Accepted Manuscript

Recent advances in microtubule-stabilizing agents

Ya-Nan Cao, Ling-Li Zheng, Dan Wang, Xiao-Xia Liang, Feng Gao, Xian-Li Zhou

PII: S0223-5234(17)30965-0 DOI: 10.1016/j.ejmech.2017.11.062 Reference: EJMECH 9937

To appear in: European Journal of Medicinal Chemistry

Received Date: 23 August 2017 Revised Date: 4 November 2017 Accepted Date: 22 November 2017

Please cite this article as: Y.-N. Cao, L.-L. Zheng, D. Wang, X.-X. Liang, F. Gao, X.-L. Zhou, Recent advances in microtubule-stabilizing agents, European Journal of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.11.062.

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. ACCEPTED MANUSCRIPT Graphic Abstract

MANUSCRIPT

ACCEPTED ACCEPTED MANUSCRIPT

Recent advances in Microtubule-stabilizing Agents

Ya-Nan Cao a,b , Ling-Li Zheng c, Dan Wang d,

Xiao-Xia Liang a,* Feng Gao a,b,* and Xian-Li Zhou b a Agronomy College, Sichuan Agriculture University, Chengdu 611130, P. R. China b School of Life Science and Engineering, Southwest Jiaotong University, Chengdu 610031, P.R. China c Department of Pharmacy, The First Affiliated Hospital of Chengdu Medical College, Chengdu 610500, P.R. China d Division of Chemistry and Structural Biology, Institute for Molecular Bioscience, the University of Queensland, Brisbane Qld 4072, Australia

* Corresponding Author Email address: [email protected] (F. Gao); [email protected] (X.-X. Liang) MANUSCRIPT

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Abstract: Highly dynamic mitotic spindle microtubules are superb therapeutic targets for a group of chemically diverse and clinically successful anticancer drugs. Microtubule-targeted drugs disrupt microtubule dynamics in distinct ways, and they are primarily classified into two groups: microtubule destabilizing agents (MDAs), such as vinblastine, colchicine, and combretastatin-A4, and microtubule stabilizing agents (MSAs), such as paclitaxel and epothilones. Systematic discovery and development of new MSAs have been aided by extensive research on paclitaxel, yielding a large number of promising anticancer compounds. This review focuses on the natural sources, structural features, mechanisms of action, structure-activity relationship (SAR) and chemical synthesis of MSAs. These MSAs mainly include paclitaxel, taccalonolides, epothilones, FR182877 (cyclostreptin), dictyostatin, discodermolide, eleutherobin and sarcodictyins, zampanolide, dactylolide, laulimalides, peloruside and ceratamines from natural sources, as well as small molecular microtubule stabilizers obtained via chemical synthesis. Then we discuss the application prospect and development of these anticancer compounds.

Keywords: Tubulin; Microtubule-stabilizing MANUSCRIPT agents; Chemotherapy; Paclitaxel; Taccalonolide; Epothilone; Structure-Activity Relationship

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1. Introduction

Microtubules (MTs) are hollow cylindrical tubes consisting of 13 aligned

protofilaments formed from repeating α- and β-tubulin heterodimers [1] . The protofilaments play a central role in pulling apart chromosomes in mitotic cell division. Cancer is a collection of related diseases involving abnormal cell growth with the potential to invade to other parts of the body. The common goal of cancer chemotherapy is to induce the apoptosis of cancer cells. Based on the behavior of tubulins in the cell mitosis, more and more drugs that interact with tubulins have been approved for clinical cancer treatment [2] . MTs have dynamical features of polymerization and de-polymerization in cell division, and they can be easily affected by factors such as low temperature, Ca 2+, and drugs, etc. Anticancer compounds that directly interact with MTs are classified into two main classes: The former, which are represented by vinblastine and colchicine, affect microtubule assembly by preventing tubulin polymerization, whereas, the latter,

like paclitaxel and epothilones, promote tubulin polymerization. The former is called microtubule destabilizing agents (MDAs), andMANUSCRIPT the latter is microtubule stabilizing agents (MSAs). This review mainly discusses MSAs. MSAs are antimitotic compounds that can promote the tubulin polymerization. As known so far, the mechanism of action thereof is that MSAs bind to tubulin firstly,

then block mitosis in the G 2/M phase in cell cycle by disturbing microtubule dynamics, and induce programmed cell apoptosis eventually [3] . In recent years, the successful development and application of MSAs have made a great contribution in clinical cancer treatment. Meanwhile, with the in-depth research, Swiss scientists have interpreted the crystal structure of those complex compounds formed from zampanolide and epothilone A with tubulin respectively by using X-ray ACCEPTEDdiffraction technique. Their work has also illuminated the mechanism of action of these compounds on tubulin, which provides a new basis for further drug

design guided by corresponding structures [4] . As we all know, MSAs mostly derive from natural products or their structure-modified derivatives whose sources are widely ranged from marine organism to terrestrial organism, or from algae to mammal. Besides, there are some

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ACCEPTED MANUSCRIPT small molecular compounds came from chemical synthesis that have similar anticancer effects to MSAs. In this review, we introduce the discovery, application and chemical synthesis of the natural MSAs originated from plants, microorganisms, and marine organisms, as well as small molecular compounds prepared by chemical synthesis, and finally make an outlook on the future applications of MSAs with anticancer effects. 2. Natural microtubule stabilizing agents

Natural MSAs refer to those compounds generated from natural products and derivatives thereof with the function of stabling the tubulin polymers and preventing microtubule de-polymerization. These compounds are generally direct extracts isolated from natural products or structure-modified derivatives thereof. But these natural MSAs are usually with low contents, and their structures are generally complex, such as diterpenes, steroids, and macrocyclic lactones, etc. The most commonly used classification criterion of these natural MSAs is according to their natural sources, which we employ herein. We classify them into three groups: obtained from plants, microorganisms, and marineMANUSCRIPT organisms, respectively.

2.1. Plants sources

2.1.1. Paclitaxel (Taxol®) Wall ′s laboratory first reported this compound isolated from the stem bark of the western yew, Taxus brevifolia Nutt , named it paclitaxel (1), and identified its diterpene

structure with a tetracyclic 17 carbon frame and 11 stereocenters [5] . In bioassay, paclitaxel shows extensive antitumor activity, especially in the treatment of ovarian cancer, breast cancer and non-small cell lung cancer, which makes it a potential broad-spectrum anticancer drug. Schiff et ACCEPTED al. [6] discovered that paclitaxel can completely inhibit human cervical cancer cell division at low concentrations of the drug (0.25 M), block human cervical cancer cells in the G2/M phase, but has no apparent effects on DNA, RNA or protein synthesis. Meanwhile, the microtubule polymers induced by paclitaxel could remain stable at a low temperature (4℃) and in the presence of CaCl 2 (4mmol). That is to say, paclitaxel induces cancer cell death by stabilizing microtubules without any effect on

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ACCEPTED MANUSCRIPT gene expression. The current studies show that the anticancer mechanism of paclitaxel is completely opposite to vinblastine. Paclitaxel is the first compound with the activity of stabling microtubules, creating a new field of anticancer drug research and making the study of paclitaxel a novel hotspot in natural medicine research in recent 30 years.

In 1992, Rao et al. [7] confirmed that the paclitaxel preferentially binds to the β-subunit of tubulin by photo-affinity labeling methods combined with gel

electrophoresis. Three years later, Rao [8] and coworkers found that paclitaxel directly assembled in the 31 amino acid residues of β-tubulin N side and the 217-231 amino acid residues of the middle piece, and this position is identified as a microtubule stabilizers binding site in later researches. Their studies of paclitaxel binding site on tubulin play a significant role in clarifying its anticancer mechanism of action. In recent years, some scientists have discussed the mechanism of action that paclitaxel prevents cancer cells proliferation, which provides reference basis for further expounding its mechanism of action. Paclitaxel can inhibit microtubule de-polymerization by binding to β-tubulin, resulting in mitotic arrest and subsequent activation of caspase-dependent apoptosis MANUSCRIPT by Bcl-2 proteins [9]. The unique pharmacological effect of paclitaxel makes it be on a fast development in its clinical research. On December 29, 1992, paclitaxel was formally approved by the US Food and Drug Administration (FDA) and the Canadian government for the treatment of ovarian cancer. Then, it was approved by FDA to be applied in the treatment of breast cancer and non-small cell lung cancer [10]. However, the original plants of paclitaxel not only grow slowly but also are in low contents (recognized the highest content of 0.069%). The content of paclitaxel is higher in taxus bark, which could be collected on destroying the plant. Therefore, the increasing needACCEPTED of paclitaxel in clinical and experimental application inevitably endangers the survival of Taxus species. At the same time, paclitaxel has poor solubility in water, which makes it difficult to cross the blood-brain barrier for patients with oral medication. On the other hand, the drug resistance and side effects have been gradually appeared accompanied by the clinical application. Some scholars held the viewpoint that the structure and organization of the axon cytoskeleton of the

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ACCEPTED MANUSCRIPT patients that are in paclitaxel treatment could be changed, which would fundamentally alter the ability of a neuron to respond to axonal loading and eventually cause neurological diseases [11]. Therefore, it has been a research hotspot that exploring semi-synthesis or total synthesis of paclitaxel as well as tissue culture, and developing new paclitaxel derivatives to solve the above problems. Due to the complex molecular structure of paclitaxel (Fig. 1) , its chemical synthesis didn’t come true until after its discovery for almost 30 years. In 1989,

