Synthesis of isochrysohermidin - Distamycin hybrids

Article abstract of DOI:10.1021/jo034326c

The synthesis of the alkylating subunit of the DNA cross-linking agent, isochrysohermidin (2), and its subsequent incorporation into conjugates with distamycin A (1) are described. The DNA binding properties of these agents were compared to that of distamycin A, using a fluorescence intercalator displacement (FID) assay.

Full text of DOI:10.1021/jo034326c

Syn th esis of Isoch r ysoh er m id in -Dista m ycin Hybr id s
Bryan K. S. Yeung and Dale L. Boger*
Department of Chemistry and The Skaggs Institute for Chemical Biology, The Scripps Research Institute,
10550 North Torrey Pines Road, La J olla, California 92037
boger@scripps.edu
Received March 11, 2003
The synthesis of the alkylating subunit of the DNA cross-linking agent, isochrysohermidin (2),
and its subsequent incorporation into conjugates with distamycin A (1) are described. The DNA
binding properties of these agents were compared to that of distamycin A, using a fluorescence
intercalator displacement (FID) assay.
In tr od u ction
selective alkylating agents (e.g. CBI,6 duocarmycin A7)
that combine the noncovalent binding selectivity inherent
in the distamycin conjugate with the alkylation selectiv-
ity to further enhance binding selectivity and affinity.
In recent studies, we described the total synthesis of
isochrysohermidin (2) and disclosed the first report of its
interstrand DNA cross-linking properties.8 Isolated from
Mercurialis perennis L., both d,l- and meso-forms were
found to occur naturally with the d,l-diastereomer un-
ambiguously identified by X-ray crystallography.9 The
dimeric N-methylcarbinolamides undergo a slow ring-
opening event during the interconversion of d,l- and
meso-2. This ring-opening reaction exposes an electro-
philic carbonyl capable of trapping nucleophiles within
the minor groove of duplex DNA. The only nucleophile
readily accessible to minor groove bound isochrysoher-
midin is believed to be the C2 amine of guanine. By
incorporation of a single carbinolamide subunit of iso-
chrysohermidin into distamycin, we sought to establish
whether it may be possible to direct a reversible (vs
irreversible)5-7 guanine alkylation near adjacent A-T
rich sites within duplex DNA (Figure 1).
Gene expression is regulated by a host of inhibitor and
enhancer proteins that selectively bind to specific se-
quences of DNA. The selective disruption of this process
by small molecules, which bind to DNA in a sequence-
specific manner, may provide access to new therapeutics.
Among this class of agents, distamycin A is one of the
most widely studied. Distamycin A, originally isolated
from Streptomyces sp.,1 is a minor groove binding agent
with sequence specificity toward A-T rich sites within
duplex DNA. Its sequence specificity and high affinity is
derived from a combination of interactions including
hydrogen bonding, van der Waals contacts, and electro-
static interactions of the cationic amidine side chain with
the phosphate backbone of DNA.2 The more recent
discovery of 2:1 complexes,3 their elaboration into side-
by-side antiparallel γ-hairpin polyamides, and the advent
of Dervan’s pairing rules with the template modifications
to selectively recognize G (Im vs Py) or A (Hp vs Py)
provides a powerful paradigm on which to design se-
quence-selective DNA binding agents.4 A number of
studies have examined the consequences of incorporating
nonselective alkylating agents into the distamycin struc-
ture (e.g. R-haloacylamides, nitrogen mustards).5 A more
limited series of studies have examined conjugates with
Resu lts a n d Discu ssion
The distamycin analogues were prepared by solution-
phase synthesis requiring only acid/base liquid-liquid
extraction protocols for the isolation and purification of
the distamycin subunits. The amidine side chain found
in the natural product was replaced with a N,N-dim-
ethylaminopropylamine side chain to facilitate the ease
of synthesis.10 This substitution is well-documented and
(1) Arcamone, F.; Penco, S.; Orezzi, P. G.; Nicolella, V.; Pirelli, A.
Nature 1964, 203, 1064.
