Proc. Natl. Acad. Sci. USAVol. 92, pp. 3642-3649, April 1995

Review

CC-1065 and the duocarmycins: Unraveling the keys to a new classof naturally derived DNA alkylating agentsDale L. Boger* and Douglas S. JohnsonDepartment of Chemistry, The Scripps Research Institute, 10666 North Torrey Pines Road, La Jolla, CA 92037

ABSTRACT Key studies defining theDNA alkylation properties and selectivity ofa new class of exceptionally potent, natu-rally occurring antitumor antibiotics in-cluding CC-1065, duocarmycin A, and duo-carmycin SA are reviewed. Recent studiesconducted with synthetic agents containingdeep-seated structural changes and the un-natural enantiomers of the natural prod-ucts and related analogs have defined thestructural basis for the sequence-selectivealkylation of duplex DNA and fundamentalrelationships between chemical structure,functional reactivity, and biological prop-erties. The agents undergo a reversible,stereoelectronicaily controlled adenine-N3addition to the least substituted carbon ofthe activated cyclopropane within selectedAT-rich sites. The preferential AT-rich non-covalent binding selectivity of the agentswithin the narrower, deeper AT-rich minorgroove and the steric accessibility to thealkylation site that accompanies deep AT-rich minor groove penetration control thesequence-selective DNA alkylation reactionand stabilize the resulting adduct. For theagents that possess sufficient reactivity toalkylate DNA, a direct relationship betweenchemical or functional stability and biolog-ical potency has been defined.

Substantial progress has been made inunderstanding the fundamental principlesresponsible for the sequence-selective rec-ognition of DNA by small organic mole-cules (1-4) including a range of naturallyoccurring antitumor antibiotics. Threefundamental issues that arise in the exam-ination of DNA binding agents are theorigin of binding affinity, binding selectiv-ity, and reaction selectivity includingDNA alkylation or cleavage. Each canindependently assert levels of control onthe sequence-selective recognition ofDNA and the relative role and origin ofthese effects remain a primary objective ofmost investigations. The emergence ofexperimental techniques for the rapid se-quencing of DNA (5-9), for the determi-nation of DNA binding selectivity andaffinity including footprinting and affinitycleavage techniques (10, 11), and for thedetermination of sites of DNA modifica-tion (12) coupled with the rapid advancesmade in the structural characterization ofDNA complexes at defined sites by x-ray

crystallography (13, 14), NMR spectros-copy (15, 16), and molecular modeling(17, 18) have advanced the understandingof the molecular interactions responsiblefor the sequence-selective recognition ofDNA. A powerful complement to suchtools in the examination of naturally de-rived DNA binding agents is the prepara-tion and subsequent examination of keypartial structures, agents containing deep-seated structural modifications or varia-tions in the natural product and theircorresponding unnatural enantiomers.Deliberate, well-conceived deep-seatedmodifications may directly address anddefine both the structural basis for thesequence selective recognition of DNAand fundamental relationships betweenstructure, functional reactivity, and bio-logical properties.CC-1065 and the Duocarmycins. One of

the newest class of agents shown to alky-late DNA includes (+)-CC-1065 (1; ref.19), (+)-duocarmycin A (2; refs. 20-24),and (+)-duocarmycin SA (3; refs. 25 and26). Given their remarkable cytotoxic po-tency, substantial efforts have been de-voted to defining their properties (27-39).In these studies, the agents have beenshown to exert their biological effectsthrough a sequence-selective alkylation ofDNA. The reversible, stereoelectronicallycontrolled adenine-N3 addition to theleast substituted cyclopropane carbon hasbeen found to occur within selected AT-rich sites in the minor groove and exten-sive efforts have been devoted to deter-mine the origin of the DNA alkylationselectivity, to establish the link betweenDNA alkylation and the ensuing biologicalproperties (40), and to define the funda-mental principles underlying the relation-ships between structure, chemical reactiv-ity, and biological activity. The limitedstudies conducted with naturally derivedduocarmycin SA (3) revealed a combina-tion of properties that make it the mostexciting of the natural products identifiedto date. In addition to lacking the delayedtoxicity characteristic of (+)-CC-1065(41), it is the most stable and most potentmember of this class of agents. As detailedherein, it is likely that this combination ofproperties is not fortuitous but rather thatthe enhanced functional stability of 3 is

directly responsible for its increased bio-logical potency.

