4′-Methyl Derivatives of 5-MOP and 5-MOA
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 22 4495
poured into 3 M HCl (500 mL) and left to stand overnight.
The resulting precipitate was filtered out, washed, and purified
by FC with 4:1 toluene/ethyl acetate as eluent to afford 11 (1.4
achieve a DNA/compound molar ratio of about 75. Aliquots
of these solutions were introduced into calibrated glass tubes,
immersed in a thermostatically controlled bath and then
irradiated for various periods of time. After irradiation, the
DNA was precipitated with NaCl and ethanol and washed with
ethanol 80%, and the pellets were dissolved in the initial
volume of buffer before radiochemical measurements were
performed.
1
g, 59%): mp 117-119 °C; H NMR (CDCl3) 7.84 (q, 1H, 4-H,
J ) 1.2), 6.69 (d, 1H, 6-H, J ) 1.9), 6.48 (d, 1H, 8-H, J ) 1.9),
3.90 (s, 3H, OMe), 2.62 (q, 2H, CH2, J ) 7.5), 2.19 (d, 3H, 3-Me,
J ) 1.2), 1.27 (t, 3H, CH3CH2, J ) 7.5); IR 2984, 2944, 1762,
1731, 1616, 1453, 1149, 1113, 1066, 822. Anal. (C14H14O5) C,
H.
In the experiments on bacterial DNA and synthetic poly-
nucleotides, buffer solutions of nucleic acids (1.5 × 10-4
were added to the labeled furocoumarins (1.85 × 10-5 M).
Irradiations were performed as above; after irradiation, the
solutions were extracted with a mixture of chloroform/isoamyl
alcohol (19:1) in order to remove the non-photobound drug and
other low molecular weight products. Afterward, the aqueous
solutions were used for radiochemical measurements.
Eva lu a tion of In ter str a n d Cr oss-Lin k s in Vitr o. After
irradiation in the presence of the tritiated furocoumarins, the
DNA solutions were thermally denatured (95 °C for 15 min)
and quickly cooled in ice. One milliliter of DNA solution was
introduced into the top of a column (0.7 cm × 4 cm) of
hydroxylapatite (Biogel type, Cat. N. 130-0420, Bio Rad
Laboratories, CA) developing with a linear gradient of a 0.05-
0.3 M phosphate buffer, pH ) 7.00. Fractions of 3.6 mL were
collected and the absorbance at 260 nm was recorded.
P r ep a r a tion of Ad d u cts. Two and half milligrams of the
furocoumarin were dissolved in 1 mL of ethanol, and this
solution was mixed with 15 mL of a solution of DNA (15 mg)
in Tris-NaCl 5 mM buffer pH 7.5. The mixture was irradiated
in a glass dish with four Philips HPW 125 lamps, arranged
two above and two below the dish, at a distance of 7 cm, for
2.5 h at room temperature. After irradiation, solid NaCl (up
to 1 M concentration) and 2 volumes of ethanol were added to
the solution; the precipitated DNA was collected on a glass
rod, washed with 80% ethanol, and dried under high vacuum.
In the case of compound 2, the pellet was dissolved in
hydrolytic buffer (CH3COONa 20 mM, NaCl 50 mM, ZnSO4 1
mM, MgCl2 10 mM, pH 4.6). Enzymatic digestion was
performed according to published procedures.28 After treat-
ment with alkaline phosphatase, the solution was filtered
through Millipore Teflon filters (2 µm) and directly injected
into a Perkin-Elmer Series 410 LC pump equipped with a LC
235 diode array detector, using a C18 column (Merck): the
eluent methanol/water (40:60 v/v) was used in an isocratic
mode for 20 min, followed by 20 min 100% methanol, always
at a flow rate of 1 mL min-1. The fractions were collected with
an automatic LKB apparatus: those corresponding to chro-
matographic peaks were submitted to spectrophotometric and
FAB analyses. Instead, DNA irradiated with compound 1 was
hydrolyzed in 0.5 N HCl at 100 °C for 1 h, neutralized, and
analyzed by HPLC using the same apparatus, as shown in
Figure 8. Larger DNA samples irradiated in the presence of
compounds 1 and 2 were hydrolyzed with HCl and extracted
with CHCl3, and the adducts were separated on TLC plates
and eluted with ethyl acetate/ethanol, 9:1.22
7-Hyd r oxy-5-m eth oxy-3-m eth ylcou m a r in (12). To cou-
marin 11 (1.7 g, 6.5 mmol) in methyl alcohol (150 mL) was
added 3 M HCl (75 mL), and the mixture was refluxed for 30
min. After cooling, the resulting precipitate was filtered out
to yield 1.25 g (93.5%) of 12: mp 260-261 °C; 1H NMR
(DMSO-d6) 10.30 (s, 1H, OH), 7.76 (q, 1H, 4-H, J ) 1.1), 6.32
(m, 2H, 6-H, 8-H), 3.85 (s, 3H, OMe), 2.02 (d, 3H, 3-Me, J )
1.2); IR 3346, 1685, 1597, 1260, 1192. Anal. (C11H10O4) C, H.
7-(Aceton yloxy)-5-m eth oxy-3-m eth ylcou m ar in (13). Pre-
pared from 12 as described for compound 8 to yield 88% of 13:
mp 148-151 °C. 1H NMR (CDCl3): 7.79 (q, 1H, 4-H, J ) 1.2),
6.38 (d,1H, 6-H, J ) 2.3), 6.29 (d, 1H, 8-H, J ) 2.3), 4.61 (s,
2H, CH2), 3.90 (s, 3H, OMe), 2.30 (s, 3H, MeCO), 2.17 (d, 3H,
3-Me, J ) 1.2). IR: 1706, 1626, 1205, 1162, 1117. Anal.
