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Synthesis 2000, No. X, x–xx ISSN 0039-7881 © Thieme Stuttgart · New York
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Synthesis 2000, No. X, x–xx ISSN 0039-7881 © Thieme Stuttgart · New York
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REVIEW 2471
Advances in Synthetic Approaches for the Preparation of Combretastatin-Based Anti-Cancer AgentsCombretastatin-Based Anticancer AgentsRohit Singh,* Harneet KaurCenter for Drug Design, Academic Health Center, University of Minnesota, Minneapolis, MN 55455, USAFax +1(612)6252633; E-mail: singh109@umn.eduReceived 13 March 2009; revised 4 May 2009
SYNTHESIS 2009, No. 15, pp 2471–2491xx.xx.2009Advanced online publication: 10.07.2009DOI: 10.1055/s-0029-1216891; Art ID: E23809SS© Georg Thieme Verlag Stuttgart · New York
Abstract: The natural product combretastatin A-4 (CA4) is a po-tent anti-cancer agent known for its antimitotic and antiangiogenicproperties. The basic structure of CA4 has inspired the design andsynthesis of a variety of medicinally active analogues that take ad-vantage of the relatively simple stilbenoid architecture of the mole-cule. Here, we examine recent advances in the synthesis of variousCA4-based analogues. A significant focus is placed on the modifi-cations to the bridging alkene moiety of the stilbene scaffold forconformationally restricting the structure in a bioactive form. An ef-fort is also made to discuss promising ring modifications and re-placements, including the incorporation of indazole and oxindolerings, as well as the design and synthesis of amino-substituted ana-logues.
1 Introduction2 New Approaches for the Synthesis of CA43 Synthetic Approaches for the Preparation of CA4 Ana-
logues3.1 Modification of the Bridging Moiety3.2 Modification of the Bridging Moiety via Heterocyclic
Functionalities3.3 Macrocyclic Rings Affording Conformational Restriction
to Combretastatin Analogues4 Amine Substituents on Aryl Rings of Combretastatin Ana-
logues5 Conclusions
Key words: natural products, drugs, combretastatin, inhibitors,antitumor agents, medicinal chemistry
1 Introduction
The discovery and development of small-molecule tubu-lin assembly inhibitors began with studies of colchicine(1, Figure 1) in the early 1930s.1 Research in this field re-ceived a major impetus in 1982 when Pettit et al. discov-ered and isolated (–)-combretastatin from Combretumcaffrum, a South African bush willow tree.2 The com-pound was found to be a potent inhibitor of cancer cellproliferation.3 Since then, a significant number of studieshave been directed towards analysis of combretastatin andrelated medicinally active compounds.4 CombretastatinA-4 (CA4) or 2-methoxy-5-[2-(3,4,5-trimethoxyphe-nyl)vinyl]phenol along with combretastatin A-1 (CA1)and their phosphate analogues CA4P and CA1P form afamily of some of the most potent combretastatins.5 After
extensive preclinical evaluation, the water-soluble pro-drug CA4P has emerged as a promising clinical candidatefor treating cancer.6,7
Tubulin is a heterodimeric protein which is present in alleukaryotic cells.8 Assembly of tubulin and disassembly ofthe polymeric form is a dynamic process which leads tothe formation of microtubules.9 The polymeric microtu-bules play an important role in the construction of the mi-totic spindle during cell division.10 The microtubules alsoperform intracellular functions such as transport andmaintaining cellular morphology.
Figure 1 Structures of colchicine, (–)-combretastatin, combretasta-tin A-4, combretastatin A-4 phosphate, combretastatin A-1 and com-bretastatin A-1 phosphate
Antimitotic drugs that inhibit growth of cancer cells byanti-tubulin action are classified into two categories: mi-crotubule-stabilizing agents and microtubule-destabiliz-ing agents. The microtubule-stabilizing agents work byinteracting directly with microtubules, enhancing the tu-bulin polymerization and hence stabilizing them againstdepolymerization. This action disturbs the dynamic pro-
OMe
O
MeO
OMe
MeO
4CA4P
P
OO–
O–
Na+
Na+
OMe
OH
MeO
OMe
MeO
2(–)-combretastatin
OH
OMe
O
MeO
OMe
MeO
6CA1P
P
OO–
O–Na+
Na+
O P
O O–
O–
Na+
Na+
OMe
OH
MeO
OMe
MeO
MeO
MeO
MeO
OMe
O
NH
O
1colchicine
3combretastatin A4 (CA4)
OMe
OH
MeO
OMe
MeO
5combretastatin A1 (CA1)
OH
2472 R. Singh, H. Kaur REVIEW
Synthesis 2009, No. 15, 2471–2491 © Thieme Stuttgart · New York
cess and is the foundation of the well-documented anti-cancer properties of drugs such as paclitaxel and docetax-el. In contrast to members of the taxoid family, the Vincaalkaloids and colchicinoids work by inhibiting the tubulinpolymerization.4f Different binding sites for Vinca alka-loids (such as vinblastine and vincristine) and colchicin-oids (such as colchicine and CA4) suggest that the twoclasses of compounds adopt different mechanisms of ac-tion.11 Furthermore, the failure of cancer chemotherapy isoften associated with an acquired resistance to variousanti-cancer drugs. The cause behind this multi-drug resis-tance characteristic has been identified as expression ofthe drug efflux transporter P-glycoprotein in increasedquantities. However, CA4 is not recognized by this glyco-protein and thus renders the protein impotent in develop-ing resistance to the drug.12
Various structural analogues of combretastatin have beensynthesized and analyzed for their anti-cancer activity.More prominent amongst them are the stilbenes, diarylethylenes, phenanthrene-based derivatives, macrocycliclactones as well as CA4-based macrocyclic derivatives.13
General structures of the above-mentioned classes of anti-cancer compounds are presented in Figure 2. Amongstthese classes, the stilbenes are more versatile and hencehave been studied with much profound interest. Addition-ally, the heterocyclic-ring-containing stilbenes provideanother sub-class with interesting steric effects worthy of
analysis. This report focuses on various stilbenoids anddiverse synthetic approaches employed to achieve thesetargeted anti-cancer compounds.
Figure 2 Various structurally diverse anti-tubulin derivatives
Rohit Singh obtained hisM.Sc. (Hons) degree fromPanjab University, India in2001. The next year, hejoined the University ofNew Orleans and obtained asecond M.Sc. degree in2003 for his work on orga-nocatalysis via N-heterocy-clic carbenes. He continuedhis research on the develop-ment of N-heterocyclic car-
benes as organic catalystsand efficient ligands in pal-ladium-catalyzed transfor-mations and obtained hisPh.D. from the University ofNew Orleans under the su-pervision of ProfessorSteven P. Nolan in 2007. Hethen joined the Center forDrug Design (University ofMinnesota) and worked onthe synthesis of combret-
astatin-based pyrazolineanti-cancer therapeuticagents. In 2008, he joinedthe group of Professor Rob-ert Vince (Director, Centerfor Drug Design, Universityof Minnesota) where he ispresently working on the de-sign and synthesis of Port-manteau inhibitors (reversetranscriptase/integrase) forthe treatment of HIV-AIDS.
Harneet Kaur obtained herM.Sc. (Hons) degree fromPanjab University, India in2001. She joined the Uni-versity of New Orleans in2002 and obtained a secondM.Sc. degree in 2003 for herwork on the development ofwell-defined N-heterocycliccarbene–copper complexesunder the supervision ofProfessor Steven P. Nolan.In 2003, she joined Profes-sor Mark L. Trudell’s group
and worked towards herPh.D. on the design and syn-thesis of tropane derivativesas potential therapeutics forcocaine abuse. After thehurricanes in New Orleans,she continued her researchat the National Institute ofDrug Abuse under the guid-ance of Dr. Amy H. New-man (NIDA – IRP AssociateDirector Translational Re-search, NIH, Baltimore).She returned to the Univer-
sity of New Orleans to fin-ish her degree in 2007. Shethen joined Professor DavidM. Ferguson’s group (Cen-ter for Drug Design andDepartment of MedicinalChemistry, University ofMinnesota) for post-doctor-al research on the synthesisof substituted heterocyclicanalogues as drug targets forcancer studies and catalyticinhibitors of human topo-isomerase-II.
Biographical Sketches
stilbene derivatives diaryl ethylene derivatives
R1
R2
R1
R2
phenanthrene derivatives
R1
R2
heterocyclic derivatives
X
R1
R2
n
OH
O
OO n
R1
R1
R2O
O
n
macrocyclic lactones CA4-macrocyclic derivatives
7 8
9 10
11 12
REVIEW Combretastatin-Based Anticancer Agents 2473
Synthesis 2009, No. 15, 2471–2491 © Thieme Stuttgart · New York
2 New Approaches for the Synthesis of CA4
Building upon their protocols for the synthesis of differentcombretastatins,14 Pettit and co-workers utilized a Wittigreaction in their first synthesis of CA4 in 1995(Scheme 1).15 To obtain the desired product, isovanillin13 was silylated by tert-butyldimethylsilyl ether followedby reduction of the aldehyde 14 with sodium borohydrideto obtain benzyl alcohol 15. The alcohol was then bromi-nated by trimethylsilyl chloride and lithium bromide. Thephosphonium bromide 17 for the key Wittig step was ob-tained by reaction of the bromide 16 with triphenylphos-phine. Phosphonium bromide 17 was coupled with 3,4,5-trimethoxybenzaldehyde to obtain a mixture of the prod-uct with Z/E stereoselectivity of 1:1.5 in 93% yield. Thecis-isomer 18 was separated from the trans-isomer bychromatography. Tetrabutylammonium fluoride (TBAF)was employed for desilylation to obtain the desired com-pound CA4 (3).
