Total syntheses of thiocoraline and BE-22179 and assessment of their DNA binding and biological properties

Article abstract of DOI:10.1021/ja003602r

Full details of the total syntheses of thiocoraline (1) and BE-22179 (2), C₂ symmetric bicyclic octadepsipeptides possessing two pendant 3-hydroxyquinoline chromophores, are described in which their relative and absolute stereochemistry were es

Full text of DOI:10.1021/ja003602r

J. Am. Chem. Soc. 2001, 123, 561-568
561
Total Syntheses of Thiocoraline and BE-22179 and Assessment of
Their DNA Binding and Biological Properties
Dale L. Boger,* Satoshi Ichikawa, Winston C. Tse, Michael P. Hedrick, and Qing Jin
Contribution from the Department of Chemistry and The Skaggs Institute for Chemical Biology,
The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037
ReceiVed October 5, 2000
Abstract: Full details of the total syntheses of thiocoraline (1) and BE-22179 (2), C2 symmetric bicyclic
octadepsipeptides possessing two pendant 3-hydroxyquinoline chromophores, are described in which their
relative and absolute stereochemistry were established. Key elements of the approach include the late-stage
introduction of the chromophore, symmetrical tetrapeptide coupling, macrocyclization of the 26-membered
octadepsipeptide conducted at the single secondary amide site following disulfide formation, and a convergent
assemblage of the tetradepsipeptide with introduction of the labile thiol ester linkage in the final coupling
reaction under near racemization free conditions. By virtue of the late-stage introduction of the chromophore
and despite the challenges this imposes on the synthesis, this approach provides ready access to a range of key
chromophore analogues. Thiocoraline and BE-22179 were shown to bind to DNA by high-affinity bisintercalation
analogous to echinomycin, but with little or no perceptible sequence selectivity. Both 1 and 2 were found to
exhibit exceptional cytotoxic activity (IC50 ) 200 and 400 pM, respectively, L1210 cell line) comparable to
echinomycin and one analogue, which bears the luzopeptin chromophore, was also found to be a potent cytotoxic
agent.
Thiocoraline (1, Figure 1) is a potent antitumor antibiotic1
isolated from Micromonospora sp. L-13-ACM2-092. It consti-
tutes the newest member of the class of naturally occurring,
2-fold symmetric bicyclic octadepsipeptides which include BE-
221792 (2), triostin A3 (3), and echinomycin4 (4), which bind
to DNA with bisintercalation.5,6 Unlike BE-22179, thiocoraline
does not inhibit DNA topoisomerase I or II, but it does inhibit
DNA polymerase R at concentrations that inhibit cell cycle
progression and clonogenicity.7 It was found to unwind double-
stranded DNA7 and was suggested to bind to DNA with
bisintercalation analogous to triostin, echinomycin, and members
of the larger cyclic decadepsipeptides including sandramycin,8,9
the luzopeptins,9,10 and the quinoxapeptins.11 The initial studies
on thiocoraline as well as BE-22179 established their two-
dimensional structures but not their relative and absolute
stereochemistry.1,2 Triostin A and echinomycin possess a
D-stereochemistry at the R-position of the amide linkage to the
quinoxaline chromophore (D-Ser) and L-stereochemistry at the
remaining stereogenic centers. We have shown that the analo-
gous centers of sandramycin8 and the quinoxapeptins,11 like the
luzopeptins,10 also incorporate D-Ser. Moreover, it was reported
that a synthetic analogue of 3 possessing an all L-stereochemistry
showed no appreciable DNA binding.12 Thus, we anticipated
that 1, as well as the structurally related 2, might possess a
similar stereochemistry including the incorporation of an unusual
chromophore bearing D-Cys. Herein, we report full details of
the first total syntheses of thiocoraline and BE-22179 which
(1) Romeo, F.; Espliego, F.; Baz, J. P.; de Quesada, T. G.; Gravalos,
D.; de la Calle, F.; Fernandez-Puentes, J. L. J. Antibiot. 1997, 50, 734. (b)
Perez Baz, J.; Canedo, L. M.; Fernandez-Puentes, J. L. J. Antibiot. 1997,
50, 738. (c) Perez Baz, J.; Millan, F. R.; De Quesada, T. G.; Gravalos, D.
G. PCT Int. Appl. WO952773, 1995; Chem. Abst. 1995, 124, 115561.
(2) Okada, H.; Suzuki, H.; Yoshinari, T.; Arakawa, H.; Okura, A.; Suda,
H. J. Antibiot. 1994, 47, 129.
(3) Shoji, J.; Katagiri, K. J. Antibiot. 1961, 14, 335. (b) Shoji, J.; Tori,
K.; Otsuka, H. J. Org. Chem. 1965, 30, 2772. (c) Otsuka, H.; Shoji, J.
Tetrahedron 1967, 23, 1535. (d) Otsuka, H.; Shoji, J.; Kawano, K.;
Kyogoku, Y. J. Antibiot. 1976, 29, 107.
(4) Corbaz, R.; Ettlinger, L.; Graumann, E.; Keller-Schierlein, W.;
Kradolfer, F.; Neipp, L.; Prelog, V.; Reusser, P.; Zahner, H. HelV. Chim.
Acta 1957, 40, 199. (b) Keller-Schierlein, W.; Prelog, V. HelV. Chim. Acta
1957, 40, 205. (c) Keller-Schierlein, W.; Mihailovic, M. Lj.; Prelog, V.
HelV. Chim. Acta 1959, 42, 305. (d) Martin, D. G.; Mizsak, S. A.; Biles,
C.; Stewart, J. C.; Baczynskyj, L.; Meulman, P. A. J. Antibiot. 1975, 28,
332. (e) Dell, A.; Williams, D. H.; Morris, H. R.; Smith, G. A.; Feeney, J.;
Roberts, G. C. K. J. Am. Chem. Soc. 1975, 97, 2497.
(5) Waring, M. J.; Wakelin, L. P. G. Nature 1974, 252, 653. (b) Wang,
A. H.-J.; Ughetto, G.; Quigley, G.; Hakoshima, T.; van der Marel, G. A.;
van Boom, J. H.; Rich, A. Science 1984, 225, 1115. (c) Quigley, G. J.;
Ughetto, G.; van der Marel, G. A.; van Boom, J. H.; Wang, A. H.-J.; Rich,
A. Science 1984, 232, 1255.
(8) Isolation: Matson, J. A.; Bush, J. A. J. Antibiot. 1989, 42, 1763.
Total synthesis: Boger, D. L.; Chen, J.-H. J. Am. Chem. Soc. 1993, 115,
11624. Boger, D. L.; Chen, J.-H.; Saionz, K. W. J. Am. Chem. Soc. 1996,
118, 1629.
(9) Boger, D. L.; Saionz, K. W. Bioorg. Med. Chem. 1999, 7, 315. (b)
Boger, D. L.; Chen, J.-H.; Saionz, K. W.; Jin, Q. Bioorg. Med. Chem. 1998,
6, 85.