2, Duocarmycin A

3, Duocarmycin SA

DNA Alkylation Selectivity. The event,sequence selectivity, quantitation, revers-ibility, and structure determination of thepredominant DNA alkylation reaction by1-3 have been defined (42-59). The alkyl-ation site identification and the assess-ment of relative selectivity were derivedthrough thermally induced depurinationand strand cleavage of labeled DNA afterexposure to the agents (Scheme I). Thealkylation selectivity for (+)-duocarmycinA (2) and (+)-duocarmycin SA (3) provednearly indistinguishable (43, 45). Each al-kylation site detected was adenine flankedby two 5' A or T bases and there proved tobe a preference for this three-base se-quence: 5'-AAA > 5'-TTA > 5'-TAA >5'-ATA. In addition, a strong preferencefor the fourth 5' base to be A or T versusG or C was observed and distinguishes thehigh- versus low-affinity sites. Table 1summarizes the consensus sequence de-

*To whom reprint requests should be ad-dressed.

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OHC> 5'

100°OC 59 wOPO20DNA+

5'

OH ---- HOPO2DNA-5'HO 2UDNA and/or O0

OPO2HDNA H O3'

+

3'-HOPO2DNA

Scheme I

rived from the evaluation of (+)-(2; ref.45) and (+)-(3; ref. 43). Although bothagents alkylated the same sites, two im-portant distinctions were defined. Theleast reactive of the agents, (+)-duocar-mycin SA (3), was found to alkylate DNAwith a greater efficiency (10-10Ox) andwith a greater selectivity among the avail-able sites. In addition, the related natu-rally occurring agents 4-7 were found topossess DNA alkylation and biologicalproperties indistinguishable from 2 thatmay be attributed to their facile cyclopro-pane ring closure under the conditions ofassay (43, 45).The study of (+)-CC-1065 revealed a

similar, but more extended, 5-bp AT-richalkylation selectivity (42, 53-59). Table 1summarizes the consensus sequence de-rived from the evaluation of (+)-CC-1065.Each site of alkylation proved to be ade-nine flanked by two 5' A or T bases. Thesequence preference for the three-baseAT-rich alkylation site was determined tofollow the order 5'-AAA = 5'-TTA >5'-TAA > 5'-ATA. In addition, the agentexhibited a strong preference for thefourth 5' base to be A or T and a weakerpreference for the fifth 5' base to be A orT. The preferences for the fourth and fifth5' bases to be A or T distinguish many of

the high- versus low-affinity sites. Thestrict AT preference within the first 3 bprepresents a combination of the initial 3'adenine-N3 alkylation site and an adja-cent 5' two-base AT site required to ac-commodate the central subunit binding.The weaker preferences for the fourth andfifth 5' bases to be A or T reflect, asobserved with the high-affinity sites, a

preferential AT-rich site for the third sub-unit bound in the minor groove. A study ofshorter and more extended analogs ofCC-1065 revealed that this AT-rich selec-tivity corresponds nicely to the length ofthe agent and, hence, binding site sizesurrounding the alkylation site required toaccommodate the agent (42, 58).Unnatural Enantiomer DNA Alkylation

Selectivity. One of the more revealingdiscoveries made possible through use ofsynthetic materials was that the unnaturalenantiomers also constitute effectiveDNA alkylating agents (42, 43, 56, 58).ent-(-)-Duocarmycin SA (3) was found toalkylate DNAbut at concentrations -1OXthat required for the natural enantiomer(43) and Table 1 summarizes its consensusalkylation sequence. Each alkylation sitedetected was adenine and essentially eachalkylation site was flanked by a 5' and 3'A or T base that exhibited the following

sequence preference: 5'-AAA > 5'-AAT> 5'-TAA > 5'-TAT. An additionalstrong preference for the second 3' basefrom the alkylation site to be A or T wasobserved. In this regard, the ent-(-)-duocarmycin SA alkylation proved analo-gous to the natural enantiomer with theexception that the binding orientation isreversed (5' -> 3') over an AT-rich 3.5-bpsite. However, while the bound conforma-tion of the natural enantiomer covers anAT-rich 3.5-bp site extending from theadenine-N3 alkylation site in the 3' -> 5'direction across the adjacent 2-3 5' bases(i.e., 5'-AAAA), the AT-rich 3.5-bp sitefor the unnatural enantiomer extends inthe reverse 5' -> 3' direction starting at

