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Bioorg Med Chem Lett. Author manuscript; available in PMC 2010 December 15.
Published in final edited form as:
PMCID: PMC2784222

Synthesis and Evaluation of a Thio Analogue of Duocarmycin SA


The design, synthesis, and preliminary evaluation of methyl 1,2,8,8a-tetrahydrocyclopropa[c]thieno[3,2-e]indol-4-one-6-carboxylate (CTI) derivatives are detailed representing a single atom change (NH→S) embedded in the duocarmycin SA alkylation subunit.

Duocarmycin SA (1, IC50 = 10 pM)1 is among the most potent members of a class of antitumor antibiotics that also include duocarmycin A (2),2 yatakemycin (3),3 and CC-1065 (4, Figure 1).4 Each derives its properties from a characteristic sequence-selective alkylation of duplex DNA,5-9 in which a stereoelectronically controlled adenine N3 addition to the least substituted carbon of the activated cyclopropane occurs within selected minor-groove AT-rich sites. Extensive investigations on the natural products in this class as well as their synthetic analogues have defined key and subtle features that contribute to their properties.9,10 Most notable of these are the structural features that contribute to the AT-rich noncovalent binding selectivity dominating the alkylation selectivity,11 those that are responsible for the source of catalysis for the DNA alkylation reaction,12,13 and those that subtly impact the unusual and intrinsic stability of their alkylation subunits.9,13-16

Figure 1
Natural products.

In a recent study,17 the rational design of a modified CC-1065 alkylation subunit was conducted with the goal of increasing its chemical stability and resulting biological potency and provided more potent analogues of the natural product. Thus, the replacement of the pyrrole NH in the CC-1065 alkylation subunit with a larger, more electron-withdrawing sulfur atom had the predicted and desired effect of reducing the solvolytic reactivity of the alkylation subunit, and ultimately increasing the cytotoxic potency of the corresponding CC-1065 analogue (IC50 = 7 pM vs 20 pM) to levels on par with duocarmycin SA (10 pM). Herein, we report the extension of these studies to an analogous alkylation subunit pyrrole NH to S modification of the duocarmycin SA alkylation subunit,18 replacing a single atom in 1 to generate 7 (Figure 2).

Figure 2
Duocarmycin SA analogues.

The synthesis of the modified alkylation subunit19,20 began with treatment of 2-hydroxy-3-methoxy-5-nitrobenzaldehyde (8) with dimethylthiocarbamoyl chloride to afford the desired carbamothioate 9 (2 equiv DABCO, 1.5 equiv dimethylthiocarbamoyl chloride, DMF, 50-60 °C, 2 h, 91%). Compound 9 was thermally rearranged to provide 10 (toluene, 110 °C, 12 h, 95%) and the dimethylformamide group was removed (4 M NaOH, then 20% HCl, MeOH, 100 °C, 5 h, 90%) to afford the free thiol 11. Alkylation of 11 with methyl bromoacetate followed by cyclization afforded benzothiophene 12 (1.5 equiv BrCH2CO2Me, 1.3 equiv K2CO3, 60 °C, 12 h, 98%). Deprotection of the methyl ether enlisting AlCl3 (13, 0.14 equiv, CH2Cl2, 40 °C, 32 h, 89%) was followed by protection of the free phenol as its benzyl ether 14 (1.2 equiv benzyl bromide, 1.2 equiv K2CO3, acetone, 60 °C, 3.5 h, 73%). The aryl nitro group was reduced to the corresponding amine 15 (3 equiv Fe, 0.6 equiv NH4Cl, MeOH/H2O, 100 °C, 4 h, 87%) and the resulting aniline was protected to provide 16 using Boc2O (5 equiv, THF, 23 °C, 12 h, 53%). Regioselective C4-iodination using N-iodosuccinimide (1.1 equiv, 1.1 equiv H2SO4, MeOH/THF, 23 °C, 4 h, 90%) afforded 17, which was alkylated with 1,3-dichloropropene (2 equiv, 3 equiv NaH, DMF, 0 °C, 4 h, 84%) to afford 18. The final ring of the alkylation subunit was constructed using a tris(trimethylsilyl)silane-mediated 5-exo-trig aryl radical–alkene cyclization (0.9 equiv TTMS, 0.2 equiv AIBN, benzene, 80 °C, 4 h, 74%) to afford 19.21 Treatment of 19 with Pd(OH)2 under an atmosphere of hydrogen gas (Pd(OH)2, 1 atm H2 gas, THF, 23 °C, 24 h, 75%) effectively cleaved the benzyl ether protecting group, affording 20. Resolution of 20 into its enantiomers was possible at this stage using a ChiralCel OD semi-preparative HPLC column (2×25 cm, 2% i-PrOH/hexane, 7 mL/min, α = 1.24).22 Spirocyclization was accomplished upon treatment of 20 with DBU (1.3 equiv DBU, MeCN, 23 °C, 2.5 h, 54%) to afford 6 (Scheme 1, natural enantiomer shown).