Scheme 1 Synthesis of CA4 via Wittig reaction by Pettit and co-workers
Harrowven and co-workers have improved the stereose-lectivity in the classic Wittig reaction for the synthesis ofCA4 and (Z)-stilbene analogues.16 The approach was in-spired from the work of Gilheany and co-workers, whodemonstrated that ortho-halide substituents in the alde-hyde and phosphonium salt components led to enhancedZ-stereoselectivities.17 As outlined in Scheme 2,
Harrowven and co-workers carried out the condensationof phosphonium salt 19 with benzaldehyde derivative 20employing potassium tert-butoxide. The reaction provid-ed (Z)-stilbenes 21 with remarkable selectivity rangingfrom 9:1 to 45:1. Removal of the ortho-bromo directinggroups by halogen–metal exchange with n-butyllithiumfollowed by quenching with water afforded the stilbenederivatives 22.
Scheme 2 Synthesis of CA4 and analogues via Wittig reaction byHarrowven and co-workers
The Taylor group devised a concise route for the synthesisof CA4 utilizing the Ramberg–Backlund reaction forobtaining the key stilbene unit as shown in Scheme 3.18
Thiol 24 was first prepared by reaction of 3,4,5-trimeth-oxybenzyl alcohol (23) and Lawesson’s reagent and sub-sequently coupled with bromide 25 using potassiumhydroxide in ethanol. The resulting sulfide was oxidizedto the sulfone 26 using m-chloroperoxybenzoic acid.
Scheme 3 Synthesis of CA4 and analogues by application ofRamberg–Backlund conditions
CHO
OMe
OH
TBSCl
CHO
OMe
OTBS
NaBH4
CH2OH
OMe
OTBS
TMSCl, LiBr
CH2Br
OMe
OTBS
Ph3P
CH2PPh3Br
OMe
OTBS
13 14
15 16
17
MeO
MeO
OMe
CHO
MeO
MeO
OMe
OMe
OTBS
93% (Z/E = 1:1.5)
18
MeO
MeO
OMe
OMe
OH
3
TBAF
93%
Br
OMe
MeO
MeOPPh3Br
CHO
X
OR
OMe
+
Br
OMe
MeO
MeO
X
OR
OMe
19 20
21a: X = Br, R = H, 96% (Z/E = 9:1)21b: X = I, R = H, 98% (Z/E = 9:1)
1. n-BuLi
2. H2OOMe
MeO
MeO
X
OR
OMe
22a: 87% (Z/E = 9:1)22b: 90% (Z/E = 9:1)
KOt-Bu
MeO
MeO
OMe
XHBr
OTBS
OMe+
23, X = O
24, X = SLawesson's reagent
1. KOH
2. m-CPBA
25
MeO
MeO
OMe
SOTBS
OMe
O Oconditions
MeO
MeO
OMe
OTBS
OMe
Chan conditions: 81% (Z/E = 1:9)Franck conditions: 72% (Z/E = 15:85)Meyers conditions: 69% (Z/E = 53:47)
26
27
(49%, 2 steps)
2474 R. Singh, H. Kaur REVIEW
Synthesis 2009, No. 15, 2471–2491 © Thieme Stuttgart · New York
The sulfone was then subjected to tandem halogenation–Ramberg–Backlund conditions as reported by Chan andco-workers (CF2Br2, KOH–Al2O3, t-BuOH, 0 °C to r.t.,12 h).19 The one-pot process furnished the O-silylatedproduct as a mixture of Z- and E-isomers (1:9) in 81%yield. CA4 was obtained by deprotection via tetrabutyl-ammonium fluoride on silica gel. The reaction was alsoperformed under Franck conditions (C2F4Br2, KOH–Al2O3, t-BuOH, D, 12 h),20 and the original halogenation–Ramberg–Backlund conditions as reported by Meyers andco-workers (CCl4, KOH, t-BuOH, H2O).21 While Franckconditions furnished the product with improved stereose-lectivity (Z/E = 15:85) in 72% yield, Meyers conditionsafforded the product with best seteroselectivity (Z/E =53:47) in 69% yield.
Alami and co-workers devised a new hydrosilylation–protodesilylation process for obtaining combretastatin-based inhibitors of tubulin assembly.22 These authors re-cently demonstrated the efficacy of heterogeneous plati-num oxide as a catalyst for the hydrosilylation of para-and ortho-substituted diarylalkynes in cis-fashion.23 Incontrast to the better-known stereoselective approach forthe semi-reduction of diarylalkynes detailed by Lindlar,24
which suffers from such shortcomings as formation of al-kane due to over-reduction and Z-to-E isomerization, thereported method presents addition of H–Si bond in stereo-selective cis-manner, and the regioselectivity of the reac-tion is independent of the choice of platinum catalyst butis instead governed by ortho-directing effects (ODE).25
The generic synthetic scheme (Scheme 4) involved the si-lylation of alkynes 28 in the presence of a platinum cata-lyst, followed by desilylation by tetrabutylammoniumfluoride in tetrahydrofuran. Optimization studies includedan analysis of the activity of various platinum sourcessuch as Pt2O, PtCl2, Pt/C, PtCl4 and Speier’s catalyst(H2PtCl6). The best conditions were found to be Pt2O withloading of 7 mol%, 1.5 equivalents of HSiOEtMe2 fol-lowed by treatment with 1.5 equivalents of tetrabutylam-monium fluoride at 0 °C. The synthetic route wasmodified to make it a one-pot process by exploiting thevolatility of HSiOEtMe2. A variety of CA4 analogueswith electron-donating as well as electron-withdrawingsubstituents were synthesized by this method. CA4 wassynthesized with a Z/E ratio of 9:1. Overall, this newmethod provided a mild, chemoselective protocol for thesynthesis of (Z)-stilbenes.
Another efficient route to CA4 and analogues uses thePerkin condensation between aldehydes and aryl acid de-rivatives mediated by acetic anhydride and triethylamine(Scheme 5).26 Commercially available 4-methoxypheny-lacetic acid (31) was first brominated to obtain the Perkincondensation precursor 3-bromo-4-methoxyphenylaceticacid (32). The aryl acid derivative 32 was then condensedwith 3,4,5-trimethoxybenzaldehyde in a reaction mediat-ed by acetic anhydride and triethylamine by heating at130 °C for five hours to furnish 1,2-diarylacrylic acid 33with excellent stereoselectivity (Z/E = 19:1). Hydroxyla-
tion was then performed to obtain the acrylic acid deriva-tive 34, which underwent decarboxylation with assistanceof quinoline and copper powder at 220 °C. CA4 was ob-tained in 71% yield via this method.
Camacho-Davila reported the first example of the utiliza-tion of a Kumada–Corriu cross-coupling reaction for thesynthesis of CA4 (Scheme 6).27 The Grignard componentof the Kumada–Corriu coupling, 38, was synthesized bybromination of the easily available guaiacol (35) in athree-step process. The free hydroxy was then protectedwith a tert-butyldimethylsilyl group followed by reactionwith magnesium to obtain the key intermediate 38. Thehalide reagent was prepared by subjecting 3,4,5-tri-methoxybenzaldehyde to Corey–Fuchs conditions (CBr4,Ph3P) followed by palladium-catalyzed reduction to ob-tain the Z-isomer 41 in a stereoselective process. The keycoupling step was performed with ferric acetylacetonateas catalyst to achieve the O-silylated product with reten-tion of stereochemistry. The final product 3 was obtained
Scheme 4 Synthesis of CA4 and analogues by a hydrosilylation–protodesilylation process
R1 R2
H-[Si], Pt catalyst
R1 R2
[Si]
R1 R2
28
29 30
TBAF
(Z/E = 9:1)
R1 = 4-MeO, 2,3,4-tri(MeO)R2 = H, 3-HO, 4-MeO
(38–97%)
(49–90%)
Scheme 5 Synthesis of CA4 by Perkin condensation
COOH
OMe
COOH
OMe
Br
OMe
MeO
MeO CHO
Br2
Ac2O, Et3N
OMe
MeO
MeO COOH
OMe
Br
1. NaOH, CuSO4
2. H+
OMe
MeO
MeO COOH
OMe
OH OMe
MeO
MeO
OMe
OH
quinoline, Cu
31 32
33
34 3
(94%)
(75%, Z/E = 19:1)
(75%)
(71%)
REVIEW Combretastatin-Based Anticancer Agents 2475
Synthesis 2009, No. 15, 2471–2491 © Thieme Stuttgart · New York
by desilylation, mediated by potassium fluoride dihy-drate, in 40% overall yield. The efficiency of the protocol,in achieving stereoselectivity in the synthesis of CA4 witha metal-catalyzed coupling reaction as key step, is note-worthy.