(10) Isolation: Konishi, M.; Ohkuma, H.; Sakai, F.; Tsuno, T.; Ko-
shiyama, H.; Naito, T.; Kawaguchi, H. J. Antibiot. 1981, 34, 148. Structure
and stereochemistry: Arnold, E.; Clardy, J. J. Am. Chem. Soc. 1981, 103,
1243. Total synthesis (luzopeptins A-C): Boger, D. L.; Ledeboer, M. W.;
Kume, M. J. Am. Chem. Soc. 1999, 121, 1098. Boger, D. L.; Ledeboer, M.
W.; Kume, M.; Searcey, M.; Jin, Q. J. Am. Chem. Soc. 1999, 121, 11375.
Luzopeptin E2: Ciufolini, M. A.; Valognes, D.; Xi, N. J. Heterocycl. Chem.
1999, 36, 1409. Ciufolini, M. A.; Valognes, D.; Xi, N. Angew. Chem., Int.
Ed. 2000, 39, 2493.
(11) Isolation: Lingham, R. B.; Hsu, A. H. M.; O’Brien, J. A.; Sigmund,
J. M.; Sanchez, M.; Gagliardi, M. M.; Heimbuch, B. K.; Genilloud, O.;
Martin, I.; Diez, M. T.; Hirsch, C. F.; Zink, D. L.; Liesch, J. M.; Koch, G.
E.; Garter, S. E.; Garrity, G. M.; Tsou, N. N.; Salituro, G. M. J. Antibiot.
1996, 49, 253. Total synthesis: Boger, D. L.; Ledeboer, M. W.; Kume,
M.; Jin, Q. Angew. Chem., Int. Ed. 1999, 38, 2424.
(6) Yoshinari, T.; Okada, H.; Yamada, A.; Uemura, D.; Oka, H.; Suda,
H.; Okura, A. Jpn. J. Cancer Res. 1994, 85, 550.
(7) Erba, E.; Bergamaschi, D.; Ronzoni, S.; Taverna, S.; Bonfanti, M.;
Catapano, C. V.; Faircloth, G.; Jimeno, J.; D’lncalci, M. Br. J. Cancer 1999,
80, 971. (b) Yoshinari, T.; Okada, H.; Yamada, A.; Uemura, D.; Oka, H.;
Okura, A. Jpn. J. Cancer Res. 1994, 85, 550.
(12) Ciardelli, T. L.; Chakravarty, P. K.; Olsen, R. K. J. Am. Chem.
Soc. 1978, 100, 7684.
10.1021/ja003602r CCC: $20.00 © 2001 American Chemical Society
Published on Web 01/04/2001

562 J. Am. Chem. Soc., Vol. 123, No. 4, 2001
Boger et al.
Figure 1.
established their relative and absolute stereochemistry,13 the
preparation of sufficient material with which further studies
could be conducted, and the extension of these studies to the
preparation of several key chromophore analogues.
DMF) or the exposure of 8 or 9 to tertiary amines (Et3N, CH2-
Cl2) led to occasional extensive â-elimination of MeSH to
provide the dehydro amino acid. Compound 11, constituting
the chromophore-bearing D-Cys residue, was prepared by the
reduction of its disulfide precursor 10 (Ph3P, 2-mercaptoethanol,
Key elements of the approach include the late-stage introduc-
tion of the chromophore, symmetrical tetrapeptide coupling,
macrocyclization of the 26-membered octadepsipeptide con-
ducted at the single secondary amide site following disulfide
formation, and a convergent assemblage of the tetradepsipeptide
with introduction of the labile thiol ester linkage in the final
coupling reaction under near racemization free conditions. By
virtue of the late-stage introduction of the chromophore and
despite the challenges this imposes on the synthesis because of
a potential intramolecular S-N acyl transfer with cleavage of
the macrocyclic thiol ester, this approach provided ready access
to a range of chromophore analogues.
Scheme 1
Tetradepsipeptide Synthesis. The convergent assemblage
of key tetradepsipeptide 16 from tripeptide 15 and N-Cbz-D-
Cys-OTce (11) along with the preparation of the three suitably
functionalized Cys residues found in 1 are summarized in
Scheme 1. Sequential S- and N-protection of N-Me-Cys-OH
(5)14 with an acetamidomethyl (Acm) group (1.5 equiv of
N-hydroxymethylacetamide, H2SO4) and BOC group (BOC2O,
62%) gave 6, the precursor to the bridging disulfide Cys residue.
Selective S-methylation of N-Me-Cys-OH (5,14 MeI, NaHCO3)
followed by BOC protection (BOC2O, NaOH, 73%) provided
7. Esterification of 7 (TMSCHN2, 89%) followed by BOC
deprotection of 8 (3 M HCl-EtOAc, 91%) provided 9, the
precursor to the second functionalized L-Cys residue. Alternative
attempts to esterify 7 under basic conditions (MeI, NaHCO3,
(13) Boger, D. L.; Ichikawa, S. J. Am. Chem. Soc. 2000, 122, 2956.
(14) Blondeau, P.; Berse, C.; Gravel, D. Can. J. Chem. 1967, 45, 49.

Syntheses of Thiocoraline and BE-22179
J. Am. Chem. Soc., Vol. 123, No. 4, 2001 563
99%) which in turn was obtained by stepwise Cbz (CbzCl,
NaHCO3) and Tce (trichloroethanol, DCC,15 HOBt,15 76%)
protection of D-cystine. The esterification reaction with trichlo-
roethanol proved sensitive to racemization and when conducted
to the absence of HOBt (33% de vs 100% de) or in the presence
of DMAP (33% de) led to extensive racemization. Coupling of
6 with 9 (EDCI,15 HOAt,15 78%) provided 12 and slightly lower
conversions were obtained with HOBt versus HOAt. BOC
deprotection of 12 (3 M HCl-EtOAc, 100%), coupling with
N-BOC-Gly-OH (EDCI, HOAt, 68%) and methyl ester hy-
drolysis of 14 (LiOH, 100%) provided 15.
Scheme 2
The key thiol esterification reaction linking the D-cysteine
derivative 11 and the tripeptide 15 was accomplished under near
racemization free conditions with use of EDCI-HOAt (83%)
in the absence of added base to afford the depsipeptide 16 (de
95:5). Much lower conversions were observed using DPPA16
or DEPC16 and Et3N due in part to competitive base-catalyzed
formation of disulfide 10. Analogous to prior reports,16 near
complete racemization was observed (16:epi-16 ) 58:42) when
the nonpolar solvent CH2Cl2 was used. In addition, the use of
base in all reactions following formation of the thiol ester 16
was found to lead to competitive â-elimination or direct cleavage
of the thiol ester and was necessarily avoided.