(C14H14O5) C, H.
M)
3,4′-Dim et h yl-5-m et h oxyfu r o[3,2-g]cou m a r in (3) a n d
3,4′-Dim eth yl-5-m eth oxyfu r o[2,3-h ]cou m a r in (4). Pre-
pared from 13 as described for compounds 1 and 2 to yield
66.5% of 4 and 22.5% of 3. Compound 3: mp 193-194 °C; 1H
NMR (CDCl3) 7.86 (q, 1H, 4-H, J ) 1.3), 7.36 (q, 1H, 5′-H, J )
1.4), 7.21 (s, 1H, 8-H), 3.98 (s, 3H, OMe), 2.40 (d, 3H, 4′-Me, J
) 1.4), 2.25 (d, 3H, 3-Me, J ) 1.3); IR 1712, 1618, 1139, 1090;
MS m/ z 244 (M+, 100), 229 (88), 201 ((M + 1)+ - CO2, 60),
115 (15). Anal. (C14H12O4) C, H. Compound 4: mp 219-220
1
°C; H NMR (CDCl3) 7.94 (q, 1H, 4-H, J ) 1.3), 7.29 (q, 1H,
5′-H, J ) 1.4), 6.79 (s, 1H, 6-H), 3.94 (s, 3H, OMe), 2.47 (d,
3H, 4′-Me, J ) 1.4), 2,21 (d, 3H, 3-Me, J ) 1.3); IR 1707, 1618,
1116, 1066; MS m/z 244 (M+, 100), 229 (43), 201 ((M + 1)+
CO2, 66), 115 (12). Anal. (C14H12O4) C, H.
-
5-MOA was synthesized according to the method Rodighiero
and Antonello (1955).15
UV spectra were recorded on a Perkin-Elmer model Lambda
5 spectrophotometer.
Nu cleic Acid s. Calf thymus DNA was purchased from
Sigma Chemical Co. (St. Louis, MO). Its hypochromicity,
determined according to Marmur and Doty,27 was over 35%.
DNA from Micrococcus lysodeikticus (Cat. D-8259) and Clostrid-
ium perfringens (Cat. D-1760), poly[dA-dT]*poly[dA-dT] (Cat.
P-0883) and poly[dG-dC]*poly[dG-dC] (Cat. P-9389) also came
from Sigma.
Ra d ioch em ica l Deter m in a tion s. Radioactivity measure-
ments were made by means of a Packard Model TRI-CARB
4000 liquid scintillation spectrometer: the efficiency of the
apparatus for counting tritium was within 35-40%.
All compounds were tritium-labeled by Amersham plc
(Amersham, Buckinghamshire, U.K.) and purified on thin
layer chromatography plates (silica gel plates Merck art. 5717,
2 mm). Plates were developed with CHCl3; purified products
showed a specific activity in the range 7.7-13.2 Ci/mol.
Equ ilibr iu m Dia lysis. Equilibrium dialysis experiments
were essentially performed following published procedures.28
To avoid binding of the drug to the membrane, all membranes
were previously saturated by putting them in a buffer solution
containing an excess of furocoumarin to reach equilibrium.
DNA concentrations expressed in phosphates ranged between
0.3 and 3 mM; initial drug concentrations were in the range
6.5-9.14 µM. In these conditions all compounds were soluble
in the buffer medium.
P h otor ever sa l of Ad d u cts. Fractions of the eluted HPLC
solutions were concentrated and irradiated in quartz cuvettes
with a mineral lamp (model UVGL 15, Ultra-Violet Products
Inc. San Gabriel, CA) placed at
a 1 cm distance. The
photosplitting reaction was followed spectrophotometrically;
the reverted solutions were concentrated and chromatographed
on silica gel plates (Merck 5715) developed with ethyl acetate/
ethanol, 8:2 (v/v).
Ma ss Sp ectr om etr y. Mass spectrometric measurements
were performed on a VG ZAB 2F double-focused, reverse-
geometry instrument, operating in FAB (Fast Atom Bombard-
ment) conditions. Glycerol solutions of the samples were
bombarded with 8 keV Xe atoms.
Ir r a d ia tion P r oced u r e. Irradiation were performed by
means of two Philips HPW 125 lamps equipped with a Philips
filter emitting over 90% at 365 nm; the DNA irradiation
intensity, determined by a potassium ferrioxalate chemical
P r od u ction of Sin glet Oxygen . The production of 1O2 by
various compounds (4.38 µM) was determined following the
spectrophotometric method proposed by Kraljic and El Mohs-
ni30 based on the bleaching of N,N-dimethyl-p-nitrosoaniline
(50 µM) by 1O2 in the presence of histidine (0.01 M) and
recording the absorbance changes at 440 nm.
actinometer,29 was 5.5 J s-1 m-2
.
P h otobin d in g to DNA in Vitr o. Small measured volumes
of concentrated ethanol solutions of the labeled compounds
were added to aqueous 0.05% calf thymus DNA (containing
10 mM TRIS, 10 mM NaCl, and 0.5 mM EDTA, pH ) 7) to
P h otobiologica l Meth od s. Cell Cu ltu r es. Ma ter ia ls.
HL60, HeLa, and A431 cells were grown respectively in RPMI