the first 5' base site preceding the ade-nine-N3 alkylation site and extendingacross the alkylation site to the first andsecond adjacent 3' bases (i.e., 5'-AAAA).The reversed binding orientation is re-

quired to permit adenine alkylation at theleast substituted cyclopropane carbon andthe offset AT-rich alkylation selectivity isthe natural consequence of the diastereo-meric relationship of the adducts. Simi-larly, ent-(-)-CC-1065 was found to alky-late DNA with a rate and efficiency thatwere comparable to the natural enanti-omer (56). Table 1 summarizes the con-sensus alkylation sequence for ent-(-)-CC-1065, which proved similar to ent-(-)-duocarmycin SA but which exhibits anextended 5-bp AT-rich selectivity (42, 56).Each alkylation site proved consistentwith 5' adenine-N3 alkylation with agentbinding in the minor groove in the 5' ->3'

direction from the alkylation site covering5 bp across an AT-rich region. All alkyla-tion sites detected proved to be adenineand nearly all of the 3' and 5' basesflanking the adenine-N3 alkylation siteproved to be A or T. Like the naturalenantiomer, there proved to be a prefer-ence for this sequence that follows theorder 5'-AAA > 5'-TAA > 5'-AAT, 5'-TAT. There also proved to be a substan-tial preference for the second and third 3'base to be A or T.Adenine-N3 Alkylation. The quantita-

tion of the adenine-N3 alkylation, confir-mation of its structure through isolationand characterization of the thermally re-leased adducts, and the search for unde-

Table 1. Consensus sequences for the DNA alkylation reactions of the natural products and their unnatural enantiomers

Agent Base* 5' 4 3 2 1 0 -1 -2 -3 -4 3'

(+)-1 A/T (56)t 67 78 94 98 100 55Consensus A/T . G/C A/T > G/C A/T A/T A R -- Y

(+)-2 and (+)-3 A/T (56)t 58 79 100 100 100 69Consensus N A/T > G/C A/T A/T A R>Y> - -

ent-(-)-3 A/T (56)t 93 100 96 73 56Consensus - A/T A A/T A/T > G/C N

ent-(-)-l A/T (56)t 88 100 93 82 73 56Consensus A/T A A/T A/T > G/C A/T > G/C N

R, purine; Y, pyrimidine.*Percentage of the indicated base located at the designated position relative to the adenine-N3 alkylation site.tPercentage composition within the DNA examined.

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- - 2 '-

4, Duocarmycin B2: X = Br5, Duocarmycin C2: X = Cl

tected alkylation sites have been con-ducted. In these studies, the adenine-N3alkylations illustrated in Scheme I by (+)-duocarmycin A, (+)-duocarmcyin SA, ent-(-)-duocarmycin SA, and (+)-CC-1065have been found to quantitatively accountfor 86-92% (46), 90-100% (43), 86-92%(43), and 80-90% (54), respectively, oftheir consumption in the presence of ex-cess DNA and to constitute the near-exclusive alkylation event. The character-ization of the adducts led to the unambig-uous assignment of the structures 8, 9, and10 in which adenine-N3 addition to theunsubstituted cyclopropane carbon of theagents was established.

Thus, all three agents provide predom-inantly or exclusively adenine-N3 adducts(.90%). However, a minor guanine-N3alkylation has been detected with 2 and toa lesser extent with 1 but only upon iso-lation of the thermally released adductfollowing treatment of DNA with excessagent (50), within oligodeoxynucleotideslacking a high-affinity adenine-N3 alkyla-tion site (51), or when the adenine alkyl-ation sites within AT-rich regions ofDNAwere protected from alkylation with high-

6, Duocarmycin B1: X = Br7, Duocarmycin Ci: X= Cl

affinity AT-rich minor groove bindingagents (52). In contrast, duocarmycin SA(3) showed no evidence of guanine-N3alkylation when subjected to similar ormore forcing conditions (43). Even undervigorous conditions, unreacted agent wasrecovered and the selectivity for ade-nine-N3 versus guanine-N3 alkylation was>25:1. This enhanced selectivity of 3 maybe attributed to its decreased reactivity.Notably, under the relevant conditions oflimiting agent, even duocarmycin A pro-vided near-exclusive adenine-N3 alkyla-tion. Since duocarmycin SA is much morepotent than A, the minor guanine-N3 al-kylation cannot be uniquely relevant tothe expression of the biological propertiesand may represent a nonproductive com-petitive event.Models of the DNA Alkylation Adducts