Holton et al. [12] completed the semi-synthesis of paclitaxel starting from a

commercially available natural product patchino. In 1994, Holton et al. [13] completed the first total synthesis of paclitaxel. Thereafter, a great number of research

groups, such as Nicolaou [14], Danishefsky [15], Wender [16], Kuwajima [17], and

Mukaiyama [18], reported the total synthesis of paclitaxel in succession. But all these synthetic methods could not be applied to produce paclitaxel in industrialization, resulting in that there are mostly the semi-synthetic products of paclitaxel in current use of paclitaxel drugs. MANUSCRIPT

Fig. 1 Chemical structures and SAR of paclitaxel and its derivatives

On the other side, it is another study direction of paclitaxel that to modify and simplify its structure based on the SAR researches. The structural modification of paclitaxel mainlyACCEPTED focuses on the 3 ′-amino substituent of the side chain, C-7 hydroxyl and C-10 acetyl. The 3 ′-substitution products mostly have high activity, some compounds even exhibit 1-3 orders of magnitude potency higher than paclitaxel

[19-20]. Docetaxel (2) , one 3′-substitution derivative of paclitaxel with successfully structural modification, was approved by the FDA for treating advanced or metastatic

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ACCEPTED MANUSCRIPT non-small cell lung cancer that failed in chemotherapy treatment in 1998 and can be

HO BnOOCO O O 11 9 OH 11 9 OCOOBn

7 o 13 3 R1, DMAP, THF, 40-60 C 7 13 3 HO 1 5 HO 1 5 H H HO O HO O BzO BzO AcO AcO 10-Deacetyl Baccatin III 7,10-diprotected baccatin III

R2, NaH, Na(TMS)2, -40oC,THF, 2.5h, 80%

HO BnOOCO O O 9 OH Boc 11 Boc 11 9 OCOOBn NH O NH O 13 7 7 3 Pd/C, H2, THF, 13 3 Ph O 5 1 RT, 5h, 98% Ph O 1 5 H H O HO O O HO O BzO AcO BzO O O AcO

AcOH/H2O(4:1, V/V), RT, 4h, 78%

HO Ph Boc O N O O 18 9 OH 11 17 O Cl O O t-BuO NH O 15 16 7 13 3 3' O 1 5 O H R1 OH HO O BzO MANUSCRIPTR2 AcO Docetaxel

Scheme 1 The semi-synthesis of Docetaxel

obtained by semi-synthesis now (Scheme 1) . Generally considered the C-7 hydroxyl

and C-10 acetyl of paclitaxel are not the essential activity groups, making it no influence on the activity of paclitaxel on sensitive tumors when these two positions are modified. However, Ojima et al. discovered that the structural modification of C-7 hydroxyl or C-10 acetyl would significantly enhance its activity on multidrug resistant tumors [21 -22] . For example, Cabazitaxel (3) , a structural modification product of docetaxel andACCEPTED whose semi-synthesis has been completed (Scheme 2), exerts weak effect when it interacts with P-glycoprotein, can more easily pass through the blood-brain barrier than docetaxel, and has been approved by the FDA for the

treatment of metastatic prostate cancer since June 2010 [23 -24] .

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HO MeO O O

11 9 OH 11 9 OSiEt3 Et SiCl, pyridine, RT, 51%; 7 3 7 13 3 13 3 HO 1 5 Et3SiO 1 5 H NaH, Mel, DMF, 76% H HO O HO O BzO AcO BzO AcO 10-Deacetyl Baccatin III

Et3N•3HF, CH2Cl2 RT, 77%

MeO MeO O O 11 9 OH 11 9 OMe o 7 NaH, MeI, DMF, 0 C, 74% 13 3 7 13 3 HO 1 5 HO H 1 5 HO O H O HO BzO AcO BzO AcO O

t-BuO N R, DCC, DMAP, COOH EtOAc, 76% O R O MeO MeO O O 9 OMe O 11 18 9 OMe O O 11 17 H 0.1 M HCl, EtOH, 7 t-BuO NH O 15 13 3 o 16 7 t-BuO N 0 C, 32% 13 3 O 1 5 3' O 1 5 O H H HO O HO O BzO OH AcO BzO AcO O MANUSCRIPTCabazitaxel Scheme 2 The semi-synthesis of Cabazitaxel Current researches about the SAR of paclitaxel indicate that the unique heterocyclic butane oxygen (an important pharmaceutical intermediate) and the C-13 side chain of paclitaxel are the necessary activity structures, and the benzoyl group at

C-2 also play an important role in anticancer activity (Fig. 1) [25-26]. In addition to docetaxel and cabazitaxel, there are several new taxane derivatives that have been completed or are ongoing in clinical trials. BMS-184476, the Phase I and II trials in non-small lung cancer and other solid tumors were completed in 2007. Larotaxel (XRP9881) went through the Phase I, II and III trials in 2011. Tesetaxel (DJ-927), applied to breastACCEPTED cancers with brain metastasis, stomach cancer, prostate cancer, advanced melanoma cancer, is ongoing the Phase I and II trials. TPI-287, applied to glioblastoma, neuroblastoma, medulloblastoma, breast cancer, brain neoplasms and central nervous system neoplasms, has been going through the Phase I and II trials, while the research of Alzheimer's Disease is ongoing the Phase I trials [27].

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2.1.2. Taccalonolides Taccalonolides are highly oxygenated pentacyclic steroids that isolated from the herbaceous tropical plant Tacca plantaginea Andre in Scheuer laboratory in 1963 (Fig.

2) [28]. In 1987, the structures of taccalonolide A (4) and taccalonolide B (5) were confirmed by Chen et al. [29] via the X-ray crystallographic method. Tina et al. [30] investigated the antiproliferative potency of taccalonolide A and taccalonolide E (6) in the drug-sensitive and multidrug-resistant cell lines in 2003. Their research indicated that both taccalonolides could arrest cancer cells into G2-M phase, Bcl-2 phosphorylation and cause apoptosis, which is similar to paclitaxel. Meanwhile, the taccalonolide A had less cross-resistance than paclitaxel, confirming that

taccalonolides represent a valuable microtubule stabilizing compound with clinical potential.

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Fig. 2 Chemical structures and SAR of taccalonolides

Some researches indicated that taccalonolides stabilize microtubules by a different mechanism from that of paclitaxel, and have superior ability to overcome multidrug resistance mechanisms than the taxanes. Susan research group reported that

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ACCEPTED MANUSCRIPT four taccalonolides A, B, E and N (7) could avoid the multidrug resistance mechanisms of the taxane family, and proved that the taccalonolides have cytotoxic potency in drug-resistant models in vitro and in vivo. They found that taccalonolides could increase the density of interphase microtubules and exert interphase microtubule bundling, caused an accumulation of cells in the G2/M phase of the cell cycle, and initiate the formation of aberrant β-tubulin spindles and mitotic arrest in cancer cell lines [31 -32] .

Besides, Susan group made a large number of researches on taccalonolides [31 ,

33 -34] . Several taccalonolide derivatives were obtained from isolation and semi-synthesis, such as taccalonolide AF (8), AJ (9), as well as AK, AL, AM, and AN from the hydrolysis reaction of taccalonolides E. Taccalonolide AF and AJ (a semi-synthetic epoxidation product of taccalonolide B) (Scheme 3) could stimulate the polymerization from purified tubulin to the same extent as paclitaxel but with a distinct mechanism from that of paclitaxel, which reveals that taccalonolides could be a new binding site on tubulin [35 -37] . MANUSCRIPT

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Scheme 3 The semi-synthesis of taccalonolides

On the taccalonolides SAR, Susan and coworkers made many deep researches that could increase the developmental possibility of the taccalonolides derivatives with higher activity (Fig. 2). Hydrolysis of the acetoxyl group at C-1 and (or) C-15

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ACCEPTED MANUSCRIPT and keeping an isovaleryl group at the C-1 position could increase the bioactivity significantly. The introduction of an epoxide group at C-22−C-23 resulted in a dramatically increased in potency, and the lactone ring at C-23−C-24 position on the taccalonolide backbone is important for biological activity [36]. Their work made a

significant contribution to the further research of the taccalinolides, and these detailed SAR data from plenty of natural and semi-synthetic taccalonolides provided important guidance for further structural modifications thereof, which indicated that taccalonolides may be a new clinical candidate. However,structural modifications of the taccalonolides mostly stays in obtaining the taccalonolises analogs via some simple reactions, and total synthesis hasn’t been reported so far. The future research can focus on finding out the activity groups based on the current SAR, so that it can provide theoretical support for their structure simplification and total synthesis.

2.2. Microorganism sources

2.2.1. Epothilones Epothilones are 16-membered bacterial MANUSCRIPTmacrolides isolated from the bacterium Sorangium cellulosum by Höfle et al. , who reported that epothilone A (10 ) and B (11 )

have antifungal and cytotoxic activity in 1993 [38]. After that, it was reported that epothilones could promote the microtubule polymerization with similar characteristics and dynamic mechanism to that of paclitaxel, and bound to tubulin on the paclitaxel microtubule binding site [39-40]. Other epothilone analogues, such as epothilone C (12 ), D (13 ), E (14 ), F (15 ) have been discovered (Fig. 3). Recently, epothilone D was

found therapeutic potential for neurodegenerative diseases such as Parkinson’s [41].

