow rate is 1.0 mL/min; the sample volume is 20 mu L; monitoring was at wavelengths of 210, 230, 254, 280 and 320 nm. Mass spectrometry was performed using a Thermo Scientific LTQ Orbitrap Discovery instrument at positive or negative ESI (electrospray ionization).

Compound 90: 4- ((tert-butyldimethylsilyl) oxy) -3,9,10, 11-tetramethoxy-6, 7-dihydrodibenzo [ a, h]Azulene-8 (5H) -ketone. Dissolve in Et at room temperature₂TBS protected benzocycloheptene analog 26(0.67g,1.7mmol) in O (10mL) was added to chlorosulfonyl isocyanate (0.15mL, 1.7mmol), and the reaction mixture was stirred at room temperature for 2 h. Adding Na at 0 deg.C₂CO₃And Na₂HPO₄Buffer (pH 7) solution, then the reaction mixture was stirred overnight to room temperature. The reaction mixture was then washed with Et₂O (3 × 20mL) extraction, combined organic phases were evaporated under reduced pressure, and the crude reaction product was purified by flash chromatography using a pre-packed 50g silica gel column [ solvent a: EtOAc; solvent B: hexane; gradient: 10% A/90% B (1CV), 10% A/90% B → 40% A/60% B (10CV), 40% A/60% B (2 CV); flow rate: 100 mL/min; monitoring at 254nm and 280nm]Product 90(0.19g,0.45mmol, 26%) was obtained as an orange oil.¹H NMR(CDCl₃,500Hz)δ7.17(1H,d,J＝12Hz),6.85(1H,d,J＝12Hz),6.65(1H,s),4.14(3H,s),3.89(3H,s),3.86(3H,s),3.83(3H,s),2.77(2H,m),2.30(2H,m),2.11(2H,m),1.02(9H,s),0.21(6H,s)。¹³C NMR(CDCl₃,150MHz)δ193.9,156.7,152.9,152.2,150.8,143.1,142.4,141.1,135.3,134.7,126.7,120.3,108.7,107.7,102.8,62.4,61.5,56.6,54.9,31.2,26.2,24.9,20.6,19.1,-3,7。

Compound 91: 4-hydroxy-3, 9,10, 11-tetramethoxy-6, 7-dihydrodibenzo [ a, h]Azulene-8 (5H) -ketone. TBS-protected cyclic ketone 90(0.39g,0.79mmol) was dissolved in THF (6mL), TBAF (0.87mL,0.87mmol) was added, and the reaction mixture was stirred at 0 ℃ for 1 h. The solution was washed with water and extracted with EtOAc (3X 20 mL). The combined organic phases were dried over sodium sulfate and evaporated under reduced pressure. The crude reaction product was purified by flash chromatography using a pre-packed 25g silica gel column [ solvent a: EtOAc; solvent B: hexane; gradient: 7% A/93% B (1CV), 7% A/93% B → 60% A/40% B (10CV), 60% A/40% B (2 CV); flow rate: 100 mL/min; monitoring at 254nm and 280nm]Phenol (0.27g,0.71mmol, 90%) was obtained as an orange solid.¹H NMR(600MHz,CDCl₃)δ7.11(1H,d,J＝6Hz),6.86(1H,d,J＝6Hz),6.65(1H,s),5.82(1H),4.14(3H,s),3.97(3H,s),3.89(3H,s),3.84(3H,s),2.77(2H,m),2.31(2H,m),2.14(2H,m)。¹³C NMR(150MHz,CDCl₃) δ 193.8,156.8,152.8,151.9,147.0,143.8,142.3,141.1,135.6,129.0,127.2,118.8,107.9,107.7,102.7,62.4,61.5,56.6,56.2,31.0,24.2, 20.4. HRMS, found 405.1315[ M + Na⁺]Theoretical value C₂₂H₂₂O₆Na:405.1309。HPLC:18.97min。

Compound 93: 3,4,9,10, 11-pentamethoxy-6, 7-dihydrodibenzo [ a, h ]]Azulene-8 (5H) -ketone. Dissolve in Et at room temperature₂To dimethoxybenzocycloheptene analog 92(50mg,0.13mmol) in O (10mL) was added chlorosulfonyl isocyanate (0.058mL, 0.13mmol), and the reaction mixture was stirred at room temperature for 2 h. At 0 deg.C, Na is added₂CO₃And Na₂HPO₄Buffer (pH 7) solution, then the reaction mixture was stirred overnight to room temperature. The reaction mixture was then washed with Et₂O (3 × 20mL) extraction, combined organic phases were evaporated under reduced pressure, and the crude reaction product was purified by flash chromatography using a pre-packed 25g silica gel column [ solvent a: EtOAc; solvent B: hexane; gradient: 10% A/90% B (1CV), 10% A/90% B → 40% A/60% B (10CV), 40% A/60% B (2 CV); flow rate: 100 mL/min; monitoring at 254nm and 280nm]Product 93(16mg,0.04mmol, 30%) was obtained as an orange solid.¹H NMR(CDCl₃,600MHz)δ7.22(1H,d,J＝6Hz),6.94(1H,d,J＝6Hz),6.76(1H,s),4.21(3H,s),3.94(3H,s),3.89(3H,s),3.87(3H,s),3.85(3H,s),2.32(6H,m,b)。¹³C NMR(CDCl₃150MHz) delta 157.7,153.5,150.6,147.5,142.0,138.7,136.5,136.2,129.1,124.3,123.9,120.8,109.9,120.3,62.0,61.3,61.28,61.24,56.5,55.8,33.0,23.8,19.6. HRMS, found 419.1467[ M + Na⁺]Theoretical value C₂₃H₂₄O₆Na⁺:419.1465。HPLC:21.10min。

Example 2 biological evaluation

Cell lines and sulforhodamine B (SRB) assay. Sulfonylrhodamine B (SRB) assay was administered to assess growth inhibition of human cancer cells as previously described. The DU-145, SK-OV-3 and NCI-H460 cancer cell lines (obtained from ATCC) were stored in T75 flasks (Corning) for up to 15 passages using high glucose DMEM supplemented with 10% fetal bovine serum (Gibco)/1% gentamicin sulfate. For these experiments, cells were trypsinized, counted, and plated at 7000-8000 cells/well in 96-well plates (Corning) at 5% CO₂Incubate at 37 ℃ for 24h in a humidified incubator under atmosphere. The compound to be tested was dissolved in DMSO to generate a stock solution of 10mg/mL, andserial dilutions were added to the plates in culture medium. Adriamycin (Sigma-Aldrich) and paclitaxel (Tokyo Chemical) were used as positive controls. After 48h of treatment, the cells were fixed with trichloroacetic acid (final concentration of 10%), washed, dried, stained with SRB dye, washed to remove excess dye, and dried. The SRB dye was dissolved and the absorbance was measured at a wavelength of 540nm using a Biotek automatic microplate reader and normalized to a value at a wavelength of 630 nm. Growth Inhibition (GI) of 50% was calculated from the absorbance data₅₀Or drug concentration that results in a 50% reduction in net protein increase).

