Synthesis and evaluation of 1,2,8, 8a-Tetrahydrocyclopropa[c]pyrrolo[3,2-e]indol-4(5H)-one, the parent alkylation subunit of CC-1065 and the duocarmycins: impact of the alkylation subunit substituents and its implications for DNA alkylation catalysis

The synthesis of 1,2,8,8a-tetrahydrocyclopropa[c]pyrrolo[3, 2-e]indol-4(5H)-one (CPI), the parent CC-1065 and duocarmycin SA alkylation subunit, is detailed. The parent CPI alkylation subunit lacks the C7 methyl substituent of the CC-1065 alkylation subunit and the C6 methoxycarbonyl group of duocarmycin SA, and their examination permitted the establishment of the impact of these natural product substituents. The studies revealed a CPI stability comparable to the CC-1065 alkylation subunit but which was 6x more reactive than the (+)-duocarmycin SA alkylation subunit, and it displayed the inherent reaction regioselectivity (4:1) of the natural products. The single-crystal X-ray structure of (+)-N-BOC-CPI depicts a near identical stereoelectronic alignment of the cyclopropane accounting for the identical reaction regioselectivity and a slightly diminished vinylogous amide conjugation relative to (+)-N-BOC-DSA suggesting that the stability distinctions stem in part from this difference in the vinylogous amide as well as alterations in the electronic nature of the fused pyrrole. Establishment of the DNA binding properties revealed that the CPI-based agents retain the identical DNA alkylation selectivities of the natural products. More importantly, the C6 methoxycarbonyl group of duocarmycin SA was found to increase the rate (12-13x) and efficiency (10x) of DNA alkylation despite its intrinsic lower reactivity while the CC-1065 C7 methyl group was found to slow the DNA alkylation rate (4x) and lower the alkylation efficiency (ca. 4x). The greater DNA alkylation rate and efficiency for duocarmycin SA and related analogues containing the C6 methoxycarbonyl is proposed to be derived from the extended length that the rigid C6 methoxycarbonyl provides and the resulting increase in the DNA binding-induced conformational change which serves to deconjugate the vinylogous amide and activate the alkylation subunit for nucleophilic attack. The diminished properties resulting from the CC-1065 C7 methyl group may be attributed to the steric impediment this substituent introduces to DNA minor groove binding and alkylation. Consistent with this behavior, the duocarmycin SA C6 methoxycarbonyl group increases biological potency while the CC-1065 C7 methyl group diminishes it.     

Yatakemycin: total synthesis, DNA alkylation, and biological properties

DNA-binding small molecules are an important source of anticancer therapeutics that display a diverse array of mechanisms of action. Synthetic studies on the new DNA-alkylating natural product yatakemycin, detailed in this Highlight, have served to reassign its structure, assign the absolute stereochemistry, and provide access to yatakemycin and a series of structural analogues for biological evaluation. Studies on the DNA alkylation properties of (+)-and ent-(-)-yatakemycin and related analogues have demonstrated the enhanced DNA alkylation properties of this class of agents and provided insight into their interaction with DNA.     

Systematic exploration of the structural features of yatakemycin impacting DNA alkylation and biological activity

A systematic examination of the impact of the yatakemycin left and right subunits and their substituents is detailed along with a study of its unique three subunit arrangement (sandwiched vs extended and reversed analogues). The examination of the ca. 50 analogues prepared illustrate that within the yatakemycin three subunit structure, the subunit substituents are relatively unimportant and that it is the unique sandwiched arrangement that substantially increases the rate and optimizes the efficiency of its DNA alkylation reaction. This potentiates the cytotoxic activity of yatakemycin and its analogues overcoming limitations typically observed with more traditional compounds in the series (CC-1065, duocarmycins). Moreover, a study of the placement of the alkylation subunit within the three subunit arrangement (sandwiched vs extended and reversed analogues) indicates that it not only has a profound impact on the rate and efficiency of DNA alkylation but also controls and establishes the DNA alkylation selectivity as well, where both enantiomers of such sandwiched agents alkylate the same adenine sites exhibiting the same DNA alkylation selectivity independent of their absolute configuration.     