In 1996, Höfle et al. [42-43] made a comprehensive report on the production, biological properties,ACCEPTED crystal structure, physical and spectroscopic data, as well as conformations in a solution of epothilone A and B, which provided basic data for the

further study of epothilones. In 2010, Altmann et al. [44] investigated the interactions of C3- and C15-modified analogs with soluble tubulin/tubulin oligomers via biochemical, computational and NMR methods, and confirmed that the aromatic side chain moiety of epothilones contributes to tubulin-binding through strong van der

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Waals interactions with the protein. In order to identify the molecular factors involved in the emergence of drug resistance induced by four tubulin mutations, Navarrete et al.

[45] investigated the structure and dynamic properties of epothilone-β-tubulin complexes with wild-type and mutated tubulin by using molecular dynamic simulations, which is helpful to conduct in-depth understanding on the epothilones’ action mechanism and SAR. Preliminary SAR showed that the ethylene oxide ring is unnecessary for activity but only stabilizes the skeleton. Modifications at C7 and C8 were harmful to the activity and the hydroxy group at C3 may not the essential substitute (Fig. 3).

the ethylene oxide ring is unnecessary for activity, it only stabilizes skeleton the modification at C7, C8 is infeasible due to more tolerant to modifications R the activity decreased O 12 S HO 8 N 15 17 very sensitive O 5 3 more tolerant to modification, but the aryl sector to modifications 1 O O requires an olefinic spacer linking the lactone at OH C15 to an aromatic subsection C3 hydroxy group is unnecessary, MANUSCRIPT but stereochemical configuration of C3 is important Epothilone A (10): R=H Epothilone B (11): R=CH3

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Fig. 3 Chemical structures and SAR of epothilones and derivatives

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Comparing with paclitaxel, epothilones have more superior antitumor properties:

1) the solubility of them is better than that of paclitaxel [42] and they are easy to

obtain 2) it’s no intracellular toxicity of paclitaxel [46]; 3) they can retain high toxicity against P-glycoprotein-expressing multidrug resistant cells [39]; 4) epothilone B has stronger antiproliferative activity than paclitaxel in the paclitaxel-sensitive cells [47]. Besides the above advantages of epothilones as MSAs, they are microbial secondary metabolites with simple structures, which makes the fermentation and chemical synthesis be the major routes for getting more epothilones products since the discovery and the biological activity was confirmed. In 2013, Li et al. [48] investigated the impact on the strains growth and epothilones production induced by different Sorangium cellulosum strains co-cultivation, and found that in co-cultivation can increase the epothilone production significantly than the pure cultures. While, it didn’t clarify that the impacted factors of epothilones biosynthetic regulatory genes. One year later, six novel epothilone A glycoside analogs were reported via enzymatic synthesis, and found that uridine diphosphate glucosyl transferase could improve the catalytic efficiency of the enzyme, which could provide a new approach to get more epothilones products [49]. MANUSCRIPT Actually, it’s limited to produce the epothilones by using enzymatic synthesis because of the long production period, low efficiency and difficult to get strains. Based on the challenges that fermentation faced, undoubtedly, the chemical modification is going to be the mainstream in epothilones’ research due to its great advantage of fast and efficiently getting epothilones products. The total synthesis of epothilone A was reported for the first time by the

Danishefsky team [50] from Columbia University via an intramolecular ester enolate-aldehyde condensation in 1996. In 1997, Nicolaou group [51] completed the total synthesisACCEPTED of epothilone A and its several analogs through an olefin metathesis cyclization approach. These two reports opened the chemical synthesis research gate of epothilones, and there had been lots of reports about the total synthesis of epothilones since that [52-56]. Though all of these synthetic methods still can not be used to produce epothilones in industrialization, they provide some structural characteristic information of epothilones, which is a benefit for the huge success in

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ACCEPTED MANUSCRIPT structural modification of these compounds.

O - N3 S

HO N

OH PdLn O O S COOH HO O N S O Pd(PPh ) 10 mol %, NaN , degassed 3 4 3 HO o N THFH2O, 45 C, 1 h, 6570% O OH O OH N3 Epothilones B O COOH o PPh3, THF, 45 C for 14 h; o 28% NH4OH, H2O, 45 C for 4 h, or H2, EtOH, PtO2, 50 wt %, 10 h; an additional 25 wt % of PtO2, 10 h, or O o PMe3, THFH2O, 25 C, 2 h, 5389% S O HO S N DPPA, NaHCO3, DMF (2.5 mM), NH 4oC, 24 h or EDCl, HOBt, MeCN HO N (0.03M), 25oC, 2h, 65% O OH O OH NH2 ixabepilone/BMS-247550 O COOH a. One semisynthesis of Ixabepilone O O S MANUSCRIPTS HO HO N onepot N O NH Pd(PPh3)4, 10 mol %, NaN3, degassed THFH O, 45oC, 20min; O OH O 2 O OH O PMe , 12 h; 3 ixabepilone/BMS-247550 Epothilones B MeCNDMF (20:1), EDClHOBt, 25oC, 412 h, 2025%. b. Another semisynthesis of Ixabepilone

Scheme 4 Two semi-synthetic methods of Ixabepilone

Ixabepilone (16 ), one lactam derivative of epothilone B (Scheme 4) developed by Bristol-Myers Squibb for the treatment of metastatic or advanced breast cancer, was approved by FDA on 16th October 2007, and it’s the first listed epothilones

derivative soACCEPTED far. In 2008, Goodin [57] found that ixabepilone shows no complete cross-resistance with other treating advanced breast cancer drugs in the treatment of metastatic or local breast cancer. On the other hand, a combination of ixabepilone and capecitabine was more effective than capetabine alone. Recently, there is a report on a combination of cetuximab and ixabepilone cures triple-negative breast cancer stem cells [58] . Thus, we can make a foresight that it will be a hotspot of clinic research that 14 / 55

ACCEPTED MANUSCRIPT to use a combination therapy replaces the monotherapy in the treatment of various cancers. Recently, epothilone derivatives, as the anticancer chemotherapy drugs, have been used in clinical trials for a wide variety of tumors, especially the prostate,

non-small cell lung, breast, colorectal, pancreatic, cervical and gastric cancer [59]. At present, Patupilone (epothilone B/ EPO960) is under clinical evaluation, as well as its four synthetic derivatives Ixabepilone ((azaepothilone B/BMS-247550), BMS-310705,

Sagopilone (17 ), epothilone D and KOS-1584(a derivative of epothilone D) [60]. Patupilone is in Phase III clinical trials of ovarian and peritoneal cancer. Ixabepilone is in Phase II clinical trials of cervical cancer and is well tolerated, but it can not be a therapy because it shows modest activity in the second- or later-line metastatic

cervical cancer cell lines [61]. Sagopilone, epothilone D, and KOS-1584 are in Phase II clinical trial, respectively for the treatment of recurrent glioblastoma and advanced metastatic breast cancer, metastatic breast cancer, and metastatic pulmonary cancer. Epothilone D (in Phase II clinical trial) and BMS-310705 (in Phase III/ IV clinical trial) were not approved for clinical use due to the negative effects such as neurotoxicity and severe diarrhea [62]. Moreover, MANUSCRIPT the pharmacokinetics and safety evaluation research of BMS-753493 started in January 2008, has been quitted in March 2010 because of its benefit-risk profile didn’t support further investigation

thereof in patients with advanced solid tumors [63]. 2.2.2. FR182877 (cyclostreptin/ WS9885B) FR182877 (cyclostreptin), formerly known as WS9885B, is a bacterial metabolic product with a unique hexacyclic structure (Fig. 4), which was generated from a strain

of Streptomyces sp. No.9885 by Fujisawa Pharmaceutical company in 1998 [64]. In vivo , FR182877 exhibits potent antitumor activity against murine ascetic tumor and solid tumor. ACCEPTEDIn 2003, Adam et al. [65] synthesized a series of rhodamine-biotin-tagged forms of (-)-FR182877 (18 ) and (+)-FR182877 (19 ), and discovered that FR182877 is a potent carboxylesterase-1 inhibitor with selective activity.