Colchicine binding assay. Measured in 0.1mL of the reaction mixture of p2³H]Inhibition of binding of colchicine to tubulin, each mixture comprising 1.0. mu.M tubulin, 5.0. mu.M³H]Colchicine (Perkin-Elmer), 5% (v/v) dimethyl sulfoxide, 1.0 or 5.0. mu.M of the indicated compounds, and a component stabilizing the colchicine-binding activity of tubulin (1.0M monosodium glutamate [ adjusted to pH 6.6 with HCl in a 2.0M stock solution)]0.5mg/mL bovine serum albumin, 0.1M glucose-1-phosphate, 1.0mM MgCl₂And 1.0mM GTP). When the colchicine binding was 40-60% complete in the control reaction mixture, incubation was performed for 10min at 37 ℃. The reaction was stopped with 2.0mL of ice-cold water and the reaction mixture was placed on ice. Each diluted sample was poured into a stack (stack) of two DEAE cellulose filters (GE Biomedical) and then 3 consecutive times into 2mL aliquots of frozen water. Excess water was removed from the filter using reduced vacuum, washed 3 times with 2mL of water, and then placed into a vial containing 5mL of the Biosafe II scintillation mixture. After 18h, the samples were counted in a Beckman scintillation counter. Samples with inhibitor were compared to samples without inhibitor and percent inhibition was determined, correcting all values for the amount of radiolabel bound to the filter in the absence of tubulin.

Inhibition of tubulin polymerization. Tubulin polymerization was assessed in 0.25mL (final volume) of reaction mixture containing 1mg/mL (10. mu.M) of purified bovine brain tubulin, 0.8M monosodium glutamate (pH 6.6), 4% (v/v) dimethyl sulfoxide, 0.4mM GTP and different compound concentrations. In addition to the GTP, the activity of the GTP,all fractions were preincubated at 30 ℃ in 0.24mL for 15 min. The assay mixture was cooled to 0 ℃ and then 10. mu.L of 0.01M GTP was added to each sample. The reaction mixture was transferred to a cuvette stored at 0 ℃ in Beckman DU-7400 and DU-7500 spectrophotometers equipped with electronic temperature controllers. The temperature was raised to 30 ℃ in about 30s and the polymerization was carried out turbidimetrically at 350nm for 20 min. Will IC₅₀Defined as the concentration of compound with a degree of polymerization inhibition of 50% after 20 min.

Blood vessels in vivo are destroyed. As detailed previously, the human prostate cancer cell line (PC3) was modified by the removal of the tumor suppressor protein PC3-DAB2IP and was further modified by firefly luciferase reporter gene transfection (PC 3). Male Copenhagen rats (originally from Charles River, propagated in UT Southwestern) were inoculated subcutaneously at 5X 10 on the right thigh⁵Compared with 30 percentMixed PC3-DAB2IP-luc cells. On the day of implantation, these rats were about 6 weeks of age and were between 100 and 120g in weight. Tumors were allowed to grow to at least 1cm in diameter before treatment began. Three rats were treated with IP (intraperitoneal) administration of VDA prodrug 89 (25 mg/mL in physiological saline). Rat 1 received 10mg/kg, 40mg/kg after 24h, and IP administered CA4P (30mg/kg, 20mg/mL in physiological saline) 4 days later as a control. Rat 2 received a single dose of 40mg/kg of VDA prodrug 89 and rat 3 received 80mg/kg of 89. IVIS used before treatment (baseline) and 4 and 24h after treatment(Perkin-Elmer) for Bioluminescence imaging (BLI). Briefly, 120mg/kg of sodium D-fluorescein salt (Gold Biotechnology, st. louis, MO) was injected subcutaneously in the anterior and posterior neck regions (foreback wick) of anesthetized rats (inhaled oxygen with 3% isoflurane), and imaged over a period of about 30 min. Fresh luciferin was administered at each time point. Use of LivingThe software analyzes the resulting light intensity time curve and measures the luminescence of the target area encompassing the tumor. All Animal procedures were performed according to the guidelines for Care and Use of Laboratory Animals (Guide for the Care and Use of Laboratory Animals) and protocols approved by the Institutional Animal Care and Use Committee of the University of Texas (University of Texas south Medical Center) (APN 2017-102168), both issued and passed by the U.S. national Institutes of Health (U.S. institute of Health).

Each of the compounds in FIG. 2 was evaluated for cytotoxicity and ability to inhibit tubulin polymerization against human cancer cell lines [ SK-OV-3 (ovary), NCI-H460 (lung), DU-145 (prostate) ]. The results are shown in tables 1 and 2 below.

TABLE 1

^an.gtoreq.3 independently determined mean values (unless otherwise indicated)

b n average of 2 independent determinations (duplicate)

Not determined ND

NR ═ incoherent (paclitaxel enhanced microtubule assembly)

TABLE 2

^an.gtoreq.3 independently determined mean values (unless otherwise indicated)

Not determined ND

Ten of the molecules evaluated were identified as strong inhibitors of tubulin assembly (IC 50)<5 μ M, cell free assay),and seven of the ten molecules had high activity (IC50 ≦ 1.2. mu.M). CA4(IC 50. mu.M) and KGP18 (Compound 27, IC 50. mu.M) were used as comparative compounds. Three of the benzocycloheptene and dihydronaphthalene analogs (compounds 33, 39) and the other compound (88) are more potent on tubulin than our dihydronaphthalene lead compound KGP03(IC 50. ltoreq.0.5. mu.M). In which the phenolic moiety in the 4-position is changed to nitrile, ethyl, CH₂The OH groups (38, 39, 47, 48), and modifications of the double bonds on the seven-membered ring, including substitution with ketone and tertiary alcohol groups, substitution with bromine groups, and increased unsaturation (24, 33, 88), all retain excellent IC for inhibiting tubulin polymerization₅₀The value is obtained. Extension of the alkyl chain (at the 4-position) by ether linkage terminated with a polar alcohol group (compound 31) and a methoxy moiety (compound 28), respectively, results in loss of inhibitory activity. Incorporation of hydrogen bond donors at the allylic position on the seven-membered ring (compounds 57 and 69) and saturation of the double bond substitutions (compounds 23 and 35) both reduced inhibition of tubulin polymerization. The observed lack of tubulin activity of compound 76 was unexpected because the semi-rigidity of the seven-membered ring was maintained by removing the one-carbon double bond from the stereocenter and the parent benzocycloheptene analog (dimethoxy on the fused aryl ring) exhibited a moderate degree of tubulin polymerization inhibition as reported in our previous studies (IC50 ═ 3.1 μ M). The lack of activity of compound 77 in inhibiting tubulin polymerization indicates that the trimethoxy side aryl ring at the benzyl position of the fused ring system is closely related to biological efficacy, at least in inhibiting tubulin polymerization. This beta-substitution with trimethoxy aryl rings has not been previously investigated.

