[125I/127I]iodoHoechst 33342: synthesis, DNA binding, and biodistribution.

Article abstract of DOI:10.1021/jm9602672

An iodinated analog of the DNA-minor-groove-binding agent Hoechst 33342 has been synthesized and evaluated for DNA binding and tumor targeting. The bis-benzimidazole ring system of the title compound was constructed from the piperazinyl terminus via a Pinner-type cyclization followed by oxidative cyclization of the diamine Schiff base. To synthesize radioiodoHoechst 33342, (trimethylstannyl)Hoechst 33342 was prepared by the same strategy and subjected to mild radioiododestannylation in the presence of lactoperoxidase. After purification by HPLC, the radiochemical was separated in carrier-free form with > 85% radiochemical yield and > 99% chemical and radiochemical purity. Fluorescence spectrometric analysis of the binding of iodoHoechst 33342 to calf thymus DNA gave an equilibrium association constant (Ka) of 2.57 x 10(7) M-1 comparable to the Ka value of Hoechst 33342. Fluorescence microscopy of viable V79 cells demonstrated that the iodinated dye stained the nuclei with avidity similar to that of the noniodinated dye. The biodistribution of [125I]-iodoHoechst 33342 in LS174T tumor-bearing athymic mice 4 h postadministration showed a tumor uptake of 3-4% injected dose per gram (ID/g), tumor/blood ratio of 6-8, and tumor/ nontumor ratios above unity for most organs. A low thyroid uptake (approximately 2% ID/g) indicated that the radiochemical did not deiodinate and was stable in vivo.

Full text of DOI:10.1021/jm9602672

4804
J . Med. Chem. 1996, 39, 4804-4809
[
125I/127I]Iod oHoech st 33342: Syn th esis, DNA Bin d in g, a n d Biod istr ibu tion
Ravi S. Harapanhalli, Larry W. McLaughlin,† Roger W. Howell,‡ Dandamudi V. Rao,‡ S. J ames Adelstein, and
Amin I. Kassis*
Department of Radiology (Nuclear Medicine), Harvard Medical School, Boston, Massachusetts 02115, Department of
Chemistry, Merkert Chemistry Center, Boston College, Chestnut Hill, Massachusetts 02167, and Department of Radiology,
Division of Radiation Research, UMDNJ -New J ersey Medical School, Newark, New J ersey 07103
Received April 4, 1996X
An iodinated analog of the DNA-minor-groove-binding agent Hoechst 33342 has been
synthesized and evaluated for DNA binding and tumor targeting. The bis-benzimidazole ring
system of the title compound was constructed from the piperazinyl terminus via a Pinner-type
cyclization followed by oxidative cyclization of the diamine Schiff base. To synthesize
radioiodoHoechst 33342, (trimethylstannyl)Hoechst 33342 was prepared by the same strategy
and subjected to mild radioiododestannylation in the presence of lactoperoxidase. After
purification by HPLC, the radiochemical was separated in carrier-free form with >85%
radiochemical yield and >99% chemical and radiochemical purity. Fluorescence spectrometric
analysis of the binding of iodoHoechst 33342 to calf thymus DNA gave an equilibrium
association constant (Ka) of 2.57 × 107 M-1 comparable to the Ka value of Hoechst 33342.
Fluorescence microscopy of viable V79 cells demonstrated that the iodinated dye stained the
nuclei with avidity similar to that of the noniodinated dye. The biodistribution of [125I]-
iodoHoechst 33342 in LS174T tumor-bearing athymic mice 4 h postadministration showed a
tumor uptake of 3-4% injected dose per gram (ID/g), tumor/blood ratio of 6-8, and tumor/
nontumor ratios above unity for most organs. A low thyroid uptake (∼2% ID/g) indicated that
the radiochemical did not deiodinate and was stable in vivo.
