Review Chemistry and biology of natural and designed enediynes

Proc. Natl. Acad. Sci. USAVol. 90, pp. 5881-5888, July 1993

Review

Chemistry and biology of natural and designed enediynesK. C. Nicolaou*, A. L. Smitht, and E. W. YueDepartment of Chemistry, The Scripps Research Institute,California at San Diego, La Jolla, CA 92093

10666 North Torrey Pines Road, La Jolla, CA 92037; and Department of Chemistry, University of

ABSTRACT Ever since the initial re-ports of the enediyne anticancer antibiot-ics in the late 1980s, researchers from anumber of disciplines have been devotingincreasing attention to their chemistry,biology, and potential medical applica-tions. Synthetic chemists and moleculardesigners have been engaged in attempts tosynthesize these molecules and to modeltheir unique architecture. Considerableefforts have been directed at understand-ing and mimicking the various processesinvolved in the targeting, activation, andDNA cleavage associated with these natu-ral products. This review summarizes themain contributions to the field, with par-ticular emphasis on work from our labo-ratories. Highlights include studies of theBergman reaction, which is central to themechanism of action of enediynes, thedesign and chemical synthesis of a numberof these systems, and biological studieswith selected molecules. Finally, the totalsynthesis of calicheamicin yI, the mostprominent member of this class of natu-rally occurring compounds, is discussed.

Nature continually serves to provide sci-entists with new ideas, often provokingus to reexamine previous studies andleading us in new directions. An exampleof such an instance which has capturedthe imagination of many scientists, our-selves included, came in 1987 with theunveiling of a new class of natural prod-ucts, the so-called "enediyne anticancerantibiotics" (Fig. 1), which possess po-tent anticancer activity and a hithertounseen biological mode of action (for aprevious review, see ref. 1). The storybegan some 15 years earlier, however,for it was in the laboratories of RobertBergman that the intriguing chemicalprocesses which provide the key to un-derstanding the remarkable activity ofthese antibiotics were first studied (2).The findings of Bergman are summarizedin Scheme I, in which the enediyne sys-tem 1 was observed to undergo cycloar-

2

8: kedarcidin chromophore 9: neocarzinostatin chromophore

FIG. 1. Naturally occurring enediyne anticancer antibiotics.

omatization when heated to give the ben-zene ring 4 with the intermediacy of thehighly reactive 1,4-benzenoid diradicalspecies 2. Masamune and coworkers (3)and Wong and Sondheimer (4) also ob-served the cycloaromatization of 10-membered ring enediynes. However, thefull significance of these simple observa-tions became apparent only when thestructures of calicheamicin -y (5, Fig. 1)and esperamicin A1 (6, Fig. 1), the firstrepresentatives of the enediyne antibiot-ics, were reported (5-8). For at the veryheart of these molecules is contained thesame enediyne unit; and therein lies themeans by which these unprecedentedmolecules exert their remarkable biolog-ical properties. Dynemicin A (7, Fig. 1)(9, 10) and kedarcidin chromophore (8,Fig. 1) (11), representing structurally dis-tinct classes of enediyne antibiotics,were subsequently reported. Neocarzi-

nostatin chromophore (9, Fig. 1) (12-14)has also been grouped with the enediyneantibiotics due to the similarity of itsstructure and mode of action.

Molecular Structures, BiologicalProperties, and Mechanisms of Action ofNaturally Occurring Enediynes

Calicheamicin y1 (5), the most prominentmember of the calicheamicins, was iso-lated from Micromonospora echinosporassp. calichensis and is a remarkable pieceof engineering by Nature, which has per-fectly constructed the molecule to endowit with its extraordinary chemical andbiological properties (15). These includeactivity in the biochemical prophage in-duction assay at concentrations < 1 pM,high antibacterial activity, and extremepotency against murine tumors such asP388 and L1210 leukemias and solid neo-plasms such as colon 26 and B16 mela-noma with optimal doses of 0.15-5 ug/kg. Calicheamicin -yl (5), along with the

*To whom reprint requests should be ad-dressed.tPresent address: Merck Sharp & Dohme Re-search Laboratories, Terlings Park, Harlow,Essex CM20 2QR, U.K.

4

SCHEME I. Bergman's original design and observation regarding the thermal cycloaroma-tization of enediynes.