Of the benzocycloheptene and dihydronaphthalene analogs studied, the most cytotoxic agents were compounds 24, 33, 38, 39, 48, 88 (e.g., 0.0314, 0.0221, 0.0648, 0.0384, 0.0403 μ M, and 0.00690 μ M for GI50 against the SK-OV-3 ovarian cancer cell line, respectively). Judicious and judicious selection of structural modifications at positions 4,8 and 9 of the parent benzocycloheptene backbone accounts for the majority of the highly potent analogs evaluated in this study. Although the strong cytotoxicity of this subset of molecules was encouraging, it is noteworthy that, although exhibiting a similar inhibitory effect on tubulin polymerization (cell-free assay), all molecules demonstrated lower cytotoxicity than the lead benzocycloheptene KGP18 and lower cytotoxicity than the natural product CA4 (with the partial exception of compound 88). These observations provide an important extension to the known SAR considerations regarding structural modification of KGP 18. As expected (and similarly observed for combretastatin a-4 phosphate), benzocycloheptene phosphate prodrug salt 89 is inactive in this cell-free assay as an inhibitor of tubulin polymerization, presumably due to the lack of phosphatase required to cleave the prodrug into its parent phenolic (biologically active) agent. Prodrug 89 is an active cytotoxic agent because of the presence of non-specific phosphatase activity in these cancer cell-based cytotoxicity assays.

Example 3 evaluation of vascular Damage

Finally, VDAs will be used in vivo, it is therefore important to understand the efficacy of vascular destruction and potential off-target toxicity in vivo. As a preliminary study, the extent of vascular injury was assessed in human prostate tumor lines in rats treated with water-soluble prodrug salt 89, compared to CA4P as a control. A number of imaging methods have been developed to non-invasively assess vascular destruction in vivo. Dynamic bioluminescence imaging (BLI) is recommended for preliminary validation of VDA activity because it provides a fast, noninvasive and simple method and allows for comparative repeated or sequential studies. BLI does require the use of transfected cells to express luciferase (luc), but these cells are generally available and we have used this method as extensively as other methods. The human prostate cancer PC3 cell line was used, in which the tumor suppressor protein DAB2IP had been removed and luciferase was introduced. BLI requires the administration of a luciferin substrate that readily passes through the membrane and is carried throughout the vasculature. The measurement of the luminescence kinetics is related to the vascular delivery of the fluorescein substrate, and thus it provides a measure of the patency rate of the blood vessels. Disruption of tumor vasculature blocks substrate delivery, thus resulting in a quantifiable decrease in bioluminescent signal. The extent of IP vascular closure was assessed using 10 and 40 or 80mg/kg IP doses of 89 and compared to the 30mg/kg dose of CA4P, at which CA4P had previously been shown to cause extensive vascular closure in rats. It should also be noted that the lead benzocycloheptene KGP18 and dihydronaphthalene KGP03, both as their corresponding water-soluble phosphate prodrug salts (KGP 265 and KGP04, respectively), as well as other structurally modified benzocycloheptene analogs, all show vascular occlusion (as evidenced by similar BLI urban and rural studies or color Doppler ultrasound).

Fig. 12 shows BLI assessment of vascular response to VDA. On the left, the heatmap is overlaid on photographs of male copenhagen rats with subcutaneous PC3-DAB2IP-luc human prostate tumor xenografts that show luminescence relative to VDA IP administration approximately 20min after D-fluorescein (120mg/kg) administration at each time point. On the right, the corresponding dynamic luminescence curves were obtained about 30min after fluorescein administration, at baseline, about 4h after VDA and 24h after VDA. A shows the results for compound 89 at 10mg/kg, indicating no vascular perturbation, but an increase in signal at 24h, consistent with rapid growth of the tumor. B shows that after 6h, administration of 40mg/kg compound 89 to the same rat produced about a 95% reduction in signal at 4h, which is consistent with massive vascular closure, and showed substantial recovery at 24 h. C shows that four days later, administration of 30mg/kg CA4P to the rat elicited a BLI response similar to that shown in B.

The prodrug 89 administered at 10mg/kg caused the least change in luminescence. The subsequent 40mg/kg dose 89 resulted in a substantial reduction in luminescence (95% reduction in signal), but was substantially recovered at 24 h. When CA4P (30mg/kg) was administered 4 days later, it elicited very similar effects in terms of the degree and longevity of BLI signal attenuation as a surrogate for vascular closure. Similar activity was observed when administered to untreated rats 40mg/kg 89, with substantial recovery at 24 h.

FIG. 13 shows the relative luminescence after VDAs administration. A shows the relative signal intensity shown after approximately 20min of subcutaneous D-fluorescein administration in the anterior and posterior neck regions of rats having PC3-DAB2IP-luc prostate tumor xenografts in the thigh. The top is baseline (no previous drug), the middle is 40mg/kg 89 h later 4h, the bottom is 89 h later 24 h. B shows the corresponding luminescence dynamics at baseline, 4h after 89 and 24h after 69. C shows the normalized BLI signal at each time point for the rats in fig. 12 and from the untreated rats in A, B, the rats in fig. 12 received 10mg/kg, 40mg/kg69 and 30mg/kg CA4P in sequence, and the untreated rats in A, B received 40mg/kg 89 (x). At 48h, the rats appeared healthy and the signal increased slightly. The dose of 80mg/kg is also well tolerated, but this higher dose does not show additional vascular damage.