Rational design, synthesis, and evaluation of key analogues of CC-1065 and the duocarmycins

The design, synthesis, and evaluation of a predictably more potent analogue of CC-1065 entailing the substitution replacement of a single skeleton atom in the alkylation subunit are disclosed and were conducted on the basis of design principles that emerged from a fundamental parabolic relationship between chemical reactivity and cytotoxic potency. Consistent with projections, the 7-methyl-1,2,8,8a-tetrahydrocyclopropa[c]thieno[3,2-e]indol-4-one (MeCTI) alkylation subunit and its isomer 6-methyl-1,2,8,8a-tetrahydrocyclopropa[c]thieno[2,3-e]indol-4-one (iso-MeCTI) were found to be 5-6 times more stable than the MeCPI alkylation subunit found in CC-1065 and slightly more stable than even the DSA alkylation subunit found in duocarmycin SA, placing it at the point of optimally balanced stability and reactivity for this class of antitumor agents. Their incorporation into the key analogues of the natural products provided derivatives that surpassed the potency of MeCPI derivatives (3-10-fold), matching or slightly exceeding the potency of the corresponding DSA derivatives, consistent with projections made on the basis of the parabolic relationship. Notable of these, MeCTI-TMI proved to be as potent as or slightly more potent than the natural product duocarmycin SA (DSA-TMI, IC50 = 5 vs 8 pM), and MeCTI-PDE2 proved to be 3-fold more potent than the natural product CC-1065 (MeCPI-PDE2, IC50 = 7 vs 20 pM). Both exhibited efficiencies of DNA alkylation that correlate with this enhanced potency without impacting the intrinsic selectivity characteristic of this class of antitumor agents.     

Total synthesis, structure revision, and absolute configuration of (+)-yatakemycin

The total synthesis of the reported structure 2 for yatakemycin, an exceptionally potent, naturally occurring antitumor agent disclosed in 2003, and its lack of correlation with the natural product are detailed. On the basis of spectroscopic distinctions between 2 and yatakemycin, the natural product structure was reformulated as 3, now bearing a thiomethyl ester versus thioacetate in the left-hand subunit. Total synthesis of 3 provided a compound nearly identical to but still subtly distinct from the natural product. A second reformulation of the yatakemycin structure as 1, incorporating the alternatively substituted right-hand subunit as well as the initial thiomethyl ester reformulation, was confirmed by total synthesis of both (+)- and ent-(-)-1 in studies that also unambiguously established the absolute configuration of the natural product.     

Duocarmycins binding to DNA investigated by molecular simulation

Duocarmycins are a potent class of antitumor agents, whose activity arises through their covalent binding to adenine nucleobases of DNA.(1-3) Here, we perform molecular dynamics (MD) and hybrid Car-Parinello QM/MM simulations to investigate aspects of duocarmycin binding to the d(pGpApCpTpApApTpTpGpApC) oligonucleotide. We focus on the derivatives (+)-duocarmycin SA (DSA) and (+)-duocarmycin SI (DSI), for which structural information of the covalent complex with the oligonucleotide is available, as well as on the related, but less reactive, NBOC-duocarmycin SA (NBOC-DSA), interacting with the same oligonucleotide. Comparison is made with adenine alkylation reaction in water performed by the smallest of these compounds (NBOC-DSA). The MD calculations suggest that, in noncovalent complexes, (i) drug binding causes a partial dehydration of the minor groove, without inducing a significant conformational changes, and (ii) DSA and DSI occupy a more favorable position for nucleophilic attack than NBOC-DSA, consistently with the lower reactivity of the latter. The QM/MM calculations, which are used to investigate the first step of the alkylation reaction, turn out to provide strongly underestimated free energy barriers. Within these approximations, our calculations suggest that an important ingredient for the experimentally observed DNA catalytic power is the polarization of the drug by the biomolecular scaffold.     