In tr od u ction
breaks in the cells treated with Hoechst 33342 but not
in those treated with Hoechst 33258. Hence, although
the two dyes are structurally similar, they differ sig-
nificantly in their biologic properties.
The bis-benzimidazole dye Hoechst 33342, 2-[2-(4-
ethoxyphenyl)-6-benzimidazolyl]-6-(1-methyl-4-pipera-
zinyl)benzimidazole (1) (Scheme 1), is a DNA-binding,
fluorescent stain used in flow cytometry studies to
quantify DNA content in live cells1 and to differentiate
cells at various locations in multicell spheroids2 and is
also used as a therapeutic agent for solid tumors.3-5 The
minor groove of the B-form of DNA offers ideal sites for
the binding of Hoechst dyes as well as other ligands
such as netropsin,6,7 distamycin,8 and CC-1065,9 and
often such binding occurs preferentially at dAdT-rich
sequences. Chen et al.10 have demonstrated that
Hoechst 33342 (1) and its parent compound Hoechst
33258 (2) specifically interrupt the breakage/reunion
reactions of mammalian DNA topoisomerase I and
nonspecifically inhibit the catalytic activities of many
DNA enzymes. Furthermore, Durand and Olive2 have
shown that Hoechst 33342 is superior to Hoechst 33258
in staining the nuclei of viable cells. Recent investiga-
tions by Denison et al.11 have confirmed these findings
and demonstrated that the uptake of Hoechst 33342 is
nearly 3-fold greater than that of its parent Hoechst
33258 under comparable conditions and that its efflux
from cells is very slow. In their study Chen et al.10
found that Hoechst 33342 possessed enhanced mem-
brane permeability and was about 2 orders of magnitude
more cytotoxic than Hoechst 33258 which they ascribed
to the ethoxy substitution on the 4-phenyl ring of the
parent compound. These authors also detected a large
number of protein-DNA cross-links and DNA strand
Hoechst 33258 (pibenzimol) has shown antitumor
activity in vivo against intraperitoneal murine L1210
and P388 leukemia and is undergoing preclinical evalu-
ation by the National Cancer Institute.12 Martin et
al.13,14 reported its radioiodination with sodium [125I]-
iodide by direct electrophilic reaction and utilized the
radiochemical in studying the radiobiologic effects of the
decaying radioiodine in the minor groove of plasmid
DNA. Our studies, however, have indicated that as a
consequence of the presence of the o-iodophenol group-
ing, 125I-labeled Hoechst 33258 is prone to excessive
deiodination both in vitro and in vivo (unpublished
data). This observation and the superior cellular uptake
and DNA binding of Hoechst 33342 prompted us to
synthesize iodoHoechst 33342 in radioactive as well as
stable form. We report herein the synthesis of 127I-
labeled iodoHoechst 33342 (3) and (trimethylstannyl)-
Hoechst 33342 (4) from the piperazinyl terminus and
the no-carrier-added synthesis of radioiodoHoechst 33342
(
125I/131I) by destannylation of the tin precursor. Due
to the absence of phenolic activation of its aromatic ring,
Hoechst 33342 could not be iodinated by direct electro-
philic reaction. DNA binding, cellular localization, and
biodistribution of [125I]iodoHoechst 33342 in tumor-
bearing nude mice have been examined.
Resu lts a n d Discu ssion
Loewe et al.15 reported the first synthesis of bis-
benzimidazole Hoechst 33258 and a wide variety of its
congeners. The synthetic strategy consisted of construc-
tion of the bis-benzimidazole ring system from the
piperazinyl terminus via two consecutive Pinner-type
* Address for correspondence: Shields Warren Radiation Labora-
tory, 50 Binney St, Boston, MA 02115.
† Boston College.
‡ UMDNJ -New J ersey Medical School.
X Abstract published in Advance ACS Abstracts, November 1, 1996.