Scheme 6 Synthesis of CA4 by Kumada–Corriu cross-coupling re-action
3 Synthetic Approaches for the Preparation of Analogues of CA4
Since the initial discovery of combretastatin and the sub-sequent determination of its absolute configuration byPettit et al. in 1987,28 a plethora of studies exploring thestructure–activity relationship (SAR) of combretastatin-based compounds have been reported. These studies havefocused on multiple substitutions on the phenyl rings(both ring A and ring B) as well as variations to the struc-ture of the bridging moiety between them (Figure 3).
The structural features of CA4 required for the robust in-hibition of tubulin assembly include a bridging moietyconnecting two phenyl rings with a cis disposition.
Although the conformation of tubulin-bound CA4 has notbeen analyzed completely, the fact that the tubulin bind-ing site is very flexible has been confirmed.29 To maintainthe structural integrity of the compounds that display inhi-bition of tubulin assembly, prevention of isomerization ofthe cis- compounds is mandatory. To achieve this goal,various methodologies have been adopted to lock thecompound in the desired cis conformation.
3.1 Modification of the Bridging Moiety
Amongst the structural modifications envisaged on theCA4 structure, modification of the bridging moiety is avery appealing option since it opens avenues for incorpo-rating a large variety of functional groups and substituentsto achieve optimum biological results.30
To impart structural rigidity to the compounds, the incor-poration of different functional groups, particularly cyclicmoieties, at the bridge connecting the two phenyl ringshas found many applications. A recent study exploitingthis postulate has resulted in the synthesis of a series of cy-clopentanone analogues of CA4.31 Jaggi and co-workersprepared 2,3-diaryl-4/5-hydroxycyclopent-2-en-1-oneanalogues of CA4 by replacing the cis double bond with4/5-hydroxycyclopentanone. The synthetic route(Scheme 7) consists of the generation of furyllithium fromthe reaction of furan with n-butyllithium in tetrahydrofu-ran and its subsequent condensation with a substitutedbenzaldehyde. The resultant 2-furylmethanol 43 was thenconverted into 2-aryl-4-hydroxycyclopentenone 44 byemploying zinc chloride as a weak Lewis acid.32 Next, thehydroxy group on 44 was protected by tert-butyldimeth-ylsilyl chloride. The protected cyclopentanone intermedi-ate was then coupled with various aryl halides viapalladium-catalyzed Heck reactions in the presence of po-tassium carbonate as base and tetrabutylammonium bro-mide (TBAB) as an additive to obtain the 4-hydroxyseries of compounds 45.
The 5-hydroxy series of compounds was prepared bytreating the cyclopentenone intermediate 46 with substi-tuted aryl halide in the presence of magnesium or n-butyl-
OMe
OH
1. Ac2O, Et3N
2. Br2
3. KOH, MeOH OMe
OH
Br
imidazole
OMe
OTBS
Br
Mg
OMe
OTBS
MgBr
35 36
37 38
MeO
MeO
OMe
CHOCBr4, Ph3P
MeO
MeO
OMe
Br
Br
MeO
MeO
OMe
BrPd(PPh3)4, Bu3SnH
39 40
41
38, Fe(acac)3, 0 °C, THFMeO
MeO
OMe
OMe
OTBS
18
KF⋅2H2O
MeO
MeO
OMe
OMe
OH
3
(overall yield 40%)
(78%, 3 steps)
(96%)
(76%, 2 steps)
(61%) (95%)
TBSCl
Figure 3 Structural features of combretastatin-based compoundsrequired for anti-tubulin activity
R1
R2
variable bridging moietycis configuration required
substituents on ring B
substituents on ring A
2476 R. Singh, H. Kaur REVIEW
Synthesis 2009, No. 15, 2471–2491 © Thieme Stuttgart · New York
lithium to get the 1,2-addition product 47. Further reactionof the tertiary alcohol with pyridinium dichromate indichloromethane afforded the desired product via allylicrearrangement of the chromate ester.33
Following the synthetic protocol, a series of 56 new com-pounds was synthesized and analyzed for biological activ-ity. Assessment of their cytotoxicity, as well as apoptoticand tubulin assembly inhibition properties, was carriedout. The in vitro cytotoxicity data indicated superior po-tential for the 5-hydroxycyclopent-2-en-1-one com-pounds 50 and 51, shown in Figure 4. These compoundsdemonstrated a very high cytotoxicity with IC50 < 2.7 nMin a panel of human cancer lines consisting of oral, larynx,ovary, colon, lung and pancreas cancer cells. Further anal-ysis of these compounds for tubulin polymerization inhi-bition activity revealed a significant difference between
50 and 51. Compound 51 was found to be more stronglyanti-tubulin with a low IC50 value of 1.75 mM.
In a separate study, Flynn and co-workers synthesized in-hibitors of tubulin assembly with structural features simi-lar to those of CA4.34 The work focused on incorporatingstructural rigidity analogous to CA4 by introducing in-danone and indenone motifs in the desired molecules. Ini-tially, aldehydes were brominated to obtaindibromostyrenes 55 and 56 (Scheme 8) which were thenconverted into lithium phenylacetylides by treatment withn-butyllithium. The lithium phenylacetylides were uti-lized for preparation of diarylpropynones 57 and 58. Next,the authors utilized their previously developed palladium-catalyzed hydrostannylation methodology for the prepara-tion of chalcones.35 Synthesis of diarylpropynones wasfollowed by the key step of palladium-catalyzed hy-drostannylation coupling to obtain the desired chalcones59. Although stereochemical retention is typically ob-served in the hydrostannylation reaction, the chalconeswere isolated as a mixture of both Z- and E-isomers. Thepresence of a catalytic amount of triphenylphosphine hasbeen acknowledged to be the reason for this stereochemi-cal outcome. Next, the indanones 60 were prepared byperforming a Nazarov cyclization of the 2-aryl- and 2-aroylchalcones using cupric triflate or methanesulfonicacid. The indanones were then oxidized to the correspond-ing indenones 61 by 2,3-dichloro-5,6-dicyanoquinone(DDQ). All indanone compounds were isolated as thetrans isomers. Various chalcones, indanones and inde-nones prepared were tested for their tubulin polymeriza-tion inhibition activity and were compared to the activityof CA4. The chalcones were determined to possess bettertubulin inhibitory properties than indanones and inde-nones and were also evaluated for activity in inhibition ofMCF-7 breast cancer cells. However, none of the com-pounds displayed inhibition of these cancer cells, whereasCA4 itself performed well against them, with activity inthe nanomolar range.
Scheme 7 Synthesis of cyclopentanone derivatives of CA4
O O
OH
MeO
MeO
OMe
1. n-BuLi
2. OMe
MeO
MeO CHO
42
MeO
MeO
OMe
O
OH
MeO
MeO
OMe
O
OH
1. TBSCl
2. Pd(OAc)2
Mg or n-BuLi
MeO X
MeO
MeO
OMe
O
OTBS
MeO
MeO
OMe
OTBS
HO
OMe
MeO
MeO
OMe
O
43
44
45
46
47 48
X
R1
R2
R1
R2
MeO
MeO
MeO
OMe
O
49MeO
TBAF
OTBS
OH
ZnCl2
PDC
(52%)
(45%)
(88%)
Figure 4 5-Hydroxycyclopent-2-en-1-one compounds 50 and 51
R3
R2
R1
R4
X
R7
MeO
MeO
MeO
OHO
OMe
R6
R5
R8
50
MeO
MeO
MeO
OHN
OMe
HO
51
REVIEW Combretastatin-Based Anticancer Agents 2477
Synthesis 2009, No. 15, 2471–2491 © Thieme Stuttgart · New York
Pinney and co-workers have attempted to incorporate thestructural features of combretastatin compounds by pre-paring benzosuberene analogues of combretastatin.36 Thesynthetic route involved the preparation of 68 and 70(Scheme 9) as key intermediates. A hydroxy group wasfirst introduced on 6-methoxy-1,2,3,4-tetrahydronaphtha-lene (62), followed by protection of the 5-hydroxy regioi-somer 63 as the isopropoxide, leading to the formation of65. The protected tetralin derivative was then regioselec-tively oxidized at the benzylic position to obtain tetralone66. The ketone was transformed into a methylene unit byperforming a Wittig reaction. Formation of 67 precededthe key ring-expansion step, which was achieved by utili-zation of cyanogen azide to obtain the important interme-diate 68. Ketone 68 was the converted into regioisomer 70by subjection to Clemmensen reduction conditions, fol-lowed by oxidation via chromium trioxide. The key inter-mediates 68 and 70 were then coupled with lithiated
trimethoxybenzene in parallel reactions. The couplingproducts were dehydrated through an acetic acid cata-lyzed process, then the hydroxy group was deprotected bythe use of a Lewis acid to obtain the desired compounds74 and 75.
