DOI: 10.1002/cmdc.201402129

The Impact of Cyclopropane Configuration on theBiological Activity of Cyclopropyl-EpothilonesFabienne Z. Gaugaz,[a] Mariano Redondo-Horcajo,[b] Isabel Barasoain,[b] J. Fernando D�az,[b]

Amanda Cobos-Correa,[c] Markus Kaufmann,[c] and Karl-Heinz Altmann*[a]

Two cis-12,13-cyclopropyl-epothilone B variants have been syn-thesized, differing only in the configuration of the stereocen-ters at C12 and C13. The syntheses were based on a commonallylic alcohol intermediate that was converted into the corre-sponding diastereomeric hydroxymethyl-cyclopropanes bymeans of stereoselective Charette cyclopropanations. Macro-cyclizations were accomplished through ring-closing metathe-sis (RCM). Substantial differences between the two compoundswere found with regard to microtubule binding affinity, anti-proliferative activity and their effects on the cellular microtu-bule network. While the analogue with the cyclopropanemoiety oriented in a corresponding way to the epoxide config-uration in natural epothilones was almost equipotent withepothilone A, the other was significantly less active. Based onthese findings, natural epothilone-like activity of cis-fused12,13-cyclopropyl-epothilone analogues is tightly linked to thenatural orientation of the cyclopropane moiety.

Epothilones A and B (Epo A and B; 1 and 2) are natural prod-ucts of myxobacterial origin with potent in vitro and in vivoantitumor activity.[1] Mechanistically, the inhibition of cancercell growth by epothilones is based on the suppression of cel-lular microtubule (MT) dynamics, which leads to cell-cyclearrest in G2/M and the induction of apoptosis.[2] Epo B (2) andseveral of its analogues have been advanced into clinical trialsin humans, and one of these compounds, the Epo B lactam ix-abepilone (also known as BMS-247550), was approved by theUS Food and Drug Administration (FDA) for breast cancertreatment in 2007 (under the trade name Ixempra).[3]

The structure–activity relationships (SAR) of epothilones hasbeen studied extensively, including the effects of changes inthe configurations of the various chiral centers.[1] While thesestudies have firmly established that the epoxide moiety assuch is dispensable for potent biological activity, rather surpris-

ingly, a firm assessment of the activity of analogues with in-verted cis-epoxide (or -cyclopropane) geometry (i.e. , analogueswith inverted stereocenters at both C12 and C13) is still lack-ing.[4] Only limited scattered data on such analogues are avail-able in the literature,[5–8] and these are not fully consistent. Forexample, Nicolaou and co-workers reported that 12,13-epi-oxa-zole-Epo B (4) inhibits the growth of ovarian carcinoma cellline 1A9 with only sevenfold lower potency than the corre-sponding “natural” 12R,13S isomer (IC50 = 1.5 nm and 0.2 nm,respectively) ; likewise, the tubulin-polymerizing activity of thetwo isomers appeared to be similar.[5] In contrast, the activityof 12,13-epi-9,10-dehydro-Epo B (5) against the human leuke-mia cell line CCRF-CEM was found by Danishefsky, Taylor andco-workers to be ca. 60-fold reduced compared with 9,10-de-hydro-Epo B (IC50 = 13.4 nm and 0.23 nm, respectively).[6] Nodata were reported for the tubulin-polymerizing activity ofthese compounds. Lastly, our own work on side-chain-modifiedepothilone analogues revealed 12S,13R analogue 6 to be morethan 600-fold less potent against human KB-31 cells than its12R,13S counterpart (IC50 = 82 nm for 6 vs. 0.13 nm for benzo-thiazole-Epo B); at the same time, compound 6 showed noeffect on tubulin polymerization under our standard assay con-ditions.[7] This latter observation raised the possibility of a tubu-lin-independent mechanism of cell growth inhibition underly-ing the still substantial inhibitory activity of 6.