5881

DH

Proc. Natl. Acad. Sci. USA 90 (1993)

FIG. 2. Computer-generated model of DNA-bound calicheamicin -yI (5) along a TCCT site.[Reprinted with permission from ref. 1 (copyright VCH, Weinheim, FRG).]

other enediyne antibiotics, is believed toexert its biological activity by damagingDNA. Indeed, it is a highly potent DNA-cleaving agent giving rise primarily tosequence-selective double-strand cuts(16).The calicheamicin yA molecule contains

two distinct structural regions. The largerof the two consists of an extended sugarresidue comprising four monosaccharideunits and., one hexasubstituted benzenering which are joined together through ahighly unusual series of glycosidic,thioester, and hydroxylamine linkages. Itis this aryloligosaccharide which serves todeliver the molecule to its target, bindingtightly in the minor groove of double-helical DNA and displaying high specific-ity for sequences such as 5'-TCCT-3' and5'-TTTT-3' (16, 17) through significanthydrophobic interactions and other forces(Fig. 2). This binding is thought to befacilitated by substantial preorganizationof the oligosaccharide into a rigid, ex-tended conformation (18). Molecularmodeling calculations by Schreiber andcoworkers (19) suggested that a signiflcantportion of the sequence selectivity for5'-TCCT-3' arises from a favorable inter-action between the large and polarizable

iodo substituent of the hexasubstitutedaromatic ring and the exocyclic aminosubstituents of the two guanines in the3'-AGGA-5' tract. Experimental evidencefrom our own laboratories supports theidea of an important role for the iodine inthe binding affinity of this oligosaccharideto DNA (20).The second region of calicheamicin yi

is its aglycon, a rigid, highly functional-ized bicyclic core termed calicheamici-none, which acts as the "warhead" ofthemolecule. The enediyne functionality ofthe molecule is locked within a rigid10-membered bridged ring awaiting acti-vation to undergo the Bergman reaction.Also forming part of the aglycon is atrisulfide, which serves as the trigger.Once the molecule is in the vicinity ofDNA (whether prior or subsequent tobinding is not known), a series of chem-ical events unfold which ultimately leadsto DNA cleavage (Scheme II) (5-8). Anucleophile (e.g., glutathione) attacks thecentral sulfur atom of the trisulfidegroup, causing the formation ofa thiolateor a thiol (10) which adds intramolecu-larly to the adjacent a,,4unsaturated ke-tone embedded within the framework ofthe aglycon. This reaction, converting a

SCHEME H. Mechanism by which calicheamicin yI (5) cleaves DNA. Nu, nucleophile.

trigonal bridgehead position to a tetrag-onal center, causes a significant changein structural geometry which imposes agreat deal of strain on the 10-memberedring. This strain is completely relieved bythe enediyne undergoing the Bergmanreaction, generating a highly reactivebenzenoid diradical (12). The calicheam-icin diradical abstracts hydrogen atomsfrom duplex DNA at the C-5' position ofthe cytidine in 5'-TCCT-3' and the C-4'position ofthe nucleotide three base pairsremoved on the 3' side of the comple-mentary strand (21, 22), leading to cleav-age of both strands of DNA. The mech-anism of action of the structurally similaresperamicins is thought to be essentiallythe same as that of the calicheamicins.Dynemicin A (7), the first member of

the dynemicin subclass of enediyne anti-biotics to be reported (9, 10), was discov-ered in the fermentation broth of Mi-cromonospora chersina. It exhibits verypotent activity against a variety of cancercell lines and significantly prolongs thelifespan of mice inoculated with P388leukemia and B16 melanoma cells. Fur-thermore, it exhibits promising in vivoantibacterial activity with low toxicity.Like the calicheamicins and esperami-cins, the dynemicins include in their mo-lecular structures a 10-membered ringwith a 1,5-diyne-3-ene bridge. They are,however, unique in combining theenediyne unit with the anthraquinonechromophore of the anthracycline anti-cancer antibiotics.A mechanism for the antitumor activ-

ity of dynemicin A (7) has been proposedwhich combines elements of the mecha-nisms of action of the calicheamicin/esperamicin, neocarzinostatin, and an-thracycline classes of antibiotics andwhich is supported by the observationthat DNA strand cleavage by dynemicinA is enhanced by the presence of thiols.In this mechanism (Scheme Im) (10), theanthraquinone nucleus intercalates intothe DNA and undergoes bioreduction(not necessarily in that order), facilitatingopening of the epoxide. This causes asignificant conformational change in themolecule and introduces considerablestrain into the enediyne system which isrelieved by the new system undergoingthe Bergman reaction, thus generatingthe DNA-damaging diradical species 17(10).