By following the presented synthetic scheme, the authorsprepared various benzosuberene analogues by incorporat-ing distinct functionalities on the aromatic rings of 74 and75, and they performed biological testing to evaluate theproducts’ activity in tubulin polymerization and non-
Scheme 8 Synthesis of CA4-based indanone and indenone deriva-tives
O H
R1
OMe
52, R1 = H
53, R1 = OH
54, R1 = Oi-Pr
i-PrBrK2CO3
55, R1 = H
Br2C H
R1
OMe
56, R1 = Oi-Pr
CBr4, Zn
OMe
MeO
MeO
1. n-BuLi
MeO
MeO
MeO
OMe
MeO
MeO
O
OMe
MeO
MeO
O
OMe
R1
57, R1 = H (100%)
58, R1 = Oi-Pr (66%)
R3
OMe
R1
Ph3P
X
O
X = H, Cl
R1 = H, Oi-PrR3 = PMP, TMP (87–93%)R3 = C(O)PMP, C(O)TMP (81–84%)
Pd2(dba)3, Ph3P, n-Bu3SnH
X
R2
MeO
R2
X = C(O)Cl, IR2 = OMe, H
59
MeSO3H, CuOTf2
O
R3
R1
OMeR1 = H, Oi-PrR3 = PMP, TMP (87–94%)R3 = C(O)PMP, C(O)TMP (87–99%)
60
MeO
MeO
MeOO
R3
R1
OMeR1 = H, Oi-PrR3 = PMP, TMPR3 = C(O)PMP, C(O)TMP
61
(59–80%)
DDQ
2. MnO2
Scheme 9 Synthesis of benzosuberene anti-tubulin compounds
MeO
1. TMEDA, s-BuLi
2. B(OMe)3
3. H2O2, AcOH
MeO
R2
R1
63, R1 = H, R2 = OH (63%)64, R1 = OH, R2 = H (23%)65, R1 = Oi-Pr, R2 = H (93%)Br
Cs2CO3,
65DDQ
(71%)MeO
Oi-Pr
X
66, X = O67, X = CH2 (68%)
MePPh3I, NaH
MeO
Oi-Pr
O
68
CNN3
(33%)
MeO
Oi-Pr
MeO
Oi-Pr
O
Zn(Hg), HCl
(19%)
AcOH⋅H2O
69 70
68 +
MeO
MeO
OMe
Br MeO
MeO
OMe
OMe
Oi-Pr
OHn-BuLi
72(66%)
MeO
MeO
OMe
OMe
Oi-Pr
73
AcOH
(100%)
AlCl3
(41%)
MeO
MeO
OMe
OMe
OH
74
70
MeO
MeO
OMe
75
OMe
OH
CrO3
62
71
(13%)
2478 R. Singh, H. Kaur REVIEW
Synthesis 2009, No. 15, 2471–2491 © Thieme Stuttgart · New York
small cell lung cancer, prostate cancer and ovarian cancercell lines. The SAR studies confirmed 75 as the most ac-tive compound. The cis disposition of the two aromaticrings analogous to CA4 potentiates the activity of thiscompound. The compound was found to have nanomolaractivity against the three cancer cell lines and is thus avery promising candidate for further studies and develop-ment as an effective anti-cancer agent.
Apart from the above-discussed elaborate syntheticschemes, a few easy-to-prepare combretastatin analogueshave also been explored. One such attempt led to the syn-thesis of carbazole sulfonamide analogues.37
The preparation of carbazole sulfonamides was precededby the synthesis of the carbazole sulfonyl chloride inter-mediates 77 (Scheme 10) from commercially availablecarbazoles 76.38 The carbazole sulfonyl chloride precur-sors were then treated with different anilines in the pres-ence of stoichiometric equivalents of triethylamine tofurnish the desired carbazole sulfonamides 78 in moderateto excellent yields.
Scheme 10 Synthesis of carbazole sulfonamide anti-tubulin com-pounds
The general structure of the synthesized compounds and afew representative examples are presented in Figure 5.The synthesized compounds were evaluated for activity inhuman leukemia cells. The SAR studies revealed that thepresence of an alkyl group on the nitrogen at the 9-posi-tion is required for strong activity in these compounds. Onreplacing carbazole with dibenzofuran (i.e., 82), a loss ofpotency was observed. Therefore, the presence of a carba-zole moiety is central to the success of this series of com-pounds. The studies also revealed that, unlike in CA4itself, the presence of the three methoxy substituents onring A is not a pharmacophoric requirement for activity inthis series of analogues, since the compounds with re-placed methoxy groups on ring A were found to be equal-ly potent.
In a different study, Alami and co-workers replaced thecis double bond of CA4 with a 1,2-diketo functionality byway of a user-friendly synthetic protocol that involved ametal-mediated cross-coupling reaction (Scheme 11).39
The diarylalkynes prepared via Sonogashira cross-cou-pling reactions were oxidized to achieve the desired benzilcompounds. The authors modified their previously report-ed oxidation protocol using iron tribromide anddimethylsulfoxide40 to include nitrile, phenols andanilines by replacing the iron catalyst with palladium(II)iodide. The modified catalytic system performed exceed-ingly well in furnishing the desired benzils with variousfunctionalities including acetates, acetamides, fluorineand heterocycles on ring B in a user-friendly protocol. Thecompounds were found to possess excellent antiprolifera-tive activity at nanomolar concentrations.
X
R1
1. ClSO3H
2. POCl3, PCl5 X
R1
ClO2S
Et3N, Py
R2
R3
R4
NH2HNR2
R3
R4
SO2
XR1
76 77
78
(56–95%)
Figure 5 General structure and representative examples of anti-tubulin carbazole sulfonamides
HNR1
R2
R3
A
B
general structure
SO2
XR4
HNMeO
MeO
OMe
SO2
NEt
HNMeO
MeO
OMe
SO2
NH
HN
MeO
SO2
NEt
HNMeO
MeO
OMe
SO2
O
79 80
81 82
Scheme 11 General synthesis of benzil analogues of combretastatin
MeO
MeO
OMe
X
+R
MeO
MeO
OMe
R
H
MeO
MeO
OMe
O
O
R
PdCl2, CuI(PPh3)2
83 84
85 86
PdI2
(31–100%)
REVIEW Combretastatin-Based Anticancer Agents 2479
Synthesis 2009, No. 15, 2471–2491 © Thieme Stuttgart · New York
3.2 Modification of the Bridging Moiety via Het-erocyclic Functionalities
In addition to the above-mentioned studies, a major vol-ume of work related to the modification of the CA4 bridg-ing moiety has focused on the use of heterocyclic rings forattaining the stereochemical congruity and structural fea-tures of CA4 which are required for optimum biologicalactivity. The presence of heteroatoms in a cyclic moietyaround the bridge not only helps in retaining the bioactiveconfiguration of the molecule, it also makes the targetedcompounds amenable to further manipulations, thus lead-ing to a broader variety of SAR studies. Some of the dif-ferent heterocyclic groups utilized for various SARstudies have included azetidinones,41 isoxazoles,42 imida-zoles,43 triazoles,4g pyrazoles,44 epoxides,44 thiophenes,45
benzo[b]thiophenes46 and furanones.47
In a recent report, Welsh and co-workers studied the in-corporation of a triazole ring at the bridge of CA4-basedanti-tubulin compounds.48 Alteration of the CA4 B-ringhas also been studied. The synthetic route consisted ofpreparation of amides 88 (Scheme 12) starting from thereaction of different amines with 3,4,5-trimethoxybenzoylchloride (87). The amides were then converted into thio-amides 89 by Curphey’s method, utilizing P4S10 and hex-amethyldisiloxane.49 The method furnished thioamideswith various B rings in quantitative yields. To obtain in-dolyl-ring-incorporated compounds, the use of Lawes-son’s reagent facilitated obtaining the desirable thioamideproduct. Next, the thioamides were treated with hydrazineat 0 °C to obtain amidrazones 90 which were cyclized bythe use of triethylorthoformate under acidic conditions.
