4808 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 24
Harapanhalli et al.
3.04-3.18 (m, 8 H, piperazinyl methylenes), 4.62 (bs, 2 H, D2O
exchangeable, -NH2), 4.90 (bs, 2 H, D2O exchangeable, -NH2),
6.58 (d, J ) 12.5 Hz, 1 H, H-6), 6.84 (dd, J ) 12.5, 3 Hz, 1 H,
H-5), 6.93 (bs, 1 H, H-3), 7.15 (dd, J ) 3, 12.5 Hz, 1 H, H-6′),
7.28 (dd, J ) 12.5, 14 Hz, 1 H, H-7′), 7.33 (bd, J ) 12.5 Hz, 1
H, H-4′). Anal. (C18H22N6‚H2O) C, H, N.
4-Eth oxyben za ld eh yd e (17). Anhydrous potassium car-
bonate was added to a stirred solution of hydroxybenzaldehyde
16 (1 g, 8.2 mmol) and iodoethane (1.56 g, 10 mmol) in 25 mL
of dry acetone. The mixture was filtered, and the residue was
thoroughly washed with acetone. The combined acetone
solution was evaporated to dryness; the residue was extracted
with chloroform, worked up as usual, and purified on a short
silica gel column to obtain an oil (yield 2.1 g): 1H NMR δ 1.38
(t, J ) 6 Hz, 3 H, -CH3), 4.15 (q, J ) 6 Hz, 2 H, -CH2-), 7.13 (d,
J ) 12 Hz, 2 H, H-3, H-5), 7.88 (d, J ) 12 Hz, 2 H, H-2, H-6).
Anal. (C9H10O2) C, H.
2-[2-(4-Eth oxyp h en yl)-6-ben zim id a zolyl]-6-(1-m eth yl-
4-p ip er a zin yl)ben zim id a zole (Hoech st 33342, 1). The
above procedure was followed with ethoxy aldehyde 17, and
pure Hoechst 33342 (1) was isolated in 80% yield.
2-[2-[3-(Tr im eth ylsta n n yl)-4-eth oxyp h en yl]-6-ben zim -
id a zolyl]-6-(1-m eth yl-4-p ip er a zin yl)ben zim id a zole ((Tr i-
m eth ylsta n n yl)Hoech st 33342, 4). The above procedure
was followed with diaminobenzimidazole 15 (16.1 mg, 0.05
mmol) and stannyl aldehyde 20 (15.9 mg, 0.0507 mmol), and
the crude material was purified by column chromatography
(yield 21 mg): mp 203 °C dec; 1H NMR δ 0.4 (s, with Sn
2
satellites, J Sn-CH ) 55 Hz, 9 H, -SnMe3), 1.40 (t, J ) 6 Hz, 3
H, -CH3), 2.21 (s, 3 H, N-CH3), 3.20-3.50 (m, 8 H, H-2′′′, H-3′′′,
H-5′′′, H-6′′′), 4.10 (q, J ) 6 Hz, 2 H, -CH2- of ethoxy), 6.88-
6.98 (m, 2 H, H-7′′, H-6), 7.13 (d, J ) 12 Hz, 1 H, H-7′), 7.45
(m, 1 H, H-6′′), 7.67 (dd, J ) 12, 14 Hz, 1 H, H-6′), 8.01 (t, J
) 3, 14 Hz, 1 H, H-4′′), 8.15-8.17 (m, 2 H, H-3, H-5), 8.30 (d,
J ) 14 Hz, 1 H, H-4′); FAB-HRMS calcd for C30H37N6OSn (M+
+ H) 617.205 084, found 617.204 200. Anal. (C30H37N6OSn)
C, H, N.