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Fig. 4 Chemical structures of FR182877

FR182877 is the only known MSA that covalently binds to tubulin [66], and there are lots of studies on the mechanism of action thereof. FR182877 can promote the tubulin polymerization, and it is active against paclitaxel- and epothilone A-resistant cells, as well as P-glycoprotein-mediated multidrug resistance in tumor cells. FR182877 exhibits the fast-binding kinetics that seen in other MSAs, although its

effect on tubulin polymerization is weaker than other MSAs [66-67]. Edler et al. got an insight of action mechanism and found that tubulin brisk assembly with FR182877 required microtubule-associated proteins (MAPs), GTP, and high temperature, which is totally opposite to that of paclitaxel. Meanwhile, FR182877-induced microtubules are more stable than the paclitaxel-induced polymer at low temperature [68]. These studies indicate that FR182877 has a unique MANUSCRIPTaction mechanism, and it has potential for clinical application. The total synthesis of FR182877 came to be a hot since its discovery. In 2001, Sorensen group reported a novel synthetic route of FR182877 including an intramolecular reductive acylation, a bromine Horner-Wadsworth-Emmons reaction

and a π-allyl Stille coupling [69]. Later on, this group focused on the biosynthesis of FR182877 and formed the structure of (+)-FR182877 from a polyunsaturated

macrocycle by successive transannular Diels-Alder reactions [70]. Their successful, potentially biomimetic route validates their biogenetic proposal and provides a chemical rationalizationACCEPTED of the complex molecular structure of FR182877 [70 -71]. In 2002, Evans and his workmates represented a cascade cycloaddition strategy for the enantioselective synthesis of (-)-FR182877 from a macrocyclic precursor by a sequence of transannular [4+2] cycloadditions [72]. One year later, they achieved the asymmetric synthesis of (-)-FR182877 with the help of two boron aldol reactions mediated by (4R)-4-benzyl-2-oxazolidinone, a region-selective Suzuki coupling,

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ACCEPTED MANUSCRIPT macrocyclization of a β-keto ester and a sequence of stereoselective transannular

Diels-Alder (IMDA) reactions [73]. Besides the reports above, Nakada group also has made lots of efforts on the synthetic study of FR182877. Their synthetic strategy is an asymmetric and highly stereoselective synthesis route. They formed the AB ring moiety of FR182877 via a

diastereoselective IMDA reaction [74] and constituted the CD ring moiety of FR182877 via highly diastereoselective intramolecular hetero-Diels–Alder (IMHDA) reaction [75-76]. Two years later, they reported an asymmetric total synthesis of (-)-FR182877 via a sequence of IMDA–IMHDA reactions and stereoselective

transformations mediated by palladium and iridium [77]. Then in 2012, this group accomplished the compound corresponded exactly to the DEF-ring moiety of

(-)-FR182877 through an inverse-electron-demand IMHDA [78].

2.3. Marine organism sources

Marine organisms are a kind of potential anticancer compound source, and many compounds from marine organisms usually have anti-inflammatory, antineoplastic activity due to their unique structures. Nowadays, MANUSCRIPT it has been a hot point that to obtain some compounds with potent biological activity from marine organisms in natural medicinal chemistry research, which makes marine pharmacochemistry be more and more popular. Herein, we classify MSAs from marine organisms into two groups according to their binding sites with tubulin: one is those that are combined with tubulin at the paclitaxel binding site, including Dictyostatin, Discodermolide, Eleutherobin, and Zampanolide; another is those that aren’t combined with tubulin at the paclitaxel binding site, including Laulimalide, Peloruside A and Ceratamines. 2.3.1. MSAs from marine organisms combined with tubulin at the paclitaxel binding site ACCEPTED 2.3.1.1. Dictyostatin Dictyostatin, a 22-membered macrolactone punctuated by 11 stereo-centers, two dienes, and a cis-1, 2-disubstituted olefin (Fig.5), was first isolated from a marine sponge in the genus Spongia from the Republic of Maldives at a 3.4×10 -7% yield by

Pettit et al. , and it was found to have potential anti-proliferative activity [79].

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Isbrucker et al. [80] isolated dictyostatin-1 (20 ) from a Lithistida sponge collected off the North Jamaican Coast and investigated its mechanism of cytotoxic activity that

was similar to that of paclitaxel. Madiraju et al. [81] studied the biochemical and cytological activity of synthetic (-)-dictyostatin (21 ) in detail, and their research results were consistent with the idea that the anti-proliferative activity of dictyostatin is similar to that of paclitaxel and discodermolide.

region appears to interact with β-tubulin C16 methyl group is essential f or its binding at or near Phe270 on β-tubulinis and is disposable,but must be "S"configuration if present C15:C16 "Z" alkene C19 "R" conf iguration well tolerated pref erred

C14 "R" configuration MANUSCRIPT26 OH C1-C21 macrolactone pref erred 16 better than open chain HO 21 methyl ester, but not necessary; C1-C19 macrolatone inactive OO 1 C9 "S" conf iguration 7 5 C2:C3 "Z" geometry required required OH OH C6,C7,C9 anti-syn arrangement least desirable; all other configurations work C2-C9 saturated analog very well retains minor activity

Fig. 5 Chemical structures and SAR of Dictyostatins

There are some reports about the total synthesis of the C9- and C16-analogues of (-)-dictyostatinACCEPTED via a versatile synthetic strategy and the SAR research, indicating that the C9 and C16 region of (-)-dictyostatin are not only the significant positions of anti-proliferative activity but also the two key positions of acting on the

paclitaxel-resistant cell lines [82-83]. In 2005, Curran group completed the total synthesis of (−)-16-Normethyldictyostatin, an important member of the dictyostatin

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ACCEPTED MANUSCRIPT family, and made its biological evaluation, confirming that C16 methyl group of the dictyostatins is essential for its binding on β-tubulin at the paclitaxel binding site [84]. In 2011, Vollmer et al. reported a new, highly convergent streamlined synthesis of 16-desmethyl-25, 26-dihydrodictyostatin and 6-epi -25, 26-dihydrodictyostatin, and the results of the anti-proliferative activities, tubulin assembly, and anti-angiogenesis assay of the two compounds suggested that the new analogs were with potential for further preclinical development [85]. Dictyostatin has been identified to have anti-proliferative activity against several cancer cells at low nanomolar concentrations, especially paclitaxel- and epothilone

B-resistant cell lines, which makes it be potential anticancer drugs [85]. Chemical synthesis and biological activity evaluation of dictyostatin were limited due to its low natural abundance and uncertain absolute stereochemistry. Until 2004, Paterson and his colleagues determined the relative and absolute stereochemistry of (-)-dictyostatin based on a combination of extensive high field NMR experiments, including J-based configuration analysis and molecular modeling. Moreover, they completed the total synthesis of (-)-dictyostatin at the first time by a modular, convergent synthesis way in 27 steps [86-87]. The same year, Curran groupMANUSCRIPT [88] reported a total synthesis of (-)-dictyostatin by coupling three pieces based on a flexible and suitable convergent synthesis method, which not only can shorten the synthetic steps but also improve the product yield. In 2006, Curran’s group obtained dictyostatin and three C6, C7-epi -dictyostatin diastereomers, and discovered that it shows equal or higher anti-proliferative effect than dictyostatin when C6-, C7- are in the same spatial conformation [89]. At the same year, Phillips and coworkers designed a convergent synthesis approach to achieve the total synthesis of (-)-dictyostatin in 26 steps, and their synthesis offered a novel synthetic strategyACCEPTED of the complex polyketide with cis-trans isomerisms via a (silyloxy)enyne cyclizations reaction [90]. In 2010, Curran and coworkers developed three new synthesis approaches to 16-desmethyl-25, 26-dihydrodictyostatin analogs, and their synthetic work marked significant advances towards the practical synthesis of dictyostatin and its analogs for both the discovery and development of the new drug, which has an important guiding

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ACCEPTED MANUSCRIPT role for the further synthesis researches [91-92]. In 2013, Stephen et al. accomplished the total synthesis of dictyostatin via three fragment syntheses, and the most advantage of their strategy is that they used an unrivalled step-economy route with one-pot procedures at several points, and each of the three fragments was prepared by commercial reagents just in 4 or 5 steps, which further promoted the synthesis efficiency of dictyostatin [93]. In summary, the common point of several different synthetic strategies above is that to divide the molecule into several major parts firstly, then synthesize a few main fragments separately, finally obtain the final target product by fragments coupling. These synthetic strategies have provided many dictyostatin analogs and make a clear understanding of the SAR of this compound (Fig.5). The difference among them is that the various divided positions and all kinds of different basic reactions, which made a difference at ultimate total synthetic steps and overall yield. Therefore, when researching the synthesis of macrocyclic target products with complex structures, it should be taken into account that to analyze its structural characteristics thoroughly, and try to use some reasonable fragments division and simple basic reactions to synthesize target products, aiming at reducing MANUSCRIPT reaction steps to improve product yield finally. 2.3.1.2. Discodermolide

Discodermolide, a polyhydroxylated lactone with 13 chirality stereo-centers (Fig. 6), was first isolated from a Caribbean marine sponge Discodermia dissoluta by

Gunasekera [94] and coworkers in 1990 and was applied as immunosuppressive agents initially. Discodermolide can stabilize microtubules and inhibit the multiplication of tumor cells effectively and interfere paclitaxel binding to microtubule [95]. Natural (+)-discodermolide (22 ) arrests cell cycle into G2/M phase, while its enantiomerACCEPTED arrests cell cycle into S phase [96]. Discodermolide has good water solubility, strong pharmacological effect, low P-glycoprotein expression quantity, and it has an inhibiting effect on paclitaxel-resistant cells at low concentrations [97]. Moreover, the pharmacological effect can be increased when

discodermolide is combined with paclitaxel [97-98].