We have recently been interested in potent analogues of cy-clopropyl-Epo B (3) with functionalized C15 side chains as po-tential drug payloads for antibody–drug conjugates (ADC).[9] In

[a] Dr. F. Z. Gaugaz, Prof. K.-H. AltmannETH Z�rich, Department of Chemistry & Applied BiosciencesInstitute of Pharmaceutical Sciences, HCI H405Vladimir-Prelog-Weg 4, 8093 Z�rich (Switzerland)E-mail : karl-heinz.altmann@pharma.ethz.ch

[b] Dr. M. Redondo-Horcajo, Dr. I. Barasoain, Dr. J. F. D�azCentro de Investigaciones Biol�gicasConsejo Superior de Investigaciones Cient�ficasRamiro de Maeztu 9, 28040 Madrid (Spain)

[c] Dr. A. Cobos-Correa, M. KaufmannNovartis Institute for Biomedical Research, 4056 Basel (Switzerland)

Supporting information for this article is available on the WWW underhttp://dx.doi.org/10.1002/cmdc.201402129.

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the context of this work, we have also performed a systematicstudy on the biological impact of the configuration of the C12and C13 stereocenters in cis-fused bicylic epothilones. Specifi-cally, we have prepared isomeric epothilone analogues 7 a and7 b, and we have assessed the in vitro antiproliferative activi-ties of these compounds across a panel of different humancancer cell lines, their microtubule binding affinity, and their ef-fects on the cellular microtubule network.

While 7 a represents a highly modified variant of Epo B (2), itis important to note that each of the three Epo B analoguesthat incorporate only one of the three modifications present in7 a simultaneously (i.e. , 9,10-dehydro-Epo B,[6] CP-Epo B (3),[9, 10]

and the 12,13-epoxide analogue of 7 a)[11] has been shown toinhibit human cancer cell growth with virtually the same po-tency as Epo B (2). Thus, we considered it highly likely that anydifferences between 7 a and 7 b would also be reflective of thedifferences in biochemical andcellular effects between Epo B(2) and its 12S,13R isomer.

The synthesis of epothiloneanalogues 7 a and 7 b is sum-marized in Schemes 1 and 2 andwas based on the stereoselectiveestablishment of the cyclopro-pane moieties by asymmetricCharette cyclopropanation[12] ofallylic alcohol 15 (Scheme 1 and2) and macrocyclic ring closureby ring-closing metathesis(RCM)[13] of dienes 21 (Scheme 2)as the key transformations.

Allylic alcohol 15 was ob-tained starting from commercial-ly available 3-nitro-4-fluoroben-zoic acid (8), which was first con-verted into ester 9 by acid-cata-lyzed esterification with metha-nol, followed by fluoridedisplacement with ethanolamineand subsequent nitro group re-duction (Scheme 1).[11] Reactionof 9 with triethyl orthoacetate,to form the imidazole ring, fol-lowed by tert-butyldiphenylsilyl(TBDPS) protection of the freehydroxy group, ester reduction

with diisobutylaluminumhydride (DIBALH) and subsequent oxi-dation of the resulting alcohol with manganese dioxide gavealdehyde 10 in 67 % overall yield from 8. Aldehyde 10 under-went asymmetric Brown allylation with (�)-allyldiisopinocam-pheylborane [(�)-allylB(Ipc)2]—prepared in situ from (�)-(Ipc)2BCl and allylmagnesium bromide[14]—to furnish homoallyl-ic alcohol 11 in good yield (80 %) and with acceptable ee(83 %). Silylation of 11 followed by oxidative cleavage of theterminal double bond by Sharpless dihydroxylation/sodium pe-riodate cleavage gave aldehyde 12 in 89 % overall yield. Thelatter was highly prone to elimination, even at lower tempera-tures, and thus was reacted with the stabilized ylide 13,[15] im-mediately after purification, to provide ester 14 in excellentyield (97 %). Subsequent reduction of the ester moiety gavethe corresponding allylic alcohol; unfortunately, however, thiscompound did not undergo regioselective cyclopropanation ofthe trisubstituted double bond.

In order to avoid the regioselectivity problem in the cyclo-propanation step, 14 was submitted to hydroboration with 9-borabicyclo(3.3.1)nonane (9-BBN), the resulting primary hy-droxy group was protected as a tert-butyldimethylsilyl (TBS)ether, and the ester moiety was reduced with DIBALH to fur-nish the desired allylic alcohol 15 (82 % based on 14) as thecommon precursor for both target structures 7 a and 7 b.

