Notes
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 7 1543
instrument. 1H NMR spectra were obtained on a Br u¨ ker
AM500 instrument with multinuclear capability. UV spectra
were obtained with Pye-Unicam SP1750 or Hitachi U2000
spectrophotometers. HPLC analysis was carried out with a
Shimadzu HPLC system equipped with an SPD 10AV dual-
wavelength photometer set for 210 nm (to detect the BSH
component) and at 309 nm (to detect nitroimidazole compo-
nent). Analytical HPLC was carried out with a Millipore 8 ×
cedure and apparatus are described elsewhere.23 Microfuge
tubes were positioned in a lucite rotator and irradiated for
different times with thermal neutrons from the BMRR at a
reactor power of 1 Megawatt (MW). Since the length of time
of neutron exposure and the thermal neutron fluence are both
proportional to dose, cell survival curves are presented in
megawatt minutes (reactor power of 1 MW × 1 min neutron
exposure ) 1 MW min) to simplify the complexities associated
with determining the “effective dose” from a mixed radiation
field.24 Experiments were carried out under euoxic conditions.
Cells were plated for clonogenic determination after irradiation
and permitted undisturbed growth for 5 days prior to counting
colonies.22 Measurements of boron uptake in cells were carried
out using the prompt γ facility of the BMRR as described
elsewhere.25 Curves fit the linear-quadratic (LQ) and single-
hit multitarget (SHMT) models equally well. Software for
analysis has previously been described.26
1
0 RCM C18 cartridge eluted at 4 mL/min with 30% methanol
in water containing 50 mM triethylammonium acetate, pH 6.
Semipreparative HPLC was carried out with a 25 × 10 RCM
C18 cartridge eluted with the same solvent at 4 mL/min.
Elemental analysis was performed by M-H-W Laboratories,
Phoenix, AZ, and were (0.4% of the calculated composition.
Ca u tion : 1,2-Dibromoethane is a potential carcinogen and
mutagen and should be handled under appropriate contain-
ment. The toxicities of the other compounds to humans is
unknown. 1-(2-Bromoethyl)-2-methyl-5-nitroimidazole (2).
Ack n ow led gm en t. This work was supported by the
Department of Energy under Grant No. DE-FG03-
90ER81011 (SBIR Phase I to D.H.S.) and under DE-
AC02-76CH00016 (B.H.L.).
2
-Methyl-5-nitroimidazole (5 g, 0.04 mol; Aldrich Chemical Co.)
was dissolved in 100 mL of N,N-dimethylformamide and
refluxed with 1,2-dibromoethane (100 mL, 1.3 mol; Aldrich)
for 4 h. After removal of solvent by rotary evaporation, the
crude mixture was fractionated on a 4- × 50-cm column of Si60
eluted with 5% methanol in dichloromethane. The band
eluting first from the column was primarily 2 with some
contamination by the major product, the 4-nitro isomer. The
solvent was removed by evaporation, and the crude 2 was
dissolved in 50% methanol (20 mL). A 5-mL aliquot was
purified by rechromatography on C-18 with 40% methanol.
Evaporation of solvent and refrigeration of the residual oil gave
pure 2 as yellow crystals (1.4 mmol, 14%): analytical HPLC
Refer en ces
(
1) Barth, R. F.; Soloway, A. H.; Fairchild R. G. Boron Neutron
Capture Therapy of Cancer. Cancer Res. 1990, 50, 1061-70.
2) Gabel, D. Present Status and Perspectives of Boron Neutron
Capture Therapy. Radiother. Oncol. 1994, 30, 199-205.
(
(3) Elliot, T. S.; Stone, J . W. Review Article: Metronidazole and the
Anaerobic Gut Flora. Aliment Pharmacol. Ther. 1990, 4, 227-
3
8.
(
4) Lau, N. P.; Piscitelli, S. C.; Wilkes L.; Danziger L. H. Clinical
Pharmacokinetics of Metronidazole and Other Nitroimidazole
Anti-Infectives. Clin. Pharmacokinet. 1992, 23, 328-64.
showed 97.5% purity; UV (methanol) λmax ) 309 nm, ꢀ ) 8900;
1
H NMR (400 MHz, DMSO-d ) δ 2.48 (s, 3 H), δ 3.83 (t, J ) 5
6
Hz, 2 H), δ 4.70 (t, J ) 5 Hz, 2 H), δ 8.05 (s, 1 H); EIMS m/ e
(
(
(5) Teicher, B. A.; Herman, T. S.; Holden, S. A.; J ones, S. M.
Addition of Misonidazole, Etanidazole, or Hyperthermia to
Treatment with Fluosol-DA/Carbogen/Radiation. J . Natl. Cancer
Inst. 1989, 81, 929-34.
+
relative intensity) 233, 235 (M , 88, 91), 187, 189 (100), 154
38), 107, 109 (96, 94).
Bis(tr ieth ylam m on iu m ) 1-[2-[(u n decah ydr o-closo-dode-
(
6) Born, J . L.; Smith, B. R.; Harper, N.; Koch, C. J . Metabolism
and Radiosensitization of 4,5-Dimethylmisonidazole, a Ring-
Substituted Analog of Misonidazole. Biochem. Pharmacol. 1992,
43, 1337-44.
cabor ato)th io]eth yl]-2-m eth yl-5-n itr oim idazole (3). Com-
pound 2 (117 mg, 0.5 mmol) was dissolved in 5 mL of argon-
purged 50% aqueous dimethylformamide. Cs
1
0
2 12
B H11SH
(
7) Adams, G. E.; Stratford, I. J . Hypoxia-Mediated Nitro-Hetero-
cyclic Drugs in the Radio- and Chemotherapy of Cancer. Bio-
chem. Pharmacol. 1986, 35, 71-76.