S0022-2623(96)00267-1 CCC: $12.00 © 1996 American Chemical Society

[
125I/ 127I]IodoHoechst 33342
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 24 4805
Sch em e 1. Synthesis of Bis-benzimidazoles by
Pinner-Type Cyclization Followed by Oxidative
Cyclization of Diamine Schiff Basea
Sch em e 2. Synthesis of Substituted Benzaldehydes for
Construction of the Second Imidazole Ringa
a
(i) Acetone/anhydrous K2CO3, stirring, room temperature, 30
h; (ii) ICl, CH2Cl2, room temperature, stirring, 17 h; (iii) (Me3Sn)2,
dioxane, (Ph3P)4Pd(0), reflux, 1 h.
in 63% yield, was reduced further to diamine 15 under
Parr hydrogenation conditions.
The second imidazole ring was constructed by heating
the diamine 15 with appropriately functionalized benz-
aldehyde (Scheme 2) in a mixture of nitrobenzene and
toluene at elevated temperature (95-100 °C) to afford
the bis-benzimidazole 1. In their 1H NMR spectra,
iodoHoechst and (trimethylstannyl)Hoechst 33342 dis-
played a total of nine aromatic protons and two highly
deshielded imidazole protons (∼δ ) 13 ppm) in addition
to the other expected signals. The (trimethylstannyl)-
Hoechst 33342 also displayed the most shielded signal
due to the trimethylstannyl group at δ ) 0.4 ppm with
the characteristic tin satellites 55 Hz apart integrating
for nine protons. Ethoxybenzaldehyde 17 was obtained
by the Williamson reaction of 16 with ethyl iodide in
acetone and anhydrous potassium carbonate (Scheme
2). The iodination of 16 with iodine monochloride in
dichloromethane afforded the monoiodo aldehyde 18
(75% yield), which was converted to ethoxy aldehyde 19
in 90% yield. Trimethylstannyl aldehyde 20, synthe-
sized from 19 using the tin exchange procedure of
Azizian et al.,16 was obtained as white crystals. The
crucial step in this synthetic methodology, however, was
to radioiodinate the tin precursor 4 via a radioiodine-
tin exchange reaction. Based on the successful iodo-
destannylation using stable iodine, radioiododestannyl-
ation of the tin precursor was carried out using sodium
a
(i) Anhydrous K2CO3, DMF, reflux, 4.5 h; (ii) H2, 10% Pd/C,
room temperature, 1.25 h; (iii) (CF3CO)2O, Py, -20 °C, 0.5 h; (iv)
10% ethanol-ether, dry HCl, -20 °C, 2 h; (v) acetic acid, 55 °C,
stirring, 12 h; (vi) nitrobenzene-toluene (4:1), 60 °C, 0.5 h; then
98-100 °C, 24-36 h.
cyclizations. In the present approach (Scheme 1), we
adapted the above procedure to construct the monoim-
idazole ring system with some improvements and
prepared the diamine 15. In brief, treatment of 5-chloro-
2-nitroaniline (7) with 1-methylpiperazine (6) in the
presence of anhydrous potassium carbonate in DMF
afforded the required piperazinylaniline 8 in nearly 60%
yield together with a byproduct, 5-(dimethylamino)-2-
nitroaniline (9). The required diamine 10 was obtained
by the catalytic hydrogenation of 8 using palladized
carbon in a Parr reactor at ambient temperature. In
our modified synthesis of ethyl benzimidate 13, amino
nitrile 11 was converted to its trifluoroacetyl derivative
12 by trifluoroacetic anhydride treatment and reacted
with hydrogen chloride to obtain ethyl benzimidate 13
in 80% yield. The latter (13) was condensed with
diamine 10 in acetic acid and the imidazole 14, obtained
[
125I]/[131I]iodide and lactoperoxidase in the presence of
small amounts of hydrogen peroxide. A representative
radio/UV HPLC profile of the radioiodination mixture
spiked with iodoHoechst 33342 (Figure 1) shows that
nearly 90% of the radioactivity eluted at a retention
time of 45 min, corresponding to the peak of iodoHoechst
33342, in chemically and radiochemically pure form.
