528 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 4
Shulman-Roskes et al.
cooled to ca. 0 °C (NaCl/wet ice bath). A balloon filled with
H2 and attached to a needle was then added to the reaction
flask through a septum. After 1 h of stirring at 0 °C under a
slight positive pressure of H2, the reaction mixture was filtered
and the filtrate was concentrated at reduced pressure and
ambient temperature to ca. 25% of its original volume. This
solution was stored overnight at -20 °C; the resultant crystals
were collected and washed with ether. Phosphoramide mus-
tard was obtained as the free acid in 44% yield (85 mg, 0.4
mmol): 1H NMR (D2O) δ 3.67 [t, 3J HH ) 7 Hz, 4H, two CH2Cl]
Each fraction was mixed with 1 mL scintillation fluor (Formula-
963, Packard Instrument Co., Meriden, CT), and radioactivity
was determined by scintillation counting.
The radioactivity in each fraction was expressed as a
fraction of the total recovered radioactivity. Assignments of
components of the chromatograms were based on consider-
ations of kinetics, UV absorption (for glutathionylated species),
literature reports of the same or similar compounds,10,25 and
cochromatography with authentic PM. Typical elution pattern
on the reverse phase column [compound (fractions)]: bishy-
droxylated 10 (6-9); monohydroxylated 8 (19-23); bisglu-
tathionylated 5 (24-28); monoglutathionylated 3 (33-37); PM
(38-41); thiosulfate monoalkylated intermediate (48-52); and
thiosulfate bisalkylated product (54-58). Note, ion pairing
between the thiosulfate adducts and the tetrabutylammonium
ion in the eluent reversed the polarity of these species.
3
3
and 3.36 [dt, J HH ) 7 Hz, J HP ) 11 Hz, 4H, two NCH2]; 31P
NMR (D2O) δ 9.4.
N,N-Bis(2-ch lor oeth yl)p h osph or odia m idic-3H Acid Cy-
cloh exyla m m on iu m Sa lt (P M-3H). Using unlabeled start-
ing materials, N,N-bis(2-chloroethyl)phosphorodiamidic acid
phenylmethyl ester [(ClCH2CH2)2NP(O)NH2(OCH2C6H5)] was
synthesized according to literature procedures.33 This material
was then randomly tritiated through a commercial source,
SibTech, Inc. (Tenafly, NJ ). All readily exchangeable labels
were removed, and the material which was returned to us had
an activity of 1.1 mCi/mg.
Ackn owledgm en t. We thank Dr. Vadappuram Chac-
ko (Department of Radiology, J ohns Hopkins University
School of Medicine) for his generous assistance with
many facets of the NMR work. We also thank Carol A.
Hartke and Sung Y. Han (Oncology Center, J ohns
Hopkins University School of Medicine) for their con-
tributions toward the synthesis of model and radiola-
beled materials. Spectra for Figure 1 were obtained with
the help of Dr. Tanya Dyakonov (Duke Comprehensive
Cancer Center); we are grateful for her time and
assistance. This work was supported in part by Public
Health Service Grants 5-RO1-CA16783 (O.M.C.),
CA51229 (M.P.G.), and CA09243-14 (training grant to
E.M.S.-R.) from the National Cancer Institute (Depart-
ment of Health and Human Services).
The solution from SibTech (15 mg of the tritiated benzyl
ester in 1.5 mL of ethanol) was transferred to a test tube (with
ethanol washings of original container) and to this was added
unlabeled benzyl ester (35 mg). [The total benzyl ester used,
then, was 50 mg or ca. 0.16 mmol.] The ethanol was
evaporated under
a stream of N2, and the residue was
dissolved in CH3OH (several milliliters). This was evaporated
under N2, and then more CH3OH was added and evaporated
again (to remove all traces of ethanol). The residue was taken
up in 0.7 mL of CH3OH and transferred to a 25-mL, three-
neck round-bottomed flask containing 10% Pd/C (3.3 mg). The
test tube was washed with more CH3OH (2 × 0.35 mL), and
this also was added to the round-bottom flask. The flask was
cooled to 5 °C (ice/water bath), and the contents were then
degassed for several minutes using a gas inlet (with stopcock)
connected to a water aspirator. The stopcock was then closed.
A balloon filled with H2 and attached to a needle was then
added to the reaction flask using a septum. The flask was
degassed a second time and then recharged with H2 via a
balloon. After 1 h of stirring at 5 °C under a slight positive
pressure of H2, the reaction mixture was filtered using CH3-
OH washes. Cyclohexylamine (0.66 mmol, 76 µL) was added
to the filtrate, and this was stirred at room temperature for 1
h. The solution was then concentrated at ambient tempera-
ture until the volume was reduced to ca. 1 mL. During storage
overnight at -20 °C, crystals formed. The mother liquor was
removed by pipet, and the crystals were washed with ether
and dried using a water aspirator. N,N-Bis(2-chloroethyl)-
phosphorodiamidic-3H acid (PM-3H) as the cyclohexylammo-
nium salt was obtained as white crystals in ca. 44% yield (ca.
0.07 mmol, 22.3 mg), 31P NMR (CD3OD) δ 12.5. A small
impurity (4% of total phosphorus intensity) was observed at δ
19.1. The activity of the product was 39 µCi/mg (ca. 13 µCi/
µmol). (Note: The “loss” of activity, relative to starting benzyl
ester, was due to the fact that in the labeling process tritium
was first incorporated into the most reactive positions, those
of the benzyl moiety. The mustard positions were more
resistant to the labeling and thus became tritiated last. The
benzyl group was subsequently removed during hydrogenation
leading to a large drop in activity. This was a drawback to
this synthetic pathway.)
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