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`DOI: 10.1002/cmdc.200600308
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`Paclitaxel And Docetaxel Resistance: Molecular
`Mechanisms and Development of New
`Generation Taxanes
`Elena Galletti, Matteo Magnani, Michela L. Renzulli, and Maurizio Botta*[a]
`
`Taxanes represent one of the most promising classes of anticanc-
`er agents. Unfortunately, their clinical success has been limited by
`the insurgence of cellular resistance, mainly mediated by the ex-
`pression of the MDR phenotype or by microtubule alterations.
`However, the remarkable relevance of paclitaxel and docetaxel in
`clinical oncology stimulated intensive efforts in the last decade to
`identify new derivatives endowed with improved activities to-
`wards resistant tumor cells, resulting in a huge number of novel
`natural and synthetic taxanes. Among them, several structurally
`different derivatives were found to exhibit a promising behavior
`
`against the MDR phenotype in terms of either MDR inhibiting
`properties, or enhanced cytotoxicity compared to parental drugs,
`or both. On the other hand, only in more recent years have the
`first taxanes retaining activity against resistant cancer cells bear-
`ing alterations of the tubulin/microtubule system emerged. This
`review describes the main molecular mechanisms of resistance to
`paclitaxel and docetaxel identified so far, focusing on the advan-
`ces achieved in the development of new taxanes potentially
`useful for the treatment of resistant tumors.
`
`Introduction
`
`Paclitaxel and docetaxel (1 and 2 in Figure 1, respectively), pro-
`genitors of the family of taxanes, are well known anticancer
`drugs currently used in clinics for the treatment of several
`kinds of tumor, including ovarian, breast, head and neck, lung,
`and prostate cancer. These agents act as microtubule stabiliz-
`ers and disrupt microtubule dynamics, thus inducing mitotic
`arrest and ultimately, cell death by apoptosis.[1]
`Despite the relevant contribution of taxanes in ameliorating
`the quality of life and overall survival of cancer patients, the
`development of cellular resistance represents a serious limita-
`
`Figure 1. Structures of paclitaxel (1) and docetaxel (2).
`
`tion to their clinical use. The two main mechanisms involved in
`resistance to taxanes are the expression of the multidrug re-
`sistance (MDR) phenotype and the alterations of their cellular
`target, namely the tubulin/microtubule system.[2, 3] Several less
`studied putative mechanisms of resistance,
`including altera-
`tions in the signaling pathways, altered regulation of the cell
`cycle and altered control of apoptosis and cell death signals,
`have also been described.[4] MDR is a term used to describe
`the ability of drug-resistant tumors to exhibit simultaneous re-
`sistance to a number of structurally and functionally unrelated
`chemotherapeutic agents.[2] The MDR phenotype is often
`mediated by the overexpression of drug efflux pumps, of
`which P-glycoprotein is the best known, that prevent the accu-
`mulation of the drugs within resistant cells. MDR was the first
`and most widely reported mechanism of resistance to taxanes;
`however, more recent studies described resistant tumor cells
`that neither overexpressed multidrug transporters nor showed
`a reduced drug accumulation, revealing that even changes of
`the microtubule structure or composition could lead to a re-
`duced sensitivity of tumor cells to antimicrotubule agents
`through alterations of microtubule dynamic properties and/or
`of drug–target interactions.[3]
`in the
`The clinical
`importance of paclitaxel and docetaxel
`treatment of solid tumors has stimulated intensive efforts to
`elucidate the molecular mechanisms of resistance to taxanes
`and to develop novel agents effective against
`resistant
`
`[a] Dr. E. Galletti, Dr. M. Magnani, Dr. M. L. Renzulli, Prof. M. Botta
`Dipartimento Farmaco Chimico Tecnologico, Università degli Studi di Siena,
`Via Alcide de Gasperi, 2, I-53100 Siena (Italy)
`Fax: (+ 39) 0577 234333
`E-mail: botta@unisi.it
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`tumors.[5, 6] Different approaches, such as identification of MDR-
`transporter inhibitors, synthesis, and evaluation of more active
`analogues, synthesis of conjugates or prodrugs as well as com-
`bined use with other drugs have been pursued to overcome
`taxane resistance.[7]
`This review will focus on the role of natural and synthetic
`taxanes in overcoming paclitaxel and docetaxel resistance. Sec-
`tion 1 will deal with multidrug transporter-mediated mecha-
`nisms of resistance; the main transporters associated with
`MDR, and the possible strategies for circumventing them will
`be briefly outlined, followed by a review on the development
`of natural and synthetic taxanes potentially useful for the treat-
`ment of MDR tumors, owing to their capability to either inhibit
`MDR efflux pumps or to bypass their action. Novel antimicrotu-
`bule taxanes which showed promising activity in the treatment
`of drug-resistant cells and currently undergoing clinical evalua-
`tion will also be reported. Section 2 will deal with mechanisms
`of resistance involving alterations of the biological target of
`taxanes; the most recent advances in identifying and elucidat-
`ing the clinical role of distinct mechanisms will be discussed,
`and the taxanes reported to retain antimitotic activity against
`cancer cells bearing changes
`in the tubulin/microtubule
`system will be described.