Fluorescence spectroscopy was used to determine the
equilibrium association constant (Ka) of the binding of
iodoHoechst 33342 and parent dyes to calf thymus DNA.
Whereas the fluorescence enhancement for the two
noniodinated bis-benzimidazoles reached the maximum
of nearly 900 units following the addition of increasing

4806 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 24
Harapanhalli et al.
Ta ble 1. Summary of Binding Studies
Ka
nucleotide/
ligand
chemical
(× 107 M-1
)
na
Hoechst 33258
Hoechst 33342
iodoHoechst 33342
2.64
2.84
2.57
0.0275
0.0282
0.0280
36
36
36
a
Number of ligands per nucleotide phosphate.
F igu r e 1. Radio/UV HPLC profile of the radioiododestannyl-
ation reaction mixture in the synthesis of [125I]iodoHoechst
33342 spiked with stable iodoHoechst 33342: (top) UV profile
and (bottom) radioactivity profile. Note the difference in
retention times of the two chemicals. Lactoperoxidase and
buffer salts eluted out in void volume.
F igu r e 3. Top: Fluorescence micrograph of viable V79 cells
stained with [127I]iodoHoechst 33342. Brightly lit areas in the
center are nuclei stained with dye. Bottom: Photomicrograph
of fluorescence image processed and converted by Spex cation
measurement system indicating that cytoplasm is devoid of
dye.
F igu r e 2. Scatchard plot of binding data for iodoHoechst
33342 and calf thymus DNA obtained from figure shown in
insert. Data were plotted as ratio of bound/free (abscissa)
versus bound fraction (ordinate) and yielded straight line with
slope ()association constant, Ka) of 2.57 × 107 M-1. Insert:
Fluorescence titration curves for bis-benzimidazoles with
increasing calf thymus DNA concentration in phosphate-
buffered saline (pH 7.4). Fluorescence maximum for all dyes
was achieved at a DNA concentration of 20 µM. Note that
enhancement in fluorescence for noniodinated dyes was identi-
cal and nearly 3 times that observed for iodoHoechst 33342:
(O) iodoHoechst 33342, (b) Hoechst 33342, and (3) Hoechst
33258. Ligand concentration was 1.69 × 10-7 M.
the DNA minor groove. The fluorescence microscopy
data (Figure 3) of viable V79 cells stained with iodo-
Hoechst 33342 demonstrated cellular uptake and local-
ization within the nucleus. Together with the DNA-
binding data, these results indicate that iodoHoechst
33342 permeates live mammalian cells and binds avidly
to their DNA. RadioiodoHoechst 33342 may, therefore,
play an important role in examining the radiobiologic
effects of radioiodine bound to the DNA minor groove.
Our results with plasmid-bound 125I-labeled iodoHoechst
33342, in which the effects of Auger electron emissions
from within the minor groove have been examined, may
be found elsewhere.17
Prompted by the reported antitumor activity of the
parent bis-benzimidazole Hoechst 33258 (pibenzimol)
against intraperitoneal murine L1210 and P388 leuke-
mia,12 the tumor-targeting potential of [125I]iodoHoechst
33342 was assessed in LS174T tumor-bearing athymic
mice 4 h after its administration (Figure 4), at which
point this molecule would be expected to have cleared
from the systemic circulation. Our results corroborate
this expectation (0.5% ID/g in blood) and indicate that
the greatest amount of the injected radioactivity was
taken up by the kidneys (22% ID/g) followed by the liver
amounts of DNA (Figure 2, insert), the corresponding
increase for the iodoHoechst 33342 was only 240 units,
although the saturation concentration of DNA was the
same (20 mM). The low fluorescence quantum yield
(∼one-third) observed for iodoHoechst 33342 compared
to noniodinated Hoechst 33258 and Hoechst 33342
appears to be due to the quenching effects of the iodine
atom present on this molecule. A plot of bound/free
ratio of ligand (abscissa) versus bound ligand (ordinate)
furnished a straight line with a slope equal to -Ka,
indicating that this ligand binds to calf thymus DNA
by only one mode of binding interaction (Figure 2). The
results summarized in Table 1 show that the Ka for all
three ligands is similar (2.6-2.8 × 107 M-1); the
presence of the bulky iodine on Hoechst 33342 does not
affect the strength of binding of this molecule within

[
125I/ 127I]IodoHoechst 33342
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 24 4807
X-ray film, and autoradiographs were developed for the
visualization of radioactivity.