The results in these examples demonstrate the effect of structural modification of the lead benzocycloheptene and dihydronaphthalene analogs on tubulin polymerization inhibition and cytotoxicity against human cancer cell lines. In this new group of molecules [ and compound (88), obtained by separate synthesis]Several promising analogues (compounds 24, 33, 38, 39, 48, 88) appeared which caused inhibition of tubulin assembly (IC)₅₀) (cell-free assay) greater than or comparable to the natural leader CA4 and our leader benzocycloheptene analogs KGP18 and KGP 156. These compounds generally show potent cytotoxicity (GI) against SK-OV-3 (ovary), NCI-H460 (lung) and DU-145 (prostate) cells in the low to mid nM range₅₀). Preliminary in vivo studies of 40mg/kg water soluble benzocycloheptene phosphate prodrug salt 89 based on BLI showed vascular disruption in PC3-DAB2IP-luc human prostate tumor xenografts (as shown in FIGS. 12 and 13), which are similar to that obtained with CA 4P.

Reference to the literature

The following patents and publications are incorporated herein by reference.

Haichan Niu,et al.,Structure Guided Design,Synthesis,and Biological Evaluation of Novel Benzosuberene Analogues as Inhibitors of Tubulin Polymerization,J.Med.Chem.2019,2019,62,5594-5615.

(1)Salmon,B.A.；Siemann,D.W.Characterizing the Tumor Response to Treatment with Combretastatin A4 Phosphate.Int.J.Radiat.Oncol.Biol.Phys.2007,68(1),211–217.https://doi.org/10.1016/j.ijrobp.2006.12.051.

(2)Hanahan,D.；Weinberg,R.A.Hallmarks of Cancer:The Next Generation.Cell 2011,144(5),646–674.https://doi.org/10.1016/j.cell.2011.02.013.

(3)Siemann,D.W.The Unique Characteristics of Tumor Vasculature and Preclinical Evidence for Its Selective Disruption by Tumor-Vascular Disrupting Agents.Cancer Treat.Rev.2011,37(1),63–74.https://doi.org/10.1016/j.ctrv.2010.05.001.

(4)Tozer,G.M.；Kanthou,C.；Baguley,B.C.Disrupting Tumour Blood Vessels.Nat.Rev.Cancer 2005,5(6),423–435.https://doi.org/10.1038/nrc1628.

(5)Kanthou,C.；Tozer,G.M.Tumour Targeting by Microtubule-Depolymerizing Vascular Disrupting Agents.Expert Opin.Ther.Targets 2007,11(11),1443–1457.https://doi.org/10.1517/14728222.11.11.1443.

(6)Denekamp,J.Endothelial Cell Proliferation as a Novel Approach to Targeting Tumour Therapy.Br.J.Cancer 1982,45(1),136–139.

(7)Denekamp,J.Review Article:Angiogenesis,Neovascular Proliferation and Vascular Pathophysiology as Targets for Cancer Therapy.Br.J.Radiol.1993,66(783),181–196.https://doi.org/10.1259/0007-1285-66-783-181.

(8)Sriram,M.；Hall,J.J.；Grohmann,N.C.；Strecker,T.E.；Wootton,T.；Franken,A.；Trawick,M.L.；Pinney,K.G.Design,Synthesis and Biological Evaluation of Dihydronaphthalene and Benzosuberene Analogs of the Combretastatins as Inhibitors of Tubulin Polymerization in Cancer Chemotherapy.Bioorg.Med.Chem.2008,16(17),8161–8171.https://doi.org/10.1016/j.bmc.2008.07.050.

(9)Horsman,M.R.；Bohn,A.B.；Busk,M.Vascular Targeting Therapy:Potential Benefit Depends on Tumor and Host Related Effects.Exp.Oncol.2010,32(3),143–148.

(10)Siemann,D.W.；Bibby,M.C.；Dark,G.G.；Dicker,A.P.；Eskens,F.A.L.M.；Horsman,M.R.；Marmé,D.；LoRusso,P.M.Differentiation and Definition of Vascular-Targeted Therapies.Clin.Cancer Res.2005,11(2 I),416–420.

(11)Mason,R.P.；Zhao,D.；Liu,L.；Trawick,M.L.；Pinney,K.G.A Perspective on Vascular Disrupting Agents That Interact with Tubulin:Preclinical Tumor Imaging and Biological Assessment.Integr.Biol.2011,3(4),375–387.https://doi.org/10.1039/C0IB00135J.

(12)Dougherty,G.J.；Chaplin,D.J.Development of Vascular Disrupting Agents.In Vascular Disruptive Agents for the Treatment of Cancer；Springer,New York,NY,2010；pp 1–27.https://doi.org/10.1007/978-1-4419-6609-4_1.

(13)Siemann,D.W.Tumor Vasculature:A Target for Anticancer Therapies.In Vascular-Targeted Therapies in Oncology；Siemann,D.W.,Ed.；Chichester,2006；pp 1-8.

(14)Monk,K.A.；Siles,R.；Hadimani,M.B.；Mugabe,B.E.；Ackley,J.F.；Studerus,S.W.；Edvardsen,K.；Trawick,M.L.；Garner,C.M.；Rhodes,M.R.；Pettit,G.R.；Pinney,K.G.Design,Synthesis,and Biological Evaluation of Combretastatin Nitrogen-Containing Derivatives as Inhibitors of Tubulin Assembly and Vascular Disrupting Agents.Bioorg.Med.Chem.2006,14(9),3231–3244.https://doi.org/10.1016/j.bmc.2005.12.033.

(15)Pettit,G.R.；Singh,S.B.；Boyd,M.R.；Hamel,E.；Pettit,R.K.；Schmidt,J.M.；Hogan,F.Antineoplastic Agents.291.Isolation and Synthesis of Combretastatins A-4,A-5,and A-6.J.Med.Chem.1995,38(10),1666–1672.https://doi.org/10.1021/jm00010a011.

(16)McGown,A.T.；Fox,B.W.Differential Cytotoxicity of Combretastatins A1 and A4 in Two Daunorubicin-Resistant P388 Cell Lines.Cancer Chemother.Pharmacol.1990,26(1),79–81.

(17)Pettit,G.R.；Moser,B.R.；Boyd,M.R.；Schmidt,J.M.；Pettit,R.K.；Chapuis,J.-C.Antineoplastic Agents 460.Synthesis of Combretastatin A-2 Prodrugs.Anticancer.Drug Des.2001,16(4–5),185–193.

(18)Pettit,G.R.；Singh,S.B.；Hamel,E.；Lin,C.M.；Alberts,D.S.；Garcia-Kendal,D.Isolation and Structure of the Strong Cell Growth and Tubulin Inhibitor Combretastatin A-4.Experientia 1989,45(2),209–211.https://doi.org/10.1007/BF01954881.

(19)Dark,G.G.；Hill,S.A.；Prise,V.E.；Tozer,G.M.；Pettit,G.R.；Chaplin,D.J.Combretastatin A-4,an Agent That Displays Potent and Selective Toxicity Toward Tumor Vasculature.Cancer Res.1997,57(10),1829–1834.