Total synthesis of the duocarmycins

The total synthesis of (+)-duocarmycin A and SA through a common indoline intermediate is described. The key reactions include selective lithiation of a 2,6-dibromoiodobenzene derivative and diastereoselective addition to a chiral nitroalkene, copper-mediated aryl amination, and addition of aryllithium to azlactones.     

Parallel synthesis and evaluation of 132 (+)-1,2,9,9a-tetrahydrocyclopropa[c]benz[e]indol-4-one (CBI) analogues of CC-1065 and the duocarmycins defining the contribution of the DNA-binding domain

The solution-phase, parallel synthesis and evaluation of a library of 132 (+)-1,2,9,9a-tetrahydrocyclopropa[c]benz[e]indol-4-one (CBI) analogues of CC-1065 and the duocarmycins containing dimeric monocyclic, bicyclic, and tricyclic heteroaromatic replacements for the DNA-binding domain are described. This systematic study revealed clear trends in the structural requirements for observation of potent cytotoxic activity and DNA alkylation efficiency, the range of which spans a magnitude of > or =10 000-fold. Combined with related studies, these results highlight that the role of the DNA-binding domain goes beyond simply providing DNA-binding selectivity and affinity (10-100-fold enhancement in properties), consistent with the proposal that it contributes significantly to catalysis of the DNA alkylation reaction accounting for as much as an additional 1000-fold enhancement in properties.     

Metal cation complexation and activation of reversed CPyI analogues of CC-1065 and duocarmycin SA: partitioning the effects of binding and catalysis

The synthesis and examination of a novel class of reversed CPyI analogues of CC-1065 and the duocarmycins are described. Capable of a unique metal cation activation of DNA alkylation, these agents allowed the effects of the DNA binding domain (10(4)-fold increase in DNA alkylation rate and efficiency) to be partitioned into two components: that derived from enhanced DNA binding affinity and selectivity (10-80-fold) and that derived from a contribution to catalysis (250-5000-fold). In addition, the reversed enantiomeric selectivity of these sequence selective DNA alkylating agents provides further strong support for a previously disclosed model where it is the noncovalent binding selectivity of the compounds, and not the alkylation subunit or the source of catalysis, that controls the DNA alkylation selectivity.     

Effective asymmetric synthesis of 1,2,9,9a-tetrahydrocyclopropa[c]benzo[e]indol-4-one (CBI)

A short, asymmetric synthesis of the 1,2,9,9a-tetrahydrocyclopropa[c]benzo[e]indol-4-one (CBI) analogue of the CC-1065 and duocarmycin alkylation subunits is detailed that employs an effective enzymatic desymmetrization reaction of prochiral diol 12 using a commercially available Pseudomonas sp. lipase. The optically active monoacetate (S)-13 is furnished in exceptional conversions (88%) and optical purity (99% ee) and serves as an intermediate for the preparation of either enantiomer of CBI. Similarly, the Pseudomonas sp. lipase resolved the racemic intermediate 19, affording advanced intermediates of CBI in good conversions and optical purity (99% ee), and provided an alternative approach to the preparation of optically active CBI derivatives.     

Stereoselective synthesis of 7,11-guaien-8,12-olides from santonin. Synthesis of podoandin and (+)-zedolactone A

Photochemical rearrangement of hydroxy ester 2, easily obtained from santonin (1), afforded butenolide 4, a good starting material for the synthesis of 7,11-guaien-8,12-olides. Compound 4 has been transformed into compound 10, which has been used for the synthesis of podoandin (5) and (+)-zedolactone A (ent-6). Regioselective elimination of the acetyl group on C10 afforded directly podoandin (5). For the synthesis of ent-6, a hydroxyl group has been regio- and stereoselectively introduced at the 4alpha-position through the 3alpha,4alpha-epoxide 15. The basic hydrolysis of the 10-acetyl group in compound 18 took place with concomitant intramolecular conjugated addition of the alkoxide to the butenolide moiety to give ether 19. Cleavage of the 7,10-oxido bridge via the lactone enolate afforded (+)-zedolactone A (ent-6). This synthesis has allowed for the establishment of the absolute stereochemistry of natural zedolactone A as the enantiomer of our synthetic product.     