Cyclic Octadepsipeptide Formation and Completion of the
Total Syntheses of Thiocoraline and BE-22179. Linear
octadepsipeptide formation was accomplished by deprotection
of the amine (3 M HCl-EtOAc, 100%) and carboxylic acid
(Zn, 90% aqueous AcOH, 99%) of 16 to provide 17 and 18,
respectively, which were coupled with formation of the second-
ary amide in the absence of added base (EDCI, HOAt, CH2Cl2,
83%) to obtain 19 (Scheme 2). Cyclization of 19 to provide
the 26-membered cyclic octadepsipeptide 23 with ring closure
conducted at the single secondary amide site was accomplished
by sequential Tce ester deprotection (Zn, 90% aqueous AcOH),
disulfide bond formation17 (I2, CH2Cl2-MeOH, 25 °C, 0.001
M, 53% for two steps), and BOC deprotection (3 M HCl-
dioxane) followed by treatment with EDCI-HOAt (0.001 M
CH2Cl2, -20 °C, 6 h, 61% for two steps). Reversing the N-BOC
deprotection and disulfide bond formation steps in this four-
step sequence resulted in lower conversions (13% overall for
four steps). To date, all attempts to effect ring closure followed
by disulfide bond formation have not been successful. Even
though the 26-membered ring macrocyclization reaction un-
constrained by the disulfide bond proceeds exceptionally well
(>50%), the subsequent disulfide bond formation (I2, CH2Cl2-
MeOH, 25 °C) within the confines of the 26-membered ring
failed to occur. Thus, the order of steps enlisted for formation
of 23 was not to improve macrocyclization via the constrained
disulfide, but rather to permit disulfide bond formation. While
it is possible this may be due to constraints within the
macrocycle destabilizing the disulfide, the lack of similar
observations with 3 and 4 suggest the origin of the difficulties
may lie with competitive intramolecular cleavage of the adjacent
thiol ester by the liberated bridging thiol within the 26-
membered macrocycle.
Removal of the Cbz-protecting group under mild conditions18
(TFA-thioanisole, 25 °C, 4 h) and coupling of the resulting
amine 24 with 3-hydroxyquinoline-2-carboxylic acid (25,19,20
EDCI, DMAP, 43%) without protection of the chromophore
phenol provided (-)-1, [R]25D -180 (c 0.11, CHCl3) [lit1 [R]25
D
-191 (c 1.1, CHCl3)], identical in all respects with the properties
reported for natural material.1 Under these conditions, a
problematic intramolecular S-N acyl migration of the liberated
amine with cleavage of the thiol ester was minimized. Treatment
of 1 with NaIO4 served to provide the corresponding bis-
sulfoxide as a mixture of diastereomers which was warmed in
CH2Cl2 (reflux, 6 h, 66% overall) to promote elimination and
provide (-)-BE-22179 (2), [R]25 -89 (c 0.01, CHCl3) [lit2
D
[R]25 -94 (c 0.44, CHCl3)], identical in all respects with the
D
properties reported for the natural material.2 The correlation of
synthetic and natural 1 and 2 confirmed the two-dimensional
structure assignments and established their relative and absolute
stereochemistries as those shown in Scheme 2.
(15) DCC ) dicyclohexylcarbodiimide; EDCI ) 1-(3-dimethylamino-
propyl)-3-ethylcarbodiimide hydrochloride; HOBt ) 1-hydroxybenzotri-
azole; HOAt ) 1-hydroxy-7-azabenzotriazole.
(16) DPPA ) diphenyl phosphorazidate; DEPC ) diethyl phosphoro-
cyanidate. (a) Yamada, S.; Yokoyama, Y.; Shioiri, T. J. Org. Chem. 1974,
39, 3302. (b) Yokoyama, Y.; Shioiri, T.; Yamada, S. Chem. Pharm. Bull.
1977, 25, 2423.
(17) Kamber, B.; Hartmann, A.; Eisler, K.; Riniker, B.; Rink, H.; Sieber,
P.; Rittel, W. HelV. Chim. Acta 1980, 63, 899.
(18) Kiso, Y.; Ukawa, K.; Akita, T. J. Chem. Soc., Chem. Commun. 1980,
101.
Interestingly, both 23 and thiocoraline (1) as well as the
related natural product analogues 26-28 adopt a single solution
1
conformation that is observed by H NMR in well-defined
(19) Prepared from methyl 3-hydroxyquinoline-2-carboxylate20 by treat-
ment with LiOH, THF-MeOH-H2O 3/1/1, 25 °C, 2 h (71%).
(20) Boger, D. L.; Chen, J.-H. J. Org. Chem. 1995, 60, 7369.

564 J. Am. Chem. Soc., Vol. 123, No. 4, 2001
Boger et al.
Scheme 3
Scheme 4
(25, EDCI, DMAP) or a protected derivative of 24 (BOC2O or
CbzCl, Et3N) were also unsuccessful.
We also examined the approach in which the bridged 26-
membered macrocycle is formed via simultaneous formation
of both secondary amides. However, intermolecular disulfide
bond formation (I2, MeOH) and sequential deprotection of Tce
and BOC group and the treatment of the resulting symmetrical
disulfide with EDCI and HOAt gave complex mixtures of
products including a range of oligomers and higher order
macrocycles in which the formation of 32 was not observed
(Scheme 3).
Finally, we also examined an approach in which the pendant
chromophore was introduced at the initial stages of the synthesis.
Thus, the coupling reaction of 15 and 3421 (EDCI, HOAt, 86%)
gave tetradepsipeptide 35 which possesses the substituted
quinoline chromophore (Scheme 4). However, elimination of
thiol ester was problematic under the conditions of BOC
deprotection (HCl or 90% aqueous TFA, 0 °C) or Tce ester
hydrolysis (Zn, 90% aqueous HOAc, 0 °C) and the following
coupling reaction which gave only a trace of the desired linear
octadepsipeptide. Presumably, this may be attributed to the
increased acidity of the R-proton of the activated N-acyl-D-Cys
derivative bearing an amide- versus carbamate-protecting group.
Analogue Synthesis. The late-stage generation of amine 24
followed by introduction of the pendant chromophore provided
the opportunity to examine chromophore analogues of 1 and 2.
Thus, the amine 24 was also coupled with quinoline-2-
carboxylic acid, quinoxaline-2-carboxylic acid (which is the
chromophore found in echinomycin and triostin A), and
3-hydroxy-6-methoxyquinoline-2-carboxylic acid10,20 (which is
the chromophore found in the luzopeptins) to afford the key
chromophore analogues 26-28 (Scheme 2).
spectra. That of synthetic 1 proved identical to the published
1H NMR spectrum of natural 1.1 In contrast, BE-22179 exhibits
a more complex, but still well defined, H NMR spectrum
1
consistent with its adoption of two unsymmetrical or four
symmetrical conformers in near equal proportions. The NMe
signals (2 NMe) and the two olefin signals (CdCHH) appear
as eight, near 1:1, well resolved singlets in the 1H NMR
1
spectrum. Importantly, the H NMR spectrum of synthetic 2
proved identical to that published for natural 2.2
DNA Binding Affinity. Apparent absolute binding constants
and apparent binding site sizes were obtained by measurement
of the fluorescence quenching upon titration of 1 and 2 with
calf thymus (CT) DNA. The excitation and emission spectra
for thiocoraline and BE-22179 were determined in aqueous
buffer (Tris-HCl, pH 7.4, 75 mM NaCl). Both thiocoraline and
BE-22179, which have the same chromophore, exhibited an
intense fluorescence in solution with enhanced excitation (380
nm) and emission (510 nm) maxima which was quenched upon
DNA binding. Moreover, the intensity of this fluorescence
greatly facilitated the measurement of fluorescence quenching
and allowed measurements to be carried out at low initial agent
concentrations of 1-10 µM where the compounds are soluble.