That Accommodate the Reversed BindingOrientation and Offset AT-Rich Selectiv-ity of the Enantiomeric Agents. The ex-amination of the natural products, theirsynthetic unnatural enantiomers, and keypartial structures not only resulted in theunusual observation that the unnaturalenantiomers constitute effective DNA al-kylating agents and potent antitumor an-

tibiotics but also has led to the emergenceof a detailed model (42, 43) of the struc-tural features responsible for their selec-tive alkylation of DNA. The characteriza-tion of 8-10, the unambiguously estab-lished absolute configuration of theagents, and the definition of the alkylationconsensus sequences for both enantio-mers of 1-3 provided the necessary infor-mation for the construction of accuratemodels of the adenine-N3 alkylation. Fig.1 illustrates models of the (+)- and ent-(-)-duocarmycin SA alkylation at a com-mon site in w794 DNA, 5'-(CTAATT),which constitutes a high-affinity site forthe unnatural enantiomer and a minor sitefor the natural enantiomer (43). Similarmodels of the (+)- and ent-(-)-CC-1065alkylations have been published (42). Inboth instances, the hydrophobic face ofthe agent is imbedded deeply in the minorgroove, the polar functionality lies on theouter face of the complex, and the boundhelical conformation of the agent comple-ments the topological curvature and pitchof the minor groove spanning an AT-rich3.5- or 5-bp site, respectively. For (+)-duocarmycin SA, the binding spans 3.5 bpstarting with the 3' adenine alkylation siteand extends in the 3' -> 5' direction overthe 2-3 adjacent 5' base pair (5'-CTAA).For ent-(-)-duocarmycin SA, the bindingsimilarly spans a 3.5-bp AT-rich site butwhich necessarily starts at the 5' baseadjacent to the alkylation site and extendsin the 5' -) 3' direction over the alkylationsite and the 1-2 adjacent 3' base pair(5'-AATT). This offset alkylation selec-tivity within an AT-rich site is the natural

FIG. 1. Comparison stick models of the (+)-duocarmycin SA (Left) and ent-(-)-duocarmycinSA (Right) alkylation at the same site within w794 DNA: duplex 5'-d(GACTAATTTTT). Thenatural enantiomer binding extends in the 3' -*5' direction from the adenine-N3 alkylation siteacross the sequence 5'-CTAA. The unnatural binding extends in the reverse 5' -* 3' directionacross the site 5'-AATT.10

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consequence of the diastereomeric rela-tionship of the adducts and the reversedbinding orientation in the minor groovewith respect to the alkylation site is re-quired to permit adenine-N3 addition tothe least substituted carbon of the elec-trophilic cyclopropane. The importance ofthe fourth base (A/T > G/C) in the bind-ing sequences is the reason this site consti-tutes a high-affinity site for the unnaturalenantiomer but only a minor site for thenatural enantiomer. Similarly, the CC-1065models (42) accommodate the more ex-tended 5-bp AT-rich binding site size andalkylation selectivity of the enantiomericagents, their reversed binding orientation inthe minor groove, and their offset AT-richselectivity relative to the alkylation site.The apparently confusing distinctions in

the relative efficiency of the natural versusunnatural enantiomer DNA alkylation re-actions were found to correlate with theinherent steric bulk surrounding the agentC7 center for which the unnatural enan-tiomers are especially sensitive, consistentwith expectations based on the models(42, 43, 60).N-Boc-DSA: A DNA Alkylating Agent