Charette cyclopropanation of 15 with ligand 16[12] gave thenaturally configured intermediate 18 a in 78 % yield as a 9:1mixture of diastereoisomers (based on 1H NMR spectroscopy)

Scheme 1. Reagents and conditions : a) SOCl2, MeOH, 0 8C!RT, 17 h, quant. ; b) ethanolamine, NEt3, CH2Cl2, RT, 24 h,quant. ; c) Pd/C, H2, MeOH, RT, 20 h, quant. ; d) triethyl orthoacetate, sulfamic acid, EtOH, reflux, 72 h, quant. ;e) TBDPSCl, imidazole, DMF, RT, 24 h, 83 %; f) DIBALH, CH2Cl2, �60 8C!RT, 4.5 h, 82 %; g) MnO2, CHCl3, reflux, 1 h,98 %; h) (1) (�)-Ipc2Cl, allyl-MgBr, Et2O, 0 8C!10 8C, 1 h; (2) 10, Et2O, �100 8C, 3.5 h, 80 % (ee = 83 %); i) TBSOTf,imidazole, CH2Cl2, 0 8C!RT, 7 h, 96 %; j) (1) AD-mix b, methanesulfonamide, tBuOH/H2O (1:1), RT, 28 h; (2) NaIO4,CH2Cl2/H2O (4:1), RT, 2 h, 93 %; k) 13, benzene, reflux, 16 h, 97 %; l) (1) 9-BBN, THF, RT, 1.5 h; (2) H2O2, aq. NaOH,RT, 1 h, 93 %; m) TBSCl, imidazole, CH2Cl2, RT, 72 h, 93 %; n) DIBALH, CH2Cl2, �78 8C!RT, 45 min, 95 %. Abbrevia-tions: DIBALH = diisobutylaluminumhydride; DMF = dimethylformamide; Ipc2Cl = chlorodiisopinocampheylborane;TBS = tert-butyldimethylsilyl ; TBDPS = tert-butyldiphenylsilyl.

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(Scheme 2);[16] the selectivity of the cyclopropanation reactionwas somewhat less satisfactory in the presence of ligand 17,which gave a 83:17 mixture of diastereoisomers in 67 %yield.[16] Neither for 18 a nor for 18 b were the isomeric mix-tures separable by flash column chromatography, and this wasalso the case for all subsequent intermediates. Scheme 2 sum-marizes the elaboration of cyclopropane 18 a into the desiredepothilone analogue 7 a. Thus, the hydroxymethyl group in18 a was transformed into the required methyl group byiodide formation and subsequent reduction with sodium boro-

hydride to furnish 19 a in 67 % overall yield. This was followedby cleavage of the primary TBS ether moiety and Grieco–Sharpless olefination,[17] which gave olefin 20 a (55 % yieldbased on 19 a). After treatment of 20 a with camphorsulfonicacid (CSA) in methanol/dichloromethane, the resulting free al-cohol was esterified with acid 22[18] under Yamaguchi condi-tions[19] to provide diene 21 a as the precursor for the crucialRCM-mediated ring closure in good yield (78 %). RCM in thepresence of Grubbs II catalyst[13] proceeded smoothly and fur-nished the desired macrolactone in 57 % yield and essentiallyas a single double bond isomer.[20] Finally, sequential treatmentof the protected macrolactone with tris(dimethylamino)sulfoni-um difluorotrimethylsilicate (TASF) and hydrogen fluoride·pyri-dine produced 7 a. Isomer 7 b was prepared from cyclopropane18 b by the same sequence of steps as for the synthesis of 7 afrom 18 a. Both 7 a and 7 b were obtained as chromatographi-cally pure isomers after HPLC purification of the material pro-duced in the final deprotection step (14 % and 5 % yield, re-spectively).