Asymmetric total synthesis of ent-(-)-roseophilin: assignment of absolute configuration

An asymmetric total synthesis of ent-(-)-roseophilin (1), the unnatural enantiomer of a novel naturally occurring antitumor antibiotic, is described. The approach enlists a room temperature heterocyclic azadiene inverse electron demand Diels-Alder reaction of dimethyl 1,2,4,5-tetrazine-3,6-dicarboxylate (7) with the optically active enol ether 6 bearing the C23 chiral center followed by a reductive ring contraction reaction for formation of an appropriately functionalized pyrrole ring in a key 1,2,4,5-tetrazine --> 1,2-diazine --> pyrrole reaction sequence. A Grubbs' ring closing metathesis reaction was utilized to close the unusual 13-membered macrocycle prior to a subsequent 5-exo-trig acyl radical-alkene cyclization that was used to introduce the fused cyclopentanone and complete the preparation of the tricylic ansa-bridged azafulvene core 32. Condensation of 32 with 33 under the modified conditions of Tius and Harrington followed by final deprotection provided (22S,23S)-1. Comparison of synthetic (22S,23S)-1 ([alpha](25)(D), CD) with natural 1 established that they were enantiomers and enabled the assignment of the absolute stereochemistry of the natural product as 22R,23R. Surprisingly, ent-(-)-1 was found to be 2-10-fold more potent than natural (+)-1 in cytotoxic assays, providing an unusually rewarding culmination to synthetic efforts that provided the unnatural enantiomer.     

Aryl amidation routes to dihydropyrrolo[3,2-e]indoles and pyrrolo[3,2-f]tetrahydroquinolines: total synthesis of the (+/-)-CC-1065 CPI subunit

CC-1065 and the related duocarmycins are members of a structurally unique family of naturally occurring molecules and remain some of the most rigorously studied antitumor compounds to date. Herein we describe a total synthesis of the (+/-)-CC-1065 CPI subunit in an overall yield of 9.3% from commercially available 5-fluoro-2-nitrophenol. The key steps of this synthesis are a Diels-Alder reaction of an o-benzoxy-monoimine quinoid and an intramolecular aryl triflate amidation, which formed the pyrrolo[3,2-f]tetrahydroquinoline intermediate en route to CPI.     

Synthesis of the combined centre- and right-hand section of the antitumour agent CC-1065

The complete centre-and right-hand section of the anti-tumour antibiotic CC-1065, known as PDE-I dimer (3), has been synthesised by coupling the pyrroloindole (6) and pyrroloindoline (5), followed by functional group transformations; the synthetic PDE-I dimer (3) was identical to material obtained from natural sources, and since natural PDE-I dimer has been converted into CC-1065, this work constitutes a formal total synthesis of the antibiotic.     

Total synthesis of (-)- and (+)-membrenone C

[reaction: see text] A synthesis of the polypropionate marine defense substance (+)-membrenone C and its enantiomer that starts from (S)-2-methyl-3-(tert-butyldimethylsilyloxy)propanal is described. Key steps include (1) additions of chiral allenylmetal reagents to effect both chain homologation and the concomitant introduction of four stereo centers, (2) a bis-intramolecular hydrosilylation-oxidation sequence to install beta-hydroxy ketone subunits, and (3) a bis-intramolecular aldol reaction to construct the two dihydropyrone termini.     