Whose Selectivity and Efficiency Are In-dependent of Absolute Stereochemistry.The accuracy of the models was revealedwhen they were found to provide a beau-tiful explanation for the unusual observa-tion that both enantiomers of simple de-rivatives of the alkylation subunit-i.e.,N-Boc-DSA (11; ref. 43) or N-Boc-CPI(12; refs. 42 and 56)-alkylate the samesites in DNA. In addition to illustratingthat the DNA alkylation reactions of (+)-and ent-(-)-11 are substantially less effi-cient (_ 104X) and less selective (selectiv-ity = 5'-AA > 5'-TA) and proceed with analtered profile than (+)- or (-)-duocar-mycin SA or CC-1065, the studies haveshown that both enantiomers of 11 alkyl-ate the same sites with essentially the sameefficiency independent of the absolute ste-reochemistry (43). Although these obser-vations appear unusual, they are the nat-ural consequence of the diastereomericrelationship of the adducts. The naturalenantiomer binds in the 3' -> 5' directionfrom the site of alkylation extending overthe adjacent 5' base. The unnatural enan-tiomer binds in the reverse 5' -> 3' orien-tation but with binding that also covers thesame adjacent 5' base. These alkylationsite models (42, 43), which are illustratedin Fig. 2, nicely accommodate the ob-served selectivity of 5'-AA > 5'-TA forboth enantiomers. The preference of5'-AA over 5'-TA (-2:1) is statisticalrather than structural in nature since thecomplementary partner strand of a 5'-ATsequence contains an identical competi-tive alkylation site. The factor controllingalkylation is simply the depth of minorgroove penetration accessible to the agentthat is required to permit adenine-N3alkylation of the electrophilic cyclopro-

FIG. 2. Comparison stick models of the (+)-N-Boc-DSA (Left) and ent-(-)-N-Boc-DSA(Right) alkylation of the same site within w794 DNA: duplex 5'-d(GACTAATTTTT). The naturalenantiomer binding extends in the 3' -* 5' direction from the adenine-N3 alkylation site acrossthe site 5'-AA. The unnatural enantiomer binding extends in the reverse 5' -* 3' direction butbinds across the same 5'-AA site.

pane. For simple agents such as 11 and 12,this is possible only when the adjacent 5'base is A or T and models of an unob-served 5'-GA alkylation support this pro-posal (42, 43). For 3 or 1, a larger 3.5- or5-bp AT-rich site surrounding the alkyla-tion site is required to permit sufficientgroove penetration for alkylation and isfurther enhanced by the preferential non-covalent binding of the agents within thenarrower, deeper AT-rich minor groove.

Reversibility of the DNA Alkylation Re-action: Binding-Driven Bonding. Al-though the duocarmycin DNA alkylationshave proven similar to CC-1065, one im-portant feature distinguishes the agents.Unlike (+)-CC-1065, which irreversiblyalkylates DNA, (+)-duocarmycin SA andA were found to reversibly alkylate DNA(43, 47). The ease of reversibility proveddependent upon the relative reactivity ofthe agent and the stability of the adduct aswell as the extent of the noncovalent bind-ing interactions. Consistent with the rela-tive reactivity of the agents and the ex-pected stability of the adducts, the (+)-duocarmycin A retroalkylation reactionwas found to be slower than that of (+)-duocarmycin SA. In addition, a faster rate

MeO2C

HN 1.

O N~-O'Bu0

I11. N-BOC-DSA

Me

HN 1O

12.O N-BOC-CP B

12. N-BOC-CPI

of retroalkylation and much lower degreeof adduct stability was observed with(+)-11 versus (+)-3 and may be attributedto the lack of the trimethoxyindole bind-ing stabilization. (+)-CC-1065 is less re-active than (+)-duocarmycin A but morereactive than (+)-duocarmycin SA. Thelack of detection of a reversible (+)-CC-1065 alkylation reaction further indicatedthat the rate of reversibility is also depen-dent on the extent of the noncovalentbinding stabilization. Consistent with thisinterpretation, analogs of CC-1065 pos-sessing the same alkylation subunit butsimpler and smaller binding subunits re-versibly alkylate DNA (57).

Thus, a dominant force stabilizing theDNA alkylation reaction is not only thecovalent bond but also the noncovalentbinding derived from hydrophobic bindingand van der Waals contacts. That is, thereversible nature of the alkylation reactionis rendered less reversible or irreversibleby noncovalent binding stabilization. Theimportance of these observations be-comes clear in the comparison of theproperties of the agents. The exceptionallypotent cytotoxic activity of the naturalproducts versus the relatively nonpotentactivity of the simple derivatives including11 and 12 may be attributed in part to thesimple event of noncovalent binding sta-bilization of the inherently reversibleDNA alkylation reaction.