`
`Maurizio Botta obtained a degree in
`Chemistry at the University of Rome in
`1974. After working as temporary assis-
`tant in Organic Chemistry at the Univer-
`sity of Rome, he got a fellowship from
`the University of New Brunswick
`(Canada), where he earned his PhD in
`Chemistry in December 1979 under the
`direction of Prof. K. Wiesner. He was in-
`vited researcher in the laboratory of
`Prof. S. Hannessian at the University of
`Montreal and in 1987 he became Associate Professor of Medicinal
`Chemistry at the Faculty of Pharmacy of the University of Siena,
`where since 2000 he is Full Professor. Since 1988 he has been re-
`sponsible, from the scientific standpoint, for about 50 research
`projects granted both by UE, MIUR, CNR, and by Pharmaceutical
`Companies, covering several fields of medicinal chemistry.
`
`Elena Galletti obtained a degree in Phar-
`maceutical Chemistry at the University
`of Siena in 2003, working on the synthe-
`sis of antifungal agents. She worked
`under the supervision of Professor Maur-
`izio Botta on the synthesis of taxane-di-
`terpenoids with potential anticancer
`and/or MDR-modulating activity and she
`earned her PhD in December 2006. In
`January 2007 she joined the medicinal
`research department of Nerviano Medi-
`cal Sciences, an oncology-focused, integrated discovery and devel-
`opment company in Milan.
`
`M. Botta et al.
`
`1. Taxane resistance associated with multidrug
`transporters
`
`The most extensively studied mechanism of resistance to tax-
`anes is the overexpression of P-glycoprotein and other multi-
`drug transporters: these are membrane proteins belonging to
`the ATP-binding cassette (ABC) family of transporters,[8, 9] and
`act as efflux pumps which extrude a large number of structur-
`ally diverse, mainly hydrophobic compounds from cells, thus
`keeping intracellular drug concentration below a cell-killing
`threshold and inducing cross-resistance to several chemically
`unrelated compounds. ABC transporters are widely distributed
`in normal tissues; although their exact physiological role is still
`to be fully elucidated, they are thought to prevent cytotoxic
`compounds in the environment and diet from entering the
`body and remove them by excretion into the bile and urine.
`
`1.1. Multidrug transporters
`
`The best known and well-studied multidrug transporters are P-
`glycoprotein (P-gp), encoded by the mdr1 gene,[10] multidrug
`resistance protein 1 (MRP1), encoded by the mrp1 gene,[11] and
`
`Matteo Magnani studied Pharmaceutical
`Chemistry at the University of Siena. He
`performed his diploma thesis research
`with Professor Maurizio Botta in the
`field of virtual library design and virtual
`screening and he obtained his under-
`graduate degree in 2004. He is currently
`a PhD student in the laboratory of Pro-
`fessor Maurizio Botta, where he con-
`ducts molecular modeling studies on
`antitubercular and anticancer com-
`pounds.
