MOLRAC Bifunctional Intercalators
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 10 2337
solution) and brine. The organic phase was separated, dried
enantiomers and the meso-compound) as a bright yellow solid: yield
179 mg (54%), mp 185-252 °C (dec).
4
(MgSO ), filtered, and concentrated in vacuo, leaving pure amine,
which was used directly without further purification.
Dimethyl (1a,2b,3a,4a,8a,9a,10b,11a,12b,13a,14a,18a,19a,-
One-Dimensional Unwinding Assay. Each potential intercalator
was incubated with pUC19 plasmid (2686 bp) in varying ratios of
drug to base pairs (37 °C, 4 h) and relaxed by topoisomerase I (20
units/100 µL total reaction volume) in Tris-HCl, pH 7.5, (50 mM),
2
0b)-6,16-(2′,2′′-Di-(9′′′-acridinylamino)ethyl)-6,16-diaza-21-ox-
3
,9 13,19 2,10 4,8 12,20 14,18
aoctacyclo[9.9.1.1. 1. 0. 0. 0.
0
]tricosane-5,7,15,17-
tetraone-1,11-dicarboxylate, 3a. Boc-amine 15 (0.15 g, 0.18
mmol) was treated with TFA/DCM (0.5 mL, 20% solution)
according to standard method 5. A reaction time of 12 h followed
by standard workup provided the intermediate amine as a bright
yellow solid that was used without further purification: yield 0.12
g (94%). The reaction of this product (0.12 g, 0.17 mmol) with
2
KCl (50 mM), MgCl (10 mM), dithiothreitol (0.5 mM), EDTA
(0.1 mM), and bovine serum albumin (200 µg/mL). The reactions
were quenched and DNA was extracted with an equal volume of
phenol/chloroform:isoamyl/alcohol (25:24:1), removing the protein
and ligand. The phases were partitioned by centrifugation (Eppen-
dorf centrifuge 5415- D, 13 000 rcf for 5 min). The top aqueous
9
-chloroacridine (0.04 g, 0.17 mmol) in dry DMF (0.8 mL) was
layer was stored at -80 °C in freezer tubes until analysis was carried
out.6
1,62,63
Aliquots of 15 µL of each sample were added to 5 µL of
performed according to standard method 1. A reaction time of 3.5
h was required, and the crude product was purified by radial
chromatography (7.5% MeOH/EtOAc) to provide the title com-
loading buffer (20% w/v sucrose, 0.05 M Tris-HCl, pH 7.5, 0.05M
EDTA, 50 µg/mL bromophenol blue) and loaded on to 1% (w/v)
agarose gels in TBE (80 mM Tris-borate, 1 mM EDTA). Gels were
run at 1.9 V/cm (15 h, 600 mL TBE). After electrophoresis, the
gels were soaked in TBE and ethidium bromide (1 µg/mL) for 2 h
and destained in several changes of water over at least an hour.
The gels were then visualized on a GDS-8000 System Transillu-
minator (UVP Inc., Upland, CA).
pound as a bright yellow solid: yield 0.09 g (58%), mp 292 °C
1
(dec). H NMR (400 MHz, CDCl
3
): δ 1.12 (2H, d, J ) 9.7 Hz,
H22a,23a); 1.93 (4H, s, H2,10,12,20); 2.54 (6H, m, H3,9,13,19,-
2
3
2b,23b); 2.81 (4H, m, H4,8,14,18); 3.44 (4H, m, H1′,1′′ or 2′,2′′);
.68 (4H, m, H1′,1′′ or 2′,2′′); 3.86 (6H, s, OCH ); 7.25 (4H, m,
3
H2′′′,7′′′ or 3′′′,6′′′); 7.53 (4H, m, H2′′′,7′′′ or 3′′′,6′′′); 7.81 (4H,
m, H1′′′,8′′′ or 4′′′,5′′′); 7.95 (4H, d, J ) 8.2 Hz, H1′′′,8′′′ or 4′′′,5′′′).