Scheme 12 Synthesis of triazole-based inhibitors of tubulin poly-merization
A series of B-ring substituted compounds were synthe-sized, including compound 96 (Figure 6) in which the B-ring was substituted with an indole group. The X-ray crys-tal structure of compound 96 was also elucidated.50 Bio-logical evaluation of the compounds revealed potentinhibition of tubulin polymerization and cytotoxicityagainst an array of cancer cell lines including multi-drug-resistant (MDR) cancer cell lines. Inhibitior–protein inter-actions were also studied by molecular modeling and dy-namics simulations. The molecular dynamics simulationson the tubulin–96 complex suggested that 96 interactscomprehensively with b-tubulin, but, in a-tubulin, it is in-volved in hydrophobic interactions with only Ala and Valresidues. The conclusions supported the general consen-sus that colchicine binds preferentially to b-tubulin.33,51
Figure 6 Structures of triazole-based inhibitors of tubulin polyme-rization
In another recently reported study, the triazole moiety wasutilized for the bridge modification in anti-tubulin com-pounds. Hansen and co-workers prepared a series of tri-azolium-CA4 analogues. Huisgen cycloaddition served asthe key reaction in the preparation of these compounds(Scheme 13).52
Initially, 3,4,5-benzaldehyde was converted into the ter-minal alkyne 98 through the use of Colvin rearrangementconditions (LDA, TMSCHN2, –78 °C).53 The magnesiumacetylide of 98 was generated by treating it with ethylmagnesium chloride. The acetylide thus generated thenreacted with azide 99 in the Huisgen cycloaddition54 as thekey reaction in the synthetic scheme. The process led tothe formation of the desired triazole derivatives of CA4 in
MeO
MeO
MeOCl
O
+ RNH2
MeO
MeO
MeONH
O
R
P4S10TMS-O-TMS
MeO
MeO
MeONH
S
R
MeO
MeO
MeONH
N
R
NH2
MeO
MeO
MeON
NNH
R
87 88
89
90 91
NH2NH2
H+
(overall yields: 69–88%)
Et3N
(MeO)3CH
MeO
MeO
MeON
NNH
OMe
MeO
MeO
MeON
NNH
N
MeO
MeO
MeON
NNH
OMe
MeO
MeO
MeON
NNH
OMe
MeO
MeO
MeON
NNH
MeMe
OH F
N
Me96
92 93
94 95
2480 R. Singh, H. Kaur REVIEW
Synthesis 2009, No. 15, 2471–2491 © Thieme Stuttgart · New York
moderate to excellent yields. Following this method, aseries of regioisomeric cis-triazole compounds was pre-pared and evaluated for cytotoxicity. After initial screen-ing against leukemia cancer cell line (K562), selectedcompounds were analyzed for their tubulin-assembly in-hibition activity. Although both 107 and 111 displayed tu-bulin assembly inhibition at micromolar concentrations,they were found to be less active than CA4. Compound111 was nevertheless tested in six human cancer lines. Itdisplayed nanomolar activity in the assays and was foundto be comparable in activity to CA4. Molecular modelingstudies revealed that the observed activity of 111 was dueto its binding affinity to the colchicine binding site of a,b-tubulin.
Scheme 13 Synthesis of triazole-based CA4 analogues with aHuisgen cycloaddition as the key reaction
In another instance of nitrogen-based heterocyclic com-pounds being utilized for bridge modification in anti-tu-bulin compounds, Moses Lee and co-workers studiedvarious pyrazoline-based CA4 derivatives (Figure 7).55
A previous study in Lee’s laboratories pertaining to pyra-zole-based CA4 derivatives revealed an unanticipated at-tenuation of bioactivity of the compounds.44 X-raycrystallographic analysis suggested that the aromaticity-induced planar structure of the pyrazole ring was the mit-igating factor in pyrazole-based compounds, as comparedto the twisted geometry of CA4.56 As a non-aromatic sub-stitute for the pyrazole ring, the authors explored the utili-zation of pyrazoline moiety in the targeted compounds. Anumber of pyrazoline derivatives were synthesized by thereaction of hydrazine hydrate with the correspondingchalcones (Scheme 14). Positive activity of N-acylated
pyrazolines in the inhibition of kinesin spindle protein(KSP) prompted the authors to synthesize N-acylated de-rivatives of CA4 and test them as potential therapeuticagents for the treatment of cancer. The synthesized com-pounds were subjected to in vitro cytotoxicity screening.
The bioactivity data indicated that pyrazoline compoundsare, indeed, suitable candidates for anti-cancer studies.The results also revealed that the presence of an acetylgroup on the pyrazoline nitrogen is detrimental to the bio-logical activity of the compounds.
MeO
MeO
MeO
H
O
MeO
MeO
MeO
LDA
TMSCHN2
1. EtMgCl
2. N3
MeO
R
MeO
MeO
MeO
N
N
N
OMe
R
97 98 99
100, R = H (83%)101, R = Br (86%)102, R = OH (79%)103, R = NO2 (67%)104, R = NH2 (80%)
MeO
MeO
MeO
N3
105
+
MeO
MeO
MeO
N
N N
OMe
R
OMe
R
107, R = H (64%)108, R = Br (67%)109, R = OH (77%)110, R = NO2 (34%)111, R = NH2 (98%)
106
EtMgCl
(91%)
Figure 7 Pyrazoline derivatives of CA4
MeO
MeO
OMe
NN
H R1
R2
R3
114: R1 = OH, R2 = OMe, R3 = H115: R1 = OMe, R2 = OMe, R3 = OMe116: R1 = OMe, R2 = OMe, R3 = H
MeO
MeO
OMe
NNR1
R2
R3
O
117: R1 = OH, R2 = OMe, R3 = H118: R1 = H, R2 = NO2, R3 = H119: R1 = H, R2 = Cl, R3 = H
MeO
NNR1
R2
O
MeO
120: R1 = NO2, R2 = OMe121: R1 = OMe, R2 = OMe
Scheme 14 Synthesis of pyrazoline-based anti-tubulin derivatives
O
MeO
MeO
OMe
OH
OMe
NH2NH2, EtOH 16 h, reflux or
NH2NH2, AcOH 3 h, reflux
MeO
MeO
OMe
NN
ROH
OMe
R = H, Ac
112
113(31–78%)
REVIEW Combretastatin-Based Anticancer Agents 2481
Synthesis 2009, No. 15, 2471–2491 © Thieme Stuttgart · New York
Pui-Kai Li and co-workers reported the use of an inhibitorof tyrosine kinase, SU5416,57 which is a receptor for thevascular endothelial growth factor, as a model for the de-sign of analogues of CA4.58 In preceding work, the au-thors reported the 2-indolinone-containing compound 122(Figure 8) as a growth inhibitor for prostate and breastcancer cell lines with nanomolar IC50 values.59 The au-thors recognized the structural congruency of SU5416,compound 122 and CA4 and decided to study the biolog-ical activity of compound 122 and analogues.
Figure 8 Structures of SU5416 and SU5416-based CA4 analogues
For the synthesis of the 3-benzylideneindolin-2-ones 123,the precursor 6-alkoxyindolin-2-ones 127 were first pre-pared by literature procedures as depicted in Scheme 15.60
Synthesis of 6-substituted 3-benzylideneindolin-2-oneswas achieved via coupling of the prepared indolin-2-ones127 and substituted benzaldehydes in the presence of pip-eridine. The coupling reaction furnished predominantlythe E-isomer of the final product. The configuration of thecompounds was assigned based upon the chemical shiftsof protons at the C-2¢ and C-6¢ positions in the phenyl ringand NOE experiments. The chemical shifts for the protonsare approximately 7.45–7.84 ppm for the E-isomers and7.85–8.53 ppm for the Z-isomers.
The synthesized compounds were tested for biological ac-tivity with a special focus on the activity of compound122. A screening of compound 122 in 53 different cancerlines revealed that the compound was very effective withGI50 below 10 nm in 46 out of the 53 cancer lines. Thecompound was found to be active in colon, renal, prostateand CNS cancer lines with concentrations of less than 10nm. To determine the effect of 122 on the progression ofcancer cells, a DNA profile comparison of pancreatic can-cer cells at different concentrations was performed. Theanalysis with podophyllotoxin, a known anti-mitotic, anti-microtubule agent as a control, revealed that 122 causedG2/M-phase cycle arrest in pancreatic cancer cells (PC-3).
The series of synthesized compounds was included in theSAR for biological activity against prostate and breast
cancer cell lines. The SAR showed that the presence ofthree methoxy groups on the benzylidene ring plays animportant role in imparting the activity to the compound.It was found that the number of methoxy groups requiredfor optimum activity is indeed three. Moreover, the threemethoxy groups are most active at 3-, 4- and 5-positions,as any alteration of number of methoxy groups or their po-sitions on the benzylidene rings diminished the com-pound’s activity. Furthermore, substitution of the threemethoxy groups with three methyl or three ethyl groupsalso led to a loss of cytotoxicity. Conceivably, this effectis due to a change in electronics of the aromatic ring aswell as an incorporation of a different hydrogen-bondingcapacity. A similar decrease in cytotoxicity was observedupon substitution of the 6¢-methoxy on the 2-indolinonering of compound 122. A complete loss of cytotoxicitywas observed upon replacement of the 6¢-methoxy withhydrogen. Replacement of the methoxy group with otherlonger and branched derivatives also resulted in a declinein cytotoxicity. Only a 6¢-ethoxy substituent maintainedactivity comparable to that of the methoxy group. Inagreement with 122 being the most cytotoxic compoundof the series, it was also established as the most potent inits ability to inhibit tubulin polymerization with an IC50 of4.5mM.