(20)Pettit,G.R.；Toki,B.；Herald,D.L.；Verdier-Pinard,P.；Boyd,M.R.；Hamel,E.；Pettit,R.K.Antineoplastic Agents.379.Synthesis of Phenstatin Phosphate.J.Med.Chem.1998,41(10),1688–1695.https://doi.org/10.1021/jm970644q.

(21)Boyland,E.；Boyland,M.E.Studies in Tissue Metabolism:The Action of Colchicine and B.Typhosus Extract.Biochem.J.1937,31(3),454–460.

(22)Woods,J.A.；Hadfield,J.A.；Pettit,G.R.；Fox,B.W.；McGown,A.T.The Interaction with Tubulin of a Series of Stilbenes Based on Combretastatin A-4.Br.J.Cancer 1995,71(4),705–711.

(23)Pettit,G.R.；Singh,S.B.；Niven,M.L.；Hamel,E.；Schmidt,J.M.Isolation,Structure,and Synthesis of Combretastatins A-1 and B-1,Potent New Inhibitors of Microtubule Assembly,Derived from Combretum Caffrum.J.Nat.Prod.1987,50(1),119–131.https://doi.org/10.1021/np50049a016.

(24)Pettit,G.R.；Grealish,M.P.；Herald,D.L.；Boyd,M.R.；Hamel,E.；Pettit,R.K.Antineoplastic Agents.443.Synthesis of the Cancer Cell Growth Inhibitor Hydroxyphenstatin and Its Sodium Diphosphate Prodrug.J.Med.Chem.2000,43(14),2731–2737.https://doi.org/10.1021/jm000045a.

(25)Herdman,C.A.；Devkota,L.；Lin,C.-M.；Niu,H.；Strecker,T.E.；Lopez,R.；Liu,L.；George,C.S.；Tanpure,R.P.；Hamel,E.；Chaplin,D.J.；Mason,R.P.；Trawick,M.L.；Pinney,K.G.Structural Interrogation of Benzosuberene-Based Inhibitors of Tubulin Polymerization.Bioorg.Med.Chem.2015,23(24),7497–7520.https://doi.org/10.1016/j.bmc.2015.10.012.

(26)Tanpure,R.P.；Harkrider,A.R.；Strecker,T.E.；Hamel,E.；Trawick,M.L.；Pinney,K.G.Application of the McMurry Coupling Reaction in the Synthesis of Tri-and Tetra-Arylethylene Analogues as Potential Cancer Chemotherapeutic Agents.Bioorg.Med.Chem.2009,17(19),6993–7001.https://doi.org/10.1016/j.bmc.2009.08.011.

(27)Pinney,K.G.；Pettit,G.R.；Trawick,M.L.；Jelinek,C.；Chaplin,D.J.The Discovery and Development of the Combretastatins.Anticancer Agents Nat.Prod.2012,27–63.

(28)Pinney,K.G.Molecular Recognition of the Colchicine Binding Site as a Design Paradigm for the Discovery and Development of Vascular Disrupting Agents.In Vascular-Targeted Therapies in Oncology；Siemann,D.W.,Ed.；Chichester,2006；pp 95-121.

(29)Siles,R.；Ackley,J.F.；Hadimani,M.B.；Hall,J.J.；Mugabe,B.E.；Guddneppanavar,R.；Monk,K.A.；Chapuis,J.-C.；Pettit,G.R.；Chaplin,D.J.；Edvardsen,K.；Trawick,M.L.；Garner,G.M.；Pinney,K.G.Combretastatin Dinitrogen-Substituted Stilbene Analogues as Tubulin-Binding and Vascular-Disrupting Agents.J.Nat.Prod.2008,71(3),313–320.https://doi.org/10.1021/np070377j.

(30)Shirali,A.；Sriram,M.；Hall,J.J.；Nguyen,B.L.；Guddneppanavar,R.；Hadimani,M.B.；Ackley,J.F.；Siles,R.；Jelinek,C.J.；Arthasery,P.；Brown,R.C.；Murrell,V.L.；McMordie,A.；Sharma,S.；Chaplin,D.J.；Pinney,K.G.Development of Synthetic Methodology Suitable for the Radiosynthesis of Combretastatin A-1(CA1)and Its Corresponding Prodrug CA1P.J.Nat.Prod.2009,72(3),414–421.https://doi.org/10.1021/np800661r.

(31)Chaplin,D.J.；III,C.M.G.；Kane,R.R.；Pinney,K.G.；Prezioso,J.A.；Edvardsen,K.Functionalized Stilbene Derivatives as Improved Vascular Targeting Agents.US7384925B2,June 10,2008.

(32)Chen,Z.；Mocharla,V.P.；Farmer,J.M.；Pettit,G.R.；Hamel,E.；Pinney,K.G.Preparation of New Anti-Tubulin Ligands Through a Dual-Mode,Addition-Elimination Reaction to a Bromo-Substitutedα,β-Unsaturated Sulfoxide.J.Org.Chem.2000,65(25),8811–8815.https://doi.org/10.1021/jo0004761.

(33)Pinney,K.G.；Bounds,A.D.；Dingeman,K.M.；Mocharla,V.P.；Pettit,G.R.；Bai,R.；Hamel,E.A New Anti-Tubulin Agent Containing the Benzo[b]Thiophene Ring System.Bioorg.Med.Chem.Lett.1999,9(8),1081–1086.https://doi.org/10.1016/S0960-894X(99)00143-2.

(34)Pinney,K.；Pettit,G.；Mocharla,V.；Mejia,M.del P.；Shirali,A.Anti-Mitotic Agents Which Inhibit Tubulin Polymerization.WO/1998/039323,September 12,1998.

(35)Pinney,K.；Sriram,M.；George,C.；Tanpure,R.Efficient Method for Preparing Functionalized Benzosuberenes.WO/2012/068284,May 25,2012.

(36)Pinney,K.；Mocharla,V.；Chen,Z.；Garner,C.；Ghatak,A.；Hadimani,M.；Kessler,J.；Dorsey,J.；Edvardsen,K.；Chaplin,D.；Prezioso,J.；Ghatak,U.Tubulin Binding Agents and Corresponding Prodrug Constructs.US20040043969A1,March 4,2004.