Analogous measurements with echinomycin could not be
conducted because of its less intense fluorescence emission and
low solubility. For the titrations, small aliquots of CT-DNA (320
µM in base pair) were added to 2 mL of a solution of the agent
(2 µM) in Tris-HCl (pH 7.4), 75 mM NaCl buffer. Additions
were carried out at 15-min intervals to allow binding equilibra-
tion. Scatchard analysis22 of the titration results was conducted
using the equation rb/c ) Kn - Krb, where rb is the number of
molecules bound per DNA nucleotide phosphate, c is the free
drug concentration, K is the apparent binding constant, and n
Alternative Approaches. Prior to implementing the success-
ful sequence, preliminary studies21 were first conducted enlisting
an FMOC-protecting group and basic deprotection conditions
versus a Cbz-protecting group on 23 (Scheme 3). Thus,
tetradepsipeptide 30 and octadepsipeptide 31 were prepared by
the procedures described for the synthesis of 16 and 19.
Cyclization of 31 to provide the bridged 26-membered cyclic
octadepsipeptide 32 was accomplished by sequential Tce ester
deprotection (Zn, 90% aqueous AcOH), BOC deprotection (3
M HCl-dioxane), and disulfide bond formation (I2, CH2Cl2-
MeOH, 25 °C, 0.001 M) followed by treatment with EDCI-
HOAt (0.001 M CH2Cl2, -20 °C, 6 h, 16% for four steps).
However, exposure of 32 to Et2NH or piperidine led to
decomposition of the macrocycle rather than clean FMOC
deprotection. Alternative treatment of 32 with other amines
including dicyclohexylamine, Et3N, or DMAP also failed to
provide the cyclic amine 24 which we attributed to the sensitivity
of the thiol ester to nucleophiles, the competitive â-elimination
induced by the deprotonation of the R-position of the Cys
residues, and a potential intramolecular S-N acyl transfer to
the liberated amine with cleavage of the thiol ester. However,
efforts to trap the liberated amine in situ to obtain 1 directly
(21) Experimental details and characterization may be found in the
Supporting Information.
(22) Scatchard, G. Ann. N. Y. Acad. Sci. 1949, 51, 660.

Syntheses of Thiocoraline and BE-22179
J. Am. Chem. Soc., Vol. 123, No. 4, 2001 565
studies of sandramycin, the luzopeptins, and quinoxapeptins,
which are larger symmetrical cyclic decadepsipeptides, revealed
that they exhibit a higher affinity for CT-DNA (KB ) 1.0-3.4
× 107 M-1). Since thiocoraline and BE-22179 possess the same
chromophore as sandramycin (KB ) 3.4 × 107 M-1), this
indicates that the differing ability to bind duplex DNA arises
from the cyclic depsipeptide, its ring size, and differing peptide
backbone and not the structure of the chromophore.
Similarly, echinomycin and triostin A bind to DNA by
bisintercalation and are the most extensively studied natural
products in these series. In contrast to sandramycin and the
luzopeptins which bind 5′-PyPuPyPu sites and exhibit the
highest affinity for 5′-CATG spanning a two-base pair 5′-AT
site,9,10 the quinoxalines bisintercalate preferentially at 5′-CG
sites (i.e., 5′-GCGT or 5′-PuPyPuPy) also spanning two base
pairs with each intercalation occurring at a PuPy versus PyPu
step. The structural distinctions between 1 and 2 versus triostin
A (3) are subtle. Beyond the different chromophores, they
include the conservative side chain CH2SCH3 versus NMe-Val
CH(Me)2 alteration and the more significant Gly versus L-Ala
(H vs Me) substitution, and the thioester versus ester (S vs O)
backbone alteration. Nonetheless, these changes abolished the
DNA binding selectivity and, as shown below, may reduce the
stability of the bisintercalation complexes.
Figure 2. (a) Fluorescence quenching of thiocoraline (excitation at
380 nm and emission at 510 nm in Tris-HCl (pH 7.4) and 75 mM
NaCl buffer solution) with increasing CT-DNA concentration. (b)
Scatchard plot of fluorescence quenching of part a. (c) Fluorescence
quenching of BE-22179 (excitation at 380 nm and emission at 510 nm
in Tris-HCl (pH 7.4) and 75 mM NaCl buffer solution) with increasing
CT-DNA concentration. (d) Scatchard plot of fluorescence quenching
of part c.
Bifunctional Intercalation. Confirmation that thiocoraline
and BE-22179 bind to DNA with bisintercalation was derived
from their ability to induce the unwinding of negatively
supercoiled DNA. This was established by their ability to
gradually decrease the agarose gel electrophoresis mobility of
supercoiled ΦX174 DNA (unwinding) at increasing concentra-
tions followed by a return to normal mobility (rewinding) at
even higher concentrations. Under the conditions we employed,
echinomycin unwound ΦX174 DNA at a 0.044 agent/base pair
ratio (Figure 3 and Table 1). Thiocoraline completely unwound
ΦX174 DNA at a higher 0.11 agent/base pair ratio, whereas
BE-22179 required even higher concentrations, producing the
unwinding at an agent/base pair ratio of 1.1. Complete rewinding
of the supercoiled DNA occurred at an agent/base pair ratio of
0.44 for thicoraline versus 0.22 for echinomycin, whereas BE-
22179 failed to produce the rewinding of ΦX174 DNA at the
concentrations examined. The thiocoraline analogue 27, which
bears the quinoxaline chromophore of echinomycin, was found
to behave in a manner indistinguishable from thiocoraline itself.
Thus, the distinctions in 1 and 2 and echinomycin detected here
appear to be related to the nature of the cyclic depsipeptide and
not the structure of the chromophore. Under these conditions,
ethidium bromide, a monointercalater, does not unwind super-
coiled DNA although it can unwind supercoiled DNA under
conditions which prevent dissociation of the bound agent during
electrophoresis. Thus, the unwinding of negatively supercoiled
DNA and the subsequent positive supercoiling of the DNA by
thiocoraline and 27, indicative of bisintercalation, were similar
although slightly less effective than echinomycin, whereas that
of BE-22179 was substantially less effective. This suggests that
BE-22179 binds with a smaller unwinding angle, with lower
stability, or with faster off-rates than echinomycin and thio-
coraline.
Table 1. Comparative DNA Binding Properties
property
KB,a M-1
thiocoraline (1) BE-22179 (2) echinomycin (4)
2.6 × 106
(1:6.5)
0.11
2.2 × 106
(1:5.8)
1.1
(2.2 × 106)d
(-)-unwinding [c]b
(+)-winding [c]c
0.044
0.22
0.44
>2.2
a Calf thymus DNA, KB ) apparent binding constant determined by
fluorescence quenching. The value in parentheses is the agent/base pair
ratio at saturated high-affinity binding and may be considered a measure
of the selectivity of binding. b Agent/base pair ratio required to unwind
negatively supercoiled ΦX174 DNA (form I to form II gel mobility,
0.9% agarose gel). c Agent/base ratio required to induce complete
rewinding or positive supercoiling of ΦX174 DNA (form II to form I
gel mobility, 0.9% agarose gel). d Binding constant established by
footprinting at a 5′-CCGC site, Figure S1 in Supporting Information.
is the number of the agent binding sites per nucleotide
phosphate. A plot of rb/c versus rb gives the association constant
(slope) and the apparent binding site size (x-intercept) for the
agents (Figure 2 and Table 1).