Noncovalent Binding Affinity and Se-lectivity. Important insights into the struc-

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13, CDPIn

14, PDE-In

>4NOtBuMe02C_

L 2 Jn15, ACDPIn

M002Ck5NOtB16, TACDPIn

tural origin of the sequence-selectiveDNA alkylation were derived from exam-ination of the binding affinity and selec-tivity of 13-16, key partial structures of1-3 that were not capable of the subse-quent covalent bond formation (61-66).Both relative and absolute DNA bindingconstants have been established and theagents were found to exhibit a substantialAT-rich minor groove binding selectivity(63, 64). In addition, CDPI3 was found tobe the optimum binding agent within theCDPI, series. The minor groove bindingof CDPI3 spans 5 bp or one-half a helixturn, which constitutes the largest siteaccessible for synchronous binding of bothends of the rigid agent. Partial boundforms of the larger agents-i.e., CDPI2-bound CDPI4-were determined to con-stitute stable noncovalent complexes. Theremoval of the hydroxy and methoxy sub-stituents (PDE-I1 -> CDPI,,) had only asmall impact on the binding affinity andno apparent impact on the AT-rich bind-ing selectivity (63).

Consequently, the studies suggestedthat (+)-CC-1065 is best represented as aselective alkylating group superimposedupon the rigid CDPI3 skeleton (63) ratherthan as a reactive alkylating agent (CPI)attached to a DNA binding agent (PDE-12). The agents exhibit a substantial pref-erence for noncovalent AT-rich minorgroove binding that is optimal with therigid trimer (CDPI3/PDE-13). Among fea-tures that contribute to minor groovebinding selectivity, two structural charac-teristics of an A-T base pair and of a runof A-T base pairs may prove important toagents that rely on stabilizing van derWaals contacts and hydrophobic binding.First, a G-C base pair possesses an aminosubstituent that extends into the minor

groove and a comparable substituent isnot present on an A-T base pair. Thissterically more accessible region adjacentto an A-T base pair permits the deeperpenetration of an agent into the minorgroove and enhances the stabilization de-rived from van der Waals contacts. Inaddition, x-ray crystallographic studies ofoligodeoxynucleotides that contain cen-tral to their structure a run of A-T basepairs revealed characteristic features in-cluding the constricted width of the AT-rich minor groove (13, 14). This narrowerAT-rich minor groove within DNA contrib-utes prominently to the noncovalent bindingselectivity of the agents that depend onstabilizing van der Waals contacts.More recently, the DNA binding proper-

ties ofACDPIn (15; n = 1-4) and TACDPIn(16; n = 1-3) have been detailed (64).Comparable to observations made withCDPIn, ACDPI3 proved to be the optimalminor groove binding agent within theseries and exhibited a selectivity for AT-versus GC-rich DNA. Similarly, TACDPI,exhibited a substantial AT-rich DNA bind-ing selectivity (n = 3; AAG' = -2.7 to -3.5kcal/mol; 1 kcal = 4.18 kJ). The comparisonof CDPIn with ACDPIn illustrated that theintroduction of a strong electronegative sub-stituent onto the peripheral face of the agentreduced the binding affinity through intro-duction of destabilizing electrostatic inter-actions with the phosphate backbone. Incontrast, the comparison with TACDPInrevealed that the introduction of a C-5 qua-ternary amine substantially enhanced thebinding affinity through introduction of sta-bilizing electrostatic interactions with thephosphate backbone (Fig. 3).

Definitive Tests ofProposed Models forthe Origin of the CC-1065 and Duocar-mycin DNA Alkylation Selectivity. In thecourse of studies on CC-1065 and theduocarmycins, the alkylation selectivity of

* Affinityn=3 > 4,5 > 2 > 1

R= NMe3 > H > NH2* Specificity

AT > GCA A GO = 2.2 - 2.7 kcal/mol

Poly dA-Poly dT A GO (250C, kcal/mol)

