Synthesis and evaluation of a thio analogue of duocarmycin SA

Article abstract of DOI:10.1016/j.bmcl.2009.10.063

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 (N to S) embedded in the duocarmycin SA alkylation subunit.

Full text of DOI:10.1016/j.bmcl.2009.10.063

Bioorganic & Medicinal Chemistry Letters 19 (2009) 6962–6965
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and evaluation of a thio analogue of duocarmycin SA
Karen S. MacMillan a, James P. Lajiness a, Carlota Lopez Cara b, Romeo Romagnoli b, William M. Robertson a,
a,
Inkyu Hwang a, Pier Giovanni Baraldi b, , Dale L. Boger
*
*
a Department of Chemistry and the Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA
b Dipartimento di Scienze Farmaceutiche,Università degli Studi di Ferrara, Via Fossato di Mortara 17/19, 44100 Ferrara, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
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 (N to S)
embedded in the duocarmycin SA alkylation subunit.
Received 2 September 2009
Revised 10 October 2009
Accepted 13 October 2009
Available online 17 October 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Duocarmycin SA
Antitumor agent
Duocarmycin SA (1, IC50 = 10 pM)1 is among the most potent
members of a class of antitumor antibiotics that also include duocar-
mycin A (2),2 yatakemycin (3),3 and CC-1065 (4,Fig. 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 cyclopro-
pane occurs within selected minor-groove AT-rich sites.
to S modificationof theduocarmycinSA alkylationsubunit,18 replac-
ing a single atom in 1 to generate 7 (Fig. 2).
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 carbamo-
thioate
9
(2 equiv DABCO, 1.5 equiv dimethylthiocarbamoyl
Extensive investigations on the natural products in this class as
well as their synthetic analogues have defined key and subtle fea-
tures that contribute to their properties.9,10 Most notable of these
are the structural features that contribute to the AT-rich noncova-
lent 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
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
morepotentanalogues ofthenaturalproduct. Thus, thereplacement
of the pyrrole NH in the CC-1065 alkylation subunit with a larger,
more electron-withdrawing sulfur atom had the predicted and de-
sired effect of reducing the solvolytic reactivity of the alkylation sub-
unit, 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 exten-
sion of these studies to an analogous alkylation subunit pyrrole NH
* Corresponding authors. Tel.: +39 0532 455921; fax: +39 0532 455953 (P.G.B.);
tel.: +1 858 784 7522; fax: +1 858 784 7550 (D.L.B.).
E-mail addresses: baraldi@unife.it (P.G. Baraldi), boger@scripps.edu (D.L. Boger).
Figure 1. Natural products.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.10.063

K. S. MacMillan et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6962–6965
6963
Figure 2. Duocarmycin SA analogues.
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 protec-
tion of the free phenol as its benzyl ether 14 (1.2 equiv benzyl bro-
mide, 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-iodosuccin-
imide (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(trimeth-
ylsilyl)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 hydro-
gen 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 Chiral-
Cel OD semi-preparative HPLC column (2 ꢀ 25 cm, 2% i-PrOH/hex-
Scheme 1.
ane, 7 mL/min,
a
= 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 enan-
tiomer shown) that was spirocyclized under mild conditions (sat-
urated aqueous NaHCO3, DMF, 57%) to afford 7.
The results of the examination of N-Boc-CTI and the correspond-
ing 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 ana-
logues displayed cytotoxic activity (L1210) nearly identical to or
slightly more potent than their DSA counterparts.
Scheme 2.
Table 1
In vitro cytotoxic activity, L1210
Compound
IC50 (pM)
(+)-5, (+)-N-Boc-DSA
6000
60,000
6000
55,000
10
100
7
60
(ꢁ)-5, ent-(ꢁ)-N-Boc-DSA
(+)-6, (+)-N-Boc-CTI
(ꢁ)-6, ent-(ꢁ)-N-Boc-CTI
(+)-1, (+)-duocarmycin SA
(ꢁ)-1, ent-(ꢁ)-duocarmycin SA
(+)-7, (+)-CTI-TMI
(ꢁ)-7, ent-(ꢁ)-CTI-TMI
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 nat-
ural enantiomers.

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K. S. MacMillan et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6962–6965
The DNA alkylation selectivity of the new analogues was exam-
DNA than its natural enantiomer, and both unnatural enantiomers
alkylated the same major site.
ined 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
50-end-labeled duplex DNA after exposure to the compounds as de-
tailed.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 enan-
tiomer, (+)-7, alkylated DNA with an efficiency not distinguishable
from (+)-duocarmycin SA, (+)-1, and appreciable alkylation was
seen at concentrations of 10ꢁ6 M (not shown). Like duocarmycin
SA, the unnatural enantiomer of 7 proved less efficient at alkylating
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 pyr-
role 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. Addi-
tionally, CTI-TMI alkylated DNA in a fashion identical to duocarmy-
cin SA, exhibiting the characteristic enantiomeric selectivities and
distinguishable enantiomeric efficiencies.
Acknowledgments
We gratefully acknowledge the financial support of the National
Institutes of Health (CA041986) and the Skaggs Institute for Chem-
ical Biology. K.S.M., J.P.L., and W.M.R. are Skaggs fellows.
Supplementary data
Full details of the synthesis of 6 and 7 and their experimental
examination are provided. Supplementary data associated with
this article can be found, in the online version, at doi:10.1016/
j.bmcl.2009.10.063.
References and notes
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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
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DNA; lanes 2–5, Sanger G, C, A, and
T sequencing standards; lane 6, (+)-
duocarmycin SA (1, 1 ꢀ 10ꢁ5 M); lane 7, (ꢁ)-duocarmycin SA (1 ꢀ 10ꢁ5 M); lanes
8 and 9, (+)-CTI-TMI and ent-(ꢁ)-CTI-TMI (7, 1 ꢀ 10ꢁ5 M).

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