Thiocoraline was found to exhibit a relatively high affinity
for duplex DNA (KB ) 2.6 × 106 M-1) with a saturating
stoichiometry of high affinity binding at a 1:6.5 agent to base
pair ratio. BE-22179, which is structurally distinct possessing
two exocyclic olefins, also displayed a similar affinity and
binding site size with CT-DNA. The high affinity binding of
one molecule per 5.8-6.5 base pairs approaches that of the
saturated limit of four base pairs assuming bisintercalation
spanning two base pairs, suggesting thiocoraline and BE-22179
bind to DNA with limited selectivity among available sites. This
proved consistent with attempts to establish a selectivity of DNA
binding by DNase I23 and MTE footprinting24 on w794 DNA,25
using protocols we successfully applied to sandramycin8 and
echinomycin, which failed to reveal a distinguishable selectivity
for 1 (Supporting Information Figures 1 and 2). Our previous
We also briefly examined the ability of 1 or 2 to cleave,
alkylate, or cross-link DNA. In particular, the electrophilic
unsaturation found in BE-22179 might be expected to serve as
an alkylation site for covalent attachment to DNA, especially
following bisintercalation binding. No evidence was found to
suggest that either 1 or 2 cleave DNA in simple assays
monitoring the conversion of supercoiled ΦX174 DNA (Form
(23) Galas, D. J.; Schmitz, A. Nucleic Acids Res. 1978, 5, 3157.
(24) Tullius, T. D.; Dombroski, B. A.; Churchill, M. A.; Kam, L. Methods
Enzymol. 1987, 155, 537.
(25) Boger, D. L.; Munk, S. A.; Zarrinmayeh, H.; Ishizaki, T.; Haught,
J.; Bina, M. Tetrahedron 1991, 47, 2661.

566 J. Am. Chem. Soc., Vol. 123, No. 4, 2001
Boger et al.
Table 3. Biological Activity
compound
L1210a (nM)
HIV-RTb (µM)
1
2
23
26
27
28
32
thiocoraline
BE-22179
0.2
0.4
900
>105
20
40
0.4
>105
0.08
0.001
0.008
30
echinomycin
sandramycin
luzopeptin A
luzopeptin B
700
2
6
3
luzopeptin C
>100
0.4
0.6
0.9
0.3
quinoxapeptin A
quinoxapeptin B
quinoxapeptin C
0.3
2
>100
a L1210 mouse leukemia cytotoxic assay. b HIV-1 reverse tran-
scriptase inhibition.
Figure 3. Agarose gel electrophoresis. (A) Lane 1, untreated super-
coiled ΦX174 DNA, 95% form I and 5% form II; lanes 2-8,
thiocoraline-treated ΦX174 DNA; lanes 9-14, echinomycin-treated
ΦX174 DNA. The [agent]-to-[base pair] ratios were 0.022 (lanes 2
and 9), 0.033 (lanes 3 and 10), 0.044 (lanes 4 and 11), 0.066 (lanes 5
and 12), 0.11 (lanes 6 and 13), 0.22 (lanes 7 and 14), and 0.44 (lane
8). (B) Lane 1, untreated supercoiled ΦX174 DNA, lanes 2-12, BE-
22179-treated ΦX174 DNA. The [agent]-to-[base pair] ratios were
0.022 (lane 2), 0.033 (lane 3), 0.044 (lane 4), 0.066 (lane 5), 0.11 (lane
6), 0.22 (lane 7), and 0.44 (lane 8), 0.44 (lane 9), 0.66 (lane 10), 1.1
(lane 11), and 2.2 (lane 12). (C) Lane 1, untreated supercoiled ΦX174
DNA, 95% form I and 5% form II; lanes 2-8, thiocoraline analogue
(27)-treated ΦX174 DNA. The [agent]-to-[base pair] ratios were 0.022
(lane 2), 0.033 (lane 3), 0.044 (lane 4), 0.066 (lane 5), 0.11 (lane 6),
0.22 (lane 7), and 0.44 (lane 8).
intercalation sites of the related bisintercalators echinomycin
(5′-PuCGPy),4 sandramycin (5′-CATG),9 and the luzopeptins
(5′-CATG).10 The binding constants were established by titration
using the fluorescence quenching that is observed upon DNA
binding. The excitation and emission spectra for thiocoraline
and BE-22179 were recorded in aqueous buffer (Tris-HCl, pH
7.4, 75 mM NaCl). To minimize fluorescence decrease due to
dissolution or photobleaching, the solutions were stirred in 4-mL
cuvettes in the dark with the minimum exposure to the excitation
beam necessary to obtain a reading. The titrations were carried
out with a 15-min equilibration time after each deoxyoligo-
nucleotide addition. Scatchard plots of thiocoraline binding to
the deoxyoligonucleotides exhibited a downward convex cur-
vature which we interpreted to indicate a high-affinity bisin-
tercalation and a lower affinity binding potentially involving
monointercalation. Using the model described by Feldman26
which assumes one ligand with two binding sites, we were able
to deconvolute the curves according to the equation
Table 2. Comparative Deoxyoligonucleotide Binding Properties
thiocoraline (1)
sandramycin
KB (106 M-1
)
KB (106 M-1
)
5′-GCGCGC
5′-GCATGC
5′-GCCGGC
5′-GCTAGC
7.0
4.3
5.9
3.0
145
230
85
r
b ) (K1(n1 - rb) + K2(n2 - rb)) +
1
85
c
2
(K (n - r ) - K (n - r ))2 + 4K K n n (1)
x
1
1
b
2
2
b
1 2 1 2
I) to relaxed (Form II) or linear (Form III) DNA under a range
of conditions. Similarly, sequencing cleavage studies conducted
with w794 DNA enlisting the thermal depurination and cleavage
detection of adenine N3 or N7 or guanine N3 or N7 alkylation
sites did not reveal alkylation by 2. However, these studies do
not exclude alkylation at nonthermally labile sites including the
guanine C2 amine. Additional assays conducted with w794
DNA following established protocols25 provided no evidence
of DNA interstrand cross-linking. These studies would detect
both thermally labile and nonthermally labile alkylation sites,
but only those engaged in interstrand cross-linking. Given the
C2 symmetric nature of 2, bisintercalation analogous to echi-
nomycin and triostin A places the two electrophilic sites in
positions to react only with the complementary strands of duplex
DNA (interstrand DNA cross-linking) and would preclude
intrastrand DNA cross-linking. Thus, these studies safely
excluded DNA cross-linking by 2 even with reaction of
nonthermally labile sites (e.g., G C2 amine), but do not rule
out monoalkylation events at nonthermally labile sites.
where K1 and K2 represent the association constants for high-
and low-affinity binding, and n1 and n2 represent the number
of bound agents per duplex for the separate binding events.
Scatchard plots of the data revealed 1:1 binding in each case.
That of the high affinity binding is consistent with binding of
a single molecule with bisintercalation surrounding a central
two base pair site. A small preference was observed for GC-
rich binding with 5′-GCGCGC and 5′-GCCGGC exhibiting the
tightest binding, but the differences are small ranging from 3
to 7 × 106 M-1 for the four deoxyoligonucleotides (Table 2).