R n= 1 2 FW3 4 5NH2 -5.1 -6.1 -8.2 -7.8H -6.6 -7.5 -9.9 -8.4 -8.6

1NMe3 -7.6 -9.8 -t1 .1I

the natural enantiomers has been attrib-uted to a sequence-dependent activationthrough C4 carbonyl protonation by astrategically positioned phosphate in theDNA backbone (35-38, 55, 56), the con-formational variability of DNA and alkyl-ation at junctions ofbentDNA (67-69), orpreferential noncovalent binding and sub-sequent alkylation within the narrower,deeper minor groove of AT-rich DNA(27-34). Central to the interpretations arethe perceived similarities (35-39, 53-56)or distinctions (27-34, 42, 43) in the alky-lation selectivity of simple derivatives in-cluding 11-12 and the natural products1-3. The former two proposals are basedon the premise that (+)-12 and (+)-1alkylate the same DNA sites and that thealkylation subunit or alkylation reactioncontrols the selectivity irrespective of non-covalent binding. In contrast, the latterproposal requires that the AT-rich non-covalent binding selectivity of the agentsand their steric accessibility to the alkyla-tion site that accompanies deep penetra-tion into the AT-rich minor groove con-trol the sequence selectivity. Notably, thislatter model accommodates nicely the re-verse and offset 3.5- or 5-bp AT-rich ad-enine-N3 alkylation selectivities of thenatural and unnatural enantiomers of 3and 1, respectively, and requires that11-12 and 1-3 exhibit distinct alkylationselectivities (42, 43, 58).

In the first key study (59, 70, 71), it wasshown that 17-20 exhibited identical alkyl-ation selectivities (5'-AA > 5'-TA) inde-pendent of their absolute configurationand identical to both enantiomers of 11and 12. In addition, the natural enantio-mers of 21-24 exhibited DNA alkylationselectivities identical to that of (+)-CC-1065 and were more selective and moreefficient than 17-20. The observation that19-20 and 23-24, which lack the C4 car-

CD:PI

ACDPI Amino group

Amino group

TACDPI Ammonium group

Ammonium group

FIG. 3. Noncovalent DNA binding agents.

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0. ,.-OMs

O0 <) R A?,>OtBu 18, R = OH OtBu

19, R = OMe17, N-BOC-Cl 20, R = H

H2N

tN21, Cl-CDPI2

HD@ N N

H

o- r = 0.924 N-BOC-DSA.--2- N-BOC-CBIo / N-BOC-CPI

_3' N-BOC-CBQ * N-BOC-DA

_; N-BOC-Cl0-6

1 2 3 4 5 6 7 8

-log k (sec-1)

0r= 0.998 DSA-CDPI,

0 . / CBI-CDPII-2- CPI-CDPI,

)-4 CBQ-CDPI,

o' Cl-CDPI,-6

1 2 3 4 5 6 7 8

-log k (sec1)

0-r=0.993 Duocarmycin SA-2- XCBI-TMI

(3' Duocarmycin A-4- CBQ-TMI

o CI-TMI0-6

1 2 3 4 5 6 7 8

-log k (sec-1)

-r= 0.987 DSA-CDPIQ~ . CBI-CDPI2_ -2- CPI-CDPI2

-4 __ CBQ-CDPI2-6 CI-CDPI2-6

1 2 3 4 5 6 7 8

-log k (sec 1)

FIG. 4. Direct correlation between chemical or functional stability and cytotoxic potency.

bonyl, alkylate the same sites as 17 or 21,respectively, established that a sequence-dependent phosphate protonation and ac-tivation cannot be the event that deter-mines the alkylation selectivity. In addi-tion, the more selective DNA alkylation by21-24/1-3 versus 17-20/11-12 indepen-dent of the nature of the electrophile wasconsistent with the noncovalent bindingmodel and inconsistent with the proposalthat alkylation is observed preferentiallyat junctions of bent DNA irrespective ofbinding selectivity.Key to the second definitive study (72)

was the disclosure that both the naturaland unnatural enantiomer of duocarmycinSA alkylate DNA efficiently (43). Oneunique feature of 3 is the C6 methyl esteron the left-hand side of the alkylationsubunit that complements the right-hand

side linking amide. This feature providesthe ability to introduce DNA binding sub-units on either side of the duocarmycin SAalkylation subunit. In the studies, both thenatural and unnatural enantiomers of theextended and reversed duocarmycin SAanalogs 25 and 26 were examined.Both (+)-DSA-CDPI2 and ent-(-)-