(2) Kopka, M. L.; Yoon, C.; Goodsell, D.; Pjura, P.; Dickerson, R. E.
Proc. Natl. Acad. Sci. U.S.A. 1985, 82, 1376.
(3) Pelton, J . G.; Wemmer, D. E. J . Am. Chem. Soc. 1990, 112, 1393.
(4) Dervan, P. B. Bioorg. Med. Chem. 2001, 9, 2215.
(5) For recent examples, see R-Haloacylamides: (a) Cozzi, P.; Beria,
I.; Caldarelli, M.; Capolongo, L.; Geroni, C.; Mongelli, N. Bioorg. Med.
Chem. Lett. 2000, 10, 1269. (b) Cozzi, P. Farmaco 2001, 56, 57. (c)
Cozzi, P.; Mongelli, N. Curr. Pharm. Des. 1998, 4, 181. (d) Krowicki,
K.; Balzarini, J .; De Clerq, E.; Newman, R. A.; Lown, J . W. J . Med.
Chem. 1988, 31, 341. (e) Baker, B. F.; Dervan, P. B. J . Am. Chem.
Soc. 1985, 107, 8266. Nitrogen mustards: (f) Wang, Y.; Wright, S. C.;
Larrick, J . W. Bioorg. Med. Chem. Lett. 2003, 13, 459. (g) Baraldi, P.
G.; Romagnoli, R.; Guadix, A. E.; Pineda de la Infantas, M. J .; Gallo,
M. A.; Espinosa, A.; Martinez, A.; Bingham, J . P.; Hartley, J . A. J .
Med. Chem. 2002, 45, 3630. (h) Cozzi, P. C.; Beria, I.; Caldarelli, M.;
Capolongo, L.; Geroni, C.; Mazzini, S.; Ragg, E. Bioorg. Med. Chem.
Lett. 2000, 10, 1653. (i) Xie, G.; Gupta, R.; Lown, J . W. Anti-Cancer
Drug Des. 1995, 10, 389. (j) Sigurdsson, S. T.; Rink, S. M.; Hopkins,
P. B. J . Am. Chem. Soc. 1993, 115, 12633. (k) Sondhi, S. M.; Praveen-
Reddy, B. S.; Lown, J . W. Curr. Med. Chem. 1997, 4, 313.
(6) (a) Chang, A. Y.; Dervan, P. B. J . Am. Chem. Soc. 2000, 122,
4856. (b) Kumar R.; Lown, J . W. Org. Lett. 2002, 4, 1851. (c) J ia, G.
F.; Iida, H.; Lown, J . W. Heterocycl. Commun. 1998, 4, 557. (d) J ia,
G.; Iida, H.; Lown, J . W. Chem. Commun. 1999, 119. (e) J ia, G.; Iida,
H.; Lown, J . W. Synlett 2000, 603. (f) Boger, D. L.; Schmitt, H.; Fink,
B. E.; Hedrick, M. P. J . Org. Chem. 2001, 66, 6654.
(7) Tao, Z.-H.; Fujiwara, T.; Saito, I.; Sugiyama, H. Angew. Chem.,
Int. Ed. 1999, 38, 650.
(8) Boger, D. L.; Baldino, C. M. J . Am. Chem. Soc. 1993, 115, 11418.
(9) Matsui, Y.; Kawabe, C.; Matsumoto, K.; Abe, K.; Miwa, T.
Phytochemistry 1989, 25, 1470.
(10) Boger, D. L.; Fink, B. E.; Hedrick, M. P. J . Am. Chem. Soc.
2000, 122, 6382.
10.1021/jo034326c CCC: $25.00 © 2003 American Chemical Society
Published on Web 05/21/2003
J . Org. Chem. 2003, 68, 5249-5253
5249

Yeung and Boger
SCHEME 2
SCHEME 3
F IGURE 1. Distamycin A (1), isochrysohermidin (2), and
hybrid agents (3-6).