(BSH, 237 mg, 0.55 mmol; Boron Biologicals, Inc., Raleigh, NC,
1
0
>
95% B enriched) was dissolved in 5 mL of the same solvent,
and the two solutions were mixed. Na
2
CO (70 mg) was added,
3
(
8) Workman, P. Pharmacokinetics of Radiosensitizing Agents. In
Pharmacokinetics of Anticancer Drugs in Humans; Ames, M. M.,
Powis, G., & Kovach, J . S., Eds.; Elsevier Science Publishers B.
V.: Amsterdam, 1983; pp 291-361.
and the reaction was stirred at 50 °C. The course of the
reaction was followed by analytical HPLC with product 3 well
separated from BSH and 2, showing retention times of 3.36,
(
9) Kedderis, G. L.; Argenbright, L. S.; Miwa G. T., Covalent
Interaction of 5-Nitroimidazoles with DNA and Protein in
vitro: Mechanism of Reductive Activation. Chem. Res. Toxicol.
1989, 2, 146-9.
4
.74, and 7.02 min, respectively. After 7 h, the reaction
mixture was evaporated to dryness and resuspended in water
10 mL), and 6 mL was fractionated on the semipreparative
column by sequential runs using 2-mL injections. Product 3
) 28.6 min) was well separated from BSH (t ) 38 min)
and 2 (t ) 50.5 min). After lyophilization, pure 3 as the bis-
triethylammonium) salt was isolated as an amorphous yellow
(
(10) Lord, E. M.; Harwell, L.; Koch, C. J . Detection of Hypoxic Cells
by Monoclonal Antibody Recognizing 2-Nitroimidazole Adducts.
Cancer Res. 1993, 53, 5721-6.
(t
R
R
R
(
11) Alvaro, R. F.; Wislocki, P. G.; Miwa, G. T.; Lu, A. Y. Drug Residue
Formation From Ronidazole, a 5-Nitroimidazole. VIII. Identifica-
tion of the 2-Methylene Position as a Site of Protein Alkylation.
Chem. Biol. Interact. 1992, 82, 21-30.
(
powder (69.9 mg, 43%): mp 128-130 °C dec; UV (H
3
NMR (as Na salt from ion-exchange resin, 500 MHz, DMSO-
d
2
O) λmax
)
H
1
20 nm, ꢀ ) 5950, (methanol) λmax ) 311 nm, ꢀ ) 6200;
+
(12) Webster, L. T. Drugs Used in the Chemotherapy of Protozoal
Infections. In The Pharmacological Basis of Therapeutics, 8th
ed.; Gilman, A., Rall, T. W., Nies, A. S., Taylor, P., Eds.;
Pergamon Press: New York, 1990; pp 999-1007.
6
) δ 0.4-1.4 (m, 11 H), 2.54 (s, 3 H), 2.57 (t, J ) 6.6 Hz, 2 H),
4
.3 (t, J ) 6.6 Hz, 2 H), 7.97 (s, 1 H), 10B-decoupling collapsed
the broad multiplet (0.4-1.4 ppm) to three singlets at 0.73 (1
H), 0.87 (5 H), and 1.08 (5 H), the NMR of the triethylammo-
nium salt was dominated by the ethyl protons, which obscured
the B-H protons, but was otherwise the same as that of the
(
13) Scobie, M.; Mahon, M. F.; Threadgill, M. D. Tumour-Targetted
Boranes. Part 2. Coupling of closo-Carboranes to Substituted
2-Nitroimidazoles via 1,3-Dipolar Cycloaddition. J . Chem. Soc.,
Perkin Trans. 1 1994, 203-210.
+
+
(14) Scobie, M.; Threadgill, M. D. Tumour-Targetted Boranes. Part
. Synthesis of Carbamate-linked Nitroimidazolyl Carboranes
Na salt; electrospray mass spectrometry m/ e 522 (MH ), 544
3
+
(MNa ) showed retention of the triethylammonium counteri-
Designed for Boron Neutron Capture Thearpy of Cancer. J .
Chem. Soc., Perkin Trans. 1 1994, 2059-2063
1
0
ons. Anal. ( B12
18 51 5 2
C H N O S) C, H, N.
In Vitr o BNCT Stu d ies. Details of the cell culture tech-
(15) Wilbur, D.; Hamlin, D.; Livesey, J .; Srivastava, R.; Laramore,
G.; Griffin, T. Preparation of Meta-carboranylpropyl-2-nitroimi-
dazole Derivatives for Application to BNCT. In Advances in
Neutron Capture Therapy; Soloway, A. H., Barth, R. F., Car-
penter, D. E., Eds.; Plenum Press: New York, 1993; pp 309-
313.
2
2
nique are described previously. Briefly, V-79 Chinese ham-
ster cells in exponential growth were incubated in DME
complete medium with varying concentrations of 3 for ∼16 h
(
concentrations shown in legend of Figure 1). After vigorously
washing the cells three times with phosphate buffer saline to
remove free extracellular drug, cells were trypsinized, har-
(16) Walton, M. I.; Bleehen, N. M.; Workman, P. Stimulation by
Localized Tumor Hyperthermia of Reductive Bioactivation of the
2-Nitroimidazole Benznidazole in Mice. Cancer Res. 1989, 49,
5
vested, and diluted to cell density of 3 × 10 cells/mL. One-
2
351-5.
milliliter cell suspensions were placed in 1.5 mL Eppendorf
microfuge tubes for irradiation over a range of doses at the
Brookhaven Medical Research Reactor. The irradiation pro-
(
17) Metzger, R. L.; Laster, R. G.; Fairchild, R. G.; Swenson, D. H.
Biological Efficacy of a Boronated Imidocaptate for Neutron
Capture. Proc. Am. Nucl. Soc. 1991, 5.