Scheme 15 Synthesis of SU5416-based CA4 analogues
Pontikis and co-workers used a metal-mediated tandemHeck–carbocyclization–Suzuki coupling protocol to pre-pare oxindole-based combretastatin analogues.61 In a pre-vious study, the authors reported the synthesis of dienederivatives 128 (Figure 9).62
To restrict the structural flexibility of the two aromaticrings on 128, the authors decided to incorporate aromaticsubstituents between the double bond via a tandem Heck–Suzuki coupling approach. Similar domino methodolgieshave been reported in stereoselective syntheses of hetero-cyclic derivatives such as indolinones,63 isoindolinones,64
indanes65 and indoles.66The key reaction for obtaining the
N H
OH
N
H
SU5416
MeO
MeO
OMe
N H
O
OMe
H
122
N R2
O
R3
H
R1
R1 = H, OMe, OEt, OPr, OBu, Oi-Pr, Oi-BuR2 = H, MeR3 = (mono-/di-/tri-) OMe, Me, Et
123
NO
R2R3
CHO
R3
piperidine, EtOHN R2
O
R3
H
R3
R1 = H, OMe, OEt, OPr, OBu, Oi-Pr, Oi-BuR2 = H, MeR3 = (mono-/di-/tri-) OMe, Me, Et
X
NO2
CH2(CO2Me)2, NaH
NO2
CO2Me
CO2Me
LiCl
(33–85%)
(56–86%) NO2
CO2Me Fe, AcOH
(60–93%)
R1R1
R1
124 125
126
127 123
X = F, Cl, Br
(25–77%)
2482 R. Singh, H. Kaur REVIEW
Synthesis 2009, No. 15, 2471–2491 © Thieme Stuttgart · New York
desired compounds involved the coupling of commercial-ly available 133 or 135 with suitable alkynamides 132 forcyclization to obtain (E)-3-arylmethyleneoxindoles 134and (E,E)-3-alkylideneoxindoles 136 (Scheme 16).
For the preparation of alkynamides, 2-iodoanilines67 (130,Scheme 16) were coupled with propynoic acid or but-2-ynoic acid using dicyclohexylcarbodiimide.68 The result-ing anilides 131 were then reacted with benzyl bromide ormethyl iodide with sodium hydride as the base to get thedesired alkynamides 132.
With the alkynamides in hand, the key step of palladium-catalyzed coupling was then performed with the respec-tive boronic acid. The process was catalyzed by 5 mol%palladium(II) acetate and 10 mol% triphenylphosphine inthe presence of a base. Use of cesium fluoride as base af-forded an excellent stereocontrol over the reaction, fur-nishing exclusively the desired E-isomer. The productwas obtained with yields as high as 80%.
Duan, Matteucci and co-workers postulated that the pres-ence of a nitrogen at the 2-position along with the oxygenof the carbonyl group could promote chelation withmetals (137) or hydrogen-bonding with water (138)(Figure 10). Such chelation would reduce the conforma-tional flexibility of the molecule, making it susceptible tomimicking a tubulin inhibitor such as CA4. To test theirtheory, they synthesized a series of 3-aroylindazoles witha focus on substitutions on the C-7 position of the indazolemoieties.69
Figure 10 Metal-chelation and hydrogen-bonding in indazole deri-vatives and general structure of the synthesized compounds
The synthesis of the desired compounds was achieved bythe coupling of 5-iodo-1,2,3-trimethoxybenzene (140)with trimethylsilylacetylene, and palladium as catalyst,followed by desilylation to give 141 (Scheme 17).70 Next,5-ethynyl-1,2,3-trimethoxybenzene (141) was coupledwith 4-iodo-3-nitroanisole in a palladium-catalyzed, mod-ified Sonogashira-type coupling to give 142 which wasreduced by iron to yield 5-methoxy-2-(3,4,5-trimethoxy-phenylethynyl)phenylamine 143. The indazole core in144 was obtained by cyclization of 143.71
The iodination of (6-methoxy-1H-indazol-3-yl)-(3,4,5-trimethoxyphenyl)methanone (144) was performed withgood regioselectivity to produce the 7-iodoindazole 145derivative with 85% yield (Scheme 18). A second Sono-gashira coupling was performed with terminal alkynes toget the desired 7-substituted indazoles 146, which werefurther derivatized to obtain 147 and 148.
All synthesized compounds were analyzed for cytotoxici-ty towards a human non-small lung cancer cell line, H460.A comparison revealed that the presence of the acetylenemoiety in the indazole compounds vastly improved thecytotoxicity. Compounds 149, 150 and 151 (Figure 11)were found to be the most active, with IC50 values of 1nM, 8 nM and 3 nM respectively.
Figure 9 General structure for diene derivatives of combretastatins
MeO
MeO
OMe
128
R2
R1
MeO
MeO
OMe
137
NHN
OMe
OMg2+
MeO
MeO
OMe
138
NHN
OMe
O H
OH
MeO
MeO
OMe
139
NHN
OMe
O
R
Scheme 16 Synthesis of oxindole derivatives of CA4 prepared viapalladium-catalyzed tandem Heck–Suzuki–Miyaura reaction
MeO
MeO
OMe
B(OH)2 MeO
MeO
OMe
R1
N
R2
R3
O
MeO
MeO
OMe
B(OH)2R1
N
R2
R3
O
MeO
OMe
MeO
NH2
DCC
130
N
131
R3X, NaH
(68–82%)
132
I
R2
I
R2
H
O
R1
I
R2
NR3
O
R1
129
132
+
Pd(OAc)2, Ph3P
132
Pd(OAc)2Ph3P
133 134
135 136
(36–58%)
(20–50%)
(55–90%)
R1
OH
O
REVIEW Combretastatin-Based Anticancer Agents 2483
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Compounds 149, 150 and 151 were further studied for ac-tivity in multi-drug-resistant cancer cell lines (MDR-1and MRP-1).13 All three compounds displayed strong an-tiproliferative activities, with IC50 values between 1.6 nMand 3.9 nM.
In addition to the nitrogen heterocycles, sulfur has alsobeen incorporated into the structural modifications toachieve optimum anti-cancer activity. In a study with thia-
diazole–linker-based combretastatin analogues, Yang,Ding and co-workers reported the design and synthesis oftwo series of 4,5-disubstituted 1,2,3-thiadiazole ana-logues of CA4 (152 and 153, Figure 12).72
The synthetic route for achieving the synthesis of thiadia-zole derivatives of the general structure 152, as shown inScheme 19, involved the phosphonation of 3,4,5-tri-methoxybenzaldehyde (154) by Pudovik reaction withdibutylamine.73155 was then protected by tetrahydropyr-an to obtain 156 before it was condensed with the desiredbenzaldehyde 157. The condensation was followed byacidic hydrolysis to obtain deoxybenzoins 158. In the fi-nal step, the substituted deoxybenzoins 158 were con-densed with toluenesulfonohydrazide in ethanol and theintermediates were treated with thionyl chloride in the keycyclization reaction to obtain the desired compounds 152.
For the synthesis of thiadiazides of general structure 153,3,4,5-trimethoxybenzaldehyde (154) was first trans-formed into its tosylhydrazone 159 and then treated withthe substituted benzaldehydes 157 to yield the desirabledeoxybenzoin 160 (Scheme 20). The key final step for cy-clization involved condensation with toluenesulfonohy-drazide followed by cyclization with thionyl chloride inchloroform to obtain the desired compounds 153.
The degree of antiproliferative activity of the complete se-ries of compounds was determined against human mye-loid leukemia cells, human colon adenocarcinoma cellsand human microvascular endothelial cell lines. The mostpotent cytotoxic compounds from the synthesized serieswere analyzed for interactions with the microtubule sys-tem and compared with the control compound CA4. Theinhibition potency in the polymerization of tubulin wasalso tested. Several compounds displayed activity thatwas equipotent to that of CA4 in the study. The effects ofthe compounds on tubulin were also confirmed via indi-rect immuno-staining.
Scheme 17 Synthesis of 3-aroylindazoles
MeO
MeO
OMe
I
TMS
1. PdCl2(PPh3)2 CuI, Et3N (89%)
2. TBAF, THF (82%)
+
140
MeO
MeO
OMe
PdCl2(PPh3)2 CuI, Et3N
OCH3
I
R
MeO
MeO
OMe
OMe
R
Fe, HClEtOH
141
142, R = NO2 (90%)143, R = NH2 (54%)
NaNO2, HCl
MeO
MeO
OMe
NHN
OMe
O
144
(80%)
Scheme 18 Synthesis of 7-substituted indazoles
144
MeO
MeO
OMe
NHN
OMe
O
I
145
NIS, AcOH R
PdCl2(PPh3)2CuI, Et3N
MeO
MeO
OMe
NHN
OMe
O
146
R
MeO
MeO
OMe
NHN
OMe
O
147
TBAF
MeO
MeO
OMe
NHN
OMe
O
NH2
NH2NH2⋅H2O146
148
(85%)
(39–98%)
(72%)
(81%)
Figure 11 7-Acetylene-substituted aroylindazole analogues of CA4
MeO
MeO
OMe
NHN
OMe
O
149, R = Me150, R = H151, R = CH2OH
R
Figure 12 Thiadiazole derivatives of CA4
MeO
MeO
MeO
152
S
NN
R1
R2
MeO
MeO
MeO
153
N
NS
R1
R2
2484 R. Singh, H. Kaur REVIEW
Synthesis 2009, No. 15, 2471–2491 © Thieme Stuttgart · New York
The authors utilized a flow cytometry assay to investigatethe thiadiazole-targeted phase of the cell cycle whichcauses disruption leading to the antiproliferative activityof the compounds. The studies displayed an increase incells in G2/M phase along with a concurrent reduction inS and G1 cells as a function of concentration of the thia-diazole compound being tested. The results were com-pared with those of the control compound CA4, indicatingthat the thiadiazole compounds lead to G2/M arrest in the
cell cycle causing the aforementioned antiproliferative ac-tivity.74
Moses Lee and co-workers synthesized thioxopyrimidineanalogues of CA4 with a concise synthetic approach(Scheme 21).75 The classic base-catalyzed Claisen–Schmidt condensation of substituted acetophenones 161and benzaldehydes 162 was performed to obtain chal-cones 163.76 The chalcones were then refluxed in ethanolin the presence of excess thiourea and potassium carbon-ate to obtain the desired series of 1,2,3,4-tetrahydro-2-thioxopyrimidine (164) analogues of CA4. Molecularmodeling studies performed on the synthesized com-pounds revealed that, indeed, the prepared thioxopyrimi-dine compounds possessed the twisted geometry essentialfor bioactivity in CA4-based compounds.56 However, incomparison to CA4, the compounds were found to be lessactive in biological studies. Nevertheless, the protocolprovided a user-friendly route of preparing CA4 ana-logues with increased solubility in aqueous media.