2-Nitr o-5-(1-m eth yl-4-pip er a zin yl)a n ilin e (8). This com-
pound was prepared per Loewe et al.:15 analysis by silica gel
TLC plates revealed a single spot, Rf ) 0.48; mp 153-154 °C
(lit.15 mp 155 °C); 1H NMR (DMSO-d6) δ 2.18 (s, 3 H, N-CH3),
2.375 (m, 4 H, piperazinyl methylenes), 3.26 (m, 4 H, piper-
azinyl methylenes), 6.18 (d, J ) 3 Hz, 1 H, H-6), 6.38 (dd, J )
12, 3 Hz, 1 H, H-4), 7.24 (bs, 2 H, -NH2), 7.78 (d, J ) 12 Hz,
1 H, H-3). Anal. (C11H16N4O2) C, H, N.
A residue from acetic acid treatment was purified on a
preparative TLC run in 0.5% methanol in dichloromethane,
1
and 9 was isolated: mp 105-107 °C; H NMR (DMSO-d6) δ
4.50 (s, 3 H, N-CH3), 4.59 (s, 3 H, N-CH3), 7.65-7.70 (m, 4 H,
H-4, H-6, -NH2), 9.50 (d, J ) 12 Hz, 1 H, H-3).
5-(1-Meth yl-4-p ip er a zin yl)-1,2-d ia m in oben zen e (10). A
solution of 8 in methanol (4 g, 17 mmol) was hydrogenated in
a Parr reactor. The dry gray product (3.95 g) was crystallized
in aqueous ethanol: mp 65-67 °C; 1H NMR (DMSO-d6) δ 2.18
(s, 3 H, N-CH3), 2.38 (m, 4 H, piperazinyl methylenes), 2.83
(m, 4 H, piperazinyl methylenes), 3.96 (bs, 2 H, -NH2), 4.38
(bs, 2 H, -NH2), 6.02 (dd, J ) 3, 12 Hz, 1 H, H-5), 6.21 (d, J )
3 Hz, 1 H, H-3), 6.40 (d, J ) 12 Hz, 1 H, H-6). Anal.
(C11H18N4) C, H, N.
F igu r e 4. Biodistribution (% ID/g) of [125I]iodoHoechst 33342
in LS174T tumor-bearing athymic mice following intravenous
injection via lateral tail vein: liver (LV), spleen (SP), kidney
(K), small intestine (SI), large intestine (LI), stomach (ST),
lungs (LU), muscle (MS), skeleton (SK), heart (H), skin (SN),
tumor (TU), bladder (BA), neck contents (NK), blood (BL),
urine (U), and brain (BR).
4-(Tr iflu or oa ceta m id o)-3-n itr oben zon itr ile (12). To a
stirred and chilled (-20 °C) solution of amino nitrile 11 in 50
mL of dry dichloromethane (2.283 g, 14 mmol) under an argon
atmosphere was added 3.54 mL of dry pyridine (42 mmol)
followed by 4.487 mL of trifluoroacetic anhydride (31 mmol)
added dropwise over 10 min. The solution was stirred for 30
min and the reaction quenched by addition of 1.3 mL of
methanol (31 mmol) while still at -20 °C. The reaction
mixture was allowed to warm to room temperature, and the
solvent was evaporated to dryness. The residue was stirred
with 50 mL of dry diethyl ether and filtered, and the pyri-
dinium-trifluoroacetate-containing residue thoroughly washed
with ether. The combined filtrate was evaporated and then
coevaporated with toluene to remove all pyridine. The pale
fluffy product (yield 2.1 g) was crystallized in benzene-hexane,
and colorless needles were obtained, mp 96-98 °C.