SCHEME 1
with 7, in the presence of EDCI and DMAP, and provided
the corresponding tripeptide 42% yield. Treatment of 14
with 4 M HCl/EtOAc removed the BOC group and
provided the corresponding HCl salt of the distamycin
subunit 15 in quantitative yield.
The trisubstituted pyrrole precursor to the isochryso-
hermidin alkylation subunit was prepared utilizing a
1,2,4,5-tetrazine f 1,2-diazine f pyrrole Diels-Alder
strategy (Scheme 3).11 An inverse electron demand Diels-
Alder reaction of 1,2,4,5-tetrazine 1612 and dimethoxy-
ketene acetal13 provided the corresponding 1,2-diazine
1714 in 84% yield. A subsequent reductive ring contrac-
tion of the 1,2-diazine was achieved by using freshly
activated zinc dust in glacial acetic acid to provide the
trisubstituted pyrrole 1814 in 67% yield. The four-carbon
tether was installed by N-alkylation of 18 with methyl
does not adversely affect minor groove binding affinity
or selectivity. The distamycin subunits 10, 12, 13, and
15 were prepared as previously detailed10 by coupling 7
with 8 in the presence of EDCI and DMAP to afford 9 in
90% yield (Scheme 1). Subsequent treatment of 9 with
anhydrous 4 M HCl/EtOAc followed by coupling with 7,
in the presence of EDCI/DMAP, provided the distamycin
core 11 in 86% yield as detailed in our prior efforts.
Subsequent treatment of peptides 9 and 11 with 4 M HCl/
EtOAc removed the BOC group and provided the corre-
sponding HCl salt of the methyl ester derivatives of the
distamycin subunits (10 and 12).
Distamycin subunits 13 and 15, incorporating the N,N-
dimethylaminopropylamine side chain, were accessed
from 9 as previously described.10 Saponification with
LiOH was followed by the addition of N,N-dimethylami-
nopropylamine and PyBOP to afford the corresponding
adduct. Subsequent treatment with 4 M HCl/EtOAc
provided 13 in 66% yield over three steps (Scheme 2).
The tripeptide 14 was prepared by coupling dipeptide 13
(11) For reviews: (a) Boger, D. L. Tetrahedron 1983, 39, 2869. (b)
Boger, D. L. Chem. Rev. 1986, 86, 781. (c) Boger, D. L. Bull. Soc. Chim.,
Belg. 1990, 99, 599. (d) Boger, D. L. Chemtracts: Org. Chem. 1996, 9,
149. (e) Boger, D. L.; Weinreb, S. M. Hetero Diels-Alder Methodology
in Organic Synthesis; Academic: San Diego, CA, 1987.
(12) Boger, D. L.; Coleman, R. S.; Panek, J . S.; Huber, F. X.; Sauer,
J . J . Org. Chem. 1985, 50, 5377.
(13) (a) Corey, E. J .; Bass, J . D.; LeMahieu, R.; Mitra, R. B. J . Am.
Chem. Soc. 1964, 86, 5570. (b) Zheng, Q. H.; Su, J . Synth. Commun.
1999, 29, 3467.
(14) (a) Boger, D. L.; Coleman, R. S.; Panek, J . S.; Yohannes, D. J .
Org. Chem. 1984, 49, 4405. (b) Boger, D. L.; Patel, M. J . Org. Chem.
1988, 53, 1405. (c) Boger, D. L.; Baldino, C. M. J . Org. Chem. 1991,
56, 6942.
5250 J . Org. Chem., Vol. 68, No. 13, 2003

Synthesis of Isochrysohermidin-Distamycin Hybrids
TABLE 1. DNA Bin d in g Affin ity of 3-6 Com p a r ed to
Dista m ycin a n d Its Der iva tives
SCHEME 4
4-bromobutyrate (K2CO3, DMF) to furnish 19 in 95%
yield. Highly selective saponification of the two sterically
and electronically more accessible methyl esters provided
the diacid 20 (99%). Initially, more elaborate, selective
protections were anticipated to be necessary to cleanly
provide 20 (e.g. use of benzyl 4-bromobutyrate). However,
such selective protections/deprotections proved unneces-
sary and simple treatment of 19 with 2 equiv of LiOH at
25 °C provided 20 in superb conversions (99%). In the
final step, a [4+2] cycloaddition of 1O2 across the pyrrole
followed by a low-temperature oxidative decarboxylation
with fragmentation of the intermediate endoperoxide
afforded the isochrysohermidin subunit 21 in 89% yield.14c
F IGURE 2. Distamycin analogues utilized in the FID assay.