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Fig. 6 Chemical structures of Discodermolide

For discodermolide, the quantity from natural separation is very limited, and it is only 0.002% weight yield isolated from the frozen marine sponge. Besides, studies on its semi-synthesis and fermentation haven’t been reported. On the contrary, there are lots of researches on its total synthesis since its biological activity was reported. In 1993, Schreiber group firstly reported the total synthesis of (-)-discodermolide (23 ) in 36 steps and the longest linear sequence had 24 steps with an overall yield of 3.2% [99]. One year later, this group reportedMANUSCRIPT the total synthesis of natural (+)-discodermolide and its enantiomer (-)-discoderm olide for the first time again, and they also evaluated biological activity and binding activity of both compounds [96]. Discodermolide synthesized by Schreiber group have a certain absolute configuration, and they have synthesized a variety of discodermolide analogs, which is significant to the SAR study of discodermolide.

In 1995, Smith group [100] reported an efficient, highly convergent and stereoselective total synthesis of (-)-discodermolide. After analyzing its structural features, they divided the molecule skeleton into three fragments (C1-C8, C9-C14, and C15-C21), and the key steps are the Witting olefination in C8-C9 and Negishi cross-couplingACCEPTED reaction at C14-C15. Later on, this group continually improved the synthetic method and formed the second-generation [101], third-generation [102] and

fourth-generation [103] total synthesis approaches. They gradually achieved the Witting olefination of double-bond at C8-C9 at atmospheric pressure, which made it more suitable for mass production and increased the overall yield to 9.0% [103]. Besides, there are other research groups studying the synthesis of discodermolide, 21 / 55

ACCEPTED MANUSCRIPT such as Myles group, Marshall group and Ian Paterson group, and so on. Both Myles group [104] and Marshall group [105] divided discodermolide skeleton into three fragments, and the former suggested dissection of the skeleton into fragments C1-C7, C9-C15 and C16-C21, while the latter divided the skeleton into fragments C1-C7, C8-C13 and C15-C24. The difference between these two dissection strategies is that Myles group added Nozaki-Kishi to the coupling reaction at C7-C8, and a coupling reaction at C15-C16 depended on ethynylation of enolates. While, Marshall group added lithium acetylide to the aldehyde reaction at C7-C8, and applied new Suzuki

cross-coupling reaction at C14-C15. Ian Paterson group [106-107] reported an original total synthesis of (+)-discodermolide based on a novel aldol-coupling strategy employed aldol reactions of chiral ketones to construct the three key subunits (C1-C6, C9-C16, C17-C24). Then this group went on study, made a modification and improvement continually, and developed an efficient, highly convergent second-generation total synthesis of (+)-discodermolide solely relied on enzymatic reaction without chiral reagents and auxiliary reagents, which was completed at an

overall yield of 7.8% over 24 linear steps in 35 steps [108-109]. Soon after, this group reported the third-generation total synthesis ofMANUSCRIPT (+)-discodermolide again, which was accomplished at an overall yield of 11.1% over 21 linear steps [110]. All of the synthesis strategies above have been highlighted in one graph (Fig. 7). Schreiber (enolate alkylation) Myles (enolate alkylation)

24 Marshall (Zhao-Wittig)

19 Smith (Negishi Pd cross-coupling) 15 Marshall (Suzuki Pd cross-coupling) OO OH OH 11 Smith (Wittig olefination) NH2 9 HO Paterson (Lithium Aldol) Schreiber (alkynyl NiCl2/CrCl2 coupling) 7 Myles (vinyl NiCl2/CrCl2 coupling) ACCEPTEDHO 5 Marshall (alkynyl lithium addition) 3 O 1 Pterson (Boron Aldol) O Fig. 7 Total synthesis strategies of (+)-Discodermolide 2.3.1.3. Eleutherobin and Sarcodictyins A & B

Eleutherobin (24 ), a new diterpene glycoside (Fig. 8) that can promote

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ACCEPTED MANUSCRIPT microtubule assembly, was isolated from a rare alcyonacean Eleutherobia sp. found

near Bennett’s Shoal in Western Australia by Lindel et al. in 1997 [111]. This compound possesses significant cytotoxicity against a wide variety of cancer cells

(IC 50 10-15nM), especially the breast, renal, ovarian and lung cancer cell lines. In addition, it shows cross-resistance against paclitaxel-resistant cell line (A2780/Tax22) and P-glycoprotein-expressing cells (HTC116 (VM46)). It shares a similar mechanism of action to that of paclitaxel and binds on microtubule where overlaps the paclitaxel binding site [112].

MANUSCRIPT

Fig. 8 ChemicalACCEPTED structures and SAR of Eleutherobin and Sarcodictyins A & B

In 1999, Ojima [113] and coworkers investigated the SAR and pharmacophore of the compounds that could promote the microtubule stability, and they thought that three regions A, B, C of eleutherobin are important regions for itself binding to tubulin (Fig. 8). Sarcodictyins A (25 ) and B (26 ) (structurally similar to eleutherobin

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ACCEPTED MANUSCRIPT but lacking the C-3 sugar moiety in C region) had IC 50 values at the 200-500 nM range [114]. The two eleutherobin analogs, caribaeoside (27 ) structurally changed in B region with a tertiary alcohol in place of an alkene, and caribaeolin (28 ) structurally changed in C region with an acetyl replaced the C-3 sugar moiety compared to

caribaeoside, both found to be much less active (IC50 20M) than eleutherobin [115]. The simplified analogs of eleutherobin (lacking the C-4/C-7 ether bridge) retained potent microtubule-stabilizing activity, though the limited cytotoxicity was three

orders of magnitude less than paclitaxel [116]. These researches indicated that this trisubstituted double bound was important for the antimitotic activity and provided

further supports to Ojima’s three regions pharmacophore model [117]. Eleutherobin has become the focus of the second-generation of MSAs due to its similar activity to paclitaxel. Although the evaluation of its activity has been in severe obstruction because the natural product is difficult to collect and the output is very

low, Nicolaou group [118-119] and Danishefsky group [120 -121] have completed the total synthesis of eleutherobin through different routes since its discovery. The main difference between the two synthetic approaches is that the establishment of tricircle and the introduction of glycosyl. MANUSCRIPT Nicolaou group finished the total synthesis of eleutherobin with (+)-carvone derivatives as the starting materials for the first time and introduced the glycosyl side chain before the ring closure. This strategy can not control the stereo-selectivity effectively leading the products to form β-glycosidic bond entirely, and they only obtained a 2.4% yield of an 8:1 mixture of β- and α-product under a strict condition control [119]. Danishefsky group developed a synthetic route staring from (R)-(-)-α-phellandrene, and they obtained the diene terpene skeleton first, then imported the carbohydrate moiety by a direct coupling reaction of C3 and β-glycosyl donor, and ACCEPTED eventually got the product at an overall yield of 0.44%.[121] In 2005, Gennari et al. [122] completed the formal total synthesis of eleutherobin through a selective protection and deprotection at 3, 4, 7, 8-hydroxy groups, and a ring-closing metathesis (RCM) reaction to form the intermediate diene.

2.3.1.4. Zampanolide and Dactylolide

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Zampanolide, a 20-membered macrolide (Fig. 9), was first isolated from marine sponge Fasciospongia rimosa of Cape Zampa in Okinawa by Tanaka and Higa in

1996 [123]. Zampanolide exhibits potent cytotoxic activity against various cancer cell lines, such as P388, A549, HT29, and MEL28 cell lines. (-)-Zampanolide (29 ) exhibits potent cytotoxic activity against SKM-1 and U937 cell lines (IC 50 1.1-2.9 nM) [124]. (-)-Dactylolide (30 ) (the zampanolide derivative lacking the N-acyl hemiaminal

side chain) exhibits GI 50 values at the nanomolar (25-99 ng/mL) range against the four cell lines HL-60, K-562, HCC-2998, and SF-539, while displays modest LC 50 values [125].

MANUSCRIPT

Fig.ACCEPTED 9 Chemical structures and SAR of Zampanolide and Dactylolide In 2009, Field and Singh et al. [126] first reported that zampanolide was a novel microtubule stabilizer with promoting tubulin assembly effects similar to that of other MSAs, blocked cells at G2/M phase in the cell cycle and made dividing cells form multiple asters. A recent study suggests that zampanolide competes with paclitaxel for covalent binding to β-tubulin at the taxane site on tubulin and overcomes

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P-glycoprotein-mediated multidrug resistance [127]. A report about the SAR of zampanolide derivatives reveals that the N-acyl hemiaminal side chain of the

molecule plays an important role to stabilize microtubule (Fig. 9) [128]. In 2014,

Kingston et al. [129] made a detailed introduction on current developments of zampanolide and its one derivative dactylolide, which has good referent value for the further study of these compounds.

MANUSCRIPT

Fig.ACCEPTED 10 Total synthesis strategies of Zampanolide and Dactylolide The total synthesis study of zampanolide and dactylolide has an early start and there are various synthetic strategies shown in one graph (Fig. 10). Smith et al.