Boc deprotection of 20 (4 N HCl/EtOAc, 23 °C, 1.5 h) followed by direct coupling of the resulting indoline hydrochloride salt with 5,6,7-trimethoxyindole-2-carboxylic acid23 (21, 1.1 equiv, 3 equiv EDCI, DMF, 23 °C, 1.5 h, 61%) afforded 22 (Scheme 2, natural enantiomer shown) that was spirocyclized under mild conditions (saturated aqueous NaHCO3, DMF, 57%) to afford 7.

The results of the examination of N-Boc-CTI and the corresponding duocarmycin SA analogue in an L1210 cytotoxic assay are summarized in Table 1 along with the results of the comparison duocarmycin SA derivatives. Both enantiomers of the CTI-based analogues displayed cytotoxic activity (L1210) nearly identical to or slightly more potent than their DSA counterparts. The natural enantiomer of CTI-TMI (7, IC50 = 7 pM) exhibited activity slightly more potent than that of duocarmycin SA (1, IC50 = 10 pM). The unnatural enantiomer of 7 exhibited the 10-fold loss of activity (IC50 = 60 pM) compared to the natural enantiomer and characteristic of duocarmycin SA itself. The Boc derivatives 6 exhibited nearly identical activity to that of N-Boc-DSA (5), with the unnatural enantiomers being 10-fold less potent than the natural enantiomers.

Table 1
In Vitro Cytotoxic Activity, L1210

The DNA alkylation selectivity of the new analogues was examined within a 150 base-pair segment of DNA utilized and described previously (w794).24 The alkylation site identification and the assessment of the relative selectivity among the available sites were obtained by thermally-induced strand cleavage of the singly 5′-end-labeled duplex DNA after exposure to the compounds as detailed.5-8 Figure 3 illustrates the alkylation selectivity of both (+)- and ent-(−)-CTI-TMI (7) alongside (+)- and ent-(−)-duocarmycin SA. Satisfyingly, each enantiomer of 7 alkylated the same site as its duocarmycin SA counterpart, displaying the same characteristic and enantiomerically distinguishable selectivity. The natural enantiomer, (+)-7, alkylated DNA with an efficiency not distinguishable from (+)-duocarmycin SA, (+)-1, and appreciable alkylation was seen at concentrations of 10−6 M (not shown). Like duocarmycin SA, the unnatural enantiomer of 7 proved less efficient at alkylating DNA than its natural enantiomer, and both unnatural enantiomers alkylated the same major site.

Figure 3
Thermally-induced strand cleavage of w794 DNA (144 bp, nucleotide no. 5238–138) after DNA–agent incubation with duocarmycin SA and CTI-TMI (24 h, 23 °C), removal of unbound agent by EtOH precipitation and 30 min thermolysis (100 ...

The CTI alkylation subunit was examined to establish whether the magnitude of the effects observed with the MeCTI alkylation subunit derived from a single atom replacement in the alkylation subunit of CC-1065 would be similarly observed with duocarmycin SA. The work presented herein demonstrates that replacing a pyrrole NH of the alkylation subunit of duocarmycin SA with a sulfur atom maintains or slightly enhances the biological potency of the natural product, but not to the extent observed with MeCTI. Additionally, CTI-TMI alkylated DNA in a fashion identical to duocarmycin SA, exhibiting the characteristic enantiomeric selectivities and distinguishable enantiomeric efficiencies.

Supplementary Material



We gratefully acknowledge the financial support of the National Institutes of Health (CA041986) and the Skaggs Institute for Chemical Biology. KSM, JPL, and WMR are Skaggs Fellows.


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Supplementary Data. Full details of the synthesis of 6 and 7 and their experimental examination are provided. The supplementary data associated with this article can be found in the online version at doi:

References and notes

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