tion to establish a binding constant (K).16 This method
is based on the loss of fluorescence derived from the
titration displacement of ethidium bromide from a DNA
of interest. The agents were examined for their ability
to bind a hairpin deoxyoligonucleotide containing a
central five base pair AT-rich binding site (AAAAA)
adjacent to capping GC base pairs relative to distamycin
A and results are summarized in Table 1.
Additionally, the binding affinities of several other
distamycin derivatives (22 and 23, Figure 2) are included
for comparison. Distamycin A binds to poly-d[A]-poly-d[T]
and the 5′-AAAAA-3′ hairpin deoxyoligonucleotide with
essentially the same affinity. Moreover, replacement of
the amidine side chain on distamycin with N,N-dimeth-
ylpropylamine (22) simplifies the synthesis and does not
adversely affect binding affinity. By contrast, substitution
at the N-terminus of distamycin analogues has more of
an impact on DNA binding affinity. Replacement of the
N-formyl group with a sterically bulky BOC group with
23 lowers the binding affinity and suggests that large
substituents at the N-terminus are not as well accom-
modated in the minor groove. Dipyrrole hybrids 3 and 4
show a 3-fold decrease in binding affinity for the hairpin
deoxyoligonucleotide. These derivatives possess one less
pyrrole subunit than distamycin and are therefore ex-
pected to be less effective noncovalent DNA binding
agents. Interestingly, both 3 and 4 exhibited an affinity
greater than expected and there is essentially no differ-
ence in binding affinity between 3 and 4 potentially
representative of a DNA alkylation event. Tripyrrole
derivative 5 lacks the C-terminal basic side chain and
has half the binding affinity of the natural product.
However, by incorporating three N-methylpyrrole sub-
units as well as the basic side chain into the hybrid 6, it
is possible to obtain a binding affinity close to that of
distamycin and its closest analogue 22. Disappointingly,
3-6 exhibited no time-dependent increase in binding
affinity indicative of a slow, reversible covalent attach-
ment to DNA. Thus, although the surprisingly effective
1
In initial efforts, the O2 was generated photochemically
in the presence of the photosensitizer, Rose Bengal.
However, since the photosensitizer was difficult to com-
pletely remove from the reaction mixtures, a resin-bound
form of Rose Bengal was used, which was found to effect
the desired transformation without any decrease in
reactivity or product yields.15
With the isochrysohermidin subunit 21 in hand, the
hybrid conjugates were prepared by coupling with the
distamycin substructures incorporating either two or
three N-methyl pyrrole subunits. Accessing isochryso-
hermidin-dipyrrole analogue 3 was achieved by treat-
ment of 10 with 21 in the presence of EDCI/DMAP to
provide 3 in 31% yield. Similarly, treatment of dipyrrole
13 followed by addition of 21 in the presence of EDCI/
DMAP provided 4 in 32% yield after purification. The
tripyrrole conjugates 5 and 6 were prepared from the
corresponding tripyrroles 12 and 15, respectively. The
tripyrrole conjugate 5 was prepared from coupling 12 and
21 in the presence of EDCI and i-Pr2NEt to provide 5 in
37% yield after purification by column chromatography.
Similarly, the tripyrrole conjugate incorporating the N,N-
dimethylpropylamine tail (6) was obtained from EDCI
and i-Pr2NEt mediated coupling of 15 and 21 to provide
6 in 32% yield (Scheme 4).