[130 -131] first completed the total synthesis of (+)-zampanolide (31 ) and (+)-dactylolide (32 ), and researched the relative and absolute configurations. The critical steps of their synthesis included: 1) the establishment of (cis)-2,

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6-bistetrahydropyrane through Petasis-Ferrier rearrangement reaction, 2) the formation of C 20 stereoisomer by stereospecific Curtius rearrangement reaction, which formed the N-acyl hemiaminal side chain of (+)-zampanolide. In 2003, Hoye et al. [132] reported a total synthesis of (-)-dactylolide and zampanolide via a novel

Ti(IV)-mediated macrolactonization of an epoxy-acid. Later, Jennings at el. [125, 133] reported the total synthesis of (-)-dactylolide and accomplished the formal synthesis of (-)-zampanolide by utilizing the formation of N-acyl hemiaminal side chain

developed by Hoye at el . In 2009, Uenishi et al. [124] achieved the total synthesis of (-)-dactylolide in 17 linear steps at an overall yield of 6.5% starting from commercial (R)-glycidol, and made it come true that (-)-zampanolide was derived synthetically from (-)-dactylolide in one step. The synthesis researches above major focus on the lactone ring part rather than

the N-acyl hemiaminal side chain. In 2011, Ghosh et al. [134] reported an enantioselective synthesis of (-)-zampanolide, and obtained stereoselectively furnished (-)-zampanolide at a 51% yield via a chiral phosphoric acid catalyzed stereoselective N-acyl aminal formation, no bis-amide byproducts, and its isomer was only 12%. This synthesis is significant to MANUSCRIPT get the N-acyl aminal derivatives of zampanolide. In 2015, Wang et al. [135] studied that a linchpin approach is the synthesis of a major fragment of zampanolide and dactylolide by using Bestmann ylide [(triphenylphosphoranylidene) ketene] to link the C16–C20 alcohol with the C3–C8 aldehyde fragment. Their approach has obvious advantages compared with other methods required multiple steps, and is a highly efficient synthetic route to obtain unsaturated polyketide derivatives strategically. Taylor et al. [136] have determined the solution conformation preferences of the macrolide core of (-)-zampanolide and (-)-dactylolide through a combination of high-field NMR experimentsACCEPTED and computational modeling, which is of great importance in the design of potent analogs by mimicking the bioactive conformation of zampanolide. 2.3.2. MSAs from marine organisms not combined with tubulin at the paclitaxel binding site.

2.3.2.1. Laulimalide and Isolaulimalide

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Laulimalide (33 ) and isolaulimalide (34 ) (an epoxy isomer of laulimalide), 18-membered macrocyclic lactones (Fig. 11), were first isolated from an Indonesian sponge Hyattella sp. and a nudibranch predator Chromodoris lochi respectively in

1988 [137]. Then Jefford et al. [138] determined the absolute configuration of laulimalide isolated from Fasciospongia rimosa that collected in Okinawan waters by

X-ray in 1996. In 1999, Mooberry et al. [139] isolated laulimalide and isolaulimalide from marine sponge Cacospongia mycofijiensis collected from Republic of the Marshall Islands, and they found that both laulimalide and isolaulimalide could promote microtubule polymerization to form short and multicore vascular bundle, and was as much as 100-fold more potent than paclitaxel against SKVLB-1 cells (a P-glycoprotein overexpressing multidrug-resistant cell line). A report indicated that laulimalide combined with docetaxel synergistically inhabited human umbilical vein endothelial cell migration [140]. While, laulimalide exhibits only minimal tumor growth inhibition in vivo and is accompanied by severe toxicity and mortality, which will limit it as a new anticancer therapeutic agent [141]. MANUSCRIPT

Fig. 11 Chemical structures and SAR of Laulimalide and Isolaulimalide

Laulimalide exhibits potent antiproliferative activity against drug-sensitive cell lines MDA-MB-435ACCEPTED and SK-OV-3, binds to tubulin at a site distinct from that of the toxoids [142-143], and has a synergistic effect on microtubule polymerization when it was combined with paclitaxel [144]. Bennett et al. [145] demonstrated that laulimalide binds to the exterior of the microtubule on β-tubulin, in a region previously unknown to support ligand binding and is well removed from the paclitaxel site via mass shift perturbation analysis and data-directed docking. The results of resistant cells culture 28 / 55

ACCEPTED MANUSCRIPT experiments provide the first cell-based evidence to support a β-tubulin–binding site

for laulimalide [146]. In 2002, Hamel et al. [142] discovered that a laulimalide analog lacking the epoxide moiety whose activity is between laulimalide and isolaulimalide in biochemical and cellular systems, which suggests that the epoxide moiety of laulimalide may be not an essential feature for the activity of this new drug family. The C19 side chain, C30 methyl group and C15 hydroxyl group are important for its activity (Fig. 11). Some researchers have acquired new, detailed information about the interactions between laulimalide and tubulin by computational methods of molecular dynamics, and they discovered that the structural changes observed in the binding site on tubulin after laulimalide binding to tubulin might explain its role in stabilizing

microtubules [147]. There are many groups who have completed the total synthesis of laulimalide

(Fig. 12). Ghosh et al. [148] first completed the total synthesis of laulimalide via a crucial olefin metathesis catalyzed by Grubbs’ catalyst in 2000. Later on, Paterson et al. [149] reported total synthesis of laulimalide in 27 steps and 2.9% overall yield starting from an unsaturated diol derived from dimethyl (R)-malate. In 2002, Mulzer group [150] described three different routes toMANUSCRIPT get deoxylaulimalide, the immediate precursor of laulimalide, and ultimately convert it into (-)-laulimalide via Sharpless asymmetric epoxidation. In 2009, Gollner [151] and coworkers reported the first synthesis of isolaulimalide, and they completed a total synthesis of laulimalide and its analogs starting from commercial materials at a 7% overall yield in 20 steps.

ACCEPTED

Fig. 12 Total synthesis strategies of Laulimalide 2.3.2.2. Peloruside A 29 / 55

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Peloruside A, a novel, polyoxygenated, pyranose ring containing 16-membered macrolide (Fig. 13), was isolated from New Zealand marine sponge Mycale henstcheli by Northcote and coworkers in 2000 [152]. It exhibits cytotoxic activity at nM range, effectively inhibits cell proliferation, and induces apoptosis in a dose-dependent

manner in murine (32D) and human (HL-60) myeloid cell lines [153]. Peloruside A alters microtubule dynamics by directly inducing tubulin polymerization in the absence of MAPs, binding to a different site on tubulin to paclitaxel, causing cells arrest at the G2/M phase of the cell cycle, and it retains activity in multidrug-resistant

cell lines and is a competitive inhibitor of laulimalide [154-155]. Current research indicates that both peloruside A and docetaxel potently inhibit the proliferation of human umbilical vein endothelial cells [156]. Comparing with paclitaxel, peloruside A not only has a greater aqueous solubility but also exhibits higher activity and better tolerability.

MANUSCRIPT

Fig. 13 Chemical structure and SAR of Peloruside A

In 2008, Schriemer [157] and coworkers proposed that peloruside A binds within a pocket on the exterior of β-tubulin at a previously unknown ligand site by using data-directed molecular docking simulations. Some researches show that peloruside A acts synergistically with a number of taxoid site agents, and this synergy probably derived, at leastACCEPTED in part, from the active drug combinations that have a lower critical concentration for tubulin than the individual drugs [158]. In 2015, Miller group revealed that the importance of four peloruside A and laulimalide binding site residues

on βI-tubulin in microtubule assembly [159]. Deciphering the binding sites of peloruside A and laulimalide on tubulin is essential for understanding the molecular mechanism and provides insight for the design of more effective antitumor agents by

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ACCEPTED MANUSCRIPT exploiting this region of tubulin.

Fig. 14 Total synthesis strategies of Peloruside A

The 16-membered lactonic ring of peloruside A is similar to that of epothilone B, and there is a number of reports about the synthesis of it and its analogs (Fig. 14). In

2003, De Brabander [160] and coworkers first reported the total synthesis of (-)-peloruside A (35 ), and determined the absolute configuration of the dextrorotatory natural product. The same year, Paterson MANUSCRIPTet al. [161] reported a stereoselective synthesis of the fragment descriptor of peloruside. Later on, Taylor et al. [162] accomplished the first total synthesis of (+)-peloruside A (36 ) with a highly convergent strategy highlighted by an aldol coupling of two complex fragments and an epoxide ring fragmentation induced by methoxymethyl ether group. In 2009,

Evans et al. [163] successfully achieved a highly convergent total synthesis of (+)-peloruside A in 22 steps (the longest linear sequence) from commercial (S)-pantolactone through the coupling of fragments based on an aldol condensation reaction.

In 2010, Altmann et al. [164] reported the stereoselective synthesis of monocyclic peloruside AACCEPTED analog and discovered that the compound is several-hundred-fold less potent than peloruside A in three human cancer cell lines. In 2012, Taylor et al. [165] synthesized a set of conformational analogs of peloruside A just in 18 steps from commercial materials by a simple esterification-based fragments coupling and a late stage RCM reaction. This efficient synthetic strategy is significant for the further research, though they didn’t make activity evaluation of conformational peloruside A

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ACCEPTED MANUSCRIPT analogs. In 2015, Brackovic et al. [166] thought the pyran ring is essential for its anti-proliferation activity by describing in detail the published syntheses of peloruside analogs and discussing the SAR available to date, which has reference value for the further study (Fig. 13). 2.3.2.3. Ceratamines

Ceratamines, a class of novel antimitotic heterocyclic alkaloids with an atypical imidazo [4, 5, d] azepine core heterocycle (Fig. 15), including ceratamine A (37 ) and ceratamine B (38 ), were isolated from Papua New Guinea marine sponge

Pseudoceratina sp. by Roberge and coworkers in 2003 [167].