3-Iod o-4-h yd r oxyben za ld eh yd e (18). To a stirred solu-
tion of hydroxybenzaldehyde 16 (1.22 g, 10 mmol) in 20 mL of
dichloromethane at ambient temperature was added a solution
of iodine monochloride (1.8 g, 11 mmol) in 1 mL of acetic acid
(17 h). The product was worked up as usual, and the residue
was purified on a silica gel column (yield 1.8 g): mp 128-130
2-[2-(3-[125I]Iod o-4-et h oxyp h en yl)-6-b en zim id a zolyl]-
6-(1-m et h yl-4-p ip er a zin yl)b en zim id a zole (R a d ioiod o-
Hoech st 33342, 3). In a vial were placed 20 µL of a solution
of 1.5 mg/mL (trimethylstannyl)Hoechst 33342 (4) in methanol,
2 mCi of sodium [125I]iodide (or sodium [131I]iodide) in 20 µL
of 0.1 M sodium hydroxide, 40 µL of 0.2 M acetate buffer (pH
4.9), 1 µL of lactoperoxidase solution (4 mg/mL), and 10 µL of
dilute hydrogen peroxide (30% solution diluted 500× in water).
The vial was vortex-mixed and incubated for 5 min, and 10
µL of dilute hydrogen peroxide was added. After a 5-min
incubation, the reaction was terminated by addition of 100 µL
of 2% tri-n-butylamine in methanol, and the mixture was
purified by HPLC on a C18 column. Mobile phase A was 10%
methanol in potassium phosphate buffer (20 mM, pH 7.0), and
phase B was 20% potassium phosphate buffer in methanol. A
linear gradient from 100% A to 100% B in 30 min at a flow
rate of 1 mL/min was employed. A radiochemical yield of 70-
80% and radiochemical purity of 99.8% were obtained.
1
°C; H NMR δ 7.15 (d, J ) 13 Hz, 1 H, H-5), 7.89 (d, J ) 13
Hz, 1 H, H-6), 8.25 (bs, 1 H, H-2), 9.68 (s, 1 H, -CHO). Anal.
(C7H5O2I) C, H, I.
3-Iod o-4-eth oxyben za ld eh yd e (19). The above procedure
was followed with iodobenzaldehyde 18 (1 g, 4 mmol), and the
crude product was purified on a silica gel column to afford
white crystals (yield 0.45 g): mp 81 °C; 1H NMR δ 1.38 (t, J )
6 Hz, 3 H, -CH3), 4.21 (q, J ) 6 Hz, 2 H, -CH2-), 7.20 (d, J )
12 Hz, 1 H, H-5), 7.91 (d, J ) 12 Hz, 1 H, H-6), 8.30 (bs, 1 H,
H-2), 9.82 (s, 1 H, -CHO); EI-HRMS calcd for C9H9O2I
275.964 732, found 275.964 670.
3-(Tr im eth ylsta n n yl)-4-eth oxyben za ld eh yd e (20).
A
solution of iodo aldehyde 19 (138 mg, 0.5 mmol), hexameth-
ylditin (250 mg, 0.76 mmol), and tetrakis(triphenylphosphine)-
palladium(0) (25 mg, 0.022 mmol) in 15 mL of anhydrous
dioxane was heated under gentle reflux in a nitrogen atmo-
sphere (1 h). The product was worked up and purified on a
silica gel column conditioned in hexane. The chemical eluted
in hexane-2% ethyl acetate (yield 130 mg): mp 104 °C; 1H
NMR δ 0.28 (s, with Sn satellites, 2J Sn-CH ) 55 Hz, 9 H, SnMe3
with tin satellites), 1.38 (t, J ) 6 Hz, 3 H, -CH3), 4.18 (q, J )
6 Hz, 2 H, -CH2-), 7.12 (d, J ) 12 Hz, 1 H, H-5), 7.90 (s, 1 H,
H-2), 7.95 (d, J ) 12 Hz, 1 H, H-6), 9.90 (s, 1 H, -CHO); 119Sn
NMR (CDCl3/Me3Sn) δ -25.2; EI-HRMS calcd for C12H18O2-
Sn 310.032 424, found 310.032 049.
2-[2-(3-Iod o-4-et h oxyp h en yl)-6-b en zim id a zolyl]-6-(1-
m eth yl-4-piper azin yl)ben zim idazole (IodoHoech st 33342,
3). A suspension of diaminobenzimidazole 15 (250 mg, 0.78
mmol) and iodo aldehyde 19 (218 mg, 0.79 mmol) in 20 mL of
nitrobenzene containing 10-15 molecular sieves was stirred
at 60 °C for 0.5 h and then at 98-100 °C (36 h). Initially a
clear dark-red solution was formed and then, by the end of 24
h, a yellow suspension. A small amount of the aldehyde 19
(20 mg) was added and the reaction continued for 12 h. The
yellow precipitate was filtered and washed thoroughly with
benzene, acetone, and hexane. The crude material was
purified on a silica gel column preconditioned in ethyl acetate.