Eth yl 4-(Tr iflu or oa ceta m id o)-3-n itr oben zim id a te Hy-
d r och lor id e (13). To a solution of the trifluoro derivative
12 in 50 mL of anhydrous ether (3 g, 105 mmol) were added
10 mL of dry ethanol and 4A molecular sieves (15-20).
Through this solution at -20 °C was bubbled dry hydrogen
chloride gently until saturation (2 h), and the solution was
kept in the freezer overnight. The crystallized imidate 13 was
filtered and thoroughly washed with chilled ether followed by
hexane and dried in vacuo (2.75 g yield). The crude amidate
was used as such for the cyclization reaction described below:
1H NMR δ 1.5 (t, J ) 6 Hz, 3 H, -CH3), 4.65 (q, J ) 6 Hz, 2 H,
-CH2-), 7.95 (d, J ) 12 Hz, 1 H, H-5), 8.08 (dd, J ) 3, 12 Hz,
1 H, H-6), 8.78 (d, J ) 3 Hz, 1 H, H-2).
2-(4-Am in o-3-n itr op h en yl)-6-(1-m eth yl-4-p ip er a zin yl)-
ben zim id a zole (14). Acetic acid (20 mL) in an RB flask was
purged with dry argon for 15 min. The diamine 10 (1.01 g,
4.9 mmol) was added followed by the imidate 13 (1.46 g, 4.68
mmol). The stirred solution was heated to 55 °C in an oil bath
for 12 h with intermittent addition of 0.55 g of imidate 13 in
three portions. Acetic acid was evaporated, the crude material
was purified by ammonia-acetic acid treatment, and the
bright-red nitro amine 14 was filtered and washed thoroughly
with 2% methanolic ether followed by ether and dried in vacuo
(yield 1.171 g): TLC Rf ) 0.35; mp 182-183 °C (lit.15 mp 183-
185 °C); 1H NMR δ 2.2 (s, 3 H, N-CH3), 2.42 (m, 4 H,
piperazinyl methylenes), 3.15 (m, 4 H, piperazinyl methylenes),
6.9 (d, J ) 12, 1 H, H-6), 7.01 (dd, J ) 12, 14 Hz, 1 H, H-7′),
7.15 (d, J ) 12 Hz, 1 H, H-6′), 7.38 (dd, J ) 12, 16 Hz, 1 H,
H-5), 7.78 (bs, 2 H, D2O exchangeable, -NH2), 8.15 (d, J ) 12
Hz, 1 H, H-4′), 8.75 (d, J ) 12 Hz, 1 H, H-3), 13.01 (bs, 1 H,
imidazole N-H). Anal. (C18H20N6O2‚H2O) C, H, N.
and spleen (∼7% each), while the subcutaneous tumor
had 3-4% ID/g. This led to tumor-to-blood ratios of 6-8
and a tumor-to-muscle ratio of ∼10. The tumor-to-
nontumor ratios for most of the organs remained above
1 except for kidneys, liver, and spleen. The radiochemi-
cal did not cross the blood-brain barrier (brain uptake
0.04%). Interestingly the activity in neck contents was
only ∼2% ID/g, reflecting the in vivo stability of this
radiochemical due to the o-iodoethoxy moiety. If this
chemical can be conjugated to a suitable carrier such
as an antibody, it may be targeted to tumors effectively.