`
`Michela Lucia Renzulli obtained a
`degree in Pharmaceutical Chemistry at
`the University of Siena in 1999, perform-
`ing her diploma thesis research in the
`field of antiviral research and solution
`and solid phase organic synthesis. She
`earned her PhD in 2002 under the su-
`pervision of Professor Maurizio Botta,
`working on synthesis of Taxuspine-deriv-
`atives as anticancer and MDR reversing
`agents. Since 2003 she is working as
`postdoctoral research assistant at the University of Siena under the
`supervision of Professor Maurizio Botta, leading a number of pro-
`jects in the field of organic and medicinal chemistry.
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`New Generation Taxanes
`
`breast cancer resistance protein (BCRP), encoded by the mxr
`gene.[12]
`lines with paclitaxel or other anti-
`Selection of cancer cell
`cancer drugs, frequently results in MDR mediated by increased
`expression of P-gp. P-gp is an ATP-dependent broad-spectrum
`multidrug efflux pump, consisting of two homologous halves
`joined by a linker region (Figure 2 a). Each half begins with a
`
`Figure 2. Structures of multidrug transporters: domain arrangement in a) P-
`gp, b) MRP1, and c) BCRP. Circles represent the ATP-binding domains; cylin-
`ders represent the segments of transmembrane domains.
`
`transmembrane domain, (which binds hydrophobic drug sub-
`strates) containing six transmembrane segments, followed by a
`hydrophilic region containing the ATP binding site.[13] Increased
`levels of P-gp are common in some tumor types and have
`been frequently associated with paclitaxel resistance: evalua-
`tion of mdr1 gene expression in a NCI 60 cell line anticancer
`drug-screening panel demonstrated a correlation of mdr1 ex-
`pression with the sensitivity profile of paclitaxel,[14] and several
`authors have detected increased levels of either mdr1 mRNA
`or P-gp itself in paclitaxel-resistant cell lines.[15, 16] Nevertheless,
`much remains to be learned about the role of the mechanisms
`of resistance mediated by P-gp and other ABC-transporters in
`different human tumors and their relevance for patients receiv-
`ing a taxane-based chemotherapy.[8, 17, 18] Besides mediating
`taxane resistance in tumor cells, P-gp may also play a signifi-
`cant role in modulating taxane absorption and tissue distribu-
`tion; in this regard, the high expression of P-gp in the intesti-
`nal mucosa has been shown to strongly limit the oral bioavail-
`ability of paclitaxel[19] and the marginal efficacy of the drug
`against primary brain tumors is consistent with its inability to
`cross the intact blood-brain barrier, where P-gp is highly ex-
`pressed.[20]
`
`MRP1 is the most studied protein of the MRP family, which
`comprises six other characterized members (MRP2, MRP3,
`MRP4, MRP5, MRP6, MRP7).[11, 21] Like P-gp, MRP1 has a core
`structure consisting of two membrane spanning domains, each
`of them being followed by an ATP-binding domain, but it also
`contains a third N-terminal transmembrane domain consisting
`of five transmembrane segments (Figure 2 b). Whereas P-gp
`targets and transports hydrophobic drugs, MRP protein recog-
`nizes hydrophilic molecules and organic anions; it also trans-
`ports neutral drugs conjugated with glutathione, glucuronide,
`or sulfate, and some anticancer agents by co-transport with
`glutathione. However, unlike P-gp, to date MRP seems to play
`a marginal role in resistance to taxanes.[22]
`As represented in Figure 2 c, BCRP consists of a N-terminal
`ATP-binding site and six transmembrane segments; it is a half-
`transporter likely to homodimerize or heterodimerize to func-
`tion.[23]
`It was initially isolated from breast cancer cell
`lines
`which demonstrated doxorubicin resistance. Although BCRP
`does not confer resistance to taxanes, noncytotoxic synthetic
`taxanes have been shown to be able to modulate BCRP-medi-
`ated drug efflux.