Scheme 21 Synthesis of 1,2,3,4-tetrahydro-2-thioxopyrimidineanalogues of CA4
With regard to oxygen-containing heterocycles, Barbierand co-workers reported on the interaction of 4-arylcou-marin analogues of combretastatins with the microtubulenetwork and analyzed their binding to tubulin.77
Compounds 165, 166 and 167 (Figure 13) were preparedby way of a method previously outlined by the authors.78
The synthesis involved treatment of 4-hydroxycoumarins168 with triflic anhydride to obtain the 4-trifluoromethyl-sulfonyloxycoumarins 169.79 Next, coupling of variousboronic acids was performed with the triflates 169 viaSuzuki–Miyaura cross-coupling protocol to obtain thedesired coumarin analogues of CA4 (Scheme 22).
Scheme 19 Synthesis of thiadiazole-containing combretastatin de-rivatives 152
MeO
MeO
MeO
154
H
O
POMeMeO
OHMeO
MeO
MeO
155
P
OHO
OMeOMe
THP, TsOHMeO
MeO
MeO
156
P
OO
OMeOMe
THP
1.NaH, 157
R2
R1
H
O
157
2. HCl
MeO
MeO
MeO
158
O
R1
R2
1. H2NNHTs
2. SOCl2
MeO
MeO
MeO
152
S
NN
R1
R2
n-Bu2NH
(79%)
(35–80%)
Scheme 20 Synthesis of thiadiazole derivatives of combretastatin
MeO
MeO
MeO
154
H
O
MeO
MeO
MeO
159
N
NaH, 157MeO
MeO
MeO
160 R1
R2
NHTs
O1. H2NNHTs
2. SOCl2
MeO
MeO
MeO
153
N
NS
R1
R2
H2NNHTsMeO
OMe
MeO
O
R2
R3
R4
R1
R5
163R = OMe, H, OH, NO2, Cl
MeO
OMe
MeO
R2
R3
R4
R1
R5
164
NHHN
S
thiourea, K2CO3
(40–90%)
R = OMe, H, OH, NO2, Cl
MeO
MeO
OMe
O R1
R2
R3
R4
R5
H
O
+
161 162
NaOH
REVIEW Combretastatin-Based Anticancer Agents 2485
Synthesis 2009, No. 15, 2471–2491 © Thieme Stuttgart · New York
Amongst the various cross-coupling reactions, the Suzu-ki–Miyaura reaction is one of the most valuable tools insynthetic chemistry.80 It has been extensively studied andreported to include both heteroaryl as well as stericallyhindered unactivated substrates.81 Barbier and co-workerstook advantage of the versatility of Suzuki–Miyaura reac-tion in a process catalyzed by palladium with copper asco-catalyst to prepare the coumarin analogues of CA4.82
The cytotoxicity of the synthesized 4-arylcoumarin com-bretastatin analogues was analyzed in human breast cells.The study revealed that compounds 165 and 166 alteredthe cell cycle progression and induced apoptosis, whilecompound 167 did not show any viable activity in bindingtubulin.
To further analyze the effects of CA4 and its coumarinanalogues on the microtubule network of human breastcancer cell line (HBL100), immunofluorescence stainingof the microtubule network was performed. Visualizationand DNA content analysis of the microtubule network re-vealed compound 165 to be more active than compound166. However, the concentration required to obtain an ac-tivity comparable to that of CA4 was a 100-fold excess.
Ampac 7.0 software was utilized to optimize compounds165, 166, 167 and CA4 with the semiempirical AM1method for molecular modeling studies. Superimpositionof CA4, 165, 166 and 167 in the tubulin colchicine site in-dicated that the methoxy group in the 7-position of ring Aplays an important role in imparting activity to the com-pounds (Figure 13). Furthermore, an increase in hydro-gen-bonding capabilities by the substituents on ring C alsoplay a vital role in imparting potency to these compoundswith respect to the anti-tubulin activity they display.
Utilizing a similar triflate–boronic acid/ester Suzuki–Miyaura route, Beletskaya, Combes and co-workers syn-thesized polymethoxylated-4-heteroarylcoumarins.83 Theprotocol employed a Pd(dppf)Cl2/K2CO3/TBAB catalyticsystem (Scheme 23) in place of the Pd(PPh)4/Na2CO3/CuIsystem used by the Barbier group.77 The authors were ableto synthesize various compounds with heteroaryl boronicacids or esters, including benzofuran, indole, pyrimidine,pyridine and quinoline analogues, with excellent yieldsranging from 71% to 98%. Evaluation of the products’biological activity in breast cancer cell lines revealed theindole-substituted coumarin analogues to be the most po-tent amongst the tested compounds.
Scheme 23 Coumarin analogues of CA4 prepared via Pd(dppf)Cl2-catalyzed Suzuki–Miyaura reaction
3.3 Macrocyclic Rings Affording Conformation-al Restriction to Combretastatin Analogues
Pettit and co-workers reported some pioneering work inthe isolation and characterization of macrocyclic diarylether lactone inhibitors of tubulin polymerization.84
Medarde, Pelaez and co-workers studied the feasibility ofretaining the structural conformation and restricting thecompound to cis-form, essentially preventing isomeriza-tion without modifying the bridging double bond.85
Medarde reported the synthesis of macrocyclic structuresin which the para-positions of the A- and B-rings of com-bretastatin are linked through a five- or six-atom-chainlinker, thereby affording conformational restriction to thecompound. Two families of compounds with differentlinkers were synthesized (Figure 14).
The two families differed in the length of the linker chainas well as in the presence or absence of an extra oxygenatom as part of the linker. The substituent X on the B-ringwas limited to H and OH. A third modification includedreplacement of the B-ring itself with the more structurally
Figure 13 Coumarin analogues of combretastatin
O OR1
R3
R2
R4
R5
A B
C
O OMeO
MeO
OH
MeO
165O OMeO
MeO
MeO
OH
MeO
166
O OMeO
MeO
MeO
OMe
OH
167
Scheme 22 Synthesis of aryl-coumarin analogues of combretastatin
O O
OH
MeO
MeO
R1
(CF3SO2)2O O O
OTf
MeO
MeO
R1
ArB(OH)2, Pd(PPh3)4
CuI, Na2CO3
O OMeO
MeO
R4
R3
R2
R1
168 169
165: R1 = H, R2 = H, R3 = OMe, R4 = OH 166: R1 = OMe, R2 = H, R3 = OMe, R4 = OH167: R1 = OMe, R2 = H, R3 = H, R4 = OMe
Et3N
O O
OTf
R1
R3
R2Het-B(OR)2, Pd(dppf)Cl2
CuI, Na2CO3
O O
Het
MeO
MeO
R1
170
172: R1 = R2 = R3 = OMe 173: R1 = OMe, R2 = H, R3 = OMe174: R1 = OMe, R2 = OMe, R3 =H175: R1 = R2 = R3 = H
171(71–98%)
2486 R. Singh, H. Kaur REVIEW
Synthesis 2009, No. 15, 2471–2491 © Thieme Stuttgart · New York
rigid indolyl group, which had shown potent activity in aprevious study.86
The synthesis of the desired compounds followed the syn-thetic strategy outlined in Scheme 24. Starting from adouble Mitsunobu reaction87 of the linker diol with thecorresponding phenolic aldehydes, the di-aldehydes 178–183 were obtained. These were then subjected to the in-tramolecular McMurry pinacol conditions88 to furnish thedesired macrocyclic CA4 analogues 184 and 185.89 How-ever, the presence of a nitro group proved detrimental to
the reaction outcome and resulted in the formation of amixture of side-products. Amongst the synthesized com-pounds, 184 resulted in the formation of two diastereo-mers, while better stereoselectivity was observed in thecase of 185 as only the trans isomer was obtained.
A conformational analysis of the compounds revealedstrict restrictions on the macrocyclic compounds as com-pared to CA4.90 The aromatic rings were found to be lessorthogonal than in colchicines and padophyllotoxin. Therotation of rings in these compounds is extremely ham-pered because of the linkers and the presence of substitu-ents on rings. These restrictions are somewhat attenuatedfor the compounds of family II as compared to those offamily I with the shorter 3-oxapentamethylene linker.Consequentially, family I compounds did not show anysignificant biological activity. Although the compoundsbelonging to family II were found to be active in display-ing anti-tubulin activity, they performed with less potencythan CA4. The authors proposed that, contrary to the de-sired increase in potency of the compounds via the use oflinkers, the presence of sterically hindered linkers in thepara-position could be the reason for low potency of thesecompounds. Although previous studies have shown thatCA4 analogues with bulky groups on the B-ring maintainmoderate to high potency,91 the presence of linkers on thepara-position of the aromatic rings of macrocyclic ana-logues of CA4 was found to be detrimental to the opti-mum activity of the compounds.