The microtubule binding affinity of epothilone analogues 7 aand 7 b was determined in a previously established fluores-cence-based displacement assay[21] that measures displacementof the fluorescent taxol derivative Flutax-2 from the taxol bind-ing site on b-tubulin (for details, see the Supporting Informa-tion). At 37 8C, Ka values of 3.71 � 108

m�1 and 6 � 105

m�1 were

determined for 7 a and 7 b, respectively (Figure S1 andTable S1 in the Supporting Information), which makes 7 b a ca.600-fold weaker microtubule binder than 7 a. At the sametime, however, these experiments clearly established specificmicrotubule binding by 7 b.

The critical tubulin concentrations in the presence of anexcess of 7 a or 7 b were 0.30 and 1.45 mm, respectively. Thus,in line with the microtubule binding data, 7 a is a stronger in-ducer of tubulin polymerization than 7 b. In comparison, thecritical tubulin concentrations for the negative (DMSO) andpositive (docetaxel) controls were 3.30 and 0.33 mm, respective-ly; the critical tubulin concentrations for Epo A (1) and Epo B(2) under identical conditions have been previously reportedas 0.46 mm (1) and 0.21 mm (2).[22]

Table 1. Antiproliferative activity of 7 a, 7 b and Epo A (1).[a]

Cell line IC50 [nm] SI[b]

7 a 7 b Epo A (7 b/7 a)

A2780 5.41�0.40 179�0.02 7.35�0.24 33A549 6.66�1.51 594�72 8.58�1.32 89H4 5.94�0.30 181�12 8.30�0.28 133HCT15 14.74�1.99 3195�454 5.26�0.26 334HT29 2.76�0.71 543�40 6.54�0.62 30LN18 4.30�0.30 6083�1031 11.04�0.68 216NCIH460 7.88�0.65 302�54 8.44�0.69 197NCIH1155 5.41�0.40 179�0.02 6.88�0.75 1413RKO 6.66�1.51 594�72 9.71�1.08 38SNU119 5.61�0.44 745�51 16.58�2.50 114

[a] Data represent the mean�SD of triplicate experiments. IC50 values for23 additional cell lines are reported in the Supporting Information. [b] Se-lectivity index (SI): IC50(7 b)/IC50(7 a).

Scheme 2. Reagents and conditions : a) (1) Et2Zn, CH2I2, CH2Cl2, 0 8C, 10 min;(2) 15, 16 (or 17), CH2Cl2, 0 8C!RT, 1 h, 18 a : 78 % (de = 80 %)/18 b : 67 %(de = 67 %); b) I2, PPh3, imidazole, CH2Cl2, 0 8C!RT, 2.5 h, 95 % (87 %);c) NaBH4, MeCN, 80 8C, 1 h, 71 % (58 %); d)CSA, CH2Cl2/MeOH (1:1), RT,75 min, 93 % (82 %); e) (1) o-NO2PhSeCN, PBu3, THF, 30 8C, 3 h; (2) H2O2,NaHCO3, 50 8C, 1.5 h, 59 % (49 %); f) CSA, CH2Cl2/MeOH (1:1), RT, 17 h, 90 %(95 %); g) (1) NEt3, 2,4,6-trichlorobenzoyl chloride, 22, toluene, RT, 1 h;(2) DMAP, 20 a (or 20 b), toluene, RT, 17 h, quant. (72 %); h) Grubbs II, tolu-ene, reflux, 4.5 h, 57 % (72 %); i) (1) TASF, DMF, 0 8C!RT, 48 h; (2) HF·pyri-dine, MeCN, RT, 16 h , 14 % after RP-HPLC purification (5 %). Yields in paren-theses refer to those obtained in the synthesis of 7 b. Abbreviations: CSA = -camphorsulfonic acid; DMAP = N,N-dimethylaminopyridine; TASF = tris(dime-thylamino)sulfonium difluorotrimethylsilicate; THF = tetrahydrofuran.