`
`1.2. Overcoming transport-based resistance: general
`overview
`
`As resistance to taxanes induced by P-gp and related MDR
`efflux pumps is one of the main obstacles to successful che-
`motherapy of cancer, several strategies for blocking the extru-
`sion of drugs and circumventing cross-resistance mediated by
`these transporters have been proposed (reviewed in refs. [24]
`and [25]), including the inhibition of transporters (engage), the
`use of cytotoxic agents that are not substrates for MDR pro-
`teins and can therefore bypass the efflux from the cell (evade),
`and approaches that take advantage of the collateral sensitivi-
`ty of MDR cells (exploit) (Figure 3).
`Several compounds have been shown to inhibit the drug
`efflux function of P-gp and therefore reverse cellular resistance
`(engage strategy, Figure 3 b). Such MDR modulators (or MDR
`reversal agents) can be co-administered together with cytotox-
`ic agents and belong to a number of different chemical
`classes,[26] which also include taxanes, as described in sec-
`tions 1.3.1 and 1.4.1. Calcium channel blockers, such as verapa-
`mil, were the first agents demonstrated to be able to reverse
`MDR[27] and constituted the first generation of MDR modula-
`tors. The unique property shared by most first generation MDR
`modulators, typically therapeutics agents already known or
`used for other purposes, was their capability to reverse MDR at
`concentrations much higher than those required for their indi-
`vidual therapeutic activity. Further investigations led to second
`and third generation modulators, which have been developed
`through structure–activity relationships and combinatorial
`chemistry approaches and are active at concentrations of
`nanomolar range.[26] However, despite the promising advances
`in preclinical models, to date clinical studies on MDR modula-
`tors have met with limited success.[25]
`P-gp mediated MDR can also be reversed by hydrophobic
`peptides which correspond to the transmembrane segments
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`Figure 3. Possible strategies for overcoming drug resistance mediated by
`multidrug transporters. a) Multidrug transporters pump out cytotoxic drugs
`(green circles) from MDR cancer cells; b) Reversal agents (yellow circle) block
`the efflux pumps, preventing cross-resistant cells from extruding the anti-
`cancer drugs; c) Cytotoxic agents that are not substrates for multidrug trans-
`porters are not extruded from resistant cells; d) The cytoprotective agent
`(magenta circles), but not the cytotoxic drug, is pumped out from resistant
`cancer cells; only in this case MDR cancer cells can be selectively killed,
`since normal cells, which do not extrude the protector molecule, remain un-
`harmed.
`
`M. Botta et al.
`
`of P-gp and interfere with the proper assembly and function-
`ing of the protein. Consistent with this idea, newly synthesized
`specific peptide inhibitors of P-gp have been recently shown
`to sensitize resistant cancer cells to chemotherapic agents,
`thus appearing to be a promising class of noncytotoxic drug
`resistance inhibitors.[28]
`Among the strategies aimed at inhibiting the activity of
`MDR transporters (still
`in the engage field), research has re-
`cently shifted to the modulation of P-gp expression, either by
`blocking the expression of mdr1 mRNA through antisense oli-
`gonucleotides[29] or hammered ribozymes,[30] or by preventing
`the P-gp biosynthesis using chemical compounds.[31, 32]
`Another approach to overcome resistance mediated by ABC-
`transporters is based on the use of drugs which are not sub-
`strates for MDR proteins (evade strategy, Figure 3 c), such as cy-
`clophosphamide, cisplatin, and epothilones.[33] The latter are
`novel tubulin targeting anticancer agents that are not recog-
`nized by P-gp, thus providing proof that new classes of antitu-
`mor drugs not interacting with MDR proteins can be devel-
`oped to improve the response to therapy. Furthermore, it has
`been demonstrated that chemical modifications of paclitaxel
`and docetaxel, MDR inducing compounds, can favorably result
`in active, but not transported, second generation taxanes,
`which will be described in section 1.4.2.