(37)Tanpure,R.P.；George,C.S.；Strecker,T.E.；Devkota,L.；Tidmore,J.K.；Lin,C.-M.；Herdman,C.A.；MacDonough,M.T.；Sriram,M.；Chaplin,D.J.；Trawick,M.L.；Pinney,K.G.Synthesis of Structurally Diverse Benzosuberene Analogues and Their Biological Evaluation as Anti-Cancer Agents.Bioorg.Med.Chem.2013,21(24),8019–8032.https://doi.org/10.1016/j.bmc.2013.08.035.

(38)Tanpure,R.P.；George,C.S.；Sriram,M.；Strecker,T.E.；Tidmore,J.K.；Hamel,E.；Charlton-Sevcik,A.K.；Chaplin,D.J.；Trawick,M.L.；Pinney,K.G.An Amino-Benzosuberene Analogue That Inhibits Tubulin Assembly and Demonstrates Remarkable Cytotoxicity.MedChemComm 2012,3(6),720–724.https://doi.org/10.1039/C2MD00318J.

(39)Hadimani,M.B.；MacDonough,M.T.；Ghatak,A.；Strecker,T.E.；Lopez,R.；Sriram,M.；Nguyen,B.L.；Hall,J.J.；Kessler,R.J.；Shirali,A.R.；Liu,L.；Garner,C.M.；Pettit,G.R.；Hamel,E.；Chaplin,D.J.；Mason,R.P.；Trawick,M.L.；Pinney,K.G.Synthesis of a 2-Aryl-3-Aroyl Indole Salt(OXi8007)Resembling Combretastatin A-4 with Application as a Vascular Disrupting Agent.J.Nat.Prod.2013,76(9),1668–1678.https://doi.org/10.1021/np400374w.

(40)Hamel,E.Antimitotic Natural Products and Their Interactions with Tubulin.Med.Res.Rev.1996,16(2),207–231.https://doi.org/10.1002/(SICI)1098-1128(199603)16:2<207::AID-MED4>3.0.CO；2-4.

(41)Wu,X.；Wang,Q.；Li,W.Recent Advances in Heterocyclic Tubulin Inhibitors Targeting the Colchicine Binding Site.Anticancer Agents Med.Chem.2016,16(10),1325-1338.https://doi.org/10.2174/1871520616666160219161921.

(42)Lee,R.M.；Gewirtz,D.A.Colchicine Site Inhibitors of Microtubule Integrity as Vascular Disrupting Agents.Drug Dev.Res.2008,69(6),352–358.https://doi.org/10.1002/ddr.20267.

(43)Gigant,B.；Cormier,A.；Dorléans,A.；Ravelli,R.B.G.；Knossow,M.Microtubule-Destabilizing Agents:Structural and Mechanistic Insights from the Interaction of Colchicine and Vinblastine with Tubulin.In Tubulin-Binding Agents:Synthetic,Structural and Mechanistic Insights；Carlomagno,T.,Ed.；Topics in Current Chemistry；Springer Berlin Heidelberg:Berlin,Heidelberg,2009；pp 259–278.https://doi.org/10.1007/128_2008_11.

(44)Sackett,D.L.Podophyllotoxin,Steganacin and Combretastatin:Natural Products That Bind at the Colchicine Site of Tubulin.Pharmacol.Ther.1993,59(2),163–228.https://doi.org/10.1016/0163-7258(93)90044-E.

(45)Wang,Y.；Zhang,H.；Gigant,B.；Yu,Y.；Wu,Y.；Chen,X.；Lai,Q.；Yang,Z.；Chen,Q.；Yang,J.Structures of a Diverse Set of Colchicine Binding Site Inhibitors in Complex with Tubulin Provide a Rationale for Drug Discovery.FEBS J.2016,283(1),102–111.https://doi.org/10.1111/febs.13555.

(46)Ji,Y.-T.；Liu,Y.-N.；Liu,Z.-P.Tubulin Colchicine Binding Site Inhibitors as Vascular Disrupting Agents in Clinical Developments.Curr.Med.Chem.2015,22(11),1348-1360.https://doi.org/10.2174/0929867322666150114163732.

(47)Nguyen,T.L.；McGrath,C.；Hermone,A.R.；Burnett,J.C.；Zaharevitz,D.W.；Day,B.W.；Wipf,P.；Hamel,E.；Gussio,R.A Common Pharmacophore for a Diverse Set of Colchicine Site Inhibitors Using a Structure-Based Approach.J.Med.Chem.2005,48(19),6107–6116.https://doi.org/10.1021/jm050502t.

(48)Chen,J.；Liu,T.；Dong,X.；Hu,Y.Recent Development and SAR Analysis of Colchicine Binding Site Inhibitors.Mini.Rev.Med.Chem.2009,9(10),1174-1190.https://doi.org/info:doi/10.2174/138955709789055234.

(49)Lu,Y.；Chen,J.；Xiao,M.；Li,W.；Miller,D.D.An Overview of Tubulin Inhibitors That Interact with the Colchicine Binding Site.Pharm.Res.2012,29(11),2943–2971.https://doi.org/10.1007/s11095-012-0828-z.

(50)Macdonough,M.T.；Strecker,T.E.；Hamel,E.；Hall,J.J.；Chaplin,D.J.；Trawick,M.L.；Pinney,K.G.Synthesis and Biological Evaluation of Indole-Based,Anti-Cancer Agents Inspired by the Vascular Disrupting Agent 2-(3’-Hydroxy-4’-Methoxyphenyl)-3-(3″,4″,5″-Trimethoxybenzoyl)-6-Methoxyindol e(OXi8006).Bioorg.Med.Chem.2013,21(21),6831–6843.https://doi.org/10.1016/j.bmc.2013.07.028.

(51)Flynn,B.L.；Gill,G.S.；Grobelny,D.W.；Chaplin,J.H.；Paul,D.；Leske,A.F.；Lavranos,T.C.；Chalmers,D.K.；Charman,S.A.；Kostewicz,E.；Shackleford,D.M.；Morizzi,J.；Hamel,E.；Jung,M.K.；Kremmidiotis,G.Discovery of 7-Hydroxy-6-Methoxy-2-Methyl-3-(3,4,5-Trimethoxybenzoyl)Benzo[b]Furan(BNC105),a Tubulin Polymerization Inhibitor with Potent Antiproliferative and Tumor Vascular Disrupting Properties.J.Med.Chem.2011,54(17),6014–6027.https://doi.org/10.1021/jm200454y.

(52)Kuo,C.-C.；Hsieh,H.-P.；Pan,W.-Y.；Chen,C.-P.；Liou,J.-P.；Lee,S.-J.；Chang,Y.-L.；Chen,L.-T.；Chen,C.-T.；Chang,J.-Y.BPR0L075,a Novel Synthetic Indole Compound with Antimitotic Activity in Human Cancer Cells,Exerts Effective Antitumoral Activity in Vivo.Cancer Res.2004,64(13),4621–4628.https://doi.org/10.1158/0008-5472.CAN-03-3474.