Molecular Design, Chemical Synthesis,and Biological Actions of Enediynes

The reports of the structures of the cali-cheamicins and esperamicins, and theirfascinating mode of action, promptedseveral groups to undertake investigationof the Bergman reaction in simple cyclicsystems. The synthesis of the monocy-clic 10-membered ring enediyne and itshomologues was the initial focus of a

5882 Review: Nicolaou et al.

Proc. Natl. Acad. Sci. USA 90 (1993) 5883

Anthraquinonenucleus

intercalates

into DNA andundergoesbioreduction

OH ~OH HN

Me

OH OH OH

DNA doublestrand cleavage

SCHEME Iml. Mechanism by which dynemicin A (7) cleaves DNA.

ciMeU-C.

H2n -W- CH)n00~~ \- J

-

1ga-h n = 1-8 20a-h 21a-h

SCHEME IV. Synthesis and cycloaromatization of monocyclic enediynes.

program in these laboratories (23, 24).The parent series of 10- through 16-membered ring enediynes (20b-h) wereconveniently prepared via the Ramberg-Backlund reaction of the correspondinga-chlorosulfones 19b-h (Scheme IV).The 10-membered ring enediyne 20breadily underwent the Bergman reactionat room temperature with a half-life of 18hr (Table 1), while the larger ringenediynes (20c-h) were found to be sta-ble. By contrast, the 9-membered ring20a could not be prepared, althoughproducts formally arising from a Berg-man reaction were identified. Compari-son of the distances cd between the ter-mini of the enediyne moiety of thesesystems and the ease with which they

underwent the Bergman reaction (Table1) showed a clear trend in which a de-creased cd distance reflected, in additionto closer intimacy between the acetylenicgroups, an increased ring torsion andhence an increased tendency to undergothe Bergman reaction in order to relievethe strain. For these simple systems acritical upper limit for the cd distance ofaround 3.2-3.3 A appeared to be requiredfor the Bergman reaction to occur at ameasurable rate at ambient tempera-tures. This finding provided a usefulmeans of predicting the thermal stabilityof compounds in subsequent work in ourlaboratories. More sophisticated calcula-tions and kinetics experiments by Snyder(25) and Magnus et al. (26) later demon-

Table 1. Calculated cd distances and stabilities of cyclic enediynesCompound n Ring size cd distance, A Stability

20a 1 9 2.84 Unknown20b 2 10 3.25 ti/2= 18 hr at 250C20c 3 11 3.61 Stable at 25°C20d 4 12 3.90 Stable at 250C20e 5 13 4.14 Stable at 250C20f 6 14 4.15 Stable at 25°C20g 7 15 4.33 Stable at 25°C20h 8 16 4.20 Stable at 25°C22 10 3.20 ti/2 = 11.8 hr at 370C25 10 3.29 ti/2= 4 hr at 500C26 10 3.34 ti/2 = 2 hr at 50°C27 10 3.42 Stable at 250C

strated that the crucial factor in deter-mining the ease with which a particularsystem undergoes the Bergman reactionis the relative strain energies of theground and transition states for the reac-tion. These findings should always beborne in mind when the empirical cddistance rule is applied.Given that the simple 10-membered

ring enediyne 20b underwent the Berg-man reaction at physiological tempera-tures at a reasonable rate, we proceededto attempt to mimic the DNA-cleavingaction of the calicheamicins and espe-ramicins by using such simple systems.The diol 22 (Scheme V) was designed inorder to endow the molecule with somedegree of water solubility and also toprovide for the option of attachment todelivery systems (24, 27). It was cor-rectly predicted from the calculated cddistance of 3.20 A that this moleculewould be sufficiently stable for isolationand handling at ambient temperatures butwould undergo the Bergman reaction atphysiological temperature at a sufficientrate to cause DNA cleavage. Thusenediyne 22 caused significant cleavageof phage 4X174 double-stranded super-coiled DNA in the absence of any addi-tives at concentrations as low as 10 ,uM at37°C, with the extent of cleavage beingdependent upon concentration, incuba-tion time, and temperature (24, 27). As acontrol, it was demonstrated that thecorresponding Bergman cyclized product24 (Scheme V) caused no DNA cleavage(24, 27). These molecules thus consti-tuted the first designed DNA-cleavingagents based upon the mechanism of ac-tion of the calicheamicins/esperamicins.Later on, the thermally reactive diols 25and 26 were similarly demonstrated toeffect DNA cleavage whereas the con-formationally locked and thermally sta-ble derivative 27 (Fig. 3) failed to cleaveDNA (28). Under basic conditions, how-ever, compound 27 became active via therelease of system 26 thus exhibiting bothDNA cleavage and cytotoxic properties.