`The exploit approach is based on the idea that drug efflux
`pumps can be exploited to selectively kill resistant cancer cells,
`while sparing sensitive normal cells; two main strategies have
`been proposed to date to take advantage of multidrug trans-
`porter overexpression in cancer cells. The first one involves the
`co-administration of a cytoprotective (antiapoptotic or cyto-
`static) agent, which is a substrate for efflux pumps, together
`with a cytotoxic agent which is not recognized by multidrug
`transporters:
`in the presence of a protective agent, normal
`cells remain unharmed, whereas resistant cells, which pump
`out the protecting agent, do succumb to cytotoxic therapy
`(Figure 3 d).[34, 35] The alternative strategy involves the use of
`anti-P-gp antibodies to destroy cells expressing P-gp, again re-
`sulting in selective killing of drug-resistant cells.[36]
`
`1.3. Natural taxanes in overcoming transport-based
`resistance
`
`Since the discovery of the promising anticancer activity of pa-
`clitaxel and some related compounds, chemical studies on
`constituents of different yew trees have resulted in the isola-
`tion of a large number of new natural taxanes. During the last
`two decades, approximately 120 taxanes with different skele-
`tons, containing 5/7/6-, 6/10/6-, 6/8/6-, or 6/12-membered ring
`systems, have been isolated from the Japanese yew, Taxus cus-
`pidata. Interestingly, some of these agents have been shown
`to reduce Ca2 +-induced depolymerization of microtubules, to
`increase cellular accumulation of vincristine in MDR tumor
`cells, and to exert significant cytotoxic activity. The structures,
`the biological activities, and the chemistry of taxanes isolated
`from T. cuspidata have been recently reviewed by Shigemori
`and Kobayashi.[37]
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`New Generation Taxanes
`
`1.3.1. Natural taxanes as MDR reversal agents
`
`As anticipated, the effects of the above mentioned natural tax-
`anes on the cellular accumulation of the antitumor drug vin-
`cristine (a P-gp substrate)
`in MDR human ovarian 2780AD
`cancer cells were examined, and the more promising com-
`pounds are reported in Figure 4: taxinine NN-1 (taxezopidine
`G, 3) showed the strongest activity in terms of vincristine accu-
`mulation in MDR tumor cell, with a value of 323 % of verapa-
`mil. Likewise, cyclotaxinine NN-2 (4) (204 %),[38] 2-deacetoxytaxi-
`nine (5) (108 %), taxuspine X (6) (105 %), and taxuspine C (tax-
`inine, 7) (104 %)[39] increased the vincristine accumulation more
`or as potently as verapamil. Moreover, taxuspine C was shown
`to reduce the binding of [3H]-azidopine (a P-gp photoreactive
`substrate) to P-gp in the adriamycin-resistant human leukemia
`line more potently than verapamil,[40] and to
`K562/ADM cell
`completely reverse the resistance to colchicine, vincristine, and
`paclitaxel in human epidermoid carcinoma KB-C2 cells, which
`overexpress P-gp;[41] when co-administered with vincristine,
`taxuspine C increased the life span of mice bearing the vincris-
`tine-resistant leukaemia cells P388/VCR.[42]
`
`Plant cell cultures of Taxus species have always been consid-
`ered a promising approach to obtain paclitaxel and related tax-
`anes in good amounts. In confirmation of that, Tsuruo and co-
`workers[43] reported the isolation of taxinine NN-11 (8) from
`callus culture of T. cuspidata cultivated on a modified Gam-
`borg’s B5 medium after stimulation with methyl
`jasmonate.
`Taxinine NN-11, whose structure is reported in Figure 4, exhibit-
`ed about twofold higher activity than verapamil towards vin-
`cristine accumulation in the MDR 2780AD cell
`line. Further
`chemical investigation on the callus cultures of T. cuspidata led
`to the isolation of the new taxane 9 (Figure 4), which is the
`9,10-isomer of taxinine NN-11 and showed 67 to 92 % activity
`of verapamil at different concentrations on the cellular accu-
`mulation of calcein (another P-gp substrate) in 2780AD cells.[44]
`Analogously, taxusin (10, Figure 4) was isolated in the course
`of investigations on secondary metabolites and production of
`useful natural product in the dark brown callus culture of T.
`cuspidata incubated under light irradiation; this taxane exhibit-
`ed stronger MDR reverse activity than verapamil, still towards
`2780AD tumor cells.[45]
`Taken together, these results demonstrate that natural tax-
`anes could be good inhibitors of
`P-gp and useful agents in over-
`coming MDR, thus stimulating
`continued efforts in searching
`for new natural or synthetic pa-
`clitaxel
`derivatives
`endowed
`with improved MDR reversal ac-
`tivity.