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The in vitro antiproliferative activity of 7 a and 7 b was as-sessed across a panel of 39 human cancer cell lines, and theIC50 values against 10 of these cell lines are summarized inTable 1 (for the complete data set, see Table S2 in the Support-ing Information). In general, the naturally configured epothi-lone analogue 7 a inhibited cancer cell growth with single todouble-digit nanomolar IC50 values and with similar potency asEpo A (1). By comparison, the activity of 7 b is 15- to 1400-foldlower; in 20 out of the 39 cell lines investigated, 7 b was more

than 50-fold less potent than 7 a. Collectively, these data high-light the importance of the natural C12,C13 configuration asa prerequisite for the potent antiproliferative activity of epothi-lones.

Flow cytometry analysis of the effects of 7 a and 7 b on thecell cycle of A549 cells revealed a concentration-dependent in-crease in the G2/M population after 20 h of treatment witheither compound (which at least for 7 a was unsurprising;Figure 1). However, while 7 a (as well as Epo A (1)) led to essen-tially complete cell cycle arrest in G2/M at a concentration aslow as 20 nm, for analogue 7 b, a significant increase in theG2/M population was only observed at concentrations of morethan 8 mm.

Cells treated with 4 nm 7 a in general had a normal microtu-bule cytoskeleton as well as nuclear morphology. Very fewcells showed small bundles in the cell periphery and multipolarmitotic spindles (Figure S3 A in the Supporting Information) ;some aberrant bipolar mitosis with lagging chromosomes inone of the spindle poles was also seen. At 20 nm 7 a, cells ininterphase had thick and short bundles of microtubules andwere mostly micronucleated. Mitotic cells were mainly multipo-lar (multiple asters) and a few showed aberrant bipolar mitosis(Figure 2 C; see also Figure S3 B in the Supporting Information).Raising the concentration to 80 nm increased the proportionof multiple asters (Figure 2 D; see also Figure S3 D in the Sup-porting Information). At higher concentrations (0.4–2 mm), mi-totic cells had multipolar spindles and the remaining cells ininterphase displayed short and very dense microtubule bun-dles. This pattern was similar to that observed with Epo A (1)-treated cells (Figure 2; see also Figure S2 in the Supporting In-formation), although the proportion of monopolar spindleswas higher for 25–100 nm Epo A (1) than 20-80 nm 7 a. Athigher concentrations, mostly multiple spindles were observedfor both compounds. Interphasic bundles induced by 80 nm–2 mm 7 a were very short and dense; for Epo A (1) these typeof bundles were only visible at concentrations of 1–2.5 mm

(Figure S2 E,F in the Supporting Information).

Figure 1. Effects of 7 a and 7 b on the cell cycle of A549 cells. A) 7 a ; B) 7 b ;C) Epo A (1). Cells in G0-G1 (&) ; cells in S phase (&) ; cells in G2/M (&). Cellswere treated with test compound for 20 h, fixed with EtOH 70 % for at least2 h, and then treated with RNAse and propidium iodide 0.01 %. Fluorescencewas quantified by flow cytometry.

Figure 2. Nuclear and microtubule effects of 7 a and 7 b in A549 cells. Cellswere treated with test compound or DMSO for 20 h. DNA was stained withHoechst 33342 (blue) and microtubules with a a-tubulin monoclonal anti-body (green). The scale bar represents 10 mm. A) untreated control ;B) 100 nm Epo A; C) 20.6 nm 7 a ; D) 83 nm 7 a ; E) 826 nm 7 b ; F) 20.6 mm 7 b.Insets show mitotic cells from the same cell preparations: A) upper right:normal spindle; B) lower left: monopolar spindle; C) upper right: multipolarspindle; D) lower left : multipolar spindle; E) upper right: monopolar spindlewith lagging chromosomes; F) lower left : monopolar spindle.

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Clear changes in microtubule organization in A549 cellswere also observed in response to treatment with 7 b, al-though at significantly higher concentrations than those re-quired for 7 a (Figure 2; see also Figure S4 in the SupportingInformation). Thus, micronucleated cells were observed at con-centrations of 7 b of 8 mm and above, together with monopolarmitotic spindles and aberrant bipolar spindles ; multipolar mi-totic spindles appeared at 20 mm and above (Figure 2 F; seealso Figure S4 C–E in the Supporting Information). A few micro-tubule bundles were detected at 7 b concentrations of 20–40 mm, while in the lower concentration range (0.8–8 mm), themicrotubule network appeared to be only slightly disorgan-ized, with a somewhat parallel alignment of microtubules(straighter microtubules) in the absence of microtubule bun-dles. (The same effect was observed at 5 nm Epo A (1) or 4 nm

7 a). These data suggest that the weak interaction of 7 b withmicrotubules in biochemical assays at high concentrationstranslates into cellular effects that would be expected for a mi-crotubule stabilizer.