DNA Bin d in g Affin ity. The DNA binding properties
of compounds 3-6 were first established by using a
fluorescence intercalator displacement (FID) assay titra-
(16) (a) Boger, D. L.; Fink, B. E.; Brunette, S. R.; Tse, W. C.; Hedrick,
M. P. J . Am. Chem. Soc. 2001, 123, 5878. (b) Boger, D. L.; Tse, W. C.
Bioorg. Med. Chem. 2001, 9, 2511.
(15) Bernasconi, C.; Cottier, L.; Descotes, G.; Nigay, H.; Pardon, J .
C.; Wisniewski, A. Bull. Soc. Chim. Fr. 1984, 7, II-323.
J . Org. Chem, Vol. 68, No. 13, 2003 5251

Yeung and Boger
0.04 mmol) and 10 (29 mg, 0.08 mmol) in anhydrous DMF (0.5
mL) and the reaction mixture was stirred at 0 °C under N2.
After 16 h, the reaction mixture was diluted with 1:1 i-PrOH-
CHCl3 (10 mL) and washed with 10% aqueous NaHCO3 (2 ×
20 mL). The organic phase was dried (Na2SO4), filtered, and
concentrated under vacuum. Chromatography (SiO2, 15:1
CHCl3-MeOH) afforded 3 (3.3 mg, 31%) as an off-white
syrup: 1H NMR (CDCl3, 400 MHz) δ 8.84 (1H, br s), 7.73 (1H,
s), 7.41 (1H, s), 7.15 (1H, s), 6.77 (1H, s), 6.67 (1H s), 5.06 (1H,
s), 3.90 (3H, s), 3.89 (3H, s), 3.85 (3H, s), 3.82 (3H, s), 3.81
(3H, s), 3.57 (1H), 2.36 (2H, t, J ) 6.4 Hz); 13C NMR (CDCl3,
100 MHz) δ 175.6, 172.6, 165.0, 163.2, 161.5, 124.6, 123.9,
123.4, 122.6, 121.0, 110.5, 106.1, 94.2, 59.8, 54.1, 51.6, 39.4,
37.1, 37.0, 34.7, 26.5; MALDI-HRFTMS m/z 554.1862 (M +
Na+, C24H29N5O9Na requires 554.1857).
behavior of 5 might suggest a covalent attachment to
DNA, the behavior of 6 relative to 22 along with the lack
of time-dependent binding affinity (data not shown)
suggests it is not observed. Although it is possible that
the covalent attachment is rapidly reversible, the intrin-
sic stability of the carbinolamide of isochrysohermidin
(t1/2 ca. 24-48 h, DMSO) suggests that is also unlikely.
Thus, although we do not yet have a good explanation
for the surprising behavior of 3-5, we are confident that
it is not derived from a stable, slowly reversible covalent
attachment to DNA.17
Exp er im en ta l Section
Com p ou n d 4. EDCI (29 mg, 0.151 mmol) was added to a
mixture of 21 (10 mg, 0.038 mmol), 13 (29 mg, 0.076 mmol),
and DMAP (18 mg, 0.151 mmol) in anhydrous DMF (0.3 mL)
and the reaction mixture was stirred at 25 °C under N2. After
24 h, the reaction mixture was diluted with 1:1 i-PrOH-CHCl3
(10 mL) and washed with H2O (2 × 10 mL). The organic phase
was dried (Na2SO4), filtered, and concentrated under vacuum.
Chromatography (RPC18-PTLC, 4:1 MeOH-50 mM HCO2NH4
buffer) afforded 4 (7 mg, 32%) as a clear syrup: 1H NMR (2:1
CD3OD-CH2Cl2, 500 MHz) δ 8.44 (1H, s), 7.15 (1H, d, J ) 1.8
Hz), 7.11 (1H, d, J ) 1.8 Hz), 6.87 (1H, d, J ) 1.8 Hz), 6.83
(1H, d, J ) 1.8 Hz), 5.18 (1H, s), 3.88 (3H, s), 3.87 (3H, s),
3.85 (3H, s), 3.78 (3H, s), 3.40 (4H, m), 3.20 (3H, m), 2.88, (6H,
s), 2.32 (2H, t, J ) 7.7 Hz), 1.97 (3H, m); 13C NMR (2:1 CD3-
OD-CH2Cl2, 125 MHz) δ 177.0, 176.9, 172.5, 159.6, 126.9,
123.3, 120.8, 120.5, 106.5, 105.9, 99.8, 94.0, 59.7, 56.6, 55.5,
43.7, 39.3, 36.6, 34.5, 30.7, 27.4, 27.3, 26.6, 26.3; MALDI-
HRFTMS m/z 602.2937 (M + H+, C28H39N7O8 requires
602.2933).