`
`1.3.2. Anticancer activity of
`natural taxanes
`
`The cytotoxic activity of natural
`taxanes isolated from T. cuspida-
`ta (mentioned above), T. yunna-
`nensis, and T. chinensis, has been
`examined against murine leuke-
`mia L1210 cells and human epi-
`dermoid carcinoma KB cells.[46]
`Some of them,
`including both
`paclitaxel-like and nonpaclitaxel-
`like compounds, exhibited a
`strong inhibitory activity against
`Ca2 +-induced depolymerization
`of microtubules, comparable to
`paclitaxel; however, despite the
`good activity against drug-sensi-
`tive cancer cells, natural taxanes
`evaluated so far have been re-
`ported to be ineffective against
`MDR cell
`lines, suggesting that,
`just like paclitaxel, these agents
`are substrates
`for multidrug
`transporters and are thereby ex-
`truded from resistant cells.
`
`Figure 4. Taxanes from T. Cuspidata endowed with inhibitory activity towards the drug efflux function of P-gp in
`MDR cells.
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`1.4. Synthetic taxanes in overcoming transport-based
`resistance
`
`Significant results have been obtained in the synthesis of new
`generation taxanes: extensive SAR studies have led to the de-
`velopment of highly efficient
`taxane-based MDR reversal
`agents (TRAs) as well as cytotoxic taxanes endowed with
`higher potencies in comparison with paclitaxel on resistant
`cells, owing to their ability to evade the MDR transporters
`(second generation taxanes).
`It is noteworthy that some of
`these newly synthesized taxanes, able to overcome MDR, are
`now in different phases of clinical development.
`
`1.4.1. Synthetic taxanes as MDR reversal agents
`
`The use of noncytotoxic chemosensitizer taxanes (MDR reversal
`agents) able to block the binding site of anticancer drugs on
`P-gp and sister proteins, thus preventing their exclusion from
`the cells, has received considerable attention as a reliable strat-
`egy to inhibit the activity of multidrug transporters.
`On the basis of the interesting activity of natural taxinines
`described above as P-gp inhibitors, the three novel taxinine
`analogues 11–13 (Figure 5) were prepared from sinenxan A (a
`
`taxuspine C analogues were synthesized by
`A series of
`Tsuruo and Kobayashi and their effect on the cellular accumu-
`lation of vincristine in MDR 2780AD cancer cells was exam-
`ined.[49] Taxinine derivatives containing a cinnamoyloxy, a ben-
`zoyloxy, a TES, or a BOM group at C2, C5, or C13 were found
`to significantly increase the cellular accumulation of vincristine
`in MDR cells, suggesting that taxinine analogues could be
`good modifiers of MDR in resistant tumor cells; the most inter-
`esting compounds, 14 and 15 (Figure 5), exhibited higher po-
`tency than verapamil.
`Very recently our group described the synthesis of a series
`of 2-deacetoxytaxinine J (5) derivatives and their evaluation as
`MDR reversal agents on the drug-resistant human breast
`cancer cell line MCF7-R, overexpressing P-gp; the most inter-
`esting results
`in terms of paclitaxel accumulation were
`ACHTUNGTRENNUNGachieved with taxinine 16 (Figure 5), bearing a benzoyl group
`at C13 position.[50]
`A wide set of TRAs based on the 10-deacetylbaccatin III
`(14-OH-DAB)
`(DAB) and 14b-hydroxy-10-deacetylbaccatin III
`skeleton has been developed during the years and their struc-
`ture–activity relationships have been recently reviewed by
`Ojima and co-workers.[51] DAB and 14-OH-DAB (17 and 18 in
`Figure 6, respectively), even though noncytotoxic by them-
`
`Figure 5. Examples of taxinine derivatives with MDR reversal activity.