(53)Liu,L.；Beck,H.；Wang,X.；Hsieh,H.-P.；Mason,R.P.；Liu,X.Tubulin-Destabilizing Agent BPR0L075 Induces Vascular-Disruption in Human Breast Cancer Mammary Fat Pad Xenografts.PLOS ONE 2012,7(8),e43314.https://doi.org/10.1371/journal.pone.0043314.

(54)Rasolofonjatovo,E.；Provot,O.；Hamze,A.；Rodrigo,J.；Bignon,J.；Wdzieczak-Bakala,J.；Desravines,D.；Dubois,J.；Brion,J.-D.；Alami,M.Conformationally Restricted Naphthalene Derivatives Type Isocombretastatin A-4 and Isoerianin Analogues:Synthesis,Cytotoxicity and Antitubulin Activity.Eur.J.Med.Chem.2012,52,22–32.https://doi.org/10.1016/j.ejmech.2012.03.001.

(55)Rasolofonjatovo,E.；Provot,O.；Hamze,A.；Rodrigo,J.；Bignon,J.；Wdzieczak-Bakala,J.；Lenoir,C.；Desravines,D.；Dubois,J.；Brion,J.-D.；Alami,M.Design,Synthesis and Anticancer Properties of 5-Arylbenzoxepins as Conformationally Restricted Isocombretastatin A-4 Analogs.Eur.J.Med.Chem.2013,62,28–39.https://doi.org/10.1016/j.ejmech.2012.12.042.

(56)Chen,Z.；O’Donnell,C.J.；Maderna,A.Synthesis of 3-Methoxy-9-(3,4,5-Trimethoxyphenyl)-6,7-Dihydro-5H-Benzo[7]Annulen-4-Ol,a Potent Antineoplastic Benzosuberene Derivative for Anti-Cancer Chemotherapy.Tetrahedron Lett.2012,53(1),64–66.https://doi.org/10.1016/j.tetlet.2011.10.145.

(57)Chen,Z.；Maderna,A.；Sukuru,S.C.K.；Wagenaar,M.；O’Donnell,C.J.；Lam,M.-H.；Musto,S.；Loganzo,F.New Cytotoxic Benzosuberene Analogs.Synthesis,Molecular Modeling and Biological Evaluation.Bioorg.Med.Chem.Lett.2013,23(24),6688–6694.https://doi.org/10.1016/j.bmcl.2013.10.039.

(58)Galli,U.；Travelli,C.；Aprile,S.；Arrigoni,E.；Torretta,S.；Grosa,G.；Massarotti,A.；Sorba,G.；Canonico,P.L.；Genazzani,A.A.；Tron,G.C.Design,Synthesis,and Biological Evaluation of Combretabenzodiazepines:A Novel Class of Anti-Tubulin Agents.J.Med.Chem.2015,58(3),1345–1357.https://doi.org/10.1021/jm5016389.

(59)Prileschajew Nikolaus.OxydationVerbindungen Mittels Organischer Superoxyde.Berichte Dtsch.Chem.Ges.1909,42(4),4811–4815.https://doi.org/10.1002/cber.190904204100.

(60)VanRheenen,V.；Kelly,R.C.；Cha,D.Y.An Improved Catalytic OsO4 Oxidation of Olefins to Cis-1,2-Glycols Using Tertiary Amine Oxides as the Oxidant.Tetrahedron Lett.1976,17(23),1973–1976.https://doi.org/10.1016/S0040-4039(00)78093-2.

(61)Gustowski,D.A.；Delgado,M.；Gatto,V.J.；Echegoyen,L.；Gokel,G.W.Electrochemical Switching in Anthraquinone-Substituted Carbon-Pivot Lariat Ethers and Podands:Chain Length Effects in Geometric and Electronic Cooperativity.J.Am.Chem.Soc.1986,108(24),7553–7560.https://doi.org/10.1021/ja00284a019.

(62)Sandmeyer Traugott.Ueber Die Ersetzung Der Amidgruppe Durch Chlor in Den Aromatischen Substanzen.Berichte Dtsch.Chem.Ges.2006,17(2),1633–1635.https://doi.org/10.1002/cber.18840170219.

(63)Sandmeyer Traugott.Ueber Die Ersetzung Der Amid-gruppe Durch Chlor,Brom Und Cyan in Den Aromatischen Substanzen.Berichte Dtsch.Chem.Ges.2006,17(2),2650–2653.https://doi.org/10.1002/cber.188401702202.

(64)Mori,A.；Mizusaki,T.；Miyakawa,Y.；Ohashi,E.；Haga,T.；Maegawa,T.；Monguchi,Y.；Sajiki,H.Chemoselective Hydrogenation Method Catalyzed by Pd/C Using Diphenylsulfide as a Reasonable Catalyst Poison.Tetrahedron 2006,62(51),11925–11932.https://doi.org/10.1016/j.tet.2006.09.094.

(65)E.Eaton,P.；R.Carlson,G.；T.Lee,J.Phosphorus Pentoxide-Methanesulfonic Acid.Convenient Alternative to Polyphosphoric Acid.J.Org.Chem.1973,38,4071-4073.https://doi.org/10.1021/jo00987a028.

(66)Tanis,V.M.；Moya,C.；Jacobs,R.S.；Little,R.D.Synthesis and Evaluation of the Bioactivity of Simplified Analogs of the Seco-Pseudopterosins；Progress Toward Determining a Pharmacophore.Tetrahedron 2008,64(47),10649–10663.https://doi.org/10.1016/j.tet.2008.09.025.

(67)Lin,W.；Wang,Q.；Xiao,Y.；He,H.；Zheng；Chen,X.；Chen,S.；WuXi AppTec.Fast Synthetic Route for 3-Aryl Substituted Propanoic Acid.China,CN 101747171 A,December 17,2008.

(68)Benner Andre；Bonifazi Alessandro；Shirataki Chikako；Temme Louisa；Schepmann Dirk；Quaglia Wilma；Shoji Osami；Watanabe Yoshihito；Daniliuc Constantin；Wünsch Bernhard.GluN2B-Selective N-Methyl-d-aspartate(NMDA)Receptor Antagonists Derived from 3-Benzazepines:Synthesis and Pharmacological Evaluation of Benzo[7]Annulen-7-amines.ChemMedChem 2014,9(4),741–751.https://doi.org/10.1002/cmdc.201300547.