Since the naturally occurring enediyneantibiotics are triggered to exert theirbiological actions by bioreductive pro-cesses, a system was designed to controlthe Bergman cyclization by a hydroqui-none = quinone redox process (29). Itwas postulated that hydroquinone sys-tems such as 28 should be more stabletoward cycloaromatization than the cor-responding quinones 29 and 30 (Fig. 3).This was indeed found to be the case.Thus the hydroquinone 28 had a half-lifeof 74 hr at 110°C, while 29 and 30 hadhalf-lives of 2.6 hr and 32 min, respec-tively at 55°C. These results were furtheremphasized by the finding that, while 28exhibited no DNA-cleaving properties,29 and 30 were able to cause DNA dam-age and death of tumor cells.

Review: Nicolaou et al.


DNAOXOH 37°C [OH ___L N OHOH OH I O H

DNA22 23 cleavage 24

SCHEME V. Enediyne 22 as a designed DNA-cleaving agent.

Me MeK,O%OH

-<OH

Me Me

25OCO'Bu OH

Me e

OCO'Bu28

Me Me<,kYOH

~*d OHMe Me26

O OHMee>

0

29

Me Me

Q0°Me Me

27O 0

Me

030

FIG. 3. Examples of designed enediynes with modulated reactivities.

This concept of activation of enediynesystems through redox processes wastaken a step further by Myers andDragovich (30), who designed the systemshown in Scheme VI. Enzyme-mediatedreduction of the anthraquinone 31 led toelimination of succinic acid followed bytautomerization and oxidation to revealthe enediyne system in 34. This thenslowly underwent the Bergman reactionat 37°C (tl2 2 days).

Several groups have synthesizedmodel systems of the calicheamicin/esperamicin enediyne core and studiedtheir Bergman cycloaromatization. No-table among the early contributions arethe studies of Danishefsky (e.g., 35, Fig.4) (31, 32), Kende (36, Fig. 4) (33), Mag-nus (e.g., 37, Fig. 4) (34, 35), andSchreiber (38, Fig. 4) (36) and their co-workers. Particularly interesting werethe observation of Danishefsky and col-laborators (37, 38) that such simpleenediyne systems can simulate the DNAcleaving properties of calicheamicin Vyiand the synthetic work of Magnus andcoworkers (34, 35) which utilized acety-lene cobalt complexes as intermediatesto arrive at the targeted enediynes.When the structure and mode of action

of dynemicin A (7) were reported in 1989(9), several groups, including ours, un-dertook programs directed toward thedesign and synthesis of models which

would mimic the mechanism of actionand facilitate the total synthesis of thishighly unusual molecule. Work fromthese laboratories culminated in the mo-lecular design, chemical synthesis, andbiological evaluation of several dynemi-cin A mimics (39-47), including thehighly potent systems 39 and 40 (SchemeVII) (42, 43). The latter system incorpo-rated what was perceived to be the cen-tral structural features responsible for thebiological actions of dynemicin A-namely, the strategic combination of theenediyne, epoxide, and nitrogen func-tionalities. Particularly important inthese systems was the installation of the2-(phenylsulfonyl)ethyl carbamate groupon the nitrogen, which acts as an easilyremovable locking device for the epoxidefunctionality and thus stabilizes the sys-tem until activation. Indeed, removal ofthis triggering device under mild basicconditions released the parent com-pounds 41 and 42, respectively, whichwere found to be rather labile, undergo-ing the cascade of reactions shown inScheme VII (41/42 -+ 43/44 -* 45/46 --

47/48 -+ 49/50). The intermediacy of the

highly reactive 1,4-benzenoid diradicals47 and 48 is presumably responsible forthe high cytotoxicity of these com-pounds, although the precise target ofthis species within the cell has not beendefined unambiguously. As expected,

31

34

32

0

OH OMe33

SCHEME VI. Myers' approach to redox activation of enediyne systems.

0SAc

HO OH

35 (Danishefsky)

SSSBn

TBSO

37 (Magnus)

0

H OH

36 (Kende)0

HO" HH...>'N. OTBS

38 (Schreiber)

FIG. 4. Calicheamicin/esperamicin modelsystems.

however, compounds 39 and 40 werefound to cleave DNA under basic condi-tions but failed to do so in acidic medium(Fig. 5) (42). The parent compound 42also cleaved DNA at physiological pH(Fig. 6). Fig. 7 exhibits a number of otherdesigned enediynes with triggering de-vices and modulated reactivity synthe-sized in these laboratories [51 (44), 52(unpublished results), 53 (45), 54 (45), 55(43), and 56-58 (46)]. Some of thesesystems demonstrated significant DNA-cleaving properties (Fig. 5) (42). Particu-larly interesting is compound 52, whichcontains a photosensitive triggering de-vice (o-nitrobenzyl carbamate) andwhich has been demonstrated (unpub-lished results) to cleave DNA upon irra-diation, as expected.