MANUSCRIPT Fig. 15 Chemical structures and SAR of Ceretamines and its analogs

The binding site of the ceratamines combined with microtubules is different from that of paclitaxel in vitro . The ceratamines have simple structures with no chiral center, making them be attractive drug leads in compared to other MSAs with complex structures [168]. In order to understand the pharmacokinetics of ceratamine A and ceratamine B, some researchers carried on metabolism studies in vitro using rat liver microsomes and identified eight metabolites by using UV and LC−MS/MS techniques

[169], which provides a better reference for studying the mechanism of action of these compounds.ACCEPTED There are plentiful researches about the total synthesis of the ceretemines since it was discovered due to the simple structure (Fig. 16), and the SAR of ceretemines is also gradually clear with the help of so many synthetic ceretemines analogs and derivatives (Fig. 15). The Andersen group reported the total synthesis of ceretemine A

[170], ceretemines analogs [171], and several ceretemines derivatives [172] in 2008, 32 / 55

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2009, and 2010, respectively. A key step in their first synthesis is that the formation of the azepine ring via an intramolecular Buchwald coupling between a vinyl bromide and an N-methyl amide, and ceratamine A was synthesized starting from tribromoimidazole. They described two synthetic approaches to ceretemine A and desbromo analogs in their second report, while only the second approach starting from tribromoimidazole was successful eventually [171]. In 2010, the Andersen group obtained several ceretemines derivatives and discovered that the synthetic compound 4 (39 ) and 33 (40 ) that both bromine atoms at C14, C16 were replaced with methyl groups and an additional methyl substituent on the amino nitrogen at C-2, are

significantly more active than the natural product [172].

In 2009, Coleman et al. [173] reported a direct synthetic route of ceretemine A and ceretemine B starting from ε-lactam in 11 and 12 steps at an overall yield of 28% and 12%, respectively. Their synthetic strategy involves a Beckmann rearrangement to form an azepine ring precursor, a Knoevenagel condensation to install the benzylic side chain, which is different from that of Andersen’s methods. In 2013, Liu [174] and coworkers accomplished the total synthesis of ceretemine A starting from 5-methoxybenzimidazole in 10 steps at 12.7% MANUSCRIPT overall yield, involving the Schmidt rearrangement to construct the azepine ring and the alkylation of lactam to introduce the C-5 benzylic side chain that different from the Coleman’s and Andersen’s methods.

ACCEPTED Fig. 16 Total synthesis strategies of Ceretamine A

3. The chemically synthesized small molecule microtubule stabilizers

The total synthesis of natural microtubule stabilizers is restricted due to their limited sources, complex structures and so on, which makes it difficult to get a large

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ACCEPTED MANUSCRIPT number of products, so as to its wide application. With the gradually in-depth study of the structure characteristics, pharmacological effects and SAR of natural microtubule stabilizers, some chemically synthesized small molecules with the ability to promote microtubule assembly are emerging. Because these compounds usually have simple structures easy to be modified, clear synthetic processes, and certain pharmacological effects foundation, they have definite research value and development space in microtubule stabilizers.

MANUSCRIPT

ACCEPTED

Fig. 17 Chemical structures of small synthetic compounds 3.1. GS-164

In 1997, Shintani et al. [175] first reported a novel synthetic compound GS-164 (Fig. 17, 41 ), which stimulated microtubule assembly in a concentration- and 34 / 55

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GTP-independent manner by a similar mechanism to that of Taxol. It had cytotoxic activity against a wide range of human tumor cell lines. Although the cytotoxicity of GS-164 against human tumor cells is 1000-fold lower than that of Taxol and GS-164 is one-tenth as active as Taxol in vitro , GS-164 has the potential of a lead compound in synthesizing clinical useful anticancer agents. In 2009, Desino et al. obtained three diastereoisomers by the reaction of tris(hydroxymethyl)aminomethane with p-fluorobenzaldehyde, and they thought that the (3R,5S,7a s)-isomer could to be a novel neuroprotective agent [176]. 3.2. Synstab A

In 1999, Mayer et al. [177] first reported synstab A (Fig. 17, 42 ), and researched the mitosis mechanism by making use of this compound. Later on, Haggerty et al.

[178] discovered that synstab A stabilizes microtubule polymerization from purified bovine brain tubulin by targeting tubulin directly in vitro and has a similar mechanism of action to that of paclitaxel, which suggests that it may be a novel microtubule stabilizer. 3.3. 4'-Methoxy-2-styrylchromone 4'-methoxy-2-styrylchromone (Fig. 17, MANUSCRIPT 43 ), a new synthetic chromone that synthesized by 2-methylchromone and 4-methoxybenzaldehyde [179] or 2'-hydroxyacetophenone and p-methoxy cinnamic acid via a Baker–Venkataraman approach [180], has been identified as a potent growth inhibitor of tumor cell lines, and it has less influence on the growth of normal cells, and various extents of tumor-specific cytotoxicity [179-181]. It could block tumor cells in the G2/M phase of the cell cycle and promote the formation of a monopolar structure causing mitotic spindles abnormal in mitosis, and it stabilized microtubules in a manner that similar to

paclitaxel, meaning that it might act as a potential MSA [182]. 3.4. DienoneACCEPTED derivatives In 2011, Luthman et al. [183] synthesized a series of dihalogenated chalcones and structurally related dienones starting from appropriate aldehyde and 3'-bromo-5'-chloro-2'-hydroxyacetophenone, and evaluated their biological activity. They found that one dienone derivative (Fig. 17, 44 ) could stabilize tubulin to the same extent as the anticancer drug docetaxel, which is the firstly reported chalcone

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ACCEPTED MANUSCRIPT with microtubule-stabilizing activity so far, meaning that it will open a new way to the development of novel small molecule MSAs. 3.5. Ketones containing aromatic side chain

Reddy [184] and coworkers reported that they synthesized a series of (Z)-1-aryl-3-arylamino-2-propen-1-ones molecules identified as ketones containing aromatic side chain and evaluated their anti-proliferative activity in the cell-based assay. They found that one of these compounds (Z)-1-(2-bromo-3, 4, 5-trimethoxyphenyl)-3-(3-hydroxy-4-methoxyphenylamino)prop-2-en-1-one (Fig. 17, 45 ), could arrest the cells in G2/M phase of cell cycle in a dose-dependent manner, causing microtubule stabilization like that paclitaxel dose, and induce apoptosis via activation of the caspase family. 3.6. Pyranochalcone derivatives

In 2013, Cao et al. [185] synthesized 25 novel pyranochalcone derivatives and evaluated their anti-proliferative activity in vitro and in vivo . Among these compounds, one molecule numbered 10i (Fig. 17, 46 ) showed the most potent activity in all investigated human tumor cell lines including multidrug-resistant phenotype. Furthermore, it could arrest the cell cycle at theMANUSCRIPT G2/M phase at 0.2 M and promote tubulin polymerization similar to paclitaxel. Their study demonstrates that this compound is supposed to be a novel MSA and provides a new molecular scaffold for the further development of antitumor agents. 3.7. α-Cyano bis(indolyl)chalcones

In 2014, Kumar et al. [186] synthesized 23 a-cyano bis(indolyl)chalcones (Fig. 17, 47 ) by a facile microwave-assisted synthesis method. The study results of anticancer activity of these compounds in vitro showed that the compounds having mono- or di-methoxy groups at various positions of indole ring were potent against multiple cancerACCEPTED cell lines. The most active compound (R 1=R 3=R 5=R 6=H, R 2=OCH 3, R4=F) exhibited selective cytotoxicity against A549 lung cancer cells in vitro in a time-dependent manner. Meanwhile, this compound was found to enhance tubulin polymerization, suggesting this class of compounds could act as MSAs [187]. 3.8. Cyclopropylamide analogs of combretastatin-A4

Combretastatin-A4, a natural stilbene isolated from Combretum caffrum , is a

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MDA binding to the colchicine domain on β-tubulin and exhibits a lower toxicity

profile than paclitaxel or the Vinca alkaloids [188]. Since the simple structure of combretastatin-A4, there are plenty of study on combretastatin-A4 analogs. In 2013, Li et al. [189] synthesized a variety of novel cyclopropylamide analogs of combretastatin-A4, which are able to stimulate tubulin polymerization with different binding mechanism from that of Combretastatin-A4. Moreover, among these compounds, one (Fig. 17, 48 ) is equally potent against paclitaxel-resistant cancer cells, arrests A549 cancer cells at the G2/M phase, and results in cellular apoptosis by disrupting microtubule dynamics finally. The research results above demonstrates that this compound represents a novel MSA, which could be used as a promising lead for the development of new antitumor agents. 4. Current progress

MSAs have received extensive development and application as novel antitumor drugs in clinic and ongoing the clinical or preclinical trials. We make a summary on the MSAs appeared in this review and divide into four classes: MSA clinical drugs, MSA under clinical trials, MSA failed to beMANUSCRIPT approved in clinic and MSA under preclinical trials (Table 1). Although some drugs used in clinical cancer treatment aren’t entirely satisfactory, such as the poor water solubility of Taxol causing the defects in use, the good treatment effects are accompanied by some side effects, for example the multidrug resistance identified as one of the biggest problems in cancer treatment, it’s still a potent chemotherapy that by applying microtubule stabilizers to block cancer cell mitosis and induce tumor cell apoptosis.