`
`readily available biosynthetic taxane),[47] and tested for their ac-
`tivity as MDR reversal agents in comparison with verapamil.[48]
`In vitro assays revealed for all the three compounds an inter-
`esting MDR reversal activity on KB/V cells, a MDR subline of
`human epidermoid cancer cells KB overexpressing P-gp; com-
`pound 12, in particular, was shown to be more potent than ve-
`rapamil. Further in vivo studies on vincristine-resistant KB/V
`tumor xenografts showed that 12 in combination with vincris-
`tine significantly inhibited the tumor growth, whereas treat-
`ment with vincristine or 12 alone did not result in growth in-
`hibition.
`
`Figure 6. Design of TRAs by insertion of hydrophobic substituents at differ-
`ent positions of DAB and 14b-OH-DAB core: a) solid arrows indicate the
`most suitable positions for enhancing the MDR reversal activity; b) hydro-
`phobic modifiers which gave the most interesting results in terms of MDR
`modulation.
`
`selves, provide the crucial components of paclitaxel and pos-
`sess several hydroxy groups that can be easily modified with
`hydrophobic side chains by esterification. In addition, 14b-OH-
`DAB, isolated from the needles of the Himalayan yew tree (T.
`wallichiana Zucc.),[52] has substantially better water solubility
`than DAB due to the presence of an extra hydroxy group at
`the C14 position; taxanes derived from 14b-OH-DAB are there-
`fore expected to have improved water solubility, bioavailability,
`and reduced hydrophobicity-related loss of efficacy.
`The extensive SAR studies of TRAs focused on two aspects,
`namely the identification of both the structural requirements
`and the most suitable position for a potential pendant group.
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`of its efficacy against the cross-resistant cancer cells MCF7-R
`and MDA-435/LCC6-MDR. Furthermore, two of these TRAs,
`namely 19 and 22, exhibited high MDR reversal activity even
`at lower concentrations, together with a much higher activity
`than verapamil and slightly better activity than cyclospor-
`ACHTUNGTRENNUNGine A.[54] To prove the mechanism of action for these agents,
`the effects of 22 on the paclitaxel uptake by the drug-resistant
`cancer cells MDA-435/LCC6-MDR in the presence and absence
`the taxane were investigated, demonstrating that 22 blocked
`the P-gp efflux mechanism, thus the anticancer drug was not
`extruded from drug-resistant cancer cells and exerted its che-
`motherapeutic effect.
`The agents described so far, similar to most of the com-
`pounds belonging to the category of TRAs, are noncytotoxic
`up to the solubility limit (approximately 30 mm), therefore they
`have excellent therapeutic indexes. Finally, it is worth noting
`that neither DAB, 14b-OH-DAB, nor the hydrophobic modifiers
`showed any MDR reversal activity at all by themselves, and
`
`New Generation Taxanes
`
`Preliminary SAR studies on structurally different classes of
`MDR reversal agents pointed out the importance of the pres-
`ence of a hydrophobic, conjugated, planar ring.[53] Accordingly,
`benzophenone, naphthalene-containing carboxylic acids, and
`other
`related hydrophobic groups have been chosen to
`modify the hydroxy groups at C2, C7, C10, and C13 positions
`of either DAB or 14b-OH-DAB. Among the hydrophobic pend-
`ant groups designed and examined, those consisting of two
`aromatic rings, spaced by a 1- or 2-atom linker and bearing a
`carbonyl or ether group, were identified as the most effective
`units (Figure 6).[51]
`Over the years, several libraries of novel TRAs have been de-
`signed, synthesized, and evaluated for their modulating capa-
`bility against P-gp, overexpressed in drug-resistant cancer cell
`lines MCF7-R and MDA-435/LCC6-MDR.[51] The results of these
`studies clearly indicated that modifications at the C7 position
`can result in strong MDR reversal activity and benzophenone
`and naphthalene appeared to be the most appropriate pend-
`ant groups. Even modification of
`the C10 hydroxy group with a
`benzophenone side chain result-
`ed in a very good reversal activi-
`ty, whereas the attachment of a
`hydrophobic side chain contain-
`ing a diphenyl ether, a diphenyl
`thioether, a benzamide, and a
`benzoate did cause a significant
`loss of activity. The effects of the
`simultaneous
`introduction
`of
`two hydrophobic side chains at
`both the C7 and C10 positions
`on the reversal activity are more
`complicated. Modification
`at
`both C7 and C10 positions with
`benzophenone proved to be
`very favorable; replacement of
`the C10 benzophenone with
`either a methyl
`formate or a
`propanoyl group was well toler-
`ated, whereas replacement with
`larger aromatic substituents or
`replacement of C7 benzophe-
`none with naphthalene resulted
`in significant
`loss of activity.