(69)Walsh,J.J.；Sha,R.；McCormack,E.M.；Hudson,G.J.；White,M.；Stack,G.D.；Moran,B.W.；Coogan,A.；Breen,E.C.Tubulin Binding Agents.US20150018566A1,August27,2012.

(70)Gilman,H.；Jones,R.G.；Woods,L.A.The Preparation of Methylcopper and Some Observations on the Decomposition of Organocopper Compounds.J.Org.Chem.1952,17(12),1630–1634.https://doi.org/10.1021/jo50012a009.

(71)Hua,J.；Sheng,Y.；Pinney,K.G.；Garner,C.M.；Kane,R.R.；Prezioso,J.A.；Pettit,G.R.；Chaplin,D.J.；Edvardsen,K.Oxi4503,a Novel Vascular Targeting Agent:Effects on Blood Flow and Antitumor Activity in Comparison to Combretastatin A-4Phosphate.Anticancer Res.2003,23(2B),1433–1440.

(72)Benham,F.J.；Fogh,J.；Harris,H.Alkaline Phosphatase Expression in Human Cell Lines Derived from Various Malignancies.Int.J.Cancer 1981,27(5),637–644.

(73)Rao,S.R.；Snaith,A.E.；Marino,D.；Cheng,X.；Lwin,S.T.；Orriss,I.R.；Hamdy,F.C.；Edwards,C.M.Tumour-Derived Alkaline Phosphatase Regulates Tumour Growth,Epithelial Plasticity and Disease-Free Survival in Metastatic Prostate Cancer.Br.J.Cancer 2017,116(2),227–236.https://doi.org/10.1038/bjc.2016.402.

(74)Gangjee,A.；Zhao,Y.；Lin,L.；Raghavan,S.；Roberts,E.G.；Risinger,A.L.；Hamel,E.；Mooberry,S.L.Synthesis and Discovery of Water Soluble Microtubule Targeting Agents That Bind to the Colchicine Site on Tubulin and Circumvent Pgp Mediated Resistance.J.Med.Chem.2010,53(22),8116–8128.https://doi.org/10.1021/jm101010n.

(75)Maguire,C.J.；Chen,Z.；Mocharla,V.P.；Sriram,M.；Strecker,T.E.；Hamel,E.；Zhou,H.；Lopez,R.；Wang,Y.；Mason,R.P.；Chaplin,D.J.；Trawick,M.L.；Pinney,K.G.Synthesis of Dihydronaphthalene Analogues Inspired by Combretastatin A-4 and Their Biological Evaluation as Anticancer Agents.MedChemComm.2018,9(10),1649-1662.https://doi.org/10.1039/C8MD00322J.

(76)Folaron,M.；Seshadri,M.Bioluminescence and MR Imaging of the Safety and Efficacy of Vascular Disruption in Gliomas.Mol.Imaging Biol.MIB Off.Publ.Acad.Mol.Imaging 2016,18(6),860–869.https://doi.org/10.1007/s11307-016-0963-8.

(77)Tumati,V.；Mathur,S.；Song,K.；Hsieh,J.-T.；Zhao,D.；Takahashi,M.；Dobin,T.；Gandee,L.；Solberg,T.D.；Habib,A.A.；Saha,D.Development of a Locally Advanced Orthotopic Prostate Tumor Model in Rats for Assessment of Combined Modality Therapy.Int.J.Oncol.2013,42(5),1613–1619.https://doi.org/10.3892/ijo.2013.1858.

(78)Zhao,D.；Richer,E.；Antich,P.P.；Mason,R.P.Antivascular Effects of Combretastatin A4 Phosphate in Breast Cancer Xenograft Assessed Using Dynamic Bioluminescence Imaging and Confirmed by MRI.FASEB J.Off.Publ.Fed.Am.Soc.Exp.Biol.2008,22(7),2445–2451.https://doi.org/10.1096/fj.07-103713.

(79)Zhao,D.；Jiang,L.；Hahn,E.W.；Mason,R.P.Tumor Physiologic Response to Combretastatin A4 Phosphate Assessed by MRI.Int.J.Radiat.Oncol.Biol.Phys.2005,62(3),872–880.https://doi.org/10.1016/j.ijrobp.2005.03.009.

(80)Zhao,D.；Chang,C.-H.；Kim,J.G.；Liu,H.；Mason,R.P.In Vivo Near-Infrared Spectroscopy and Magnetic Resonance Imaging Monitoring of Tumor Response to Combretastatin A-4-Phosphate Correlated with Therapeutic Outcome.Int.J.Radiat.Oncol.Biol.Phys.2011,80(2),574–581.https://doi.org/10.1016/j.ijrobp.2010.12.028.

(81)Galbraith,S.M.；Maxwell,R.J.；Lodge,M.A.；Tozer,G.M.；Wilson,J.；Taylor,N.J.；Stirling,J.J.；Sena,L.；Padhani,A.R.；Rustin,G.J.S.Combretastatin A4 Phosphate Has Tumor Antivascular Activity in Rat and Man as Demonstrated by Dynamic Magnetic Resonance Imaging.J.Clin.Oncol.Off.J.Am.Soc.Clin.Oncol.2003,21(15),2831–2842.https://doi.org/10.1200/JCO.2003.05.187.

(82)Vichai,V.；Kirtikara,K.Sulforhodamine B Colorimetric Assay for Cytotoxicity Screening.Nat.Protoc.2006,1(3),1112–1116.https://doi.org/10.1038/nprot.2006.179.

(83)Monks,A.；Scudiero,D.；Skehan,P.；Shoemaker,R.；Paull,K.；Vistica,D.；Hose,C.；Langley,J.；Cronise,P.；Vaigro-Wolff,A.；Gray-Goodrich,M.；Campbell,H.；Mayo,J.；Boyd,M.Feasibility of a High-Flux Anticancer Drug Screen Using a Diverse Panel of Cultured Human Tumor Cell Lines.JNCI J.Natl.Cancer Inst.1991,83(11),757–766.https://doi.org/10.1093/jnci/83.11.757.

(84)Hamel,E.；Lin,C.M.Stabilization of the Colchicine-Binding Activity of Tubulin by Organic Acids.Biochim.Biophys.Acta BBA-Gen.Subj.1981,675(2),226–231.https://doi.org/10.1016/0304-4165(81)90231-2.

(85)Hamel,E.Evaluation of Antimitotic Agents by Quantitative Comparisons of Their Effects on the Polymerization of Purified Tubulin.Cell Biochem.Biophys.2003,38(1),1–22.https://doi.org/10.1385/CBB:38:1:1.