Dimethyl (1a,2b,3a,4a,8a,9a,10b,11a,12b,13a,14a,18a,19a,-
Unwinding Angle and Intrinsic Association Constant Deter-
mination by Two-Dimensional Gels. Topoisomer separation and
5
0
20b)-6-(4′-(4′′′-Acridinyl)-4′-keto-3′-azabutyl)-16-(2′′-(9′′′′-acridi-
subsequent analysis were carried as described by Zeman et al.
3,9 13,19 2,10 4,8
nylamino)ethyl)-6,16-diaza-21-oxaoctacyclo [9.9.1.1. 1. 0. 0.
Circular DNA (pUC19) was relaxed by topoisomerase I in the
presence of various concentrations of potential intercalators at a
constant ratio of DNA base pair to molecules of ligand. The ligand-
DNA mixtures were incubated (37 °C, 4 h) with topoisomerase I
(20 units/100 µL total reaction volume) in Tris-HCl, pH 7.5, (50
1
2,20 14,18
0
.
0
]tricosane-5,7,15,17-tetraone-1,11-dicarboxylate, 3b.
The alkene 12 (96 mg, 0.25 mmol) and epoxide 20 (102 mg, 0.18
mmol) were combined in a sealed tube with acetonitrile (1.6 mL)
and heated (140 °C) for 5 h according to standard method 2. The
crude product was purified by radial chromatography (10% MeOH/
2
mM), KCl (50 mM), MgCl (10 mM), dithiothreitol (0.5 mM),
EtOAc) to yield the title compound as a pale yellow solid: yield
EDTA (0.1 mM), and bovine serum albumin (200 µg/mL). The
reactions were quenched (phenol/chloroform/isoamyl alcohol; vol.
ratio 25:24:1), DNA extracted, and stored at -80 °C until gel
analysis. For analysis by two-dimensional electrophoresis, samples
were thawed and the aqueous phase was concentrated to a volume
of approximately 20 µL (Vivaspin concentrator). Aliquots were
loaded on to agarose gels (1%) and run as described above. After
the first dimension had been run (23 h, TBE, 1.9V/cm), gels were
soaked (2 h on a shaker, TBE containing 1.1 µg/mL chloroquine)
and rotated 90° before running the second dimension (19 h, TBE
+ 1.1 µg/mL chloroquine, 1.9V/cm). Gels were stained with
ethidium bromide and visualized by UV.
1
1
1
2
03 mg (60%), mp 198 °C (dec). H NMR (400 MHz, CDCl
.17 (2H, d, J ) 9.3 Hz, H22a,23a); 2.25 (2H, s, H2,10 or 12,20);
.29 (2H, s, H2,10 or 12,20); 2.58 (2H, m, H3,9 or 13,19); 2.60
3
): δ
(
2H, m, H22b,23b); 2.64 (2H, m, H3,9 or 13,19); 2.94 (2H, m,
H4,8 or 14,18); 2.97 (2H, m, H4,8 or 14,18); 3.67-3.82 (8H, m,
H1′,2′,1′,2′′); 3.86 (6H, s, OCH
3
); 7.15 (2H, t, J ) 9.8 Hz, H2′′′′,7′′′′
or 3′′′′,6′′′′); 7.38 (1H, t, J ) 10.2 Hz, H2′′′); 7.50 (3H, m, H2′′′′,7′′′′
or 3′′′′,6′′′′ and 7′′′); 7.71-7.82 (5H, m, H1′′′,8′′′,6′′′,1′′′′,8′′′′ or
4
(
9
′′′′,5′′′′); 7.88 (2H, d, J ) 11.6 Hz, H1′′′′,8′′′′ or 4′′′′,5′′′′); 8.10
1H, d, J ) 11.7 Hz, H5′′′); 8.52 (1H, s, H 9′′′); 8.73 (1H, d, J )
.1 Hz, H 3′′′); 11.93 (1H, s (br), CONH)
Tetramethyl (1a,2b,3a,4b,5a,6a,10a,11a,12b,13a,14b,15a,16b,-
Two-Dimensional Data Analysis. Data were analyzed using
Kaleidagraph Software (Synergy Software, Reading, PA). The band
intensities for individual topoisomer bands were quantified using
Labworks Software (UVP Inc., Upland, CA). The integrated
intensities for each topoisomer were plotted to give a Boltzmann
1
7a,18b,19a,20a,24a,25a,26b,27a,28b)-8,22-(2′,2′′-Di-(9′′′-acridinylamino)-
3
,13 5,11 17,27
ethyl)-8,22-diaza-30,32-dioxadodecacyclo [13.13.1.1. 1. 1.