`Modifications at the C2 or C13
`position
`gave
`poor
`results.
`These findings strongly corrobo-
`rated the idea that hydropho-
`bicity is not
`the only feature
`necessary for an efficient MDR
`reversal activity, and that there
`is a specific binding site for
`TRAs on P-gp with rather strict
`steric/shape requirements.
`When TRAs 19–22 (Figure 7)
`were co-administered at 1.0 mm,
`paclitaxel
`recovered 95–99.8 %
`
`Figure 7. Examples of TRAs. Worthy of note, TRAs 23–28 are able to modulate the P-gp, MRP1 and BCRP efflux
`pumps.
`
`ChemMedChem 2007, 2, 920 – 942
`
` 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
`
`www.chemmedchem.org
`
`927
`
`

`
`MED
`
`M. Botta et al.
`
`therefore the MDR reversal activity was unique to the combi-
`nation of their structures.[54]
`The majority of
`initial studies concerning MDR reversal
`agents have only focused on modulators of P-gp protein. How-
`ever, the true measure of the efficacy of a MDR reversal agent
`is represented by its ability to inhibit the drug efflux mediated
`by a broad spectrum of ABC transporters. On the basis of such
`a remark, among the large number of synthesized TRAs the
`best twenty compounds in terms of MDR reversal activity in
`combination with paclitaxel against the MDA435/LCC6-MDR
`cell line, overexpressing P-gp, were chosen to assess their ca-
`pability to also block the MRP1 and BCRP efflux pumps. Ac-
`cordingly, the efflux of mitoxantrone (which was demonstrated
`to be a substrate for P-gp, MRP1, and BCRP transporters)[55]
`was evaluated on drug-resistant human myelogenous leuke-
`mia and myeloma cell lines overexpressing MRP1 (HL60-ADR),
`P-gp (8226-Dox6), and BCRP (8226-MR20).
`Interestingly, the
`four agents 23–26, plus the newly synthesized 27 and 28
`(Figure 7) provided very good results, being able to strongly
`modulate not only P-gp, but also the other MDR-associated
`ABC transporters.[51, 56]
`In the course of studies towards new taxanes to be em-
`ployed in overcoming transport-mediated MDR resistance,
`modifications of the taxane skeleton led to the C-aromatic tax-
`anes, evaluated as MDR reversal agents by Tsuruo and co-
`workers.[57] Starting from the C-aromatic taxanes 29 a and 29 b,
`derivatives 29 c–i (Figure 8) were designed taking into account
`that the hydrophobicity of a molecule appeared to be impor-
`tant for P-gp affinity, as aromatic functional groups were incor-
`porated into most of the known active compounds. According-
`ly, a benzoyl group was chosen and linked to the hydroxy
`groups of 29 a and 29 b in most of the synthesized derivatives.
`The MDR reversal activity of the newly synthesized C-aromatic
`taxanes 29 c–i was then evaluated as an enhancing effect of
`vincristine accumulation in ovarian MDR cancer cells 2780AD,
`using verapamil as a positive control. The intermediate 29 a ex-
`hibited weak activity compared to verapamil, and most of the
`functional group transformations (29 c–e) proved ineffective.
`However, a significant enhancement of the activity was ob-
`served for the benzoate derivatives, as the monobenzoate 29 g
`and especially the C2-benzoate 29 h exhibited the same poten-
`cy as verapamil. Additional incorporation of a benzoyl group in
`29 f and 29 i was again ineffective in enhancing activity. These
`results indicated that an aromatic functional group on the B-
`ring may play an important role in the interaction with P-gp,
`though it was not clear why the benzoyl group m