4 Amine Substituents on Aryl Rings of Com-bretastatin Analogues
The effect of the presence of various substituents on thearyl rings of the CA4 structure has been studied. In thesecases the absence of a functionality to lock the compoundin the cis form makes the structure susceptible to a possi-bly detrimental isomerization. However, the presence ofthe various substituents helps in docking the drug mole-cule at the colchicine binding site, thereby making theseanalogues potent for inhibition of tubulin assembly.
Following a report by Pinney and co-workers detailing thesynthesis of various nitrogen-containing combretastatinderivatives including compound 188,92 Liou and co-work-ers synthesized a series of amino-combretastatins andevaluated their anti-tumor activity.93
The synthetic strategy for the preparation of these com-pounds involved a Wittig reaction between nitro-substi-tuted benzaldehyde 187 or 190 (Scheme 25) and 3,4,5-trimethoxybenzyl triphenylphosphonium chloride (186)or 4-methoxybenzyl triphenylphosphonium bromide(191) as the key reaction. The Wittig reaction was fol-lowed by the reduction of the nitro group using zinc inacetic acid to furnish 188 and 189, and 192–194, respec-tively.
Figure 14 Macrocyclic analogues of combretastatin
MeO
OMeO
O
X
Me
Me
Linker
family I family II
MeO
OMe
O
O
X
O
MeO
OMe
O
O
X
Scheme 24 Synthesis of macrocyclic analogues of CA4
OH
CHO
OMeX
YOHHO
176
DBAD or DIAD, Ph3P
O
CHO
OMeX
YOH
177
DBAD or DIAD, Ph3P
OHOHC
Z
O
CHO
OMeX
YO
178: X = OMe, Y = O, Z = H (80%)179: X = OMe, Y = (CH2)2, Z = H (62%)180: X = NO2, Y = O, Z = H (20%)181: X = NO2, Y = (CH2)2, Z = H (25%)182: X = OMe, Y = O, Z = NO2 (47%)183: X = OMe, Y = (CH2)2, Z = NO2 (62%)
Z
CHO
(64–89%)
MeO
OMe
O
O
H
Y
OH
OH[TiCl4]-Zn
178 184: Y = Ocis (11%) trans (35%)
[TiCl4]-Zn179 185: Y = (CH2)2
trans (23%)
[TiCl4]-Zn180, 181, 182, 183 NR
REVIEW Combretastatin-Based Anticancer Agents 2487
Synthesis 2009, No. 15, 2471–2491 © Thieme Stuttgart · New York
Scheme 25 Synthesis of aminocombretastatin analogues
For the synthesis of compounds 198 and 200(Scheme 26), the ylide 196 was first prepared via reactionof triphenylphosphine with 1-bromomethyl-3,4,5-tri-methoxy-2-nitrobenzene (195). Wittig reaction of 197 and199 with 196 was followed by reduction with zinc andacetic acid to obtain the desired compounds 198 and 200.
The synthesized compounds were tested against five hu-man cancer cell lines as well as multi-drug-resistant celllines for anti-tumor activity. Compounds 189, 192, 198
and 200 displayed significant antiproliferative activitywith IC50 values ranging from 11 to 55 nM. Upon beinginvestigated for in vitro tubulin polymerization inhibitoryactivities and colchicine binding activities, compounds189, 198 and 200 displayed activity comparable to that ofCA4. The compounds were found to be superior to col-chicine.
In a separate study, Pinney and co-workers elaboratedupon their previous extensive work to present the synthe-sis and biological studies of similar amino-combretastatincompounds.94 Once again, the key step for the strategyadopted by the authors involved a Wittig reaction. Theprecursors for Wittig reaction were prepared by the stepsoutlined in Scheme 27.
Scheme 27 Synthesis of precursors for Wittig reaction leading tothe preparation of amino-combretastatins
3,4,5-Trimethoxybenzyl alcohol (201) was brominatedwith phosphorous tribromide followed by treatment withtriphenylphosphine to yield 3,4,5-trimethoxybenzyl tri-phenylphosphonium bromide 202. To prepare nitrobenz-aldehyde precursors 205 and 206, benzyl bromide 20392
was hydrolyzed by refluxing it in acetone–water mix-ture.95 The benzyl alcohol thus obtained was treated withpyridinium chlorochromate to obtain the benzaldehydeintermediate 204.96Nitration of 20497 gave a mixture of ni-trobenzaldehydes 205 and 20698 which were separated bycolumn chromatography.
Precursor 202 was treated with sodium hydride to gener-ate ylide 207 (Scheme 28) which was then treated with205 or 206 under Wittig reaction conditions.92c This yield-ed both Z- and E-isomers of the nitro-substituted com-pounds 208. The stereoisomers were separated by flashchromatography and the nitro groups were reduced byzinc and acetic acid, to provide the desired amino-substi-
MeO
MeO
MeO
P+Ph3
H
O NO2
MeO
MeO
MeO
NH2
Cl–
+1. n-BuLi
2. Zn, AcOH
186
R1
R2
MeO
H Ph3P+
+
190
ONO2
Br–
OMe
187
191
R1
R2
MeO
NH2
OMe
R1
R2
R1
R2
188: R1 = H, R2 = OMe (41%)189: R1 = OMe, R2 = H
1. n-BuLi
2. Zn, AcOH
192: R1 = R2 = OMe (39%)193: R1 = H, R2 = OMe194: R1 = OMe, R2 = H (40%)
(Z/E = 3:1)
(Z/E = 3:1)
Scheme 26 Synthesis of 198 and 200
H
O
MeO
MeO
MeO
1. n-BuLi
2. Zn, AcOH
199
OMe
NH2
OMe(34%, Z/E = 3:1)
MeO
MeO
MeO
Br
195
NO2
MeO
MeO
MeO
P+Ph3
NO2
196
Br–
Ph3P
196 +
NO2
NH2
200
H
O
MeO
MeO
MeO
1. n-BuLi
2. Zn, AcOH
197
OMe
OH
OMe(30%, Z/E = 3:1)
196 +
OTBS
NH2
198
MeO
OMe
OMe
OH
MeO
OMe
OMe
PPh3Br
1. PBr3 (77%)
2. Ph3P (77%)
201 202
OMe
Br
NO2
OMe
H
NO2
O
1. Me2O, H2O (91%)
2. PCC, Celite (97%)
203 204
204HNO3
H2SO4
OMe
H
NO2
O
OMe
H
NO2
O
O2N NO2
+
205 206
(42%) (47%)
2488 R. Singh, H. Kaur REVIEW
Synthesis 2009, No. 15, 2471–2491 © Thieme Stuttgart · New York
tuted combretastatin analogues 209. Biological testing ofthe compounds that revealed di-amino substitution on the2¢- and 3¢-positions of the B-ring provided the most potentanti-tumor activity.
5 Conclusions
A survey of recent studies highlighting significant ad-vances in the development of various structural motifs andmethodologies for the synthesis of anti-cancer stilbenoidshas been presented. Structural requirements outlining pro-found effects on the desired biological activity have beenstudied in these reports. The structural features targeted inthese studies have included optimization of molecularvolume produced by additional atoms between the aro-matic rings corresponding to the original structural tem-plate of CA4, restrictions imposed upon the aryl rings,and variation in functional group compatibility.
Diverse synthetic transformations (such as Wittig reac-tion, Suzuki–Miyaura coupling, and Huisgen cycloaddi-tion) have been used to achieve the synthesis ofbiologically active compounds with the incorporation ofthese structural features. The variation of synthetic strate-gies has allowed for the efficient preparation of CA4analogues with the desired stereo-, regio- and chemo-selectivity.
While these developments underscore the significance ofefforts being directed towards finding better methodolo-gies with a goal of achieving biologically active anti-cancer combretastatin-based compounds, further research
aimed towards achieving greater cytotoxicity and anti-mitotic activity in CA4 analogues is foreseeable.
Acknowledgment
The authors wish to thank Professor Robert Vince (Director, CDD-UMN) for his constant support and invaluable advice. The authorsalso thank Professor David M. Ferguson and Professor Courtney C.Aldrich for helpful discussions.
References
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Scheme 28 Synthesis of di- and tri-amino analogues of combreta-statin
MeO
OMe
OMe
PPh3Br
202
MeO
OMe
OMe
PPh3
207
207
OMe
H
R1
O
R3 R2+
OMe
R1
R3
208
R2MeO
MeO
MeO
R1, R2, R3 = NO2, H205, 206
OMe
R1
R3
209
R2MeO
MeO
MeO
R1, R2, R3 = NO2, H
Zn, AcOH
(45–52%)
(43–61%)
NaH
CH2Cl2
REVIEW Combretastatin-Based Anticancer Agents 2489
Synthesis 2009, No. 15, 2471–2491 © Thieme Stuttgart · New York
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