ACCEPTED

Table 1 Description of the MSAs used in this review

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MSAs clinical drugs

Name Type of Cancer Treatment Developing Status Reference

ovarian cancer, breast cancer, Paclitaxel approved to be used in [6] [10] non-small cell lung cancer, human (Taxol®) clinic by FDA in 1992 [199] cervical cancer advanced or metastatic non-small approved to be used in Docetaxel [23-24] [199] cell lung cancer clinic by FDA in 1998 approved to be used in Cabazitaxel metastatic prostate cancer [23-24] [199] clinic by FDA in 2010

Ixabepilone drug-resistant/refractory metastatic approved to be used in [60-61] [199] (BMS-247550) or locally advanced breast cancer clinic by FDA in 2007

MSAs under clinical trials

Name Type of Cancer Treatment Clinical Phase Reference

non-small lung cancer and other the Phase I and II trials BMS-184476 MANUSCRIPT[27] [199] advanced solid tumors were completed in 2007 has been went through the Larotaxel advanced or metastatic breast cancer, Phase I, II and III trials in [27] [199] (XRP9881) breast cancer, pancreatic cancer 2011 breast cancers with brain metastasis, Tesetaxel ongoing the Phase I and II stomach cancer, prostate cancer, [27] [199] (DJ-927) trials advanced melanoma cancer glioblastoma, neuroblastoma, has been went through the medulloblastoma, breast cancer, Phase I and II trials, TPI-287 ACCEPTED [27] [199] brain neoplasms, central nervous Alzheimer's Disease I is system neoplasms ongoing ovarian cancer, peritoneal cancer, has been went through the Patupilone [60-61] [199] fallopian tube cancer Phase III clinical trials

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recurrent glioblastoma, advanced metastatic breast cancer, brain and central nervous system tumors, has been went through the Sagopilone [60-62] [199] non-small cell lung cancer, Phase II clinical trial melanoma, prostate cancer, ovarian cancer has been went through the KOS-1584 non-small cell lung cancer [60-62] Phase II clinical trial

MSA failed to be approved in clinic

Name Type of Cancer Treatment Reason of Termination Reference

metastatic breast cancer, colorectal neurotoxicity and severe epothilone D cancer, breast cancer, lung cancer, [60-62] diarrhea prostate cancer ovarian cancer, bladder cancer, neurotoxicity and severe BMS-310705 non-small cell lung cancer, gastric [62] MANUSCRIPTdiarrhea cancer, breast cancer

its benefit risk profile

BMS-753493 advanced solid tumors didn’t support further [63] investigation thereof

Discodermolide advanced solid malignancies lung toxicity [31]

MSA under preclinical trials

Name Type of Cancer Treatment Study Model Reference Peloruside A ACCEPTED Lung and breast tumor xenograft athymic nu/nu mice [3] [8] human breast cancer, and Laulimalide athymic NCr-nu/nu mice [3] [8] fibrosarcoma xenograft

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As is well known, there are 3 different inhibitor binding sites in microtubule: paclitaxel site, vincristine site and colchicine site. The microtubule stabilizers can influence the MT protein dynamics by binding to microtubule on the paclitaxel site. There is no doubt that the stabilization binding sites are important for understanding the mechanism of action and designing more simple and effective anticancer compounds in the future. Perhaps we can think about that focusing on the discovery of new potential compounds, the modification of the known MSAs, the stabilization binding sites, and mechanism of action of these compounds, so that we can design more and more potential molecule compounds by using organic chemistry and medicinal chemistry. At present, some scholars have reasonably designed a paclitaxel nanoparticle CBT-paclitaxel according to a biocompatible condensation reaction and enzymatic self-assembly, aiming at overcoming the multidrug resistance. The experiments of this paclitaxel nanoparticle in vitro exhibit a 4.5-fold increase than paclitaxel itself in

anti-multidrug resistance effects and in vivo that is 1.5-fold [190], suggesting a new strategy for overcoming such defects. However, lots of MSAs are mostly used in adult cancer treatment and aren’t as widely used as MANUSCRIPT the MDAs (mainly refer to vincristine) in childhood leukemia treatment. Reynolds et al. [191] have discovered that cabazitaxel shows superior activity than docetaxel when the both are used at a similar dose in the childhood cancer preclinical models, and these results will promote the research of applying MSAs to the childhood cancer treatment. Foreseeably, the study on combination therapy of two or multiple drugs will gradually increase in order to avoid some shortcomings of monotherapy or improve the curative effects. For instance, it can be equivalent to reduce the blood toxicity and overcome the chemotherapy drug resistance defects when high-affinity paclitaxel analog CTX-40ACCEPTED or the covalent binder zampanolide is combined with daunomycin in vitro , which is of great importance to the acute myelogenous leukemia treatment [192]. Ixabepilone combined with capecitabine has better therapeutic effects than the

capecitabine monotherapy in the metastatic breast cancer treatment [57]. The combination therapy of monoclonal antibody cetuximab with ixabepilone may be

effective in a small subset of triple negative breast cancer stem cells [58]. A

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ACCEPTED MANUSCRIPT combination treatment of MSAs (patupilone and paclitaxel) with antiangiogenic agents (everolimus and bevacizumab) can overcome tumor cell-linked MSA resistance, and this therapeutic schedule should be considered as a new strategy for

MSA-refractory tumor entities [193]. We can believe with no doubt that the combination therapy of two drugs or even more than three drugs will become the development trend of cancer treatment in future. Maybe we should consider to evaluate the mutual effects and drawbacks of several microtubule stabilizers rather than just one compound. 5. Future prospects

Most of the MSAs are natural extracts acquired from natural products with low contents, it could not meet the modern requirements if only rely on the separation and extraction. Besides, the natural extracts they usually have complex structures and load some non-pharmacophores, making it difficult to be applied in clinic. Therefore, it is an excellent way to solve the quantity problem and save the cost to get lots of molecules with effective biological activity quickly by chemical synthetic methods or simplify natural molecular structures. In 2014,MANUSCRIPT Kingston et al. [194] introduced a series of structurally simplified paclitaxel analogs with bioactive activity, and analyzed their active groups and pharmacophores, having referential meaning for the further design of paclitaxel analogs with simple structures and outstanding therapeutic effects. While on the other hand, it usually requires a long period and high cost for screening the synthetic molecules with specific therapeutic effects by the pharmacological researches and passing through clinical trials of the synthetic compounds with certain structures and activities. Thus it will be a feasible method to make a total synthesis, semi-synthesis or modification on the molecular structures of existing naturalACCEPTED MSAs after analyzing the structural characteristics thereof, which will not only get some compounds with specific targets quickly, but also can improve the efficiency of drug screening, save the production cost and reduce the development cycle. Besides, perhaps we can consider that using some natural products with explicit pharmacophores, such as the side chain of Taxol, and small molecules, for example, benzimidazole, benzoxazole and benzothiazole with a hydroxyl group, to make a

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ACCEPTED MANUSCRIPT coupling in organic synthesis, then study their activity to get some potential anticancer compounds in the future designing of MSAs. Undoubtedly, MSAs will be more and more widely applied in future cancer treatments, even they might alter microtubule dynamics in neurodegenerative diseases, such as Alzheimer’s disease, and have the therapeutic potential of these modern diseases [195]. Benbow et al. found that paclitaxel has significant effects in both dorsal root ganglia neuronal nuclei and axons and induces pronounced neurodegenerative effects [196]. Brunden et al. made a detailed review that the evidence of MT abnormalities in a number of neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis and traumatic brain injury, which suggested that MT-stabilizing drugs hold promise for the potential

treatment of several neurodegenerative diseases [197]. Some researchers thought that peloruside A might be an attractive therapeutic candidate for the cancer treatment,

neurodegenerative tauopathies and autoimmune diseases [198], which would create a new research field of these compounds. One of the major challenges in the field of natural product drug development is that the limited natural supply of the compounds MANUSCRIPT and the difficulty of total synthesis. With the help of the researches on pharmacology, toxicology and SAR and so on, it will speed up the development and application of this kind of anticancer drugs. It will be a future development direction of these compounds that to confirm the pharmacophore of MSAs with better therapeutic effects, then to simplify the complex structures or direct synthesize the compounds with similar structures and pharmacophores by natural products combined with chemical synthesis, finally to develop more novel, effective MSAs with low toxicity even without side effects. In a word, the unclear bioactivity, SAR and mechanism of action of some MSAs need to be studied in-depthACCEPTED in the future, so do the structure simplification and chemistry synthesis.

ACKNOWLEDGMENTS Financial support from NSFC (No. 31570341), Key Technology Program from Sichuan Province, China (No. 2015SZ0105) and Key Program from Education 42 / 55

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Department of Sichuan Province (No. 16ZA0290) are greatly appreciated.

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ACCEPTED MANUSCRIPT Highlights:
Recent advances in the development of microtubule stabilizers were summarized in detail.
Natural microtubule stabilizers show significant applications in anti-cancer drug discovery.
Chemical synthesized small molecule compounds exhibit promising potency in the development of tubulin-binding drugs.

MANUSCRIPT

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