Com p ou n d 5. EDCI (35 mg, 0.18 mmol) was added to a
mixture of 21 (12 mg, 0.04 mmol), 12 (39 mg, 0.09 mmol), and
i-Pr2NEt (16 µL, 0.09 mmol) in anhydrous DMF (0.5 mL). The
reaction mixture was stirred under N2 at 0 °C for 3 h and
allowed to warm to 25 °C. After 18 h, the reaction mixture
was diluted with 1:1 i-PrOH-CHCl3 (10 mL) and washed with
H2O (2 × 10 mL). The organic phase was dried (Na2SO4),
filtered, and concentrated under vacuum. Chromatography
(SiO2, 12:1 CHCl3-MeOH) afforded 5 (11 mg, 37%) as an off-
white syrup: 1H NMR (1:1 CD3OD-CD2Cl2, 500 MHz) δ 7.68
(1H, s), 7.34 (1H, d, J ) 2.2 Hz), 7.18 (1H, d, J ) 2.2 Hz), 7.11
(1H, d, J ) 1.8 Hz), 6.91 (1H, d, J ) 2.2 Hz), 6.87 (1H, d, J )
2.2 Hz), 6.80 (1H, d, J ) 1.8 Hz), 5.12 (1H, s), 3.89 (3H, s),
3.88 (3H, s), 3.87 (3H, s), 3.84 (3H, s), 3.78 (3H,s), 3.77 (3H,
s), 3.45 (1H, dt, J ) 14.5, 7.0 Hz), 3.16 (1H, dt, J ) 14.5, 7.0
Hz), 2.32 (2H, t, J ) 7.5 Hz), 1.86 (2H, m); 13C NMR (1:1, CD3-
OD-CD2Cl2, 125 MHz) δ 174.7, 173.3, 172.0, 169.1, 162.7,
160.8, 124.2, 124.1, 123.2, 122.8, 122.6, 122.0, 120.5, 120.3,
120.1, 109.8, 105.8, 105.3, 93.7, 88.6, 78.8, 59.4, 51.4, 38.9, 36.9,
36.7, 34.1, 30.3, 26.0; MALDI-HRFTMS m/z 653.2459 (M+,
C30H35N7O10 requires 653.2440).
Com p ou n d 6. EDCI (31 mg, 0.180 mmol) was added to a
mixture of 21 (11 mg, 0.04 mmol), 15 (39 mg, 0.08 mmol), and
i-Pr2NEt (14 µL, 0.090 mmol) in anhydrous DMF (0.5 mL).
The reaction mixture was stirred under N2 at 0 °C for 3 h and
allowed to warm to 25 °C. After 24 h, the reaction mixture
was diluted with 1:1 i-PrOH-CHCl3 (10 mL) and washed with
10% aqueous NaHCO3 (2 × 10 mL). The organic phase was
dried (Na2SO4), filtered, and concentrated under vacuum.