Table 2 (42) shows the cytotoxicity ofcompound 40 against a range of cell lines.Significant differences are observed, withcytotoxicities ranging from 1 ,uM for thehighly resistant melanoma cell lines to 10fM for the highly sensitive MOLT-4 leu-kemia cell line. Particularly significant isthe high cytotoxicity of this compoundagainst the multiple-drug-resistant TCAF-DAX cell line (IC50 = 1.7 nM) and therelatively low cytotoxicity against a num-ber of normal cell lines. Furthermore,preliminary in vivo studies using 40 and atritiated analogue of40 with mice infectedwith leukemia and solid tumors showedencouraging results (unpublished results).

Interestingly, treatment of MOLT-4cells under appropriate conditions withenediyne 40 followed by observation ofcell morphology and cell death revealedthe phenomenon of programmed celldeath (apoptosis) (48) as the prevailingcause of cell destruction (49). Further-more, competition experiments usingenediynes with relatively low toxicitiesresulted in the identification of certaininhibitors of apoptosis. Specifically, themethoxy enediyne 55 (Fig. 7), which dis-played diminished tendency to undergothe Bergman reaction, exhibited inhibi-tion of the cytotoxic action of compound40. Thus, when MOLT-4 cells were pre-incubated with enediyne 55 at 0.1 mM for1 hr prior to treatment with the cytotoxiccompound 40, a dramatic reduction (fac-



hS"

Base: J

cd = 3.64A R39: R = H40: R = OCH2CH20H

DNA

DNAcleavage

r

cd = 3.10 - 3.20A49: R = H 47: R = H 45: R = H50: R = OCH2CH20H 48: R = OCH2CH20H 46: R = OCH2CH20H

SCHEME VII. Design of dynemicin models with triggering devices and their postulatedmechanism of action.

tor of _105) in the cytotoxicity of 40 wasobserved. Similar reductions (factors of1O2-104) were observed in the cytotoxic-ities of the naturally occurring enediynesdynemicin A (9) and calicheamicin 'y (5)in the presence of the methoxy enediyne55. Particularly intriguing was the obser-vation that 55 inhibited apoptotic mor-phology of cell death by powerful induc-ers of apoptosis such as actinomycin Dand cycloheximide, although cell viabil-ity was not affected in these cases (49).

Further insight into the remarkablecell-type selectivity of these designedenediynes was obtained by comparingthe cytotoxicities of enantiomericallypure compounds (+)-39 and (-)-39(Scheme VII) (50) and the methyl-substituted compounds 56-58 (Fig. 7)

pH 6.0 pH 7.0 pH 7.4 pH 8.5a r-*i n-i r--i r-i_compound: - 40 - 40 - 40 - 40

I1:lane: 1 2 3 4 5 6 7 8

bcompound: - 41 39 40 55 54 51 49

III

(46). These experiments demonstrateddramatic differences in potencies de-pending on the enantiomeric form of theenediyne 39 and the degree and stereo-chemistry of methyl substitution in com-pounds 56-58 and raised intriguing ques-tions: Is there an intracellular receptorfor these enediynes other than DNA?Could this putative receptor serve as acapturing and delivery system for theseenediynes to specific sequences ofDNA?Is there a prevailing biological mecha-nism in certain cell types which facilitatesthe /3elimination? These questions raiseeven more interesting issues concerningthe regulation of cell death. Exploitationof such observations and elucidation ofthe mechanism of action of these agentsmay lead to new approaches to drugdesign.

In addition to our own efforts in thedynemicin area, a number of othergroups have contributed to the designand synthesis of model systems. Notableamong these contributions are those ofSchreiber (59, Fig. 8) (51), Wender (60,

51

Ph'.1 J0' Ph" -' .O N

0. ~0'

53 54

050 0OH 00 H H

h"SI Ph Y,)< OAN0Ph0 0 ~~~~~~~R,R,

55 56: RI =Me; R2=H57: RI =H; R2 MO58: RI = R2 = Me

FIG. 7. Designed enediynes with triggeringdevices and modulated reactivity.