Thus, consistent with the results of footprinting and other related
studies herein, the binding of 1 with the deoxyoligonucleotides
exhibited little selectivity.
Biological Properties. Table 3 summarizes the biological
properties of echinomycin, thiocoraline, and BE-22179 along
with those of precursor 23 and their analogues. Thiocoraline
and BE-22179 exhibit exceptionally potent cytotoxic activity
in the L1210 assays (IC50 ) 200 and 400 pM, respectively)
being slightly less potent than echinomycin. Compounds 23 and
32 lacking both chromophores and containing the Cbz- and
FMOC-protecting groups were inactive and >105 times less
DNA Binding Selectivity. The preceding studies suggested
that thiocoraline binds to DNA with high affinity, but with little
or no selectivity. Consequently, we examined the binding of 1
with a set of four duplex deoxyoligonucleotides, 5′-GCGC-3′
where ) TA, AT, GC, CG, incorporating the high affinity
(26) Feldman, H. A. Anal. Biochem. 1972, 48, 317.

Syntheses of Thiocoraline and BE-22179
J. Am. Chem. Soc., Vol. 123, No. 4, 2001 567
The residue was diluted with EtOAc (100 mL), and the organic phase
was washed with 1 N aqueous HCl (100 mL), saturated aqueous
NaHCO3 (100 mL), and saturated aqueous NaCl (50 mL). The organic
phase was dried (Na2SO4), filtered, and concentrated in vacuo. Flash
chromatography (SiO2, 3 × 15 cm, 20% EtOAc-hexane eluent)
afforded 1021 (1.23 g, 1.6 mmol, 76%) as a colorless oil.
potent than thiocoraline. Analogue 28, which bears the same
chromophore as the luzopeptins, also exhibited potent activity
while 26, lacking the quinoline C3 phenol, and 27, bearing the
quinoxaline chromophore of echinomycin and triostin A,
exhibited less potent cytotoxic activity. In addition, thiocoraline,
like echinomycin, was found to be only a weak inhibitor of
HIV-1 reverse transcriptase.
Most notable of these observations is that both thiocoraline
and BE-22179 are exceptionally potent cytotoxic agents joining
the small group of compounds that exhibit IC50’s at subnano-
molar or low picomolar concentrations (200-400 pM).
N-Cbz-D-Cys-OTce (11). A solution of 10 (771 mg, 1.0 mmol) in
10 mL of THF was treated with Ph3P (262 mg, 1.0 mmol), 2-mercap-
toethanol (70 µL, 1.0 mmol), and water (180 µL, 10 mmol), and the
reaction mixture was stirred at 50 °C for 5 h before being concentrated
in vacuo. Flash chromatography (SiO2, 3 × 18 cm, 20% EtOAc-hexane
eluent) afforded 1121 (764 mg, 1.98 mmol, 99%) as a colorless oil.
N-BOC-NMe-L-Cys(Acm)-NMe-L-Cys(Me)-OMe (12). A solution
of 6 (l.75 g, 5.74 mmol) in CH2Cl2 (57 mL) was treated sequentially
with HOAt (781 mg, 5.74 mmol) and EDCI (1.10 g, 5.74 mmol), and
the mixture was stirred at 0 °C for 15 min. A solution of 9 (935 mg,
5.74 mmol) was added, and the reaction mixture was stirred for an
additional 12 h. The reaction mixture was poured onto 1 N aqueous
HCl (100 mL) and extracted with EtOAc (2 × 100 mL). The combined
organic phases were washed with saturated aqueous NaHCO3 (100 mL)
and saturated aqueous NaCl (50 mL), dried (Na2SO4), filtered, and
concentrated in vacuo. Flash chromatography (SiO2, 3 × 15 cm, EtOAc
eluent) afforded 1221 (2.01 g, 4.46 mmol, 78%) as a white foam.
Experimental Section
N-BOC-NMe-L-Cys(Acm)-OH (6). A solution of NMe-L-Cys-OH
hydrochloride salt (5, 1.35 g, 10.0 mmol) and acetamidomethanol (13.4
g, 15 mmol) in water (5 mL) was treated with concentrated HCl (0.64
mL), and the reaction mixture was stirred at 25 °C for 12 h. The reaction
mixture was concentrated in vacuo. The residue was dissolved in 100
mL of THF-H2O (1:1), and the resulting solution was brought to pH
8 by adding 1 N aqueous NaOH. Di-tert-butyl dicarbonate (2.62 g,
12.0 mmol) was added, and the reaction mixture was stirred at 25 °C
for 12 h, maintaining a pH 8. The reaction mixture was poured onto 1
N aqueous HCl (150 mL) and extracted with CHCl3 (3 × 100 mL).
The combined organic phases were dried (Na2SO4), filtered, and
concentrated in vacuo. Flash chromatography (SiO2, 3 × 15 cm, 4%
MeOH-CHCl3 eluent) afforded 621 (1.89 g, 6.21 mmol, 62%) as a
white foam.
N-BOC-NMe-L-Cys(Me)-OH (7). A solution of NMe-L-Cys-OH
hydrochloride salt (5, 1.35 g, 10.0 mmol) in 100 mL of THF-H2O
(1:1) was sequentially treated with NaHCO3 (1.68 g, 20.0 mmol) and
MeI (0.65 mL, 10.5 mmol), and the reaction mixture was stirred at 25
°C for 3 h. The reaction mixture was brought to pH 8 by adding 1 N
aqueous NaOH. Di-tert-butyl dicarbonate (2.62 g, 12.0 mmol) was
added, and the reaction mixture was stirred at 25 °C for 12 h,
maintaining a pH 8. The reaction mixture was poured onto 1 N aqueous
HCl (150 mL) and extracted with CHCl3 (3 × 100 mL). The combined
organic phases were dried (Na2SO4), filtered, and concentrated in vacuo.
Flash chromatography (SiO2, 3 × 15 cm, 2% MeOH-CHCl3 eluent)
afforded 721 (1.89 g, 7.63 mmol, 76%) as a colorless oil.
N-BOC-NMe-L-Cys(Me)-OMe (8). Trimethylsilyl diazomethane
(2.0 M hexane solution, 3.70 mL, 0.74 mmol) was added dropwise to
a solution of 7 (1.86 g, 7.40 mmol) in 100 mL of benzene-MeOH
(5:1) at 0 °C. Following the addition, the reaction mixture was
concentrated in vacuo. Flash chromatography (SiO2, 3 × 15 cm, 20%
EtOAc-hexane eluent) afforded 821 (1.77 g, 6.73 mmol, 91%) as a
colorless oil.
NMe-L-Cys(Me)-OMe (9). Compound 8 (1.32 g, 5.0 mmol) was
treated with 5 mL of 3 M HCl-EtOAc and the mixture was stirred at
25 °C for 30 min before the volatiles were removed in vacuo. The
residual HCl was removed by adding Et2O (10 mL) to the hydrochloride
salt followed by its removal in vacuo. The residue was dissolved in
CHCl3 (200 mL), and the organic layer was washed with saturated
aqueous NaHCO3 (100 mL) and saturated aqueous NaCl (100 mL).