1
9,25 2,14 4,12 6,10 16,28 18,26 20,24
1
.
0. 0. 0. 0.
0.
0
]tritriacontane-7,9,21,23-tet-
raone-3,13,17,27-tetracarboxylate, 4a. The alkene 12 (0.36 g, 0.95
mmol) and bis-epoxide 282
6,34,35
distribution from which ∆Lk was calculated by fitting to the
(0.19 g, 0.47 mmol) were combined
c
in a sealed tube with acetonitrile (5.5 mL) and heated (140 °C) for
h according to standard method 2. The crude product was purified
by radial chromatography (10% MeOH/EtOAc) to yield the bis-
following equation
6
0
I ) IM exp[-w(∆Lk - ∆Lk )]
(1)
c
c
acridine (4a), which was recrystallized (DCM/Pet) as a bright
1
yellow solid: yield 0.22 g (41%), mp 276 °C (dec). H NMR (400
I and IM are, respectively, integrated band intensity and maximum
band intensity at the peak of the topoisomer distribution; w is the
MHz, CDCl ): δ 0.31 (4H, s, H2,14,16,28); 1.09 (2H, d, J ) 10.2
3
Hz, H31a,33a); 1.25 (2H, s, H1,15); 1.44 (2H, s, H29); 2.09 (4H,
c
width of the distribution; and ∆Lk and ∆Lk are the average most
0
c
s, H4,12,18,26); 2.49 (4H, s, H5,11,19,25); 2.52 (2H, d, J ) 10.2
Hz, H31b,33b); 2.89 (4H, s, H6,10,20,24); 3.70 (12H, s, OCH );
3
.15 (4H, m, H1′,1′′ or 2′,2′′); 4.22 (4H, m, H1′,1′′ or 2′,2′′); 7.39
abundant topoisomer in the Boltzmann population for a reaction
with ligand and for the control reaction containing no ligand,
4
(
respectively. φ , the apparent unwinding angle for each reaction
ap,
4H, t, J ) 7.6 Hz, H2′′′,7′′′ or 3′′′,6′′′); 7.68 (4H, t, J ) 7.6 Hz,
was calculated using
H2′′′,7′′′ or 3′′′,6′′′); 8.01 (4H, d, J ) 7.9 Hz, H1′′′,8′′′ or 4′′′,5′′′);
8
.09 (4H, d, J ) 8.8 Hz, H1′′′,8′′′ or 4′′′,5′′′).
ND
0
φ
) 360
(∆Lk - ∆Lk )
(2)
Tetramethyl (1a,2b,3a,4b,5a,6b,8a,9b,10a,11b,12a,13b,14a,-
5b,16a,17b,19a,20b,21a,22b)-6,17-(2′,2′′-Di-(9′′′-acridinylami-
ap
( )
c
c
N
L
1
3
,10 5,8 14,21 16,19 2,11 4,9
no)ethyl)-24,26-dioxadecacyclo [10.10.1.1. 1. 1. 1. 0. 0.
with N
D
equal to the number of plasmid molecules and N
L
equal to
13,22 15,20
0.
0
]heptacosane-3,10,14,21- tetracarboxylates, 4b/c. The
the number of ligands. The unwinding angle was calculated by
fitting the data to
alkene 30 (200 mg, 0.64 mmol) and epoxide 28 (130 mg, 0.32
mmol) were combined in a sealed tube with THF (9.0 mL) and
heated (140 °C) for 6 h according to standard method 2. The crude
products were purified by radial chromatography (5% MeOH/
EtOAc) to yield adducts 4b/c as a mixture of isomers (both
φ
ap
φ
) φ -
(3)
ap
[
(K [C - (2n - 1)C )
]
a
N
T