In summary, the data collected here for epothilone ana-logues 7 a and 7 b clearly demonstrate that the orientation ofthe cyclopropane ring in cis-fused 12,13-cylclopropyl-epothi-lones has a profound impact on the biological activity of therespective isomers. Low nanomolar antiproliferative activityagainst solid tumor cell lines in vitro was only observed for thenatural orientation of the three-membered ring. At the sametime, the effects of 7 b on cell cycle and microtubule organiza-tion together with biochemical data on microtubule bindingstrongly suggest that the antiproliferative activity of this com-pound is related to its interactions with the cellular microtu-bule network, at least to a significant extent. Given the similari-ties in the profile of 7 a and Epo A (1), it seems likely that theconclusions derived here for 7 a and 7 b also hold true for12,13-cis-epoxide-based epothilone analogues with oppositeconfigurations at C12/C13.

Experimental Section

For experimental details on the synthesis of 7 a/7 b and their bio-chemical and biological profiling see the Supporting Information.

Acknowledgements

K.H.A. and F.Z.G. are indebted to the Swiss National Science Foun-dation (SNF) (project 205320-117594) and ETH Z�rich for gener-ous financial support. J.F.D. acknowledges financial support bythe Ministerio de Economia of Spain and from the Comunidad deMadrid (projects BIO2013-42984-R and S2010/BMD-2457 BIPPED,respectively). Raphael Schiess is gratefully acknowledged for pro-viding acid 22. The authors also thank Kurt Hauenstein for excel-lent technical assistance and Louis Bertschi and the entire ETHZ-LOC MS-Service for HRMS spectra acquisition. Finally, the authorsare grateful to Dr. Philipp Krastel, Novartis Institute for Biomedi-cal Research (Basel, Switzerland) for establishing the contact withACC.

Keywords: cancer · epothilones · inhibitors · microtubules ·structure–activity relationships · stereoselective synthesis

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[16] Based on an ee of 83 % for 15, the dr of 9:1 for the cyclopropanationproduct(s) 18 a translates into a product ratio of ca. 90:8:2:0.15 for thecorresponding (12R,13S,15S), (12R,13S,15R), (12S,13R,15S), and(12S,13R,15R) isomers; for 18 b (67 % de) the product ratio for the(12S,13R,15S), (12S,13R,15R), (12R,13S,15S), and (12R,13S,15R) isomerscan be calculated as ca. 82:8:9:1. The absolute configuration (atC12,C13) of the different products has not been strictly proven but isinferred from the known and well-established stereochemical preferen-ces of the catalysts 16 and 17. The stereochemical assignments are alsosupported by the potent biological activity of 7 a.

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[20] No sign of a cis-isomer was detectable in the NMR spectra of the puri-fied product. However, we cannot exclude that minor amounts of thecis-isomer had been removed in the purification process.

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Received: April 17, 2014Published online on && &&, 0000

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COMMUNICATIONS

F. Z. Gaugaz, M. Redondo-Horcajo,I. Barasoain, J. F. D�az, A. Cobos-Correa,M. Kaufmann, K.-H. Altmann*

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The Impact of CyclopropaneConfiguration on the BiologicalActivity of Cyclopropyl-Epothilones

Up is better than down: Cyclopropyl-epothilones 7 a and 7 b were synthe-sized via a ring-closing metathesis(RCM)-based macrocyclization. Substan-tial differences between the two com-pounds were found with regard to mi-crotubule binding affinity, antiprolifera-tive activity, and effects on cellular mi-crotubules. Natural epothilone-like activ-ity was only observed for the naturallyconfigured stereoisomer 7 a.

� 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ChemMedChem 0000, 00, 1 – 6 &7&

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