Synthesis of a novel pentagastrin-drug conjugate for a targeted tumor therapy

The synthesis of the novel pentagastrin seco-CBI conjugate 3, which is based on the highly cytotoxic antitumor antibiotic (+)-duocarmycin SA (1), is reported. A key step in the synthesis is the palladium-catalyzed carbonylation of aryl bromide 7 to give the benzyl ester 16, which is transformed into the new seco-CBI derivative 21 bearing a carboxylic acid ester moiety. Subsequent transformation of 21 into an activated ester followed by the introduction of beta-alanine and tetragastrin led to the new pentagastrin drug 3 that contains a peptide moiety for targeting cancer cells expressing CCK-B/gastrin receptors.     

Selective metal cation activation of a DNA alkylating agent: synthesis and evaluation of methyl 1,2,9, 9a-Tetrahydrocyclopropa[c]pyrido[3,2-e]indol-4-one-7-carboxylate (CPyI)

The synthesis of methyl 1,2,9,9a-tetrahydrocyclopropa[c]pyrido[3, 2-e]indol-4-one-7-carboxylate (CPyI) containing a one carbon expansion of the C ring pyrrole found in the duocarmycin SA alkylation subunit and its incorporation into analogues of the natural product are detailed. The unique 8-ketoquinoline structure of CPyI was expected to provide a tunable means to effect activation via selective metal cation complexation. The synthesis of CPyI was based on a modified Skraup quinoline synthesis followed by a 5-exo-trig aryl radical cyclization onto an unactivated alkene with subsequent TEMPO trap or 5-exo-trig aryl radical cyclization onto a vinyl chloride for synthesis of the immediate precursor. Closure of the activated cyclopropane, accomplished by an Ar-3' spirocyclization, provided the CPyI nucleus in 10 steps and excellent overall conversion (29%). The evaluation of the CPyI-based agents revealed an intrinsic stability comparable to that of CC-1065 and duocarmycin A but that it is more reactive than duocarmycin SA and the CBI-based agents (3-4x). A pH-rate profile of the addition of nucleophiles to CPyI demonstrated that an acid-catalyzed reaction is observed below pH 4 and that an uncatalyzed reaction predominates above pH 4. The expected predictable activation of CPyI by metal cations toward nucleophilic addition was found to directly correspond to established stabilities of the metal complexes with the addition product (Cu(2+) > Ni(2+) > Zn(2+) > Mn(2+) > Mg(2+)) and provides the opportunity to selectively activate the agents upon addition of the appropriate Lewis acid. This tunable metal cation activation of CPyI constitutes the first example of a new approach to in situ activation of a DNA binding agent complementary to the well-recognized methods of reductive, oxidative, or photochemical activation. Resolution and synthesis of a full set of natural product analogues and subsequent evaluation of their DNA alkylation properties revealed that the CPyI analogues retain identical DNA alkylation sequence selectivity and near-identical DNA alkylation efficiencies compared to the natural products. Consistent with past studies and even with the deep-seated structural change in the alkylation subunit, the agents were found to exhibit potent cytotoxic activity that directly correlates with their inherent reactivity.     

Total synthesis of (+)-yatakemycin

A convergent total synthesis of (+)-yatakemycin was accomplished in a 20-step sequence in an overall yield of 13%. The synthesis features the regioselective ring opening of (S)-epichlorohydrin with 2,6-dibromophenyllithium species, the mild copper-mediated aryl amination utilizing the combination of CuI and CsOAc, and the efficient deprotection of benzyl groups of aryl benzyl ether with BCl3 in the presence of pentamethylbenzene.     

First total synthesis of (-)- and (+)-6-O-desmethylantofine

The first total synthesis of (-)-6-O-desmethylantofine (A) and its unnatural enantiomer (+)-6-O-desmethylantofine (B) is described. The synthetic route is efficient and practical with easily available glutamic acid dimethyl ester hydrochloride as the chiral material under mild conditions.