Chromatography (RPC18-PTLC, 6:1 MeOH-50 mM HCO2NH4
buffer) afforded 4 (7 mg, 32%) as a clear syrup: 1H NMR (2:1
CD3OD-CD2Cl2, 500 MHz) δ 7.17 (1H, br s), 7.09 (1H, s), 6.92
(1H, s), 6.86 (1H, s), 6.91 (1H, br s), 6.82 (1H, s), 6.79 (1H, s),
5.09 (1H, s), 3.90 (3H, s), 3.88 (12H, br s), 3.39 (4H, br t, J )
6.3 Hz), 3.13 (2H, m), 3.05 (2H, br t, J ) 7.7 Hz), 2.80 (6H, br
s), 2.31 (2H, m), 1.96 (2H, m), 1.83 (2H, m), 0.877 (2H, br t, J
) 6.6 Hz); 13C NMR (1:1, CD3OD-CD2Cl2, 125 MHz) δ 173.0,
170.3, 169.4, 168.1, 167.0, 164.9, 161.4, 124.6, 123.8, 123.4,
Dim eth yl 3-Meth oxy-1-[(3-m eth oxyca r bon yl)p r op yl]-
1H-p yr r ole-2,5-d ica r boxyla te (19). Methyl 4-bromobutyrate
(309 µL, 2.44 mmol) was added to a solution of 1814 (281 mg,
1.32 mmol) and K2CO3 (455 mg, 3.29 mmol) in anhydrous DMF
(20 mL). The reaction mixture was warmed at 80 °C and
stirred under N2. After 4 h, the reaction mixture was cooled
to 25 °C, poured into H2O (100 mL), and extracted with CH2-
Cl2 (4 × 100 mL). The combined organic layers were dried (Na2-
SO4), filtered, and concentrated under vacuum. Chromatog-
raphy (SiO2, 50% EtOAc-hexanes) afforded 19 (399 mg, 96%)
as a white solid: mp 74-75 °C; 1H NMR (CDCl3, 400 MHz) δ
6.52 (1H, s), 4.81 (2H, t, J ) 7.3 Hz), 3.85 (3H, s), 3.82 (6H, s),
3.64 (3H, s), 2.31 (2H, t, J ) 7.6 Hz), 2.04 (2H, quint, J ) 7.9
Hz); 13C NMR (CDCl3, 100 MHz) δ 173.2, 161.1, 160.6, 152.5,
124.1, 112.8, 101.0, 57.9, 51.7, 51.5, 51.4, 45.4, 31.0, 26.7;
MALDI-HRFTMS m/z 336.1056 (M + Na+, C14H19NO7 requires
336.1054).
1-(4-Bu tyr ic a cid )-4-m eth oxy-5-m eth oxyca r bon yl-1H-
p yr r ole-2-ca r boxylic Acid (20). LiOH‚H2O (117 mg, 2.80
mmol) was added to a stirred solution of 19 (399 mg, 1.27
mmol) in a 2:1:1 solution of THF:MeOH:H2O (8 mL). After 20
h, the mixture was partitioned between Et2O and H2O. The
aqueous layer was acidified with the addition of 5% aqueous
HCl (pH 3.0) and extracted with EtOAc (4 × 20 mL). The
combined EtOAc layers were dried (Na2SO4), filtered, and
concentrated under vacuum to afford diacid 20 (358 mg, 99%)
as a white solid: mp 193-195 °C; 1H NMR (CDCl3, 400 MHz)
δ 6.61 (1H, s), 4.79 (2H, t, J ) 7.3 Hz), 3.81 (3H, s), 3.79 (3H,
s), 2.25 (2H, t, J ) 7.4 Hz), 2.04 (2H, quint, J ) 7.9 Hz); 13C
NMR (CDCl3, 100 MHz) δ 176.7, 163.1, 162.8, 154.2, 126.3,
113.8, 102.5, 58.3, 51.6, 46.3, 31.9, 28.1; MALDI-HRFTMS m/z
308.0744 (M + Na+, C12H15NO7 requires 308.0741).