Fig. 8) (52), Magnus (61, Fig. 8) (53), andIsobe (62, Fig. 8) (54).

Inspired by the molecular architectureand the mechanism of action of neocarzi-nostatin chromophore (9), a number ofgroups have designed, synthesized, andstudied a variety of model systems. Aselection of these model compounds cov-ering the rather wide spectrum of struc-tural types synthesized in this field isshown in Fig. 9 [63 (55), 64 (56), 65 (57),66 (58), 67 (59), and 68 (60)].

Designed Enediynes Tethered to DeliverySystems

Compared with the naturally occurringcompounds (e.g., calicheamicin Ay), thedesigned enediynes reported so far ex-hibit significantly lower potencies asDNA-cleaving agents. In the absence ofsuitable delivery systems these observa-

Table 2. Cytotoxicities of designed enediyne 40 against a panel of tumor cell lines andnormal cell lines

Cell type Cell line IC50, Mlane: 1 2 3 4 5 6 7 8

FIG. 5. (a) DNA cleavage by enediyne 40.(b) DNA cleavage by enediynes 39-41, 51, 54,and 55 and Bergman product 49. For condi-tions, see ref. 42. [Reproduced with permis-sion from ref. 42 (copyright 1992, AAAS,Washington, DC).]

1 2 3 4 5 6

Form II

Form IlIlForm I

FIG. 6. DNA cleavage by enediyne 42. Forconditions see ref. 47. [Reproduced with per-mission from ref. 47 (copyright Am. Chem.Soc., Washington, DC).]

Tumor cell linesMelanomaColon carcinomaOvarian carcinomaBreast carcinomaLung carcinomaPancreatic carcinomaT-cell leukemia

Promyelocytic leukemiaT-cell leukemia

Normal cell linesBone marrowHuman mammary epithelial cellsNormal human dermal fibroblastChinese hamster ovary

*Multiple-drug-resistant cell line.

HN

Nu

R

M-14HT-29Ovcar-3MCF-7

UCLA P-3Capan-1TCAF

TCAF-DAX*HL-60MOLT-4

HNBMHMECNHDFCHO

1.6 x1.6 x7.8 x7.8 x9.8 x3.1 x1.1 x1.7 x3.6 x2.0 x

5.0 x6.3 x5.0 x3.1 x

10-610-610-710-710-810-910-910-910-1110-1

10-610-610-6



tions are not surprising, but they didprompt several attempts to improve theDNA-cleaving properties of these mole-cules by tethering them to suitable mol-ecules known to bind DNA. The hybridmolecules shown in Fig. 10 are represen-tative examples ofthe work carried out inthis area [69 (1), 70 (1), 71 (61), 72 (62), 73(63), and 74 (64)]. Despite these efforts,however, the increase in potency oftheseagents as DNA cleavers is not as dra-matic as expected. Reasons for the lackof success in this area so far may be dueto the requirement for precise docking ofthe enediyne into the DNA site in orderfor the generated radicals to abstract hy-drogen from the DNA backbone. As inthe case of the natural enediynes, muchfine tuning and evolution needs to bedone before we can reach sophisticatedmolecular designs with highly potent andefficient DNA-cleaving profiles. Such de-signs may be accelerated by molecularmodeling and human ingenuity.

Total Synthesis of Naturally OccurringEnediynes: Calicheamicin yiWhen the structures of calicheamicin iy,(5) and esperamhicin A1 (6) were revealedto the scientific community, our group,like many other synthetic groups aroundthe world, recognized the formidablesynthetic challenge presented by theseunprecedented molecular systems andundertook the task of achieving the totalsynthesis of calicheamicin Vyi Such adaring endeavor would require carefulplanning in order to overcome the con-siderable difficulties inherent in con-structing a molecule of such complexity.The total synthesis of calicheamicin VyIwould not only require control of theabsolute stereochemistry at 19 chiral cen-ters but also require insight into the po-tential instability and reactivity of themultitude of functionalities which com-prise the molecule, in order to judge thecorrect timing for their introduction. Notunreasonably, the strategy adopted byour group was one in which suitablyadvanced precursors to the aglycon andoligosaccharide fragments of the mole-

59 (Schreiber)

61 (Magnus)

cule would be coupled together in thefinal stages of the synthesis (Fig. 11).However, degradation studies on the nat-ural product by other groups had failed tofurnish either the intact aglycon or oligo-saccharide due to their inherent instabil-ities, and so there would be no opportu-nity for establishing the correct condi-tions necessary for achieving this finalunion of the two domains without firstcarrying out the mammoth task of theirseparate syntheses.