In view of the fact that the parent Hoechst 33258 is
already undergoing clinical trials,12 it is worthwhile to
examine the antitumor activity of the newly synthesized
iodoHoechst 33342. The pursuit of such studies is also
justified since the substitution by stable iodine (127I) may
sometimes lead to improvements in the biologic action
of certain drugs, as exemplified by 4′-iodo-4′-deoxydoxo-
rubicin, a chemical that has shown greater anticancer
activity and less cardiac toxicity than doxorubicin.18
Exp er im en ta l Section
The reagents and chemicals were obtained from Aldrich
Chemical Co. (Milwaukee, WI). Calf thymus DNA and lac-
toperoxidase (EC 1.11.1.7) were purchased from Sigma Chemi-
cal Co. (St. Louis, MO). Carrier-free sodium [125I]iodide (0.63
TBq/mg) and sodium [131I]iodide (0.44 TBq/mg) were obtained
from Amersham Corp. (Arlington Heights, IL). Elemental
analyses were carried out by Atlantic Microlabs (Atlanta, GA).
Melting points were determined on a Fisher-J ohns melting
point apparatus (Pittsburgh, PA) and are uncorrected. NMR
spectra were obtained on either a Varian XL-300 or a Varian
XL-500 instrument. Electron impact (EI) and direct chemical
ionization fast atom bombardment (DCI/FAB; H) mass spectra
were recorded on a ZAB 7070 mass spectrometer at the
University of Illinois, Urbana Campaigna. The UV spectra
were recorded on a Hitachi UV/vis spectrophotometer (Lambda
3B), and fluorescence spectra were obtained from a Perkin-
Elmer fluorescence spectrophotometer (LS-50B). TLC analy-
ses were carried out on either silica gel plates (Baker-flex, IB2-
F; J . T. Baker Inc., Phillipsburg, NJ ) or reversed phase TLC
plates (Uniplate RPSF; Analtech, Newark, DE). Silica gel
plates were run in a mobile phase consisting of methanol:
ethanol:tri-n-butylamine (20:10:0.3), whereas the reversed
phase plates were run in 40% methanol in aqueous ammonia
(2.2%). The plates were examined under UV and exposed to
2-(3,4-Dia m in op h e n yl)-6-(1-m e t h yl-4-p ip e r a zin yl)-
ben zim id a zole (15). The nitro amine 14 (1.1 g) was reduced
as above, and a dark gray solid (15) was obtained: mp 264-
266 °C (lit.15 mp 268 °C); 1H NMR δ 2.22 (s, 3 H, N-CH3),

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

[
125I/ 127I]IodoHoechst 33342
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 24 4809
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(Nicholson Precision Instruments, Gaithersburg, MD) mounted
on a Nikon Diaphot fluorescence microscope.
Biod istr ibu tion of [125I]Iod oHoech st 33342 in LS174T
Tu m or -Bea r in g Ath ym ic Mice. Human colonic adenocar-
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tained as monolayers in DMEM. To provide single-cell
suspensions for injection, the cells were trypsinized, gently
pipetted, and repeatedly passed through a syringe with a 22-
gauge needle. Athymic Nu/Nu female mice (4-5 weeks old;
Harlan Sprague-Dawley, Indianapolis, IN) were injected
subcutaneously in the flank with 106 cells/0.1 mL of PBS/
mouse. Each mouse received an intravenous injection (lateral
tail vain) of 5 µCi of carrier-free radioiodoHoechst 33342 in
0.1 mL of PBS. The mice were sacrificed at 4 h, and organs
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Ack n ow led gm en t. We wish to thank S. B. Rajur,
Ph.D., for recording some of the NMR spectra and
Burton Fine, M.D., and Kathleen Hansen, B.S., for help
with the fluorescence micrographs of V79 cells. R.S.H.
is the recipient of an institutional NRSA fellowship. This
work has been supported in part by NIH grants CA
15523 (A.I.K. and S.J .A.), CA 32877 (D.V.R.), CA 54891
(R.W.H.), and GM 32065 (L.W.M.).
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