Isoch r ysoh er m id in Su bu n it 21. A 3:1 solution of CH3-
CN-H2O (40 mL) was added to a quartz flask charged with
20 (23 mg, 0.08 mmol) and Rose Bengal resin (7.0 mg, 0.0006
mmol).18 The solution was irradiated under a Hanovia high-
pressure mercury lamp (450 W) through a uranium yellow
glass filter (transmits <330 nm) with a steady stream of O2
bubbled through the solution. After 3 h, a small amount of
activated charcoal was added to remove any solubilized Rose
Bengal and the solution filtered through Celite and rinsed with
MeOH. The solvent was concentrated under vacuum to afford
21 (21.8 mg, 86% yield) as a transparent glass: 1H NMR
(CDCl3, 400 MHz) δ 5.14 (1H, s), 4.62 (1H, s), 3.79 (3H, s),
3.77 (3H, s), 3.68 (1H, dt, J ) 14.4, 6.6 Hz), 3.08 (1H, dt, J )
14.5, 6.6 Hz), 2.32 (2H, t, J ) 7.2 Hz), 1.79 (2H, quint, J ) 7.3
Hz); 13C NMR (CDCl3, 100 MHz) δ 172.8, 170.9, 170.8, 167.3,
94.8, 63.6, 59.0, 53.4, 40.2, 25.2, 23.6; MALDI-HRFTMS m/z
296.0728 (M + Na+, C11H15NO7 requires 296.0741).
Com p ou n d 3. 1-(3-Dimethylaminopropyl)-3-ethylcarbodi-
imide hydrochloride (EDCI) (33 mg, 0.17 mmol) and DMAP
(11 mg, 0.08 mmol) were added to a mixture of 21 (12 mg,
(17) Cytotoxic activity: L1210 IC50 ) 67 (1), 119 (3), 165 (4), >200
(5), and >200 µM (6).
(18) Concentration of resin-bound Rose Bengal was determined
according to ref 15 (0.09 mol of Rose Bengal/g of support).
5252 J . Org. Chem., Vol. 68, No. 13, 2003

Synthesis of Isochrysohermidin-Distamycin Hybrids
123.3, 120.9, 120.7, 120.5, 106.5, 106.4, 105.9, 56.7, 47.4, 47.3,
43.8, 41.1, 37.1, 37.0, 36.7, 34.4, 30.7, 27.4, 27.3, 26.6, 25.3,
23.7, 23.1; MALDI-HRFTMS m/z 723.3337 (M + H+, C34H45N9O9
requires 723.3340).
generated where ∆F/[free agent] was plotted vs ∆F. The slope
of the region immediately preceding complete saturation of the
system provided -K.16
∆Fx
1
X
Deter m in a tion of DNA Bin d in g Con sta n ts. A 3-mL
quartz cuvette was loaded with Tris buffer (0.1 M Tris, 0.1 M
NaCl, pH 8) and ethidium bromide (0.44 × 10-5 M final
concentration). The fluorescence was measured (excitation 545
nm, emission 595 nm, EtBr) and normalized to 0% relative
fluorescence. The 5′-AAAAA-3′ hairpin deoxyoligonucleotide
was added (1.5 µM, 12 µM in base pair final concentration),
and the fluorescence measured again and normalized to 100%
relative fluorescence. A solution of the agent (3 µL, 0.1 mM in
DMSO) was added, and the fluorescence measured following
5 min of incubation at 23 °C. Subsequent addition of 3-µL
aliquots of the agent was continued until the system reached
saturation and the fluorescence remained constant with suc-
cessive compound additions.
) fraction of DNA - agent complex
(1)
(2)
(3)
(
)
∆Fsat
∆Fx
1
X
1 -
) fraction of free agent
(
)
[
]
∆Fsat
∆Fx
[DNA]T X -
) [free agent]
[
]
∆Fsat
Ack n ow led gm en t. We gratefully acknowledge the
financial support of the National Institutes of Health
(CA 41986, CA 42056) and the Department of the Army
(DAMD17-02-1-0135).
Sca tch a r d An a lysis of th e Titr a tion Cu r ve. The ∆F was
plotted versus molar equivalents of agent and the ∆Fsat was
determined mathematically by solving the simultaneous equa-
tions representing the pre- and postsaturation regions of the
titration curve. Utilizing eqs 1-3, a Scatchard plot was
Su p p or tin g In for m a tion Ava ila ble: 1H NMR of all new
compounds 3-6 and 19-21. This material is available free of
charge via the Internet at http://pubs.acs.org.
J O034326C
J . Org. Chem, Vol. 68, No. 13, 2003 5253