The organic phase was dried (Na2SO4), filtered, and concentrated in
vacuo to give 921 (746 mg, 91%) as a colorless oil which was used
directly in the next reaction without further purification.
N-BOC-Gly-NMe-L-Cys(Acm)-NMe-L-Cys(Me)-OMe (14). A
sample of 12 (2.01 g, 4.46 rnmol) was treated with 4.5 mL of 3 M
HCl-EtOAc and the mixture was stirred at 25 °C for 30 min before
the volatiles were removed in vacuo. The residual HCl was removed
by adding Et2O (10 mL) to the hydrochloride salt 13 followed by its
removal in vacuo. After this procedure was repeated three times, 1.96
g of 13 (100%) was obtained and used directly in the following reaction
without further purification.
A solution of N-BOC-Gly-OH (773 mg, 4.46 mmol) and hydro-
chloride salt 13 (1.96 g, 4.46 mmol) in CH2Cl2 (45 mL) was treated
sequentially with HOAt (909 mg, 6.69 mmol), EDCI (1.26 g, 6.69
mmol), and NaHCO3 (549 mg, 6.69 mmol), and the reaction mixture
was stirred at 0 °C for 12 h. The reaction mixture was poured onto 1
N aqueous HCl (100 mL) and extracted with EtOAc (2 × 100 mL).
The combined organic phase was washed with saturated aqueous
NaHCO3 (100 mL) and saturated aqueous NaCl (50 mL), dried (Na2-
SO4), filtered, and concentrated in vacuo. Flash chromatography (SiO2,
5 × 14 cm, 20% acetone-EtOAc eluent) afforded 1421 (1.54 g, 3.03
mmol, 68%) as a white foam.
N-BOC-Gly-NMe-L-Cys(Acm)-NMe-L-Cys(Me)-OH (15). Lithium
hydroxide monohydrate (92 mg, 2.31 mmol) was added to a solution
of 14 (394 mg, 0.77 mmol) in 10 mL of THF-MeOH-H2O (3:1:1) at
0 °C, and the resulting reaction mixture was stirred at 25 °C for 1.5 h.
The reaction mixture was poured onto 1 N aqueous HCl (100 mL) and
extracted with CHCl3 (3 × 50 mL). The combined organic phases were
dried (Na2SO4), filtered, and concentrated in vacuo to give 1521 (393
mg, 100%) as a white foam which was used without further purification.
N-Cbz-D-Cys[N-BOC-Gly-NMe-L-Cys(Acm)-NMe-L-Cys(Me)]-
OTce (16). A solution of 15 (393 mg, 0.77 mmol) in DMF (8 mL)
was treated sequentially with HOAt (150 mg, 0.92 mmol) and EDCI
(183 mg, 0.92 mnol), and the mixture was stirred at -20 °C for 15
min. A solution of 11 (300 mg, 0.77 mmol) was added, and the reaction
mixture was stirred for an additional 4 h. The reaction mixture was
poured onto 1 N aqueous HCl (100 mL) and extracted with EtOAc
(100 mL). The combined organic phase was washed with saturated
aqueous NaHCO3 (100 mL) and saturated aqueous NaCl (50 mL), dried
(Na2SO4), filtered, and concentrated in vacuo. Flash chromatography
(SiO2, 3 × 15 cm, 33% EtOAc-hexane eluent) afforded 1621 (551
mg, 0.64 mmol, 83%) as a white foam and epi-16 (28 mg, 0.032 mnol,
4%) as a white foam.
(N-Cbz-D-Cys-OTce)2 (10). A solution of D-cystine (500 mg, 2.1
mmol) and NaOH (352 mg, 8.4 mmol) in 20 mL of THF-H2O (1:1)
was treated with CbzCl (0.63 mL, 4.4 mmol), and the reaction mixture
was stirred at 25 °C for 1 h. The reaction mixture was diluted with
water (50 mL) and washed with CHCl3 (3 × 50 mL). The aqueous
phase was acidified with 6 N aqueous HCl (50 mL) and extracted with
CHCl3 (3 × 50 mL). The combined organic phases were dried (Na2-
SO4), filtered, and concentrated in vacuo. The residue was dissolved
in pyridine (20 mL), and HOBt (840 mg, 6.3 mmol) and trichloroethanol
(0.69 mL, 5.3 mmol) were added. The mixture was cooled to -20 °C
and treated with DCC (1.29 g, 6.3 mmol), and the resulting mixture
was stirred at -20 °C under Ar for 24 h. The white precipitate of DCU
was removed by filtration, and the filtrate was concentrated in vacuo.
N-Cbz-D-Cys[N-Cbz-D-Cys(N-BOC-Gly-NMe-L-Cys(Acm)-NMe-
L-Cys(Me))-Gly-NMe-L-Cys(Acm)-NMe-L-Cys(Me)]-OTce (19). Com-
pound 16 (432 mg, 0.5 mmol) was treated with 5.0 mL of 3 M HCl-
EtOAc, and the mixture was stirred at 25 °C for 30 min before the
volatiles were removed in vacuo. The residual HCl was removed by
adding Et2O (10 mL) to the hydrochloride salt 17 followed by its
removal in vacuo. After repeating this procedure three times, 429 mg

568 J. Am. Chem. Soc., Vol. 123, No. 4, 2001
Boger et al.
of 17 (100%) was obtained and used directly in the following reaction
without further purification.
and the reaction mixture was stirred at 25 °C for 30 min. The
hydrochloride salt 24 was added, and the reaction mixture was stirred
at 25 °C for 3 d. The reaction mixture was poured onto 1 N aqueous
HCl (5 mL) and extracted with EtOAc (2 × 5 mL). The combined
organic phases were washed with saturated aqueous NaCl (3 mL), dried
(Na2SO4), filtered, and concentrated in vacuo. PTLC (SiO2, CHCl3:
EtOAc:HOAc ) 10:20:0.3 eluent) afforded 1 (6.5 mg, 5.5 µmol, 43%)
A solution of 16 (432 mg, 0.5 mmol) in 90% aqueous AcOH (15
mL) was treated with Zn (1.62 g, 25 mmol), and the resulting suspension
was stirred at 0 °C for 2 h. The zinc was removed by filtration, and
the filtrate was concentrated in vacuo. The residue was poured onto 1
N aqueous HCl (50 mL) and extracted with CHCl3 (3 × 50 mL). The
combined organic phase was dried (Na2SO4), filtered, and concentrated
in vacuo to give 18 (430 mg, 100%) as a white foam which was
employed directly in the next reaction without further purification.
A solution of 17 (429 mg, 0.5 mmol) and 18 (430 mg, 0.5 mmol) in
CH2Cl2 (5.0 mL) was treated sequentially with HOAt (98 mg, 0.6 mmol)
and EDCI (119 mg, 0.6 mmol), and the reaction mixture was stirred at
0 °C for 6 h. The reaction mixture was poured onto 1 N aqueous HCl
(50 mL) and extracted with EtOAc (2 × 50 mL). The combined organic
phases were washed with saturated aqueous NaHCO3 (50 mL) and
saturated aqueous NaCl (30 mL), dried (Na2SO4), filtered, and
concentrated in vacuo. Flash chromatography (SiO2, 4 × 15 cm, 20%
acetone-EtOAc eluent) afforded 1921 (613 mg, 0.42 mmol, 83%) as a
white foam.