The product was eluted in a mixture of 15% methanol and 0.4%
tri-n-butylamine in ethyl acetate. Following evaporation, the
residue was dissolved in 2 mL of methanol and cooled in an
ice bath. A methanolic solution of hydrogen chloride (2 M)
was added, and the precipitate was filtered and washed with
methanol:acetone, 1:1, followed by acetone and dried in
vacuo: mp 233 °C dec; 1H NMR δ 1.42 (t, J ) 6 Hz, 3 H, -CH3),
2.21 (s, 3 H, N-CH3), 3.10-3.50 (m, 8 H, H-2′′′, H-3′′′, H-5′′′,
H-6′′′), 4.20 (q, J ) 6 Hz, 2 H, -CH2- of ethoxy), 6.9-7.0 (m, 2
H, H-7′′, H-6), 7.20 (d, J ) 12 Hz, 1 H, H-7′), 7.40 (bd, J ) 12
Hz, 1 H, H-6′′), 7.70 (bd, J ) 12 Hz, 1 H, H-6′), 8.05 (bs, 1 H,
H-4′′), 8.20 (d, J ) 12 Hz, 1 H, H-5), 8.30 (bs, 1 H, H-4′), 8.64
(s, 1 H, H-3); FAB-HRMS calcd for C27H28N6OI (M+ + H)
579.136 937, found 579.137 700. Anal. (C27H27N6OI‚H2O) C,
H, N.
Equ ilibr iu m Associa tion Con sta n ts of Hoech st 33258,
Hoech st 33342, a n d Iod oHoech st 33342. Calf thymus DNA
(Sigma Chemical Co.) was dissolved in phosphate-buffered
saline (PBS), pH 7.4, and the solution was filtered through a
0.22-µm filter. Based on the molecular weight (1.9 × 1012) and
the ꢀ/phosphate of 6490,19,20 the concentration of DNA was
calibrated to 50 µg/mL (2.63 × 10-14 M ≡ 1.6 × 10-4
M
phosphate) for an OD280 of 1 unit. From this stock solution,
dilutions were prepared ((7.9 × 10-7) - (6.312 × 10-5) M). To
cuvettes containing 2 mL each of these solutions was added
either Hoechst 33258, Hoechst 33342, or iodoHoechst 33342
in 25 µL of methanol to a final ligand concentration of 1.69 ×
10-7 M. Following mixing and a 5-min equilibration, the
fluorescence intensity (FI) in each cuvette was measured in a
thermostated (25 °C) fluorescence spectrophotometer (Perkin-
Elmer, LS-50B) at 335 nm excitation and 450 nm emission
with slit widths of 10 nm. The data were prepared as a
Scatchard plot (Figure 2) in the form of FI versus DNA
phosphate concentration by adapting the procedure of Pea-
cocke21 developed for UV absorption spectroscopy. Using the
ratio of fluorescence in the presence of excess DNA to that in
the absence of DNA (36.2 for Hoechst 33258, 14.63 for Hoechst
33342, and 14.85 for iodoHoechst 33342), the concentration
of unbound ligand was calculated.
Cellu la r Loca liza tion of Iod oHoech st 33342 in V79
Cells by F lu or escen ce Micr oscop y. V79 cells in monolay-
ers were grown in tissue culture dishes containing circular
cover slips in 5 mL of Dulbecco’s modified minimum essential
medium (DMEM; Gibco BRL, Grand Island, NY) supplemented
with 10% fetal calf serum, 2 mM L-glutamine, 50 000 units/L
penicillin-streptomycin, and 100 mM nonessential amino
acids. A solution of 0.1 mg/mL iodoHoechst was prepared in
DMEM, 100 µL of this solution was added, and the culture
dishes were incubated for 1 h in an incubator at 37 °C. The
medium was decanted, and the cells were washed three times
with 2 mL of cold PBS. The coverslips containing cells were
transferred to a DSC 200 Duorak-Stotler culture chamber