DSA-CDPI2 (25) were found to alkylateDNA with the same selectivity as (+)- andent-(-)-CC-1065 (1), respectively. Thenatural enantiomer of the reversed agent(+)-CDPI2-DSA (26) was found to alkyl-ate DNA with the same selectivity asent-(-)-DSA-CDPI2 and ent-(-)-CC-1065 (72). This incorporation and conver-sion of a natural enantiomer of the duo-carmycin SA alkylation subunit into anagent that exhibits the DNA alkylationselectivity of a typical unnatural enantio-mer by simple reversal of the orientationof the DNA binding subunits of the agentare only consistent with a model where theAT-rich noncovalent binding selectivityand depth of minor groove penetrationsurrounding the alkylation site are con-

trolling the sites of alkylation. Moreover,the observations are inconsistent with al-ternative models based on the premisethat the natural enantiomer alkylationsubunit controls the alkylation selectivity.Similarly, the unnatural enantiomer of thereversed agent ent-(-)-CDPI2-DSA (26)was found to alkylate the same sites as(+)-DSA-CDPI2 (25) and (+)-CC-1065,typical natural enantiomers. Thus, thisreversal of the enantiomeric alkylationselectivity that accompanies the simplereversal of agent orientation is generaland confirms that the same fundamentalrecognition features are operative for boththe natural and unnatural enantiomers.Modified Alkylation Subunits: Defini-

tion of a Fundamental Relationship Be-tween Functional Reactivity and Biologi-cal Activity. In addition to the total syn-thesis of the natural products CC-1065 (1;refs. 73-75), duocarmycin A (2; ref. 76),and duocarmycin SA (3; refs. 77-79), andthe extensive number of investigations di-rected at the subunits of the natural prod-ucts (33), studies that have provided ana-logs (80, 81) and agents containing deep-seated structural modifications haveproven unusually valuable in defining therelationships between structure, func-tional reactivity, and biological properties(82-97). The acid-catalyzed activation ofthe DNA alkylation reaction led to theintuitive proposal that there may exist adirect relationship between the reactivityand cytotoxic activity of the agents andestablished the expectation that the bio-logical potency may be enhanced as theelectrophilic reactivity is increased (81).However, studies conducted with theagents 27-32, which were prepared bysynthesis employing technology devel-oped in the natural product total synthe-ses, revealed the reverse relationship andthat the agents possessing the greateststability may be expected to exhibit themost potent cytotoxic activity. Moreover,a well-defined direct relationship betweensolvolysis stability (pH 3) and biological

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potency (IC50, L1210) was observed andproved to be general with both simple andadvanced analogs of the natural products(Fig. 4; refs. 84, 87, 88, 91, and 94).

This fundamental correlation betweenthe functional reactivity of the agents andtheir biological potency should prove use-ful in the predictable design of new ana-logs. For agents that possess sufficientreactivity to effectively alkylate duplexDNA, the chemically more stable agentsmay be expected to constitute the biolog-ically more potent agents. Presumably,this may be attributed to the more effec-tive delivery of the more stable agents totheir intracellular target.

Conclusions. The ability to formulatehypotheses and test the model with care-fully designed agents whose evaluationwill directly address the question providesdetailed information on the structural or-igin of their properties. Almost withoutfail, subtle but important features notapparent from the initial models revealthemselves under such scrutiny. In addi-tion to the observations detailed herein,such studies have identified and definedthe structural origin of the distinguishingefficiency of DNA alkylation by enantio-meric agents (42, 43, 60), the structuralorigins of the unusual stability of thenatural alkylation subunits (94), the ste-reoelectronic control that dictates nucleo-philic addition to the least substitutedcyclopropane carbon (94), subtle struc-tural features that can modulate reactivityof the alkylation subunits (94, 95), the SN2versus SN1 mechanism of cyclopropanering opening (94), and extensions of thestudies to rationally designed DNA cross-linking agents (96, 97) and prodrugs. Fu-ture studies with additional structural an-alogs of CC-1065 and the duocarmycinswill continue to provide further insightsinto the origin of their properties. Nodoubt this will continue to lead to ratio-nally designed synthetic agents whoseproperties overcome inherent limitations

of the natural products themselves andfurther define subtle relationships be-tween their structure, functional reactiv-ity, and biological properties.

We gratefully acknowledge the financial sup-port of the National Institutes of Health(CA41986 and CA55276).

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