In tackling the synthesis of the oligo-saccharide portion of calicheamicin yly,Nicolaou and coworkers were fortunatein being able to draw upon their experi-ence in the field of sugar chemistry, en-abling them to recognize the most chal-lenging features of the fragment and de-sign their synthetic strategy around thesolution of these problems. They per-ceived that the B ring (Fig. 11), with itssynthetically demanding 2-deoxy-,8-hydroxylamino glycosidic linkage and4-thio substituent, should be at the center

OCOtBu c

HO63 (Myers) 64 (h

HO'. MeM

66(Hirama) 67T(S

66 (Hirama) 67 (T

of their thoughts. This led them to under-take several model studies for the con-struction of the B ring which culminatedin providing a strategy (Scheme VIII) forthe pivotal step of the synthesis of theoligosaccharide fragment (65). Thus,heating the thionoimidazolide 75 in re-fluxing toluene effected a clean allylictransposition of functionality on the Bring via a Ferrier-like rearrangement asillustrated by the arrows in Scheme VIII.This [3,3] sigmatropic rearrangement si-multaneously introduced the 4-thio sub-stituent at C-4 with the correct stereo-chemistry and deoxygenated the 2-posi-tion. Nicolaou et al. (65) were thus able toreport the first total synthesis of the oli-gosaccharide portion of calicheamicin VyItoward the end of 1990. Significantly, theefficient and convergent nature of thisstrategy provided a means of readily ob-taining the multi-gram quantities of suit-ably advanced precursors to the oligo-saccharide which would be needed inattempts to reach calicheamicin yI itself.

DLOH LY=S=. OH

Nicolaou) 65 (Nicolaou)0PPh2

7D + OH

OH7oshima) 68 (Nicolaou)

FIG. 9. Selected neocarzinostatin models and related systems.

MeO N0.N

OH

60 (Wender)

0

EtO N

MMe

OH

62 (Isobe)

FIG. 10. Enediynes tethered to delivery systems.


FIG. 8. Dynemicin model systems.


Aglycon fragment

0HO~... 0

Me O MeSSS _ HCO2Me

ct NO~~y~OMeoH ~HO oMeOMe H ~~~~~~~~~Strategic glycosideHO

e

M Me_Na1:j bond for coulingOH MeO j the aglycon and

oligosaccharidefragments of the

Oligosaccharide fragment target molecule.

FIG. 11. Retrosynthetic analysis of calicheamicin Ay (5).

m0 0

BzOw vOMEM77

0

NHCO2MeHO.... =

OH

MeSSS

80

m0 0,Ns

BzOKj'OMEM

78several steps

n0 0

several 4 NPhthsteps ll

TESO7 CHO

/C02Me

79

Furthermore, this strategy provided themeans by which several modified oligo-saccharides became available for bindingstudies with DNA fragments (20).By this time thoughts were turning

toward the synthesis of the other portionof calicheamicin AyI its aglycon. Thissynthesis would require a strategy whichwould provide large amounts of a suit-ably advanced precursor in homochiralform (i.e., as a single enantiomer). Sev-eral groups had spent the previous 4years examining the chemistry of theaglycon and synthetic approaches towardits various structural features. Danishef-sky (31, 32), Kende (33), Magnus (34, 35),and Schreiber (36) and their colleagueswere among the first of many to demon-strate that the construction ofthe bicyclicframework of the aglycon is not as daunt-ing as at first appeared. Meanwhile, Mag-nus et al. (66) had studied the chemistryof the allylic trisulfide group, and Dan-ishefsky and coworkers (67), in a mag-nificent achievement, had succeeded inassembling the full functionality toachieve the first synthesis of calicheam-icinone (80) as its racemate. Thus at thebeginning of 1991, we embarked upon anew approach to the aglycon whichwould hopefully provide the molecule inenantiomerically pure form and in suffi-cient quantities for coupling to the oligo-saccharide and completing the total syn-thesis of calicheamicin 'y. Indeed, prog-ress was rapid, since we were able tobenefit during the latter stages of oursynthesis from the lessons learned by ourpeers, allowing us to complete the firstenantioselective synthesis of the aglycontowards the end of the same year (68). Atthe heart of our strategy was an intramo-lecular dipolar cycloaddition reaction ofthe nitrile oxide 77 (Scheme IX), in whichchirality had already been established, togive the adduct 78. This strategy not onlyled directly to the efficient installation of