1
as a white solid which exibited a H NMR spectrum identical to the
chart published for authentic 1.1,21
BE-22179 (2). A sample of 1 (1.0 mg, 0.85 µmol) in 30% aqueous
acetone (400 µL) was treated with NaIO4 (0.4 mg, 8.5 µmol), and the
reaction mixture was stirred at 25 °C for 12 h before being quenched
by adding aqueous Na2S2O3. The mixture was concentrated in vacuo,
and the residue was extracted with EtOAc (2 × 2 mL). The combined
organic phases were washed with saturated aqueous NaCl (3 mL), dried
(Na2SO4), filtered, and concentrated in vacuo to give the crude
sulfoxides. A solution of the crude sulfoxides in CH2Cl2 (400 µL) was
warmed at reflux for 6 h, and the volatiles were removed in vacuo.
PTLC (SiO2, CHCl3:EtOAc:HOAc ) 10:20:0.3 eluent) afforded 2 (0.6
mg, 0.56 µmol, 66%) as a pale yellow solid which exibited a 1H NMR
spectrum identical to the chart published for authentic 2.2,21
[N-(2-Quinoline carboxyl)-D-Cys-Gly-NMe-L-Cys-NMe-L-Cys-
(Me)]2 (Cysteine Thiol) Dilactone (26). In the same manner as that
described for 1, the reaction of 23 (5.0 mg, 4.6 µmol) with quinoline-
2-carboxylic acid (4.0 mg, 23.0 µmol), EDCI (4.5 mg, 23.0 µmol),
and DMAP (2.8 mg, 23.0 µmol) in CH2Cl2 (300 µL) and purification
by PTLC (SiO2, CHCl3:EtOAc:HOAc ) 10:20:0.3 eluent) afforded 2621
(2.8 mg, 2.4 µmol, 52%) as a white foam.
[N-(2-Quinoxaline carboxyl)-D-Cys-Gly-NMe-L-Cys-NMe-L-Cys-
(Me)]2 (Cysteine Thiol) Dilactone (27). In the same manner as that
described for 1, the reaction of 23 (5.0 mg, 4.6 µmol) with quinoxaline-
2-carboxylic acid (4.0 mg, 23.0 µmol), EDCI (4.5 mg, 23.0 µmol),
and DMAP (2.8 mg, 23.0 µmol) in CH2Cl2 (300 µL) and purification
by PTLC (SiO2, CHCl3:EtOAc:HOAc ) 10:20:0.3 eluent) afforded 2721
(2.0 mg, 2.2 µmol, 47%) as a white foam.
[N-(3-Hydroxy-6-methoxy-2-quinoline carboxyl)-D-Cys-Gly-NMe-
L-Cys-NMe-L-Cys(Me)]2 (Cysteine Thiol) Dilactone (28). In the
manner similar to that described for 1, the reaction of 23 (5.0 mg, 4.6
µmol) with 3-hydroxy-6-methoxy-quinoline-2-carboxylic acid10,20 (4.0
mg, 23.0 µmol), EDCI (4.5 mg, 23.0 µmol), and DMAP (2.8 mg, 23.0
µmol) in CH2Cl2 (300 µL) and purification by PTLC (SiO2, CHCl3:
EtOAc:HOAc ) 10:20:0.3 eluent) afforded 2821 (2.5 mg, 2.4 µmol,
51%) as a white foam.
N-Cbz-D-Cys[N-Cbz-D-Cys(N-BOC-Gly-NMe-L-Cys-NMe-L-Cys-
(Me)]-Gly-NMe-L-Cys-NMe-L-Cys(Me)]-OH (21). A solution of 19
(500 mg, 0.34 mmol) in 90% aqueous AcOH (15 mL) was treated with
Zn (1.08 g, 17.0 mmol), and the resulting suspension was stirred at 0
°C for 2 h. The zinc was removed by filtration, and the filtrate was
concentrated in vacuo. The residue was poured onto 1 N aqueous HCl
(100 mL) and extracted with CHCl3 (3 × 50 mL). The combined
organic phase was dried (Na2SO4), filtered, and concentrated in vacuo.
The residue in CH2Cl2 (100 mL) was added dropwise to a solution of
iodine (868 mg, 3.4 mmol) in 340 mL of CH2Cl2-MeOH (10:1), and
the reaction mixture was stirred at 25 °C for 2 h. The reaction mixture
was cooled in an ice bath, and 5% aqueous Na2S2O3 was added until
the color of iodine disappeared. The mixture was washed with 1 N
aqueous HCl (50 mL) and saturated aqueous NaCl (30 mL), dried (Na2-
SO4), filtered, and concentrated in vacuo. Flash chromatography (SiO2,
3 × 16 cm, 10% MeOH-CHCl3 eluent) afforded 2121 (201 mg, 0.17
mmol, 49%, typically 49-53%) as a pale yellow foam.
[N-Cbz-D-Cys-Gly-NMe-L-Cys-NMe-L-Cys(Me)]2
(Cysteine
Thiol) Dilactone (23). A sample of 21 (180 mg, 0.15 mmol) was treated
with 1.5 mL of 3 M HCl-dioxane, and the mixture was stirred at 25
°C for 30 min before the volatiles were removed in vacuo. The residual
HCl was removed by adding Et2O (5 mL) to the hydrochloride salt
followed by its removal in vacuo. The residue in CH2Cl2 (150 mL)
was treated sequentially with HOAt (122 mg, 0.75 mmol) and EDCI
(149 mg, 0.75 mmol), and the reaction mixture was stirred at 0 °C for
6 h. The reaction mixture was poured onto 1 N aqueous HCl (50 mL)
and extracted with EtOAc (2 × 50 mL). The combined organic phase
was washed with saturated aqueous NaHCO3 (50 mL) and saturated
aqueous NaCl (30 mL), dried (Na2SO4), filtered, and concentrated in
vacuo. Flash chromatography (SiO2, 4 × 15 cm, 25% EtOAc-hexane
eluent) afforded 2321 (84 mg, 77 µmol, 52%, typically 52-61%) as a
white solid.
Thiocoraline (1). A sample of 23 (14.0 mg, 12.9 µmol) was treated
with 2 mL of TFA-thioanisole (10:1) and the reaction mixture was
stirred at 25 °C for 6 h before being concentrated in vacuo. The residue
was treated with 3 M HCl-EtOAc, and the volatiles were removed in
vacuo to give the hydrochloride salt.
A solution of 25 (11.9 mg, 64.5 µmol) and DMAP (7.7 mg, 64.5
µmol) in CH2Cl2 (1 mL) was treated with EDCl (12.6 mg, 64.5 µmol),
Acknowledgment. We gratefully acknowledge the financial
support of the National Institute of Health (CA41101), the
Skaggs Institute for Chemical Biology, and the Japan Society
for Promotion of Sciences (S.I.)
Supporting Information Available: Characterization data
for 6-12, 14-16, 19, 21, 23, 1, 2, 26-28, experimental details
and characterization data on 29-35, and experimental details
for the DNA binding studies are provided including two figures
illustrating the DNase footprinting of 1 and echinomycin (PDF).
This material is available free of charge via the Internet at
http://pubs.acs.org.
JA003602R