Me 0 0 ,oA Me 0N 110°C N

vN4N~~~~_- OR

\=J R = SituMe275 76

SCHEME VIII. Ferrier-like rearrange-ment strategy utilized in the synthesis of thecalicheamicin 'y oligosaccharide.

the full functionality of the aglycon butalso allowed ultimate control of its abso-lute stereochemistry to provide cali-cheamicinone (80) as well as an advancedprecursor, 82 (Scheme X), suitable forcoupling with the oligosaccharide frag-ment and thus opening the way for fur-ther progress.Now that both domains of calicheam-

icin y' were available, it remained for usto couple them together and complete thesynthesis. By this time Nicolaou et al.(69) had already demonstrated the syn-thetic utility of the carbohydrate precur-sor 81 (Scheme X) as a coupling partnerin model studies, but the question re-mained as to whether it would be possibleto find a precursor to the aglycon whichwas suitable for coupling to the oligosac-charide and yet sufficiently advanced toallow the final unveiling of its features.Examination of molecular models indi-cated that it would be sterically demand-ing to bring the two pieces together,although only the required f3glycosidiclinkage would be formed if it was possibleat all. Indeed, at this very time Danishef-sky and coworkers (70) were reportingdifficulty in their attempts to couple asufficiently advanced precursor of theaglycon to their oligosaccharide, whichthey had also synthesized by this time,although they were able to couple animmature version of the aglycon. How-ever, our fears were unfounded, for the

SCHEME IX. Strategy for the enantioselec-tive synthesis of calicheamicinone (80) andother calicheamicin precursors. MEM,2-methoxyethoxymethyl; Phth, phthaloyl; Bz,benzoyl.

aglycon precursor 82 coupled smoothlyand stereoselectively with 81 (SchemeX), and the careful choice we had made ofprotecting groups for the various sensi-tive functionalities of the entire moleculemeant that several steps later we wereable to unveil the final target in its fullglory to complete the synthesis of cali-cheamicin 'y (5) (71), the first total syn-thesis of any member of the enediyneclass of natural products.

Concluding Remarks and FutureProspects

Much original work has been carried outin the area of enediynes since 1987, whenthe first structures of the naturally occur-ring calicheamicins and esperamicinswere disclosed. Even though some of thefundamental chemistry had been knownprior to the 1987 reports on these novelanticancer antibiotics, it took a bold les-son from Nature again to show us the wayto bring together chemistry and biologyin arriving at such elegant molecular sys-tems and with such specific biologicalactions. Research in this field is continu-ing unabated in chemistry, biology, and

Me 0

MeN 0TESO... .

Me TEHBzO NHCO2MeMe ~ OMe FMOC OyNHMeo OTES MeO --O

81 82[carbohydrate precursor] [aglycon precursor]

I several steps

0HO..._ 2M

Me 0 MeSSS NHCO2MeMe

O S~MeO HO lm

HO ~~HO~lme 7o OMe H.N.-IHO_ Me,_N147Meo OH MeO

5: calicheamicin y1I

SCHEME X. Coupling of oligosaccharide and aglycon fragments and completion of the totalsynthesis of calicheamicin y' (5).



medicine, with some compounds alreadyin clinical trials.

In chemistry, research has taken sev-eral directions, including molecular de-sign and chemical synthesis. Severalmodel systems related to the naturalproducts have been synthesized, demon-strating new synthetic principles andstrategies. Designed enediynes demon-strated abilities to cleave DNA and ex-hibited selective cytotoxicity against tu-mor cells versus normal cells (39, 42).Enediynes have been implicated in thepuzzling but important phenomenon ofprogrammed cell death (apoptosis) (49).And the total synthesis ofthe most prom-inent and complex member of theenediyne class, calicheamicin yI (5), hasbeen achieved (71).The next phase of research in the

enediyne field will undoubtedly includefurther synthetic attempts at the natu-rally occurring targets, new designedenediynes with sophisticated mecha-nisms of in vitro and in vivo activation,and attachment of these systems to suit-able delivery systems. Targeting devicesmay include antibodies, oligonucleo-tides, oligosaccharides, peptides and pro-teins, DNA intercalators, DNA groovebinders, hormones, and other ligands.Hybrid molecules between enediyne"molecular warheads" and such deliverysystems should provide new insights intobiological phenomena and may facilitatedrug design and development.

We thank our many talented associates,whose names appear in the original articlescited below, for their invaluable contributionsto this research effort. Our work was finan-cially supported by the National Institutes ofHealth, the National Science Foundation, TheScripps Research Institute, and the Universityof California, San Diego.

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Documents

Review Chemistry and biology of natural and designed enediynes